Design, synthesis, and bioactivity evaluation of novel isoxazole-amide derivatives containing an acylhydrazone moiety as new active antiviral agents

Article abstract of DOI:10.3390/molecules24203766

As a continuation of our efforts to discover and develop “me-better” active molecules, in this study, a series of novel isoxazole-amide derivatives containing an acylhydrazone moiety were synthesized and evaluated for their antiviral activities against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). Antiviral bioassays indicated that some of the target compounds exhibited better in vivo antiviral activities against TMV and CMV than those of Ningnanmycin (NNM). Especially, the compound 7t exhibited the best curative, protection, and inactivation activities against TMV and CMV which were superior to those of NNM. Meanwhile, our present work also revealed that compound 7t could enhance the defense-related enzyme activity and increase the chlorophyll content in tobacco leaves to induce resistance and enhance plant tolerance to TMV infection.

Full text of DOI:10.3390/molecules24203766

molecules
Article
Design, Synthesis, and Bioactivity Evaluation of
Novel Isoxazole-Amide Derivatives Containing an
Acylhydrazone Moiety as New Active
Antiviral Agents
1
, ,†
1,2, ,†
and Yin-Ju He 1
Zai-Bo Yang * , Pei Li *
1
School of Chemistry and Chemical Engineering, Qiannan Normal University for Nationalities,
Duyun 558000, China; heyinju2007@163.com
Qiandongnan Engineering and Technology Research Center for Comprehensive Utilization of National
Medicine, Kaili University, Kaili 556011, China
2
*
Correspondence: yzb1976110@sohu.com (Z.-B.Y.); pl19890627@126.com (P.L.);
Tel.: +86(854)873-7046 (Z.-B.Y.)
†
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Antonio Carta
Received: 2 September 2019; Accepted: 18 October 2019; Published: 19 October 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: As a continuation of our efforts to discover and develop “me-better” active molecules,
in this study, a series of novel isoxazole-amide derivatives containing an acylhydrazone moiety
were synthesized and evaluated for their antiviral activities against tobacco mosaic virus (TMV) and
cucumber mosaic virus (CMV). Antiviral bioassays indicated that some of the target compounds
exhibited better in vivo antiviral activities against TMV and CMV than those of Ningnanmycin (NNM).
Especially, the compound 7t exhibited the best curative, protection, and inactivation activities against
TMV and CMV which were superior to those of NNM. Meanwhile, our present work also revealed
that compound 7t could enhance the defense-related enzyme activity and increase the chlorophyll
content in tobacco leaves to induce resistance and enhance plant tolerance to TMV infection.
Keywords: isoxazole-amide; acylhydrazone; synthesis; antiviral activity
1
. Introduction
Tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are two significant plant viruses
and can cause significant economic losses in various crops, including tobacco, tomato, pepper,
cucumbers, and a number of ornamental flowers [ ]. Ribavirin, Curcumin, and Ningnanmycin
NNM) are successful antiviral agents that are widely used for preventing plant viruses, however,
1,2
(
they showed unsatisfactory cure rates (30%–60%) [3]. Therefore, appreciable efficiency for controlling
TMV and CMV remain unavailable and development of new effective antiviral agents remains a
significant challenge.
In recent years, literature revealed that isoxazole-amide, an important scaffold for synthesis
of various active molecules and their derivatives, had a wide range of biological activities, such as
antifungal [
Meanwhile, literature revealed that acylhydrazone derivatives had a wide range of biological activities,
such as antibacterial [ ], fungicidal [10], insecticidal [11 12], and antiviral [13] activity. Specifically,
in our previous work, we reported a series of novel isoxazole-amide derivatives containing an
acylhydrazone moiety (Figure 1) with potent antiviral activity against TMV [ ]. In addition, the phenyl
ring especially with electron-withdrawing groups, a privileged structure, represents a key motif in
4], herbicidal [4], antiviral [5], anticancer [6], antibacterial [7], and insecticide [8] activity.
9
,
5
Molecules 2019, 24, 3766; doi:10.3390/molecules24203766
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3766
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medicinal chemistry because of its ability to improve pharmacological activities including antiviral [14],
antifungal [15], antibacterial [16,17], insecticidal [18], and nematicidal [17,19] activity.
Figure 1. Design route of the target compounds.
As a continuation of our efforts to discover and develop “me-better” active molecules, in this study,
we aimed to replace the pyrazole ring with a phenyl ring with electron-withdrawing groups, shown in
Figure 1, to build some new isoxazole-amide derivatives containing an acylhydrazone moiety as novel
active antiviral agents. Antiviral bioassays indicated that some of the target compounds exhibited
better in vivo antiviral activities against TMV and CMV than those of NNM. Especially, the compound
7t exhibited the best curative, protection, and inactivation activities against TMV and CMV which
were superior to those of NNM. Meanwhile, our present work also revealed that compound 7t could
enhance the defense-related enzyme activity and increase the chlorophyll content in tobacco leaves to
induce resistance and enhance plant tolerance to TMV infection.
2
. Results
2
.1. Chemistry
Using 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic acid as the starting material,
as shown in Scheme 1, a series of novel isoxazole-amide derivatives containing an acylhydrazone
moiety 7a
–
7v were synthesized in five steps with yields of 87%
−
93%. The physical characteristics,
1
13
IR, H NMR, C NMR, ESI-MS, and elemental analysis data for all the target compounds 7a
–7v are
shown below.
Scheme 1. Synthetic route of the target compounds 7a–7v.

