Design, Synthesis, and Fungicidal Evaluation of Novel Pyrazole-furan and Pyrazole-pyrrole Carboxamide as Succinate Dehydrogenase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.7b01251

The identification of novel succinate dehydrogenase (SDH) inhibitors represents one of the most attractive directions in the field of fungicide research and development. During our continuous efforts to pursue inhibitors belonging to this class, some structurally novel pyrazole-furan carboxamide and pyrazole-pyrrole carboxamide derivatives have been discovered via the introduction of scaffold hopping and bioisosterism to compound 1, a remarkably potent lead obtained by pharmacophore-based virtual screening. As a result of the evaluation against three destructive fungi, including Sclerotinia sclerotiorum, Rhizoctonia solani, and Pyricularia grisea, a majority of them displayed potent fungicidal activities. In particular, compounds 12I-i, 12III-f, and 12III-o exhibited excellent fungicidal activity against S. sclerotiorum and R. solani comparable to that of commercial SDHI thifluzamide and 1.

Full text of DOI:10.1021/acs.jafc.7b01251

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Article
Design, Synthesis, Fungicidal Evaluation of Novel Pyrazole-furan and
Pyrazole-pyrrole Carboxamide as Succinate Dehydrogenase Inhibitors
Ting-Ting Yao, Douxin Xiao, Zhong-Shan Li, Jingli Cheng, Shaowei
Fang, Yongjun Du, Jinhao Zhao, Xiaowu Dong, and Guonian Zhu
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01251 • Publication Date (Web): 15 Jun 2017
Downloaded from http://pubs.acs.org on June 19, 2017
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Design, Synthesis, Fungicidal Evaluation of Novel Pyrazole-furan and
Pyrazole-pyrrole Carboxamide as Succinate Dehydrogenase Inhibitors
TingꢀTing Yao,†, § DouꢀXin Xiao,†, § ZhongꢀShan Li,† JingꢀLi Cheng,† ShaoꢀWei
Fang,† YongꢀJun Du,† JinꢀHao Zhao,*, † XiaoꢀWu Dong,*, ‡ and GuoꢀNian Zhu†
§ These authors contributed equally to this study
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†Institute of Pesticide and Environmental Toxicology, Ministry of Agriculture Key Lab
of Molecular Biology of Crop Pathogens and Insects, Zhejiang University, Hangzhou
310029, P. R. China
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‡ZJUꢀENS Joint Laboratory of Medicinal Chemistry, Zhejiang Province Key
Laboratory of AntiꢀCancer Drug Research, College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou, 310058, P. R. China
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*Institute of Pesticide and Environmental Toxicology, Zhejiang University, Kaixuan
Road 268, Hangzhou, 310029, P. R. China. Tel: 086ꢀ571ꢀ86971923. Fax:
086ꢀ571ꢀ86971923. Eꢀmail: jinhaozhao@zju.edu.cn (JinꢀHao Zhao).
*College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, P. R.
China. Tel (Fax): 086ꢀ571ꢀ88981051. Eꢀmail: dongxw@zju.edu.cn (XiaoꢀWu Dong).
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ABSTRACT
The identification of novel succinate dehydrogenase (SDH) inhibitors represents one
of the most attractive directions in the field of fungicide research and development.
During our continuous efforts to pursue inhibitors belonging to this class, some
structurally novel pyrazoleꢀfuran carboxamide and pyrazoleꢀpyrrole carboxamide
derivatives have been discovered via introducing scaffold hopping and bioisosterism
to compound 1, a remarkably potent lead obtained by pharmacophoreꢀbased virtual
screening. As a result of the evaluation against three destructive fungi, including
Sclerotinia sclerotiorum, Rhizoctonia solani and Pyricularia grisea, a majority of
them displayed potent fungicidal activities. In particular, compound 12I-i, 12III-f and
12III-o exhibited excellent fungicidal activity against S. sclerotiorum and R. solani
comparable to that of commercial SDHI thifluzamide and 1.
KEYWORDS: succinate dehydrogenase inhibitors, scaffold hopping, molecular
docking, synthesis, fungicidal activity
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INTRODUCTION
Fungicides are of value for global food security, attributed to its important role in
controlling the growth and reproduction of plant pathogens. Among the 55 classes of
fungicides listed by the Fungicide Resistance Action Committee (FRAC), succinate
dehydrogenase inhibitors (SDHI) have been extensively employed to combat
destructive plant fungi, such as Sclerotinia sclerotiorum, Rhizoctonia solani and
Botrytis cinerea.1ꢀ3 The SDHI exert their biological activities by disrupting the
mitochondrial respiration chain and the Krebs cycle.4, 5 Benefiting from its unique
mode of action, the SDHI has no crossꢀresistance with other commercial fungicides,
and have aroused a considerable research interest in exploiting fungicides of this
class.6ꢀ10 To date, 19 commercial SDHI fungicides have been approved for plant
protection since the first launch of carboxin in 1966. However, the unrestricted usage
of SDHI fungicides over several decades has resulted in the development of resistance
in fungi.11ꢀ14 Therefore, novel SDHIs with improved fungicidal potency are still in
clear demand.
