Design and synthesis of N-alkyl-N'-substituted 2,4-dioxo-3,4- dihydropyrimidin-1-diacylhydrazine derivatives as ecdysone receptor agonist

Article abstract of DOI:10.1016/j.bmc.2013.05.010

Based on the discovery of thymine as an ecdysteroid agonist, a series of 1,4-disubstituted diacylhydrazine derivatives with a thymine moiety were designed and synthesized. The activities of these compounds against Spodoptera litura (Fabricius) were evaluated by the insect immersion method. Results showed that compound 2h with an N-cyclohexylmethyl substituent exhibits the most potent agonist activity with a median lethal concentration of 23.21 μg/mL. This compound also caused malformation of molting larvae and adults. Compound 2h was further demonstrated as an ecdysteroid agonist by reporter gene assay on the Spodoptera frugiperda cell line (Sf9 cells). A molecular docking study indicated that hydrophobic interactions and the formation of hydrogen bonds between the compounds and the ecdysone receptor play critical roles in promoting the binding affinity of the compound. The structure of compound 2h may serve as a favorable template for the development of new ecdysteroid agonists with a pyrimidinedione moiety.

Full text of DOI:10.1016/j.bmc.2013.05.010

Bioorganic & Medicinal Chemistry 21 (2013) 4687–4697
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of N-alkyl-N0-substituted 2,4-dioxo-3,4-dihy-
dropyrimidin-1-diacylhydrazine derivatives as ecdysone receptor
agonist
Xiu Liu a,b, Ling Zhang a,b,c, Jin-Guo Tan a,b, Han-Hong Xu a,b,
⇑
a State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, PR China
b Key Laboratory of Natural Pesticide and Chemical Biology of the Ministry of Education, South China Agricultural University, Guangzhou 510642, PR China
c College of Life Science and Technology, Jinan University, Guangzhou 510632, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the discovery of thymine as an ecdysteroid agonist, a series of 1,4-disubstituted diacylhydrazine
derivatives with a thymine moiety were designed and synthesized. The activities of these compounds
against Spodoptera litura (Fabricius) were evaluated by the insect immersion method. Results showed
that compound 2h with an N-cyclohexylmethyl substituent exhibits the most potent agonist activity
Received 19 April 2013
Revised 7 May 2013
Accepted 8 May 2013
Available online 16 May 2013
with a median lethal concentration of 23.21 lg/mL. This compound also caused malformation of molting
larvae and adults. Compound 2h was further demonstrated as an ecdysteroid agonist by reporter gene
assay on the Spodoptera frugiperda cell line (Sf9 cells). A molecular docking study indicated that hydro-
phobic interactions and the formation of hydrogen bonds between the compounds and the ecdysone
receptor play critical roles in promoting the binding affinity of the compound. The structure of compound
2h may serve as a favorable template for the development of new ecdysteroid agonists with a pyrimi-
dinedione moiety.
Keywords:
Diacyhydrazine
Thymine
Synthesis
Insecticidal activity
Molecular docking
Ecdysteroid agonist
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
The crystal structures of the ligand-binding domains (LBD) of
EcR from Heliothis virescens (HvEcR) have been reported in complex
20-Hydroxyecdysone (20E; Fig. 1) and juvenile hormones regu-
late insect growth and development. 20E, a biologically active
metabolite of steroid hormones, is found in most insects and regu-
lates molting and metamorphosis as well as cell differentiation and
reproduction.1 The action of 20E is dependent upon its bond with a
heterodimeric complex composed of the ecdysone receptor (EcR)
and ultraspiracle (USP).2–4 The complex triggers cascades of molt-
ing-related gene expressions upon ligand binding, which leads to
insect developmental transitions.5–7 Therefore, agonists or antago-
nists of 20E are potential insect growth regulator insecticides.
RH-5849 (Fig. 1), which features a scaffold of N-t-butyl-N,N0-
dibenzoyl-hydrazine (DBH), was first discovered and demon-
strated as a nonsteroidal ecdysone agonist in the 1980s.8 This ago-
nist could halt feeding in lepidopteran larvae by forcing an
ultimately lethal and developmentally premature molt.8–10 A series
of DBH derivatives were synthesized decades later.11–16 Of these
derivatives, three compounds, namely, tebufenozide,17,18 meth-
oxyfenozide,19 and chromafenozide,20 (Fig. 1) have been commer-
cialized for agricultural applications to control lepidopteran pests,
such as armyworm and leaf roller.21,22
with ecdysteroid ponasterone A (PonA; one of the most potent
ecdysteroids) and a non-steroidal agonist BYI06830 (a DBH analog)
(Fig. 1).23 As elucidated in the crystal structures, the binding pock-
ets of HvEcR LBD for PonA and DBH are only partially overlapped.
The t-butyl moiety of DBH shares the same space as the terminal i-
propyl moiety of the side chain of PonA. However, the ring moiety
of DBH away from the t-butyl group is oriented in an extra pocket
that is not occupied by PonA. The difference between the binding
pockets of PonA and DBH provides new insights by which to im-
prove the agonist activity of DBH by modifying its ring moiety
away from the t-butyl group.
Structure–activity relationship studies have demonstrated that
hydrogen bonding between the NH group of DBH and amino acid
residues of the LBD of EcR plays a critical role in biological activ-
ity.23,24 As indicated in 3D QSAR studies, electrostatic and steric ef-
fects of the substituent on the ring affect the binding affinity of
DBHs. Moreover, the hydrophobic parameters of the ligands, such
as logP, are also important to the binding affinity.25,26 The hydro-
phobic t-butyl group interacting with the hydrophobic amino acid
residues of the receptor is a key factor affecting the logP of DBHs.
Consequently, replacing the t-butyl group with a bulky alkyl group
is a strategy for increasing the hydrophobic property of a molting
hormone agonist.
⇑
Corresponding author. Tel.: +86 20 85285127; fax: +86 20 38604926.
E-mail address: hhxu@scau.edu.cn (H.-H. Xu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.010

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X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
OH
OH
OH
O
HO
HO
HO
HO
O
O
N
OH
OH
N
H
O
HO
HO
O
O
BYI06830
20E
PonA
O
N
NH
O
N
NH
O
N
NH
N
NH
O
O
O
O
O
O
O
RH-5849
RH-5992 (tebufenozide)
RH-2485 (methoxyfenozide)
ANS-118 (chromafenozide)
Figure 1. The structure of DBHs and ecdysteroids.
In the present study, a series of structurally novel agonists for
EcR were designed and synthesized from DBH. These agonists fea-
tured substitution of the ring moiety of DBH away from the t-butyl
group with thymine and substitution of the t-butyl group of DBH
with hydrogen or an alkyl or cycloalkyl group (Fig. 2). The activities
of the synthesized compounds against the important agricultural
pest Spodoptera litura (Fabricius) were assayed, and ecdysone ago-
nist activity was evaluated by reporter gene assay on the Spodop-
tera frugiperda cell line (Sf9 cells). The binding mode of the active
compound 2h was studied by molecular docking.
ecdysone response element (ERE) derived from the Drosophila mel-
anogaster hsp27 gene and a basal actin promoter derived from
Bombyx mori, followed by a gfp gene and a termination signal. In
this study, the plasmid was transfected into Sf9 cells in which
endogenous EcR and USP were detected.29 The results showed that
all of the pyrimidinedione derivatives, except 5-bromouracil, could
induce transcription of the reporter gene of the transfected cells at
25
lg/mL.
Figure 3A shows green fluorescence in the transiently transfec-
ted cells after incubation with thymine. Tebufenozide, as a positive
control, can also induce green fluorescence. The fluorescence inten-
sities of the cells treated with thymine and 5-fluorouracil showed
2. Results and discussion
similarities, but were weaker than that of tebufenozide at 0.5
mL (Fig. 3B).
lg/
2.1. Reporter gene assay of pyrimidinedione derivatives and
structure design
Reporter gene assay indicated that thymine and 5-fluorouracil
as EcR agonists induce transcription of the reporter gene. Consider-
ing the high cytotoxicity of 5-fluorouracil to normal human cells,
thymine was selected to design new agonists with the DBH scaf-
fold. In this study, a series of compounds were synthesized with
a thymine moiety replacing the phenyl ring away from the t-butyl
group of DBH. The t-butyl group of DBH was also replaced with dif-
ferent alkyl groups to increase the hydrophobic property of the de-
signed compounds (Fig. 2).
