Natural product-based pesticide discovery: Design, synthesis and bioactivity studies of N-amino-maleimide derivatives

Article abstract of DOI:10.3390/molecules23071521

Natural products are an important source of pesticide discovery. A series of N-amino-maleimide derivatives containing hydrazone group were designed and synthesized based on the structure of linderone and methyllinderone which were isolated from Lindera erythrocarpa Makino. According to the bioassay results, compounds 2 and 3 showed 60% inhibition against mosquito (Culex pipiens pallens) at 0.25 μg·mL^?1. Furthermore, the results of antifungal tests indicated that most compounds exhibited much better antifungal activities against fourteen phytopathogenic fungi than linderone and methyllinderone and some compounds exhibited better antifungal activities than commercial fungicides (carbendazim and chlorothalonil) at 50 μg·mL^?1. In particular, compound 12 exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against 11 phytopathogenic fungi) and compounds 12 and 14 displayed 60.6% and 47.9% inhibitory activity against Rhizoctonia cerealis at 12.5 μg·mL^?1 respectively. Furthermore, compound 17 was synthesized, which lacks N-substituent at maleimide and its poor antifungal activity against Sclerotinia sclerotiorum and Rhizoctonia cerealis at 50 μg·mL^?1 showed that the backbone structure of N-amino-maleimide derivatives containing hydrazone group was important to the antifungal activity.

Full text of DOI:10.3390/molecules23071521

molecules
Article
Natural Product-Based Pesticide Discovery:
Design, Synthesis and Bioactivity Studies of
N-Amino-Maleimide Derivatives
Xiangmin Song 1, Chunjuan Liu 2, Peiqi Chen 2, Hao Zhang 2 and Ranfeng Sun 1,
*
1
Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China;
18898958572@163.com
School of Chemical & Chemical Engineering, Inner Mongolia University, Hohhot 010021, China;
17330926558@163.com (C.L.); chenpq17@lzu.edu.cn (P.C.); haozhang@imu.edu.cn (H.Z.)
Correspondence: srf18@hainu.edu.cn; Tel.: +86-898-6619-2906
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 31 May 2018; Accepted: 21 June 2018; Published: 24 June 2018
Abstract: Natural products are an important source of pesticide discovery. A series of
N-amino-maleimide derivatives containing hydrazone group were designed and synthesized based
on the structure of linderone and methyllinderone which were isolated from Lindera erythrocarpa
Makino. According to the bioassay results, compounds
2 and 3 showed 60% inhibition against
mosquito (Culex pipiens pallens) at 0.25
that most compounds exhibited much better antifungal activities against fourteen phytopathogenic
fungi than linderone and methyllinderone and some compounds exhibited better antifungal
µ
g
·
mL−1. Furthermore, the results of antifungal tests indicated
activities than commercial fungicides (carbendazim and chlorothalonil) at 50
µg
mL−1. In particular,
·
compound 12 exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against
11 phytopathogenic fungi) and compou−nd1 s 12 and 14 displayed 60.6% and 47.9% inhibitory activity
µg mL respectively. Furthermore, compound 17 was synthesized,
against Rhizoctonia cerealis at 12.5
which lacks N-substituent at maleimide and its poor antifungal activity against Sclerotinia sclerotiorum
·
and Rhizoctonia cerealis at 50
derivatives containing hydrazone group was important to the antifungal activity.
µ
g
·
mL−1 showed that the backbone structure of N-amino-maleimide
Keywords: maleimide; linderone; methyllinderone; antifungal activity
1. Introduction
Chitin is a unique component of the fungal cell wall and shells of crustaceans, but it is absent
in vertebrates, mammals, and humans [1]. Chitin synthase is thus an attractive molecular target
for developing fungicides and insecticides. Natural products are an important source for drug and
pesticide discovery. Lindera species (Lauraceae) have rich chemical compositions and pharmacological
activities [
(Figure 1), were isolated from Lindera erythocarpa Makino (Lauraceae) and exhibited inhibitory
activity against chitin synthase 2 (CaCHS2p) with IC50 value of 23.3, 21.4
mL−1 respectively,
which was better than polyoxin D (70.0 ]. In 2015,
mL−1) and nikkomycin Z (176.0 mL−1) [
Seok-Hee Lee et al. reported that methyllinderone ( ) and methyllucidone ( ) (Figure 1) exhibited
juvenile hormone antagonistic activity against Aedes aegypti [ ]. Sheng-Yang Wang et al. reported that
linderone ( ), methyllinderone ( ), lucidone ( ) and methyllucidone ( ) (Figure 1) displayed good
anti-inflammatory activity [5].
