Design, synthesis and fungicidal evaluation of novel pyraclostrobin analogues

Article abstract of DOI:10.1016/j.bmc.2018.01.004

A series of novel pyraclostrobin derivatives were designed and prepared as antifungal agents. Their antifungal activities were tested in vitro with five important phytopathogenic fungi, namely, Batrylis cinerea, Phytophthora capsici, Fusarium sulphureum, Gloeosporium pestis and Sclerotinia sclerotiorum using the mycelium growth inhibition method. Among these compounds, 5s displayed IC₅₀ value of 0.57 μg/mL against Batrylis cinerea and 5k-II displayed IC₅₀ value of 0.43 μg/mL against Sclerotinia sclerotiorum, which were close to that of the positive control pyraclostrobin (0.18 μg/mL and 0.15 μg/mL). Other compounds 5f, 5k-II, 5j, 5m and 5s also exhibited strong antifungal activity. Further enzymatic assay demonstrated compound 5s inhibited porcine bc₁ complex with IC₅₀ value of 0.95 μM. The statistical results from an integrated computational pipeline demonstrated the predicted total binding free energy for compound 5s is the highest. Consequently, compound 5s with the biphenyl-4-methoxyl side chain could serve as a new motif as inhibitors of bc₁ complex and deserve to be further investigated.

Full text of DOI:10.1016/j.bmc.2018.01.004

Accepted Manuscript
Design, Synthesis and Fungicidal Evaluation of Novel Pyraclostrobin Ana-
logues
Lili Wang, Shuangshuang Zhao, Xiaotian Kong, Lingling Cao, Sheng Tian,
Yonghao Ye, Chunhua Qiao
PII:
S0968-0896(17)32279-4
https://doi.org/10.1016/j.bmc.2018.01.004
BMC 14158
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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5 January 2018
8 January 2018
Please cite this article as: Wang, L., Zhao, S., Kong, X., Cao, L., Tian, S., Ye, Y., Qiao, C., Design, Synthesis and
Fungicidal Evaluation of Novel Pyraclostrobin Analogues, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.01.004
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Design, Synthesis and Fungicidal Evaluation of Novel Pyraclostrobin
Analogues
Lili Wang,†ǁ Shuangshuang Zhao§ǁ, Xiaotian Kong,¤ Lingling Cao,§ Sheng
Tian,†* Yonghao Ye*§, Chunhua Qiao*†
† Jiangsu Key Laboratory of Preventive and Translational Medicine for
Geriatric Diseases,
College of Pharmaceutical Sciences, Soochow University，Suzhou 215123, P.
R. China.
§ College of Plant Protection, State & Local Joint Engineering Research
Center of Green Pesticide Invention and Application, Nanjing Agricultural
University, Nanjing 210095, P.R. China.
¤ Institute of Functional Nano and Soft Materials (FUNSOM), Soochow
University, Suzhou, Jiangsu, 215123, P. R. China
ǁ these two authors contributed equally
Corresponding author: Yonghao Ye: Phone 86 25 84399753, E-mail:
yeyh@njau.edu.cn; Sheng Tian, Email: stian@suda.edu.cn; Chunhua Qiao:
Phone 86 512 65882092, Email: qiaochunhua@suda.edu.cn
Abstract: A series of novel pyraclostrobin derivatives were designed and
prepared as antifungal agents. Their antifungal activities were tested in vitro
with five important phytopathogenic fungi, namely, Batrylis cinerea,
Phytophthora capsici, Fusarium sulphureum, Gloeosporium pestis and
1

