A new exploration towards aminothiazolquinolone oximes as potentially multi-targeting antibacterial agents: Design, synthesis and evaluation acting on microbes, DNA, HSA and topoisomerase IV

Article abstract of DOI:10.1016/j.ejmech.2019.06.046

This work did a new exploration towards aminothiazolquinolone oximes as potentially multi-targeting antimicrobial agents. A class of novel hybrids of quinolone, aminothiazole, piperazine and oxime fragments were designed for the first time, conveniently synthesized as well as characterized by ¹H NMR, ¹³C NMR and HRMS spectra. Biological activity showed that some of the synthesized compounds exhibited good antimicrobial activities in comparison with the reference drugs. Especially, O-methyl oxime derivative 10b displayed excellent inhibitory efficacy against MRSA and S. aureus 25923 with MIC values of 0.009 and 0.017 mM, respectively. Further studies indicated that the highly active compound 10b showed low toxicity toward BEAS-2B and A549 cell lines and no obvious propensity to trigger the development of bacterial resistance. Quantum chemical studies have also been conducted and rationally explained the structural features essential for activity. The preliminarily mechanism exploration revealed that compound 10b could not only exert efficient membrane permeability by interfering with the integrity of cells, bind with topoisomerase IV–DNA complex through hydrogen bonds and π-π stacking, but also form a steady biosupramolecular complex by intercalating into DNA to exert the efficient antibacterial activity. The supramolecular interaction between compound 10b and human serum albumin (HSA) was a static quenching, and the binding process was spontaneous, where hydrogen bonds and van der Waals force played vital roles in the supramolecular transportation of the active compound 10b by HSA.

Full text of DOI:10.1016/j.ejmech.2019.06.046

Accepted Manuscript
A new exploration towards aminothiazolquinolone oximes as potentially multi-
targeting antibacterial agents: Design, synthesis and evaluation acting on microbes,
DNA, HSA and topoisomerase IV
Liang-Liang Wang, Narsaiah Battini, Rammohan R. Yadav Bheemanaboina,
Mohammad Fawad Ansari, Jin-Ping Chen, Yun-Peng Xie, Gui-Xin Cai, Shao-Lin
Zhang, Cheng-He Zhou
PII:
S0223-5234(19)30573-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.046
EJMECH 11449
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 March 2019
Revised Date: 15 June 2019
Accepted Date: 16 June 2019
Please cite this article as: L.-L. Wang, N. Battini, R.R. Yadav Bheemanaboina, M.F. Ansari, J.-P.
Chen, Y.-P. Xie, G.-X. Cai, S.-L. Zhang, C.-H. Zhou, A new exploration towards aminothiazolquinolone
oximes as potentially multi-targeting antibacterial agents: Design, synthesis and evaluation acting on
microbes, DNA, HSA and topoisomerase IV, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.06.046.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A new exploration towards aminothiazolquinolone oximes as potentially multi-targeting antibacterial
agents: Design, synthesis and evaluation acting on microbes, DNA, HSA and topoisomerase IV
Liang-Liang Wang1, Narsaiah Battini1,†, Rammohan R. Yadav Bheemanaboina1,†, Mohammad Fawad
Ansari1,‡, Jin-Ping Chen1, Yun-Peng Xie1, Gui-Xin Cai1,*, Shao-Lin Zhang2,*, Cheng-He Zhou1,*
1
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R.
China.
2
School of Pharmaceutical Sciences, Chongqing Key Laboratory of Natural Product Synthesis and Drug
Research, Chongqing University, Chongqing, 401331, P. R. China.
† Postdoctoral researchers from CSIR-Indian Institute of Integrative Medicine (IIIM), India
‡ Postdoctoral researcher from Jamia Millia Islamia, India.
* Corresponding Address:
Tel.: +86-23-68254967; fax: +86-23-68254967; E-mail: gxcai@swu.edu.cn (Gui-Xin Cai);
zhangsl@cqu.edu.cn (Shao-Lin Zhang); zhouch@swu.edu.cn (Cheng-He Zhou).

