Antibacterial cyclic peptide and use thereof

Abstract

The application provides an antibacterial cyclic peptide having a structure shown in formula I or a stereoisomer, a mixture, or a pharmaceutically acceptable salt thereof. Test results show that the antibacterial cyclic peptide has high antibacterial activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antimicrobial cyclic peptide and its applications. Background Technology

[0002] Antibiotics are primarily secondary metabolites produced by bacteria, fungi, or other microorganisms, or synthetic analogues. Antibiotics are widely used for various infectious diseases; however, the overuse of traditional antibiotics in clinical practice has led to the development of many drug-resistant bacterial strains.

[0003] Cationic antimicrobial peptides represent a new class of antibiotics. While the exact mechanisms of action of cationic antimicrobial peptides are not fully understood, all cationic amphiphilic antimicrobial peptides interact with the cell membrane. The cell membrane is a primary target for antimicrobial peptides; aggregation of antimicrobial peptide molecules on the cell membrane leads to increased permeability and impairs the cell membrane's barrier function. Such resistance in microorganisms requires substantial changes in the lipid composition of the cell membrane; therefore, resistance resulting from antimicrobial peptides targeting these membrane activities is unlikely.

[0004] Alpha-helical and β-sheet antimicrobial peptides are the two major classes of cationic antimicrobial peptides. β-sheet antimicrobial peptides include cyclic polypeptides anchored by intramolecular disulfide bonds, as well as polypeptides with covalent bonds from the N-terminus to the C-terminus, such as bacitracin S and bacitracin. Alpha-helical antimicrobial peptides are more linear molecules that exist in a disordered structure in aqueous media, but they exhibit an amphiphilic helical state through interactions with hydrophobic cell membranes, such as mothropol, magnatenin, and meliostepin.

[0005] Antimicrobial peptides, as a novel type of antibiotic, are less likely to induce drug resistance in microorganisms and have broad application prospects. However, antimicrobial peptides derived directly from organisms are exclusive to other organisms and are difficult to apply directly to humans; that is, these types of antimicrobial peptides are highly toxic to humans and cannot achieve the goal of becoming pharmaceuticals. Therefore, the market potential for artificially synthesized peptide drugs is enormous. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an antimicrobial cyclic peptide and its application, wherein the prepared antimicrobial cyclic peptide has high antibacterial activity.

[0007] Indolicidine (Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2) is an antimicrobial peptide isolated from bovine neutrophil cytoplasmic granules. Composed of 13 amino acids, it is one of the smallest known natural linear antimicrobial peptides. Indolicidin has a broad antimicrobial spectrum, exhibiting strong antimicrobial activity against a variety of aerobic Gram-negative bacteria, Gram-positive bacteria, and fungi. This invention, based on the polypeptide sequence of indolicidine, replaces the amino acids with similar-property amino acids and a small number of modifiable functional amino acids. These functional amino acids are then linked through a cyclic chemical process to form a new antimicrobial peptide with similar or higher antimicrobial activity to indolicidine. Furthermore, cyclization enhances the stability of the antimicrobial peptide.

[0008] To achieve the above objectives, the present invention provides an antimicrobial cyclic peptide having the structure shown in Formula I or its stereoisomers, mixtures, or pharmaceutically acceptable salts:

[0009]

[0010] Wherein, R1 is selected from H, Leu (with the OH group removed from the main chain carboxyl group), Ile (with the OH group removed from the main chain carboxyl group), Ala (with the OH group removed from the main chain carboxyl group), NH2-(CH2)4CO-, NH2-(CH2)6CO-, NH2-(CH2)8CO-, and NH2-(CH2). 10 CO-, NH2-(CH2) 12 CO- or NH2-(CH2) 14 CO-, NH2-CH2CH2OCO-, NH2-(CH2CH2O)2CO- or NH2-(CH2CH2O)3CO-;

[0011] R2 is selected from Ile residue, Leu residue, Ala residue, Phe residue, or Trp residue;

[0012] R3 is selected from Ile residue, Leu residue, Ala residue, Phe residue, or Trp residue;

[0013] R4 is selected from basic natural or basic non-natural amino acid residues.

