Discovery of Novel Pyrazole Carboxylate Derivatives Containing Thiazole as Potential Fungicides

Article abstract of DOI:10.1021/acs.jafc.1c01189

Inspired by commercially established fluxapyroxad as the lead compound of novel efficient antifungal ingredients, novel pyrazole carboxylate derivatives containing a flexible thiazole backbone were successfully designed, synthesized, and detected for their in vitro and in vivo biological activities against eight agricultural fungi. The antifungal bioassay results showed that compound 24 revealed excellent bioactivities against Botrytis cinerea and Sclerotinia sclerotiorum, with median effective concentrations (EC50) of 0.40 and 3.54 mg/L, respectively. Compound 15 revealed remarkable antifungal activity against Valsa mali, with an EC50 value of 0.32 mg/L. For in vivo fungicide control against B. cinerea and V. mali, compounds 3 and 24 at 25 mg/L, respectively, displayed prominent efficacy on cherry tomatoes and apple branches. Molecular docking results demonstrated that compound 15 could form an interaction with several crucial residues of succinate dehydrogenase (SDH), and the in vitro enzyme assay indicated that the target compound 15 displayed an inhibitory effect toward SDH, with an IC50 value of 82.26 μM. The experimental results indicated that phenyl pyrazole carboxylate derivatives displayed a weak antifungal property and low activity compared to the other title substituent pyrazole carboxylate derivatives. Compounds 3, 15, and 24 are promising antifungal candidates worthy of further fungicide development due to their prominent effectiveness.

Full text of DOI:10.1021/acs.jafc.1c01189

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Article
Discovery of Novel Pyrazole Carboxylate Derivatives Containing
Thiazole as Potential Fungicides
Dongguo Xia, Xiang Cheng, Xiaohang Liu, Chengqi Zhang, Yunxiao Wang, Qiaoyun Liu, Qi Zeng,
Niqian Huang, Yao Cheng, and Xianhai Lv*
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ABSTRACT: Inspired by commercially established fluxapyroxad as the lead compound of novel efficient antifungal ingredients,
novel pyrazole carboxylate derivatives containing a flexible thiazole backbone were successfully designed, synthesized, and detected
for their in vitro and in vivo biological activities against eight agricultural fungi. The antifungal bioassay results showed that
compound 24 revealed excellent bioactivities against Botrytis cinerea and Sclerotinia sclerotiorum, with median effective concentrations
(EC50) of 0.40 and 3.54 mg/L, respectively. Compound 15 revealed remarkable antifungal activity against Valsa mali, with an EC50
value of 0.32 mg/L. For in vivo fungicide control against B. cinerea and V. mali, compounds 3 and 24 at 25 mg/L, respectively,
displayed prominent efficacy on cherry tomatoes and apple branches. Molecular docking results demonstrated that compound 15
could form an interaction with several crucial residues of succinate dehydrogenase (SDH), and the in vitro enzyme assay indicated
that the target compound 15 displayed an inhibitory effect toward SDH, with an IC50 value of 82.26 μM. The experimental results
indicated that phenyl pyrazole carboxylate derivatives displayed a weak antifungal property and low activity compared to the other
title substituent pyrazole carboxylate derivatives. Compounds 3, 15, and 24 are promising antifungal candidates worthy of further
fungicide development due to their prominent effectiveness.
KEYWORDS: pyrazole, thiazole, antifungal, Botrytis cinerea, Valsa mali, molecular docking
Encouraged by its remarkable antifungal activity, fluxapyroxad
was selected as the lead compound and the substituted
pyrazole “core” as the original pharmacophore fragment was
reserved.18 Inspired by the classical design strategy of
bioisosterism,19 the common amide bond was substituted by
the ester linkage between the pyrazole fragment and
pharmacophore moiety. Notably, pyrazole carboxylate deriva-
tives are much less frequently found than pyrazole carbox-
amide derivatives in pesticide development, which may
indicate that pyrazole carboxylate derivatives in agrochemicals
have still not been explored to their full scope in crop
protection lead discovery and optimization. Keeping in mind
the tremendous contributions of thiazole as the privileged
scaffold in medicinal and agrochemical exploration, as shown
in Scheme 1B, the thiazole backbone was widely employed in
antifungal,20 antibacterial,21 anticancer,22 insecticidal,23 and
anti-inflammatory agents.24 Thiazole derivatives with such
broad spectrum biological activities have sparked enormous
interest in the design and synthesis of compounds over the past
few years.25 The aromatic structure enables thiazole to be
modified into compounds with various biological activities.
