Discovery, Structural Optimization, and Mode of Action of Essramycin Alkaloid and Its Derivatives as Anti-Tobacco Mosaic Virus and Anti-Phytopathogenic Fungus Agents

Article abstract of DOI:10.1021/acs.jafc.9b06006

Plant diseases seriously affect crop yield and quality and are difficult to control. Marine natural products (MNPs) have become an important source of drug candidates with new biological mechanisms. Marine natural product essramycin (1) was found to have

Full text of DOI:10.1021/acs.jafc.9b06006

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Agricultural and Environmental Chemistry
Discovery, Structural Optimization and Mode of Action of
Essramycin Alkaloid and Its Derivatives as Anti-Tobacco
Mosaic Virus (TMV) and Anti-phytopathogenic-Fungus Agents
tienan wang, shan yang, Hongyan Li, Aidang Lu, Ziwen Wang, Yingwu Yao, and Qingmin Wang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b06006 • Publication Date (Web): 16 Dec 2019
Downloaded from pubs.acs.org on December 17, 2019
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Page 1 of 53
Journal of Agricultural and Food Chemistry
Discovery, Structural Optimization and Mode of Action of Essramycin
Alkaloid and Its Derivatives as Anti-Tobacco Mosaic Virus (TMV) and
Anti-phytopathogenic-Fungus Agents
†
†
†
†,
‡,
†,
Tienan Wang , Shan Yang , Hongyan Li , Aidang Lu *, Ziwen Wang *, Yingwu Yao *,
§
,
Qingmin Wang *
†National-Local Joint Engineering Laboratory for Energy Conservation in Chemical Process Integration
and Resources Utilization, School of Chemical Engineering and Technology, Hebei University of
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 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;
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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 diseases seriously affect crop yield and quality and are difficult to
control. Marine natural products (MNPs) have become an important source of drug
candidates with new biological mechanisms. Marine natural product essramycin (1) was
found to have good anti-tobacco mosaic virus (TMV) and anti-phytopathogenic-fungus
activities for the first time. A series of essramycin derivatives were designed, synthesized
and evaluated for their bio-activity. Most of these compounds exhibited higher antiviral
effects than control ribavirin. Compounds 7e and 8f displayed higher antiviral activities
than ningnanmycin (the most widely used antiviral agent at present), thus emerged as novel
antiviral lead compounds. As the lead compound, 7e was selected for further antiviral
mechanism research. The results indicated that 7e could inhibit virus assembly and promote
20S disk protein aggregation. Fungicidal activity tests against 14 kinds of phytopathogenic
fungi revealed that essramycin analogues displayed broad-spectrum fungicidal activities.
Compound 5b displayed more than 50% inhibition rate against most of the 14 kinds of
phytopathogenic fungi at 50 μg/mL. Current research lays a solid foundation for the
application of essramycin alkaloids in crop protection.
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KEYWORDS: marine natural product, essramycin, alkaloid, anti-TMV activity,
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fungicidal activity, mode of action
INTRODUCTION
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Plant diseases caused by plant viruses and pathogens have been recognized as a
worldwide threat to the agricultural industry. Tobacco mosaic virus (TMV) is the first virus
to be found, which is considered the most destructive disease of plant in the world. Because
of its clear research, TMV is often used as a model virus in the development of antiviral
agents. It has been found that TMV can infect more than 400 crops. As the widely used
antiviral agents, ribavirin (less than 50% inhibitory effect at 500 μg/mL) and ningnanmycin
(50-60% inhibitory effect at 500 μg/mL) (Figure 1) only gave moderate anti-TMV effects.
How to control TMV is still a very challenging task.1
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Natural products are still an important area to provide drug candidates with novel
structure and unique mechanism.2-4 However, the shortcomings of limited compound
availability, poor solubility, and metabolic instability of natural products have always
3
limited their direct application as drugs. Till now, only a small amount of natural products
are used directly as anti plant virus agents.5-6 Structural optimization based on natural
products is an important method to overcome these shortcomings.7-13 Essramycin (Figure
1), obtained from the culture broth of the marine Streptomyces sp., is the first isolated
triazolopyrimidine natural product. The bio-activity research revealed that essramycin
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possesses antibacterial activity.14-16 As the core framework, triazolopyrimidine is a well-
known scaffold in agricultural and medicinal chemistry. These compounds have broad-
1
7
spectrum biological activities, such as anticancer activity , phosphodiesterase inhibition
activity18 and anti-tubercular activity . Triazolopyrimidine derivatives also have
19
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0
21
22
remarkable antibacterial activity , anti-epileptic activity , anti-mycobacterial activity ,
antimalarial activity23-25. As the widely used antiviral agent, ribavirin also contains triazole
skeleton. Research revealed that ribavirin triphosphate (RTP) can interact with various viral
RNA polymerases.26,27 Essramycin and ribavirin contain the same core structural unit
(Figure 1). Whether they have the same biological activity and mechanism of action is
worth to further study.
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Based on the above findings, natural product essramycin was selected as the parent
structure. A series of essramycin analogues were designed (Figure 2), synthesized and
evaluated for their anti-TMV and fungicidal activities. The preliminarily antiviral
mechanism research was also carried out by transmission electron microscope (TEM).
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MATERIALS AND METHODS
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Instruments and Chemicals. The melting points of the products were determined on an
X-4 binocular microscope (Gongyi Yuhua Instrument Co., China) and the thermometer
1
3
was not corrected. NMR spectra were acquired with a Bruker 400 MHz (100 MHz for C)
instrument at room temperature. Chemical shifts were measured relative to residual solvent
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1
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7
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0
1
peaks of CDCl ( H: δ = 7.26 ppm; C: δ = 77.0 ppm) or d -DMSO ( H: δ = 2.50 ppm;
3
6
13C: δ = 39.5 ppm) with tetramethylsilane as internal standards. The following
abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet,
t = triplet, m = multiplet, and bs = broad singlet. All first-order splitting patterns were
assigned on the basis of multiplet appearance. Splitting patterns that could not be easily
interpreted were designated multiplet (m) or broad (br). HRMS data were obtained with an
FT-ICR MS spectrometer (Ionspec, 7.0 T). Procedures for the preparation of compounds
1, 2, 2-(5-amino-1H-1,2,4-triazol-3-yl)acetic acid (3), methyl 5-amino-1H-1,2,4-triazole-
3-carboxylate (4b) and 5 (Figure 3) can been seen in Supporting Information. 5-Amino-
4H-1,2,4-triazole-3-carboxylic acid (4a, Figure 3) was bought from Bide Pharmatech Ltd.
