Discovery of Novel Thiazole Carboxamides as Antifungal Succinate Dehydrogenase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.8b06935

To contribute molecular diversity for novel fungicide development, a series of novel thiazole carboxamides were rationally designed, synthesized, and characterized with the succinate dehydrogenase (SDH) as target. Bioassay indicated that compound 6g showed the similar excellent SDH inhibition as that of Thifluzamide with IC₅₀ of 0.56 mg/L and 0.55 mg/L, respectively. Some derivatives displayed improved in vitro fungicidal activities against Rhizoctonia cerealis and Sclerotinia sclerotiorum with EC₅₀ of 1.2-16.4 mg/L and 0.5-1.9 mg/L. Surprisingly, 6g showed promising in vitro fungicidal activities against R. cerealis and S. sclerotiorum with EC₅₀ of 6.2 and 0.6 mg/L, respectively, which was superior to Thifluzamide with the EC₅₀ of 22.1 and 4.4 mg/L, respectively. Additionally, compounds 6c and 6g displayed excellent in vivo fungicidal activities against S. sclerotiorum on Brassica napus L. leaves with protective activity of 75.4% and 67.3% at 2.0 mg/L, respectively, while Thifluzamide without activity at 5.0 mg/L. Transcriptomic analysis of S. sclerotiorum treated with 6g by RNA sequencing indicated the down-regulation of succinate dehydrogenase gene SDHA and SDHB, and the inhibition of the TCA-cycle.

Full text of DOI:10.1021/acs.jafc.8b06935

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Agricultural and Environmental Chemistry
Discovery of Novel Thiazole Carboxamides as
Antifungal Succinate Dehydrogenase Inhibitors
Xiaofeng Guo, Bin Zhao, Zhijin Fan, Dong-yan Yang, Nailou Zhang, Qifan
Wu, Bin Yu, Shuang Zhou, Tatiana Kalinina, and Nataliya P. Belskaya
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06935 • Publication Date (Web): 23 Jan 2019
Downloaded from http://pubs.acs.org on January 25, 2019
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Page 1 of 43
Journal of Agricultural and Food Chemistry
Discovery of Novel Thiazole Carboxamides as Antifungal
Succinate Dehydrogenase Inhibitors
†
,†
,†,‡
†
†
Xiaofeng Guo , Bin Zhao* , Zhijin Fan* , Dongyan Yang , Nailou Zhang , Qifan
†
†
†
§
§
Wu , Bin Yu , Shuang Zhou , Tatiana A. Kalinina , Natalia P. Belskaya
†State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry,
Nankai University, Tianjin 300071, P. R. China
‡Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),
Nankai University, Tianjin 300071, P. R. China.
§The Ural Federal University Named after the First President of Russia B. N.
Yeltsin, Yeltsin UrFU 620002, Ekaterinburg, Russia.
*
Address correspondence to these authors at State Key Laboratory of
Elemento-Organic Chemistry, College of Chemistry, Nankai University, No. 94,
Weijin Road, Nankai District, Tianjin 300071, P. R. China (telephone
+
86-13920714666; Fax:+86 022-23503620; e-mail: fanzj@nankai.edu.cn for Zhijin
Fan or binzhao@nankai.edu.cn for Bin Zhao).
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Abstract
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To contribute molecular diversity for novel fungicide development, a series of novel
thiazole carboxamides were rationally designed, synthesized and characterized with
the succinate dehydrogenase (SDH) as target. Bioassay indicated that compound 6g
showed the similar excellent SDH inhibition as that of thifluzamide with IC of 0.56
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mg/L and 0.55 mg/L, respectively. Some derivatives displayed improved in vitro
fungicidal activities against Rhizoctonia cerealis and Sclerotinia sclerotiorum with
EC50 of 1.2-16.4 mg/L and 0.5-1.9 mg/L, respectively, which was better than
thifluzamide with its EC of 22.1 mg/L and 4.4 mg/L, respectively. Surprisingly, 6g
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showed promising in vitro fungicidal activities against R. cerealis and S. sclerotiorum
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with EC of 6.2 and 0.6 mg/L, respectively, which was superior to thifluzamide with
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the EC50 of 22.1 and 4.4 mg/L, respectively. Additionally, compounds 6c and 6g
displayed excellent in vivo fungicidal activities against S. sclerotiorum on Brassica
napus L. leaves with protective activity of 75.4% and 67.3% at 2.0 mg/L, respectively,
while thifluzamide without activity at 5.0 mg/L. Transcriptome analysis of S.
sclerotiorum treated with 6g by RNA sequencing indicated the down-regulation of
succinate dehydrogenase gene SDHA and SDHB, and the inhibition of TCA-cycle.
