Novel pyrazole-hydrazone derivatives containing an isoxazole moiety: Design, synthesis, and antiviral activity

Article abstract of DOI:10.3390/molecules23071798

In this study, a series of novel pyrazole-hydrazone derivatives containing an isoxazole moiety were synthesized. Antiviral bioassays indicated that some of the title compounds exhibited better in vivo antiviral activities against tobacco mosaic virus (TMV). In particular, compounds 6a, 6c and 6q exhibited the best curative activity, protection activity, and inactivation activity against TMV, respectively, which were superior to those of Ningnanmycin. This study demonstrated that this series of novel pyrazole-hydrazone derivatives containing an isoxazole amide moiety could effectively control TMV.

Full text of DOI:10.3390/molecules23071798

molecules
Article
Novel Pyrazole-Hydrazone Derivatives Containing an
Isoxazole Moiety: Design, Synthesis,
and Antiviral Activity
Zaibo Yang *, Pei Li and Xiuhai Gan 3
1
,
2
1
School of Chemistry and Chemical Engineering, Qiannan Normal University for Nationalities,
Guizhou, Duyun 558000, China
Qiandongnan Engineering and Technology Research Center for Comprehensive Utilization of National
2
Medicine, Kaili University, Guizhou, Kaili 556011, China; pl19890627@126.com
College of Chemistry and Life Science, Guizhou Normal College, Guizhou, Guiyang 550018, China;
3
gxh200719@163.com
*
Correspondence: qnsyyzb27001@sgmtu.edu.cn; Tel.: +86-85-4873-7046
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 19 June 2018; Accepted: 18 July 2018; Published: 20 July 2018
Abstract: In this study, a series of novel pyrazole-hydrazone derivatives containing an isoxazole
moiety were synthesized. Antiviral bioassays indicated that some of the title compounds exhibited
better in vivo antiviral activities against tobacco mosaic virus (TMV). In particular, compounds 6a, 6c
and 6q exhibited the best curative activity, protection activity, and inactivation activity against TMV,
respectively, which were superior to those of Ningnanmycin. This study demonstrated that this series
of novel pyrazole-hydrazone derivatives containing an isoxazole amide moiety could effectively
control TMV.
Keywords: pyrazole-hydrazone; isoxazole; antiviral activity; tobacco mosaic virus
1
. Introduction
Plant viruses cause diseases in a wide range of crop plant species and annually contribute to an
estimated $600 billion in economic loss worldwide [1]. In recent years, progress has been made in
screening for high-activity antiviral agents in natural products in China. For example, Ningnanmycin,
isolated from Strepcomcesnoursei var. Xichangensis, was found to be more effective than existing
products in the treatment of plant viruses such as tobacco mosaic virus (TMV) and cucumber mosaic
virus (CMV) [2]. Meanwhile, Ribavirin, a successful antiviral agent, is widely used to prevent TMV
disease. However, the use of Ningnanmycin and Ribavirin in field trials is unsuitable because of
its unsatisfactory curative rates and high control costs [3]. Therefore, identification of new effective
antiviral agents remains a significant challenge [4].
In recent years, literature revealed that pyrazole derivatives had a wide range of biological
activities, such as antibacterial [ ], fungicidal [ 10], insecticidal [10 13], antiviral [14 15],
and herbicidal [16 17] activities. In our previous work, we reported a series of pyrazole derivatives
5–
7
8
–
–
,
,
which had better insecticidal activity against Plutella xylostella [18]. Meanwhile, hydrazone, a highly
efficient pharmacophore, has attracted more and more attention in the pesticides areas due to its
broad-activities, such as antibacterial [19], fungicidal [20,21], insecticidal [20,22,23], and antiviral [20,24]
activities. A few of the pyrazole-hydrazone derivatives have also been reported and possess
various biological activities such as antibacterial [25], antifungal [21], anticancer [26], antioxidant [27],
anti-inflammatory [28], antitumor and antiangiogenesis [29] and antiplatelet activities [30]. In addition,
isoxazole, a five membered heterocyclic ring, was an important scaffold for synthesis of various
Molecules 2018, 23, 1798; doi:10.3390/molecules23071798
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1798
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natural compounds and their congeners and had a broad spectrum of pharmacological activities like
antibacterial [31], antifungal [9,32,33], insecticide [34,35], and antiviral [36] activities.
Motivated by the above-mentioned findings and in continuation of our investigation, to discover
new potentially active agents, in this study, a series of novel pyrazole-hydrazone derivatives
containing an isoxazole moiety were designed and synthesized using a 3-substituted phenyl-5-methyl
isoxazole-4-carboxylic acid as the starting material. The antiviral bioassay results indicated that most
of the title compounds exhibited excellent in vivo antiviral activities against TMV. To the best of our
knowledge, it is the first report on pyrazole-hydrazone derivatives containing an isoxazole moiety
with potent antiviral activity against TMV.
2
. Results and Discussion
2
.1. Chemistry
Using 3-(2-chlorophenyl)-5-methyl isoxazole-4-carboxylic acid and 3-(2,6-dichlorophenyl)-5-
methyl isoxazole-4-carboxylic acid as the starting materials, as shown in Scheme 1, the target
compounds 6a 6r were obtained in five steps with yields of 85.9–91.2%. The physical characteristics, IR,
H-NMR, C-NMR, and elemental analysis data for all the synthesized compounds are shown below.
–
1
13
1
13
The H-NMR and C-NMR for all the synthesized compounds are shown in Supplementary Materials.
Scheme 1. Synthetic route of the title compounds 6a–6r.
