Dehydrozingerone Inspired Discovery of Potential Broad-Spectrum Fungicidal Agents as Ergosterol Biosynthesis Inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b04231

A series of dehydrozingerone derivatives were synthesized, and their fungicidal activities and action mechanism against Colletotrichum musae were evaluated. The bioassay result showed that most compounds exhibited excellent fungicidal activity in vitro at 50 μg mL^-1. Compounds 13, 16, 18, 19, and 27 exhibited broad-spectrum fungicidal activity; especially, compounds 19 and 27 were found to have more potent fungicidal activity than azoxystrobin. The EC₅₀ values of compounds 19 and 27 against Rhizoctonia solani were 0.943 and 0.161 μg mL^-1 respectively. Moreover, compound 27 exhibited significant in vitro bactericidal activity against Xanthomonas oryzae pv. oryzae, with an EC₅₀ value of 11.386 μg mL^-1, and its curative effect (49.64%) and protection effect (51.74%) on rice bacterial blight disease was equivalent to that of zhongshengmycin (42.90%, 40.80% respectively). Compound 27 could also effectively control gray mold (87.10%, 200 μg mL^-1) and rice sheath blight (100%, 200 μg mL^-1 82.89%, 100 μg mL^-1) in vivo. Preliminary action mechanism study showed that compound 27 mainly acted on the cell membrane and significantly inhibited ergosterol biosynthesis in Colletotrichum musae.

Full text of DOI:10.1021/acs.jafc.9b04231

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Agricultural and Environmental Chemistry
Dehydrozingerone Inspired Discovery of Potential Broad-
spectrum Fungicidal Agents as Ergosterol Biosynthesis Inhibitors
Xiangmin Song, Xinyue Zhu, Ting Li, Cai Liang, Meng Zhang,
Zhan Hu, Yu Shao, Li Yang, Jun Tao, and Ranfeng Sun
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04231 • Publication Date (Web): 18 Sep 2019
Downloaded from pubs.acs.org on September 19, 2019
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Dehydrozingerone Inspired Discovery of
Potential Broad-spectrum Fungicidal Agents
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as Ergosterol Biosynthesis Inhibitors
Xiangmin Song,1,# Xinyue Zhu,1,# Ting Li,3 Cai Liang,1 Meng Zhang,1 Yu Shao,2 Jun
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Tao,3 and Ranfeng Sun,*,1
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Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and
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Pests, Ministry of Education, College of Plant Protection, Hainan University, Haikou,
570228, People’s Republic of China
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2Danzhou Tobacco Company, Hainan Provincial Branch of CNTC, Danzhou, 571700,
People’s Republic of China
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3Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources,
Haikou, 570228, People’s Republic of China
*Corresponding Author, Telephone/Fax: +86-898-66192906. E-mail: srf18@hainanu.edu.cn.
#These authors contributed equally to this work.
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ABSTRACT: A series of dehydrozingerone derivatives were synthesized, and their fungicidal
activities and action mechanism against Colletotrichum musae were evaluated. The bioassay result
showed that most compounds exhibited excellent fungicidal activity in vitro at 50 μg mL-1.
Compounds 13, 16, 18, 19 and 27 exhibited broad-spectrum fungicidal activity, especially,
compounds 19 and 27 were found and presented more potent fungicidal activity than
azoxystrobin. The EC50 values of compounds 19 and 27 against Rhizoctonia solani were 0.943 μg
mL-1 and 0.161 μg mL-1 respectively. Moreover, compound 27 exhibited significant in vitro
bactericidal activity against Xanthomonas oryzae pv.oryzae, with an EC50 value of 11.386 μg mL-1
and its curative effect (49.64%) and protection effect (51.74%) on rice bacterial blight disease was
equivalent to that of zhongshengmycin (42.90%, 40.80% respectively). Compound 27 could also
effectively control gray mold (87.10%, 200 μg mL-1) and rice sheath blight (100%, 200 μg mL-1;
82.89%, 100 μg mL-1) in vivo. Preliminary action mechanism study showed that compound 27
mainly acted on the cell membrane and significantly inhibited the ergosterol biosynthesis in
Colletotrichum musae.
KEYWORDS: dehydrozingerone; fungicidal activity; action mechanism; ergosterol
biosynthesis inhibitors
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INTRODUCTION
In the past few years, the “Research and Development” costs and number of screening
compounds to bring a new active ingredient to market have been rising significantly in the
discovery of agrochemicals1. Searching for active lead compounds from natural products and then
structural modification or derivatization has proven to be a successful way to discover pesticides
with new modes of action, such as pyrethroid insecticides,2 neonicotinoid insecticides3 and
strobilurins fungicides4. The fresh rhizome of ginger (zingiber officinale Rosc.) is recognized as a
plant resource for both medicine and food by Ministry of Health of the People's Republic of
China. Dehydrozingerone (A, Figure 1) is one of the chemical constituents in ginger, which was
first isolated from Aframomum giganteum in 1976,5 Subsequently, its pharmacological activities
such as antifungal activity (the values of MIC against Aspergillus oryzae, Aspergillus flavus,
Aspergillus niger, Aspergillus ochraceus and Fusarium oxysporum were 755~911 μM
respectively6), anticancer activity (increasing oxidative stress significantly which was associated
with hydrogen peroxide in PC12 cells7), antioxidant activity (IC50 = 57.0±2.5 μM8) and
anti-Alzheimer's activity9 have been reported.
However, there are few reports on the use of dehydrozingerone as pesticides. At the same time,
we noticed that its analogue, cinnamaldehyde (B, Figure 1), which was isolated from
Cinnamomum cassia Presl, has been reported to be used in the control of Rhizoctonia solani and
Sclerotinia homeocarpa Bennett10. Five kinds of cinnamamide analogues (C, D, E, F, G, Figure
1) were isolated from the seeds of clausena lansium (Lour.) Skeels. in 2018, and their nematicidal
activities against Meloidogyne incognita were 0%, 21.4%, 71.5%, 20.2% and 16.3% at 2.5 mg
mL-1, respectively.11
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Therefore, we decided to separate the dehydrozingerone molecule into two parts, benzene ring
and ketone parts (Figure 1), and study the structure-activity relationship of different functional
groups. In order to reduce the cost of synthesis as much as possible, bioactivity-guided mixed
synthesis and evaluation were carried out in our labs, then a total of 27 dehydrozingerone
derivatives were synthesized and their fungicidal activity tests were completed. Moreover, the in
vivo fungicidal activity for compound 27 against Botrytis cinereal and R. solani and the
preliminary action mechanism of compound 27 against Colletotrichum musae were evaluated.
