Synthesis and biological evaluation of a class of quinolone triazoles as potential antimicrobial agents and their interactions with calf thymus DNA

Article abstract of DOI:10.1016/j.bmcl.2013.03.118

A novel series of quinolone triazoles were synthesized and characterized by IR, NMR, MS and HRMS spectra. All the newly prepared compounds were screened for their antimicrobial activities against seven bacteria and four fungi. Bioactive assay manifested that most of new compounds exhibited good or even stronger antibacterial and antifungal activities against the tested strains including multi-drug resistant MRSA in comparison with reference drugs Norfloxacin, Chloromycin and Fluconazole. The preliminary interactive investigations of compound 6b with calf thymus DNA by fluorescence and UV-vis spectroscopic methods revealed that compound 6b could effectively intercalate DNA to form compound 6b-DNA complex which might block DNA replication and thus exert its antimicrobial activities.

Full text of DOI:10.1016/j.bmcl.2013.03.118

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3267–3272
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a class of quinolone triazoles
as potential antimicrobial agents and their interactions with calf
thymus DNA
Sheng-Feng Cui, Yu Ren, Shao-Lin Zhang, Xin-Mei Peng, Guri L. V. Damu , Rong-Xia Geng,
⇑
Cheng-He Zhou
Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of quinolone triazoles were synthesized and characterized by IR, NMR, MS and HRMS spec-
tra. All the newly prepared compounds were screened for their antimicrobial activities against seven bac-
teria and four fungi. Bioactive assay manifested that most of new compounds exhibited good or even
stronger antibacterial and antifungal activities against the tested strains including multi-drug resistant
MRSA in comparison with reference drugs Norfloxacin, Chloromycin and Fluconazole. The preliminary
interactive investigations of compound 6b with calf thymus DNA by fluorescence and UV–vis spectro-
scopic methods revealed that compound 6b could effectively intercalate DNA to form compound 6b–
DNA complex which might block DNA replication and thus exert its antimicrobial activities.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 17 January 2013
Revised 21 March 2013
Accepted 27 March 2013
Available online 4 April 2013
Keywords:
Quinolone
Triazole
Antibacterial
Antifungal
DNA
Quinolones are one of the most widely prescribed antimicrobial
agents as they are generally well tolerated with excellent safety
profile, favorable pharmacokinetic characteristics and broad anti-
bacterial spectrum against genitourinary infections and common
respiratory tract pathogens. It is well known that quinolones tar-
geting bacterial type II topoisomerases can bind with the en-
zyme–DNA binary complex to form ternary complexes, thereby
blocking DNA replication and leading to bacterial cells’ death.1
Since the first generation of quinolone drugs was found to be active
in the early 1960s, four generations of quinolones have been clin-
ically used in succession, such as Nalidixicacid, Pipemidicacid,
Lomefloxacin, Moxifloxacin and so on.2
Nevertheless, their extensive use and narrow antibacterial spec-
trum especially low activity against Gram-positive pathogens,
including staphylococci, streptococci, and enterococci have greatly
affected the therapeutic efficacy in clinic.3 Furthermore, their
shortcomings including phototoxicity, lipid peroxidation, photo-
hemolysis and bad water-solubility also limit the administrable
mode.4 This situation stimulates the further structural modifica-
tion of quinolones. Especially the N-1 position and benzene ring
in quinolones are adaptable sites for regulating physicochemical
property and greatly influencing the antimicrobial potency, spec-
trum and safety.5 Two successful examples are Ciprofloxacin and
Norfloxacin with cyclopropyl and ethyl groups respectively at the
N-1 position, which display different bioavailability and antibacte-
rial spectrum. Moreover, the substituents on benzene ring of quin-
olones, such as Ciprofloxacin and Moxifloxacin, can also make
contributions to their bioactivities.6
F
O
F
COOH
F
N
N
N
N
N
N
H
OH
WY-1
Our recent work indicated that the introduction of triazolyl eth-
anol moiety in the C-7 side chain of ciprofloxacin could effectively
inhibit the growth of all the tested bacteria and fungi. The clina-
floxacin derivative WY-1 with a 2,4-difluorophenyl group dis-
played more potent antimicrobial efficacy (MIC = 0.25–1 lg/mL)
than its positive control.7 When the triazole ring was introduced
into N-1 position of quinolones, the resulting quinolone triazoles
also showed great potential to treat the drug-resistant bacterial
infections.8 These results clearly pointed out that the hybrids of
antibacterial quinolones with antifungal triazole moiety could
not only remarkably enhance the antimicrobial activities, but also
broaden the antimicrobial spectrum.