Molecules 2019, 24, 3766
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(
E)-N-(5-chloro-2-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)phenyl)-3-(2,6-dichlorophenyl)-5-
◦
1
methylisoxazole-4-carboxamide (7a). White solid; mp 257
DMSO-d , ppm)
−
258 C; yield 90%; H NMR (500 MHz,
δ
: 12.15 (s, 1H, isoxazole–CONH), 11.21 (s, 1H, Ar–CONH), 8.41 (s, 1H, –CH=N–),
6
8
.35 (d, 1H, J = 1.50 Hz, Ar–H), 7.82 (dd, 2H, J = J = 8.5 Hz, Ar–H), 7.62–7.49 (m, 6H, Ar–H), 7.37
1
2
13
(dd, 1H, J = 2.50 Hz, J = 2.00 Hz, Ar–H), 2.89 (s, 3H, –CH ); C NMR (125 MHz, DMSO-d , ppm)
1 2 3 6
δ
: 172.05, 164.19, 162.84, 159.17, 158.45, 148.41, 140.09, 135.46, 135.01, 133.45, 132.89, 130.80, 129.60,
−1
1
1
1
29.47, 128.97, 127.22, 123.87, 121.23, 120.11, 113.80, 13.33; IR (KBr, cm )
699.29, 1683.86, 1653.00, 1635.64, 1616.35, 1595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08,
456.26, 1429.25, 1411.89, 1398.39, 1361.74, 1338.60, 1313.52, 1253.73; Anal. Calcd. for C H Cl N O :
ν: 3444.87, 3176.76, 2951.09,
25
16
4
4
+
3
C 53.41%, H 2.87%, N 9.97%, Found: C 53.47%, H 3.14%, N 10.30%; MS (ESI) m/z: 563.2 ([M + H] ).
(
E)-N-(2-(2-(4-bromobenzylidene)hydrazine-1-carbonyl)-5-chlorophenyl)-3-(2,6-dichlorophenyl)-5-
◦
1
methylisoxazole-4-carboxamide (7b). White solid; mp 259
DMSO-d , ppm)
−
260 C; yield 91%; H NMR (500 MHz,
δ
: 12.15 (s, 1H, isoxazole–CONH), 11.19 (s, 1H, Ar–CONH), 8.39 (s, 1H, –CH=N–),
6
8
.35 (d, 1H, J = 2.50 Hz, Ar–H), 7.85 (d, 1H, J = 8.50 Hz, Ar–H), 7.73–7.68 (m, 4H, Ar–H), 7.61 (d, 2H, J
=
3.50 Hz, Ar–H), 7.51 (q, 1H, Ar–H), 7.37 (dd, 1H, J = 2.00 Hz, J = 1.50 Hz, Ar–H), 2.89 (s, 3H, –CH );
1 2 3
13
C NMR (125 MHz, DMSO-d , ppm)
δ: 172.06, 164.19, 162.85, 159.17, 158.45, 148.50, 140.07, 137.31,
6
1
35.01, 133.78, 132.89, 132.51, 130.81, 129.69, 128.97, 127.21, 124.30, 123.88, 121.24, 120.12, 113.80, 13.33;
−1
IR (KBr, cm
) ν: 3444.87, 3261.63, 3066.82, 1699.29, 1683.86, 1670.35, 1653.00, 1645.28, 1591.27, 1570.06,
1
1
558.48, 1541.12, 1519.91, 1506.41, 1489.08, 1456.26, 1429.25, 1398.39, 1361.74, 1300.02, 1257.29, 1234.44,
195.87; Anal. Calcd. for C H BrCl N O : C 49.49%, H 2.66%, N 9.23%; Found: C 49.57%, H 3.04%,
25
16
3
4
3
+
N 9.54%; MS (ESI) m/z: 607.7 ([M + H] ).
(
E)-N-(5-chloro-2-(2-(2-fluorobenzylidene)hydrazine-1-carbonyl)phenyl)-3-(2,6-dichlorophenyl)-5-
◦
1
methylisoxazole-4-carboxamide (7c). White solid; mp 248
DMSO-d , ppm)
−
249 C; yield 91%; H NMR (500 MHz,
δ
: 12.19 (s, 1H, isoxazole–CONH), 11.21 (s, 1H, Ar–CONH), 8.67 (s, 1H, –CH=N–),
6
8
.35 (d, 1H, J = 1.50 Hz, Ar–H), 7.98 (t, 1H, J = 6.00 Hz, J = 7.50 Hz, Ar–H), 7.87 (d, 1H, J = 8.50 Hz,
1
2
Ar–H), 7.61 (d, 2H, J = 8.00 Hz, Ar–H), 7.56–7.50 (m, 2H, Ar–H), 7.39–7.32 (m, 3H, Ar–H), 2.89 (s, 3H,
CH3); 13C NMR (125 MHz, DMSO-d , ppm)
: 172.03, 164.15, 160.47, 159.18, 158.46, 142.48, 140.10,
37.37, 135.00, 132.91, 130.80, 128.98, 127.20, 126.93, 125.63, 123.93, 122.09, 121.28, 120.09, 116.74, 116.58,
–
δ
6
1
1
1
1
−
1
13.81, 13.32; IR (KBr, cm )
595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1456.126, 1436.97, 1417.68, 1398.39, 1361.74,
338.60, 1311.59, 1247.94, 1224.80; Anal. Calcd. for C H Cl FN O : C 55.02%, H 2.95%, N 10.27%,
ν: 3444.87, 3265.49, 1699.29, 1683.86, 1670.25, 1653.00, 1635.64, 1616.35,
25
16
3
4
3
+
Found: C 55.47%, H 3.22%, N 10.62%; MS (ESI) m/z: 546.8 ([M + H] ).
E)-N-(5-chloro-2-(2-(4-fluorobenzylidene)hydrazine-1-carbonyl)phenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7d). White solid; mp 245–246 C; yield 92%; H NMR (500 MHz,
DMSO-d , ppm) : 12.07 (s, 1H, isoxazole–CONH), 11.21 (s, 1H, Ar–CONH), 8.38 (s, 1H, –CH=N–), 8.32
d, 1H, J = 1.50 Hz, Ar–H), 7.57 (d, 2H, J = 8.00 Hz, Ar–H), 7.47 (q, 1H, Ar–H), 7.34–7.28 (m, 3H, Ar–H),
δ
6
(
2
8
.85 (s, 3H, –CH3); 13C NMR (125 MHz, DMSO-d , ppm)
5.88 Hz), 148.62, 140.09, 137.28, 135.00, 132.88, 131.10, 130.77, 130.11, 130.04, 128.98, 127.23, 123.86,
δ: 172.03, 164.84, 164.15, 162.88, 158.81 (d, J =
6
−1
1
1
1
5
21.19, 120.05, 116.67, 116.49, 113.80, 13.33; IR (KBr, cm
)
ν
: 3444.87, 3265.49, 1699.29, 1683.86, 1670.25,
653.00, 1635.64, 1616.35, 1595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1456.26, 1436.97,
417.68, 1398.39, 1361.74, 1338.60, 1307.74, 12759.29, 1234.44; Anal. Calcd. for C H Cl FN O : C
25
16
3
4
3
+
5.02%, H 2.95%, N 10.27%, Found: C 55.51%, H 3.35%, N 10.37%; MS (ESI) m/z: 546.8 ([M + H] ).
E)-N-(5-chloro-2-(2-(4-methylbenzylidene)hydrazine-1-carbonyl)phenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7e). White solid; mp 247–248 C; yield 90%; H NMR (500 MHz,
DMSO-d , ppm)
δ
: 12.05 (s, 1H, isoxazole–CONH), 11.29 (s, 1H, Ar–CONH), 8.39 (s, 1H, –CH=N–),
6
8
=
.36 (d, 1H, J = 2.50 Hz, Ar–H), 7.86 (d, 1H, J = 8.50 Hz, Ar–H), 7.69–7.59 (m, 4H, Ar–H), 7.51 (t, 1H, J
1
13
8.00 Hz, J = 8.50 Hz, Ar–H), 7.38–7.30 (m, 3H, Ar–H), 2.90 (s, 3H, –CH ), 2.37 (s, 3H, –CH );
C
2
3
3
NMR (125 MHz, DMSO-d , ppm) δ: 171.97, 164.06, 159.13, 158.52, 149.87, 140.95, 140.10, 137.22, 134.99,
6
−1
1
3
32.88, 131.76, 130.77, 130.09, 128.96, 127.85, 127.25, 123.83, 120.07, 113.82, 21.62, 13.34; IR (KBr, cm ) ν:
444.87, 3265.49, 1699.29, 1683.86, 1670.25, 1653.00, 1635.64, 1616.35, 1595.13, 1575.84, 1558.48, 1541.12,

