Synthesis and Biological Activities of Some Novel ( E )-Alpha-( methoxyimino ) benzeneacetate Derivatives with Modified 1 , 2 , 4-Triazole Moiety

To find new strobilurin analogues with high activity against resistant pathogens, a series of (E)-α-(methoxyimino)benzeneacetate derivatives containing 1,2,4-triazole Schiff base side chainwere designed and synthesized.Their structures were confirmed by IR, H NMR, and C NMR, ESI-HRMS, or elemental analyses. Bioassays indicated that most of the target compounds showed moderate to good fungicidal activities against Rhizoctonia solani, Botrytis cinerea Pers., Fusarium graminearum, Cotton rhizoctoniosis, and Blumeria graminis. For example, compounds 6g and 6j exhibited promising antifungal activity against Rhizoctonia solani, Botrytis cinerea Pers., and Fusarium graminearum. Compounds 6c, 6l, and 6m had higher fungicidal activities against Blumeria graminis at the concentration of 50 μg/mL; inhibitory rate is 91.41%, 92.13%, and 91.77%, respectively.


Introduction
The strobilurins, derived from fermentations of Strobilurus tenacellus by Anke and coworkers in 1977, are one of the most important classes of agricultural fungicides [1].Their primary mechanism of action is the inhibition of mitochondrial respiration by blocking electron transfer at the ubiquinol oxidation center (Q o site) of the cytochrome  1 complex (complex III) [2].
Strobilurin derivatives have attracted significant attention of the agricultural chemists owing to their outstanding characteristics and unique mode of action, broader antifungal spectrum, long-lasting effects, high antifungal activity, and low toxicity toward mammalian cells [3][4][5][6].The strobilurins were first commercialized in 1996 with the launch of azoxystrobin and kresoxim-methyl (Figure 1) [7].Till date, over ten strobilurin derivatives are commercially available [8][9][10].However, following the use of strobilurin fungicides in a short period of field applications, significant increase in resistance to fungicide has been observed [11].
Recently, significant research efforts focusing on structural modification of strobilurins have been devoted to overcoming the above-mentioned problem.Moreover, according to the literature, the methoxyiminoacetate is an effective pharmacophore which is indispensable for antifungal activity of strobilurin fungicides.The aromatic bridge helps to stabilize the molecule and the molecule also exhibits photo stability.Therefore, numerous studies have reported that modification of the side chain is the most effective method to obtain novel strobilurin derivatives with higher biological activities [6,[12][13][14].

Experimental
2.1.General Information.All melting points were determined on an XT-4A apparatus and are uncorrected.The IR spectra (KBr disks) were taken on a Bruker Equinox 55 spectrophotometer.The 1 H NMR spectra were measured on a Bruker Advance 600 spectrometer for DMSO-d6 solutions using TMS as internal standard.Elemental analyses were determined on a Flash-1112 series elemental analyzer.All the reagents used were AR grade.Molecular weights of monomers were determined by high resolution mass spectroscopy (ESI-HRMS, Bruker daltonics apexultra 7.0 tesla Fourier transform ion cyclotron resonance mass spectrometer).The completion of reactions was monitored by TLC.

General
Procedure for the Synthesis of Benzohydrazide (2) [23].A mixture of ethyl benzoate 1 (0.1 mol) and hydrazine hydrate (0.1 mol) in ethanol (30 mL) was stirred vigorously for 6 h at room temperature.The mixture was filtered, and the solid was washed with cold water, dried, and recrystallized from ethanol to give intermediate 2, white crystal, yield: 90.6%, m.p.: 112-114 ∘ C. [24].Potassium hydroxide (0.15 mol) was dissolved in absolute ethanol (100 mL) followed by the addition of benzohydrazide 2 (0.1 mol).The resulting solution was cooled in ice.Subsequently, carbon disulfide (0.15 mol) was added dropwise, and the reaction mixture was stirred for 15 h at room temperature.The precipitated potassium dithiocarbazinate was collected by filtration.The precipitate was further washed with anhydrous ether (100 mL), dried, and used directly without purification for the subsequent reaction.

General Method for the Synthesis of Potassium Dithiocarbazinate (3)
2.4.Synthesis of 4-Amino-3-phenyl-5-thiol-1,2,4-triazole (4) [25].Potassium dithiocarbazinate 3 (0.5 mol) was added to hydrazine hydrate (0.15 mmol) and refluxed for 6 h with occasional shaking.The color of the reaction mixture changed to green with the evolution of hydrogen sulfide gas (lead acetate paper test and odor).The reaction mixture was cooled to room temperature and diluted with water.On acidification with concentrated hydrochloric acid, the corresponding triazole was precipitated.It was filtered, washed thoroughly with cold water, and recrystallized from ethanol to give 4-amino-       the presence of absolute ethanol and potassium hydroxide to afford intermediate potassium acylhydrazine dithioformate 3.This salt underwent ring closure with an excess of 85% hydrazine hydrate to give intermediate 4, which was used directly without further purification for the subsequent reaction.Subsequently, compound 4 was allowed to react with appropriate aromatic aldehydes and 2 to 3 drops of glacial acetic acid to produce series of compounds 5 in moderate yield according to the method reported in the literature [18].Finally, a series of the target compounds 6 were obtained by the reaction of corresponding compounds 5 with (E)-methyl-2-(2-(bromomethyl)phenyl)-2-(methoxyimino)acetate in the presence of base following the literature method [26].The structures of the desired compounds were confirmed by 1 H NMR, 13 C NMR, IR, and ESI-HRMS.

Biological Evaluation.
The fungicidal activities of the series of title compounds 6 were tested at a concentration of 50 g mL −1 by a modified method described in the literature [27].The five fungi used in the fungicidal bioassay, Rhizoctonia solani, Botrytis cinerea Pers., Fusarium graminearum, Cotton rhizoctoniosis, and Blumeria graminis, were tested by mycelium growth rate method.The commercial agricultural fungicide kresoxim-methyl was used as a standard.The results of preliminary bioassays are listed in Table 1.
Interestingly, the fungicidal activities of the synthesized compounds 6 were influenced by the position of substituted group on the benzene ring.The sequence of fungicidal activity against Rhizoctonia solani, Botrytis cinerea Pers., and Fusarium graminearum was as follows: o-substitutedbenzylidene derivatives > m-substituted-benzylidene derivatives > p-substituted-benzylidene derivatives.Surprisingly, for the p-substituted benzylidene, the fungicidal activity against Blumeria graminis was significantly enhanced.

Conclusion
A series of novel (E)--methoxyimino-benzeneacetate derivatives were synthesized.They were characterized by IR, 1 H NMR, 13 C NMR, and ESI-HRMS.All the synthesized compounds were screened for their antifungal activity by mycelium growth rate method.The antifungal tests indicated that compounds 6g and 6j exhibited promising antifungal activity against Rhizoctonia solani, Botrytis cinerea Pers., and Fusarium graminearum.Moreover, compounds 6c, 6l, and 6m exhibited higher fungicidal activities against Blumeria graminis.Therefore, this study demonstrated that methoxyiminoacetate derivatives containing 1,2,4-triazole Schiff base side chain acted as promising candidates for developing novel fungicides.This study provides an impetus to the further exploration of antifungal compounds.Further research involving design, synthesis, and structure optimizations is still in progress.
3.1.Synthetic Chemistry.Scheme 1 shows the schematic representation of the synthetic route for the preparation of target compounds.Compound 2 was prepared by the reaction between ethyl benzoate 1 and 85% hydrazine hydrate in ethanol at room temperature in excellent yield (90.6%).Further, compound 2 was heated with carbon disulfide in