Design, synthesis, and biological activity of novel myricetin derivatives containing amide, thioether, and 1,3,4-thiadiazole moieties

Article abstract of DOI:10.3390/molecules23123132

A series of myricetin derivatives containing amide, thioether, and 1,3,4-thiadiazole moieties were designed and synthesized, and their antiviral and antibacterial activities were assessed. The bioassays showed that all the title compounds exhibited potent in vitro antibacterial activities against Xanthomonas citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo). In particular, the compounds 5a, 5f, 5g, 5h, 5i, and 5l, with EC₅₀ values of 11.5–27.3 μg/mL, showed potent antibacterial activity against Xac that was better than the commercial bactericides Bismerthiazol (34.7 μg/mL) and Thiodiazole copper (41.1% μg/mL). Moreover, the in vivo antiviral activities against tobacco mosaic virus (TMV) of the target compounds were also tested. Among these compounds, the curative, protection, and inactivation activities of 5g were 49.9, 52.9, and 73.3%, respectively, which were better than that of the commercial antiviral Ribavirin (40.6, 51.1, and 71.1%, respectively). This study demonstrates that myricetin derivatives bearing amide, thioether, and 1,3,4-thiadiazole moieties can serve as potential alternative templates for the development of novel, highly efficient inhibitors against plant pathogenic bacteria and viruses.

Full text of DOI:10.3390/molecules23123132

molecules
Article
Design, Synthesis, and Biological Activity of Novel
Myricetin Derivatives Containing Amide, Thioether,
and 1,3,4-Thiadiazole Moieties
Xianghui Ruan †, Cheng Zhang †, Shichun Jiang, Tao Guo, Rongjiao Xia, Ying Chen, Xu Tang and
Wei Xue *
Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou University,
Huaxi District, Guiyang 550025, China; rxh9105@163.com (X.R.); zhangcheng6954@163.com (C.Z.);
15885526512@163.com (S.J.); guotao9405163@163.com (T.G.); xrjiao@163.com (R.X.); 15286114381@163.com (Y.C.);
tx1173182020@163.com (X.T.)
* Correspondence: wxue@gzu.edu.cn; Tel./Fax: +86-851-8829-2090
† These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 12 October 2018; Accepted: 12 November 2018; Published: 29 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of myricetin derivatives containing amide, thioether, and 1,3,4-thiadiazole
moieties were designed and synthesized, and their antiviral and antibacterial activities were assessed.
The bioassays showed that all the title compounds exhibited potent in vitro antibacterial activities
against Xanthomonas citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo).
In particular, the compounds 5a
showed potent antibacterial activity against Xac that was better than the commercial bactericides
Bismerthiazol (34.7 g/mL) and Thiodiazole copper (41.1% g/mL). Moreover, the in vivo antiviral
, 5f, 5g, 5h, 5i, and 5l, with EC50 values of 11.5–27.3 µg/mL,
µ
µ
activities against tobacco mosaic virus (TMV) of the target compounds were also tested. Among these
compounds, the curative, protection, and inactivation activities of 5g were 49.9, 52.9, and 73.3%,
respectively, which were better than that of the commercial antiviral Ribavirin (40.6, 51.1, and 71.1%,
respectively). This study demonstrates that myricetin derivatives bearing amide, thioether, and 1,3,4-
thiadiazole moieties can serve as potential alternative templates for the development of novel, highly
efficient inhibitors against plant pathogenic bacteria and viruses.
Keywords: myricetin; amide; 1,3,4-oxadiazole; thioether; bioactivity
1. Introduction
In recent years, pathogenic bacteria and plant viruses targeting agricultural crops have become
increasingly difficult to control, leading to enormous losses in worldwide crop production each year.
As such, the development of environmentally friendly pesticides with high selectivity, high efficiency,
low toxicity, and suitable degradation are important challenges to both chemists and biologists [
Natural products and extracts, derived from the biosphere, have been widely applied for use in
medicines [ ], pesticides [ ], cosmetics [ ], and food products in ancient and modern societies around
the world [ ]. Recently, many natural products have shown promise as antimicrobials and antivirals [ ],
1].
