Synthesis and Biological Activity of Novel Succinate Dehydrogenase Inhibitor Derivatives as Potent Fungicide Candidates

Article abstract of DOI:10.1021/acs.jafc.9b05751

In searching for novel fungicidal leads, the novel bioactive succinate dehydrogenase inhibitor (SDHI) derivatives were designed and synthesized by the inversion of carbonyl and amide groups. Bioassay indicated that compound 5i stood out with a broad spectrum of in vitro activity against five fungi. Its EC₅₀ value (0.73 μg/mL) was comparable to that of boscalid (EC₅₀ of 0.51 μg/mL) and fluxapyroxad (EC₅₀ of 0.19 μg/mL) against Sclerotinia sclerotiorum. For Rhizoctonia cerealis, 5i and 5p with EC₅₀ values of 4.61 and 6.48 μg/mL, respectively, showed significantly higher activity than fluxapyroxad with the EC₅₀ value of 16.99 μg/mL. In vivo fungicidal activity of 5i exhibited an excellent inhibitory rate (100%) against Puccinia sorghi at 50 μg/mL, while the positive control boscalid showed only a 70% inhibitory rate. Moreover, 5i showed promising fungicidal activity with a 60% inhibitory rate against Rhizoctonia solani at 1 μg/mL, which was better than that of boscalid (30%). Compound 5i possessed better in vivo efficacy against P. sorghi and R. solani than boscalid. Molecular docking showed that even the carbonyl oxygen atom of 5i was far from the pyrazole ring. It could also form hydrogen bonds toward the hydroxyl hydrogen and amino hydrogen of TYR58 and TRP173 on SDH, respectively, which consisted of the positive control fluxapyroxad. Fluorescence quenching analysis and SDH enzymatic inhibition studies also validated its mode of action. Our studies showed that 5i was worthy of further investigation as a promising fungicide candidate.

Full text of DOI:10.1021/acs.jafc.9b05751

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Functional Structure/Activity Relationships
Synthesis and Biological Activity of Novel Succinate Dehydrogenase
Inhibitor Derivatives as Potent Fungicide Candidates
Dong-yan Yang, Bin Zhao, Zhijin Fan, Bin Yu, Nailou Zhang, Zheng-
Ming Li, Yilin Zhu, jinghui Zhou, Tatiana Kalinina, and Tatiana Glukhareva
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b05751 • Publication Date (Web): 07 Nov 2019
Downloaded from pubs.acs.org on November 8, 2019
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Synthesis and Biological Activity of Novel Succinate Dehydrogenase Inhibitor
Derivatives as Potent Fungicide Candidates
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Dongyan Yang†, Bin zhao†, Zhijin Fan†,*, Bin Yu†, Nailou Zhang†, Zhengming Li †,*, Yilin Zhu†, Jinghui
Zhou†, Tatiana A. Kalinina‡, Tatiana V. Glukhareva‡
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† State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, No. 94,
Weijin Road, Tianjin 300071, P. R. China
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‡ The Ural Federal University Named after the First President of Russia B. N. Yeltsin, Yeltsin UrFU
620002, Ekaterinburg, Russia
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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 nkzml@vip.163.com for Zhengming Li).
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Abstract
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In searching for novel fungicidal leads, the novel bioactive succinate dehydrogenase
inhibitor (SDHI) derivatives were designed and synthesized by the inversion of carbonyl and
amide groups. Bioassay indicated that compound 5i was outstood with broad-spectrum of in vitro
activity against five fungi, its EC50 value (0.73 μg/mL) was comparable to boscalid ( EC50 of 0.51
μg/mL) and fluxapyroxad (EC50 of 0.19 μg/mL) against Sclerotinia sclerotiorum. For
Rhizoctonia cerealis, 5i and 5p with the EC50 of 4.61 μg/mL and 6.48 μg/mL respectively,
showed significant higher activity than fluxapyroxad with the EC50 of 16.99 μg/mL. In vivo
fungicidal activity of 5i exhibited excellent inhibitory rate (100%) against Puccinia sorghi at 50
μg/mL, while the positive control boscalid showed only 70% of inhibitory rate. Moreover, 5i
showed promising fungicidal activity with 60% inhibitory rate against Rhizoctonia solani at 1
μg/mL, which was better than boscalid (30%). Compound 5i possessed better in vivo efficacy
against P. sorghi and R. solani than boscalid. Molecular docking showed, even the carbonyl
oxygen atom of 5i was far from pyrazole ring, it could also form hydrogen bonds towards the
hydroxyl hydrogen, amino hydrogen of TYR58 and TRP173 on SDH, respectively, which
consisted with the positive control fluxapyroxad. Fluorescence quenching analysis and SDH
enzymatic inhibition studies also validated its mode of action. Our studies showed that 5i was
worthy for further investigation as a promising fungicide candidate.
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Key words：SDHIs, amide inversion, molecular docking, fungicidal activity, rice disease
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Introduction
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Plant diseases cause a dramatic decline in crop quality and yield, farmers suffer huge
economic losses.1, 2 Treating crops by fungicides with different mode of action to against plant
disease damage is always necessary.3 Succinate dehydrogenase (SDH) inhibitors are widely used
to protect crops from all kinds of plant pathogens attack.4-7 As an important membrane complex
in the tricarboxylic acid cycle in mitochondrial respiratory chain, SDH is an ideal fungicide
target.8, 9 SDHIs disrupt the respiration of the pathogen and inhibit its growth by inhibiting the
oxidation of succinate to fumarate, and thus cause the pathogen death.10, 11
Among the SDHIs, pyrazole amide derivatives have a central place in fungicidal
chemistry because of their broad spectrum of high efficiency, low toxicity and structure diversity.