Keyword: Succinate dehydrogenase inhibitor, Fungicide, Thiazole carboxamide,
Molecular
docking
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Introduction
Succinate dehydrogenase (SDH), also known as succinate-ubiquinone reductase
(SQR) or respiratory protein complex II, is involved in the tricarboxylic acid cycle
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and mitochondrial electron transport. Its inhibitors mainly bind to the ubiquinone
pocket of SDH and disrupt the mitochondrial respiration chain, thus leading to the
pathogen death.2-3 Succinate dehydrogenase inhibitors (SDHIs) have been
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commercialized since the earliest launching of carboxin as fungicide in 1966. To date,
19 commercial SDHI fungicides belonging to diverse chemical types, such as
oxathilin carboxamides (carboxin), benzamides (fluopyram), pyridine carboxamides
(boscalid), thiazole carboxamides (thifluzamide), furan carboxamides (fenfuram),
thiophene carboxamide (isofetamid) and pyrazole carboxamides (fluxapyroxad,
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pydiflumetofen) have been used for plant protection (Figure 1). SDHI becomes the
hotspot of the novel fungicide development due to its novel mode of action and
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broad-spectrum of activity. However, with the frequent and extensive application of
SDHI fungicides, many resistant fungi have been reported.7-9 Therefore, it’s of great
benefit to discover novel SDH inhibitor with improved potency.
It’s clear that the SDHI possesses a substituted five- or six-membered ring core,
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an amide bond and an amine moiety. In terms of commercial SDHI, the most
represented type contains the pyrazole, the substituents on the ring core are generally
halogen, alkyl and haloalkyl, the current efforts mainly focus on the modification of
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amine moiety.10-13 Therefore, discovery of novel carboxyl core moiety would be
another important direction with great chances for the novel SDHI development.
Different heterocycles always display different biological functions,14,15 both the
N and S containing heterocycles such as thiazole derivatives exhibit various
biological activity,16-18 for example, ethaboxam is a tubulin polymerization and
oxidative respiration inhibitor used for oomycetes control,19 thiazopyr inhibit cell
division by disrupting spindle microtubule formation and is used as herbicide for
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pre-emergence annual grass and some broad-leaved weeds control, while thiacloprid
acts as an agonist of the nicotinic acetylcholine receptor in the insect central nervous
system and is used by foliar application against sucking and biting insects.21 Our
group discovers different piperdinyl thiazole and isothiazole derivatives with different
fungicidal activity and mode of actions.22-24
To continue our sustainable studies on five-membered heterocyclic based novel
pesticide development with various novel lead structures and mode of action, here,
homology modeling and structure-based molecular design strategies were used for
novel SDHI development (Figure 2). We firstly built the three-dimensional (3D)
protein models of SDH from Sclerotinia sclerotiorum using SWISS-MODEL. By
molecular docking of fluxapyroxad with S. sclerotiorum SDH (SsSDH), 200
compounds with different five-membered heterocyclic carboxyl core moieties were
designed according to the structure characteristics of reported SDHI, pesticide
molecular design principles and experience. After docking these ligands into SsSDH,
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six candidates were identified (Figure S1). 4-Hydroxy thiazole was identified as
carboxyl core moiety according to the binding affinity energies and synthesis
feasibility. Herein, a novel series of thiazole carboxamide derivatives containing
hydroxy or alkoxy substituent at the 4-position of thiazole were designed and
synthesized for biologically evaluation. Pesticide target discovery and target
validation of the new lead compound were the same important tasks for novel
pesticide development.25 Docking and structure activity relationship (SAR) studies
were further conducted to validate the key structural features responsible for their
fungicidal potency. Transcriptome analysis of the highly active compound 6g was
also performed by RNA sequencing analysis for their target validation.
Materials and Methods
Equipment and materials: Melting points of new compounds were determined
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on an X-4 melting point apparatus. H NMR (400 MHz) and C NMR (101 MHz)
spectra were taken on Bruker Avance 400 MHz spectrometer in CDCl or DMSO-d
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solution. High resolution mass spectra (HRMS) were obtained by using a 7.0 T
FTICR-MS instrument. Crystal structure was recorded on a Bruker SMART
1000CCD diffraction meter.
Synthesis of Ethyl 4-Hydroxy-2-methyl Thiazole-5-carboxylate (2).
Compound 2 was synthesized according to the revisions of previously reported
procedures.26 Pyridine (73.2 g, 0.78 mol) was added to a solution of compound 1
(20.0 g, 0.26 mol) in ethanol (250 mL). Diethyl 2-bromomalonate was then added
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and the mixture was heated at 78°C for 2 h. After cooling of the mixture, the solvent
was removed by rotary evaporation and the crude residue was dissolved in ethyl
acetate (200 mL). The solution was washed with HCl (1 mol/L, 150 mL × 2) and
saturated brine (100 mL), the organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated under reduced pressure. The residue was recrystallized
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from ethanol to give white crystal of compound 2 (39.0 g 78%), m.p.: 37-39°C; H
NMR (400 MHz, CDCl ) δ 4.36 (q, J = 7.1 Hz, 2H, OCH ), 2.67 (s, 3H,
3
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thiazole-CH ), 1.37 (t, J = 7.1 Hz, 3H, OCH -CH ); C NMR (101 MHz, CDCl ) δ
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170.25, 169.02, 165.71, 95.96, 61.52, 20.29, 14.31; HRMS (ESI) m/z calcd for
+
C H NO S [M + H] : 188.0376; found: 188.0374.
