Design, Synthesis, and Anti-ToCV Activity of Novel 4(3 H)-Quinazolinone Derivatives Bearing Dithioacetal Moiety

Article abstract of DOI:10.1021/acs.jafc.0c00086

Tomato chlorosis virus (ToCV) has caused great harm to the production of tomato worldwide. To develop efficient anti-ToCV agents, some novel 4(3H)-quinazolinone derivatives containing dithioacetal were designed and synthesized, and their anti-ToCV activities were evaluated by microscale thermophoresis (MST) using ToCV coat protein (ToCV-CP) as a new target. The results showed that some compounds had a strong binding capacity to ToCV-CP. In particular, compounds C5 and C22 have an excellent binding capacity to ToCV-CP, with binding constant values of 0.24 and 0.25 μM, respectively. Additionally, reduced ToCV-CP gene expression levels of 81.05 and 87.59% could be achieved when tomato was treated with compounds C5 and C22, respectively, which were obviously higher than the levels after ningnanmycin (NNM) treatment (43.88%) and lead compound Xiangcaoliusuobingmi (XCLSBM) treatment (63.56%). Therefore, this work indicates that 4(3H)-quinazolinone derivatives containing dithioacetal moiety can be used as novel anti-ToCV agents.

Full text of DOI:10.1021/acs.jafc.0c00086

Subscriber access provided by BIU Pharmacie | Faculté de Pharmacie, Université Paris V
Agricultural and Environmental Chemistry
Design, synthesis and anti-ToCV activity of novel 4(3H)-
quinazolinone derivatives bearing dithioacetal moiety
Guangcheng Zu, Xiuhai Gan, Dandan Xie, Huanyu Yang,
Awei Zhang, Shaoyuan Li, Deyu Hu, and Baoan Song
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c00086 • Publication Date (Web): 23 Apr 2020
Downloaded from pubs.acs.org on April 24, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 25
Journal of Agricultural and Food Chemistry
1
2
3
4
5
6
7
8
9
Design, synthesis and anti-ToCV activity of novel 4(3H)-
quinazolinone derivatives bearing dithioacetal moiety
Guangcheng Zu, Xiuhai Gan, Dandan Xie, Huanyu Yang, Awei Zhang, Shaoyuan Li,
Deyu Hu*, Baoan Song*
Current address: State Key Laboratory Breeding Base of Green Pesticide and
Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural
Bioengineering, Ministry of Education, Guizhou University, Huaxi District, Guiyang
550025, China.
*Address of the corresponding author
10
11
Fax: 0086-851-83622211; E-mail: dyhu@gzu.edu.cn; songbaoan22@yahoo.com.
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 25
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
ABSTRACT: Tomato chlorosis virus (ToCV) has caused great harm to the
production of tomato worldwide. To develop efficient anti-ToCV agents, some novel
4(3H)-quinazolinone derivatives containing dithioacetal were designed and
synthesized, and their anti-ToCV activities were evaluated by microscale
thermophoresis (MST) using ToCV coat protein (ToCV-CP) as a new target. The
results showed that some compounds had strong binding capacity to ToCV-CP. In
particular, compounds C5 and C22 have excellent binding capacity to ToCV-CP, with
binding constant values of 0.24 and 0.25 μM, respectively. Additionally, reduced
ToCV-CP gene expression levels of 81.05% and 87.59% could be achieved when
tomato was treated with compounds C5 and C22, respectively, which were obviously
higher than those levels after Ningnanmycin (NNM) treatment (43.88%) and lead
compound Xiangcaoliusuobingmi (XCLSBM) treatment (63.56%). Therefore, this
work indicates that 4(3H)-quinazolinone derivatives containing dithioacetal moiety
can be used as novel anti-ToCV agents.
KEYWORDS: Tomato chlorosis virus, 4(3H)-quinazolinone, dithioacetal, MST, coat
28
29
protein, anti-ToCV activity
2
ACS Paragon Plus Environment

