Investigation of novel pesticides with insecticidal and antifungal activities: Design, synthesis and SAR studies of benzoylpyrimidinylurea derivatives

Article abstract of DOI:10.3390/molecules23092203

In order to find pesticides with insecticidal and antifungal activities, a series of novel benzoyl pyrimidinylurea derivatives were designed and synthesized. All target compounds were identified by ¹H-NMR spectroscopy and HRMS. Insecticidal and antifungal activity of these compounds were evaluated and the structure-activity relationships (SAR) were clearly and comprehensively illustrated. Compound 7, with low toxicity to zebrafish (LC₅₀ = 378.387 μg mL^?1) showed 100% inhibition against mosquito (Culex pipiens pallens) at 0.25 μg mL^?1. Both compounds 19 and 25 exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against 13 phytopathogenic fungi), which were better than those of the commercial pesticide pyrimethanil (>50% inhibitory activities against eight phytopathogenic fungi). Furthermore, compounds 19 and 25 exhibited protective activity against Sclerotinia sclerotiorum on leaves of Brassica oleracea L. during in vivo experiments.

Full text of DOI:10.3390/molecules23092203

molecules
Article
Investigation of Novel Pesticides with Insecticidal
and Antifungal Activities: Design, Synthesis and
SAR Studies of Benzoylpyrimidinylurea Derivatives
Peiqi Chen 1,2,3, Xiangmin Song 1,2, Yongmei Fan 1,2, Weihao Kong 1,2, Hao Zhang 3
and Ranfeng Sun 1,2,
*
ID
1
Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, Hainan University,
Haikou 570228, China; chenpq17@lzu.edu.cn (P.C.); 18898958572@163.com (X.S.);
yongmeifan@126.com (Y.F.); 15799032481@163.com (W.K.)
Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
School of Chemical & Chemical Engineering, Inner Mongolia University, Hohhot 010021, China;
haozhang@imu.edu.cn
2
3
*
Correspondence: srf18@hainu.edu.cn; Tel.: +86-898-6619-2906
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 9 August 2018; Accepted: 28 August 2018; Published: 31 August 2018
Abstract: In order to find pesticides with insecticidal and antifungal activities, a series of novel
benzoyl pyrimidinylurea derivatives were designed and synthesized. All target compounds
were identified by 1H-NMR spectroscopy and HRMS. Insecticidal and antifungal activity of
these compounds were evaluated and the structure-activity relationships (SAR) were clearly and
comprehensively illustrated. Compound
showed 100% inhibition against mosquito (Culex pipiens pallens) at 0.25
7
, with low toxicity to zebrafish (LC50 = 378.387
µ )
g mL−1
µ
g mL−1. Both compounds
19 and 25 exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against 13
phytopathogenic fungi), which were better than those of the commercial pesticide pyrimethanil
(>50% inhibitory activities against eight phytopathogenic fungi). Furthermore, compounds 19 and
25 exhibited protective activity against Sclerotinia sclerotiorum on leaves of Brassica oleracea L. during
in vivo experiments.
Keywords: benzoylpyrimidinylurea derivatives; antifungal activity; insecticidal activity; embryo
toxicity; zebrafish
1. Introduction
Plant disease and pest control are at least as old as agriculture because there is a need to keep
crops free of pests and phythopatogenic fungi. Benzoylphenylureas (BPUs) have been developed
as chitin synthesis inhibitors because of their unique mode of action coupled with a high degree of
activity on targeted pests and low toxicity to non-target organisms [1,2]. The first commercial product
Dimilin (diflubenzuron) (Figure 1), introduced in the market at 1975, exhibits broad spectrum larvicidal
activities against caterpillars (Pieris brassicae), mosquitoes (Aedes aegypti), houseflies (Musca domestica),
desert locust (Schistocera gregaria) and cotton strainers (Dysdercus superstitiosus) [
years, we and our co-workers have designed and synthesized several series of benzoylphenylureas
and , Figure 1) and most compounds exhibited excellent insecticidal activities against oriental
armyworm (Mythimna separata) or mosquito (Culex pipiens pallens) [ ]. Interestingly, compounds
and (Figure 1) with a 2,2,2-trifluoroethyl group (R = CH2CF3) substituent, displayed much better
3]. In the past few
(A
B
4
–8
C
D
insecticidal activities against oriental armyworm than other derivatives.
Molecules 2018, 23, 2203; doi:10.3390/molecules23092203
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2203
2 of 25
Figure 1. Chemical structures of benzoylurea compounds.
At the same time, a class of phenylurea compounds (E, Figure 2) containing a pyrimidine ring
attracted our attention. When bearing two 2,2,2-trifluoroethyl groups on the pyrimidine ring most of
these compounds have good insecticidal activity performance against second-instar corn earworm
(Helicoverpa zea) and beet armyworm (Spodoptera exigua) at 50 µg mL−1
.
Figure 2. Chemical structures of compounds with pyrimidine groups.

Molecules 2018, 23, 2203
3 of 25
More importantly, the 2-aminopyrimidinyl unit is commonly found in the chemical structure
of antifungal compounds. For example, the antifungal activity of commercial pyrimethanil
(Figure 2) against Alternaria kikuchiana Tanaka was 100% at 0.1
EC50 = 1.03 µg mL−1) exhibited better antifungal activity against Phytophthora capsici than
dimethomorph (EC50 = 4.26 (Figure 2) with excellent antifungal
g mL−1) (Figure 2) [13]. Compound
activity (EC50 = 0.84 µg mL−1) against Phytophthora capsici was discovered in 2011 by Nam et al. [14].
Based on our previous works [ ] and the literature [ 14], in order to find new pesticides with
µ [9–12]. Compound F
g mL−1
(
µ
G
4–8
9–
powerful insecticidal and antifungal properties, we were interested in introducing pyrimidine group
into the benzoylurea backbone to design a series of benzoylpyrimidinylurea derivatives (Figure 3).
Therefor target compounds 1–38 were synthesized and their insecticidal and antifungal activities were
evaluated. At the same time, the aquatic toxicity of compounds
embryo acute toxicity test (OECD TG 236).
7
and 25 were evaluated by a zebrafish
F
O
O
OCH2CF3
N
N
N N
H
H
N
N
OCH2CF3
F
NH
O
N
R
F
O
F
O
O
S
OCH2CF3
N
N
N
OCH2CF3
N
N
H
H
H
H
excellent insecticidal activities
excellent antifungal activities
excellent insecticidal activities
R1
R3
R2
O
X
N
N
N
N
R
H
H
B
C
A
X = O, S, NH
Figure 3. Design strategies of the target benzoylpyrimidinylurea derivatives.
2. Results
2.1. Synthesis
In order to comprehensively analyze the structure-activity relationship of compounds,
the chemical structures of the benzoylpyrimidinylureas was separated into three units, namely,
the benzoyl ring (
2-aminopyrimidines Ia
target compounds (Schemes 1 and 2).
A
), pyrimidine ring (
B
) and urea bridge (
C
) (Figure 3).
Substituted
–
p
were synthesized and used as starting materials for the synthesis of the

Molecules 2018, 23, 2203
4 of 25
Scheme 1. Synthetic route for substituted 2-aminopyrimidines Ia–c, Id–f and Ig–m.

Molecules 2018, 23, 2203
5 of 25
Scheme 2. Synthetic route for substituted 2-aminopyrimidines In, Io and Ip.
In a first series of compounds, intermediate Ia was retained and the benzoyl moiety was altered
resulting in compounds 1 to 10 (Scheme 3).
Scheme 3. Synthetic route for compounds 1–10.

Molecules 2018, 23, 2203
6 of 25
Secondly, different substituents were changed on the pyrimidine ring and the compounds 11–30
with substituted 2-aminopyrimidines were obtained (Scheme 4).
Scheme 4. Synthetic route for compounds 11–30.
Thirdly, we replaced the oxygen atom on the urea bridge with sulfur or nitrogen to gain
compounds 31 and 32 (Scheme 5).

Molecules 2018, 23, 2203
7 of 25
Scheme 5. Synthetic route for compounds 31 and 32.
Finally, the benzoyl ring was exchanged by a six- or five-membered nitrogen heterocycles to
compare the effects between benzene ring and heterocycles on the bioactivity of the compounds,
therefor compounds 33–37 were synthesized (Scheme 6). To illustrate the role of the carbonyl
group of the benzoyl group in the biological activity of the target compounds, compound 38 was
synthesized from 2,6-dichloroaniline (Scheme 6). All reactions gave excellent yields and the NMR data
of compounds proved the proposed structures. Among them, compounds with trifluoromethyl groups
have a characteristic quartet peak with 13C-19F couplings in the NMR spectra. The spectra are shown
in Figures S1–S101 in the Supporting Information.

Molecules 2018, 23, 2203
8 of 25
Scheme 6. Synthetic route for compounds 33–38.
2.2. Bioassays
2.2.1. Toxicity against Mosquito (Culex pipiens pallens)
Table 1 shows the larvicidal activities of the compounds
1–
25
,
31
,
37 and fipronil against
mosquito. The bioassay results indicated that eighteen compounds (1–7, 9–10, 13–14, 16, 18, 20,
22 24 31) exhibited more than 50% larvicidal activities against mosquito and eleven of them reached
–
,

Molecules 2018, 23, 2203
9 of 25
100% at 10
By comparing the chemical structure of compounds
larvicidal activitives decreased (100%, 60% mortality at 2
µ
g mL−1, especially, compound
7
which showed 100% mortality even at 0.25
(2-Cl), (2-Br) and (2-I), we found that the
g mL−1, 50% mortality at 10 g mL−1
µ .
g mL−1
2
3
4
µ
µ
respectively) as the radius of the halogen atom at the 2-position on the benzoyl ring increased.
Next, we found that when the 2,6-position on the benzoyl ring was substituted by two halogens
or electron-rich groups, it was not conducive to larvicidal activity, for example, the larva mortalities of
compounds
and 20% at 5 µg mL−1 respectively.
8 (2-OCH3), 9 (2,4-di-Cl) and 10 (2,6-di-Cl) were 20% at 10 µ µ
g mL−1, 40% at 2 g mL−1
Table 1. Larvicidal Activities against Oriental Armyworm and Mosquito of Compounds 1–25, 31, 37.
Larvicidal Activity (%)
−1
−1
)
Compd.
Oriental Armyworm (µg mL
)
Mosquito (µg mL
600
200
100
10
5
2
1
0.5
0.25
0.1
a
1
2
3
4
5
6
7
8
9
45
5
-
-
-
60
-
-
-
-
-
40
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
100
100
100
50
100
100
100
-
60
100
60
-
-
20
100
-
40
-
-
-
70
60
-
-
-
-
-
-
-
-
-
-
-
-
-
-
20
-
-
-
-
100
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
20
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
100
-
-
-
100
-
-
-
100
70
65
15
65
10
100
40
20
5
100
-
-
-
-
50
-
100
100
20
100
100
10
100
100
-
100
20
-
-
100
100
-
0
-
-
-
-
-
-
100
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
100
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
100
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
31
37
Fipronil
20
60
50
30
5
100
100
20
100
5
70
10
80
5
65
60
65
30
100
15
100
30
35
30
10
20
30
15
20
40
100
20
-
-
-
-
-
-
-
-
-
-
-
-
-
20
-
-
100
-
100
100
-
100
50
-
100
-
-
100
100
100
a—no test data.
However, electron-withdrawing groups in the para position enhanced the bioactivity of the
compounds, for example, the larva mortality of compound (4-CF3) reached 100% at 0.25 .
g mL−1
7
µ
When the 4,6-position on the pyrimidine ring were substituted with haloalkoxyl, alkoxy or alkyl groups,
the larvicidal activity decreased successively, at the same time the longer the alkoxy chain, the larvicidal
activity was better. For example, the mortalities of compounds 11 (4,6-di-CH3), 12 (4,6-di-OCH3), 13
(4,6-di-OEt) and
2
(4,6-di-OCH2CF3) were 10% at 10
µ
g mL−1, 20% at 10
µ
g mL−1, 70% at 2 µg mL−1
and 14
,
100% at 0.5
µ
g mL−1, respectively. Similarly, the structure-activity relationships of compounds
2
told us that a trifluoroethoxy group was more helpful to increase the mortality activities than s halogen
group. Furthormore it was easy to find that the urea bridge unit was important to the larvicidal activity.
For example compound 31 containing a thiourea unit (50% mortality at 1 µg mL−1) exhibited lower
larvicidal activity than compound 2 (100% mortality at 0.5 µ
g mL−1). Finally, when a pyridine ring

