Amino acid directed phthalide analogs and uses thereof

Abstract

The present application belongs to the field of pharmaceutical chemistry, and relates to the prevention and treatment of peanut stemphylium leaf blight and peanut root rot, in particular to amino acid-oriented phthalide analogues and a preparation method and application thereof. The phthalide analogues have a structural general formula as shown in formula A or formula B: and formula B, wherein R and R' are selected from natural amino acids or unnatural amino acids, and the unnatural amino acids are substitution products of 4-NO2, 4-F or 4-Cl of amino acids. The results of bioactivity determination show that all the compounds have Sclerotium rolfsii mycelium growth inhibition activity, wherein the 5-substituted phthalide of phenylalanine and the 5-substituted phthalide of arginine in formula A have obvious bacteriostatic activity, and meanwhile, the results of pot experiments show that the 5-substituted phthalide of phenylalanine has good control effect. Therefore, the amino acid-oriented phthalide analogues have good development and application prospects.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to the prevention and control of peanut white mold disease and peanut root rot disease, especially to amino acid-directed phthalide analogues, their preparation methods and applications. Background Technology

[0002] Peanuts are widely distributed in my country. The main peanut-producing areas are Shandong, eastern Liaoning, the Leizhou Peninsula in Guangdong, the Huang-Huai-He River region, and the coastal hills and sandy areas of southeastern China. However, the prevalence and severity of peanut diseases have consistently constrained yield increases. Peanut diseases are often caused by pathogens and occur on the roots or leaves of plants, such as root rot, crown rot, damping-off, white mold, early leaf spot, brown spot, and late leaf spot. When two or more diseases occur simultaneously, they can cause 50%-70% yield losses. For example, statistics on peanut diseases in 2009 showed that early leaf spot caused losses of approximately US$326 million, rust caused losses of approximately US$467 million, and late leaf spot caused losses of approximately US$599 million. Among these, peanut white mold, caused by *Sclerotium sclerotiorum*, is a devastating soil-borne fungal disease. This disease is becoming increasingly serious in various peanut-producing areas of my country, and its distribution area is expanding year by year. Preliminary investigations show that in peanut-producing areas of Henan Province, the disease incidence rate in general fields is 10%-30%, reaching over 40% in severely affected fields. Under normal circumstances, yield losses in infected fields are 10%-20%, while severely affected fields suffer losses exceeding 50%, sometimes even resulting in total crop failure. This has become a significant factor restricting the safe production of peanuts in the province. Currently, the control of peanut white mold mainly relies on chemical methods. A search on the China Pesticide Information Network (http: / / www.chinapesticide.org.cn) reveals that the number of chemical agents registered for peanut white mold control in my country is relatively small, mainly consisting of succinate dehydrogenase inhibitors such as thifluzamide, fluopyram, and boscalid. These fungicides have a certain degree of toxicity to non-target organisms, and long-term use can easily lead to the development of pathogen resistance. Therefore, there is an urgent need to find new, green control agents.

[0003] Searching for potential highly active fungicides from natural products is feasible and of great significance. Currently registered fungicidal active ingredients such as carvacrol, eugenol, and allicin are all derived from plants. Our research group has long been engaged in the study of the antibacterial activity of plant-derived agricultural active substances, and has found that phthalide compounds in Ligusticum chuanxiong essential oil have good in vitro and in vivo antibacterial activity against white mold (ZL202110982319.9). Plant essential oils have good environmental compatibility and systemic properties. Research on their absorption and translocation properties in peanut plants is beneficial for better understanding their application in the control of peanut diseases. Amino acids are essential substances for maintaining normal growth and development in plants, and amino acid pesticides are highly effective in controlling many plant diseases, pests, and weeds. Amino acid fungicides are characterized by high efficiency, low toxicity, no environmental pollution, broad antibacterial spectrum, and can promote plant growth. Active amino acids as fungicides include amino acids and their hydrochlorides, amino acid metal complexes, N-acyl amino acids, amino acid esters, and fungicides containing amino acid structures. Meanwhile, plants contain abundant amino acid transport proteins that combine amino acid groups with pesticide active molecules in different ways to form amino acid-pesticide couplings. These couplings are then transported to the phloem by the abundant amino acid transport proteins in the plant, thereby improving the distribution of pesticides within the crop and enhancing their targeting. Summary of the Invention

[0004] This invention proposes an amino acid-directed phthalide analogue, its preparation method, and its application. By using the phthalide structure as a backbone and derivatizing it with amino acids, a novel, safe, and efficient amino acid-directed phthalide bactericide can be obtained.

[0005] The technical solution of this invention is implemented as follows:

[0006] Amino acid-directed phthalide analogs, characterized in that they have a general structural formula as shown in Formula A or Formula B:

[0007] In the formula:

[0008] R and R' are selected from natural amino acids or non-natural amino acids. Non-natural amino acids are substitution products of amino acids with 4-NO2, 4-F or 4-Cl.

[0009] The bridging bond between the amino acid and phthalide is located at position 4, 5, or 6 on the benzene ring.

[0010] The bridging bond in Formula A is an amide bond formed between a carboxyl phthalide and an amino acid; the bridging bond in Formula B is an amide bond formed between an amino phthalide and an amino acid.

[0011] Preferably, in Formula A, R is selected from phenylalanine and arginine substituted at the 5-position; preferably, in Formula B, R' is selected from phenylalanine substituted at the 6-position.

