Sulfonate derivatives bearing an amide unit: design, synthesis and biological activity studies

Pest disasters which occurs on crops is a serious problem that not only cause crop yield loss or even crop failure but can also spread a number of plant diseases.Sulfonate derivatives have been widely used in insecticide and fungicide research in recent years. On this basis, a series of sulfonate derivatives bearing an amide unit are synthesized and the biological activities are evaluated. The bioassay results showed that compounds A8, A13, A16, B1, B3, B4, B5, B10, B12 − 20, C3, C5, C9, C10, C14, C15, C17 and C19 showed 100% activity at a concentration of 500 µg/mL against the Plutella xylostella (P. xylostella). Among them, B15 which contains a thiadiazole sulfonate structure still shows 100% activity at 50 µg/mL concentration against P. xylostella and had the lowest median lethal concentration (LC50) (7.61 µg/mL) among the target compounds. Further mechanism studies are conducted on compounds with better insecticidal activity. Molecular docking results shows that B15 formed hydrophobic interactions π-π and hydrogen bonds with the indole ring of Trp532 and the carboxyl group of Asp384, respectively, with similar interaction distances or bond lengths as those of diflubenzuron. Moreover, chitinase inhibition assays are performed to further demonstrate its mode of action. In addition, the anti-bacterial activity of the series of compounds is also tested and the results showed that the series of compounds has moderate biological activity against Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), with inhibition rates of 91%, 92% and 92%, 88% at the concentration of 100 µg/mL, respectively. Our study indicates that B15 can be used as a novel insecticide for crop protection. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13065-024-01151-0.


Introduction
Agricultural pests are the most important impact factors that threaten crops.They often cause serious quality and yield losses in agricultural production [1,2].Become potential "carriers" while feeding on susceptible plants, spreading more serious diseases to the plants.Among agricultural pests, the P. xylostella is one of the most challenging pests to control, infesting cruciferous crops and causing up to $4.5 billion in losses annually [3].Since crops are threatened not only by agricultural pests but also by crop pathogens, the focus of addressing these issues is on effective disease and pest management.
Therefore, different types of pesticides such as fungicides/bactericides, insecticides, are applied to manage the corresponding pests and diseases [4].However, 4.6 million tons of pesticides are used worldwide every year, with about 90% of them unable to be used properly and ending up in vulnerable farming systems, leading to an excessive build-up of harmful residues in crops [5][6][7][8][9][10][11].Hence, the development of novel insecticidal agents is particularly important to reduce impacts on agroecosystem diversity.
Sulfonate derivatives have been widely studied owning to their strong affinity for lipids, which can easily cross the plant cuticle to perform their biological activities [12,13].Aryl sulfonate esters have been widely used for agricultural and pharmaceutical research due to their antiviral [14,15], antibacterial [16], antifungal [17], insecticidal [18], and anticancer [19] properties.And more aryl sulfonate compounds with high efficacy in insecticidal, antibacterial, and antiviral activities can be obtained by structural modification [20,21], such as, genite, irosustat, chlorfenson and nimrod, etc.In recent years, 1,3,4-thiadiazole derivatives and amide derivatives have become a major focus for the development of novel bactericides, insecticides, and fungicides [22][23][24][25][26], and a number of commercial agents containing thiadiazole/ amide structures have been discovered and commercialized, such as bismerthiazol, thiodiazole copper, boscalid, diflubenzuron, and chlorantraniliprole, etc. [21].In view of the above, a series of novel sulfonate derivatives containing 1,3,4-thiadiazole/amide backbone was designed and synthesized for further development of efficient and active lead compounds (Fig. 1) and evaluated for their biological activities.To our delight, most of the title compounds showed average biological activity against bacteria but most of the target compounds exhibited moderate to excellent insecticidal activity.The high insecticidal activity of the compounds was validated by further preliminary mechanism of action studies and molecular docking.

