Discovery of Pyrazine-Carboxamide-Diphenyl-Ethers as Novel Succinate Dehydrogenase Inhibitors via Fragment Recombination

Article abstract of DOI:10.1021/acs.jafc.0c05646

The discovery of novel succinate dehydrogenase inhibitors (SDHIs) has attracted great attention worldwide. Herein, a fragment recombination strategy was proposed to design new SDHIs by understanding the ligand-receptor interaction mechanism of SDHIs. Thre

Full text of DOI:10.1021/acs.jafc.0c05646

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Discovery of Pyrazine-Carboxamide-Diphenyl-Ethers as Novel
Succinate Dehydrogenase Inhibitors via Fragment Recombination
Hua Li, Meng-Qi Gao, Yan Chen, Yu-Xia Wang, Xiao-Lei Zhu,* and Guang-Fu Yang*
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ABSTRACT: The discovery of novel succinate dehydrogenase inhibitors (SDHIs) has attracted great attention worldwide. Herein,
a fragment recombination strategy was proposed to design new SDHIs by understanding the ligand−receptor interaction mechanism
of SDHIs. Three fragments, pyrazine from pyraziflumid, diphenyl-ether from flubeneteram, and a prolonged amide linker from
pydiflumetofen and fluopyram, were identified and recombined to produce a pyrazine-carboxamide-diphenyl-ether scaffold as a new
SDHI. After substituent optimization, compound 6y was successfully identified with good inhibitory activity against porcine SDH,
which was about 2-fold more potent than pyraziflumid. Furthermore, compound 6y exhibited 95% and 80% inhibitory rates against
soybean gray mold and wheat powdery mildew at a dosage of 100 mg/L in vivo assay, respectively. The results of the present work
showed that the pyrazine-carboxamide-diphenyl-ether scaffold could be used as a new starting point for the discovery of new SDHIs.
KEYWORDS: succinate dehydrogenase, fragment, molecular docking, fungicide, diphenyl-ether
surface plasmon resonance (SPR).20,21 Therefore, a variety of
INTRODUCTION
■
alternative biophysical methods have been used to detect the
Succinate dehydrogenase (SDH, EC 1.3.5.1, also known as
binding of such fragments.22 In our laboratories, we developed
succinate-ubiquinone oxidoreductase or complex II) is one of
a methodology for screening fragments, known as pharmaco-
the most important fungicidal targets and is a functional part of
phore-linked fragment virtual screening (PFVS), which
the tricarboxylic acid cycle and linked to the mitochondrial
identifies high potent inhibitors for the bc1 complex and
electron transport chain.1,2 SDH catalyzes the oxidation of
SDH.23,24 Recently, our group also identified potent and
succinate to fumarate followed by reduction of ubiquinone to
bioselective inhibitors for protoporphyrinogen oxidase using a
ubiquinol. Inhibiting SDH will cause the organism not to
fragment deconstruction method.22 The common character-
synthesize ATP normally and then die.3−5 Due to its crucial
istic of PFVS and fragment deconstruction methods is
role in the life cycle, 23 commercial carboxamide fungicides
fragment identification and linking. The hypothesis for
targeting SDH have been launched to date. Among them, the
fragment linking is that the binding mode of the fragments is
sale of pyrazole carboxamide fungicides significantly promoted
conserved upon modification of the fragment, and when two
market development of SDH inhibitors (SDHIs) due to high
fragments are linked together, their orientation in their
fungicidal activity and broad-spectrum properties.6 However,
respective binding district should remain the same.22,25
according to the results from the Fungicide Resistance Action
In this study, we report the molecular design of new SDHIs
Committee (FRAC), many plant pathogens have developed
using a fragment identification and recombination strategy,
resistance toward existing SDHI fungicides due to their long-
which includes five steps: (1) to uncover the ligand−receptor
interaction mechanisms of four representative SDHIs,
including pyraziflumid, flubeneteram, pydiflumetofen, and
fluopyram (Figure 1), by integrating molecular docking,
molecular dynamics, and binding energy calculations; (2) to
identify three active fragments, pyrazine from pyraziflumid,
diphenyl-ether from flubeneteram, and prolong amide linker
from pydiflumetofen and fluopyram, which occupied three
different sub-pockets in the SDH binding site; (3) to combine
three active fragments and produce four virtual compounds
term abuse and high usage rate.7−10 Therefore, it is particularly
important and urgent to design new SDHI fungicides.