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Synthesis of Ethyl 4-Alkoxy-2-methyl Thiazole-5-carboxylate (3). To a
solution of compound 2 (11.0 g, 0.059 mol) in acetone (200 mL), CH I or alkyl
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bromide (0.118 mol) was added, followed by the addition of Ag O (14.98 g, 0.065
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mol). The reaction mixture was stirred at room temperature for 8 h. The suspension
was filtered and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography with petroleum ether (60-90°C fraction):EtOAc
(30:1-20:1) as eluent to afford compounds 3.
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Data for 3a: yellow oil; yield, 75%; H NMR (400 MHz, CDCl ) δ 4.21 (q, J =
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7.0 Hz, 2H, OCH ), 4.05 (s, 3H, OCH ), 2.55 (s, 3H, thiazole-CH ), 1.25 (t, J = 7.0
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Hz, 3H, OCH -CH ); C NMR (101 MHz, CDCl ) δ 167.66, 165.43, 161.38, 99.93,
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60.64, 58.12, 20.08, 14.38; HRMS (ESI) m/z calcd for C H NO S [M + H] :
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202.0533; found: 202.0536.
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Data for 3b: yellow oil; yield, 50%; H NMR (400 MHz, CDCl ) δ 5.01 (d, J =
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2.4 Hz, 2H, C-CH ), 4.21 (q, J = 7.1 Hz, 2H, OCH ), 2.56 (s, 3H, thiazole-CH ), 2.44
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(t, J = 2.3 Hz, 1H, CH), 1.26 (t, J = 7.1 Hz, 3H, CH -CH ); C NMR (101 MHz,
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CDCl ) δ 167.73, 163.18, 161.00, 101.54, 78.52, 75.19, 60.76, 57.97, 20.09, 14.33;
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HRMS (ESI) m/z calcd for C H NO S [M + H] : 226.0538; found: 226.0534.
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Synthesis of Ethyl 4-Methoxy-2-methyl Thiazole-5-carboxylic Acid (4).
Compound 2 was synthesized according to the revisions of previously reported
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procedures. To a round bottom flask containing compound 3 (5.87 mg, 29.17 mmol)
and KOH (30 mL of a 2 mol/L solution in methanol) was added. The solution was
then heated at 65°C for 2h and the solvent was removed under reduced pressure. The
residue was added water and acidified to about pH 2 with 2 mol/L HCl. The
suspension was then filtered and filter cake was dried to obtain the compound 4 (4.09
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g, 81%) as yellow powder, m.p.: 181-183°C; H NMR (400 MHz, DMSO-d ) δ 3.97
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(s, 3H, OCH ), 2.59 (s, 3H, thiazole-CH ); C NMR (101 MHz, DMSO-d ) δ 167.97,
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164.03, 161.86, 100.23, 57.65, 19.73; HRMS (ESI) m/z calcd for C H NO S [M + H] :
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174.0220; found: 174.0220.
General Synthesis Procedures of the Compounds 5a-5q. To a solution of
compounds 4 (0.2 g, 1.15 mmol) in DMF (4 mL), N,N-diisopropylethylamine
(DIPEA, 0.57 mL, 3.45 mmol) and (3-hydroxy-3H-1,2,3-thiazolo[4,5-b]pyridinato-O)
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tri-1-pyrrolidinylphosphonium hexafluorophosphate (PyAOP, 0.66 g, 1.27 mmol)
were added at 0°C. The mixture was stirred at 0°C for 15 mins and the corresponding
amine (1.04 mmol) was added. The reaction was warmed to room temperature or
75°C according to the corresponding amine and stirred until TLC analysis showed
complete consumption of amine. The reaction mixture was dissolved in water (50 mL)
and extracted with dichloromethane (25 mL × 3). The combined organic layer was
washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered
and concentrated under educed pressure. The residue was purified by silica gel
column chromatography with petroleum ether (60-90°C fraction):EtOAc (35:1-2:1) as
eluent to afford the title compounds 5a-5r (25-98%).
General Synthesis Procedures of the Compounds 6a-6n. The compound
5a-5q (0.60 mmol) was dissolved in dry dichloromethane (10 mL) and cooled to
-20°C. The solution of BCl in dichloromethane (1 mol/L, 2 mL, 2.0 mmol) was
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dropwised and the mixture was stirred for 0.5-3 h. The reaction was quenched by the
addition of aqueous saturated NaHCO (10 mL) and extracted with dichloromethane
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(10 mL × 3). The combined organic layer was washed with saturated brine (20 mL),
dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
The residue was recrystallized from methanol to give the title compounds 6a-6n
(32-99%).
Single Crystal X-ray Diffraction Data Collection
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Single crystal X-ray diffraction data of compounds 5l and 6d were collected on a
Rigaku Saturn 724 CCD system using Mo Kα radiation. Unique reflections with Rint
and I > 2σ(I) were collected for refinements. The structure was directly solved by
SHELXS-97 program and hydrogens were introduced in idealized positions according
to the theoretical models. The derived atomic parameters were refined through full
matrix least-squares.
