Design, synthesis and antifungal activity of novel fenfuram-diarylamine hybrids

Article abstract of DOI:10.1016/j.bmcl.2016.11.026

Ten novel fenfuram-diarylamine hybrids were designed and synthesized. And their antifungal activities against four phytopathogenic fungi have been evaluated in vitro and most of the compounds demonstrated a significant antifungal activities against Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 5e exhibited the most potent antifungal activity against R. solani with an EC₅₀value of 0.037 mg/L, far superior to the commercially available fungicide boscalid (EC₅₀= 1.71 mg/L) and lead fungicide fenfuram (EC₅₀= 6.18 mg/L). Furthermore, scanning electron microscopy images showed that the mycelia on treated media grew abnormally with tenuous, wizened and overlapping colonies compared to the negative control. Molecular docking studies revealed that compound 5e featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 3-chlorophenyl group in compound 5e formed a CH-π interaction with B/Trp-206 and a Cl-π interaction with D/Tyr-128, rendering compound 5e more active than fenfuram against SDH.

Full text of DOI:10.1016/j.bmcl.2016.11.026

Accepted Manuscript
Design, synthesis and antifungal activity of a fenfuram-diarylamine hybrid
Hongyu Wang, Xuheng Gao, Xiaoxiao Zhang, Hong Jin, Ke Tao, Taiping Hou
PII:
S0960-894X(16)31169-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.026
BMCL 24421
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
5 May 2016
21 October 2016
11 November 2016
Please cite this article as: Wang, H., Gao, X., Zhang, X., Jin, H., Tao, K., Hou, T., Design, synthesis and antifungal
activity of a fenfuram-diarylamine hybrid, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/
10.1016/j.bmcl.2016.11.026
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Design,
synthesis
and
antifungal
activity
of
a
fenfuram-diarylamine hybrid
Hongyu Wang, Xuheng Gao, Xiaoxiao Zhang, Hong Jin *, Ke Tao, Taiping Hou *
Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education,
College of Life Sciences, Sichuan University, Chengdu, 610064, China
*Corresponding authors:
E-mail: jinhong@scu.edu.cn; houtplab@scu.edu.cn
Phone No: 86-28-85415611
1 / 16

ABSTRACT
Ten novel fenfuram-diarylamine hybrids were designed and synthesized. And their
antifungal activities against four phytopathogenic fungi have been evaluated in vitro
and most of the compounds demonstrated a significant antifungal activity against
Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 5e exhibited the most
potent antifungal activity against R. solani with an EC50 value of 0.037 mg/L, far
superior to the commercially available fungicide boscalid (EC50 = 1.71 mg/L) and
lead fungicide fenfuram (EC50 = 6.18 mg/L). Furthermore, scanning electron
microscopy images show that the mycelia on treated media grew abnormally
compared to the negative control with tenuous, wizened and overlapping colonies.
Molecular docking studies revealed that compound 5e features a higher affinity for
succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the
3-chlorophenyl group in compound 5e forms a CH-π interaction with B/Trp-206 and a
Cl-π interaction with D/Tyr-128, rendering compound 5e more active than fenfuram
against SDH.
Keywords: Fenfuram-diarylamine hybrid; Synthesis; Antifungal activity; Molecular
docking
2 / 16

Plant diseases have been recognized as a worldwide threat to crop production.1
The uses of fungicides are, and will remain, critical for the effective controls of most
plant diseases in agriculture and have contributed greatly to higher crop yields and
quality benefits in China and other countries worldwide.2,3 Fungicides based on
carboxylic amides, as one class of the most important classes of agrochemical
fungicides, have been intensively studied across the globe to fight highly destructive
plant pathogens, including Rhizoctonia spp., Sclerotinia spp. and others.4-7 The first
carboxylic amide fungicide used for crop protection was carboxin (Uniroyal). Newly
discovered compounds include fenfuram (Bayer), mepronil (Kumiai), boscalid
(BASF), penflufen (Bayer) and fluxapyroxad (BASF) (Figure 1).8The initial narrow
biological spectrums of these compounds were broadened with progressive
modification of the chemical structures. By comparison of all chemical structures of
these commercial carboxylic amides, Dehne found that they indeed shared common
chemical features, essential for fungicidal activity.9 This finding is most notably due
to the fact that these compounds bind to their biological target in a similar fashion.
The common target receptor for carboxylic amide fungicides is succinate
dehydrogenase (SDH).10 Most of these compounds act as SDH inhibitors (SDHIs) and
disrupt the mitochondrial tricarboxylic acid cycle and respiration chain of various
fungi species.11-13
3 / 16

