Metronidazole containing pyrazole derivatives potently inhibit tyrosyl-tRNA synthetase: design, synthesis, and biological evaluation

Article abstract of DOI:10.1111/cbdd.12793

As an important enzyme in bacterial protein biosynthesis, tyrosyl-tRNA synthetase (TyrRS) has been an absorbing therapeutic target for exploring novel antibacterial agents. A series of metronidazole-based antibacterial agents has been synthesized and iden

Full text of DOI:10.1111/cbdd.12793

Received: 16 November 2015
DOI: 10.1111/cbdd.12793
Revised: 24 February 2016
Accepted: 14 May 2016
R E S E A R C H A R T I C L E
Metronidazole containing pyrazole derivatives potently inhibit
tyrosyl-tRNA synthetase: design, synthesis, and biological
evaluation
Long-Wang Chen† | Peng-Fei Wang† | Dan-Jie Tang | Xiang-Xiang Tao |
Ruo-Jun Man | Han-Yue Qiu | Zhong-Chang Wang | Chen Xu | Hai-Liang Zhu
State Key Laboratory of Pharmaceutical
Biotechnology, Nanjing University, Nanjing, China
As an important enzyme in bacterial protein biosynthesis, tyrosyl-tRNA synthetase
(TyrRS) has been an absorbing therapeutic target for exploring novel antibacterial
agents. A series of metronidazole-based antibacterial agents has been synthesized
Correspondence
Zhong-Chang Wang, wangzhongchang2006@163.
and identified as TyrRS inhibitors with low cytotoxicity and significant antibacte-
rial activity, especially against Gram-negative organisms. Of the compounds
com; Chen Xu xuchn@nju.edu.cn; Hai-Liang Zhu
and zhuhl@nju.edu.cn
obtained, 4f is the most potent agent which inhibited the growth of Pseudomonas
aeruginosa ATCC 13525 (MIC = 0.98 μg/mL) and exhibited TryRS inhibitory
activity (IC50 = 0.92 μm). Docking simulation was performed to further understand
its potency. Membrane-mediated apoptosis in P. aeruginosa was verified by flow
cytometry.
K E Y W O R D S
antibacterial agent, apoptosis, docking simulation, Pseudomonas aeruginosa,
tyrosyl-tRNA synthetase
Bacterial infection has been a consistent and relentless threat
to human health which has contributed innumerable finan-
cial burden to the healthcare system for a long time.[1] In
addition, resistance to antibiotics, a widespread and thorny
clinical problem, needs urgent attention due to a scarcity of
new therapeutic agents.[2,3] Very recently, aminoacyl-tRNA
synthetases (aaRSs) which exist in all creatures including the
prokaryotes and eukaryotes have attracted impressive inter-
est in antibacterial drug discovery.[4] Theoretically speaking,
aaRSs are essential enzymes catalyzing a vital process in
which amino acids are loaded to the cognate tRNAs.[5] It will
lead to the failure of protein synthesis and cell growth arrest
once they were inhibited in the cell. Moreover, because of the
difference of significant evolutionary divergence between the
human beings and microbes, aaRSs in the bacteria could be
the targets selectively inhibited.[6,7] It is noteworthy that the
tyrosyl-tRNA synthetase (TyrRS), a member of aaRSs fam-
ily, has obtained increasing attention nowadays and may be
a promising target for antibacterial agents.[7] Nevertheless,
there is still no marketed drug targeting TyrRS[8] while our
group had developed one kind of inhibitors with diaryl het-
erocycle scaffold (Fig. S1) and another series of inhibitors
based on metronidazole skeleton (Fig. S2), both of which
could selectively suppress it.[9,10]
Pyrazole, as a part of many natural and synthetic com-
pounds and employed as the central building blocks for the
synthesis of compound libraries,[11] exerts beneficial effects in
the prevention of many diseases, including antimicrobial,[12]
antitumor,[13] anti-inflammatory,[14] antidepressant,[15] and
other activities. Nitroimidazole derivatives are provided with
broad spectrum of biological activities,[16] especially antimi-
crobial activity. Among all the nitroimidazoles, metronidazole
was used broadly in the treatment of infections induced by bac-
teria and other pathogenic protozoan parasites.[17] It is the truth
†These two authors equally contributed to this manuscript.
|
Chem Biol Drug Des 2016; 1–8
wileyonlinelibrary.com/cbdd
© 2016 John Wiley & Sons A/S.
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R2
O
O
OH
CO2CH3
R1
R1
N
OCH3
N
R1
i
ii
O
1a-1j
2a-2y
R2
R2
N
OH
N
SCHEME 1 The synthetic routes of compounds 4a–
y. Reagents and conditions: (i) oxalic acid dimethyl ester,
NaOCH3, CH3OH, reflux, 5 h; (ii) phenylhydrazine, diluted
hydrochloric acid, CH3OH, reflux, 6 h; (iii) KOH, H2O,
CH3OH, reflux, 2 h; (iv) EDC, HOBT, DMAP, CH2Cl2,
45°C, overnight
R1
iii
iv
R1
N
N
CH3
N
O
O
N
O
3a-3y
4a-4y
N
O2
that the metronidazole derivatives in organisms can produce
electrophilic substances through biological reduction which
can destroy proteins and nucleic acids; thus, the structure of
metronidazole is widely applied in medicinal chemistry as
an important pharmacophore.[18] In recent years, researchers
increasingly tended to focus on the modification of the hydroxy
group of metronidazole to screen more valuable drugs.[19] In
our previous work, several pyrazole derivatives (Fig. S1) have
been validated to inhibit aaRSs well. Encouraged by the find-
ings, we have further confirmed that their efficacy mainly owes
to the diaryl heterocycle containing pyrazole.[9] Furthermore,
our group lately synthesized a class of compounds with met-
ronidazole analogs targeting TyrRS (Fig. S2) and evaluated
for their antibacterial activities.[10] Considering that multiple
pharmacophores and complex scaffold are the common fea-
tures of most antibacterial drugs,[20] we made the structural
optimization of introducing the metronidazole on the pyra-
zole scaffold based on previous studies in this work.[19] The
following docking simulation was carried out to predict the
binding mode and affinity of the new compounds and enzyme.
The results showed that the affinity of the new compounds
was improved; thus, it was of interest to synthesize these com-
pounds and evaluate their bioactivity.
pathway depicted in Scheme 1, and the structures of the
desired compounds are shown in Table S1. The starting
chalcone derivatives, compounds 1 (1a–j), were synthesized
from the reaction of acetophenone with dimethyl oxalate
in the methanol mixed with sodium methoxide. Reaction
of compound 1 with phenylhydrazine afforded the inter-
mediates 2 (2a–y). Afterward, the intermediates 2 were
hydrolyzed by KOH and drops of water in the methanol
to give the last intermediate compounds 3 (3a–y), which
contain a carboxyl group. Compounds 4a–y hereafter were
prepared from the reaction of compounds 3 with metro-
nidazole in dichloromethane. The reactions were monitored
by thin layer chromatography (TLC), and the crude products
were purified by column chromatography and recrystalliza-
tion with ethanol. All of the target compounds gave sat-
isfactory analytical and spectroscopic data, that is, 1H
NMR, EI-MS, which are in accordance with their depicted
structures. All of these compounds were reported for the
first time. Furthermore, the crystal structure of compound
4l was determined by single crystal X-ray diffraction
analysis in Figure 1, and its crystal data, data collection,
and refinement parameters for the compound 4l were listed
in Table S2.
In this research, we reported a sequence of metronidazole
compounds bearing a pyrazole ring for the sake of finding
new efficient antibacterial agents. We thereafter evaluated the
antimicroorganism activity of them and also discussed the
possible mechanism of their antibacterial action.
1
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RESULTS AND DISCUSSION
1.1 | Chemistry
All the novel metronidazole-pyrazole derivatives (4a–y)
described herein were synthesized following the synthetic
FIGURE 1 Crystal structure diagram of compound 4l.