Data for N-(4-(2-(4-chlorobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-3-(2-chlorophenyl)-5-
◦
1
methylisoxazole-4-carboxamide (6a). White solid; m.p. 141–143 C; yield 87.9%; H-NMR (500 MHz,
Dimethylsulfoxide-d6 (DMSO-d ), ppm) 11.54 (s, 1H, isoxazole–CONH–), 10.32 (s, 1H,
pyrazole–CONH–), 8.31 (s, 1H, –N=CH–), 8.01 (s, 1H, pyrazole-H), 7.74–7.47 (m, 8H, Ar–H), 3.65 (s,
δ
:
6
13
3
H, pyrazole–CH ), 2.85 (s, 3H, –CH ). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.53, 160.66, 158.43,
3
3
6
1
1
1
47.02, 145.78, 144.08, 140.42, 140.78, 139.24, 137.88, 137.67, 132.84, 132.15, 130.25, 130.13, 129.97, 127.94,
−
1
27.50, 127.26, 113.72, 109.57, 36.66, 12.99. IR (KBr, cm
) ν: 3404.36, 3282.84, 2933.73, 1716.65, 1699.29,
683.86, 1653.00, 1635.64, 1627.92, 1595.13, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1473.62,

Molecules 2018, 23, 1798
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1
458.18, 1436.97, 1411.89, 1375.25, 1303.88, 1253.75, 1219.01. Anal. Calc. for C H Cl N O : C, 55.55%;
23 18 2 6 3
H, 3.65%; N, 16.90%; Found: C, 55.81%; H, 3.79%; N, 17.08%.
Data for 3-(2-chlorophenyl)-N-(4-(2-(4-bromobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-5-
◦
1
methylisoxazole-4-carboxamide (6b). White solid; m.p. 236–237 C; yield 90.1%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.57 (s, 1H, isoxazole–CONH–), 10.35 (s, 1H, pyrazole–CONH–), 8.29 (s, 1H,
N=CH–), 8.01 (s, 1H, pyrazole-H), 7.85–7.85 (d, 1H, J = 3.45 Hz, Ar–H), 7.66–7.45 (m, 7H, Ar–H), 3.66
δ
6
–
13
(s, 3H, pyrazole–CH ), 2.86 (s, 3H, –CH ). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.46, 160.67,
3
3
6
1
1
1
1
3
58.38, 145.62, 137.80, 134.21, 132.87, 132.38, 132.36, 132.17, 132.16, 131.80, 130.14, 129.37, 129.19, 127.96,
−
1
27.93, 123.57, 113.57, 109.52, 36.55, 12.99. IR (KBr, cm
683.86, 1662.64, 1653.00, 1635.64, 1608.63, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1473.62,
458.18, 1436.97, 1429.25, 1398.39, 1319.31, 1301.95. Anal. Calc. for C H ClBrN O : C, 50.99%; H,
) ν: 3444.87, 326.91, 2933.73, 1716.65, 1699.29,
23
18
6
3
.35%; N, 15.51%; Found: C, 51.09%; H, 3.39%; N, 15.55%.
Data for 3-(2-chlorophenyl)-N-(4-(2-(2-bromobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-5-
◦
1
methylisoxazole-4-carboxamide (6c). White solid; m.p. 181–183 C; yield 89.2%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.69 (s, 1H, isoxazole–CONH–), 10.30 (s, 1H, pyrazole–CONH–), 8.61 (s, 1H,
N=CH–), 7.97 (s, 1H, pyrazole–H), 7.91 (d, 1H, J = 7.45 Hz, Ar–H), 7.63 (d,1H, J = 8.00 Hz, Ar–H),
.54–7.38 (m, 5H, A–H), 7.31 (t, 1H, J = 14.85 Hz, Ar–H), 3.59 (s, 3H, pyrazole–CH ), 2.79 (s, 3H, –CH ).
δ
6
–
7
3
3
1
3
C-NMR (125 MHz, DMSO-d , ppm)
δ
: 160.56, 158.40, 158.18, 152.48, 147.87, 144.98, 137.96, 137.71,
6
1
1
1
1
1
33.63, 132.75, 132.10, 131.86, 130.07, 128.58, 127.88, 127.71, 127.63, 123.88, 113.69, 113.58, 112.30, 36.61,
−
1
2.91. IR (KBr, cm
575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1473.62, 1458.18, 1436.97, 1411.89, 1398.39, 1361.74,
338.60, 1319.31, 1298.09, 1253.73, 1220.94. Anal. Calc. for C H ClBrN O : C, 50.99%; H, 3.35%; N,
) ν: 3446.79, 3298.28, 2933.73, 1716.65, 1699.29, 1683.86, 1670.35, 1653.00, 1635.64,
23
18
6
3
5.51%; Found: C, 51.06%; H, 3.41%; N, 15.57%.
Data for 3-(2-chlorophenyl)-N-(4-(2-(2-chlorobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-5-
◦
1
methylisoxazole-4-carboxamide (6d). White solid; m.p. 170–171 C; yield 88.7%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.64 (s, 1H, isoxazole–CONH–), 10.28 (s, 1H, pyrazole–CONH–), 8.66 (s, 1H,
N=CH–), 7.97 (s, 1H, pyrazole–H), 7.53–7.37 (m, 8H, Ar–H), 3.59 (s, 3H, pyrazole–CH ), 2.79 (s, 3H,
δ
6
3
–
CH3). 13C-NMR (125 MHz, DMSO-d , ppm)
δ: 171.52, 160.65, 158.51, 142.75, 140.37, 138.09, 137.70,
6
1
3
33.55, 132.82, 132.16, 131.88, 131.73, 130.46, 130.14, 128.17, 127.95, 127.84, 127.33, 127.14, 113.71, 109.36,
−
1
6.66, 12.97. IR (KBr, cm
) ν: 3444.87, 3286.70, 2933.73, 1716.65, 1695.43, 1683.86, 1670.35, 1653.00,
1
1
1
635.64, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1498.69, 1473.62, 1458.18, 1436.97, 1419.61, 1398.39,
361.74, 1340.53, 1319.31, 1251.80, 1219.01. Anal. Calc. for C H Cl N O : C, 55.55%; H, 3.65%; N,
23
18
2
6
3
6.90%; Found: C, 55.70%; H, 3.79%; N, 17.24%.
