Novel 3-Aminothiazolquinolones: Design, Synthesis, Bioactive Evaluation, SARs, and Preliminary Antibacterial Mechanism

Article abstract of DOI:10.1021/acs.jmedchem.5b01678

A series of novel 3-aminothiazolquinolones as analogues of quinolone antibacterial agents were designed and synthesized in an effort to circumvent quinolone resistance. Among these 3-aminothiazolquinolones, 3-(2-aminothiazol-4-yl)-7-chloro-6-(pyrrolidin-1-yl) quinolone 12b exhibited potent antibacterial activity, low cytotoxicity to hepatocyte cells, strong inhibitory potency to DNA gyrase, and a broad antimicrobial spectrum including against multidrug-resistant strains. This active molecule 12b also induced bacterial resistance more slowly than norfloxacin. Analysis of structure-activity relationships (SARs) disclosed that the 2-aminothiazole fragment at the 3-position of quinolone plays an important role in exerting antibacterial activity. Molecular modeling and experimental investigation of aminothiazolquinolone 12b with DNA from a sensitive methicillin-resistant Staphylococcus aureus (MRSA) strain revealed that the possible antibacterial mechanism might be related to the formation of a compound 12b-Cu²⁺-DNA ternary complex in which the Cu²⁺ ion acts as a bridge between the backbone of 3-aminothiazolquinolone and the phosphate group of the nucleic acid.

Full text of DOI:10.1021/acs.jmedchem.5b01678

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Article
Novel 3-Aminothiazolquinolones: Design, Synthesis, Bioactive
Evaluation, SARs and Preliminary Antibacterial Mechanism
Sheng-Feng Cui, Dinesh Addla, and Cheng-He Zhou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01678 • Publication Date (Web): 26 Apr 2016
Downloaded from http://pubs.acs.org on April 27, 2016
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Novel 3ꢀAminothiazolquinolones: Design,
Synthesis, Bioactive Evaluation, SARs and
Preliminary Antibacterial Mechanism
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ShengꢀFeng Cui , Dinesh Addla , ChengꢀHe Zhou *
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Institute of Bioorganic & Medicinal Chemistry, Key Laboratory of Applied Chemistry of
Chongqing Municipality, School of Chemistry and Chemical Engineering, Southwest University,
Chongqing 400715, China
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Department of Public Security Technology, Railway Police College, Zhengzhou 450053, China
KEYWORDS: Thiazole, Quinolone, Antibacterial, HSA, DNA
ABSTRACT: A series of novel 3ꢀaminothiazolquinolones as analogs of quinolone antibacterial
agents were designed and synthesized in an effort to circumvent quinolone resistance. Among
these 3ꢀaminothiazolquinolones, 3ꢀ(2ꢀaminothiazolꢀ4ꢀyl)ꢀ7ꢀchloroꢀ6ꢀ(pyrrolidinꢀ1ꢀyl) quinolone
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2b exhibited potent antibacterial activity, low cytotoxicity to hepatocyte cells, strong inhibitory
potency to DNA gyrase and a broad antimicrobial spectrum including against multidrugꢀresistant
strains. This active molecule 12b also induced bacterial resistance more slowly than Norfloxacin.
Analysis of structureꢀactivity relationships (SARs) disclosed that the 2ꢀaminothiazole fragment at
the 3ꢀposition of quinolone plays an important role in exerting antibacterial activity. Molecular
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modeling and experimental investigation of aminothiazolquinolone 12b with DNA from a
sensitive methicillinꢀresistant Staphylococcus aureus (MRSA) strain revealed that the possible
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antibacterial mechanism might be related to the formation of a compound 12bꢀCu ꢀDNA ternary
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complex, in which the Cu
ion acts as a bridge between the backbone of 3ꢀ
aminothiazolquinolone and the phosphate group of the nucleic acid.
1. INTRODUCTION
Quinolones with a 3ꢀcarboxyl benzopyridone skeleton are among the most important firstꢀline
antibacterial drugs because of their good treatment effectiveness, broad antibacterial spectra and
suitable pharmacokinetics. Since this 3ꢀcarboxyl benzopyridone skeleton was discovered in the
1960s, structural modifications based on the skeleton have become an extremely attractive area
of research, and many quinolones such as Levofloxacin, Sparfloxacin, Grepafloxacin,
Trovafloxacin, Gatifloxacin and Moxifloxacin have been successfully and widely used in the
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clinic to treat genitourinary infections and common respiratory tract pathogens. The structureꢀ
activity relationships (SARs) of quinolone antibacterial drugs have been clearly summarized and
elucidated, disclosing that the special 3ꢀcarboxylic benzopyridone skeleton plays an important
role in antimicrobial activity. Based on many valuable SAR studies, the 3ꢀcarboxyl and 4ꢀ
carbonyl groups of quinolone can form stable complexes with DNAꢀgyrase or DNAꢀ
topoisomerase IV via a waterꢀmetal ion bridge to trigger irreparable DNA breakage, ultimately
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resulting in the death of the bacterial cell. However, due to the wide use and even abuse of
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quinolone drugs in the clinic, the chelating ability of the carbonyl and carboxyl moieties tends to
damage the bacterial DNA and trigger an errorꢀprone signal to the DNA repair system, which
leads to specific mutations in gyrase and topoisomerase IV and an increase in quinoloneꢀresistant
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bacterial strains. Moreover, many of the severe side effects of quinolone drugs such as
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gastroenteritis, vomiting and cartilage damage have been shown to be mainly induced by the
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carboxyl moieties. Therefore, structural modification at the Cꢀ3 position should be a promising
pathway to change the classic binding mode of quinolone to the enzymeꢀDNA complex and
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overcome the resistance, as well as the side effects caused by the carboxyl group.
It is well known that 2ꢀaminothiazole, as an quite important aromatic fiveꢀmembered
heterocycle, is present in a variety of clinical drugs, such as the antibacterial drugs
cephalosporins Cefodizime, Cefoselis and Cefmenoxime, the anticancer drug Dasatinib and the
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antiꢀinflammatory drug Meloxicam.
