Multi-targeting exploration of new 2-aminothiazolyl quinolones: Synthesis, antimicrobial evaluation, interaction with DNA, combination with topoisomerase IV and penetrability into cells

Article abstract of DOI:10.1016/j.ejmech.2016.10.011

A series of new potentially multi-targeting antimicrobial 2-aminothiazolyl quinolones were designed, synthesized and characterized by¹H NMR,¹³C NMR, IR, MS and HRMS spectra. Bioactive assay manifested that some of the prepared compounds showed moderate to good antibacterial and antifungal activities. Noticeably, compound 10f could effectively inhibit the growth of B. typhi and MRSA with MIC values of 1 and 8 μg/mL, respectively. Experimental results revealed that compound 10f was membrane-active and had the ability to rapidly kill the tested strains and effectively prevent the development of bacterial resistance. Moreover, this compound also exhibited low toxicity against L929 cells. Molecular docking indicated that compound 10f could bind with topoisomerase IV–DNA complexes through hydrogen bonds and hydrophobic interactions. Quantum chemical studies were also performed on 10f to understand the structural features essential for activity. The preliminary mechanism research suggested that compound 10f could intercalate into calf thymus DNA to form a steady supramolecular complex which might block DNA replication to exert the powerful bioactivities.

Full text of DOI:10.1016/j.ejmech.2016.10.011

European Journal of Medicinal Chemistry 124 (2016) 935e945
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Multi-targeting exploration of new 2-aminothiazolyl quinolones:
Synthesis, antimicrobial evaluation, interaction with DNA,
combination with topoisomerase IV and penetrability into cells
,
, 2
Yu Cheng a, Srinivasa Rao Avula a 1, Wei-Wei Gao a, Dinesh Addla a
,
Vijai Kumar Reddy Tangadanchu a 2, Ling Zhang a, Jian-Mei Lin b **, Cheng-He Zhou a
,
,
, *
a Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing Municipality, School of Chemistry and Chemical
Engineering, Southwest University, Chongqing 400715, China
b Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu 610072, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new potentially multi-targeting antimicrobial 2-aminothiazolyl quinolones were designed,
synthesized and characterized by 1H NMR, 13C NMR, IR, MS and HRMS spectra. Bioactive assay man-
ifested that some of the prepared compounds showed moderate to good antibacterial and antifungal
activities. Noticeably, compound 10f could effectively inhibit the growth of B. typhi and MRSA with MIC
Received 29 June 2016
Received in revised form
20 September 2016
Accepted 6 October 2016
Available online 7 October 2016
values of 1 and 8 mg/mL, respectively. Experimental results revealed that compound 10f was membrane-
active and had the ability to rapidly kill the tested strains and effectively prevent the development of
bacterial resistance. Moreover, this compound also exhibited low toxicity against L929 cells. Molecular
docking indicated that compound 10f could bind with topoisomerase IVeDNA complexes through
hydrogen bonds and hydrophobic interactions. Quantum chemical studies were also performed on 10f to
understand the structural features essential for activity. The preliminary mechanism research suggested
that compound 10f could intercalate into calf thymus DNA to form a steady supramolecular complex
which might block DNA replication to exert the powerful bioactivities.
Keywords:
Aminothiazole
Quinolone
Antibacterial
Antifungal
Molecular modeling
Quantum chemical studies
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
Levofloxacin, Moxifloxacin, and Sparfloxacin have shown to be the
most successful economically and clinically with good oral effect,
Quinolones are one of the most commonly prescribed and
wholly synthetic classes of antibacterial drugs all around the world
and have been extensively used to combat with a number of bac-
terial infections, including gastrointestinal infections, respiratory
and urinary tract diseases [1]. Since the nalidixic acid, the first
marketed antimicrobial agent of quinolone family, was introduced
into the clinic about 50 years ago as a byproduct of chloroquine
synthesis, four generations of quinolones have been developed
successfully. Especially fluoroquinolone family such as
broad antimicrobial spectrum, achievable doses in clinic and
comparatively easy synthesis [2,3]. This kind of drugs have been
revealed to target DNA gyrase or topoisomerase IV by binding with
the enzymeeDNA binary complexes to form ternary supramolec-
ular complexes, interfering DNA replication and eventually
bringing about bacterial-cell death. Molecular mode of action has
demonstrated that the keto-carboxyl system at C-3 and C-4 posi-
tions of quinolones can facilitate quinoloneetopoisomerase bind-
ing in a manner of a wateremetal ion bridge [4,5]. However, along
with the wide clinical use even overuse of these drugs, quinolone
resistance increasingly caused widespread concern. Some re-
searches revealed that it might be ascribed to the specific muta-
tions of amino acids in gyrase and topoisomerase IV that disrupted
the carbonyl group of C-3 position, water and metal ion bridge and
weakened interactions between quinolones and these enzymes
[6e8]. Furthermore, some side effects caused by quinolones are
also in relation to the carbonyl group of C-3 position such as loss of
* Corresponding author.
** Corresponding author.
E-mail addresses: feiyu127@126.com (J.-M. Lin), zhouch@swu.edu.cn
(C.-H. Zhou).
1
Postdoctoral fellow from CSIR-Central Drug Research Institute, Lucknow
226001, India.
2
Postdoctoral fellow from CSIR-Indian Institute of Chemical Technology,
Hyderabad 500007, India.
http://dx.doi.org/10.1016/j.ejmech.2016.10.011
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

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Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
metal ions and gastrointestinal reactions [2]. All of those impact the
clinical utility of this important class of drugs. Therefore, the
structural modification of the C-3 position in quinolone nucleus is a
promising strategy to improve the biological activities and over-
come the drug-resistance. Previously, much effort had been focused
on the N-1, C-6 and C-7 positions of quinolones by incorporating
various types of electron-rich systems such as nitrogen heterocy-
cles and many important achievements had been made [9,10].
Nevertheless, to the best of our knowledge, the structural modifi-
cation of quinolone ring at C-3 position is relatively less in com-
parison to other positions. Based on above mentions, quinolone-
like drugs were designed in which the carboxyl group of C-3 po-
sition would be replaced by other isosteric active groups that no
longer depended on the wateremetal ion bridge mediated
interaction.
2-Aminothiazolyl group as an important structural fragment has
been expeditiously expanding in the design and synthesis of drugs
[11]. Numerous 2-aminothiazole drugs have been successfully
developed and widely used for clinical treatment of various dis-
eases such as antibacterial, anticancer, antiparasitic, anti-
inflammatory [12,13]. Especially, as antimicrobial agents, lots of 2-
aminothiazole derivatives like Ceftazidime, Cefuzonam, Car-
umonam and Aztreonam play an important role in fighting against
infective diseases [14,15]. The clinical success of 2-aminothiazole
drugs has spawned an array of new bioactive molecules. One of
the most common approaches is to construct new bioactive agents
based on the existed drugs by introducing 2-aminothiazolyl group
to improve biological activities. All these findings clearly demon-
strate that 2-aminothiazole derivatives should be promising can-
didates for developing new antimicrobial agents.
