A small molecule active peptide and its preparation method and application

Abstract

The present invention discloses a small molecule active peptide and its preparation method and application, relating to the field of antimicrobial peptide technology. The small molecule active peptide of the present invention includes a compound shown in Formula I, or a stereoisomer, tautomer, nitrogen oxide, solvate, hydrate, metabolite, pharmaceutically acceptable salt, pharmaceutically acceptable ester, pharmaceutically acceptable ether or prodrug of the compound shown in Formula I: #imgabs0# wherein R1 and R2 are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylamino, haloalkyl or cycloalkyl, or R1 and R2 are linked to form a cycloalkyl. The small molecule active peptide of the present invention is obtained by modifying the TP4 polypeptide sequence. Compared with TP4, it has fewer amino acids, low synthesis cost, and relatively higher stability and safety. In addition, it has excellent antibacterial effect and anti-resistance ability.

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and in particular to a small molecule active peptide and a preparation method and application thereof. Background Art

[0002] Helicobacter pylori is a spiral-shaped Gram-negative bacterium that resides in the stomach and duodenal bulb. It resides stably and permanently in the gastric antrum. It produces urease, which breaks down urea to produce ammonia. It also secretes cytotoxins, causing inflammation and immune responses. Helicobacter pylori infection is an infectious disease with a global infection rate of up to 50%. It is closely associated with the development of indigestion, gastritis, peptic ulcers, and gastric cancer. Eradication of Helicobacter pylori can reduce gastric mucosal inflammation, promote ulcer healing, and reduce the risk of gastric cancer.

[0003] In the relevant technology, antibiotics are commonly used drugs for the treatment of Helicobacter pylori, and they have achieved good clinical therapeutic effects. However, long-term use can easily lead to a gradual increase in the resistance rate of the bacteria. In the treatment of Helicobacter pylori, for primary infection, the resistance rate of clarithromycin and levofloxacin is usually 20% to 40%, and the resistance rate of metronidazole is 60% to 90%; for secondary infection, the resistance rate of clarithromycin is 70% to 80%, the resistance rate of levofloxacin is 60% to 70%, and the resistance rate of metronidazole is >90%. However, with the continuous use of drugs such as amoxicillin, furazolidone, and tetracycline, the resistance rate of Helicobacter pylori has also quietly increased, making it difficult for the success rate of triple or even quadruple eradication treatment of Helicobacter pylori containing metronidazole to reach Class C (85% to 89%).

[0004] Antimicrobial peptides are small, biologically active polypeptides widely found in organisms. They typically consist of 12 to 50 amino acid residues and lack highly conserved sequences. Most are short, amphipathic, and highly cationic molecules. Currently, proven mechanisms of action for antimicrobial peptides include cell membrane targeting, cell wall targeting, intracellular targeting, and dual or multiple mechanisms. However, the most common mechanism of action for antimicrobial peptides is through direct activity on pathogen cell membranes. This is because many antimicrobial peptides possess a net positive charge, which facilitates their ability to interact with cell membranes. Electrostatic interactions between cationic antimicrobial peptides and anionic bacterial membranes stabilize their binding to the cell membrane. This binding can lead to disruption of membrane potential, altered membrane permeability, and leakage of cellular contents, ultimately leading to bacterial cell death. Because antimicrobial peptides primarily target the relatively conserved cell membrane, they possess a strong ability to resist drug resistance, a significant advantage over antibiotics.

[0005] Tilapia piscidin 4 (TP4, amino acid sequence: FIHHIIGGLFSAGKAIHRL IRRRRR, molecular weight: 2982 g / mol) is an antimicrobial peptide isolated from tilapia (Tilapia spp.). It exhibits potent antibacterial and bactericidal activity against Helicobacter pylori (H. pylori) and shows strong potential for treating H. pylori infection. However, in hemolytic assays, TP4 exhibits a 100% hemolytic rate at a concentration of 100 μg / mL. This high hemolytic activity, large molecular weight, and high production costs limit its clinical application in humans.

[0006] Therefore, it is very necessary to develop new small molecule active peptides that can target drug-resistant Helicobacter pylori strains. Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a small molecule active peptide and its preparation method and application. The small molecule active peptide has excellent antibacterial activity against Helicobacter pylori strains and is highly safe and substantially non-toxic to cells as shown in cytotoxicity tests.

[0008] In a first aspect, the present invention provides a small molecule active peptide comprising a compound of Formula I, or a stereoisomer, tautomer, nitrogen oxide, solvate, hydrate, metabolite, pharmaceutically acceptable salt, pharmaceutically acceptable ester, pharmaceutically acceptable ether, or prodrug of the compound of Formula I:

[0009]

[0010] Wherein, R1 and R2 are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylamino, haloalkyl or cycloalkyl, or R1 and R2 are connected to form a cycloalkyl.

[0011] The small molecule active peptide according to the embodiment of the present invention has at least the following beneficial effects:

[0012] (1) Low cost and good stability. The small molecule active peptide of the present invention is obtained based on the modification of the TP4 (Tilapia piscidin 4) polypeptide sequence. Compared with TP4, it has fewer amino acids (reduced from 25 to 3), which greatly reduces the difficulty and cost of synthesis. At the same time, the present invention also introduces D-amino acids on its basis, which helps to improve the stability of the compound.

[0013] (2) Excellent antibacterial effect. The small molecule active peptide of the present invention has a structure similar to arginines arranged continuously at the end of TP4. Under physiological conditions, the active peptide carries a positive charge of 4 (TP4 is 7), and has shown excellent antibacterial ability in antibacterial activity tests against Helicobacter pylori. For example, when R1 and R2 are long alkyl chains with 13 to 15 carbon atoms, the minimum inhibitory concentration (MIC) of the active peptide against Helicobacter pylori ATCC43504 strain is 0.25 μg / mL.

[0014] (3) High safety. The small molecule active peptide of the present invention has the characteristics of low hemolysis, and the cytotoxicity test shows that the half-toxification concentration of the active peptide cell CC is 50 >200ug / mL indicates that there is basically no cytotoxicity and high safety.

[0015] (4) Excellent anti-proteolytic effect. The small molecule active peptide of the present invention introduces a D-amino acid structure, which makes the active small peptide have a strong ability to resist protease hydrolysis such as trypsin / pepsin.

[0016] In some embodiments of the present invention, R1 and R2 are independently selected from C 1～18 Alkyl, C 2～18 Alkenyl, C 2～18 Alkynyl, C 1～18 Alkoxy, C 1～18 Alkylamino, halogenated C 1～18 Alkyl or ring C 3～18 Alkyl, or, R1 and R2 are linked to form a ring C 3～18 alkyl.

[0017] In some embodiments of the present invention, R1 and R2 are independently selected from C 6～18 Alkyl, C 6～18 Alkenyl, C 6～18 Alkynyl, C 6～18 Alkoxy, C 6～18 Alkylamino, halogenated C 6～18 Alkyl or ring C 6～18 Alkyl, or, R1 and R2 are linked to form a ring C 6～18 alkyl.

[0018] In some embodiments of the present invention, R1 and R2 are independently selected from C 6～18 Alkyl, C 6～18 Alkenyl, C 6～18 Alkynyl or C 6～18 Alkylamino.

[0019] In some embodiments of the present invention, R1 and R2 are independently selected from C 12～16 alkyl.

[0020] In some embodiments of the present invention, R1 and R2 are independently selected from C 13～15 alkyl.

[0021] In some embodiments of the present invention, R1 and R2 are independently selected from C 14 or C 15 alkyl.

[0022] In some embodiments of the present invention, the compound represented by Formula I is selected from any one of the compounds represented by Formula I-1 to Formula I-18 below:

[0023]

[0024]

[0025]

[0026] In some embodiments of the present invention, "pharmaceutically acceptable salts" refer to salts of compounds of Formula I of the present invention, prepared by reacting the compounds having specific substituents discovered herein with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of base in a neat solution or a suitable inert solvent. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the free form of such compounds with a sufficient amount of acid in a neat solution or a suitable inert solvent.

