Synthesis, biological evaluation, and structure-activity relationship study of novel stilbene derivatives as potential fungicidal agents

Article abstract of DOI:10.1021/jf5052893

A total of 22 novel stilbene derivatives containing the 1,3,4-oxadiazole moiety and trimethoxybenzene were designed and synthesized. Their chemical structures were characterized by ¹H and ¹³C nuclear magnetic resonance, infrared, and high-resolution mass spectrometry. Bioassay results revealed that some of the title compounds showed potent in vivo fungicidal activities against three phytopathogenic fungi (Pseudoperonospora cubensis, Colletotrichum lagenarium, and Septoria cucurbitacearum) from cucurbits at 600 ～g/mL. Notably, compounds 4b, 4d, 4i, 4k, and 4l exhibited a broad spectrum and remarkably high activities against those fungi, some of which even showed a comparable control efficacy to that of the commercial fungicides. Three-dimensional quantitative structure-activity relationship based on comparative molecular field analysis with good predictive ability (q² = 0.516; r² = 0.920) was reasonably discussed. For the first time, the present work suggested that the stilbene derivatives containing the 1,3,4-oxadiazole moiety could be developed as potential fungicides for crop protection.

Full text of DOI:10.1021/jf5052893

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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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Page 1 of 33
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 Yatꢀsen University, Guangzhou, 510006, China
*Corresponding author, (Tel: + 86ꢀ20ꢀ 8711 0234; Fax: + 86ꢀ20ꢀ8711 0234;
Eꢀmail: he16221@163.com).
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ABSTRACT
Twentyꢀtwo novel stilbene derivatives containing the 1,3,4ꢀoxadiazole moiety and
the trimethoxybenzene were designed and synthesized. Their chemical structures
were characterized by 1H NMR, 13C NMR, IR, and HRMS. Bioassay results revealed
that some of the title compounds showed potent in vivo fungicidal activities against
three phytopathogenic fungi (Pseudoperonospora cubensis, Colletotrichum
lagenarium, and Septoria cucurbitacearum) from cucurbits at 600 ꢁg/mL. Notably,
compounds 4b, 4d, 4i, 4k and 4l exhibited broadꢀspectrum and remarkably high
activities against those fungi, some of which even showed a comparable control
efficacy to that of the commercial fungicides. Threeꢀdimensional quantitative
structure–activity relationship (3DꢀQSAR) based on comparative molecular field
analysis (CoMFA) with good predictive ability (q2=0.516, r2=0.920) was reasonably
discussed. For the first time, the present work suggested that the stilbene derivatives
containing 1,3,4ꢀoxadiazole moiety could be developed as potential fungicides for
crop protection.
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KEYWORDS: stilbene derivatives, 1,3,4ꢀoxadiazole, 3DꢀQSAR, fungicidal activity,
synthesis
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INTRODUCTION
Plant diseases caused by pathogenic fungi are a severe threat to fruits and
vegetables, which limit the crop production worldwide. Cucumber downy mildew
(Pseudoperonospora cubensis) and cucumber anthracnose (Colletotrichum
lagenarium) are two main devastating diseases found in cucumber, which are
widespread under both greenhouse and open field cultivation responsible for yield
reductions and food decays.1ꢀ4 For example, C. lagenarium can cause premature
plant death by reducing the photosynthetic surface area of 29–42%, resulting in
cucumber yield losses of 6–48%.5 Fungicide applications are still one of the most
effective solutions for controlling plant diseases in the current agricultural system.
However, conventional synthetic pesticides are far from satisfaction because of their
limited efficacy, and the increasing incidence of fungicide resistance.6, 7 Therefore,
the search for alternative improved fungicides remains a challenging task in
agrochemical industry.
Natural productꢀbased libraries are expected to provide a rich source for new
fungicides discovery.8 Stilbenoid transꢀResveratrol is a phytoalexin being produced
in response to environmental stresses such as pathogenic infections.9,
The
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fungicidal activity of stilbenes against plant fungal diseases was first reported by
Langcake and Pryce.11 And later, many naturally and synthetically derived stilbenes
were reported to exhibit remarkable biological properties.12ꢀ15 Biological screening
has revealed that the transꢀolefin structure of stilbene skeleton plays an important
role in their bioactivity,16 and applications of those stilbene derivatives appeared to
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be dependent on the electronꢀdonating or ꢀwithdrawing nature of their functional
groups.17 However, little information is available concerning the fungcidal activity of
stilbenes against cucurbit phytopathogens such as C. lagenarium. On the other hand,
1,3,4ꢀOxadiazoles are known as the biologically active nitrogenꢀcontaining
heterocyclic molecules. Derivatives of 1,3,4ꢀoxadiazole are also shown to possess
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various activities such as antibacterial,18,
antifungal,20,
insecticidal,22
antiꢀinflammatory,23 and anticancer,24 which are widely used as a scaffold in
agricultural and medicinal chemistry. All these findings inspired us to modify the
bioactive stilbene skeleton with functional motifs as potential agrochemical leads for
plant disease control.
In the light of the above reports, we herein designed and synthesized a series of
novel stilbene derivatives via an efficient approach (Figure 1). Their in vivo
fungicidal activities against P. cubensis, C. lagenarium, and S. cucurbitacearum
(Septoria cucurbitacearum from Cucurbita moschata Duch.) were evaluated for the
first time. To further study the influence of different substituents on fungicidal
activity of stilbene derivatives, we performed the CoMFA modeling to gain insights
into a deeper understanding of the quantitative structure–activity relationships.
MATERIALS AND METHODS
All chemicals and reagents were commercially available and used without
further purification. All solvents were dried and redistilled prior to use. Melting
points were determined on an OPM100 OptiMelt apparatus and were uncorrected.