2]. Methyllinderone (A) and linderone (B), which were classic inhibitors of chitin synthetase
µ
g
·
·
µg
·
µg
3
A
C
4
D
A
E
C
Molecules 2018, 23, 1521; doi:10.3390/molecules23071521
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1521
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Figure 1. Chemical structures of methyllinderone (
lucidone (E).
A), linderone (B), methyllucidone (C), linderone (D),
Interestingly, it was found that compounds containing a maleimide fragment also have chitin
synthase and -1,3-glucan synthase inhibitory activity according to the literature. For example,
compounds and (Figure 2) inhibited -1,3-glucan synthase with IC50 value of 8.5, 17.3
mL−1
respectively [ ]. In 2007, Atul R. Gholap et al. reported compound (Figure 2) containing maleimide
β
F
6
G
β
µg
·
,
H
group performed over 91% inhibitory against chitin synthase, which exhibited comparable activity
with nikkomycin Z (90%) [7].
Figure 2. Structures of compounds with maleic amide fragment.

Molecules 2018, 23, 1521
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Therefore, we focused on introducing maleimide fragments into natural products to obtain more
efficient fungicides and insecticides. A series of N-amino-maleimide derivatives containing hydrazone
group were designed based on the structure of methyllinderone, linderone and acylhydrazone
derivatives with excellent antifungal activity (Figure 3) [
synthesized and their biological activities were evaluated.
8]. Then target compounds 1–16 were
Figure 3. Design strategy for N-amino-maleimide derivatives.
2. Results
2.1. Synthesis
Different aryl-substituted unsaturated ketones, which were synthesized according to the
literature [ ], and N-amino-maleimide, which were produced in situ without purification [10],
are refluxed in dry EtOH with a catalytic amount of p-toluenesulfonic acid to produce a variety
of N-amino-maleimide derivatives containing hydrazone group (compounds 16, Scheme 1) and
9
1–
NMR spectral information was listed (Figures S1–S34). Although the reaction produced good yields,
the maleic anhydride and hydrazine hydrate cyclized to form a six-membered ring isomer (Figure 4)
which is in accordance with previous report [10].
Scheme 1. General synthetic route for compounds 1–16.

Molecules 2018, 23, 1521
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Figure 4. Synthesis of six-membered ring isomers.
At the same time, linderone and methyllinderone were synthesized according to the literature
(Scheme 2) [11].
Scheme 2. Synthetic route for linderone and methyllinderone.
Finally, 3,4-dichloro-1H-pyrrole-2,5-dione (17) was synthesized to further illustrate the active
functional group of compounds 1–16 (Scheme 3) [10].
Scheme 3. Synthetic route for compound 17.

Molecules 2018, 23, 1521
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2.2. Bioassays
2.2.1. Stomach Toxicity against Oriental Armyworm (Mythimna separata)
It can be seen from Table 1 that some compounds have considerable insecticidal activities against
oriental armyworm and seemed to display some electronic effects. For example, comparing the
insecticidal activities of compounds
is unsubstituted, the electron-rich substituents of maleimide unit will significantly enhance the
insecticidal activity of the compound, the insecticidal activity values of compounds (X = H),
6 (X = 2-CH3) 10 (X = 2,3-Cl) are 25%, 65% and 20% respectively. When comparing two sets of
the compounds substituted in maleinimide fragment (X = H) or (X = 2-CH3), it was found that
they displayed different structure–activity relationships (SARS). For example, when the maleimide
units are unsubstituted (X = H), compound (R = 3,5-CF3) and (R = 2,4-Cl) displayed superior
insecticidal activities (60%, 50% respectively) than compound (R = 4-NO2) and (R = 2-F) (10%,
25% respectively), whereas compound (R = H) exhibited the best insecticidal activities when the
1, 6, 10, we can easily find that when the phenyl ring (R = H)
1
,
1
–5
6–8
2
3
4
5
6
substituent of maleimide units is methyl group (X = 2-CH3). In general, the insecticidal activity of the
meta-substitute on the phenyl ring is superior to that of the ortho and para-substitute.
Table 1. Larvacidal activities against oriental armyworm and mosquito of compounds 1–8, 10.
−1
Larvicidal Activity (%) at Concn (µg·mL
Oriental Armyworm Mosquito
)
Compd.