Figure 1. The carboxylic amides fungicides.
Diarylamines represent an important structural motif for many bioactive
compounds used in the agrochemical field over the years.14Their derivatives feature
significant biological activities, including fungicidal, insecticidal, acaricidal,
rodenticidal and herbicidal activities.15-19 Therefore, diarylamines may represent a
promising bioactive motif to integrate with other pharmacophores. To extend the
research on the development of novel carboxylic amide derivatives as fungicides,
fenfuram was applied as a lead molecule and diarylamines were introduced in order to
replace the phenyl group in fenfuram based on the principle of “splicing-up” bioactive
substructures.20 A series of novel fenfuram-diarylamine hybrids were designed and
synthesized (Figure 2). Bioassay studies have been carried out and demonstrated that
some target molecules exhibited good antifungal activities and may be useful as
potential lead compounds. To the best of our knowledge, this is the first time the
antifungal activities of all synthetic compounds shown in this paper have been
studied.
4 / 16

Figure 2. Design strategy of the target compounds.
Scheme 1, Scheme 2 and Table 1 summarize the synthesis and chemical structures
of the fenfuram-diarylamine hybrids. The key intermediates 4a-j shown in Scheme 1
were obtained by classic condensation reactions and reduction with Fe/HCl. And the
intermediate 2-methylfuran-3-carboxylic acid shown in Scheme 2 was synthesized in
good yield following reported procedures and was characterized by 1H NMR.
Scheme 1. Synthesis of 2-amine-diarylamines 4a-j.
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Scheme 2. Synthesis of target compounds 5a-j.
The results of the in vitro antifungal activity of compounds 5a-j, boscalid and
fenfuram at a dosage of 20 mg/L against Rhizoctonia solani, Sclerotinia sclerotiorum,
Botrytis cinerea and Fusarium oxysporum are listed in Table 1. Here, the antifungal
activities are expressed as the inhibition percentage. Although it seems impossible to
devise an obvious structure activity relationship from the data shown in Table 1, it
was found that the target compounds exhibited fungicidal activity to varying extent
against four fungi. Most of the target compounds showed very strong activity against
R. solani and S. sclerotiorum, however, poor activity against B. cinerea and F.
oxysporum could be observed. For example, compound 5a displayed a much higher
fungicidal activity against R. solani and S. sclerotiorum (90.34 and 88.55 %) than B.
cinerea and F. oxysporum (59.34 and 27.43 %).
Table 1
Antifungal activities of fenfuram-diarylamine hybrid at 20 mg L-1
Inhibition ratea (%)
Compound
R1
R.solani
S.sclerotiorum
B. cinerea
F. axysporum
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H
4-F
5a
90.34
81.12
89.82
86.43
90.93
83.18
74.95
83.72
55.60
57.12
75.6
88.55
79.45
82.88
79.98
78.64
76.37
61.26
86.58
87.90
80.96
65.61
54.85
59.34
53.96
52.53
43.87
41.06
53.70
15.21
47.07
53.12
47.94
21.44
34.43
27.43
29.86
18.77
7.62
5b
2,4-F2
3,4-F2
3-Cl
4-Cl
2,4-Cl2
4-Br
4-CH3
4-OCH3
-
5c
5d
5e
26.07
44.21
13.25
35.38
24.17
19.75
9.34
5f
5g
5h
5i
5j
Fenfuram
Boscalid
-
70.5
44,58
aAverage of three replicates.
By comparing the different substitute groups of compounds 5a-j it was found that
compound 5i or 5j (R1 = 4-CH3 or 4-OCH3, electron-donating group; 55.60 or
57.12 %) showed a poorer fungicidal activity against R. solani than the other
compound 5b, 5f, or 5h (R1 = 4-F, 4-Cl or 4-Br, electron-donating group; 81.12, 83.18,
or 83.72 %). And the position of substitute groups of comparing compounds 5b, 5e, 5f
and 5h, it also was seen that compound with substitute group in third position was
better than it in fourth position, such as compound 5e (R1 = 3-Cl, 90.93 %) had better
antifungal activity against against R. solani than compounds 5b (R1 = 4-F, 81.12 %),
5f (R1 = 4-Cl, 83.18 %) and 5h (R1 = 4-Br, 83.72 %).
To analyze the antifungal activities of the compounds, compounds 5a and 5e
exhibiting stronger antifungal activities against R. solani were selected for further
studies. The corresponding EC50 values were listed in Table 2. Compounds 5a and 5e
exhibited a higher antifungal activity against R. solani than boscalid or fenfuram, and
7 / 16

their EC50 values were found to be much smaller than boscalid or fenfuram. For
example, compound 5e (R1 = 3-Cl) exhibited the greatest antifungal activity in the
group.
Table 2
EC50 values of some compounds with excellent effect against R.solani
Compound
5a
Regression equation
y=5.6455+0.9906x
y=6.045+0.7278x
y=4.3706+0.7957x
y=4.7886+0.9048x
Value of regression
EC50 (mg/L)
0.223
0.95
0.96
0.037
5e
fenfuram
boscalid
0.97
0.98
6.18
1.71
And the SEM images (Figure 3) clearly showed R.solani cultivated on media
without addition of any drugs feature dense, sturdy, and smooth mycelia with a fine
morphology. In contrast, the morphology of the mycelia of R. solani changed when
cultured on media with addition of 0.037 mg/L of compound 5e or boscalid. The
mycelia grew abnormally with a comparatively tenuous, wizened, and overlapping
colony, with the surface being rough and less ramified.
A
8 / 16

