Novel coumarin-pyrazole carboxamide derivatives as potential topoisomerase II inhibitors: Design, synthesis and antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2018.07.059

The identification of novel Topoisomerase II (Topo II) inhibitors is one of the most attractive directions in the field of bactericide research and development. In our ongoing efforts to pursue the class of inhibitors, six series of 70 novel coumarin-pyrazole carboxamide derivatives were designed and synthesized. As a result of the evaluation against four destructive bacteria, including Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella. Compound 8III-k (MIC = 0.25 mg/L) showed considerable inhibitory activity than ciprofloxacin (MIC = 0.5 mg/L) against Escherichia coli and 8V-c (MIC = 0.05 mg/L) exhibited excellent antibacterial activity than ciprofloxacin (MIC = 0.25 mg/L) against Salmonella. The selected compounds (8III-k, 8V-c and 8V-k) exhibit potent inhibition against Topo II and Topo IV with IC₅₀ values (9.4–25 mg/L). Molecular docking model showed that the compounds 8V-c and 8V-k can bind well to the target by interacting with amino acid residues. It will provide some valuable information for the commercial Topo II inhibiting bactericides.

Full text of DOI:10.1016/j.ejmech.2018.07.059

Accepted Manuscript
Novel coumarin-pyrazole carboxamide derivatives as potential topoisomerase II
inhibitors: Design, synthesis and antibacterial activity
Hao Liu, Zi-Li Ren, Wei Wang, Jie-Xiu Gong, Ming-Jie Chu, Quan-Wei Ma, Jie-Chun
Wang, Xian-Hai Lv
PII:
S0223-5234(18)30624-X
10.1016/j.ejmech.2018.07.059
EJMECH 10594
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 June 2018
Revised Date: 19 July 2018
Accepted Date: 25 July 2018
Please cite this article as: H. Liu, Z.-L. Ren, W. Wang, J.-X. Gong, M.-J. Chu, Q.-W. Ma, J.-C. Wang,
X.-H. Lv, Novel coumarin-pyrazole carboxamide derivatives as potential topoisomerase II inhibitors:
Design, synthesis and antibacterial activity, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.07.059.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel Coumarin-Pyrazole Carboxamide derivatives as potential
Topoisomerase II Inhibitors: Design, synthesis and antibacterial
activity
Hao Liu,† Zi-Li Ren,† Wei Wang,† Jie-Xiu Gong,† Ming-Jie Chu,† Quan-Wei Ma,†
Jie-Chun Wang, † Xian-Hai Lv*,†,§
†School of Science, Anhui Agricultural University, 230036, Hefei, People’s Republic of China
§State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093, Nanjing,
People’s Republic of China
*Tel./Fax: +86-551-6578-6906. E-mail: lvxianhai@ahau.edu.cn.

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Abstract: The identification of novel Topoisomerase II (Topo II) inhibitors is one of the most
attractive directions in the field of bactericide research and development. In our ongoing efforts
to pursue the class of inhibitors, six series of 70 novel coumarin-pyrazole carboxamide
derivatives were designed and synthesized. As a result of the evaluation against four destructive
bacteria, including Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and
Salmonella. Compound 8III-k (MIC=0.25 mg/L) showed considerable inhibitory activity than
ciprofloxacin (MIC=0.5 mg/L) against Escherichia coli and 8V-c (MIC=0.05 mg/L) exhibited
excellent antibacterial activity than ciprofloxacin (MIC=0.25 mg/L) against Salmonella. The
selected compounds (8III-k, 8V-c and 8V-k) exhibit potent inhibition against Topo II and Topo
IV with IC50 values (9.4–25mg/L). Molecular docking model showed that the compounds 8V-c
and 8V-k can bind well to the target by interacting with amino acid residues. It will provide some
valuable information for the commercial Topo II inhibiting bactericides.
Keywords: topoisomerase II inhibitors, coumarin, pyrazole, molecular docking, 3D-QSAR,
antibacterial activity

