Synthesis, fungicidal activity and mode of action of 4-phenyl-6-trifluoromethyl-2-aminopyrimidines against Botrytis cinerea

Article abstract of DOI:10.3390/molecules21070828

Anilinopyrimidines are the main chemical agents for management of Botrytis cinerea. However, the drug resistance in fungi against this kind of compounds is very serious. To explore new potential fungicides against B. cinerea, a series of 4-phenyl-6-trifluoromethyl-2-amino-pyrimidine compounds (compounds III-1 to III-22) were synthesized, and their structures were confirmed by ¹H-NMR, IR and MS. Most of these compounds possessed excellent fungicidal activity. The compounds III-3 and III-13 showed higher fungicidal activity than the positive control pyrimethanil on fructose gelatin agar (FGA), and compound III-3 on potato dextrose agar (PDA) indicated high activity compared to the positive control cyprodinil. In vivo greenhouse results indicated that the activity of compounds III-3, III-8, and III-11 was significantly higher than that of the fungicide pyrimethanil. Scanning electron micrography (SEM) and transmission electron micrography (TEM) were applied to illustrate the mechanism of title compounds against B. cinerea. The title compounds, especially those containing a fluorine atom at the ortho-position on the benzene ring, could maintain the antifungal activity against B. cinerea, but their mechanism of action is different from that of cyprodinil. The present study lays a good foundation for us to find more efficient reagents against B. cinerea.

Full text of DOI:10.3390/molecules21070828

molecules
Article
Synthesis, Fungicidal Activity and Mode of Action of
4-Phenyl-6-trifluoromethyl-2-aminopyrimidines
against Botrytis cinerea
Chunhui Liu 1, Zining Cui 2,*, Xiaojing Yan 3, Zhiqiu Qi 1, Mingshan Ji 1 and Xinghai Li 1,
*
1
Department of Pesticide Science, Plant Protection College, Shenyang Agricultural University,
Shenyang 110866, Liaoning, China; 18809855316@163.com (C.L.); syqizhiqiu@sina.com (Z.Q.);
jimingshan@163.com (M.J.)
2
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, China
Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant
Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; yanxiaojing@caas.cn
Correspondence: ziningcui@scau.edu.cn (Z.C.); xinghai30@163.com (X.L.); Tel./Fax: +86-20-8528-8229 (Z.C.);
Tel.: +86-24-8834-2018 (X.L.); Fax: +86-24-8848-7148 (X.L.)
3
*
Academic Editor: Derek J. McPhee
Received: 13 May 2016; Accepted: 21 June 2016; Published: 24 June 2016
Abstract: Anilinopyrimidines are the main chemical agents for management of Botrytis cinerea.
However, the drug resistance in fungi against this kind of compounds is very serious. To explore new
potential fungicides against B. cinerea, a series of 4-phenyl-6-trifluoromethyl-2-amino-pyrimidine
compounds (compounds III-1 to III-22) were synthesized, and their structures were confirmed
by 1H-NMR, IR and MS. Most of these compounds possessed excellent fungicidal activity.
The compounds III-3 and III-13 showed higher fungicidal activity than the positive control
pyrimethanil on fructose gelatin agar (FGA), and compound III-3 on potato dextrose agar (PDA)
indicated high activity compared to the positive control cyprodinil. In vivo greenhouse results
indicated that the activity of compounds III-3, III-8, and III-11 was significantly higher than that
of the fungicide pyrimethanil. Scanning electron micrography (SEM) and transmission electron
micrography (TEM) were applied to illustrate the mechanism of title compounds against B. cinerea.
The title compounds, especially those containing a fluorine atom at the ortho-position on the benzene
ring, could maintain the antifungal activity against B. cinerea, but their mechanism of action is
different from that of cyprodinil. The present study lays a good foundation for us to find more
efficient reagents against B. cinerea.
Keywords: aminopyrimidines; fungicide; structure-activity relationship; mode of action
1. Introduction
Pyrimidine is an important structural motif to enhance the bioactivity in many agrochemicals,
including insecticides, herbicides and fungicides [
pyrimidine motifs, such as pyrimethanil, cyprodinil, and mepanipyrim [
of the main chemical prevention and control reagents against B. cinerea, but they are prone to developing
fungicidal resistance [ ]. Strains of B. cinerea highly resistant to pyrimethanil have been found in
many countries, such as France [ ], America [ ], Australia [ ], Spain [ ], Greece [ ], Israel [ ], and
China [10 11]. Leroux et al. have identified three resistant phenotypes towards anilinopyrimidines (e.g.,
cyprodinil, mepanipyrim and pyrimethanil) since 1999 [ ]. Subsequently, Latorre et al. reported that
cyprodinil resistant isolates of B. cinerea showed cross resistance to the anilinopyrimidines fungicides
1,2
]. There are many kinds of fungicides containing
2,3]. These fungicides are one
3
4
5
6
7
8
9
,
4
Molecules 2016, 21, 828; doi:10.3390/molecules21070828
www.mdpi.com/journal/molecules

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mepanipyrim and pyrimethanil [12]. Moyano et al. collected resistance to pyrimethanil data in
B. cinerea populations on vegetable crops in Spain and found that three of 42 isolates tested in 1992
(7%) and four of 40 isolates tested in 2000 (10%), were resistant to pyrimethanil [7]. Latorre and
Torres reported isolates having simultaneous resistance to anilinopyrimidines, DMIs, phenylpyrroles
and hydroxyanilides in B. cinerea populations from grapevines in Chile [13]. Fungicide resistance is
a serious problem that questions the sustainability of the current gray mold control strategy, which
relies almost exclusively on fungicides with single-site modes of action [13]. Besides, Yin et al. reported
that the resistance factors (RF) of pyrimethanil-resistant isolates from strawberry ranged from 53.0
to 320.1 in Zhejiang Province, China [10]. In addition, the overall resistance frequency of 750 isolates
collected in 2012 for cyprodinil was 27% and the frequency of 1060 isolates collected in 2013 was 17%
from strawberry fields of seven southern U.S. states [5,14]. Liu et al. also found that in all of the isolates
from tomato tested in Henan Province, China, 86% were resistant to pyrimethanil [11]. The data show
the development of resistance to anilinopyrimidines in B. cinerea increases rapidly.
Toshihiro et al. [15] systematically investigated the structure-activity relationship (SAR) of
aminopyrimidines and found that the introduction of aniline to the 2-position, methyl to the 4-position,
propargyl, methyl, cyclopropane and other electron-donating groups to the 6-position, respectively, could
lead to higher fungicidal activities. 6-Trifluoromethyl pyrimidinamine derivatives were synthesized, and
the results showed that they had no fungicidal activity against B. cinerea. Trifluoromethyl is an active
group widely used in drug molecule design [16]. Some 4-trifluoromethyl pyrimidines possessed
pharmaceutical bioactivities [17], for example, 2-(2,6-dihalophenyl)-3-pyrimidinyl-1,3-thiazolidin-4-
one analogues (Figure 1–1) [18] and (4-(4-(methylsulfonyl) phenyl)-6-(trifluoromethyl)-2-pyrimidinyl)
amines (Figure 1–2) [19] belong to the 2-amino-4-phenyl-6-trifluoromethyl pyrimidine class of
compounds, and can act as anti-HIV-1 agents and potent and selective cyclooxygenase-2 inhibitors,
respectively. There have been no reports however on the agricultural fungicidal activity of 4-substituted
phenyl-6-trifluoromethyl-2-aminopyrimidines.
CF3
CF3
Cl
N
N
Cl
H2N
N
N
N
S
SO2Me
O
2
1
Figure 1. 2-amino-4-phenyl-6-trifluoromethyl pyrimidine class of compounds.
In this paper 4-substituted phenyl-6-trifluoromethyl-2-aminopyrimidines were synthesized to
explore new potential fungicides against B. cinerea. Studies on the synthetic methodology for preparing
4-phenyl-6-trifluoromethyl-2-aminopyrimidines have been reported. Reports on substituent changes
on the benzene ring are scarse, for example, the only modifications on the benzene ring performed
to-date involved substitution of the hydrogen atom at the 4-position with methyl [20], fluorine [21,22],
methoxy and chlorine [23]. Furthermore, there are no reports on fungicidal activity. The structure and
location of the substituent groups on the title compounds underwent various changes, which have
a great influence on fungicidal activity of compounds. Compared to pyrimidinamine fungicides, the
title compounds possess trifluoromethyl and substituted phenyl groups at position 6 and position 4 of
the pyrimidine ring, respectively. The 2-anilino substitutent was transformed into an amino group on
the pyrimidine ring (Figure 2). These compounds were then evaluated for their antifungal activities
and further study on the mode of action.