MATERIALS AND METHODS
Instruments. NMR spectra were completed in CDCl3 or DMSO-d6 solution by Bruker avance
III 500 spectrometer (Bruker Daltonics, Bremen, Germany). HRMS data were obtained on Agilent
6210 ESI/TOF MS (Agilent Technologies Inc., USA). HPLC data was obtained on Thermo
UltiMate3000 (Thermo Fisher Scientific, USA). Transmission Electron Microscope (TEM) data
were obtained on HITACHI-HT7700. X-4 binocular microscope melting point apparatus (Beijing
Tech Instruments Co., Beijing, China) was used to determine the melting points of all compounds.
Synthesis. All anhydrous solvents were dried and purified by standard techniques before use.
Chemical materials at analytical grade were purchased from commercial sources TCI (Shanghai,
China) or Aladdin (Shanghai, China).
General procedure for the synthesis of compounds 1-24. Compounds 1–24 were synthesized
according to literature.12, 13 Trifluoroethanoicacid (1 mmol) was added dropwise into a solution of
morpholine (1 mmol) in Et2O (10 mL) at 0oC, then the solution was stirred vigorously for one
hour at room temperature. The white precipitate was filtered out, then dissolved in a mixture of
substituted benzaldehydes (5 mmol) and substituted acetone (5 mL). The mixture was refluxed for
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48 h and then cooled to room temperature. It was poured into saturated NaHCO3 solution (20 mL)
and was extracted by EtOAc (20 mL), then organic layer was collected and washed with saturated
brine (20 mL). After dried by anhydrous Na2SO4, the organic layer was evaporated under reduced
pressure. The residue was puried by flash column chromatography (gel, petroleum ether/EtOAc)
to give compounds 1–24.
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(E)-4-(3,4-dimethoxyphenyl)but-3-en-2-one (1). White solid, mp 85-86 C, yield = 53%. H
NMR (500 MHz, CDCl3) δ 7.45 (d, J = 16.2 Hz, 1H, CH=CH), 7.11 (dd, J = 8.3, 1.9 Hz, 1H, Ph),
7.06 (d, J = 1.9 Hz, 1H, Ph), 6.87 (d, J = 8.3 Hz, 1H, Ph), 6.59 (d, J = 16.2 Hz, 1H, CH=CH), 3.91
(s, 6H, OCH3), 2.36 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 198.3, 151.4, 149.3, 143.5,
127.3, 125.3, 123.0, 111.1, 109.6, 56.0, 55.9, 27.4. HRMS (ESI): m/z calcd for C12H15O3 [M+H]+:
207.1016, found 207.1018.
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(E)-4-(2-fluorophenyl)but-3-en-2-one (2). Yellow solid, mp 81-82 C, yield = 85%. H NMR
(500 MHz, CDCl3) δ 7.64 (d, J = 16.4 Hz, 1H, CH=CH), 7.54 (td, J = 7.6, 1.7 Hz, 1H, Ph), 7.37 –
7.32 (m, 1H, Ph), 7.17 – 7.13 (m, 1H, Ph), 7.08 (ddd, J = 10.7, 8.3, 1.2 Hz, 1H, Ph), 6.75 (d, J =
16.4 Hz, 1H, CH=CH), 2.37 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 198.3, 161.3 (d, J =
253.7 Hz), 135.6 (d, J = 3.5 Hz), 132.0 (d, J = 8.8 Hz), 129.2 (d, J = 5.4 Hz), 128.7 (d, J = 2.8
Hz), 124.6 (d, J = 3.6 Hz), 122.5 (d, J = 11.5 Hz), 116.2 (d, J = 21.9 Hz), 27.5. HRMS (ESI): m/z
calcd for C10H10FO [M+H]+: 165.0710, found 165.0714.
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(E)-4-(2-chlorophenyl)but-3-en-2-one (3). Brown oil, yield = 83%. 1H NMR (500 MHz, CDCl3)
δ 7.93 (d, J = 16.4 Hz, 1H, CH=CH), 7.63 (dd, J = 7.5, 1.9 Hz, 1H, Ph), 7.42 (dd, J = 7.8, 1.4 Hz,
1H, Ph), 7.33 (dd, J = 7.4, 1.9 Hz, 1H, Ph), 7.31 – 7.29 (m, 1H, Ph), 6.66 (d, J = 16.4 Hz, 1H,
CH=CH), 2.42 (s, 3H, CH3). HRMS (ESI): m/z calcd for C10H10ClO [M+H]+: 181.0415, found
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181.0413. H-NMR spectrum was consistent with the literature.14
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(E)-4-(2-bromophenyl)but-3-en-2-one (4). Yellow oil, yield = 69%. H NMR (500 MHz,
CDCl3) δ 7.88 (d, J = 16.3 Hz, 1H, CH=CH), 7.61 (d, J = 8.0 Hz, 2H, Ph), 7.33 (t, J = 7.4 Hz, 1H,
Ph), 7.24 (td, J = 7.7, 1.6 Hz, 1H, Ph), 6.61 (d, J = 16.3 Hz, 1H, CH=CH), 2.42 (s, 3H, CH3). 13C
NMR (125 MHz, CDCl3) δ 198.3, 141.9, 134.4, 133.5, 131.4, 129.8, 127.8, 127.8, 125.6, 27.2.
HRMS (ESI): m/z calcd for C10H10BrO [M+H]+: 224.9910, found 224.9915.
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(E)-4-(2-nitrophenyl)but-3-en-2-one (5). Brown oil, yield = 53%. H NMR (500 MHz, CDCl3)
δ 8.07 (d, J = 8.4 Hz, 1H, Ph), 7.98 (d, J = 16.2 Hz, 1H, CH=CH), 7.68 – 7.63 (m, 2H, Ph), 7.56
(ddd, J = 8.6, 7.0, 2.0 Hz, 1H, Ph), 6.57 (d, J = 16.2 Hz, 1H, CH=CH), 2.43 (s, 3H, CH3). HRMS
(ESI): m/z calcd for C10H10NO3 [M+H]+: 192.0655, found 192.0651. H-NMR spectrum was
consistent with the literature.15
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(E)-4-(2-(trifluoromethyl)phenyl)but-3-en-2-one (6). Brown oil, yield = 75%. H NMR (500
MHz, CDCl3) δ 7.88 (d, J = 16.2 Hz, 1H, CH=CH), 7.72 (d, J = 7.9 Hz, 2H, Ph), 7.58 (t, J = 7.6
Hz, 1H, Ph), 7.49 (t, J = 7.5 Hz, 1H, Ph), 6.64 (d, J = 16.1 Hz, 1H, CH=CH), 2.41 (s, 3H, CH3).