All reagents were of analytical reagent grade or chemically pure. Among the synthesized
compounds, 1–6, 7a–7c, 8a, 8b, 8d, 8e, 8g, 9b, 9c and 9e are reported compounds and their
data were according with reference. The in vitro TMV rod assembly inhibition and 20S CP
disk assembly inhibition were tested via transmission electron microscopy (Tecnai G2 F20).
7
2
Procedures for the Preparation of Compounds 6, 7a–7c.15,16
7
7
7
7
3
4
5
6
The solution of corresponding 3-substituted ethyl acetoacetate (2 mmol, 1.0 equiv.) and 3,
4a or 4b (2 mmol, 1.0 equiv.) in CH COOH (20 mL) was refluxed for 10 h. The solvent
3
was evaporated in vacuo and the residue was purified by recrystallization with ether to give
compounds 6, 7a−7c.
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2-(5-Methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)acetic acid (6): white
1
solid, 97% yield; mp 257−259 °C; H NMR (DMSO-d , 400 MHz): 12.90 (br s, 2H, NH),
6
1
3
5.82 (s, 1H, C=CH), 3.73 (s, 2H, CH ), 2.32 (s, 3H, CH ); C NMR (DMSO-d , 100 MHz):
2
3
6
170.7, 159.2, 156.0, 151.8, 151.3, 98.7, 35.4, 19.1; HR-MS (ESI): Calcd for C H N O
3
8
9
4
+
+
[M+H] 209.0669, found (ESI ) 209.0672.
8
8
8
8
8
2
3
4
5
6
5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (7a): white solid, 65% yield; mp
1
266−268 °C; H NMR (DMSO-d , 400 MHz): 13.20 (br s, 1H, NH), 8.18 (s, 1H, N=CH),
6
1
3
5.82(s, 1H, C=CH), 2.31 (s, 3H, CH ); C NMR (DMSO-d , 100 MHz): 155.8, 151.8,
3
6
+
+
151.6, 150.6, 98.1, 18.6; HR-MS (ESI): Calcd for C H N O [M+H] 151.0614, found (ESI )
6
7
4
151.0611.
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8
8
9
9
9
7
8
9
0
1
2
5-Propyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (7b): white solid, 91% yield; mp
1
117−120 °C; H NMR (DMSO-d , 400 MHz): 13.08 (br s, 1H, NH), 8.20 (s, 1H, N=CH),
6
5.84 (s, 1H, C=CH), 2.55 (t, J = 7.5 Hz, 2H, CH CH CH ), 1.62−1.71 (m, 2H,
2
2
3
1
3
CH CH CH ), 0.90 (t, J = 7.3 Hz, 3H, CH ); C NMR (DMSO-d , 100 MHz): 156.0, 155.2,
2
2
3
3
6
+
151.9, 150.7, 97.6, 34.1, 21.2, 13.2; HR-MS (ESI): Calcd for C H N O [M+H] 179.0927,
8
11
4
+
found (ESI ) 179.0923.
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9
9
3
4
5
Methyl 5-methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-carboxylate (7c):
1
white solid, 98% yield; mp 232−234 °C; H NMR (DMSO-d , 400 MHz): 13.39 (br s, 1H,
6
1
3
NH), 5.94 (s, 1H, C=CH), 3.91 (s, 3H, OCH ), 2.35 (s, 3H, CCH ); C NMR (DMSO-d ,
3
3
6
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100 MHz): 160.0, 155.6, 153.0, 152.7, 151.1, 98.7, 52.6, 18.8; HR-MS (ESI): Calcd for
+
+
C H N O [M+H] 209.0669, found (ESI ) 209.0668.
8
9
4
3
9
9
8
9
Procedure for the Preparation of Compound 7d.
Compound 7c (0.84 g, 4 mmol, 1.0 equiv.) and hydrazine hydrate (80% solution in water,
1
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1
1
1
1
00
01
02
03
04
05
1.6 mL, 40 mmol, 10 equiv.) were dissolved in ethanol (20 mL). The mixture was refluxed
for 16 h. The suspension was cooled to room temperature and filtered. Compound 7d was
1
obtained as a white solid, 98% yield; mp 213−214 °C; H NMR (DMSO-d , 400 MHz):
6
9.79 (br s, 1H, CNHC=N), 7.27 (br s, 3H, NHNH ), 5.54 (s, 1H, C=CH), 2.20 (s, 3H, CH );
2
3
1
3
C NMR (DMSO-d , 100 MHz): 162.3, 159.7, 158.1, 157.9, 155.0, 95.4, 24.2; HR-MS
6
+
+
(ESI): Calcd for C H N O [M+H] 209.0781, found (ESI ) 209.0783.
7
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6
2
1
06
Procedure for the Preparation of Compound 7e.
1
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08
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To a solution of 7c (0.97 g, 5.0 mmol, 1.0 equiv.) in tetrahydrofuran (40 mL) and water (8
mL) was added lithium hydroxide hydrate (0.36 g, 15.0 mmol). The reaction mixture was
stirred at room temperature for 12 h, then the mixture was evaporated in vacuo and the
residue was diluted with ice water (20 mL) and adjusted to pH = 3 by 4 M HCl solution.
The solution was refrigerated at 0 °C for 10 h. Compound 7e was collected by filtration as
1
a white solid, 91% yield; mp 187−190 °C; H NMR (DMSO-d , 400 MHz): 13.40 (br s,
6
1
3
1H, NH), 5.90 (s, 1H, C=CH), 2.33 (s, 3H, CH ); C NMR (DMSO-d , 100 MHz): 160.4,
3
6
+
155.2, 153.5, 152.0, 150.5, 98.0, 18.2; HR-MS (ESI): Calcd for C H N O [M+H]
7
7
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3
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15
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195.0514, found (ESI ) 195.0518.
1
Procedures for the Preparation of Compounds 8a–8g.
The solution of corresponding 5 (2 mmol, 1.0 equiv.), ethyl acetoacetate (2.4 mmol, 1.2
1
1
1
17
18
19
equiv.) in CH COOH (2 mL) was refluxed for 6 h. After cooling, ethanol (15 mL) was
3
added to the solution. The precipitate was filtered to obtain the target compounds 8a–8g.
1
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1
1
1
20
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23
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2-(Benzylthio)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one: white solid (8a): 95%
1
yield; mp 214−217 °C; H NMR (DMSO-d , 400 MHz): 13.18 (s, 1H, NH), 7.44−7.46 (m,
6
2H, ArH), 7.26−7.34 (m, 3H, ArH), 5.81 (s, 1H, C=CH), 4.43 (s, 2H, CH ), 2.29 (s, 3H,
2
1
3
CH ); C NMR (DMSO-d , 100 MHz): 162.0, 154.8, 151.1, 150.7, 137.4, 128.8, 128.5,
3
6
+
127.3, 98.5, 34.5, 18.5; HR-MS (ESI): Calcd for C H N OS [M+H] 273.0805, found
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3
13
4
+
(ESI ) 273.0809.