Sclerotinia sclerotiorum using the mycelium growth inhibition method. Among
these compounds, 5s displayed IC50 value of 0.57 µg/mL against Batrylis
cinerea and 5k-II displayed IC50 value of 0.43 µg/mL against Sclerotinia
sclerotiorum, which were close to that of the positive control pyraclostrobin
(0.18 µg/mL and 0.15 µg/mL). Other compounds 5f, 5k-II, 5j, 5m and 5s also
exhibited strong antifungal activity. Further enzymatic assay demonstrated
compound 5s inhibited porcine bc1 complex with IC50 value of 0.95 µM. The
statistical results from an integrated computational pipeline demonstrated the
predicted total binding free energy for compound 5s is the highest.
Consequently, compound 5s with the biphenyl-4-methoxyl side chain could
serve as a new motif as inhibitors of bc1 complex and deserve to be further
investigated.
Keywords: antifungal activity; pyraclostrobin analogues; phytopathogenic
fungi; cytochrome bc1 complex inhibitor; molecular docking
1. Introduction
Pyraclostrobin (Figure 1) belongs to the strobilurin type of fungicide.1
Pyraclostrobin was believed to bind to the Qo site of a membrane-bound
homodimeric cytochrome bc1 complex (EC 1.10.2.2, also known as complex
Ⅲ of mitochondrial respiration) and blocked the electron transfer between
cytochrome b and cytochrome c1, resulting in the inhibition of the
mitochondrial respiration chain and the reduced production of ATP which is
essential for the proper function of fungal cell.2 Ever since being introduced to
the market by BASF, 3 pyraclostrobin has been playing critical roles in crop
2

protection. After extensive application for over two decades, fungi
phytopathogens with moderate to high resistance to pyraclostrobin have been
reported.4-5 Therefore, development of pyraclostrobin analogues for the
treatment of resistant pathogens has been the efforts for the agricultural
chemists.
Figure 1. Chemical structure of pyraclostrobin
As shown in Figure 1, the structure of pyraclostrobin could be divided into
three parts: the pharmacophore, the phenyl bridge and the side chain. The
pharmacophore moiety exists in a number of highly potent strobilurin type
fungicides, 6-17 variation at the side chain is observed in several marketed
agents, while modification of the phenyl linker is rarely reported.18-21
Considering phenyl is commonly replaced by its bioisostere pyridinyl, and the
synthesis of N-ortho substituted pyridine analogues could be easily achieved
by nucleophilic aromatic substitution reaction, we herein designed and
synthesized a series of pyraclostrobin derivatives bearing the pyridinyl linker,
and the side chain was accordingly investigated. The fungicidal activities of
these compounds were evaluated against five important plant pathogen strains
including Batrylis cinerea, Phytophthora capsici, Fusarium sulphureum,
Gloeosporium pestis and Sclerotinia sclerotiorum in vitro, and the inhibitory
capability against porcine bc1 complex were evaluated. Finally, the
3

structure-activity relationships (SARs) were also analyzed using integrated
computational strategy including molecular docking, MM/GBSA binding free
energy calculation and binding free energy decomposition.
2. Results and discussion
2.1 Chemistry. The synthesis of the target molecules 5a-u and 6a-b was
started from the commercially available 2-chloro-3-nitropyridine (Scheme 1).
To
prepare
1a-v,
the
essential
reaction
intermediates
and
4'-chloro-(1,1'-biphenyl)-4-ol,
3',4'-dichloro-(1,1'-biphenyl)-4-ol
4-(pyridin-3-yl)phenol were prepared by Suzuki coupling reactions according
procedures.22-23
to
reported
Reaction
intermediate
(1-(4-chlorophenyl)-1H-pyrazol-3-yl)methanol was synthesized employing the
reported method through a three-step procedure.24 Reduction of the
commercially available 5-(2,5-dichlorophenyl)furan-2-carbaldehyde using
NaBH4
afforded
the
alcohol
intermediate
(5-(2,5-dichlorophenyl)furan-2-yl)methanol. All other side chain fragments
were from commercial sources. Nucleophilic aromatic substitution reaction of
2-chloro-3-nitropyridine with the above reaction intermediates using different
base afforded the corresponding products 1a-v. Treatment of 1k with NaH in
anhydrous THF followed by addition of iodomethane provided intermediate 2k.
Reduction of 1a-j, 1l-v and 2k using stannous chloride in the presence of
sodium acetate afforded the crude product hydroxylamines 3a-v, which were
immediately treated with methyl chloroformate at –10 ℃ to give the
corresponding 4a-v in good yields (60-83%). Methylation of 4a-v with
iodomethane in a sealed tube under basic condition (potassium carbonate in
acetone) afforded the target molecules 5a-v in 66-91% yields. The quaternary
4

