Novel sulfonamides against Botrytis cinerea with no positive cross-resistance to commercial fungicides: Design, synthesis and SAR study

Article abstract of DOI:10.1016/j.bmcl.2019.126859

Thirty-four novel compounds were synthesized using chesulfamide (N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexyl sulfonamide), a high-profile fungicide, as the lead compound, and their structures were characterized by ¹H NMR, ¹³C NMR, MS and elemental analysis. Additionally, the structure of (1S,2R)-2-((3-bromophenethyl)amino)-N-(4-chloro-2-trifluoromethylphenyl)cyclohexane-1-sulfonamide (IV-9) was confirmed by X-ray single crystal diffraction. The mycelium inhibition tests, spore germination inhibition tests, tomato pot tests and field trials were performed against strains of B. cinerea. Bioassay results showed that most of target compounds had good fungicidal activity against B. cinerea, in particular, IV-9 exhibited similar or superior effects to procymidone, boscalid and pyrisoxazole in all in vitro and in vivo tests. Moreover, there was no positive cross-resistance found between the compound IV-9 and eight commercial fungicides (azoxystrobin, boscalid, chlorothalonil, diethofencarb, fludioxonil, procymidone, pyrimethanil and pyrisoxazole) in the cross-resistance validation test performed by an innovative method.

Full text of DOI:10.1016/j.bmcl.2019.126859

Journal Pre-proofs
Novel sulfonamides against Botrytis cinerea with no positive cross-resistance
to commercial fungicides: design, synthesis and SAR study
Nan Cai, Lu He, Kai Wang, Zhihui Feng, Zining Cui, Mingshan Ji, Zhiqiu Qi,
Peiwen Qin, Xinghai Li
PII:
S0960-894X(19)30833-9
DOI:
https://doi.org/10.1016/j.bmcl.2019.126859
BMCL 126859
Reference:
Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
2 August 2019
30 October 2019
25 November 2019
Please cite this article as: Cai, N., He, L., Wang, K., Feng, Z., Cui, Z., Ji, M., Qi, Z., Qin, P., Li, X., Novel
sulfonamides against Botrytis cinerea with no positive cross-resistance to commercial fungicides: design, synthesis
and SAR study, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126859
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Novel sulfonamides against Botrytis cinerea with no positive cross-resistance to
commercial fungicides: design, synthesis and SAR study
Nan Cai a, Lu He a, Kai Wang a, Zhihui Feng a, Zining Cui b, Mingshan Ji a, Zhiqiu Qi
a, Peiwen Qin a, Xinghai Li a, *
a
Department of Pesticide Science, Plant Protection College, Shenyang Agricultural
University, Shenyang 110866, Liaoning, China.
b
State Key Laboratory for Conservation and Utilization of Subtropical
Agro-bioresources, Integrative Microbiology Research Centre, Guangdong Province
Key Laboratory of Microbial Signals and Disease Control, South China Agricultural
University, Guangzhou 510642, Guangdong, China.
Corresponding Author. Shenyang Agricultural University, Dongling Road 120,
Shenhe District, Shenyang, China. E-mail address: xinghai30@163.com (Xinghai Li)

Abstract
Thirty-four novel compounds were synthesized using chesulfamide
(N-(2-trifluoromethyl-4-chlorophenyl)-2-oxocyclohexyl sulfonamide), a high-profile
fungicide, as the lead compound, and their structures were characterized by 1H NMR,
13C NMR, MS and elemental analysis. Additionally, the structure of
(1S,2R)-2-((3-bromophenethyl)amino)-N-(4-chloro-2-trifluoromethylphenyl)cyclohex
ane-1-sulfonamide (IV-9) was confirmed by X-ray single crystal diffraction. The
mycelium inhibition tests, spore germination inhibition tests, tomato pot tests and
field trials were performed against strains of B. cinerea. Bioassay results showed that
most of target compounds had good fungicidal activity against B. cinerea, in
particular, IV-9 exhibited similar or superior effects to procymidone, boscalid and
pyrisoxazole in all in vitro and in vivo tests. Moreover, there was no positive
cross-resistance found between the compound IV-9 and eight commercial fungicides
(azoxystrobin, boscalid, chlorothalonil, diethofencarb, fludioxonil, procymidone,
pyrimethanil and pyrisoxazole) in the cross-resistance validation test performed by an
innovative method.
Key words: cycloalkylsulfonamides; reductive amination; Botrytis cinerea;
fungicidal activity; SAR; cross resistance
Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant
pathogen with a necrotrophic lifestyle attacking over 200 dicotyledonous crops
worldwide, which main threaten the mature or senescent tissues and the products in

