Discovery of membrane active benzimidazole quinolones-based topoisomerase inhibitors as potential DNA-binding antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2016.01.052

A series of novel benzimidazole quinolones as potential antimicrobial agents were designed and synthesized. Most of the prepared compounds exhibited good or even stronger antimicrobial activities in comparison with reference drugs. The most potent compound 15m was membrane active and did not trigger the development of resistance in bacteria. It not only inhibited the formation of biofilms but also disrupted the established Staphylococcus aureus and Escherichia coli biofilms. It was able to inhibit the relaxation activity of E. coli topoisomerase IV at 10 μM concentration. Moreover, this compound also showed low toxicity against mammalian cells. Molecular modeling and experimental investigation of compound 15m with DNA suggested that this compound could effectively bind with DNA to form a steady 15m-DNA complex which might further block DNA replication to exert the powerful bioactivities.

Full text of DOI:10.1016/j.ejmech.2016.01.052

Accepted Manuscript
Discovery of membrane active quinolone-benzimidazoles based topoisomerase
inhibitors as potential DNA-binding antimicrobial agents
Ling Zhang, Dinesh Addla, Jeyakkumar Ponmani, Ao Wang, Dan Xie, Ya-Nan Wang,
Shao-Lin Zhang, Rong-Xia Geng, Gui-Xin Cai, Shuo Li, Cheng-He Zhou
PII:
S0223-5234(16)30060-5
10.1016/j.ejmech.2016.01.052
EJMECH 8342
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 October 2015
Revised Date: 25 January 2016
Accepted Date: 28 January 2016
Please cite this article as: L. Zhang, D. Addla, J. Ponmani, A. Wang, D. Xie, Y.-N. Wang, S.-L. Zhang,
R.-X. Geng, G.-X. Cai, S. Li, C.-H. Zhou, Discovery of membrane active quinolone-benzimidazoles
based topoisomerase inhibitors as potential DNA-binding antimicrobial agents, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.01.052.
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ACCEPTED MANUSCRIPT
Discovery of membrane active quinolone-benzimidazoles based topoisomerase inhibitors as potential
DNA-binding antimicrobial agents
a
a,
†
a,
‡
a
a
a
Ling Zhang , Dinesh Addla , Jeyakkumar Ponmani , Ao Wang , Dan Xie , Ya-Nan Wang , Shao-Lin
Zhang , Rong-Xia Geng , Gui-Xin Cai , Shuo Li and Cheng-He Zhou
a
a
a
b,*
a,*
a
Key Laboratory of Applied Chemistry of Chongqing Municipality, Institute of Bioorganic & Medicinal
Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China.
b
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, People's Republic
of China.
†
Postdoctoral fellow from Indian Institute of Chemical Technology, Hyderabad, 500607, India
‡
Ph. D candidate from India
*
Corresponding Authors:
E-mail: zhouch@swu.edu.cn (Cheng-He Zhou), lishuo@cqut.edu.cn (Shuo Li)
A novel series of membrane active quinolone benzimidazoles were synthesized and screened for their
antimicrobial activities. Molecular modeling and experimental investigation with DNA suggested the
possible antibacterial mechanism.

ACCEPTED MANUSCRIPT
Title page
Title:
Discovery of membrane active benzimidazole quinolones-based topoisomerase inhibitors as potential
DNA-binding antimicrobial agents
Author names and affiliations:
a
a, †
a, ‡
a
a
a
Ling Zhang , Dinesh Addla , Jeyakkumar Ponmani , Ao Wang , Dan Xie , Ya-Nan Wang , Shao-Lin
a
a
a
b,
a,
Zhang , Rong-Xia Geng , Gui-Xin Cai , Shuo Li * and Cheng-He Zhou *
a
Key Laboratory of Applied Chemistry of Chongqing Municipality, Institute of Bioorganic & Medicinal
Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China.
b
School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, People's Republic
of China.
†
Postdoctoral fellow from Indian Institute of Chemical Technology, Hyderabad, 500607, India
‡
Ph. D candidate from India
*
Corresponding Authors:
E-mail: zhouch@swu.edu.cn (Cheng-He Zhou), lishuo@cqut.edu.cn (Shuo Li)
Abstract:
A series of novel benzimidazole quinolones as potential antimicrobial agents were designed and
synthesized. Most of the prepared compounds exhibited good or even stronger antimicrobial activities in
comparison with reference drugs. The most potent compound 15m was membrane active and did not
trigger the development of resistance in bacteria. It not only inhibited the formation of biofilms but also
disrupted the established Staphylococcus aureus and Escherichia coli biofilms. It was able to inhibit the
relaxation activity of E. coli topoisomerase IV at 10 ꢀM concentration. Moreover, this compound also
showed low toxicity against mammalian cells. Molecular modeling and experimental investigation of
compound 15m with DNA suggested that this compound could effectively bind with DNA to form a steady
15m-DNA complex which might further block DNA replication to exert the powerful bioactivities.
1

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Keywords:
Quinolone; Benzimidazole; anti-MRSA; DNA; Topoisomerase
2

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1. Introduction
Bacterial topoisomerases are able to catalyze changes in the topology of DNA and are clinically
validated targets for antibacterial drug discovery. The complexity of reactions in DNA replication catalyzed
by DNA topoisomerase offers multiple opportunities for therapeutic intervention. Fluoroquinolones, a
clinically important kind of synthetic antibacterial agents, primarily target gyrase or topoisomerase in
bacteria to stabilize the cleavage complex at specific sites on DNA, then block DNA replication and finally
lead to bacterial cells death [1-3]. However, the emergence of resistance to quinolones has become one of
the most paramount public health threats in recent years and much effort has been focusing on this topic [4].
Some researches [5] show that an important reason to induce the resistance to quinolones may be ascribed
to the gene mutations in partial disruption of the water-metal ion bridge to hinder interactions of quinolone
with the catalytic site of enzyme. Further structure activity relationship (SAR) reveals that the groups at
C–7 position of quinolones are vicinity to the Arg456 residue in the topo IV–DNA complex whose
mutation causes quinolone resistance. Meanwhile, the C–7 substituents in quinolone are capable to
influence the cell permeability [6] which were also in relation with bacterial resistance [7]. Therefore, the
structural modification of the C–7 position in quinolone nucleus is a promising strategy to extend the
antibacterial spectrum and overcome the drug-resistance [8-10].
Benzimidazole with special benzene-fused imidazole ring prevalently present in a range of bioactive
agents (e.g. albendazole, mebendazole, candesartan, astemizole) [11,12] has drawn much attention in
medicinal chemistry especially antimicrobial aspect in very recent years [13,14]. Benzimidazole derivatives
have been revealed to be specific topoisomerase inhibitors. It could bind to the minor groove of DNA, trap
the reversible cleavable complex and produce highly specific single-strand DNA breakage [15].
Particularly, the C–2 alkylations of the benzimidazole nucleus have been actively investigated. Moreover,
benzimidazoles as purine-like mimics are sometimes addressed as 1,3-dideaza-purines and are always used
as model compounds to compete with purines [16]. These benzimidazole types of purine mimics have the
potential to kill the bacterial strains or inhibit their growth through inhibition of the synthesis of nucleic
acids and proteins inside the bacterial cell wall with distinct mechanism from that of common antibacterial
drugs. All the above observations reveal that the development of benzimidazoles as novel antibacterial
agents may be served as a good strategy to exploit new anti-infective agents with better activity and less
bacterial resistance.
In view of this situation and as an extension of our previous work [17], it is of great interest for us to
combine quinolones through their C–7 position with benzimidazole ring to generate a new structural type
3

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of potentially antimicrobial hybrids and investigate their effect on antimicrobial activity (Figure 1). It is
expected for these hybrids of two different type of topoisomerase inhibitors to have large potentiality in the
treatment of disease-caused bacteria. Design of structures for target compounds was mainly from three
aspects: (I) Structural modification on the benzimidazole nucleus; (II) Structural change on the quinolone
ring; (III) The variation of linker between benzimidazole and quinolone nucleus. The related considerations
were given as follows:
(1) Much literature evidenced that linkers could exert large effect on biological potency by regulating the
lipid-water partition coefficient and binding affinity to target enzyme [18-21], thus various linkers between
benzimidazole and quinolone nucleus in target compounds were incorporated, including no linker and
flexible aliphatic chain such as different alkyl chains (e.g. one carbon -CH -, two carbon -CH –CH -
2
2
2
groups);
2) It is well known that amino moiety is an important structural fragment prevalently present in
(
numerous clinical drugs, in which it could form hydrogen bonds, coordinate with metal ions and accept
protons and/or perform quaternization to not only beneficially regulate physicochemical properties of
desired molecules but also helpfully interact with various enzymes and receptors in biological system.
Therefore, the -CH –NH- amino moiety as bioisostere of -CH –CH - linker was introduced into target
2
2
2
compounds (Scheme 4). The N-Mannich reaction was successfully developed to achieve this goal in this
paper. It is believed that this N-Mannich functional group could influence the bioactivities by the
enhancement of cell permeability [22];
(3) Schiff bases (-C=N-) as important structural fragment in many antibacterial agents had the capacity to
modulate the pharmacokinetic properties and enhance the antimicrobial efficacy [23]. As a comparison
with the above -CH –NH- group, the rigid Schiff bases (-C=N-) was incorporated into target compounds
2
(Scheme 4) to investigate its effect on antimicrobial activities;
(4) Numerous works revealed that substituents could significantly influence the bioactivities [24-26].
Especially, it was proposed that proper lipophilicity was a very important factor in the quinolones intestinal
absorption. Rationally, various aliphatic chains with different lengths and substituted phenyl moieties
including chloro, fluoro and nitro groups were further modified at the selected most active compounds
based on the preliminary active screening (Scheme 3);
(5) The substituted groups on quinolone nucleus were also considered to investigate the effects of the
substituents in the quinolone nucleus on antibacterial activities.
Figure 1
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The designed structures of this series of novel benzimidazole quinolones are shown in Schemes 1–4. All
the newly synthesized compounds were characterized by spectral analysis and evaluated for their
antimicrobial activities in vitro against four Gram-positive bacteria, six Gram-negative bacteria and five
fungi according to the National Committee for Clinical Laboratory Standards (NCCLS). Bactericidal
kinetic assay, antibiofilm activity, bacterial membrane permeabilization, antibacterial resistance study and
cytotoxicity as well as the preliminary antimicrobial mechanism with topoisomerase and DNA to highly
active compound were also investigated.
2
Results and discussion
2.1. Chemistry
The synthetic route of target benzimidazole quinolones was outlined in Schemes 1–4. Initially, N-formyl
quinolone 2 was selected as intermediate to construct the target benzimidazole-quinolone hybrid in which
the 2-position of benzimidazole is directly linked with the piperazine ring in quinolone, and it could be
conveniently obtained by the treatment of commercially available quinolone 1a with formamide at 70 ºC.
Its structure was confirmed by spectra and also supported by single crystal X-ray diffraction analysis
(
Figure 2, supporting information). However, the reaction of N-formyl derivative 2 with
benzene-1,2-diamine was difficult via cyclization to produce benzimidazoles. This might be attributed to
the undesired acid effect of carboxyl group to the acid-sensitive amino material. To confirm this hypothesis,
quinolones 1a–b were esterified to afford the corresponding esters 3a–b in good yields, and further treated
with formamide to give N-formyl intermediates 4a–b in high yields. The following cyclization in
1,4-dioxane with anhydrous copper sulfate was able to occur smoothly to access the desired bezimidazoles
5a–b, and then were further hydrolyzed at 100 ºC in the presence of sodium hydroxide solution (3%) to
afford the target benzimidazole quinolones 6a–b with yields of 85.2–87.8% (Scheme 1).
Scheme 1
The benzimidazole-quinolone hybrids 13–16 with different linkers and substituents were synthesized
from haloalkyl benzimidazoles 8a–f, 10a–h and 12a–g which were prepared from o-phenylene diamines
and halogen-substituted carboxylic acids. The intermediates 2-chloromethyl benzimidazoles 8a–f were
easily obtained by the direct reaction of a series of o-phenylene diamine or its substituted benzene
derivatives respectively with chloroacetic acid or chloropropionic acid. The alkylation of o-phenylene
diamine with alkyl halides and then cyclization with chloroacetic acid produced the desired intermediates
5

