Natural Products for Drug Discovery: Discovery of Gramines as Novel Agents against a Plant Virus

Article abstract of DOI:10.1021/acs.jafc.8b06859

Plant viral diseases seriously affect crop yield and quality. The natural product gramine (1) and its simple structural analogues 2-35 were synthesized from indoles, amines, and aldehydes in one step. The antiviral effects of these alkaloids were evaluated systematically. Most of these compounds were found to have higher antiviral effects than commercial ribavirin for the first time. Especially compounds 22, 30, and 31 exhibited significantly higher effects than ningnanmycin, thereby emerging as novel antiviral leads for further optimization. The preliminary implementation indicated that these compounds likely inhibit the assembly of tobacco mosaic virus (TMV) by cross-linking TMV capsid protein. Gramine analogues were also found to have broad-spectrum fungicidal effects. Although gramine has been reported to have influence on germination and development of Erysiphe graminis, these compounds displayed no fungicidal effects against Blumeria graminis f. sp. tritici on wheat in our test. Some of these compounds also exhibited certain insecticidal activities.

Full text of DOI:10.1021/acs.jafc.8b06859

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Agricultural and Environmental Chemistry
Natural Products for Drug Discovery: Discovery of
Gramines as Novel Agents against a Plant Virus
Aidang Lu, Tienan Wang, Hao Hui, Xiaoye Wei, Weihao Chui, Chunlv Zhou,
Hongyan Li, Ziwen Wang, Jincheng Guo, Dejun Ma, and Qingmin Wang
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 07 Feb 2019
Downloaded from http://pubs.acs.org on February 7, 2019
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Journal of Agricultural and Food Chemistry
Natural Products for Drug Discovery: Discovery of Gramines as Novel
Agents against a Plant Virus
Aidang Lu†,*, Tienan Wang†, Hao Hui†, Xiaoye Wei†, Weihao Cui†, Chunlv Zhou†,
Hongyan Li†, Ziwen Wang‡,*, Jincheng Guo§, Dejun Ma§, Qingmin Wang§,*
†School of Chemical Engineering and Technology, Hebei University of Technology,
Hebei Collaborative Innovation Center of Modern Marine Chemical Technology, Tianjin 300130,
China
‡Tianjin Key Laboratory of Structure and Performance for Functional Molecules, MOE Key
Laboratory of Inorganic–Organic Hybrid Functional Material Chemistry, College of Chemistry,
Tianjin Normal University, Tianjin Normal University, Tianjin 300387, China;
§State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic
Chemistry, College of Chemistry, Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Nankai University, Tianjin 300071, China
* To whom correspondence should be addressed. For Aidang Lu, E-mail: luaidang@hebut.edu.cn;
Phone:
0086-22-60302812;
Fax:
0086-22-60204274;
For
Ziwen
Wang,
E-mail:
hxxywzw@tjnu.edu.cn; Phone: 0086-22-23766531; Fax: 0086-22-23766531; For Prof. Qingmin
Wang, E-mail: wangqm@nankai.edu.cn; Phone: 0086-22-23503952; Fax: 0086-22-23503952.
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ABSTRACT: Plant viral diseases seriously affect crop yield and quality. Natural product
gramine (1) and its structure simple analogues 2–35 were synthesized from indoles,
amines and aldehydes by one-step. The antiviral effects of these alkaloids were evaluated
systematically. Most of these compounds were found to have higher antiviral effects than
commercial ribavirin for the first time. Especially for compounds 22, 30 and 31 exhibited
significantly higher effects than ningnanmycin, since emerged as novel antiviral leads for
further optimization. The preliminary implementation indicated that these compounds
likely inhibit the assembly of TMV by crosslinking TMV CP. Gramine analogues were
also found to have broad-spectrum fungicidal effects. Although gramine has been
reported to have influence on germination and development of Erysiphe graminis, these
compounds displayed no fungicidal effects against Blum eria graminis f. sp.tritici on
wheat in our test. Some of these compounds also exhibited certain insecticidal activities.
