Design, synthesis, insecticidal evaluation and molecular docking studies of cis-nitenpyram analogues bearing diglycine esters

Article abstract of DOI:10.1007/s11426-012-4748-x

Based on the strategies of receptor structure-guided neonicotinoid design, a series of novel cis-nitenpyram analogues bearing diglycine esters were designed and synthesized. Preliminary bioassays indicated that the insecticidal spectra of the target compounds were expanded compared with our previous work, while all the target compounds presented excellent insecticidal activities against Nilaparvata lugens and Aphis medicagini at 100 mg/L. Among these analogues, 6b showed 100% mortality against Nilaparvata lugens (LC₅₀ = 0.163 mg/L) and 90% against Aphis medicagini at 4 mg/L. SARs suggested that the insecticidal potency of our designed cis-nitenpyram analogues was dual-controlled by the size and species of the ester groups. The molecular docking simulations revealed that the structural uniqueness of these analogues may lead to a unique molecular recognition and binding mode compared with the previously designed compounds. Introduction of the peptide bond gave rise to more significant hydrogen bonds between the nitenpyram analogues bonding with the amino acid residues of insect nAChRs. The docking results explained the SARs observed in vitro, and shed light on the novel insecticidal mechanism of these cis-nitenpyram analogues.

Full text of DOI:10.1007/s11426-012-4748-x

SCIENCE CHINA
Chemistry
February 2013 Vol.56 No.2: 159–168
doi: 10.1007/s11426-012-4748-x
• ARTICLES •
Design, synthesis, insecticidal evaluation and molecular docking
studies of cis-nitenpyram analogues bearing diglycine esters
CHEN YanXia1, SUN ChuanWen1,2*, WEN XiaXia1 & ZHANG WangGeng2
1College of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China
2Jiangsu Institute of Ecomones Co., Ltd., Jintan 213200, China
Received April 24, 2012; accepted June 5, 2012; published online August 31, 2012
Based on the strategies of receptor structure-guided neonicotinoid design, a series of novel cis-nitenpyram analogues bearing
diglycine esters were designed and synthesized. Preliminary bioassays indicated that the insecticidal spectra of the target com-
pounds were expanded compared with our previous work, while all the target compounds presented excellent insecticidal ac-
tivities against Nilaparvata lugens and Aphis medicagini at 100 mg/L. Among these analogues, 6b showed 100% mortality
against Nilaparvata lugens (LC50 = 0.163 mg/L) and 90% against Aphis medicagini at 4 mg/L. SARs suggested that the insec-
ticidal potency of our designed cis-nitenpyram analogues was dual-controlled by the size and species of the ester groups. The
molecular docking simulations revealed that the structural uniqueness of these analogues may lead to a unique molecular
recognition and binding mode compared with the previously designed compounds. Introduction of the peptide bond gave rise
to more significant hydrogen bonds between the nitenpyram analogues bonding with the amino acid residues of insect nAChRs.
The docking results explained the SARs observed in vitro, and shed light on the novel insecticidal mechanism of these
cis-nitenpyram analogues.
cis-nitenpyram analogues, hydrogen bonds, diglycine ester, synthesis, insecticidal activity, molecular docking, nAChRs,
peptide bond
toxicity, broad insecticidal spectra, and good systemic
properties [5, 6]. However, the new mode of action, fre-
quent applications in the field of pest control and structural
1
Introduction
Recently, neonicotinoids began to replace pyrethroids, chlo-
rinated hydrocarbons, organophosphates (OPs), carbamates
and several other types of insecticides to control insect pests
on major crops. Due to their efficient mode of action (MoA),
neonicotinoids showed little cross-resistance to the conven-
tional long-established insecticides, and they make up ap-
proximately one-fourth of the world insecticide market [1, 2].
As potent agonists, they selectively act on the insect nicotinic
acetylcholine receptors (nAChRs), i.e., their molecular tar-
get site [3]. Neonicotinoids represented by imidacloprid [4]
are the most important class of insecticides over the past
three decades because of their potency, low mammalian
similarity among neonicotinoids have led to the acquisition
of resistance and cross-resistance in a range of species such
as Plutella xylostella, Tetranychus cinnabarinus and Aphis
medicaginis, Nilaparvata lugens [2, 7–13]. Especially, the
brown planthopper, Nilaparvata lugens, a major rice pest in
many parts of Asia, has developed strong resistance to im-
idacloprid [2, 9, 14]. As a result, development of new neon-
icotinoids with high insecticidal activities against resistance
strains is highly desirable.
It is well-known that the structure optimization of com-
mercial neonicotinoids is one of the effective strategies to
manage tactics [15–17]. In our previous work [18], we have
focused our attention on designing novel neonicotinoids
with cis-configuration. Starting from nitenpyram, three lin-
*Corresponding author (email: willin@shnu.edu.cn)
© Science China Press and Springer-Verlag Berlin Heidelberg 2012
chem.scichina.com www.springerlink.com

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Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
ear amino acids with different chain length were introduced,
and various ester groups were applied (Figure 1). To this
end, a new series of cis-configuration nitenpyram analogues 3,
4, 5 were synthesized by introducing 1, 2, 3, 6-tetrahydro-
pyrimidine with flexible ester arms. These compounds have
exhibited good insecticide activities against Nilaparvata
lugens. The structure-activity relationships implied that the
different insecticidal potency was dual-controlled by the
length of the ester arm and the size of the ester group. In ad-
dition, the molecular docking simulations revealed that the
insecticidal potency depended greatly on the number of sig-
nificant hydrogen bonds which are formed by the nitenpyram
analogues and the amino acid residues of insect nAChRs.