2
3,
4
5
6
7
including the quercetin, genistein, and silybin, which belong to the flavonoids group of compounds
and have demonstrated inhibitory activity against the DNA virus herpes type 1 (HSV-1) and RNA
virus parainfluenza type 3 (PI-3) [8]. Consequently, our research has focused on the properties of
natural products as a starting point for the design of pesticide molecules.
Myricetin (Figure 1) belongs to a class of natural flavonoids that is widely found in vegetables,
fruits, teas, and medicine herbs [9,10]. Over the last decade, many researchers have shown that
Molecules 2018, 23, 3132; doi:10.3390/molecules23123132
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3132
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natural myricetin exhibits various biological activities, including antibacterial [11], antiviral [12],
antioxidant [ 13], anti-inflammatory [14], and anticancer [15 17] properties. Most of these prior studies
have assessed the properties of myricetin itself, but its derivatives have rarely been studied [18 21].
9
,
–
–
Recently, in our prior work, some myricetin derivatives bearing 1,3,4-thiadiazole [18], Schiff-base [20],
and acid amide [21] moieties were rationally synthesized and assessed for their potential application
as novel anticarcinogens and bactericides.
The compound 1,3,4-thiadiazole contains an important nitrogenous pharmacophore and exhibits
different biological activities, such as antimicrobial, antidepressant, and anti-tumor activities.
The 1,3,4-thiadiazole ring is very weak due to the induction of sulfur atoms, which makes it easy for it
to unlink in alkaline aqueous solutions; however, it is relatively stable in acidic aqueous solutions [22].
The ring readily reacts with a nucleophile to generate diverse derivatives due to its electronic state [23].
Commercial bactericides and antivirals bearing 1,3,4-thiadiazoles have been developed and are widely
available, and these include the agricultural bactericidal agents Bismerthiazol and Thiodiazole copper
(Figure 1).
Based on these considerations, we designed and synthesized a series of novel myricetin
derivatives containing amide, thioether, and 1,3,4-thiadiazole moieties. The derivatives were
evaluated via turbidimetry tests and screened against three plant pathogens Xanthomonas citri (Xac),
Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo). In addition, the ability of these
compounds to inhibit tobacco mosaic virus (TMV) was also evaluated in vivo via the half-leaf method
at 500 µg/mL, with the commercial antiviral Ribavirin serving as a positive control (Figure 1).
Figure 1. The structures of myricetin and selected commercial drugs.
2. Results and Discussion
2.1. Chemistry
The synthesis protocol was divided into five steps (Scheme 1). Firstly, 2-amino-5-mercapto-
1,3,4-thiadiazole (intermediate
Secondly, intermediates 2a 2p were synthesized from intermediate
substitution reaction. Thirdly, the intermediates 3a 3p were synthesized via a substitution reaction by
treating intermediates 2a 2p with chloroacetyl chloride in the presence of the base K2CO3. Fourthly,
myricitrin was converted to intermediate by methylation and deglycosidation. Finally, the 3-OH on
intermediate underwent a substitution reaction with the halogen atom of intermediates 3a 3p to
1) was obtained according to the previously reported method [24].
–
1
and various benzyl chlorides via
–
–
4
4
–
generate the target compounds 5a–5p.
1
The structures of all the target compounds (5a–
5p) were verified by H-NMR, 13C-NMR,
and HRMS. The spectra data are provided in the experiments below. The 1H-NMR results showed
that all the aryl protons presented as multiplets at approximately 8.65–6.40 ppm, and the characteristic
-O-CH2- and -S-CH2- groups between the myricetin scaffold and 1,3,4-thiadiazole heterocycle
were observed at approximately 4.36–4.91 ppm. In the 13C-NMR spectra, the typical shifts near
165–169, 150–155, and 31–32 ppm reveal the presence of -N=C- and C=O, the carbon atoms of the
1,3,4-thiadiazole, and -S-CH2- groups.

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Scheme 1. Synthesis of the title compounds 5a–5p.
2.2. Antiviral Activities of the Title Compounds against Plant Pathogens In Vivo
Antibacterial activities of myricetin derivatives containing the amide, thioether, and 1,3,4-
oxadiazole moieties (5a 5p) against the plant pathogens Xac, Rs, and Xoo were evaluated in vitro using
a turbidimeter method [25 27]. The standard drug Myricetin (MYR) and the commercial bactericides
–
–
Thiodiazole copper (TC) and Bismerthiazol (BT) were chosen as control agents. The results are shown
in Table 1. Most of the target compounds exhibited similar or slightly enhanced inhibition activities
against the pathogens Xac, Rs, and Xoo at the two concentrations tested (100 and 50 µg/mL).
