Novel hybrids of metronidazole and quinolones: Synthesis, bioactive evaluation, cytotoxicity, preliminary antimicrobial mechanism and effect of metal ions on their transportation by human serum albumin

European Journal of Medicinal Chemistry 86 (2014) 318e334

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Novel hybrids of metronidazole and quinolones: Synthesis, bioactive evaluation, cytotoxicity, preliminary antimicrobial mechanism and effect of metal ions on their transportation by human serum albumin Sheng-Feng Cui, Li-Ping Peng, Hui-Zhen Zhang, Syed Rasheed 1, Kannekanti Vijaya Kumar 2, Cheng-He Zhou* Institute of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People's Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 March 2014 Received in revised form 22 August 2014 Accepted 23 August 2014 Available online 24 August 2014

A novel series of hybrids of metronidazole and quinolones as antimicrobial agents were designed and synthesized. Most prepared compounds exhibited good or even stronger antimicrobial activities in comparison with reference drugs. Furthermore, these highly active metronidazoleequinolone hybrids showed appropriate ranges of pKa, log P and aqueous solubility to pharmacokinetic behaviors and no obvious toxicity to A549 and human hepatocyte LO2 cells. Their competitive interactions with metal ions to HSA revealed that the participation of Mg2þ ion in compound 7deHSA association could result in a concentration increase of free compound 7d. Molecular modeling and experimental investigation of compound 7d with DNA suggested that possible antibacterial mechanism might be in relation with multiple binding sites between bioactive molecules and topo IVeDNA complex. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Quinolone Metronidazole Imidazole DNA Antibacterial HSA

1. Introduction Quinolones are an important kind of synthetic antibacterial agents as they are generally well tolerated with excellent safety profile, favorable pharmacokinetic characteristics, broad antibacterial spectrum and good treatment effectiveness [1e3]. Since the first generation of quinolones were discovered in 1960s, four generations of quinolones have been successfully developed in succession and a large number of quinolone drugs, such as norfloxacin, moxifloxacin, ofloxacin, temafloxacin, have been successfully and widely used in clinic to treat genitourinary infections and common respiratory tract pathogens. Furthermore, the antibacterial mechanism of quinolones has been elucidated clearly. These drugs primarily target type II topoisomerases or DNA gyrase in Gramnegative bacteria to stabilize the cleavage complex at specific sites on DNA, then block DNA replication and finally lead to a lethal lesion [4e5]. However, in recent years the emergence and spread of * Corresponding author. E-mail addresses: [email protected], [email protected] (C.-H. Zhou). 1 Postdoctoral fellow from Department of Chemistry, University of Hyderabad, India. 2 Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India. http://dx.doi.org/10.1016/j.ejmech.2014.08.063 0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

genitourinary infections, which have evolved mechanisms of resistance to quinolones, are becoming one of the most paramount public health threats. A growing medical concern on mechanisms of resistance to quinolones has drawn much attention, and much effort has been focusing on this topic [6]. Some researches [7] show that the resistance to quinolones is principally due to mutations in either ParC breakage-reunion or ParE TOPRIM domains of topo IV (topoisomerase IV) and in the analogous gyrase domains [8]. Further structure activity relationship (SAR) reveals that the groups at N-1 position of quinolones are in close proximity to the conserved ParC helix a4 residues Ser79 and Asp83 in the topo IVeDNA complex, whose mutation causes quinolone resistance in pneumococci and other bacteria [9]. Therefore, the structural modification of quinolones at N-1 position is a promising strategy to overcome the quinolone resistance. Many kinds of functional groups such as ethyl, cyclopropyl and various heterocyclic moieties have been successfully introduced into N-1 position, and produced many effective clinical quinolone drugs [10]. In recent years, azole heterocyclic compounds have been paid special attention due to their potential applications as medicinal agents [11e12], and an increasing effort has been directing towards their possible medicinal potentiality [13e16]. Particularly, the

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nitro-containing imidazoles are found to be a kind of important imidazole antimicrobial derivatives, and many of them such as benznidazole, secnidazole, metronidazole and ornidazole have been extensively used in clinic to treat anaerobic infections [17]. Especially the structurally simple metronidazole as an effective synthetic drug introduced in 1960 possesses strong inhibitory efficacies against Gram-negative anaerobic bacteria like Helicobacter pylori and protozoa such as Giardia, Lamblia and Entomoeba histolytic [18]. These outstanding achievements encourage numerous researchers to focus on their research and development in nitroimidazoles with potential medicinal application. The mechanism researches indicate that the nitro fragment of metronidazole plays a significant role through the metabolic activation in exerting biological activities [19]. Furthermore, a lot of researches disclose that the reactive metabolic intermediates formed in microorganisms by reducing nitro group in nitroimidazoles can covalently bind with DNA and trigger the adverse effect [20]. The sterical protection of nitro group in metronidazole has been proved to be an effective strategy to improve the metabolism and physicochemical property of such compounds [21]. Moreover, the incidence of resistance in anaerobic bacteria is still very low. Therefore, the structural modification of metronidazole has been an increasingly attractive topic. Our previous work has reported that the incorporation of nitroimidazoles into berberine not only enhance the antimicrobial activities, but also broaden antimicrobial spectrum. This type of hybrids of berberine and nitroimidazoles exhibited anti-Gramnegative efficacies with low MIC values ranging from 4 to 8 mg/ mL. They were also proven to effectively bind with biomacromolecules like DNA and Human Serum Albumin (HSA) [22]. In view of the above considerations and as an extension of our previous work [23e28], herein we introduced the nitroimidazole moieties into N-1 position of quinolones to generate a novel series of hybrids of metronidazole and quinolone. Rational design and relevant synthons were shown in Fig. 1. The nitroimidazole groups at N-1 position of quinolones were expected to exert noncovalent interactions with DNA base, ParC breakage-reunion or ParE TOPRIM domains of topo IVeDNA complex to overcome the resistance and broaden the antimicrobial spectrum. Therefore, the antimicrobial

319

activities for all newly synthesized compounds were evaluated in vitro against nine bacteria including Methicillin-Resistant Staphylococcus aureus N315 (MRSA) and five fungi [29]. Cytotoxicity, ionization constants (pKa), aqueous solubility and partition coefficients of some highly active target compounds were also evaluated to predict their pharmacokinetics behaviors. The effects of metal ions on their transportation by HSA were also investigated in order to preliminarily evaluate their transportation, distribution, and metabolism by fluorescence and UVevis absorption spectroscopy on molecular level. Furthermore, flexible ligandereceptor docking investigation was undertaken to rationalize the antibacterial activity and understand the possible mechanism of the hybrids. The comparative study of preliminary interaction between compound 8d and norfloxacin separately with calf thymus DNA were also evaluated to further predict the binding ability between these hybrids and topo IVeDNA complex. 2. Results and discussion 2.1. Chemistry The target hybrids were synthesized according to the synthetic route outlined in Scheme 1. Commercial ethoxymethylene malonic ester (EMME) was reacted with a series of substituted phenylamines in ethanol to afford intermediates 1aeh in almost quantitative yields. The obtained compounds were then cyclized in phenoxybenzene under reflux to produce the desired quinolones 2aeh in good yields ranging from 49.0% to 60.0%, which were further N-alkylated by commercial racemic 2-(chloromethyl)oxirane to yield ethyl 1-(oxiran-2-ylmethyl) quinoline-3-carboxylate derivatives 3aeh in high yields (81.2e95.0%). The epoxy rings of compounds 3aeh were opened by 4-nitroimidazole and 2-methyl5-nitroimidazole respectively in ethanol using sodium bicarbonate as base to produce the corresponding racemates 4aeh and 5aeh in satisfactory yields ranging from 80.2% to 98.5%, and then the latter were further hydrolyzed by 3% sodium hydroxide at 100  C to afford the target quinoloneemetronidazole hybrids 6aeh and 7aeh in excellent yields. In the preparation of compounds 3aeh, it was found that the solvents and base exerted remarkable effect on their yields. In particular, 2-(chloromethyl)oxirane as both solvent and reactant led to relatively high yield in contrast to other common solvents such as ethanol, methanol and acetonitrile. It was found that suitable base in the synthesis of intermediates 3aeh was potassium carbonate, since other bases resulted in the low yields of target compounds, and formation of various by-products was observed. 2.2. Analysis of spectra All the new compounds were characterized by IR, 1H NMR, 13C NMR, MS and HRMS spectra. Their spectral analyses were consistent with the assigned structures and listed in the experimental section. The mass spectra for new compounds gave a major fragment of [MþH]þ or [MþNa]þ according to their molecular formula.

Fig. 1. Design of novel target hybrids of metronidazole and quinolones.

2.2.1. 1H NMR spectra In 1H NMR spectra, it was noticed that the protons of hydroxyl group and tertiary carbon in compounds 4e7 appeared as multiplet peaks at d 5.20e5.95 ppm and d 4.67e4.87 ppm respectively due to the adjacency of chiral tertiary carbon and the electronwithdrawing character of quinolone backbones and imidazole rings. For compounds 3aeh, because of the effect of electronwithdrawing quinolones and epoxy rings, the protons of CH2 linked to N-1 position gave large downfield chemical shifts at d 4.40e5.04 ppm. The CH proton on the epoxy ring exhibited a

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Scheme 1. Synthetic route of hybrids of metronidazole and quinolones. a Reagents and conditions: (i) ethoxymethylene malonic ester, ethanol, reflux, 2 h; (ii) phenoxybenzene, reflux, 1 h; (iii) 2-(chloromethyl)oxirane, K2CO3, 120  C, 3 h; (iv) 4-nitroimidazole, K2CO3, ethanol, reflux, 2 h; (v) 2-methyl-5-nitroimidazole, K2CO3, ethanol, reflux, 2 h; (vi) 3% NaOH, 100  C, 2 h.

multiplet signal at d 3.35e3.46 ppm in DMSO-d6 because of the chiral carbon and the presence of strong electrophilic groups, and the same reasons resulted in large downfield chemical shifts (2.54e2.86 ppm) for the CH2 protons on the epoxy ring. 2.2.2. 13C NMR spectra The 13C NMR spectral analyses were consistent with the assigned structures. No large differences were found in 13C chemical shifts for C-2, C-3, C-4 position and quaternary carbon of quinolone backbones. The chemical shifts of the tertiary carbon followed a decreased order: epoxy quinolone hydrochloride (68.1e68.9 ppm) > 4-nitroimidazole derivatives (d 60.2e60.8 ppm) z 2-methyl-5-nitroimidazole derivatives (d 60.6e60.8 ppm) > epoxy quinolone derivatives (d 57.2e57.7 ppm). Additionally, all the other aromatic and aliphatic carbons also appeared at the appropriate chemical shift regions. 2.3. Biological activity 2.3.1. Antibacterial activity The antibacterial results revealed that most of the prepared compounds could effectively inhibit the growth of all the tested bacteria in vitro. The synthesized intermediates 3aeh with oxiran2-ylmethyl group at N-1 position of quinolone showed moderate activities against the tested bacterial strains (Supporting Information). Moreover, the E. typhosa strain was quite sensitive to the synthetic intermediates 3aeh with MIC values of 1e8 mg/mL, which were more effective than standard drug chloromycin (MIC ¼ 32 mg/mL) and almost equipotent to norfloxacin (MIC ¼ 4 mg/mL). It was worthwhile to note that 6,8-dichloro-1(oxiran-2-ylmethyl) quinolone 3f and 8-fluoro-1-(oxiran-2ylmethyl) quinolone 3g exhibited significant activity against most of the tested Gram-positive strains except for M. luteus and S. dysenteriae.