600
10
5
2
1
0.5
0.25
a
1
2
3
4
5
6
7
8
10
25
60
50
10
25
65
40
5
50
-
-
-
-
-
60
60
-
-
-
-
20
-
100 100 100 100 100
100 100 100 100 100
40
30
50
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
100
20
100 100 100 100 100
100 100 40
- no test data.
20
-
-
a
2.2.2. Toxicity against Mosquito (Culex pipiens pallens)
Table 1 showed the larvacidal activities of the compounds
1
–
8
,
10 against mosquito. The bioassay
) exhibited excellent larvicidal activities
(X = H, R = 3,5-CF3) and (X = H, R = 2,4-Cl) which showed
mL−1. This seems to indicate that the substitution of the meta position
results indicated that some compounds (compounds
against mosquito, especially compounds
60% mortality even at 0.25
2, 3 and 8
2
3
µg
·
on the phenyl ring or the substitution of the ortho and para positions will have a greater effect on the
activity of the compounds.
2.2.3. In Vitro Antifungal Activity
The fungicidal results of compounds 1–17 are listed in Tables 2 and 3. Most compounds
exhibited much better antifungal activities against fourteen phytogenic fungi than linderone and
methyllinderone and some compounds exhibited better antifungal activities than commercial
fungicides (carbendazim and chlorothalonil). All the compounds except compound
showed more than 50% inhibition rate against Rhizoctonia cerealis at 50
mL−1 and six compounds
10 13 14 16) showed better antifungal activities than linderone (58.2%) and methyllinderone (74.5%),
in particular compounds 12 14, and 15 with 98.2%, 100.0%, 90.9% of inhibition rate respectively.
When the concentration was adjusted to 25
mL−1 and 12.5 mL−1, the inhibition rate of
1, 4–6 and 9
µg
·
(
–
,
–
,
µg
·
µg
·

Molecules 2018, 23, 1521
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compound 12 against Rhizoctonia cerealis was 95.8%, 60.6% and the inhibition rate of compound 14
against Rhizoctonia cerealis was 77.5%, 47.9% respectively (Table 3). Compound 10 14 and
16 showed more than 50% inhibition rate against Botryospuaeria berengeriana at 50
mL−1, and five
of them (compound 10 and 14) were superior to linderone (57.1%) and methyllinderone
(8.2%), for example, compound exhibited 89.3% inhibitory activity. Compound 10 12 and 14 16
showed more than 60% inhibition rate against Sclerotinia sclerotiorum at 50
mL−1, which were even
superior to chlorothalonil (<50%). Compound 11 12 15 and 16 showed more than 50% inhibition
rate against Cercospora arachidicola at 50
mL−1, which were even superior to carbendazim (<50%).
Compound 12 15 and 16 displayed more than 50% inhibition rate against Helminthosporium maydis at
50 12
mL−1, which were superior to linderone (27.8%) and methyllinderone (38.9%). Compound 10
and 15 16 showed more than 50% inhibition rate against Botrytis cinerea at 50
mL−1, which were
superior to linderone (27.8%) and methyllinderone (38.9%). Compound 10 12 and 14 16 showed
more than 50% inhibition rate against Rhizoctonia solani at 50
mL−1, which were superior to
linderone (28.4%) and methyllinderone (24.7%). Compound 12 and 14 16 showed more than 50%
inhibition rate against Phytophthora capsica at 50
mL−1, which were superior to linderone (27.8%) and
2
,
4
,
6,
7,
,
µg
·
2, 4, 6,
4
–
–
µg
·
8,
,
,
µg
·
4,
,
µg
·
–
–
µ
g
·
2,
7,
,
–
µg
·
–
µg
·
methyllinderone (27.8%). In particular, compound 14 exhibited broad-spectrum fungicidal activities
against test phytogenic fungi and even displayed much better antifungal activities against five fungi
(Botrytis cinereal, Fusarium oxysporum f.sp. cucumerinum, Cercospora arachidicola, Fusarium moniliforme
and Phytophthora capsica) than carbendazim (<50%). At the same time, we found that the substituents
of maleimide have an important effect on the antifungal activity, for example, compound 12 (X = 2,3-Cl,
R = 2,4-Cl) exhibits more power and broad-spectrum antifungal activity than compounds
3 (X = H,
R = 2,4-Cl), (X = 2-CH3, R = 2,4-Cl). Finally, in order to prove whether the maleimide has antifungal
8
activity as reported in the literature [12], we synthesized compound 17 and then tested their inhibitory
activities against Sclerotinia sclerotiorum and Rhizoctonia cerealis, which was 21.4%, 23.6% respectively
at 50
µg 1–16 are
mL−1 (Table 3). This result indicates that the antifungal activities of compounds
·
dependent by the entire pesticide molecule rather than the maleimide fragment.