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anilinopyrimidines
title compounds
,
,
Figure 2. Design strategy for the title compounds.
2. Results and Discussion
2.1. Synthesis of 4-Phenyl-6-trifluoromethyl-2-aminopyrimidines
The syntheses of the title compounds are simple and their yields are good (63%–90%). The synthetic
route of the title compounds is shown in Scheme 1.
CF3
O
O
O
N
a
b
CF3
H2N
N
CH3
R2
R2
R2
Ⅰ
Ⅱ
Ⅲ
Scheme 1. Synthetic route of title compounds. Reagents and Conditions: (a) CF3COOC2H5/CH3ONa,
reflux, 2 h; (b) guanidine hydrochloride/CH3ONa, reflux, 6 h;
We found the title compounds containing trifluoromethyl group at 6-position, such as
compound III-3, had very high fungicidal activity. From the structural perspective (Figure 3), the
major diference between anilinopyrimidine fungicides and the title compounds relies on their core
framework, which is based on pyrimidinamine and on phenylpyrimidine motifs, respectively.
No activity
Compound III-3 (High activity)
Figure 3. Compared to core framework of anilinopyrimidines and compound III-3.
As shown in Table 1, the structure-activity relationships can be summarized in three points.
First, for the single substituted benzene structure, whether it is for electron-donating or electron-
withdrawing groups, the activity of ortho-position is better than that of the para-position. Second, the
activity of compounds containing halogen is generally higher than that of compounds containing
the methyl group and the methoxy group. Third, as far as halogen is concerned, the activity of an
ortho-position substituent is higher than that of a para-position one. Especially for fluorine at the
ortho-position, compounds showed excellent activity. For example, on FGA, compared with the EC50
and EC80 values of the positive control pyrimethanil, the relative toxicity of compound III-3 was

Molecules 2016, 21, 828
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30, 15, respectively, but on PDA, the relative toxicities of compound III-3 were 13, 46, respectively.
In addition, for the benzene ring containing multiple substituents, the activity of ortho-position and
fluorine atom-containing compounds is still relatively high. The related literature reports that fluorine
can be used in medicinal chemistry to modulate the physicochemical properties, such as lipophilicity or
basicity. In addition, a fluorine substituent can lead to a change in the preferred molecular conformation
and improve the metabolic stability. Due to the major successes of fluorinated compounds in medicinal
chemistry, it may be predicted that the number of fluorine containing drugs on the market will continue
to increase [24–26]. The experimental results proved that the role of the fluorine is consistent with
previous reports. Fluorine will also play a continuing role in agricultural chemicals [27].
Table 1. The fungicidal activity of compounds III-1–III-22 against B. cinerea.
FGA
EC50/(µg/mL)
PDA
EC80/(µg/mL)
R2
Compound
EC80/(µg/mL)
EC50/(µg/mL)
>103
125.5
3.9
III-1
III-2
III-3
H
2-CH3
2-F
48.1
5.3
0.8
>100
18.1
5.1
223.5
20.3
0.6
III-4
III-5
III-6
III-7
III-8
III-9
2-Cl
2-Br
2-CF3
2-OCH3
2-OH
4-OCH3
4-CH3
4-CH2CH3
2,4-(CH3)2
4-F
12.1
26.1
49.0
27.2
42.3
>100
18.0
2.6
3.1
1.3
55.5
4.6
0.3
/
3.3
>100
17.2
1.1
0.3
32.9
24.0
36.9
48.8
133.1
99.0
100.1
>100
72.1
14.8
14.6
6.3
>100
38.8
19.6
/
>100
>100
>100
50.4
18.5
177.8
76.2
3.3
35.4
17.8
95.1
9.4
71.5
>103
88.8
>103
>103
>103
>103
>103
>103
>103
>103
>103
555.3
>103
277.8
441.4
>103
>103
175.8
180.1
403.9
348.4
846.9
202.2
16.3
135.6
497.1
155.6
>103
46.5
69.4
3.4
42.9
808.7
120.5
17.8
7.5
III-10
III-11
III-12
III-13
III-14
III-15
III-16
III-17
III-18
III-19
III-20
III-21
III-22
cyprodinil
pyrimethanil
4-Cl
4-Br
4-CF3
3,4-Cl2
3-F, 4-OCH3
3,4-F2, 5-OCH3
3-CF3, 4-OCH3
3,5-(CF3)2
3, 4-F2
2.2. Fungicidal Activity and Mode of Action of 4-Phenyl-6-trifluoromethyl-2-aminopyrimidines
In this paper, twenty-two compounds were subjected to in vitro and in vivo fungicidal activity
screening using the mycelium inoculation method (Tables 1 and 2). Highly active compound III-3 was
selected, which fungicidal activity was significantly higher than that of pyrimethanil and cyprodinil.
Finally, the mode of action of compound III-3 was studied. With respect to the mode of action of
pyrimethanil, cyprodinil and compound III-3, they had both similarities and differences. They were
similar in both protective and therapeutic effects. Although pyrimethanil and cyprodinil both belong
to the pyrimidinamine fungicides, their modes of action are inhibition of methionine biosynthesis [28
]
and the bioactivity of cytoderm hydrolytic enzymes, respectively [29]; they also had significantly
different fungicidal activities in different culture media. Pyrimethanil only exhibits higher fungicidal
activity in fructose gelatin agar (FGA) culture medium, but cyprodinil can have higher activity in
potato dextrose agar (PDA) culture media. In a screening against five pyrimethanil-resistant strains of
B. cinerea, cyprodinil showed higher activity, but compound III-3 showed the highest activity. Further
studies were performed to understand the mode of action of compound III-3 and whether cyprodinil
and compound III-3 have different mechanisms of action. The effects of compound III-3 treatment on