13C NMR (125 MHz, CDCl3) δ 198.2, 139.0, 133.4, 132.2, 131.2, 129.8, 129.3 – 128.3 (m), 127.9,
126.3 (q, J = 5.6 Hz), 125.3 (d, J = 57.7 Hz), 124.0 (d, J = 273.9 Hz), 27.1. HRMS (ESI): m/z
calcd for C11H10F3O [M+H]+: 215.0678, found 216.0675.
(E)-4-(o-tolyl)but-3-en-2-one (7). Yellow oil, yield = 82%. 1H NMR (500 MHz, CDCl3) δ 7.82
(d, J = 16.1 Hz, 1H, CH=CH), 7.57 (d, J = 7.6 Hz, 1H, Ph), 7.29 (t, J = 7.3 Hz, 1H, Ph), 7.22 (t, J
= 7.4 Hz, 2H, Ph), 6.65 (d, J = 16.1 Hz, 1H, CH=CH), 2.45 (s, 3H, Ph- CH3), 2.39 (s, 3H, CH3).
13C NMR (125 MHz, CDCl3) δ 198.4, 140.9, 137.9, 133.4, 130.9, 130.2, 128.1, 126.4, 126.4, 27.8,
19.8. HRMS (ESI): m/z calcd for C11H13O [M+H]+: 161.0961, found 161.0959.
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(E)-4-(2-methoxyphenyl)but-3-en-2-one (8). Yellow solid, mp 48-50 oC, yield = 76%. 1H NMR
(500 MHz, CDCl3) δ 7.91 – 7.85 (m, 1H, CH=CH), 7.53 (dd, J = 7.7, 1.6 Hz, 1H, Ph), 7.39 – 7.33
(m, 1H, Ph), 6.96 (t, J = 7.5 Hz, 1H, Ph), 6.91 (d, J = 8.3 Hz, 1H, Ph), 6.74 (d, J = 16.5 Hz, 1H,
CH=CH), 3.89 (s, 3H, OCH3), 2.38 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 199.1, 158.3,
138.7, 131.8, 128.3, 127.8, 123.3, 120.8, 111.1, 55.5, 27.2. HRMS (ESI): m/z calcd for C11H13O2
[M+H]+: 177.0910, found 177.0913.
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(E)-4-(2-hydroxyphenyl)but-3-en-2-one (9). Yellow solid, mp 136-138 C, yield = 92%. H
NMR (500 MHz, CDCl3) δ 7.85 (d, J = 16.4 Hz, 1H, CH=CH), 7.47 (dd, J = 8.0, 1.4 Hz, 1H, Ph),
7.28 – 7.24 (m, 1H, Ph), 7.04 (d, J = 16.4 Hz, 1H, CH=CH), 6.97 – 6.90 (m, 2H, Ph), 2.43 (s, 3H,
CH3). 13C NMR (125 MHz, CDCl3) δ 201.1, 156.0, 140.7, 131.9, 129.7, 127.8, 121.5, 120.7,
116.6, 26.9. HRMS (ESI): m/z calcd for C10H11O2 [M+H]+: 163.0754, found 163.0752.
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(E)-4-(3-chlorophenyl)but-3-en-2-one (10). Yellow oil, yield = 82%. H NMR (500 MHz,
CDCl3) δ 7.51 (s, 1H, Ph), 7.42 (d, J = 16.3 Hz, 1H, CH=CH), 7.40 – 7.26 (m, 3H, Ph), 6.69 (d, J
= 16.3 Hz, 1H, CH=CH), 2.37 (s, 3H, CH3). HRMS (ESI): m/z calcd for C10H10ClO [M+H]+:
181.0415, found 181.0417. H-NMR spectrum was consistent with the literature.16
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(E)-4-(4-chlorophenyl)but-3-en-2-one (11). White solid, mp 49-51 C, yield = 82%. H NMR
(500 MHz, CDCl3) δ 7.49 – 7.46 (m, 2H, Ph), 7.43 (s, 1H, CH=CH), 7.39 – 7.34 (m, 2H, Ph), 6.68
(d, J = 16.3 Hz, 1H, CH=CH), 2.37 (s, 3H, CH3). HRMS (ESI): m/z calcd for C10H11ClO [M+H]+:
181.0415, found 181.0421. H-NMR spectrum was consistent with the literature.16
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(E)-4-phenylbut-3-en-2-one (12). Brown oil, yield = 83%. H NMR (500 MHz, CDCl3) δ 7.54
(dd, J = 6.6, 3.2 Hz, 2H, Ph), 7.50 (s, 1H, CH=CH), 7.41 – 7.37 (m, 3H, Ph), 6.72 (d, J = 16.3 Hz,
1H, CH=CH), 2.38 (s, 3H, CH3). HRMS (ESI): m/z calcd for C10H11O [M+H]+: 147.0804, found
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147.0801. H-NMR spectrum was consistent with the literature.15
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(E)-4-(2,3-dichlorophenyl)but-3-en-2-one (13). Yellow solid, mp 78-79 C, yield = 79%. H
NMR (500 MHz, CDCl3) δ 7.90 (d, J = 16.3 Hz, 1H, CH=CH), 7.50 (ddd, J = 18.1, 7.9, 1.3 Hz,
2H, Ph), 7.23 (t, J = 7.9 Hz, 1H, Ph), 6.63 (d, J = 16.3 Hz, 1H, CH=CH), 2.41 (s, 3H, CH3). 13C
NMR (125 MHz, CDCl3) δ 198.0, 139.1, 135.1, 134.0, 133.1, 131.7, 130.6, 127.5, 125.7, 27.4.
HRMS (ESI): m/z calcd for C10H9Cl2O [M+H]+: 215.0025, found 215.0027.
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(E)-4-(2,4-dichlorophenyl)but-3-en-2-one (14). Yellow solid, mp 80-81 C, yield = 89%. H
NMR (500 MHz, CDCl3) δ 7.84 (d, J = 16.3 Hz, 1H, CH=CH), 7.56 (d, J = 8.5 Hz, 1H, Ph), 7.44
(d, J = 2.0 Hz, 1H, Ph), 7.29 – 7.26 (m, 1H, Ph), 6.64 (d, J = 16.3 Hz, 1H, CH=CH), 2.40 (s, 3H,
CH3). 13C NMR (125 MHz, CDCl3) δ 198.0, 137.9, 136.6, 135.6, 131.3, 130.0, 129.8, 128.3,
127.7, 27.4. HRMS (ESI): m/z calcd for C10H8Cl2ONa [M+Na]+: 236.9844, found 236.9848.