[0012] The compounds of formula A and formula B provided by this invention are synthesized using methods for solid-phase organic synthesis and liquid-phase synthesis of polypeptides, respectively, with the following specific steps:

[0013] The preparation steps of the phthalide analogue shown in Formula A are as follows:

[0014] (1) Rink Amide-AM resin was activated with dichloromethane and then a 50% (V / V) dichloromethane solution of DBU (1,8-diazabicycloundec-7-ene) was added. After the deprotection reaction, the resin was washed with DMF and DCM to obtain the activated resin.

[0015] (2) Add Fmoc-AA-OH, HBTU, HOBt and DIEA to the activated resin in step (1), then dissolve them in DMF and stir at room temperature to react. Remove the reaction solution, wash with DMF and DCM, then add a dichloromethane solution of DBU to carry out the deprotection reaction, and wash with DMF to obtain AA-Rink Amide-AM.

[0016] (3) Add 5-carboxyphthalide, HBTU, HOBt and DIEA to AA-Rink Amide-AM in step (2), then dissolve in DMF and stir at room temperature to react, remove the reaction solution, wash with DMF and DCM to obtain 5-carboxyphthalide-AA-Rink Amide-AM.

[0017] (4) After cleaving the 5-carboxyphthalimide-amino acid-RinkAmide-AM from step (3) with phenol, water, anisole and TFA as cleaving reagents, filter to remove TFA, add anhydrous diethyl ether, centrifuge to obtain a white precipitate, and separate and purify to obtain the phthalimide analog shown in formula A.

[0018] In the above Fmoc-AA-OH, AA refers to an amino acid selected from any one of glycine, alanine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, aspartic acid, glutamic acid, threonine, arginine, or phenylalanine substituted with 4-NO2, 4-F, or 4-Cl.

[0019] In step (2), the equivalent ratio of activated resin, Fmoc-AA-OH, HBTU, HOBt and DIEA is in the range of 1:3-4:3-4:3-4:6-8;

[0020] In step (3), the equivalent ratio of AA-Rink Amide-AM, 5-carboxyphthalide, HBTU, HOBt and DIEA is in the range of 1:3-4:3-4:3-4:6-8;

[0021] In step (4), the volume ratio of phenol, water, anisole and TFA is 1:2:1:36.

[0022] The preparation steps of the phthalide analogue shown in Formula B are as follows:

[0023] 1) Dissolve Boc-AA`-OH, 4-aminophthalide, HOBt and DIEA in DMF and stir at room temperature until the reaction is complete;

[0024] 2) Add water to the reaction solution obtained in step 1) until a viscous solid precipitates, then discard the supernatant and wash the solid with water;

[0025] 3) The solid treated in step 2) is dissolved in DCM, washed with water to remove the aqueous phase, and then the DCM is removed by rotary evaporation to obtain an oily product.

[0026] 4) Add hydrochloric acid solution to the oily product, stir until the reaction is complete, evaporate the water by rotary evaporation, separate and purify the crude product, remove acetonitrile from the obtained solution, and freeze-dry to obtain the phthalide analog shown in formula B.

[0027] The AA' mentioned above refers to phenylalanine.

[0028] In step 1), the equivalent ratio of Boc-AA`-OH, 4-aminophthalide, HOBt, and DIEA is in the range of 1:3-4:3-4:3-4:6-8.

[0029] The drug containing the amino acid-directed phthalide analogue of this application further includes a carrier selected from one or more of diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants, and stabilizers.

[0030] The application of the above-mentioned phthalide analogues or drugs in the prevention and control of peanut white mold and peanut root rot.

[0031] The in vitro bioassay of the compounds of this invention was performed using the mycelial growth rate method to test the in vitro activity of compounds A and B against Sclerotium rolfsii, Fusarium haematococca, Fusarium oxysporum, and Fusarium proliferatum.

[0032] The in vivo bioassay of the compounds of this invention was conducted using a pot experiment for control. All bioactivity assays of this invention were performed in accordance with the patent: Application of ligustilide and its contained plant essential oils in the control of peanut white mold disease (ZL202110982319.9).

[0033] When necessary, one or more carriers acceptable for pesticide formulations may be added to the drug, including conventional diluents, excipients, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants, stabilizers, etc., used in pesticide formulations. The resulting drug can be in various dosage forms, such as powders, microemulsions, baits, microcapsules, emulsifiable concentrates, etc.

[0034] The present invention has the following beneficial effects:

[0035] 1. This invention relates to a class of amino acid-directed phthalide analogs, using carboxylphthalide or aminophthalide as a lead compound. Through an active substructure splicing method, natural and non-natural amino acids are introduced to modify the compounds, resulting in a class of directed phthalide derivatives. Some of these compounds exhibit superior in vitro activity compared to the lead carboxylphthalide or aminophthalide, while also possessing certain in vivo activity. The compounds of this invention are safe, efficient, and simple to synthesize, demonstrating potential for future development and application.

[0036] 2. The bioactivity assay results of the compounds in this application show that some compounds have good inhibitory activity against the mycelial growth of *Sclerotium sclerotiorum*. Specifically, the phenylalanine-substituted phthalide and arginine-substituted phthalide in Formula A exhibit significant antibacterial activity. Furthermore, pot experiments show that the phenylalanine-substituted phthalide compound has good preventative efficacy. The amino acid-guided compounds of this invention promote root growth in peanut plants. Therefore, these amino acid-guided phthalide compounds have excellent development and application prospects.

[0037] 3. The amino acid-directed phthalide compound of formula A in this application is synthesized using a polypeptide solid-phase method. The use of DBU as a deprotecting agent in the synthesis process was screened to be 50% (V / V), which effectively avoids the use of piperidine, a precursor chemical. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 The A1 compound prepared in Example 1 1 1H NMR (400MHz) spectrum.

