An N-ester salicylamide compound, its preparation method and uses

Abstract

This invention provides N-ester salicylamide compounds and N-ester-1,3-benzoxazine-2,4-dione compounds, their preparation methods, and uses. These compounds are prepared by reacting amino acids and alcohols as raw materials under the action of thionyl chloride to generate amino acid ester hydrochloride, which is then reacted with salicylic acid under the action of oxalyl chloride to obtain N-ester salicylamide. Further, the N-ester salicylamide is reacted with triphosgene to obtain N-ester-1,3-benzoxazine-2,4-dione compounds. The raw materials used in the synthesis of these compounds are inexpensive and readily available, and the synthesis method is simple. Simultaneously, these compounds exhibit good inhibitory activity against crop pathogens and antitumor activity, particularly showing significant inhibitory activity against Fusarium head blight, rice blast fungus, Phytophthora blight, Sclerotinia sclerotinia, Botrytis cinerea, and Sheath blight, and significant activity against HCCLM3 liver cancer cells.

Description

Technical Field

[0001] This invention relates to a pharmaceutical compound, specifically to an N-ester salicylamide compound with bactericidal activity, an N-ester-1,3-benzoxazine-2,4-dione compound, and their preparation methods and uses, belonging to the field of pharmaceutical technology. Background Technology

[0002] Salicylamide compounds are a hot topic in pharmaceutical and pesticide research due to their broad bioactivity and wide application in medicine, pesticides, fine chemicals, and military fields. Salicylamide compounds possess excellent bioactivity and can be used as pathogen inhibitors, fungicides, herbicides, and plant growth regulators. In 1998, Macielag M J et al. reported the screening of chlorosalicylate and tetrachlorosalicylate based on salicylanilides, finding their antibacterial activity against methicillin-resistant microorganisms, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus faecalis (VREF). In 2013, Pauk K et al. synthesized and characterized three series of salicylanilides and 2-hydroxy-N-[1-(2-hydroxyaniline)-1-oxoalkyl-2-yl]benzamide compounds. The compounds were tested for antibacterial activity against seven bacteria and three mycobacteria. Some compounds showed antibacterial activity comparable to or higher than standard ampicillin, ciprofloxacin, or isoniazid, and exhibited high bioactivity against Staphylococcus aureus (60.03 lmol / L), Mycobacterium marineum (60.40 lmol / L), Bacillus cereus (0.09 lmol / L), and Mycobacterium kansaiense (1.58 lmol / L). In 2015, Sulzer-Mosse S et al. discovered that N-thiazol-4-acylsalicylic acid amides exhibited high activity against important plant pathogens such as late blight of potato and tomato, downy mildew of grapes, and yellow mold. In 2021, Krátky et al. designed and synthesized propargylamine-substituted salicylamide compounds, finding that they inhibited cholinesterase, with the highest selectivity and activity against BuChE (butyrylcholinesterase). In 2023, Kaur et al. designed and synthesized acyl salicylamide compounds containing urea structural units. Activity tests showed that they had inhibitory activity against cyclooxygenase (COX-2) and had the potential to become nonsteroidal anti-inflammatory drugs.

[0003] However, to our knowledge, ester-substituted salicylamide compounds are not commonly found in previously reported literature. Therefore, we designed ester-substituted salicylamide compound IV and investigated the inhibitory activity of these compounds against crop pathogens and tumor cells.

[0004] Benzooxazine diones possess antifungal, antibacterial, antituberculosis, and anti-inflammatory activities, and can also be used to treat osteoclasts and as potential antipsychotic drugs. In 1999, Waisser et al. reported a series of 1,3-benzoxazine-2,4(3H)-diones, and activity tests showed that they had significant inhibitory activity against tuberculous (non-tuberculous) mycobacteria (MIC in the range of 4-250 μmol / L). Furthermore, they also showed some antibacterial activity against Mycobacterium kansas and Mycobacterium avium. In 2008, Kowalski et al. synthesized a class of 1,3-benzoxazine diones and investigated their affinity for serotonin receptors. Test results showed that these compounds had binding affinity for 5-HT1A, 5-HT2A, and 5-HT7 receptors. In 2016, Sun et al. designed and synthesized a series of urethane-substituted 3-benzyl-1,3-benzoxazine-2,4-diones. Cellular experiments showed that the compounds effectively inhibited the expression of ERK1 / 2 pathway and EV71 VP1, and the EV71 (Viagra 71)-induced rhabdomyosarcoma cell pathogenesis effect. In 2021, Lawal's group reported fluorine- and chlorine-containing 1,3-benzoxazine dione compounds, and bioactivity tests showed that they had broad-spectrum anti-proliferative activity and selective cytotoxicity against drug-sensitive and multidrug-resistant melanoma, kidney, central nervous system, and colon cancer. In 2022, Hammouda et al. reported a class of triazolyl-substituted 1,3-benzoxazine dione compounds, finding that the compounds had superior antibacterial activity against Staphylococcus aureus, Pseudomonas lutea, and Pseudomonas aeruginosa compared to the control drugs tetracycline and ampicillin; their activity against Escherichia coli was superior to the commercial antibiotic tetracycline; and their activity against Candida albicans and Candida cluximab was superior to the control drug ampicillin.

[0005] However, the overall antibacterial and antitumor effects of these compounds on crops are still insufficient (relatively low), and there is still much room for improvement. Furthermore, research has revealed that studies on ester-substituted 1,3-benzoxazine dione compounds and their antibacterial activities are rare in previous literature. Therefore, we designed and synthesized N-ester-1,3-benzoxazine-2,4-dione compounds containing both ester and benzoxazine dione structures, using N-ester salicylamide compounds as raw materials. We then investigated the inhibitory activities of these compounds against Fusarium head blight, Phytophthora blight, rice blast fungus, Sclerotinia sclerotinia, Botrytis cinerea, and Sheath blight fungus, as well as their inhibitory activity against HCCLM3 liver cancer cells. Summary of the Invention

[0006] This invention addresses the excellent bioactivity of ester-substituted salicylamides and benzoxazine-2,4-diones, as well as their preparation methods and uses, for antibacterial purposes in crops. These compounds are simple to prepare, use readily available raw materials, and exhibit excellent antibacterial activity against crop pathogens, particularly against Fusarium head blight, Phytophthora blight, rice blast fungus, sclerotinia sclerotinia, gray mold, and sheath blight, thus significantly improving crop yield. Furthermore, these compounds also demonstrate good inhibitory activity against liver cancer cells.

[0007] According to a first embodiment of the present invention, an N-ester salicylamide compound is provided.

[0008] An N-ester salicylamide compound having the general structural formula (IV),

[0009]

[0010] In formula (IV), R1 is a C1-C6 alkyl, benzyl, or substituted benzyl group; R is one of H, C1-C6 alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, methylthioethyl, or substituted methylthioethyl.

[0011] Preferably, R1 is a C1-C3 alkyl or benzyl group; R is one of H, C1-C4 alkyl, phenyl, chlorophenyl, benzyl, hydroxybenzyl, or methylthioethyl.

[0012] Preferably, R1 is CH3, CH2CH3, or benzyl; R is one of H, C1-C4 alkyl, phenyl, 3-chlorophenyl, 4-chlorophenyl, 4-hydroxybenzyl, or methylthioethyl.

[0013] Preferably, the compound having the general structural formula (IV) is specifically selected from one or more of the following compounds:

[0014] N-(2-ethyl phenylacetyl)salicylamide:

[0015]

[0016] N-(2-(3-chlorophenyl)methyl acetate)salicylic acid amide:

[0017]

[0018] N-(2-(3-chlorophenyl)ethyl acetate)salicylic acid amide:

[0019]

[0020] N-(2-(4-chlorophenyl)methyl acetate)salicylic acid amide:

[0021]

[0022] N-(2-(4-chlorophenyl)ethyl acetate)salicylic acid amide:

[0023]

[0024] N-(3-(4-hydroxyphenyl)propionate methyl)salicylic acid amide:

[0025]

[0026] N-((4-methylthio)butyrate methyl ester) salicylamide

[0027]

[0028] N-(benzyl acetate) salicylamide:

[0029]

[0030] According to a second embodiment of the present invention, a method for preparing N-ester salicylamide compounds is provided.

[0031] A method for preparing N-ester salicylamide compounds having the general structural formula (IV), or a method for preparing N-ester salicylamide compounds having the general structural formula (IV) as described in the first embodiment: the method specifically includes the following steps:

[0032] S1) An amino acid with structural formula (I) is reacted with an alcohol with structural formula (II) in the presence of thionyl chloride to obtain an amino acid ester hydrochloride with structural formula (III):

[0033]

[0034] S2) Salicylic acid is reacted with oxaloyl chloride to obtain salicyl chloride, which is then reacted with an amino acid ester hydrochloride having structural formula (III) to obtain an N-ester salicylamide compound having general structural formula (IV):

[0035]

[0036] In the formula, R1 is a C1-C6 alkyl, benzyl, or substituted benzyl group; R is one of H, C1-C6 alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, methylthioethyl, or substituted methylthio.

[0037] Preferably, R1 is a C1-C3 alkyl or benzyl group; R is one of H, C1-C4 alkyl, phenyl, chlorophenyl, benzyl, hydroxybenzyl, or methylthioethyl.

[0038] Preferably, the amino acid is glycine, phenylglycine, chlorophenylglycine, tyrosine, leucine, or methionine.

[0039] Preferably, the alcohol is methanol, ethanol or benzyl alcohol.