Table 2. Fungicidal activity of title compounds 1–16 and Linderone, Methyllinderone at 50 µg·mL
Inhibition Rate (%)
−1
.
Compd.
a
A.S
G.Z P.I S.S B.C R.S F.O C.A B.B R.C H.M C.L F.M P.C
b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
25.0
33.3
8.3
25
8.3
17.2 17.6 8.9 15.4 6.2 11.6 36.7 26.8 34.9 32.7 10.0 18.2
20.7 23.5 37.5 34.6 54.3 23.3 26.7 66.1 50.0 32.7 22.5 30.3
24.1 17.6 17.9 26.9 37.0 32.6 46.7 35.7 57.0 34.7 32.5 57.6
37.9 29.4 26.8 34.6 18.5 27.9 46.7 89.3 29.1 61.2 27.5 39.4
10.3 17.6 8.9 7.7 12.3 14.0 36.7 25.0 31.4 30.6 5.0 15.2
13.8 11.8 32.1 19.2 30.9 16.3 13.3 62.5 23.3 20.4 15.0 30.3
10.3 23.5 21.4 19.2 53.1 20.9 36.7 51.8 58.1 26.5 22.5 30.3
17.2 17.6 39.3 23.1 45.7 25.6 56.7 48.2 64.0 34.7 32.5 27.3
44.8 23.5 5.4 3.8 14.8 16.3 30.0 7.1 40.7 26.5 17.5 21.2
44.8 58.8 75.0 73.1 66.7 39.5 46.7 75.0 79.1 44.9 47.5 48.5
-
-
-
-
-
-
-
-
-
-
25
16.7
16.7
33.3
41.7
27.8
44.4
44.4
44.4
44.4
44.4
27.8
38.9
39.5 18.2 63.4 52.4 43.2 45.7 50.0 14.3 89.1 47.2 40.7 46.2 33.3
47.4 50.0 87.5 71.4 67.9 65.7 65.0 42.9 98.2 63.9 70.4 61.5 55.6
36.8 27.3 26.8 47.6 42 51.4 35.0 34.7 61.8 27.8 18.5 38.5 27.8
39.5 54.5 60.6 47.6 67.9 45.7 25.0 67.3 100.0 55.6 44.4 46.2 69.4
44.7 63.6 66.2 52.4 63.0 45.7 50.0 36.7 90.9 52.8 51.9 46.2 69.4
42.1 45.5 63.4 52.4 61.7 45.7 55.0 51.0 89.1 52.8 37.0 53.8 63.9
31.6 27.3 66.2 47.6 28.4 51.4 30.0 57.1 74.5 27.8 33.3 26.9 27.8
31.6 27.3 42.3 42.9 24.7 34.3 20.0 8.2 58.2 38.9 33.3 38.5 27.8
16
Linderone
Methyllinderon

Molecules 2018, 23, 1521
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Table 2. Cont.
carbendazim
chlorothalonil
<50
73.3
100 100 100 <50 100 <50 <50 <50 100 100 100 <50 <50
<50 86.4 <50 100 100 100 73.3 100 100 91.3 91.3 100 100
a
A.S: Alternaria solani; G.Z: Gibberella zeae; 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;
B.B: Botryosphaeria berengeriana; R.C: Rhizoctonia cerealis; H.M: Helminthosporium maydis; C.L: Colleetotrichum lagenarium;
b
F.M: Fusarium moniliforme; P.C: Phytophthora capsica. - no test data.
Table 3. Fungicidal activity of compounds 12, 14, 17 against S. sclerotiorum and R. cerealis.
Inhibition Rate (%)
Compd.
S. sclerotiorum
R. cerealis
−1
−1
−1
−1
50 µg·mL
50 µg·mL
25 µg·mL
12.5 µg·mL
a
12
14
17
-
-
95.8
77.5
-
60.6
47.9
-
-
-
23.6
a
21.4
- no test data.
3. Materials and Methods
3.1. Instruments
NMR spectra were obtained at 500 MHz using a Bruker avance III 500 spectrometer
(Bruker Daltonics, Bremen, Germany) in CDCl3 or DMSO-d6 solution with tetramethylsilane as the
internal standard. HRMS data were obtained on an Ultraflex III MALDI-TOF/TOF (Bruker Daltonics).
The melting points were determined on an X-4 binocular microscope melting point apparatus
(Beijing Tech Instruments Co., Beijing, China) and are uncorrected.