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N-alkyl benzimidazoles 10a–h in high yields. N-Halobenzyl derivatives 12a–g were provided in
quantitative yields by the reaction of o-phenylene diamine with a series of halobenzyl halides, and then
treatment with chloroacetic acid (Scheme 2). These prepared benzimidazole halides 8a–f, 10a–h and 12a–g
respectively were further coupled with the piperazine in quinolones 1a–b to smoothly produce the target
benzimidazole-quinolone hybrids 13–16 (Scheme 3).
Scheme2
Scheme 3
The -CH –NH- bridged benzimidazole quinolone hybrids 17a–b were conveniently prepared in the
2
yields of 29.4–31.5% via Mannich reaction starting from quinolones 1a–b and paraformaldehyde (Scheme
4). Shiffbase-linked hybrids 18a–b were smoothly obtained in 39.5–41.4% yields by condensation reaction
of N-formyl quinolone esters 4a–b with 2-aminobenzimidazole. However, these prepared esters 18a–b
could not be hydrolyzed to produce desired target carboxyl derivatives, which might be related with the
base-sensitive shiffbases (Scheme 4). It was also noteworthy that the direct reaction of N-formyl quinolone
2
with 2-aminobenzimidazole via condensation was difficult to go.
1
13
All the newly synthesized compounds were characterized by H NMR, C NMR, IR, MS and HRMS
spectra. Their spectral analyses were consistent with the assigned structures and the spectral data were
given in the experimental section.
Scheme 4
2.2. Biological activity
All the newly prepared compounds were evaluated for antimicrobial activities in vitro against four
Gram-positive bacteria (Staphylococcus aureus ATCC25923, Methicillin-resistant Staphylococcus aureus
N315, Bacillus subtilis ATCC6633 and Micrococcus luteus ATCC4698), six Gram-negative bacteria
(Escherichia coli DH52, Escherichia coli JM109, Shigella dysenteriae, Pseudomonas aeruginosa
ATCC27853, Bacillus proteus ATCC13315 and Salmonella enterica ATCC14028) and five fungi (Candida
albicans ATCC10231, Candida mycoderma ATCC9888, Candida utilis ATCC9950, Saccharomyces
cerevisia ATCC9763 and Aspergillus flavus ATCC204304) using two-fold broth dilution method in 96-well
microtest plates recommended by NCCLS [27]. The biological tests were carried out in triplicate. Minimal
inhibitory concentration (MIC, ꢀg/mL) was defined as the lowest concentration of the tested new
compounds that completely inhibited the growth of strains. Currently available antimicrobial drugs such as
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chloromycin, norfloxacin, ciprofloxacin and fluconazole were used as standard drugs. Clinafloxacin, a
newly synthesized fluoroquinolone antimicrobial agent that has been demonstrated to be more active than
currently available fluoroquinolones with expanded activity against most bacteria was also selected as
standard [10a].
The obtained results (depicted in Table 1 and supporting information: Table S1) revealed that most of the
prepared benzimidazole quinolones could effectively inhibit the growth of all the tested strains in vitro.
Compounds 13a and 13f with methylene linker between benzimidazole and the piperazine ring in
quinolone gave low MIC values in the range of 0.125–2 ꢀg/mL which exhibited obvious stronger
antimicrobial efficacy in comparison with norfloxacin (MIC = 1–16 ꢀg/mL) and ciprofloxacin (MIC =
0
.25–1 ꢀg/mL). In contrast to this, the directly linked ones 6a–b (MIC = 0.5–8 ꢀg/mL), and CH CH
2 2
(
compounds 16a–b: MIC = 0.5–128 ꢀg/mL), CH NH (compounds 17a–b: MIC = 0.25–16 ꢀg/mL) orC=N
2
(compounds 18a–b: MIC = 2–128 ꢀg/mL) bridged derivatives all gave generally bigger MIC values. This
fact that either the decrease or increase of the linker length was unfavorable for the bioactivity,
demonstrated the proper length of linker exerted an important influence in enhancing bioactivity, and the
methylene linker was the most suitable one. Further, the replacement of one CH moiety in the CH CH
2
2
2
linker (compounds 16a–b) by NH group (compounds 17a–b) was beneficial for the activity.
The introduction of chloro, fluoro, bromo or nitro group into the 5(6)-position of benzimidazole nucleus
resulted in lower activity and they contributed to the antibacterial activities in the order of chloro ꢀ fluoro
ꢀ
nitro ꢀ bromo group. Among them, chloro modified derivative 13h showed obvious increase in
inhibiting the growth of pathogenic bacterial strains (MIC = 0.25–0.5 ꢀg/mL) in comparison with reference
drug chloromycin (MIC = 8–32 ꢀg/mL) and superior to norfloxacin (MIC = 1–16 ꢀg/mL), ciprofloxacin
(MIC = 1–2 ꢀg/mL) and clinafloxacin (MIC = 0.25–1 ꢀg/mL) respectively.
The further structure–activity relationship demonstrated that the significant effect of the substituents at
the N-position of benzimidazoles on biological activity. Most of N-alkyl derivatives 14a–p displayed
moderate antibacterial activity against the tested bacteria. In comparison with other bacteria, they were
relatively less sensitive to E. coli JM109 and S. aureus. Among all the alkyl derivatives, compounds 14e
and 14m with octyl group gave the best bioactivity with MIC values in the range of 0.5–32 ꢀg/mL. The
replacement of octyl group by ethyl or propyl fragment resulted in low activity towards the tested strains.
Moreover, when the alkyl substituents were extended to decyl, dodecyl and octadecyl groups, compounds
showed relatively weaker inhibitory activity. The short alkyl chains with poor lipophilicity and long alkyl
ones with high lipophilicity in these compounds might make them unfavorable to be delivered to the
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binding sites.
In comparison with the alkyl derivatives 14a–p, most of halobenzyl ones 15a–n exerted relatively better
activities in inhibiting the growth of the tested strains. Notably, in this series of halobenzyl derivatives,
2
,4-dichlorobenzyl hybrid 15m gave the best antibacterial efficiencies with MIC values of 0.0312 to 8
g/mL towards the corresponding bacteria. Noticeably, this compound demonstrated effective bioactivity
against M. luteus (MIC = 0.0625 ꢀg/mL), B. proteus (MIC = 0.0312 ꢀg/mL), and S. enterica (MIC =
ꢀ
0.0312 ꢀg/mL) which were 128– and 1025–fold more potent than the reference drug chloromycin, and
32-fold more active than norfloxacin and ciprofloxacin. Particularly, it was even 8–, 16– and 8–fold more
active than clinafloxacin. Besides, compound 15i with 3-chlorophenyl group also displayed fairly good
antibacterial activities with the MIC values in the range of 0.125–8 ꢀg/mL in comparison with reference
drug chloromycin (MIC = 8–32 ꢀg/mL) and superior to norfloxacin (MIC = 1–16 ꢀg/mL), ciprofloxacin
(MIC = 1–2 ꢀg/mL) and clinafloxacin (MIC = 0.25–1 ꢀg/mL) respectively. The replacement of
chlorobenzyl moiety by fluorobenzyl group (compounds 15j and 15k) resulted in unfavorable potency
against all the tested strains at the concentrations of 0.25–256 ꢀg/mL. The substitution of chlorine atom in
compound 15m by nitro group to give derivative 15n was not beneficial for the antibacterial activities.
These results manifested that 2,4-dichlorophenyl derivative 15m should be worthy to be further
investigated as potential antimicrobial agent.
Excitedly, the directly linked benzimidazole hybrid 6b, methylene bridged ones 13f and 13h, halobenzyl
derivatives 15a, 15f, 15i–j and 15m, NH CH linked ones 17a–b all exhibited good biological activity
2
2
against MRSA with quite low MIC values of 0.125–0.5 ꢀg/mL which were more active than chloromycin
MIC = 16 ꢀg/mL), norfloxacin (MIC = 8 ꢀg/mL), ciprofloxacin (MIC = 2 ꢀg/mL) and clinafloxacin (MIC
1 ꢀg/mL). Especially, methylene bridged 13f (MIC = 0.125 ꢀg/mL), 15a (MIC = 0.125 ꢀg/mL), 15f
(
=
(
MIC = 0.125 ꢀg/mL) and 15m (MIC = 0.125 ꢀg/mL) exerted superior anti-MRSA activity to norfloxacin
MIC = 8 ꢀg/mL), ciprofloxacin (MIC = 2 ꢀg/mL) and clinafloxacin (MIC = 1 ꢀg/mL). These implied that
(
this new type of benzimidazole-quinolone hybrids should have the possibility to be developed as
anti-MRSA agents.
In addition, the in vitro antifungal evaluation were also evaluated for these prepared benzimidazole
quinolone hybrids and revealed that they exhibited moderate antifungal activities against most of the tested
fungal strains (supporting information).
Table 1
2.3 Bactericidal kinetic assay
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In order to understand the antibacterial potency of the prepared compounds, time−kill kinetics
experiments of highly active benzimidazole quinolone hybrid 15m against MRSA were performed. As
shown in Figure 3, it revealed more than 3 log (CFU/mL) reduction in the number of viable bacteria within
an hour at a concentration of 6 × MIC. The result manifested that this compound had rapid-bactericidal
activity.
Figure 3
2.4 Antibiofilm activity
Bacteria with biofilms are inherently insensitive to antimicrobial agents and they are much more resistant
to conventional antibiotic treatment [7]. Moreover, biofilms account for more than 80% of microbial
infections in humans. Compound with the ability not only to inhibit bacterial biofilm formation but also to
disperse established biofilms would be an ideal agent to tackle infections caused by bacteria. Both
Gram-positive MRSA and Gram-negative E. coli with biofilms are known to cause many infections in
humans. Hence the ability of compound 15m to inhibit both MRSA and E. coli DH52 biofilm formation
was evaluated. Further, the ability of target compound to disperse established MRSA and E. coli DH52
biofilms was also investigated.
2.4.1 Biofilm inhibition
Compound 15m was found to be an effective inhibitor for both MRSA and E. coli DH52 biofilm
formation. The IC (the concentration of the compounds that inhibits 50% biofilm development) values of
5
0
15m against MRSA and E. coli DH52 biofilm formation were found to be 0.125 and 0.25 ꢀg/mL,
respectively (Figure 4), which made it a promising antibiofilm agent.
Figure 4
2.4.2 Biofilm disruption
Compound 15m was selected to evaluate the efficiency to eradicate the established MRSA and E. coli
DH52 biofilms. Matured MRSA biofilm (developed for 24 h) having an initial count of 11.8 log CFU/mL
1
0
of bacteria was treated at four different concentrations (0.125, 0.25, 1 and 2 ꢀg/mL). After the treatment,
the cell viabilities in biofims decreased to 10.5, 9.6, 5, and 0.1 log CFU/mL at 0.125, 0.25, 1 and 2 ꢀg/mL,
1
0
whereas cell viability in untreated biofilm increased to 13.6 log10 CFU/mL. Thus, the EC50 (the
concentration of compound that reduces 50% bacterial titer of a preformed biofilm) value of the biocide
was 0.76 ꢀg/mL. More importantly, the biocide at the highest tested concentration (2 ꢀg/mL) showed about
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zero cell viability in 24 h MRSA biofilm indicating nearly complete eradication of established biofilm
(
Figure 5A). Matured E. coli biofilm (developed for 48 h) having an initial count of 16.2 log CFU/mL of
10
bacteria was similarly treated at four different concentrations (0.25, 0.5, 2, and 4 ꢀg/mL respectively). After
the treatment, the cell viabilities in biofims for the different concentrations decreased to 12.2, 10.1, 7.2, and
3
.6 log CFU/mL at 0.25, 0.5, 2, and 4 ꢀg/mL respectively (Figure 5B). Thus, the EC value of compound
1
0
5
0
15m was 1.5 ꢀg/mL. Moreover, this compound was able to reduce viable bacteria even in 48 h grown
matured E. coli biofilm. The above results clearly indicated the biocide ability to disperse the biofilms and
hence to kill the microorganism present in the biofilm.
Figure 5
2.5 Bacterial membrane permeabilization
Natural and synthetic membrane active antibacterial agents offer a hope to solve the problem of bacterial
resistance as the membrane-active nature imparts low propensity for the development of resistance. The
ability of compound 15m to permeabilize the bacterial cell membrane was studied using propidium iodide
(PI) (supporting information). This dye can pass through the membrane of compromised bacterial cells and
fluoresces upon binding to the DNA [28,29]. As can be seen from Figure 6A and Figure 6B, the tested
compound was efficient in permeabilizing the membranes of both Gram-positive (MRSA) and
Gram-negative (E. coli DH52) bacteria.
Figure 6
2.6 Resistance study
The high-level resistance of norfloxacin to MRSA strains was observed, so we investigated the ability of
MRSA to develop drug resistance against the most potent compound 15m. We exposed a standard strain of
MRSA towards increasing concentrations of compound 15m from sub-MIC for sustained passages and
determined the MIC values of compound 15m for each passage of MRSA. The freshly diluted MRSA
5
strains (1.0 × 10 CFU) in the broth medium in the presence of 0.08 ꢀg/mL (2/3 MIC) of compound 15m
were cultured at 37 ºC for 12 h on a shaker bed at 90 rpm, and tested its sensitivity to 0.125 ꢀg/mL (the
original MIC) of compound 15m for each passage of MRSA. After 12 passages in the presence of
two-thirds of the MIC of compound 15m, bacterial resistance to the original MIC of compound 15m (0.125
ꢀg/mL) did not emerge, and the MIC values just changed between 0.125 ꢀg/mL and 0.5 ꢀg/mL. To make
comparative analysis, parallel cultures were exposed to 2-fold dilutions of norfloxacin as a positive control.
By contrast, MRSA quickly developed resistance to norfloxacin, showing resistance to the original MIC (8
1
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ꢀg/mL) after 6 passages. This assay indicated that bacteria MRSA did not develop resistance easily against
compound 15m as they did against norfloxacin [30].
2.7 Inhibition of DNA Relaxation Activity of EcTopo IV.
The DNA relaxation assay was done for the newly synthesized compound 15m. In this experiment,
superhelical plasmid DNA was incubated with EcTopo IV at increasing concentrations of the prepared
compound (1, 5, 10, 25, and 50 ꢀM) and DNA relaxation products were then resolved by gel
electrophoresis on 1% agarose gel. Compound 15m was able to inhibit the relaxation activity of EcTopo IV
at 10 ꢀM concentration (Figure S1, supporting information).
2.8 Cytotoxicity.
The cytotoxic activities of compound 15m were tested by Cell Counting Kit-8 (CCK8) method. The in
vitro cytotoxicity was evaluated in HEK293 (human embryonic kidney 293) cells. As shown in Figure 7,
compound 15m showed relative low toxicity to normal human embryonic kidney cells at high
concentration, and the IC value was 106 ꢀg/mL.
5
0
Figure 7
2.9 Molecular modeling
To rationalize the observed antibacterial activity and understand the possible mechanism of the target
benzimidazole quinolones, a flexible ligand receptor docking investigation was undertaken. The crystal
structure data (topoisomerase IV–DNA complex) were obtained from the protein data bank (PDB code:
NA1059), which was representative target to investigate the antibacterial mechanism of quinolones [5,31].
Target compound 15m and clinafloxacin were selected to dock with the topoisomerase IV–DNA complex.
According to the docking evaluation, hybrid 15m possessed high total score (14.30) even superior to
clinafloxacin (score: 12.45) against topoisomerase IV–DNA complex. Interactions of compound 15m and
clinafloxacin with topoisomerase IV–DNA receptor were shown in Figure 8(i-iii). The docking result of
compound 15m with topoisomerase IV–DNA complex might rationalize the possible antibacterial
mechanism. The carboxyl group of this molecule was in close vicinity to the residue Arg117 of the
topoisomerase IV–DNA complex. The prepared molecule 15m could also form hydrogen bonds with Ser79
of topoisomerase IV–DNA complex through the hydrogen atom of carboxyl group. Furthermore,
compound 15m could intercalate into superhelical DNA of the enzyme–DNA complex (Figure 8 (iii) and
supporting information: Figure S2). This cooperative binding might be beneficial to stabilize the
1
1