Molecules 2016, 21, 828
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mycelial shape and the ultrastructure of B. cinerea have both similarities and differences. Scanning
electron microscopy (SEM) analysis displayed that the mycelia after compound III-3 and cyprodinil
treatment appeared branched to different degrees. The growth points of mycelia appeared deformed
to different degrees, but the growth point deformation after compound III-3 treatment was more
serious than that caused by cyprodinil treatment. Cyprodinil treatment caused serious swelling of
mycelia at 3.125 µg/mL, suggesting their mechanisms of action may be different. Transmission electron
microscopy (TEM) analysis indicated that the ultrastructure of B. cinerea changed significantly with
cyprodinil and compound III-3 treatment. Nevertheless, the effect of compound III-3 on mitochondria
is more significant than that of cyprodinil. These results also suggested that the mode of action of
compound III-3 may be different from that of cyprodinil, however, further biochemical evidence, such
as their cystathionine lyase and synthase activities, need further research to prove their mechanisms of
action are different.
Table 2. Control efficiency of compounds III-1–III-22 against B. cinerea at 750 µg/mL (mycelium
inoculation method).
Compound
R2
Control Efficiency on Leaves of Cucumber (%) (˘SEM)
III-1
III-2
III-3
III-4
III-5
III-6
III-7
III-8
III-9
H
2-CH3
2-F
2-Cl
2-Br
2-CF3
2-OCH3
2-OH
4-OCH3
4-CH3
4-CH2CH3
2,4-(CH3)2
4-F
n
n
98 ˘ 2 a
73 ˘ 6 c
79 ˘ 5 bc
59 ˘ 8 c
71 ˘ 7 c
95 ˘ 3 ab
52 ˘ 18 bcde
69 ˘ 8 ab
79 ˘ 4 ab
44 ˘ 19 de
68 ˘ 6 ab
n
III-10
III-11
III-12
III-13
III-14
III-15
III-16
III-17
III-18
III-19
III-20
III-21
III-22
cyprodinil
pyrimethanil
4-Cl
4-Br
4-CF3
20 ˘ 28 e
45 ˘ 18 de
n
3,4-Cl2
3-F, 4-OCH3
3,4-F2, 5-OCH3
3-CF3, 4-OCH3
3,5-(CF3)2
3,4-F2
24 ˘ 25 e
n
25 ˘ 9 e
n
52 ˘ 14 bcde
64 ˘ 9 ab
47 ˘ 10 cde
The letters a–e denoted the results of difference significance analysis. Means followed by the same letter within
the same column are not significantly different (p > 0.05, Fisher1s LSD multiple comparison test). n: no activity.
As shown in Table 1, most of the compounds showed high in vitro fungicidal activity on FGA
culture medium. Based on EC50 and EC80 values, compound III-3 had high activity, whose EC50 value
was less than 1.0 µg/mL. Compound III-3 exhibited higher activity than pyrimethanil and cyprodinil.
The fungicidal activity of all the compounds was generally low on PDA medium, but the trend was
consistent with that on FGA.
As shown in Table 2, twenty-two compounds showed fungicidal activity in vivo against B. cinerea
at 750 µg/mL. Among them, the activities of compounds III-3, III-8, III-10, III-11 and III-13 were
higher than that of the positive control pyrimethanil, and were equivalent to that of the fungicide
cyprodinil. In general, the in vitro and in vivo activities showed good consistency.
As shown in Table 3, according to the EC50 or EC80 values and relative toxicity values,
compound III-3 strongly inhibited the mycelia growth compared to pyrimethanil and cyprodinil.

Molecules 2016, 21, 828
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The activity of compound III-3 was the highest against all the pyrimethanil-resistant strains.
In addition, some irregularities were caused in colonial growth of B. cinerea in the presence of
compound III-3.
Table 3. The fungicidal activity of compound III-3 against resistent strains of B. cinerea (FGA).
Resistent Strains
of B. cinerea
Relative
Toxicity (EC50
Relative
Toxicity (EC80)
Compound
EC50 (µg/mL)
EC80 (µg/mL)
)
III-3
cyprodinil
pyrimethanil
1.0
9.7
36.1
35.8
3.7
1.0
32.2
60.9
92.2
2.9
1.5
1.0
CY-12
HLD-18
HLD-15
FS-09
III-3
cyprodinil
pyrimethanil
3.8
25.4
44.4
11.7
1.7
1.0
20.6
56.8
145.4
7.1
2.6
1.0
III-3
cyprodinil
pyrimethanil
1.42
34.05
16.11
11.3
0.5
1
10.96
87.29
132.64
12.1
1.52
1
III-3
cyprodinil
pyrimethanil
1.19
7.59
9.67
8.1
1.3
1
22.29
49.79
1146.44
51.4
23.0
1
III-3
cyprodinil
pyrimethanil
2.74
8.06
23.37
8.5
2.9
1
23.72
54.9
190.77
8.0
3.5
1
DL-11
As shown in Figure 4, compound III-3 had inhibitory effect on spore production of B. cinerea.
The inhibition rate reached more than 90%, which was equivalent to pyrimethanil at 80 g/mL.
With decreased concentrations (20 g/mL and 5 g/mL), the sporulation inhibition rate of compound III-3
µ
µ
µ
against B. cinerea decreased, while the inhibitory effect was better than that of pyrimethanil.
100
80
60
III-3
40
20
0
pyrimethanil
0
20
40
60
80
100
Concentration (μg /mL)
Figure 4. Effect of compound III-3 on the spore production of B. cinerea (FGA).
As shown in Figure 5, compound III-3 had a stronger inhibitory effect on the germination of
B. cinerea at high concentration, but the overall activity was lower than that of pyrimethanil. At the
concentration of 100 µg /mL, the spore germination inhibition rate of compound III-3 was 91.80%,
which was at the same level as the commercial fungicide pyrimethanil. With decreasing concentration,
the spore germination inhibition rate of compound III-3 decreased significantly.

Molecules 2016, 21, 828
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120
100
80
60
40
20
0
III-3
pyrimethanil
0
20
40
60
80
100
120
Concentration (μg /mL)
Figure 5. Effect of compound III-3 on the spore germination of B cinerea.
As shown in Figure 6, compound III-3 completely inhibited the sclerotia of B. cinerea. However, as
the concentration decreased, the sclerotia-producing ability of cyprodinil increased. At the concentration
of 5 µg /mL, the inhibition rate of cyprodinil is negative, which means it has no inhibition. The result
showed that inhibitory effect of compound III-3 on sclerotia production was better than that
of cyprodinil.
Ⅲ-3
100 cyprodinil
140
100
100
100
100
83
120
100
80
58
60
40
20
0
40
20
10
5
-20
-40
-60
-13
Concentration of compounds (μg/mL)
Figure 6. Effect of compound III-3 on the sclerotia production of B cinerea.
As shown in Figure 7a,b, the results indicate that compound III-3 had good prevention and
treatment effect against B. cinerea in cucumber and tomato. Overall, its preventive effect was better
than the treatment effect. The activity of compound III-3 was higher than that of pyrimethanil.
Control efficiency of compound III-3 against B. cinerea on leaves of cucumber was shown in
Figure 7a. The plants treated by pyrimethanil displayed obvious water soaked lesions, and the lesions
were not only more numnerous but also larger, even with a perforated phenomenon and leaf curl and
wilting phenomena were also seen. In addition, the plants treated by compound III-3 presented edge
onset on plant leaves, inconsistent lesion sizes and low incidence in stems and did not appear to show
plant wilting and lodging.
Figure 7b shows the control efficiency of compound III-3 against B. cinerea in tomato. Compound III-3
had excellent prevention and treatment effects, and the activity was higher than that of pyrimethanil
at 100 µg/mL.

