Discovery of Potent and Novel Quinazolinone Sulfide Inhibitors with Anti-ToCV Activity

Article abstract of DOI:10.1021/acs.jafc.0c00686

A series of novel quinazolinone sulfide derivatives containing a dithioacetal moiety were designed and synthesized using Tomato chlorosis virus coat protein (ToCVCP) as a potential drug target, and the inhibitory effect of ToCV was systematically evaluate

Full text of DOI:10.1021/acs.jafc.0c00686

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Agricultural and Environmental Chemistry
Discovery of Potent and Novel Quinazolinone
Sulfide Inhibitors with Anti-ToCV Activity
Leilei Ran, Huanyu Yang, Liangzhi Luo, Maoxi Huang, and Deyu Hu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c00686 • Publication Date (Web): 16 Apr 2020
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Discovery of Potent and Novel Quinazolinone Sulfide Inhibitors with
Anti-ToCV Activity
Leilei Ran, Huanyu Yang, Liangzhi Luo, Maoxi Huang, Deyu Hu*
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
*Author to whom correspondence should be addressed; E-mail: dyhu@gzu.edu.cn;
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ABSTRACT
A series of novel quinazolinone sulfide derivatives containing a dithioacetal moiety
was designed and synthesized using Tomato chlorosis virus coat protein (ToCVCP) as
a potential drug target, and the inhibitory effect of ToCV was systematically
evaluated in vitro and in vivo. The experimental results showed that most of the
compounds presented strong affinity. Notably, the binding abilities of compounds D8
and D16 to ToCVCP both reached micromolar level, which were 0.19 and 0.83 μM,
respectively. The relative expression level of ToCVCP gene were detected by real
time quantitative polymerase chain reaction (qPCR) in Nicotiana benthamiana.
Compounds D8 and D16 significantly reduced the relative expression level of
ToCVCP gene by 93.34% and 83.47%, respectively, which were better than
conventional antiviral agents. This study laid a good foundation for the structural
design and modification of quinazolinone sulfide derivatives as anti-ToCV drugs.
KEYWORDS: quinazolinone sulfide derivatives, dithioacetal moiety, Tomato
chlorosis virus, coat protein, Nicotiana benthamiana, antiviral activity.
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INTRODUCTION
Vegetables are an essential part of human daily life but are always endangered by
plant virus diseases. Cucumber mosaic virus, Potato virus Y, Tobacco mosaic virus,
and Tomato chlorosis virus (ToCV) are common plant virus diseases. The usual
phenomenon of vegetables after infection with these viruses is a sharp decline in yield
and quality.1–3 Among them, ToCV is a relatively special plant virus. In 1998, a new
virus named ToCV was brought to light in tomatoes growing in greenhouses in
Columbia, Baker, Florida, Colorado, and others.4 This virus has swept across dozens
of countries, such as China, the United States, Brazil, and France, at an amazing pace
during the following two decades.5–8 From the bottom up, the tomato will show some
symptoms similar to malnutrition once it is infected with ToCV. The main
manifestation is that the leaves gradually become fragile, the mature leaves at the
bottom of the tomatoes become slightly greenish, and the middle and above leaves
become yellow and green.9 ToCV causes the early aging of tomatoes and seriously
reduces the yield.10 The chief route of transmission of ToCV is through whitefly.11
Therefore, ToCV is generally inhibited by controlling the numbers of whitefly to
block the transmission vector. However, there are still no antiviral drugs that can
effectively inhibit ToCV. This is a key scientific problem that needs to be solved
urgently.