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1 INTRODUCTION
Bacterial DNA topoisomerase II (Topo II, EC 5.99.1.3) has been extensively exploited as a
target in antimicrobial chemotherapy.[1] As a subclass of the topoisomerase family, Topo II are
found in all organisms from bacteria to human, and even in some viruses.[2-4] DNA Topo II
enables cells to escape this fate by cutting the DNA duplex, passing the cleavage body through
the second DNA duplex and connecting the cleaved DNA. There are two major structural types
of Topo II inhibitors, the quinolones and the coumarins. However, mutations in the quinolone
binding site of Topo II result in resistance to quinolones.[5, 6] Novobiocin as a representative of
coumarin antibiotics, has a good inhibitory effect on DNA topoisomerase. Coumarins form a vast
class of natural products widely occurring as secondary plant metabolites.[7, 8] More than 1800
different natural coumarins have been discovered and described to date, many of them exhibiting
high levels of biological activity, such as insecticidal,[9] antioxidant,[10] anticancer,[11, 12]
anti-HIV,[13] antifungal,[14] antibacterial,[15] antimicrobial,[16] and antibiotic activities.[17, 18]
Because of its high bioactivities and naturally occurring scaffold, some efforts focus on the
development of novel coumarin-based therapeutic agents.[19-21]
From the other hand, increasing bacterial resistance has led to the demand for new
bactericides.[22] In addition, the toxicity of the novel bactericides and whether they are good for
the environment account for a large proportion of current drug design. In recently years, pyrazole
as an antimicrobial agent has a wide range of antimicrobial activity after the discovery of natural
pyrazole C-glycosides such as pyrazolofuran, and is therefore of great interest.[23] Consequently,
several pyrazole derivatives that exhibited antimicrobial activity are reported by Tanitame and
coworkers.[24, 25] Therefore, such medicinal properties associated with these two heterocycles

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render them as useful structural units in drug research. Our current research efforts are to find
natural product-based antibacterial agents as Topo II inhibitors.
We chose novobiocin as primer molecule, introduced the pyrazole ring and binding it to
coumarin with an amide bond to design the target scaffold. In our previous work,[26] we found
that the introduction of a benzene ring as a linker on the pyrazole ring can increase the number of
the degree of combination of the compound and the target, finally got the target scaffold (scheme
1). In order to generate a new class of Topo II inhibitors, six series totaling 70 compounds were
used for testing the antibacterial activity against two gram-positive bacteria, namely
Staphylococcus aureus and Listeria monocytogenes and two gram-negative bacteria, namely
Escherichia coli and Salmonella. In addition, molecular docking analysis and structure-activity
relationship (SAR) studies were performed on all derivatives to determine critical structural
factors responsible for their antibacterial efficacy.
B
N
NH2
N
A
Linker
A
O
O
O
H
N
N
C
N
B
Novobiocin
Scheme 1. Design of the target scaffold

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2 MATERIALS AND METHODS
2.1 General methods
All chemicals were purchased from Energy, Meryer, and Aladdin Chemicals and were used as
1
received. H NMR and 13C NMR spectra were carried out using Agilent DD2 600 Hz
spectrometer with CDCl3 or DMSO as the solvent and tetramethylsilane as the internal standard.
ESI-MS spectra were carried out on a Mariner System 5304 mass spectrometer. Elemental
analyses were performed on a CHN-O-Rapid instrument. Quantitative structure−activity
relationship (QSAR) analyses and molecular docking were performed with Discovery Studio3.5.
Crystal structure determination of compounds was carried out on a Bruker D8 VENTURE
PHOTON.
2.2 General procedure for the synthesis of coumarin-3-carboxylic acids (4)
2-hydroxybenzaldehydes (1) (0.02 mol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (2) (0.02
mol) were first reacted at room temperature in the presence of a catalytic amount of piperidinyl
acetate and then refluxed in ethanol. The product then crystallizes in the solvent. After filtration and
washing with ethanol, coumarin-3-carboxylic acid (4) was obtained in comparable yield and purity.
Figure 1. Synthetic route for intermediate 4. Reagents and conditions: (a) piperidine, acetic acid,
EtOH, 25∘C; (b) reflux, 2 h.
2.3 General procedure for the synthesis of ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate
derivatives (5)