As shown in Figure 1, furametpyr was invented by Sumitomo Chemical in 1986,12 and then,
penflufen was developed by Bayer Cropscience in 2006.13 In recent decades, several pyrazole
contained fungicides, such as sedaxane,14 bixafen,15 fluxapyroxad,16 benzovindiflupyr,17
pydiflumetofen18 and so on, have made a significant contribution to the crop protection against
plant diseases. All these representative compounds containing pyrazole moiety show outstanding
fungicidal activity. The wide practical interest in this important pyrazole moiety motivates
continuous efforts of the exploitation of efficient compounds.19-21
In research for some commercially available aromatic amide fungicides, we found
mebenil and benodanil, which could be seen as a couple of mirror compounds.22 Many
documents reported the lead modification of SDHIs, but the design strategy by inversion of
carbonyl and amide groups was rarely used in the studies of novel pyrazole fungicide
development. On the basis of the development of fungicides targeted at SDH and prompted by
the excellent biological activity of pyrazole carboxamide fungicides, we attempted to investigate
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the structure of pyrazole-contained derivatives, and target compounds were designed and
synthesized by inversion of carbonyl and amide groups of fluxapyroxad (Figure 2). All of the
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target compounds were identified by H NMR, elemental analysis and HRMS. The in vitro
fungicidal activities of these compounds were evaluated against eight common plant pathogenic
fungi and in vivo fungicidal activities of these compounds were tested against three different
crop diseases. Furthermore, the enzymatic activity and fluorescence quenching analysis of 5i to
SDH were investigated to validate its mode of action.
MATERIALS AND METHODS
Equipment and Materials
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Melting points were determined using an X-4 melting point apparatus and were
uncorrected (Beijing Tech Instruments Co., Beijing, China). 1H NMR spectra was obtained using
a Bruker Avance 400 MHZ spectrometer at 400 MHz in deuterochloroform (CDCl3) and
tetramethylsilane (TMS) as internal standards (Bruker, Switzerland). Elemental analyses were
performed on a Vario EL III elemental analysis instrument (Elementar, Germany). High
resolution mass spectrometry data were obtained with an Agilent 6520 Q-TOF LC/MS
instrument equipped with electrospray ionization (ESI) source (Agilent, America). Crystal
structure was recorded by a Rigaku 007 Saturn 70 diffraction meter (Rigaku MSC, Japan). All
solvents were of analytical grade. All of the yields were not optimized. The synthesis procedure
of intermediate 4 and all the corresponding data could be found in the Supplementary
Information.
Molecular Docking. The molecular structures were drawn by ChemBioDraw Ultra 14.0
and energetically minimized by using Chemdraw 3D Pro MM2 minimized procedure, then saved
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as mol2 file. The structure of SDH from Gallus gallus (pdb code: 2FBW)23 was downloaded
from RCSB PDB database and the docking program was performed with SYBYL-X 6.91. The
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detail docking procedure was conducted·according to the reported literature.24
Synthetic Procedures
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General Synthesis Procedure for Intermediate (1). The Methyl 2-iodobenzoate (3.6mmol),
substituted phenylboronic acid (4.5mmol) and dichloro-bis(triphenyl-phosphine)palladium(II)
(5%mmol) were added to a three-neck round-bottom flask equipped with a magnetic stir bar and
a reflux condenser. Under an atmosphere of argon, 15 mL of THF was injected to the mixture by
syringe, and then 1mol/L Na2CO3 was added in one portion. The flask was put into an oil bath
and heated to 65 °C for 12h. After completion, the flask was cooled to room temperature; 30ml
water was added and then extracted with CH2Cl2 (3×15 mL). The organic layer was combined
and dried over anhydrous Na2SO4. Solvent was removed under the reduced pressure. The residue
was subjected to silica gel chromatography using PE and EtOAc (50:1, v/v) as an eluent to afford
intermediate (1) and used for the next step without further purification.
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General Synthesis Procedure for Intermediate (2).
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A solution of 1 (2.8mmol) dissolving in 10 mL THF was added in NaOH (28mmol, in
10mL H2O). The mixture was heated to 80℃ and monitored by TLC until the reaction was
complete. Solvent was removed under the reduced pressure. The residue was acidified by 2mol/L
HCl and precipitation was appeared. The precipitated solid was filtered out and recrystallized in
CH2Cl2 and hexane to give pure products in an 88-99% yield.
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Data for 3',4',5'-trifluoro-[1,1'-biphenyl]-2-carboxylic acid (2a). Yield 95%; white solid;
mp 155-156 °C. 1H NMR (400 MHz, CDCl3) δ 10.80 (s, 1H), 8.03 (dd, J=7.8, 1.4 Hz, 1H), 7.60
(td, J =7.5, 1.4 Hz, 1H), 7.49 (td, J =7.7, 1.3 Hz, 1H), 7.30 (dd, J =7.6, 1.3 Hz, 1H), 6.99 – 6.87
(m, 2H).
Data for 3',4'-dichloro-[1,1'-biphenyl]-2-carboxylic acid (2b). Yield 96%; white solid; mp
173-174 °C. 1H NMR (400 MHz, CDCl3) δ 9.59 (s, 1H), 7.97 (d, J =7.8 Hz, 1H), 7.60 (t, J =7.5
Hz, 1H), 7.47 (t, J =7.6 Hz, 1H), 7.41 (d, J =8.4 Hz, 2H), 7.32 (d, J =7.6 Hz, 1H), 7.13 (dd, J
=8.2, 2.0 Hz, 1H).
Data for 4'-chloro-[1,1'-biphenyl]-2-carboxylic acid (2c). Yield 99%; white solid; mp 170-
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171°C. H NMR (400 MHz, CDCl3) δ 10.92 (s, 1H), 7.98 (dd, J =7.8, 1.4 Hz, 1H), 7.57 (td, J
=7.6, 1.4 Hz, 1H), 7.44 (td, J =7.6, 1.3 Hz, 1H), 7.39 – 7.30 (m, 3H), 7.29 – 7.22 (m, 2H).
Data for 4'-(tert-butyl)-5-fluoro-[1,1'-biphenyl]-2-carboxylic acid (2d). Yield 88%; white
1
solid; mp 149-150 °C. H NMR (400 MHz, CDCl3) δ 7.97 (dd, J =8.4, 5.9 Hz, 1H), 7.40 (d, J
=8.1 Hz, 2H), 7.27 (s, 1H), 7.25 (d, J =1.8 Hz, 1H), 7.12 – 7.03 (m, 2H), 1.35 (s, 9H).
Data for 3',4',5,5'-tetrafluoro-[1,1'-biphenyl]-2-carboxylic acid (2e). Yield 95%; white
solid; mp 186-187 °C. 1H NMR (400 MHz, CDCl3) δ 8.11 (dd, J =8.8, 5.7 Hz, 1H), 7.20 (ddd, J
=8.8, 7.7, 2.6 Hz, 1H), 7.03 (dd, J =8.9, 2.6 Hz, 1H), 6.99 – 6.90 (m, 2H).