ToCV belongs to Crinivirus in the family of Closteroviridae. It is composed of two
single-stranded sense RNA. RNA 1 can encode proteins that participate in the viral
RNA replication based on the homology of other viral duplicating factors and consists
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of four open reading frames, including domains of proteases, helicases, and
methyltransferases. RNA 2 consists of nine open reading frames, which contain genes
encoding a heat shock protein 70 (HSP70) and two proteins that participate in
wrapping viral RNA, which are called coat protein (CP) and minor coat protein
(mCP).12–14 The virus in the Closteroviridae family has a “rattlesnake” structure.15
Most of the RNA is wrapped by CP, and only 5% RNA is wrapped by mCP. CP plays
a significant part in viruses of the Closteroviridae family. If the CP is lost, the virions
will not be able to effectively assemble and will hamper the movement and migration
of cells.16,17 Therefore, taking ToCVCP as a potential drug target to screen anti-ToCV
activity compounds is helpful in finding alternative drugs that effectively inhibit
ToCV.
As a class of natural products, quinazolinone alkaloids are the main structural units of
traditional Chinese medicines such as dichroine, isofebrifugin, and tryptanthrin. These
components mainly exist in Dichroa febrifuga Lour of Saxifragaceae plants.18 And it
has gained much importance in recent years due to their excellent biological activity,
environmental friendliness, and other characteristics. Considering these advantages, a
commercial antifungal with a unique mechanism of action, systemicity, and
harmlessness was developed.19 Therefore, it demonstrates that quinazolinones have
great potential for commercialization and environmentally friendly advantage.
Quinazolinone is also an important lead compound and shows good activity against
plant viruses. Previous studies have found that quinazolinone compounds exhibit
good inhibitory activity against three types of viruses: TMV, CMV, and PVY.20–22
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Furthermore, it also processes antibacterial,23 anticancer,24 anticonvulsant25,
analgesic,26 and anti-inflammatory activities.27 In our previous research, we found that
dithioacetal derivatives have excellent anti-plant virus activity for the first time.28 In
another study, our research group synthesized a large number of compounds with
anti-plant virus activity, for example, methacrylate-containing29 and indole-containing
dithioacetal compounds.30 Therefore, we conclude that dithioacetal is an excellent
pharmacophore. A new and reliable model for screening anti-ToCV activity was
established in previous studies, and the anti-ToCV activity of a series of glycoside
derivatives containing dithioacetals was evaluated in tomato by using this method.35
On this basis, the goal of our research was to introduce dithioacetal into the
quinazolinone framework and designed and synthesized new quinazolinone sulfide
derivatives containing dithioacetal moiety. (Figure 1) The interaction of this target
compounds with ToCVCP was tested with ToCVCP as a potential drug target in vitro,
and Nicotiana benthamiana was used as a new anti-ToCV drug screening model plant
for verification in vivo for the first time. To the best of our knowledge, this is the first
time that quinazolinone sulfide inhibitors of ToCV were developed to support the
possibility of ToCVCP as a potential drug target.
Figure 1
Materials and Methods
General information
All original chemicals were purchased from legitimate commercial suppliers and can
be used directly. CDCl3 or DMSO-d6 was utilized as a solvent, and the spectrum data
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of the target compounds were recorded by using a Bruker DPX 400 MHz or JEOL-
ECX500 MHz (JEOL, Tokyo, Japan) nuclear magnetic resonance (NMR) instrument.
The melting points of the compounds were measured by using a WRX-4 microscope
melting points apparatus (Shanghai Yisi Instrument Equipment Co., Ltd., China).
High-resolution mass spectrometry data of compounds were recorded on a Thermo
Scientific Q Exactive (Thermo) instrument.
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General steps to synthesize compounds
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Intermediates A1–A731 and B32 were synthesized according to the methods reported
in the literature. In the presence of 3 equiv K2CO3, 1 equiv of intermediates A1–A7
was refluxed with 1 equiv of intermediate B in acetonitrile to acquire C1–C7. The
synthetic route of the target compounds D1–D28 was shown in Figure 2. Different
types of thiol (3.00 mmol) were condensed with intermediates C1–C7 in the presence
of NaHSO4·SiO2 (0.01 mmol) with dichloromethane (25 mL) as a solvent and stirred
at room temperature. NaHSO4·SiO2 was removed by filtration after completing the
reaction, and the solvent was evaporated. The remaining solid was separated by silica
gel column chromatography with petroleum ether/ethyl acetate (volume ratio 3:1) to
obtain the pure product. The characterization data for all of the synthesized
compounds are given in the Supporting Information.