Data for 3-(2-chlorophenyl)-5-methyl-N-(1-methyl-4-(2-(4-methylbenzylidene)hydrazinecarbonyl)-1H- pyrazol
◦
1
-
5-yl)isoxazole-4-carboxamide (6e). White solid; m.p. 221–223 C; yield 89.8%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.38 (s, 1H, isoxazole–CONH–), 10.27 (s, 1H, pyrazol–CONH–), 8.24 (s, 1H,
N=CH–), 7.97 (s, 1H, pyrazole–H), 7.55–7.42 (m, 6H, Ar–H), 7.22 (s, 1H, Ar–H), 7.20 (s, 1H, Ar–H), 3.62
δ
6
–
(
1
3
s, 3H, pyrazole–CH ), 2.81(s, 3H, –CH ), 2.28 (s, 3H, Ar–CH ). C-NMR (125 MHz, DMSO-d , ppm)
3 3 3 6
δ
: 193.16, 171.80, 171.53, 163.39, 160.66, 158.43, 147.02, 145.78, 144.08, 140.42, 140.28, 140.08, 139.24,
1
1
1
1
1
37.88, 137.67, 134.52, 132.84, 132.15, 130.25, 130.13, 129.97, 129.65, 127.94, 127.50, 127.26, 113.72, 109.57,
−
1
08.03, 36.66, 21.55, 12.99. IR (KBr, cm
653.00, 1635.64, 1602.85, 1577.77, 1558.48, 1541.12, 1521.84, 1506.41, 1473.62, 1456.26, 1436.97, 1409.96,
398.39, 1375.25, 1301.95, 1246.02, 1219.01. Anal. Calc. for C H ClN O : C, 60.44%; H, 4.44%; N,
) ν: 3444.87, 3203.76, 3030.17, 2933.73, 1699.29, 1683.86, 1662.64,
24
21
6
3
7.62%; Found: C, 60.53%; H, 4.48%; N, 17.73%.
Data for 3-(2-chlorophenyl)-N-(4-(2-(2-fluorobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-5-
◦
1
methylisoxazole-4-carboxamide (6f). White solid; m.p. 180–182 C; yield 88.3%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.62 (s, 1H, isoxazole–CONH–), 10.36 (s, 1H, pyrazole–CONH–), 8.57 (s, 1H,
N=CH–), 8.02 (s, 1H, pyrazole–H), 7.93–7.90 (t, 1H, J = 16.60 Hz, Ar–H), 7.60–7.28(m, 7H, Ar–H), 3.66
δ
6
–

Molecules 2018, 23, 1798
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13
(
s, 3H, pyrazole–CH ), 2.85 (s, 3H, –CH ). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.25, 160.15
3
3
6
(
d, 1C, 2-F-Ph-C), 158.14, 139.27, 137.76, 137.41, 132.54, 132.04, 131.88, 129.86, 127.65, 127.54, 126.77,
−
1
1
26.49, 125.21, 122.11, 116.33, 116.18, 113.43, 109.10, 36.42, 12.69. IR (KBr, cm
933.73, 1699.29, 1683.86, 1653.00, 1635.64, 1616.35, 1602.85, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41,
489.08, 1473.62, 1436.97, 1419.61, 1398.39, 1379.10, 1363.67, 1319.31, 1242.16, 1215.15. Anal. Calc. for
C H ClFN O : C, 57.45%; H, 3.77%; N, 17.48%; Found: C, 57.51%; H, 3.96%; N, 17.53%.
) ν: 3444.87, 3167.12,
2
1
23
18
6
3
Data for 3-(2-chlorophenyl)-5-methyl-N-(1-methyl-4-(2-(thiophen-2-ylmethylene)hydrazine carbonyl)-1H
◦
1
-
pyrazol-5-yl)isoxazole-4 -carboxamide (6g). White solid; m.p. 225–227 C; yield 89.6%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.44 (s, 1H, isoxazole–CONH–), 10.32 (s, 1H, pyrazole–CONH–), 8.54 (s, 1H,
N=CH–), 8.21 (s, 1H, pyrazole–H), 7.98 (s, 1H, Ar–H), 7.65–7.14 (m, 7H, Ar–H, thiophen–H), 3.65
δ
6
–
(
13
s, 3H, pyrazole–CH ), 2.85 (s, 3H, –CH ). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.34, 160.60,
3
3
6
1
1
1
1
1
58.32, 142.16, 139.72, 137.72, 137.62, 132.80, 132.16, 131.25, 130.75, 130.14, 129.26, 128.85, 128.57, 128.38,
−
1
27.96, 113.68, 109.11, 36.64, 12.99. IR (KBr, cm
635.64, 1595.13, 1577.77, 1558.48, 1541.12, 1521.84, 1506.41, 1498.69, 1473.62, 1436.97, 1419.61, 1398.39,
375.25, 1317.38, 1300.02, 1251.80, 1224.80. Anal. Calc. for C H ClN O S: C, 53.79%; H, 3.65%; N,
) ν: 3446.79, 3263.56, 2929.87, 1699.29, 1683.86, 1653.00,
21
17
6
3
7.92%; Found: C, 54.08%; H, 4.02%; N, 17.96%.
Data for 3-(2-chlorophenyl)-N-(4-(2-(2,4-dichlorobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)
◦
1
-
5-methylisoxazole-4-carboxamide (6h). White solid; m.p. 189–190 C; yield 87.9%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.77 (s, 1H, isoxazole–CONH–), 10.39 (s, 1H, pyrazole–CONH–), 8.67 (s, 1H,
N=CH–), 8.03 (s, 1H, pyrazole–H), 7.80 (d, 1H, J = 8.00 Hz, Ar–H), 7.63–7.47 (m, 6H, Ar–H), 3.66
δ
6
–
(
13
s, 3H, pyrazole–CH ), 2.85 (s, 3H, –CH ). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.53, 160.76,
3
3
6
1
1
1
1
58.52, 141.59, 138.08, 137.68, 135.37, 135.20, 134.23, 132.76, 132.19, 132.08, 131.22, 130.17, 129.94, 128.61,
−
1
28.53, 127.98, 113.75, 109.32, 108.41, 36.62, 13.00. IR (KBr, cm
) ν: 3444.87, 3284.77, 3186.40, 2929.87,
699.29, 1683.86, 1674.21, 1653.00, 1635.64, 1581.63, 1570.06, 1543.05, 1521.84, 1506.41, 1489.05, 1473.62,
458.18, 1436.97, 1419.61, 1398.39, 1386.82, 1363.67, 1319.31, 1300.02, 1251.80, 1222.87. Anal. Calc. for
C H Cl N O : C, 51.95%; H, 3.22%; N, 15.80%; Found: C, 51.99%; H, 3.48%; N, 15.93%.