retail chain. [1,2] The fungus is considered as a typical necrotroph, which co-opts
programmed cell death pathways in the host to accomplish infection. [2] Besides, B.
cinerea has a high risk of development of resistance due to its genetic plasticity, and
under selective pressure, the proportion of resistant strains inclines to increase in
populations after the acquired benefits of resistant over the wild-type phenotype.
[1,3,4] Moreover, during the past decades, many strains of B. cinerea with
multiple-resistance to conventional fungicides have appeared on various crops
worldwide. [5-9] Therefore, the risk of drug-free rotation drives us to search for new
candidates to control this plant disease.
Sulfonamides have received extensive attention by chemists and biologists
owing to their eminent biological activities, such as anti-cancer [10,11], antimicrobial
[12-14], treatment of chronic pain [15] and inhibition of certain enzymes [16-18]. It is
worth mentioning that the antimicrobial sulfonamides exhibit a broad-spectrum effect,
covering all organisms that synthesize folic acid de novo through the folate pathway
(they inhibit the enzyme dihydropteroate synthase (DHPS)). [19,20] Rather, the
biosynthetic pathway exists in many pathogenic microorganisms but absent in
mammals. Regrettably, commercial varieties of sulfonamide fungicides, like
tolnifanide, cyazofamid and amisulbrom, are less used in the field owing to their
action characteristics are sometimes not obvious enough. [21] Therefore, it is
meaningful to develop the sulfonamide fungicides with more excellent properties.

Our team dedicated in the synthesis and fungicidal activities research of
sulfonamides, especially the cycloalkylsulfonamides. Previously, our team reported
several series of 2-oxyalkylsulfonamides (Figure 1, A-D) which showed excellent
fungicidal activity [22-25], and the potential of chesulfamide as a fungicidal lead
compound was found therein. Chesulfamide, a cycloalkylsulfonamide fungicide, can
effectively control B. cinerea with strong preventive, therapeutic and osmotic activity.
[26] Using chesulfamide as the lead structure, we reduced the oxo group to the
hydroxyl group [27] to make relative esters [28,29] and replaced the hydroxyl group
with amino group [30], which enabled the introduction of aromatic and heterocyclic
rings [31] (Figure 2). Some of these compounds exhibited more pronounced
fungicidal activities than chesulfamide. These achievements motivated us to further
modify the structure of the lead compound to obtain the potential antifungal agents
with superb properties. Hence, based on structure-activity relationship in our previous
experiments, we designed to modify the lead compound with an active substituted
aromatic ethylamine group, which contained in the commercial fungicides like
fluopyram and mandipropamid (Figure 3). Moreover, the substituted ethylamine was
attached to the lead structure with a flexible carbon chain, which allowed for
abundant conformations and increased probability of binding to the targets. In this
paper, we designed and synthesized a series of 2-substituted ethylamino-N-substituted
phenyl cycloalkylsulfonamides in a simplified synthesis method compared to our
previous studies. [30] The comprehensive bioassays against B. cinerea of all target

compounds were evaluated and the structure-activity relationship (SAR) was analyzed.
In addition, we performed the cross-resistance tests between the optimal compound
IV-9 and eight commonly used commercial fungicides against B. cinerea with a novel
method.
O
O
S
O
R5
O
O
O
N
O
O
R3
O
S
O
R6
H
S
(CH2)n
N
N
H
SO2NR1R2
OR4
H
Cl
A
B
C
D
Figure 1. Reported 2-oxyalkylsulfonamide structures.
Figure 2. The designed strategy for the target compounds.
O
O
Cl
F3C
O
N
O
CF3
N
N
H
H
O
Cl
mandipropamid
fluopyram
Figure 3. Commercial fungicides containing the structure of aromatic ethylamine.
The synthetic route of target compounds is shown in Scheme 1. The synthesis of
key immediate III was done according to the reported method [32]. Then, the
immediate III was subjected to reductive amination to give the target compounds