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KEYWORDS: natural product, gramine analogues, anti-TMV activity, fungicidal
activity
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INTRODUCTION
In the past 60 years, the population has more than doubled. The demand for more
fast increasing food production is needed.1,2 Plant diseases can cause a significant
economic and environmental impact on agricultural communities. Tobacco Mosaic Virus
(TMV), named after its first discovery in tobacco, is a well-studied plant virus. It is
reported that TMV can infect more than 400 crops including tobacco, pepper, cucumber,
banana and ornamental flowers.3 Ningnanmycin (Figure 1), perhaps the most effective
antiviral agent, only gave 50-60% control effects at 500 μg/mL. Ribavirin (Figure 1),
currently widely used antiviral agent, conveys an anti-TMV effect of less than 50% at
500 μg/mL. In fact, once a plant is infected with TMV, there is no agent to cure it
completely. Therefore, it is urgently needed to find agents with novel structures and
unique mechanisms of action.4
Nitrogen-containing heterocyclic compounds often exhibit a variety of biological
activities and are widely concerned in the field of medicine and pesticides. Among them,
compounds containing indole skeleton have aroused great interest of chemists,5 and have
been reported to have various kinds of activities, such as antibacterial activity, anticancer
activity, antiviral activity, protein kinase inhibitory effect.6-12 Gramine (Figure 1) is an
indole alkaloid widely found in raw plants and in coal tar.12-14 This alkaloid has various
kinds of activities, such as regulation of bronchial smooth muscle activity, regulation of
vasodilation activity and controlling blood pressure activity.15 It can control
mitochondrial energy metabolism in rat liver and beef heart and displayed weak
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acetylocholinesterase inhibitory effect.16,17 Recent studies have shown that gramine can
act as a 5-HT2A receptor and thus exhibits vasodilatory activity.15,18 In agriculture,
gramine was reported to have influence on germination and development of Erysiphe
graminis.19 However, there is no report on the antiviral activity of gramine in plants.
In our previous work, indole alkaloid topsentin A (Figure 1) and its derivatives,20
were found to have good anti-TMV activities. As another indole-containing natural
alkaloid, gramine has more simple structure than topsentin A. As a continuation of our
work, various of gramine analogues were designed (Figure 2), synthesized and
systematically evaluated for their antiviral, anti-phytopathogenic fungi and insecticidal
activities.
MATERIALS AND METHODS
Instruments. The melting points of the target compounds were determined on an X-4
binocular microscope (Gongyi Yuhua Instrument Co., China). NMR spectra were
acquired with a Bruker 400 MHz (100 MHz for 13C) instrument at room temperature.
Chemical shifts were measured relative to residual solvent peaks of CDCl3 (1H: δ = 7.26
13
ppm; C: δ = 77.0 ppm) with tetramethylsilane as internal standards. HRMS data were
obtained with an FT-ICR MS spectrometer (Ionspec, 7.0 T). Analytical TLC was
performed on silica gel GF 254. Column chromatographic purification was performed
using silica gel. The in vitro TMV rod assembly inhibition and 20S CP Disk assembly
inhibition were tested on transmission electron microscopy (Tecnai G2 F20).
General Synthesis. The synthetic route is given in Figure 3.
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The detailed operation steps and experimental data of gramines 1–35 can be found in
the Supporting Information.
Biological Assay. Each group of tests was repeated three times at 25±1 °C. Activity
results were given as a percentage scale of 0−100 (0: no activity; 100: total inhibited).
Detailed bioassay procedures for the anti-TMV,21 fungicidal22-24 and insecticidal22
activities were described in literature, also can be seen in Supporting Information.
Mode of Action Studies.
In vitro TMV rod assembly inhibition：TMV was purified by Leberman method.25
TMV RNA purification was performed by RNApure virus kit (CoWin Biosciences) and
TMV capsid protein (TMV CP) was purified by the acetic acid method.26 Before
assembling, 20S CP Disk was obtained by hatching CP (20 mg/mL) in pH 7.0 phosphate
buffer (0.1 M) at 20 °C for 12 h. Then, in vitro TMV assembly experiments were carried
out by successive adding the phosphate buffer 5 μL (0.1 M, pH 7.0), 20S Disk 4 μL (2
mg/mL) and TMV RNA 1 μL (200 ng/μL). The mixture was hatched at 20 °C for 12 h
and could be then transferred into the copper grid for transmission electron microscopy
(TEM) assay. The assembly reaction mixture (5 μL) was mixed with 0.1 M phosphate
buffer 5 μL (pH 7.0) and dropped onto the copper film waiting for 5 minutes. After the
incubation, the droplet was removed by filter paper and negatively stained by 2%
phosphotungstic acid (pH 7.0) for three minutes. After removing the staining agent, the
copper was placed at 37 ℃ for 2 h for drying. The morphology of the reconstituted TMV
rods was imaged at 200 keV on a CCD camera. For the inhibition experiments with the
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target compounds, in vitro TMV reconstitution inhibition experiments were performed by
successive adding phosphate buffer 4.8 μL (0.1 M, pH 7.0), 20S Disk 4 μL (2 mg/mL),
TMV RNA 1 μL (200 ng/μL) and DMSO 0.2 μL or the target compound (10 μM). Repeat
each experiment at least three times to ensure the reliability of the data.
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In vitro 20S CP Disk assembly inhibition: For the inhibition tests with the compounds,
TMV CP was first adjusted with 0.1 M phosphate buffer (pH 7.0) to 20.4 mg/mL. In vitro
20S CP Disk assembly experiments were carried out by adding 9.8 μL TMV CP (20.4
mg/mL) and 0.2 μL DMSO or the compound (10 μM). The assembly experiment was
hatched at 20 °C for 12 h. The morphology of the 20S CP Disk was imaged via TEM at
200 keV on a CCD camera. Repeat each experiment at least three times to ensure the
reliability of the data.