Over the past several decades, some new techniques, such as
high-performance computation, structure biology, and artificial
intelligence, have been widely applied in structure-based drug
and pesticide discovery.11−14 Fragment-based drug discovery
(FBDD) has been recognized as a successful method for hit
identification and lead conception. Then, FBDD has been
successfully used in many systems and yielded very promising
results.15,16 Some articles have summarized fragment-to-lead
(F2L) success stories published during 2017 and 2018.17,18
The core step in the FBDD process is to identify fragments
with a low molecular weight (<300 g/mol) that weakly bind
the target protein.19 Due to the low binding affinity of
fragments, they are usually difficult to detect using bioassay-
based screening methods, such as high-throughput X-ray
crystallography, nuclear magnetic resonance (NMR), and
Received: September 1, 2020
Revised: October 29, 2020
Accepted: November 4, 2020
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© XXXX American Chemical Society
A

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Figure 1. Design of virtual compounds. Pyraziflumid, pydiflumetofen, and fluopyram are commercial SDH fungicides. Flubeneteram is a candidate
SDH fungicide and is not currently on the market. Virtual-1, virtual-2, virtual-3, and virtual-4 were designed based on the above compounds with
different amine bond lengths.
Molecular docking was performed using Autodock4.2.27 The grid
(virtual-1, virtual-2, virtual-3, and virtual-4); (4) to synthesize a
center was set according to the UBIQUINONE ligand in 1ZOY. The
grid box was set as 42 × 34 × 50, and the grid space was set at 0.375
Å. The default values were used for all other parameters. The 256
series of pyrazine-carboxamide-diphenyl-ether scaffold based
on virtual-3 and virtual-4 binding modes and evaluate their
structural−activity relationship in vitro and in vivo; and (5) one
hit compound 6y was identified with an IC50 value of 0.83 μM
conformations were acquired with the conformation search method of
the Lamarckian Genetic Algorithm (LGA)27 and then subjected to
against porcine SDH, which was about 2-fold more potent than
further energy minimization and short molecular dynamics (MD)
simulation using Amber16.28,29 First, the ligand was minimized with
the protein fixed. Second, only the backbone atoms of the protein
were fixed. Last, minimization was carried out with all the atoms
without any constraint. Each step of minimization was performed by
means of 1000 cycle steepest descent and 2000 cycle conjugate
gradient method, with a convergence criterion of 0.01 kcal/mol/Å.
The energy minimization was followed by an additional 20 ps MD
simulation, which was carried out at 300 k by applying the Langevin
thermostat. The coordinate file was recorded every 1 ps. The last
frame of the MD simulation was minimized with the backbone atoms
of proteins fixed. Finally, the minimized complex structure was used
to calculate the binding energy, making use of the molecular
mechanics Possion−Boltzmann surface area (MM/PBSA) method.24
The final structure was selected based on the binding energy and
binding mode of commercial carboxamide fungicides obtained from
our previous study.30
Enzymatic Assay. The preparation of SDH from a porcine heart
was the same as previously reported.31 The enzymatic activities of
SDH were analyzed in a reaction mixture as reported previously.32
The inhibition rates of 26 samples were measured at a 10 μM
concentration. To test the IC50 value, reactions were carried out in the
presence of varying concentrations of the inhibitor.33,34 The
commercial fungicide pyraziflumid was chosen as a positive control.
In Vitro Fungicidal Activity. Compounds were evaluated in
mycelia growth inhibition tests against Phytophthora capsici, Pythium
dissimile, B. fuckeliana, Gibberella zeae, and Zymoseptoria tritici (rich
media) in artificial media and Uromyces viciae-fabae on bean leaf
disks.35 All testing was done by Syngenta AG and undertaken on 96-
well microtiter plates. Each testing was carried out in triplicate.36
Three commercial fungicides were chosen as positive controls.