For the tested compounds 4aeh and 5aeh bearing nitroimidazole fragment (Supporting Information), most of them showed better antibacterial activities than intermediates 3aeh, and displayed more potent against Gram-negative bacteria than Grampositive bacteria. 4-Nitroimidazolyl quinolones 4aeb and 2methyl-5-nitroimidazolyl compounds 5aeb and 5d exhibited good activities against the tested Gram-negative bacteria including P. aeruginosa, E. coli DH52, S. dysenteriae and E. typhosa strains with low MIC values in the range of 4e16 mg/mL. Notably, compound 5d with trifluoromethyl group at 7-position of quinolone ring gave stronger antimicrobial efficacy and broad antimicrobial spectrum in comparison with norfloxacin and chloromycin, which was quite sensitive to all the tested bacterial strains except for B. subtilis with MIC values of 2e8 mg/mL. However, compared to 2-methyl-5nitroimidazolyl quinolone 5d, the corresponding 4nitroimidazolyl derivative 4d gave low inhibitory concentrations to these tested bacterial strains especially towards P. aeruginosa, E. coli DH52, S. dysenteriae and E. typhosa (MIC ¼ 16e128 mg/mL). Furthermore, except for 6,8-difluoro quinolone 4a and 6,8-dichloro quinolone 4f and 5f, all the other target compounds could effectively inhibit the growth of MRSA at the concentrations of 2e16 mg/ mL, which were more effective than clinical drug chloromycin (MIC ¼ 16 mg/mL). It was also observed (Table 1) that metronidazoleequinolone hybrids 6aeh and 7aeh displayed stronger antibacterial efficacy than their precursor carboxylic esters 4aeh and 5aeh. The hydrolysis of carboxylic esters, which produced the corresponding carboxylic acids, could enhance antimicrobial efficiency and broaden the antimicrobial spectrum. As shown in Fig. 2, compound 7b exhibited better activities against M. luteus, MRSA, P. aeruginosa and E. coli DH52 than its precursor 5b. Compound 7d in vitro with MIC value of 1 mg/mL against MRSA was eight fold more active than compound 5d. Furthermore, compound 6aeh and 7aeh showed moderate to good anti-S. pneumoniae activity with MIC values from

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Table 1 In vitro antibacterial activities for compounds 6e7 expressed as MIC (mg/mL).a,b,c Compds

6a 6b 6c 6d 6e 6f 6g 6h 7a 7b 7c 7d 7e 7f 7g 7h A B

Gram-positive bacteria

Gram-negative bacteria

S. pneumoniae

M. luteus

MRSA

S. aureus

B. subtilis

P. aeruginosa

E. coli DH52

S. dysenteriae

E. typhosa

16 32 8 16 16 8 32 64 16 4 8 4 8 16 32 64 16 1

8 8 16 8 8 8 4 256 2 1 2 1 64 16 4 512 8 2

16 8 8 4 8 16 4 16 2 4 2 1 4 32 4 8 16 8

8 8 16 4 16 8 16 64 16 8 8 4 32 16 8 32 16 0.5

8 16 16 4 8 4 4 16 4 8 16 4 8 8 16 32 8 1

4 4 8 8 4 8 8 128 2 2 4 4 16 8 2 512 32 1

4 4 8 2 32 8 4 64 4 1 8 0.5 2 32 4 64 32 16

8 16 8 4 16 8 8 16 4 8 1 0.5 16 16 8 32 32 4

2 4 8 4 2 16 32 32 16 8 32 8 128 32 4 8 32 4

a

Minimum inhibitory concentrations were determined by micro broth dilution method for microdilution plates. A ¼ chloromycin, B ¼ norfloxacin. c S. pneumoniae, Streptococcus pneumoniae; M. luteus, Micrococcus luteus ATCC 4698; MRSA, Methicillin-Resistant Staphylococcus aureus N315; S. aureus, Staphylococcus aureus ATCC 25923; B. subtilis, Bacillus subtilis ATCC 6633; P. aeruginosa, Pseudomonas aeruginosa; E. Coli DH52, Escherichia coli; S. dysenteriae, Shigella dysenteriae; E. typhosa, Eberthella typhosa. b

2.3.2. Antifungal activity The in vitro antifungal evaluation (Supporting Information) revealed that the intermediates 3aeh with oxiran-2-ylmethyl group at N-1 position of quinolones exhibited better antifungal activities against most of the tested fungal strains than these hybrids of metronidazole and quinolone. Most of the prepared hybrids with MIC values of 64e512 mg/mL showed moderate to bad antifungal activities against the tested fungal strains. The substitution of metronidazole group in quinolones caused remarkable decrease or total loss of antifungal properties. However, hybrids 6b (MIC ¼ 1, 4 mg/mL) and 7b (MIC ¼ 2, 2 mg/mL) could effectively

16

Compound 5b Compound 7b Compound 5d Compound 7d

14 12

M.luteus

B.subtilis

10

MIC / µg/mL

4 to 64 mg/mL. Especially, compound 7b and 7d (MIC ¼ 4 mg/mL) exhibited much better inhibitory potency against S. pneumoniae strain than chloromycin (MIC ¼ 16 mg/mL). Compound 7d possessed stronger antimicrobial activities and broader antimicrobial spectrum than other target compounds, which was four fold more active against M. luteus, B. subtilis, E. coli DH52 and S. dysenteriae in comparison with its precursor 5d. The above results suggested that the antimicrobial efficacies should be related to substituents on benzene ring of quinolones and nitroimidazole ring to some extent. Among this series of hybrids, compounds with fluoro substituent on benzene ring exerted the most important influence on antimicrobial activities. 6,8-Difluoro quinolone 7a, 7-trifluoromethyl 7d, 6-fluoro 7c, 7-chloro-6-fluoro 7e and 8-fluoro 7g could effectively inhibit the growth of most tested bacterial strains at the concentrations of 0.5e16 mg/mL, which were more effective than 6,8-dichloro quinolone 7f and 6methoxyl derivative 7c. Especially 7-trifluoromethyl derivative 7d showed significant inhibition against all the tested strains with low inhibitory concentrations, which were comparable or even superior to the reference drugs chloromycin and norfloxacin. To our surprise, 6-methyl quinolones 7b and 6b also exhibited good activity in comparison to standard drugs. Generally, the antibacterial activities of these hybrids followed the order of 2-methyl-5nitroimidazolyl compounds > 4-nitroimidazolyl derivatives. In contrast to 4-nitroimidazolyl compounds 6aeh, the corresponding 2-methyl-5-nitroimidazolyl quinolones 7aeh showed better antibacterial activities against all the tested strains.

S.aureus

P.aeruginosa S.dysenteriae

8 6 4

MRSA

E.typhosa E.coli DH52

2 0

Fig. 2. The comparison of antibacterial data between compounds 5b and 7b, compounds 5d and 7d respectively.

inhibit the growth of A. flavus and B. yeast strains, which gave superior inhibitory potency to the first-line antifungal drug fluconazole (MIC ¼ 256, 16 mg/mL). 1-(Oxiran-2-ylmethyl)-quinolones 3aeh showed efficient inhibition against B. yeast, C. albicans, and C. mycoderma. The fungal strain of A. flavus was more sensitive to compounds 3aeh with quite low MIC values (MIC ¼ 0.5e16 mg/mL) than fluconazole (MIC ¼ 256 mg/mL). Especially the activities of compounds 3d, 3f, and 3g against A. flavus strain were 512-fold more potent than reference drug fluconazole. Moreover, all the quinolone intermediates 3aeh possessed moderate to good antifungal activities against B. yeast, C. albicans and C. mycoderma, which were comparable or even better in comparison with the reference drug fluconazole.

2.3.3. Cell toxicity The highly bioactive hybrids 6b, 7b and 7d were further examined for their cytotoxic properties on A549 cell line and

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normal human hepatocyte LO2 by means of CKK-8 Kit and MTT respectively, and the absorbance of viable cells (450 nm and 490 nm) increased linearly as cells proliferate. Compounds 6b, 7b and 7d were dissolved in DMSO to prepare the stock solutions, and then the tested compounds were prepared in medium to obtain the required concentrations of 256, 128, 64, 32, 16 and 8 mg/mL. Cells were cultured for 24 h in these solutions. The cell viability was examined by microplate reader. Cytotoxicity results (Fig. 3) showed that the cell viability of all the three compounds was at least more than 83% within concentration of 256 mg/mL, which indicated all the tested compounds did not affect the viability of A549 cell line within the concentration of 256 mg/mL. Especially, 6-methyl hybrid 7b showed the lowest cell toxicity among the tested compounds. Moreover, with the increase of concentrations of the tested compounds, there were no obviously reducing trends to the cell viability. As shown in Table 2, the toxicity assay suggested nontoxicity for compound 7d on normal human hepatocyte LO2 within the concentration of 128 mg/mL (Inhibition rate < 17.5%). 2.3.4. Analysis of pKa values The ionization constant of drug is an important physicochemical parameter to significantly influence pharmacokinetic behaviors including absorption, distribution, metabolism, and excretion [30e31]. The pKa values are usually used to estimate the extent of ionization of drug molecules at different pH values, which are of fundamental importance in the consideration of their interaction with biological membranes. For instance, compounds with minimally one charged with a pKa <4 for acid and correspondingly a pKa >10 for base will be charged at physiological pH and display slower diffusion rate to across biological membranes such as the bloodebrain barrier [32]. The hybrids of metronidazole and quinolone should have two pKa values, one for the proton on the nitrogen of metronidazole and the other one for the carboxyl hydrogen of quinolone ring. Because the metronidazole groups and quinolone ring in these hybrid molecules do not possess one large pep conjugated system, the changes of absorbance in different pH buffer solutions should be two relatively independent processes and take place on different maximum absorption wavelength respectively, which followed the reaction formulas (Fig. 4).

Compound 7d Compound 6d Compound 7b

120

Table 2 The inhibition rate of target compound 7d on human hepatocyte LO2 tested by MTT method, each data represents an average of three parallels. Concentration (mg/mL)

OD values

0 8 16 32 64 128

0.732 0.706 0.683 0.657 0.621 0.604

± ± ± ± ± ±

0.132 0.102 0.094 0.096 0.076 0.054

Inhibition rate (%) 0 3.6 6.7 10.3 15.2 17.5

Fig. 4 clearly showed the changes observed in the UV spectrum of compound 7d in different buffer solutions. With the increase of pH from 3.0 to 9.4, the molecular formula changed from I to II, and then to III. As shown in Fig. 5, there were two parts of spectral differences (from 245 to 272 and from 308 to 344 nm), which resulted from the proton on the nitrogen of metronidazole and carboxyl hydrogen of quinolone ring respectively. Herein the spectral differences from 308 to 344 nm were chosen to analyze the pKa values of target hybrids. Data analysis included normalization of the raw scans (Abs450 nm ¼ 0), and then calculated the spectral difference between the acid spectra and the spectra obtained at every other pH (Fig. 6). The wavelengths of maximum positive and negative deviations were determined graphically, and the absolute values of the absorbance difference at the chosen wavelengths were summed. The total absorbance difference was then plotted versus pH (Fig. 7), and the data were fit to Equation (1) to obtain the pKa values.

Absorbance total ¼


 εНА  εА  10ðpHpKaÞ  ½St  1 þ 10ðpHpKaÞ

(1)

Where εHA and εA are the extinction coefficients of the acid and base forms of compound respectively, and [St] is the total compound concentration. When using absorbance differences, the εHA and εA are simply the minima and maxima of the curve. As shown in Table 3, the pKa values of compounds 7aeh displayed an appropriate range (5.257e6.430) to pharmacokinetic behaviors. Since the electron density on the aromatic rings could be reduced by the electron withdrawing groups such as Cl, F and CF3 groups, the pKa values of these compounds decreased accordingly. Meanwhile, due to their electron donating ability of methyl and methoxyl groups, compounds 7b and 7h could destabilize anionic form, therefore decrease the acidic character and show high pKa values.

Cell viability (%)

100

80

60

40

20

0

01

82

3 16

4 32

5 64

6 128

7 256

Concentration of compounds (μg/mL) Fig. 3. Cytotoxic assay of target compounds 6d, 7b and 7d on A549 tested by CCK-8 Kit. Each data bar represents an average of three parallels, and error bars indicate one standard deviation from the mean.

2.3.5. Analysis of partition coefficient and solubility Physical characteristics of drugs such as lipophilicity, hydrophilicity and solubility have been suggested to play a decisional role in determining where drugs are distributed and how rapidly they are metabolized and excreted in the body. The ideal distribution coefficient is a moderate intermediate between hydrophilic and hydrophobic to evaluate the pharmacokinetic and pharmacodynamic properties. The partition coefficient (log P) is frequently used to define the lipophilic character of a drug. Therefore, the lipophilicity/hydrophilicity expressed as octanol/water partition coefficient was determined experimentally by traditional saturation shake flask method combining with UVevis spectrophotometric approach (Supporting Information). As given in Table 4, the tested compounds had suitable partition coefficient that was favorable to permeate through biological membrane and to be delivered to the binding sites. Moreover, for the tested compound 7aeh, most of them were hydrophilic. Especially, compound 7d, as expected, was

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323

Fig. 4. Structural transposition of compound 7d in different buffer solution from 3.0 to 9.4.

0.5

Normalized data pH

0.3

0.2

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5

7.0 7.5 8.0 8.5 8.8 9.1 9.4

0.1

0.0 270

300

330

360

390

420

Wavelength (nm) Fig. 5. UV spectra (l ¼ 258e470 nm) of compound 7d in different aqueous buffer solutions ranging from pH 3.0 to 9.4. The absorbances are normalized to zero for l ¼ 460 nm.

