Biological activity evaluation and action mechanism of chalcone derivatives containing thiophene sulfonate

Article abstract of DOI:10.1039/c9ra05349b

A series of novel chalcone derivatives containing a thiophene sulfonate group were designed and synthesized. The structures of all title compounds were determined by ¹H-NMR, ¹³C-NMR and HRMS. Antibacterial bioassays indicated that, compound 2l demonstrated excellent antibacterial activities against Xanthomonas axonopodis pv. citri (Xac), with an EC₅₀ value of 11.4 μg mL^-1, which is significantly superior to those of bismerthiazol (BT) (51.6 μg mL^-1) and thiodiazole-copper (TC) (94.7 μg mL^-1). Meanwhile, the mechanism of action of compound 2l was confirmed by using scanning electron microscopy (SEM). In addition, compound 2e showed remarkable inactivation activity against Tobacco mosaic virus (TMV), with an EC₅₀ value of 44.3 μg mL^-1, which was superior to that of ningnanmycin (120.6 μg mL^-1). Microscale thermophoresis (MST) also showed that the binding of compounds 2e and 2h to Tobacco mosaic virus coat protein (TMV-CP) yielded K_d values of 0.270 and 0.301 μmol L^-1, which are better than that of ningnanmycin (0.596 μmol L^-1). At the same time, molecular docking studies for 2e and 2h with TMV-CP (PDB code: 1EI7) showed that the compound was embedded well in the pocket between the two subunits of TMV-CP in each case. These results suggested that chalcone derivatives containing a thiophene sulfonate group may be considered as activators in the design of antibacterial and antiviral agents.

Full text of DOI:10.1039/c9ra05349b

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Biological activity evaluation and action
mechanism of chalcone derivatives containing
Cite this: RSC Adv., 2019, 9, 24942
thiophene sulfonate†
Tao Guo, Rongjiao Xia, Mei Chen, Jun He, Shijun Su, Liwei Liu, Xiangyang Li
*
and Wei Xue
A series of novel chalcone derivatives containing a thiophene sulfonate group were designed and
synthesized. The structures of all title compounds were determined by 1H-NMR, 13C-NMR and HRMS.
Antibacterial bioassays indicated that, compound 2l demonstrated excellent antibacterial activities
against Xanthomonas axonopodis pv. citri (Xac), with an EC50 value of 11.4 mg mLꢀ1, which is significantly
superior to those of bismerthiazol (BT) (51.6 mg mLꢀ1) and thiodiazole-copper (TC) (94.7 mg mLꢀ1).
Meanwhile, the mechanism of action of compound 2l was confirmed by using scanning electron
microscopy (SEM). In addition, compound 2e showed remarkable inactivation activity against Tobacco
mosaic virus (TMV), with an EC50 value of 44.3 mg mLꢀ1, which was superior to that of ningnanmycin
(120.6 mg mLꢀ1). Microscale thermophoresis (MST) also showed that the binding of compounds 2e and
2h to Tobacco mosaic virus coat protein (TMV-CP) yielded Kd values of 0.270 and 0.301 mmol Lꢀ1, which
are better than that of ningnanmycin (0.596 mmol Lꢀ1). At the same time, molecular docking studies for
2e and 2h with TMV-CP (PDB code: 1EI7) showed that the compound was embedded well in the pocket
between the two subunits of TMV-CP in each case. These results suggested that chalcone derivatives
containing a thiophene sulfonate group may be considered as activators in the design of antibacterial
and antiviral agents.
Received 12th July 2019
Accepted 6th August 2019
DOI: 10.1039/c9ra05349b
rsc.li/rsc-advances
bacteria and plant viruses.15–17 However, prolonged use and
abuse of traditional antibacterial drugs lead to increased
1. Introduction
resistance of pathogenic genes, which not only enhances the
resistance of target pathogens but is also harmful to the envi-
ronment and health of the plant.18–20 Therefore, the develop-
ment of new antibacterial and antiviral agents is an important
task in pesticide science.
