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Synthesis, Biological Evaluation and Structure-Activity Relationship Study of Novel Stilbene Derivatives as Potential Fungicidal Agents Daohang He, Weilin Jian, Xianping Liu, Huifang Shen, and Shaoyun Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5052893 • Publication Date (Web): 16 Jan 2015 Downloaded from http://pubs.acs.org on January 21, 2015
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Journal of Agricultural and Food Chemistry
Synthesis, Biological Evaluation and Structure-Activity Relationship Study of Novel Stilbene Derivatives as Potential Fungicidal Agents Daohang He,†∗ Weilin Jian,† Xianping Liu,† Huifang Shen,‡ and Shaoyun Song§ †
School of Chemistry and Chemical Engineering, South China University of
Technology, Guangzhou 510640, China ‡
Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences,
Guangzhou 510640, China §
State Key Lab of Biocontrol, Sun Yat-sen University, Guangzhou, 510006, China
*Corresponding author, (Tel: + 86-20- 8711 0234; Fax: + 86-20-8711 0234; E-mail: [email protected]).
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ABSTRACT
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Twenty-two novel stilbene derivatives containing the 1,3,4-oxadiazole moiety and
3
the trimethoxybenzene were designed and synthesized. Their chemical structures
4
were characterized by 1H NMR, 13C NMR, IR, and HRMS. Bioassay results revealed
5
that some of the title compounds showed potent in vivo fungicidal activities against
6
three
7
lagenarium, and Septoria cucurbitacearum) from cucurbits at 600 µg/mL. Notably,
8
compounds 4b, 4d, 4i, 4k and 4l exhibited broad-spectrum and remarkably high
9
activities against those fungi, some of which even showed a comparable control
10
efficacy to that of the commercial fungicides. Three-dimensional quantitative
11
structure–activity relationship (3D-QSAR) based on comparative molecular field
12
analysis (CoMFA) with good predictive ability (q2=0.516, r2=0.920) was reasonably
13
discussed. For the first time, the present work suggested that the stilbene derivatives
14
containing 1,3,4-oxadiazole moiety could be developed as potential fungicides for
15
crop protection.
phytopathogenic
fungi
(Pseudoperonospora
cubensis,
Colletotrichum
16 17
KEYWORDS: stilbene derivatives, 1,3,4-oxadiazole, 3D-QSAR, fungicidal activity,
18
synthesis
19 20 21 22 2
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INTRODUCTION
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Plant diseases caused by pathogenic fungi are a severe threat to fruits and
25
vegetables, which limit the crop production worldwide. Cucumber downy mildew
26
(Pseudoperonospora
27
lagenarium) are two main devastating diseases found in cucumber, which are
28
widespread under both greenhouse and open field cultivation responsible for yield
29
reductions and food decays.1-4 For example, C. lagenarium can cause premature
30
plant death by reducing the photosynthetic surface area of 29–42%, resulting in
31
cucumber yield losses of 6–48%.5 Fungicide applications are still one of the most
32
effective solutions for controlling plant diseases in the current agricultural system.
33
However, conventional synthetic pesticides are far from satisfaction because of their
34
limited efficacy, and the increasing incidence of fungicide resistance.6, 7 Therefore,
35
the search for alternative improved fungicides remains a challenging task in
36
agrochemical industry.
cubensis)
and
cucumber
anthracnose
(Colletotrichum
37
Natural product-based libraries are expected to provide a rich source for new
38
fungicides discovery.8 Stilbenoid trans-Resveratrol is a phytoalexin being produced
39
in response to environmental stresses such as pathogenic infections.9,
40
fungicidal activity of stilbenes against plant fungal diseases was first reported by
41
Langcake and Pryce.11 And later, many naturally and synthetically derived stilbenes
42
were reported to exhibit remarkable biological properties.12-15 Biological screening
43
has revealed that the trans-olefin structure of stilbene skeleton plays an important
44
role in their bioactivity,16 and applications of those stilbene derivatives appeared to 3
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be dependent on the electron-donating or -withdrawing nature of their functional
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groups.17 However, little information is available concerning the fungcidal activity of
47
stilbenes against cucurbit phytopathogens such as C. lagenarium. On the other hand,
48
1,3,4-Oxadiazoles are known as the biologically active nitrogen-containing
49
heterocyclic molecules. Derivatives of 1,3,4-oxadiazole are also shown to possess
50
various activities such as antibacterial,18,
51
anti-inflammatory,23 and anticancer,24 which are widely used as a scaffold in
52
agricultural and medicinal chemistry. All these findings inspired us to modify the
53
bioactive stilbene skeleton with functional motifs as potential agrochemical leads for
54
plant disease control.
19
antifungal,20,
21
insecticidal,22
55
In the light of the above reports, we herein designed and synthesized a series of
56
novel stilbene derivatives via an efficient approach (Figure 1). Their in vivo
57
fungicidal activities against P. cubensis, C. lagenarium, and S. cucurbitacearum
58
(Septoria cucurbitacearum from Cucurbita moschata Duch.) were evaluated for the
59
first time. To further study the influence of different substituents on fungicidal
60
activity of stilbene derivatives, we performed the CoMFA modeling to gain insights
61
into a deeper understanding of the quantitative structure–activity relationships.
62 63
MATERIALS AND METHODS
64
All chemicals and reagents were commercially available and used without
65
further purification. All solvents were dried and redistilled prior to use. Melting
66
points were determined on an OPM100 OptiMelt apparatus and were uncorrected. 4
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FT-IR spectra were measured as KBr pellets on a Bruker Vector 33. 1H NMR and 13C
68
NMR spectra were recorded in CDCl3 on a Bruker AVANCE-400 MHz NMR
69
spectrometer using TMS as an internal standard. Mass spectra were obtained with a
70
Bruker Esquire HCTplus spectrometer (APCI/ESI). Purity of the compounds were
71
confirmed by thin layer chromatography (TLC) on silica gel ‘G’-coated glass plates
72
and spots were visualized under UV irradiation.
73
General Synthetic Procedures for Title Compounds 4a-4v. The title
74
compounds were synthesized according to our previously reported procedure.25 The
75
desired starting materials were prepared by esterification of 4-methylbenzoic acid
76
followed by treatment with hydrazine hydrate in absolute ethanol. The data for target
77
compounds 4a-4v is shown below, and the detailed procedures for intermediates 1-3
78
and target compounds are available in Supporting Information.
79
Data for 4a: light yellow solid; yield 62.5%; m.p.: 156-157°C; 1H-NMR (400
80
MHz, CDCl3) δ 8.13 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.67 (d, J = 8.3 Hz, 2H, C6H4
81
3,5-H), 7.56 (d, J = 7.5 Hz, 2H, C6H5 2,6-H), 7.45 – 7.36 (m, 4H, C6H5 3,5-H C6H2
82
2,6-H ), 7.32 (t, J = 7.3 Hz, 1H, C6H5 4-H), 7.25 (d, J = 16.3 Hz, 1H, CH=CH), 7.15
83
(d, J = 16.3 Hz, 1H, CH=CH), 4.00 (s, 6H, OCH3), 3.96 (s, 3H, OCH3);
84
(101 MHz, CDCl3) δ 164.42, 153.73, 141.28, 140.76, 136.71, 131.07, 128.81, 128.29,
85
127.39, 127.28, 127.00, 126.80, 122.56, 119.00, 104.31, 61.04, 56.45; HRMS (ESI):
86
m/z cacld for C25H23N2O4: (M+H)+ 415.1652, found 415.1650.
13
C-NMR
87
Data for 4b: yellow solid; yield 76.9%; m.p.: 180-181°C; 1H-NMR (400 MHz,
88
CDCl3) δ 8.11 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.63 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 5
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7.51 (m, 2H, C6H4 2,6-H), 7.36 (s, 2H, C6H2 2,6-H), 7.18 (d, J = 16.3 Hz, 1H,
90
CH=CH), 7.13-6.97 (3H, C6H4 3,5-H, CH=CH ), 3.99 (s, 6H, OCH3), 3.95 (s, 3H,
91
OCH3);
92
140.57, 132.91, 129.78, 128.34, 127.27, 127.16, 126.91, 122.56, 118.93, 115.79,
93
115.68, 104.31, 61.02, 56.44; HRMS (ESI): m/z cacld for C25H22FN2O4 (M+H)+
94
433.1558, found 433.1556.
13
C-NMR (101 MHz, CDCl3) δ 164.41, 164.35, 163.92, 153.72, 141.30,
95
Data for 4c: light yellow solid; yield 69.6%; m.p.: 178-179°C; 1H-NMR (400
96
MHz, CDCl3) δ 8.14 (d, J = 8.2 Hz, 2H, C6H4 2,6-H), 7.70 (t, 3H, C6H4 3,5-H, C6H4
97
5-H), 7.64 (d, J = 16.3 Hz, 1H, CH=CH), 7.42 (d, J = 7.8 Hz, 1H, C6H4 2-H), 7.37 (s,
98
2H, C6H2 2,6-H), 7.32 – 7.21 (m, 2H, C6H4 3,4-H), 7.12 (d, J = 16.3 Hz, 1H,
99
CH=CH), 3.98 (s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
100
164.46, 164.33, 153.72, 141.29, 140.40, 134.80, 133.74, 129.95, 129.89, 129.14,
101
127.32, 127.29, 127.02, 126.99, 126.58, 122.98, 118.96, 104.30, 61.03, 56.44;
102
HRMS (ESI): m/z cacld for C25H22ClN2O4 (M+H)+ 449.1263, found 449.1257.
103
Data for 4d: light yellow solid; yield 80.6%; m.p.: 176-177°C; 1H-NMR (400
104
MHz, CDCl3) δ 8.11 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 8.4 Hz, 2H, C6H4
105
3,5-H), 7.52 (s, 1H, C6H4 5-H), 7.38 (d, J = 7.5 Hz, 1H, C6H4 2-H), 7.35 (s, 2H,
106
C6H2 2,6-H), 7.29 (t, J = 7.7 Hz, 1H, C6H4 3-H), 7.26 (s, 1H, C6H4 4-H), 7.13 (s, 2H,
107
CH=CH), 3.96 (s, 6H, OCH3), 3.92 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
108
164.45, 164.28, 153.71, 141.29, 140.12, 138.58, 134.77, 129.99, 129.45, 128.76,
109
128.11, 127.28, 127.13, 126.53, 125.01, 122.95, 118.94, 104.29, 61.02, 56.43;
110
HRMS (ESI): m/z cacld for C25H22ClN2O4 (M+H)+ 449.1263, found 449.1257. 6
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Data for 4e: light yellow solid; yield 72.5%; m.p.: 209-210°C; 1H-NMR (400
112
MHz, CDCl3) δ 8.09 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.62 (d, J = 8.3 Hz, 2H, C6H4
113
3,5-H), 7.45 (d, J = 8.5 Hz, 2H, C6H4 2,6-H), 7.34 (s, 2H, C6H2 2,6-H), 7.33 (d, J =
114
8.5 Hz, 2H, C6H4 3,5-H), 7.15 (d, J = 16.3 Hz, 1H, CH=CH), 7.07 (d, J = 16.3 Hz,
115
1H, CH=CH), 3.97 (s, 6H, OCH3), 3.94 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3)
116
δ 164.43, 164.32, 153.71, 141.30, 140.34, 135.21, 133.89, 129.64, 128.97, 127.94,
117
127.27, 127.02, 122.77, 118.93, 104.30, 61.02, 56.43; HRMS (ESI): m/z cacld for
118
C25H22ClN2O4 (M+H)+ 449.1263, found 449.1260.
