Verlamelin cyclic peptide derivative / isomer, preparation method thereof and application of verlamelin cyclic peptide derivative / isomer in resisting growth of plant pathogenic fungi

Abstract

The invention discloses verlamelin cyclic peptide derivatives / isomers as well as a preparation method and application of the verlamelin cyclic peptide derivatives / isomers in resisting growth of plant pathogenic fungi. The preparation method is simple in process, low in price, suitable for large-scale production and reliable and stable in source. The verlamelin A and the derivative / isomer thereof have good antifungal activity, so that the verlamelin A and the derivative / isomer thereof can be used for preparing antifungal drugs, are applied to research of plant pathogenic fungi and have great popularization and application potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pesticide chemical industry, and particularly relates to a verlamelin cyclic peptide derivative / isomer, a preparation method thereof and application of the verlamelin cyclic peptide derivative / isomer in resisting growth of plant pathogenic fungi. BACKGROUND

[0002] Plant pathogenic fungi can cause destructive diseases in natural habitats and agricultural environments, thereby threatening the biodiversity of plants and global food security. Fungi are the biggest biological threat to crop production, causing extremely serious damage to yield and quality, and resulting in a crop loss of up to 50% in some developing countries. With the widespread use of antibacterial drugs, the drug resistance of plant pathogenic fungi is increasing, and therefore it is crucial to find and develop new candidate drugs against fungi. Natural cyclic peptide verlamelin A is a cyclic hexalipopeptide compound composed of L-valine, L-glutamine, L-proline, D-alanine, D-tyrosine, D-isothreonine and a 5-hydroxytetradecanoic acid chain. Studies have shown that natural cyclic peptide verlamelin A has good antibacterial activity against plant pathogenic fungi such as apple black spot (Alternaria alternate), tomato early blight (Alternaria solani), peanut sclerotium blight (Rhizoctonia solani) and rice blast fungus (Pyricularia oryaza HNM1003). However, natural cyclic peptide verlamelin A is prepared by fungal culture, and the process is complex and difficult. The target compound can be synthesized efficiently and cheaply by solid-phase synthesis. Through structural modification, the activity and physicochemical properties of the derivative / isomer can be improved, and highly efficient anti-plant pathogenic fungal compounds can be found. Since the compounds have a wide range of and high efficiency of anti-plant pathogenic fungal activity, they can provide protection for agricultural production and food abundance. SUMMARY

[0003] The application provides a chemical preparation method of natural cyclic peptide verlamelin A and derivatives or isomers thereof (a total of 6 compounds), which is prepared by solid-phase synthesis method and can be completed in 3 days, and the synthesis is efficient.

[0004] The application provides a compound 5R-OH-verlamelin A, 5S-glu-verlamelin A (gul-verlamelin A) and 5R-OH-glu-verlamelin A with enhanced anti-plant pathogenic fungal activity.

[0005] The verlamelin A and derivatives or isomers thereof have the following structural formula:

[0006]

[0007] The preparation method of the natural cyclic peptide verlamelin A and its isomer 5R-OH-verlamelin A comprises the following steps:

[0008] 1) Compound I (N-Fmoc-D-tyrosine-O-tert-butyl ether) is loaded on the resin through the carboxyl group, the Fmoc protecting group is removed, and then FMOC-L-glutamine, FMOC-L-proline, FMOC-D-alanine, and FMOC-D-allothreonine are sequentially subjected to condensation reaction under the action of a condensation reagent, and the Fmoc protecting group is removed after each condensation reaction, to finally obtain intermediate III:

[0009]

[0010] 2) The product of hydrolysis of the racemic tetradecanoic acid lactone IV under alkaline conditions is reacted with allyl bromide to obtain intermediate V, the hydroxyl group of intermediate V is reacted with FMOC-L-valine under the action of a condensation reagent, and the allyl group is removed in the presence of a palladium reagent to obtain intermediate VI:

[0011]