[0040] Figure 2 The A1 compound prepared in Example 1 13 C NMR (101MHz) plot.

[0041] Figure 3 The image shows a high-resolution mass spectrum (HRMS) of the A1 compound prepared in Example 1.

[0042] Figure 4 The B1 compound prepared in Example 13 1 1H NMR (400MHz) spectrum.

[0043] Figure 5 The B1 compound prepared in Example 13 13 C NMR (101MHz) plot.

[0044] Figure 6 The image shows a high-resolution mass spectrometry (HRMS) spectrum of the B1 compound prepared in Example 13.

[0045] Figure 7 The potted plant control effect of A1 and polyoxin on peanut white mold disease. Detailed Implementation

[0046] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0047] The compounds of formula A and formula B in this application were prepared using a solid-phase polypeptide synthesis method. The deprotecting agent for the Fmoc group was optimized.

[0048] The structural formula of the lead compound phthalide used in this application is as follows:

[0049]

[0050] The following description is based on specific embodiments:

[0051] Example 1

[0052] This example illustrates the preparation method of compound A1:

[0053]

[0054] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM (dichloromethane) for 3h, add 50% (V / V) DBU in dichloromethane solution to deprotect for 10min, wash 3 times each with DMF (N,N-dimethylformamide) and DCM, and monitor the reaction with Kaiser's reagent.

[0055] (2) Phe incorporation: Add Fmoc-Phe-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0056] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0057] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0058] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0059] The resulting products 1 The H NMR (400MHz) spectrum is as follows: Figure 1 As shown, 13 The C NMR (101MHz) spectrum is as follows: Figure 2 The high-resolution mass spectrometry (HRMS) image is shown below. Figure 3 As shown.

[0060] Compound A1: 1 H NMR (400MHz, DMSO-d6) δ8.84(d,J=8.5Hz,1H),8.03(s,1H),7.99–7.89(m,2H),7.65(s,1H),7.35(d,J=8.2Hz,2H),7.26(t,J=7.5Hz, 2H),7.21–7.12(m,2H),5.46(s,2H),4.69(ddd,J=10.6,8.5,4.0Hz,1H),3.15(dd,J=13.8,4.1Hz,1H),2.99(dd,J=13.8,10.8Hz,1H). 13C NMR(101MHz,DMSO-d6)δ173.56,170.53,165.84,147.77,139.89,138.91,129.58,1 28.58,128.55,127.54,126.71,125.27,122.54,70.46,55.41,37.71.HRMS(ESI):C 18 H 16 N₂O₄, [M+Na] + Calculated value: 347.1008; Measured value: 347.1014.

[0061] Example 2

[0062] This embodiment describes a method for preparing compound A2:

[0063] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0064] (2) Phe(4-NO2) incorporation: Add Fmoc-Phe(4-NO2)-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0065] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0066] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0067] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0068] Compound A2: 1H NMR (400MHz, DMSO-d6) δ8.94(d,J=8.6Hz,1H),8.19–8.12(m,2H),8.03(s,1H),7.94(q,J=7.9Hz,2H),7.72(s,1H),7.66–7.60(m ,2H),7.25(s,1H),5.46(s,2H),4.76(ddd,J=10.6,8.5,3.9Hz,1H),3.29(dd,J=13.6,4.0Hz,1H),3.13(dd,J=13.7,11.0Hz,1H). 13 C NMR(101MHz,DMSO-d6)δ172.99,170.51,165.95,147.79,147.36,146.67,139.68,1 30.91,128.58,127.62,125.31,123.70,122.56,70.47,54.80,37.51.HRMS(ESI):C 18 H 15 N3O6,[M+Na] + Calculated value: 392.0859; Measured value: 392.0862.

[0069] Example 3

[0070] This embodiment describes a method for preparing compound A3:

[0071] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0072] (2) Phe(4-F) incorporation: Add Fmoc-Phe(4-F)-OH (4 equivalents), HBTU (4 equivalents), HOBt (4 equivalents), and DIEA (8 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0073] (3) Add 5-carboxyphthalide: Add 5-carboxyphthalide (3.5 equivalents), (3.5 equivalents), HOBt (3.5 equivalents), DIEA (7 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0074] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0075] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0076] Compound A3: 1 H NMR (400MHz, DMSO-d6) δ8.84(d,J=8.5Hz,1H),8.03(s,1H),7.94(q,J=8.0Hz,2H),7.65(s,1H),7.37(dd,J=8.3,5.6Hz,2H),7.19(s,1H ),7.09(t,J=8.9Hz,2H),5.46(s,2H),4.66(ddd,J=10.4,8.6,4.0Hz,1H),3.13(dd,J=13.8,4.0Hz,1H),2.97(dd,J=13.8,10.8Hz,1H). 13 C NMR(101MHz,DMSO-d6)δ173.41,170.53,165.87,162.58,160.18,158.99,158.62,147.78,139.84,135.05,1 35.02,131.43,131.35,128.56,127.56,125.28,122.53,115.35,115.15,70.46,55.41,36.87.HRMS(ESI):C 18 H 15 FN₂O₄,[M+Na) + Calculated value: 365.0914; Measured value: 365.0912.

