The methodology for the synthesis of the title compounds 5a–5s was presented in Scheme 1. The title compound 5 was obtained by the cyclization, substitution, hydrazinolysis and condensation reactions with 36%–53% yields over four steps. The structures of the title compound 5 were confirmed by ¹H NMR, ¹³C NMR, ¹⁹F NMR and HRMS, and all analytical data were consistent with the assigned structures. The Supplementary Material contain the ¹H NMR, ¹³C NMR, ¹⁹F NMR and HRMS spectra of the target compound 5 (Supplementary Figures S1–S66).

o-Amino phenol (3 g, 30 mmol), benzyl triethyl ammonium chloride (TEBA, 6.27 g, 30 mmol) and dichloromethane (20 ml) was added to a 100 ml reaction flask equipped with a magnetic stir bar. After the mixture was stirred for 10 min, sodium hydrogen carbonate (9.18 g, 108 mmol) was added step into mixture. The resultant mixture was allowed to stir at 0°C, and chloroacetyl chloride (3.66 g, 32.4 mmol) was added via syringe over a period of 15 min. Then, the reactant mixture was refluxed at 40°C for 6–8 h. After the reactants were consumed (TLC analysis), the resulting crude residue was poured into ice water (20 ml). The crude products were collected by filtration, washed with water, and recrystallized from methanol to obtain intermediate 2 (Benarjee et al., 2022).

The intermediate 2 (1.49 g, 10 mmol) and K₂CO₃ (1.38 g, 10 mmol) were added into a 100 ml reaction flask with 20 ml dimethylformamide (DMF). Then, ethyl bromoacetate (1.84 g, 11 mmol) was dropwise added via syringe and the reaction mixture was allowed to stir at rt for 12 h. After the reactants were consumed (TLC analysis), the resulting crude residue was extracted with ethyl acetate (3 × 20 ml). The organic layer was washed with water, dried over anhydrous Na₂SO₄, concentrated under reduced pressure to obtain intermediate 3 (Safakish et al., 2020).

Intermediate 3 was dissolved in 10 ml ethanol and heated at around 60°C about 10 min. Then, hydrazine hydrate (4 equiv.) was dropwise added via syringe to this mixture. The reaction mixture was refluxed for 6–8 h, and then allowed to pour into ice water. The crude residue was filtered to yield the intermediate 4 (Zhang et al., 2022).

Intermediate 4 was dissolved in 10 ml ethanol, and the solution was gently heated at around 60°C. Then, corresponding substituent aldehyde (1 equiv.) in 5 ml ethanol was dropwise added via syringe into this solution. The reaction mixture was refluxed for 2–4 h, and allowed to cool to room temperature. The crude residue was filtered and recrystallized from toluene to get the title compound 5.

The nuclear magnetic resonance (NMR) spectroscopy is a practical strategy to identify the cis-trans isomers in acylhydrazones (Lopes et al., 2013). The analysis of the ¹H NMR spectra displayed that title compounds 5a–5s exist as Z/E isomers, and the ratio of Z/E isomers can be confirmed via ¹H NMR based on the previously reported literature method (Palla, 1986). The chemical shift of ¹H NMR spectrum of the title compounds 5a–5s show slightly narrow singlets for N–H group at 11.26–11.84 ppm. The characteristic singlets of imine hydrogens appear between 7.38 and 8.46 ppm with the cis-trans isomers, which control the Z/E isomers ratio of the title compound 5 synthesized from our approach is 3:1 according to previously reported literatures (Munir et al., 2021). The methylene moiety of 1,4-benzoxazin-3-one ring appeared at around 4.66 and 5.05 ppm also presents 3:1 Z/E isomers ratio, which is consistent with the Z/E isomers ratio of imine hydrogens. ¹³C NMR exhibited peaks at around 165.0 and 168.0 ppm for carbonyl groups of N-acylhydrazone and 1,4-benzoxazin-3-one ring portion. The signal of imine carbon is meant to appear at 142.8–145.1 ppm. For example, the signal of imine hydrogens of the title compound 5a appears at 8.06 and 8.24 ppm, with the Z/E isomers ratio of 3:1. The chemical shift of methylene moiety of 1,4-benzoxazin-3-one ring appeared at 5.08 and 4.67 ppm, also with the Z/E isomers ratio of 3:1. Finally, the ESI-HRMS spectrum revealed an obvious signal at 332.1006, which was assigned to the [M + Na]⁺ species of the target compound 5a.