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quinolone–DNA enzyme complex, which might be responsible for the strong inhibitory efficacy of
compound 15m against MRSA. Since clinafloxacin suffered the exposed severe side effects including
phototoxicity and photohemolysis, these benzimidazole quinolones are worthy to be further investigated as
potential candidates for infective chemotherapy.
Figure 8
2.10 Interactions with DNA
Based on the good activities of target compounds and the high score results of molecular modeling, the
incorporation of benzimidazole might improve the interaction between target compounds and DNA of
topoisomerase IV–DNA complexes. So the interaction between the most active compound 15m and calf
thymus DNA (CT DNA, a DNA model with medical importance, low cost and ready availability properties)
was evaluated to further explore the preliminary antimicrobial mechanism. In contrast to this interaction
between compound 15m and DNA, the reference drug norfloxacin was employed to investigate the
interaction with CT DNA.
Figure 9
With a fixed concentration of DNA, UV–vis absorption spectra were recorded with the increasing
amount of compound 15m. As shown in Figure 9, 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 15m. Meanwhile the phenomenon displayed that the absorption value of
simply sum of free DNA and free compound 15m was a little greater than the measured value of
15m–DNA complex. These meant a weak hypochromic effect existed between DNA and compound 15m.
Furthermore, the intercalation of the aromatic chromophore of compound 15m into the helix and the strong
overlap of π-π* states of the large π-conjugated system with the electronic states of DNA bases were
consistent with the observed spectral changes [32,33].
On the basis of variations in the absorption spectra of DNA upon binding to 15m, equation (1) can be
utilized to calculate the binding constant (K).
0
A
ξC
ξC
1
=
+
×
(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 15m at 260 nm, ξ
C
and ξ - are the absorption coefficients of compound 15m and 15m–DNA complex respectively. The plot
D C
0
0
of A /(A-A ) versus 1/[compound 15m] is constructed by using the absorption titration data and linear
1
2

ACCEPTED MANUSCRIPT
4
fitting (Figure 10), yielding the binding constant, K = 4.30 ×10 L/mol, R = 0.999, SD = 0.24 (R is the
correlation coefficient. SD is standard deviation).
Figure 10
As shown in Figure 11, there were some differences between norfloxacin and compound 15m. The
hyperchromism of DNA–norfloxacin complex was observed. With the gradually increasing concentration
of compound 15m or norfloxacin, the DNA–compound 15m complex showed obviously stronger spectral
change than DNA–norfloxacin complex, which suggested the new benzimidazole quinolone gave better
interaction with DNA. Therefore, this type of interaction with DNA might improve binding ability between
these compounds and topo–DNA complex.
Figure 11
3. Conclusion
In conclusion, a series of benzimidazole quinolones 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 bacterial strains especially against MRSA even superior to reference drugs.
Structure–activity relationship indicated that lengths of linker exerted obvious influence in enhancing
bioactivity, and the methylene linker was the most active one. The prepared compound 15m showed
significant inhibition against all the tested bacterial strains with low inhibitory concentrations (MIC =
0.0312–8 ꢀg/mL). Additionally, it not only inhibited the formation of biofilm but also dispersed the
established bacterial biofilms. Further, it also displayed low toxicity against mammalian cells and did not
trigger development of resistance in bacteria. Thus, this membrane-active antibacterial compound could be
potentially used as therapeutic agent to treat bacterial infections caused by multidrug-resistant bacteria.
Moreover, the most active compound 15m was able to inhibit the relaxation activity of E. coli
topoisomerase IV at 10 ꢀM concentration. Further molecular modeling indicated the good antibacterial
activity of the prepared compound 15m against quinolone-resistant bacterial strains and the possible
mechanism might be explained by the noncovalent interaction between compound 15m and topo IV–DNA
complex, especially these hydrogen bonds between molecule and Ser79 (resistance mutation region of topo
IV–DNA complex). The preliminary interactive investigations of compound 15m with CT DNA revealed
that it gave better interaction with DNA than norfloxacin, which might improve binding ability between
this compound with topo IV–DNA complex.
1
3

ACCEPTED MANUSCRIPT
4. Experimental protocols
4.1 General methods
Melting points were recorded on X-6 melting point apparatus and uncorrected. TLC analysis was done
using pre-coated silica gel plates. FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer
-1
(
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 fluorescence spectra were recorded on F-7000
Spectrofluorimeter (Hitachi, Tokyo, Japan) equipped with 1.0 cm quartz cells, the widths of both the
excitation and emission slit were set as 2.5 nm. The UV spectrum was recorded at room temperature on a
TU-2450 spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz
cells. Tris(hydroxymethyl) aminomethane (Tris) and concentrated HCl were analytical purity. Sample
masses were weighed on a microbalance with a resolution of 0.1 mg. All other chemicals and solvents were
commercially available, and were used without further purification.
4.1.1. 1-Ethyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (2)
Norfloxacin 1a (2.000 g, 0.006 mol) was stirred in formamide (50 mL) at 70 ꢁ for 2 h. After the mixture
was kept in the cold overnight, the formed solid was filtered off and recrystallized with methanol to give
the pure target compound 2 as pale solid (1.457 g). Yield: 70%; mp: > 250 °C (literature [21] mp:
-
1
2
1
1
92–293 °C). IR (KBr, cm ) ν: 3055 (aromatic C-H), 2937, 2813 (CH , CH ), 1726, 1721 (C=O), 1615,
3 2
1
559, 1538, 1517, 1447 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.31 (s, 1H, COOH), 8.96 (s,
6
H, quinolone 2-H), 8.13 (s, 1H, CHO), 7.94 (d, J = 13.1 Hz, 1H, quinolone 5-H), 7.23 (d, J = 7.2 Hz, 1H,
quinolone 8-H), 4.60 (q, J = 7.0 Hz, 2H, quinolone N-CH ), 3.61 (dd, J = 10.5, 6.8 Hz, 4H, piperazine
2
N-CH ), 3.35 (d, J = 5.2 Hz, 2H, piperazine N-CH ), 3.31–3.28 (m, 2H, piperazine N-CH ), 1.43 (t, J = 7.1
2
2
2
1
3
Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.7, 165.4, 165.9, 159.1, 154.8, 145.2, 142.7,
2
3
6
+
1
38.3, 118.3, 106.0, 105.3, 99.7, 48.5, 46.1, 44.4, 42.0, 40.5, 34.8, 8.6 ppm; MS (m/z): 348 [M+H] ;
+
HRMS (TOF) calcd. for C H FN O : [M+H] , 348.1360; found, 348.1367.
1
7
18
3
4
4.1.2. 1-Ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid methyl ester (3a)
Compound 3a was prepared according to the literature procedure starting from norfloxacin (3.190 g,
1
4