Data for 3',4'-dichloro-5-fluoro-[1,1'-biphenyl]-2-carboxylic acid (2f). Yield 94%; white
solid; mp 182-183°C. 1H NMR (400 MHz, CDCl3) δ 8.08 (td, J =8.3, 6.9, 3.5 Hz, 1H), 7.46 (dd,
J =8.4, 3.5 Hz, 1H), 7.41 (q, J =2.2 Hz, 1H), 7.15 (dddd, J =10.5, 6.8, 4.8, 2.0 Hz, 2H), 7.02 (dq,
J =9.2, 2.6 Hz, 1H).
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Data for 5-fluoro-[1,1'-biphenyl]-2-carboxylic acid (2g). Yield 98%; white solid; mp 111-
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112°C. H NMR (400 MHz, CDCl3) δ11.34 (s, 1H), 7.99 (dd, J =8.6, 5.8 Hz, 1H), 7.38 (dd, J
=5.5, 1.8 Hz, 3H), 7.30 (dt, J =6.7, 1.6 Hz, 2H), 7.14 – 7.02 (m, 2H).
Data for 4'-chloro-5-fluoro-[1,1'-biphenyl]-2-carboxylic acid (2h). Yield 93%; white solid;
mp 150-151 °C. 1H NMR (400 MHz, CDCl3) δ 10.87 (s, 1H), 8.06 (dd, J =8.7, 5.8 Hz, 1H), 7.42
– 7.36 (m, 2H), 7.28 – 7.22 (m, 2H), 7.15 (ddd, J =8.8, 7.8, 2.6 Hz, 1H), 7.04 (dd, J =9.2, 2.6 Hz,
1H).
General Synthesis Procedure for Intermediate (3).
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Compound 2 (3 mmol) and CH2Cl2 (30 mL) was added to a 100 mL single-neck round
bottom flask and stirred in an ice bath. To the mixture, 2 drops of DMF was added and 6 mmol
of oxalyl chloride solution was added dropwise. The mixture was slowly rise to room
temperature, after stirring at room temperature for 2 h, the mixture was concentrated under the
reduced pressure to remove excess oxalyl chloride solution and solvent to afford intermediate (3)
and used for the next step without further purification.
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General Synthesis Procedure for target compound (5).
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In a 100 mL one-neck round bottom flask, 3 mL of compound 3 was added and dissolved
in 30 mL of dry CH2Cl2. After the addition of 6 mmol of triethylamine, the mixture was stirred
for 10 min. in an ice bath. Compound 4 (3 mmol) was dissolved in 5 mL of dry CH2Cl2 and
added dropwise to the reaction system, then the mixture was stirred for 30 min in an ice bath, and
stirred at room temperature for 4 h. After the reaction was completed, 15 mL water was added to
the reaction solution and extracted with CH2Cl2 (3×15 mL). The organic layer was combined and
washed with brine, dried over anhydrous sodium sulfate, filtered. The solvent was removed
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under reduced pressure. The residue was subjected to silica gel chromatography using PE and
EtOAc (10:1-2:1, v/v) as an eluent to afford the target compounds 5a-5u (46-90%).
Data for N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-4'-(trifluoromethyl)-[1,1'-
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biphenyl]-2-carboxamide (5a). Yield 49%; white solid; mp 153-154 °C. H NMR (400 MHz,
CDCl3) δ 8.25 (s, 1H), 7.81 (dd, J =7.6, 1.5 Hz, 1H), 7.67 (d, J =8.1 Hz, 2H), 7.60 (td, J =7.5, 1.5
Hz, 1H), 7.56 – 7.49 (m, 3H), 7.43 (dd, J =7.6, 1.4 Hz, 1H), 7.11 (s, 1H), 3.90 (s, 3H). Elemental
anal. calcd for C19H13F6N3O: C, 55.21; H, 3.17; N, 10.17. Found: C, 54.98; H, 3.52; N, 9.92.
HRMS (ESI) m/z calcd for C19H14F6N3O (M+ H)+ 414.1036, found 414.1035.
Data for N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-4'-(trifluoromethyl)-[1,1'-
1
biphenyl]-2-carboxamide (5b). Yield 61%; white solid; mp 132-133°C. H NMR (400 MHz,
CDCl3) δ 8.19 (s, 1H), 7.80 (dd, J=7.6, 1.5 Hz, 1H), 7.66 (d, J=8.1 Hz, 2H), 7.59 (td, J=7.5, 1.5
Hz, 1H), 7.55 – 7.47 (m, 3H), 7.42 (dd, J=7.5, 1.4 Hz, 1H), 7.30 (s, 1H), 6.48 (t, J=54.6 Hz, 1H),
3.86 (s, 3H). Elemental anal. calcd for C19H14F5N3O: C, 57.73; H, 3.57; N, 10.63. Found: C,
57.09; H, 3.26; N, 10.63. HRMS (ESI) m/z calcd for C19H15F5N3O+ (M+ H)+ 396.1130, found
396.1131.
Data for 4'-methyl-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-biphenyl]-2-
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carboxamide (5c). Yield 68%; white solid; mp 115-116°C. H NMR (400 MHz, CDCl3) δ 8.26
(s, 1H), 7.82 (dd, J=7.7, 1.5 Hz, 1H), 7.51 (td, J=7.5, 1.5 Hz, 1H), 7.43 (td, J=7.5, 1.4 Hz, 1H),
7.38 (dd, J=7.6, 1.4 Hz, 1H), 7.28 – 7.22 (m, 2H), 7.18 (d, J=7.9 Hz, 2H), 7.08 (s, 1H), 3.86 (s,
3H), 2.33 (s, 3H). Elemental anal. calcd for C19H16F3N3O: C, 63.51; H, 4.49; N, 11.69. Found: C,
63.06; H, 4.64; N, 11.56. HRMS (ESI) m/z calcd for C19H17F3N3O (M+ H)+ 360.1318, found
360.1322.
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Data for N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-4'-methyl-[1,1'-biphenyl]-2-
carboxamide (5d). Yield 79%; white solid; mp 140-141 °C. 1H NMR (400 MHz, CDCl3) δ 8.21
(s, 1H), 7.82 (dd, J=7.7, 1.5 Hz, 1H), 7.53 (td, J=7.5, 1.5 Hz, 1H), 7.49 – 7.36 (m, 2H), 7.34 –
7.25 (m, 3H), 7.21 (d, J=7.9 Hz, 2H), 6.39 (t, J=54.7 Hz, 1H), 3.85 (s, 3H), 2.36 (s, 3H).
