Expedient discovery for novel antifungal leads targeting succinate dehydrogenase: Pyrazole-4-formylhydrazide derivatives bearing a diphenyl ether fragment

Article abstract of DOI:10.1021/acs.jafc.0c03736

The pyrazole-4-carboxamide scaffold containing a flexible amide chain has emerged as the molecular skeleton of highly efficient agricultural fungicides targeting succinate dehydrogenase (SDH). Based on the above vital structural features of succinate dehydrogenase inhibitors (SDHI), three types of novel pyrazole-4-formylhydrazine derivatives bearing a diphenyl ether moiety were rationally conceived under the guidance of a virtual docking comparison between bioactive molecules and SDH. Consistent with the virtual verification results of a molecular docking comparison, the in vitro antifungal bioassays indicated that the skeleton structure of title compounds should be optimized as an N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide scaffold. Strikingly, N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives 11o against Rhizoctonia solani, 11m against Fusarium graminearum, and 11g against Botrytis cinerea exhibited excellent antifungal effects, with corresponding EC₅₀ values of 0.14, 0.27, and 0.52 μg/mL, which were obviously better than carbendazim against R. solani (0.34 μg/mL) and F. graminearum (0.57 μg/mL) as well as penthiopyrad against B. cinerea (0.83 μg/mL). The relative studies on an in vivo bioassay against R. solani, bioactive evaluation against SDH, and molecular docking were further explored to ascertain the practical value of compound 11o as a potential fungicide targeting SDH. The present work provided a non-negligible complement for the structural optimization of antifungal leads targeting SDH.

Full text of DOI:10.1021/acs.jafc.0c03736

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Article
Expedient Discovery for Novel Antifungal Leads Targeting Succinate
Dehydrogenase: Pyrazole-4-formylhydrazide Derivatives Bearing a
Diphenyl Ether Fragment
Xiaobin Wang, An Wang, Lingling Qiu, Min Chen, Aimin Lu, Guohua Li, Chunlong Yang,*
and Wei Xue*
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ABSTRACT: The pyrazole-4-carboxamide scaffold containing a flexible amide chain has emerged as the molecular skeleton of
highly efficient agricultural fungicides targeting succinate dehydrogenase (SDH). Based on the above vital structural features of
succinate dehydrogenase inhibitors (SDHI), three types of novel pyrazole-4-formylhydrazine derivatives bearing a diphenyl ether
moiety were rationally conceived under the guidance of a virtual docking comparison between bioactive molecules and SDH.
Consistent with the virtual verification results of a molecular docking comparison, the in vitro antifungal bioassays indicated that the
skeleton structure of title compounds should be optimized as an N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide scaffold.
Strikingly, N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives 11o against Rhizoctonia solani, 11m against Fusarium
graminearum, and 11g against Botrytis cinerea exhibited excellent antifungal effects, with corresponding EC50 values of 0.14, 0.27, and
0.52 μg/mL, which were obviously better than carbendazim against R. solani (0.34 μg/mL) and F. graminearum (0.57 μg/mL) as
well as penthiopyrad against B. cinerea (0.83 μg/mL). The relative studies on an in vivo bioassay against R. solani, bioactive evaluation
against SDH, and molecular docking were further explored to ascertain the practical value of compound 11o as a potential fungicide
targeting SDH. The present work provided a non-negligible complement for the structural optimization of antifungal leads targeting
SDH.
KEYWORDS: pyrazole-4-formylhydrazine, diphenyl ether, crop protection, fungicide, lead optimization, succinate dehydrogenase inhibitor
tion, which implicitly indicates the optimization importance of
amide chain fragments for developing novel carboxamide
fungicides with higher efficacies and broader biological
spectra.16−18
1. INTRODUCTION
The electron transfers along mitochondrial respiratory enzyme
complexes generate a vital proton gradient across the cellular
membrane, which activates adenosine 5′-triphosphate (ATP)
synthase that translates adenosine 5′-diphosphate into ATP.1,2
The above oxidative phosphorylation on cellular mitochondria
produces essential energies for maintaining normal physio-
logical and biochemical reactions within aerobic eukaryotes.3,4
In this pivotal biochemical process, succinate dehydrogenase
(SDH), recognized as the crucial component of respiratory
enzyme complexes, catalyzes succinate oxidation coupling with
electron transfers from succinate to ubiquinone.5−7 Over the
past half a century, practical explorations have confirmed that
inhibiting the normal physiological function of SDH within
phytopathogenic fungi could be an important strategy to
effectively control the fungal infections in grains, vegetables,
and fruits.8−10 During the development process of succinate
dehydrogenase inhibitors (SDHIs) that structurally possess a
common carboxamide framework, a stable pyrazole-4-carbox-
amide fragment has emerged as the indispensable dominant
scaffold (Figure 1) that leads to a significant improvement in
the antifungal effects of constructed carboxamides against
phytopathogens.9,11−15 Currently, introducing a flexible amide
chain into pyrazole-4-carboxamide templates (e.g., isoflucy-
pram, pyrapropoyne, and pydiflumetofen) is considered as an
innovative exploitation strategy for SDHI structural optimiza-
Hydrazides exist as nitrogenous fragments in numerous
bioactive compounds that present antibacterial,19 antifungal,20
antiviral,21 insecticidal,22 herbicidal,23 anticancer,24 anticoagu-
lant,25 anti-inflammatory,26 and antimalarial27 properties.
Among them, formylhydrazide skeletons are widely utilized
as the ideal bioisosteres of carboxamide scaffolds for
developing novel agrochemical candidates due to their more
effective combinations with various biological enzymes than a
carboxamide moiety within living organisms.28−30 As repre-
sentative fungicides bearing a formylhydrazide fragment,
miexiuyihao and famoxadone (Figure 2) were applied as
efficacious agricultural measures to control fungal diseases and
reduce crop losses.31,32 During the last decade, extensive
studies on the structural optimizations of carboxamide
derivatives documented that replacing the formylamide
Received: June 14, 2020
Revised: October 25, 2020
Accepted: November 10, 2020
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A

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Figure 1. Commercialized SDHIs bearing a pyrazole-4-carboxamide fragment as agricultural fungicides.