Page 3 of 25
Journal of Agricultural and Food Chemistry
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
INTRODUCTION
Tomato chlorosis virus (ToCV), a plant RNA virus transmitted by whitefly, was first
discovered in the United States in 1998, and it belongs to the family Closteroviridae,
genus Crinivirus.1,2 ToCV has a wide range of hosts; it not only infects tomato,
pepper, potato and other Solanaceae plants but also infects 25 other plant species
from eight different families. After being infected with ToCV, the tomato mainly
exhibited developmental delay, leaf brittleness, interveinal chlorosis, and limited
necrotic flecking.3-6 In recent years, ToCV has occurred in more than 20 countries
around the world and caused enormous economic losses.7-11 Currently, effective
agents to control this plant virus disease are still lacking, resulting in a serious decline
in tomato quality and yield. Therefore, the development of efficient anti-ToCV agents
is urgently needed.
The genome of ToCV consists of two genomic positive-sense RNAs, RNA1 and
RNA2, with a total genome length of 16.8 kb,12,13 encoding four and nine open
reading frames (ORFs) 14,15. RNA2 encodes multiple functional proteins, of which, the
coat protein (CP) and the minor coat protein (mCP) are mainly involved in virus
assembly, coating, and transporting. Coat proteins are essential for the intercellular
translocation of this type virus.16 The loss of CP can prevent the virus from forming a
complete viable virion.17,18 Therefore, it is an important pathway for development of
anti-ToCV agents using ToCV-CP as a target protein.19
In our previous work, we discovered and reported the anti-plant virus activity of the
dithioacetal structure, which has good antiviral activity against tobacco mosaic virus
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 25
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
(TMV), potato virus Y (PVY), cucumber mosaic virus (CMV) and ToCV.19-21 In
addition, we established an efficient and convenient screening method for anti-ToCV
agents using ToCV-CP as a target protein and some glycoside-containing dithioacetal
derivatives were synthesized with good antiviral activity against ToCV.19 As an
important class of nitrogen-containing heterocyclic compounds, 4(3H)-
quinazolinones are widely found in natural products with a range of significant
biological activities. Our group found that some 4(3H)-quinazolinone derivatives
showed good antiviral activity against CMV and TMV.22-24 However, the biological
activity of this class of compounds against ToCV has not been reported. In this work,
a series of 4(3H)-quinazolinone derivatives containing dithioacetal (Figure 1) were
designed, synthesized, and evaluated for their anti-ToCV activities in vitro and in
vivo. The findings will provide guidance for the design, synthesis and development of
anti-ToCV agents.
Figure 1
MATERIALS AND METHODS
Chemicals. Analytically pure reagents used in the experiments did not require
further drying or purification.
13
1
Instruments. H NMR and C NMR spectra of the compounds were obtained
using a Bruker DPX 400 MHz (Bruker, Germany) and a JEOL-ECX500 MHz (JEOL,
Tokyo, Japan) in CDCl3 or DMSO-d6 solution. HRMS was performed with a Thermo
Scientific Q Exactive (Thermo Scientific, USA). The melting points of the compounds
were measured using WRX-4 equipment.
4
ACS Paragon Plus Environment