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FTꢀIR spectra were measured as KBr pellets on a Bruker Vector 33. 1H NMR and 13C
NMR spectra were recorded in CDCl3 on a Bruker AVANCEꢀ400 MHz NMR
spectrometer using TMS as an internal standard. Mass spectra were obtained with a
Bruker Esquire HCTplus spectrometer (APCI/ESI). Purity of the compounds were
confirmed by thin layer chromatography (TLC) on silica gel ‘G’ꢀcoated glass plates
and spots were visualized under UV irradiation.
General Synthetic Procedures for Title Compounds 4a-4v. The title
compounds were synthesized according to our previously reported procedure.25 The
desired starting materials were prepared by esterification of 4ꢀmethylbenzoic acid
followed by treatment with hydrazine hydrate in absolute ethanol. The data for target
compounds 4a-4v is shown below, and the detailed procedures for intermediates 1-3
and target compounds are available in Supporting Information.
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Data for 4a: light yellow solid; yield 62.5%; m.p.: 156ꢀ157°C; HꢀNMR (400
MHz, CDCl3) δ 8.13 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH), 7.67 (d, J = 8.3 Hz, 2H, C6H4
3,5ꢀH), 7.56 (d, J = 7.5 Hz, 2H, C6H5 2,6ꢀH), 7.45 – 7.36 (m, 4H, C6H5 3,5ꢀH C6H2
2,6ꢀH ), 7.32 (t, J = 7.3 Hz, 1H, C6H5 4ꢀH), 7.25 (d, J = 16.3 Hz, 1H, CH=CH), 7.15
(d, J = 16.3 Hz, 1H, CH=CH), 4.00 (s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13CꢀNMR
(101 MHz, CDCl3) δ 164.42, 153.73, 141.28, 140.76, 136.71, 131.07, 128.81, 128.29,
127.39, 127.28, 127.00, 126.80, 122.56, 119.00, 104.31, 61.04, 56.45; HRMS (ESI):
m/z cacld for C25H23N2O4: (M+H)+ 415.1652, found 415.1650.
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Data for 4b: yellow solid; yield 76.9%; m.p.: 180ꢀ181°C; HꢀNMR (400 MHz,
CDCl3) δ 8.11 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH), 7.63 (d, J = 8.3 Hz, 2H, C6H4 3,5ꢀH),
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7.51 (m, 2H, C6H4 2,6ꢀH), 7.36 (s, 2H, C6H2 2,6ꢀH), 7.18 (d, J = 16.3 Hz, 1H,
CH=CH), 7.13ꢀ6.97 (3H, C6H4 3,5ꢀH, CH=CH ), 3.99 (s, 6H, OCH3), 3.95 (s, 3H,
OCH3); 13CꢀNMR (101 MHz, CDCl3) δ 164.41, 164.35, 163.92, 153.72, 141.30,
140.57, 132.91, 129.78, 128.34, 127.27, 127.16, 126.91, 122.56, 118.93, 115.79,
115.68, 104.31, 61.02, 56.44; HRMS (ESI): m/z cacld for C25H22FN2O4 (M+H)+
433.1558, found 433.1556.
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Data for 4c: light yellow solid; yield 69.6%; m.p.: 178ꢀ179°C; HꢀNMR (400
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MHz, CDCl3) δ 8.14 (d, J = 8.2 Hz, 2H, C6H4 2,6ꢀH), 7.70 (t, 3H, C6H4 3,5ꢀH, C6H4
5ꢀH), 7.64 (d, J = 16.3 Hz, 1H, CH=CH), 7.42 (d, J = 7.8 Hz, 1H, C6H4 2ꢀH), 7.37 (s,
2H, C6H2 2,6ꢀH), 7.32 – 7.21 (m, 2H, C6H4 3,4ꢀH), 7.12 (d, J = 16.3 Hz, 1H,
CH=CH), 3.98 (s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ
164.46, 164.33, 153.72, 141.29, 140.40, 134.80, 133.74, 129.95, 129.89, 129.14,
127.32, 127.29, 127.02, 126.99, 126.58, 122.98, 118.96, 104.30, 61.03, 56.44;
HRMS (ESI): m/z cacld for C25H22ClN2O4 (M+H)+ 449.1263, found 449.1257.
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Data for 4d: light yellow solid; yield 80.6%; m.p.: 176ꢀ177°C; HꢀNMR (400
MHz, CDCl3) δ 8.11 (d, J = 8.4 Hz, 2H, C6H4 2,6ꢀH), 7.64 (d, J = 8.4 Hz, 2H, C6H4
3,5ꢀH), 7.52 (s, 1H, C6H4 5ꢀH), 7.38 (d, J = 7.5 Hz, 1H, C6H4 2ꢀH), 7.35 (s, 2H,
C6H2 2,6ꢀH), 7.29 (t, J = 7.7 Hz, 1H, C6H4 3ꢀH), 7.26 (s, 1H, C6H4 4ꢀH), 7.13 (s, 2H,
CH=CH), 3.96 (s, 6H, OCH3), 3.92 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ
164.45, 164.28, 153.71, 141.29, 140.12, 138.58, 134.77, 129.99, 129.45, 128.76,
128.11, 127.28, 127.13, 126.53, 125.01, 122.95, 118.94, 104.29, 61.02, 56.43;
HRMS (ESI): m/z cacld for C25H22ClN2O4 (M+H)+ 449.1263, found 449.1257.