2. Results and discussion
2.1. Chemistry
The target 2-aminothiazolyl quinolones 7aeh and 10aef were
synthesized via multi-step reactions from commercially available
ethyl acetoacetate, 2,4-difluoroaniline, aliphatic bromides, aro-
matic halides and bromine. The synthetic process was outlined in
Scheme 1. Ethyl 2-(ethoxymethylene)-3-oxobutanoate 2 was easily
prepared by the condensation reaction of ethyl acetoacetate 1 and
triethoxymethane with acetic anhydride. The synthesized com-
pound 2 was reacted with 2,4-difluoroaniline in the absence of
solvent to afford ethyl 2-((2,4-difluorophenylamino)methylene)-3-
oxobutanoate 3 in almost 80% yield, and which was then further
cyclized in phenoxybenzene under reflux to produce the desired
intermediate 3-acetyl quinolone 4 in moderate yield. Compound 4
was N-aralkylated or alkylated by aromatic halides and aliphatic
bromides respectively to give N-1 aralkyl quinolones 5aeh and
alkyl derivatives 8aef in good yields of 70e80%, which were then
brominated by bromine in acetic acid to yield corresponding 3-(2-
bromoacetyl) quinolones 6aeh and 9aef. The cyclization of bro-
moacetyl group at C-3 position of compounds 6aeh and 9aef with
commercial thiourea in ethanol at 60 ꢀC readily gave the target
aralkyl 2-aminothiazolyl quinolones 7aeh and alkyl derivatives
10aef with moderate yields ranging from 50% to 60%. All the new
compounds were confirmed by 1H NMR, 13C NMR, IR, MS and HRMS
spectra.
2.2. Biological activity
Our previous work had reported that the incorporation of azoles
such as imidazole, triazole, tetrazole and benzimidazole fragments
into N-1 and C-7 positions of quinolones not only enhanced the
antimicrobial activities, but also broadened antimicrobial spectrum
[16]. In view of the above observations, considering the importance
of 2-aminothiazolyl fragment and quinolone compounds, and as an
extension of our ongoing research on bioactive heterocycles [17],
we incorporated 2-aminothiazolyl group into the C-3 position of
quinolone skeleton and changed substituents at N-1 position to
generate a series of new 2-aminothiazolyl quinolones, and inves-
tigated their antimicrobial potency [18,19]. All target molecules
would be screened against Gram-positive bacterial, Gram-negative
bacterial and fungal strains including MRSA (Methicillin-Resistant
Staphylococcus aureus). To further assess the prospective of the
most active compound as new antibacterial drug, timeekill kinetic
assay, bacterial resistance development research, cytotoxicity and
quantum chemical studies would be done. 2-Aminothiazole moiety
provides special five-membered aromatic structure that can easily
bind with various enzymes and receptors in organisms through
noncovalent interactions such as hydrogen bonds, coordination
bonds, hydrophobic effect as well as van der Waals force etc. [20]
and thus show multi-targeting ability toward multiple biological
active sites. In fact, plenty of biochemical and pharmacological
studies have revealed that 2-aminothiazoles can easily interact
with DNA and other biomacromolecules because of unique struc-
ture with both electron-donating groups (NH2, -S-) and an
electron-accepting group (C]N) [21]. Moreover, the multi-
targeting drugs, which simultaneously interact with the different
targets and effectively inhibit resistant pathways, can provide a
more effective approach for infectious diseases [22]. Therefore, in
this work the multi-targeting exploration for new 2-aminothiazolyl
quinolones including the membrane permeabilization capability,
interaction with calf thymus DNA and molecular docking with
topoisomerase IVeDNA of the most active compound would also be
performed.
The in vitro antimicrobial activity for all the target compounds
were evaluated for four Gram-positive bacteria (Staphylococcus
aureus ATCC25923, Methicillin-Resistant Staphylococcus aureus
N315 (MRSA), Bacillus subtilis ATCC6633 and Micrococcus luteus
ATCC4698), six Gram-negative bacteria (Escherichia coli DH52,
Escherichia coli JM109, Pseudomonas aeruginosa ATCC27853,
Shigella dysenteriae, Bacillus proteus ATCC13315 and Bacillus typhi)
and five fungi (Candida albicans ATCC76615, Candida mycoderma,
Candida utilis, Aspergillus flavus and Beer yeast) using two folds
serial dilution technique in 96ewell micro-test plates recom-
mended by National Committee for Clinical Laboratory Standards
(NCCLS) [23] with the positive control of clinically antimicrobial
drugs Chloromycin, Norfloxacin and Fluconazole. The antibacterial
and antifungal data were summarized in Tables 1 and 2.
As depicted in Table 1, most of target compounds gave moderate
to good antibacterial activities against the tested stains in vitro.
Preliminary active screening showed N-1 propargyl modified 2-
aminothiazolyl quinolone 10f displayed relatively good broad-
spectrum and efficient antibacterial activities in comparison with
other compounds. The anti-B. typhi potency of compound 10f
(MIC ¼ 1
mg/mL) was the strongest among the target compounds,
which was 32-fold and 4-fold more potent than reference drugs
Chloromycin and Norfloxacin, respectively. Furthermore, MRSA was
also quite sensitive to compound 10f (MIC ¼ 8
mg/mL) comparable
to Norfloxacin. Moreover, this compound also exhibited equal or
better activity in contrast to the two reference drugs against
S. dysenteriae and P. aeruginosa. These indicated that 2-
aminothiazolyl quinolone 10f had the potency to be a lead com-
pound in the development of more effective board-spectrum
antimicrobial agents. Continual study found that the length of the
carbon chain also had a certain effect on the antibacterial activity.
The shorter carbon chain was more conducive to exerting anti-
bacterial activity. Amongst compounds 10aee, propyl derivative
10a gave good antibacterial activity with MIC values in the range of
4e64
mg/mL, especially against S. dysenteriae (MIC ¼ 4
mg/mL),

Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
937
N
O
N
O
O
N
O
N
NH2
F
Br
F
F
COCH3
S
vi
v
R1
R1
R1
R2
R3
F
R2
R3
F
R2
R3
F
6a-h
7a-h
5a-h
iv
O
H3C
EtO
F
H3C
COCH3
EtO2C
F
F
COCH3
O
O
O
ii
iii
i
N
H
EtO
O
N
H
EtO
F
F
1
2
4
3
vii
N
O
O
O
O
NH2
F
Br
F
COCH3
S
ix
viii
N
N
R4
N
R4
F
R4
F
10a-f
F
8a-f
9a-f
a, R4 = CH2CH2CH3
b, R4 = CH2(CH2)3CH3
c, R4 = CH2(CH2)5CH3
d, R4 = CH2(CH2)8CH3
e, R4 = CH2(CH2)10CH3
CH C CH
8-10:
5-7: a, R1 = F, R2 = H, R3 = H; e, R1 = H, R2 = Cl, R3 = H;
b, R1 = H, R2 = F, R3 = H; f, R1 = H, R2 = H, R3 = Cl;
c, R1 = H, R2 = H, R3 = F; g, R1 = H, R2 = H, R3 = OCH3;
d, R1 = Cl, R2 = H, R3 = H; h, R1 = H, R2 = H, R3 = NO2
f, R4
=
2
Scheme 1. Synthetic route of 2-aminothiazolyl quinolones.
Reagents and conditions: (i) triethyl orthoformate, acetic anhydride, 130 ꢀC; (ii) 2,4-difluoroaniline, 130 ꢀC; (iii) phenoxybenzene, reflux; (iv) aromatic halides, DMF, K2CO3, 100 ꢀC;
(v) bromine, acetic acid, rt; (vi) thiourea, ethanol, 60 ꢀC; (vii) aliphatic bromides, DMF, K2CO3, 100 ꢀC; (viii) bromine, acetic acid, rt; (ix) thiourea, ethanol, 60 ꢀC.
