Discovery of novel triazole-containing pyrazole ester derivatives as potential antibacterial agents

Article abstract of DOI:10.3390/molecules24071311

To develop new antibacterial agents, a series of novel triazole-containing pyrazole ester derivatives were designed and synthesized and their biological activities were evaluated as potential topoisomerase II inhibitors. Compound 4d exhibited the most potent antibacterial activity with Minimum inhibitory concentration (MIC) alues of 4 μg/mL, 2 μg/mL, 4 μg/mL, and 0.5 μg/mL against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella gallinarum, respectively. The in vivo enzyme inhibition assay 4d displayed the most potent topoisomerase II (IC₅₀ = 13.5 μg/mL) and topoisomerase IV (IC₅₀ = 24.2 μg/mL) inhibitory activity. Molecular docking was performed to position compound 4d into the topoisomerase II active site to determine the probable binding conformation. In summary, compound 4d may serve as potential topoisomerase II inhibitor.

Full text of DOI:10.3390/molecules24071311

molecules
Article
Discovery of Novel Triazole-Containing Pyrazole
Ester Derivatives as Potential Antibacterial Agents
Ming-Jie Chu 1,†, Wei Wang 1,†, Zi-Li Ren 1, Hao Liu 1, Xiang Cheng 1, Kai Mo 1, Li Wang 1,
Feng Tang 2 and Xian-Hai Lv 1,3,
*
1
School of Science, Anhui Agricultural University, Hefei 230036, China; chumingjie@ahau.edu.cn (M.-J.C.);
weiwang133@163.com (W.W.); renzilix@163.com (Z.-L.R.); 13167720906@163.com (H.L.);
xiangcheng215b@163.com (X.C.); 18256549679@163.com (K.M.); Wangli-hx@ahau.edu.cn (L.W.)
International Center for Bamboo and Rattan, 8 Fu Tong East Street, Beijing 100714, China;
fengtang@icbr.ac.cn
2
3
Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences,
Nanyang Technological University, Singapore 637371, Singapore
*
Correspondence: lvxianhai@ahau.edu.cn; Tel.: +86-551-6578-6906
†
These authors contributed equally to this work and should be considered co-first authors.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 January 2019; Accepted: 1 April 2019; Published: 3 April 2019
Abstract: To develop new antibacterial agents, a series of novel triazole-containing pyrazole ester
derivatives were designed and synthesized and their biological activities were evaluated as potential
topoisomerase II inhibitors. Compound 4d exhibited the most potent antibacterial activity with
Minimum inhibitory concentration (MIC) alues of 4 µg/mL, 2 µg/mL, 4 µg/mL, and 0.5 µg/mL
against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella gallinarum,
respectively. The in vivo enzyme inhibition assay 4d displayed the most potent topoisomerase
II (IC50 = 13.5 µg/mL) and topoisomerase IV (IC50 = 24.2 µg/mL) inhibitory activity. Molecular
docking was performed to position compound 4d into the topoisomerase II active site to determine
the probable binding conformation. In summary, compound 4d may serve as potential topoisomerase
II inhibitor.
Keywords: pyrazole; triazole; ester; antibacterial; topoisomerase II inhibitor
1. Introduction
Bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are ubiquitous and essential
enzymes that have critical roles in the fundamental biological processes of replication, transcription,
recombination, repair, and chromatin remodeling [1,2]. The action of topoisomerases is to change
the spatial structure of DNA by catenation and decatenation of duplex DNA rings, relaxation of
supercoiled DNA, and in the use of DNA topoisomerase II introduction of negative supercoils into
DNA in an energy-dependent reaction [
attention as a selected target for finding potent antibacterial agents [
quinolone antibacterial agents have been marketed and are now widely used for the treatment of
bacterial infectious diseases [ ]. Specifically, sparfloxacin has proven to be a very successful inhibitor
of bacterial DNA topoisomerase II [ ]. However, the abuse of antibacterial has led to an increase in
3]. Therefore, bacterial DNA topoisomerase II has drawn much
4–6]. For example, a lot of synthetic
7,8
9
bacterial resistance, especially in immunocompromised patients [10]. Our current research efforts are
to find novel antibacterial agents as DNA topoisomerase II inhibitors.