ACCEPTED MANUSCRIPT
0.010 mol), thionyl chloride (15 mL) and methanol (150 mL). Yield: 67%; mp: 190−191 °C. (literature [34]
mp: 189−190 °C)
4
.1.3. 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid methyl
ester (3b)
Compound 3b was prepared according to the literature procedure starting from ciprofloxacin (3.310 g,
.010 mol), thionyl chloride (15 mL) and methanol (150 mL). Yield: 69%. (literature [35] mp: 272−274 °C)
0
4.1.4. 1-Ethyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid methyl
ester (4a)
Compound 3a (3.330 g, 0.010 mol) was stirred in formamide (50 mL) at 70 ꢁ for 2 h. After the mixture
was kept in the cold overnight, the formed solid was filtered off and recrystallized with methanol to give
-1
the pure target compound 4a as pale solid (3.480 g). Yield: 96.4%; mp: 148−150 °C; IR (KBr, cm ) ν: 3055
(
aromatic C-H), 2938, 2812 (CH , CH ), 1725, 1720 (C=O), 1614, 1555, 1536, 1507, 1448 (aromatic
3
2
1
frame); H NMR (600 MHz, CDCl ) δ: 8.47 (s, 1H, quinolone 2-H), 8.13 (s, 1H, CHO), 8.11 (d, J = 13.0
3
Hz, 1H, quinolone 5-H), 6.78 (d, J = 6.7 Hz, 1H, quinolone 8-H), 4.23 (q, J = 7.2 Hz, 2H, quinolone
N-CH ), 3.93 (s, 3H, OCH ), 3.81–3.77 (m, 2H, piperazine N-CH ), 3.63–3.60 (m, 2H, piperazine N-CH ),
2
3
2
2
3
.30–3.26 (m, 2H, piperazine N-CH ), 3.23–3.20 (m, 2H, piperazine N-CH ), 1.55 (t, J = 7.2 Hz, 3H,
2
2
1
3
CH CH ) ppm; C NMR (151 MHz, CDCl ) δ: 176.8, 168.2, 161.6, 157.9, 155.9, 149.2, 143.7, 131.2,
2
3
3
+
1
19.5, 109.0, 105.5, 99.8, 47.2, 46.2, 44.7, 44.2, 40.7, 35.0, 9.6 ppm; MS (m/z): 362 [M+H] ; HRMS (TOF)
+
calcd. for C H FN O : [M+H] , 362.1516; found, 362.1518.
1
8
21
3
4
4.1.5. 1-Cyclopropyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
methyl ester (4b)
Compound 4b was prepared according to the procedure described for compound 4a starting from
compound 3b (3.450 g, 0.010 mol) and formamide (50 mL). The target compound 4b (3.570 g) was
-1
obtained as yellow solid. Yield: 95.8%; mp: 149−150 °C; IR (KBr, cm ) ν: 3085 (aromatic C-H), 2958,
1
2
818 (CH , CH ), 1726, 1721 (C=O), 1611, 1557, 1536, 1505, 1445 (aromatic frame); H NMR (600 MHz,
3
2
CDCl ) δ: 8.45 (s, 1H, quinolone 2-H), 8.14 (s, 1H, CHO), 8.09 (d, J = 12.9 Hz, 1H, quinolone 5-H), 6.71
3
(
d, J = 6.6 Hz, 1H, quinolone 8-H), 3.87 (m, 1H, cyclopropyl-CH), 3.90 (s, 3H, OCH ), 3.79–3.75 (m, 2H,
3
piperazine N-CH ), 3.62–3.60 (m, 2H, piperazine N-CH ), 3.30–3.25 (m, 2H, piperazine N-CH ),
2
2
2
3
.22–3.19 (m, 2H, piperazine N-CH ), 1.36 (d, J = 6.2 Hz, 2H, cyclopropyl-CH ), 1.23 (d, J = 6.8 Hz, 2H,
2
2
1
3
cyclopropyl-CH ) ppm; C NMR (151 MHz, CDCl ) δ: 175.4, 168.8, 163.7, 158.1, 155.5, 149.1, 144.1,
2
3
1
5

ACCEPTED MANUSCRIPT
+
1
32.5, 119.4, 109.1, 105.3, 99.7, 47.3, 46.1, 44.8, 44.3, 40.8, 35.1, 9.9 ppm; MS (m/z): 374 [M+H] ;
+
HRMS (TOF) calcd. for C H FN O : [M+H] , 374.1516; found, 374.1518.
1
9
21
3
4
4.1.6.
7-(4-(1H-Benzimidazol-2-yl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquino
line-3-carboxylic acid methyl ester (5a)
To a stirred solution of benzene-1,2-diamine (0.300 g, 2.770 mmol) and copper sulfate (0.005 g, 0.030
mmol) in 1,4-dioxane (15 mL), a solution of compound 4a (1.000 g, 2.770 mmol) in 1,4-dioxane (5 mL)
was added dropwise during 15 min and heated at reflux for 24 h. After the reaction was completed
(monitored by TLC, chloroform/methanol (30/1, V/V), the mixture was filtered. After the 1,4-dioxane 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 5a as white power (0.520 g). Yield:
-
1
4
1
2
1.7%; mp: 155−157 °C; IR (KBr, cm ) ν: 3025 (aromatic C-H), 2945, 2825 (CH , CH ), 1721 (C=O),
3 2
1
622, 1563, 1553, 1545, 1435 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 8.09 (s, 1H, quinolone
6
-H), 7.82–7.78 (m, 1H, quinolone 5-H), 7.46 (ddd, J = 8.8, 6.0, 3.2 Hz, 2H, benzimidazole 4,7-H), 7.13
(ddd, J = 38.6, 5.8, 3.1 Hz, 2H, benzimidazole 5,6-H), 6.38 (s, 1H, quinolone 8-H), 4.72 (m, 2H, quinolone
N-CH ), 3.58 (s, 3H, OCH ), 3.34 (s, 4H, piperazine N-CH ), 2.48 (s, 4H, piperazine N-CH ), 1.51 (s, 3H,
2
3
2
2
1
3
CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.8, 166.4, 154.2, 152.6, 151.7, 148.4, 145.6, 139.7,
2
3
6
+
1
22.1, 119.0, 111.5, 111.3, 107.3, 106.8, 79.7, 52.8, 49.8, 49.1, 36.4, 14.0 ppm; MS (m/z): 450 [M+H] ;
+
HRMS (TOF) calcd. for C H FN O : [M+H] , 450.1941; found, 450.1942.
2
4
25
5
3
4.1.7.
7-(4-(1H-Benzimidazol-2-yl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro
quinoline-3-carboxylic acid methyl ester (5b)
Compound 5b was prepared according to the procedure described for compound 5a starting from
compound 4b (1.000 g, 2.680 mmol) and formamide (50 mL). The target compound 5b (0.530 g) was
-1
obtained as yellow solid. Yield: 42.8%; mp: 157−158 °C; IR (KBr, cm ) ν: 3085 (aromatic C-H), 2958,
1
2
829 (CH , CH ), 1721 (C=O), 1611, 1557, 1536, 1505, 1445 (aromatic frame); H NMR (600 MHz,
3
2
DMSO-d ) δ: 8.17 (s, 1H, quinolone 2-H), 7.90 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.58−7.50 (m, 2H,
6
benzimidazole 4,7-H), 7.15 (m, 2H, benzimidazole 5,6-H), 6.58 (s, 1H, quinolone 8-H), 3.80 (m, 1H,
cyclopropyl-CH), 3.57 (s, 3H, OCH ), 3.35 (s, 4H, piperazine N-CH ), 2.49 (s, 4H, piperazine N-CH ),
3
2
2
1
3
1
.35 (d, J = 6.1 Hz, 2H, cyclopropyl-CH ), 1.21 (d, J = 6.3 Hz, 2H, cyclopropyl-CH ) ppm; C NMR (151
2
2
MHz, DMSO-d ) δ: 176.7, 166.3, 154.1, 152.2, 151.7, 148.4, 145.6, 139.7, 122.1, 119.0, 111.5, 111.3,
6
+
1
07.3, 106.8, 52.7, 49.5, 49.2, 35.3, 8.0 ppm; MS (m/z): 462 [M+H] ; HRMS (TOF) calcd. for
+
C H FN O : [M+H] , 462.1941; found, 462.1945.
2
5
25
5
3
1
6

ACCEPTED MANUSCRIPT
4.1.8.
7-(4-(1H-Benzimidazol-2-yl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid (6a)
To a stirred solution of aqueous solution sodium hydroxide (3%, 20 mL) was added compound 4a (0.449
g, 0.001 mol). The mixture was stirred at 100 °C for 2 h. After the reaction was completed (monitored by
TLC, chloroform/ methanol 30/1, V/V), the solution was treated with formic acid to approximately reach
pH 7. The mixture was filtered and washed with water for three times to give compound 6a (0.371 g) as
-
1
white solid. Yield: 85.2%; mp: 157−158 °C; IR (KBr, cm ) ν: 3085 (aromatic C-H), 2948, 2831 (CH ,
3
1
CH ), 2945, 1721 (C=O), 1611, 1557, 1536, 1505, 1445 (aromatic frame); H NMR (600 MHz, DMSO-d )
2
6
δ: 15.28 (s, 1H, COOH), 12.25 (s, 1H, benzimidazole-NH), 8.99 (s, 1H, quinolone 2-H), 8.02 (d, J = 13.1
Hz, 1H, quinolone 5-H), 7.50 (s, 2H, benzimidazole 4,7-H), 7.22 (d, J = 6.6 Hz, 1H, quinolone 8-H), 7.12
(
s, 2H, benzimidazole 5,6-H), 4.66–4.58 (m, 2H, quinolone N-CH ), 3.61 (s, 4H, piperazine N-CH ), 3.27
2
2
1
3
(
s, 4H, piperazine N-CH ), 1.46 (t, J = 6.9 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ:
2 2 3 6
176.7, 166.5, 153.2, 152.7, 151.5, 148.2, 145.7, 140.1, 123.2, 118.3, 111.6, 111.2, 106.9, 106.5, 79.6, 49.8,
+
+
4
9.0, 36.3, 14.1 ppm; MS (m/z): 436 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 436.1785;
23 23 5 3
found, 436.1787.
4.1.9.
7-(4-(1H-Benzimidazol-2-yl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro
quinoline-3-carboxylic acid (6b)
Compound 6b was prepared according to the procedure described for compound 6a starting from
compound 5b (0.461 g, 0.001 mol) and aqueous solution sodium hydroxide (3%, 20 mL). The target
-1
compound 6b (0.390 g) was obtained as yellow solid. Yield: 87.8%; mp: 158−159 °C; IR (KBr, cm ) ν:
1
3
018 (aromatic C-H), 2839 (CH ), 1720 (C=O), 1625, 1542, 1478, 1467 (aromatic frame); H NMR (600
2
MHz, DMSO-d ) δ: 15.26 (s, 1H, COOH), 12.15 (s, 1H, benzimidazole-NH), 8.58 (s, 1H, quinolone 2-H),
6
8.03 (d, J = 12.3 Hz, 1H, quinolone 5-H), 7.49 (s, 2H, benzimidazole 4,7-H), 7.22 (d, J = 6.5 Hz, 1H,
quinolone 8-H), 7.11 (s, 2H, benzimidazole 5,6-H), 3.80 (m, 1H, cyclopropyl-CH), 3.62 (s, 4H, piperazine
N-CH ), 3.26 (s, 4H, piperazine N-CH ), 1.32 (d, J = 6.2 Hz, 2H, cyclopropyl-CH ), 1.09 (d, J = 6.1 Hz,
2
2
2
1
3
2
1
H, cyclopropyl-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 174.9, 163.5, 153.4, 151.9, 151.1, 148.2,
2 6
44.9, 140.1, 123.2, 118.3, 111.6, 111.2, 106.9, 106.5, 79.6, 50.1, 49.0, 35.2, 8.1 ppm; MS (m/z): 448
+
+
[
M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 448.1785; found, 448.1787.
24 24 5 3
4.1.10. General procedures for the preparation of intermediates (8–12)
The intermediates 8–12 were prepared according to the previously reported methods [36].
4.1.11.
7-(4-((1H-Benzimidazol-2-yl)methyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydro
1
7