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Intermediates 5 was synthesized based on the literature of Stephane L.et al.[27] Dissolve
phenylhydrazine hydrochloride with 1.5 equivalents of sodium acetate in ethanol, stir and reflux
for 1 hour, filter to obtain filtrate, then add ethyl (E)-2-cyano-3-ethoxyacrylate, stir and reflux for
1 hour, then the solution was rotary evaporated until the volume of the solution was reduced by
half. After cooling, solids intermediate 5 can be precipitated, then filtered under vacuum. The
resulting crude products are subjected to recrystallization purification.
Figure 2. Synthetic route for intermediates 5,6 and 7. Reagents and conditions: (a) ethyl
(E)-2-cyano-3-ethoxyacrylate, H2O, NaOH, EtOH, 3 h, reflux; (b) H2O, NaOH, EtOH, 3 h,
reflux; (c) 2-(ethoxymethyl)malononitrile, H2O, NaOH, EtOH, 3 h, reflux.
2.4 General procedure for the synthesis of 5-amino-1-phenyl-1H-pyrazole-4-carboxylic acid
derivatives (6)
Intermediates 6 was synthesized according to the literature by ML Bender.[28]
Intermediates 5 was dissolved in a solution of ethanol and potassium hydroxide and refluxed for
2 h. After cooling, the pH of the mixture was adjusted to 5-6 by the dropwise addition of 10%
HCl solution to give an intermediate 6, which was filtered under vacuum and recrystallized from
ethanol to give the pure products.
2.5 General procedure for the synthesis of 5-amino-1-phenyl-1H-pyrazole-4-carbonitrile

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derivatives (7)
Intermediates 7 were synthesized according to the literature by Harden et al.[29] A stirred
mixture of p-substituted phenylhydrazine hydrochloride was dissolved in EtOH, then the pH was
adjusted to alkaline by the addition of 10% NaOH solution to remove the hydrochloride, which
were then refluxed for 3 h with 2-(ethoxymethyl)malononitrile in ethanol. Finally, the mixture is
cooled at room temperature, and the intermediates 7 were filtered under vacuum and
recrystallized from ethanol to give the pure products.
Figure 3. Synthetic routes for target compounds 8I−VI. Reagent and condition: (a) POCl3,
pyridine, 40∘C.
2.6 General procedure for the synthesis of
2-oxo-N-(1-phenyl-1H-pyrazol-5-yl)-2H-chromene-3-carboxamide derivatives(8I-8VI)
Intermediate 4 (0.001 mol) and 5 (0.001 mol) were dissolved in pyridine, phosphorus
oxychloride is slowly added dropwise under ice bath conditions, then at 40-60° C reacted for 5-8
hours. Finally, the mixture was poured into 30 mL saturated Na2CO3 solution and stirred well.
After the mixture was acidified, the saturated CuSO4 solution was used to remove pyridine,
which greatly improved the purity and yield of the product. The data for compounds 8I-8VI are
provided in the supporting information.

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2.7 Biological assay
2.7.1 Minimum inhibitory concentration (MIC)[30]
The in-vitro antibacterial activity for synthesized compounds 8I-a−8VI-l were evaluated using
the agar-dilution method.[31, 32] Twofold serial dilutions of the compounds and reference drugs
(ciprofloxacin and novobiocin) were 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, 0.25, 0.125, 0.05 mg/L, and the MIC values
were calculated separately.
2.7.2 Enzyme Inhibitory effects against Topo II and Topo IV
The in vivo antibacterial activity of the target compounds was carried out on by the methods of
Sato et al.[33] and Peng and Marians[34].
2.8 3D(Three-dimensional)-QSAR analysis
The three-dimensional(3D) Quantitative structure–activity relationship was performed with
Discovery Studio3.5, using genetic function algorithm (GFA).[35]
2.9 Molecular docking
The crystal structure of bacterial DNA Topo II (PDB code:2xcs) was downloaded from the
RCSB Protein Data Bank. The molecular docking procedures were performed by using
CDOCKER protocol for receptor–ligand interactions section of Discovery Studio3.5.[36]