Chen et al.
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Changing the position of the methyl group from the para-
position (4g) to the ortho-position (4p) significantly enhanced
the level of TyrRS inhibition. However, over 20-fold loss of
enzyme inhibitory activity was observed when the 2-methyl
group was replaced by fluoro atom (4t). On the other hand,
the exchange of the fluoro atom from the ortho- to the meta-
position resulted in an equally potent compound (4x), while the
replacement of the methyl group to the meta-position (4v) led to
a fourfold increase in potency. All the results suggest that com-
pounds with electron-donating groups showed better inhibitory
activities than those with electron-withdrawing groups.
1.2 | Inhibitory activities of metronidazole-
pyrazole derivatives against TyrRS
All the synthesized compounds (4a–y) were tested for inhibi-
tory activity against TyrRS to obtain a further understanding
of the structure–activity relationships (SAR). The IC50s of
these compounds are presented in Table S3. As we can
see, the compound 4f with 4-methoxy substituent in the
benzene ring fragment (A) exerted the highest inhibitory
activity against TyrRS with an IC50 of 0.92 μm and afforded
over 60-fold improvement over the unsubstituted analog
(4r). In comparison with 4r, most substituted compounds
were more potent, indicating that the modification on the
aryl rings seems to be beneficial to enzyme inhibition.
In the case of compounds (4q, 4u) with identical R2 showed
great difference of activity against TyrRS, while compounds
(4p, 4q) with same R1 led to scarce alternation in potency. It
TABLE 1 Minimum inhibitory activity (MIC) of the synthetic compounds against microbes
Compounds
MICa (μg/mL)
Gram-positive
Gram-negative
Staphylococcus aureus
Pseudomonas aeruginosa
ATCC 6538
Bacillus subtilis ATCC 6633
Escherichia coli ATCC 35218
ATCC 13525
4a
31.25
62.5
7.82
7.82
31.25
62.5
4b
31.25
125
62.5
31.25
62.5
4c
125
4d
31.25
62.5
3.91
125
62.5
15.63
15.63
1.96
15.63
31.25
0.98
4e
31.25
3.91
4f
4g
62.5
62.5
31.25
31.25
7.82
4h
62.5
15.63
15.63
7.82
15.63
31.25
62.5
62.5
15.63
62.5
62.5
31.25
125
62.5
62.5
4i
31.25
15.63
7.82
7.82
4j
15.63
3.91
7.82
4k
1.96
4l
31.25
62.5
3.91
3.91
4m
31.25
31.25
15.63
3.91
31.25
15.63
15.63
3.91
4n
62.5
4o
31.25
15.63
32.25
31.25
31.25
125
4p
4q
7.82
3.91
4r
31.25
7.82
31.25
15.63
62.5
4s
4t
125
4u
62.5
15.63
31.25
62.5
125
62.5
31.25
15.63
15.63
62.5
62.5
4v
15.63
15.63
62.5
7.82
4w
15.63
31.25
31.25
7.82
4x
4y
62.5
31.25
7.82
Penicillin G
Chloramphenicol
Metronidazole
1.96
3.91
62.5
0.98
3.91
3.91
0.98
62.5
31.25
62.5
aValues are the average of three independent experiments run in triplicate.

