Facile Synthesis of Novel Vanillin Derivatives Incorporating a Bis(2-hydroxyethyl)dithhioacetal Moiety as Antiviral Agents

Article abstract of DOI:10.1021/acs.jafc.7b01035

A series of vanillin derivatives incorporating a bis(2-hydroxyethyl)dithioacetal moiety was designed and synthesized via a facile method. A plausible reaction pathway was proposed and verified by computational studies. Bioassay results demonstrated that target compounds possessed good to excellent activities against potato virus Y (PVY) and cucumber mosaic virus (CMV), of which, compound 6f incorporating a bis(2-hydroxyethyl)dithioacetal moiety, exhibited the best curative and protection activities against PVY and CMV in vivo, with 50% effective concentration values of 217.6, 205.7 μg/mL and 206.3, 186.2 μg/mL, respectively, better than those of ribavirin (848.0, 808.1 μg/mL and 858.2, 766.5 μg/mL, respectively), dufulin (462.6, 454.8 μg/mL and 471.2, 465.4 μg/mL, respectively), and ningnanmycin (440.5, 425.3 μg/mL and 426.1, 405.3 μg/mL, respectively). Current studies provide support for the application of vanillin derivatives incorporating bis(2-hydroxyethyl)dithioacetal as new antiviral agents.

Full text of DOI:10.1021/acs.jafc.7b01035

Subscriber access provided by UNIV OF ARIZONA
Article
Facile Synthesis of Novel Vanillin Derivatives Incorporating
a Bis(2-hydroxyethyl)dithioacetal Moiety as Antiviral Agents
Jian Zhang, Lei Zhao, Chun Zhu, Zengxue Wu, Guoping Zhang,
Xiuhai Gan, Dengyue Liu, Jianke Pan, De-Yu Hu, and Bao-An Song
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 May 2017
Downloaded from http://pubs.acs.org on May 29, 2017
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 free 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 accessible to all
readers and 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.
Journal of Agricultural and Food Chemistry 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 35
Journal of Agricultural and Food Chemistry
1
2
Facile Synthesis of Novel Vanillin Derivatives Incorporating a Bis(2ꢀ
hydroxyethyl)dithioacetal Moiety as Antiviral Agents
3
4
5
6
7
8
9
Jian Zhang,† Lei Zhao,† Chun Zhu,§ Zengxue Wu,† Guoping Zhang,† Xiuhai Gan,†
Dengyue Liu,† Jianke Pan,† Deyu Hu,*† and Baoan Song* †
†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
§School of Chemistry and Chemical Engineering, Guizhou University, Huaxi District,
Guiyang 550025, China
10
11
12
*Corresponding author (Tel.: 86ꢀ851ꢀ83620521; Fax: 86ꢀ851ꢀ83622211; Eꢀmail:
songbaoan22@yahoo.com)
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 35
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
ABSTRACT:
A
series of vanillin derivatives incorporating
a
bis(2ꢀ
hydroxyethyl)dithioacetal moiety was designed and synthesized via a facile method. A
plausible reaction pathway was proposed and verified by computational studies. Bioassay
results demonstrated that target compounds possessed good to excellent activities against
potato virus Y (PVY) and cucumber mosaic virus (CMV), of which, compound 6f
incorporating a bis(2ꢀhydroxyethyl)dithioacetal moiety, exhibited the best curative and
protection activities against PVY and CMV in vivo, with 50% effective concentration
values of 217.6, 205.7 ꢀg/mL and 206.3, 186.2 ꢀg/mL, respectively, better than those of
ribavirin (848.0, 808.1 ꢀg/mL and 858.2, 766.5 ꢀg/mL, respectively), dufulin (462.6,
454.8 ꢀg/mL and 471.2, 465.4 ꢀg/mL), and ningnanmycin (440.5, 425.3 ꢀg/mL and 426.1,
405.3 ꢀg/mL, respectively). Current studies provide support for the application of vanillin
derivatives incorporating bis(2ꢀhydroxyethyl)dithioacetal as new antiviral agents.
KEYWORDS: vanillin, bis(2ꢀhydroxyethyl)dithioacetal, antiviral activity, potato virus Y,
cucumber mosaic virus
2
ACS Paragon Plus Environment

Page 3 of 35
Journal of Agricultural and Food Chemistry
28
INTRODUCTION
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Potato virus Y (PVY) and cucumber mosaic virus (CMV) are important destructive plant
pathogens. They can infect various crops, such as potato, cucumber, tobacco, pepper, and
other economic corps, and cause significant economic loss.1−3 To date, few antiviral
agents can effectively control PVY or CMV because various aphids, which are difficult to
control with pesticides, can rapidly inoculate plants.2,4 Ningnanmycin, dufulin and
ribavirin (Figure 1) are widely used for preventing plant viruses. However, their field
efficacies are unsatisfactory.5−7 In recent years, biologists have found several compounds
with good antiviral activities,8−11 but few virucides have yet been used in the field. Thus,
a continuing need for the development of new, highly active antiviral agents exists.
Natural products are important sources for the development of new drugs, featuring
unique modes of action, low mammalian toxicity, easy decomposition, environmental
friendliness, desirable biological activities, and specificity to target species.12,13 Vanillin
(4ꢀhydroxyꢀ3ꢀmethoxybenzaldehyde), a natural product derived from orchids (Vanilla
planifolia, V. pompona, or V. tahitiensis),14 has attracted the attention of biologists for
several reasons. First, vanillin as a flavoring compound, possesses general bioꢀsafety and
has extensive uses in the food, nutraceutical, beverage, and pharmaceutical industries.15,16
Second, vanillin possesses a simple chemical structure (Figure 1), which may reduce the
possibility of difficult synthesis to some extent. Third, vanillin has desirable biological
activities,15 such as antitumor,17–19 antioxidant,20,21 antimicrobial,22,23 antifungal,24–26
antiinflammatory,27 antimutagenic,28 and antiproliferative29 activities. In addition, vanillin
derivatives possess desirable antifungal30 and antibacterial31 activities. However, studies
on the application of vanillin derivatives in antiviral pesticides have not been reported.
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 35
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
Ketene dithioacetal derivatives also demonstrate various biological properties, such as
antiviral,32 antibacterial,33 antifungal,34,35 and insecticidal properties.36 Isoprothiolane is a
commercial fungicide containing
a
ketene dithioacetal moiety. Bis(2ꢀ
hydroxyethyl)dithioacetals display a hydrogenꢀbonded infinite supraꢀmolecular helical
structure, which is similar to biological systems,37 and have attracted considerable interest
because of their biocompatibility.38
Considering the biosecurity, simple chemical structure, and extensive bioactivities of
vanillin and the biocompatibility of bis(2ꢀhydroxyethyl)dithioacetal, we synthesized new
simple chemical structures with antiviral activities (Figure 2). These structures include
bis(2ꢀhydroxyethyl)dithioacetals, vanillin derivatives incorporating
a
bis(2ꢀ
hydroxyethyl)dithioacetal moiety, and vanillin derivatives incorporating a dithioacetal
moiety. Their antiviral activities against PVY and CMV were evaluated by using the halfꢀ
leaf method. The structureꢀactivity relationships (SARs) were investigated on the basis of
biological activity.
MATERIALS AND METHODS
General Information. All reagents were purchased from Aladdin Chemicals Co.
(Shanghai, China) and were used without further purification. All solvents used in the
reactions without further drying and purification. Reaction progress was monitored by
thinꢀlayer chromatography on silica gel GF254 with UV detection. Melting points were
uncorrected and determined on a WRXꢀ4 monocular microscope (Shanghai Yice
Apparatus & Equipment Co., Ltd., Shanghai, China), and the thermometer was
uncorrected. 1H NMR spectra were obtained by using ECXꢀ500 and Varian Mercury plus
500 MHz spectrometers (JEOL, Tokyo, Japan) with CDCl3 or D2O as a solvent. Chemical
4
ACS Paragon Plus Environment

