Design, synthesis and insecticidal activity of benzenesulfonamide derivatives containing various alkynyl, alkenyl and cyclopropyl groups in para position

Abstract Celangulin V is a natural β-dihydrofuran sesquiterpene polyester with anti Mythimna separate activity and unique mechanism of action. Further study showed that its target was the H subunit of V-ATPase in the midgut of M. separate. Thus, combined with the previous work, thirty-two benzene sulfonamide derivatives were systematically synthesised to discover efficient and low-budget insecticidal candidates for the H subunit of V-ATPase. Screening results showed that compounds C2, C4, C5, C6 and C8 could significantly cause death of tested third-instar larvae of M. separate, and provided the corresponding LC50 values of 0.844, 0.953, 0.705, 0.599 and 0.887 mg/mL, which were extremely better than Celangulin V (LC50 = 11.5 mg/mL). The docking results indicated that this novel framework might target H subunit of V-ATPase. Given these excellent bioactivity results, this kind of sulfonamide framework could provide a suitable point for exploring highly efficient insecticidal agents. Graphical Abstract


Introduction
The constant growth of pests caused widespread destruction of vegetables and fruits, and seriously threatened the output and quality of agricultural production worldwide (Liu et al. 2008;Tang et al. 2011;Tang and Hou 2011;Liu et al. 2014).In view of the present treatment and prevention programs toward insect attack, chemical control measures using insecticides were one of the most efficient governance methods, and had been extensively developed due to low toxicity in the plant and benefits to farmers (Xu et al. 2003;Zheng et al. 2003;Tang et al. 2011).With the increasing use and abuse of traditional insecticide, the resistance of pests had been strengthened (Yang et al. 1995;Cao et al. 2002).Thus, developing and researching novel, simple molecular structures possessing highly powerful bioactivity and unique mechanism of action as replaceable insecticide were needed.
Considering above factors, a large number of studies had been selected comprehensively to search for highly efficient molecule as insecticidal surrogates, in which the biological effects of different key fragment had been elaborately concerned and explored (Napal and Palacios 2015;Ganiger et al. 2018;Hay et al. 2020).Especially, the skeleton of sulfamide displayed wide spectrum of pharmic activities, including antibacterial, anticancer, insecticidal, antiviral, antitumor and antifungal activity (Bolli et al. 2012;Suthagar and Fairbanks 2016;Devendar and Yang 2017;Jun et al. 2018;Jun and Xie 2021;Zhao et al. 2021).What is more, a large number of sulfonamide derivatives were synthesised and evaluated their biological activity in our previous work, and derivatives of NH substituted by n-butyl, isobutyl, n-propyl and cyclopropyl had better insecticidal activity than other compounds (Yang et al. 2018;Gang et al. 2020;Yang et al. 2020).Further research showed that sulfonamide derivatives and Celangulin V might act on the same target of the H subunit of V-ATPase in the midgut of M. separate (Zhao et al. 2021).
Given our previous works, a series of derivatives containing alkynyl, alkenyl and cyclopropyl groups in para position were synthesised based on N-butyl-4-propargyloxybenzenesulfonamide as the lead compound in this paper.Subsequently, the insecticidal activity and structure-activity relationship of the target compounds were evaluated and explored to provide ideas for the synthesis of novel insecticides (Figure S1).

Chemistry
The target compounds were synthesised by three different routes (Figure S2).The purchased drugs and reagents (Shanghai Titan Scientific Co., Ltd) in the synthesis were analytically pure without further purification.Specific synthesis of method, raw materials proportioning, reaction time and treatment method for each step were shown in supporting information.Melting points of the target compounds were determined on a micro melting point apparatus (Equipment Co., Ltd.Of Shanghai). 1 H and 13 C NMR spectra were determined in CDCl 3 at 400 MHz for 1 H and 101 MHz for 13 C (Bruker AMX-500 spectrometer).The NMR data for all the compounds were recorded in the support information.