Fungicidal Activities
The fungicidal activities of synthesized compounds against A. s: Alternaria
solani; B. c: Botrytis cinerea; C. a: Cercospora arachidicola; G. z: Gibberella zeae;
P. i: Phytophthora infestans (Mont) de Bary; P. p: Physalospora piricola; P. s:
Pellicularia sasakii; R. c: Rhizoctonia cerealis; S. s: Sclerotinia sclerotiorum were
evaluated in vitro at 50 mg/L for the preliminary screening according to the
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previously reported procedures. The commercial SDHI fungicide thifluzamide was
selected as positive control. Compounds with good inhibitory activities were further
evaluated for their median effective concentration (EC ) values detection by the
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established procedures.27 All experiments were tested and repeated for three times.
Data were presented as the mean ± standard deviation.
The in vivo fungicidal activity of the target compounds against S sclerotiorum
was carried out on Brassica napus L. leaves. For protective activity assay, healthy
leaves of Brassica napus L. were sprayed with target the compounds (2.0 mg/L)
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sclerotiorum. For curative activity assay, healthy leaves of B. napus L. were sprayed
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with the target compounds (2.0 mg/L) respectively and then cultivated at 25 C for 24
h after inoculation with S sclerotiorum. For inactivation activity assay, S sclerotiorum
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treated with the target compounds (2.0 mg/L) respectively and then cultivated at 25 C
for 0.5 h before inoculation. The results were observed as diameters of the lesion after
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cultivation at 25 C for 36 h. Thifluzamide (5.0, 10.0 mg/L) was chosen as the positive
control. The disease control efficacy (%) was calculated as (c - d) / (c - 0.5) × 100,
where c is the diameter (cm) of the blank, and d is the diameter (cm) of the treatment.
Each treatment was performed for three times.
SDH Enzymatic Inhibition Activities
The SDH enzymatic activity was determined using the succinate dehydrogenase
assay kit (Solarbio, BC0950). S. sclerotiorum grew in Fries medium for 5 days and
then treated with various concentrations of the chosen active compounds. The SDH
enzymatic activity was determined after treatment with compounds for 24 h and the
absorbance at 600 nm was obtained by a microplate reader. Compounds were tested at
6 different concentrations at 0.05 - 20 mg/L and each compound was repeated for
three times. The IC values were calculated by GraphPad Prim version 6.02.
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The Modeling and Molecular Docking Analysis
The 3D structure profile of SsSDH (A Subunit, SS1G_07864; B Subunit,
SS1G_04384; C Subunit, SS1G_01661; D Subunit, SS1G06173) were modelled using
SWISS-MODEL with default parameters. Protein structure of individual subunits
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were built by YASARA and evaluated by Ramachandran plot, then precise structures
of the subunits were align to SsSDH structure profile which modeled by
SWISS-MODEL in order to form the accurate final structure. And the ligand binding
center of SsSDH was identified by Autodock Tools28-29 with carboxin, the ligand of
succinate dehydrogenase. The structures of ligands were optimized with the ligand
minimization protocol. The molecular docking analysis was performed by the
Autodock Tools with default values. The structure of complexes which analyzed by
docking were showed by Ligplot.30
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RNA Sequencing and Data Analysis
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S. sclerotiorum was grown in PDA at 25 C for 1 week, and subsequently treated
with compound 6g at 0.1 mg/L for 36 hours, with DMF used as control. Total RNA of
samples was extracted using TRIzol Reagent (Invitrogen, USA). cDNA libraries were
constructed using a Truseq stranded mRNA kit (Illumina, San Diego, USA).
Sequencing was conducted on an Illumina MiSeq sequencing system using the HiSeq
4000 SBS Kit (Illumina, San Diego, USA) following the manufacturer’s instructions
and S. sclerotiorum genome database was used as a reference for mapping the short
reads. The count data were normalized to generate effective library using Trimmed
Means of Means (TMM) values. Statistical analysis was performed with these data
using a generalized linear model linked to the negative binomial distribution
performed using the EdgeR package. The different expression genes were identified
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with FDR ≤ 0.05 and fold change ＞ 1.5 (or fold change < 0.67). The Kyoto
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Encyclopedia of Genes and Genomes (KEGG) pathway for each gene were extracted.
07 Results and Discussion
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Chemical Synthesis
The synthetic route for the compounds 5a-5r and 6a-6n was described in Figure
3. Thioamide 1 was reacted with diethyl 2-bromomalonate to yield the
4-hydroxythiazole 2, which underwent silver oxide mediated methylation to provide
ether 3. Hydrolysis of intermediate 3 in the solution of potassium hydroxide in
methanol obtained acid 4, which was coupled with various amines to give the
corresponding amides 5a-5r in the yield of 25-98%. The compounds 6a-6n were
obtained by demethylation of compounds 5a-5q in a yield of 38-99%. The chemical
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structures of all the title compounds 5a-5r and 6a-6n were confirmed by H NMR,
13C NMR and HRMS, All the physical and chemical properties of the target
compounds were shown in the supporting information.
The molecule structure of the target compounds 5l (CCDC: 1877458) and 6d
(CCDC: 1877459) were further identified by single crystal X-ray analysis as shown in
Figure 4 and Table S1. Both the X-ray detected structures of 5l and 6d were accorded
with its theoretical molecular structures.
Fungicidal Activities and SAR Discussion
To evaluate the fungicidal activities of all the target compounds, the in vitro
fungicidal activity determination against nine representative plant pathogens, A.