ammonium salt 5i was obtained at the methylation step. Compounds 6a-b
were prepared through the classical click chemistry (copper-catalyzed
azide–alkyne cycloaddition). Treatment of 5v with the two azido compounds
1-(azidomethyl)-4-chlorobenzene and 1-azido-4-chlorobenzene25-26 in the
presence of hydrazine hydrate and copper(II) sulfate gave 6a-b. All prepared
compounds were analyzed by high-pressure liquid chromatography to ensure
the purity (> 98%) before submission for biological evaluation.
(a): NaH, PhCH3; or: 18-crown-6, KOH, PhCH3, rt.; or: t-BuOK, THF, rt.; or
Cs2CO3, DMF; (b): NaH, CH3I,THF, rt.; (c): SnCl2 . 2H2O, NaOAc, THF/ MeOH,
H2O, 0oC; (d): methyl carbonochloridate, NaHCO3, CH2Cl2, -10oC; (e): CH3I,
K2CO3, acetone, rt., 12h; (f): CuSO4 . 5H2O, N2H4. H2O (50%, w/w), H2O, rt.,
15 min.
Scheme 1. Synthetic route of the target molecules 5a-v and 6a-b
2.2 Antifungal activity.
As shown in Table 1, the prepared twenty
three target compounds were divided into two different types. For type I
compounds, the aryl ring is directly attached to the pyridone oxygen, while type
IA (5a-c) distinguished from IB (5d-k) by the presence of only one aryl ring at
the side chain. For type II compounds, the pyridone oxygen is attached to the
aromatic ring through a methylene linker. In term of antifungal activity, all type
5

IA compounds displayed poor antifungal activity. By comparison, Type IB
compounds generally displayed improved antifungal activity. Specifically,
compound 5k-II, with N-methylcarbazole at the side chain, exhibited a broad
spectrum of antifungal activity against all five tested phytopathogens. The
inhibition rate (25 µg/mL) against Fusarium sulphureum and Sclerotinia
sclerotiorum were 73% and 98%, comparable to that of the positive control
pyraclostrobin (78% and 100%). Replacement of one or two hydrogen atoms
with chlorine at the side chain lead to significantly decreased inhibition
percentage, from 100% (5f) to 31% (5h) against Sclerotinia sclerotiorum,
suggesting the chlorine atom on the side chain of type IB compounds could not
be accommodated. Compound 5i, with the quaternary ammonium side chain,
exhibited less than 10% antifungal activity against all five tested fungi
phytopathogens; while high inhibitory activity of 5f and 5k-II concluded that the
electron rich hydrophobic side chain is favorable for the compounds antifungal
activity. Compounds with benzyloxyl group at the 2-position of the pyridine ring
were grouped as typeⅡ, while type IIA (5l-r) with a single aryl ring at the side
chain distinguished from type IIB (5s-5u, 6a-ab) with biaryl rings. As shown in
Table 1, nine out of twelve typeⅡ compounds displayed moderate to good
antifungal activity. Compound 5m demonstrated the highest inhibitory potency
against all tested fungi phytopathogens (inhibition rate between 43-100% at 25
µg/mL) in this group. Compound 5b devoid of the methylene linker exhibited
much lower antifungal activity, suggesting more flexible side chain is favorable
for the antifungal activity. In addition, halogen atom on the phenyl ring in
typeⅡA compounds could significantly increase the antifungal activity (5l vs
5m-q), this is different from the type I compounds. Moreover, the chlorine atom
6