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Figure 2
Preparation of ToCVCP
According to a previously reported method,33 after the bacterial solution was cultured
at 37 °C for 3 hours in Luria–Bertani medium, it was immediately induced with 1 mM
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IPTG and continued to be cultured for 4 hours. Then proteins were purified by affinity
chromatography using a HisTrap™ column (GE Healthcare, USA). The protein was
verified by using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) after being desalted. The result was obtained after Coomassie Blue
staining and decolorization.
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Interaction of compounds with ToCVCP
According to the literature,34 NanoTemper Monolith NT.115 (Germany) instrument
was utilized to study the binding affinities between a series of compounds and
ToCVCP through microscale thermophoresis (MST). Xiangcaoliusuobingmi and
Ningnanmycin were token as positive antiviral agents. Measurements were repeated
three times in average.
Anti-ToCV activity in vivo pot experiment
As one of the hosts of ToCV, N. benthamiana can be easily infected with ToCV.
Therefore, N. benthamiana that was suspected to be infected with ToCV was utilized
as experimental material. All samples were tested by using polymerase chain reaction
(PCR). The primer sequence used in PCR was based on means reported by our
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research group as before.
After N. benthamiana was infected with ToCV, the
compound was formulated into 500 μg/mL solution (2 mg of compound + 30 μL
DMF + 4 mL of 1% Tween water), and the solution was evenly smeared on the
leaves. N. benthamiana was placed into the artificial climate room alone. Samples
were taken before and after drug treatment and frozen at −80 °C after being fast
frozen by liquid nitrogen. Lastly, the ToCVCP gene expression level was quantified
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by real time quantitative polymerase chain reaction, thereby indirectly obtaining the
inhibitory effect of the target compounds on ToCV.
RESULTS AND DISCUSSION
Chemistry
The target compounds were obtained through four steps reaction by using anthranilic
acid and vanillin as starting materials. The synthetic route of D1–D28 is shown in
Figure 2. First, intermediates A1–A7 were obtained through cyclization between
anthranilic acid and various substituted phenyl isothiocyanates. Vanillin was
converted to intermediate B through a nucleophilic substitution reaction. Then,
intermediates A1–A7 reacted with intermediate B by a nucleophilic substitution to
obtain intermediates C1–C7. Intermediates C1–C7 were condensed with different
thiols to obtain the target compounds. All compounds were characterized by 1H NMR,
13C NMR, and HRMS.
Purification of ToCVCP
The desalted ToCVCP was determined by 12% SDS-PAGE, and the results are
presented in Figure 3A. An obvious band was observed in lane 1 at the 30 Kd
position and was the same location based on previous reports.35
Figure 3A
Figure 3B
Analysis of binding constants between compounds and ToCVCP
As can be seen from Table 1 and Figure 4, the results of the MST study showed that
the great majority of compounds exhibited good affinity with ToCVCP. The
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compounds with strong affinities to ToCVCP were D3, D8, D11, D19, and D25. The
dissociation constants (Kd) were 0.22, 0.19, 0.23, 0.27, and 0.39 μM, respectively,
which were slightly better than that of the antiviral agent Xiangcaoliusuobingmi (0.83
μM) and significantly better than that of the positive control agent Ningnanmycin
(1.53 μM). The Kd values of compounds D2, D12, and D16 also reached micromolar
level of 0.73, 0.72, and 0.81 μM, respectively, which were slightly better than that of
the control agent Ningnanmycin (1.53 μM) and equivalent to that of
Xiangcaoliusuobingmi (0.83 μM).
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Structure-activity relationships (SARs)
Structure-activity relationships were analyzed based on the preliminary results. The
type and position of the substituent of the title compounds greatly influenced their
binding ability. The R3 moiety has a greater effect on the affinity of the compound.
When R1 and R2 are the same and R3 is hydrophilic group, the affinity is improved.