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RESULTS AND DISCUSSION
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Chemistry.
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Synthesis of the Compounds 1−35. Indole group was selected as template for structural
diversification. By introducing a series of functional groups into the side chain, we
investigated the effects of electronic effect, steric hindrance, hydrophilicity and
lipophilicity on the activity. The Mannich reaction in the presence of either protic or
Lewis acids14 is considered as powerful carbon-carbon bond forming processes to afford
3-substituted indoles. Compounds 1−35 were prepared by using the depicted procedures
in Figure 3. Substituted indoles were reacted with corresponding aldehydes and amines to
obtain 1−35 in 53%−97% yields. As the new compounds, 7−9, 12, 14−16, 18 and 24−35
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were identified by nuclear magnetic resonance (NMR) and high-resolution mass
spectrometer (HR-MS). Other known compounds were compared with literature.
Compound 22 is a known compound, but no physical properties available. So the NMR
and data of HR-MS of 22 were also given.
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Phytotoxic Activity. The phytotoxic activities of compounds 1−35 were first tested by
using the test plants which indicated that compounds 1−35 showed no phytotoxic
activities at 500 μg/mL. No local lesion appear on the plant leaves. The detailed test
procedures also can be seen in Supporting Information.
Antiviral Activity. The in vitro and in vivo anti-TMV activities of gramine and its
analogues were listed in Tables 1 and 2. The commercial plant virucides ribavirin and
ningnanmycin were selected as the controls.
In Vitro Anti-TMV Activity. Gramine analogues 1−35 were first tested in vitro
anti-TMV activities using the conventional half-leaf method.21 As shown in Table 1,
compounds 2, 4, 8−10, 15, 17, 22, 27, 30, 31 and 35 displayed significantly higher
antiviral activities than ribavirin. Among the compounds, 22, 30 and 31 exhibited higher
TMV inhibition effects than ningnanmycin. The mainly difference among 1−4 lies in the
substituents in indole ring. Gramine analogues 2−4 displayed significantly higher
antiviral activities than gramine (1), which showed that the introduction of substituent
groups at 1-position, 5-position and 6-position of indole ring are favorable for antiviral
activity. Bromination of 5-position is more favorable than 6-position (antiviral effect: 2 >
3). Gramine analogues 5−12 displayed higher antiviral activities than gramine (1), further
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revealed that the substituents of N,N-dimethylmethanamine region in gramine are
favorable for antiviral activity. Among analogues 5−12, compounds 8, 9 and 10 exhibited
much higher antiviral activities than the others, which indicated that the substituents of
N,N-dimethylmethanamine region in gramine exhibited synergistic effect. The mainly
difference among 6, 13 and 19−21 lies in the replacement of N,N-dimethyl group in
gramine. Compound 13 exhibited higher TMV inhibitory effects than the others, which
showed that the introduction of 2-amine pyridine group is favorable for antiviral activity.
Among derivatives 13−18, compounds 15 and 17 exhibited much higher antiviral
activities than the others. Based on the above structure-activity relationships, 5-bromine
and 1-substituent gramine analogues 22−35 were further designed. Among derivatives
22−24, compound 22 exhibited much higher TMV inhibitory effects than the others,
which indicated that the methylation of gramine is favorable for antiviral activity. The
mainly difference among 25−35 lies in the aryl substituents. Compounds 25, 26, 28, 29,
32, and 34 showed similar level antiviral activities with ribavirin. Compound 33
displayed relatively lower antiviral activity than the others. Gramine analogues 27, 30, 31
and 35 exhibited significantly higher antiviral activities than ribavirin, especially for 30
and 31 (antiviral effect: 30 and 31 > ningnanmycin).
In Vivo Anti-TMV Activity. The in vivo antiviral tests including inactivation activity,
curative activity and protection activity against TMV is carried out using reported
method.21 As depicted in Table 2, target compounds also showed higher in vivo TMV
inhibitory effects than ribavirin. Compounds 22, 30 and 31 showed significantly higher
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TMV inhibitory effects than ningnanmycin, since emerged as novel antiviral leads for
further optimization. Unlike in vitro anti-TMV test results, gramine analogues 1, 4−6, 14,
15 and 18 exhibited relatively higher in vivo TMV inhibitory effects than in vitro. The
protection effects of compounds 15, 17 and 30 relatively higher than inactivation effect
and curative effect, which indicated that compounds 15, 17 and 30 may have a certain
inducible antiviral activities. The other structure-activity relationships in vivo are similar
to that of in vitro.