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FIGURE 2 Ligand interaction diagram of
compound 4f with TyrRS using Discovery Studio
program with the essential amino acid residues at the
binding site are tagged in circles.
FIGURE 3 The 3D model structure of
compound 4f binding model with TyrRS.
illustrates that ring A seems to be more suitable for further
subtilis ATCC 6633, Staphylococcus aureus ATCC 6538)
and Gram-negative bacteria (Escherichia coli ATCC 35218,
Pseudomonas aeruginosa ATCC 13525). The results are
presented in Table 1. By and large, the obtained compounds
showed more sensitivity to Gram-negative bacterial strains
rather than Gram-positive bacterial strains, especially against
P. aeruginosa. Compound 4f and 4k were found to exhibit
impressive activity against both Gram-positive and
modification with a substituent containing more diverse groups.
1.3 | Antibacterial activity
Encouraged by the results of TyrRS inhibitory assays, all
the synthetic compounds were evaluated for their antimi-
crobial activities against two Gram-positive bacteria (Bacillus

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FIGURE 4 Annexin-V/PI dual-immuno-fluorescence staining after treatment with different concentrations of 4f revealed significantly increased
number of apoptotic cells (measured with Annexin-V+/PI+ cells). (A) Cells treated with 0, 0.49, 0.98 and 1.96 μg/mL 4f for 14 h were collected and
processed for analysis. (B) The percentage of apoptotic cells were calculated after the treatment of 4f. Image is representative of three independent
experiments.
FIGURE 5 Effects of 4f on membrane potential. Membrane potential (ΔΨm) was analyzed using Rho123. The P. aeruginosa cells were treated with
the designed concentration of 4f (0, 0.49, 0.98, 1.96 μg/mL) at 37 °C for 14 h. P. aeruginosa cells were incubated with Rho123 (1 μM) for 30 min and then
harvested for analysis by flow cytometry. (A) reflected the reduction of ΔΨm. (B) presented the offset of the peaks.
Gram-negative bacterial strains and 4f which presented the
most active enzyme inhibition also showed the highest
potency of antibacterial activity with MIC of 0.98 μg/mL
against P. aeruginosa and 1.96 μg/mL against E. coli, which
is close to marketed antibiotics. When it comes to the MIC
values against Gram-negative bacteria (especially against