ACCEPTED MANUSCRIPT
Title page
Title:
A new exploration towards aminothiazolquinolone oximes as potentially multi-targeting antibacterial
agents: Design, synthesis and evaluation acting on microbes, DNA, HSA and topoisomerase IV
Author Names and Affiliations:
Liang-Liang Wang1, Narsaiah Battini1,†, Rammohan R. Yadav Bheemanaboina1,†, Mohammad Fawad
Ansari1,‡, Jin-Ping Chen1, Yun-Peng Xie1, Gui-Xin Cai1,*, Shao-Lin Zhang2,*, Cheng-He Zhou1,*
1
Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of Chongqing
Municipality, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R.
China.
2
School of Pharmaceutical Sciences, Chongqing Key Laboratory of Natural Product Synthesis and Drug
Research, Chongqing University, Chongqing, 401331, P. R. China.
† Postdoctoral researchers from CSIR-Indian Institute of Integrative Medicine (IIIM), India.
‡ Postdoctoral researcher from Jamia Millia Islamia, India.
* Corresponding Address:
Tel.: +86-23-68254967; fax: +86-23-68254967; gxcai@swu.edu.cn (Gui-Xin Cai); zhangsl@cqu.edu.cn
(Shao-Lin Zhang); zhouch@swu.edu.cn (Cheng-He Zhou).
Abstract:
This work did a new exploration towards aminothiazolquinolone oximes as potentially multi-targeting
antimicrobial agents. A class of novel hybrids of quinolone, aminothiazole, piperazine and oxime fragments
were designed for the first time, conveniently synthesized as well as characterized by 1H NMR, 13C NMR
and HRMS spectra. Biological activity showed that some of the synthesized compounds exhibited good
antimicrobial activities in comparison with the reference drugs. Especially, O-methyl oxime derivative 10b
displayed excellent inhibitory efficacy against MRSA and S. aureus 25923 with MIC values of 0.009 and
0.017 mM, respectively. Further studies indicated that the highly active compound 10b showed low toxicity
toward BEAS-2B and A549 cell lines and no obvious propensity to trigger the development of bacterial
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resistance. Quantum chemical studies have also been conducted and rationally explained the structural
features essential for activity. The preliminarily mechanism exploration revealed that compound 10b could
not only exert efficient membrane permeability by interfering with the integrity of cells, bind with
topoisomerase IV–DNA complex through hydrogen bonds and π-π stacking, but also form a steady
biosupramolecular complex by intercalating into DNA to exert the efficient antibacterial activity. The
supramolecular interaction between compound 10b and human serum albumin (HSA) was a static
quenching, and the binding process was spontaneous, where hydrogen bonds and Van der Waals force
played vital roles in the supramolecular transportation of the active compound 10b by HSA.
Keywords:
Quinolone; Oxime; Thiazole; Antimicrobial; Molecular docking.
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1. Introduction
Quinolones are one of the most important first-line antibacterial agents with high oral bioavailability, good
pharmacokinetic properties and excellent curative effects, which have been employed as an effective
weapon to struggle against morbidity and mortality caused by microbial pathogens [1]. This kind of drugs
could target DNA gyrase or topoisomerase IV by binding with the enzyme–DNA binary complexes via a
water-metal ion bridge to trigger irrepairable DNA breakage, interfering DNA replication and ultimately
bringing about bacterial cell death [2]. However, due to the excessive use and even abuse of this kind of
drugs in clinical therapy, the resistance of pathogenic bacteria is increasing seriously, and some serious
side effects like gastroenteritis, vomiting and cartilage damage also have gradually emerged, which have
brought great challenge for public health, underscoring the need to develop more effective new quinolone
derivatives with profound potentiality to overcome the side effects and drug-resistance [3]. In recent years,
the chemical modification on quinolone skeleton by various types of medicinally important structural
fragments has attracted great attention and led to a large number of new marketed quinolone-based
antibacterial agents with excellent antibacterial activity and pharmacokinetic profiles [4].
It has been found that the structural modification at C-3, C-6 or C-7 positions of the quinolone ring is
generally the most essential and efficient strategy [5]. It was considered that the carboxyl group of C-3
position was related to the side-effects of the quinolone [6], which might be attributed to the conjugation of
the benzopyridone skeleton to the carboxyl moiety to some extent. Therefore, the substitution of the
carboxyl group at the C-3 position by other isosteric groups might be a promising alternative pathway to
change the classic binding mode of quinolone to the enzyme–DNA complex, and eventually reduce or
overcome the resistance as well as the side effects [7]. In addition, the modification of the C-6 and C-7
positions also has a significant effect on the biological and pharmacological properties of quinolones,
especially the substitution at the C-7 position could greatly affect the inhibitory activity of topoisomerase
and cell membrane permeabilization, and ultimately impact the solubility, bioactivity, and
pharmacokinetics [8]. These strongly promoted much effort to focus on the structural modification of these
sites in the quinolone ring by incorporating various types of functional fragments.
Azoles are five-membered nitrogen-containing heterocyclic compounds that played an important role in
the field of medicinal chemistry [9], particularly azole-based antimicrobial agents [10]. The incorporation
of azole rings into quinolone has resulted in the discovery of lots of quinolone-based azoles derivatives
with strong bioactivity and high safety profile [11]. Aminothiazole as the most representative azole has
been attracting interest of medicinal chemists in drug development due to its unique structure with
five-membered aromatic heterocyclic core containing nitrogen and sulfur atoms, which make its
derivatives possess good binding ability to interact with their functional targets [12]. The hybridization of
weakly basic 2-aminothiazole fragment with quinolones might be a rationale choice to construct more
effective candidates.
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Oxime group (-C=N-O-) is an important fragment in antimicrobial drugs like cefodizime, cefoselis and
cefmenoxime (antibacterial) as well as oxiconazole (antifungal), which was characterized with both
electron accepting group (C=N) and donating group (-O-), could easily bind with various biomolecules like
nucleic acid DNA, RNA, or some important enzymes through non-covalent interactions [13]. In addition,
the introduction of oxime fragments into quinolone led to the discovery of quinolone oxime derivatives
[14], most of them showed excellent antibacterial potentiality and pharmacokinetic characteristics, which
further emphasized the importance of the oxime fragments with respect to biological activity [15],
therefore, the structural modification of oximes toward quinolone is still an attractive topic.
Piperazine ring is a beneficial structural fragment for bioactivity because of its unique structure that can
easily form hydrogen bonds or ionic bonds to regulate the lipid-water partition coefficient (logP) and
acid-base balance constant of drugs, and prevalently presents in a large variety of potent marketed
antimicrobial drugs like linezolid and ciprofloxacin [16]. Much work has shown that the incorporation of a
functionalized polar alkaline piperazinyl group into quinolone not only significantly improves the water
solubility but also is helpful for enhancing the antibacterial activity [5]. Furthermore, considering that the
piperazine moiety possesses enough structural flexibility for product optimization, it is typically used as an
attractive linker to develop more innovative multi-functional quinolone hybrids with better biological
activities, bioavailability and lower toxicity [17].
Considering all the above facts and in continuation of our previous works on the development of
2-aminothiazolyl quinolones [18], here we reported the design, synthesis and biological activity of a class
of novel hybrids of quinolone, aminothiazole, piperazine and oxime fragments, in which the acidic
carboxyl group at the C-3 position was replaced by a weak basic 2-aminothiazole moiety as an isosteric
group, and various functional oxime fragments were incorporated into the C-6 or C-7 positions of the
quinolone ring through a piperazine bridge (Fig. 1). This series of new multi-component hybrids might
possess multi-targeting antibacterial potentiality. Thus
a
series of piperazine-bridged
aminothiazolquinolone oximes with various substituents were prepared, and their chemical structures were
1
characterized by H NMR, 13C NMR and HRMS spectra. All the synthesized aminothiazolquinolone
oximes were evaluated for their antimicrobial ability. The highly active compounds were also investigated
for the cytotoxic activity, bacterial resistance and drug combination use. Furthermore, the possible
antibacterial mechanism was preliminarily explored, including biofilm disruption assay, molecular docking
and biosupramolecular interactions with DNA. Additionally, the ability evaluation for the supramolecular
transportation of the active compound by human serum albumin (HSA) was performed to study its
absorption, distribution and metabolism.
Fig. 1
2. Results and discussion
2.1. Chemistry
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The synthetic routes for the desired series of C-6 or C-7 substituted aminothiazolquinolone oximes 10a-c,
11, 15a-e, 22a-b, 23a-d and imine derivative 10d were shown in Schemes 1–3. First, the ethyl
2-(ethoxymethylene)-3-oxobutanoate
2
was obtained from the condensation of commercial
triethoxymethane 1, ethyl acetoacetate and acetic anhydride, further reacted with 3-chloro-4-fluoroaniline
and then cyclized in phenoxybenzene under reflux to give the 3-acetyl quinolone derivative 4 in 53.3%
yield. The prepared intermediate 4 went through N-alkylation by bromoethane in acetonitrile, bromination
via bromine in acetic acid and then cyclization with thiourea in ethanol to afford aminothiazolyl quinolone
derivative 7 in 70.9% yield. The substitution of the chlorine atom at the C-7 position of 2-aminothiazolyl
quinolone 7 with piperazine in NMP at 125 ºC under nitrogen atmosphere produced the
7-piperazine-substituted quinolone 8 with the yield of 33.2%. The piperazine derivative 8 was further
reacted with 1-chloropropan-2-one to get the corresponding carbonyl compound 9 in 65.3% yield. The
oximation of the carbonyl derivative 9 with a series of alkoxyamines in acetonitrile at 80 ºC with
potassium carbonate as base produced the corresponding oximes 10a-c with moderate to good yields
ranging from 56.5% to 67.3%. Under similar conditions, the imine compound 10d was also obtained in
52.6% yield. The oxime derivative 10a was further modified by 2,4-dichlorobenzyl chloride to yield the
target compound 11 in 50.9% yield (Scheme 1).
Scheme 1
The 7-substituted quinolone oximes 15a-e were synthesized according to Scheme 2. The desired
intermediates 13a-e were efficiently prepared by the acetylation of substituted benzenes 12a-e with
chloroacetyl chloride at room temperature for 20 h, and further subjected to condensation with
O-methylhydroxylamine hydrochloride in ethanol to afford various phenyl substituted O-methyloxime
intermediates 14a-e in moderate yields of 50.4–70.1%. Subsequently, the desired 7-substituted oxime
derivatives 15a-e were conveniently prepared by the N-alkylation of quinolone derivative 8 with various
phenyl substituted O-methyloximes 14a-e with yields ranging from 54.6% to 60.4%.
Scheme 2
With the aim to investigate the effect of different substituted oxime fragments at the C-6 position of
quinolone skeleton on antimicrobial activity, a set of 6-substituted quinolone oxime derivatives 22a-b and
phenyl oximes 23a-d were synthesized by directly coupling with piperazine moiety at C-6 position of
quinolone as shown in Scheme 3. The nucleophilic aromatic substitution of intermediate 5 with the
piperazinyl moiety in DMSO at 140 ºC using triethylamine as base gave 6-substituted quinolone 16 and
7-substituted quinolone by-product with 41.7 and 55.1% yields, respectively. The 6-substituted derivative
16 was protected by Boc group, and the product was brominated with cupric bromide in ethyl acetate to
yield corresponding 3-(2,2-dibromoacetyl)quinolone 18 in 50.1% yield. Subsequent cyclization with
thiourea in ethanol afforded 6-substituted 3-aminothiazolquinolone 19, which was further removed the Boc
group to produce the desired compound 20. A similar procedure described for the synthesis of compounds
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10a-d, was used for the preparation of target molecules 22a-b starting from intermediate 20. Compound 20
was reacted with 1-chloropropan-2-one and then condensed with hydroxylamine hydrochloride or
O-methylhydroxylamine hydrochloride to generate target compounds 22a-b in 54.2‒61.8% yields. The
target 6-substituted oxime derivatives 23a–d were efficiently prepared from the reaction of the
intermediate 20 and phenyl O-methyl oximes 14a-d with yields ranging from 59.0% to 63.7%.
Scheme 3
2.1.1. The mechanism for the construction of thiazole ring.
The plausible mechanism for the construction of thiazole ring takes place via the Hantzsch thiazole
synthesis, the thiazole derivative (E) was formed by condensation of α-haloketone or α-dihaloketone (A)
and thiourea (B1). This reaction occurs because of the thiourea as tautomer (B2) and forms an intermediate
(C). The intermediate (C) undergoes intramolecular cyclization to form a five-membered cyclic
intermediate (D), followed by dehydration/elimination of bromic acid of cyclic intermediate (D) to give
final molecule (E) (Scheme 4).
Scheme 4
2.1.2. Analysis of configuration (E or Z)
There are usually two configurations of oxime fragment, Z and E, so it was necessary to identify the
geometries of the target oxime derivatives. Therefore, the single crystal of oxime derivative 22b was
obtained and X-ray diffraction analysis showed that the oxime geometry was E configuration (Fig. 2 and
Fig. S1). This is because the Z configuration is generally unstable and easily converted to the E
configuration. Furthermore, the NOE experiment with final compound (15b) was also performed to
confirm the configuration but no correlation was observed between the crucial protons of –OCH3 and
N–CH2 and the results revealed that the oxime geometry should possess similar configuration to compound
22b (Supplementary NOESY spectrum).
Fig. 2
2.2. Biological Activity
All the synthesized aminothiazolquinolone oxime derivatives were evaluated for their in vitro antibacterial
and antifungal activities according to Clinical and Laboratory Standards Institute (CLSI) by means of the
two folds serial dilution technique, and the clinical drug chloromycin, norfloxacin and fluconazole were
used as the positive controls [19]. The minimum inhibitory concentration (MIC) is defined as the lowest
concentration (mM) of target molecules that could completely inhibit the growth of bacteria or fungi. The
antibacterial and antifungal data were summarized in Table 1 and Table S1.
2.2.1. Antibacterial activity
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The in vitro antibacterial evaluation in Table 1 demonstrated that some of the prepared oxime derivatives
exhibited good inhibitory activity against the tested strains. Especially, 7-substituted quinolone oxime
derivative 10b not only displayed broad antibacterial spectrum among all the tested strains, but also could
effectively inhibit the growth of the MRSA strain with low MIC value of 0.009 mM, which was 5-fold and
2-fold more potent than the positive controls of chloromycin (0.050 mM) and norfloxacin (0.025 mM).
However, the structure–activity relationship (SAR) data revealed that the methoxy fragment of oxime
derivative 10b was replaced by hydroxy or ethoxy group, the corresponding hydroxy and ethoxy
derivatives 10a and 10c gave moderate MIC values in the range of 0.034–0.288 mM showing relatively
weak biological activity in comparison with compound 10b, which indicated that either decrease or
increase of the alkyl chain length of the alkoxy fragment could not effectively improve their biological
activity. In addition, we have also found if the alkyl group of the oxime fragment was substituted by the
2,4-dichlorobenzyl group, the resulting compound 11 also significantly lose its biological activity. To
further investigate the effect of oxime fragments on the biological activity of this class of compounds, the
O-methyl oxime was substituted with semicarbazide to afford corresponding imine derivative 10d.
Although it showed a broad spectrum of antibacterial activity, the biological activity was significantly
reduced. After direct comparison of the activities of compounds 9 and 10b, it was also found that for most
of the tested strains, the biological activity of O-methyl oxime derivative 10b was significantly better than
that of carbonyl derivative 9. These results indicated that the oxime fragment played an important role in
the antibacterial activity, especially the introduction of the O-methyl oxime fragment could greatly
optimize the biological activity.
Table 1
Much research has reported that halogen-containing phenyl moieties are beneficial for biological
activity and membrane permeability and could exert large effect on the rate of absorption and transport of
drugs [20]. In our work, with the aim to enrich the chemical diversity as well as to further explore the
SAR, a series of halogenated phenyl O-methyl oximes 15a-e were synthesized and evaluated for their
antibacterial activities. Among them, the 4-chlorophenyl-O-methyl oxime derivative 15b exhibited the best
activities against the tested bacteria with MIC values ranging from 0.029 to 0.461 mM. Especially, it could
effectively inhibit the growth of MRSA strain with MIC value of 0.029 mM, which was approximately
2-fold more potent than chloromycin (MIC = 0.050 mM) and equivalent to norfloxacin (MIC = 0.025
mM). However, when the chlorine atom was replaced by an electron-withdrawing fluorine or
electron-donating methoxy group, the biological activity of the resulted 4-fluorophenyl derivative 15a and
4-methoxyphenyl derivative 15c decreased, indicating that the electronic effect on the biological activity of
the compound is limited. The replacement of 4-fluorophenyl moiety or 4-chlorophenyl group by
2,4-dichlorophenyl and 2,4-difluorophenyl groups, respectively, afforded corresponding O-methyl oxime
derivatives 15d and 15e with weaker activities, which indicated that the position of chlorine or fluorine
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atom on phenyl ring played a crucial role for biological activity and the mono-substituted fluorobenzene or
chlorobenzene was more beneficial to antimicrobial activity in contrast with difluorophenyl or
dichlorophenyl derivatives.
Previous research work including our and other groups has shown that the substitution at the C-6
position of quinolone skeleton also had a significant effect on biological activity [18]. As shown in Table 1,
both of the 6-substituted hydroxyl oxime 22a and O-methyl oxime 22b showed good biological activity.
The hydroxyl oxime derivative 22a could significantly inhibit the growth of S. aureus 25923 and E. coli
with relatively lower MIC values of 0.017 and 0.069 mM, respectively, while O-methyl oxime derivative
22b could effectively inhibit the growth of MRSA and S. aureus with the same concentrations of 0.067
mM. However, among the phenyl O-methyl oxime fragments incorporated derivatives 23a-d, except that
the 4-fluorophenyl oxime derivative 23a exhibited good biological activity with the MIC values ranging
from 0.014 to 0.461 mM against the tested strains, all the other compounds significantly lose the biological
activity or showed even no activity compared to the corresponding 7-substituted counterparts. These
results indicated that the introduction of the oxime fragments played an essential role in the antibacterial
activity, and it was more effective to be connected at the C-7 position of quinolone.
2.2.2. Antifungal activity
The antifungal evaluation was also carried out and the results revealed that some target oxime derivatives
exhibited good potency against the tested fungi. As depicted in Table S1, O-methyl oxime derivative 10b
displayed the best antifungal activity, which not only could completely inhibit the growth of C. albicans
ATCC 90023 with MIC value of 0.017 mM, but also exerted much stronger inhibitory activity against A.
fumigatus (MIC = 0.035 mM) than fluconazole (MIC = 0.836 mM). It was worthy to note that oxime
derivative 10a exhibited equivalent inhibitions against C. parapsilosis ATCC 22019, C. albicans as well as
C. albicans ATCC 90023 with same MIC value of 0.072 mM. Furthermore, O-ethyl oxime derivative 10d
showed MIC value of 0.263 mM against A. fumigatus, which was 3-fold more active than reference drug
fluconazole. In addition, it was found that the hydroxylamine fragment incorporated quinolone derivative
22a possessed considerable potentiality with MIC values ranging from 0.069 to 0.278 mM. Noticeably, in
series of halobenzyl O-methyl oxime derivatives, compounds 15b, 15e and 23a all exhibited moderate to
good antifungal activity against the tested fungi. Especially, derivative 23a bearing 4-fluorobenzyl
fragment showed relatively lower MIC value (0.029 mM) against A. fumigatus than fluconazole. These
results manifested that this kind of oxime derivatives should have great potentiality for the further
development as new antifungal drugs.
2.3. Combination use effects of the O-methyl oxime derivative 10b with reference drugs. (MIC = µg/mL)
Combination therapy is a widely used method for treating various infectious diseases caused by pathogenic
bacteria, which can effectively overcome drug resistance and enhance antimicrobial activity [21]. Herein,
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drug combination studies of active compound 10b with clinical antibacterial cefixime and antifungal
fluconazole against microorganism were performed, and the combination effects were determined by the
calculated fraction inhibitory concentration (FIC) index [22]. The results showed in Table 2 indicated that
the treated strains with combined drugs were more susceptible than their individual use, and their
combined effects mainly exhibited synergistic and additive effects. Particularly, the combination of
compound 10b with cefixime resulted in excellent antibacterial activity against S. aureus with low MIC
value of 8.72 µM, which was 4-fold than itself alone. Moreover, compound 10b also exhibited a
significantly improved antibacterial efficacy against drug-resistant MRSA and K. pneumonia when it was
combined with cefixime. It was worth noting that the combination of compound 10b with clinical
antifungal fluconazole also gave good results against all the tested fungi, while C. tropicals exhibited
additive sensitivity in this combination use. These results revealed that the combination uses of compound
10b with cefixime or fluconazole could effectively enhance antimicrobial activity, overcome drug
resistance and broaden antimicrobial spectrum.
Table 2
2.4. The propensity to induce bacterial resistance
The development of microbial resistance has posed great challenge to human health, and therefore the
assessment of resistance to promising drug candidates comes to be a critical criteria in the drug discovery
process [23]. Herein, the probability of the highly active compound 10b to elicit resistance in bacteria was
performed against MRSA strain with the clinical antibacterial norfloxacin as a positive control. The results
shown in Fig. 3 demonstrated that there was no obvious substantial variation in susceptibility of MRSA to
compound 10b even after 16 passages, while MIC values of norfloxacin toward MRSA increased around
16-fold after the same time, indicating that it was more difficult for active molecule 10b to trigger the
development of bacterial resistance than reference drug norfloxacin. Furthermore, when compound 10b
was combined with cefixime, no drug resistance was obviously generated (green line), which was
consistent with the results of the above-mentioned drug combination, which enhanced the antibacterial
potentiality of compound 10b and overcome the drug resistance.
Fig. 3
2.5. Cytotoxicity assay
Studies have shown that there is a good correlation between extracellular cytotoxicity of chemical
substances and animal mortality as well as blood drug concentration in human death [24]. The cytotoxic
effects of the highly active compound 10b toward mammalian cell lines (A549 and BEAS-2B cells) were
also investigated via MTT assay, and doxorubicin with the concentration of 0.50 µM was used as a positive
control. As shown in Fig. 4, after treatment with different concentrations of 10b for 24 hours, the viability
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of both cell lines remained above 75% even at the concentrations up to 139.52 µM, suggesting that this
molecule possessed less toxicity toward mammalian cell lines.
Fig. 4
2.6. Cell integrity disruption of MRSA induced by active molecule.
To further investigate the possible antibacterial mechanism of this kind of compounds, the ability of
compound 10b to perturb the bacterial cell membrane was studied [25]. The fluorescent nucleic acid dye
propidium iodide (PI) was employed to stain the DNA of bacteria, because it could successfully penetrate
the cells with compromised or damaged membranes and emit fluorescence upon binding to the DNA [26].
As shown in Fig. 5, there was almost no change in fluorescence intensity in untreated bacteria, while the
fluorescence intensity of experimental group increased sharply after the addition of the compound 10b and
it became steady after 100 min. The observed results demonstrated that this molecule had great potentiality
to permeabilize the cell membrane by interfering with the integrity of the membrane.
Fig. 5
2.7. Molecular docking study
Bacterial topoisomerase IV and DNA gyrase have been known as prominent targets for quinolone classes
of antibiotics, which could control the topological state of DNA during the processes of transcription,
replication and recombination [27]. Considering the membrane activity results obtained above, we deduced
that the highly active compound 10b might attack and cause damage to bacterial cell membrane and then
entered the cells to further disrupt the functions of topoisomerase IV or DNA gyrase in the cytoplasm,
which would in turn lead to cell death. Therefore, a flexible ligand-receptor docking investigation was
carried out with topoisomerase IV–DNA complex and gyrase–DNA complex (PDB code: 2XKK and
4DUH, respectively) as representative targets to rationalize the action mechanism of compound 10b [28].
Docking results (Fig. 6) showed that there was a hydrogen bond between the oxygen atom of O-methyl
oxime fragment and DA-20 residue with a distance of 1.8 Å. The NH2 group at the 2-position of thiazole
ring could interact with the ASP-1083 residue through hydrogen bond with the distance of 2.1 Å (Fig. 6A).
In addition, the alkyl interaction can also be found between the piperazine ring and ARG-418 residue,
which further revealed that the introduction of piperazine ring was beneficial. Furthermore, the aromatic
ring of quinolone could interact with base DT-15 of DNA via π-π stacking (Fig. 6B). The docking model
with DNA gyrase indicated that the nitrogen atom on the aminothiazole ring and the oxygen atom of the
quinolone ring can form multiple hydrogen bonds with the Arg 136 residue, which might be beneficial for
the stability of the 10b-gyrase–DNA complex (Fig. S2). Furthermore, the nitrogen atom of O-methyl
oxime fragment also took part in the non-covalent coordination with Asn-46 residue by a hydrogen bond,
which further revealed that nitrogen atom played a crucial role in drug molecules. The docking evaluation
gave the lowest binding energy of -7.93 kcal/mol between compound 10b and topoisomerase IV. All these
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non-covalent interactions indicated that compound 10b could disturb the synthesis of bacterial DNA
through binding to DNA gyrase or topoisomerase IV to exert good antibacterial activity.
Fig. 6
2.8. Electronic properties
It is well known that the frontier molecular orbitals (FMO), namely the highest occupied molecular orbital
(HOMO) and the lowest unoccupied molecular orbital (LUMO), play an extremely important role in the
ligand–receptor interactions process because of the reason that the HOMOs and LUMOs are related to the
potential and ability of molecules to donate or accept electrons. The concept is usually extended into
drug-receptor binding systems to study the pharmacokinetic properties of drugs [29]. As shown in Table 3,
The HOMOs of O-methyl oxime derivatives 10b, 15b and 23a were primarily concentrated on the
quinolone skeleton and the 2-aminothiazole fragment, indicating that these positions might be active sites
and biological interactions may occur between these sites and positively charged molecules. Besides, it
was worth noting that LUMOs were also mainly localized on the quinolone ring, manifesting that the
quinolone ring could not only interact with the electron donor, but also facilitate nucleophilic attacks.
However, the substituted oxime fragments and piperazine ring did not directly contribute to any HOMOs
and LUMOs, suggesting that these groups might mainly play roles in regulating the physicochemical
properties of the molecules. Furthermore, it was found that compound 10b shown best antibacterial
activity possessed the highest energy gap value (∆E) of 4.024 eV, which made it more reactivity and less
stability.
Table 3
Molecular electrostatic potential (MEP) surface can usually effectively indicate the distribution of
electron density on the molecular surface, where the positive isosurfaces (blue) represent the electron
deficit regions and the negative isosurfaces (red) signify the electron rich regions, which might be
responsible for the formation of hydrogen bond and interaction between these sites and biological targets
in biological systems. As shown in Figure 7, the positive charge regions (in blue) mainly located on the
amino group of thiazole ring (NH2) and the electronegative area (in red) primarily concentrated on the
oxygen atom of the quinolone ring, which was a good explanation why it was easier for amino and oxygen
to form hydrogen bonds with topoisomerase IV and DNA gyrase (Fig. 7 and Fig. S2), which was excellent
agreement with docking study.
Fig. 7
2.9. Biosupramolecular interactions between highly active molecule 10b and DNA
Deoxyribonucleic acid (DNA), a significant informative molecule with the function of encoding genetic
instructions, has been considered as a validated drug target for the extensive exploration of newly
11