Hybridization was a common method of drug development and design [11]. It was based
on combining two or more different biologically active moieties in a single molecule to obtain the
corresponding conjugated hybrid molecules [12]. These hybrid molecules may exhibit better activities
Molecules 2019, 24, 1311; doi:10.3390/molecules24071311
www.mdpi.com/journal/molecules

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than their precursors [13]. The novel synthesized heterocyclic molecules can work by the same or
different mechanisms of action compared to the precursors [14]. Nitrogen containing heterocyclic
may be potential antibacterial drugs which inhibit bacterial topoisomerases [15], such as triazoles [16],
quinolones [17], oxazolopyridines, aminopyrazinamides, and pyrazole [18–20]. Recently, Plech [21]
and Lal [22] reported triazole derivatives as potential topoisomerase II inhibitors, which exhibited
greater antibacterial activities than ciprofloxacin (Figure 1).
Figure 1. Recently discovered triazole derivatives as DNA topoisomerase inhibitors.
As is well known, prolonged use of antibacterial agents can lead to environmental problems
and residual toxicity [23,24]. However, compound containing ester groups, can easily be hydrolyzed
to produce low toxicity compounds. In addition, it can increase its soluble fat in vivo and enhance
antimicrobial activity. Furthermore, it has multiple charges, enabling them to interact with the
bacterial cell surface more strongly than their monomeric counterparts. Recently, Haldar et al. [25
reported that two compounds were linked by an ester group, and the antibacterial activities of the new
compounds were improved compared with the previous compounds. In the previous research work [ ],
]
8
we discovered a novel Topoisomerase II (Topo II) inhibitors compound 3, which showed considerable
inhibitory activity than ciprofloxacin. In the search for a class of new Topoisomerase II inhibitors,
we chose pyrazole as primer molecule, introduced a triazole structure, and used the ester group as
a linker to obtain a new scaffold (Figure 2) [26,27]. After that, a series of novel triazole-containing
pyrazole ester derivatives were designed and synthesized. Initially, all of the synthesized compounds
were used against two fungi, but the results of the antifungal activity were not ideal. Therefore,
four bacteria were used to test the antibacterial activity of these compounds, two Gram-positive
bacteria, namely Staphylococcus aureus and Listeria monocytogenes, and two Gram-negative bacteria,
namely Escherichia coli and Salmonella gallinarum. In addition, molecular docking analysis studies
were performed on all derivatives to determine critical structural factors responsible for their
antibacterial efficacy.

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Figure 2. The design of the target scaffold.
2. Results and Discussion
2.1. Chemistry
A series of triazole-containing pyrazole ester derivatives were prepared and the route listed in
Scheme 1. We dissolved substituted phenylhydrazine with ethanol, then added ethylacetoacetate.
Preparation of 1a
we added KMnO4 as an oxidation reagent to obtain 2a
–
d
was with Vilsmeier–Haack Reagent (N,N-Dimethylformamide/POCl3). Then,
Compounds 3a were obtained
–
d.
–d
from phenylhydrazine hydrochloride and urea, we used formic acid under acidic conditions.
The preparation of the target compounds was carried out using the reported method [28]. We analyzed
all prepared compounds using spectral and elemental methods, which showed that all compounds
fully complied with the structure in Table 1.
◦
Scheme 1. General synthesis of compounds 4a
–
m
. Reagents and conditions: (
a
) H2O, ethanol, 60 C;
◦
◦
◦
(
b
) N,N-Dimethylformamide(DMF), POCl3, 90 C, 5h; (
c
) KMnO4, 70–80 C; (
d
) HCl, 135 C, H2SO4,
◦
90 C; (e) Dicyclohexylcarbodiimide(DCC), 4-dimethylaminopyridine(DMAP), DMF, rt.

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Table 1. Minimum inhibitory concentration (MIC) of all compounds against bacteria.