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Preliminary Mode of Action. The preliminary mode of action of compounds 15 and 22
were evaluated via TEM with RNA inhibitor antofine27 and CP disks assembly inhibitor
NK020928 as controls. The TMV is 300 nm length rod-shaped particle. It contains a
single-stranded RNA consisting of about 6400 nucleotides. Its coat protein contains 2130
protein subunits, each of which is composed of 158 amino acids and spirally arranged
around RNA molecules.29 The test results revealed that 20S CP Disk and TMV RNA can
assemble into TMV rod effectively (Figure 4, B). The use of a small amount of DMSO
does not affect assembly (Figure 4, C). Compounds 15, 22, antofine and NK0209 can
significantly inhibit the assembly of TMV rod (Figure 4, D−G). Further 20S CP Disk
assembly inhibition tests were carried out to evaluate the interaction of these compounds
with TMV CP. The 20S CP Disk can obtained by incubation of TMV CP at 20 °C for 12
h (Figure 5, A). The use of a small amount of DMSO has no impact on assembly of 20S
CP Disk (Figure 5, B). As the TMV RNA inhibitor, antofine displayed no impact on 20S
CP Disk assembly
(Figure 5, C). CP disks assembly inhibitor NK0209 showed
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significantly impact on 20S CP Disk. Although the 20S CP Disk can assemble efficiently,
aggregation and fusion of a large number of CP Disks was detected (Figure 5, D). Just
like NK0209, compounds 15 and 22 also can effectively induce CP Disks aggregation
and fusion (Figure 5, E, F). Compound 22 with higher in vivo TMV inhibitory effect than
15 did exhibit stronger polymerization-promoting ability (As shown in Figure 5, the
number of 20S CP Disk in F is significantly lower than that in E). The above results
indicated that these compounds likely inhibit virus assembly by crosslinking TMV CP.
Fungicidal Activity. Gramines 1−35 were simultaneously determined for fungicidal
activity with chlorothalonil and azoxystrobin as controls.
In Vitro Fungicidal Activity. The in vitro fungicidal effects of gramines 1−35 were
obtained by using mycelial growth tests22 with 14 plant pathogens as artificial media.
Gramines 1−35 exhibited broad-spectrum fungicidal activities (Table 3). What’s more,
these compounds displayed high bioselective. Compounds 17 and 23 displayed about
similar level antifungal activities against Alternaria solani with chlorothalonil. Most of
these compounds showed good antifungal activities against Phytophthora capsici and
Physalospora piricola, especially for compounds 17 and 30 (antifungal activity against
Phytophthora capsici: 30 > 90%; antifungal activity against Physalospora piricola: 17
and 30 ≥ 90%). Compound 8 displayed more than 60% antifungal activity against
Rhizoctonia cerealis.
In Vivo Fungicidal Activity. Gramines 1−35 were further tested fungicidal activities in
vivo.22 The pathogens tested in this screen were Sclerotinia sclerotiorum on rape,
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Rhizoctonia cerealis on cerealis, Botrytis cinerea on cucumber, Phytophthora capsici on
capsici, Corynespora cassiicola on cucumber with protection and Blum eria graminis f.
sp.tritici on wheat. The results (Table 3) showed that lots of compounds displayed more
than 30% inhibitory effect. Although gramine has been reported to have influence on
germination and development of Erysiphe graminis,19 all compounds displayed no
fungicidal activity against Blum eria graminis f. sp.tritici on wheat in our test.
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Insecticidal Activity. Some compounds showed certain insecticidal activity. Compounds
13, 22 and 32 exhibited broad-spectrum insecticidal activities against Mythimna separate,
Helicoverpa armigera, Ostrinia nubilalis and Culex pipiens pallens.
In summary, natural product gramine and its derivatives were prepared and tested for
their TMV inhibitory effects for the first time. Most of these structure simple compounds
showed higher TMV inhibitory effects than ribavirin. Compounds 22, 30 and 31 with
significantly higher TMV inhibitory effects than ningnanmycin emerged as new antiviral
leads for further research. The introduction of substituents at 1-position, 5-position and
6-position of indole ring are favorable for TMV inhibitory effect. Bromination of
5-position is more favorable than 6-position. Compounds 15, 17 and 30 may have a
certain inducible antiviral activities. The preliminary mode of action studies revealed that
these compounds likely inhibited TMV assembly by crosslinking TMV CP. Further
fungicidal activity test showed that these compounds exhibited broad-spectrum fungicidal
activity. Compounds 17 and 23 displayed about similar level antifungal activities against
Alternaria solani with chlorothalonil. Insecticidal activity bio-assay showed that gramine
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and its derivatives also displayed a certain insecticidal activity. Current study provides
strong data support for the application of gramines in plant protection.
ASSOCIATED CONTENT
Supporting Information
The detailed bio-assay procedures. The preparation procedures and the spectra data of
compounds 1−35. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(A.L.)
0086-22-60204274
*(Z.W.) E-mail:
0086-22-23766531.