Page 5 of 25
Journal of Agricultural and Food Chemistry
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
General Procedure for the Synthesis of the Intermediates A1-A3 and B1-B9.
Intermediates A1-A3 were synthesized according to the methods reported in the
literature.25 Intermediates A1-A3 (2.35 mmol), substituted p-hydroxybenzaldehyde
(2.35 mmol) and CH3CN (20 mL) were added to a 50 mL three-necked flask, with
K2CO3 (2.82 mmol) as an acid-binding agent. The mixing system was stirred at room
temperature for 10 minutes, and KI (0.23 mmol) was then added. The mixture was
allowed to react under reflux for 8 to 12h. After the reaction completed (as monitored
by TLC), the solvent was removed under vacuum, the residue was then diluted with
water, dichloromethane (50 mL×3) was added for extraction, and the two layers were
separated. The organic layer was dried and concentrated to obtain the crude products,
which were recrystallized with anhydrous ethanol to obtain the intermediates B1-B9.
Figure 2
General Procedure for Preparation of the Title Compounds C1-C27. The
intermediates B1-B9 (1.34mmol) were added to a round bottom flask containing
substituted mercaptan (2.68 mmol) and NaHSO4·SiO2 (1.34 mmol) in CH2Cl2 solvent
(10 mL). Upon reaction completion, the solvent was removed under vacuum, the
residue was dissolved with water, dichloromethane (30 mL×3) was added for
extraction, and the two layers were separated. The collected dichloromethane layer
was evaporated, and then purified by flash chromatography with ethyl acetate
/petroleum ether (1:3, v/v) to obtain the title Compounds C1-C27 (Figure 2).
Purification of Tomato Chlorotic Virus Coat Protein (ToCV-CP). The
ToCV-CP was cloned, expressed and purified according to the method described in
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 25
96
97
the literature.19
Evaluation of Anti-ToCV Activity in vitro. Binding of compounds with
ToCV-CP was analyzed through microscale thermophoresis (MST) to obtain Kd
values by methods described in the literature.26,27 Meanwhile, NNM, the lead
compound XCLSBM and Ribavirin (RIB) were used as positive controls. All tests
were duplicated three times.
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
Evaluation of Anti-ToCV Activity in vivo. We used tomato samples (Shouguang,
Shandong) infected with ToCV to start in vivo experiments. All of the samples were
confirmed to be infected with ToCV by polymerase chain reaction (PCR). The PCR
primer sequences used are shown in Table 1. Anti-ToCV activity was then assessed in
vivo according to the previously described method.19 The primers used in quantitative
real-time PCR are shown in Table 1. A MiniBEST Plasmid Purification Kit 4.0
(Takara) was used to extract the total RNA of samples, and a Goldenstar RT6 cDNA
Synthesis Kit (Tsingke) was used when the extracted total RNA was reverse
transcribed into cDNA. The quantitative real-time PCR was performed using the Fast
qPCR mix SYBR Green kit (Tsingke).
RESULTS AND DISCUSSION
Chemistry. Figure 2 shows the synthetic routes of 4(3H)-quinazolinone derivatives
bearing dithioacetal groups. Methanol was used as a solvent, sodium was added,
substituted anthranilic acid and chloroacetonitrile as raw materials, and the mixture
was stirred at room temperature for 4-6 h to obtain intermediates A1-A3. The second
step of the reaction used acetonitrile as a solvent, K2CO3 as a catalyst, intermediates
6
ACS Paragon Plus Environment

Page 7 of 25
Journal of Agricultural and Food Chemistry
118
A1-A3 and substituted 4-hydroxybenzaldehyde under reflux for 8-12 h to obtain
intermediates B1-B9. Finally, with dichloromethane as a solvent and NaHSO4·SiO2 as
a catalyst, intermediates B1-B9 and substituted mercaptan were reacted at room
temperature to obtain the target compounds C1-C27. The chemical structures of these
compounds were identified by HRMS, and NMR (Supporting Information).
Figure 3
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
Anti-ToCV Activity of Compounds in vitro. The results of MST experiments are
shown in Table 2, which indicates that the target compounds of this series have good
binding affinity to ToCV-CP, with most of the compounds binding at the micromolar
level. In particular, compounds C5 and C22 showed excellent binding capacity to
ToCV-CP, with Kd values of 0.24 and 0.25 μM, which are significantly better than
that of RIB (15.62 μM) and slightly superior to those of NNM (0.44 μM) and lead
compound XCLSBM (0.36 μM). Compounds C9, C10, C12, and C19 showed good
binding capacity to ToCV-CP, with Kd values of 0.52, 1.63, 1.66, and 1.02 μM,
respectively, which are preferred over that of RIB, but second to those of NNM and
lead compound XCLSBM (Figure 3).
Structure-activity relationships (SARs). The in vitro binding affinities of ToCV-CP
with target compounds showed that when R2 was a chlorine atom
(electron-withdrawing group), these compounds had the worst anti-ToCV activity; for
example, C10 > C13 > C16 (H > 2-OCH3 > 2-Cl); C11 > C14 > C17 (H > 2-OCH3 >
2-Cl); C12 > C15 > C18 (H > 2-OCH3 > 2-Cl); C20 > C23 > C26 (H > 2-OCH3 >
2-Cl); C5 > C2> C8 (2-OCH3 > H > 2-Cl); C22 > C19 > C25 (2-OCH3 > H > 2-Cl);
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 25
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
however, this rule is not followed by all compounds. For example, C9 > C3 > C6
(2-Cl > H > 2-OCH3) and C27 > C21 > C24 (2-Cl > H> 2-OCH3). The changes of the
substituents of R1 have a slight effect on the anti-ToCV activity, as fluorine, chlorine
and bromine atoms are all electron-withdrawing groups; for example, C19 > C10 >
C1 (6-Br > 6-Cl > 6-F), but C20 > C2 > C11 (6-Br > 6-F > 6-Cl).
Figure 4
Anti-ToCV Activity of Compounds C5 and C22 in vivo. After confirming
tomato infection of ToCV by polymerase chain reaction (PCR), the PCR reaction
produced an expected site amplicon (774 bp), as shown in Figure 4. The side branches
of tomato infected with ToCV were cut off and soaked it in tap water for 5 days, and
then situated in a 50 μg/mL compound solution for 7 days. The result showed that the
expression levels of the ToCV-CP gene in different compound treatment groups
showed large differences. As shown in Figure 5, 81.05% and 87.59% reductions in
ToCV-CP gene expression levels could be achieved in the groups treated with
compounds C5 and C22 in the tomato sample, respectively, which were obviously
superior to the water (CK) treatment, the NNM treatment (43.88%) and the lead
compound XCLSBM treatment (63.56%) counterparts. The results of in vivo
experiments showed that the expression level of ToCV-CP in tomato can be
significantly reduced by compounds C5 and C22, which have certain controlling
effects on tomato chlorotic virus.
Figure 5
In summary, twenty-seven novel 4(3H)-quinazolinone derivatives bearing
8
ACS Paragon Plus Environment