Elemental anal. calcd for C19H17F2N3O: C, 66.85; H, 5.02; N, 12.31. Found: C, 66.93; H, 5.43;
N, 12.23. HRMS (ESI) m/z calcd for C19H18F2N3O (M+ H)+ 342.1412, found 342.1414.
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Data
for
3',4',5'-trifluoro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-
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biphenyl]-2-carboxamide (5e). Yield 73%; white solid; mp 150-151 °C. H NMR (400 MHz,
CDCl3) δ 8.23 (s, 1H), 7.73 (dd, J=7.6, 1.5 Hz, 1H), 7.55 (dtd, J=22.9, 7.5, 1.5 Hz, 2H), 7.37 (dd,
J=7.5, 1.4 Hz, 1H), 7.22 (s, 1H), 7.09 – 6.96 (m, 2H), 3.92 (s, 3H). Elemental anal. calcd for
C18H11F6N3O: C, 54.14; H, 2.78; N, 10.52. Found: C, 54.20; H, 2.96; N, 10.35. HRMS (ESI) m/z
calcd for C18H12F6N3O (M+ H)+ 400.0879, found 400.0882.
Data
for
N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-3',4',5'-trifluoro-[1,1'-
1
biphenyl]-2-carboxamide (5f). Yield 88%; white solid; mp 142-143°C. H NMR (400 MHz,
CDCl3) δ 8.21 (s, 1H), 7.76 (dd, J =7.5, 1.6 Hz, 1H), 7.62 – 7.48 (m, 2H), 7.42 (s, 1H), 7.38 (dd,
J =7.5, 1.4 Hz, 1H), 7.11 – 6.99 (m, 2H), 6.61 (t, J =54.5 Hz, 1H), 3.89 (s, 3H). Elemental anal.
calcd for C18H12F5N3O: C, 56.70; H, 3.17; N, 11.02. Found: C, 56.52; H, 3.27; N, 11.02. HRMS
(ESI) m/z calcd for C18H13F5N3O (M+ H)+ 382.0973, found 382.0975.
Data for N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-[1,1'-biphenyl]-2-carboxamide
(5g). Yield 90%; white solid; mp 87-88 °C. 1H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H), 7.84 (dd,
J=7.7, 1.4 Hz, 1H), 7.57 (td, J=7.5, 1.5 Hz, 1H), 7.49 (td, J=7.5, 1.4 Hz, 1H), 7.46 – 7.34 (m,
6H), 7.27 (s, 1H), 6.41 (t, J=54.8 Hz, 1H), 3.86 (s, 3H). Elemental anal. calcd for C18H15F2N3O:
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C, 66.05; H, 4.62; N, 12.84. Found: C, 65.68; H, 4.63; N, 12.86. HRMS (ESI) m/z calcd for
C18H16F2N3O (M+ H)+ 328.1256, found 328.1258.
Data for 3',4'-dichloro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-biphenyl]-
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2-carboxamide (5h). Yield 66%; white solid; mp 117-118 °C. H NMR (400 MHz, CDCl3) δ
8.25 (s, 1H), 7.77 (dd, J=7.7, 1.4 Hz, 1H), 7.65 – 7.37 (m, 5H), 7.28 – 7.14 (m, 2H), 3.93 (s, 3H).
Elemental anal. calcd for C18H12Cl2F3N3O: C, 52.20; H, 2.92; N, 10.14.Found: C,51.90; H,2.78;
N,10.21. HRMS (ESI) m/z calcd for C18H13Cl2F3N3O (M+ H)+ 414.0382, found 414.0381.
Data for 3',4'-dichloro-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-[1,1'-biphenyl]-
2-carboxamide (5i). Yield 72%; white solid; mp 100-101 °C. 1H NMR (400 MHz, CDCl3) δ 8.18
(s, 1H), 7.76 (dd, J=7.6, 1.4 Hz, 1H), 7.60 – 7.47 (m, 3H), 7.44 (d, J=8.3 Hz, 1H), 7.41 – 7.34
(m, 2H), 7.21 (dd, J=8.3, 2.1 Hz, 1H), 6.56 (t, J=54.6 Hz, 1H), 3.86 (s, 3H). Elemental anal.
calcd for C18H13Cl2F2N3O: C, 54.57; H, 3.31; N, 10.61. Found: C, 54.16; H, 3.50; N, 10.45.
HRMS (ESI) m/z calcd for C18H14Cl2F2N3O (M+ H)+ 396.0477, found 396.0470.
Data for 4'-chloro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-biphenyl]-2-
1
carboxamide (5j). Yield 46%; white solid; mp 139-140 °C. H NMR (400 MHz, CDCl3) δ 8.26
(s, 1H), 7.79 (dd, J =7.6, 1.4 Hz, 1H), 7.52 (dtd, J =30.2, 7.6, 1.4 Hz, 2H), 7.42 – 7.35 (m, 3H),
7.35 – 7.29 (m, 2H), 7.14 (s, 1H), 3.90 (s, 3H). Elemental anal. calcd for C18H13ClF3N3O: C,
56.93; H, 3.45; N, 11.06. Found: C, 56.83; H, 3.95; N, 10.82. HRMS (ESI) m/z calcd for
C18H14ClF3N3O (M+ H)+ 380.0772, found 380.0772.
Data for 4'-chloro-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-[1,1'-biphenyl]-2-
1
carboxamide (5k). Yield 82%; white solid; mp 128-129°C. H NMR (400 MHz, CDCl3) δ 8.19
(s, 1H), 7.77 (dd, J=7.6, 1.5 Hz, 1H), 7.51 (dtd, J=28.9, 7.5, 1.4 Hz, 2H), 7.41 – 7.29 (m, 6H),
6.50 (t, J=54.6 Hz, 1H), 3.85 (s, 3H). Elemental anal. calcd for C18H14ClF2N3O: C, 59.76; H,
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3.90; N, 11.62. Found: C, 59.28; H, 4.02; N, 11.48. HRMS (ESI) m/z calcd for C18H15ClF2N3O
(M+ H)+ 362.0866, found 362.0872.
