An amphiphilic bicyclic peptide and its applications

Abstract

This invention provides an amphiphilic bicyclic peptide and its applications. The amphiphilic bicyclic peptide has an amino acid sequence as shown in general formula (1). The bicyclic antimicrobial peptide of this invention exhibits good antimicrobial activity against a variety of drug-resistant bacterial strains.

Description

Technical Field

[0001] This invention relates to the field of cyclic peptide technology, and more particularly to an amphiphilic bicyclic peptide and its applications. Background Technology

[0002] In recent years, the overuse of antibiotics has led to a continuous increase in bacterial resistance. This phenomenon has severely limited the clinical application of traditional antibiotics, making the treatment of many bacterial infections extremely difficult. Therefore, developing antibiotic alternatives against drug-resistant bacterial infections has become an urgent problem to be solved. Antimicrobial peptides, as an important component of the body's innate immune defense system, possess broad-spectrum antibacterial activity and are less likely to induce bacterial resistance, thus showing promising application prospects in antibiotic alternatives.

[0003] Most antimicrobial peptides have a linear structure, which makes them easily degraded by ubiquitous proteases in the body when administered in vivo, resulting in poor in vivo antimicrobial efficacy. Compared to linear peptides, cyclic peptides have a more compact structure between their amino acid residues, which, through steric hindrance, effectively reduces the recognition and degradation of peptide bonds by proteases. Therefore, antimicrobial peptides containing cyclic structures generally exhibit higher in vivo stability and better in vivo antimicrobial efficacy.

[0004] Currently, research on linear antimicrobial peptides is quite in-depth and extensive, but research and development on cyclic peptide drugs is relatively limited. The main reason is that the synthesis of cyclic peptides involves multiple orthogonal protecting groups and multiple steps, making the synthesis process quite difficult. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an amphiphilic bicyclic peptide and its applications, which is a novel bicyclic peptide compound with good antibacterial activity.

[0006] In one aspect, the present invention provides an amphiphilic bicyclic peptide or a pharmaceutically acceptable salt thereof, said amphiphilic bicyclic peptide having an amino acid sequence as shown in general formula (1):

[0007]

[0008] Wherein, X1 is a basic amino acid or an amino acid derivative with an amino group in its side chain, X2 is a hydrophobic amino acid or a hydrophobic amino acid derivative, n and m may be the same or different numbers, n = 0 to 10, m = 0 to 10; Linker is a linker arm, X3 and X4 may be the same or different, X3 and X4 are linkers used to connect the two amino acids at the two ends of the cyclic peptide, C1 and C2 are cysteine ​​or non-natural amino acids with a thiol group in their side chain.

[0009] According to an embodiment of the present invention, X1 may be selected from one of arginine (Arg, R), lysine (Lys, K), histidine (His, H), homoarginine, or ornithine.

[0010] According to an embodiment of the present invention, X2 may be selected from one of phenylalanine (Phe, F), alanine (Ala, A), leucine (Leu, L), methionine (Met, M), isoleucine (Ile, I), tryptophan (Trp, W), proline (Pro, P), valine (Val, V), alanine (Ala, A), naphthalenealanine, 6-aminohexanoic acid, or γ-aminobutyric acid.

[0011] According to an embodiment of the present invention, the linker may be selected from one of several amino acids (e.g., glycine, alanine, etc.), fatty acid chains (e.g., C6–C18 fatty acids), or polyethylene glycol (PEG).

[0012] According to an embodiment of the present invention, the linker is selected from 2-10 amino acids, such as 2-4 amino acids, 5-7 amino acids, or 8-10 amino acids. In one embodiment of the present invention, the linker is selected from 2-10 glycines. In another embodiment of the present invention, the linker is selected from 3 glycines.

[0013] According to an embodiment of the present invention, X3 or X4 is used to connect the -NH2- group of the N-terminal amino acid and the -SH- group of the C-terminal amino acid of the cyclic peptide.

[0014] According to an embodiment of the present invention, X3 or X4 includes at least one reactive group that forms a chemical bond with a thiol group of C1 or C2, the chemical bond including a thioether bond, a disulfide bond or other form of covalent bond.

[0015] According to embodiments of the present invention, the reactive group includes a haloacyl group, a haloalkyl group, a mercapto group, a carboxyl group, an alcohol group, or an imine group. Preferably, the reactive group is a haloacyl group, and more preferably a chloroacetyl group.

[0016] According to an embodiment of the present invention, X3 or X4 preferably comprises a thioether bond formed by a chloroacetyl group and a thiol group (-SH) of cysteine ​​(Cys, C).

[0017] In one embodiment of the present invention, X3 or X4 is selected from the following structures:

[0018]

[0019] According to an embodiment of the present invention, X1 is selected from arginine (Arg, R).

[0020] According to an embodiment of the present invention, X1 is selected from F(Lys, K).

[0021] According to an embodiment of the present invention, X1 is selected from histidine (His, H).

[0022] According to an embodiment of the present invention, X2 is selected from tryptophan.

[0023] According to an embodiment of the present invention, X2 is selected from phenylalanine.

[0024] According to an embodiment of the present invention, X2 is selected from valine.

[0025] According to an embodiment of the present invention, X2 is selected from leucine or isoleucine.

[0026] According to an embodiment of the present invention, X2 is selected from alanine.

[0027] According to an embodiment of the present invention, the bicyclic peptide includes a C-terminal group, which may be selected from a carboxyl group or an amide group.

[0028] According to an embodiment of the present invention, the amino acid sequence of the bicyclic peptide of the amphiphilic bicyclic peptide is shown in the table below.

[0029]

[0030] According to an embodiment of the present invention, the amphiphilic bicyclic peptide has the amino acid sequence shown in SEQ ID NO:1.