Special difference

0.30

pH

0.15

7.0 7.5 8.0 8.5 8.8 9.1 9.4

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5

2.3.6. The effect of metal ions on the transportation of compound 7d by HSA It is well known that HSA is a kind of important and principal extracellular protein in circulatory system, which is closely related to the overall absorption, transportation, distribution, metabolism of drugs and has an immense impact on the efficacy of drugs in human body [33]. Therefore, the investigations of interactions between bioactive small molecules and HSA are not only beneficial to provide a proper understanding of the pharmacokinetic properties

4

0.00

Spectral difference plot -0.15

250

300

350

400

Wavelength (nm) Fig. 6. Plot of the spectral difference between different solutions of compound 7d. The maximum positive deviation occurred at 344 nm while the maximum negative deviation occurred at 308 nm.

the most hydrophilic one because of the trifluoromethyl group on 7-position of quinolone. However, because the chloro and methoxyl groups on benzene ring of quinolones might increase the lipophilicity of compounds, compounds 7f and 7h with these groups gave considerably larger lipophilicity in contrast to other compounds. Based on the results of antibacterial activities and log P

Aborbance difference

Absorbance

0.4

values, it was suggested that the target compounds with suitable log P values between 0.25 and 0.18, such as compounds 7b, 7d and 7e, showed better inhibition activities than others. Aqueous solubility is also a governing property for how small molecules interact with bimolecular and living systems, which plays a critical role in every stage of drug discovery and development. Incorrect estimation of solubility can lead to erroneous interpretation of in vitro assay results and weaken structureeactivity relationship analysis. Thereby the aqueous solubility of target compounds was also given in this work (Table 4). In general, all the tested compounds showed good aqueous solubility ranging from 0.353 to 0.792 mg/mL. These obtained results were almost consistent with the antibacterial activities. The compounds with good aqueous solubility showed low inhibitory concentrations against bacterial strains. For example, due to the hydrogen bonds between water and fluoro and imidazole groups, compounds 7a (0.57 mg/mL) and 7d (0.79 mg/mL) gave good aqueous solubility. Meanwhile both of them exhibited significant antibacterial activities.

pKa Determination

3

2

1

0 3.0

4.5

6.0

pH

7.5

9.0

Fig. 7. The total absorbance difference (the sum of the absolute absorbance difference values at 244 and 308 nm at each pH) was plotted against solution pH. The data was then fit to equation.

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compound 7d in the blood, and thus improved its antimicrobial efficacy [39]. The altered binding constants might be explained by the conformational changes of HSA structure caused by binding to metal ions or interaction between compound 7d and metal ions, which in turn affected protein binding to drugs.

Table 3 The pKa values of compounds 7aeh.a Compds 7a

7b

7c

7d

7e

7f

7g

7h

l (nm) 308/

308/ 342 6.430 0.990

308/ 343 5.179 0.990

308/ 344 5.599 0.994

308/ 344 5.145 0.991

308/ 344 5.257 0.992

308/ 343 5.121 0.991

308/ 344 5.718 0.994

pKa Ra a

344 5.326 0.991

3. Molecular modeling

R is the correlation coefficient.

of drugs, but also instructive to the design, modification and screening of drug molecules [34]. Furthermore, some metal ions in plasma, when drugs bind reversibly to HSA and are delivered to the binding sites, can have an affect on the transposition ability of HSA. The 4-oxo and 3-carboxyl groups of quinolones endow them with excellent chelating properties with metal ions [35]. These chelating abilities can not only influence the in vivo antimicrobial activity, but also interfere with their pharmacokinetic properties when co-administration of other drugs and/or some food that contain metal ions [36]. So it is considerably reasonable for us to further investigate the effect of metal ions including major elements like Ca2þ, Kþ, Mg2þ and Ni2þ ions on the binding constant of these compoundeHSA complex by fluorescence and UVevis absorption spectroscopy on molecular level. Fluorescence quenching is considered as an effective approach to investigate the transportation ability of HSA to small molecules. Much literature discloses that the emission of Trp 214 (the single tryptophan of HSA at residue 214) may be blue shifted if the group is buried within a native protein, and its emission may shift to longer wavelengths (red shift) when protein is unfolded [37,38]. The effect of compound 7d on HSA fluorescence intensity at 286 K was shown in Fig. 8. Evidently, a progressive decrease in the fluorescence intensity was caused by quenching, accompanied with the blue shift wavelength (from 345.0 to 356.0 nm) in the albumin spectrum. These suggested an increased hydrophobicity of the region surrounding the single Try-214 residue. The numbers of binding sites and the equilibrium binding constants can also be calculated according to the Scatchard Equation (2). The result showed the compound 7d gave suitable binding constant (Fig. 9, K ¼ 5.15  104 L/mol)

. r Df ¼ nKb  rKb

(2)

Where Df is the molar concentration of free small molecules, r is the moles of small molecules bound per mole of protein, n is binding sites multiplicity per class of binding sites, and Kb is the equilibrium binding constant. The Scatchard plots and the Kb are listed in Table 5 The effect of metal ions on the transportation of compound 7d by HSA was evaluated by Kb/K0. As shown in Table 5, the values of Kb/K0 were tested ranging from 0.91 to 1.51, which indicated the metal ions including Ca2þ, Kþ, Mg2þ and Ni2þ ions had an obvious effect on the transportation by HSA. Clearly, the value of Kb/K0 in the presence of Ni2þ ion was approximated to 1.00, which suggested the Ni2þ ion caused little impact on the transportation of compound 7d by HSA. However the presence of metal ions such as Ca2þ and Kþ ones considerably increased the binding constants of compound 7deHSA complex (Kb/K0 ¼ 1.43, 1.51), thus causing compound 7d to be slowly cleared from blood, which may lead to more doses of compound 7d to achieve the desired therapeutic effectiveness (Fig. 10). While the participation of Mg2þ ion reduced the binding constants of compound 7deHSA complex, suggesting the presence of these metal ions could decreased the concentration of free compound 7d, shorten the storage time and half-life of

To further rationalize the observed antibacterial activity and understand the possible mechanism of the hybrids, a flexible ligand receptor docking investigation was undertaken. The crystal structure data (Streptococcus pneumoniae topoisomerase IVeDNA complexes) was obtained from the protein data bank [40], which was a representative protein to investigate the antibacterial mechanism of quinolones [41]. Target compounds 6aeh and 7aeh were selected to dock with the topo IVeDNA complex. According to the docking results, 7-trifluoromethyl hybrid of metronidazole and quinolone 7d possessed the best score (5.800) and lowest energy (6.093) among these target compounds. It was also observed that 2-methyl-5-nitroimidazolyl quinolones 7aeh got higher scores than 4-nitroimidazolyl quinolones 6aeh. Furthermore, the substituents on benzene ring of quinolone could affect the scores of docking results, which followed the order of 7-trifluoromethyl quinolones > 6-fluoro-7-chloro derivatives >6,8-difluoro derivatives > 6-fluoro derivatives. These docking results of the target hybrids were almost in accordance with the observed in vivo antibacterial activities except for 6-methyl hybrids. As shown in Fig. 11, the docking result of compound 7d and topo IVeDNA complex might rationalize the possible antibacterial mechanism. The hydroxyl group and nitrogen atom of imidazole were in close vicinity to the phosphate of the topo IVeDNA phosphodiester bond, which formed two hydrogen bonds with distance of 2.9 Å. Furthermore, the carboxyl and nitro groups of this molecule were also located closest to DNA base and interacted through hydrogen bonds with distance of 2.5 Å and 2.6 Å respectively. These noncovalent bonds could interact with DNA base and might block DNA replication. Moreover, the nitro and oxygen atoms at nitro group of imidazole were in close proximity to conserve ParC helix a4 residues Ser79 (with closest distances to the side chain atoms of 3.1 Å and 3.2 Å respectively), whose mutation caused quinolone resistance in pneumococci and other bacteria. Therefore these hydrogen bonds at resistance mutation region might be the important reason that compound 7d gave strong inhibitory efficacy against strains of quinolone-resistant bacteria such as MRSA. 4. The interaction with DNA Based on the results of molecular modeling, the incorporating of metronidazole might improve the interaction between target compounds and DNA of topoisomerase IVeDNA complexes. So the interaction between the strongest active hybrid 7d and calf thymus deoxyribonucleic acid (DNA) was evaluated to further explore preliminary antimicrobial mechanism. In contrast to this interaction between compound 7d and DNA, the reference drug norfloxacin was employed to investigate the interaction with calf thymus DNA. The binding studies on molecular level were carried out by UVevis spectroscopic method. In absorption spectroscopy, hypochromism and hyperchromism were very important spectral features to distinguish the change of DNA double-helical structure [42]. With a fixed concentration of DNA, UVevis absorption spectra were recorded as increasing amount of compound 7d. As shown in Fig. 12, UVevis spectra displayed that the maximum absorption peak of DNA at 260 nm exhibited proportional increase and slight blue shift along with the increasing concentration of

S.-F. Cui et al. / European Journal of Medicinal Chemistry 86 (2014) 318e334 Table 4 Log P values and solubility of compounds 7aeh. Compds

7a

7b

7c

7d

7e

7f

7g

7h

Log P 0.21 0.03 0.13 0.25 0.18 0.22 0.13 0.24 Solubility (mg/mL) 0.57 0.39 0.38 0.79 0.43 0.35 0.39 0.40

2000

Fluorescence intensity

HSA 1500

a

k

1000

325

compound 7d. Meanwhile, the phenomenon, that the absorption value of simply sum of free DNA and free compound 7d was less than the measured value of DNAecompound 7d complex, was observed in the inset of Fig. 12. The interesting observed hyperchromism accompanied by a slight blue shift (7 nm) suggested a conformational change in the DNA duplex. Furthermore, it was noticed that this hyperchromism was becoming increasingly strong along with the increasing concentration of compound 7d. This change was characteristic of non-covalent interactions between the complexes and DNA resulting in some uncoiling of the DNA helix and the exposure of some previously embedded DNA bases. The observed spectral effects could be explained by the electrostatic interaction of the complexes with DNA. Based on the variations in these spectra, Equation (3) could be used to calculate the intrinsic binding constant (K).

A0 xC xC 1 ¼ þ  A  A0 xDC  xC xDC  xC K½7d

500 -5

Compound 7d /10 mol /L 0 320

340

360

380

400

Wavelength(nm) Fig. 8. Emission spectra of HSA in the presence of various concentrations of compound 7d. c(HSA) ¼ 1.0  105 mol/L; c(compound 7d)/(105 mol/L), aek from 0.0 to 2.0 at increments of 0.20; blue dash line shows the emission spectrum of compound 7d only; T ¼ 286 K, lex ¼ 295 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(3)

A0 and A represent the absorbance of DNA in the absence and presence of compound 7d at 260 nm, xC and xDeC were the absorption coefficients of compound 7d and DNAecompound 7d complex respectively. The plot of A0/(A  A0) versus 1/[compound 7d] was constructed by using the absorption titration data and linear fitting, yielding the binding constant, K ¼ 1.434  104 L/mol, R ¼ 0.9997, SD ¼ 0.12 (R is the correlation coefficient. SD is standard deviation). As shown in Fig. 13, the similar hyperchromism of DNAenorfloxacin complex was also observed. However, there were some differences between norfloxacin and compound 7d. With the gradually increasing concentration of compound 7d or norfloxacin, the DNAecompound 7d complex showed stronger hyperchromism than DNAenorfloxacin complex. These suggested that the new metronidazoleequinolone hybrids have better interaction with DNA. Therefore, this type of interaction with DNA might improve binding ability between these hybrids and topo IVeDNA complex.

2+

HSA+Ca

2000

Fig. 9. Emission spectra of HSA in the presence of various concentrations of compound 7d. c(HSA) ¼ 1.0  105 mol/L; c(compound 7d)/(105 mol/L), aek from 0.0 to 2.0 at increments of 0.20; blue dash line shows the emission spectrum of compound 7d only; T ¼ 286 K, lex ¼ 295 nm. R is the correlation coefficient. S.D. is standard deviation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 5 Effect of metal ions on the binding constants of compound 7deHSA complex. 4

Systems HSAecompound HSAecompound HSAecompound HSAecompound HSAecompound

7d 7deCa2þ 7deNi2þ 7deMg2þ 7deKþ

10 Kb (L/mol)

Kb/K0

5.15 3.61 5.07 5.63 3.42

1.00 1.43 1.02 0.91 1.51

a

R

b

0.991 0.990 0.991 0.990 0.995

Fluorescence intensentiy

HSA 1500

a

1000

k 500

Compound 7d only 0 320

340

360

380

400

Wavelength (nm) Fig. 10. Emission spectra of HSA þ Ca2þ in the presence of various concentrations of compound 7d. c(HSA) 1.0  105 M; c(Ca2þ)/c(HSA) ¼ 1/1; c(compound 7d)/(105 M), aek: from 0.0 to 2.0 at increments of 0.2; dash line shows the emission spectrum of compound 7d only; T ¼ 298 K, lex ¼ 295 nm. The inset corresponds to the Scatchard plot.

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preliminary interactive investigations of compound 7d with calf thymus DNA revealed that these hybrids gave better interaction with DNA than norfloxacin, which might improve binding ability between these hybrids with topo IVeDNA complex. 6. Experimental 6.1. General methods

Fig. 11. Three-dimensional conformations of compound 7d docked in topoisomerase IVeDNA complexes.