[0077] Example 4

[0078] This embodiment describes a method for preparing compound A4:

[0079] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0080] (2) Phe(4-Cl) incorporation: Add Fmoc-Phe(4-Cl)-OH (4 equivalents), HBTU (4 equivalents), HOBt (4 equivalents), and DIEA (8 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0081] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0082] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0083] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0084] Compound A4: 1 H NMR(400MHz, DMSO-d6)δ8.90(dd,J=16.4,8.7Hz,1H),8.10–7.91(m,3H),7.69(d,J=16.7Hz,1H),7 .43–7.23(m,5H),5.55–5.45(m,2H),4.80–4.58(m,1H),3.20–3.13(m,1H),3.01(d,J=13.0Hz,1H). 13 C NMR(101MHz,DMSO-d6)δ173.30,170.52,165.87,147.79,139.79,137.96,131.46,1 31.41,128.57,128.50,127.58,125.29,122.55,70.47,55.20,37.03.HRMS(ESI):C 18 H 15 ClN₂O₄,[M+Na] + Calculated value: 381.0618; Measured value: 381.0625.

[0085] Example 5

[0086] This embodiment describes a method for preparing compound A5:

[0087] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0088] (2) Tyr inoculation: Add Fmoc-Tyr-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0089] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0090] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0091] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0092] Compound A5: 1 H NMR(400MHz, DMSO-d6)δ9.09(s,1H),8.68(d,J=8.4Hz,1H),7.96(s,1H),7.87(q,J=8.0Hz,2H),7.52(s,1H),7.05(d,J=8.2Hz,3H), 6.55(d,J=8.0Hz,2H),5.39(s,2H),4.51(ddd,J=10.3,8.4,4.0Hz,1H),2.94(dd,J=13.8,4.1Hz,1H),2.79(dd,J=13.8,10.6Hz,1H). 13C NMR(101MHz,DMSO-d6)δ173.70,170.55,165.79,156.16,147.78,139.96,130.50,1 28.91,128.58,127.51,125.27,122.55,115.35,70.47,55.77,36.96.HRMS(ESI):C 18 H 16 N₂O₅, [M+Na] + Calculated value: 363.0957; Measured value: 363.0958.

[0093] Example 6

[0094] This embodiment describes a method for preparing compound A6:

[0095] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0096] (2) Tyr inoculation: Add Fmoc-Tyr-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0097] (3) Pro: Add Fmoc-Pro-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0098] (4) Add 5-carboxyphthalide: Add 5-carboxyphthalide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0099] (5) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, vacuum dry and detect by mass spectrometry.

[0100] (6) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0101] Compound A6: 1 H NMR(400MHz, DMSO-d6)δ9.18(s,1H),8.10–7.54(m,4H),7.44–6.51(m,6H),5.48(s,1H),5.44–5.25(m,1H),4.53–4. 08(m,2H),3.51–3.35(m,2H),3.06–2.74(m,1H),2.61(td,J=13.8,7.2Hz,1H),2.15–1.98(m,1H),1.85–1.55(m,3H). 13 C NMR(101MHz,DMSO-d6)δ173.40,171.33,170.57,168.37,156.22,156.20,147.80,142.25,130.52,13 0.28,128.34,125.41,122.21,115.29,70.49,60.93,54.34,50.23,36.50,29.60,25.11.HRMS(ESI):C 23 H 23 N3O6,[M+Na] + Calculated value: 460.1485; Measured value: 460.1484.

[0102] Example 7

[0103] This embodiment describes a method for preparing compound A7:

[0104] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0105] (2) Arg inoculation: Add Fmoc-Arg-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0106] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0107] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0108] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0109] Compound A7: 1 H NMR (400MHz, DMSO-d6) δ8.75(d,J=7.9Hz,1H),8.15(s,1H),8.07(d,J=8.0Hz,1H),7.9 6(d,J=8.0Hz,1H),7.66(t,J=5.7Hz,1H),7.53(s,1H),7.30(s,1H),7.15(s,2H),6.97 (s,1H),5.49(s,2H),4.42(td,J=8.5,4.9Hz,1H),3.13(q,J=6.7Hz,2H),1.83(td,J=1 0.5,9.7,4.6Hz,1H),1.72(dd,J=14.3,9.6Hz,1H),1.56(td,J=15.7,13.7,8.0Hz,2H). 13 CNMR(101MHz,DMSO-d6)δ173.87,170.57,166.13,159.72,159.42,157.30,147.77,139.94, 128.79,127.56,125.25,122.69,118.92,115.96,70.46,53.62,29.16,25.92.HRMS(ESI):C15 H 19 N5O4, [M+H] + Calculated value: 334.1515; Measured value: 334.1516.

[0110] Example 8

[0111] This embodiment describes a method for preparing compound A8:

[0112] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0113] (2) Inoculation with Gln: Add Fmoc-Gln-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0114] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0115] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0116] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0117] Compound A8: 1H NMR (400MHz, DMSO-d6) δ8.82(d,J=7.7Hz,1H),8.15(s,1H),8.07(d,J=8.0Hz,1H),7.95(d,J=8.0Hz,1H),7.47(s,1H), 7.36(s,1H),7.10(s,1H),6.85(s,1H),5.49(s,2H),4.36(td,J=8.7,4.9Hz,1H),2.27–2.12(m,2H),2.11–1.84(m,2H). 13 CNMR(101MHz,DMSO-d6)δ174.47,173.78,170.58,165.93,147.79,139.98,128.72,127.54,125.26,122.65,70.48,53.93,32.16,27.69.HRMS(ESI):C 14 H 15 N3O5, [M+Na] + Calculated value: 328.0909; Measured value: 328.0909.