[0014] R5 is selected from Trp residue, Leu residue, Phe residue, Lys residue, Arg residue, His residue, D-Phe residue, or (2-Me)Phe residue;

[0015] R6 is selected from basic natural or basic non-natural amino acid residues.

[0016] R7 is selected from Trp residue, Leu residue, Phe residue, Chg residue, Phg residue, Cha residue, 1-Nal residue, 2-Nal residue, high phenylalanine residue, Lys residue, Arg residue, His residue, D-Phe residue or (2-Me)Phe residue;

[0017] R8 is selected from Trp residue, Leu residue, Phe residue, Chg residue, Phg residue, Cha residue, 1-Nal residue, 2-Nal residue, high phenylalanine residue, Lys residue, Arg residue, His residue, Pro residue, D-Phe residue or (2-Me)Phe residue;

[0018] R9 is selected from Trp residue, Pro residue, Ala residue, Leu residue, Val residue, Met residue, Glu residue, Chg residue, Phg residue, Cha residue, 1-Nal residue, 2-Nal residue, high phenylalanine residue, Lys residue, Arg residue, His residue, D-Phe residue, or (2-Me)Phe residue;

[0019] R 10 Selected from basic natural or basic non-natural amino acid residues;

[0020] R 11 Selected from basic natural or basic non-natural amino acid residues;

[0021] R 12 Selected from basic natural or basic non-natural amino acid residues;

[0022] R 13 Selected from NH2, Lys (remaining group after removing one hydrogen atom from the main chain amino group), Arg (remaining group after removing one hydrogen atom from the main chain amino group), His (remaining group after removing one hydrogen atom from the main chain amino group), -NH(CH2)4CONH2, -NH(CH2)6CONH2, -NH(CH2)8CONH2, -NH(CH2) 10 CONH2、-NH(CH2) 12 CONH2、-NH(CH2) 14 CONH2, -NHCH[(CH2)4NH2]CONH2, NHCH2CH2OCONH2-, NH(CH2CH2O)2CONH2- or NH(CH2CH2O)3CONH2-;

[0023] R 14 Selected from Cys residues, Hcy residues, or Pen residues;

[0024] In this invention, the amino acid residues mentioned above refer to the groups remaining after removing the carboxyl and amino groups from the corresponding amino acids;

[0025] R 15 Selected from C3-C7 methylene groups, and R 14 The thiol group forms an -S-bond.

[0026] Optionally, R1 is selected from H, NH2-(CH2)6CO-, NH2-(CH2)8CO-, or NH2-(CH2). 10 CO-; preferably H.

[0027] Optionally, R2 is a Leu residue or an Ile residue; preferably an Ile residue.

[0028] Optionally, R3 is a Leu residue or an Ile residue; preferably a Leu residue.

[0029] In this invention, R4 can be a basic natural or basic non-natural amino acid residue. Optionally, R4 is selected from Pro residue, Ala residue, Leu residue, Val residue, Met residue, Glu residue, Ile residue, Chg residue, Phg residue, 2-aminoisobutyric acid residue, (N-Me)Ala residue, Lys residue, Arg residue, His residue, Dab residue, or Orn residue; preferably Pro residue, Ala residue, Leu residue, Val residue, Met residue, Glu residue, Lys residue, or Arg residue; more preferably Pro residue, Ala residue, or Lys residue.

[0030] Optionally, R5 is selected from Trp residues, Leu residues, Phe residues, Lys residues, D-Phe residues, or (2-Me)Phe residues; preferably Trp residues, Leu residues, Phe residues, or Lys residues.