B
C
Figure 3. Scanning electron micrographs of negative control (A) and treated
colony with boscalid (B) and compound 5e (C) of 20 mg/L.
Meanwhile, the theoretical binding mode of compound 5e or fenfuram to SDH is
shown in Figures 4, 5 and 6. Compound 5e fitted in the cavity composed of subunits
B, C and D (Figure 4). The phenyl group in compound 5e bound to the hydrophobic
pocket was surrounded by residues B/Pro-202, B/Ile-251, C/Ile-77 and C/Trp-73,
while the 2-methylfuryl moiety in compound 5e, located at another hydrophobic
pocket, was surrounded by residues B/Trp-205, B/Trp-206, C/Phe-64 and C/Trp-73. A
detailed analysis showed that a π-π interaction was observed between the phenyl
group of compound 5e and side chain of residue C/Trp-73. Fenfuram also fitted in the
cavity composed of subunits B, C and D (Figure 5) and shared a similar binding mode
with compound 5e (Figure 6). The main difference between 5e and fenfuram was that
the 3-chlorophenyl group in compound 5e formed a CH-π interaction with B/Trp-206
and a Cl-π interaction with D/Tyr-128, which rendered compound 5e more active
against SDH than fenfuram.
9 / 16

Figure 4. The theoretical binding mode between compound 5e and SDH, and the
result was shown by PyMoL 1.7.6.
10 / 16

Figure 5. The theoretical binding mode of between Fenfuram and SDH, and the
result was shown by PyMoL 1.7.6.
11 / 16

Figure 6. The theoretical binding mode of compound 5e and Fenfuram to SDH,
and the result was shown by PyMoL 1.7.6 (overlap).
In addition, via energy calculations of 5e or fenfuram and SDH’s interaction, the
estimated binding energies were determined to be -7.9 kcal/mol for compound 5e, and
-6.2 kcal/mol for fenfuram, respectively. Docking results revealed that the two
compounds from the screen might be potential SDH inhibitors. Furthermore, the
different free binding energies of compound 5e and fenfuram suggested that
compound 5e featured a higher affinity for SDH than fenfuram, consistent with the
results of the in vitro antifungal activity assay (Table 2). The molecular simulations
described above provided a rational explanation of the interactions between the
compound 5e or fenfuram with SDH and therefore offered valuable insights for the
future development of SDH inhibitors.
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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 5e and
fenfuram were selected and tested against SDH enzyme in vitro from mitochondria of
R. solani. As demonstrated in Table 3, the selected compound 5e (IC50 = 0.13 µg/mL)
showed higher inhibition abilities against SDH enzyme than fenfuram (IC50 = 0.81
µg/mL). And this indicated that the inhibition ability of compound 5e was 6-fold
higher than fenfuram. It proved that the SDH enzyme is one of the important action
targets of title compounds.
Table 3
IC50 (µg/mL) values of fungal SDH inhibition activity (in vitro).
compound
5e
R. solani
0.13±0.0174
0.81±0.3918
fenfuram
In summary, a series of fenfuram-diarylamine hybrids were designed,
synthesized, and screened for their antifungal activity against four phytopathogenic
fungi. Compounds 5a and 5e exhibited EC50 values of 0.223 and 0.037 mg/L against
R. solani, respectively, superior to the positive controls. The marked changes in the
SEM images when comparing the negative control with the treated media in
combination with molecular docking studies provided further insights into the
interactions between the ligand and the receptor proteins. The fenfuram-diarylamine
hybrids exhibited broad antifungal activities and the selective potent inhibition against
R. solani may offer a promising lead compound for the potential development of
fungicides.
13 / 16

Acknowledgments
This study was supported by National Natural Science Foundation of China
(31272068), National Key Research and Development Program of China (Grant No.
2016YFC0502004-2), Hi-Tech Research and Development of China (863 program,
2011AA10A202-3), National Key Technology R&D Program of China
(2011BAE06B01-23) and Applied Basic Research Program of Sichuan Province
(2014JY0063) for financial support.
Supplementary data
Supplementary data associated with this article can be found in the online version.
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Graphical abstract
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