This observation provides us a rational guide to design nov-
el cis-configuration nitenpyram analogues. In this context, it
is very important to increase the number of significant hy-
drogen bonds between the nitenpyram analogues and the
amino acid residues of insect nAChRs for the purpose of
structure optimization or design.
In order to increase the number of significant hydrogen
bonds between the nitenpyram analogues and the amino
acid residues of insect nAChRs, based on our previous work
(Figure 1), we inseted the peptide bond to the flexible ester
arm of the nitenpyram analogues 5, and designed and syn-
thesized another novel nitenpyram analogues 6 as shown in
Figure 2. The first reason to introduce the organic segment
peptide bond is that it contains more electronegative atoms
such as oxygen atoms and nitrogen atoms which can form
more significant hydrogen bonds with the amino acid resi-
dues of insect nAChRs, which may result in improving the
insecticidal activities. The second reason is that peptides are
involved in important physiological and biochemical func-
tions such as neuro transmission, neuro modulation, and act
as hormones in receptor mediated signal transduction [19].
The increasing interests regarding the manifold actions of
the bioactive peptides have made their structural studies an
important aspect of research in pharmacology and medical
sciences [20]. During drug development, identification of an
‘active’ part of a large peptide is critical for further improv-
ing its pharmacology.
As expected, preliminary bioassays indicated that not
only the insecticidal spectra of the target compounds were
expanded when compared with our previous work, but also
all the target compounds presented excellent insecticidal
activities against Nilaparvata lugens and Aphis medicagini
at 100 mg/L. Among these analogues, 6b showed 100%
mortality against Nilaparvata lugens (LC50 = 0.163 mg/L)
and 90% against Aphis medicagini at 4 mg/L. Their structure-
activity relationships indicated that insecticide activities
were dual-controlled by the size and species of the ester
group. To further investigate their binding interactions, mo-
lecular docking simulations were carried out. The results
showed that the active analogues exhibited significant hy-
drogen bonding interactions with the amino acid residues of
insect nAChRs, where the peptide bonds displayed an im-
portant role. These results confirmed our ideas. The docking
results explained the structure-activity relationships ob-
served in vitro, and shed a light on the novel insecticidal
Figure 1 Reaction of nitenpyram with different lengths of amino acid alkyl esters in our previous work.
Figure 2 cis-nitenpyram analogues containing different diglycine esters.

Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
161
mechanism of these new analogues, which may provide some
useful information for future design of new insecticides.
to dramatically reduce reaction time, increase product yields,
and enhance product purities by reducing unwanted side
reactions [24].
2 Chemistry
3 Experimental
To prepare a series of cis-nitenpyram analogues bearing the
diglycine ester, an efficient synthesis approach was devel-
oped as depicted in Scheme 1. Diglycine 1 was synthesized
according to Ref. [21], and was then converted to the inter-
mediate of diglycine hydroxyalkyl ester hydrochloride ac-
cording to the procedures given in Ref. [22]. Nitenpyram,
which was prepared based on the procedures previously
reported [23], was reacted with formaldehyde and a variety
of substituted diglycine ester hydrochloride in ethanol to
afford the desired compounds 6a–6n with cis-configuration
fixed by 1, 2, 3, 6-tetrahydropyrimidine bearing the di-
glycine ester. Parallel to our previous work [18], the
microwave-assisted synthesis was also extended to the pre-
sent work. As compared to the conventional synthetic
methods, the controlled microwave heating had been shown
3.1 Materials and apparatus
Unless otherwise noted, reagents and solvents were of ana-
lytical reagent grade or were chemically pure and used as
received without further purification. H NMR spectrum
1
(CDCl3) was recorded on a Bruker AVANCE-400 MHz
with TMS as an internal standard. Coupling constants (J
values) are in Hertz. The IR spectra were obtained from
KBr discs in the range of 4000–400 cm1 on a Nicolet
5DXFT-IR spectrophotometer. Combustion analyses for
elemental composition were conducted on a Perkin-Elmer
2400 instrument. All microwave experiments were per-
formed using a YL8023B1 microwave reactor possessing a
single-mode microwave cavity producing controlled irradia-
tion at 2.45 GHz.
Scheme 1 Synthesis of the target compounds (6a–6n). Reagents and conditions: a) Boc2O, NaOH/THF/H2O, rt; b) N2 atmosphere, CDI/THF, 60 °C; c)
CF3COOH/CH2Cl2, 5 °C;d) ethanamine; e) 1, 1, 1-trichloro-2-nitroethane/CHCl3, 2–7 °C; f) methanamine, 3–7 °C; g) diglycine ester hydrochloride, HCHO,
Et3N/EtOH, 60–75 °C.

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Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
3.2 Insecticidal activity assay
a negative control. All experiments were carried out in three
replicates for the purpose of statistic requirements, and the
results are shown in Table 1.
The insecticidal activities of compounds 6a–6n were meas-
ured against Nilaparvata lugens and Aphis medicagini ac-
cording to the standard test [25] with a slight modification.