The successful development of a variety of clinical 2ꢀ
aminothiazole drugs has motivated extensive efforts to construct further bioactive molecules
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based on this fragment. Structural modifications of clinical drugs by introducing a 2ꢀ
aminothiazole group is one of the most convenient and rewarding methods to exploit new
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medicinal agents. These 2ꢀaminothiazoleꢀmodified drugs can generally overcome severe
resistance and possess strong bioactivity, a high safety profile and good binding ability with their
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functional targets. For example, the thirdꢀgeneration cephalosporin Cefdinir, the fourthꢀ
generation cephalosporin Cefepime and the fifthꢀgeneration cephalosporin Tigemonam all have
good inhibitory potency against bacterial strains, including resistant ones, and have been widely
used in the clinic to treat various types of infections (Figure 1). Furthermore, some 2ꢀ
aminothiazole derivatives have been shown to be highly successful inhibitors of bacterial DNA
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gyrase, with the same targets as antibacterial quinolone drugs. Therefore, the structural
modification of quinolone by introducing a 2ꢀaminothiazole fragment should be a highly
attractive topic in the antibacterial field.
In our previous works, the introduction of azoles, such as triazoles, imidazoles, and
nitroimidazoles, into the quinolone backbone was shown to not only improve antimicrobial
potencies but also widen the antimicrobial spectrum, including against methicillinꢀresistant
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Staphylococcus aureus (MRSA).
These hybrids of azoles and quinolones exhibit good
antibacterial activity with low Minimum Inhibitory Concentration (MIC) values and strong
binding ability to the DNAꢀtopoisomerase IV complex. In view of the above considerations and
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as an extension of our previous work,
herein we for the first time replaced the carboxyl group
of quinolone with a 2ꢀaminothiazole fragment to produce a series of novelꢀstructure 3ꢀ(2ꢀ
aminothiazolꢀ4ꢀyl) quinolones. The 2ꢀaminothiazole introduced at the 3ꢀposition of quinolone
was expected to change the classic binding model of quinolone with enzymeꢀDNA complexes to
overcome the resistance and side effects caused by the carboxyl group. Therefore, the
antimicrobial activities in vitro for the target 3ꢀaminothiazolquinolones and some intermediates
were evaluated against eight bacterial strains including MRSA. The SARs, cytotoxicity,
inhibitory activity of DNA gyrase and the development of resistance by MRSA and methicillinꢀ
and quinoloneꢀresistant Staphylococcus aureus ATCC 700699 (MQRSA) to the highly active
molecule were also evaluated. The binding behavior of the highly active molecule to human
serum albumin (HSA) was investigated by fluorescence spectroscopy to preliminarily study its
absorption, distribution, and metabolism. Moreover, the molecular modeling and experimental
investigation of highly active 3ꢀaminothiazolquinolone with genomic DNA isolated from a
sensitive MRSA strain were further studied to explore the possible antibacterial mechanism of
these new 3ꢀaminothiazolquinolones.
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Substitution
COOH
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OH
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S
N
H H
H
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RO
Clinical antibacterial cephalosporins
Antibacterial quinolones
Novel hybrids
NH2
NH2
NH2
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Figure 1. Design of novel 3ꢀ(2ꢀaminothiazolꢀ4ꢀyl) quinolones.
2. CHEMISTRY
The synthetic routes of the target 3ꢀ(2ꢀaminothiazolꢀ4ꢀyl) quinolones are outlined in Schemes
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–3. Ethyl 2ꢀ(ethoxymethylene)ꢀ3ꢀoxobutanoate 1 was easily prepared by the reaction of
commercial triethoxymethane, ethyl 3ꢀoxobutanoate and propionic anhydride. The synthesized
intermediate 1 was reacted with a series of substituted phenylamines in the absence of solvent to
afford phenylamino butanoates 2a–i in almost quantitative yields, and then the latter were further
cyclized in phenoxybenzene under reflux conditions to produce the desired 3ꢀacetyl quinolones
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a–i in moderate yields ranging from 42.3% to 56.1%. Further Nꢀalkylation of compounds 3a–i
by bromoethane gave 3ꢀacetylꢀ1ꢀethyl derivatives 4a–i. Further bromination of 4a–i by bromine
in acetic acid produced the corresponding 3ꢀ(2ꢀbromoacetyl) quinolones 5a–i. The cyclization of
the bromoacetyl groups at the 3ꢀpositions of compounds 5a–i with commercial thiourea in
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ethanol at 60 °C readily yielded the target 3ꢀ(2ꢀaminothiazolꢀ4ꢀyl) quinolones 6a–i in high yields
ranging from 76.2% to 86.5% (Scheme 1).
a
Scheme 1. Synthetic route of quinolone thiazoles 6a−i
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Reagents and conditions: (i) ethyl 3ꢀoxobutanoate, propionic anhydride, reflux, 2 h; (ii)
substituted aniline, 130 °C, 10 min; (iii) phenoxybenzene, reflux, 1.5 h; (iv) bromoethane, DMF,
K CO , 100 °C, 12 h; (v) bromine, acetic acid, 0−40 °C, 6 h; (vi) thiourea, ethanol, 60 °C, 3 h.
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As shown in Schemes 2 and 3, the target 3ꢀaminothiazolquinolones 9a–e and 12a–e were
prepared starting from the intermediate 7ꢀchloroꢀ6ꢀfluoroquinolone 4e. The substitution of
compound 4e with alicyclic amines in DMSO at 130 °C using triethylamine as a base afforded a
mixture of 7ꢀposition substituted quinolones 7a–d with relatively high yields of 40.5–49.1% and
6ꢀposition substituted quinolones 10a–d in slightly lower yields of 31.6–35.4%. It was observed
that 6ꢀposition substitution by pyrrolidine gave higher yields than piperidine and morpholine.
The 7ꢀalicyclic amino compounds 7a–d and 6ꢀalicyclic amino derivatives 10a–d were reacted
with bromine in acetic acid to produce the corresponding 3ꢀ(2ꢀbromoacetyl) quinolones 8a–d and
11a–d, respectively, and then further cyclizations with thiourea in ethanol at 60 °C produced
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target aminothiazolquinolones 9a–d and 12a–d, respectively. The deprotection of the Boc groups
in compounds 9d and 12d in a solution of trifluoroacetic acidꢀCH Cl produced the
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corresponding target compounds 9e and 12e, respectively. All the structures of the new
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compounds were confirmed by H NMR, C NMR, MS, HRMS and IR spectra. Furthermore,
1,3,5ꢀtrioxane was employed to check the purity of the target compounds using the quantitative
nuclear magnetic resonance (QNMR) method. The results indicated that all the target compounds
possess a purity of at least 95%, and their spectral data are provided in the experimental section.