Chemical synthesis
The synthetic route of target compounds is shown in Scheme 1. 4-hydroxybenzhydrazideproduce intermediate A-1 by nucleophilic substitution reaction with acyl chloride, and intermediate A-1 is sulfonated with sulfonyl chloride to produce target compounds A 1 -A 18 (Scheme 1A).4-hydroxybenzhydrazide produce amino thiourea compounds by addition reaction with potassium thiocyanide under acidic condition, and intermediate B-1 produce target compounds

Insecticidal activity
The insecticidal activities of the target compounds are shown in Table 1, where diflubenzuron is used as a positive control.It can be seen that most of the target compounds show excellent insecticidal activities against both P. xylostella and Myzus persicae.Among them, compounds A 8 , A 13 , A 16  In order to assess the insecticidal potency, the compounds that showed more than 90% mortality at 50 µg/ mL concentration are further investigated to obtain their half lethal concentration (LC 50 ).As shown in  15 is the compound with the highest mortality against Plutella xylostella with an LC 50 value of 7.61 µg/mL, which was about three times more active than that of diflubenzuron.

Changes of chitinase, juvenile hormone and ecdysteroids in P. xylostella
The growth and development of insects are mainly affected by chitinase, and the interaction of juvenile hormone and ecdysteroids also affects the activity and content of chitinase, which in turn affects the metamorphosis and development of insects.In order to further evaluate the possible insecticidal mechanism of action of compound B 15 , it firstly is observed the morphological changes of P. xylostella feeding on drug-containing leaves, and then tested the effects of compound B 15 on chitinase, juvenile hormone and ecdysteroids of P. xylostella.It can be seen that P. xylostella in the treated group after feeding on the drug-containing leaves over time the death of the worms is accompanied by body stiffness, as compared to that of diflubenzuron as a positive control (Fig. 2).To further explore the possible mechanism of action of the title compound in killing the insects, we test the changes in chitinase, juvenile hormone and ecdysteroids in P. xylostella after feeding on the drug-containing leaves (Fig. 3).The inhibition of P. xylostella chitinase activity by compound B 15 shows a tendency of increasing and then decreasing, and the inhibition of chitinase activity is most obvious at 12 h, whereas the inhibition of chitinase activity by diflubenzuron is almost non-existent (Fig. 3a).The effect of compound B 15 on juvenile hormones appears to follow the same trend as its effect on chitinase activity (Fig. 3b).The levels of ecdysteroids are higher in both compound B 15 and diflubenzuron-treated P. xylostella than in the blank control group (CK), and there is no inhibition of ecdysteroids by compound B 15 and diflubenzuron (Fig. 3c).Chitinase plays an important role in the growth and development of insects, and this result suggests that compound B 15 may prevent P. xylostella from completing the normal metamorphosis and development process by inhibiting the activity of chitinase, leading to its death.

Molecular docking
Chitin is essential in insect growth and development, and it has been shown that the title compound disrupts chitinase activity, which in turn may cause dysregulation of chitin synthesis and catabolism in insects; therefore, the chitinase-related protein (PDB ID: 6JMA) has been selected to be used for molecular docking.In order   Å and 2.9 Å) with ASP384 as well as the nitro group on the benzene ring with ARG239 (2.9 Å and 3.1 Å), which is two more than that of the diflubenzuron.The benzene ring forms a π-π stacking interaction with ARG439 and GLN466 (4.8 Å and 4.5 Å), respectively.All these differences explain the more active insecticidal activity of B 15 against the P. xylostella than that of diflubenzuron.

In vitro antibacterial activity
In order to explore the effect of the synthesized target compounds beyond insecticides, antibacterial activity   experiments are carried out.The antibacterial activities of the title compounds in vitro are shown in Table 3.Most of the compounds show moderate inhibitory activity against the tested bacteria.Among them, some of the compounds show a good inhibitory activity against Xoo and Xoc, and compounds A 4 and C 4 both showed more than 90% inhibitory activity against Xoo at a concentration of 100 µg/mL.4).