[0027] In some embodiments of the present invention, the pharmaceutically acceptable salts include but are not limited to trifluoroacetate, hydrochloride, hydrobromide, nitrate, phosphate, sulfate, bisulfate, arylsulfonate, benzoate, formate, acetate, propionate, maleate, fumarate, tartrate, succinate, succinate, citrate, alkylsulfonate and arylsulfonate.

[0028] In some embodiments of the present invention, the alkyl sulfonate includes but is not limited to methyl sulfonate and ethyl sulfonate.

[0029] In some embodiments of the present invention, the aryl sulfonate includes but is not limited to benzenesulfonate and p-toluenesulfonate.

[0030] Preferably, the pharmaceutically acceptable salt is trifluoroacetate.

[0031] The second aspect of the present invention provides a method for preparing the small molecule active peptide as described in the first aspect, comprising the following steps:

[0032] The compound represented by formula II is reacted with the compound represented by formula III, and then subjected to a deprotection treatment to obtain the product, wherein the structure of formula II is as follows:

[0033]

[0034] R1 and R2 are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylamino, haloalkyl or cycloalkyl, or R1 and R2 are linked to form a cycloalkyl;

[0035] The structural formula of the compound shown in Formula III is as follows:

[0036]

[0037] Said X1 and X2 are independently selected from amino protecting groups, and said X1 and X2 are the same or different.

[0038] The preparation method according to the embodiment of the present invention has at least the following beneficial effects: the preparation method of the small molecule active peptide of the present invention is simple, has high yield, low cost, and is suitable for industrial production.

[0039] In some embodiments of the present invention, R1 and R2 in the compound represented by Formula II are independently selected from C 1～18 Alkyl, C 2～18 Alkenyl, C 2～18 Alkynyl, C 1～18 Alkoxy, C 1～18 Alkylamino, halogenated C 1～18 Alkyl or ring C 3～18 Alkyl, or, R1 and R2 are linked to form a ring C 3～18 alkyl.

[0040] In some embodiments of the present invention, R1 and R2 in the compound represented by Formula II are independently selected from C 6～18 Alkyl, C 6～18 Alkenyl, C 6～18 Alkynyl, C 6～18 Alkoxy, C 6～18 Alkylamino, halogenated C 6～18 Alkyl or ring C 6～18 Alkyl, or, R1 and R2 are linked to form a ring C 6～18 alkyl.

[0041] In some embodiments of the present invention, R1 and R2 in the compound represented by Formula II are independently selected from C 6～18 Alkyl, C 6～18 Alkenyl, C 6～18 Alkynyl or C 6～18 Alkylamino.

[0042] In some embodiments of the present invention, R1 and R2 in the compound represented by Formula II are independently selected from C 6～18 alkyl.

[0043] In some embodiments of the present invention, R1 and R2 in the compound represented by Formula II are independently selected from C 12～16 Alkyl; preferably C 14 alkyl.

[0044] In some embodiments of the present invention, the method for preparing the compound represented by Formula II comprises:

[0045] The compound represented by formula IV is reacted with the compound represented by formula V, and then subjected to a deprotection treatment to obtain the compound, wherein the structure of formula IV is as follows:

[0046]

[0047] wherein R1 and R2 are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylamino, haloalkyl or cycloalkyl, or R1 and R2 are linked to form a cycloalkyl;

[0048] The structural formula of the compound represented by formula V is as follows:

[0049]

[0050] wherein X1 and X2 are independently selected from amino protecting groups, and X1 and X2 are the same or different.

[0051] In some embodiments of the present invention, X1 and X2 are independently selected from any one of a Boc group, a Cbz group, a Fmoc group, an Alloc group, a Teoc group, a methyl(ethyl)oxycarbonyl group, a Pht group, a Tos group, a Tfa group, a Trt group, a PMB group, and a Bn group.

[0052] In some embodiments of the present invention, the deprotection treatment comprises treatment with trifluoroacetic acid or dichloromethane.

[0053] The third aspect of the present invention provides a pharmaceutical composition comprising the small molecule active peptide as described in any one of the first aspects, and pharmaceutically acceptable excipients.

[0054] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.

[0055] According to some embodiments of the invention, the excipient comprises water.

[0056] According to some embodiments of the invention, the filler comprises at least one of starch and sucrose.

[0057] According to some embodiments of the invention, the binder comprises at least one of a cellulose derivative, an alginate, a gelatin and a polyvinyl pyrrolidone.

[0058] According to some embodiments of the invention, the humectant comprises glycerol.

[0059] According to some embodiments of the invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0060] According to some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.

[0061] According to some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0062] According to some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0063] According to some embodiments of the invention, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0064] According to some embodiments of the present invention, the dosage form of the drug is any conventional dosage form in the art.

[0065] According to some embodiments of the present invention, the dosage form of the drug is in the form of solid, semi-solid or liquid, and can be an aqueous solution, a non-aqueous solution or a suspension.

[0066] According to some embodiments of the present invention, the dosage form of the drug is tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, dropping pills, dry extracts, injections or infusions.

[0067] According to some embodiments of the present invention, the drug may be administered by a conventional method in the art, including but not limited to oral administration.

[0068] The fourth aspect of the present invention provides use of the small molecule active peptide according to any one of the first aspect or the pharmaceutical composition according to any one of the third aspect in the preparation of antibacterial drugs.

[0069] Other features and advantages of the present invention will be set forth in the description that follows.

[0070] Definition of terms

[0071] "Alkyl" refers to a straight or branched saturated carbon chain of 1-20 carbon atoms, wherein the alkyl group may be independently substituted with one or more substituents, including but not limited to hydroxy, amino, halogen, cyano, alkoxy, alkylamino, alkenyl, alkynyl, nitro, etc.

[0072] "Alkoxy" refers to a group connected to the main carbon chain through an oxygen atom, including but not limited to methoxy, ethoxy, propoxy, butoxy, etc. Alkoxy groups can be independently substituted with one or more substituents, including but not limited to hydroxy, amino, halogen, cyano, alkoxy, alkyl, alkenyl, alkynyl, nitro, etc.

[0073] "Alkylamino" refers to a group connected to the main carbon chain via a nitrogen atom, including but not limited to methylamino, ethylamino, propylamino, butylamino, etc. Alkylamino groups may be independently substituted with one or more of the above substituents.

[0074] "Alkenyl" refers to a linear or branched hydrocarbon group containing double bonds of 2-20 carbon atoms, wherein at least one position is unsaturated, including but not limited to vinyl (-CH=CH2), allyl (-CH2CH=CH2), etc. The alkenyl group may be independently substituted with one or more of the above substituents.

[0075] "Alkynyl" refers to a linear or branched hydrocarbon group containing a triple bond of 2-20 carbon atoms, wherein at least one position is unsaturated, including but not limited to ethynyl (-C≡CH), propargyl (-CH2C≡CH), etc. The alkynyl group may be independently substituted with one or more of the above substituents.

[0076] "Cycloalkyl" refers to a monovalent or polyvalent, non-aromatic, saturated or partially unsaturated ring containing no heteroatoms, including monocyclic rings of 3-20 carbon atoms or bicyclic rings of 7-20 carbon atoms. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-enyl, 1-cyclopentyl-2-enyl, 1-cyclopentyl-3-enyl, cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, and cyclododecyl. Cycloalkyl groups may be independently substituted with one or more substituents, including, but not limited to, hydroxy, amino, halogen, cyano, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, and nitro.

[0077] "Halo" means that the group followed by it is substituted with halogen, and the number of substitutions can be one or more, wherein halogen refers to F, Cl, Br or I.

[0078] "Isomers" are compounds that have the same chemical formula but different arrangements of the atoms.

[0079] "Metabolite" refers to a pharmaceutically active product produced by the in vivo metabolism of a compound of Formula I or a salt thereof. Such a product may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, glucuronidation, enzymatic cleavage, or the like of the administered compound. Thus, the present invention includes metabolites of compounds of the invention, including compounds produced by methods in which a compound of the invention is contacted with a mammal for a period of time sufficient to yield a metabolite thereof. DETAILED DESCRIPTION

[0080] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0081] The terms "preferably," "more preferably," and the like, used herein refer to embodiments of the present invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the present invention.

[0082] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein are understood to include any and all subranges subsumed therein.