⇑
Corresponding author. Tel./fax: +86 23 68254967.
E-mail address: zhouch@swu.edu.cn (C.-H. Zhou).
Postdoctoral fellow from Indian Institute of Chemical Technology (IICT), India.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.118

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S.-F. Cui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3267–3272
In view of the above observations,9 herein we incorporated
fungal strains except C. utilis. Almost all the new compounds
(MIC = 0.5–8 g/mL) showed comparable or even superior activity
against E. typhosa to standard drugs Norfloxacin (4 g/mL) and
Chloromycin (32 g/mL). Especially compound 6d with a trifluoro-
methyl group at the 7-position of quinolone backbone gave stron-
ger antibacterial efficacies and broader bioactive spectrum than
Norfloxacin and Chloromycin with quite low MIC values.
It was known that MRSA was one of the most virulent organ-
isms that showed severe multi-drug resistance to numerous cur-
rently available agents. Excitingly, all the new compounds except
for compounds 5a, 5d, 5g, 6e and 6g could effectively inhibit the
triazolyl ethanol fragment into the N-1 position of quinolones
and changed different substituents on benzene ring of quinolones
to generate a novel class of quinolone triazoles. The prepared hy-
brids might be expected to exhibit potential against bacterial and
fungal strains including drug-resistant microorganism. Therefore,
their antibacterial and antifungal activities for all newly synthe-
sized compounds were evaluated in vitro against three Gram-posi-
tive bacteria including Methicillin-Resistant Staphylococcus aureus
N315 (MRSA), four Gram-negative bacteria and four fungi. The pre-
liminary antimicrobial mechanism was investigated by evaluating
the interaction of the prepared highly active compound with calf
thymus DNA.
The target quinolone triazole compounds were synthesized
according to the synthetic route outlined in Scheme 1. Commer-
cially available diethyl malonate was reacted with triethoxyme-
thane in the presence of anhydrous zinc chloride to produce
diethyl 2-(ethoxymethylene) malonate 1 in an almost quantitative
yield of 97%, and then compound 1 was further treated with a ser-
ies of substituted phenylamines in ethanol to afford diethyl
2-((phenylamino)methylene)malonate derivatives 2a–g, which
were cyclized in oxydibenzene under reflux to give the desired
quinolones 3a–g.10 The latter was further N-alkylated by commer-
cial 2-(chloromethyl)oxirane to yield racemic ethyl 1-(oxiran-2-yl-
methyl) quinoline-3-carboxylate derivatives 4a–g in high yields
(82–91%), subsequently the epoxy ring was opened by 1,2,4-tria-
zole in ethanol using sodium bicarbonate as base to produce race-
mates 5a–g, and then were further hydrolyzed in water by 3%
sodium hydroxide at 100 °C to afford the corresponding target
quinolone triazoles 6a–g with excellent yields. All the new com-
pounds were confirmed by 1H NMR, 13C NMR, IR, MS and HRMS
spectra.
l
l
l
growth of MRSA at the concentrations of 0.5–16
were more effective than clinical Chloromycin (MIC = 16
It was specially noticed that compounds 5f, 6c and 6d with MIC
values of 1 g/mL were 8- and 16-fold more active than Norfloxa-
cin (MIC = 8 g/mL) and Chloromycin respectively. Especially 7-tri-
fluoromethyl intermediate 4d (MIC = 0.5 g/mL), as new
lg/mL, which
lg/mL).
l
l
l
a
potential anti-MRSA agent, was 16- and 32-fold more active than
both of standard drugs Norfloxacin and Chloromycin respectively.
The antifungal evaluation in vitro revealed that most of the
newly prepared compounds exhibited completely different results
in comparison with their antibacterial activities. Quinolones 4a–g
with oxiran-2-ylmethyl group at N-1 position showed good inhib-
itory efficiency against all the tested fungal strains except for C.
utilis, which gave comparable or superior inhibitory potency to
the first-line antifungal drug Fluconazole. Especially compounds
4a, 4c, 4d, 4f and 4g could efficiently inhibit the growth of A. flavus
strain in vitro with MIC values ranging from 0.5 to 1
lg/mL, which
were 256-fold more potent than Fluconazole (MIC = 256
lg/mL).