3.2. General Synthesis
All anhydrous solvents were dried and purified by standard techniques. Chemical materials
purchased from Aladdin (Shanghai, China) or TCI (Shanghai, China) for analytical grade.
3.3. General Procedure for the Synthesis of Compounds 1–16
Unsaturated aldehydes or ketones and N-amino-maleimide products were prepared according to
the literature [9,10]. A mixture of unsaturated aldehydes or ketones (3.0 mmol), N-amino-maleimide
products (2 mmol) and p-toluenesulfonic acid (0.2 mmol) in dry ethanol (5 mL) was heated under
reflux for 12 h with stirring. The organic layer 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 1–16.
◦
1-(((E)-4-phenylbut-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (
yield = 67%. 1H-NMR (CDCl3)
1
). Pale yellow solid, mp 159–161 C,
δ
7.37–7.29 (m, 5H), 6.89 (d, J = 10.2 Hz, 1H), 6.80 (d, J = 10.2 Hz,
1H), 6.06 (s, 1H), 5.39 (s, 1H), 2.53 (s, 3H); 13C-NMR (CDCl3)
δ 154.1, 154.0, 137.0, 136.3, 135.8, 134.2,
128.9, 127.2, 110.8, 77.4, 77.1, 76.8, 15.2; HRMS (ESI): m/z calcd for C14H12N2O2 [M + H]+: 241.0977,
found 241.0972.
1-(((E)-4-(3,5-bis(trifluoromethyl)phenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (
2
). Pale yellow solid,
◦
mp 122–125 C, yield = 57%. 1H-NMR (CDCl3)
δ
7.84 (s, 1H), 7.78 (s, 2H), 6.94 (d, J = 10.2 Hz,
1H), 6.83 (d, J = 10.2 Hz, 1H), 6.17 (s, 1H), 5.36 (s, 1H), 2.57 (s, 3H); 13C-NMR (CDCl3)
δ 154.3, 154.0,

Molecules 2018, 23, 1521
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138.8, 138.6, 136.9, 133.9, 127.8, 123.0, 108.4, 77.3, 77.1, 76.8, 65.7, 15.2; HRMS (ESI): m/z calcd for
C16H10N2O2F6 [M + H]+: 377.0719, found 377.0699.
1-(((E)-4-(2,4-dichlorophenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (
3
). Pale yellow solid,
7.40 (s, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 8.4 Hz,
1H), 6.95 (d, J = 10.2 Hz, 1H), 6.86 (d, J = 10.2 Hz, 1H), 6.38 (s, 1H), 5.39 (s, 1H), 2.48 (s, 3H);
13C-NMR (CDCl3)
153.9, 137.7, 136.7, 134.8, 133.7, 132.9, 131.8, 130.1, 127.9, 108.6, 77.3, 77.1, 76.8, 15.1;
HRMS (ESI): m/z calcd for C14H10N2O2Cl2 [M + H]+: 308.0113, found 308.0096.
◦
1
mp 143–144 C, yield = 61%. H-NMR (CDCl3)
δ
δ
◦
1-(((E)-4-(4-nitrophenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (
4
). Pale yellow solid, mp 172–174 C,
1
yield = 52%. H-NMR (CDCl3)
δ
8.19 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 6.92 (d, J = 10.2 Hz,
154.1, 153.9,
1H), 6.82 (d, J = 10.2 Hz, 1H), 6.13 (s, 1H), 5.36 (s, 1H), 2.53 (s, 3H); 13C-NMR (CDCl3)
δ
148.1, 142.9, 138.3, 136.8, 133.9, 128.2, 124.2, 109.0, 77.4, 77.2, 76.9, 15.2; HRMS (ESI): m/z calcd for
C14H11N3O4 [M + H]+: 285.0750, found 285.0760.
◦
1-(((E)-4-(2-fluorophenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (
yield = 63%. 1H-NMR (CDCl3)
6.92 (d, J = 10.2 Hz, 1H), 6.84 (d, J = 10.2 Hz, 1H), 6.29 (s, 1H), 5.38 (s, 1H), 2.49 (s, 3H); 13C-NMR
(CDCl3) 161.23, 159.3, 154.0, 137.4, 136.5, 133.8, 130.3, 127.7, 124.6, 116.2, 116.0, 109.3, 77.4, 77.1, 76.8,
15.1; HRMS (ESI): m/z calcd for C14H11N2O4F [M + H]+: 258.0799, found 258.0771.