[0040] Preferably, step S1) specifically involves dissolving an amino acid having general structural formula (I) in an alcohol having general structural formula (II), adding thionyl chloride, and heating the reaction solution to react and obtain an amino acid ester hydrochloride having structural formula (III).

[0041] In this invention, in step S1), the molar ratio of the amino acid having general structural formula (I) to the alcohol having general structural formula (II) added to the reaction is 1:1-10, preferably 1:2-8, for example 1:5. The molar ratio of the amino acid having general structural formula (I) to thionyl chloride added to the reaction is 1:1-10, preferably 1:2-5, for example 1:3. The reaction temperature is 20 to 80°C, preferably 30 to 60°C. The reaction time is 2-24 h, preferably 3-12 h.

[0042] Preferably, step S2) specifically involves: dissolving salicylic acid in a solvent, adding oxaloyl chloride, optionally adding or not adding N,N-dimethylformamide (DMF), stirring the reaction, and obtaining an acyl chloride mixture. An amino acid ester hydrochloride having structural formula (III) is dissolved in an ester solution with K2CO3 to obtain a mixed solution containing the amino acid ester hydrochloride. The acyl chloride mixture is added to the mixed solution containing the amino acid ester hydrochloride, reacted, and separated to obtain an N-ester salicylamide compound having general structural formula (IV).

[0043] In this invention, the acyl chloride mixture is prepared in-house using a self-synthesized salicyl chloride solution. The salicyl chloride is reacted with an amino acid ester hydrochloride having structural formula (III) to obtain an N-ester salicylamide compound having general structural formula (IV).

[0044] In this invention, the addition of N,N-dimethylformamide serves to increase solvent polarity and promote the reaction.

[0045] In this invention, the purpose of adding alkali in step S2) is twofold: first, to remove hydrochloric acid from the carbamate hydrochloride; and second, to neutralize the hydrochloric acid generated by the reaction of acyl chloride and amino acid ester, thereby promoting the reaction.

[0046] Preferably, the solvent is an organic solvent, preferably dichloromethane (DCM). The mass concentration of salicylic acid solvent in the solvent is 0.2-10%, preferably 0.3-8%, and more preferably 0.5-5%.

[0047] Preferably, the molar ratio of oxaloyl chloride to salicylic acid is 0.2-10:1, more preferably 0.5-8:1, and even more preferably 1-5:1.

[0048] Preferably, the volume ratio of N,N-dimethylformamide (DMF) to salicylic acid is 1:0.02-0.5, and more preferably 1:0.03-0.4.

[0049] Preferably, in the mixed solution containing amino acid ester hydrochloride, the molar ratio of amino acid ester hydrochloride to K2CO3 is 1:0.1-3, more preferably 1:0.2-2, and even more preferably 1:0.3-1.

[0050] Preferably, the ester solution is an ethyl acetate solution, preferably a mixture of ethyl acetate and water in a volume ratio of 0.5-10:1, and more preferably a mixture of ethyl acetate and water in a volume ratio of 1-5:1.

[0051] Preferably, the acyl chloride mixture is added to the mixed solution containing amino acid ester hydrochloride, such that the molar ratio of amino acid ester hydrochloride to salicylic acid is 1:0.2-5, preferably 1:0.5-3, for example 2:1.

[0052] In this invention, the reaction temperature for obtaining the acyl chloride mixture is 10-50°C, preferably 15-40°C, more preferably 20-35°C; for example, room temperature. The reaction time is 1-48 h, preferably 2-24 h, more preferably 3-12 h.

[0053] In this invention, before adding the acyl chloride mixture to the mixed solution containing amino acid ester hydrochloride, the amino acid ester hydrochloride is first cooled to below zero degrees Celsius. The reaction temperature between the acyl chloride mixture and the mixed solution containing amino acid ester hydrochloride is -20 to 0 degrees Celsius, preferably -10 to 0 degrees Celsius. The reaction time is 0.2 to 48 hours, preferably 0.5 to 24 hours, and more preferably 1 to 12 hours.

[0054] In this invention, the separation is performed by filtration, vacuum filtration, or extraction; preferably, brine is used for extraction and separation.

[0055] Preferably, the present invention further includes drying the extracted organic phase, preferably using anhydrous Na2SO4.

[0056] Preferably, the present invention further includes a solvent removal process for the dried product, preferably by vacuum spin-drying.

[0057] Preferably, step S2) is as follows: Salicylic acid is added to a round-bottom flask, followed by dichloromethane (DCM), and then oxalyl chloride is added dropwise. The mixture is stirred at room temperature, and N,N-dimethylformamide (DMF) is added to the reaction system. The reaction continues at room temperature to obtain the acyl chloride. Phenylglycine ethyl ester hydrochloride and K₂CO₃ are dissolved in a 2:1 mixture of EA and H₂O, cooled to 0°C, and then the acyl chloride is poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture is stirred at 0°C. After the reaction is complete, the mixture is extracted with saturated brine, and the aqueous phase is extracted with EA. The organic phases are combined, dried over anhydrous Na₂SO₄, and the solvent is removed under reduced pressure. A yellow solid is obtained by column chromatography.

[0058] According to a third embodiment of the present invention, the use of an N-ester salicylamide compound having the general structural formula (IV) is provided.

[0059] The use of an N-ester salicylamide compound having the general structural formula (IV) as described in the first embodiment or an N-ester salicylamide compound having the general structural formula (IV) prepared by the method described in the second embodiment, wherein the N-ester salicylamide compound having the general structural formula (IV) is used for antibacterial purposes on crops; specifically, for inhibiting one or more of Fusarium head blight, rice blast fungus, Phytophthora blight, Sclerotinia sclerotiorum, Botrytis cinerea, and Sheath blight; preferably, the N-ester salicylamide compound having the general structural formula (IV) is used to inhibit Sclerotinia sclerotiorum.

[0060] According to a fourth embodiment of the present invention, the use of an N-ester salicylamide compound having the general structural formula (IV) is provided.

[0061] The use of an N-ester salicylamide compound having the general structural formula (IV) as described in the first embodiment or an N-ester salicylamide compound having the general structural formula (IV) prepared by the method described in the second embodiment, wherein the N-ester salicylamide compound having the general structural formula (IV) is used to prepare a crop antibacterial drug, specifically for preparing a drug that inhibits one or more of the following pathogens: Fusarium head blight, rice blast fungus, Phytophthora blight, Sclerotinia sclerotinia, Botrytis cinerea, and Sheath blight; preferably, the N-ester salicylamide compound having the general structural formula (IV) is used to prepare a drug that inhibits Sclerotinia sclerotinia.

[0062] According to a fifth embodiment of the present invention, the use of an N-ester salicylamide compound having the general structural formula (IV) is provided.

[0063] The use of an N-ester salicylamide compound having general structural formula (IV) as described in the first embodiment or an N-ester salicylamide compound having general structural formula (IV) prepared by the method described in the second embodiment, wherein the N-ester salicylamide compound having general structural formula (IV) is used to prepare an antitumor drug, preferably for preparing a drug for inhibiting liver cancer cells, more preferably for inhibiting the activity of HCCLM3 in liver cancer cells.

[0064] According to a sixth embodiment of the present invention, an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is provided.

[0065] An N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V),

[0066]

[0067] In formula (V), R1 is a C1-C6 alkyl, benzyl, or substituted benzyl group; R is one of H, C1-C6 alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, methylthioethyl, or substituted methylthioethyl.

[0068] Preferably, R1 is a C1-C3 alkyl or benzyl group; R is one of H, C1-C4 alkyl, phenyl, chlorophenyl, benzyl, hydroxybenzyl, or methylthioethyl.

[0069] Preferably, R1 is CH3, CH2CH3, or benzyl; R is one of H, C1-C4 alkyl, phenyl, 3-chlorophenyl, 4-chlorophenyl, 4-hydroxybenzyl, or methylthioethyl.

[0070] An N-ester-1,3-benzoxazine-2,4-dione compound having general structural formula (V) is prepared by reacting an N-ester-salicylamide compound having general structural formula (IV) with triphosgene.

[0071]

[0072] In formula (V), R1 is a C1-C6 alkyl, benzyl, or substituted benzyl group; R is one of H, C1-C6 alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, methylthioethyl, or substituted methylthioethyl.

[0073] Preferably, R1 is a C1-C3 alkyl or benzyl group; R is one of H, C1-C4 alkyl, phenyl, chlorophenyl, benzyl, hydroxybenzyl, or methylthioethyl.

[0074] Preferably, R1 is CH3, CH2CH3, or benzyl; R is one of H, C1-C4 alkyl, phenyl, 3-chlorophenyl, 4-chlorophenyl, 4-hydroxybenzyl, or methylthioethyl.

[0075] Preferably, the N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is selected from one or more of the following compounds:

[0076] N-(ethyl acetate)-1,3-benzoxazine-2,4-dione:

[0077]

[0078] N-(2-ethyl phenylacetyl)-1,3-benzoxazine-2,4-dione:

[0079]

[0080] N-(2-(4-chlorophenyl)acetic acid methyl ester)-1,3-benzoxazine-2,4-dione:

[0081]

[0082] N-(2-(4-chlorophenyl)ethyl acetate)-1,3-benzoxazine-2,4-dione:

[0083]

[0084] N-((4-methyl)valerate)-1,3-benzoxazine-2,4-dione:

[0085]

[0086] N-(4-methylthio)butyrate methyl 1,3-benzoxazine-2,4-dione

[0087]

[0088] N-(benzyl acetate)-1,3-benzoxazine-2,4-dione

[0089]

[0090] According to a seventh embodiment of the present invention, a method for preparing N-ester-1,3-benzoxazine-2,4-dione compounds having the general structural formula (V) is provided.