Table 1. Antibacterial activities of the target compounds (5a–5p) against plant pathogenic bacteria in vitro.
Inhibition Rate a/%
Compd.
Xac
100 µg/mL
Rs
Xoo
50 µg/mL
100 µg/mL
50 µg/mL
100 µg/mL
50 µg/mL
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
75.7 ± 5.3
54.7 ± 1.9
51.3 ± 4.9
39.3 ± 1.4
44.4 ± 2.8
75.0 ± 5.9
89.2 ± 1.9
74.3 ± 2.8
89.5 ± 6.5
41.5 ± 4.1
73.5 ± 6.7
75.3 ± 5.3
72.6 ± 4.8
55.3 ± 2.1
55.6 ± 7.7
67.4 ± 6.8
40.7 ± 4.8
73.3 ± 8.2
67.7 ± 3.7
64.3 ± 3.1
53.5 ± 1.1
18.9 ± 2.6
34.7 ± 1.7
15.2 ± 5.3
60.3 ± 3.6
79.5 ± 1.6
68.3 ± 5.6
68.7 ± 2.7
29.2 ± 3.0
51.6 ± 2.7
55.3 ± 4.1
35.2 ± 1.7
47.9 ± 1.4
46.8 ± 7.0
51.1 ± 7.0
31.1 ± 3.0
55.0 ± 4.5
44.3 ± 4.0
70.4 ± 7.5
41.5 ± 7.5
68.7 ± 6.5
35.3 ± 3.0
78.0 ± 1.5
33.5 ± 9.3
29.7 ± 0.8
41.5 ± 0.3
85.6 ± 4.9
66.3 ± 2.0
28.9 ± 2.1
34.9 ± 4.5
41.4 ± 7.3
52.5 ± 2.9
33.2 ± 2.3
36.7 ± 4.0
57.1 ± 7.5
69.7 ± 1.9
35.3 ± 3.8
50.2 ± 6.5
16.4 ± 0.9
63.4 ± 4.6
37.0 ± 2.0
60.5 ± 3.1
3.0 ± 1.7
20.6 ± 0.7
38.5 ± 2.0
64.8 ± 1.7
59.6 ± 4.9
25.6 ± 1.1
31.2 ± 4.9
34.7 ± 5.8
49.9 ± 4.2
8.3 ± 0.8
35.0 ± 1.3
44.2 ± 3.4
57.6 ± 3.1
18.9 ± 4.4
c
59.1 ± 2.3
51.7 ± 2.6
62.7 ± 2.8
59.1 ± 2.6
69.8 ± 8.2
68.4 ± 3.5
65.6 ± 5.8
69.5 ± 1.7
78.3 ± 6.9
44.4 ± 5.2
48.3 ± 2.8
54.1 ± 4.0
55.3 ± 2.8
44.8 ± 0.7
77.6 ± 4.3
47.8 ± 7.5
68.5 ± 8.0
71.1 ± 3.8
36.1 ± 1.3
54.4 ± 2.9
44.2 ± 3.4
44.1 ± 6.7
58.0 ± 1.7
62.0 ± 3.0
50.4 ± 2.5
61.4 ± 1.5
33.7 ± 2.8
53.0 ± 1.8
36.9 ± 3.9
47.5 ± 2.5
36.8 ± 1.7
50.2 ± 1.0
43.8 ± 3.4
42.3 ± 1.6
31.8 ± 2.6
53.2 ± 9.4
36.5 ± 2.9
35.8 ± 2.1
MYR b
BT c
TC c
a
b
Average of three replicates. The lead compound of myricetin. The commercial agricultural bactericides,
Bismerthiazol, and Thiodiazole copper were used in a comparison of antibacterial activity.

Molecules 2018, 23, 3132
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Among the title compounds, the inhibitory rates of compounds 5a
excellent antibacterial activities against Xac at 100
respectively, which were better than those of the commercial bactericides BT (73.3%) and TC (67.7%).
