Design and synthesis of aminothiazolyl norfloxacin analogues as potential antimicrobial agents and their biological evaluation

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Graphical Abstract

Design and synthesis of aminothiazolyl norfloxacin analogues as potential antimicrobial agents and their

biological evaluation

1

1,†

2,*

Liang-Liang Wang , Narsaiah Battini , Rammohan R. Yadav Bheemanaboina , Shao-Lin Zhang ,

1

,*

Cheng-He Zhou

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.

*

Corresponding Address:

Tel.: +86-23-68254967; fax: +86-23-68254967; E-mail: zhouch@swu.edu.cn (Cheng-He Zhou);

zhangsl@cqu.edu.cn (Shao-Lin Zhang).

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Title page

Title:

Design and synthesis of aminothiazolyl norfloxacin analogues as potential antimicrobial agents and their

biological evaluation

Author Names and Affiliations:

1

1,†

2,*

Liang-Liang Wang , Narsaiah Battini , Rammohan R. Yadav Bheemanaboina , Shao-Lin Zhang ,

1

,*

Cheng-He Zhou

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.

*

Corresponding Address:

Tel.: +86-23-68254967; fax: +86-23-68254967; zhouch@swu.edu.cn (Cheng-He Zhou);

zhangsl@cqu.edu.cn (Shao-Lin Zhang).

Abstract:

A series of aminothiazolyl norfloxacin analogues as a new type of potential antimicrobial agents were

synthesized and screened for their antimicrobial activities. Most of the prepared compounds exhibited

excellent inhibitory efficiencies. Especially, norfloxacin analogue II-c displayed superior antimicrobial

activities against K. pneumoniae and C. albicans with MIC values of 0.005 and 0.010 mM to reference

drugs, respectively. This compound not only showed broad antimicrobial spectrum, rapid bactericidal

efficacy and strong enzymes inhibitory potency including DNA gyrase and chitin synthase (CHS), low

toxicity against mammalian cells and no obvious propensity to trigger the development of bacterial

resistance, but also exerted efficient membrane permeability, and could effectively intercalate into K.

pneumoniae DNA to form a steady supramolecular complex, which might block DNA replication to exhibit

their powerful antimicrobial activity. Quantum chemical studies were also performed to explain the high

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antimicrobial activities. Molecular docking showed that compound II-c could bind with gyrase–DNA and

topoisomerase IV–DNA through hydrogen bonds and π-π stacking.

Keywords:

Norfloxacin; Thiazole; Antimicrobial; Drug combination; DNA gyrase; Chitin synthase

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1

. Introduction

Infectious diseases continue to be a leading threat to human health, and the rapid development of bacterial

resistance to current antibiotic chemotherapies has rendered lots of therapy weapons less effective [1]. It is

anticipated that antibiotic resistance is going to cause more than 10,000,000 deaths per year by the year

2

050, posing a formidable challenge for disease treatment as pathogens become resistant to clinical drugs

[2]. The World Health Organization has launched a global action plan calling on all countries to take

measures towards drug-resistant microbes, and the discovery of efficacious and safer antimicrobials with

new or multiple mechanisms of action has been an urgent need to combat resistant strains [3,4].

Norfloxacin bearing a 3-carboxyl benzopyridone skeleton is one of the commonly used

fluoroquinolones with high oral bioavailability, eminent pharmacokinetic properties and excellent curative

effects [5], since the nalidixic acid as the first member of quinolones was introduced in the 1960s into the

clinic for the treatment of uncomplicated urinary tract infections caused by Escherichia coli, continuous

structural modification has been made worldwide [6–8]. Norfloxacin as one of the third generation of

quinolone antibacterial drugs was highlighted, which exerted powerful biological activities by binding with

DNA–gyrase or DNA–topoisomerase IV via a water-metal ion bridge to form ternary supramolecular

complexes, interfering DNA replication and eventually bringing bacterial cell death [9]. However, due to

the overuse and even misuse of norfloxacin in clinical therapy, the chelating ability of the carbonyl and

carboxyl moieties is inclined to trigger an error-prone signal to the DNA repair system, which leads to

specific mutations in DNA gyrase and topoisomerase IV, as well as gives rising to norfloxacin-resistant

bacterial strains, whereby decreasing norfloxacin’s therapeutic effectiveness [10]. Moreover, many of the

several side-effects of norfloxacin such as gastroenteritis, vomiting and cartilage damage are also in

relation to the carboxyl group of C-3 position. All of these have already caused profound concern around

the world, requiring the development of alternative antibacterial strategies. To the best of our knowledge, a

practical approach to overcome quinolones resistance is to exploit new analogues or modify existing drugs

with limited or no cross resistance, which has resulted in the successful development of a large number of

quinolone antibacterial drugs [11,12]. Therefore, the structural modification at the C-3 position of

norfloxacin is supposed to be a promising strategy to conquer the resistance as well as the side effects.

Aminothiazole is a beneficial bioactive fragment and prevalently presents in a variety of clinically

antibacterial drugs such as cephalosporins, sulfathiazole and so on. The successful development of

numerous clinical 2-aminothiazole drugs has provoked extensive studies to construct more bioactive

molecules on the basis of this fragment in the field of antimicrobials [13,14]. Our previous work has

revealed that the incorporation of azoles such as imidazole [15–17], triazole [18], tetrazole [19], and

benzimidazole [20] into quinolones can significantly increase the antimicrobial efficiency and broaden the

antimicrobial spectrum. Especially, 2-aminothiazolyl quinolones (Leads A and B, Fig. 1) exhibited potent

antibacterial activity, low cytotoxicity to hepatocyte cells, strong inhibitory potency to DNA gyrase, and

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broad antimicrobial spectrum including against multidrug-resistant strains [21,22]. They exerted the

2

+

antimicrobial activity by forming compound–Cu –DNA ternary supramolecular complex, in which the

2

+

Cu ion acts as a bridge between the phosphate group of the nucleic acid and the backbone of

2

-aminothiazolyl quinolone, different from the classic binding mode depending on the water–metal ion

bridge mediated interaction. In addition, the amino, nitrogen and sulfur atoms of 2-aminothiazole moiety

participated in the non-covalent coordination, making the ternary supramolecular complex more stable.

The new kind of mechanism is favorable to overcome the resistance as well as the side effects caused by

the carboxyl group. These stimulating properties have made the structural modification of 2-aminothiazole

fragment toward quinolones become a continuously attractive topic [23].

Fig. 1

In view of the above observations, taking into account the side effects caused by the carboxyl group of

norfloxacin and the essential role of 2-aminothiazole fragment in quinolones, as an extension of our

previous work on the development of 2-aminothiazolyl quinolones [21,22], we incorporated

2

-aminothiazole fragment into the C-3 position of norfloxacin and changed the substituents at N-4 position

of the piperazine ring to generate a series of new aminothiazolyl norfloxacin analogues (Fig. 2). Among

them, the choice of the substituents was mainly based on the following reasons:

(I) It is well known that the alkyl chain could exert large effect on biological potency by regulating the

lipid-water partition coefficient and binding affinity to target enzymes [24,25]. Therefore, various alkyl

chains with different lengths were incorporated into piperazine ring to investigate the effects on

antimicrobial activities.

(II) Unsaturated bonds tend to form electron-deficient reactive intermediates or compounds with active

electrophilic groups in the body, thereby covalently bind to electron-rich groups (such as amino, mercapto,

hydroxyl and phosphoric acid groups, etc.) in the biomacromolecule (DNA, RNA, or some important

enzymes), rendering it inactive or making DNA molecules break [26,27].

(

III) Halogen-containing phenyl moieties are beneficial for liposolubility and membrane permeability,

can effectively influence the rate of absorption and transport of drugs [28,29].

IV) Hydroxyethyl fragment plays a crucial role in the phase of exerting biological effect and is widely

(

present in many medicinal drugs such as disinfectant ethanol, antibacterial metronidazole, antifungal

fluconazole and so on [30,31].

(V) Carbonyl group is able to bind with the amino group of proteins and other functional moieties like

sulfhydryl, hydroxyl and carboxyl groups present in the nucleic acids, leading to protein denaturation and

coagulation, thus causing microbial death. For instance, formaldehyde, glutaraldehyde and

o-phthalaldehyde have been longly used as disinfectants with large antimicrobial potentiality [32].

(VI) Amide bonds as the backbone of proteins exert pivotal role, which could readily bind with a variety

of enzymes, receptors, DNA and RNA in biological system via weak interactions such as coordination

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bonds, hydrogen bonds, ion-dipole, Van der Waals force and so on [33].

The newly prepared aminothiazolyl norfloxacin analogues were screened for their antimicrobial

activities in vitro. Enzyme inhibitory activities, bacterial resistance, bactericidal kinetics, and cytotoxicity

of the highly active compounds were also evaluated. Additionally, the further possible antimicrobial

mechanism was investigated through biofilm disruption assay, quantum chemical study, molecular docking

and interactions with DNA isolated from the sensitive resistant strains.

Fig. 2

2

. Results and discussion

2

.1. Chemistry

A series of aminothiazolyl norfloxacin analogues 8, 9, 11 and I–VIII were synthesized via multistep

reactions starting from commercial ethyl acetoacetate, triethyl orthoformate, 3-chloro-4-fluoroaniline,

bromoethane and bromine according to Schemes 1–6. Condensation of ethyl acetoacetate 1,

triethoxymethane and acetic anhydride gave compound 2 in 80.0% yield, which was further reacted with

3

-chloro-4-fluoroaniline in the absence of solvent to produce 3-chloro-4-fluorophenylamino butanoate 3

with quantitative yield, and then the latter was further cyclized in phenoxybenzene under reflux condition

to yield the intermediate 3-acetyl quinolone 4 in moderate yield. The N-alkylation of intermediate 3-acetyl

quinolone 4 with bromoethane afforded N-ethylquinolone 5 in good yield of 80.4%. Further bromination of

compound 5 by bromine in acetic acid produced the corresponding 3-(2-bromoacetyl) quinolone 6, which

o

was then cyclized with thiourea in ethanol at 80 C via typical Hantsch thiazole synthesis to give

2

-aminothiazolyl quinolone 7 in 78.3% yield [34]. Compound 7 was treated with piperazine in

o

1

-methyl-2-pyrrolidinone (NMP) at 130 C under nitrogen atmosphere to afford the aminothiazolyl

norfloxacin analogue 8 in low yield of 21.4%. Under the same reaction condition, compound 8 was reacted

with intermediate 7 to produce the bisthiazole derivative 9 with the yield of 37.6%. The N-alkylation of

aminothiazolyl norfloxacin analogue 8 with a series of saturated and unsaturated alkyl bromides in

o

acetonitrile at 80 C with potassium carbonate as base readily gave corresponding aliphatic derivatives I–II

with moderate to good yields ranging from 43.1% to 71.6% (Scheme 1). The phenyl series III-a–d were

also obtained in 60.3% to 65.4% yields under the similar reaction condition starting from compound 8 and

the substituted benzyl halides (Scheme 2).

Scheme 1

Scheme 2

With the aim to investigate the vital effect of hydroxyethyl fragment on the antimicrobial activity,

compound 8 was further modified by bromoethanol to generate the target aminothiazolyl norfloxacin

analogue IV-a in 54.4% yield. In addition, the reduction of compound VI-a by sodium borohydride

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resulted in hydroxyethyl derivative IV-b with the high yield of 81.3% (Scheme 3).

