Novel chalcone derivatives containing a 1,2,4-triazine moiety: Design, synthesis, antibacterial and antiviral activities

Article abstract of DOI:10.1039/c9ra00618d

A series of novel chalcone derivatives containing the 1,2,4-triazine moiety were synthesized and their structures were confirmed by ¹H NMR, ¹³C NMR and elemental analyses. Antiviral bioassays revealed that most of the compounds exhibited good antiviral activity against tobacco mosaic virus (TMV) at a concentration of 500 μg mL^-1. The designated compound 4l was 50% effective in terms of curative and protective activities against TMV with 50% effective concentrations (EC₅₀) of 10.9 and 79.4 μg mL^-1, which were better than those of ningnanmycin (81.4 and 82.2 μg mL^-1). Microscale thermophoresis (MST) also showed that the binding of compound 4l to coat protein (TMV-CP) yielded a K_d value of 0.275 ± 0.160 μmol L^-1, which was better than that of ningnanmycin (0.523 ± 0.250 μmol L^-1). At the same time, molecular docking studies for 4l with TMV-CP (PDB code:1EI7) showed that the compound was embedded well in the pocket between the two subunits of TMV-CP. Meanwhile, compound 4a demonstrated excellent antibacterial activities against Ralstonia solanacearum (R. solanacearum), with an EC₅₀ value of 0.1 μg mL^-1, which was better than that of thiodiazole-copper (36.1 μg mL^-1) and bismerthiazol (49.5 μg mL^-1). The compounds act by causing folding and deformation of the bacterial cell membrane as observed using scanning electron microscopy (SEM). The chalcone derivatives thus synthesized could become potential alternative templates for novel antiviral and antibacterial agents.

Full text of DOI:10.1039/c9ra00618d

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PAPER
Novel chalcone derivatives containing a 1,2,4-
triazine moiety: design, synthesis, antibacterial and
antiviral activities†
Cite this: RSC Adv., 2019, 9, 6011
Xu Tang,‡ Shijun Su,‡ Mei Chen,‡ Jun He,‡ Rongjiao Xia,‡ Tao Guo,‡ Ying Chen,‡
*
Cheng Zhang,‡ Jun Wang‡ and Wei Xue
‡
A series of novel chalcone derivatives containing the 1,2,4-triazine moiety were synthesized and their
1
structures were confirmed by H NMR, 13C NMR and elemental analyses. Antiviral bioassays revealed that
most of the compounds exhibited good antiviral activity against tobacco mosaic virus (TMV) at
a concentration of 500 mg mLꢀ1. The designated compound 4l was 50% effective in terms of curative
and protective activities against TMV with 50% effective concentrations (EC50) of 10.9 and 79.4 mg mLꢀ1
,
which were better than those of ningnanmycin (81.4 and 82.2 mg mLꢀ1). Microscale thermophoresis
(MST) also showed that the binding of compound 4l to coat protein (TMV-CP) yielded a Kd value of
0.275 ꢁ 0.160 mmol Lꢀ1, which was better than that of ningnanmycin (0.523 ꢁ 0.250 mmol Lꢀ1). At the
same time, molecular docking studies for 4l with TMV-CP (PDB code:1EI7) showed that the compound
was embedded well in the pocket between the two subunits of TMV-CP. Meanwhile, compound 4a
demonstrated excellent antibacterial activities against Ralstonia solanacearum (R. solanacearum), with an
EC50 value of 0.1 mg mLꢀ1, which was better than that of thiodiazole-copper (36.1 mg mLꢀ1) and
bismerthiazol (49.5 mg mLꢀ1). The compounds act by causing folding and deformation of the bacterial
cell membrane as observed using scanning electron microscopy (SEM). The chalcone derivatives thus
synthesized could become potential alternative templates for novel antiviral and antibacterial agents.
Received 24th January 2019
Accepted 6th February 2019
DOI: 10.1039/c9ra00618d
rsc.li/rsc-advances
through the use of some agrochemicals.5,6 So far, some tradi-
tional commercial viricides and bactericides, such as ribavirin,
1. Introduction
ningnanmycin, dufulin, thiodiazole-copper and bismerthiazol,
are available to be used in the control of plant viral and bacterial
diseases. However, the application of these traditional viricides
and bactericides are limited by their negative impacts on the
environment and the rapid emergence of resistance.7–9 Thus,
exploring novel lead compounds with potent agricultural
bioactivities is very crucial in the agricultural chemistry.10
Recent works have emphasized that the skeleton of chal-
cones has structural characteristics typical of diaryl enone
scaffold, and are an important class of natural and synthetic
bioactive compounds, which exhibit a broad spectrum of
pharmacological activities,11–15 including antifungal,16 antibac-
terial,17 antiviral,18 inhibitors,19 anticancer,20 antiparasitic,21
anti-Alzheimer's disease,22 antitrichomonal,23 anti-trypanosoma
cruzi24 activities. Meanwhile, Verma et al. demonstrated that
a series of chalcones derivatives could be used to control plant
viruses.25 In our previous study, we had demonstrated the
synthesis of a series of (E)-3-(3,4-dimethoxyphenyl)-1-(4-(2-
(quinazolin-4-ylthio)ethoxy)phenyl)prop-2-en-1-one and (E)-3-
(4-(2-(4-((E)-3-(4-uorophenyl) acryloyl) phenoxy)ethoxy)-3-
methoxy phenyl)acrylic acid derivatives (Fig. 1 1a, 1b), which
exhibited excellent antiviral activities.26,27
Plant bacterial diseases, such as citrus canker, rice bacterial leaf
blight and tobacco bacterial wilt, caused by Xanthomonas axo-
nopodis pv. citri (Xac), Xanthomonas oryzae pv. oryzae (Xoo) and
Ralstonia solanacearum (R. solanacearum), respectively, can be
very serious and difficult to manage in agricultural produc-
tion.1,2 Tobacco mosaic virus (TMV), a classic pathogenic virus,
has been extensively studied, and being a virus causes massive
loss to crops and plants. This virus has been found to
commonly infect nine plant families and at least 125 individual
species including tobacco, tomato, pepper, potato, and
cucumber, and its infection is extremely difficult to control
under eld conditions.3,4 Therefore, it is necessary to protect
these crops against infestation and destruction by the bacterial
and viral infections, and this has been effectively achieved
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/c9ra00618d
‡ These authors contributed to this work equally.
