Synthesis and insecticidal activities of 4-(propargyloxy) benzenesulfonamide derivatives

Abstract A series of 4-(propargyloxy) benzenesulfonamide derivatives with different substituents on the benzene ring were synthesized and evaluated for their insecticidal activity. Some of the compounds showed good insecticidal activity against Mythimna separata, and the LC50 value of the most active compound B2.5 was 0.235 mg/ml. Ultrastructural changes in the midgut epithelial cells of Mythimna separata were observed using transmission electron microscopy, and severe structural damage was found in microvilli, mitochondria and rough endoplasmic reticulum. It indicates that the possible site of action of these benzenesulfonamides is the cytoplasmic membrane and endomembrane system of the midgut epithelial cells. The above provides a basis for the development of novel insecticidal active compounds with a novel mechanism of action. Graphical Abstract


Introduction
Stable crop production is necessary for the population of any region [1], but agricultural pests seriously affect crop production and pose a serious threat to agricultural production and food security [2][3][4]. Currently, chemical pesticides are still the main means of pest control [5]. However, the environmental pollution and human health problems associated with the heavy use of chemical pesticides are increasing, and the resistance of agricultural pests is growing rapidly [6][7][8][9]. Therefore, it is important to create new target pesticides.
Natural products have been an important source of insecticidal active compounds [10]. Wu et al. have been working on the active ingredients of Celastrus angulatus Maxim, and celangulin V was isolated. It's a typical representative of b-dihydrofuran sesquiterpenoids with insecticidal activity [11][12][13]. Subsequent studies revealed that its target protein was the V-ATPase of Mythimna separata [14][15][16]. We applied the computer-aided drug design (CADD) method to synthesize a class of benzenesulfonamides with insecticidal activity on this basis, and the results indicated that propargyloxy and sulfonamide were the main active groups, and the insecticidal activity was only when the amino group in sulfonamide was aliphatic amine [17,18]. Aromatic sulfonamides with naphthalene ring as the parent ring were also reported subsequently, some of which were found to have good insecticidal activity [19].
Based on the previous research work, to further investigate the effect of different substituents on the benzene ring on the insecticidal activity of such compounds, the substituents are halogen atoms [20,21], which are commonly found in pesticides, and methyl and amino groups. We synthesized 141 propargyloxy benzenesulfonamides (133 of which were not reported), evaluated their activity against M. separata and discussed structure-activity relationships. The possible sites of action of these compounds were analyzed by transmission electron microscopy (TEM).

Chemistry
The synthesis of compounds B1.1-B1.27 is shown in Scheme 1. Firstly, compound 1.1 reacted with bromopropyne in methanol under alkaline environment to form ether and then reacted with PCl 5 to form sulfonyl chloride [22]. Finally, the sulfonyl chloride was reacted with different aliphatic amines to obtain the target products. As shown in Scheme 3, compound 3.1 was reacted with di-tert-butyldicarbonate, thereby protecting the amino group. The next steps were similar to Scheme 1 and finally Then it reacted with excess of chlorosulfonic acid at 0 C to form the corresponding sulfonyl chloride and finally reacted with different aliphatic amines to obtain the product [23]. The R group in the compound is shown in Figure 1.
All compounds were purified by silica gel column chromatography or neutral alumina column chromatography and were characterized by 1 H NMR and 13 C NMR spectra. Among them, compounds B6.3 (CCDC 2131976) and B7.7 (CCDC 2131975) were subjected to X-ray single crystal diffraction ( Figure 2).

Evaluation of insecticidal activity
The insecticidal activity of all compounds against the third-instar M. separata was evaluated by the leaf-dipping method for 24 h. Celangulin V was used as a positive control and acetone as a blank control.  As shown in Table 1, some compounds of the B1, B2, B3, B6 and B7 series showed good insecticidal activity against M. separata at a concentration of 5 mg/ml. Among the B4 series compounds, only compounds B4.3 and B4.5 showed more than 50% lethality against M. separata, while the rest of the compounds showed low or even no activity against M. separata. The B5 series compounds were inactive against M. separata. Among them, compounds B1.3, B1.5, B1.6, B1.7, B1.13, B1.16, B1.24, B2.5, B2.6, B2.24 and B2.25 showed mortality rates of more than 90% against M. separata. To further evaluate the activity, the LC 50 values of these 11 compounds were determined. All of these compounds had low LC 50 values compared to celangulin V, with B2.5 having the lowest LC 50 value (0.235 mg/ml). This result suggests that only the introduction of fluorine atoms increases the insecticidal activity, but unfortunately this enhancement is small.
During the insecticidal activity test, a series of test insect symptoms could be observed (Figure 3). At first, the test insects were knocked down by anesthesia after feeding on the poisonous leaves, and the insects were stiff and immobile. Subsequently, the insects started to wriggle and twitch, and water gradually started to emerge from the tail of the insects, and finally water loss led to the atrophy of the insects and death. This is similar to the symptoms caused by celangulin V in M. separata. Therefore, it can be inferred that the sites of action of these compounds are the same as celangulin V.