Molecules 2019, 24, 3766
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1
1
1
521.84, 1506.41, 1489.08, 1473.62, 1456.26, 1436.97, 1429.25, 1398.39, 1375.25, 1338.60, 1317.38, 1253.73,
234.44; Anal. Calcd. for C H Cl N O : C 57.64%, H 3.53%, N 10.34%, Found: C 57.87%, H 3.78%, N
2
6
19
3
4
3
+
0.32%; MS (ESI) m/z: 542.8 ([M + H] ).
(
E)-N-(5-chloro-2-(2-(2-chlorobenzylidene)hydrazine-1-carbonyl)phenyl)-3-(2,6-dichlorophenyl)-5-
◦
1
methylisoxazole-4-carboxamide (7f). White solid; mp 241–243 C; yield 91%; H NMR (500 MHz,
DMSO-d , ppm) : 12.28 (s, 1H, isoxazole–CONH), 11.21 (s, 1H, Ar–CONH), 8.84 (s, 1H, –CH=N–),
.35 (d, 1H, J = 2.50 Hz, Ar–H), 8.06 (q, 1H, Ar–H), 7.89 (d, 1H, J = 8.50 Hz, Ar–H), 7.61 (d, 1H, J =
δ
6
8
8
13
.00 Hz, Ar–H), 7.57–7.48 (m, 4H, Ar–H), 7.38 (dd, J = J = 1.50 Hz, Ar–H), 2.89 (s, 3H, –CH );
C
1
2
3
NMR (125 MHz, DMSO-d , ppm)
δ: 172.01, 164.23, 159.19, 158.49, 145.65, 140.12, 137.45, 135.01, 133.99,
6
1
32.88, 132.43, 131.76, 130.85, 130.56, 128.97, 128.30, 127.51, 127.24, 123.90, 121.30, 120.07, 113.94, 13.32;
−1
IR (KBr, cm
) ν: 3444.87, 3265.49, 1699.29, 1683.86, 1670.25, 1653.00, 1635.64, 1616.35, 1595.13, 1575.84,
1
1
5
558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1473.62, 1456.26, 1436.97, 1411.89, 1398.39, 1361.74, 1354.03,
338.60, 1317.38, 1249.87; Anal. Calcd. for C H Cl N O : C 53.41%, H 2.87%, N 9.97%, Found: C
25
16
4
4
3
+
3.43%, H 3.05%, N 10.22%; MS (ESI) m/z: 563.2 ([M + H] ).
E)-N-(2-(2-(2-bromobenzylidene)hydrazine-1-carbonyl)-5-chlorophenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7g). White solid; mp 245
DMSO-d , ppm) : 12.32 (s, 1H, isoxazole–CONH), 11.22 (s, 1H, Ar–CONH), 8.80 (s, 1H, –CH=N–),
.35 (s, 1H, Ar–H), 8.04 (d, 1H, J = 7.50 Hz, Ar–H), 7.90 (d, 1H, J = 8.00 Hz, Ar–H), 7.74–7.37 (m, 7H,
−
247 C; yield 92%; H NMR (500 MHz,
δ
6
8
13
Ar–H), 2.90 (s, 3H, –CH3); C NMR (125 MHz, DMSO-d , ppm)
δ
: 171.98, 164.24, 159.18, 158.50,
6
1
1
1
1
47.99, 140.10, 137.40, 135.01, 133.81, 133.25, 132.88, 132.67, 130.87, 128.97, 128.93, 128.77, 127.88,
27.25, 124.38, 123.89, 121.29, 120.09, 113.82, 13.34; IR (KBr, cm ) ν: 3446.79, 3307.92, 1699.29, 1683.86,
670.25, 1653.00, 1635.64, 1616.35, 1595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1456.26,
436.97, 1429.25, 1411.89, 1398.39, 1361.74, 1338.60, 1313.52, 1276.88, 1247.94, 1234.44; Anal. Calcd. for
−
1
C H BrCl N O : C 49.49%, H 2.66%, N 9.23%, Found: C 49.88%, H 2.75%, N 9.65%; MS (ESI) m/z:
25
16
3
4
+
3
6
07.7([M + H] ).
E)-N-(5-chloro-2-(2-(furan-2-ylmethylene)hydrazine-1-carbonyl)phenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7h). White solid; mp 173
DMSO-d , ppm) : 12.05 (s, 1H, isoxazole–CONH), 11.27 (s, 1H, Ar–CONH), 8.34 (d, 1H, J = 2.50 Hz,
Ar–H), 8.30 (s, 1H, –CH=N–), 7.91 (d, 1H, J = 1.00 Hz, Ar–H), 7.84 (d, 1H, J = 9.00 Hz, Ar–H), 7.62 (d,
−
174 C; yield 89%; H NMR (500 MHz,
δ
6
2
3
H, J = 8.00 Hz, Ar–H), 7.53 (q, 1H, Ar–H), 7.37 (dd, 1H, J = J = 2.50 Hz, furyl–H), 7.02 (d, 1H, J =
1 2
13
.50 Hz, furyl–H), 6.68 (q, 1H, furyl–H), 2.90 (s, 3H, –CH3); C NMR (125 MHz, DMSO-d , ppm) δ:
6
1
1
1
1
71.81, 164.07, 159.11, 158.62, 149.60, 146.23, 140.05, 139.33, 137.26, 134.96, 132.89, 130.71, 128.96, 127.32,
−
1
23.88, 121.21, 120.08, 115.17, 113.81, 112.89, 13.32; IR (KBr, cm
) ν: 3419.79, 3265.49, 1716.65, 16.95.43,
683.86, 1662.64, 1653.00, 1635.64, 1616.35, 1595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08,
473.62, 1456.26, 1436.97, 1429.25, 1398.39, 1361.74, 1338.60, 1313.52, 1249.87, 1233.44; Anal. Calcd. for
C H Cl N O : C 53.36%, H 2.92%, N 10.82%, Found: C 53.59%, H 3.25%, N 10.65%; MS (ESI) m/z:
23
15
3
4
4
+
5
18.7([M + H] ).
E)-N-(5-chloro-2-(2-(2,3-dichlorobenzylidene)hydrazine-1-carbonyl)phenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7i). White solid; mp 231–232 C; yield 93%; H NMR (500 MHz,
DMSO-d , ppm) : 12.36 (s, 1H, isoxazole–CONH), 11.17 (s, 1H, Ar–CONH), 8.86 (s, 1H, –CH=N–), 8.34
d, 1H, J = 1.50 Hz, Ar–H), 8.02 (d, 1H, J = 7.00 Hz, Ar–H), 7.88 (d, 1H, J = 8.50 Hz, Ar–H), 7.76 (d, 1H,
J = 8.00 Hz, Ar–H), 7.62 (d, 2H, J = 8.00 Hz, Ar–H), 7.54–7.50 (m, 2H, Ar–H), 7.39 (dd, 1H, J = 2.50 Hz,
δ
6
(
1
13
J = 2.00 Hz, Ar–H), 2.89 (s, 3H, –CH ); C NMR (125 MHz, DMSO-d , ppm)
2
δ
: 172.03, 164.28, 159.20,
3
6
1
1
1
1
58.46, 145.36, 140.08, 137.47, 135.00, 134.22, 132.99, 132.90, 132.47, 130.86, 129.18, 128.98, 127.20, 126.11,
−1
23.95, 113.80, 13.32; IR (KBr, cm )
635.64, 1616.35, 1595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1473.26, 1456.26, 1436.97,
419.61, 1398.39, 1361.74, 1350.17, 1313.52, 1288.45, 1246.02, 1226.73; Anal. Calcd. for C H Cl N O :
ν: 3446.79, 3265.49, 1716.65, 1699.29, 1683.86, 1670.25, 1653.00,
25
15
5
4
3
+
C 50.32%, H 2.53%, N 9.39%, Found: C 50.47%, H 2.95%, N 9.28%; MS (ESI) m/z: 597.7 ([M + H] ).

Molecules 2019, 24, 3766
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(
E)-N-(2-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)-4-fluorophenyl)-3-(2,6-dichlorophenyl)-5-
◦
1
methylisoxazole-4-carboxamide (7j). White solid; mp 258–259 C; yield 90%; H NMR (500 MHz,
DMSO-d , ppm) : 12.08 (s, 1H, isoxazole–CONH), 10.77 (s, 1H, Ar–CONH), 8.38 (s, 1H, –CH=N–),
.14 (q, 1H, Ar–H), 7.96 (s, 1H, Ar–H), 7.79 (d, 2H, J = 9.00 Hz, Ar–H), 7.66 (dd, 1H, J = 2.50 Hz,
δ
6
8
1
J = 3.50 Hz, Ar–H), 7.58 (q, 4H, Ar–H), 7.51 (q, 1H, Ar–H), 7.45–7.41 (m, 1H, Ar–H), 2.86 (s, 3H,
2
13
–
CH3); C NMR (125 MHz, DMSO-d , ppm)
δ: 171.54, 163.56, 162.84, 159.00 (d, J = 15.50 Hz), 158.47,
6
1
1
1
57.13, 148.26, 135.44, 135.01, 134.67, 133.45, 132.80, 129.59, 129.47, 128.93, 127.35, 124.73, 119.47, 119.29,
−
1
15.89, 115.70, 113.94, 13.25; IR (KBr, cm )
ν: 3444.87, 3248.13, 1683.86, 1653.00, 1616.35, 1595.13,
558.48, 1521.84, 1448.54, 1417.68, 1404.18, 1386.82, 1328.95, 1267.23, 1244.09, 1213.23. Anal. Calcd. for
C H FCl N O : C 55.02%, H 2.95%, N 10.27%, Found: C 55.36%, H 3.06%, N 10.43%; MS (ESI) m/z:
25
16
3
4
3
+
5
46.8 ([M + H] ).
E)-N-(2-(2-(4-bromobenzylidene)hydrazine-1-carbonyl)-4-fluorophenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7k). White solid; mp 265–267 C; yield 90%; H NMR (500 MHz,
DMSO-d , ppm) : 12.08 (s, 1H, isoxazole–CONH), 10.75 (s, 1H, Ar–CONH), 8.36 (s, 1H, –CH=N–),
.13 (q, 1H, Ar–H), 7.73–7.69 (m, 4H, Ar–H), 7.59 (d, 2H, J = 8.00 Hz, Ar–H), 7.50 (q, 1H, Ar–H),
.44–7.40 (m, 1H, Ar–H), 2.86 (s, 3H, –CH3); 13C NMR (125 MHz, DMSO-d , ppm)
: 171.64, 163.69,
62.84, 159.04, 158.71 (d, J = 58.38 Hz), 157.95, 157.13, 148.34, 135.00, 134.65, 133.78, 132.80, 132.50,
δ
6
8
7
δ
6
1
1
3
−
1
32.48, 129.68, 129.66, 128.93, 128.91, 127.35, 124.73, 124.27, 119.47, 113.94, 13.19; IR (KBr, cm )
444.87, 3248.13, 1683.86, 1670.35, 1653.00, 1635.64, 1589.34, 1558.48, 1521.84, 1458.18, 1417.68, 1361.74,
319.31, 1271.09, 1244.09, 1201.65; Anal. Calcd. for C H BrCl FN O : C 50.87%, H 2.73%, N 9.49%,
ν:
1
25
16
2
4
+
3
Found: C 51.08%, H 2.96%, N 9.78%; MS (ESI) m/z: 591.2 ([M + H] ).
(
E)-3-(2,6-dichlorophenyl)-N-(4-fluoro-2-(2-(2-fluorobenzylidene)hydrazine-1-carbonyl)phenyl)-5-
◦
1
methylisoxazole-4-carboxamide (7l). White solid; mp 248
−
249 C; yield 89%; H NMR (500 MHz,
DMSO-d , ppm)
δ
: 12.11 (s, 1H, isoxazole–CONH), 10.78 (s, 1H, Ar–CONH), 8.65 (s, 1H, –CH=N–),
.16 (q, 1H, Ar–H), 7.98 (s, 1H, Ar–H), 7.69 (d, 1H, J = 7.00 Hz, Ar–H), 7.60 (d, 1H, J = 7.00 Hz, Ar–H),
.52 (q, 1H, Ar–H), 7.42 (d, 1H, J = 9.00 Hz, Ar–H), 7.33 (t, 1H, J = 8.00 Hz, J = 9.00 Hz, Ar–H), 2.87 (s,
6
8
7
3
1
2
H, –CH3); 13C NMR (125 MHz, DMSO-d , ppm)
δ: 171.64, 163.52, 158.72 (d, J = 61.63 Hz), 157.17 (d,
6
J = 56.00 Hz), 142.35, 135.02, 134.71, 133.00, 132.93, 132.79, 128.93, 128.59, 128.49, 127.35, 126.94, 125.60,
−
1
1
1
1
5
24.82, 124.63, 122.02, 119.36, 116.55, 115.69, 113.96, 13.24; IR (KBr, cm )
ν: 3444.87, 3244.27, 1683.86,
670.35, 1653.00, 1635.64, 1616.35, 1589.34, 1558.48, 1521.84, 1458.18, 1436.97, 1411.89, 1361.74, 1319.31,
305.81, 1238.30, 1228.66; Anal. Calcd. for C H F Cl N O : C 56.73%, H 3.05%, N 10.58%, Found: C
7.05%, H 3.14%, N 10.91%; MS (ESI) m/z: 530.3 ([M + H] ).
E)-3-(2,6-dichlorophenyl)-N-(4-fluoro-2-(2-(4-fluorobenzylidene)hydrazine-1-carbonyl)phenyl)-5-
25
16
2
2
4
+
3
(
◦
1
methylisoxazole-4-carboxamide (7m). White solid; mp 234
DMSO-d , ppm) : 12.03 (s, 1H, isoxazole–CONH), 10.80 (s, 1H, Ar–CONH), 8.39 (s, 1H, –CH=N–),
.15 (q, 1H, Ar–H), 7.83 (q, 2H, Ar–H), 7.71 (dd, 1H, J = J = 3.00 Hz, Ar–H), 7.60 (d, 2H, J = 3.00 Hz,
−
235 C; yield 91%; H NMR (500 MHz,
δ
6
8
1
2
Ar–H), 7.50 (q, 1H, Ar–H), 7.44–7.40 (m, 1H, Ar–H), 7.34 (t, 2H, J = 8.50 Hz, J = 9.50 Hz, Ar–H), 2.87
1
2
(
s, 3H, –CH3); 13C NMR (125 MHz, DMSO-d , ppm)
δ
: 171.64, 164.86, 162.20 (d, J = 79.88 Hz), 158.69
6
(
d, J = 56.00 Hz), 157.11, 148.50, 135.01, 134.70, 132.80, 131.90, 130.10, 130.03, 128.93, 127.35, 124.73,
−1
1
1
1
5
24.66, 119.43, 119.26, 116.66, 116.49, 115.87, 113.94, 13.25; IR (KBr, cm )
ν: 3444.87, 3244.27, 1683.86,
670.35, 1653.00, 1635.64, 1602.85, 1558.48, 1541.12, 1521.84, 1506.41, 1458.18, 1436.97, 1361.74, 1317.38,
273.02, 1238.30, 1211.30; Anal. Calcd. for C H F Cl N O : C 56.73%, H 3.05%, N 10.58%, Found: C
25
16
2
2
4
+
3
6.68%, H 3.32%, N 10.69%; MS (ESI) m/z: 530.3 ([M + H] ).
E)-3-(2,6-dichlorophenyl)-N-(4-fluoro-2-(2-(4-methylbenzylidene)hydrazine-1-carbonyl)phenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7n). White solid; mp 267–269 C; yield 87%; H NMR (500 MHz,
DMSO-d , ppm) : 11.96 (s, 1H, isoxazole–CONH), 10.82 (s, 1H, Ar–CONH), 8.36 (s, 1H, –CH=N–),
.16 (q, 1H, Ar–H), 7.66 (t, 3H, J = 6.00 Hz, J = 8.00 Hz, Ar–H), 7.58 (t, 1H, J = 9.00 Hz, J = 8.50 Hz,
δ
6
8
1
2
1
2
Ar–H), 7.44–7.40 (m, 1H, Ar–H), 7.31 (d, 1H, J = 8.00 Hz, Ar–H), 2.87 (s, 3H, –CH ), 2.37 (s, 3H, –CH );
3
3
13
C NMR (125 MHz, DMSO-d , ppm)
δ
: 171.60, 163.44, 158.70 (d, J = 48.88 Hz), 157.10, 149.77, 140.92,
6
135.00, 134.74, 132.78, 131.78, 130.09, 128.92, 127.83, 127.81, 127.38, 124.55, 119.38, 119.21, 115.85, 115.65,

