Design, Synthesis, and Antifungal Activities of Novel Aromatic Carboxamides Containing a Diphenylamine Scaffold

Article abstract of DOI:10.1021/acs.jafc.9b00151

A series of novel N-(2-(phenylamino)-4-fluorophenyl)-pyrazole-4-carboxamides 1-15 and aromatic carboxamides with a diphenylamine scaffold 16-29 were designed, synthesized, and evaluated for their antifungal activities. In vitro experiments showed that com

Full text of DOI:10.1021/acs.jafc.9b00151

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Functional Structure/Activity Relationships
Design, Synthesis and Antifungal Activities of Novel
Aromatic Carboxamides Containing Diphenylamine Scaffold
Aigui Zhang, Ying Yue, Jian Yang, Jiaxing Shi, Ke Tao, Hong Jin, and Taiping Hou
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00151 • Publication Date (Web): 12 Apr 2019
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Journal of Agricultural and Food Chemistry
Design, Synthesis and Antifungal Activities of Novel Aromatic
Carboxamides Containing Diphenylamine Scaffold
Aigui Zhang, Ying Yue, Jian Yang, Jiaxing Shi, Ke Tao, Hong Jin,* and Taiping Hou
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,
College of Life Sciences, Sichuan University, Chengdu 610065, Sichuan, P.R. China
*E-mail address: jinhong@scu.edu.cn (H. Jin)
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ABSTRACT:
a
series
of
novel
N-(2-(phenylamino)-4-fluorophenyl)-pyrazole-4-carboxamides 1-15 and aromatic
carboxamides with diphenylamine scaffold 16-29 were designed, synthesized and
evaluated for their antifungal activities. In vitro experiments showed that compound 6
(EC50 = 0.03 mg/L) was superior to bixafen (EC50 = 0.04 mg/L) against Rhizoctoinia
solani, and compound 6 (IC50 = 1.41 mg/L) was close to bixafen (IC50 = 1.22 mg/L)
against succinate dehydrogenase from R. solani. Additionally, in vivo pot experiments
showed that compounds 6 (EC50 = 1.93 mg/L) was better than bixafen (EC50 = 3.72
mg/L) and close to thifluzamide (EC50 = 1.83 mg/L) against R. solani. And that in
vivo field trials showed that compound 6 at 200 g ai ha-1 had 64.10 % control efficacy
against rice sheath blight after 21 days with two sprayings, close to thifluzamide
(71.40 %). Furthermore, molecular docking showed that compound 6 anchor in the
binding site of SDH.
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KEYWORDS: Aromatic carboxamides, bioisosteric modification, structure-activity
relationships, antifungal activity, molecular docking
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INTRODUCTION
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Carboxamides inhibiting succinate dehydrogenase (SDH), namely succinate
dehydrogenase inhibitors (SDHIs), play an important role in plant protection against
many phytopathogenic fungi.1,2 Among this family, Carboxin was the first
commercial product launched by Uniroyal Chemical Co. in 1966.3 Subsequently a
number of Carboxin-related carboxamides had been found with good efficacy in the
control of plant diseases, such as Benodanil (BASF), Fenfuram (Bayer), Bixafen
(Bayer), Thifluzamide (Dow AgroSciences) and Boscalid (BASF) (Figure 1).4,5 All of
these SDHIs have a common prototypical pharmacophoric scheme, which includes a
conserved amide function, a structurally diverse carboxyl “core” and amine moiety
(Figure 1). According to the cocrystal structure of SDH from porcine heart,6 avian,7
and Escherichia coli,8,9 the carboxyl “core” buries deep into the ubiquinone binding
site (Q-site) and contributes predominantly to the binding affinity of SDHIs. Current
efforts mainly focused on the modification of the carboxyl “core” and amine moiety
of SDHIs.10-18
Over the past few years, our group mainly focused on optimization of the amine
moiety on SDHIs and discovered some good compounds with antifungal
activities.19-22 For example, by fluxapyroxad being as a lead compound, the
diarylamines were introduced to replace the biphenyl group in fluxapyroxad, some
pyrazole carboxamides with diarylamine-modified scaffold were synthesized, and
compound 1c presented the highest in vitro fungicidal activities against
Rhizoctoinia solani (R. solani) with EC50 value of 0.005 mg/L.19
Here, to develop the novel carboxamide fungicides, bixafen was applied as a lead
compound and the substituted phenylamine group were introduced to replace the
biphenyl
group
by
splicing-up.
A
series
of
novel
N-(2-(phenylamino)-4-fluorophenyl)-pyrazole-4-carboxamides
1-15
containing
diphenylamine scaffold were designed and synthesized (Figure 2). Subsequently, in
vitro antifungal activities were performed to evaluate their antifungal activities against
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R. solani, Phytophthora infestans (P. infestans), Botryosphaeria dothidea (B.
dothidea), Gibberella zeae (G. zeae), Fusarium oxysporum f. sp. vasinfectum (F.
oxysporum f. sp. vasinfectum), Alternaria alternate (A. alternata) and Fusarium
oxysporum f. sp. niveum (F. oxysporum f. sp. niveum). In addition, in order to further
study antifungal activities of aromatic carboxamides with the diphenylamine scaffold,
based on the above selected compound 6 with the highest antifungal activities, some
carboxyl “cores” were introduced and a series of novel aromatic carboxamides 16-29
with the diphenylamine scaffold were synthesized and evaluation for their in vitro
antifungal activities against R. solani. And, inhibitory activity against SDH, in vivo
pot tests against R. solani, field trials against rice sheath blight and molecular docking
of compound 6 were evaluated.
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MATERIALS AND METHODS
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Chemicals. All reagents and solvents were commercially available and used
directly without further purification.
