Small molecule active peptide as well as preparation method and application thereof

Abstract

The invention discloses a small-molecule active peptide as well as a preparation method and application thereof, and relates to the technical field of antibacterial peptides. The small molecule active peptide comprises a compound shown in a formula I, or a stereoisomer, a tautomer, a nitrogen oxide, a solvate, a hydrate, a metabolite, a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, a pharmaceutically acceptable ether or a prodrug of the compound shown in the formula I: # imgabs0 #, r1 and R2 are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylamino, halogenated alkyl or naphthenic base, or R1 and R2 are connected to form naphthenic base. The small-molecule active peptide is obtained by modifying a TP4 polypeptide sequence, has fewer amino acids compared with TP4, is low in synthesis cost and relatively high in stability and safety, and has excellent antibacterial effect and drug resistance.

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 bacterium that exists in the stomach and duodenal bulb. It is a Gram-negative bacterium that has long-term and stable residence in the gastric antrum. It can decompose urea to produce ammonia by producing urease. In addition, it can secrete 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 related to the occurrence of indigestion, gastritis, peptic ulcers and gastric cancer. The eradication of Helicobacter pylori can reduce gastric mucosal inflammation, promote ulcer healing, and reduce the risk of gastric cancer.

[0003] In the related art, 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 drug resistance rate of the bacteria. In the treatment of Helicobacter pylori, for primary infection, the resistance rates of clarithromycin and levofloxacin are usually 20% to 40%, and the resistance rate of metronidazole is 60% to 90%; for secondary infection, the resistance rates of clarithromycin are 70% to 80%, the resistance rates of levofloxacin are 60% to 70%, and the resistance rates of metronidazole are all >90%. However, with the continuous use of drugs such as amoxicillin, furazolidone, and tetracycline, the resistance rate of Helicobacter pylori is also quietly increasing, resulting in the success rate of triple or even quadruple eradication treatment of Helicobacter pylori containing metronidazole being difficult to reach Class C (85% to 89%).

[0004] Antimicrobial peptides are small molecule polypeptides with biological activity that are widely present in organisms. They are usually composed of 12 to 50 amino acid residues, have no highly conserved sequences, and are mostly short, amphipathic, and highly cationic molecules. The currently confirmed mechanisms of action of antimicrobial peptides include cell membrane targeting mechanism, cell wall targeting mechanism, intracellular targeting mechanism, and dual or multiple mechanisms. However, the most common mechanism of action of antimicrobial peptides is through their direct activity on the cell membrane of pathogens. This is because many antimicrobial peptides have a net positive charge. The charge of antimicrobial peptides helps their ability to interact with the cell membrane. The electrostatic interaction between cationic antimicrobial peptides and anionic bacterial membranes stabilizes the binding of antimicrobial peptides to the cell membrane. This binding can lead to the destruction of membrane potential, changes in membrane permeability, and leakage of cell contents, ultimately causing the death of pathogenic cells. Since the main target of antimicrobial peptides is the cell membrane with a relatively conservative structure, they have good resistance to drug resistance, which is 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, which has a strong antibacterial and bactericidal effect on Helicobacter pylori and shows a strong potential for treating Helicobacter pylori infection. However, in the hemolysis test, when the concentration of TP4 was 100 μg / mL, its hemolysis rate reached 100%. Due to its excessive hemolytic activity, large molecular weight and high production cost, its clinical application in humans is limited.

[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 a preparation method and application thereof, the small molecule active peptide has excellent antibacterial activity against Helicobacter pylori strains, and the cytotoxicity test shows that it is highly safe and has little toxicity to cells.

[0008] In the first aspect of the present invention, a small molecule active peptide is provided, 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 the basis of the peptide, 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 arginine continuously arranged at the end of TP4. Under physiological conditions, the positive charge of the active small peptide is 4 (TP4 is 7), and it shows excellent antibacterial ability in the antibacterial activity test against Helicobacter pylori. For example, when R1 and R2 are 13-15 carbon alkyl long chains, the minimum inhibitory concentration (MIC) of the active small peptide against Helicobacter pylori ATCC43504 strain is 0.25μg / 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 toxic concentration of the active small peptide cell is CC 50 >200ug / mL means 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 super 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:

[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 from compounds with specific substituents discovered by the present invention and relatively non-toxic 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 pure 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 pure solution or a suitable inert solvent.

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

[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 benzene sulfonate and p-toluene sulfonate.

[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 structural formula 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] The X1 and X2 are independently selected from amino protecting groups, and the 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, in the compound represented by formula II, 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.