[0031] In this invention, R6 can be a basic natural or basic non-natural amino acid residue. Optionally, R6 is selected from Lys residue, Arg residue, His residue, Dab residue, Orn residue, Ala residue, 2-aminoisobutyric acid residue, (N-Me)Ala residue, Leu residue, Phe residue, Trp residue, or Pro residue. Preferably, R6 is selected from Lys residue, Arg residue, Leu residue, Trp residue, Ala residue, Orn residue, Dab residue, or Pro residue. More preferably, it is Lys residue, Trp residue, or Pro residue.

[0032] Optionally, R7 is selected from Trp residues, Leu residues, Phe residues, Lys residues, D-Phe residues, or (2-Me)Phe residues.

[0033] Optionally, R8 is selected from Trp residues, Leu residues, Phe residues, Lys residues, Pro residues, D-Phe residues, or (2-Me)Phe residues.

[0034] Optionally, R9 is selected from Trp residues, Leu residues, Lys residues, D-Phe residues, or (2-Me)Phe residues.

[0035] In this invention, the R 10 It can be a basic natural or basic non-natural amino acid residue; optionally, the R... 10 The residue is selected from Pro residue, Ala residue, Trp residue, Leu residue, Phe residue, Lys residue, Arg residue, His residue, Val residue, Met residue, Glu residue, Ile residue, Chg residue, Phg residue, 2-aminoisobutyric acid residue, (N-Me)Ala residue, Dab residue, Orn residue, D-Phe residue or (2-Me)Phe residue, preferably Pro residue, Ala residue, Leu residue, Val residue, Met residue, Glu residue, Lys residue or Arg residue; more preferably Pro residue, Ala residue or Lys residue.

[0036] In this invention, the R 11 It can be a basic natural or basic non-natural amino acid residue, preferably a Lys residue, an Arg residue, a His residue, a Dab residue or an Orn residue; more preferably a Lys residue or an Arg residue, and most preferably an Arg residue.

[0037] In this invention, the R 12 It can be a basic natural or basic non-natural amino acid residue, preferably a ys residue, an Arg residue, a His residue, a Dab residue or an Orn residue; more preferably a Lys residue, an Arg residue, a His residue, a Dab residue or an Orn residue; even more preferably a Lys residue or an Arg residue, and most preferably an Arg residue.

[0038] Optionally, the R 11 With R 12 Independently selected from Lys residues or Arg residues; preferably Arg residues.

[0039] Optionally, the R 13 Selected from NH2, Lys (remaining group after removing one hydrogen atom from the main chain amino group), Arg (remaining group after removing one hydrogen atom from the main chain amino group), -NH(CH2)6CONH2 or -NHCH[(CH2)4NH2]CONH2.

[0040] Optionally, the R 14Selected from Cys residues, Hcy residues, or Pen residues.

[0041] Unless otherwise specified, this invention, except for R1 and R 13 In addition, the residues in other groups are the groups remaining after removing the carboxyl and amino groups from amino acids.

[0042] R 15 Selected from C3-C7 methylene groups, and R 14 The thiol group forms an -S-bond.

[0043] Optionally, the R 15 It has any of the following structures:

[0044]

[0045] Indicates the connection location.

[0046] The residues mentioned above in this invention refer to the amino and carboxyl groups that form bonds between amino acids in the protein sequence through dehydration. Since some groups of an amino acid participate in the formation of peptide bonds, the remaining structural part is called a residue. That is, the group remaining after removing the carboxyl and amino groups from an amino acid. Taking Leu as an example, the Leu residue in this invention refers to the group remaining after removing the carboxyl and amino groups from Leu, specifically with the following structure:

[0047]

[0048] Indicates the connection location.

[0049] The structures of the remaining amino acid residues can be deduced similarly.