As a well established lead optimization strategy, scaffold hopping is frequently
utilized for discovering compounds with structural novelty, enhanced potency,
improved physicochemical properties or more favorable absorption, distribution,
metabolism, and excretion (ADME) profiles.15ꢀ19 In respect to pesticide design,
scaffold hopping is also widely introduced to modify the lead compounds. For
example, scaffold hopping of Fipronil and human adrenergic ligand clonidine
contributed to the successful identification of a novel 7ꢀpyrazolopyridine
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GABA/GluCl insecticide along with the thioureas and isothioureas insecticides.20,21 In
addition to the rationality, another factor that should be noted is the synthetic
accessibility when employing scaffold hopping strategy in compound design. In this
article, we report a computerꢀaided scaffold hopping approach to a novel structural
series of pyrazoleꢀfuran and pyrazoleꢀpyrrole carboxamides as SDHIs.
We recently disclosed a novel lead structure of SDH inhibitor by pharmacophore
based virtual screening. Our previous work at pharmacophore based virtual screening
has culminated in the discovery of a promising lead, the pyrazoleꢀphenyl carboxamide
derivative 1 (Figure 1), which displayed excellent activities against S. sclerotiorum
(EC50 = 1.9 mg/L), R. solani (EC50 = 3.8 mg/L) and P. grisea (EC50 = 4.1 mg/L), as
well as potent enzymatic inhibition of porcine SDH (IC50 = 1.07 µM).22 As a part of
our continuous work in pursuit of SDHI inhibitors with novel structure and high
efficacy, we initiated a structural optimization campaign focusing on introducing
scaffold hopping to 1 in silico. Taking the molecular docking results and synthetic
accessibility into consideration, a series of pyrazoleꢀfuran carboxamide and
pyrazoleꢀpyrrole carboxamide derivatives were identified as the target compound for
chemical synthesis (Scheme 1). Their fungicidal activities were evaluated against S.
sclerotiorum, R. solani and P. grisea. Furthermore, docking analysis and
structureꢀactivity relationship (SAR) studies were extensively performed on all of the
derivatives to identify key structural features responsible for their fungicidal potency.
MATERIALS AND METHODS
Instruments and Chemicals All reagents and solvents were commercially available
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and used directly without further purification. Reactions were monitored by thinꢀlayer
chromatography (TLC). Target compounds were purified by column chromatography
using silica gel. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded
at a Bruker AVANCE III spectrometer in CDCl3 or DMSOꢀd6 solution, with SiMe4
(TMS) serving as the internal standard. Chemical shift values (δ) were listed in parts
per million (ppm). ESI mass spectral data were obtained from AccurateꢀMass QꢀTOF
spectrometer and QUATTRO PREMIER XE. The melting points were determined on
an Xꢀ4 binocular microscope melting point apparatus (Beijing Tech Instruments Co.,
Beijing, China) and are uncorrected.
Synthesis of methyl 4,5ꢀdibromofuranꢀ2ꢀcarboxylate (3).23 The aluminum chloride
(70.5 g, 0.53 mol) was added to a solution of 2 (31.5 g, 0.25 mol) in chloroform (250
mL) at ꢀ10 °C under nitrogen. Br2 (79.5 g, 0.1 mol) was then added dropwise over 1 h,
°
and the resulting mixture was maintained at 23 C for an additional 3 h. The reaction
mixture was poured into water (200 mL). The mixture was extracted with
dichloromethane (100 mL × 3), and the combined organic phases were further washed
with 10% aqueous sodium thiosulfate, saturated sodium bicarbonate, brine, dried over
anhydrous sodium sulfate and concentrated under reduced pressure. Purification by
crystallization with hexane afforded the title compound as a paleꢀyellow solid. Yield
°
1
78%; m.p. = 39ꢀ40 C. H NMR (500 MHz, CDCl3) δ 7.19 (s, 1H, furanꢀH), 3.90 (s,
3H, OCH3). 13C NMR (125 MHz, CDCl3) δ 157.31, 143.33, 140.07, 121.93, 98.62,
52.35.
Synthesis of methyl 4,5ꢀdibromofuranꢀ2ꢀcarboxylate (4). A solution of 3 (28.4 g, 0.1
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mol) in anhydrous THF (200 mL) was purged with nitrogen and cooled to ꢀ40 C.
Isopropylmagnesium chloride (2 N in THF, 76 mL) was added dropwise, and the
resulting mixture was stirred for 2 h. The reaction mixture was quenched with water
(100 mL), and the precipitates were filtered off. The organic layer was separated and
the aqueous layer was extracted with ethyl acetate (50 mL × 3). The combined organic
phases were washed with brine, dried over anhydrous sodium sulfate and purified on
°
1
silica gel to afford 4 as a yellow solid. Yield 89%; m.p. = 39ꢀ40 C. H NMR (500
MHz, CDCl3) δ 7.58 (d, J = 0.8 Hz, 1H, furanꢀH), 7.18 (d, J = 0.8 Hz, 1H, furanꢀH),
3.91 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3) δ 158.04, 143.77, 141.15, 119.96,
108.89, 52.05.