[0040] In some embodiments of the present invention, in the compound represented by formula II, 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.

[0041] In some embodiments of the present invention, in the compound represented by formula II, R1 and R2 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, in the compound represented by formula II, R1 and R2 are independently selected from C 6～18 alkyl.

[0043] In some embodiments of the present invention, in the compound represented by formula II, R1 and R2 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 product, 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 connected to form a cycloalkyl;

[0048] The structural formula of the compound shown in 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 aspect, 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, semisolid or liquid, and can be an aqueous solution, non-aqueous solution or 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, pellets, dry extracts, injections or infusions.

[0067] According to some embodiments of the present invention, the administration method of the drug can be a conventional administration 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 as described in any one of the first aspect or the pharmaceutical composition as described in 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 which 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 by one or more substituents, including but not limited to hydroxy, amino, halogen, cyano, alkoxy, alkylamino, alkenyl, alkynyl, nitro, etc.

[0072] "Alkoxy" means connected to the main carbon chain through an oxygen atom, including but not limited to methoxy, ethoxy, propoxy, butoxy, etc. And the alkoxy group 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. And the alkylamino group may be independently substituted with one or more of the above substituents.

[0074] "Alkenyl" refers to a straight 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. wherein the alkenyl group may be independently substituted by one or more of the above substituents.

[0075] "Alkynyl" refers to a straight or branched triple-bond hydrocarbon group 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 by one or more of the above substituents.

[0076] "Cycloalkyl" refers to a monovalent or polyvalent, non-aromatic, saturated or partially unsaturated ring, and does not contain heteroatoms, including a monocyclic ring of 3-20 carbon atoms or a bicyclic ring of 7-20 carbon atoms. It includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-alkenyl, 1-cyclopentyl-2-alkenyl, 1-cyclopentyl-3-alkenyl, cyclohexyl, 1-cyclohexyl-1-alkenyl, 1-cyclohexyl-2-alkenyl, 1-cyclohexyl-3-alkenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, etc. And the cycloalkyl group can be independently substituted with one or more substituents, which include but are not limited to hydroxy, amino, halogen, cyano, alkoxy, alkylamino, alkyl, alkenyl, alkynyl, nitro, etc.

[0077] "Halo" means that the group following it is substituted with halogen, and the number of substitutions may 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, etc. of the administered compound. Thus, the present invention includes metabolites of the compounds of the present invention, including compounds produced by a process of contacting a compound of the present invention with a mammal for a period of time sufficient to yield a metabolite thereof. DETAILED DESCRIPTION

[0080] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments 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 words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0082] When a numerical range is disclosed herein, the above range is considered to be 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 features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included 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, the description with reference to the terms "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 schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0085] In the embodiment of the present invention, the mass spectrum is detected by using an Agilent 6470A LC-MS / MS triple quadrupole liquid chromatography-mass spectrometer, and the nuclear magnetic hydrogen spectrum is detected by using an AVANCEIII 500MHz 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 conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0088] General preparation method:

[0089] The present invention provides an active tripeptide for resisting Helicobacter pylori and a preparation method thereof. The general structural formula of the active tripeptide is R1R2-(D)-Lys-(D)-Orn2, wherein R1 and R2 are independently alkyl, alkenyl, alkynyl, alkoxy, alkylamino, halogenated alkyl or cycloalkyl, or R1 and R2 are connected to form a cycloalkyl; preferably, a long-chain or branched saturated alkane containing 6 to 18 carbon atoms, and (D)-Lys and (D)-Orn are both non-natural D-type amino acids. The synthetic route of 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, and the tert-butyloxycarbonyl (Boc) protecting group is removed under trifluoroacetic acid conditions to obtain a compound B, which is then subjected to a substitution reaction with N,N'-bis-Boc-D-ornithine p-nitrophenolate to obtain a compound C, and the tert-butyloxycarbonyl (Boc) protecting group is removed under trifluoroacetic acid conditions to obtain a compound D, which is then subjected to a substitution reaction with 1,3-di-BOC-2-(trifluoromethylsulfonyl)guanidine to obtain a compound E, and finally all tert-butyloxycarbonyl (Boc) protecting groups are removed under trifluoroacetic acid conditions to obtain a 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 a preparation method thereof. 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] Add N,N'-bis-Boc-D-ornithine (1 mol), p-nitrophenol (1 mol), and 4-dimethylaminopyridine (DMAP, 10 mmol) to a reaction bottle containing 2.5 liters of dichloromethane, stir at room temperature to dissolve, cool to -5°C to -10°C, and add N,N'-dicyclohexylcarbodiimide (DCC, 1.1 mol) dissolved in 500 ml of dichloromethane while controlling the temperature not higher than -5°C. After the addition, keep stirring at -5°C to -10°C for 0.5 hour, then warm to room temperature (about 29°C) and continue stirring to react for 10 hours. The reaction was monitored by thin layer chromatography (TLC). A certain amount of diatomaceous earth was placed in the sand core funnel. The reaction solution was filtered under reduced pressure through the sand core funnel and dried. The filter cake was washed with 200 ml of dichloromethane. The filtrate was combined and concentrated under reduced pressure until no liquid was evaporated. 500 ml of dichloromethane was added to the concentrate to stir and dissolve. Then 2.5 liters of n-heptane were added. A white solid was precipitated under stirring. The stirring and beating was continued for 2 hours. The suspension was filtered under reduced pressure with a Buchner funnel, the filter cake was washed with 400 ml of n-heptane, dried, and the filter cake was dried in a 60°C forced air oven to obtain an off-white solid N,N'-bis-Boc-D-ornithine p-nitrophenolate (301.51 g, yield 66%, HPLC purity 96%).