[0012] 3) Intermediate III of step 1) and intermediate VI of step 2) are condensed under the action of a condensation reagent, and then the Fomc group is removed and the resin is removed to obtain compound VII:

[0013]

[0014] 4) Compound VII of step 3) is subjected to ring closure under the action of a ring closure reagent, and the protecting group is removed under acidic conditions to obtain a mixture of verlamelin A and the non-corresponding isomer 5R-OH-verlamelin A with R configuration of the long side chain 5-OH, and then the pure product is obtained by HPLC preparation and separation:

[0015]

[0016] Preferably, in step 1), the resin is Wang resin, 2-CTC resin, etc.; and the condensation reagent in step 1) is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N,N'-diisopropyl carbodiimide (DIC), 2-(7-azobenzenetriazole)-tetramethyl urea hexafluorophosphate (HATU), (7-azabenzotriazole-1-oxyl) tripyrrolidine phosphate (pyAOP), 1H-benzotriazole-1-yl oxy tripyrrolidine hexafluorophosphate (pyBOP), etc.

[0017] Preferably, in step 1), the molar ratio of N-Fmoc-D-ornithine-O-tert-butyl ether (compound I) to FMOC-L-glutamine, FMOC-L-proline, FMOC-D-alanine, FMOC-D-allothreonine is 1:(1-1.5):(1-1.5):(1-1.5):(1-1.5), respectively.

[0018] Preferably, in step 1), the molar ratio of the condensing reagent to compound I is (1.5-2.5):1 in each condensation reaction.

[0019] Preferably, in step 1), the Fmoc protecting group is removed by using an organic base after each condensation reaction, and the organic base is piperidine, diethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, etc. Further preferably, the molar ratio of the organic base to compound I is (140-250):1.

[0020] Preferably, in step 2), the molar ratio of racemic tetradecanoic acid lactone IV to FMOC-L-valine, condensing reagent, palladium reagent is 1:(1-1.5):(1-1.5):(0.1-0.5).

[0021] Preferably, in step 2), LiOH, NaOH, KOH, etc. are added to form an alkaline condition; the condensing reagent in step 2) is DIC, EDC, tetramethylchloro uric acid hexafluorophosphate, etc.; the palladium reagent in step 2) is palladium acetate, tetraphenylphosphine palladium base, etc.

[0022] Preferably, in step 2), the allyl group is removed to obtain intermediate VI in the presence of palladium reagent and phenylsilane.

[0023] Preferably, in step 3), the molar ratio of intermediate III to intermediate VI, condensing reagent is 1:(2-2.5):(1.5-2.5).

[0024] Preferably, in step 3), an organic base is used to remove the Fomc group, and trifluoroethanol is used to remove the resin. Further, the molar ratio of the organic base to intermediate III is (140-250):1. More preferably, the organic base is Et2NH.

[0025] Preferably, in step 3), the condensing reagent is 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide hydrochloride (EDC), N,N'-diisopropyl carbodiimide (DIC), 2-(7-azobenzotriazole)-tetramethyl uric acid hexafluorophosphate (HATU), (7-azabenzotriazole-1-oxyl)tripyrrolidinophosphonium hexafluorophosphate (pyAOP), 1H-benzotriazole-1-yl oxytripyrrolidinyl hexafluorophosphate (pyBOP), etc.

[0026] Preferably, in step 4), the cyclization reagent comprises (7-azabenzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (pyAOP), N-hydroxy-7- azabenzotriazol (HOAt) and N,N-diisopropylethylamine; the molar ratio of compound VII to pyAOP, HOAt is 1: (5-10) : (1-3).

[0027] Preferably, in step 4), the acid condition is formed by adding dilute hydrochloric acid, trifluoroacetic acid, methanesulfonic acid, etc.

[0028] Preferably, in step 4), the HPLC condition is as follows: the chromatographic column is nanochrom C18 column, the mobile phase is methanol / water, the water phase contains 0.4% formic acid, and the flow rate is 2.5 mL / min. Further, the chromatographic column specification is 250 mm, 5 μm, the mobile phase ratio is methanol / water = 80 / 20 (v / v), the water phase contains 0.4% formic acid, the injection volume is 80 μL, and the HPLC instrument is Hitachi; verlamelin A is eluted prior to 5R-OH-verlamelin A.