IV-1 to IV-22. After analyzed the structure-activity relationship, it was determined
the most optimal structure of R1 as 3-Br-C6H4-. Afterwards, the structures of R2 and
the cyclohexane were tried to optimize and obtained the target compounds IV-23 to
IV-34 by the same synthesis method.
NH2
R2
O
O
SO3
O
O
ClCOCOCl, DMF
CH2Cl2, r.t, 4h.
SO3K
CH2ClCH2Cl,
K2CO3
Et3N, CH2Cl2, 2h
n
n
I
II
NH2
Ti(OiPr)4, EtOH, r.t, 3h.
R1
R1
1.
O
O
S
HN
O
S
O
O
R2
R2
N
N
2. NaBH4, r.t, 3h.
H
H
n
n
III
IV
Structural optimization of R1
R2 = 2-CF3-4-Cl, n = 1,
R1 = 2-F-C6H4-, 3-F-C6H4-, 2-Cl-C6H4-, 3-Cl-C6H4-, 4-Cl-C6H4-, 2,5-Cl-C6H3-,
3,4-Cl-C6H3-, 2-Br-C6H4-, 3-Br-C6H4-, 4-Br-C6H4-, 4-CH3-C6H4-, 3,4-CH3-C6H4-,
3-CF3-C6H4-, 4-CF3-C6H4-, 2-OCH3-C6H4-, 3-OCH3-C6H4-, 4-OCH3-C6H4-,
3,4-OCH3-C6H3-, 4-OCF3-C6H4-, 4-OH-C6H4-, 4-pyridine, 2-morpholine;
Structural optimization of R2
R1 = 3-Br-C6H4-, n =1,
R2 = 2-F, 3-F, 4-F, 2-Cl, 3-Cl, 4-Cl, 2-Br, 3-Br, 4-Br, 2,4,5-Cl;
Structural optimization of n
R1 = 3-Br-C6H4-, R2 = 2-CF3-4-Cl, n = 2, 3.
Scheme 1. The synthetic route of target compounds.
In our previous study, the carbonyl group was generally reduced to an amine
group by introducing ammonia gas into the ethanol solution of immediate III and
titanium (IV) isopropoxide, then, followed by a series of N-alkylation reactions for
further structural derivation [30-33]. Rather, in this paper, we had optimized the
synthetic route. This improved method reduced the reaction steps, shortened the
reaction time, and easiness of product purification with higher yield.
1
13
The structure of target compounds were characterized by H NMR, C NMR,
MS and elemental analysis. Additionally, the structure of IV-9 was confirmed by the
X-ray single crystal diffraction (CCDC No. 1867015, Figure 4). The spatial structure

of cyclohexane in IV-9 was the chair conformation, the sulfonamide moiety on the
equatorial bond position and the phenylethylamine moiety connected to the
cyclohexane by an axial bond. The first chiral carbon atom (C1) existed as the R
configuration and the second chiral carbon atom (C6) had the S configuration. Due to
the structural similarity of this series of compounds, it was inferred that all target
compounds had the same configuration as IV-9.
Figure 4. X-ray single crystal structure of IV-9.
The results for mycelium inhibition of all target compounds against three strains
of B. cinerea (DL-11, KZ-9, 5055) are shown in Table 1. The results showed that
IV-2, IV-9 and IV-16 had higher fungicidal activities against DL-11 with the EC50
values of 1.96, 1.39 and 2.07 μg mL−1 than procymidone, boscalid, pyrisoxazole and
the lead compound chesulfamide with 14.02, 2.11, 2.08 and 8.27 μg mL−1,
respectively. In addition, 13 compounds had lower EC50 values than 5 μg mL−1
against KZ-9. Based on the results, 13 compounds with excellent fungicidal activities
were tested on 5055, and the result also confirmed the high fungicidal activity.