The compounds obtained by substituting thiazole on their
INTRODUCTION
■
Pathogenic fungus has been regarded as a worldwide threat for
plant growth and food security. Botrytis cinerea is a
commercially necrotrophic pathogenic fungus, affecting
devastating diseases and significant crop losses in a wide
variety of plant species.1,2 The complex interactions between
this necrotrophic pathogenic fungus, the plants it infects, and
significant crop losses caused by B. cinerea on over 1400
species of cultivated plants attract particular attention from the
main global agrochemical researchers and companies.3
Pyrazole carboxamide derivatives showed a broad spectrum
of antifungal activities as the succinate dehydrogenase inhibitor
(SDHI),4−7 and some of them reached the market in the past
decade, such as penthiopyrad, penflufen, sedaxane, bixafen,
fluxapyroxad, and pydiflumetofen (Scheme 1A).8,9 Despite the
remarkable effectiveness of commercial SDHI fungicides to
agricultural production and food security in the past decade,
invasive fungal diseases continue to cause global agricultural
production reduction because these commercial SDHI
fungicides possess similar structural skeletons, which may
easily lead to drug resistance with continuous drug
therapy.10−12 Novel pharmacophores should be urgently
explored to avoid SDH-based fungi cross-resistance.
Received: February 26, 2021
Revised: June 11, 2021
Accepted: July 7, 2021
Published: July 19, 2021
It is well known that most SDHI fungicides consist of a
common amide function, a structurally diverse “core” moiety
attached to the carbonyl of the amide bond and a substituted
amine moiety.13−15 Fluxapyroxad as a classical SDHI fungicide
is a commercially available agricultural pesticide against various
necrotrophic pathogenic fungi for crop protection.16,17
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Scheme 1. (A−C) Design Strategy of the Target Compounds
General Procedures for Preparing the Target Compounds 1−12.
To a flame-dried round-bottom flask were successively added
intermediates a−l (1 mmol),1-methyl-3-(difluoromethyl)-1H-pyra-
zole-4-carboxylic acid (1.5 mmol),1-ethyl-3(3-dimethylpropylamine)-
carbodiimide (1.5 mmol),4-dimethylaminopyridine (1.5 mmol),and
moderate triethylamine followed by dichloromethane (10 mL). The
resulting solution was then stirred overnight at ambient temperature.
The reaction mixture was diluted with dichloromethane and
successively washed three times with diluted hydrochloric acid and
sodium bicarbonate solution. The combined aqueous layers were
washed with dichloromethane. The combined organic layers were
dried over Na2SO4, filtered, and concentrated in vacuo. The crude
products were recrystallized from ethanol or purified by column
chromatography (ethyl acetate/hexanes) to obtain the pure target
compounds 1−12.
General Procedures for Preparing the Target Compounds 13−
24. To a flame-dried round bottom flask were added intermediates a−
l (1 mmol), 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylic
acid (1.5 mmol), 1-ethyl-3(3-dimethylpropylamine)carbodiimide (1.5
mmol), 4-dimethylaminopyridine (1.5 mmol), and moderate triethyl-
amine followed by dichloromethane (10 mL). The reaction mixture
was diluted with dichloromethane and successively washed three
times with diluted hydrochloric acid and sodium bicarbonate solution.
The combined aqueous layers were washed with dichloromethane.
The combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuo. The crude products were recrystallized from
ethanol or purified by column chromatography (ethyl acetate/
hexanes) to obtain the pure target compounds 13−24.