[0050] Optionally, the antimicrobial cyclic peptide has any of the following structures or stereoisomers, mixtures, or pharmaceutically acceptable salts:

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] The antimicrobial cyclic peptides provided by this invention are not limited to the above-described structures. Any antimicrobial cyclic peptide may have an amidated or unamidated C-terminus, and / or an acetylated or unacetylated N-terminus, and / or one or more L-configured amino acids in the structure may be replaced with D-configured amino acids. The resulting antimicrobial cyclic peptide structure is also within the scope of protection of this invention.

[0059] The C-terminus mentioned above refers to the end of the antimicrobial cyclic peptide that has a -COOH group. The amidation structure mentioned above is an amidation structure formed by linking the -COOH end of the peptide with Rink Amide resin or MBHA resin and then cleaving it.

[0060] The N-terminus mentioned above refers to the end of the antimicrobial cyclic peptide that has -NH2, and the acetylated structure mentioned above is an acetylated structure formed by the reaction of acetic anhydride with -NH2.

[0061] The present invention does not impose any particular limitation on the preparation method of the above-mentioned antimicrobial cyclic peptides, their stereoisomers, mixtures, and pharmaceutically acceptable salts, and any conventional method known to those skilled in the art can be used, including but not limited to solid-phase peptide synthesis. For example, [Stewart J. My Young JD, "Solid Phase Peptide Synthesis, 2nd edition", (1984), Pierce Chemical Company, Rockford, Illinois; Bodanzsky M. y Bodanzsky A., "The practice of Peptide Synthesis", (1994), Springer Verlag, Berlin; Lloyd Williams P. et al., "Chemical Approaches to the Synthesis of Peptides and Proteins", (1997), CRC, Boca Raton, FL, USA], synthesis in solution, enzymatic synthesis [Kullmann W. "Proteases as catalysts for enzymic syntheses of opioidpeptides", (1980), J. Biol. Chem., 255(17), 8234-8238] or any combination thereof.

[0062] Some compound intermediates can also be obtained by fermentation with genetically engineered or unmodified bacterial strains intended to produce the desired sequence, or by controlled hydrolysis of a protein of animal, fungal, or preferably plant origin containing at least the desired peptide sequence. For example, the compounds of the present invention can be produced using nucleic acid sequences encoding the amino acid sequence of the polypeptide described herein, and optionally with appropriate amino acid modifications.

[0063] By way of example only, methods for obtaining the polypeptide compounds, stereoisomers thereof, mixtures, and pharmaceutically acceptable salts of the present invention may include the following stages:

[0064] - Couple N-terminal protected and C-terminal free amino acids with N-terminal free and C-terminal protected or bound amino acids to a solid support;

[0065] - Eliminate the groups protecting the N-terminus;

[0066] - Repeat the coupling procedure and remove the groups protecting the N-terminus until the desired peptide sequence is obtained;

[0067] - Eliminate groups protecting the C-terminus or cleave solid supports;

[0068] Preferably, the C-terminus is bound to a solid support and the process is carried out in a solid phase, thus comprising coupling an N-terminus-protected and C-terminus-free amino acid with an N-terminus-free and C-terminus-bound amino acid to the polymer support; removing the N-terminal protecting group; and repeating the procedure as many times as necessary to obtain a compound of the desired length, ultimately a compound synthesized from the subsequent cleavage of the original polymer support.

[0069] Throughout the synthesis, the functional groups of the amino acid side chains are conveniently protected with temporary or permanent protective groups and can be deprotected simultaneously or orthogonally with the process of cleaving the peptide from the polymer carrier.

[0070] Alternatively, solid-phase synthesis can be carried out using a convergent strategy: coupling the peptide to a polymer carrier or to a peptide or amino acid pre-bound to a polymer carrier. Convergent synthesis strategies are widely known to those skilled in the art and described in Lloyd-Williams P. et al., “Convergent Solid-Phase Peptide Synthesis”, (1993), Tetrahedron, 49(48), 11065-11133.

