Synthesis and Fungicidal Activity of 1-(Carbamoylmethyl)-2-aryl-3,1-benzoxazines

A series of new 1-(carbamoylmethyl)-2-aryl-3,1-benzoxazines were prepared in moderate to good yields by BF3·OEt2-catalyzed reactions of aromatic aldehydes with 2-(N-substituted carbamoylmethylamino)benzyl alcohols. The structures of the target compounds were confirmed by IR, 1H-NMR, 13C-NMR, and elemental analyses. The fungicidal activities of the target compounds against plant fungi were preliminarily evaluated, and some of them exhibited good activity.


Chemistry
The synthetic route to the title compounds 5a-r is shown in Scheme 1. The key intermediate 2-(N-substituted carbamoylmethylamino)benzyl alcohols 3 (also named as: N-substituted 2-(2-(hydroxymethyl)phenylamino)acetamide) were prepared by selective N-alkylation of 2-aminobenzyl alcohol with N-substituted 2-bromoacetamide 2. The reaction exclusively occurred at the nitrogen atom giving products 3a-f ( 1 H-NMR and 13 C-NMR Data in Supplementary materials) in 65-73% yields when the reaction was carried out in a mixture solvent of N,N-dimethyl formamide (DMF) and tetrahydrofuran (THF) (v/v = 1:2) with potassium carbonate as base. Then, we began to synthesize the target products 5a-r ( 1 H-NMR and 13 C-NMR Data in Supplementary materials). The preparation of 5a was selected as model to optimize the reaction conditions. Firstly, reaction of 2-(N-(2-methylphenyl)carbamoylmethylamino)benzyl alcohol 3a with 3-nitrobenzaldehyde 4a in the presence of BF 3 ·OEt 2 (10% mol) in THF under 65 • C gave the desired product 5a in 35 yields (No. 1, Table 1). By optimizing the conditions, the yield was improved to 55% (No. 6). Under the same conditions, compounds 5b-r were synthesized in 40-85% yields. As shown in Table 1, for compounds with an amide nitrogen connected to the benzyl group, a higher yield was achieved than for those with a phenyl group (No. [16][17][18] Table 1), and moreover, the reaction yields of the former depended on the position of the nitro group on the benzene ring in order of para > ortho > meta. But, the yields for those with an amide nitrogen connected with an aryl group presented the order of meta > para > ortho.

Chemistry
The synthetic route to the title compounds 5a-r is shown in Scheme 1. The key intermediate 2-(N-substituted carbamoylmethylamino)benzyl alcohols 3 (also named as: N-substituted 2-(2-(hydroxymethyl)phenylamino)acetamide) were prepared by selective N-alkylation of 2-aminobenzyl alcohol with N-substituted 2-bromoacetamide 2. The reaction exclusively occurred at the nitrogen atom giving products 3a-f ( 1 H-NMR and 13 C-NMR Data in Supplementary materials) in 65-73% yields when the reaction was carried out in a mixture solvent of N,N-dimethyl formamide (DMF) and tetrahydrofuran (THF) (v/v = 1:2) with potassium carbonate as base. Then, we began to synthesize the target products 5a-r ( 1 H-NMR and 13 C-NMR Data in Supplementary materials). The preparation of 5a was selected as model to optimize the reaction conditions. Firstly, reaction of 2-(N-(2-methylphenyl) carbamoylmethylamino)benzyl alcohol 3a with 3-nitrobenzaldehyde 4a in the presence of BF3·OEt2 (10% mol) in THF under 65 °C gave the desired product 5a in 35 yields (No. 1, Table 1). By optimizing the conditions, the yield was improved to 55% (No. 6). Under the same conditions, compounds 5b-r were synthesized in 40-85% yields. As shown in Table 1, for compounds with an amide nitrogen connected to the benzyl group, a higher yield was achieved than for those with a phenyl group (No. [16][17][18] Table 1), and moreover, the reaction yields of the former depended on the position of the nitro group on the benzene ring in order of para > ortho > meta. But, the yields for those with an amide nitrogen connected with an aryl group presented the order of meta > para > ortho.  The structures of the products were established on the basis of their spectroscopic data (IR, 1 H-NMR, 13 C-NMR) and elemental analysis. All compounds exhibit characteristic signals appropriately (see experimental section). This can be illustrated with compound 5a. In IR spectra, strong absorption at 1661 cm −1 corresponds to the stretching vibration of the C=O group; and absorption at 1586 and1536 cm −1 to the C=C bond. A singlet at 5.88 ppm observed in 1 H-NMR spectra corresponds to OCHN proton of the benzoxazine ring. Particularly, the OCH 2 protons within the benzoxazine ring absorb as two doublets at 4.74 and 4.97 ppm instead of singlet. These are characteristic signals indicating the formation of the benzoxazine ring [28]. Interestingly, the NCH 2 CO protons also absorb as two doublets at 3.88 and 4.13 ppm. A singlet at 2.06 ppm corresponds to CH 3 protons. In 13 C-NMR, a signal at 167.30 ppm corresponds to the C=O carbon, and signals at 88.59 and 64.74 ppm stand for the OCHN and OCH 2 carbons, respectively. CH 3 carbon absorbs at 17.37 ppm.