General Procedures for Preparing Intermediate q. Compound o
is commercially available. Using compound 2 as the starting material,
the reaction mixtures were added to a solution of DMF and POCl3
and cooled to 0 °C with an ice bath to obtain intermediate p. To a
100 mL round-bottom flask, intermediate p and KMnO4 dissolved in
H2O were mixed and reacted for 4 h at room temperature. A solution
of 75% aqueous HCl was added slowly and dropwise with an addition
funnel under vigorous stirring. After completion of 75% aqueous HCl,
the white solids precipitated out from the reaction mixtures. The
organic extracts were filtered from the reaction mixtures, dried to
obtain intermediate q, and used for the next step without further
purification.
rings have great worth in the pharmaceutical and agrochemical
industries. Therefore, we envisaged that the rigid biphenyl
group was modified by attachment of flexible substituents
containing the thiazole backbone, which may improve the
bioactivity of the designed compounds. The benzene ring is
significant in drug design, and different substituted phenyl
groups improve a variety of the target molecules. Additionally,
naphthyl or isopropyl also was introduced to the target
compounds. Three series of novel pyrazole carboxylate
derivatives containing thiazole were designed, synthesized,
and detected for their in vitro and in vivo biological activities
against eight agricultural fungi.
MATERIALS AND METHODS
■
Chemicals and Instruments. Unless otherwise stated, all the
solvents and reagents were purchased from Energy, Meryer, and
Aladdin Chemicals and were analytically or chemically pure. Using
tetramethylsilane as the internal standard, H NMR and 13C NMR
1
spectra were carried out by using an Agilent DD2 600 Hz
spectrometer with CDCl3 as the solvent. Electrospray ionization−
mass spectroscopy (ESI-MS) spectra were carried out on a Mariner
System 5304 mass spectrometer.
Chemical Synthesis. The synthetic route of the target
compounds 1−12, 13−24, and 25−36 is shown in Scheme 2.
General Procedures for Preparing Intermediates a−l.26,27 4,5-
Dimethylthiazole (10 mmol) was added in a flame-dried round-
bottom flask under nitrogen conditions, then tetrahydrofuran (THF,
45 mL) was added, and the solution was cooled to −78 °C.
Whereafter, n-butyl lithium (1.9 M, 12 mmol) was added dropwise to
the cold reaction mixture. The resulting dark red solution was stirred
for 45 min at −78 °C. The appropriate aldehyde (30 mmol) was then
added, causing the solution to turn yellow, and the reaction was
stirred for 1 h at −78 °C. The reaction was warmed to room
temperature and stirred for 5 h. After completion of the reaction step,
water was added to quench the reaction. The reaction mixture was
diluted with ethyl acetate and washed with saturated salt water. The
aqueous layer was extracted two additional times with ethyl acetate.
The combined organic extracts were dried over sodium sulfate,
filtered, and concentrated in vacuo. The resulting residue was purified
by recrystallization (ethyl acetate/hexanes).
General Procedures for Preparing the Target Compounds 25−
36. To a flame-dried round bottom flask were added intermediates a−
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Scheme 2. Synthetic Route of the Target Compounds
l (1 mmol), intermediate q (1.5 mmol), 1,3-dicyclohexylcarbodiimide
(2 mmol), and 4-dimethylaminopyridine (0.1 mmol) followed by
dichloromethane (10 mL). The resulting solution was then stirred
overnight at ambient temperature. After filtration of the reaction
mixture, the filtrate was diluted with dichloromethane and washed
three times with saturated salt solution. The combined aqueous layers
were washed with dichloromethane. The combined organic layers
were dried over Na2SO4, filtered, and concentrated in vacuo. The
crude products were recrystallized from ethanol or purified by column
chromatography (ethyl acetate/hexanes) to obtain the pure target
compounds 25−36.
In Vitro Antifungal Activity Test. The selected plant pathogenic
fungi, B. cinerea, Fusarium verticillioide, Fusarium oxysporum, Valsa
mali, Sclerotinia sclerotiorum, Alternaria alternata, Fusarium graminea-
rum, and Physalospora piricola, were supplied by the Laboratory of
Plant Disease Control, Anhui Agricultural University (Hefei, China).