Molecules 2018, 23, 2203
10 of 25
(compound 37) was employed instead of a benzene ring (compound
2
), the larvicidal activities were
reduced greatly (15% mortality of compound 37 at 10
0.5
g mL−1). Therefore, it was concluded that electron-withdrawing groups (such as a trifuloromethyl
µ 2 at
g mL−1 and 100% mortality of compound
µ
group) on the para position of the benzoyl ring, alkoxy groups (especially haloalkoxyl groups, such as
a trifluoroethoxy group) and a urea bridge unit favored the larvicidal activities.
2.2.2. Stomach Toxicity against Oriental Armyworm (Mythimna separata)
The bioactivity results indicated that most compounds exhibited certain insecticidal activities
against oriental armyworm as listed in Table 1. Firstly, we can easily see from compounds
2 (2-Cl) and
5
(4-Cl) or (2-CF3) and
6
7
(4-CF3) and their larva moralities (5%, 65%, 15% and 65% at 600 µg mL−1
respectively) that para-substituents of the benzoyl ring result in better insecticidal activities than
ortho-substituents. Secondly, it was figured out that both the radius and electronegativity of the
halogen atom at the 2-position on the benzoyl ring influenced the larvicidal activities, for example,
compound
compound
than compound
3
4
(2-Br, 100% mortality at 200
(2-I, 70% mortality at 600
g mL−1) which exhibited much better larvicidal activity
(2-Cl, 5% mortality at 600
g mL−1). At the same time, it also pointed out that
µ
g mL−1) was a more effective insecticidal agent than
µ
2
µ
chlorine atom on the pyrimidine ring was beneficial to improve the insecticidal activity of compounds,
for example, the larva mortalities of compounds 14 (4,6-Cl), 15 (4-Cl, 6-CH3) and 16 (4-Cl, 6-OCH3)
were 50%, 30%, 5% at 600
on the urea bridge with a sulfur atom, the insecticidal activities of the compounds increased greatly,
µ
g mL−1 respectively. Interestingly, when we replaced the oxygen atom
as seen by comparing the insecticidal activities of compound 2 (5% mortality of at 600 µ
g mL−1) and
compound 31 (100% mortality at 600 µg mL−1).
2.2.3. In Vitro Antifungal Activity
The fungicidal results are listed in Table 2. Most of the derivatives showed antifungal activity
against 14 kinds of phytopathogenic fungi. We first determined the antifungal activities of compounds
(2-Cl), (2-Br), (2-I), (4-Cl), (2-CF3), (4-CF3) and (2-OCH3, 5-Cl) and we found that ortho-halogen
substitutents on the benzoyl ring were beneficial for antifungal activity. Among them, compound
2
3
4
5
6
7
8
2
was better than the others. At this moment, our design strategy was divided into two parts. One was
to maintain 2-chloro group on the benzoyl ring and a series of differently substituted compounds
(such as compounds 11–22) at the 4,6-position on the pyrimidine ring were synthesized. The other
was that we continued to explore the effect of the number of halogen atoms on the benzoyl ring for
the antifungal activity, followed by the synthesis of compounds with different substitutions at the
4,6-position on pyrimidine ring, such as compounds 1, 9, 10 and 23–30.
From the first part of strategy, we reached three important conclusions. Firstly, compounds with
alkoxy groups had better antifungal activity than those bearing an alkyl group, and if the hydrogen
atom on the alkoxy group was substituted with a halogen atom, the antifungal activity of compounds
would be further enhanced. For example, the antifungal activity order of compounds
13 was compound (4,6-di-OCH2CF3) > compound 13 (4,6-di-OEt) > compound 12 (4,6-di-OCH3) >
compound 11 (4,6-di-CH3) (Table 2). Secondly, chlorine atoms were beneficial to increase the antifungal
activity of the compounds. For example, the antifungal activity order of compounds 14 15 and 16 was
compound 14 (4,6-Cl) > compound 15 (4-Cl, 6-CH3) > compound 16 (4-Cl, 6-OCH3) (Table 2).
2, 11, 12 and
2
,

Molecules 2018, 23, 2203
11 of 25
−1
Table 2. Fungicidal Activity of Compounds 1–38 against Phytopathogenic Fungi at 50 µg mL
.
Inhibition Rate (%)
Compd.
A.S a
F.G
P.I
P.C
S.S
B.C
R.S
F.C
C.H
P.P
R.C
B.M W.A F.M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
16.7
16.7
55.6
44.4
33.3
16.7
12.5
16.7
33.3
55.6
44.4
44.4
38.9
44.4
33.3
27.8
38.9
33.3
66.7
55.6
33.3
22.2
27.8
11.1
55.6
44.4
22.2
16.7
11.1
31.3
8.3
3.4
5.9
17.2
41.9
36.8
31.6
15.8
38.7
25.8
32.3
31.6
31.6
10.5
26.3
26.3
31.6
68.4
26.3
26.3
15.8
73.7
26.3
27.8
19.4
26.3
26.3
52.6
61.1
19.4
27.8
27.8
27.3
24.0
22.2
40.9
4.5
10.7
62.5
53.5
41.9
10.5
44.6
10.7
14.3
53.5
58.1
11.6
18.6
16.3
20.9
16.3
14.0
10.5
11.6
83.7
14.0
19.6
53.6
10.5
11.6
90.7
82.1
26.8
53.6
17.9
33.3
5.4
7.7
6.2
35.8
38.5
30.8
11.5
35.8
12.3
6.2
32.7
32.7
17.3
9.6
19.2
7.7
11.5
11.5
21.2
9.6
11.6
41.9
53.6
50.0
21.4
34.9
16.3
14.0
39.3
57.1
21.4
25.0
25.0
14.3
10.7
21.4
17.9
14.3
60.7
39.3
37.1
37.1
7.1
25.0
60.7
48.6
34.3
14.3
31.4
16.7
8.3
17.1
36.7
10.0
6.7
16.7
46.7
47.8
47.8
21.7
43.3
30.0
16.7
47.8
52.2
17.4
30.4
30.4
43.5
17.4
17.4
30.4
30.4
60.9
47.8
40.0
30.0
26.1
30.4
69.6
60.0
35.0
5.0
26.8
91.1
62.8
81.4
41.9
57.1
44.6
26.8
76.7
58.1
51.2
46.5
97.7
60.5
51.2
55.8
32.6
51.2
88.0
76.7
44.9
51.0
67.4
67.4
84.2
51.0
65.3
6.1
11.6
89.5
94.9
93.2
69.5
83.7
23.3
17.4
88.1
94.9
67.8
62.7
69.5
71.2
67.8
67.8
47.5
62.7
93.2
84.7
87.3
87.3
59.3
71.2
94.9
92.7
89.1
58.2
72.7
66.2
27.3
56.4
78.4
20.3
37.8
78.4
89.1
98.2
95.7
16.3
55.1
63.3
76.7
16.7
51.0
16.3
14.3
70.0
66.7
20.0
33.3
40.0
30.0
20.0
20.0
20.0
20.0
70.0
66.7
50.0
44.4
3.3
13.3
66.7
55.6
47.2
22.2
27.8
28.9
29.6
19.4
44.7
31.6
15.8
68.4
61.1
63.9
22.2
12.5
47.5
55.6
63.0
22.2
45.0
22.5
27.5
37.0
51.9
25.9
33.3
48.1
22.2
25.9
33.3
33.3
33.3
66.7
59.3
44.4
29.6
37.0
14.8
59.3
48.1
40.7
14.8
14.8
21.4
7.4
27.3
57.6
50.0
45.5
13.6
48.5
21.2
18.2
50.0
68.2
13.6
13.6
22.7
18.2
13.6
18.2
13.6
22.7
63.6
50.0
50.0
46.2
18.2
9.1
63.6
53.8
53.8
26.9
30.8
27.3
17.9
30.8
40.9
18.2
18.2
54.5
65.4
61.5
25.0
44.8
56.0
68.0
16.0
37.9
10.3
41.4
40.0
56.0
24.0
36.0
28.0
24.0
36.0
20.0
16.0
32.0
64.0
36.0
31.6
31.6
12.0
12.0
44.0
31.6
31.6
18.4
5.3
35.3
57.1
57.1
7.1
34.6
44.8
37.9
24.1
34.6
11.5
11.5
27.6
51.7
27.6
13.8
27.6
58.6
44.8
10.3
20.7
20.7
75.9
10.3
47.6
23.8
10.3
17.2
89.7
47.6
23.8
14.3
19.0
12.5
31.0
14.3
18.8
18.8
18.8
31.3
47.6
71.4
87.5
23.5
23.5
17.6
28.6
50.0
10.7
14.3
28.6
28.6
21.4
21.4
21.4
7.1
64.3
42.9
36.4
18.2
14.3
10.7
57.1
54.5
27.3
27.3
4.5
21.1
20.7
27.3
31.6
15.8
15.8
31.6
50.0
50.0
21.1
46.2
11.5
16.0
33.3
9.6
9.6
57.7
30.9
18.5
6.2
6.2
37.9
6.6
25.0
45.5
9.5
26.5
56.4
13.2
8.2
89.7
66.7
46.2
79.5
22.4
42.9
96.2
45.5
15.8
21.1
27.3
31.8
27.3
36.4
44.7
47.4
36.4
11.1
31.3
43.8
12.5
37.5
44.4
44.4
75.0
23.2
29.4
33.3
11.8
49.0
81.7
37.5
100
6.2
15.0
50.0
27.3
18.2
50.0
40.0
65.0
94.4
18.5
21.4
14.3
10.7
57.1
59.3
70.4
23.8
33
34
35
36
37
38
22.7
18.2
12.1
30.3
34.6
67.9
93.9
4.5
27.3
55.6
55.6
90.9
56.7
54.3
65.7
28.0
Pyrimethanil
a
A.S: Alternaria solani; F.G: Fusarium graminearum; P.I: Phytophthora infestans; S.S: Sclerotinia sclerotiorum; B.C: Botrytis
cinereal; R.S: Rhizoctonia solani; F.O: Fusarium oxysporum f.sp.cucumerinum; C.A: Cercospora arachidicola; P.P: Physalospora
piricola; R.C: Rhizoctonia cerealis; B.M: Bipolaris maydis; W.A: Watermelon-anthracnose; F.M: Fusarium moniliforme; P.C:
Phytophthora capsica.
Thirdly, when the 4,6-positions of the pyrimidine ring were substituted by a chlorine atom
and trifluoroethoxy group (such as compound 17), the antifungal activity was weakened instead
of enhanced. Therefore, we further fixed the 4-position of the pyrimidine ring with a chlorine
atom or trifluoroethoxy group, and a series of compounds with nitrogen-substituted group at
6-position of the pyrimidine ring were synthesized, such as compounds 18 (4-Cl, 6-morpholine),
19 (4-Cl, 6-N’,N’-diethyl), 21 (4-OCH2CF3, 6-pyrrolidinyl) and 22 (4-OCH2CF3, 6-N’-hexyl) (Table 2).
Fortunately, compound 19 exhibited broad-spectrum fungicidal activity (>60% inhibitory activities
against 13 phytopathogenic fungi), which was better than the inhibitory activities of commercial
pyrimethanil (>50% against eight phytopathogenic fungi, Table 2).
As another part of our strategy, we found that the antifungal activity of 10 (2,6-Cl on the
benzoyl ring) is superior to compounds
9 (2,4-Cl on the benzoyl ring, Table 2). Compound 25
exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against 13 phytopathogenic
fungi). Especially notable was the inhibition activity of compound 25 against Rhizoctonia cerealis,
Phytophthora capsica, Botrytis cinereal and Physalospora piricola. reached 94.9%, 90.7%, 89.7% and 84.2%