Fungi. The plant pathogenic fungi, R. solani, P. infestans, B. dothidea, G. zeae, F.
oxysporum f. sp. vasinfectum, A. alternata and F. oxysporum f. sp. niveum were
kindly provided by College of Life Sciences, Sichuan University, China. These fungi
were grown on potato dextrose agar (PDA) plates at 28 °C and maintained at 4 °C
with periodic subcultivations.
Instruments. The melting points were determined on an X-4 micro-melting point
apparatus (Beijing second optical instrument factory, P.R. China) and were
1
13
uncorrected. H and C NMR spectra were recorded in DMSO-d6 as the solvent by
using a Bruker 400 MHz NMR spectrometer (Bruker Co., Switzerland), using
tetramethylsilane as the internal standard. Thin-layer chromatography (TLC) was
performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd., P. R. China).
Column chromatography purification was performed over silica gel (300-400 mesh,
Qingdao Marine Chemical Ltd., P. R. China). MS data were obtained by using a
MALDI-TOF/TOF mass spectrometer (Bruker Co., Switzerland).
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Synthesis. The synthetic routes of the target compounds 1-15 and 16-29 were
outlined in Figure 3.19,23
General synthetic procedure for intermediate c. Anhydrous KF (20 mmol) was
added to 2,4-difluoro-1-nitrobenzene a (20 mmol) and substituted aniline b (30
mmol), and the resulting mixtures were heated at 160 ºC for 14 h. The reaction
mixtures were then quenched with water at room temperature, extracted with ethyl
acetate (3 x 30 mL). The organic phase was wash with brine, dried over MgSO4, then
concentrated under reduced pressure. The residue was subjected to flash column
chromatography to give the product c.
General synthetic procedure for intermediate d. Compound c (15 mmol), reductive
iron powder (15 mmol), NH4Cl (45 mmol) and ethanol aqueous solution (75 %, 60
mL) were added in a flask. The reaction proceeded with refluxing for 3 h at 90 °C.
When the reaction finished, the mixtures were cooled to room temperature and
extracted with CH2Cl2 (3 x 20 mL) and the organic phase was evaporated in vacuum
to obtain compound d.
General synthetic procedure for the target compounds 1-15 and 16-29.
Intermediate d (10 mmol), EDCI (10 mmol), DMAP (0.1 mmol) and compound e (10
mmol) were dissolved in DCM (CH2Cl2, 30 mL). The mixture was stirred at room
temperature for 6 h. The compounds 1-15 were purified via column chromatography.
Similarly, compounds 16-29 were synthesized by using the same methods. The
physical data of compounds 1-29 in detail were included in the Supporting
Information.
Biological Assay. In vitro antifungal activities of the target compounds. All
synthesized compounds were screened for their in vitro antifungal activities against
seven fungi, including R. solani, P. infestans, F. oxysporum f. sp. vasinfectum, B.
dothidea, G. zeae, A. alternate and F. oxysporum f. sp. niveum by the mycelium
growth rate method.19,24 The antifungal activities of the target compounds 1-29 in
detail were included in the Supporting Information.
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Assay of SDH’s inhibitory activity. Isolation of R. solani. Fungus mitochondrial
was isolated according to a previously reported method.11,24The isolation of R. solani
in detail was included in the Supporting Information.
Activity inhibition of Succinate: Ubiquinone/DCPIP. Activity inhibition of
Succinate was carried according to a previously reported method.11,24The activity
inhibition of the target compound 6 in detail was included in the Supporting
Information.
In vivo pot tests for the target compounds 6, 9 and 15 against R. solani. The in vivo
antifungal activities of 6, 9 and 15 against R. solani were tested according to the
procedure described previously.25The pot tests of the target compounds 6, 9 and 15 in
detail were included in the Supporting Information.
Field trials for compound 6 in controlling rice sheath blight. A rice field naturally
infected by R. solani was selected to perform the field trial by using the standard
method.25 Each treatment took up in three plots and was distributed randomly in each
field. The disease severity was scored by using the following scale.26,27The field trials
of compound 6 in detail were included in the Supporting Information.
Homology modeling and molecular docking. Homology modeling. We applied
SDH from avian (PDB ID:1YQ3) as the template, and the homology of the SDH
amino acid sequence of R. solani was aligned. Homology modeling of SDH from R.
solani was carried out using MODELER 9.15 (http://salilab. org/modeller/). The
homology modeling in detail was included in the Supporting Information.
Molecular docking. Molecular docking studies were performed to investigate the
binding mode between compound and the SDH by using Autodockvina 1.1.2.28 The
3D structure of the compounds were drawn by ChemBioDraw Ultra 14.0 and
ChemBio3D Ultra 14.0 softwares and minimized by MM2 using ChemBio3D Ultra
14.0 software. The AutoDockTools 1.5.6 package was employed to generate the
docking input files.29,30The molecular docking in detail was included in the
Supporting Information.
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RESULTS AND DISCUSSION
Chemistry. The
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syntheses
of
the
N-(2-(phenylamino)-4-fluorophenyl)-pyrazole-4-carboxamides 1-15 and aromatic
carboxamides 16-29 were outline in Figure 3. They were obtained by the reactions i -
iii, and MS, 1H NMR and 13C NMR of the target compounds were applied to confirm
their structures.
In Figure 3, the syntheses and chemical structures of the target compounds were
detailed. Initially, compounds c were obtained by condensation reaction of
compounds a and b, and then were transformed into the corresponding compound d
through reduction reaction. The key aromatic acid e reacted with the primary amine d
under EDCI and DMAP immediately to afford the target compounds 1-15.23
Compounds 16-29 were synthesized by using the same method as described.