Synthesis of 2,2,2ꢀtrichloroꢀ1ꢀ(1ꢀmethylꢀ1Hꢀpyrrolꢀ2ꢀyl)ethanꢀ1ꢀone (6).24 A solution
of 5 (30 g, 0.37 mol) in dichloromethane (100 mL) was purged with nitrogen and
°
cooled to ꢀ10 C. 2,2,2ꢀtrichloroacetyl chloride (66 g, 0.37 mol) was dissolved in
dichloromethane (100 mL) and added dropwise, and the resulting mixture was
°
maintained at 23 C overnight. The solvent was evaporated under vacuum, and the
residue was purified by silica column to afford 6 as a yellow solid. Yield 85%; m.p. =
°
1
60ꢀ61 C. H NMR (500 MHz, CDCl3) δ 7.52 (dd, J = 4.4, 1.6 Hz, 1H, pyrroleꢀH),
7.10 – 6.81 (m, 1H, pyrroleꢀH), 6.24 (dd, J = 4.4, 2.4 Hz, 1H, pyrroleꢀH), 3.99 (s, 1H,
CH3). 13C NMR (125 MHz, CDCl3) δ 172.81, 133.62, 123.97, 121.77, 108.86, 96.30,
38.48.
Synthesis of 1ꢀ(4ꢀBromoꢀ1ꢀmethylꢀ1Hꢀpyrrolꢀ2ꢀyl)ꢀ2,2,2ꢀtrichloroethanꢀ1ꢀone (7).
NBS (14.4 g, 0.09 mol) was added portionꢀwise to a solution of 6 (18 g, 0.08 mol) in
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anhydrous tetrahydrofuran (160 mL) at ꢀ10 °C. The resulting mixture was warmed to
°
23 C and stirred overnight. The reaction mixture was concentrated under reduced
pressure, and the crude residue was dissolved in ethyl acetate (150 mL). The solution
was washed sequentially with saturated aqueous sodium carbonate (50 mL × 3) and
brine (50 mL × 2). The organic layer was dried over anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure to afford the title compound as an
offꢀwhite solid. Yield 77%; m.p. = 97ꢀ98 °C. 1H NMR (500 MHz, CDCl3) δ 7.47 (d, J
= 1.6 Hz, 1H, pyrroleꢀH), 6.97 (d, J = 1.4 Hz, 1H, pyrroleꢀH), 3.96 (s, 3H, CH3). 13C
NMR (125 MHz, CDCl3) δ 172.45, 132.84, 124.62, 122.15, 96.15, 95.72, 38.76.
Synthesis of methyl 4ꢀbromoꢀ1ꢀmethylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate (8). The sodium
methoxide (5.2 g, 0.09 mol) was added to an iceꢀcooled solution of 7 (20.0 g, 0.06
°
mol) in anhydrous methanol (320 mL). The resulting mixture was warmed to 23 C
and stirred for 1 h. The reaction mixture was quenched with HCl (1 N, 30 mL), and
extracted with ethyl acetate (30 mL × 3). The combined organic layer washed with
brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced
pressure to afford the title compound as a white solid. Yield 90%; m.p. = 63ꢀ64 °C. 1H
NMR (500 MHz, CDCl3) δ 6.91 (d, J = 1.9 Hz, 1H, pyrroleꢀH), 6.78 (d, J = 1.9 Hz,
1H, pyrroleꢀH), 3.91 (s, 3H, CH3), 3.82 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ
160.82, 128.76, 122.88, 119.22, 95.06, 51.27, 36.91.
General procedure for the synthesis of 9I and 9II.
A
solution of
1ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀ dioxaborolanꢀ2ꢀyl)ꢀ1Hꢀpyrazole (7.5 g, 0.036
mol), K3PO4•3H2O (12.0 g, 0.045 mmol) and bromide 4 or 8 (0.03 mol) in
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N,Nꢀdimethylformamide
(100
mL)
was
stirred
under
nitrogen.
Tetrakis(triphenylphosphine)palladium (3.5 g, 0.003 mol) was added, and the mixture
was heated at 100 °C for 10 h. The reaction mixture was dissolved in water (300 mL),
and extracted with ethyl acetate (150 mL × 3). The combined organic layer was
washed with brine (50 mL × 2), dried over anhydrous sodium sulfate, and
concentrated under vacuum to afford the title compounds. The crude product was
purified by flash silica chromatography to obtain the title compound.
Data for 9I: Yellow solid; Yield 67%; m.p. = 62ꢀ64 °C. 1H NMR (500 MHz, CDCl3) δ
7.43 (d, J = 2.0 Hz, 1H, furanꢀH), 7.20 (d, J = 3.6 Hz, 1H, pyrazoleꢀH), 6.60 (d, J =
3.6 Hz, 1H, pyrazoleꢀH), 6.56 (d, J = 2.0 Hz, 1H, furanꢀH), 4.07 (s, 3H, NCH3), 3.86
(s, 3H, OCH3). 13C NMR (125 MHz, CDCl3) δ 158.75, 148.21, 143.93, 138.49,
132.59, 119.26, 109.60, 106.41, 77.29, 77.03, 76.78, 51.90, 38.84.