Molecules 2018, 23, 2203
12 of 25
at 50 µg mL−1 respectively, which was comparable to commercial pyrimethanil (Table 2). Unlike the
low activity of compound 17 (2-Cl), the broad-spectrum antifungal activity of compound 25 indicated
that 2,6-dichloro substitution on the benzoyl ring was very important for the antifungal activity.
However, when we replaced the ethyl group on the nitrogen with benzyl group (compound 29
compared with compound 27) or the 5-position of the pyrimidine ring was substituted with 2-butyl
group (compound 30), the antifungal activity decreased significantly (Table 2). This result indicated
that groups with large steric hindrance were not conducive to antifungal activity.
In the investigation of the urea bridge unit, a benzoylthiourea derivative (compound 31) and a
benzoylguanidine derivative (compound 32) displayed lower antifungal activities than compound
2
and compound 27, respectively (Table 2). Although the phenylurea compound 38 also showed better
antifungal activity (>50% inhibitory activities against 10 phytopathogenic fungi), its antifungal activity
was lower and its antifungal spectrum smaller than those of compound 19. Finally, because of the
broad spectrum and powerful antifungal activity of compounds 19 and 25, the values of EC50 for
compounds 19 and 25 against Sclerotinia sclerotiorum, Botrytis cinereal, Physalospora piricola, Rhizoctonia
cerealis were determined and are listed in Table 3.
Table 3. Inhibitory Effects of Compounds 19 and 25 against Phytopathogenic Fungi.
−1
R b
Compd.
19
Phytopathogens
EC50 (µg mL
)
Toxic Regression Eq
S.S a
P.P
R.C
B.C
10.268
15.891
14.744
17.175
y = −2.048 + 2.025x
y = −3.842 + 3.198x
y = −4.221 + 3.612x
y = −1.600 + 1.295x
0.9757
0.9778
0.9844
0.9762
S.S
P.P
R.C
B.C
7.072
4.604
13.231
7.050
y = −1.436 + 1.691x
y = −1.128 + 1.701x
y = −3.657 + 3.260x
y = −2.941 + 3.467x
0.9752
0.9597
0.9695
0.9618
25
a
b
S.S: Sclerotinia sclerotiorum; B.C: Botrytis cinereal; P.P: Physalospora piricola; R.C: Rhizoctonia cerealis;
R:
regression coefficient.
2.2.4. In Vivo Antifungal Bioassay against Sclerotinia sclerotiorum
The protective effets of compounds 19 and 25 and carbendazim (positive control) against
S. sclerotiorum on detached Brassica oleracea L. leaves were evaluated. The results showed that both
compounds 19 and 25 exhibited protective activity on leaves of Brassica oleracea L. (Table 4).
Table 4. In vivo Protective Effects of Compounds 19 and 25 against Sclerotinia sclerotiorum.
−1
Compd.
19
Concn (µg mL
)
Protection Effect (%)
3000
1500
500
55.7
35.2
34.1
3000
1500
500
83.0
35.2
20.5
25
Carbendazim
500
100
This protective effect was enhanced with the increasing concentration of the compounds
(experimental photos shown in Figure S102 in the Supporting Information). For example, the values
of the protective effect for compound 19 were 55.7% at 3 mg mL−1, 35.2% at 1.5 mg mL−1, 34.1% at
0.5 mg mL−1 and the protective effect values for compound 25 were 83.0% at 3 mg mL−1, 35.2% at
−1
1.5 mg mL−1, 20.5% at 0.5 mg mL (Table 4). Interestingly, although compound 19 has better in vitro
antifungal activity than compound 25, it appears that compound 25 has a better protective effect.

Molecules 2018, 23, 2203
13 of 25
2.2.5. Zebrafish Embryo Toxicity Assay
The fish embryo acute toxicity test (FET) with zebrafish embryos, which is a general model in
ecotoxicology and toxicology had been established in our labs [15 16]. The mortality in the positive
control was 40% greater than 30% and survival in the negative control was 100%. As shown in Table 5,
compound has low toxicity to zebrafish embryos (LC50 = 378.387
g mL−1), which was what we
,
7
µ
like to see because of its excellent larvicidal activity to mosquito. At the same time, compound 25
which exhibited broad-spectrum antifungal activity also possesses low toxicity to zebrafish embryos
(
LC50 = 21.668 µg mL−1). Fortunately, both compounds
7 and 25 did not affect zebrafish embryo
hatching, induce the pericardial cysts and produce abnormality (such as malformation and coagulation)
during the zebrafish embryo acute toxicity test.
Table 5. The Zebrafish Embryo Acute Toxicity of Compounds 7 and 25.
−1
Compd.
7
Time (h)
LC50 (µg mL
)
Toxic Regression Eq
R
95% Confidence Limits
96
24
48
72
96
378.387
38.187
30.435
26.886
21.668
y = −4.378 + 1.698x
y = −2.193 + 1.387x
y = −2.142 + 1.444x
y = −2.274 + 1.591x
y = −2.131 + 1.595x
0.9617
0.8718
0.9154
0.9508
0.9762
325.159–369.971
23.899–67.569
18.065–48.014
16.271–39.781
11.777–31.651
25
3. Materials and Methods
3.1. Instruments
1H-NMR and 13C-NMR spectra were obtained at 500/126 MHz using a Bruker Avance III
500 spectrometer (Bruker Daltonics, Bremen, Germany) in CDCl3 or DMSO-d6 solution with
tetramethylsilane as the internal standard. Chemical shift values (δ) are given in ppm. HRMS data
were obtained on FTICR-MS (Ionspec 7.0 T, Lebrilla League, Davis, CA, USA) high resolution mass
spectrometer and LQC Advantage MAX multi-stage ion mass spectrometer (Agilent Technologies
Inc., CA, USA). The melting points were determined on an X-4 binocular microscope melting
point apparatus (Beijing Tech Instruments Co., Beijing, China) and are uncorrected. Yields were
not optimized.
3.2. General Synthesis
All anhydrous solvents were dried and purified by standard techniques. Chemicals (analytical
grade) were purchased from Aladdin (Shanghai, China).
3.3. General Synthesis Procedure for Substituted 2-Aminopyrimidines Ia–c
Intermediates Ia–c were prepared according to the literature [17]. A solution of NaH (0.35 g,
14.6 mmol) in dry THF (12 mL) was cooled to 0 ◦C under N2, then a substituted alcohol (15.2 mmol)
was added dropwise. The mixture was stirred for 15 min while maintaining the temperature at 0 ◦C.
Next, 2-amino-4,◦6-dichloropyrimidine (1.0 g, 6.1 mmol) was added to the solution. The reaction was
continued at 62 C for 15 h. Then the mixture was cooled to ambient temperature, and quenched
with 1 mL of 1 M hydrochloric acid solution. The mixture was diluted with EtOAc (20 mL), washed
twice with a saturated NaHCO3 solution (20 mL) and brine (20 mL), dried with anhydrous Na2SO4
and evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography
(EtOAc/petroleum ether) to afford compounds Ia–c.
1
4,6-Bis(2,2,2-trifluoroethoxy)pyrimidin-2-amine (Ia). Yellow oil, yield = 94%. H-NMR (CDCl3)
(s, 1H, ArH), 4.96 (s, 2H, NH2), 4.57 (q, J = 8.6 Hz, 4H, OCH2CF3).
δ
5.58
1
4,6-Dimethoxypyrimidin-2-amine (Ib). White solid, mp 87–89 ◦C, yield = 91%. H-NMR (CDCl3)
δ
5.46
(s, 1H, ArH), 5.09 (s, 2H, NH2), 3.84 (s, 6H, OCH3).

Molecules 2018, 23, 2203
14 of 25
1
4,6-Diethoxypyrimidin-2-amine (Ic). White solid, mp 174–176 ◦C, yield = 94%. H-NMR (CDCl3)
δ 5.42
(s, 1H, ArH), 4.95 (s, 2H, NH2), 4.23 (q, J = 7.1 Hz, 4H, OCH2CH3), 1.35 (t, J = 7.1 Hz, 6H, OCH2CH3).
3.4. General Synthesis Procedure for Substituted 2-Aminopyrimidines Id–f
Intermediates Id
–
f
were prepared by a method similar to that for intermediates Ia
–
c
. A solution of
◦
NaH (5.8 mmol) in dry THF (12 mL) was cooled to 0 C under N2, then a substituted alcohol (6.1 mmol)
was added dropwise. The mixture was stirred for 15 min while maintaining the temperature at 0 ◦C.
Next, 2-amino-4-chloro-6- substituted-pyrimidine (5.8 mmol) was added to the solution. The reaction
was continued at 62 ◦C for 15 h. Then the mixture was cooled to ambient temperature, and quenched
with 1 mL of 1 M hydrochloric acid solution. The mixture was diluted with EtOAc (20 mL), washed
twice with a saturated NaHCO3 solution (20 mL) and brine (20 mL), dried with anhydrous Na2SO4
and evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography
(EtOAc/petroleum ether) to afford compounds Id–f.
1
4-Chloro-6-methoxypyrimidin-2-amine (Id). White solid, mp 165–167 ◦C, yield = 84%. H-NMR (CDCl3)
δ 6.04 (s, 1H, ArH), 5.35–5.14 (m, 2H, NH2), 3.81 (s, 3H, OCH3).
◦
4-Methyl-6-(2,2,2-trifluoroethoxy)pyrimidin-2-amine (Ie). White solid, mp 109–111 C, yield = 69%.
1H-NMR (CDCl3)
(s, 3H, CH3).
δ 6.42 (s, 1H, ArH), 5.50 (s, 2H, NH2), 4.60 (q, J = 8.4 Hz, 2H, OCH2CF3), 2.18
◦
1
4-Chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-amine (If). White solid, mp 82–83 C, yield = 76%. H-NMR
(CDCl3) δ 6.23 (s, 1H, ArH), 5.59 (s, 2H, NH2), 4.70 (q, J = 8.4 Hz, 2H, OCH2CF3).
3.5. General Synthesis Procedure for Substituted 2-Aminopyrimidines Ig–m
Intermediates Ig–m were prepared according to [18]. A suspension of CuI (5.8 mg, 0.03
mmol), K2CO3 (0.41 g, 2.9 mmol), the corresponding amine (0.21 g, 2.4 mmol) and substituted
2-aminopyrimidines (0.4 g, 2.4 mmol) in◦2 mL DMF was stirred for 30 min at room temperature
and then the mixture was heated to 110 C. At the end of the reaction, as monitored by TLC, the
reaction mixture was cooled to room temperature and diluted with 20 mL of EtOAc, washed twice
with a saturated NaHCO3 solution (20 mL) and brine (20 mL), dried with anhydrous MgSO4 and
evaporated in vacuo. Finally, the residue was purified by silica gel column chromatography to afford
compound Ig–m.
◦
4-Chloro-6-morpholinopyrimidin-2-amine (Ig). White solid, mp 210–212 C, yield = 79%. 1H-NMR
(CDCl3) 5.94 (s, 1H, ArH), 5.00 (s, 2H, NH2), 3.79–3.68 (m, 4H, morpholine), 3.55 (t, J = 4.9 Hz, 4H,
morpholine).
δ
1
6-Chloro-N4,N4-diethylpyrimidine-2,4-diamine (Ih). White solid, mp 104–106 ◦C, yield = 81%. H-NMR
(CDCl3) 5.84 (s, 1H, ArH), 5.04 (s, 2H, NH2), 3.42 (s, 4H, CH2CH3), 1.15 (t, J = 7.1 Hz, 6H, CH2CH3).
δ
◦
N4-(Pyrrolidin-1-yl)-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Ii). White solid, mp 118–120 C,
1
yield = 51%. H-NMR (CDCl3)
δ
5.21 (s, 1H, ArH), 4.82 (s, 2H, NH2), 4.67 (q, J = 8.7 Hz, 2H, OCH2CF3),
168.7,
3.38 (s, 4H, tetrahydropyrrole), 1.93 (d, J = 5.9 Hz, 4H, tetrahydropyrrole). 13C-NMR (CDCl3)
δ
163.3, 162.0, 124.9, 122.7, 62.0, 61.7, 61.4, 61.1, 46.5, 25.3. HRMS (ESI): m/z calcd for C10H13F3N4O
[M + H]+: 263.1120, found 263.1136.
1
N4-Hexyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Ij). Yellow oil, yield = 53%. H-NMR (CDCl3)
δ
5.24 (s, 1H, ArH), 5.13 (s, 1H, NH), 5.06 (s, 2H, NH2), 4.65 (q, J = 8.6 Hz, 2H, OCH2CF3), 3.12
(q, J = 6.7 Hz, 2H, N–CH2(CH2)4CH3), 1.77–1.42 (m, 2H, N–(CH2)4CH2CH3), 1.44–1.11 (m, 6H,
N–CH2(CH2)3CH2CH3), 0.88 (t, J = 6.8 Hz, 3H, N–(CH2)5CH3). 13C-NMR (CDCl3)
169.4, 165.6,
δ
162.1, 124.7, 122.5, 62.0, 61.7, 61.5, 61.2, 41.8, 31.5, 29.2, 26.6, 22.6, 14.0. HRMS (ESI): m/z calcd for
C10H19F3N4O [M + H]+: 293.1589, found 293.1593.