Scheme 3

Compounds V-a–b with a carbonyl group directly linked with the piperazine ring and one

methylene-linked carbonyl derivatives VI-a–b were easily prepared as shown in Scheme 4. Firstly,

carbonyl compound V-a was conveniently obtained in 86.2% yield by the treatment of compound 8 with

o

formamide at 75 C, and then the analogue 8 was reacted with halides of carbonyl compounds to generate

the target aminothiazolyl norfloxacin analogues V-b and VI-a–b with moderate to good yields ranging

from 51.6% to 71.0%.

Scheme 4

The amide derivatives VII-a–d were prepared from commercially available aliphatic amines (Scheme 5).

The intermediate chloroacetamides 10a–d in 50.1–71.9% yields were easily synthesized in acetonitrile by

the N-acylation of commercial aliphatic amines with chloroacetyl chloride in presence of potassium

carbonate at room temperature, and were further reacted with compound 8 to yield the target

aminothiazolyl quinolones VII-a–d with moderate yields in the range of 59.3–64.1%.

Scheme 5

In order to investigate whether the positional change of the piperazine fragment from the 7-position of

quinolone skeleton to the 6-position would retain or improve the antimicrobial activity, the 6-substituted

piperazine derivative 11 and its analogues VIII-a–b were designed and synthesized as shown in Scheme 6.

The intermediate 7 was reacted with piperazine in NMP to produce the corresponding piperazine derivative

o

1

1, and then further N-alkylation with alkyl bromides in acetonitrile at 80 C produced target

2

-aminothiazolyl quinolones VIII-a–b with the yields ranging from 51.2% to 73.3%. All the structures of

1

13

the target molecules were characterized by H NMR, C NMR and HRMS spectra.

Scheme 6

2

.2. Biological Activity

All the synthesized 2-aminothiazolyl norfloxacin analogues were screened for their antibacterial and

antifungal activities by the two folds serial dilution technique in 96-well micro-test plates according to

Clinical and Laboratory Standards Institute (CLSI) [35]. Norfloxacin, clinafloxacin and fluconazole were

employed as standard drugs for evaluation of antimcorbial activity. Minimal inhibitory concentration

(MIC, mM) was defined as the lowest concentration of the tested target compounds that completely

inhibited the growth of strains. The results of antibacterial and antifungal activities were summarized in

Tables 1–2 and Table S1 (Supporting Information), respectively.

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.2.1. Antibacterial activity

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The antibacterial evaluation in vitro (Tables 1 and 2) displayed that most of the prepared norfloxacin

analogues could effectively inhibit the growth of all the tested strains. Remarkably, cyanomethyl derivative

II-c displayed greater antibacterial activity against K. pneumoniae with MIC value of 0.005 mM, which

were 5-fold and 2-fold more potent than reference drugs chloromycin (0.025 mM) and norfloxacin (0.013

mM), respectively. Furthermore, molecule II-c showed 2.5-fold inhibitory potency against E. coli ATCC

2

5922 than norfloxacin with MIC value of 0.025 mM. Among all the tested strains, compound II-c gave

low MIC values in the range of 0.005–0.039 mM. These results indicated that norfloxacin analogue II-c

had the potency to be further studied.

The structure-activity relationships (SARs) study demonstrated that the substituents at the N-4 position

of piperazine ring put forth a significant impact on biological activities. Among alkyl derivatives I-a–h and

II-a–c, the butyl modified derivative I-d not only remarkably improved antimicrobial activities toward all

the tested strains in comparison with corresponding unsubstituted aminothiazolyl norfloxacin analogue 8,

but also gave broader antibacterial spectrum and better activities with low MIC values of 0.005–0.149

mM. Noticeably, compound I-d exhibited the best anti-K. pneumoniae activity with MIC value of 0.005

mM, which was superior to the standard drugs chloromycin (MIC = 0.025 mM) and norfloxacin (MIC =

0

.013 mM). Moreover, when the alkyl substituents were extended to decyl, octyl, and dodecyl groups,

corresponding compounds I-f–h displayed weak or no obvious antibacterial activity. Similarly, the ethyl

and propyl substituted compounds I-b and I-c were also less beneficial for the antibacterial efficiency even

at high concentration. This fact pointed out that either decrease or increase of the alkyl chain length was

unfavourable for the bioactivity, and only a suitable length of alkyl chain in piperazine ring was necessary

for better antibacterial activity. Surprisingly, the replacement of propyl chain by the unsaturated alkyl

groups to produce alkenyl, alkynyl and cyanomethyl derivatives II-a–c remarkably improved antimicrobial

activities in comparison with corresponding propyl analogue I-c. Especially, compounds II-b and II-c

displayed better antibacterial activity against S. aureus ATCC 29213 and K. pneumoniae with the same

low MIC value of 0.005 mM, respectively, which were more effective than reference drugs chloromycin

and norfloxacin. These results suggested that the introduction of unsaturated groups exhibited significant

effect on inhibiting the growth of the tested bacterial strains.

Among phenyl ones, 4-chlorophenyl derivative III-b gave the best activities against the tested

Gram-positive bacteria with MIC values of 0.016–0.064 mM. Particularly, it could inhibit the growth of

MRSA with MIC value of 0.016 mM, which was equivalent inhibitory activity to norfloxacin. However,

the substitution of 4-chlorophenyl moiety by 2,4-dichlorophenyl or 3,4-dichlorophenyl group afforded

compounds III-a and III-d with weaker activities, which indicated that the position of chlorine atom on

phenyl ring displayed significant effects on the biological activity. Additionally, the replacement of

4

-chlorophenyl moiety by 4-fluorophenyl group to yield compound III-c resulted in good bioactivity

against E. coli ATCC 25922 with MIC value of 0.033 mM. Probably because fluorine atom could easily

and efficiently form non-covalent forces thereby being helpful for the biological transportation and

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distribution in organism. Furthermore, compounds IV-a and IV-b bearing a hydroxyl group also gave

good antibacterial activity, which might be attributed to the formation of hydrogen bonds between

compounds and biologically important species. However, piperazine-bridged bisthiazole derivative 9

showed poor or even no activity in comparison to other compounds, which might be on account of the

excessive molecular weight.

Compounds V-a–b with a carbonyl group directly linked with the piperazine ring and one

methylene-linked carbonyl compounds VI-a–b gave moderate antibacterial activities against the tested

stains. In these carbonyl derivatives, the 2-acetyl modified molecule V-b and methylene-bridged carbonyl

compound VI-b could completely inhibit the growth of P. aeruginosa ATCC 27853 and S. aureus ATCC

2

5923 at relatively low concentrations of 0.010 mM and 0.017 mM, respectively, which were equal or

better activity in contrast to the two reference drugs. However, carbonyl compound V-a showed relatively

weak biological activity, which might be related to its poor water solubility. The replacement of carbonyl

moiety with amide groups generated methylene-bridged amide derivatives VII-a–d with good potencies

against Gram-positive bacteria (MIC = 0.015–0.249 mM). Specifically, diethanolamine derivative VII-c

showed better MIC value against S. aureus ATCC 25923 (0.008 mM) and stronger antibacterial potency

against E. faecalis (0.015 mM) and S. aureus ATCC 29213 (0.062 mM).

In terms of the position of the piperazine ring on the quinolone skeleton, although the 6-substituted

piperazine derivatives VIII-a–b showed good biological activities against MRSA with relatively lower

MIC values (0.072 and 0.067 mM, respectively), both compounds lost significantly in activity compared

with the corresponding 7-substituted counterparts VI-a–b against all of other tested strains. This result

indicated that the position of piperazine ring plays a crucial role for antimicrobial activity and it must be

attached at the 7-position of quinolone.

Table 1

Table 2

2

.2.2. Antifungal activity

The antifungal evaluation in Table S1 revealed that most of target compounds exhibited similar inhibitory

tendency to their antibacterial activity, though relatively weaker than the latter. The alkyl substituted

analogues I–II displayed moderate to good antifungal activity against most of the tested fungi.

Predominantly, compound I-d with butyl chain generally showed profound effect against C. tropicals with

MIC value of 0.009 mM. The substitution of butyl chain by unsaturated cyanomethyl fragment, which

generated compound II-c, resulted in better efficiency against A. fumigatus with MIC value of 0.019 mM,

it could also effectively inhibit the growth of C. albicans with low MIC value of 0.010 mM. In addition,

the carbonyl compound V-b and amide derivative VII-c also gave strong bioactivities against A. fumigatus

and C. albicans ATCC 90023. These suggested that this new type of aminothiazolyl analogues should have

great potential in new antifungal drug development.

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2

.3. DNA gyrase inhibitory activity

Bacterial DNA gyrase, a member of bacterial type IIA topoisomerase enzymes, controls the topological

state of DNA during processes of transcription, replication and recombination, and has been known as a

validated target of aminocoumarin and quinolone classes of antibiotics [36–38]. Aminothiazolyl

norfloxacin analogue II-c and the standard drug norfloxacin were selected to investigate their inhibitory

activity against the DNA gyrase from E. coli. The results in Table 3 revealed that compound II-c exhibited

good inhibitory potency of DNA gyrase (IC₅₀= 16.7 µM), which was more effective than the reference

drug norfloxacin (IC₅₀= 18.6 µM), indicating that the replacement of the carboxyl moiety with the weak

basic 2-aminothiazole fragment could exert similar antibacterial mechanism to norfloxacin by targeting

DNA gyrase.

Table 3

2

.4. Chitin synthase (CHS) inhibitory activity

Chitin is the unique fungus cell wall component and its biosynthesis is mediated by CHS，when the

synthesis is interrupted fungal cell becomes osmotically unstable as it maintains cell wall integrity.

Therefore, CHS is considered as a promising target for the development of selective novel antifungal

agents [39]. Herein, some highly active antifungal compounds were further evaluated for their in vitro

chitin synthase inhibitory activities in comparison with commercially available polyoxin B [40]. Table 4

revealed that the tested compounds displayed moderate to high inhibition ratios on chitin biosynthesis at

concentration of 300 µg/mL. Comparing the results of all the tested compounds, compounds II-c, III-b

and V-b whose inhibition ratios were greater than 50% can be identified as promising chitin synthase

inhibitors for further evaluation of their IC values (Figures S2 and S3). The experimental results implied

50

that aminothiazolyl norfloxacin analogues II-c and V-b could inhibit the chitin synthase enzyme with

higher inhibitory activity (IC = 0.18 and 0.15 mM, respectively), which might be an encouraging start in

5

0

the discovery of antimicrobial fluoroquinolone agents with the ability to indirectly target fungal cell walls

by the inhibition of CHS.

Table 4

2

.5. Drug resistance development

The resistance of bacteria has caused great concern and becomes a serious challenge to the drug

development. Therefore, the evaluation of novel promising drug candidates for inducing bacterial

resistance is of great significance [41]. Herein, the ability of K. pneumoniae to develop drug resistance

against the highly active compound II-c was tested with the reference drug norfloxacin as a positive

control. As can be seen from Fig. 3, it indicated that the susceptibility of K. pneumoniae to compound II-c

remained nearly unaffected even after 10 passages, while the MIC value of norfloxacin toward K.

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pneumoniae got dramatically increased after several passages, indicating that K. pneumoniae was more

difficult to develop resistance against compound II-c than reference drug norfloxacin.

Fig. 3

2

.6. Bactericidal kinetics

In order to examine the antibacterial potency of the target compounds, a time-kill kinetic experiment of the

highly active molecule II-c against K. pneumoniae was performed. As shown in Fig. 4, it revealed that

there was more than 2.5 Log (CFU/mL) reduction in the number of viable bacteria within one hour at a

concentration of 4 × MIC. This result manifested that the compound had a rapid killing effect toward K.

pneumoniae.