Bioassays
The insecticidal activities of target compounds were carried out by using the leafdipping method with the third-instar larvae of M. separate starved for 24 h.In this experiment, Celangulin V and acetone were used as the positive and blank controls, respectively.Fresh wheat leaves (5 mm Â 5 mm) were soaked in the 10 mg/mL solution of prepared compounds.The leaves containing the compound were eaten by 12 larvae of M. separate in a 12-well cell plate.The tested third-instar larvae were cultured under constant temperature and humidity, and their deaths were recorded after 48 h.While the drug concentrations, for example, 1, 0.8, 0.6, 0.4, 0.2 mg/mL, were used for LC 50 calculation.The mortality (I) was gained by following the formula: I (%) ¼ T/ 12 Â 100 (T is the number of larvae that die).And the related LC 50 values were analysed by using SPSS 22.0 software.Three sample replications were used in this study.The initial screening concentration of designed derivatives was set at 10 mg/mL and the mortality rate was greater than 50% before the test was continued.
Further bioactivity screening results were shown in Table S2 and expressed by LC 50 values.Clearly, compounds C2, C4-C6 and C8 provided similar bioactivity than the lead compound (LC50 ¼ 0.689 mg/mL) against third-instar larvae of M. separate, with the corresponding values of 0.844, 0.953, 0.705, 0.599 and 0.887 mg/mL, respectively.The LC 50 values of aforesaid compounds were lower than Celangulin V (LC50 ¼ 11.5 mg/mL).Especially, the LC 50 value of compound C6 was nearly twenty times that of Celangulin V.
As shown in the Figure S3, a series of symptoms of the tested insects were carefully observed and studied in the course of the experiment.Summed up as follows, the tested third-instar larvae of M. separate gradually went through three stages after eating the poison-laden leaves.Firstly, the poisonous leaves were first eaten by the tested insect, the symptoms of the bug with paralysis and stiffness obviously appeared.Secondly, the tail of the insect gradually lost water and a yellow liquid flowed out.Finally, the tested insects dehydrated completely until death.According to the above phenomenon, the death symptoms caused by some target compounds were similar to those of the lead compound and Celangulin V. Thus, it could be inferred that the mechanism of action of designed compounds could be the same as celangulin V.

Structure-activity relationship
The structure À activity relationship was potentially concluded from the Tables S1 and  S2.
(1) After oxygen atom was replaced by -NH-or an ethyl group is added to the alkyne-H, the activity of the compound decreased greatly.(2) Compounds A9-A12 had one less carbon atom than compounds A13-16, but the insecticidal capacity of compounds A9-A12 was more effective.(3) The comparison of compounds B1-B4 with compounds B5-B8 showed that compound with one more propargyl have better activity.(4) Acetylene groups and trimethyl acetylene were introduced into 4 positions of the benzene ring to effectively enhance the bioactivity.Given the above analysis, compound C6 with highest biological activities could be considered as new lead compound for exploring further.

Docking simulation
The structure of V-ATPase H subunit was created in the previous work. 21Molecular docking for V-ATPase H subunit of M. separate was performed using the Sybyl 2.0 software.The docking results were visually analysed using Pymol 2.5.Based on the molecular docking study (Figure S4), compound C6 could entirely locate in the binding pocket of the V-ATPase H subunit of M. separate.Interestingly, the effective sulfamide core of compound C6 could form three hydrogen bonds with the side-chain amino acid residues A/ASN231 (distance ¼ 1.8 Å), A/ILE236 (distances ¼ 1.9 Å) and A/ LYS232 (distance ¼ 2.5 Å, Figure S4B), which was same from the side-chain amino acid and which was different from the distance of the hydrogen bonds of the lead compound (distance of 1.9 Å and 2.0 Å against A/ASN231 and A/LYS232, respectively, Figure S4A).This outcome validated that the sulfamide fragment could act as an important tool for binding target proteins of the V-ATPase H subunit.

Conclusion
In summary, several designed derivatives were synthesised and screened for insecticidal activity.The results showed that the certain compounds were conferred with markedly enhanced insecticidal behaviors toward third-instar larvae of M. separate (C2, C4, C5, C6 and C8, LC50 values within 0.599-0.953mg/mL).This effect was superior to Celangulin V (LC50 ¼ 11.5 mg/mL).Pharmacophore studies revealed that the synergistic combination of acetenyl and (trimethylsilyl)ethynyl skeleton improved the biological actions.Molecular docking of C6 with V-ATPase H subunit indicated that novel framework might target V-ATPase H subunit.Given this facile structural modification and the valuable biological behaviors, we anticipated that this novel framework needed to be further studied as a suitable template for discovering and developing pesticide.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
We acknowledge the financial support provided by the National Natural Science Foundation of China [21977083].