MIC (µg/mL)
Compounds
R1
R2
S.a a
L.m a
E. c a
S.e a
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
4e
H
F
Cl
64
64
64
16
32
>128
64
32
16
>128
64
32
>128
64
32
32
32
>128
64
32
>128
>128
16
16
64
64
16
8
64
64
64
64
4
16
64
64
32
16
64
64
0.5
32
CH3
H
F
Cl
CH3
H
H
H
H
F
H
F
Cl
CH3
H
16
>128
>128
2
64
4
32
4
64
32
4f
4g
4h
4i
4j
4k
4l
F
F
F
F
Cl
Cl
CH3
CH3
>128
64
8
>128
64
4
>128
64
16
64
64
>128
64
32
>128
64
4
>128
64
32
64
32
Cl
CH3
F
Cl
H
8
64
4
32
F
32
32
4m
DMSO
CIP b
64
>128
0.125
32
>128
1
64
>128
0.5
32
>128
0.25
Cl
CH3
a
Abbreviations: Staphylococcus aureus (ATCC-12600); Listeria monocytogenes (ATCC-15313); Escherichia coli
b
(ATCC-25922); Salmonella gallinarum (ATCC-9184). Ciprofloxacin.
2.2. Antibacterial Activity and Structure-Activity Relationships (SAR) Discussion
In vitro antimicrobial activity of the prepared compounds against four bacteria was evaluated by
the conventional agar-dilution method. Ciprofloxacin was selected as a reference standard. The results
of the in-vitro antibacterial activity screening of the test compounds are summarized in Table 1.
The results revealed that most of the synthetic compounds exhibited antibacterial activities
Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella gallinarum, demonstrating
the rationality of our design strategy. As can be seen from Table 1, among all the intermediate
compounds, compound 2d exhibited the most potent antibacterial activity with MIC values of
16
µg/mL against Staphylococcus aureus, but among the tested target compounds, three compounds
(i.e., 4d
,
4g, and 4k) were found to display improved antibacterial activities against Staphylococcus
aureus compared with (16 mg/L), with MIC values ranging from 4 to 8 mg/L. The eye-catching finding
was that the target products greater excellent antibacterial activity than intermediate compounds.
Among them, compounds 4d and 4k displayed most potent activity with MIC values of 4, 2, 4, and
0.5 µg/mL and 4, 8, 4, and 4 µg/mL against Staphylococcus aureus, Listeria monocytogenes, Escherichia
coli, and Salmonella gallinarum, which were similar to the broad-spectrum antibiotic Ciprofloxacin),
indicating that they possess antibacterial activity. Compound 4a showed antibacterial activities
against Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Salmonella gallinarum, Then,
we introduced different substituent groups on benzene ring in 4a, the introduction of methyl at R1
displayed significantly enhanced effects compared with that of other compounds. Compound 4d
exhibited best antibacterial activity than others. When R2 position was substituted with fluorine,
methyl substituted at the R1 site was beneficial to antibacterial activity of the synthesized compounds
(e.g., 4h). When the R2 position was substituted with methyl, electron-donating substituents (e.g., 4k
at R1 showed more potent activities than those with electron-withdrawing (e.g., 4l 4m). When R1 and
R2 were simultaneously substituted by electron-withdrawing, their activities were poor compared
)
,

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with other compounds. (e.g., 4j
,
4f
,
4i). Considering the above SAR results, these findings suggest that
the antibacterial potency of designed compounds could be ascribed to a combination of factors, such as
the R1 position was substituted with electron-donating groups, which may enhance antibacterial
activity, and that the R2 position was substituted with hydrogen and the compound may enhance
antibacterial activity.
2.3. Inhibitory Effects against DNA Gyrase and Topoisomerase IV
In order to determine the relationship between compounds and antibacterial activity, the inhibitory
activity of selected compounds (4d and 4k) against DNA gyrase and topoisomerase IV isolated from
Escherichia coli was examined. As shown in Table 2, 4d showed more potent inhibition than 4k against
the two enzymes. The same exhibited the same tendency as the MIC data, suggesting that compound
4d may serve as a potential topoisomerase II inhibitor.