In Vivo Fungicidal Activity. According to the published pesticide
bioassay testing method in Shenyang Sinochem Agrochemicals R&D
Co., Ltd. (Shenyang, China),37 13 target compounds were evaluated
for protective activities in the greenhouse against wheat powdery
mildew and soybean gray mold. The commercial fungicide
pyraziflumid was chosen as a positive control.
pyraziflumid (IC50 = 1.52 μM). Further biological assays
showed that compound 6y had excellent fungicidal potency
toward Botryotinia fuckeliana in vitro and soybean gray mold in
vivo. Computational simulations revealed that compared to the
commercial fungicide pyraziflumid, compound 6y showed a
more favorable VDW interaction with SDH, which led to its
higher activity. The present study provides an example of an
application of computational design of pesticide molecules.
MATERIALS AND METHODS
■
Chemistry. All reagents and solvents were commercially available
and used directly without further purification. Reactions were
monitored by thin-layer chromatography (TLC). Target compounds
1
were purified by column chromatography using silica. The H NMR
(600, 500, or 400 MHz) and 13C NMR (125 MHz) spectra were
recorded on a Bruker Avance 500 spectrometer (Bruker Inc., Billerica,
MA), Mercury Plus 400 MHz, or 600 MHz spectrometer (Varian,
Palo Alto, CA) in DMSO-d6 or CDCl3 solution, with SiMe4 (TMS) as
the internal standard. Mass spectrometry (MS) data were obtained
with a DSQII GC−MS (Thermo Fisher, Austin, TX) instrument with
an electrospray ionization (ESI) source. High-resolution mass spectra
(HRMS) were determined with an Agilent 6224 time-of-flight liquid
chromatograph mass spectrometer (Agilent Technologies, Santa
Clara, CA), which was equipped with a 250 mm × 4.6 mm i.d., 5
μm, Eclipse XDB-C18 (Agilent Technologies, Santa Clara, CA)
column. The melting points were determined on a BuCHI B-545
̈
melting point apparatus and were uncorrected. Detailed synthetic
routes and characterization data for all synthesized compounds are
given in the Supporting Information.
Molecular Model. The 3D structures of the newly synthesized
compounds were built using SYBYL and then minimized with the
steepest descent method and conjugate gradient method, both with
2000 steps with a convergence criterion of 0.001 kcal/mol/Å.26 The
crystal structure of porcine SDH (PDB ID: 1ZOY)2 was chosen as the
receptor.
B
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Figure 2. (A and B) Binding mode of pyraziflumid with SDH. Projection_1 and projection_2 in B are the projections of the inner phenyl in
pyraziflumid. (C) Relative positions of pyraziflumid (green line), pydiflumetofen (magenta stick), and fluopyram (yellow stick) when they bound
with the SDH binding site. (D) Relative positions of pyraziflumid, pydiflumetofen, fluopyram, and flubeneteram bound with the SDH binding site.
For clarity, the pyrazine ring (green stick) in pyraziflumid, prolonged amide bond (gray and yellow stick) in pydiflumetofen and fluopyram, and
diphenyl-ether (magenta stick) in flubeneteram. (E) Binding mode of compound 6y with SDH. (F) Overlay of pyraziflumid (yellow stick) and 6y
(magenta stick) when bound with SDH. Only the key residues are shown. The red and green lines represent an Hbond. All these figures were built
by a Pymol program.
docking followed by molecular dynamics and MM/PBSA
calculations was performed for pyraziflumid. As shown in
Figure 2A, pyraziflumid showed a conserved binding mode
with other carboxamide SDH fungicides. The pyrazine ring in
pyraziflumid, similar to other acid moieties in SDHIs, bound to
the bottom of the SDH binding site and formed a cation−π
interaction with the residue of C_R46. The carbonyl oxygen in
pyraziflumid formed an Hbond with D_Y91 and B_W173, its
biphenyl moiety extended toward the entrance of the SDH
binding site, then its inner phenyl bounded by C_I30, C_I43,
and C_W35, and its tail phenyl formed the edge-to-edge π−π
interaction with D_Y91. Here, we noticed that the ring plane
of inner phenyl in pyraziflumid was misaligned with C_I30 and
C_W35 (Figure 2B), which could be further structurally
optimized to increase its activity. Collectively, these results
indicated that a pyrazine ring as an acid moiety would be a
good starting point to design new SDHIs.
