4906
G. P. Lahm et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4898–4906
off on 4-week-old soybean plants. Once the plants had
4.6 g of a white solid. Yield 81 %; mp 195–196 ꢁC; IR
(Nujol) mmax 3347, 1690, 1680, 1582, 1547, 1463, 1418,
dried, a leaf (or trifoliate) was excised from the treated
plant. The leaves were cut into 24 pieces and placed singly
into a 5.5 cm by 3.5 cm cell of a 16-well plastic tray
(Mullinix Packages, Inc.). Each cell also contained a 2.5
square of moistened chromatography paper (Whatman
No. 3MM) to prevent desiccation. One third instar larvae
of Spodoptera frugiperda was placed into each cell. A total
of 16 insects were tested per rate. Larval mortality was
assessed at 96 h post-infestation. Percent control was
evaluated [(#dead/total number of insects) · 100] and
LC50s were calculated.
1
1376, 1306 cmÀ1; H NMR (400 MHz CDCl3) d 1.17 (d,
6H, J = 6.53 Hz), 2.13 (s, 3H), 4.15 (m, 1H), 5.98 (d, 1H,
J = 7.90 Hz), 7.13 (s, 1H), 7.16 (s, 1H), 7.30 (dd, 1H,
J = 2.25, 12.69 Hz), 7.55 (s, 1H), 7.88 (dd, 1H, 1.56, 8.10
Hz), 8.49 (dd, 1H, J = 1.46, 4.68 Hz), 10.34 (s, 1H); 13C
NMR (100 MHz, DMSO) d 18.28, 22.69, 41.58, 106.97,
121.46 (q, JC–F 268.57 Hz), 126.16, 127.64, 128.54, 131.63,
131.68, 131.87, 137.24, 139.23, 139.80, 139.94, 142.36 (q,
JC–F 38.31 Hz), 147.80, 149.13, 156.11, 165.50; HRMS
(APESI, M+) C21H18Cl2F3 N5O2: m/z calcd 500.0868, m/z
found 500.0851 (M+).
15. Embryonic neurons from the American cockroach, Per-
iplaneta americana, were cultured onto glass coverslips
following published methods.22 Cells grown 7–14 days in
culture were treated for 30 min with the calcium fluoro-
probe, Fura-2 AM, (2 lM), and 0.02% pluronic acid in
saline having the following composition (mM): NaCl 190,
KCl 3.1, CaCl2 9.0, and Tris 10.0, pH 7.2. Fluoroprobe
loaded cells were then rinsed with saline and placed in a
recording chamber perfused with saline. Calcium radio-
imaging studies were conducted by monitoring fluores-
cence emission images (510 nm) with cells alternately
excited at 340 and 380 nm using a fluorescence microscope
coupled with the MetaFluorTM imaging system (Universal
Imaging, West Chester, PA). As a positive control, cells
were treated with the ryanodine receptor agent, caffeine
(20 mM), prior to test compound challenges (3 min
duration). Caffeine exhibits a calcium mobilization thresh-
old of 5.5 mM in this assay. Intracellular free calcium
concentrations ([Ca2+]i) were calculated using the Gry-
nkiewicz equation with calcium mobilization threshold
values defined as the concentration required to stimulate a
25 nM mean increase in basal [Ca2+]i.23 Only those cells
responsive to caffeine were included for data analysis.
16. McCullagh, P.; Nelder, J. A., 2nd ed. In Generalized Linear
Models; Chapman and Hall: London, 1989, pp 1–511.