1
1
1
1
1
1
26
27
28
29
30
31
5-Methyl-2-((4-methylbenzyl)thio)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (8b): white
1
solid, 91% yield; mp 248−250 °C; H NMR (DMSO-d , 400 MHz): 13.18 (s, 1H, NH),
6
7.32 (d, J = 7.8 Hz, 2H, ArH), 7.12 (d, J = 7.7 Hz, 2H, ArH), 5.81 (s, 1H, C=CH), 4.38 (s,
1
3
2H, CH ), 2.29 (s, 3H, CCH ), 2.26 (s, 3H, ArCH ); C NMR (DMSO-d , 100 MHz):
2
3
3
6
162.0, 154.8, 151.1, 150.7, 136.5, 134.2, 129.0, 128.8, 98.5, 34.3, 20.7, 18.5; HR-MS (ESI):
+
+
Calcd for C H N OS [M+H] 287.0961, found (ESI ) 287.0957.
1
4
15
4
1
1
32
33
2-((3-Methoxybenzyl)thio)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one
(8c):
1
white solid, 91% yield; mp 153−155 °C; H NMR (DMSO-d , 400 MHz): 13.19 (s, 1H,
6
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34
35
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NH), 7.23 (t, J = 7.8 Hz, 1H, ArH), 7.05 (s, 1H, , ArH), 7.01 (d, J = 7.5 Hz, 1H, ArH), 6.83
(d, J = 9.4 Hz, 1H, ArH), 5.81 (s, 1H, C=CH), 4.40 (s, 2H, CH ), 3.73 (s, 3H, OCH ), 2.29
2
3
1
3
(s, 3H, CCH ); C NMR (DMSO-d , 100 MHz): 162.0, 159.2, 154.8, 151.1, 150.7, 138.9,
3
6
129.5, 121.0, 114.5, 12.8, 98.5, 55.0, 34.5, 18.5; HR-MS (ESI): Calcd for C H N O S
1
4
15
4
2
+
+
[M+H] 303.0910, found (ESI ) 303.0914.
1
1
1
1
1
1
1
39
40
41
42
43
44
45
2-((4-Fluorobenzyl)thio)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (8d): white
1
solid, 93% yield; mp 214−217 °C; H NMR (DMSO-d , 400 MHz): 13.19 (s, 1H, NH),
6
7.50 (t, J = 8.0 Hz, 2H, ArH), 7.16 (t, J = 8.8 Hz, 2H, ArH), 5.81 (s, 1H, C=CH), 4.43 (s,
1
3
2H, CH ), 2.29 (s, 3H, CCH ); C NMR (DMSO-d , 100 MHz): 161.9, 161.4 (d, J = 243.4
2
3
6
Hz), 154.8, 151.2, 150.7, 133.8 (d, J = 2.8 Hz), 130.8 (d, J = 8.2 Hz), 115.2 (d, J = 21.3
+
Hz), 98.5, 33.6, 18.5; HR-MS (ESI): Calcd for C H FN OS [M+H] 297.0710, found
1
3
12
4
+
(ESI ) 297.0708.
1
1
1
1
1
1
46
47
48
49
50
51
2-((4-Chlorobenzyl)thio)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (8e): white
1
solid, 90% yield; mp 248−251 °C; H NMR (DMSO-d , 400 MHz): 13.19 (s, 1H, NH),
6
1
3
7.36−7.48 (m, 4H, ArH), 5.81 (s, 1H, C=CH), 4.42 (s, 2H, CH ), 2.28 (s, 3H, CCH ); C
2
3
NMR (DMSO-d , 100 MHz): 161.7, 154.8, 151.1, 150.7, 136.8, 131.9, 130.7, 128.4, 98.5,
6
+
+
33.7, 18.5; HR-MS (ESI): Calcd for C H ClN OS [M+H] 307.0415, found (ESI )
1
3
12
4
307.0413.
1
52
2-((2-Chloro-4-fluorobenzyl)thio)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one
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(8f): white solid, 94% yield; mp 216−219 °C; H NMR (DMSO-d , 400 MHz): 13.21 (s,
6
54
55
56
57
58
59
1H, NH), 7.66 (t, J = 8.1 Hz, 1H, ArH), 7.50 (d, J = 8.5 Hz, 1H, ArH), 7.21 (t, J = 8.3 Hz,
1
3
1H, ArH), 5.83 (s, 1H, C=CH), 4.50 (s, 2H, CH ), 2.29 (s, 3H, CCH ); C NMR (DMSO-d ,
2
3
6
100 MHz): 162.5, 161.4, 160.0, 154.8, 151.0 (d, J = 44.3 Hz), 134.0 (d, J = 10.7 Hz), 132.5
(d, J = 9.0 Hz), 131.1 (d, J = 3.3 Hz), 116.8 (d, J = 25.2 Hz), 114.5 (d, J = 21.1 Hz), 98.6,
+
+
32.0, 18.5; HR-MS (ESI): Calcd for C H ClFN OS [M+H] 325.0321, found (ESI )
1
3
11
4
325.0325.
1
1
1
1
1
1
60
61
62
63
64
65
5-Methyl-2-((4-nitrobenzyl)thio)-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (8g): white
1
solid, 94% yield; mp 256−259 °C; H NMR (DMSO-d , 400 MHz): 13.21 (s, 1H, NH),
6
8.18 (d, J = 8.5 Hz, 2H, ArH), 7.73 (d, J = 8.5 Hz, 2H, ArH), 5.82 (s, 1H, C=CH), 4.56 (s,
1
3
2H, CH ), 2.28 (s, 3H, CCH ); C NMR (DMSO-d , 100 MHz): 161.4, 154.8, 151.2, 150.8,
2
3
6
146.6, 146.0, 130.0, 123.5, 98.6, 33.7, 18.5; HR-MS (ESI): Calcd for C H1 N O S
1
3
2
5
3
+
+
[M+H] 318.0655, found (ESI ) 318.0658.
1
66
Procedures for the Preparation of Compounds 9a–9o, 10.
1
1
1
1
1
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68
69
70
71
The mixture of corresponding 5 (2 mmol, 1.0 equiv.), ethyl acetoacetate or acetylacetone
(2.4 mmol, 1.2 equiv.), substituted aldehydes (1.0 mmol, 1.0 equiv.) in DMF (1.0 mL) was
heated in oil bath at 140 °C for 2 h. After cooling, water (5 mL) was added to the solution.
The precipitate was filtered and washed with ether to obtain the desired products 9a–9o,
10.