[0071] Using standard procedures and conditions known in the prior art, the process of the present invention can include, in a non-discriminatory sequence, additional C-terminal deprotection and / or cleavage of the peptide from the polymer carrier; thereafter these terminal functional groups can be modified. Optional C-terminal modification can be performed when the peptide compound of Formula I is immobilized to the polymer carrier or once the peptide compound has been separated from the polymer carrier.

[0072] Those skilled in the art will readily understand that the deprotection / cleavage steps of the C-terminus and N-terminus and their subsequent derivatization can be performed in different sequences according to processes known in the prior art.

[0073] In some specific embodiments of the present invention, the above-mentioned antimicrobial cyclic peptides are prepared according to the following method:

[0074] 1) Starting from amide resin, Fmoc protected amino acid, coupling reagent and organic base, the Fmoc protected amino acid-amide resin coupling product is obtained by reacting in a protective agent.

[0075] 2) A fully protected linear peptide was synthesized by sequentially coupling amino acids with protecting groups using a solid-phase method.

[0076] 3) Add a shearing agent to shear the linear peptide from the resin, while removing the protecting groups of amino acid residues. Vacuum freeze-dry to obtain crude linear peptide, which is then purified to obtain pure linear peptide.

[0077] 4) The above-mentioned linear polypeptide, cross-linking agent and organic base are combined in the liquid phase to obtain antimicrobial cyclic peptide.

[0078] Optionally, the crude linear polypeptide obtained in step 3) is purified as follows:

[0079] Use C4, C8 or C 18 A reversed-phase chromatography column was used to prepare linear polypeptides by AB linear elution using high-performance liquid chromatography.

[0080] Preferably, the elution rate of the AB linear elution is 1-20 mL / min; wherein, mobile phase A is an aqueous solution containing 0.01%-0.5% TFA, and mobile phase B is acetonitrile containing 0.01%-0.5% TFA.

[0081] Optionally, the antimicrobial cyclic peptide obtained in step 4) undergoes the following purification process:

[0082] Use C4, C8 or C 18 A reversed-phase column was used to prepare the pure antimicrobial cyclic peptide by AB linear elution using high-performance liquid chromatography.

[0083] Preferably, the elution rate of the AB linear elution is 1-20 mL / min; wherein, mobile phase A is an aqueous solution containing 0.01%-0.5% TFA, and mobile phase B is acetonitrile containing 0.01%-0.5% TFA.

[0084] The present invention provides a composition comprising the above-described antimicrobial cyclic peptide and an acceptable excipient.

[0085] In this invention, the above composition may be a pharmaceutical composition or a health product composition.

[0086] In this invention, the excipients include, but are not limited to, carriers, diluents, excipients or auxiliaries well known to those skilled in the art.

[0087] Preferably, the carrier of the present invention includes, but is not limited to, sterile water, saline, buffer, phosphate-buffered saline, buffered sodium chloride, plant salt, minimum essential culture medium (MEM), MEM containing HEPES, etc.

[0088] In the above-described compositions of the present invention, the polypeptide compounds may exist alone, or in mixtures of two or more, or may be more tightly associated through compounding, crystallization, ionic bonding, or covalent bonding.

[0089] This invention provides the use of the above-mentioned antimicrobial cyclic peptide or the above-mentioned composition in the preparation of a medicament for controlling microbial infections.

[0090] Optionally, the microbial infection is one or more of the following: wound infection, acne, diabetic foot ulcer, athlete's foot, onychomycosis, tinea, boils, impetigo, cellulitis, staphylococcal scalded skin syndrome, septic arthritis, and bacteremia.

[0091] Based on this, the present invention provides a drug for controlling microbial infection, comprising the above-mentioned antimicrobial cyclic peptide or the above-mentioned composition.

[0092] Compared with existing technologies, this invention provides an antimicrobial cyclic peptide having the structure shown in Formula I or its stereoisomers, mixtures, or pharmaceutically acceptable salts. The antimicrobial activity of the above-mentioned antimicrobial cyclic peptide was tested using methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus epidermidis (MRSE). The test results showed that it has high antimicrobial activity. Detailed Implementation

[0093] To further illustrate the present invention, the antimicrobial cyclic peptides provided by the present invention and their applications are described in detail below with reference to embodiments.