For example, D2 and D28 > D1, D4 and D27; D12 > D6 and D7; D15 and D16 >
D14; D19 > D18; D22 > D20 and D21; D25 > D24 and D23. But compounds D10
and D26 do not follow this rule. In addition, branched-chain compounds (Isopropyl
and Tert-butyl) show lower affinity than straight-chain compounds, such as D3 > D5,
D4 > D27. When R1 and R3 are the same and R3 is a hydrophobic group, the stronger
the electron withdrawing ability of R2, the compound shows better affinity. For
example, D8 > D1, D11 > D4, with the exception D9 < D3. But in general, when R1 is
F and R3 is a hydrophilic group (2-hydroxyethyl, 2,3-dihydroxypropyl), it is beneficial
to enhance the binding ability of the compound with ToCVCP.
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Figure 4
Effects of D8 and D16 on ToCV in N. benthamiana
N. benthamiana was suspected to be infected with ToCV through several symptoms.
Then, molecular biology testing using reverse transcription PCR revealed that the
target band appeared at 700–1000 bp after PCR amplification (Figure 3B), which was
consistent with the theoretical value. (774 bp)35The results indicated that the all
samples had been infected with ToCV. Then, the compounds were prepared into a
500 μg/mL solution. The solution was spread evenly on the leaves and left in a
climate chamber after drug treatment. Obvious differences in ToCVCP gene
expression were observed between the drug-treated and control groups. The ToCV
gene expression level in N. benthamiana was obviously lower than that of CK (30 μL
DMF + 4 mL 1% Tween water) after treatment with compounds D8 and D16. The
compound D8 evidently reduced the relative expression of ToCVCP gene by 93.34%
(Figures 5 and 6). Xiangcaoliusuobingmi and NNM control groups also showed good
ToCV inhibitory activity, reaching 88.31% and 68.14%, respectively (Figures 5 and
6), but lesser than the D8 treatment groups. The relative expression level of the
ToCVCP gene in the compound D16 treatment group was also significantly reduced
by 83.47%, which was slightly lower to Xiangcaoliusuobingmi (88.31%) but was
evidently better than NNM (68.14%). The pot experiment in vivo demonstrated that
compounds D8 and D16 can obviously decrease the relative expression level of
ToCVCP gene in N. benthamiana.
In summary, our research goal was to find drugs that have high inhibitory effect on
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ToCV. For this purpose, we designed and synthesized novel quinazolinone sulfide
derivatives containing dithioacetal structure. The interactions between ToCVCP and
the target compounds were tested, and the anti-ToCV activity of compounds with
excellent affinity to ToCVCP was assessed in N. benthamiana. The results showed
that the interplay of target compounds D8 and D16 with ToCVCP can achieve strong
bonding and were consistent with the test results in vivo, which uncommonly reduced
the relative expression level of ToCVCP gene in N. benthamiana. Therefore,
compounds D8 and D16 can be utilized as anti-ToCV alternative drugs for further
drug development. This study laid the foundation for the synthesis of anti-ToCV
drugs.
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CONTENT ASSOCIATED
Supporting Information
1H NMR ,13C NMR, and HRMS spectrum of intermediates C1–C7 and the title
compounds D1–D28 are shown in the Supplementary Information.
AUTHOR INFORMATION
Corresponding Authors
*E-mail for Deyu Hu: dyhu@gzu.edu.cn; *Phone: 851-8829-2170. Fax: 86-851-8829-
2170.
ORCID
Deyu Hu: 0000-0001-7843-371X
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Leilei Ran: 0000-0003-3126-4175
Huanyu Yang: 0000-0001-5842-9166
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Liangzhi Luo: 0000-0001-5493-9791
Maoxi Huang: 0000-0002-3837-6703
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ACKNOWLEDGMENT
This study was supported by the National Natural Science Foundation of China (No.
21867002), National Key Research and Development Program of China
(2018YFD0200100), and Program of Introducing Talents of Discipline to Universities
of China (111 Program, D20023).