Figure 2. Design strategy of title compounds.
fragment in bioactive leads with a formylhydrazide skeleton
could significantly improve their inhibition effects and greatly
broaden their biological spectra against phytopathogenic
fungi.29,30,33−35 Recently, inhibition of the normal physio-
logical function of cellular SDH was revealed as the underlying
action mechanism of formylhydrazide derivatives against
agricultural fungi.29,36
Diphenyl ether derivatives have not only emerged as
versatile secondary metabolites exhibiting antibacterial,37
anticancer,38 anti-inflammatory,39 antidiabetic,40 and osteo-
genic41 bioactivities, but also are proverbially utilized as
valuable industrial products, including flame retardants,
fungicides, herbicides, insecticides, perfumes, coatings, preser-
vatives, and so on.42−45 Remarkably, subsequent explorations
on the application developments of diphenyl ethers unraveled
that introducing this special lipophilic group into bioactive
leads could effectively improve the permeability of obtained
molecules within the cellular membranes of phytopathogenic
fungi.46 As a bioactive diphenyl ether derivative blocking
electron transfers along the cellular respiratory chain,
metominostrobin (Figure 2) was arduously developed as an
agricultural fungicide after long-term studies on structural
optimizations of natural strobilurins.47 Meanwhile, integrations
of lipophilic diphenyl ether and bioactive 1,2,4-triazole
fragments in a single molecular structure ingeniously
constructed the commercialized fungicides difenoconazole
and mefentrifluconazole (Figure 2), which inhibit ergosterol
biosynthesis within agricultural fungi.48,49 Recently, diphenyl
ether fragments have aroused our interest again due to their
existence in the novel fungicide aminopyrifen (Figure 2),
which perturbs the remodeling and formation of cell wall
polymers in phytopathogenic fungi.50
Considering the structural features of the commercialized
SDHIs bearing a pyrazole-4-carboxamide scaffold as well as the
B
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a
Scheme 1. Synthesis of Title Compounds 11a−t
a
Reagents and conditions: (a) NaNO2 (1.2 equiv), HCl, and 0 °C; (b) SnCl2 (3.0 equiv), HCl, and 0 °C; (c) NaOH (1.2 equiv), H2O, and reflux;
(d) triethyl orthoformate (2.0 equiv), acetic anhydride, and reflux; (e) R2NHNH2 (3.0 equiv), reflux; (f) LiOH (1.5 equiv), THF, and reflux; (g)
5% HCl, pH = 2.0; (h) R2NHNH2 (1.0 equiv), EtOH, and reflux; (i) POCl3 (25.0 equiv), dimethylformamide (DMF), and reflux; (j) NaOH, pH
= 7.0; (k) KMnO4 (1.5 equiv), H2O, and 80 °C; (l) 5% HCl, pH = 2.0; (m) TBTU (1.5 equiv), NEt3 (1.5 equiv), and CH2Cl2; (n) SOCl2 (5.0
equiv), reflux; and (o) NEt3 (1.5 equiv), CH2Cl2.
(AB Sciex, America). A Thermo Nicolet 380 FT-IR spectrometer
(Thermo Nicolet Corporation, America) scanned the KBr flake
carrier containing a title compound to obtain a relevant infrared (IR)
spectrum.
vital applications of formylhydrazide and diphenyl ether
fragments on searching for novel agricultural fungicides, the
crucial aims of this work are mainly to: (i) construct novel
potential SDHIs by ingeniously integrating formylhydrazide
and diphenyl ether fragments in the molecular structures of
commercialized pyrazole-4-carboxamides, as shown in Figure
2, (ii) generate a series of pyrazole-4-formylhydrazine
derivatives bearing a diphenyl ether fragment (Scheme 1)
and evaluate their in vitro and in vivo antifungal effects against
Rhizoctonia solani, Fusarium graminearum, Botrytis cinerea, and
Colletotrichum capsici, and (iii) analyze the structure−activity
relationship of title compounds against the above-tested strains
and further investigate the interaction modes of title
compounds against SDH. To the best of our knowledge, this
is the first report on the design, synthesis, and antifungal
activity of pyrazole-4-formylhydrazine derivatives bearing a
diphenyl ether fragment as potential SDHIs.
2.2. General Synthesis Procedures for Intermediates 4. In an
ice bath, sodium nitrite (12.00 mmol) dissolved in distilled water
(3.00 mL) was dropwise added into concentrated hydrochloric acid
(5.00 mL) containing a substituted phenoxyaniline (10.00 mmol) and
then, the resulting mixture was stirred for 1 h. Subsequently, the
above solution containing an intermediate 2 was slowly added into
concentrated hydrochloric acid (7.50 mL) containing stannous
chloride (30.00 mmol) in an ice bath. After the above mixture was
stirred at 0 °C for 1 h, the solid that emerged was filtered and washed
with distilled water (30.00 mL), cooled alcohol (30.00 mL), and
cooled ether (30.00 mL) to obtain a substituted phenoxy phenyl-
hydrazine hydrochloride 3. The intermediate 3 (10.00 mmol) mixed
with sodium hydroxide (12.00 mmol) in distilled water (50.00 mL)
was extracted with dichloromethane (30.00 mL × 3), and the
obtained organic phase was removed under reduced pressure to
generate a substituted phenoxy phenylhydrazine 4.