[0083] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0084] In the description of the present invention, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the exemplary expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0085] In an embodiment of the present invention, mass spectrometry was performed using an Agilent 6470A LC-MS / MS triple quadrupole liquid chromatography-mass spectrometer, and nuclear magnetic resonance spectroscopy was performed using an AVANCE III 500 MHz instrument.

[0086] In an embodiment of the present invention, the following active tripeptides against Helicobacter pylori are all salts of the compounds shown, specifically, trifluoroacetate salts of the compounds shown.

[0087] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0088] General preparation method:

[0089] The present invention provides an active tripeptide for use against Helicobacter pylori and a method for preparing the same. The active tripeptide has the general structural formula R1R2-(D)-Lys-(D)-Orn2, wherein R1 and R2 are independently alkyl, alkenyl, alkynyl, alkoxy, alkylamino, haloalkyl, or cycloalkyl, or R1 and R2 are linked to form a cycloalkyl; preferably, they are long-chain or branched saturated alkanes containing 6 to 18 carbon atoms, and (D)-Lys and (D)-Orn are both non-natural D-amino acids. The synthetic route for the active tripeptide is as follows:

[0090]

[0091] The specific synthesis steps are as follows:

[0092] First, N,N'-bis-Boc-D-lysine is condensed with an amine R1R2-NH, wherein R1 and R2 are independently long-chain or branched saturated alkanes containing 6 to 18 carbon atoms, to obtain an amide compound A. The tert-butyloxycarbonyl (Boc) protecting group is removed under trifluoroacetic acid conditions to obtain compound B, which is then subjected to a substitution reaction with N,N'-bis-Boc-D-ornithine p-nitrophenolate to obtain compound C. The tert-butyloxycarbonyl (Boc) protecting group is removed under trifluoroacetic acid conditions to obtain compound D, which is then subjected to a substitution reaction with 1,3-di-BOC-2-(trifluoromethylsulfonyl)guanidine to obtain compound E. Finally, all tert-butyloxycarbonyl (Boc) protecting groups are removed under trifluoroacetic acid conditions to obtain the final active tripeptide, wherein R1 and R2 are independently long-chain or branched saturated alkanes containing 6 to 18 carbon atoms.

[0093] Example 1: Compound LS-1 and its preparation method

[0094] This embodiment provides an active tripeptide (Compound LS-1) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-1 is as follows:

[0095]

[0096] The preparation process of the above compound LS-1 is as follows:

[0097]

[0098] The specific preparation method includes the following contents.

[0099] 1. Preparation of N,N'-bis-Boc-D-ornithine p-nitrophenolate:

[0100] The synthetic route of N,N'-bis-Boc-D-ornithine p-nitrophenolate of this embodiment is as follows:

[0101]

[0102] The specific preparation steps are as follows:

[0103] N,N'-bis-Boc-D-ornithine (1 mol), p-nitrophenol (1 mol), and 4-dimethylaminopyridine (DMAP, 10 mmol) were added to a reaction flask containing 2.5 L of dichloromethane. The mixture was stirred at room temperature until dissolved. The temperature was then lowered to -5°C to -10°C. A solution of N,N'-dicyclohexylcarbodiimide (DCC, 1.1 mol) in 500 ml of dichloromethane was added while controlling the temperature not to exceed -5°C. After the addition, the mixture was stirred at -5°C to -10°C for 0.5 hour, then the mixture was warmed to room temperature (about 29°C) and the reaction was continued with stirring for 10 hours. The reaction was monitored by thin-layer chromatography (TLC). A fritted funnel was padded with diatomaceous earth and the reaction solution was filtered under reduced pressure through the funnel and drained. The filter cake was washed with 200 ml of dichloromethane. The combined filtrates were concentrated under reduced pressure until no liquid was distilled off. 500 ml of dichloromethane was added to the concentrate and stirred to dissolve. 2.5 L of n-heptane was then added, and a white solid precipitated under stirring. Stirring was continued for 2 hours. The suspension was filtered under reduced pressure through a Buchner funnel, and the filter cake was washed with 400 ml of n-heptane and drained. The filter cake was dried in a forced-air oven at 60°C to obtain N,N'-bis-Boc-D-ornithine p-nitrophenolate as an off-white solid (301.51 g, 66% yield, 96% HPLC purity).

[0104] The hydrogen spectrum data of N,N'-bis-Boc-D-ornithine p-nitrophenolate are as follows:

[0105] 1H NMR(500MHz, CDCl3):1.40(s,9H),1.43(s,9H),1.45-1.64(m,2H),1.82-1. 90(m,1H),1.92-2.03(m,1H),2.95-3.03(m,2H),4.13-4.27(m,1H),6.80(br t,1H),7.55(brd,1H),7.24-7.27(m,2H),8.20-8.24(m,2H).

[0106] 2. Preparation of compound LS-1A:

[0107] Dissolve N,N'-bis-Boc-D-lysine (3.46 g, 10 mmol) in N,N-dimethylformamide (DMF, 30 ml), cool to 0-5°C in an ice-water bath and stir, add 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PyBOP, 6.24 g, 12 mmol), stir for 0.5 hour, add triethylamine (3.04 g, 30 mmol), stir for 5 minutes, then add di-n-hexylamine (2.04 g, 11 mmol), keep stirring at 0-5°C for 1 hour, then move to room temperature (about 28°C) and react for 5 hours. The reaction was monitored by thin layer chromatography (TLC), and 70 ml of water was added to the reaction system, stirred for 5 minutes, and extracted four times with an organic mixed solvent (volume ratio of n-heptane: methyl tert-butyl ether = 3:1), using 60 ml each time. The upper organic phase extracts were combined, and the organic phase was concentrated and purified by silica gel column chromatography (eluent volume ratio: n-heptane: ethyl acetate = 8:1) to obtain compound LS-1A (4.16 g, yield: 81%, HPLC purity: 92%) as an off-white flocculent solid.

[0108] 3. Preparation of compound LS-1B:

[0109] Compound LS-1A (2.57 g, 5 mmol) was dissolved in trifluoroacetic acid (TFA, 20 ml) and stirred at room temperature (about 26°C) for 3 hours. The reaction was monitored by thin layer chromatography (TLC) to complete. The reaction system was concentrated by rotary evaporation under reduced pressure until no trifluoroacetic acid was evaporated. 25 ml of tetrahydrofuran was added to dilute and dissolve the concentrate. The concentrated solution was again evaporated under reduced pressure until no solvent was evaporated to obtain compound LS-1B (HPLC purity: 90%) as a light yellow viscous solid. Further mass spectrometry results showed that the measured m / z value was 313.3 in electrospray ionization (ESI+) mode, and the theoretical calculated value was 313.3.

[0110] 4. Preparation of compound LS-1C:

[0111] The obtained compound LS-1B was dissolved in 30 ml of N,N-dimethylformamide (DMF), cooled to 0-5°C in an ice-water bath and stirred. Triethylamine (4.05 g, 40 mmol) and N,N'-bis-Boc-D-ornithine p-nitrophenolate (5.44 g, 12 mmol) were added. The mixture was stirred at 0-5°C for 10 minutes and then brought to room temperature (about 26°C) for 4 hours. The reaction was monitored by thin layer chromatography (TLC). 1 ml of aqueous ammonia was added to the reaction system, stirred for 0.5 hour, 80 ml of water was added, stirred for 5 minutes, and extracted four times with an organic mixed solvent (volume ratio of n-heptane: methyl tert-butyl ether = 3:1), using 40 ml each time. The upper organic phase extracts were combined, and the organic phase was concentrated and purified by silica gel column chromatography (eluent volume ratio: n-heptane: methyl tert-butyl ether = 1:4) to obtain compound LS-1C (3.63 g, yield: 77%, HPLC purity: 97%) as an off-white solid.