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Scheme 2. Synthetic route of quinolone thiazoles 9a−e
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Reagents and conditions: (vii) DMSO, triethylamine, alicyclic amines, 130 C, 24 h; (viii)
bromine, acetic acid, 0−40 C, 4 h; (ix) thiourea, ethanol, reflux, 3 h; (xiii) CF COOH, CH Cl .
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Scheme 3. Synthetic route of quinolone thiazoles 12a−e
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. BIOLOGICAL EVALUATION AND DISCUSSION
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.1. In Vitro Antibacterial Activity. The antibacterial results (Table 1) reveal that most of the
intermediates and target compounds display biological activity in vitro against the eight tested
bacterial strains. Most of the intermediates 3ꢀacetylꢀ1ꢀethyl quinolones 4a–i, 7a–d and 10a–d, a
series of products substituted at the 6ꢀ, 7ꢀ and 8ꢀpositions of the 3ꢀacetyl quinolone skeleton,
showed weak activities against the eight bacteria. Nevertheless, 6ꢀfluoroꢀ7ꢀmorpholino quinolone
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c exhibited much stronger antimicrobial efficacy against Gramꢀnegative bacterial stains B.
proteus and E. typhosa, with low MIC values of 2.7 and 2.3 µg/mL, than Norfloxacin (MIC =
3.3 and 5.3 µg/mL) and Chloromycin (MIC = 32.0 and 26.6 µg/mL). 6ꢀFluoroꢀ7ꢀ(pyrrolidinꢀ1ꢀ
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yl) quinolone 7b also showed good antiꢀS. aureus activity, with an MIC value of 5.1 µg/mL.
The introduction of 2ꢀaminothiazole at the 3ꢀposition of quinolone yielded compound series 6.
None of the tested strains were sensitive to 6,8ꢀdifluoro quinolone thiazole 6a or 8ꢀfluoro
quinolone thiazole 6c, which suggested that the fluorine modification at the 8ꢀposition of
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quinolone is not beneficial for antibacterial activity. To further study the SARs of this new class
of quinolone thiazoles, electronꢀwithdrawing (CF , F, Cl) and electronꢀdonating groups (methyl,
3
methoxyl) were introduced at the 6ꢀ and 7ꢀpositions of this quinolone scaffold. These
modifications produce a clear relationship between the antibacterial activity and the different
substituents at the 6ꢀ and 7ꢀpositions of quinolone. The electronꢀwithdrawing groups are effective
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at enhancing antiꢀGramꢀnegative bacterial activities. Specifically, 7ꢀtrifluoromethyl derivative 6d
showed good MIC values (1.7–13.3 µg/mL) against all tested Gramꢀnegative strains, including B.
proteus, P. aeruginosa, E. typhosa, and E. coli DH52, with much stronger antibacterial potency
than reference drugs. Both 6,7ꢀdifluoro aminothiazolquinolone 6b and 6ꢀfluoro derivative 6g
exhibit superior or comparable inhibitory potency against the tested Gramꢀnegative bacteria
compared to Chloromycin and Norfloxacin. 6,7ꢀDifluoro compound 6b could not only efficiently
inhibit the growth of B. proteus (MIC = 0.6 µg/mL) and E. typhosa (MIC = 1.2 µg/mL), with
activities 22ꢀ and 4ꢀfold greater in comparison to Norfloxacin, but it also displayed inhibitory
potency against Gramꢀpositive bacteria including M. luteus and S. aureus at low concentrations.
On the other hand, 6ꢀfluoro aminothiazolquinolone 6g showed slightly weaker antibacterial
activity against the tested strains than the 6,7ꢀdifluoro derivative 6b, which suggests that the
substituents at the 7ꢀposition of the quinolone ring should have an important effect on the
antibacterial activity.
7ꢀChloroꢀ6ꢀfluoro quinolone thiazole 6e not only exhibits effective inhibition toward the
growth of Gramꢀnegative bacteria but also enhances antiꢀGramꢀpositive bacterial potency. It
exhibited good antiꢀMRSA activity (MIC = 8.0 µg/mL), which was more effective than clinical
Norfloxacin and Chloromycin, with MIC values of 10.7 and 21.3 µg/mL, respectively.
Compared to electronꢀwithdrawing groups, quinolone thiazoles 6h and 6i with electronꢀ
donating methyl and methoxyl groups showed better antiꢀGramꢀpositive bacterial potency,
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especially against S. aureus strains. Generally, the target 3ꢀ(2ꢀaminothiazolꢀ4ꢀyl) quinolones
showed much higher inhibitory potency against all tested strains than most of their corresponding
intermediates. This phenomenon suggests that the aminothiazolyl substituents at the 3ꢀposition of
quinolones should play an important role in exerting antibacterial potency. On the other hand, the
electronꢀdonating groups at the 6ꢀ and 7ꢀpositions of quinolone were helpful for improving the
antiꢀGramꢀpositive bacterial efficacy, whereas the electronꢀwithdrawing groups were beneficial
to the antiꢀGramꢀnegative bacterial potency.
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9
0
Based on these obtained results, both electronꢀdonating and electronꢀwithdrawing groups were
incorporated into the aminothiazolquinolone backbone, and the nucleophilic substitution at the 6ꢀ
and 7ꢀpositions of the 7ꢀchloroꢀ6ꢀfluoroquinolone nucleus 4e provided some important results.
By introducing piperidine, pyrrolidine, morpholine and piperazine into the 6ꢀ and 7ꢀpositions, the
7ꢀposition substituted series 7 and 6ꢀposition substituted series 10 were obtained, which showed
weak inhibitory potency toward most of the evaluated bacteria. Nevertheless, intermediates 7a−d
and 10b showed good inhibitory activity against S. aureus and MRSA strains. The active
molecule 7a, with a piperidineꢀ1ꢀyl group at the 7ꢀposition of quinolone, and 7ꢀchloroꢀ6ꢀ
(pyrrolidinꢀ1ꢀyl) quinolone 10b gave especially low inhibitory concentrations (MIC = 17.8 and
16.0 µg/mL) against the MRSA strain, with stronger activity than the antibacterial Chloromycin
(
MIC = 21.3 µg/mL) and slightly weaker than Norfloxacin (MIC = 10.7 µg/mL).