5). Pale yellow solid, mp 135–137 C,
δ
7.31–7.26 (m, 1H), 7.10 (ddd, J = 29.9, 16.0, 7.0 Hz, 3H),
δ
3-methyl-1-(((E)-4-phenylbut-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione
mp 143–144 ◦C, yield = 68%. 1H-NMR (CDCl3)
7.38–7.28 (m, 5H), 6.71 (d, J = 26.2 Hz, 1H),
6.04 (s, 1H), 5.35 (d, J = 21.6 Hz, 1H), 2.53 (d, J = 8.0 Hz, 3H), 2.13 (d, J = 30.7 Hz, 3H); 13C-NMR
(6). Pale yellow solid,
δ
(CDCl3)
δ 155.1, 154.4, 146.0, 144.4, 137.0, 136.2, 132.2, 130.4, 129.0, 128.6, 127.4, 127.1, 111.0, 110.6,
77.4, 77.1, 76.9, 16.9, 16.4, 15.4, 15.2; HRMS (ESI): m/z calcd for C15H14N2O2 [M + H]+: 277.0974,
found 277.0969.
1-(((E)-4-(3,5-bis(trifluoromethyl)phenyl)but-3-en-2-ylidene)amino)-3-methyl-1H-pyrrole-2,5-dione (
7
). Pale
1
yellow solid, mp 143–144 ◦C, yield = 68%. H-NMR (CDCl3)
δ 7.83 (s, 1H), 7.77 (d, J = 5.8 Hz, 2H),
6.74 (d, J = 40.7 Hz, 1H), 6.15 (s, 1H), 5.33 (d, J = 21.8 Hz, 1H), 2.56 (d, J = 9.1 Hz, 3H), 2.16 (d, J = 37.4 Hz,
3H); 13C-NMR (CDCl3)
δ
155.4, 155.0, 154.7, 154.3, 146.9, 144.3, 139.1, 138.7, 132.8, 132.4, 132.1, 130.0,
127.8, 124.2, 122.9, 122.0, 108.7, 107.9, 77.4, 76.9, 65.8, 65.4, 17.0, 16.4, 15.6, 15.4; HRMS (ESI): m/z calcd
for C17H12N2O2F6 [M + H]+: 391.0875, found 391.0904.
1-(((E)-4-(2,4-dichlorophenyl)but-3-en-2-ylidene)amino)-3-methyl-1H-pyrrole-2,5-dione (
8
). Pale yellow solid,
1
mp 143–144 ◦C, yield = 68%. H-NMR (CDCl3)
δ 7.39 (t, J = 2.0 Hz, 1H), 7.21 (ddd, J = 8.3, 4.9, 2.1 Hz,
1H), 7.01 (dd, J = 12.4, 8.4 Hz, 1H), 6.76 (d, J = 25.1 Hz, 1H), 6.37 (d, J = 20.2 Hz, 1H), 5.35 (d, J = 37.7 Hz,
1H), 2.47 (d, J = 5.7 Hz, 3H), 2.16 (d, J = 32.3 Hz, 3H); 13C-NMR (CDCl3)
δ
155.0, 154.3, 146.6, 144.1,
137.8, 134.8, 133.2, 132.9, 132.6, 132.2, 130.3, 129.9, 128.2, 127.9, 108.9, 108.1, 77.4, 77.1, 76.9, 17.0, 16.4,
15.3, 15.1; HRMS (ESI): m/z calcd for C15H12N2O2Cl2 [M + H]+: 323.0348, found 323.0355.
4,5,6,7-tetrach◦loro-2-(((E)-4-phenylbut-3-en-2-ylidene)amino)isoindoline-1,3-dione (
7.31 (d, J = 16.5 Hz, 1H), 7.17 (d, J = 16.4 Hz, 1H), 2.20 (s, 3H); 13C-NMR (CDCl3)
δ 176.1, 159.6, 140.5,
9). Pale yellow solid,
δ 7.56 (d, J = 7.2 Hz, 2H), 7.47–7.34 (m, 3H),
mp 212–213 C, yield = 58%. 1H-NMR (CDCl3)
135.2, 130.0, 129.1, 127.6, 127.1, 126.9, 77.4, 77.1, 76.8, 16.4; HRMS (ESI): m/z calcd for C18H10N2O2Cl4
[M + H]+: 448.9388, found 448.9383.