Molecules 2016, 21, 828
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Protective
action
Curative
action
Protective
action
b
a
77.2
77.0
69.1
85.8
90
80
70
60
50
40
30
20
10
0
100
64.4
56.9
Curative
action
90
80
70
60
50
40
30
20
10
0
14.6
10.8
Compound Name
Compound Name
Figure 7. Control efficiency of compound III-3 against B. cinerea (
a) Control efficiency of compound
III-3 against B. cinerea on leaves of cucumber (spore inoculation method
)
; (b) control efficiency of
compound III-3 against B. cinerea on leaves of tomato (spore inoculation method).
2.3. Scanning Electron Microscopic Analysis
The results are shown in Figure 8. The mycelia of blank control were plump and had good
extensibility (Figure 8 CK). Nevertheless, the mycelia after compound III-3 and cyprodinil treatment
for 3 days at 0.78
(Figure 8 cy-1). The mycelia with compound III-3 treatment appear branched (Figure 8 III-3-1).
As shown in Figure 8 III-3-2 and III-3-3, after compound III-3 treatment at 3.125 g/mL mycelia show
µg/mL are different. The growth point of mycelia display a slight deformity
µ
serious deformity at the growth points. With the increase of concentration, the number of damaged
growth points obviously increased. Unlike compound III-3 treatment, cyprodinil treatment caused
serious swelling of mycelia at 3.125 µg/mL (Figure 8 cy-2), and produced branches at 12.5 µg/mL
(Figure 8 cy-3). The results show that the effects of different concentrations on the hyphae are different.
The effects of compound III-3 treatment on the mycelial morphology of B. cinerea are manifested in the
destruction of the growth points and the increase in the amount of hyphal branching. However, the
effects of cyprodinil treatment are manifested in swelling of mycelia and hyphal branching, but not
much. The preliminary results thus indicate that they differ in their mode of action.
CK
III-3-1
cy-1
Figure 8. Cont.

Molecules 2016, 21, 828
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III-3-3
III-3-2
cy-3
cy-2
Figure 8. Effect of compound III-3 treatment on mycelial morphology of B. cinerea. CK: The blank
control; III-3-1: Compound III-3 treatment for 3 days at 0.78 g/mL; III-3-2: Compound III-3 treatment
for 3 days at 3.125 g/mL; III-3-3: Compound III-3 treatment for 3 days at 12.5 g/mL; cy-1: Cyprodinil
treatment for 3 days at 0.78 g/mL; cy-2: Cyprodinil treatment for 3 days at 3.125 g/mL; cy-3:
µ
µ
µ
µ
µ
Cyprodinil treatment for 3 days at 12.5 µg/mL.
2.4. Transmission Electron Microscopy Analysis
The results are shown in Figure 9. The cell walls of hyphae are regular and smooth without
chemical treatment (Figure 9 CK-1, CK-2). Cell nuclear, mitochondria and other organelles are evenly
distributed in the cytoplasm (Figure 9 CK-1, CK-2). As shown in Figure 9 CK-2, the cell membrane
is clearly visible and intact. When treated with compound III-3 for 5 days, the cell membrane
disappears at 40
µg/mL (Figure 9 III-3-1) and mitochondrial matrix appears swollen at 20
µg/mL
(Figure 9 III-3-3). After compound III-3 treatment for 5 days at 40
µg/mL, the mycelium shows
vacuoles (Figure 9 III-3-2). In addition, the cytoplasm is not uniform (Figure 9 III-3-4). It is found that
compound III-3 affects the ultrastructure of B. cinerea, and causes abnormal alterations such as uneven
distribution of the cytoplasm, mitochondrial matrix swelling, membrane structural damage and the
formation of vacuoles.
As seen from Figure 9 cy-1, cy-2, the positive control cyprodinil treatment at 40 µg/mL causes serious
cavitation. The above results show that effects of compound III-3 and cyprodinil on the ultrastructure
of B. cinerea are mostly the same. The difference is that compound III-3 causes mitochondrial matrix
swelling, but cyprodinil does not. It is necessary to further study their different action sites.
CK-2
CK-1
Cell nucleus
Cell wall
III-3-2
III-3-1
cavitation
septum
Figure 9. Cont.

Molecules 2016, 21, 828
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III-3-3
III-3-4
mitochondrion
cytoplasm
1 4
cy-1
cy-2
cavitation
cytoplasm
Figure 9. Effect of compound III-3 treatment on the ultrastructure of B. cinerea. CK-1: The blank
control after 5 days; CK-2: The blank control after 5 days; III-3-1: Compound III-3 treatment for 5 days
at 40 µg/mL
;
III-3-2: Compound III-3 treatment for 5 days at 40
µg/mL; III-3-3: Compound III-3
treatment for 5 days at 20
µg/mL; III-3-4: Compound III-3 treatment for 5 days at 20
µg/mL; cy-1:
Cyprodinil treatment for 5 days at 20 µg/mL; cy-2: Cyprodinil treatment for 5 days at 40 µg/mL.
3. Materials and Methods
3.1. Instruments
Infrared (IR) spectra were recorded in potassium bromide disks on a Spectrum 65 spectrophotometer
(Perkin Elmer, MA, USA). Nuclear magnetic resonance (NMR) spectra were recorded in CDCl3 unless
indicated otherwise with Bruker 300 MHz or 600 MHz spectrometers (Bruker, NASDAQ, USA), using
tetramethylsilane (TMS) as an internal standard. GC-MS: 6890-5973N GC-MS (Agilent, Palo Alto,
CA, USA), chromatographic column HP-5MS 5% phenyl methyl siloxane 30 m
ˆ
250
µ
m
ˆ
0.25 µm,
carrier gas: He; flow rate: 1 mL/min; column temperature, temperature programmed: the initial
temperature of 60 ˝C rose to˝200 ˝C at rate of 20 ˝C/min, and then rose to 24˝0 ˝C at rate of 5 ˝C/min
˝
˝
˝
˝
(60 C–20 C/min-200 C–5 C/min-240 C); injection port temperature: 230 C; Shunt model sample:
shunt ratio 40:1; assist line temperature: 285 ˝C. Ion source: 230 ˝C; quadrupole: 150 ˝C; ionization
mode: EI; acquisition methods: scanning; scan quality range: 35–520 mAU. The solvents and reagents
were used as received or were dried prior to use as needed.
3.2. Synthetic Procedures
3.2.1. General Synthetic Procedure for 4,4,4-Trifluoro-1-phenylbutane-1,3-dione Compounds II
Referring to Scheme 1, to the appropriate acetophenone derivative (0.05 mol) and ethyl
trifluoroacetate (0.075 mol) in methanol (20 mL), sodium methoxide solution (0.1 mol of Na + 15 mL of
CH3OH) was added dropwise at room temperature, and the mixture was refluxed for 2 h. After the
methanol was evaporated under vacuum, the residue was dissolved in ethyl acetate (50 mL), washed
with 5% HCl (25 mL) and water (25 mL), and dried over sodium sulfate. After the solvent was
evaporated under vacuum, the corresponding compound II was obtained.
3.2.2. General Procedure for the Preparation of Title Compounds III
Compounds III were synthesized according to the method given in [16]. To the solution of sodium
methoxide (0.09 mol of Na + 10 mL of CH3OH), guanidine carbonate (0.08 mol) was added, and the
mixture was refluxed for 30 min. The appropriate compound II (0.01 mol) was added, then reacted for
˝
6 h. Acetic acid was added dropwise to the solution till pH = 4–5 at 0–5 C. The reaction solution was
filtered, and the filter cake was washed with water (15 mL 2). The crude product was recrystallized
ˆ

