Synthesis and insecticidal activity study of novel anthranilic diamides analogs containing a diacylhydrazine bridge as effective Ca2+ modulators

Article abstract of DOI:10.1111/cbdd.13349

Anthranilic diamides is a class of insecticides target at ryanodine receptors (RyRs). To discover potent insecticides targeting at RyRs, a series of novel anthranilic diamides with a diacylhydrazine bridge were designed and synthesized. Their insecticidal activities were evaluated and a preliminary structure-activity relationship (SAR) was summarized. In particular, compound 5g exhibited good lethality against oriental armyworm (Mythimna separata) at a concentration of 5?mg/L. The calcium imaging experimental results indicated that the compound 5g can serve as effective insect Ca²⁺ level modulators by disrupting the cellular calcium homeostasis in Mythimna separata (Walker) and Spodoptera exigua (Hübner), which probably activated the RyRs on the ER membrane.

Full text of DOI:10.1111/cbdd.13349

Synthesis and insecticidal activity study of novel anthranilic diamides analogues containing a
diacylhydrazine bridge as effective Ca2+ modulators
Yunyun Zhou,b Wei Wei,c Liangliang Zhu,b Yuxin Li,*a Zhengming Li*a
a. State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, College of
Chemistry, Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071, China
b. State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan
University, No. 2005, Songhu Road, Yangpu District, Shanghai, 200433, China
c. WuXi AppTec (Tian Jin) Co., Ltd., 168 Nan Hai Road, Binhai New District, Tianjin, 300457, China
*
Corresponding author. E-mail: nkzml@vip.163.com; liyx128@nankai.edu.cn
Abstract: Anthranilic diamides is a class of insecticides target at ryanodine receptors (RyRs). To
discover potent insecticides targeting at RyRs, a series of novel anthranilic diamides with a
diacylhydrazine bridge were designed and synthesized. Their insecticidal activities were evaluated and
a preliminary structure-activity relationship (SAR) was summarized. In particular, compound 5g
exhibited good lethality against oriental armyworm (Mythimna separata) at a concentration of 5 mg/L.
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The calcium-imaging experimental results indicated that the compound 5g can serve as effective insect
Ca2+ level modulators by disrupting the cellular calcium homeostasis in Mythimna separata (Walker)
and Spodoptera exigua (Hübner), which probably activated the RyRs on the ER membrane.
Keywords: anthranilic diamides, diacylhydrazine, Ryanodine receptors, Ca2+ modulator
The anthranilic diamides are a new category of insecticides. In particular, two of representative
compounds chlorantraniliprole and cyantraniliprole were widely applied over the world due to their
ultra-high efficiency, low toxicity, broad insecticidal spectra.[1, 2] They have also been verified as the
modulators of the insect Ryanodine receptors (RyRs), a non-voltage calcium channel on the
endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) with different binding sites.[3] The binding of
the anthranilic diamides would cause the unregulated release of Ca2+ from ER/SR lumen and the
subsequent break of the intracellular calcium homeostasis, which led to further uncontrolled
contraction of skeletal muscle and the final death of the insect.[4-6]
During the past decade, chemical biologists have been devoted to clarify the structure-activity
relationship (SAR) of anthranilic diamide insecticides. Many new compounds with high insecticidal
activities were found through structural optimization of chlorantraniliprole.[7-10] Most research focused
on the aliphatic amide moiety (Fig. 1a, part A), the functional groups including cyano[11], hydrazine[8]
and heterocyclic[12,13] groups were found to be highly effective. Yet exploration on amide bridge moiety
(Fig. 1a, part B) were relatively rare and there has not been sufficiently comprehensive research on it.
In our previous studies, the acylhydrazine group was found to be a potential effective substituent.[14,15]
Actually, the acylhydrazine fragment have been reported to display a variety of biological activities,
such as antimicrobial,[16, 17] insecticidal[18, 19], anti-HIV[20, 21], anti-HCV[22], antitumor[23, 24], analgesic[25]
antitrypanosomal[26], antioxidant[27]. In addition, some diacylhydrazines (Fig. 1b) such as tebufenozide
are potent ecdysone receptor agonists with high insect growth regulatory activity.[28,29]
,
In hope of finding novel potent lead compounds, a series of diamdes was designed and
synthesized by introducing a diacylhydrazine moiety to modify the amide bridge (Fig. 1a, part B), the
strategy of which was seldom reported.[30] We also attempted to replace the 1-(3-chloropyridyl)
pyrazole moiety (Fig. 1a, part C) with substituted phenyl group for further SAR study. Their
insecticidal activities against oriental armyworms (Mythimna separata) were tested, some compounds
exhibited excellent insecticidal activities. The structure–activity relationship (SAR) was discussed as
well. The calcium imaging technique was also adopted to investigate the effects on calcium channels in
the central neurons of M. separata and S. exigua.