[0029] The present application also provides a preparation method of the above-mentioned compound glu-verlamelin A and its non-corresponding isomer 5R-OH-glu-verlamelin A, which is to replace FMOC-L-glutamine in step 1) of the preparation method of the above-mentioned natural cyclic peptide verlamelin A and its isomer 5R-OH-verlamelin A with FMOC-L-glutamic acid, and the remaining steps are the same, and step 4) is used to synthesize a mixture of glu-verlamelin A and 5R-OH-glu-verlamelin A, and then the pure product is obtained by HPLC preparation separation. Preferably, the HPLC condition is as follows: the chromatographic column is nanochrom C18 column, the mobile phase is methanol / water, the water phase contains 0.4% formic acid, and the flow rate is 2.5 mL / min. Further, the chromatographic column specification is 250 mm, 5 μm, the mobile phase ratio is methanol / water = 80 / 20 (v / v), the water phase contains 0.4% formic acid, the injection volume is 80 μL, and the HPLC instrument is Hitachi; glu-verlamelin A is eluted prior to 5R-OH-glu-verlamelin A.

[0030] The application also provides a preparation method of the above-mentioned compound arg-verlamelin A and its non-enantiomeric 5R-OH-arg-verlamelin A, which is to replace FMOC-L-glutamine in step 1) of the preparation method of the above-mentioned natural cyclic peptide verlamelin A and its isomer 5R-OH-verlamelin A with FMOC-L-arginine, and the remaining steps are the same, and a mixture of arg-verlamelin A and 5R-OH-arg-verlamelin A is synthesized in step 4), and then pure products are obtained by HPLC preparation separation. Preferably, the HPLC conditions are as follows: the chromatographic column is a nanochrom C18 column, the mobile phase is methanol / water, the water phase contains 0.4% formic acid, and the flow rate is 2.5 mL / min. Further, the chromatographic column specification is 250 mm, 5 μm, the mobile phase ratio is methanol / water = 75 / 25 (v / v), the water phase contains 0.4% formic acid, the injection volume is 80 μL, and the Hitachi liquid chromatograph is used; arg-verlamelin A is eluted before 5R-OH-arg-verlamelin A.

[0031] The application also provides application of the above-mentioned compounds 5R-OH-verlamelin A, glu-verlamelin A and 5R-OH-glu-verlamelin A in resisting growth of plant pathogenic fungi.

[0032] Preferably, the plant pathogenic fungi are plant pathogenic fungi of apple black spot (caused by Alternaria alternate), tomato early blight (caused by Alternaria solani), peanut sclerotium blight (caused by Rhizoctonia solani), rice blast (caused by Pyricularia oryaza HNM 1003), corn ear rot (caused by Gibberella zeae) and wheat scab (caused by Gibberella sanbinetti).

[0033] Some reports currently consider that verlamelin A and derivatives / isomers have good antifungal activity. The chemical preparation method of verlamelin A and derivatives / isomers of the application has not been reported in the literature, and therefore the chemical synthesis of such compounds cannot be studied. In addition, the method of fungal culture separation in the literature is very complex, and the total yield is very low. Therefore, it is of great significance and great practical value to study the preparation method of such verlamelin A and derivatives / isomers and their antifungal activity.

[0034] The application has the following beneficial effects:

[0035] 1) The present application provides derivatives or isomers 5R-OH-verlamelin A, glu-verlamelin A, 5R-OH-glu-verlamelin A of verlamelin A, which have strong inhibitory activity on the pathogenic fungi of apple black spot and tomato early blight, are significantly enhanced relative to natural products verlamelin A, are expected to be used for preparing antifungal drugs after subsequent research and development, and have great application potential.