Molecules 2016, 21, 828
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from methanol to give pure compound III-1 to III-22. The nuclear magnetic resonance (NMR), infrared
(IR), and mass spectrum (MS) data were as follows:
˝
˝
4-Phenyl-6-trifluoromethyl-2-aminopyrimidine (III-1): White crystals, yield 89%, m.p. 130–131 C (130–132 C)
1
(Rawal et al. [18], 2007). H-NMR (CDCl3, 300 MHz),
δ
(ppm): 5.47 (br, 2H, NH2), 7.35 (s, 1H, py-H),
7.47–8.06 (m, 5H, C6H5). IR (KBr), ν (cm´1): 3323, 3208, 1640, 1600. MS (EI), m/z: 239 (M+, 100%).
4-(2-Methylphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-2): White crystals, yield 76%, m.p. 135–136 ˝C
1H-NMR (CDCl3, 300 MHz),
δ (ppm): 2.53 (s, 3H, CH3), 5.45 (br, 2H, NH2), 6.29 (s, 1H, py-H), 7.25–7.56
(m, 4H, C6H4). IR (KBr), ν (cm´1): 3310, 3163, 1677, 1633. MS (EI), m/z: 253 (M+, 82%).
4-(2-Fluorophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-3): White crystals, yield 83%, m.p. 116–117 ˝C,
1H-NMR (CDCl3, 600 MHz),
δ
(ppm): 5.45 (br, 2H, NH2), 7.18–8.10 (m, 5H, C6H4 + py-H); IR (KBr),
ν
(cm´1): 3324, 3210, 1634, 1585. MS (EI), m/z: 257 (M+, 100%).
4-(2-Chlorophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-4): White crystals, yield 79%, m.p. 118–120 ˝C,
1H-NMR (CDCl3, 600 MHz),
(ppm): 5.57 (s, 2H, NH2), 7.27–7.74 (m, 5H, C6H4 + py-H); IR (KBr),
(cm´1): 3326, 3210, 1635, 1584. MS (EI), m/z: 272.9 (M+, 100%).
δ
ν
4-(2-Bromophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-5): White crystals, yield 77%, m.p. 138–140 ˝C,
1H-NMR (CDCl3, 600 MHz),
(ppm): 5.14 (s, 2H, NH2), 7.25–7.71 (m, 5H, C6H4 + py-H); IR (KBr),
(cm´1): 3325, 3212, 1634, 1555. MS (EI), m/z: 316.9 (M+, 100%).
δ
ν
4-(2-Trifl˝uoromethylphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-6): White crystals, yield 76%, m.p.
100–101 C, 1H-NMR (CDCl3, 600 MHz),
δ (ppm): 5.40 (s, 2H, NH2), 7.25–7.89 (m, 5H, C6H4 + py-H);
IR (KBr), ν (cm´1): 3505, 3331, 1633, 1472. MS (EI), m/z: 306.9 (M+, 100%).
4-(2-Methoxyphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-7): Light yellow crystals, yield 75%, m.p.
95–97 ˝C, 1H-NMR (CDCl3, 600 MHz),
δ (ppm): 5.43–5.45 (d, 2H, NH2), 7.02–7.89 (m, 5H, C6H4 +
py-H), 3.91 (s, 3H, OCH3); IR (KBr), ν (cm´1): 3334, 3202, 1638, 1584. MS (EI), m/z: 268.9 (M+, 100%).
4-(2-Hydroxyphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-8): Light yellow solid, yield 80%, m.p.
180–185 ˝C, 1H-NMR (CDCl3, 600 MHz),
δ
(ppm): 5.29(s, 2H, NH2), 6.96–7.81(m, 5H, C6H4 + py-H),
13.05 (s,1H, OH); IR (KBr), ν (cm´1): 3318, 3189, 1646, 1591. MS (EI), m/z: 255 (M+, 100%).
˝
4-(4-Methoxyphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-9): White crystals, yield 82%, m.p. 193–194 C
(192–193 C) (Bonacorso et al. [23], 2008). H-NMR (CDCl3, 300 MHz),
˝
1
δ (ppm): 3.88 (s, 3H, CH3), 5.40
(br, 2H, NH2), 7.00 (d, 2H, J = 8.81 Hz, C6H2), 7.28 (s, 1H, py-H), 8.03 (d, 2H, J = 8.81 Hz, C6H2). IR
(KBr), ν (cm´1): 3333, 3219, 1632, 1596. MS (EI), m/z: 269 (M+, 100%).
˝
4-(4-Methylphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-10): White crystals, yield 87%, m.p. 180–181 C.
1H-NMR (CDCl3, 300 MHz),
(ppm): 2.43 (S, 3H, CH3), 5.37 (br, 2H, NH2), 7.30 (d, 2H, J = 8.10 Hz,
δ
C6H2), 7.32 (s, 1H, py-H), 7.94 (d, 2H, J = 8.10 Hz, C6H2). IR (KBr),
ν
(cm´1): 3313, 3178, 1640, 1596.
MS (EI), m/z: 253 (M+, 100%).
˝
4-(4-Ethylphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-11): Yellow crystals, yield 90%, m.p. 152–153 C.
1H-NMR (CDCl3, 300 MHz),
(ppm): 1.27 (t, 3H, J = 7.50 Hz, CH3), 2.72 (t, 2H, J = 7.50 Hz, CH2), 5.38
δ
(br, 2H, NH2), 7.32 (s, 1H, py-H), 7.33 (d, 2H, J = 8.40 Hz, C6H2), 7.97(d, 2H, J = 8.40 Hz, C6H2). IR
(KBr), ν (cm´1): 3323, 3203, 1640, 1596. MS (EI), m/z: 267 (M+, 100%).
4-(2,4-Dimethylphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-12): White crystals, yield 72%, m.p.
˝
1
128–129 C. H-NMR (CDCl3, 600 MHz),
δ (ppm): 2.32 (s, 3H, CH3), 2.36 (s, 3H, CH3), 7.04 (s,
1H, py-H), 7.11–7.39 (m, 3H, C6H3), 7.32 (br, 2H, NH2). IR (KBr),
(EI), m/z: 267 (M+, 77%).
ν
(cm´1): 3323, 3208, 1631, 1590. MS
˝
4-(4-Fluorophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-13): White crystals, yield 77%, m.p. 168–169 C
1
(158 ˝C) (Sareen et al. [22] 1993). H-NMR (CDCl3, 300 MHz),
δ
(ppm): 5.46 (br, 2H, NH2), 7.15–7.22