23
17
3
6
3
Data for 3-(2-chlorophenyl)-5-methyl-N-(1-methyl-4-(2-(4-nitrobenzylidene)hydrazinecarbonyl)-1H-pyrazol-
◦
1
5
-yl)isoxazole-4-carboxamide (6i). White solid; m.p. 155–156 C; yield 89.7%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.79 (s, 1H, isoxazole–CONH–), 10.37 (s, 1H, pyrazole–CONH–), 8.42 (s, 1H,
N=CH–), 8.31 (s, 1H, pyrazole–H), 8.29 (s, 1H, Ar–H), 8.14–7.92 (m, 2H, Ar–H), 7.61–7.46 (m, 5H,
δ
6
–
13
Ar–H), 3.67 (s, 3H, pyrazole–CH ), 2.86 (s, 3H, –CH ). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.53,
3
3
6
1
1
2
60.62, 158.46, 148.23, 144.36, 141.23, 137.77, 132.81, 132.17, 132.09, 130.15, 130.13, 129.40, 128.37, 128.24,
−
1
27.96, 127.83, 124.65, 124.56, 113.70, 109.35, 36.66, 12.97. IR (KBr, cm
929.87, 1699.29, 1683.86, 1674.21, 1653.00, 1635.64, 1627.92, 1587.42, 1570.06, 1558.48, 1541.12, 1521.84,
508.33, 1489.05, 1473.62, 1458.18, 1436.97, 1411.89, 1398.39, 1375.25, 1344.38, 1317.38, 1253.73, 1215.15.
Anal. Calc. for C H ClN O : C, 54.39%; H, 3.57%; N, 19.30%; Found: C, 54.52%; H, 3.74%; N, 19.36%.
) ν: 3444.87, 3246.20, 3064.89,
1
23
18
7
5
Data for 3-(2-chlorophenyl)-N-(4-(2-(furan-2-ylmethylene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-5
◦
1
-
methylisoxazole-4-carboxamide (6j). White solid; m.p. 147–149 C; yield 86.4%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.45 (s, 1H, isoxazole–CONH–), 10.36 (s, 1H, pyrazole–CONH–), 8.21 (s, 1H,
N=CH–), 7.85 (s, 1H, pyrazole–H), 7.61–7.47 (m, 5H, Ar-H, furan–H), 6. 91 (t, 1H, J = 13.15 Hz,
δ
6
–
13
furan–H), 6.63 (s, 1H, furan–H), 3.65 (s, 3H, pyrazole–CH ), 2.85 (s, 3H, –CH ). C-NMR (125 MHz,
3
3
DMSO-d , ppm) δ: 171.48, 163.12, 160.69, 158.35, 149.97, 145.59, 137.85, 137.60, 136.76, 133.51, 132.81,
6
−
1
1
3
1
32.17, 130.15, 127.96, 127.84, 113.86, 113.72, 112.70, 109.55, 36.63, 12.96. IR (KBr, cm
)
ν
: 3462.22,
265.49, 2929.87, 1699.29, 1683.86, 1647.12, 1653.00, 1635.64, 1627.92, 1575.84, 1570.06, 1558.48, 1541.12,
521.84, 1496.76, 1489.05, 1473.62, 1436.97, 1419.61, 1398.39, 1361.74, 1319.31, 1300.02, 1253.73, 1213.23.
Anal. Calc. for C H ClN O : C, 55.70%; H, 3.78%; N, 18.56%; Found: C, 55.78%; H, 4.03%; N, 18.89%.
21
17
6
4
Data for 3-(2-chlorophenyl)-5-methyl-N-(1-methyl-4-(2-(3,4,5-trimethoxybenzylidene)hydrazine carbonyl)-1H
◦
1
-
pyrazol-5-yl)isoxazole-4-carboxamide (6k). White solid; m.p. 206–207 C; yield 91.2%; H-NMR (500 MHz,

Molecules 2018, 23, 1798
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DMSO-d , ppm)
δ
: 11.44 (s, 1H, isoxazole–CONH–), 10.28 (s, 1H, pyrazole–CONH–), 8.26 (s, 1H,
6
–
3
N=CH–), 7.80 (s, 1H, pyrazole–H), 7.60–7.47 (m, 4H, Ar–H), 6.99 (s, 1H, Ar–H), 6.97 (s, 1H, Ar–H),
13
.83 (s, 6H, –OCH ), 3.70 (s, 3H, pyrazole–CH ), 3.66 (s, 3H, –OCH ), 2.85 (s, 3H, –CH ). C-NMR
3
3
3
3
(
125 MHz, DMSO-d , ppm)
δ
: 171.58, 161.01, 160.59, 158.45, 158.23, 153.73, 153.71, 147.08, 146.67, 137.70,
6
1
3
36.96, 132.82, 132.17, 130.42, 130.15, 127.95, 127.81, 114.14, 110.70, 109.58, 104.72, 104.44, 60.65, 56.48,
−
1
6.68, 13.02. IR (KBr, cm
) ν: 3444.87, 3226.91, 31867.40, 3066.82, 2929.87, 1699.29, 1683.86, 1668.43,
1
1
1
647.21, 1616.35, 1577.77, 1558.48, 1541.12, 1527.62, 1506.41, 1489.05, 1473.62, 1448.54, 1417.68, 1381.03,
354.03, 1317.38, 1301.95, 1238.30, 1203.58. Anal. Calc. for C H ClN O : C, 56.47%; H, 4.56%; N,
26
25
6
6
5.20%; Found: C, 56.86%; H, 4.84%; N, 15.47%.