Data for 5-fluoro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-biphenyl]-2-
carboxamide (5l). Yield 61%; white solid; mp 82-83 °C. 1H NMR (400 MHz, CDCl3) δ 8.25 (s,
1H), 7.86 (dd, J =8.6, 5.8 Hz, 1H), 7.40 (qq, J =7.1, 2.6, 1.8 Hz, 5H), 7.20 – 7.00 (m, 3H), 3.88
(s, 3H). Elemental anal. calcd for C18H13F4N3O: C, 59.51; H, 3.61; N, 11.57. Found: C, 59.47; H,
4.03; N, 11.60. HRMS (ESI) m/z calcd for C18H14F4N3O (M+ H)+ 364.1068, found 364.1070.
Data for N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-5-fluoro-[1,1'-biphenyl]-2-
carboxamide (5m). Yield 66%; white solid; mp 95-96°C. 1H NMR (400 MHz, CDCl3) δ 8.21 (s,
1H), 7.86 (dd, J =8.6, 5.7 Hz, 1H), 7.52 – 7.33 (m, 5H), 7.25 (s, 1H), 7.22 – 7.10 (m, 2H), 6.41
(t, J =54.8 Hz, 1H), 3.86 (s, 3H). Elemental anal. calcd for C18H14F3N3O: C, 62.61; H, 4.09; N,
12.17. Found: C, 62.03; H, 3.93; N, 12.27. HRMS (ESI) m/z calcd for C18H15F3N3O (M+ H)+
346.1162, found 346.1164.
Data for 3',4',5,5'-tetrafluoro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-
1
biphenyl]-2-carboxamide (5n). Yield 61%; white solid; mp 155-156 °C. H NMR (400 MHz,
CDCl3) δ 8.22 (s, 1H), 7.76 (dd, J=8.6, 5.6 Hz, 1H), 7.22 (td, J=8.2, 2.6 Hz, 1H), 7.16 (s, 1H),
7.08 (dd, J=9.0, 2.6 Hz, 1H), 7.03 (dd, J=7.6, 6.2 Hz, 2H), 3.92 (s, 3H). Elemental anal. calcd for
C18H10F7N3O: C, 51.81; H, 2.42; N, 10.07. Found: C, 51.86; H, 2.60; N, 9.98. HRMS (ESI) m/z
calcd for C18H11F7N3O (M+ H)+ 418.0785, found 418.0778.
Data for N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-3',4',5,5'-tetrafluoro-[1,1'-
1
biphenyl]-2-carboxamide (5o). Yield 60%; white solid; mp 146-147 °C. H NMR (400 MHz,
CDCl3) δ 8.19 (s, 1H), 7.79 (dd, J=8.6, 5.6 Hz, 1H), 7.37 (s, 1H), 7.22 (td, J=8.3, 2.6 Hz, 1H),
7.07 (ddd, J=17.8, 8.3, 4.4 Hz, 3H), 6.61 (t, J=54.5 Hz, 1H), 3.89 (s, 3H). Elemental anal. calcd
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for C18H11F6N3O: C, 54.14; H, 2.78; N, 10.52. Found: C, 54.01; H, 2.85; N, 10.69. HRMS (ESI)
m/z calcd for C18H12F6N3O (M+ H)+ 400.0879, found 400.0871.
Data for 3',4'-dichloro-5-fluoro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-
1
biphenyl]-2-carboxamide (5p). Yield 48%; white solid; mp 117–118 °C. H NMR (400 MHz,
CDCl3) δ 8.22 (s, 1H), 7.80 (dd, J=8.6, 5.6 Hz, 1H), 7.54 – 7.44 (m, 2H), 7.21 (ddd, J=8.3, 6.5,
2.3 Hz, 2H), 7.10 (dd, J=9.1, 2.6 Hz, 2H), 3.91 (s, 3H). Elemental anal. calcd for
C18H11Cl2F4N3O: C, 50.02; H, 2.57; N, 9.72. Found: C, 49.25; H, 2.59; N, 9.73. HRMS (ESI)
m/z calcd for C18H12Cl2F4N3O (M+ H)+ 432.0288, found 432.0285.
Data for 3',4'-dichloro-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-5-fluoro-[1,1'-
1
biphenyl]-2-carboxamide (5q). Yield 53%; white solid; mp 111-112 °C. H NMR (400 MHz,
CDCl3) δ 8.16 (s, 1H), 7.77 (dd, J=8.6, 5.6 Hz, 1H), 7.51 (d, J=2.1 Hz, 1H), 7.46 (d, J=8.2 Hz,
1H), 7.35 (s, 1H), 7.23 – 7.14 (m, 2H), 7.08 (dd, J=9.1, 2.6 Hz, 1H), 6.55 (t, J=54.5 Hz, 1H),
3.86 (s, 3H).Elemental anal. calcd for C18H12Cl2F3N3O: C, 52.20; H, 2.92; N, 10.14. Found: C,
51.80; H, 4.06; N, 10.17. HRMS (ESI) m/z calcd for C18H13Cl2F3N3O (M+ H)+ 414.0382, found
414.0386.
Data
for
4'-chloro-5-fluoro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-
1
biphenyl]-2-carboxamide (5r). Yield 50%; white solid; mp 132-133°C. H NMR (400 MHz,
CDCl3) δ 8.24 (s, 1H), 7.81 (dd, J=8.6, 5.7 Hz, 1H), 7.46 – 7.28 (m, 4H), 7.17 (td, J=8.3, 2.6 Hz,
1H), 7.12 – 7.04 (m, 2H), 3.90 (s, 3H). Elemental anal. calcd for C18H12ClF4N3O: C, 54.35; H,
3.04; N, 10.56. Found: C, 54.18; H, 2.95; N, 10.59. HRMS (ESI) m/z calcd for C18H13ClF4N3O
(M+ H)+ 398.0678, found 398.0672.
Data
for
4'-chloro-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-5-fluoro-[1,1'-
1
biphenyl]-2-carboxamide (5s). Yield 61%; white solid; mp 129-130 °C. H NMR (400 MHz,
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CDCl3) δ 8.17 (s, 1H), 7.79 (dd, J=8.6, 5.7 Hz, 1H), 7.42 – 7.27 (m, 5H), 7.16 (td, J=8.2, 2.6 Hz,
1H), 7.08 (dd, J=9.3, 2.6 Hz, 1H), 6.49 (t, J=54.6 Hz, 1H), 3.85 (s, 3H). Elemental anal. calcd for
C18H13ClF3N3O: C, 56.93; H, 3.45; N, 11.06. Found: C, 57.03; H, 3.55; N, 11.12. HRMS (ESI)
m/z calcd for C18H14ClF3N3O (M+ H)+ 380.0772, found 380.0769.