Surprisingly, the 6-methyl quinolone triazole 6b displayed not only
good antibacterial activities against all the tested bacterial strains,
but also excellent antifungal activities against A. flavus, B. yeast, and
All synthesized quinolone triazoles were evaluated in vitro for
their antimicrobial activities against three Gram-positive bacteria
(Micrococcus luteus ATCC 4698, MRSA and Staphylococcus aureus
ATCC25923), four Gram-negative bacteria (Pseudomonas aerugin-
osa, Escherichia coli DH52, Shigella dysenteriae, and Eberthella typh-
osa) and four fungi (Candida utilis, Aspergillus flavus, Beer yeast, and
Candida albicans) by two folds serial dilution technique recom-
mended by National Committee for Clinical Laboratory Standards
(NCCLS) with the positive control of clinically antimicrobial drugs
Norfloxacin, Chloromycin and Fluconazole. The antimicrobial tests
were carried out for three times. The obtained results as depicted
in Table 1 revealed that quinolone triazoles 5a–g and 6a–g could
effectively inhibit the growth of most tested bacterial strains, while
compounds 4a–g exhibited good activities against all the tested
C. albicans in low inhibitory concentrations (MIC = 0.5–4 lg/mL).
Based on these observations and analysis, compound 6b might
act different target site from standard drugs Fluconazole (cyto-
chrome P450) and Norfloxacin (topoisomerase IV), which suggested
this compound might inhibit bacterial and fungal strains growth
by different mechanisms of action.
Generally the substituents in quinolone B ring could affect anti-
microbial potency. Compound 4b with electron-donating methyl
substituent exhibited poor activities against M. luteus, E. coli
DH52 and S. dysenteriae (MIC = 256, 128 and 128 lg/mL, respec-
tively). On the other hand, electron-withdrawing halogen groups
especially fluorine moiety in 6-, 7- and 8-position of quinolones ex-
erted great influences on antibacterial activities. Trifluoromethyl
quinolone triazole 6d and 7-chloro-6-fluoro quinolone 6e showed
O
R1
COOEt
EtO
H
N
EtOOC
R3
R2
R2
R3
COOEt
COOEt
O
O
iii
i
ii
COOEt
EtO
N
H
R1
3a-g
1
2a-g
EtO
iv
O
N
O
N
O
R3
R2
HOOC
R3
R2
EtOOC
EtOOC
R3
R2
vi
v
N
R1
R1
6a-g
R1
4a-g
N
N
N
O
N
5a-g
OH
OH
N
N
2~6: a, R1 = F, R2 = H, R3 = F; b, R1 = H, R2 = H, R3 = Me;
c, R1 = H, R2 = H, R3 = F; d, R1 = H, R2 = CF3, R3 = H;
e, R1 = H, R2 = Cl, R3 = F; f, R1 = Cl, R2 = H, R3 = Cl;
g, R1 = F, R2 = H, R3 = H
Scheme 1. Reagents and conditions: (i) triethoxymethane, acetic anhydride, ZnCl2, N2, reflux, 4 h; (ii) substituted phenylamines, EtOH, reflux, 2 h; (iii) Ph2O, reflux, 1 h; (iv)
2-(chloromethyl)oxirane, K2CO3, 120 °C, 3 h; (v) triazole, K2CO3, EtOH, reflux, 2 h; (vi) 3% NaOH, 100 °C, 2 h.