5. Conclusion In conclusion, starting from commercially available materials, a novel class of hybrids of metronidazole and quinolone were designed and synthesized in good yields by a convenient and efficient procedure. All the new compounds were characterized by IR, 1 H NMR, 13C NMR, MS, and HRMS spectra. The in vitro antimicrobial evaluation against nine bacterial and five fungal strains revealed that most of the newly synthesized compounds exhibited good antibacterial and antifungal activities against the tested strains in comparison with the reference drugs chloromycin, norfloxacin and fluconazole. The prepared hybrids 7b and 7d showed significant inhibition against all the tested bacterial strains with low inhibitory concentrations (MIC ¼ 0.5e8 mg/mL). Especially, nitroimidazole derivatives 7aed and 7g showed comparable or superior potency against MRSA to reference drugs. Furthermore, 1-(oxiran-2ylmethyl)-quinolones 3aeh exhibited better antifungal activities against most of the tested fungal strains including A. flavus, B. yeast, C. albicans and C. mycoderma than fluconazole. Structure activity relationship indicated that compounds with fluoro substituent on benzene ring exerted important effect on antimicrobial activities. Moreover, the antibacterial activities of these hybrids were also affected by the substituents on N-1 position of quinolone, which followed the order of 2-methyl-5-nitroimidazolyl derivatives > 4nitroimidazolyl derivatives. Cell toxicity suggested most of the target compounds did not show obvious cytotoxicity to A459 and human hepatocyte LO2 cell lines. The pKa values of target hybrids were also evaluated ranging from 5.145 to 6.430, which showed appropriate range to pharmacokinetic behaviors. The target compounds also gave suitable log P values ranging from 0.25 to 0.22 and good aqueous solubility (0.35e0.79 mg/mL). The metal ions’ influence on their transportation by HSA revealed that the participation of Mg2þ ion in compound 7deHSA association could result in the concentration increase of free compound 7d, shorten the storage time and half-life of compound 7d in the blood, and thus may improve its antimicrobial efficacy. Further molecular modeling indicated the good antibacterial activity of the prepared hybrids against quinolone-resistant bacterial strains and the possible mechanism might be explained by the noncovalent interaction between molecular 7d and topo IVeDNA complex, especially these hydrogen bonds between molecule and ParC helix a4 residues Ser79 (resistance mutation region of topo IVeDNA complex). The

Melting points were recorded on X-6 melting point apparatus and uncorrected. TLC analysis was done using pre-coated silica gel plates. FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer (Bio-Rad, Cambridge, MA, USA) using KBr pellets in the 400e4000 cm1 range. NMR spectra were recorded on a Bruker AV 300 spectrometer using TMS as an internal standard. The chemical shifts were reported in parts per million (ppm), the coupling constants (J) were expressed in hertz (Hz) and signals were described as singlet (s), doublet (d), triplet (t), as well as multiplet (m). The mass spectra were recorded on LCMS-2010A and the high-resolution mass spectra (HRMS) were recorded on an IonSpec FT-ICR mass spectrometer with ESI resource. All fluorescence spectra were recorded on F-7000 Spectrofluorimeter (Hitachi, Tokyo, Japan) equipped with 1.0 cm quartz cells, the widths of both the excitation and emission slit were set as 2.5 nm, and the excitation wavelength was 295 nm. The UV spectrum was recorded at room temperature on a TU-2450 spectrophotometer (Puxi Analytic Instrument Ltd. of Beijing, China) equipped with 1.0 cm quartz cells. HSA, CKK kit and MTT was obtained from SigmaeAldrich (St. Louis, MO, USA). Tris, NaCl and HCl were analytical purity. HSA was dissolved in TriseHCl buffer solution (0.05 M Tris, 0.15 M NaCl, pH ¼ 7.4). Sample masses were weighed on a microbalance with a resolution of 0.1 mg. All other chemicals and solvents were commercially available, and were used without further purification. 6.1.1. General procedures for the preparation of intermediates (1aeh) and (2aeh) Compounds 1aeh and 2aeh were prepared according to the literature procedures [43]. 6.1.2. Synthesis of ethyl 6,8-difluoro-1-(oxiran-2-ylmethyl)-4-oxo1,4-dihydroquinoline-3-carboxylate (3a) A mixture of compound 2a (0.253 g, 1.0 mmol) and potassium carbonate (0.151 g, 1.2 mmol) was stirred under reflux in 2(chloromethyl)oxirane (15 mL) for 3 h. When the reaction was completed (monitored by TLC, eluent, chloroform/methanol 70/1, V/V), the mixture was cooled to room temperature, and then the excess 2-(chloromethyl)oxirane was evaporated under reduced pressure. After water was added, the resulting mixture was extracted with chloroform (3  20 mL). The combined organic phase was dried over anhydrous Na2SO4, and then the solvent was evaporated. The residue was purified via silica gel column chromatography (eluent, chloroform/methanol ¼ 70/1, V/V) to give compound 3a (0.294 g) as light yellow solid. Yield: 95.1%; m.p: >250  C. IR (KBr, cm1) n: 3131 (AreH), 3016 (]CeH), 2988, 2902 (CH2, CH3), 1683, 1641 (C]O), 1609, 1559, 1493 (aromatic frame); 1 H NMR (300 MHz, DMSO-d6): d 8.51 (s, quinolone-2-H, 1H), 7.84e7.93 (m, quinolone-5-H, 1H), 7.79e7.81 (m, quinolone-7-H, 1H), 4.85e4.90 (m, NeCH2, 1H), 4.45e4.50 (m, NeCH2, 1H), 4.26 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.46 (s, OeCH, 1H), 2.54e2.55 (m, OeCH2, 2H), 1.29 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 171.0, 164.5, 160.2, 157.0, 154.6, 152.2, 151.3, 131.9, 130.3, 110.3, 60.4, 57.2, 50.3, 44.9, 14.7 ppm; MS (ESI): m/z 310 [MþH]þ; HRMS (ESI) calcd. for C15H13F2NO4 [MþH]þ, 310.0813; found, 310.0811.

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0.6

Absorbance

f 0.4

Absorbance

0.5

120.1, 110.5, 111.1, 61.5, 57.4, 50.6, 45.1, 22.1, 14.7 ppm; MS (ESI): m/z 288 [MþH]þ; HRMS (ESI) calcd. for C16H17NO4 [MþH]þ, 288.1236; found, 288.1234.

DNA+compound 7d DNA-compound 7d complex

0.40 0.38 0.36 0.34 0.32

0.3

a

4

8

-6

12

16

20

10 (mol/L)

0.2 DNA

0.1 0.0 250

300

350

Wavelength (nm) Fig. 12. UV absorption spectra of DNA with different concentrations of compound 7d (pH ¼ 7.4, T ¼ 288 K). Inset: comparison of absorption at 260 nm between the DNAecompound 7d complex and the sum values of free DNA and free compound 7d. c(DNA) ¼ 7.61  105 mol/L, and c(compound 7d) ¼ 0e20  106 mol/L for curves aef respectively at increment 4  106.

0.8

DNA + norfloxacin DNA-norfloxacin complex

Absorbance

0.7

0.6

0.5

0.4 4

8

12

16

327

20

-6

10 (mol/L) Fig. 13. Comparison of absorption at 260 nm between the DNAenorfloxacin complex and the sum values of free DNA and free norfloxacin. c(DNA) ¼ 7.61  105 mol/L, and c(norfloxacin) ¼ 0e20  106 mol/L at increment of 4  106.

6.1.3. Synthesis of ethyl 6-methyl-1-(oxiran-2-ylmethyl)-4-oxo-1,4dihydroquinoline-3-carboxylate (3b) Compound 3b was prepared according to the procedure depicted for compound 3a, starting from compound 2b (0.231 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chloromethyl)oxirane (15 mL). The product 3b (0.253 g) was obtained as white solid. Yield: 88.2%; m.p: >250  C; IR (KBr, cm1) n: 3128 (AreH), 3060 (]CeH), 2989, 2905 (CH2, CH3), 1740, 1680 (C]O), 1608, 1555, 1495 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.85 (s, quinolone-2-H, 1H), 8.19 (s, quinolone-5-H, 1H), 7.98 (s, quinolone-7-H, 1H), 7.83 (d, J ¼ 3.1 Hz, quinolone-8-H, 1H), 4.51e4.89 (m, NeCH2, 2H), 4.26 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.45 (m, OeCH, 1H), 2.55e2.84 (m, OeCH2, 2H), 2.36 (s, quinolone-6CH3, 3H), 1.31 (t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 174.0, 166.2, 134.7, 150.2, 121.4, 131.0, 125.9,

6.1.4. Synthesis of ethyl 6-fluoro-1-(oxiran-2-ylmethyl)-4-oxo-1,4dihydroquinoline-3-carboxylate (3c) Compound 3c was prepared according to the procedure depicted for compound 3a, starting from compound 2c (0.235 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chloromethyl)oxirane (15 mL). The product 3c (0.262 g) was obtained as white solid. Yield: 90.0%; m.p: >250  C. IR (KBr, cm1) IR (KBr, cm1) n: 3131 (AreH), 3015 (]CeH), 2988, 2902 (CH2, CH3), 1725, 1641 (C]O), 1609, 1559, 1493 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.49 (s, quinolone-2-H, 1H), 8.11 (d, J ¼ 8.0 Hz, quinolone-5-H, 1H), 7.66e7.75 (m, quinolone-7-H, 1H), 7.45e7.51 (m, quinolone-8-H, 1H), 4.51e4.89 (m, NeCH2, 2H), 4.28 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.46 (s, OeCH, 1H), 2.54e2.75 (m, OeCH2, 2H), 1.31 (t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 172.0, 164.7, 151.1, 150.8, 135.5, 131.1, 129.1, 125.2, 120.3, 110.6, 60.4, 57.7, 50.6, 45.1, 14.7 ppm; MS (ESI): m/z 292 [MþH]þ; HRMS (ESI) calcd. for C15H14FNO4 [MþH]þ, 292.0985; found, 292.0984. 6.1.5. Synthesis of ethyl 1-(oxiran-2-ylmethyl)-4-oxo-7(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate (3d) Compound 3d was prepared according to the procedure depicted for compound 3a, starting from compound 2d (0.285 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chloromethyl)oxirane (15 mL). The product 3d (0.302 g) was obtained as white solid. Yield: 88.3%; m.p: >250  C; IR (KBr, cm1) n: 3129 (AreH), 3061 (]CeH), 2989, 2906 (CH2, CH3), 1730, 1635 (C]O), 1609, 1556, 1494 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.75 (s, quinolone-2-H, 1H), 8.45 (d, J ¼ 8.0 Hz, quinolone-5-H, 1H), 8.27 (s, quinolone-8-H, 1H), 7.82 (d, J ¼ 3.2 Hz, quinolone-6-H, 1H), 4.45e5.04 (m, NeCH2, 2H), 4.29 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.41 (m, OeCH, 1H), 2.64e2.87 (m, OeCH2, 2H), 1.32 (t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.8, 167.2, 151.1, 137.3, 129.7, 122.1, 124.5, 132.2, 126.5, 120.7, 112.1, 60.5, 57.4, 50.6, 45.1, 14.7 ppm; MS (ESI): m/z 342 [MþH]þ; HRMS (ESI) calcd. for C16H14F3NO4 [MþH]þ, 342.0953; found, 342.0950. 6.1.6. Synthesis of ethyl 7-chloro-6-fluoro-1-(oxiran-2-ylmethyl)-4oxo-1,4-dihydroquinoline-3-carboxylate (3e) Compound 3e was prepared according to the procedure depicted for compound 3a, starting from compound 2e (0.270 g, 1 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chloromethyl) oxirane (15 mL). The product 3e (0.294 g) was obtained as white solid. Yield: 90.5%; m.p: >250  C; IR (KBr, cm1) n: 3127 (AreH), 3015 (]CeH), 2989, 2902 (CH2, CH3), 1731, 1642 (C]O), 1617, 1555, 1497 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.64 (s, quinolone-2-H, 1H), 8.28 (d, J ¼ 3.7 Hz, quinolone-5-H, 1H), 8.06 (s, quinolone-8-H, 1H), 4.45e4.92 (m, 2H), 4.27 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.41 (m, OeCH, 1H), 2.64e2.86 (m, OeCH2, 2H), 1.31 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSOd6): d 174.8, 167.5, 151.1, 139.7, 132.3, 129.3, 127.4, 128.7, 124.5, 114.1, 60.5, 57.7, 50.6, 45.1, 14.7 ppm; MS (ESI): m/z 326 [MþH]þ; HRMS (ESI) calcd. for C15H13ClFNO4 [MþH]þ, 326.0595; found, 326.0593. 6.1.7. Synthesis of ethyl 6,8-dichloro-1-(oxiran-2-ylmethyl)-4-oxo1,4-dihydroquinoline-3-carboxylate (3f) Compound 3f was prepared according to the procedure depicted for compound 3a, starting from compound 2f (0.286 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chloromethyl)oxirane (15 mL). The product 3f (0.284 g) was obtained as white solid. Yield: 83.1%; m.p: >250  C; IR (KBr, cm1) n: 3125 (AreH), 3015 (]CeH), 2989, 2902 (CH2, CH3), 1725, 1634 (C]O),

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1608, 1557, 1498 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.51 (s, quinolone-2-H, 1H), 8.08e8.19 (m, quinolone-5-H, 1H), 7.69 (s, quinolone-8-H, 1H), 4.48e4.93 (m, 2H, NeCH2), 4.24 (q, J ¼ 7.2 Hz, OeCH2CH3, 2H), 3.46 (s, OeCH, 1H), 2.54e2.84 (m, OeCH2, 2H), 1.29 (t, J ¼ 7.0 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 172.0, 164.6, 153.8, 151.2, 150.5, 131.1, 125.9, 123.0, 120.6, 111.0, 60.5, 57.7, 50.6, 45.1, 14.7 ppm. MS (ESI): m/z 342 [MþH]þ; HRMS (ESI) calcd. for C15H13Cl2NO4 [MþH]þ, 342.0300; found, 342.0303.