5-Fluorouracil is an anticancer drug that acts on thymidylate
synthetase and inhibits the proliferation and division of cancer
cells.27 This drug may regulate the growth and development of in-
sects. To explore new lead compounds as insect growth regulators
acting on EcR, 5-fluorouracil, together with four other pyrimidines
derivatives, namely, uracil, thymine, trifluorothymine, and 5-bro-
mouracil, were assayed using a cell-based reporter gene system
to evaluate whether or not they act on the receptor. Swevers
et al.28 first reported the cell-based high-throughput screening sys-
tem used in this study on Bm-5 cell lines for screening out ecdy-
steroid agonists and antagonists from plant extracts and libraries
of synthetic compounds. The reporter plasmid contains an
2.2. Chemistry
Schemes 1 and 2 show the synthetic procedures for the de-
signed compounds. The intermediate (thymine-1-yl) acetic acid
O
HN
N
N
N
H
O
O
H
O
Thymine
Tebufenozide
SlEcR LBD
O
O
R2
N
O
R2
N
O
R1
R1
N
NH
N
n
NH
N
H
N
H
O
O
O
O
I
II
n = 1 or 2
Figure 2. The schematic diagram for the design of the target compounds.

X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
4689
Figure 3. The reporter gene assay for pyrimidinedione derivatives. (A) Observation of induction of green fluorescence. CK1 and CK2 showed the cells untreated with
compounds by ordinary inverted microscopy and fluorescence microscopy respectively. Cells treated with tebufenozide and thymine, were observed by fluorescence
microscopy shown at right. (B) The intensities of fluorescence were recorded on the Multilabel Counter Victor 3V. The concentration of tebufenozide was 0.5
lg/mL. Those of
the pyrimidinedione derivatives were 25 g/mL. The values were the means of three replicates and the error bars present the SE. The same letters on histograms indicate no
l
significant difference of the means at p <0.05 by Duncan’s multiple-range test.
O
O
O
NHNH2
O
R1
NH
R1
1.1 R: KOH, S: H2O
H
N
R2
N
O
NH
OH
O
N
+
Br
+
O
O
N
H
O
N
N
O
1.2 HCl
H
O
R: EDCI, S:DMF
HO
3
R1 R2
H
4
1a
1b OH
H
H
Scheme 1. Synthetic procedure for compounds 1a and 1b.
(4) was prepared using a previously reported method.30 Amidation
of 4 with benzoylhydrazine and 4-hydroxyl-benzoylhydrazine
afforded the target compounds diacylhydrazine 1a and 1b. The
EDC coupling protocol and not the BOP reagent/HOBT coupling
protocol appeared to be the best route for obtaining the desired
compounds. This result is consistent with that reported by Thom-
son et al.31
1-Carbethoxyalkylthymines 5a–5c, synthesized from thymine,
were reacted with hydrazine hydrate to afford 1-acetylhydrazino-
thymines 6a–6c.32 Then, condensation of 6a–6c with aldehyde or

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X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
O
n
X
1.1 aldehydes or ketone,
O
OEt
R
O
O
5
EtOH, reflux
NH2NH2, EtOH
Y
NH
R
NH
OEt
1.2 NaBH4, THF
H2N
DBU, DMF
O
N
H
3
6
O
R1
R1
Cl
O
R
O
O
R1
MeONa/MeOH
O
N
R2
O
NH
N
N
R2
HN
R
H
N
H
Et3N, DMAP, CH2Cl2
R
R2
R1 = OAcO, 3,5-di-Me
2a-2m
R1 = OH
3a-3c
7
O
O
O
R2 = cyclohexyl, cyclohexyl-methly, 2-hexyl, 4-heptyl,
NH
NH
NH
R =
,
,
X
Y
H
H
n
1
2
1
N
O
N
O
N
O
1 Cl
2 Br
3 Cl CH3
T1
T2
T3
Scheme 2. General procedure for the synthesis of compounds 2a–2m and 3a–3c.
ketone and subsequent reduction of the products in the presence of
sodium borohydride gave the key intermediates 7a–7h.33 Conden-
sation of 7a–7h with substituted benzoyl chloride yielded the tar-
get compounds 2a–2m,34 and deacetylation of 2j, 2l, and 2m
afforded 3a–3c. All of the diacylhydrazines were synthesized for
the first time and characterized by 1H and 13C NMR and HR-MS
(ESI).
Compounds 7a, 7c, 7e and 7h, all of which contain amide bonds,
were isolated as a mixture of two isomers, similar to a report by
Dueholm et al.35 The ratio of the major to minor isomers was
3:1, and two sets of signals were observed in the 1H and 13C
NMR spectra of the compounds. However, the spectra of interme-
diates 7b, 7d, 7f, and 7g as well as those of the target compounds
showed only one set of signals because of steric hindrance. This re-
sult is similar to findings reported by Toya et al.24
2.3. Insecticidal activities
The insecticidal activities of diacylhydrazines 1a–1b, 2a–2l, and
3a–3c against S. litura at 100 lg/mL were primarily assayed by the
insect immersion method with tebufenozide as a positive control;
the results of this assay are summarized in Table 1. Most of the
compounds exhibited low activities after 48 h of treatment. How-
ever, after 96 h of treatment, the mortalities of half of the com-
pounds were higher than 50%. After 144 h of treatment, the
mortalities of all tested compounds, except 1a, 2d, 3a, and 3b, were
higher than 60%. The activities of these synthesized compounds
against S. litura larvae increased in a time-dependent manner.
For example, the compound 2h did not exhibit activity after 48 h
of treatment, but mortality rates sharply increased to 72.0% after
96 h and 97.0% after 144 h. Meanwhile, the phenotypes of the trea-
ted larvae were observed. Typical symptoms, such as abnormal
molting and malformation of pupae and adults, were observed un-
der tebufenozide and target compound treatment (Fig. 4).
Figure 4. The abnormal molting, malformation of pupae and adults of S. litura
caused by 2h and tebufenozide.
with 1a and 1b, most of the N-substituted derivatives 2a–2l, except
2d, were more potent. Among compounds 3a–3c, all of which
feature a 4-OH phenyl moiety, only 3c exhibited excellent activity
with a mortality of 90.6% after 144 h of treatment. Comparing 2j
(4-OAc, 83.0%) with 3a (4-OH, 12.6%) and 2l (4-OAc, 64.8%) with
3b (4-OH, 26.1%), all of which feature an N-cyclohexylmethyl
The activity of 1b, which features a hydrogen atom on the nitro-
gen of acylhydrazine and a 4-OH group on the phenyl ring, was
moderate. The activity of 1a was even lower than that of 1b; 1a
features a 4-H atom in place of the 4-OH group of 1b. Compared

X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
4691
Table 1
The effect of the compounds on S. litura larvae by insect immersion method at the concentration of 100
l
g/mL
O
O
O
R1
NH
NH
NH
O
R =
,
,
O
N
O
N
O
N
O
N
R2
N
R
H
T1
T2
T3
Compounds
Corrected mortalitiesA (%) (means SE)
96 h
48 h
144 h
1a (R = T1, R1 = H, R2 = H)
1b (R = T1, R1 = H, R2 = OH)
2a (R = T1, R1 = 3,5-dimethyl, R2 = 4-heptyl)
2b (R = T1, R1 = OAc, R2 = 4-heptyl)
10.0 10.0 b
0.0 0.0 b
0.0 0.0 b
11.7 3.0 b
8.1 4.2 b
0.0 0.0 b
7.0 3.5 b
0.0 0.0 b
13.0 13.0 b
0.0 0.0 b
3.0 3.0 b
11.7 11.7 b
8.9 8.9 b
0.0 0.0 b
2. 4 2.4 b
0.0 0.0 b
3.3 3.3 b
1.8 1.8 b
87.8 6.2 a
13.7 8.8 gh
28.7 6.2 efgh
49.7 6.2 cde
58.8 12.0 cde
68.6 13.2 bcd
7.0 3.5 h
85.9 7.1 ab
43.6 1.8 cdef
52.0 11.1 cde
72.0 8.4 abc
44.3 11.3 cdef
54.3 8.7 cde
66.0 12.3 bcd
35.2 7.7 defg
7.8 4.2 h
25.7 2.3 gh
64.4 5.0 efgh
72.8 5.0 cde
69.4 9.8 cde
87.8 6.5 abcde
35.4 9.5 fg
89.3 6.4 abcd
74.9 3.6 cde
65.7 8.2 def
97.0 3.0 ab
75.4 4.1 cde
83.0 11.5 bcde
84.7 9.7 abcde
64.8 5.2 ef
2c (R = T3, R1 = OAc, R2 = 4-heptyl)
2d (R = T1, R1 = 3,5-dimethyl, R2 = cyclohexyl)
2e (R = T1, R1 = OAc, R2 = cyclohexyl)
2f (R = T1, R1 = 3,5-dimethyl, R2 = cyclohexyl)
2g (R = T2, R1 = OAc, R2 = cyclohexyl)
2h (R = T1, R1 = OAc, R2 = cyclohexylmethyl)
2i (R = T1, R1 = 3,5-dimethyl, R2 = cyclohexylmethyl)
2j (R = T2, R1 = OAc, R2 = cyclohexylmethyl)
2k (R = T1, R1 = 3,5-dimethyl, R2 = cyclohexylmethyl)
2l (R = T3, R1 = OAc, R2 = cyclohexylmethyl)
3a (R = T2, R1 = OH, R2 = cyclohexylmethyl)
3b (R = T3, R1 = OH, R2 = cyclohexylmethyl)
3c (R = T1, R1 = OH, R2 = 2-hexyl)
12.6 8.4 h
15.4 4.8 fgh
73.0 9.4 abc
26.0 8.0 efgh
93.9 3.1 a
26.1 3.9 gh
90.6 5.3 ab
37.9 6.8 fg
Thymine
Tebufenozide
100.0 0.0 a
A
The same letters in the same column indicate no significant difference of the means at p <0.05 by Duncan’s multiple-range test.