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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 thermophoresis instrument
(NanoTemper Technologies GmbH, Germany). The uores-
cence spectroscopy of the compounds interacting with TMV CP
was determined by FluoroMax-4 uorescence spectrometer
(HORIBA Scientic, France). The molecular docking was per-
formed by using DS-CDocker implemented in Discovery Studio
(version 4.5). All reagents and solvents were purchased from
Chinese Chemical Reagent Company and were of chemically
pure analytical grade reagents. The synthetic route to chalcone
derivatives containing 1,2,4-triazine moiety is shown in Scheme
1. The intermediates 1, 2 and 3 were prepared according to the
methods already reported in the literature.26,27,41
Fig. 1 Compounds previously reported against tobacco mosaic virus
(TMV).
1,2,4-triazine and their derivatives are an important class of
heterocyclic compounds and thus have been reported in a wide
spectrum of biological activities,28–31 including antith-
rombotic,32 anti-oxidant,33 anticancer,34 antiplatelet,35 throm-
boxane
synthetase
inhibition,36
anti-inammatory,37
antimalarial38 and a-glucosidase inhibition.39 It has also been
reported that a minor modication in the structure of 1,2,4-
triazine can lead to quantitative, as well as qualitative changes
in the biological activities.40 Fu et al. reported potent inhibitory
effect of 1,2,4-triazine and their derivatives against three cell
lines (MGC-803, EC-109 and PC-3) compared to 5-uorouracil.34
However, there are only few studies that focus on 1,2,4-triazine
moiety to be potentially utilize them as an antibacterial and
antiviral scaffold. Based on these, it is of urgent importance to
search for novel effective agro-chemicals that have unique
mechanism of action. In this work, 5,6-disubstituted-1,2,4-
triazine group was introduced into the 4-position of chalcone
nucleus to build a new molecular structure and the potency of
which was tested in terms of biological activity (Fig. 2). The
synthesized compounds were evaluated for their antibacterial
and antiviral activities.
2.2. General procedure for the synthesis of intermediates 1
and 2
As shown in Scheme 1, using 1-(4-hydroxyphenyl) ethan-1-one
or 1-(2-hydroxyphenyl)ethan-1-one and different substituted
benzaldehyde as the initial materials in ethanol under ice-bath
conditions in the presence of different substituted chalcone,
intermediate 2 was favourably prepared according to the
1
previously reported procedure.26,27 The H NMR, 13C NMR and
HMRS spectra of the intermediate 2 have been provided in the
ESI.†
2.3. General procedure for the synthesis of intermediate 3
A general synthesis of 5,6-diphenyl-1,2,4-triazine-3-thiol or 5,6-
dimethyl-1,2,4-triazine-3-thiol is shown in Scheme 1. Benzil (10
mmol) or biacetyl (10 mmol) with thio-semicarbazide (11 mmol)
was reuxed in acetic acid (20 mL) and water (35 mL), to obtain
the corresponding 5,6-diphenyl-1,2,4-triazine-3-thiol or 5,6-
dimethyl-1,2,4-triazine-3-thiol (3).41
2. Experimental
2.1. Methods and materials
Melting points of the synthesized compounds (4a–4w) were
measured using XT-4 Binocular Microscope (Beijing Tech.
Instrument, China) and were uncorrected. Using DMSO-d6 as
the solvent and TMS as an internal standard H and 13C NMR
1
spectra of the target compounds were recorded using JEOL-ECX
500 NMR spectrometer (JEOL, Japan). HRMS data were obtained
using Thermo Scientic Q Exactive Hybrid Quadrupole Mass
2.4. General procedure for the synthesis of title compounds
4a–4w
The synthetic route to the title compounds, (E)-1-(4-(2-((5,6-
diphenyl-1,2,4-triazin-3-yl)thio)ethoxy)phenyl)-3-(2-substituted
aromatic groups)prop-2-en-1-one or (E)-1-(2-(2-((5,6-diphenyl-
1,2,4-triazin-3-yl)thio)ethoxy)phenyl)-3-(2-substituted aromatic
groups)prop-2-en-1-one derivatives (4a–4w) are shown in
Scheme 1. A mixture of intermediate 3 (10 mmol) and anhy-
drous potassium carbonate (30 mmol) in dimethylformamide
(DMF, 40 mL) was stirred at room temperature for 1 h. Then, the
intermediate 2 (12 mmol) was added to the above solution and
stirred at room temperature for 6–8 h. Upon completion of
reaction (indicated by TLC), the mixture was extracted, the
solvent was removed under vacuum, and the crude product was
then separated by column chromatography with petroleum
ether/ethyl acetate (3 : 1 v/v) to obtain the designated title
1
compounds 4a–4w at different yields. The H NMR, 13C NMR
and HMRS spectra of the designated title compounds 4a–4w are
also provided in the ESI.†
Fig. 2 Design route of the title compounds 4a–4w.