The bacterial and virus infections of crops have become some of
the world's most important agricultural problems, and the
threat they pose is not only about agricultural production but
also about human health.1–3 Pathogens that cause bacterial
diseases, such as Xanthomonas oryzae pv. oryzae (Xoo), Ralstonia
solanacearum (Rs) and Xanthomonas axonopodis pv. citri (Xac)4–6
are the world's most important and well-known plant
bacteria.7–11 These bacteria can actively suppress the crop yields
worldwide. Besides, Tobacco mosaic virus (TMV), one of the
world's most severe pathogenic viruses can infect a variety of
crops, such as tobacco, cucumbers and other economic crops,
resulting in a signicant loss of crops each year.12–14 To over-
come these serious problems, antibacterial drugs such as bis-
merthiazol (BT), thiodiazole-copper (TC), and ningnanmycin
have been widely developed and used to reduce the infection by
Recent studies have shown that chalcone readily binds to
receptors of different structures to form chalcone derivatives
due to its exible structure.21–23 Its derivatives have bacteri-
cidal,24,25 insecticidal,26,27 anticancer,28 antiviral,29,30 and anti-
inammatory31 activities, thus demonstrating a wide range of
applications in chemical and biological research. Thiophene
sulfonate plays a prominent role in the studies and applications
of organic chemistry.32,33 As a biologically important active
intermediate, it is oen used to synthesize thiophene sulfonate
compounds with various biological activities.34–36 Therefore,
thiophene sulfonate and its derivatives have a wide range of
applications in industry, agriculture, pharmaceuticals and have
a broad spectrum of biological activities, such as insecti-
cidal,37,38 anti-inammatory,39,40 antiviral,41,42 and anticancer.43
However, to the best of our knowledge, there have been no
reports on chalcone-based thiophene sulfonates so far. To nd
excellent biological activities and environment-friendly small
organic molecules, thiophene sulfonate structure with excellent
State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Center for Research and Development of Fine Chemicals,
Guizhou University, Guiyang, 550025, China. E-mail: wxue@gzu.edu.cn; Fax: +86-
851-88292090; Tel: +86-851-88292090
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra05349b
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biological activity is combined with the structure of chalcone by
active splicing to design and synthesize a series of novel mole-
cules. Through biological activity screening, it is expected to
nd chalcone compounds with high anti-plant viral and anti-
bacterial activities, which lays a theoretical foundation for the
creation of new pesticides.
2.3. General procedure for the synthesis of title compounds
2a–2v
A
solution of intermediate 1 (6.5 mmol) and 2-thio-
phenesulfonyl chloride (5.5 mmol) in acetonitrile (50 mL) was
stirred until dissolved, the reaction mixture was reuxed at
90 ꢁC for 10 h. Aer completion of the reaction, the whole
reaction system was poured into water, extracted with ethyl
acetate, the solvent was removed under depressurization, and
the residue was puried by column chromatography on silica
gel with a mixture (v(petroleum ether) : v(ethyl acetate) ¼ 3 : 1)
2. Experimental
2.1. Instrument, chemicals and reagents
1
to yield the title compounds 2a–2v.45 The H-NMR, 13C-NMR,
19F-NMR and HMRS spectra of the designated title
The molecular docking was performed by using DS-CDocker
implemented in Discovery Studio (version 4.5). Melting points
of the synthesized compounds (2a–2v) were measured using XT-
4 Binocular Microscope (Beijing Tech. Instrument, China)
without correction. 1H-NMR, 13C-NMR and 19F-NMR spectra
were obtained on a Bruker Ascend-400 spectrometer (Bruker
Optics, Switzerland) and DMSO-d6 or CDCl3 were used as
a solvent and TMS was used as an internal standard. HRMS data
were obtained using Thermo Scientic Q Exactive Hybrid
Quadrupole Mass Spectrometer (Thermo Scientic Inc., St
Louis, MO, USA). The microscale thermophoresis (MST) of the
compounds to check the interaction with TMV-CP (Tobacco
mosaic virus coat protein) was determined using a micro ther-
mophoresis instrument (NanoTemper Technologies GmbH,
Germany). All reagents and solvents were purchased from
Chinese Chemical Reagent Company and were chemically pure
analytical grade reagents. The synthetic route of chalcone
derivatives containing thiophene sulfonate is shown in Scheme
1. The intermediate 1 were prepared according to the methods
already reported in the literature.44
compounds 2a–2v are also provided in the ESI.†
3. Results and discussion
3.1. Spectral properties
The structures of all the compounds were conrmed using 1H-
NMR, 13C-NMR and HRMS. In 1H-NMR spectra, multiplet
signals at d 8.27–7.26 ppm indicate the presence of protons in
olenic bonds and aromatic nuclei. The 13C-NMR spectra of the
title compounds exhibited a characteristic (C]O) signal at
d 186.79–188.77 ppm. The 13C-NMR spectra of the title
compounds exhibited a characteristic (thiophene-2-C) signal at
d 142.22–145.08 ppm. The strong presence of [M + H]+ ions
indicates that the title compounds are in the steady state.