Table 1
Antibacterial data as MIC (
,
b
m
g/mL) for compounds 7aeh and 10aef.a
Compds
Gram-positive bacteria
Gram-negative bacteria
MRSA
S. aureus
B. subtilis
M. luteus
E. coli (DH52)
E. coli (JM109)
S. dysenteriae
P. aeruginosa
B. proteus
B. typhi
7a
7b
7c
7d
7e
7f
7g
7h
64
32
16
64
128
64
256
128
32
32
64
32
128
64
128
256
256
16
256
256
512
512
8
64
64
128
16
64
128
64
64
128
16
128
256
128
128
32
64
128
32
128
32
64
64
32
64
32
32
64
256
16
32
1
64
128
16
128
256
128
128
128
64
64
128
64
128
32
32
32
64
4
64
128
16
128
256
4
16
32
64
128
8
128
64
64
32
64
128
64
64
16
16
32
64
32
16
32
16
128
32
64
256
256
32
256
128
64
64
32
128
256
32
8
128
128
256
256
256
128
128
32
64
64
256
64
16
32
16
128
32
32
64
64
16
16
64
256
64
1
10a
10b
10c
10d
10e
10f
Chloromycin
Norfloxacin
32
128
256
128
8
16
8
16
2
32
4
8
2
16
16
32
8
32
4
1
a
Minimal inhibitory concentrations were determined by micro broth dilution method for microdilution plates.
MRSA, Methicillin-Resistant Staphylococcus aureus (N315); S. aureus, Staphylococcus aureus (ATCC25923); B. subtilis, Bacillus subtilis; M. luteus, Micrococcus luteus
b
(ATCC4698); E. coli (DH52), Escherichia coli (DH52); E. coli (JM109), Escherichia coli (JM109); S. dysenteriae, Shigella dysenteriae, P. aeruginosa, Pseudomonas aeruginosa
(ATCC27853); B. proteus, Bacillus proteus (ATCC13315); B. typhi, Bacillus typhi.
which was 4-fold more active than reference drugs Chloromycin
and Norfloxacin. However, when the propyl group was extended to
pentyl, octyl, decyl or more longer alkyl chains, the antibacterial
activity decreased gradually. The fact demonstrated that suitable
side chain was necessary for better antibacterial activities in drug
design. In respect to the aromatic ones, fluorobenzyl compounds
7aec were more active than chlorobenzyl derivatives 7def. Espe-
cially, compound 7c could greatly inhibit the growth of the
S. dysenteriae (MIC ¼ 4
mg/mL). Surprisingly, electronic character-
istic of moieties had little effect on the antibacterial activity. The
activities of compound 7g with electro-donating OCH3 moiety and
7h with electro-withdrawing NO2 moiety were not much different
against most strains and both showed similar weak bioactivity.
The antifungal evaluation results in Table 2 showed that most of
target compounds had similar bioactivity to their antibacterial ac-
tivities. All 2-aminothiazolyl quinolones possessed comparative

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Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
Table 2
media and serially diluted concentrations of the tested compound
Antifungal data as MIC (m
g/mL) for compounds 7aeh and 10aef.a
10f. The experiment was repeated for 15 days. Results in Fig. 1
indicated that the MIC for compound 10f showed no obvious
change, whereas that of Norfloxacin began to significantly increase
after 5 passages against MRSA. All demonstrated that MRSA was
more difficult to develop resistance against compound 10f than
clinical Norfloxacin.
Compds
Fungi
C. albicans
C. mycoderma
C. utilis
A. flavus
B. yeast
7a
7b
7c
7d
7e
7f
7g
7h
64
64
32
128
128
512
256
32
32
64
32
64
128
16
64
32
16
64
64
256
256
128
32
32
256
64
32
8
128
64
64
32
64
32
64
64
64
64
64
32
16
64
64
256
8
64
64
32
128
64
128
64
64
16
128
16
64
64
256
256
512
256
64
64
64
128
64
32
2.4. Bactericidal kinetic assay
To determine the antibacterial potency of the synthesized
compounds, the viability of exponentially growing MRSA against
highly active 2-aminothiazolyl quinolone 10f was checked by
timeekill kinetics experiment. As can be seen from Fig. 2, it
revealed more than 3 log (CFU/mL) reduction in the number of
viable bacteria within an hour at a concentration of 4 ꢁ MIC. The
experimental result manifested that this compound had rapidly
killing effect against MRSA [26].
10a
10b
10c
10d
10e
10f
Fluconazole
128
8
16
1
4
8
256
a
C. albicans, Candida albicans (ATCC76615); C. mycoderma, Candida mycoderma;
C. utilis, Candida utilis; A. flavus, Aspergillus flavus; B. yeast, Beer yeast.
2.5. Cytotoxicity
even better anti-A. flavus (MIC ¼ 8e256
m
g/mL) efficiency to Flu-
conazole (MIC ¼ 256
m
g/mL), especially compound 10f (MIC ¼ 8
mg/
Biocompatibility is one of the most important properties to be
considered for active drug candidates. Fig. 4 showed the in vitro
cytotoxicity of compound 10f against L929 (normal mouse fibro-
blast) cells, which was evaluated by the MTT assay. The experi-
mental results indicated that compound 10f showed relatively low
toxicity to L929 cells at high concentration, and the IC50 value was
mL), which was 32-fold more active than Fluconazole. Furthermore,
target molecule 10f also exhibited moderate to good inhibitory
activity against other fungi with MICs between 8 and 32 mg/mL. The
antifungal activities of compounds 7aec were stronger than that of
compounds 7deh, which confirmed that fluorine atom on the
benzyl moieties should be of special importance in microbial in-
hibition probably due to its easy and efficient formation of non-
covalent forces which could be helpful for the biological trans-
portation and distribution in organism. Even though alkyl
substituted 2-aminothiazolyl quinolones 10aee displayed moder-
ate antifungal activity against most of the tested fungi, compounds
10a and 10c exhibited good activity against B. yeast with the MIC
289 mg/mL which demonstrated the favorable biocompatibility of
compound 10f [27].
2.6. Bacterial membrane permeabilization
It is very tough for reassembly process of bacterial cell mem-
brane lipids to resist membrane-active antibacterial agents, which
can weaken propensity for the development of drug resistance [28].
Hence, bacterial membrane has been considered as a particularly
interesting and valuable antibacterial target and expected to solve
the problem of bacterial resistance. The ability of compound 10f to
permeabilize the bacterial cell membrane was studied using pro-
pidium iodide (PI). This dye can pass through the membrane of
compromised bacterial cells and fluoresces upon binding to the
DNA [29,30]. As we can see in the Fig. 3A and Fig. 3B, the tested
compound was efficient in permeabilizing the membranes of both
Gram-positive (MRSA) and Gram-negative (B. typhi) bacteria.
value of 16 mg/mL, which was comparable to Fluconazole.
2.3. Resistance study
The resistance problem is a serious threat to the use of clinical
drugs and high-level resistance of Norfloxacin to MRSA strains has
been observed [24,25]. Therefore, the ability of MRSA to develop
drug resistance against the most active compound 10f was exam-
ined and reference Norfloxacin was chosen as a positive control.
The standard strain of MRSA was exposed to compound 10f from
MIC for sustained passages and the new MIC values were deter-
mined every 24 h after propagation of MRSA cultures with fresh
Fig. 2. Bactericidal kinetics of 2-aminothiazolyl quinolone 10f at 4 ꢁ MIC against
MRSA. The data obtained are from two independent experiments performed in
triplicate.
Fig. 1. Evaluation of resistance development against 2-aminothiazolyl quinolone 10f in
bacterial strain MRSA.

Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
939
The nitrogen atom of thiazole ring was in close vicinity to the
residue Arg418 of topoisomerase IV and base DA20 of DNA through
hydrogen bonds with distance of 3.4 and 3.2 Å, respectively. The
prepared molecule 10f could also form hydrogen bonds with
Arg418 of topoisomerase IVeDNA complex through the oxygen
atom of carbonyl group. Furthermore, compound 10f could also
interact with base DA16 of DNA by
p-p stacking. All of these
binding might be favorable to stabilize the quinolone-
eenzymeeDNA ternary complexes, which might be responsible for
the good inhibitory efficacy of compound 10f against the tested
strains.