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Scheme 1 Synthetic route to title compounds 4a–4w.
(45.7%). In addition, 4b, 4g, 4k, 4l, 4u and 4v compounds
exhibited signicant protective activities against TMV at 500 mg
mLꢀ1, with 58.7%, 55.5%, 56.8%, 58.6%, 62.7% and 56.2%,
respectively, which were even better than that of ningnanmycin
(53.4%). The inactivation activity of the compound 4k at 500 mg
mLꢀ1 was 86.8%, which was also better than that of ningnan-
mycin (77.3%).
To compare the antiviral activity of the synthesized
compounds (4a–4w), EC50 values of 4g, 4k, 4l and 4u were
calculated and have been summarized in Table 2. It could be
seen that compounds 4g, 4k, 4l and 4u exhibited higher curative
activity than ningnanmycin (81.5 mg mLꢀ1), with EC50 values of
17.5, 21.8, 10.9 and 72.2 mg mLꢀ1, respectively. Compounds 4l
and 4u exhibited the best protective activity against TMV, with
EC50 values of 79.4 and 52.4 mg mLꢀ1, respectively, which were
better than that of ningnanmycin (82.3 mg mLꢀ1). Notably,
compound 4k was found to exhibit signicant inactivation
activity (2.5 mg mLꢀ1), which was similar to ningnanmycin (1.1
mg mLꢀ1). It can be implied from these results that most of
these novel chalcone derivatives containing 1,2,4-triazine
moiety could be further studied as a potential alternative
template in the search for novel antiviral agents.
3. Results and discussion
3.1. Spectral properties
1H NMR, 13C NMR and HRMS spectra of the target compounds
are provided in the ESI.† In 1H NMR spectra, multiplet signals at
d 8.71–6.33 ppm indicate the presence of protons in olenic
bonds and aromatic nuclei, and a singlet at d 4.59–4.20 ppm
and 3.80–3.40 ppm reveals the presence of –OCH2– and –SCH2–
groups, respectively. The absorption signals at d 192.94–187.20
in 13C NMR spectra conrm the presence of –C]O group, while
the absorption signals at d 67.99–55.53 and 55.52–29.31 ppm in
13C NMR spectra conrm the presence of –OCH2– and –SCH2–
groups, respectively. In the HRMS spectra of the title
compounds, the characteristic absorption signals of [M + H]+
ions were obtained, which agree with their molecular weights.
3.2. Antiviral activity of the title compounds against TMV
Using N. tabacun L. leaves under the same age as that of test
subjects, the curative, protective and inactivation activities
against TMV (in vivo) at a concentration of 500 mg mLꢀ1 were
evaluated by the half-leaf blight spot method9,26,27,42 and the
obtained results are shown in Table 1. The preliminary bioassay
results demonstrated that the inhibitory rates of title
compounds (4a–4w) against TMV ranged from 30.6 to 65.6% in
3.3. Structure–activity relationship (SAR) of the title
compounds against TMV
terms of their curative activities, while their protective and An analysis of Tables 1 and 2 reveals that variations in the
inactivation activities ranged from 34.3 to 62.7% and 23.3 to structural features of chalcone derivatives containing 1,2,4-
86.8%, respectively. Among them, the designated compounds, triazine moiety could play a role in their anti-TMV activities.
4g, 4j, 4k, 4l, 4o, 4u and 4w afforded better curative activities First, the 4-F-Ph, 4-methylthiazole and 4-Cl-Ph groups at –R
against TMV at 500 mg mLꢀ1, with 54.9%, 57.1%, 55.3%, 62.7%, position greatly improved the curative activities of the title
61.4, 65.6% and 60.6%, respectively. These compounds were compounds against TMV. For instance, the curative activities of
found to exhibit better antiviral activities than ningnanmycin the target compounds 4l (R ¼ 4-F-Ph), 4u (R ¼ 4-Cl-Ph) and 4o (R
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Table 1 Antiviral activities of the target compounds against TMV in vivo at 500 mg mLꢀ1
Compd.