[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, 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 completed by monitoring with thin layer chromatography (TLC), and 70 ml of water was added to the reaction system, which was stirred for 5 minutes. The mixture was 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 separated 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), stirred at room temperature (about 26°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, 25 ml of tetrahydrofuran was added to dilute and dissolve the concentrate, and the concentrated solution was evaporated under reduced pressure again until no solvent was evaporated to obtain compound LS-1B (HPLC purity: 90%), which was a light yellow viscous solid. Further mass spectrometry results showed that the measured m / z value was 313.3 under electrospray ionization (ESI+) mode, and the theoretical calculated value was 313.3.

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

[0111] The compound LS-1B obtained above was dissolved in 30 ml of N,N-dimethylformamide (DMF), cooled to 0-5°C in an ice-water bath with stirring, triethylamine (4.05 g, 40 mmol) and N,N'-bis-Boc-D-ornithine p-nitrophenolate (5.44 g, 12 mmol) were added, and the mixture was stirred at 0-5°C for 10 minutes, then moved to room temperature (about 26°C) and reacted 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, and the upper organic phase extracts were combined. After the organic phase was concentrated, it was separated 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 the thin layer chromatography (TLC). The reaction system was concentrated by vacuum rotary evaporation until no trifluoroacetic acid was evaporated, 15 ml of dichloromethane was added to the concentrate to dissolve the concentrate, and vacuum rotary evaporation was performed until no solvent was evaporated, and 15 ml of dichloromethane was added to the concentrate again to dissolve the concentrate, and vacuum rotary evaporation was performed 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] The compound LS-1D obtained above was dissolved in 25 ml of dichloromethane, and N,N-diisopropylethylamine (3.23 g, 25 mmol) and 1,3-di-BOC-2-(trifluoromethylsulfonyl)guanidine (3.52 g, 9 mmol) were added, and the reaction was stirred at room temperature (about 23-30°C) for 72 hours. After the reaction was completed by thin layer chromatography (TLC), 30 ml of 8% citric acid aqueous solution was added, stirred for 5 minutes, separated, the lower organic phase was separated, and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel column chromatography (eluent volume ratio: n-heptane: ethyl acetate = 5:1) to obtain compound LS-1E (1.78 g, yield: 59%, HPLC purity: 97%), which was 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), stirred at room temperature (about 30°C) for 3 hours, and the reaction was completed by monitoring the thin layer chromatography (TLC). The reaction system was concentrated by rotary evaporation under reduced pressure until no trifluoroacetic acid was distilled out. 50 ml of methyl tert-butyl ether was added to the concentrate to precipitate a solid, cooled to 0-5°C in an ice-water bath and stirred for 2 hours, and filtered under reduced pressure using a sand core funnel under nitrogen protection. 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 (0.98 g, yield: 84%, HPLC purity: 96%) as a light yellow solid.