Molecules 2019, 24, 3766
6 of 17
−
1
1
1
13.95, 21.61, 13.26; IR (KBr, cm )
ν
: 3444.87, 3246.20, 1683.86, 1668.43, 1637.56, 1610.56, 1558.48,
521.84, 1508.33, 1458.18, 1429.25, 1413.82, 1363.67, 1313.52, 1274.95, 1238.30, 1199.72; Anal. Calc. for
C H FCl N O : C 59.44%, H 3.65%, N 10.66%, Found: C 59.58%, H 3.55%, N 10.85%; MS (ESI) m/z:
26
19
2
4
3
+
5
26.4 ([M + H] ).
E)-N-(2-(2-(2-chlorobenzylidene)hydrazine-1-carbonyl)-4-fluorophenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7o). White solid; mp 250–251 C; yield 88%; H NMR (500 MHz,
DMSO-d , ppm) : 12.20 (s, 1H, isoxazole–CONH), 10.77 (s, 1H, Ar–CONH), 8.81 (s, 1H, –CH=N–),
.12 (d, 1H, J = 5.00 Hz, Ar–H), 8.04 (s, 1H, Ar–H), 7.70 (d, 1H, J = 6.50 Hz, Ar–H), 7.60–7.44 (m, 7H,
δ
6
8
13
Ar–H), 2.87 (s, 3H, –CH3); C NMR (125 MHz, DMSO-d , ppm)
δ
: 171.59, 163.58, 158.73 (d, J = 59.63
6
Hz), 145.51, 135.01, 134.68, 133.96, 132.79, 132.42, 131.75, 130.57, 128.93, 128.82, 128.29, 127.50, 127.36,
−
1
1
1
1
5
24.91, 124.73, 119.55, 119.38, 115.92, 115.72, 113.97, 13.24; IR (KBr, cm )
653.00, 1635.64, 1616.35, 1558.48, 1541.12, 1521.84, 1506.41, 1456.26, 1429.25, 1398.39, 1361.74, 1319.31,
269.16, 1236.37, 1197.79; Anal. Calcd. for C H FCl N O : C 55.02%, H 2.95%, N 10.27%, Found: C
ν: 3444.87, 3244.27, 1683.86,
25
16
3
4
3
+
5.14%, H 3.15%, N 10.36%; MS (ESI) m/z: 546.8 ([M + H] ).
E)-N-(2-(2-(2-bromobenzylidene)hydrazine-1-carbonyl)-4-fluorophenyl)-3-(2,6-dichlorophenyl)-5-
methylisoxazole-4-carboxamide (7p). White solid; mp 249–250 C; yield 89%; H NMR (500 MHz,
DMSO-d , ppm) : 12.23 (s, 1H, isoxazole–CONH), 10.77 (s, 1H, Ar–CONH), 8.77 (s, 1H, –CH=N–),
.13 (q, 1H, Ar–H), 8.03 (dd, 1H, J = 1.00 Hz, J = 1.50 Hz, Ar–H), 7.74–7.69 (m, 2H, Ar–H), 7.59 (d, 2H,
J = 8.00 Hz, Ar–H), 7.53–7.49 (m, 2H, Ar–H), 7.44–7.39 (m, 2H, Ar–H), 2.86 (s, 3H, –CH3); C NMR
125 MHz, DMSO-d , ppm) : 171.57, 163.60, 159.11, 158.96, 158.51, 158.74 (d, J = 57.25 Hz), 147.83,
35.01, 134.67, 133.81, 133.25, 132.79, 132.66, 128.93, 128.77, 127.88, 127.37, 124.92, 124.92, 124.71, 124.35,
(
◦
1
δ
6
8
1
2
13
(
δ
6
1
1
1
1
5
−1
19.54, 119.36, 115.93, 115.74, 113.97, 13.26; IR (KBr, cm
635.64, 1608.63, 1591.27, 1558.48, 1541.12, 1521.84, 1506.41, 1458.18, 1429.25, 1417.68, 1363.67, 1321.24,
280.73, 1244.09, 1207.44; Anal. Calcd. for C H BrCl FN O : C 50.87%, H 2.73%, N 9.49%, Found: C
) ν: 3444.87, 3224.98, 3066.82, 1683.86, 1653.00,
25
16
2
4
3
+
1.03%, H 2.74%, N 9.76%; MS (ESI) m/z: 591.2 ([M + H] ).
E)-3-(2,6-dichlorophenyl)-N-(4-fluoro-2-(2-(furan-2-ylmethylene)hydrazine-1-carbonyl)phenyl)-5-
methylisoxazole-4-carboxamide (7q). Brown solid; mp 232–234 C; yield 87%; H NMR (500 MHz,
DMSO-d , ppm) : 11.92 (s, 1H, isoxazole–CONH), 10.77 (s, 1H, Ar–CONH), 8.23 (s, 1H, –CH=N–),
.10 (q, 1H, Ar–H), 7.86 (d, 1H, J = 1.50 Hz, Ar–H), 7.61 (dd, 1H, J = 2.50 Hz, J = 3.00 Hz, Ar–H),
(
◦
1
δ
6
8
7
1
2
.56 (d, 2H, J = 8.00 Hz, furyl–H), 7.47 (q, 1H, furyl–H), 7.40–7.36 (m, 1H, Ar–H), 2.83 (s, 3H, –CH3);
13
C NMR (125 MHz, DMSO-d , ppm)
δ
: 171.47, 163.44, 158.74 (d, J = 37.00 Hz), 157.12, 149.63, 146.19,
6
1
1
1
1
5
39.25, 134.98, 134.70, 132.79, 128.91, 127.44, 124.82, 124.75, 124.64, 119.43, 119.26, 115.78, 115.59, 115.08,
−1
13.95, 112.87, 13.24; IR (KBr, cm )
595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1458.18, 1429.25, 1409.95, 1394.53, 1338.60, 1319.31,
271.09, 1242.16, 1199.72; Anal. Calcd. for C H FCl N O : C 55.11%, H 3.02%, N 11.18%, Found: C
ν: 3444.87, 3224.98, 3066.82, 1683.86, 1653.00, 1635.64, 1608.63,
23
15
2
4
4
+
5.43%, H 3.15%, N 11.57%; MS (ESI) m/z: 502.3 ([M + H] ).
E)-N-(2-(2-(2,4-dichlorobenzylidene)hydrazine-1-carbonyl)-4-fluorophenyl)-3-(2,6-dichlorophenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7r). White solid; mp 231–233 C; yield 92%; H NMR (500 MHz,
DMSO-d , ppm) : 12.23 (s, 1H, isoxazole–CONH), 10.75 (s, 1H, Ar–CONH), 8.75 (s, 1H, –CH=N–),
.13 (q, 1H, Ar–H), 8.05 (d, 1H, J = 8.50 Hz, Ar–H), 7.75 (d, 1H, J = 2.00 Hz, Ar–H), 7.69 (dd, 1H, J = J
δ
6
8
1
2
=
3.00 Hz, Ar–H), 7.64 (q, 2H, Ar–H), 7.50 (q, 2H, Ar–H), 7.45–7.42 (m, 1H, Ar–H), 2.86 (s, 3H, –CH3);
13
C NMR (125 MHz, DMSO-d , ppm)
δ: 171.62, 163.59, 158.73 (d, J = 64.38 Hz), 144.42, 136.02, 135.02,
6
1
1
1
1
34.69, 134.65, 132.78, 130.89, 130.03, 128.92, 128.69, 127.36, 124.95, 124.89, 124.89, 124.68, 119.61, 119.43,
−
1
15.88, 115.69, 113.95, 13.24; IR (KBr, cm )
ν: 3444.87, 3089.11, 1683.86, 1674.21, 1653.00, 1635.64,
616.35, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1473.62, 1458.18, 1436.97, 1416.68, 1361.74, 1338.60,
319.31, 1271.09, 1240.23, 1211.30; Anal. Calcd. for C H Cl FN O : C 51.75%, H 2.61%, N 9.66%,
25
15
4
4
+
3
Found: C 51.83%, H 3.03%, N 9.78%; MS (ESI) m/z: 581.2 ([M + H] ).