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(E)-4-(2,5-dichlorophenyl)but-3-en-2-one (15). White solid, mp 83-84 C, yield = 99%. H
NMR (500 MHz, CDCl3) δ 7.81 (d, J = 16.3 Hz, 1H, CH=CH), 7.59 (d, J = 2.4 Hz, 1H, Ph), 7.35
(d, J = 8.6 Hz, 1H, Ph), 7.28 (dd, J = 8.6, 2.5 Hz, 1H, Ph), 6.65 (d, J = 16.3 Hz, 1H, CH=CH),
2.41 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 197.7, 137.7, 137.7, 134.2, 133.2, 131.2, 131.0,
130.3, 127.4, 27.6. HRMS (ESI): m/z calcd for C10H9Cl2O [M+H]+: 215.0025, found 215.0022.
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(E)-4-(2,6-dichlorophenyl)but-3-en-2-one (16). Yellow oil, yield = 79%. H NMR (500 MHz,
CDCl3) δ 7.59 (d, J = 16.7 Hz, 1H, CH=CH), 7.36 (d, J = 8.1 Hz, 2H, Ph), 7.19 (t, J = 8.1 Hz, 1H,
Ph), 6.80 (d, J = 16.7 Hz, 1H, CH=CH), 2.41 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 198.1,
136.8, 135.0, 134.9, 132.1, 129.9, 128.8, 27.6. HRMS (ESI): m/z calcd for C10H8Cl2ONa
[M+Na]+: 236.9844, found 236.9847.
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(E)-4-(2-chloro-4-fluorophenyl)but-3-en-2-one (17). Yellow solid, mp 77-79 C, yield = 92%.
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1H NMR (500 MHz, CDCl3) δ 7.85 (d, J = 16.3 Hz, 1H, CH=CH), 7.63 (dd, J = 8.8, 6.0 Hz, 1H,
Ph), 7.17 (dd, J = 8.3, 2.6 Hz, 1H, Ph), 7.02 (td, J = 8.3, 2.6 Hz, 1H, Ph), 6.61 (d, J = 16.3 Hz, 1H,
CH=CH), 2.40 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 198.0, 163.4 (d, J = 254.7 Hz), 138.0,
136.0 (d, J = 10.3 Hz), 129.3 (d, J = 2.1 Hz), 129.1 (d, J = 3.8 Hz), 128.9 (d, J = 9.1 Hz), 117.6 (d,
J = 24.8 Hz), 114.9 (d, J = 21.7 Hz), 27.3. HRMS (ESI): m/z calcd for C10H9ClFO [M+H]+:
199.0320, found 199.0321.
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(E)-4-(2-chloro-6-fluorophenyl)but-3-en-2-one (18). Brown oil, yield = 90%. H NMR (500
MHz, CDCl3) δ 7.71 (d, J = 16.6 Hz, 1H, CH=CH), 7.25 (dd, J = 3.8, 1.6 Hz, 2H, Ph), 7.06 – 7.01
(m, 1H, Ph), 6.95 (d, J = 16.6 Hz, 1H, CH=CH), 2.39 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ
198.7, 161.9 (d, J = 256.7 Hz), 136.2 (d, J = 5.1 Hz), 133.5, 133.4 (d, J = 10.8 Hz), 130.9 (d, J =
10.4 Hz), 126.0 (d, J = 3.5 Hz), 121.7 (d, J = 13.9 Hz), 115.0 (d, J = 23.3 Hz), 27.9. HRMS (ESI):
m/z calcd for C10H8ClFONa [M+Na]+: 221.0140, found 221.0137.
(E)-4-(2,3,6-trichlorophenyl)but-3-en-2-one (19). Yellow oil, yield = 75%. 1H NMR (500 MHz,
CDCl3) δ 7.52 (d, J = 16.6 Hz, 1H, CH=CH), 7.38 (d, J = 8.7 Hz, 1H, Ph), 7.31 (d, J = 8.7 Hz, 1H,
Ph), 6.72 (d, J = 16.6 Hz, 1H, CH=CH), 2.42 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 197.7,
136.7, 135.5, 134.2, 133.2, 132.7, 132.5, 130.5, 129.0, 27.8. HRMS (ESI): m/z calcd for
C10H8Cl3O [M+H]+: 248.9635, found 248.9639.
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(E)-4-(2,4,6-trichlorophenyl)but-3-en-2-one (20). Yellow solid, mp 101-103 C, yield = 72%.
1H NMR (500 MHz, CDCl3) δ 7.52 (d, J = 16.6 Hz, 1H, CH=CH), 7.39 (s, 2H, Ph), 6.80 (d, J =
16.6 Hz, 1H, CH=CH), 2.41 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 197.8, 135.7, 135.5,
135.1, 135.0, 130.7, 128.9, 27.8. HRMS (ESI): m/z calcd for C10H7Cl3ONa [M+Na]+: 270.9455,
found 270.9457.
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(E)-4-(perfluorophenyl)but-3-en-2-one (21). Yellow oil, yield = 79%. H NMR (500 MHz,
CDCl3) δ 7.46 (d, J = 16.6 Hz, 1H, CH=CH), 7.00 (d, J = 16.6 Hz, 1H, CH=CH), 2.39 (s, 3H,
CH3). 13C NMR (125 MHz, CDCl3) δ 197.4, 146.7, 144.6, 138.8, 133.6, 133.6, 133.5, 133.5,
133.5, 133.4, 126.4, 126.4, 126.4, 110.0, 110.0, 28.4. HRMS (ESI): m/z calcd for C10H5F5ONa
[M+Na]+: 259.0153, found 259.0159.
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(E)-1-(2,4-dichlorophenyl)pent-1-en-3-one (22). Yellow solid, mp 92-94 C, yield = 78%. H
NMR (500 MHz, CDCl3) δ 7.87 (d, J = 16.3 Hz, 1H, CH=CH), 7.56 (d, J = 8.5 Hz, 1H, Ph), 7.44
(d, J = 2.1 Hz, 1H, Ph), 7.29 – 7.25 (m, 1H, Ph), 6.67 (d, J = 16.3 Hz, 1H, CH=CH), 2.73 (q, J =
7.3 Hz, 2H, CH2), 1.17 (t, J = 7.3 Hz, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 200.5, 136.7,
136.4, 135.7, 131.5, 130.0, 128.8, 128.3, 127.6, 33.9, 8.1. HRMS (ESI): m/z calcd for C11H11Cl2O
[M+H]+: 229.0181, found 229.0186.