Molecules 2016, 21, 828
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(m, 2H, C6H2), 7.29 (s, 1H, py-H), 8.03–8.09 (m, 2H, C6H2). IR (KBr),
MS (EI), m/z: 257 (M+, 100%).
ν
(cm´1): 3323, 3188, 1650, 1591.
˝
4-(4-Chlorophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-14): White crystals, yield 87%, m.p. 202–203 C
1
(181–183 ˝C) (Bonacorso et al. [23], 2008). H-NMR (CDCl3, 300 MHz),
δ
(ppm): 7.40 (br, 2H, NH2),
7.56 (s, 1H, py-H), 7.61 (d, 2H, J = 8.40 Hz, C6H2), 8.21 (d, 2H, J = 8.40 Hz, C6H2). IR (KBr),
3318, 3186, 1642, 1596. MS (EI), m/z: 273 (M+, 100%).
ν
(cm´1):
˝
4-(4-Bromophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-15): Yellow crystals, yield 63%, m.p. 222–223 C.
1H-NMR (CDCl3, 300 MHz),
(ppm): 3.33 (s, 3H, CH3), 7.41 (br, 2H, NH2), 7.52 (s, 1H, py-H), 7.75 (d,
δ
2H, J = 8.40 Hz, C6H2), 8.13 (d, 2H, J = 8.40Hz, C6H2); IR (KBr),
(EI), m/z: 319 (M+, 100%).
ν
(cm´1): 3318, 3203, 1641, 1596. MS
4-(4-Trifl˝uoromethylphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-16): White crystals, yield 86%, m.p.
181–182 C. 1H-NMR (CDCl3, 300 MHz),
= 8.40 Hz, C6H2), 8.38 (d, 2H, J = 8.40 Hz, C6H2); IR (KBr),
m/z: 307 (M+, 100%).
δ (ppm): 7.50 (br, 2H, NH2), 7.64 (s, 1H, py-H), 7.91 (d, 2H, J
ν
(cm´1): 3323, 3213, 1646, 1586. MS (EI),
4-(3,4-Di˝chlorophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-17): White crystals, yield 69%, m.p.
1
197–198 C. H-NMR (CDCl3, 300 MHz),
δ (ppm): 7.47 (br, 2H, NH2), 7.67 (s, 1H, py-H), 7.81 (d,
1H, J = 8.40 Hz, C6H), 8.19 (dd, 1H, J = 8.40, 2.10 Hz, C6H), 8.46 (d, 1H, J = 2.10Hz, C6H). IR (KBr),
(cm´1): 3323, 3208, 1646, 1586. MS (EI), m/z: 307 (M+, 100%).
ν
4-(3-Fluoro-4-methoxyphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-18): White crystals, yield 64%, m.p.
˝
175–176 C. 1H-NMR (CDCl3, 300 MHz),
δ (ppm): 3.96 (s, 3H, OCH3), 5.42 (br, 2H, NH2), 7.04–7.88 (m,
4H, C6H3 + py-H). IR (KBr), ν (cm´1): 3333, 3213, 1631, 1596. MS (EI), m/z: 287 (M+, 100%).
4-(3,4-Difluoro-5-methoxyphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-19): White crystals, yield 73%,
˝
m.p. 198–199 C. 1H-NMR (CDCl3, 300 MHz),
δ
(ppm): 4.10 (s, 3H, OCH3), 5.45 (br, 2H, NH2), 7.22 (s,
1H, py-H), 7.64 (d, 2H, J = 9.90 Hz, C6H2); IR (KBr),
ν
(cm´1): 3328, 3213, 1641, 1591. MS (EI), m/z: 305
(M+, 100%).
4-(3-Trifluoromethyl-4-methoxyphenyl)-6-trifluoromethyl-2-aminopyrimidine (III-20): White crystals, yield 78%,
m.p. 168–169 ˝C. 1H-NMR (CDCl3, 300 MHz),
δ (ppm): 3.99 (s, 3H, OCH3), 7.38 (s, 2H, NH2), 7.42–8.51
(m, 4H, C6H3 + py-H). IR (KBr), ν (cm´1): 3318, 3213, 1641, 1596. MS (EI), m/z: 337 (M+, 100%).
4-(3,5-Bis(trifluoromethyl)phenyl)-6-trifluoromethyl-2-aminopyrimidine (III-21): White crystals, yield 83%,
m.p. 142–143 ˝C. 1H-NMR (CDCl3, 300 MHz),
δ
(ppm): 5.49 (br, 2H, NH2), 7.38 (s, 1H, py-H), 8.03 (s,
1H, C6H1), 8.50 (s, 2H, C6H2); IR (KBr),
ν
(cm´1): 3326, 3217, 1644, 1591. MS (EI), m/z: 375 (M+, 100%).
4-(3,4-Difluorophenyl)-6-trifluoromethyl-2-aminopyrimidine (III-22): White crystals, yield 65%, m.p.
138–140 ˝C. 1H-NMR (CDCl3, 600 MHz),
(ppm): 5.41 (br, 2H, NH2), 7.27 (s, 1H, py-H), 7.26–7.31
δ
(m, 1H, C6H1), 7.78–7.80 (m, 1H, C6H1), 7.93–7.97 (m, 1H, C6H1). IR (KBr),
1591. MS (EI), m/z: 275 (M+, 100%).
ν
(cm´1): 3323, 3209, 1645,
3.3. Bioassay of Fungicidal Activity
Fungicidal activity of the title compounds was evaluated using the method given in references [30–32].
3.3.1. In Vitro Fungicidal Activity of Compounds III-1–III-22 against B. cinerea
(The Mycelium Growth Test)
The B. cinerea strain was isolated from damaged tomato parts from a greenhouse in Shenyang,
Liaoning Province, China, in April 2013, and cultured on potato dextrose agar (PDA) for many
generations. The culture media were fructose gelatin agar (FGA) [33] and potato dextrose agar (PDA).
The final concentrations of compounds were 50, 12.5, 3.13, 0.78
µg/mL on FGA, and 100, 25, 6.25,
1.56, 0.39 g/mL on PDA. The commercial reference fungicides were pyrimethanil and cyprodinil.
µ

Molecules 2016, 21, 828
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The EC50 and EC80 values of compounds III-1 to III-22 were estimated using logit analysis, and the
results given in Table 1.
3.3.2. In Vivo Fungicidal Activity of Compounds III-1–III-22 against B. cinerea
(Mycelium Inoculation Method)
In vivo, the concentration of compounds was 750 µg/mL. The results of compounds III-1 to III-22
against B. cinerea by pot culture test method (mycelium inoculation method) [32,34] in greenhouse
were shown in Table 2.
3.4. Mode of Action
High activity compound III-3 was screen out by the above method and chosen for mode of
action studies.
3.4.1. Toxicity Test of Compound III-3 Against Five Pyrimethanil-resistant Strains of B. cinerea
CY-12, HLD-18, HLD-15, FS-09 and DL-11were five pyrimethanil-resistant strains of B. cinerea,
which were respectively isolated from damaged tomato parts from a greenhouse in Chaoyang,
Huludao, Fushun and Dalian, Liaoning Province, China, in April 2014. The effect of compound III-3
against pyrimethanil-resistant strains of B. cinerea was assessed on FGA, and the results are shown in
Table 3.
3.4.2. Spore Yield of B. cinerea Test Method
The concentration gradient was 80, 20, 5 µg/mL. The plugs of B. cinerea were inoculated on PDA,
and they were cultured for ten days at 23 ˝C. After a large number of spores were produced in the
blank control, the conidial suspensions were prepared by seeding conidia in a 0.05% Tween 80 solution
with 2 mL sterile water. Spores were determined directly on the hemocytometer. Spore production
was calculated according to Equation (1). The spore yield inhibition rate was calculated according to
Equation (2):
Q “ Q0 ˆ 106{mL
(1)
G1
I “ p1 ´
q ˆ 100%
(2)
G0
where Q is spore production quantity in per milliliter conidial suspension, Q0 is the average number
of spores each small lattice. I is the spore yield inhibition rate, G0 is the average spore production in
the blank test, G1 is the average spore production in the presence of compounds.
3.4.3. The Spore Germination Test Method
The effect of compound III-3 on spore germination against B. cinerea was determined in concave
slides by liquid drop method. The method was given in reference [32]. Concentration gradient was
100, 50, 25, 12.5, 6.25 µg/mL. The commercial fungicide pyrimethanil was used as the positive control,
and the results were given in Figure 5.
3.4.4. Effect of Compound III-3 on the Sclerotia Production of B. cinerea
Concentration gradient was 40, 20, 10, 5 µg/mL. The commercial fungicide cyprodinil was used
as the positive control, and the results are given in Figure 6.
3.4.5. In Vivo Protective and Therapeutic Effects of Compound III-3 Against B. cinerea
(Spore Inoculation method)
The spore inoculation method was given in reference [29]. Preventive and therapeutic effects of
the compound III-3 were tested against B. cinerea in cucumber (200 µg/mL) and tomato (100 µg/mL).
The results are shown in Figure 7.

