Design, synthesis, bioactivity and mechanism of dithioacetal derivatives containing dioxyether moiety

Article abstract of DOI:10.1016/j.bmcl.2019.06.030

The present work designed and synthesized a series of dithioacetal derivatives containing dioxyether, as well as evaluated their antiviral activities against tobacco mosaic virus (TMV). Bioassays demonstrated that the target compounds showed excellent ant

Full text of DOI:10.1016/j.bmcl.2019.06.030

Accepted Manuscript
Design, Synthesis, Bioactivity and Mechanism of Dithioacetal Derivatives Con-
taining Dioxyether Moiety
Yanju Wang, Jian Zhang, Fangcheng He, Xiuhai Gan, Baoan Song, Deyu Hu
PII:
S0960-894X(19)30410-X
https://doi.org/10.1016/j.bmcl.2019.06.030
BMCL 26508
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
18 May 2019
13 June 2019
19 June 2019
Please cite this article as: Wang, Y., Zhang, J., He, F., Gan, X., Song, B., Hu, D., Design, Synthesis, Bioactivity and
Mechanism of Dithioacetal Derivatives Containing Dioxyether Moiety, Bioorganic & Medicinal Chemistry
Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.030
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Design, Synthesis, Bioactivity and Mechanism of Dithioacetal
Derivatives Containing Dioxyether Moiety
Yanju Wang,† Jian Zhang,† Fangcheng He,† Xiuhai Gan,† Baoan Song,*† Deyu Hu*†
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
Fax: 0086-851-88292170; E-mail: dyhu@gzu.edu.cn; songbaoan22@yahoo.com
1

ABSTRACT: The present work designed and synthesized a series of dithioacetal
derivatives containing dioxyether, as well as evaluated their antiviral activities against
tobacco mosaic virus (TMV). Bioassays demonstrated that the target compounds
showed excellent anti-TMV activities in vivo and in vitro. Compound 24c has excellent
anti-TMV activities, and its curative, protective and inactivating activities for TMV
were 50.9%, 58.9% and 81.8%, respectively, which are obviously superior to those of
ribavirin (50.2%, 41.3% and 69.5%, respectively). Moreover, the EC50 of the
inactivating activities of the anti-TMV of compound 24c is 67.9 mg/L, which is superior
to that of ribavirin (149.5 mg/L). Transmission electron microscopy showed that
compound 24c caused great damage to the morphology of TMV particles, causing
fracture and bending. Molecule docking model revealed that this compound formed five
conventional hydrogen bonds with the active sites of amino acids GLN57, ASN73,
TYR139, and SER138. Furthermore, the test results of Fluorescence titration and
microscale thermophoresis showed that compound 24c has a strong binding force with
TMV coat protein (TMV CP), with an association constant (Ka) of 1.04×105 L/mol and
dissociation constant (Kd) of 1.6 ± 0.6 μM. These results indicate that the dithioacetal
derivatives containing dioxyether are worthy of further research and development as
novel antiviral agents.
KEYWORDS: dithioacetal derivatives, vanillin, dioxyether moiety, antiviral activities,
Transmission electron microscopy, interaction, molecule docking
2

Plant virus disease seriously endangers the growth of crops, causing significant
economic losses to agricultural production and seriously affecting the development of
agricultural industries.1 Among them, tobacco mosaic virus (TMV) is a common plant
virus that mainly harm crops, such as cucumber, tomato, potato, tobacco and pepper.
Given the absolute parasitism of the virus in plant and the shortage of an integral
immune metabolic system, the prevention and control of plant virus disease is currently
a major problem. The current antiviral agents for controlling plant virus diseases are
principally ningnanmycin and ribavirin, but the former has poor field control effect and
is costly, while the antiviral activities of the latter is poor.2,3 A broad-spectrum and
efficient agent that can control plant virus disease has not yet been developed.
Natural products are of great interest to biologists because of their low toxicity, easy
degradation, environmental friendliness, specific targets, and special modes of
action.4,5,6 The pesticide research based on natural products has become one of the
hotspots of pesticide development and utilization in recent years.7-9 Vanillin (3-
methoxy-4-hydroxybenzaldehyde) (Fig. 1) is a flavor component of vanilla beans. It
can be found in beets, vanilla beans, benzoin gum, Peru balsam, tolu balsam, and so on.
It is an important spice with a strong aroma and a widely used edible flavor. It can be
found in vanilla seeds and can also be artificially synthesized. Vanillin has extensive
biological activities, such as antibacterial,10,11 anticancer,12 antioxidant,13 antifungal,14
anti-inflammatory,15 anti-tumor,16 antimutagenic,17 and antiproliferative,18 antiviral
activities,19 and so on.
Our group reported for the first time vanillin derivatives containing dithioacetal and
3