Data for 4'-(tert-butyl)-5-fluoro-N-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)-[1,1'-
1
biphenyl]-2-carboxamide (5t). Yield 73%; white solid; mp 125-126 °C. H NMR (400 MHz,
CDCl3) δ 8.30 (s, 1H), 7.90 (dt, J=8.5, 4.2 Hz, 1H), 7.45 (dd, J=8.5, 2.2 Hz, 2H), 7.31 (dd, J=8.6,
2.3 Hz, 2H), 7.19 – 6.99 (m, 3H), 3.89 (s, 3H), 1.32 (s, 9H). Elemental anal. calcd for
C22H21F4N3O: C, 63.00; H, 5.05; N, 10.02 Found: C, 62.72; H, 5.29; N, 9.95. HRMS (ESI) m/z
calcd for C22H22F4N3O (M+ H)+ 420.1694, found 420.1699.
Data for 4'-(tert-butyl)-N-(3-(difluoromethyl)-1-methyl-1H-pyrazol-4-yl)-5-fluoro-[1,1'-
1
biphenyl]-2-carboxamide (5u). Yield 75%; white solid; mp 115-116 °C. H NMR (400 MHz,
CDCl3) δ 8.24 (s, 1H), 7.90 (dd, J=8.6, 5.8 Hz, 1H), 7.47 (d, J=8.1 Hz, 2H), 7.35 (d, J=8.1 Hz,
2H), 7.27 (s, 1H), 7.20 – 7.08 (m, 2H), 6.36 (t, J=54.8 Hz, 1H), 3.87 (s, 3H), 1.35 (s,
9H).Elemental anal. calcd for C22H22F3N3O: C, 65.82; H, 5.52; N, 10.47. Found: C, 65.65; H,
5.47; N, 10.55. HRMS (ESI) m/z calcd for C22H23F3N3O (M+ H)+ 402.1788, found 402.1792.
X-ray Diffraction. Compound 5i was recrystallized by a slow evaporation from a solution
of dichloromethane/n-hexane (ʋ/ʋ=2:5) to afford a single crystal suitable for X-ray
crystallography for structure validation. Cell dimensions and intensities were measured using a
Rigaku 007 Saturn 70 diffractometer with graphite monochromated Mo Kα radiation. Compound
5i: Monoclinic, a=10.994 (2) Å, b=19.344 (4) Å, c=8.4094 (17) Å, U=1759.4(6) Å3, T=113(2),
space group P2(1)/c. A total of 18736 reflections were measured, of which 4184 were unique
(Rint=0.0768) in the range of 1.883 <2Θ< 27.893°(-14 ≤ h ≤ 13, -25 ≤ k ≤ 25, -11 ≤ l ≤ 11), and
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2882 observed reflections with I > 2σ (I) were used in the refinement on F2. The structure of 5i
was solved by direct method with the SHELXTL-97 program and all of the non-H atoms were
refined anisotropically by full matrix least-squares to give the Final R indices R1=0.0599,
wR2=0.1903. The atomic coordinates of 5i were deposited at the Cambridge Crystallographic
Data Centre (CCDC) and CCDC-1942098 contained the supplementary crystallographic data for
this paper. The crystallographic data of 5i can be download from the CCDC and the Supporting
Information here.
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Bioassays
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In vitro fungicidal activity. The fungicidal activity of the target compounds was tested in
vitro against Alternaria solani, Botrytis cinerea, Gibberella zeae, Phytophthora infestans (Mont)
de Bary, Physalospora piricola, Pellicularia sasakii, Rhizoctonia cerealis and Sclerotinia
sclerotiorum by using mycelium growth rate test.25 The commercial SDHIs boscalid and
fluxapyroxad were selected as positive control. The synthesized compounds and controls were
dissolved in DMSO to prepare the 20 mg/mL stock solution before diluting by PDA. Target
compounds at a concentration 50 μg/mL were used for the initial preliminary screening with
three replicates. Their relative inhibitory rate (I, %) was calculated according to the following
equation:
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I (%) = [(C-T)/(C-4)]*100
I, inhibitory rate; C, colony diameter of control (mm); T, colony diameter of treatment
(mm).
The compounds with excellent inhibitory rate were selected for the determination of EC50
values. The 20 mg/mL stock solutions were diluted by PDA to obtain a series of concentrations
for the same procedures as described in the above experiments. The EC50 data were calculated
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according to the concentration and the probit of the corresponding inhibitory rate respectively.
The results were summarized in Table 1 and Table 2.
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In vivo fungicidal activity. The in vivo fungicidal activities of the target compounds
against Erysiphe graminis (wheat white powder), Puccinia sorghi Schw (corn rust) and
Rhizoctonia solani (rice sheath blight) were evaluated with three replicates by using pot bioassay
as reported previously.26,27 The biological assay was carry out by Shenyang Sinochem
Agrochemicals R&D Co. Ltd. and the results were summarized in Table 3 and Table 4.
Fluorescence quenching analysis of succinate dehydrogenase. Succinate
dehydrogenase of R. cerealis was extracted according to operation instructions (BC0955,
solarbio, Beijing) for the assay. Fluorescence quenching analysis was performed with F-4500
fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Fluorescence emission spectra was
recorded at excitation wavelength of 280 nm with a wavelength ringing from 300-390 nm at 4°C,
and the emission band-width was set at 10 nm with medium sensitive. Compound 5i with 0 to 4
mg/L was added into the succinate dehydrogenase for fluorescence determination, with boscalid,
fluxapyroxad and tricyclazole as control. The fluorescence intensity under the treatment was
equal which in SDH and compound interaction minus fluorescence intensity of compound itself.
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SDH enzymatic activity. The SDH enzyme activity of 5i was determined by using a
succinate dehydrogenase assay kit (Solabio, BC0950) and assessed as reported previously.28 R.
cerealis was grown in sterilized Fries medium for 5 d and then treated with different
concentrations of 5i and fluxapyroxad, respectively. The SDH enzyme activity was measured
after 24 h of treatment with the selected compounds, and the absorbance value was recorded at
600 nm by using a microplate reader. Compounds were tested at 6 different concentrations with
three replicates and the IC50 value was calculated by GraphPad Prim version 6.02.