3.2. Antibacterial activity of title compounds against Xac,
Xoo and Rs in vitro
The in vitro antibacterial activities of the title compounds 2a–2v
against three phytopathogenic bacteria (Xac, Xoo and Rs) were
tested by using turbidimeter.46–48 Commercial agricultural
bactericides (TC and BT) were used as the control, as shown in
Table 1, and most of the compounds exhibited signicant
2.2. General procedure for the synthesis of intermediate 1
At rst, aqueous sodium hydroxide solution (5% NaOH, 5 antibacterial activities against Xac, Xoo and Rs at 100 or 50 mg
mmol) was added to a round-bottomed ask containing 1-(4- mLꢀ1
Among these, the antibacterial activities of the
.
hydroxyphenyl)ethan-1-one (4 mmol) and differently compounds 2e, 2l and 2p against Xac at 100 mg mLꢀ1 were 77.2,
substituted aldehydes (6 mmol). The mixture was stirred at 95.2 and 79.6%, respectively, which exceeded both TC (57.2%)
ambient temperature for 12 h. The resulting dark-yellow and BT (65.3%). Compounds 2a, 2f, 2j, 2l, 2m, 2o, 2q and 2u
mixture was acidied by HCl (5% HCl, 5 mmol) aer the reac- against Xoo at 50 mg mLꢀ1 were 65.6, 60.8, 46.4, 58.0, 58.4, 58.5,
tion was completed. Finally, the mixture was ltered under 56.9 and 62.2%, respectively, which were better compared to TC
vacuum, and the residue was dried to yield intermediate 1.
(37.2%) and BT (45.2%). In particular, the antibacterial
Scheme 1 Synthetic route of title compounds 2a–2v.
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Table 1 Antibacterial activities of title compounds (2a–2v) against plant pathogens Xac, Xoo and Rs in vitro
Xac (%)
Xoo (%)
Rs (%)
100 mg
Compounds
R
mLꢀ1
50 mg mLꢀ1
100 mg mLꢀ1
50 mg mLꢀ1
100 mg mLꢀ1
50 mg mLꢀ1
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
Ph
67.5 ꢂ 1.7
59.3 ꢂ 2.5
45.3 ꢂ 3.1
51.6 ꢂ 0.9
77.2 ꢂ 1.1
66.5 ꢂ 1.5
63.5 ꢂ 1.5
52.9 ꢂ 2.2