Page 5 of 35
Journal of Agricultural and Food Chemistry
74
shifts (δ) were given in parts per million (ppm) and were measured downfield from
internal tetramethylsilane. 13C NMR spectra were recorded by using a JEOL ECX–500
spectrometer (125 MHz) with CDCl3 or D2O as a solvent. Infrared (IR) spectra were
recorded on a VECTOR 22 spectrometer (Bruker, Karlsruhe, Germany) using KBr disks.
HRMS data were measured on Thermo Scientific Q Exactive (Thermo Scientific,
Missour, MO). Density functional theory (DFT) calculations were carried out using the
Gaussian 09 package.
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
General Procedure for Preparation of Bis(2-hydroxyethyl)dithioacetals, 3a–3t.
ZrCl4 (2.2 mg, 0.01 mmol) was added to the mixture of aldehyde (1.0 mmol) and 2ꢀ
mercaptoethanol (140 ꢁL, 2.0 mmol) under solventꢀfree conditions, and the mixture was
stirred for 5–30 min at room temperature. After completing the reaction, 10 mL of water
was added into the above reaction mixture. Then, the mixture was extracted with 10 mL
of dichloromethane. The organic layer was dried over anhydrous Na2SO4. Finally,
dichloromethane was removed in a rotary evaporator and the crude product was purified
by column chromatography and recrystallization using hexane/EtOAc (1:2, v/v). The
representative data for 3a is shown below.
2,2'ꢁ(phenylmethylene)bis(2ꢁhydroxyethyl)dithioacetal (3a): Yield: 231 mg (95%);
1
white solid; m.p. 58–60 °C, lit.37 59 °C; IR (KBr): 3306 cm−1, (–OH group); H NMR
(CDCl3, 500 MHz) δ 7.45 (dd, J = 8.2, 1.0 Hz, 2H, Ar–H), 7.39–7.32 (m, 2H, Ar–H),
7.30 (dd, J = 5.0, 3.7 Hz, 1H, Ar–H), 5.07 (s, 1H, –SCHS–), 3.73 (t, J = 5.8 Hz, 4H, –
OCH2–), 2.84 (dt, J = 14.0, 5.7 Hz, 2H, –SCH2–), 2.71 (dt, J = 14.0, 5.9 Hz, 2H, –SCH2–),
2.37 (s, 2H, –OH, D2O exchangeable); 13C NMR (CDCl3, 125 MHz) δ 139.9 (1C), 128.8
(2C), 128.2 (1C), 127.6 (2C), 61.25 (2C), 53.0 (1C), 35.5 (2C); HRMS (ESI) m/z for
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 35
97
98
C11H16O2NaS2 [M+Na]+ calcd. 267.0484, found: 267.0482.
General Procedure for the Preparation of Vanillin Derivatives, 5a–5n. A solution
of vanillin (10 mmol) and substituted benzyl chloride (10 mmol) in acetonitrile (40 mL)
was heated under reflux for 6 h in the presence of K2CO3 (100 mol%) and KI (5 mol%).
The mixture was cooled down to room temperature, and the solvent was removed in
vacuo. The residue was washed with water and recrystallized using ethanol affording
vanillin derivatives. The representative data for 5a is shown below.
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
4ꢁ(benzyloxy)ꢁ3ꢁmethoxybenzaldehyde (5a): Yield: 232 mg (96%); white solid; m.p.
1
55–57 °C; H NMR (CDCl3, 500 MHz): δ 9.80 (s, 1H, –CHO), 7.43 (t, J = 4.9 Hz, 3H,
Ar–H), 7.38 (ddd, J = 6.1, 4.3, 2.4 Hz, 3H, Ar–H), 7.35–7.28 (m, 1H, Ar–H), 6.98 (d, J =
13
8.2 Hz, 1H, Ar–H), 5.24 (s, 2H, –OCH2–), 3.93 (s, 3H, –OCH3); C NMR (CDCl3, 125
MHz): δ 191.0 (1C), 153.8 (1C), 150.2 (1C), 136.1 (1C), 130.4 (1C), 128.8 (2C), 128.3
(1C), 127.3 (2C), 126.7 (1C), 112.5 (1C), 109.4 (1C), 71.0 (1C), 56.1 (1C).
General Procedure for Synthesis of Vanillin Derivatives Incorporating Bis(2-
hydroxyethyl)dithioacetal, 6a–6n. ZrCl4 (2.2 mg, 0.01 mmol) was added to the solution
of vanillin derivatives (1.0 mmol) and 2ꢀmercaptoethanol (140 ꢁL, 2.0 mmol) in THF (2
mL). Then, the mixture was stirred for 5–30 min at room temperature. After completing
the reaction, the solvent was removed in vacuo. CH2Cl2 (10 mL) and H2O (10 mL) were
added to the resulting mixture, and the two layers were separated. The organic phase was
dried over anhydrous Na2SO4, filtered, and then concentrated to obtain the crude product,
the crude product was purified by column chromatography and recrystallization using
hexane/EtOAc (1:2, v/v). The representative data for 6a is shown below.
2,2'ꢁ(((4ꢁ((4ꢁchlorobenzyl)oxy)ꢁ3ꢁmethoxyphenyl)methylene) bis(2ꢁhydroxyethyl)dithioꢁ
6
ACS Paragon Plus Environment