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3 RESULTS AND DISCUSSION
3.1 Chemistry
The chemical synthesis routes of 8I-a−8VI-l are shown in Figure 1−3. For the synthesis of
intermediate 4(Figure 1), 2-hydroxybenzaldehydes (1) and 2,2-dimethyl-1,3-dioxane-4,6-dione
(2) were first reacted at room temperature in the presence of a catalytic amount of piperidinyl
acetate and then refluxed in ethanol. The synthesis process is very easy because of the good
solubility in the solvent and the very fast crystal formation. After filtration and washing with
ethanol, coumarin-3-carboxylic acids derivatives (4) were obtained with considerable yields and
purity. The intermediates 5 and 7 were synthesized by stirring and refluxing the mixture of
p-substituted
phenyl
hydrazine
hydrochlorides
and
ethyl
(E)-2-cyano-3-ethoxyacrylate/ethoxymethylenemalononitrile in the ethanol medium respectively.
Intermediate 5 can be hydrolyzed to get 6. The target compound under mild conditions by using
POCl3 in pyridine amine and the acid converted to the amide by convenient and efficient one-pot
process is obtained. We optimized the post-treatment conditions of the products, using saturated
Na2CO3 to liberate pyridine. After the system was acidified, the saturated CuSO4 was used to
remove pyridine, which greatly improved the purity and yield of the product. The chemical
structures of all synthesized compounds were determined by 1H NMR, 13C NMR, and MS. The
crystal data and checkcif files for compounds 8IV-a, 8V-f and 8V-g are listed in supporting
information, the crystal structures are shown in Figure 4. Crystallographic data (excluding
structure factors) for the structure were deposited with the Cambridge Crystallographic Data
Center as supplementary publication No. CCDC-1842674 (8IV-a), No. CCDC-1842675(8V-f),
No. CCDC-1842676(8V-g).

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Figure 4. The crystal structures of 8IV-a, 8V-f and 8V-g displacement ellipsoids.
3.3 Antibacterial activity and SAR Discussion
All newly synthesized compounds were evaluated for in-vitro antibacterial activity against two
gram-positive bacteria, namely Staphylococcus aureus and Listeria monocytogenes and two
gram-negative bacteria, namely Escherichia coli and Salmonella. Novobiocin and ciprofloxacin
were used as a reference standard. The results of the in-vitro antibacterial activity screening of
the test compounds are summarized in Table 1.
Most of the derivatives exhibited moderate to potent antibacterial activities against
Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella, demonstrating
the rationality of our design strategy. First, we discuss the activities of compounds against
gram-positive bacteria. Among them, nine compounds (i.e., 8II-f, 8III-k, 8III-a, 8V-a, 8V-l,
8V-I, 8V-g, 8VI-g and 8VI-l) were found to display improved antibacterial activities against

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Staphylococcus aureus compared with novobiocin (MIC = 2 mg/L), with MIC values ranging
from 0.5 to 2 mg/L. Particularly, compound 8III-k, determined as the most potent inhibitor
against Staphylococcus aureus with a MIC value of 0.5 mg/L, showed four-fold higher inhibitory
activity than novobiocin. However, the MIC value of ciprofloxacin is better than it. Three
derivatives (i.e., 8II-c, 8III-k and 8V-c) exhibited improved antibacterial activity against Listeria
monocytogenes in comparison to ciprofloxacin (MIC = 1 mg/L), with MIC values ranging from
0.5 to 1 mg/L. In these derivatives, compound 8V-c, showed antibacterial activity against
Listeria monocytogenes with a MIC value at 0.5 mg/L. Obviously, it was better than
ciprofloxacin, but much worse than novobiocin. Then, the data on the activity of compounds
inhibiting gram-negative bacteria are discussed below. Among them, sixteen derivatives were
found to show improved antibacterial activities against Escherichia coli compared with
novobiocin (MIC = 4 mg/L), with MIC values ranging from 0.25 to 4 mg/L. Especially,
compound 8III-k, identified as the most potent inhibitor against Escherichia coli with a MIC
value at 0.25 mg/L, showed 16-fold higher inhibitory activity than novobiocin and two-fold than
ciprofloxacin. Five derivatives (i.e., 8II-g, 8IV-g, 8V-c, 8V-i and 8V-k) were found to display
improved antibacterial activities against Salmonella compared with ciprofloxacin (MIC = 0.25
mg/L), with MIC values ranging from 0.05 to 0.25 mg/L. Noticeably, compound 8V-c, identified
as the most potent inhibitor against Salmonella with a MIC value at 0.05 mg/L, showed five-fold
higher inhibitory activity than novobiocin and ten-fold than ciprofloxacin.