lene linker, were the three most potent compounds. Differences in
the final activities of these three compounds and tebufenozide
were not significant. The median lethal concentrations (LC50) of
the potent compounds (2e, 2h, and 3c) were further determined
(Table 2). Compound 2h, which features a thymine moiety, was
the most potent, with an LC50 of 23.2 lg/mL after 144 h of treat-
ment. The LC50 of the tested compounds increased in the order of
2h < 3c < 2e.
The results thus far indicate that a bulky alkyl substituent on
the nitrogen of the acylhydrazine is beneficial to agonist activity.
Electrostatic and steric effects of the substituent on the phenyl ring
also affect this activity. These results are similar to the structure–
activity relationship of DBHs.25,26 In addition, the rigidity of the lin-
ker between thymine and acylhydrazine, not its flexibility, is favor-
able to the activity of the agonist.
2.4. Growth inhibition activities
Considering the regulation of ecdysteroid agonists on growth,
development, and metamorphosis, the effects of the most potent
compound, 2h, on the weight gain of larvae after a 60 h treatment,
the duration of the larval and pupal stages, and the eclosion rate of
the treated insects were evaluated by the feed-poison method at a
Figure 5. Reporter gene assay to evaluate molting hormone activity of the potent
compounds. Compounds 2e, 2h, and 3c were assayed at 25
lg/mL, and tebufenoz-
ide was assayed at 0.5 g/mL. The values were the means of three replicates and the
l
error bars present the SE. The same letters on histograms indicate no significant
difference of the means at p <0.05 by Duncan’s multiple-range test.
concentration of 25 lg/mL. The results are summarized in Tables 3
and 4. The weight gain of larvae was significantly inhibited by
compound 2h, which showed an inhibition rate of 56.7%. The ef-
fects of compound 2h on the duration of the larval and pupal stage
were not significant, but the rate of adult emergence was only
30.3% after treatment by compound 2h. These results demon-
strated that compound 2h has potent inhibitory activities on
growth and development.
substituent, we can conclude that introduction of 4-OAc to the
phenyl ring could lead to more potent compounds than those
obtained from introduction of a 4-OH group. The activities of 2d–
2g, which feature N-cyclohexyl substituents, decreased in the
order of 2e (89.3%) > 2f (74.9%) > 2g (65.7%) > 2d (35.4%). The activ-
ities of derivatives with 4-OAc or 3,5-dimethyl on the phenyl ring
also depended on the linker between the thymine and acylhydr-
azine groups. The activity of derivatives with a methylene linker
was 2e (4-OAc) > 2g (3,5-dimethyl). By contrast, the activity of
derivatives with an ethylidene moiety was 2f (3,5-dimethyl) > 2g
(4-OAc). Compounds 2e, 2h, and 3c, all of which feature a methy-
2.5. Reporter gene assay of the target compounds
To confirm the agonist activity of these compounds, reporter
gene assay was conducted on compounds 2e, 2h, and 3c,28 and

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X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
Table 2
The LC50 of compounds against S. litura lavae by insect immersion method
Compounds
Times (h)
Regression equation
LC50
(lg/mL)
95% Confidence intervals
Correlation coefficient
2e
96
144
Y = ꢀ3.17 + 4.18x
Y = ꢀ0.79 + 3.41x
89.5
46.7
78.6–102.0
41.6–59.3
0.985
0.953
2h
96
144
Y = ꢀ0.75 + 3.27x
Y = 1.14 + 2.82x
57.3
23.2
47.7–68.7
17.5–30.7
0.986
0.977
3c
96
144
Y = ꢀ0.99 + 3.20x
Y = 0.20 + 3.02x
73.9
38.8
60.8–89.7
30.4–49.6
0.997
0.971
Tebufenozide
96
144
Y = 2.84 + 1.94x
Y = 2.76 + 2.94x
13.1
5.8
10.0–17.5
4.7–7.0
0.991
0.992
Table 3
The effect of compound 2h on the growth of S. litura
interactions were also observed in the 2d–receptor complex. The
N-cyclohexylmethyl group of 2h was located in the lipophilic pock-
et surrounded by residues Trp567, Ile380, Leu559, Leu563, Pro560,
Leu522, and Phe377. The 4-OAc phenyl moiety of 2h was located in
the lob surrounded by residues Leu541, Asp460, and Gln544; the
4-ethyl phenyl moiety of tebufenozide was also located in this
lob. The thymine moiety of 2h as well as the 3,5-dimethyl phenyl
moiety of tebufenozide were oriented in the same pocket sur-
rounded by residues Tyr444, Ser418, Met421, Val425, and
Met422 in the SlEcR LBD. However, the binding pocket of the
LBD of the thymine moiety of 2d overlapped with that of the 4-
ethyl phenyl group of tebufenozide. The hydrophobic cyclohexyl
group on the nitrogen atom of the hydrazine group of 2d was
placed in the same cavity accommodating the hydrophobic t-butyl
group of tebufenozide and surrounded by the hydrophobic resi-
dues Phe377, Ile380, Val425, Leu552, and Leu559 (Fig. 6). The bind-
ing modes of 2d and 2h within the LBD of SlEcR provide detailed
structural insights into the interaction between the compounds
and the receptor. The formation of hydrogen bonds in the li-
gand–receptor complex and the hydrophobic interaction are both
important for the binding affinity.24
Treatment
Weight (mg)
60 h
Weight gain (mg)
Inhibition (%)
56.7 6.4
0 h
Control
3.2 0.2
4.1 0.5
11.8 1.0
7.6 0.6
8.7 1.1
3.5 0.2
2ha
a
The concentration of compound 2h was 25 lg/mL.
Table 4
The effect of compound 2h on the growth and development of S. litura
Entry Duration of lavaeA (d) Duration of pupae (d) Rate of eclosion (%)
CKB
2hC
17.3 1.2 a
19.7 0.8 a
10.9 0.5 a
12.0 0.9 a
93.3 3.3 a
30.3 3.0 b
A
B
C
CK is the black control not containing the compounds.
The concentration of compound 2h was 25 g/mL.
The values are means SE. The same letters indicate no significant difference of
l
the means at p <0.05 by Duncan’s multiple-range test.
their induced fluorescence intensity was detected (Fig. 5). The fluo-
rescence intensities of these compounds at 25 lg/mL were signifi-
3. Conclusion
cantly higher than that of the negative control after correction for
autofluorescence but lower than that of tebufenozide. This result
suggests that the compounds act on EcR as agonists and induce
transcription of the reporter gene.
In summary, a series of structurally novel diacylhydrazine
derivatives with a thymine moiety substituting the ring away from
t-butyl were synthesized. Among these derivatives, compounds 2e,
2h, and 3c exhibited potent insecticidal activities against S. litura
2.6. Molecular modeling and docking study
larvae with an LC50 of 23.2–46.7 lg/mL. These active compounds
functioned as ecdysteroid agonists, causing abnormal metamor-
phosis in insects and inducing transcription of the reporter gene.
These results conform to our initial assumption that diacylhydra-
zines with pyrimidinedione may be used to develop new insect
growth regulators acting on EcR. The results of this work may serve
as a reference for future studies that aim to explore lead com-
pounds featuring the DBH scaffold with a thymine moiety and with
improved activities.