3,4-dichloro-1-(((E)-4-phenylbut-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (10). Pale yellow solid,
◦
1
mp 152–153 C, yield = 62%. H-NMR (CDCl3) δ 7.39–7.31 (m, 5H), 6.10 (s, 1H), 5.49 (s, 1H), 2.56 (s, 3H);

Molecules 2018, 23, 1521
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13C-NMR (CDCl3)
δ
149.5, 149.2, 139.4, 137.9, 137.3, 134.6, 129.3, 129.0, 127.7, 111.6, 77.4, 77.1, 76.9, 15.3;
HRMS (ESI): m/z calcd for C14H10N2O2Cl2 [M + H]+: 308.0113, found 308.0093.
1-(((E)-4-(3,5-bis(trifluoromethyl)phenyl)but-3-en-2-ylidene)amino)-3,4-dichloro-1H-pyrrole-2,5-dione (11).
◦
1
Pale yellow solid, mp 152–153 C, yield = 60%. H-NMR (CDCl3) δ 7.87 (s, 1H), 7.81 (s, 2H), 6.22 (s, 1H),
5.47 (s, 1H), 2.60 (s, 3H); 13C-NMR (CDCl3)
123.5, 109.3, 77.4, 77.2, 76.9, 15.5; HRMS (ESI): m/z calcd for C16H8N2O2F6Cl2 [M + H]+: 466.9759,
δ 150.0, 149.2, 140.2, 139.2, 137.8, 137.5, 132.7, 132.4, 128.4,
found 466.9751.
3,4-dichloro-1-(((E)-4-(2,4-dichlorophenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (12). Pale yellow solid,
◦
mp 189–191 C, yield = 62%. 1H-NMR (CDCl3)
δ
7.42 (d, J = 2.0 Hz, 1H), 7.27–7.25 (m, 1H),
7.05 (d, J = 8.4 Hz, 1H), 6.45 (s, 1H), 5.47 (s, 1H), 2.52 (s, 3H); 13C-NMR (CDCl3)
δ
155.0, 154.3, 146.6,
144.1, 137.8, 134.8, 133.2, 132.9, 132.6, 132.2, 130.3, 129.9, 128.2, 127.9, 108.9, 108.1, 77.4, 77.1, 76.8, 16.4,
15.3, 15.1; HRMS (ESI): m/z calcd for C14H8N2O2Cl4 [M + H]+: 398.9232, found 398.9234.
3,4-dichloro-1-(((E)-4-(4-nitrophenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (13). Pale yellow solid,
◦
1
mp 161–163 C, yield = 41%. H-NMR (CDCl3)
δ 8.22 (s, 2H), 7.52 (d, J = 8.5 Hz, 2H), 6.20 (s, 1H),
5.48 (s, 1H), 2.57 (s, 3H); 13C-NMR (CDCl3)
124.2, 109.0, 77.4, 77.2, 76.9, 15.2; HRMS (ESI): m/z calcd for C14H9N3O4Cl2 [M + H]+: 352.9956,
δ 154.1, 153.9, 148.1, 142.9, 138.3, 136.8, 133.9, 128.2,
found 352.9970.
3,4-dichloro-1-(((E)-4-(2-fluorophenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (14). Pale yellow solid,
1
mp 140–141 ◦C, yield = 64%. H-NMR (CDCl3)
δ
7.33–7.29 (m, 1H), 7.18–7.13 (m, 2H), 7.07 (s, 1H)
,
6.33 (s, 1H), 5.47 (s, 1H), 2.53 (d, J = 1.5 Hz, 3H); 13C-NMR (CDCl3)
δ
161.5, 159.5, 149.3, 139.7,
137.7, 130.8, 128.2, 124.8, 122.0, 116.4, 116.2, 110.2, 77.4, 77.1, 76.9, 15.3; HRMS (ESI): m/z calcd for
C14H9N2O2Cl2F [M + H]+: 326.0019, found 325.9990.
3,4-dichloro-1◦-(((E)-4-(p-tolyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (15). Pale yellow solid,
mp 123–124 C, yield = 65%. 1H-NMR (CDCl3)
δ 7.22 (d, J = 7.8 Hz, 2H), 7.17 (d, J = 7.7 Hz, 2H),
6.08 (s, 1H), 5.47 (s, 1H), 2.56 (s, 3H), 2.33 (s, 3H); 13C-NMR (CDCl3)
δ 149.6, 149.3, 139.4, 138.1, 137.3,
131.6, 129.7, 127.8, 111.8, 77.4, 77.2, 76.9, 29.8, 15.4; HRMS (ESI): m/z calcd for C15H12N2O2Cl2 [M + H]+
:
345.0168, found 345.0170.