Table 1. Inhibitory effect of target compounds on mycelium growth of B. cinerea.
Structure
EC50 (95% Confidence Limit) (μg mL−1)
Compd.
n
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
3
1
1
1
1
1
1
1
1
1
1
/
R1
R2
DL-11
KZ-9
5055
IV-1
2-F-C6H4-
3-F-C6H4-
2-Cl-C6H4-
3-Cl-C6H4-
4-Cl-C6H4-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-CF3-4-Cl-
2-F-
8.98(4.04-19.95)
1.96(0.77-5.01)
2.43(1.15-5.14)
3.76(1.85-7.62)
3.60(1.93-6.68)
3.39(2.03-5.66)
3.50(1.88-6.52)
9.01(5.40-15.02)
1.39(0.57-3.40)
2.39(1.15-4.95)
3.44(1.63-7.26)
6.71(4.29-10.51)
3.78(1.61-8.88)
5.07(2.15-11.96)
9.68(4.90-19.15)
2.07(0.81-5.32)
4.68(2.52-8.70)
6.84(3.36-13.92)
7.38(4.32-12.59)
15.26(10.05-23.19)
31.95(10.27-99.36)
39.07(12.25-124.58)
49.00(9.85-243.68)
5.92(3.53-9.92)
17.17(7.99-36.90)
21.45(11.76-39.13)
21.07(11.49-38.64)
17.48(8.86-34.47)
20.40(9.40-44.25)
18.97(8.57-42.01)
15.80(7.93-31.47)
19.02(7.52-48.07)
20.70(8.82-48.60)
18.79(8.24-42.84)
8.27(3.75-18.26)
14.02(6.00-32.74)
2.11(1.07-4.14)
2.08(1.41-3.06)
10.70(3.70-30.92)
2.03(0.82-5.00)
3.48(1.18-10.21)
4.05(2.16-7.59)
3.88(2.47-6.09)
3.99(2.62-6.08)
4.06(2.21-7.45)
3.04(1.25-7.41)
2.92(1.44-5.91)
2.18(1.06-4.51)
5.34(2.82-10.11)
4.08(2.62-6.34)
3.41(0.42-28.07)
3.28(1.53-7.05)
6.69(3.73-11.98)
5.16(0.08-341.76)
5.10(2.74-9.50)
6.60(4.08-10.69)
4.59(3.16-6.67)
19.04(10.59-34.21)
20.71(5.41-79.32)
18.40(4.63-73.09)
36.98(3.02-452.59)
5.85(1.45-23.66)
13.23(2.67-65.58)
20.79(3.07-140.71)
20.54(6.66-63.41)
31.07(4.98-193.92)
15.30(3.18-73.64)
21.08(5.04-88.10)
10.30(4.50-23.59)
21.84(5.26-90.66)
9.84(4.96-19.51)
13.75(5.20-36.35)
8.11(3.97-16.58)
7.39(2.10-26.05)
2.09(1.16-3.75)
1.81(1.20-2.75)
/
IV-2
/
IV-3
3.17(1.82-5.50)
IV-4
／
IV-5
3.94(1.73-9.01)
IV-6
2,5-Cl-C6H3-
3.4-Cl-C6H3-
2-Br-C6H4-
3-Br-C6H4-
4-Br-C6H4-
4-CH3-C6H4-
3,4-CH3-C6H3-
3-CF3-C6H4-
4-CF3-C6H4-
2-OCH3-C6H4-
3-OCH3-C6H4-
4-OCH3-C6H4-
3,4-OCH3-C6H3-
4-OCF3-C6H4-
4-OH-C6H4-
4-pyridin-
2-morpholino-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
3-Br-C6H4-
/
2.90(1.24-6.75)
IV-7
/
IV-8
/
IV-9
1.90(0.67-5.37)
IV-10
IV-11
IV-12
IV-13
IV-14
IV-15
IV-16
IV-17
IV-18
IV-19
IV-20
IV-21
IV-22
IV-23
IV-24
IV-25
IV-26
IV-27
IV-28
IV-29
IV-30
IV-31
IV-32
IV-33
IV-34
chesulfamide
procymidone
boscalid
pyrisoxazole
／
3.27(1.26-8.44)
／
2.89(1.51-5.52)
4.45(2.11-9.37)
/
1.56(0.38-6.45)
／
/
／
/
/
/
/
4.67(2.55-8.55)
/
3-F-
/
4-F-
/
2-Cl-
/
3-Cl-
9.57(4.59-19.94)
/
4-Cl-
2,4,5-Cl-
2-Br-
5.83(3.85-8.82)
/
3-Br-
7.12(4.30-11.79)
9.95(5.45-18.17)
8.34(4.63-15.02)
1.66(1.08-2.54)
1.60(0.49-5.24)
1.13(0.73-1.73)
4-Br-
/
/
/
/
/
/
/
/
/
/
The target compounds exhibited mediocre inhibition effect on spore

