Design and synthesis of new norfloxacin-1,3,4-oxadiazole hybrids as antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA)

Article abstract of DOI:10.1016/j.ejps.2019.104966

Toward the search of new antibacterial agents to control methicillin-resistant Staphylococcus aureus (MRSA), a class of new norfloxacin-1,3,4-oxadiazole hybrids were designed and synthesized. Antibacterial activities against drug-sensitive bacteria S. aureus and clinical drug resistant isolates of MRSA were evaluated. Compound 5k exhibited excellent antibacterial activities against S. aureus (MIC: 2 μg/mL) and MRSA1–3 (MIC: 0.25–1 μg/mL). The time-kill kinetics demonstrated that compound 5k had an advantage over commonly used antibiotics vancomycin in killing S. aureus and MRSA. Moreover, compound 5k could inhibit the bacteria and destroy their membranes in a short time, and showed very low cytotoxicity to NRK-52E cells. Some interesting structure-activity relationships (SARs) were also discussed. These results indicated that these norfloxacin-1,3,4-oxadiazole hybrids could be further developed into new antibacterial agents against MRSA.

Full text of DOI:10.1016/j.ejps.2019.104966

Accepted Manuscript
Design and synthesis of new norfloxacin-1,3,4-oxadiazole hybrids
as
antibacterial
agents
against
methicillin-resistant
Staphylococcus aureus (MRSA)
Yong Guo, Ting Xu, Chongnan Bao, Zhiyan Liu, Jiangping Fan,
Ruige Yang, Shangshang Qin
PII:
DOI:
Article Number:
Reference:
S0928-0987(19)30229-5
https://doi.org/10.1016/j.ejps.2019.104966
104966
PHASCI 104966
To appear in:
European Journal of Pharmaceutical Sciences
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Revised date:
Accepted date:
20 April 2019
27 May 2019
20 June 2019
Please cite this article as: Y. Guo, T. Xu, C. Bao, et al., Design and synthesis of new
norfloxacin-1,3,4-oxadiazole hybrids as antibacterial agents against methicillin-resistant
Staphylococcus aureus (MRSA), European Journal of Pharmaceutical Sciences,
https://doi.org/10.1016/j.ejps.2019.104966
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ACCEPTED MANUSCRIPT
Design and synthesis of new norfloxacin-1,3,4-oxadiazole
hybrids as antibacterial agents against methicillin-resistant
Staphylococcus aureus (MRSA)
Yong Guo*, Ting Xu, Chongnan Bao, Zhiyan Liu, Jiangping Fan, Ruige Yang* and
Shangshang Qin*.
Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education,
School of Pharmaceutical Sciences, Zhengzhou University, No. 100, KeXue Avenue,
Zhengzhou, 450001, Henan Province, PR China
*Corresponding
guoyong_122@zzu.edu.cn
qinshangshang@126.com (S. Qin).
authors:
Tel.
/fax:
+86
371
67781902,
(R.
e-mail:
Yang);
(Y.
Guo);
yrggg@zzu.edu.cn.
1

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Abstract:
Towards the search of new antibaterial agents to control methicillin-resistant
Staphylococcus aureus (MRSA), a class of new norfloxacin-1,3,4-oxadiazole hybrids
were designed and synthesized. Antibacterial activities against drug-sensitive
bacteria S. aureus and clinical drug resistant isolates of MRSA were evaluated.
Compound 5k exhibited excellent antibacterial activities against S. aureus (MIC: 2
μg/mL) and MRSA1-3 (MIC: 0.25-1 μg/mL). The time-kill kinetics demonstrated
that compound 5k had an advantage over commonly used antibiotics vancomycin in
killing S. aureus and MRSA. Moreover, compound 5k could inhibit the bacteria and
destroy their membranes in a short time, and showed very low cytotoxicity to
NRK-52E cells. Some interesting structure-activity relationships (SARs) were also
discussed. These results indicated that these norfloxacin-1,3,4-oxadiazole hybrids
could be further developed into new antibacterial agents against MRSA.
Keywords:
Norfloxacin;
1,3,4-Oxadiazole;
Antibacterial
activity;
Structure-activity relationship; Cytotoxic activity.