Fig. 4

2

.7. Cytotoxicity

Cytotoxicity is one of the most essential criteria to be considered for active drug candidates [42]. The

bioactive molecule II-c was further evaluated for its toxicity toward normal mammalian cells (RAW 264.7)

via colorimetric cell proliferation MTT assay. The cytotoxic results (Fig. 5) revealed that the cell viability

of compound II-c was at least 80.25% even though the concentration up to 128 µg/mL, which indicated

that this molecule exhibited relatively low toxicity toward mammalian cells. In addition, it also concluded

that the antimicrobial activities exerted by aminothiazolyl norfloxacin analogues were not due to the

cytotoxic effects.

Fig. 5

2

.8. Bacterial membrane permeabilization

Bacterial membrane has been considered as a particularly valuable antibacterial target and is expected to

solve the problem of bacterial resistance by the method of destroying bacterial cell membrane through

active molecules [43]. The clinical drug-resistant K. pneumoniae was chosen to investigate the bacterial

membrane disruption ability of highly active compound II-c using propidium iodide (PI), a common dye

that can successfully pass through the membrane of compromised bacterial cells and emit fluorescence

upon binding to the DNA. As shown in Fig. 6, the fluorescence intensity of the mixture of II-c and

PI-treated K. pneumoniae exhibited rapid augment and became steady after one hour, while the control

group almost kept constant, which demonstrated that compound II-c could efficiently permeate the

membrane of K. pneumoniae.

Fig. 6

2

.9. Analysis of ClogP values on antimicrobial activity

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Hydrophobic/lipophilic properties possess remarkable effects on various biological processes of bioactive

molecules including transportation, distribution, metabolism and secretion [44]. Lipid/water partition

coefficients (ClogP) as one of the most important factors have been extensively employed to predict the

bioactivity of target molecules [45]. The theoretically calculated values of logP (ClogP) for all the target

compounds were calculated using ChemBioOffice 2014 (Cambridge Soft, Massachusettes, USA), and the

obtained results were shown in Table 5. Obviously, different substituents had a great influence on ClogP

of the predicted compounds and the values ranged from 0.38 to 6.97. The ClogP values of target

compounds generally increased with the increasing length of alkyl groups, and the introduction of short

alkyl chains and unsaturated polar moieties dramatically reduced the values of ClogP. As shown in Fig. 7,

the relationship of all the target compounds between ClogP and antibacterial effect against drug-resistant K.

pneumoniae revealed that the evaluated compounds with lower values of ClogP (< 2) displayed better

antimicrobial activities, but the compounds with high values of ClogP (> 4) usually showed poor

inhibitory activities, which might be explained by the possibility that higher lipophilic compounds were

unfavourable for being delivered to the binding sites in organism, and manifested the significant role of

suitable hydrophilicity for the antibacterial activities.

Table 5

Fig. 7

2

.10. Molecular docking study

A flexible ligand-receptor docking was successfully performed to rationalize the observed antibacterial

activity and understand the possible mechanism of the aminothiazolyl norfloxacin analogues. The crystal

structure data’s (topoisomerase IV–DNA complex and gyrase–DNA complex) were obtained from the

protein data bank (PDB code: 2XKK and 4DUH, respectively), which were representative targets to

investigate the antibacterial mechanism [46]. Target compound II-c was selected to dock with the

topoisomerase IV–DNA complex and gyrase–DNA complex, and the docking modes with the lowest

binding energy (-10.43 kcal/mol and -6.69 kcal/mol, respectively) were shown in Fig. 8. The oxygen atom

of carbonyl group could interact with the ARG-1123 residue through hydrogen bonds with the distance of

2

.8 Å. The NH group at 2-position of the thiazole ring in molecule II-c was in close vicinity to ASP-397

2

residue, forming a hydrogen bond with a distance of 2.0 Å. Furthermore, compound II-c could also form

hydrogen bond with the DA-14 residue through nitrogen atom of cyano group, which revealed that

nitrogen atom played an important role in drug molecules (Fig. 8A). The sulfur atom in the

2

-aminothiazole moiety also participated in the non-covalent coordination with ARG-136 residue by

forming a hydrogen bond with a distance of 2.2 Å (Fig. 8B). Besides, the aromatic fragment of compound

II-c could interact with base DT-15 and DA-16 of DNA through π-π stacking, respectively (Fig. 8C). It

was noteworthy that hydrophobic interactions also existed between the hydrophobic residues and the active

sites (Fig. 8D). All of these non-covalent might be favorable to stabilize the compound II-c–enzyme–DNA

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supramolecular complex, which further accounted for the good inhibitory efficacy of compound II-c

against the tested strains.

Fig. 8

2

.11. Quantum chemical studies

Computational methods in structure-activity relationships (SARs) are reported to predict important

pharmacokinetic properties and have yielded promising results for correlation of biological activity [47]. It

is known that intermolecular interactions were dominated by the frontier molecular orbitals (FMO), in

which E_H_O_M_Ois related with the potential of a molecule to donate electrons, whereas E_L_U_M_Orepresents the

ability of a molecule to accept electrons [48]. Extending the concept into drug-receptor binding systems

(Table 6), HOMOs of II-c, III-b and V-b were mainly focused on the quinolone ring and 2-aminothiazole

ring, manifesting that these locations might be active sites and biological interactions could take place

between positively charged molecules and these sites. Besides, it was observed that the substituents at the

piperazine ring of quinolone did not directly contribute to the HOMO and LUMO, which suggested that

these groups might be primarily used to regulate the physicochemical properties. In addition, the LUMOs

of these molecules were mainly focused on the benzopyridone skeleton where nucleophilic attacks might

be favorable. Among the drug-receptor binding systems, the extents of 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 are inversely related to the energy gap between the interacting orbitals. More HOMO energy and

lesser LUMO energy in the drug molecule result in larger stabilizing interactions [49]. Therefore, the

orbital energy of both HOMOs and LUMOs and their gaps for compounds II-c, III-b and V-b were

calculated shown in Table 7 and the results suggested that compound II-c, exhibiting the highest

antibacterial activities, had the lowest energy gap (∆E) of 3.964 eV.

Table 6

Table 7

The molecular electrostatic potential (MEP) surface generally gives an indication of the charged surface

area [50], which might be responsible for the hydrophilicity of molecules and can be used to explain

electrostatic interaction between compounds and biological targets and the orientation of drug candidates

for their activity [51]. Herein, the MEP maps of target compounds II-c, III-b and V-b were investigated

and the results (Table 6) suggested that an electronegative area (in red) can be observed at the oxygen atom

of quinolone ring and nitrogen atom of aminothiazolyl ring, which might indicate the capability of

hydrogen bond formation with share of the oxygen atom of benzopyridone skeleton and nitrogen atoms of

thiazole ring, thus easily interacting with various enzymes and receptors in biological systems. In terms of

their substituents, compounds II-c and V-b possessed more negative charged regions (in red) on the

carbonyl and nitrile, which implied the better biological activity of compounds II-c and V-b than III-b.

1

2

ACCEPTED MANUSCRIPT

The results obtained from quantum chemical studies are in accordance with the binding mode obtained

from above docking study.

2

.12. Interactions between compound II-c and DNA

DNA is a renowned drug target with multiple sites for drug interaction and has attracted considerable

attention for the rational design and construction of novel and effective DNA-targeting drugs [52]. Herein,

the binding behavior of compound II-c (exerting good inhibition against bacteria and fungi strains) with

DNA isolated from K. pneumoniae strains was studied to explore the possible antimicrobial mechanism of

action on a molecular level in vitro with neutral red (NR) dye as a spectral probe by using UV-vis

spectroscopic method.

2

.12.1. Absorption spectra of DNA in the presence of compound II-c

The absorption spectroscopy as one of the most excellent techniques is extensively employed in

DNA-binding studies [53]. In the absorption spectroscopy, hyperchromism and hypochromism have been

regarded to be vital spectral features to distinguish the changes of DNA double-helical structure [54],

which were respectively generated from the breakage of DNA duplex secondary structure and the

stabilization of DNA duplex by either intercalation binding mode or electrostatic effects of small molecules

[55].

The UV-vis absorption spectra were recorded with the sequentially and proportionately increasing

amount of compound II-c at a fixed concentration of DNA. As shown in Fig. 9, the UV-vis spectra

displayed that the maximum absorption peak of DNA at 260 nm exhibited proportional increase and a

slight red shift with the increasing concentration of molecule II-c. At the same time, the absorption value

of simply the sum of free DNA and free compound II-c was slightly lower than the measured value of the

II-c–DNA complex (inset of Fig. 9), indicating that a weak hyperchromism effect existed between DNA

and compound II-c. Furthermore, the intercalation of the chromophore fragment of compound II-c into the

DNA helix and the strong overlap of π-π states of the large π-conjugated system with the electronic states

of DNA bases were in accordance with the observed spectral changes.

Fig. 9

By keeping all the above observations in absorption spectra of DNA, intrinsic binding constant (K) for

the selected compound II-c was calculated using the following equation [56].

0

A

ξ_C

1

=

+

×

0

A− A

ξ_D₋_C− ξ_Cξ_D₋_C− ξ_CK[Q]

0

A and A stand for the absorbance of DNA in the absence and presence of compound II-c at 260 nm, ξ_C

and ξ_D_–_Crefer to the absorption coefficients of compound II-c and compound II-c–DNA complex,

0

respectively. The plot of A /(A–A ) versus 1/[compound II-c] was constructed by using the absorption

4

titration data and linear fitting (Fig. 10), yielding the binding constant, K = 1.43 × 10 L/mol, R = 0.999,

1

3

ACCEPTED MANUSCRIPT

SD = 0.046 (R is correlation coefficient. SD is standard deviation).

Fig. 10

2

.12.2. Absorption spectra of NR interactions with DNA

NR is a convenient planar phenazine dye with the features of low toxicity, high stability and convenient

application, which has been demonstrated that the binding mode of NR with DNA is intercalation [57].

Therefore, NR was chosen as a spectral probe to investigate the binding mode of compound II-c with

DNA. In present work, the absorption spectra of NR dye upon the addition of DNA were shown in Fig. S4.

With the increasing concentration of DNA, the absorption peak of the NR at around 460 nm showed

gradual decrease, and a new band at around 530 nm was developed, which could be attributed to the

formation of the new DNA–NR complex. The isosbestic point at 504 nm also demonstrated the formation

of DNA–NR complex.

2

.12.3. Absorption spectra of competitive interaction of compound II-c and NR with DNA

The absorption spectrum (Fig. 11) showed a competitive binding between NR and II-c with DNA. With

the increasing concentration of compound II-c, the maximum absorption of the DNA–NR complex at

around 530 nm decreased, but an apparent intensity increase occurred in the developing band at around

4

60 nm, which were opposite with the absorption of NR–DNA complex at the same wavelength. This

suggested that compound II-c could intercalate into the double helix of DNA by competitively substituting

NR in NR–DNA complex, which further blocked DNA replication and thus exerted the antimicrobial

activities. Moreover, the increase of absorbance around 276 nm further provided the evidence for

intercalation of compound II-c into K. pneumoniae DNA.