germination at the concentrations of 10 and 50 μg mL−1 (Table 2). But with increasing
to 100 μg mL−1 of specific compounds, they completely inhibited spore germination.
Resultantly, the increase in concentration was accompanied by a proportional increase
in the inhibition of spores. In comparison with the positive controls, the target
compounds were not typical respiratory inhibitor like boscalid. While, part of
compounds can almost completely inhibit spore germination under higher
concentration conditions, and have the same or higher inhibition effect to
procymidone and pyrisoxazole. Therefore, it can be preliminarily inferred that the
mode of action of the target compounds should be inhibit the mycelium growth or the
synthesis of certain substances.
Table 2. Inhibition rate of target compounds on spore germination of B. cinerea.
Inhibition rate (%)
50 μg mL-1
52.33
Inhibition rate (%)
50 μg mL-1
27.11
Compd.
Compd.
10 μg mL-1
8.74
100 μg mL-1
10 μg mL-1
11.46
17.85
20.31
20.58
29.88
31.68
18.51
4.08
100 μg mL-1
IV-1
/
IV-20
/
IV-2
2.86
51.06
/
IV-21
37.75
/
IV-3
17.97
10.29
1.87
38.40
/
IV-22
53.94
/
IV-4
70.76
98.25
IV-23
40.76
/
IV-5
49.65
/
IV-24
33.59
96.08
IV-6
0.30
55.49
/
IV-25
59.94
94.50
IV-7
1.68
23.24
/
IV-26
41.52
/
IV-8
5.54
40.17
/
IV-27
46.92
/
IV-9
3.25
52.61
96.67
IV-28
21.30
-8.08
11.02
22.19
34.36
39.99
13.13
11.81
6.61
57.93
93.40
IV-10
IV-11
IV-12
IV-13
IV-14
IV-15
IV-16
IV-17
IV-18
IV-19
2.34
24.84
/
IV-29
5.90
/
1.54
56.86
92.65
IV-30
44.05
/
1.49
29.82
/
IV-31
35.03
/
8.71
49.77
95.21
IV-32
53.36
/
2.18
34.64
/
/
/
/
/
/
IV-33
58.64
94.78
/
-1.27
10.39
31.73
6.07
22.19
IV-34
47.39
56.29
chesulfamide
procymidone
boscalid
pyrisoxazole
39.73
87.66
91.00
97.25
92.97
22.19
42.08
40.28
96.55
28.9
93.82
10.62
36.64
23.66

Hereafter, compounds IV-1 to IV-22 were tested for their in vivo fungicidal
activity on tomato seedlings, the leading compound chesulfamide and the commercial
fungicides were used as the positive control (Table 3). The obtained results from
bioassay study demonstrated that the control efficacy of 4 compounds (IV-5, IV-9,
IV-16 and IV-18) on tomato seedlings reached over 80%. Moreover, IV-9 was the
best compound with the control efficacy of 90.27%, which were superior to all
positive controls.
Table 3. In vivo fungicidal activities of target compounds in pot tests at 200 μg mL−1.
Control efficacy %
(On tomato leaves)
35.63
Control efficacy %
(On tomato leaves)
35.32
Compd.
Compd.
IV-1
IV-20
IV-2
49.77
IV-21
54.24
IV-3
28.05
IV-22
78.35
IV-4
42.68
IV-23
29.81
IV-5
86.32
IV-24
30.52
IV-6
72.39
IV-25
30.18
IV-7
77.74
IV-26
40.70
IV-8
35.40
IV-27
66.44
IV-9
90.27
IV-28
1.46
IV-10
IV-11
IV-12
IV-13
IV-14
IV-15
IV-16
IV-17
IV-18
IV-19
48.84
IV-29
11.14
78.88
IV-30
43.61
67.75
IV-31
51.09
60.53
IV-32
14.01
62.75
IV-33
75.33
66.30
IV-34
60.91
85.64
chesulfamide
procymidone
boscalid
pyrisoxazole
59.98
66.47
52.44
81.38
71.70
74.43
66.73
After the in vitro and in vivo bioassay tests, the optimal structure of R1 was
determined to be 3-Br-C6H4-. In order to improve the fungicidal activity, R2 and the
carbon number of cyclohexane were modified respectively. But it was regrettable that