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
1H NMR, 1H nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance;
HRMS,
thiogalactopyranosid; ToCV, tomato chlorosis virus; CP, coat protein; MST,
microscale thermophoresis; Kd, dissociation constant; qPCR, real
high-resolution
mass
spectrometry;
IPTG,
isopropyl-β-D-
time quantitative polymerase chain reaction.
REFERENCES
(1) Tomlinson, J. A. Epidemiology and control of virus diseases of vegetables. Ann.
Appl. Biol. 1987, 110, 661−681.
(2) Bos L. Crop losses caused by viruses. Crop Prot. 1982, 1, 263−282.
(3) Scholthof, K. B. G.; Adkins, S.; Czosnek, H.; Palukaitis, P.; Jacquot, E.; Hohn, T.;
ACS Paragon Plus Environment

Page 13 of 26
Journal of Agricultural and Food Chemistry
244
Hohn, B.; Saunders, K.; Candresse, T.; Ahlquist, P.; Hemenway, C.; Foster, G. D.
Top 10 plant viruses in molecular plant pathology. Mol. Plant Pathol. 2011, 12,
938−954.
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
(4) 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.
(5) Tsai, W. S.; Shih, S. L.; Green, S. K.; Hanson, P. First report of the occurrence of
tomato chlorosis virus and tomato infectious chlorosis virus in Taiwan. Plant Dis.
2004, 88, 311.
(6) Barbosa, J. C.; Teixeira, A. P. M.; Moreira, A. G.; Camargo, L. E. A.; Rezende, J.
A. M. First report of tomato chlorosis virus infecting tomato crops in Brazil. Plant
Dis. 2008, 92, 1709−1709.
(7) Sundaraj.; Srinivasan, R.; Webster, C. G.; Adkins, S.; Riley, D. First report of
tomato chlorosis virus in Georgia. Plant Dis. 2011, 95, 881−881.
(8) Dalmon, A.; Bouyer, S.; Cailly, M.; Girard, M.; Jacquemond, M. First report of
tomato chlorosis virus and tomato infectious chlorosis virus in tomato crops in
France. Plant Dis. 2005, 89, 1243−1243.
(9) Wintermantel, W. M.; Wisler, G. C. Vector specificity, host range, and genetic
diversity of tomato chlorosis virus. Plant Dis. 2006, 90, 814−819.
(10) Velasco, L.; B, Simon.; Janssen, D.; Cenis, J. L. Incidences and progression of
tomato chlorosis virus disease and tomato yellow leaf curl virus disease in tomato
under different greenhouse covers in southeast spain. Ann. Appl. Biol. 2008, 153,
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 26
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
335−344.
(11) Kataya, A. R. A.; Stavridou, E.; Farhan, K.; Livieratos, I. C.; Nucleotide
sequence analysis and detection of a Greek isolate of Tomato chlorosis virus. Plant
Pathol. 2008, 57, 819–824.
(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.
(13) 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.
(14) Lozano, G.; Moriones, E.; Navas-Castillo, J. Complete sequence of the RNA1 of
a European tomato chlorosis virus. Arch. Virol. 2007, 152, 839−841.
(15) Agranovsky, A. A.; Lesemann, D. E.; Maiss, E.; Hull, R.; Atabekov, J. G.
"Rattlesnake" structure of a filamentous plant RNA virus built of two capsid
proteins. Proc. Natl. Acad. Sci. 1995, 92, 2470−2473.
(16) 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.
(17) 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.
(18) Fairbairn, J. W.; Lou, T. C. A pharmacognostical study of dichroa febrifuga lour.
ACS Paragon Plus Environment

Page 15 of 26
Journal of Agricultural and Food Chemistry
288
A chinese antimalarial plant. J.ꢀPharm.ꢀPharmacol. 1950, 2, 162−177.
(19) Wang, G. Y.; Zhou, Y. G.; Wang, H.; Kong, F. L. Synthesis of fungicide
fluquinconazole. Agrochemicals 2014, 53, 93−95.