Fungicidal Activity Assay
According to standard method NY/T1156. 5-2006 [29], the in vitro fungicidal activities of the prepared compounds 5a-r were evaluated, adopting the mycelium growth rate test method. The fungicidal activity was tested against Sclerotonia sclerotiorum, Botrytis cinerea, Rhizoctonia solani, Gibberella zeae and Phytophythora capsic at 25 µg/mL, and against Magnaporthe oryzae at 50 µg/mL. The activities were expressed as inhibition rate (%), and the results are summarized in Table 2. All compounds exhibited certain activities against the tested fungi, and some showed good activities. Compounds 5d and 5g showed 60.1% and 54.5% activity against M. oryzae, respectively, which are all higher than that of chlorothalonil (53.8%). The activity of compound 5o (51.7%) is closed to that of chlorothalonil. Compound 5i showed 71.9% activity against S. sclerotiorum at 25 µg/mL, which is close to that of chlorothalonil (84.9%). In addition, the compounds with the amide nitrogen atom connected with an aryl group were more active than those with a benzyl group. For S. sclerotiorum and R. solani, when the substituent at the 4-position of the benzene ring was connected with the amide nitrogen, the compounds with a NO 2 group at the 4-position of the benzene ring exhibited higher activities than those with a NO 2 group at 3-or 2-position.

Materials and Reagents
All solvents were dried by standard procedure. Aromatic aldehydes, 2-aminobenzyl alcohol, and substituted anilines were commercially available. Infrared spectra were recorded on a Nicolet-6700 FT-IR. 1 H-and 13 C-NMR spectra were recorded on Bruker Avance-500 MHz spectrometer. Elemental analysis was measured on PE 2400 II CHNS instrument. Melting points were determined on a WRS-1B digital melting point instrument and uncorrected.

Fungicidal Activity Testing
The in vitro inhibition of mycelium in the agar culture medium caused by the title compounds against six phytopathogenic fungi: Sclerotonia sclerotiorum, Botrytis cinerea, Rhizoctonia solani, Gibberella zeae, Phytophythora capsic and Magnaporthe oryzae was performed. Referring to the standard method NY/T1156.5-2006, antifungal activity assays adopted the mycelium growth rate test method. Chlorothalonil was used as a reference compound. A stock solution of every test compound was prepared in acetone and then diluted to the required test concentrations (500 µg/mL) with sorporl-144 (concentration: 200 µg/mL). Solutions of the test compounds (1 mL) were added to potato dextrose agar (PDA) medium (9 mL, 45 • C) to provide the final concentration of 25 µg/mL, but 50 µg/mL for Magnaporthe oryzae. The mixed medium without sample was used as the blank control. The inocula, 4 mm in diameter, were removed from the margins of actively growing colonies of mycelium and placed in the centers of the above plates. 4 replicates were performed per treatment. Percentages of growth inhibition were calculated by comparing the mean value of the diameters of the mycelia in the test plates after placement in a 24 • C biochemical incubator thermostat for 3 days. The inhibition percent was calculated according to the following equation: where I is the inhibition rate, D 1 is the average diameter of mycelia the blank test, and D 0 is the average diameter of mycelia in the presence of compounds. The results are given in Table 2.

Conclusions
In summary, we have prepared a series of novel 1-(carbamoylmethyl)-2-aryl-3,1-benzoxazines by aza-acetalizations of 2-(N-substituted carbamoylmethylamino)benzyl alcohols with aromatic aldehydes in the presence of BF 3 ·OEt 2 . The fungicidal activities of the prepared compounds against plant fungi were preliminarily evaluated, and some compounds showed good activities. Compounds 5d and 5g showed higher activity against M. Oryzae than chlorothalonil. The activity of compound 5i against S. Sclerotiorum is close to that of chlorothalonil.