46.5 ꢂ 2.9
52.5 ꢂ 2.7
59.7 ꢂ 1.2
95.2 ꢂ 0.7
44.4 ꢂ 1.5
40.3 ꢂ 2.8
58.3 ꢂ 0.5
79.6 ꢂ 0.5
55.6 ꢂ 2.5
59.4 ꢂ 1.4
52.3 ꢂ 2.1
46.3 ꢂ 0.8
52.4 ꢂ 1.2
56.2 ꢂ 1.8
57.2 ꢂ 1.3
65.3 ꢂ 2.8
53.4 ꢂ 1.6
22.6 ꢂ 3.4
23.4 ꢂ 2.9
36.7 ꢂ 1.8
44.3 ꢂ 2.1
44.6 ꢂ 2.4
55.2 ꢂ 1.9
36.8 ꢂ 0.9
32.4 ꢂ 3.5
25.6 ꢂ 1.7
32.6 ꢂ 1.8
72.3 ꢂ 1.1
25.8 ꢂ 1.7
22.4 ꢂ 1.3
38.7 ꢂ 1.1
56.4 ꢂ 1.1
18.7 ꢂ 1.4
21.4 ꢂ 0.2
38.7 ꢂ 1.6
26.1 ꢂ 3.8
32.5 ꢂ 1.4
31.1 ꢂ 1.9
27.8 ꢂ 3.8
54.9 ꢂ 5.5
72.4 ꢂ 1.7
62.6 ꢂ 1.3
52.9 ꢂ 3.6
48.6 ꢂ 2.1
50.5 ꢂ 1.4
88.1 ꢂ 1.7
43.2 ꢂ 1.3
53.3 ꢂ 0.9
36.0 ꢂ 2.1
77.3 ꢂ 0.4
43.2 ꢂ 0.6
82.3 ꢂ 1.1
65.3 ꢂ 2.4
51.4 ꢂ 2.5
72.3 ꢂ 1.5
44.6 ꢂ 1.8
68.2 ꢂ 1.1
44.6 ꢂ 1.8
55.6 ꢂ 0.4
36.7 ꢂ 1.9
83.3 ꢂ 0.8
56.7 ꢂ 2.4
50.2 ꢂ 0.9
64.9 ꢂ 3.9
65.6 ꢂ 1.4
43.8 ꢂ 2.4
37.5 ꢂ 2.9
29.5 ꢂ 4.6
37.6 ꢂ 1.6
60.8 ꢂ 2.6
39.4 ꢂ 1.6
35.7 ꢂ 3.7
24.3 ꢂ 1.4
46.4 ꢂ 2.6
32.9 ꢂ 1.2
58.0 ꢂ 0.6
58.4 ꢂ 1.5
22.6 ꢂ 1.6
58.5 ꢂ 1.6
34.2 ꢂ 0.9
56.9 ꢂ 1.8
34.2 ꢂ 0.9
41.8 ꢂ 2.7
25.9 ꢂ 4.5
62.2 ꢂ 1.0
43.4 ꢂ 2.5
37.2 ꢂ 3.2
45.2 ꢂ 2.0
76.6 ꢂ 1.4
47.1 ꢂ 3.7
53.3 ꢂ 2.9
47.5 ꢂ 1.0
71.1 ꢂ 2.4
67.8 ꢂ 0.6
58.3 ꢂ 1.5
52.6 ꢂ 3.8
40.8 ꢂ 5.6
56.7 ꢂ 2.4
73.7 ꢂ 7.9
44.3 ꢂ 3.5
91.4 ꢂ 1.6
67.1 ꢂ 1.9
92.4 ꢂ 1.2
85.2 ꢂ 0.5
57.2 ꢂ 1.3
52.8 ꢂ 1.6
50.6 ꢂ 1.7
45.3 ꢂ 3.2
66.3 ꢂ 0.3
42.3 ꢂ 1.3
45.2 ꢂ 4.3
53.7 ꢂ 3.6
48.8 ꢂ 1.4
40.3 ꢂ 1.2
48.9 ꢂ 2.5
36.8 ꢂ 2.5
52.3 ꢂ 2.4
55.1 ꢂ 1.6
48.8 ꢂ 1.3
33.0 ꢂ 0.8
33.5 ꢂ 1.9
43.4 ꢂ 2.5
56.3 ꢂ 2.0
21.3 ꢂ 1.2
77.5 ꢂ 1.3
53.3 ꢂ 2.1
63.3 ꢂ 2.0
56.6 ꢂ 2.0
47.2 ꢂ 2.6
32.2 ꢂ 1.3
42.5 ꢂ 1.8
37.6 ꢂ 2.4
58.2 ꢂ 0.9
28.3 ꢂ 2.5
20.6 ꢂ 2.7
32.2 ꢂ 2.8
4-Cl-Ph
2-Cl-Ph
3-Cl-Ph
2-F-Ph
3-F-Ph
4-F-Ph
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-Py
3-NO2-Ph
3-CH3-Ph
4-CH3-Ph
3,4-di-OCH3-Ph
2,4-di-Cl-Ph
3,4-di-Cl-Ph
C3H7-Ph
2-Th
2s
2t
2u
2v
TCa
BTa
4-Py
3,4-di-CH3-Ph
2-OCH3-Ph
—
—
a
The commercial agricultural antibacterial agents thiodiazole copper (TC) and bismerthiazol (BT) were used as control agents.
