Synthesis and antifungal activity of 2H-1,4-benzoxazin-3(4H)-one derivatives

ABSTRACT A series of 2-alkyl-2H-1,4-benzoxazin-3(4H)-ones (4a-l) was easily synthesized by two-step process involving O-alkylation of 2-nitrophenols with methyl 2-bromoalkanoates and next “green” catalytic reductive cyclization of the obtained 2-nitro ester intermediates (3a-l). Further, 6,7-dibromo (5a-c) and N-acetyl (6) derivatives were prepared by bromination and acetylation of unsubstituted 2-alkyl-2H-1,4-benzoxazin-3(4H)-ones (4a-c). The novel compounds (3a-l, 4d-l, 5a-c and 6) were fully characterized by spectroscopic methods (MS, 1H and 13C NMR). 2-Alkyl-2H-1,4-benzoxazin-3(4H)-ones (4a-l, 5a-c and 6) were screened for antifungal activity. Preliminary assays were performed using two methods: in vitro against seven phytopathogenic fungi—Botrytis cinerea, Phythophtora cactorum, Rhizoctonia solani, Phoma betae, Fusarium culmorum, Fusarium oxysporum and Alternaria alternata—and in vivo against barley powdery mildew Blumeria graminis. The tested compounds displayed moderate to good antifungal activity at high concentration (200 mg L−1). The most potent compounds were 2-ethyl-2H-1,4-benzoxazin-3(4H)-one (4a), 2-ethyl-7-fluoro-2H-1,4-benzoxazin-3(4H)-one (4g) and 4-acetyl-2-ethyl-2H-1,4-benzoxazin-3(4H)-one (6), which completely inhibited the mycelial growth of seven agricultural fungi at the concentration of 200 mg L−1 in the in vitro tests. Moreover, 2-ethyl-2H-1,4-benzoxazin-3(4H)-one (4a) and 4-acetyl-2-ethyl-2H-1,4-benzoxazin-3(4H)-one (6) were also screened for antifungal activity at concentrations of 100 mg L−1 and 20 mg L−1. In the concentration of 100 mg L−1, the N-acetyl derivative (6) completely inhibited the growth of three strains of fungi (F. culmorum, P. cactorum and R. solani), while 2-ethyl-2H-1,4-benzoxazin-3(4H)-one (4a) completely inhibited only R. solani strain. At the concentration of 20 mg L−1, compound 6 showed good activity only against P. cactorum strain (72%).


Antifungal bioassay
The fungicidal activity of the prepared compounds was tested in vitro against seven species of plant pathogenic fungi using a mycelia growth inhibition method. [40] The test was performed by applying the agar disks with the fungi mycelia taken from the edge of a good fungal culture to Petri dishes containing PDA medium mixed with the tested substances at a concentration of 200 mg L ¡1 , 100 mg L ¡1 or 20 mg L ¡1 . The target compounds were first dissolved in acetone (15% v/v relative to the medium) and then added to the molten agar after autoclaving. Control plates (without compounds) contained PDA with acetone only. After a few days, depending on the growth of mycelium in the control combination, the linear growth of each strain was measured. The fungicidal activity of the tested compounds was expressed as a percentage inhibition of mycelium compared to the control combination. Percentage inhibition was calculated as (1 -A/B) £ 100%, where A represents a colony diameter in Petri dishes with tested compounds and B is the mean colony diameter in control dishes without compounds. Each measurement consisted of at least three replicates.
In vivo test was preformed against wheat powdery mildew B. graminis f.sp. tritici, on seedlings of common wheat plants (Triticum aestivum, L.) winter cultivar Kobra in the first leaf development stage under greenhouse conditions. The plants were sprayed with acetone solution of the tested compounds at a concentration of 1,000 mg L ¡1 containing 0.0125% Tween-20 as surfactant. The inoculation with powdery mildew was performed after 2 h. Further vegetation proceeded in a controlledenvironment chamber under the following conditions: 14 h photoperiod of daylight, the air temperature 20 § 1 C/day, 16 § 1 C/night and relative humidity 80 § 5%. The efficacy for B. graminis control was assessed after six days by standard methods (visual scoring of the percentage of leaf area infected according to assessment keys presented in the EPPO Standard PP 1/26, and efficacy calculation according to the Abbot formula). [41] The commercial agricultural fungicides carbendazim, propineb, metiram and flusilazol were used for the comparison of activity.