119
Data for 4f: light yellow solid; yield 62.7%; m.p.: 193-194°C; 1H-NMR (400
120
MHz, CDCl3) δ 8.15 (d, J = 7.8 Hz, 2H, C6H4 2,6-H), 7.70 (d, J = 7.8 Hz, 2H, C6H4
121
3,5-H), 7.65 (d, J = 8.5 Hz, 1H, C6H3 5-H), 7.56 (d, J = 16.3 Hz, 1H, CH=CH), 7.44
122
(d, J = 2.0 Hz, 1H, C6H3 2-H), 7.38 (s, 2H, C6H2 2,6-H), 7.30 (d, J = 2.0 Hz, 1H,
123
C6H3 3-H), 7.11 (d, J = 16.3 Hz, 1H, CH=CH), 4.01 (s, 6H, OCH3), 3.96 (s, 3H,
124
OCH3);
125
134.19, 133.44, 130.38, 129.72, 127.45, 127.39, 127.36, 127.30, 125.85, 123.20,
126
118.89, 104.36, 61.05, 56.48; HRMS (ESI): m/z cacld for C25H21Cl2N2O4 (M+H)+
127
483.0873, found 483.0874.
13
C-NMR (101 MHz, CDCl3) δ 164.52, 164.27, 153.74, 141.36, 140.09,
128
Data for 4g: light green solid; yield 71.0%; m.p.: 211-212°C; 1H -NMR (400 MHz,
129
CDCl3) δ 8.12 (d, J = 8.0 Hz, 2H, C6H4 2,6-H), 7.63 (m, 3H, C6H4 3,5-H, C6H4 2-H),
130
7.47 – 7.41 (m, 1H, C6H4 3-H), 7.40 – 7.31 (m, 3H, C6H2 2,6-H, C6H4 6-H), 7.15 –
131
7.06 (m, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.96 (s, 3H, OCH3);
132
MHz, CDCl3) δ 164.50, 164.24, 153.73, 141.33, 139.85, 136.84, 132.98, 131.85, 7
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130.68, 129.22, 128.36, 128.31, 127.32, 127.18, 125.86, 123.14, 118.91, 104.31,
134
61.04, 56.46; HRMS (ESI): m/z cacld for C25H21Cl2N2O4 (M+H)+ 483.0873, found
135
483.0875.
136
Data for 4h: light yellow solid; yield 72.6%; m.p.: 176-177°C; 1H-NMR (400
137
MHz, CDCl3) δ 8.16 (d, J = 8.2 Hz, 2H, C6H4 2,6-H), 7.72 (d, J = 8.2 Hz, 3H, C6H4
138
3,5-H, C6H4 5-H), 7.67 – 7.58 (m, 3H, CH=CH, C6H4 2-H), 7.43 – 7.32 (m, 3H,
139
C6H2 2,6-H, C6H4 3-H), 7.18 (t, J = 7.0 Hz, 1H, C6H4 4-H), 7.10 (d, J = 16.2 Hz, 1H,
140
CH=CH), 4.01 (s, 6H, OCH3), 3.97 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
141
164.47, 164.33, 153.72, 141.28, 140.34, 136.51, 133.20, 130.07, 129.67, 129.38,
142
127.66, 127.33, 127.31, 126.82, 124.41, 123.00, 118.96, 104.31, 61.04, 56.45;
143
HRMS (APCI): m/z cacld for C25H22BrN2O4 (M+H)+ 493.0757, found 493.0759.
144
Data for 4i: yellow solid; yield 80.8%; m.p.: 213-214°C; 1H-NMR (400 MHz,
145
CDCl3) δ 8.09 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.62 (d, J = 8.3 Hz, 2H, C6H4 3,5-H),
146
7.49 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.38 (d, J = 8.4 Hz, 2H, C6H4 3,5-H), 7.34 (s,
147
2H, C6H2 2,6-H), 7.13 (d, J = 16.4 Hz, 1H, CH=CH), 7.08 (d, J = 16.4 Hz, 1H,
148
CH=CH), 3.97 (s, 6H, OCH3), 3.94 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
149
164.44, 164.31, 153.72, 141.32, 140.32, 135.64, 131.93, 129.70, 128.21, 128.06,
150
127.29, 127.04, 122.78, 122.09, 118.90, 104.31, 61.03, 56.45; HRMS (APCI): m/z
151
cacld for C25H22BrN2O4 (M+H)+ 493.0757, found 493.0754.
152
Data for 4j: light green solid; yield 77.6%; m.p.: 205-206°C; 1H-NMR (400 MHz,
153
CDCl3) δ 8.11 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.98 (d, J = 8.2 Hz, 1 H, C6H4 5-H),
154
7.76 (d, J = 7.7 Hz, 1 H, C6H4 2-H), 7.73 – 7.59 (m, 4H, C6H4 3,5-H, CH=CH, C6H4 8
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3-H), 7.43 (t, J = 7.7 Hz, 1H, C6H4 4-H), 7.35 (s, 2H, C6H2 2,6-H), 7.08 (d, J = 16.1
156
Hz, 1H, CH=CH), 3.98 (s, 6H, OCH3), 3.94 (s, 3H, OCH3);
157
CDCl3) δ 164.51, 164.18, 153.71, 148.02, 141.30, 139.74, 133.25, 132.44, 132.37,
158
128.53, 128.25, 127.57, 127.30, 125.83, 124.87, 123.45, 118.86, 104.28, 61.01,
159
56.43; HRMS (APCI): m/z cacld for C25H22N3O6 (M+H)+ 460.1503, found
160
460.1505.
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C-NMR (101 MHz,
161
Data for 4k: light yellow solid; yield 87.5%; m.p.: 236-237°C; 1H-NMR (400
162
MHz, CDCl3) δ 8.39 (s, 1H, C6H4 6-H), 8.13 (m, 3H, C6H4 3,5-H, C6H4 4-H), 7.82
163
(d, J = 7.8 Hz, 1H, C6H4 2-H), 7.68 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 7.55 (t, J = 8.0
164
Hz, 1H, C6H4 3-H), 7.36 (s, 2H, C6H2 2,6-H), 7.25 (s, 2H, CH=CH), 3.98 (s, 6H,
165
OCH3), 3.94 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ 164.55, 164.19, 153.74,
166
148.77, 141.39, 139.58, 138.52, 132.48, 130.41, 129.73, 128.33, 127.39, 127.36,
167
123.42, 122.59, 121.10, 118.83, 104.35, 61.04, 56.46; HRMS (APCI): m/z cacld for
168
C25H22N3O6 (M+H)+ 460.1503, found 460.1507.
169
Data for 4l: light green solid; yield 88.1%; m.p.: 271-272°C; 1H NMR (400 MHz,
170
CDCl3) δ 8.26 (d, J = 8.8 Hz, 2H, C6H4 3,5-H), 8.18 (d, J = 8.3 Hz, 2H, C6H4 2,6-H),
171
7.74 - 7.65 (m, 4H, C6H4 2,6-H C6H4 3,5-H), 7.38 (s, 2H, C6H2 2,6-H), 7.30 (d, J =
172
5.7 Hz, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (APCI): m/z
173
cacld for C25H22N3O6 (M+H)+ 460.1503, found 460.1507.
174
Data for 4m: light yellow solid; yield 60.9%; m.p.: 145-146°C; 1H-NMR (400
175
MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 2H, C6H4 2,6-H), 7.66 (d, J = 8.1 Hz, 2H, C6H4
176
3,5-H), 7.61 (d, J = 4.5 Hz, 1H, C6H4 2-H), 7.58 (d, J = 3.9 Hz, 1H, C6H4 5-H), 7.35 9
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(s, 2H, C6H2 2,6-H), 7.27 (m, 1H, C6H4 4-H), 7.14 (d, J = 16.5 Hz, 1H, CH=CH),
178
6.98 (t, J = 7.5 Hz, 1H, C6H4 3-H), 6.91 (d, J = 8.3 Hz, 1H, CH=CH), 3.97 (s, 6H,
179
OCH3), 3.94 (s, 3H, OCH3), 3.90 (s, 3H, OCH3);
180
164.51, 164.35, 157.16, 153.70, 141.45, 141.20, 129.37, 127.75, 127.19, 127.01,
181
126.68, 125.95, 125.77, 122.25, 120.81, 119.07, 111.02, 104.27, 61.03, 56.43, 55.54;
182
HRMS (ESI): m/z cacld for C26H25N2O5 (M+H)+ 445.1758, found 445.1761.
13
C-NMR (101 MHz, CDCl3) δ
183
Data for 4n: light yellow solid; yield 61.5%; m.p.: 131-132°C; 1H-NMR (400
184
MHz, CDCl3) δ 8.11 (d, J = 7.2 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 7.3 Hz, 2H, C6H4
185
3,5-H), 7.37 (s, 2H, C6H2 2,6-H), 7.34 – 7.26 (m, 1H, C6H4 3-H), 7.19 (d, J = 16.3
186
Hz, 1H, CH=CH), 7.16 - 7.03 (m, 3H, C6H4 2-H, C6H4 5-H, CH=CH), 6.86 (d, J =
187
8.1 Hz, 1H, C6H4 4-H), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3), 3.86 (s, 3H, OCH3);
188
13
189
138.10, 131.00, 129.76, 127.61, 127.29, 127.03, 122.39, 119.50, 118.73, 113.94,
190
112.06, 104.36, 61.03, 56.45, 55.27; HRMS (ESI): m/z cacld for C26H25N2O5
191
(M+H)+ 445.1758, found 445.1765.
C-NMR (101 MHz, CDCl3) δ 164.37, 164.34, 159.96, 153.71, 141.39, 140.74,
192
Data for 4o: light yellow solid; yield 77.3%; m.p.: 190-191°C; 1H-NMR (400
193
MHz, CDCl3) δ 8.08 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.61 (d, J = 8.3 Hz, 2H, C6H4
194
3,5-H), 7.47 (d, J = 8.7 Hz, 2H, C6H4 2,6-H), 7.35 (s, 2H, C6H2 2,6-H), 7.18 (d, J =
195
16.3 Hz, 1H, CH=CH), 6.99 (d, J = 16.3 Hz, 1H, CH=CH), 6.90 (d, J = 8.7 Hz, 2H,
196
C6H4 3,5-H), 3.97 (s, 6H, OCH3), 3.92 (s, 3H, OCH3), 3.83 (s, 3H, OCH3); 13C-NMR
197
(101 MHz, CDCl3) δ 164.48, 164.34, 159.83, 153.70, 141.21, 141.12, 130.59, 129.49,
10
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128.09, 127.22, 126.68, 125.22, 122.09, 119.05, 114.26, 104.26, 61.02, 56.43, 55.33;
199
HRMS (APCI): m/z cacld for C26H25N2O5 (M+H)+ 445.1758, found 445.1767.
200
Data for 4p: light green solid; yield 81.1%; m.p.: 187-188°C; 1H-NMR (400 MHz,
201
CDCl3) δ 8.11 (d, J = 8.1 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 8.1 Hz, 2H, C6H4 3,5-H),
202
7.36 (s, 2H,C6H2), 7.15 (d, J = 16.2 Hz, 1H, CH=CH), 7.03 (d, J = 16.2 Hz, 1H,
203
CH=CH), 6.76 (s, 2H, C6H2 2,6-H), 3.97 (s, 6H, OCH3), 3.92 (d, 9H, OCH3), 3.87 (s,
204
3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ 164.40, 164.39, 153.70, 153.47, 141.28,
205
140.64, 138.56, 132.40, 130.99, 127.25, 126.88, 126.79, 122.46, 118.96, 104.28,
206
103.97, 61.00, 60.96, 56.43, 56.17; HRMS (ESI): m/z cacld for C28H29N2O7 (M+H)+
207
505.1969, found 505.1970.