ACCEPTED MANUSCRIPT
quinoline-3-carboxylic acid (13a)
A mixture of norfloxacin (1.920 g, 0.006 mol), potassium carbonate (0.830 g, 0.006 mol) and potassium
iodide (0.100 g, 0.006 mol) in acetonitrile (100 mL) was stirred at 50 °C for 1 h. After the mixture was
cooled to room temperature, compound 8a (1.000 g, 0.006 mol) was added. The reaction mixture was then
heated at 50 °C for 3 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 13a as white
-1
power (1.120 g). Yield: 41.7%; mp: 155−157 °C; IR (KBr, cm ) ν: 3108 (aromatic C-H), 2939, 2870 (CH ,
3
1
CH ), 1719 (C=O), 1635, 1588, 1478, 1457 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.34 (s,
2
6
1H, COOH), 12.35 (s, 1H, benzimidazole-NH), 8.94 (s, 1H, quinolone 2-H), 7.90 (d, J = 13.3 Hz, 1H,
quinolone 5-H), 7.50 (s, 2H, benzimidazole 4,7-H), 7.17 (s, 1H, quinolone 8-H), 7.16ꢂ7.12 (m, 2H,
benzimidazole 5,6-H), 4.58 (q, J = 6.8 Hz, 2H, quinolone N-CH ), 3.84 (s, 2H, benzimidazole-CH ), 3.37 (s,
2
2
1
3
4
H, piperazine N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.41 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR
2
2
2
3
(
151 MHz, DMSO-d ) δ: 176.8, 166.4, 154.2, 152.6, 151.7, 148.4, 145.6, 139.7, 122.1, 119.0, 111.5, 111.3,
6
+
1
07.3, 106.8, 79.7, 60.2, 55.7, 52.8, 49.8, 49.1, 14.0 ppm; MS (m/z): 450 [M+H] ; HRMS (TOF) calcd. for
+
C H FN O : [M+H] , 450.1941; found, 450.1940.
2
4
25
5
3
4.1.12.
Ethyl-6-fluoro-7-(4-((6-fluoro-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (13b)
Compound 13b was prepared according to the procedure described for compound 13a starting from
compound 8b (0.554 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.415 g, 0.003
mol) and potassium iodide (0.050 g, 0.003 mol). The target compound 13b (0.558 g) was obtained as white
-1
solid. Yield: 39.8%; mp: 157−158 °C; IR (KBr, cm ) ν: 3078 (aromatic C-H), 2942, 2862 (CH , CH ),
3
2
1
1
728 (C=O), 1614, 1540, 1501, 1442 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.21 (s, 1H,
6
COOH), 8.64 (s, 1H, quinolone 2-H), 7.87 (d, J = 13.3 Hz, 1H, quinolone 5-H), 7.55–7.49 (m, 1H,
quinolone 8-H), 7.47 (dd, J = 8.1, 4.7 Hz, 1H, benzimidazole 7-H), 7.03 (dd, J = 18.8, 9.2 Hz, 2H,
benzimidazole 4,5-H), 4.57 (q, J = 6.7 Hz, 2H, quinolone N-CH ), 3.88 (s, 2H, benzimidazole CH ),
2
2
3
.33–3.30 (m, 4H, piperazine N-CH ), 2.69 (s, 4H, piperazine N-CH ), 1.40 (t, J = 7.0 Hz, 3H, CH CH )
2 2 2 3
1
3
ppm; C NMR (151 MHz, DMSO-d ) δ: 176.4, 165.9, 156.1, 151.6, 151.5, 148.5, 145.6, 141.4, 137.6,
6
+
1
22.7, 118.1, 110.5, 108.3, 106.2, 79.6, 60.1, 52.6, 49.1, 48.0, 13.9 ppm; MS (m/z): 468 [M+H] ; HRMS
+
(
TOF) calcd. for C H F N O : [M+H] , 468.1847; found, 468.1846.
2
4
24
2
5
3
1
8

ACCEPTED MANUSCRIPT
4.1.13.
7-(4-((6-Chloro-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (13c)
Compound 13c was prepared according to the procedure described for compound 13a starting from
compound 8c (0.603 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.415 g, 0.003
mol) and potassium iodide (0.050 g, 0.003 mol). The target compound 13c (0.582 g) was obtained as white
-1
solid. Yield: 40.1%; mp: 157−159 °C; IR (KBr, cm ) ν: 3082 (aromatic C-H), 2955, 2861 (CH , CH ),
3
2
1
1
718 (C=O), 1615, 1545, 1525, 1493 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.33 (s, 1H,
6
COOH), 12.54 (s, 1H, benzimidazole-NH), 8.93 (s, 1H, quinolone 2-H), 7.89 (d, J = 13.3 Hz, 1H,
quinolone 5-H), 7.55 (d, J = 24.3 Hz, 2H, quinolone 8-H, benzimidazole 7-H), 7.20−7.14 (m, 2H,
benzimidazole 4,5-H), 4.58 (d, J = 6.6 Hz, 2H, quinolone N-CH ), 3.86 (s, 2H, benzimidazole-CH ), 3.38 (s,
2
2
1
3
4
H, piperazine N-CH ), 2.72 (s, 4H, piperazine N-CH ), 1.42 (t, J = 6.1 Hz, 3H, CH CH ) ppm; C NMR
2
2
2
3
(
151 MHz, DMSO-d ) δ: 176.4, 166.9, 156.1, 152.4, 151.0, 147.5, 146.6, 143.4, 137.5, 123.7, 118.2, 110.4,
6
+
1
07.3, 105.7, 78.6, 61.1, 52.7, 49.9, 48.3, 14.0 ppm; MS (m/z): 485 [M+H] ; HRMS (TOF) calcd. for
+
C H ClFN O : [M+H] , 484.1552; found, 484.1554.
2
4
24
5
3
4.1.14.
7-(4-((6-Bromo-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (13d)
Compound 13d was prepared according to the procedure described for compound 13a starting from
compound 8d (0.735 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.415 g, 0.003
mol) and potassium iodide (0.050 g, 0.003 mol). The target compound 13d (0.558 g) was obtained as
-
1
yellow solid. Yield: 35.2%; mp: 159−160 °C; IR (KBr, cm ) ν: 3079 (aromatic C-H), 2940, 2861 (CH ,
3
1
CH ), 1729 (C=O), 1617, 1540, 1504, 1442 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.25 (s,
2
6
1H, COOH), 12.51 (s, 1H, benzimidazole-NH), 8.94 (s, 1H, quinolone 2-H), 7.86 (d, J = 13.2 Hz, 1H,
quinolone 5-H), 7.55 (d, J = 24.2 Hz, 2H, quinolone 8-H, benzimidazole 7-H), 7.21–7.13 (m, 2H,
benzimidazole 4,5-H), 4.53 (d, J = 6.2 Hz, 2H, quinolone N-CH ), 3.81 (s, 2H, benzimidazole-CH ), 3.29 (s,
2
2
1
3
4
H, piperazine N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.40 (t, J = 6.1 Hz, 3H, CH CH ) ppm; C NMR
2
2
2
3
(
151 MHz, DMSO-d ) δ: 177.4, 167.8, 157.2, 153.4, 151.0, 148.3, 145.6, 143.2, 136.3, 124.7, 117.1, 111.4,
6
+
1
06.4, 105.5, 78.7, 61.2, 52.8, 49.8, 46.3, 14.1 ppm; MS (m/z): 529 [M+H] ; HRMS (TOF) calcd. for
+
C H BrFN O : [M+H] , 528.1047; found, 528.1046.
2
4
24
5
3
4.1.15.
1-Ethyl-6-fluoro-7-(4-((6-nitro-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (13e)
Compound 13e was prepared according to the procedure described for compound 13a starting from
1
9

ACCEPTED MANUSCRIPT
compound 8e (0.635 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.415 g, 0.003
mol) and potassium iodide (0.050 g, 0.003 mol). The target compound 13e (0.536 g) was obtained as
-
1
yellow solid. Yield: 36.1%; mp: 158−159 °C; IR (KBr, cm ) ν: 3080 (aromatic C-H), 2949, 2862 (CH ,
3
1
CH ), 1719 (C=O), 1613, 1542, 1522, 1492 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.24 (s,
2
6
1H, COOH), 12.52 (s, 1H, benzimidazole-NH), 8.93 (s, 1H, quinolone 2-H), 7.87 (d, J = 13.1 Hz, 1H,
quinolone 5-H), 7.45 (d, J = 22.1 Hz, 2H, quinolone 8-H, benzimidazole 7-H), 7.20–7.14 (m, 2H,
benzimidazole 4,5-H), 4.54 (d, J = 6.1 Hz, 2H, quinolone N-CH ), 3.82 (s, 2H, benzimidazole-CH ), 3.28 (s,
2
2
1
3
4
H, piperazine N-CH ), 2.70 (s, 4H, piperazine N-CH ), 1.41 (t, J = 5.9 Hz, 3H, CH CH ) ppm; C NMR
2
2
2
3
(
151 MHz, DMSO-d ) δ: 177.5, 167.7, 157.4, 152.8, 151.0, 147.9, 145.6, 144.1, 142.3, 141.7, 133.1, 117.4,
6
+
1
16.4, 115.5, 110.4, 107.5, 103.5, 61.2, 53.8, 49.8, 48.5, 46.3, 14.1 ppm; MS (m/z): 495 [M+H] ; HRMS
+
(
TOF) calcd. for C H FN O : [M+H] , 495.1792; found, 495.1793.
24 24 6 5
4.1.16.
7-(4-((1H-Benzimidazol-2-yl)methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (13f)
Compound 13f was prepared according to the procedure described for compound 13a starting from
compound 8a (1.000 g, 0.006 mol), ciprofloxacin (1.986 g, 0.006 mol), potassium carbonate (0.830 g,
0.006 mol) and potassium iodide (0.100 g, 0.006 mol). The target compound 13f (1.324 g) was obtained as
-
1
yellow solid. Yield: 47.8%; mp: 157−158 °C; IR (KBr, cm ) ν: 3079 (aromatic C-H), 2862 (CH ), 1727
2
1
(
C=O), 1619, 1540, 1514, 1442 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.20 (s, 1H, COOH),
6
8
4
.66 (s, 1H, quinolone 2-H), 7.90 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.58−7.50 (m, 3H, benzimidazole
,7-H, quinolone 8-H), 7.17 (s, 2H, benzimidazole 5,6-H), 3.88 (s, 2H, quinolone N-CH ), 3.81 (s, 1H,
2
cyclopropyl-CH), 3.39 (s, 4H, piperazine N-CH ), 2.76 (s, 4H, piperazine N-CH ), 1.31 (d, J = 5.9 Hz, 2H,
2
2
1
3
cyclopropyl-CH ), 1.18 (s, 2H, cyclopropyl-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.8, 166.4,
2
2
6
1
4
4
54.2, 152.6, 151.7, 148.4, 145.6, 139.7, 122.1, 119.0, 111.5, 111.3, 107.3, 106.8, 79.7, 60.2, 55.7, 52.8,
+
+
9.8, 49.1, 36.4, 8.0 ppm; MS (m/z): 462 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] ,
2
5
25
5
3
62.1941; found, 462.1942.
.1.17. 1-Cyclopropyl-6-fluoro-7-(4-((6-fluoro-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
4
dihydroquinoline-3-carboxylic acid (13g)
Compound 13g was prepared according to the procedure described for compound 13a starting from
compound 8b (0.554 g, 0.003 mol), ciprofloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g,
0.003 mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 13g (0.515 g) was obtained as
-
1
white solid. Yield: 35.8%; mp: 158−159 °C; IR (KBr, cm ) ν: 3042 (aromatic C-H), 2856 (CH ), 1726
2
2
0