Molecules 2018, 23, 2203
15 of 25
N4,N4-Diethyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Ik). Yellow oil, yield = 49%. 1H-NMR
(CDCl3) 5.32 (s, 1H, ArH), 4.78 (s, 2H, NH2), 4.72–4.61 (m, 2H, OCH2CF3), 3.41 (q, J = 7.0 Hz, 4H,
δ
CH2CH3), 1.13 (t, J = 7.1 Hz, 6H, CH2CH3). 13C-NMR (CDCl3)
δ 170.4, 169.3, 163.9, 124.9, 81.7, 62.3,
62.0, 61.7, 61.4, 42.1, 12.9. HRMS (ESI): m/z calcd for C10H15F3N4O [M + H]+: 265.1276, found 265.1280.
1
N4-Benzyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Il). Yellow oil, yield = 55%. H-NMR (CDCl3)
δ
7.36–7.15 (m, 5H, Ph), 5.66 (s, 1H, ArH), 5.22 (s, 1H, NH), 5.02 (s, 2H, NH2), 4.59 (q, J = 8.6 Hz, 2H,
OCH2CF3), 4.31 (d, J = 6.0 Hz, 2H, PhCH2). 13C-NMR (CDCl3)
δ
169.4, 165.6, 162.2, 138.2, 128.7, 127.5,
127.1, 126.9, 124.7, 122.5, 120.3, 62.0, 61.7, 61.4, 61.2, 45.6. HRMS (ESI): m/z calcd for C13H13F3N4O
[M + H]+: 299.1120, found 299.1119.
1
N4-Benzyl-N4-ethyl-6-(2,2,2-trifluoroethoxy)pyrimidine-2,4-diamine (Im). Yellow oil, yield = 43%. H-NMR
(DMSO-d6)
δ 7.65–7.03 (m, 5H, Ph), 6.23 (s, 2H, NH2), 5.31 (s, 1H, ArH), 4.86 (q, J = 9.2 Hz, 2H,
OCH2CF3), 4.69 (s, 2H, PhCH2), 3.41 (s, 2H, CH2CH3), 1.04 (t, J = 7.0 Hz, 3H, CH2CH3). 13C-NMR
(DMSO-d6)
δ 169.6, 165.1, 163.1, 129.4, 127.9, 127.7, 126.1, 123.9, 75.6, 61.3, 61.1, 42.7, 13.4. HRMS (ESI):
m/z calcd for C15H17F3N4O [M + H]+: 327.1433, found 327.1429.
3.6. Synthesis of 5-(sec-Butyl)-4,6-dichloropyrimidin-2-amine (Intermediate In)
Intermediate In was prepared according to [19]. Elemental sodium (0.30 g, 13.0 mmol) was
added into absolute ethanol (7 mL) under N2 while being intensively stirred with a magnetic stirrer.
After all the sodium was dissolved and the reaction mixture was cooled to room temperature,
guanidine hydrochloride (0.49 g, 5.1 mmol) was added under intensive stirring, followed by the
diethyl sec-butylmalonate (1.9 g, 4.6 mmol). The reaction mixture was further stirred intensively at
room temperature. After another 4 h, absolute ethanol (5 mL) was added and the reaction mixture was
refluxed for 1 h. Afterward, ethanol was evaporated on a vacuum rotary evaporator and water (12 mL)
was added to the reaction mixture. After stirring, the product was dissolved. The obtained mixture was
subsequently neutralized by acetic acid and then this mixture was heated under reflux for 10 min and
then cooled to room temperature. This heating and cooling was repeated twice to get a well-filterable
solid. The solid was filtered off, washed with water (2
×
50 mL), ethanol (2
×
50 mL), and acetone
(2 × 50 mL). The product was dried under high vacuum for 2 days. Subsequently, the aboveobtained
solid (4.6 mmol) was suspended under N2 in a solution of (chloro-methylene)dimethyl ammonium
chloride (4.8 g, 37.1 mmol) in chloroform (19 mL). The reaction mixture was subsequently heated at
reflux for 4 h, during which the starting material was completely dissolved. The reaction mixture was
cooled to the room temperature, poured into ice and rapidly neutralized with a saturated aqueous
NaHCO3 solution. The obtained mixture was quickly transferred into a separatory funnel and
immediately extracted with chloroform (3
×
20 mL). The organic layers were combined together,
dried over MgSO4, filtered and concentrated down on a rotary evaporator. This crude residue was
dissolved in the mixture of ethanol (9 mL) and 37% aqueous HCl (0.9 mL). The reaction mixture was
heated at 50 ◦C for 2 h. After that, water (14 mL) was added and the reaction mixture was stirred for
10 min. The precipitated product was filtered off and washed with a water/ethanol mixture (v/v = 1/1
,
2
×
5 mL), 5 % aqueous solution of NaHCO3 (5 mL). The product was subsequently recrystallized
from aqueous ethanol, filtered off, washed with a water/ethanol mixture (v/v = 1/1, 5 mL) and the
solid recrystallized from ethanol to give intermediate In as a white solid, mp 157–159 ◦C, yield = 73%.
1H-NMR (CDCl3)
δ 5.98 (d, J = 6.5 Hz, 2H, NH2), 3.27 (dq, J = 6.9, 3.7, 2.9 Hz, 1H, CH), 1.95–1.75 (m,
1H, CHCH2), 1.63 (dddd, J = 16.0, 9.0, 4.7, 2.3 Hz, 1H, CHCH2), 1.24 (dt, J = 7.4, 2.3 Hz, 3H, CHCH3),
0.77 (ddd, J = 9.1, 5.7, 2.1 Hz, 3H, CHCH2CH3).
3.7. Synthesis of 5-(sec-Butyl)-6-chloro-N4,N4-diethylpyrimidine-2,4-diamine (Intermediate Io)
Intermediate Io was prepared by a method similar to that used for compounds Ig
–
m
. Yellow oil,
1
yield = 42%. H-NMR (DMSO-d6)
δ 6.44 (s, 2H), 3.23 (dd, J = 13.7, 6.9 Hz, 2H), 3.05 (dd, J = 13.7, 7.0 Hz,
2H), 2.79 (q, J = 7.4 Hz, 1H), 1.79–1.61 (m, 2H), 1.31 (d, J = 7.1 Hz, 3H), 1.05 (t, J = 7.0 Hz, 6H), 0.71 (t,

Molecules 2018, 23, 2203
16 of 25
J = 7.4 Hz, 3H). 13C-NMR (DMSO-d6)
δ
170.8, 160.8, 159.7, 114.1, 45.8, 34.4, 28.3, 19.0, 14.0, 13.6. HRMS
(ESI): m/z calcd for C12H21ClN4 [M + Na]+: 279.1352, found 279.1356.
3.8. Synthesis of 5-(sec-Butyl)-4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-amine (Intermediate Ip)
Intermediate Ip was prepared by a method similar to that used for compounds Id
–
f
. White solid,
7.00 (s, 2H, NH2), 4.98 (q, J = 9.0 Hz, 2H, OCH2CF3),
3.02 (dt, J = 9.3, 6.7 Hz, 1H, CH), 1.76–1.62 (m, 1H, CHCH2), 1.56 (ddd, J = 13.6, 7.6, 6.4 Hz, 1H, CHCH2),
1.18 (d, J = 7.1 Hz, 3H, CHCH3), 0.75 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C-NMR (DMSO-d6)
167.6,
◦
1
mp 53–54 C, yield = 40%. H-NMR (DMSO-d6)
δ
δ
160.9, 160.7, 109.8, 62.8, 62.5, 62.2, 62.0, 36.5, 27.9, 19.4, 13.3. HRMS (ESI): m/z calcd for C10H13ClF3N3O
[M + H]+: 284.0777, found 284.0799.
3.9. General Synthesis Procedure for Target Compounds 1–30
Compounds 1–30 were synthesized according to [20,21]. Substituted benzamide (5.0 mmol) and
1,2-dichloroethane (10 mL) were added to a 100 mL three-necked flask under N2. The reaction mixture
was cooled to 0 ◦C and oxalyl chloride (1.27 g, 10.0 mmol) was added dropwise under stirring, then
the mixture was stirred at room temperature for 1 h, left standing at 60 ◦ C for 3 h and refluxed for
1 h. The solvent and excess oxalyl chloride were evaporated under reduced pressure to give a yellow
transparent liquid. Anhydrous dichloromethane (5 mL) was added to the residue, then a substituted
2-aminopyrimidine (2.5 mmol) was added to the system and reacted at room temperature for 12 h.
The solvent was removed under reduced pressure and the residue was purified by flash column
chromatography on silica gel to give compounds 1–30.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-difluorobenzamide (
δ 11.65 (s, 1H, NH), 10.77 (s, 1H, NH), 7.76–7.57 (m,
1H, Ph), 7.26 (t, J = 8.3 Hz, 2H, Ph), 6.40 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR
1
). White solid, mp
1
163–164 ◦C, yield = 83%. H-NMR (DMSO-d6)
(DMSO-d6)
δ 170.9, 160.6, 156.3, 148.9, 134.2, 125.6, 123.4, 113.2, 113.0, 86.8, 63.7, 63.4, 63.1, 62.8. HRMS
(ESI): m/z calcd for C16H10F8N4O4 [M + H]+: 475.0652, found 475.0641.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-chlorobenzamide (
2
). White solid, mp
173–175 ◦C, yield = 95%. 1H-NMR (DMSO-d6)
δ
11.47 (s, 1H, NH), 10.96 (s, 1H, NH), 7.65 (dd,
J = 7.6, 1.6 Hz, 1H, Ph), 7.61–7.52 (m, 2H, Ph), 7.48 (td, J = 7.3, 1.7 Hz, 1H, Ph), 6.39 (s, 1H, ArH), 5.07 (q,
J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6)
δ 170.9, 169.2, 156.3, 149.0, 135.6, 133.1, 130.7, 130.0,
128.2, 125.7, 123.4, 86.8, 63.6, 63.3, 63.0, 62.8. HRMS (ESI): m/z calcd for C16H11ClF6N4O4 [M + H]+:
473.0451, found 473.0440.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-bromobenzamide (
3
). White solid, mp
1
110–112 ◦C, yield = 77%. H-NMR (DMSO-d6)
δ
11.46 (s, 1H, NH), 10.96 (s, 1H, NH), 7.73 (d, J = 7.9 Hz
,
1H, Ph), 7.62 (dd, J = 7.4, 1.7 Hz, 1H, Ph), 7.57–7.42 (m, 2H, Ph), 6.39 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz
δ 170.8, 170.0, 156.2, 149.0, 137.8, 133.7, 133.0, 129.8, 128.6, 125.6,
119.5, 86.8, 63.6, 63.3, 63.0, 62.7. HRMS (ESI): m/z calcd for C16H11BrF6N4O4 [M + Na]+: 538.9766,
,
4H, OCH2CF3). 13C-NMR (DMSO-d6)
found 538.9769.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-iodobenzamide (
115–116 ◦C, yield = 88%. 1H-NMR (DMSO-d6)
11.42 (s, 1H, NH), 11.01 (s, 1H, NH), 7.95 (dd,
J = 7.9, 1.0 Hz, 1H, Ph), 7.72–7.38 (m, 2H, Ph), 7.28 (td, J = 7.6, 1.9 Hz, 1H, Ph), 6.39 (s, 1H, ArH), 5.07
(q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6)
171.7, 170.9, 156.3, 149.1, 141.7, 140.2, 132.9,
4
).
White solid, mp
δ
δ
129.2, 129.1, 125.7, 125.4, 93.9, 86.8, 63.6, 63.4, 63.1, 62.8. HRMS (ESI): m/z calcd for C16H11F6IN4O4
[M + Na]+: 586.9627, found 586.9603.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-4-chlorobenzamide (
5
). White solid, mp
11.42 (s, 1H, NH), 11.22 (s, 1H, NH), 8.02 (d, J = 8.5 Hz
2H, Ph), 7.60 (d, J = 8.6 Hz, 2H, Ph), 6.36 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR
◦
1
162–163 C, yield = 94%. H-NMR (DMSO-d6)
δ
,