discovered that xiangcaoliusuobingmi (Fig. 1) has excellent antiviral activities against
19,20
CMV and PVY.
Subsequently, our group designed and synthesized a series of
dithioacetal derivatives with prominent antiviral activities against CMV, PVY, and
TMV.21,22 However, no compounds with high inactivating activities for TMV were
found. Hence, in the present study, target compounds of dithioacetal derivatives
containing dioxyether were designed (Fig. 1) and synthesized (Fig. 2), and their
activities against TMV were evaluated via half-leaf method in an attempt to find a new,
efficient, and broad-spectrum anti-plant virus compounds as precursors for further
derivatization and studying their mechanism of action.
Fig. 1. Design of the target compounds
Fig. 2. Synthetic route of the target compounds 1c–27c
The anti-TMV activities of the target compounds (Table 1) showed that the protective
effect of 3c, 6c and 8c (60.2%, 62.9% and 60.9%, respectively) are better than that of
4

ribavirin (41.3%). The curative effects of 6c and 22c (68.5% and 65.8%, respectively)
are superior to that of ribavirin (50.2%). Moreover, the inactivating activities of
compounds 9c, 12c and 24c (82.8%, 84.3% and 81.8%, respectively) are better than
that of ribavirin (69.5%), and the EC50 of inactivating activities of compounds 6c, 7c,
9c, 11c, 12c, 13c and 24c (95.6, 110.3, 80.1, 140.3, 71.5, 139.6 and 67.9 mg/L,
respectively) are superior to that of ribavirin (149.5 mg/L).
Structure-activity relationships (SARs). The activities of the target compounds
against TMV showed that the type and position of substituents have a great impact on
the anti-TMV curative, protective and inactivating activities. The curative and
protective activities are increased when R1 is H or electron-donating group; for example,
the anti-TMV curative activities of the compounds decrease in the following order 6c >
14c > 18c > 1c (H > 4-OCH3 > 2-OCH3-4-CH2-CH=CH2 > 2-OCH3), while the anti-
TMV protective activities decrease in the following order 6c > 8c > 3c > 24c (H > 4-
OCH3 > 2-OCH3-4-CH2=CH-CH3 > 4-Br). However, not all of compounds follow this
rule. For example, the curative and protective activities of compound 24c is superior to
those of 11c, that is, 4-Br > 2-OCH3. The compounds demonstrate better inactivating
activities when the substituent R1 is H or an electron-withdrawing group, as in the
following order 12c > 24c > 7c > 13c (H > 4-Br > 4-NO2 > 4-F). However, not all
compounds follow this rule; for example, 10c >7c (2-OCH3-5-COCH3 > 4-NO2). In
addition, the value of n is also variable for the anti-TMV activity of the compound.
Usually, when n=1, the compound has better anti-TMV curative, protective and
inactivating activities; for example, the curative activities of the compounds decrease
5

in the following order: 6c > 12c, 5c > 13c, 7c > 15c, 20c > 26c, 24c > 27c, but some
exceptions have occurred, as in 11c > 1c, 14c > 8c, 16c > 9c, 25c > 21c. The general
variation rule of protective activities is as follows: 1c > 11c, 5c > 13c, 6c > 12c, 7c >
15c, 8c > 14c, 9c > 16c, 20c > 26c, 21c > 25c, 24c > 27c, but with one exception of 10c
> 4c.
Table 1. Antiviral activities of target compounds 1c-27c against TMV in vivo at 500 mg/L and
in vitro
In vivo
effect
In vitro
effect Kd
(μM)
a
Compd.
protective
activitya
（%）
curative
activitya
（%）
inactivating
activitya
（%）
EC50 for TMV
inactivating activitya
（mg/L）
346.8±3.7
172.4±4.3
163.3±2.8
718.8±2.1
160.6±1.9
95.6±1.9
1c
2c
50.3±1.4
52.1±8.8
60.2±4.2
27.3±3.3
48.8±9.2
62.9±6.1
42.9±6.4
60.9±5.2
43.6±9.9
47.8±8.9
55.6±8.7
46.0±7.1
28.4±6.7
35.8±5.3
40.0±6.3
30.7±6.3
40.9±6.3
41.4±7.2
44.6±2.8
43.6±4.5
48.4±9.4
57.2±2.1
52.7±7.5
52.9±11.1
38.3±9.7
51.2±9.2
68.5±2.1
55.4±4.12
43.7±6.4
31.5±3.6
32.1±7.6
23.1±6.2
41.4±9.5
50.9±8.7
59.3±9.3
44.3±11.3
56.5±11.4
52.6±9.7
58.9±9.9
42.5±6.4
51.0±1.8
44.8±2.5
53.3±3.4
68.0±0.3
65.4±8.9
37.2±4.0
65.8±7.4
77.9±2.7
76.6±0.7
52.9±7.4
82.8±5.6
66.9±0.8
77.9±2.5
84.3±0.2
72.0±5.3
55.7±4.6
67.1±4.4
81.1±5.4
44.8±2.4
58.5±0.4
65.0±2.8
57.0±1.1
54.9±0.5
150.4±27.1
85.6±26.5
77.2±35.5
244.2±155.0
72.1±3.4
3c
4c
5c
6c
14.4±4.9
7c
110.3±2.5
393.9±3.4
80.1±2.0
14.5±3.2
8c
166.2±45.5
10.8±7.5
9c
10c
11c
12c
13c
14c
15c
16c
17c
18c
19c
20c
21c
160.5±4.6
140.3±2.1
71.5±3.4
63.1±31.1
44.3±4.5
4.4±1.4
139.6±5.2
331.8±2.7
178.3±4.3
245.6±3.6
685.3±5.3
253.4±2.1
190.3±6.2
263.5±5.1
360.2±4.6
22.1±9.8
140.4±37.1
88.3±17.4
116.9±63.9
198.1±73.5
120.5±65.4
101.9±67.4
123.7±59.7
149.5±48.9
6