Fig. 11

2

.12.4. Cleavage of drug-resistant K. pneumoniae DNA

The cleaving agents of nucleic acid have attracted extensive attention due to their potential applications in

the fields of molecular biological technology and drug development [58]. Encouraged by the intercalation

result, with the aim to investigate the different action modes of compound II-c toward K. pneumoniae

o

DNA, the DNA-cleaving activity was also studied under physiological conditions (T = 37 C and pH = 7.4).

To our disappointment, there was the same result between the control (Lane A) and the experimental group

(

Lane B), indicating that our compound could not effectively cleave DNA but the intercalation was the

predominant type of action mode (Fig. S5).

2

.13. Drug combination study

Combination therapy currently is an increasingly prevalent method to improve treatment efficiency and

bioavailability, reduce or eliminate side effects and even combat drug resistance via different modes of

1

4

ACCEPTED MANUSCRIPT

action [59]. In the present work, the drug combination study was investigated in vitro between highly

active molecule II-c and clinical cefathiamidine by fraction inhibitory concentration (FIC) index [60]. The

results in Table 8 indicated that the combination of compound II-c with cefathiamidine mainly exhibited

synergistic and additive effects on Gram-positive bacteria showing no obvious antagonistic effects.

Particularly, active molecule II-c in combination use gave rise to improving antibacterial efficacy of

cefathiamidine by 2-fold (MIC value from 0.004 mM to 0.002 mM) toward K. pneumonia. Meanwhile, the

MIC value of II-c was dropped by 5-fold in this combination (MIC value from 0.005 mM to 0.001 mM). It

was worth noting that the combination could effectively inhibit the growth of drug-resistant MRSA. These

results indicated that the combination of cefathiamidine and fluoroquinolone derivative II-c was of great

significance in the treatment of increasingly serious bacterial infections. Cefathiamidine inhibits bacterial

cell wall mucin synthetase, where fluoroquinolones inhibit DNA gyrase and topoisomerase. The

dual-targeting bactericidal effect of the two classes of drugs may be the mechanism for their synergistic

bactericidal effect. These results provide an efficacious approach to maximize the antimicrobial

efficiencies via different action modes.

Table 8

3

. Conclusion

A series of new aminothiazolyl norfloxacin analogues as potentially antimicrobial agents have been

1

13

successfully synthesized by efficient protocols, and their structures were characterized by H NMR,

C

NMR and HRMS spectra. The in vitro biological evaluation revealed that some target compounds

exhibited good to better antimicrobial activities. SAR study suggested that unsaturated alkyl at N-4

position of the piperazine ring exerted a significant impact on biological activity. Particularly, norfloxacin

analogue II-c with cyanomethyl fragment displayed superior antimicrobial activities against K.

pneumoniae and C. albicans with MIC values of 0.005 mM and 0.010 mM, respectively. Moreover, this

compound showed broad antimicrobial spectrum, rapid bactericidal efficacy and strong enzymes inhibitory

activities including DNA gyrase and chitin synthase, and it also possessed efficient membrane permeability

and low toxicity against mammalian cells with lower propensity to trigger the development of bacterial

resistance than norfloxacin. Further preliminarily mechanism exploration indicated that compound II-c

could not only bind with gyrase–DNA and topoisomerase IV–DNA through hydrogen bonds and π-π

stacking, but also could form compound II-c–DNA supramolecular complex by intercalating into DNA of

resistant K. pneumoniae to exert the powerful bioactivities. The combination use of the active molecule

II-c with cefathiamidine was found to be an efficacious approach to maximize the antimicrobial

efficiencies via different action modes. On the basis of these results, it might be concluded that

aminothiazolyl norfloxacin analogue II-c should have great potential in the development of new

antimicrobial agents and further studies including in vivo bioactive evaluation are in progress.

1

5

ACCEPTED MANUSCRIPT

4

. Experimental

4

.1. General methods

All chemicals and solvents were commercial and used without further purification. The compound masses

were weighed on a microbalance with a resolution of 0.1 mg. TLC (Thin-layer chromatography) analysis

was performed through pre-coated silica gel plates. Column chromatography carried out by silica gel

1

13

(

#100–200). H and C NMR spectra were recorded on Bruker AVANCE III 600 MHz spectrometer using

DMSO-d as solvent, or TMS as internal standard. The chemical shifts were reported in parts per million

6

(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 mass spectra (MS) were recorded on

LCMS-2010A and the high-resolution mass spectra (HRMS) were recorded on an IonSpec FT–CR mass

spectrometer with ESI resource. Melting point (mp) was measured on a melting point apparatus (X-6 type).

The purity was determined by quantitative nuclear magnetic resonance (QNMR) method using

1

,3,5-trioxane as the internal standard. The results indicated that all the target compounds possessed purity

95%, and the test method for purity of target compounds were provided in Supporting Information.

≥

4

.1.1. General procedures for the synthesis of intermediates (2−7)

The intermediates 2−7 were prepared according to the previously reported procedures [21].

.1.2. General procedures for the synthesis of intermediates (10a d)

The intermediates 10a−d were prepared according to the reported procedures [61].

.1.3. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one (8)

4

−

4

To a solution of intermediate 7 (4.86 g, 15.00 mmol) in NMP (20 mL) was added piperazine (3.87 g, 45.00

o

mmol). Then the mixture was stirred at 130 C 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 x 100 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 8 (1.19 g) as yellow solid. Yield: 21.3%; mp: >

o

1

2

50 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.7 Hz, 1H,

6

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-NH ), 4.38 (q, J = 7.0 Hz, 2H, CH CH ), 3.15 (t, J = 4.02 Hz, 4H, piperazine-2,2-N-(CH ) ),

2

3

2 2

1

3

2

.91 (t, J = 4.38 Hz, 4H, piperazine-3,3-N-(CH ) ), 1.38 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151

2 2 2 3

MHz, DMSO-d ) 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,

6

4

9.1, 48.4, 45.7, 14.8 ppm.

4

.1.4. Synthesis of 3-(2-aminothiazol-4-yl)-6-(4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4

-dihydroquinolin-7-yl)piperazin-1-yl)-7-chloro-1-ethylquinolin-4(1H)-one (9)

1

6

ACCEPTED MANUSCRIPT

To a solution of intermediate 8 (0.56 g, 1.50 mmol) in NMP (10 mL) was added intermediate 7 (0.97 g,

o

3

.00 mmol). Then the mixture was stirred at 130 C for 24 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). 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 9 (0.38 g) as yellow solid. Yield: 37.6%; mp: >

o

1

2

7

50 C; H NMR (600 MHz, DMSO-d ) δ 8.72 (s, 1H, 6-F-quinolone-2-H), 8.53 (s, 1H,

6

-Cl-quinolone-2-H), 8.14 (d, J = 9.4 Hz, 1H, 6-F-quinolone-5-H), 8.09 (d, J = 5.5 Hz, 1H,

-Cl-quinolone-5-H), 8.01 (s, 1H, 6-F-quinolone-8-H), 7.69 (s, 1H, 6-F-thiazole-4-H), 7.64 (s, 1H,

-Cl-thiazole-4-H), 6.98 (s, 2H, 6-F-thiazole-2-NH ), 6.92 (s, 1H, 7-Cl-quinolone-8-H), 6.90 (s, 2H,

2

-Cl-thiazole-2-NH ), 4.40 (q, J = 6.9 Hz, 2H, 6-F-CH CH ), 4.00 (q, J = 6.7 Hz, 2H, 7-Cl-CH CH ), 2.98

2

3

2

3

(

s, 4H, 6-F-piperazine-2,2-N-(CH ) ), 2.85 (s, 4H, 6-F-piperazine-3,3-N-(CH ) ), 1.31 (t, J = 7.1 Hz, 3H,

2 2 2 2

1

3

6

1

-F-CH CH ), 1.06 (t, J = 7.1 Hz, 3H, 7-Cl-CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 173.3,

2 3 2 3 6

72.9, 167.3, 167.1, 158.8, 157.1, 146.9, 144.9, 144.5, 143.52 (s), 142.6, 138.2, 135.5, 135.1, 129.0, 126.2,

24.9, 117.0, 115.6, 115.3, 111.9, 111.8, 104.8, 104.1, 53.8, 49.1, 48.6, 48.3, 46.3, 14.9, 14.2 ppm; HRMS

-

(

ESI) calcd. for C H ClFN O S [M - H] , 675.1533; found, 675.1792.

3

2

30

8

2 2

4

.1.5. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-methylpiperazin-1-yl)quinolin-4(1H)-one

(I-a)

To a stirred suspension of potassium carbonate (103.66 mg, 0.75 mmol) in acetonitrile (15 mL) was added

o

intermediate 8 (186.73 mg, 0.50 mmol). The mixture was stirred at 60 C for 1.5 h, and then cooled to

room temperature. Iodomethane (106.46 mg, 0.75 mmol) was added, and the resulting mixture was stirred

at room temperature for 5 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 x 50 mL).

The organic extracts were dried over anhydrous sodium sulfate and concentrated. The crude product was

purified via silicagel column chromatography (eluent, chloroform/methanol (V/V) = 25/1) to afford

o

1

corresponding I-a (83.50 mg) as yellow solid. Yield: 43.1%; mp: > 250 C; H NMR (600 MHz,

DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.7 Hz, 1H, quinolone-5-H), 7.64 (s, 1H,

6

thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.91 (s, 2H, thiazole-2-NH ), 4.38 (q, J = 7.0 Hz,

2

H, CH CH ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 2.54 (s, 4H, piperazine-3,3-N-(CH ) ), 2.27 (s, 3H,

2 3 2 2 2 2

1

3

piperazine-CH ), 1.39 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1,

3

2

3

6

1

51.7, 144.9, 144.2, 142.6, 136.1, 121.5, 114.9, 111.9, 105.4, 103.9, 55.3, 54.9, 50.1, 49.1, 48.4, 46.1, 14.8

+

ppm; HRMS (ESI) calcd. for C H FN OS [M + H] , 388.1607; found, 388.1604.

1

9

22

5

4

.1.6. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-7-(4-ethylpiperazin-1-yl)-6-fluoroquinolin-4(1H)-one

I-b)

To a stirred suspension of potassium carbonate (103.66 mg, 0.75 mmol) in acetonitrile (15 mL) was added

(

17

ACCEPTED MANUSCRIPT

o

intermediate 8 (186.73 mg, 0.50 mmol). The mixture was stirred at 60 C for 1.5 h, and then cooled to

room temperature. Bromoethane (81.73 mg, 0.75 mmol) was added slowly, and the resulting mixture was

o

stirred at 80 C 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 x 50 mL).

The organic extracts were dried over anhydrous sodium sulfate and concentrated. The crude product was

purified via silicagel column chromatography (eluent, chloroform/methanol (V/V) = 25/1) to afford target

o

1

compound I-b (141.33 mg) as yellow solid. Yield: 70.4%; mp: 245–247 C; H NMR (600 MHz,

DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.7 Hz, 1H, quinolone-5-H), 7.63 (s, 1H,

6

thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.92 (s, 2H, thiazole-2-NH ), 4.38 (q, J = 7.0 Hz,

2

H, CH CH ), 3.23 (t, J = 4.2 Hz, 4H, piperazine-2,2-N-(CH ) ), 2.56 (s, 4H, piperazine-3,3-N-(CH ) ),

2 3 2 2 2 2

2

.40 (q, 7.1 Hz, 2H, piperazine-N-CH CH ), 1.39 (t, J = 7.1 Hz, 3H, CH CH ), 1.05 (t, J = 7.2 Hz, 3H,

2

3

2

3

1

3

piperazine-N-CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.9, 144.2, 142.6,

2

3

6

1

36.1, 121.5, 114.9, 111.7, 105.3, 103.9, 52.6, 52.1, 50.2, 49.1, 48.4, 14.8, 12.4 ppm; HRMS (ESI) calcd.

