Design, synthesis and antifungal evaluation of novel pyrazole carboxamides with diarylamines scaffold as potent succinate dehydrogenase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.08.001

Sixteen novel pyrazole carboxamides with diarylamines scaffold were designed, synthesized and characterized in detail via ¹H NMR, ¹³C NMR and ESI-HRMS. Preliminary bioassays showed that some of the target compounds exhibited good ant

Full text of DOI:10.1016/j.bmcl.2018.08.001

Accepted Manuscript
Design, synthesis and antifungal evaluation of novel pyrazole carboxamides
with diarylamines scaffold as potent succinate dehydrogenase inhibitors
Aigui Zhang, Jingya Zhou, Ke Tao, Taiping Hou, Hong Jin
PII:
S0960-894X(18)30645-0
https://doi.org/10.1016/j.bmcl.2018.08.001
BMCL 25981
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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31 March 2018
19 June 2018
1 August 2018
Please cite this article as: Zhang, A., Zhou, J., Tao, K., Hou, T., Jin, H., Design, synthesis and antifungal evaluation
of novel pyrazole carboxamides with diarylamines scaffold as potent succinate dehydrogenase inhibitors,
Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.08.001
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Design, synthesis and antifungal evaluation of novel pyrazole
carboxamides with diarylamines scaffold as potent succinate
dehydrogenase inhibitors
Aigui Zhang1, Jingya Zhou1, Ke Tao, Taiping Hou, Hong Jin*
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,
College of Life Sciences, Sichuan University, Chengdu 610065, Sichuan, P.R.China
*Corresponding Author at: Key Laboratory of Bio-Resource and Eco-Environment of
Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065,
Sichuan, P.R.China
1 These authors contributed equally to this work.
E-mail address: jinhong@scu.edu.cn (H. Jin).
1

ABSTRACT
Sixteen novel pyrazole carboxamides with diarylamines scaffold were designed,
1
synthesized and characterized in detail via H NMR, 13C NMR and ESI-HRMS.
Preliminary bioassays showed that some of the target compounds exhibited good
antifungal activity against Rhizoctonia solani, Fusarium oxysporum,
Phytophthora infestans and Fusarium graminearum. Among them, compound 1c
exhibited the highest antifungal activities against R. solani in vitro with EC50 value of
0.005 mg/L, superior to the commercially available fungicide fluxapyroxad (EC50 =
0.033 mg/L). And compound 1c (IC50 = 0.034 mg/L) showed higher inhibition
abilities against succinate dehydrogenase than fluxapyroxad (IC50 = 0.037 mg/L). This
study suggests that compound 1c could be regarded as a potential succinate
dehydrogenase inhibitor.
Keywords: Pyrazole carboxamides, Synthesis, Succinate dehydrogenase inhibitors,
Antifungal activities
2

Succinate dehydrogenase (SDH, EC 1.3.5.1, also known as complex II), which
catalyzes the oxidation of succinate to fumarate in the mitochondrial matrix, is the
only enzyme complex simultaneously involved in the respiration chain and Krebs
cycle.1-3Due to its crucial role in life processes, SDH has been particularly appreciated
as a promising target for agrochemical discovery.4-6 To date, 19 structurally diverse
SDH inhibitor (SDHI) fungicides have been successfully developed and shown
potential for plant protection. As one of the most important categories of the SDHIs,
the substituted pyrazole carboxamide fungicides have been intensively employed
throughout the world to fight again slighly destructive plant pathogens, such as
Botrytis cinerea, Sclerotinia spp., Leveillula taurica and Sphaerotheca
macularis.7They share a prototypical pharmacophoric scheme, which consists of a
substituted pyrazole carboxyl “core”, a conserved amide function and an amine
moiety (Fig.1).8According to the cocrystal structure of the SDH from porcine heart,9
avian,10and Escherichia coli,11 the substituted pyrazole carboxyl “core” buries deep
into the ubiquinone binding site (Q-site) and contributes predominantly to the binding
affinity of SDHI fungicide. So lots of companies, institutes and universities have
carried on the substantial work. They kept the substituted pyrazole carboxyl “core”
and amide function unchanged, and the amine moieties have been structurally diverse.
For example, Yang et al. discovered if the biphenyl group in bixafen was replaced
with the diphenyl ether group, the novel substituted pyrazole carboxamide fungicides
showed good antifungal activities.12
3