Table 2 EC50 values of the title compounds against plant pathogenic bacteria in vitro
Bacterial
Xac
Compounds
R
Toxic regression equation
r
EC50/(mg mLꢀ1
)
2e
2l
2p
TCa
BTa
2a
2f
2j
2l
2m
2o
2q
2u
TCa
BTa
2a
2e
2f
2k
2-F-Ph
y ¼ 1.2997x + 2.9498
y ¼ 1.5536x + 2.7744
y ¼ 1.1393x + 3.3968
y ¼ 1.1576x + 2.7122
y ¼ 1.0698x + 3.1679
y ¼ 0.8781x + 3.8623
y ¼ 1.4287x + 3.1693
y ¼ 1.4836x + 2.5829
y ¼ 1.4387x + 2.9155
y ¼ 1.0376x + 3.3799
y ¼ 1.1362x + 3.2891
y ¼ 1.3694x + 2.8072
y ¼ 1.6774x + 2.5248
y ¼ 1.2058x + 2.2869
y ¼ 0.9913x + 3.1606
y ¼ 1.3445x + 2.8696
y ¼ 1.2021x + 3.0881
y ¼ 1.4532x + 2.6183
y ¼ 1.4622x + 2.6783
y ¼ 1.3326x + 3.5835
y ¼ 0.9189x + 3.5445
y ¼ 1.9357x + 2.2953
y ¼ 1.7032x + 2.4391
y ¼ 1.0373x + 3.3816
y ¼ 1.4682x + 1.8274
y ¼ 1.2581x + 2.4918
0.9862
0.9872
0.9801
0.9859
0.9831
0.9842
0.9869
0.9756
0.9778
0.9891
0.9981
0.9876
0.9928
0.9971
0.9802
0.9812
0.9907
0.9729
0.9975
0.9814
0.9775
0.9886
0.9758
0.9864
0.9758
0.9746
46.5
11.4
27.1
94.7
51.6
19.8
19.1
42.6
28.1
21.8
32.0
39.9
29.8
97.8
71.7
38.4
38.9
43.6
38.7
11.6
38.4
25.0
31.9
36.3
78.8
98.6
3-NO2-Ph
2,4-di-Cl-Ph
—
—
Ph
Xoo
3-F-Ph
4-Br-Ph
3-NO2-Ph
3-CH3-Ph
3,4-di-OCH3-Ph
3,4-di-Cl-Ph
3,4-di-CH3-Ph
—
—
Ph
2-F-Ph
3-F-Ph
Rs
2-Py
2m
2n
2o
2p
2u
TCa
BTa
3-CH3-Ph
4-CH3-Ph
3,4-di-OCH3-Ph
2,4-di-Cl-Ph
3,4-di-CH3-Ph
—
—
a
The commercial agricultural antibacterial agents thiodiazole copper (TC) and bismerthiazol (BT) were used as control agents.
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Fig. 1 SEM images for Xac after incubated using different concentrations of compound 2l, (A) 0 mg mLꢀ1, (B) 50 mg mLꢀ1 and (C) 100 mg mLꢀ1
Scale bar for (A), (B) and (C) are 2 mm.
.
activities of the compounds 2m, 2o and 2p against Rs at 100 and which were signicantly superior to TC (97.8 mg mLꢀ1) and BT
50 mg mLꢀ1 were 91.4 and 77.5%, 92.4 and 63.3%, 85.2 and (71.7 mg mLꢀ1). Compounds 2a, 2e, 2f, 2k, 2m, 2n, 2o, 2p and 2u
56.6%, respectively, which were signicantly superior to TC exhibited notable antibacterial activities against Rs, with EC50
(45.2 and 20.6%) and BT (53.7 and 32.2%).
values of 38.4, 38.9, 43.6, 38.7, 11.6, 38.4, 25.0, 31.9 and 36.3 mg
The antibacterial activities of the compounds were further mLꢀ1, respectively, which were much better than TC (98.6 mg
conrmed by determining their EC50 values, and the obtained mLꢀ1) and BT (78.8 mg mLꢀ1). These results indicate that such
results are shown in Table 2. Compounds 2e, 2l and 2p compounds should be further studied as potential alternative
exhibited remarkable antibacterial activities against Xac, with templates in the search for novel antibacterial agents.
EC50 values of 46.5, 11.4, and 27.1 mg mLꢀ1, which were much
better compared to TC (94.7 mg mLꢀ1) and BT (51.6 mg mLꢀ1).