The test analogues were dissolved in DMF and serially di-
luted with water containing Triton X-80 (0.1 mg/L) to ob-
tain the required concentrations. The insects were reared at
25 (±1) °C, and groups of 10 were transferred to glass Petri
dishes and sprayed with the aforementioned solutions using
a Potter sprayer. Assessments were made after 72 h by the
number and size of the live insects relative to those in the
negative control, and evaluations are based on a percentage
scale of 0–100. The mortality rates were subjected to probit
analysis [26]. The reference compounds were nitenpyram,
while water containing Triton X-80 (0.1 mg/L) was used as
3.3 General procedure
3.3.1 General synthetic procedures for diglycine
NaOH (33 mmol, 0.87 g), followed by Boc2O (11 mmol,
2.4 g) was added to a stirred solution of glycine (12 mmol,
0.90 g) in THF/H2O (30 mL of each solvent) at room tem-
perature and the resulting mixture was stirred for 18 h. THF
was removed under vacuum and the aqueous layer was ex-
tracted with CH2Cl2 (20 mL). The aqueous layer was acidi-
fied with hydrochloric acid (1 mol/L) to pH 4, and then ex-
tracted with CH2Cl2 (4 × 15 mL). The organic phase was
combined and dried over Na2SO4 and the solvent was evap-
Table 1 Insecticidal activities of cis-nitenpyram analogues (6a–6n) against Nilaparvata lugens and Aphis medicagini
Mortality (%) at different concentrations (mg/L)
Compd.
R
Nilaparvata lugens
Aphis medicagini
100
100
20
4
100
100
20
4
6a
6b
100
100
98
100
100
88
100
100
100 a)
100
100
90
85
6c
100
100
95
95
90
88
85
75
6d
6e
100
100
100
88
80
72
100
100
95
70
63
50
6f
85
6g
6h
100
100
90
90
75
60
95
90
80
70
55
48
6i
100
80
50
90
65
42
6j
100
90
80
60
45
40
85
80
60
53
35
24
6k
6l
100
100
75
100
84
70
6m
100
100
100
100
70
80
100
100
80
90
65
75
6n
5b
O
100
100
85
70
100 b)
90
70
34
nitenpyram
100
100
100
100
a) LC50 = 0.163 mg/L; b) LC50 = 0.129 mg/L.

Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
163
orated under vacuum. The resulting crude Boc-glycine was
used in the next step without further purification.
N-ethoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridinylmethyl)
ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahydropyrimi-
dinyl)]acetamide (6b)
A 100 mL round bottom flask was charged with
Boc-glycine (10 mmol, 1.75g), carbonyldimidazole (6
mmol, 0.97 g) and freshly distilled THF (20 mL) under the
nitrogen atmosphere and was heated to 60 °C for 1 h. The
mixture was cooled to room temperature and glycine (10
mmol, 0.75 g) was added by syringe. The mixture was
stirred for 3–4 h to get the intermediates solution, then the
intermediates solution was concentrated and subjected to
flash column chromatography on silica gel to afford
Boc-diglycine (ethyl acetate/petroleum ether (v/v)= 3/2 as
eluent).
CF3CO2H (15 mmol) was added dropwise to the solution
of Boc-diglycine (10 mmol, 2.48g) in CH2Cl2 (10 mL) in
ice bath. The mixture was stirred at 5 °C and the progress
of the reaction was tracked by TLC. The digycline that
broke off from the Boc was obtained. Methanol (10 mL)
was added to the reaction mixture and the reaction mixture
was concentrated under reduced pressure. This process was
repeated four times in order to remove the residual
CF3CO2H, and the resulting oil liquid was used in the next
step without further purification.
Yellow oil, yield 78.9%; 1H NMR (CDCl3, 400 MHz)
 8.33 (d, J = 2.3 Hz, 1H, Py-H), 7.71 (dd, J = 8.2, 2.5 Hz,
1H, Py-H), 7.40 (t, J = 5.2 Hz, 1H, NH), 7.35 (d, J = 8.2 Hz,
1H, Py-H), 4.51 (d, J = 15.0 Hz, 1H, Py-CH2), 4.22 (dd, J =
11.6, 4.6 Hz, 2H, CH2COO), 4.12 (dd, J = 9.3, 4.3 Hz,
2H,OCH2), 4.06 (d, J = 5.6 Hz, 1H, Py-CH2), 3.823.71 (m,
4H), 3.30 (d, J = 9.0 Hz, 1H), 3.26 (d, J = 3.6 Hz, 2H,
NCH2CO), 3.10 (s, 3H, NCH3), 3.02–2.94 (m, 1H), 1.31 (t,
J = 7.1 Hz, 3H, OCH2CH3), 1.22 (t, J = 7.1 Hz, 3H,
NCH2CH3). FTIR(KBr, cm1) vmax: 3210 (N–H), 2951, 2872,
1744, 1541 (CO), 1544 (NO2), 1303 (vas (C–O–C)), 1251
(va (C–O–C)). Anal. calcd for C19H27ClN6O5: C, 50.16; H, 5.98;
N, 18.47. Found: C, 50.18; H, 5.96; N, 18.50. ESI-MS (M +
H) m/z: 454.17.
N-propoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridinylmethyl)
ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahydropyrimi-
dinyl)]acetamide (6c)
Yellow oil, yield 75.8%; 1H NMR (CDCl3, 400 MHz)
 8.32 (d, J = 1.5 Hz, 1H, Py-H), 7.70 (dd, J = 8.1, 2.1 Hz,
1H,P y-H), 7.40 (s, 1H, NH), 7.34 (d, J = 8.2 Hz, 1H, Py-H),
4.51 (d, J = 14.9 Hz, 1H, Py-CH2), 4.20 (d, J = 15.0 Hz, 1H,
Py-CH2), 4.12 (s, 2H, NHCH2), 3.81 (t, J = 8.0 Hz, 2H,
COO CH2), 3.77–3.66 (m, 4H), 3.35–3.27 (m, 1H), 3.25 (d,
J = 2.8 Hz, 2H, NCH2CO), 3.07 (s, 3H, NCH3), 2.96 (td, J =
14.1, 7.1 Hz, 1H), 1.70 (dt, J = 14.2, 7.1 Hz, 2H, CH2CH3),
1.21 (t, J = 5.7 Hz, 3H, NCH2CH3), 0.96 (t, J = 7.3 Hz, 3H,
CH2CH2CH3). FTIR (KBr, cm1) vmax: 3211 (N–H), 2951,
2872, 1744, 1540 (C=O), 1546 (NO2), 1302 (vas (C–O–C)),
1250 (va (C–O–C)). Anal. calcd for C20H29ClN6O5: C, 51.23; H,
6.23; N, 17.92. Found: C, 51.18; H, 6.32; N, 17.88. ESI-MS
(M + H) m/z: 469.19.