Page 7 of 35
Journal of Agricultural and Food Chemistry
1
120
acetal (6a): Yield: 393 mg (95%); white solid; m.p. 80–82 °C; H NMR (500 MHz,
CDCl3) δ 7.40–7.30 (m, 4H, Ar–H), 7.04 (d, J = 2.1 Hz, 1H, Ar–H), 6.88 (dd, J = 8.2, 2.1
Hz, 1H, Ar–H), 6.76 (d, J = 8.2 Hz, 1H, Ar–H), 5.09 (s, 2H, –OCH2–), 5.02 (s, 1H, –
SCHS–), 3.90 (s, 3H, –OCH3), 3.73 (q, J = 5.9 Hz, 4H, –OCH2–), 2.83 (dt, J = 14.0, 5.7
Hz, 2H, –SCH2–), 2.70 (dt, J = 14.0, 5.9 Hz, 2H, –SCH2–), 2.28 (s, 2H, –OH, D2O
exchangeable); 13C NMR (125 MHz, CDCl3) δ 150.0 (1C), 147.9 (1C), 135.5 (1C), 133.8
(1C), 133. (1C), 128.9 (2C), 128.7 (2C), 120.0 (1C), 113.4 (1C), 110.9 (1C), 70.3 (1C),
61.4 (2C), 56.1 (1C), 53.0 (1C), 35.8 (2C); HRMS (ESI) m/z for C19H24O4NaS2 [M+Na]+
calcd. 403.1008, found. 403.1007.
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
General Procedure for Synthesis of Vanillin Derivatives Incorporating Dithioacetal,
7–11. A mixture of 1.0 mmol vanillin derivatives and 2.0 mmol 4ꢀchlorothiophenol (or
2.0 mmol 4ꢀfluorothiophenol, 2.0 mmol furfurylmercaptan, 2.0 mmol ethanethiol, and 1.0
mmol dithioglycol) in THF solvent (2 mL) was stirred at room temperature. After
complete dissolution, ZrCl4 (2.2 mg, 0.01 mmol) was added to the mixture. The solution
was stirred for 5–30 min. After completing the reaction, the solvent was removed in
vacuo, treated with H2O (10 mL) and CH2Cl2 (10 mL), and the two layers were separated.
Then, the organic layer was dried over anhydrous Na2SO4. Finally, dichloromethane was
removed in a rotary evaporator and the crude product was purified by column
chromatography and recrystallization using hexane/EtOAc (4:1, v/v). The representative
data for 7 is shown below.
((4ꢁ((4ꢁchlorobenzyl)oxy)ꢁ3ꢁmethoxyphenyl)methylene)bis((4ꢁchlorophenyl)sulfane) (7):
Yield: 393 mg (95%); white solid; mp 80–82 °C; IR (KBr): 3065.4, 3002.3, 2961.8,
2930.9, 2876.9, 1515.1, 1465.0, 1411.9, 1385.9, 1333.8, 1264.3, 1240.2, 1144.8, 1092.7,
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 35
1
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
1035.8, 997.2, 812.0, 732.9 cm–1; H NMR (500 MHz, CDCl3) δ 7.37–7.33 (m, 4H, Ar–
H), 7.25 (d, J = 2.2 Hz, 1H, Ar–H), 7.24–7.23 (m, 3H, Ar–H), 7.22–7.21 (m, 3H, Ar–H),
7.20 (d, J = 2.1 Hz, 1H, Ar–H), 6.88 (d, J = 1.7 Hz, 1H, Ar–H), 6.71 (dd, J = 7.7, 5.1 Hz,
2H, Ar–H), 5.29 (s, 1H, –SCHS–), 5.08 (s, 2H, –OCH2–), 3.82 (s, 3H, –OCH3); 13C NMR
(125 MHz, CDCl3) δ 149.7 (1C), 147.8 (1C), 135.4 (1C), 134.4 (2C), 134.3 (4C), 133.9
(1C), 132.5 (2C), 132.1 (1C), 129.1 (4C), 128.9 (2C), 128.8 (2C), 120.3 (1C), 113.5 (1C),
111.2 (1C), 70.3 (1C), 60.6 (1C), 56.0 (1C); HRMS (ESI) m/z for C27H21O2Cl3NaS2
[M+Na]+ calcd. 568.9941, found. 568.9930.
DFT calculations. All geometric structures involved in the reactions were fully
optimized without any symmetry constraints using B3LYP hybrid functional in
combination with the effective core potential coupled with the LANL2DZ basis set for
the Zr atom and the allꢀelectron 6ꢀ31G(d) basis set for other atoms (B3LYP/(LANL2DZ,
6ꢀ31G(d))). Subsequently, frequencies were computed at the same level to confirm the
nature of stationary point on energy surfaces.
Antiviral Biological Assay. Antiviral Biology against PVY. PVY purification and antiꢀ
PVY activity evaluation of the compounds were performed as previously described.39
Antiviral Biology against CMV. CMV purification and antiꢀCMV activity evaluation of
the compounds were performed as previously reported.40, 41
RESULTS AND DISCUSSION
Chemistry. Optimization of Reaction Conditions. We investigated the ZrCl4ꢀcatalyzed
condensation of benzaldehyde (1a) with 2ꢀmercaptoethanol. When benzaldehyde (1a)
was treated with a catalytic amount of ZrCl4 ([Zr] = 20 mol%) and 2ꢀmercaptoethanol
(200 mol%) at room temperature using CH2Cl2, toluene, hexane or xylene as a solvent,
8
ACS Paragon Plus Environment