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
(20) Luo, H.; Liu, J. J.; Jin, L. H.; Hu, D.Y.; Chen, Z.; Yang, S.; Wu, J.; Song, B. A.
Synthesis and antiviral bioactivity of novel (1E,4E)-1-aryl-5-(2-(quinazolin-4-
yloxy)phenyl)-1,4-pentadien-3-one derivatives. Eur. J. Med. Chem. 2013, 63,
662−669.
(21) 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.
(22) Xie, D. D.; Shi, J.; Zhang, A. W.; Lei, Z. W.; Zu, G. C.; Fu, Y.; Gan, X. H.; Yin,
L. M.; Song, B. A.; Hu, D. Y. Syntheses, antiviral activities and induced resistance
mechanisms of novel quinazoline derivatives containing a dithioacetal moiety.
Bioorg. Chem. 2018, 80, 433−443.
(23) Wang, X.; Li, P.; Li, Z. N.; Yin, J.; He, M.; Xue, W.; Chen, Z. W.; Song, B. A.
Synthesis and bioactivity evaluation of novel arylimines containing 3-aminoethyl-2-
[(p-trifluoromethoxy) aniline]-4(3H)-quinazolinone moiety. J. Agric. Food Chem.
2013, 61, 9575−9582.
(24) Ahmed, M. F.; Youns, M.; Belal, A. Design, synthesis, molecular docking and
anti-breast cancer activity of novel quinazolinones targeting estrogen
receptor. Acta. Pol. Pharm. 2016, 73, 115−127.
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311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
(25) Rajasekaran, A.; Rajamanickam, V.; Darlinquine, S. Synthesis of some new
thioxoquinazolinone derivatives and a study on their anticonvulsant and antimicrobial
activities. Eur. Rev. Med. Pharmacol. Sci. 2013, 17, 95−104.
(26) Kumar, A.; Sharma, S.; Archana, B. K.; Sharma, S.; Panwar, H. ； Singh, T.;
Srivastava, V. K. Some new 2,3,6-trisubstituted quinazolinones as potent anti-
inflammatory, analgesic and cox-ii inhibitors. Bioorg. Med. Chem. 2003, 11,
5293−5299.
(27) Farag, A. A.; Khalifa, E. M.; Sadik, N. A.; Abbas, S. Y.; Al-Sehemi, A. G.;
Ammar, Y. A. Synthesis, characterization, and evaluation of some novel 4(3H)-
quinazolinone derivatives as anti-inflammatory and analgesic agents. Med. Chem.
Res. 2013, 22, 440−452.
(28) Zhang, J.; Zhao, L.; Zhu, C.; Wu, Z. X.; Zhang, G. P.; Gan, X. H.; Liu, D. Y.;
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.
(29) Chen, J.; 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.
(30) Wei, C. L.; Zhang, J.; Shi, J.; Gan, X. H.; Hu, D. Y.; Song, B. A. Design,
Synthesis, Antiviral Bioactivity, and Defense Mechanisms of Novel Dithioacetal
Derivatives Bearing a Strobilurin Moiety. J. Agric. Food Chem. 2019, 67, 13882-
ACS Paragon Plus Environment

Page 17 of 26
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332
13891.
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
(31) Mohamed, M. A.; Ayyad, R. R.; Shawer, T. Z.; Abdel-Aziz, A. A. M.; El-Azab,
A. S. Synthesis and antitumor evaluation of trimethoxyanilides based on 4(3H)-
quinazolinone scaffolds. Eur. J. Med. Chem. 2016, 112, 106−113.
(32) Konken, C. P.; Haufe, G.; Brömmeld, K.; Bernhard, W.; Schäfers, M.; Wagnera,
S.; Hugenberg, V. Development of symmetric O-BODIPYs with different optical
properties as building blocks for the synthesis of ligands for multimodal
imaging. Dyes and Pigments 2018, 158, 88−96.
(33) Jacquemond, M.; Verdin, E.; Dalmon, A.; Guilbaud, L.; Gognalons, P.