ꢀFluoroꢀ7ꢀmorpholino aminothiazolquinolone 9c exhibited only good antiꢀS. aureus (MIC =
.8 µg/mL) and antiꢀB. proteus (MIC = 1.7 µg/mL) potency. The desired targets 6ꢀfluoroꢀ7ꢀBocꢀ
6
0
piperazinyl aminothiazolquinolone 9d and 6ꢀfluoroꢀ7ꢀpiperazinyl aminothiazolquinolone 9e
showed low MIC values against the tested Gramꢀnegative bacteria but demonstrated moderate
antiꢀGramꢀpositive activity. Meanwhile, 6ꢀfluoroꢀ7ꢀ(piperidinꢀ1ꢀyl) aminothiazolquinolone 9a
and 6ꢀfluoroꢀ7ꢀ(pyrrolidinꢀ1ꢀyl) compound 9b also displayed disappointing results, with poor
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antibacterial activity against the tested bacteria. Generally, compounds 9a−d did not achieve the
desired results. However, a turning point in our work was achieved by testing 7ꢀchloroꢀ6ꢀ
(pyrrolidineꢀ1ꢀyl) aminothiazolquinolone 12b, which was previously obtained as a byproduct and
10
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12
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displayed a remarkable inhibitory potency against all tested strains. Notably, the tested Gramꢀ
negative bacteria including E. Coli DH52, E. typhosa, P. aeruginosa and B. proteus were quite
sensitive to 7ꢀchloroꢀ6ꢀ(pyrrolidinꢀ1ꢀyl) aminothiazolquinolone 12b, with low MIC values of
0
.5–2.0 µg/mL, which was superior antibacterial activity to that of Chloromycin and Norfloxacin.
The antiꢀGramꢀpositive bacterial activity against M. luteus, S. aureus and B. subtilis (MIC = 2.7,
.6, and 1.3 µg/mL, respectively) was also comparable or even better in contrast to clinical
0
Norfloxacin. Excitingly, the target compound 12b exhibited remarkable biological activity
toward MRSA at quite a low concentration (MIC = 0.8 µg/mL), being 13ꢀ and 27ꢀfold more
active than Norfloxacin and Chloromycin, respectively. Unfortunately, 7ꢀchloroꢀ6ꢀ(piperidinꢀ1ꢀyl)
compound 12a, 7ꢀchloroꢀ6ꢀmorpholin compound 12c, 7ꢀchloroꢀ6ꢀBocꢀpiperazinyl compound
12d and 7ꢀchloroꢀ6ꢀpiperazinyl compound 12e showed poor or even no activity against most of
the tested bacterial strains, which suggested that the stereospecific blockade of the sixꢀmembered
ring at the 6ꢀposition of quinolone should be negative with respect to the antibacterial potency.
a,b
Table 1. Antibacterial MIC values of compounds 6, 7, 9, 10 and 12 (µg/mL)
Gramꢀpositive bacterial strains
Gramꢀnegative bacterial strains
Compounds
M. luteus
MRSA
S. aureus
B. subtilis
P. aeruginosa
E. coli
B. proteus
E. typhosa
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1
1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
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5
5
5
5
5
5
5
5
6
6
a
256
5.3
NA
213.3
NA
NA
0.8
NA
26.7
13.3
63.9
10.6
2.7
NA
78.6
24.0
13.3
1.6
64
256
NA
0.6
NA
1.2
6b
6
c
2.7
NA
9.3
256
13.3
3.3
NA
6
d
NA
53.2
8.0
127.9
NA
5.3
1.7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6e
26.6
16
6.7
2.6
6
f
NA
3.0
2.3
21.2
24.0
13.3
4.7
3.3
2.7
6
6
g
18.7
42.7
5.4
NA
128.0
18.8
2.4
3.3
13.3
21.3
2.7
10.7
2.7
21.3
8.0
h
256
10.7
64.4
12.7
244.5
NA
6i
128.8
17.8
50.9
256
0.8
24.1
121.2
122.2
2.3
7
7
a
10.1
244.4
128.0
NA
8.9
22.8
12.7
256
NA
40.6
43.3
256
20.3
33.1
2.7
b
5.1
7c
53.3
16.0
64.0
32.0
0.8
7d
9a
9b
64.0
53.3
17.3
32.0
10.7
32.1
128.0
16.0
128.0
24.6
0.8
21.3
42.7
53.3
18.7
42.7
10.7
35.4
10.7
NA
128.0
128.0
9.3
16.0
37.3
128.0
1.7
42.7
74.7
42.7
14.7
5.3
NA
42.7
42.7
10.7
8.0
106.7
13.3
32.0
64.2
37.3
10.7
53.3
49.0
2.7
9
c
6.0
9
d
64.0
16.0
37.3
18.7
NA
10.7
16.0
13.3
26.7
NA
2.6
9
e
4.0
2.0
10.7
NA
10a
0b
0c
12a
2b
2c
2d
42.7
9.3
170.7
64.0
13.3
34.4
1.0
1
18.7
37.3
17.2
2.0
1
42.7
34.4
0.5
61.2
0.6
19.6
1.3
117.8
0.8
1
1
127.7
53.2
32.0
13.3
2.7
255.3
32.0
NA
0.8
255.3
21.3
2.0
170.2
8.0
127.7
10.7
128.0
13.8
4.3
NA
95.7
NA
1
127.7
8.0
32.0
6.4
12e
10.7
32.0
1.7
8.0
Chloromycin
Norfloxacin
21.3
10.7
16.0
0.8
26.6
1.3
32.0
13.3
26.6
5.3
a
Micrococcus luteus ATCC 4698, M. luteus; MethicillinꢀResistant Staphylococcus aureus
N315, MRSA; Staphylococcus aureus ATCC 25923, S. aureus; Bacillus subtilis ATCC 6633, B.
subtilis; Pseudomonas aeruginosa ATCC 9027, P. aeruginosa; Escherichia coli ATCC 25922,
E. Coli; Bacillus proteus ATCC 13315, B. proteus; Eberthella typhosa ATCC 14028, E.
b
typhosa. NA, no activity.
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3
.2. Cell Toxicity and DNA Gyrase Inhibitory Activity. The highly bioactive 3ꢀ
aminothiazolquinolone 12b was further evaluated for its toxicity against PC 12 cancer and
normal human hepatocyte LO2 cells using the colorimetric cell proliferation MTT assay.