Data for 9II: White solid; m.p. = 52ꢀ53 °C. 1H NMR (500 MHz, CDCl3) δ 7.44 (d, J =
1.9 Hz, 1H, furanꢀH), 7.04 (d, J = 2.0 Hz, 1H, pyrazoleꢀH), 6.95 (d, J = 1.9 Hz, 1H,
pyrazoleꢀH), 6.23 (d, J = 1.9 Hz, 1H, furanꢀH), 3.97 (s, 3H, PyrazoleꢀCH3), 3.93 (s,
3H, pyrroleꢀCH3), 3.84 (s, 3H, OCH3). 13C NMR (125 MHz, CDCl3) δ 161.37, 138.33,
137.35, 127.73, 123.09, 116.57, 112.97, 104.64, 51.25, 37.57, 36.96.
General procedure for the synthesis of 10I and 10II. The aqueous sodium hydroxide
(3 N, 65 mL) added to a solution of 9I or 9II (0.02 mol) in methanol (20 mL). The
reaction mixture was stirred at 23 °C for 5 h. The solvent was removed by rotary
evaporation, and the crude residue was redissolved in 20 mL water. The solution was
further acidified with hydrochloric acid (3 N) until pH = 1, and extracted with ethyl
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acetate (10 mL × 3). The collected organics was further washed with brine, dried over
anhydrous sodium sulfate, and then concentrated to yield the title acid.
1
Data for 10I: Yellow solid; Yield 60%; m.p. = 172ꢀ174 °C. H NMR (500 MHz,
DMSO) δ 7.59 (s, 1H, furanꢀH), 7.33 (d, J = 3.6 Hz, 1H, pyrazoleꢀH), 7.05 (d, J = 3.6
13
Hz, 1H, furanꢀH), 4.05 (s, 3H, CH3). C NMR (125 MHz, CDCl3) δ 159.03, 145.15,
144.30, 137.11, 137.04, 128.91, 118.88, 112.63, 108.52.
1
Data for 10II: Yellow solid; Yield 68%; m.p. = 207ꢀ208 °C. H NMR (500 MHz,
DMSO) δ 7.44 (d, J = 2.0 Hz, 1H, pyrazoleꢀH), 7.35 (d, J = 1.9 Hz, 1H, pyrroleꢀH),
7.05 (d, J = 2.1 Hz, 1H, pyrroleꢀH), 6.33 (d, J = 1.9 Hz, 1H, pyrazoleꢀH), 3.89 (s, 6H,
pyrazoleꢀCH3, pyrroleꢀCH3). 13C NMR (125 MHz, CDCl3) δ 161.82, 137.62, 137.09,
128.11, 123.38, 115.84, 111.97, 103.99, 37.68, 36.55.
General procedure for the synthesis of 11I and 11II. NCS (1.5 g, 0.011 mol) was
added to an iceꢀcooled solution of 10I or 10II (0.01 mol) in dry THF/DMF (6/9 mL).
The resulting mixture was warmed to 80 °C and stirred for 2 h. The reaction mixture
was then diluted with ethyl acetate (20 mL), washed with saturated aqueous sodium
bicarbonate and brine, dried over anhydrous sodium sulfate, and then concentrated
under vacuum. Purification by flash silica chromatography afforded the title
compound.
1
Data for 11I: White solid; Yield 85%; m.p. >200 °C. H NMR (500 MHz, DMSO) δ
7.59 (s, 1H, pyrazoleꢀH), 7.33 (d, J = 3.6 Hz, 1H, furanꢀH), 7.05 (d, J = 3.6 Hz, 1H,
furanꢀH), 4.05 (s, 3H, CH3). 13C NMR (125 MHz, CDCl3) δ 159.03, 145.15, 144.30,
137.11, 137.04, 128.91, 118.88, 112.63, 108.52.
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Data for 11II: White solid; Yield 87%; m.p. = 187ꢀ188 °C. H NMR (500 MHz,
DMSO) δ 7.55 (s, 1H, pyrazoleꢀH), 7.50 (d, J = 2.0 Hz, 1H, pyrroleꢀH), 7.11 (d, J =
2.0 Hz, 1H, pyrroleꢀH), 3.93 (s, 3H, pyrazoleꢀCH3), 3.85 (s, 3H, pyrroleꢀCH3). 13C
NMR (125 MHz, DMSO) δ 161.70, 136.21, 133.70, 129.31, 123.54, 116.45, 108.49,
106.16, 38.64, 36.68.