Molecules 2019, 24, 3766
7 of 17
(
E)-N-(2-(2-(2,3-dichlorobenzylidene)hydrazine-1-carbonyl)-4-fluorophenyl)-3-(2,6-dichlorophenyl)-5-
◦
1
methylisoxazole-4-carboxamide (7s). White solid; mp 176–177 C; yield 90%; H NMR (500 MHz,
DMSO-d , ppm) : 12.27 (s, 1H, isoxazole–CONH), 10.73 (s, 1H, Ar–CONH), 8.82 (s, 1H, –CH=N–),
.11 (q, 1H, Ar–H), 8.01 (dd, 1H, J = J = 1.50 Hz, Ar–H), 7.76 (dd, 1H, J = J = 1.00 Hz, Ar–H), 7.69
δ
6
8
1
2
1
2
(
7
dd, 1H, J = 2.50 Hz, J = 3.00 Hz, Ar–H), 7.60 (d, 2H, J = 7.50 Hz, Ar–H), 7.52–7.49 (m, 2H, Ar–H),
.44 (t, 1H, J = 6.00 Hz, J = 8.50 Hz, Ar–H), 2.86 (s, 3H, –CH ); C NMR (125 MHz, DMSO-d , ppm)
1 2 3 6
1 2
13
δ
: 171.61, 163.62, 158.74 (d, J = 66.76 Hz), 157.13, 145.21, 135.00, 134.62, 134.23, 132.99, 132.81, 132.46,
31.76, 129.18, 128.94, 128.82, 127.34, 126.11, 124.90, 124.78, 119.44, 115.90, 115.67, 113.96, 13.24; IR (KBr,
1
cm
−
1
) ν: 3444.87, 1734.01, 1716.05, 1683.86, 1674.21, 1653.00, 1635.64, 1616.35, 1575.84, 1558.48, 1541.12,
1
1
9
521.84, 1506.41, 1489.05, 1473.62, 1458.18, 1436.97, 1429.25, 1416.61, 1361.74, 1338.60, 1319.31, 1271.09,
226.21; Anal. Calcd. for C H Cl FN O : C 51.75%, H 2.61%, N 9.66%, Found: C 51.93%, H 2.87%, N
25
15
4
4
3
+
.70%; MS (ESI) m/z: 581.2 ([M + H] ).
E)-3-(2,6-dichlorophenyl)-N-(4-fluoro-2-(2-(thiophen-2-ylmethylene)hydrazine-1-carbonyl)phenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7t). Brown solid; mp 246–248 C; yield 87%; H NMR (500 MHz,
DMSO-d , ppm) : 11.96 (s, 1H, isoxazole–CONH), 10.76 (s, 1H, Ar–CONH), 8.58 (s, 1H, –CH=N–),
.14 (q, 1H, Ar–H), 7.74 (d, 1H, J = 4.50 Hz, Ar–H), 7.64 (dd, 1H, J = J = 3.00 Hz, Ar–H), 7.60 (d, 1H, J
δ
6
8
1
2
=
1.50 Hz, Ar–H), 7.59 (s, 1H, thienyl–H), 7.54–7.49 (m, 2H, Ar–H), 7.44–7.40 (m, 1H, thienyl–H), 7.18
: 171.70, 163.28, 158.98 (d,
J = 63.25 Hz), 157.10, 144.65, 139.19, 135.01, 134.67, 132.81, 132.25, 130.11, 128.94, 128.55, 128.41, 127.32,
(
q, 1H, thienyl–H), 2.86 (s, 3H, –CH3); 13C NMR (125 MHz, DMSO-d , ppm)
δ
6
−1
1
1
1
5
24.75, 124.61, 119.39, 119.22, 115.82, 115.63, 113.95, 13.27; IR (KBr, cm )
683.86, 1674.21, 1653.00, 1635.64, 1608.13, 1589.34, 1558.48, 1541.12, 1521.84, 1506.41, 1489.05, 1473.62,
458.18, 1436.97, 1409.96, 1373.32, 1319.31, 1271.09, 1238.30; Anal. Calcd. for C H Cl FN O S: C
3.40, H 2.92, N 10.83, Found: C 53.62%, H 3.05%, N 11.18%; MS (ESI) m/z: 518.4 ([M + H] ).
E)-3-(2,6-dichlorophenyl)-N-(4-fluoro-2-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)phenyl)-5-
ν: 3444.87, 3186.40, 3047.53,
23
15
2
4
3
+
(
◦
1
methylisoxazole-4-carboxamide (7u). Light yellow solid; mp 269–271 C; yield 89%; H NMR (500 MHz,
DMSO-d , ppm) : 12.29 (s, 1H, isoxazole–CONH), 10.70 (s, 1H, Ar–CONH), 8.48 (s, 1H, –CH=N–),
.34 (d, 1H, J = 7.50 Hz, Ar–H), 8.12 (q, 1H, Ar–H), 8.03 (d, 2H, J = 7.50 Hz, Ar–H), 7.96 (s, 1H, Ar–H),
.67 (dd, 1H, J = 2.50 Hz, J = 3.00 Hz, Ar–H), 7.60 (d, 2H, J = 8.00 Hz, Ar–H), 7.52–7.42 (m, 1H, Ar–H),
δ
6
8
7
2
1
2
13
.74 (s, 3H, –CH3); C NMR (125 MHz, DMSO-d , ppm) δ: 171.68, 163.77, 162.83, 158.73 (d, J = 76.30
6
Hz), 158.42, 157.21, 148.61, 146.93, 140.76, 135.01, 134.57, 132.80, 128.94, 128.76, 127.33, 125.03, 124.68,
−
1
1
1
1
24.45, 119.59, 119.42, 115.98, 115.78, 113.95, 13.25; IR (KBr, cm ) ν: 3481.51, 3265.49, 3186.40, 3047.53,
683.86, 1674.21, 1653.00, 1635.64, 1616.35, 1595.31, 1558.48, 1541.12, 1521.84, 1506.41, 1489.05, 1458.18,
436.97, 1417.68, 1361.74, 1342.46, 1317.38, 1303.88, 1271.09, 1242.16, 1213.21, 1203.58; Anal. Calcd. for
C H FCl N O : C 53.97%, H 2.90%, N 12.59%, Found: C 54.13%, H 3.04%, N 12.81%; MS (ESI) m/z:
25
16
2
5
5
+
5
57.3 ([M + H] ).
E)-3-(2,6-dichlorophenyl)-N-(4-fluoro-2-(2-(2-nitrobenzylidene)hydrazine-1-carbonyl)phenyl)-5-
(
◦
1
methylisoxazole-4-carboxamide (7v). Light yellow solid; mp 266–268 C; yield 89%; H NMR (500 MHz,
DMSO-d , ppm) : 12.26 (s, 1H, isoxazole–CONH), 10.71 (s, 1H, Ar–CONH), 8.59 (s, 1H, –CH=N–),
.49 (d, 1H, J = 6.00 Hz, Ar–H), 8.30 (s, 1H, Ar–H), 8.15 (q, 2H, Ar–H), 8.12 (q, 1H, Ar–H), 7.78 (q,
δ
6
8
13
1H, Ar–H), 7.67–7.44 (m, 5H, Ar–H), 2.85 (s, 3H, –CH3); C NMR (125 MHz, DMSO-d , ppm) δ:
6
171.71, 163.74, 162.84, 158.71 (d, J = 77.50 Hz), 157.20, 148.82, 147.00, 136.38, 135.02, 134.57, 134.08,
1
cm
32.81, 131.11, 128.93, 127.33, 125.13, 124.99, 121.69, 119.54, 119.37, 115.97, 115.78, 113.95, 13.27; IR (KBr,
−
1
) ν: 3444.87, 3265.49, 3066.82, 1683.86, 1674.21, 1653.00, 1635.64, 1616.35, 1595.31, 1575.84, 1558.48,
1
1
541.12, 1521.84, 1506.41, 1489.05, 1473.62, 1456.26, 1436.97, 1417.68, 1398.39, 1350.17, 1317.38, 1271.09,
238.30, 1201.68; Anal. Calcd. for C H FCl N O : C 53.97%, H 2.90%, N 12.59%, Found: C 54.05%,
25
16
2
5
5
+
H 3.23%, N 12.71%; MS (ESI) m/z: 557.3 ([M + H] ).