[0091] A method for preparing N-ester-1,3-benzoxazine-2,4-dione compounds having the general structural formula (V), or a method for preparing N-ester-1,3-benzoxazine-2,4-dione compounds having the general structural formula (V) as described in the sixth embodiment: the method specifically includes the following steps:

[0092] S1) An amino acid with structural formula (I) is reacted with an alcohol with structural formula (II) in the presence of thionyl chloride to obtain an amino acid ester hydrochloride with structural formula (III):

[0093]

[0094] S2) Salicylic acid is reacted with oxaloyl chloride to obtain salicyl chloride, which is then reacted with an amino acid ester hydrochloride having structural formula (III) to obtain an N-ester salicylamide compound having general structural formula (IV):

[0095]

[0096] S3) Reaction of N-ester salicylamide compounds with general structural formula (IV) with triphosgene yields N-ester-1,3-benzoxazine-2,4-dione compounds with general structural formula (V):

[0097]

[0098] In the formula, R1 is a C1-C6 alkyl, benzyl, or substituted benzyl group; R is one of H, C1-C6 alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, methylthioethyl, or substituted methylthioethyl.

[0099] Preferably, R1 is a C1-C3 alkyl or benzyl group; R is one of H, C1-C4 alkyl, phenyl, chlorophenyl, benzyl, hydroxybenzyl, or methylthioethyl.

[0100] Preferably, the amino acid is glycine, phenylglycine, chlorophenylglycine, tyrosine, leucine, or methionine.

[0101] Preferably, the alcohol is methanol, ethanol or benzyl alcohol.

[0102] Preferably, step S1) specifically involves dissolving an amino acid having general structural formula (I) in an alcohol having general structural formula (II), adding thionyl chloride, and heating the reaction solution to react and obtain an amino acid ester hydrochloride having structural formula (III).

[0103] In this invention, in step S1), the molar ratio of the amino acid having general structural formula (I) to the alcohol having general structural formula (II) added to the reaction is 1:1-10, preferably 1:2-8, for example 1:5. The molar ratio of the amino acid having general structural formula (I) to thionyl chloride added to the reaction is 1:1-10, preferably 1:2-5, for example 1:3. The reaction temperature is 20 to 80°C, preferably 30 to 60°C. The reaction time is 2-24 h, preferably 3-12 h.

[0104] Preferably, step S2) specifically involves: dissolving salicylic acid in a solvent, adding oxaloyl chloride, optionally adding or not adding N,N-dimethylformamide (DMF), stirring the reaction, and obtaining an acyl chloride mixture. An amino acid ester hydrochloride having structural formula (III) is dissolved in an ester solution with K2CO3 to obtain a mixed solution containing the amino acid ester hydrochloride. The acyl chloride mixture is added to the mixed solution containing the amino acid ester hydrochloride, reacted, and separated to obtain an N-ester salicylamide compound having general structural formula (IV).

[0105] In this invention, the acyl chloride mixture is prepared in-house using a self-synthesized salicyl chloride solution. The salicyl chloride is reacted with an amino acid ester hydrochloride having structural formula (III) to obtain an N-ester salicylamide compound having general structural formula (IV).

[0106] In this invention, the addition of N,N-dimethylformamide serves to increase solvent polarity and promote the reaction.

[0107] In this invention, the purpose of adding alkali in step S2) is twofold: first, to remove hydrochloric acid from the carbamate hydrochloride; and second, to neutralize the hydrochloric acid generated by the reaction of acyl chloride and amino acid ester, thereby promoting the reaction.

[0108] Preferably, the solvent is an organic solvent, preferably dichloromethane (DCM). The mass concentration of salicylic acid solvent in the solvent is 0.2-10%, preferably 0.3-8%, and more preferably 0.5-5%.

[0109] Preferably, the molar ratio of oxaloyl chloride to salicylic acid is 0.2-10:1, more preferably 0.5-8:1, and even more preferably 1-5:1.

[0110] Preferably, the volume ratio of N,N-dimethylformamide (DMF) to salicylic acid is 1:0.02-0.5, and more preferably 1:0.03-0.4.

[0111] Preferably, in the mixed solution containing amino acid ester hydrochloride, the molar ratio of amino acid ester hydrochloride to K2CO3 is 1:0.1-3, more preferably 1:0.2-2, and even more preferably 1:0.3-1.

[0112] Preferably, the ester solution is an ethyl acetate solution, preferably a mixture of ethyl acetate and water in a volume ratio of 0.5-10:1, and more preferably a mixture of ethyl acetate and water in a volume ratio of 1-5:1.

[0113] Preferably, the acyl chloride mixture is added to the mixed solution containing amino acid ester hydrochloride, such that the molar ratio of amino acid ester hydrochloride to salicylic acid is 1:0.2-5, preferably 1:0.5-3, for example 2:1.

[0114] In this invention, the reaction temperature for obtaining the acyl chloride mixture is 10-50°C, preferably 15-40°C, more preferably 20-35°C; for example, room temperature. The reaction time is 1-48 h, preferably 2-24 h, more preferably 3-12 h.

[0115] In this invention, before adding the acyl chloride mixture to the mixed solution containing amino acid ester hydrochloride, the amino acid ester hydrochloride is first cooled to below zero degrees Celsius. The reaction temperature between the acyl chloride mixture and the mixed solution containing amino acid ester hydrochloride is -20 to 0 degrees Celsius, preferably -10 to 0 degrees Celsius. The reaction time is 0.2 to 48 hours, preferably 0.5 to 24 hours, and more preferably 1 to 12 hours.

[0116] In this invention, the separation is performed by filtration, vacuum filtration, or extraction; preferably, brine is used for extraction and separation.

[0117] Preferably, the present invention further includes drying the extracted organic phase, preferably using anhydrous Na2SO4.

[0118] Preferably, the present invention further includes a solvent removal process for the dried product, preferably by vacuum spin-drying.

[0119] Preferably, step S2) is as follows: Salicylic acid is added to a round-bottom flask, followed by dichloromethane (DCM), and then oxalyl chloride is added dropwise. The mixture is stirred at room temperature, and N,N-dimethylformamide (DMF) is added to the reaction system. The reaction continues at room temperature to obtain the acyl chloride. Phenylglycine ethyl ester hydrochloride and K₂CO₃ are dissolved in a 2:1 mixture of EA and H₂O, cooled to 0°C, and then the acyl chloride is poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture is stirred at 0°C. After the reaction is complete, the mixture is extracted with saturated brine, and the aqueous phase is extracted with EA. The organic phases are combined, dried over anhydrous Na₂SO₄, and the solvent is removed under reduced pressure. A yellow solid is obtained by column chromatography.

[0120] Preferably, step S3) specifically involves: adding an N-ester salicylamide compound having the general structural formula (IV) and an alkaline solution to a reaction vessel, adding a solvent to obtain reactant I. Dissolving triphosgene in the solvent to obtain reactant II. Adding reactant II to reactant I to carry out the reaction; after the reaction is complete, isolating an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V).

[0121] Preferably, the alkaline solution is triethylamine.

[0122] Preferably, the solvent is an organic solvent, preferably tetrahydrofuran.

[0123] Preferably, the molar ratio of the N-ester salicylamide compound having the general structural formula (IV) to the alkaline solution is 1:0.5-10, more preferably 1:0.8-8, and even more preferably 1:1-5, for example 1:2.

[0124] Preferably, in reactant I, the mass concentration of the N-ester salicylamide compound having the general structural formula (IV) in the solvent is 0.2-30%, preferably 0.5-20%, and more preferably 1-10%.

[0125] Preferably, the mass concentration of triphosgene in reactant II is 0.1-50%, more preferably 0.3-30%, and even more preferably 0.5-20%.

[0126] Preferably, the molar ratio of the N-ester salicylamide compound having the general formula (IV) to triphosgene is 1:0.2-5, more preferably 1:0.5-4, and even more preferably 1:0.6-3.

[0127] Preferably, the step of dissolving triphosgene in a solvent is carried out under nitrogen protection.

[0128] In this invention, the step of adding reactant II to reactant I is carried out by adding reactant II dropwise to reactant I through a constant pressure dropping funnel under stirring in an ice-water bath.

[0129] In this invention, the reaction between reactant II and reactant I is carried out at 25-80°C for 1-48 hours, preferably at 30-75°C for 2-24 hours, and more preferably at 35-70°C for 3-12 hours.

[0130] In this invention, the separation specifically involves: after the reaction is completed, cooling and depressurizing to remove the solvent, filtering the obtained solid with dichloromethane, further depressurizing to remove the solvent in the organic phase, and then obtaining the product by column chromatography.

[0131] According to an eighth embodiment of the present invention, the use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is provided.

[0132] The use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) as described in the sixth embodiment, or an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) prepared by the method described in the seventh embodiment, for the purpose of using the N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) for antibacterial purposes on crops; specifically for inhibiting one or more of Fusarium head blight, rice blast fungus, Phytophthora blight, Sclerotinia sclerotinia, Botrytis cinerea, and Sheath blight.

[0133] Preferably, the N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is used to inhibit rice blast fungus.

[0134] According to a ninth embodiment of the present invention, the use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is provided.