Conclusion
In summary, 58 novel series of sulfonate derivatives containing an amide backbone were designed and synthesized.They have been evaluated for their antibacterial and insecticidal activities.The results of the bioactivity tests indicated that the compounds show fair antibacterial activity, but most of them showed good insecticidal activity against the P. xylostella.Among them, compound B 15 showed the best insecticidal activity against P. xylostella with LC 50 of 7.61 µg/mL, which is much lower than the control agent diflubenzuron.Further preliminary studies on the insecticidal mechanism indicated that compound B 15 may exert its insecticidal activity by inhibiting chitinase and juvenile preserving hormone in the body of the P. xylostella, and this result was also consistent with the molecular docking experiments.Therefore, compound B 15 may be a promising insecticide candidate.

Equipment and materials
Melting points of new compounds were determined on an X-4 digital display micro melting point apparatus (Henan, Gongyi Yuhua Instrument Co., Ltd, China) and were uncorrected.The NMR spectra of the title compounds were obtained on a JEOL-ECX500 apparatus (JEOL, Japan) or a Bruker Biospin AG-400 apparatus (Bruker Corporation, Germany) using tetramethylsilane as the internal standard.High-resolution mass spectra were recorded with a Thermo Scientific UltiMate 3000 spectrometer (Thermo Fisher Scientific, USA).Optical density was recorded on a Cytation™ 5 multi-mode readers (BioTek Instruments, USA).All chemical reagents were obtained from commercial suppliers.All strains used (Xoo, Xac, Psa and Xoc) and the test insects (Plutella xylostella and Myzus persicae) were provided by the laboratory of Guizhou University.All ELISA kits were obtained from commercial suppliers (Jiangsu Meimian Industrial Co., Ltd, China).