Fig.1. Representative pyrazole carboxamide fungicides inhibiting succinate
dehydrogenase and their prototypical pharmacophore.
Many bioactive compounds with diarylamines have been used in the agrochemical
field over the years.13Diarylamine derivatives feature significant biological activities,
including fungicidal, insecticidal, acaricidal, rodenticidal and herbicidal activities.
14–18
Therefore, diarylamine may represent a promising bioactive moiety to integrate
with other pharmacophore. Recently our research group discovered when the phenyl
group in fenfuram (a commercial fungicide) was replaced with the diarylamine, the
novel fenfuram-diarylamine hybrid exhibited better antifungal activities than
fenfuram.19
Inspired by these researches, to extend the researches on the development of novel
amide derivatives as fungicides,19-21fluxapyroxad was applied as a lead molecule and
4

the diarylamines were introduced in order to replace the substituted biphenyl group in
fluxapyroxad based on the principle of “splicing-up” bioactive substructures. A series
of novel pyrazole carboxamides with diarylamines scaffold were designed and
synthesized (Fig. 2). Subsequently, in vitro antifungal activities were performed to
evaluate the fungicidal activities against the selected four phytopathogenic fungi
Rhizoctonia solani (R. solani), Fusarium oxysporum (F. oxysporum),
Phytophthora infestans (P. infestans) and Fusarium graminearum (F. graminearum).
Furthermore, SDH inhibition activities in vitro and molecular docking of target
compounds were used in order to verify their potential mechanism.
Fig. 2. The strategy for design of novel substituted pyrazole carboxamides with
diarylamines scaffold.
Scheme 1 and Table 2 detail the syntheses and chemical structures of the target
compounds. Initially, compounds 5 were obtained by condensation reaction and
transformed into the corresponding compounds
6
through reduction
reactions.13Meanwhile, the important intermediate compound 3 was prepared via
chlorination of the key compound 2 (Scheme 1).19 Finally, the compound 3 was
reacted with the compounds 6 through classical approach to afford a series of novel
pyrazole carboxamide fungicides, namely compounds 1a-1p.19
5

Scheme 1. Synthesis of the target compounds. Reagents and conditions: (a)
substituted anilines, KF, 160 C, 15 h; (b) Fe/NH4Cl, C2H5OH (75%), 2 h; (c)
SOCl2/CH2Cl2, 80C, 3 h; (d) CH2Cl2, Et3N, 0 C-r.t., 3 h.
The results of the in vitro antifungal activities of the target compounds 1a-1p and
fluxapyroxad at a dosage of 20 mg/L against R. solani, F. oxysporum, P. infestans and
F. graminearum were listed in Table 1. Here, the antifungal activities were expressed
as the inhibition percentage. It was found that the target compounds exhibited
fungicidal activities against four fungi. Almost all target compounds showed very
strong activities against R. solani, however, poor antifungal activities against F.
oxysporum, P. infestans and F. graminearum could be observed. For example, except
compounds 1a, 1f, 1k, 1m and 1n, another eleven compounds displayed much higher
fungicidal activities against R. solani (over 90% Inhibition rate) and were better than
fluxapyroxad.
Table 1
Preliminary antifungal activities of target compounds at 20 mg/L in vitro.
Inhibition rate (%)
Compound
R1
R2
R.solani F.oxysporum
P.infestans
23.00
F.graminearum
30.01
CN
H
80.09
92.90
35.13
56.52
1a
1b
CH3
2-CH3
43.67
29.38
6