Molecules 2016, 21, 828
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3.4.6. Preparation of Scanning Electron Microscopy Samples
The method was given in reference [35
,
36]. Spore suspensions (1 mL, 5
ˆ
105 spores/mL) were
inoculated for PD cultivation on medium which contained compound III-3. Final concentrations
were 0.78, 3.125, 12.5 g/mL. The commercial fungicide cyprodinil was used as positive control.
µ
The samples were shook culture at 23 ˝C, 140 r/min. Then the mycelia were randomly selected after
72 h. The samples were fixed in 3%–4% glutaraldehyde and washed with phosphate buffer solution
(PBS pH 6.8) 4–6 times for each time at intervals of 20–30 min to remove the debris. Then the samples
were dehydrated with acetone series (30%, 50%, 70%, 80%, 90%, 95% and 100%) at intervals of 30 min,
which were dehydrated 100% acetone three times. Finally, the samples were treated by CO2 critical
point drying, sticky sample, and sputter coated with a thin layer of gold. The ultrastructure of samples
was observed using a BCPCAS4800 scanning electronic microscope (Hitachi, Tokyo, Japan).
3.4.7. Preparation of Transmission Electron Microscopy Samples
Culture and pretreatment of samples were the same as the method of Section 3.4.6. Final
concentrations of compound III-3 and cyprodinil were 20, 40 µg/mL. The mycelia were randomly
selected after 120 h. The uttrastructure of samples was observed using a Hitachi transmission electron
microscope (Hitachi, Tokyo, Japan).
4. Conclusions
In summary, based on our previous structure-activity relationship studies, a series of
4-phenyl-6-trifluoromethyl-2-aminopyrimidines were designed and synthesized. The title compounds
exhibited high in vitro and in vivo fungicidal activity against B. cinerea, not only against the sensitive
strain but also against resistant strains. Compound III-3 had strong inhibition effect on the
mycelium growth, spore production, spore germination and sclerotia production of B. cinerea, which
indicated excellent prevention and treatment effects on gray mold. The preliminary structure-activity
relationships demonstrated that the substituents at the ortho-position of phenyl ring favored the
bioactivity, especially a fluorine atom. The effects of compound III-3 on the mycelial morphology
and ultrastructure of B. cinerea were different from that of cyprodinil. Further research is necessary to
understand its site of action.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
7/828/s1.
Acknowledgments: This work was supported by the National Natural Science Foundation of China (31101466
and 31570122), the National Key Project for Basic Research (973 Project, 2015CB150600), the Key Laboratory of
Pesticide Chemistry and Application, Ministry of Agriculture (MOAPCA201009), the Natural Science Foundation
of Liaoning Province (2015020766), the Specialty and Innovation Project from the Department of Education in
Guangdong Province (No. 2014KTSCX031), the Pearl River S & T Nova Program of Guangzhou (201506010029),
the Special Fund for the Research and Construction of Public Service Ability in Guangdong (2014A020210019).
Author Contributions: X.L. conceived and designed the experiments; C.L. performed the experiments; Z.C. and
C.L. analyzed the data; .X.Y., Z.Q. and M.J. contributed reagents/materials/analysis tools; C.L. and Z.C. wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
Sandmann, G. Bleaching activities of substituted pyrimidines and structure-activity comparison to related
heterocyclic derivatives. Pestic. Biochem. Phys. 2001, 70, 86–91. [CrossRef]
Leroux, P. Chemical control of Botrytis and its resistance to chemical fungicides. Springer Neth. 2007, 195–222.
[CrossRef]
Rosslenbroich, H.J.; Stuebler, D. Botrytis cinerea—History of chemical control and novel fungicides for its
management. Crop Prot. 2000, 19, 557–561. [CrossRef]