Preparation procedure of intermediate compounds
Synthesis of intermediate A-1: following the method described in the literature [27], 4-hydroxybenzhydrazide (65.7 mmol) and Et 3 N (131.4mmol) were added to a clean 250 mL three-necked flask with tetrahydrofuran (130 mL), at 25 ℃.Then 2,6-difluorobenzoyl chloride (65.7 mmol) was added to the mixture at ice bath.The reaction would be accomplished quickly in 5 min (monitored by TLC).Then the reaction mixture was quenched by adding 100 mL H 2 O and extracted with ethyl acetate (100mL × 3).The collected organic phase was extracted with NaOH solution and the water phase was adjusted to pH = 4-5 with diluted hydrochloric acid.Then ethyl acetate was added to extract and concentrate to get the crucial solid, which was then recrystallized with anhydrous ethanol to obtain white crystal intermediate A-1 (yield, 76%).Synthesis of intermediate B-1: following the method described in the literature [28], 4-hydroxybenzhydrazide (65.7 mmol) was added into a clean 250 mL three-necked flask with 120 mL of 1 M HCl solution at room temperature.After stirring for 15 min, all the solid was dissolved.Then potassium thiocyanate (131.4 mmol) was added and the mixture was heated at 100 °C for 8 h until the reaction completion as monitored by TLC.The system was cooled to room temperature and a large amount of white solid appeared.The solid product was filtered and washed by water for three times and ethanol once, and then recrystallized in anhydrous ethanol to obtain intermediate B-1, with yield of 57.6%.
Synthesis of intermediate B-2: following the method in the literature [28], intermediate B-1 (28.4 mmol) was added into a clean 50 mL three-neck flask at 25 ℃ followed with H 2 SO 4 (20 mL) while stirring.After 6 h the reaction was completed.Then the reaction mixture was slowly poured into crushed ice, and 25% ammonia water was added to adjust the solution to pH = 8-10 during stirring and a large amount of white solid precipitated.The precipitate was filtered and washed with water three times and dried.The solid was then recrystallized in anhydrous ethanol to obtain intermediate B-2, with yield of 32.8%.
Synthesis of intermediate B-3: following the method in the literature [29],intermediate B-2 (9.3 mmol) and Et 3 N (18.6 mmol) were added to 20 mL tetrahydrofuran in a clean 50 mL three-neck flask and stirred for 15 min at 25 ℃.Next in an ice bath, 2,6-difluorobenzoyl chloride (9.3 mmol) was slowly added and then and the mixture was stirred at room temperature till reaction was completed according to TLC.Then the reaction mixture was quenched by adding 20 mL H 2 O and extracted with ethyl acetate (20 mL× 3).The collected organic phase was extracted with  NaOH solution, and the water phase was adjusted to pH = 4-5 with diluted hydrochloric acid.Then ethyl acetate was added to extract and concentrate to get the crucial solid, which was then recrystallized with anhydrous ethanol to obtain intermediate B-3, with yield of 61.4%.Synthesis of intermediate C-1: following the published methods [28], 4-hydroxybenzhydrazide (32.8 mmol), KOH (49.2 mmol), and 80 mL of ethanol were added sequentially to a 150 mL three-neck flask at room temperature.The solids were dissolved after 10 min stirring.The CS 2 (49.2 mmol) was slowly added into the mixture in an ice bath.The mixture was stirred overnight at room temperature.The reaction solution was becoming, and till the reaction completed as indicated by TLC.Solid products appeared in the solution, filtered and washed by ethanol to obtain intermediate C-1, with yield 92%.The intermediate C-1 was subjected directly to the next reaction without recrystallization.
Synthesis of Intermediate C-2: following the published methods [28], intermediate C-1 (7.5 mmol) was slowly added to the ice-cold H 2 SO 4 (15 mL) in 50 mL three-neck flask while stirring.The reaction was kept stirring for 4 h at an ice-water bath and poured into crushed ice.The newly appeared precipitate product was filtered to dissolve in a 20% NaOH aqueous solution.After insoluble substances were excluded, and the solution was acidified with diluted hydrochloric acid and got a precipitate, which was then filtered, washed by water and dried to obtain intermediate C-2, with a yield of 76.1%.
Synthesis of intermediate C-3: following the method described in the literature [30], 2,6-difluoroaniline (38.7 mmol) was added into 80 mL of 1,4-dioxanein a 150 mL three-necked flask and dissolved after stirring for 10 min.Then bromoacetyl bromid (50.3 mmol) early dissolved in dry dioxane (15 mL), was added dropwise to the above solution at 0 ℃ then mixture was stirred at room temperature for 5 h.Finally, the reaction mixture was poured into cold water, precipitate appeared, which was then filtered, washed by water and dried to obtain intermediate C-3, with a yield of 64%.
Synthesis of intermediate C-4: as reported [31], a mixture of thiazole intermediate C-2 (15.6 mmol), anhydrous potassium carbonate (23.4 mmol), and the corresponding amide intermediate C-3 (17.2 mmol) was added to dimethylformamide (80 mL) in a 150 mL three-necked flask at 25 ℃.The mixture was stirred for 4 h (monitored by TLC).Then, the reaction solution was poured into 80 mL cold water, and got  For all target compounds, the final synthetic procedure was an esterification between the substituted benzene sulfonyl chloride and A-1, B-3 or C-4 respectively.And the reaction condition was completely same.Here, compound A series was taken for example to illustrate the reaction procedure.Intermediate A-1 (1.7 mmol) and triethylamine (5.1 mmol) were added and dissolved in 20 mL of dichloromethane in a clean 50 mL three-necked flask at room temperature.To the flask, different substituted benzene sulfonyl chloride (2.1 mmol) was added accordingly.The mixture was stirred overnight till the reaction was completed (monitored by TLC).The reaction was quenched with 20 mL water.Then ethyl acetate was added to extract and concentrate to get the crude solid, which was purified by the column chromatography (dichloromethane: methanol = 50:1, v/v) and obtained the title compounds A 1 -A 18 with a yield in the range of 70-96%.For title compound B 1 -B 20, the yield ranged 74-90% and C 1 -C 20 68-95%.