S.-F. Cui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3267–3272
3269
Table 1
In vitro antimicrobial data as MIC (lg/mL) for compounds 4–6
Compds
Fungi
B. yeast
Gram-positive bacteria
Gram-negative bacteria
C. utilis
A. flavus
C. albicans
M. luteus
MRSA
S. aureus
P. aeruginosa
E. coli DH52
S. dysenteriae
E. typhosa
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
5g
6a
6b
>512
256
256
>512
>512
>512
>512
>512
>512
64
1
16
1
0.5
8
0.5
0.5
64
256
512
8
64
64
2
8
4
8
4
512
64
512
64
512
32
32
512
4
512
256
256
128
256
>512
>512
16
4
2
16
4
64
2
2
8
16
4
0.5
8
2
2
32
2
8
32
4
1
128
8
8
1
1
64
4
64
16
8
16
256
32
256
128
1
64
32
128
64
32
0.5
256
4
2
16
1
4
4
32
2
16
8
8
2
8
8
32
1
>512
128
128
64
64
64
32
16
8
>512
128
64
256
128
>512
512
32
4
64
32
32
128
8
16
32
0.5
1
64
4
1
32
2
2
8
2
8
2
2
0.5
4
1
0.5
1
4
0.5
1
8
1
4
4
32
512
128
64
>512
>512
2
16
8
2
4
8
2
2
16
32
2
16
64
8
1
2
1
64
128
32
64
8
512
32
8
2
64
1
16
32
16
>512
>512
1
64
32
16
64
1
64
2
32
128
1
64
64
32
16
0.5
>512
4
16
32
32
4
8
2
32
32
8
8
64
4
128
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
>512
8
16
0.5
512
256
0.5
512
512
128
256
128
>512
>512
256
6c
6d
6e
6f
16
64
32
4
6g
Chloromycin
Norfloxacin
Fluconazole
8
2
>512
32
16
>512
4
>512
>512
>512
C. utilis, Candida utilis; A. flavus, Aspergillus flavus; B. yeast, Beer yeast; C. albicans, Candida albicans; M. luteus, Micrococcus luteus ATCC 4698; MRSA, Methicillin-Resistant
Staphylococcus aureus N315; S. aureus, Staphylococcus aureus ATCC25923; P. aeruginosa, Pseudomonas aeruginosa; E. coli DH52, Escherichia coli; S. dysenteriae, Shigella
dysenteriae; E. typhosa, Eberthella typhosa.
comparable or even superior activities to Chloromycin and Nor-
floxacin against all the tested strains. The results suggested that
the introduction of hydroxyl and triazolyl moieties as well as elec-
tron-donating groups could improve the antibacterial efficiencies
to some extent.
DNA+6f
DNA+6b
DDNNAA--666bfbcocmomplepxlex
0.66
0.60
0.54
0.48
g
a
0.6
0.4
0.2
0.0
Thus a preliminary antimicrobial mechanism was explored by
evaluating the interaction between the strongest active compound
6b and calf thymus deoxyribonucleic acid (DNA). DNA is the infor-
mational molecule encoding the genetic instructions used in the
development and function of almost all known living organisms.
In recent years, DNA as a therapeutic target has attracted consider-
able attention in biomedical science. These binding studies with
small molecules are important and helpful for developing novel
and more efficient drugs. Calf thymus DNA was selected as DNA
model because of its medical importance, low cost and ready avail-
ability properties. Therefore, in order to investigate the possible
mechanism of antimicrobial action, the binding studies between
the highly active compound 6b and calf thymus DNA on molecular
level were carried out by UV–vis spectroscopic and fluorescent
methods.11
In absorption spectroscopy, hypochromism and hyperchromism
are very important spectral features to distinguish the change of
DNA double-helical structure. Due to the strong interaction be-
tween the electronic states of intercalating chromophore and that
of the DNA base, the observed large hypochromism strongly sug-
gests a close proximity of the aromatic chromophore to the DNA
bases.12 With a fixed concentration of DNA, UV–vis absorption
spectra were recorded with increasing amount of compound 6b.
As shown in Figure 1, UV–vis 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
compound 6b. Meanwhile the phenomenon, that the absorption
value of simply sum of free DNA and free compound 6b was a little
greater than the measured value of DNA–compound 6b complex,
was observed in the inset of Figure 1. This meant that a weak hypo-
chromic effect existed between DNA and compound 6b. Moreover
1
2
3
4
5
6
7
-6
10 [66bf ]
DNA
240
260
280
300
320
340
Wavelength (nm)
Figure 1. UV absorption spectra of DNA with different concentrations of compound
6b (pH 7.4, T = 288 K). Inset: comparison of absorption at 260 nm between the
DNA–compound 6b complex and the sum values of free DNA and free compound
6b. c(DNA) = 7.61 ꢁ 10ꢀ5 mol/L, and c(compound 6b) = 0–6.72 ꢁ 10ꢀ6 mol/L for
curves a–g respectively at increment 1.12 ꢁ 10-6
.
the intercalation of the aromatic chromophore of compound 6b
into the helix and the strong overlap of
⁄ states of the large
-conjugated system with the electronic states of DNA bases were
p–p
p
consistent with the observed spectral changes. Based on the varia-
tions in these absorption spectra, equation (1) could be used to cal-
culate the intrinsic binding constant (K):
A0
nC
nC
1
¼
þ
ꢁ
ð1Þ
A ꢀ A0
A
nDꢀC ꢀ nC nDꢀC ꢀ nC K½6bꢂ
0 and A represent the absorbance of DNA in the absence and pres-
ence of compound 6b at 260 nm, nC and nD–C were the absorption
coefficients of compound 6b and DNA–compound 6b complex
respectively. The plot of A0/(A–A0) versus 1/[compound 6b] was

3270
S.-F. Cui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3267–3272
constructed by using the absorption titration data and linear fitting,
yielding the binding constant, K = 1.43 ꢁ 103 L/mol, R = 0.998,
SD = 0.17 (R is the correlation coefficient. SD is standard deviation).