The activities of the above target compounds against Xac at 50 g/mL were 64.3, 60.3, 79.5, 68.3,
68.7, and 55.3% respectively, which were better than those of BT (55.0%) and TC (44.3%). Moreover,
the inhibition rates of compounds 5a 5e, and 5i at 100 g/mL against Rs were 70.4, 68.7, 78.0,
and 85.6%, respectively, which exceeded that of the BT (69.7%). Compounds 5c 5e, and 5i at 50 g/mL
showed 63.4, 60.5, and 64.8% inhibition rates, respectively, which were superior to that of BT (57.6%).
Furthermore, the inhibitory activities of all the target compounds at 100 g/mL against Xoo were better
,
5f,
5g
,
5h
,
5i, and 5l possessed
µg/mL were 75.7, 75.0, 89.2, 74.3, 89.5, and 75.3%,
µ
,
µ
,
µ
µ
than the BT (71.1%) and similar to TC (36.1%). Notably, compound 5i showed comparable or stronger
antibacterial activities against Xac, Rs, and Xoo when compared with the two commercial bactericides.
Thus, compound 5i was regarded as the most promising antibacterial agent and used for further
evaluation. According to our previous work on myricetin derivatives containing 1,3,4-thiadiazole [18],
when the R group is CH3, Ph, 2-CH3-Ph, and 4-CH3O-Ph, the antibacterial activities against Xoo of our
present work is better than previous work, when R group is 2,4-diCl-Ph, 4-CH3O-Ph, and 2-Cl-thiazolyl,
the antibacterial activities against Rs is better than previously.
Based on previously described bioassays methods [28], some of the compounds with excellent
antibacterial activities were tested at five halved concentrations (100, 50, 25, 12.5, and 6.25 µg/mL)
in order to obtain their corresponding EC50 values. The screening results are shown in Table 2.
All of the tested compounds exhibited better antibacterial activities against the pathogens Xac
and Rs when compared with BT and TC. In addition, compounds 5e
values of 34.0, 44.9, and 31.2 g/mL, respectively, which were better than BT and TC (45.3 and
105.4 g/mL, respectively). Compared with previous work [18], when the R group is CH3, 2-Cl-Ph,
, 5f, and 5i exhibited EC50
µ
µ
2,4-diCl-Ph, and 1,3,4-thiadiazole myricetin derivatives exhibited excellent inhibitory effect on Rs at
low concentrations.
Table 2. EC50 values of the title compounds against plant pathogenic bacteria in vitro.
Bacteria
Compd.
R
Toxic Regression Equation
r
EC50/(µg/mL)
5a
5f
5g
5h
5i
CH3
2-F-Ph
4-F-Ph
2-CH3-Ph
3-CH3-Ph
4-CF3-Ph
-
y = 1.2309x + 3.2852
y = 1.1496x+3.3598
y = 1.3129x + 3.5997
y = 1.3011x + 3.1703
y = 1.5172x + 3.0628
y = 1.0060x + 3.5803
y = 1.2497x + 3.0744
y = 1.1587x + 3.1306
0.9814
0.9978
0.9928
0.9882
0.9740
0.9869
0.9755
0.9920
24.7 ± 2.7
26.7 ± 1.3
11.5 ± 1.8
25.5 ± 3.2
18.9 ± 2.6
27.3 ± 2.9
34.7 ± 1.3
41.1 ± 3.7
Xac
5l
BT a
TC a
-
5a
5c
5e
CH3
2-Cl-Ph
2,4-diCl-Ph
3-CH3-Ph
y = 1.0299x + 3.4622
y =0.7749x + 3.8771
y = 1.7574x + 2.1963
y = 2.0968x + 1.7437
y = 1.2725x + 2.6703
y = 1.2681x + 2.3203
0.9675
0.9861
0.9954
0.9873
0.9839
0.9927
31.3 ± 2.5
28.1 ± 4.2
39.4 ± 3.4
35.7 ± 1.6
67.2 ± 2.2
112.5 ± 4.7
Rs
5i
BT a
TC a
-
-
5e
5f
5h
2,4-diCl-Ph
2-F-Ph
2-CH3-Ph
3-CH3-Ph
4-CF3-Ph
-
y = 1.3410x + 2.9457
y = 1.3885x + 2.7900
y = 2.0971x + 1.1978
y = 1.3586x + 2.9710
y = 1.8004x + 1.9332
y = 1.6145x + 1.7430
y = 1.8721x + 1.8993
0.9951
0.9839
0.9918
0.9891
0.9792
0.9887
0.9830
34.0 ± 3.2
44.9 ± 3.5
65.0 ± 2.1
31.2 ± 2.4
50.5 ± 5.7
45.3 ± 3.0
105.1 ± 4.6
5i
Xoo
5o
BT a
TC a
-
a
The commercial agricultural antibacterial agents Thiodiazole copper (TC) and Bismerthiazol (BT) were used as
control agents.