3.3.2 General synthetic procedures for target compounds
6a–6n
A mixture of nitenpyram (2.71 g, 10.0 mmol), diglycine
ester hydrochloride (10.0 mmol), Et3N (1.7 mL), and for-
maldehyde (1.95 mL, 37%) in ethanol (20 mL) was heated
to 65 °C for 5 min in a microwave reactor and stirred for 20
min at the temperature. The reaction mixture was concen-
trated under reduced pressure and treated with 20 mL of
water. Then, the solution was extracted three times with
ethyl acetate (3 × 30 mL), and the combined extracts were
dried over MgSO4. The organic phase was evaporated under
reduced pressure, and the residue was subjected to flash
column chromatography on silica gel, eluting with ethyl
acetate/petroleum ether (v/v = 3:1) to afford pure products.
N-(1-methyl)ethoxycarbonylmethyl-2-[[4-(6-chloro-3-pyri-
dinylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetra-
hydropyrimidinyl)]acetamide (6d)
Yellow oil, yield 76.7%; 1H NMR (CDCl3, 400 MHz)
 8.33 (d, J = 2.3 Hz, 1H, Py-H), 7.71 (dd, J = 8.2, 2.5 Hz,
1H, Py-H), 7.40 (s, 1H, NH), 7.35 (d, J = 8.2 Hz, 1H, Py-H),
4.51 (d, J = 15.0 Hz, 1H, Py-CH2), 4.27–4.22 (m, 2H,
CH2COO), 4.18 (dd, J = 14.0, 7.0 Hz, 2H, NCH2COO),
4.14–4.09 (m, 1H, OCH(CH3)2), 4.05 (d, J = 9.9 Hz, 1H,
Py-CH2), 3.85–3.66 (m, 4H ), 3.30 (d, J = 7.6 Hz, 1H), 3.26
(d, J = 3.6 Hz, 2H, NCH2CO), 3.10 (s, 3H, NCH3), 2.96 (dt,
J = 14.1, 7.1 Hz, 1H), 1.31 (dd, J = 9.0, 5.3 Hz, 3H,
NCH2CH3), 1.29 (d, J = 6.9 Hz, 2H, CH2CH3), 1.19–1.26
(m, 6H, CH(CH3)2). FTIR (KBr, cm1) vmax: 3217 (N–H),
2952, 2873, 1749, 1541 (CO), 1540(NO2), 1301 (vas (C–O–C)),
1255 (va (C–O–C)). Anal. calcd for C20H29ClN6O5: C, 51.23; H,
6.23; N, 17.92. Found: C, 51.20; H, 6.25; N, 17.86. ESI-MS
(M + H) m/z: 469.19.
N-methoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridinylmethyl)
ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahydropyrimi-
dinyl)]acetamide (6a)
Yellow oil, yield 76.4%; 1H NMR (CDCl3, 400 MHz)
 8.34 (d, J = 2.2 Hz, 1H, Py-H), 7.71 (dd, J = 8.2, 2.5 Hz,
1H, Py-H), 7.39 (d, J = 5.1 Hz, 1H,NH), 7.35 (s, 1H, Py-H),
4.52 (d, J = 14.9 Hz, 1H, Py-CH2), 4.21 (d, J = 15.0 Hz, 1H,
Py-CH2), 4.14 (q, J = 5.1 Hz, 2H, NCH2CO), 3.85–3.72 (m,
7H), 3.35–3.28 (m, 1H, NCH2), 3.26 (d, J = 3.2 Hz, 2H,
NCH2CONH), 3.10 (s, 3H, NCH3), 3.03–2.93 (m, 1H, NCH2),
1.23 (q, J = 7.2 Hz, 3H, NCH2CH3). FTIR (KBr, cm1)
vmax: 3200 (NH), 2951, 2872, 1744, 1540 (CO), 1546
(NO2), 1303 (vas (C–O–C)),1251 (va (C–O–C)). Anal. calcd. for
C18H25ClN6O5: C, 49.04; H, 5.72; N, 19.06. Found: C, 49.11;
H, 5.68; N, 19.10. ESI-MS (M + H) m/z: 441.16.