In vitro antifungal activities of compounds 1-15 and structure-activity relationships
analysis. The antifungal activities of 15 target compounds were shown in Tables 1
and 2, and bixafen was selected as the positive control at a dosage of 20 mg/L by the
mycelium growth rate method.19,24The results in Table 1 was the preliminary
screening of antifungal activities of the target compounds 1-15 and in Table 2 was the
further screening of the target compounds against R. solani.
As shown in Table 1, most of the synthesized compounds exhibited good antifungal
activities against R. solani. Among them, 12 target compounds displayed good
antifungal activities (inhibition rate ＞85 %) at 20 mg/L. For example, compounds 2,
6, 7, 9, 11 and 12 displayed much higher antifungal activities against R. solani than
bixafen (90.46 %). In addition, the introduced substituents of compounds 1-15
included halogen, methoxyl, trifluoromethoxyl, cyano and so on. As for F. oxysporum
f. sp. vasinfectum (Fov.), two compounds 3 (67.46 %) and 12 (64.49 %) have slightly
higher antifungal activities than lead compound bixafen (63.49 %). And for the B.
dothidea, compound 15 (83.73 %) showed relatively higher activities than bixafen
(82.54 %). But for the F. oxysporum f. sp. niveum (Fon.), only compound 9 (53.23 %)
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displayed higher activities than bixafen (43.55 %).
To analyze the structure-activity relationships (SARs) of the target compounds 1-15
against R. solani, all compounds 1-15 were selected for further studies. Their EC50
values were listed in Table 2. All compounds showed moderate to good activities with
EC50 of 0.03 mg/L to 0.88 mg/L. Among the chloro-substituted compounds 2, 3, 4, 5
and 6, compound 6 (R = 3-Cl-4-Me) showed the best antifungal activity against R.
solani with EC50 value of 0.03 mg/L, higher than that of bixafen (0.04 mg/L).
Compound 3 (R = 3,4-Cl2) also showed good antifungal activity against R. solani
(EC50 = 0.08 mg/L). Compounds 4 (R = 2,4-Cl2) and 5 (R = 2-Cl-4-Br) exhibited
relatively weak activities, even lower than compound 1 (R = H). Bromo-substituted
compounds also showed good activities. For example, compounds 9 (R =
2-CH3-3-Br) displayed higher activity with EC50 value of 0.04 mg/L. Meanwhile,
compounds 12 (R = 3,5-F2) and 15 (R = 3,4,5-F3) also exhibited good activities (0.07
mg/L and 0.05 mg/L, respectively). For the other compounds 10 (R = 3-OCH3), 11 (R
= 3-I), 13 (R = 3-CN) and 14 (R = 3-OCF3), relatively weak activities were observed.
Inhibitory of SDH. In order to investigate whether the SDH is a potential target
enzyme of the target compounds or not, assay of inhibitory activity of the fungal SDH
was performed.24 Compound 6 was selected and tested against SDH from
mitochondria of R. solani. The lead compound bixafen was used as a control. As
demonstrated in Table 3, the selected compound 6 (IC50 = 1.41 mg/L) showed good
inhibitory activity against SDH, close to bixafen’s inhibitory activity (IC50 = 1.22
mg/L). It proved that the SDH may be one of the important targets of the compound 6.
In vitro antifungal activities of compounds 16-29 and structure-activity
relationships analysis. The carboxyl “core” is one of the prototypical pharmacophoric
scheme of aromatic carboxamides and contributes primarily to the binding affinity of
the SDHIs. Fortunately, compound 6 showed excellent in vitro antifungal activities
against R. solani and in vitro inhibitory activity against SDH. These results indicated
that compound 6 could be the novel potential lead compound against R. solani. So, in
order to further study antifungal activities of aromatic carboxamides with the
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diphenylamine scaffold, based on compound 6 as the lead compound, some carboxyl
“cores” were introduced such as pyridyl, furyl, phenyl and thienyl. Antifungal
activities of various aromatic carboxamides 16-29 were evaluated. Preliminary in
vitro antifungal activities of aromatic carboxamides against R. solani were
summarized in Table 4. In compounds 16-29, only compounds 18, 25, 26 and 29
displayed promising antifungal activities (Inhibition rate ≥ 85 %) against R. solani
at a dosage of 20 mg/L, and compound 18 (Inhibition rate = 91.67 %) was higher than
that of compound 6 (96.17 %) and bixafen (90.44 %).
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To analyze the structure-activity relationships (SARs) of the target compounds
16-29 against R. solani, all compounds 16-29 were tested for further studies. The
results were showed in Table 5. For compounds with pyridyl moieties, compound 18
(R = 2-chloro-3-pyridyl) owned the higher activity than compounds 16 and 17. For
example, compound 18 showed the higher activity (EC50 = 0.34 mg/L) than
compound 17 (EC50 = 0.37 mg/L). In compounds 19-23 with phenyl moieties,
compounds 22 (R = 2-methylphenyl) and 23 (R = 2-trifluoromethylphenyl) exhibited
the higher activity against R. solani with EC50 valve of 0.82 mg/L and 0.84 mg/L. In
addition, compound 20 (R = 2-chlorophenyl) and 21 (R = 2-iodophenyl) exhibited
relatively low activity with EC50 values of 1.72 mg/L and 1.66 mg/L, respectively. In
compounds 24-29, compound 29 (R = thiazole) exhibited the highest activity (EC50 =
0.31 mg/L) against R. solani. But for 27 (R = 2-thiophene) and 28 (R = 3-thiophene),
the two compounds revealed lower activities. Similar results also appeared on
compounds 24 (R = 2-furyl) and 25 (R = 3-furyl). However, compound 26 (R =
2-methyl-3-furyl) showed relatively good antifungal activity (EC50 = 0.35 mg/L).