[0135] The use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) as described in the sixth embodiment, or an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) prepared by the method described in the seventh embodiment, for the preparation of antibacterial drugs for crops, specifically for the preparation of drugs that inhibit one or more of the following pathogens: Fusarium head blight, rice blast fungus, Phytophthora blight, Sclerotinia sclerotinia, Botrytis cinerea, and Sheath blight.

[0136] Preferably, the N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is used to prepare a drug for inhibiting rice blast fungus.

[0137] According to a tenth embodiment of the present invention, the use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is provided.

[0138] The use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) as described in the sixth embodiment, or an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) prepared by the method described in the seventh embodiment, for the preparation of an antitumor drug, preferably for the preparation of a drug that inhibits liver cancer cells, more preferably for the preparation of a drug that inhibits the activity of HCCLM3 liver cancer cells.

[0139] In this invention, an amino acid ester salt solution is obtained by reacting amino acids with alcohols, and a salicylic acid acyl chloride solution is obtained by reacting salicylic acid with oxaloyl chloride. Then, the amino acid ester salt solution is reacted with the salicylic acid acyl chloride solution to obtain a novel N-ester salicylamide compound with general structural formula (IV). This type of compound has good inhibitory effects on crop pathogens and can be used for direct antibacterial treatment of crops or for preparing antibacterial drugs for crops. This type of compound also has good inhibitory effects on liver cancer cells.

[0140] The inventors further experimented by using an N-ester salicylamide compound with general structural formula (IV) as an intermediate, and reacting it with triphosgene to obtain a novel N-ester-1,3-benzoxazine-2,4-dione compound with general structural formula (V). This class of compounds also exhibits good inhibitory effects against crop pathogens and can be used for direct antibacterial activity against crops or for preparing antibacterial drugs for crops. The inhibitory effect of this class of compounds on rice blast fungus reached 100%. This class of compounds also shows good inhibitory effects on liver cancer cells.

[0141] Through experimental research, N-ester salicylamide compounds with general structural formula (IV) and N-ester-1,3-benzoxazine-2,4-dione compounds with general structural formula (V) were prepared. The prepared drugs were used in antibacterial and antitumor experiments on crops, and both showed good inhibitory activity. The prepared compounds can be used directly for antibacterial or antitumor purposes on crops, or they can be used to prepare antibacterial drugs for crops or antitumor drugs.

[0142] Compared with the prior art, the present invention has the following beneficial technical effects:

[0143] 1. The N-ester salicylamide compound with general structural formula (IV) prepared by the present invention is a novel compound, and the compound has very good antibacterial and antitumor activities; in particular, it has a significant inhibitory effect on pathogens such as Fusarium head blight, Phytophthora blight, rice blast fungus, sclerotinia sclerotinia, gray mold, and sheath blight, and also has a significant activity against HCCLM3 liver cancer cells.

[0144] 2. The N-ester-1,3-benzoxazine-2,4-dione compound with general structural formula (V) prepared by this invention is a novel compound, and this compound has very good antibacterial and antitumor activities in crops; in particular, it has a significant inhibitory effect on pathogens such as Fusarium head blight, Phytophthora blight, rice blast fungus, sclerotinia sclerotinia, gray mold, or sheath blight, and also has a significant activity against HCCLM3 liver cancer cells.

[0145] 3. The preparation methods of N-ester salicylamide compounds with general structural formula (IV) and N-ester-1,3-benzoxazine-2,4-dione compounds with general structural formula (V) provided by the present invention have the advantages of inexpensive and readily available raw materials, simple synthesis methods, high yield, and easy separation and purification of products. Attached Figure Description

[0146] Figure 1 This is the general structural formula of the N-ester salicylamide compound having the general structural formula (IV) described in this invention.

[0147] Figure 2 This is a synthetic route diagram for N-ester salicylamide compounds having the general structural formula (IV) described in this invention.

[0148] Figure 3 This is a structural diagram of the N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) described in this invention.

[0149] Figure 4 This is a synthetic route diagram for N-ester-1,3-benzoxazine-2,4-dione compounds having the general structural formula (V) described in this invention. Detailed Implementation

[0150] The technical solution of the present invention will be illustrated below with examples. The scope of protection sought by the present invention includes, but is not limited to, the following embodiments.

[0151] The structural formula of the intermediate product prepared by this invention is as follows:

[0152]

[0153]

[0154] The chemical reagents used in the various embodiments of this invention are sourced from the following:

[0155] Tianjin Kemei Chemical Reagent Co., Ltd.: N,N-Dimethylformamide (CAS: 68-12-2).

[0156] Shanghai Mairui Chemical Technology Co., Ltd.: Glycine methyl ester hydrochloride (CAS: 5680-79-5), DL-phenylglycine (CAS: 2835-06-5), DL-p-chlorophenylglycine (CAS: 6212-33-5), DL-m-chlorophenylglycine (CAS: 7292-71-9), L-leucine (CAS: 26782-71-8), L-tyrosine (CAS: 60-18-4), methionine (CAS: 63-68-3), salicylic acid (CAS: 69-72-7), oxaloyl chloride (CAS: 79-37-8), triphosgene (CAS: 32315-10-9), thionyl chloride (CAS: 7719-09-7), anhydrous potassium carbonate (CAS: 584-08-7).

[0157] Guangdong Guanghua Technology Co., Ltd.: Anhydrous methanol (CAS: 67-56-1), dichloromethane (CAS: 75-09-2), ethyl acetate (CAS: 141-78-6).

[0158] Xilong Chemical Co., Ltd.: Triethylamine (CAS: 121-44-8), Tetrahydrofuran (CAS: 109-99-9).

[0159] Hunan Huihong Reagent Co., Ltd.: Anhydrous ethanol (CAS: 64-17-5).

[0160] Sinopharm Chemical Reagent Co., Ltd.: Glycine (CAS: 56-40-6),

[0161] Example A1

[0162] Preparation of phenylglycine ethyl ester hydrochloride:

[0163] DL-phenylglycine (0.1511 g, 1 mmol) and anhydrous ethanol (0.2304 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain DL-phenylglycine ethyl ester hydrochloride.

[0164] Preparation of N-(2-ethyl phenylacetyl)salicylamide:

[0165]

[0166] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. Phenylglycine ethyl ester hydrochloride (0.431 g, 2 mol) and K₂CO₃ (0.166 g, 1.2 mol) were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by evaporation under reduced pressure. A yellow solid was obtained by column chromatography.

[0167] 1 H NMR(400MHz, CDCl3) δ11.96(s,1H),7.50(dd,J=8.0,1.6Hz,1H),7.47–7.33(m,7H),6.96(dd,J=8.4,1.2H z,1H),6.86(ddd,J=8.3,7.3,1.2Hz,1H),5.70(d,J=6.6Hz,1H),4.32–4.14(m,2H),1.24(t,J=7.1Hz,3H).

[0168] 13 C NMR (101MHz, CDCl3) δ170.79,169.32,161.74,136.30,134.73,129.17,128.84,127.30,125.92,118.90,118.71,113.82,62.46,56.62,14.10.

[0169] Example A2

[0170] Preparation of DL-3-chlorophenylglycine methyl ester hydrochloride:

[0171] DL-3-chlorophenylglycine (0.1856 g, 1 mmol) and anhydrous methanol (0.1602 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain DL-3-chlorophenylglycine methyl ester hydrochloride.

[0172] Preparation of N-(2-(3-chlorophenyl)acetic acid methyl ester) salicylamide:

[0173]

[0174] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was then added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. 0.472 g (2 mol) of DL-3-chlorophenylglycine methyl ester hydrochloride and 0.166 g (1.2 mol) of K₂CO₃ were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and then slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by evaporation under reduced pressure. A white solid was obtained after chromatography.

[0175] 1 H NMR (400MHz, CDCl3) δ11.83(s,1H),7.52(dd,J=8.0,1.6Hz,1H),7.43(tdd,J=10.5,6.1,2.3Hz,3H),7.34– 7.31(m,3H),6.97(dd,J=8.4,1.2Hz,1H),6.88(td,J=7.6,1.2Hz,1H),5.69(d,J=6.4Hz,1H),3.80(s,3H).

[0176] 13 C NMR (101MHz, CDCl3) δ170.76,169.37,161.77,138.17,135.08,134.94,130. 45,129.15,127.45,125.95,125.61,119.02,118.79,113.61,56.07,53.51.

[0177] Example A3

[0178] Preparation of DL-3-chlorophenylglycine ethyl ester hydrochloride:

[0179] DL-3-chlorophenylglycine (0.1856 g, 1 mmol) and anhydrous ethanol (0.2304 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain DL-3-chlorophenylglycine ethyl ester hydrochloride.

[0180] Preparation of N-(2-(3-chlorophenyl)ethyl acetate)salicylamide:

[0181]

[0182] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was then added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. DL-3-chlorophenylglycine ethyl ester hydrochloride (0.496 g, 2 mol) and K₂CO₃ (0.166 g, 1.2 mol) were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by evaporation under reduced pressure. A yellow solid was obtained after chromatography.

[0183] 1 H NMR (400MHz, CDCl3) δ11.87(s,1H),7.55–7.45(m,2H),7.45–7.38(m,2H),7.31(d,J=1.3Hz,3H),6.97(d d,J=8.4,1.2Hz,1H),6.92–6.84(m,1H),5.67(d,J=6.5Hz,1H),4.35–4.15(m,2H),1.25(t,J=7.2Hz,3H).