Data for 3-(2-chlorophenyl)-5-methyl-N-(1-methyl-4-(2-(propan-2-ylidene)hydrazinecarbonyl)-1H-pyrazol-5-yl)
◦
1
isoxazole-4-carboxamide (6l). White solid; m.p. 189–191 C; yield 87.2%; H-NMR (500 MHz, DMSO-d ,
6
ppm)
δ
: 10.16 (s, 1H, isoxazole–CONH–), 9.28 (s, 1H, pyrazole–CONH–), 7.86 (s, 1H, pyrazole–H),
7
2
.60–7.47 (m, 4H, Ar–H), 4.34 (s, 3H, –CH ), 3.61 (s, 3H, pyrazole–CH ), 2.85 (s, 3H, isoxazole–CH ),
3
3
3
.50 (s, 3H, –CH3). 13C-NMR (125 MHz, DMSO-d , ppm)
δ
: 170.80, 161.37, 159.91, 159.61, 136.57, 135.96,
6
1
cm
32.09, 131.43, 131.38, 129.43, 129.38, 127.22, 127.15, 112.97, 108.85, 35.82, 24.84, 18.36, 12.22. IR (KBr,
−
1
) ν: 3444.87, 3066.82, 1699.29, 1683.86, 1668.43, 1653.00, 1635.64, 1616.35, 1577.77, 1558.48, 1541.12,
1521.84, 1506.41, 1489.05, 1458.18, 1473.62, 1429.25, 1411.89, 1386.82, 1338.60, 1313.52, 1279.88, 1236.73.
Anal. Calc. for C H ClN O : C, 55.01%; H, 4.62%; N, 20.26%; Found: C, 55.35%; H, 4.98%; N, 20.52%.
19
19
6
3
Data for 3-(2-chlorophenyl)-5-methyl-N-(1-methyl-4-(2-(2-methylpropylidene)hydrazinecarbonyl)-1H-pyrazol
◦
1
-
5-yl)isoxazole-4-carboxamide (6m). White solid; m.p. 212–214 C; yield 88.3%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.14 (s, 1H, isoxazole–CONH–), 9.27 (s, 1H, pyrazole–CONH–), 7.85 (s, 1H,
δ
6
pyrazole–H), 7.60–7.47 (m, 5H, –C=NH–, Ar–H), 4.32 (s, 3H, CH ), 3.61 (s, 3H, pyrazole–CH ), 2.85
3
3
13
(
s, 4H, –CH–, –CH ), 2.50 (s, 3H, –CH ). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.15, 166.20,
3
3
6
1
61.71, 160.25, 136.94, 136.32, 132.43, 131.76, 131.67, 129.74, 127.56, 127.47, 125.47, 119.61, 113.32, 109.20,
−
1
3
6.14, 30.18, 20.12, 12.57. IR (KBr, cm
635.64, 1610.56, 1577.77, 1558.48, 1541.12, 1521.84, 1506.41, 1489.05, 1458.18, 1473.62, 1458.18, 1429.25,
411.89, 1386.82, 1338.60, 1278.81, 1249.87, 1201.65. Anal. Calc. for C H ClN O : C, 56.01%; H, 4.94%;
) ν: 3325.28, 3304.06, 3066.82, 1699.29, 1683.86, 1674.21, 1653.00,
1
1
20
21
6
3
N, 19.60%; Found: C, 56.19%; H, 5.13%; N, 20.01%.
Data for 3-(2-chlorophenyl)-5-methyl-N-(1-methyl-4-(2-propylidenehydrazinecarbonyl)-1H-pyrazol-5-yl)
◦
1
isoxazole-4-carboxamide (6n). White solid; m.p. 199–201 C; yield 89.6%; H-NMR (500 MHz, DMSO-d ,
6
ppm)
δ
: 11.01 (s, 1H, isoxazole–CONH–), 10.23 (s, 1H, pyrazole–CONH–), 7.91 (s, 1H, pyrazole–H),
7
2
.60–7.39 (m, 5H, –C=NH–, Ar–H), 3.62 (s, 3H, pyrazole–CH ), 2.82 (s, 3H, isoxazole–CH ), 2.25 (q,
3
3
3
1
H, J = 14.90 Hz, –CH –), 1.05 (t, 3H, J = 14.90 Hz, –CH ). C-NMR (125 MHz, DMSO-d , ppm) δ:
2
3
6
1
72.81, 166.66, 161.98, 153.62, 146.78, 138.90, 137.94, 134.20, 133.51, 131.48, 129.28, 126.80, 121.48, 116.59,
−
1
3
8.00, 27.25, 14.25, 12.62. IR (KBr, cm
635.64, 1615.63, 1577.77, 1558.48, 1541.12, 1521.84, 1506.41, 1489.05, 1473.62, 1456.26, 1436.97, 1417.68,
398.39, 1338.60, 1309.31, 1269.81, 1238.74, 1207.29. Anal. Calc. for C H ClN6O : C, 55.01%; H, 4.62%;
) ν: 3325.28, 3304.06, 3066.82, 1699.29, 1683.86, 1674.21, 1653.00,
1
1
19
19
3
N, 20.26%; Found: C, 55.36%; H, 4.87%; N, 20.54%.
Data for 3-(2,6-dichlorophenyl)-N-(4-(2-(4-chlorobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)
◦
1
-
5-methylisoxazole-4-carboxamide (6o). White solid; m.p. 168–170 C; yield 91.3%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.56 (s, 1H, isoxazole–CONH), 10.26 (s, 1H, pyrazole–CONH), 8.32 (s, 1H,
CH=N–), 8.03–8.01 (d, 1H, J = 9.70 Hz, pyrazole–H), 7.74–7.51 (m, 7H, Ar–H), 3.61 (s, 3H,
δ
6
–
13
pyrazole–CH ), 2.92 (s, 3H, isoxazole–CH ). C NMR (125 MHz, DMSO-d , ppm)
δ
: 171.84, 163.47,
3
3
6
1
1
1
1
3
59.92, 158.61, 158.40, 145.62, 142.82, 140.37, 137.72, 134.95, 134.91, 133.86, 132.91, 129.47, 129.14, 128.96,
−
1
28.93, 127.28, 127.12, 113.60, 109.62, 36.42, 13.17. IR (KBr, cm
683.86, 1653.00, 1635.64, 1616.35, 1605.89, 1575.84, 1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1473.62,
448.54, 1411.89, 1398.39, 1319.31, 1301.95, 1215.15. Anal. Calc. for C H Cl N O : C, 51.95% H,
) ν: 3444.87, 3176.76, 2951.09, 1699.29,
23
17
3
6
3
.22%; N, 15.80%; Found: C, 51.98%; H, 3.59; N, 15.96%.

