Discovery of Î²-Carboline Oxadiazole Derivatives as Fungicidal Agents

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Agricultural and Environmental Chemistry

Discovery of #-Carboline Oxadiazole Derivatives as Fungicidal Agents Against Rice Sheath Blight Zhi-Jun Zhang, Zhiyan Jiang, Qi Zhu, and Guo-Hua Zhong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02124 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

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Journal of Agricultural and Food Chemistry

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Discovery of β-Carboline Oxadiazole Derivatives as

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Fungicidal Agents Against Rice Sheath Blight

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Zhi-Jun Zhang, Zhi-Yan Jiang, Qi Zhu and Guo-Hua Zhong*

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Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education,

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Key Laboratory of Crop Integrated Pest Management in South China, Ministry of

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Agriculture, South China Agricultural University, Guangzhou, 510642, P.R. China.

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ACS Paragon Plus Environment


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Abstract

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A series of β-carboline oxadiazoles were synthesized and their fungicidal activities

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and mechanism of action against rice sheath blight caused by Rhizoctonia solani was

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evaluated. The results showed that all of these compounds exhibited significant in

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vitro fungicidal activity. Significantly, compound 5i (EC50 = 4.2 µg/mL) displayed the

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best efficacy and superior fungicidal activity compared with validamycin A (EC50 =

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197.6 µg/mL). Moreover, in vivo test also demonstrated that 5i could effectively

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control rice sheath blight and showed higher in vivo protective and curative activities

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against R. solani than validamycin A. Preliminary mechanism studies revealed that 5i

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caused the loss of mitochondrial membrane potential, ROS accumulation, cell

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membrane destruction and DNA synthesis interference. These findings indicated that

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compound 5i displayed superior fungicidal activities against R. solani, and could be a

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potential fungicidal candidate against rice sheath blight.

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Keywords: β-carboline; oxadiazole; fungicidal activity; rice sheath blight

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INTRODUCTION

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Rice sheath blight, caused by Rhizoctonia solani, is one of the most important

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rice fungal diseases and has caused great yield losses in all temperate and tropical

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rice-growing regions throughout the globe.1 Each year, up to 50% decrease of rice

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grain yield has been attributed to the sheath blight when susceptible cultivars are

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planted in the United States.2,3 In China, the sheath blight affects ~15-20 million

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hectares of paddy-irrigated rice and causes a yield loss of 6 million tons of rice grains

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per year.4 Fungicides are the important measure to control rice sheath blight.5 For

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example, in China, validamycin A (jinggangmycin) has been widely used for

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controlling sheath blight disease for over 30 years.6 Unfortunately, the extensive and

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continuous use of a single fungicide increases the risk of resistance development of

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the pathogen.7 Therefore, it is necessary to develop new green fungicides against rice

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sheath blight.

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Owing to higher selectivity to control diseases and pests and the less adverse side

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effects on the environment, bioactive natural products from plant origin can be used

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as ideal lead compounds to develop biorational alternatives compared with synthetic

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agrochemicals.8,9 The β-carboline alkaloids, including harmine, harmaline and

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harmane, were the main biologically active alkaloids of medicinal plants, Peganum

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harmala,10 Grewia bicolor11 and Eurycoma longifolia.12 These compounds have

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received research attention for their interesting fungicidal activities, as well as their

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neurophysiological and pharmacological effects.13,14 In recent years, several reports

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demonstrated that β-carboline alkaloids showed marginal fungicidal activity against R.



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solani,15 indicating that β-carboline is a promising lead compound to develop novel

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fungicidal agents against rice sheath blight.16 In our previous work,17 we introduced

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urea, acylurea and acylthiourea groups into the 3-position to synthesize a series of

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β-carboline derivatives and found that β-carboline benzoylurea displayed the best in

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vitro fungicidal activity against R. solani with EC50 value of 69.5 µg/mL, which was

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8-fold more potent that harmine. However, its in vivo protective effect at a

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concentration of 100 µg/mL was only 36.2%, and was half of that of validamycin A

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(63.8%).