Synthetic routes for compound series 5 were shown in Scheme 1. Intermediates 2 were obtained
by treated anthranilic acid derivatives with triphosgene, after that intermediates 3 were synthesized via
a nucleophilic substitution at carbonyl group of 2 using hydrazine hydrate at room temperature. At first,
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the compound 4 was prepared with trimethylamine as acid binding reagent and dichloromethane as the
solvent. The yield was low with a couple of byproducts, because there were two competitive reactions
between NH2 of aniline and benzoyl hydrazine in the reaction process. With the improvement of
reaction conditions, compound 3 were selectively acylated by 1-(3-chloropyridyl) pyrazole-5-carbonyl
chlorides in a THF/pyridine system through controlling 1.0 equivalent of the reagent and pyridyl
pyrazole chlorides very slowly added to the mixture. Finally, compounds 5 were prepared in good
yields by another acylation reaction of the aniline derivatives 4 with corresponding acyl chlorides.
Because of multiple reaction sites in the compound 4, it was necessary to keep the reaction temperature
at 0 during the acylation process to increase the yield and minimize the byproducts. The substituents
of compounds 4a-h and 5a-u were summarized in Table 1.
Table 1. Substituent pattern of compounds 4a-h and 5a-u.
Compd. R1
R2
Br
Br
Br
R3
Compd. R1
R2
R3
4a
4b
4c
4d
4e
4f
3-CH3
-
5h
5i
3-CH3,5-Cl Br
3-CH3,5-Cl Br
3-CH3,5-Cl Br
3-CH3,5-Cl Br
3-CH3,5-Cl Br
OCH2CH3
OCH(CH3)2
COOCH3
4-Cl
5-Cl
-
-
5j
3-CH3,5-Cl Br
3-CH3,5-Cl CF3
3-CH3,5-Cl Cl
3-CH3,5-Br Br
-
5k
5l
COOCH2CH3
COOCH2CH=CH2
-
-
5m
5n
5o
5p
5q
5r
5s
5t
3-CH3,5-Cl CF3 OCH3
4g
4h
5a
5b
5c
5d
5e
5f
-
3-CH3,5-Cl CF3 OCH2CH3
3-CH3,5-Cl CF3 OCH(CH3)2
3-CH3,5-Cl CF3 COOCH3
3-CH3,5-I
3-CH3
3-CH3
4-Cl
Br
Br
Br
Br
Br
Br
Br
-
OCH3
OCH2CH3
OCH3
OCH2CH3
OCH3
OCH2CH3
OCH3
3-CH3,5-Cl Cl
3-CH3,5-Br Br
3-CH3,5-Br Br
OCH3
OCH3
4-Cl
OCH2CH3
OCH3
5-Cl
3-CH3,5-I
3-CH3,5-I
Br
Br
5-Cl
5u
OCH2CH3
5g
3-CH3,5-Cl Br
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Table 2. Insecticidal activities of compounds 4 and 5 against oriental armyworms
Larvicidal activity (%) at conc. (mg/L) Larvicidal activity (%) at conc. (mg/L)
Compd.
Compd.
200
40
100
50
10
5
200
50
100
50
10
5
4a
4b
4c
4d
4e
4f
5j
50
5k
5l
100
100
100
100
40
40
40
70
60
30
100
30
100
100
60
5m
5n
5o
5p
5q
5r
5s
100
40
100
20
4g
4h
5a
5b
5c
5d
5e
5f
10
60
100
100
100
45
100
100
60
40
50
10
75
10
5t
20
5u
5v
5w
65
10
10
10
30
5g
5h
5i
100
100
60
100
30
100
100
60
controla 100
controlb 100
100
100
100
100
100
100
100
70
a: chlorantraniliprole; b: tebufenozide
The insecticidal activities of compounds 4 and 5 against oriental armyworms were evaluated and
the results were shown in Table 2. Chlorantraniliprole and tebufenozide were selected as positive
controls. A part of compounds 4 and 5 exhibited good insecticidal activity at 100 mg/L concentration
especially 4f and 5g, which showed lethality rates of 20 % at 10 mg/L and 60 % at 5 mg/L,
respectively. The potency of compound 5g was comparable to tebufenozide (70 % at 5 mg/L),
however, was less effective than chlorantraniliprole.
The preliminary SAR was summarized. Activities varied significantly with respect to the
structures. The substitution pattern of R1 was crucial to the insecticidal activity of title compounds. The
compounds bearing 3-CH3, 5-Cl substituted phenyl ring displayed superior activities (4d, 4f, 5g and
5q) than those containing Br (4i and 5s), I (5t and 5u) or H (4a and 5a). The position of R2 exhibited
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minor effect on the insecticidal activity and could tolerate some different groups, in which Br and Cl
(4d, 4f, 5g and 5q) are slightly better than CF3 (5m and 5n). In addition, we observed that the volume
of the R3 group was an important factor for the activity. The methyl ester (5g, 5q and 5r) showed
preferable efficacy than other compounds. The compounds containing ethyl esters (5n and 5s) showed
little loss of activities. And the activities decreased significantly along with the volume augmentation
of the substituent groups such as isopropyl (5i and 5o) or methoxalyl group (5j, 5k, 5l and 5p). We
attempted to replace the complicated 1-(3-chloropyridyl) pyrazole moiety (Fig. 1a, part C) with more
easy available phenyl analogues (5v and 5w) but the compounds displayed limited insecticidal
activities. These results indicated that the pyridyl-pyrazole moiety played an important role in
insecticidal activity.