To validate the binding mode of the potent compounds toward
the LBD of SlEcR, a docking simulation was performed using the
CDOCKER protocol of Discovery Studio 2.5 software (DS 2.5, Accel-
rys, Co. Ltd, San Diego, CA, USA). The active site of the compounds
was defined as the ligand-binding site with a radius of 10 Å. Each
compound was docked as described, and the pose with the highest
–CDOCKER interaction energy was considered the best. After re-
docking BYI06830 to the SlEcR LBD, the root mean square deviation
(RMSD) of the internal ligand to the receptor was determined to be
0.61 Å by the CDOCKER algorithm, which indicates the molecular
docking procedure is reliable. Compound 2h, which showed the
most potent activity in in vivo tests and the in vitro reporter gene
assay, exhibited high affinity to SlEcR LBD and ranked first in terms
of CDOCKER interaction energy (Table 1S).
To obtain more insights into the ligand–receptor interaction,
the binding modes of compounds 2d and 2h as well as tebufenoz-
ide docked to the active site of SlEcR are shown in (Fig. 6). At least
seven residues in the LBD were involved in the interactions be-
tween the ligands and the receptor. Compound 2h and tebufenoz-
ide could potentially form hydrogen bonds with Thr384, Tyr449,
and Asn545, which correspond to Thr343, Tyr408, and Asn504 in
the LBD of HvEcR interacting with the substituted CONHNH group
of diacylhydrazine BYI06830.23 These hydrogen-bonding
4. Experimental protocols
4.1. Chemistry
All reagents and solvents were purchased from the commercial
company. Dichloromethane and THF were dehydrated according to
standard methods before use. Melting points (mp) were recorded
on a RY-1G melting point apparatus uncorrected. 1H NMR and
13C NMR spectra were obtained with Bruker AV-600 instrument
with deuteriodimethyl sulfoxide (DMSO-d6) as solvent. Chemical
shifts were reported in d (part per million) values with tetrameth-
ylsilane (TMS) as an internal standard and coupling constants J
were given in Hz. High resolution electro-spray mass spectra
(HR-ESI-MS) were recorded on Bruker maXis 4G ESI-Q-TOF

X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
4693
Figure 6. The interaction mode of tebufenozide (A and B), compounds 2h (C and D) and 2d (E and F) within the binding site of SlEcR LBD. For simplicity, the protein residues
around 4 Å of ligand binding site are depicted and labeled. Carbon is green, oxygen atom is red, nitrogen atom is light blue, and hydrogen atom is white. Green dotted line
represents hydrogen bond. Ligands in a scaled ball and stick model and residues in a line model except those forming hydrogen bond (stick model) with the ligand
compounds.
instrument. Analytical thin-layer chromatography (TLC) was car-
ried out on precoated plates (silica gel GF254), and spots were
visualized by ZF-20D ultraviolet (UV) analyzer.
275–277 °C. 1H NMR (600 MHz, DMSO-d6) d 7.83 (dd, J = 8.4,
1.2 Hz, 2H, phenyl-CH (2, 6)), 7.51–7.54 (m, 1H, phenyl-CH (4)),
7.46–7.42 (m, 2H, phenyl-CH (3, 5)), 7.40 (d, J = 1.2 Hz, 1H, thy-
mine-CH(6)), 4.44 (s, 2H, CH2CONH), 1.76 (d, J = 1.1 Hz, 3H, thy-
mine-CH3). 13C NMR (150 MHz, DMSO-d6) d 167.5, 166.7, 165.3,
151.7, 142.7, 132.9, 132.5, 129.0, 128.0, 109.3, 48.7, 12.0. HRMS
(ESI): calcd for C14H15N4O4 (M+H)+ 303.1093, found 303.1095.
4.1.1. General procedure for the preparation of (thymine-1-yl)
acetic acid (4)
(Thymine-1-yl) acetic acid 4 was prepared according to the pre-
viously reported method.30 The product was yielded as white solid,
yield 80%, mp 253–255 °C; 1H NMR (600 MHz, DMSO-d6) d 11.33
(s, 1H, thymine-NH (3)), 7.49 (d, J = 1.2 Hz, 1H, thymine-CH (6)),
4.36 (s, 2H, CH2), 1.75 (d, J = 1.1 Hz, 3H, thymine-CH3). 13C NMR
(150 MHz, DMSO-d6) d 169.6, 164.4, 151.0, 141.8, 108.3, 48.4, 11.9.
4.1.2.2. 4-Hydroxy-N0-(2-(5-methyl-2,4-dioxo-3,4-dihydropyr-
imidin-1(2H)-yl)acetyl)benzohydrazide (1b).
White pow-
der, yield 90%, mp 240–242 °C. 1H NMR (600 MHz, DMSO-d6) d
11.33 (s, 1H, thymine-NH (3)), 10.81 (br s, 1H, CH2CONH), 10.42
(br s, 1H, phenyl-OH), 8.76 (dd, J = 4.4, 1.6 Hz, 2H, phenyl-CH (2,
6)), 7.76 (dd, J = 4.5, 1.6 Hz, 2H, phenyl-CH (3, 5)), 7.51 (d,
J = 1.2 Hz, 1H, thymine-CH (6)), 4.46 (s, 2H, CH2CONH), 1.76 (d,
J = 1.0 Hz, 3H, thymine-CH3). 13C NMR (150 MHz, DMSO-d6) d
166.6, 165.1, 164.4, 160.7, 150.9, 142.2, 129.4, 122.9, 115.0,
108.1, 48.0, 11.9. HRMS (ESI): calcd for C14H15N4O5 (M+H)+
319.1042, found 319.1039.
4.1.2. General procedure for the synthesis of the title
compounds 1a and 1b
N-(3-Dimethylaminopropyl)-N0-ethylcarbodiimide hydrochlo-
ride (EDCꢁHCl, 1.2 mmol) was added into a solution of (thymine-
1-yl) acetic acid (4, 1 mmol) and ArCONHNH2 (1 mmol) in 8 mL
dimethylformamide (DMF). The solution was stirred at room tem-
perature for 12 h, and then it was evaporated under reduced pres-
sure and purified by silic gel chromatograph with CHCl3/MeOH
(9:1–5:1, v/v) to afford 1a and 1b.36
4.1.3. General synthetic procedure for the synthesis of 1-
carbethoxyalkylthymines 5
To
a solution of thymine (10 mmol) and 1,8-diazabicy-
4.1.2.1. N0-(2-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
clo[5.4.0]undec-7-ene (DBU, 15 mmol) in 10 mL anhydrous DMF
was added ethyl chloroacetate, ethyl 3-bromopropionate, or ethyl
yl)acetyl)benzohydrazide (1a).
White powder, yield 86%, mp

4694
X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
2-chloropropionate (12 mmol) with stirring under reflux for 3 h.
The mixture was extracted with dichloromethane, washed with
saturated brine and water subsequently, dried with anhydrous so-
dium sulfate. The solvent was evaporated and the residue was
purified by silic gel chromatography with CHCl3/MeOH (10:1, v/
v) to yield the products 5a–5c. Compounds 5a and 5b have been
reported previously in the literature.32,37 The data are as follows:
successively with water and brine, dried over anhydrous sodium
sulfate. After concentrated in vacuo, the residue was purified by
silica gel column chromatography by CHCl3/MeOH (10:1–5:1, v/
v) to give the intermidate 7 as white solid. The data of these com-
pounds are shown following.
4.1.5.1. N0-(Heptan-4-yl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyr-
imidin-1(2H)-yl)acetohydrazide (7a).
Yield 65%, mp 164–
4.1.3.1. Ethyl 2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
166 °C. 1H NMR (600 MHz, DMSO-d6) d 11.26 (s, 0.75H, thymine-
NH (3)) and 11.20 (s, 0.25H, thymine-NH (3)), 9.51 (s, 0.75H,
CONHNH) and 8.71 (s, 0.25H, CONHNH), 7.43 (s, 0.75H, thymine-
CH (6)) and 7.39 (s, 0.25H, thymine-CH (6)), 4.63 (d, J = 4.7 Hz,
1H, CONHNH), 4.51 (s, 0.5H, CH2CONH) and 4.25 (s, 1.5H,
CH2CONH), 2.65 (m, 0.75H, heptyl-CH (1)) and 2.60 (m, 0.25H, hep-
tyl-CH (1)), 1.75 (s, 3H, thymine-CH3), 1.40–1.20 (m, 8H, heptyl-
4 ꢂ CH2), 0.89–0.84 (m, 6H, heptyl-2 ꢂ CH3). 13C NMR (150 MHz,
DMSO-d6) d 170.5 (0.25C) and 165.8 (0.75C), 164.5 (0.25C) and
164.4 (0.75C), 151.0 (0.25C) and 150.9 (0.75C), 142.7 (0.25C)
and 142.3 (0.75C), 107.9 (0.75C) and 107.6 (0.25C), 59.1 (0.25C)
and 58.5 (0.75C), 48.2 (0.75C) and 48.1 (0.25C), 34.2 (0.75C) and
33.6 (0.25C), 18.4 (0.25C) and 18.2 (0.75C), 14.2 (1C), 11.9 (1C).