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P. aeruginosa) and the corresponding IC50 values against
TyrRS, a distinct correlation was found in almost all cases
(Table 1 and Table S3), which indicates that the antibacte-
rial activity may due to their inhibition of TyrRS.
percentage of apoptotic cells significantly increased when
concentration up to 1.96 μg/mL. The results consequently
came to the conclusion that compound 4f could induce
bacterial cell apoptosis well.
1.4 | Molecular docking
1.6 | Effects of 4f on membrane-mediated
apoptosis pathways
For the purpose of giving an explanation and understand-
ing of the excellent activity observed, we examined the
interaction of the most potent inhibitor 4f with TyrRS
(1JIJ.pdb). All docking runs were applied CDOCKER Dock
protocol of Discovery Studio 3.5. The docking model
obtained in this study indicated that 4f docks favorably
into the active site of TyrRS (Figures 2 and 3) through
five hydrogen bonds with Tyr36 (distance = 2.4 Å), Tyr170
(distance = 2.4 Å), Cys37 (distance = 2.1 Å), Lys84
(distance = 2.5 Å), and Arg88 (distance = 2.3 Å) resi-
dues. There were three important sections participating to
form hydrogen bonds: nitrogen atom in nitroimidazole
moiety and oxygen atom of nitro group were both responded
to tyrosines; furthermore, methoxy group in the benzene
ring contributed to form two hydrogen bonds with Lys84
and Arg88, respectively; finally, the ester bond connected
the former two structures took part in the last hydrogen
bond with cysteine. Besides, the higher binding affinity
of 4f might also be accounted for by some weak interac-
tions, such as π bonds, van der Waals forces, and others.
The above-mentioned interactions tightly anchoring 4f to
the active site of TyrRS probably explain its excellent
inhibitory activity. As depicted in Figure S3, the CDOCK
interaction energy of 4f was −59.27 kcal/mol, which is
the highest one of all. Our modeling results revealed that
compound 4f possessed an important influence on the
interactions of the protein–ligand complex and was crucial
to the potency of TyrRS inhibitory activity. Therefore, 4f
could be a potential inhibitor of TyrRS.
We measured the disruption of cell membrane potential
(ΔΨm) using Rho123 stain to test whether compound 4f
could induce apoptosis of P. aeruginosa through destroy-
ing the cell membrane.[24] As depicted in Figure 5, com-
pared with control group, P. aeruginosa cells which were
treated with 0.49, 0.98, 1.96 μg/mL of compound 4f for
14 h are all exposing the collapse of ΔΨm. The observa-
tions indicated that compound 4f might induce irreversible
programed cell death phenomenon through membrane-
mediated apoptosis pathway.
1.7 | Cytotoxicity
To explore potential antibiotic, all the compounds were
evaluated by the MTT assay for their toxicity against the
human renal epithelial cells which was defined by the
median cytotoxic concentration (CC50) data. As shown in
Table S3, most of the analogs possessed low
cytotoxicity.
2
|
CONCLUSION
Faced with the shortage of new therapeutic drugs to
antibiotic-resistant bacteria, battery of novel
a
metronidazole-pyrazole derivatives targeted to TyrRS was
synthesized and characterized. Meanwhile, their biological
activities were evaluated. Antimicrobial activity and TyrRS
inhibitory activity have identified that compound 4f pos-
sessed the most potent inhibition activity against B. subtilis,
S. aureus, E. coli, and P. aeruginosa, especially the
Gram-negative bacteria (with MIC of 1.96 and 0.98 μg/
mL) and inhibition of enzyme (with IC50 of 0.92 μm).
The cytotoxicity test employing human renal epithelial
cells indicated these derivatives were highly safe. A poten-
tial binding model with docking simulation of compound
4f into TyrRS active site afforded a good understanding
and explanation of its impressive performance. Analysis
of the binding model showed that compound 4f was sta-
bilized by five hydrogen bonds. The following experiments
including apoptosis and disruption of the membrane poten-
tial demonstrated 4f exerts proapoptotic effects through
a membrane-mediated apoptosis pathway. These findings
bring the truth that compound 4f would be a potential
antibacterial agent for further research to light.
1.5 | 4f caused the apoptosis of P. aeruginosa
in a dose-dependent manner
Earlier studies have showed that phosphatidylserine expo-
sure, chromosome condensation, and DNA fragmentation,
which are markers of apoptosis, could be observed in
microbes treated with bactericidal antibiotic.[21–23] To fur-
ther explore whether the apoptosis was involved in the
antibacterial effect of target compound, P. aeruginosa cell
apoptosis induced by different concentrations of compound
4f (0.49, 0.98, 1.96 μg/mL) was performed. Annexin V
and propidium iodide (PI) double staining by flow cytom-
etry was conducted in this research. As shown in Figure 4,
almost no apoptotic cells were found in the control group
and a certain percentage of apoptotic cells were discovered
with low concentrations of compound 4f; however, the

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