Norfloxacin was selected as a positive control to reduce the impact of substituents at the Nꢀ
position of the quinolones. The cell viability against 3ꢀaminothiazolquinlone 12b of both PC 12
and human hepatocyte LO2 cells was more than 88%, which suggested that this molecule
showed low toxicity to the cells at concentrations below 512 µg/mL (Figure 2). On the whole, the
cell viability against 3ꢀaminothiazolquinlone 12b was higher than that against Norfloxacin at
concentrations of 128, 256 and 512 µg/mL, which indicated that the new 3ꢀaminothiazolquinlone
showed lower cell toxicity than the positive control Norfloxacin.
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135
120
105
Hepatocyte LO2
PC 12
90
75
60
45
30
15
0
8
1
2
16
3
3
2
6
4
4
1
5
28
2
6
56
512
7
Concentration of compound 12b (ꢀg/mL)
Figure 2. Cytotoxicity assay with 3ꢀaminothiazolquinlone 12b in the PC 12 and human
hepatocyte LO2 cell lines tested by MTT methodology. Each data bar is an average of three
replicates.
To investigate inhibitory activity against the target enzymes of quinolones at a molecular level,
3ꢀaminothiazolquinlone 12b and Norfloxacin were selected to test their DNA gyrase inhibitory
activity. Table 2 shows that 3ꢀaminothiazolquinlone 12b could inhibit the supercoiling activity of
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2
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DNA gyrase with a low IC50 value of 11.5 µM, which is better inhibitory potency than the
standard drug Norfloxacin (IC = 18.2 µM).
50
Table 2. Inhibition of DNA gyrase activity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comps
DNA gyrase supercoiling (IC , µM)
50
1
2b
11.5
18.2
Norfloxacin
3.3 Development of Resistance to 3ꢀAminothiazolquinlone 12b. Resistance to quinolones
2
6
among Gramꢀpositive cocci has emerged in recent years. The mutagenic properties of
quinolones raises the possibility of the resistance even in smaller populations of bacteria exposed
to low concentrations of quinolones, which may promote the ease of selection of lowꢀlevel
resistance mutations. Thus, the bacterial resistance against compound 12b was also studied by
investigating the resistance development rates of MRSA and MQRSA strains. Both strains were
exposed to subꢀMIC concentrations of 3ꢀaminothiazolquinlone 12b for sustained passages, and
then the MIC values of compound 12b were determined against each passage of the MQRSA and
2
7
5
MRSA strains. The freshly diluted MRSA and MQRSA strains (1.0 × 10 CFU) in the broth
medium were cultured in 0.5 µg/mL (2/3 MIC) of compound 12b at 37 °C for 12 h on a shaker
bed at 90 rpm, and the sensitivity of each strain passage to compound 12b was tested. After 12
passages of MQRSA and MRSA strains in the subꢀMIC concentration of compound 12b,
bacterial resistance to the original MIC of compound 12b (0.8 µg/mL) did not emerge, and the
MIC values just changed between 0.5 µg/mL and 1.2 µg/mL (Figure 3). Meanwhile, after 7
passages, compound 12b also showed good antiꢀMQRSA activity, with MIC values between 2
µg/mL and 8 µg/mL. On the other hand, to reduce the impact of substituents at the Nꢀposition of
the quinolones, Norfloxacin, bearing the same ethyl group at the Nꢀposition as compound 12b,
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was chosen as a positive control. The MRSA and MQRSA strains quickly developed resistance
to Norfloxacin (MIC = 266.7 µg/mL and 512 µg/mL) and showed strong resistance to the
original MIC (10.7 µg/mL and 16 µg/mL) after just one passage. The Norfloxacin acquired 32ꢀ
fold potency compared to the original MIC after six passages. This assay indicated that the
MRSA and MQRSA strains did not develop significant resistance against 3ꢀ
aminothiazolquinlone 12b as easily as they did against the positive control Norfloxacin.
Therefore, this result indicated that the 3ꢀaminothiazolquinlone was helpful for overcoming the
resistance to clinical quinolones.
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3.0
2.5
2.0
1.5
2
4
6
8
10
12
Passages of MRSA strain
Figure 3. Development of MRSA resistance to compound 12b, each MIC value of every passage
was an average of three parallels.
3.4 StructureꢀActivity Relationship. Structural features play a remarkable role in the
antibacterial potency of these aminothiazolquinolones, especially the substituents at the 3ꢀ, 6ꢀ, 7ꢀ
and 8ꢀpositions of the quinolone skeleton. The following discussions encompass the correlation
of each physical property discussed previously with the antibacterial activity.
3.4.1. Partition Coefficient and Molecular Shape Analysis. The physical characteristics of
drugs govern many types of biological processes including absorption, distribution, and
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secretion. Therefore, it is quite necessary to acquire a good knowledge of these physical
2
9
characteristics such as lipophilicity and hydrophilicity. To achieve ideal pharmacokinetic and
pharmacodynamic properties in a drug, the distribution coefficient should be intermediate
between hydrophilic and hydrophobic. The partition coefficient is frequently used to define the
lipophilic character of a drug. The theoretically calculated value of log P (ClogP) was calculated
using the commercial ChemBioOffice 2010 (Cambridge Soft, Massachusettes, USA) and is
presented in Table 3. In general, all the target compounds were lipophilic (ClogP = 2.88−4.58).
The ClogP values of the quinolone thiazole derivatives 6a−i were related to the number of
fluorine and chlorine atoms in the benzene rings of the quinolones. The ClogP values followed
the order of 7ꢀtrifluoromethyl quinolone (6d) > 6,8ꢀdifluoro derivative (6a) and 6,7ꢀdifluoro
derivative (6b) > 6ꢀfluoro derivative (6g) and 8ꢀfluoro derivative (6c). In comparison to 6,7ꢀ
difluoro quinolone thiazole 6b, the 7ꢀchloroꢀ6ꢀfluoro derivative 6e showed greater lipophilicity,
which suggests that the introduction of a chloro group into the quinolone backbone resulted in a
stronger lipophilicity than that of a fluoro group. Therefore, 6,8ꢀdichloro quinolone thiazole 6f
was the most lipophilic member of series 6. It was also observed that compound 9e, with a
piperazinꢀ1ꢀyl group at the 7ꢀposition of quinolone, possessed the lowest calculated log P value
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(ClogP
=
2.82) among all the target compounds. 7ꢀChloroꢀ6ꢀ(pyrrolidinꢀ1ꢀyl)
4.16) showed stronger lipophilicity than
aminothiazolquinolone 12b (ClogP
=
aminothiazolquinolones 6a−h. 7ꢀChloroꢀ6ꢀ(piperidinꢀ1ꢀyl) aminothiazolquinolone 12a provided
the most hydrophobic surface and the highest value of ClogP (ClogP = 4.58) of all the target
compounds..
