M. Mao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 42–46
43
The larvicidal activity of compounds 6a–o against oriental
armyworms is summarized in Table 1. The bioassay results indi-
cated that most compounds have excellent larvicidal activities
against oriental armyworm. For example, the larvicidal activities
of 6a, 6e, 6h and 6i against oriental armyworm at 1.0 mg LÀ1 were
60%, 80%, 50%, 20%, respectively. Activities varied significantly
depending upon the types of substituents on the 3-position pyra-
zole. Compared with 3-Br and 3-CF3 in pyrazole, compounds with
3-OCH2CF3 substituents showed higher insecticidal activities
against oriental armyworm, with the sequence of 6h > 6a > 6e,
6i > 6f > 6b and 6o > 6m > 6n, which suggests that the introduction
of the 2,2,2-trifluoroethoxy groups in the 3-position of pyrazole
has a positive effect on the larvicidal activities. Furthermore, differ-
ent substituents in benzene ring had various influence on activity.
When R was fixed as Br, the bioactivity of compounds with differ-
ent X indicated the sequence of Cl > Br > CN > I > NO2 > H, while
compounds with 3-CF3 and 3-OCH2CF3 in pyrazole showed a sim-
ilar trend. However, the compounds with cyano group in 4-posi-
tion of the benzene ring did not exhibited higher activities as we
expected. For example, the larvicidal activities of 6m and 6n at a
concentration of 10 mg LÀ1 were 70% and 20%, respectively. In
addition, the introduction of nitro group at the 5-position of the
benzene ring led to a significant decrease in activity, such as 6l.
The larvicidal activity of compounds 6a–o against diamondback
moth were evaluated as shown in Table 2. Most of them had excel-
lent larvicidal activity against diamondback moth. In particular,
compounds 6l and 6o had around 86% mortality at the concentra-
tion of 0.1 mg/L, approaching closer to chlorantraniliprole. Surpris-
ingly, compound 6d (X = 4-I) showed good activity against
diamondback moth (86% death rate at 1 mg/L).
H
N
Br
N
H
N
Br
N
O
O
H3C
Cl
H
N
H3C
NC
H
N
N
N
Cl
Cl
O
O
Me
N
Me
N
A
B
H
N
Br
R
O
H
N
CN
NC
N
H
N
N
N
N
Cl
O
Cl
O
Cl
Me
N
Me
N
X
C
D
Figure 1. Chemical structures of compounds A–D.
due to the strong electron-withdrawing cyano group resulting in a
poor reactivity of amino moiety. Instead, 5m was obtained via re-
flux in acetonitrile (Scheme 3).15 The nitro-containing intermedi-
ates (5l, 5j, 5k) were synthesized as shown in Scheme 4, and the
compounds (5a, 5c, 5g) were treated with fuming HNO3 in concen-
trated H2SO4 to yield corresponding nitro-containing products in
good yields and high regioselectivity.16
The target compounds 6a–m were synthesized from 5a–m as
shown in Schemes 2–4. We attempted to treat 5a–m with
hydroxylamine hydrochloride in DMF at 50–55 °C to afford the
corresponding cyano-containing products with CN in the ortho-
position. Unfortunately, the aldehyde group of compound 5 were
converted into oxime. In the presence of iodine and aqueous NH3
in THF, the target compounds 6a–m were achieved with satisfac-
tory yields and purity.17
Figure 2 illustrated the change of [Ca2+]i versus recording time
when the neurons were treated with 6b, 6c, 6e, 6h, 6k, 6l, 6n
and chlorantraniliprole. The peak of [Ca2+
] were elevated to
i
117.38 4.21% (n = 18), 111.71 3.29% (n = 13), 119.29 3.47%
(n = 13), 114.63 4.11% (n = 9), 114.43 3.78% (n = 9),
109.23 2.37% (n = 9) and 122.06 2.54% (n = 18) of the initial va-
lue when the cells were treated with 1000 mg/L of 6b, 6c, 6e, 6h,
6k, 6l, 6n and chlorantraniliprole, respectively. Compared with
the control (99.91 2.56%), these compounds induced [Ca2+]i in-
crease without extracellular Ca2+. It indicated that compounds
could activate the calcium release channel in the endoplasmic
reticulum (ER) membrane. Figure 2 also indicated that the re-
The alternative synthetic route to prepare the target com-
pounds 6m–o with cyano group in the 4-position of the benzene
ring from 8a–c as shown in Scheme 5. Intermediates 7a–c and
8a–c were synthesized following the previously reported proce-
dure with minor improvements.4,18 No reaction occurred using
2,4,6-trichloro-1,3,5-triazine as dehydrant to synthesize 6m–o
from 8a–c.19 However, the dehydration reaction proceeded
smoothly with thionyl dichloride and the products were obtained
in excellent yields.20
corded [Ca2+
bioactivities.
]
(F/F0) had a good positive correlation with
i
Most of the intermediates were determined by 1H NMR, and all
new target compounds were characterized with 1H NMR, 13C NMR
and elemental analysis (or HRMS) (see Supplementary data). Com-
pounds 6b was selected to further investigate the IR spectrum
characterization of this kind of compounds. The characteristic
As shown in Figure 3, brief application of compound 6h contin-
ued to stimulate a transient elevation in [Ca2+]i in the absence of
external calcium. Reintroduction of standard saline allowed de-
pleted calcium stores to become refilled and thereby available for
the next 6h challenge but resulted in an attenuated response.
To test why compound 6h and chlorantraniliprole can cause
[Ca2+]i elevation, the primary cultured neurones were dyed loading
stretching vibration
m
(C„N) appears at 2235 cmÀ1
.
with fluo-5 N. Figure 4 illustrated the change of [Ca2+
] versus
i
recording time when the neurons were treated with 6h and chlo-
rantraniliprole. Compound 6h and chlorantraniliprole decrease
[Ca2+]i to 95.12 2.06% (n = 12) and 90.34 3.64% (n = 18), respec-
tively. These data indicated that [Ca2+]i decreased by 1000 mg/L of
6h and chlorantraniliprole. It means that compound 6h and chlo-
rantraniliprole could deliver calcium from endoplasmic reticulum
(ER) to cytoplasm.
There were two kinds of calcium release channels in the ER
membrane, namely RyR and IP3R Ca2+ channels. To test which
pathway was involved in the elevation of [Ca2+]i, the primary cul-
tured neurones were dyed loading with fluo-5 N (low-affinity cal-
cium indicator, accurately tracks the dynamic changes in calcium
in the ER and SR), and then incubated with 2-aminoethoxydiphe-
OH
COOH
CHO
NH2
NH2
NH2
i
ii
X
X
X
1a-d
2a-d
CHO
3a-d
CHO
NH2
iii
NH2
1a:X=H; 1b:X=Cl; 1c:X=Br; 1d:X=I
2a:X=H; 2b:X=Cl; 2c:X=Br; 2d:X=I
3a:X=H; 3b:X=Cl; 3c:X=Br; 3d:X=I
I
NC
3d
3e
Scheme 1. Reagents and conditions: (i) LiAlH4, THF, 0 °C, then NaSO4Á10H2O, room
temperature; (ii) MnO2, CH2Cl2, room temperature; (iii) CuCN, DMF, 140 °C
nyl borate (2-APB 50 lM, a chemical that acts to inhibit both IP3