208
Data for 4q: light yellow solid; yield 67.3%; m.p.: 182-183°C; 1H-NMR (400
209
MHz, CDCl3) δ 8.21 (d, J = 8.2 Hz, 1H, naphthalene-H), 8.11 (d, J = 8.2 Hz, 2H,
210
C6H4 2,6-H), 7.97 (d, J = 16.0 Hz, 1H, CH=CH), 7.86 (d, J = 7.9 Hz, 1H,
211
naphthalene-H), 7.81 (d, J = 8.2 Hz, 1H, naphthalene-H), 7.74 (d, J = 7.1 Hz, 1H,
212
naphthalene-H), 7.70 (d, J = 8.2 Hz, 2H, C6H4 3,5-H), 7.59 – 7.45 (m, 3H,
213
naphthalene-H), 7.33 (s, 2H, C6H2 2,6-H), 7.14 (d, J = 16.0 Hz, 1H, CH=CH), 3.97
214
(s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ 164.39, 164.35,
215
153.68, 141.23, 140.89, 134.22, 133.75, 131.33, 130.25, 128.72, 128.65, 127.93,
216
127.26, 127.14, 126.32, 125.96, 125.65, 123.82, 123.52, 122.66, 118.96, 104.25,
217
61.02, 56.41; HRMS (APCI): m/z cacld for C29H25N2O6 (M+H)+ 465.1809, found
218
465.1806.
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Data for 4r: yellow solid; yield 76.5%; m.p.: 191-192°C; 1H-NMR (400 MHz,
220
CDCl3) δ 8.46 (s, 1H, anthracen-H), 8.41-8.33(m, 2H, anthracen-H), 8.24 (d, J = 8.3
221
Hz, 2H, C6H4 2,6-H), 8.14 – 8.01 (m, 3H, CH=CH, anthracen-H), 7.85 (d, J = 8.3 Hz,
222
2H, C6H4 3,5-H), 7.57-7.49 (m , 4H, anthracen-H), 7.42 (s, 2H, C6H2 2,6-H), 7.05 (d,
223
J = 16.6 Hz, 1H, CH=CH), 4.02 (s, 6H, OCH3), 3.98 (s, 3H, OCH3); HRMS (ESI):
224
m/z cacld for C33H27N2O4 (M+H)+ 515.1965, found 515.1964.
225
Data for 4s: yellow solid; yield 70.1%; m.p.: 212-213°C; 1H-NMR (400 MHz,
226
CDCl3) δ 8.02 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.54 (d, J = 8.3 Hz, 2H, C6H4 3,5-H),
227
7.39 (d, J = 8.7 Hz, 2H, C6H4 2,6-H), 7.31 (s, 2H, C6H2 2,6-H), 7.11 (d, J = 16.2 Hz,
228
1H, CH=CH), 6.87 (d, J = 16.2 Hz, 1H, CH=CH), 6.67 (d, J = 8.6 Hz, 2H, C6H4
229
3,5-H), 3.94 (s, 6H, OCH3), 3.91 (s, 3H, OCH3), 2.95 (s, 6H, CH3); 13C-NMR (101
230
MHz, CDCl3) δ 164.60, 164.23, 153.68, 150.44, 141.73, 141.14, 131.25, 128.01,
231
127.17, 126.36, 122.81, 121.45, 119.13, 112.32, 104.23, 61.02, 56.43, 40.34; HRMS
232
(ESI): m/z cacld for C27H28N3O4 (M+H)+ 458.2074, found 458.2071.
233
Data for 4t: light yellow solid; yield 72.8%; m.p.: 196-197°C; 1H-NMR (400
234
MHz, CDCl3) δ 8.64 (d, J = 4.6 Hz, 1H, pyridine-H), 8.15 (d, J = 8.1 Hz, 2H, C6H4
235
2,6-H), 7.78 – 7.67 (m, 4H, C6H4 3,5-H pyridine-H, CH=CH), 7.45 (d, J = 7.6 Hz,
236
1H, pyridine-H), 7.37 (s, 2H, C6H2 2,6-H), 7.31 (d, J = 16.9 Hz, 1H, CH=CH), 7.26
237
– 7.19 (m, 1H, pyridine-H), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); 13C NMR (101
238
MHz, CDCl3) δ 164.51, 164.32, 154.73, 153.73, 149.43, 141.27, 139.91, 137.04,
239
131.80, 129.62, 127.68, 127.30, 123.33, 122.65, 118.96, 104.29, 61.03, 56.44;
240
HRMS (ESI): m/z cacld for C24H22N3O4 (M+H)+ 416.1605, found 416.1610. 12
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Data for 4u: light green solid; yield 69.3%; m.p.: 182-183°C; 1H NMR (600 MHz,
242
CDCl3) δ 8.77 (d, J = 2.0 Hz, 1H, pyridine-H), 8.53 (d, J = 4.8 Hz, 1H, pyridine-H),
243
8.14 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.88 (d, J = 7.9 Hz, 1H, pyridine-H), 7.68 (d, J
244
= 8.4 Hz, 2H, C6H4 3,5-H), 7.37 (s, 2H, C6H2 2,6-H), 7.33 (q, 1H, pyridine-H), 7.20
245
(s, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (ESI): m/z cacld
246
for C24H22N3O4 (M+H)+ 416.1605, found 416.1606.
247
Data for 4v: light green solid; yield 70.2%; m.p.: 221-222°C; 1H NMR (600 MHz,
248
CDCl3) δ 8.63 (d, J = 4.6 Hz, 2H, pyridine-H), 8.18 (d, J = 8.4 Hz, 2H, C6H4 2,6-H),
249
7.71 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 7.44 (d, J = 4.7 Hz, 2H, pyridine-H), 7.37 (d, J
250
= 16.2 Hz, 3H, C6H2 2,6-H, CH=CH), 7.16 (d, J = 16.3 Hz, 1H, CH=CH), 4.00 (s,
251
6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (ESI): m/z cacld for C24H22N3O4 (M+H)+
252
416.1605, found 416.1603.
253
In Vivo Bioassays. Pot culture test method was adopted to evaluate fungicidal
254
activities of the title compounds against P. cubensis, C. lagenarium, and S.
255
cucurbitacearum in vivo at 600 µg/mL according to the references with slight
256
modifications.26,
257
Carbendazim WP were evaluated as controls against the above mentioned fungi at
258
the same condition. All compounds were dissolved in dimethylformamide (DMF),
259
and then distilled water (containing 0.1% Tween 80) was added to the solution to
260
generate a tested concentration of 600 µg/mL, in each of which the final
261
concentration of DMF was < 1% (v/v). P. cubensis sporangia were obtained by
262
washing off the leaves of highly infected cucumber plants that were kept overnight
27
Two commercial fungicides, 80% Mancozeb WP and 80%
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(above 80% relative humidity). C. lagenarium and S. cucurbitacearum were cultured
264
on the potato dextrose agar (PDA). Spores of those two fungal pathogens were
265
removed from the PDA plates. All of the spore suspensions were adjusted to the
266
desired concentrations prior to use. Germination was conducted by soaking cucurbit
267
seeds in water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °C in
268
an incubator.
269
When the cucurbit plants used for inoculations were at the stage of two seed
270
leaves, tested compounds and commercial fungicides were sprayed with a
271
hand-sprayer on the upper leaf surface of seed leaves at the standard concentration of
272
600 µg/mL. Distilled water without compounds or commercial fungicides treatments
273
was set as a blank control. After the solution on leaves were air-dried, inoculations of
274
P. cubensis, C. lagenarium and S. cucurbitacearum were carried out by spraying a
275
spore suspension with the concentration of 1×105, 1×106, 1×106 spores/mL of water,
276
respectively. Then the plants were maintained at 24 ± 1 °C and above 80% relative
277
humidity. Three replicates were used per treatment, and the experiment was repeated
278
three times.
279
The fungicidal activity was evaluated visually when the non-treated cucurbit plant
280
(blank control) fully developed symptoms. The percentage of diseased area on
281
inoculated treated leaves was assessed and compared to that of non-treated ones to
282
determine the average disease index. The relative control efficacy of compounds
283
compared to the blank assay was calculated via the following equation:
284
I (%) = [(CK−PT)/CK] ×100% 14
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Where I is the relative control efficacy, CK is the average disease index during the
286
blank assay, and PT is the average disease index after treatment during testing.
287
3D-QSAR Analysis. All molecular modeling studies were performed using the
288
Tripos SYBYL 7.3.5 software package.28 The comparative molecular field analysis
289
(CoMFA) model was derived from the antifungal activity of 22 compounds (17
290
compounds for training sets, 5 compounds for test sets, respectively) against C.
291
lagenarium at 600 µg/mL. The fungicidal activity was converted into D values by
292
the formula D=log{[I/(100−I)] −MW},29 where I is the percent control efficacy and
293
MW is the molecular weight of the tested compounds. Compound 4k was chosen as
294
a template for superimposition because of the highest control efficacy. Each structure
295
was built using the SKETCH option and fully geometry-optimized using a conjugate
296
gradient procedure based on the TRIPOS force field and Gasteiger and Hückel
297
charges.
298
The steric and electrostatic interaction fields for CoMFA were calculated using
299
the SYBYL default parameters: 2.0 Å grid points spacing, a sp3 carbon probe atom
300
with a charge of +1.0 and a van der Waals radius of 1.52 Å. Steric and electrostatic
301
fields were scaled by the CoMFA-STD method in SYBYL with default cut-off
302
energy of 30 kcal/mol. A partial least-squares (PLS) approach was used to derive the
303
3D QSAR model, in which the CoMFA descriptors were used as independent
304
variables and the experimental D values were used as dependent variables. The
305
cross-validation with the leave-one-out (LOO) option and the SAMPLS program
306
was carried out to obtain the optimal number of components (N) and cross-validated 15
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307
coefficient (q2). After the optimal number of components was determined, a
308
non-cross-validated analysis was performed without column filtering to obtain
309
regression coefficients (r2) and its standard error (S) and F-test value (F) for the
310
model evaluation.
311
RESULTS AND DISCUSSION
312
Synthesis. The synthetic route of the title compounds is outlined in Figure 1. A
313
series of novel stilbene derivatives containing an 1,3,4-oxadiazole moiety were
314
synthesized in good yields by bromination of N-Bromosuccinimide (NBS), Arbuzov
315
reaction, and Wittig−Horner reaction. The key step involved the formation of the
316
stilbenes which were efficiently generated by the reaction of phosphonate 3 with
317
various aldehydes via Wittig–Horner reaction under mild reaction conditions. The
318
chemical structures of the target compounds were confirmed by FT-IR, 1H NMR, 13C
319
NMR, and HRMS. Notably, the 1H NMR spectra of target compounds show two fine
320
doublets as well as coupling constant (16.7-16.0 Hz) corresponding to the olefinic
321
protons (CH=CH) from trans-stilbene, which are further confirmed by the FT-IR
322
spectra.30 All spectral and analytical data are consistent with the assigned structures.
323
Fungicidal Activity. The in vivo fungicidal activity of compounds 4a-4v against
324
P. cubensis, C. lagenarium and S. cucurbitacearum at a concentration of 600 µg/mL
325
are listed in Table 1. The results of bioassays are compared with those obtained from
326
two commercial fungicides. It was worthy to note that all of the tested compounds
327
were found safe for the cucurbit plants. As indicated in Table 1, some of the title
328
compounds showed broad spectrum and comparable fungicidal activities against the 16
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tested fungi. Four main conclusions were drawn from the comparison of the
330
fungicidal activities of title compounds against test fungi to those of commercial
331
fungicides: (a) Compounds 4s exhibited a significant inhibition effect against
332
cucumber P. cubensis with control efficacy of 71.38 ± 1.47%, which was comparable
333
to that of 80% Mancozeb WP (70.13 ± 3.25%). While compounds 4d, 4l and 4v
334
showed relatively high antifungal activity against cucumber P. cubensis (65.01 ±
335
1.81 and 63.42 ± 4.60%, respectively) compared with the fungicide 80% Mancozeb
336
WP. (b) Compounds 4b, 4d, 4f, 4i, 4k and 4l showed prominent activity against C.