22c
23c
32.9±6.6
29.6±2.8
58.9±8.8
28.3±0.2
35.0±7.6
35.6±5.3
41.3±2.4
65.8±3.1
47.0±9.5
50.9±2.7
48.8±8.5
34.2±9.8
50.3±2.7
50.2±3.1
45.4±1.1
63.2±5.0
81.8±2.2
57.9±7.2
66.4±0.97
56.9±1.7
69.5±3.2
683.5±5.3
240.9±6.4
67.9±3.1
190.5±80.4
108.6±62.7
1.6±0.6
24c
25c
265.8±4.3
180.9±2.6
260.8±1.9
149.5±2.3
136.4±54.7
95.1±36.2
121.8±76.1
57.6±23.7
26c
27c
Ribavirinb
Footnotes: a Average of three replicates; b Ribavirin was used as the control.
The morphological observation of TMV granules by transmission electron
microscope (TEM) (Fig. 3) revealed that normal TMV particles were straight, short
rod-shaped in structure, and few particles were broken (Fig. 3A). At a concentration of
200 mg/L, TMV particles treated with ribavirin were broken (Fig. 3B), but the TMV
particles treated with 24c had more severe rupture (Fig. 3C). This finding indicates that
compound 24c has a certain destructive effect on the morphology of TMV particles.
Fig. 3. Effect of compounds CK (A), Ribavirin (B), and 24c (C) on the morphology of TMV
particles.
Molecular docking was performed using Discovery Studio 4.5 software to investigate
the mode of binding of the compounds with TMV CP. Compounds 24c, 6c and 4c were
selected to dock with TMV CP and showed prominent to poor inactivating activities
against TMV. As shown in the Fig. 4, the binding of the compounds with TMV CP can
be attributed to the interaction of compounds with hydrogen and non-hydrogen bonds
of TMV CP. First, five conventional hydrogen bonds were formed between compound
24c and the active sites of amino acids GLN57, ASN73, TYR139, and SER138 with
7

distance of 2.68, 2.92, 2.62, 2.11, and 3.07 Å, respectively. Among them, compound
24c formed two conventional hydrogen bonds with TYR139 (2.62 and 2.11 Å,
respectively), one carbon hydrogen bond with LYS268 (3.00 Å), and one halogen with
GLU222 (2.85 Å), as well as six alkyl or pi-alkyl with VAL75 (5.36 Å), VAL260 (5.15
Å), LYS253 (4.24 and 4.20 Å), VAL251 (5.03 Å), ALA218 (4.30 Å). Second,
compound 6c formed three conventional hydrogen bonds with the active sites of amino
acids GLN257 (2.24 Å), ASN73 (1.81 and 2.76 Å), one pi-pi T-shaped with TYR139
(5.80 Å), five alkyl or pi-alkyl with LYS253 (4.72 Å), LYS268 (4.70 Å), VAL75 (4.24
Å), PRO263 (4.57 Å) and ALA74 (5.27 Å). Third, three conventional hydrogen bonds
were formed between compound 4c and the active sites of the amino acids GLY137
(1.65 Å), ASN73 (2.65 and 2.81 Å), one carbon hydrogen bond with THR259 (2.80 Å),
one pi-pi stacked with TYR139 (4.24 Å), three alkyl or pi-alkyl with VAL260 (4.86 Å),
PRO263 (4.34 Å) and VAL75 (4.46 Å). Finally, the molecular docking results of
ribavirin with TMV CP showed that ribavirin formed three conventional hydrogen
bonds with LYS268 (2.48 and 1.72 Å) and ARG134 (2.03 Å), seven carbon hydrogen
bonds with THR136 (2.64 Å), PRO254 (2.50 Å), GLU222 (2.68 Å), GLU131 (2.73 Å),
TRP217 (2.26 Å) and SER255 (2.14 Å), one pi-alkyl with VAL75 (4.54 Å), and one
unfavorable donor-donor with ASP219 (1.08 Å).
8