Table 2. Inhibitory effects of selected compounds against DNA gyrase and topoisomerase IV.
IC50 (µg/mL)
Compd.
Gyrase a
Topo IV b
4d
4k
13.5
25.7
0.25
24.2
33.1
6.9
CIP c
a
c
Topoisomerase II supercoiling activity; b Topoisomerase IV decatenation activity. Ciprofloxacin.
2.4. Docking Analysis
In order to gain a better understanding on the potency of the synthesized compounds and guide
further structure, we conducted activity relationships studies. All of the derivatives were docked
into the active site of Topo II (PDB entry: 2xcs) in the binding model of compound 4d and Topo II.
The skeleton of compound 4d was embedded in the binding pocket, showing that the pose of 4d
into the Topo II-binding site had a suitable shape that was complementary to the binding pocket,
which means that our design strategy was rational. E:Dc11, F:Dc11, and F:Dg10 established
interactions with a benzene ring of compound 4d (distance: 5.01 Å, 4.18 Å, 5.31 Å), E:Dc11 established
interactions with triazole ring of compound 4d (distance: 4.60 Å), Arg 1122 established cation–
interactions with pyrazole ring of compound 4d (distance: 5.26 Å) (Figure 3).
π–π
π
–π
π
Figure 3.
(A) Binding model of 4d (2D diagram). (B) The π–π interactions are displayed as yellow
solid lines. The cation–π interactions are displayed as green solid lines.

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3. Experimental Section
3.1. Materials and Methods
All chemicals were purchased from Energy, Meryer, and Aladdin Chemicals and were used as
1
received. H-NMR spectra analyses were carried out using anAgilent DD2 600 Hz spectrometer with
CDCl3 as the solvent and tetramethyllsilane as the internal standard. ESI-MS (Electrospray Ionisation
Mass Spectrometry) spectra were carried out on a Mariner System 5304 mass spectrometer. Elemental
analyses were performed on a CHN–O–Rapid instrument. Molecular docking was performed with
Discovery Studio 3.5 [29]. The 13C-NMR and ESI-MS spectrum of compounds can be found in the
Supplementary Materials.
3.1.1. General Procedure for the Synthesis of 5-Chloro-1-aryl-3-methyl-1H-pyrazole-4-carboxylic
Acids 2a–d
Intermediates 2a–d were obtained from Reference [30]. Dissolve para-substituted phenyl
hydrazine (0.025 mol) with anhydrous ethanol ethyl acetoacetate (0.025 mol) was added portion
wise, stirred, and refluxed for 5 h, then the solution was rotary evaporated to form a solid, which
was dissolved in DMF (25 mL) and phosphorus oxychloride (20 mL) of cold mixed solution and
◦
stirred at 85 C for 2 h. The resulting product was poured into ice-cold water and the solid was
isolated by filtration to give a yellow solid, which was oxidized by KMnO4 solution and stirred at
70–80 ◦C. After cooling to room temperature, the pH was adjusted to alkaline by the addition of 10%
NaOH solution, and the solution was filtered, HCl solution was added to the solution and solid 2a–d
eventually separated out. The resulting crude product was recrystallized from anhydrous ethanol to
give the pure product.
3.1.2. General Pathway for Prepare of 1-phenyl-1H-1,2,4-triazol-3-ol 3a–d
Intermediates 3a–d were synthesized according to Bin sun et al. [31]. Phenylhydrazine (0.2 mol)
and urea (0.25 mol) were dissolved in water (50 mL) and 30% hydrochloric acid (64.8 g) was added for
4 h at 135 ◦C. Add formic acid (0.3 mol) and 98% concentrated sulfuric acid (4.8 g) at 90 ◦C for 6 h.
Finally, the product was cooled to room temperature, filtered, and washed with water to neutral and
dried to give solid 3a–d.