(300 MHz, DMSO-d6): d 8.58 (s, quinolone-2-H, H), 8.42 (s, imidazole-5-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.41e7.44 (m, quinolone-5-H, 1H), 7.24e7.28 (m, quinolone-7-H, 1H), 5.84 (s, eOH, 1H), 4.76 (m, CH, 1H), 4.21e4.71 (m, eCH2e, 6H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 172.3, 164.8, 152.9, 149.6, 147.8, 145.2, 138.6, 135.8, 131.5, 129.0, 126.9, 123.0, 109.9, 68.4, 60.8, 60.4, 51.1, 14.7 ppm; MS (ESI): m/z 423 [MþH]þ; HRMS (ESI) calcd. for C18H16F2N4O6 [MþH]þ, 423.1116; found, 423.1114.

6.1.8. Synthesis of ethyl 8-fluoro-1-(oxiran-2-ylmethyl)-4-oxo-1,4dihydroquinoline-3-carboxylate (3g) Compound 3g was prepared according to the experimental procedure described for compound 3a, starting from compound 2g (0.235 g, 1 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2(chloromethyl)oxirane (15 mL). The product 3g (0.257 g) was obtained as yellow solid. Yield: 88.3%; m.p: >250  C; IR (KBr, cm1) n: 3127 (AreH), 3015 (]CeH), 2989, 2902 (CH2, CH3), 1735, 1638 (C] O), 1612, 1555, 1495 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 8.49 (s, quinolone-2-H, 1H), 8.11 (d, J ¼ 9.3 Hz, quinolone-5-H, 1H), 7.66e7.75 (m, quinolone-7-H, 1H), 7.45e7.51 (m, quinolone-6H, 1H), 4.51e4.89 (m, NeCH2, 2H), 4.28 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.46 (m, OeCH, 1H), 2.57e2.75 (m, OeCH2, 2H), 1.31 (t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 172.0, 164.6, 153.8, 152.2, 150.5, 131.1, 129.1, 123.0, 120.3, 110.6, 60.4, 57.5, 50.6, 45.1, 14.7 ppm; MS (ESI): m/z 292 [MþH]þ; HRMS (ESI) calcd. for C15H14FNO4 [MþH]þ, 292.0985; found, 292.0982.

6.1.11. Synthesis of ethyl 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1yl)propyl)-6-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylate (4b) Compound 4b was prepared according to the procedure depicted for compound 4a, starting from compound 3b (0.287 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 4-nitro-1Himidazole (0.113 g, 1.0 mmol). The product 4b (0.334 g) was obtained as white solid. Yield: 83.5%; m.p: >250  C; IR (KBr, cm1) n: 3350 (OeH), 3122 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1609, 1555, 1497, 1401 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.82 (s, quinolone-2-H, 1H), 8.41 (s, imidazole-5-H, 1H), 8.18 (s, quinolone-5-H, 1H), 7.98 (s, quinolone-7-H, 1H), 7.83e7.86 (m, quinolone-8-H and imidazole-2-H, 2H), 5.84 (m, eOH, 1H), 4.73 (m, eCH, 1H), 4.17e4.43 (m, eCH2e, 6H), 2.36 (s, quinolone-6-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 173.6, 161.2, 153.1, 151.2, 149.5, 140.4, 137.7, 135.1, 131.2, 124.5, 121.2, 115.3, 110.5, 69.0, 60.7, 60.3, 52.6, 25.6, 14.7 ppm; MS (ESI): m/z 401 [MþH]þ; HRMS (ESI) calcd. for C19H20N4O6 [MþH]þ, 401.1461; found, 401.1463.

6.1.9. Synthesis of ethyl 6-methoxy-1-(oxiran-2-ylmethyl)-4-oxo1,4-dihydroquinoline-3-carboxylate (3h) Compound 3h was prepared according to the procedure depicted for compound 3a, starting from compound 2h (0.247 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-(chloromethyl)oxirane (15 mL). The product 3h (0.246) was obtained as white solid. Yield: 81.2%; m.p: >250  C; IR (KBr, cm1) n: 3128 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1720, 1641 (C]O), 1616, 1557, 1498 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.57 (s, quinolone-2-H, 1H), 7.89 (d, J ¼ 3.4 Hz, quinolone-5-H, 1H), 7.68 (s, quinolone-7-H, 1H), 7.39e7.43 (m, quinolone-8-H, 1H), 4.43e4.85 (m, NeCH2, 2H), 4.26 (q, J ¼ 7.1 Hz, OeCH2CH3, 2H), 3.87 (s, quinolone-6-OCH3, 3H), 3.35 (s, OeCH, 1H), 2.57e2.85 (m, OeCH2, 2H), 1.29 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 172.9, 165.2, 157.0, 151.2, 131.1, 129.3, 123.5, 120.1, 108.8, 107.2, 60.2, 57.7, 55.9, 50.6, 45.1, 14.7 ppm; MS (ESI): m/ z 304 [MþH]þ; HRMS (ESI) calcd. for C16H17NO5 [MþH]þ, 304.1185; found, 304.1183. 6.1.10. Synthesis of ethyl 6,8-difluoro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (4a) To a stirred suspension of potassium carbonate (0.151 g, 1.2 mmol) in ethanol was added 4-nitro-1H-imidazole (0.113 g, 1.0 mmol). The mixture was stirred at 60  C for 1.5 h, and then was cooled to room temperature. Compound 3a (0.309 g, 1.0 mmol) was added, and the mixture was stirred on reflux for 2 h. After the reaction came to end (monitored by TLC, eluent, chloroform/methanol, 20/1, V/V), the solvent was evaporated and the residue was treated with water and extract with chloroform (3  20 mL). The combined organic extracts was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified via silica gel column chromatography (eluent, chloroform/methanol ¼ 20/1, V/V) to afford compound 4a (0.408 g) as yellow solid. Yield: 96.7%; m.p: >250  C. IR (KBr, cm1) n: 3443 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771, 1676 (C]O), 1614, 1564, 1508, 1399 (aromatic frame); 1H NMR

6.1.12. Synthesis of ethyl 6-fluoro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (4c) Compound 4c was prepared according to the procedure depicted for compound 4a, starting from compound 3c (0.291 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 4-nitro-1Himidazole (0.113 g, 1.0 mmol). The product 4c (0.350 g) was obtained as white solid. Yield: 86.6%; m.p: >250  C; IR (KBr, cm1) n: 3412 (OeH), 3128 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.43 (s, quinolone-2-H, H), 8.41 (s, imidazole-5-H, 1H), 8.12 (d, J ¼ 3.2 Hz, quinolone-5-H, 1H), 7.85 (s, imidazole-2-H, 1H), 7.64e7.72 (m, quinolone-7-H, 1H), 7.46e7.50 (m, quinolone-8-H, 1H), 5.78 (d, J ¼ 3.2 Hz, eOH, 1H), 4.67e4.82 (m, eCH, 1H), 4.04e4.39 (m, eCH2e, 6H), 1.29 (t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 173.3, 164.8, 151.3, 147.8, 145.2, 138.6, 131.5, 126.9, 129.0, 127.9, 122.8, 119.0, 109.9, 68.4, 60.8, 60.2, 51.1, 14.7 ppm; MS (ESI): m/z 427 [MþNa]þ; HRMS (ESI) calcd. for C18H17FN4O6 [MþNa]þ, 427.1030; found, 427.1028. 6.1.13. Synthesis of ethyl 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1yl)propyl)-4-oxo-7-(trifluoromethyl)-1,4-dihydroquinoline-3carboxylate (4d) Compound 4d was prepared according to the procedure depicted for compound 4a, starting from compound 3d (0.341 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 4-nitro-1Himidazole (0.113 g, 1.0 mmol). The product 4d (0.364 g) was obtained as white solid. Yield: 80.2%; m.p: >250  C; IR (KBr, cm1) n: 3441 (OeH), 3130 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.68 (s, quinolone-2-H, 1H), 8.44 (s, quinolone-5-H, 1H), 8.41 (s, imidazole-5-H, 1H), 8.19 (s, quinolone-8-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.79 (d, J ¼ 8.4 Hz, quinolone-6-H, 1H), 5.83 (m, eOH, 1H), 4.75 (m, eCHe, 1H), 4.17e4.43 (m, eCH2e, 6H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz,

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DMSO-d6): d 173.6, 164.8, 121.6, 147.8, 145.2, 139.4, 138.6, 135.5, 129.4, 129.9, 126.9, 121.2, 110.1, 109.9, 68.4, 60.8, 60.2, 51.1, 14.7 ppm; MS (ESI): m/z 455 [MþH]þ; HRMS (ESI) calcd. for C19H17F3N4O6 [MþH]þ, 455.1187; found, 455.1186. 6.1.14. Synthesis of ethyl 7-chloro-6-fluoro-1-(2-hydroxy-3-(4nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3carboxylate (4e) Compound 4e was prepared according to the procedure depicted for compound 4a, starting from compound 3e (0.326 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 4-nitro-1Himidazole (0.113 g, 1.0 mmol). The product 4e (0.348 g) was obtained as white solid. Yield: 79.5%; m.p: >250  C; IR (KBr, cm1) n: 3434 (OeH), 3125 (AreH), 3016 (]CeH), 2998, 2912 (CH2, CH3), 1728, 1684 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.57 (s, quinolone-2-H, 1H), 8.42 (s, imidazole-5-H, 1H), 8.23 (d, J ¼ 4.3 Hz, quinolone-5-H, 1H), 8.05 (s, quinolone-8-H, 1H), 7.87 (s, imidazole-2-H, 1H), 5.76 (s, eOH, 1H), 4.75 (m, eCHe, 1H), 4.16e4.55 (m, eCH2e, 6H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.4, 164.8, 150.3, 147.8, 145.2, 141.8, 138.6, 127.9, 131.5, 129.0, 123.9, 115.0, 109.9, 68.4, 60.8, 60.4, 51.0, 14.7 ppm; MS (ESI): m/z 439 [MþH]þ; HRMS (ESI) calcd. for C18H16ClFN4O6 [MþH]þ, 439.0821; found, 439.0820. 6.1.15. Synthesis of ethyl 6,8-dichloro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (4f) Compound 4f was prepared according to the procedure depicted for compound 4a, starting from compound 3f (0.326 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 4-nitro-1Himidazole (0.113 g, 1.0 mmol). The product 4f (0.369 g) was obtained as white solid. Yield: 81.2%; m.p: >250  C. IR (KBr, cm1) n: 3439 (OeH), 3127 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1721, 1684 (C]O), 1621, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.51 (s, quinolone-2-H, 1H), 8.41 (s, imidazole-5-H, 1H), 8.23 (s, quinolone-5-H, 1H), 8.08e8.19 (m, quinolone-7-H, 1H), 7.69 (s, imidazole-2-H, 1H), 5.20e5.25 (m, eOH, 1H), 4.73e4.80 (m, eCHe, 1H), 4.28e4.56 (m, eCH2e, 6H), 1.29 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 175.2, 163.7, 153.1, 146.7, 136.2, 152.1, 151.4, 148.4, 134.1, 132.1, 131.2, 124.8, 110.5, 69.0, 60.8, 60.4, 52.5, 14.8 ppm; MS (ESI): m/z 455 [MþH]þ; HRMS (ESI) calcd. for C18H16Cl2N4O6 [MþH]þ, 455.0525; found, 455.0521. 6.1.16. Synthesis of ethyl 8-fluoro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (4g) Compound 4g was prepared according to the procedure depicted for compound 4a, starting from compound 3g (0.291 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 4-nitro-1Himidazole (0.113 g, 1.0 mmol). The product 4g (0.398 g) was obtained as white solid. Yield: 98.5%; m.p: >250  C; IR (KBr, cm1) n: 3417 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1614, 1557, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.43 (d, J ¼ 1.2 Hz, quinolone-2-H, 1H), 8.41 (s, imidazole-5-H, H), 8.12 (m, quinolone-5-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.63e7.71 (m, quinolone-7-H, 1H), 7.43e7.50 (m, quinolone-6-H, 1H), 5.73 (s, eOH, 1H), 4.75 (m, eCHe, 1H), 4.15e4.43 (m, eCH2e, 6H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13 C NMR (75 MHz, DMSO-d6): d 173.3, 164.8, 152.9, 147.8, 145.2, 138.6, 126.9, 131.5, 129.0, 125.6, 122.8, 109.9, 119.0, 68.4, 60.8, 60.2, 51.1, 14.7 ppm; MS (ESI): m/z 427 [MþNa]þ; HRMS (ESI) calcd. for C18H17FN4O6 [MþNa]þ, 427.1030; found, 427.1028.