Molecules 2018, 23, 1798
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Data for 3-(2,6-dichlorophenyl)-N-(4-(2-(4-fluorobenzylidene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)
◦
1
-
5-methylisoxazole-4-carboxamide (6p). White solid; m.p. 154–156 C; yield 90.6%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.51 (s, 1H, isoxazole–CONH–), 10.25 (s, 1H, pyrazol–CONH–), 8.33 (s, 1H,
CH=N–), 8.05 (d, 1H, J = 18.35 Hz, pyrazole–H), 7.78–7.57 (m, 5H, Ar–H), 7.31 (t, 2H, J = 17.75 Hz,
δ
6
–
13
Ar–H), 3.62 (s, 3H, pyrazole–CH ), 2.92 (s, 3H, isoxazole–CH ). C-NMR (125 MHz, DMSO-d , ppm)
3
3
6
δ
: 171.60, 171.21, 163.90, 162.81, 161.93, 159.29, 157.87 (d, 1C, 4-F-Ph-C), 145.21, 142.34, 139.74, 138.51,
1
37.08, 134.29, 132.26, 130.87, 129.00, 128.30, 126.66, 115.88, 115.71, 112.96, 109.02, 107.44, 35.81, 12.54.
−
1
IR (KBr, cm
) ν: 3446.79, 3068.75, 2951.09, 1699.29, 1683.86, 1653.00, 1635.64, 1627.92, 1602.85, 1575.84,
1558.48, 1541.12, 1521.84, 1506.41, 1489.08, 1473.62, 1448.54, 1419.61, 1398.39, 1319.31, 1298.09, 1234.44,
1
213.23. Anal. Calc. for C H Cl FN O : C, 53.61%; H, 3.33%; N, 16.31%; Found: C, 53.69%; H, 3.75%;
23
17
2
6
3
N, 16.51%.
Data for 3-(2,6-dichlorophenyl)-N-(4-(2-(furan-2-ylmethylene)hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)
◦
1
-
5-methylisoxazole-4-carboxamide (6q). White solid; m.p. 255–257 C; yield 86.5%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.46 (s, 1H, isoxazole–CONH–), 10.29 (s, 1H, pyrazole–CONH–), 8.22 (s, 1H,
CH=N–), 7.99 (d, 1H, pyrazole–H), 7.85 (s, 1H, Ar–H), 7.64–7.52 (m, 3H, Ar–H, pyrazole–H, furan–H),
.91 (d, 1H, J = 9.75 Hz, furan–H), 6.63 (s, 1H, furan–H), 3.62 (s, 3H, pyrazole–CH ), 2.93 (s, 3H,
δ
6
–
6
3
13
isoxazole–CH3). C-NMR (125 MHz, DMSO-d , ppm)
δ
: 171.82, 163.26, 159.95, 158.32, 149.98, 145.59,
6
1
1
1
40.80, 139.31, 137.64, 136.81, 134.92, 133.62, 132.90, 128.94, 127.28, 113.83, 112.70, 109.67, 107.80, 36.44,
−
1
3.16. IR (KBr, cm
) ν: 3446.79, 3068.75, 2953.02, 1689.64, 1653.00, 1635.64, 1602.85, 1558.48, 1541.12,
521.84, 1506.41, 1489.08, 1473.62, 1431.18, 1409.96, 1398.39, 1330.88, 1317.38, 1255.66, 1220.94. Anal.
Calc. for C H Cl N O : C, 51.76%; H, 3.31%; N, 17.25%; Found: C, 51.84%; H, 3.36%; N, 17.28%.
21
16
2
6
4
Data for 3-(2,6-dichlorophenyl)-5-methyl-N-(1-methyl-4-(2-(thiophen-2-ylmethylene)hydrazinecarbonyl) -1H
o
1
-
pyrazol-5-yl)isoxazole-4-carboxamide (6r). White solid; m.p. 248–251 C; yield 85.9%; H-NMR (500 MHz,
DMSO-d , ppm) : 11.43 (s, 1H, isoxazole–CONH–), 10.22 (s, 1H, pyrazole–CONH–), 8.54 (s, 1H,
CH=N–), 7.98 (s, 1H, pyrazole–H), 7.65–7.49 (m, 5H, Ar–H, thiophen–H), 7.13 (s, 1H, thiophen–H),
δ
6
–
3
13
.61(s, 3H, pyrazole–CH ), 2.91 (s, 3H, isoxazole–CH ). C-NMR (125 MHz, DMSO-d , ppm) δ: 172.35,
3 3 6
171.82, 163.26, 159.95, 158.64, 158.45, 158.32, 149.98, 149.87, 145.59, 140.80, 139.31, 137.69, 137.64, 136.81,
1
34.92, 133.62, 132.90, 128.94, 128.64, 127.28, 113.83, 113.61, 113.36, 112.70, 109.67, 107.80, 36.72, 13.16.
−
1
IR (KBr, cm
) ν: 3446.79, 3265.49, 2953.02, 1699.29, 1683.86, 1653.00, 1635.64, 1595.13, 1575.84, 1558.48,
1541.12, 1521.84, 1506.41, 1489.08, 1473.62, 1458.18, 1436.97, 1398.39, 1373.32, 1315.45, 1294.24, 1251.80,
1
222.87. Anal. Calc. for C H Cl N O S: C, 50.11%; H, 3.20%; N, 16.70%; Found: C, 50.28%; H, 3.43%;
21
16
2
6
3
N, 16.76%.
2
.2. Biological Evaluations
Table 1 shows that most of the title compounds exhibited good antiviral activity against TMV
in vivo. Especially, among the title compounds evaluated, compounds 6a and 6h exhibited good
curative activity against TMV, with inhibition rates of 56.2% and 55.6% at 500 g/mL, respectively,
which were similar to that of Ningnanmycin (54.6%). Meanwhile, compounds 6c 6d 6n, and 6k
exhibited significant protection activity against TMV, with inhibition rates of 66.2%, 64.5%,64.2%,
and 64.7% at 500 g/mL, respectively, which were equal to that of Ningnanmycin (63.8%). In addition,
compounds 6c 6o 6p 6q, and 6r revealed excellent inactivation activities against TMV, with inhibition
µ
,
,
µ
,
,
,
rates of 91.4%, 92.3%, 91.7%, 93.2%, and 92.6% at 500
of Ningnanmycin (92.5%).
µg/mL, respectively, which were similar to that

Molecules 2018, 23, 1798
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Table 1. Antiviral activity of the title compounds against TMV at 500 µg/mL in vivo.
