3418 J. Agric. Food Chem., Vol. 58, No. 6, 2010
Sun et al.
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]benzamide (3a). Yield, 72.5%; mp
235-237 °C. 1H NMR (DMSO-d6), δ 9.97 (s, 1H, NNHCO), 8.36 (d,
J = 2.4 Hz, 1H, Py-H), 7.78 (dd, J1 = 3.2 Hz, J2 = 8.0 Hz, 1H, Py-H),
7.47-7.57 (m, 5H, Ph-H), 7.36 (d, J = 8.0 Hz, 1H, Py-H), 4.54 (d, J =
16.0, 1H, Py-CH2), 4.21 (d, J = 16.0 Hz, 1H, Py-CH2), 3.79-3.83 (m, 4H),
3.10-3.17 (m, 1H), 2.95 (s, 3H, NCH3), 2.84-2.92 (m, 1H), 1.13 (t, J =
7.2 Hz, 3H, NCH2CH3). IR (KBr, cm-1): 3252, 3077, 1682, 1541, 1311,
752. Anal. calcd for C20H23ClN6O3: C, 55.75; H, 5.38; N, 19.50. Found: C,
55.67; H, 5.31; N, 19.58.
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]-2-bromo-5-methoxybenzamide (3h). Yield,
72.6%; mp 206-208 °C. 1H NMR (DMSO-d6): δ 9.94 (s, 1H, NNHCO),
8.32 (d, J = 2.4 Hz, 1H, Py-H), 7.75 (dd, J1 = 3.0 Hz, J2 = 8.0 Hz, 1H,
Py-H), 7.50-7.74 (m, 3H, Ph-H), 7.32 (d, J = 8.2 Hz, 1H, Py-H), 4.51
(d, J = 16.0, 1H, Py-CH2), 4.21 (d, J = 16.0 Hz, 1H, Py-CH2), 3.83-3.87
(m, 4H), 3.80 (s, 3H, OCH3), 3.10-3.16 (m, 1H), 2.95 (s, 3H, NCH3),
2.80-2.92 (m, 1H), 1.12 (t, J = 7.2 Hz, 3H, NCH2CH3). IR (KBr, cm-1):
3254, 3082, 1685, 1543, 1309, 1260, 754. Anal. calcd for C21H24BrClN6O4:
C, 46.72; H, 4.48; N, 15.57. Found: C, 46.78; H, 4.43; N, 15.52.
X-ray Crystallography. The yellow crystal of the title compound 2b
(grown from a mixed solution of petroleum ether/ethyl acetate) was
mounted on a glass fiber in a random orientation, with approximate
dimensions of 0.20 mm ꢀ 0.13 mm ꢀ 0.10 mm. The data were collected
by a Bruker Smart Apex CCD diffractometer with a graphite-monochro-
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]-4-chlorobenzamide (3b). Yield, 70.2%;
mp 221-222 °C. 1H NMR (DMSO-d6): δ 9.95 (s, 1H, NNHCO), 8.34
(d, J = 2.0 Hz, 1H, Py-H), 7.76 (dd, J1 = 3.4 Hz, J2 = 8.4 Hz, 1H, Py-H),
7.55-7.82 (m, J = 8.0 Hz, J = 8.0 Hz, 4H, Ph-H), 7.34 (d, J = 8.0 Hz, 1H,
Py-H), 4.53 (d, J = 16.0, 1H, Py-CH2), 4.23 (d, J = 16.0 Hz, 1H, Py-CH2),
3.80-3.85 (m, 4H), 3.12-3.18 (m, 1H), 2.97 (s, 3H, NCH3), 2.81-2.91 (m,
1H), 1.10 (t, J = 7.2 Hz, 3H, NCH2CH3). IR (KBr, cm-1): 3254, 3078,
1680, 1540, 1310, 810. Anal. calcd for C20H22Cl2N6O3: C, 51.62; H, 4.77;
N, 18.06. Found: C, 51.71; H, 4.78; N, 18.14.