2.8. Quantum chemical studies
It is known that intermolecular interactions were dominated by
the frontier molecular orbitals (FMO), namely the highest occupied
molecular orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO). Extending the concept into drugereceptor binding
systems, the major contribution to binding involves the interaction
between the HOMO of the drug with the LUMO of the receptor and
that between LUMO of the drug with the HOMO of the receptor. The
extents of these stabilizing interactions are inversely related to the
energy gap between the interacting orbitals. Higher HOMO energy
and lower LUMO energy in the drug molecule result in larger sta-
bilizing interactions [32]. Therefore, the orbital energies of both
HOMO and LUMO and their gaps calculated for compounds 7c, 10a,
and 10f were reported in Table 3. It was noteworthy that the most
Fig. 3. Relative cell viabilities of compound 10f in L929 cells.
2.7. Molecular docking
To rationalize the observed antibacterial activity and under-
stand the possible mechanism of the 2-aminothiazolyl quinolones,
a flexible ligand receptor docking investigation was undertaken.
The crystal structure data (topoisomerase IVeDNA complex) was
obtained from the protein data bank (PDB code: 2XKK), which was
representative target to investigate the antibacterial mechanism
[31]. Target compound 10f was selected to dock with the topo-
isomerase IVeDNA complex.
active compound 10f had the lowest energy gap (DE) of 3.422 eV.
According to the docking evaluation, 2-aminothiazolyl quino-
lone 10f possessed a moderate total score (5.60) against topo-
isomerase IVeDNA complex. Interaction of compound 10f with
topoisomerase IVeDNA receptor was shown in Fig. 5 (ieii). The
docking result of target compound 10f with topoisomerase IVeDNA
complex might rationalize the possible antibacterial mechanism.
We also obtained plots of the HOMOs and LUMOs of 7c, 10a and
10f to analyze the main atomic contributions for these orbitals. The
results displayed in Table 4 illustrated that HOMOs of 7c, 10a and
10f were mainly located in the 2-aminothiazole ring and quinolone
ring, manifesting that those rings might be active sites and bio-
logical interactions could take place between positively charged
Fig. 4. Bacterial membrane permeabilization of compound 10f (12 ꢁ MIC) against: (A) MRSA; (B) B. typhi.
Fig. 5. (i) Stereoview of conformation of compound 10f docked in topoisomerase IVeDNA complex. (ii) Three-dimensional conformation of compound 10f docked in topoisomerase
IVeDNA complex.

940
Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
Table 3
useful techniques in DNA-binding studies. In absorption spectros-
copy, hypochromism and hyperchromism are very important
spectral features to distinguish the change of DNA double-helical
structure. Due to the strong interaction between the electronic
states of intercalating chromophore and that of the DNA bases, the
observed large hypochromism strongly suggests a close proximity
of the aromatic chromophore to the DNA bases [36].
Energies of both HOMO and LUMO and their gaps (in eV) calculated for compounds
7c, 10a and 10f.
Compds
EHOMO
ELUMO
DE
7c
10a
10f
ꢂ5.055
ꢂ4.977
ꢂ4.961
ꢂ1.563
ꢂ1.497
ꢂ1.539
3.492
3.480
3.422
With a fixed concentration of DNA, UVevis absorption spectra
were recorded with sequentially and proportionately addition of
compound 10f. As shown in Fig. 6, UVevis spectra displayed that
the maximum absorption peak of DNA at 260 nm was gradually
enhanced, accompanied by a slightly red shift during titration with
the increasing concentration of compound 10f. Meanwhile, the
absorption value of 10feDNA complex was a little higher than that
of the simply sum of free DNA and free compound 10f, which was
observed in the inset of Fig. 6. These indicated a weak hyper-
chromism effect existed between DNA and compound 10f. This
hyperchromism effect on the spectra of 10feDNA mixtures and the
slight red spectral shift may be attributed to the intercalation
compound as well as the charge attraction of prepared molecule
10f to DNA base, forming binary complexes. Furthermore, the
intercalation of the aromatic fragment of compound 10f into the
molecules and these sites. It was also found that N-1 position
substituted group did not contribute directly to HOMOs which
indicated these groups might be primarily used to adjust the
physicochemical characterizations. Furthermore, the LUMOs of
these molecules were mainly focused on the quinolone ring in
which nucleophilic attacks might be favorable. In an attempt to
further understand the lower antimicrobial activities of compounds
7c and 10a and the higher antimicrobial activity of 10f, molecular
electrostatic potentials (MEPs) had been performed to examine the
similarities and differences in electrostatic binding characteristic of
the surface of the molecules (Table 4). Comparison of the electro-
static maps of 7c, 10a and 10f revealed that compound 7c had an
increased positive charge regions (in blue) located on the amino
(NH2), while 10f possessed more negative charge regions (in red)
on the oxygen atom of quinolone ring and nitrogen atoms of ami-
nothiazolyl ring. It might indicate capability of hydrogen bond
formation with share of nitrogen atoms of thiazole ring and the
oxygen atom of quinolone ring. This was in agreement with the
binding mode obtained from above docking study (Fig. 5).
helix and the large overlap of p-p* states in the large p-conjugated
system with the electronic states of DNA bases were consistent
with the observed spectral changes.
Based on the variations in the absorption spectra of DNA upon
binding to 10f, equation (1) can be utilized to calculate the binding
constant (K).
2.9. Interactions with calf thymus DNA
A0
A ꢂ A0
xC
xDꢂC
xC
xC xDꢂC
1
K½Qꢃ
¼
þ
ꢁ
(1)
ꢂ
ꢂ
xC
Deoxyribonucleic acid (DNA) is quite often the main cellular
target for studies with small molecules of biological importance.
Recently, DNA as the therapeutic target in biomedical field for the
rational design and construction of new and efficient drugs has
attracted considerable attention [33,34]. Calf thymus DNA was al-
ways selected as DNA model because of its medical importance, low
cost and ready availability properties [35]. To investigate the pre-
liminary mechanism of antimicrobial action, the in vitro binding
studies between the biological active compound 10f and calf
thymus DNA on molecular level were carried out by UVevis
spectroscopy.
A0 and A represent the absorbance of DNA in the absence and
presence of compound 10f at 260 nm, xC and xDeC are the absorption
coefficients of compound 10f and compound 10f¡DNA complex
respectively. The plot of A0/(AeA0) versus 1/[compound 10f] is
constructed by using the absorption titration data and linear fitting
(Supporting Information: Fig. S1), yielding the binding constant,
K ¼ 0.538 ꢁ 104 L/mol, R ¼ 0.998, SD ¼ 0.135 (R is the correlation
coefficient. SD is standard deviation).
2.9.2. Absorption spectra of NR interaction with DNA
Neutral Red (NR) is a planar phenazine dye and structurally
similar to other planar dyes, for example, those of the acridine,
2.9.1. Absorption spectra of DNA in the presence of compound 10f
The application of absorption spectroscopy is one of the most
Table 4
Plots of the HOMO, LUMO and MEPs of compounds 7c, 10a and 10f.
Compds HOMO
LUMO
MEPs
7c
10a
10f
Fig. 6. UV absorption spectra of DNA with different concentrations of compound 10f
(pH ¼ 7.4, T ¼ 290 K). Inset: comparison of absorption at 260 nm between the com-
pound 10fꢂDNA complex and the sum values of free DNA and free compound 10f.
c(DNA) ¼ 5.22 ꢁ 10ꢂ5 mol/L, and c(compound 10f) ¼ 0e9.00 ꢁ 10ꢂ5 mol/L for curves
aꢂh respectively at increment 1.28 ꢁ 10ꢂ5
.

Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
941
thiazine, and xanthene kind. Compared with other common
probes, NR showed lower toxicity, higher stability and convenient
application. It has been demonstrated that the binding mode of NR
with DNA is an intercalation binding in recent years [37]. Therefore,
NR was employed as a spectral probe to study the binding mode of
10f with DNA in the present work.
The absorption spectra of the NR dye upon the addition of DNA
are showed in Fig. 7. It could be observed that the absorption peak
of the NR at around 460 nm showed gradual decrease with the
increasing concentration of DNA, and a new band at around 530 nm
developed. This was attributed to the formation of the new
DNAeNR complex. An isosbestic point at 504 nm provided evi-
dence of DNAeNR complex formation.
2.9.3. Absorption spectra of competitive interaction of compound
10f and NR with DNA
Fig. 8. UV absorption spectra of the competitive reaction between 10f and NR with
DNA. c(DNA)
¼
4.32
ꢁ
10ꢂ5 mol/L, c(NR)
¼
2
ꢁ
10ꢂ5 mol/L, and c(compound
Fig. 8 displayed the absorption spectra of a competitive binding
between NR and 10f with DNA. As shown, with the increasing
concentration of 10f, an apparent intensity increase was observed
in the developing band around 460 nm. Compared with the ab-
sorption around 460 nm of free NR in the presence of the increasing
concentrations of DNA (Fig. 7), the absorbance at the same wave-
length exhibited the reverse process (inset of Fig. 8). The results
suggested that compound 10f intercalated into the double helix of
DNA by substituting for NR in the DNAeNR complex. In addition,
increase of absorbance at 276 nm provided evidence for interca-
lation of compound 10f into DNA. The DNA-cleaving activity of
compound 10f was also studied under physiological conditions
(5 h, 37 ꢀC). Experiment results revealed that compound 10f could
not cleave DNA, but it could intercalate into DNA as the predomi-
nant type of action mode between active molecule 10f and DNA.
10f) ¼ 0e1.75 ꢁ 10ꢂ5 mol/L for curves aꢂh respectively at increment 0.25 ꢁ 10ꢂ5
.
(Inset) Absorption spectra of the system with the increasing concentration of 10f in the
wavelength range of 350e500 nm absorption spectra of competitive reaction between
compound 10f and NR with DNA.
growth of B. typhi and MRSA with MIC values of 1 and 8 mg/mL,
respectively. Furthermore, it did not trigger development of resis-
tance in bacteria and displayed low toxicity against L929 cells.
Additionally, it could efficiently permeate membrane of Gram-
positive (MRSA) and Gram-negative (B. typhi). Molecular docking
indicated that compound 10f could bind with topoisomerase
IVeDNA not only through hydrogen bonds between molecule and
residue Arg418 of topoisomerase IV but also through p-p stacking
between molecule and base DA16 of DNA. Quantum chemical
studies indicated the capability of hydrogen bond formation with
share of nitrogen atoms of thiazole ring and the oxygen atom of
quinolone ring. This was in agreement with the binding mode
obtained from above docking study. The preliminary research
revealed that compound 10f could intercalate into calf thymus DNA
to form a steady complex which might block DNA replication to
exert the powerful bioactivities.
3. Conclusion
In conclusion, a series of new 2-aminothiazolyl quinolones were
successfully designed and synthesized as multi-targeting anti-in-
fectious agents through a convenient and efficient procedure. Their
structures were confirmed by 1H NMR, 13C NMR, MS, IR and HRMS
spectra. The in vitro antimicrobial evaluation revealed that some of
the synthesized 2-aminothiazolyl quinolones could effectively
inhibit the growth of some tested strains. Structureeactivity rela-
tionship suggested that short aliphatic chains at N-1 position
exerted a positive impact on biological activity. The most active
compound 10f with propargyl group could effectively inhibit the
4. Experimental protocols
4.1. General methods
Melting points were recorded on Xe6 melting point apparatus
and were uncorrected. TLC analysis was done using pre-coated
silica gel plates. FT-IR spectra were carried out on Bruker RFS100/
S spectrophotometer (Bio-Rad, Cambridge, MA, USA) using KBr
pellets in the 400e4000 cmꢂ1 range. 1H NMR and 13C NMR spectra
were recorded on a Bruker AV 300 spectrometer using TMS as an
internal standard. The chemical shifts were reported in parts per
million (ppm), the coupling constants (J) were expressed in hertz
(Hz) and signals were described as singlet (s), doublet (d), triplet (t)
as well as multiplet (m). The mass spectra were recorded on LCMS-
2010A and the high resolution mass spectra (HRMS) were recorded
on an IonSpec FTICR mass spectrometer with ESI resource. UV
spectra were recorded at room temperature on a TU-2450 spec-
trophotometer (Puxi Analytic Instrument Ltd. of Beijing, China)
equipped with 1.0 cm quartz cells. Calf thymus DNA and NR were
obtained from Sigma-Aldrich (St. Louis, MO, USA). Tris, NaCl, HCl
were analytical purity. Sample masses were weighed on a micro-
balance with a resolution of 0.1 mg. All chemicals and solvents were
commercially available and were used without further purification.
Fig. 7. UV absorption spectra of NR in the presence of DNA at pH 7.4 and room tem-
perature. c(NR) ¼ 2 ꢁ 10ꢂ5 mol/L, and c(DNA) ¼ 0e4.32 ꢁ 10ꢂ5 mol/L for curves aꢂj
respectively at increment 0.48 ꢁ 10ꢂ5 mol/L.
4.1.1. Synthesis of ethyl 2-(ethoxymethylene)-3-oxobutanoate (2)
A
mixture of compound 1 (10.41 g, 0.08 mol), triethyl

942
Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
orthoformate (14.23 g, 0.10 mol) and acetic anhydride (20.42 g,
0.20 mol) was heated at 130 ꢀC for 4 h. The resulting EtOH,
CH3CO2CH2CH3 and remaining materials were eliminated under
reduced pressure and compound 2 (9.68 g) was obtained in 65%
yield.
to give compound 7a (2.90 g) as yellow solid. Yield: 75%; mp:
220e222 ꢀC; IR (KBr, cmꢂ1
: 3430, 3297 (NeH), 3068 (aromatic
CeH), 2925 (CH2), 1715 (C]O), 1619, 1572, 1540, 1498, 1456 (aro-
matic frame); 1H NMR (600 MHz, DMSO-d6, ppm):
8.78 (s, 1H,
quinolone-2-H), 7.89 (d, J ¼ 8.6 Hz, 1H, quinolone-5-H), 7.73e7.63
(m, 2H, quinolone-7-H, thiazole-4-H), 7.36 (dd, J ¼ 13.3, 6.5 Hz, 1H,
FPh-3-H), 7.29e7.25 (m, 1H, FPh-4-H), 7.12 (t, J ¼ 7.5 Hz, 1H, FPh-5-
H), 6.96 (s, 2H, thiazole-2-NH2), 6.91 (d, J ¼ 7.5 Hz, 1H, FPh-6-H),
5.77 (s, 2H, quinolone-1-N-CH2); 13C NMR (151 MHz, DMSO-d6,
)
n
d
4.1.2. Synthesis of ethyl 2-((2,4-difluorophenylamino) methylene)-
3-oxobutanoate (3)
Compound 2 (18.62 g, 0.10 mol) was heated at 130 ꢀC for 0.5 h,
and then 2,4-difluoroaniline (12.91 g, 0.10 mol) was added into the
mixture. After the reaction was completed (monitored by TLC,
eluent, petroleum/ethyl acetate 20/3, V/V) about 1 h, the mixture
was cooled to room temperature, and then the generated ethanol
was evaporated under reduced pressure. The residue was purified
via silica gel column chromatography (eluent, petroleum/ethyl ac-
etate 20/3, V/V) to give compound 3 (15.15 g) as light yellow solid
with 60% yield.
ppm):
d 172.2, 167.4, 160.5, 158.9, 158.7, 157.1, 153.4, 151.7, 146.6,
143.9, 130.4, 130.1, 127.5, 125.7, 125.4, 124.9, 116.1, 115.3, 109.0, 107.4,
105.4, 55.3; HRMS (ESI) calcd. for
410.0551; found, 410.0554.