the change did not lead to an increase in activity in all in vitro tests (Table 1 and
Table 2). The in vivo bioassays of compounds IV-23 to IV-34 were carried out in the
same method described (Table 3). It was obvious that no satisfactory compounds had
been screened out in these trials. This result could be explained by the fact that
introduction of trifluoromethyl group improved the physicochemical properties of the
compounds, such as lipophilicity, metabolic stability, activity, etc [34], which guided
the direction for the further optimization.
Based on the in vitro and in vivo bioassay results, the relationship between
chemical structures and fungicidal activity was determined. As for the R1 moiety, we
summarized the following rules as (1) the activity of the benzene ring is superior to
that of the heterocyclic rings; (2) in general, the activity of the electron-withdrawing
group on the benzene ring is superior to that of the electron-donating group; (3) the
chlorine or bromine atoms on the benzene ring show better activity than that of
fluorine atoms; (4) Comparing the compound IV-3 to IV-5 with the compounds IV-6
and IV-7, it can be seen that there is no significant difference in activity between the
mono- and di-chlorine atom substitutions on the benzene ring. For the R2 moiety, the
trifluromethyl group is the functional group, and the loss of it leads to a significant
decrease in activity. The activities of IV-9 containing 6-membered ring is superior to
that of compounds IV-23 and IV-24 containing 7- and 8-membered ring respectively.
Based on all experiment results, we selected the best performing compound IV-9
for field trials in tomato greenhouses. We conducted three trials at different times and
test pilots in Liaozhong District, Shenyang City, Liaoning Province, China. The

overall control efficacy of IV-9 (80%) was better than that of the positive control
boscalid (66%), and the combination of the two showed the best performance (86%).
Table 4. Control efficacy of compound IV-9 in field.
Control efficacy (%)
Compd.
1st trial
91
2nd trial
74
3rd trial
75
average
80
IV-9
boscalid
69
86
43
66
IV-9:boscalid (1:1)
100
87
72
86
For the purpose of conducting further meaningful research in practical
application and determining the cross-resistance relationship between compound IV-9
and commercial fungicides more accurately, we cultured the resistance of the B.
cinerea strain using the compound IV-9 instead of the conventional method [35]. The
strain B0510, which is sensitive to the compound IV-9 (EC50 = 0.21 μg mL−1, MIC
(Minimum Inhibitory Concentration) = 75 μg mL−1), was selected as the test strain.
Inoculated 5 mm mycelium dishes in the center of fresh molten PDA medium were
amended with low concentration of compound IV-9. When the mycelium growth
reached to a diameter of about 5 cm, the mycelium were punched and transferred to a
medium with a higher concentration of compound IV-9. The strain B0510R (EC50 =
8.52 μg mL−1, MIC = 400 μg mL−1) was obtained by culturing the B0510 for 30
generations under the condition of the concentration of compound IV-9 was gradually
increased. Additionally, the sensitive strain B0510 and the resistant strain B0510R
both had strong pathogenicity on cucumber leaves in the infection test. Compound
IV-9 and eight commercial fungicides (azoxystrobin, boscalid, chlorothalonil,
diethofencarb, fludioxonil, procymidone, pyrimethanil and pyrisoxazole) were tested