ACCEPTED MANUSCRIPT
promising DNA-targeting antimicrobial drugs [30]. Due to the medical importance, low cost and ready
availability properties of calf thymus DNA, it was always selected as the DNA model to explore the
preliminary antibacterial action mechanism of active candidate molecules [31]. Herein, the in vitro binding
behavior of the active compound 10b with calf thymus DNA was performed at a molecular level using NR
(neutral red dye) as a spectral probe through UV-vis spectroscopy.
2.9.1. Absorption spectra of DNA in the presence of compound 10b
The UV-vis absorption spectrum of the molecule is a commonly useful spectral analysis method based on
the absorption spectrum generated by intramolecular electronic transitions, and it is widely used in DNA
binding studies [32]. The hyperchromaticity and hypochromaticity as important spectral features are
usually emplyed to identify the changes of DNA double helix structure, which are generally resulted from
the strong interaction of electronic states between an aromatic chromophore and the base pairs of DNA
[33]. As shown in Fig. 8, the UV-vis spectra showed that the absorption peak of DNA at 260 nm gave a
proportional increase and slight red shift with the sequentially and proportionately increasing amount of
compound 10b under a fixed concentration of DNA. Furthermore, it was observed (inset of Fig. 8) that the
measured value of the 10b–DNA complex was a little higher than the absorption value of the simply sum
of free DNA and free compound 10b, which illustrated that a weak hyperchromic effect existed between
DNA and molecule 10b. This hyperchromic effect and the red shift phenomenon might be the consequence
of electrostatic interaction between 10b and the DNA base, which provided the convincing evidences for
the constructional change of DNA duplex and the formation of binary complexes.
A0
A− A0 ξD−C − ξC ξD−C − ξC K[Q]
ξC
ξC
1
=
+
×
(1)
On the basis of the variations in these spectra, the binding constant K could be calculated by using
equation (1) [34], yielding K = 2.51 × 104 L/mol, R = 0.995, SD = 0.077 (R and SD stand for the
correlation coefficient and standard deviation, respectively). The plot of A0/(A–A0) versus 1/[compound
10b] was constructed and showed in Fig. S3.
Fig. 8
2.9.2. The competitive interaction of active molecule 10b and NR with DNA
Neutral red (NR) bearing a similar structure to other planar dyes like acridine, thiazine and xanthene has
the advantages of lower toxicity, higher stability and more convenient application as compared to other
common probes, and it has been proved to bind with DNA by the way of intercalation [35]. Therefore, NR
was selected as a spectral probe to study the binding mode of compound 10b with DNA in the present
work, and the absorption spectrum of competitive binding between NR and 10b with DNA was shown in
Fig. 9. It was found that there was an obvious decrease of the absorption spectra around 530 nm along with
the increasing concentration of 10b, which was a reverse process as compared with the absorption of free
12