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(E)-1-(2,4-dichlorophenyl)hex-1-en-3-one (23). Yellow solid, mp 96-97 C, yield = 68%. H
NMR (500 MHz, CDCl3) δ 7.86 (d, J = 16.3 Hz, 1H, CH=CH), 7.56 (d, J = 8.5 Hz, 1H, Ph), 7.43
(d, J = 2.1 Hz, 1H, Ph), 7.26 (dd, J = 8.5, 2.1 Hz, 1H, Ph), 6.66 (d, J = 16.3 Hz, 1H, CH=CH),
2.67 (t, J = 7.3 Hz, 2H, CH2), 1.71 (h, J = 7.4 Hz, 2H, CH2CH2CH3), 0.98 (t, J = 7.4 Hz, 3H,
CH3). 13C NMR (125 MHz, CDCl3) δ 200.1, 136.8, 136.4, 135.7, 131.5, 130.0, 129.1, 128.3,
127.6, 42.6, 17.7, 13.8. HRMS (ESI): m/z calcd for C12H13Cl2O [M+H]+: 243.0338, found
243.0333.
(E)-1-(2,4-dichlorophenyl)-4,4-dimethylpent-1-en-3-one (24). Yellow solid, mp 103-104 oC, yield
= 92%. 1H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 15.6 Hz, 1H, CH=CH), 7.58 (d, J = 8.5 Hz, 1H,
Ph), 7.43 (d, J = 2.1 Hz, 1H, Ph), 7.28 – 7.23 (m, 1H, Ph), 7.08 (d, J = 15.6 Hz, 1H, CH=CH),
1.23 (s, 9H, t-butyl). 13C NMR (125 MHz, CDCl3) δ 204.0, 137.9, 136.5, 136.4, 132.3, 130.5,
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128.8, 127.8, 124.0, 43.7, 26.6. HRMS (ESI): m/z calcd for C13H15Cl2O [M+H]+: 257.0494, found
257.0488.
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General procedure for the synthesis of compounds 25-26. Compounds 25–26 were
synthesized according to literature17. Sodium methoxide (28% in MeOH, 2 mmol) was added to a
stirred solution of substituted benzaldehydes (2.1 mmol) and trimethyl phosphonoacetate (2
o
mmol) in dimethylformamide (10 mL) at 0 C. After the mixture was stirred for 2 h, the reaction
mixture was poured into water to give crystals. Then the crystals were filtered as white solid. The
crude was used for the next step without further purification.
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methyl (E)-3-(2,4-dichlorophenyl)acrylate (25). White solid, mp 113-115 C, yield = 92%. H
NMR (500 MHz, CDCl3) δ 8.01 (d, J = 16.0 Hz, 1H, CH=CH), 7.55 (d, J = 8.5 Hz, 1H, Ph), 7.44
(d, J = 2.1 Hz, 1H, Ph), 7.28 – 7.25 (m, 1H, Ph), 6.41 (d, J = 16.0 Hz, 1H, CH=CH), 3.82 (s, 3H,
OCH3). 13C NMR (125 MHz, CDCl3) δ 166.7, 139.4, 136.4, 135.5, 131.3, 130.0, 128.3, 127.6,
120.9, 51.9. HRMS (ESI): m/z calcd for C10H9Cl2O2 [M+H]+: 230.9974, found 230.9976.
methyl (E)-3-(2,3,6-trichlorophenyl)acrylate (26). White solid, mp 122-125 oC, yield = 95%. 1H
NMR (500 MHz, CDCl3) δ 7.72 (d, J = 16.4 Hz, 1H, CH=CH), 7.38 (d, J = 8.7 Hz, 1H, Ph), 7.31
(d, J = 8.7 Hz, 1H, Ph), 6.51 (d, J = 16.4 Hz, 1H, CH=CH), 3.84 (s, 3H, OCH3). 13C NMR (125
MHz, CDCl3) δ 166.3, 138.3, 134.0, 133.2, 132.7, 132.5, 130.4, 129.0, 127.3, 52.1. HRMS (ESI):
m/z calcd for C10H8Cl3O2 [M+H]+: 264.9584, found 264.9589.
Synthesis of (E)-4-(2,4-dichlorophenyl)-1,1,1-trifluorobut-3-en-2-one (27). Compound 27 was
synthesized according to literature18. CsF (0.01 mmol) was added to a mixture of intermediate 25
(1 mmol) and TMS-CF3 (1.25 mmol) in ethylene glycol diethyl ether (10 mL) at room
temperature. The reaction was stirred for 5 h and then was carried out over 3 h by using 4 N HCl
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(5 mL). The reaction solution extracted by ethyl acetate (20 mL) and washed twice with saturated
sodium bicarbonate solution (20 mL), brine (20 mL). The organic layer was dried over anhydrous
sodium sulfate and concentrated under vacuum. Finally, silica gel column chromatography
(petroleum ether/ EtOAc) was used to afford compound 27 as a white solid, mp 76-78 oC, yield =
82%. 1H NMR (500 MHz, CDCl3) δ 8.32 (d, J = 15.9 Hz, 1H, CH=CH), 7.68 (d, J = 8.5 Hz, 1H,
Ph), 7.51 (d, J = 2.1 Hz, 1H, Ph), 7.33 (dd, J = 8.5, 2.0 Hz, 1H, Ph), 6.99 (d, J = 15.9 Hz, 1H,
CH=CH). 13C NMR (125 MHz, CDCl3) δ 179.7 (d, J = 35.8 Hz), 180.4 – 179.0 (m), 144.3, 138.5,
137.1, 130.5, 130.1, 128.8, 127.9, 118.5 (d, J = 290.7 Hz), 116.2 (d, J = 290.7 Hz), 113.9 (d, J =
290.7 Hz). HRMS (ESI): m/z calcd for C10H5Cl2F3ONa [M+Na]+: 290.9562, found 290.9564.
Synthesis of dehydrogenosterone (A). Natural product dehydrogenosterone (A) was
synthesized according to literature19. Vanillin (2 mmol) and acetone (2 mL) were dissolved in
ethanol (5 mL), added with 5 mL of 5% NaOH aqueous solution, reacted at room temperature for
1.5 h, diluted with 20 mL of distilled water, cooled to 0 °C in an ice bath, and neutralized with 2
M HCl. The reaction solution extracted by ethyl acetate (20 mL) and washed twice with sodium
bicarbonate solution (20 mL), brine (20 mL). The organic layer was dried over anhydrous sodium
sulfate and concentrated under vacuum. Finally, silica gel column chromatography (petroleum
ether/ EtOAc) was used to afford compound A as a yellow solid, mp 129-130 oC, yield = 64%. 1H
NMR (500 MHz, CDCl3) δ 7.45 (d, J = 16.2 Hz, 1H, CH=CH), 7.09 (dd, J = 8.2, 1.9 Hz, 1H, Ph),
7.06 (d, J = 1.9 Hz, 1H, Ph), 6.93 (d, J = 8.2 Hz, 1H, Ph), 6.59 (d, J = 16.2 Hz, 1H, CH=CH), 5.90
(s, 1H, OH), 3.94 (s, 3H, OCH3), 2.37 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 198.5, 148.3,
146.9, 143.8, 126.9, 124.9, 123.5, 114.9, 109.4, 56.0, 27.3. HRMS (ESI): m/z calcd for C11H13O3
[M+H]+: 193.0859, found 193.0855.