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Table 1. Chemical Structures of Compounds 8I-VI and MIC Values against S. aureus, L.
monocytogenes, E. coli and Salmonella.
Compd.
R1
R2
R3
R4
MIC(mg/L)
L.ma E.ca
64 64
S.aa
S.ea
8I-a
8I-b
H
H
H
H
H
F
COOEt
COOEt
16
2
＞128
16
＞128
＞128
64
＞128
16
8I-c
8I-d
H
H
H
H
Cl
COOEt
COOEt
64
32
CH3
＞128
16
＞128
64
＞128
32
8I-e
8I-f
H
H
H
H
H
F
CN
CN
64
64
＞128
＞128
＞128
16
8I-g
8I-h
H
H
H
H
Cl
CN
CN
4
8
32
＞128
32
32
32
CH3
＞128
8I-i
8I-j
H
H
H
H
H
F
COOH
COOH
＞128
＞128
＞128
＞128
＞128
＞128
32
＞128
32
8I-k
8I-l
H
H
H
H
Cl
COOH
COOH
32
64
64
64
CH3
＞128
16
＞128
64
8II-a
8II-b
Br
Br
H
H
H
F
COOEt
COOEt
16
64
16
4
＞128
＞128
8II-c
8II-d
8II-e
Br
Br
Br
H
H
H
Cl
COOEt
COOEt
CN
4
1
2
8
32
2
CH3
H
32
32
＞128
＞128
＞128
＞128
＞128
8II-f
Br
H
F
CN
2
64
64
8II-g
8II-h
Br
Br
H
H
Cl
CN
CN
4
16
8
8
0.25
64
CH3
＞128
＞128
8II-i
Br
H
H
COOH
＞128
＞128
＞128
＞128

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8II-j
Br
H
F
COOH
32
4
32
＞128
8II-k
8II-l
Br
Br
H
H
Cl
COOH
COOH
32
64
32
4
32
CH3
64
＞128
64
＞128
8III-a
8III-b
Cl
Cl
H
H
H
F
COOEt
COOEt
4
16
64
64
64
＞128
＞128
8III-c
8III-d
Cl
Cl
H
H
Cl
COOEt
COOEt
2
4
32
2
4
32
CH3
64
＞128
8III-e
8III-f
8III-g
8III-h
Cl
Cl
Cl
Cl
H
H
H
H
CH3
F
CN
CN
CN
CN
16
32
4
32
32
8
32
32
4
64
64
64
Cl
CH3
＞128
＞128
＞128
＞128
＞128
8III-i
8III-j
Cl
Cl
H
H
H
F
COOH
COOH
32
32
32
64
32
32
＞128
4
8III-k
8III-l
Cl
Cl
H
H
Cl
COOH
COOH
0.5
0.5
16
0.25
CH3
＞128
＞128
＞128
8IV-a
8IV-b
NO2
NO2
H
H
H
F
COOEt
COOEt
＞128
＞128
＞128
＞128
32
8
64
＞128
64
8IV-c
8IV-d
NO2
NO2
H
H
Cl
COOEt
COOEt
32
32
64
CH3
＞128
＞128
＞128
＞128
8IV-e
8IV-f
NO2
NO2
H
H
H
F
CN
CN
＞128
＞128
＞128
＞128
＞128
16
＞128
8
＞128
4
＞128
0.125
＞128
64
8IV-g
8IV-h
NO2
NO2
H
H
Cl
CN
CN
CH3
＞128
64
＞128
＞128
64
8IV-i
8IV-j
NO2
NO2
H
H
H
F
COOH
COOH
＞128
＞128
32
＞128
32
＞128
＞128
8IV-k
8IV-l
NO2
NO2
H
H
Cl
COOH
COOH
32
4
32
64
CH3
＞128
＞128