In vitro antifungal activities of novel pyrazole carboxylate derivatives
containing thiazole against B. cinerea, F. verticillioide, F. oxysporum, V.
mali, S. sclerotiorum, A. alternata, F. graminearum, and P. piricola were
detected at 10 mg/L according to the reported mycelial growth
inhibition methods by using the commercial fungicides boscalid and
carbendazim as positive controls.28,29 Each compound was dissolved
in DMSO to prepare the 10,000 mg/L stock solution. Percentage
inhibition was calculated by the following equation: percentage
inhibition (%) = (1 − PT/CK) × 100, where PT is the mean colony
diameter with compounds and CK is the mean colony diameter
without tested compounds. For the potent compounds with an
average inhibitory rate >70% at 10 mg/L, their median effective
concentrations (EC50) against B. cinerea, V. mali, and S. sclerotiorum
were further measured at concentrations of 16, 8, 4, 2, 1, and 0.5 mg/
L. All experiments were replicated three times, and the statistical
analyses of the antifungal bioassay were performed using SPSS
software version 26.0.
In Planta Fungicidal Activities. For the Protective Activity
against B. cinerea In Vivo. Based on the preceding test of in vitro
antifungal activity, the potent compounds 3, 24, and 27 were further
carried out in vivo on cherry tomatoes, according to reported common
methods.30,31 The selected compounds and positive controls boscalid
and carbendazim in 0.1 mL of DMSO were dissolved in 10 mL of
deionized water at 25 mg/L. Boscalid and carbendazim were used as
positive controls because they are specific commercial fungicides for
controlling B. cinerea. Each sample measured in triplicate was sprayed
evenly onto the cherry tomatoes, which had been already washed and
treated with water and 75% aqueous ethyl alcohol. After 24 h,
pathogen was inoculated on cherry tomatoes. DMSO (1%) in 10 mL
of water was set up as the blank control. All the treated samples were
then placed into an illumination incubator in 25 °C and 100% relative
humidity for 7 days. Each treatment was performed three times as
technical replicates.
For the Protective Activity against V. mali In Vivo. Based on the
preceding test of in vitro antifungal activity, compounds 3, 15, and 24
were further measured in vivo on apple branches, according to a
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j
Table 1. In Vitro Fungicidal Activities (Inhibition Rate) of the Target Compounds at 10 mg/L
a
b
c
d
e
f
Values are the mean standard deviation (SD) of three replicates. B. c: B. cinerea. F. v: F. verticillioide. F. o: F. oxysporum. V. m: V. mali. S. s: S.
g
h
i
j
sclerotiorum. A. a: A. alternata. F. g: F. graminearum. P. p: P. piricola. -: No activity.
reported common approach.29 Fresh and clean apple branches were
prepared by artificial removal of the epidermis after disinfection. The
target compound (25 mg/L) was then applied to the wound, and after
drying, V. mali was inoculated at the wound and cultured for 7 days in
an incubator at 25 °C under moisturizing conditions (100%
humidity) before evaluation of the symptoms. Boscalid and
carbendazim were employed as the positive control. Each treatment
was performed three times as technical replicates.
3.5. The crystal structure of SDH (PDB entry: 2fbw) was downloaded
from the RCSB Protein Data Bank. After preparing protein to get
protein that can be used for docking, defining the active site of the
receptor protein from current selection. Compound 15 and boscalid
were imported into Discovery Studio for ligand preparation. The
molecular docking procedures were performed by using the
CDOCKER protocol for the receptor−ligand interaction section of
Discovery Studio 3.5.
Scanning Electron Microscopy (SEM) Observations. SEM
observations on the hyphae of V. mali were carried out according to
the reported methods.32,33 Mycelial disks were cut from the periphery
of the colony grown on potato dextrose agar (PDA) containing EC50
(0.32 mg/L) of compound 15 and 0.1% DMSO (blank control). The
samples were fixed in a 4 °C stationary liquid (5% glutaraldehyde, 0.1
M phosphate buffer) at room temperature for 12 h, were washed with
0.1 M phosphate buffer for 20 min, and were dehydrated successively
with 30, 50, 70, 80, 95, and 100% ethanol and 100% acetone at 4 °C
temperature. After drying at critical points by CO2 and gold coating,
the mycelial cell wall morphology was carried out under SEM
(HITACHI, S-4800, Japan) at an accelerating voltage of 3.0 kV.