General procedure for the synthesis of 12-I~12-IV.25 A mixture of 10I, 10II, 11I or
11II (0.5 mmol), HATU (209 mg, 0.55 mmol), N, Nꢀdiisopropylethylamine (129 mg,
1 mmol) and corresponding amine (0.75 mmol) in acetonitrile (20 mL) was warmed
to 60 °C and stirred overnight. The reaction mixture was concentrated under reduced
pressure. The crude residue was diluted with dichloromethane (20 mL) and washed
with water (10 mL × 3). The organic layer was then dried over anhydrous sodium
sulfate, and then concentrated under vacuum. Purification by flash silica
chromatography afforded the title compounds. H NMR, 13C NMR and HRMS
1
spectral data are supplied in the Supporting Information.
Fungicidal Assay. Sclerotinia sclerotiorum, Rhizoctonia solani, Pyricularia grisea
and susceptible cole leaves were provided by the Institute of Pesticide and
Environment Toxicology, Zhejiang University. The fungicidal activities of the
synthetic compounds were tested in vitro against abovementioned fungi according to
the reported mycelia growth inhibition method.9 Each compound was dissolved in
DMSO to prepare the 10 mg/mL stock solution. Compounds were firstly tested at a
concentration of 100 mg/L for primary screening. Percentage inhibition was
calculated by the following equation: Percentage inhibition (%) = (1 – PT / CK) × 100,
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where PT is the mean colony diameter with compounds and CK is the mean colony
diameter without tested compounds. The commercial fungicide thifluzamide was
employed as positive control. Each treatment was performed for three replicates. The
inhibition rate of the potent compounds were further tested at the concentration of 100,
50, 25, 12.5, 6.25, 3.12, 1.56 µg/mL, and the corresponding EC50 values were
calculated using SPSS Statistics v17.0.
The in vivo fungicidal activity of the target compounds against S. sclerotiorum was
carried out on cole leaves.9 Healthy cole leaves were sprayed with target compounds
(200 µg/mL) and then cultivated at 25 °C for 24 h before inoculation with S.
sclerotiorum. Results were observed as diameters of lesion after cultivation at 25 °C
for 36 h. Thifluzamide was employed as positive control. Each treatment was
performed for three replicates. The preventative rate was expressed as (1 ꢀ c/d) × 100,
where c is the diameter of the treatment and d is the diameter of the negative control.
Molecular Docking. Due to the absence of crystal structure of SDH from fungi, a
homology model of Rhizoctonia solani SDH (RsSDH) was built and validated in our
previous study. The constructed homology model of RsSDH was used for the
subsequent docking study. The structures of small molecules were optimized with the
Ligand Minimization protocol. Molecular docking study was performed by the
Libdock module implemented in Discovery Studio 2.5.26 The active site of SDH was
derived from the copied ligand flutolanil in RsSDH model. Ten random conformations
were generated for each ligand. The rest of the parameters were set to the default
values. The optimal pose was selected in terms of docking score and visual
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inspection.
RESULTS AND DISCUSSION
Rational design. According to our previous study, the pyrazoleꢀbenzoic “core” of
lead compound 1 entered deep into the Qꢀsite of receptor protein, contributing
preliminarily to its binding affinity. To explore novel SDHIs and make better
interaction with the Qꢀsite, herein, we introduced scaffold hopping to the
pyrazoleꢀbenzoic “core” of lead compound 1. It was well known that pyrazole was a
favorable pharmacophore for SDHI fungicides.4ꢀ7 Thus, we retained the pyrazole
group and replaced the phenyl linkage with fiveꢀ or other sixꢀmembered ring systems
(Scheme 1). From our assessment, pyrrole (L1) and furan (L2) isomers can maintain
similar binding pose to lead compound 1. Considering the favorable binding mode
and synthetic tractability, pyrazoleꢀpyrrole and pyrazoleꢀfuran acid cores were
selected for chemical synthesis and structureꢀactivity relationship study.
Chemical Synthesis. The synthetic route for 12I-a~12IV-e was shown in Figure 1~3.
For the synthesis of bromide 4 (Figure 2), the commercial furoate (2) was firstly
brominated to provide dibromoꢀfuroate 3, which was further treated with
isopropylmagnesium chloride to afford monoꢀbromide 4. For the synthesis of bromide
8 (Figure 3), commercial methylpyrazole (5) was coupled with 2,2,2ꢀtrichloroacetyl
chloride to generate 6, and this was followed by bromination using NBS, leading to
bromide 7 in good yield. Treatment of intermediate 7 with sodium methoxide in
MeOH furnished the target bromide 8. The bromide 4 and 8 were subsequently
coupled with pyrazoleborate to provide 9I and 9II. Hydrolysis of 9I and 9II under
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basic conditions provided the corresponding acids 10I and 10II. The exposure of 10I
and 10II to NCS resulted in the smooth chlorination of reactive pyrazole ring to
afford 11I and 11II. The chlorine substituted acids were further coupled with different
amines to afford desired derivatives 12I-a~12IV-e (Figure 4). The chemical structures
of all prepared compounds were confirmed by 1H NMR, 13C NMR and HRMS.