Molecules 2018, 23, 2203
17 of 25
(DMSO-d6)
δ 170.8, 168.0, 156.4, 149.4, 139.1, 132.1, 131.1, 129.6, 125.6, 86.4, 63.6, 63.3, 63.0, 62.8. HRMS
(ESI): m/z calcd for C16H11ClF6N4O4 [M + H]+: 473.0451, found 473.0442.
N-((4,6-bis(◦2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2-(trifluoromethyl)benzamide (
6). White solid,
δ 11.55 (s, 1H, NH), 10.91 (s, 1H, NH), 7.88 (d,
mp 51–53 C, yield = 68%. 1H-NMR (DMSO-d6)
J = 7.7 Hz, 1H, Ph), 7.85–7.71 (m, 3H, Ph), 6.40 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3).
13C-NMR (DMSO-d6)
δ 170.8, 170.0, 156.3, 149.0, 134.8, 133.5, 131.9, 129.3, 127.4, 127.1, 126.8, 126.5,
126.3, 125.6, 123.4, 86.7, 63.6, 63.3, 63.0, 62.8. HRMS (ESI): m/z calcd for C17H11F9N4O4 [M + H]+:
507.0715, found 507.0711.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-4-(trifluoromethyl)benzamide (
7
). White solid,
11.61 (s, 1H, NH), 11.15 (s, 1H, NH), 8.17 (d,
J = 8.1 Hz, 2H, Ph), 7.90 (d, J = 8.1 Hz, 2H, Ph), 6.38 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 4H, OCH2CF3).
13C-NMR (DMSO-d6)
170.9, 167.9, 156.4, 149.3, 137.4, 133.7, 133.5, 130.1, 126.5, 126.5, 125.8, 125.6, 86.5,
63.7, 63.4, 63.1, 62.8. HRMS (ESI): m/z calcd for C17H11F9N4O4 [M + H]+: 507.0715, found 507.0713.
◦
mp 167–169 C, yield = 93%. 1H-NMR (DMSO-d6)
δ
δ
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-5-chloro-2-methoxybenzamide ( ). White solid,
8
◦
mp 164–166 C, yield = 95%. 1H-NMR (DMSO-d6)
δ
11.04 (s, 1H, NH), 10.98 (s, 1H, NH), 7.65 (d,
J = 2.7 Hz, 1H, Ph), 7.61 (dd, J = 8.9, 2.8 Hz, 1H, Ph), 7.23 (d, J = 8.9 Hz, 1H, Ph), 6.39 (s, 1H, ArH),
5.06 (q, J = 8.9 Hz, 4H, OCH2CF3), 3.89 (s, 3H, CH3). 13C-NMR (DMSO-d6) δ 170.9, 167.3, 156.6, 156.3,
148.9, 133.9, 130.1, 125.4, 125.1, 115.3, 86.7, 63.5, 63.3, 63.0, 62.7, 57.5. HRMS (ESI): m/z calcd for
C17H13ClF6N4O5 [M + H]+: 503.0557, found 503.0535.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,4-dichlorobenzamide (
9
). White solid, mp
11.49 (s, 1H, NH), 10.88 (s, 1H, NH), 7.79 (d, J = 1.9 Hz
1H, Ph), 7.68 (d, J = 8.2 Hz, 1H, Ph), 7.58 (dd, J = 8.2, 2.0 Hz, 1H, Ph), 6.40 (s, 1H, ArH), 5.06 (q,
◦
1
109–110 C, yield = 92%. H-NMR (DMSO-d6)
δ
,
J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6)
δ 170.8, 168.3, 156.3, 149.0, 136.9, 134.6, 132.0, 131.4,
130.3, 128.5, 125.6, 86.8, 63.6, 63.3, 63.0. HRMS (ESI): m/z calcd for C16H10Cl2F6N4O4 [M + Na]+:
528.9881, found 528.9866.
N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (10). White solid, mp
1
62–64 ◦C, yield = 81%. H-NMR (DMSO-d6)
δ
11.68 (s, 1H, NH), 10.80 (s, 1H, NH), 7.82–7.39 (m, 3H,
170.9, 156.2, 148.8,
Ph), 6.41 (s, 1H, ArH), 5.08 (q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6)
δ
131.4, 129.2, 127.8, 125.6, 123.4, 121.2, 100.5, 86.9, 63.7, 63.4, 63.1, 62.9. HRMS (ESI): m/z calcd for
C16H10Cl2F6N4O4 [M + Na]+: 528.9881, found 528.9866.
2-Chloro-N-((4,6-dimethylpyrimidin-2-yl)carbamoyl)benzamide (11). White solid, mp 186–188 ◦C,
1
yield = 87%. H-NMR (DMSO-d6)
δ
12.51 (s, 1H, NH), 10.61 (s, 1H, NH), 7.64 (dd, J = 7.6, 1.6 Hz, 1H,
Ph), 7.61–7.52 (m, 2H, Ph), 7.47 (td, J = 7.3, 1.5 Hz, 1H, Ph), 6.98 (s, 1H, ArH), 2.33 (s, 6H, Ar-CH3).
13C-NMR (DMSO-d6)
δ 168.8, 167.5, 157.6, 149.8, 136.3, 132.9, 130.8, 130.6, 129.8, 128.3, 116.0, 24.2.
HRMS (ESI): m/z calcd for C14H13ClN4O2 [M + Na]+: 327.0625, found 327.0629.
2-Chloro-N-((4,6-dimethoxypyrimidin-2-yl)carbamoyl)benzamide (12). White solid, mp 155–157 ◦C,
1
yield = 89%. H-NMR (DMSO-d6)
δ
11.87 (s, 1H, NH), 10.67 (s, 1H, NH), 7.62 (dd, J = 7.6, 1.6 Hz, 1H,
Ph), 7.59–7.50 (m, 2H, Ph), 7.46 (td, J = 7.3, 1.5 Hz, 1H, Ph), 5.97 (s, 1H, ArH), 3.82 (s, 6H, Ar-OCH3).
13C-NMR (DMSO-d6)
δ 172.4, 168.4, 156.9, 149.4, 136.3, 132.8, 130.6, 130.5, 129.7, 128.3, 85.1, 55.2. HRMS
(ESI): m/z calcd for C14H13ClN4O4 [M + Na]+: 359.0523, found 359.0524.
2-Chloro-N-((4,6-diethoxypyrimidin-2-yl)carbamoyl)benzamide (13). White solid, mp 113–115 ◦C,
1
yield = 91%. H-NMR (DMSO-d6)
δ
11.92 (s, 1H, NH), 10.61 (s, 1H, NH), 7.65 (dd, J = 7.6, 1.5 Hz, 1H,
Ph), 7.57 (qd, J = 8.1, 4.1 Hz, 2H, Ph), 7.52–7.44 (m, 1H, Ph), 5.87 (s, 1H, ArH), 4.20 (q, J = 7.0 Hz, 4H,
OCH2CH3), 1.26 (t, J = 7.1 Hz, 6H, OCH2CH3). 13C-NMR (DMSO-d6)
δ
171.9, 167.9, 156.9, 149.4, 136.3,
135.8, 132.8, 130.6, 129.7, 128.3, 85.3, 64.5, 63.6, 15.2, 15.0. HRMS (ESI): m/z calcd for C16H17ClN4O4
[M + Na]+: 387.0836, found 387.0851.

Molecules 2018, 23, 2203
18 of 25
2-Chloro-N-((4,6-dichloropyrimidin-2-yl)carbamoyl)benzamide (14). White solid, mp 156–158 ◦C,
yield = 88% 11.52 (s, 1H, NH), 11.24 (s, 1H, NH), 7.69 (s, 1H, ArH), 7.65
1H-NMR (DMSO-d6)
(dd, J = 7.6, 1.5 Hz, 1H, Ph), 7.57 (qd, J = 8.1, 1.7 Hz, 2H, Ph), 7.49 (td, J = 7.2, 1.9 Hz, 1H, Ph). 13C-NMR
(DMSO-d6) 169.0, 162.5, 157.4, 148.8, 133.1, 130.8, 130.8, 130.0, 128.3, 117.0. HRMS (ESI): m/z calcd for
C12H7Cl3N4O2 [M + Na]+: 366.9532, found 366.9549.
.
δ
δ
◦
2-Chloro-N-((4-chloro-6-methylpyrimidin-2-yl)carbamoyl)benzamide (15). White solid, mp 126–128 C,
yield = 93% 11.89 (s, 1H, NH), 10.98 (s, 1H, NH), 7.65 (dd, J = 7.6, 1.6 Hz,
1H-NMR (DMSO-d6)
1H, Ph), 7.63–7.51 (m, 2H, Ph), 7.47 (td, J = 7.3, 1.5 Hz, 1H, Ph), 7.32 (s, 1H, ArH), 2.40 (s, 3H, CH3).
.
δ
13C-NMR (DMSO-d6)
δ 171.4, 169.1, 168.3, 161.4, 157.5, 149.2, 138.1, 131.5, 130.5, 129.6, 127.9, 116.4, 24.2.
HRMS (ESI): m/z calcd for C13H10Cl2N4O2 [M + Na]+: 347.0079, found 347.0064.
2-Chloro-N-((4-chloro-6-methoxypyrimidin-2-yl)carbamoyl)benzamide (16). White solid, mp 166–167 ◦C,
1
yield = 87%. H-NMR (DMSO-d6)
δ
11.64 (s, 1H, NH), 10.98 (s, 1H, NH), 7.65 (d, J = 7.5 Hz, 1H, Ph),
7.56 (d, J = 8.5 Hz, 2H, Ph), 7.48 (t, J = 7.3 Hz, 1H, Ph), 6.82 (s, 1H, ArH), 3.91 (s, 3H, OCH3). 13C-NMR
(DMSO-d6) δ 171.9, 168.6, 160.8, 157.2, 149.0, 135.7, 132.9, 130.6, 129.9, 128.2, 102.6, 55.7. HRMS (ESI):
m/z calcd for C13H10Cl2N4O3 [M + Na]+: 363.0028, found 363.0013.
2-Chloro-N-((4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (17). White solid, mp
1
144–146 ◦C, yield = 88%. H-NMR (DMSO-d6)
δ 11.51 (s, 1H, NH), 11.07 (s, 1H, NH), 7.65 (dd, J = 7.5,
1.5 Hz, 1H, Ph), 7.62–7.51 (m, 2H, Ph), 7.48 (td, J = 7.3, 1.7 Hz, 1H, Ph), 7.09 (s, 1H, ArH), 5.10 (q,
J = 8.8 Hz, 2H, OCH2CF3). 13C-NMR (DMSO-d6)
δ
170.0, 169.1, 161.8, 156.9, 148.9, 135.6, 133.1, 130.8,
130.0, 128.2, 125.5, 123.3, 103.0, 63.8, 63.5, 63.2, 62.9. HRMS (ESI): m/z calcd for C14H9Cl2F3N4O3
[M + Na]+: 430.9901, found 430.9887.
2-Chloro-N-((4-chloro-6-morpholinopyrimidin-2-yl)carbamoyl)benzamide (18). White solid, mp 147–149 ◦C,
1
yield = 85%. H-NMR (DMSO-d6)
δ
11.78 (s, 1H, NH), 10.59 (s, 1H, NH), 7.62 (dd, J = 7.6, 1.6 Hz, 1H,
Ph), 7.59–7.50 (m, 2H, Ph), 7.46 (td, J = 7.3, 1.5 Hz, 1H, Ph), 6.71 (s, 1H, ArH), 3.58 (dd, J = 23.7, 4.9 Hz,
8H, morpholine). 13C-NMR (DMSO-d6)
168.2, 163.6, 157.0, 149.3, 136.1, 132.9, 130.7, 130.6, 129.8,
128.3, 97.9, 66.6, 60.7. HRMS (ESI): m/z calcd for C16H15Cl2N5O3 [M + Na]+: 418.0450, found 418.0441.
δ
2-Chloro-◦N-((4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)benzamide (19).
white solid, mp
δ 11.97 (s, 1H, NH), 10.46 (s, 1H, NH), 7.60 (dd,
151–153 C, yield = 83%. 1H-NMR (DMSO-d6)
J = 7.6, 1.6 Hz, 1H, Ph), 7.58–7.49 (m, 2H, Ph), 7.45 (td, J = 7.3, 1.6 Hz, 1H, Ph), 6.49 (s, 1H, ArH), 3.44
(s, 4H, CH2CH3), 1.19–0.91 (m, 6H, CH2CH3). 13C-NMR (DMSO-d6)
δ
168.1, 162.1, 159.4, 157.2, 149.6,
136.5, 132.7, 130.5, 130.5, 129.7, 128.3, 97.1, 43.3, 13.4. HRMS (ESI): m/z calcd for C16H17Cl2N5O2 [M +
Na]+: 404.0657, found 404.0642.
2-Chloro-N-((4-methyl-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (20). White solid, mp
1
56–58 ◦C, yield = 82%. H-NMR (DMSO-d6)
δ
12.13 (s, 1H, NH), 10.79 (s, 1H, NH), 7.72–7.63 (m, 1H,
Ph), 7.63–7.52 (m, 2H, Ph), 7.49 (td, J = 7.3, 1.6 Hz, 1H, Ph), 6.71 (s, 1H, ArH), 5.07 (q, J = 8.9 Hz, 2H,
OCH2CF3), 2.34 (s, 3H, CH3). 13C-NMR (DMSO-d6)
δ
171.4, 170.3, 169.3, 168.1, 161.4, 157.0, 149.4, 136.0,
132.9, 130.7, 129.9, 128.2, 102.1, 62.8, 62.5, 62.2, 61.9, 24.1. HRMS (ESI): m/z calcd for C15H12ClF3N4O3
[M + Na]+: 411.0448, found 411.0442.
2-Chloro-N-((4-(pyrrolidin-1-yl)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (21). White
◦
1
solid, mp 121–122 C, yield = 81%. H-NMR (DMSO-d6)
δ 12.55 (s, 1H, NH), 10.28 (s, 1H, NH), 7.61 (d,
J = 7.4 Hz, 1H, Ph), 7.58–7.50 (m, 2H, Ph), 7.46 (td, J = 7.3, 1.7 Hz, 1H, Ph), 5.62 (s, 1H, ArH), 4.97 (q,
J = 9.0 Hz, 2H, OCH2CF3), 3.25 (s, 2H, tetrahydropyrrole), 3.02 (s, 2H, tetrahydropyrrole), 1.97–1.82
(m, 2H, tetrahydropyrrole), 1.77–1.60 (m, 2H, tetrahydropyrrole). 13C-NMR (DMSO-d6)
δ 168.7, 167.3,
161.8, 156.7, 149.8, 136.9, 132.5, 130.5, 130.3, 129.4, 128.3, 125.9, 81.1, 62.2, 61.9, 61.6, 47.3, 25.7. HRMS
(ESI): m/z calcd for C18H17ClF3N5O3 [M + H]+: 444.1050, found 444.1047.
2-Chloro-N-((4-(hexylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (22). White solid,
◦
1
mp 126–128 C, yield = 83%. H-NMR (DMSO-d6)
δ 12.40 (s, 1H, NH), 10.28 (s, 1H, NH), 7.85–7.36 (m,