Molecules 2019, 24, 3766
8 of 17
2
.2. Biological Evaluations
In this study, the curative, protection, and inactivation activities of the target compounds 7a
against TMV and CMV in vivo were performed according to the previously reported literature
references [20 21]. Among the target compounds evaluated, as shown in Figure 2 and Table S1,
compounds 7d 7h 7m 7q, and 7t exhibited good curative activity against TMV, with inhibition rates
of 62.9%, 64.5%, 65.1%, 67.0%, and 68.8%, respectively, which were even better than that of NNM
55.6%); compounds 7d 7h 7m 7q, and 7t exhibited significantly greater protection activity against
TMV, with inhibition rates of 67.2%, 69.6%, 69.7%, 72.7%, and 74.8%, respectively, compared with
NNM (63.8%); compounds 7m 7q, and 7t showed superior inactivation activity, with inhibition rates
of 95.3%, 96.3%, and 96.9%, respectively, compared with NNM (92.5%). Meanwhile, compounds 7h
–7v
,
,
,
,
(
,
,
,
,
,
7
m,
7q, and 7t showed remarkable curative activity against CMV, with inhibition rates of 58.8%, 58.6%,
60.8%, and 65.9%, respectively, which were even better than that of NNM (54.7%); compounds 7q
and 7t showed remarkable protection activity against CMV, with inhibition rates of 67.8% and 70.2%,
respectively, which were better than that of NNM (62.6%); compounds 7q and 7t showed remarkable
inactivation activity against CMV, with inhibition rates of 93.8% and 94.6%, respectively, which were
better than that of NNM (91.0%). Preliminary bioassay results showed that compound 7t revealed the
best curative, protection, and inactivation activities against TMV and CMV at 500 mg/L (Figures S1
and S2) compared with those of NNM and the other target compounds.
Figure 2. The preliminary in vivo antiviral activities against tobacco mosaic virus (TMV) and cucumber
mosaic virus (CMV) of the target compounds 7a
–
7v at 500 mg/L. (
A
) Curative activity against TMV,
) Curative activity against
(B
) Protection activity against TMV, ( ) Inactivation activity against TMV, (D
C
CMV, (E) Protection activity against CMV, (F) Inactivation activity against CMV. * p < 0.05, ** p < 0.01.
Vertical bars refer to mean ± SD (n = 3).
Based on the preliminary bioassays, the 50% effective concentration (EC ) values of curative,
50
protection, and inactivation activities against TMV and CMV of the target compounds were tested and
are listed in Figure 3 and Tables S2 and S3. Bioassay results showed that some of the target compounds
showed moderate to good antiviral activities against TMV and CMV. Especially, Figure 3 and Table S2
showed that compounds 7d
, 7h, 7m, 7q, 7r, and 7t exhibited excellent curative activity against TMV,
with the EC50 values of 238.9, 195.5, 189.9, 179.6, 266.8, and 168.5 mg/L, respectively, which were even

Molecules 2019, 24, 3766
9 of 17
better that of NNM (286.4 mg/L); compounds 7d
,
7h
,
7m
,
7q, and 7t revealed better protection activity
against TMV, with the EC50 values of 187.2, 172.6, 176.5, 169.2, and 157.6 mg/L, than that of NNM
198.2 mg/L); compounds 7h 7q, and 7t demonstrated remarkable inactivation activity against TMV,
with the EC50 values of 43.1, 36.9, and 33.7 mg/L, which were superior to that of NNM (46.3 mg/L).
Meanwhile, Figure 3 and Table S3 also showed that compounds 7d 7h 7m 7q 7r, and 7t exhibited
excellent curative activity against CMV, with the EC50 values of 276.5, 226.4, 248.5, 227.3, 268.5, and
97.9 mg/L, respectively, which were even better that of NNM (297.3 mg/L); compounds 7d 7h 7m
7r, and 7t revealed better protection activity against CMV, with the EC50 values of 247.8, 215.8,
98.3, 179.7, 236.9, and 168.4 mg/L, than that of NNM (263.4 mg/L); compounds 7h 7m 7q, and 7t
(
,
,
,
,
,
1
,
,
,
7q,
1
,
,
demonstrated remarkable inactivation activity against CMV, with the EC50 values of 53.7, 51.8, 47.6,
and 45.8 mg/L, which were superior to that of NNM (62.8 mg/L).
Figure 3. The EC50 values of the target compounds 7a–7v against TMV and CMV in vivo. (
activity against TMV, ( ) Protection activity against TMV, ( ) Inactivation activity against TMV,
) Curative activity against CMV, ( ) Protection activity against CMV, ( ) Inactivation activity against
A) Curative
B
C
(D
E
F
CMV. * p < 0.05, ** p < 0.01. Vertical bars refer to mean ± SD (n = 3).
2
.3. Effect on Chlorophyll Contents
a
b
Figure 4 showed that the chlorophylls included chlorophyll a (C ) and chlorophyll b (C ), and total
t
chlorophyll content (C ) contents of tobacco plant decreased gradually with TMV inoculation from day
1
to day 5. However, in TMV-inoculated tobacco leaves treated with compound 7t, chlorophyll contents
increased from day 1 to day 5 and reached the highest value at day 5. Meanwhile, it is a remarkable
fact that variation in trends of 7t + TMV treatment surpassed that of NNM + TMV treatment.