˚
mated Mo Ka radiation (k = 0.71073 A), using an j-ω scan mode in the
range of 2.47 e θ e 26.49° at 298(2) K. Empirical absorption correction was
applied, and a total of 13121 reflections including 4511 unique ones (Rint
=
0.1048) were measured. The structure was solved by direct methods and
refined by full-matrix least-squares techniques on F2 using the SHELXTL
program package (16). All of the nonhydrogen atoms were refined
anisotropically, and hydrogen atoms were located at their idealized posi-
tions. The final R = 0.0746, wR = 0.1586 {w = 1/[σ2(Fo2) þ (0.0749P)2 þ
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]-2-chlorobenzamide (3c). Yield, 68.9%;
mp 208-210 °C. 1H NMR (DMSO-d6): δ 9.94 (s, 1H, NNHCO), 8.32
(d, J = 2.4 Hz, 1H, Py-H), 7.75 (dd, J1 = 3.0 Hz, J2 = 8.0 Hz, 1H, Py-H),
7.50-7.80 (m, 4H, Ph-H), 7.32 (d, J = 8.2 Hz, 1H, Py-H), 4.51 (d, J =
16.0, 1H, Py-CH2), 4.21 (d, J = 16.0 Hz, 1H, Py-CH2), 3.82-3.87 (m, 4H),
3.10-3.16 (m, 1H), 2.95 (s, 3H, NCH3), 2.80-2.92 (m, 1H), 1.12 (t, J =
7.2 Hz, 3H, NCH2CH3). IR (KBr, cm-1): 3253, 3080, 1679, 1541, 1309,
749. Anal. calcd for C20H22Cl2N6O3: C, 51.62; H, 4.77; N, 18.06. Found:
C, 51.69; H, 4.71; N, 18.11.
2
0.0000P], where P = (Fo þ 2Fc2)/3, S = 1.097, (Δ/σ)max = 0.004, (ΔF)max
3
˚
= 0.385, and (ΔF)min = -0.227 e/A }. The structural plots were drawn with
SHELXTL-97 software package. Other details of the structure have been
deposited with the Cambridge Crystallographic Data Centre, CCDC740000.
Biology Assay. The bioassay was measured according to the standard
test (17) with a slight modification, and all analogues were tested against
N. legen to evaluate their insecticidal activities. The compounds were
dissolved in dimethyl formamide (DMF) and serially diluted with water
containing Triton X-80 (0.1 mg/L) to get the required test concentrations.
All experiments were carried out in three replicates according to statistical
requirements. The insects were reared at 25 ((1) °C, 60 ((2)% relative
humidity, and 12 h light photoperiod. Groups of 12 were transferred to
glass Petri dishes and sprayed with the aforementioned solutions using a
Potter sprayer. After they were air dried, they were kept in a special room
for normal cultivation. Assessments were made after 72 h by the number of
killed and size of live insects relative to that in the negative control, and
evaluations were based on a percentage scale of 0-100, in which 100 was
total kill and 0 was no activity. The mortality rates were subjected to probit
analysis (18). All results are shown in Table 2. The reference compound
was nitenpyram, and water containing DMF (0.5 mg/L) and Triton X-80
(0.1 mg/L) was used as a negative control.
Experimental Protocol of Docking Study. The high nAChR
inhibitory activity of compound 2d was chosen to understand the
ligand-protein interactions in detail, and AutoDock 4.0 (19) was used
to carry out the molecular modeling study. Because the amino acids
forming the active pockets are both structurally and functionally consis-
tent in the diverse nAChRs and AchBPs, the crystal structure of the
Lymnaea stagnalis AchBP (Ls-AChBP) complexed with imidacloprid
(protein data bank code: 2zju) (20) was used as the template to construct
the models. The receptor was prepared for docking by the addition of
hydrogen atoms and the removal of cocrystallized molecules. The putative
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]-4-methylbenzamide (3d). Yield, 72.8%;
mp 219-220 °C. 1H NMR (DMSO-d6): δ 9.93 (s, 1H, NNHCO), 8.31 (d,
J = 2.0 Hz, 1H, Py-H), 7.76 (dd, J1 = 3.2 Hz, J2 = 8.2 Hz, 1H, Py-H),
7.42-7.64 (m, J = 8.2 Hz, J = 8.2 Hz, 4H, Ph-H), 7.34 (d, J = 8.0 Hz, 1H,
Py-H), 4.53 (d, J = 16.2, 1H, Py-CH2), 4.23 (d, J = 16.2 Hz, 1H, Py-CH2),
3.82-3.85 (m, 4H), 3.13-3.17 (m, 1H), 2.97 (s, 3H, NCH3), 2.81-2.94 (m,
1H), 2.36 (s, 3H, Ph-CH3), 1.15 (t, J = 7.2 Hz, 3H, NCH2CH3). IR (KBr,
cm-1): 3251, 3080, 1682, 1541, 1308, 805. Anal. calcd for C21H25ClN6O3:
C, 56.69; H, 5.66; N, 18.89. Found: C, 56.75; H, 5.73; N, 18.95.