2.3. General Synthesis Procedures for Intermediates 8.
Intermediates 8a−d were successfully synthesized using the reported
procedures, with minor modifications.4 Acetic anhydride (30 mL)
containing a substituted ethyl acetoacetate (100.00 mmol) and
triethyl orthoformate (200.00 mmol) was stirred under reflux for 7 h
and then, superfluous acetic anhydride was removed under reduced
pressure. After the above yellow liquid including an intermediate 6
was slowly added into the ethyl acetate (50.00 mL) containing
substituted hydrazine (300.00 mmol) in an ice bath, the obtained
solution was stirred under reflux for 3 h and was then extracted with
ethyl acetate (50.00 mL × 3). After that, the obtained organic phase
was removed under reduced pressure to obtain a substituted ethyl 1H-
pyrazole-4-carboxylate 7. Subsequently, the tetrahydrofuran (20.00
2. MATERIALS AND METHODS
2.1. Chemicals and Instruments. All used solvents and reagents
without further purification were analytical reagent grade. Thin-layer
chromatography (TLC) on silica gel GF254 was used to monitor
chemical reactions. The melting points (mp) of all synthesized
compounds were tested using an uncorrected SMP50 Automatic
Melting Point Apparatus from Bibby Scientific LTD (Staffordshire,
United Kingdom). The synthesized compounds dissolved in a
DMSO-d6 or CDCl3 solvent were detected on a BRUKER 400
NMR spectrometer (Bruker Corporation, Germany) to generate
1
corresponding H and 13C NMR spectra at room temperature. Mass
spectral studies on pyrazole-4-formylhydrazine derivatives were
performed on a Triple TOF 5600 plus LC/MS/MS spectrometer
C
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mL) containing an intermediate 7 (10.00 mmol) and 10% lithium
hydroxide (15.00 mmol) was stirred under reflux for 3 h and then,
superfluous tetrahydrofuran was removed under reduced pressure.
After the remaining solution was acidified with 5% hydrochloric acid
until the pH values reached 2.0, the yellow solid formed in water was
filtered and washed with distilled water to generate a substituted 1H-
pyrazole-4-carboxylic acid 8. Meanwhile, the relevant synthetic
procedures for intermediates 8e and 8f were reported in detail in
our previous work.51
2.4. General Synthesis Procedures for Title Compounds
11a−q. Dichloromethane (30 mL) containing a substituted 1H-
pyrazole-4-carboxylic acid 8 (2.00 mmol), a substituted phenoxy
phenylhydrazine 4 (2.00 mmol), triethylamine (3.00 mmol), and O-
(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate
(TBTU, 3.00 mmol) was stirred for 2 h at room temperature. After
the above reaction was completed, the superfluous solvent was
removed under reduced pressure and the resulting mixture was
separated by column chromatography (Vpetroleum ether:Vethyl acetate = 1:2)
to produce a title compound. The obtained pyrazole-4-formylhy-
drazine derivatives bearing a diphenyl ether fragment were structurally
characterized by Fourier-transform infrared spectroscopy (FT-IR), 1H
NMR, 13C NMR, and high-resolution mass spectrometry (HRMS)
spectra that are collected and presented in the Supporting
Information.
of the fungal colonies was measured and the data were statistically
analyzed. The title compounds, which had inhibitions that exceeded
50% at 10 μg/mL, were further tested for their antifungal effects at
five double-declining concentrations to calculate the corresponding
EC50 values using SPSS 11.5 software. The in vivo anti-R. solani effects
of title compounds were determined on rice leaves (yiyou 186) using
a detached leaf assay, which is a widely used testing method for
investigating the practical application of bioactive molecules as
potential agricultural fungicides.31,34,36,57 Although the SDHI
fungicides boscalid and penthiopyrad exhibited outstanding anti-R.
solani effects in the in vitro bioassays, they are not registered and
recommended for effectively controlling the fungal diseases caused by
R. solani. Therefore, the agricultural fungicides validamycin and
carbendazim, which are registered and recommended for effectively
controlling the fungal diseases caused by R. solani,53,58 were selected
as positive controls to investigate the practical application of
constructed compounds as potential anti-R. solani fungicides.
2.7. In Vitro SDH Inhibition Assay. The succinate dehydrogen-
ase assay kit (BC0955) purchased from Beijing Solarbio Science &
Technology Co., Ltd was used to determine the in vitro inhibitory
effects of title compound 11o against SDH, with the commercialized
SDHIs boscalid and penthiopyrad as positive controls.5,13 The
mitochondrial suspension containing fungal SDH was collected
from the mycelia of R. solani that had been incubated in potato
dextrose medium for 5 days.14,17 An inhibitor dissolved in 2 μL
DMSO was added into 190 μl specific substrates that are provided by
the succinate dehydrogenase assay kit (BC0955). After adding 10 μL
mitochondrial suspensions into the above specific substrates, the
absorbance values of the resulting mixtures were monitored at 595 nm
to determine the inhibition effects of corresponding treatments
against fungal SDH.9−11,14,29
2.8. Molecular Docking Study. The three-dimensional con-
formations of title compounds were drawn using SYBYL 2.0 software
(Shanghai Tri-I. Biotech. Inc., China).2,4,30,51 Molecular energy
optimizations of three-dimensional conformations with a convergence
criterion of 0.01 kcal/mol were obtained using Powell and Conj Grad
conjugate gradient algorithms, Tripos force field, and the Gasteiger−
Huckel charge.2,4,30,51,59 The SDH crystal structure (PDB code:
2FBW) was downloaded from the RCSB Protein Data Bank (https://
www.rcsb.org) and was treated using Discovery Studio 4.5 to remove
the superfluous nonprotein compositions from the crystal structure of
2FBW.9−11,17 The above three-dimensional structures of the title
compounds and the crystal structure of 2FBW were further treated
using AutoDockTools 1.5.6 to generate docking input files.4,5,7,8 The
binding energies between the title compounds and 2FBW were
calculated using Autodock 4.2.6.4,12,14 The obtained three-dimen-
sional binding modes between title compounds and 2FBW were
determined using PyMol 1.7.6 and were further treated using
Discovery Studio 4.5 to obtain the corresponding two-dimensional
binding modes.4,7−15,60
2.5. General Synthesis Procedures for Title Compounds
11r−t. Thionyl chloride (10.00 mmol) containing a substituted 1H-
pyrazole-4-carboxylic acid 8 (2.00 mmol) was stirred under reflux for
3 h. After the superfluous thionyl chloride was removed under
reduced pressure, the obtained residue dissolved in dichloromethane
(15 mL) was stirred with dichloromethane (15 mL) containing (4-(4-
chlorophenoxy)phenyl)hydrazine (10.00 mmol) and triethylamine
(15.00 mmol) for 1 h in an ice bath. Then, the superfluous solvent
was removed under reduced pressure, and the resulting mixture was
subsequently separated by column chromatography
(Vpetroleum ether:Vethyl acetate = 2:1) to produce a title compound. The
obtained pyrazole-4-formylhydrazine derivatives bearing a diphenyl
1
ether fragment were structurally characterized by FT-IR, H NMR,
13C NMR, and HRMS spectra, which are collected and presented in
the Supporting Information.