Insecticidal assay
The insecticidal activity of compounds against P. xylostella and Myzus persicae was evaluated according to our previously reported methods [21].After soaked in a solution of 500 and 50 µg/mL of compounds A 1 -A 18 , B 1 -B 20 and C 1 -C 20 , diflubenzuron (positive control), or no compound (blank control) for 10s, the fresh cabbage discs (6 cm in diameter) were air-dried and placed in 9 cm petri dishes lined with wet filter paper.Ten P. xylostella/Myzus persicae (third instar larvae) were placed in the petri dishes and the survival insect individual was recorded after 72 h.The mortality rate was determined by the number of survival larvae comparing to the initial number.Compounds that were active at this concentration were further tested at 5 lower gradient concentrations (6.25, 12.5, 25.00, 50.00 and 100.00 µg/mL) to get LC 50 .Each treatment was repeated three times.The data were analyzed with probit analysis to obtain LC 50 values All experiments were conducted at 25 °C using laboratory reared test insects and were repeated according to statistical requirements.The death rate was estimated by calculating the ratio of the number of insects killed to the initial number.The adjusted mortality rate (%) is as follows: Adjusted mortality rate (%) = (death rate in the treatment group -death rate in the blank control group)/(1death rate in the blank control group) × 100.

Effect of highly active compound on chitinase, juvenile hormone and ecdysterorids inP. xylostella
Fresh cabbage discs (6 cm in diameter) were first soaked in a solution of 50µg/mL of highly active compound for 10 s, air-dried and placed in a 9 cm petri dish lined with wet filter paper.Ten third instar larvae of P. xylostella were placed in the petri dishes and samples were taken and stored at − 80 °C after 12, 24, 36 and 48 h of feeding by P. xylostella.Chitinase, juvenile hormone, and ecdysterorids activities were determined by MicroplateReader using commercial ELISA kits following the manufacturer's instructions.Each experiment was repeated three times.The blank control group was treated with distilled water containing 0 µg/mL of the compounds, and the positive control was selected as diflubenzuron that is a commercially available insecticide.

Molecular docking
A molecular docking station was built using the Ledock program according to the literature [32], the crystal structure of chitinase (PDB: 6JMA) of the P. xylostella was downloaded from the Protein Data Bank (https:// www.pdb.org,accessed on 10 February 2023) and was processed with Pymol [33].The molecular structures of highly active compound and diflubenzuron were drawn using ChemBioDraw Ultra 14.0 software and were optimized to minimize energy.A 17.5 × 15.3 × 14.7 docking box was generated with the Carboxin Standard in the protein as the center, and the docking station generated 20 ligand conformations with an RMSD less than 1.0 Å.The docking results were visualized in 3D by the Pymol software v.2.4.0.

Antibacterial activity assay
According to previously reported procedures [21], the title compounds A 1 -A 18 , B 1 -B 20 , C 1 -C 20 were tested in vitro against Xoo, Xoc, Xac and Psa at concentrations of 100 and 50 µg/mL respectively.The commercial fungicides Bismerthiazol (BT), Zinc Thiazole (TZ) and Thiodiazole copper (TC) were selected as positive controls.The solution containing no compound was set as negative control (CK).Compounds that were active at this concentration were further tested at 5 lower gradient concentrations (6.25, 12.50, 25.00, 50.00 and 100.00 µg/mL) to get EC 50 .Each treatment was repeated three times.The inhibitory effect of compounds on bacteria was calculated based on optical density values using the following formula.I (%) = (CK-T)/T × 100.I (%) denotes the inhibition rate.CK denotes the OD value of the treatment with solvent with no tested compound.T denotes the OD value of the treatment in tested compound group.