Page 9 of 35
Journal of Agricultural and Food Chemistry
166
the desired product bis(2ꢀhydroxyethyl)dithioacetal derivative (3a) was observed in trace.
However, when the solvent was replaced with THF, the desired product bis(2ꢀ
hydroxyethyl)dithioacetal derivative (3a) was obtained with a yield of 96%. This result
may be associated with the catalytic activity of ZrCl4, which could be activated when
ZrCl4 coordinated with THF.42,43 Then, benzaldehyde (1a) was reacted with 2 equiv of 2ꢀ
mercaptoethanol at room temperature in the presence of 20 mol% ZrCl4 under solventꢀ
free conditions. This reaction gave bis(2ꢀhydroxyethyl)dithioacetal derivative (3a) as the
sole product in 98% yield. To further optimize the amount of ZrCl4, several trial reactions
involving different catalyst amounts were examined. ZrCl4 catalyst (1 mol%) was
sufficient for the condensation of benzaldehyde (1a) with 2ꢀmercaptoethanol.
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
Synthesis of Bis(2ꢁhydroxyethyl)dithioacetals (3a–3t). The scope of the reaction was
investigated under the optimized conditions (Figure 3). All substituted aldehydes, 1a–1t,
using ZrCl4 rapidly reacted, producing to bis(2ꢀhydroxyethyl)dithioacetals 3a–3r in good
to excellent isolated yields of 81%–96%. The reactivities of benzaldehyde and aromatic
aldehydes containing electronꢀdonating and electronꢀwithdrawing substituents in the ring
were investigated. Electronꢀrich aromatic aldehydes (1b–1f) exhibited excellent yields in
a short reaction time, and electronꢀwithdrawing aromatic aldehydes (1m–1p) produced
slightly lower yields in a longer reaction time. Similarly, 2ꢀnaphthaldehyde,
cinnamaldehyde, 2ꢀfuraldehyde, and 2ꢀthiophenealdehyde were also converted to bis(2ꢀ
hydroxyethyl) dithioacetals (3q–3t) with high yields in a short reaction time.
Synthesis of Vanillin Derivatives Incorporating Bis(2ꢁhydroxyethyl)dithioacetal (6a–
6n), and Dithioacetal (7–11). Substitution of vanillin with different substituted benzyl
chloride produced vanillin derivatives 5a–5n. Condensation of 5a–5n with 2ꢀ
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 35
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
mercaptoethanol yielded corresponding bis(2ꢀhydroxyethyl)dithioacetals 6a–6n.
Condensation of 5a with other sulfydryl compounds produced corresponding
dithioacetals 7–11 (Figure 4).
1
Studies on the reaction mechanism. H NMR spectra. To understand the reaction
mechanism, ZrCl4ꢀcatalyzed reactions of benzaldehyde and 2ꢀmercaptoethanol were
performed. The 1H NMR spectra show that after 2ꢀmercaptoethanol reacted with ZrCl4,
the hydroxyl hydrogen appeared at δ 4.68, whereas it appeared at δ 3.06 in 2ꢀ
mercaptoethanol. However, no chemical shift change in sulfydryl hydrogen was observed.
The reaction was highly exothermic. Moreover, no change occurred when ZrCl4 reacted
with benzaldehyde. Thus, we presumed that ZrCl4 initially reacted with 2ꢀ
mercaptoethanol to form a Zr···O complex, which resulted in exothermic and chemical
shift change. To further observe the coordination mode, ZrCl4 was reacted with 2ꢀ
mercaptothanol in different molar ratios. Experimental results demonstrated that ZrCl4
was coordinated with 2ꢀmercaptothanol in a 1:2 molar ratio. A plausible reaction pathway
based on experimental results above is illustrated as shown in Figure 5.
DFT calculations. DFT results indicated that the coordination of one oxygen of 2ꢀ
mercaptoethanol with ZrCl4 formed the pentacoordinate CM1 with a binding energy of –
17.08 kcal/mol, and the coordination of two oxygens of 2ꢀmercaptoethanol with ZrCl4 in
the paraꢀposition formed the sexadentate CM2 with a binding energy of –31.72 kcal/mol.
However, the coordination of two oxygens of 2ꢀmercaptoethanol with ZrCl4 in the orthoꢀ
position formed the sexadentate CM3 with a binding energy of –35.07 kcal/mol, which is
more stable than the other coordination modes. The reaction of 2ꢀmercaptoethanol with
benzaldehyde was investigated on the basis of the optimized possible coordination mode
10
ACS Paragon Plus Environment

Page 11 of 35
Journal of Agricultural and Food Chemistry
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
(CM3). Our calculations demonstrated that the reaction proceeded via two steps. In the
first step, the addition of a thiol of the complex A (CM3) to benzaldehyde formed the
intermediate B (Figure 5), with a Gibbs energy barrier of 34.57 kcal/mol. In the second
step, the substitution of another free 2ꢀmercaptoethanol to the hydroxyl of intermediate B
by its mercapto group formed the intermediate C (Figure 5). This step was easily carried
out, with a Gibbs energy barrier of only 13.99 kcal/mol. Obviously, the first step is the
rateꢀdetermining step. Further DFT studies indicated that the coordination of methylene
chloride with intermediate B formed a weak hydrogen bonding compound, and its
binding energy was greater by 1.17 kcal/mol than that of intermediate C, indicating that
free 2ꢀmercaptoethanol had difficulty reacting with intermediate B. This phenomenon
may explain the effect of methylene chloride as a solvent on the reaction.
Antiviral Activity. In Vivo AntiꢁPVY Activity. As shown in Table 1, bis(2ꢀhydroxyꢀ
ethyl)dithioacetals 3a–3t exhibited good antiviral activities against PVY in vivo. Among
these compounds, 3f derived from vanillin possessed good curative and protective
activities with values of 45.2% and 46.5%, respectively, at 500 ꢀg/mL, better than those
of ribavirin (40.5% and 43.2%). Vanillin derivatives incorporating a bis(2ꢀ
hydroxyethyl)dithioacetal moiety possessed good to excellent activities at 500 ꢀg/mL.
Compounds 6d–6g and 6i displayed potent curative activities, with values of 53.7%,
56.7%, 61.3%, 58.7%, and 63.1%, respectively, which were significantly greater than
those of ribavirin (40.5%), dufulin (48.2%), and ningnanmycin (50.9%). Compounds 6d–
6g showed significant protective activities against PVY, with values of 54.6%, 58.4%,
63.4%, and 59.6% at 500 ꢀg/mL, which were greater than those of ribavirin (43.2%),
dufulin (49.5%), and ningnanmycin (52.2%). Interestingly, these compounds could still
11
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 35
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
maintain potent antiviral activities even at low concentrations. However, the activities of
the vanillin derivatives incorporating dithioacetal (7–11) did not exceed that of the
vanillin derivative incorporating bis(2ꢀhydroxyethyl)dithioacetal (6f).
To further confirm the antiviral activities and to study the SARs of the target
compounds, the EC50 values of some compounds were investigated on the basis of
previous bioassays, and the results are listed in Table 2. Compounds 6d–6g and 6i
exhibited remarkable curative activities against PVY, with EC50 values of 357.5, 308.4,
217.6, 245.1, and 197.4 µg/mL, which were superior to those of ribavirin (848.0 µg/mL),
dufulin (462.6 ꢀg/mL), and ningnanmycin (440.5 µg/mL). Compounds 6d–6g showed
significant protective activities against PVY, with EC50 values of 352.4, 278.7, 205.7, and
266.7 ꢀg/mL, respectively, which were better than those of ribavirin (808.1 ꢀg/mL),
dufulin (454.8 ꢀg/mL), and ningnanmycin (425.3 ꢀg/mL).
In Vivo AntiꢁCMV Activity. AntiꢀCMV activity results showed that most of the target
compounds displayed obvious inhibitory effects (Table 2). Compounds 6d–6h exhibited
excellent curative activities against CMV, with EC50 values of 300.6, 270.5, 206.3, 271.6,
and 366.7 µg/mL, which were superior to those of ribavirin (858.2 µg/mL), dufulin
(471.2 ꢀg/mL), and ningnanmycin (426.1 µg/mL). In addition, compounds 6d–6h
exhibited significant protective effects against CMV, with EC50 values of 298.4, 260.8,
186.2, 265.3, and 358.6 µg/mL, respectively, which were better than those of ribavirin
(766.5 µg/mL), dufulin (465.4 ꢀg/mL), and ningnanmycin (405.3 µg/mL). The antiꢀCMV
activity is consistent with the trend of antiꢀPVY activity.
SARs. Among simple bis(2ꢀhydroxyethyl)dithioacetals 3a–3t, electronꢀwithdrawing
aromatic bis(2ꢀhydroxyethyl)dithioacetals (3m–3q) exhibited higher activities than
12
ACS Paragon Plus Environment