Molecules 2018, 23, 2203
19 of 25
5H, Ph), 5.62 (s, 1H, ArH), 4.93 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.05 (s, 2H, N–CH2(CH2)4CH3), 1.46–1.07
(m, 8H, N–CH2(CH2)4CH3), 0.86 (t, J = 6.8 Hz, 3H, N(CH2)5CH3). 13C-NMR (DMSO-d6)
168.0, 165.1,
δ
157.1, 151.8, 149.8, 136.7, 132.6, 130.5, 129.6, 128.3, 125.9, 123.6, 82.2, 62.1, 61.8, 41.3, 32.0, 29.6, 27.0, 23.1,
14.9. HRMS (ESI): m/z calcd for C20H23ClF3N5O3 [M + Na]+: 496.1339, found 496.1357.
◦
2,4-Dichloro-N-((4,6-dimethylpyrimidin-2-yl)carbamoyl)benzamide (23). White solid, mp 185–186 C,
yield = 81%
Ph), 7.67 (d, J = 8.2 Hz, 1H, Ph), 7.57 (dd, J = 8.2, 2.0 Hz, 1H, Ph), 6.99 (d, J = 2.7 Hz, 1H, ArH), 2.35 (s,
. δ 12.50 (s, 1H, NH), 10.60 (s, 1H, NH), 7.77 (d, J = 2.0 Hz, 1H,
1H-NMR (DMSO-d6)
6H, CH3). 13C-NMR (DMSO-d6)
δ 168.8, 167.0, 157.6, 149.8, 136.5, 135.4, 131.8, 131.1, 130.3, 128.5, 116.1,
24.3. HRMS (ESI): m/z calcd for C14H12Cl2N4O2 [M + Na]+: 361.0235, found 361.0208.
2,4-Dichloro-N-((4-methyl-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (24). White solid,
◦
mp 155–157 C, yield = 81%. 1H-NMR (DMSO-d6)
δ
12.14 (s, 1H, NH), 10.77 (s, 1H, NH), 7.76 (d,
J = 2.0 Hz, 1H, Ph), 7.70 (d, J = 8.3 Hz, 1H, Ph), 7.64–7.54 (m, 1H, Ph), 6.71 (s, 1H, ArH), 5.07 (q,
J = 8.9 Hz, 2H, OCH2CF3), 2.37 (s, 3H, CH3). 13C-NMR (DMSO-d6)
170.4, 169.3, 167.5, 157.1, 149.5,
δ
136.7, 135.0, 132.0, 131.2, 130.3, 128.5, 125.7, 102.2, 62.9, 62.6, 62.3, 62.0, 24.2. HRMS (ESI): m/z calcd for
C15H11Cl2F3N4O3 [M + Na]+: 445.0058, found 445.0064.
2,6-Dichloro-N-((4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (25). White solid,
◦
mp 125–126 C, yield = 87%. 1H-NMR (-DMSO-d6)
δ
11.64 (s, 1H, NH), 10.90 (s, 1H, NH), 7.59
(d, J = 8.9 Hz, 2H, Ph), 7.56–7.48 (m, 1H, Ph), 7.12 (s, 1H, ArH), 5.11 (q, J = 8.9 Hz, 2H, OCH2CF3).
13C-NMR (DMSO-d6)
δ 170.0, 165.0, 161.8, 156.8, 148.7, 137.7, 132.1, 131.9, 129.0, 125.4, 103.2, 63.6, 63.3,
63.0. HRMS (ESI): m/z calcd for C14H8Cl3F3N4O3 [M + Na]+: 464.9512, found 464.9502.
2,6-Dichloro-N-((4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)benzamide (26). White solid, mp
◦
169–171 C, yield = 81%. 1H-NMR (DMSO-d6)
δ
12.42 (s, 1H, NH), 10.44 (s, 1H, NH), 7.61–7.47
(m, 3H, Ph), 6.54 (s, 1H, ArH), 3.46 (s, 4H, CH2CH3), 1.12 (s, 6H, CH2CH3). 13C-NMR (DMSO-d6)
δ
165.0, 164.0, 160.9, 158.5, 136.7, 131.1, 130.9, 128.1, 96.2, 42.6, 25.8, 12.4. HRMS (ESI): m/z calcd for
C16H16Cl3N5O2 [M + H]+: 416.0448, found 416.0444.
2,6-Dichloro-N-((4-(diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)benzamide (27). White
◦
solid, mp 140–142 C, yield = 87%. 1H-NMR (DMSO-d6)
δ
12.84 (s, 1H, NH), 10.22 (s, 1H, NH),
8.60–6.40 (m, 3H, Ph), 5.84 (s, 1H, ArH), 4.96 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.46 (s, 4H, CH2CH3), 1.12
(s, 6H, CH2CH3). 13C-NMR (DMSO-d6)
δ 169.2, 168.2, 163.3, 156.7, 149.8, 138.1, 131.5, 130.6, 129.7,
127.9, 80.4, 62.6, 62.3, 62.0, 61.7, 43.2, 13.4. HRMS (ESI): m/z calcd for C18H18Cl2F3N5O3 [M + Na]+:
502.0636, found 502.0635.
N-((4-(Benzylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (28). White
◦
solid, mp 76–78 C, yield = 80%. 1H-NMR (DMSO-d6)
δ
12.46 (d, J = 369.0 Hz, 1H, NH), 10.27 (s,
1H, NH), 8.25 (s, 1H, Ar-NH), 7.82–7.40 (m, 3H, Ph), 7.42–7.14 (m, 5H, Ph), 5.70 (s, 1H, ArH), 4.93 (q,
J = 9.0 Hz, 2H, OCH2CF3), 4.39 (s, 2H, N–CH2Ph). 13C-NMR (DMSO-d6)
164.0, 155.9, 151.0, 148.6,
δ
138.7, 136.7, 131.1, 130.8, 129.9, 128.3, 128.0, 127.9, 127.0, 126.1, 124.8, 122.6, 120.4, 81.6, 61.6, 61.3,
61.0, 60.7, 44.2, 30.6, 25.7. HRMS (ESI): m/z calcd for C21H16Cl2F3N5O3 [M + Na]+: 536.0480, found
536.0485.
N-((4-(Benzyl(ethyl)amino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (29).
1
White solid, mp 61–62 ◦C, yield = 75%. H-NMR (DMSO-d6)
δ
12.72 (s, 1H, NH), 10.28 (s, 1H, NH),
7.71–7.08 (m, 8H, Ph), 5.85 (s, 1H, ArH), 4.95 (d, J = 9.0 Hz, 2H, OCH2CF3), 4.74 (s, 2H, N–CH2Ph), 3.41
(d, J = 52.6 Hz, 2H, CH2CH3), 1.08 (s, 3H, CH2CH3). 13C-NMR (DMSO-d6) δ 169.3, 167.5, 166.0, 165.2,
164.7, 156.6, 156.3, 133.7, 131.1, 129.8, 129.5, 129.0, 128.9, 128.1, 125.8, 123.6, 81.1, 62.5, 62.2, 61.9, 60.7,
30.9, 15.0. HRMS (ESI): m/z calcd for C23H20Cl2F3N5O3 [M + H]+: 542.0974, found 542.0971.
N-((5-(Sec-butyl)-4-chloro◦-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-2,6-dichlorobenzamide (30).
White solid, mp 37–38 C, yield = 73%. 1H-NMR (DMSO-d6)
δ 11.64 (s, 1H, NH), 10.81 (s, 1H,
NH), 7.76–7.39 (m, 3H, Ph), 5.16 (q, J = 8.8 Hz, 2H, OCH2CF3), 3.21 (dt, J = 9.2, 6.8 Hz, 1H, CH),

Molecules 2018, 23, 2203
20 of 25
1.90–1.74 (m, 1H, CHCH2CH3), 1.68 (dt, J = 13.8, 6.7 Hz, 1H, CHCH2CH3), 1.29 (d, J = 7.1 Hz, 3H,
CHCH3), 0.83 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C-NMR (DMSO-d6) δ 167.7, 166.7, 160.4, 154.0, 148.7,
131.2, 129.1, 125.6, 123.4, 118.3, 63.9, 63.6, 63.3, 63.0, 31.6, 27.4, 18.6, 13.2. HRMS (ESI): m/z calcd for
C18H16Cl3F3N4O3 [M + Na]+: 521.0138, found 521.0157.
3.10. Synthesis of N-((4,6-bis(2,2,2-Trifluoroethoxy)pyrimidin-2-yl)carbamothioyl)-2-chlorobenzamide (31)
Compound 31 was prepared by the method given in [22]. 2-Chlorobenzoic acid (5 mmol) and
thionyl chloride (10 mL) were added to a 100 mL three-necked flask under N2. The reaction mixture
was refluxed for 3 h. The excess thionyl chloride was evaporated under reduced pressure to give a
colorless transparent liquid. Anhydrous acetonitri◦le (5 mL) was added to the residue, then KSCN
(5 mmol) was added to the system and reacted at 80 C for 1 h. The reaction mixture was cooled to the
room temperature, filtered and neutralized with triethylamine. Then intermediate Ia (5 mmol) and
TBAB (10 mmol) were added into reaction mixture. The mixture was heated to reflux for 4 h. When the
reaction was completed, the reaction mixture was cooled to room temperature, diluted with 20 mL of
EtOAc, washed twice with brine (20 mL), dried with anhydrous Na2SO4 and evaporated in vacuo.
Finally, the residue was purified by silica gel column chromatography to afford compound 31 as a
1
yellow solid, mp 93–95 ◦C, yield = 53%. H-NMR (DMSO-d6)
δ
12.84 (s, 1H, NH), 12.45 (s, 1H, NH),
7.71–7.62 (m, 1H, Ph), 7.56 (dd, J = 6.5, 1.7 Hz, 2H, Ph), 7.51–7.42 (m, 1H, Ph), 6.51 (s, 1H, ArH), 5.11
(q, J = 8.9 Hz, 4H, OCH2CF3). 13C-NMR (DMSO-d6)
δ
178.3, 170.8, 168.3, 156.2, 135.2, 133.3, 131.1,
130.7, 130.3, 128.2, 125.6, 123.4, 88.2, 63.9, 63.6, 63.3, 63.0. HRMS (ESI): m/z calcd for C16H11ClF6N4O3S
[M + Na]+: 511.0042, found 511.0015.
3.11. Synthesis of 2,6-Dichloro-N-((4-(diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamothioyl)-
benzamide (32a)
Compound 32a was synthesized by a method similar to that used for compound 31. Yellow solid,
◦
mp 42–43 C, yield = 59%. 1H-NMR (DMSO-d6)
δ
12.84 (s, 1H, NH), 10.22 (s, 1H, NH), 7.93–7.14
(m, 3H, Ph), 5.84 (s, 1H, ArH), 4.96 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.46 (s, 4H, CH2CH3), 1.12 (s, 6H,
CH2CH3). 13C-NMR (DMSO-d6)
δ 169.2, 168.2, 163.2, 156.7, 149.8, 132.5, 131.5, 130.6, 129.7, 127.9, 80.4,
62.6, 62.3, 62.0, 61.7, 43.2, 13.4. HRMS (ESI): m/z calcd for C18H18Cl2F3N5O2S [M + Na]+: 518.0408,
found 518.0407.
3.12. Synthesis of 2,6-Dichloro-N-(N-(4-(diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)-
carbamimidoyl)benzamide (32)
Compound 32 was synthesized by the method described in [23]. A solution of compound 32a
(0.37 g, 0.75 mmol) and EDCI (0.28 g, 1.49 mmol) was stirred at 0 ◦C for 30 min and
hexamethyldisilazide (HMDS, 1.2 g, 7.4 mmol) was added. After the mixture was stirred for 3 h
under 0 ◦C, the mixture was raised to room temperature and stood for 24 h. The organic layer was
diluted with EtOAc (20 mL), washed twice with a saturated NaHCO3 solution (20 mL) and brine
(20 mL), dried with anhydrous Na2SO4 and evaporated in vacuo. Finally, the residue was purified by
◦
silica gel column chromatography to afford compound 32 as a white solid, mp 166–168 C, yield = 81%.
1H-NMR (DMSO-d6)
δ
11.25 (s, 1H, NH), 9.56 (s, 1H, NH), 9.48 (s, 1H, NH), 7.46 (d, J = 8.1 Hz, 2H, Ph),
7.35 (dd, J = 8.8, 7.5 Hz, 1H, Ph), 5.85 (s, 1H, ArH), 4.98 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.69–3.40 (m, 4H,
CH2CH3), 1.13 (t, J = 7.0 Hz, 6H, CH2CH3). 13C-NMR (DMSO-d6)
176.3, 169.0, 163.2, 160.1, 157.5,
δ
140.6, 131.0, 130.6, 129.0, 128.9, 126.0, 123.7, 80.8, 62.7, 62.4, 62.2, 43.5, 13.6. HRMS (ESI): m/z calcd for
C18H19Cl2F3N6O2 [M + Na]+: 501.0796, found 501.0791.
3.13. Synthesis of the Target Compounds 33–38
Compounds 33–38 were prepared by a method similar to that used for compounds 1–30.