ACCEPTED MANUSCRIPT
NR at the same wavelength [31]. In addition, a gradually increasing absorption at around 276 nm was also
observed in the spectra. These apparent spectral changes suggested that 10b could effectively intercalate
into the double helix of DNA by substituting NR in the DNA–NR complex, which might block bacterial
DNA replication to exert powerful antibacterial activity.
Fig. 9
2.10. Supramolecular transportation of the highly active molecule 10b by human serum albumin (HSA)
To understand the pharmacological and pharmacodynamic behavior of drugs, the interaction between
drugs and HAS was done using Fluorescence quenching mechanism [36]. As shown in Fig. 10, it could be
found that HSA had a strong fluorescence emission peak at around 343 nm (red line), whereas in the same
wavelength range, compound 10b only showed a weak fluorescence intensity (blue line), which could be
almost ignored. In addition, under the fixed HSA concentration, the intensity of maximum emission peak
gave a gradual decrease with the increasing concentrations of 10b, but the maximum emission wavelength
of HSA remained unchanged (black lines), indicating that Trp-214 did not undergo any polarity change,
which further suggested that 10b might interact with HSA through hydrophobic regions located in the HSA.
And the fluorescence quenching behavior disclosed that the supramolecular interaction between compound
10b and human serum albumin (HSA) was a static quenching rather than dynamic quenching, and the
binding process was spontaneous, where hydrogen bonds and Van der Waals force played vital roles in the
supramolecular transportation of the active compound 10b by HSA through forming a stable 1:1
supramolecular complex (Supporting Information).
Fig. 10
3. Conclusion
This work explored a class of novel hybrids of quinolone, aminothiazole, piperazine and oxime fragments
as potentially multi-targeting antimicrobial agents for the first time. A series of piperazine-bridged
aminothiazolquinolone oximes have been successfully developed, and tested for their in vitro antimicrobial
activities. Some of the synthesized compounds exhibited moderate to good antimicrobial activities. SAR
study revealed that the positions of the oxime fragments at the quinolone skeleton exerted a significant
impact on biological activity. Especially, C-7 substituted O-methyl oxime derivative 10b displayed
excellent inhibitory efficacy against MRSA and S. aureus 25923 with MIC values of 0.009 and 0.017 mM,
respectively. Further experiments indicated that this molecule showed low toxicity against A549 and
BEAS-2B cell lines and no obvious propensity to trigger the development of bacterial resistance. Quantum
chemical studies have rationally explained the structural features essential for activity. The preliminarily
mechanism exploration revealed that compound 10b could effectively permeate bacterial membrane,
interact with topoisomerase IV through hydrogen bonds and π-π stacking, and intercalate into DNA to
form a stable supramolecular complex, which might be responsible for the powerful bioactivity.
13

ACCEPTED MANUSCRIPT
Furthermore, the active molecule 10b could be effectively transported by HSA via hydrogen bonds and
Van der Waals force, and the transportation mechanism was static rather than dynamic. These results
revealed that O-methyl oxime derivative 10b could be served as a promising candidate in the development
of potent antimicrobial agents.
4. Experimental
4.1. General methods
All the chemicals, reagents and organic solvents were commercially available and used directly without
further purification. The samples were weighed on a microbalance with a resolution of 0.1 mg. Thin-layer
chromatography (TLC) analysis were carried out through pre-coated silica gel plates. Column
chromatography was performed by silica gel (#100–200). NMR spectra were recorded on Bruker AVANCE
III 600 MHz spectrometer using DMSO-d6 as solvent, or TMS as internal standard. The chemical shifts
were reported in parts per million (ppm), the coupling constants (J) were expressed in hertz unit (Hz) and
signals were described as singlet (s), doublet (d), triplet (t), broad (br) as well as multiplet (m). The
high-resolution mass spectra (HRMS) were recorded on IonSpec FT–CR mass spectrometer with ESI
resource. Melting point (mp) was measured on a melting point apparatus (X-6 type). All fluorescence
spectra were recorded on F-7000 spectrofluorimeter (Hitachi, Tokyo, Japan) and UV spectra were obtained
through TU-2450 spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China), both of which were
equipped with 1.0 cm quartz cells. The microbial strains were provided by Sichuan Provincial People's
Hospital (Chengdu, China) and the neutral red (NR) and DNA were purchased from Sigma-Aldrich (St.
Louis, MO, USA). Quantitative nuclear magnetic resonance (QNMR) was used to determine the purity of
the target compounds using 1,3,5-trioxane as the internal standard. The results indicated that all the target
compounds possessed purity ≥ 95%.
4.1.1. General experimental procedures for the synthesis of the desired intermediates (2–7).
The desired intermediates 2–7 were prepared according to the previously reported methods [18].
4.1.2. General experimental procedures for the synthesis of the desired intermediates (13a-e and 14a-e)
The desired intermediates 13a-e and 14a-e were prepared according to the previously reported methods
[37].
4.1.3. Synthesis of 3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (8).
A mixture of intermediate 7 (4.86 g, 15.00 mmol) and piperazine (3.87 g, 45.00 mmol) in 1-methyl-
2-pyrrolidinone (NMP) (20 mL) was stirred at 130 ºC for 24 h. After completion of the reaction, the
solvent was evaporated under reduced pressure and the residue was diluted with water (200 mL), and then
extracted with chloroform (3 × 100 mL). The combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated. The crude product was purified via silica gel column chromatography
(eluent, chloroform/ methanol (V/V) = 15/1) to afford compound 8 (1.19 g) as yellow solid. Yield: 21.3%;
14