After further analysis of the structure-activity relationships of the compounds 16-29
and 6, one interesting finding was that compound with ortho-substituent group on the
aromatic generally displayed higher antifungal activity than its corresponding
compounds. But different substituents influenced their corresponding antifungal
activities. This possible reason for this phenomenon might be that ortho-substituent of
the aromatic favored the interaction between the compound and the target enzyme
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SDH. Unfortunately, no compounds displayed more active than lead compound 6 and
bixafen. The results demonstrated that, among all synthesized aromatic carboxamides
with
the
diphenylamine
scaffold,
compound
6,
3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, had the best antifungal
activity against R.solani. One possible answer for this phenomenon may be that the
pyrazole unit favor the interaction between ligand and protein.
In vivo antifungal activities for compounds 6, 9 and 15 against R. solani. To further
know the antifungal activity of the synthesized compounds against R. solani, in vivo
fungicidal activities of compounds 6, 9 and 15 with high in vitro activities were
evaluated by pot experiments in a greenhouse environment according to a previously
reported procedure.25-27 The lead compound bixafen and thifluzamide, a commercial
fungicide targeting rice sheath blight, were selected as positive control (in vivo
fungicidal activities were listed in supplementary data). All three compounds
exhibited ≥ 90 % control against tested fungi at 20 mg/L, which was superior to
bixafen (80 %), but lower than that of thifluzamide (100 %). Even at a lower dosage
of 2.5 mg/L, compounds 6 and 9 still displayed ≥ 55 % control against R. solani. In
addition, compound 6 and 9 showed more promising antifungal activity than bixafen
at all tested dosages. EC50 values of tested compounds against R.solani were showed
in Table 6. All three compounds exhibited promising activities with EC50 values of
1.93 mg/L, 2.29 mg/L and 6.13 mg/L, respectively. Among them, compound 6 and 9
exhibited higher control against R. solani than the lead compound bixafen (EC50 =
3.72 mg/L). However, compound 6 displayed promising activity (1.93 mg/L), similar
to thifluzamide (1.88 mg/L). The results suggested that these molecules could
potentially control the disease caused by R. solani and field trials were carried out at
the next step.
Field trials for compound 6 controlling rice sheath blight. To further study the
potential of compound 6 controlling rice sheath blight, field trials were carried out in
Table 7.25-27 Rice plants at fruiting stage naturally infected by R. solani were used.
Applications of compounds 6 and thifluzamide at 200 g ai. ha-1 had 48.80 % and
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57.19 % control efficacy after the first spraying at the seventh day, respectively. The
control efficacy would respectively go up to 64.10 % and 71.40 % after 14 days after
the second spraying for compounds 6 and thifluzamide. The result showed that
compound 6 was a potential fungicide.
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Molecular docking studies. In an effort to explain the possible antifungal
mechanism of these aromatic carboxamides containing the diphenylamine scaffold,
the compound 6 was docked into the binding site of the SDH and the theoretical
binding mode between compound 6 and SDH was shown in Figure 4. The compound
6 fit in the gap composed of subunit B, C and D (Figure 4). The phenyl group in the
middle of compound 6 was stretched into the hydrophobic pocket that consisted of the
residues B/Pro-202, B/Ile-251, C/Ile-77 and C/Trp-73, while the methylpyrazole
group of compound 6 located at another hydrophobic pocket, surrounded by the
residues B/Trp-205, B/Trp-206, C/Phe-64 and C/Trp-73, forming a stable
hydrophobic binding (Figure 4). Detailed analysis showed that the
3-chloro-4-methylphenyl group of compound 6 formed π-π stacking interaction and
cation-π interaction with the residues D/Tyr-128 and C/Arg-80, respectively. In
addition, the CH-π interactions were observed between the methylpyrazole group in
the terminal of 6 and sidechains of the residues B/Trp-206, C/Phe-64 and C/Trp-73
(Figure 4). Besides, a Cl-π interaction was formed between compound 6 and the
residue B/His-249 of SDH (Figure 4). All these interactions helped compound 6 to
anchor in the binding site of SDH.
To explain the activity order of bixafen and compound 6 against the SDH,
bixafen was then docked into the binding pocket of the SDH, and the theoretical
binding mode between bixafen and the SDH were shown in Figure 5. The interaction
between bixafen and the SDH was nearly the same as the analog 6 (Figure 5).
Compared with bixafen, the methylpyrazole group in the terminal of the compound 6
formed extra CH-π interactions with the residues B/Trp-206, C/Phe-64 and C/Trp-73.
Furthermore, the 3-chloro-4-methylphenyl group of compound 6 formed π-π stacking
interaction with the residue D/Tyr-128, while bixafen not. Another difference was that
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an extra Cl-π interaction was formed between compound 6 and the residue B/His-249
of SDH. Compared with 6, the oxygen atom in the amido bond of bixafen formed an
extra hydrogen bond (bond length: 3.4 Å) interaction with B/Trp-206, however, the
hydrogen bond was very weak (Figure 6). All these differences made compound 6
was more active than bixafen (Figure 6).
The estimated binding energies were -8.1 kcal·mol−1 for bixafen, and -8.5
kcal·mol−1 for compound 6, respectively. The docking results revealed that compound
6 might be potential SDHI. The above molecular simulations gave us rational
explanation of the interactions between bixafen, compound 6 and SDH, which
provided valuable information for further discovery of SDHIs.