Molecules 2018, 23, 2203
21 of 25
5-Chloro-N-((4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)-1-methyl-1H-pyrazole-4-carboxamide (33).
1
White solid, mp 96–97 ◦C, yield = 88%. H-NMR (DMSO-d6)
δ
11.27 (s, 1H, NH), 10.73 (s, 1H, NH),
8.30 (s, 1H, ArH), 6.50 (s, 1H, ArH), 3.86 (s, 3H, N–CH3), 3.43 (s, 4H, CH2CH3), 1.11 (t, J = 7.0 Hz, 6H,
CH2CH3). 13C-NMR (DMSO-d6)
δ 162.7, 162.0, 159.0, 157.1, 149.7, 140.2, 131.8, 113.0, 97.3, 42.9, 37.6,
13.6. HRMS (ESI): m/z calcd for C14H17Cl2N7O2 [M + H]+: 386.0899, found 386.0929.
N-((5-(sec-Butyl)-4-chloro-6-(diethylamino)pyrimidin-2-yl)carbamoyl)-5-chloro-1-methyl-1H-pyrazole-4-car
◦
1
boxamide (34). White solid, mp 159–161 C, yield = 75%. H-NMR (DMSO-d6) δ 11.10 (s, 1H, NH), 10.87
(s, 1H, NH), 8.34 (s, 1H, ArH), 3.86 (s, 3H, N-CH3), 3.23 (dq, J = 14.0, 7.0 Hz, 2H, CHCH2CH3), 2.79 (q,
J = 7.3 Hz, 1H, CH), 1.78 (dtd, J = 15.2, 13.6, 7.3 Hz, 2H, CH2CH3), 1.39 (d, J = 7.2 Hz, 3H, CHCH3),
1.22 (t, J = 2.8 Hz, 2H, CH2CH3), 1.15 (t, J = 7.0 Hz, 6H, CH2CH3), 0.73 (t, J = 7.4 Hz, 3H, CHCH2CH3).
13C-NMR (DMSO-d6)
δ 169.4, 162.3, 158.9, 153.5, 149.7, 140.2, 131.9, 119.1, 112.7, 45.7, 37.5, 35.2, 27.9,
18.4, 13.8, 13.4. HRMS (ESI): m/z calcd for C18H25Cl2N7O2 [M + Na]+: 464.1344, found 464.1363.
5-Chloro-N-((4-chloro-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)-1-methyl-1H-pyrazole-4-carboxamide
1
(
35). White solid, mp 181–183 ◦C, yield = 85%. H-NMR (DMSO-d6)
δ
11.40 (s, 1H, NH), 11.10 (s, 1H,
NH), 8.37 (s, 1H, ArH), 7.07 (s, 1H, ArH), 5.11 (q, J = 8.9 Hz, 2H, OCH2CF3), 3.86 (s, 3H, N–CH3).
13C-NMR (DMSO-d6)
δ 169.9, 162.8, 161.8, 156.8, 149.4, 140.3, 132.2, 112.3, 102.9, 63.5, 63.2, 62.9, 37.5.
HRMS (ESI): m/z calcd for C12H9Cl2F3N6O3 [M + H]+: 434.9963, found 434.9975.
N-((5-(sec-Butyl)-4,6-dichloropyrimidin-2-yl)carbamoyl)-2-chloronicotinamide (36). White solid, mp
◦
139–140 C, yield = 86%. 1H-NMR (DMSO-d6)
δ 11.49 (s, 1H, NH), 10.99 (s, 1H, NH), 8.55 (dd,
J = 4.9, 1.9 Hz, 1H, ArH), 8.09 (dd, J = 7.6, 1.9 Hz, 1H, ArH), 7.58 (dd, J = 7.6, 4.8 Hz, 1H, ArH), 3.40 (dt,
J = 9.0, 7.1 Hz, 1H, CH), 2.01–1.85 (m, 1H, CHCH2CH3), 1.73 (dt, J = 13.9, 7.1 Hz, 1H, CHCH2CH3),
1.33 (d, J = 7.2 Hz, 3H, CHCH3), 0.81 (t, J = 7.4 Hz, 3H, CHCH2CH3). 13C-NMR (DMSO-d6)
δ 167.9,
154.5, 152.1, 148.9, 146.8, 139.1, 132.4, 129.8, 124.0, 37.1, 27.1, 18.2, 13.3. HRMS (ESI): m/z calcd for
C15H14Cl3N5O2 [M + Na]+: 424.0111, found 424.0112.
2-Chloro-N-((4-(diethylamino)-6-(2,2,2-trifluoroethoxy)pyrimidin-2-yl)carbamoyl)nicotinamide (37). White
1
solid, mp 124–126 ◦C, yield = 85%. H-NMR (DMSO-d6)
δ
12.33 (s, 1H, NH), 10.30 (s, 1H, NH), 8.53
(dd, J = 4.9, 1.9 Hz, 1H, ArH), 8.06 (dd, J = 7.6, 1.9 Hz, 1H, ArH), 7.55 (dd, J = 7.6, 4.9 Hz, 1H, ArH), 5.84
(s, 1H, ArH), 4.97 (q, J = 9.0 Hz, 2H, OCH2CF3), 3.47 (s, 4H, CH2CH3), 1.11 (t, J = 7.0 Hz, 6H, CH2CH3).
13C-NMR (DMSO-d6)
δ 169.2, 167.3, 163.1, 156.7, 151.5, 146.5, 138.6, 133.5, 125.9, 124.0, 80.5, 62.6, 62.3,
62.1, 61.8, 43.4, 13.5. HRMS (ESI): m/z calcd for C17H18ClF3N6O3 [M + Na]+: 469.0979, found 469.0966.
◦
1-(4-Chloro-6-(diethylamino)pyrimidin-2-yl)-3-(2,6-dichlorophenyl)urea (38). White solid, mp 98–99 C,
1
yield = 78%. H-NMR (DMSO-d6)
δ
10.90 (s, 1H, NH), 10.11 (s, 1H, NH), 7.58 (d, J = 8.1 Hz, 2H, Ph),
7.37 (t, J = 8.1 Hz, 1H, Ph), 6.47 (s, 1H, ArH), 3.33 (s, 4H, CH2CH3), 2.50 (s, 6H, CH2CH3). 13C-NMR
(DMSO-d6)
δ 161.3, 158.9, 157.5, 151.8, 133.9, 132.8, 129.5, 129.0, 95.6, 43.1, 12.8. HRMS (ESI): m/z calcd
for C15H16Cl3N5O [M + H]+: 388.0499, found 388.0488.
3.14. Insecticidal Biological Assay
All bioassays were performed on representative test organisms reared in the laboratory.
◦
The bioassay was repeated at 25
±
1 C according to statistical requirements. Assessments were made
on a dead/alive basis and mortality rates were corrected using Abbott’s formula [24]. Evaluations are
based on a percentage scale of 0–100 in which 0 = no activity and 100 = total kill.
3.14.1. Toxicity against Mosquito (Culex pipiens pallens)
The toxicities of compounds
1–25, 31 and 37 against mosquito were evaluated according to the
reported procedure [25 26]. One milliliter of different concentrated dilutions of each compound was
,
added to 99 mL of water to obtain different concentrations of tested solution. Then 20 fourth-instar
mosquito larvae were put into the solution. Percentage mortalities were evaluated 8 day after treatment.

Molecules 2018, 23, 2203
22 of 25
For comparative purposes, fipronil was tested under the same conditions, and each test was performed
in triplicate.
3.14.2. Stomach Toxicity against Oriental Armyworm (Mythimna separata)
The stomach toxicities of compounds 1–25, 31, 37 and the contrast fipronil against oriental
armyworm were evaluated by foliar application using the reported procedure [27]. For the foliar
armyworm tests, individual corn leaves were placed on moistened pieces of filter paper in Petri dishes.
The leaves were then sprayed with the test solution and allowed to dry. The dishes were infested
with 10 fourth-instar Oriental armyworm larvae. Percentage mortalities were evaluated 3 days after
treatment. Each treatment was performed three times
3.15. In Vitro Antifungal Bioassay
The antifungal activities were screened and evaluated by the poison plate technique [28]. All final
compounds were dissolved in DMF (0.1 mL) before mixing with potato dextrose agar (PDA; 9.9 mL).
The compounds were tested at a concentration of 50 µ
g mL−1. All fungi were cultivated in PDA
at 27 ± 1 ◦C for 4 days to make new mycelium for the identification of antifungal activity. Then,
mycelia dishes of approximately 5 mm diameter were cut from the culture medium. A mycelium
was obtained using a germ-free inoculation needle and i◦noculated in the middle of the PDA plate
aseptically. The inoculated plates were incubated at 27
±
1 C for 5 days. DMF in sterile distilled water
served as the negative control, whereas hymexazol served as the positive control. Each treatment
condition consisted of three replicates. Radial growth of the fungal colonies was measured, and the
data were statistically analyzed. Inhibitory effects of the test compounds in vitro on these fungi were
calculated by the formula I (%) = [(C
growth on untreated PDA, T represents the diameter of fungi on treated PDA, and I represents the
−
T)/(C
−
0.5)]
×
100, where C represents the diameter of fungal
inhibition rate.
3.16. In Vivo Antifungal Bioassay against Sclerotinia sclerotiorum
The requisite amounts of compounds 19 and 25 was disolved in sterile Tween 80 (0.1%, v/v)
solution to give different concentration test solution (500, 1500 and 3000 µ
g mL−1). For protective
activity assay [29], fresh leaves were sprayed with these solution (10 mL for each leave) until liquid
flowed on surface at 24 h before inoculation. A colonized mycelial plug (5 mm in diameter) from
a 5-day-old PDA culture of carbendazim-resistant Sclerotinia sclerotiorum was placed on the surface
◦
of fresh leaves. Inoculated leaves were placed at 25 C with 80% relative humidity for disease
development. After 5 days, the average lesion diameter was determined by measuring each lesion
in two perpendicular directions. The lengths of the long and short axes were averaged and disease
control efficacy was calculated as follows: disease control efficacy (%) = (lesion diameter in the water
control-lesion diameter in the treatment)/lesion diameter in the water control × 100 [30].
3.17. Zebrafish Embryo Toxicity Assay
Zebrafish wild-type AB (Danio rerio AB) was introduced from the National Zebrafish Center
(Wuhan, China). The zebrafish for experiment was cultured by the five-layer zebrafish culture system
of Beijing Aisheng Technology Development Co., Ltd. (Beijing, China) to the fifth generation. Animals
◦
were housed in feeding system (pH 6.5–7.5, oxygen content >85%, water temperture 28 C, conductivity
500
±
50 µ
s cm−1, Light-dark ratio 14 h/10 h). Natural mortality during feeding was less than 1%.
Fish were fed twice a day with Artemia salina, the total weight of feeding was 5
±
1% of zebrafish body
weight. For this experiment, a sexually mature 8-month zebrafish were used. Males and females were
maintained separately until the night before the spawning at a ratio of 1:2 and the light-dark ratio was
controlled at 14 h/10 h. Embryos were obtained by naturally mate [31].
Toxicological analyses were based on approved standard OECD TG 236: Fish Embryo Toxicity
(FET) Test [32]. 20 embryos were distributed in each of different concentrations of the compounds