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RESULTS AND DISCUSSION
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Molecular Docking Analysis. Novel SDHI derivatives are rationally designed by
reversing of –CO-NH– group and we speculate whether these compounds also target at SDH and
form hydrogen bonds at the pocket. Molecular docking of selected compounds and fluxapyroxad
were carried out for evaluation its potent mode of action of target compounds. All reported SDHs
are highly reserved in spatial structure, subunit and electron transport pathway, and even the
ubiquinone binding site in the prokaryotes and eukaryotes is also highly reserved.29 For these
reasons, avian (Gallus gallus) respiratory complex II with carboxin bound (pdb code: 2FBW)
was chose for molecular docking study. In order to illustrate the binding mode between ligand
and receptor, 5i was selected as an example. As shown in Figure 3, 5i located in the same site
and adopted a similar binding conformation as compared to fluxapyroxad in the three-
dimensional schematic diagrams. Compound 5i bounded to the receptor protein, the carboxyl
oxygen formed two hydrogen bonds with the amino acid residue TRP173 and TYR58,
respectively. The binding mode was similar to that of fluxapyroxad, this indicated that, the target
compounds also possessed strong interaction with SDH.
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Chemical Synthesis. The synthetic procedures for the target compounds are described in
Scheme 1. The Suzuki-Miyaura cross-coupling of aryl halide and aryl boronic acid was
conducted under argon at 80 ℃ to produce the key intermediate 1.30 Intermediate 1 was then
reacted with NaOH via a direct ester hydrolysis under 80 ℃ to yield the compound 2. Compound
2 was reacted with oxalyl chloride in CH2Cl2 with DMF to afford intermediate 3. Intermediate 3
was then reacted with pyrazole amine 4 in CH2Cl2 in the presence of Et3N to afford target
compound 5 with moderate to good yields. To further validate the structure of the title
compounds, the structure of 5i was further identified by X-ray diffraction studies (see Figure. 4).
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Fungicidal Activity. The results of in vitro and in vivo fungicidal activities of the target
compounds (5a to 5u) were summarized in Table 1-4. In general, most of the compounds
displayed considerable to excellent fungicidal activities against eight phytopathogens in vitro at
50 μg/mL. (Table 1). No obvious difference of fungicidal activity between 3-CF3 substituent
prazole compounds and 3-CF2H substituent prazole compounds was observed. Most of these
compounds were highly active against B. cinerea, R. cerealis and S. sclerotiorum. As shown in
Table 1, 5c, 5d and 5j showed over 90% of inhibitory rate against B. cinereal, which were
comparable to boscalid and fluxapyroxad. For G. zeae, 5i and 5j showed better activity than
positive controls. Compound 5i, 5p, 5q, 5s displayed higher activity against P. infestans than
boscalid and fluxapyroxad, 5h, 5i, 5j, 5p, 5q, 5r, 5s exhibited excellent activities against P.
sasakii, which were better than boscalid and fluxapyroxad. Eight compounds showed over 86%
of inhibitory rate against R. cerealis, boscalid and fluxapyroxad only showed 35% and 86% of
inhibitory rate, respectively. All compounds exhibited excellent activities against S. sclerotiorum.
Several compounds with superior fungicidal activities were selected for the calculation of
EC50 value. As shown in Table 2, several compounds showed good activity against Botrytis
cinerea, but not as effective as that of the positive controls. Compounds 5i and 5q displayed
excellent activities against P. infestans and P. sasakii with the EC50 values lower than the
positive control fluxapyroxad. Nine compounds exhibited better fungicidal activity against R.
cerealis than fluxapyroxad, especially, 5i showed excellent fungicidal activity with the EC50
value of 4.61 μg/mL, which was more active than that of fluxapyroxad with its EC50 value of
16.99 μg/mL. The EC50 values of all target compounds were tested against S. sclerotiorum, 5i
also showed the highest fungicidal activity with its EC50 value of 0.73 μg/mL, which was
comparable to that of boscalid with its EC50 value of 0.51 μg/mL. Therefore, 5i exhibited
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promising fungicidal activity against five fungi with broader spectrum of fungicidal activity and
could be an alternative to SDHIs candidate.
The in vivo fungicidal activity of the target compounds was tested against E. graminis, P.
sorghi and R. solani for comparison with the commercial fungicide boscalid and fluxapyroxad
(Table 3). Most of the compounds exhibited excellent fungicidal activity against P. sorghi and R.
solani at the concentration 200 μg/mL. Compound 5c, 5l, 5o, 5t displayed 100% of inhibitory
rate against E. graminis and similar activity as that of boscalid and fluxapyroxad. Compound 5d,
5f, 5g, 5h, 5i, 5g exhibited more than 90% of excellent fungicidal activity against P. sorghi,
which was comparable to those of boscalid and fluxapyroxad. Compound 5g, 5h, 5i, 5t displayed
over 80% of inhibitory rate against R. solani.
Some compounds with higher inhibitory rate were selected for further greenhouse in vivo
fungicidal activity evaluation. As shown in Table 4, 5g and 5h were not as effective as 5i against
P. sorghi and R. solani. Compound 5i and fluxapyroxad exhibited 100% inhibitory rate against P.
sorghi at the concentration of 50 μg/mL, they were better than boscalid (70%). Furthermore,
compound 5i showed excellent inhibitory rate (60%) against R. solani at 1μg/mL, which was
better than boscalid (30%) at the same concentration. In general, 5i presented excellent activity
against R. cerealis in vitro and was highly effective against R. solani in vivo. Both in vitro and in
vivo activity determination indicated that, 5i could be a promising candidate against R. solani for
rice disease control.
SDH Enzymatic Inhibition Activity. Compound 5i with promising fungicidal activity
was selected and evaluated for SDH enzymatic inhibition determination to its target site
validation. As shown in Table 5, 5i exhibited very good SDH inhibition with an IC50 of 0.60
μg/mL, while the IC50 of fluxapyroxad was 0.29 μg/mL, 5i showed the same level of inhibitory
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activity as fluxapyroxad. Compound 5i with carbonyl group adjacent to benzene ring contrast to
fluxapyroxad which carbonyl group was adjacent to pyrazole could also exhibit excellent SDH
enzymatic inhibition activity and formed strong interaction with SDH.