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Data for 4e: light yellow solid; yield 72.5%; m.p.: 209ꢀ210°C; HꢀNMR (400
MHz, CDCl3) δ 8.09 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH), 7.62 (d, J = 8.3 Hz, 2H, C6H4
3,5ꢀH), 7.45 (d, J = 8.5 Hz, 2H, C6H4 2,6ꢀH), 7.34 (s, 2H, C6H2 2,6ꢀH), 7.33 (d, J =
8.5 Hz, 2H, C6H4 3,5ꢀH), 7.15 (d, J = 16.3 Hz, 1H, CH=CH), 7.07 (d, J = 16.3 Hz,
1H, CH=CH), 3.97 (s, 6H, OCH3), 3.94 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3)
δ 164.43, 164.32, 153.71, 141.30, 140.34, 135.21, 133.89, 129.64, 128.97, 127.94,
127.27, 127.02, 122.77, 118.93, 104.30, 61.02, 56.43; HRMS (ESI): m/z cacld for
C25H22ClN2O4 (M+H)+ 449.1263, found 449.1260.
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Data for 4f: light yellow solid; yield 62.7%; m.p.: 193ꢀ194°C; HꢀNMR (400
MHz, CDCl3) δ 8.15 (d, J = 7.8 Hz, 2H, C6H4 2,6ꢀH), 7.70 (d, J = 7.8 Hz, 2H, C6H4
3,5ꢀH), 7.65 (d, J = 8.5 Hz, 1H, C6H3 5ꢀH), 7.56 (d, J = 16.3 Hz, 1H, CH=CH), 7.44
(d, J = 2.0 Hz, 1H, C6H3 2ꢀH), 7.38 (s, 2H, C6H2 2,6ꢀH), 7.30 (d, J = 2.0 Hz, 1H,
C6H3 3ꢀH), 7.11 (d, J = 16.3 Hz, 1H, CH=CH), 4.01 (s, 6H, OCH3), 3.96 (s, 3H,
OCH3); 13CꢀNMR (101 MHz, CDCl3) δ 164.52, 164.27, 153.74, 141.36, 140.09,
134.19, 133.44, 130.38, 129.72, 127.45, 127.39, 127.36, 127.30, 125.85, 123.20,
118.89, 104.36, 61.05, 56.48; HRMS (ESI): m/z cacld for C25H21Cl2N2O4 (M+H)+
483.0873, found 483.0874.
Data for 4g: light green solid; yield 71.0%; m.p.: 211ꢀ212°C; 1H ꢀNMR (400 MHz,
CDCl3) δ 8.12 (d, J = 8.0 Hz, 2H, C6H4 2,6ꢀH), 7.63 (m, 3H, C6H4 3,5ꢀH, C6H4 2ꢀH),
7.47 – 7.41 (m, 1H, C6H4 3ꢀH), 7.40 – 7.31 (m, 3H, C6H2 2,6ꢀH, C6H4 6ꢀH), 7.15 –
7.06 (m, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13CꢀNMR (101
MHz, CDCl3) δ 164.50, 164.24, 153.73, 141.33, 139.85, 136.84, 132.98, 131.85,
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130.68, 129.22, 128.36, 128.31, 127.32, 127.18, 125.86, 123.14, 118.91, 104.31,
61.04, 56.46; HRMS (ESI): m/z cacld for C25H21Cl2N2O4 (M+H)+ 483.0873, found
483.0875.
Data for 4h: light yellow solid; yield 72.6%; m.p.: 176ꢀ177°C; 1HꢀNMR (400
MHz, CDCl3) δ 8.16 (d, J = 8.2 Hz, 2H, C6H4 2,6ꢀH), 7.72 (d, J = 8.2 Hz, 3H, C6H4
3,5ꢀH, C6H4 5ꢀH), 7.67 – 7.58 (m, 3H, CH=CH, C6H4 2ꢀH), 7.43 – 7.32 (m, 3H,
C6H2 2,6ꢀH, C6H4 3ꢀH), 7.18 (t, J = 7.0 Hz, 1H, C6H4 4ꢀH), 7.10 (d, J = 16.2 Hz, 1H,
CH=CH), 4.01 (s, 6H, OCH3), 3.97 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ
164.47, 164.33, 153.72, 141.28, 140.34, 136.51, 133.20, 130.07, 129.67, 129.38,
127.66, 127.33, 127.31, 126.82, 124.41, 123.00, 118.96, 104.31, 61.04, 56.45;
HRMS (APCI): m/z cacld for C25H22BrN2O4 (M+H)+ 493.0757, found 493.0759.
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Data for 4i: yellow solid; yield 80.8%; m.p.: 213ꢀ214°C; HꢀNMR (400 MHz,
CDCl3) δ 8.09 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH), 7.62 (d, J = 8.3 Hz, 2H, C6H4 3,5ꢀH),
7.49 (d, J = 8.4 Hz, 2H, C6H4 2,6ꢀH), 7.38 (d, J = 8.4 Hz, 2H, C6H4 3,5ꢀH), 7.34 (s,
2H, C6H2 2,6ꢀH), 7.13 (d, J = 16.4 Hz, 1H, CH=CH), 7.08 (d, J = 16.4 Hz, 1H,
CH=CH), 3.97 (s, 6H, OCH3), 3.94 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ
164.44, 164.31, 153.72, 141.32, 140.32, 135.64, 131.93, 129.70, 128.21, 128.06,
127.29, 127.04, 122.78, 122.09, 118.90, 104.31, 61.03, 56.45; HRMS (APCI): m/z
cacld for C25H22BrN2O4 (M+H)+ 493.0757, found 493.0754.
