Peanut endogenous peptide hypogin derivatives and applications thereof

Abstract

The present application belongs to the field of pharmaceutical chemistry, and particularly relates to Hypogin derivative and application thereof. The present application firstly simplifies the structure of Hypogin endogenous peptide, finds an active fragment, and designs and synthesizes Hypogin derivatives of series I. The Hypogin derivatives with better activity in series I are combined with cinnamic acid to design and synthesize compounds of series II. Research finds that the compound II-1 can better inhibit the hypha growth of Sclerotium rolfsii, and it is found that I-12 affects the signal pathway of PI3K of Sclerotium rolfsii, and the potential target is GPCR, RTK and PI3K of Sclerotium rolfsii. The cytotoxicity experiment shows that II-1 has low hepatocyte toxicity of human LO2, and conforms to the development direction of green bactericides. The Hypogin derivative has bacteriostatic activity, can be used as a basis for development of small peptide bacteriostatic agent, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology and relates to peanut endogenous peptides. Background Technology

[0002] Peanuts are one of the world's most important oilseed crops, playing a vital role in global oil production. In recent years, peanut white mold disease caused by *Sclerotium rolfsii* Sacc. has severely damaged peanuts, significantly restricting production and causing substantial economic losses to the peanut industry. Currently, the control of peanut diseases mainly relies on chemical control, but this method suffers from problems such as pathogen resistance, pesticide residues, and environmental pollution. Today, with increasing awareness of personal health and environmental protection, people's pesticide choices have changed, with a growing preference for green pesticides that leave less residue, are safer for humans and animals, and cause less environmental pollution.

[0003] In recent years, peptides have become a research hotspot in the field of plant protection due to their wide range of raw material sources, excellent activity, and ideal environmental compatibility. They are widely used in various aspects such as insecticidal, antibacterial, herbicidal, immune activation, and plant growth regulation. Developing antimicrobial peptides for peanut white mold disease is in line with the development direction of green pesticides. Many experts at home and abroad have found that peanut peptides have a variety of good functional activities, such as antioxidant, free radical scavenging, antibacterial and bacteriostatic effects, and enhanced immunity. Chen Guitang et al. found that peanut peptides have strong antioxidant effects (Chen Guitang, Zhao Liyan, Cong Tao, et al. Preparation of peanut peptides and their antioxidant effects on oxidative damage model mice [J]. Food Science, 2007, (03): 324-327.). Zhang Yuhao et al. showed through experiments that peanut short peptides with a molecular weight of less than 1 kDa can significantly inhibit the activity of angiotensin-converting enzyme (Zhang Yuhao, Ma Liang, Wang Qiang. Study on the antihypertensive activity of peanut short peptides [J]. Food Science, 2008, (06): 399-403.). Chen Tong et al. found that the polypeptide Hypogin (HYP) produced by the hydrolysis of neutral proteinase A has an inhibitory effect on Escherichia coli and Fusarium graminearum, can inhibit fungal growth, and can inhibit HIV reverse transcription (Chen Tong, Wang Changqing, Bai Yunyun, et al. Study on the antibacterial effect of enzymatic hydrolysis polypeptide of peanut meal [J]. Agricultural Products Processing, 2015, (07): 23-25.).

[0004] With the development of science and technology, more methods will be applied to the preparation and research of antimicrobial peptides, and their application fields will continue to expand, including food preservation, agricultural development, medicine, and cosmetics. Patent CN202411350080.3 discloses a peanut cysteine-rich peptide, its encoding gene, an antibacterial agent, and its applications. A novel antimicrobial peptide family member was identified and extracted from a peanut transcriptome library and named peanut cysteine-rich peptide. The cysteine-rich peptide prepared using a solid-phase chemical synthesis method exhibited broad-spectrum antimicrobial activity, with its polypeptide fragments showing strong inhibitory and killing abilities against various common pathogenic bacteria and crop molds. Peanut antimicrobial peptides are expected to become an important natural antimicrobial substance in the future, bringing more benefits to human health and life, and their research and application will continue to deepen and develop. Summary of the Invention

[0005] To address the above problems, this invention proposes a peanut endogenous peptide Hypogin derivative and its applications.