ACCEPTED MANUSCRIPT
1
mp: > 250 ºC; H NMR (600 MHz, DMSO-d6) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.7 Hz, 1H,
quinolone-5-H), 7.63 (s, 1H, thiazole-4-H), 7.01 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.92 (s, 2H,
thiazole-2-NH2), 4.38 (q, J = 7.0 Hz, 2H, CH2CH3), 3.15 (t, J = 4.02 Hz, 4H, piperazine-2,2-N-(CH2)2),
2.91 (t, J = 4.38 Hz, 4H, piperazine-3,3-N-(CH2)2), 1.38 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151
MHz, DMSO-d6) δ 172.9, 167.1, 151.8, 144.9, 144.7, 142.6, 136.1, 121.5, 114.9, 111.9, 105.3, 103.9, 51.2,
49.1, 48.4, 45.7, 14.8 ppm.
4.1.4.
Synthesis
of
3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-oxopropyl)piperazin-1-
yl)quinolin-4(1H)-one (9).
A mixture of intermediate 8 (186.73 mg, 0.50 mmol) and potassium carbonate (103.66 mg, 0.75 mmol) in
acetonitrile (25 mL) was stirred at 60 ºC for 1.5 h, and then cooled to room temperature. To the reaction
system was added slowly 1-Chloropropan-2-one (69.39 mg, 0.75 mmol), and the resulting mixture was
stirred at 80 ºC for 6 h. After the completion of reaction, the solvent was evaporated under reduced
pressure and the residue was diluted with water (100 mL), and then extracted with chloroform (3 × 50 mL).
The organic extracts were dried over anhydrous sodium sulfate and concentrated. The crude product was
purified by silicagel column chromatography (eluent, chloroform/methanol (V/V) = 25/1) to afford target
compound 9 (143.24 mg) as yellow solid. Yield: 66.7%; mp: 163–165 ºC; 1H NMR (600 MHz, DMSO-d6)
δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.63 (s, 1H, thiazole-4-H), 7.05
(d, J = 7.3 Hz, 1H, quinolone-8-H), 6.90 (s, 2H, thiazole-2-NH2), 4.38 (q, J = 7.1 Hz, 2H, CH2CH3), 3.28
(s, 2H, CH2COCH3), 3.27–3.22 (m, 4H, piperazine-2,2-N-(CH2)2), 2.66–2.60 (m, 4H,
piperazine-3,3-N-(CH2)2), 2.11 (s, 3H, CH2COCH3), 1.39 (t, J = 7.2 Hz, 3H, CH2CH3) ppm; 13C NMR
(151 MHz, DMSO-d6) δ 207.1, 172.9, 167.1, 151.7, 144.9, 144.2, 142.7, 136.1, 121.6, 114.9, 111.8, 105.5,
103.9, 67.7, 52.9, 50.2, 49.1, 48.4, 28.1, 14.8 ppm; HRMS (ESI) calcd. for C21H24FN5O2S [M + H]+,
430.1713; found, 430.1710.
4.1.5. Synthesis of (E)-3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-(hydroxyimino)propyl)piperazin-
1-yl)quinolin-4(1H)-one (10a).
A mixture of intermediate 9 (214.75 mg, 0.50 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
hydroxylamine hydrochloride (52.11 mg, 0.75 mmol) in acetonitrile (25 mL) was stirred at 80 ºC for 6 h.
After the completion of reaction, the solvent was evaporated under reduced pressure and the residue was
diluted with water (100 mL), and then extracted with chloroform (3 × 50 mL). The organic extracts were
dried over anhydrous sodium sulfate and concentrated. The crude product was purified via silica gel
column chromatography (eluent, chloroform/methanol (V/V) = 25/1) to afford target compound 10a
(149.58 mg) as light yellow solid. Yield: 67.3%; mp: > 250 ºC; 1H NMR (600 MHz, DMSO-d6) δ 10.58 (s,
1H, OH), 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.63 (s, 1H, thiazole-4-H),
7.05 (d, J = 7.1 Hz, 1H, quinolone-8-H), 6.92 (s, 2H, thiazole-2-NH2), 4.38 (dd, J = 13.8, 6.7 Hz, 2H,
CH2CH3), 3.24 (s, 4H, piperazine-2,2-N-(CH2)2), 3.04 (s, 2H, CH2C(N-OH)CH3), 2.54 (s, 4H,
15

ACCEPTED MANUSCRIPT
piperazine-3,3-N-(CH2)2), 1.81 (s, 3H, CH3), 1.38 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz,
DMSO-d6) δ 172.9, 167.1, 153.4, 144.9, 144.2, 142.7, 136.1, 121.6, 114.9, 111.7, 105.6, 103.9, 62.1, 52.9,
50.2, 48.4, 14.8, 12.7 ppm; HRMS (ESI) calcd. for C21H26FN6O2S [M + H]+, 445.1822; found, 445.1815.
4.1.6. Synthesis of (E)-3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-(methoxyimino)propyl)piperazin
-1-yl)quinolin-4(1H)-one (10b).
Compound 10b was prepared according to the experimental procedure described for compound 10a
starting from intermediate 9 (214.75 mg, 0.50 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
O-methylhydroxylamine hydrochloride (41.76 mg, 0.50 mmol). The target compound 10b (141.11 mg)
was obtained as light yellow solid. Yield: 61.6%; mp: 230–232 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.59
(s, 1H, quinolone-2-H), 7.86 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.63 (s, 1H, thiazole-4-H), 7.05 (d, J =
7.1 Hz, 1H, quinolone-8-H), 6.92 (s, 2H, thiazole-2-NH2), 4.38 (dd, J = 13.9, 6.9 Hz, 2H, CH2CH3), 3.78 (s,
3H, OCH3), 3.24 (s, 4H, piperazine-2,2-N-(CH2)2), 3.03 (s, 2H, CH2C(N-OCH3)CH3), 2.55 (s, 4H,
piperazine-3,3-N-(CH2)2), 1.84 (s, 3H, CH3), 1.38 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz,
DMSO-d6) δ 172.9, 167.1, 155.4, 153.4, 144.9, 144.2, 142.6, 136.1, 121.6, 114.9, 111.7, 105.5, 103.9, 61.6,
61.4, 52.9, 50.2, 49.7, 48.4, 14.8, 13.3 ppm; HRMS (ESI) calcd. for C22H27FN6O2S [M + H]+, 459.1978;
found, 459.1974.
4.1.7. Synthesis of (E)-3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-(methoxyimino)propyl)piperazin
-1-yl)quinolin-4(1H)-one (10c).
Compound 10c was prepared according to the experimental procedure described for compound 10a
starting from intermediate 9 (214.75 mg, 0.50 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
O-ethylhydroxylamine hydrochloride (73.16 mg, 0.75 mmol). The target compound 10c (133.40 mg) was
1
obtained as yellow solid. Yield: 56.5%; mp: 202–204 ºC; H NMR (600 MHz, DMSO-d6) δ 8.59 (s, 1H,
quinolone-2-H), 7.86 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.63 (s, 1H, thiazole-4-H), 7.04 (d, J = 7.2 Hz,
1H, quinolone-8-H), 6.91 (s, 2H, thiazole-2-NH2), 4.38 (q, J = 7.1 Hz, 2H, CH2CH3), 4.04 (q, J = 7.0 Hz,
2H, OCH2CH3), 3.24 (s, 4H, piperazine-2,2-N-(CH2)2), 3.04 (s, 2H, CH2C(N-OCH2CH3)CH3), 2.55 (s, 4H,
piperazine-3,3-N -(CH2)2), 1.85 (s, 3H, CH3), 1.39 (t, J = 7.2 Hz, 3H, N-CH2CH3), 1.19 (t, J = 7.0 Hz, 3H,
OCH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 172.9, 167.1, 154.9, 153.4, 151.8, 144.9, 142.7, 136.1,
121.6, 114.9, 111.9, 105.6, 103.9, 68.7, 61.8, 52.9, 50.2, 48.4, 15.1, 14.8, 13.4 ppm; HRMS (ESI) calcd. for
C23H29FN6O2S [M + H]+, 473.2135; found, 473.2133.
4.1.8. Synthesis of (E)-2-(1-(4-(3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7
-yl)piperazin-1-yl)propan-2-ylidene)hydrazine-1-carboxamide (10d).
Compound 10d was prepared according to the experimental procedure described for compound 10a
starting from intermediate 9 (214.75 mg, 0.50 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
aminourea hydrochloride (83.65 mg, 0.75 mmol). The target compound 10d (127.87 mg) was obtained as
1
white solid. Yield: 52.6%; mp: > 250 ºC; H NMR (600 MHz, DMSO-d6) δ 9.06 (s, 1H, N-NHCONH2),
16

ACCEPTED MANUSCRIPT
8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.04 (d,
J = 7.2 Hz, 1H, quinolone-8-H), 6.91 (s, 2H, thiazole-2-NH2), 6.24 (s, 2H, N-NHCONH2), 4.38 (q, J = 7.0
Hz, 2H, CH2CH3), 3.24 (s, 4H, piperazine-2,2-N-(CH2)2), 3.07 (s, 2H, CH2C(N- NHCONH2)CH3), 2.55 (s,
4H, piperazine-3,3-N-(CH2)2), 1.87 (s, 3H, CH3), 1.39 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151
MHz, DMSO-d6) δ 172.9, 167.1, 157.6, 153.4, 151.8, 144.9, 142.7, 136.1, 121.6, 114.9, 111.7, 105.5,
103.9, 64.9, 52.9, 50.3, 48.4, 14.9, 14.7 ppm; HRMS (ESI) calcd. for C22H27FN8O2S [M + H]+, 487.2040;
found, 487.2036.
4.1.9. Synthesis of (E)-3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-(methoxyimino)propyl)piperazin
-1-yl)quinolin-4(1H)-one (11).
A mixture of compound 10a (222.09 mg, 0.50 mmol) and potassium carbonate (103.66 mg, 0.75 mmol) in
acetonitrile (25 mL) was stirred at 60 ºC for 1.5 h, and then cooled to room temperature. To the reaction
system was added 2,4-Dichlorobenzyl chloride (293.21 mg, 1.50 mmol), and the resulting mixture was
stirred at 80 ºC for 12 h. After the completion of reaction, the solvent was evaporated under reduced
pressure and the residue was diluted with water (50 mL), and then extracted with chloroform (3 × 20 mL).
The organic extracts were dried over anhydrous sodium sulfate and concentrated. The crude product was
purified via silicagel column chromatography (eluent, dichloromethane/methanol (V/V) = 50/2) to afford
1
target compound 11 (153.25 mg) as yellow solid. Yield: 50.9%; mp: > 250 ºC; H NMR (600 MHz,
DMSO-d6) δ 8.60 (s, 1H, quinolone-2-H), 7.87 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H,
thiazole-4-H), 7.62 (s, 1H, 2,4-diCl-Ph-3-H), 7.45 (s, 2H, 2,4-diCl-Ph-4,5-2H), 7.04 (d, J = 7.2 Hz, 1H,
quinolone-8-H), 6.94 (s, 2H, thiazole-2-NH2), 5.14 (s, 2H, 2,4-2ClPh-CH2), 4.38 (q, J = 7.0 Hz, 2H,
CH2CH3), 3.24 (s, 4H, piperazine-2,2-N-(CH2)2), 3.07 (s, 2H, CH2C(N-OCH2- 2,4-2Cl-Ph)CH3), 2.54 (s,
4H, piperazine-3,3-N-(CH2)2), 1.93 (s, 3H, -CH3), 1.39 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151
MHz, DMSO-d6) δ 172.9, 167.1, 153.4, 144.8, 142.7, 136.1, 135.2, 133.7, 133.5, 132.2, 131.9, 131.7,
129.1, 127.7, 121.6, 114.8, 111.9, 105.5, 103.9, 71.9, 67.9, 52.8, 50.1, 48.4, 14.8, 14.3 ppm; HRMS (ESI)
calcd. for C28H29Cl2FN6O2S [M + H]+, 603.1512; found, 603.1486.
4.1.10.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-(4-fluorophenyl)-2-
(methoxyimino)ethyl)piperazin-1-yl)quinolin- 4(1H)-one (15a).
A mixture of compound 8 (186.57 mg, 0.50 mmol) and potassium carbonate (103.66 mg, 0.75 mmol) in
acetonitrile (25 mL) was stirred at 60 ºC for 1.5 h, and then cooled to room temperature. Intermediate 14a
(150.78 mg, 0.75 mmol) was added, and the resulting mixture was stirred at 80 ºC for 10 h. After the
completion of reaction, the solvent was evaporated under reduced pressure and the residue was diluted
with water (50 mL), and then extracted with chloroform (3 × 20 mL). The organic extracts were dried over
anhydrous sodium sulfate and concentrated. The crude product was purified via silica gel column
chromatography (eluent, dichloromethane/methanol (V/V) = 50/2) to afford target compound 15a (171.68
1
mg) as yellow solid. Yield: 63.8%; mp: 230–232 ºC; H NMR (600 MHz, DMSO-d6) δ 8.58 (s, 1H,
17