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1. Introduction
Bacterial infection by Staphylococcus aureus is a major health threat during the
practice of medical procedures, such as surgery and organ transplants. This health
threat may worsen because of drug-resistant S. aureus, such as methicillin-resistant S.
aureus (MRSA) (Bassetti et al., 2013; Dai et al., 2018). MRSA, associated with high
morbidity and mortality in both humans and animals, is the most common bacteria
(VanEperen and Segreti 2016). It can cause pneumonia sepsis and skin infections and
has been proved to be highly resistant to many antibiotics (Woods and Colice 2014;
Enright et al., 2002; Mihu et al., 2015).
In 2017, the World Health Organization (WHO) officially ranked MRSA as one
of the 12 deadliest drug-resistant bacteria (Hua et al., 2018). Moreover, researchers
have also observed that MRSA strains become resistant to vancomycin that is
regarded as the last-resort antibiotic for the treatment of MRSA infections (Mandal
et al., 2017). Therefore, there is an urgent need to discover new antibiotics against
this major human pathogen.
Norfloxacin (NFX, Fig.1) belonging to a third generation broad-spectrum
fluoroquinolone group antibiotic, has been used widely for treatment of various
bacterial infections in human beings and animals. However, the repeated and overuse
of norfloxacin has resulted the growing incidence of resistance, which has become a
serious problem (Kumar et al., 2018; Wang et al., 2018). Hence, there are still new
norfloxacin derivatives need to develop since the continuous demand of new
antibacterial agents against drug-resistant pathogens such as MRSA and
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vancomycin-resistant S. aureus (VRSA). There is no doubt that the ADME/Tox
(absorption, distribution, metabolism, elimination and toxicity) drug properties, are
crucial to a drug candidate in clinic (El-Zahabi et al., 2019). By considering the
absorption and solubility properties, so we want to introduce some small
N-heterocyclics in piperazine ring rather than in carboxylic group. In addition, the
molecular weight of target compounds after the introduction of N-heterocyclics
should be as small as 500 Da to conform to Lipinski Rule of Five (Lipinski et al.,
1997). 1,3,4-Oxadizaole is often used as a small pharmacophore in drug design due
to presence of azole (–N=C–O–) group which increases the hydrophobicity
(Andreani et al., 2001). This could benefit the ability of drug to reach the infection
site by transmembrane diffusion (Bajaj et al., 2015). 1,3,4-Oxadizaoles also exhibit a
wide range of biological activities such as antibacterial, anti-inflammatory,
anticancer, insecticidal and antifungal activities (Xu et al., 2012; Sahin et al., 2002;
Guo et al., 2016, 2017; Dai et al., 2017; Zou et al., 2002).
Fig. 1. Illustration of design strategy for norfloxacin-1,3,4-oxadiazole hybrids.
A great deal of research has reported that some nitrogen-containing
heterocycles, in combination with quinolones (I–III, Fig.1), show good antibacterial
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activities, especially against some drug-resistant bacteria, such as
methicillin-resistant S. aureus (MRSA), with excellent activity and low cytotoxicity
(Wang et al., 2018; Zhang et al., 2016; Cianchetta et al., 2004). However, as far as
we know, the combination of 1,3,4-oxadizaole and the norfloxacin has been never
investigated. In view of above mentioned facts, we hypothesized that combining the
structural features of norfloxacin and 1,3,4-oxadiazole moieties with the intention of
owning beneficial effect on the antibacterial activity. In the current study, we have
prepared a number of norfloxacin linked 1,3,4-oxadiazoles 5a–t and evaluated their
anti-MRSA and cytotoxic properties.
2. Materials and methods
2.1. Instrument and materials
A digital melting-point apparatus was used to determine melting points which
1
were uncorrected (Beijing Tech Instrument Co., Ltd.). H/13C NMR spectra were
carried out in CDCl3 on a Bruker Avance (400 MHz) spectrometer using
tetramethylsilane (TMS) as the internal standard (Bruker, Bremerhaven, Germany).
Electrospray ion trap mass spectrometry (ESI-MS) were carried out with an Agilent
1100 LC/MSD SL instrument (Agilent, Palo Alto, USA). Infrared spectra (IR) were
recorded on a PE-1710 FT-IR spectrometer (Perkin-Elmer, Waltham, MA, USA).