3,4-dichloro-1-(((E)-4-(2,6-dichlorophenyl)but-3-en-2-ylidene)amino)-1H-pyrrole-2,5-dione (16). Pale yellow solid,
◦
1
mp 212–213 C, yield = 59%. H-NMR (CDCl3) δ 7.40 (d, J = 7.7 Hz, 1H), 7.28–7.19 (m, 2H), 6.97 (s, 1H),
5.34 (s, 1H), 2.53 (s, 3H); 13C-NMR (CDCl3)
128.2, 107.0, 77.4, 77.2, 76.9, 15.3; HRMS (ESI): m/z calcd for C14H8N2O2Cl4 [M + H]+: 398.9232,
δ 149.5, 149.2, 139.8, 139.6, 137.2, 136.0, 133.6, 130.4, 129.2,
found 398.9260.
3.4. Synthesis of (E)-2-(1-hydroxy-3-phenylallylidene)-4,5-dimethoxycyclopent-4-ene-1,3-dione (Linderone)
This compound was prepared according to literature [11]: yield = 84%. NMR data consistent with
1
literature [11]: H-NMR (CDCl3)
4.17 (s, 3H).
δ 11.56 (s, 1H), 7.67 (s, 2H), 7.62 (m, 2H), 7.41 (m, 3H), 4.22 (s, 3H),
3.5. Synthesis of (E)-4,5-dimethoxy-2-(1-methoxy-3-phenylallylidene)cyclopent-4-ene-1,3-dione
(Methyllinderone)
Methyllinderone was prepared from linderone according to the literature [11]. Yield = 93%;
1H-NMR (CDCl3)
δ
7.94 (d, J = 15.6 Hz, 1H), 7.55–7.65 (m, 2H), 7.52 (d, J = 15.6 Hz, 1H), 7.30–7.40
(m, 3H), 4.19 (s, 6H), 4.10 (s, 3H).

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3.6. General Procedure for the Synthesis of Copound 17
3,4-dichloro-1H-pyrrole-2,5-dione (17). The compound 17 was prepared according to the
1
literature [12]. Orange crystal powder, mp 178–179 ◦C, yield = 82%. H-NMR (500 MHz, DMSO-d6)
δ
11.73 (s, 1H); 13C-NMR (126 MHz, DMSO-d6)
δ 164.48, 133.26. HRMS (ESI): m/z calcd for C4HNO2Cl2
[M + H]+: 163.9312, found 163.9306.
3.7. 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 [13]. Evaluations are
based on a percentage scale of 0–100 in which 0 = no activity and 100 = total kill.
3.7.1. Stomach Toxicity against Oriental Armyworm (Mythimna separata)
The stomach toxicities of compounds 1–8, 10 against oriental armyworm were evaluated by foliar
application using the reported procedure [14]. 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.
3.7.2. Toxicity against Mosquito (Culex pipiens pallens)
The toxicities of compounds
1–8, 10 against mosquito were evaluated according to the reported
procedure [15 16]. 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 days after treatment.
Each test was performed in triplicate.
3.8. In Vitro Antifungal Bioassay
The antifungal activities were screened and evaluated by the poison plate technique [17].
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
µg
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.
4. Conclusions
A series of novel N-amino-maleimide derivatives containing hydrazone group were designed and
synthesized and their insecticidal and antifungal activities were evaluated and discussed. The results of
bioassays indicated that some compounds (compounds
2, 3 and 8) possessed excellent activities against
mosquito and compounds and exhibited considerable activities against oriental armyworm.
2,
3
6
In particular, some compounds exhibited better antifungal activities against fourteen phytogenic fungi
than linderone and methyllinderone. This shows that using natural active substance as a first lead

Molecules 2018, 23, 1521
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structure, designing and synthesizing its derivatives is a good choice for discovering active pesticide
molecules. Because compound 12 exhibited best broad-spectrum antifungal activities, it makes sense
to further explore its fungicidal mechanism, chitinase inhibitory activity in vitro and discover the
fungicide candidate.
Supplementary Materials: The following are available online, Figures S1–S34, NMR spectral information of
compounds 1–17.
Author Contributions: R.S. conceived and designed the experiments; X.S., C.L. and P.C. performed the
experiments, analyzed the data; X.S. drafted the manuscript; R.S. revised the manuscript; H.Z. gave some
suggestion to the experiment. 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 Provincial
Natural Science Foundation Innovation Research Team Project (2016CXTD002).
Conflicts of Interest: The authors declare no conflict of interest
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Sample Availability: Not available.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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