+

for C H FN OS [M + H] , 402.1758; found, 402.1764.

20

24

5

4

.1.7. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-propylpiperazin-1-yl)quinolin-4(1H)-one

I-c)

Compound I-c was prepared according to the procedure described for compound I-b starting from

-bromopropane (92.25 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound I-c (148.76 mg) was obtained as yellow solid. Yield:

(

1

(

o

1

7

1.6%; mp: 170–172 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6

6

Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.92 (s, 2H,

thiazole-2-NH ), 4.38 (q, J = 6.9 Hz, 2H, N-CH CH ), 3.23 (s, 4H, piperazine-2,2-N-(CH ) ), 2.56 (s, 4H,

2

3

2 2

piperazine-3,3-N-(CH ) ), 2.32 (t, J = 7.3 Hz, 2H, CH CH CH ), 1.51–1.45 (m, 2H, CH CH CH ), 1.39 (t,

2

3

2

3

1

3

J = 7.1 Hz, 3H, N-CH CH ), 0.89 (t, J = 7.3 Hz, 3H, CH CH CH ) ppm; C NMR (151 MHz, DMSO-d )

2

3

2

3

6

δ 172.9, 167.1, 151.7, 144.9, 144.3, 142.6, 136.1, 121.5, 114.9, 111.7, 105.4, 103.9, 60.1, 53.1, 50.3, 48.4,

+

1

9.9, 14.8, 12.2 ppm; HRMS (ESI) calcd. for C H FN OS [M + H] , 416.1920; found, 416.1916.

2

1

26

5

4

.1.8. Synthesis of 3-(2-aminothiazol-4-yl)-7-(4-butylpiperazin-1-yl)-1-ethyl-6-fluoroquinolin-4(1H)-one

(

I-d)

Compound I-d was prepared according to the procedure described for compound I-b starting from

-bromobutane (102.77 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound I-d (149.06 mg) was obtained as yellow solid. Yield:

1

(

o

1

6

9.4%; mp: 143–145 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6

6

Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.92 (s, 2H,

thiazole-2-NH ), 4.38 (q, J = 7.0 Hz, 2H, N-CH CH ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 2.60 (s, 4H,

2

3

2 2

piperazine-3,3-N-(CH ) ), 2.39 (s, 2H, CH (CH ) CH ), 1.49–1.44 (m, 2H, CH CH CH CH ), 1.39 (t, J =

2

2 2

3

2

3

1

8

ACCEPTED MANUSCRIPT

7

.1 Hz, 3H, N-CH CH ), 1.36–1.29 (m, 2H, CH CH CH CH ), 0.90 (t, J = 7.3 Hz, 3H, CH (CH ) CH )

2

3

2

3

2

2 2

3

13

ppm;. C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 153.4, 144.9, 144.2, 142.6, 136.1, 121.5, 114.9,

6

111.8, 105.4, 103.9, 57.8, 52.9, 50.1, 48.4, 28.7, 20.5, 14.8, 14.3 ppm; HRMS (ESI) calcd. for

+

C H FN OS [M + H] , 430.2077; found, 430.2071.

22

28

5

4

.1.9. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-hexylpiperazin-1-yl)quinolin-4(1H)-one

I-e)

Compound I-e was prepared according to the procedure described for compound I-b starting from

-bromohexane (123.81 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound I-e (149.87 mg) was obtained as yellow solid. Yield:

(

1

(

o

1

6

5.5%; mp: 163–165 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6

6

Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.91 (s, 2H,

thiazole-2-NH ), 4.38 (q, J = 7.0 Hz, 2H, CH CH ), 3.22 (s, 4H, piperazine-2,2-N-(CH ) ), 2.55 (s, 4H,

2

3

2 2

piperazine-3,3-N-(CH ) ), 2.34 (t,

J

=

7.0 Hz, 2H, CH (CH ) CH ), 1.47–1.43 (m, 2H,

2 2 4 3

2

CH CH (CH ) CH ), 1.38 (t, J = 7.1 Hz, 3H, CH CH ), 1.31–1.28 (m, 6H, CH CH (CH ) CH ), 0.87 (t, J

2

2 3

3

2

3

2

2 3

3

13

=

6.6 Hz, 3H, CH CH (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.9,

2 2 2 3 3 6

1

44.3, 142.6, 136.1, 121.5, 114.9, 111.7, 105.4, 103.9, 58.2, 53.1, 50.3, 49.1, 48.4, 31.7, 27.1, 26.7, 22.6,

+

4.8, 14.4 ppm; HRMS (ESI) calcd. for C H FN OS [M + H] , 458.2390; found, 458.2385.

2

4

32

5

4

.1.10. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-octylpiperazin-1-yl)quinolin-4(1H)-one

(

I-f)

Compound I-f was prepared according to the procedure described for compound I-b starting from

-bromooctane (144.85 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound I-f (155.90 mg) was obtained as light yellow solid. Yield:

1

(

o

1

6

4.2%; mp: 188–190 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6

6

Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.02 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.91 (s, 2H,

thiazole-2-NH ), 4.38 (q, J = 7.0 Hz, 2H, CH CH ), 3.22 (s, 4H, piperazine-2,2-N-(CH ) ), 2.54 (s, 4H,

2

3

2 2

piperazine-3,3-N-(CH ) ), 2.32 (t, J = 7.3 Hz, 2H, CH CH (CH ) CH ), 1.46–1.44 (m, 2H,

2

2 5

3

CH CH (CH ) CH ), 1.38 (t, J = 7.1 Hz, 3H, CH CH ), 1.26–1.21 (m, 10H, CH CH (CH ) CH ), 0.86 (t, J

2

2 5

3

2

3

2

2 5

3

1

3

=

6.9 Hz, 3H, CH CH (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.8, 144.9,

2 2 2 5 3 6

1

2

44.3, 142.6, 136.1, 121.5, 114.9, 111.8, 105.4, 103.9, 58.2, 53.1, 50.3, 48.4, 31.7, 29.4, 29.2, 27.4, 26.7,

+

2.5, 14.8, 14.4 ppm; HRMS (ESI) calcd. for C H FN OS [M + H] , 486.2703; found, 486.2697.

2

6

36

5

4

.1.11. Synthesis of 3-(2-aminothiazol-4-yl)-7-(4-decylpiperazin-1-yl)-1-ethyl-6-fluoroquinolin-4(1H)-one

(

I-g)

Compound I-g was prepared according to the procedure described for compound I-b starting from

-bromodecane (165.89 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound I-g (144.10 mg) was obtained as light yellow solid. Yield:

1

(

1

9

ACCEPTED MANUSCRIPT

o

1

5

6.1%; mp: 180–182 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6

6

Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.91 (s, 2H,

thiazole-2-NH ), 4.38 (q, J = 6.9 Hz, 2H, CH CH ), 3.23 (s, 4H, piperazine-2,2-N-(CH ) ), 2.57 (s, 4H, ,

2

3

2 2

piperazine-3,3-N-(CH ) ), 2.35 (s, 2H, CH CH (CH ) CH ), 1.47–1.45 (m, 2H, CH CH (CH ) CH ), 1.38

2

2 7

3

2

2 7

3

(

t, J = 7.1 Hz, 3H, CH CH ), 1.27–1.25 (m, 14H, CH CH (CH ) CH ), 0.86 (t, J = 6.9 Hz, 3H,

2 3 2 2 2 7 3

1

3

CH CH (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.9, 144.2, 142.6,

2

2 7

3

6

1

2

36.1, 121.5, 114.9, 111.7, 105.4, 103.9, 58.1, 52.9, 50.2, 49.1, 48.4, 31.8, 29.6, 29.5, 29.4, 29.2, 27.4,

+

2.5, 14.8, 14.4 ppm; HRMS (ESI) calcd. for C H FN OS [M + H] , 514.3016; found, 514.3009.

2

8

40

5

4

.1.12. Synthesis of 3-(2-aminothiazol-4-yl)-7-(4-dodecylpiperazin-1-yl)-1-ethyl-6-fluoroquinolin-4(1H)

-

one (I-h)

Compound I-h was prepared according to the procedure described for compound I-b starting from

-bromododecane (186.93 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound I-h (147.10 mg) was obtained as white solid. Yield: 54.3%;

1

(

o

1

mp: 184–186 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.5 Hz, 1H,

6

quinolone-5-H), 7.63 (s, 1H, thiazole-4-H), 7.02 (d, J = 6.7 Hz, 1H, quinolone-8-H), 6.91 (s, 2H,

thiazole-2-NH ), 4.38 (q, J = 6.7 Hz, 2H, CH CH ), 3.22 (s, 4H, piperazine-2,2-N-(CH ) ), 2.55 (s, 4H,

2

3

2 2

piperazine-3,3-N-(CH ) ), 2.33 (t, J = 6.5 Hz, 2H, CH CH (CH ) CH ), 1.47–1.44 (m, 2H,

2

2 9

3

CH CH (CH ) CH ), 1.38 (t, J = 6.7 Hz, 3H, CH CH ), 1.28–1.24 (m, 18H, CH CH (CH ) CH ), 0.85 (t, J

2

2 9

3

2

3

2

2 9

3

1

3

=

6.1 Hz, 3H, CH CH (CH ) CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.9,

2 2 2 9 3 6

1

2

5

44.3, 142.6, 136.1, 121.5, 114.9, 111.8, 105.4, 103.9, 58.2, 53.1, 50.3, 49.1, 48.4, 31.7, 29.5,29.4, 29.2,

+

7.4, 26.7, 22.5, 14.8, 14.4 ppm; HRMS (ESI) calcd. for C H FN OS [M + H] , 542.3329; found,

3

0

44

5

42.3320.

4

.1.13. Synthesis of 7-(4-allylpiperazin-1-yl)-3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoroquinolin-4(1H)-one

(II-a)

Compound II-a was prepared according to the procedure described for compound I-b starting from

3

8

5

-bromoprop-1-ene (90.73 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound

(186.73 mg, 0.50 mmol). The target compound II-a (110.41 mg) was obtained as white solid. Yield:

o

1

3.4%; mp: 225–227 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6

6

Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.90 (s, 2H,

thiazole-2-NH ), 5.86 (s, 1H, CH CH=CH ), 5.24 (d, J = 17.1 Hz, 1H, CH CH=CH ), 5.17 (d, J = 10.2 Hz,

2

1

6

H, CH CH=CH ), 4.38 (q, J = 7.0 Hz, 2H, CH CH ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 3.04 (d, J =

2 2 2 3 2 2

.0 Hz, 2H, CH CH=CH ), 2.58 (s, 4H, piperazine-3,3-N-(CH ) ), 1.39 (t, J = 7.1 Hz, 3H, CH CH ) ppm;

2

2 2

2

3

1

3

C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.3, 142.6, 135.9, 121.5, 118.2, 114.9, 111.9,

6

+

1

05.4, 103.9, 61.1, 55.3, 52.9, 50.2, 49.1, 48.4, 14.8 ppm; HRMS (ESI) calcd. for C H FN OS [M + H] ,

2

1

24

5

4

14.1764; found, 414.1759.