Optical density (OD) of the bacterial suspension was recorded using a Microplate
Reader at 450 nm (Synergy H1 Hybrid Microplate Reader, BioTek Instruments, Inc.
VT, USA). Different aromatic acids and Norfloxacin were purchased from Macklin
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Biochemical Inc. (Shanghai, China). Hydrazine hydrate, chloroacetyl chloride and
phosphorus oxychloride were obtained from Aladdin Inc. (Shanghai, China). Other
reagents were of analytically grade also purchased from commercial resources.
Anhydrous solvents were dried and purified according to standard methods before
use. Analytical thin-layer chromatography (TLC) and preparative thin-layer
chromatography (PTLC) were prepared by silica gel plates using silica gel GF254
(Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). Methicillin-susceptible
bacterial strain S. aureus (ATCC 29213) was purchased from the American Type
Culture Collection (ATCC, Manassas, VA, USA) and three non-duplicate clinical
methicillin-resistant S. aureus isolates (MRSA1-3) were recovered from sputum
specimen of different patients from an affiliated hospital of Zhengzhou University.
2.2. Chemical synthesis
2.2.1. Synthesis of norfloxacin-1,3,4-oxadiazole hybrids 5a–t.
A mixture of the compound 1 (0.3 mmol, 95.8 mg), K2CO3 (1.8 mmol, 248.4
mg), 2-(chloromethyl)-5-substitued-1,3,4-oxadiazoles 4a–t (0.72 mmol) and KI
(0.12 mmol, 19.9 mg) in acetone was refluxed. When the reaction was complete
according to TLC analysis, the solvent was removed in vacuo and the residue was
dissolved in MeOH and filtered. The filtrate was concentrated and then purified by
PTLC to afford 5a–t in 88-99% yield.
2.3. Biological activity
2.3.1. Antibacterial activity
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MIC, as the minimum concentration of compounds resulting in inhibition of
obvious growth of bacteria, was determined by the standard broth microdilution
method according to the Clinical and Laboratory Standards Institute (CLSI).
Cation-adjusted Muller-Hinton broth (MHB) was commonly used as the test medium.
Single colony were cultured at 37 °C in constant temperature for 3-5 h to give about
108 CFU/mL of bacterial. Bacterial cultures were diluted 100 times to 106 CFU/mL
with MHB. All the final compounds were dissolved in DMSO. The storage liquid
concentration should be 25600 µg/mL, which were diluted to give 256 µg/mL with
Milli-Q water. The wells of 96 well plate were filled with 100 µL two-fold diluted
tested compounds (256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 µg/mL) and
100 µL bacterial suspension. The control experiments included 200 µL of media
(negative contrast) and 200 µL of bacterial solution (positive contrast). The antibiotic
vancomycin was used as positive control drug. The plates were incubated at 37°C for
16-20 h, and then observed the results. The bacterial solution with the lowest
turbidity in the MIC experiment was plated (20 µL) on the agar plates, then
incubated at 37°C for 20-24 h. The concentration which bacterial growth was not
observed indicated the MBC of the respective compounds.
2.3.2. Bactericidal time-kill kinetics
The single colony of S. aureus and MRSA-2 were cultured in 1 mL fresh MHB
overnight. The bacterial suspension was diluted 1:10000 with fresh MHB and
incubated at 37 °C, with shaking at 225 rpm for 2.5 h to obtain early
exponential-phase cells (~106 CFU/mL). The suspension was then incubated with
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different concentrations of compound (6 µg/mL and 8 µg/mL) and Vancomycin (12
µg/mL) for 0, 1, 2, 4, 6, 8 and 10 h respectively. At each specific time, 100 µL
samples were removed and washed three times with PBS. Diluted the samples 105
fold with PBS serially and spotted 10 µL of diluent on agar plates. After incubating
the plates overnight (~18 h) at 37 °C, observed the growth of bacteria and counted
the colonies on the plates.