1
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Ethyl 2-(benzylthio)-5-methyl-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6-
1
1
1
1
1
1
1
73
74
75
76
77
78
carboxylate (9a): white solid, 90% yield; mp 171−173 °C; H NMR (DMSO-d , 400 MHz):
6
10.85 (s, 1H, NH), 7.19−7.35 (m, 10H, ArH), 6.22 (s, 1H, CH), 4.23 (d, J = 13.2 Hz, 1H,
SCH ), 4.16 (d, J = 13.2 Hz, 1H, SCH ), 3.92−3.97 (m, 2H, OCH CH ), 2.40 (s, 3H, CCH ),
2
2
2
3
3
1
3
1.04 (t, J = 7.1 Hz, 3H, OCH CH ); C NMR (DMSO-d , 100 MHz): 165.0, 157.7, 147.7,
2
3
6
146.3, 141.8, 138.0, 128.7, 128.4, 128.2, 128.0, 127.0, 97.6, 59.5, 59.4, 34.7, 18.4, 13.8;
+
+
HR-MS (ESI): Calcd for C H N O S [M+H] 407.1536, found (ESI ) 407.1533.
2
2
23
4
2
1
79
Ethyl 2-(benzylthio)-5-methyl-7-(p-tolyl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-6
1
1
1
1
1
1
1
1
80
81
82
83
84
85
86
-carboxylate (9b): white solid, 90% yield; mp 157−159 °C; H NMR (DMSO-d , 400 MHz):
6
10.84 (s, 1H, NH), 7.24−7.27 (m, 2H, ArH), 7.19−7.20 (m, 3H, ArH), 7.09−7.14 (m, 4H,
ArH), 6.17 (s, 1H, CH), 4.23 (d, J = 13.2 Hz, 1H, SCH ), 4.16 (d, J = 13.2 Hz, 1H, SCH ),
2
2
3.92−3.97 (m, 2H, OCH CH ), 2.39 (s, 3H, CCH ), 2.26 (s, 3H, ArCH ), 1.06 (t, J = 7.1
2
3
3
3
1
3
Hz, 3H, OCH CH ); C NMR (DMSO-d , 100 MHz): 165.5, 158.1, 148.2, 146.6, 139.5,
2
3
6
138.4, 137.7, 129.4, 129.2, 128.7, 127.5, 127.4, 98.2, 59.9, 59.7, 35.3, 21.1, 18.8, 14.3;
+
+
HR-MS (ESI): Calcd for C H N O S [M+H] 421.1693, found (ESI ) 421.1688.
2
3
25
4
2
1
87
Ethyl 2-(benzylthio)-7-(4-methoxyphenyl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
1
1
1
88
89
90
pyrimidine-6-carboxylate (9c): white solid, 92% yield; mp 170−172 °C; H NMR (DMSO-
d6, 400 MHz): 10.82 (s, 1H, NH), 7.25−7.27 (m, 2H, ArH), 7.19−7.21 (m, 3H, ArH), 7.14
(d, J = 8.7 Hz, 2H, ArH), 6.87 (d, J = 8.7 Hz, 2H, ArH), 6.17 (s, 1H, CH), 4.23 (d, J = 13.2
1
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Journal of Agricultural and Food Chemistry
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1
1
1
1
91
Hz, 1H, SCH ), 4.16 (d, J = 13.2 Hz, 1H, SCH ), 3.92−3.96 (m, 2H, OCH CH ), 3.72 (s,
2
2
2
3
1
3
92
93
94
95
3H, OCH ), 2.39 (s, 3H, CCH ), 1.06 (t, J = 7.1 Hz, 3H, OCH CH ); C NMR (DMSO-
3 3 2 3
d6, 100 MHz): 165.5, 159.4, 158.0, 148.1, 146.4, 138.5, 134.5, 129.2, 128.7, 127.5, 114.2,
+
98.3, 59.9, 59.4, 55.5, 35.2, 18.8, 14.4; HR-MS (ESI): Calcd for C H N O S [M+H]
2
3
25
4
3
+
437.1642, found (ESI ) 437.1645.
1
96
Ethyl 2-(benzylthio)-7-(4-fluorophenyl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
1
1
1
2
2
2
2
2
97
98
99
00
01
02
03
04
pyrimidine-6-carboxylate (9d): white solid, 89% yield; mp 153−156 °C; H NMR (DMSO-
d6, 400 MHz): 10.90 (s, 1H, NH), 7.24−7.30 (m, 4H, ArH), 7.13−7.20 (m, 5H, ArH), 6.24
(s, 1H, CH), 4.23 (d, J = 13.2 Hz, 1H, SCH ), 4.16 (d, J = 13.2 Hz, 1H, SCH ), 3.90−4.00
2
2
1
3
(m, 2H, OCH CH ), 2.40 (s, 3H, CCH ), 1.03 (t, J = 7.1 Hz, 3H, OCH CH ); C NMR
2
3
3
2
3
(DMSO-d , 100 MHz): 165.4, 162.1 (d, J = 244.1 Hz), 158.3, 148.0, 147.0, 138.7, 138.5,
6
129.6 (d, J = 8.4 Hz), 129.2, 128.7, 127.5, 115.6 (d, J = 15.9 Hz), 97.8, 59.9, 59.3, 35.2,
+
+
18.9, 14.3; HR-MS (ESI): Calcd for C H FN O S [M+H] 425.1442, found (ESI )
2
2
22
4
2
425.1447.
2
05
Ethyl 2-(benzylthio)-7-(4-chlorophenyl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
2
06
07
08
09
pyrimidine-6-carboxylate (9e): white solid, 90% yield; mp 150−152 °C; H NMR (DMSO-
d6, 400 MHz): 10.92 (s, 1H, NH), 7.56 (d, J = 8.5 Hz, 2H, ArH), 7.19−7.25 (m, 7H, ArH),
6.22 (s, 1H, CH), 4.23 (d, J = 13.2 Hz, 1H, SCH ), 4.14 (d, J = 13.3 Hz, 1H, SCH ),
2
2
3.92−3.99 (m, 2H, OCH CH ), 2.40 (s, 3H, CCH ), 1.03 (t, J = 7.1 Hz, 3H, OCH CH );
2
3
3
2
3
1
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1
3
2
2
2
10
11
12
C NMR (DMSO-d , 100 MHz): 165.4, 158.3, 148.1, 147.2, 141.4, 138.5, 133.0, 131.6,
6
129.2, 128.9, 128.7, 127.5, 97.6, 60.0, 59.4, 35.2, 18.9, 14.4; HR-MS (ESI): Calcd for
+
+
C H ClN O S [M+H] 441.1147, found (ESI ) 441.1153.