[0006] The technical solution of this invention is implemented as follows:

[0007] On the one hand, the present invention proposes a peanut endogenous peptide Hypogin derivative, wherein the Hypogin derivative is selected from any of the following:

[0008] (1) The 4-peptide or 5-peptide fragments that make up the Hypogin polypeptide.

[0009] (2) Polypeptide-carboxylic acid compounds formed by carboxylation of compounds with the structure of case (1).

[0010] Preferably, the Hypogin derivative in case (1) above is one or more of KSPYY-NH2, QKKTE-NH2, NPQAQ-NH2, RQLQS-NH2, DDQEP-NH2, AKLK-NH2, PAKLK-NH2, SDDQE-NH2, QRQLQ-NH2, ENPQA-NH2, YQKKT-NH2 and KSPY-NH2.

[0011] Preferably, the carboxylic acid in case (2) above is cinnamic acid.

[0012] Preferably, the polypeptide-carboxylic acid compound in the above case (2) is cinnamic acid-KSPY-NH2 or cinnamic acid-RQLQ-NH2.

[0013] Secondly, the application of the aforementioned peanut endogenous peptide Hypogin derivative in the preparation of drugs for the prevention and treatment of white rot.

[0014] Preferably, the above-mentioned white sclerotium disease is caused by infection with *Sclerotium sclerotiorum*.

[0015] Thirdly, the present invention also proposes a drug in which the active ingredient contains one or more of the above-mentioned peanut endogenous peptide Hypogin derivatives.

[0016] Preferably, the above-mentioned drugs also contain excipients that give the drugs their corresponding pharmaceutical formulation type.

[0017] Preferably, the above-mentioned agent type is any one of powder, wettable powder, soluble powder, granules, water emulsion, microemulsion, aqueous solution, suspension, dispersible oil suspension, microcapsule and emulsifiable concentrate.

[0018] Fourthly, the aforementioned drugs are used as agents for the prevention and treatment of peanut white mold disease.

[0019] The present invention has the following beneficial effects:

[0020] 1. This invention designs and optimizes the structure of the peanut endogenous polypeptide Hypogin to obtain a highly active Hypogin polypeptide fragment. Further, using an active substructure splicing method, a highly active natural carboxylic acid is introduced to modify the compound, resulting in a polypeptide-carboxylic acid compound. The modified compound exhibits superior in vitro activity compared to the lead compound. The compounds of this invention are safe, efficient, environmentally friendly, and simple to synthesize, possessing potential for future development and application.

[0021] 2. The Hypogin derivatives series I and II provided by this invention both exhibited antibacterial activity against *Sclerotium truncatum*, with compounds I-12 and II-1 showing the best antibacterial effects, with inhibition rates of 56.61% and 50.20%, respectively. This invention selected II-1 for non-target human hepatocyte LO2 toxicity testing, and the results showed that the Hypogin derivatives have extremely low cytotoxicity. The Hypogin derivatives provided by this invention are novel green bactericide candidate compounds, characterized by a small peptide fragment containing 4-5 amino acids, exhibiting low or no toxicity to humans and the environment. Attached Figure Description

[0022] 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.

[0023] Figure 1 For I-1 compound 1 1H NMR (400M) spectrum.

[0024] Figure 2 This is the high-resolution mass spectrum (HRMS) of compound I-1.

[0025] Figure 3 For compound II-1 1 1H NMR (400M) spectrum.

[0026] Figure 4 This is the high-resolution mass spectrum (HRMS) of compound II-1.

[0027] Figure 5 The antibacterial activity of compound I-12 against Sclerotium riberi was determined.

[0028] Figure 6 The antibacterial activity of compound II-1 against Sclerotium ribentum was demonstrated.

[0029] Figure 7 The effects of three peptides on the relative expression levels of phosphatidylinositol 3-kinase (PI3K) mRNA in *Sclerotinia ribonucleoides*.