Page 13 of 35
Journal of Agricultural and Food Chemistry
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
electronꢀrich aromatics (3b–3e), and the hydroxy group is favorable for activity (3f > 3a–
3e, 3g–3t). To study the influence of the substitutions at the –OH position and the SARs
under the substituents, vanillin derivatives incorporating bis(2ꢀhydroxyethyl)dithioacetal
6a–6n were prepared. Bioassay results indicated that a benzyl ether could influence the
antiviral effect. Among these compounds 6a–6n, halogen atomꢀsubstituted benzyl ether
containing compounds 6d–6i exhibited better antiviral activities, and chlorineꢀsubstituted
benzyl ethers are the most favorable for activity (6d–6g > 6h and 6i), especially with the
chlorine atom at the paraꢀposition (6f > 6d, 6e, and 6g). To further investigate the effect
of different sulfur nucleophilic reagents on the antiviral potency after the substituent
group at the –OH position of vanillin was determined, compounds 7–11 were synthesized
and their antiviral potency was tested. Compared with compounds 7–11, 6f exhibited
higher activity. This phenomenon indicates that the infinite helical structure of bis(2ꢀ
hydroxyethyl)dithioacetal is favourable for antiviral activity. The main difference in
compounds 7–11 lies in the sulfur nucleophilic reagent. Compounds 7 and 9 displayed
stronger antiviral activities than compounds 10 and 11. The results indicate that the aryl
group is more suitable for antiviral activities than the alkyl group. The antiviral activities
of vanillin derivative 5f were considerably weaker than those of 6d–6f incorporating
dithioacetal. This result indicates that bis(2ꢀhydroxyethyl)dithioacetal is indispensable for
the antiviral activities of these compounds.
In summary,
a
series of vanillin derivatives incorporating
a
bis(2ꢀ
hydroxyethyl)dithioacetal moiety was designed and synthesized via a new and efficient
method. Experimental results indicate that the O···Zr···O complex plays an important
role in the formation of bis(2ꢀhydroxyethyl)dithioacetals, which was further verified by
13
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 35
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
DFT calculations. Bioassay results indicated that most compounds exhibited broadꢀ
spectrum and good antiviral activities against PVY and CMV. Compound 6f displayed
superior antiviral activity to the commercial ribavirin, dufulin, and ningnanmycin. These
results revealed that vanillin derivatives incorporating bis(2ꢀhydroxyethyl)dithioacetals
with an infinite helical structure can be considered as new antiviral agents. Field trials and
further investigations on structural optimization, mode of action, and other functions in
agrochemicals are currently underway.
ASSOCIATED CONTENT
Supporting Information
1
Characterization data, H, 13C NMR spectra, and highꢀresolution mass spectra for
products 3a–3t, 5a–5n, 6a–6n, and 7–11 are provided. Supplementary data associated
with this article can be found in the online version at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(D.Y. H) Eꢀmail: dyhu@gzu.edu.cn
*(B.A. S) Eꢀmail: songbaoan22@yahoo.com
Funding
The authors gratefully acknowledge the National Natural Science Foundation of China
(Nos. 21362004, 21562013) and Subsidy Project for Outstanding Key Laboratory of
Guizhou Province in China (20154004).
Notes
The authors declare no competing financial interest.
ABRREVIATIONS
14
ACS Paragon Plus Environment