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Continuing our efforts on fungicidal agent discovery for the control of rice

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sheath blight, and considering that oxadiazole groups are highly efficient

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pharmacophores, with particularly fungicidal effect,18,19 and widely used in drug and

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pesticide molecular design, here we synthesized a series of β-carboline oxadiazole

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derivatives and systematically20 evaluated their in vitro and in vivo fungicidal

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activities against R. solani. Moreover, the preliminary mechanism of action of the

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most effective fungicidal candidate against R. solani was also evaluated.

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MATERIALS AND METHODS

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General. All reactions were performed with commercially available reagents and

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solvents without further purification and monitored by analytical thin-layer

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chromatography with silica gel plates using silica gel 60 GF254 (Qingdao Haiyang

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Chemical Co., Ltd., Qingdao, China). Melting points were performed using a XT-4

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melting point apparatus (Beijing Tech Instrument Co., Beijing, China). 1H-NMR and

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C NMR spectra were recorded at 600 MHz on an Bruker Avance 600 spectrometer


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(Bruker Company, Karisruhe, Gemany). High-resolution mass spectrometry (HRMS)

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data was recorded using an Uplc1290-6540B Q-TOF LC/MS instrument (Agilent Co.,

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Santa Clara, CA, USA) via direct injection. The key intermediates 1, compounds 10

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and 11 were synthesized using the procedure reported previously,21,22 and the synthesis

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and data of compounds 2a, 2d-2x, 3a-3c, 4a, 4b, 5a-5c, 5i and 5n-5p could be seen in

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our previous work.23

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General Synthetic Procedure for Compounds 2b, 2c and 2y-2ac. To a solution of

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1 (1 mmol) in absolute ethanol (10 mL) were added KOH (84 mg, 1.5 mmol) and CS2

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(0.3 mL, 5 mmol) and the reaction mixture was then heated to reflux for 24 h. After

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removal of the solvent, the water was added and the mixture was acidified with dilute

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hydrochloric acid. After filtration, the product was obtained by recrystallization from

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ethanol.

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Data for Compound 2b. Yield: 59%; mp: 257-259 °C; 1H NMR (600 MHz,

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DMSO-d6) δ: 14.69 (s, 1H, NH), 12.08 (s, 1H, 9-NH), 8.71 (s, 1H, 4-H), 8.40 (d, J =

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7.9 Hz, 1H, 5-H), 7.66 (d, J = 8.2 Hz, 1H, 8-H), 7.60 (t, J = 8.1 Hz, 1H, 7-H), 7.31 (t,

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J = 7.8 Hz, 1H, 6-H), 3.19-3.11 (m, 2H, 1'-CH2), 1.91-1.83 (m, 2H, 2'-CH2), 1.02 (t, J

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= 7.3 Hz, 3H, 3'-CH3); 13C NMR (150 MHz, DMSO-d6) δ: 177.5, 161.3, 146.8, 140.8,

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135.2, 130.2, 128.6, 127.3, 122.2, 121.0, 120.1, 113.1, 112.3, 35.4, 21.5, 13.9; HRMS

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Calcd for 311.0961 (C16H14N4OS, [M+H]+), found 311.0962.

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General Synthetic Procedure for Compounds 5d-h and 5j-m. To a mixture of 1 (302

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mg, 1 mmol) and the appropriate carboxylic acid (1 mmol) was added 5 mL

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phosphoryl chloride. After refluxed for 6 h, the excessive phosphoryl chloride was



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removed, and the residue was diluted with crushed ice, and neutralized with 5%

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NaOH solution. The residue was filtrated and recrystallized from methanol to give

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target compounds.