3.1.3. General Procedure for Synthesis of 1-phenyl-1H-1,2,4-triazol-3-yl 5-chloro-3-methyl-1-phenyl-
1H-pyrazole-4-carboxylate 4a–m
Compounds 2a
–d (1 mmol) was stirred with triethylamine (1.5 mmol) into DMF (10 mL) medium,
then a mixture of DCC (1 mmol) and DMAP (1 mmol) was added in the reaction system, stirred at
25 ◦C for 1 h. The mixture of intermediate
3 (1.2 mmol) and DMF (6 mL) was added in the reaction,
stirred at 25 ◦C for 3 h. Finally, the product was extracted from chloroform with water, hydrochloric
acid (0.2 mol/L), sodium hydroxide (2 mol/L), saturated sodium chloride successively, and then dried,
concentrated, and purified by preparative thin layer chromatography (PE: EA = 8:1) [27].
3.1.4. 1-phenyl-1H-1,2,4-triazol-3-yl5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate (4a)
White solid, yield 62%; mp 138–140 ◦C; 1H-NMR (600 MHz, CDCl3)
δ 8.00 (s, 1H, Triazole-H),
7.53 (s, 6H, Ph-H), 7.08 (d, J = 8.0 Hz, 1H, Ph-H), 6.98 (t, J = 8.6 Hz, 1H, Ph-H), 6.93 (d, J = 4.4 Hz,
1H, Ph-H), 6.88 (d, J = 8.2 Hz, 1H, Ph-H), 2.55 (s, 3H, CH3). MS (ESI) calculated for C19H15ClN5O2
[
M + H]+, 380.1, found 380.1; Anal. Calcd for C19H14ClN5O2: C, 60.09; H, 3.72; N, 18.44%; Found: C,
60.12; H, 3.79; N, 18.33%.

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3.1.5. 1-phenyl-1H-1,2,4-triazol-3-yl5-chloro-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4b)
◦
White solid, yield 54%; mp166–168 C; 1H-NMR (600 MHz, CDCl3)
δ
7.95 (s, 1H, Triazole-H), 7.51
(dd, J = 8.8, 4.7 Hz, 2H, Ph-H), 7.28 (s, 1H, Ph-H), 7.25 (s, 1H, Ph-H), 7.22 (t, J = 8.4 Hz, 2H, Ph-H),
6.98–6.91 (m, 3H, Ph-H), 2.54 (s, 3H, CH3).MS (ESI) calculated for C19H14ClFN5O2 [M + H]+, 398.1,
found 398.1; Anal. Calcd for C19H13ClFN5O2: C, 57.37; H, 3.29; N, 17.61%; Found: C, 57.41; H, 3.31;
N, 17.73%.
3.1.6. 1-phenyl-1H-1,2,4-triazol-3-yl5-chloro-1-(4-chlorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4c)
White solid, yield 57%; mp 172–173 ◦C; 1H-NMR (600 MHz, CDCl3)
δ 7.92 (s, 1H, Triazole-H),
7.49 (d, J = 2.3 Hz, 4H, Ph-H), 7.28 (s, 1H, Ph-H), 7.25 (s, 1H, Ph-H), 6.98–6.90 (m, 3H, Ph-H), 2.54
(s, 3H, CH3) MS (ESI) calculated for C19H14Cl2N5O2 [M + H]+,414.1,found 414.1; Anal. Calcd for
C19H13Cl2N5O2: C, 55.09; H, 3.16; N, 16.91%; Found: C, 55.13; H, 3.24; N, 17.05%.
3.1.7. 1-phenyl-1H-1,2,4-triazol-3-yl5-chloro-3-methyl-1-(p-tolyl)-1H-pyrazole-4-carboxylate (4d)
◦
White solid, yield 52%; mp 154–156 C; 1H-NMR (600 MHz, CDCl3)
δ8.42 (s, 1H, Triazole-H), 7.57
(d, J = 8.4 Hz, 6H, Ph-H), 7.49 (d, J = 8.3 Hz, 1H, Ph-H), 7.31 (t, J = 8.2 Hz, 2H, Ph-H), 2.62 (s, 3H, CH3),
2.42 (s, 3H, CH3). [M + H]+). MS (ESI) calculated for C20H17ClN5O2 [M + H]+, 394.1, found 394.1; Anal.