In Vitro Enzyme Inhibition Assay. The in vitro inhibitory effects
of title compounds 3, 15, and 24 and boscalid against fungal succinate
dehydrogenase (SDH) were determined by a succinate dehydrogen-
ase assay kit that was purchased from the Nanjing Jiancheng
Bioengineering Institute. After being incubated in PDA for 72 h,
the mycelia of B. cinerea and V. mali were collected for preparing the
tested mitochondrial suspension. The tested compounds were added
into working solution according to the operating instruction of the
purchased SDH assay kit. After adding mitochondrial suspensions
containing fungal SDH into the mentioned-above mixture solution,
the absorbance values of the obtained mixtures were monitored at 600
nm to calculate median inhibitory concentration (IC50) against fungal
SDH.34
RESULTS AND DISCUSSION
■
Chemistry. To develop novel fungicides by integrating the
substituent pyrazole “core” and the active scaffold of thiazole
into one molecule, three series of novel pyrazole carboxylate
compounds were successfully designed, synthesized, and well
characterized. General procedures for preparing the target
compounds 1−36 are depicted in Scheme 2. According to the
reported procedure,26 using different substituted benzaldehyde
and 4,5-dimethylthiazole as the starting materials, intermedi-
ates a−l were prepared in a yield of 75−90% through
reduction reaction. Target compounds 1−36 were obtained
through the classical esterification reactions of the correspond-
ing intermediates a−l. The chemical structures of all the
synthesized compounds were confirmed by H NMR, 13C
NMR, and MS. It should be noted in this context that, to the
best of our knowledge, all of the target compounds are first
reported.
In Vitro Antifungal Activities. In vitro antifungal activities
of novel pyrazole carboxylate derivatives containing thiazole
against B. cinerea, F. verticillioide, F. oxysporum, V. mali, S.
sclerotiorum, A. alternata, F. graminearum, and P. piricola were
detected at 10 mg/L according to a reported mycelial growth
inhibition method by using boscalid and carbendazim as
1
Molecular Docking. Molecular docking of compound 15 and
boscalid to SDH was performed with the software Discovery Studio
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positive controls. As listed in Table 1, the results indicated that
majority of the newly synthesized compounds displayed
remarkable inhibitory effects toward B. cinerea and V. mali,
and even several of these were superior to the commonly used
fungicides boscalid and carbendazim. Among all the synthe-
sized compounds, compounds 3, 4, 14, 15, 21, and 24 showed
remarkable fungicide activity against more than half of the
tested fungi, with an inhibitory rate of over 50% at 10 mg/L.
For the outstanding compounds with an average inhibition
>70% at 10 mg/L, their median effective concentrations
(EC50) against B. cinerea, V. mali, and S. sclerotiorum were
measured according to the reference standard by using
boscalid and carbendazim as positive controls. As listed in
Table 2, compounds 3, 24, and 27 revealed excellent antifungal
effects toward B. cinerea, with EC50 values of 0.77, 0.40, and
0.87 mg/L, respectively. Compounds 3, 15, and 24 displayed
prominent antifungal effects toward V. mali, with EC50 values
of 0.67, 0.32, and 0.57 mg/L, respectively. To evaluate
inhibitory effects of the outstanding compounds mentioned
above, these compounds were further tested for their in vivo
antifungal activities.
In Planta Fungicidal Activities. Based on the preceding
test results of in vitro antifungal activity, compounds 3, 24, and
27 at 25 mg/L were further tested for in vivo protective activity
against B. cinerea on cherry tomatoes. As shown in Figure 1A,
Table 2. In Vitro Antifungal EC50 Value against B. cinerea, V.
mali, and S. sclerotiorum of the Target Compounds
95%
EC50
(mg/L)
confidence
regression
equation
fungi
compd
interval
r
B. c
3
0.77
1.75
2.25
1.18
3.50
4.38
2.15
2.17
3.17
0.40
0.87
0.79
0.91
0.67
2.86
3.83
0.32
0.57
13.91
4.22
3.54
0.96
0.50
0.40−1.13 y = 1.805x +
0.991
0.209
4
1.19−2.30 y = 1.739x − 0.985
0.421
9
1.52−3.01 y = 1.488x − 0.998
0.523
11
14
15
18
19
21
24
27
0.75−1.59 y = 1.896x − 0.985
0.132
2.79−4.35 y = 2.061x − 0.989
1.122
Figure 1. In planta fungicidal activities. (A) In vivo fungicidal activities
against B. cinerea on cherry tomatoes. (B) In vivo fungicidal activities
against V. mali on apple branches.