Curative activitya (%)
Protective activitya (%)
Inactivation activitya (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
49.4 ꢁ 0.8
40.4 ꢁ 0.8
30.6 ꢁ 0.4
48.3 ꢁ 1.3
56.4 ꢁ 1.0
55.6 ꢁ 2.3
54.9 ꢁ 4.6
53.6 ꢁ 2.6
54.9 ꢁ 9.7
57.1 ꢁ 1.1
55.3 ꢁ 0.6
62.7 ꢁ 0.3
45.2 ꢁ 4.3
44.1 ꢁ 1.2
61.4 ꢁ 1.4
37.8 ꢁ 1.7
44.8 ꢁ 0.5
41.7 ꢁ 1.8
43.1 ꢁ 0.5
56.1 ꢁ 0.4
65.6 ꢁ 0.1
50.7 ꢁ 1.8
60.6 ꢁ 0.5
45.7 ꢁ 2.6
46.3 ꢁ 7.9
58.7 ꢁ 1.3
34.3 ꢁ 4.0
44.6 ꢁ 4.4
41.7 ꢁ 4.6
38.7 ꢁ 2.4
55.5 ꢁ 4.9
46.9 ꢁ 2.8
42.2 ꢁ 0.5
51.7 ꢁ 0.1
56.8 ꢁ 0.9
58.6 ꢁ 1.3
39.5 ꢁ 3.2
51.1 ꢁ 0.3
55.9 ꢁ 0.3
44.4 ꢁ 2.3
47.4 ꢁ 4.8
20.1 ꢁ 0.8
27.3 ꢁ 1.0
51.3 ꢁ 1.9
62.7 ꢁ 0.7
56.2 ꢁ 2.3
50.8 ꢁ 0.1
53.4 ꢁ 2.4
60.9 ꢁ 2.4
43.8 ꢁ 4.2
23.3 ꢁ 1.6
55.2 ꢁ 1.8
46.3 ꢁ 0.1
50.8 ꢁ 1.9
64.6 ꢁ 0.8
51.6 ꢁ 1.7
52.1 ꢁ 2.5
62.9 ꢁ 0.3
86.8 ꢁ 1.0
62.6 ꢁ 2.0
51.1 ꢁ 1.1
23.9 ꢁ 9.2
23.3 ꢁ 0.1
40.1 ꢁ 1.7
43.2 ꢁ 4.0
54.6 ꢁ 2.9
51.2 ꢁ 2.2
46.2 ꢁ 7.6
51.6 ꢁ 0.7
38.8 ꢁ 2.6
49.6 ꢁ 0.9
77.3 ꢁ 1.6
Ningnanmycinb
a
b
Average of three replicates. A commercial agricultural antiviral agent ningnanmycin was used for comparison of antiviral activities.
¼ 4-methylthiazole) were 62.7%, 65.6% and 61.4%, respectively, ningnanmycin (77.3%). Second, when R1 was substituted by a –
which were better than that of other substituent groups. In CH3 group, the corresponding compound 4g (R1 ¼ Me, R ¼ 3,4-
addition, when –R was 2-O-Me-Ph, 2,4-di-OMe-Ph, 3-NO2-Ph, 4- di-OMe) had better curative, protection and inactivation effect
F-Ph, 4-methylthiazole and 4-Cl-Ph groups, the protective against TMV than the compound bearing a –Ph (4c) group.
activities of the corresponding compounds 4b, 4g, 4k, 4l, 4o, 4u When R was substituted with a 3,4-di-OMe-Ph, 3-NO2-Ph, 4-F-Ph
and 4w at 500 mg mLꢀ1 were 58.7%, 55.5%, 56.8%, 58.6%, or 4-Cl-Ph group, the obtained compounds exhibited better
55.9%, 62.7% and 56.2%, respectively, which were better than curative, protection and inactivation activities against TMV. For
that of ningnanmycin (53.4%). Furthermore, when –R was 3- instance, the curative, protection and inactivation effects of the
NO2-Ph group, the inactivation activity of the compound 4k at target compounds 4g (R ¼ 3,4-di-OMe-Ph, R1 ¼ –Me), 4k (3-NO2-
500 mg mLꢀ1 was 86.8%, which was better than that of Ph, R1 ¼ –Ph), 4l (4-F-Ph, R1 ¼ –Ph) and 4u (4-Cl-Ph, R1 ¼ –Ph)
Table 2 EC 50 values of the 4g, 4k, 4l and 4u against TMV in vivoa
Compd.
Against TMV
Regression equation
r2
EC50
4g
Curative activity
y ¼ 0.3961x + 4.5075
y ¼ 0.4213x + 3.9441
y ¼ 0.5973x + 4.0952
y ¼ 0.3432x + 4.5403
y ¼ 0.2912x + 4.2541
y ¼ 0.2248x + 4.9103
y ¼ 0.3387x + 4.6487
y ¼ 0.3195x + 4.3930
y ¼ 0.5142x + 4.1551
y ¼ 0.4456x + 4.1718
y ¼ 0.4467x + 4.2318
y ¼ 0.2934x + 4.4604
y ¼ 0.4415x + 4.1563
y ¼ 0.4732x + 4.0939
y ¼ 0.4591x + 4.9815
0.9707
0.9534
0.9654
0.9006
0.9415
0.9939
0.9347
0.9078
0.9541
0.9690
0.9440
0.9156
0.9720
0.9097
0.9056
17.5 ꢁ 0.34
Protection activity
Inactivation activity
Curative activity
Protection activity
Inactivation activity
Curative activity
Protection activity
Inactivation activity
Curative activity
Protection activity
Inactivation activity
Curative activity
320.8 ꢁ 0.19
32.7 ꢁ 0.03
21.8 ꢁ 0.05
364.3 ꢁ 0.21
2.5 ꢁ 0.04
4k
4l
10.9 ꢁ 0.02
79.4 ꢁ 0.22
43.9 ꢁ 0.03
72.2 ꢁ 4.04
52.4 ꢁ 0.06
68.9 ꢁ 0.19
81.4 ꢁ 0.28
82.2 ꢁ 0.05
1.1 ꢁ 0.02
4u
Ningnanmycinb
Protection activity
Inactivation activity
a
b
Average of three replicates. A commercial agricultural antiviral agent ningnanmycin was used for comparison of antiviral activities.