2
2
22
4
2
2
13
Ethyl 2-(benzylthio)-7-(4-bromophenyl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
2
2
2
2
14
15
16
17
18
19
20
pyrimidine-6-carboxylate (9f): white solid, 93% yield; mp 143−147 °C; H NMR (DMSO-
d6, 400 MHz): 10.91 (s, 1H, NH), 7.53 (d, J = 8.4 Hz, 2H, ArH), 7.14−7.24 (m, 7H, ArH),
6.22 (s, 1H, CH), 4.23 (d, J = 13.3 Hz, 1H, SCH ), 4.15 (d, J = 13.3 Hz, 1H, SCH ),
2
2
3.91−3.98 (m, 2H, OCH CH ), 2.40 (s, 3H, CCH ), 1.04 (t, J = 7.1 Hz, 3H, OCH CH );
2
3
3
2
3
1
3
C NMR (DMSO-d , 100 MHz): 165.4, 158.4, 148.1, 147.2, 141.7, 138.4, 131.8, 129.8,
6
129.2, 128.7, 127.5, 121.7, 97.5, 60.0, 59.5, 35.2, 18.9, 14.3; HR-MS (ESI): Calcd for
+
+
C H BrN O S [M+H] 485.0641 found (ESI ) 485.0643.
2
2
22
4
2
2
21
Ethyl 2-(benzylthio)-7-(furan-2-yl)-5-methyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
2
2
2
2
22
23
24
25
26
27
28
pyrimidine-6-carboxylate (9g): white solid, 82% yield; mp 195−197 °C; H NMR (DMSO-
d6, 400 MHz): 10.92 (s, 1H, NH), 7.56 (s, 1H, CH), 7.31−7.32 (m, 2H, ArH), 7.21−7.27
(m, 3H, ArH), 6.39−6.40 (m, 1H, ArH), 6.33 (m, 2H, ArH), 4.27 (d, J = 13.2 Hz, 1H, SCH2),
4.20 (d, J = 13.2 Hz, 1H, SCH ), 3.96−4.07 (m, 2H, OCH CH ), 2.38 (s, 3H, CCH ), 1.10
2
2
3
3
1
3
(t, J = 7.1 Hz, 3H, OCH CH ); C NMR (DMSO-d , 100 MHz): 165.3, 158.3, 153.5, 148.5,
2
3
6
147.5, 143.2, 138.4, 129.3, 128.8, 127.6, 111.0, 108.0, 95.4, 59.9, 53.3, 35.2, 18.9, 14.4;
+
+
HR-MS (ESI): Calcd for C H N O S [M+H] 397.1329, found (ESI ) 397.1327.
2
0
21
4
3
1
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Journal of Agricultural and Food Chemistry
2
29
Ethyl 2-(benzylthio)-5-methyl-7-(thiophen-2-yl)-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
2
2
2
2
30
31
32
33
34
35
36
pyrimidine-6-carboxylate (9h): white solid, 95% yield; mp 170−172 °C; H NMR (DMSO-
d6, 400 MHz): 10.98 (s, 1H, NH), 7.43−7.45 (m, 1H, ArH), 7.31−7.33 (m, 2H, ArH),
7.21−7.27 (m, 3H, ArH), 6.94−6.96 (m, 2H), 6.53 (s, 1H, CH), 4.26 (d, J = 13.3 Hz, 1H,
SCH ), 4.21 (d, J = 13.3 Hz, 1H, SCH ), 3.96−4.07 (m, 2H, OCH CH ), 2.39 (s, 3H, CCH ),
2
2
2
3
3
1
3
1.10 (t, J = 7.1 Hz, 3H, OCH CH ); C NMR (DMSO-d , 100 MHz): 164.8, 157.9, 147.7,
2
3
6
145.0, 137.9, 128.8, 128.3, 127.1, 126.8, 125.9, 125.6, 97.6, 59.5, 54.3, 34.8, 18.3, 13.9;
+
+
HR-MS (ESI): Calcd for C H N O S [M+H] 413.1100, found (ESI ) 413.1101.
2
0
21
4
2 2
2
37
Ethyl 2-(benzylthio)-5-methyl-7-propyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine
1
2
2
2
2
2
2
2
2
38
39
40
41
42
43
44
45
-6-carboxylate (9i): white solid, 75% yield; mp 172−174 °C; H NMR (DMSO-d , 400
6
MHz): 10.56 (s, 1H, NH), 7.36−7.38 (m, 2H, ArH), 7.23−7.30 (m, 3H, ArH), 5.27 (t, J =
3.92 Hz, 1H, CH), 4.30 (d, J = 13.4 Hz, 1H, SCH ), 4.25 (d, J = 13.4 Hz, 1H, SCH ),
2
2
4.05−4.17 (m, 2H, OCH CH ), 2.30 (s, 3H, CCH ), 1.71−1.80 (m, 1H, ), 1.54−1.61 (m,
2
3
3
1H), 1.23 (t, J = 7.1 Hz, 3H, OCH CH ), 1.68−1.24 (m, 1H, CH CH CH ), 0.75−0.82 (m,
2
3
2
2
3
1
3
4H, CH CH CH ); C NMR (DMSO-d , 100 MHz): 165.7, 157.6, 149.0, 147.6, 138.5,
2
2
3
6
129.2, 128.8, 127.6, 96.8, 60.0, 56.0, 38.0, 35.5, 18.9, 17.2, 14.6, 14.0; HR-MS (ESI):
+
+
Calcd for C H N O S [M+H] 373.1693, found (ESI ) 373.1697.
1
9
25
4
2
2
2
46
47
Ethyl 5-methyl-2-((4-methylbenzyl)thio)-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
pyrimidine-6-carboxylate (9j): white solid, 95% yield; mp 157−160 °C; H NMR (DMSO-
1
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2
2
2
2
2
2
48
49
50
51
52
53
d6, 400 MHz): 10.87 (s, 1H, NH), 6.99−7.33 (m, 10H, ArH), 6.21 (s, 1H, CH), 4.18 (d, J =
12.8 Hz, 1H, SCH ), 4.12 (d, J = 12.7 Hz, 1H, SCH ), 3.93−3.96 (m, 2H, CH CH ), 2.40
2
2
2
3
1
3
(s, 3H, CCH ), 2.23 (s, 3H, ArCH ), 1.04 (t, J = 5.6 Hz, 3H, CH CH ); C NMR (DMSO-d ,
3
3
2
3
6
100 MHz): 165.0, 157.7, 147.6, 146.3, 141.8, 136.2, 134.8, 128.8, 128.6, 128.4, 127.9,
127.0, 97.6, 59.5, 59.4, 34.5, 20.6, 18.4, 13.8; HR-MS (ESI): Calcd for C H N O S
2
3
25
4
2
+
+
[M+H] 421.1693, found (ESI ) 421.1697.