2.6. Antifungal Effects of Title Compounds In Vitro and In
Vivo. The tested strains B. cinerea, R. solani, F. graminearum, and C.
capsici were provided by the State & Local Joint Engineering Research
Center of Green Pesticide Invention and Application at Nanjing
Agricultural University. In the in vitro bioassays conducted in our
present work, the commercial agricultural fungicides carbendazim,
boscalid, and penthiopyrad were selected as the positive controls to
evaluate the practical value of constructed molecules as potential
fungicide candidates. Carbendazim is the broad-spectrum systemic
fungicide that is currently used for effectively controlling the fungal
diseases caused by R. solani and F. graminearum (two important fungi
that were bioassayed in our present work).52−54 As the widely used
SDHI fungicides that were registered for effectively controlling the
fungal diseases caused by B. cinerea (an important fungus that was
bioassayed in our present work),55,56 boscalid and penthiopyrad are
widely utilized as positive controls to evaluate the application
potential of the constructed molecules as potential fungicides
targeting fungal SDH.2,7,9−11,13
The in vitro antifungal effects of title compounds against the above
phytopathogenic fungi were evaluated using a mycelium growth rate
method that is briefly described as follows:2,5,7−16,29−36 100 μL
dimethylsulfoxide (DMSO) dissolved in a tested compound was
added into 45 mL of potato dextrose agar (PDA). After shaking well,
the obtained mixture was equally divided and poured into three nine-
centimeter Petri plates. Equal DMSO and the commercial agricultural
fungicides carbendazim, boscalid, and penthiopyrad were used as the
blank and positive controls, respectively. Then, a mycelia dish with a
diameter of 5 mm was aseptically inoculated in the center of the above
PDA plate with three replicates. The inoculated plates were incubated
at 25 1 °C for 3−7 days in a dark environment. After the mycelium
diameter of the blank control reached 7.0−7.5 cm, the radial growth
3. RESULTS AND DISCUSSION
3.1. Molecular Design and Virtual Verification.
Numerous studies focusing on the structural optimizations of
fungal SDHIs have been carried out during the past half a
century. These studies have shown that highly efficient and
broad-spectrum SDHIs bearing a carboxamide framework
should feature two vital molecular characteristics in chemical
structures. One structural feature is that the aromatic acid part
of carboxamide structures should be optimized as a pyrazole-4-
carboxylic acid fragment; the other is the existence of a flexible
amide chain on pyrazole-4-carboxamide templates, which was
perfectly incorporated into some commercialized agricultural
SDHIs, including isoflucypram, pyrapropoyne, and pydiflume-
tofen. Considering the above two important features in SDHI
structures, in the present work, we tactfully conceived a
pyrazole-4-formylhydrazine scaffold that has better structural
flexibility than the aryl amide fragment in commercialized
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Figure 3. Virtual molecular docking comparisons of title compound 11e (A), title compound 11p (B), title compound 11t (C), and penthiopyrad
(D) with SDH (PDB code: 2FBW).
carboxamide derivatives. Concurrently, a lipophilic diphenyl
ether fragment, which facilitates the permeability of existing
fungicides within the cellular membranes of phytopathogens,
was ingeniously introduced into the obtained pyrazole-4-
formylhydrazine scaffold to construct three types of potential
SDHIs that are different in molecular structures (Figure 2).
Compared with all commercialized SDHI fungicides bearing a
pyrazole-4-carboxamide fragment, the antifungal molecules
constructed in our present work display two crucial character-
istics in molecular structures. One feature is creatively turning
the amide link in the SDHI leads into a novel hydrazide
fragment; the other is the location difference of the diphenyl
ether fragment that was introduced into the hydrazide group of
the constructed pyrazole-4-carbohydrazine scaffold.
Increasing studies have documented that the discovery of
bioactive leads targeting functional proteins was greatly
promoted by the verification of virtual molecular docking
against constructed molecular skeletons.4,11,61−64 Therefore,
before being synthesized, the feasibility of designed com-
pounds as potential SDHIs was rationally verified by a virtual
molecular docking comparison. As shown in Figure 3A−C, the
pyrazole-4-formylhydrazine fragment in three types of designed
molecules tightly bound with SDH in an identical pocket by
approximately the same conformations, which closely
resembled the binding conformation of the pyrazole-4-
carboxamide fragment in the commercialized SDHI penthio-
pyrad (Figure 3D). As shown in Figure 3A, the phenoxy
fragment of the diphenyl ether moiety in title compound 11e
did not interact obviously with the ambient amino acid
residues at the binding pocket, which gave a weaker binding
energy of −6.455 kcal/mol. Meanwhile, the binding con-
formation of the diphenyl ether moiety in Figure 3B was very
similar to that of the 2-(4-methylpentan-2-yl)thiophene
fragment that is shown in Figure 3D. Additionally, as shown
in Figure 3B,C, although the diphenyl ether moiety of title
compound 11t possessed similar binding conformation to that
of title compound 11p, the distorted conformation of the
pyrazole ring in Figure 3C led to the weaker interaction of title
compound 11t with SDH than that of title compound 11p
with SDH. Notably, the binding energy of title compound 11p
with SDH was calculated as −7.613 kcal/mol, which was
superior to that of penthiopyrad (−6.510 kcal/mol) with SDH,
which implicitly indicated that the title compound 11p had a
better inhibition effect against SDH than penthiopyrad.