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Figure 4. Effects of compound 7t on chlorophyll a (C
a
) (A
), chlorophyll b (C ) (
B), and total chlorophyll
b
content (C ) ( ) contents in tobacco leaves. Vertical bars refer to mean
t
C
±
SD (n = 3). CK, negative control.
2
.4. Effect on Defensive Enzyme Activities
Figure 5A shows that superoxide dismutase (SOD) activity of 7t + TMV treatment group, which
was higher than those of CK, CK + TMV, and NNM + TMV groups, increased at day 1 to day 3 and
reached the highest value at day 3. Figure 5B shows that peroxidase (POD) activity of 7t + TMV
treatment group, which was higher than those of CK, CK + TMV, and NNM + TMV groups, increased
at day 1 to day 5 and reached the highest value at day 5. Meanwhile, Figure 5C shows that catalase
(
CAT) activity of 7t + TMV treatment group, which was higher than those of CK and CK + TMV groups
but lower than that of NNM + TMV group, increased at day 1 to day 3 and reached the highest value
at day 3. In addition, Figure 5D shows that phenylalanine ammonia lyase (PAL) activity of 7t + TMV
treatment group, which was higher than those of CK, CK + TMV, and NNM + TMV groups, increased
at day 1 to day 5 and reached the highest value at day 5.
Figure 5. Effects of compound 7t on superoxide dismutase (SOD) (
CAT) ( ), and phenylalanine ammonia lyase (PAL) ( ) activity in tobacco leaves. Vertical bars refer to
mean ± SD (n = 3). CK, negative control.
A), peroxidase (POD) (B), catalase
(
C
D

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3
. Discussion
3
.1. Structure–Activity Relationship (SAR) Analysis
As an extension of this approach to discover and develop “me-better” active molecules, in this
study, the SAR were analyzed based on the antiviral activities of the target compounds against TMV
and CMV. SAR analysis showed that the type and position of the substituent group R at the phenyl
1
ring and the type of the substituent group R had an important effect on the antiviral activities of the
2
target compounds. First, compared with the same substituent on the R substituent group, the antiviral
2
activities against TMV and CMV of the corresponding compounds with the –F substituent group at
4
-position are higher than the –Cl substituent group at 5-position in the order of 7j
> 7a, 7k > 7b, 7g >
7
h
, and 7s 7i. Second, compared with the same substituent on the R substituent group, the smaller
>
1
electron-withdrawing groups at the 2- or 4-position of phenyl are beneficial to the improvement of
antiviral activities against TMV and CMV in the order of 7d
and 7l 7o
>
7a
>
7b
,
7c
>
7f
> 7g, 7m > 7j > 7k,
>
>
7p. Third, compared with the same substituent on the R and R substituent groups,
1
2
the antiviral activities against TMV and CMV of the corresponding compounds with the R substituent
2
group at 4-position of phenyl are higher than those at 2-position in the order of 7d
> 7c and 7m > 7l.
Meanwhile, when replacing the pyrazole ring with the phenyl ring, the corresponding compounds
synthesized in this work exhibited excellent bioactivities against TMV compared with the bioassay
results of the compounds that were reported in our previous work [5].
3
.2. Physiological and Biochemical Responses of Tobacco
Plants face infection by various aggressive pathogens over their lifetime. To protect themselves
during pathogenic infections, plants have evolved unique immune systems and developed various
defense responses; such defense mechanisms include those involving chlorophyll content and defense
enzyme activity [22
alterations. Photosynthesis is a special, and most basic, life process of green plants providing them
with necessary growth and energy [24 25]. Chlorophyll is the photosynthetic pigment of green plants
,23]. That is, virus-infected plants develop strong physiological and biochemical
,
whose content is closely related to photosynthesis, the extent of bacterial infection in plants leading to
proliferation and destruction of the plant chloroplasts, and factors retarding the synthesis of chlorophyll
causing leaf chlorosis [26
photosynthesis to enhance plant defense against viral infection [28
,
27]. Meanwhile, the increased levels of total chlorophyll were able to improve
30]. Therefore, the chlorophyll
–
contents, shown in Figure 4, demonstrate that compound 7t may increase chlorophyll content to
improve photosynthesis, and thereby enhance plant host resistance to viral diseases.
Disease resistance in plants is associated with activation of a wide array of defense responses that
slow down or halt infection at certain stages of the host–pathogen interaction. The defense mechanisms
include pre-existing physical and chemical barriers that interfere with pathogen establishment. Other
methods of protection rely on inducible defense responses in the form of enzymes that are activated
upon infection or plant activators [31–35]. The interaction between the pathogen or plant activators
and the host plant induces some changes in primary activity of enzymes, particularly SOD, POD, CAT,
and PAL, etc. SOD, a key enzyme that resists biological oxidation in the plant, catalyzes the reduction
2
−
2−
of superoxide anions (O ) to hydrogen peroxide (H O ). The diminished capacity for O removal
2
2
ensues decreased ability of progeria cells to minimize oxidative damage and may be a key factor in
disease. It plays a critical role in the defense of cells against the toxic effects of oxygen radicals [36].
Antioxidant defense machinery protects plant cells from oxidative damage induced by reactive oxygen
species (ROS) to reflect the state of photosynthesis. POD can induce the biosynthetic pathway of
salicylic acid (SA), lignin, and phytoalexins, which can activate systemic acquired resistance (SAR) and
promote cell-wall reinforcement and pathogen inhibition [37,38]. CAT can catalyze decomposition of
hydrogen peroxide to water and oxygen. This process can protect cells against oxidative damage from
ROS [39]. PAL is a catalytic enzyme involved in biosynthesis of phenylpropanoids to cinnamic acid,
which can produce SA for defense against pathogens [40]. Therefore, we analyzed defensive enzyme

Molecules 2019, 24, 3766
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activities of 7t-treated tobacco, the results of defensive enzyme activity assay, shown in Figure 5.
demonstrate that compound 7t may improve disease resistance of tobacco through induction defensive
responses in the form of enzymes.
4
. Materials and Methods
4
.1. General Methods
The melting points of the products were determined on an XT-4 binocular microscope (Beijing
Tech Instrument Co., Beijing, China) and were not corrected. The IR spectra were recorded on a Bruker
1
13
VECTOR 22 spectrometer (Bruker Optics Inc. Billerica, MA, USA) in KBr disk. H NMR and
C
NMR (solvent DMSO-d ) spectral analyses were performed on a JEOL-ECX 500 NMR spectrometer
6
(
JEOL Ltd., Tokyo, Japan) at room temperature using tetramethylsilane (TMS) as an internal standard.
Elemental analysis was performed on an Elementar Vario-III CHN analyzer (Elementar, Frankfurt,
Germany). Mass spectral studies were conducted on an Agilent 5973 organic mass spectrometer
(
Agilent Technologies, Palo Alto, Canada). Microwave experiments were carried out using a CEM
®
Discover Labmate microwave apparatus (Discover LabMate instrument, CEM Corporate, Matthews,
NC, USA). Analytical TLC was performed on silica gel GF254 (200–300 mesh). All solvents were dried
by standard methods in advance and distilled before use.
4
.2. General Procedure for the Preparation of the Key Intermediate 6
As shown in Scheme 1, 3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carboxylic acid (
1
, 20 mmol)
. Then,
◦
was added to distillated SOCl (50 mL) and reacted at 80 C for 3
−
5 h to obtain intermediate
2
2
intermediate
, 20 mmol) in anhydrous THF (100 mL) and TEA (1 mL) and the reaction mixture was stirred at
room temperature for 1 2 h. The reaction mixture was poured into cold 5.0% dilute HCl solution
200 mL). The solid obtained was filtered, washed with water, and dried under vacuum to give
the crude product which was further recrystallized and dried to give intermediate . A mixture of
intermediate (20 mmol) and Ac O (200 mmol) was heated under reflux for 3 4 h. The solvent was
removed under reduced pressure and then the residue was washed with water, dried under vacuum,
and recrystallized from ethanol to give intermediate . Finally, 80% hydrazine hydrate (12 mmol) was
added to a solution of intermediate 5 (10 mmol) in THF (50 mL) and reacted at room temperature for
2 h. The solvent was removed under reduced pressure, and the residue was washed with water
2 (20 mmol) was added dropwise to a stirred solution of substituted o-aminobenzoate
(
3
−
(
4
4
−
2
5
1
−
and anhydrous ethanol to give the crude product, then recrystallized from ethanol and dried under
vacuum to give the key intermediate 6.
4
.3. Preparation of the Target Compounds 7a−7v
To a 50 mL round-bottom flask with the intermediate
6 (1.0 mmol) and anhydrous ethanol (5 mL),
the solutions of different substituted aromatic aldehydes (1.2 mmol) in anhydrous ethanol (5 mL) with
a few drops of glacial acetic acid were added dropwise, and then the round-bottom flask was sealed
◦
and placed in the synthetic reactor and irradiated in a microwave at 90 C and 150 W for 1
−
2 h. Upon
completion of the reaction (monitored by TLC), the mixture was concentrated under vacuum, filtered,
and recrystallized from a mixture of ethanol and DMF (1:1 in volume) to obtain the target compounds
7
4
4
a−7v.
.4. In Vivo Antiviral Activity Test
.4.1. Purification of TMV and CMV
The TMV and CMV viruses, multiplied in Nicotiana tabacum (N. tabacum) cv. K326, were purified
by Gooding’s and Zhou’s methods, respectively [41
60 nm by using an ultraviolet spectrophotometer.
,42]. The absorbance values were estimated at
2