All synthesized 1,4-benzoxazin-3-one derivatives in this work were screened for in vitro fungicidal activities against five types of plant pathogenic fungi including G. zeae, P. sasakii, P. infestans, C. wilt and P. capsica at the concentration of 50 μg/ml through the classical mycelium growth rate method according to the previously established procedure (see Supplementary Materials for full procedure details). Commercial agrochemicals hymexazol and carbendazim were used as positive fungicides against fungal strains. The obtained results are depicted in Table 1. The results of preliminary screening indicated that some of synthesized 1,4-benzoxazin-3-one derivatives also had moderate to good activities to inhibit the growth of G. zeae, P. sasakii, P. infestans and C. wilt fungal strains, but almost all the title compounds failed to exhibit effective inhibition effects against P. capsica in this work. Firstly, for G. zeae, compounds 5L and 5o exhibited excellent bioactive with the inhibitory rates 76.37% and 76.14%, respectively, which were superior to hymexazol (49.47%). Secondly, compounds 5L and 5q displayed stronger inhibitory activities against P. sasakii, with the inhibition rates of 64.38% and 73.32%, respectively, than that of hymexazol (60.70%). Meanwhile, compound 5r exhibited the excellent inhibitory activity against P. infestans, with the inhibition rate of 82.62%, which was better than hymexazol (72.86%) and carbendazim (58.57%). In addition, compound 5p had 71.33% inhibition rate against C. wilt, which was better than hymexazol (49.88%).

To further validate the antifungal activities of the title compounds, the EC₅₀ values of some title compounds against G. zeae, P. sasakii, P. infestans, and C. wilt were tested and the result data were demonstrated in Table 2. Our results indicated that compounds 5L and 5o exhibited commendable in vitro antifungal activities against G. zeae, with the EC₅₀ values of 20.06, and 23.17 μg/ml, respectively, which are nearly double effective than that of hymexazol (40.51 μg/ml). Meanwhile, compound 5q displayed outstanding inhibitory activity against P. sasakii, with the EC₅₀ value of 26.66 μg/ml, respectively, which is better than hymexazol (32.77 μg/ml). Furthermore, compound 5r also had excellent inhibitory activity against P. infestans, with EC₅₀ value of 15.37 μg/ml, which is double more efficient compared to that of carbendazim (34.41 μg/ml). Then, the EC₅₀ values of compounds 5b and 5p against to C. wilt were calculated, just the EC₅₀ of 5p (26.76 μg/ml) was slightly close to that of carbendazim (26.08 μg/ml).

For compounds 5a-5g (no substituents attached on 1,4-benzoxazin-3-one skeleton), only compound 5e showed better activity against P. infestans, with 3-Br attached aryl of acylhydrazone, the introduction of other substituents, including 4-F-Ph, 2-F-Ph, 3-F-Ph, 4-Br-Ph and 2-furan could not obviously affect the antifungal activities. Then, some of target compounds showed better activity against fungal strains, when introduction of methyl attached on 1,4-benzoxazin-3-one skeleton (for compounds 5h-5n), for example, compound 5L (3-Br attached aryl of acylhydrazone) exhibited bioactive with the inhibitory rates 76.37% and 64.38% against G. zeae and P. sasakii, respectively. However, target compounds (5o – 5t), with 6-Cl attached on 1,4-benzoxazin-3-one skeleton, exhibited better antifungal activity than other substituents attached on 1,4-benzoxazin-3-one skeleton, such as compound 5o, 5p, 5q and 5s were exhibited excellent bioactive with the inhibitory rates 76.14%, 71.33%, 73.32% and 82.62% against G. zeae, C. wilt, P. sasakii and P. infestans, with EC₅₀ value of 23.17, 26.76, 26.66 and 15.37 μg/ml, respectively. Especially, compound 5s had the best activity against P. infestans fungal strain. The replacement of the benzene ring (5o) to alkyl such as ethyl (5t) group could not improve the antifungal activity.