Compounds
Curative Activity (%) a
Protection Activity (%) a
Inactivation Activity (%) a
3
a
20.6 ± 3.3
21.5 ± 1.9
56.2 ± 1.3
48.3 ± 1.5
51.4 ± 2.1
47.1 ± 1.4
44.4 ± 1.7
47.7 ± 2.7
50.9 ± 3.3
55.6 ± 2.0
43.3 ± 3.1
52.8 ± 2.2
51.3 ± 2.6
54.7 ± 2.1
33.6 ± 2.4
27.5 ± 2.5
23.3 ± 1.1
40.7 ± 3.2
21.9 ± 2.2
48.9±2.0
54.6±1.6
a
30.1 ± 2.3
28.9 ± 3.0
60.3 ± 2.1
56.5 ± 3.2
66.2 ± 2.5
64.5 ± 1.7
45.6 ± 1.2
59.3 ± 1.1
47.8 ± 3.4
64.2 ± 1.9
55.3 ± 2.8
39.4 ± 2.4
64.7 ± 2.9
56.9 ± 1.2
40.7 ± 1.8
35.8 ± 2.3
56.2 ± 2.3
49.3 ± 3.1
36.3±1.0
42.7 ± 3.2
39.4 ± 2.2
73.3 ± 1.4
81.5 ± 2.5
91.4 ± 2.0
78.2 ± 2.1
61.4 ± 2.0
71.8 ± 2.1
74.1 ± 1.5
84.9 ± 2.6
73.6 ± 2.1
65.9 ± 2.2
81.5 ± 2.3
66.7 ± 3.2
54.3 ± 2.1
46.5 ± 2.6
92.3 ± 1.8
91.7 ± 1.5
93.2 ± 1.9
92.6 ± 2.2
92.5 ± 1.3
3
6
b
a
6
b
6
c
6
6
d
e
6
f
6
g
6
h
6
6
i
j
6
k
6
l
6
6
m
n
6
6
6
o
p
q
6
r
62.6 ± 1.2
63.8 ± 2.9
Ningnanmycin
Average of three replicates.
As shown in Table 2, compounds 6a, 6h and 6k displayed excellent curative activities against TMV,
with the 50% effective concentration (EC ) values of 240.8, 255.4 and 267.4
µg/mL, which were even
50
better than that of Ningnanmycin (286.4
6d
48.4, 184.9, 189.7 and 176.5
Furthermore, compounds 6q and 6r, as shown in Table 2, exhibited the best inactivation activity,
with the EC50 values of 37.3 and 45.1 g/mL, which were even better than that of Ningnanmycin
46.3 µg/mL).
µg/mL). Meanwhile, as can be seen from Table 2, compounds
6
1
c
,
,
6h and 6j demonstrated excellent protection activities against TMV, with the EC50 values of
g/mL, which were superior to that of Ningnanmycin (198.2 g/mL).
µ
µ
µ
(
Table 2. The EC50 values of the testing compounds against TMV in vivo.
EC50 (µg/mL) a
Compounds
Curative Activity
Protection Activity
Inactivation Activity
6
a
240.8 ± 2.2
457.4 ± 3.4
342.6 ± 4.3
516.9 ± 2.5
505.8 ± 3.3
362.4 ± 1.9
255.4 ± 2.3
315.2 ± 3.6
343.7 ± 4.2
267.4 ± 3.1
/
287.4 ± 2.3
369.6 ± 3.2
148.4 ± 3.3
184.9 ± 2.0
323.8 ± 3.5
/
172.3 ± 2.1
102.0 ± 1.9
62.2 ± 1.6
128.3 ± 2.7
194.6 ± 2.8
150.9 ± 4.0
83.9 ± 3.1
/
98.0 ± 2.6
247.4 ± 4.5
56.4 ± 1.7
64.2 ± 2.3
37.3 ± 1.9
45.1 ± 2.4
46.3 ± 1.9
6
b
6
c
6
d
6
f
6
g
6
h
189.7 ± 2.7
6
6
i
j
/
176.5 ± 2.8
342.5 ± 3.6
381.8 ± 3.6
509.5 ± 3.2
/
6
6
k
o
6
6
p
q
632.3 ± 5.3
/
6
r
455.3 ± 4.6
286.4 ± 2.2
a
224.8 ± 1.9
Ningnanmycin
198.2 ± 2.1
Average of three replicates.

Molecules 2018, 23, 1798
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As an extension of this approach, the structure-activity relationships (SAR) were analyzed on
1
2
the basis of the activity values in Tables 1 and 2. First, when R and R substituent groups were
3
2
-Cl and H, respectively, R was substituted with 4-Cl-Ph, 2,4-diCl-Ph, and 3,4,5-triMe-Ph groups,
the corresponding target compounds 6a
,
6h and 6k exhibited excellent curative activity against TMV,
1
which was superior to those of Ningnanmycin and the other target compounds. Second, when R
2
3
and R were substituted with 2-Cl and H groups, respectively, R was 2-Br-Ph, 2-Cl-Ph, 2,4-diCl-Ph,
and Furyl groups, the corresponding target compounds 6c 6d 6h and 6j exhibited remarkable
protection activity against TMV than those of Ningnanmycin and the other target compounds. Third,
,
,
1
2
3
when R , R , and R substituent groups were 2,6-diCl, H, and Furyl, the corresponding compound
revealed the best inactivation activity which was even better than those of Ningnanmycin and
the other target compounds. Finally, as shown in Table 1, compound showed better anti-TMV
activity than those of compound . Therefore, we found that, when the presence of the –Cl at
-position or 2,6-positions of phenyl at R substituent group, the presence of small groups (–H) at R
6
q
6
3
1
2
2
substituent group, the corresponding compounds presented good antiviral activities. We also found
that introducing a hydrazine moiety into the target compounds could enhance the anti-TMV activity,
which were also observed in previous studies of Li et al. and Yang [20,24].
3
. Experimental
3
.1. General Methods
The melting points of the products were determined on a XT-4 binocular microscope (Beijing
Tech Instrument Co., Beijing, China) and were not corrected. The IR spectra were recorded on a
1
Bruker VECTOR 22 spectrometer (Bruker Optics Inc. Billerica, MA, USA) in KBr disk. H-NMR and
13
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 analyser (Elementar, Frankfurt,
Germany). 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). Column chromatographic purification was carried out
using silica gel. Commercial reagents were used as received, unless otherwise indicated. All solvents
were dried by standard methods in advance and distilled before use.