337
lagenarium as compared to the control 80% Carbendazim WP. In particularly, the
338
activity of compounds 4d and 4k were 82.43 ± 4.66 and 83.82 ± 2.69%, respectively,
339
which were similar to that of 80% Carbendazim WP (81.57 ± 2.19%). (c) Most of
340
the
341
cucurbitacearum. Notably, compounds 4b, 4h and 4i, whose efficacy rates were
342
72.05 ± 0.96, 69.34 ± 0.93 and 67.68 ± 1.43%, respectively, were found to be almost
343
the same activity level as that of 80% Carbendazim WP (69.02 ± 0.47%). (d) Some
344
compounds showed significant bioactivity variances against the different fungi. For
345
example, compound 4g exhibited moderate activity (44.81 ± 3.85%) against C.
346
lagenarium,
347
cucurbitacearum was only 8.03 ± 2.78%, and even showed a negative effect on P.
348
cubensis. These results indicated that compounds 4b, 4d, 4i, 4k and 4l were the most
349
promising candidates for crop protection because of their broad spectrum and
350
remarkably high activities against all tested fungi.
compounds
exhibited
whereas
the
fair
to
control
moderate
efficacy
of
control
efficacy
compound
17
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4g
against
against
S.
S.
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351
CoMFA Analysis. The statistical results of CoMFA model are summarized in
352
Table 2. The internal validation of LOO cross-validated q2 and non-cross validated
353
coefficient r2 are commonly applied as the criterion of robustness and predictive
354
ability of a QSAR model. The statistically recommendatory values for a satisfactory
355
QSAR model are q2 > 0.5 and r2 > 0.8.31 As listed in Table 2, a good predictive
356
CoMFA model with the LOO cross-validated coefficient q2=0.516 and the
357
correlation coefficient r2=0.920 was established. The predicted D values are
358
generally in good agreement with the experimental data as shown in Figure 2. The
359
standard error (S=0.114) and F-test value (F=25.186) further validate the developed
360
model.
361
The contribution steric and electrostatic fields are 56.2 and 43.8% for the QSAR
362
model, respectively. CoMFA contour maps are displayed in Figure 3 to visualise how
363
steric and electrostatic fields contribute to the bioactivity of test compounds. As
364
shown in steric field contour map (Figure 3a). The green contour surrounding
365
meta-position of the benzene ring indicates that a bulky group would be favorable
366
for higher fungicidal activity, while the yellow contour represents region of
367
unfavorable steric effect. For example, compounds 4k and 4d bearing bulkier
368
substituents at the meta-position displayed higher fungicidal activity. For
369
electrostatic contours map (Figure 3b), the negatively charged favorable red regions
370
are
371
electron-withdrawing substituents enhance the bioactivity, whereas a positively
372
charged blue region around para-position is favored. Such as compound 4o
observed
at
meta-position
of
the
benzene
18
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indicating
that
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373
(para-position)
displayed
higher
activity
than
those
of
compounds
374
(ortho-position) and 4n (meta-position). Analysis of CoMFA contour plots revealed
375
that substitutions at the meta-position, the bulky groups and electron-withdrawing
376
groups (e.g., -NO2, -Cl) are favorable to increase the bioactivity.
4m
377
The results obtained from 3D-QSAR provided reliable clues for mechanistic
378
study and designing of optimized stilbene derivatives. On the basis of the in vivo
379
bioassay and 3D-QSAR analysis, it can be concluded that the variances (substituted
380
positions, electronic properties and steric effects) among substituents (Ar) on
381
stilbenes showed a significant relationship with fungicidal activity against the three
382
fungi. Furthermore, considering that the bioassay of title compounds was evaluated
383
in vivo, effects of metabolism and/or biotransformation within the plants or fungi
384
may contribute to the significant variations in fungicidal potency. It is reported that
385
the electron-withdrawing substituents of the phenyl moiety seem to enhance the
386
potency, which may be related to the fact that they tend to retard mechanisms of
387
oxidative metabolism occurring on (or close to) the benzene ring.32 This assumption
388
is consistent with the higher bioactivity of the title compounds with
389
electron-withdrawing substituents at meta-position proposed by the 3D-QSAR
390
analysis.
391
Discovery of phytopathogenic fungicidal agents with new modes of action and
392
eco-friendly properties is of great importance for crop protection. 33 In the present
393
study, 22-novel stilbene derivatives containing the 1,3,4-oxadiazole moiety and the
394
trimethoxybenzene were designed and synthesized. The approach was proved to be 19
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395
an efficient method to implement structural modification by combination of several
396
functional fragments. Bioassay results indicated that compounds 4b, 4d, 4i, 4k and
397
4l exhibited broad spectrum fungicidal activity in vivo against all tested fungi, some
398
of which were comparable with the commercial fungicides. Further field evaluations
399
on their biological efficacies, as well as crop toxicities and safety, are necessary
400
before they can be developed as fungicide candidates.
401 402
ASSOCIATED CONTENT
403
Supporting Information Available:
404
Synthetic procedures for intermediates 1-3 and target compounds. This material is
405
available free of charge via the Internet at http://pubs.acs.org.
406 407
Notes
408
The authors declare no competing financial interest.
409 410
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(20) Chen, Q.; Zhu, X. L.; Jiang, L. L.; Liu, Z. M.; Yang, G. F., Synthesis, antifungal
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activity and CoMFA analysis of novel 1,2,4-triazolo[1,5-a]pyrimidine derivatives.
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Eur. J. Med. Chem. 2008, 43, 595-603.
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(21) Zhang, M. Z.; Mulholland, N.; Beattie, D.; Irwin, D.; Gu, Y. C.; Chen, Q.; Yang,
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G. F.; Clough, J. Synthesis and antifungal activity of 3-(1,3,4-oxadiazol-5-yl)-indoles
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and 3-(1,3,4-oxadiazol-5-yl)methyl-indoles. Eur. J. Med. Chem. 2013, 63, 22-32.
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(22) Shi, W.; Qian, X. H.; Zhang, R.; Song, G. H. Synthesis and quantitative
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structure-activity relationships of new 2,5-disubstituted-1,3,4-oxadiazoles. J. Agric.
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Food Chem. 2001, 49, 124−130.
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(23) Akhter, M.; Husain, A.; Azad, B.; Ajmal, M. Aroylpropionic acid based
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2,5-disubstituted-1,3,4-oxadiazoles: synthesis and their anti-inflammatory and
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analgesic activities. Eur. J. Med. Chem. 2009, 44, 2372-2378.
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(24) Zhang, S.; Luo, Y.; He, L. Q.; Liu, Z. J.; Jiang, A. Q.; Yang, Y. H.; Zhu, H. L.,
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Synthesis, biological evaluation, and molecular docking studies of novel
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1,3,4-oxadiazole derivatives possessing benzotriazole moiety as FAK inhibitors with
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anticancer activity. Bioorg. Med. Chem. 2013, 21, 3723-9.
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(25) Li, X. W.; He, D. H. Synthesis and optical properties of novel anthracene-based
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stilbene derivatives containing an 1,3,4-oxadiazole unit. Dyes Pigm. 2012, 93,
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1422-1427.
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(26) Tu, S.; Xu, L. H.; Ye, L. Y.; Wang, X.; Sha, Y.; Xiao, Z. Y. Synthesis and
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fungicidal activities of novel indene-substituted oxime ether strobilurins. J. Agric.
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Food Chem. 2008, 56, 5247-5253.
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(27) Cui, Z. N.; Shi, Y. X.; Zhang, L.; Ling, Y.; Li, B. J.; Nishida, Y.; Yang, X. L.
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Synthesis and fungicidal activity of novel 2,5-disubstituted-1,3,4-oxadiazole
495
derivatives. J. Agric. Food Chem. 2012, 60, 11649-11656.
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(28) Sybyl Molecular Modeling Software Packages, V 7.3.5, TRIPOS Inc., St Louis,
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2008.
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(29) Zhu, Y. Q.; Wu, C.; Li, H. B.; Zou, X. M.; Si, X. K.; Hu, F. Z.; Yang, H. Z.
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Design, synthesis, and quantitative structure-activity relationship study of herbicidal
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analogues of pyrazolo[5,1-d][1,2,3,5]tetrazin-4(3H)ones. J. Agric. Food Chem. 2007,
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55, 1364-1369. 24
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(30) Lu, H. X.; He, D. H. Asymmetric 1,3,4-oxadiazole derivatives containing
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naphthalene and stilbene units: synthesis, optical and electrochemical properties.
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Spectrochim. Acta A Mol. Biomol. Spectrosc. 2014, 124, 91-96.
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(31) Golbraikh, A.; Tropsha, A. Beware of q2! J. Mol. Graph. Model. 2002, 20,
506
269-276.
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(32) Li, M.; Liu, C. L.; Yang, J. C.; Zhang, J. B.; Li, Z. N.; Zhang, H.; Li, Z. M.
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Synthesis and biological activity of new (E)-alpha-(Methoxyimino)benzeneacetate
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derivatives containing a substituted pyrazole ring. J. Agric. Food Chem. 2010, 58,
510
2664-2667.
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(33) Qian, X. H.; Lee, P. W.; Cao, S. China: Forward to the green pesticides via a
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basic research program. J. Agric. Food Chem. 2010, 58, 2613−2623.
513 514
Funding
515
This work was financially supported by the Fundamental Research Funds for the
516
Central Universities (No.2012ZM0035) and Guangdong Provincial Natural Science
517
Foundation (No.04300531)
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FIGURES CAPTIONS Figure 1. General Synthetic Route for Target Compounds. 4a: Ar=phenyl; 4b: Ar=4-F phenyl; 4c: Ar=2-Cl phenyl; 4d: Ar=3-Cl phenyl; 4e: Ar=4-Cl phenyl; 4f: Ar=2,4-di-Cl phenyl; 4g: Ar=3,4-di-Cl phenyl; 4h: Ar=2-Br phenyl; 4i: Ar=4-Br phenyl; 4j: Ar=2-NO2 phenyl; 4k: Ar=3-NO2 phenyl; 4l: Ar=4-NO2 phenyl; 4m: Ar=2-methoxy phenyl; 4n: Ar=3-methoxy phenyl; 4o: Ar=4-methoxy phenyl; 4p: Ar=3,4,5-trimethoxy phenyl; 4q: Ar=1-naphthyl; 4r: Ar=9- anthryl; 4s: Ar=4- N,N-dimethylaniline; 4t: Ar=2- pyridyl; 4u: Ar=3- pyridyl; 4v: Ar=4- pyridyl. Figure 2. Plot of Experimental Values Versus Predicted Values for Training and Test Sets Based on CoMFA Model. Figure 3. Graphical Representation of CoMFA Analysis for Compound 4k. (a) Steric contour plots: Sterically favored areas are represented by green polyhedra, and sterically disfavored areas are represented by yellow polyhedra. (b) Electrostatic contour plots: Positive charge favored areas are represented by red polyhedra, and positive charge disfavored areas are represented by blue polyhedra.
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TABLES Table 1. Chemical Structures and Fungicidal Activities of Compounds 4a-4v at 600 µg/mL in Vivo.
Compd.
Ar
P. cubensis
control efficacy (%)a C. lagenarium S. cucurbitacearum
4a
58.77 ± 1.97
50.03 ± 4.01
55.17 ± 4.22
4b
52.20 ± 2.59
65.14 ± 3.52
72.05 ± 0.96
4c
32.66 ± 6.18
31.48 ± 4.10
9.34 ± 0.36
4d
65.01 ± 1.81
82.43 ± 4.66
32.03 ± 7.57
4e
36.96 ± 5.57
47.78 ± 1.15
4f
38.52 ± 4.06
63.83 ± 5.38
27.54 ± 8.20
4g
4.08 ± 4.17
44.81 ± 3.85
8.03 ± 2.78
19.08 ± 1.29
4h
8.37 ± 3.03
26.38 ± 1.16
69.34 ± 0.93
4i
54.87 ± 3.55
64.77 ± 2.10
67.68 ± 1.43
4j
48.34 ± 0.62
56.07 ± 3.45
35.36 ± 2.85
4k
50.13 ± 3.15
83.82 ± 2.69
46.83 ± 4.50
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Compd.