Serological and molecular detection of tomato chlorosis virus and tomato infectious
chlorosis virus in tomato. Plant Pathol. 2008, 58, 210−220.
(34) 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.
(35) Xie, D. D.; Zhang, J.; Yang, H.; Liu, Y.; 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.
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Information of Figure
Figure 1. Design of target compounds.
Figure 2. Synthetic route of the title compounds D1–D28.
Figure 3. (A) ToCVCP was identified by 12% SDS-PAGE. (B) Identified results of
samples used in vivo through PCR.
Figure 4. Interaction of target compounds with ToCVCP using MST.
Figure 5. Anti-ToCV activity of compounds D8 and D16 were tested in vivo on N.
benthamiana at 500 μg/mL. XCLSBM and Ningnanmycin were used as control.
Figure 6. Effect of compounds on the inhibition of ToCVCP gene expression levels
in N. benthamiana.
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Table 1. Binding constants of title compounds (Kd) with ToCVCP
R2
O
N
R1
O
N
S
R3
S
Compd.
Kd value (μM)
O
S
R3
R1
6-F
6-F
R2
H
H
R3
-Ethyl
-2-hydroxyethyl
D1
D2
24.01±14.37
0.73±0.17
D3
6-F
H
-Propyl
0.22±0.15
D4
D5
D6
D7
D8
D9
D10
D11
D12
D14
D15
D16
D18
D19
D20
D21
D22
6-F
6-F
6-Cl
6-Cl
6-F
6-F
6-F
6-F
6-Cl
6-CH3
6-CH3
6-CH3
6-Cl
6-Cl
6-Br
6-Br
6-Br
6-F
H
H
H
-Butyl
-Isopropyl
-Ethyl
-Propyl
-Ethyl
10.18±7.59
1.33±0.55
9.72±4.77
6.34±3.38
0.19±0.09
1.97±1.23
7.02±3.79
0.23±0.09
0.72±0.64
41.14±33.94
2.61±1.84
0.81±0.27
68.43±11.56
0.27±0.17
252.75±132.38
180.25±48.12
1.54±0.90
118.63±104.51
7.05±2.62
0.39±0.13
7.13±3.45
84.92±39.53
2.21±1.56
0.83±0.16
1.53±0.48
H
4-Cl
4-Cl
4-Cl
4-Cl
H
4-CH3
4-CH3
4-CH3
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
-Propyl
-2-hydroxyethyl
-Butyl
-2-hydroxyethyl
-Propyl
-2-hydroxyethyl
-2,3-dihydroxypropyl
-Propyl
-2-hydroxyethyl
-Ethyl
-Propyl
-2-hydroxyethyl
-Ethyl
D23
D24
D25
D26
D27
D28
4-OCH3
4-OCH3
4-OCH3
4-Cl
6-F
6-F
6-F
6-F
-Propyl
-2-hydroxyethyl
-2,3-dihydroxypropyl
-Tert-butyl
H
H
6-F
-2,3-dihydroxypropyl
XCLSBMb
Ningnanmycinc
363
364
aAverage of three replicates; XCLSBMb and Ningnanmycinc were used as positive
control.
365
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Figure 1. Design of target compounds.
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Journal of Agricultural and Food Chemistry
Figure 2. Synthetic route of the title compounds D1–D28.
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Figure 3. (A) ToCVCP was identified by 12% SDS-PAGE. (B) Identified results of samples used in vivo
through PCR.
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Figure 4. Interaction of target compounds with ToCVCP using MST.
276x178mm (300 x 300 DPI)
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Figure 5. Anti-ToCV activity of compounds D8 and D16 were tested in vivo on N. benthamiana at 500
μg/mL. XCLSBM and Ningnanmycin were used as control.
156x80mm (300 x 300 DPI)
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Figure 6. Effect of compounds on the inhibition of ToCVCP gene expression levels in N. benthamiana.
236x122mm (300 x 300 DPI)
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TOC Graphic
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