[0036] 2) The present application provides a preparation method of verlamelin A and its derivatives / isomers (a total of 6 compounds), which is simple in process, low in price, suitable for large-scale production, and reliable and stable in source. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a liquid phase diagram of natural product verlamelin A.

[0038] Figure 2 is a liquid phase diagram of chemically synthesized verlamelin A in Example 1.

[0039] Figure 3 is a liquid phase diagram of natural product verlamelin A as a control mixed with chemically synthesized verlamelin A in Example 1. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0041] Example 1: Synthesis of verlamelin A and 5R-OH-verlamelin A

[0042]

[0043] The preparation method of verlamelin A and 5R-OH-verlamelin A comprises the following steps:

[0044] Step one, compound 1 (N-FMOC-D-Tyrosine-O-t-butyl ether) was weighed into anhydrous dichloromethane 30 ml (DCM) and N,N-diisopropyl ethylamine (DIPEA) 1 ml, dissolved, reacted with 1 g of CTC-resin swelled in anhydrous dichloromethane overnight in a solid-phase synthesis tube at room temperature for 3 h to obtain compound 2, wherein compound 1 was weighed at 1-1.5 equivalents per gram of resin; compound 2 was capped by adding 2 ml of N,N-diisopropyl ethylamine, 4 ml of methanol and 34 ml of anhydrous dichloromethane, reacted at room temperature for 30 min, then 15 ml of acetonitrile, 15 ml of diethylamine were added and reacted at room temperature for 40 min to remove the protecting group to obtain compound 3; 1.25 equivalents of FMOC-L-Gln, 2.5 equivalents of 1-hydroxybenzotriazole (HOBT), 2.5 equivalents of N,N-diisopropyl carbodiimide (DIC) and 2.5 equivalents of N,N-diisopropyl ethylamine (DIPEA) were dissolved in 30 ml of N,N-dimethylformamide (DMF), added to the solid-phase synthesis tube containing compound 3, and reacted at room temperature for 3 h to obtain compound 4, wherein the molar ratio of compound 3 to FMOC-L-Gln, 1-hydroxybenzotriazole (HOBT), N,N-diisopropyl carbodiimide (DIC), N,N-diisopropyl ethylamine is 1:1.25:2.5:2.5:2.5; the protecting group of compound 4 was removed by adding acetonitrile and diethylamine to react at room temperature for 40 min to obtain compound 5, the volume ratio of diethylamine to acetonitrile is 1:1, and the molar ratio of diethylamine to compound 1 is 200:1; 1.25 equivalents of FMOC-L-Pro, 2.5 equivalents of 1-hydroxybenzotriazole (HOBT), 2.5 equivalents of N,N-diisopropyl carbodiimide (DIC) were dissolved in 30 ml of N,N-dimethylformamide (DMF), added to the solid-phase synthesis tube containing compound 5, and reacted at room temperature for 3 h to obtain compound 6, wherein the molar ratio of compound 5 to FMOC-L-Pro, 1-hydroxybenzotriazole (HOBT), N,N-diisopropyl carbodiimide (DIC) is 1:1.25:2.5:2.5; the protecting group of compound 6 was removed by adding acetonitrile and diethylamine to react at room temperature for 40 min to obtain compound 7, the volume ratio of diethylamine to acetonitrile is 1:1, and the molar ratio of diethylamine to compound 1 is 200:1; 1.25 equivalents of FMOC-D-Ala, 2.5 equivalents of 1-hydroxybenzotriazole (HOBT), 2.5 equivalents of N,N-diisopropyl carbodiimide (DIC) were dissolved in 30 ml of N,N-dimethylformamide (DMF), added to the solid-phase synthesis tube containing compound 7, and reacted at room temperature for 3 h to obtain compound 8, the molar ratio of compound 7 to FMOC-D-Ala, 1-hydroxybenzotriazole (HOBT), N,N-diisopropyl carbodiimide (DIC) is 1:1.25:2.5:2.5; the compound 8 is added with acetonitrile, diethylamine, and the protecting group is removed after reaction at room temperature for 40 min to obtain compound 9, the volume ratio of diethylamine to acetonitrile is 1:1, and the molar ratio of diethylamine to compound 1 is 200:1; 1.25 equivalents of FMOC-D-allo-Thr, 2.5 equivalents of 1-hydroxybenzotriazole (HOBT), and 2.5 equivalents of N,N-diisopropylcarbodiimide (DIC) are dissolved in 30 ml of N,N-dimethylformamide (DMF) and added to the solid-phase synthesis tube containing compound 9, and reaction is carried out at room temperature for 3 h to obtain compound 10, the molar ratio of compound 9 to FMOC-D-allo-Thr, 1-hydroxybenzotriazole (HOBT), and N,N-diisopropylcarbodiimide (DIC) is 1:1.25:2.5:2.5; the compound 10 is added with acetonitrile, diethylamine, and the protecting group is removed after reaction at room temperature for 40 min to obtain compound 11, the volume ratio of diethylamine to acetonitrile is 1:1, and the molar ratio of diethylamine to compound 1 is 200:1.