[0184] 13 C NMR (101MHz, CDCl3) δ170.27,169.34,161.75,138.34,134.99,134.89,130.37, 129.03,127.38,125.95,125.56,119.00,118.77,113.64,62.79,56.10,14.10.

[0185] Example A4

[0186] Preparation of DL-4-chlorophenylglycine methyl ester hydrochloride:

[0187] DL-4-chlorophenylglycine (0.1856 g, 1 mmol) and anhydrous methanol (0.1602 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain DL-4-chlorophenylglycine methyl ester hydrochloride.

[0188] Preparation of N-(2-(4-chlorophenyl)acetic acid methyl ester) salicylamide:

[0189]

[0190] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. DL-4-chlorophenylglycine methyl ester hydrochloride (0.472 g, 2 mol) and K₂CO₃ (0.166 g, 1.2 mol) were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by evaporation under reduced pressure. A yellow solid was obtained after chromatography.

[0191] 1 H NMR(400MHz, CDCl3)δ11.84(s,1H),7.51(dd,J=8.0,1.6Hz,1H),7.47–7.42(m,1H),7.42–7.38(m,1H),7.37 –7.32(m,4H),6.96(dd,J=8.4,1.2Hz,1H),6.87(td,J=7.7,1.2Hz,1H),5.68(d,J=6.4Hz,1H),3.78(s,3H).

[0192] 13 C NMR (101MHz, CDCl3) δ170.92,169.36,161.74,134.91,134.89,134.75,129.39,128.71,125.91,119.00,118.78,113.62,55.97,53.44.

[0193] Example A5

[0194] Preparation of DL-4-chlorophenylglycine ethyl ester hydrochloride:

[0195] DL-4-chlorophenylglycine (0.1856 g, 1 mmol) and anhydrous ethanol (0.2304 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain DL-4-chlorophenylglycine ethyl ester hydrochloride.

[0196] Preparation of N-(2-(4-chlorophenyl)ethyl acetate)salicylamide:

[0197]

[0198] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. DL-4-chlorophenylglycine ethyl ester hydrochloride (0.496 g, 2 mol) and K₂CO₃ (0.166 g, 1.2 mol) were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation under reduced pressure. A yellow solid was obtained after chromatography.

[0199] 1 H NMR(400MHz, CDCl3)δ11.89(s,1H),7.54–7.45(m,2H),7.45–7.32(m,5H),7.07 –6.78(m,2H),5.66(d,J=6.3Hz,1H),4.41–4.05(m,2H),1.25(t,J=7.1Hz,3H).

[0200] 13 C NMR (101MHz, CDCl3) δ170.44,169.33,161.73,134.93,134.87,134.75,129.32,128.65,125.91,118.98,118.76,113.65,62.72,56.01,14.09.

[0201] Example A6

[0202] Preparation of L-tyrosine methyl ester hydrochloride:

[0203] L-tyrosine (0.1812 g, 1 mmol) and anhydrous methanol (0.1602 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain L-tyrosine methyl ester hydrochloride.

[0204] Preparation of N-(3-(4-hydroxyphenyl)propionic acid methyl ester) salicylamide:

[0205]

[0206] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. L-tyrosine methyl ester hydrochloride (0.391 g, 2 mol) was dissolved in a 2:1 mixture of EA and H2O with K2CO3 (0.166 g, 1.2 mol). The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na2SO4, and the solvent was removed by evaporation under reduced pressure. A yellow oil was obtained by column chromatography.

[0207] 1 H NMR (400MHz, CDCl3) δ7.38(ddd,J=8.6,7.2,1.6Hz,1H),7.33(dd,J=8.1,1.6Hz,1H),6.99–6.93(m,3H),6.88(d,J=7.5Hz,1H) ,6.82(ddd,J=8.2,7.2,1.2Hz,1H),6.75–6.70(m,2H),5.01(dt,J=7.6,5.5Hz,1H),3.78(s,3H),3.16(dd,J=5.6,3.7Hz,2H).

[0208] 13 C NMR (101MHz, CDCl3) δ172.27,169.56,161.45,155.28,134.73,130.53,127.14,125.92,119.11,118.62,115.78,113.94,53.43,52.83,37.10.

[0209] Example A7

[0210] Preparation of methionine methyl ester hydrochloride:

[0211] Methionine (0.149 g, 1 mmol) and anhydrous methanol (0.1602 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain methionine methyl ester hydrochloride.

[0212] Preparation of N-((4-methylthio)butyrate methyl ester) salicylamide:

[0213]

[0214] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. Methionine methyl ester hydrochloride (0.399 g, 2 mol) was dissolved in a 2:1 mixture of EA and H2O with K2CO3 (0.166 g, 1.2 mol). The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na2SO4, and the solvent was removed by evaporation under reduced pressure. A yellow oil was obtained by column chromatography.

[0215] 1 H NMR (400MHz, CDCl3) δ12.05(s,1H),7.46(dd,J=8.1,1.6Hz,1H),7.39(ddd,J=8.6,7.2,1.6Hz,1H),7.31(d,J=7.5Hz,1H),6.95(dd,J=8.3,1 .1Hz,1H),6.88–6.80(m,1H),4.88(td,J=7.2,5.0Hz,1H),4.26(q,J=7.1Hz,2H),2.59(t,J=7.3Hz,2H),2.11(s,5H),1.31(t,J=7.1Hz,3H).

[0216] 13C NMR (100MHz, CDCl3) δ171.95,169.87,161.68,134.65,125.86,118.90,118.64,113.82,62.14,51.89,31.34,30.14,15.62,14.27.

[0217] Example A8

[0218] Preparation of glycine benzyl ester hydrochloride:

[0219] Glycine (0.0751 g, 1 mmol) and benzyl alcohol (0.2304 g, 5 mmol) were added to a round-bottom flask, and thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C. The reaction solution was then heated under reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain glycine benzyl ester hydrochloride.

[0220] Preparation of N-(2-ethyl phenylacetyl)salicylamide:

[0221]

[0222] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. 0.403 g (2 mol) of glycine benzyl ester hydrochloride and 0.166 g (1.2 mol) of K₂CO₃ were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by evaporation under reduced pressure. A white solid was obtained by column chromatography.

[0223] 1 H NMR (400MHz, CDCl3) δ12.01(s,1H),7.47–7.34(m,7H),6.97(dd,J=8.4,1.2H z,2H),6.85(td,J=7.6,7.1,1.2Hz,1H),5.24(s,2H),4.25(d,J=5.0Hz,2H).

[0224] 13C NMR (100MHz, CDCl3) δ170.09,169.76,161.52,134.98,134.62,128.74,128.72,128.46,125.77,118.86,118.58,113.69,67.61,41.45.

[0225] Crop antibacterial activity test

[0226] The bactericidal activity of N-(2-ethyl phenylacetate)salicylamide, N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylamide, N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylamide, N-(2-(4-chlorophenyl)acetic acid methyl ester)salicylamide, N-(2-(4-chlorophenyl)acetic acid methyl ester)salicylamide, N-(3-(4-hydroxyphenyl)propionic acid methyl ester)salicylamide, N-((4-methylthio)butyrate methyl ester)salicylamide, and N-(benzyl acetate)salicylamide was tested using an in vitro method.

[0227] Fusarium head blight, Phytophthora blight, rice blast fungus, sclerotinia sclerotinia, gray mold, and sheath blight fungus were used as test materials for fungicidal activity testing. The test agents were dissolved in acetone and then diluted to 500 g / mL with 200 g / mL Sorporl-144 emulsifier. Under aseptic conditions, 1 mL of the compound solution was pipetted into a sterilized Petri dish, followed by 9 mL of sterile PDA culture medium. The mixture was then stirred to prepare the appropriate concentration of drug-containing plates. Under aseptic conditions, mycelial cakes were cut from the edge of the colony using a 4 mm diameter sterile punch. After the culture medium solidified, the mycelial cakes were inoculated into the center of the drug-containing plate using an inoculator and incubated at a suitable temperature. A blank control was prepared without the added agent. Each treatment was incubated in an incubator at 24±1℃. After 72 hours, the diameter of the colonies was observed and measured. The diameter of each colony was measured vertically once using the cross-sectional method, and the average value was taken.

[0228] Growth inhibition rate (%) = (control colony diameter - treated colony diameter) × 100 / (control colony diameter - 4 mm).

[0229] The drug concentration was 50 μg / mL. The results of the bactericidal activity test are shown in Table 1.

[0230] Table 1. Results of antibacterial activity tests of N-ester salicylamide compounds.

[0231]

[0232] As shown in Table 1, the target compounds exhibited moderate to good inhibitory activity against the tested pathogens. Specifically, N-(2-ethylphenylacetate)salicylamide showed an inhibition rate of up to 75.8% against *Sclerotinia sclerotinia*; N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylamide showed an inhibition rate of up to 71.1% against *Phytophthora*; N-(2-(3-chlorophenyl)acetic acid ethyl ester)salicylamide showed an inhibition rate of up to 73.9% against *Botrytis cinerea*; N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylamide showed an inhibition rate of up to 88.9% against *Sclerotinia sclerotinia*; N-(2-(4-chlorophenyl)acetic acid methyl ester)salicylamide showed an inhibition rate of 89.9% against *Sclerotinia sclerotinia*; N-(2-(4-chlorophenyl)acetic acid ethyl ester)salicylamide showed an inhibition rate of 92.9% against *Sclerotinia sclerotinia*; and N-(3-(4-hydroxyphenyl)propionate methyl ester)salicylamide showed an inhibition rate of 85.0% against *Sclerotinia sclerotinia*.