3.53−5.48 y = 2.058x − 0.990
1.320
1.42−2.92 y = 1.445x − 0.998
0.481
cherry tomatoes of control showed that the mycelia developed
white filamentous sectors on the surface of cherry tomatoes
without pesticide application. The tested compounds exhibited
a significant biocontrol effect toward B. cinerea and were almost
equal to positive controls, where merely the inoculated
position was slightly sunken.
1.44−2.93 y = 1.461x − 0.985
0.490
2.38−4.09 y = 1.690x − 0.993
0.846
0.12−0.74 y = 1.542x +
0.977
0.985
0.971
0.972
0.976
0.608
0.30−1.45 y = 1.139x +
According to the preceding screening results, compounds 3,
15, and 24 at 25 mg/L were further tested for in vivo protective
activity against V. mali on apple branches. As shown in Figure
1B, under the fungal infection conditions, the experimental
results demonstrated that the mycelia developed white
filamentous sectors over the whole wound on the apple
branch for CK. Among all the tested compounds, compound 3
could significantly inhibit V. mali to expand from the
inoculation site to the surroundings of the strains at 25 mg/
L, even slightly superior to boscalid and carbendazim. In
addition, the other tested compounds showed prominent
inhibitory potency toward V. mali on apple branches. The
results demonstrated that compound 3 at 25 mg/L was a
highly effective antifungal candidate toward V. mali in planta.
Scanning Electron Microscopy (SEM) Analysis. The
mycelia morphology changes of each treated V. mali were
observed in the appropriate size by scanning electron
microscopy (SEM). As shown in Figure 2, normal mycelial
morphological characteristics of V. mali in the untreated
groups (Figure 2A,B) were relatively smooth and regular,
whereas V. mali mycelia treated with compound 15 (Figure
2C,D) at 0.32 mg/L significantly shrunk. These SEM
0.072
boscalid
0.32−1.28 y = 1.327x +
0.134
carbendazim
0.29−1.51 y = 1.658x +
0.068
V. m
3
0.31−1.05 y = 1.648x +
0.285
4
2.20−3.59 y = 1.913x − 0.991
0.872
14
3.07−4.77 y = 2.055x − 0.977
1.200
15
0.02−0.78 y = 0.928x +
0.960
0.980
0.461
24
0.22−0.93 y = 1.578x +
0.389
boscalid
carbendazim
24
10.13−22.63 y = 1.795x − 0.964
2.052
3.55−5.14 y = 1.674x − 0.980
1.047
S. s
2.90−4.30 y = 2.343x − 0.977
1.286
boscalid
carbendazim
0.68−1.25 y = 1.789x +
0.994
0.995
0.029
0.02−1.01 y = 2.253x +
0.668
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Figure 2. Scanning electron micrographs of the hyphae of V. mali grown on PDA with DMSO or compound 15 at 25 °C. (A, B) Untreated control;
(C, D) after 72 h of compound 15 at 0.32 mg/L (EC50) treatment.
observations revealed that compound 15 caused a certain
degree of damage to the cell wall of V. mali.
ARG43, and TRP173 in the semi-open ellipsoid region on
succinate dehydrogenase, and the pyridine fragment of
boscalid occupies the cavity of the SDH enzyme inside.
Compound 15 formed a hydrogen bond interaction with
residues TYR58 and SER39, and the benzene ring of
compound 15 formed a π−π interaction with the residue
ARG43.
In Vitro Enzyme Assay. For antifungal activity of the
target compounds in vitro, compounds 3, 15, and 24 exhibited
the highest activities against B. cinerea and V. mali, respectively.