F
F
H
4,5-F2
4-CH3
5-Cl
6-Cl
H
92.92
93.66
93.81
85.25
91.59
93.07
95.28
93.36
83.78
91.45
83.33
89.68
94.54
95.13
91.44
42.26
82.09
44.87
21.84
63.48
54.43
53.04
61.39
29.04
74.26
31.83
36.87
66.60
52.75
49.62
31.33
40.00
29.17
15.33
61.17
32.83
51.67
60.00
18.33
43.33
21.17
36.67
60.83
42.83
39.26
38.46
50.82
35.81
16.88
43.51
36.32
43.63
43.76
19.04
48.30
24.97
28.37
45.40
23.77
38.24
1c
1d
Cl
1e
Cl
1f
Cl
1g
CH3
Cl
1h
4-F
2-Cl
H
1i
Cl
1j
CH(CH3)2
OCF3
OCH3
CF3
Cl
1k
H
1l
H
1m
H
1n
4-Cl
H
1o
1p
Br
fluxapyroxad
To analyze the antifungal activities of the target compounds with excellent effects
against R. solani, the compounds 1a-1p were selected for further studies. Their EC50
values were listed in Table 2. Compounds 1b, 1c, 1d, 1f, 1g, 1j, 1n and 1p with EC50
values of 0.005-0.028 mg/L showed significant antifungal activities, much more
superior to fluxapyroxad (EC50 = 0.033 mg/L).
Table 2
EC50 (mg/L) values of target compounds against R. solani.
Compound
EC50 (mg/L)
0.146
Compound
EC50 (mg/L)
0.054
1a
1b
1c
1d
1i
0.023
0.006
1j
1k
1l
0.005
0.033
0.027
0.033
7

0.069
0.016
0.009
0.214
0.016
0.034
0.028
1e
1f
1m
1n
1o
1g
0.050
0.033
1h
1p
fluxapyroxad
In order to investigate whether the SDH is a potential target enzyme of title
compounds or not, the fungal SDH inhibition assay was performed. Compound 1c and
1j were selected and tested against SDH in vitro from mitochondria of R. solani. As
demonstrated in Table 3, the selected compound 1c (IC50 = 0.034 mg/L) showed
higher inhibition abilities against SDH than fluxapyroxad (IC50 = 0.037 mg/L). It
further proved that the SDH probably is one of the important action targets of the
novel pyrazole carboxamides.
Table 3
IC50 (mg/L) values of target compounds’SDH inhibition activities in vitro.
Compound
R. solani
0.034
1c
0.040
1j
0.037
Fluxapyroxad
To further survey whether the SDH is a potential target enzyme of novel pyrazole
carboxamides with diarylamines scaffold or not, the binding mode of compound 1c
and Fluxapyroxad to SDH was shown in Figs. 3-4. The compound 1c fit in the gap
composed of subunit B, C and D (Fig.3) and the phenyl group in the middle of
compound 1c 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 2-methylpyrazole scaffold of
compound 1c 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.
Detailed analysis showed that a π-π stacking interaction was observed between the
8

phenyl group of compound 1c and sidechain of residue C/Trp-73. The fluxapyroxad
also fit in the gap composed of subunit B, C and D and shared a similar binding mode
with compound 1c (Fig. 4). The docking results revealed that the compound 1c might
be potential SDHI.
Fig.3. The binding mode between compound 1c (white sticks) and SDH by PyMoL
1.7.6.
Fig. 4. The binding mode of compound 1c (white sticks) and fluxapyroxad (rose red
sticks) to SDH by PyMoL 1.7.6 (overlap).
9

In summary, a series of novel substituted pyrazole carboxamides were designed and
synthesized. Preliminary bioassays showed that some of the target compounds
exhibited good antifungal activities against Rhizoctonia solani, Fusarium oxysporum,
Phytophthora infestans and Fusarium graminearum. Among them, compound 1c
exhibited the highest antifungal activity against R. solaniin vitro with EC50 value of
0.005 mg/L, superior to the lead compound fluxapyroxad (EC50 = 0.033 mg/L). And
compound 1c (IC50 = 0.034 mg/L) showed higher inhibition ability against SDH than
compound fluxapyroxad (IC50 = 0.037 mg/L) and it may be a promising lead
compound for the potential development of fungicide.
Acknowledgment
The authors would like to acknowledge the National Key Research and
Development Program of China (2016YFC0502004) for the financial support.
A. Supplementary data
Supplementary data associated with this article can be found, in the online version,
at
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Highlights
1. Sixteen novel substituted pyrazole carboxamides with diarylamines scaffold were
designed and synthesized.
2. Compound 1c exhibited the highest antifungal activities against R. solani in vitro,
superior to fluxapyroxad.
3. Compound 1c showed higher inhibition abilities against SDH than fluxapyroxad.
12

Graphical Abstract
13