In
summary,
a
series
of
N-(2-(phenylamino)-4-fluorophenyl)-pyrazole-4-carboxamides 1-15 were designed,
synthesized and evaluated for their fungicidal activities against seven phytopathogenic
fungi (R. solani, P. infestans, F. oxysporum f. sp. vasinfectum, B. dothidea, G. zeae, A.
alternate and F. oxysporum f. sp. niveum). In vitro experiments showed that
compounds 3, 6, 9, 12 and 15 showed significant antifungal activities with EC50
values of 0.03-0.08 mg/L. Among them, compounds 6 (EC50 = 0.03 mg/L) and 9
(EC50 = 0.04 mg/L) were superior to the lead compound bixafen (EC50 = 0.04 mg/L)
and compound 6 had good inhibitory activity against SDH (IC50 = 1.41 mg/L), close
to bixafen’s inhibitory activity (IC50 = 1.22 mg/L). Then, based on compound 6,
aromatic carboxamides with diphenylamine scaffold 16-29 were synthesized and
evaluated for their fungicidal activities against R. solani. Structure-activity
relationship analysis showed that pyrazole moiety was the most important carboxyl
“core” and displayed high fungicidal activities. In addition, in vivo pot experiments
showed that compounds 6 and 9 had promising activities against R. solani with EC50
values of 1.93 mg/L and 2.29 mg/L, respectively, higher than that of the lead
compound bixafen (EC50 = 3.72 mg/L). And, compound 6 displayed similar activity
(EC50= 1.93 mg/L) to thifluzamide (1.88 mg/L). Further field trials showed that
compound 6 at 200 g ai. ha-1 had 64.10 % control efficacy against rice sheath blight
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after the second spraying at the 14th day. Additional computational simulations
revealed the possible binding mode of the target compound 6 to the SDH. It was
believed that the results of the current studies could support the development of new
potent and antifungal drug-like candidates. Further synthesis optimization and
toxicological studies are currently in progress.
304
305
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307
308
309
CONFLICT OF INTEREST
The authors declare that they have no competing interests.
310
311
312
313
314
315
ACKNOWLEDGMENTS
This study was supported by the National Key Research and Development Program of
China (2016YFC0502004) for the financial support.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications
website at DOI:
316
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REFERENCES
(1) Kulka, M.; Schmeling B. V. Carboxin fungicide and related compounds, In: Lyr,
H., (Eds), modern selective fungicides-properties, applications, mechanisms of
action. 2nd Ed., Gustav Fischer Verlag, Germany and New York, 1995, 8,
133-147.
(2) Xiong, L.; Shen, Y. Q.; Jiang, L. N.; Zhu, X. L.; Yang, G. F. Succinate
dehydrogenase: an ideal target for fungicide discovery. ACS Symposium Series
2015, 175-194.
(3) Yoshikawa, Y.; Katsuta, H.; Kishi, J.; Yanase. Y. Structure-activity relationship of
carboxin-related carboxamides as fungicide. J. Pestic. Sci. 2011, 36, 347-356.
(4) Walter, H. Fungicidal succinate-dehydrogenase-inhibiting carboxamides, In:
Lamberth C.; Dinges J. (Eds), bioactive carboxylic compound classes:
pharmaceuticals and agrochemicals. Wiley-VCH, Weinheim, 2016, 405-425.
(5) Rheinheimer, J. Succinate dehydrogenase inhibitors: anilides. In: Krämer W.;
Schirmer U.; Jeschke P.; Witschel M. (Eds), Modern crop protection
compounds. 2nd edn. Wiley-VCH, Weinheim, 2012, 627-645.
(6) Inaoka, D. K.; Shiba, T.; Sato, D.; Balogun, E. O.; Sasaki, T.; Nagahama, M.;
Oda, M.; Matsuoka, S.; Ohmori, J.; Honma, T.; Inoue, M.; Kita, K.; Harada, S.
Structural insights into the molecular design of flutolanil derivatives targeted for
fumarate respiration of parasite mitochondria. Int. J. Mol. Sci. 2015, 16,
15287-15308.
(7) Huang, L. S.; Sun, G.; Cobessi, D.; Wang, A. C.; Shen, J. T.; Tung, E. Y.;
Anderson, V. E.; Berry, E.A. 3-Nitropropionic acid is a suicide inhibitor of
mitochondrial respiration that, upon oxidation by complex II, forms a covalent
adduct with a catalytic base arginine in the active site of the enzyme. J. Biol.
Chem. 2006, 281, 5965-5972.
(8) Ruprecht, J.; Yankovskaya, V.; Maklashina, E.; Iwata, S.; Cecchini, G. Structure
of Escherichia coli succinate: quinone oxidoreductase with an occupied and
empty quinone-binding site. J. Biol. Chem. 2009, 284, 29836-29846.
14 / 33
ACS Paragon Plus Environment

Page 15 of 33
Journal of Agricultural and Food Chemistry
346
(9) Ruprecht, J.; Iwata, S.; Rothery, R. A.; Weiner, J. H.; Maklashina, E.; Cecchini, G.
Perturbation of the quinone-binding site of complex II alters the electronic
properties of the proximal 3Fe-4S iron-sulfur cluster. J. Biol. Chem. 2011, 286
12756-12765.
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
(10) Xiong, L.; Li, H.; Jiang, L. N.; Ge, J. M.; Yang, W. C.; Zhu, X. L.; Yang, G. F.
Structure-based discovery of potential fungicides as succinate ubiquinone
oxidoreductase inhibitors. J. Agric. Food Chem. 2017, 65, 1021-1029.
(11) Ye, Y. H.; Ma, L.; Dai, Z. C.; Xiao, Y.; Zhang, Y. Y.; Li, D. D.; Wang, J. X.;
Zhu, H. L. Synthesis and antifungal activity of nicotinamide derivatives as
succinate dehydrogenase inhibitors. J. Agric. Food Chem. 2014, 62, 4063-4071.