C
19H12F3N3OS [MþNa]þ,
4.1.7. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-(3-fluorobenzyl)
quinolin-4(1H)-one (7b)
Compound 7b was prepared according to the procedure
described for compound 7a starting from 6b (4.10 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 7b (3.09 g) was
obtained as yellow solid. Yield: 80%; mp: 222e224 ꢀC; IR (KBr,
4.1.3. Synthesis of 3-acetyl-6,8-difluoroquinolin-4(1H)-one (4)
Diphenylether (50 mL) was heated to 250 ꢀC and compound 3
(5.80 g, 0.02 mol) was slowly added. The mixture was kept under
reflux for 1.5 h. After the ethanol evaporated during the reaction, a
brown solid was formed. When the reaction was completed, the
solid was filtered, and then washed with petroleum ether to
remove the diphenylether. The crude target compound 4 (2.00 g)
was obtained in 45% yield.
cmꢂ1
)
n
: 3423, 3301 (NeH), 3061 (aromatic CeH), 2924 (CH2), 1715
(C]O), 1617, 1579, 1535, 1499, 1455 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.74 (s, 1H, quinolone-2-H), 7.90 (d,
d
J ¼ 8.7 Hz, 1H, quinolone-5-H), 7.72e7.64 (m, 2H, quinolone-7-H,
thiazole-4-H), 7.39 (dd, J ¼ 14.2, 7.8 Hz, 1H, FPh-4-H), 7.12 (t,
J ¼ 8.5 Hz,1H, FPh-5-H), 7.01 (d, J ¼ 9.8 Hz,1H, FPh-2-H), 6.95 (s, 2H,
thiazole-2-NH2), 6.90 (d, J ¼ 7.8 Hz, 1H, FPh-6-H), 5.73 (s, 2H, qui-
4.1.4. General synthesis of aralkyl quinolones (5a-h) and alkyl
derivatives (8a-f)
nolone-1-N-CH2); 13C NMR (151 MHz, DMSO-d6, ppm):
d 172.2,
167.4, 163.8, 162.0, 158.7, 157.1, 153.3, 151.6, 146.3, 143.9, 131.4, 130.3,
125.8, 121.8, 115.3, 114.8, 113.1, 113.0, 109.0, 107.5, 105.4, 59.2; HRMS
(ESI) calcd. for C19H12F3N3OS [MþH]þ, 388.0731; found, 388.0733.
To a stirred suspension of potassium carbonate (1.51 g, 0.01 mol)
in N,N-dimethylformamide (25 mL) was added intermediate 4
(2.23 g, 0.01 mol). The mixture was stirred at 60 ꢀC for 1.5 h, and
then cooled to room temperature. Corresponding aliphatic bro-
mides or aromatic halides (0.01 mol) were added, and the resulting
mixture was stirred at 100 ꢀC for 12 h. After the reaction came to an
end, the solvent was evaporated under reduced pressure and the
residue was treated with water, and then extracted with chloro-
form. The organic extracts were dried over anhydrous sodium
sulfate and concentrated. The crude product was purified via silica
gel column chromatography (eluent, chloroform) to afford corre-
sponding compounds 5a-h and 8a-f as yellow solid with yields of
70e80%.
4.1.8. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-(4-fluorobenzyl)
quinolin-4(1H)-one (7c)
Compound 7c was prepared according to the procedure
described for compound 7a starting from 6c (4.10 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 7c (2.70 g) was
obtained as yellow solid. Yield: 70%; mp: 221e223 ꢀC; IR (KBr,
cmꢂ1
)
n
: 3462, 3292 (NeH), 3076 (aromatic CeH), 2925 (CH2), 1716
(C]O), 1613, 1571, 1537, 1500, 1465 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.76 (s, 1H, quinolone-2-H), 7.89 (d,
d
J ¼ 8.7 Hz, 1H, quinolone-5-H), 7.72e7.64 (m, 2H, quinolone-7-H,
thiazole-4-H), 7.17 (d, 4H, FPh-2,3,5,6-H), 6.99 (s, 2H, thiazole-2-
NH2), 5.69 (s, 2H, quinolone-1-N-CH2); 13C NMR (151 MHz,
4.1.5. General synthesis of 3-(2-bromoacetyl)-6,8-difluoroquinolin-
4-ones (6a-h) and (9a-f)
DMSO-d6, ppm): d 172.1, 167.4, 162.8, 161.2, 158.6, 157.0, 153.3, 151.7,
Compounds 5a-h or 8a-f (0.01 mol) were respectively dissolved
146.4, 143.7, 134.0, 130.4, 128.2, 125.8, 116.2, 115.1, 109.0, 107.5,
105.3, 59.1; HRMS (ESI) calcd. for C19H12F3N3OS [MþH]þ, 388.0731;
found, 388.0732.
in glacial acetic acid, and bromine (1.60 g, 0.01 mol) was added to
ꢀ
the mixture at 0 C. The resulting system was stirred overnight at
room temperature, evaporated and partitioned between an
aqueous solution of saturated sodium bicarbonate and diethylether.
The organic phase was purified by silica gel chromatography using
a gradient of petroleum ether and diethylether to afford 6a-h and
9a-f with yields of 50e60%.
4.1.9. 3-(2-Aminothiazol-5-yl)-1-(2-chlorobenzyl)-6,8-
difluoroquinolin-4(1H)-one (7d)
Compound 7d was prepared according to the procedure
described for compound 7a starting from 6d (4.26 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 7d (2.90 g) was
obtained as yellow solid. Yield: 72%; mp: 218e220 ꢀC; IR (KBr,
4.1.6. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-(2-fluorobenzyl)
quinolin-4(1H)-one (7a)
cmꢂ1
)
n
: 3445, 3290 (NeH), 3073 (aromatic CeH), 2924 (CH2), 1720
(C]O), 1620, 1575, 1536, 1498, 1440 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.72 (s, 1H, quinolone-2-H), 7.91 (d,
A mixture of compound 6a (4.10 g, 0.01 mol) and thiourea
(0.76 g, 0.01 mol) was stirred in ethanol (30 mL) at 60 ꢀC for 6 h.
When the reaction was completed, the mixture was cooled to room
temperature, and then the solvent was evaporated under reduced
pressure. After ethyl acetate was added, the resulting mixture was
washed with saturated sodium bicarbonate solution. The organic
phase was dried over anhydrous sodium sulfate and concentrated
under reduced pressure. The residue was purified via silica gel
column chromatography (eluent, chloroform/methanol 50/1, V/V)
d
J ¼ 8.6, 1H, quinolone-5-H), 7.71e7.64 (m, 2H, quinolone-7-H,
thiazole-4-H), 7.56 (d, J ¼ 7.9 Hz, 1H, ClPh-3-H), 7.33 (t, J ¼ 7.4 Hz,
1H, ClPh-4-H), 7.24 (t, J ¼ 7.6 Hz,1H, ClPh-5-H), 6.93 (s, 2H, thiazole-
2-NH2), 6.72 (d, J ¼ 7.7 Hz, 1H, ClPh-6-H), 5.77 (s, 2H, quinolone-1-
N-CH2); 13C NMR (151 MHz, DMSO-d6, ppm):
d 172.2, 167.3, 158.9,
158.7, 153.3,151.6, 146.4, 143.9, 135.5, 131.1, 130.3, 130.1, 129.6, 128.3,
126.3, 125.9, 115.4, 109.0, 107.4, 105.4, 57.8; HRMS (ESI) calcd. for

Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
943
C19H12ClF2N3OS [MþH]þ, 404.0436; found, 404.0433.