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8V-a
8V-b
H
H
N(Et)2
N(Et)2
H
F
COOEt
COOEt
64
＞128
1
64
＞128
0.5
64
＞128
2
64
＞128
0.05
8V-c
8V-d
H
H
N(Et)2
N(Et)2
Cl
COOEt
COOEt
CH3
＞128
＞128
16
＞128
＞128
8V-e
8V-f
H
H
N(Et)2
N(Et)2
H
F
CN
CN
4
32
64
32
64
64
＞128
8
8V-g
8V-h
H
H
N(Et)2
N(Et)2
Cl
CN
CN
2
2
16
CH3
＞128
＞128
＞128
＞128
8V-i
H
N(Et)2
N(Et)2
N(Et)2
N(Et)2
H
H
COOH
COOH
COOH
COOH
COOEt
COOEt
COOEt
2
32
2
4
8
32
4
0.25
16
8V-j
H
F
16
2
8V-k
8V-l
H
Cl
0.125
64
H
CH3
H
64
32
8
64
32
16
64
16
4
8VI-a
8VI-c
8VI-d
CH3
CH3
CH3
16
H
Cl
8
H
CH3
32
64
＞128
64
＞128
8
8VI-e
8VI-f
CH3
CH3
H
H
H
F
CN
CN
64
8
＞128
＞128
＞128
＞128
8VI-g
8VI-i
8VI-j
8VI-k
8VI-l
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
Cl
H
CN
2
16
32
32
16
4
32
4
2
COOH
COOH
COOH
COOH
64
64
16
32
32
32
16
F
Cl
CH3
8
64
＞128
＞128
CIPb
NBc
0.125
2
1
0.5
4
0.25
0.5
0.25
aAbbreviations: Staphylococcus aureus (ATCC-12600); Listeria monocytogenes(ATCC-15313); Escherichia
coli(ATCC-25922); Salmonella(ATCC-9184). bCiprofloxacin. c Novobiocin.
It can be observed from Table 1 that chlorine substituted on the benzene ring at the R3 site
was beneficial to antibacterial activity of the compounds when substitution groups on the
benzopyran ring were determined. Intriguingly, the introduction of diethylamino (8V series) at
R2 showed noticeable enhanced effects compared with other series. For Staphylococcus aureus,
when R1 position was substituted with other moieties except nitro, the antibacterial activities

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were increased. When R4 is cyano group, there are not much different among them. When R4 is
carboxyl or ester group, 8III series exhibit a very good activity than other series. For Listeria
monocytogenes, when R4 position was substituted with ester group or carboxyl, 8II-c and 8III-k
displayed improved antibacterial activities than others respectively. When R4 is cyano group,
there are not much different among them. For Escherichia coli, when R1 position was substituted
with other moieties, the antibacterial activities were increased. And when R1 is chlorine, 8III-k
exhibit best antibacterial activity than others. However, when R1 is other moieties, their
performance is general. For Salmonella, when R4 is carboxyl or ester group, their antibacterial
activities are less than ideal. When R1 is bromine and R4 is cyano group, 8II-g showed excellent
antibacterial effect. Next, we discuss the structure-activity relationship between the 8IV and 8V
series. The overall antibacterial activities of 8IV series are not ideal except 8IV-g against
Salmonella. Compared with other series, we speculate that the antibacterial activity is decreased
because of the introduction of nitro groups. In addition, 8V series displayed significantly
enhanced effects against Salmonella than other series. Their difference lies in the introduction of
diethylamino so that we believe that diethylamino can act as a special pharmacophore for
Salmonella inhibition. Every nutshell has a concave and convex side. From the data we observed
that the introduction of fluorine and methyl groups resulted in a decrease in antibacterial activity.
Taking into account the above SAR results, these findings indicate that the antibacterial efficacy
of the designed compounds may be attributed to a combination of factors, such as chlorine
substitution at the benzene ring which is attached to pyrazole ring, changes in substituents on the
benzopyran ring and diversity of substituents on pyrazole ring.

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3.4 Inhibitory effects against Topo II and Topo IV
In order to determine the relationships by which the chlorine, diethylamino, ester group and
carboxyl group substituents inducing their antibacterial activity, the inhibitory activity of selected
compounds (8III-k , 8V-c and 8V-k) against topoisomerase II and topoisomerase IV isolated
from E. coli was examined[33, 34]. As shown in Table 2, compounds 8V-c and 8V-k having the
same three substituents but 8V-c showed more potent inhibition than 8V-k against the two
enzymes. Compounds 8III-k showed the best suppression effect among them. We suspect that
the reason for this difference is that 8III-k contains two chlorine groups. On the other hand, there
was a good correlation between the MICs and the IC50s (Tables 1 and 2), suggesting that
inhibition of the Topo II and Topo IV by the chlorine, diethylamino, ester group and carboxyl
group substituents suppresses bacterial cell growth. These results indicate that the chlorine,
diethylamino, ester and carboxyl substituents put a great deal of pressure on the survival of the
bacteria.
Table 2. Inhibitory effects of selected compounds against topoisomerase II and
topoisomerase IV
Compd.
IC50(mg/L)
Topo II a
0.125
0.25
Topo IV b
8III-k
8V-c
8V-k
CIP
8
8
0.25
16
8
0.5
NB
0.5
4
a Topoisomerase II supercoiling activity. b Topoisomerase IV decatenation activity.
3.5 3D Quantitative Structure−Activity Relationships
In this study, a total of 3D-QSAR models for four different bacteria were established (see the
Support Information for the remaining models). Here, salmonella is used as a target and