ACCEPTED MANUSCRIPT
quinolone-2-H), 7.86 (d, J = 7.3 Hz, 1H, quinolone-5-H), 7.84 (d, J = 2.8 Hz, 1H, 4-F-Ph-2-H), 7.83 (d, J
= 2.0 Hz, 1H, 4-F-Ph-6-H), 7.64 (s, 1H, thiazole-4-H), 7.23 (t, J = 8.9 Hz, 2H, 4-F-Ph-3,5-2H), 7.01 (d, J =
7.2 Hz, 1H, quinolone-8-H), 6.93 (s, 2H, thiazole-2-NH2), 4.36 (q, J = 7.1 Hz, 2H, CH2CH3), 3.94 (s, 3H,
OCH3), 3.72 (s, 2H, CH2C(N-OCH3)), 3.15 (s, 4H, piperazine-2,2-N-(CH2)2), 2.62 (s, 4H,
piperazine-3,3-N-(CH2)2), 1.36 (d, J = 7.0 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ
172.9, 167.4, 167.1, 163.9, 162.3, 153.9, 151.8, 144.8, 144.2, 142.7, 136.0, 132.2, 132.0, 129.4, 129.1,
121.7, 115.6, 115.4, 114.8, 111.8, 105.6, 103.9, 62.3, 53.2, 51.2, 50.2, 49.1, 48.4, 14.8 ppm; HRMS (ESI)
calcd. for C27H28F2N6O2S [M + H]+, 539.2041; found, 539.2040.
4.1.11.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-7-(4-(2-(4-chlorophenyl)-2-(methoxyimino)ethyl)
piperazin-1-yl)-1-ethyl-6-fluoroquinolin -4(1H)-one (15b).
Compound 15b was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14b (162.75 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 8 (186.73 mg, 0.50 mmol). The target compound 15b (174.84 mg) was obtained as light yellow
solid. Yield: 63.1%; mp: 220–222 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.57 (s, 1H, quinolone-2-H), 7.85
(d, J = 13.6 Hz, 1H, quinolone-5-H), 7.81 (s, 1H, 4-Cl-Ph-2-H), 7.80 (s, 1H, 4-Cl-Ph-6-H), 7.63 (s, 1H,
thiazole-4-H), 7.46 (s, 1H, 4-Cl-Ph-3-H), 7.45 (s, 1H, 4-Cl-Ph-5-H), 7.00 (d, J = 7.2 Hz, 1H,
quinolone-8-H), 6.86 (s, 2H, thiazole-2-NH2), 4.34 (q, J = 7.1 Hz, 2H, CH2CH3), 3.94 (s, 3H, OCH3), 3.72
(s, 2H, CH2C(N-OCH3), 3.15 (s, 4H, piperazine-2,2-N-(CH2)2), 2.61 (s, 4H, piperazine-3,3-N-(CH2)2), 1.36
(t, J = 7.1 Hz, 3H, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 172.9, 167.1, 153.9, 153.4, 151.8,
144.9, 144.2, 142.7, 136.0, 134.3, 128.9, 128.6, 121.6, 114.9, 111.8, 111.7, 105.6, 103.9, 62.4, 53.0, 51.1,
50.2, 49.1, 48.4, 14.8 ppm; HRMS (ESI) calcd. for C27H28ClFN6O2S [M + H]+, 555.1745; found,
555.1740.
4.1.12.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-(methoxyimino)-2-(4-
methoxyphenyl)ethyl)piperazin-1-yl)quinolin -4(1H)-one (15c).
Compound 15c was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14c (159.79 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 8 (186.73 mg, 0.50 mmol). The target compound 15c (162.59 mg) was obtained as yellow solid.
Yield: 59.1%; mp: 197–199 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.57 (s, 1H, quinolone-2-H), 7.85 (d, J =
13.6 Hz, 1H, quinolone-5-H), 7.74 (d, J = 2.0 Hz, 1H, 4-CH3O-Ph-2-H), 7.73 (s, 1H, 4-CH3O-Ph-6-H),
7.63 (s, 1H, thiazole-4-H), 7.01 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.95 (s, 1H, 4-CH3O-Ph-3-H), 6.94 (s,
1H, 4-CH3O-Ph-5-H), 6.91 (s, 2H, thiazole-2-NH2), 4.35 (q, J = 7.1 Hz, 2H, CH2CH3), 3.91 (s, 3H,
-OCH3), 3.78 (s, 3H, Ph-OCH3), 3.69 (s, 2H, CH2C(N-OCH3), 3.15 (s, 4H, piperazine-2,2-N-(CH2)2), 2.62
(s, 4H, piperazine-3,3-N-(CH2)2), 1.35 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6)
δ 172.9, 167.1, 160.5, 154.2, 153.4, 151.8, 144.9, 144.2, 142.7, 136.0, 128.5, 121.6, 114.9, 114.0, 111.8,
18

ACCEPTED MANUSCRIPT
111.7, 105.6, 103.9, 62.1, 55.7, 53.1, 51.1, 50.3, 48.4, 14.8 ppm; HRMS (ESI) calcd. for C28H31FN6O3S [M
+ H]+, 551.2241; found, 551.2236.
4.1.13. Synthesis of (E)-3-(2-Aminothiazol-4-yl)-7-(4-(2-(2,4-dichlorophenyl)-2-(methoxyimino)ethyl)-
piperazin-1-yl)-1-ethyl-6-fluoro quinolin-4(1H)-one (15d)
Compound 15d was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14d (188.23 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 8 (186.73 mg, 0.50 mmol). The target compound 15d (177.61 mg) was obtained as yellow solid.
Yield: 60.4%; mp: 140–142 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.57 (s, 1H, quinolone-2-H), 7.83 (d, J =
13.6 Hz, 1H, quinolone-5-H), 7.68 (d, J = 2.0 Hz, 1H, 2,4-diCl-Ph-6-H), 7.62 (s, 1H, thiazole-4-H), 7.50 (s,
1H, 2,4-diCl-Ph-3-H), 7.48 (d, J = 2.0 Hz, 1H, 2,4-diCl-Ph-5-H), 6.96 (d, J = 7.3 Hz, 1H, quinolone-8-H),
6.92 (s, 2H, thiazole-2-NH2), 4.35 (q, J = 7.1 Hz, 2H, CH2CH3), 3.91 (s, 3H, -OCH3), 3.70 (s, 2H,
CH2C(N-OCH3), 3.03 (s, 4H, piperazine-2,2-N-(CH2)2), 2.55 (d, J = 3.9 Hz, 4H, piperazine-3,3-N-(CH2)2),
1.35 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 172.9, 167.1, 156.6, 153.3,
151.7, 144.9, 144.1, 142.6, 136.0, 134.3, 133.9, 132.8, 129.3, 127.5, 121.6, 114.9, 111.7, 105.5, 103.9, 62.4,
54.2, 53.4, 50.1, 48.3, 14.9 ppm; HRMS (ESI) calcd. for C27H27Cl2FN6O2S [M + H]+, 589.1356; found,
589.1348.
4.1.14. Synthesis of (E)-3-(2-Aminothiazol-4-yl)-7-(4-(2-(2,4-difluorophenyl)-2-(methoxyimino)ethyl)-
piperazin-1-yl)-1-ethyl-6-fluoro- quinolin-4(1H)-one (15e).
Compound 15e was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14e (164.27 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 8 (186.73 mg, 0.50 mmol). The target compound 15e (151.84 mg) was obtained as yellow solid.
Yield: 54.6%; mp: 144–146 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.57 (s, 1H, quinolone-2-H), 7.83 (d, J =
13.6 Hz, 1H, quinolone-5-H), 7.69–7.65 (m, 1H, 2,4-diF-Ph-6-H), 7.63 (s, 1H, thiazole-4-H), 7.31–7.27 (m,
1H, 2,4-diF-Ph-5-H), 7.13 (td, J = 8.4, 2.4 Hz, 1H, 2,4-diF-Ph-3-H), 6.97 (d, J = 7.3 Hz, 1H, quinolone
-8-H), 6.91 (s, 2H, thiazole-2-NH2), 4.35 (d, J = 7.1 Hz, 2H, CH2CH3), 3.92 (s, 3H, -OCH3), 3.68 (s, 2H,
CH2C(N-OCH3)), 3.06 (s, 4H, piperazine-2,2-N-(CH2)2), 2.56–2.52 (m, 4H, piperazine-3,3-N-(CH2)2), 1.35
(t, J = 7.2 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 172.9, 167.1, 162.1, 153.9, 153.3,
151.7, 144.9, 144.1, 142.6, 136.0, 131.9, 129.2, 121.6, 114.9, 111.8, 105.5, 104.6, 103.9, 62.4, 53.5, 53.2,
50.1, 48.3, 14.8 ppm; HRMS (ESI) calcd. for C27H27F3N6O2S [M + H]+, 557.1947; found, 557.1940.
4.1.15. Synthesis of 3-Acetyl-7-chloro-1-ethyl-6-(piperazin-1-yl)quinolin-4(1H)-one (16).
A mixture of compound 5 (4.00 g, 14.98 mmol), triethylamine (6.06 g, 59.92 mmol), and piperazine (5.16
g, 59.92 mmol) was stirred at 140 ºC in DMSO (30 mL) for 12 h. After completion of the reaction, the
solvent was evaporated under reduced pressure and the residue was diluted with water (200 mL), and then
extracted with chloroform (3 × 100 mL). The organic extracts were dried over anhydrous sodium sulfate
19