Data for 4j: light green solid; yield 77.6%; m.p.: 205ꢀ206°C; 1HꢀNMR (400 MHz,
CDCl3) δ 8.11 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH), 7.98 (d, J = 8.2 Hz, 1 H, C6H4 5ꢀH),
7.76 (d, J = 7.7 Hz, 1 H, C6H4 2ꢀH), 7.73 – 7.59 (m, 4H, C6H4 3,5ꢀH, CH=CH, C6H4
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3ꢀH), 7.43 (t, J = 7.7 Hz, 1H, C6H4 4ꢀH), 7.35 (s, 2H, C6H2 2,6ꢀH), 7.08 (d, J = 16.1
Hz, 1H, CH=CH), 3.98 (s, 6H, OCH3), 3.94 (s, 3H, OCH3); 13CꢀNMR (101 MHz,
CDCl3) δ 164.51, 164.18, 153.71, 148.02, 141.30, 139.74, 133.25, 132.44, 132.37,
128.53, 128.25, 127.57, 127.30, 125.83, 124.87, 123.45, 118.86, 104.28, 61.01,
56.43; HRMS (APCI): m/z cacld for C25H22N3O6 (M+H)+ 460.1503, found
460.1505.
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Data for 4k: light yellow solid; yield 87.5%; m.p.: 236ꢀ237°C; HꢀNMR (400
MHz, CDCl3) δ 8.39 (s, 1H, C6H4 6ꢀH), 8.13 (m, 3H, C6H4 3,5ꢀH, C6H4 4ꢀH), 7.82
(d, J = 7.8 Hz, 1H, C6H4 2ꢀH), 7.68 (d, J = 8.3 Hz, 2H, C6H4 3,5ꢀH), 7.55 (t, J = 8.0
Hz, 1H, C6H4 3ꢀH), 7.36 (s, 2H, C6H2 2,6ꢀH), 7.25 (s, 2H, CH=CH), 3.98 (s, 6H,
OCH3), 3.94 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ 164.55, 164.19, 153.74,
148.77, 141.39, 139.58, 138.52, 132.48, 130.41, 129.73, 128.33, 127.39, 127.36,
123.42, 122.59, 121.10, 118.83, 104.35, 61.04, 56.46; HRMS (APCI): m/z cacld for
C25H22N3O6 (M+H)+ 460.1503, found 460.1507.
Data for 4l: light green solid; yield 88.1%; m.p.: 271ꢀ272°C; 1H NMR (400 MHz,
CDCl3) δ 8.26 (d, J = 8.8 Hz, 2H, C6H4 3,5ꢀH), 8.18 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH),
7.74 ꢀ 7.65 (m, 4H, C6H4 2,6ꢀH C6H4 3,5ꢀH), 7.38 (s, 2H, C6H2 2,6ꢀH), 7.30 (d, J =
5.7 Hz, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (APCI): m/z
cacld for C25H22N3O6 (M+H)+ 460.1503, found 460.1507.
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Data for 4m: light yellow solid; yield 60.9%; m.p.: 145ꢀ146°C; HꢀNMR (400
MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 2H, C6H4 2,6ꢀH), 7.66 (d, J = 8.1 Hz, 2H, C6H4
3,5ꢀH), 7.61 (d, J = 4.5 Hz, 1H, C6H4 2ꢀH), 7.58 (d, J = 3.9 Hz, 1H, C6H4 5ꢀH), 7.35
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(s, 2H, C6H2 2,6ꢀH), 7.27 (m, 1H, C6H4 4ꢀH), 7.14 (d, J = 16.5 Hz, 1H, CH=CH),
6.98 (t, J = 7.5 Hz, 1H, C6H4 3ꢀH), 6.91 (d, J = 8.3 Hz, 1H, CH=CH), 3.97 (s, 6H,
OCH3), 3.94 (s, 3H, OCH3), 3.90 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ
164.51, 164.35, 157.16, 153.70, 141.45, 141.20, 129.37, 127.75, 127.19, 127.01,
126.68, 125.95, 125.77, 122.25, 120.81, 119.07, 111.02, 104.27, 61.03, 56.43, 55.54;
HRMS (ESI): m/z cacld for C26H25N2O5 (M+H)+ 445.1758, found 445.1761.
1
Data for 4n: light yellow solid; yield 61.5%; m.p.: 131ꢀ132°C; HꢀNMR (400
MHz, CDCl3) δ 8.11 (d, J = 7.2 Hz, 2H, C6H4 2,6ꢀH), 7.64 (d, J = 7.3 Hz, 2H, C6H4
3,5ꢀH), 7.37 (s, 2H, C6H2 2,6ꢀH), 7.34 – 7.26 (m, 1H, C6H4 3ꢀH), 7.19 (d, J = 16.3
Hz, 1H, CH=CH), 7.16 - 7.03 (m, 3H, C6H4 2ꢀH, C6H4 5ꢀH, CH=CH), 6.86 (d, J =
8.1 Hz, 1H, C6H4 4ꢀH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3), 3.86 (s, 3H, OCH3);
13CꢀNMR (101 MHz, CDCl3) δ 164.37, 164.34, 159.96, 153.71, 141.39, 140.74,
138.10, 131.00, 129.76, 127.61, 127.29, 127.03, 122.39, 119.50, 118.73, 113.94,
112.06, 104.36, 61.03, 56.45, 55.27; HRMS (ESI): m/z cacld for C26H25N2O5
(M+H)+ 445.1758, found 445.1765.
1
Data for 4o: light yellow solid; yield 77.3%; m.p.: 190ꢀ191°C; HꢀNMR (400
MHz, CDCl3) δ 8.08 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH), 7.61 (d, J = 8.3 Hz, 2H, C6H4
3,5ꢀH), 7.47 (d, J = 8.7 Hz, 2H, C6H4 2,6ꢀH), 7.35 (s, 2H, C6H2 2,6ꢀH), 7.18 (d, J =
16.3 Hz, 1H, CH=CH), 6.99 (d, J = 16.3 Hz, 1H, CH=CH), 6.90 (d, J = 8.7 Hz, 2H,
C6H4 3,5ꢀH), 3.97 (s, 6H, OCH3), 3.92 (s, 3H, OCH3), 3.83 (s, 3H, OCH3); 13CꢀNMR
(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;
HRMS (APCI): m/z cacld for C26H25N2O5 (M+H)+ 445.1758, found 445.1767.