(12) Li, S.; Li, D.; Xiao, T.; Zhang, S.; Song, Z.; Ma, H. Design, synthesis, fungicidal
activity, and unexpected docking model of the first chiral boscalid analogues
containing oxazolines. J. Agric. Food Chem. 2016, 64, 8927-8934.
(13) Yao, T. T.; Xiao, D. X.; Li, Z. S.; Cheng, J. L.; Fang, S. W.; Du, Y. J.; Zhao, J.
H.; Dong, X. W.; Zhu, G. N. A. Design, synthesis, and fungicidal evaluation of
novel pyrazole-furan and pyrazole-pyrrole carboxamide as succinate
dehydrogenase inhibitors. J. Agric. Food Chem. 2017, 65, 5397-5403.
(14) Xiong, L.; Zhu, X. L.; Gao, H. W.; Fu, Y.; Hu, S. Q.; Jiang, L. N. Yang, W. C.;
Yang, G. F. Discovery of potent succinate-ubiquinone oxidoreductase inhibitors
via pharmacophore-linked fragment virtual screening approach. J. Agric. Food
Chem. 2016, 64, 4830-4837.
(15) Guo, X.; Zhao, B.; Fan, Z.; Yang, D.; Zhang, N.; Wu, Q.; Yu, B.; Zhou, S.;
Kalinina, T. A.; Belskaya, N. P. Discovery of novel thiazole carboxamides as
antifungal succinate dehydrogenase inhibitors. J. Agric. Food Chem. 2019, 67,
1647-1655.
(16) Zhang, L.; Li, W.; Xiao, T.; Song, Z.; Csuk, R.; Li, S. Design and discovery of
novel chiral antifungal amides with 2-(2-oxazolinyl)aniline as a promising
pharmacophore. J. Agric. Food Chem. 2018, 66, 8957-8965.
(17) Walter, H.; Lamberth, C.; Corsi, C. Synthesis of fungicidally active succinate
15 / 33
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Page 16 of 33
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
dehydrogenase inhibitors with novel difluoromethylated heterocyclic acid
moieties. Monatsh. Chem. 2018, 149, 791-799.
(18) Ren, Z. L.; Liu, H.; Jiao, D.; Hu, H. T.; Wang, W.; Gong, J. X.; Wang, A. L.;
Cao, H. Q.; Lv, X. H. Design, synthesis, and antifungal activity of novel
cinnamon-pyrazole carboxamide derivatives. Drug Develop. Res. 2018, 79,
307-312.
(19) Zhang, A. G.; Zhou, J. Y.; Tao, K.; Hou, T. P.; Jin, H. Design, synthesis and
antifungal evaluation of novel pyrazole carboxamide s with diarylamines
scaffold as potent succinate dehydrogenase inhibitors. Bioorg. Med. Chem. Lett.
2018, 28, 3042-3045.
(20) Wang, H. Y.; Gao, X. H.; Zhang, X. X.; Jin, H.; Tao, K.; Hou, T. P. Design,
synthesis and antifungal activity of novel fenfuram-diarylamine hybrids. Bioorg.
Med. Chem. Lett. 2017, 27, 90-93.
(21) Wen, F.; Jin, H.; Tao, K.; Hou, T. P. Design, synthesis and antifungal activity of
novel furancarboxamide derivatives. Eur. J. Med. Chem. 2016, 120, 244-251.
(22) Pu, T.; Wang, H. Y.; Liu, Y.; Jin, H.; Hou, T. P. Design ， synthesis and
antifungal activity of novel 2-methyl-3-furancarboxamide derivatives. J. Sichuan
Univ. (Nat. Sci. Ed) 2017, 54, 178-184.
(23) Han, S. Y.; Kim, Y. A. Recent development of peptide coupling reagents in
organic synthesis. Tetrahedron 2004, 60, 2447-2467.
(24) Zeun, R.; Scalliet, G.; Oostendorp, M. Biological activity of sedaxane a novel
broad-spectrum fungicide for seed treatment. Pest Manag. Sci. 2013, 69,
527−534
(25) Chen, Y.; Yao, J.; Yang, X.; Zhang, A. F.; Gao, T. C. Sensitivity of Rhizoctonia
solani causing rice sheath blight to fluxapyroxad in China. Eur. J. Plant. Pathol.
2014, 140, 419-428.
(26) Yang, D.; Wang, B.; Wang, J.; Chen, Y.; Zhou, M. G. Activity and efficacy of
Bacillus subtilis strain NJ-18 against rice sheath blight and Sclerotinia stem rot
of rape. Biol. Control 2009, 51, 61-65.
16 / 33
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(27) Gu, B. G.; Liu, N. C.; Wu, X. P.; Zhu, Q. H. Pesticide-Guidelines for the field
efficacy trials(I)-Fungicides against sheath blight of rice. Pesticide-Guidelines
for the Field Efficacy Trials (I), National Standards of China, GB/T 17980.
20-2000.
405
406
407
408
409
410
411
412
413
414
415
(28) Trott, O.; Olson A. J. AutoDock Vina: improving the speed and accuracy of
docking with a new scoring function, efficient optimization, and multithreading.
J. Comput. Chem. 2010, 31, 455-461.
(29) Sanner, M. F. Python: a programming language for software integration and
development. J. Mol. Graph. Model. 1999, 17, 57-61.
(30) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell,
D. S.; Olson, A. J. AutoDock 4 and AutoDockTools 4: Automated docking with
selective receptor flexibility. J. Comput. Chem. 2009, 30, 2785-2791.
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422
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424
425
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Figure captions
Figure 1. Representative SDHIs and their prototypical pharmacophore.