HRMS (ESI): calcd for C14H25N4O3 (M+H)+ 297.1926, found 297.1927.
1(2H)-yl)acetate (5a).
White solid, yield 85%, mp 136–138 °C.
4.1.3.2. Ethyl 3-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)propanoate (5b).
142 °C.
White solid, yield 90%, mp 140–
4.1.3.3. Ethyl 2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)propanoate (5c). White solid, yield 87%, mp 105–
107 °C. 1H NMR (600 MHz, DMSO-d6) d 11.35 (s, 1H, thymine-NH
(3)), 7.56 (d, J = 1.1 Hz, 1H, thymine-CH (6)), 4.96 (q, J = 7.3 Hz,
1H, CH3CH), 4.12 (qd, J = 7.1, 1.3 Hz, 2H, CH3CH2), 1.78 (d,
J = 1.0 Hz, 3H, thymine-CH3), 1.51 (d, J = 7.3 Hz, 3H, CH3CH), 1.17
(t, J = 7.1 Hz, 3H, CH3CH2). 13C NMR (150 MHz, DMSO-d6) d 170.3,
164.2, 150.9, 139.7, 109.0, 61.4, 54.6, 15.4, 14.1, 12.1.
4.1.5.2. N0-(Heptan-4-yl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyr-
4.1.4. General procedure for the synthesis of 1-
acetylhydrazinothymines 6
A mixture of 5 (10 mmol), hydrazine hydrate (3 mL) and etha-
nol (6 mL) was heated under reflux for 4 h. Then the mixture was
cooled to room temperate and the product was filtered off and
recrystallized from ethanol to give 6a–6c.
imidin-1(2H)-yl)propanehydrazide (7b).
Yield 70%, mp
138–140 °C. 1H NMR (600 MHz, DMSO-d6) d 11.25 (s, 1H, thy-
mine-NH (3)), 9.59 (s, 1H, CONHNH), 7.54 (s, 1H, thymine-CH
(6)), 5.03 (q, J = 7.3 Hz, 1H, CH3CHCONH), 4.64 (br s, 1H, CONHNH),
2.66 (m, 1H, heptyl-CH (1)), 1.78 (s, 3H, thymine-CH3), 1.44 (d,
J = 7.3 Hz, 3H, CH3CHCONH), 1.34–1.19 (m, 8H, heptyl-4 ꢂ CH2),
0.89–0.80 (m, 6H, heptyl-3 ꢂ CH3). 13C NMR (150 MHz, DMSO-d6)
d 168.7, 163.9, 150.9, 138.6, 108.2, 58.4, 51.1, 34.3, 34.2, 18.3,
18.2, 16.7, 14.3, 14.2, 12.1. HRMS (ESI): calcd for C15H27N4O3
(M+H)+ 311.2083, found 311.2085.
4.1.4.1.
yl)acetohydrazide (6a).
2-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
White solid, yield 90%, mp 245–
247 °C. 1H NMR (600 MHz, DMSO-d6) d 11.26 (s, 1H, thymine-NH
(3)), 9.29 (s, 1H, CONHNH2), 7.42 (s, 1H, thymine-CH (6)), 4.24 (s,
2H, CH2CONH), 1.75 (s, 3H, thymine-CH3), 1.23 (s, 2H, CONHNH2).
13C NMR (150 MHz, DMSO) d 166.5, 164.6, 151.0, 142.3, 108.2,
48.2, 12.0.
4.1.5.3. N0-Cyclohexyl-2-(5-methyl-2,4-dioxo-3,4-dihydropyrim-
idin-1(2H)-yl)acetohydrazide (7c).
Yield 82%, mp 290–
292 °C. 1H NMR (600 MHz, DMSO-d6) d 11.25 (s, 0.75H, thymine-
NH (3)) and 11.19 (s, 0.25H, thymine-NH (3)), 9.50 (s, 0.75H,
CONHNH) and 8.69 (s, 0.25H, CONHNH), 7.44 (s, 0.75H, thymine-
CH (6)) and 7.40 (s, 0.25H, thymine-CH (6)), 4.80 (d, J = 3.48 Hz
0.25H, CH2CONHNH) and 4.67(br s, 0.75H, CH2CONHNH), 4.52 (s,
0.5H, CH2CONH) and 4.25 (s, 1.5H, CH2CONH), 2.62 (br m, 0.75H,
cyclohexyl-CH (1)) and 2.55 (br m, 0.25H, cyclohexyl-CH (1)),
1.80 (d, 0.5H, cyclohexyl-CH2), 1.75 (s, 3H, thymine-CH3), 1.73 (br
d, 1.5H, cyclohexyl-CH2), 1.66 (br d, 2H, cyclohexyl-CH2), 1.59–
1.55 (m, 0.25H, cyclohexyl-CH2) and 1.54–1.49 (m, 0.75H, cyclo-
hexyl-CH2), 1.25–1.10 (m, 3H, cyclohexyl-CH2) and 1.07–0.94 (m,
4.1.4.2.
yl)propanehydrazide (6b).
3-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
White solid, yield 93%, mp 178–
180 °C. 1H NMR (600 MHz, DMSO-d6) d 11.22 (s, 1H, thymine-NH
(3)), 9.13 (s, 1H, CONHNH2), 7.41 (s, 1H, thymine-CH (6)), 3.82 (t,
J = 6.5 Hz, 2H, CH2CH2CONH), 2.39 (t, J = 6.5 Hz, 2H, CH2CH2CONH),
1.72 (s, 3H, thymine-CH3), 1.23 (s, 2H, CONHNH2). 13C NMR
(150 MHz, DMSO-d6) d 169.1, 164.4, 150.8, 141.9, 108.1, 44.5,
32.48, 12.0.
4.1.4.3.
yl)propanehydrazide (6c).
2-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
White solid, yield 93%, mp 172–
2H, cyclohexyl-CH2). 13C NMR (150 MHz, DMSO-d6)
d 170.5
(0.25C) and 165.9 (0.75C), 164.5 (0.25C) and 164.4 (0.75C), 151.0
(0.25C) and 150.9 (0.75C), 142.8 (0.25C) and 142.4 (0.75C), 107.9
(0.75C) and 107.6 (0.25C), 58.4 (0.25C) and 57.7 (0.75C), 48.2
(0.25C) and 48.1 (0.75C), 25.7, 24.2 (0.75C) and 23.9 (0.25C), 11.9
174 °C. 1H NMR (600 MHz, DMSO-d6) d 11.25 (s, 1H, thymine-NH
(3)), 9.38 (s, 1H, CONHNH2), 7.53 (s, 1H, thymine-CH (6)), 5.00 (q,
J = 7.3 Hz, 1H, CH3CHCO), 4.31 (s, 2H, CONHNH2), 1.78 (s, 3H, thy-
mine-CH3), 1.43 (d, J = 7.3 Hz, 3H, CH3CHCO). 13C NMR (150 MHz,
DMSO-d6) d 169.1, 164.0, 151.0, 138.7, 108.4, 51.2, 17.0, 12.2.
(0.75C) and 11.8 (0.25C). HRMS (ESI): calcd for
C13H21N4O3
(M+H)+ 281.1613, found 281.1620.
4.1.5. General procedure for the synthesis of intermediates 7
A mixture of 6 (3 mmol), alkyl/cycloalkyl ketene or aldehyde
(3 mmol), and anhydrous ethanol (10 mL) was refluxed for 3 h.