[0112] 5. Preparation of compound LS-1D:

[0113] Compound LS-1C (1.88 g, 2 mmol) was added to a mixed solvent of 5 ml of dichloromethane and 10 ml of trifluoroacetic acid (TFA), stirred at room temperature (about 30°C) for 3 hours, and the reaction was completed by monitoring with thin layer chromatography (TLC). The reaction system was concentrated by rotary evaporation under reduced pressure until no trifluoroacetic acid was evaporated. 15 ml of dichloromethane was added to the concentrate to dissolve the concentrate, and the concentrate was evaporated under reduced pressure until no solvent was evaporated. 15 ml of dichloromethane was added to the concentrate again to dissolve the concentrate, and the concentrate was evaporated under reduced pressure until no solvent was evaporated to obtain compound LS-1D (HPLC purity: 94%), which was a light yellow viscous solid. Further mass spectrometry results showed that under electrospray ionization (ESI + ) mode, the measured m / z value was 541.5, and the theoretical calculated value was 541.5.

[0114] 6. Preparation of compound LS-1E:

[0115] Compound LS-1D obtained above was dissolved in 25 ml of dichloromethane, and N,N-diisopropylethylamine (3.23 g, 25 mmol) and 1,3-bis-BOC-2-(trifluoromethylsulfonyl)guanidine (3.52 g, 9 mmol) were added. The mixture was stirred at room temperature (approximately 23-30°C) for 72 hours. After completion of the reaction, monitored by thin-layer chromatography (TLC), 30 ml of 8% (wt%) citric acid solution was added, and the mixture was stirred for 5 minutes. The layers were separated, and the lower organic phase was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-heptane:ethyl acetate = 5:1 by volume) to obtain compound LS-1E (1.78 g, yield: 59%, HPLC purity: 97%) as a light yellow solid.

[0116] 7. Preparation of target compound LS-1:

[0117] Compound LS-1E (1.51 g, 1 mmol) was added to a mixed solvent of 5 ml of dichloromethane and 10 ml of trifluoroacetic acid (TFA) and stirred at room temperature (approximately 30°C) for 3 hours. The reaction was monitored for completion by thin-layer chromatography (TLC). The reaction system was then concentrated by rotary evaporation under reduced pressure until no more trifluoroacetic acid was removed. 50 ml of methyl tert-butyl ether was added to the concentrate to precipitate a solid. The mixture was cooled to 0-5°C in an ice-water bath and stirred for 2 hours. The mixture was then filtered under reduced pressure using a fritted funnel under nitrogen. The filter cake was washed twice with 30 ml of methyl tert-butyl ether, dried, and dried in a vacuum oven at 80°C to obtain the target compound LS-1 as a light yellow solid (0.98 g, yield: 84%, HPLC purity: 96%).

[0118] The mass spectrometry results of the target compound LS-1 showed that the + ) mode, the measured m / z value is 709.7, and the theoretical calculated value is 709.6. The H NMR spectrum data of the target compound LS-1 are as follows (chemical shift value δ: Hz):

[0119] 1 H NMR(500MHz,DMSO-d6):0.88(t,6H),1.19-1.24(m,18H),1.38-1.61(m,10H),1.70(m,2H),3.0 5-3.13(m,6H),3.22(m,2H),3.31(m,2H),4.12(m,1H),4.23(m,1H),4.61(m,1H),7.16-7.54(br s,12H),7.68(m,2H),7.74(t,1H),7.90(d,1H),8.23(t,1H),8.39(d,1H).

[0120] After deuterium exchange, the following peaks disappeared: 7.16-7.54 (br s, 12H), 7.68 (m, 2H), 7.74 (t, 1H), 7.90 (d, 1H), 8.23 (t, 1H), 8.39 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0121] Example 2: Compound LS-2 and its preparation method

[0122] This embodiment provides an active tripeptide (Compound LS-2) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-2 is as follows:

[0123]

[0124] The preparation process of the above compound LS-2 is as follows:

[0125]

[0126] The specific preparation method includes the following contents.

[0127] 1. Preparation of N,N'-bis-Boc-D-ornithine p-nitrophenolate:

[0128] The preparation method of N,N'-bis-Boc-D-ornithine p-nitrophenolate in this example is referenced to Example 1.

[0129] 2. Preparation of compound LS-2A:

[0130] Dissolve N,N'-bis-Boc-D-lysine (3.46 g, 10 mmol) in N,N-dimethylformamide (DMF, 30 ml), cool to 0-5°C in an ice-water bath and stir, add 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate (PyBOP, 6.24 g, 12 mmol), stir for 0.5 hour, add triethylamine (3.04 g, 30 mmol), stir for 5 minutes, then add di-n-heptylamine (2.35 g, 11 mmol), keep stirring at 0-5°C for 1 hour, then move to room temperature (about 29°C) and react for 5.5 hours. The reaction was monitored by thin layer chromatography (TLC), and 80 ml of water was added to the reaction system, stirred for 5 minutes, and extracted four times with an organic mixed solvent (volume ratio of n-heptane: methyl tert-butyl ether = 3:1), using 60 ml each time. The upper organic phase extracts were combined, and the organic phase was concentrated and purified by silica gel column chromatography (eluent volume ratio: n-heptane: ethyl acetate = 8:1) to obtain compound LS-2A (4.23 g, yield: 78%, HPLC purity: 94%) as an off-white flocculent solid.

[0131] 3. Preparation of compound LS-2B:

[0132] Compound LS-2A (2.71 g, 5 mmol) was dissolved in trifluoroacetic acid (TFA, 25 ml) and stirred at room temperature (about 28°C) for 3 hours. The reaction was completed by monitoring with thin layer chromatography (TLC). The reaction system was concentrated by rotary evaporation under reduced pressure until no trifluoroacetic acid was evaporated. 25 ml of tetrahydrofuran was added to dilute and dissolve the concentrate. The concentrated solution was again evaporated under reduced pressure until no solvent was evaporated to obtain compound LS-2B (HPLC purity: 91%) as a light yellow viscous solid. Further mass spectrometry results showed that under electrospray ionization (ESI + ) mode, the measured m / z value was 341.3, and the theoretical calculated value was 341.3.

[0133] 4. Preparation of compound LS-2C:

[0134] The compound LS-2B obtained above was dissolved in 32 ml of N,N-dimethylformamide (DMF), cooled to 0-5°C in an ice-water bath with stirring, and triethylamine (4.05 g, 40 mmol) and N,N'-bis-Boc-D-ornithine p-nitrophenolate (5.44 g, 12 mmol) were added. The mixture was stirred at 0-5°C for 10 minutes and then brought to room temperature (about 28°C) for 4.5 hours. The reaction was monitored by thin layer chromatography (TLC). 1.2 ml of aqueous ammonia was added to the reaction system, stirred for 0.5 hour, 80 ml of water was added, stirred for 5 minutes, and extracted four times with an organic mixed solvent (volume ratio of n-heptane: methyl tert-butyl ether = 3:1), using 45 ml each time. The upper organic phase extracts were combined, and the organic phase was concentrated and purified by silica gel column chromatography (eluent volume ratio: n-heptane: methyl tert-butyl ether = 1:4) to obtain compound LS-2C (3.40 g, yield: 70%, HPLC purity: 95%) as an off-white solid.

[0135] 5. Preparation of compound LS-2D:

[0136] Compound LS-2C (1.94 g, 2 mmol) was added to a mixed solvent of 5 ml of dichloromethane and 10 ml of trifluoroacetic acid (TFA), stirred at room temperature (about 29°C) for 4 hours, and the reaction was completed by monitoring with thin layer chromatography (TLC). The reaction system was concentrated by rotary evaporation under reduced pressure until no trifluoroacetic acid was distilled out, 20 ml of dichloromethane was added to the concentrate to dissolve the concentrate, and the concentrate was evaporated under reduced pressure until no solvent was distilled out. 20 ml of dichloromethane was added to the concentrate again to dissolve the concentrate, and the concentrate was evaporated under reduced pressure until no solvent was distilled out to obtain compound LS-2D (HPLC purity: 91%), which was a light yellow viscous solid. Further mass spectrometry results showed that under electrospray ionization (ESI + ) mode, the measured m / z value was 597.4, and the theoretical calculated value was 597.5.