[0030] Figure 8 The diagram shows the molecular docking of compound I-12; (a) represents rhodopsin from blackleg fungus, (b) represents RET from yeast, and (c) represents molecular docking of PI3K from yeast.

[0031] Figure 9 The toxicity of compound II-1 and thifluzamide to human LO2 cells. Detailed Implementation

[0032] 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.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0034] This invention uses Hypogin as a lead compound to design and optimize the structure of the peanut endogenous polypeptide Hypogin, and obtains a highly active Hypogin polypeptide fragment using a solid-phase polypeptide synthesis method. Furthermore, by employing an active substructure splicing method and introducing a highly active natural carboxylic acid, the compound is modified to obtain a polypeptide-carboxylic acid compound. The in vitro activity of the modified compound is superior to that of the lead compound.

[0035] This invention provides peanut endogenous polypeptide Hypogin and its derivatives, the structure of the Hypogin derivative series I is shown below:

[0036]

[0037]

[0038] The structure of the Hypogin derivative series II is shown in the figure below:

[0039]

[0040] The mass spectrometry data for Series I and Series II are shown in Table 1:

[0041] Table 1 Mass spectrometry data of Hypogin derivatives series I and II

[0042]

[0043] The polypeptide compounds in this application are synthesized using a solid-phase polypeptide synthesis method, with further improvements:

[0044] 1. The usage ratio (volume ratio) of 1,8-diazabicycloundec-7-ene (DBU) as the deprotecting agent was optimized to 20%; 2. The post-treatment was simplified by blowing away trifluoroacetic acid with N2 gas and optimizing the method to use a rotary evaporator to evaporate most of the solvent, thus avoiding the damage of TFA to the respiratory tract of experimental personnel.

[0045] Example 1

[0046] This example illustrates the preparation method of compound I-1:

[0047] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, place the resin in a 25 mL peptide synthesizer, add 10 mL of dichloromethane (DCM), shake for 30 minutes, add 15 mL of 20% (V / V) 1,8-diazabicycloundec-7-ene (DBU) dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0048] 2. Add 3 molar equivalents of Fmoc-L-Tyr-OH, then add 6 molar equivalents of diisopropylethylamine (DIEA), and dissolve in 15 mL of dimethylformamide (DMF). Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0049] 3. Add 3 molar equivalents of Fmoc-L-Tyr-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of dimethylformamide (DMF). Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0050] 4. Add 3 molar equivalents of Fmoc-L-Pro-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0051] 5. Add 3 molar equivalents of Fmoc-L-Ser-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0052] 6. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0053] 7. Preparation of cutting solution: 90.0% trifluoroacetic acid (TFA); 10.0% water. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0054] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0055] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-1.

[0056] Example 2

[0057] This example illustrates the preparation method of compound I-2:

[0058] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0059] 2. Add 3 molar equivalents of Fmoc-L-Glu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0060] 3. Add 3 molar equivalents of Fmoc-L-Thr-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0061] 4. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0062] 5. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, dissolve in 15 mL of DMF, and shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, wash 3 times alternately with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0063] 6. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0064] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0065] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0066] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-2.

[0067] Example 3

[0068] This example illustrates the preparation method of compound I-3:

[0069] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0070] 2. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0071] 3. Add 3 molar equivalents of Fmoc-L-Ala-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0072] 4. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0073] 5. Add 3 molar equivalents of Fmoc-L-Pro-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0074] 6. Add 3 molar equivalents of Fmoc-L-Asn-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0075] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0076] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0077] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-3.

[0078] Example 4

[0079] This example illustrates the preparation method of compound I-4:

[0080] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0081] 2. Add 3 molar equivalents of Fmoc-L-Ser-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0082] 3. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0083] 4. Add 3 molar equivalents of Fmoc-L-Leu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0084] 5. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0085] 6. Add 3 molar equivalents of Fmoc-L-Arg-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0086] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0087] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0088] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-4.