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6.1.17. Synthesis of ethyl 7-chloro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-6-methoxy-4-oxo-1,4-dihydroquinoline-3carboxylate (4h) Compound 4h was prepared according to the procedure depicted for compound 4a, starting from compound 3h (0.303 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 4-nitro-1Himidazole (0.113 g, 1.0 mmol). The product 4h (0.338 g) was obtained as white solid. Yield: 81.2%; m.p: >250  C; IR (KBr, cm1) n: 3413 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.51 (s, quinolone-2-H, H), 8.41 (s, imidazole-5-H, 1H), 7.85 (s, quinolone-5-H, 1H), 7.82 (s, imidazole-2-H, 1H), 7.67 (d, J ¼ 7.3 Hz, quinolone-7-H, 1H), 7.43 (m, quinolone-8-H, 1H), 5.75 (d, J ¼ 3.7 Hz, eOH, 1H), 4.82 (d, J ¼ 3.6 Hz, eCHe, 1H), 4.08e4.51(m, eCH2e, 6H), 3.87 (s, quinolone-6-OCH3, 3H), 1.28 (t, J ¼ 7.0 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 172.3, 164.8, 147.7, 146.1, 145.3, 138.6, 135.6, 126.9, 122.3, 120.8, 110.5, 109.0, 68.5, 60.8, 60.2, 55.9, 51.1, 25.7, 14.7 ppm; MS (ESI): m/z 417 [MþH]þ; HRMS (ESI) calcd. for C19H20N4O7 [MþH]þ, 417.1410; found, 417.1410. 6.1.18. Synthesis of ethyl 6,8-difluoro-1-(2-hydroxy-3-(2-methyl-5nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3carboxylate (5a) To a stirred suspension of potassium carbonate (0.151 g, 1.2 mmol) in ethanol was added 2-methyl-5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The mixture was stirred at 60  C for 1.5 h. When the reaction was cooled to room temperature, compound 3a (0.309 g, 1.0 mmol) was added and stirred on reflux for 2 h. After the reaction came to end (monitored by TLC, eluent, chloroform/ methanol, 20/1, V/V), the solvent was evaporated and the residue was treated with water and extract with chloroform (3  20 mL). The combined organic extracts was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified via silica gel column chromatography (eluent, chloroform/methanol ¼ 20/1, V/V) to give compound 5a (0.371 g) as yellow solid. Yield: 85.2%; m.p: >250  C. IR (KBr, cm1) n: 3443 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771, 1676 (C]O), 1614, 1564, 1508, 1399 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.58 (s, quinolone-2-H, H), 8.32 (s, imidazole-4-H, 1H), 7.41e7.46 (m, quinolone-5-H, 1H), 7.24e7.28 (m, quinolone-7-H, 1H), 5.82 (s, eOH, 1H), 4.73 (m, eCHe, 1H), 4.21e4.71 (m, eCH2e, 6H), 2.41 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 173.4, 164.8, 152.8, 151.3, 149.6, 145.2, 138.6, 135.7, 132.5, 131.5, 129.0, 123.0, 109.9, 68.4, 60.8, 60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/ z 437 [MþH]þ; HRMS (ESI) calcd. for C19H18F2N4O6 [MþH]þ, 437.1273; found, 437.1270. 6.1.19. Synthesis of ethyl 1-(2-hydroxy-3-(2-methyl-5-nitro-1Himidazol-1-yl)propyl)-6-methyl-4-oxo-1,4-dihydroquinoline-3carboxylate (5b) Compound 5b was prepared according to the procedure depicted for compound 5a, starting from compound 3a (0.287 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5b (0.367 g) was obtained as white solid. Yield: 88.4%; m.p: >250  C; IR (KBr, cm1) n: 3350 (OeH), 3122 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1609, 1555, 1497, 1401 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.51 (s, quinolone-2-H, H), d 8.31 (s, imidazole-4-H, H), 7.87 (s, quinolone-5-H, 1H), 7.67 (d, J ¼ 8.3 Hz, quinolone-7-H, 1H), 7.42 (m, quinolone-8-H, 1H), 5.85 (s, eOH, 1H), 4.83 (d, J ¼ 13.2 Hz, eCHe, 1H), 4.08e4.50 (m, eCH2e, 6H), 3.87 (s, quinolone-6-CH3, 3H), 2.41 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6):

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d 176.3, 164.8, 149.9, 146.1, 134.6, 131.3, 133.5, 128.0, 132.8, 124.9, 120.8, 119.0, 110.5, 68.4, 60.8, 60.2, 51.1, 25.7, 21.7, 14.7 ppm; MS (ESI): m/z 414 [MþH]þ; HRMS (ESI) calcd. for C20H22N4O6 [MþH]þ, 415.1612; found, 415.11610. 6.1.20. Synthesis of ethyl 6-fluoro-1-(2-hydroxy-3-(2-methyl-5nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3carboxylate (5c) Compound 5c was prepared according to the procedure depicted for compound 5a, starting from compound 3c (0.291 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5c (0.345 g) was obtained as white solid. Yield: 82.3%; m.p: >250  C; IR (KBr, cm1) n: 3412 (OeH), 3128 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.43 (d, J ¼ 1.2 Hz, quinolone-2-H, 1H), 8.32 (s, imidazole-4-H, H), 8.12 (d, J ¼ 3.4 Hz, quinolone-5-H, 1H), 7.64e7.72 (m, quinolone-7-H, 1H), 7.46e7.50 (m, quinolone-8H, 1H), 5.88 (s, eOH, 1H), 4.82 (m, eCHe, 1H), 4.04e4.39 (m, eCH2e, 6H), 2.41 (s, imidazole-2-CH3, 3H), 1.29 (t, J ¼ 7.2 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 173.3, 164.8, 151.3, 149.8, 149.8, 134.6, 131.9, 131.5, 129.0, 127.9, 122.8, 119.0, 109.9, 68.4, 60.8, 60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/z 441 [MþNa]þ; HRMS (ESI) calcd. for C19H19FN4O6 [MþNa]þ, 441.1186; found, 441.1183. 6.1.21. Synthesis of ethyl 1-(2-hydroxy-3-(2-methyl-5-nitro-1Himidazol-1-yl)propyl)-4-oxo-6-(trifluoromethyl)-1,4dihydroquinoline-3-carboxylate (5d) Compound 5d was prepared according to the procedure depicted for compound 5a, starting from compound 3d (0.341 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5d (0.418 g) was obtained as white solid. Yield: 89.3%; m.p: >250  C; IR (KBr, cm1) n: 3441 (OeH), 3130 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.70 (s, quinolone-2-H, 1H), 8.45 (s, imidazole-4-H, 1H), 8.28 (m, quinolone-5-H, 2H), 7.80 (d, J ¼ 8.0 Hz, quinolone-6-H, 1H), 7.43 (d, J ¼ 3.0 Hz, quinolone-8-H, 1H), 5.94 (s, eOH, 1H), 4.81 (d, J ¼ 14.4 Hz, eCHe, 1H), 4.11e4.39 (m, eCH2e, 6H), 3.42 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 173.6, 164.8, 151.7, 149.8, 145.2, 142.3, 139.4, 134.6, 135.5, 129.4, 132.6, 121.2, 110.1, 109.9, 68.4, 60.7, 60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/z 469 [MþH]þ; HRMS (ESI) calcd. for C20H19F3N4O6 [MþH]þ, 469.1335; found, 469.1334. 6.1.22. Synthesis of ethyl 7-chloro-6-fluoro-1-(2-hydroxy-3-(2methyl-5-nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4dihydroquinoline-3-carboxylate (5e) Compound 5e was prepared according to the procedure depicted for compound 5a, starting from compound 3e (0.326 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5e (0.381 g) was obtained as white solid. Yield: 84.3%; m.p: >250  C; IR (KBr, cm1) n: 3434 (OeH), 3125 (AreH), 3016 (]CeH), 2998, 2912 (CH2, CH3), 1728, 1684 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.57 (s, quinolone-2-H, 1H), 8.30 (s, imidazole-4-H, 1H), 8.23 (d, J ¼ 3.3 Hz, quinolone-5-H, 1H), 8.05 (s, quinolone-8-H, 1H), 5.86 (s, eOH, 1H), 4.78 (m, eCHe, 1H), 4.16e4.54 (m, eCH2e, 6H), 3.42 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.4, 164.8, 150.3, 149.8, 145.2, 141.8, 134.6, 132.6, 131.5, 129.0, 123.9, 115.0, 109.9, 68.4, 60.8, 60.2, 51.0, 21.7, 14.7 ppm; MS (ESI): m/ z 453 [MþH]þ; HRMS (ESI) calcd. for C19H18ClFN4O6 [MþH]þ, 453.0977; found, 453.0976.

6.1.23. Synthesis of ethyl 6,8-dichloro-1-(2-hydroxy-3-(2-methyl5-nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3carboxylate (5f) Compound 5f was prepared according to the procedure depicted for compound 5a, starting from compound 3f (0.341 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5f (0.381 g) was obtained as white solid. Yield: 81.2%; m.p: >250  C. IR (KBr, cm1) n: 3439 (OeH), 3127 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1721, 1684 (C]O), 1621, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.62 (s, quinolone-2-H, 1H), 8.31 (s, imidazole-4-H, 1H), 7.69 (d, J ¼ 2.1 Hz, quinolone-5-H, 1H), 7.38 (d, J ¼ 2.1 Hz, quinolone-7-H, 1H), 5.80 (s, eOH, 1H), 4.78 (m, eCHe, 1H), 4.15e4.48 (m, eCH2e, 6H), 2.41 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 174.4, 164.8, 149.8, 147.8, 139.8, 136.5, 134.6, 132.7, 131.5, 129.0, 131.1, 122.6, 109.9, 68.5, 60.8, 60.2, 51.0, 21.7, 14.7 ppm; MS (ESI): m/ z 469 [MþH]þ; HRMS (ESI) calcd. for C19H18Cl2N4O6 [MþH]þ, 469.0982; found, 469.0977.

6.1.24. Synthesis of ethyl 8-fluoro-1-(2-hydroxy-3-(2-methyl-5nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3carboxylate (5g) Compound 5g was prepared according to the procedure depicted for compound 5a, starting from compound 3g (0.291 g, 1 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl-5nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5g (0.349 g) was obtained as white solid. Yield: 83.4%; m.p: >250  C; IR (KBr, cm1) n: 3417 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1614, 1557, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.43 (d, J ¼ 1.2 Hz, quinolone-2-H, 1H), 8.31 (s, imidazole-4-H, 1H), 8.12 (m, quinolone-5-H, 1H), 7.63e7.72 (m, quinolone-7-H, 1H), 7.43e7.50 (m, quinolone-6-H, 1H), 5.83 (s, eOH, 1H), 4.78 (m, eCHe, 1H), 4.15e4.43 (m, eCH2e, 6H), 2.41 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.1 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 174.0, 164.8, 152.9, 149.9, 146.2, 134.6, 132.7, 131.5, 129.0, 125.6, 122.8, 109.9, 119.0, 68.4, 60.6, 60.2, 51.1, 21.7, 14.7 ppm; MS (ESI): m/z 441 [MþNa]þ; HRMS (ESI) calcd. for C19H19FN4O6 [MþNa]þ, 441.1186; found, 441.1185.

6.1.25. Synthesis of ethyl 1-(2-hydroxy-3-(2-methyl-5-nitro-1Himidazol-1-yl)propyl)-6-methoxy-4-oxo-1,4-dihydroquinoline-3carboxylate (5h) Compound 5h was prepared according to the procedure depicted for compound 5a, starting from compound 3h (0.303 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 2-methyl5-nitro-1H-imidazole (0.127 g, 1.0 mmol). The product 5h (0.347 g) was obtained as white solid. Yield: 80.6%; m.p: >250  C; IR (KBr, cm1) n: 3413 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 8.53 (s, quinolone-2-H, H), 8.34 (s, imidazole-4-H, H), 7.93 (d, J ¼ 9.3 Hz, quinolone-5-H, 1H), 7.67 (d, J ¼ 2.7 Hz, quinolone-7-H, 1H), 7.40 (m, quinolone-8-H, 1H), 5.95 (s, eOH, 1H), 4.70 (d, J ¼ 12.7 Hz, eCHe, 1H), 4.12e4.35 (m, eCH2e, 6H), 3.87 (s, quinolone-6-OCH3, 3H), 2.41 (s, imidazole-2-CH3, 3H), 1.28 (t, J ¼ 7.0 Hz, OeCH2CH3, 3H) ppm; 13C NMR (75 MHz, DMSOd6): d 172.3, 164.8, 147.7, 145.3, 134.6, 135.6, 126.9, 132.7, 122.3, 120.8, 110.5, 109.0, 68.4, 60.6, 60.2, 55.9, 51.1, 25.7, 21.7, 14.7 ppm; MS (ESI): m/z 431 [MþH]þ; HRMS (ESI) calcd. for C20H22N4O7 [MþH]þ, 431.1567; found, 431.1566.