[0118] Example 9

[0119] This embodiment describes a method for preparing compound A9:

[0120] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0121] (2) Glu inoculation: Add Fmoc-Glu-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0122] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0123] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0124] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0125] Compound A9: 1 H NMR (400MHz, DMSO-d6) δ12.16(s,1H),8.71(d,J=7.8Hz,1H),8.15(s,1H),8.07(d,J=8.0Hz,1H),7.95(d,J=7.9Hz,1H),7. 49(s,1H),7.13(s,1H),5.49(s,2H),4.40(td,J=8.7,4.9Hz,1H),2.38–2.25(m,2H),2.13–1.99(m,1H),1.99–1.85(m,1H). 13 C NMR(101MHz,DMSO-d6)δ174.47,173.63,170.57,166.13,147.77,139.92,128.80,127.55,125.25,122.71,70.48,53.44,31.01,27.33.HRMS(ESI):C 14 H 14 N₂O₆,[M+Na] + Calculated value: 329.0750; Measured value: 329.0751.

[0126] Example 10

[0127] This embodiment describes a method for preparing compound A10:

[0128] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0129] (2) Asn incorporation: Add Fmoc-Asn-OH (3 equivalents), HBTU (3 equivalents), HOBt (3 equivalents), and DIEA (6 equivalents) to 5 mL of DMF. Stir at room temperature for 4 hours, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 minutes, and wash 3 times with DMF.

[0130] (3) Add 5-carboxyphthalimide: Add 5-carboxyphthalimide (3 equivalents), (3 equivalents), HOBt (3 equivalents), DIEA (6 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0131] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0132] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0133] Compound A10: 1 H NMR (400MHz, DMSO-d6) δ8.80(d,J=7.8Hz,1H),8.11(s,1H),8.03(d,J=8.0Hz,1H),7.96(dd,J=8.0,1.6Hz,1H),7 .43(s,1H),7.35(s,1H),7.12(s,1H),6.93(s,1H),5.49(s,2H),4.72(td,J=8.1,5.4Hz,1H),2.68–2.52(m,2H). 13 C NMR (101MHz, DMSO-d6) δ173.40,172.14,170.57,165.75,147.80,139.99,128.69,127.54,125.29,122.61,70.49,51.13,37.45.HRMS(ESI):C 13 H 13 N3O5, [M+Na] + Calculated value: 314.0753; Measured value: 314.0754. Example 11

[0134] This embodiment describes a method for preparing compound A11:

[0135] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5ml DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0136] (2) Asp inoculation: Add Fmoc-Asp-OH (4 equivalents), HBTU (4 equivalents), HOBt (4 equivalents), and DIEA (8 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0137] (3) Add 5-carboxyphthalide: Add 5-carboxyphthalide (4 equivalents), (4 equivalents), HOBt (4 equivalents), DIEA (8 equivalents), dissolve in 5 mL DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0138] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0139] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0140] Compound A11: 1 H NMR (400MHz, DMSO-d6) δ12.32(s,1H),8.90(d,J=7.8Hz,1H),8.12(s,1H),8.04(d,J=8.0Hz,1H),7.96(d,J=8.0Hz,1H),7.4 8(s,1H),7.17(s,1H),5.49(s,2H),4.75(td,J=8.4,5.0Hz,1H),2.80(dd,J=16.4,5.0Hz,1H),2.67(dd,J=16.5,8.9Hz,1H). 13CNMR(101MHz,DMSO-d6)δ172.89,172.41,170.57,165.87,147.77,139.90,128.76,127.57,125.28,122.67,70.48,50.79,36.51.HRMS(ESI):C 13 H 12 N₂O₆,[M+Na] + Calculated value: 315.0593; Measured value: 315.0587.

[0141] Example 12

[0142] This embodiment describes a method for preparing compound A12:

[0143] (1) Resin activation: Weigh 440mg Rink Amide-AM resin, activate it with 5mL DCM for 3h, add 50% (V / V) DBU dichloromethane solution to deprotect for 10min, wash with DMF and DCM 3 times each, and monitor the reaction with Kaiser's reagent.

[0144] (2) Ala inoculation: Add Fmoc-Ala-OH (4 equivalents), HBTU (4 equivalents), HOBt (4 equivalents), and DIEA (8 equivalents) to 5 mL of DMF. Stir at room temperature for 4 h, remove the reaction solution, and wash 3 times each with DMF and DCM. Monitor the reaction with Kaiser's reagent. Then add 50% (v / v) DBU dichloromethane solution for deprotection for 20 min, and wash 3 times with DMF.

[0145] (3) Add 5-carboxyphthalide: Add 5-carboxyphthalide (4 equivalents), (4 equivalents), HOBt (4 equivalents), DIEA (8 equivalents), dissolve in 5 mL of DMF, stir at room temperature for 4 h, remove the reaction solution, wash 3 times each with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0146] (4) Resin cutting: Add 0.25 mL phenol, 0.50 mL water, 0.25 mL anisole sulfide and 9.00 mL TFA to the resin, stir at room temperature for 2.5 h, filter to remove TFA, add 30 mL anhydrous ether, centrifuge at 3000 r / min for 5 min to obtain white precipitate, dry under vacuum and detect by mass spectrometry.

[0147] (5) The crude product was separated and purified by HPLC. After removing acetonitrile from the collected solution, the pure product was obtained by lyophilization. The chromatographic column was a C18 semi-preparative column.