To further understand the interaction between compound 6b
and DNA, the absorption spectra of competitive interaction of com-
pound 6b were investigated. Compared with other common
probes, Neutral Red (NR) showed lower toxicity, higher stability
and convenient application. Furthermore, it has been sufficiently
demonstrated by spectrophotometric and electrochemical tech-
niques that NR can bind to DNA by an intercalative mode.13 There-
fore NR was employed as a spectral probe to investigate the
binding mode of compound 6b with DNA in this work. The absorp-
tion peak of the NR around 272 nm gradually decreased by increas-
ing the concentration of DNA, which suggested the formation of
new DNA–NR complex (Supplementary data). Figure 2 displayed
the absorption spectra of a competitive binding between NR and
compound 6b with DNA. As the gradually increasing concentration
of compound 6b, an apparent intensity increase was observed
around 272 nm. Compared with the absorption around 272 nm of
NR–DNA complex, the absorbance at the same wavelength (inset
of Fig. 2) exhibited the reverse process. In addition to that, the
absorbance at 255 and 327 nm and extensive broadening were also
observed in the spectra. These various spectral changes were con-
sistent with the intercalation of compound 6b into DNA by substi-
tuting for NR in the DNA–NR complex.
Measurement of fluorescence was also carried out to prove the
interaction between compound 6b and DNA. With a fixed concen-
tration of DNA, fluorescence spectra were recorded with increasing
amount of compound 6b. The fluorescence intensity of the peak
around 376 nm increased gradually with increasing the concentra-
tion of compound 6b. Furthermore, the fluorescence spectrum of
compound 6b in the absence of DNA was also recorded under
the same conditions (Supporting Information). Figure 3 showed
that there were apparent differences about max fluorescence
intensity between them. At the same concentration of compound
6b, the system max fluorescence intensity in absence of DNA was
higher than the system in presence of DNA, which meant the bind-
ing of compound 6b with DNA helix was found to strongly quench
the fluorescence of compound 6b. From the experimental results, it
was concluded that the chromophore of compound 6b could inter-
calate into the DNA helix.
2500
2000
1500
1000
500
DNA-compound 6b
compound 6b
0.5
1.0
1.5
2.0
2.5
3.0
Compound 6b (mol L-110-5)
Figure 3. Relative fluorescence intensity for compound 6b at different concentra-
tions with or without DNA. c(DNA) = 7.18 ꢁ 10ꢀ6 mol/L, and c(compound 6b) = 0–
8.0 ꢁ 10ꢀ5 mol/L for curves a–t, respectively at increment 0.4 ꢁ 10ꢀ5
.