Page 9 of 25
Journal of Agricultural and Food Chemistry
162
dithioacetal moiety were designed and synthesized. The results showed that most of
the compounds had good binding capacity to ToCV-CP. In particular, compounds C5
and C22 had excellent Kd to ToCV-CP, with values of 0.24 and 0.25 μM, respectively.
The in vivo experiments further indicate that the ToCV-CP gene expression levels are
reduced when tomatoes are treated with compounds C5 and C22. Our present work
indicated that novel 4(3H)-quinazolinone derivatives containing dithioacetal moiety
can be used as novel anti-ToCV agents to control this plant virus disease.
163
164
165
166
167
168
169
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 25
170
171
172
173
174
175
176
177
178
179
180
ASSOCIATED CONTENT
Supporting information
1
13
Characterization data, H and C NMR spectra, and HRMS for the title compounds
C1−C27 are shown in the Supplementary Information. The data of the interactions
between C1−C27 and ToCV-CP are also listed in the Supporting Information.
AUTHOR INFORMATION
Corresponding Authors
*E-mail for Baoan Song: songbaoan22@yahoo.com. *Phone: 86-851-88292170.
Fax: 86-851-83622211.
*E-mail: dyhu@gzu.edu.cn. Tel.: 851-8829-2170. Fax: 86-8518829-2170.
ORCID
181
182
183
184
185
186
187
188
Baoan Song: 0000-0002-4237-6167
Deyu Hu: 0000-0001-7843-371X
Guangcheng Zu: 0000-0003-4677-2957
Xiuhai Gan: 0000-0002-4070-0824
Dandan Xie: 0000-0002-2531-1038
Huanyu Yang: 0000-0001-5842-9166
Awei Zhang: 0000-0003-0479-9705
Shaoyuan Li: 0000-0001-6280-9129
10
ACS Paragon Plus Environment