2

0

ACCEPTED MANUSCRIPT

4

.1.14. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(prop-2-yn-1-yl)piperazin-1-yl)quinolin-

(1H)-one (II-b)

Compound II-b was prepared according to the procedure described for compound I-b starting from

3

-bromo-1-propyne (89.22 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound

8

(186.73 mg, 0.50 mmol). The target compound II-b (141.76 mg) was obtained as light yellow solid.

o

1

Yield: 68.9%; mp: 235–237 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.87 (d, J =

6

1

3.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.05 (d, J = 7.1 Hz, 1H, quinolone-8-H), 6.92 (s,

H, thiazole-2-NH ), 4.39 (q, J = 6.9 Hz, 2H, CH CH ), 3.37 (s, 2H, CH C≡CH), 3.26 (s, 4H,

2

3

2

piperazine-2,2-N-(CH ) ), 3.21 (s, 1H, CH C≡CH), 2.67 (s, 4H, piperazine-3,3-N-(CH ) ), 1.39 (t, J = 7.1

2

2 2

1

3

Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.9, 144.1, 142.7, 136.1,

2

3

6

1

21.6, 114.9, 111.9, 105.5, 103.9, 79.6, 76.3, 51.4, 50.1, 49.1, 48.4, 46.5, 14.8 ppm; HRMS (ESI) calcd. for

+

C H FN OS [M + H] , 412.1607; found, 412.1604.

21

22

5

4

.1.15.

yl)piperazin-1-yl)acetonitrile (II-c)

Compound II-c was prepared according to the procedure described for compound I-b starting from

Synthesis

of

2-(4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7

-

2

-chloroacetonitrile (56.62 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound

8

(186.73 mg, 0.50 mmol). The target compound II-c (128.49 mg) was obtained as light yellow solid.

o

1

Yield: 62.3%; mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.87 (d, J =

6

1

3.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.05 (d, J = 7.1 Hz, 1H, quinolone-8-H), 6.92 (s,

H, thiazole-2-NH ), 4.39 (q, J = 6.9 Hz, 2H, CH CH ), 3.37 (s, 2H, CH C≡CH), 3.26 (s, 4H,

2

3

2

piperazine-2,2-N-(CH ) ), 3.21 (s, 1H, CH C≡CH), 2.67 (s, 4H, piperazine-3,3-N-(CH ) ), 1.39 (t, J = 7.1

2

2 2

1

3

Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.9, 143.9, 142.7, 136.0,

2

3

6

1

21.7, 116.2, 114.9, 111.9, 105.7, 104.0, 51.5, 49.9, 48.4, 45.5, 14.8 ppm; HRMS (ESI) calcd. for

+

C H FN OS [M + H] , 413.1560; found, 413.1555.

20

21

6

4

.1.16.

fluoroquinolin-4(1H)-one (III-a)

Compound III-a was prepared according to the procedure described for compound I-b starting from

,4-dichloro-1-(chloromethyl)benzene (146.60 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75

mmol) and compound 8 (186.73 mg, 0.50 mmol). The target compound III-a (174.11 mg) was obtained as

Synthesis

of

3-(2-aminothiazol-4-yl)-7-(4-(2,4-dichlorobenzyl)piperazin-1-yl)-1-ethyl-6-

2

o

1

light yellow solid. Yield: 65.4%; mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H,

6

quinolone-2-H), 7.87 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.61 (d, J = 1.1 Hz,

1

3

H, Ph-3-H), 7.57 (d, J = 8.3 Hz, 1H, Ph-5-H), 7.45 (dd, J = 8.2, 1.4 Hz, 1H, Ph-6-H), 7.05 (d, J = 7.2 Hz,

H, quinolone-8-H), 6.94 (s, 2H, thiazole-2-NH ), 4.38 (q, J = 7.0 Hz, 2H, CH CH ), 3.64 (s, 2H, Ph-CH ),

2

3

2

.25 (s, 4H, piperazine-2,2-N-(CH ) ), 2.65 (s, 4H, piperazine-3,3-N-(CH ) ), 1.38 (t, J = 7.1 Hz, 3H,

2

2 2

1

3

CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 153.4, 144.8, 144.3, 142.7, 136.1, 135.2,

2

3

6

2

1

ACCEPTED MANUSCRIPT

1

34.75, 132.8, 132.6, 129.2, 127.7, 121.7, 114.9, 111.9, 105.6, 103.9, 58.4, 55.4, 52.9, 50.3, 49.1, 48.4,

+

4.8 ppm; HRMS (ESI) calcd. for C H Cl FN OS [M + H] , 532.1141; found, 532.1140.

2

5

24

2

5

4

.1.17. Synthesis of 3-(2-aminothiazol-4-yl)-7-(4-(4-chlorobenzyl)piperazin-1-yl)-1-ethyl-6-fluoroquinolin

-

4(1H)-one (III-b)

Compound III-b was prepared according to the procedure described for compound I-b starting from

-chloro-4-(chloromethyl)benzene (120.77 mg, 0.75 mol), potassium carbonate (103.66 mg, 0.75 mmol)

and compound 8 (186.73 mg, 0.50 mmol). The target compound III-b (150.15 mg) was obtained as light

1

o

1

yellow solid. Yield: 60.3%; mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H),

6

7

3

.86 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.42–7.39 (m, 4H, ClPh-2,3,5,6-4H),

.04 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.94 (s, 2H, thiazole-2-NH ), 4.37 (q, J = 7.0 Hz, 2H, CH CH ),

2

3

.56 (s, 2H, ClPh-CH ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 2.59 (s, 4H, piperazine-3,3-N-(CH ) ), 1.38

2

2 2

1

3

(

t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 153.4, 144.9, 144.2,

2

3

6

1

42.7, 136.0, 131.2, 128.7, 121.7, 114.9, 111.9, 105.6, 103.9, 61.3, 55.3, 52.7, 50.1, 49.1, 48.4, 14.8 ppm;

+

HRMS (ESI) calcd. for C H ClFN OS [M + H] , 498.1531; found, 498.1530.

2

5

25

5

4

.1.18. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(4-fluorobenzyl)piperazin-1-yl)quinolin

4(1H)-one (III-c)

Compound III-c was prepared according to the procedure described for compound I-b starting from

-(chloromethyl)-4-fluorobenzene (108.43 mg, 0.75 mol), potassium carbonate (103.66 mg, 0.75 mmol)

and compound 8 (186.73 mg, 0.50 mmol). The target compound III-c (151.45 mg) was obtained as yellow

-

1

o

1

solid. Yield: 62.9%; mp: 230–232 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86

6

(d, J = 13.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.38 (t, 2H, FPh-3,5-2H), 7.17 (t, J = 8.6

Hz, 2H, FPh-2,4-2H)), 7.03 (d, J = 7.1 Hz, 1H, quinolone-8-H), 6.93 (s, 2H, thiazole-2-NH ), 4.37 (q, J =

2

6

.9 Hz, 2H, CH CH ), 3.55 (s, 2H, FPh-CH ), 3.23 (s, 4H, piperazine-2,2-N-(CH ) ), 2.57 (s, 4H,

2 3 2 2 2

1

3

piperazine-3,3-N-(CH ) ), 1.37 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9,

2

3

6

1

5

4

67.1, 153.4, 144.9, 142.7, 136.1, 131.2, 121.6, 115.5, 115.3, 114.9, 111.8, 111.7, 105.5, 104.0, 61.4, 55.3,

+

2.7, 50.3, 49.1, 48.4, 14.8 ppm; HRMS (ESI) calcd. for C H F N OS [M + H] , 482.1826; found,

2

5

25

2

5

82.1825.

4

.1.19.

Synthesis

of

3-(2-aminothiazol-4-yl)-7-(4-(3,4-dichlorobenzyl)piperazin-1-yl)-1-ethyl-6-

fluoroquinolin-4(1H)-one (III-d)

Compound III-d was prepared according to the procedure described for compound I-b starting from

,2-dichloro-4-(chloromethyl)benzene (146.60 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75

mmol) and compound 8 (186.73 mg, 0.50 mmol). The target compound III-d (163.73 mg) was obtained as

1

o

1

yellow solid. Yield: 61.5%; mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H),

6

7

.86 (d, J = 13.5 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.62–7.57 (m, 2H, Ph-2,5-2H), 7.35

(

d, J = 7.7 Hz, 1H, Ph-6-H), 7.04 (d, J = 4.3 Hz, 1H, quinolone-8-H)), 6.93 (s, 2H, thiazole-2-NH ), 4.37

2

ACCEPTED MANUSCRIPT

(

q, J = 6.6 Hz, 2H, CH CH ), 3.58 (s, 2H, CH ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 2.59 (s, 4H,

2

3

2

2 2

1

3

piperazine-3,3-N-(CH ) ), 1.38 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9,

2

3

6

1

5

67.1, 153.4, 144.9, 142.7, 136.1, 131.4, 131.0, 130.8, 129.7, 121.7, 114.9, 111.9, 105.6, 104.0, 60.7, 55.3,

+

2.7, 50.3, 48.4, 14.8 ppm; HRMS (ESI) calcd. for C H Cl FN OS [M + H] , 532.1141; found,

2

5

24

2

5

32.1143.

4

.1.20. Synthesis of 3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-hydroxyethyl)piperazin-1-yl)quinolin

-

4(1H)-one (IV-a)

Compound IV-a was prepared according to the procedure described for compound I-b starting from

-bromoethan-1-ol (93.72 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound IV-a (113.56 mg) was obtained as white solid. Yield:

2

(

o

1

5

1

4.4%; mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.87 (d, J = 13.6 Hz,

6

H, quinolone-5-H), 7.63 (s, 1H, thiazole-4-H), 7.04 (d, J = 6.9 Hz, 1H, quinolone-8-H), 6.92 (s, 2H,

thiazole-2-NH ), 4.39 (q, J = 6.8 Hz, 2H, CH CH ), 3.60 (s, 2H, CH CH OH), 3.28 (s, 4H,

2

3

2

piperazine-2,2-N-(CH ) ), 2.72 (d, J = 16.4 Hz, 4H, piperazine-3,3-N-(CH ) ), 2.63–2.53 (m, 2H,

2

2 2

1

3

CH CH OH), 1.39 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1,

2

3

6

1

51.7, 144.9, 143.9, 142.7, 136.1, 121.6, 114.9, 111.9, 105.5, 103.9, 60.2, 53.1, 49.6, 49.1, 48.4, 14.8 ppm;

+

HRMS (ESI) calcd. for C H FN O S [M + H] , 418.1713; found, 418.1707.

2

0

24

5

2

4

.1.21.

Synthesis

of

3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-hydroxypropyl)piperazin-1-

yl)quinolin-4(1H)-one (IV-b)

Compound VI-a (214.76 mg, 0.50 mmol) was dissolved in methanol (5 mL), and sodium borohydride

o

(

37.83 mg, 1.00 mmol) was slowly added to the mixture at 0 C. Then the resulting mixture was allowed to

stir for 1 h at room temperature. 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 x

5

0 mL). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated. The

crude product was purified via silicagel column chromatography (eluent, chloroform/methanol (V/V) =

o

1

2

5/1) to afford corresponding IV-b (175.42 mg) as light yellow solid. Yield: 81.3%; mp: 245–247 C; H

NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.63

6

(

s, 1H, thiazole-4-H), 7.03 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.92 (s, 2H, thiazole-2-NH ), 4.38 (q, J =

2

7

.0 Hz, 2H, CH CH ), 4.34 (s, 1H, OH), 3.85–3.78 (m, 1H, CH(OH)CH ), 3.23 (s, 4H,

2 3 3

piperazine-2,2-N-(CH ) ), 2.63 (s, 4H, piperazine-3,3-N-(CH ) ), 2.36–2.32 (m, 1H, CH CH(OH)CH ),

2

2 2

2

3

2

.28–2.23 (m, 1H, CH CH(OH)CH ), 1.38 (t, J = 7.1 Hz, 3H, CH CH ), 1.08 (d, J = 6.1 Hz, 3H,

2 3 2 3

1

3

CH(OH)CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 151.7, 144.9, 144.3, 142.6, 136.1,

3

6

1

21.5, 114.9, 111.8, 105.4, 103.9, 66.2, 63.8, 53.6, 50.3, 48.4, 22.3, 14.8 ppm; HRMS (ESI) calcd. for

+

C H FN O S [M + H] , 432.1869; found, 432.1865.