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Biological Assay. The in vitro antifungal activity of the target compounds was determined by the
mycelium growth rate test which has been described in our previous studies20 and the detailed
experimental method was also given in the Supporting Information.
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In Vivo Protective Activity against Botrytis cinereal on Tomato Leaves. The requisite amounts
of compound 27 was diluted to 200 µg mL−1 with Tween 80 (0.1%, v/v) solution, and then 10 mL
for each tomato leave was sprayed with the solution until liquid flowed on surface before
inoculation. A suspension of Botrytis cinereal spores at a concentration of 5 x 106 cfu mL-1 was
prepared after filtration through 4 layers of gauze, and then sprayed on tomato leaves. All the
treated plants were placed into illumination incubator (20oC, 85% relative humidity) and
replicated for 3 plants, Azoxystrobin (Yuanye, Shanghai, China) was used as a positive control.
After 5 days, the disease was observed and graded.21 The grading standards were as follows:
Grade 0: without disease spot.
Grade 1: 3 diseased spots in one leaf.
Grade 3: 4~6 diseased spots in one leaf.
Grade 5；7~10 diseased spots in one leaf.
Grade 7: 11~20 diseased spots in one leaf,parts are densely packed.
Grade 9: single leaf stick spot dense leaf area of more than a quarter.
∑
(incidence number of all levels × representative value of this level)
Disease index =
Control effect =
× 100
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total number of investigated leaves × highest value
control disease index ― treatment disease index
× 100%
control disease index
In Vivo Protective Activity against Rice Sheath Blight. In order to evaluate the protective
activity against rice sheath blight, the rice plants were inoculated with R. solani., which were
treated with different concentrations of target compound solution (200, 100, 50, 25 µg mL−1), and
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Azoxystrobin (Yuanye, Shanghai, China) was used as a positive control.22 Each treated with 20
plants, after 5 days the control effect is calculated by the formula:
A0 ― A1
control efficacy (%) =
× 100
A0
where A0 and A1 are the diameters of the lesion without or with treatment.
In Vitro Antibacterial Activities Against Xanthomonas oryzae pv.oryzae. According to the
literature method,23 an experimental system consists of nutrient broth (NB) media (4 mL) , 1 mL
of the test compounds (final concentration was 100 and 50 μg mL-1) and 40 μL of Xoo bacterium
solution prior to storage at 28 ± 1 °C for 24-48 h at 180 rpm, commercial bactericides
zhongshengmycin was used as a positive control. The in vitro inhibitory rates I (%) were
calculated by the following formula:
Inhibitory rate I (%)=(C-T)/C×100
Where T represents the corrected absorbance value (OD600) of the treated NB media and C
represents the corrected absorbance value (OD600) of the untreated NB media.
In Vivo Antibacterial Activity Test24. The in vivo antibacterial activity of compound 27 against
Xoo strains was studied by the leaf-cutting method. Rice strain IR24 (susceptible) was used as
hosts for antibacterial activity test, and zhongshengmycin was used as the positive control. Fully
unfolded leaves were inoculated by dipping sterile scissors into a freshly diluted bacterial
suspension (OD600=0.8, approximately 4×109 CFU mL-1) and cutting the tips. Each tested strain
Xoo was inoculated with at least 25 leaves and graded (curative activity test was to inoculate for
48 h before spraying, protective activity test was spray for 48h before inoculation). Symptoms
were scored by measuring lesion lengths after 14 days after inoculation and expressed as mean
lesion lengths ± SD. The whole experiment was repeated three times independently. The control
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efficiency I (%) of treatment and protection activities are calculated by the following formula:
control efficiency I (%)=(C-T)/C×100
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Where C is the disease index of the negative control group, and T is the disease index of the
treatment group.
Transmission Electron Microscopy. According to the literature method,25 the hyphal ultrastructure
of C. musae treated with compound 27 was observed under HT7700(HITACHI) Transmission
Electron Microscope (TEM) and the detailed experimental method was also given in the
Supporting Information.
Detection of Cell Membrane Permeability26. After incubated 4 days, the hyphae of C. musae
was collected and washed with double distilled water. Mycelium (1 g) was placed in 20 mL
centrifuge tube containing compound 27 at a concentration of 6 µg mL-1. And then gradually
measure the conductivity at 0 (J0), 1, 2, 4, 6, 8 h (J1). After boiling, cool and measure
conductivity (J2). Finally, the permeability is calculated by the formula:
J1 - J0
P (%) =
× 100
J2 - J0
Where P represents permeability.
Detection of Ergosterol Content27. Putting the mycelia dishes (d=5 mm) of C. musae on PDA
medium culture 7d after being produced spores with sterile water elution, mixture concentration
was about 1×105 spores suspension, inoculate 100 μL spore suspension in 200 mL YG (yeast
extract 5 g and glucose 5 g)culture, 150 r/min shaking culture under the condition of 25 oC for 5d
placed 4 layer gauze buchner funnel, mycelium was dried under aseptic conditions, The same
amount of wet heavy mycelium was added to 150 mL sterile water containing the agent, repeated
three times for each sample, they were cultured at 150 r/min under the condition of 25 oC for 3d.
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In the same way dry mycelium culture, mycelium was dried and then ground under the condition
o
of 60 C. The saponification treatment was carried out with 0.124 g respectively. Add dried
mycelium to 10 mL mixture of methanol and chloroform (3:1), room temperature for 24 h.
successively, add water, chloroform, and contains 20 mol/L KCl 0.5 mol/L phosphate buffer 10
mL each, stratification, the extraction of chloroform phase on the rotary evaporation apparatus 60
o
oC dry. Add 10 mL of methanol and ethanol mixture containing 1.4 mol/L KOH, 60 C
saponification for 1 h, adding petroleum ether (boiling range is 60-90 oC) each 10 mL, extraction
separation, enrichment in the rotary evaporation apparatus for petroleum ether layer, dissolve the
precipitation with ethanol, the capacity to 10 mL, through the cylinder microporous membrane
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filter (ф 0.45 μm), 0 C refrigerator storage back up. Under the condition of chromatography
determination of ergosterol in various samples of response values (absorption peak area),
calculating the concentration of ergosterol in samples, and calculate the ergosterol content in
mycelia.