Molecules 2018, 23, 3132
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2.3. Antiviral Activities of the Title Compounds against TMV In Vivo
At the concentration of 500
tested in vivo against TMV using the half-leaf method [
For comparison, the commercial antiviral agent ribavirin (RBV) was chosen as the positive control for
TMV. The results indicated that compounds 5a 5p showed various degrees of antiviral activities against
TMV. Some of these compounds displayed promising curative and inactivation activities, with curative
and inactivation rates ranging from 22.3 to 49.9% and 51.5 to 76.7%, respectively. The compounds 5f
5g 5i 5l, and 5n showed 42.2, 49.9, 44.2, 45.3 and 43.1% curative effects at 500 g/mL, respectively,
µ
g/mL, the antiviral activities of the target compounds 5a−5p were
7,
29], and the results are listed in Table 3.
–
,
,
,
,
µ
which were better than that of RBV (40.6%). Compounds 5g and 5j with values of 52.9 and 54.5%
showed protective activity against TMV. Moreover, the inactivation activities of all the compounds were
similar to that of RBV (71.1%). Compared to our previous work on myricetin derivatives containing
1,3,4-thiadiazole [18], when the R group is Me or 2-F-Ph, the anti-TMV curative activities of present
work is better than that of past work. When the R group is CH3, 2-F-Ph and 4-CH3O-Ph, the anti-TMV
protective effect of this work is superior to that of the previous work.
Table 3. Antiviral activities of the test compounds against TMV in vivo at 500 µg/mL.
Inhibition Rate a/%
Compd.
R
Curative
Protection
Inactivation
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
CH3
Ph
2-Cl-Ph
4-Cl-Ph
2,4-diCl-Ph
2-F-Ph
38.0 ± 6.3
26.0 ± 4.9
27.1 ± 4.1
35.6 ± 4.9
22.3 ± 1.8
42.2 ± 8.9
49.9 ± 5.0
31.0 ± 6.4
44.2 ± 5.3
30.6 ± 1.0
39.9 ± 4.2
45.3 ± 4.1
33.2 ± 4.8
43.1 ± 3.3
33.8 ± 7.6
35.9 ± 3.7
36.7 ± 6.3
40.6 ± 2.5
40.0 ± 6.9
36.0 ± 6.0
40.8 ± 7.1
42.2 ± 7.3
43.2 ± 4.6
49.2 ± 8.4
52.9 ± 5.2
34.7 ± 5.7
45.3 ± 7.2
54.5 ± 5.7
46.4 ± 5.9
49.9 ± 7.3
32.2 ± 8.8
36.1 ± 8.6
44.8 ± 5.9
47.7 ± 2.4
42.3 ± 6.5
51.1 ±2.3
53.6 ± 5.4
71.5 ± 5.1
72.9 ± 5.1
62.0 ± 2.8
76.7 ± 3.5
61.2 ± 5.9
73.3 ± 2.8
58.1 ± 4.6
57.0 ± 4.6
63.5 ± 4.9
60.2 ± 3.9
59.5 ± 6.2
52.5 ± 5.5
62.6 ± 3.3
65.3 ± 2.7
64.6 ± 4.1
51.5 ± 3.7
71.1 ± 4.2
4-F-Ph
2-CH3-Ph
3-CH3-Ph
4-CH3O-Ph
3-CF3-Ph
4-CF3-Ph
3-pyridyl
4-pyridyl
2-Cl-5-thiazolyl
4-NO2-Ph
-
5o
5p
MYR b
RBV c
-
a Average of three replicates. b The lead compound of myricetin(MYR). c The commercial antiviral agent Ribavirin (RBV).