2
54
Ethyl 5-methyl-2-((3-methoxybenzyl)thio)-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
2
2
2
2
2
55
56
57
58
59
60
61
62
pyrimidine-6-carboxylate (9k): white solid, 93% yield; mp 151−153 °C; H NMR (DMSO-
d6, 400 MHz): 10.89 (s, 1H, NH), 7.21−7.32 (m, 5H, ArH), 7.11 (t, J = 7.9 Hz, 1H, ArH),
6.88 (s, 1H, ArH), 6.82 (d, J = 7.3 Hz, 1H, ArH), 6.77 (d, J = 7.9 Hz, 1H, ArH), 6.21 (s,
1H, CH), 4.20 (d, J = 13.4 Hz, 1H, SCH ), 4.16 (d, J = 13.1 Hz, 1H, SCH ), 3.93−3.97 (m,
2
2
2H, CH CH ), 3.67 (s, 3H, OCH ), 2.40 (s, 3H, CCH ), 1.03 (t, J = 7.0 Hz, 3H, CH CH );
2
3
3
3
2
3
1
3
C NMR (DMSO-d , 100 MHz): 164.9, 159.1, 157.8, 147.7, 146.3, 141.8, 139.3, 129.3,
6
128.4, 127.9, 127.0, 120.9, 114.2, 112.6, 97.7, 59.5, 59.4, 54.9, 34.8, 18.3, 13.8; HR-MS
+
+
(ESI): Calcd for C H N O S [M+H] 437.1642, found (ESI ) 437.1639.
2
3
25
4
3
2
63
Ethyl 5-methyl-2-((4-fluorobenzyl)thio)-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
64
65
66
pyrimidine-6-carboxylate (9l): white solid, 91% yield; mp 179−181 °C; H NMR (DMSO-
d6, 400 MHz): 10.87 (s, 1H, NH), 7.22−7.34 (m, 7H, ArH), 6.99 (t, J = 8.7 Hz, 2H, ArH),
6.21 (s, 1H, CH), 4.22 (d, J = 13.4 Hz, 1H, SCH ), 4.14 (d, J = 13.4 Hz, 1H, SCH ),
2
2
1
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Journal of Agricultural and Food Chemistry
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3
2
2
2
2
2
67
3.93−3.96 (m, 2H, CH CH ), 2.40 (s, 3H, CCH ), 1.02 (t, J = 7.0 Hz, 3H, CH CH ); C
2 3 3 2 3
68
69
70
71
NMR (DMSO-d , 100 MHz): 165.0, 161.4 (d, J = 243.1 Hz), 157.5, 147.7, 146.3, 141.8,
6
134.3 (d, J = 2.6 Hz), 130.7 (d, J = 8.1 Hz), 128.4, 128.0, 127.0, 114.9 (d, J = 21.3 Hz),
+
97.6, 59.5, 59.4, 33.8, 18.4, 13.8; HR-MS (ESI): Calcd for C H FN O S [M+H]
2
2
22
4
2
+
425.1442, found (ESI ) 425.1447.
2
72
Ethyl 5-methyl-2-((4-chlorobenzyl)thio)-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
2
2
2
2
73
74
75
76
77
78
79
pyrimidine-6-carboxylate (9m): white solid, 93% yield; mp 108−111 °C; H NMR (CDCl ,
3
400 MHz): 10.58 (s, 1H, NH), 7.32 (s, 5H, ArH), 7.05−7.07 (m, 4H, ArH), 6.34 (s, 1H,
CH), 4.23 (d, J = 13.4 Hz, 1H, SCH ), 4.04−4.08 (m, 2H, CH CH ), 4.02 (d, J = 13.4 Hz,
2
2
3
1
3
1H, SCH ), 2.57 (s, 3H, CCH ), 1.13 (t, J = 7.1 Hz, 3H, CH CH ); C NMR (DMSO-d ,
2
3
2
3
6
100 MHz): 164.6, 157.0, 147.3, 146.0, 141.5, 136.9, 131.2, 130.2, 128.1, 127.8, 127.6,
+
126.7, 97.2, 59.2, 59.0, 33.5, 18.0, 13.5; HR-MS (ESI): Calcd for C H ClN O S [M+H]
2
2
22
4
2
+
441.1447, found (ESI ) 441.1145.
2
80
Ethyl 5-methyl-2-((2-chloro-4-fluorobenzyl)thio)-7-phenyl-4,7-dihydro-[1,2,4]triazolo
1
2
2
2
2
2
81
82
83
84
85
[1,5-a]pyrimidine-6-carboxylate (9n): white solid, 94% yield; mp 195−198 °C; H NMR
(CDCl , 400 MHz): 10.78 (s, 1H, NH), 7.32 (s, 5H, ArH), 6.96−7.03 (m, 2H, ArH), 6.50
3
(t, J = 6.5 Hz, 1H, ArH), 6.34 (s, 1H, ArH), 4.31 (d, J = 13.7 Hz, 1H, SCH ), 4.13 (d, J =
2
13.6 Hz, 1H, SCH ), 4.04−4.07 (m, 2H, CH CH ), 2.57 (s, 3H, CCH ), 1.13 (t, J = 6.9 Hz,
2
2
3
3
1
3
3H, CH CH ); C NMR (DMSO-d , 100 MHz): 164.9, 161.0 (d, J = 247.4 Hz), 157.0,
2
3
6
1
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2
2
2
2
86
87
88
89
147.7, 146.3, 141.8, 133.8 (d, J = 10.6 Hz), 132.2 (d, J = 8.5 Hz), 131.7 (d, J = 3.1 Hz),
128.4, 128.0, 127.1, 116.6 (d, J = 25.1 Hz), 114.0 (d, J = 21.0 Hz), 97.6, 59.6, 59.4, 32.2,
+
+
18.3, 13.8; HR-MS (ESI): Calcd for C H ClFN O S [M+H] 459.1052, found (ESI )
2
2
21
4
2
459.1047.
2
90
Ethyl 5-methyl-2-((4-nitrobenzyl)thio)-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]
1
2
2
2
2
2
2
2
2
91
92
93
94
95
96
97
98
pyrimidine-6-carboxylate (9o): white solid, 91% yield; mp 172−175 °C; H NMR (DMSO-
d6, 400 MHz): 10.86 (s, 1H, NH), 8.00 (d, J = 8.5 Hz, 2H, ArH), 7.48 (d, J = 8.5 Hz, 2H,
ArH), 7.27−7.36 (m, 3H, ArH), 7.20−7.22 (m, 2H, ArH), 6.20 (s, 1H, CH), 4.34 (d, J =
14.0 Hz, 1H, SCH ), 4.26 (d, J = 13.9 Hz, 1H, SCH ), 3.92−3.96 (m, 2H, CH CH ), 2.39
2
2
2
3
1
3
(s, 3H, CCH ), 1.02 (t, J = 7.1 Hz, 3H, CH CH ); C NMR (DMSO-d , 100 MHz): 164.9,
3
2
3
6
162.3, 156.9, 147.8, 146.5, 146.3, 141.8, 129.9, 128.4, 127.9, 127.0, 123.2, 97.6, 59.5, 59.4,
+
+
33.8, 18.3, 13.8; HR-MS (ESI): Calcd for C H N O S [M+H] 452.1387, found (ESI )
2
2
22
5
4
452.1389.