Fungicidal Activities and SAR Discussion. The fungicidal activities of all the target
compounds were evaluated against three destructive fungi (S. sclerotiorum, R. solani
and P. grisea). The commercial SDHI fungicide thifluzamide (TH) was used as the
positive control, and the results are provided in Table 1. Most of the derivatives
exhibited moderate to potent fungicidal activities against S. sclerotiorum, R. solani
and P. grisea, demonstrating the rationality of our scaffold hopping strategy. Among
them, five newly synthesized derivatives (i.e. 12I-b, 12I-i, 12III-g, 12III-h and
12III-q) were found to display improved fungicidal activities against S. sclerotiorum
compared with thifluzamide (EC50 = 33.2 µM) and lead structure 1 (EC50 = 5.5 µM),
with EC50 values ranging from 2.2 to 5.1 µM. In particular, compound 12I-i,
identified as the most potent inhibitor against S. sclerotiorum with an EC50 value of
2.2 µM, showed 15ꢀfold higher inhibitory activity than thifluzamide. Furthermore,
eight derivatives (i.e. 12III-a, 12III-e, 12III-f, 12III-i, 12III-l, 12III-o, 12III-p and
12III-q) exhibited improved fungicidal activity against R. solani in comparison to
lead compound 1 (EC50 = 11.0 µM), with EC50 values ranging from 0.5 to 10.6 µM.
However, replacement of the phenyl core by pyrrole or furan group led to the
unexpected decrease in bioactivity against P. grisea.
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As shown in Table 1, substitution on the pyrazole ring at A site was detrimental to
fungicidal activity of the synthesized compounds. Notably, 12I and 12III with
chlorine substituted pyrazole displayed remarkably better activities than the
corresponding unsubstituted ones (12II and 12IV). As for the chlorine substituted
pyrazoleꢀfuran series 12I, the influence of substituents and their positions at aniline
moiety was investigated. Modification at the ortho, meta and para position of aniline
with electronꢀwithdrawing or donating groups (12I-b~r) did not significantly augment
the fungicidal activity as compared to 12I-a. Furthermore, among the single
substituted aniline analogues, substituents in the orthoꢀpositions were more favorable
for their fungicidal activities compared with the meta and paraꢀsubstituted ones. For
example, 12I-b, 12I-e and 12I-i possessed better efficacy than corresponding meta
and paraꢀsubstituted ones. As for the chlorine substituted pyrazoleꢀpyrrole series
12III, similar phenomenon was observed for the monoꢀsubstituted aniline derivatives.
In particular, the orthoꢀmethoxy substituted derivative (12III-f) displayed the most
potent fungicidal activities, with EC50 values of 3.5, 1.4 and 129.3 µM against S.
sclerotiorum, R. solani and P. grisea, respectively. Meanwhile, replacing aniline
moiety with flexible or rigid amine group was also investigated. Intriguingly, the
introduction of flexible 4ꢀ(tertꢀbutyl) benzylamine moiety (12III-o) displayed
significantly enhanced effects compared with 12III-a. Considering the above SAR
results, these findings suggested that the fungicidal potency of designed compounds
could be ascribed to a combination of factors, such as chlorine substitution at pyrazole
ring, replacement of phenyl core and variation of amine part.
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In vivo Fungicidal Activities. From the results of the in vitro test, compound 12III-f
was identified as the most promising candidates for further study, and its in vivo
fungicidal activity was determined in the greenhouse for the control of S. sclerotiorum
infected cole. As shown in Figure 5, the untreated negative control (pathogen only)
resulted in 100% disease incidence. To our delight, 12III-f afforded a preventative
rate of 60.4% against S. sclerotiorum at the concentration of 200 mg/L. In contrast,
leaves treated with thifluzamide at the same concentration only resulted in a
preventative rate of 32.6%. The results further demonstrated the practical potential of
this novel compound for crop protection.
Docking Analysis. In order to elucidate the mechanism of newly designed SDH
inhibitors and explain the SAR in details, all the derivatives were docked into the
active site of RsSDH. The detail interactions of two representative derivatives 12III-f
and 12I-g with RsSDH were provided in Figure 6. In the docked complex of
compound 12III-f and RsSDH (Figure 6(A)), the pyrazoleꢀpyrrole “core” of 12III-f
buried deep in the Qꢀstie and mainly interacted with Arg358 through Piꢀcation
interaction. Moreover, the amide function of 12III-f formed hydrogen bond with the
side chain of Trp206, which was crucial to the binding of inhibitors and SDH. The
orthꢀmethoxy substituted phenyl moiety was orientated towards the entrance of Qꢀsite,
and interacted with nearby amino acids through hydrophobic interaction. All these
favorable interactions may result in the potent fungicidal activity of 12III-f. As shown
in Figure 6(B), the binding mode of 12I-g was similar to 12III-f. The pyrazoleꢀfuran
“core” of 12I-g embedded deep into the Qꢀsite, forming Piꢀcation interaction with
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Arg358. However, the introduction of methoxy group to the para position of phenyl
moiety resulted in the repulsion interaction with Phe342. Thus, the amide function of
12I-g failed to form Hꢀbond with Tyr206, which may account for its remarkable
decrease in bioactivity. Moreover, it was observed that the pyrazole moiety had
adopted a noncoplanar conformation with the linkage group (pyrrole or furan). Thus,
we speculated that the introduction of chlorine favored the active conformation and
thus led to the enhanced bioactivity of 12I and 12III. Furthermore, the relative
weaker fungicidal potency against P. grisea of these newly designed compounds may
attribute to the sequential differences of SDH from specific plant pathogens.