2.3.3. Biofilm disruption ability
The ability of the title compounds to destroy the membranes of MRSA-2 and S.
aureus was determined with DAPI and PI fluorescent dyes. Single colony was
cultured at 37 °C in constant temperature for 3-5 h to give about 108 CFU/mL of
bacterial solution. Bacterial solution was diluted 100 times to 106 CFU/mL with
MHB. The bacterial solution with 8 µg/mL of the tested compounds were incubated
at 37 °C for 2 h. Cell pellets from the centrifuge (4000 × g, 15 min, 4 °C) were
washed with PBS, and incubated with DAPI (1 µg/mL) and PI (2 µg/mL) in the dark
at 0 °C for 10 min. The 10 µL of suspension was spotted on chamber slides after
washing three times with PBS, observed under fluorescence microscope.
2.3.4. Cytotoxicity assay against NRK-52E cells
CCK-8 assay was performed to detected compound 5k cytotoxicity using normal
rat kidney tubular epithelial (NRK-52E) cells which was purchased from ATCC.
Briefly, 5 × 103 NRK-52E cells in 100 μL medium were seed to each of 96-well plates.
o
After 24 h incubation at 37 C, the culture medium was removed and replaced with
fresh medium (100 μL) containing the compounds 5k in different concentration. The
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negative control was only added fresh medium. At the end of the treatment (24 h), the
medium was discarded and washed twice with the new culture medium, then added
100 μL new medium containing 5% CCK-8 to each well. The cells were cultured at 37
oC for a further 4 h and then the absorbance at 450 nm was measured using a
Microplate Reader. The results were calculated with the formula: Cell inhibitory rate
(%) = 1-(ODexperiment - ODblank)/ (ODnegative control - ODblank) × 100%. Each compound
was tested three times in each of the cell lines.
3. Materials and methods
3.1. Chemistry
As shown in Scheme 1, the key intermediates 4a–t were prepared in four steps
starting from commercially aromatic acids (Dai et al., 2017). Firstly, different
aromatic acids reacted with EtOH to obtain the aromatic acid ethyl esters, respectively,
then these esters were hydrazinolysised in EtOH at reflux to get aromatic hydrazines
2a–t. Chloroacetylation of 2a–t to afford the unsymmetrical bis(hydrazide) 3a–t. At
last, treatment of 3a–t with POCl3 at 90–100 oC to obtain the
2-(chloromethyl)-5-substitued-1,3,4-oxadiazoles 4a–t. With these key intermediates
4a–t in hand, the compound 1 reacted with 4a–t at presence of K2CO3 and KI to
obtain the title compounds 5a–t smoothly. The structures of 5a–t were characterized
by 1H NMR, 13C NMR, IR, mp and ESI-MS.
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Scheme 1. Synthetic route for new norfloxacin-1,3,4-oxadiazole hybrids 5a–t.
3.2. Biological activity and structure–activity relationship
3.2.1. Antibacterial activity
The antibacterial activity of these new norfloxacin-1,3,4-oxadiazole hybrids
5a–t was examined in vitro against Gram-positive bacteria S. aureus (ATCC 29213)
and three drug-resistant bacteria MRSA1-3 (clinical isolated strains). The results
were represented as the minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC). The third generation fluoroquinolone norfloxacin
and the glycopeptide antibiotic vancomycin were used as the positive control drugs.