[0137] 6. Preparation of compound LS-2E:

[0138] Compound LS-2D obtained above was dissolved in 25 ml of dichloromethane, and N,N-diisopropylethylamine (3.23 g, 25 mmol) and 1,3-bis-BOC-2-(trifluoromethylsulfonyl)guanidine (3.52 g, 9 mmol) were added. The mixture was stirred at room temperature (approximately 20-30°C) for 80 hours. After completion of the reaction, monitored by thin-layer chromatography (TLC), 32 ml of 8% (wt.%) citric acid solution was added, and the mixture was stirred for 5 minutes. The layers were separated, and the lower organic phase was separated and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: n-heptane:ethyl acetate = 5:1 by volume) to obtain compound LS-2E (1.91 g, yield: 62%, HPLC purity: 96%) as a light yellow solid.

[0139] 7. Preparation of compound LS-2:

[0140] Compound LS-2E (1.54 g, 1 mmol) was added to a mixed solvent of 5 ml of dichloromethane and 10 ml of trifluoroacetic acid (TFA) and stirred at room temperature (approximately 22°C) for 5 hours. The reaction was monitored for completion by thin-layer chromatography (TLC). The reaction system was then concentrated by rotary evaporation under reduced pressure until no trifluoroacetic acid was distilled off. 55 ml of methyl tert-butyl ether was added to the concentrate to precipitate a solid. The mixture was cooled to 0-5°C in an ice-water bath and stirred for 2.5 hours. The mixture was then filtered under reduced pressure using a sand-core funnel under nitrogen protection. The filter cake was washed twice with 40 ml of methyl tert-butyl ether, dried, and dried in a vacuum oven at 80°C to obtain compound LS-2 (1.06 g, yield: 89%, HPLC purity: 95%) as a light yellow solid.

[0141] The mass spectrometry results of the target compound LS-2 showed that the + ) mode, the measured m / z value is 737.6, and the theoretical calculated value is 737.6. The H NMR spectrum data of the target compound LS-2 are as follows (chemical shift value δ: Hz):

[0142] 1 H NMR(500MHz,DMSO-d6):0.83(t,6H),1.16-1.28(m,20H),1.34-1.55(m,12H),1.70(m,2H),3.0 2-3.10(m,6H),3.24(m,2H),3.30(m,2H),4.06(m,1H),4.20(m,1H),4.57(m,1H),7.12-7.57(br s,12H),7.63(m,2H),7.70(t,1H),7.94(d,1H),8.20(t,1H),8.32(d,1H).

[0143] After deuterium exchange, the following peaks disappeared: 7.12-7.57 (br s, 12H), 7.63 (m, 2H), 7.70 (t, 1H), 7.94 (d, 1H), 8.20 (t, 1H), 8.32 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0144] Example 3: Compound LS-3 and its preparation method

[0145] This embodiment provides an active tripeptide (Compound LS-3) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-3 is as follows:

[0146]

[0147] The preparation method of the compound LS-3 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-octylamine, and the remaining steps are the same.

[0148] The mass spectrometry results of the target compound LS-3 showed that the + ) mode, the measured m / z value is 765.6, and the theoretical calculated value is 765.6. The H NMR spectrum data of the target compound LS-3 are as follows (chemical shift value δ: Hz):

[0149] 1 H NMR(500MHz,DMSO-d6):0.87(t,6H),1.10-1.27(m,22H),1.30-1.52(m,12H),1.73(m,4H),3.1 1-3.16(m,6H),3.22(m,2H),3.33(m,2H),3.98(m,1H),4.21(m,1H),4.49(m,1H),7.03-7.40(br s,12H),7.43(m,2H),7.65(t,1H),7.86(d,1H),8.12(t,1H),8.28(d,1H).

[0150] After deuterium exchange, the following peaks disappeared: 7.03-7.40 (br s, 12H), 7.43 (m, 2H), 7.65 (t, 1H), 7.86 (d, 1H), 8.12 (t, 1H), 8.28 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0151] Example 4: Compound LS-4 and its preparation method

[0152] This embodiment provides an active tripeptide (Compound LS-4) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-4 is as follows:

[0153]

[0154] The preparation method of the compound LS-4 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-nonylamine, and the remaining steps are the same.

[0155] The mass spectrometry results of the target compound LS-4 showed that the + ) mode, the measured m / z value is 793.7, and the theoretical calculated value is 793.6. The H NMR spectrum data of the target compound LS-4 are as follows (chemical shift value δ: Hz):

[0156] 1H NMR(500MHz,DMSO-d6):0.89(t,6H),1.08-1.33(m,24H),1.36-1.59(m,16H),1.78(m,2H),3.1 6-3.23(m,6H),3.29(m,2H),3.32(m,2H),4.05(m,1H),4.26(m,1H),4.54(m,1H),7.23-7.56(br s,12H),7.61(m,2H),7.68(t,1H),7.80(d,1H),8.17(t,1H),8.33(d,1H).

[0157] After deuterium exchange, the following peaks disappeared: 7.23-7.56 (br s, 12H), 7.61 (m, 2H), 7.68 (t, 1H), 7.80 (d, 1H), 8.17 (t, 1H), 8.33 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0158] Example 5: Compound LS-5 and its preparation method

[0159] This embodiment provides an active tripeptide (Compound LS-5) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-5 is as follows:

[0160]

[0161] The preparation method of the compound LS-5 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-decylamine, and the remaining steps are basically the same.

[0162] The mass spectrometry results of the target compound LS-5 showed that the + ) mode, the measured m / z value is 821.6, and the theoretical calculated value is 821.7. The H NMR spectrum data of the target compound LS-5 are as follows (chemical shift value δ: Hz):

[0163] 1 H NMR(500MHz,DMSO-d6):0.80(t,6H),1.18-1.39(m,26H),1.42-1.57(m,18H),1.73(m,2H),3.1 0-3.21(m,6H),3.26(m,2H),3.36(m,2H),4.13(m,1H),4.25(m,1H),4.50(m,1H),7.02-7.45(br s,12H),7.48(m,2H),7.70(t,1H),7.82(d,1H),8.08(t,1H),8.26(d,1H).

[0164] After deuterium exchange, the following peaks disappeared: 7.02-7.45 (br s, 12H), 7.48 (m, 2H), 7.70 (t, 1H), 7.82 (d, 1H), 8.08 (t, 1H), 8.26 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0165] Example 6: Compound LS-6 and its preparation method

[0166] This embodiment provides an active tripeptide (Compound LS-6) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-6 is as follows:

[0167]

[0168] The preparation method of the compound LS-6 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-undecylamine, and the remaining steps are the same.

[0169] The mass spectrometry results of the target compound LS-6 showed that the + ) mode, the measured m / z value is 849.8, and the theoretical calculated value is 849.7. The H NMR spectrum data of the target compound LS-6 are as follows (chemical shift value δ: Hz):

[0170] 1 H NMR(500MHz,DMSO-d6):0.85(t,6H),1.10-1.47(m,30H),1.52-1.63(m,16H),1.78(m,4H),3.1 8-3.29(m,6H),3.34(m,2H),3.41(m,2H),4.04(m,1H),4.20(m,1H),4.45(m,1H),7.12-7.39(br s,12H),7.42(m,2H),7.66(t,1H),7.88(d,1H),8.15(t,1H),8.36(d,1H).

[0171] After deuterium exchange, the following peaks disappeared: 7.12-7.39 (br s, 12H), 7.42 (m, 2H), 7.66 (t, 1H), 7.88 (d, 1H), 8.15 (t, 1H), 8.36 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0172] Example 7: Compound LS-7 and its preparation method

[0173] This embodiment provides an active tripeptide (Compound LS-7) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-7 is as follows:

[0174]

[0175] The preparation method of the compound LS-7 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-dodecylamine, and the remaining steps are the same.

[0176] The mass spectrometry results of the target compound LS-7 showed that the + ) mode, the measured m / z value is 877.7, and the theoretical calculated value is 877.7. The H NMR spectrum data of the target compound LS-7 are as follows (chemical shift value δ: Hz):

[0177] 1 H NMR(500MHz,DMSO-d6):0.91(t,6H),1.15-1.52(m,28H),1.55-1.64(m,20H),1.72(m,6H),3.0 9-3.22(m,6H),3.30(m,2H),3.43(m,2H),4.00(m,1H),4.27(m,1H),4.53(m,1H),7.15-7.36(br s,12H),7.40(m,2H),7.56(t,1H),7.77(d,1H),8.12(t,1H),8.27(d,1H).