Most target compounds possess better in curative, protective, and inactivation activities against
TMV in vivo when compared to RBV. Remarkably, compound 5g was superior to RBV in regard to all
three activities and, thus, was noted as a promising lead compound. As Figure 2 shows, the number
of local lesion on the leaf treated with either agent (RBV or 5g) was significantly less than that
without treatment. In addition, the area of the smeared with compound was smaller. Regarding the
inhibitory mechanism of myricetin on TMV, we are studying it and will report it in subsequent studies.
The inhibition rate of the compound was calculated according to the following formula. The average
of three replicates were conducted for each sample.
Inhibition rate (%) = [(local lesion number of control (not treated with compound)—local lesion
number smeared with compound)/ local lesion number of control ×100%.

Molecules 2018, 23, 3132
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Figure 2. Tobacco leaf morphology effects of the RBV and 5g against TMV in vivo. (Left leaf: Not
treated with compound, right leaf: Smeared with compound).
3. Experimental
Instruments. The melting points (m.p.) of the title compounds were determined when left
untouched on an XT-4-MP apparatus (Beijing Tech Instrument, Beijing, China). The 1H-NMR and
13C-NMR spectra were recorded on a JEOL-ECX 500 NMR spectrometer (JEOL, Tokyo, Japan) at
ambient temperature operating at 500 MHz for 1H-NMR and 126 MHz for 13C-NMR, using CDCl3
or DMSO-d6 or (CD3)2CO as solvents and TMS as an internal standard. Chemical shifts were given
in
δ relative to TMS. The HRMS spectrogram was performed on a Thermo Scientific Q Exactive
(Thermo, Waltham, MA, USA). Unless noted, all reagents were of analytical grade or chemically pure
and purchased from Tansoole (Shanghai, China). All anhydrous solvents were dried and purified
immediately prior to use in accordance with standard techniques.
3.1. Chemistry
3.1.1. Synthesis Procedure for Intermediate 1
Intermediate 1 was synthesized as detailed in a prior report [30]. A mixture of CS2 (120 mmol),
anhydrous K2CO3 (50 mmol), and thiosemicarbazide (100 mmol) in absolute ethanol (120 mL)
was stirred under reflux for one hour and then further heated on a steam bath for four hours.
Upon completion of the reaction (as indicated by TLC), the solvent was removed, and the residue
was dissolved in water, after dropwise addition of 10% HCl, to afford a white solid. The solid was
filtered and washed with cold water. Then, the crude product was filtered, washed, and dried to obtain
◦
◦
5-amino-1,3,4-thiadiazole-2-thiol (1) 10.64 g with a 80% yield. m.p. 234–236 C (References 233 C [30]).
3.1.2. General Procedure for Preparation of Intermediates 2a–6p
Intermediate
2 was synthesized as detailed in a prior report [31] with minor modification.
Intermediate (3.8 mmol) and triethylamine (7.6 mmol) were added into 30 mL of pyridine.
1
The mixture was incubated ambient temperature for 0.5 h. Then, bromoethane (3.9 mmol) was added to
the reaction system and the mixture was treated at room temperature for four h. After the reaction was
completed (as indicated by TLC), 100 mL of deionized water was added to the mixture and left to stand
for 10 min. The mixture was then filtered, and the solid was washed with water and subsequently dried
◦
under vacuum. Intermediate 2a was obtained 0.55 g at a yield of 89%. m.p. 136–137 C. Intermediates
2b–2p were prepared following a similar procedure.
3.1.3. General Procedure for Preparation of Intermediates 3a−3p
Compounds 3a–3p were synthesized according to known methods [32]. K2CO3 (4.5 mmol) were
added to a solution of intermediate 2a (1.8 mmol) in dichloromethane (30 mL). The resulting mixture
was then stirred at room temperature for 30 min. Next, a solution prepared by dissolving chloroacetyl

Molecules 2018, 23, 3132
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chloride (2.7 mmol) in 5 mL dichloromethane was added to the reaction system in a drop wise fashion.
The mixture was then stirred at ambient temperature for a further eight hours. After completion
of the reaction, the mixture was refluxed for 30 min, and the solvent was removed under reduced
pressure. Then, water (20 mL) was added to the mixture to afford a white solid, which was filtered
and recrystallized from methanol/water (1:3/v:v). Intermediate 3a was obtained 0.34 g with a yield of
77%. m.p. 166–168 ◦C. Following a similar procedure, intermediates 3b–3p were prepared.