[0233] Antitumor activity test

[0234] Cell growth assay (CCK8 assay) was performed by adjusting the suspension of hepatocellular carcinoma cells (HCCLM3) to 5 × 10⁻⁶ cells / mL. 4 / mL (adjust suspension cells to 10) 5 Cells were seeded into 96-well plates at a concentration of 100 μL / well, with 5000 cells / well. After 4 h of seeding, the antitumor activity of N-(2-ethylphenylacetyl)salicylic acid amide, N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylic acid amide, N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylic acid amide, N-(2-(4-chlorophenyl)acetic acid methyl ester)salicylic acid amide, N-(3-(4-hydroxyphenyl)propionic acid methyl ester)salicylic acid amide, N-((4-methylthio)butyrate methyl ester)salicylic acid amide, and N-(benzyl acetate)salicylic acid amide was tested. 100 μL of culture medium containing different concentrations of the compound was added to each well, with six replicates for each concentration. Wells without cells served as blank controls, wells with cells but without the compound served as negative controls for the compound, and 5-fluorouracil served as a positive control for the compound. Incubate at 37℃ in 5% CO2 for 24 h, then add 10 μL of CCK8 staining solution to each well and continue incubation. After 4 h, transfer to 37℃ and shake for 5-10 min. Measure the absorbance (OD) value of each well at 450 nm using a microplate reader. The cell growth inhibition rate is calculated using the following formula:

[0235] Cell survival inhibition rate (%) = (OD value of negative control group - OD value of experimental group) × 100% / (OD value of negative control group - OD value of blank control group).

[0236] The drug concentration was 50 μmol / L. The results of the antitumor activity test are shown in Table 2.

[0237] Table 2. Results of antitumor activity tests on N-ester salicylamide compounds.

[0238] N-(2-ethyl phenylacetyl)salicylamide 96.8 N-(2-(3-chlorophenyl)methyl acetate)salicylic acid amide 96.6 N-(2-(3-chlorophenyl)ethyl acetate)salicylamide 99.2 N-(2-(4-chlorophenyl)methyl acetate)salicylic acid amide 89.3 N-(2-(4-chlorophenyl)ethyl acetate)salicylamide 94.8 N-(3-(4-hydroxyphenyl)propionic acid methyl ester) salicylamide 90.5 N-((4-methylthio)butyrate methyl ester) salicylamide 95.7 N-(benzyl acetate) salicylamide 96.1 5-Fluorouracil (positive control drug) 99.9

[0239] Table 2 shows that the target compounds exhibited good inhibitory activity against the tested pathogens. Specifically, N-(2-ethyl phenylacetate)salicylic acid amide showed an inhibition rate of up to 96.8% against liver cancer cells; N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylic acid amide showed an inhibition rate of up to 96.6% against liver cancer cells; and N-(2-(3-chlorophenyl)acetic acid methyl ester)salicylic acid amide showed an inhibition rate of up to 99.2% against liver cancer cells, which is close to the 99.9% inhibition rate of the positive control drug 5-fluorouracil against liver cancer cells. N-(2-(4-chlorophenyl)acetic acid methyl ester)salicylic acid methyl ester)salicylic acid amide showed an inhibition rate of up to 99.2% against liver cancer cells, which is close to the 99.9% inhibition rate of the positive control drug 5-fluorouracil against liver cancer cells. The inhibition rate of N-(2-(4-chlorophenyl)ethyl acetate) salicylamide against liver cancer cells was as high as 89.3%; the inhibition rate of N-(2-(4-chlorophenyl)ethyl acetate) salicylamide against liver cancer cells was 94.8%; the inhibition rate of N-(3-(4-hydroxyphenyl)propionate methyl) salicylamide against liver cancer cells was 90.5%; the inhibition rate of N-((4-methylthio)butyrate methyl) salicylamide was 95.7%; and the inhibition rate of N-(benzyl acetate) salicylamide was 96.1%.

[0240] Example B1

[0241] Preparation of glycine ethyl ester hydrochloride:

[0242] Glycine (0.0751 g, 1 mmol) and anhydrous ethanol (0.2304 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain glycine ethyl ester hydrochloride.

[0243] Preparation of N-(ethyl acetate)salicylamide:

[0244]

[0245] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was then added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. Glycine ethyl ester hydrochloride (0.251 g, 2 mol) and K₂CO₃ (0.166 g, 1.2 mol) were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by evaporation under reduced pressure. N-(ethyl acetate) salicylamide was obtained by column chromatography.

[0246] 1 H NMR (400MHz, CDCl3) δ8.09 (dd, J=7.8, 1.7Hz, 1H), 7.73 (ddd, J=8.4, 7.4, 1.7Hz, 1H), 7.39 (td, J=7.6 ,1.0Hz,1H),7.32(dd,J=8.4,1.0Hz,1H),4.77(s,2H),4.25(q,J=7.1Hz,2H),1.30(t,J=7.1Hz,3H).

[0247] 13 C NMR (100MHz, CDCl3) δ167.11,160.29,152.77,148.03,136.64,128.41,125.80,116.75,113.93,62.13,43.17,14.25.

[0248] Synthesis of N-(ethyl acetate)-1,3-benzoxazine-2,4-dione:

[0249]

[0250] Weigh 0.669 g (3 mmol) of N-(ethyl acetate-based) salicylamide and 0.606 g (6 mmol) of triethylamine into a 100 mL round-bottom flask, and dissolve them in 30 mL of tetrahydrofuran. Under nitrogen protection, slowly add 0.579 g (1.95 mmol) of triphosgene dissolved in tetrahydrofuran to the reaction flask through a constant-pressure dropping funnel with stirring in an ice-water bath. After all the solution has been added, transfer the flask to a 65 °C oil bath and continue the reaction for 4 h. After the reaction is complete, cool and remove the solvent under reduced pressure. Filter the obtained solid with dichloromethane. After further removing the solvent from the organic phase under reduced pressure, separate the solid by column chromatography to obtain a white solid with a yield of 74.3%; melting point (mp): 118.0–119.7 °C.

[0251] 1 H NMR (400MHz, CDCl3) δ8.09 (dd, J=7.8, 1.7Hz, 1H), 7.73 (ddd, J=8.4, 7.4, 1.7Hz, 1H), 7.39 (td, J=7.6 ,1.0Hz,1H),7.32(dd,J=8.4,1.0Hz,1H),4.77(s,2H),4.25(q,J=7.1Hz,2H),1.30(t,J=7.1Hz,3H).

[0252] 13 C NMR (100MHz, CDCl3) δ167.11,160.29,152.77,148.03,136.64,128.41,125.80,116.75,113.93,62.13,43.17,14.25.

[0253] Example B2

[0254] Synthesis of N-(2-ethyl phenylacetyl)-1,3-benzoxazine-2,4-dione:

[0255]

[0256] Weigh 0.897 g (3 mmol) of N-(2-ethylphenylacetyl)salicylic acid amide and 0.606 g (6 mmol) of triethylamine prepared in Example A1 into a 100 mL round-bottom flask, and dissolve them in 30 mL of tetrahydrofuran. Under nitrogen protection, slowly add 0.579 g (1.95 mmol) of triphosgene dissolved in tetrahydrofuran to the reaction flask through a constant-pressure dropping funnel with stirring in an ice-water bath. After all the solution has been added, transfer the flask to a 65 °C oil bath and continue the reaction for 4 h. After the reaction is complete, cool and remove the solvent under reduced pressure. Filter the obtained solid with dichloromethane. After further removing the solvent from the organic phase under reduced pressure, separate the solid by column chromatography to obtain a white solid with a yield of 17.9% and a melting point (mp): 96.5–984 °C.

[0257] 1 H NMR (400MHz, CDCl3) δ7.88–7.81(m,2H),7.70(dd,J=7.8,1.7Hz,1H),7.41(t,J=7.7Hz,2H),7.35–7.22(m, 2H), 7.07 (dd, J=8.4, 1.1Hz, 1H), 6.93 (td, J=7.6, 1.1Hz, 1H), 4.44 (q, J=7.1Hz, 2H), 1.51 (t, J=7.1Hz, 3H).

[0258] 13 C NMR (100MHz, CDCl3) δ157.26,153.06,152.60,131.74,130.40,128.71,126.99,125.36,125.10,119.46,117.28,115.23,111.13,70.37,15.31.

[0259] Example B3

[0260] Synthesis of N-(2-(4-chlorophenyl)acetic acid methyl ester)-1,3-benzoxazine-2,4-dione:

[0261]

[0262] Weigh 0.957 g (3 mmol) of N-(2-(4-chlorophenyl)acetic acid methyl ester) salicylamide and 0.606 g (6 mmol) of triethylamine prepared in Example A4 into a 100 mL round-bottom flask, and dissolve them in 30 mL of tetrahydrofuran. Under nitrogen protection, slowly add 0.579 g (1.95 mmol) of triphosgene dissolved in tetrahydrofuran to the reaction flask through a constant-pressure dropping funnel with stirring in an ice-water bath. After all the solution has been added, transfer the flask to a 65 °C oil bath and continue the reaction for 4 h. After the reaction is complete, cool and remove the solvent under reduced pressure. Filter the obtained solid with dichloromethane. After further removing the solvent from the organic phase under reduced pressure, separate the solid by column chromatography to obtain a yellow solid with a yield of 15.3% and a melting point (mp): 115.4–117.1 °C.