[0045] The specific synthesis route is as follows:

[0046]

[0047]

[0048] Step two, compound 12 is dissolved in methanol, hydrolysis is carried out by adding sodium hydroxide and water, and compound 13 is generated after reaction for 4 h, wherein the molar ratio of compound 12 to sodium hydroxide is 1:5; compound 13 is reacted with allyl bromide and potassium carbonate for 4 h to generate compound 14, wherein the molar ratio of compound 13 to allyl bromide and potassium carbonate is 1:4:1.5; compound 14 is dissolved in acetonitrile, and condensation reaction is carried out by adding FMOC-L-Val, 1-methylimidazole (NMI), and tetramethylchloroformamidinium hexafluorophosphate (TCFH), and compound 15 is obtained after reaction for 3 h, wherein the molar ratio of compound 14 to FMOC-L-Val, 1-methylimidazole, and tetramethylchloroformamidinium hexafluorophosphate is 1:1.5:2.1:1.1; compound 15 is dissolved in anhydrous dichloromethane, tetra-triphenylphosphine palladium and phenylsilane are added, the allyl group is removed, and compound 16 is obtained, wherein the molar ratio of compound 15 to tetra-triphenylphosphine palladium and phenylsilane is 1:0.1:2; the synthesis route is as follows:

[0049]

[0050]

[0051] Step three, compound 11 was reacted with a mixture solution of compound 16, 1- hydroxybenzotriazole, N,N-diisopropylcarbodiimide dissolved in N,N-dimethylformamide overnight to obtain compound 17, wherein the molar ratio of compound 11 to compound 16, 1- hydroxybenzotriazole, N,N-diisopropylcarbodiimide was 1:2.5:2.5:2.5; compound 17 was reacted with acetonitrile, diethylamine to remove the protecting group for 40 min to obtain compound 18, wherein the volume ratio of acetonitrile to diethylamine was 1:1, and the molar ratio of diethylamine to compound 11 was 200:1; a mixed solution of trifluoroethanol (TFE) and dichloromethane was added to compound 18 to remove the resin to obtain compound 19, wherein the volume ratio of trifluoroethanol (TFE) to dichloromethane (DCM) was 1:4; the synthetic route is as follows:

[0052]

[0053]