Calcd for C20H16ClN5O2: C, 61.00; H, 4.10; N, 17.78%; Found: C, 61.05; H, 4.18; N, 17.68%.
3.1.8. 1-(4-fluorophenyl)-1H-1,2,4-triazol-3-yl5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-
carboxylate (4e)
White solid, yield 47%; mp 187–189 ◦C; 1H-NMR (600 MHz, CDCl3)
δ7.97 (s, 1H, Triazole-H),
7.58–7.43 (m, 6H, Ph-H), 6.98–6.91 (m, 3H, Ph-H), 2.55 (s, 3H, CH3). MS (ESI) calculated for
C19H14ClFN5O2 [M + H]+, 398.1, found 398.1; Anal. Calcd for C19H13ClFN5O2: C, 57.37; H, 3.29; N,
17.61%; Found: C, 57.43; H, 3.30; N, 17.57%.
3.1.9. 1-(4-fluorophenyl)-1H-1,2,4-triazol-3-yl5-chloro-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4f)
White solid, yield 75%; mp 69–71 ◦C; 1H-NMR (600 MHz, CDCl3)
δ 8.03 (s, 1H, Triazole-H), 7.89
(d, J = 8.2 Hz, 2H, Ph-H), 7.51 (dd, J = 8.8, 4.7 Hz, 2H, Ph-H), 7.35 (d, J = 8.1 Hz, 2H, Ph-H), 7.20 (d,
J = 8.2 Hz, 2H, Ph-H), 2.54 (s, 3H, CH3). MS (ESI) calculated for C19H13ClF2N5O2 [M + H]+, 416.1,
found 416.1; Anal. Calcd for C19H12ClF2N5O2: C, 54.89; H, 2.91; N, 16.84%; Found: C, 54.96; H, 3.10;
N, 16.77%.
3.1.10. 1-(4-fluorophenyl)-1H-1,2,4-triazol-3-yl5-chloro-1-(4-chlorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4g)
White solid, yield 66%; mp 253–254 ◦C; 1H-NMR (600 MHz, CDCl3)
δ 8.43 (s, 1H, Triazole-H),
7.65 (dd, J = 8.2, 4.0 Hz, 2H, Ph-H), 7.54–7.48 (m, 4H, Ph-H), 7.21 (t, J = 8.2 Hz, 2H, Ph-H), 2.61 (s,
3H, CH3). MS (ESI) calculated for C19H13Cl2FN5O2 [M+H]+, 432.0, found 432.0; Anal. Calcd for
C19H12Cl2FN5O2: C, 52.80; H, 2.80; N, 16.20%; Found: C, 52.87; H, 2.77; N, 16.28%.
3.1.11. 1-(4-fluorophenyl)-1H-1,2,4-triazol-3-yl5-chloro-3-methyl-1-(p-tolyl)-1H-pyrazole-4-
carboxylate (4h)
◦
White solid, yield 71%; mp 99–101 C; 1H-NMR (600 MHz, CDCl3)
δ 8.41 (s, 1H, Triazole-H), 7.66
(dd, J = 8.1, 4.5 Hz, 2H, Ph-H), 7.42 (d, J = 7.6 Hz, 2H, Ph-H), 7.31 (d, J = 7.9 Hz, 2H, Ph-H), 7.21 (t,
J = 8.0 Hz, 2H, Ph-H), 2.60 (s, 3H, CH3), 2.43 (s, 3H. CH3). MS (ESI) calculated for C20H16ClFN5O2
[
M + H]+, 412.1, found 412.1; Anal. Calcd for C20H15ClFN5O2: C, 58.33; H, 3.67; N, 17.01%; Found: C,
58.41; H, 3.59; N, 17.09%.