Moreover, in our previous study, the diphenyl-ether in
flubeneteram was proven as a good fragment extending toward
the entrance of the SDH binding site, forming a T−π
interaction with the indole ring of C_W35.24,40 To continue
our research, the diphenyl-ether was selected as the hydro-
phobic side fragment in this work. Here, we noticed that the
distance between the pyrazine ring and diphenyl-ether in the
SDH binding site was about 5.1 Å, which was larger than one
amide bond length (3.8 Å) (Figure 2S, Supporting
Information).
RESULTS AND DISCUSSION
■
Fragment Identification and Recombination. Finding
novel fragments as starting points for optimization is a major
challenge in FBDD. The structures of the fragments binding to
the protein can be used to design new compounds with
increased affinity and novelty. The chemical structure of
commercial SDH fungicides always consists of three fragments,
including an acid moiety, hydrophobic side moiety, and amide
linker, indicating three fragment directions for designing novel
SDHI.21,38 According to our previous study, an acid moiety
bound to the bottom of the SDH binding site formed a
cation−π interaction with C_R46, an amide linker located at
the mouth of the SDH binding site formed a hydrogen bond
(Hbond) with B_W173 and D_Y91, and a hydrophobic side
moiety extended outside of the SDH binding site formed
hydrophobic interaction with some residues, such as C_W35,
D_Y91, and C_I30, among others (Figure 1S, Supporting
Information).30
Some active fragments from commercial fungicides are good
choices for designing SDHIs. The pyrazine-carboxamide
fungicide pyraziflumid with structural novelty was developed
by Nihon Nohyaku Co., Ltd. in 2017 with excellent biological
performance and no cross resistance with the existing
fungicides.39 The main difference between pyraziflumid and
other SDH fungicides is its pyrazine ring in the acid moiety.
Understanding the binding mechanism of these highly potent
inhibitors can help to uncover the binding “hot spots” and
identify the regions contributing to activity. Hence, molecular
For the linker between the pyrazine ring and diphenyl-ether,
the prolonged amide bond with one or two methylene groups
C
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a
Scheme 1. Synthetic Route for the Target Compounds 6a−6z
a
Reagents and conditions: (a) K2CO3, DMF, reflux; (b) NH2OH·HCl, EtOH, reflux; Zn/HCl, room temperature; (c) 5, HATU/DIPEA, CH2Cl2,
room temperature.
Table 1. Chemical Structures, Inhibitory Activities against Porcine SDH, and the Binding Energy between Compounds with
SDH (kcal/mol)
no.
n
R
inhibitory rate (%)/IC50 (μM)a
ΔEvdw
ΔEele
ΔGpol
ΔGnp
ΔGcal
ΔGexp
b
6a
6b
6c
6d
6e
6f
6g
6h
6i
6g
6k
6l
6m
6n
6o
6p
6q
6r
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
2-F
2-Br
23.61%
48.43%
29.27%
31.42%
32.45%
32.85%
26.16%
59.68%
54.83%
43.27%
63.37%
31.34%
2-Me
3-Cl
4-Me
4-Br
2,3-F2
2,3-Cl2
2-Cl-4-F
2,4-Cl2
2-Cl-4-CF3
3,4-F2
−36.44
−24.27
45.12
−3.80
−19.39
3,4-Cl2
2-F
52.45%
4.79 0.12
1.25 0.12
3.04 0.01
5.40 0.12
3.40 0.01
2.72 0.12
0.90 0.01
4.18 0.11
1.14 0.12
2.67 0.11
1.23 0.12
0.83 0.01
3.49 0.12
1.52 0.11
−39.86
−40.83
−40.99
−39.92
−40.62
−40.42
−42.40
−40.31
−40.44
−38.30
−41.73
−45.88
−40.01
−41.68
−19.45
−20.55
−18.91
−17.67
−22.59
−20.94
−17.70
−20.25
−20.63
−18.39
−16.59
−19.80
−18.59
−19.77
37.98
36.78
37.77
36.17
40.59
37.63
34.12
38.49
36.02
33.03
33.65
38.80
36.01
36.57
−3.85
−3.82
−3.86
−3.60
−3.84
−3.62
−3.78
−3.78
−3.72
−3.43
−3.75
−3.94
−3.57
−3.49
−25.19
−28.42
−25.99
−25.01
−26.45
−27.34
−29.76
−25.85
−28.77
−27.08
−28.42
−30.83
−26.16
−28.37
−7.30
−8.10
−7.57
−7.23
−7.50
−7.64
−8.30
−7.38
−8.15
−7.65
−8.11
−8.34
−7.49
−7.98
2-Cl
2-Br
4-F
2,3-F2
6s
6t
2-Cl-3-F
2,3-Cl2
3,4-F2
2,4-Cl2
2-Cl-4-F
2-Cl-4-CF3
2-Br-4-Cl
2-Cl-5-F
6u
6v
6w
6x
6y
6z
pyraziflumid
a
The compound inhibitory rate was first tested at 10 μM concentration. If the inhibitory rate was over 70%, then the IC50 value would be calculated
b
with eight different concentration gradients. Not tested.