E-mail:
luaidang@hebut.edu.cn;
hxxywzw@tjnu.edu.cn;
Phone:
Phone:
0086-22-60302812;
0086-22-23766531;
Fax:
Fax:
*(Q.W.) E-mail: wangqm@nankai.edu.cn. Phone: 0086-22-23503952. Fax:
0086-22-23503952
Funding
This study was supported by Natural Science Fund of China (21772145, 21732002,
21672117, 21501042).
Notes
The authors declare no competing financial interest.
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(19) Wippich, C.; Wink, M. Biological properties of alkaloids. Influence of quinolizidine
alkaloids and gramine on the germination and development of powdery mildew,
Erysiphe graminis f. sp. Hordei. Experientia 1985, 41, 1477–1479.
(20) Ji, X. F.; Wang, Z. W.; Dong, J.; Liu, Y. X.; Lu, A. D.; Wang, Q. M. Discovery of
topsentin alkaloids and their derivatives as novel antiviral and anti-phytopathogenic
fungus agents. J. Agric. Food Chem., 2016, 64, 9143− 9151.
(21) Wang, Z. W.; Wei, P.; Wang, L. Z.; Wang, Q. M. Design, synthesis, and
anti-tobacco mosaic virus (TMV) activity of phenanthroindolizidines and their
analogues. J. Agric. Food Chem. 2012, 60, 10212−10219.
(22) Zhao, H. P.; Liu, Y. X.; Cui, Z. P.; Beattie, D.; Gu, Y. C.; Wang, Q. M. Design,
synthesis, and biological activities of arylmethylamine substituted chlorotriazine and
methylthiotriazine compounds. J. Agric. Food Chem. 2011, 59, 11711−11717.
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295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
(23)Lv, P.; Chen, Y. L.; Zhao, Z.; Shi, T. Z.; Wu, X. W.; Xue, J. Y.; Li, Q. X.; Hua, R. M.
Design, synthesis, and antifungal activities of 3-acyl thiotetronic acid derivatives:
New fatty acid synthase inhibitors. J. Agric. Food Chem. 2018, 66, 1023–1032.
(24)Lv, P.; Chen, Y. L.; Shi, T. Z.; Wu, X. W.; Li, Q. X.; Hua, R. M. Synthesis and
fungicidal activities of sanguinarine derivatives. Pestic. Biochem. Phys. 2018, 147,
3–10.
(25) Leberman, R. Isolation of plant viruses by means of simple coacervates. Virol 1966,
30, 341–347.
(26) Fraenkel Conrat, H.; Williams, R. C. Reconstitution of active tobacco mosaic virus
fromits inactive protein and nucleic acid components. Proc Natl Acad Sci U S A
1955, 41, 690–698.
(27) Xi, Z.; Zhang, R. Y.; Yu, Z. H.; Ouyang, D. The interaction between tylophorine B
and TMV RNA. Bioorg. Med. Chem. Lett. 2006, 16, 4300–4304.
(28) Li, X. Y.; Hao, G. F.; Wang, Q. M.; Chen, Z.; Ding, Y.; Yu, L.; Hu, D. Y.; Song, B.
A. Ningnanmycin inhibits tobacco mosaic virus virulence by binding directly to its
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(29) Butler, P. J. G. The current picture of the structure and assembly of tobacco mosaic
virus. J. Gen. Virol. 1984, 65, 253–279.
Figure Captions
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Journal of Agricultural and Food Chemistry
Figure 1. Structures of Ningnanmycin, Ribavirin, Gramine and Topsentin A.
Figure 2. Design of Gramine Analogues.
Figure 3. Synthesis of Gramine Analogues 1–35.
Figure 4. TMV Rod Assembly Inhibition of Compounds 15, 22, NK0209 and Antofine.
(A) 20S CP Disk (50 nm scale bar); (B) 20S CP Disk + RNA (100 nm scale bar); (C)
20S CP Disk + RNA + DMSO (200 nm scale bar); (D) 20S CP Disk + RNA + antofine
(200 nm scale bar); (E) 20S CP Disk + RNA + NK0209 (200 nm scale bar); (F) 20S CP
Disk + RNA + 15 (200 nm scale bar); (G) 20S CP Disk + RNA + 22 (100 nm scale
bar).
Figure 5. 20S CP Disk Assembly Inhibition of Compounds 15, 22, NK0209 and Antofine
(100 nm scale bar). (A) CP; (B) CP + DMSO; (C) CP + antofine; (D) CP + NK0209; (E)
CP + 15; (F) CP + 22.
Table 1. In Vitro Antiviral Activities of Compounds 1−35, Ribavirin and Ningnanmycin
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against TMV.