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solani, B. cinerea, C. arachidicola, G. zeae, P. infestans, P. piricola, P. sasakii, R.
cerealis and S. sclerotiorum, were initially carried out at the concentration of 50 mg/L.
As shown in Table 1, the target compounds 5i, 5o, 5p and 5q exhibited > 50% activity
against 5-7 different kinds of fungi, and the results indicated that derivatives with a
flexible amine such as benzylamine (5o), pyridylmethylamine (5p) and
phenylethanamine (5q) had a broader fungicidal spectrum. On the contrary, the target
compounds with more rigid amines, naphthylamine (5k) and tetrahydronaphthyl
amine (5l) displayed very weak fungicidal activities against nine fungi tested. In
addition, most of the compounds displayed promising fungicidal activities against S.
sclerotiorum, B. cinerea and R. cerealis at 50 mg/L. Compound 5j exhibited > 90%
activity against the above three fungi, compounds 5i, 5n, 6a, 6c, 6d, 6f, 6g and 6i
exhibited > 90% activity against both S. sclerotiorum and R. cerealis. Compounds 5a,
5b, 5c, 5d, 5f, 5g, 5q, 6b, 6e, 6j, 6k, 6m and 6n exhibited > 90% activity against S.
sclerotiorum, whereas compounds 5m and 5p exhibited > 90% activity against B.
cinerea.
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To further determine the fungicidal potency and probe the SAR of these novel
thiazole carboxamide derivatives, their EC values were tested against B. cinerea, R.
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cerealis and S. sclerotiorum, the results were shown in Table 2. In general,
compounds bearing a hydroxyl group at 4-position of thiazole showed better
fungicidal activities than their corresponding alkoxyl-substituted analogues. For
example, 6c (EC50 = 13.2, 1.3 mg/L) was more active than 5c (EC50 = 108.5, 20
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mg/L); 6d (EC = 4.7, 9.8 mg/L) was more active than 5f (EC = 59.4, 17.0 mg/L);
5
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50
6f (EC = 1.2, 2.3 mg/L) was more active than 5h (EC > 100 mg/L, > 100 mg/L); 6l
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(EC = 40.3, 4.8 mg/L) was more active than 5o (EC > 100 mg/L, = 23.4 mg/L)
5
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50
against R. cerealis and S. sclerotiorum, respectively. Compound 5i (with
3,4-F -phenyl group at the ortho-position of aniline) showed better fungicidal activity
2
than 5h (with 3,4,5-F -phenyl group at the same position), which indicated that the
3
3,4-F -phenyl group at the ortho-position of aniline was more favorable for the
2
hydrophobic interactions with SDH. As for hydroxyl-substituted thiazole series 6, we
initially explored the effect of substituents on aniline moiety, especially substituents at
the ortho-positions. The results indicated that introduction of bulky alkyl groups (6c)
and halophenyl groups (6d, 6f and 6g) at the ortho-position of aniline were favorable
for the fungicidal activities against S. sclerotiorum and R. cerealis, but not all the
fungi showed this rule. Among them, 6f (EC = 1.2, 2.3 and 33.7 mg/L) and 6g (EC
50
5
0
= 6.2, 0.6 and 42.3 mg/L) displayed the most potent fungicidal activities against R.
cerealis, S. sclerotiorum and B. cinerea, respectively. In addition, substituting the
aniline moiety with other amines, including flexible alkyl amines, naphthyl amine and
cyclic amines, was also studied. The results indicated that, naphthylamine (5k) and
tetrahydronaphthyl amine (5l) substituents decreased the fungicidal activities of the
target compounds.
Among these novel derivatives, compounds 5i, 5j, 5n, 6a, 6c, 6d, 6f, 6g and 6i
displayed improved fungicidal activities with EC50 of 1.2 - 16.4 mg/L against R.
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cerealis as compared to thifluzamide with EC50 of 22.1 mg/L. Surprisingly,
compound 6f showed the highest fungicidal activity against R. cerealis with an EC50
value of 1.2 mg/L, which was about 18-times more active than that of the
thifluzamide. As for S. sclerotiorum, compounds 5a, 6b, 6c, 6f, 6i, 6g and 6k
exhibited improved fungicidal activities with EC of 0.5 - 1.9 mg/L as compared to
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5
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the thifluzamide with EC of 4.4 mg/L. In particular, compound 5a with EC of 0.5
5
0
50
mg/L and compound 6g with EC of 0.6 mg/L displayed 7 - 9 times higher inhibitory
5
0
activity than thifluzamide against S. sclerotiorum. While, compounds 5j, 5m, 5o and
6n showed comparable activity with EC50 of 11.3 - 13.1 mg/L as compared to the
thifluzamide with EC of 10.4 mg/L against B. cinerea.