Page 11 of 25
Journal of Agricultural and Food Chemistry
189
ACKNOWLEDGMENTS
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
This work was supported by the National Natural Science Foundation of China (no.
21867002 and 21732002).
NOTES
The authors declare no competing financial interest.
ABBREVIATIONS
1H NMR, 1H nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance;
HRMS, High-resolution mass spectrometry; NNM, Ningnanmycin; RIB, Ribavirin;
XCLSBM, Xiangcaoliusuobingmi; ToCV, tomato chlorosis virus; CP, coat protein;
MST, microscale thermophoresis; ToCV-CP, Tomato chlorosis virus coat protein.
REFERENCES
(1) Wisler, G. C.; Li, R. H.; Liu, H. Y.; Lowry, D. S.; Duffus, J. E. Tomato chlorosis
virus: a new whitefly-transmitted, phloem-limited, bipartite closterovirus of
tomato. Phytopathology 1998, 88, 402-409.
(2) Martelli, G. P.; Agranovsky, A. A.; Barjoseph, M.; Boscia, D.; Candresse, T.;
Coutts, R. H. A.; Dolja, V. V.; Falk, B. W.; Gonsalves, D.; Jelkmann, W.;
Karasev, A. V.; Minafra, A. Namba, S.; Vetten, H. J.; Wisler, G. C.; Yoshikawa,
N. The family Closteroviridae revised．Arch. Virol. 2002, 147, 2039-2044
(3) Tsai, W. S.; Shih, S. L.; Green, S. K.; Hanson, P.; Liu, H. Y. First report of the
occurrence of tomato chlorosis virus and tomato infectious chlorosis virus in
Taiwan. Plant Dis. 2004, 88, 311.
(4) Juarez, M.; Martinez, O.; Jorda, C. Current status and newly discovered natural
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 25
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
hosts of tomato infectious chlorosis virus and tomato chlorosis virus in Spain.
Plant Dis. 2004, 88, 82.
(5) Fortes, I. M.; Navas-Castillo, J. Potato, an experimental and natural host of the
crinivirus tomato chlorosis virus. Eur. J. Plant Pathol. 2012, 134, 81-86.
(6) Wintermantel, W. M.; Wisler, G. C. Vector specificity, host range, and genetic
diversity of tomato chlorosis virus. Plant Dis. 2006, 90, 814-819.
(7) Bese, G.; Boke, K.; Krizbai, L. First report of tomato chlorosis virus in tomato
from Huangary. Plant Dis. 2011, 95, 363.
(8) Sedyo, H.; Tomohide, N.; Haruki, S.; Hiroki, A.; Seiichi, O. Yellowing disease of
tomato caused by Tomato infectious chlorosis virus newly recognized in Japan. J.
Gen. Plant Pathol. 2003, 69, 61-64.
(9) Navas-Castillo, J.; Camero, R.; Bueno, M.; Moriones, E. Severe yellowing
outbreaks in tomato in Spain associated with infection of tomato chlorosis virus.
Plant Dis. 2000, 84, 835-837.
(10) Segev, L.; Wintermantel, W. M.; Polston, J. E.; Lapidot, M. First report of
tomato chlorosis virus in Israel. Plant Dis. 2004, 88, 1160
(11) Arruabarrena, A.; Rubio, L.; Gonzalez-Arcos, M.; Maeso, D.; Fonseca, M. E. N.;
Boiteux, L. S. First report of tomato chlorosis virus infecting tomato crops in
Urugay. Plant Dis. 2014, 98, 1445.
(12) Wintermantel, W. M.; Wisler, G. C.; Anchieta, A. G.; Liu, H. Y.; Karasev, A. V.;
Tzanetakis, I. E. The complete nucleotide sequence and genome organization of
tomato chlorosis virus. Arch. Virol. 2005. 150, 2287-2298.
12
ACS Paragon Plus Environment