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3.1.12. 1-(4-chlorophenyl)-1H-1,2,4-triazol-3-yl5-chloro-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4i)
White solid, yield 51%; mp 84–86 ◦C; 1H-NMR (600 MHz, CDCl3)
δ 8.46 (s, 1H, Triazole-H), 7.64
(d, J = 8.6 Hz, 2H, Ph-H), 7.55–7.54 (m, 2H, Ph-H), 7.50 (d, J = 8.6 Hz, 2H, Ph-H), 7.23–7.20 (m, 2H,
Ph-H), 2.61 (s, 3H, CH3). MS (ESI) calculated for C19H13Cl2FN5O2 [M + H]+, 432.0, found 432.0; Anal.
Calcd for C19H12Cl2FN5O2: C, 52.80; H, 2.80; N, 16.20%; Found: C, 52.86; H, 2.74; N, 16.11%.
3.1.13. 1-(4-chlorophenyl)-1H-1,2,4-triazol-3-yl5-chloro-1-(4-chlorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4j)
◦
1
White solid, yield 56%; mp 72–74 C; H-NMR (600 MHz, CDCl3)
δ 8.42 (s, 1H, Triazole-H),
7.55–7.53 (d, J = 3.1 Hz, 4H, Ph-H), 7.31 (dd, J = 8.9, 4.7 Hz, 2H, Ph-H), 7.20 (t, J = 8.4 Hz, 2H, Ph-H),
2.42 (s, 3H, CH3). MS (ESI) calculated for C19H13Cl3N5O2 [M + H]+, 448.0, found 448.0; Anal. Calcd
for C19H12Cl3N5O2: C, 50.86; H, 2.70; N, 15.61%; Found: C, 50.91; H, 2.64; N, 15.58%.
3.1.14. 1-(p-tolyl)-1H-1,2,4-triazol-3-yl-5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carboxylate (4k)
White solid, yield 72%; mp 135–138 ◦C; 1H-NMR (600 MHz, CDCl3)
δ 8.43 (s, 1H, Triazole-H),
7.57–7.46 (m, 7H, Ph-H), 7.31-7.40 (d, J = 8.2 Hz, 2H, Ph-H), 2.62 (s, 3H, CH3), 2.42 (s, 3H, CH3).MS
(ESI) calculated for C20H17ClN5O2 [M + H]+, 394.1, found 394.1; Anal. Calcd for C20H16ClN5O2: C,
61.00; H, 4.10; N, 17.78%; Found: C, 61.08; H, 4.15; N, 17.84%.
3.1.15. 1-(p-tolyl)-1H-1,2,4-triazol-3-yl5-chloro-1-(4-fluorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4l)
White solid, yield 46%; mp 141–143 ◦C; 1H-NMR (600 MHz, CDCl3)
δ 8.43 (s, 1H, Triazole-H),
7.55–7.53 (m, 4H, Ph-H), 7.31 (d, J = 8.2 Hz, 2H, Ph-H), 7.23-7.21 (t, J = 8.5 Hz, 2H, Ph-H), 2.61 (s, 3H,
CH3), 2.42 (s, 3H, CH3). MS (ESI) calculated for C20H16ClFN5O2 [M + H]+, 412.1, found 412.1; Anal.
Calcd for C20H15ClFN5O2: C, 58.33; H, 3.67; N, 17.01%; Found: C, 58.42; H, 3.74; N, 17.07%.
3.1.16. 1-(p-tolyl)-1H-1,2,4-triazol-3-yl5-chloro-1-(4-chlorophenyl)-3-methyl-1H-pyrazole-4-
carboxylate (4m)
White solid, yield 49%; mp 174–176 ◦C; 1H-NMR (600 MHz)
δ 7.97 (s, 1H, Triazole-H), 7.53 (d,
J = 4.2 Hz, 4H, Ph-H), 7.49 (dd, J = 8.9, 4.5 Hz, 1H, Ph-H), 7.00–6.91 (m, 3H, Ph-H), 2.55 (s, 3H, CH3).