[0118] The mass spectrometry results of the target compound LS-1 showed that under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

[0122] This embodiment provides an active tripeptide (compound LS-2) against Helicobacter pylori and a preparation method thereof. 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 completed by monitoring with thin layer chromatography (TLC), 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, and the upper organic phase extracts were combined. After the organic phase was concentrated, it was separated 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), stirred at room temperature (about 28°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, 25 ml of tetrahydrofuran was added to dilute and dissolve the concentrate, and the concentrated solution was evaporated again under reduced pressure until no solvent was evaporated to obtain compound LS-2B (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 is 341.3, and the theoretical calculated value is 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, triethylamine (4.05 g, 40 mmol) and N,N'-bis-Boc-D-ornithine p-nitrophenolate (5.44 g, 12 mmol) were added, and the temperature was kept at 0-5°C with stirring for 10 minutes, then moved to room temperature (about 28°C) and reacted 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, and the upper organic phase extracts were combined. After the organic phase was concentrated, it was separated 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 the thin layer chromatography (TLC). The reaction system was concentrated by vacuum rotary evaporation until no trifluoroacetic acid was evaporated, 20 ml of dichloromethane was added to the concentrate to dissolve the concentrate, and vacuum rotary evaporation was performed until no solvent was evaporated, and 20 ml of dichloromethane was added to the concentrate again to dissolve the concentrate, and vacuum rotary evaporation was performed until no solvent was evaporated 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] The compound LS-2D obtained above was dissolved in 25 ml of dichloromethane, and N,N-diisopropylethylamine (3.23 g, 25 mmol) and 1,3-di-BOC-2-(trifluoromethylsulfonyl)guanidine (3.52 g, 9 mmol) were added, and the reaction was stirred at room temperature (about 20-30°C) for 80 hours. After the reaction was completed by thin layer chromatography (TLC), 32 ml of 8% citric acid aqueous solution was added, stirred for 5 minutes, separated, the lower organic phase was separated, and concentrated under reduced pressure. The concentrated residue was separated and purified by silica gel column chromatography (eluent volume ratio: n-heptane: ethyl acetate = 5:1) to obtain compound LS-2E (1.91 g, yield: 62%, HPLC purity: 96%), which was 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), stirred at room temperature (about 22°C) for 5 hours, and the reaction was completed by monitoring the thin layer chromatography (TLC). The reaction system was concentrated by rotary evaporation under reduced pressure until no trifluoroacetic acid was distilled out. 55 ml of methyl tert-butyl ether was added to the concentrate to precipitate solids, cooled to 0-5°C in an ice-water bath and stirred for 2.5 hours, filtered under reduced pressure with a sand core funnel under nitrogen protection, the filter cake was washed twice with 40 ml of methyl tert-butyl ether, and dried, and the filter cake was dried in a vacuum oven at 80°C to obtain a light yellow solid compound LS-2 (1.06 g, yield: 89%, HPLC purity: 95%).

[0141] The mass spectrometry results of the target compound LS-2 showed that under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

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

[0146]

[0147] The preparation method of the above 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

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

[0153]

[0154] The preparation method of the above 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

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

[0160]

[0161] The difference between the preparation method of the above compound LS-5 and Example 1 is that di-n-hexylamine is replaced by di-n-decylamine, and the other steps are basically the same.

[0162] The mass spectrometry results of the target compound LS-5 showed that under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

[0166] This embodiment provides an active tripeptide (compound LS-6) against Helicobacter pylori and a preparation method thereof. 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

[0173] This embodiment provides an active tripeptide (compound LS-7) against Helicobacter pylori and a preparation method thereof. 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

[0180] This embodiment provides an active tripeptide (compound LS-8) against Helicobacter pylori and a preparation method thereof. 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

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

[0188]

[0189] The preparation method of the above 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

[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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

[0201] This embodiment provides an active tripeptide (compound LS-11) against Helicobacter pylori and a preparation method thereof. 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 under electrospray ionization (ESI + ) mode, the measured value of m / z is 989.8, and the theoretical calculated value is 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

[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-heptadecylammine, and the remaining steps are the same.

[0211] The mass spectrometry results of the target compound LS-12 showed that under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

[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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

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

[0223]

[0224] The preparation method of the above 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

[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 above 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

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

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

[0237]

[0238] The preparation method of the above 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 under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

[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 under electrospray ionization (ESI +) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

[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 di-isooctylamine, and the remaining steps are the same.

[0253] The mass spectrometry results of the target compound LS-18 showed that under electrospray ionization (ESI + ) mode, the measured value of m / z 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 heavy water exchange, the following signal 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), which proved to be active hydrogen on the nitrogen atom.