binding of molecule with DNA. To investigate the binding mecha-
nism, iodide quenching studies were introduced in this work. Gen-
erally, small molecules have three modes via diverse non-covalent
interactions to bind with double-helix DNA including electrostatic
interaction with the anionic sugar phosphate backbone of DNA,
groove binding with the DNA groove, and intercalation with DNA
base pairs. Among the three bonding modes, the intercalative bind-
ing and groove binding are the most effective interactions mode to
block DNA replication in comparison to electrostatic interaction.14
In the presence of anionic quencher, DNA base pairs around the
intercalators can hinder the accessibility of fluorescent probe to
quencher. While the electrostatic repelling between DNA phos-
phate backbone and anionic quenchers will provide protection
for the intercalated species. Therefore, if the binding mode is inter-
calation, the molecule should be protected from being quenched by
anionic quencher. The value of quenching constants (KSV) of the
intercalative bound molecule should be lower than that of the mol-
ecule bound to DNA by groove binding.15 Negatively charged Iꢀ ion
was selected for this purpose. The values of KSV of compound 6b by
Iꢀ ion in the absence and presence of DNA were calculated to be
7.65 L/mol (R = 0.997, SD = 0.05) and 6.53 L/mol (R = 0.998,
SD = 0.04), respectively (Fig. 4). The results suggested that the
Steady-state quenching experiments by highly negatively
charged quenchers can provide further information about the
3.0
0.7
1.2
i
Compound 6b
Compound 6b-DNA
0.6
a
2.5
i
0.9
0.5
i
2.0
1.5
1.0
270
275
280
285
a
0.6
0.3
Wavelength/nm
a
i
DNA+NR
a
0
5
10
15
20
25
240
260
280
300
320
340
Wavelength (nm)
KI (mol/L, 10-2)
Figure 2. UV absorption spectra of competitive reaction between compound 6b and
NR with DNA. c(DNA) = 6.04 ꢁ 10ꢀ5 mol/L, c(NR) = 2 ꢁ 10ꢀ5 mol/L, and c(compound
Figure 4. Fluorescence quenching plot of compound 6b by KI in the absence and
presence of DNA (pH 7.4, T = 288 K). c(compound 6b) = 1 ꢁ 10ꢀ5 mol/L, and
c(DNA) = 7.18 ꢁ 10ꢀ5 mol/L
6b) = 0–8.0 ꢁ 10ꢀ5 mol/L for curves a–i, respectively at increment 1.0 ꢁ 10ꢀ5
.

S.-F. Cui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3267–3272
3271
imidazole, benzimidazole and their derivatives) as well as other
substituents on skeleton quinolone ring are now in progress. All
these will be discussed in future paper.
70
60
50
40
30
20
10
0
DNA-NR
a
o
Acknowledgments
a
This work was partially supported by National Natural Science
Foundation of China [(No. 21172181), the Research Fund for Inter-
national Young Scientists from International (Regional) Cooperation
and Exchange Program (Nos. 81250110089, 81250110554)], the
Key Program of Natural Science Foundation of Chongqing
(CSTC2012jjB10026), the Specialized Research Fund for the Doctoral
Program of Higher Education of China (SRFDP 20110182110007),
the Research Funds for the Central Universities (XDJK2011D007,
XDJK2012B026).
o
Compound 6b only
-10
630
660
690
720
750
780
Wavelength (nm)
References and notes
Figure 5. Fluorescence spectra of DNA–NR in the presence and absence of
compound 6b at different concentrations (pH 7.4, T = 288 K). c(DNA) = 7.18 ꢁ
10ꢀ6 mol/L, and c(compound 6b) = 0–6.0 ꢁ 10ꢀ5 mol/L for curves a–o respectively
at increment 0.4 ꢁ 10ꢀ5. Blue line shows the emission spectrum of compound 6b
only.
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binding mode of compound 6b with DNA was of intercalation
nature.
In further examination the NR was introduced as a fluorescence
probe to investigate the binding characteristics of compound 6b
with DNA.16 Figure 5 showed the emission spectra of the DNA–
NR system in the presence and absence of compound 6b. The only
emission spectrum of 6b indicated that it did not posses significant
fluorescence features, thus the effect of compound 6b on fluores-
cence of DNA-NR complex would be negligible. When the concen-
tration of compound 6b was increasing gradually, a remarkable
fluorescence decrease in DNA–NR system was observed around
640 nm. Furthermore, an obvious intensity increase was observed
around 726 nm. This phenomenon suggested that compound 6b
could compete with NR for the intercalation sites in DNA and lead
to a significant decrease at 640 nm and a remarkable increase at
726 nm. These results meant that compound 6b could intercalate
into the helix of DNA.
In conclusion, a novel class of quinolone triazoles were designed
and synthesized in good yields via an easy, convenient and efficient
synthetic route. All the new compounds were characterized by 1H
NMR, 13C NMR, MS, IR and HRMS spectra.17 The in vitro antimicro-
bial activities of these quinolone triazoles and their intermediates
were evaluated against seven bacterial and four fungal strains.18
The biological results revealed that most of the newly synthesized
compounds exhibited good antibacterial and antifungal activities
against most of the tested strains in comparison to the reference
drugs Chloromycin, Norfloxacin and Fluconazole. Quinolone tria-
zoles 6d and 6e showed significant inhibition against all tested
bacterial strains with low inhibitory concentrations in range of
10. Vandurm, P.; Guiguen, A.; Cauvin, C.; Georges, B.; Van, K. L.; Michaux, C.;
Cardona, C.; Mbembac, G.; Mouscadet, J. F.; Hevesi, L.; Lint, C. V.; Wouters, J.