Molecules 2019, 24, 3766
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Virus concentration (mg/L) = (A
×
dilution ratio)/E0.1% 260 nm, where E represents the
1 cm
is 3.1.
260
extinction coefficient for TMV, E0
.1%
260 nm
1
cm
4
.4.2. Curative Activity of the Target Compounds against TMV In Vivo
Growing Nicotiana tabacum L. (N. tabacum L.) leaves of the same age were selected and inoculated
−
3
with TMV (concentration of 6
×
10 mg/L) by dipping and brushing the whole leaves, which had
previously been scattered with silicon carbide. The leaves were washed with water after inoculation
for 0.5 h. Then, the solutions of the target compounds were smeared on the left side of the leaves,
respectively, instead, the solvent was smeared on the right side as the control. Finally, the number of
local lesions was counted and recorded 3–4 d after inoculation. All target compounds were tested with
three repetitions to ensure the veracity of the results.
4
.4.3. Protection Activity of the Target Compounds against TMV In Vivo
The target compounds solutions were first smeared on the left side of the N. tabacum L. leaves,
and the solvents were smeared on the right side as the control sample. After 12 h, the crude TMV
−
3
(
concentration of 6
×
10 mg/L) purified from the leaves of N. tabacum cv. K326 was inoculated on
whole leaves, which had previously been scattered with silicon carbide. After 0.5 h, the leaves were
washed with water and then dried in room temperature. The number of local lesions was recorded
3–4 d after inoculation. All target compounds were tested with three repetitions to ensure the veracity
of the results.
4
.4.4. Inactivation Activity of the Target Compounds against TMV In Vivo
The virus was inhibited by mixing with the same volume of the compound solution for 0.5 h.
The right side of the N. tabacum L. leaves was inoculated with the compound solvent, and the virus
−
3
(
concentration of 6
×
10 mg/L) inoculated on the left side was used as the control. All of the leaves
had been previously scattered with silicon carbide. The number of local lesions was recorded 3–4 d
after inoculation. All target compounds were tested with three repetitions to ensure the veracity of
the results.
4
.4.5. Curative Activity of the Target Compounds against CMV In Vivo
The leaves were selected from Chenopodium amaranticolor (C. amaranticolor) plants of the same age.
−
3
Crude CMV, at the same concentration (6
×
10 mg/L), was inoculated with a brush on whole leaves,
which had previously been sprinkled with silicon carbide. Subsequently, the leaves were washed with
water and dried after inoculation for 0.5 h. The compound solution was then smeared on the right side,
and the solvent was smeared on the left side as the control. The number of local lesions was recorded
3–4 d after inoculation. All target compounds were tested with three repetitions to ensure the veracity
of the results.
4
.4.6. Protection Activity of the Target Compounds against CMV In Vivo
The leaves of C. amaranticolor plants of the same age were selected. The compound solution was
smeared on the right side of the leaves. Meanwhile, the solvent was smeared on the left side as the
−
3
control. After 12 h, crude CMV, at the same concentration (6
×
10 mg/L), was inoculated with a
brush on whole leaves, which had previously been sprinkled with silicon carbide. The number of
local lesions was then recorded 3–4 d after inoculation. All target compounds were tested with three
repetitions to ensure the veracity of the results.
4
.4.7. Inactivation Activity of the Target Compounds against CMV In Vivo
The virus was inhibited by mixing with the same volume of the compound solution for 0.5 h.
The right side of C. amaranticolor leaves was inoculated with the mixture and CMV, diluted to a suitable

Molecules 2019, 24, 3766
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−
3
concentration (6
×
10 mg/L), inoculated on the left side as the control. All leaves had previously been
sprinkled with silicon carbide. The number of local lesions was then recorded 3–4 d after inoculation.
All target compounds were tested with three repetitions to ensure the veracity of the results.
The inhibition rates of the target compounds were calculated according to the following formula
(“av.” means average):
Inhibition rate (%) = [(av. local lesion number of control (not treated with compound) – av.
local lesion number smeared with compound)/av. local lesion number of control (not treated with
compound)] × 100%
On the basis of the preliminary biological activity, the EC5 values were also determined and
0
calculated via the software SPSS 17.0 (SPSS, Chicago, IL). Meanwhile, statistical analysis was performed
by Student’s t-test via the software SPSS 17.0 (SPSS, Chicago, IL), “*” and “**” indicate the values of
antiviral activity of the target compounds with significant difference compared with those of NNM at
p < 0.05 and p < 0.01, respectively.
4
.5. Physiological and Biochemical Analysis
4
.5.1. Plant Growth and Sample Collection
Similarly grown N. tabacum L. K326 was selected at the 7-leaf stage, and compound 7t solution,
at a concentration of 500 mg/L, was smeared on whole leaves. Meanwhile, solvent and NNM were
used as negative control (CK) and positive control, respectively. After smearing for 12 h, the leaves
were inoculated with the TMV virus and then cultivated in a greenhouse. Four treatments were
adopted: CK, CK + TMV, NNM + TMV, and 7t + TMV. Tissue samples were collected at 1, 3, 5, and 7 d
after inoculation treatment for assays on chlorophyll content and defensive enzyme activities assay.
Measurements were performed in triplicate.
4
.5.2. Chlorophyll Content Test
In this study, chlorophyll contents of samples were measured using a modified reported
method [43]. Test samples in triplicate of each treatment were sliced into small uniform pieces
by a hole puncher while avoiding the midrib. Samples (50 mg) were placed in 5 mL of a cold solution
of a 1:1 mixture of 85% acetone and 85% ethanol (v/v). Samples were homogenized, incubated for
◦
0
6
.5 h at 35 C, and centrifuged for 15 min at 6500 rpm. Absorbance spectra were recorded at 663 and
45 nm for C
a
and C , respectively, against a solution as reference. C
a
, C , and C
t
were calculated by
b
b
the following equations:
Ca (mg/L) = 0.0127OD663 - 0.00269OD645
(1)
C (mg/L) = 0.0229OD - 0.00468OD663
(2)
(3)
b
645
Ct (mg/L) = Ca + Cb
4
.5.3. Determination of Defensive Enzyme Activity
Activities of SOD, POD, CAT, and PAL were measured and calculated with enzyme assay reagent
kits in accordance with the manufacturer’s instructions (Suzhou Comin Bioengineering Institute,
Suzhou, China).
5
. Conclusions
In this study, a total of 22 novel isoxazole-amide derivatives containing an acylhydrazone moiety
were designed and synthesized. Antiviral activity results showed that compound 7t exhibited the best
curative, protection, and inactivation activities against TMV and CMV and which were even better
than those of NNM. Meanwhile, we also found that compound 7t could enhance the defense-related

Molecules 2019, 24, 3766
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enzyme activity and increase the chlorophyll content in tobacco leaves to induce resistance and enhance
plant tolerance to TMV infection. Furthermore, according to the requirements of pesticide registration
in China, further field studies on the biological efficacies, crop safety, and toxicities of compound 7t as
an antiviral candidate will be performed in our next work.
Supplementary Materials: The following are available online, Table S1. The preliminary in vivo antiviral activities
against TMV and CMV of the target compounds 7a–7v at 500 mg/L. Table S2. The EC50 values of the target
compounds 7a–7v against TMV in vivo. Table S3. The EC50 values of the target compounds 7a–7v against CMV
in vivo. Figure S1. The antiviral activity of compound 7t against TMV at 500 mg/L. Left: smeared with solvent;
Right: smeared with compound 7t or NNM. Figure S2. The antiviral activity of compound 7t against CMV at
5
00 mg/L. Left: smeared with solvent; Right: smeared with compound 7t or NNM.
Author Contributions: Experiment, P.L. and Y.-J.H.; writing—original draft preparation, Z.-B.Y., P.L., and Y.-J.H.;
project administration, Z.-B.Y.
Funding: This research was funded by the National Natural Science Foundation of China, grant number 21466031;
Youth Science and Technology Talent Growth Program of Guizhou Province’s Department of Education, grant
number Qian Jiaohe KY [2018] 350; Construction Project of Engineering Research Center of Ethnic Medicinal Plant
Resources Development in Guizhou Universities and Colleges, grant number Qian Jiaohe KY [2014] 227; Guizhou
Province Botany Provincial Key Supporting Subject Special Research Program, grant number ZDXK [2016] 23;
Guizhou Province Qiannan Normal University for Nationalities Chemistry First-class Subject Construction Project
Support Program, grant number QNSYL2017001; Special Fund for Achievement Conversion of School and
Enterprise in Guizhou Province Qiannan Normal University for Nationalities, grant number QNSY2018CG003;
Guizhou Province Education Quality Promotion Engineering Project, grant number KY [2014] 228.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 7a–7v are available from the authors.
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