Page 13 of 25
Journal of Agricultural and Food Chemistry
233
(13) Wisler, G. C.; Duffus, J. E.; Liu, H. Y.; Li, R. H. Ecology and epidemiology of
whitefly-transmitted closteroviruses. Plant Dis. 1998, 82, 270-280.
(14) Karasev, A. V. Genetic diversity and evolution of closteroviruses. Annu. Rev.
Phytopathol. 2000, 38, 293-324.
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
(15) Lozano, G.; Moriones, E.; Navas-Castillo, J. Complete nucleotide sequence of
the RNA2 of the crinivirus tomato chlorosis virus. Arch. Virol. 2006, 151,
581-587.
(16) Alzhanova, D. V.; Hagiwara, Y.; Peremyslov, V. V.; Dolja, V. V. Genetic
analysis of the cell-to-cell movement of beet yellows closterovirus. Virology
2000, 268, 192-200.
(17) Satyanarayana, T.; Gowda, S.; Mawassi, M.; Albiach-Marti, M. R.; Ayllon, M.
A.; Robertson. C.; Garnsey, S. M.; Dawson, W. O. Closterovirus encoded
HSP70 homolog and p61 in addition to both coat proteins function in efficient
virion assembly. Virology 2000, 278, 253-265.
(18) Tian, T.; Rubio, L.; Yeh, H.; Crawford, B.; Falk, B. W. Lettuce infectious
yellows virus: in vitro acquisition analysis using partially purified virions and the
whitefly Bemisia tabaci. J. Gen. Virol. 1999, 80, 1111-1117.
(19) Xie, D. D.; Zhang, J.; Yang, H. Y.; Liu, Y. W.; Hu, D. Y.; Song, B. A. First
anti-ToCV activity evaluation of glucopyranoside derivatives containing a
dithioacetal moiety through a novel ToCVCP-oriented screening method. J.
Agric. Food Chem. 2019, 67, 7243-7248
(20) Zhang, J.; Zhao, Lei.; Zhu, C.; Wu, Z. X.; Zhang, G. P.; Gan, X. H.; Liu, D. Y.;
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 25
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
Pan, J. K.; Hu, D. Y.; Song, B. A. Facile synthesis of novel vanillin derivatives
incorporating a bis(2-hydroxyethyl)dithioacetal moiety as antiviral agents. J.
Agric. Food Chem. 2017, 65, 4582-4588.
(21) Chen, Jin.; Shi, J.; Yu, L.; Liu, D. Y.; Gan, X. H.; Song, B. A.; Hu, D. Y. Design,
synthesis, antiviral bioactivity, and defense mechanisms of novel dithioacetal
derivatives bearing a strobilurin moiety. J. Agric. Food Chem. 2018, 66,
5335-5345.
(22) Gao, X. W.; Cai, X. J.; Yan, K.; Song, B. A.; Gao, L. L.; Chen, Z. Synthesis and
antiviral bioactivities of 2-aryl-or2-methyl-3-(substituted-benzalamino)-4(3H)
-quinazolinone derivatives. Molecules 2007, 12, 2621-2642.
(23) Ma, J.; Li, P.; Li, X. Y.; Shi, Q. C.; Wan, Z. H.; Hu, D. Y.; Jin, L. H.; Song, B. A.
Synthesis and antiviral bioactivity of novel 3-((2-((1E, 4E))-3-oxo-5-arylpenta-1,
4-dien-1-yl) phenoxy) methyl)-4(3H)-quinazolinone derivatives. J. Agric. Food
Chem. 2014, 62, 8928-8934.
(24) Chen, M. H.; Li, P.; Hu, D. Y.; Zeng, S.; Li, T. X.; Jin, L. H.; Xue, W.; Song, B.
A. Synthesis, antiviral activity, 3D-QSAR, and interaction mechanisms study of
novel malonate derivatives containing quinazolin-4(3H)-one moiety. Bioorg.
Med. Chem. Lett. 2016, 26, 168-173.
(25) Li, H. Z.; He, H. Y.; Han, Y. Y.; Gu, X.; He, L.; Qi, Q. R.; Zhao, Y. L.; Yang, L.
A General Synthetic Procedure for 2-chloromethyl-4(3H)quinazolinone
derivatives and their utilization in the preparation of novel anticancer agents with
4-anilinoquinazoline scaffolds. Molecules 2010, 15, 9473-9485.
14
ACS Paragon Plus Environment

Page 15 of 25
Journal of Agricultural and Food Chemistry
277
(26) Ran, L. L.; Ding, Y.; Luo, L. Z.; Gan, X. H.; Li, X. Y.; Chen, Y. Z.; Hu, D. Y.;
Song, B. A. Interaction research on an antiviral molecule that targets the coat
protein of southern rice black-streaked dwarf virus. Int. J. Biol. Macromol. 2017,
103, 919-930.
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
(27) Zhou, D. G.; Xie, D. D.; He, F. C.; Song, B. A.; Hu, D. Y. Antiviral properties
and interaction of novel chalcone derivatives containing a purine and
benzenesulfonamide moiety. Bioorg. Med. Chem. Lett. 2018, 28, 2091-2097.
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 25
299
300
301
302
303
304
305
306
307
308
309
310
311
Figure Information
Figure 1. Design of the title compounds.
Figure 2. Synthesis route of the title compounds C1-C27.
Figure 3. Microscale thermophoresis (MST) results of the title compounds.
Figure 4. Results of samples tested in vivo through polymerase chain reaction (PCR).
Figure 5. Effects of compounds on the inhibition of ToCV-CP levels in tomato in
vivo.
312
313
314
315
316
317
318
319
320
321
322
323
16
ACS Paragon Plus Environment