Eur. J. Med. Chem. 2011, 46, 1749.
11. Berdis, A. J. Biochemistry 2008, 47, 8253.
12. (a) Zhang, G.; Fu, P.; Wang, L.; Hu, M. J. Agric. Food Chem. 2011, 59, 8944; (b)
Kanakis, C. D.; Nafisi, S.; Rajabi, M.; Shadaloi, A.; Tarantilis, P. A.; Polissiou, M.
G.; Bariyanga, J.; Tajmir-Riahi, H. A. Spectroscopy 2009, 23, 29.
13. Ni, Y.; Dua, S.; Kokot, S. Anal. Chim. Acta. 2007, 584, 19.
14. Clark, C. C.; Marton, A.; Meyer, G. J. Inorg. Chem. 2005, 44, 3383.
15. Akbay, N.; Seferog˘lu, Z.; Gök, E. J. Fluoresc. 2009, 19, 1045.
16. Li, X. L.; Hu, Y. J.; Wang, H.; Yu, B. Q.; Yue, H. L. Biomacromolecules 2012, 13, 873.
17. Experimental: melting points were recorded on X-6 melting point apparatus
and were uncorrected. TLC analysis was done using pre-coated silica gel plates.
FT-IR spectra were carried out on Bruker RFS100/S spectrophotometer (Bio-
Rad, Cambridge, MA, USA) using KBr pellets in the 400–4000 cmꢀ1 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) are expressed in hertz (Hz) and singlet (s),
doublet (d) and triplet (t), broad (br) as well as multiplet (m). The mass spectra
(MS) were recorded on LCMS–2010A and the high-resolution mass spectra
(HRMS) were recorded on an IonSpec FT-ICR mass spectrometer with ESI
resource.
1–64 lg/mL. Intermediates 4a–g gave better antifungal activities
against all the tested fungal strains except C. utilis than Fluconaz-
ole. Especially compound 6b showed potent antibacterial and anti-
fungal efficacy against all the tested strains. The specific
interaction of compound 6b with DNA was studied by fluorescence
and UV–vis absorption spectroscopy. The experimental results dis-
played that compound 6b could intercalate DNA to form com-
pound 6b–DNA complex which might further block DNA
replication to exert their powerful antibacterial and antifungal
activities. Further researches, including the quenching mechanism
of fluorescence of DNA by the compound 6b, the thermodynamic
parameters, binding sites, the in vivo bioactive evaluation along
with toxicity investigation and the effect factors on antimicrobial
activities such as other heterocyclic azole rings (benzotriazole,
Synthesis of ethyl 6,8-difluoro-1-(oxiran-2-ylmethyl)-4-oxo-1,4-dihydroquinoline-
3-carboxylate (4a).
potassium carbonate
A
mixture of compound
(0.151 g, 1.2 mmol)
3
(0.253 g, 1.0 mmol) and
was stirred in 2-
(chloromethyl)oxirane (15 mL) under reflux for 3 h. After the reaction was
completed, the mixture was cooled to room temperature, the excess 2-
(chloromethyl) oxirane was evaporated under reduced pressure, and water
was added. The residue was extracted with chloroform (3 ꢁ 20 mL), the
combined organic phase was dried over anhydrous sodium sulfate, and then
the residue was purified by column chromatography (eluent, chloroform/
methanol 70:1, V/V) to give the desired compound 4a (0.294 g) as light yellow
solid. Yield: 94.7%; mp >250 °C. IR (KBr, cmꢀ1): 3131 (Ar-H), 3016 (@C–H),
2988, 2902 (CH2, CH3), 1683, 1641 (C@O), 1609, 1559, 1493 (aromatic frame),
1370, 1237, 1085, 1028, 981, 852; 1H NMR (300 MHz, DMSO-d6): d 8.51 (s, 1H,
C-2 quinolone), 7.84–7.93 (m, 1H, C-5 quinolone), 7.79–7.81 (m, 1H, C-7

3272
S.-F. Cui et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3267–3272
quinolone), 4.85–4.90 (m, 1H, O-CH), 4.45–4.50 (m, 2H, N-CH2), 4.26 (q, 2H,
128.57, 123.16, 110.06, 68.23, 60.90, 60.35, 52.58, 14.73. MS (ESI): m/z 379
[M+H]+; HRMS (ESI) calcd for C17H16F2N4O4 [M+H]+, 379.1140; found,
379.1142.