Ar
P. cubensis
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control efficacy (%)a C. lagenarium S. cucurbitacearum
4l
63.42 ± 4.60
66.72 ± 2.83
32.23 ± 1.57
4m
25.28 ± 5.99
33.12 ± 1.66
48.91 ± 1.54
4n
44.13 ± 4.56
37.90 ± 5.83
47.62 ± 3.37
4o
58.21 ± 5.91
56.93 ± 3.04
40.51 ± 4.43
4p
49.57 ± 1.12
61.92 ± 2.69
7.05 ± 1.39
4q
36.01 ± 0.51
37.98 ± 5.48
51.18 ± 4.76
4r
15.3 ± 2.77
14.29 ± 5.50
53.09 ± 4.36
4s
71.38 ± 1.47
30.26 ± 2.38
44.84 ± 4.22
4t
18.38 ± 1.59
52.88 ± 1.06
45.14 ± 6.89
4u
42.67 ± 2.51
37.58 ±1.98
40.27 ± 2.23
4v
62.35 ± 3.09
42.61 ± 1.92
34.24 ± 4.43
Fungicidesb
70.13 ± 3.25 A
81.57 ± 2.19 B
69.02 ± 0.47 B
a
b
Values represent means of three independent replicates ± SD.
Control fungicides: A, 80% Mancozeb WP; B, 80% Carbendazim WP.
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Table 2. The Statistical Parameters of CoMFA Model. Contribution (%)
CoMFA
N
q2
r2
S
F
compound
steric
electrostatic
5
0.516
0.920
0.114
25.186
4k
56.2
43.8
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FIGURES
Figure 1
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Figure 2
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Figure 3
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Table of Contents (TOC) Graphic:
(3.2×1.8 in.)
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Article
Synthesis, Biological Evaluation and Structure-Activity Relationship Study of Novel Stilbene Derivatives as Potential Fungicidal Agents Daohang He, Weilin Jian, Xianping Liu, Huifang Shen, and Shaoyun Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5052893 • Publication Date (Web): 16 Jan 2015 Downloaded from http://pubs.acs.org on January 21, 2015
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Synthesis, Biological Evaluation and Structure-Activity Relationship Study of Novel Stilbene Derivatives as Potential Fungicidal Agents Daohang He,†∗ Weilin Jian,† Xianping Liu,† Huifang Shen,‡ and Shaoyun Song§ †
School of Chemistry and Chemical Engineering, South China University of
Technology, Guangzhou 510640, China ‡
Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences,
Guangzhou 510640, China §
State Key Lab of Biocontrol, Sun Yat-sen University, Guangzhou, 510006, China
*Corresponding author, (Tel: + 86-20- 8711 0234; Fax: + 86-20-8711 0234; E-mail: [email protected]).
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1
ABSTRACT
2
Twenty-two novel stilbene derivatives containing the 1,3,4-oxadiazole moiety and
3
the trimethoxybenzene were designed and synthesized. Their chemical structures
4
were characterized by 1H NMR, 13C NMR, IR, and HRMS. Bioassay results revealed
5
that some of the title compounds showed potent in vivo fungicidal activities against
6
three
7
lagenarium, and Septoria cucurbitacearum) from cucurbits at 600 µg/mL. Notably,
8
compounds 4b, 4d, 4i, 4k and 4l exhibited broad-spectrum and remarkably high
9
activities against those fungi, some of which even showed a comparable control
10
efficacy to that of the commercial fungicides. Three-dimensional quantitative
11
structure–activity relationship (3D-QSAR) based on comparative molecular field
12
analysis (CoMFA) with good predictive ability (q2=0.516, r2=0.920) was reasonably
13
discussed. For the first time, the present work suggested that the stilbene derivatives
14
containing 1,3,4-oxadiazole moiety could be developed as potential fungicides for
15
crop protection.
phytopathogenic
fungi
(Pseudoperonospora
cubensis,
Colletotrichum
16 17
KEYWORDS: stilbene derivatives, 1,3,4-oxadiazole, 3D-QSAR, fungicidal activity,
18
synthesis
19 20 21 22 2
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INTRODUCTION
24
Plant diseases caused by pathogenic fungi are a severe threat to fruits and
25
vegetables, which limit the crop production worldwide. Cucumber downy mildew
26
(Pseudoperonospora
27
lagenarium) are two main devastating diseases found in cucumber, which are
28
widespread under both greenhouse and open field cultivation responsible for yield
29
reductions and food decays.1-4 For example, C. lagenarium can cause premature
30
plant death by reducing the photosynthetic surface area of 29–42%, resulting in
31
cucumber yield losses of 6–48%.5 Fungicide applications are still one of the most
32
effective solutions for controlling plant diseases in the current agricultural system.
33
However, conventional synthetic pesticides are far from satisfaction because of their
34
limited efficacy, and the increasing incidence of fungicide resistance.6, 7 Therefore,
35
the search for alternative improved fungicides remains a challenging task in
36
agrochemical industry.
cubensis)
and
cucumber
anthracnose
(Colletotrichum
37
Natural product-based libraries are expected to provide a rich source for new
38
fungicides discovery.8 Stilbenoid trans-Resveratrol is a phytoalexin being produced
39
in response to environmental stresses such as pathogenic infections.9,
40
fungicidal activity of stilbenes against plant fungal diseases was first reported by
41
Langcake and Pryce.11 And later, many naturally and synthetically derived stilbenes
42
were reported to exhibit remarkable biological properties.12-15 Biological screening
43
has revealed that the trans-olefin structure of stilbene skeleton plays an important
44
role in their bioactivity,16 and applications of those stilbene derivatives appeared to 3
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be dependent on the electron-donating or -withdrawing nature of their functional
46
groups.17 However, little information is available concerning the fungcidal activity of
47
stilbenes against cucurbit phytopathogens such as C. lagenarium. On the other hand,
48
1,3,4-Oxadiazoles are known as the biologically active nitrogen-containing
49
heterocyclic molecules. Derivatives of 1,3,4-oxadiazole are also shown to possess
50
various activities such as antibacterial,18,
51
anti-inflammatory,23 and anticancer,24 which are widely used as a scaffold in
52
agricultural and medicinal chemistry. All these findings inspired us to modify the
53
bioactive stilbene skeleton with functional motifs as potential agrochemical leads for
54
plant disease control.
19
antifungal,20,
21
insecticidal,22
55
In the light of the above reports, we herein designed and synthesized a series of
56
novel stilbene derivatives via an efficient approach (Figure 1). Their in vivo
57
fungicidal activities against P. cubensis, C. lagenarium, and S. cucurbitacearum
58
(Septoria cucurbitacearum from Cucurbita moschata Duch.) were evaluated for the
59
first time. To further study the influence of different substituents on fungicidal
60
activity of stilbene derivatives, we performed the CoMFA modeling to gain insights
61
into a deeper understanding of the quantitative structure–activity relationships.
62 63
MATERIALS AND METHODS
64
All chemicals and reagents were commercially available and used without
65
further purification. All solvents were dried and redistilled prior to use. Melting
66
points were determined on an OPM100 OptiMelt apparatus and were uncorrected. 4
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FT-IR spectra were measured as KBr pellets on a Bruker Vector 33. 1H NMR and 13C
68
NMR spectra were recorded in CDCl3 on a Bruker AVANCE-400 MHz NMR
69
spectrometer using TMS as an internal standard. Mass spectra were obtained with a
70
Bruker Esquire HCTplus spectrometer (APCI/ESI). Purity of the compounds were
71
confirmed by thin layer chromatography (TLC) on silica gel ‘G’-coated glass plates
72
and spots were visualized under UV irradiation.
73
General Synthetic Procedures for Title Compounds 4a-4v. The title
74
compounds were synthesized according to our previously reported procedure.25 The
75
desired starting materials were prepared by esterification of 4-methylbenzoic acid
76
followed by treatment with hydrazine hydrate in absolute ethanol. The data for target
77
compounds 4a-4v is shown below, and the detailed procedures for intermediates 1-3
78
and target compounds are available in Supporting Information.
79
Data for 4a: light yellow solid; yield 62.5%; m.p.: 156-157°C; 1H-NMR (400
80
MHz, CDCl3) δ 8.13 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.67 (d, J = 8.3 Hz, 2H, C6H4
81
3,5-H), 7.56 (d, J = 7.5 Hz, 2H, C6H5 2,6-H), 7.45 – 7.36 (m, 4H, C6H5 3,5-H C6H2
82
2,6-H ), 7.32 (t, J = 7.3 Hz, 1H, C6H5 4-H), 7.25 (d, J = 16.3 Hz, 1H, CH=CH), 7.15
83
(d, J = 16.3 Hz, 1H, CH=CH), 4.00 (s, 6H, OCH3), 3.96 (s, 3H, OCH3);
84
(101 MHz, CDCl3) δ 164.42, 153.73, 141.28, 140.76, 136.71, 131.07, 128.81, 128.29,
85
127.39, 127.28, 127.00, 126.80, 122.56, 119.00, 104.31, 61.04, 56.45; HRMS (ESI):
86
m/z cacld for C25H23N2O4: (M+H)+ 415.1652, found 415.1650.
13
C-NMR
87
Data for 4b: yellow solid; yield 76.9%; m.p.: 180-181°C; 1H-NMR (400 MHz,
88
CDCl3) δ 8.11 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.63 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 5
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7.51 (m, 2H, C6H4 2,6-H), 7.36 (s, 2H, C6H2 2,6-H), 7.18 (d, J = 16.3 Hz, 1H,
90
CH=CH), 7.13-6.97 (3H, C6H4 3,5-H, CH=CH ), 3.99 (s, 6H, OCH3), 3.95 (s, 3H,
91
OCH3);
92
140.57, 132.91, 129.78, 128.34, 127.27, 127.16, 126.91, 122.56, 118.93, 115.79,
93
115.68, 104.31, 61.02, 56.44; HRMS (ESI): m/z cacld for C25H22FN2O4 (M+H)+
94
433.1558, found 433.1556.
13
C-NMR (101 MHz, CDCl3) δ 164.41, 164.35, 163.92, 153.72, 141.30,
95
Data for 4c: light yellow solid; yield 69.6%; m.p.: 178-179°C; 1H-NMR (400
96
MHz, CDCl3) δ 8.14 (d, J = 8.2 Hz, 2H, C6H4 2,6-H), 7.70 (t, 3H, C6H4 3,5-H, C6H4
97
5-H), 7.64 (d, J = 16.3 Hz, 1H, CH=CH), 7.42 (d, J = 7.8 Hz, 1H, C6H4 2-H), 7.37 (s,
98
2H, C6H2 2,6-H), 7.32 – 7.21 (m, 2H, C6H4 3,4-H), 7.12 (d, J = 16.3 Hz, 1H,
99
CH=CH), 3.98 (s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
100
164.46, 164.33, 153.72, 141.29, 140.40, 134.80, 133.74, 129.95, 129.89, 129.14,
101
127.32, 127.29, 127.02, 126.99, 126.58, 122.98, 118.96, 104.30, 61.03, 56.44;
102
HRMS (ESI): m/z cacld for C25H22ClN2O4 (M+H)+ 449.1263, found 449.1257.