Data for 4p: light green solid; yield 81.1%; m.p.: 187ꢀ188°C; 1HꢀNMR (400 MHz,
CDCl3) δ 8.11 (d, J = 8.1 Hz, 2H, C6H4 2,6ꢀH), 7.64 (d, J = 8.1 Hz, 2H, C6H4 3,5ꢀH),
7.36 (s, 2H,C6H2), 7.15 (d, J = 16.2 Hz, 1H, CH=CH), 7.03 (d, J = 16.2 Hz, 1H,
CH=CH), 6.76 (s, 2H, C6H2 2,6ꢀH), 3.97 (s, 6H, OCH3), 3.92 (d, 9H, OCH3), 3.87 (s,
3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ 164.40, 164.39, 153.70, 153.47, 141.28,
140.64, 138.56, 132.40, 130.99, 127.25, 126.88, 126.79, 122.46, 118.96, 104.28,
103.97, 61.00, 60.96, 56.43, 56.17; HRMS (ESI): m/z cacld for C28H29N2O7 (M+H)+
505.1969, found 505.1970.
1
Data for 4q: light yellow solid; yield 67.3%; m.p.: 182ꢀ183°C; HꢀNMR (400
MHz, CDCl3) δ 8.21 (d, J = 8.2 Hz, 1H, naphthaleneꢀH), 8.11 (d, J = 8.2 Hz, 2H,
C6H4 2,6ꢀH), 7.97 (d, J = 16.0 Hz, 1H, CH=CH), 7.86 (d, J = 7.9 Hz, 1H,
naphthaleneꢀH), 7.81 (d, J = 8.2 Hz, 1H, naphthaleneꢀH), 7.74 (d, J = 7.1 Hz, 1H,
naphthaleneꢀH), 7.70 (d, J = 8.2 Hz, 2H, C6H4 3,5ꢀH), 7.59 – 7.45 (m, 3H,
naphthaleneꢀH), 7.33 (s, 2H, C6H2 2,6ꢀH), 7.14 (d, J = 16.0 Hz, 1H, CH=CH), 3.97
(s, 6H, OCH3), 3.96 (s, 3H, OCH3); 13CꢀNMR (101 MHz, CDCl3) δ 164.39, 164.35,
153.68, 141.23, 140.89, 134.22, 133.75, 131.33, 130.25, 128.72, 128.65, 127.93,
127.26, 127.14, 126.32, 125.96, 125.65, 123.82, 123.52, 122.66, 118.96, 104.25,
61.02, 56.41; HRMS (APCI): m/z cacld for C29H25N2O6 (M+H)+ 465.1809, found
465.1806.
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Data for 4r: yellow solid; yield 76.5%; m.p.: 191ꢀ192°C; HꢀNMR (400 MHz,
CDCl3) δ 8.46 (s, 1H, anthracenꢀH), 8.41ꢀ8.33(m, 2H, anthracenꢀH), 8.24 (d, J = 8.3
Hz, 2H, C6H4 2,6ꢀH), 8.14 – 8.01 (m, 3H, CH=CH, anthracenꢀH), 7.85 (d, J = 8.3 Hz,
2H, C6H4 3,5ꢀH), 7.57ꢀ7.49 (m , 4H, anthracenꢀH), 7.42 (s, 2H, C6H2 2,6ꢀH), 7.05 (d,
J = 16.6 Hz, 1H, CH=CH), 4.02 (s, 6H, OCH3), 3.98 (s, 3H, OCH3); HRMS (ESI):
m/z cacld for C33H27N2O4 (M+H)+ 515.1965, found 515.1964.
1
Data for 4s: yellow solid; yield 70.1%; m.p.: 212ꢀ213°C; HꢀNMR (400 MHz,
CDCl3) δ 8.02 (d, J = 8.3 Hz, 2H, C6H4 2,6ꢀH), 7.54 (d, J = 8.3 Hz, 2H, C6H4 3,5ꢀH),
7.39 (d, J = 8.7 Hz, 2H, C6H4 2,6ꢀH), 7.31 (s, 2H, C6H2 2,6ꢀH), 7.11 (d, J = 16.2 Hz,
1H, CH=CH), 6.87 (d, J = 16.2 Hz, 1H, CH=CH), 6.67 (d, J = 8.6 Hz, 2H, C6H4
13
3,5ꢀH), 3.94 (s, 6H, OCH3), 3.91 (s, 3H, OCH3), 2.95 (s, 6H, CH3); CꢀNMR (101
MHz, CDCl3) δ 164.60, 164.23, 153.68, 150.44, 141.73, 141.14, 131.25, 128.01,
127.17, 126.36, 122.81, 121.45, 119.13, 112.32, 104.23, 61.02, 56.43, 40.34; HRMS
(ESI): m/z cacld for C27H28N3O4 (M+H)+ 458.2074, found 458.2071.
1
Data for 4t: light yellow solid; yield 72.8%; m.p.: 196ꢀ197°C; HꢀNMR (400
MHz, CDCl3) δ 8.64 (d, J = 4.6 Hz, 1H, pyridineꢀH), 8.15 (d, J = 8.1 Hz, 2H, C6H4
2,6ꢀH), 7.78 – 7.67 (m, 4H, C6H4 3,5ꢀH pyridineꢀH, CH=CH), 7.45 (d, J = 7.6 Hz,
1H, pyridineꢀH), 7.37 (s, 2H, C6H2 2,6ꢀH), 7.31 (d, J = 16.9 Hz, 1H, CH=CH), 7.26
– 7.19 (m, 1H, pyridineꢀH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); 13C NMR (101
MHz, CDCl3) δ 164.51, 164.32, 154.73, 153.73, 149.43, 141.27, 139.91, 137.04,
131.80, 129.62, 127.68, 127.30, 123.33, 122.65, 118.96, 104.29, 61.03, 56.44;
HRMS (ESI): m/z cacld for C24H22N3O4 (M+H)+ 416.1605, found 416.1610.