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Table 1. MIC/MBC (µg/mL) of norfloxacin-1,3,4-oxadiazole hybrids 5a–t.a
S. aureus
MRSA-1
MRSA-2
MRSA-3
Compound
MIC MBC MIC MBC MIC MBC
MIC MBC
4
4
1
2
2
2
2
4
2
2
2
2
2
2
4
2
4
1
2
1
1
2
4
4
1
2
4
2
2
4
2
2
4
2
8
8
8
2
4
1
4
1
1
2
4
2
0.5
4
1
2
2
2
4
1
0.5
2
2
2
1
1
1
1
1
4
2
0.5
4
1
4
4
4
8
1
1
2
2
4
8
1
2
1
1
2
1
1
2
1
4
0.5
1
1
2
2
2
0.5
1
0.5
1
0.5
0.5
1
1
1
1
2
1
2
1
2
2
1
2
2
1
2
1
1
1
0.5
2
1
2
1
1
4
1
4
2
2
2
2
1
2
1
4
4
0.5
2
1
2
2
5a
5b
5c
5d
5e
2
0.5
0.5
0.5
1
1
1
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
0.25
1
0.25
0.5
0.5
0.5
1
1
0.5
1
2
8
1
2
2
1
1
2
8
1
1
2
5s
5t
4
1
2
1 b
VAN c
2
1
1
a Experimental data was repeated three times. b 1 (norfloxacin). c VAN (vancomycin)
As shown in Table 1, most target compounds displayed the equal or better
antibacterial activity than vancomycin and the precursor norfloxacin. It was worth
mentioning that the compounds 5c, 5r and 5t exhibited two-fold superior to
vancomycin (MIC = 2 µg/mL) against S. aureus. Besides, twelve target compounds
(5d–5g, 5i–5n, 5p and 5s) showed the equal antibacterial activity as the positive
control vancomycin. The antibacterial ability of these compounds against three
clinical isolated drug-resistant bacteria MRSA was further detected. The range of
MIC values for these compounds 5a–5t were 0.25-4 µg/mL against MRSA1-3. In
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particular, compound 5k displayed the most potent activity against MRSA1-3 (MIC
= 0.5, 0.25 and 1 µg/mL, respectively), which was 2-4 folds more effective than the
positive control vancomycin (MIC = 2-4 µg/mL) and norfloxacin (MIC = 1-2
µg/mL). In addition, compounds 5c–h and 5j–s also showed the equal or better
antibacterial activity than vancomycin and norfloxacin against MRSA1-3. For the
MBC evaluation, the MBCs of all the target compounds ranged from 0.5-8 µg/mL
(Table 1). According to the previous research (Pankey and Sabath, 2004; Levison
2004), the antimicrobial drug could be considered bacteriostatic when the MBC/MIC
is ≥ 4, while being bactericidal when the MBC/MIC is ≤ 4. In addition, to the clinical
isolated strain, when it showed MBC/MIC of ≥ 32, it could be considered resistant or
tolerant to the tested antibacterial compounds (Rose et al., 2012). As depicted in
Table 1, the MBC/MIC ratios of all title compounds against MRSA1-3 were ≤ 4.
This result demonstrated that the norfloxacin-1,3,4-oxadiazole hybrids had potent
and stable bactericidal effect. Meanwhile, the preliminary structure-activity
relationships (SARs) study were also explored. When R was 2-chlorophenyl, the
corresponding product 5c showed more potent activity than that of other
2-substituted phenyl. [e.g., 5c: R = (2-Cl)Ph vs. 5b: R = (2- F)Ph, 5d: R = (2-Br)Ph,
5e: R = (2-CH3)Ph and 5f: R = (2-OCH3)Ph]. When R were 3-substituted phenyl
groups, the title compound with R = 3-(OCH3)Ph exhibited excellent antibacterial
activity. When R were 4-substituted phenyl groups, R = 4-(OCH3)Ph could lead to
more potent title compound than that of other 4-substituted phenyl groups.
3.2.2. Bactericidal time-kill kinetics
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Fig. 2. Time-kill curves of pathogens by compound 5k. (a) S. aureus were cultured
to early exponential phase, and experiment groups include compound 5k (at 12
µg/mL and 16 µg/mL), Van (at 12 µg/mL) and sterile water; (b) MRSA2 were
cultured to early exponential phase, and challenged with compound 5k (at 1.5 µg/mL
and 2 µg/mL) and Van (at 12 µg/mL) sterile water. Each data was repeated three
times.
The time-kill kinetics of compound 5k was conducted to investigate the speed
of bactericidal effect against MRSA2 and S. aureus. Compound 5k displayed
favorable bacterial activity. As shown in Fig. 2.(a) and (b), compound 5k can
completely kill S. aureus at 6 h at 8 × MIC and 8 h at 6 × MIC. The compound 5k
showed more superior bactericidal activity against MRSA2 than observed for S.
aureus. At the concentration of 8 × MIC and 6 × MIC, the colonies of MRSA2
dropped from 106 to 100 CFU/mL after 6 h, whereas, vancomycin (6 × MIC) could
not eradicate MRSA2 and S. aureus at 6 h and remain a steady trend. Moreover, the
groups of 5k (6 × MIC and 8 × MIC) samples were both clear after 12 h treatment,
but the groups of control sample were turbid [Fig. 3.(a) and (b)]. Thus, these results
indicated that the compound 5k has an advantage over commonly used antibiotics
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vancomycin in killing S. aureus and MRSA.