[0178] After deuterium exchange, the following peaks disappeared: 7.15-7.36 (br s, 12H), 7.40 (m, 2H), 7.56 (t, 1H), 7.77 (d, 1H), 8.12 (t, 1H), 8.27 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0179] Example 8: Compound LS-8 and its preparation method

[0180] This embodiment provides an active tripeptide (Compound LS-8) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-8 is as follows:

[0181]

[0182] The preparation method of the compound LS-8 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-tridecylamine, and the remaining steps are the same.

[0183] The mass spectrometry results of the target compound LS-8 showed that the + ) mode, the measured m / z value is 905.9, and the theoretical calculated value is 905.8. The H NMR spectrum data of the target compound LS-8 are as follows (chemical shift value δ: Hz):

[0184] 1H NMR(500MHz,DMSO-d6):0.84(t,6H),1.05-1.40(m,34H),1.43-1.66(m,20H),1.80(m,4H),3.0 2-3.16(m,6H),3.25(m,2H),3.40(m,2H),4.11(m,1H),4.32(m,1H),4.56(m,1H),7.04-7.49(br s,12H),7.53(m,2H),7.59(t,1H),7.70(d,1H),8.16(t,1H),8.38(d,1H).

[0185] After deuterium exchange, the following peaks disappeared: 7.04-7.49 (br s, 12H), 7.53 (m, 2H), 7.59 (t, 1H), 7.70 (d, 1H), 8.16 (t, 1H), 8.38 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0186] Example 9: Compound LS-9 and its preparation method

[0187] This embodiment provides an active tripeptide (Compound LS-9) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-9 is as follows:

[0188]

[0189] The preparation method of the compound LS-9 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-tetradecylamine, and the remaining steps are the same.

[0190] The mass spectrometry results of the target compound LS-9 showed that the + ) mode, the measured m / z value is 933.7, and the theoretical calculated value is 933.8. The H NMR spectrum data of the target compound LS-9 are as follows (chemical shift value δ: Hz):

[0191] 1 H NMR(500MHz,DMSO-d6):0.85(t,6H),1.15-1.33(m,44H),1.35-1.65(m,16H),1.72(m,2H), 3.03-3.15(m,6H),3.26(m,2H),3.35(m,2H),4.09-4.19(m,2H),4.60(m,1H),6.90-7.71(br s,12H),7.79(m,2H),7.84(t,1H),7.88(d,1H),8.21(t,1H),8.32(d,1H).

[0192] After deuterium exchange, the following peaks disappeared: 6.90-7.71 (br s, 12H), 7.79 (m, 2H), 7.84 (t, 1H), 7.88 (d, 1H), 8.21 (t, 1H), 8.32 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0193] Example 10: Compound LS-10 and its preparation method

[0194] This embodiment provides an active tripeptide (Compound LS-10) against Helicobacter pylori and a preparation method thereof. The structural formula of the compound LS-10 is as follows:

[0195]

[0196] The preparation method of the compound LS-10 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-pentadecylamine, and the remaining steps are the same.

[0197] The mass spectrometry results of the target compound LS-10 showed that the + ) mode, the measured m / z value is 961.8, and the theoretical calculated value is 961.8. The H NMR spectrum data of the target compound LS-10 are as follows (chemical shift value δ: Hz):

[0198] 1 H NMR(500MHz,DMSO-d6):0.82(t,6H),1.07-1.39(m,42H),1.43-1.66(m,20H),1.77(m,4H),3.1 0-3.18(m,6H),3.24(m,2H),3.30(m,2H),3.99(m,1H),4.26(m,1H),4.53(m,1H),7.01-7.64(br s,12H),7.75(m,2H),7.86(t,1H),7.95(d,1H),8.15(t,1H),8.20(d,1H).

[0199] After deuterium exchange, the following peaks disappeared: 7.01-7.64 (br s, 12H), 7.75 (m, 2H), 7.86 (t, 1H), 7.95 (d, 1H), 8.15 (t, 1H), 8.20 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0200] Example 11: Compound LS-11 and its preparation method

[0201] This embodiment provides an active tripeptide (Compound LS-11) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-11 is as follows:

[0202]

[0203] The preparation method of the compound LS-11 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-hexadecylamine, and the remaining steps are the same.

[0204] The mass spectrometry results of the target compound LS-11 showed that the + ) mode, the measured m / z value was 989.8, and the theoretical calculated value was 989.9. The H NMR spectrum data of the target compound LS-11 are as follows (chemical shift value δ: Hz):

[0205] 1 H NMR(500MHz,DMSO-d6):0.88(t,6H),1.14-1.35(m,46H),1.40-1.56(m,18H),1.74(m,6H),3.0 0-3.15(m,6H),3.20(m,2H),3.33(m,2H),4.12(m,1H),4.20(m,1H),4.62(m,1H),7.06-7.55(br s,12H),7.67(m,2H),7.80(t,1H),7.98(d,1H),8.12(t,1H),8.37(d,1H).

[0206] After deuterium exchange, the following peaks disappeared: 7.06-7.55 (br s, 12H), 7.67 (m, 2H), 7.80 (t, 1H), 7.98 (d, 1H), 8.12 (t, 1H), 8.37 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0207] Example 12: Compound LS-12 and its preparation method

[0208] This embodiment provides an active tripeptide (Compound LS-12) against Helicobacter pylori and a preparation method thereof. The structural formula of the compound LS-12 is as follows:

[0209]

[0210] The preparation method of the compound LS-12 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-heptadecylamine, and the remaining steps are the same.

[0211] The mass spectrometry results of the target compound LS-12 showed that the + ) mode, the measured m / z value is 1018.0, and the theoretical calculated value is 1017.9. The H NMR spectrum data of the target compound LS-12 are as follows (chemical shift value δ: Hz):

[0212] 1 H NMR(500MHz,DMSO-d6):0.80(t,6H),1.02-1.41(m,50H),1.45-1.68(m,22H),1.81(m,2H),3.1 3-3.18(m,6H),3.26(m,2H),3.38(m,2H),4.17(m,1H),4.24(m,1H),4.55(m,1H),7.02-7.59(br s,12H),7.64(m,2H),7.83(t,1H),7.89(d,1H),8.17(t,1H),8.35(d,1H).

[0213] After deuterium exchange, the following peaks disappeared: 7.02-7.59 (br s, 12H), 7.64 (m, 2H), 7.83 (t, 1H), 7.89 (d, 1H), 8.17 (t, 1H), 8.35 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0214] Example 13: Compound LS-13 and its preparation method

[0215] This embodiment provides an active tripeptide (Compound LS-13) against Helicobacter pylori and a preparation method thereof. The structural formula of the compound LS-13 is as follows:

[0216]

[0217] The preparation method of the compound LS-13 is different from that of Example 1 in that di-n-hexylamine is replaced by di-n-octadecylamine, and the remaining steps are the same.

[0218] The mass spectrometry results of the target compound LS-13 showed that the + ) mode, the measured m / z value is 1045.9, and the theoretical calculated value is 1045.9. The H NMR spectrum data of the target compound LS-13 are as follows (chemical shift value δ: Hz):

[0219] 1H NMR(500MHz,DMSO-d6):0.87(t,6H),1.12-1.39(m,50H),1.43-1.65(m,26H),1.72(m,2H),3.0 5-3.15(m,6H),3.21(m,2H),3.34(m,2H),4.10(m,1H),4.29(m,1H),4.50(m,1H),6.98-7.55(br s,12H),7.60(m,2H),7.78(t,1H),7.85(d,1H),8.24(t,1H),8.38(d,1H).

[0220] After deuterium exchange, the following peaks disappeared: 6.98-7.55 (br s, 12H), 7.60 (m, 2H), 7.78 (t, 1H), 7.85 (d, 1H), 8.24 (t, 1H), 8.38 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0221] Example 14: Compound LS-14 and its preparation method

[0222] This example provides an active tripeptide (Compound LS-14) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-14 is as follows:

[0223]

[0224] The preparation method of the compound LS-14 is different from that of Example 1 in that di-n-hexylamine is replaced by tridecyltetradecylamine, and the remaining steps are the same.