[0263] 1 H NMR (400MHz, CDCl3) δ7.76–7.67(m,3H),7.41–7.29(m,3H),7.07(dd,J=8.4,1.1Hz,1H),6.94(td,J=7.5,1.1Hz,1H),4.18(d,J=0.8Hz,3H).

[0264] 13 C NMR (100MHz, CDCl3) δ157.16,153.77,152.35,132.51,131.89,128.88,128.82,126.27,125.35,119.55,117.32,113.34,110.90,60.37.

[0265] Example B4

[0266] Synthesis of N-(2-(4-chlorophenyl)ethyl acetate)-1,3-benzoxazine-2,4-dione:

[0267]

[0268] Weigh 0.999 g (3 mmol) of N-(2-(4-chlorophenyl)ethyl acetate)salicylic acid amide and 0.606 g (6 mmol) of triethylamine prepared in Example A5 into a 100 mL round-bottom flask, and dissolve them in 30 mL of tetrahydrofuran. Under nitrogen protection, slowly add 0.579 g (1.95 mmol) of triphosgene dissolved in tetrahydrofuran to the reaction flask through a constant-pressure dropping funnel with stirring in an ice-water bath. After all the solution has been added, transfer the flask to a 65 °C oil bath and continue the reaction for 4 h. After the reaction is complete, cool and remove the solvent under reduced pressure. Filter the obtained solid with dichloromethane. After further removing the solvent from the organic phase under reduced pressure, separate the solid by column chromatography to obtain a yellow solid with a yield of 18.1% and a melting point (mp): 107.4–109.5 °C.

[0269] 1 H NMR(400MHz, CDCl3)δ7.80–7.73(m,2H),7.70(dd,J=7.8,1.7Hz,1H),7.42–7.30(m,3H),7.07(d d,J=8.4,1.1Hz,1H),6.95(td,J=7.5,1.2Hz,1H),4.47(q,J=7.1Hz,2H),1.53(t,J=7.1Hz,3H).

[0270] 13 C NMR (100MHz, CDCl3) δ157.19,153.09,152.58,132.49,131.89,128.95,128.89,126.29,125.38,119.54,117.32,114.17,110.96,70.34,15.32.

[0271] Example B5

[0272] Preparation of L-leucine methyl ester hydrochloride:

[0273] L-leucine (0.1318 g, 1 mmol) and anhydrous methanol (0.1602 g, 5 mmol) were added to a round-bottom flask. Thionyl chloride (0.3569 g, 3 mmol) was slowly added dropwise at 0 °C, and the reaction solution was heated to reflux for 4–5 h. After the reaction was complete, the solution was concentrated under reduced pressure to obtain L-leucine methyl ester hydrochloride.

[0274] Preparation of N-((4-methyl)valerate)salicylamide:

[0275]

[0276] Salicylic acid (0.138 g, 1 mol) was added to a 150 mL round-bottom flask, followed by 20 mL of dichloromethane (DCM). Oxaloyl chloride (0.254 g, 2 mol) was added dropwise, and the mixture was stirred at room temperature for 0.5 h. One drop of N,N-dimethylformamide (DMF) was then added to the reaction system, and the reaction was continued at room temperature for 6 h to obtain the acyl chloride. L-leucine methyl ester hydrochloride (0.363 g, 2 mol) and K₂CO₃ (0.166 g, 1.2 mol) were dissolved in a 2:1 mixture of EA and H₂O. The mixture was cooled to 0 °C, and the acyl chloride was poured into a constant-pressure dropping funnel and slowly added dropwise to the reaction system. The mixture was stirred at 0 °C for 2 h. After the reaction was complete, the mixture was extracted with saturated brine (50 mL × 2 times), and the aqueous phase was extracted with EA (50 mL × 3 times). The organic phases were combined, dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation under reduced pressure. N-((4-methyl)valerate)salicylic acid amide was obtained by column chromatography.

[0277] 1 H NMR (400MHz, CDCl3) δ12.07(s,1H),7.43(dd,J=8.0,1.6Hz,1H),7.37(ddd,J=8.6,7.2,1.6Hz,1H),6.95–6.88(m,2H),6. 83(ddd,J=8.2,7.3,1.2Hz,1H),4.83(td,J=8.3,4.1Hz,1H),3.79(s,3H),1.82–1.60(m,3H),0.97(dd,J=6.1,2.9Hz,6H).

[0278] 13 C NMR (100MHz, CDCl3) δ173.69,169.87,161.66,134.61,125.76,118.82,118.66,113.83,52.72,50.83,41.62,25.05,22.93,22.04.

[0279] Synthesis of N-((4-methyl)pentanoic acid ethyl ester)-1,3-benzoxazine-2,4-dione:

[0280]

[0281] N-((4-methyl)valerate)salicylic acid ester-based salicylamide (0.837 g, 3 mmol) and triethylamine (0.606 g, 6 mmol) were weighed and added to a 100 mL round-bottom flask, followed by the addition of 30 mL of tetrahydrofuran for dissolution. Under nitrogen protection, triphosgene (0.579 g, 1.95 mmol) dissolved in tetrahydrofuran was slowly added dropwise to the reaction flask through a constant-pressure dropping funnel with stirring in an ice-water bath. After all the addition was complete, the mixture was transferred to a 65 °C oil bath and the reaction was continued for 4 h. After the reaction was completed, the mixture was cooled and the solvent was removed under reduced pressure. The obtained solid was filtered through dichloromethane. After further removing the solvent from the organic phase under reduced pressure, the solid was separated by column chromatography to obtain a yellow oil with a yield of 45.7%.

[0282] 1 H NMR(400MHz, CDCl3) δ7.68(dd,J=7.8,1.7Hz,1H),7.33–7.24(m,1H),7.03(dd,J=8.3,1.2Hz,1H),6.91(td,J=7.6,1.2Hz,1 H), 4.25 (q, J = 7.1Hz, 2H), 2.33 (d, J = 7.1Hz, 2H), 2.01 (dp, J = 13.5, 6.7Hz, 1H), 1.43 (t, J = 7.0Hz, 3H), 0.96 (d, J = 6.7Hz, 6H).

[0283] 13 C NMR (100MHz, CDCl3) δ157.23,153.85,152.88,131.35,125.17,119.31,117.13,116.11,111.49,70.92,33.39,27.87,22.44,15.22.

[0284] Example B6

[0285] Synthesis of N-((4-methylthio)butyrate methyl ester)-1,3-benzoxazine-2,4-dione:

[0286]

[0287] Weigh 0.849 g (3 mmol) of N-((4-methylthio)butyrate methyl)salicylic acid amide and 0.606 g (6 mmol) of triethylamine prepared in Example A7 into a 100 mL round-bottom flask, and dissolve them in 30 mL of tetrahydrofuran. Under nitrogen protection, slowly add 0.579 g (1.95 mmol) of triphosgene dissolved in tetrahydrofuran to the reaction flask through a constant-pressure dropping funnel with stirring in an ice-water bath. After all the solution has been added, transfer the flask to a 65 °C oil bath and continue the reaction for 4 h. After the reaction is complete, cool and remove the solvent under reduced pressure. Filter the obtained solid with dichloromethane. After further removing the solvent from the organic phase under reduced pressure, separate the solid by column chromatography to obtain a white solid with a yield of 38.7% and a melting point (mp): 66.9–69.4 °C.

[0288] 1 H NMR (400MHz, CDCl3) δ8.08(dd,J=7.9,1.7Hz,1H),7.73(ddd,J=8.4,7.4,1.7Hz,1H),7.39(td,J=7.6,1.0Hz,1H),7.30(dd,J=8 .3,1.0Hz,1H),5.68(dd,J=8.9,5.0Hz,1H),3.73(s,3H),2.70–2.60(m,1H),2.60–2.54(m,2H),2.41–2.31(m,1H),2.07(s,3H).

[0289] 13 C NMR (100MHz, CDCl3) δ169.18,160.53,152.72,147.57,136.65,128.51,125.78,116.65,113.88,54.11,52.92,30.99,27.74,15.43.

[0290] Example B7

[0291] Synthesis of N-(benzyl acetate)-1,3-benzoxazine-2,4-dione:

[0292]

[0293] Weigh 0.855 g (3 mmol) of N-(benzyl acetate) salicylamide and 0.606 g (6 mmol) of triethylamine prepared in Example A8 into a 100 mL round-bottom flask, and dissolve them in 30 mL of tetrahydrofuran. Under nitrogen protection, slowly add 0.579 g (1.95 mmol) of triphosgene dissolved in tetrahydrofuran to the reaction flask through a constant-pressure dropping funnel with stirring in an ice-water bath. After all the solution has been added, transfer the flask to a 65 °C oil bath and continue the reaction for 4 h. After the reaction is complete, cool and remove the solvent under reduced pressure. Filter the obtained solid with dichloromethane. After further removing the solvent from the organic phase under reduced pressure, separate the solid by column chromatography to obtain a white solid with a yield of 57.5% and a melting point (mp): 111.4–113.7 °C.

[0294] 1 H NMR (400MHz, CDCl3) δ8.09 (dd, J = 7.8, 1.8Hz, 1H), 7.78–7.66 (m, 1H), 7.44–7.29 (m, 7H), 5.23 (s, 2H), 4.83 (s, 2H).