3.1.4. General Procedure for Preparation of Intermediate 4
Preparation of the intermediate
4
has been previously described [19]. The mixture of myricitrin
◦
(10 mmol), CH3I (130 mmol), and K2CO3 (12 mmol) was dissolved in DMF (60 mL), and stirred at 40 C
for two days until the reaction was complete (as indicated by TLC analysis). The reaction mixtures
were then filtered, and the filtrate was dissolved in 100 mL water and finally extracted three times
with dichloromethane (30 mL
×
3), combined the dichloromethane, and concentrated under reduced
pressure. The concentrated solution was diluted with 40 mL of absolute ethanol, stirred, and refluxed
for one hour. The concentrated hydrochloric acid (9 mL) was slowly added to the above obtained for two
hours in reflux. The solid was precipitated from the clear solution. After cooling to ro◦ om temperature,
the reaction mixture was filtered, and the obtained solid product was dried at 40 C for two hours.
Intermediate 4 was obtained 2.38 g at a yield of 75%. m.p. 228–230 ◦C (References 227–229 ◦C [19]).
3.1.5. General Procedure for Preparation of Title Compounds 5a−5p
K2CO3 (2.1 mmol) was added to a solution of intermediate
4 (0.7 mmol) in DMF (10 mL).
The mixture was stirred at room temperature for 0.5 h. Then, a solution prepared by dissolving
intermediate 3a (0.7 mmol) in five mL DMF was add◦ed to the reaction system in a dropwise fashion.
The reaction mixture was then allowed to stand at 105 C for four h. Upon completion of the reaction as
indicated by TLC (ethyl acetate/methyl alcohol, 15:1/v:v), the mixture was diluted with water (100 mL)
and extracted with dichloromethane (30 mL
dried over anhydrous Na2SO4, filtered, concentrated in vacuo, and the crude product was purified by
×
3). The combined dichloromethane extracts were
column chromatography (silica gel) using ethyl acetate/methanol (10:1/v:v) as the eluent to afford a
pure target compound. Compound 5a was obtained 0.31 g at a yield of 75%. Compounds 5b–5p were
obtained by the same method. Representative data regarding 5a are listed below. Data for compounds
5b–5p are given in the Supporting Information.
5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)-3-O-(N-(5-((ethyl)thio)-1,3,4-thiadiazol-2-yl) acetamido-2-yl)-4H-
◦
chromen-4-one (5a). White solid; yield: 75%; m.p. 223–225 C; 1H-NMR (500 MHz, CDCl3)
δ: 12.71
(s, 1H, NH), 7.24 (s, 2H, Ar-H), 6.54 (d, J = 2.2 Hz, 1H, Ar-H), 6.40 (d, J = 2.2 Hz, 1H, Ar-H), 4.48 (s,
2H, O-CH2), 3.98 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.94 (s, 6H, OCH3), 3.93 (s, 3H, OCH3), 3.27 (q,
J = 7.4 Hz, 2H, S-CH2), 1.44 (t, J = 7.4 Hz, 3H, CH3); 13C-NMR (126 MHz, CDCl3)
δ: 174.27, 167.50,
164.93, 161.18, 160.43, 158.98, 158.03, 154.18, 153.48, 140.82, 140.77, 124.62, 108.56, 105.82, 96.36, 92.62,
72.53, 61.09, 56.49, 55.98, 28.71, 14.72. ESI-HRMS (m/z), calcd. for C26H27N3O9S2 [M + H] + 590.1262,
found 590.1252.