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Data for Compound 5d. Yield: 67%; mp: 182-184 °C; 1H NMR (600 MHz,

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DMSO-d6) δ: 11.93 (s, 1H, 9-NH), 8.98 (s, 1H, 4-H), 8.47 (d, J = 7.9 Hz, 1H, 5-H),

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8.06 (d, J = 7.0 Hz, 2H, Ar-H), 7.70 (d, J = 8.2 Hz, 1H, 8-H), 7.67 (t, J = 7.6 Hz, 2H,

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Ar-H), 7.64-7.58 (m, 2H, 7-H, Ar-H), 7.34 (t, J = 7.1 Hz, 1H, 6-H), 2.97 (t, J = 7.4 Hz,

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2H), 1.88-1.80 (m, 2H), 1.02 (t, J = 7.4 Hz, 3H, CH3);

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DMSO-d6) δ: 166.8, 164.6, 142.6, 141.7, 137.3, 133.9, 132.2, 129.7, 129.1, 128.9,

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128.6, 122.2, 120.9, 120.4, 113.9, 112.8, 26.6, 19.5, 13.4; HRMS Calcd for 355.1553

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(C22H18N4O, [M+H]+), found 355.1556.

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C NMR (150 MHz,

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General Synthetic Procedure for Compounds 6a-h. To a slurry of derivatives 1 (1

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mmol) in absolute ethanol (10 mL) was added dropwise corresponding aldehyde (1.1

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mmol) and acetic acid (1 mL), and the reaction mixture was then refluxed for 6-8 h.

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After the complete consumption of 1, the mixture was cooled to room temperature,

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and solid was precipitated out. The intermediates 12a-12h was filtrated without

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further purification.

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A solution of 12a-12h (0.5 mmol) in acetic anhydride (2 mL) was heated to

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reflux. After 2 h, the reaction mixture was cooled to room temperature, and the

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excessive acetic anhydride was removed, and the residue was poured into crushed ice.

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The residue was filtrated and recrystallized from ethanol to give the target compounds

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6a-h.


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Data for Compound 6a. Yield: 64%; mp: 247-249 °C; 1H NMR (600 MHz,

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DMSO-d6) δ: 12.04 (s, 1H, 9-NH), 11.92 (brs, 1H, -OH), 8.82 (s, 1H, 2′-H), 8.71 (s,

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1H, 4-H), 8.39 (d, J=7.8 Hz, 1H, 5-H), 7.66 (d, J=7.8 Hz, 1H, 8-H), 7.61 (q, J=7.8 Hz,

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1H, Ar-H), 7.58 (m, 1H, 7-H), 7.48 (m, 1H, Ar-H), 7.30 (m, 1H, Ar-H), 7.27 (t, J=9.0

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Hz, 1H, 6-H), 7.21 (d, J=9.0 Hz, 1H, Ar-H), 2.91 (s, 3H, 1-CH3), 2.46 (s, 3H,

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3′-COCH3).

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141.1, 138.1, 136.1, 132.5, 128.4, 127.7, 126.9, 126.3, 125.8, 123.4, 122.2, 121.4,

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120.0, 113.3, 112.2, 20.9, 20.8; HRMS Calcd for 387.1452 (C22H18N4O3, [M+H]+),

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found 387.1453.

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C NMR (151 MHz, DMSO-d6) δ: 169.4, 161.3, 157.0, 148.9, 143.2,

Synthetic details and data of the other β-carboline derivatives in Figure 1 are

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provided in the Supporting Information.

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Biological Assay

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Mycelial Growth Inhibition assay. R. solani strain GD-118 was obtained from

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our laboratory. The fungicidal activity of the target compounds against R. solani was

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assessed according to our previous method.17

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In vivo Protective and Curative Activities against Rice Sheath Blight. The rice

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cultivar 'Ruanhuayou 1179' was cultivated and used to evaluate the protective and

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curative activities against rice sheath blight with validamycin A as a positive control.17

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The rice plants were treated with target compounds by spraying with the compounds

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at concentrations of 200 and 100 µg/mL, respectively, and then inoculated with R.

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solani 24 h later. However, for the curative activity, the plants were firstly inoculated

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with R. Solani for 24 h and then sprayed with target compounds (200 and 100 µg/mL).



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All the treatments were replicated for 20 plants. The control efficacies were calculated

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as follow after 7 days inoculation.