Figure 2. Design of
novel
N-(2-(phenylamino)-4-fluorophenyl)-pyrazole-4-carboxamides 1-15 and aromatic
carboxamides 16-29.
Figure 3. General synthesis of the target compounds 1-15 and 16-29. Reaction
conditions: (i) KF, 160 C, 14 h; (ii) Fe/NH4Cl, C2H5OH (75 %), 2 h; (iii)
EDCI/DMAP, DCM, rt, 6 h.
Figure 4. The theoretical binding mode between compound 6 and SDH and the result
was shown by PyMoL 1.7.6.
Figure 5. The theoretical binding mode between bixafen and SDH and the result was
shown by PyMoL 1.7.6.
Figure 6. The theoretical binding mode between compound 6, bixafen and SDH, and
the result was shown by PyMoL 1.7.6 (overlap).
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Table 1. Preliminary in vitro antifungal activities of the target compounds 1-15 at 20
mg/L.
Inhibition rate (%)
Compd
Rs
Pi
Fov
Bd
Gz
Aa
Fon
1
86.03±0.25
91.91±0.74
83.09±0.73
89.12±0.31
86.77±0.29
96.17±0.29
94.41±0.22
88.53±0.31
91.47±0.31
86.18±0.30
93.23±0.15
95.29±0.29
83.82±0.73
81.17±0.17
89.27±0.23
90.44±0.25
31.25±0.72
50.78±0.78
56.72±0.16
60.94±0.41
27.19±0.48
56.87±0.48
40.62±0.78
64.38±0.65
66.10±0.16
23.75±0.16
50.78±0.78
39.84±0.68
18.59±0.15
22.81±0.31
45.00±0.31
67.81±0.31
29.36±0.79
57.14±0.92
67.46±0.79
50.00±0.79
17.46±00.92
61.90±0.92
51.59±1.21
22.22±1.83
61.11±1.21
31.75±0.92
57.93±0.79
64.49±0.40
23.80±0.09
67.46±1.21
42.86±0.46
63.49±0.46
64.28±0.79
68.25±0.94
61.11±1.21
77.78±0.46
42.86±0.92
68.25±0.92
68.25±0.92
48.78±2.21
80.95±0.92
43.65±0.79
64.28±0.79
59.52±0.46
30.43±1.32
50.79±0.92
83.73±0.92
82.54±0.46
39.35±0.95
38.40±0.00
61.32±0.83
62.75±0.83
48.90±0.48
64.66±1.26
55.56±2.21
63.23±1.26
65.62±0.00
22.64±3.60
71.35±0.29
60.84±1.72
19.77±2.19
47.71±1.24
63.23±0.48
86.63±1.91
54.97±0.58
57.89±0.88
60.53±0.87
64.91±1.34
26.32±0.88
49.12±2.03
53.51±0.51
60.52±0.88
68.42±0.35
27.00±0.26
59.65±1.34
58.77±0.16
21.93±0.71
37.72±0.11
72.81±0.13
91.89±0.55
29.84±0.81
31.45±0.80
41.13±0.81
37.10±0.93
33.87±0.93
34.68±0.58
30.65±0.93
36.29±1.23
53.23±0.93
28.23±0.81
32.16±1.02
31.45±0.81
19.35±0.93
33.18±1.35
29.03±0.04
43.55±1.86
2
3
4
5
6
7
8
9
10
11
12
13
14
15
bixafen
Rs, R. solani; Pi, P. infestans; Fon, F. oxysporum f. sp. vasinfectum; Bd, B. dothidea;
Gz, G. zeae; Aa, A. alternate; Fov, F. oxysporum f. sp. niveum.
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Table 2. In vitro EC50 values of the target compounds 1-15 against R. solani.
Compd
Regression
EC50 (mg/L)
95% confidence
interval
R
equation
1
Y=0.6389X+5.3657
Y=0.6701X+5.4918
Y=0.4779X+5.5377
Y=0.6785X+5.3570
Y=0.6522X+5.2150
Y=0.6991X+6.0547
Y=0.7481X+5.7382
Y=0.7165X+5.2503
Y=0.5080X+5.7142
Y=0.6159X+5.4744
Y=0.7905X+5.4637
Y=0.7113X+5.8094
Y=0.8024X+5.0461
Y=0.4982X+5.2208
Y=0.4381X+5.5859
Y=0.8124X+6.1105
0.27
0.18
0.08
0.30
0.47
0.03
0.10
0.45
0.04
0.17
0.26
0.07
0.88
0.36
0.05
0.04
0.2098-0.3415
0.1461-0.2331
0.0586-0.0959
0.2411-0.3676
0.3647-0.6007
0.0214-0.0449
0.0659-0.1612
0.3460-0.5785
0.0305-0.0505
0.1629-0.1769
0.1439-0.4664
0.0528-0.1005
0.3911-1.9628
0.2782-0.4672
0.0288-0.0733
0.0357-0.0517
0.9972
0.9977
0.9981
0.9980
0.9974
0.9940
0.9928
0.9968
0.9982
0.9999
0.9846
0.9967
0.9714
0.9968
0.9940
0.9952
2
3
4
5
6
7
8
9
10
11
12
13
14
15
bixafen
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Table 3. In vitro inhibitory activity of compound 6 against SDH.
Compd
Regression
equation
IC50 (mg/L)
95% confidence
interval
R
6
Y=0.5869X+4.9121
Y=0.6841X+4.9404
1.41
1.22
0.6401-3.1144
0.6855-2.1783
0.9427
0.9648
bixafen
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Table 4. Preliminary in vitro antifungal activities of aromatic carboxamides 16-29
against R. solani at 20 mg/L.
Ar
No. Inhibition rate (%)
Ar
No.