Concn
(μg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
Inhibition
compd
Concn
(μg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
Inhibition
rate (%)a
16±1
0
Compd
rate (%)a
23±1
20
1
2
0
51±2
21
30±2
0
20±2
39±1
22
61±2
27±1
35±3
10±2
40±1
18±2
45±1
18±1
39±3
11±2
54±2
19±1
43±2
16±1
46±2
15±2
62±1
29±1
67±2
31±2
44±1
18±2
30±1
0
3
5±1
48±2
23
4
31±1
33±2
24
5
4±1
25±2
25
6
0
43±1
26
7
16±2
52±2
27
8
23±2
49±1
28
9
15±1
51±2
29
10
11
12
13
14
15
16
17
19±1
35±2
30
0
46±1
31
14±1
36±2
32
0
35±2
33
13±1
52±2
34
42±2
16±2
51±1
20±2
41±1
15±2
58±2
23±2
17±1
36±1
35
5±2
50±1
Ribavirin
22±2
46±1
18
19
Ningnanmycin
13±2
19±2
aAverage of three replicates; All results are
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Journal of Agricultural and Food Chemistry
expressed as mean ± SD.
100
0
Table 2. In Vivo Antiviral Activities of Compounds 1−35, Ribavirin and Ningnanmycin
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against TMV.
Concn
Inactivation
effect (%)a effect (%)a effect (%)a
Curative
Protection
Concn
Inactivation
effect (%)a
21±2
0
Curative
effect (%)a effect (%)a
Protection
Compd
Compd
(μg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
(μg/mL)
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
500
100
32±1
0
26±1
0
37±2
0
18±2
0
26±1
0
1
2
20
57±1
29±1
42±2
6±2
53±2
22±1
47±2
12±1
53±2
29±1
42±2
7±1
51±1
27±1
41±2
8±1
30±2
0
31±1
0
30±2
6±2
21
64±2
30±2
32±1
0
60±1
31±2
29±2
0
65±2
35±1
35±2
0
3
22
59±2
27±1
46±2
12±1
33±2
0
54±2
33±1
39±2
17±2
31±2
0
4
23
41±2
12±2
48±3
18±2
40±2
11±2
57±1
21±2
47±2
22±2
45±1
18±2
61±2
28±1
68±2
34±2
44±3
11±2
32±1
0
38±1
8±2
45±2
13±2
44±2
16±1
41±2
16±2
56±1
23±2
46±2
27±1
42±2
16±1
65±2
33±1
66±2
35±2
40±2
12±1
26±2
0
5
24
32±2
0
42±1
14±2
38±1
7±2
6
25
52±1
26±2
57±2
31±1
45±4
7±1
45±1
16±1
53±1
28±1
48±2
9±1
41±2
9±1
7
26
61±1
23±1
41±2
13±1
49±2
27±1
36±2
10±1
34±2
0
52±2
24±2
44±3
20±1
40±2
12±1
59±1
27±2
65±1
32±1
45±1
15±2
28±1
0
8
27
9
28
55±1
13±2
43±1
6±1
51±1
24±2
36±1
8±1
10
11
12
13
14
15
16
17
29
30
41±1
6±2.0
36±1
0
39±2
11±2
34±2
0
31
38±2
0
32
44±2
10±1
60±2
13±1
43±2
10±2
56±4
15±2
53±1
29±2
33±2
48±1
21±1
55±2
19±2
47±1
18±1
52±2
20±1
51±2
31±1
27±2
42±2
15±1
64±1
32±1
39±2
6±1
33
34
41±3
12±2
52±2
20±1
38±1
11±1
58 ± 1
27 ± 2
37±3
15±2
50±3
18±2
36±2
14±1
56 ± 1
25 ± 2
42±2
17±1
53±2
23±2
39±1
10±1
58 ± 2
30 ± 1
35
60±1
25±1
58±1
25±1
23±2
Ribavirin
Ningnanmycin
18
19
a Average of three replicates; All results are expressed as mean ± SD.
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Journal of Agricultural and Food Chemistry
100
0
0
0
Table 3. In Vitro Fungicidal Activities of Compounds 1–35 and Chlorothalonil against 14
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Kinds of Fungi.