Page 15 of 35
Journal of Agricultural and Food Chemistry
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
PVY, potato virus Y; CMV, cucumber mosaic virus; DFT, density functional theory; SAR,
structure–activity relationship.
REFERENCES
(1) Karasev, A. V.; Gray, S. M. Continuous and emerging challenges of potato virus Y in
potato. Annu. Rev. Phytopathol. 2013, 51, 571−586.
(2) Boquel, S.; Zhang, J.; Goyer, C.; Giguère, M. A.; Clark, C.; Pelletier, Y. Effect of
insecticideꢀtreated potato plantson aphid behavior and potato virus Y acquisition.
Pest Manag. Sci. 2015, 71, 1106–1112.
(3) Jacquemond, M. Cucumber mosaic virus. Adv. Virus Res. 2012, 84, 439–504.
(4) Vinchesi, A. C.; Rondon, S. I.; Goyer, A. Priming potato with thiamin to control
potato virus Y. Am. J. Potato Res. 2017, 94, 120–128.
(5) Wang, Z. W.; Wang, L.; Ma, S.; Liu, Y. X.; Wang, L. Z.; Wang, Q. M. Design,
synthesis, antiviral activity, and SARs of 1,4ꢀAminophenanthroindolizidines. J.
Agric. Food Chem. 2012, 60, 5825–5831.
(6) Long, C. W.; Li, P.; Chen, M. H.; Dong, L. R.; Hu, D. Y.; Song, B. A. Synthesis,
antiꢀtobacco mosaic virus and cucumber mosaic virus activity, and 3DꢀQSAR study
of novel 1,4ꢀpentadienꢀ3ꢀone derivatives containing 4ꢀthioquinazoline moiety. Eur. J.
Med. Chem. 2015, 102, 639−647.
(7) Ji, X. F.; Z. Wang, W.; Dong, J.; Liu, Y. X.; Lu, A. D.; Wang, Q. M. Discovery of
topsentin alkaloids and their derivatives as novel antiviral and antiꢀphytopathogenic
fungus agents. J. Agric. Food Chem.2016, 64, 9143−9151.
(8) Sidwell, R. W.; Huffman, J. H.; Khare, G. P.; Allen, L. B.; Witkowski, J. T.; Robins,
R. K. Broadꢀspectrum antiviral activity of virazole: 1ꢀβꢀDꢀRibofuranosylꢀ1,2,4ꢀ
15
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 35
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
triazoleꢀ3ꢀcarboxamide. Science 1972, 177, 705−706.
(9) Li, Y. M.; Zhang, Z. K.; Jia, Y. T.; Shen, Y. M.; He, H. P.; Fang, R. X.; Chen, X. Y.;
Hao, X. J. 3ꢀAcetonylꢀ3ꢀhydroxyoxindole: a new inducer of systemic acquired
resistance in plants. Plant Biotechnol. J. 2008, 6, 301−308.
(10) 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 3ꢀ((2ꢀ((1E, 4E)ꢀ3ꢀoxoꢀ5ꢀarylpentaꢀ1,4ꢀ
dienꢀ1ꢀyl)phenoxy)methyl)ꢀ4(3H)ꢀquinazolinone derivatives. J. Agric. Food Chem.
2014, 62, 8928−8934.
(11) Chen, L. W.; Xie, J. L.; Song, H. J.; Liu, Y. X.; Gu, Y. C.; Wang, L. Z.; Wang, Q. M.
Design, synthesis, and biological activities of spirooxindoles containing
acylhydrazone fragment derivatives based on the biosynthesis of alkaloids derived
from tryptophan. J. Agric. Food Chem. 2016, 64, 6508−6516.
(12) Qian, X. H.; Lee, P. W.; Cao, S. China: forward to the green pesticides via a basic
research program. J. Agric. Food Chem. 2010, 58, 2613−2623.
(13) Seiber, J. N. Sustainability and agricultural and food chemistry. J. Agric. Food Chem.
2011, 59, 1−2.
(14) Walton, N. J.; Mayer, M. J.; Narbad, A. Molecules of interest: vanillin.
Phytochemistry 2003, 63, 505–515.
(15) Sinha, A. K.; Sharma, U. K.; Sharma, N. A comprehensive review on vanilla flavor:
Extraction, isolation and quantification of vanillin and others constituents. Int. J.
Food Sci. Nutr. 2008, 59, 299−326.
(16) Harshvardhan, K.; Suri, M.; Goswami, A.; Goswami, T. Biological approach for the
production of vanillin from lignocellulosic biomass (Bambusa tulda). J. Clean. Prod.
16
ACS Paragon Plus Environment

Page 17 of 35
Journal of Agricultural and Food Chemistry
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
2017, 149, 485−490.
(17) Lirdprapamongkol, K.; Sakurai, H.; Kawasaki, N.; Choo, M. K.; Saitoh, Y.; Aozuka,
Y.; Singhirunnusorn, P.; Ruchirawat, S.; Svasti, J.; Saiki, I. Vanillin suppresses in
vitro invasion and in vivo metastasis of mouse breast cancer cells. Eur. J. Pharm. Sci.
2005, 25, 57−65.
(18) Bhanawase, S. L.; Yadav, G. D. Novel silica encapsulated CuꢀAl hydrotalcite
catalyst: Oxidative decarboxylation of vanillyl mandelic acid to vanillin in water at
atmospheric pressure. Ind. Eng. Chem. Res. DOI: 10.1021/acs.iecr.6b04982.
(19) Jantaree, P.; Lirdprapamongkol, K.; Kaewsri, W.; Thongsornkleeb, C.;
Choowongkomon, K.; Atjanasuppat, K.; Ruchirawat, S.; Svasti, J. Homoꢀdimers of
vanillin and apocynin decrease metastatic potential of human cancer cells by
inhibiting the FAK/PI3K/Akt signaling pathway. J. Agric. Food Chem. 2017, 65,
2299−2306.
(20) Tai, A.; Sawano, T.; Yazama, F.; Ito, H. Evaluation of antioxidant activity of
vanillin by using multiple antioxidant assays. Biochim. Biophys. Acta Gen. Subj.
2011, 1810, 170–177.
(21) Dalmolin, L. F.; Khalil, N. M.; Khalil, R. M. Delivery of vanillin by poly(lacticꢀacid)
nanoparticles: Development, characterization and in vitro evaluation of antioxidant
activity. Mater. Sci. Eng., C 2016, 62, 1−8.
(22) Cerruti, P.; Alzamora, S. M.; Vidales, S. L. Vanillin as an antimicrobial for
producing shelfꢀstable strawberry puree. J. Food Sci. 1997, 62, 608−610.
(23) Fitzgerald, D. J.; Stratford, M.; Gasson, M. J.; Ueckert, J.; Bos, A.; Narbad, A. Mode
of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 35
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
and Listeria innocua. J. Appl. Microbiol. 2004, 97, 104−133.
(24) Cerrutti, P.; Alzamora, S. M. Inhibitory effects of vanillin on some food spoilage
yeasts in laboratory media and fruit purees. Int. J. Food Microbiol. 1996, 29,
379−386.
(25) Fitzgerald, D. J.; Stratford, M.; Gasson, M. J.; Narbad, A. The potential application
of vanillin in preventing yeast spoilage of readyꢀtoꢀdrink beverages. J. Food Prot.
2004, 67, 391−395.
(26) Fitzgerald, D. J.; Stratford, M.; Gasson, M. J.; Narbad, A. Structureꢀfunction
analysis of the vanillin molecule and its antifungal properties. J. Agric. Food Chem.
2005, 53, 1769−1775.
(27) Galgani, J. E.; Nunez, B.; Videla, L. A.; Vanillin suppresses Kupffer cellꢀrelated
colloidal carbonꢀinduced respiratory burst activity in isolated perfused rat liver:
antiinflammatory implications. Food Funct. 2012, 3, 1319–1323.
(28) Keshava, C.; Keshava, N.; Whong, W.ꢀZ.; Nath, J.; Ong, T.ꢀM. Inhibition of
methotrexateꢀinduced chromosomal damage by vanillin and chlorophyllin in V79
cells. Teratog., Carcinog., Mutagen. 1998, 17, 313−326.
(29) Roberto, C. G.; Sarah, K. W.; Martina, H.; Lisa, L.; Gisbert, S.; Manfred, S. Z.;
Ewgenij, P.; Birgit, S. Vanillinꢀderived antiproliferative compounds influence Plk1
activity. Bioorg. Med. Chem. Lett. 2014, 24, 5063–5069.
(30) Lu, S. Y.; Zhang, Y. M. Synthesis and antibacterial activities of βꢀcarboline oxime
ester compounds. Hecheng Huaxue 2011, 19, 769−772.
(31) Ahluwalia, V.; Garg, N.; Kumar, B.; Suresh Walia, S.; Sati, O. P. Synthesis,
antifungal activity and structureꢀactivity relationships of vanillin oximeꢀNꢀOꢀ
18
ACS Paragon Plus Environment