[0225] The mass spectrometry results of the target compound LS-14 showed that the + ) mode, the measured m / z value is 919.7, and the theoretical calculated value is 919.8. The H NMR spectrum data of the target compound LS-14 are as follows (chemical shift value δ: Hz):

[0226] 1 H NMR(500MHz,DMSO-d6):0.82(t,6H),1.03-1.44(m,40H),1.49-1.68(m,16H),1.78(m,4H),3.1 5-3.25(m,6H),3.29(m,2H),3.38(m,2H),4.15(m,1H),4.25(m,1H),4.52(m,1H),6.95-7.58(br s,12H),7.62(m,2H),7.76(t,1H),7.80(d,1H),8.27(t,1H),8.31(d,1H).

[0227] After deuterium exchange, the following peaks disappeared: 6.95-7.58 (br s, 12H), 7.62 (m, 2H), 7.76 (t, 1H), 7.80 (d, 1H), 8.27 (t, 1H), 8.31 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0228] Example 15: Compound LS-15 and its preparation method

[0229] This embodiment provides an active tripeptide (Compound LS-15) against Helicobacter pylori and a preparation method thereof. The structural formula of the compound LS-15 is as follows:

[0230]

[0231] The preparation method of the compound LS-15 is different from that of Example 1 in that di-n-hexylamine is replaced by tridecylpentadecylamine, and the remaining steps are the same.

[0232] The mass spectrometry results of the target compound LS-15 showed that the + ) mode, the measured m / z value is 933.8, and the theoretical calculated value is 933.8. The H NMR spectrum data of the target compound LS-15 are as follows (chemical shift value δ: Hz):

[0233] 1 H NMR(500MHz,DMSO-d6):0.88(t,6H),1.06-1.40(m,40H),1.45-1.59(m,20H),1.74(m,2H),3.0 4-3.21(m,6H),3.28(m,2H),3.35(m,2H),4.10(m,1H),4.22(m,1H),4.57(m,1H),6.99-7.65(br s,12H),7.73(m,2H),7.78(t,1H),7.88(d,1H),8.25(t,1H),8.36(d,1H).

[0234] After deuterium exchange, the following peaks disappeared: 6.99-7.65 (br s, 12H), 7.73 (m, 2H), 7.78 (t, 1H), 7.88 (d, 1H), 8.25 (t, 1H), 8.36 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0235] Example 16: Compound LS-16 and its preparation method

[0236] This embodiment provides an active tripeptide (Compound LS-16) against Helicobacter pylori and its preparation method. The structural formula of the compound LS-16 is as follows:

[0237]

[0238] The preparation method of the compound LS-16 is different from that of Example 1 in that di-n-hexylamine is replaced by tetradecylpentadecylamine, and the remaining steps are the same.

[0239] The mass spectrometry results of the target compound LS-16 showed that the + ) mode, the measured m / z value is 947.8, and the theoretical calculated value is 947.8. The H NMR spectrum data of the target compound LS-16 are as follows (chemical shift value δ: Hz):

[0240] 1 H NMR(500MHz,DMSO-d6):0.84(t,6H),1.11-1.43(m,42H),1.48-1.67(m,20H),1.80(m,2H),3.0 4-3.21(m,6H),3.28(m,2H),3.35(m,2H),4.10(m,1H),4.22(m,1H),4.57(m,1H),6.99-7.65(br s,12H),7.73(m,2H),7.78(t,1H),7.88(d,1H),8.25(t,1H),8.36(d,1H).

[0241] After deuterium exchange, the following peaks disappeared: 6.99-7.65 (br s, 12H), 7.73 (m, 2H), 7.78 (t, 1H), 7.88 (d, 1H), 8.25 (t, 1H), 8.36 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0242] Example 17: Compound LS-17 and its preparation method

[0243] This embodiment provides an active tripeptide (Compound LS-17) against Helicobacter pylori and a preparation method thereof. The structural formula of the compound LS-17 is as follows:

[0244]

[0245] The preparation method of the above compound LS-17 is different from that of Example 1 in that di-n-hexylamine is replaced by tetradecylhexadecylamine, and the remaining steps are the same.

[0246] The mass spectrometry results of the target compound LS-17 showed that the +) mode, the measured m / z value is 961.9, and the theoretical calculated value is 961.8. The H NMR spectrum data of the target compound LS-17 are as follows (chemical shift value δ: Hz):

[0247] 1 H NMR(500MHz,DMSO-d6):0.82(t,6H),1.05-1.52(m,46H),1.55-1.70(m,16H),1.78(m,4H),3.0 1-3.24(m,6H),3.29(m,2H),3.38(m,2H),4.05(m,1H),4.28(m,1H),4.50(m,1H),7.06-7.69(br s,12H),7.72(m,2H),7.81(t,1H),7.92(d,1H),8.16(t,1H),8.30(d,1H).

[0248] After deuterium exchange, the following peaks disappeared: 7.06-7.69 (br s, 12H), 7.72 (m, 2H), 7.81 (t, 1H), 7.92 (d, 1H), 8.16 (t, 1H), 8.30 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0249] Example 18: Compound LS-18 and its preparation method

[0250] This embodiment provides an active tripeptide (Compound LS-18) against Helicobacter pylori and a preparation method thereof. The structural formula of the compound LS-18 is as follows:

[0251]

[0252] The preparation method of the above compound LS-18 is different from that of Example 1 in that di-n-hexylamine is replaced by diisooctylamine, and the remaining steps are the same.

[0253] The mass spectrometry results of the target compound LS-18 showed that the + ) mode, the measured m / z value is 765.5, and the theoretical calculated value is 765.6. The H NMR spectrum data of the target compound LS-18 are as follows (chemical shift value δ: Hz):

[0254] 1H NMR(500MHz,DMSO-d6):0.83(t,6H),0.88(t,6H),1.05-1.52(m,14H),1.55-1.70(m,14H),1.73(m,2H),3.01-3 .12(m,2H),3.15-3.21(m,4H),3.32(m,2H),3.40(m,2H),4.00(m,1H),4.25(m,1H),4.57(m,1H),7.02-7.63(br s,12H),7.69(m,2H),7.83(t,1H),7.95(d,1H),8.10(t,1H),8.39(d,1H).

[0255] After deuterium exchange, the following peaks disappeared: 7.02-7.63 (br s, 12H), 7.69 (m, 2H), 7.83 (t, 1H), 7.95 (d, 1H), 8.10 (t, 1H), 8.39 (d, 1H), indicating that they were active hydrogen atoms on nitrogen atoms.

[0256] Test Example 1: Anti-Helicobacter pylori activity test

[0257] This test example tested the anti-Helicobacter pylori activity of the above-mentioned compounds, and the specific method is as follows:

[0258] First, use an inoculating loop to gently pick up a colony of Helicobacter pylori (deposit number ATCC43504) and place it in 2 mL of normal saline. Mix well to prepare a 0.5 McFarland turbidimetric bacterial suspension. Dilute with normal saline to a concentration of approximately 1 × 10 8 CFU / mL, set aside. Then, accurately weigh each test compound and dissolve it in DMSO to a concentration of 640 μg / mL as a stock solution, which is then sterilized by filtration. Use sterile water for sequential gradient dilutions to obtain a series of drug solutions with concentrations of 640 μg / mL, 320 μg / mL, 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL, set aside.

[0259] Take 1 mL of the above drug solution to be tested and add it to a sterile plate, add 9 mL of blood agar medium (50 ° C), and mix immediately to obtain the final concentrations of the drug solution to be tested of 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL of blood agar plates. Inoculate 10 5CFU / spot of Helicobacter pylori was inoculated on each drug-containing plate, and three plates without drug were inoculated as blank controls. Different concentrations of TP4 (CAS: 1429184-62-2, purchased from Shanghai Hongpeptide Biotechnology Co., Ltd., HPLC purity: 98%) were inoculated as a control group. The plates were cultured at 37°C for 72h, the results were taken out for observation, and the growth status was recorded.

[0260] Criteria: Results were visually observed, with a "+" indicating bacterial growth and no inhibition; a "-" indicating no growth and inhibition. The lowest concentration that inhibited visible bacterial growth was designated as the minimum inhibitory concentration (MIC) of the compound. Each experiment was repeated three times, and the average value was calculated.

[0261] The results of the anti-Helicobacter pylori activity test are shown in Table 1.