Fungicidal activities (%)a/ 50 μg/mL
Compd
A.Sb
26±1
30±1
35±2
30±1
22±1
30±2
43±1
26±1
30±1
26±1
39±1
43±2
22±2
26±1
13±2
52±1
61±2
B.Cb
12±1
19±2
15±2
19±2
20±2
29±1
32±1
7±1
B.Mb
6±1
C.Hb
7±2
F.Cb
14±1
22±1
17±1
14±1
22±1
25±1
25±1
22±1
19±2
19±2
22±1
30±1
28±1
25±1
22±1
42±1
39±1
F.Gb
27±1
50±1
35±2
31±2
12±2
15±1
12±1
11±2
8±1
F.Mb
25±1
21±1
25±1
21±2
4±1
P.Cb
7±2
P.Ib
P.Pb
28±1
69±1
19±1
28±1
33±1
58±1
33±2
25±1
58±1
44±1
33±1
69±1
56±2
28±1
53±1
64±1
92±1
R.Cb
13±2
42±1
41±1
18±1
34±2
25±1
35±1
63±1
32±1
32±1
41±2
41±1
32±2
35±2
25±1
42±1
35±2
R.Sb
12±1
46±2
25±1
12±1
12±1
12±1
46±1
8±1
S.Sb
W.Ab
18±1
33±1
37±2
18±1
37±1
30±1
18±2
26±1
26±2
15±1
19±2
26±2
22±2
33±1
30±2
33±1
30±2
1
2
14±1
38±1
38±1
19±1
28±2
24±2
14±1
14±1
24±1
14±2
28±2
28±2
24±1
24±1
24±1
47±2
47±2
14±2
24±1
19±1
29±2
29±2
24±1
29±1
33±1
33±1
29±2
33±1
33±1
24±1
19±1
14±1
12±1
38±1
18±1
15±1
15±1
18±1
18±1
15±1
18±1
21±1
18±1
18±1
21±1
21±1
21±1
24±1
33±2
27±2
11±1
11±1
11±1
7±2
52±1
52±1
45±1
24±1
20±1
62±1
17±1
41±1
24±2
62±1
62±2
41±1
17±1
24±1
55±1
45±1
3
4
5
6
15±1
18±1
11±1
18±1
4±1
8±1
7
17±2
12±2
8±1
8
9
15±1
12±1
12±1
37±2
20±1
12±1
12±1
49±1
58±2
33±1
21±2
8±1
10
11
12
13
14
15
16
17
19±1
27±1
15±1
8±2
17±1
12±1
25±1
4±1
18±1
33±1
15±1
30±2
18±2
33±1
37±1
37±1
4±1
15±1
4±1
12±1
12±1
12±1
29±1
33±1
4±1
38±1
46±1
46±2
58±1
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Journal of Agricultural and Food Chemistry
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
35±2
17±2
56±2
43±1
35±2
62±2
55±2
16±1
19±2
11±1
23±2
18±2
51±1
10±2
31±1
21±1
26±2
32±2
73±2
42±1
32±1
44±1
37±1
44±1
50±2
46±1
25±2
33±2
16±2
25±1
32±2
45±1
9±1
24±1
24±1
36±2
33±2
30±1
37±1
32±2
30±1
28±2
22±1
21±2
25±1
39±2
15±2
28±2
32±2
30±1
32±2
91±1
30±1
18±1
18±1
15±1
19±2
36±2
28±2
14±2
34±2
15±2
19±2
31±2
33±1
33±2
34±1
23±1
25±2
29±2
73±2
28±2
22±1
28±1
25±1
25±2
45±1
35±1
24±2
42±2
26±2
22±1
42±2
42±2
24±2
51±2
31±2
21±1
22±1
100
15±2
11±2
39±1
11±1
15±1
38±2
29±2
10±2
19±2
10±1
9±2
29±1
12±1
12±1
12±2
13±2
32±2
26±1
12±1
28±2
13±2
11±2
22±1
23±2
15±2
12±2
11±2
13±1
19±2
100
62±1
27±1
58±2
42±1
58±1
82±2
75±2
52±2
62±2
58±2
55±1
51±2
92±2
42±2
72±1
62±1
65±2
76±1
100
14±1
14±1
62±1
38±1
40±2
53±1
42±1
15±1
28±2
16±1
24±2
26±2
41±1
20±2
32±2
26±2
32±1
38±2
86±1
36±1
50±1
58±1
36±2
64±2
86±2
74±2
53±2
67±1
56±2
46±1
55±2
90±1
38±1
68±2
63±2
59±2
72±2
100
31±1
17±2
31±1
21±1
25±1
42±2
38±1
25±1
38±2
21±1
23±1
26±2
30±1
12±2
24±1
21±1
22±1
26±1
100
42±1
12±2
46±1
42±1
43±2
43±1
35±1
30±2
42±2
13±2
31±1
34±2
32±2
11±2
23±1
16±2
17±2
19±2
100
24±1
14±1
29±1
24±2
33±2
32±2
28±1
22±2
33±2
19±1
11±2
10±1
37±1
23±2
8±2
26±1
26±1
33±1
30±2
35±1
37±2
29±2
26±2
30±2
26±2
9±1
18±2
20±1
8±1
25±2
39±2
12±2
11±1
14±1
10±2
26±1
86±1
25±2
15±2
24±1
22±1
100
21±2
9±2
13±2
9±1
14±1
16±2
<50
24±2
<50
Chlorothalonilc
b
aAverage of three replicates; All results are expressed as mean ± SD. A.S, Alternaria solani; B.C, Botrytis cinerea.;
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B.M, Bipolaris maydis; C.H, Cercospora arachidicola Hori; F.C, Fusarium oxysporium f. sp. cucumeris; F.G,
Fusarium graminearum; F.M, Fusarium moniliforme; P.C, Phytophthora capsici; P.I, Phytophthora infestans; P.P,
Physalospora piricola; R.C, Rhizoctonia cerealis; R.S, Rhizoctonia solani; S.S, Sclerotinia sclerotiorum; W.A,
watermelon anthracnose. c The commercial agricultural fungicide was used for comparison of antifungal activity.