Statistical analyses
All trials were performed in triplicate, and the results of each trial are shown as mean ± standard deviation (SD).Analyses of variance (ANOVAs) were performed using SPSS Statistics 24.0 with and without the assumption of equal variance (with P > 0.05 taken as the significance level for both).Significant differences between treatment groups are indicated using various lowercase letters.

B 1 -
B 20 (Scheme 1B) by cyclization reaction.Intermediate B-2 is formed by cyclization of intermediate B-1, and intermediate B-3 is sulfonated with sulfonyl chloride to form target compounds B 1 -B 20 .The synthesis route of target compounds C 1 -C 20 is shown in Scheme 1C.4-hydroxybenzhydrazidereacts with carbon disulfide in a condensation reaction to form intermediate C-1 under alkaline conditions.Intermediate C-1 is cyclized to form intermediate C-2, intermediate C-2 and C-3 react with nucleophilic substitution to form intermediate C-4, intermediate C-4 and sulfonyl chloride react with sulfonation to form the target compound C 1 -C 20 .The chemical structures of all the target compounds, A 1 -A 18 , B 1 -B 20 and C 1 -C 20 , have been confirmed by 1 1 H NMR, 13 13 C NMR spectroscopy and high-resolution mass spectrometry.Physicochemical properties are provided in supporting information.

Fig.
Fig. 1 Design strategy for target compounds to verify the possible insecticidal mechanism of the title compound B 15 , it is docked at the theoretical binding site of the chitinase protein, diflubenzuron is used as a positive control.The interactions of B 15 and diflubenzuron with the chitinase protein are shown in Fig.4.In the docking complex of diflubenzuron and chitinase protein (Fig.4A) the two N atoms of the amide bond form two hydrogen bonds (2.8 Å and 2.9 Å) with the key residue ASP384, which is essential for the binding of the inhibitor and chitinase protein.The benzene ring forms a π-π stacking interaction with residues TRP532 and ASP384 (4.4 Å and 4.5 Å).As shown in Fig.4B, the thiazole ring and amide bond of B 15 forms two hydrogen bonds(3.2

15 , C 14 , C 15 , C 17 and C 19 show
insecticidal activity against the P. xylostella of more than 80% at 50 µg/mL, notably compound B 15 still show 100% mortality against P. xylostella at both 50 and 500 µg/mL concentrations.

Table 2 ,
B 15 , C 14 , C 15 , C 17 and C 19 show better insecticidal activity against Plutella xylostella with LC 50 values ranging from 7 to 19 µg/mL as compared to diflubenzuron with LC 50 value of 24.08 µg/mL.Among them, B

Table 1
Insecticidal activity of title compounds A 1 -A 18 , B 1 -B 20 and C 1 -C 20 against P. xylostella and Myzus persicae

Table 2
LC 50 values of some compounds with excellent insecticidal activity against the P. xylostella a All results are expressed as mean ± SD.Fig. 2 Morphological changes of the P. xylostella feeding on the drug-containing leaves at different time periods a All results are expressed as mean ± SD.

Table 1
(continued) Then, we test the EC 50 values of those title compounds that showed better activity against Xoo and Xoc, the EC 50 values of antibacterial activity of compound A 4 and compound C 4 against Xoo are 24.87 and 30.04 µg/mL, respectively.The EC 50 values of compounds A 4 , A 15 , A 16 , B 7 , C 4 and C 7 of that inhibitory activity against Xoc are 30.12,39.59, 18.75, 67.9, 43.85 and 54.56 µg/mL, respectively (Table

Table 3
In vitro antibacterial activity results of the title compounds

Table 4
EC 50 values of some compounds with better inhibitory activity against Xoo, Xoc a All results are expressed as mean ± SD.aAll results are expressed as mean ± SD.