[0262] Table 1: Anti-Helicobacter pylori activity test results

[0263]

[0264] The above test results show that the active small peptide compounds of the present invention have good anti-Helicobacter pylori activity, wherein when the R1 and R2 groups are independently CH3(CH2) 12 -、CH3(CH2) 13 - or CH3(CH2) 14 -, it has relatively better anti-Helicobacter pylori activity, with the MIC value as low as 0.25μg / mL.

[0265] Test Example 2: In vitro hemolytic activity test

[0266] This test example uses the three active tripeptides LS-9, LS-10, and LS-14 with the best antibacterial activity as research compounds and TP4 as the control compound to test their in vitro hemolytic activity. The specific steps are as follows:

[0267] (1) Centrifuge sterile sheep blood at 3000 rpm for 5 minutes, rinse with PBS buffer three times, repeat the centrifugation operation once, discard the supernatant, and retain the red blood cells.

[0268] (2) 0.1 mL of red blood cell solution was diluted with 9.9 mL of PBS buffer to a final red blood cell concentration of 1%. 200 μL of a solution of a tripeptide or TP4 at a concentration of 1024, 512, 256, 128, 64, 32, 16, 8, 4, or 2 μg / mL was mixed with 200 μL of the diluted red blood cell solution and incubated at 37°C for 1 hour. The mixture was then centrifuged at 4000 rpm for 5 minutes. The suspension was transferred to a 96-well ELISA plate and the absorbance of the suspension was measured at a wavelength of 414 nm. The hemolysis rate of each well was calculated. 0.01 mol / L PBS buffer was used as a negative control, and 0.1% Triton-X100 (polyethylene glycol octylphenyl ether) was used as a positive control. The hemolysis rate experiment was repeated three times independently, and the average value was taken.

[0269] Here, hemolysis rate (%) = (absorbance of test tube - absorbance of negative control tube) / (absorbance of positive control tube - absorbance of negative control tube) × 100%. The lower the hemolysis rate, the less hemolytic toxicity the compound has.

[0270] The hemolysis rate test results are shown in Table 2.

[0271] Table 2: Hemolysis rate test results

[0272]

[0273] The above test results show that when the test concentration reaches 32 μg / mL (128 times the MIC), the hemolytic rates of the active tripeptides LS-9, LS-10 and LS-14 of the present invention are still less than 5%, while the hemolytic rate of the natural antimicrobial peptide TP4 has reached 100%. It can be seen that the hemolytic toxicity of the compounds LS-9, LS-10 and LS-14 of the present invention is very low and has a high safety. In addition, it can be inferred from the data in the table that the half-maximal hemolytic concentration HC50 of the three compounds LS-9, LS-10 and LS-14 is greater than 512 μg / mL, which shows that their hemolytic activity is low and their relative safety is high.

[0274] Test Example 3: Anti-protease enzymatic ability test

[0275] This test example uses the three active tripeptides LS-9, LS-10, and LS-14 with the best antibacterial activity as research compounds and TP4 as the control compound to test their ability to resist protease hydrolysis. The specific steps are as follows:

[0276] Trypsin, chymotrypsin, pepsin, and proteinase K solutions were incubated with TP4, LS-9, LS-10, and LS-14, respectively, in a 37°C water bath. The final concentration of the protease in the incubation solution was 0.5 mg / mL, and the incubation time was 8 hours. After the incubation was terminated, the incubation solution was inactivated at 60°C for 15 minutes to inactivate the protease. The minimum inhibitory concentration (MIC value) of the antimicrobial peptides treated with each protease against Helicobacter pylori ATCC43504 was then determined according to the antimicrobial activity assay method of Test Example 1. The control group was a group not treated with the antimicrobial peptides.

[0277] The results of the anti-protease enzymatic ability test are shown in Table 3.

[0278] Table 3: Antimicrobial activity of antimicrobial peptides after treatment with different proteases

[0279]

[0280]

[0281] The above test results show that the natural antimicrobial peptide TP4 essentially lost its antimicrobial activity after being treated with trypsin, chymotrypsin, pepsin, and proteinase K solutions for 8 hours, demonstrating low stability against protease hydrolysis. However, under the same conditions, the antimicrobial peptides LS-9, LS-10, and LS-14 of the present invention, containing D-amino acids and modified with long-chain saturated alkyl groups, maintained the same minimum inhibitory concentration (MIC) or only doubled their MIC after being treated with trypsin, chymotrypsin, pepsin, and proteinase K solutions for 8 hours, respectively. This demonstrates that the active small peptides of the present invention possess superior stability against protease hydrolysis compared to the natural antimicrobial peptide TP4.

[0282] In summary, the present invention provides an active small peptide against Helicobacter pylori, its preparation method and application. The active small peptide is obtained based on the modification of the TP4 polypeptide sequence. Compared with TP4, it has fewer amino acids (reduced from 25 to 3), which greatly reduces the difficulty and cost of synthesis. At the same time, the present invention also introduces D-amino acids on its basis, which helps to improve the stability of the compound. In addition, the antibacterial activity, hemolytic activity and anti-protease enzymatic stability tests show that the active small peptide of the present invention has excellent anti-Helicobacter pylori activity. When the R1 and R2 groups are independently CH3(CH2) 12 -、CH3(CH2) 13 - or CH3(CH2) 14 -, the MIC value is as low as 0.25μg / mL, and it has low hemolysis, high safety, and good stability. After being treated with trypsin, chymotrypsin, pepsin, proteinase K, etc., it still maintains good antibacterial activity.

[0283] While the embodiments of the present invention have been described in detail above, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A small molecule active peptide, characterized in that: It is a compound shown in formula I: ； wherein R1 and R2 are independently selected from n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane and 2,2-dimethylbutane, n-heptane, 2-methylhexane, 3-methylhexane, 2,4-dimethylpentane, 2,3-dimethylpentane, 2,2-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, n-octane, 2,2-dimethylhexane, 3,3-dimethylhexane, 2,3-dimethylhexane, 2,4-dimethylhexane, 2,5-dimethylhexane, 3,4-dimethylhexane, 3-ethylhexane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane or n-octadecane.

2. The small molecule active peptide according to claim 1, characterized in that: The compound represented by formula I is selected from any one of the compounds represented by formula I-1 to formula I-18: 。 3. A method for preparing a small molecule active peptide according to claim 1, characterized in that: The following steps are involved: The compound represented by formula II is reacted with the compound represented by formula III, and then subjected to a deprotection treatment to obtain the product, wherein the structure of formula II is as follows: ； wherein R1 and R2 are independently selected from n-hexane, 2-methylpentane, 3-methylpentane, 2,3-dimethylbutane and 2,2-dimethylbutane, n-heptane, 2-methylhexane, 3-methylhexane, 2,4-dimethylpentane, 2,3-dimethylpentane, 2,2-dimethylpentane, 3,3-dimethylpentane, 3-ethylpentane, 2,2,3-trimethylbutane, n-octane, 2,2-dimethylhexane, 3,3-dimethylhexane, 2,3-dimethylhexane, 2,4-dimethylhexane, 2,5-dimethylhexane, 3,4-dimethylhexane, 3-ethylhexane, n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, n-pentadecane, n-hexadecane, n-heptadecane or n-octadecane; The structural formula of the compound shown in Formula III is as follows: ； In the formula, X1 and X2 are independently selected from amino protecting groups, and X1 and X2 are the same or different.

4. The preparation method according to claim 3, characterized in that The deprotection treatment includes treatment with trifluoroacetic acid or dichloromethane.

5. The preparation method according to claim 3, characterized in that The X1 and X2 are independently selected from any one of a Boc group, a Cbz group, a Fmoc group, an Alloc group, a Teoc group, a methyl(ethyl)oxycarbonyl group, a Pht group, a Tos group, a Tfa group, a Trt group, a PMB group, and a Bn group.

6. A pharmaceutical composition, characterized in that: The invention comprises the small molecule active peptide as claimed in claim 1 or 2, and pharmaceutically acceptable excipients.

7. Use of the small molecule active peptide according to claim 1 or 2 or the pharmaceutical composition according to claim 6 in the preparation of an anti-Helicobacter pylori drug.

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