[0148] Compound A12: 1H NMR (400MHz, DMSO-d6) δ8.75(d,J=7.4Hz,1H),8.14(s,1H),8.07(d,J=8.0Hz,1H),7.95(d,J=8 .0Hz,1H),7.46(s,1H),7.06(s,1H),5.49(s,2H),4.43(p,J=7.1Hz,1H),1.35(d,J=7.1Hz,3H). 13 C NMR (101MHz, DMSO-d6) δ174.62,170.57,165.64,147.77,139.98,128.75,127.50,125.23,122.68,70.47,49.48,18.41.HRMS(ESI):C 12 H 12 N₂O₄, [M+Na] + Calculated value: 271.0695; Measured value: 271.0695.

[0149] Example 13

[0150]

[0151] This example illustrates the preparation method of compound B1:

[0152] (1) Synthesis of B1: Add 4-aminophthalide (1 equivalent), Boc-Phe-OH (4 equivalent), N,N'-dicyclohexylcarbodiimide (6 equivalent), and 4-dimethylaminopyridine (0.05 equivalent) to 30 mL of DCM and stir at room temperature for 12 h until the reaction is complete.

[0153] (2) Dissolve the reactants in 10 mL of 90% trifluoroacetic acid and stir at room temperature for 1 h. Remove a large amount of trifluoroacetic acid by blowing nitrogen, and then add ice-cold anhydrous diethyl ether. The white solid precipitate that appears is the crude product.

[0154] (3) Use semi-preparative solution to separate and purify the compound, and obtain the final product by freeze drying.

[0155] The resulting products 1 The H NMR (400M) spectrum is as follows: Figure 4 The 13C NMR (101MHz) spectrum is shown below. Figure 5 The high-resolution mass spectrometry (HRMS) image is shown below. Figure 6 As shown.

[0156] Compound B1: 1H NMR (400MHz, DMSO-d6) δ10.68(d,J=4.4Hz,1H),8.50(s,2H),7.76(d,J=7.8Hz,1H),7.70(d,J=7.5Hz,1H),7.62(t,J=7.7 Hz,1H),7.36(dd,J=8.4,6.2Hz,2H),7.33–7.28(m,3H),5.24–5.10(m,2H),4.30(d,J=7.4Hz,1H),3.17(d,J=7.1Hz,2H). 13 CNMR(101MHz,DMSO-d6)δ170.62,167.57,159.31,158.99,158.68,139.25,135.14,132.45,130.42, 130.03,129.07,127.76,127.66,126.85,122.18,118.94,115.97,69.68,54.39,37.53.HRMS(ESI):C 17 H 16 N₂O₃, [M+H] + Calculated value: 297.1239; Measured value: 297.1242.

[0157] Example 14

[0158] This embodiment describes a method for preparing compound B2:

[0159] (1) Synthesis of B2: Add 6-aminophthalide (1 equivalent), Boc-Phe-OH (4 equivalent), N,N'-dicyclohexylcarbodiimide (6 equivalent), and 4-dimethylaminopyridine (0.05 equivalent) to 30 mL of DCM and stir at room temperature for 12 h until the reaction is complete.

[0160] (2) Dissolve the reactants in 10 mL of 90% trifluoroacetic acid and stir at room temperature for 1 h. Remove a large amount of trifluoroacetic acid by blowing nitrogen, and then add ice-cold anhydrous diethyl ether. The white solid precipitate that appears is the crude product.

[0161] (3) Use semi-preparative solution to separate and purify the compound, and obtain the final product by freeze drying.

[0162] Compound B2: 1H NMR(400MHz,DMSO-d6)δ10.76(s,1H),8.37(s,2H),8.07(d,J=1.8Hz,1H),7.66–7.56(m,2H),7 .24(d,J=6.8Hz,2H),7.23–7.17(m,3H),5.32(s,2H),4.14(t,J=7.1Hz,1H),3.16–3.01(m,2H). 13 C NMR (101MHz, DMSO-d6) δ170.94,167.77,159.27,158.96,143.19,139.04,135.13,129.92,129. 02,127.71,126.16,126.07,124.11,118.98,116.02,115.17,70.34,54.81,37.48.HRMS(ESI):C 17 H 16 N₂O₃, [M+H] + Calculated value: 297.1239; Measured value: 297.1245. Application example 1: Virulence of *Sclerotium sclerotiorum* hyphae growth.

[0163] The mycelial growth rate method was used for determination. 0.12 g of the compound prepared in Examples 1-14 was dissolved in 0.2 mL of DMSO and 0.2 mL of Tween-80, and 9.6 mL of sterile water was added to bring the volume to 10 mL to prepare a stock solution of 12000 mg / L, where the volume ratio of DMSO to Tween-80 was 2%. This solution was then diluted with sterile water to prepare six concentrations: 6000, 4000, 2000, 1000, 500, and 250 mg / L. 1 mL of each concentration was added to 9 mL of PDA medium to prepare six drug-containing media with final concentrations of 600, 400, 200, 100, 50, and 25 mg / L. A drug-free medium containing 0.05% DMSO and 0.05% Tween-80 was used as a control. Each concentration was repeated three times. The colonies were incubated in the dark at 28°C and the diameter of each treatment was measured using the cross-sectional method after 3 days.

[0164] According to the formula: mycelial growth inhibition rate = [(control colony diameter - mycelial cake diameter) - (treatment colony diameter - mycelial cake diameter)] / (control colony diameter - mycelial cake diameter) × 100;

[0165] The inhibition rates of the compounds prepared in Examples 1-15 on the growth of *Sclerotium oliguriae* hyphae were calculated, and the effective median concentration (EC50) was determined using probability value analysis. 50 The test results are shown in Table 1.

[0166] Table 1. Structure, physical properties, and in vitro inhibitory activity against *Sclerotium guillezei* of Formulas A and B.