5
0
In Vivo Fungicidal Activities
According to the results of the in vitro fungicidal assay, compounds 5i, 6c, 6f
and 6g were chosen and further evaluated their greenhouse fungicidal activity in vivo
for controlling the S. sclerotiorum in B. napus L. As shown in Figure 5, compounds
6c and 6g showed promising protective activity of 75.4% and 67.3% (Table 3)
respectively at a concentration of 2.0 mg/L, while compounds 5i (2.0 mg/L), 6f (2.0
mg/L) and the positive control thifluzamide (10.0 mg/L) with negligible protective
activity. In curative activity assay, tested compounds 5i, 6c, 6f and 6g displayed
improved efficacy of 17.1 – 56.7% at the concentration of 2.0 mg/L than thifluzamide
(5.0 mg/L) with curative activity of 14.9%. In inactivation activity assay, compounds
6c and 6g showed promising protective activity of 83.9% and 86.1% respectively at
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the concentration of 2.0 mg/L, which were similar to that of thifluzamide with
inactivation activity of 92.5% at the concentration of 5.0 mg/L. The results further
indicated that, compounds 6c and 6g had great potential for the novel fungicide
development.
SDH Enzymatic Inhibition Activities
Target discovery and identification is an essential basis for novel pesticide
development and success opportunity improvement.25, 31 Enzymatic bioassay of
compounds 5i, 5j, 6c, 6f and 6g with promising fungicidal activities were selected and
further evaluated for SDH enzymatic inhibition. As showed in Table 4, compounds 5i,
5j, 6c, 6f and 6g displayed SDH inhibitory activities with IC values of 2.27, 10.77,
5
0
0.89, 2.28 and 0.56 mg/L respectively, this trend was very similar to the results of
their fungicidal activity against S. sclerotiorum bioassay. Compound 6g exhibited the
best inhibitory activity with IC50 value of 0.56 mg/L at the same level of the
thifluzamide with IC of 0.55 mg/L.
5
0
Docking Analysis
In order to elucidate the possible mechanism of newly synthesized thiazole
carboxamide derivatives and further explain the SAR, the 3D structure of SsSDH was
modeled and the docking analysis was performed. Ramachandran plot showed that the
structure of SsSDH was a good quality of model (Figure S2), which had a better
overlap with template proteins (2WQY, Figure S3). All of the derivatives were
docked into the active site of SsSDH and the docking scores were shown in Table S3.
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The detailed interactions of two representative derivatives (6g and 5l) with SsSDH
were provided in Figure 6. Active compound 6g (Figure 6A) showed the similar
binding mode with that of commercial fluxapyroxad (Figure 2). The amide group of
6g and fluxapyroxad formed a hydrogen bond with the side chain of Trp230.
Moreover, the halophenyl moiety at the ortho-position of aniline was deeply buried
into the active site by hydrophobic interactions. While all the favorable interactions
were absent from the docked complex of inactive compound 5l and SsSDH (shown in
Figure 6B), which resulted in the sharply decreased fungicidal activity of 5l. In order
to confirm the accuracy of the molecular docking, combining the homology analysis
results of SsSDH and 2WQY (data not shown) with the single crystal data of 2WQY.
Trp172 ， Trp173, Trp32 and Pro169 were identified as the binding site between
carboxin and protein, Arg43 was the binding site of Heme. It was consistent with the
docking results, the same binding site in SsSDH were Trp229, Trp230, Trp81, Pro146
and Arg88, which were 6g and Heme binding site respectively (Figure 6A).
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The RNA-seq Analysis of S. sclerotiorum Treated with 6g
The different expression genes (DGEs) affected by 6g was identified by RNA
sequencing. Totally 2831 DGEs with fold changes > 1.5 (or < 0.67) were identified in
6g vs CK. Among these DGEs, there were 1562 up-regulated genes and 1269
down-regulated genes. As indicated in Figure 7, the expression of succinate
dehydrogenase gene SDHA and SDHB were down-regulated. Because of the activity
down-regulation of the SDH, the whole TCA-cycle in fungi was inhibited, and thus
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led to the down-regulation of aconitase (ACO1 and ACO2), isocitrate dehydrogenase
(IDH1, IDH2 and IDH3), 2-oxoglutarate dehydrogenase (OGDH1), succinate-CoA
ligase (LSC1), fumarate hydratase (FUMC), and malate dehydrogenase (MDH2).
While, the pathway for citric acid formation by oxaloacetate in TCA cycle was
inhibited, and thus resulting in significant up-regulation of the phosphoenolpyruvate
carboxykinase (PCK1) which converted oxaloacetate into phosphoenolpyruvate and
carbon dioxide, and the down–regulation of the pyruvate carboxylase (PYC1) which
converted pyruvate into oxaloacetate. However, the expression of pyruvate kinase
(PK) and citrate synthase (CS) were not significantly changed.
To further identify the function of the biological pathways in S. sclerotiorum
which affected by 6g, the KEGG pathway was performed for functional enrichment
classification of the 2381 DGEs. As shown in Figure 8, the biological process in the
pentose and the glucuronate interconversion, starch and sucrose metabolism, and TCA
cycle had a significant enrichment change. Additionally, the pyruvate metabolism,
fatty acid degradation, and amino acid metabolism were also changed, which was
reported to be related to TCA cycle.32
Abbreviations Used
SDH, succinate dehydrogenase; EC , median effective concentration; DIPEA,
5
0
N,N-diisopropylethylamine; PyAOP, (3-hydroxy-3H-1,2,3-thiazolo[4,5-b]pyridinato
-O)tri-1-pyrrolidinylphosphonium hexafluorophosphate; TMM, Trimmed Means of
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Means values; KEGG. Kyoto Encyclopedia of Genes and Genomes; DGEs, different
expression genes; IDH, isocitrate dehydrogenase; OGDH1, 2-oxoglutarate
dehydrogenase; LSC, succinate-CoA ligase; FUMC, fumarate hydratase; MDH,
malate dehydrogenase; PCK, phosphoenolpyruvate carboxykinase; PYC, pyruvate
carboxylase; PK, pyruvate kinase; CS, citrate synthase.