[0295] 13 C NMR (100MHz, CDCl3) δ167.08,160.25,152.73,147.99,136.67,135.04,128.78,128.68,128.44,128.39,125.81,116.74,113.87,67.78,43.14.

[0296] Crop antibacterial activity test

[0297] The bactericidal activity of N-(ethyl acetate)-1,3-benzoxazine-2,4-dione, N-(2-ethyl phenylacetate)-1,3-benzoxazine-2,4-dione, N-(2-(4-chlorophenyl)acetic acid methyl ester)-1,3-benzoxazine-2,4-dione, N-(2-(4-chlorophenyl)acetic acid ethyl ester)-1,3-benzoxazine-2,4-dione, N-((4-methyl)valerate ethyl ester)-1,3-benzoxazine-2,4-dione, N-((4-methylthio)butyrate methyl ester)-1,3-benzoxazine-2,4-dione, and N-(benzyl acetate)-1,3-benzoxazine-2,4-dione was tested using an in vitro method.

[0298] Using *Fusarium head blight*, *Phytophthora*, *Magnapordica oryzae*, *Magnapordica rice*, *Sclerotinia sclerotiorum*, *Botrytis cinerea*, or *Sheath blight* as test materials for fungicidal activity testing, the test agent was dissolved in acetone and then diluted to a 500 g / mL solution with 200 g / mL sorporl-144 emulsifier. Under aseptic conditions, 1 mL of the compound solution was pipetted into a sterilized Petri dish, followed by 9 mL of sterile PDA culture medium. The mixture was then stirred to prepare the appropriate concentration of drug-containing plates. Under aseptic conditions, mycelial cakes were cut from the edge of the colony using a 4 mm diameter sterile punch. After the culture medium solidified, the mycelial cakes were inoculated into the center of the drug-containing plate using an inoculator and incubated at a suitable temperature. A blank control was prepared without the added agent. Each treatment was incubated in an incubator at 24±1℃. After 72 hours, the diameter of the colonies was observed and measured. The diameter of each colony was measured vertically once using the cross-sectional method, and the average value was taken.

[0299] Growth inhibition rate (%) = (control colony diameter - treated colony diameter) × 100 / (control colony diameter - 4 mm).

[0300] The drug concentration was 50 μg / mL. The results of the bactericidal activity test are shown in Table 3 below.

[0301] Table 3. Antibacterial activity results of N-ester-1,3-benzoxazine-2,4-dione compounds (inhibition rate / %)

[0302]

[0303]

[0304] Table 3 shows that the target compounds exhibit good inhibitory activity against the tested pathogens. Specifically, N-(ethyl acetate)-1,3-benzoxazine-2,4-dione showed an inhibition rate of 100.0% against *Pythium oryzae* and 86.1% and 82.6% against *Phytophthora blight* and *Sheath blight*, respectively; N-(ethyl acetate 2-phenylacetate)-1,3-benzoxazine-2,4-dione showed an inhibition rate of 70.7% against *Sheath blight*; and N-((4-methyl)valerate)-1,3-benzoxazine-2,4-dione showed an inhibition rate of 70.8% against *Sclerotinia sclerotinia*.

[0305] Antitumor activity test

[0306] Cell growth assay (CCK8 assay) was performed by adjusting the suspension of hepatocellular carcinoma cells (HCCLM3) to 5 × 10⁻⁶ cells / mL. 4 / mL (adjust suspension cells to 10) 5Cells were seeded into 96-well plates (100 μL / well), 5000 cells / well. After 4 h of seeding, the antitumor activity of N-(ethyl acetate)-1,3-benzoxazine-2,4-dione, N-(2-(4-chlorophenyl)acetic acid methyl ester)-1,3-benzoxazine-2,4-dione, N-(2-(4-chlorophenyl)acetic acid ethyl ester)-1,3-benzoxazine-2,4-dione, N-((4-methyl)valerate)acetic acid methyl ester)-1,3-benzoxazine-2,4-dione, N-((4-methylthio)butyrate methyl ester)-1,3-benzoxazine-2,4-dione, and N-(benzyl acetate)-1,3-benzoxazine-2,4-dione was tested. Add 100 μL of culture medium containing different concentrations of the compound to each well, with six replicates for each concentration. Wells without cells serve as blank controls, wells with cells but without the compound serve as negative controls for the compound, and 5-fluorouracil serves as a positive control for the compound. Incubate at 37°C and 5% CO2 for 24 h, then add 10 μL of CK8 staining solution to each well and continue incubation. After 4 h, transfer to 37°C and shake for 5-10 min. Measure the absorbance (OD) value of each well at 450 nm using a microplate reader. The cell growth inhibition rate is calculated using the following formula:

[0307] Cell survival inhibition rate (%) = (OD value of negative control group - OD value of experimental group) × 100% / (OD value of negative control group - OD value of blank control group).

[0308] The drug concentration was 50 μmol / L. The results of the antitumor activity test are shown in Table 4.

[0309] Table 4. Results of antitumor activity tests on N-ester-1,3-benzoxazine-2,4-dione compounds.

[0310]

[0311] Table 4 shows that the target compounds exhibited good inhibitory activity against the tested liver cancer cells. Specifically, N-(ethyl acetate)-1,3-benzoxazine-2,4-dione showed an inhibition rate of up to 95.5% against liver cancer cells; N-(2-ethyl phenylacetate)-1,3-benzoxazine-2,4-dione showed an inhibition rate of up to 95.9% against liver cancer cells; N-(2-(4-chlorophenyl)methyl acetate)-1,3-benzoxazine-2,4-dione showed an inhibition rate of up to 89% against liver cancer cells; and N-(2-(4-chlorophenyl)ethyl acetate)-1,3-benzoxazine-2,4-dione showed an inhibition rate of up to 89% against liver cancer cells. The inhibition rate of N-((4-methyl)valerate)-1,3-benzoxazine-2,4-dione against liver cancer cells reached 74.3%, N-((4-methyl)valerate)-1,3-benzoxazine-2,4-dione against liver cancer cells reached 84.5%, N-((4-methylthio)butyrate)-1,3-benzoxazine-2,4-dione against liver cancer cells reached 95.4%, and N-(benzyl acetate)-1,3-benzoxazine-2,4-dione against liver cancer cells reached 97.8%.

Claims

1. An N-ester-1,3-benzoxazin-2,4-dione compound having the general structure (V) ###0001### characterized in that: The general structural formula (V) is: ； In the formula, R1 is a C1-C3 alkyl or benzyl group; R is one of C1-C4 alkyl, phenyl, chlorophenyl, benzyl, hydroxybenzyl, or methylthioethyl.

2. The compound of claim 1, wherein: R1 is CH3, CH2CH3, or benzyl; R is one of C1-C4 alkyl, phenyl, 3-chlorophenyl, 4-chlorophenyl, 4-hydroxybenzyl, or methylthioethyl.

3. An N-ester-based-1,3-benzoxazin-2,4-dione compound having the general structure (V) ###0002### characterized in that: The N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) is selected from one of the following compounds: N-(2-ethyl phenylacetyl)-1,3-benzoxazine-2,4-dione: ； N-(2-(4-chlorophenyl)acetic acid methyl ester)-1,3-benzoxazine-2,4-dione: ； N-(2-(4-chlorophenyl)ethyl acetate)-1,3-benzoxazine-2,4-dione: ； N-((4-methyl)valerate)-1,3-benzoxazine-2,4-dione: ； N-((4-methylthio)butyrate methyl ester)-1,3-benzoxazine-2,4-dione ； N-(benzyl acetate)-1,3-benzoxazine-2,4-dione: 。 4. A method for preparing N-ester-1,3-benzoxazine-2,4-dione compounds having the general structural formula (V), characterized in that: The method specifically includes the following steps: S1) An amino acid with structural formula (I) is reacted with an alcohol with structural formula (II) in the presence of thionyl chloride to obtain an amino acid ester hydrochloride with structural formula (III): ； S2) Salicylic acid is reacted with oxaloyl chloride to obtain salicyl chloride, which is then reacted with an amino acid ester hydrochloride having structural formula (III) to obtain an N-ester salicylamide compound having general structural formula (IV): ； S3) Reaction of N-ester salicylamide compounds with general structural formula (IV) with triphosgene yields N-ester-1,3-benzoxazine-2,4-dione compounds with general structural formula (V): ； In the formula, R1 is a C1-C3 alkyl or benzyl group; R is one of C1-C4 alkyl, phenyl, chlorophenyl, benzyl, hydroxybenzyl, or methylthioethyl.

5. The method according to claim 4, characterized in that: The amino acid is glycine, phenylglycine, chlorophenylglycine, tyrosine, leucine, or methionine.

6. The method according to claim 4, characterized in that: The alcohol is methanol, ethanol, or benzyl alcohol.

7. Use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) as described in any one of claims 1-3, characterized in that: The N-ester-1,3-benzoxazine-2,4-dione compounds having the general structural formula (V) are used to prepare antibacterial drugs for crops, specifically for preparing drugs that inhibit one or more of the following pathogens: Fusarium head blight, rice blast fungus, Phytophthora blight, Sclerotinia sclerotinia, Botrytis cinerea, and Sheath blight.

8. The use according to claim 7, characterized in that: The N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) was used to prepare a drug for inhibiting rice blast fungus.

9. Use of an N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) as described in any one of claims 1-3, characterized in that: The N-ester-1,3-benzoxazine-2,4-dione compound having the general structural formula (V) was used to prepare a drug for inhibiting liver cancer cells.

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