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Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
N-butoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridinylmethyl)
ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahydropyrimi-
dinyl)]acetamide (6e)
N-pentoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridinylmethyl)
ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahydropyrimi-
dinyl)]acetamide (6h)
Yellow oil, yield 75.7%; 1H NMR (CDCl3, 400 MHz)
 8.33 (d, J = 2.4 Hz, 1H, Py-H), 7.71 (dd, J = 8.2, 2.5 Hz,
1H, Py-H), 7.39 (t, J = 5.2 Hz, 1H, NH), 7.35 (d, J = 8.2 Hz,
1H, Py-H), 4.51 (d, J = 15.0 Hz, 1H, Py-CH2), 4.22 (s, 1H,
Py-CH2), 4.17 (t, J = 6.8 Hz, 2H, NHCH2), 3.81 (t, J = 8.3
Hz, 2H, COOCH2), 3.74 (dt, J = 14.0, 6.3 Hz, 4H), 3.34–
3.27 (m, 1H), 3.26 (d, J = 3.5 Hz, 2H, NCH2CO), 3.10 (s,
3H, NCH3), 2.97 (dd, J = 14.1, 7.1 Hz, 1H), 1.67 (dt, J =
13.8, 6.9 Hz, 2H, COOCH2CH2), 1.38–1.32 (m, 4H,
CH2CH2CH3), 1.23–1.20 (m, 3H, NCH2CH3), 0.95–0.89 (m,
3H, CH2CH3). FTIR (KBr, cm1) vmax: 3210 (N–H), 2952,
2870, 1750, 1541 (CO), 1548 (NO2), 1312 (vas (C–O–C)),
1260 (va (C–O–C)). Anal. calcd for C22H33ClN6O5: C, 53.17; H,
6.69; N, 16.91. Found: C, 53.20; H, 6.65; N, 16.97. ESI-MS
(M + H) m/z: 497.22.
Yellow oil, yield 78.8%; 1H NMR (CDCl3, 400 MHz)
 8.30 (d, J = 1.4 Hz, 1H, Py-H), 7.72 (dd, J = 8.0, 2.0 Hz,
1H,P y-H), 7.41 (s, 1H, NH), 7.34 (d, J = 8.0 Hz, 1H, Py-H),
4.50 (d, J = 14.9 Hz, 1H, Py-CH2), 4.21 (d, J = 14.6 Hz, 1H,
Py-CH2), 4.12 (s, 2H, NHCH2), 3.81 (t, J = 8.0 Hz, 2H,
COOCH2), 3.78–3.65 (m, 4H), 3.32–3.25(m, 1H), 3.24 (d,
J = 2.8 Hz, 2H, NCH2CO), 3.07 (s, 3H, NCH3), 2.96 (td, J =
14.1, 7.1 Hz, 1H), 1.70–1.33 (m, 4H, CH2CH2), 0.96 (t, J =
7.3 Hz, 3H, CH2CH2CH3). FTIR (KBr, cm1) vmax: 3210
(N–H), 2952, 2870, 1751, 1542 (C=O), 1546 (NO2), 1312
(vas (C–O–C)), 1252 (va (C–O–C)). Anal. calcd for C21H31ClN6O5:
C, 52.22; H, 6.47; N, 17.40. Found: C, 52.25; H, 6.49; N,
17.48. ESI-MS (M + H) m/z: 483.20.
N-(1-methyl)propoxycarbonylmethyl-2-[[4-(6-chloro-3-pyri-
dinylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetra-
hydropyrimidinyl)]acetamide (6f)
N-(2-methyl)butoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridi-
nylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahy-
dropyrimidinyl)]acetamide (6i)
Yellow oil, yield 75.3%; 1H NMR (CDCl3, 400 MHz)
 8.33 (s, 1H, Py-H), 7.71 (d, J = 8.1 Hz, 1H, Py-H), 7.40 (t,
sJ = 14.9 Hz, 1H, Py-CH2), 4.20 (d, J = 15.1 Hz, 1H,
Py-CH2), 4.13 (t, J = 5.0 Hz, 2H, NHCH2COO), 3.95 (d, J =
6.7 Hz, 2H, OCH2), 3.80 (d, J = 4.5 Hz, 2H), 3.75 (d, J =
7.5 Hz, 2H), 3.34–3.28 (m, 1H), 3.25 (d, J = 2.8 Hz, 2H,
NCH2), 3.09 (s, 3H, NCH3), 2.97 (dq, J = 14.6, 7.4 Hz, 1H),
2.02–1.92 (m, 1H, CH(CH3)2), 1.21 (t, J = 7.1 Hz, 3H,
NCH2CH3), 0.95 (d, J = 6.6 Hz, 6H, CH(CH3)2). FTIR (KBr,
cm1) vmax: 3213 (N–H), 2950, 2874, 1750, 1540 (CO),
1542 (NO2), 1310 (vas (C–O–C)), 1256 (va (C–O–C)). Anal. calcd
for C21H31ClN6O5: C, 52.22; H, 6.47; N, 17.40. Found: C,
52.20; H, 6.45; N, 17.45. ESI-MS (M + H) m/z: 483.20.
Yellow oil, yield 72.8%; 1H NMR (CDCl3, 400 MHz)
 8.33 (d, J = 2.0 Hz, 1H, Py-H), 7.73 (dd, J = 8.1, 2.4 Hz,
1H, Py-H), 7.40 (s, 1H, NH), 7.33 (d, J = 8.0 Hz, 1H, Py-H),
4.53 (d, J = 15.3 Hz, 1H, Py-CH2), 4.27–4.22 (m, 2H,
CH2COO), 4.20 (dd, J = 14.0, 7.0 Hz, 2H, NCH2COO),
4.13–4.09 (m, 1H,OCH), 4.05 (d, J = 9.9 Hz, 1H, Py-CH2),
3.85–3.66 (m, 4H), 3.30 (d, J = 7.6 Hz, 1H), 3.26 (d, J = 3.6
Hz, 2H, NCH2CO), 3.10 (s, 3H, NCH3), 2.96 (dt, J = 14.1,
7.1 Hz, 1H), 1.31 (dd, J = 9.0, 5.3 Hz, 3H, NCH2CH3), 1.29
(m, 2H, CHCH2), 1.10 (m, 2H, CH2CH3), 1.19 (d, J = 7.0
Hz, 3H, CHCH3). 0.96 (t, J = 6.9 Hz, CH3). FTIR (KBr,
cm1) vmax: 3215 (N–H), 2950, 2871, 1749, 1541 (C=O),
1540 (NO2), 1305 (vas (C–O–C)), 1251 (va (C–O–C)). Anal. calcd
for C22H33ClN6O5: C, 53.17; H, 6.69; N, 16.91. Found: C,
53.20; H, 6.89; N, 16.93. ESI-MS (M + H) m/z: 497.22.