was taken into consideration. Here, the commercial fungicides,
pydiflumetofen and fluopyram, with different novel prolonged
strategies for the amide bond (Figure 1), were selected as
representative compounds to study the interaction with the
target SDH.41,42 As shown in Figure 3S (Supporting
Information), both of them formed an Hbond with B_W173
and D_Y91 and a cation−π interaction with C_R46.
Compared to the classical amide bond linker, the N-alkyl
amide linker in pydiflumetofen is also located at the mouth of
the SDH binding site, and then the difference was that the
prolonged amide linker in pydiflumetofen resulted in its
substituted phenyl ring extending further outside of the
binding site to form π−π interaction with C_W35 rather
than pyraziflumid (Figure 2C). The same thing occurred to the
ethanamine linker in fluopyram (Figure 2C). These results
indicated that the prolonged amide bond could adjust the
D
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interaction of the hydrophobic side moiety as an inhibitor with
SDH. Our previous study showed that as more interaction
occurs between the hydrophobic side moiety in the inhibitor
and SDH, the stronger the activity of the inhibitor.32
activity (2,4-Cl2, IC50 = 1.14 μM) than 6x (2-Cl-4-CF3, IC50
1.23 μM) and 6w (2-Cl-4-F, IC50 = 2.67 μM).
=
To further understand SAR, molecular docking was used to
explore the inhibition mechanism of compounds 6n−6z. As
shown in Table 1, the correlation coefficient (R2) between
ΔGcal (calculated binding energies) and ΔGexp (ΔGexp = −RT
× ln IC50) was high, up to 0.95, which indicated that the
binding mode was reasonable and reliable. The binding mode
of the representative compound 6y (Figure 2E) was similar to
pyraziflumid, forming a cation−π interaction with C_R46 and
a Hbond with B_W173 and D_Y91. The pyrazine ring in 6y
showed nearly the same position as that in pyraziflumid.
Different from pyraziflumid (Figure 2F), the amide bond
extension in 6y led to its diphenyl-ether moiety extending
toward the binding site entrance of SDH, forming a stronger
sandwich hydrophobic interaction with C_I30 and C_I43 than
pyraziflumid (Figure 2F). This resulted in a more favorable
VDW interaction with SDH (ΔEvdw = −45.88 kcal/mol for
compound 6y vs −41.68 kcal/mol for pyraziflumid, Table 1).
Then, we also noticed that the ΔEvdw values for compounds
6n−6z were around −38.30 to −42.40 kcal/mol, lower than 6k
(−36.44 kcal/mol), indicating that one methyl introduction (n
= 1) into the scaffold of the target was unfavorable for VDW
interaction between the ligand and SDH.
As shown in Table 1, one interesting thing was that the
increase in ΔEele was not able to enhance the activity of the
compound. For example, compound 6r had the highest ΔEele
(−22.59 kcal/mol) but its IC50 value (3.40 μM) was not good.
However, the influence of ΔEvdw was remarkable. In general,
the stronger the ΔEvdw between the compound and SDH, the
higher the activity of the compound, such as in compounds 6t,
6k, and 6y. These results indicated that ΔEvdw is very
important for designing new SDH inhibitors.
We note that the activity of compound 6y was still lower
than pydiflumetofen (0.13 μM, data was not shown) and
flubeneteram (0.11 μM). Nevertheless, the scaffold of
compound 6y, especially for its amide bond with one
methylene extension, provides new insight into designing
new SDHIs.