2
3
3
3
3
3
99
00
01
02
03
04
(2-(Benzylthio)-5-methyl-7-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidin-6-
1
yl)ethanone (10): white solid, 87% yield; mp 203−205 °C; H NMR (DMSO-d , 400 MHz):
6
10.85 (s, 1H, NH), 7.25−7.33 (m, 7H, ArH), 7.19−7.21 (m, 3H, ArH), 6.42 (s, 1H, CH),
4.25 (d, J = 13.3 Hz, 1H, SCH ), 4.17 (d, J = 13.3 Hz, 1H, SCH ), 2.42 (s, 3H, CCH ), 2.13
2
2
3
1
3
(s, 3H, C=OCH ); C NMR (DMSO-d , 100 MHz):195.2, 158.2, 147.9, 146.1, 141.8,
3
6
138.5, 129.1, 129.2, 128.9, 128.8, 127.8, 127.5, 108.2, 59.9, 35.2, 31.0, 19.9; HR-MS (ESI):
1
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Journal of Agricultural and Food Chemistry
+
+
3
05
Calcd for C H N OS [M+H] 377.1431, found (ESI ) 377.1437
21 21 4
3
3
06
07
Biological Assay. Each bioassay was repeated three times at 25±1 °C. Activity results were
estimated according to a percentage scale of 0−100 (0: no activity; 100: total inhibited).
2
8
29-31
3
3
08
09
Detailed bioassay procedures for the anti-TMV and and fungicidal
activities were
described in literature and also can be found in the Supporting Information.
3
3
3
10
11
12
Mode of Action Studies. The detailed procedures of in vitro TMV rod and 20S CP disk
1
3
assembly inhibition was described in literature and also can been found in the Supporting
Information.
3
3
13
14
RESULTS AND DISCUSSION
Chemistry.
3
3
3
3
3
3
3
3
15
16
17
18
19
20
21
22
Synthesis of the Compounds 1−10. Procedures for the preparation of compounds 1, 2, 3,
4b and 5a−5g (Figure 3) can been seen in Supporting Information. Essramycin (1) and 2-
(5-amino-4H-1,2,4-triazol-3-yl)-1-phenylethanone (2) were synthesized according to the
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method reported by Moody with modification. Methyl ester 4b was obtained in good
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yield by esterification of 4a with methanol in the presence of SOCl . Condensation of 3-
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amino-5-thiol-1,2,4-triazole and corresponding alkyl bromides in the presence of triethyl
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amine to give triazoles 5a−5g in excellent yields. Compounds 6, 7a−7c and 8a−8g were
prepared by condensation of corresponding 3-amino-1,2,4-triazoles and corresponding
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ethyl acetoacetates in acetic acid (Figure 4). The tautmoeric structures (Figure 5) of a large
variety of 1,2,4-triazolo[1,5-a]pyrimidines have been determined in literature.33,34 The
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structures of 1, 6−8 were validated as the lactam tautomeric forms via comparison of H
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NMR, C NMR with literature. Compounds 7a and 7b were obtained as 2-decarboxylic
1,2,4-triazolo[1,5-a]pyrimidines rather than design products. In order to determine the
structures of 7a and 7b, the single crystal of 7a was obtained (Figure 6, CCDC number:
1948036), which enabled the configuration of the product to be 2-decarboxylic 1,2,4-
triazolo[1,5-a]pyrimidine. The configuration of 7b was assigned by analogy. Compound
7d was obtained by hydrazinolysis of 7c with hydrazine hydrate in 98% yield (Figure 4).
Compound 7e was gained by hydrolysis of 7c with lithium hydroxide hydrate with 91%
yield (Figure 7).
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Compounds 9a−9o and 10 were obtained in excellent yields (Figure 8). The Biginelli-
like heterocyclization reaction of aldehyde, β-dicarbonyl compound and 3-alkylthio-5-
amino-1,2,4-triazole was used.
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Phytotoxic Activity. Compounds 1−10 were first tested for their phytotoxic activities
against the test plant. The results indicated that compounds 1−10 showed no phytotoxic
activities at 500 μg/mL. There was no lesions on the tobacco leaves.
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Antiviral Activity. The inhibition ratio of essramycin and its analogues against TMV
are shown in Tables 1 and 2 with the commercial plant virucides ribavirin and
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ningnanmycin as the controls.
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In Vitro Anti-TMV Activity. The natural alkaloid essramycin (1) and its analogues 2−10
were first investigated for their in vitro inhibition ratio against TMV using conventional
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half-leaf method. Essramycin alkaloids were found to have good antiviral activities for
the first time. Most of these compounds exhibited higher antiviral activities than ribavirin
(Table 1). Compounds 7e and 8f exhibited significantly higher TMV inhibition effects than
ningnanmycin (the most widely used antiviral agent at present), thus emerged as novel
antiviral lead compounds. Essramycin (1) gave 39% inhibition ratio at 500 μg/mL, which
about similar to that of ribavirin. Interestingly, pyrimidine ring opening compound 2
exhibited better activity than essramycin (1). The biological activity was further improved
after the phenyl was converted into carboxyl group (inhibitory effect: 3 > 2). Further
reducing one methylene of compound 3 leaded to a decrease in activity (inhibitory effect:
3 > 4a). However, 4b, a carboxylic ester compound, exhibited the same level of biological
activity as compound 3. Above results indicate that minor changes of groups at 2-position
can lead to significant changes in biological activity. A series of sulfur-containing
functional groups were introduced into the 2-site to further investigate the structure-activity
relationship in this region. Compounds 5a−5c showed similar level of activities with 3 and
higher than essramycin (1). Compounds 5d, 5e containing p-fluorophenyl and p-
chlorophenyl exhibited the same biological activities as 1. Among compounds 5a−5g,
compound 5f containing 4-fluoro-2-chlorophenyl showed the best activity, while
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compound 5g containing p-nitrophenyl showed the lowest activity, indicated that the
electron-withdrawing group was unfavorable to the activity. The 1,2,4-triazolo[1,5-
a]pyrimidine compound 6 displayed the same activity as 3 but higher than compound 1.