Then the solution was concentrated and cooled to separate the
condensation product hydrazones with crystallization from etha-
nol. To the suspension of the above step product (2 mmol) of anhy-
drous THF (6 mL), sodium borohydride (4 mmol) was added under
ice bath and stirred at room temperature for 5 h. The excess so-
dium borohydride was decomposed with 1 N hydrochloric
acid. The mixture was extracted with dichloromethane, washed
4.1.5.4. N0-Cyclohexyl-3-(5-methyl-2,4-dioxo-3,4-dihydropyrim-
idin-1(2H)-yl)propanehydrazide (7d). Yield 80%, mp 135–
137 °C. 1H NMR (600 MHz, DMSO-d6) d 11.23 (s, 1H, thymine-NH
(3)), 9.36 (d, J = 4.6 Hz, 1H, CONHNH), 7.34 (d, J = 1.1 Hz, 1H, thy-
mine-CH (6)), 4.66 (br s, 1H, CONHNH), 3.83 (t, J = 6.5 Hz, 2H,
CH2CH2CONH), 2.40 (t, J = 6.5 Hz, 2H, CH2CH2CONH), 1.71 (d,
J = 0.9 Hz, 3H, thymine-CH3), 1.68–1.60 (m, 4H, cyclohexyl-
2 ꢂ CH2), 1.53–1.47 (m, 1H, cyclohexyl-CH (1)), 1.18–1.03 (m, 4H,
cyclohexyl-2 ꢂ CH2), 1.01–0.90 (m, 2H, cyclohexyl-CH2). 13C NMR
(150 MHz, DMSO-d6) d 168.7, 164.6, 150.9, 142.2, 108.2, 58.0,

X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
4695
44.8, 32.7, 31.0, 25.8, 24.2, 12.2. HRMS (ESI): calcd for C14H23N4O3
(M+H)+ 295.1770, found 295.1771.
55.1 (0.75C), 48.4 (0.75C) and 48.3 (0.25C), 33.9 (0.25C) and 33.8
(0.75C), 27.8 (0.25C) and 27.6 (0.75C), 22.5 (0.25C) and 22.4
(0.75C), 18.2 (0.75C) and 18.1 (0.25C), 14.1 (1C), 12.0 (1C). HRMS
(ESI): calcd for C13H23N4O3 (M+H)+ 283.1770, found 283.1770.
4.1.5.5. N0-(Cyclohexylmethyl)-2-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)acetohydrazide (7e).
Yield 75%, mp
186–188 °C. 1H NMR (600 MHz, DMSO) d 11.26 (s, 0.75H, thy-
mine-NH (3)) and 11.19 (s, 0.25H, thymine-NH (3)), 9.50 (s,
0.75H, CONHNH) and 8.68 (s, 0.25H, CONHNH), 7.43 (d,
J = 0.9 Hz, 0.75H, thymine-CH (6)) and 7.39 (d, J = 0.9 Hz, 0.25H,
thymine-CH (6)), 4.67 (br s, 1H, CONHNH), 4.52 (s, 0.5H, CH2CON-
HNH) and 4.25 (s, 1.5H, CH2CONHNH), 2.64–2.59 (m, 0.5H, CON-
HNHCH2) and 2.57–2.53 (m, 1.5H, CONHNHCH2), 1.81 (br m,
0.25H, cyclohexyl-CH (1)) and 1.79 (br m, 0.75H, cyclohexyl-CH
(1)), 1.74 (d, J = 1.1 Hz, 3H, thymine-CH3), 1.73–1.63 and 1.58–
1.50 (m, 4H, cyclohexyl-2 ꢂ CH2), 1.22–1.09 (m, 4H, cyclohexyl-
2 ꢂ CH2), 1.07–0.95 (m, 2H, cyclohexy-CH2). 13C NMR (150 MHz,
DMSO) d 170.5 (0.25C) and 165.9 (0.75C), 164.6 (0.25C) and
164.5 (0.75C), 151.1 (0.25C) and 151.0 (0.75C), 142.8 (0.25C) and
142.4 (0.75C), 108.0 (0.75C) and 107.6 (0.25C), 58.5 (0.25C) and
57.8 (0.75C), 48.3 (0.75C) and 48.1 (0.25), 35.6 (0.75C) and 35.4
(0.25C), 30.8 (0.75C) and 30.6 (0.25), 25.7, 24.2 (0.25) and 24.0
(0.75C), 11.9 (1C). HRMS (ESI): calcd for C13H21N4O3 (M+H)+
295.1770, found 295.1770.
4.1.6. General synthetic procedure for diacylhydrazine
derivatives 2a–2m and 3a–3c
A mixture of 7 (1 mmol), triethylamine (1.5 mmol) and 4-
dimethylaminopyridine (0.05 mmol) in anhydrous CH2Cl2
(10 mL) was stirred in an ice bath, and substituted benzoyl chloride
(1.1 mmol) in CH2Cl2 (5 mL) was added. After stirring for 3 h at
room temperature and another 2–5 h at 35–40 °C, the reaction
mixture was extracted with CHCl3. The combined organic layer
was washed with aqueous sodium bicarbonate, brine and water,
and then dried over anhydrous magnesium sulfate. After evapo-
rated, the residue was purified by silica gel column chromatogra-
phy by petroleum ether/ethyl acetate to afford the target
compounds 2a–2m.34,16 And then, the products 2j, 2l, and 2m were
deacetylation to give the target compounds 3a–3c. Their physico-
chemical properties and the spectral data are provided in Supple-
mentary data.
4.2. Biological assay
4.1.5.6. N0-(Cyclohexylmethyl)-3-(5-methyl-2,4-dioxo-3,4-dihy-
Uracil (99%), thymine (99%), 5-fluorouracil (99%), trifluorothy-
mine (95%) and 5-bromouracil (99%) were purchased from Aladdin
Reagent Co., Ltd. Tebufenozide (95%) was purchased from Jingbo
Agrochemicals Technology Co.
The larvae of S. litura were obtained from Guangzhou Bio-con-
trol Station, China reared on artificial diet in the laboratory at
25 1 °C with 60–70% relative humidity.
dropyrimidin-1(2H)-yl)propanehydrazide (7f).
Yield 71%,
mp 121–123 °C. 1H NMR (600 MHz, DMSO-d6) d 11.23 (s, 1H, thy-
mine-NH (3)), 9.36 (s, 1H, CONHNH), 7.36 (d, J = 1.0 Hz, 1H, thy-
mine-CH (6)), 3.82 (t, J = 6.5 Hz, 2H, CH2CH2CONH), 3.32 (s, 1H,
CONHNH), 2.43 (d, J = 6.8 Hz, 2H, CONHNHCH2), 2.37 (t,
J = 6.5 Hz, 2H, CH2CH2CONH), 1.71 (d, J = 0.8 Hz, 3H, thymine-
CH3), 1.68 (m, 1H, cyclohexyl-CH (1)), 1.66–1.56 (m, 4H, cyclo-
hexyl-2 ꢂ CH2), 1.18–1.07 (m, 4H, cyclohexyl-2 ꢂ CH2), 0.85–0.76
(m, 2H, cyclohexyl-CH2). 13C NMR (150 MHz, DMSO-d6) d 168.4,
164.4, 150.7, 141.9, 108.0, 57.6, 44.5, 35.6, 32.5, 30.8, 29.5, 26.2,
4.2.1. Toxicity against S. litura evaluated by immersing insect
method
The second instar larvae of S. litura were selected and starved
for 4 h.38 The stock solutions of the compounds were prepared
by DMSO, and diluted to test solution with water containing 0.1%
Tween 80 before use. The larvae were immersed into the test solu-
tion for 10 s, dried with filter paper for the surplus water, intro-
duced into Petri dishes and reared on artificial diet. The larvae
received the solution without compounds were treated as negative
control and with tebufenozide as the positive control. The cor-
rected mortalities were calculated after treatment with 48 h,
96 h, and 144 h. The median lethal concentration (LC50) was ob-
tained by the log concentration versus mortality probability.
25.5, 25.0, 12.0. HRMS (ESI): calcd for
309.1926, found 309.1930.