Fig. 4 Molecule docking results of compounds 24c (A), 6c (B), 4c (C), and Ribavirin (D) with
TMV CP
The interaction of compounds with TMV CP was studied through FT and MST. Ka
and Kd values were obtained by FT and MST, respectively, as shown in Table 2 and
Figs. 5－6. The results showed that the compound 24c has powerful adhesion to TMV
CP, with Ka = 1.04×105 L/mol and Kd = 1.6 ± 0.6 μM. Compound 6c and TMV CP
exhibited moderate binding force strength, with Ka = 0.85×103 L/mol and Kd = 14.4 ±
4.9 μM. Ribavirin showed a binding force slightly lower than that of 6c, with Ka =
1.04×103 L/mol and Kd = 57.6 ± 23.7 μM. Compound 4c showed the lowest binding
force, with Ka = 8.30×102 L/mol and Kd = 244.2 ± 155.0 μM. These binding constants
are consistent with the inactivating activities of EC50 of target compounds against TMV.
Table 2 Interaction results of target compounds with TMV CP
Compd.
Ka a (L/mol)
8.30×102
0.85×103
1.04×105
1.04×103
Kd a (μM)
244.2±155.0
14.4±4.9
EC50 for TMV inactivating activity a（mg/L）
4c
6c
718.8±2.1
95.6±1.9
67.9±3.1
149.5±2.3
24c
1.6±0.6
Ribavirin b
57.6±23.7
Footnotes: a Average of three replicates; b Ribavirin was used as the control.
9

Fig. 5 FT test results of compounds 24c (A), 6c (B), 4c (C), and Ribavirin (D) with TMV CP.
Fig. 6 Microscale thermophoresis (MST) test results of compounds 24c (A), 6c (B), 4c (C) and
Ribavirin (D) with TMV CP.
In this study, we designed and synthesized a series of dithioacetal derivatives
containing dioxyether, as well as evaluated their anti-TMV activities. The biological
activities test results indicated that most of the target compounds have excellent anti-
TMV curative, protective, and inactivating activities. Especially, compounds 6c, 9c,
12c and 24c have excellent anti-TMV inactivating activities with EC50 values of 95.6,
80.1, 71.5 and 67.9 mg/L, respectively. TEM showed that compound 24c greatly
damaged the morphology of TMV particles, causing fracture and bending, and it has a
10

certain influence on the shape and size of TMV. Based on the inactivating activities
against the TMV of target compounds, the mode of interaction between target
compounds and TMV CP was studied by molecular docking, in which compound 24c
was found to have excellent binding ability. In order to derivate molecular docking
results, FT and MST were used to further study the interaction of compounds with TMV
CP. Compound 24c demonstrated significant binding with TMV CP, with Ka =
1.04×105 L/mol and Kd = 1.6 ± 0.6 μM. This study laid the foundation for the study of
antiviral agents.
Acknowledgements
This work has received the financial support of the National Natural Science
Foundation of China (No. 21867002) and Subsidy Project for Outstanding Key
Laboratory of Guizhou Province in China (20154004).
Supporting Information
The synthesis, physical analysis, 1H NMR spectra, 13C NMR spectra and high resolution
mass spectrum (HRMS) of compounds 1b-16b and 1c-27c can be found in the
supplementary data.
Abbreviations
TLC, thin layer chromatography; EC50, half-maximal effective concentration; 1H
NMR, 1H nuclear magnetic resonance; 13C NMR, 13C nuclear magnetic resonance;
TMS, tetramethylsilane; TMV, tobacco mosaic virus; PVY, potato mosaic virus; CMV,
cucumber mosaic virus; THF, tetrahydrofuran; SARs, Structure-activity relationships;
TEM, Transmission electron microscopy; FT, fluorescence titration; MST, micro-
11

thermal surge; Ka, association constant; Kd, dissociation constant.
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