103
Data for 4d: light yellow solid; yield 80.6%; m.p.: 176-177°C; 1H-NMR (400
104
MHz, CDCl3) δ 8.11 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 8.4 Hz, 2H, C6H4
105
3,5-H), 7.52 (s, 1H, C6H4 5-H), 7.38 (d, J = 7.5 Hz, 1H, C6H4 2-H), 7.35 (s, 2H,
106
C6H2 2,6-H), 7.29 (t, J = 7.7 Hz, 1H, C6H4 3-H), 7.26 (s, 1H, C6H4 4-H), 7.13 (s, 2H,
107
CH=CH), 3.96 (s, 6H, OCH3), 3.92 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
108
164.45, 164.28, 153.71, 141.29, 140.12, 138.58, 134.77, 129.99, 129.45, 128.76,
109
128.11, 127.28, 127.13, 126.53, 125.01, 122.95, 118.94, 104.29, 61.02, 56.43;
110
HRMS (ESI): m/z cacld for C25H22ClN2O4 (M+H)+ 449.1263, found 449.1257. 6
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Data for 4e: light yellow solid; yield 72.5%; m.p.: 209-210°C; 1H-NMR (400
112
MHz, CDCl3) δ 8.09 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.62 (d, J = 8.3 Hz, 2H, C6H4
113
3,5-H), 7.45 (d, J = 8.5 Hz, 2H, C6H4 2,6-H), 7.34 (s, 2H, C6H2 2,6-H), 7.33 (d, J =
114
8.5 Hz, 2H, C6H4 3,5-H), 7.15 (d, J = 16.3 Hz, 1H, CH=CH), 7.07 (d, J = 16.3 Hz,
115
1H, CH=CH), 3.97 (s, 6H, OCH3), 3.94 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3)
116
δ 164.43, 164.32, 153.71, 141.30, 140.34, 135.21, 133.89, 129.64, 128.97, 127.94,
117
127.27, 127.02, 122.77, 118.93, 104.30, 61.02, 56.43; HRMS (ESI): m/z cacld for
118
C25H22ClN2O4 (M+H)+ 449.1263, found 449.1260.
119
Data for 4f: light yellow solid; yield 62.7%; m.p.: 193-194°C; 1H-NMR (400
120
MHz, CDCl3) δ 8.15 (d, J = 7.8 Hz, 2H, C6H4 2,6-H), 7.70 (d, J = 7.8 Hz, 2H, C6H4
121
3,5-H), 7.65 (d, J = 8.5 Hz, 1H, C6H3 5-H), 7.56 (d, J = 16.3 Hz, 1H, CH=CH), 7.44
122
(d, J = 2.0 Hz, 1H, C6H3 2-H), 7.38 (s, 2H, C6H2 2,6-H), 7.30 (d, J = 2.0 Hz, 1H,
123
C6H3 3-H), 7.11 (d, J = 16.3 Hz, 1H, CH=CH), 4.01 (s, 6H, OCH3), 3.96 (s, 3H,
124
OCH3);
125
134.19, 133.44, 130.38, 129.72, 127.45, 127.39, 127.36, 127.30, 125.85, 123.20,
126
118.89, 104.36, 61.05, 56.48; HRMS (ESI): m/z cacld for C25H21Cl2N2O4 (M+H)+
127
483.0873, found 483.0874.
13
C-NMR (101 MHz, CDCl3) δ 164.52, 164.27, 153.74, 141.36, 140.09,
128
Data for 4g: light green solid; yield 71.0%; m.p.: 211-212°C; 1H -NMR (400 MHz,
129
CDCl3) δ 8.12 (d, J = 8.0 Hz, 2H, C6H4 2,6-H), 7.63 (m, 3H, C6H4 3,5-H, C6H4 2-H),
130
7.47 – 7.41 (m, 1H, C6H4 3-H), 7.40 – 7.31 (m, 3H, C6H2 2,6-H, C6H4 6-H), 7.15 –
131
7.06 (m, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.96 (s, 3H, OCH3);
132
MHz, CDCl3) δ 164.50, 164.24, 153.73, 141.33, 139.85, 136.84, 132.98, 131.85, 7
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130.68, 129.22, 128.36, 128.31, 127.32, 127.18, 125.86, 123.14, 118.91, 104.31,
134
61.04, 56.46; HRMS (ESI): m/z cacld for C25H21Cl2N2O4 (M+H)+ 483.0873, found
135
483.0875.
136
Data for 4h: light yellow solid; yield 72.6%; m.p.: 176-177°C; 1H-NMR (400
137
MHz, CDCl3) δ 8.16 (d, J = 8.2 Hz, 2H, C6H4 2,6-H), 7.72 (d, J = 8.2 Hz, 3H, C6H4
138
3,5-H, C6H4 5-H), 7.67 – 7.58 (m, 3H, CH=CH, C6H4 2-H), 7.43 – 7.32 (m, 3H,
139
C6H2 2,6-H, C6H4 3-H), 7.18 (t, J = 7.0 Hz, 1H, C6H4 4-H), 7.10 (d, J = 16.2 Hz, 1H,
140
CH=CH), 4.01 (s, 6H, OCH3), 3.97 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
141
164.47, 164.33, 153.72, 141.28, 140.34, 136.51, 133.20, 130.07, 129.67, 129.38,
142
127.66, 127.33, 127.31, 126.82, 124.41, 123.00, 118.96, 104.31, 61.04, 56.45;
143
HRMS (APCI): m/z cacld for C25H22BrN2O4 (M+H)+ 493.0757, found 493.0759.
144
Data for 4i: yellow solid; yield 80.8%; m.p.: 213-214°C; 1H-NMR (400 MHz,
145
CDCl3) δ 8.09 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.62 (d, J = 8.3 Hz, 2H, C6H4 3,5-H),
146
7.49 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.38 (d, J = 8.4 Hz, 2H, C6H4 3,5-H), 7.34 (s,
147
2H, C6H2 2,6-H), 7.13 (d, J = 16.4 Hz, 1H, CH=CH), 7.08 (d, J = 16.4 Hz, 1H,
148
CH=CH), 3.97 (s, 6H, OCH3), 3.94 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ
149
164.44, 164.31, 153.72, 141.32, 140.32, 135.64, 131.93, 129.70, 128.21, 128.06,
150
127.29, 127.04, 122.78, 122.09, 118.90, 104.31, 61.03, 56.45; HRMS (APCI): m/z
151
cacld for C25H22BrN2O4 (M+H)+ 493.0757, found 493.0754.
152
Data for 4j: light green solid; yield 77.6%; m.p.: 205-206°C; 1H-NMR (400 MHz,
153
CDCl3) δ 8.11 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.98 (d, J = 8.2 Hz, 1 H, C6H4 5-H),
154
7.76 (d, J = 7.7 Hz, 1 H, C6H4 2-H), 7.73 – 7.59 (m, 4H, C6H4 3,5-H, CH=CH, C6H4 8
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3-H), 7.43 (t, J = 7.7 Hz, 1H, C6H4 4-H), 7.35 (s, 2H, C6H2 2,6-H), 7.08 (d, J = 16.1
156
Hz, 1H, CH=CH), 3.98 (s, 6H, OCH3), 3.94 (s, 3H, OCH3);
157
CDCl3) δ 164.51, 164.18, 153.71, 148.02, 141.30, 139.74, 133.25, 132.44, 132.37,
158
128.53, 128.25, 127.57, 127.30, 125.83, 124.87, 123.45, 118.86, 104.28, 61.01,
159
56.43; HRMS (APCI): m/z cacld for C25H22N3O6 (M+H)+ 460.1503, found
160
460.1505.
13
C-NMR (101 MHz,
161
Data for 4k: light yellow solid; yield 87.5%; m.p.: 236-237°C; 1H-NMR (400
162
MHz, CDCl3) δ 8.39 (s, 1H, C6H4 6-H), 8.13 (m, 3H, C6H4 3,5-H, C6H4 4-H), 7.82
163
(d, J = 7.8 Hz, 1H, C6H4 2-H), 7.68 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 7.55 (t, J = 8.0
164
Hz, 1H, C6H4 3-H), 7.36 (s, 2H, C6H2 2,6-H), 7.25 (s, 2H, CH=CH), 3.98 (s, 6H,
165
OCH3), 3.94 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ 164.55, 164.19, 153.74,
166
148.77, 141.39, 139.58, 138.52, 132.48, 130.41, 129.73, 128.33, 127.39, 127.36,
167
123.42, 122.59, 121.10, 118.83, 104.35, 61.04, 56.46; HRMS (APCI): m/z cacld for
168
C25H22N3O6 (M+H)+ 460.1503, found 460.1507.
169
Data for 4l: light green solid; yield 88.1%; m.p.: 271-272°C; 1H NMR (400 MHz,
170
CDCl3) δ 8.26 (d, J = 8.8 Hz, 2H, C6H4 3,5-H), 8.18 (d, J = 8.3 Hz, 2H, C6H4 2,6-H),
171
7.74 - 7.65 (m, 4H, C6H4 2,6-H C6H4 3,5-H), 7.38 (s, 2H, C6H2 2,6-H), 7.30 (d, J =
172
5.7 Hz, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (APCI): m/z
173
cacld for C25H22N3O6 (M+H)+ 460.1503, found 460.1507.
174
Data for 4m: light yellow solid; yield 60.9%; m.p.: 145-146°C; 1H-NMR (400
175
MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 2H, C6H4 2,6-H), 7.66 (d, J = 8.1 Hz, 2H, C6H4
176
3,5-H), 7.61 (d, J = 4.5 Hz, 1H, C6H4 2-H), 7.58 (d, J = 3.9 Hz, 1H, C6H4 5-H), 7.35 9
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(s, 2H, C6H2 2,6-H), 7.27 (m, 1H, C6H4 4-H), 7.14 (d, J = 16.5 Hz, 1H, CH=CH),
178
6.98 (t, J = 7.5 Hz, 1H, C6H4 3-H), 6.91 (d, J = 8.3 Hz, 1H, CH=CH), 3.97 (s, 6H,
179
OCH3), 3.94 (s, 3H, OCH3), 3.90 (s, 3H, OCH3);
180
164.51, 164.35, 157.16, 153.70, 141.45, 141.20, 129.37, 127.75, 127.19, 127.01,
181
126.68, 125.95, 125.77, 122.25, 120.81, 119.07, 111.02, 104.27, 61.03, 56.43, 55.54;
182
HRMS (ESI): m/z cacld for C26H25N2O5 (M+H)+ 445.1758, found 445.1761.
13
C-NMR (101 MHz, CDCl3) δ
183
Data for 4n: light yellow solid; yield 61.5%; m.p.: 131-132°C; 1H-NMR (400
184
MHz, CDCl3) δ 8.11 (d, J = 7.2 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 7.3 Hz, 2H, C6H4
185
3,5-H), 7.37 (s, 2H, C6H2 2,6-H), 7.34 – 7.26 (m, 1H, C6H4 3-H), 7.19 (d, J = 16.3
186
Hz, 1H, CH=CH), 7.16 - 7.03 (m, 3H, C6H4 2-H, C6H4 5-H, CH=CH), 6.86 (d, J =
187
8.1 Hz, 1H, C6H4 4-H), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3), 3.86 (s, 3H, OCH3);
188
13
189
138.10, 131.00, 129.76, 127.61, 127.29, 127.03, 122.39, 119.50, 118.73, 113.94,
190
112.06, 104.36, 61.03, 56.45, 55.27; HRMS (ESI): m/z cacld for C26H25N2O5
191
(M+H)+ 445.1758, found 445.1765.