Although the above results generated by virtual molecular
docking are not exactly suitable for real situations, it still
greatly encouraged us to explore the practical value of
constructed molecules as the potential fungicides targeting
SDH.
3.2. Effective Construction. The synthetic route to
pyrazole-4-formylhydrazine derivatives bearing a diphenyl
ether fragment is shown in Scheme 1. A substituted
phenoxyaniline was oxidized by sodium nitrite to generate an
intermediate 2 that was then reacted with stannous chloride in
concentrated hydrochloric acid to synthesize a substituted
phenoxy phenylhydrazine hydrochloride 3. The above
intermediate 3 was reacted with sodium hydroxide in distilled
water to produce a substituted phenoxy phenylhydrazine 4.
The nucleophilic reaction of substituted ethyl acetoacetate
with triethyl orthoformate created an intermediate 6 that was
reacted with substituted hydrazine to obtain a substituted ethyl
1H-pyrazole-4-carboxylate 7. After the reaction of an
intermediate 7 with 10% lithium hydroxide was completed in
tetrahydrofuran, the resulting residue was acidized with
hydrochloric acid to obtain a substituted 1H-pyrazole-4-
carboxylic acid 8. The cyclization reaction of substituted
ethyl acetoacetate with substituted hydrazine was carried out
under reflux to generate a substituted 2,4-dihydro-3H-pyrazol-
3-one 9 that was reacted with phosphorus oxychloride under
reflux to synthesize a substituted 5-chloro-1H-pyrazole-4-
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a
Table 1. Inhibition Effects (%) of Title Compounds 11a−t against Phytopathogenic Fungi at 10 μg/mL
a
b
Average of three replicates. Agricultural fungicides boscalid, penthiopyrad, and carbendazim were used to compare antifungal effects with title
compounds.
carbaldehyde 10. After the above intermediate 10 was oxidized
by potassium permanganate, the resulting mixture was acidized
with hydrochloric acid to obtain a substituted 5-chloro-1H-
pyrazole-4-carboxylic acid 8e or 8f. The condensation of an
intermediate 4 with an intermediate 8 was catalyzed by TBTU
in the dichloromethane containing triethylamine to generate a
substituted N′-(2-phenoxyphenyl)-1H-pyrazole-4-carbohydra-
zide or N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide.
The chlorination of an intermediate 8 with thionyl chloride
synthesized a substituted 1-methyl-1H-pyrazole-4-carbonyl
chloride that expediently reacted with (4-(4-chlorophenoxy)-
phenyl)hydrazine to yield a substituted N-(4-(4-
chlorophenoxy)phenyl)-1-methyl-1H-pyrazole-4-carbohydra-
zide.
2956 cm−1 were attributed to the stretching vibrations of an
−NHNH− or −NH2 fragment. Simultaneously, the FT-IR
signals ranging from 1682 to 1612 cm−1 confirmed the
1
presence of a CO fragment in title compounds. In the H
NMR spectra of title compounds, the protons located at
benzene rings and methyl groups were, respectively, recognized
by the signals at 7.56−6.73 ppm and 3.95−2.25 ppm.
Meanwhile, the characteristic signals of the −CONHNH−
group in title compounds 11a−q were clearly presented by the
two peaks at 10.27−9.73 and 8.16−7.36 ppm in their 1H NMR
spectra and the singlet at 163.61−159.85 ppm in their 13C
NMR spectra. Moreover, the −NH2 fragment in title
compounds 11r−t yielded a singlet with 2H integral areas at
5.57−5.43 ppm in their corresponding 1H NMR spectra. In the
HRMS spectra of title compounds, the detection values of [M
+ H]+ ion absorption signals were consistent with their
calculated values.
3.3. Spectral Characteristic of Title Compounds.
Three categories of designed compounds were structurally
1
characterized by FT-IR, H NMR, 13C NMR, and HRMS
spectra. Their structural characteristics were carefully analyzed
and are described below. In the FT-IR spectra of title
compounds, the obvious absorption peaks located at 3411−
3.4. In Vitro Antifungal Activity Screening of Title
Compounds. A reported mycelium growth rate technique
was used as a typical tested method to explore the in vitro
F
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antifungal effects of title compounds against the phytopatho-
genic fungi including R. solani, F. graminearum, B. cinerea, and
C. capsici.2,5,7−16,29−36 Aiming to contrast the antifungal effects
of title compounds against the tested fungi, the agricultural
fungicides carbendazim, boscalid, and penthiopyrad were used
as positive controls under the same conditions. The
preliminary bioassay results in Table 1 show that most of the
title compounds, especially those derivatives bearing an N′-(4-
phenoxyphenyl)-1H-pyrazole-4-carbohydrazide skeleton, effec-
tively inhibited the in vitro mycelium growth of R. solani, B.
cinerea, and F. graminearum, with the inhibition rates
surpassing 50% at 10 μg/mL. For example, the anti-R. solani
effects of title compounds 11g−p ranged from 81.1 to 94.5% at
10 μg/mL, which were obviously better than that of boscalid
(80.3%). Meanwhile, title compounds 11g, 11h, 11m, 11n,
11o, and 11q exhibited impressive anti-B. cinerea effects at 10
μg/mL, with the corresponding inhibition rates of 74.4, 75.5,
82.2, 77.9, 79.7, and 82.5%, which were similar to
penthiopyrad (79.0%). Remarkably, the anti-F. graminearum
effects of all N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydra-
zide derivatives (11g−q) exceeded 87.1% at 10 μg/mL, which
was superior to those of other designed compounds (3.5−
59.4%), boscalid (30.6%), and penthiopyrad (30.9%).