To further investigate the antifungal mechanism of the title
compounds, compounds 3, 15, and 24 were detected for in
vitro enzyme inhibition experiments. As shown in Table 3, the
In summary, this current accomplished study highlighted
pyrazole carboxylate derivatives containing thiazole assisted by
the lead compound fluxapyroxad for the discovery of promising
agrochemicals. From the in vitro bioassay results, majority of
novel pyrazole carboxylate derivatives containing thiazole
displayed prominent efficacy against the tested phytopatho-
genic fungi and structure−activity relationships were inves-
tigated. As shown in Table 1, the experimental results indicated
that the introduction of the phenyl group at the pyrazole ring
displayed a weak antifungal property and low activity
compared to the other title substituent pyrazole carboxylate
derivatives. This work demonstrated that the design strategy of
new fungicides was a rational approach by optimized
substituent group at the R position of the thiazole backbone
to improve efficacy and specificity; especially, the introduction
of the m-chlorophenyl group at the R site remarkably improved
effectiveness and selectivity for treatment of phytopathogenic
fungi, exemplified by compounds 3, 15, and 27. Among all the
pyrazole carboxylate derivatives with the trifluoromethyl group,
compound 24 demonstrated that the profitably conglomer-
ation of the isobutyl group and thiazole backbone exhibited the
most excellent inhibitory effect against B. cinerea and V. mali.
Furthermore, the in vitro enzyme assay indicated that the target
Table 3. IC50 Values of Fungal SDH Inhibition Activity
IC50 (μM)
compd
B. c
>100
V. m
>100
3
15
24
>100
>100
82.26 2.55
>100
boscalid
18.56 1.30
25.22 1.51
experimental results indicated that compound 15 showed
inhibitory activity against SDH enzymes, with an IC50 value of
82.26 μM, and was inferior to boscalid. In addition,
compounds 3 and 24 showed no inhibitory effect toward
SDH enzymes of B. cinerea and V. mali, and these compounds
may show in vitro inhibitory activities against B. cinerea and V.
mali by other antifungal mechanisms.
Molecular Docking Analysis. The molecular docking
model of the representative compound 15 and boscalid with
SDH is shown in Figure 3. The binding mode of the positive
drug boscalid with SDH indicated that boscalid could form an
interaction with several crucial residues including TYR58,
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Figure 3. Binding modes of compound 15 (A, B) and boscalid (C, D) with SDH (3D and 2D diagram).
compound 15 displayed an inhibitory effect toward SDH, with
an IC50 value of 82.26 μM, and molecular docking results
demonstrated that compound 15 could form an interaction
with several crucial residues of SDH. Compounds 3 and 24
showed no inhibitory activity against SDH despite their in vitro
remarkable antifungal activity, which was worthy of further
investigation to explore about their mechanism of inhibiting
growth of phytopathogenic fungi. Considering prominent
fungicidal effectiveness against phytopathogenic fungi, com-
pounds 3, 15, and 24 are potential antifungal candidates
worthy of further fungicide development.
Chengqi Zhang − School of Plant Protection, Anhui
Agricultural University, Hefei 230036, China
Yunxiao Wang − School of Science, Anhui Agricultural
University, Hefei 230036, China
Qiaoyun Liu − School of Science, Anhui Agricultural
University, Hefei 230036, China
Qi Zeng − School of Science, Anhui Agricultural University,
Hefei 230036, China
Niqian Huang − School of Science, Anhui Agricultural
University, Hefei 230036, China
Yao Cheng − School of Science, Anhui Agricultural University,
Hefei 230036, China
ASSOCIATED CONTENT
■
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.1c01189
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.1c01189.
Notes
The authors declare no competing financial interest.
Chemical structures of all the synthesized compounds as
confirmed by 1H NMR, 13C NMR, and MS, report of all
of the target compounds, and more details of general
procedures, yields, and physical and characterization
data of all the synthesized compounds (PDF)
ACKNOWLEDGMENTS
■
This work was supported by Anhui Province Natural Science
Funds for Distinguished Young Scholars (2008085J09). We
are grateful to Prof. Hailiang Zhu (State Key Laboratory of
Pharmaceutical Biotechnology, Nanjing University, Nanjing)
for computational molecular docking assistance.
AUTHOR INFORMATION
Corresponding Author
Xianhai Lv − School of Science, Anhui Agricultural University,
Hefei 230036, China; orcid.org/0000-0001-8535-0015;
Phone: +86-551-6578-6096; Email: lvxianhai@
ahau.edu.cn
■
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Dongguo Xia − School of Science, Anhui Agricultural
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Xiang Cheng − School of Science, Anhui Agricultural
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Xiaohang Liu − School of Plant Protection, Anhui Agricultural
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