[0089] Example 5

[0090] This example illustrates the preparation method of compound I-5:

[0091] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0092] 2. Add 3 molar equivalents of Fmoc-L-Pro-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0093] 3. Add 3 molar equivalents of Fmoc-L-Glu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0094] 4. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0095] 5. Add 3 molar equivalents of Fmoc-L-Asp-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0096] 6. Add 3 molar equivalents of Fmoc-L-Asp-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0097] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0098] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0099] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-5.

[0100] Example 6

[0101] This example illustrates the preparation method of compound I-6:

[0102] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0103] 2. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0104] 3. Add 3 molar equivalents of Fmoc-L-Leu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0105] 4. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0106] 5. Add 3 molar equivalents of Fmoc-L-Ala-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0107] 6. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0108] 7. After removing the solvent from the lysate using a rotary evaporator, wash six times with 15 mL of diethyl ether, and then dry in a vacuum drying oven to obtain the crude peptide sequence.

[0109] 8. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-6.

[0110] Example 7

[0111] This example illustrates the preparation method of compound I-7:

[0112] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0113] 2. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0114] 3. Add 3 molar equivalents of Fmoc-L-Leu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0115] 4. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0116] 5. Add 3 molar equivalents of Fmoc-L-Ala-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0117] 6. Add 3 molar equivalents of Fmoc-L-Pro-OH, then add 6 molar equivalents of DIEA, dissolve in 15 mL of DMF, and shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, wash 3 times alternately with DMF and DCM, and monitor the reaction with Kaiser's reagent.

[0118] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0119] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0120] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-7.

[0121] Example 8

[0122] This example illustrates the preparation method of compound I-8:

[0123] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0124] 2. Add 3 molar equivalents of Fmoc-L-Glu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0125] 3. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0126] 4. Add 3 molar equivalents of Fmoc-L-Asp-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0127] 5. Add 3 molar equivalents of Fmoc-L-Asp-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0128] 6. Add 3 molar equivalents of Fmoc-L-Ser-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0129] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0130] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0131] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-8.

[0132] Example 9

[0133] This example illustrates the preparation method of compound I-9:

[0134] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0135] 2. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0136] 3. Add 3 molar equivalents of Fmoc-L-Leu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0137] 4. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0138] 5. Add 3 molar equivalents of Fmoc-L-Arg-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0139] 6. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0140] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0141] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0142] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-9.

[0143] Example 10

[0144] This example illustrates the preparation method of compound I-10:

[0145] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0146] 2. Add 3 molar equivalents of Fmoc-L-Ala-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0147] 3. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0148] 4. Add 3 molar equivalents of Fmoc-L-Pro-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0149] 5. Add 3 molar equivalents of Fmoc-L-Asn-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0150] 6. Add 3 molar equivalents of Fmoc-L-Glu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0151] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0152] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0153] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-10.

[0154] Example 11

[0155] This example illustrates the preparation method of compound I-11:

[0156] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0157] 2. Add 3 molar equivalents of Fmoc-L-Thr-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0158] 3. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0159] 4. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0160] 5. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0161] 6. Add 3 molar equivalents of Fmoc-L-Tyr-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0162] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0163] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0164] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-11.

[0165] Example 12

[0166] This example illustrates the preparation method of compound I-12:

[0167] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0168] 2. Add 3 molar equivalents of Fmoc-L-Tyr-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0169] 3. Add 3 molar equivalents of Fmoc-L-Pro-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0170] 4. Add 3 molar equivalents of Fmoc-L-Ser-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0171] 5. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0172] 6. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0173] 7. After removing the solvent from the lysate using a rotary evaporator, wash six times with 15 mL of diethyl ether, and then dry in a vacuum drying oven to obtain the crude peptide sequence.

[0174] 8. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product I-12.

[0175] Example 13

[0176] This example illustrates the preparation method of compound II-1:

[0177] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0178] 2. Add 3 molar equivalents of Fmoc-L-Tyr-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0179] 3. Add 3 molar equivalents of Fmoc-L-Pro-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0180] 4. Add 3 molar equivalents of Fmoc-L-Ser-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0181] 5. Add 3 molar equivalents of Fmoc-L-Lys-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 2 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0182] 6. Add 3 molar equivalents of cinnamic acid, then 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0183] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0184] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0185] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product II-1.