3.2. Biological Assays
The antibacterial activities of compounds 5a
China), Rs (Guizhou University, China), and Xoo (Nanjing Agricultural University, China) were
evaluated based on the turbidimeter test [25 27]. The test compounds were dissolved in 150
of dimethyl sulfoxide (DMSO) and diluted with water containing Tween 20 (0.1%) to obtain final
concentrations of 100 and 50 g/mL. Pure DMSO in sterile distilled water served as a blank control,
–5p against Xac (Shanghai Jiao Tong University,
–
µL
−
µ
whereas TC and BT served as positive controls. Approximately one mL of liquid sample was added
to the nontoxic nutrient broth (NB; three g of beef extract, five g of peptone, one g of yeast powder,
10 g of glucose, and one L of distilled water, Adjust pH = 7.0–7.2 with 10% NaOH) liquid medium in

Molecules 2018, 23, 3132
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four mL tubes. Then, approximately 40
µ
L of NB containing bacterium Xac/Rs/Xoo was added to five
mL of solvent NB containing the test compounds or TC/BT. The above test tubes were incubated at
30
1 ◦C with continuous shaking at 180 rpm for 1–3 days. Bacterial growth was monitored with a
spectrophotometer by measuring the optical density at 600 nm (OD600), given by corrected turbidity
values = ODbacterium ODno bacterium. The relative inhibitory rate (I%) of the circle mycelium compared
with a blank assay was calculated as follows: I = (Ctur tur)/Ctur 100%. The Ctur represents the
±
−
−
T
×
corrected turbidity value of bacterial growth in untreated NB (blank control), and Ttur is the corrected
turbidity value of bacterial growth in treated NB. Similarly, the excellent antibacterial activities of
the target compounds and TC/BT were further assessed against the bacteria Xac, Rs, and Xoo at
five double-declining concentrations (100, 50, 25, 12.5, and 6.25 µg/mL) to obtain their EC50 values,
which were statistically determined using probit analysis within the software package SPSS 17.0.
The listed EC50 values in Table 2 are the average of three trials.
The curative, protection, and inactivation activities against TMV of the target compounds were
tested using previously reported methods [7,29]. The commercial compound RBV was used as a
comparison and an average of three replications were conducted for each sample.
4. Conclusions
In conclusion, a series of myricetin derivatives containing amide, 1,3,4-oxadiazole, and thioether
moieties were developed as potential antibacterial and antiviral agents, and sixteen novel compounds
were synthesized. The bioassay results indicated that some of the target compounds possessed excellent
antibacterial activities against the plant pathogens Xoo, Rs, and Xoo. For example, compounds 5a
5h 5i, and 5l showed strong antibacterial activity against Xac, with the EC50 values of 11.5–27.3
which are significantly better than the commercial bactericides BT (34.7 g/mL) and TC (41.1%
,
µ
5f
g/mL,
g/mL).
,
5g
,
,
µ
µ
An anti-TMV activity test for the target compounds was performed, and among these compounds,
the curative, protection and inactivation activities of 5g were 49.9%, 52.9%, and 73.3%, respectively,
which are better than that of the commercial antiviral RBV (40.6, 51.1, and 71.1%, respectively). It is
noteworthy that most of the myricetin derivatives obtained by derivatization had better antibacterial
and antiviral activities than those of myricetin. The above studies showed that myricetin derivatives
containing amide, 1,3,4-oxadiazole, and thioether moieties could serve as promising candidates for
the development of new and more efficient agricultural bactericides against bacterial pathogens and
plant viruses.
On the basis of the previous work [18], the hydrophilic amide structure was introduced to improve
the water-solubility of target compounds and enrich the structure type and quantity of myricetin
derivatives. Extending the linking group between myricetin and the 1,3,4-thiadiazole also made the
molecules more flexible and provided some preliminary structure-activity relationships. The bioassays
indicated that 1,3,4-thiadiazole and myricetin could not bind to the target protein of Rs, Xoo, and TMV
by extending the linking group between 1,3,4-thiadiazole and myricetin.
Supplementary Materials: The supplementary materials containing the yield relationship, 1H-NMR, 13C-NMR,
HRMS dates and spectra of the title compounds 5a–5p are available online.
Author Contributions: W.X. conceived and designed the experiments; X.R. and C.Z. performed the experiments
and analyzed the data; S.J. and T.G. evaluated the antibacterial activities of the title compounds; R.X., Y.C. and
X.T. assessed the antiviral activities of the title compounds; X.R. wrote the paper; and W.X. reviewed the paper.
All authors read and approved the final manuscript.
Funding: This research was funded by the National Key Research and Development Program of China
(2017YFD0200506), the National Nature Science Foundation of China (21867003), the Special Fund for Outstanding
Scientific—Technological Candidates of Guizhou Province (201535), Major Program of Innovation Research
Team of Educational Commission of Guizhou Province (2016113), Science and Technology Platform for Talent of
Guizhou Province (20165608), and Science and Technology Project of Guizhou Province (20172956).
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2018, 23, 3132
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).