C
15H25N4O3 (M+H)+
4.1.5.7. N0-(Cyclohexylmethyl)-2-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)propanehydrazide (7g). Yield 73%,
mp 122–124 °C. 1H NMR (600 MHz, DMSO-d6) d 11.25 (s, 1H, thy-
mine-NH (3)), 9.60 (s, 1H, CONHNH), 7.52 (s, 1H, thymine-CH (6)),
6.53 (s, 1H, CONHNH), 4.98 (q, J = 7.3 Hz, 1H, CH3CHCONH), 1.78 (s,
3H, thymine-CH3), 1.72 (d, J = 12.4 Hz, 2H, CONHNHCH2), 1.69–
1.55 (m, 4H, cyclohexyl-2 ꢂ CH2), 1.43 (d, J = 7.3 Hz, 3H,
CH3CHCONH), 1.36 (m, 1H, cyclohexyl-CH (1)), 1.25–1.01 (m, 4H,
cyclohexyl-2 ꢂ CH2), 0.84 (m, 2H, cyclohexyl-CH2). 13C NMR
(150 MHz, DMSO-d6) d 168.5, 163.9, 150.9, 138.6, 108.3, 57.5,
51.2, 35.8, 30.8, 26.2, 25.5, 16.5, 12.1. HRMS (ESI): calcd for
4.2.2. Larval growth inhibition and regulation assayed by feed-
poison method
The effect of these compounds on growth of S. litura was evalu-
ated referring to the reported procedure with slight modification.39
Each compound was dissolved in acetone as a stock solution, and
diluted to the required concentration with acetone as test solu-
tions. The poisoned diet was prepared with the test solution. Every
10 pre-starved (4 h) larvae were weighted and introduced into
each dish, reared on the poisoned diet. Acetone (without the tested
compound) was used as the negative control and tebufenozide was
as the positive control. After feeding 24 h, the poisoned diet was re-
placed with fresh diet and the larvae were reared till pupation and
adult emergence. The weight of the treated larvae was recorded
after a 60 h treatment. The weight gain, duration of larval and pu-
pal stages, and adult emergence rate were recorded.
C
15H25N4O3 (M+H)+ 309.1926, found 309.1925.
4.1.5.8. N0-(Hexan-2-yl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyr-
imidin-1(2H)-yl)acetohydrazide (7h). Yield 59%, mp 163–
165 °C. 1H NMR (600 MHz, DMSO-d6) d 11.26 (s, 0.75H, thymine-
NH (3)) and 11.20 (s, 0.25H, thymine-NH (3)), 9.54 (s, 0.75H,
CONHNH) and 8.70 (s, 0.25H, CONHNH), 7.44 (s, 0.75H, thymine-
CH (6)) and 7.40 (s, 0.25H, thymine-CH (6)), 4.51 (s, 0.5H,
CH2CONH) and 4.26 (s, 1.5H, CH2CONH), 2.78 (dt, J = 12.7, 6.3 Hz,
0.75H, CONHNH) and 2.72 (s, 0.25H, CONHNH), 1.75 (s, 3H, thy-
mine-CH3), 1.50–1.44 (m, 0.25H, hexyl-CH) and 1.43–1.34 (m,
0.75H, hexyl-CH), 1.32–1.22 (m, 4.5H, hexyl-2 ꢂ CH2) and 1.19–
1.26 (m, 1.5H, hexyl-CH2), 0.97 (d, J = 6.3 Hz, 1H, hexyl-CH3) and
0.92 (d, J = 6.3 Hz, 2H, hexyl-CH3), 0.86 (q, J = 7.0 Hz, 3H, hexyl-
CH3). 13C NMR (150 MHz, DMSO-d6) d 170.6 (0.25C) and 166.2
(0.75C), 164.7, 151.2 (0.25C) and 151.1 (0.75C), 143.0 (0.25C) and
142.5 (0.75C), 108.5 (0.25C) and 108.3 (0.75C), 55.3 (0.25C) and
4.2.3. Agonistic activity determined by reporter gene assay
Sf9 cells (derived from ovary of S. frugiperda and obtained from
Dr. Yang Cao, College of Animal Science, South China Agricultural
University, China) were used for the reporter gene assay. Cells were

4696
X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
cultured in HyClone SFX-Insect serum-free insect culture medium
(Thermo Fisher Scientific, China) supplying with 5% fetal calf serum
at 27.5 °C. A reporter plasmid pBmbA/hsp27/gfp, referred as ERE-
b.act-GFP, was composed of seven copies of the ERE derived from
D. melanogaster hsp27 promoter, and a promoter region of actin
A3 gene of B. mori, which was followed by an EGFP coding gene.
This plasmid was constructed for detecting ecdysone agonists-
dependent activation of transcription by Dr. Swevers.28
The plasmid was purified by Omega Endo-free Plasmid Midi Kit
(Omega Bio-Tek, Inc., China) after amplified in Escherichia coli
according to the manufacturer. Then the plasmids were merged
with MegaTran 1.0 (OriGene, Beijing, China) as complex and gently
added into the cells (5 ꢂ 104 per well) in each well of 24-well
plates. After incubation for 24 h, the test compound (dissolved
with DMSO) was diluted with culture medium and added into
the cells for another 24 h incubation. The induced GFP fluorescence
on the living cells was observed directly using Olympus CKX41 in-
verted microscope (Aizu, Japan). The micrographs were collected
with ProgRes CF digital camera system (Jenoptik, Germany). Then,
cells in each well were collected and were gently washed twice
300 K. Finally, ten molecular docking poses saved for each ligand
were ranked according to their dock score function. The pose with
the highest –CDOCKER interaction energy was considered as the
best one, whereas the –CDOCKER energy between compounds
and key residues of LBD of SlEcR was also calculated. After end of
molecular docking, the types of interactions of the docked LBD of
SlEcR with ligand were analyzed. Accuracy testing was performed
by calculating the root mean square deviation (RMSD) after re-
docking the ligand BYI06830 with the same algorithm into the
SlEcR LBD.
Acknowledgments
The authors thank Dr. Luc Swevers and Dr. Kostas Iatrou (Biosci-
ences and Applications of National Centre for Scientific Research
‘Demokritos’, Greece) for providing the plasmid, and financial sup-
ports from the National Natural Science Foundation of China
(Grant No. 31171886), the Specialized Research Fund for the Doc-
toral Program of Higher Education of China (Grant No.
20114404110020) and the Foundation of Department of Science
and Technology of Guangdong Province of China (Grant No.
2012B020309004).
with 250 lL PBS and transferred to black 96-well plates at a den-
sity of 3 ꢂ 104 cells/mL. Fluorescence intensity of the cells was re-
corded by a 1420 Multilabel Counter Victor 3V (Perkinelmer,
Massachusetts, America), and corrected by auto-fluorescence and
background fluorescence in the absence of compounds.40
Supplementary data
Supplementary data (data of the compounds 2a–2m and 3a–3c)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2013.05.010.
4.2.4. Statistical analysis
All the experiments were repeated at least three times. Data
were expressed as the means SE and analyzed by Duncan’s multi-
ple-range test (DMRT). The level of significance was set at p <0.05.
References and notes
4.3. Molecular modeling and docking study
1. Gilbert, L. I. Mol. Cell. Endocrinol. 2004, 215, 1.
2. Yao, T. P.; Forman, B. M.; Jiang, Z.; Cherbas, L.; Chen, J. D.; McKeown, M.;
Cherbas, P.; Evans, R. M. Nature 1993, 366, 476.
3. Thomas, H. E.; Stunnenberg, H. G.; Stewart, A. F. Nature 1993, 362, 471.
4. Koelle, M. R.; Talbot, W. S.; Segraves, W. A.; Bender, M. T.; Cherbas, P.; Hogness,
D. S. Cell 1991, 67, 59.
5. Swevers, L.; Iatrou, K. In Ecdysone: Structure and Function; Smagghe, G., Ed.;
Springer, 2009; p 127.
6. Nakagawa, Y.; Henrich, V. C. FEBS J. 2009, 276, 6128.
The ligand binding domain (LBD) of S. litrua EcR (SlEcR) was
constructed on the Swiss Model website (http://swissmodel.exp-
asy.org/).41–43 The primary sequence of SlEcR was derived from
the previously reported reference (NCBI accession number:
ABX79143).44 The sequence of SlEcR was aligned with HvEcR with
an identity of 87% (shown in Supplementary data Fig. 1S).45 The
chian D of the crystal-structure of the ligand binding domain of
HvEcR (PDB ID is 1R20) was selected as the template for homology
modeling building.23,46 The constructed modeling was evaluated
by QMEAN, with the value of 0.63, as a reasonable modeling.47
The downloaded structure was qualified and employed for the
docking study.