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6.1.26. Synthesis of 6,8-difluoro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-4-oxo-1,4 dihydroquinoline-3-carboxylic acid (6a) To a stirred solution of aqueous solution NaOH (3%, 20 mL) was added compound 4a (0.422 g, 1.0 mmol). The mixture was stirred at 100  C for 2 h. After the reaction was completed (monitored by TLC, eluent, chloroform/methanol ¼ 15/1, V/V), the solution was treated with formic acid to reach pH ¼ 7. The mixture was filtered and washed with water for three times to give compound 6a (0.337 g) as white solid. Yield: 85.2%; m.p: >250  C. IR (KBr, cm1) n: 3443 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771, 1676 (C]O), 1614, 1564, 1508, 1399 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 14.83 (s, eCOOH, 1H), 8.56 (s, quinolone-2H, 1H), 8.42 (s, imidazole-5-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.41e7.43 (m, quinolone-5-H, 1H), 7.24e7.29 (m, quinolone-7-H, 1H), 5.83 (s, eOH, 1H), 4.76 (m, eCHe, 1H), 4.21e4.71 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6): d 173.3, 165.7, 152.9, 149.6, 147.8, 145.2, 138.6, 135.8, 131.5, 129.0, 126.9, 123.0, 110.3, 68.4, 60.8, 51.1 ppm; MS (ESI): m/z 395 [MþH]þ; HRMS (ESI) calcd. for C16H12F2N4O6 [MþH]þ, 395.0803; found, 395.0801. 6.1.27. Synthesis of 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-yl) propyl)-6-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6b) Compound 6b was prepared according to the procedure depicted for compound 6a, starting from compound 4b (0.400 g, 1.0 mmol). The product 6b (0.325 g) was obtained as white solid. Yield: 87.3%; m.p: >250  C; IR (KBr, cm1) n: 3350 (OeH), 3122 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1609, 1555, 1497, 1401 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 14.54 (s, eCOOH, 1H), 8.51 (s, quinolone-2-H, 1H), d 8.40 (s, imidazole-5-H, 1H), 7.87 (s, quinolone-5-H, 1H), 7.82 (s, imidazole2-H, 1H), 7.65 (d, J ¼ 9.3 Hz, quinolone-7-H, 1H), 7.42 (m, quinolone8-H, 1H), 5.75 (s, eOH, 1H), 4.76 (d, J ¼ 13.2 Hz, eCHe, 1H), 4.08e4.51 (m, eCH2e, 4H), 3.87 (s, quinolone-6-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.8, 165.4, 147.8, 146.1, 138.6, 131.3, 128.0, 126.8, 124.9, 120.8, 110.6, 119.0, 68.4, 60.8, 51.1, 25.7 ppm; MS (ESI): m/z 373 [MþH]þ; HRMS (ESI) calcd. for C16H16N4O6 [MþH]þ, 373.1148; found, 373.1148. 6.1.28. Synthesis of 6-fluoro-1-(2-hydroxy-3-(4-nitro-1H-imidazol1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-carboxylic acid (6c) Compound 6c was prepared according to the procedure depicted for compound 6a, starting from compound 4c (0.404 g, 1.0 mmol). The product 6c (0.348 g) was obtained as white solid. Yield: 92.6%; m.p: >250  C; IR (KBr, cm1) n: 3412 (OeH), 3128 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 14.63 (s, eCOOH, 1H), 8.53 (s, quinolone-2-H, 1H), 8.42 (s, imidazole-5-H, 1H), 8.13 (d, J ¼ 3.8 Hz, quinolone-5-H, 1H), 7.85 (s, imidazole-2-H, 1H), 7.64e7.70 (m, quinolone-7-H, 1H), 7.46e7.50 (m, quinolone-8-H, 1H), 5.84 (s, eOH, 1H), 4.67e4.80 (m, eCHe, 1H), 4.04e4.39 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.3, 165.7, 151.3, 147.8, 145.2, 138.6, 126.9, 131.5, 129.0, 127.9, 122.8, 118.0, 110.3, 68.4, 60.8, 51.1 ppm; MS (ESI): m/z 399 [MþNa]þ; HRMS (ESI) calcd. for C16H13FN4O6 [MþNa]þ, 399.1717; found, 399.1716. 6.1.29. Synthesis of 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-yl) propyl)-4-oxo-6-(trifluoromethyl)-1,4-dihydroquinoline-3carboxylic acid (6d) Compound 6d was prepared according to the procedure depicted for compound 6a, starting from compound 4d (0.454 g, 1.0 mmol). The product 6d (0.339 g) was obtained as white solid. Yield: 79.5%; m.p: >250  C; IR (KBr, cm1) n: 3441 (OeH), 3130

331

(AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 15.43 (s, eCOOH, 1H), 8.68 (s, quinolone-2-H, 1H), 8.44 (s, quinolone-5-H, 1H), 8.41 (s, imidazole-5-H, 1H), 8.19 (s, quinolone6-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.79 (d, J ¼ 8.4 Hz, quinolone8-H, 1H), 5.83 (s, eOH, 1H), 4.76 (m, eCHe, 1H), 4.17e4.43 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.5, 165.7, 151.6, 147.8, 145.2, 139.4, 138.6, 135.5, 129.4, 129.9, 126.9, 121.2, 110.1, 109.9, 68.4, 60.8, 51.1 ppm; MS (ESI): m/z 427 [MþH]þ; HRMS (ESI) calcd. for C17H13F3N4O6 [MþH]þ, 427.0865; found, 427.0864. 6.1.30. Synthesis of 7-chloro-6-fluoro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-carboxylic acid (6e) Compound 6e was prepared according to the procedure depicted for compound 6a, starting from compound 4e (0.438 g, 1.0 mmol). The product 6e (0.362 g) was obtained as white solid. Yield: 88.3%; m.p: >250  C; IR (KBr, cm1) n: 3434 (OeH), 3125 (AreH), 3016 (]CeH), 2998, 2912 (CH2, CH3), 1728, 1684 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 14.83 (s, eCOOH, 1H), 8.57 (s, quinolone-2-H, 1H), 8.45 (s, imidazole-5-H, 1H), 8.25 (d, J ¼ 5.3 Hz, quinolone-5-H, 1H), 8.09 (s, quinolone-8-H, 1H), 7.87 (s, imidazole-2-H, 1H), 5.87 (s, eOH, 1H), 4.78 (m, eCHe, 1H), 4.12e4.56 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6): d 177.2, 165.6, 150.3, 147.8, 145.2, 141.8, 138.6, 127.9, 131.5, 129.0, 123.9, 115.0, 110.0, 68.4, 60.7, 51.0 ppm; MS (ESI): m/z 411 [MþH]þ; HRMS (ESI) calcd. for C16H12ClFN4O6 [MþH]þ, 411.0508; found, 411.0507. 6.1.31. Synthesis of 6,8-dichloro-1-(2-hydroxy-3-(4-nitro-1Himidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6f) Compound 6f was prepared according to the procedure depicted for compound 6a, starting from compound 4f (0.455 g, 1.0 mmol). The product 6f (0.346 g) was obtained as white solid. Yield: 81.1%; m.p: >250  C. IR (KBr, cm1) n: 3439 (OeH), 3127 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1721, 1684 (C]O), 1621, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d15.23 (s, eCOOH, 1H), 8.44 (s, quinolone-2-H, 1H), 8.40 (s, imidazole-5-H, 1H), 8.07e8.15 (m, quinolone-5-H, 1H), 7.87 (s, imidazole-2-H, 1H), 7.68 (s, quinolone-7-H, 1H), 5.83 (s, eOH, 1H), 4.82e4.87 (m, eCHe, 1H), 4.16e4.29 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6): d 177.3, 166.1, 147.8, 145.2, 139.8, 138.6, 136.5, 130.8, 131.5, 129.0, 126.9, 122.6, 109.3, 68.5, 60.8, 51.0 ppm; MS (ESI): m/z 427 [MþH]þ; HRMS (ESI) calcd. for C16H12Cl2N4O6 [MþH]þ, 427.0212; found, 427.0210. 6.1.32. Synthesis of 8-fluoro-1-(2-hydroxy-3-(4-nitro-1H-imidazol1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (6g) Compound 6g was prepared according to the procedure depicted for compound 6a, starting from compound 4g (0.404 g, 1 mmol). The product 6g (0.338 g) was obtained as white solid. Yield: 89.7%; m.p: >250  C; IR (KBr, cm1) n: 3417 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1614, 1557, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 14.65 (s, eCOOH, 1H), 8.43 (d, J ¼ 1.5 Hz, quinolone-2-H, 1H), 8.40 (s, imidazole-5-H, H), 8.12 (m, quinolone-5-H, 1H), 7.86 (s, imidazole-2-H, 1H), 7.60e7.70 (m, quinolone-7-H, 1H), 7.43e7.53 (m, quinolone-6-H, 1H), 5.78 (s, eOH, 1H), 4.75 (m, eCHe, 1H), 4.15e4.43 (m, eCH2e, 4H) ppm; 13C NMR (75 MHz, DMSO-d6): d 175.3, 165.6, 152.9, 147.8, 145.2, 138.6, 126.9, 131.5, 129.0, 125.6, 122.8, 110.3, 109.0, 68.4, 60.8, 51.1 ppm; MS (ESI): m/z 399 [MþNa]þ; HRMS (ESI) calcd. for C16H13FN4O6 [MþNa]þ, 399.1717; found, 399.1716.

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6.1.33. Synthesis of 1-(2-hydroxy-3-(4-nitro-1H-imidazol-1-yl) propyl)-6-methoxy-4-oxo-1,4 -dihydroquinoline-3-carboxylic acid (6h) Compound 6h was prepared according to the procedure depicted for compound 6a, starting from compound 4h (0.416 g, 1.0 mmol). The product 6h (0.302 g) was obtained as white solid. Yield: 77.8%; m.p: >250  C; IR (KBr, cm1) n: 3413 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 14.43 (s, eCOOH, 1H), 8.58 (s, quinolone-2-H, 1H), 8.43 (s, imidazole-5-H, 1H), 7.87 (s, quinolone-5-H, 1H), 7.80 (s, imidazole2-H, 1H), 7.67 (d, J ¼ 8.3 Hz, quinolone-7-H, 1H), 7.43 (m, quinolone8-H, 1H), 5.85 (s, eOH, 1H), 4.82 (m, eCHe, 1H), 4.08e4.51(m, eCH2e, 4H), 3.87 (s, quinolone-6-OCH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 174.8, 165.6, 147.7, 146.1, 145.3, 138.6, 135.7, 126.9, 122.3, 120.8, 110.3, 109.0, 68.4, 60.8, 55.9, 51.1, 25.7 ppm; MS (ESI): m/z 389 [MþH]þ; HRMS (ESI) calcd. for C17H16N4O7 [MþH]þ, 389.1097; found, 389.1097. 6.1.34. Synthesis of 6,8-difluoro-1-(2-hydroxy-3-(2-methyl-5nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3carboxylic acid (7a) Compound 7a was prepared according to the procedure depicted for compound 6a, starting from compound 5a (0.436 g, 1.0 mmol). The product 7a (0.367 g) was obtained as white solid. Yield: 90.0%; m.p: >250  C; IR (KBr, cm1) n: 3443 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1771, 1676 (C]O), 1614, 1564, 1508, 1399 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 14.67 (s, eCOOH, 1H), 8.59 (s, quinolone-2-H, 1H), 8.31 (s, imidazole-4-H, 1H), 7.41e7.46 (m, quinolone-5-H, 1H), 7.24e7.28 (m, quinolone-7-H, 1H), 5.82 (s, eOH, 1H), 4.73 (m, eCHe, 1H), 4.21e4.65 (m, eCH2e, 4H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 175.4, 165.5, 152.8, 151.3, 149.6, 145.2, 138.6, 135.7, 132.5, 131.5, 129.0, 123.0, 110.4, 68.4, 60.8, 51.1, 21.7 ppm; MS (ESI): m/z 409 [MþH]þ; HRMS (ESI) calcd. for C17H14F2N4O6 [MþH]þ, 409.3210; found, 409.3209. 6.1.35. Synthesis of 1-(2-hydroxy-3-(2-methyl-5-nitro-1Himidazol-1-yl)propyl)-6-methyl-4-oxo-1,4 -dihydroquinoline-3carboxylic acid (7b) Compound 7b was prepared according to the procedure depicted for compound 6a, starting from compound 5b (0.414 g, 1.0 mmol). The product 7b (0.341 g) was obtained as white solid. Yield: 88.3%; m.p: >250  C; IR (KBr, cm1) n: 3350 (OeH), 3122 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1609, 1555, 1497, 1401 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 15.38 (s, eCOOH, 1H), 8.51 (s, quinolone-2-H, 1H), d 8.34 (s, imidazole-4-H, H), 7.86 (s, quinolone-5-H, 1H), 7.67 (d, J ¼ 5.1 Hz, quinolone-7-H, 1H), 7.42 (m, quinolone-8-H, 1H), 5.85 (s, eOH, 1H), 4.83 (m, eCHe, 1H), 4.08e4.53 (m, eCH2e, 4H), 3.87 (s, quinolone6-CH3, 3H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.9, 165.9, 149.9, 146.1, 134.6, 131.3, 133.5, 128.0, 132.8, 124.9, 120.8, 110.3, 119.0, 68.4, 60.8, 51.1, 25.7, 21.7 ppm; MS (ESI): m/z 387 [MþH]þ; HRMS (ESI) calcd. for C18H18N4O6 [MþH]þ, 387.1305; found, 387.1303. 6.1.36. Synthesis of 6-fluoro-1-(2-hydroxy-3-(2-methyl-5-nitro1H-imidazol-1-yl)propyl)-4-oxo-1,4 -dihydroquinoline-3-carboxylic acid (7c) Compound 7c was prepared according to the procedure depicted for compound 6a, starting from compound 5c (0.418 g, 1.0 mmol). The product 7c (0.347 g) was obtained as white solid. Yield: 89.0%; m.p: >250  C; IR (KBr, cm1) n: 3412 (OeH), 3128 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-

d6): d 8.52 (s, quinolone-2-H, 1H), 8.32 (s, imidazole-4-H, H), 8.12 (d, J ¼ 7.8 Hz, quinolone-5-H, 1H), 7.64e7.72 (m, quinolone-7-H, 1H), 7.46e7.50 (m, quinolone-8-H, 1H), 5.88 (s, eOH, 1H), 4.82 (m, eCHe, 1H), 4.04e4.39 (m, eCH2e, 4H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 175.3, 165.0, 151.3, 149.8, 149.7, 134.6, 131.9, 131.5, 129.0, 127.9, 122.8, 119.0, 110.0, 68.4, 60.8, 51.1, 21.7 ppm; MS (ESI): m/z 413 [MþNa]þ; HRMS (ESI) calcd. for C17H17FN4O4 [MþNa]þ, 413.0873; found, 413.0872.