[0094] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified, specific conditions in the embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0095] Peptide synthesis was performed using the standard Fmoc solid-phase method. Rink Amide resin was selected, and the peptide chain was elongated from the C-terminus to the N-terminus.

[0096] Protected amino acids include: Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-(2-Me)Phe-OH, Fmoc-Pro-OH, Fmoc-Trp(Boc)-OH, Fmoc-Val-OH, Fmoc-Met-OH, Fmoc -Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-His(Boc)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Hcy(Trt)-OH, Fmoc-Pen(Trt)-OH, Fmoc-Orn(Boc)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Abu-OH, Fmoc-Hph-OH, Fmoc-Chg-OH, Fmoc-Phg-OH, Fmoc-Cha-OH, Fmoc-1-Nal-OH, Fmoc-2-Nal-OH, Fmoc-Aib-OH, Fmoc-(N-Me)Ala-OH, Fmoc-D-Cys(Trt)-OH, Fmoc-D-Ala-OH, Fmoc-D-Leu-OH, Fmoc-D-Ile-OH, Fmoc-D-Phe-OH, Fmoc-D-Pro-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH. The condensing agent is HBTU / HOBt / DIEA. The deprotecting agent is piperidine / DMF solution. The crude peptide is dissolved in water and then lyophilized for storage. The peptides were separated and purified using medium-pressure liquid chromatography or high-performance liquid chromatography (HPLC), with a purity greater than 85%. The molecular weight of the peptide sequence was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The purified linear peptides were cross-linked with a cross-linking agent in the presence of an activated base to form a ring. The resulting reaction solution was then separated and purified using HPLC, with a purity greater than 90%.

[0097] Peptide sequence synthesis:

[0098] The synthesis conditions are as follows:

[0099] Protect amino acids (natural or non-natural): 2M DMF solution;

[0100] Condensing agent: DMF solution of 3M HBTU / HOBt;

[0101] Activating base: 2M DIEA in DMF solution;

[0102] Deprotecting agent: 20% v / v piperidine in DMF solution;

[0103] Crosslinking agent activated alkaline solution: 6 equivalents of crosslinking agent (1,7-dibromoheptane, 1,6-dibromohexane, 1,5-dibromopentane) of purified linear peptide and DIEA in acetonitrile / water (volume ratio 1:1).

[0104] Examples 1-50: Preparation of compounds 1-50

[0105] 1. Deprotection: Weigh 2.3g (1mmol) of Rink Amide resin and place it in the peptide synthesis reactor. Then, prepare the deprotection reagent according to the above concentration and add it to the resin. React at room temperature, dry under vacuum, add piperidine / DMF again, react at room temperature, dry under vacuum, and wash with DMF until the test is qualified.

[0106] 2. Condensation reaction: Amino acids and condensing agents are added to DMF for activation under ice bath conditions, followed by the addition of an activating base to obtain an activated solution. Finally, the activated solution is added to the resin and reacted at room temperature. The resin is then colored with 5% ninhydrin colorimetric reagent. After the resin changes color, the solvent is removed and the resin is washed with DMF. After passing the test, the solvent is removed, indicating that the condensation reaction is complete.

[0107] 3. Repeat the above deprotection and condensation reactions until peptide chain synthesis is complete, to obtain a peptide resin containing a complete polypeptide sequence structure.