Structure-activity relationship
The experimental results showed that the insecticidal activity of the compounds varied considerably with the change of substituents. When an amino group was introduced on the benzene ring, the compounds were essentially inactive, and when a methyl group was introduced, the activity was reduced. When chlorine and bromine atoms were introduced on the benzene ring, the activity of the compounds decreased. When fluorine atoms were introduced, the insecticidal activity was comparable to that of the B1 series compounds, and only some compounds (e.g. B2.5) showed an increase in activity. Therefore, it can be inferred that the electronegativity of the substituents on the benzene ring is positively correlated with the activity, and the less electronegativity of the substituents, the lower the insecticidal activity of the compounds. This may be due to the electronegativity of the introduced group, which leads to a change in its ability to bind to the target protein and thus affects the change in the activity of the compound. The activity of the compound is also significantly related to the structure of the sulfonamide group. The insecticidal activity of the sulfonamides is prominent when the amino group in the sulfonamide is a primary amine with four carbon atoms. And the activity of the compounds was generally better when the amino group was cyclopropylmethylamine. As shown in Table 2, the LC 50 values of compounds B1.24 and B2.24 were 0.386 mg/ml and 0.342 mg/ml. This may be related to the hydrophobic property and van der Waals force interaction of the sulfonamide tail within the active cavity of the target protein.

TEM analysis
These benzenesulfonamides have many similarities to celangulin V in terms of symptom presentation and should be a digestive toxicant, so we hypothesized that the location of action might be on the midgut cells, as with celangulin V [24]. Therefore, the ultrastructure of the midgut cells of M. separata was observed by TEM.
The midgut epithelial cells of M. separata in the blank control group had closely arranged and well-structured microvilli (Figure 4(a,b)); the inner and outer membranes of mitochondria were intact and the ridges were clearly visible ( Figure 5(a)); the rough endoplasmic reticulum was neatly arranged and well textured, with a large number of ribosomes attached to the surface (Figure 6(a)). After treatment with compound B1.5, the number of microvilli in midgut cells began to decrease and the arrangement was gradually disorganized (Figure 4(c,d)); the inner and outer membranes of mitochondria were separated and appeared bright white cavities, and the   inner ridges were disordered and blurred ( Figure 5(b)); the coarse endoplasmic reticulum showed a large number of bright white cavities, vesiculation was serious, and ribosomes were detached ( Figure 6(b)). From this, we speculated that the site of action was the cytoplasmic membrane and endomembrane system of the midgut epithelial cells, leading to perforation of the intestinal wall and massive loss of body fluid, which eventually led to the death of M. separata.
Overall, a series of sulfonamides with different substituents on the benzene ring were synthesized and evaluated for insecticidal activity, and compound B2.5 was enhanced by about two orders of magnitude compared to the positive control, celangulin V. The structure-activity relationships indicated that the introduction of electron-donating groups reduced the activity of the compounds, with the best activity for primary amines with amino substituents of three to four carbon atoms. The observation by TEM showed that these benzenesulfonamides may function by disrupting the microvilli, mitochondria and rough endoplasmic reticulum of the intestinal epithelial cells in M. separata. These show the potential of such compounds for the development of novel pesticides.

General experimental procedures
Melting points were determined using a WRS-1B melting point apparatus (Shanghai YiCe Apparatus and Equipment Co., Ltd., Shanghai, China) without correction. 1 H NMR and 13 C NMR spectra were recorded utilizing a Bruker AMX-400 or AMX-500 spectrometer. M. separata were provided by the Key Laboratory of Botanical Pesticide R&D in Shanxi Province (Northwest A&F University, Yangling, China). All reagents are from commercial sources and no further purification is required. Silica gel (200-300 mesh) (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) and neutral aluminum oxide (200-300 mesh) (Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) were used to perform the column chromatography. The progress of the reaction was monitored by thin-layer chromatography (TLC) using GF 254 silica gel plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China).

General synthesis procedures
The specific synthesis of the compounds is described in the Supporting Information.

Biological assay
The compound and positive control drug were configured into a 5 mg/ml solution with acetone. Fresh wheat leaves (5 mm Â 5 mm) were cut and dipped into the solution for 3 s and then removed to evaporate the acetone. Next, the toxin-laden leaves were placed into 12-well cell plates, and one third-instar M. separata that had been starved for 24 h was placed in each well in three replicates. The state and mortality of the test insects were recorded over 24 h at 25 C.
The compounds and positive control drugs were prepared in six concentrations in equal gradients and the mortality rate of the test insects was maintained between 20% and 80%. The data were obtained and the LC 50 values were calculated using SPSS 22.0 software.

TEM imaging
The newly molted sixth-instar M. separata were placed individually in petri dish and starved for 24 h. In the experimental group, B1.5 was dissolved in acetone (0.4 mg/ ml) and 1 ll of the solution was applied uniformly on fresh wheat leaves (5 mm Â 5 mm); the blank control was treated with acetone. The larvae showing symptoms and the blank control larvae at the same time were selected after feeding. Subsequently, ultrathin slices were prepared by Life Science Research Core Services of Northwest A&F University. Finally, the analysis was observed by TECNAI G2 SPIRIT BIO TEM.

Disclosure statement
No potential conflict of interest was reported by the author(s).