3.3. Structure–activity relationships of antibacterial
Compounds 2a, 2f, 2j, 2l, 2m, 2o, 2q and 2u exhibited excellent
activities
antibacterial activities against Xoo, with EC50 values of 19.8,
19.1, 42.6, 28.1, 21.8, 32.0, 39.9 and 29.8 mg mLꢀ1, respectively
Tables 1 and 2 show that the changes in the substituted groups
could signicantly impact the inhibitory effects against plant
Table 3 Antiviral activities of the test compounds against TMV in vivo at 500 mg mLꢀ1
Compounds
R
Curative activitya (%)
Protective activitya (%)
Inactivation activitya (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Ph
72.5 ꢂ 2.5
46.4 ꢂ 2.6
52.4 ꢂ 1.2
45.3 ꢂ 3.8
76.2 ꢂ 1.2
48.5 ꢂ 0.9
52.3 ꢂ 1.8
73.5 ꢂ 2.1
26.9 ꢂ 1.8
48.4 ꢂ 0.8
45.6 ꢂ 1.6
74.8 ꢂ 1.7
72.6 ꢂ 1.9
30.3 ꢂ 1.6
65.3 ꢂ 0.8
52.8 ꢂ 2.7
52.1 ꢂ 2.0
27.2 ꢂ 1.6
48.7 ꢂ 4.3
43.9 ꢂ 2.8
49.3 ꢂ 1.9
53.0 ꢂ 1.8
53.3 ꢂ 1.2
67.0 ꢂ 2.1
58.3 ꢂ 0.9
29.9 ꢂ 1.0
51.9 ꢂ 2.2
70.2 ꢂ 1.9
35.3 ꢂ 2.2
58.1 ꢂ 0.4
68.8 ꢂ 4.9
54.6 ꢂ 4.4
61.9 ꢂ 2.5
42.9 ꢂ 1.9
69.8 ꢂ 1.6
52.8 ꢂ 0.7
35.4 ꢂ 2.9
60.2 ꢂ 1.8
40.9 ꢂ 1.6
46.2 ꢂ 2.8
41.6 ꢂ 1.9
32.1 ꢂ 1.9
57.6 ꢂ 1.7
36.3 ꢂ 1.8
51.1 ꢂ 2.6
62.6 ꢂ 1.3
80.1 ꢂ 1.7
53.8 ꢂ 2.2
62.3 ꢂ 1.8
59.7 ꢂ 2.3
87.3 ꢂ 0.7
60.2 ꢂ 1.5
58.7 ꢂ 0.6
81.1 ꢂ 1.9
65.3 ꢂ 3.1
58.9 ꢂ 1.7
66.1 ꢂ 1.8
75.3 ꢂ 2.4
69.2 ꢂ 0.7
45.3 ꢂ 2.7
79.8 ꢂ 2.1
35.2 ꢂ 1.9
55.1 ꢂ 3.3
58.1 ꢂ 0.9
54.2 ꢂ 1.7
56.2 ꢂ 3.8
61.2 ꢂ 0.9
45.2 ꢂ 1.4
78.3 ꢂ 1.5
4-Cl-Ph
2-Cl-Ph
3-Cl-Ph
2-F-Ph
3-F-Ph
4-F-Ph
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-Py
3-NO2-Ph
3-CH3-Ph
4-CH3-Ph
3,4-di-OCH3-Ph
2,4-di-Cl-Ph
3,4-di-Cl-Ph
C3H7-Ph
2-Th
2q
2r
2s
2t
2u
2v
4-Py
3,4-di-CH3-Ph
2-OCH3-Ph
—
Ningnanmycinb
a
b
Average of three replicates. The commercial antiviral agent ningnanmycin.
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Table 4 EC50 values of some compounds against TMV
Compounds
R
Toxic regression equation
r
EC50a/(mg mLꢀ1
)
Curative
2a
2e
2h
2l
2m
2o
Ph
2-F-Ph
2-Br-Ph
3-NO2-Ph
3-CH3-Ph
3,4-di-OCH3-Ph
y ¼ 1.5764x + 1.2033
y ¼ 1.2516x + 2.1833
y ¼ 1.6378x + 0.9941
y ¼ 1.5526x + 1.2543
y ¼ 1.3411x + 1.6219
y ¼ 1.2168x + 1.9170
y ¼ 1.1975x + 1.8793
y ¼ 1.3994x + 1.4998
y ¼ 1.3560x + 1.7053
y ¼ 1.2375x + 1.9576
y ¼ 1.5374x + 1.2315
y ¼ 1.2081x + 1.9784
y ¼ 1.0219x + 3.0517
y ¼ 1.0379x + 3.2916
y ¼ 0.9258x + 3.3392
y ¼ 0.9448x + 3.2055
y ¼ 1.0209x + 2.8752
0.9948
0.9797
0.9981
0.9958
0.9740
0.9829
0.9956
0.9745
0.9767
0.9913
0.9901
0.9804
0.9985
0.9894
0.9973
0.9935
0.9818
256.1
178.0
279.2
258.5
330.3
341.7
403.7
317.1
269.0
287.4
282.6
317.0
80.6
Ningnanmycinb
Protection
2a
2e
2h
2l
Ph
2-F-Ph
2-Br-Ph
3-NO2-Ph
Ningnanmycinb
Inactivation
2a
2e
2h
2o
Ph
2-F-Ph
2-Br-Ph
3,4-di-OCH3-Ph
44.3
62.2
79.3
120.6
Ningnanmycinb
a
b
Average of three replicates. The commercial antiviral agent ningnanmycin.
bacteria, and analysis on the structure–activity relationships is
discussed below. For instance, the designated compounds 2e (R
¼ 2-F-Ph), 2l (R ¼ 3-NO2-Ph) and 2p (R ¼ 2,4-di-Cl-Ph) exhibited
signicant anti Xac at 100 mg mLꢀ1, with the inhibition rates of
77.2, 95.2 and 79.6%, respectively. These compounds were
found to be more active compared to other tested compounds.