Molecules 2016, 21, 828
15 of 16
4.
5.
6.
Pierre, L.; Florence, C.; Denis, D.; Michel, G. Patterns of cross-resistance to fungicides in Botryotinia
Fuckeliana (Botrytis cinerea) isolates from french vineyards. Crop Prot. 1999, 18, 687–697.
Fernandez-Ortuno, D.; Grabke, A.; Bryson, P.K.; Amiri, A.; Peres, N.A.; Schnabel, G. Fungicide resistance
profiles in Botrytis cinerea from strawberry fields of seven southern U.S. states. Plant Dis. 2014, 98, 825–833.
Sergeeva, V.; Nair, N.G.; Verdanega, J.R.; Shen, C.; Barchia, I.; Spooner-Hart, R. First report of
anilinopyrimidine-resistant phenotypes in Botrytis cinerea on grapevines in Australia. Australas. Plant Path.
2002, 31, 299–300. [CrossRef]
7.
8.
Moyano, C.; Gómez, V.; Melgarejo, P. Resistance to pyrimethanil and other fungicides in Botrytis cinerea
populations collected on vegetable crops in Spain. J. Phytopathol. 2004, 152, 484–490. [CrossRef]
Petsikos-Panayotarou, N.; Markellou, E.; Kalamarakis, A.E.; Kyriakopoulou, D.; Malathrakis, N.E. In vitro
and in vivo activity of cyprodinil and pyrimethanil on Botrytis cinerea isolates resistant to other botryticides
and selection for resistance to pyrimethanil in a greenhouse population in Greece. Eur. J. Plant Pathol. 2003
109, 173–182. [CrossRef]
,
9.
Korolev, N.; Mamiev, M.; Zahavi, T.; Elad, Y. Screening of Botrytis cinerea isolates from vineyards in Israel for
resistance to fungicides. Eur. J. Plant Pathol. 2011, 129, 591–608. [CrossRef]
10. Yin, D.; Chen, X.; Hamada, MS.; Yu, M.; Yin, Y.; Ma, Z. Multiple resistance to QoIs and other classes of
fungicides in Botrytis cinerea populations from strawberry in Zhejiang Province, China. Eur. J. Plant Pathol.
2015, 141, 169–177. [CrossRef]
11. Liu, S.-M.; Che, Z.-P.; Chen, G.-Q. Multiple-fungicide resistance to carbendazim, diethofencarb, procymidone,
and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan Province, Chin. Crop Prot. 2016
84, 56–61. [CrossRef]
,
12. Latorre, B.A.; Spadaro, I.; Rioja, M.E. Occurrence of resistant strains of Botrytis cinerea toanilinopyrimidine
fungicides in table grapes in Chile. Crop Prot. 2002, 21, 957–961. [CrossRef]
13. Bernardo, A.N.L.; René, T. Prevalence of isolates of Botrytis cinerea resistant to multiple fungicides in Chilean
vineyards. Crop Prot. 2012, 40, 49–52.
14. Li, X.-P.; Fernandez-Ortuno, D.; Chen, S.-N.; Grabke, A.; Luo, C.-X.; Bridges, W.-C.; Schnabel, G.
Location-specific fungicide resistance profiles and evidence for stepwise accumulation of resistance in
Botrytis cinerea. Plant Dis. 2014, 98, 1066–1074. [CrossRef]
15. Toshihiro, N.; Katsumi, M.; Shinichiro, M.; Ichiro, M. Synthesis and structure-activity study of fungicidal
anilinopyrimidines leading to mepanipyrim (KIF-3535) as an anti-Botrytis agent. Pest Manag. Sci. 2003, 60,
399–407.
16. Nie, J.; Guo, H.C.; Cahard, D.; Ma, J.A. Asymmetric construction of stereogenic carbon centers featuring
a trifluoromethyl group from prochiral trifluoromethylated substrates. Chem. Rev. 2011, 111, 455–529.
[CrossRef] [PubMed]
17. Saijo, R.; Watanabe, G.; Kurihara, K.; Kawase, M. Fluorinated
β-diketo phosphorus ylides: Their
cyclocondensation with amidines affording 4-trifluoromethyl- and 4-perfluoroalkyl-substituted pyrimidines.
Heterocycles 2014, 89, 2334–2345. [CrossRef]
18. Rawal, R.K.; Tripathi, R.; Katti, S.B. Pannecouque C, De Clercq E, Synthesis and evaluation of 2-(2,6-
dihalophenyl)-3-pyrimidinyl-1, 3-thiazolidin-4-one analogues as anti-HIV-1 agents. Bioorg. Med. Chem. 2007
15, 3134–3142. [CrossRef] [PubMed]
,
19. Swarbrick, M.E.; Beswick, P.J.; Gleave, R.J.; Green, R.H.; Bingham, S.; Bountra, C.; Carter, M.C.; Chambers, L.J.;
Chessell, I.P.; Clayton, N.M.; et al. Identification of (4-(4-(methylsulfonyl) phenyl)-6-(trifluoromethyl)-
2-pyrimidinyl) amines and ethers as potent and selective cyclooxygenase-2 inhibitors. Bioorg. Med.
Chem. Lett. 2009, 19, 4504–4508. [CrossRef] [PubMed]
20. Nishiwaki, T. Studies on heterocyclic chemistry. IV. Preparation of several trifluoromethyl-substituted
heterocyclic compounds and observation of metastable ion in the mass spectre of trifluoromethyl-pyrazoles.
Bull. Chem. Soc. Jpn. 1969, 42, 3024–3026. [CrossRef]
21. Guan, H.-P.; Tang, X.-Q.; Luo, B.-H.; Hu, C.-M. Convenient synthesis of fluoroalkyl-substituted heterocycles
from 1-fluoroalkyl-2-iodoalkenes. Synthesis 1997, 12, 1489–1494. [CrossRef]
22. Sareen, V.; Jain, S. Studies in fluorinated 1,3-diketones and related compounds. Part XVII. Synthesis and
spectroscopic studies of some new 5-alkyl-3-fluoroaryl-1-pentafluoroaryl-pyrazoles and 2-amino-6-alkyl
(perfluoroalkyl)-4-aryl pyrimidines. J. Fluorine Chem. 1993, 63, 265–269. [CrossRef]

Molecules 2016, 21, 828
16 of 16
23. Bonacorso, H.G.; Ferla, A.; Cechinel, C.A.; Zanatta, N.; Martins, M.A. One-pot synthesis of N2-aminoprotected
6-substituted and cycloalka[d] 4-trifluoromethyl-2-acetylaminopyrimidines. J. Heterocyclic Chem. 2008, 45,
483–487. [CrossRef]
24. Hagmann, W.K. The many roles for fluorine in medicinal chemistry. J. Med. Chem. 2008, 51, 4359–4369.
[CrossRef] [PubMed]
25. Böhm, H.J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.; Stahl, M. Fluorine in medicinal chemistry.
ChemBioChem 2004, 5, 637–643. [CrossRef] [PubMed]
26. Purser, S.; Moore, P.R.; Swallow, S.; Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 2008, 37,
320–330. [CrossRef] [PubMed]
27. Jeschke, P. The unique role of fluorine in the design of active ingredients for modern crop protection.
ChemBioChem 2004, 5, 570–589. [CrossRef] [PubMed]
28. Fritz, R.; Lanen, C.; Colas, V.; Leroux, P. Inhibition of methionine biosynthesis in Botrytis cinerea by the
anilinopyrimidine fungicide pyrimethanil. Pestic. Sci. 1997, 49, 40–46. [CrossRef]
29. Milling, R.J.; Richardson, C.J. Mode of action of the anilinopyrimidines fungicidepyrimethanil. 2. Effects on
enzyme secretion in Botrytis cinerea. Pestic. Sci. 1995, 45, 43–48. [CrossRef]
30. Li, X.-H.; Cui, Z.-N.; Chen, X.-Y.; Wu, D.-C.; Qi, Z.-Q.; Ji, M.-S. Synthesis of 2-acyloxycyclohexylsulfonamides
and evaluation on their fungicidal activity. Int. J. Mol. Sci. 2013, 14, 22544–22557. [CrossRef] [PubMed]
31. Li, X.-H.; Pan, Q.; Cui, Z.-N.; Ji, M.-S.; Qi, Z.-Q. Synthesis and fungicidal activity of N-(2,4,5-trichlorophenyl)-
2-oxo- and 2-hydroxycycloalkylsulfonamides. Lett. Drug Des. Discov. 2013, 10, 353–359. [CrossRef]
32. Li, X.-H.; Ji, M.-S.; Qi, Z.-Q.; Li, X.-W.; Shen, Y.-X.; Gu, Z.-M.; Zhang, Y.; Wei, S.-H.; Wang, Y.-Z.;
Wang, D.-Q. Synthesis of 2-amino-6-oxocyclohexenylsulfonamides and them activity against Botrytis cinerea.
Pest Manag. Sci. 2011, 67, 986–992. [CrossRef] [PubMed]
33. Chapeland, F.; Fritz, R.; Lanen, C.; Gredt, M.; Leroux, P. Inheritance and mechanisms of resistance to
anilinopyrimidine fungicides in Botrytis cinerea (Botryotinia fukeliana). Pestic. Biochem. Physiol. 1999, 64,
85–100. [CrossRef]
34. Potted plant test for fungicide control Botrytis cinerea Pers. In Ministry of Agriculture of the People’s Republic of
China; Agriculture Press: Beijing, China, 2008.
35. Kang, Z.-S. Ultrastructure of Plant Pathogenic Fungi; China Science and Technology Press: Beijing, China, 1996;
pp. 7–11.
36. Cui, Z.-N.; Li, Y.-S.; Hu, D.-K.; Tian, H.; Jiang, J.-Z.; Wang, Y.; Yan, X.-J. Synthesis and fungicidal activity of
novel 2,5-disubstituted-1,3,4-thiadiazole derivatives containing 5-phenyl-2-furan. Sci. Rep. 2016, 6, 20204.
[CrossRef] [PubMed]
Sample Availability: Samples of the compounds are available from the authors.
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