RESULTS AND DISCUSSION
Synthesis. According to the Scheme 1, compounds 1-27 and A were synthesized with yields of
1
53-95%. Compounds 3, 5, 10, 11, 12 were known compounds and our H-NMR data are
consistent with those reported in the literature. In addition, data of HRMS for them were
supplemented in the article. These data are sufficient to confirm the correct structure of these
compounds.
In Vitro Antifungal Activity. A clear structure-activity relationship was shown in Table 1.
Firstly, to investigate whether the substituents on the phenyl ring played an important role in the
fungicidal activity, the methoxylation of the hydroxyl group on the phenyl ring of
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dehydrozingerone (A) was carried out to obtain compound 1, and it was found that a slight
modification of the sturcture lead to the fungicidal activity improvement. Then substitution with
electron-withdrawing groups (compounds 2, 3, 4, 5) and electron-rich groups (compounds 7, 8, 9)
on the benzene ring were completed, electron-withdrawing groups were benificial to antifungal
activity, especially compound 3 (2-Cl) and compound 4 (2-Br). Considering that chlorine atom
was more economical than bromine atom, we decided to choose chlorine atom to continue our
research.
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The antifungal acitivities of compounds 3 (2-Cl), 10 (3-Cl) and 11 (4-Cl) showed that different
substitution position of one chlorine atom on the benzene ring had indistinct effects on the
antifungal activity, however, an increase in the number of chlorine atom on the benzene ring could
improve the antifungal activity, for example, the antifungal activities of compounds 3 (2-Cl), 14
(2,4-diCl), 20 (2,4,6-triCl) verified the rule (Table 1). Subsequent compounds 13 (2,3-diCl), 14
(2,4-diCl), 15 (2,5-diCl), 16 (2,6-diCl) and 19 (2,3,6-triCl), 20 (2,4,6-triCl) brought us another
good news that when multiple chlorine atoms were in the ortho position with each other, it would
be more beneficial to improve the antifungal activity of the compound. Therefore, there was no
doubt that the compound substituted with five halogen atoms on the benzene ring would exhibit
higher antifungal activity, but the antifungal activity of compound 21 (2,3,4,5,6-pentaF) gave us a
great disappointment. Fortunately, compounds 14 (2,4-diCl), 17 (2-Cl-4-F) or compounds 16
(2,6-diCl), 18 (2-Cl-6-F) immediately gave the reason for the failure which was that the effect of
fluorine atoms on the antifungal activity was weaker than the chlorine atom. Summarizing this
part of the study, we have come to the conclusion that the more the ortho-substituted chlorine
atom on the benzene ring, the stronger the antifungal activity. So far, compound 19 (2,3,6-triCl)
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has the best broad-spectrum antifungal activity, the antifungal activity against all phytopathogenic
fungi is over 80%
Secondly, in order to investigate whether the carbonyl side chain part has an effect on the
antifungal activity, alkyl groups with different steric hindrance sizes were selected. According to
the Table 1, we analyzed the antifungal activity of compounds 14, 22, 23 and 24 in turn, and found
that the antifungal activity of the compound gradually decreased as the carbon chain increased or
the steric hindrance increased. When a methoxy-substituted, named compound 25 or 26 was
synthesized as electron-rich group substituted compound, the antifungal activity was remarkably
lowered compared with the compound 14 or 19. Thus, a trifluoromethyl group was introduced into
the compound 27, because of its electron-withdrawing effect and small steric hindrance. It was
exciting that compound 27 exhibited powerful antifungal activity and its inhibition rate for all
phytopathogenic fungi was over 90%.
In order to understand the antifungal activity of these compounds more clearly and intuitively,
we determined the EC50 values of compounds with more than 85% inhition rate at 50 μg mL-1 and
the result was showed in Table S1. From the Table S1, it was found that the EC50 values of
compounds 13, 16, 18, 19 and 27 against phytopathogenic fungi were 3.377~56.770 μg mL-1, and
especially compounds 19 and 27 showed the best broad-spectrum antifungal activity in a series of
dehydrozingerone derivatives. So we further tested the EC50 values of compounds 19 and 27
against R. solani, Fusarium graminearum and B. cinereal in Table 2. It was showed that
compounds 19 and 27 exhibited excellent inhibitory activity against R. solani, the EC50 values
were 0.943 μg mL-1 and 0.161 μg mL-1 respectively.
It was apparent that the fungicidal activity of compound 19 was comparable to that of the
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trifluoromethyl compound 27. Therefore, we want to carry out the trifluoromethylation of
compound 19 in order to further enhance the fungicidal activity of the compound. Unfortunately,
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we failed to complete this synthesis after trying a variety of methods28, 29
.
In Vivo Antifungal Activity against Botrytis cinereal on Tomato Leaves. As illustrated in
Table 3, a protective effect of 87.10% was achieved when compound 27 was treated for 5 days at
a concentration of 200 μg mL-1. However, the protective activity of the positive control
azoxystrobin reached 74.19% at a concentration of 200 μg mL-1. Therefore, compound 27
exhibited significant in vivo antifungal activity against B. cinereal which was comparable with
azoxystrobin.
In Vivo Antifungal Activity against Rice Sheath Blight. The EC50 value of compound 27 was
0.161 μg mL-1, so we selected it to further evaluate its in vivo protective effects against R. solani.
As shown in Table 3, the in vivo protective activity of compound 27 against R. solani were 100%
and 82.89% respectively with the concentrations of 200 and 100 μg mL-1, which was not
significantly different from azoxystrobin (100 μg mL-1, 80.53%).
In Vitro Antibacterial Activity. The Rice bacterial leaf blight is one of the most serious
disease in tropical areas caused by Xanthomonas oryzae pv. oryzae (Xoo).24 In our present study,
we have found that compound 27 has excellent antifungal activity against R. solani. Therefore, the
in vitro antibacterial activity of compound 27 against Xoo was worth to determine and the result
was showed in Table 4. Based on the experimental results, we found that the antibacterial activity
of compound 27 at 100 and 50 μg mL-1 were 88.10%, 80.88%, which was comparable with the
positive control zhongshengmycin (86.09%, 85.11% respectively). The EC50 value of compound
27 was 11.386 μg mL-1, while the positive control zhongshengmycin was 14.496 μg mL-1.
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Comparing compounds 14 and 24, we found that increased steric hindrance of substituted alkyl
groups on the carbonyl side chain significantly reduced the bactericidal activity. Meanwhile, due
to trifluoromethyl group, the bactericidal activity of compound 27 was 7 times higher than that of
compound 14.