[0167]

[0168]

[0169] Table 1 shows that: Compound A can inhibit the growth of *Sclerotium regia* hyphae, and the target compound A1 (EC) introduced by phenylalanine and arginine... 50 =332.21 mg / L) and A7 (EC 50 =379.41 mg / L) showed good inhibitory activity, while the activities of tyrosine-guided compound A5 and proline-tyrosine-guided compound A6 (EC) were significantly lower. 50 >600 mg / L) showed low inhibitory activity. The introduction of highly active amino acids was beneficial to enhancing activity, while the introduction of low-activity amino acids was detrimental to improving the activity of the target compound. The structure-activity relationship is shown in Table 1: (1) Electron-withdrawing groups at the four positions of the benzene ring of phenylalanine were detrimental to activity (A2-A4); (2) Amides at the end of the branched chain were more beneficial to activity than carboxylic acids (comparison of A8 and A9, comparison of A10 and A11); (3) The length of the side chain had little effect on activity. These structure-activity relationships provide guidance for the design and synthesis of subsequent compounds.

[0170] The inhibitory effects of 13 L / D configuration amino acids on the mycelial growth activity of *Sclerotium truncatum* were detected at a concentration of 600 mg / L. The bioactivity was determined by the mycelial growth rate method, as described above.

[0171] Table 2. Inhibitory activity of 13 L / D configuration amino acids against the mycelial growth of *Sclerotium regiatum* at a concentration of 600 mg / L.

[0172]

[0173]

[0174] Note: Data in the table are mean ± standard deviation.

[0175] Table 2 shows that the 13 L / D amino acids exhibited poor inhibitory activity against *Sclerotium riberi*. Combined with the bioassay results in Table 1, it indicates that structurally combining the highly active L-phenylalanine, L-arginine, L-glutamine, L-glutamic acid, L-asparagine, L-aspartic acid, and L-alanine (at a concentration of 600 mg / L) with the lead compound is beneficial for activity enhancement. Introducing the less active proline and tyrosine resulted in poor activity enhancement.

[0176] Application Example 2: Toxicity of the compound against the hyphal growth of Fusarium solanum, Fusarium oxysporum, and Fusarium moniliforme.

[0177] Taking compounds A1, A7, A8, and A10 as examples, and using the lead compound phthalide and the commercially available agent polyoxin as controls, the mycelial growth rate method was used to test their inhibitory activity on the mycelial growth of *Haematonectria haematococca*, *Fusarium oxysporum*, and *Fusarium proliferatum* at a concentration of 600 mg / L. The results are shown in Table 3.

[0178] Table 3. Antibacterial activity of some compounds of the present invention against Fusarium at a concentration of 600 mg / L.

[0179]

[0180] Note: Data in the table are mean ± standard deviation.

[0181] Table 3 shows that the selected compounds have certain inhibitory activity against Fusarium, with analogs A1 and A8 showing the best activity. This indicates that these compounds have certain activity in preventing and controlling peanut root rot caused by Fusarium solani, Fusarium oxysporum, and Fusarium moniliforme, and therefore have further application and development value.

[0182] Application Example 3: The effect of the compound on potted plant control of peanut white mold disease

[0183] The effect of A1 on the control of peanut white mold disease in potted plants was tested using the spray method.

[0184] The peanut variety used in the experiment was Yuhua 9326. Plump and uniformly sized peanut seeds were selected and placed in a pot. Sterile water was added, submerging one-third of the seeds, and the pot was placed in an incubator to soak for half a day. Then, substrate soil and vermiculite were mixed in a 3:1 volume ratio, and sterile water was added to adjust the soil moisture content to 70%. The mixture was then placed in a pot, and the germinated seeds were sown with the radicle facing down.

[0185] Artificial inoculation: One week after sowing, select healthy plants of uniform growth and size. Inoculate each plant with one mycelial cake at the base of the stem and cover with soil. Once mycelial growth is observed on the peanut plants and soil, spray the plant roots and surrounding soil with 600 mg / L A1 and 300 mg / L polyoxin, 10 mL of solution per plant. Sterile water containing 0.1% DMSO and 0.1% Tween-80 serves as a control. Each treatment consisted of 15 plants, replicated three times. Disease severity was assessed 5 and 7 days after application, and the disease index and control efficacy for different treatments were calculated. The results are shown in Table 4. (Control efficacy diagram shown). Figure 7As shown, after five days of treatment with A1 at a concentration of 600 mg / L, the plants grew well compared to the control, with a small amount of mycelium on the ground. Treatment with the commercially available polyoxin at 300 mg / L resulted in good plant growth, but with more mycelium on the ground, indicating a weaker control effect compared to A1 at 600 mg / L. After seven days of treatment with 600 mg / L A1, compared to 300 mg / L polyoxin, the peanut plants treated with 600 mg / L A1 showed better growth, indicating that compound A1 has good in vivo activity.

[0186] Table 4. Potted plant control efficacy against peanut white mold disease.

[0187]

[0188] Note: Data in the table are mean ± standard deviation.

[0189] As shown in Table 4, the control efficacy of A1 at a concentration of 600 mg / L was 53.69% and 57.75% at 5 and 7 days after application, respectively, which was better than that of the control agent polyoxin at 300 mg / L, indicating that A1 has development potential in the control of peanut white mold.