ACCEPTED MANUSCRIPT
and concentrated. The crude product was purified via silica gel column chromatography (eluent,
chloroform/methanol (V/V) = 15/1) to afford compound 16 (1.95 g) as yellow solid. Yield: 39.2%.
4.1.16. Synthesis of Tert-butyl4-(3-acetyl-7-chloro-1-ethyl-4-oxo-1,4-dihydroquinolin-6-yl)piperazine-1-
carboxylate (17).
A mixture of compound 16 (1.50 g, 4.50 mmol), triethylamine (0.91 g, 9.01 mmol), and di-tert-butyl
pyrocarbonate (1.96 g, 9.01 mmol) was stirred at room temperature in dichloromethane (100 mL) for 12 h.
After completion of the reaction, the solvent was evaporated under reduced pressure and the residue was
diluted with water (100 mL), and then extracted with chloroform (3 × 50 mL). The organic extracts were
dried over anhydrous sodium sulfate and concentrated. The crude product was purified via silica gel
column chromatography (eluent, ethyl acetate/ petroleum ether (V/V) = 1/3) to afford compound 17 (1.53
1
g) as light yellow solid. Yield: 78.6%; mp: 243–245 ºC; H NMR (600 MHz, DMSO-d6) δ 8.60 (s, 1H,
quinolone-2-H), 8.00 (s, 1H, quinolone-5-H), 7.94 (s, 1H, quinolone-8-H), 4.43 (q, J = 7.1 Hz, 2H,
CH2CH3), 3.52 (s, 4H, piperazine-3,3-N-(CH2)2), 3.03–2.99 (m, 4H, piperazine-2,2-N-(CH2)2), 2.61 (s, 3H,
COCH3), 1.44 (s, 9H, Boc-CH3), 1.34 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6)
δ 196.3, 174.2, 154.4, 148.4, 146.6, 135.4, 134.2, 129.0, 119.7, 118.1, 117.6, 79.6, 51.5, 48.6, 31.6, 28.6,
14.9 ppm; HRMS (ESI) calcd. for C22H28ClN3O4 [M + H]+, 434.1847; found, 434.1842.
4.1.17. Synthesis of Tert-butyl 4-(7-chloro-3-(2,2-dibromoacetyl)-1-ethyl-4-oxo -1,4-dihydroquinolin-6-
yl)piperazine-1-carboxylate (18).
To a solution of compound 17 (1.00 g, 2.31 mmol) in ethyl acetate (150 mL) was added cupric bromide
(1.03 g, 4.62 mmol). The mixture was then stirred at 78 ºC for 12 h. After completion of the reaction, the
solvent was evaporated under reduced pressure and the residue was washed with methanol and
dichloromethane (200 mL, (V/V) = 2/1). The organic solvents were dried over anhydrous sodium sulfate
and concentrated. The crude product was purified via silica gel column chromatography (eluent, ethyl
acetate/ petroleum ether (V/V) = 1/8) to afford compound 18 (0.83 g) as yellow solid. Yield: 61.2%; mp: >
1
250 ºC; H NMR (600 MHz, CDCl3) δ 8.64 (s, 1H, quinolone-2-H), 8.12 (s, 1H, quinolone-5-H), 7.88 (s,
1H, quinolone-8-H), 7.59 (s, 1H, COCHBr2), 4.31 (q, J = 7.2 Hz, 2H, CH2CH3), 3.63 (d, J = 4.2 Hz, 4H,
piperazine-3,3-N-(CH2)2), 3.11–3.08 (m, 4H, piperazine-2,2-N-(CH2)2), 1.61 (t, J = 7.2 Hz, 3H, CH2CH3),
1.50 (s, 9H，Boc-CH3) ppm; 13C NMR (151 MHz, CDCl3) δ 185.6, 174.0, 154.8, 149.2, 147.7, 135.5, 134.6,
128.6, 118.6, 118.4, 112.6, 80.1, 51.4, 49.6, 44.6, 28.5, 14.6 ppm; HRMS (ESI) calcd. for
C22H26Br2ClN3O4 [M + H]+, 590.0057; found, 590.0053.
4.1.18. Synthesis of Tert-butyl 4-(3-(2-aminothiazol-4-yl)-7-chloro-1-ethyl-4-oxo -1,4-dihydroquinolin-6-
yl)piperazine-1-carboxylate (19).
A mixture of intermediate 18 (0.60 g, 1.02 mmol) and thiourea (93.04 mg, 1.22 mmol) was stirred at 80 ºC
in ethanol (100 mL) for 4 h. After completion of the reaction, the solvent was evaporated under reduced
pressure and the residue was diluted with water (100 mL), and then extracted with chloroform (3 x 50 mL).
20

ACCEPTED MANUSCRIPT
The organic extracts were dried over anhydrous sodium sulfate and concentrated. The crude product was
purified via silica gel column chromatography (eluent, chloroform/methanol (V/V) = 50/1) to afford
compound 19 (0.30 g) as yellow solid. Yield: 59.2%; mp: > 250 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.63
(s, 1H, quinolone-2-H), 7.97 (s, 1H, quinolone-5-H), 7.96 (s, 1H quinolone-8-H), 7.67 (s, 1H,
thiazole-4-H), 6.95 (s, 2H, thiazole-2-NH2), 4.38 (q, J = 6.9 Hz, 2H, CH2CH3), 3.53 (s, 4H,
piperazine-3,3-N-(CH2)2), 3.00 (s, 4H, piperazine-2,2-N-(CH2)2), 1.44 (s, 9H, Boc-CH3), 1.36 (t, J = 7.1
Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 173.1, 167.2, 154.5, 145.3, 144.7, 142.8, 134.8,
133.6, 126.6, 118.8, 117.3, 115.4, 104.2, 79.5, 51.7, 48.3, 28.6, 15.1 ppm; HRMS (ESI) calcd. for
C23H28ClN5O3S [M + H]+, 490.1680; found, 490.1674.
4.1.19. Synthesis of 3-(2-Aminothiazol-4-yl)-7-chloro-1-ethyl-6-(piperazin-1-yl)quinolin-4(1H)-one(20).
A mixture of compound 19 (0.25 g, 1.02 mmol) and hydrochloric acid (1 mL) in dichloromethane (100 mL)
was stirred at room temperature for 4 h. When the reaction was completed, the solvent was evaporated
under reduced pressure. After chloroform was added, the resulting mixture was washed with aqueous
solution of sodium hydroxide (3 × 20 mL). The organic extracts were dried over anhydrous sodium sulfate
and concentrated. The crude product was purified via silica gel column chromatography (eluent,
chloroform/methanol (V/V) = 15/1) to afford compound 20 (0.14 g) as yellow solid. Yield: 71.4%; mp:
1
241–243 ºC; H NMR (600 MHz, DMSO-d6) δ 8.63 (s, 1H, quinolone-2-H), 7.96 (s, 2H, quinolone-5-H,
quinolone-8-H), 7.66 (s, 1H, thiazole-4-H), 6.94 (s, 2H, thiazole-2-NH2), 4.39 (q, J = 7.1 Hz, 2H, CH2CH3),
3.01 (s, 4H, piperazine-2,2-N-(CH2)2), 2.98 (d, J = 3.5 Hz, 4H, piperazine-3,3-N-(CH2)2), 1.36 (t, J = 7.2
Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 173.2, 167.1, 145.8, 144.8, 142.8, 134.7, 133.6,
126.6, 118.9, 116.9, 115.4, 104.2, 52.3, 48.3, 45.7, 15.1 ppm; HRMS (ESI) calcd. for C18H20ClN5OS [M +
H]+, 390.1155; found, 390.1154.
4.1.20. Synthesis of 3-(2-Aminothiazol-4-yl)-7-chloro-1-ethyl-6-(4-(2-oxopropyl)piperazin-1-yl)quinolin
-4(1H)-one (21).
Compound 21 was prepared according to the experimental procedure described for compound 15a starting
from 1-chloropropan-2- one (69.39 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 20 (194.95 mg, 0.50 mmol). The target compound 21 (163.45 mg) was obtained as yellow solid.
Yield: 73.3%; mp: 248–250 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.63 (s, 1H, quinolone-2-H), 7.98 (s, 1H,
quinolone-5-H), 7.95 (s, 1H, quinolone-8-H), 7.66 (s, 1H, thiazole-4-H), 6.94 (s, 2H, thiazole-2-NH2), 4.38
(q, J = 7.1 Hz, 2H, CH2CH3), 3.28 (s, 2H, CH2COCH3), 3.06 (s, 4H, piperazine-2,2-N-(CH2)2), 2.65 (s, 4H,
piperazine-3,3-N- (CH2)2), 2.12 (s, 3H, CH2COCH3), 1.36 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR
(151 MHz, DMSO-d6) δ 207.3, 173.2, 167.1, 145.5, 144.8, 142.8, 134.7, 133.5, 126.6, 118.9, 116.9, 115.4,
104.2, 67.7, 53.2, 51.7, 48.3, 28.1, 15.1 ppm; HRMS (ESI) calcd. for C21H24ClN5O2S [M + H]+, 446.1417;
found, 446.1416.
21