21

26

5

2

4

.1.22.

Synthesis

of

4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-

2

3

ACCEPTED MANUSCRIPT

yl)piperazine-1-carbaldehyde (V-a)

o

Intermediate 8 (186.73 mg, 0.50 mmol) was stirred in formamide (10 mL) at 75 C for 3 h. After the

mixture was kept in the cold overnight, the formed solid was filtered off and recrystallized with methanol

o

1

to give the pure target compound V-a (172.89 mg) as off-white solid. Yield: 86.2%. mp: > 250 C; H

NMR (600 MHz, DMSO-d ) δ 8.60 (s, 1H, quinolone-2-H), 8.12 (s, 1H, CHO), 7.89 (d, J = 13.3 Hz, 1H,

6

quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.09 (d, J = 7.1 Hz, 1H, quinolone-8-H), 6.93 (s, 2H,

thiazole-2-NH ), 4.39 (q, J = 6.9 Hz, 2H, CH CH ), 3.63–3.57 (m, 4H, piperazine-2,2-N-(CH ) ), 3.24 (s,

2

3

2 2

2

H, piperazine-3,3-N-(CH ) ), 3.19 (s, 2H, piperazine-3,3-N-(CH ) ), 1.39 (t, J = 7.1 Hz, 3H, CH CH )

2 2 2 2 2 3

1

3

ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 167.1, 161.4, 151.7, 144.8, 144, 142.8, 135.9, 122, 114.9,

6

1

11.8, 106.2, 104.1, 51.2, 49.9, 49.1, 48.4, 45.1, 14.9 ppm; HRMS (ESI) calcd. for C H FN O S [M +

19 20 5 2

+

H] , 402.1400; found, 402.1398.

4

.1.23. Synthesis of 7-(4-acetylpiperazin-1-yl)-3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoroquinolin-4(1H)-one

(V-b)

To a stirred suspension of potassium carbonate (103.66 mg, 0.75 mmol) in acetonitrile (15 mL) was added

o

intermediate 8 (186.73 mg, 0.50 mmol). The mixture was stirred at 60 C for 1.5 h, and then cooled to

room temperature. Acetyl chloride (58.87 mg, 0.75 mmol) was added, and the resulting mixture was stirred

at room temperature for 4 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 x 50 mL).

The combined organic extracts were dried over anhydrous sodium sulfate and concentrated. The crude

product was purified via silicagel column chromatography (eluent, chloroform/methanol (V/V) = 25/1) to

o

1

afford corresponding V-b (147.38 mg) as light yellow solid. Yield: 71.0%; mp: > 250 C; H NMR (600

MHz, DMSO-d ) δ 8.60 (s, 1H, quinolone-2-H), 7.89 (d, J = 13.4 Hz, 1H, quinolone-5-H), 7.64 (s, 1H,

6

thiazole-4-H), 7.07 (d, J = 7.0 Hz, 1H, quinolone-8-H), 6.93 (s, 2H, thiazole-2-NH ), 4.39 (q, J = 6.6 Hz,

2

H, CH CH ), 3.65 (s, 4H, piperazine-2,2-N-(CH ) ), 3.24 (s, 2H, piperazine-3,3-N-(CH ) ), 3.18 (s, 2H,

2 3 2 2 2 2

1

3

piperazine-3,3-N-(CH ) ), 2.07 (s, 3H, COCH ), 1.39 (t, J = 7.0 Hz, 3H, CH CH ) ppm; C NMR (151

2

3

2

3

MHz, DMSO-d ) δ 172.9, 168.8, 167.1, 151.7, 144.7, 144.1, 142.8, 136.1, 121.9, 114.9, 111.8, 105.9,

6

+

1

03.9, 65.4, 50.6, 50.2, 49.1, 48.4, 46.1, 14.8 ppm; HRMS (ESI) calcd. for C H FN O S [M + H] ,

2

0

22

5

2

4

16.1556; found, 416.1552.

4

.1.24. Synthesis of

3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-7-(4-(2-oxopropyl)piperazin-1-

yl)quinolin-4(1H)-one (VI-a)

Compound VI-a was prepared according to the procedure described for compound I-b starting from

-chloropropan-2-one (69.39 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and

compound 8 (186.73 mg, 0.50 mmol). The target compound VI-a (143.24 mg) was obtained as yellow

1

o

1

solid. Yield: 66.7%; mp: 163–165 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86

6

(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),

2

4

ACCEPTED MANUSCRIPT

6

.90 (s, 2H, thiazole-2-NH ), 4.38 (q, J = 7.1 Hz, 2H, CH CH ), 3.28 (s, 2H, CH COCH ), 3.27–3.22 (m,

2

3

2

3

4

H, piperazine-2,2-N-(CH ) ), 2.66–2.60 (m, 4H, piperazine-3,3-N-(CH ) ), 2.11 (s, 3H, CH COCH ), 1.39

2

2 2

2

3

1

3

(

t, J = 7.2 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 207.1, 172.9, 167.1, 151.7, 144.9,

2

3

6

1

44.2, 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 C H FN O S [M + H] , 430.1713; found, 430.1710.

2

1

24

5

2

4

.1.25. Synthesis of ethyl 2-(4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7

-

yl)piperazin-1-yl)acetate (VI-b)

Compound VI-b was prepared according to the procedure described for compound I-b starting from ethyl

-chloroacetate (91.91 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound 8

186.73 mg, 0.50 mmol). The target compound VI-b (118.56 mg) was obtained as yellow solid. Yield:

2

(

o

1

5

1.6%; mp: 198–200 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.5

6

Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.05 (d, J = 7.1 Hz, 1H, quinolone-8-H), 6.92 (s, 2H,

thiazole-2-NH ), 4.38 (q, J = 6.8 Hz, 2H, N-CH CH ), 4.12 (q, J = 7.1 Hz, 2H, OCH CH ), 3.32 (s, 2H,

2

3

2

3

N-CH COCH CH ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 2.73 (s, 4H, piperazine-3,3-N-(CH ) ), 1.39 (t,

2

3

2 2

1

3

J = 7.0 Hz, 3H, N-CH CH ), 1.21 (t, J = 7.1 Hz, 3H, OCH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ

2

3

2

3

6

1

5

72.9, 170.4, 167.1, 151.7, 144.9, 144.2, 142.7, 136.1, 121.6, 114.9, 111.9, 105.5, 103.9, 60.3, 58.8, 52.2,

+

0.2, 48.4, 14.8, 14.6 ppm; HRMS (ESI) calcd. for C H FN O S [M + H] , 460.1819; found, 460.1820.

2

26

5

3

4

.1.26.

Synthesis

of

2-(4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-

yl)piperazin-1-yl)-N,N-dimethylacetamide (VII-a)

To a stirred suspension of potassium carbonate (103.66 mg, 0.75 mmol) in acetonitrile (15 mL) was added

o

intermediate 8 (186.73 mg, 0.50 mmol). The mixture was stirred at 60 C for 1.5 h, and then cooled to

room temperature. 2-Chloro-N,N-dimethylacetamide (10a) (91.17 mg, 0.75 mmol) was added, and the

o

resulting mixture was stirred at 80 C 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 x 50 mL). The organic extracts were dried over anhydrous sodium sulfate and

concentrated. The crude product was purified via silicagel column chromatography (eluent,

chloroform/methanol (V/V) = 25/1) to afford corresponding VII-a (119.91 mg) as yellow solid. Yield:

o

1

5

2.3%; mp: 211–213 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H, quinolone-2-H), 7.86 (d, J = 13.6

6

Hz, 1H, quinolone-5-H), 7.63 (s, 1H, thiazole-4-H), 7.05 (d, J = 7.2 Hz, 1H, quinolone-8-H), 6.91 (s, 2H,

thiazole-2-NH ), 4.39 (q, J = 7.1 Hz, 2H, CH CH ), 3.36 (s, 2H, N-CH -CO), 3.24 (s, 4H,

2

3

2

piperazine-2,2-N-(CH ) ), 3.16 (s, 3H, N-CH ), 3.03 (s, 3H, N-CH ), 2.66 (s, 4H, piperazine-3,3-N-(CH ) ),

2

3

2 2

1

3

1

.38 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 168.2, 167.1, 151.7,

2 3 6

44.9, 144.1, 142.7, 136.1, 121.6, 114.9, 111.9, 105.4, 103.6, 60.3, 52.8, 50.2, 49.1, 48.4, 40.6, 14.9 ppm;

+

HRMS (ESI) calcd. for C H FN O S [M + H] , 459.1978; found, 459.1976.

2

27

6

2

4

.1.27.

Synthesis

of

2-(4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-

2

5

ACCEPTED MANUSCRIPT

yl)piperazin-1-yl)-N,N-diethylacetamide (VII-b)

Compound VII-b was prepared according to the procedure described for compound VII-a starting from

-chloro-N,N-diethylacetamide (10b) (112.21 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75

mmol) and compound 8 (186.73 mg, 0.50 mmol). The target compound VII-b (155.83 mg) was obtained

2

o

1

as yellow solid. Yield: 64.1%; mp: 246–248 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H,

6

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 = 7.2 Hz,

1

H, quinolone-8-H), 6.91 (s, 2H, thiazole-2-NH ), 4.39 (q, J = 7.0 Hz, 2H, CH CH ), 3.41 (q, J = 6.6 Hz,

2 2 3

2

H, N-CH CH ), 3.29–3.21 (m, 8H, piperazine-2,2-N-(CH ) and N-CH CO-N-CH CH ), 2.66 (s, 4H,

2

3

2 2

2

3

piperazine-3,3-N-(CH ) ), 1.38 (t, J = 7.1 Hz, 3H, CH CH ), 1.16 (t, J = 7.0 Hz, 3H, N-CH CH ), 1.03 (t, J

2

3

2

3

1

3

=

7.0 Hz, 3H, N-CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 168.2, 167.1, 151.7, 144.9,

2 3 6

1

44.1, 142.7, 136.1, 121.6, 114.9, 111.9, 105.5, 103.9, 60.6, 52.9, 50.2, 49.1, 48.4, 41.8, 14.9, 13.4 ppm;

+

HRMS (ESI) calcd. for C H FN O S [M + H] , 487.2291; found, 487.2287.

2

4

31

6

2

4

.1.28.