[0108] 4. Peptide Resin Cleavage: Weigh 7g of the synthesized peptide resin and place it in a 250mL round-bottom flask. Place the flask in an ice bath and stir magnetically. Prepare a lysis buffer by adding 10mL of lysis buffer per 1g of peptide resin [lysis buffer (volume percentage): trifluoroacetic acid: triisopropylsilane: ethylenedithiol: water = 92.5:2.5:2.5:2.5]. TFA needs to be pre-cooled in an ice bath for 30min or stored in a refrigerator before use. Add the prepared lysis buffer to the peptide resin under ice bath conditions, stir magnetically, and react for 30min under ice bath conditions. Then remove the ice bath and continue stirring at room temperature for 180min. After the reaction is complete, filter the reaction solution through a G4 sintered glass funnel, concentrate by rotary evaporation, and then add ice-cold methyl tert-butyl ether at a volume ratio of 1:10. A white precipitate will form. Centrifuge for 10min (5000 rpm), remove the supernatant, take the precipitate, dissolve it in 50mL of double-distilled water, and freeze-dry to obtain 1.23g of crude peptide.

[0109] 5. Purification of linear crude peptide: The crude peptide was purified by medium-pressure or high-performance liquid chromatography (HPLC). A C18 column was used, and the eluent consisted of acetonitrile, water, and a small amount of trifluoroacetic acid. Specific procedures: Weigh 1.00 g of the crude peptide, add 20 mL of water and 5 mL of acetonitrile to dissolve the solid, centrifuge for 10 min (5000 rpm), and load the supernatant. The column was pre-equilibrated with 200 mL of 5% acetonitrile / water / 0.1% trifluoroacetic acid solution. After loading, continue rinsing with 200 mL of 5% acetonitrile / water / 0.1% trifluoroacetic acid solution, and analyze the eluent components using HPLC. Based on the HPLC results, gradually increase the acetonitrile concentration until the purified peptide peak is eluted. Combine the eluents, remove most of the solvent by rotary evaporation, lyophilize the pure peptide, and confirm the molecular weight (greater than 85%) using HPLC and MALDI-TOF-MS.

[0110] 6. Linear peptide crosslinking and purification: Take 0.1g of linear peptide and react it with the crosslinking agent in 10mL of activated alkaline solution at room temperature for 4-24h. After the reaction is complete, add 20mL of double-distilled water and load the sample. The chromatographic column is pre-equilibrated with 200mL of 5% acetonitrile / water / 0.1% trifluoroacetic acid solution. After loading the sample, continue to wash with 200mL of 5% acetonitrile / water / 0.1% trifluoroacetic acid solution, and analyze the eluent components by high-performance liquid chromatography (HPLC). Gradually increase the acetonitrile content according to the HPLC results until the main peak of the purified peptide is eluted. Combine the eluents, remove most of the solvent by rotary evaporation, lyophilize the pure peptide, and confirm the molecular weight by HPLC (greater than 95%) and MALDI-TOF-MS.

[0111] The prepared antimicrobial cyclic peptide compounds are shown in Table 1 below.

[0112] Table 1 List of synthetic antimicrobial cyclic peptide compounds

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] Example 51: In vitro bacterial drug susceptibility test (MIC) of antimicrobial cyclic peptides

[0122] Screening for antimicrobial cyclic peptides is performed through in vitro bacterial drug susceptibility testing. The antimicrobial efficacy of antimicrobial cyclic peptides can be evaluated by determining the sensitivity or resistance level of bacteria to them in vitro.

[0123] 1. Experimental Procedure

[0124] 1.1 Test strains

[0125] The test strains were methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-resistant Staphylococcus epidermidis (MRSE). The quality control strain was Staphylococcus aureus ATCC 29213. The test strains were cultured in MHA medium at 35–37°C for 24 h.

[0126] Preparation of bacterial culture: Before the experiment, each bacterial strain was purified by streaking agar plates into single colonies. After incubation at 35–37°C, single colonies were picked and adjusted to approximately 0.5 McFarland units (about 10) using the McFarland turbidimetric method. 8 (CFU / mL). Dilute this bacterial suspension 100-fold and use the agar dilution method. Inoculate the diluted bacterial suspension onto prepared MH agar plates using a multi-point inoculation device. Dilute this bacterial suspension 1000-fold and use the micro-broth method.