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were 54.9%, 55.5% and 64.6%, 55.3%, 56.8% and 86.8%, 62.7%, between GLU131, ASN73, ALA74 and GLN257 and other
58.6% and 62.6%, 65.6%, 62.7% and 51.6%, respectively, which subunits of TMV-CP.44 Moreover, there are also notable effects
were better than that of ningnanmycin (45.7%, 53.4% and of hydrogen bonding, p-sulfur, p-alkyl, and alkyl between the
77.3%) (Fig. 3).
molecules and the above residues in TMV-CP as depicted in
Fig. 5. For instance, ASN73 showed strong hydrogen bond with
˚
4l (2.35 A), and triazine demonstrates p-alkyl interaction with
3.4. Binding sites of 4l, 4d, 4o and ningnanmycin to TMV-CP
the residue VAL-74. There was also p-alkyl interaction between
the phenyl group and the residue VAL-74. These interactions
between molecules and TMV-CP are likely to weaken the inter-
action of two subunits of TMV-CP, thereby preventing the self-
assembly of TMV particle, as well as the binding capability
with TMV-CP. The results of molecular docking studies were
consistent with the experimental results (protection and inac-
tivation activities) and support that the chalcone derivatives
may be potential lead structures for developing novel anti-TMV
agents.
To further analyze the interactions between the title compounds
and TMV coat protein (TMV CP), the microscale thermophoresis
(MST) analysis was used.43 The MST results as summarized in
Fig. 4 and Table 3 show that the binding of compounds 4l, 4d,
4o and ningnanmycin to TMV CP protein yielded Kd values of
0.275 ꢁ 0.160 mmol Lꢀ1, 81.744 ꢁ 22.310 mmol Lꢀ1, 245.285 ꢁ
63.860 mmol Lꢀ1 and 0.523 ꢁ 0.250 mmol Lꢀ1, respectively. As
indicated in MST, 4l and ningnanmycin share strong affinity,
while 4d shares moderate affinity, which is contrary to 4o as it
shares weak affinity. These results exhibited that the combining
capacity in the following order of 4l > ningnanmycin > 4d > 4o is
consistent with the trends of the screening of antiviral activities.
Based on the experimental results it can be predicted that
compound 4l may interact with TMV CP. As depicted in Fig. 4
and Table 3, the bioactivity was mainly determined by the
electrostatic interactions, and the activity could have been
enhanced by the presence of aromatic ring which is rich in
electrons for absorption. For example, when R was substituted
with a 4-F-Ph group, the corresponding compound (4l) exhibi-
ted a stronger combining capacity, with Kd values of 0.275 ꢁ
0.160 mmol Lꢀ1, as compared to that of compounds 4d (81.744 ꢁ
22.310 mmol Lꢀ1) and 4o (245.285 ꢁ 63.860 mmol Lꢀ1), which
was substituted with a 2,4-di-OMe-Ph or 4-methylthiazole.
3.6. Antibacterial activities
The antibacterial activities of the title compounds against Xoo,
R. solanacearum and Xac were evaluated at 100 mg mLꢀ1 using
a turbidimeter.2,9,46 The commercial bactericide, bismerthiazol
and thiadiazole-copper were tested as a control under the same
conditions, and the obtained results are summarized in Table 4.
It can be observed from this table that most of the target
compounds exhibited considerable antibacterial effects against
Xoo, R. solanacearum and Xac. For instance, compounds 4c, 4d,
4g and 4n presented antibacterial activities against Xoo at 100
mg mLꢀ1, with the inhibition rates of 62.9%, 60.5%, 61.8% and
69.0%, respectively, which are better than that of bismerthiazol
(56.1%) and thiadiazole-copper (32.7%). The inhibition rates of
compounds 4c, 4d, 4g and 4n against Xoo at 50 mg mLꢀ1 were
3.5. Molecular docking of 4l and TMV-CP
Molecular docking studies (Fig. 5) for 4l with TMV-CP (PDB 44.9%, 39.9%, 44.5% and 42.3%, respectively, which were better
code:1EI7)44 have shown that the compound was well- than that of thiadiazole-copper (27.2%) and similar to bismer-
embedded between the two subunits of TMV-CP (including thiazol (49.3%). Moreover, compounds 4a, 4j, 4l, 4n, 4r, 4s and
residues of GLU131, LYS268, GLN257, ALA74, ASN73, PHE12 4w presented excellent antibacterial activities against R. sol-
and TYR139). Previous reports have demonstrated that these anacearum at 100 mg mLꢀ1, with the inhibition rates of 93.7%,
residues play important roles in the self-assembly of TMV 87.9%, 88.6%, 99.9%, 96.7%, 85.1% and 94.2%, respectively,
particles.44,45 Some hydrophobic aromatic components and which are higher as compared to bismerthiazol (52.1%) and
clusters such as PHE12, LYS268 and TRY139, play a key role in thiadiazole-copper (80.2%). Compounds 4a, 4j, 4l, 4n, 4r, 4s and
the continuous hydrophobic interactions within each ring of 4w revealed excellent antibacterial activities against R. sol-
this assembly.45 In addition, there are strong interactions anacearum at 50 mg mLꢀ1 with the inhibition rates of 74.2%,
Fig. 3 Tobacco leaf morphology effects of the ningnanmycin and 4l against TMV in vivo. (Right leaf: not treated with compound; left leaf:
smeared with compound).
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Fig. 4 Microscale thermophoresis (MST) of 4l (A); 4d (B); 4o (C) and ningnanmycin (D).
70.0%, 82.9%, 74.8%, 71.1%, 80.3% and 87.0%, respectively.