J ¼ 8.6 Hz, 2H, O2NPh-2,6-H), 6.98 (s, 2H, thiazole-2-NH2), 5.77 (s,
2H, quinolone-1-N-CH2); 13C NMR (151 MHz, DMSO-d6, ppm):
4.1.10. 3-(2-Aminothiazol-5-yl)-1-(3-chlorobenzyl)-6,8-
difluoroquinolin-4(1H)-one (7e)
d 175.4, 167.8, 160.2, 158.6, 156.2, 154.5, 152.5, 148.6, 144.6, 134.1,
131.2, 128.2, 125.8, 116.2, 114.9, 109.2, 107.6, 105.4, 59.0; HRMS (ESI)
Compound 7e was prepared according to the procedure
described for compound 7a starting from 6e (4.26 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 7e (3.02 g) was
obtained as yellow solid. Yield: 75%; mp: 214e216 ꢀC; IR (KBr,
calcd. for C19H12F2N4O3S [MþH]þ, 414.0598; found, 414.0597.
4.1.14. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-propyl quinolin-
4(1H)-one (10a)
cmꢂ1
)
n
: 3422, 3286 (NeH), 3071 (aromatic CeH), 2925 (CH2), 1714
(C]O), 1614, 1572, 1528, 1493, 1437 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.76 (s, 1H, quinolone-2-H), 7.90 (d,
Compound 10a was prepared according to the procedure
described for compound 7a starting from 9a (3.43 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compoundꢀ10a (2.43 g) was
d
J ¼ 8.5 Hz, 1H, quinolone-5-H), 7.72e7.65 (m, 2H, quinolone-7-H,
thiazole-4-H), 7.31 (d, J ¼ 8.3 Hz, 2H, ClPh-2,4-H), 7.14 (m, 2H, ClPh-
5,6-H), 6.97 (s, 2H, thiazole-2-NH2), 5.71 (s, 2H, quinolone-1-N-
obtained as yellow solid. Yield: 76%; mp: 110e112 C; IR (KBr, cmꢂ1
)
n
: 3438, 3369 (NeH), 3046 (aromatic CeH), 2968, 2927, 2874 (CH3,
CH2), 1711 (C]O), 1611, 1576, 1534, 1495, 1434 (aromatic frame); 1H
NMR (600 MHz, DMSO-d6, ppm): 8.60 (s, 1H, quinolone-2-H), 7.88
CH2); 13C NMR (151 MHz, DMSO-d6, ppm):
d
172.1, 167.4, 158.6,
d
157.1, 153.2,151.6, 146.4,143.9,136.9,132.7,131.6, 130.3,129.3,128.8,
128.0, 125.7, 115.2, 109.0, 107.5, 105.4, 59.2; HRMS (ESI) calcd. for
C
(d, J ¼ 7.5 Hz, 1H, quinolone-5-H), 7.76 (dd, J ¼ 11.8, 8.3 Hz, 1H,
quinolone-7-H), 7.66 (s, 1H, thiazole-4-H), 7.00 (s, 2H, thiazole-2-
NH2), 4.35 (t, J ¼ 5.8 Hz, 2H, CH2CH2CH3), 1.82e1.78 (m, 2H,
CH2CH2CH3), 0.91 (t, J ¼ 7.1 Hz, 3H, CH2CH2CH3); 13C NMR
19H12ClF2N3OS [MþH]þ, 404.0436; found, 404.0437.
4.1.11. 3-(2-Aminothiazol-5-yl)-1-(4-chlorobenzyl)-6,8-
difluoroquinolin-4(1H)-one (7f)
(151 MHz, DMSO-d6, ppm): d 171.9, 167.3, 158.4, 156.9, 153.5, 151.7,
145.8, 144.0, 130.3, 125.4, 114.6, 109.0, 107.5, 105.0, 58.9, 24.1, 11.1;
HRMS (ESI) calcd. for C15H13F2N3OS [MþH]þ, 322.0826; found,
322.0827.
Compound 7f was prepared according to the procedure
described for compound 7a starting from 6f (4.26 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 7f (3.22 g) was
obtained as yellow solid. Yield: 80%; mp: 215e217 ꢀC; IR (KBr, cmꢂ1
)
4.1.15. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-pentylquinolin-
4(1H)-one (10b)
n
: 3428, 3290 (NeH), 3072 (aromatic CeH), 2926 (CH2), 1716 (C]
O), 1633, 1580, 1518, 1495, 1447 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.76 (s, 1H, quinolone-2-H), 7.88 (d,
Compound 10b was prepared according to the procedure
described for compound 7a starting from 9b (3.71 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 10b (2.61 g) was
obtained as yellow solid. Yield: 75%; mp: 165e167 ꢀC; IR (KBr,
d
J ¼ 8.6 Hz, 1H, quinolone-5-H), 7.73e7.65 (m, 2H, quinolone-7-H,
thiazole-4-H), 7.41 (t, J ¼ 8.4 Hz, 2H, ClPh-3,5-H), 7.14 (t, J ¼ 8.4 Hz,
2H, ClPh-2,6-H), 6.96 (s, 2H, thiazole-2-NH2), 5.71 (s, 2H, quino-
cmꢂ1
)
n
: 3394, 3306 (NeH), 3066 (aromatic CeH), 2954, 2927, 2861
(CH3, CH2), 1720 (C]O), 1610, 1574, 1534, 1495, 1434 (aromatic
frame); 1H NMR (600 MHz, DMSO-d6, ppm):
8.59 (s, 1H, quino-
lone-1-N-CH2); 13C NMR (151 MHz, DMSO-d6, ppm):
d 172.1, 167.4,
158.6,157.1,153.2,151.6,146.3,143.9,136.9,132.7,130.3,129.3,127.9,
125.8, 115.2, 109.0, 107.5, 105.4, 59.3; HRMS (ESI) calcd. for
d
lone-2-H), 7.87 (d, J ¼ 7.5 Hz, 1H, quinolone-5-H), 7.77 (dd, J ¼ 11.8,
8.3 Hz, 1H, quinolone-7-H), 7.66 (s, 1H, thiazole-4-H), 6.98 (s, 2H,
thiazole-2-NH2), 4.35 (t, J ¼ 5.8 Hz, 2H, CH2CH2(CH2)2CH3), 1.77 (m,
2H, CH2CH2(CH2)2CH3), 1.30 (m, 4H, CH2CH2(CH2)2CH3), 0.86 (t,
J ¼ 6.3 Hz, 3H, CH2CH2(CH2)2CH3); 13C NMR (151 MHz, DMSO-d6,
C
19H12ClF2N3OS [MþH]þ, 404.0436; found, 404.0438.