[0186] Example 14

[0187] This example illustrates the preparation method of compound II-2:

[0188] 1. Weigh 1.0 g of Rink Amide-AM resin with a degree of substitution of 0.69 mmol / g, put the resin into a 25 mL peptide synthesizer, add 10 mL of DCM, shake for 30 minutes, add 15 mL of 20% (V / V) DBU in dichloromethane solution to deprotect for 20 minutes, add 10 mL of DCM to wash 3 times, and filter out the solvent DCM by vacuum filtration.

[0189] 2. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0190] 3. Add 3 molar equivalents of Fmoc-L-Leu-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0191] 4. Add 3 molar equivalents of Fmoc-L-Gln-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0192] 5. Add 3 molar equivalents of Fmoc-L-Arg-OH, then add 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 3 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0193] 6. Add 3 molar equivalents of cinnamic acid, then 6 molar equivalents of DIEA, and dissolve in 15 mL of DMF. Shake for 4 hours. After the reaction is complete, wash 3 times alternately with DMF and DCM. Add 15 mL of 20% (v / v) DBU in dichloromethane solution to deprotect for 20 min, and wash 3 times alternately with DMF and DCM. Monitor the reaction with Kaiser's reagent.

[0194] 7. Preparation of cutting fluid: TFA 90.0%; water 10.0%. Place the resin containing the crude product into a 50mL flask and cut for 2.5 hours. Then add 20mL of 90.0% TFA aqueous solution.

[0195] 8. After removing the solvent from the lysate using a rotary evaporator, wash it six times with 15 mL of diethyl ether, and then dry it in a vacuum drying oven to obtain the crude peptide sequence.

[0196] 9. The crude peptide was separated and purified by semi-preparative liquid chromatography, and the purified solution was freeze-dried to obtain the target product II-2.

[0197] Application Example 1

[0198] Virulence assay of Hypogin derivatives against the hyphal growth of *Sclerotium truncatum*:

[0199] The antibacterial activity of series I and series II strains against *Sclerotium tumefaciens*, the causal agent of peanut white mold, was tested using the mycelial growth rate method. The tested strain, *Sclerotium tumefaciens*, was first activated until the mycelium reached two-thirds of the petri dish before inoculation. A Hypogin derivative was dissolved in 0.1% DMSO and 0.1% Tween 80, and sterile water was added to prepare a 4 g / L stock solution. The final concentration of the drug-containing medium for series I was 400 mg / L, and for series II it was 200 mg / L. The prepared solutions were poured into sterile 90 mm disposable petri dishes. PDA plates containing 0.1% DMSO and 0.1% Tween 80 were used as controls. Activated 5 mm mycelial discs were inoculated into the drug-containing PDA medium, and the petri dishes were incubated in the dark at 28°C. After 2 days, the colony diameter (mm) for each treatment was measured twice using the cross-sectional method, and the average value was used to represent the size of the mycelial zone. Each treatment was repeated in triplicate, and the experiment was performed twice. The formula for calculating the mycelial growth inhibition rate is as follows:

[0200] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / (Control colony diameter - 5 mm) × 100%

[0201] The experimental results are shown in Table 2:

[0202] Table 2. Inhibitory activity of Series I on the growth of *Sclerotium regia* mycelia at a concentration of 400 mg / L in vitro.

[0203]

[0204] Note: Data in the table are mean ± standard deviation. Different letters indicate significant differences between different treatments (Waller-Duncan, P<0.05).

[0205] As shown in Table 2, at a concentration of 400 mg / L, some of the I-series compounds exhibited inhibitory activity against the mycelial growth of *Sclerotium guillezei*. Among them, compound I-12 (structure KSPY-NH2) showed the most significant inhibitory effect on *Sclerotium guillezei* growth, with an inhibition rate of 56.61%. Its antibacterial activity is as follows: Figure 5 As shown; the second was compound I-4 (structure RQLQS-NH2), with an antibacterial rate of 41.28%.