N-(2-methyl)propoxycarbonylmethyl-2-[[4-(6-chloro-3-pyri-
dinylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetra-
hydropyrimidinyl)]acetamide (6g)
N-(3-methyl)butoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridi-
nylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahy-
dropyrimidinyl)]acetamide (6j)
Yellow oil, yield 76.3%; 1H NMR (CDCl3, 400 MHz)
 8.30 (d, J = 2.1 Hz, 1H, Py-H), 7.74 (dd, J = 8.0, 2.5 Hz,
1H, Py-H), 7.42 (s, 1H, NH), 7.34 (d, J = 8.0 Hz, 1H, Py-H),
4.52 (d, J = 15.3 Hz, 1H, Py-CH2), 4.28–4.23 (m, 2H,
CH2COO), 4.19 (dd, J = 14.2, 7.1 Hz, 2H, NCH2COO),
4.13–4.09(m, 1H, OCH), 4.05 (d, J = 9.9 Hz, 1H, Py-CH2),
3.85–3.66 (m, 4H ), 3.30 (d, J = 7.6 Hz, 1H), 3.26 (d, J =
3.6 Hz, 2H, NCH2CO), 3.10 (s, 3H, NCH3), 2.96 (dt, J =
14.1, 7.1 Hz, 1H), 1.31 (dd, J = 9.0, 5.3 Hz, 3H, NCH2CH3),
1.29 (d, J = 6.9 Hz, 2H, CH2CH3), 1.19 (d, J = 7.0 Hz, 3H,
Yellow oil, yield 76.4%; 1H NMR (CDCl3, 400 MHz)
 8.30 (s, 1H, Py-H), 7.70 (d, J = 8.2 Hz, 1H, Py-H), 7.42 (t,
J = 5.2 Hz, 1H, NH), 7.34 (d, J = 8.0 Hz, 1H, Py-H), 4.52 (d,
J = 14.8 Hz, 1H, Py-CH2), 4.22 (d, J = 15.2 Hz, 1H,
Py-CH2), 4.12 (t, J = 5.1 Hz, 2H, NHCH2COO), 3.92 (d, J =
6.6 Hz, 2H, OCH2), 3.80 (d, J = 4.5 Hz, 2H), 3.75 (d, J =
7.5 Hz, 2H), 3.34–3.28 (m, 1H), 3.25 (d, J = 2.8 Hz, 2H,
NCH2), 3.09 (s, 3H, NCH3), 2.97 (dq, J = 14.6, 7.4 Hz, 1H),
2.01–1.83 (m, 1H, CH(CH3)2), 1.53–1.22 (m, 2H, CH2CH),
1.21 (t, J = 7.1 Hz, 3H, NCH2CH3), 1.01 (d, J = 6.6 Hz, 6H,
CH(CH3)2). FTIR(KBr, cm1) vmax: 3213 (N–H), 2950, 2874,
1750, 1540 (CO), 1542 (NO2), 1310 (vas (C–O–C)), 1256
(va (C–O–C)). Anal. calcd for C22H33ClN6O5: C, 53.17; H, 6.69;
CHCH3). 0.96 (t, J = 6.9 Hz, CH3). FTIR (KBr, cm1) vmax
:
3218 (N–H), 2952, 2873, 1749, 1541 (CO), 1542 (NO2), 1305
(vas(C–O–C)), 1250 (va(C–O–C)). Anal. calcd for C21H31ClN6O5: C,
52.22; H, 6.47; N, 17.40. Found: C, 52.20; H, 6.50; N, 17.43.
ESI-MS (M + H) m/z: 483.20.

Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
165
N, 16.91. Found: C, 53.15; H, 6.71; N, 16.94. ESI-MS (M +
H) m/z: 497.22.
N-(2-tetrahydrofuryl)methoxycarbonylmethyl-2-[[4-(6-chloro-
3-pyridinylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-
tetrahydropyrimidinyl)]acetamide (6n)
Yellow oil, yield 72.3%; 1H NMR (CDCl3, 400 MHz)
 8.35 (s, 1H, Py-H), 7.71 (d, J = 8.2 Hz, 1H, Py-H), 7.39 (d,
J = 6.1 Hz, 1H, NH), 7.33 (s, 1H, Py-H), 7.30–6.19 (m, 3H,
tetrahydrofury-H), 5.20 (s, 2H, COOCH2), 4.50 (d, J = 15.0
Hz, 1H, Py-CH2), 4.22 (s, 1H, Py-H), 4.20–4.14 (m, 2H,
NHCH2), 3.83–3.69 (m, 4H), 3.29 (d, J = 6.7 Hz, 1H), 3.25
(d, J = 2.7 Hz, 2H, NCH2), 3.06 (s, 3H, NCH3), 2.97 (dq, J =
13.9, 7.0 Hz, 1H), 1.21 (t, J = 7.1 Hz, 3H, NCH2CH3). FTIR
(KBr, cm1) vmax: 3211 (N–H), 2950, 2870, 1755, 1542
(C=O), 1546 (NO2), 1312 (vas (C–O–C)), 1260 (va (C–O–C)). Anal.
calcd for C20H26ClN6O6: C, 49.85; H, 5.44; N, 17.44. Found:
C, 49.87; H, 5.42; N, 17.46. ESI-MS (M + H) m/z: 511.20.
