Synthesis process optimization and field trials of insecticide candidate NKY-312

NKY-312 is a highly active insecticide candidate with a simple structure. In order to carry out field trials and toxicity tests, its scale preparation is urgently needed, but the final step of the original synthetic route is a low-yielding sulfonylation reaction that generates a high proportion of a bissulfonylated by-product, its foliar contact activities against bean aphid (80% at 100 mg/kg) is significantly lower than that of NKY-312 (100% at 5 mg/kg), and uses pyridine as the solvent. In this work, we developed a highly selective (4-dimethylaminopyridine)-catalyzed monosulfonylation reaction that avoids the use of pyridine as a solvent and shows a much higher yield (98% yield with 98% HPLC purity) than the original reaction (68%). Then, we carried out the field trials and toxicity tests. In field experiments, the activities of NKY-312 against rice planthopper and wheat aphid were equal to pymetrozine and imidacloprid respectively.

. We found that the reaction of 1 and p-toluenesulfonyl chloride (2) in DCM gave the highest yield of the desired monosulfonylation product (58%, entry 1), and the NKY-312:3 ratio was > 50:1. Using DCM as the solvent, we then screened various organic and inorganic bases (entries 6-10), but no improvements were observed. Specifically, reactions with K 2 CO 3 , Na 2 CO 3 , and triethylamine as the base gave mainly the bissulfonylated by-product, the structure of which was confirmed by single crystal X-ray analysis (Fig. 4) 30 . Then we varied the reaction temperature. When both the addition of the sulfonyl chloride and the subsequent reaction were carried out at 0 °C, the solubility of the raw materials was poor, and the reaction time had to be extended to 18 h (entry 11). When both the sulfonyl chloride addition and the subsequent reaction were carried out at room temperature, the target product was obtained in 51% yield (entry 12).
Screening of amounts of dmap, pyridine, solvent, and 2. Having optimized the solvent, base, and temperature, we explored various other reaction parameters (Table 2). First, we varied the amount of DMAP (entries 1-4) and found that the highest yield was obtained with 1 mol % DMAP (entry 3). Next we screened various concentrations of 1 (entries 3 and 5-8). Increasing the concentration turned out to be beneficial: the yield of NKY-312 was highest (89%) when the concentration of 1 was 0.5 mol/L (entry 5). Then we varied the amount of sulfonyl chloride 2 (entries 5 and 9-11) and found that increasing the amount was deleterious; it is possible that the excess sulfonyl chloride increased formation of the bissulfonylated by-product, leading to a corresponding decrease in the yield of NKY-312. Finally, we screened the amount of pyridine (entries 5 and 12 -14) and found that 2.5 equiv was optimal, affording NKY-312 in 98% yield (entry 13).

Scaled-up monosulfonylation reaction.
Using the optimized conditions, we carried out a reaction of 30 g of 1 (Fig. 5) and obtained 64.8 g of NKY-312 (98% yield) with 98% HPLC purity; recrystallization from 1 L methanol afforded 60.2 g of NKY-312 (91% yield) with > 99% HPLC purity (determined by an external standard method; details are provided in Figure S3).

Control experiment.
To study the mechanism of this DMAP-catalyzed monosulfonylation reaction, some control experiments were performed. When p-toluene sulfonyl chloride reacted with DMAP (The mole ratio is 1:1) in deuterium chloroform at room temperature, 1-tosyl-4-dimethylaminopyridinium chloride can be gen-   www.nature.com/scientificreports/ erated in situ (Fig. 6a). We synthesized 1-tosyl-4-dimethylaminopyridinium chloride according to a literature procedure 31 , and we confirmed its structure by means of NMR spectroscopy (see the supporting information for details). When the synthesized salt was allowed to react with NKY-312 under the standard conditions, no bissulfonylated by-product was obtained, which indicates that the salt did not react with the monosulfonylation product ( Fig. 6b).

Proposed reaction mechanism.
On the basis of control experiments and literature precedents 31 , we propose the mechanism outlined in Fig. 7. First, p-toluene sulfonyl chloride reacted with DMAP to form 4, and second, the nucleophilic substrate 1 attacks 4 to release the product NKY-312 and generates DMAP•HCl (which is a very fast process). With the help of pyridine, DMAP•HCl reacts with p-toluene sulfonyl chloride to regener- Table 1. Screening of solvent, base, and reaction temperature a . a Reaction conditions, unless otherwise noted: p-toluenesulfonyl chloride (2, 5.25 mmol) was added dropwise over the course of 10 min to a solution of 1 (5 mmol), DMAP (10 mol %), and base (1.5 equiv) in solvent (20 mL) at 0 °C, and the reaction was then allowed to proceed for 10 h at room temperature.     Field trials. After the completion of process optimization and sample preparation, NKY-312 was employed to evaluate its insecticidal activities against rice planthopper and wheat aphid in field trials using pymetrozine and imidacloprid as controls respectively. The results exhibited that NKY-312 showed the same efficacy as the controls. Tables 3 and 4 showed part of the results of the field trials.
In summary, we have developed a process for DMAP-catalyzed monosulfonylation of 1 to obtain insecticide candidate NKY-312. This process, which afforded NKY-312 in 98% yield, with 98% HPLC purity, was highly selective for the monosulfonylation product, did not use pyridine as a solvent, and afforded a higher yield than the previously reported synthesis of this compound. In addition, the process could be used to prepare more than 60 g of NKY-312. After completing the process optimization, we conducted the field trials and toxicity tests. In field experiments, the activities of NKY-312 against rice planthopper and wheat aphid were equal to pymetrozine and imidacloprid respectively. This compound has a very good prospect in commercial development.

Methods
General. All reagents were obtained from commercial suppliers and used as received, assuming 100% purity.
Preparation of 4-amino-6-methyl-4,5-dihydro-1,2,4-triazin-3(2H)-one (1). Concentrated HCl (12 mol/L, 36.8 mL, 0.45 mol) was added dropwise to a solution of N-(6-methyl-3-oxo-2,5-dihydro-1,2,4-triazin-4(3H)-yl)acetamide (50 g, 0.30 mol) in methanol (500 mL) at room temperature, and the resulting mixture was heated at reflux until the reaction was complete, as indicated by TLC (15:1 DCM/methanol). After the reaction solution cooled to room temperature, the pH was adjusted to 7 with 50% sodium NaOH solution, and the solvent was removed by evaporation in vacuo. The residue was taken up in 125 mL of ethanol, and the solvent was again removed in vacuo. Finally, the residue was taken up in 700 mL of acetonitrile, undissolved precipitates (salts) were removed by filtration, and the filtrate was concentrated in vacuo to give 1 (35.0 g, 93%) as a light yellow solid. Mp 120-122 °C. 1  Preparation of 1-tosyl-4-dimethylaminopyridinium chloride(4). 1-Tosyl-4-dimethylaminopyridinium chloride was prepared according to a literature procedure 32 . Briefly, DMAP (767 mg, 6.29 mmol) was dissolved in dry EtOAc (40 mL), and the solution was cooled with an ice-water bath. p-Toluenesulfonyl chloride (996 mg, 5.22 mmol) in dry EtOAc (12.5 mL) was added by means of a syringe. The reaction mixture was allowed to warm to room temperature and kept at that temperature for at least 22 h. The product was filtered from the solution, washed thoroughly with diethyl ether (50 mL × 3), and dried in vacuo to afford 1.30 g (79%) of a bright white solid (mp 128-130 °C). The spectral data is consistent with the literature data.