on the phenyl ring is more appropriate than the fluorine atom, as demonstrated
by comparing the activity of 5m vs 5p, and 5o vs 5q. Compound 5r with
pyridine ring at side chain displayed significantly decreased antifungal activity,
and low inhibition activity similar to that of compound 5i was observed. In
general, the typeⅡB compounds (5s-u, 6a-b) displayed antifungal activity
close to that of typeⅡA compounds. Compound 5s in this group exhibited the
highest inhibitory potency against Batrylis cinerea, Gloeosporium pestis and
Sclerotinia sclerotiorum, with inhibition percentage ranges from 72% to 81% at
25 µg/mL. Replacement of the phenyl ring with other five-member heterocycle
(5t-u, 6a-b) reduced the antifungal activity.
Table 1. Antifungal activity of the prepared compounds in vitro
Percentage of inhibition (25 µg/mL)
NO.
Batrylis
cinerea
Phytophthora
capsici
Fusarium
Gloeosporium
Sclerotinia
sulphureum pestis
sclerotiorum
R =
5a
8
2
-
17
-
Type
5b
5c
5d
42
6
4
13
-
23
22
66
12
-
ⅠA
6
79
34
33
100
5e
30
18
8
36
55
Type
Ⅰ
5f
82
64
46
4
47
35
17
-
50
29
19
10
37
70
54
41
8
100
64
31
-
5g
5h
5i
Type
ⅠB
5j
67
32
53
89
5k-II
85
50
73
50
98
5l
15
78
71
10
43
35
0
29
51
45
23
Type
Type
5m
5n
55
37
100
53
Ⅱ
ⅡA
7

5o
5p
64
76
20
21
27
25
43
27
41
38
5q
5r
60
28
81
13
1
16
1
29
13
78
31
-
5s
33
44
72
5t
64
61
48
23
24
8
30
14
8
64
41
21
40
34
3
Type
5u
6a
6b
ⅡB
28
9
21
78
32
87
33
100
100
100
-：No antifungal effect. The antifungal activity was determined using the mycelia
growth inhibitory rate method.
To analyze the antifungal effect more accurately, the IC50 values were
further determined for compounds with inhibitory percentage higher than 50%
at 25 µg/mL. The results were shown in Table 2. Compound 5f and 5k-II
exhibited a broad spectrum of antifungal activity against the five tested
phytopathogens. Especially, the IC50 values of 5f (0.98 µg/mL against Batrylis
cinerea) and 5k-II (0.43 µg/mL against Sclerotinia sclerotiorum) were close to
that of positive control pyraclostrobin (0.18 µg/mL and 0.15 µg/mL).
Additionally, compound 5s displayed strong antifungal activity against Batrylis
cinerea with an IC50 value 0.57 µg/mL, which is comparable to that of
pyraclostrobin (0.18 µg/mL). Generally, these compounds demonstrated
higher fungicidal activity against Batrylis cinerea and Sclerotinia sclerotiorum
than other three fungi phytopathogens.
8

Table 2. IC50 values of some selected compounds against the five tested
phytopathogens
IC50 SD(µg/mL)a
NO.
Batrylis
cinerea
Phytophthora
capsici
Fusarium
Gloeosporium
pestis
Sclerotinia
sclerotioru
sulphureum
R =
5d
5f
2.93 0.03 12.92 0.15
0.98 0.02 7.51 0.07
>25
>25
3.51 0.11
0.64 0.03
25.00 0.30 25.61 0.27
5j
>25
>25
>25
>25
1.73 0.05
0.43 0.02
5k-II
1.18 0.13 24.78 0.37
2.43 0.13
24.56 0.11
5m
5n
5p
5s
19.28 0.3 >25
10.82 0.1 >25
20.09 0.2 >25
0.57 0.01 >25
>25
>25
>25
>25
>25
>25
>25
ND
1.46 0.04
>25
>25
7.18 0.09
0.18 0.05 0.005 0.00
0.36 0.03
0.03 0.00
0.15 0.01
aValues are the mean standard deviation (SD) of three replicates. ND: not determined.
2.3 Inhibition effect against bc1 complex. To further understand the
inhibition mechanism, representative compounds were selected for
cytochrome bc1 complex assay. The activity of the complex in the
succinate-cytochrome
c
reductase (SCR) was determined using
decylubiquinol (DBH2) and cytochrome c1 as substrates.27
As shown in Table 3, compound 5s exhibited the lowest IC50 value (0.95
µM) against porcine SCR, which is lower than the positive control penthiopyrad
(95% at 100 µM, IC501.56 µM). In contrast, compound 5f without the
methylene linker displayed only 11% inhibitory activity at 100 µM, suggesting
the flexible side chain is favorable for the enzymatic inhibition activity. This
result is consistent with the cell based assay. The same trend was confirmed
by comparing the inhibitory activity of 5m (79%) with 5b (< 10%). Comparing
the inhibitory activity of compound 5b (< 10%) with 5d-5k-II (11-43%), it could
9