N-hexoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridinylmethyl)
ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahydropyrimi-
dinyl)]acetamide (6k)
1
Yellow oil, yield 75.3%; H NMR (400 MHz, DMSO-d6)
 8.33 (d, J = 2.1 Hz, 1H, Py-H), 7.71 (dd, J = 8.2, 2.4 Hz,
1H, NH), 7.38 (t, J = 5.4 Hz, 1H, Py-H), 7.35 (d, J = 8.2 Hz,
1H, Py-H), 4.51 (d, J = 14.9 Hz, 1H, Py-CH2), 4.22 (s, 1H,
Py-CH2), 4.18 (d, J = 6.8 Hz, 2H, NHCH2), 4.14–4.09 (m,
2H, COOCH2), 3.78 (dd, J = 16.0, 6.1 Hz, 4H), 3.33–3.27
(m, 1H), 3.25 (d, J = 3.1 Hz, 2H, NCH2CO), 3.10 (s, 3H,
NCH3), 1.40–1.28 (m, 8H, (CH2)4CH3), 1.22 (t, J = 7.1 Hz,
3H, NCH2CH3), 0.90 (t, J = 6.7 Hz,3H, (CH2)4CH3). FTIR
(KBr, cm1) vmax: 3212 (N–H), 2953, 2872, 1753, 1544
(CO), 1542 (NO2), 1310 (vas (C–O–C)), 1262 (va (C–O–C)). Anal.
calcd for C21H30ClN6O5: C, 52.33; H, 6.27; N, 17.44. Found:
C, 52.30; H, 6.25; N, 17.46. ESI-MS (M + H) m/z: 511.24.
4 Results and discussion
4.1 Evaluation of insecticidal activities
By analyzing the data of Table 1, we found that all the de-
signed analogues have better insecticidal performance than
5b which had the best insecticidal activities in the previous
work. Most of our designed analogues exhibited excellent
insecticide activities against Nilaparvata lugen and Aphis
medicagini at 100 mg/L (Table 1), while the target com-
pounds had higher insecticidal activities against Nilaparvata
lugen than Aphis medicagini at a lower dose. Among these
analogues, 6a and 6b afforded the best in vitro inhibitory
activities and had 98% and 100% mortality at 4 mg/L, re-
spectively. The LC50 value of 6b was 0.163 mg/L, which
was more comparable to nitenpyram (LC50 = 0.129 mg/L)
than 5b.
The insecticidal activities showed significant differences
with the change in the size of the ester groups R. When R
was smaller, the insecticidal potency was improved. As for
the different R groups, their insecticidal activities increased
in the order: ethyl (6b) > methyl (6a) > propyl (6c) > butyl
(6e) > pentyl (6h) > hexyl (6k). These observations suggest
that the size of the ester group is one of the important fac-
tors that influence the potency of the nitenpyram analogues.
As for the species of the R groups, the insecticidal activities
of the target compounds were higher when R was a linear
aliphatic alkyl group than those when R was a branched
aliphatic alkyl group. As an example, the insecticidal activi-
ties of the target compounds increased in the order: n-propyl
(6c) > isopropyl (6d), n-butyl (6e) > sec-butyl (6g) >
iso-butyl (6f), n-pentyl (6h) > sec-pentyl (6i) > iso-pentyl
(6j).
N-phenmethoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridin-
ylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahy-
dropyrimidinyl)]acetamide (6l)
1
Yellow oil, yield 71.7%; H NMR (400 MHz, DMSO)
 8.32 (s, 1H, Py-H), 7.70 (d, J = 8.2 Hz, 1H, Py-H), 7.41 (d,
J = 6.0 Hz, 1H, NH), 7.40–7.34 (m, 5H, Ph-H), 7.33 (s, 1H,
Py-H), 5.20 (s, 2H, COOCH2), 4.50 (d, J = 15.0 Hz, 1H,
Py-CH2), 4.22 (s, 1H, Py-H), 4.20–4.14 (m, 2H, NHCH2),
3.83–3.69 (m, 4H), 3.29 (d, J = 6.7 Hz, 1H), 3.25 (d, J = 2.7
Hz, 2H, NCH2), 3.06 (s, 3H, NCH3), 2.97 (dq, J = 13.9, 7.0
Hz, 1H), 1.21(t, J = 7.1 Hz, 3H, NCH2CH3). FTIR (KBr,
cm1) vmax 3213 (N–H), 2954, 2871, 1750, 1541 (C=O),
1549 (NO2), 1680, 1580, 1500 (C=C), 1312 (vas (C–O–C)),
1260 (va (C–O–C)). Anal. calcd for C22H24ClN6O5: C, 54.16; H,
4.96; N, 17.22. Found: C, 54.20; H, 4.93; N, 17.25. ESI-MS
(M + H) m/z: 517.19.