Synthesis

of

2-(4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-

yl)piperazin-1-yl)-N,N-bis(2-hydroxyethyl)acetamide (VII-c)

Compound VII-c was prepared according to the procedure described for compound VII-a starting from

2

-chloro-N,N-bis(2-hydroxyethyl)acetamide (10c) (136.21 mg, 0.75 mmol), potassium carbonate (103.66

mg, 0.75 mmol) and compound 8 (186.73 mg, 0.50 mmol). The target compound VII-c (163.36 mg) was

o

1

obtained as yellow solid. Yield: 63.0%; mp: 173–175 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H,

6

quinolone-2-H), 7.87 (d, J = 13.6 Hz, 1H, quinolone-5-H), 7.64 (s, 1H, thiazole-4-H), 7.05 (d, J = 7.2 Hz,

1

3

H, quinolone-8-H), 6.93 (s, 2H, thiazole-2-NH ), 4.68 (s, 1H，OH), 4.39 (q, J = 7.0 Hz, 2H, CH CH ),

2

3

.58 (t, J = 5.3 Hz, 2H, N-CH CH OH), 3.53 (t, J = 5.0 Hz, 2H, N-CH CH OH), 3.50 (s, 2H, N-CH -CO),

2

.36 (t, J = 6.2 Hz, 4H, N-(CH CH OH) ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 2.68 (s, 4H,

2

2 2

1

3

piperazine-3,3-N-(CH ) ), 1.39 (t, J = 7.1 Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ

2

3

6

1

5

72.9, 169.5, 167.1, 151.7, 144.9, 144.2, 142.7, 136.8, 121.6, 114.9, 111.9, 105.5, 103.9, 60.4, 59.7, 59.2,

+

2.9, 50.9, 50.1, 48.7, 48.4, 14.8 ppm; HRMS (ESI) calcd. for C H FN O S [M + H] , 519.2190; found,

2

4

31

6

4

19.2185.

4

.1.29.

Synthesis

of

2-(4-(3-(2-aminothiazol-4-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-

yl)piperazin-1-yl)-N,N-diisopropylacetamide (VII-d)

Compound VII-d was prepared according to the procedure described for compound VII-a starting from

2

-chloro-N,N-diisopropylacetamide (10d) (132.82 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75

mmol) and compound 8 (186.73 mg, 0.50 mmol). The target compound VII-d (147.77 mg) was obtained

o

1

as white solid. Yield: 59.2%; mp: > 250 C; H NMR (600 MHz, DMSO-d ) δ 8.59 (s, 1H,

6

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,

1

H, quinolone-8-H), 6.90 (s, 2H, thiazole-2-NH ), 4.39 (q, J = 7.0 Hz, 2H, CH CH ), 4.32–4.24 (m, 1H,

2 2 3

CH(CH ) ), 3.38–3.43 (m, 1H, CH(CH ) ), 3.24 (s, 4H, piperazine-2,2-N-(CH ) ), 3.15 (s, 2H,

3

2

3 2

2 2

2

6

ACCEPTED MANUSCRIPT

N-CH -CO), 2.61 (s, 4H, piperazine-3,3-N-(CH ) ), 1.38 (t, J = 7.1 Hz, 3H, CH CH ), 1.32 (d, J = 6.6 Hz,

2

2 2

2

3

1

3

6

1

4

5

H, CH(CH ) ), 1.17 (d, J = 6.5 Hz, 6H, CH(CH ) ) ppm; C NMR (151 MHz, DMSO-d ) δ 172.9, 168.1,

3 2 3 2 6

67.1, 151.7, 144.9, 144.2, 142.6, 136.1, 121.5, 114.9, 111.9, 105.4, 103.9, 63.4, 55.3, 52.8, 50.3, 49.1,

+

8.9, 48.3, 45.2, 20.9, 20.8, 14.9 ppm; HRMS (ESI) calcd. for C H FN O S [M + H] , 515.2604; found,

2

6

35

6

2

15.2598.

4

.1.30. Synthesis of 3-(2-aminothiazol-4-yl)-7-chloro-1-ethyl-6-(piperazin-1-yl)quinolin-4(1H)-one (11)

Compound 11 was prepared according to the procedure described for compound 8 starting from

intermediate 7 (4.86 g, 15.00 mmol), piperazine (3.87 g, 45.00 mmol) in NMP (20 mL). The target

o

1

compound 11 (0.83 g) was obtained as yellow solid. Yield: 14.2%; mp: 241–243 C; H NMR (600 MHz,

DMSO-d ) δ 8.63 (s, 1H, quinolone-2-H), 7.96 (s, 2H, quinolone-5-H, quinolone-8-H), 7.66 (s, 1H,

6

thiazole-4-H), 6.94 (s, 2H, thiazole-2-NH ), 4.39 (q, J = 7.1 Hz, 2H, CH CH ), 3.01 (s, 4H,

2

3

piperazine-2,2-N-(CH ) ), 2.98 (d, J = 3.5 Hz, 4H, piperazine-3,3-N-(CH ) ), 1.36 (t, J = 7.2 Hz, 3H,

2

2 2

1

3

CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 173.2, 167.1, 145.8, 144.8, 142.8, 134.7, 133.6, 126.6,

2

3

6

+

1

18.9, 116.9, 115.4, 104.2, 52.3, 48.3, 45.7, 15.1 ppm; HRMS (ESI) calcd. for C H ClN OS [M + H] ,

18 20 5

3

90.1155; found, 390.1154.

4

.1.31. Synthesis of 3-(2-aminothiazol-4-yl)-7-chloro-1-ethyl-6-(4-(2-oxopropyl)piperazin-1-yl)quinolin

-

4(1H)-one (VIII-a)

Compound VIII-a was prepared according to the procedure described for compound I-b starting from

-chloropropan-2-one (69.39 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and

compound 11 (194.95 mg, 0.50 mmol). The target compound VIII-a (163.45 mg) was obtained as yellow

1

o

1

solid. Yield: 73.3%; mp: 248–250 C; H NMR (600 MHz, DMSO-d ) δ 8.63 (s, 1H, quinolone-2-H), 7.98

6

(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-NH ), 4.38 (q, J = 7.1 Hz, 2H, CH CH ), 3.28 (s, 2H, CH COCH ), 3.06 (s, 4H,

2

3

2

3

piperazine-2,2-N-(CH ) ), 2.65 (s, 4H, piperazine-3,3-N-(CH ) ), 2.12 (s, 3H, CH COCH ), 1.36 (t, J = 7.1

2

2 2

2

3

1

3

Hz, 3H, CH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 207.3, 173.2, 167.1, 145.5, 144.8, 142.8, 134.7,

2

3

6

1

33.5, 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

+

C H ClN O S [M + H] , 446.1417; found, 446.1416.

21

24

5

2

4

.1.32. Synthesis of ethyl 2-(4-(3-(2-aminothiazol-4-yl)-7-chloro-1-ethyl-4-oxo-1,4-dihydroquinolin-6-

yl)piperazin-1-yl)acetate (VIII-b)

Compound VIII-b was prepared according to the procedure described for compound I-b starting from

ethyl-2-chloroacetate (91.91 mg, 0.75 mmol), potassium carbonate (103.66 mg, 0.75 mmol) and compound

1

1 (194.95 mg, 0.50 mmol). The target compound VIII-b (121.85 mg) was obtained as light yellow solid.

o

1

Yield: 51.2%; mp: 195–198 C; H NMR (600 MHz, DMSO-d ) δ 8.62 (s, 1H, quinolone-2-H), 7.97 (s, 1H,

6

quinolone-5-H), 7.95 (s, 1H, quinolone-8-H), 7.66 (s, 1H, thiazole-4-H), 6.93 (s, 2H, thiazole-2-NH ), 4.38

2

(

d, J = 6.7 Hz, 2H, N-CH CH ), 4.12 (q, J = 6.2 Hz, 2H, OCH CH ), 3.33 (s, 2H, N-CH COCH CH ), 3.05

2 3 2 3 2 2 3

27

ACCEPTED MANUSCRIPT

(

s, 4H, piperazine-2,2-N-(CH ) ), 2.75 (s, 4H, piperazine-3,3-N-(CH ) ), 1.36 (t, J = 6.4 Hz, 3H,

2

2 2

1

3

N-CH CH ), 1.22 (t, J = 7.1 Hz, 3H, OCH CH ) ppm; C NMR (151 MHz, DMSO-d ) δ 177.9, 175.1,

2

3

2

3

6

1

71.9, 150.2, 149.5, 147.6, 139.4, 138.3, 131.4, 123.6, 121.7, 120.1, 108.9, 65.1, 63.6, 57.2, 56.5, 53.0,

+

9.8, 19.4 ppm; HRMS (ESI) calcd. for C H ClN O S [M + H] , 476.1523; found, 476.1524.

2

26

5

3

Acknowledgments

This work was partially supported by National Natural Science Foundation of China (No. 21672173), the

Research Fund for International Young Scientists from International (Regional) Cooperation and Exchange

Program (No. 21850410447), 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).

References

[1] I. Levin-Reisman, I. Ronin, O. Gefen, I. Braniss, N. Shoresh, N.Q. Balaban, Antibiotic tolerance facilitates the evolution

of resistance, Science 355 (2017) 826–830.

[2] M. Sprenger, K. Fukuda, New mechanisms, new worries, Science 351 (2016) 1263–1264.

[3] E.D. Brown, G.D.Wright, Antibacterial drug discovery in the resistance era, Nature 529 (2016) 336–343.

[4] S.L. Silver, Challenges of antibacterial discovery, Clin. Microb. Rev. 24 (2011) 71–109.

[5] K.C. Fang, Y.L. Chen, J.Y. Sheu, T.C. Wang, C.C. Tzeng, Synthesis, antibacterial, and cytotoxic evaluation of certain

7

-substituted norfloxacin derivatives, J. Med. Chem. 43 (2000) 3809–3812.

[

6] G.S. Bisacchi, Origins of the quinolone class of antibacterials: An expanded “discovery story”, J. Med. Chem. 58 (2015)

4

874–4882.

7] L.A. Mitscher, Bacterial topoisomerase inhibitors: Quinolone and pyridone antibacterial agents, Chem. Rev. 105 (2005)

59–592.

5

8] R. Neelarapu, J.R. Maignan, C.L. Lichorowic, A. Monastyrskyi, T.S. Mutka, A.N. LaCrue, L.D. Blake, D. Casandra, S.

Mashkouri, J.N. Burrows, P.A. Willis, D.E. Kyle, R. Manetsch, Design and synthesis of orally bioavailable piperazine

substituted 4(1H)-quinolones with potent antimalarial activity: Structure-activity and structure-property relationship

studies, J. Med. Chem. 61 (2018) 1450–1473.

[

9] K.M.G. O'Connell, J.T. Hodgkinson, H.F. Sore, M. Welch, G.P.C. Salmond, D.R. Spring, Combating multidrug-resistant

bacteria: Current strategies for the discovery of novel antibacterials, Angew. Chem. Int. Ed. 52 (2013) 10706–10733.

10] I. Laponogov, M.K. Sohi, D.A. Veselkov, X.S. Pan, R. Sawhney, A.W. Thompson, K.E. McAuley, L.M. Fisher, M.R.

Sanderson, Structural insight into the quinolone-DNA cleavage complex of type IIA topoisomerases, Nat. Struct. Mol.

Biol. 16 (2009) 667–669.

[

[11] Y.Q. Hu, S. Zhang, Z. Xu, Z.S. Lv, M.L. Liu, L.S. Feng, 4-Quinolone hybrids and their antibacterial activities, Eur. J.

Med. Chem. 141 (2017) 335–345.

2

8

Article Doi

Design and synthesis of aminothiazolyl norfloxacin analogues as potential antimicrobial agents and their biological evaluation

DOI: 10.1016/j.ejmech.2019.01.072

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Article abstract of DOI:10.1016/j.ejmech.2019.01.072

Full text of DOI:10.1016/j.ejmech.2019.01.072

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