C-NMR (101 MHz, CDCl3) δ 164.37, 164.34, 159.96, 153.71, 141.39, 140.74,
192
Data for 4o: light yellow solid; yield 77.3%; m.p.: 190-191°C; 1H-NMR (400
193
MHz, CDCl3) δ 8.08 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.61 (d, J = 8.3 Hz, 2H, C6H4
194
3,5-H), 7.47 (d, J = 8.7 Hz, 2H, C6H4 2,6-H), 7.35 (s, 2H, C6H2 2,6-H), 7.18 (d, J =
195
16.3 Hz, 1H, CH=CH), 6.99 (d, J = 16.3 Hz, 1H, CH=CH), 6.90 (d, J = 8.7 Hz, 2H,
196
C6H4 3,5-H), 3.97 (s, 6H, OCH3), 3.92 (s, 3H, OCH3), 3.83 (s, 3H, OCH3); 13C-NMR
197
(101 MHz, CDCl3) δ 164.48, 164.34, 159.83, 153.70, 141.21, 141.12, 130.59, 129.49,
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128.09, 127.22, 126.68, 125.22, 122.09, 119.05, 114.26, 104.26, 61.02, 56.43, 55.33;
199
HRMS (APCI): m/z cacld for C26H25N2O5 (M+H)+ 445.1758, found 445.1767.
200
Data for 4p: light green solid; yield 81.1%; m.p.: 187-188°C; 1H-NMR (400 MHz,
201
CDCl3) δ 8.11 (d, J = 8.1 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 8.1 Hz, 2H, C6H4 3,5-H),
202
7.36 (s, 2H,C6H2), 7.15 (d, J = 16.2 Hz, 1H, CH=CH), 7.03 (d, J = 16.2 Hz, 1H,
203
CH=CH), 6.76 (s, 2H, C6H2 2,6-H), 3.97 (s, 6H, OCH3), 3.92 (d, 9H, OCH3), 3.87 (s,
204
3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ 164.40, 164.39, 153.70, 153.47, 141.28,
205
140.64, 138.56, 132.40, 130.99, 127.25, 126.88, 126.79, 122.46, 118.96, 104.28,
206
103.97, 61.00, 60.96, 56.43, 56.17; HRMS (ESI): m/z cacld for C28H29N2O7 (M+H)+
207
505.1969, found 505.1970.
208
Data for 4q: light yellow solid; yield 67.3%; m.p.: 182-183°C; 1H-NMR (400
209
MHz, CDCl3) δ 8.21 (d, J = 8.2 Hz, 1H, naphthalene-H), 8.11 (d, J = 8.2 Hz, 2H,
210
C6H4 2,6-H), 7.97 (d, J = 16.0 Hz, 1H, CH=CH), 7.86 (d, J = 7.9 Hz, 1H,
211
naphthalene-H), 7.81 (d, J = 8.2 Hz, 1H, naphthalene-H), 7.74 (d, J = 7.1 Hz, 1H,
212
naphthalene-H), 7.70 (d, J = 8.2 Hz, 2H, C6H4 3,5-H), 7.59 – 7.45 (m, 3H,
213
naphthalene-H), 7.33 (s, 2H, C6H2 2,6-H), 7.14 (d, J = 16.0 Hz, 1H, CH=CH), 3.97
214
(s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13C-NMR (101 MHz, CDCl3) δ 164.39, 164.35,
215
153.68, 141.23, 140.89, 134.22, 133.75, 131.33, 130.25, 128.72, 128.65, 127.93,
216
127.26, 127.14, 126.32, 125.96, 125.65, 123.82, 123.52, 122.66, 118.96, 104.25,
217
61.02, 56.41; HRMS (APCI): m/z cacld for C29H25N2O6 (M+H)+ 465.1809, found
218
465.1806.
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Data for 4r: yellow solid; yield 76.5%; m.p.: 191-192°C; 1H-NMR (400 MHz,
220
CDCl3) δ 8.46 (s, 1H, anthracen-H), 8.41-8.33(m, 2H, anthracen-H), 8.24 (d, J = 8.3
221
Hz, 2H, C6H4 2,6-H), 8.14 – 8.01 (m, 3H, CH=CH, anthracen-H), 7.85 (d, J = 8.3 Hz,
222
2H, C6H4 3,5-H), 7.57-7.49 (m , 4H, anthracen-H), 7.42 (s, 2H, C6H2 2,6-H), 7.05 (d,
223
J = 16.6 Hz, 1H, CH=CH), 4.02 (s, 6H, OCH3), 3.98 (s, 3H, OCH3); HRMS (ESI):
224
m/z cacld for C33H27N2O4 (M+H)+ 515.1965, found 515.1964.
225
Data for 4s: yellow solid; yield 70.1%; m.p.: 212-213°C; 1H-NMR (400 MHz,
226
CDCl3) δ 8.02 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.54 (d, J = 8.3 Hz, 2H, C6H4 3,5-H),
227
7.39 (d, J = 8.7 Hz, 2H, C6H4 2,6-H), 7.31 (s, 2H, C6H2 2,6-H), 7.11 (d, J = 16.2 Hz,
228
1H, CH=CH), 6.87 (d, J = 16.2 Hz, 1H, CH=CH), 6.67 (d, J = 8.6 Hz, 2H, C6H4
229
3,5-H), 3.94 (s, 6H, OCH3), 3.91 (s, 3H, OCH3), 2.95 (s, 6H, CH3); 13C-NMR (101
230
MHz, CDCl3) δ 164.60, 164.23, 153.68, 150.44, 141.73, 141.14, 131.25, 128.01,
231
127.17, 126.36, 122.81, 121.45, 119.13, 112.32, 104.23, 61.02, 56.43, 40.34; HRMS
232
(ESI): m/z cacld for C27H28N3O4 (M+H)+ 458.2074, found 458.2071.
233
Data for 4t: light yellow solid; yield 72.8%; m.p.: 196-197°C; 1H-NMR (400
234
MHz, CDCl3) δ 8.64 (d, J = 4.6 Hz, 1H, pyridine-H), 8.15 (d, J = 8.1 Hz, 2H, C6H4
235
2,6-H), 7.78 – 7.67 (m, 4H, C6H4 3,5-H pyridine-H, CH=CH), 7.45 (d, J = 7.6 Hz,
236
1H, pyridine-H), 7.37 (s, 2H, C6H2 2,6-H), 7.31 (d, J = 16.9 Hz, 1H, CH=CH), 7.26
237
– 7.19 (m, 1H, pyridine-H), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); 13C NMR (101
238
MHz, CDCl3) δ 164.51, 164.32, 154.73, 153.73, 149.43, 141.27, 139.91, 137.04,
239
131.80, 129.62, 127.68, 127.30, 123.33, 122.65, 118.96, 104.29, 61.03, 56.44;
240
HRMS (ESI): m/z cacld for C24H22N3O4 (M+H)+ 416.1605, found 416.1610. 12
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Data for 4u: light green solid; yield 69.3%; m.p.: 182-183°C; 1H NMR (600 MHz,
242
CDCl3) δ 8.77 (d, J = 2.0 Hz, 1H, pyridine-H), 8.53 (d, J = 4.8 Hz, 1H, pyridine-H),
243
8.14 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.88 (d, J = 7.9 Hz, 1H, pyridine-H), 7.68 (d, J
244
= 8.4 Hz, 2H, C6H4 3,5-H), 7.37 (s, 2H, C6H2 2,6-H), 7.33 (q, 1H, pyridine-H), 7.20
245
(s, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (ESI): m/z cacld
246
for C24H22N3O4 (M+H)+ 416.1605, found 416.1606.
247
Data for 4v: light green solid; yield 70.2%; m.p.: 221-222°C; 1H NMR (600 MHz,
248
CDCl3) δ 8.63 (d, J = 4.6 Hz, 2H, pyridine-H), 8.18 (d, J = 8.4 Hz, 2H, C6H4 2,6-H),
249
7.71 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 7.44 (d, J = 4.7 Hz, 2H, pyridine-H), 7.37 (d, J
250
= 16.2 Hz, 3H, C6H2 2,6-H, CH=CH), 7.16 (d, J = 16.3 Hz, 1H, CH=CH), 4.00 (s,
251
6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (ESI): m/z cacld for C24H22N3O4 (M+H)+
252
416.1605, found 416.1603.
253
In Vivo Bioassays. Pot culture test method was adopted to evaluate fungicidal
254
activities of the title compounds against P. cubensis, C. lagenarium, and S.
255
cucurbitacearum in vivo at 600 µg/mL according to the references with slight
256
modifications.26,
257
Carbendazim WP were evaluated as controls against the above mentioned fungi at
258
the same condition. All compounds were dissolved in dimethylformamide (DMF),
259
and then distilled water (containing 0.1% Tween 80) was added to the solution to
260
generate a tested concentration of 600 µg/mL, in each of which the final
261
concentration of DMF was < 1% (v/v). P. cubensis sporangia were obtained by
262
washing off the leaves of highly infected cucumber plants that were kept overnight
27
Two commercial fungicides, 80% Mancozeb WP and 80%
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(above 80% relative humidity). C. lagenarium and S. cucurbitacearum were cultured
264
on the potato dextrose agar (PDA). Spores of those two fungal pathogens were
265
removed from the PDA plates. All of the spore suspensions were adjusted to the
266
desired concentrations prior to use. Germination was conducted by soaking cucurbit
267
seeds in water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °C in
268
an incubator.
269
When the cucurbit plants used for inoculations were at the stage of two seed
270
leaves, tested compounds and commercial fungicides were sprayed with a
271
hand-sprayer on the upper leaf surface of seed leaves at the standard concentration of
272
600 µg/mL. Distilled water without compounds or commercial fungicides treatments
273
was set as a blank control. After the solution on leaves were air-dried, inoculations of
274
P. cubensis, C. lagenarium and S. cucurbitacearum were carried out by spraying a
275
spore suspension with the concentration of 1×105, 1×106, 1×106 spores/mL of water,
276
respectively. Then the plants were maintained at 24 ± 1 °C and above 80% relative
277
humidity. Three replicates were used per treatment, and the experiment was repeated
278
three times.
279
The fungicidal activity was evaluated visually when the non-treated cucurbit plant
280
(blank control) fully developed symptoms. The percentage of diseased area on
281
inoculated treated leaves was assessed and compared to that of non-treated ones to
282
determine the average disease index. The relative control efficacy of compounds
283
compared to the blank assay was calculated via the following equation:
284
I (%) = [(CK−PT)/CK] ×100% 14
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Where I is the relative control efficacy, CK is the average disease index during the
286
blank assay, and PT is the average disease index after treatment during testing.
287
3D-QSAR Analysis. All molecular modeling studies were performed using the
288
Tripos SYBYL 7.3.5 software package.28 The comparative molecular field analysis
289
(CoMFA) model was derived from the antifungal activity of 22 compounds (17
290
compounds for training sets, 5 compounds for test sets, respectively) against C.
291
lagenarium at 600 µg/mL. The fungicidal activity was converted into D values by
292
the formula D=log{[I/(100−I)] −MW},29 where I is the percent control efficacy and
293
MW is the molecular weight of the tested compounds. Compound 4k was chosen as
294
a template for superimposition because of the highest control efficacy. Each structure
295
was built using the SKETCH option and fully geometry-optimized using a conjugate
296
gradient procedure based on the TRIPOS force field and Gasteiger and Hückel
297
charges.
298
The steric and electrostatic interaction fields for CoMFA were calculated using
299
the SYBYL default parameters: 2.0 Å grid points spacing, a sp3 carbon probe atom
300
with a charge of +1.0 and a van der Waals radius of 1.52 Å. Steric and electrostatic
301
fields were scaled by the CoMFA-STD method in SYBYL with default cut-off
302
energy of 30 kcal/mol. A partial least-squares (PLS) approach was used to derive the
303
3D QSAR model, in which the CoMFA descriptors were used as independent
304
variables and the experimental D values were used as dependent variables. The
305
cross-validation with the leave-one-out (LOO) option and the SAMPLS program
306
was carried out to obtain the optimal number of components (N) and cross-validated 15
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coefficient (q2). After the optimal number of components was determined, a
308
non-cross-validated analysis was performed without column filtering to obtain
309
regression coefficients (r2) and its standard error (S) and F-test value (F) for the
310
model evaluation.