carbendazim (0.34 μg/mL). Meanwhile, as can be seen from
Table 3, title compounds 11k, 11i, 11m, and 11q dramatically
inhibited F. graminearum in vitro, with corresponding EC50
values of 0.32, 0.54, 0.27, and 0.46 μg/mL, which were
superior to carbendazim (0.57 μg/mL). In addition, the anti-B.
cinerea EC50 values of title compounds 11g and 11q were
found to be 0.52 and 0.71 μg/mL, as shown in Table 3, which
exceeded that of penthiopyrad (0.83 μg/mL). Interestingly, the
antifungal activity screening presented in Tables 1−3 indicates
that the skeleton structure of title compounds should be
optimized as an N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbo-
hydrazide scaffold, which is consistent with the virtual
verification results that are presented in Figure 3.
3.5. Structure−Activity Relationships (SARs). As can be
seen from Tables 1−3, the inhibition effects of title
compounds against phytopathogenic fungi were undeniably
affected by the structural transformations on molecular
skeletons and substituent groups. Based on the in vitro
bioassay results, some SAR rules were analyzed and are
described below. First, Table 1 shows that pyrazole-4-
formylhydrazine derivatives containing a 4-phenoxyphenyl
fragment, such as 11g, 11h, 11i, 11j, 11k, and 11l, exhibited
better antifungal effects against all tested fungi than their
isomerides bearing a 2-phenoxyphenyl substructure (11a, 11b,
11c, 11d, 11e, and 11f). Second, as shown in Table 1, the
inhibition effects of N′-phenyl-1H-pyrazole-4-carbohydrazides
against tested fungi declined sharply by converting their
molecular skeleton into an N-phenyl-1H-pyrazole-4-carbohy-
drazide scaffold. For instance, the antifungal effects of N′-
phenyl-1H-pyrazole-4-carbohydrazides 11m and 11p against R.
solani, B. cinerea, and F. graminearum at 10 μg/mL were
obviously better than those of the corresponding N-phenyl-1H-
pyrazole-4-carbohydrazides 11s and 11t. Third, the antifungal
EC50 values in Tables 2 and 3 indicate that all N′-(4-
phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives, ex-
cept 11m, presented better antifungal effects against R. solani
than against F. graminearum and B. cinerea. Fourth, replacing a
CH3 or CHF2 group at the R1 position of N′-(4-
phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives
with a CF3 fragment will generally enhance the anti-R. solani
effects of the obtained compounds. For example, the anti-R.
solani EC50 values of title compounds 11i, 11k, 11n, and 11p
(R1 = CF3), respectively, reached 0.25, 0.29, 0.31, and 0.17 μg/
mL, which were obviously better than those of title compounds
11g, 11l, and 11q (R1 = CH3; 0.38, 0.41, and 0.17 μg/mL) as
well as 11h and 11m (R1 = CHF2; 0.31 and 0.39 μg/mL).
Fifth, the anti-R. solani activities of N′-(4-phenoxyphenyl)-1H-
pyrazole-4-carbohydrazide derivatives could be further im-
proved after the methyl group at the R2 position was changed
into a hydrogen atom. For instance, the anti-R. solani effects of
title compounds 11i and 11n (R2 = CH3) with the EC50 values
of 0.25 and 0.31 μg/mL were lower than those of title
compounds 11j and 11o (R2 = H), which had EC50 values of
0.21 and 0.14 μg/mL. Sixth, when the R4 group of N′-(4-
phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives was
substituted as a chlorine atom, most of the obtained
compounds exhibited better anti-R. solani effects than the
title compounds bearing a hydrogen atom at the above-
mentioned R4 position. As shown in Table 2, the anti-R. solani
EC50 values of title compounds 11o, 11p, and 11q (R4 = Cl)
were 0.14, 0.17, and 0.17 μg/mL, which were obviously better
than those of title compounds 11j, 11k, and 11l (R4 = H; 0.21,
0.29, and 0.41 μg/mL).
Subsequently, the EC50 values of some title compounds with
significant antifungal activities against R. solani, F. graminearum,
and B. cinerea were further evaluated and are presented in
Tables 2 and 3. As shown in Table 2, the anti-R. solani EC50
values of title compounds 11h, 11i, 11j, 11k, 11n, 11o, 11p,
and 11q were 0.31, 0.25, 0.21, 0.29, 0.31, 0.14, 0.17, and 0.17
μg/mL, respectively, which were observably better than those
of the commercialized fungicides boscalid (2.21 μg/mL) and
a
Table 2. Anti-R. solani EC50 Values of Title Compounds
b
compound
11a
regression equation
r
EC50 (μg/mL)
y = 1.90x + 3.29
y = 1.67x + 3.20
y = 1.77x + 3.08
y = 1.75x + 2.97
y = 1.57x + 3.09
y = 1.82x + 3.12
y = 1.17x + 5.79
y = 1.26x + 5.64
y = 1.20x + 5.51
y = 1.06x + 5.63
y = 1.63x + 5.87
y = 1.26x + 5.48
y = 1.27x + 5.51
y = 1.36x + 5.69
y = 0.91x + 5.77
y = 0.77x + 5.59
y = 0.88x + 5.68
y = 1.39x + 3.94
y = 1.89x + 3.55
y = 2.12x + 3.47
y = 1.06x + 4.64
y = 0.48x + 5.59
y = 5.09x + 7.38
0.99
0.94
0.94
0.88
0.93
0.98
0.99
0.99
0.96
0.99
0.98
0.98
0.99
0.99
0.98
0.98
0.99
0.99
0.99
0.97
0.99
0.98
0.93
7.91 1.66
11.87 4.46
12.17 5.41
14.37 6.57
16.31 8.69
10.67 2.63
0.38 0.13
0.31 0.13
0.25 0.21
0.21 0.18
0.29 0.24
0.41 0.11
0.39 0.32
0.31 0.27
0.14 0.09
0.17 0.12
0.17 0.13
5.83 1.00
5.85 0.62
5.30 1.31
2.21 1.84
0.06 0.03
0.34 0.22
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
11r
11s
11t
boscalid
penthiopyrad
c
c
c
carbendazim
a
b
Average of three replicates. All results are expressed as the mean
c
standard error (SE). The agricultural fungicides boscalid, penthio-
pyrad, and carbendazim were used to compare antifungal activities
with title compounds.