[0054] Step four, compound 19 was dissolved in anhydrous dichloromethane, and a highly diluted solution of N-hydroxy-7-azabenzotriazole (HOAT), (7-azabenzotriazol-1- yloxy)tripyrrolidinophosphonium hexafluorophosphate (pyAOP) and N,N- diisopropylethylamine was added for cyclization, and the reaction was carried out overnight to obtain compound 20, wherein the molar ratio of compound 19 to condensing agent N- hydroxy-7-azabenzotriazole (HOAT), (7-azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (pyAOP) and N,N-diisopropylethylamine (DIPEA) was 1:3:10:20; a mixed solution of trifluoroacetic acid and dichloromethane was added to compound 20 to remove the protecting group to obtain compound 21, wherein the volume ratio of trifluoroacetic acid to dichloromethane was 1:4; the compound 21 was a mixture of verlamelin A and its isomer 5R-OH-verlamelin A, which was separated by high performance liquid chromatography. The liquid chromatography conditions were as follows: nanochrom C18 column, 250 mm, 5 μm, the mobile phase was methanol / water = 80 / 20 (v / v), the water phase contained 0.4% formic acid, the flow rate was 2.5 mL / min, the injection volume was 80 μL, the Hitachi liquid phase instrument, the liquid phase retention time of verlamelin A was 19 min, and the liquid phase retention time of 5R-OH-verlamelin A was 25.5 min, and the yield was 5.4% and 6.1%, respectively; the synthetic route is as follows:

[0055]

[0056] The compound synthesized in Example 1 was subjected to mass spectrometry. The mass spectrometry data of compound verlamelin A and 5R-OH-verlamelin A were: HRMS-ESI (m / z): Calcd for C 48 H 71 N7O 11 [M+H] + 885.5212, Found 885.5276; HRMS-ESI (m / z): Calcd for C 48 H 71 N7O 11 [M+H] + 885.5212, Found 885.5266.

[0057] Example 2: Synthesis of glu-verlamelin A and 5R-OH-glu-verlamelin A

[0058]

[0059] The method for preparing glu-verlamelin A and 5R-OH-glu-verlamelin A was the same as that in Reference Example 1, except that in Step 1, FMOC-L-glutamine was replaced by FMOC-L-glutamic acid, and other steps were the same as in Example 1. A mixture of glu-verlamelin A and 5R-OH-glu-verlamelin A was synthesized in Step 4, and then the pure products were obtained by HPLC preparation separation. The HPLC conditions were: the chromatographic column was a nanochrom C18 column with a size of 250 mm and a particle size of 5 μm, the mobile phase was methanol / water = 80 / 20 (v / v), the water phase contained 0.4% formic acid, the flow rate was 2.5 mL / min, the injection volume was 80 μL, and the Hitachi liquid chromatograph was used; the retention time of glu-verlamelin A was 23 min, and that of 5R-OH-glu-verlamelin A was 31 min, and the yields were 2.9% and 4.3%, respectively.

[0060] The mass spectrometry data of glu-verlamelin A and 5R-OH-glu-verlamelin A were: HRMS-ESI (m / z): Calcd for C 45 H 70 N6O 12 [M+H] + 886.5052, Found 886.5135; HRMS-ESI (m / z): Calcd for C 45 H 70 N6O12 [M+H] + 886.5052, Found 886.5122.

[0061] Example 3: Synthesis of arg-verlamelin A and 5R-OH-arg-verlamelin A

[0062]

[0063] The method for preparing arg-verlamelin A and 5R-OH-arg-verlamelin A was the same as that in Reference Example 1, except that in Step 1, FMOC-L-glutamine was replaced by FMOC-L-arginine, and other steps were the same as those in Example 1. A mixture of arg-verlamelin A and 5R-OH-arg-verlamelin A was synthesized in Step 4, and then the pure products were obtained by HPLC preparation separation. The HPLC conditions were as follows: the chromatographic column was a nanochrom C18 column with a specification of 250 mm x 5 μm, the mobile phase was methanol / water = 75 / 25 (v / v), the water phase contained 0.4% formic acid, the flow rate was 2.5 mL / min, the injection volume was 80 μL, and the Hitachi liquid chromatograph was used; the retention time of arg-verlamelin A was 15 min, and the retention time of 5R-OH-arg-verlamelin A was 22 min; the yields were 3% and 2.1%, respectively.

[0064] The mass spectrometry data of arg-verlamelin A and 5R-OH-arg-verlamelin A were as follows: HRMS-ESI (m / z): Calcd for C 45 H 70 N6O 12 [M+H] + 913.5637, Found 913.5681; HRMS-ESI (m / z): Calcd for C 45 H 70 N6O 12 [M+H] + 913.5637, Found 913.5704.