Inhibition rate (%)
78.50±0.48
16
17
18
19
20
21
22
77.92±0.50
84.07±0.30
91.67±0.98
73.50±0.44
70.54±0.70
72.40±0.22
79.00±0.41
23
CF3
N
24
25
26
27
28
29
73.97±0.79
85.00±0.41
85.54±0.18
65.00±0.41
78.49±0.92
87.29±0.58
N
O
N
Cl
Cl
O
CH3
O
S
I
S
F3C
N
CH3
S
H3C
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Table 5. In vitro EC50 values of aromatic carboxamides 16-29 against R.solani.
Compd
Regression
EC50 (mg/L)
95% confidence
interval
R
equation
16
17
Y=0.6545X+4.9358
Y=0.7770X+5.3367
Y=0.5376X+5.2536
Y=0.7175X+4.7113
Y=0.4888X+4.8850
Y=0.5890X+4.8705
Y=0.5521X+5.0469
Y=0.5300X+5.0392
Y=0.6753X+4.7793
Y=0.8555X+4.9614
Y=0.6204X+5.2857
Y=0.6638X+4.5703
Y=0.6136X+4.9251
Y=0.6095X+5.3140
Y=0.6991X+6.0547
Y=0.8124X+6.1105
1.25
0.37
0.34
2.53
1.72
1.66
0.82
0.84
2.12
1.11
0.35
4.44
1.32
0.31
0.03
0.04
1.0798-1.4548
0.3012-0.4513
0.2788-0.4087
2.1945-2.9077
1.3389-2.2066
1.2955-2.1249
0.6419-1.0534
0.6636-1.0718
1.7772-2.5337
0.9580-1.2847
0.2835-0.4230
3.7196-5.2987
1.1279-1.5555
0.2620-0.3559
0.0214-0.0449
0.0357-0.0517
0.9967
0.9968
0.9972
0.9972
0.9964
0.9964
0.9967
0.9969
0.9954
0.9969
0.9969
0.9964
0.9962
0.9980
0.9940
0.9952
18
19
20
21
22
23
24
25
26
27
28
29
6
bixafen
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Table 6. In vivo EC50 values of the target compounds against R. solani.
Comp
Regression
EC50 (mg/L)
95% confidence
interval
R
equation
6
9
Y=1.4566X+4.5841
Y=1.7438X+4.3726
Y=2.5126X+3.0216
Y=1.3155X+4.2488
1.93
2.29
6.13
3.72
1.88
1.4034-2.6541
2.0223-2.5929
5.4688-6.8682
3.1194-4.4460
0.8897-3.9685
0.9794
0.9962
0.9950
0.9885
0.9026
15
bixafen
thifluzamide Y=3.8126X+3.9556
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Table 7. Relative control efficacy of compound 6 against rice sheath blight in field
trials.
7th day after the first 14th day after the second
spraying
disease
index
1.10
spraying
disease
index
1.08
concentration
(g ai ha-1)
compd
control
control
efficacy (%)
48.80±1.21
44.05±2.46
57.19±1.73
56.10±1.68
----
efficacy (%)
64.10±1.55
59.78±3.56
71.40±1.60
69.40±2.48
----
6
200
100
1.19
1.20
thifluzamide 200
100
0.92
0.86
0.94
0.92
control
0
2.15
3.01
Data are means ± SE.
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O
S
N
H
O
O
I
Carboxin
O
N
H
N
H
amide bond
O
Benodanil
Fenfuram
O
R
N
H
amine moiety
O
Br
O
OCF3
carboxyl "core"
F3C
N
H
N
N
H
N
Cl
S
Br
Thifluzamide
Boscalid
F
Cl
O
F2HC
N
N
H
N
Bixafen
Cl
Cl
Figure 1. Representative SDHIs and their prototypical pharmacophore.
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F
O
F2HC
N
N
N
F
H
F2HC
O
F
O
N
F2HC
N
N
H
Cl
NH
N
Splicing-up
Cl
N
N
H
NH
Bixafen
Cl
F
R
1-15
6
H2N
NH
Bioisosteric
modification
R
F
O
Diphenylamines
Ar
N
H
NH
Cl
16-29
Figure
2.
Design
of
novel
N-(2-(phenylamino)-4-fluorophenyl)-pyrazole-4-carboxamides 1-15 and aromatic
carboxamides 16-29.
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F
F
F
NH2
O2N
H2N
(ii)
(i)
+
NH
NH
O2N
R
F
R
R
a
b
d
c
F
F
O
F2HC
F2HC
COOH
H2N
N
(iii)
H
NH
N
NH
N
N
+
N
R
R
e
d
1 R=H
2 R=2-Cl
3 R=3,4-Cl2
4 R=2,4-Cl2
9 R=2-CH3-3-Br
10 R=3-OCH3
11 R=3-I
12 R=3,5-F2
5 R=2-Cl-4-Br 13 R=3-CN
6 R=3-Cl-4-Me 14 R=3-OCF3
7 R=3-Br
15 R=3,4,5-F3
8 R=2-CH3-5-Br
F
F
O
Ar
N
H
H2N
(iii)
+
ArCOOH
NH
NH
Cl
Cl
Ar=
N
N
16
N
Cl
Cl
20
17
19
24
18
O
O
25
I
CF3
CH3
21
22
23
F3C
N
S
CH3
S
O
S
H3C
29
27
28
26
Figure 3. General synthesis of the target compounds 1-15 and 16-29. Reaction
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Journal of Agricultural and Food Chemistry
conditions: (i) KF, 160 C, 14 h; (ii) Fe/NH4Cl, C2H5OH (75 %), 2 h; (iii)
EDCI/DMAP, DCM, rt, 6 h.
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