[0206] Table 3. Inhibition of *Sclerotium regia* mycelial growth in series II at a concentration of 200 mg / L.

[0207]

[0208] Table 3 shows that compound II-1, modified using I-12 as the lead compound, exhibits an antibacterial rate of 50.20% at a concentration of 200 mg / L. Its antibacterial activity is as follows: Figure 6 As shown, compound II-2 has an antibacterial rate of 27.93%. Therefore, II-1 can be used as a secondary leader for further structural optimization.

[0209] Application Example 2:

[0210] Mechanism of action of the target compound:

[0211] Table 4. Determination of PI3K content (pmol / L) by enzyme-linked immunosorbent assay (ELISA). n=4)

[0212]

[0213] Note: Values ​​are mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences.

[0214] The effect of two peptides at a concentration of 800 mg / L (Table 4) on the expression level of the PI3K gene in *Sclerotium sclerotiorum* hyphae was tested. Figure 7 ).Depend on Figure 7 It can be seen that the relative expression level of PI3K gene mRNA decreased after treatment with I-4 and I-12. Enzyme-linked immunosorbent assay (ELISA) revealed that treatment with I-12 significantly reduced the PI3K content in the hyphae of *Sclerotium sclerotiorum* (Table 4). Molecular docking experiments showed that I-12 had good docking activity with fungal GPCRs, RTK, and PI3K. Figure 8 This indicates that the potential targets of HYP derivative I-12 are GPCRs, RTKs, and PI3Ks in *Sclerotium regranense*.

[0215] Application Example 3

[0216] Cytotoxicity assays of Hypogin derivative II-1:

[0217] LO2 cells in good growth condition were digested to prepare a single-cell suspension, and the cell density was adjusted to 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates. After 24 h of cell culture, the medium was replaced with serum-free high-glucose DMEM and cultured for another 24 h to starve the cells and stop their division. The serum-free medium was then discarded, and the appropriate drug was added to each well for incubation for 24 h. After the drug treatment period, the drug-containing medium was discarded, and 100 μL of fresh serum-free high-glucose DMEM and 20 μL of MTT were added to each well. The plates were then incubated for another 4 h. The supernatant was carefully removed, and 100 μL of DMSO was added to each well to dissolve the crystals. The plates were then shaken for 10 min to ensure complete dissolution of the crystals. The OD value at 492 nm was measured using an enzyme-linked immunosorbent assay (ELISA) analyzer.

[0218] The formula for calculating cytotoxicity is: Cell death rate = (1 - Sample absorbance / Blank absorbance) × 100%

[0219] Depend on Figure 9 The results showed that compound II-1 exhibited low toxicity to normal human liver cells (LO2 cells). LO2 cells treated with 400 mg / L of compound II-1 showed a cell death rate of only 2.89%. However, at the same concentration, LO2 cells treated with the commercially available fungicide thifluzamide showed a mortality rate of 97.33%. This indicates that the Hypogin derivative of this invention has low or no toxicity, is harmless to humans, animals, and the environment, aligns with the trend of sustainable development in green agriculture, and has promising prospects for development and application.

[0220] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a peanut endogenous peptide Hypogin derivative in the preparation of a drug for preventing and treating white rot, characterized in that: The peanut endogenous peptide Hypogin derivative is selected from either KSPY-NH2 or cinnamic acid-KSPY-NH2.

2. The application according to claim 1, characterized in that: The white rot disease mentioned above is caused by peanuts being infected with Sclerotium sclerotiorum.

3. The application of a pharmaceutical composition as a drug for preventing and treating peanut white mold disease, characterized in that: The pharmaceutical composition comprises a peanut endogenous peptide Hypogin derivative and excipients that give the drug the corresponding pharmaceutical type; The peanut endogenous peptide Hypogin derivative is selected from either KSPY-NH2 or cinnamic acid-KSPY-NH2.

4. The application according to claim 3, characterized in that: The drug formulation type is any one of powder, granules, emulsion, microemulsion, aqueous solution, suspension, microcapsule, and emulsifiable concentrate.

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