Results and discussion
Chemistry After analysis of existing methods and considering availability of starting materials, as well as selectivity and efficiency of available processes for the synthesis of the target compounds, we decided to use a two-step method involving O-alkylation of 2-nitrophenols (1a-d) with methyl 2-bromoalkanoates (2a-c), followed by catalytic reductive cyclization of the obtained methyl 2-(2-nitrophenoxy)alkanoate intermediates (3a-l) (Scheme 1). The method starting from 2-aminophenols and 2bromo esters although seemed to be simpler, however, the pilot synthesis of 2-ethyl-2H-1,4-benzoxazin-3(4H)-one by this method led to a mixture of products, due to the presence of two possible centers of reactivity in the starting 2-aminophenol (amine and hydroxy group).
The O-alkylation process was carried out by heating the starting materials in N,N-dimethylformamide at 92-94 C in the presence of potassium bicarbonate as a base and provided the desired 2-(2-nitrophenoxy)alkanoates (3a-l) in yields of 79-93% (Table 1).

Fungicidal activity
The fungicidal activity of the tested compounds was expressed as percentage of mycelial growth inhibition of pathogenic fungi (in vitro assays) or percentage of wheat leaf area infected (in vivo assays) with respect to the control. Table 3 presents the results of both antifungal tests.
The results of antifungal activity of the synthesized 2-alkyl-2H-1,4-benzoxazin-3(4H)-ones have allowed to determine the preliminary structure-activity relationships of this type of compounds. It was observed that increasing the length of the hydrophobic alkyl chain at 2-position of 2H-1,4-benzoxazin-3(4H)one reduces the antifungal activity of the tested compounds. Therefore, further elongation of the alkyl chain is not desired. An interesting observation is that the presence of halogens, in particular bromine, is not required for good antifungal activity and 2-ethyl-2H-1,4-benzoxazin-3(4H)-one, without any halogens, was found to be more active than its derivatives possessing such substituents on the benzene ring. Among the halogen substituents, fluorine at 7 position of 2-ethyl-2H-1,4-benzoxazin-3(4H)-one and chlorine at 6,8 positions gave a positive effect. Moreover, the substitution of the hydrogen atom of NH group with acetyl group results in increased activity. Compounds 4a, 4g, 4j and 6 were chosen to further pot experiments and next to crop field trials. Further synthesis and structural optimization studies are ongoing in laboratory. None of the tested compounds showed acceptable antifungal effect in the in vivo test against wheat powdery mildew B. graminis on seedlings of winter wheat cultivar Kobra. -97

Conclusion
We have presented herein an efficient and "green" synthesis of a series of 2-alkyl-2H-1,4-benzoxazin-3(4H)-ones designed as potential antifungal agents. The results of the preliminary screening tests showed some inhibitory activity against seven different strains of phytopathogenic fungi and allowed us to select the most potent derivatives representing a model structure for further modifications to optimize the antifungal activity. It seems to be advisable to focus on 1,4-benzoxazin-3(4H)-ones that are analogs of allelochemicals and therefore they may be safe to non-target organisms. Syntheses of new compounds, their toxicological studies and the determination of detailed structure-activity relationships are the subjects of our further studies in this area.

Funding
This work was co-financed by the European Union within the European Social Fund and the State Budget, Human Capital Operational Program, Priority VIII, Measure 8.2. Transfer of knowledge, Sub-measure 8.2.2. Regional Innovation Strategies, the system project implemented by the Voivodeship Labour Office in Szczecin "An investment in knowledge driven innovation in the region -II edition." Table 3. Antifungal activity of 2-alkyl-2H-1,4-benzoxazin-3(4H)-ones against various phytopathogenic fungi.