4.1.12. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-(4-methoxy benzyl)
quinolin-4(1H)-one (7g)
Compound 7g was prepared according to the procedure
described for compound 7a starting from 6g (4.21 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 7g (3.10 g) was
ppm): d 171.9, 167.3, 158.4, 156.8, 153.4, 151.8, 145.6, 144.1, 130.3,
125.3, 114.7, 108.9, 107.4, 105.0, 57.6, 30.5, 28.5, 22.2, 14.2; HRMS
(ESI) calcd. for C17H17F2N3OS [MþNa]þ, 372.0958; found, 372.0959.
obtained as yellow solid. Yield: 78%; mp: 216e218 ꢀC; IR (KBr, cmꢂ1
)
n
: 3429, 3307 (NeH), 3061 (aromatic CeH), 2932, 2840 (CH3, CH2),
1723 (C]O), 1639, 1591, 1511, 1496, 1463 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.76 (s, 1H, quinolone-2-H), 7.88 (d,
4.1.16. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-heptylquinolin-
4(1H)-one (10c)
d
Compound 10c was prepared according to the procedure
described for compound 7a starting from 9c (3.99 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 10c (2.86 g) was
obtained as yellow solid. Yield: 76%; mp: 160e162 ꢀC; IR (KBr,
J ¼ 8.6 Hz, 1H, quinolone-5-H), 7.73e7.65 (m, 2H, quinolone-7-H,
thiazole-4-H), 7.06 (t, J ¼ 8.5 Hz, 2H, CH3OPh-2,6-H), 6.99 (s, 2H,
thiazole-2-NH2), 6.90 (t, J ¼ 8.5 Hz, 2H, CH3OPh-3,5-H), 5.62 (s, 2H,
quinolone-1-N-CH2), 3.70 (s, 3H, OCH3); 13C NMR (151 MHz, DMSO-
cmꢂ1
)
n
: 3408, 3308 (NeH), 3060 (aromatic CeH), 2955, 2927, 2856
(CH3, CH2), 1701 (C]O), 1588, 1568, 1531, 1497, 1437 (aromatic
frame); 1H NMR (600 MHz, DMSO-d6, ppm):
8.59 (s, 1H, quino-
d6, ppm):
d 172.1, 167.4, 159.3, 158.7, 157.0, 153.4, 151.7, 146.3, 144.0,
130.4, 129.5, 127.6, 125.8, 115.1, 114.7, 109.0, 107.5, 105.3, 59.4, 55.6;
HRMS (ESI) calcd. for C20H15F2N3O2S C20H15F2N3O2S [MþH]þ,
400.0931; found, 400.0933.
d
lone-2-H), 7.87 (d, J ¼ 7.5 Hz, 1H, quinolone-5-H), 7.76 (dd, J ¼ 11.8,
8.3 Hz, 1H, quinolone-7-H), 7.66 (s, 1H, thiazole-4-H), 6.99 (s, 2H,
thiazole-2-NH2), 4.36 (t, J ¼ 5.8 Hz, 2H, CH2CH2(CH2)4CH3), 1.76 (m,
2H, CH2CH2(CH2)4CH3), 1.29e1.23 (m, 8H, CH2CH2(CH2)4CH3), 0.84
(t, J ¼ 6.9 Hz, 3H, CH2CH2(CH2)4CH3); 13C NMR (151 MHz, DMSO-d6,
4.1.13. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-(4-nitrobenzyl)
quinolin-4(1H)-one (7h)
Compound 7h was prepared according to the procedure
described for compound 7a starting from 6h (4.36 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 7h (3.39 g) was
obtained as yellow solid. Yield: 82%; mp: 230e232 ꢀC; IR (KBr,
ppm): d 171.9, 167.3, 158.4, 156.8, 153.4, 151.8, 145.7, 144.1, 130.3,
125.3, 114.7, 108.9, 107.3, 105.0, 57.7, 31.6, 30.8, 28.7, 26.3, 22.4, 14.3;
HRMS (ESI) calcd. for C19H21F2N3OS [MþNa]þ, 400.1271; found,
400.1274.
cmꢂ1
)
n
: 3425, 3296 (NeH), 3065 (aromatic CeH), 2924 (CH2), 1720
(C]O), 1635, 1582, 1521, 1498, 1453 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.78 (s, 1H, quinolone-2-H), 7.88 (d,
4.1.17. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-nonylquinolin-
4(1H)-one (10d)
d
J ¼ 8.6, 1H, quinolone-5-H), 7.75e7.65 (m, 2H, quinolone-7-H,
Compound 10d was prepared according to the procedure
described for compound 7a starting from 9d (4.41 g, 0.01 mol) and
thiazole-4-H), 7.60 (t, J ¼ 8.6 Hz, 2H, O2NPh-3,5-H), 7.28 (t,

944
Y. Cheng et al. / European Journal of Medicinal Chemistry 124 (2016) 935e945
thiourea (0.76 g, 0.01 mol). The target compound 10d (3.27 g) was
obtained as yellow solid. Yield: 78%; mp: 140e142 ꢀC; IR (KBr,
References
cmꢂ1
)
n
: 3405, 3305 (NeH), 3073 (aromatic CeH), 2925, 2853 (CH3,
CH2), 1713 (C]O), 1637, 1587, 1529, 1497, 1437 (aromatic frame); 1H
NMR (600 MHz, DMSO-d6, ppm): 8.60 (s, 1H, quinolone-2-H), 7.87
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4.1.18. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-undecyl quinolin-
4(1H)-one (10e)
ꢀ
Compound 10e was prepared according to the procedure
described for compound 7a starting from 9e (4.69 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compoundꢀ10e (3.44 g) was
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)
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6919e6929;
n
: 3416, 3306 (NeH), 3075 (aromatic CeH), 2924, 2853 (CH3, CH2),
1717 (C]O), 1606, 1569, 1530, 1497, 1438 (aromatic frame); 1H NMR
(600 MHz, DMSO-d6, ppm): 8.60 (s, 1H, quinolone-2-H), 7.86 (d,
d
J ¼ 7.5 Hz, 1H, quinolone-5-H), 7.75 (dd, J ¼ 11.8, 8.3 Hz, 1H, qui-
nolone-7-H), 7.68 (s, 1H, thiazole-4-H), 6.98 (s, 2H, thiazole-2-NH2),
4.36 (t, J ¼ 5.8 Hz, 2H, CH2CH2(CH2)9CH3), 1.81e1.72 (m, 2H,
CH2CH2(CH2)9CH3), 1.30e1.18 (m, 18H, CH2CH2(CH2)9CH3), 0.84 (t,
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ꢁ
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d 171.9, 167.3, 158.4, 156.9, 153.5, 151.8, 145.7, 144.1, 130.3,
125.4, 114.7, 108.9, 107.5, 105.0, 57.7, 31.7, 30.7, 29.5, 29.4, 29.3, 29.2,
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[MþH]þ, 448.2234; found, 448.2237.
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4.1.19. 3-(2-Aminothiazol-5-yl)-6,8-difluoro-1-(prop-2-ynyl)
quinolin-4(1H)-one (10f)
Compound 10f was prepared according to the procedure
described for compound 7a starting from 9f (3.39 g, 0.01 mol) and
thiourea (0.76 g, 0.01 mol). The target compound 10f (2.53 g) was
obtained as yellow solid. Yield: 80%; mp: 126e128 ꢀC; IR (KBr,
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cmꢂ1
)
n
: 3448, 3299 (NeH), 3231 (alkynyl CeH), 3083 (aromatic
CeH), 2929 (CH2), 2113 (C^C), 1723 (C]O), 1596, 1572, 1527, 1492,
1443 (aromatic frame); 1H NMR (600 MHz, DMSO-d6, ppm)
8.71
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d
(s, 1H, quinolone-2-H), 7.87 (d, J ¼ 7.5 Hz, 1H, quinolone-5-H), 7.77
(dd, J ¼ 11.8, 8.3 Hz, 1H, quinolone-7-H), 7.65 (s, 1H, thiazole-4-H),
6.98 (s, 2H, thiazole-2-NH2), 5.32 (s, 2H, CH2C^CH), 3.59 (s, 1H,
CH2C^CH); 13C NMR (151 MHz, DMSO-d6, ppm):
d 172.1, 167.4,
158.8, 157.1, 153.4, 151.8, 145.2, 143.7, 130.1, 125.2, 115.3, 109.1, 107.4,
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340.0332; found, 340.0330.
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This work was partially supported by National Natural Science
Foundation of China (Nos. 21672173 and 21372186), the Research
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(Regional) Cooperation and Exchange Program of NSFC (No.
81450110451), Chongqing Special Foundation for Postdoctoral
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