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Journal of Agricultural and Food Chemistry
Table 4. In Vivo Fungicidal Activities of Compounds 1–35 and Azoxystrobin against 6
Kinds of Fungi.
Inhibition rate (%)b/ 200 μg/mL
compd
S.S a
20±2
18±2
16±1
16±2
15±2
16±2
11±1
9±2
R.C
B.C
P.C
C.C
B.G
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
22±1
22±2
19±2
15±2
18±1
22±2
30±2
13±1
12±2
16±1
22±2
19±2
8±2
28±2
11±2
23±2
16±2
13±2
19±1
16±2
19±2
28±2
25±2
8±1
26±2
21±1
15±2
27±1
31±2
23±2
17±2
16±2
23±1
17±2
35±2
39±2
11±1
9±2
32±1
25±2
37±2
13±2
28±2
35±1
36±2
18±1
16±2
15±2
12±2
40±2
16±2
28±2
17±1
41±2
41±2
12±2
28±1
15±2
21±1
12±2
35±1
26±2
26±1
39±2
22±1
1
2
3
4
5
6
7
8
9±2
9
11±2
29±2
26±1
12±2
28±1
25±2
9±2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
20±1
12±2
23±1
19±2
27±2
19±2
28±2
19±1
19±2
19±1
19±2
20±1
15±2
17±1
25±2
13±1
8±2
27±2
32±1
21±2
27±1
12±2
23±1
11±2
15±1
33±2
22±1
22±2
32±1
22±2
15±2
35±2
45±2
11±1
21±2
11±1
13±2
20±1
43±2
26±1
25±2
36±1
15±2
18±2
29±2
11±1
18±2
9±1
20±2
27±1
11±2
25±1
21±2
15±1
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11±2
26±1
21±2
9±1
20±1
12±2
12±1
12±2
29±1
23±2
22±1
25±2
27±1
25±2
17±1
25±2
27±1
18±2
22±1
23±2
36±1
15±2
36±1
35±2
26±1
21±2
16±1
46±2
16±1
36±2
38±1
16±2
0
0
0
0
0
0
0
28
29
30
31
32
33
34
21±2
25±1
16±2
15±1
100
22±2
100
19±1
100
15±2
85±2
29±1
80±1
0
35
azoxystrobinc
81±2
a
S.S, Sclerotinia sclerotiorum (rape-protection); R.C, Rhizoctonia cerealis; B.C, Botrytis cinerea.
(cucumber-protection); P.C, Phytophthora capsici; C.C, Corynespora cassiicola (cucumber-protection); B. G, Blum
eria graminis f. sp.tritici (wheat-protection). b Average of five replicates. c The dilution of azoxystrobin is 1000 times.
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Journal of Agricultural and Food Chemistry
Table 5. Larvacidal Activities of Compounds 1–35 and Rotenone against Oriental
Armyworm (Mythimna Separate), Cotton Bollworm (Helicoverpa Armigera), Corn Borer
(Ostrinia Nubilalis) and Mosquito (Culex Pipiens Pallens).
Larvacidal activities (motality %) at concn (μg/mL)
compd
M. Separat
H. armigera O. Nubilalis
C. Pipiens Pallens
600
0
600
0
600
0
10
0
1
2
0
0
0
0
3
0
5
20
35
25
25
25
30
0
20
40
20
80
40
20
20
60
20
20
80
40
4
20
20
0
35
15
20
10
15
0
5
6
7
0
8
15
0
9
10
11
12
13
14
0
20
0
20
5
0
20
30
0
20
45
15
10
50
10
27
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Journal of Agricultural and Food Chemistry
Page 28 of 32
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
0
10
0
5
0
20
20
40
20
20
20
30
35
40
30
20
30
35
30
10
60
45
60
0
15
10
10
5
15
10
15
0
20
0
20
0
10
5
25
10
35
25
15
0
20
30
15
0
30
15
10
0
0
30
10
15
0
10
15
15
0
20
10
0
20
10
30
20
0
30
20
45
10
0
45
30
30
0
0
0
28
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Page 29 of 32
Journal of Agricultural and Food Chemistry
35
20
30
75
10
60
50
Rotenone
100
100
Figure 1.
H
N
CONH2
O
O
NH2
N
C
O
N
H
N
O
N
N
O
HOH2C
N
N
N
N
NH2
HO
H
H
O
N
OH
CH2OH
N
N
H
N
HO
OH
H
H
O
topsentin A
ningnanmycin
gramine
ribavirin
Figure 2.
N
Br
N
N
R
N
N
H
N
H
gramine
22
gramine analogues
Novel and simple antiviral agents discovery
Hordeum vulgare L.
Figure 3.
29
ACS Paragon Plus Environment