N-phenethoxycarbonylmethyl-2-[[4-(6-chloro-3-pyridin-
ylmethyl)ethylamino]-3-methyl-5-nitro-1-(1,2,3,6-tetrahydro-
pyrimidinyl)]acetamide (6m)
1
Yellow oil, yield 74.9%; H NMR (400 MHz, DMSO)
 8.30 (s, 1H, Py-H), 7.78 (d, J = 8.2 Hz, 1H, Py-H), 7.40 (d,
J = 6.1 Hz, 1H, NH), 7.42–7.32 (m, 5H, Ph-H), 7.34 (s, 1H,
Py-H), 5.21 (s, 2H, COOCH2), 4.53 (d, J = 15.0 Hz, 1H,
Py-CH2), 4.23 (s, 1H, Py-H), 4.20–4.14 (m, 2H, NHCH2),
3.83–3.69 (m, 4H), 3.30 (d, J = 6.6 Hz, 1H), 3.24 (d, J = 2.5
Hz, 2H, NCH2), 3.03 (s, 3H, NCH3), 2.97 (dq, J = 13.9, 7.0
Hz, 1H), 2.83 (t, J = 2.3 Hz, 2H, Ph-CH2), 1.21 (t, J = 7.1
Hz, 3H, NCH2CH3). FTIR (KBr, cm1) vmax: 3214 (N–H),
2953, 2870, 1754, 1540 (CO), 1545 (NO2), 1684, 1582,
1508 (C=C), 1313 (vas (C–O–C)), 1261 (va (C–O–C)). Anal. calcd
for C23H26ClN6O5: C, 55.04; H, 5.22; N, 16.74. Found: C,
55.06; H, 5.00; N, 16.75. ESI-MS (M + H) m/z: 531.20.
Furthermore, the nitenpyram analogues containing aro-
matic esters (6l, 6m) had lower activities than the saturated
aliphatic esters (6b, 6c), and the insecticidal activities of
tetrahydrofuryl ester (6n) which contains an oxygen atom
was relatively high. These results insecticidal activities fur-

166
Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
ther suggested that small structure differences could lead to
large differences in the overall activities, which implies
further possibilities of lead compound development.
ing site of analogue 6b was different from that of the ana-
logues 3, 4, 5 with a more rational way.
As illustrated in Figure 3(b), analogue 6b exhibits two
hydrogen bonds via its nitro O (30) and O (31) with the
backbone H and side chain H of Tyr192, respectively, and
the N (17) of its tetrahydropyrimidine interacts with the side
chain H of Gln55. Besides, its binding conformation exhib-
ited three important hydrogen bonds between the O (22) of
the diglycine ester and H–O of Tyr164, H–O of Gln155,
respectively. In addition, another important hydrogen bond
between HN (23) of its peptide bond and H–S of Met114
was displayed. Other interactions in this area may be medi-
ated via water(s) as these residues are near the protein sur-
face. These observations have also explained why the ana-
logue 6b attained the highest score.
Furthermore, most of the other active analogues (6a, 6c,
and 6n) shared a quite similar binding mode with 6b, and
many of them exhibited more than four hydrogen bonds
with different amino acids of the active pocket between the
nAChRs subunits, which is consistent with their high insec-
ticidal activities. However, consistent with the SARs ob-
served in vitro, the binding interactions of analogues with
low inhibitory potency were not satisfactory, so did their
docking scores. No significant formation of hydrogen bonds
and hydrophilic or hydrophobic contact could be found in
their best binding conformations, such as the predicted
binding mode of compound 6k (Figure 3(c)). Our docking
results coincided well with the experimental activities,
which indicated that different insecticidal potency depended
on the number of significant hydrogen bonds formed by the
nitenpyram analogue binding with the amino acid residues.
Thereby, the binding model proposed here may provide an
alternative way close to the actual binding features of these
4.2 Molecular docking study
To further explore the structural features of the target com-
pounds for better activities, models of these new compounds–
receptor complexes were investigated by docking studies
with CDOCK [27]. Since the amino acids formed at the
active sites are both structurally and functionally consistent
in the diverse nAChRs and AchBPs, the published crystal
structure of a Lymnaea stagnalis-AChBP (Ls-AChBP)
co-crystallized with imidacloprid (PDB ID: 2zju) [28] was
used as the template of receptor. The docking study was
carried out through the graphical user interface
CDOCKTOOLS. The only modification was the number of
docking runs that was set to 200 (previously 100) for high-
er accuracy.
The scoring function of the docking program ranked the
compounds in the same general order observed experimen-
tally (data not shown), and all the active analogues exhibit-
ed significant hydrogen bonding interactions with the
nAChRs target. As expected, the most potent compound 6b
is nicely accommodated within the subunit interfacial bind-
ing pocket between the two faces of adjacent subunits (Fig-
ure 3(a)), with its backbone and chains nicely nestled. The
binding conformation of 6b in this docking simulation re-
vealed an intriguing molecular binding mode at the active
site of nAChRs, with a very low docked energy. As com-
pared with our previous work, the binding conformation of
analogue 6b in this docking simulation showed the similar
binding mode. However, unexpectedly, the molecular bind-
Figure 3 Binding site interactions of analogue 6b with the extracellular domain of nAChRs (protein data bank code: 2zju). (a) Compound 6b is bound into
the subunit interfacial binding pocket between two faces of adjacent subunits. For clarity, only two of five subunits are extracted and shown from the pen-
tameric nAChRs structure, and the corresponding interfacial binding pocket of interest is displayed; (b) zoomed-in view of the interactions between com-
pound 6b and amino acids from the active site of the receptor; (c) the predicted binding mode of compound 6k with relatively low activity. Key H-bonds are
indicated by green dotted lines.

Chen YX, et al. Sci China Chem February (2013) Vol.56 No.2
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sion of Shanghai (105405503400), the Innovation Project of Shanghai
Education Commission (12YZ078), the Leading Academic Discipline Pro-
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