Molecular docking was performed with CDOCKER protocol in
Discovery Studio version 2.5 (DS 2.5, Accelrys, Co., Ltd, San Diego,
CA) with the CHARMm force field48,49 to evaluate the potential
molecular binding mode between the synthesized compounds
1a–1b, 2a–2l, 3a–3c and LBD of SlEcR. The CDOCKER algorithm
employed a strategy involving the generation of several initial li-
gand orientations in the active site of target protein followed by
molecular dynamics (MD)-based simulated annealing, and final
refinement by energy minimization. The three dimensional struc-
ture of SlEcR LBD constructed as statement above, was refined with
CHARMm and defined as a receptor. The site sphere was defined
based on the ligand binding location of BYI06830 with a radius
of 10 Å. The 3D structures of the synthesized compounds were gen-
erated with ChemBioOffice 2010 and optimized with MM2 meth-
od.50 After refinement with CHARMm, the compounds were
docked into the protein. A set of 10 different orientations of every
compound were randomly generated. Once the ligand had been
docked into the active site, a simulated annealing method was con-
ducted consisting of a heating phase from 300 K to 700 K with
2000 steps, followed by a cooling phase back to 300 K with 5000
steps. The energy threshold for van der Waals force was set at
7. King-Jones, K.; Thummel, C. S. Genetics 2005, 6, 311.
8. Wing, K. D.; Slawecki, R. A.; Carlson, G. R. Science 1988, 241, 470.
9. Wing, K. D. Science 1988, 241, 467.
10. Hsu, A. C. In Synthesis and Chemistry of Agrochemicals II; Baker, D. R., Fenyes, J.
G., Moberg, W. K., Eds.; American Chemical Society, 1991; Vol. 443, p 478.
11. Birru, W.; Fernley, R. T.; Graham, L. D.; Grusovin, J.; Hill, R. J.; Hofmann, A.;
Howell, L.; James, P. J.; Jarvis, K. E.; Johnson, W. M.; Jones, D. A.; Leitner, C.;
Liepa, A. J.; Lovrecz, G. O.; Lu, L.; Nearn, R. H.; O’Driscoll, B. J.; Phan, T.; Pollard,
M.; Turner, K. A.; Winkler, D. A. Bioorg. Med. Chem. 2010, 18, 5647.
12. Holmwood, G.; Schindler, M. Bioorg. Med. Chem. 2009, 17, 4064.
13. Mao, C. H.; Wang, K. L.; Wang, Z. W.; Ou, X. M.; Huang, R. Q.; Bi, F. C.; Wang, Q.
M. Bioorg. Med. Chem. 2008, 16, 488.
14. Zhao, P. L.; Li, J.; Yang, G. F. Bioorg. Med. Chem. 1888, 2007, 15.
15. Nakagawa, Y.; Takahashi, K.; Kishikawa, H.; Ogura, T.; Minakuchi, C.;
Miyagawa, H. Bioorg. Med. Chem. 2005, 13, 1333.
16. Ke, S.; Qian, X.; Liu, F.; Wang, N.; Fan, F.; Li, Z.; Yang, Q. Eur. J. Med. Chem. 2009,
44, 2985.
17. Dhadialla, T. S.; Jansson, R. K. Pestic. Sci. 1999, 55, 357.
18. Heller, J.; Mattioda, H.; Klein, E.; Sagenmuller, A. In Proceedings, Brighton Crop
Protection Conference, Pests and Diseases: Brighton, 1992.
19. Carlson, G. R.; Dhadialla, T. S.; Hunter, R.; Jansson, R. K.; Jany, C. S.; Lidert, Z.;
Slawecki, R. A. Pest. Manage. Sci. 2001, 57, 115.
20. Sawada, Y.; Yanai, T.; Nakagawa, H.; Tsukamoto, Y.; Yokoi, S.; Yanagi, M.; Toya,
T.; Sugizaki, H.; Kato, Y.; Shirakura, H.; Watanabe, T.; Yajima, Y.; Kodama, S.;
Masui, A. Pest. Manage. Sci. 2003, 59, 36.
21. Dhadialla, T. S.; Retnakaran, A.; Smagghe, G. In Insect Growth and Development-
Disrupting Insecticides. Elsevier: London, 2010, p 121.
22. Nakagawa, Y.; Harada, T. In Advanced Technologies for Managing Insect Pests;
Ishaaya, I., Palli, S. R., Horowitz, A. R., Eds.; Springer: Netherlands, 2013; p 135.
23. Billas, I. M.; Iwema, T.; Garnier, J. M.; Mitschler, A.; Rochel, N.; Moras, D. Nature
2003, 426, 91.
24. Toya, T.; Yamaguchi, K.; Endo, Y. Bioorg. Med. Chem. 2002, 10, 953.
25. Wheelock, C. E.; Nakagawa, Y.; Harada, T.; Oikawa, N.; Akamatsu, M.; Smagghe,
G.; Stefanou, D.; Iatrou, K.; Swevers, L. Bioorg. Med. Chem. 2006, 14, 1143.
26. Fujita, T.; Nakagawa, Y. SAR QSAR Environ. Res. 2007, 18, 77.
27. Dai, Z. K.; Yang, X. S.; Yu, L. M. Zhongguo Zhong Xi Yi Jie He Za Zhi 2011, 31, 1117.

X. Liu et al. / Bioorg. Med. Chem. 21 (2013) 4687–4697
4697
28. Swevers, L.; Kravariti, L.; Ciolfi, S.; Xenou-Kokoletsi, M.; Ragoussis, N.;
Smagghe, G.; Nakagawa, Y.; Mazomenos, B.; Iatrou, K. FASEB J. 2004, 18, 134.
29. Chen, J. H.; Turner, P. C.; Rees, H. H. Insect Biochem. Mol. Biol. 2002, 32, 657.
30. Katritzky, A. R.; Narindoshvili, T. Org. Biomol. Chem. 2008, 6, 3171.
31. Thomson, S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman, C. F.; Luzzio, M. J.;
Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.; Noble, S. A. Tetrahedron 1995,
51, 6179.
32. Ali, O. M.; Amer, H. H.; Abdel-Rahman, A. Synthesis (Stuttgart) 2007, 2823.
33. Ke, S. Y.; Qian, X. H.; Liu, F. Y.; Wang, N.; Yang, Q.; Li, Z. Eur. J. Med. Chem. 2009,
44, 2113.
39. Zhou, Y. Y.; Luo, S. H.; Yi, T. S.; Li, C. H.; Luo, Q.; Hua, J.; Liu, Y.; Li, S. H. J. Agric.
Food Chem. 2011, 59, 6004.
40. Soin, T.; Swevers, L.; Mosallanejad, H.; Efrose, R.; Labropoulou, V.; Iatrou, K.;
Smagghe, G. J. Insect Physiol. 2008, 54, 429.
41. Arnold, K.; Bordoli, L.; Kopp, J.; Schwede, T. Bioinformatics 2006, 22, 195.
42. Schwede, T.; Kopp, J.; Guex, N.; Peitsch, M. C. Nucleic Acids Res. 2003, 31, 3381.
43. Guex, N.; Peitsch, M. C. Electrophoresis 1997, 18, 2714.
44. Pascal, E. J.; Valentine, S. A.; Brown, J. A.; Cockrell, A. S.; Johnson, B. D.
WO02061102A2, 2002, 319.
45. Corpet, F. Nucleic Acids Res. 1988, 16, 10881.
34. Sawada, Y.; Yanai, T.; Nakagawa, H.; Tsukamoto, Y.; Tamagawa, Y.; Yokoi, S.;
Yanagi, M.; Toya, T.; Sugizaki, H.; Kato, Y.; Shirakura, H.; Watanabe, T.; Yajima,
Y.; Kodama, S.; Masui, A. Pest. Manage. Sci. 2003, 59, 49.
35. Dueholm, K. L.; Egholm, M.; Behrens, C.; Christensen, L.; Hansen, H. F.; Vulpius,
T.; Petersen, K. H.; Berg, R. H.; Nielsen, P. E.; Buchardt, O. J. Org. Chem. 1994, 59,
5767.
36. Hickman, D. T.; Sreenivasachary, N.; Lehn, J. Helv. Chim. Acta 2008, 91, 1.
37. Mokhir, A. A.; Richert, C. Nucleic Acids Res. 2000, 28, 4254.
38. Yoon, K. S.; Strycharz, J. P.; Baek, J. H.; Sun, W.; Kim, J. H.; Kang, J. S.;
Pittendrigh, B. R.; Lee, S. H.; Clark, J. M. Insect Mol. Biol. 2011, 20, 687.
46. Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.;
Shindyalov, I. N.; Bourne, P. E. Nucleic Acids Res. 2000, 28, 235.
47. Benkert, P.; Biasini, M.; Schwede, T. Bioinformatics 2011, 27, 343.
48. Wu, G.; Robertson, D. H.; Brooks, C. R.; Vieth, M. J. Comput. Chem. 2003, 24,
1549.
49. Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.;
Karplus, M. J. Comput. Chem. 1983, 4, 187.
50. ChemBioOffice Ultra Version 12.0, CambridgeSoft Inc., Cambridge, USA; 2010.