G
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a
Table 3. Antifungal EC50 Values of Title Compounds 11g−q against F. graminearum and B. cinerea
F. graminearum
r
B. cinerea
r
b
b
compound
11g
11k
11i
11j
11k
11l
regression equation
EC50 (μg/mL)
regression equation
EC50 (μg/mL)
y = 1.57x + 5.27
y = 1.58x + 5.79
y = 2.05x + 5.54
y = 1.31x + 5.11
y = 1.64x + 5.24
y = 1.36x + 5.10
y = 1.56x + 5.86
y = 1.46x + 5.28
y = 1.40x + 5.19
y = 1.23x + 4.22
y = 1.30x + 5.43
/
0.96
0.98
0.98
0.98
0.99
0.97
0.99
0.95
0.87
0.93
0.97
/
0.67 0.48
0.32 0.24
0.54 0.44
0.82 0.65
0.71 0.58
0.84 0.62
0.27 0.25
0.64 0.44
0.73 0.38
4.26 0.52
0.46 0.34
/
y = 0.96x + 5.27
y = 0.71x + 4.80
y = 0.67x + 4.83
y = 0.90x + 4.71
y = 0.61x + 4.59
y = 0.82x + 4.76
y = 1.20x + 4.69
y = 0.97x + 4.68
y = 0.85x + 4.87
/
0.99
0.98
0.92
0.99
0.98
0.99
0.99
0.99
0.98
/
0.52 0.11
1.89 0.41
1.76 0.73
2.10 0.44
4.59 3.64
1.97 1.60
1.81 1.51
2.11 1.81
1.41 0.48
/
11m
11n
11o
11p
11q
boscalid
y = 0.91x + 5.14
y = 1.22x + 5.32
y = 0.70x + 5.06
y = 3.54x + 6.35
0.99
0.99
0.99
0.98
0.71 0.58
0.54 0.50
0.83 0.67
0.42 0.31
c
c
penthiopyrad
carbendazim
/
/
0.99
/
c
y = 8.64x + 7.10
0.57 0.51
a
b
c
Average of three replicates. All results are expressed as the mean SE. The agricultural fungicides boscalid, penthiopyrad, and carbendazim
were used to compare antifungal activities with title compounds.
Figure 4. In vivo anti-R. solani preventative efficacies of bioactive compounds at 200 μg/mL.
Figure 5. Inhibition effects of bioactive molecules against fungal SDH.
3.6. In Vivo Anti-R. solani Control Efficacies of
Bioactive Compounds on Rice Leaves. The bioassay
results in Tables 1−3 clearly show that the in vitro mycelium
growth of R. solani, B. cinerea, and F. graminearum could be
effectively inhibited by the title compounds (11g−q) that
utilized an N′-(4-phenoxyphenyl)-1H-pyrazole-4-carbohydra-
zide scaffold as their skeleton structure. In the in vitro bioassays
conducted in our present work, the antifungal effect of title
compound 11o against R. solani was evaluated as the best
among that of all title compounds. Although lower than that of
penthiopyrad (0.06 μg/mL), the in vitro anti-R. solani EC50
value of title compound 11o remarkably reached 0.14 μg/mL,
which was approximately 2- and 15-fold higher than those of
the commercialized fungicides carbendazim (0.34 μg/mL) and
boscalid (2.21 μg/mL), respectively. Encouraged by the above
results, the in vivo anti-R. solani effect of title compound 11o
was evaluated on rice leaves (yiyou 186) using a detached leaf
assay to investigate its practical application as a potential
agricultural fungicide.31,34,36,57 As shown in Figure 4, the in
vivo anti-R. solani control efficacy of title compound 11o was
73.25% at 200 μg/mL, which was obviously better than that of
carbendazim under the same conditions (59.81%). The above
in vivo bioassay result laid a significant foundation for the
structural optimization of N′-(4-phenoxyphenyl)-1H-pyrazole-
4-carbohydrazide derivatives as potential agricultural fungi-
cides.
3.7. Inhibitory Effects against Fungal SDH In Vitro.
The title compound 11o exhibiting excellent anti-R. solani
effects in vitro and in vivo was selected as a representative
molecule to determine its inhibitory effects against the SDH
that was collected from the mycelia of R. solani. Under the
same conditions, the commercialized SDHIs boscalid and
penthiopyrad were used as positive controls for comparing the
inhibitory effects of title compound 11o against the above-
mentioned SDH. As shown in Figure 5, the IC50 values of
penthiopyrad, title compound 11o, and boscalid were,
respectively, calculated as 0.79 μM (0.29 μg/mL), 3.99 μM
(1.58 μg/mL), and 13.37 μM (4.57 μg/mL). The above
H
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Figure 6. Molecular docking modes of title compound 11o (A−C) and penthiopyrad (D−F) with SDH (PDB code: 2FBW).
bioactive evaluations against SDH provided a non-negligible
basis that powerfully ascertains the feasibility of designed
compounds as potential SDHIs.