[0256] Test Example 1: Anti-Helicobacter pylori Activity Test

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

[0258] First, use an inoculation loop to gently pick up a colony of Helicobacter pylori (deposit number ATCC43504) in 2 mL of normal saline and mix well to prepare a 0.5 McFarland turbidimetric bacterial suspension. Dilute with normal saline to a concentration of about 1 × 10 8 CFU / mL, for later use. Then accurately weigh each compound to be tested, dissolve it in DMSO to a concentration of 640μg / mL as a stock solution, and filter to sterilize. Use sterile water to perform gradient dilutions in sequence to obtain a series of drug solutions with concentrations of 640μg / mL, 320μg / mL, 160μg / mL, 80μg / mL, 40μg / mL, 20μg / mL, 10μg / mL, 5μg / mL, 2.5μg / mL, and 1.25μg / mL for later use.

[0259] Take 1 mL of the above drug solution to be tested, add it to a sterile plate, add 9 mL of blood agar medium (50°C), mix it immediately, and get 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] Judgment criteria: The results were observed with the naked eye, where "+" means that the bacteria grew and were not inhibited; "-" means that the bacteria did not grow and were inhibited. The lowest concentration that inhibited the visible growth of bacteria was recorded as the minimum inhibitory concentration (MIC value) of the compound. Each group of experiments was repeated three times and the average value was taken.

[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] In this test example, the three active tripeptides LS-9, LS-10, and LS-14 with the best antibacterial activity were used as research compounds, and TP4 was used as the control compound to detect their in vitro hemolytic activity. The specific steps were 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 with 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] Wherein, 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 lower the hemolytic toxicity of the compound.

[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 hemolysis rates of the active tripeptides LS-9, LS-10 and LS-14 of the present invention are still less than 5%, while the hemolysis 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 small and has high safety. In addition, it can be inferred from the data in the table that the half hemolytic concentration HC50 of the three compounds LS-9, LS-10 and LS-14 is greater than 512 μg / mL, which shows that their hemolysis 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 co-incubated with TP4, LS-9, LS-10, and LS-14 at 37°C in a water bath, respectively, wherein 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 stopped, the incubation solution was inactivated at 60°C for 15 minutes to inactivate the protease. Then, the minimum inhibitory concentration (MIC value) of the antimicrobial peptides treated with each protease against Helicobacter pylori ATCC43504 was determined according to the antimicrobial activity determination method of Detection Example 1, wherein 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 has basically lost its antimicrobial activity after being treated with trypsin, chymotrypsin, pepsin and proteinase K solution for 8 hours, showing low anti-protease enzymatic stability. Under the same conditions, the antimicrobial peptides LS-9, LS-10 and LS-14 containing D-amino acids and modified with long-chain saturated alkyl of the present invention, after being treated with trypsin, chymotrypsin, pepsin and proteinase K solution for 8 hours, still maintain the same minimum inhibitory concentration MIC or the MIC is only doubled, and the antimicrobial activity is well maintained, indicating that the active small peptide of the present invention has better anti-protease enzymatic stability than the natural antimicrobial peptide TP4.

[0282] In summary, the present invention provides an active small peptide against Helicobacter pylori and 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-type amino acids on the basis of it, which helps to improve the stability of the compound. In addition, through the detection of antibacterial activity, hemolytic activity and anti-protease enzymatic stability, it can be seen that the active small peptide of the present invention has better 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] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A small molecule active peptide, characterized in that: Including the compound of formula I, or the stereoisomer, tautomer, nitrogen oxide, solvate, hydrate, metabolite, pharmaceutically acceptable salt, pharmaceutically acceptable ester, pharmaceutically acceptable ether or prodrug of the compound of formula I: 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.

2. The small molecule active peptide according to claim 1, characterized in that: 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; Preferably, said 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; More preferably, said R1 and R2 are independently selected from C 6～18 Alkyl, C 6～18 Alkenyl, C 6～18 Alkynyl or C 6～18 Alkylamino.

3. The small molecule active peptide according to claim 1, characterized in that: The 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.

4. 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:

5. A method for preparing a small molecule active peptide as claimed in 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 structural formula of formula II is as follows: 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 structural formula of the compound shown in Formula III is as follows: The X1 and X2 are independently selected from amino protecting groups, and the X1 and X2 are the same or different.

6. The preparation method according to claim 5, characterized in that: The deprotection treatment includes treatment with trifluoroacetic acid or dichloromethane.

7. The preparation method according to claim 5, 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.

8. A pharmaceutical composition, characterized in that: The invention comprises the small molecule active peptide as claimed in any one of claims 1 to 4, and pharmaceutically acceptable excipients.

9. Use of the small molecule active peptide according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 8 in the preparation of antibacterial drugs.

10. The use according to claim 9, characterized in that: The antibacterial drug is a drug against Helicobacter pylori.

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