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Acknowledgment
This work was supported in part by the National Natural Science Foundation of China
(No. 31571991, No. 31872007 and No. 3181101739), the Tianjin Natural Science
Foundation (No. 18JCZDJC33500), the China Postdoctoral Science Foundation (No.
2017M611156), and the International Science & Technology Cooperation Program
of China (No. 2014DFR41030). We gratefully thank Huabao Chen in College of
Agronomy, Sichuan Agricultural University for the in vivo experiments of the
compounds against S. sclerotiorum on Brassica napus L. leaves.
Supporting Information
1
13
H NMR, C NMR data for the target compounds; Crystal data of 5l and 6d; EC
5
0
value of target compounds. This material is available free of charge via the Internet at
http: //pubs.acs.org.
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mitochondrial
pyruvate
transport.
Mol.
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FIGURE CAPTIONS
Figure 1. Representative chemical structure of commercialized succinate
dehydrogenase inhibitor (SDHI) fungicides.
Figure 2. Molecular design of the target compounds.
Figure 3. General synthetic procedure for the target compounds 5a-5r and 6a-6n.
o
Reagents and conditions: (i) diethyl 2-bromomalonate, pyridine, ethanol, 78 C, 5 h; (ii)
1
CH I or R Br, Ag O, acetone, r.t. 10 h; (iii) 2 mol/L KOH in MeOH, 2 mol/L HCl,
3
2
o
65 C, 2 h; (iv) DIPEA, PyAOP, amine, DMF, r.t. 10 h; (v) 2 mol/L BCl in CH Cl ,
3
2
2
o
CH Cl -20 C, 2 h.
2
2,
Figure 4. X-ray crystal structure of compounds 5l (A) and 6d (B).
Figure 5. In vivo activity of target compounds against S. sclerotiorum infected cole
leaves. (A) Protective activity. (B) Curative activity. (C) Inactivation activity.
Figure 6. Binding modes of 6g (A) and 5l (B) with S. sclerotiorum SDH.
Figure 7. The expression of metabolic enzyme related genes in TCA cycle in S.
sclerotiorum treated with 6g. Citrate synthase, CS1 (SS1G_04899). Aconitase, ACO1
(SS1G_11047), ACO2 (SS1G_10635). Isocitrate dehydrogenase, IDH1
(SS1G_04924), IDH2 (SS1G_09958), IDH3 (SS1G_04160). 2-Oxoglutarate
dehydrogenase, OGDH1 (SS1G_05934). Succinate-CoA ligase, LSC1 (SS1G_07524).
Succinate dehydrogenase, SDH1 (SS1G_07864), SDH2 (SS1G_04384). Fumarate
hydratase, FUMC (SS1G_05243). Malate dehydrogenase, MDH2 (SS1G_13825).
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Phosphoenolpyruvate carboxykinase, PCK1 (SS1G_02281). Pyruvate carboxylase,
PYC1 (SS1G_12839). Pyruvate kinase, PK (SS1G_04568).
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Figure 8. The DEGs enriched in the biological pathway.
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Table 1. In Vitro Fungicidal Activities (Inhibition Rate / %) of the Target Compounds at 50 mg/L
Compd
A.sb
B.c
C.a
G.z
P.i
P.p
P.s
R.c
S.s
5
a
20.6±0.8 55.8±2.8
14.7±1.1 27.8±1.0
29.4±4.2 86.7±0.2
17.6±4.8 51.1±4.7
38.2±0.1 69.4±1.1
9.8±2.2
5.9±1.5
2.9±0.7 18.2±2.1 10.5±4.5
8.8±1.5 36.4±2.2 13.2±4.8
0
0
8.2±1.7
95.4±1.5
91.8±0.5
90.1±3.3
91.3±3.4
82.9±0.2
90.7±2.1