Computational methods can be used to predict important pharmacokinetic properties of drug
candidates such as the ability to permeate biological membranes and have been used to
30
rationalize biological activity. The molecular electrostatic potential (MEP) surface gives an
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indication of the charged surface area and hydrophilicity of compounds. Therefore, MEP maps of
the target compounds were generated to investigate key structural features such as steric and
electrostatic interactions, hydrogen donor/acceptor properties and lipophilicity. Table 4 shows a
comparison of the MEP surfaces that were generated for a selection of compounds at the neutral
state via the DFTꢀB3LYP theoretical computation.
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Table 3. ClogP values of compounds 6a−i, 9a−e and 12a−e.
Compds.
ClogP
3.32
3.32
3.16
3.93
3.72
4.12
3.16
Compds.
ClogP
2.88
3.49
4.21
3.76
3.45
3.93
2.82
Compds.
12a
ClogP
4.58
4.16
3.45
4.33
3.22
6
a
6h
6i
6b
12b
6
c
9a
9b
9c
9d
9e
12c
6
d
12d
6
e
12e
6
f
6g
In the comparison to the MEP maps of the two modes of quinolone derivatives, series 4 and
series 6 were taken as examples. The most noticeable difference came from the 2ꢀaminothiazole
fragment at the 3ꢀposition of the quinolone backbone. In the optimized structures of series 6,
sulfur atoms with lone pairs were in similar positions to the carbonyl groups at the 3ꢀposition of
the quinolone drugs and also had a weak ability to interact with the enzyme. On the other hand,
the amino fragments at the 2ꢀposition of the thiazole rings were electronically available with their
lone pairs, which were probably oriented toward the outer part of the molecules and therefore
were accessible to interact with their surroundings. Because of the πꢀπ conjugative effect and
steric hindrance, the nitrogen lone pairs at the 3ꢀposition of the thiazole rings were buried in the
structures and only partially available for interaction with solvents or other molecules. One
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4
4
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6
additional electronegative site could be observed at the 1ꢀposition of quinolone because of the
ethyl group.
Table 4. Comparison of the MEP surfaces of some target 3ꢀaminothiazolquinolones
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Compared to the MEP surfaces of target compounds 6a−i, the electropositively charged zones
of the carbonyl groups at the 4ꢀposition of quinolone thiazoles 9a−e and 12a−e are slightly
decreased, as a result of the introduction of alicyclic amines at the 6ꢀ and 7ꢀpositions of the
quinolone rings. The introduction of amines also led to the exchange of spatial positions between
the 1ꢀposition sulfur atom and 3ꢀposition nitrogen atom in the thiazole ring, which resulted in
positively charged surface areas for the nitrogen atoms of compounds 9a−e and 12a−e. The
electron density associated with the amino group at the 2ꢀposition of the thiazole ring also
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experienced a considerable decrease, which resulted from the inductive and electronic changes
generated by the introduction of amines at the 6ꢀ and 7ꢀpositions of the quinolone rings. The
different substitution positions of the alicyclic amino groups in quinolones 9a−e and 12a−e could
affect their conformations and electrostatic distributions. The introduction of alicyclic amines at
the 6ꢀposition of the quinolone backbones demonstrated a slightly smaller influence on the
electron density of the carbonyl groups and thiazole rings than that at the 7ꢀposition.
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3.4.2. Antibacterial Activity, Substituents in 6ꢀ, 7ꢀ and 8ꢀPositions. Based on the structural
features and the antibacterial activity data, the SARs are summarized in Figure 4. Generally, the
target 3ꢀ(2ꢀaminothiazolꢀ4ꢀyl) quinolones showed much higher inhibitory potency against all the
tested strains than most of the corresponding intermediates, especially against Gramꢀnegative
bacteria. It seems that the 2ꢀaminothiazole fragments at the Cꢀ3 position of the quinolones are
important for antibacterial potency and are feasible to replace the carboxyl groups of quinolone
drugs. The poor antibacterial potency of 6,8ꢀdifluoroquinolone thiazole 6a, 8ꢀfluoroquinolone
derivative 6c and 6,8ꢀdichloro derivative 6e might suggest that modification at the 8ꢀposition is
not favorable to improve antibacterial potency. Strong electronꢀwithdrawing groups like the
trifluoromethyl moiety at the 6ꢀ and 7ꢀpositions of quinolone could significantly increase the
inhibitory potency against Gramꢀnegative bacteria but reduce the antiꢀGramꢀpositive bacterial
activity. It was beneficial to introduce some electronꢀdonating substituents such as methyl,
methoxyl or alicyclic amino groups, particularly for antiꢀGramꢀpositive bacteria activity. The
stereospecific blockade of substituents at the 6ꢀposition of quinolone was found to be another
influential factor. The pyrrolidinꢀ1ꢀyl group was more effective than the other fragments,
including chloro, fluoro, piperidineꢀ1ꢀyl and morpholinyl moieties. To our surprise, 7ꢀ
chloroquinolones showed better inhibitory potency than 7ꢀfluoroquinolones.
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0
Figure 4. Primary summary of SAR of 3ꢀ(2ꢀaminothiazolꢀ4ꢀyl) quinolones.
3.4.3. Antibacterial Activity, Lipophilicity and Hydrophilicity. The effect on the
antibacterial activity of the overall balance between the lipophilicity and hydrophilicity of 3ꢀ(2ꢀ
aminothiazolꢀ4ꢀyl) quinolones was investigated by examining the calculated lipophilicity. As
shown in Figures 5−8, the compounds with low lipophilicity (ClogP < 3.3) exhibited high MIC
values, which indicated that the introduction of hydrophilic groups like single fluorine,
morpholinyl and methoxyl groups into the benzene rings of quinolone was detrimental to their
antibacterial action. Nevertheless, in the case of high lipophilicity, the target compounds with
high ClogP values (ClogP > 4.2) showed moderate to poor potency against S. aureus, P.
aeruginosa and E. typhosa strains. These highly lipophilic substituents might result in significant
decreases in antibacterial activity. Compared with other strains, the MRSA strain was more
sensitive to the target compounds with high ClogP values (4.16, 4.21 and 4.58). The increase in
lipophilicity might be beneficial for improving activity against this drugꢀresistant strain. Finally,
it was very interesting that the ClogP values of the compounds with the best activity ranged from
3.3 to 4.2. The target aminothiazolquinolones with ClogP values in this range showed much
better antibacterial activity in comparison with others.