These rates were also higher compared to bismerthiazol (44.2%)
and thiadiazole-copper (48.3%). In addition, compounds 4a, 4c,
4d, 4e, 4j, 4m and 4n at 100 and 50 mg mLꢀ1, exhibited better
antibacterial activities against Xac than bismerthiazol (70.5%
and 33.6%) and thiodiazole-copper (75.9% and 29.5%), with the
values of 81.1% and 66.5%, 81.2% and 60.9%, 83.3% and
60.6%, 93.1% and 40.0%, 96.8% and 48.6%, 88.0% and 62.7%
and 87.7% and 79.4%, respectively.
Table 3 The dissociation constant of 4l, 4d, 4o and ningnanmycin
with TMV-coat protein (TMV-CP)
Compd.
Kd (mmol Lꢀ1
)
4l
4d
4o
0.275 ꢁ 0.16
81.744 ꢁ 22.31
245.285 ꢁ 63.86
0.523 ꢁ 0.25
Ningnanmycin
Fig. 5 The binding mode of compound 4l docked with TMV-CP.
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Table 4 Inhibition effect of the title compounds against Xoo, R. solanacearum and Xacb
Inhibition (%)
Xoo
R. solanacearum
Xac
100 mg
Compd.
mLꢀ1
50 mg mLꢀ1
100 mg mLꢀ1
50 mg mLꢀ1
100 mg mLꢀ1
50 mg mLꢀ1
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
35.4 ꢁ 5.4
47.9 ꢁ 2.0
62.9 ꢁ 4.8
60.5 ꢁ 2.4
25.4 ꢁ 5.4
27.9 ꢁ 2.0
61.8 ꢁ 5.7
18.8 ꢁ 3.5
18.0 ꢁ 2.6
13.9 ꢁ 3.0
27.3 ꢁ 7.1
17.5 ꢁ 3.9
17.4 ꢁ 4.3
69.0 ꢁ 4.2
17.2 ꢁ 4.6
15.6 ꢁ 4.2
26.5 ꢁ 1.0
35.4 ꢁ 3.3
19.2 ꢁ 4.6
12.2 ꢁ 0.8
42.1 ꢁ 6.2
56.1 ꢁ 7.3
37.2 ꢁ 0.9
32.7 ꢁ 8.2
34.7 ꢁ 2.7
44.9 ꢁ 2.3
39.9 ꢁ 2.5
22.7 ꢁ 8.2
24.7 ꢁ 2.7
44.5 ꢁ 3.6
24.0 ꢁ 5.6
11.2 ꢁ 3.0
17.5 ꢁ 3.9
11.2 ꢁ 4.3
12.5 ꢁ 3.9
15.1 ꢁ 4.6
42.3 ꢁ 1.2
15.1 ꢁ 4.6
11.1 ꢁ 1.2
25.2 ꢁ 2.1
28.8 ꢁ 4.3
13.2 ꢁ 4.6
11.6 ꢁ 3.6
31.0 ꢁ 6.0
49.3 ꢁ 5.4
27.2 ꢁ 3.2
93.7 ꢁ 4.5
63.4 ꢁ 6.2
72.2 ꢁ 3.2
78.5 ꢁ 3.0
74.7 ꢁ 0.5
70.3 ꢁ 1.6
83.5 ꢁ 4.5
71.9 ꢁ 2.1
83.7 ꢁ 2.8
87.9 ꢁ 2.2
80.3 ꢁ 4.4
88.6 ꢁ 4.5
81.2 ꢁ 7.6
99.9 ꢁ 2.6
72.5 ꢁ 2.4
63.9 ꢁ 8.3
80.3 ꢁ 0.6
96.7 ꢁ 2.7
85.1 ꢁ 3.6
75.8 ꢁ 3.0
94.2 ꢁ 2.6
52.1 ꢁ 3.4
80.2 ꢁ 4.0
74.2 ꢁ 0.2
62.2 ꢁ 7.0
62.7 ꢁ 6.2
77.7 ꢁ 1.6
53.8 ꢁ 3.4
57.6 ꢁ 6.6
81.8 ꢁ 1.8
70.3 ꢁ 3.2
79.8 ꢁ 2.0
79.0 ꢁ 2.3
79.7 ꢁ 1.5
82.9 ꢁ 0.8
72.6 ꢁ 4.7
74.8 ꢁ 1.9
68.7 ꢁ 1.8
58.0 ꢁ 14.3
70.9 ꢁ 2.8
71.1 ꢁ 1.5
80.3 ꢁ 3.4
61.7 ꢁ 3.2
87.0 ꢁ 3.5
44.2 ꢁ 3.9
48.3 ꢁ 4.8
81.1 ꢁ 3.5
45.9 ꢁ 7.2
81.2 ꢁ 2.7
83.3 ꢁ 5.4
93.1 ꢁ 9.0
56.4 ꢁ 2.4
63.0 ꢁ 1.2
75.4 ꢁ 3.3
53.0 ꢁ 1.0
96.8 ꢁ 4.4
70.2 ꢁ 3.3
59.4 ꢁ 2.2
88.0 ꢁ 6.2
87.7 ꢁ 4.1
39.3 ꢁ 6.0
64.5 ꢁ 1.1
39.8 ꢁ 1.8
41.8 ꢁ 2.6
65.1 ꢁ 4.1
63.9 ꢁ 1.2
70.7 ꢁ 2.1
70.5 ꢁ 1.5
75.9 ꢁ 1.5
66.5 ꢁ 1.7
39.9 ꢁ 4.8
60.9 ꢁ 2.0
60.6 ꢁ 1.7
40.0 ꢁ 3.0
51.3 ꢁ 4.1
49.3 ꢁ 3.7
48.4 ꢁ 0.6
33.2 ꢁ 9.3
48.6 ꢁ 3.3
40.7 ꢁ 3.8
48.0 ꢁ 5.3
62.7 ꢁ 3.8
79.4 ꢁ 1.3
27.7 ꢁ 6.0
50.2 ꢁ 10.5
29.1 ꢁ 3.1
40.9 ꢁ 1.4
45.8 ꢁ 9.1
51.7 ꢁ 2.7
65.7 ꢁ 4.9
33.6 ꢁ 1.7
29.5 ꢁ 4.3
4s
4t
4w
BTa
TCa
a
b
BT: bismerthiazol; TC: thiadiazole-copper; average of three replicates. A commercial agricultural antibacterial agent bismerthiazol and
thiadiazole-copper were used for comparison of antibacterial activities.