for concentration gradient tests on B0510 and B0510R at concentrations of 10, 2.5,
0.625 and 0.15625 μg mL−1 on PDA, respectively. The plates were sealed with
parafilm and incubated under a regular 12:12 h light/dark regimen at 23℃ for 72 h.
The radical growth diameters were measured and EC50 values were calculated. These
fungicides contain different mechanism of action, such as respiration inhibitor, single
sterol biosynthesis inhibitor, methionine biosynthesis inhibitor, acting on signal
transduction or tubulin assembly and multisite inhibitor. As can be seen from Table 5,
the resistance factor of two strains to compound IV-9 was 40.57 (Resistance Factor =
EC50resistant / EC50sensitive). Rather, there was no significant difference between the two
strains in EC50 values of all fungicides.
Table 5. Inhibition effect of commercial fungicides on B. cinerea B0510 and
B0510R.
EC50 (95% Confidence Limit) (μg mL−1)
Compd.
B0510
B0510R
IV-9
0.21(0.05-0.92)
2.70(1.00-7.29)
1.15(0.48-2.75)
0.36 (0.13-1.32)
3.30(0.67-16.32)
0.19(0.12-0.30)
2.54(0.91-7.09)
0.36(0.22-0.62)
0.66(0.37-1.17)
8.52(2.13-30.45)
3.37(1.00-11.30)
1.41(0.52-3.82)
1.12(0.64-1.95)
3.54(1.60-7.86)
0.26(0.14-0.47)
2.47(1.09-5.62)
0.41(0.18-0.91)
0.38(0.18-0.80)
azoxystrobin
boscalid
chlorothalonil
diethofencarb
fludioxonil
procymidone
pyrimethanil
pyrisoxazole
In conclusion, 34 novel compounds were designed and synthesized, and their in
vitro and in vivo fungicidal activities were evaluated against strains of B. cinerea.
IV-9 performed best in this series of compounds, which was similar or superior to all
the positive control procymidone, boscalid and pyrisoxazole in both in vitro and in

vivo bioassays. The control efficacy of IV-9 in field trials was 80%, which showed
higher activity than boscalid (66%). Moreover, there was no positive cross-resistance
relationship between compound IV-9 and the eight commonly used commercial
fungicides, which provided references for the development of sulfonamides and the
creation of new pesticides.
Acknowledgements
This research was supported in part by the National Key Technology Research
and Development Program (No. 2017YFD0200504), the Shenyang Young and
Middle-aged Science and Technology Innovation Talents Support Project
(RC170518), the Basic research project of higher education institutions of Liaoning
Province (No. 01032017004) and the Scientific Research Project of Department of
Education of Liaoning Province (LSNYB201611). Thanks to the Laboratory of
Pesticide Molecule Design and Discovery of China Agricultural University for
supporting us the SYBYL software. We are grateful to Maqsood Ahmed for providing
us English editing.
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Declaration of interests
√☐The authors declare that they have no known competing financial interests
or personal relationships that could have appeared to influence the work
reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

Novel sulfonamides against Botrytis cinerea with no positive cross-resistance to
commercial fungicides: design, synthesis and SAR study
Nan Cai a, Lu He a, Kai Wang a, Zhihui Feng a, Zining Cui b, Mingshan Ji a, Zhiqiu Qi
a, Peiwen Qin a, Xinghai Li a, *
a
Department of Pesticide Science, Plant Protection College, Shenyang Agricultural
University, Shenyang 110866, Liaoning, China
b
State Key Laboratory for Conservation and Utilization of Subtropical
Agro-bioresources, Integrative Microbiology Research Centre, Guangdong Province
Key Laboratory of Microbial Signals and Disease Control, South China Agricultural
University, Guangzhou 510642, Guangdong, China
Cl
O
O O
S
Cl
HN
Br
O O
S
N
H
N
CF3
H
CF3
Optimal target compound IV-9
Lead compound (chesulfamide)
Bioassay
Structure optimization
R1
The performance was better than the positive
controls procymidone, boscalid and pyrisoxazole
in the mycelium inhibition tests, tomato pot tests.
Moreover, the control efficacy of IV-9 in field trials
was 80%, which showed higher activity than
boscalid (66%).
HN
O O
S
R2
N
H
n = 1, 2, 3
Synthesis
n
no positive cross-
Meaningfully, there was
Target compounds
resistance
between IV-9 and azoxystrobin,
boscalid, chlorothalonil, diethofencarb, fludioxonil,
procymidone, pyrimethanil and pyrisoxazole,
respectively.