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60
9
6
3
0
0
0
0
2.8
3.2
3.6
4.0
4.4
Clog P
Figure 5. Calculated partition coefficients vs antibacterial activity against S. aureus strain. The
most active compounds (low MIC) present partition coefficients in the range of ClogP from 3.3
to 3.6.
1000
800
600
400
200
0
3
.0
3.3
3.6
3.9
4.2
4.5
Clog P
Figure 6. Calculated partition coefficients vs antibacterial activity against MRSA strain. The
most active compounds (low MIC) present partition coefficients in the range of ClogP from 3.45
to 4.58.
The hydrophilicity of these compounds was studied via MEP surfaces. Among target
compounds 6a−i, those compounds with electronꢀwithdrawing groups at the 6ꢀ and 7ꢀpositions of
quinolone presented MEP surfaces with increased electronegatively charged areas, especially the
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lone pair of the amino group, which was beneficial to antiꢀGramꢀnegative bacterial potency.
However, those compounds with electronꢀdonating groups demonstrated MEP surfaces with
reduced electronegatively charged areas, which were helpful for antiꢀGramꢀnegative bacterial
potency. For the compounds of series 9 and 12, the introduction of alicyclic amines at the 6ꢀ and
0
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7ꢀpositions of the quinolone rings resulted in changes to the thiazole rings’ conformation, which
decreased the positively charged surface areas at the 3ꢀ and 4ꢀpositions of the quinolones
compared to series 6. These introductions also affected the surfaces of the amino groups, which
showed reduced electronegatively charged areas. The strong antibacterial potency and broad
antibacterial spectrum, including against MRSA and MQRSA, may be explained by these
differences between series 6 and compound 12b.
1000
500
120
00
1
80
60
40
20
0
3.0
3.3
3.6
3.9
4.2
4.5
Clog P
Figure 7. Calculated partition coefficients vs antibacterial activity against P. aeruginosa strain.
The most active compounds (low MIC) present partition coefficients in the range of ClogP from
3.45 to 4.2.
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6
4
2
0
0
0
0
3.0
3.3
3.6
3.9
4.2
4.5
Clog P
Figure 8. Calculated partition coefficients vs antibacterial activity against E. typhosa strain. The
most active compounds (low MIC) present partition coefficients in the range of ClogP from 3.45
to 4.2.
3
.5 Binding Behavior with HSA. It is well known that HSA is an important extracellular
protein in the circulatory system and is closely related to drug absorption, distribution, and
3
1
metabolism. Drugsꢀalbumin binding behaviors are important during drug discovery and lead
optimization as strong or weak binding may reduce the bioavailability and/or increase the drug’s
in vivo halfꢀlife. Therefore, investigation of these binding behaviors is not only helpful for
understanding the pharmacokinetic properties but also instructive of the design of drug
molecules. Herein, highly active molecule 12bꢀalbumin binding was investigated by fluorescence
spectroscopy on the molecular level to preliminarily study the absorption, distribution and
metabolism.
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HSA
a
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0
300
0
-6
[Compound 12b]/10 mol /L
3
30
360
390
420
Wavelength (nm)
Figure 9. Emission spectra of HSA with different concentrations of compound 12b. c(compound
ꢀ
6
ꢀ5
1
2b)/(10 M), a−j: from 0−8.56 at increments of 1.07; c(HSA) = 1.0 × 10 M; the red and blue
dashed lines are the emission spectrum of compound 12b only and HSA only, respectively; T =
298 K, λ = 295 nm.
ex
3
.5.1. Fluorescence Quenching Mechanism. Two major methods (equilibrium dialysis and
fluorescence competition experiments) are usually employed to study drug binding sites in
3
2,33
albumin.
Fluorescence competition is generally accepted as a good approach to explore the
3
4
binding behaviors between small molecules and HSA. The tryptophan residue Trp 214 in HSA
is a dominant fluorophore capable of fluorescence quenching that absorbs near 280 nm and emits
near 340 nm. The emission of this fluorophore may be blueꢀshifted if the group is buried within a
native protein, and its emission may shift to a longer wavelength (redꢀshift) when the protein is
unfolded. Hence, changes in the fluorescence intensity can reflect the interaction of small
3
5
molecules with Trpꢀ214 in HSA.
With a fixed amount of HSA, the fluorescence changes of HSA (T = 298 K, λ = 295 nm) with
ex
increasing concentrations of compound 12b were determined. The red dashed line in Figure 9 is
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the only emission spectrum of the active molecule 12b, which indicates that its fluorescence
intensity is very weak and could be negligible in comparison with the fluorescence of HSA at the
excitation wavelength. The maximum emission peak of HSA appeared at 356 nm in the
fluorescence spectra, exhibiting a proportional decrease and slight red shift from 356 nm to 359
nm as the concentration of compound 12b increased. According to the following wellꢀknown
SternꢀVolmer equation (1), the fluorescence quenching data of HSA could be analyzed:
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F0
=
1+K [C]
(1)
SV
F
In this equation the F is the steadyꢀstate fluorescence intensity of HSA alone, and F represents
0
its fluorescence in the presence of compound 12b. KSV and [C] are the SternꢀVolmer quenching
constant and the concentration of compound 12b, respectively. K was obtained by performing a
SV
linear regression of a plot of F /F vs [C], and the results are shown in Table 5.