In Vitro Fungicidal Activity. To determine whether the
target compounds had fungicidal activity, compounds 6n−6z
were assessed on six plant pathogens, namely, Ph. capsici, Py.
dissimile, B. fuckeliana, G. zeae, Z. tritici, and U. viciae-fabae.
The results are summarized in Table 2 and expressed as
general assessment criteria for biological assays.
As shown in Table 2, all tested compounds did not show
good fungicidal activity against Ph. capsici, Py. dissimile, G. zeae,
Z. tritici, and U. viciae-fabae. In contrast, most compounds
showed an over 80% inhibitory rate activity against B.
fuckeliana, except 6x, with a 62% inhibitory rate at 20 mg/L.
Furthermore, compounds 6o−6r, 6u, 6y, and 6z had a 100%
control effect against B. fuckeliana, which was better than
pyraziflumid (90%). Compound 6y also showed an 80%
inhibitory rate against Z. tritici at a concentration of 20 mg/L,
indicating its potential broad-spectrum property.
In Vivo Fungicidal Activities. It is known that gray mold
caused by B. fuckeliana can cause heavy losses in many crop
yields worldwide, such as grapes, soybean, cucumber, and
strawberry.45 Here, soybean gray mold (SGM) and wheat
powdery mildew (WPM) were selected as the target diseases
to evaluate the biological activity of the tested compounds in
the greenhouse, and the commercial fungicide pyraziflumid
was selected as a positive control. As shown in Table 3, most
Subsequently, four virtual compounds (virtual-1, virtual-2,
virtual-3, and virtual-4) were designed based on the relative
position of the pyrazine ring, diphenyl-ether, and prolonged
amide bond in the SDH binding site (Figure 2D). Then, four
of them were docked to the SDH binding site. As shown in
Figure 4S, the ring plane of the pyrazine fragment in virtual-1
did not form a good cation−π interaction with C_R46, and
virtual-2 just formed one Hbond with B_W173. However,
virtual-3 and virtual-4 showed the conserved binding modes
with that of the commercial fungicides, indicating that they
could be selected as the new starting points for SDHIs and
subjected to subsequent synthesis. Meanwhile, the synthesis
processes of virtual-3 and virtual-4 were simpler than those of
virtual-1 and virtual-2 as described in the following Chemistry
section.
Chemistry. To enrich the structural−activity relationship
(SAR) of virtual-3 (equal to 6k) and virtual-4 (equal to 6x), a
series of pyrazine-carboxamide-diphenyl-ether compounds
(6a−6z) were synthesized. According to Scheme 1, com-
pounds 6a−6m were synthesized with yields of 37−66% by
only three steps. The substituted phenol 2 and 2′-
fluoroacetophenone 1 were selected as the starting materials
and then employed under the presence of K2CO3 in DMF to
produce intermediate 3.43 Intermediate 3 reacted with
NH2OH·HCl to afford oxime and then was restored to key
intermediate amine 4 under a zinc duct.44 Finally, compound
5, 3-(trifluoromethyl)pyrazine-2-carboxylic acid, was obtained
according to reported methods39 and then reacted with amine
4 to afford the target compounds 6a−6m by the condensing
agent HATU/DIPEA catalyst. The compounds 6n−6z were
synthesized with 2-fluorobenzaldehyde as a starting material,
and their yields were about 32−62%.
Structural−Activity Relationship. As shown in Table 1,
6a−6m showed weak enzymatic inhibition activity with an
inhibitory rate ranging from 23.61 to 63.37% against porcine
SDH at 10 μM. To understand its low activity, compound 6k
was redocked followed by MM/PBSA calculations. The
binding energy with SDH was −19.39 kcal/mol, which was
larger than the binding energy of pyraziflumid with SDH
(−28.37 kcal/mol). Here, we noticed that the VDW
interaction energy of 6k with SDH was −36.44 kcal/mol
(Table 1), which was lower than pyraziflumid (−41.68 kcal/
mol) and may explain its low activity.