However, when R was substituted with Ph, 3-F-Ph, 4-Br-Ph, 3-
NO2-Ph, 3,4-di-OCH3-Ph, and 3,4-di-CH3-Ph groups, the activi-
ties of the corresponding compounds 2a, 2f, 2j, 2l, 2o and 2u,
against Xoo at 100 mg mLꢀ1 were 72.4, 88.1, 77.3, 82.3, 72.3 and
83.3%, respectively, which were higher than that of bismer-
thiazol (64.9%) and thiadiazole-copper (50.2%). Notably, when
R groups was Ph (2a), 3-F-Ph (2e), 2-Py (2k), 3-CH3-Ph (2m), 3,4-
di-OCH3-Ph (2o) and 2,4-di-Cl-Ph (2p) excellent biological
activity was found anti Rs at 100 mg mLꢀ1, inhibition rates of
76.6, 71.1, 73.7, 91.4, 92.4 and 85.2%, respectively.
3.4. Scanning electron microscopy (SEM) study
Following the antibacterial activities of the compounds, as
shown in Tables 1 and 2, the antibacterial mechanism of Xac
was further studied through SEM analysis.49 It can be seen that
the compound destroys the cell membrane, and more impor-
tantly, as the concentration of the compound increases, the
damage of the cell membrane becomes more serious. For
example, when the concentration was 50 mg mLꢀ1, a part of the
cell membrane begins to destroy (Fig. 1B). However, when the
concentration was further increased to 100 mg mLꢀ1, most of the
cell membrane was damaged, and only a small number of cells
was present (Fig. 1C). In contrast, in the absence of treatment
with compound 2l, the cells were smooth and well stacked with
an intact membrane (Fig. 1A). These SEM images further
conrm that compound 2l destroys bacterial cells and may
eventually kill the cells.
Fig. 2 Tobacco leaf morphology effects of the ningnanmycin and 2e against TMV in vivo. (Right leaf: not treated with compound; left leaf:
smeared with compound).
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Fig. 3 Microscale thermophoresis (MST) results of compounds 2e, 2h, 2n and ningnanmycin.
results that most of these novel chalcone derivatives containing
thiophene sulfonate could be further studied as a potential alter-
native template in the search for novel antiviral agents (Fig. 2).
3.5. Antiviral activity of title compounds against TMV in vivo
Using the same age tobacco leaves as the test subjects, the in
vivo healing and protective activities of TMV at 500 mg mLꢀ1
were assessed by the half-blight spot method.50–52 For this,
a commercial agricultural antiviral agent ningnanmycin was
used as a control, and the obtained preliminary bioassay results
are shown in Table 3. The compounds exhibited signicant
antiviral activity against TMV. Among them, compounds 2a, 2e,
2h, 2l, 2m and 2o showed excellent curative activities against
TMV, with the inhibition of 72.5, 84.2, 73.5, 81.8, 72.6 and
75.3%, which were better than ningnanmycin (53.3%). The
protective activities of 2a, 2e, 2h and 2l (70.0, 75.2, 71.8 and
72.8%, respectively) against TMV were more effective than
ningnanmycin (62.6%). In particularly, compounds 2a, 2e, 2h
and 2o showed excellent inactivation activities against TMV,
with the inhibition of 80.1, 87.3, 81.1 and 79.8%, which were
better than ningnanmycin (78.3%).