Page 19 of 35
Journal of Agricultural and Food Chemistry
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
alkanoates. Nat. Prod. Commun. 2012, 7, 1635−1638.
(32) Lhassani, M.; Chavignon, O.; Chezal, J. M.; Teulade, J. C.; Chapat, J. P.; Snoeck, R.;
Andrei, G.; Balzarini, J.; Clercq, E. D.; Gueiffier, A. Synthesis and antiviral activity
of imidazo[1,2ꢀa]pyridines. Eur. J. Med. Chem. 1999, 34, 271−274.
(33) Sakthikumar, L.; Kavitha, J.; Mahalakshmi, R. Synthesis, characterization and
antibacterial activity of alkyl, benzyl and chloro substituted benzyl derivatives of
nitroketene dithioacetals. Der Pharma Chemica 2014, 6, 294−298.
(34) Koga, H.; Tsuji, Y.; Kanai, K. I. K.; Majima, T.; Uchida, T. K. K.; Yamaguchi, H. In
vitro antifungal activity of luliconazole against clinical isolates from patients with
dermatomycoses. J. Infect. Chemother. 2006, 12, 163−165.
(35) Koga, H.; Nanjoh, Y.; Makimura, K.; Tsuboi, R. In vitro antifungal activities of
luliconazole, a new topical imidazole. Med. Mycol. 2009, 47, 640−647.
(36) Pandey, S.; Suryawanshi, S.; Gupta, S.; Srivastava, V. Chemotherapy of
leishmaniasis part II: synthesis and bioevaluation of substituted arylketene
dithioacetals as antileishmanial agents. Eur. J. Med. Chem. 2005, 40, 751−756.
(37) Laskar, A. L.; Begum, N. A.; Mir, M. H.; Rohman, M. R.; Khan, A. T. Nickel(II)
chloride hexahydrate catalyzed reaction of aromatic aldehydes with 2ꢀ
mercaptoethanol: formation of supramolecular helical assemblage of the product.
Tetrahedron Lett. 2013, 54, 5839−5844.
(38) Prins, L J.; Huskens, J.; deJong, F.; Timmerman, P.; Reinhoudt, D. N. Complete
asymmetric induction of supramolecular chirality in a hydrogenꢀbonded assembly.
Nature 1999, 398, 498.
(39) Iannelli, D.; Apice, L. D.; Cottone, C.; Viscardi, M.; Scala, F.; Zoina, A.; Sorbo, G.
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 35
419
420
421
422
423
424
425
426
427
428
429
430
431
D.; Spigno, P.; Capparelli, R. Simultaneous detection of cucumber mosaic virus,
tomato mosaic virus and potato virus Y by flow cytometry. J. Virol. 1997, 69,
137−145.
(40) Scott, H. Purification of cucumber mosaic virus. Virology 1963, 20, 103−106.
(41) Song, B. A.; Zhang, H. P.; Wang, H.; Yang, S.; Jin, L. H.; Hu, D. Y.; Pang, L. L.;
Xue, W. Synthesis and antiviral activity of novel chiral cyanoacrylate derivatives. J.
Agric. Food Chem. 2005, 53, 7886−7891.
(42) Manzer, L. E.; Deaton, J.; Sharp, P.; Schrock, R. R. Tetrahydrofuran complexes of
selected early transition metals. Inorganic Syntheses 1982, 21, 135−140.
(43) Proto, A.; Capacchione, C.; Motta, O.; Carlo, F. C. ZrCl4 as catalyst for olefins and
styrene polymerization: effect of ethereal donors on the activity and stereospecificity.
Macromolecules 2003, 36, 5942−5946.
20
ACS Paragon Plus Environment