0
The values of the constant KSV at three different temperatures (298, 303, and 310 K) were
calculated from SternꢀVolmer plots and are listed in Table 5. The quenching constant KSV varies
inversely with the temperature, which suggests that groundꢀstate complex formation between
compound 12b and HSA (static quenching) governs the quenching mechanism of this complex.
a
Table 5. SternꢀVolmer constants of interaction between HSA and compound 12b at pH 7.4
ꢀ4
Temperature (K)
KSV (L/mol ×10 )
R
SD
2
3
3
98
03
10
1.43
1.39
1.26
0.996
0.993
0.996
0.06
0.06
0.05
a
R and SD are the correlation coefficient and standard deviation, respectively
3.5.2. Binding Sites and Constants. The static quenching data could be analyzed by the
following modified SternꢀVolmer formulas (2):
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F0
1
1
1
=
+
ꢁ
F
f K [Q]
fa
a
a
(
2)
ꢁ
F is the fluorescence difference of HSA in the presence and absence of compound 12b, and fa
0
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and K are the fraction of accessible fluorescence and the effective quenching constant for the
a
accessible fluorophore, respectively. F /ꢁF depends on the reciprocal value of the concentration
0
ꢀ
1
ꢀ1
[
Q] , which is linear with the slope on the basis of the (f K ) value. Figure 10 shows modified
a a
SternꢀVolmer plots, and the results are listed in Table 6.
The Scatchard equation (3) could be used to calculate the equilibrium binding constant (Kb)
and the number of binding sites (n):
r
=
nK ꢀ rKb
(3)
b
Df
D and r are the molar concentration of free small molecules and the moles of small molecules
f
bound per mole of protein, respectively. K is the equilibrium binding constant, and n is the
b
binding site multiplicity per class of binding sites. Figure 11 shows the Scatchard plots, and the
K and n values are listed in Table 6.
b
1
0.5
3
10 K
303 K
288 K
9
7
6
.0
.5
.0
4.5
3
.0
1.5
0.2
0.4
0.6
0.8
-1
-5
Compound 12b [M , 10
]
Figure 10. Modified SternꢀVolmer plots for compound 12bꢀHSA complex.
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.10
298 K
303 K
310 K
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0
.08
.07
0
0
.1
0.2
0.3
0.4
0.5
r
Figure 11. Scatchard plots of compound 12bꢀHSA complex.
The binding constants and sites of the compound 12bꢀHSA system at 288 K, 303 K, and 310K
are listed in Table 6. The values of K and K decrease with increasing temperature, which is
a
b
similar to the dependence of KSV on the temperature. The binding site n was approximately equal
to 1, which suggests that the compound 12bꢀHSA complex should have one binding site. The
binding constants of the compound 12bꢀHSA complex were also suitable. Based on these results,
compound 12b could be transported by HSA.
Table 6. Binding sites and constants for compound 12bꢀHSA complex in pH 7.4 solution
Modified SternꢀVolmer formula method
Scatchard formula method
Temperature
K)
(
5
5
K (L/mol × 10 )
R
SD
K (L/mol × 10 )
b
R
SD
n
a
288
303
310
1.47
1.39
1.37
0.9999
0.9998
0.9996
0.03
0.05
0.08
0.726
0.701
0.656
0.997 0.002
0.992 0.002
0.991 0.001
1.4
1.4
1.4
3
.6 Preliminary Antibacterial Mechanism of 3ꢀAminothiazolquinolones. Although a large
amount of biological data has indicated that the functional targets of quinolones are
topoisomerase IV and DNA gyrase, many studies of antibacterial mechanisms have dismissed
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these two enzymes as targets and point to DNA as the direct binding species. Generally, this type
of drug does not bind directly to DNA gyrase or DNA topoisomerase IV at their inhibitory
concentration but rather specifically to a saturable site on the supercoiled DNA in a highly
0
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36
cooperative manner. On the basis of previous observations and evidence, a model for the
ternary complex has been proposed, in which magnesium or copper ions act as linkers between
3
7
the groups at the 3ꢀ and 4ꢀ positions of quinolone and the DNA phosphate group. Therefore, the
in vitro enzyme inhibition assay against DNA gyrase or DNA topoisomerase IV is not enough to
illustrate the antibacterial mechanism of quinolone drugs. Molecular modeling and the
interaction between the active molecule and bacterial DNA is an important and helpful strategy
for investigating preliminary antimicrobial mechanism. In our work, DNA was isolated from
MRSA bacteria that were most sensitive to 3ꢀaminothiazolquinolone 12b, and the purity was
checked by gel electrophoresis (Figure 12) and absorption spectroscopy. The concentration of
MRSA DNA in stock solution was determined by UV absorption at 260 nm using a molar
ꢀ1
ꢀ1
absorption coefficient ξ260 = 6600 L mol cm (expressed as molarity of phosphate groups)
according to the BouguerꢀLambertꢀBeer law. The purity of MRSA DNA was checked by
monitoring the ratio of the absorbance at 260 nm to that at 280 nm. The solution gave a ratio of >
1.8 at A260/A280, which indicated that the DNA was sufficiently free from protein.
Figure 12. Purity of MRSA DNA checked by gel electrophoresis. The left is marker, and the
right is the MRSA DNA
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3
.6.1. Molecular Docking Study. A docking investigation was undertaken to explore the
possible mechanism of action of aminothiazolquinolones. Crystals of topoisomerase IVꢀDNA
3
8,39
and gyraseꢀDNA complexes were selected as representative targets in the protein data bank.
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The resolutions of the crystals were checked and determined to be not higher than 2 Å.
Aminothiazolquinolone 12b was used to dock with the gyraseꢀDNA and topoisomerase IVꢀDNA
complexes.
Figure 13. 3D conformations of 3ꢀaminothiazolquinolone 12b docked in the gyraseꢀDNA
complex (left) and the topoisomerase IVꢀDNA complex (right).
The docking evaluation gave good total scores (5.68 and 6.46) for aminothiazolquinolone 12b
against topoisomerase IVꢀDNA and gyraseꢀDNA complexes, which might rationalize the
antibacterial mechanism of aminothiazolquinolones. As shown in Figure 13, the carbonyl
fragment of the aminothiazolquinolones 12b was adjacent to the ASPꢀ83 residue in the
topoisomerase IVꢀDNA complex, forming a hydrogen bond with a distance of 2.9 Å. The amino
group at the 2ꢀposition of the thiazole ring was close to conserve the ParC helix a4 residues Serꢀ
79 (with the closest distances to the side chain atoms of 2.1 Å), whose mutation causes quinolone
resistance in bacteria. Similar hydrogen bonds could also form between aminothiazolquinolone
12b and the Serꢀ1084 and DTꢀ8 base pairs of the gyraseꢀDNA complex. These hydrogen bonds
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