Based on these preliminary results of bioassays, EC50 values solanacearum and anti Xac activities than their corresponding
of some of the compounds showed excellent antibacterial anti Xoo activities as could be observed from the results pre-
activities against Xoo, Xac and R. solanacearum as indicated in sented in Table 4. The antibacterial activities of the title
Table 5. As can be seen from Table 5, compounds 4a and 4e compounds were also affected by the nature or identity of
exhibited satisfactory in vitro antibacterial activities against different groups attached to R. For instance, the designated
Xac, with the EC50 values of 53.6 and 67.0 mg mLꢀ1, which were compounds 4a (R ¼ –NO2-Ph), 4j (R ¼ 4-OMe-Ph), 4l (R ¼ 4-F-
higher than that of bismerthiazol (153.7 mg mLꢀ1) and Ph), 4n (R ¼ N,N-di-Me-Ph), 4r (R ¼ isopropyl-Ph) and 4w (R
thiodiazole-copper (77.8 mg mLꢀ1) (Table 5). Also, compound ¼ 4-methylthiazole) exhibited signicant anti R. solanacearum
4g was found to exhibit higher antibacterial activity against effects at 100 mg mLꢀ1, with the inhibition rates of 93.7%,
Xoo (76.7 mg mLꢀ1), than bismerthiazol (94.5 mg mLꢀ1). On the 87.9%, 88.6%, 99.9%, 96.7% and 94.2%, respectively. These
other hand, as shown in Table 5, the title compounds 4a–4w compounds were found to be more active compared to other
exhibited in vitro antibacterial activities against R. sol- tested compounds. However, when R was substituted with 4-
anacearum, with the EC50 values of 0.1–93.9 mg mLꢀ1. The NO2-Ph, 2,4-di-OMe-Ph, phenyl, 4-OMe-Ph, 2-F-Ph and N,N-di-
target compounds 4a, 4g, 4j, 4l, 4s, 4t and 4v showed excellent Me-Ph groups, the activities of the corresponding compounds
in vitro antibacterial activities against R. solanacearum, with 4a, 4d, 4e, 4j, 4m and 4n, against Xac at 100 mg mLꢀ1 were
the EC50 values of 0.1, 0.1, 0.3, 0.4, 0.2, 0.1 and 2.7 mg mLꢀ1
,
81.1%, 83.3%, 93.1%, 96.8%, 88.0% and 87.7%, respectively,
respectively. These values were comparable with thiodiazole- which were higher than that of bismerthiazol (70.5%) and
copper (36.1 mg mLꢀ1) and bismerthiazol (49.5 mg mLꢀ1) as thiadiazole-copper (75.9%).
shown in Table 5.
3.8. SEM study
3.7. Structure–activity relationships (SAR) of antibacterial
activities
In order to further explore the mechanism of antibacterial
action against R. solanacearum, scanning electron microscopic
Table 4 reveals that the antibacterial activities of the title (SEM) studies were carried out using the designated compound
compounds were considerably affected by the structural varia- 4a and the observed SEM micrographs are shown in Fig. 6. It
tions in the chalcone derivatives that contain 1,2,4-triazine could be noted that the compounds act by causing the bacterial
moiety. Notably, all the title compounds exhibited better anti R. cell membranes to wrinkle. Wrinkling of the cell membranes
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Table 5 EC50 values of some title compounds against Xoo, Xac and R. solanacearumb
Tested bacteriasl
Compd.