[0190] Application Example 4: The growth-promoting effect of compounds on peanuts

[0191] The tested variety was Yuhua 9326. Yuhua 9326 seeds of uniform size and plumpness were selected and soaked in a 0.3% hydrogen peroxide solution in the dark for 6 hours. After soaking, the seeds were rinsed 2-3 times with deionized water. The seeds were then evenly spread on a tray containing absorbent cotton and allowed to germinate in the dark for 3-5 days. When the cotyledons were fully expanded and small lateral roots had sprouted, robust seedlings of uniform size were transplanted into 12-cell hydroponic boxes (20cm long, 15cm wide, and 7cm high) containing 1.4L of deionized water for 3 days to equilibrate. Twenty seedlings were transplanted per pot. After equilibration, the experimental treatment was performed. A 600mg / L concentration of the compound of this invention and a water control were set up. Three replicates were set up, with 10 peanut plants in each replicate. Each replicate had two sampling and measurement times: 16 days and 24 days.

[0192] After the experiment, peanut seedlings from each treatment were collected for testing. Ten seedlings were taken from each replicate of each treatment, and the following measurements were taken: taproot length, main stem height, first lateral branch length, stem diameter, aboveground fresh weight, underground fresh weight, aboveground dry weight, underground dry weight, and total leaf dry weight. (See Table 5)

[0193] Table 5 shows the growth-promoting effects of compounds in Formula A at a concentration of 600 mg / L on peanuts.

[0194]

[0195] Note: Data in the table are mean ± standard deviation.

[0196] Table 5 shows that the entire series promotes the growth of peanut stems and roots, with A7, A8, and A10 exhibiting the most significant growth-promoting effects. This indicates that amino acid-directed phthalide compounds have a growth-promoting effect on peanut plants and show promising development potential in the control of peanut white mold disease.

[0197] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An amino acid-directed phthalide analogue, characterized in that, Having a general structural formula as shown in formula A or formula B: ； In formula A, R is Phe-NH2, Arg-NH2, Gln-NH2, Asn-NH2, or Ala-NH2; ； In formula B, R` represents Phe; R or R' is connected to the benzene ring via an amide bond.

2. The method for preparing the amino acid-directed phthaloyl analogue according to claim 1, characterized in that, The preparation steps of the phthalide analogue shown in Formula A are as follows: (1) Rink Amide-AM resin was activated with dichloromethane, and a dichloromethane solution of 50% V / V DBU was added to carry out the deprotection reaction. The resin was washed with DMF and DCM to obtain activated resin. (2) Add Fmoc-AA-OH, HBTU, HOBt and DIEA to the activated resin in step (1), then dissolve them in DMF and stir at room temperature to react. Remove the reaction solution, wash with DMF and DCM, and then add a 50% V / V DBU dichloromethane solution for deprotection reaction. After washing with DMF, AA-Rink Amide-AM is obtained; AA in Fmoc-AA-OH is selected from the following amino acids: Phe, Arg, Gln, Asn or Ala; (3) Add 5-carboxyphthalide, HBTU, HOBt and DIEA to AA-Rink Amide-AM in step (2), then dissolve in DMF and stir at room temperature to react, remove the reaction solution, wash with DMF and DCM to obtain 5-carboxyphthalide-AA-Rink Amide-AM; (4) After cleaving the 5-carboxyphthalimide-amino acid-RinkAmide-AM from step (3) with phenol, water, anisole and TFA as cleaving reagents, filter to remove TFA, add anhydrous diethyl ether, centrifuge to obtain a white precipitate, and then lyophilize and freeze to obtain the phthalimide analog shown in formula A.

3. The method for preparing amino acid-directed phthalide analogs according to claim 2, characterized in that: In step (2), the equivalent ratio of activated resin, Fmoc-AA-OH, HBTU, HOBt and DIEA is in the range of 1:3-4:3-4:3-4:6-8; in step (3), the equivalent ratio of AA-Rink Amide-AM, 5-carboxyphthalide, HBTU, HOBt and DIEA is in the range of 1:3-4:3-4:3-4:6-8; in step (4), the volume ratio of phenol, water, anisole and TFA is 1:2:1:

36.

4. The method for preparing the amino acid-directed phthaloyl analogue according to claim 1, characterized in that, The preparation steps of the phthalide analogue shown in Formula B are as follows: 1) Dissolve Boc-Phe-OH, 6-aminophthalide, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine in DCM and stir at room temperature until complete. 2) Dissolve the reactants in 90% trifluoroacetic acid and stir at room temperature for 1 h. Remove a large amount of trifluoroacetic acid by blowing nitrogen, and then add ice-cold anhydrous diethyl ether. The white solid precipitate that appears is the crude product. 3) The crude product was separated and purified using a semi-preparative solution, and the phthalide analogue shown in Formula B was obtained by freeze drying.

5. The method for preparing amino acid-directed phthalide analogs according to claim 4, characterized in that, In step 1), the equivalent ratio of Boc-Phe-OH, 6-aminophthalide, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine is in the range of 1:4:6:0.

05.

6. The use of the amino acid-directed phthalide analogue of claim 1 in the preparation of a drug for preventing and treating peanut white mold disease.

7. The use of the amino acid-directed phthaloyl analogue of Formula A as described in claim 1 in the preparation of a drug for preventing and controlling peanut root rot.

8. A drug, characterized in that: It includes the amino acid-directed phthaloyl analogue as described in claim 1.

9. The medicament according to claim 8, characterized in that: It also includes one or more of the following: diluents, excipients, fillers, binders, wetting agents, absorption promoters, lubricants, and stabilizers.

10. The medicament according to claim 9, characterized in that: The dosage form of the drug is any one of powder, microemulsion, microcapsule, and emulsifiable concentrate.

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