[0065] Antifungal activity of the compound in Example 4

[0066] Plant pathogenic fungi of apple black spot (caused by Alternaria alternate), tomato early blight (caused by Alternaria solani), peanut sclerotium blight (caused by Rhizoctonia solani), rice blast (caused by Pyricularia oryaza HNM1003), corn Gibberella disease (caused by Gibberella zeae), wheat Gibberella disease (caused by Gibberella sanbinetti) and the like were selected to determine the activity of verlamelin A and its derivatives / isomers. The MIC microdilution method was used to determine the antibacterial activity, and the experimental steps were as follows:

[0067] 1. The test strain was inoculated in a potato glucose agar solid culture plate in advance, and cultured at 25°C in an environment;

[0068] 2. A part of the test strain was added to a potato glucose aqueous liquid medium, and the bacterial liquid was cultured in a 25°C environment on a shaking table;

[0069] 3. High-pressure sterilization gun head, EP tube, sterile water, and corresponding liquid medium were prepared;

[0070] 4. The test antibacterial drug and positive drug powder (nystatin) were weighed, dissolved in DMSO to prepare a 0.64 μg / μl storage solution, and used in the experiment;

[0071] 5. In the clean bench, the test drug storage solution was gradiently diluted in the EP tube according to the half dilution method, a total of 11 gradients. The drug solution was added to each drug well of the sterile 96-well plate according to the gradient, and was diluted to 100 μl with the addition of the corresponding liquid medium 80 μl, and was mixed uniformly;

[0072] 6. The test bacterial liquid was added to the EP tube, and then sterile water was added to dilute, shake and mix, and 100 μl of the bacterial liquid was added to the 96-well plate, and the OD value was adjusted to about 0.1 using an enzyme marker;

[0073] 7. The diluted test bacterial liquid was taken 100 μl and added to the 96-well plate, and the total volume was diluted to 200 μl, and was mixed uniformly;

[0074] 8. The 96-well plate was placed in a 25°C environment for 18-24 hours, and 10% DMSO sterile culture medium solution was used as a blank control. The growth inhibition curve of the fungus was analyzed by visual observation and determination of the OD value, and the minimum inhibitory concentration (MIC) was determined.

[0075] The determination results are shown in the following table.

[0076] Table 1. Antifungal MIC of verlamelin A and its derivatives / isomers

[0077]

[0078] “-”: activity not evaluated.

[0079] As can be seen from the above table, verlamelin A and its derivatives have inhibitory activity against plant pathogenic fungi, in particular, glu-verlamelin A, 5R-OH-glu-verlamelin A have strong inhibitory activity against apple black spot (Alternaria alternate) and tomato early blight (Alternaria solani), relative to the antibacterial performance of natural product verlamelin A, the antibacterial activity of some fungi is obviously enhanced, and it is expected to be used for the preparation of antifungal drugs after subsequent research and development.

Claims

1. Verlamelin A derivatives or their isomers 5R-OH-verlamelin A, glu-verlamelin A and 5R-OH-glu-verlamelin A, having the following structural formulas:

2. A method for simultaneously preparing verlamelin A and the compound 5R-OH-verlamelin A according to claim 1, wherein 5R-OH-verlamelin A is a diastereomer of verlamelin A, characterized in that: The following steps are involved: 1) N-Fmoc-D-tyrosine-O-tert-butyl ether is loaded on the resin through the carboxyl group, the Fmoc protecting group is removed, and then condensation reaction is carried out with FMOC-L-glutamine, FMOC-L-proline, FMOC-D-alanine, and FMOC-D-allothreonine in sequence under the action of a condensation reagent, and the Fmoc protecting group is removed after each condensation reaction to finally obtain 2) Under alkaline conditions, the hydrolysis product reacts with allyl bromide to obtain Then it reacts with FMOC-L-valine under condensation reagent conditions and removes the allyl group in the presence of palladium reagent to obtain 3) The step 1) and step 2) Condensation occurs under the action of a condensation reagent, followed by removal of the Fomc group and the resin to obtain 4) Change the The compound undergoes cyclization under the action of a cyclization reagent and removes the protecting group under acidic conditions to obtain a mixture of verlamelin A and its isomer 5R-OH-verlamelin A, which is then separated and prepared by HPLC to obtain a pure product.