Regression equation
r2
EC50 (mg mLꢀ1
)
Xac
4a
4c
y ¼ 1.1509x + 3.0094
y ¼ 0.3984x + 3.9390
y ¼ 0.3020x + 4.3584
y ¼ 0.5608x + 3.9757
y ¼ 0.3926x + 4.1415
y ¼ 1.7056x + 1.7748
y ¼ 0.7929x + 3.0664
y ¼ 0.9379x + 3.2319
y ¼ 0.7539x + 3.4235
y ¼ 1.5696x + 1.8988
y ¼ 0.9843x + 3.3369
y ¼ 0.3081x + 5.3419
y ¼ 1.3629x + 3.9942
y ¼ 0.6011x + 3.9125
y ¼ 0.8807x + 3.5862
y ¼ 0.9347x + 4.2405
y ¼ 0.3225x + 4.7271
y ¼ 0.4227x + 5.4036
y ¼ 0.3235x + 4.3949
y ¼ 2.0693x + 2.9527
y ¼ 0.5553x + 5.2478
y ¼ 1.5277x + 3.5958
y ¼ 0.4623x + 5.1549
y ¼ 1.3267x + 2.5265
y ¼ 0.6065x + 3.9014
y ¼ 2.1930x + 2.0260
y ¼ 1.1930x + 3.0290
y ¼ 0.2018x + 4.6019
y ¼ 2.3888x + 1.4419
y ¼ 0.4483x + 5.3221
y ¼ 0.4271x + 5.3495
y ¼ 0.6484x + 3.7942
y ¼ 0.6851x + 4.7031
y ¼ 0.9945x + 3.4690
y ¼ 1.0223x + 3.2674
y ¼ 0.7337x + 3.8570
0.9084
0.9265
0.9155
0.9644
0.9072
0.9161
0.9622
0.9558
0.9573
0.9551
0.9475
0.9157
0.9383
0.9449
0.9583
0.9546
0.9655
0.9696
0.9113
0.9540
0.9379
0.9378
0.9939
0.9339
0.9395
0.9601
0.9301
0.9719
0.9198
0.9351
0.9695
0.9415
0.9378
0.9266
0.9095
0.9042
53.6 ꢁ 4.5
460.4 ꢁ 2.4
133.2 ꢁ 4.7
67.0 ꢁ 4.9
153.7 ꢁ 5.9
77.8 ꢁ 2.3
274.5 ꢁ 5.0
76.7 ꢁ 6.2
123.3 ꢁ 6.3
94.5 ꢁ 4.3
48.9 ꢁ 8.3
0.1 ꢁ 3.6
4d
4e
BTa
TCa
4c
Xoo
RS
4g
4n
BTa
TCa
4a
4b
4c
4d
4e
1.0 ꢁ 4.2
64.4 ꢁ 5.6
42.5 ꢁ 8.1
6.5 ꢁ 5.1
4f
7.02 ꢁ 2.8
0.1 ꢁ 0.4
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
74.2 ꢁ 6.1
9.8 ꢁ 2.1
0.3 ꢁ 0.6
8.3 ꢁ 0.9
0.4 ꢁ 4.5
73.2 ꢁ 6.1
64.8 ꢁ 4.8
24.3 ꢁ 5.2
22.7 ꢁ 4.8
93.9 ꢁ 7.8
30.8 ꢁ 5.1
0.2 ꢁ 0.2
4s
4t
0.1 ꢁ 0.5
4u
4v
4w
BTa
TCa
72.4 ꢁ 5.5
2.7 ꢁ 3.5
34.6 ꢁ 2.8
49.5 ꢁ 8.5
36.1 ꢁ 3.9
a
b
BT: bismerthiazol; TC: thiadiazole-copper. A commercial agricultural antibacterial agent bismerthiazol and thiadiazole-copper were used for
comparison of antibacterial activities.
became more obvious with an increase in the concentration of (Fig. 6C). In contrast, without any treatment with compound 4a,
compound 4a. For instance, when the concentration was 12.5 the bacterial cells were smooth and the cell membrane was
mg mLꢀ1, the cell membrane began to wrinkle (Fig. 6B). intact (Fig. 6A). The results from SEM provide clear evidence
Whereas, a further increase in the concentration to 50 mg mLꢀ1
,
that the compound 4a exhibited antibacterial effects on the cell,
it results in the development of folds in the cell membrane and this effect might eventually results in cell death.
Fig. 6 SEM images for R. solanacearum after incubated in different concentration of compound 4a (A) 0 mg mLꢀ1, (B) 12.5 mg mLꢀ1, and (C)
50 mg mLꢀ1. Scale bar for (A, B and C) are 2 mm.
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4. Conclusions
With the aim to develop novel, highly-efficient and environ-
mentally benign agrochemicals, 1,2,4-triazine scaffold was
introduced into chalcone structure to synthesize chalcone
derivatives, and also their in vivo antibacterial and antiviral
activities were evaluated. The obtained bioassay results revealed
that some of the title compounds exhibited excellent antibac-
terial and antiviral activities and notably they were better than
the commercial agents. Compound 4l exhibited signicant
curative and protective activities against tobacco mosaic virus
(TMV), with the EC50 values of 10.9 mg mLꢀ1 and 79.4 mg mLꢀ1
,
which was better than that of ningnanmycin (81.4 mg mLꢀ1 and
82.2 mg mLꢀ1). Microscale thermophoresis (MST) showed that
the binding of compound 4l to coat protein (TMV-CP) yielded Kd
values of 0.275 ꢁ 0.160 mmol Lꢀ1, which was better than that of
ningnanmycin (0.523 ꢁ 0.250 mmol Lꢀ1). Molecular docking
studies for 4l with TMV-CP (PDB code: 1EI7) showed that the
compound was embedded well in the pocket between the two
subunits of TMV-CP. Compounds 4a, 4g, 4j, 4s, 4l and 4t
demonstrated excellent antibacterial activities against R. sol-
anacearum, with the EC50 values of 0.1, 0.1, 0.3, 0.46 and 0.1 mg
mLꢀ1, which were better than that of thiodiazole-copper (36.1
mg mLꢀ1) and bismerthiazol (49.5 mg mLꢀ1). The observed
antibacterial activity could be due to folding and deformation of
the bacterial cell membrane by the addition of compound as
observed using SEM. All these results support that the chalcone
derivatives containing 1,2,4-triazine moiety possess antibacte-
rial effects, and thus could be further studied as potential
alternative templates in the search for novel antiviral and
antibacterial agents.
Conflicts of interest
The authors declare that they have no competing interests.
25 V.
S.
Verma,
Zentralbl.
Bakteriol.,
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The authors gratefully acknowledge the nancial support of the
National Key Research and Development Program of China (No.
2017YFD0200506) and Science and Technology Project of
Guizhou Province (No. 20185781, 20171028).
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