95.7±1.8
53.8±3.0
95.0±2.9
91.6±2.3
46.4±2.2
0
5
b
48.2±0.2
50.6±1.1
47.4±1.7
51.0±1.2
46.0±4.2
80.2±2.9
12.2±4.2
96.9±0.8
95.2±0.8
38.2±4.9
51.6±1.9
85.1±0.7
90.1±1.5
81.0±1.1
57.4±1.1
70.7±3.9
93.5±0.3
94.3±3.2
78.8±4.6
90.7±2.0
5
c
13.7±4.9 26.5±4.7 18.2±2.4 13.2±4.5
13.7±1.9 5.9±4.7 18.2±0.7 13.2±2.4
37.3±4.9 11.8±3.7 22.7±2.1 26.3±4.1
7.3±3.6
0
5
d
5
e
7.3±2.0
5
f
0
73.8±3.6
10.0±4.5 41.7±2.1
5.3±2.1 31.3±0.2
10.6±4.9 10.1±0.3 11.5±0.7 11.5±0.1 10.3±1.0
15.7±4.5 10.5±3.5 18.0±0.5 12.5±3.9 11.3±4.7
5
g
5
h
15.4±4.2
88.2±3.9 58.8±0.6 22.7±0.1 5.3±2.1
3.9±2.7 23.5±4.0 11.4±5.0
5.4±2.3
7.7±4.0
0
10.3±2.6
12.2±2.2
0
5
i
67.6±1.1 50.0±4.0
20.6±2.4 92.0±3.4
29.4±2.2 41.7±2.2
29.4±0.3 11.1±4.4
23.5±3.2 90.0±2.1
5
j
0
5
k
21.6±3.8 17.6±5.8 13.6±1.2 26.3±1.9 12.2±2.7
5
l
25.5±4.9 23.5±5.1 11.4±0.1 13.2±2
0
0
5
m
29.4±1.6
6.7±3.4
0
18.2±4.4
0
0
80.8±4.0
91.5±3.3
83.6±3.2
86.4±4.5
90.9±1.0
64.0±1.4
93.1±0.8
98.3±0.9
95.6±3.7
5
n
0.0±0.7
81.5±1.2
5.0±2.2
18.0±1.2
3.4±3.0
5
o
p
q
52.9±4.5 41.7±0.6
47.1±2.9 92.0±1.0
88.2±2.0 69.4±3.0
54.9±4.3 54.2±4.3 18.2±4.9 26.3±1.2 12.2±2.6
39.2±5.1 67.6±3.6 27.3±2.4 52.6±4.8 12.2±2.5
68.6±3.1 70.6±0.5 54.5±0.3 46.1±1.7 36.6±0.6
5
5
5
r
0
22.2±2.8
80.8±0.4 25.0±3.8 23.8±2.5 5.8±3.6
20.0±5.4
6
a
23.5±4.5 47.5±1.6
10.5±3.9 68.3±0.8
23.5±1.7 55.6±3.0
13.7±2.4
13.3±4.2
5.9±3.8
5.9±4.1 20.5±4.6 26.3±4.4
5.0±8.5 7.7±1.0 4.9±2.5
8.8±4.6 27.3±1.4 44.7±3
0
6.9±4.9
0
6
b
6
c
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Page 29 of 43
Journal of Agricultural and Food Chemistry
6
d
15.8±4.5 60.6±0.2
21.1±0.7 60.0±2.2
15.8±3.7 65.0±4.0
47.1±0.8 60.0±3.5
17.6±1.2 44.4±3.8
31.6±2.8 40.6±2.9
23.5±1.5 63.3±3.9
47.4±0.2 40.6±4.5
15.8±2.4 42.5±3.6
10.5±4.9 55.3±4.6
10.5±4.1 63.8±4.9
89.5±2.8 86.3±0.6
26.7±1.3 15.0±1.3 38.5±1.4 14.8±2.7 17.2±3.6
95.2±1.0
36.6±3.6
98.0±1.9
91.0±3.6
50.6±0.6
90.2±0.3
73.8±4.4
17.1±3.7
81.0±1.7
57.1±2.6
82.2±3.6
76.6±2.2
33.6±6.2
38.1±5.8
90.4±3.9
91.6±2.3
95.7±3.5
100.0
6
e
33.5±3.5
26.7±3.8
5.3±3.1
0
0
18.0±3.4 27.6±2.8
6
f
19.2±3.8 8.2±8.0 10.3±0.5
6
g
19.6±4.3 23.5±4.0 31.8±1.6 39.5±3.1 12.2±0.2
6
h
7.8±1.2
8.8±3.0 13.6±1.6 26.3±0.4
0
83.9±4.8
90.8±1.4
91.6±3.6
90.1±0.1
79.5±3.4
93.9±3.5
95.4±4.2
90.1±4.4
88.2±0.9
84.0±4.3
6
i
10.5±3.4 15.0±3.1 11.5±3.1 3.3±7.1
17.2±4.3
7.3±0.7
3.4±1.9
6
j
33.3±5.1
3.3±3.6
2.9±3.1 18.2±0.5
20.0±4.0 11.5±3.2 4.9±3.8
5.5±3.3
0
6
k
6
l
10.3±2.5
7.7±2.1 27.9±4.2 17.2±2.9
6
m
10.1±0.7 15.1±2.5 5.8±0.4
3.3±3.9 5.4±4.9 3.8±3.4
6.6±0.1
8.2±0.4
13.8±4.2
17.2±3.7
6
n
Thifluzamide
Fluxapyroxad 38.0±1.9
Isopyrazam 27.0±2.6 64.0±2.3
20.1±1.3 80.0±3.2 23.1±1.9 27.9±2.9 27.6±0.3
100.0
100.0
0
0
0
0
38.4±0.9
50.3±4.6
0
0
48.0±1.1
aValues are the mean ± standard deviation (SD) of three replicates
bA. s: Alternaria solani; B. c: Botrytis cinerea; C. a: Cercospora arachidicola; G. z: Gibberella zeae; P. i: Phytophthora infestans (Mont) de Bary; P. p:
Physalospora piricola; P. s: Pellicularia sasakii; R. c: Rhizoctonia cerealis; and S. s: Sclerotinia sclerotiorum.
2
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