OCH2CH3), 2.54–2.55 (m 2H, OCH2) 1.29 (t, J = 7.1 Hz, 3H, OCH2CH3); 13C NMR
(75 MHz, DMSO-d6): d 170.95, 164.50, 160.24, 156.97, 154.61, 152.22, 151.25,
131.92, 130.43, 110.32, 60.37, 57.21, 50.26, 44.87, 14.71; MS (ESI): m/z 310
[M+H]+; HRMS (ESI) calcd for C15H13F2NO4 [M+H]+, 310.0813; found, 310.0811.
Synthesis of ethyl 6,8-difluoro-1-(2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-4-
Synthesis of 6,8-difluoro-1-(2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-4-oxo-
1,4-dihydroquinoline-3- carboxylic acid (6a) To a stirred solution of sodium
hydroxide (3%, 20 mL) was added compound 5a. The mixture was stirred at
100 °C for 2 h. After the reaction was completed, the mixture was treated with
formic acid to adjust pH to 7, and then the suspension was filtered and washed
with water for three times to give the target compound 6a (0.282 g) as light
yellow solid. Yield: 80.5%; mp >250 °C; IR (KBr, cmꢀ1): 3420 (O–H), 3130 (Ar-
H), 3016 (@C–H), 2996, 2903 (CH2, CH3), 1730, 1665 (C@O), 1630, 1565, 1507
(aromatic frame), 1323, 1296, 1112, 1019, 997, 893, 804; 1H NMR (300 MHz,
DMSO-d6): d 14.95 (s, 1H, COOH), 8.95 (s, 1H triazole 3-H), 8.57 (s, 1H, C-2
quinolone), 8.07 (s, 1H, triazole 5-H), 7.59–7.62 (m, 1H, C-5 quinolone), 7.46–
7.49 (m, 1H, C-7 quinolone), 5.77 (m, 1H, OH), 5.05 (br, 1H, O-CH), 4.36–
4.84(m, 4H, –CH2–); 13C NMR (75 MHz, DMSO-d6): d 173.21, 164.64, 155.31,
153.69, 153.01, 152.43, 150.21, 145.32, 131.23, 128.57, 123.16, 110.06, 68.21,
60.35, 52.58, MS (ESI): m/z 351 [M+H]+; HRMS (ESI) calcd for C15H12F2N4O4
[M+H]+, 351.0827; found, 351.0830.
oxo-1,4-dihydroquinoline-3- carboxylate (5a). To
a stirred suspension of
potassium carbonate (0.151 g, 1.2 mmol) in ethanol was added 1,2,4-triazole
(0.691 g, 1.0 mmol). The mixture was stirred at 60 °C for 1 h. The reaction was
cooled to room temperature, compound 4a (0.309 g, 1.0 mmol) was added at
the room temperature and stirred for 2 h under reflux. After the reaction came
to end, solvent was evaporated and the residue was extracted with chloroform
(3 ꢁ 20 mL). The combined organic extracts were dried over anhydrous sodium
sulfate and concentrated under reduced pressure. The crude product was
purified by column chromatography (eluent, chloroform/methanol 30:1, V/V)
to afford the compound 5a (0.408 g) as yellow solid. Yield: 85.2%; mp >250 °C.
IR (KBr, cmꢀ1): 3443 (O–H), 3129 (Ar-H), 3016 (@C–H), 2989, 2902 (CH2, CH3),
1771, 1676 (C@O), 1614, 1564, 1508 (aromatic frame), 1318, 1254, 1099, 1021,
961, 853; 1H NMR (300 MHz, DMSO-d6): d 8.58 (s, 2H, C-2 quinolone and
triazole 3-H), 8.07 (s, 1H, triazole 5-H), 7.41–7.44 (m, 1H, C-5 quinolone), 7.24–
7.28 (m, 1H, C-7 quinolone), 5.66 (m, 1H, OH), 4.97 (m, 1H, O-CH), 4.19–4.69
(m, 6H, –CH2–), 1.26 (t, J = 7.1 Hz, 3H, OCH2CH3); 13C NMR (75 MHz, DMSO-d6):
18. National Committee for Clinical Laboratory Standards Approved standard
Document. M27-A2, Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts, National Committee for Clinical Laboratory
Standards, Wayne, PA, 2002.
d
172.23, 164.73, 155.22, 153.65, 153.14, 152.78, 150.26, 145.42, 131.23,