Compounds 7a and 7b with a hydrogen atom, 7c with methyl formyl, and 7d with
formylhydrazine showed a similar level of activities as ribavirin. Compared with 7a,
substitution of the n-propyl group (7b) in 5-position of 1,2,4-triazolo[1,5-a]pyrimidine
resulted in sharp decline of anti-TMV activity, which indicated that addition of a lipophilic
group on 5-position of 1,2,4-triazolo[1,5-a]pyrimidine is inadvisable. The COOH
containing compound 7e exhibited higher activity than compounds 6, 4a and ningnanmycin.
Compound 8a containing –SCH Ph showed about similar level of TMV inhibition effect
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with compounds 6 and 4a. Encouraged by these results, essramycin analogues 8b−8g
containing kinds of S-benzyl groups were further designed. Compounds with electron-
withdrawing groups (8d, 8e and 8g) at the S-benzyl ring showed relatively lower activities
than 8a. However, 8f with electron-withdrawing groups 4-F and 2-Cl performed excellent
anti-TMV activity. Compounds 8b and 8c with electron-donating groups at S-benzyl ring
displayed about similar level of activities as compound 8a. From the above results, we can
see that biological activity is very sensitive to structural changes. In order to further
investigate the effect of substituent change at other sites on biological activity, compounds
9a−9o and 10 were designed and synthesized. The results proved that the 5-methyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one is important core structure for activity
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(inhibitory effect: 8a > 9a; 8b > 9j; 8c > 9k; 8d > 9l; 8f > 9n; 8g > 9o). Compounds 9b−9d
showed slightly higher activities than 9a. The activities of 9e, 9f and 9i are higher than that
of ribavirin. Compounds 9g, 9h, 9j−9o and 10 displayed relatively low activities.
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In Vivo Anti-TMV Activity. All of target compounds and a part of intermediates were
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investigated in vivo antiviral activities against TMV using our reported method. As shown
in Table 2, most of the target compounds also exhibited higher in vivo TMV inhibitory
effects than commercial ribavirin. Compounds 3, 5f, 6, 8c and 9e showed about similar
level of TMV inhibitory effects as ningnanmycin. Compounds 7e and 8f displayed
significantly higher TMV inhibitory effects than ningnanmycin. Unlike the data of in vitro
anti-TMV activity, the curative effects were slightly decreased. Other structure-activity
relationships are consistent with in vitro activity. The EC value of compound 7e for
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protection effect was further investigated to further confirm the activity data. As shown in
Table 3, compound 7e displayed 197 μg/mL EC value which is lower than that of ribavirin
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(711 μg/mL) and ningnanmycin (204 μg/mL).
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Preliminary Mode of Action
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With lead compounds in hand, we began to study their mechanism of action. Compound
7e was selected to investigate preliminary mode of action with RNA inhibitor antofine35
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and CP disk assembly inhibitor NK0209 as controls. As shown in Figure 9, Figure 9A
and Figure 9B revealed that 20S CP disk and TMV rod can be formed effectively. The use
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of a small amount of DMSO did not affect virus assembly (Figure 9C). The bits and pieces
of TMV rod in Figure 9D and Figure 9E revealed that antofine and NK0209 can obviously
inhibit the assembly of TMV rods. From Figure 9E, we can also see a large number of 20S
CP disk, which further proved that antofine had no effect on protein. By comparing the
Figures 9D, 9E and 9F, we can see that compound 7e has a better inhibitory effect on virus
assembly than controls antofine and NK0209. Further 20S CP disk assembly inhibition
tests were carried out to evaluate the interaction of compound 7e with TMV CP. As
depicted in Figure 10, the 20S CP disk can be gained by hatching TMV CP at 20 °C for 12
h (Figure 10A). The use of small amount of DMSO did not affect the formation of 20S CP
disk (Figure 10B). Antofine has no impact on 20S CP disk assembly (Figure 10C). As CP
disk assembly inhibitor, NK0209 exhibited obviously impact on the 20S CP disk, which
can induce fusion and aggregation of 20S CP disks (Figure 10D). Comparing with Figure
10D, there are larger number of CP disks aggregated and fused in Figures 10E and 10F.
The above results suggested that compound 7e may inhibit viral assembly by fusing 20S
CP disks, thus exerting its antiviral activity.
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Fungicidal Activity. The fungicidal activities of compounds 1−10 on 14 kinds of plant
fungi at 50 μg/mL were evaluated with commercial fungicides chlorothalonil and
carbendazim as controls (Table 4). All of the compounds displayed broad spectrum
fungicidal activities at 50 μg/mL. Compound 5b displayed more than 50% inhibition rate
against most of the 14 kinds of phytopathogenic fungi at 50 μg/mL. Compounds 5a−5c and
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8e displayed higher fungicidal activities than carbendazim against Botrytis cinerea. The
fungicidal activity of 5b against Cercospora arachidicola Hori is higher than that of
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commercial fungicides chlorothalonil and carbendazim. Compounds 5b and 9n displayed
higher fungicidal activities than chlorothalonil and carbendazim against Rhizoctonia
cerealis. Compound 5b with broad spectrum and high effect fungicidal activity emerged
as new fungicidal lead compound.
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In summary, marine natural product essramycin (1) was found to have good anti-TMV
activity for the first time. A series of [1,2,4]triazolo[1,5-a]pyrimidines were designed,
synthesized and evaluated for their anti-TMV activities systematically. Most of these
compounds exhibited higher antiviral activities than ribavirin. Compounds 3, 5f, 6, 8c and
9e showed about similar level of TMV inhibitory effects as ningnanmycin. Compounds 7e
and 8f exhibited significantly higher TMV inhibition effects than ningnanmycin, thus
emerged as novel antiviral lead compounds. The structure-activity relationship study
revealed that the biological activity is very sensitive to structural changes for these
compounds. Compound 7e was selected to further investigate preliminary mode of action.
The results revealed that compound 7e may inhibit viral assembly by fusing 20S CP disks,
thus exerting its antiviral activity. All of the compounds also displayed broad spectrum
fungicidal activities against 14 kinds of phytopathogenic fungi at 50 μg/mL. Compound 5b
with broad spectrum and high effect fungicidal activity emerged as new fungicidal lead
compound. Current work demonstrated that these simple essramycin analogues could be
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considered as potential candidates for the development of novel plant virus and
fungi inhibitors in the future.
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SUPPORTING INFORMATION
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Detailed preparation procedures of 1−5, detailed bio-assay procedures and the spectra data
of compounds 1−10 are provided in Supporting Information. This material is available free
of charge via the Internet at http://pubs.acs.org.
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Funding
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This study was supported by Natural Science Fund of China (21772145, 21732002,
21672117) and the Research Innovation Program for Postgraduate of Hebei Province
(CXZZSS219014).
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Notes
The authors declare no competing financial interest.
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