Fig.3. Time-kill curves of pathogens by compound 5k. (a) S. aureus were cultured to
early exponential phase and experiment groups include compound 5k (at 12 µg/mL
and 16 µg/mL), Van (at 12 µg/mL) and sterile water; (b) MRSA2 were cultured to
early exponential phase and challenged with compound 5k (at 1.5 µg/mL and 2
µg/mL), Vancomycin (at 12 µg/mL) and sterile water.
3.2.3. Biofilm disruption ability
The most efficient compound 5k was detected for its capability to breach
membranes of S. aureus and MRSA2. As is known to all,
4′,6-diamidino-2-phenylindole (DAPI) could tint membranes of bacterial cells and
display blue fluorescence irrespective of cell viability. Whereas, propidium iodide
(PI), a DNA intercalator, its red fluorescence could only be observed when cell
membranes are destroyed. As shown in Fig. 4, the control experiments S. aureus and
MRSA2 displayed blue fluorescence in the absence of compound 5k (Fig. 4. A1 and
B1). However, no red fluorescence was displayed in the PI channel (Fig. 4. A3 and
B3). After the bacterial solution was incubated with 5k at 37 °C for 2 h, the red
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fluorescence was observed under the PI channel in both S. aureus and MRSA2
groups (Fig. 4. A4 and B4). These results demonstrated that the membranes of S.
aureus and MRSA2 were destroyed by 5k. The compound 5k could inhibit the
bacteria and destroy their membranes in a short time, which caused irreversible
damage to the bacteria.
Fig.4. Fluorescence micrographs of S. aureus and MRSA2 that were disposed or not
disposed with 8 µg/mL of compound 5k for 2 h. (A1) control, S. aureus, DAPI
stained; (A2) 5k, S. aureus, DAPI stained; (A3) control, S. aureus PI stained; (A4)
5k, S. aureus, PI stained; (B1) control, MRSA2, DAPI stained; (B2) 5k, MRSA2,
DAPI stained; (B3) control, MRSA2, PI stained; (B4) 5k, MRSA2, PI stained.
3.2.4. Cytotoxicity assay against NRK-52E cells
Finally, the cytotoxic activity of compound 5k was mensurated by Cell
Counting Kit-8 (CCK-8) (Guo et al., 2019). The IC50 values of 5k was >200 µg /mL
which showed very low cytotoxicity to normal rat kidney tubular epithelial
(NRK-52E) cells. This result indicated that the compound 5k was not toxic in the
range of effective blood drug concentration.
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4. Conclusion
In summary, we have prepared a class of new norfloxacin-1,3,4-oxadiazole
hybrids 5a–t and evaluated their antibacterial activities against S. aureus and MRSA.
The antibacterial assays showed that compound 5k had excellent antibacterial
activities against S. aureus (MIC: 2 μg/mL) and MRSA1-3 (MIC: 0.25-1 μg/mL).
The compound 5k had an advantage over commonly used antibiotics vancomycin in
killing S. aureus and MRSA in the time-kill kinetics study. In addition, the
compound 5k could destroy the bacteria membranes in a short time, and also showed
very low cytotoxicity with IC50 of >200 µg /mL. The preliminary SARs indicated
that when R was 2-chlorophenyl/3-(OCH3)Ph/4-(OCH3)Ph group, it could lead to
more potent target compounds than that of other substituted phenyl groups. Thus,
these norfloxacin-1,3,4-oxadiazole hybrids have potential antibacterial effects
against S. aureu and MRSA infections. Considering the ADME properties of drugs
and Lipinski Rule of Five, further optimization of these norfloxacin-1,3,4-oxadiazole
hybrids toward enhanced the probability of the success of drug candidates is ongoing
in our lab, which will be reported in due course.
Acknoledgements
The anthors acknowledge financial support of this research by Natural Science
Foundation of Hennan province of China (No. 182300410025), the China
Postdoctoral Science Foundation (2019M652585), Key Science and Technology
Program of Henan Province, China (192102110054) and Startup Research Fund of
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Zhengzhou University (145- 32211180). We are deeply thankful to Dr. Bing Zhao for
the NMR tests.
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