Previously, it was reported that insecticidal diamides targeted the ryanodine receptors (RyRs),
resulting in the calcium disorder of the cells.5 To explore the insecticidal mechanism of our novel
active structures, we turn to study their impacts on the cellular calcium homeostasis of the central
neurons of M. separata and S. exigua., using calcium imaging technique with Fluo-3 AM (for cytosolic
calcium) or Fluo-5N AM (for ER lumen calcium) as fluorescent dyes.
Fig. 2 showed that the application of 1000 mg/L 5g and 5h to isolated M. separata central neurons
could obviously increase the cytosolic calcium concentration. The peak of [Ca2+]i was increased to
104.91 1.57 % (n = 9) and 103.33 1.24 % (n = 9) of the initial value after the neurons were treated
with compounds 5g and 5h, respectively. Thus, calcium released from the calcium store elevated the
cytosolic calcium levels. In addition, the extent of calcium change was positively correlated with the
insecticidal activities of the compounds. In addition, it was consistent with our expectation that
tebufenozide did not activate ryanodine receptor. Fig. 3 showed that the impact of 5h on calcium
response almost unchanged after the neurons had been pre-incubated with tebufenozide, demonstrating
tebufenozide has no adverse or positive effects on the activation of the ryanodine receptor by our
compounds and there was no synergistic effect between compound 5h and tebufenozide. These results
indicated that these novel compounds indeed have the ability to deliver calcium from endoplasmic
reticulum to cytoplasm.
We speculated that the elevated cytosolic calcium was released from the ER lumen, the internal
calcium store. Therefore, the effect of compound 5g on the calcium level of the ER lumen in S. exigua
central neuron was determined and the result was shown in Fig. 4. We observed that 5g could cause
continuous release of calcium from the ER lumen at the concentration of 200 mg/L. 7 min later, the
[Ca2+]i was decreased to 61 %. In other words, 39 % [Ca2+]i was released through the ryanodine
receptor (or three trisphosphate receptor), causing the calcium to be overloaded in the cytoplasm. It
also showed from the fluorescence photo that the fluorescence intensity was obviously reduced after
the compound 5g was added, because of the decrease of calcium in ER lumen. This indicated that
compound 5g was an effective calcium modulator for insect cells which targeted RyRs to exhibit this
biological effect.
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In conclusion, a novel series of anthranilic diamides containing diacylhydrazine bridge was
synthesized and their insecticidal SAR against oriental armyworm was preliminarily summarized. The
bioassays showed that compound 5g exhibited good lethality against oriental armyworm at 5 mg/L.
Calcium imaging technique was applied to detect cellular calcium level for evaluating the impact of 5g
on the cellular calcium homeostasis in the central neurons of M. separata and S. exigua. The results
indicated that compounds 5g could effectively modulate the calcium level in the insect cells, possibly
by activating the ryanodine receptor on the ER.
Figure 1. Chemical structures of anthranilic diamide, Tebufenozide and the target compounds.
Figure 2. Effects of compounds 5g, 5h and tebufenozide on [Ca2+]i in the central neurons of M. separata at 1000
mg/L concentration. The central neurons of M. separata third-instar larvae were dyed by Fluo-3 AM.
Figure 3. Effects of 5h at 1000 mg/L concentration on [Ca2+]i in the central neurons of M. separata when the
neurons were preincubated by tebufenozide. The central neurons of M. separata third-instar larvae were dyed by
loading with Fluo-3 AM.
Figure 4. Effects of compounds 5g on [Ca2+]i in the central neurons of S. exigua at 200 mg/L concentration. The
central neurons of S. exigua third-instar larvae were dyed by Fluo-5N AM.
Figure 5. Fluorescence photos of compounds 5g on [Ca2+]i in the central neurons of S. exigua at 200 mg/L
concentration. The central neurons of S. exigua third-instar larvae were dyed by Fluo-5N AM. The neurons in the
left one show the fluorescence before the compound 5g was added; the right one shows the fluorescence of the
neurons affected 7 min by compound 5g (the excitation wavelength was 488 nm).
Scheme 1. Synthetic routes of compounds 5a-w. Reagents and conditions: (i) triphosgene, THF, rt; (ii)
NH2NH2·H2O, EtOH; (iii) 1-(3-chloropyridyl) pyrazole-5-carbonyl chlorides, pyridine, THF, ice bath; (iv)
chloroformates, pyridine, THF, ice bath; (v) benzoyl chloride, pyridine, THF, ice bath; (vi) methyl chloroformate;
pyridine, THF, ice bath.
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Acknowledgements
This work was supported by the NSFC/China (21502229) and Tianjin Natural Science Foundation of
China(No.16JCYBJC29400).
Conflict of interest
The authors declare no competing financial interest.
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Supplementary data
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