Page 17 of 25
Journal of Agricultural and Food Chemistry
324
Table 1. Primers used in this study
Primer
ToCP F
ToCP R
qToCP-F2
qToCP-R
2
Sequence (5'-3')
Purpose
ATGGAGAACAGTGCCGTTGC
TTAGCAACCAGTTATCGATGC
TAGATGATGGCGTAGATGAC
ToCV identificationa
ToCV qPCRb
CTAGTGGAGTGTACCTTCAAT
qActin-F
qActin-R
TGCCATTCTCCGTCTTGACT
TGCAGTCTCGAGTTCCTGTT
Tomato reference genesb
325
326
327
a Primer sequences were reported by Segev et al.10
b
Primer sequences were provided by Zhou Tao of China Agricultural University
Friendship.
328
329
330
331
332
333
334
335
336
337
338
339
340
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 25
341
342
343
Table 2. Compound structures and their binding constants (Kd) with the ToCV coat
protein
O
NH
R1
R2
O
N
S
Compound
Kd value (μM)
R3
S
R3
R1
R2
R3
C1
C2
6-F
H
H
H
CH2CH3
3.49±2.27
5.67±2.92
6-F
CH2CH2CH3
CH(CH3)2
CH2CH3
C3
6-F
3.03+1.42
C4
6-F
2-OCH3
2-OCH3
2-OCH3
2-Cl
15.44±5.99
0.24±0.07
C5
6-F
CH2CH2CH3
CH(CH3)2
CH2CH3
C6
6-F
33.95±20.84
11.56±7.00
59.65±23.49
0.52±0.09
C7
6-F
C8
6-F
2-Cl
CH2CH2CH3
CH(CH3)2
CH2CH3
C9
6-F
2-Cl
C10
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Cl
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
6-Br
H
1.63±0.73
C11
H
CH2CH2CH3
CH(CH3)2
CH2CH3
9.98+3.08
C12
H
1.66±0.54
C13
2-OCH3
2-OCH3
2-OCH3
2-Cl
10.43±6.81
67.60±30.87
28.74±10.69
15.22±3.64
73.11±52.75
42.91±29.93
1.02+0.39
C14
CH2CH2CH3
CH(CH3)2
CH2CH3
C15
C16
C17
2-Cl
CH2CH2CH3
CH(CH3)2
CH2CH3
C18
2-Cl
C19
H
C20
H
CH2CH2CH3
CH(CH3)2
CH2CH3
4.82±3.13
C21
H
25.50±8.79
0.25±0.06
C22
2-OCH3
2-OCH3
2-OCH3
2-Cl
C23
CH2CH2CH3
CH(CH3)2
CH2CH3
42.69±22.76
48.65±32.42
9.64±8.79
C24
C25
C26
2-Cl
CH2CH2CH3
CH(CH3)2
44.61±24.51
10.71±4.15
0.44±0.26
C27
2-Cl
ningnanmycina
Ribavirina
XCLSBMb
15.62±7.15
0.36±0.24
344
345
aCommercially available agrichemicals were used as controls.
bLead compound was reported by Zhang et al.20
18
ACS Paragon Plus Environment

Page 19 of 25
Journal of Agricultural and Food Chemistry
346
347
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 25
Figure 1. Design of the title compounds.
379x82mm (300 x 300 DPI)
ACS Paragon Plus Environment

Page 21 of 25
Journal of Agricultural and Food Chemistry
Figure 2. Synthesis route of the title compounds C1-C27.
197x113mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 25
Figure 3. Microscale thermophoresis (MST) results of the title compounds.
279x165mm (200 x 200 DPI)
ACS Paragon Plus Environment

Page 23 of 25
Journal of Agricultural and Food Chemistry
Figure 4. Results of samples tested in vivo through polymerase chain reaction (PCR).
61x83mm (96 x 96 DPI)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 25
Figure 5. Effects of compounds on the inhibition of ToCV-CP levels in tomato in vivo.
192x122mm (150 x 150 DPI)
ACS Paragon Plus Environment

Page 25 of 25
Journal of Agricultural and Food Chemistry
TOC Graphic
ACS Paragon Plus Environment