However, the activity of compounds 6n−6z showed
significant improvement over 6a−6m. Moreover, the IC50
values of compounds 6o, 6t, 6v, and 6x were 1.25, 0.90,
1.14, and 1.23 μM, respectively, which showed higher activities
than pyraziflumid (IC50 = 1.52 μM). Remarkably, compound
6y, bearing 2-Br-4-Cl, displayed the highest activity with an
IC50 value of 0.83 μM, with approximately 2-fold improved
potency compared with pyraziflumid. The SARs of compounds
6n−6z are summarized as follows: (1) Monosubstituted
compounds, a 2-position substituent, had a positive effect on
the activities compared to a 4-position substituent. For
example, 6n (2-F, IC50 = 4.79 μM) showed a higher activity
than 6q (4-F, IC50 = 5.40 μM). (2) Disubstituted compounds
always had better activity than monosubstituted compounds.
(3) For 2,4-disubstituted compounds, 6v showed a higher
E
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Table 2. Fungicidal Activity of Target Compounds In Vitro
Table 4. EC50 and EC90 Values (mg/L) for Target
Compounds against Soybean Gray Mold
a
a
a
a
a
a
Uvf
100
Pd
Bf
Gz
Pc
20
Zt
b
b
b
b
b
b
no.
EC50
EC90
regression equation
r
no.
2
20
95
20
20
c
6p
6o
6y
6q
76.51
72.04
52.28
57.24
60.96
139.74
128.97
87.61
96.90
107.06
−4.2278 + 4.8987x
−4.4124 + 5.0671x
−4.8227 + 5.7163x
−4.8523 + 5.6052x
−4.3515 + 5.2389x
0.94
0.95
0.93
0.94
0.91
6n
6o
6p
6q
6r
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
100
100
100
90
85
100
90
pyraziflumid
6s
6t
1
2
control effects against B. fuckeliana at a dosage of 20 mg/L in
vitro and over 90% control effects against SGM at 100 mg/L in
vivo, which were also better than that of pyraziflumid. Further
EC50 and EC90 values also indicated that both of them were
superior to pyraziflumid. The computational simulations
revealed that the van der Waals interactions between
compounds and SDH played an important role in adjusting
its activity, which may provide insight into designing new
SDHIs. The above results also indicated that the amide bond
with one methylene extension is a novel active fragment for
designing SDHIs.
6u
6v
6w
6x
6y
6z
27
0
2
1
1
2
1
80
62
0
0
100
100
90
100
100
80
0
pyraziflumid
azoxystrobin
prochloraz
100
100
0
1
0
100
0
100
0
100
100
100
a
Uvf, U. viciae-fabae; Pd, Py. dissimile; Bf, B. fuckeliana; Gz, G. zeae;
b
c
Pc, Ph. capsici; Zt, Z. tritici. Dose in mg/L. The data are the mean of
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c05646.
three replicates.
■
sı
compounds did not show good activity against WPM. As
expected, for SGM at 100 mg/L, compounds 6p, 6r, 6u, and
6z showed over a 80% control effect, and compounds 6o, 6q,
and 6y showed over a 90% control effect, all of which were
better than pyraziflumid (70%). Notably, compound 6y had a
95% control effect against SGM and an 80% control effect
against WPM at a concentration of 100 mg/L. Compound 6q
had nearly the same control effect against SGM and WPM as
6y. Then, the EC50 and EC90 were tested against SGM for
some potential compounds. The results are shown in Table 4
(Table 1S, Supporting Information), and we can see that
compounds 6y and 6q exhibited better EC50 and EC90 values
than pyraziflumid. These results indicated that compounds 6y
and 6q have potential broad-spectrum features and thus may
be used as candidates for further development.