6.1.37. Synthesis of 1-(2-hydroxy-3-(2-methyl-5-nitro-1Himidazol-1-yl)propyl)-4-oxo-6-(trifluoromethyl) -1,4dihydroquinoline-3-carboxylic acid (7d) Compound 7d was prepared according to the procedure depicted for compound 6a, starting from compound 5d (0.468 g, 1.0 mmol). The product 7d (0.396 g) was obtained as white solid. Yield: 90.0%; m.p: >250  C; IR (KBr, cm1) n: 3441 (OeH), 3130 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1708, 1682 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 15.69 (s, eCOOH, 1H), 8.65 (s, quinolone-2-H, 1H), 8.45 (s, quinolone-5-H, 1H), 8.30 (m, quinolone-6-H and imidazole-4-H, 2H), 7.80 (m, quinolone-8-H, 1H), 5.94 (s, eOH, 1H), 4.81 (m, eCHe, 1H), 4.17e4.43 (m, eCH2e, 4H), 3.42 (s, imidazole-2-CH3, 3H) ppm; 13 C NMR (75 MHz, DMSO-d6): d 175.6, 164.8, 121.6, 149.8, 145.2, 139.4, 134.6, 135.5, 129.4, 129.9, 132.6, 121.2, 110.1, 109.9, 68.4, 60.2, 51.1, 21.7 ppm; MS (ESI): m/z 441 [MþH]þ; HRMS (ESI) calcd. for C18H15F3N4O6 [MþH]þ, 441.1202; found, 441.1203.

6.1.38. Synthesis of 7-chloro-6-fluoro-1-(2-hydroxy-3-(2-methyl-5nitro-1H-imidazol-1-yl)propyl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid (7e) Compound 7e was prepared according to the procedure depicted for compound 6a, starting from compound 5e (0.452 g, 1.0 mmol). The product 7e (0.388 g) was obtained as white solid. Yield: 91.5%; m.p: >250  C; IR (KBr, cm1) n: 3434 (OeH), 3125 (AreH), 3016 (]CeH), 2998, 2912 (CH2, CH3), 1728, 1684 (C]O), 1631, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 8.57 (s, quinolone-2-H, 1H), 8.30 (s, imidazole-4-H, 1H), 8.23 (d, J ¼ 3.3 Hz, quinolone-5-H, 1H), 8.05 (s, quinolone-8-H, 1H), 5.86 (s, eOH, 1H), 4.78 (m, eCHe, 1H), 4.26e4.57 (m, eCH2e, 4H), 3.42 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 176.4, 165.9, 150.3, 149.8, 145.2, 141.8, 134.6, 132.6, 131.5, 129.0, 123.9, 115.0, 109.9, 68.4, 60.7, 51.0, 21.7 ppm; MS (ESI): m/z 425 [MþH]þ; HRMS (ESI) calcd. for C17H14ClFN4O6 [MþH]þ, 425.0664; found, 425.0662.

6.1.39. Synthesis of 6,8-dichloro-1-(2-hydroxy-3-(2-methyl-5nitro-1H-imidazol-1-yl)propyl)-4-oxo 1,4-dihydroquinoline-3carboxylic acid (7f) Compound 7f was prepared according to the procedure depicted for compound 6a, starting from compound 5f (0.468 g, 1.0 mmol). The product 7f (0.391 g) was obtained as white solid. Yield: 81.2%; m.p: >250  C; IR (KBr, cm1) n: 3439 (OeH), 3127 (AreH), 3016 (]CeH), 2996, 2903 (CH2, CH3), 1721, 1684 (C]O), 1621, 1555, 1497, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 15.42 (s, eCOOH, 1H), 8.48 (s, quinolone-2-H, 1H), 8.30 (s, imidazole-4-H, 1H), 8.02e8.15 (m, quinolone-5-H, 1H), 7.68 (s, quinolone-7-H, 1H), 5.87 (s, eOH, 1H), 4.87 (m, eCHe, 1H), 4.16e4.32 (m, eCH2e, 4H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 175.3, 164.9, 149.8, 147.8, 139.8, 136.5, 134.6, 132.7, 131.5, 129.0, 131.1, 122.6, 110.0, 68.5, 60.8, 51.0, 21.7 ppm; MS (ESI): m/z 441 [MþH]þ; HRMS (ESI) calcd. for C17H14Cl2N4O6 [MþH]þ, 441.0369; found, 441.0368.

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6.1.40. Synthesis of 8-fluoro-1-(2-hydroxy-3-(2-methyl-5-nitro1H-imidazol-1-yl)propyl)-4-oxo -1,4-dihydroquinoline-3-carboxylic acid (7g) Compound 7g was prepared according to the procedure depicted for compound 6a, starting from compound 5g (0.418 g, 1.0 mmol), potassium carbonate (0.151 g, 1.2 mmol) and 1H-1,2,4triazole (0.691 g, 1.0 mmol). The product 7g (0.398 g) was obtained as white solid. Yield: 86.3%; m.p: >250  C; IR (KBr, cm1) n: 3417 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1614, 1557, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSO-d6): d 14.58 (s, eCOOH, 1H), 8.51 (s, quinolone-2H, 1H), 8.31 (s, imidazole-4-H, H), 8.12 (m, quinolone-5-H, 1H), 7.65e7.72 (m, quinolone-7-H, 1H), 7.43e7.50 (m, quinolone-6-H, 1H), 5.87 (s, eOH, 1H), 4.74 (m, eCHe, 1H), 4.15e4.43 (m, eCH2e, 4H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSOd6): d 176.0, 165.3, 152.9, 149.9, 146.2, 134.6, 132.7, 131.5, 129.0, 125.6, 122.8, 109.5, 119.0, 68.4, 60.8, 51.1, 21.7 ppm; MS (ESI): m/z 413 [MþNa]þ; HRMS (ESI) calcd. for C17H17FN4O4 [MþNa]þ, 413.0873; found, 413.0872. 6.1.41. Synthesis of 1-(2-hydroxy-3-(2-methyl-5-nitro-1Himidazol-1-yl)propyl)-6-methoxy-4-oxo-1,4-dihydroquinoline-3carboxylic acid (7h) Compound 7h was prepared according to the procedure depicted for compound 6a, starting from compound 5h (0.430 g, 1.0 mmol). The product 7h (0.326 g) was obtained as white solid. Yield: 81.2%; m.p: >250  C; IR (KBr, cm1) n: 3413 (OeH), 3129 (AreH), 3016 (]CeH), 2989, 2902 (CH2, CH3), 1713, 1682 (C]O), 1611, 1556, 1494, 1400 (aromatic frame); 1H NMR (300 MHz, DMSOd6): d 15.41 (s, eCOOH, 1H), 8.57 (s, quinolone-2-H, H), 8.34 (s, imidazole-4-H, H), 7.93 (s, quinolone-5-H, 1H), 7.67 (d, J ¼ 4.2 Hz, quinolone-7-H, 1H), 7.40 (m, quinolone-8-H, 1H), 5.95 (s, eOH, 1H), 4.70 (m, eCHe, 1H), 4.12e4.36 (m, eCH2e, 4H), 3.87 (s, quinolone6-OCH3, 3H), 2.41 (s, imidazole-2-CH3, 3H) ppm; 13C NMR (75 MHz, DMSO-d6): d 174.9, 165.4, 147.7, 145.3, 134.6, 135.7, 126.9, 132.7, 122.3, 120.8, 110.6, 109.0, 68.4, 60.8, 55.9, 51.1, 25.7, 21.7 ppm; MS (ESI): m/z 403 [MþH]þ; HRMS (ESI) calcd. for C18H18N4O7 [MþH]þ, 403.1254; found, 403.1254. 6.2. Antibacterial and antifungal assays Minimal inhibitory concentration (MIC, mg/mL) is defined as the lowest concentration of target compounds that completely inhibit the growth of bacteria, by means of standard two-fold serial dilution method in 96-well microtest plates according to the National Committee for Clinical Laboratory Standards (NCCLS). The tested microorganism strains were provided by the School of Pharmaceutical Sciences, Southwest University and the College of Pharmacy, Third Military Medical University. Chloromycin, Norfloxacin and Fluconazole were used as control drugs. DMSO with inoculation bacterial not medicine was used as positive control to ensure that the solvent had no effect on bacteria growth. All the bacteria and fungi growth was monitored visually and spectrophotometrically, and the experiments were performed in triplicate. The MIC values in mg/mL were summarized in Table 1 and Supporting Information. 6.2.1. Antibacterial assays The prepared compounds 3e7 were evaluated for their antibacterial activities against Gram-positive bacteria (S. aureus ATCC 6538, Methicillin-resistant Staphylococcus aureus N315 (MRSA) and B. subtilis ATCC 21216), Gram-negative bacteria (E. coli ATCC 8099, P. aeruginosa ATCC 27853 and B. proteus ATCC 13315). The bacterial suspension was adjusted with sterile saline to a concentration of 1  105 CFU. Initially the compounds were dissolved in DMSO to

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prepare the stock solutions, then the tested compounds and reference drugs were prepared in MuellereHinton broth (Guangdong huaikai microbial sci. & tech co., Ltd, Guangzhou, Guangdong, China) to obtain the required concentrations of 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 mg/mL. These dilutions were inoculated and incubated at 37  C for 24 h. 6.2.2. Antifungal assays The newly synthesized compounds 3e7 were evaluated for their antifungal activities against C. albicans ATCC 76615, A. fumigatus ATCC 96918, C. utilis, S. cerevisia and A. flavus. A spore suspension in sterile distilled water was prepared from one day old culture of the fungi growing on Sabouraud agar (SA) media. The final spore concentration was 1e5  103 spore mL1. From the stock solutions of the tested compounds and reference antifungal drug Fluconazole, dilutions in sterile RPMI 1640 medium (Neuronbc Laboraton Technology CO., Ltd, Beijing, China) were made resulting in eleven wanted concentrations (0.25e256 mg/mL) of each tested compound. These dilutions were inoculated and incubated at 35  C for 24 h. 6.3. Cytotoxicity assays A549 cells and human hepatocyte LO2 cells were plated in 96well plates in 100 mL of medium and allowed to attach for 24 h, and then 100 mL of medium containing an appropriate dilution of the test compounds was added. Cells and compounds were incubated at 37  C in 5% CO2 humidified incubator for 2 days. Cell viability was determined by using CCK-8 kit and MTT respectively. Absorbance of converted dye was measured in an ELISA plate reader at the wavelength of 450 nm and 490 nm respectively. The cytotoxicity of the compounds was tested for three times and calculated as the reduction percentage of viable cells observed in the drug-free control cell culture. 6.4. Assays of pKa values Buffer stock solutions of 500 mM were prepared and adjusted to the final pH as follows: acetic acidesodium acetate (pH ¼ 4.0, 4.5, and 5.0), MES (pH ¼ 5.5, 6.0, and 6.5), HEPES (pH ¼ 7.0, 7.5, and 8.0), and CHES (pH ¼ 8.5, 8.8, 9.1, and 9.4). Analyte compound stock solutions were prepared at 4.0  103 mol/L in DMSO. Quartz cell was loaded with 2 mL of buffer solutions. Then, 50 mL of the compound stock solutions were added to the quartz cell with a micropipette (the resulting analyte solution was premixed with the micropipette), which was incubated at room temperature and shaken for 10 min before the reading was performed. UV-spectra scans were recorded between 210 nm and 400 nm (1 nm resolution). 6.5. Assays of effect of metal ions to compoundseHSA complex All fluorescence spectra were recorded on F-7000 Spectrofluorimeter (Hitachi, Tokyo, Japan) equipped with 1.0 cm quartz cells, the widths of both the excitation and emission slit were set as 2.5 nm, and the excitation wavelength was 295 nm. Fluorescence spectra were recorded at 298 K in the range of 300e450 nm. HSA was obtained from SigmaeAldrich (St. Louis, MO, USA). Tris, NaCl, HCl, CaCl2, KCl, MgCl2, and NiCl2 were analytical purity. HSA was dissolved in TriseHCl buffer solution (0.05 M Tris, 0.15 M NaCl, pH ¼ 7.4). Sample masses were weighed on a microbalance with a resolution of 0.1 mg. All other chemicals and solvents were commercially available, and were used without further purification.

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