ACCEPTED MANUSCRIPT
1
(
C=O), 1618, 1546, 1525, 1491, 1445 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.32 (s, 1H,
6
COOH), 8.63 (s, 1H, quinolone 2-H), 7.88 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.56–7.51 (m, 1H,
quinolone 8-H), 7.48 (dd, J = 8.0, 4.6 Hz, 1H, benzimidazole 7-H), 7.03 (dd, J = 18.8, 9.2 Hz, 2H,
benzimidazole 4,5-H), 3.88 (s, 2H, benzimidazole CH ), 3.74 (dd, J = 8.3, 5.1 Hz, 1H, cyclopropyl-CH),
2
3
.32–3.30 (m, 4H, piperazine N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.30 (t, J = 6.7 Hz, 2H,
2
2
1
3
cyclopropyl-CH ), 1.16 (s, 2H, cyclopropyl-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.5, 166.3,
2
2
6
1
4
4
56.2, 152.5, 151.5, 147.4, 145.5, 140.5, 138.2, 121.9, 117.1, 111.5, 111.3, 107.3, 106.8, 79.7, 60.2, 52.8,
+
+
9.8, 49.1, 35.4, 8.0 ppm; MS (m/z): 480 [M+H] ; HRMS (TOF) calcd. for C H F N O : [M+H] ,
2
5
24
2
5
3
80.1847; found, 480.1848.
.1.18. 7-(4-((6-Chloro-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
4
dihydroquinoline-3-carboxylic acid (13h)
Compound 13h was prepared according to the procedure described for compound 13a starting from
compound 8c (0.603 g, 0.003 mol), ciprofloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g,
0.003 mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 13h (0.497 g) was obtained as
-
1
white solid. Yield: 33.4%; mp: 158−160 °C; IR (KBr, cm ) ν: 3029 (aromatic C-H), 2876 (CH ), 1718
2
1
(
C=O), 1629, 1541, 1498, 1457 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.29 (s, 1H, COOH),
6
1
2.34 (s, 1H, benzimidazole-NH), 8.88 (s, 1H, quinolone 2-H), 7.85 (d, J = 13.3 Hz, 1H, quinolone 5-H),
.71 (d, J = 6.2 Hz, 1H, quinolone 8-H), 7.55 (d, J = 24.3 Hz, 1H, benzimidazole 7-H), 7.28–7.13 (m, 2H,
7
benzimidazole 4,5-H), 3.88 (s, 2H, benzimidazole-CH ), 3.70 (s, 1H, cyclopropyl-CH), 3.36 (s, 4H,
2
piperazine N-CH ), 2.70 (s, 4H, piperazine N-CH ), 1.29 (d, J = 6.4 Hz, 2H, cyclopropyl-CH ), 1.12 (s, 2H,
2
2
2
1
3
cyclopropyl-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.4, 167.3, 155.3, 151.4, 150.3, 146.3, 145.6,
2
6
140.5, 138.1, 120.8, 118.0, 111.5, 110.4, 106.3, 105.3, 78.8, 61.2, 53.7, 49.8, 48.3, 35.3, 8.1 ppm; MS (m/z):
+
+
4
96 [M+H] ; HRMS (TOF) calcd. for C H ClFN O : [M+H] , 496.1552; found, 496.1553.
2
5
24
5
3
4.1.19.
7-(4-((6-Bromo-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (13i)
Compound 13i was prepared according to the procedure described for compound 13a starting from
compound 8d (0.737 g, 0.003 mol), ciprofloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g,
0.003 mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 13i (0.557 g) was obtained as
-
1
white solid. Yield: 34.4%; mp: 157−158 °C; IR (KBr, cm ) ν: 3085 (aromatic C-H), 2886 (CH ), 1721
2
1
(
C=O), 1611, 1557, 1536, 1505, 1445 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.22 (s, 1H,
6
COOH), 12.04 (s, 1H, benzimidazole-NH), 8.58 (s, 1H, quinolone 2-H), 7.65 (d, J = 13.2 Hz, 1H,
2
1

ACCEPTED MANUSCRIPT
quinolone 5-H), 7.71 (d, J = 6.1 Hz, 1H, quinolone 8-H), 7.51 (d, J = 24.1 Hz, 1H, benzimidazole 7-H),
7
3
1
1
.25–7.15 (m, 2H, benzimidazole 4,5-H), 3.86 (s, 2H, benzimidazole-CH ), 3.71 (s, 1H, cyclopropyl-CH),
2
.35 (s, 4H, piperazine N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.25 (d, J = 6.3 Hz, 2H, cyclopropyl-CH ),
2
2
2
1
3
.12 (s, 2H, cyclopropyl-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.5, 167.4, 156.2, 152.5, 150.4,
2
6
46.7, 145.2, 142.5, 138.2, 121.8, 117.8, 111.6, 110.3, 107.3, 105.7, 78.7, 61.5, 52.5, 49.7, 48.5, 35.5, 8.1
+
+
ppm; MS (m/z): 541 [M+H] ; HRMS (TOF) calcd. for C H BrFN O : [M+H] , 540.1047; found,
2
5
24
5
3
540.1049.
4.1.20.
1-Cyclopropyl-6-fluoro-7-(4-((6-nitro-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (13j)
Compound 13j was prepared according to the procedure described for compound 13a starting from
compound 8e (0.635 g, 0.003 mol), ciprofloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g,
0.003 mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 13j (0.492 g) was obtained as
-
1
white solid. Yield: 32.4%; mp: 159−160 °C; IR (KBr, cm ) ν: 3027 (aromatic C-H), 2836 (CH ), 1722
2
1
(
C=O), 1624, 1564, 1555, 1542, 1436 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.27 (s, 1H,
6
COOH), 13.16 (s, 1H, benzimidazole-NH), 9.06 (d, J = 4.9 Hz, 1H, quinolone 2-H), 8.71 (s, 1H, quinolone
-H), 8.47 (d, J = 24.4 Hz, 1H, benzimidazole 7-H), 8.15 (s, 1H, benzimidazole 4,5-H), 7.95 (s, 1H,
5
benzimidazole 4,5-H), 7.61 (d, J = 7.3 Hz, 1H, quinolone 8-H), 4.84 (s, 2H, quinolone N-CH ), 3.87 (s, 1H,
2
cyclopropyl-CH), 3.45 (s, 4H, piperazine N-CH ), 2.81 (s, 4H, piperazine N-CH ), 1.37 (d, J = 6.5 Hz, 2H,
2
2
1
3
cyclopropyl-CH ), 1.24 (d, J = 6.8 Hz, 2H, cyclopropyl-CH ) ppm; C NMR (151 MHz, DMSO-d ) δ:
2
2
6
176.5, 167.5, 156.3, 152.6, 150.3, 147.5, 145.7, 144.2, 142.5, 141.6, 135.2, 118.8, 116.8, 115.4, 110.3,
+
1
07.3, 102.7, 61.6, 52.4, 49.6, 48.2, 35.2, 8.1 ppm; MS (m/z): 507 [M+H] ; HRMS (TOF) calcd. for
+
C H FN O : [M+H] , 507.1792; found, 507.1790.
2
5
24
6
5
4.1.21.
1-Ethyl-7-(4-((1-ethyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14a)
Compound 14a was prepared according to the procedure described for compound 13a starting from
compound 10a (0.558 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14a (0.980 g) was obtained as white
-
1
solid. Yield: 66.3%; mp: 150−152 °C; IR (KBr, cm ) ν: 3018 (aromatic C-H), 2961, 2862, 2849 (CH ,
3
1
CH ), 1718 (C=O), 1621, 1542, 1463, 1454 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.37 (s,
2
6
1H, COOH), 8.98 (s, 1H, quinolone 2-H), 7.94 (d, J = 13.1 Hz, 1H, quinolone 5-H), 7.63 (dd, J = 24.8, 7.9
Hz, 2H, benzimidazole 4,7-H), 7.25 (ddd, J = 20.1, 13.5, 7.1 Hz, 3H, quinolone 8-H, benzimidazole 5,6-H),
2
2

ACCEPTED MANUSCRIPT
4
.64–4.58 (m, 2H, benzimidazole N-CH ), 4.42 (dd, J = 13.8, 6.7 Hz, 2H, benzimidazole CH ), 3.95 (s, 2H,
2
2
quinolone CH ), 3.38 (s, 4H, piperazine N-CH ), 2.75 (s, 4H, piperazine N-CH ), 1.45 (dd, J = 14.5, 7.2 Hz,
2
2
2
1
3
6
1
1
4
H, CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 171.8, 167.2, 160.4, 154.5, 151.1, 148.4, 145.9, 140.6,
3 6
37.6, 132.9, 129.0, 116.7, 112.2, 108.7, 106.6, 65.5, 60.3, 52.8, 49.8, 44.7, 30.5, 21.2, 19.1, 15.3, 14.5,
+
+
4.1 ppm; MS (m/z): 478 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 478.2254; found,
2
6
29
5
3
78.2257.
4.1.22.
1-Ethyl-6-fluoro-4-oxo-7-(4-((1-propyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic acid (14b)
Compound 14b was prepared according to the procedure described for compound 13a starting from
compound 10b (0.647 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14b (0.930 g) was obtained as white
-1
solid. Yield: 61.3%; mp: 152−153 °C; IR (KBr, cm ) ν: 3118 (aromatic C-H), 2960, 2873, 2852 (CH ,
3
1
CH ), 1719 (C=O), 1655, 1578, 1488, 1449 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.16 (s,
2
6
1
2
3
H, COOH), 8.97 (s, 1H), 7.96 (dd, J = 12.9, 6.3 Hz, 1H), 7.87 (dd, J = 45.1, 7.9 Hz, 2H), 7.55–7.47 (m,
H), 7.27 (d, J = 6.5 Hz, 1H), 4.67 (s, 2H), 4.63 (d, J = 6.8 Hz, 2H), 4.44 (t, J = 7.3 Hz, 2H), 3.60 (s, 4H),
1
3
.34 (s, 4H), 1.93–1.87 (m, 2H), 1.46 (t, J = 7.1 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H) ppm; C NMR (151 MHz,
DMSO-d ) δ: 176.6, 166.5, 154.1, 148.9, 144.9, 137.6, 133.9, 125.1, 120.2, 118.3, 116.4, 114.5, 112.6,
6
+
1
11.8, 107.7, 106.4, 52.5, 51.4, 49.5, 48.2, 46.1, 22.7, 14.6, 11.0 ppm; MS (m/z): 492 [M+H] ; HRMS
+
(
TOF) calcd. for C H FN O : [M+H] , 492.2411; found, 492.2415.
27 31 5 3
4.1.23.
1-Ethyl-6-fluoro-4-oxo-7-(4-((1-pentyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic acid (14c)
Compound 14c was prepared according to the procedure described for compound 13a starting from
compound 10c (0.710 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14c (0.940 g) was obtained as white
-
1
solid. Yield: 58.6%; mp: 156−157 °C; IR (KBr, cm ) ν: 3029 (aromatic C-H), 2942, 2852, 2842 (CH ,
3
1
CH ), 1717 (C=O), 1624, 1540, 1497, 1456 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.33 (s,
2
6
1H, COOH), 8.94 (s, 1H, quinolone 2-H), 7.90 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.58 (dd, J = 29.8, 7.9
Hz, 2H, benzimidazole 4,7-H), 7.26−7.14 (m, 3H, quinolone 8-H, benzimidazole 5,6-H), 4.57 (q, J = 7.0
Hz, 2H, benzimidazole N-CH ), 4.33−4.29 (m, 2H, benzimidazole CH ), 3.89 (d, J = 5.3 Hz, 2H, quinolone
2
2
CH ), 3.32 (s, 4H, piperazine N-CH ), 2.70 (s, 4H, piperazine N-CH ), 1.87−1.80 (m, 2H, CH (CH ) CH ),
2
2
2
2
2 2
3
1
3
1
.44−1.33 (m, 7H, CH (CH ) CH ), 0.88 (t, J = 6.9 Hz, 3H, quinolone CH ) ppm; C NMR (151 MHz,
2 2 2 3 3
2
3