To compare the antiviral activity of the synthesized compounds
(2a–2v), EC50 values of 2a, 2e, 2h, 2l, 2m and 2o were calculated and
have been summarized in Table 4. It could be seen that compounds
2a, 2e, 2h and 2l exhibited higher curative activity than ningnan-
mycin (403.7 mg mLꢀ1), with EC50 values of 256.1, 178.0, 279.2 and
258.5 mg mLꢀ1, respectively. Compounds 2e and 2l exhibited the
best protective activity against TMV, with EC50 values of 269.0 and
282.6 mg mLꢀ1, respectively, which were better than that of ning-
3.6. Structure–activity relationships of antiviral activities
As indicated in Tables 3 and 4, most of the thiophene sulfonate
chalcone derivatives showed signicant antiviral activities
against TMV, and some of the structure–activity relationships
can be analyzed and summarized. First, the Ph, 2-F-Ph, 2-Br-Ph,
3-NO2-Ph and 3-CH3-Ph groups at –R position greatly improved
the curative activities of the title compounds against TMV. For
instance, the curative activities of the target compounds 2a, 2e,
2h, 2l and 2m, which were better than that of other substituent
groups. In addition, when –R was Ph, 2-F-Ph, 2-Br-Ph and 3-NO2-
Ph groups, the protective activities of the corresponding
compounds 2a, 2e, 2h and 2l at 500 mg mLꢀ1 were better than
that of ningnanmycin. When R was with Ph (2a), 2-F-Ph (2e), 2-
Br-Ph (2h) and 3,4-di-OCH3-Ph (2o) groups, the corresponding
compounds presented excellent inactivation activity against
TMV. For instance, some target compounds the curative,
protection and inactivation effects which were better than that
of ningnanmycin following the order of 2e (R ¼ 2-F-Ph) > 2h (R
¼ 2-Br-Ph) > 2a (R ¼ Ph). The detailed values corresponding to
these results can be seen in Table 3.
nanmycin (317.0 mg mLꢀ1). Among them, compounds 2e and 2h 3.7. Binding sites of 2e, 2h, 2n and ningnanmycin to TMV-
exhibited the best inactivation activity against TMV, with EC50 values CP
of 44.3 and 62.2 mg mLꢀ1, respectively, which were better than that
To further analyze the interactions between the title compounds
and TMV coat protein (TMV-CP), the microscale
of ningnanmycin (120.6 mg mLꢀ1). It can be indicated from these
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Fig. 4 Molecular docking studies of compounds 2e (A and B), 2h (C and D) and ningnanmycin (E and F).
thermophoresis (MST) analysis was used.53,54 The MST results as trends of the screening of antiviral activities. Based on the
summarized in Fig. 3 show that the binding of compounds 2e, experimental results it can be predicted that compounds 2e and
2h, 2n and ningnanmycin to TMV-CP protein yielded Kd values 2h may interact with TMV-CP. As depicted in Fig. 3 the bioac-
of 0.270 ꢂ 0.190 mmol Lꢀ1, 0.301 ꢂ 0.121 mmol Lꢀ1, 56.401 ꢂ tivity was mainly determined by the electrostatic interactions,
11.298 mmol Lꢀ1 and 0.596 ꢂ 0.338 mmol Lꢀ1, respectively. As and the activity could have been enhanced by the presence of
indicated in MST, 2e or 2h with ningnanmycin share strong aromatic ring which is rich in electrons for absorption. For
affinity, which is contrary to 2n as it shares weak affinity. These example, when R was substituted with 2-F-Ph and 2-Br-Ph
results exhibited that the combining capacity in the following groups, the corresponding compounds 2e and 2h exhibited
order of 2e > 2h > ningnanmycin > 2n is consistent with the a stronger combining capacity, with Kd values of 0.270 ꢂ 0.190
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mmol Lꢀ1 and 0.301 ꢂ 0.121 mmol Lꢀ1, as compared to that of
Acknowledgements
11.298 mmol Lꢀ1), which was
compound 2h (56.401
substituted with a 4-CH3-Ph.
ꢂ
This research was funded by the National Key Research and
Development Program of China (No. 2017YFD0200506) and the
Science and Technology Project of Guizhou Province (No.
20185781).
3.8. Molecular docking of 2e or 2h and TMV-CP
To identify the 2e and 2h recognition sites in TMV-CP (PDB
code: 1EI7), we performed molecular docking using the Gold
method with 200 cycles. As depicted in Fig. 4, the three
compounds were well-embedded in the activity pocket (ARG-46,
THR-42, ARG-90, GLN-34, THR-37, etc.) between the two
subunits of TMV-CP. Among them, ARG-46 had strong
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ꢀ
ꢀ
hydrogen bond with 2e (1.620 A and 1.653 A), C]O (2e)
demonstrates three hydrogens with the THR-42 (O–H ¼ 2.332
ꢀ
ꢀ
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In summary, with the aim of developing a novel, highly efficient,
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