17. All new compounds gave satisfactory spectral data consis-
tent with their structures. Selected spectral data for com-
19. Compound DP-17: mp 232–232 ꢁC; IR (Nujol) mmax 3309,
1
1672, 1617, 1587, 1536, 1486, 1462, 1379, 1301 cmÀ1; H
NMR (400 MHz CDCl3) d 1.20 (d, 6H, J = 6.53 Hz), 2.20
(s, 3H), 4.17 (m, 1H), 5.95 (d, 1H, J = 7.61 Hz), 7.15 (t,
1H, J = 7.61 Hz), 7.23 (m, 2H), 7.31 (s, 1H), 7.40 (m, 2H),
7.44 (m, 1H), 7.54 (m, 1H), 10.30 (s, 1H); 13C NMR
(100 MHz, DMSO) d 17.87, 22.08, 40.77, 106.39, 120.93
(q, JC–F 269.68 Hz), 125.79, 126.70, 127.78, 128.93, 129.51,
130.31, 131.06, 131.60, 132.28, 134.70, 135.92, 137.59,
139.49, 141.34 (q, JC–F 39.33 Hz), 155.54, 166.40; HRMS
(APESI, M+) C22H20ClF3N4O2: m/z calcd 465.1305, m/z
found 465.1298 (MÀ).
20. Compound DP-18: mp 232–232 ꢁC; IR (Nujol) mmax 3326,
1
1672, 1629, 1592, 1528, 1492, 1462, 1378, 1306 cmÀ1; H
NMR (400 MHz CDCl3) d 1.13 (d, 6H, J = 6.53 Hz), 4.13
(m, 1H), 5.96 (d, 1H, J = 7.90 Hz), 7.11 (t, 1H, J = 7.81 Hz),
7.26 (m, 2H), 7.38 (m, 2H), 7.46 (m, 2H), 7.57 (s, 1H), 10.10
(s, 1H); 13C NMR (100 MHz, DMSO) d 22.03, 40.90,
106.71, 120.89 (q, JC–F 268.01 Hz), 127.01, 127.74, 128.37,
128.94, 129.51, 130.44, 130.62, 131.07, 131.09, 132.17,
137.45, 137.57, 138.89, 141.33 (q, JC–F 39.33 Hz), 155.60,
165.12; HRMS (APESI, M+) C21H17Cl2F3N4O2: m/z calcd
485.0759, m/z found 485.0755.
21. Compound DP-22: mp 191–192 ꢁC; IR (Nujol) mmax 3265,
1
1657, 1627, 1598, 1546, 1460, 1416, 1375, 1305 cmÀ1; H
NMR (400 MHz CDCl3) d 1.18 (d, 6H, J = 6.83 Hz), 2.19
(s, 3H), 4.18 (m, 1H), 5.97 (d, 1H, J = 8.78 Hz), 7.21 (m,
1H), 7.23 (m, 2H), 7.40 (m, 1H), 7.41 (s, 1H), 7.85 (dd, 1H,
J = 1.76, 8.19 Hz), 8.48 (dd, 1H, J = 1.75, 4.97 Hz), 10.43
(s, 1H); 13C NMR (100 MHz, DMSO) d 18.49, 22.77,
41.43, 106.85, 121.49 (q, JC–F 267.13 Hz), 126.48, 127.54,
127.62, 128.55, 132.24, 132.69, 135.67, 136.63, 139.92,
140.02, 142.36 (q, JC–F 38.72 Hz), 147.80, 149.19, 156.09,
166.96; HRMS (APESI, M+) C21H19ClF3N5O2: m/z calcd
466.1258, m/z found 466.1253.
pounds DP-17, DP-18, DP-22, DP-23 are reported.18–21
.
18. Synthesis of DP-23: To a solution of 6-chloro-2-[1-(3-
chloro-2-pyridinyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-
8-methyl-4H-3,1-benzoxazin-4-one (5.0 g, 11.3 mmol) in
tetrahydrofuran (35 mL) was added dropwise isopropyl-
amine (2.9 mL, 34.0 mmol) in tetrahydrofuran (10 mL) at
room temperature. The reaction mixture was warmed until
all solids had dissolved and stirred an additional 5 min, at
which point thin layer chromatography on silica gel
confirmed completion of the reaction. The tetrahydrofu-
ran was evaporated under reduced pressure, and the
residual solid was purified by chromatography on silica
gel, followed by trituration with ether/hexane to afford
22. Lees, G.; Beadle, D. J.; Botham, R. P.; Kelly, J. S. J. Insect
Physiol. 1985, 31, 135.
23. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem.
1985, 260, 3440.