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Data for 4u: light green solid; yield 69.3%; m.p.: 182ꢀ183°C; 1H NMR (600 MHz,
CDCl3) δ 8.77 (d, J = 2.0 Hz, 1H, pyridineꢀH), 8.53 (d, J = 4.8 Hz, 1H, pyridineꢀH),
8.14 (d, J = 8.4 Hz, 2H, C6H4 2,6ꢀH), 7.88 (d, J = 7.9 Hz, 1H, pyridineꢀH), 7.68 (d, J
= 8.4 Hz, 2H, C6H4 3,5ꢀH), 7.37 (s, 2H, C6H2 2,6ꢀH), 7.33 (q, 1H, pyridineꢀH), 7.20
(s, 2H, CH=CH), 3.99 (s, 6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (ESI): m/z cacld
for C24H22N3O4 (M+H)+ 416.1605, found 416.1606.
Data for 4v: light green solid; yield 70.2%; m.p.: 221ꢀ222°C; 1H NMR (600 MHz,
CDCl3) δ 8.63 (d, J = 4.6 Hz, 2H, pyridineꢀH), 8.18 (d, J = 8.4 Hz, 2H, C6H4 2,6ꢀH),
7.71 (d, J = 8.3 Hz, 2H, C6H4 3,5ꢀH), 7.44 (d, J = 4.7 Hz, 2H, pyridineꢀH), 7.37 (d, J
= 16.2 Hz, 3H, C6H2 2,6ꢀH, CH=CH), 7.16 (d, J = 16.3 Hz, 1H, CH=CH), 4.00 (s,
6H, OCH3), 3.95 (s, 3H, OCH3); HRMS (ESI): m/z cacld for C24H22N3O4 (M+H)+
416.1605, found 416.1603.
In Vivo Bioassays. Pot culture test method was adopted to evaluate fungicidal
activities of the title compounds against P. cubensis, C. lagenarium, and S.
cucurbitacearum in vivo at 600 µg/mL according to the references with slight
modifications.26, 27 Two commercial fungicides, 80% Mancozeb WP and 80%
Carbendazim WP were evaluated as controls against the above mentioned fungi at
the same condition. All compounds were dissolved in dimethylformamide (DMF),
and then distilled water (containing 0.1% Tween 80) was added to the solution to
generate a tested concentration of 600 µg/mL, in each of which the final
concentration of DMF was < 1% (v/v). P. cubensis sporangia were obtained by
washing off the leaves of highly infected cucumber plants that were kept overnight
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(above 80% relative humidity). C. lagenarium and S. cucurbitacearum were cultured
on the potato dextrose agar (PDA). Spores of those two fungal pathogens were
removed from the PDA plates. All of the spore suspensions were adjusted to the
desired concentrations prior to use. Germination was conducted by soaking cucurbit
seeds in water for 2 h at 50 °C and then keeping the seeds moist for 24 h at 28 °C in
an incubator.
When the cucurbit plants used for inoculations were at the stage of two seed
leaves, tested compounds and commercial fungicides were sprayed with a
handꢀsprayer on the upper leaf surface of seed leaves at the standard concentration of
600 ꢁg/mL. Distilled water without compounds or commercial fungicides treatments
was set as a blank control. After the solution on leaves were airꢀdried, inoculations of
P. cubensis, C. lagenarium and S. cucurbitacearum were carried out by spraying a
spore suspension with the concentration of 1×105, 1×106, 1×106 spores/mL of water,
respectively. Then the plants were maintained at 24 ± 1 °C and above 80% relative
humidity. Three replicates were used per treatment, and the experiment was repeated
three times.
The fungicidal activity was evaluated visually when the nonꢀtreated cucurbit plant
(blank control) fully developed symptoms. The percentage of diseased area on
inoculated treated leaves was assessed and compared to that of nonꢀtreated ones to
determine the average disease index. The relative control efficacy of compounds
compared to the blank assay was calculated via the following equation:
I (%) = [(CK
−
PT)/CK] ×100%
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Where I is the relative control efficacy, CK is the average disease index during the
blank assay, and PT is the average disease index after treatment during testing.
3D-QSAR Analysis. All molecular modeling studies were performed using the
Tripos SYBYL 7.3.5 software package.28 The comparative molecular field analysis
(CoMFA) model was derived from the antifungal activity of 22 compounds (17
compounds for training sets, 5 compounds for test sets, respectively) against C.
lagenarium at 600 µg/mL. The fungicidal activity was converted into D values by
the formula D=log{[I/(100−I)] −MW},29 where I is the percent control efficacy and
MW is the molecular weight of the tested compounds. Compound 4k was chosen as
a template for superimposition because of the highest control efficacy. Each structure
was built using the SKETCH option and fully geometryꢀoptimized using a conjugate
gradient procedure based on the TRIPOS force field and Gasteiger and Hückel
charges.
The steric and electrostatic interaction fields for CoMFA were calculated using
the SYBYL default parameters: 2.0 Å grid points spacing, a sp3 carbon probe atom
with a charge of +1.0 and a van der Waals radius of 1.52 Å. Steric and electrostatic
fields were scaled by the CoMFAꢀSTD method in SYBYL with default cutꢀoff
energy of 30 kcal/mol. A partial leastꢀsquares (PLS) approach was used to derive the
3D QSAR model, in which the CoMFA descriptors were used as independent
variables and the experimental D values were used as dependent variables. The
crossꢀvalidation with the leaveꢀoneꢀout (LOO) option and the SAMPLS program
was carried out to obtain the optimal number of components (N) and crossꢀvalidated
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coefficient (q2). After the optimal number of components was determined, a
nonꢀcrossꢀvalidated analysis was performed without column filtering to obtain
regression coefficients (r2) and its standard error (S) and Fꢀtest value (F) for the
model evaluation.