Page 21 of 35
Journal of Agricultural and Food Chemistry
432
433
434
435
436
437
438
439
FIGURE CAPTIONS
Figure 1 Chemical structures of Ningnanmycin, Dufulin, Ribavirin and Vanillin.
Figure 2 Design of the target compounds.
Figure 3 Synthesis of bis(2ꢀhydroxyethyl)dithioacetals using ZrCl4 as a catalyst.
Figure
4
Synthetic route of vanillin derivatives incorporating
a
bis(2ꢀ
hydroxyethyl)dithioacetal moiety.
Figure 5 Postulated reaction mechanism.
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 35
Table 1 Inhibitory Effect of Target Compounds against PVY and CMV at 500 ꢀg/mLa
440
AntiꢀPVY
curative protection
effect (%) effect (%)
AntiꢀCMV
curative protection
effect (%) effect (%)
AntiꢀPVY
AntiꢀCMV
curative protection
effect (%) effect (%)
compd
compd
curative
protection
effect (%)
effect (%)
11.6±2.1
12.6±3.2
13.1±2.4
24.6±2.4
23.4±1.7
45.2±0.5
38.3±2.3
41.6±1.8
34.2±1.5
35.7±1.6
32.7±1.6
35.8±2.2
25.4±2.2
26.6±1.7
28.6±2.7
28.3±1.7
35.8±1.9
38.3±2.4
39.2±2.0
41.0±2.6
45.1±1.3
42.8±1.5
10.5±2.3
13.1±2.6
14.1±2.9
23.6±3.5
22.1±1.6
46.5±1.8
35.2±2.7
42.3±3.3
35.8±2.6
37.3±2.2
32.4±2.7
32.5±3.1
26.6±2.8
25.8±1.5
29.2±1.7
27.7±1.9
36.2±2.4
39.6±3.1
38.7±3.2
41.6±1.8
46.1±1.9
43.2±1.7
12.4±3.6
14.3±2.8
14.1±3.7
23.7±2.8
23.8±2.3
45.4±1.7
34.3±3.1
43.5±2.6
36.4±1.9
35.1±2.8
33.1±1.4
34.1±2.3
26.2±2.1
25.7±2.6
28.5±2.3
27.3±2.4
37.6±3.1
38.2±2.7
39.1±2.5
40.6±3.5
46.7±1.9
41.8±4.2
13.1±2.5
15.2±1.6
14.9±2.8
23.6±2.5
26.4±2.8
47.8±2.3
37.3±1.4
42.4±2.1
37.3±2.8
36.1±1.9
34.9±1.7
34.3±1.9
27.2±2.6
26.5±1.9
27.1±2.6
27.6±2.1
36.4±2.6
40.5±2.4
39.8±1.6
42.4±3.1
46.8±1.6
42.1±3.5
36.5±1.5
38.2±2.8
53.7±1.6
56.7±1.3
61.3±1.1
58.7±2.1
50.3±2.3
63.1±2.5
39.7±2.1
37.3±1.2
34.7±2.0
43.3±2.6
39.7±2.4
55.6±1.2
47.6±2.4
46.8±2.5
44.8±3.7
43.1±2.9
40.5±2.2
50.9±2.3
48.2±1.7
38.0±1.6
39.5±1.5
54.6±2.3
58.4±1.4
63.4±1.5
59.6±2.5
47.7±1.7
48.3±1.3
38.4±2.2
36.4±2.3
35.9±2.0
44.3±3.1
37.4±1.5
56.4±2.2
48.3±3.1
47.2±3.7
45.1±3.0
44.2±3.2
43.2±2.5
52.2±2.0
49.5±1.3
38.5±4.1
41.1±3.4
56.9±2.3
58.7±2.6
62.3±2.6
58.5±3.1
55.7±1.8
47.2±2.2
36.7±3.2
35.9±2.2
36.1±1.9
44.3±2.2
40.7±2.6
55.7±3.6
56.4±2.9
47.6±2.0
45.5±2.1
44.9±3.7
39.4±2.5
51.3±1.7
47.6±1.4
40.0±3.2
39.1±2.6
57.6±3.8
60.4±3.2
65.4±3.3
59.4±2.2
56.7±2.8
49.3±1.9
38.1±3.4
36.4±3.3
38.2±2.3
43.3±2.6
38.4±4.2
57.5±2.8
49.6±3.4
48.5±3.6
46.2±1.9
45.1±2.6
42.3±1.8
53.8±2.3
49.4±2.1
3a
3b
3c
3d
3e
3f
6b
6c
6d
6e
6f
6g
3g
3h
3i
6h
6i
6j
3j
6k
3k
3l
6l
6m
3m
3n
3o
3p
3q
3r
3s
3t
6n
7
8
9
10
11
Ribavirinb
Ningnanmycinc
Dufulind
5f
6a
441
442
aAverage of three replicates; bRibavirin, cningnanmycin, and ddufulin were used as the control.
22
ACS Paragon Plus Environment

Page 23 of 35
Journal of Agricultural and Food Chemistry
443
Table 2 EC50 Values of Target Compounds against PVY and CMVa
EC50 for AntiꢀPVY
EC50 for AntiꢀCMV
compd
curative effect
protection effect
(ꢀg/mL)
curative effect
(ꢀg/mL)
protection effect
(ꢀg/mL)
(ꢀg/mL)
726.7±4.8
708.9±3.2
828.8±3.4
1051.5±3.8
872.8±2.1
357.5±4.4
308.4±4.4
217.6±3.6
245.1±2.0
398.4±2.4
197.4±3.9
737.3±4.0
817.3±3.6
1069.2±3.2
691.8±1.4
839.4±3.5
347.4±2.4
848.0±2.8
440.5±2.0
462.6±3.8
679.7±2.1
644.4±3.5
598.6±4.8
841.1±2.6
790.4±1.6
352.4±5.2
278.7±5.2
205.7±2.4
266.7±2.2
521.4±1.9
502.2±2.7
920.9±3.3
1042.3±4.2
1039.3±3.5
605.4±1.8
839.3±4.0
348.0±2.1
808.1±5.6
425.3±2.4
454.8±2.6
716.8±2.4
652.3±4.4
816.5±3.5
895.2±3.3
824.6±2.1
300.6±3.3
270.5±3.9
206.3±4.3
271.6±1.8
366.7±2.1
516.7±2.9
968.7±3.6
982.3±3.8
965.1±3.1
603.5±3.2
676.3±3.2
346.3±2.8
858.2±3.0
426.1±4.5
471.2±4.2
658.4±3.1
657.9±5.1
807.6±2.2
857.8±3.6
885.1±2.5
298.4±4.0
260.8±3.7
186.2±4.5
265.3±2.5
358.6±2.6
467.6±2.3
921.4±3.4
961.5±2.6
906.2±2.4
657.3±2.7
724.3±2.6
324.8±4.6
766.5±3.4
405.3±3.2
465.4±3.4
3f
5f
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
7
Ribavirinb
Ningnanmycinc
Dufulind
aAverage of three replicates; bRibavirin, cningnanmycin, and ddufulin were used as the control.
444
445
23
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 35
446
447
Figure 1
448
24
ACS Paragon Plus Environment

Page 25 of 35
Journal of Agricultural and Food Chemistry
449
450
Figure 2
451
25
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 35
452
453
Figure 3
454
26
ACS Paragon Plus Environment

Page 27 of 35
Journal of Agricultural and Food Chemistry
455
456
Figure 4
457
27
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 35
458
459
460
Figure 5
28
ACS Paragon Plus Environment

Page 29 of 35
Journal of Agricultural and Food Chemistry
Table of Contents Graphic
461
462
29
ACS Paragon Plus Environment