MS (ESI) calculated for C20H16Cl2N5O2 [M + H]+, 428.1, found 428.1; Anal. Calcd for C20H15Cl2N5O2:
C, 56.09; H, 3.53; N, 16.35%; Found: C, 56.13; H, 3.61; N, 16.44%.
3.2. In Vitro Antibacterial Activity
3.2.1. Medium
The solid media Luria-Bertani (LB)-Broth-Agar-Medium (tryptone 10 g/L, yeast 5 g/L,
NaCl 10 g/L, agar 15 g/L, and distilled water 1000 mL, adjusted to pH 7.4 was used for testing
the antibacterial activity.
3.2.2. Minimum Inhibitory Concentration (MIC)
The in vitro antibacterial activity for synthesized compounds 4a–m were evaluated using the
agar-dilution method [32 34]. Two-fold serial dilutions of the compounds and reference drugs
–
(ciprofloxacin) was prepared in LB-Broth-Agar-Medium. Drugs (10.0 mg) were dissolved in DMSO
(1 mL) and the solution was diluted with water (9 mL). Further progressive double dilution with
melted LB-Broth-Agar-Medium was performed to obtain the required concentrations of 128, 64, 32,
16, 8, 4, 2, 1, 0.5 µg/mL, and the MIC values were calculated separately. The bacterial inocula were
prepared by suspending 24 h-old bacterial colonies from LB-Broth-Agar-Medium in 0.85% saline.
The inocula were adjusted to 0.5 McFarland Standard (1.56108 CFU/mL). The suspensions were then

Molecules 2019, 24, 1311
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diluted in 0.85% saline to give 107 CFU/mL. Petri dishes were spot-inoculated with 1
µL of each of
the prepared bacterial suspensions (104 CFU/spot) and incubated at 37 ◦C for 24 h. At the end of the
incubation period, the MIC was determined, which is the lowest concentration of the test compound
that resulted in no visible growth on the plate. A control test was also performed with test medium
supplemented with DMSO at the same dilutions as used in the experiment in order to ensure that the
solvent had no influence on bacterial growth.
3.3. Enzyme Inhibition Experimental
The in vitro antibacterial activity of the target compounds was carried out on by the methods
of Sato et al. [35] and Peng and Marians [36]. First, the E. coli suspension is extracted to obtain a
crude enzyme solution. After the purification step, the selected compounds are determined by gel
electrophoresis to obtain data of different gradient concentrations, thereby calculating the IC50 values.
3.4. Molecular Docking
The crystal structures of bacterial DNA topoisomerase II (PDB entry: 2xcs) was downloaded from
the RCSB (Research Collaboratory for Structural Bioinformatic) Protein Data Bank. The molecular
docking procedure was performed using CDOCKER protocol for receptor–ligand interactions section
of DS 3.5 [29].
4. Conclusions
A series of novel triazole-containing pyrazole ester derivatives 4a
evaluated for their biological activities. Compound 4d showed significant antibacterial activities
with MIC values of 4, 2, 4, and 0.5 g/mL against Staphylococcus aureus, Listeria monocytogenes,
Escherichia coli, and Salmonella gallinarum. Furthermore, compound 4d displayed the most potent
Topo II (IC50 = 13.5 µg/mL) and topoisomerase IV (IC50 = 24.2 g/mL) inhibitory activity. This study
–m were synthesized and
µ
µ
suggests that compound 4d can be used as a potential topoisomerase II inhibitor and this finding
will lay the foundation for further structural modification and development of novel topoisomerase
II inhibitors.
Supplementary Materials: The following are available online.
Author Contributions: Conceptualization, X.-H.L.; methodology, M.-J.C. and W.W.; software, Z.-L.R. and X.C.;
writing—original draft preparation, L.W. and H.L.; writing—review and editing, F.T. and K.M.; funding acquisition,
X.-H.L.
Funding: This work was supported by The Basic Science Research Fund Program of ICBR (1632017005), Natural
Science Foundation of Education Committee of Anhui Province (KJ2018A0162), and National Key R&D Program
of China (Project No. 2017YFD0600805). We are grateful to Jeffrey for checking our manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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