RESULTS AND DISCUSSION
Synthesis. The synthetic route of the title compounds is outlined in Figure 1. A
series of novel stilbene derivatives containing an 1,3,4ꢀoxadiazole moiety were
synthesized in good yields by bromination of NꢀBromosuccinimide (NBS), Arbuzov
reaction, and Wittig−Horner reaction. The key step involved the formation of the
stilbenes which were efficiently generated by the reaction of phosphonate 3 with
various aldehydes via Wittig–Horner reaction under mild reaction conditions. The
chemical structures of the target compounds were confirmed by FTꢀIR, 1H NMR, 13C
NMR, and HRMS. Notably, the 1H NMR spectra of target compounds show two fine
doublets as well as coupling constant (16.7-16.0 Hz) corresponding to the olefinic
protons (CH=CH) from transꢀstilbene, which are further confirmed by the FTꢀIR
spectra.30 All spectral and analytical data are consistent with the assigned structures.
Fungicidal Activity. The in vivo fungicidal activity of compounds 4a-4v against
P. cubensis, C. lagenarium and S. cucurbitacearum at a concentration of 600 µg/mL
are listed in Table 1. The results of bioassays are compared with those obtained from
two commercial fungicides. It was worthy to note that all of the tested compounds
were found safe for the cucurbit plants. As indicated in Table 1, some of the title
compounds showed broad spectrum and comparable fungicidal activities against the
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tested fungi. Four main conclusions were drawn from the comparison of the
fungicidal activities of title compounds against test fungi to those of commercial
fungicides: (a) Compounds 4s exhibited a significant inhibition effect against
cucumber P. cubensis with control efficacy of 71.38 ± 1.47%, which was comparable
to that of 80% Mancozeb WP (70.13 ± 3.25%). While compounds 4d, 4l and 4v
showed relatively high antifungal activity against cucumber P. cubensis (65.01 ±
1.81 and 63.42 ± 4.60%, respectively) compared with the fungicide 80% Mancozeb
WP. (b) Compounds 4b, 4d, 4f, 4i, 4k and 4l showed prominent activity against C.
lagenarium as compared to the control 80% Carbendazim WP. In particularly, the
activity of compounds 4d and 4k were 82.43 ± 4.66 and 83.82 ± 2.69%, respectively,
which were similar to that of 80% Carbendazim WP (81.57 ± 2.19%). (c) Most of
the compounds exhibited fair to moderate control efficacy against S.
cucurbitacearum. Notably, compounds 4b, 4h and 4i, whose efficacy rates were
72.05 ± 0.96, 69.34 ± 0.93 and 67.68 ± 1.43%, respectively, were found to be almost
the same activity level as that of 80% Carbendazim WP (69.02 ± 0.47%). (d) Some
compounds showed significant bioactivity variances against the different fungi. For
example, compound 4g exhibited moderate activity (44.81 ± 3.85%) against C.
lagenarium, whereas the control efficacy of compound 4g against S.
cucurbitacearum was only 8.03 ± 2.78%, and even showed a negative effect on P.
cubensis. These results indicated that compounds 4b, 4d, 4i, 4k and 4l were the most
promising candidates for crop protection because of their broad spectrum and
remarkably high activities against all tested fungi.
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CoMFA Analysis. The statistical results of CoMFA model are summarized in
Table 2. The internal validation of LOO crossꢀvalidated q2 and nonꢀcross validated
coefficient r2 are commonly applied as the criterion of robustness and predictive
ability of a QSAR model. The statistically recommendatory values for a satisfactory
QSAR model are q2 > 0.5 and r2 > 0.8.31 As listed in Table 2, a good predictive
CoMFA model with the LOO crossꢀvalidated coefficient q2=0.516 and the
correlation coefficient r2=0.920 was established. The predicted D values are
generally in good agreement with the experimental data as shown in Figure 2. The
standard error (S=0.114) and Fꢀtest value (F=25.186) further validate the developed
model.
The contribution steric and electrostatic fields are 56.2 and 43.8% for the QSAR
model, respectively. CoMFA contour maps are displayed in Figure 3 to visualise how
steric and electrostatic fields contribute to the bioactivity of test compounds. As
shown in steric field contour map (Figure 3a). The green contour surrounding
metaꢀposition of the benzene ring indicates that a bulky group would be favorable
for higher fungicidal activity, while the yellow contour represents region of
unfavorable steric effect. For example, compounds 4k and 4d bearing bulkier
substituents at the metaꢀposition displayed higher fungicidal activity. For
electrostatic contours map (Figure 3b), the negatively charged favorable red regions
are observed at metaꢀposition of the benzene ring, indicating that
electronꢀwithdrawing substituents enhance the bioactivity, whereas a positively
charged blue region around paraꢀposition is favored. Such as compound 4o
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(paraꢀposition) displayed higher activity than those of compounds 4m
(orthoꢀposition) and 4n (metaꢀposition). Analysis of CoMFA contour plots revealed
that substitutions at the metaꢀposition, the bulky groups and electronꢀwithdrawing
groups (e.g., -NO2, -Cl) are favorable to increase the bioactivity.