In summary, we described the rational scaffold hopping and bioisosteric
modification of lead structure 1. Facilitated by molecular docking analysis, a series of
pyrazoleꢀfuran and pyrazoleꢀpyrrole carboxamide derivatives were designed and
subsequently synthesized on the basis of a systemic SAR study. Furthermore, their
fungicidal activities were evaluated against S. sclerotiorum, R. solani and P. grisea,
demonstrating that most of these compounds displayed moderate to good fungicidal
activities. Most surprisingly, compound 12I-i, 12III-f and 12III-o exhibited improved
fungicidal activity against S. sclerotiorum and R. solani comparable to that of
commercial SDHI fungicide thifluzamide and lead structure 1. SAR analysis and
molecular docking simulations revealed that the promising fungicidal potency of our
designed compounds could be ascribed to structural alteration, including the chlorine
substitution at pyrazole ring, replacement of phenyl core and modification of amine
part of lead structure 1. Thereby, the rationality and efficiency of our molecular
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347
docking assisted scaffold hopping approach in the development of novel fungicidal
agents was fully supported in our study, and the finding will lay the foundation for
further structural modification and development of novel SDH inhibitors.
SUPPORTING INFORMATION
1H NMR, 13C NMR and HRMS for the target compounds.
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369
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Figure captions
Figure 1. Lead structure 1 and its binding mode.
Scheme 1. Design of the target scaffolds.
Figure 2. The synthetic route for intermediate 4. Reagents and conditions: (a) AlCl3,
Br2, CHCl3, ꢀ10~23 °C, 4 h; (b) isopropylmagnesium chloride, THF, ꢀ40 °C, 2 h.
Figure 3. The synthetic route for intermediate 8. Reagents and conditions: (a)
2,2,2ꢀtrichloroacetyl chloride, DCM, ꢀ10~23 °C, overnight; (b) NBS, THF,ꢀ10~23°C,
10 h; (c) sodium methoxide, MeOH, 0~23°C, 1 h.
Figure 4. The synthetic route for target compounds 12I~IV. Reagents and conditions:
(a) 1ꢀmethylꢀ5ꢀ(4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolanꢀ2ꢀyl) ꢀ1Hꢀpyrazole, Pd(PPh3)4,
H3PO4•3H2O, DMF, 100 °C, 10 h; (b) NaOH, MeOH/H2O, 23 °C, 5 h; (c) NCS,
THF/DMF, 0~80 °C, 2 h; (d) HATU, N, Nꢀdiisopropylethylamine, MeCN, 60 °C,
overnight.
Figure 5. In vivo activity of 12III-f against S. sclerotiorum infected cole leaves.
Figure 6. The binding mode of 12III-f (A) and 12I-g (B).
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Table 1. Chemical structures of compounds 12I~IV and their EC50 values against S.
sclerotiorum, R. solani and P. grisea, respectively.
X
N
O
N
HN R
A
EC50 (µM)a
Rsb
No.
X
A
R
Ssb
Pgb
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Ph
5.6±0.1
5.1±1.5
17.3±1.0
8.3±0.6
14.5±2.3
6.0±1.4
>600
37.8±0.5
33.9±0.4
27.4±0.2
109.0±1.8
>600
12I-a
12I-b
12I-c
12I-d
12I-e
12I-f
12I-g
12I-h
12I-i
2ꢀClꢀPh
3ꢀClꢀPh
100.5±0.7
4ꢀClꢀPh
36.6±0.5 323.3±14.2
23.8±0.4 365.0±25.0
2ꢀMeOꢀPh
3ꢀMeOꢀPh
4ꢀMeOꢀPh
3,4ꢀMeO2ꢀPh
2ꢀCF3OꢀPh
4ꢀCF3OꢀPh
2ꢀCF3ꢀPh
4ꢀCF3ꢀPh