be concluded that the presence of biaryl rings is more favorable than single
aryl ring at the side chain, this conclusion is apparently also supported by the
activity difference between compound 5m (79%) vs 5s (99.9%).
Table 3. Percentage of inhibition, IC50 values and predicted total binding free
energy of selected compounds against porcine SCR
%inhibition (100 µM)
IC50 SD (µM)a
∆Gpredc (kcal/mol)
NO.
R =
b
5b
5d
<10%
41%
/
-15.60
-35.81
/
5e
13%
/
3.30
5f
11%
22%
/
/
-23.84
-15.10
5g
5k-II
43%
/
-18.69
5m
5s
79%
32.14 0.11
0.95 0.012
-37.87
-44.17
99.9%
5t
55%
38.84 0.78
-34.15
-35.85
99.9%
1.76 0.17 nM
95%
1.56 0.12
/
aValues are the mean standard deviation (SD) of three replicates. bNot tested. cThe predicted
binding free energies between each compound and cytochrome bc1 complex (PDB ID: 3TGU,
εin = 1.0)
2.4 Structure-activity relationships (SARs) discussion In order to
demonstrate the detected biological activity difference, the representative
compounds listed in Table 3 were docked into the binding pocket of
cytochrome bc1 complex using standard precision (SP) and extra precision
(XP) scoring functions in Glide docking. The statistical results from the Glide
docking do not have satisfactory prediction capability for ranking the biological
activities of the studied chemicals. Thus, the binding free energy
10

calculation/decomposition were employed to analyze the interaction patterns
between each compound and cytochrome bc1 complex. The predicted
conformations of these compounds from XP model of Glide docking were
retrieved and optimized using MM/GBSA. The detailed procedure of
MM/GBSA was reported in the previous studies.28-31 The modified GB model
developed by Onufriev (GBOBC1) was adopted by setting the interior (solute)
dielectric constant to 1.32 As can be seen in Table 3, the binding free energies
calculated by MM/GBSA has better capability than the docking scores to rank
the bioactivities of this series of compounds. The compound with highest
inhibition activity (compound 5s) had the lowest binding free energy (-44.17
kcal/mol). The aligned binding poses and interaction patterns for compounds
5s, 5f and positive control compound, pyraclostrobin were depicted in Figure
2.
11

Figure 2. (a) The aligned predicted binding poses for compound 5s (colored in green), 5f
(colored in purple) and positive control compound, pyraclostrobin (colored in red) in the
binding pocket of cytochrome bc1 complex. The interaction patterns predicted by Glide
docking and MM/GBSA minimization for (b) positive control compound, pyraclostrobin; (c)
compound 5s; (d) compound 5f.
To uncover the contribution of key residues for inhibitor binding
quantitatively, the total binding free energies for compounds 5s, 5f and
pyraclostrobin were further decomposed into individual residue contributions.
The inhibitor-residues interaction spectra for three compounds were illustrated
in Figure 3. Apparently, minor chemical modifications could induce different
binding poses for in the binding pocket. For example, the pyridine ring in
compound 5s can form stable arene-H interaction with residue proline 271
12