In Vivo Antibacterial Activity. As illustrated in Table 5 and Figure 2, compound 27 displayed
good in vivo protective activity (51.74%) and curative activity (49.64%) against Xoo. At the same
concentration, the protective and curative activities of zhongshengmycin reached 40.80% and
42.90%, respectively. Therefore, compound 27 were more effective to reduce rice bacterial blight
than zhongshengmycin.
Effect of Compound 27 on the Hyphae Morphology. Compound 27 exhibited best fungicidal
activity against R. solani. However, R. solani is a typical fungus that spreads disease through the
soil and it is difficult to produce zoospores with a small size, which is not conducive to
experimental observation. One compound should work the same way for different
phytopathogenic fungi, so we chose C. musae which had easy-to-obtain conidia and noticeable
spindle-shaped structure as the model strains to investigate the action mechanism of compound 27.
The soluble protein content,30 reducing sugar content26 and chitinase activity30 in the mycelia were
found to be non-responsive to compound 27 (experimental details were given in the Supporting
Information from Figure S52-S55), the effect of compound 27 on the ultrastructural
characteristics of hypha was studied by Transmission Electron Microscope (TEM). As shown in
Figure 3, the cell morphology of hypha was normal in the control study (panels a to d), their cell
walls (CW) and membranes were distinct and uniform (panels a and d ), organelles were abundant
in cytoplasm (panels b and c ), such as mitochondria (M) and vacuoles (V). In contrast, after
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treatment with compound 27, their cell walls get thickened abnormally (panels a and e) and cell
membranes invaginated with an obvious plasmolysis (panels d and h). Moreover, organelles of the
hypha were also disturbed and abnormal changes occurred when treated by compound 27 (panels
b and f), such as the number of vacuole increased from one to three in one cell (panels a and e),
the nucleus was deformed by the compressing of extra vacuoles (panels f and g), the mitochondria
were swollen, exhibited decreased matrix density, developed flocculent dense bodies in the matrix
space, and the cell tissue fluid exudates obviously (panels e of Figure 3).
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Effect of Compound 27 on the Cell Membrane Permeability. Severe cytoplasmic separation
and cytoplasmic exudation of hyphae treated with compound 27 were observed under TEM.
Therefore, in order to confirm whether compound 27 acted on the cell membrane of C. musae, the
permeability of the fungal cell membrane can be explained by measuring the conductivity (relative
seepage ratio) of fungal culture medium.26 To confirm the effect of compound 27 on the cell
membrane permeability, we evaluated changes in the conductivity of mycelia, when treated with
compound 27 (6 µg mL-1), the relative seepage ratio of cell membrane was always higher than that
of control (Figure 4). After C. musae was treated with compound 27 (6 µg mL-1), the relative
permeability rate of cell membrane was always higher than that of control. After 4 h treatment
with compound 27 (6 μg mL-1), the relative permeability was found to be 20.5%, meanwhile
control was 10.4%. After 8 h treatment, the relative permeability of compound 27 increased by
98.90% compared with the control, the results showed that the cell membrane was destroyed faster
than that of control when C. musae was treated with compound 27. The results can be attributed to
the fact that compound 27 attacks the membrane of C. musae (Figure 4). This may be the result of
plasma leakage in hyphae cells, which eventually leads to increase conductivity.26
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Analysis of ergosterol content in hyphae of C. musae. Ergosterol was an important
component of cell membranes, and ergosterol synthesis inhibitors were common broad-spectrum
antifungal drugs31. As shown in Table 6 (High Performance Liquid Chromatography (HPLC) test
details will be given in the Supporting Information), different treatments the hyphae of C. musae
for 3 d had significant effects on ergosterol content. The content of ergosterol in the control
hyphae was 0.48 mg/g. However, when C. musae was treated with compound 27, the content of
ergosterol was almost 77.08% lower than that of the control. When C. musae was treated with
diniconazole, the content of ergosterol was almost 93.75% lower than that of the control. The
Ducan's variance showed that the content of ergosterol in mycelia treated with different agents
was significantly different from that in control, the inhibitory effect of compound 27 on the
ergosterol content in mycelia was not significantly different from that of DMIs fungicide
diniconazole, which confirmed that the inhibition of mycelial ergosterol biosynthesis was one of
the main mechanism of compound 27. It was well known that diniconazole inhibits the
biosynthesis of ergosterol by inhibiting 14α-demethylation.32 Although compound 27 and
diniconazole had similar inhibitory effects on the biosynthesis of the ergosterol of mycelium,
whether compound 27 achieved fungicidal activity by inhibiting 14α-demethylation remains to be
further studied.
In summary, a series of dehydrozingerone derivatives were synthesized, and the in vitro and in
vivo fungicidal activity were evaluated, and the primary action mechanism of compound 27
against C. musae were investigated. Most of compounds showed significant in vitro fungicidal
activity. And compared with dehydrozingerone (compound A), their activity increased by 5-10
times. Notably, compounds 13, 16, 18, 19 and 27 exhibited broad-spectrum fungicidal activity,
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which were as potent or more potent than pyrimethanil and azoxystrobin. Especially, the EC50
values of compounds 19 and 27 against R. solani were 0.943 μg mL-1 and 0.161 μg mL-1.
Moreover, compound 27 also showed excellent antibacterial activity against Xoo (EC50 = 11.386
μg mL-1). The in vivo bioassay also showed that compound 27 could effectively control gray mold
and rice sheath blight caused by B. cinereal and R. solani respectively. The preliminary action
mechanism study showed that compound 27 displayed fungicidal activity through acting on the
cell membrane to increase cell membrane permeability, resulting in cytoplasmic exudation of
mycelium. One possible reason was that compound 27 inhibited the ergosterol synthesis of
mycelium. Next work will focus on detailed mechanism study and structural optimization, and the
important progress will be reported in the future.
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ACKNOWLEGEMENT
This work was financially supported by the National Science Foundation of China (No.
21462028), the Startup Foundation for Introduced Talents of Hainan University (kyqd1640) and
Annual Science and Technology Project of Hainan Provincial Tobacco Company of CNTC (No.
2018003).
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FIGURE CAPTIONS
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Figure 1. Design strategies of target dehydrozingerone derivatives
Figure 2. Curative and Protection activities of compound 27 against rice bacterial leaf blight
under greenhouse conditions.
Figure 3. Transmission electron micrographs of C. musae hyphae in (a-d) untreated control and
(e-h) 6 μg mL-1 of compound 27-treated
Figure 4. Relative seepage ratio of the hyphae suspensions during different time exposures to
compound 27
Scheme 1. General synthetic route for compounds 1-27 and A.
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Figure 1. Design strategies of target dehydrozingerone derivatives.
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Figure 2. Curative and Protection activities of compound 27 against rice bacterial leaf blight
under greenhouse conditions.
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