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Control efficacy (%) =

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A0 - A1 × 100 A0

where A0 and A1 are the diameter of the lesion without or with treatment.

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Morphological Observation. Scanning Electron Microscopy (SEM).24 R. solani

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mycelia tips (0.6 cm) were cut from the edge of the colony after treated with 0 and

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100 µg/mL 5i for 72 h. After treated with 2.5% of glutaraldehyde at 4 °C, the samples

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were washed three times with 0.1 M PBS, and fixed with 1% w/v osmium tetraoxide

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solution. The samples were washed with 0.1 M PBS before being dehydrated with a

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series of ethanol solutions (30%, 50%, 70%, 80%, 90% and anhydrous ethanol). After

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drying at critical point and gold spraying, the samples were observed using a scanning

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electron microscope XL-30-ESEM (FEI, Eindhoven, the Netherlands).

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Transmission Electron Microscopy (TEM). The dehydrated mycelial blocks were

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embedded in resin at 70 °C for 24 h and then cut into thin sections. After being

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double-stained with uranyl acetate and lead citrate, the samples were observed with a

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Tecnai G2 12 transmission electron microscope (FEI).

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Sclerotia Germination Inhibition assay. R. solani were cultured for 21 days to

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obtain sclerotia.25 The culture media containing various concentrations of compound

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5i were prepared, and 15 sclerotia were placed on the culture and each concentration

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consisted of three replicates. Validamycin A served as the positive control. All the

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treatments were incubated at 25 °C for 24 h and then the inhibition rate was

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calculated.


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Sclerotia Formation Inhibition assay. R. solani mycelia tips (0.6 cm) on PDA

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medium amended with 10, 50 and 100 µg/mL 5i were cultured at 25 °C for 21 days.

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Validamycin A served as the positive control. The sclerotia formed were collected and

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dried at 60 °C for 24 h. The weight of sclerotia and the inhibitory rate were calculated.

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DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) Staining. R. solani

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mycelia tips (0.6 cm) were treated with 0 and 100 µg/mL 5i for 48 h and then placed

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on a sterile slide in a 9 cm culture dish, respectively. After being cultured at 25 °C for

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24 h, the PDA medium was removed carefully and the hyphae were stained with 10

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µM DCFH-DA solution (Beyotime, Shanghai, China). The hyphae were incubated at

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37 °C for 30 min in the darkness, and then washed twice with PBS. A cover slip was

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placed on the hyphae and the samples were observed and photographed using an

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Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan).

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Hoechst 33258 Staining.26 The hyphae were fixed with stain-fixative at 4 °C

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overnight, washed twice with PBS, and then stained with 1 mL Hoechst 33258

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solution (Beyotime) at 25 °C for 10 min. After being rinsed twice with PBS, the

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samples were observed and photographed using fluorescence microscopy.

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Rhodamine 123 Staining. The hyphae was stained with 1 mL 1 µM Rhodamine

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123 solution (Beyotime). After incubation at 37 °C for 30 min in the darkness, the

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hyphae were washed twice with PBS, and a cover slip was then placed on the hyphae.

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The samples were observed and photographed using fluorescence microscopy.

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Detection of Cell Membrane Permeability. The eight R. solani mycelial disks

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(0.6 cm) were placed in 250 mL PD liquid medium with 200 rpm shaking at 25 °C for



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three days. The mycelia were filtered and washed with sterile distilled water. The

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fresh mycelia (1 g) were added into 20 and 100 µg/mL 5i solutions which were diluted

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with 50 mL sterile distilled water. After 0, 1, 2, 4, 6, 15, 26 and 48 h, the conductivity

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was determined with a DDSJ-308F conductivity detector (Shanghai INESA Scientific

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Instrument Co. Ltd., Shanghai, China), respectively.

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Statistical Analysis. Results were expressed as the mean ± standard deviation

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(SD) and analyzed using the SPSS version 13.0. A paired Student’s t-test was used to

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determine the significant differences between two groups. p

Discovery of Î²-Carboline Oxadiazole Derivatives as Fungicidal Agents

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