ACCEPTED MANUSCRIPT
DMSO-d ) δ: 176.6, 166.5, 154.2, 152.5, 151.1, 148.9, 145.9, 142.4, 137.7, 135.9, 122.7, 121.5, 119.4,
6
111.6, 110.5, 107.8, 106.2, 54.9, 52.8, 49.9, 49.6, 43.9, 29.6, 29.0, 22.3, 14.7, 14.4 ppm; MS (m/z): 520
+
+
[
M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 520.2724; found, 520.2722.
29 35 5 3
4.1.24.
1-Ethyl-6-fluoro-7-(4-((1-hexyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14d)
Compound 14d was prepared according to the procedure described for compound 13a starting from
compound 10d (0.777 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14d (0.936 g) was obtained as white
-
1
solid. Yield: 56.6%; mp: 154−156 °C; IR (KBr, cm ) ν: 3080 (aromatic C-H), 2945, 2852, 2829 (CH ,
3
1
CH ), 1719 (C=O), 1615, 1540, 1525, 1435 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.31 (s,
2
6
1
H, COOH), 8.93 (s, 1H, quinolone 2-H), 7.87 (d, J = 13.1 Hz, 1H, quinolone 5-H), 7.58 (dd, J = 29.5, 7.7
Hz, 2H, benzimidazole 4,7-H), 7.25–7.11 (m, 3H, quinolone 8-H, benzimidazole 5,6-H), 4.56 (q, J = 6.8 Hz,
H, benzimidazole N-CH ), 4.34–4.28 (m, 2H, benzimidazole CH ), 3.89 (d, J = 5.3 Hz, 2H, quinolone
2
2
2
CH ), 3.33 (s, 4H, piperazine N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.86–1.81 (m, 2H, CH (CH ) CH ),
2
2
2
2
2 3
3
1
3
1
.45–1.32 (m, 6H, CH (CH ) CH ), 0.92 (d, J = 7.4 Hz, 3H, CH (CH ) CH ) ppm; C NMR (151 MHz,
2 2 3 3 2 2 3 3
DMSO-d ) δ: 176.7, 166.4, 152.8, 151.3, 148.3, 144.8, 141.7, 138.8, 135.8, 122.5, 121.7, 118.5, 111.9,
6
111.7, 107.5, 106.7, 56.1, 52.7, 49.8, 44.6, 36.3, 31.6, 29.4, 25.5, 22.5, 14.7, 13.5 ppm; MS (m/z): 534
+
+
[
M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 534.2880; found, 534.2884.
30 37 5 3
4.1.25.
1-Ethyl-6-fluoro-7-(4-((1-octyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14e)
Compound 14e was prepared according to the procedure described for compound 13a starting from
compound 10e (0.864 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14e (0.775 g) was obtained as white
-
1
solid. Yield: 43.6%; mp: 156−157 °C; IR (KBr, cm ) ν: 3015 (aromatic C-H), 2952, 2869, 2840 (CH ,
3
1
CH ), 1718 (C=O), 1626, 1545, 1498, 1457 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.17 (s,
2
6
1
H, COOH), 8.65 (s, 1H, quinolone 2-H), 7.89 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.57 (dt, J = 17.5, 8.5
Hz, 3H, benzimidazole 4,7-H, quinolone 8-H), 7.21 (dt, J = 33.0, 7.2 Hz, 2H, benzimidazole 5,6-H), 4.29
dt, J = 25.8, 7.5 Hz, 2H, benzimidazole N-CH ), 3.88 (s, 2H, benzimidazole CH ), 3.32 (s, 4H, piperazine
(
2
2
N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.86–1.79 (m, 2H, CH (CH ) CH ), 1.40–1.17 (m, 10H,
2
2
2
2 5
3
13
CH (CH ) CH ), 0.84 (t, J = 6.7 Hz, 3H, CH (CH ) CH ), 0.78 (t, J = 6.3 Hz, 3H, quinolone CH ) ppm; C
2
2 5
3
2
2 5
3
3
NMR (151 MHz, DMSO-d ) δ: 176.8, 166.3, 154.3, 154.0, 152.6, 151.1, 145.5, 142.4, 122.5, 121.8, 111.5,
6
2
4

ACCEPTED MANUSCRIPT
110.7, 107.3, 106.7), 57.1, 54.9, 52.8, 49.9, 44.0, 43.8, 36.3, 31.7, 29.9, 26.9, 26.7, 14.4, 7.9 ppm; MS
+
+
(
m/z): 562 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 562.3193; found, 562.3195.
32 41 5 3
4.1.26.
7-(4-((1-Decyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14f)
Compound 14f was prepared according to the procedure described for compound 13a starting from
compound 10f (0.951 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14f (0.791 g) was obtained as white
-
1
solid. Yield: 42.4%; mp: 163−164 °C; IR (KBr, cm ) ν: 3090 (aromatic C-H), 2948, 2867, 2850 (CH ,
3
1
CH ), 1718 (C=O), 1630, 1540, 1500, 1459 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.31 (s,
2
6
1
H, COOH), 8.94 (s, 1H, quinolone 2-H), 7.90 (d, J = 13.0 Hz, 1H, quinolone 5-H), 7.57 (dd, J = 31.5, 7.9
Hz, 2H, benzimidazole 4,7-H), 7.20 (dt, J = 17.8, 7.0 Hz, 3H, quinolone 8-H, benzimidazole 5,6-H), 4.57
d, J = 6.8 Hz, 2H, benzimidazole N-CH ), 4.30 (t, J = 7.3 Hz, 2H, quinolone N-CH ), 3.88 (s, 2H,
(
2
2
benzimidazole CH ), 3.32 (s, 4H, piperazine N-CH ), 2.70 (s, 4H, piperazine N-CH ), 1.86–1.79 (m, 2H,
2
2
2
1
3
CH (CH ) CH ), 1.42−1.09 (m, 17H, CH (CH ) CH ), 0.74 (d, J = 5.3 Hz, 3H, CH ) ppm; C NMR (151
2
2 7
3
2
2 7
3
3
MHz, DMSO-d ) δ: 176.6, 166.5, 154.1, 151.1, 148.8, 145.8, 142.5, 137.6, 135.9, 122.5, 121.7, 119.4,
6
111.5, 110.6, 106.1, 54.9, 52.7, 49.9, 49.5, 49.0, 44.0, 31.6, 29.9, 29.4, 29.2, 26.9, 22.5, 14.7, 14.3 ppm; MS
+
+
(
m/z): 590 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 590.3506; found, 590.3505.
34 45 5 3
4.1.27.
7-(4-((1-Dodecyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14g)
Compound 14g was prepared according to the procedure described for compound 13a starting from
compound 10g (1.038 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14g (0.867 g) was obtained as white
-
1
solid. Yield: 44.4%; mp: 169−170 °C; IR (KBr, cm ) ν: 3092 (aromatic C-H), 2982, 2878, 2834 (CH ,
3
1
CH ), 1727 (C=O), 1626, 1520, 1489, 1466, 1451 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ:
2
6
1
5.31 (s, 1H, COOH), 8.95 (s, 1H, quinolone 2-H), 7.91 (dd, J = 18.0, 12.4 Hz, 1H, quinolone 5-H), 7.57
dd, J = 27.9, 7.2 Hz, 2H, benzimidazole 4,7-H), 7.19 (dd, J = 23.8, 15.7 Hz, 3H, quinolone 8-H,
benzimidazole 5,6-H), 4.57 (s, 2H, quinolone N-CH ), 4.29 (s, 2H, benzimidazole N-CH ), 3.88 (s, 2H,
(
2
2
benzimidazole CH ), 3.31 (s, 4H, piperazine N-CH ), 2.70 (s, 4H, piperazine N-CH ), 1.83 (s, 2H,
2
2
2
CH CH (CH ) CH ), 1.38–1.06 (m, 21H, CH CH (CH ) CH , CH ), 0.78 (d, J = 5.7 Hz, 3H,
2
2
2 9
3
2
2
2 9
3
3
1
3
CH CH (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.6, 166.5, 154.2, 151.2, 148.9, 145.9,
2
2
2 9
3
6
142.5, 137.6, 136.1, 122.5, 121.7, 119.2, 111.8, 110.7, 107.6, 106.1, 55.0, 52.9, 50.0, 49.4, 43.9, 31.6, 29.8,
2
5