bond with the title compound 11o (2.06 Å) and penthiopyrad
(2.18 Å), and ILE 218 connected the aromatic nucleus in title
compound 11o (4.75 Å) and penthiopyrad (5.07 Å) by a π−
alkyl interaction. Meanwhile, title compound 11o and
penthiopyrad both interacted with ARG 43 by a π−cation
interaction (4.16 and 4.16 Å) and a π−alkyl interaction (4.37
and 3.79 Å). To some extent, the above-mentioned similar
interactions of title compound 11o/penthiopyrad with the
crucial residues on SDH might be pivotal factors to guarantee
their inhibition efficiencies against phytopathogenic fungi in
vitro and in vivo. Furthermore, as clearly shown in Figure 6C,
the para-substituted chlorine atom at the benzene ring of title
compound 11o formed two alkyl interactions with the residues
ARG 43 (4.61 Å) and HIS 42 (4.58 Å), which might be the
potential reason why their antifungal bioactive expression is
different from other pyrazole-4-formylhydrazine derivatives
and penthiopyrad.
In summary, based on the vital structural features of
bioactive molecules targeting fungal SDH, 20 pyrazole-4-
formylhydrazine derivatives with three categories of structural
skeletons were conceived, synthesized, and characterized by
FT-IR, 1H NMR, 13C NMR, and HRMS analyses. The
feasibility of designed compounds as potential SDHIs was
subsequently verified by a virtually molecular docking
comparison and bioactive evaluations against phytopathogenic
fungi in vitro and in vivo. Consistent with the virtual verification
results of a molecular docking comparison, the in vitro
antifungal bioassays indicated that the skeleton structure of
title compounds should be optimized as an N′-(4-phenox-
yphenyl)-1H-pyrazole-4-carbohydrazide scaffold. Among N′-
3.8. Molecular Docking Study. A semi-open ellipsoid
region on succinate dehydrogenase was formed by several
crucial residues including tyrosine at the 58 position (TYR 58),
histidine at the 42 position (HIS 42), isoleucine at the 218
position (ILE 218), arginine at the 43 position (ARG 43),
histidine at the 216 position (HIS 216), tyrosine at the 173
position (TYR 173), isoleucine at the 40 position (ILE 40),
histidine at the 105 position (HIS 105), and so on. Numerous
crystallographic studies have confirmed that the above-
mentioned protein cavity has emerged as the active pocket
of the commercialized pyrazole-4-carboxamides targeting
fungal SDH.2−4,6−15,17,18 Based on the important structural
features of bioactive SDHI, the title compound 11o was
conveniently synthesized as the potential SDHI that exhibited
excellent antifungal effects against phytopathogenic fungi in
vitro and in vivo. To further explore their plausible binding
modes with fungal SDH, molecular docking of title compound
11o with the potential targeting protein (PDB code: 2FBW)
was conducted with the commercialized SDHI penthiopyrad as
a reference compound for comparison in the same
investigation.
As can be seen from Figure 6, title compound 11o and
penthiopyrad were embedded well in the active protein pocket
on SDH via approximately the same conformations that
connected with circumjacent residues by a conventional
hydrogen bond, π−cation, π−alkyl, alkyl, and π−sulfur
interactions. For example, TYR 58 showed a strong hydrogen
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(4-phenoxyphenyl)-1H-pyrazole-4-carbohydrazide derivatives,
compounds 11o against R. solani, 11m against F. graminearum,
and 11g against B. cinerea exhibited striking antifungal effects,
with corresponding EC50 values of 0.14, 0.27, and 0.52 μg/mL,
which were obviously better than those of carbendazim against
R. solani (0.34 μg/mL) and F. graminearum (0.57 μg/mL) as
well as penthiopyrad against B. cinerea (0.83 μg/mL).
Furthermore, the relative studies on an in vivo anti-R. solani
bioassay, bioactive evaluation against SDH, and molecular
docking were further explored to ascertain the practical value
of compound 11o as a potential fungicide targeting SDH. The
present work laid a non-negligible foundation for further
structural optimization of N′-(4-phenoxyphenyl)-1H-pyrazole-
4-carbohydrazide derivatives targeting fungal SDH.
Education, Nanjing Agricultural University, Nanjing 210095,
China
Guohua Li − Jiangsu Key Laboratory of Pesticide Science,
College of Sciences and Key Laboratory of Integrated
Management of Crop Diseases and Pests, Ministry of
Education, Nanjing Agricultural University, Nanjing 210095,
China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c03736
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the grants from the
National Natural Science Foundation of China (No.
31772209), the Fundamental Research Funds for the Central
Universities of China (No. KYTZ201604), and the Post-
graduate Research & Practice Innovation Program of Jiangsu
Province (No. KYCX19_0602).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c03736.
Physical and spectroscopic data of all conceived
pyrazole-4-formylhydrazine derivatives bearing a diphen-
yl ether moiety; all of the copies of FT-IR, 1H NMR, 13
C
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Corresponding Authors
Chunlong Yang − Jiangsu Key Laboratory of Pesticide Science,
College of Sciences and Key Laboratory of Integrated
Management of Crop Diseases and Pests, Ministry of
Education, Nanjing Agricultural University, Nanjing 210095,
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Authors
Xiaobin Wang − Jiangsu Key Laboratory of Pesticide Science,
College of Sciences and Key Laboratory of Integrated
Management of Crop Diseases and Pests, Ministry of
Education, Nanjing Agricultural University, Nanjing 210095,
China; Key Laboratory of Green Pesticide and Agricultural
Bioengineering, Ministry of Education, Guizhou University,
Guiyang 550025, China
An Wang − Jiangsu Key Laboratory of Pesticide Science,
College of Sciences, Nanjing Agricultural University, Nanjing
210095, China
Lingling Qiu − Jiangsu Key Laboratory of Pesticide Science,
College of Sciences, Nanjing Agricultural University, Nanjing
210095, China
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College of Sciences and Key Laboratory of Integrated
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