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Fluorescence Quenching Analysis of SDH. The interaction of protein and small
molecules could be revealed by fluorescence quenching analysis. Fluorescence quenching of the
SDH by 5i, boscalid and fluxapyroxad were conducted to validate interactions between protein
and compounds. As shown in Figure 5C, with the increase of the concentration of 5i, the
fluorescence intensity for SDH gradually quenched, it was similar to that of the positive control
boscalid and fluxapyroxad (Figure 5A, 5B), there showed no significant fluorescence changes
when treated by a negative control tricyclazole, a melanin synthesis inhibitor31 (Figure 5D).
Boscalid and fluxapyroxad, carboxamide fungicides, were SDHIs.32 Pesticide target discovery
and validation is a most important basis for novel pesticide development.33 Compound 5i
exhibited the similar fluorescence quenching rules as that of boscalid and fluxapyroxad, which
indicated that they might act at the same target site.
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In summary, a series of novel SDHIs derivatives were designed and synthesized by
inversion of carbonyl and amide groups of fluxapyroxad. Bioassay discovered highly active 5i
with broad-spectrum of outstanding in vitro fungicidal activity and better in vivo efficacy against
P. sorghi and R. solani. Fluorescence quenching analysis for SDH target validation indicated that,
5i exhibited the similar properties as that of the two SDHI positive controls boscalid and
fluxapyroxad, these results revealed that, the designed compounds might also act at SDH. Our
results provided a new clue for the molecular design and development of the novel highly active
SDHIs.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
1H NMR spectra for all compounds and HRMS spectra for target compounds
Crystal data of 5i (CIF)
Corresponding Authors
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*Phone +86 022-23499464. Fax:+86 022-23503620. E-mail: fanzj@nankai.edu.cn (Z.F.), or
nkzml@vip.163.com (Z.L.)
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ORCID
Dongyan Yang: 0000-0001-9752-9186
Bin Zhao: 0000-0002-0383-097X
Zhijin Fan: 0000-0001-5565-0949
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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This work was supported in part by the National Natural Science Foundation of China (No.
31872007 and No. 31571991), the Tianjin Natural Science Foundation (No. 18JCZDJC33500),
the International Science & Technology Cooperation Program of China (No. 2014DFR41030),
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The Fundamental Research Funds for the Central Universities, Nankai University (No.
63191743), The Tianjin Development Program for Innovation and Entrepreneurship and The
Russian Foundation for Basic Research (No. 18-316-20018).
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Dedicated to the 100th anniversary of Nankai University and the 100th birthday
anniversary of Professor Ruyu Chen.
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REFERENCES
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535
O
F2HC
O
O
O
F3C
S
N
N
N
N
N
N
H
N
N
N
N
N
N
H
H
H
O
Cl
F
Furametpyr
Penflufen
F2HC
Penthiopyrad
F
Sedaxane
O
F2HC
O
O
F2HC
N
N
N
N
N
N
H
H
N
N
N
H
F
F
Cl
F
Cl
Cl
Isopyrazam
O
Bixafen
O
Fluxapyroxad
Cl
Cl
F2HC
O
F2HC
F2HC
Cl
N
O
N
N
N
N
H
N
N
N
N
Cl
F
Cl
Pydiflumetofen
Benzovindiflupyr
Isoflucypram
536
537
Figure 1. Representative chemical structures of pyrazole contained SDHIs
538
O
H
N
N
H
O
I
Mebenil
Benodanil
R3
O
HF2C
R1
H
C
N
H
N
C
O
N
N
N
N
Inversion
CO-NH
H3C
H3C
F
F
R2
Target Compounds
F
Fluxapyroxad
539
540
Figure 2. The design strategy of the target compounds.
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541
542
Figure 3. Binding modes of 5i (A) and fluxapyroxad (B) with SDH.
543
544
545
546
Figure 4. Crystal structure for 5i by X-ray diffraction determination. (CCDC number: 1942098)
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A
B
260
215
170
125
80
260
215
a
a
e
170
125
e
80
35
35
-10
290
-10
290
340
390
440
490
340
390
440
490
Wavelength (nm)
Wavelength (nm)
C
D
280
220
160
100
40
290
240
190
140
90
a
e
a
e
40
-10
290
-20
290
340
390
440
490
340
390
440
490
Wavelength (nm)
Wavelength (nm)
547
548
549
550
Figure 5. Fluorescence spectra of succinate dehydrogenase: A, boscalid; B, fluxapyroxad; C, compound 5i; D,
tricyclazole “a to e” were “0 to 4 mg/L”.
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R1
R1
O
I
HO
OH
B
Pd(PPh3)2Cl2
COOMe
COOH
R4
NaOH
O
+
R1
EtOH/H2O
THF/H2O
R4
R2
R2
R2
R4
R3
R3
2
R3
1
R1
R1
R1
R5
R5
NH2
H
N
N
(COCl)2
N
COOH
R4
4
COCl
R4
C
O
N
N
DMF/CH2Cl2
Et3N/CH2Cl2
R2
R2
R2
R4
R3
2
R3
3
R3
5
551
Compd R1 R2
R3
CF3
CF3
CH3
CH3
F
R4
H
H
H
H
F
R5
Compd R1 R2 R3 R4
R5
Compd R1 R2 R3 R4
R5
5a
5b
5c
5d
5e
5f
H
H
H
H
H
H
H
H
H
H
H
F
CF3
5h
5i
H
H
H
H
F
H
H
H
H
H
H
F
Cl Cl
CF3
5o
5p
5q
5r
5s
F
F
F
F
F
F
F
F
F
F
CHF2
CF3
CHF2
CF3
Cl Cl CHF2
H
H
H
H
H
H
Cl Cl
5j
Cl
Cl
H
H
F
H
H
H
H
F
CF3
CHF2
CF3
Cl Cl CHF2
CHF2
CF3
5k
5l
Cl
Cl
H
H
H
H
CF3
CHF2
CF3
F
F
F
CHF2
CHF2
5m
5n
F
CHF2
CF3
5t
tBu
tBu
5g
H
H
H
F
5u
CHF2
552
553
554
Scheme 1 The synthetic route of target compounds
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