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]-6-chloro-2-fluorobenzamide (3e). Yield,
70.5%; mp 213-215 °C. 1H NMR (DMSO-d6): δ 9.92 (s, 1H, NNHCO),
8.35 (d, J = 2.0 Hz, 1H, Py-H), 7.78 (dd, J1 = 3.2 Hz, J2 = 8.4 Hz, 1H,
Py-H), 7.60-7.76 (m, 3H, Ph-H), 7.33 (d, J = 8.0 Hz, 1H, Py-H), 4.53
(d, J = 16.4, 1H, Py-CH2), 4.23 (d, J = 16.4 Hz, 1H, Py-CH2), 3.85-3.89
(m, 4H), 3.12-3.18 (m, 1H), 2.92 (s, 3H, NCH3), 2.83-2.94 (m, 1H), 1.14
(t, J = 7.0 Hz, 3H, NCH2CH3). IR (KBr, cm-1): 3256, 3079, 1683, 1542,
1310, 808. Anal. calcd for C20H21Cl2FN6O3: C, 49.70; H, 4.38; N, 17.39.
Found: C, 49.78; H, 4.30; N, 17.32.
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]-3,5-dichlorobenzamide (3f). Yield, 71.3%;
mp 209-211 °C. 1H NMR (DMSO-d6): δ 9.93 (s, 1H, NNHCO), 8.33 (d,
J = 2.0 Hz, 1H, Py-H), 7.76 (dd, J1 = 3.0 Hz, J2 = 8.2 Hz, 1H, Py-H),
7.58-7.73 (m, 3H, Ph-H), 7.34 (d, J = 8.0 Hz, 1H, Py-H), 4.52 (d, J =
16.2, 1H, Py-CH2), 4.22 (d, J = 16.2 Hz, 1H, Py-CH2), 3.81-3.87 (m, 4H),
3.11-3.17 (m, 1H), 2.93 (s, 3H, NCH3), 2.81-2.92 (m, 1H), 1.16 (t, J =
7.0 Hz, 3H, NCH2CH3). IR (KBr, cm-1): 3257, 3083, 1684, 2544, 1308,
823, 703. Anal. calcd for C20H21Cl3N6O3: C, 48.06; H, 4.24; N, 16.82.
Found: C, 48.12; H, 4.32; N, 16.88.
˚
active binding site was characterized by selecting all residues within a 12 A
radius of the original binding substrate in the X-ray structure. Each ligand
€
was iteratively minimized and assigned the Gasteiger-Huckel charges.
The studied compounds were flexibly docked automatically in the
active site of nAChR. The AMBER force field was used to calculate a
three-dimensional grid of interaction energies for the target nAChR by
AutoGrid (Component of the AutoDock 4.0 program), and these grids
were precomputed to store the electrostatic and van der Waals values.
Default values were used for all docking parameters with 20 independent
docking runs for each ligand. Intermolecular energy, torsional free energy,
and intermolecular hydrogen bonds were included to evaluate their
binding free energy. Cluster analysis was performed on the docked results
N-[(4Z)-4-[[(6-Chloro-3-pyridinyl)methyl]ethylamino]-3-methyl-5-nitro-
1,2,3,6-tetrahydropyrimidin-1-yl]-4-trifluoromethylbenzamide (3g). Yield,
65.4%; mp 206-208 °C. 1H NMR (DMSO-d6): δ 9.92 (s, 1H, NNHCO),
8.32 (d, J = 2.4 Hz, 1H, Py-H), 7.74 (dd, J1 = 3.2 Hz, J2 = 8.2 Hz, 1H, Py-
H), 7.68-7.75 (m, J = 8.0 Hz, J = 8.0 Hz, 4H, Ph-H), 7.32 (d, J = 8.2 Hz,
1H, Py-H), 4.51 (d, J = 16.0, 1H, Py-CH2), 4.21 (d, J = 16.0 Hz, 1H, Py-
CH2), 3.80-3.86 (m, 4H), 3.14-3.16 (m, 1H), 2.93 (s, 3H, NCH3),
2.80-2.95 (m, 1H), 1.13 (t, J = 7.0 Hz, 3H, NCH2CH3). IR (KBr, cm-1):
3258, 3081, 1682, 1543, 1310, 810. Anal. calcd for C21H22ClF3N6O3: C,
50.56; H, 4.44; N, 16.85. Found: C, 50.51; H, 4.49; N, 16.78.
˚
using a root-mean-square deviation (rmsd) tolerance of 0.75 A. For each
cluster, the conformation with the lowest binding energy in the binding site
was chosenfor further analysis and comparison. Accelrys DS visualizer 2.5
[Accelrys Inc., San Diego, CA (2009)] was used for molecular modeling to
determine their binding orientations and interactions.