3. The method according to claim 2, characterized in that The resin described in step 1) is Wang resin or 2-CTC resin; the condensation reagent described in step 1) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate or 1H-benzotriazol-1-yloxytripyrrolidinophosphine hexafluorophosphate; after each condensation reaction in step 1), an organic base is used to remove the Fmoc protecting group, and the organic base is piperidine, diethylamine or 1,8-diazabicyclo[5.4.0]undec-7-ene.

4. The method according to claim 2, characterized in that In step 1), the molar ratios of N-Fmoc-D-tyrosine-O-tert-butyl ether to FMOC-L-glutamine, FMOC-L-proline, FMOC-D-alanine, and FMOC-D-allothreonine are 1:(1-1.5):(1-1.5):(1-1.5):(1-1.5), respectively; in each condensation reaction in step 1), the molar ratio of the condensation reagent to N-Fmoc-D-tyrosine-O-tert-butyl ether is (1.5-2.5):

1.

5. The method according to claim 2, characterized in that In step 2), The molar ratio of FMOC-L-valine, condensation reagent and palladium reagent is 1:(1-1.5):(1-1.5):(0.1-0.5); the condensation reagent is N,N'-diisopropylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or tetramethyl chlorourea hexafluorophosphate; and the palladium reagent is palladium acetate or tetrakistriphenylphosphine palladium base.

6. The method according to claim 2, characterized in that In step 3), and The molar ratio of the condensation reagent is 1:(2-2.5):(1.5-2.5); the condensation reagent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-diisopropylcarbodiimide, 2-(7-azobenzotriazole)-tetramethyluronium hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate or 1H-benzotriazole-1-yloxytripyrrolidinophosphine hexafluorophosphate; and in step 3), an organic base is used to remove the Fomc group.

7. The method according to claim 2, characterized in that In step 4), the cyclization reagent includes (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate, N-hydroxy-7-azobenzotriazole and N,N-diisopropylethylamine; The molar ratio of (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate and N-hydroxy-7-azobenzotriazole is 1:(5-10):(1-3); in step 4), acidic conditions are formed by adding dilute hydrochloric acid, trifluoroacetic acid or methanesulfonic acid; the HPLC conditions in step 4) are: the chromatographic column is a nanochrom C18 column, the mobile phase is methanol / water, the aqueous phase contains 0.4% formic acid, and the flow rate is 2.5 mL / min.

8. A method for simultaneously preparing the compound glu-verlamelin A and 5R-OH-glu-verlamelin A according to claim 1, wherein 5R-OH-glu-verlamelin A is a diastereomer of glu-verlamelin A, characterized in that: The method is to replace FMOC-L-glutamine in step 1) of the method according to any one of claims 2 to 7 with FMOC-L-glutamic acid, and the remaining steps are the same to synthesize and separate glu-verlamelin A and 5R-OH-glu-verlamelin A.

9. A method for simultaneously preparing the compounds arg-verlamelin A and 5R-OH-arg-verlamelin A, wherein 5R-OH-arg-verlamelin A is a diastereomer of arg-verlamelin A, characterized in that: In the method according to any one of claims 2 to 7, FMOC-L-glutamine in step 1) is replaced with FMOC-L-arginine, and the remaining steps are the same to synthesize and separate arg-verlamelin A and 5R-OH-arg-verlamelin A; the structural formulas of the compounds arg-verlamelin A and 5R-OH-arg-verlamelin are as follows:

10. Use of the compounds 5R-OH-verlamelin A, glu-verlamelin A and 5R-OH-glu-verlamelin A according to claim 1 in inhibiting the growth of plant pathogenic fungi.

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