(Figure 2d and Figure 3). Whereas, this interaction cannot be observed
between pyraclostrobin and cytochrome bc1 complex. The result
demonstrated that the activity difference was not simply caused by the pyridine
ring. Similar phenomenon can also be observed for compounds 5s and 5f
(Figures 2c and 2d). The predicted total binding free energies for compounds
5s, 5f and pyraclostrobin were -44.17, -23.84 and -35.85 kcal/mol, respectively.
The major differences among three compounds, especially between
compounds 5s and 5f were the contributions of six key residues including
Met125, Phe129, Gly143, Pro271, Glu272 and Phe275. The differences for
these residues between the compounds 5s and 5f were higher than 1.0
kcal/mol. This observation is consistent with the finding derived from Glide
docking. As can be shown in Figure 1c and 1d, the important interactions
between the inhibitor and residue such as Met125, Glu272 and Phe275 were
broken by replacing the R group (4-methylene-1,1'-biphenyl) of compound 5s
with the R group (1,1'-biphenyl) of compound 5f. Consequently, retention the
interaction between pyridine in compound 5s and proline 271, while
modification the R group of this series of compounds to achieve improved
interaction with positive key residues, like Met125 and Phe 275 (Figure 3)
might produce more potent inhibitors in the next lead optimization stage.
13

Figure 3. The inhibitor-residues interaction spectra for compounds 5s, 5f and
positive control compound, pyraclostrobin.
Finally, the observed decreased potency for several compounds in cellular
relative to enzymatic assay is noticed. It was understood to be the
consequence of compound permeability, or could be the competition with
endogenous substrates, et al. This so called rightward shift in potency is
commonly reported. 33
3. Conclusion
In summary, a series of novel pyraclostrobin analogues were designed and
prepared using a convenient synthetic route. The in vitro antifungal assay
result indicated that eight out of twenty-three compounds displayed significant
inhibitory activity against five selected phytopathogenic fungal strains and their
antifungal activities are comparable to that of positive control pyraclostrobin.
Although only one analogue reported herein was found to possess lower IC50
against porcine SCR than pyraclostrobin, our results confirmed that some
analogues, especially typeⅡseries, inhibited the activities of SCR from porcine
heart mitochondria. The combined computational strategy was applied to
calculate binding free energy and key favorable residues for inhibitor binding
were discerned. On the basis of our present experiment results, several
suggestions for further structural modification of the pyridinyl linker
pyraclostrobin analogues could be put forward: hydrophobic side chain is
critical for the compound antifungal activity, biaryl rings at the side chain is
preferable, appropriate flexibility is beneficial to the inhibitory activity against
bc1 complex. Finally, the antifungal activity of these compounds against
pyraclostrobin resistant phytopathogenic fungi are under investigation and
14

deserves further exploration.
Acknowledgement: the National Key Research and Development
Program of China (2017YFD0200506). We also thank Prof. Guangfu Yang
from Central China Normal University for the SCR experiments.
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19

Design, Synthesis and Fungicidal Evaluation of Novel Pyraclostrobin Analogues
‖
‖
Lili Wang,† Shuangshuang Zhao§ , Xiaotian Kong,¤ Lingling Cao,§ Sheng Tian,†* Yonghao
Ye*§, Chunhua Qiao*†
† Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases,
College of Pharmaceutical Sciences, Soochow University，Suzhou 215123, P. R. China.
§ College of Plant Protection, State & Local Joint Engineering Research Center of Green
Pesticide Invention and Application, Nanjing Agricultural University, Nanjing 210095, P.R.
China.
¤ Institute of Functional Nano and Soft Materials (FUNSOM), Soochow University, Suzhou,
Jiangsu, 215123, P. R. China
Bridge
Bridge
Side Chain
N
Ar
O
N
N
O
Cl
Side Chain
O
N
O
N
O
O
O
O
Pyraclostrobin
Pharmacophore
Pharmacophore
20

Research highlight:
► Twenty three novel pyraclostrobin derivatives were designed and prepared as
antifungal agents; ► compounds 5s and 5k displayed similar IC50 to positive control
pyraclostrobin against certain fungal pathogen; ►The SAR of these compounds at the
side chain position was investigated.
21