ACCEPTED MANUSCRIPT
4.1.21. Synthesis of (E)-3-(2-Aminothiazol-4-yl)-7-chloro-1-ethyl-6-(4-(2-(hydroxyimino)propyl)piperazin
-1-yl)quinolin-4(1H)-one (22a).
Compound 22a was prepared according to the experimental procedure described for compound 10a
starting from intermediate 21 (222.98 mg, 0.50 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
hydroxylamine hydrochloride (52.11 mg, 0.75 mmol). The target compound 22a (124.69 mg) was obtained
as yellow solid. Yield: 54.2%; mp: > 250 ºC; 1H NMR (600 MHz, DMSO-d6) δ 10.55 (s, 1H, OH), 8.63 (s,
1H, quinolone-2-H), 7.98 (s, 1H, quinolone-5-H), 7.95 (s, 1H, quinolone-8-H), 7.66 (s, 1H, thiazole-4-H),
6.92 (s, 2H, thiazole-2-NH2), 4.38 (q, J = 7.1 Hz, 2H, CH2CH3), 3.06 (s, 4H, piperazine-2,2-N-(CH2)2),
3.04 (s, 2H, CH2C(N-OH)CH3), 2.55 (s, 4H, piperazine-3,3-N -(CH2)2), 1.83 (s, 3H, -CH3), 1.37 (d, J = 7.1
Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 173.2, 167.1, 145.5, 144.8, 142.8, 134.6, 133.5,
132.0, 129.1, 126.6, 118.9, 116.9, 115.4, 104.2, 62.2, 53.2, 51.8, 49.1, 48.3, 15.1, 12.7 ppm; HRMS (ESI)
calcd. for C21H25ClN6O2S [M + H]+, 461.1526; found, 461.1524.
4.1.22.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-7-chloro-1-ethyl-6-(4-(2-(methoxyimino)propyl)
piperazin-1-yl)quinolin-4(1H)-one (22b).
Compound 22b was prepared according to the experimental procedure described for compound 10a
starting from intermediate 21 (222.98 mg, 0.50 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
O-methylhydroxylamine hydrochloride (62.64 mg, 0.75 mmol). The target compound 22b (146.52 mg)
was obtained as light yellow solid. Yield: 61.8%; mp: > 250 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.62 (s,
1H, quinolone-2-H), 7.98 (s, 1H, quinolone-5-H), 7.93 (s, 1H, quinolone-8-H), 7.66 (s, 1H, thiazole-4-H),
6.88 (s, 2H, thiazole-2-NH2), 4.37 (q, J = 7.1 Hz, 2H, CH2CH3), 3.78 (s, 3H, OCH3), 3.29 (s, 2H,
CH2C(N-OCH3)CH3), 3.06 (s, 4H, piperazine-2,2-N-(CH2)2), 2.56 (s, 4H, piperazine-3,3-N- (CH2)2), 1.85
(s, 3H, -CH3), 1.36 (t, J = 7.1 Hz, 3H, CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 173.2, 167.1,
155.5, 145.5, 144.8, 142.8, 134.7, 133.5, 126.6, 118.9, 116.9, 115.4, 104.2, 61.7, 61.4, 53.2, 51.8, 49.1,
48.2, 15.0, 13.3 ppm; HRMS (ESI) calcd. for C22H27ClN6O2S [M + H]+, 475.1683; found, 475.1683.
4.1.23.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-7-chloro-1-ethyl-6-(4-(2-(4-fluorophenyl)-2-
(methoxyimino)ethyl)piperazin-1-yl)quinolin- 4(1H)-one (23a).
Compound 23a was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14a (112.21 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 20 (194.55 mg, 0.50 mmol). The target compound 23a (166.53 mg) was obtained as yellow
solid. Yield: 60.1%; mp: 131–133 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.61 (s, 1H, quinolone-2-H), 7.94
(s, 1H, quinolone-5-H), 7.92 (s, 1H, quinolone-8-H), 7.86 (dd, J = 8.5, 5.7 Hz, 2H, 4-F-Ph-2,6-2H), 7.64 (s,
1H, thiazole-4-H), 7.24 (t, J = 8.8 Hz, 2H, 4-F-Ph-3,5-2H), 6.93 (s, 2H, thiazole-2-NH2), 4.37 (dd, J = 13.6,
6.6 Hz, 2H, CH2CH3), 3.94 (s, 3H, -OCH3), 3.72 (s, 2H, CH2C(N-OCH3), 2.97 (s, 4H,
piperazine-2,2-N-(CH2)2), 2.63 (s, 4H, piperazine-3,3-N-(CH2)2), 1.35 (t, J = 7.0 Hz, 3H, CH2CH3) ppm;
13C NMR (151 MHz, DMSO-d6) δ 173.1, 167.1, 162.3, 153.9, 145.4, 144.8, 142.8, 134.7, 133.6, 131.9,
22

ACCEPTED MANUSCRIPT
129.4, 126.6, 118.8, 116.9, 115.7, 115.6, 104.2, 62.3, 53.3, 51.7, 51.3, 48.2, 15.1 ppm; HRMS (ESI) calcd.
for C27H28ClFN6O2S [M + H]+, 555.1745; found, 555.1741.
4.1.24.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-7-chloro-6-(4-(2-(4-chlorophenyl)-2-
(methoxyimino)ethyl)piperazin-1-yl)-1-ethylquinolin-4(1H)-one (23b).
Compound 23b was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14b (162.75 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 20 (194.55 mg, 0.50 mmol). The target compound 23b (181.59 mg) was obtained as yellow
solid. Yield: 63.7%; mp: 244–246 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.61 (s, 1H, quinolone-2-H), 7.93
(s, 1H, quinolone-5-H), 7.92 (s, 1H, quinolone-8-H), 7.83 (s, 1H, 4-Cl-Ph-2-H), 7.82 (s, 1H, 4-Cl-Ph-6-H),
7.65 (s, 1H, thiazole-4-H), 7.47 (s, 1H, 4-Cl-Ph-3-H), 7.46 (s, 1H, 4-Cl-Ph-5-H), 6.91 (s, 2H,
thiazole-2-NH2), 4.37 (q, J = 7.0 Hz, 2H, CH2CH3), 3.95 (s, 3H, -OCH3), 3.72 (s, 2H, CH2C(N-OCH3),
2.97 (s, 4H, piperazine-2,2-N-(CH2)2), 2.62 (s, 4H, piperazine-3,3-N-(CH2)2), 1.35 (t, J = 7.1 Hz, 3H,
CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 173.1, 167.1, 153.9, 145.4, 144.8, 142.8, 134.7, 134.3,
133.6, 128.9, 128.7, 126.6, 118.8, 116.9, 115.4, 104.2, 62.4, 53.3, 51.7, 51.1, 48.2, 15.1 ppm; HRMS (ESI)
calcd. for C27H28Cl2N6O2S [M + H]+, 571.1450; found, 571.1445.
4.1.25.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-7-chloro-1-ethyl-6-(4-(2-(methoxyimino)-2-
(4-methoxyphenyl)ethyl)piperazin-1-yl) quinolin -4(1H)-one (23c).
Compound 23c was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14c (159.79 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 20 (194.55 mg, 0.50 mmol). The target compound 23c (170.71 mg) was obtained as light
yellow solid. Yield: 60.3%; mp: 139–141 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.61 (s, 1H,
quinolone-2-H), 7.93 (s, 2H, quinolone-5-H, quinolone-8-H), 7.75 (d, J = 8.8 Hz, 2H, 4-CH3O-Ph-2,6-2H),
7.65 (s, 1H, thiazole-4-H), 6.96 (d, J = 8.8 Hz, 2H, 4-CH3O-Ph-3,5-2H), 6.92 (s, 2H, thiazole-2-NH2), 4.37
(q, J = 7.1 Hz, 2H, CH2CH3), 3.91 (s, 3H, -OCH3), 3.79 (s, 3H, Ph-OCH3), 3.69 (s, 2H, CH2C(N-OCH3),
2.98 (s, 4H, piperazine-2,2-N-(CH2)2), 2.63 (s, 4H, piperazine-3,3-N-(CH2)2), 1.35 (t, J = 7.1 Hz, 3H,
CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 173.1, 167.1, 160.5, 154.2, 145.5, 144.8, 142.8, 134.7,
133.6, 128.5, 127.9, 126.6, 118.8, 116.9, 115.4, 114.1, 104.2, 62.1, 55.6, 53.3, 51.8, 51.2, 48.2, 15.1 ppm;
HRMS (ESI) calcd. for C28H31ClN6O3S [M + H]+, 567.1945; found, 567.1935.
4.1.26.
Synthesis
of
(E)-3-(2-Aminothiazol-4-yl)-7-chloro-6-(4-(2-(2,4-dichlorophenyl)-2-
(methoxyimino)ethyl)piperazin-1-yl)-1-ethylquinolin-4(1H)-one (23d).
Compound 23d was prepared according to the experimental procedure described for compound 15a
starting from intermediate 14e (188.23 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and
compound 20 (194.55 mg, 0.50 mmol). The target compound 23d (178.21 mg) was obtained as yellow
solid. Yield: 59.0%; mp: 137–139 ºC; 1H NMR (600 MHz, DMSO-d6) δ 8.61 (s, 1H, quinolone-2-H), 7.92
(s, 1H, quinolone-5-H), 7.86 (s, 1H, quinolone-8-H), 7.68 (d, J = 1.9 Hz, 1H, 2,4-diCl-Ph-6-H), 7.64 (s, 1H,
23

ACCEPTED MANUSCRIPT
thiazole-4-H), 7.50 (s, 1H, 2,4-diCl-Ph-3-H), 7.49 (d, J = 1.9 Hz, 1H, 2,4-diCl-Ph-5-H), 6.92 (s, 2H,
thiazole-2-NH2), 4.36 (q, J = 7.1 Hz, 2H, CH2CH3), 3.91 (s, 3H, -OCH3), 3.68 (s, 2H, CH2C(N-OCH3)),
2.84 (s, 4H, piperazine-2,2-N-(CH2)2), 2.56 (s, 4H, piperazine-3,3-N-(CH2)2), 1.34 (t, J = 7.1 Hz, 3H,
CH2CH3) ppm; 13C NMR (151 MHz, DMSO-d6) δ 173.1, 167.1, 156.7, 145.3, 144.8, 142.8, 134.7, 134.3,
134.0, 133.5, 132.8, 129.3, 127.5, 126.5, 118.8, 116.9, 115.4, 104.2, 62.4, 54.0, 53.5, 51.7, 48.2, 15.0 ppm;
HRMS (ESI) calcd. for C27H27Cl3N6O2S [M + H]+, 605.1060; found, 605.1027.
Acknowledgments
This work was partially supported by National Natural Science Foundation of China (Nos. 21672173,
21850410447, 21807008), China Postdoctoral Science Foundation (2018M633313), Chongqing Special
Foundation for Postdoctoral Research Proposal (Nos. Xm2017184, XmT2018082), and Program for
Overseas Young Talents from State Administration of Foreign Experts Affairs, China (Nos.
WQ2017XNDX047, WQ20180083).
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