Molecules 2018, 23, 2203
23 of 25
to be analyzed, a positive control (3,4-dichloroaniline), a negative control (water) and a solvent
control (10% Hank’s solution). All of the treatments were cared at 26 ◦C 1 ◦C, light/dark cycle of
14/10 h. The entire experiment was carried out for 96 h. All of the data were analyzed by SPSS 19.0
IBM Corporation, New York, NY, USA), LC50 was corrected taking into account control mortality
±
(
with Abbott’s formula [15].
4. Conclusions
In summary, a series of novel benzoylpyrimidinylurea derivatives were designed and synthesized.
Although we did not find any compounds which possessed both insecticidal and antifungal as efficient
as we expected, compounds
7 and 25 still exhibited excellent larvicidal activity against mosquito
(Culex pipiens pallens) and broad-spectrum antifungal activity against fourteen phytopathogenic fungi,
respectively. High efficiency, low toxicity and environmentally friendly pesticides are consistent
with the requirements of sustainable agricultural development. Therefore, compounds
7 and 25
with low toxicity to zebrafish will be potential lead compounds to develop green mosquitocides and
broad-spectrum fungicides.
Supplementary Materials: The following are available online. Figures S1–S101.
Author Contributions: R.S. conceived and designed the experiments; P.C., X.S. and W.K. performed the
experiments, analyzed the data; X.S. drafted the manuscript; R.S. revised the manuscript; Y.F. and H.Z. gave some
suggestion to the experiment.; P.C and X.S contributed equally to this work. All authors have read and approved
the final manuscript.
Funding: This work was financially supported by the National Science Foundation of China (No. 21462028),
Startup Foundation for Outstanding Young Scientists of Hainan University (kyqd1640) and Hainan Province
Natural Science Foundation of China (No. 20163040).
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Ishaaya, I.; Degheele, D. Insecticides with Novel Modes of Action: Mechanisms and Application; Springer:
Berlin/Heidelberg, Germany, 1998; p. 289, ISBN 9783540630586.
Oberlander, H.; Silhacek, D.L. Mode of action of insect growth regulators in lepidopteran tissue culture.
Pestic. Sci. 1998, 54, 300–302. [CrossRef]
Sun, R.F.; Liu, C.J.; Zhang, H.; Wang, Q.M. Benzoylurea Chitin Synthesis Inhibitors. J. Agric. Food Chem. 2015
,
63, 6847–6865. [CrossRef] [PubMed]
Sun, R.F.; Lu, M.Y.; Chen, L.; Li, Q.S.; Song, H.B.; Bi, F.C.; Huang, R.Q.; Wang, Q.M. Design, synthesis,
bioactivity, and structure-activity relationship (SAR) studies of novel benzoylphenylureas containing oxime
ether group. J. Agric. Food Chem. 2008, 56, 11376–11391. [CrossRef] [PubMed]
5.
Sun, R.F.; Li, Y.Q.; Lu, M.Y.; Xiong, L.X.; Wang, Q.M. Synthesis, larvicidal activity, and SAR studies of new
benzoylphenylureas containing oxime ether and oxime ester group. Bioorg. Med. Chem. Lett. 2010, 20,
4693–4699. [CrossRef] [PubMed]
6.
7.
Sun, R.F.; Zhang, Y.L.; Bi, F.C.; Wang, Q.M. Design, synthesis, and bioactivity study of novel benzoylpyridazyl
ureas. J. Agric. Food Chem. 2009, 57, 6356–6361. [CrossRef] [PubMed]
Sun, R.F.; Li, Y.Q.; Xiong, L.X.; Liu, Y.X.; Wang, Q.M. Design, synthesis, and insecticidal evaluation of
new benzoylureas containing isoxazoline and isoxazole group. J. Agric. Food Chem. 2011, 59, 4851–4859.
[CrossRef] [PubMed]
8.
Sun, R.F.; Wang, Z.W.; Li, Y.Q.; Xiong, L.X.; Liu, Y.X.; Wang, Q.M. Design, synthesis, and insecticidal
evaluation of new benzoylureas containing amide and sulfonate groups based on the sulfonylurea receptor
protein binding site for diflubenzuron and glibenclamide. J. Agric. Food Chem. 2013, 61, 517–522. [CrossRef]
[PubMed]
9.
Niyaz, N.M.; Hunter, R.; Johnson, T.C.; Trullinger, T.K.; Brown, A.V.; Bryan, K. Preparation of Substituted
N-azinyl-N'-aryl Ureas as Insecticides. U.S. Patent 20090093486A1, 9 April 2009.
10. Gewehr, M. Fungicidal Mixtures Comprising Quinoline Fungicides. WO2018050421A1, 22 March 2018.

Molecules 2018, 23, 2203
24 of 25
11. Eberlin, A.R.; Frampton, C.S. A metastable polymorphic form of the antifungal anilinopyrimidine active
pyrimethanil. Acta Crystallogr. Sect. E Crystallogr. Commun. 2017, 73, 886–889. [CrossRef] [PubMed]
12. Li, Y.; Liu, S.; Qin, Y.; Xing, R.; Chen, X.; Li, K.; Li, P. Synthesis of novel pyrimethanil grafted chitosan
derivatives with enhanced antifungal activity. BioMed Res. Int. 2016, 2016, 8196960. [CrossRef] [PubMed]
13. Choi, W.S.; Nam, S.W.; Ahn, E.K.; Park, B.S.; Lee, S.E.; Kim, T.J.; Choi, I.Y. Synthesis and fungicidal activity of
N-[4-(4-fluoro)phenyl-2-piperidin-4-yl-thiazol-5-yl]pyrimidin-2-yl-N-phenylamines on Phytophthora capsici.
J. Korean Soc. Appl. Biol. Chem. 2010, 53, 206–214. [CrossRef]
14. Nam, S.W.; Choi, I.Y.; Choi, W.S. Synthesis and antifungal activity of 5-[2-(Alkylamino)pyrimidin-4-yl]-4-
phenylthiazol-2-cycloalkylamine derivatives on Phytophthora capsici. J. Korean Soc. Appl. Biol. Chem. 2011, 54,
395–402. [CrossRef]
15. Fan, Y.M.; Feng, Q.; Lai, K.H.; Huang, W.K.; Zhang, C.H.; Li, Q.X. Toxic effects of indoxacarb enantiomers on
the embryonic development and induction of apoptosis in zebrafish larvae (Danio rerio). Environ. Toxicol.
2017, 32, 7–16. [CrossRef] [PubMed]
16. Fan, Y.M.; Miao, W.G.; Lai, K.H.; Huang, W.K.; Song, R.X.; Li, Q.X. Developmental toxicity and inhibition
of the fungicide hymexazol to melanin biosynthesis in zebrafish embryos. Pestic. Biochem. Phys. 2018, 147,
139–144. [CrossRef] [PubMed]
17. Zhang, J.; Jin, G.Y.; Xiao, S.; Wu, J.; Cao, S. Novel synthesis of 1,4,5-trisubstituted 1,2,3-triazoles via a
one-pot three-component reaction of boronic acids, azide, and active methylene ketones. Tetrahedron 2013
69, 2352–2356. [CrossRef]
,
18. Sreedhar, B.; Arundhathi, R.; Reddy, P.L.; Kantam, M.L. CuI Nanoparticles for C-N and C-O Cross Coupling
of Heterocyclic Amines and Phenols with Chlorobenzenes. J. Org. Chem. 2009, 74, 7951–7954. [CrossRef]
[PubMed]
19. Jansa, P.; Holy, A.; Dracinsky, M.; Kolman, V.; Janeba, Z.; Kostecka, P.; Kmonickova, E.; Zidek, Z. 5-Substituted
2-amino-4,6-dihydroxypyrimidines and 2-amino-4,6-dichloropyrimidines. Synthesis and inhibitory effects
on immune-activated nitric oxide production. Med. Chem. Res. 2014, 23, 4482–4490. [CrossRef]
20. He, H.F.; Wang, X.Y.; Shi, L.Q.; Yin, W.Y.; Yang, Z.W.; He, H.W.; Liang, Y. Synthesis, antitumor activity and
mechanism of action of novel 1,3-thiazole derivatives containing hydrazide-hydrazone and carboxamide
moiety. Bioorg. Med. Chem. Lett. 2016, 26, 3263–3270. [CrossRef] [PubMed]
21. Lei, P.; Xu, Y.; Du, J.; Yang, X.L.; Yuan, H.Z.; Xu, G.F.; Ling, Y. Design, synthesis and fungicidal activity
of N-substituted benzoyl-1,2,3,4-tetrahydroquinolyl-1-carboxamide. Bioorg. Med. Chem. Lett. 2016, 26,
2544–2546. [CrossRef] [PubMed]
22. Mawnai, I.L.; Adhikari, S.; Kaminsky, W.; Kollipara, M.R. Synthesis of strained complexes of arene d6 metals
with benzoylthiourea and their spectral studies. J. Organomet. Chem. 2018, 869, 26–36. [CrossRef]
23. Shinada, T.; Umezawa, T.; Ando, T.; Kozuma, H.; Ohfune, Y. A new entry for the synthesis of N-acyl-N
'-
substituted guanidines. Tetrahedron Lett. 2006, 47, 1945–1947. [CrossRef]
24. Abbott, W.S. A method of computing the effectiveness of an insecticide. J. Am. Mosq. Control Assoc. 1987, 3,
302–303. [CrossRef] [PubMed]
25. Zhao, Q.Q.; Li, Y.Q.; Xiong, L.X.; Wang, Q.M. Design, Synthesis and Insecticidal Activity of Novel
Phenylpyrazoles Containing a 2,2,2-Trichloro-1-alkoxyethyl Moiety. J. Agric. Food Chem. 2010, 58, 4992–4998.
[CrossRef] [PubMed]
26. Labbe, P.; Berticat, C.; Berthomieu, A.; Unal, S.; Bernard, C.; Weill, M.; Lenormand, T. Forty years of erratic
insecticide resistance evolution in the mosquito Culex pipiens. PLoS Genet. 2007, 3, 2190–2199. [CrossRef]
[PubMed]
27. Zhao, Q.Q.; Shang, J.; Liu, Y.X.; Wang, K.Y.; Bi, F.C.; Huang, R.Q.; Wang, Q.M. Synthesis and insecticidal
activities of novel N-sulfenyl-N'-tert-butyl-N,N'-diacylhydrazines. 1. N-alkoxysulfenate derivatives. J. Agric.
Food Chem. 2007, 55, 9614–9619. [CrossRef] [PubMed]
28. Song, X.M.; Liu, C.J.; Chen, P.Q.; Zhang, H.; Sun, R.F. Natural Product-Based Pesticide Discovery: Design,
Synthesis and Bioactivity Studies of N-Amino-Maleimide Derivatives. Molecules 2018, 23, 1521. [CrossRef]
[PubMed]
29. Hou, Y.; Mao, X.; Lin, S.; Song, X.; Duan, Y.; Wang, J.; Zhou, M. Activity of a novel succinate dehydrogenase
inhibitor fungicide pyraziflumid against Sclerotinia sclerotiorum. Pestic. Biochem. Phys. 2018, 145, 22–28.
[CrossRef] [PubMed]

Molecules 2018, 23, 2203
25 of 25
30. Kuang, J.; Hou, Y.P.; Wang, J.X.; Zhou, M.G. Sensitivity of Sclerotinia sclerotiorum to fludioxonil: In vitro
determination of baseline sensitivity and resistance risk. Crop Prot. 2011, 30, 876–882. [CrossRef]
31. Bridi, D.; Altenhofen, S.; Gonzalez, J.B.; Reolon, G.K.; Bonan, C.D. Glyphosate and Roundup® alter
morphology and behavior in zebrafish. Toxicology 2017, 392, 32–39. [CrossRef] [PubMed]
32. Kluever, N.; Vogs, C.; Altenburger, R.; Escher, B.I.; Scholz, S. Development of a general baseline toxicity
QSAR model for the fish embryo acute toxicity test. Chemosphere 2016, 164, 164–173. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds are not available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).