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compound 8V-k is taken as the example to show the structure−activity relationships. The pMIC
values were calculated on the basis of the in vitro activity experiment results and we get the linear
regression equation through GFA, r2 value is 0.964 which explains the accuracy and reliability of
the experimental results. The 3D-CoMFA contour maps are shown in Figure 5. In the electrostatic
map Figure 5(A), the red region indicates that the stronger the negative charge of the substituent
in this region is, the more favorable the activity of the compound, and the blue region indicates
that the stronger the positive charge of the substituent in this region is, the more favorable the
activity of the compound. In the steric map Figure 5(B), the yellow region indicates that the
increase of substituents in this region is not conducive to the activity of the compound, while the
blue region is the opposite. It can be observed that the electrostatically favorable zone is located in
the pyrazole ring, while the electrostatically unfavorable zone covers the space around the
carboxyl group. In the steric contour map, the sterically favored region covers a small space near
the phenyl ring. The 3D-CoMFA contour maps provide valuable clues for further structural
optimization of this class of new Topo II inhibitors.
Figure 5. Electrostatic map (A) and steric map (B) for the CoMFA model.
3.6 Molecular Docking Analysis

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After the previous bioactivity test, in order to further obtain the relationship between the
structure of the compounds and the inhibition of bacterial activity, all synthesized compounds
were molecularly docked using Discover Studio 3.5. The detailed interactions of representative
derivative compounds 8V-c and 8V-k with Topo II are shown in Figure 6. In the binding models
of compound 8V-c and 8V-k, their scaffolds and amino acid residues linked together tightly,
suggesting the pose of 8V-c and 8V-k into the Topo II-binding site which revealed that it has
suitable shape complementarity with the binding pocket, which means that the rationality of our
design scheme. As show in the Figure 6, Dg10 established π-π interactions between benzene and
furan ring, Arg1122 established cation-π interactions with furan ring. The simulated docking
results of 8V-c and 8V-k are the same, which may account for their antibacterial effect are almost
the same.
Figure 6. (A) Binding model of 8V-c. (B) Binding model of 8V-k. The π-π interactions are
displayed as yellow solid line, cation-π interactions are displayed as rose-carmine solid line

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4. CONCLUSIONS
All of the novel coumarin-pyrazole carboxamide derivatives were synthesized and evaluated.
Furthermore, their antibacterial activity against Staphylococcus aureus, Listeria monocytogenes,
Escherichia coli and Salmonella, indicating that most of compounds exhibited moderate to
potent antibacterial activities. Most surprisingly, 8II-g,8IV-g, 8V-c and 8V-k exhibited excellent
antibacterial activity against Salmonella and compound 8III-k showed considerable inhibitory
activity against three bacteria except Salmonella. SAR analysis and molecular docking
simulations show that the promising antibacterial efficacy of our designed compounds can be
attributed to structural changes including the substitution of chlorine on the benzene and
benzofuran rings and modification of the pyrazole ring. Therefore, the rationality of the
molecules we designed was fully confirmed in experiments. These results demonstrate that the
chlorine groups at different substitution positions and the modification on the pyrazole ring have
a very good effect on the antibacterial effect. We are further modifying the coumarin-pyrazole
scaffold to obtain more effective Topo II and Topo IV inhibitors and looking for valuable
information for further structural optimization and development of novel Topo II inhibitors.
ACKNOWLEDGEMENT
This work was financially supported by National Natural Science Foundation of China
(No.21302002) and National Key R&D Program of China (Project No. 2017YFD0600805).

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Highlights
1. Structure-based design and synthesis of novel coumarin-pyrazole carboxamide derivatives are
reported
2. Molecular docking and 3D-QSAR were carried out to verify the rationality of the design and
the reliability of the activity data of the compounds.
3. In vitro and in vivo studies reveal that some of the reported compounds possessed good
antibacterial activities.