ACCEPTED MANUSCRIPT
+
+
2
9.1, 26.7, 22.5, 14.6, 14.3 ppm; MS (m/z): 618 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] ,
3
6
49
5
3
6
18.3819; found, 618.3822.
.1.28. 1-Ethyl-6-fluoro-7-(4-((1-octadecyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
4
dihydroquinoline-3-carboxylic acid (14h)
Compound 14h was prepared according to the procedure described for compound 13a starting from
compound 10h (1.298 g, 0.003 mol), norfloxacin (1.000 g, 0.003 mol), potassium carbonate (0.414 g, 0.003
mol) and potassium iodide (0.498 g, 0.003 mol). The target compound 14h (0.770 g) was obtained as white
-
1
solid. Yield: 35.4%; mp: 191−193 °C; IR (KBr, cm ) ν: 3020 (aromatic C-H), 2964, 2848, 2825 (CH ,
3
1
CH ), 1718 (C=O), 1625, 1544, 1494, 1451 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.31 (s,
2
6
1
2
H, COOH), 8.82 (s, 1H, quinolone 2-H), 7.87 (s, 2H, quinolone 5-H), 7.80 (s, 1H, quinolone 8-H), 7.54 (s,
H, benzimidazole 4,7-H), 7.13 (s, 2H, benzimidazole 5,6-H), 4.53 (s, 2H, quinolone N-CH ), 4.33 (s, 2H,
2
benzimidazole N-CH ), 3.75 (s, 2H, benzimidazole CH ), 3.29 (s, 4H, piperazine N-CH ), 2.68 (s, 4H,
2
2
2
piperazine N-CH ), 1.83 (s, 2H, CH CH (CH ) CH ), 1.38–1.06 (m, 33H, CH CH (CH ) CH , CH ), 0.78
2
2
2
2 15
3
2
2
2 15
3
3
1
3
(
d, J = 5.7 Hz, 3H, CH CH (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.7, 166.4, 152.5,
2 2 2 15 3 6
148.2, 145.3, 139.7, 136.6, 122.5, 121.6, 118.4, 117.1, 114.8, 113.2, 52.6, 49.8, 49.3, 46.3, 31.6, 29.1, 29.0,
+
+
2
7.0, 22.2, 14.1 ppm; MS (m/z): 702 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 702.4758;
42 61 5 3
found, 702.4759.
4.1.29.
1-Cyclopropyl-7-(4-((1-ethyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14i)
Compound 14i was prepared according to the procedure described for compound 13a starting from
compound 10a (0.360 g, 0.002 mol), ciprofloxacin (1.000 g, 0.002 mol), potassium carbonate (0.299 g,
0.002 mol) and potassium iodide (0.332 g, 0.002 mol). The target compound 14i (0.617 g) was obtained as
-
1
white solid. Yield: 63.0%; mp: 150−152 °C; IR (KBr, cm ) ν: 3090 (aromatic C-H), 2971, 2842, 2821 (CH ,
3
1
CH ), 1725 (C=O), 1626, 1520, 1498, 1477, 1450 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ:
2
6
1
2
2
4
5.28 (s, 1H, COOH), 8.76 (s, 1H, quinolone 2-H), 8.01 (d, J = 11.1 Hz, 1H, quinolone 5-H), 7.69 (d, J =
4.6 Hz, 3H, benzimidazole 4,7-H, quinolone 8-H), 7.32 (d, J = 34.7 Hz, 2H, benzimidazole 5,6-H), 4.40 (s,
H, benzimidazole N-CH ), 4.01 (s, 2H, benzimidazole CH ), 3.88 (s, 1H, quinolone N-CH), 3.39–3.34 (m,
2
2
H, piperazine N-CH ), 2.83 (s, 4H, piperazine N-CH ), 0.90 (d, J = 47.8 Hz, 7H, CH , cyclopropyl-CH )
2
2
3
2
1
3
ppm; C NMR (151 MHz, DMSO-d ) δ: 173.8, 167.3, 160.5, 154.6, 151.0, 147.4, 145.8, 140.5, 137.5,
6
133.8, 129.0, 116.5, 112.4, 108.6, 106.5, 65.6, 61.2, 53.1, 49.7, 44.6, 30.4, 21.5, 19.4, 15.5, 14.6, 8.1 ppm;
+
+
MS (m/z): 490 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 490.2254; found, 490.2256.
2
7
28
5
3
2
6

ACCEPTED MANUSCRIPT
4.1.30.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-((1-propyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic acid (14j)
Compound 14j was prepared according to the procedure described for compound 13a starting from
compound 10b (0.417 g, 0.002 mol), ciprofloxacin (1.000 g, 0.002 mol), potassium carbonate (0.299 g,
0.002 mol) and potassium iodide (0.332 g, 0.002 mol). The target compound 14j (0.591 g) was obtained as
-
1
white solid. Yield: 58.7%; mp: 153−154 °C; IR (KBr, cm ) ν: 3039 (aromatic C-H), 2948, 2848 (CH ,
3
1
CH ), 1721 (C=O), 1627, 1542, 1497, 1452 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.18 (s,
2
6
1
H, COOH), 8.64 (s, 1H, quinolone 2-H), 7.88 (d, J = 13.3 Hz, 1H, quinolone 5-H), 7.58 (dd, J = 19.4, 8.0
Hz, 3H, quinolone 8-H, benzimidazole 4,7-H), 7.20 (dt, J = 15.0, 7.2 Hz, 2H, benzimidazole 5,6-H),
.31–4.27 (m, 2H, benzimidazole N-CH ), 3.88 (s, 2H, benzimidazole CH ), 3.80 (dt, J = 10.5, 3.6 Hz, 1H,
4
2
2
cyclopropyl-CH), 3.33 (s, 4H, piperazine N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.86 (dd, J = 14.9, 7.4
2
2
Hz, 2H, CH CH CH ), 1.30 (q, J = 6.6 Hz, 2H, cyclopropyl-CH ), 1.16 (q, J = 6.8 Hz, 2H,
2
2
3
2
1
3
cyclopropyl-CH ), 0.94 (d, J = 7.4 Hz, 3H, CH CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ: 176.8,
2
2
2
3
6
1
4
5
66.5, 152.6, 148.4, 145.5, 139.8, 136.0, 122.4, 121.7, 119.5, 111.4, 110.8, 107.3, 106.8, 54.9, 52.6, 49.8,
+
+
5.3, 36.3, 23.2, 11.7, 8.1 ppm; MS (m/z): 504 [M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] ,
2
8
31
5
3
04.2411; found, 504.2413.
.1.31. 1-Cyclopropyl-6-fluoro-4-oxo-7-(4-((1-pentyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-1,4-
4
dihydroquinoline-3-carboxylic acid (14k)
Compound 14k was prepared according to the procedure described for compound 13a starting from
compound 10c (0.473 g, 0.002 mol), ciprofloxacin (1.000 g, 0.002 mol), potassium carbonate (0.299 g,
0.002 mol) and potassium iodide (0.332 g, 0.002 mol). The target compound 14k (0.602 g) was obtained as
-
1
white solid. Yield: 56.6%; mp: 154−155 °C; IR (KBr, cm ) ν: 3033 (aromatic C-H), 2932, 2853, 2820 (CH ,
3
1
CH ), 1728 (C=O), 1604, 1513, 1476 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.18 (s, 1H,
2
6
COOH), 8.64 (s, 1H, quinolone 2-H), 7.89 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.61–7.52 (m, 3H,
quinolone 8-H, benzimidazole 4,7-H), 7.21 (dt, J = 34.3, 7.4 Hz, 2H, benzimidazole 5,6-H), 4.34–4.28 (m,
2
H, benzimidazole N-CH ), 3.88 (s, 2H, benzimidazole CH ), 3.79 (dd, J = 8.7, 5.2 Hz, 1H, quinolone
2
2
N-CH), 3.32–3.30 (m, 4H, piperazine N-CH ), 2.71 (s, 4H, piperazine N-CH ), 1.87–1.81 (m, 2H,
2
2
CH (CH ) CH ), 1.36 (dd, J = 8.7, 5.3 Hz, 4H, CH (CH ) CH ), 1.30 (t, J = 6.7 Hz, 2H, cyclopropyl-CH ),
2
2 2
3
2
2 2
3
2
1
3
1
1
5
.16 (s, 2H, cyclopropyl-CH ), 0.88 (t, J = 6.7 Hz, 3H, CH ) ppm; C NMR (151 MHz, DMSO-d ) δ:
2
3
6
76.9, 166.4, 151.2, 148.5, 142.5, 139.6, 135.9, 122.6, 121.8, 119.5, 111.5, 110.7, 107.3, 106.8, 54.9, 52.7,
+
0.0, 43.9, 36.4, 29.7, 29.0, 22.4, 14.4, 8.0 ppm; MS (m/z): 532 [M+H] ; HRMS (TOF) calcd. for
2
7

ACCEPTED MANUSCRIPT
+
C H FN O : [M+H] , 532.2724; found, 532.2726.
3
0
35
5
3
4.1.32.
1-Cyclopropyl-6-fluoro-7-(4-((1-hexyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14l)
Compound 14l was prepared according to the procedure described for compound 13a starting from
compound 10d (0.501 g, 0.002 mol), ciprofloxacin (1.000 g, 0.002 mol), potassium carbonate (0.299 g,
0.002 mol) and potassium iodide (0.332 g, 0.002 mol). The target compound 14l (0.499 g) was obtained as
-
1
white solid. Yield: 45.7%; mp: 154−155 °C; IR (KBr, cm ) ν: 3011 (aromatic C-H), 2935, 2843 (CH , CH ),
3
2
1
1
8
727 (C=O), 1600, 1515, 1478 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.18 (s, 1H, COOH),
6
.65 (s, 1H, quinolone 2-H), 7.90 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.61–7.53 (m, 3H, quinolone 8-H,
benzimidazole 4,7-H), 7.21 (dt, J = 33.9, 7.4 Hz, 2H, benzimidazole 5,6-H), 4.34–4.28 (m, 2H,
benzimidazole N-CH ), 3.89 (s, 2H, benzimidazole CH ), 3.79 (dd, J = 6.5, 3.1 Hz, 1H, quinolone N-CH),
2
2
3
.26 (s, 4H, piperazine N-CH ), 2.72 (s, 4H, piperazine N-CH ), 1.83 (dt, J = 15.2, 7.7 Hz, 2H,
2
2
CH (CH ) CH ), 1.38 (dd, J = 14.7, 6.9 Hz, 2H, CH CH (CH ) CH ), 1.34–1.25 (m, 4H,
2
2 3
3
2
2
2 2
3
1
3
CH CH (CH ) CH ), 1.19–1.13 (m, 4H, cyclopropyl-CH ), 0.84 (t, J = 7.0 Hz, 3H, CH ) ppm; C NMR
2
2
2 2
3
2
3
(
151 MHz, DMSO-d ) δ: 176.8, 166.3, 152.7, 151.1, 148.4, 145.5, 142.5, 139.6, 135.9, 122.6, 121.8, 119.4,
6
111.5, 110.7, 107.4, 106.8, 55.6, 52.8, 49.9, 44.5, 36.3, 31.5, 29.6, 26.5, 22.3, 14.3, 7.9 ppm; MS (m/z): 546
+
+
[
M+H] ; HRMS (TOF) calcd. for C H FN O : [M+H] , 546.2880; found, 546.2881.
31 37 5 3
4.1.33.
1-Cyclopropyl-6-fluoro-7-(4-((1-octyl-1H-benzimidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (14m)
Compound 14m was prepared according to the procedure described for compound 13a starting from
compound 10e (0.558 g, 0.002 mol), ciprofloxacin (1.000 g, 0.002 mol), potassium carbonate (0.299 g,
0.002 mol) and potassium iodide (0.332 g, 0.002 mol). The target compound 14m (0.514 g) was obtained
-1
as white solid. Yield: 44.8%; mp: 157−158 °C; IR (KBr, cm ) ν: 3012 (aromatic C-H), 2952, 2842 (CH ,
3
1
CH ), 1720 (C=O), 1614, 1555, 1507, 1448 (aromatic frame); H NMR (600 MHz, DMSO-d ) δ: 15.33 (s,
2
6
1H, COOH), 8.95 (s, 1H, quinolone 2-H), 7.92 (d, J = 13.2 Hz, 1H, quinolone 5-H), 7.56 (dt, J = 30.4, 9.5
Hz, 2H, benzimidazole 4,7-H), 7.25–7.15 (m, 3H, quinolone 8-H, benzimidazole 5,6-H), 4.61–4.54 (m, 2H,
benzimidazole N-CH ), 4.28 (dt, J = 23.7, 7.5 Hz, 2H, benzimidazole CH ), 3.88 (s, 1H, quinolone N-CH),
2
2
3
.33 (s, 4H, piperazine N-CH ), 2.72 (d, J = 27.4 Hz, 4H, piperazine N-CH ), 1.86–1.79 (m, 2H,
2
2
1
3
CH (CH ) CH ), 1.40–1.23 (m, 10H, CH (CH ) CH ), 0.77 (d, J = 6.4 Hz, 3H, CH ) ppm; C NMR (151
2
2 5
3
2
2 5
3
3
MHz, DMSO-d ) δ: 176.5, 167.1, 151.2, 148.9, 145.9, 142.5, 138.2, 135.9, 122.5, 121.7, 119.5, 110.7,
6
107.6, 106.6, 72.8, 70.0, 60.6, 54.9, 52.8, 50.0, 49.5, 44.0, 31.5, 29.8, 29.2, 26.8, 22.5, 14.8, 14.3 ppm; MS
2
8