3ꢀMeꢀPh
34.7±0.3
>600
417.1±10.8 261.0±9.4
151.7±2.5 253.7±27.0 199.3±0.7
2.2±1.6
13.8±3.2
17.2±1.6
16.9±1.7
27.4±1.6
>600
146.3±4.5 175.3±2.6
12I-j
12I-k
12I-l
35.1±1.3
44.7±1.1
302.6±8.8
253.5±8.3
80.1±1.8
88.4±25.2 150.1±2.7
12I-m
12I-n
12I-o
12I-p
4ꢀMeꢀPh
16.2±2.2
13.9±1.2
8.9±0.6
41.4±0.3 225.0±13.3
2,5ꢀMe2ꢀPh
3ꢀNO2ꢀPh
36.1±0.5
22.9±1.1
>600
>600
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O
O
O
O
O
O
Cl
Cl
H
4ꢀcyanoꢀPh
4ꢀcyanomethylꢀPh
Ph
12.9±1.7
13.0±2.8
21.8±0.1
31.7±1.2
380.2±5.6
>600
12I-q
12I-r
298.2±7.2 102.5±2.9 191.5±10.7
12II-a
12II-b
12II-c
12II-d
H
3ꢀClꢀPh
141.5±1.7 398.3±20.3
>600
37.8±0.7
>600
H
4ꢀClꢀPh
36.1±1.1
170.8±2.8
10.8±0.8
5.7±1.0
85.2±0.2
88.8±1.6
5.4±1.0
H
2ꢀMeOꢀPh
Ph
12III-a NMe Cl
334.2±22.8
NMe Cl
NMe Cl
NMe Cl
NMe Cl
2ꢀClꢀPh
12.3±1.8 313.8±21.8
37.2±3.9 254.6±14.6
12III-b
12III-c
12III-d
12III-e
3ꢀClꢀPh
14.6±0.1
34.1±0.3
106.6±0.5
3.5±0.1
4ꢀClꢀPh
91.1±7.8
7.3±0.4
1.4±3.4
37.1±0.5
20.9±2.1
8.3±1.5
11.1±1.6
294.9±8.7
210.6±7.3
129.3±2.9
82.9±5.5
3,5ꢀCl2ꢀPh
2ꢀMeOꢀPh
3ꢀMeOꢀPh
4ꢀMeOꢀPh
3,4ꢀMeO2ꢀPh
2ꢀCF3ꢀPh
12III-f NMe Cl
NMe Cl
NMe Cl
NMe Cl
NMe Cl
81.8±1.9
4.9±1.2
12III-g
12III-h
12III-i
12III-j
209.4±6.4
140.3±3.5
141.0±5.3
8.8±1.1
68.3±3.4
12III-k NMe Cl
4ꢀCF3ꢀPh
130.6±9.3 285.3±6.9 129.0±3.8
>600 10.6±1.1 389.6±4.8
275.3±14.1 77.1±14.5 192.3±10.1
NMe Cl
NMe Cl
NMe Cl
NMe Cl
4ꢀcyanomethylꢀPh
2ꢀ(4'ꢀClꢀphenyl)ꢀPh
PhCH2
12III-l
12III-m
12III-n
12III-o
9.4±1.0
7.5±1.6
9.9±0.1
43.5±4.2
0.5±0.4
9.6±0.5
157.2±7.4
196.4±4.4
145.5±3.0
4ꢀtꢀBuꢀPhCH2
12III-p NMe Cl 6ꢀClꢀPyridinꢀ3ꢀylꢀCH2
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NMe Cl
12III-r NMe Cl
4ꢀClꢀPhCH2CH2
1ꢀnaphthalen
Ph
2.9±1.5
9.3±1.0
109.2±5.0
81.9±4.2
>600
12III-q
76.7±1.9
274.4±0.9
NMe
NMe
NMe
NMe
H
H
H
H
H
431.9±23.9 59.9±1.6
12IV-a
12IV-b
12IV-c
12IV-d
2ꢀMeOꢀPh
56.4±2.6
84.9±3.0
118.6±6.2
132.4±3.2
33.2±0.8
88.9±1.4
82.9±4.9
27.4±2.3
273.2±7.7
2ꢀ(4’ꢀClꢀphenyl)ꢀPh
PhCH2
86.7±7.9 339.6±14.5
12IV-e NMe
4ꢀtꢀBuꢀPhCH2
45.9±1.7
285.6±0.2
33.4±4.2
THc
0.09±0.01
aValues are the mean ± standard deviation (SD) of three replicates. bAbbreviations: Ss:
Sclerotinia sclerotiorum; Rs: Rhizoctonia solani; Pg: Pyricularia grisea.
cThifluzamide.
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Figure 1.
Scheme 1.
HN
O
Cl
N
O
N
N
N
N
N
H
N
H
Scaffold hopping
N
N
R
R
O
Cl
O
A
Linkage
Bioisosteric modification
A
A = Cl or H
Linkage candidate
O
S
S
O
N
N
N
N
L1
L2
L3
L4
L5
L6
L7
L8
Br
Br
O
O
a
O
b
Br
O
O
O
O
O
O
2
3
4
Figure 2.
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Journal of Agricultural and Food Chemistry
Br
Br
Cl
Cl
O
Cl
Cl
Cl
Cl
c
O
b
a
N
N
N
N
O
O
5
6
7
8
Figure 3.
O
O
O
O
R
O
OH
X
X
X
O
X
d
a
b
N
N
N
N
N
A
A
N
Br
4 X = O
9I X = O
9II X = NMe
10I X = O A = H
11I X = O A = Cl
12I
12II
X = O A= H
X = O A= Cl
c
c
8
X = NMe
10II X = NMe A = H
11II
12III X = NMe A= H
12IV X = NMe A= Cl
X = NMe A = Cl
Figure 4.
Figure 5.
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Figure 6.
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Journal of Agricultural and Food Chemistry
Graphic for table of contents
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