In summary, we implemented a fragment recombination
strategy for new SDHI design. Computational analysis
demonstrates that three active fragments, pyrazine ring,
diphenyl-ether, and prolonged amide bond, occupied three
different sub-pockets in the SDH binding site and could be
combined to a novel scaffold of SDHI. After substitute
optimizations, a hit compound 6y, N-(2-(2-bromo-4-
chlorophenoxy)benzyl)-3-(trifluoromethyl)pyrazine-2-carboxa-
mide, was identified with an IC50 value 0.83 μM, which was
about 2-fold more potent than pyraziflumid. Biological
experiments showed that compounds 6q and 6y had 100%
Relative position, binding mode, decomposed VDW
interaction energy, control effect picture, enzymatic
assay, in vitro and in vivo fungicidal assay, leaf-piece
assay, synthetic routes, and H and 13C NMR spectro-
1
grams (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Xiao-Lei Zhu − Key Laboratory of Pesticide & Chemical
Biology of Ministry of Education, International Joint Research
Center for Intelligent Biosensor Technology and Health of
Ministry of Science and Technology, Central China Normal
University, Wuhan 430079, People’s Republic of China;
orcid.org/0000-0002-5672-5209; Email: xlzhu@
mail.ccnu.edu.cn
Guang-Fu Yang − Key Laboratory of Pesticide & Chemical
Biology of Ministry of Education, International Joint Research
Center for Intelligent Biosensor Technology and Health of
Ministry of Science and Technology, Central China Normal
University, Wuhan 430079, People’s Republic of China;
Collaborative Innovation Center of Chemical Science and
Engineering, Tianjin 300071, People’s Republic of China;
Phone: 86-27-67867800; Email: gfyang@
mail.ccnu.edu.cn; Fax: 86-27-67867141
Table 3. Fungicidal Activity of Target Compounds In Vivo
a
a
a
a
no.
SGM (100 mg/L)
WPM (100 mg/L)
no.
SGM (100 mg/L)
WPM (100 mg/L)
b
6n
6o
6p
6q
6r
6s
6t
0
NT
30
40
85
30
NT
NT
6u
6v
6w
6x
6y
6z
80
0
20
90
85
90
85
20
0
NT
NT
NT
80
0
98
20
10
95
80
70
pyraziflumid
a
b
SGM, soybean gray mold; WPM, wheat powdery mildew. Not tested.
F
https://dx.doi.org/10.1021/acs.jafc.0c05646
J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry
pubs.acs.org/JAFC
Article
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Authors
Hua Li − Key Laboratory of Pesticide & Chemical Biology of
Ministry of Education, International Joint Research Center for
Intelligent Biosensor Technology and Health of Ministry of
Science and Technology, Central China Normal University,
Wuhan 430079, People’s Republic of China
Meng-Qi Gao − Key Laboratory of Pesticide & Chemical
Biology of Ministry of Education, International Joint Research
Center for Intelligent Biosensor Technology and Health of
Ministry of Science and Technology, Central China Normal
University, Wuhan 430079, People’s Republic of China
Yan Chen − Key Laboratory of Pesticide & Chemical Biology
of Ministry of Education, International Joint Research Center
for Intelligent Biosensor Technology and Health of Ministry of
Science and Technology, Central China Normal University,
Wuhan 430079, People’s Republic of China
Yu-Xia Wang − Key Laboratory of Pesticide & Chemical
Biology of Ministry of Education, International Joint Research
Center for Intelligent Biosensor Technology and Health of
Ministry of Science and Technology, Central China Normal
University, Wuhan 430079, People’s Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c05646
(14) Wu, F.-X.; Wang, F.; Yang, J.-F.; Jiang, W.; Wang, M.-Y.; Jia, C.-
Y.; Hao, G.-F.; Yang, G.-F. AIMMS suite: A web server dedicated for
prediction of drug resistance on protein mutation. Briefings Bioinf.
2020, 20, 318−328.
Funding
The research was supported in part by the National Key
Research and Development Program of China
(2018YFD0200100), the Key Research and Development
Program of Hubei Province (2020BBA052), and the National
Natural Science Foundation of China (no.21977035,
21837001, and 21772057).
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design: From experimental to computational approaches. Curr. Med.
Chem. 2012, 19, 5128−5147.
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the aspartic protease endothiapepsin: Fragment-based drug design
facilitated by dynamic combinatorial chemistry. Angew. Chem., Int. Ed.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(17) Erlanson, D. A.; de Esch, I. J. P.; Jahnke, W.; Johnson, C. N.;
Mortenson, P. N. Fragment-to-lead medicinal chemistry publications
in 2018. J. Med. Chem. 2020, 63, 4430−4444.
This paper is dedicated to Professor Youyou Tu, the 2015
Nobel Prize Laureate of Physiology or Medicine, on the
occasion of her 90th birthday.
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