The results obtained from 3DꢀQSAR provided reliable clues for mechanistic
study and designing of optimized stilbene derivatives. On the basis of the in vivo
bioassay and 3DꢀQSAR analysis, it can be concluded that the variances (substituted
positions, electronic properties and steric effects) among substituents (Ar) on
stilbenes showed a significant relationship with fungicidal activity against the three
fungi. Furthermore, considering that the bioassay of title compounds was evaluated
in vivo, effects of metabolism and/or biotransformation within the plants or fungi
may contribute to the significant variations in fungicidal potency. It is reported that
the electronꢀwithdrawing substituents of the phenyl moiety seem to enhance the
potency, which may be related to the fact that they tend to retard mechanisms of
oxidative metabolism occurring on (or close to) the benzene ring.32 This assumption
is consistent with the higher bioactivity of the title compounds with
electronꢀwithdrawing substituents at metaꢀposition proposed by the 3DꢀQSAR
analysis.
Discovery of phytopathogenic fungicidal agents with new modes of action and
ecoꢀfriendly properties is of great importance for crop protection. 33 In the present
study, 22ꢀnovel stilbene derivatives containing the 1,3,4ꢀoxadiazole moiety and the
trimethoxybenzene were designed and synthesized. The approach was proved to be
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an efficient method to implement structural modification by combination of several
functional fragments. Bioassay results indicated that compounds 4b, 4d, 4i, 4k and
4l exhibited broad spectrum fungicidal activity in vivo against all tested fungi, some
of which were comparable with the commercial fungicides. Further field evaluations
on their biological efficacies, as well as crop toxicities and safety, are necessary
before they can be developed as fungicide candidates.
ASSOCIATED CONTENT
Supporting Information Available:
Synthetic procedures for intermediates 1-3 and target compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
Notes
The authors declare no competing financial interest.
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Funding
This work was financially supported by the Fundamental Research Funds for the
Central Universities (No.2012ZM0035) and Guangdong Provincial Natural Science
Foundation (No.04300531)
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FIGURES CAPTIONS
Figure 1. General Synthetic Route for Target Compounds.
4a: Ar=phenyl; 4b: Ar=4ꢀF phenyl; 4c: Ar=2ꢀCl phenyl; 4d: Ar=3ꢀCl phenyl; 4e:
Ar=4ꢀCl phenyl; 4f: Ar=2,4ꢀdiꢀCl phenyl; 4g: Ar=3,4ꢀdiꢀCl phenyl; 4h: Ar=2ꢀBr
phenyl; 4i: Ar=4ꢀBr phenyl; 4j: Ar=2ꢀNO2 phenyl; 4k: Ar=3ꢀNO2 phenyl; 4l:
Ar=4ꢀNO2 phenyl; 4m: Ar=2ꢀmethoxy phenyl; 4n: Ar=3ꢀmethoxy phenyl; 4o:
Ar=4ꢀmethoxy phenyl; 4p: Ar=3,4,5ꢀtrimethoxy phenyl; 4q: Ar=1ꢀnaphthyl; 4r:
Ar=9ꢀ anthryl; 4s: Ar=4ꢀ N,Nꢀdimethylaniline; 4t: Ar=2ꢀ pyridyl; 4u: Ar=3ꢀ pyridyl;
4v: Ar=4ꢀ pyridyl.
Figure 2. Plot of Experimental Values Versus Predicted Values for Training and Test
Sets Based on CoMFA Model.
Figure 3. Graphical Representation of CoMFAAnalysis 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
ꢁg/mL in Vivo.
control efficacy (%)a
C. lagenarium
Compd.
4a
Ar
P. cubensis
S. cucurbitacearum
58.77 ± 1.97
50.03 ± 4.01
55.17 ± 4.22
52.20 ± 2.59
32.66 ± 6.18
65.14 ± 3.52
31.48 ± 4.10
72.05 ± 0.96
4b
4c
ꢀ9.34 ± 0.36
65.01 ± 1.81
36.96 ± 5.57
38.52 ± 4.06
82.43 ± 4.66
47.78 ± 1.15
63.83 ± 5.38
32.03 ± 7.57
ꢀ19.08 ± 1.29
27.54 ± 8.20
4d
4e
4f
4g
ꢀ4.08 ± 4.17
44.81 ± 3.85
8.03 ± 2.78
8.37 ± 3.03
54.87 ± 3.55
48.34 ± 0.62
26.38 ± 1.16
64.77 ± 2.10
56.07 ± 3.45
69.34 ± 0.93
67.68 ± 1.43
35.36 ± 2.85
4h
4i
4j
50.13 ± 3.15
83.82 ± 2.69
46.83 ± 4.50
4k
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control efficacy (%)a
C. lagenarium
Compd.
Ar
P. cubensis
S. cucurbitacearum
63.42 ± 4.60
66.72 ± 2.83
32.23 ± 1.57
4l
25.28 ± 5.99
33.12 ± 1.66
48.91 ± 1.54
4m
44.13 ± 4.56
58.21 ± 5.91
37.90 ± 5.83
56.93 ± 3.04
47.62 ± 3.37
40.51 ± 4.43
4n
4o
49.57 ± 1.12
61.92 ± 2.69
7.05 ± 1.39
4p
4q
4r
36.01 ± 0.51
15.3 ± 2.77
37.98 ± 5.48
14.29 ± 5.50
51.18 ± 4.76
53.09 ± 4.36
4s
4t
71.38 ± 1.47
18.38 ± 1.59
42.67 ± 2.51
30.26 ± 2.38
52.88 ± 1.06
37.58 ±1.98
44.84 ± 4.22
45.14 ± 6.89
40.27 ± 2.23
4u
62.35 ± 3.09
42.61 ± 1.92
34.24 ± 4.43
4v
Fungicidesb
70.13 ± 3.25 A
81.57 ± 2.19 B
69.02 ± 0.47 B
a Values represent means of three independent replicates ± SD.
b Control fungicides: A, 80% Mancozeb WP; B, 80% Carbendazim WP.
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Table 2. The Statistical Parameters of CoMFA Model.
Contribution (%)
compound steric electrostatic
56.2 43.8
N
q2
r2
S
F
CoMFA
5
0.516 0.920 0.114 25.186
4k
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