[0127] 1.2 Test Procedure

[0128] The antimicrobial peptide concentration was set at a 2-fold dilution within the range of 0.008–128 μg / mL. 1 mL of the antimicrobial peptide solution was added to a sterile Petri dish, followed by 14 mL of melted 50°C MHA medium, and mixed thoroughly. The final antimicrobial peptide concentrations in each dish were 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015, and 0.008 μg / mL, respectively. After cooling, bacteria were inoculated using a multi-spot inoculation device, the dishes were capped, and incubated on MHA medium at 35–37°C for 24 hours. After incubation, the lowest sample concentration where no bacterial growth was observed was determined visually; this was the minimum inhibitory concentration (MIC). A bacterial control without any sample and a blank medium control were also established.

[0129] Based on the above method, the results of the in vitro bacterial drug susceptibility test are shown in Table 2, where serial number 51 is the natural antimicrobial peptide Indolicidine.

[0130] Table 2. Minimum inhibitory concentrations (MICs) of the antimicrobial cyclic peptides against three types of bacteria (μg / mL)

[0131]

[0132]

[0133]

[0134] As shown in Table 2, the various antimicrobial cyclic peptides prepared in this invention exhibited in vitro antibacterial activity.

[0135] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. An antimicrobial cyclic peptide, characterized in that, Having any of the following structures or their pharmaceutically acceptable salts: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 2. The antimicrobial cyclic peptide according to claim 1, characterized in that, The C-terminus of any of the antimicrobial cyclic peptides shown in the structure is an amidated or unamidated structure, and / or the N-terminus is an acetylated or unacetylated structure, and / or one or more L-configured amino acids in the structure are replaced with D-configured amino acids.

3. A method for preparing the antimicrobial cyclic peptide according to any one of claims 1 to 2, comprising the following steps: 1) Starting from amide resin, Fmoc protected amino acid, coupling reagent and organic base, the Fmoc protected amino acid-amide resin coupling product is obtained by reacting in a protecting agent. 2) A fully protected linear peptide was synthesized by sequentially coupling amino acids with protecting groups using a solid-phase method. 3) Add a shearing agent to shear the linear peptide from the resin, while removing the protecting groups of amino acid residues. Vacuum freeze-dry to obtain crude linear peptide, which is then purified to obtain pure linear peptide. 4) The above-mentioned linear polypeptide, cross-linking agent and organic base are combined in the liquid phase to obtain antimicrobial cyclic peptide.

4. The preparation method according to claim 3, characterized in that, The crude linear polypeptide prepared in step 3) is then purified as follows: Use C4, C8 or C 18 A reversed-phase chromatography column was used to prepare linear polypeptides by AB linear elution using high-performance liquid chromatography. The antimicrobial cyclic peptide prepared in step 4) undergoes the following purification process: Use C4, C8 or C 18 A reversed-phase chromatographic column was used to prepare the pure antibacterial cyclic peptide by AB linear elution using high-performance liquid chromatography. The elution rate of the AB linear elution is 1-20 mL / min; wherein, mobile phase A is an aqueous solution containing 0.01%-0.5% TFA, and mobile phase B is acetonitrile containing 0.01%-0.5% TFA.

5. A composition comprising the antimicrobial cyclic peptide according to any one of claims 1 to 2 and an acceptable excipient.

6. The use of the antimicrobial cyclic peptide according to any one of claims 1 to 2 or the composition according to claim 5 in the preparation of a medicament for controlling microbial infections; The microbial infection is one or more of the following: staphylococcal infection causing wound infection, acne, diabetic foot ulcer, boil, impetigo, cellulitis, scalded skin syndrome, septic arthritis, and bacteremia.

7. A medicament for controlling microbial infection, comprising the antimicrobial cyclic peptide according to any one of claims 1 to 2 or the composition according to claim 5; The microbial infection is caused by Staphylococcus.

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