3
.2. General Procedure for the Preparation of the Intermediate 5
As shown in Scheme 1, 3-substituted phenyl-5-methylisoxazole-4-carboxylic acid (0.02
◦
mol) was added to distillated SOCl2 (50 mL) and reacted at 80 C for 5 h to obtain
intermediate
-amino-1-methyl-1H-pyrazole-4-carboxylic acid (0.02 mol) in anhydrous tetrahydrofuran (THF,
00 mL) and trimethylamine, the reaction mixture was stirred at room temperature for 2.0 h.
2. Then, intermediate 2 (0.02 mol) was added dropwise to a stirred solution of
5
1
The reaction mixture was poured into cold 5.0% dilute HCl solution (200 mL). The solid obtained
was filtered, washed several times with water, dried to give the crude product which was further
recrystallized and dried to give intermediate 3. A mixture of intermediate 3 (0.02 mol) and acetic
anhydride (0.2 mol) was heated under reflux for 4.0 h. The solvent was removed under reduced
pressure. The residue was washed with water. The separated solid was collected by filtration,
washed with water, dried, and recrystallized and dried to give intermediate
4. To a solution of
intermediate (10 mmol) in THF (50 mL), 6 mL of 80% hydrazine hydrate was added slowly at
4
room temperature. Then, the mixture was further reacted at room temperature for 2 h. The solvent
was removed under reduced pressure, and the residue was washed with water and anhydrous
ethanol to give the crude product, then recrystallized by ethanol and dried under vacuum to give the
key intermediate 3-(2-chlorophenyl)-N-(4-(hydrazinecarbonyl)-1-methyl-1H-pyrazol-5-yl)-5-methyl
isoxazole-4-carboxamide 5.

Molecules 2018, 23, 1798
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3
.3. General Procedure for the Preparation of the Target Compounds 6a–6r
As shown in Scheme 1, different aldehydes and ketones (1 mmol) were added to a well-stirred
solution of (1 mmol) in ethanol (8 mL), and then a few drops of glacial acetic acid were added.
5
The resulting mixture was refluxed for 2 h to afford a solid, and then filtered and recrystallized from a
mixture of ethanol and DMF (1:1 in volume) to gain the title compounds 6a–6r.
3
.4. In Vivo Antiviral Activity Test
3
.4.1. Purification of TMV
Using the Gooding method [37], the virus was multiplied in N. tabacum cv. K326 and purified.
The absorbance values were estimated at 260 nm using an ultraviolet spectrophotometer. The virus
concentration was calculated by the following formula, where E represents the extinction coefficient
0
.1%
260nm
for TMV, E
is 3.1:
1cm
0.1%
260nm
Virus concentration (mg/mL) = (A260 × dilution ratio)/E
.4.2. Protection Activity of the Title Compounds against TMV in Vivo
The compound solution was smeared on the left side of growing N. tabacum L. leaves of the same
1cm
3
age. The solvent without compound solution was smeared on the right side of the leaves to serve as
the control. The leaves, which were previously scattered with silicon carbide, were inoculated with
−
3
the virus after 12 h using a brush dipped in 6
×
10 mg/mL TMV and subsequently washed with
water and rubbed softly along the nervature once or twice. After three to four days of the inoculation,
the local lesions were counted. Three replications were reproduced for each compound.
3
.4.3. Curative Activity of the Title Compounds against TMV in Vivo
The leaves were inoculated with TMV (concentration of 6
−
3
×
10 mg/mL) by dipping and
brushing the whole leaves, which were previously scattered with silicon carbide. The leaves were
then washed with water after inoculation for 0.5 h. The compound solution was smeared on the left
side of the leaves, and the solvent without compound solution was smeared on the right side for
control. The number of local lesions was counted and recorded three to four days after the inoculation.
Three replications were reproduced for each compound.
3
.4.4. Inactivation Activity of the Title Compounds against TMV in Vivo
The virus was inhibited after it was mixed with a compound solution of the same volume for
30 min. The right side of the N. tabacum L. leaves was then inoculated with the solvent and virus
mixture for control. All of the leaves were previously scattered with silicon carbide. The number of
local lesions was recorded three to four days after the inoculation. Three replications were reproduced
for each compound.
The inhibition rates of the compounds were calculated according to the following formula:
Inhibition rate (%) = [(average local lesion number of control (not treated with
compounds) − average local lesion number smeared with compounds)/average
local lesion number of control (not treated with compounds)] × 100%
4
. Conclusions
In summary, a series of novel pyrazole-hydrazone derivatives containing an isoxazole moiety
were obtained with moderate yields and their antiviral activities against TMV were examined by
a half-leaf method. Bioassay results revealed that some of the target compounds exhibited better
antiviral activity against TMV in vivo than Ningnanmycin. Among the title compounds, compound

Molecules 2018, 23, 1798
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6
6
a
c
exhibited better curative activity against TMV than that of Ningnanmycin. Meanwhile, compound
showed remarkable protection activity against TMV, which was superior to that of Ningnanmycin.
Moreover, compound 6q exhibited better inactivation activity against TMV which were superior to
that of Ningnanmycin. The antibacterial tests showed that introducing a hydrazine moiety into the
target compounds could enhance the anti-TMV activity; meanwhile, when the presence of the –Cl at
1
2
-position or 2,6-positions of phenyl in the R substituent group, the presence of small groups (–H) in
2
the R substituent group, the corresponding compounds presented good antiviral activities. Therefore,
this study demonstrated that this series of pyrazole-hydrazone derivatives containing an isoxazole
moiety can be considered for further development as a new class of tobacco protection agents.
Supplementary Materials: The H-NMR and 13C-NMR for all the synthesized compounds are available online.
1
Author Contributions: Writing—Original Draft Preparation, Z.Y.; Writing—Review and Editing, P.L. and X.G.;
Project Administration, Z.Y.
Funding: This research was funded by the National Natural Science Foundation of China (Grant No. 21466031).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 6a–6r are available from the authors.
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