311
RESULTS AND DISCUSSION
312
Synthesis. The synthetic route of the title compounds is outlined in Figure 1. A
313
series of novel stilbene derivatives containing an 1,3,4-oxadiazole moiety were
314
synthesized in good yields by bromination of N-Bromosuccinimide (NBS), Arbuzov
315
reaction, and Wittig−Horner reaction. The key step involved the formation of the
316
stilbenes which were efficiently generated by the reaction of phosphonate 3 with
317
various aldehydes via Wittig–Horner reaction under mild reaction conditions. The
318
chemical structures of the target compounds were confirmed by FT-IR, 1H NMR, 13C
319
NMR, and HRMS. Notably, the 1H NMR spectra of target compounds show two fine
320
doublets as well as coupling constant (16.7-16.0 Hz) corresponding to the olefinic
321
protons (CH=CH) from trans-stilbene, which are further confirmed by the FT-IR
322
spectra.30 All spectral and analytical data are consistent with the assigned structures.
323
Fungicidal Activity. The in vivo fungicidal activity of compounds 4a-4v against
324
P. cubensis, C. lagenarium and S. cucurbitacearum at a concentration of 600 µg/mL
325
are listed in Table 1. The results of bioassays are compared with those obtained from
326
two commercial fungicides. It was worthy to note that all of the tested compounds
327
were found safe for the cucurbit plants. As indicated in Table 1, some of the title
328
compounds showed broad spectrum and comparable fungicidal activities against the 16
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tested fungi. Four main conclusions were drawn from the comparison of the
330
fungicidal activities of title compounds against test fungi to those of commercial
331
fungicides: (a) Compounds 4s exhibited a significant inhibition effect against
332
cucumber P. cubensis with control efficacy of 71.38 ± 1.47%, which was comparable
333
to that of 80% Mancozeb WP (70.13 ± 3.25%). While compounds 4d, 4l and 4v
334
showed relatively high antifungal activity against cucumber P. cubensis (65.01 ±
335
1.81 and 63.42 ± 4.60%, respectively) compared with the fungicide 80% Mancozeb
336
WP. (b) Compounds 4b, 4d, 4f, 4i, 4k and 4l showed prominent activity against C.
337
lagenarium as compared to the control 80% Carbendazim WP. In particularly, the
338
activity of compounds 4d and 4k were 82.43 ± 4.66 and 83.82 ± 2.69%, respectively,
339
which were similar to that of 80% Carbendazim WP (81.57 ± 2.19%). (c) Most of
340
the
341
cucurbitacearum. Notably, compounds 4b, 4h and 4i, whose efficacy rates were
342
72.05 ± 0.96, 69.34 ± 0.93 and 67.68 ± 1.43%, respectively, were found to be almost
343
the same activity level as that of 80% Carbendazim WP (69.02 ± 0.47%). (d) Some
344
compounds showed significant bioactivity variances against the different fungi. For
345
example, compound 4g exhibited moderate activity (44.81 ± 3.85%) against C.
346
lagenarium,
347
cucurbitacearum was only 8.03 ± 2.78%, and even showed a negative effect on P.
348
cubensis. These results indicated that compounds 4b, 4d, 4i, 4k and 4l were the most
349
promising candidates for crop protection because of their broad spectrum and
350
remarkably high activities against all tested fungi.
compounds
exhibited
whereas
the
fair
to
control
moderate
efficacy
of
control
efficacy
compound
17
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against
against
S.
S.
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351
CoMFA Analysis. The statistical results of CoMFA model are summarized in
352
Table 2. The internal validation of LOO cross-validated q2 and non-cross validated
353
coefficient r2 are commonly applied as the criterion of robustness and predictive
354
ability of a QSAR model. The statistically recommendatory values for a satisfactory
355
QSAR model are q2 > 0.5 and r2 > 0.8.31 As listed in Table 2, a good predictive
356
CoMFA model with the LOO cross-validated coefficient q2=0.516 and the
357
correlation coefficient r2=0.920 was established. The predicted D values are
358
generally in good agreement with the experimental data as shown in Figure 2. The
359
standard error (S=0.114) and F-test value (F=25.186) further validate the developed
360
model.
361
The contribution steric and electrostatic fields are 56.2 and 43.8% for the QSAR
362
model, respectively. CoMFA contour maps are displayed in Figure 3 to visualise how
363
steric and electrostatic fields contribute to the bioactivity of test compounds. As
364
shown in steric field contour map (Figure 3a). The green contour surrounding
365
meta-position of the benzene ring indicates that a bulky group would be favorable
366
for higher fungicidal activity, while the yellow contour represents region of
367
unfavorable steric effect. For example, compounds 4k and 4d bearing bulkier
368
substituents at the meta-position displayed higher fungicidal activity. For
369
electrostatic contours map (Figure 3b), the negatively charged favorable red regions
370
are
371
electron-withdrawing substituents enhance the bioactivity, whereas a positively
372
charged blue region around para-position is favored. Such as compound 4o
observed
at
meta-position
of
the
benzene
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ring,
indicating
that
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373
(para-position)
displayed
higher
activity
than
those
of
compounds
374
(ortho-position) and 4n (meta-position). Analysis of CoMFA contour plots revealed
375
that substitutions at the meta-position, the bulky groups and electron-withdrawing
376
groups (e.g., -NO2, -Cl) are favorable to increase the bioactivity.
4m
377
The results obtained from 3D-QSAR provided reliable clues for mechanistic
378
study and designing of optimized stilbene derivatives. On the basis of the in vivo
379
bioassay and 3D-QSAR analysis, it can be concluded that the variances (substituted
380
positions, electronic properties and steric effects) among substituents (Ar) on
381
stilbenes showed a significant relationship with fungicidal activity against the three
382
fungi. Furthermore, considering that the bioassay of title compounds was evaluated
383
in vivo, effects of metabolism and/or biotransformation within the plants or fungi
384
may contribute to the significant variations in fungicidal potency. It is reported that
385
the electron-withdrawing substituents of the phenyl moiety seem to enhance the
386
potency, which may be related to the fact that they tend to retard mechanisms of
387
oxidative metabolism occurring on (or close to) the benzene ring.32 This assumption
388
is consistent with the higher bioactivity of the title compounds with
389
electron-withdrawing substituents at meta-position proposed by the 3D-QSAR
390
analysis.
391
Discovery of phytopathogenic fungicidal agents with new modes of action and
392
eco-friendly properties is of great importance for crop protection. 33 In the present
393
study, 22-novel stilbene derivatives containing the 1,3,4-oxadiazole moiety and the
394
trimethoxybenzene were designed and synthesized. The approach was proved to be 19
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395
an efficient method to implement structural modification by combination of several
396
functional fragments. Bioassay results indicated that compounds 4b, 4d, 4i, 4k and
397
4l exhibited broad spectrum fungicidal activity in vivo against all tested fungi, some
398
of which were comparable with the commercial fungicides. Further field evaluations
399
on their biological efficacies, as well as crop toxicities and safety, are necessary
400
before they can be developed as fungicide candidates.
401 402
ASSOCIATED CONTENT
403
Supporting Information Available:
404
Synthetic procedures for intermediates 1-3 and target compounds. This material is
405
available free of charge via the Internet at http://pubs.acs.org.
406 407
Notes
408
The authors declare no competing financial interest.
409 410
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Funding
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This work was financially supported by the Fundamental Research Funds for the
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Central Universities (No.2012ZM0035) and Guangdong Provincial Natural Science
517
Foundation (No.04300531)
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Journal of Agricultural and Food Chemistry
FIGURES CAPTIONS Figure 1. General Synthetic Route for Target Compounds. 4a: Ar=phenyl; 4b: Ar=4-F phenyl; 4c: Ar=2-Cl phenyl; 4d: Ar=3-Cl phenyl; 4e: Ar=4-Cl phenyl; 4f: Ar=2,4-di-Cl phenyl; 4g: Ar=3,4-di-Cl phenyl; 4h: Ar=2-Br phenyl; 4i: Ar=4-Br phenyl; 4j: Ar=2-NO2 phenyl; 4k: Ar=3-NO2 phenyl; 4l: Ar=4-NO2 phenyl; 4m: Ar=2-methoxy phenyl; 4n: Ar=3-methoxy phenyl; 4o: Ar=4-methoxy phenyl; 4p: Ar=3,4,5-trimethoxy phenyl; 4q: Ar=1-naphthyl; 4r: Ar=9- anthryl; 4s: Ar=4- N,N-dimethylaniline; 4t: Ar=2- pyridyl; 4u: Ar=3- pyridyl; 4v: Ar=4- pyridyl. Figure 2. Plot of Experimental Values Versus Predicted Values for Training and Test Sets Based on CoMFA Model. Figure 3. Graphical Representation of CoMFA Analysis for Compound 4k. (a) Steric contour plots: Sterically favored areas are represented by green polyhedra, and sterically disfavored areas are represented by yellow polyhedra. (b) Electrostatic contour plots: Positive charge favored areas are represented by red polyhedra, and positive charge disfavored areas are represented by blue polyhedra.
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TABLES Table 1. Chemical Structures and Fungicidal Activities of Compounds 4a-4v at 600 µg/mL in Vivo.
Compd.
Ar
P. cubensis
control efficacy (%)a C. lagenarium S. cucurbitacearum
4a
58.77 ± 1.97
50.03 ± 4.01
55.17 ± 4.22
4b
52.20 ± 2.59
65.14 ± 3.52
72.05 ± 0.96
4c
32.66 ± 6.18
31.48 ± 4.10
9.34 ± 0.36
4d
65.01 ± 1.81
82.43 ± 4.66
32.03 ± 7.57
4e
36.96 ± 5.57
47.78 ± 1.15
4f
38.52 ± 4.06
63.83 ± 5.38
27.54 ± 8.20
4g
4.08 ± 4.17
44.81 ± 3.85
8.03 ± 2.78
19.08 ± 1.29
4h
8.37 ± 3.03
26.38 ± 1.16
69.34 ± 0.93
4i
54.87 ± 3.55
64.77 ± 2.10
67.68 ± 1.43
4j
48.34 ± 0.62
56.07 ± 3.45
35.36 ± 2.85
4k
50.13 ± 3.15
83.82 ± 2.69
46.83 ± 4.50
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Compd.
Ar
P. cubensis
Page 28 of 33
control efficacy (%)a C. lagenarium S. cucurbitacearum
4l
63.42 ± 4.60
66.72 ± 2.83
32.23 ± 1.57
4m
25.28 ± 5.99
33.12 ± 1.66
48.91 ± 1.54
4n
44.13 ± 4.56
37.90 ± 5.83
47.62 ± 3.37
4o
58.21 ± 5.91
56.93 ± 3.04
40.51 ± 4.43
4p
49.57 ± 1.12
61.92 ± 2.69
7.05 ± 1.39
4q
36.01 ± 0.51
37.98 ± 5.48
51.18 ± 4.76
4r
15.3 ± 2.77
14.29 ± 5.50
53.09 ± 4.36
4s
71.38 ± 1.47
30.26 ± 2.38
44.84 ± 4.22
4t
18.38 ± 1.59
52.88 ± 1.06
45.14 ± 6.89
4u
42.67 ± 2.51
37.58 ±1.98
40.27 ± 2.23
4v
62.35 ± 3.09
42.61 ± 1.92
34.24 ± 4.43
Fungicidesb
70.13 ± 3.25 A
81.57 ± 2.19 B
69.02 ± 0.47 B
a
b
Values represent means of three independent replicates ± SD.
Control fungicides: A, 80% Mancozeb WP; B, 80% Carbendazim WP.
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Table 2. The Statistical Parameters of CoMFA Model. Contribution (%)
CoMFA
N
q2
r2
S
F
compound
steric
electrostatic
5
0.516
0.920
0.114
25.186
4k
56.2
43.8
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FIGURES
Figure 1
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Figure 2
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Figure 3
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Table of Contents (TOC) Graphic:
(3.2×1.8 in.)
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