[0031] According to an embodiment of the present invention, the amphiphilic bicyclic peptide has the amino acid sequence shown in SEQ ID NO:2.

[0032] According to an embodiment of the present invention, the amphiphilic bicyclic peptide has the amino acid sequence shown in SEQ ID NO:3.

[0033] According to an embodiment of the present invention, the amphiphilic bicyclic peptide further includes a modified amphiphilic bicyclic peptide derivative.

[0034] According to embodiments of the present invention, the modified amphiphilic bicyclic peptide derivative comprises one or more modifications selected from the following: N-terminal and / or C-terminal modification; replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacement of one or more peptide bonds with substitution bonds; peptide backbone length modification; replacement of the hydrogen on the α-carbon of one or more amino acid residues with another chemical group; and post-synthetic bisorthogonal modification of amino acids (such as cysteine, lysine) with suitable amine, thiol, carboxylic acid, and phenol reactive reagents.

[0035] According to an embodiment of the present invention, the amphiphilic bicyclic peptide has antibacterial activity.

[0036] According to an embodiment of the present invention, the minimum inhibitory concentration (MIC, in μM) of the amphiphilic bicyclic peptide is 5-50 μM.

[0037] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-described amphiphilic bicyclic peptide or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0038] According to embodiments of the present invention, the excipients include, but are not limited to, commonly used pharmaceutically acceptable excipients including, but not limited to, fillers such as lactose and microcrystalline cellulose; binders such as povidone (PVP) and hydroxypropyl methylcellulose (HPMC); disintegrants such as crospovidone and crospovidone sodium carboxymethyl cellulose; lubricants such as magnesium stearate and talc; flow aids such as colloidal silica; solvents such as water or ethanol for preparing solutions or suspensions; and antioxidants such as ascorbic acid.

[0039] The pharmaceutical compositions of the present invention can be administered orally, parenterally, or via inhalation spray. The term "parenterical" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intratendon sheath, and intracranial injection techniques.

[0040] The pharmaceutical compositions of the present invention can be administered orally in any orally acceptable dosage form, including, but not limited to, capsules, tablets, suspensions, and solutions.

[0041] Thirdly, the present invention provides a method for synthesizing the above-mentioned amphiphilic bicyclic peptide or its pharmaceutically acceptable salt, the method comprising preparing a linear polypeptide fragment using a solid-phase synthesis method, performing a cyclization reaction under liquid-phase conditions to obtain a cyclic peptide fragment, and achieving the linkage of the cyclic peptide fragment by a liquid-phase amide condensation method.

[0042] According to an embodiment of the present invention, the linear polypeptide fragment is synthesized using a solid-phase synthesis method, with Rink-amide resin and Fmoc-Gly-CTC resin as supports. In the cyclization step, the monocyclic structure is constructed through the formation of thioether bonds. The connection between monocyclic fragments is achieved by liquid-phase amide condensation. The polypeptide is prepared by reversed-phase high-performance liquid chromatography.

[0043] Fourthly, the present invention provides the application of the above-mentioned amphiphilic bicyclic antimicrobial peptide or its pharmaceutically acceptable salt in the preparation of drugs against drug-resistant bacteria. The antimicrobial peptide can be used to inhibit and / or kill drug-resistant strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Klebsiella pneumoniae.

[0044] The present invention has the following beneficial effects:

[0045] This invention designs a novel class of amphiphilic bicyclic antimicrobial peptides with a novel structure based on the structure-activity relationship of antimicrobial peptides. This provides a new direction for the design of cyclic antimicrobial peptides and helps to promote the development and application of cyclic antimicrobial peptide drugs. The bicyclic antimicrobial peptides of this invention exhibit good antimicrobial activity against a variety of drug-resistant bacterial strains. Attached Figure Description

[0046] Figure 1A This is the mass spectrum of the bicyclic peptide P-01; Figure 1B This is the mass spectrum of the bicyclic peptide P-02; Figure 1C This is the mass spectrum of the bicyclic peptide P-03;

[0047] Figure 2A The reverse high performance liquid chromatogram of the bicyclic peptide P-01 is shown. Figure 2B The reverse high-performance liquid chromatogram of the bicyclic peptide P-02 is shown. Figure 2C This is a reversed-phase high-performance liquid chromatogram of the bicyclic peptide P-03. Detailed Implementation

[0048] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0049] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0050] "Hydrophobic" amino acid residues are those that tend to repel water. They have low or no affinity for water molecules but high affinity for oil molecules. Hydrophobic substances tend to have low or no solubility in water or an aqueous phase and are generally nonpolar, but tend to have high solubility in an oil phase. Examples of hydrophobic amino acids include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), phenylalanine (Phe), methionine (Met), and tryptophan (Trp). In some embodiments, the hydrophobic amino acid residue may include tryptophan.

[0051] "Amphiphilic peptide" refers to a peptide that has both hydrophilic and hydrophobic functional groups. In some embodiments, the secondary structure may place hydrophobic and hydrophilic amino acid residues on opposite sides of the amphiphilic peptide (e.g., inside versus outside when the peptide is in a solvent such as water).

[0052] The term "basic amino acid" refers to an amino acid that has a basic side chain at neutral pH. Basic amino acids have a sufficiently high pKa that they tend to bind to protons, thereby acquiring a positive charge in the process. In some embodiments, the basic amino acid includes a nitrogen-containing side chain that either binds to (and protonates) a proton or releases the proton (and deprotonates). In some embodiments, the basic amino acid may be in NH2 (deprotonated) and NH3. + Between (protonated) forms or between NH (deprotonated) and NH2 + (Protonated) forms or between N (deprotonated) and NH + Balance between (protonated) forms. For basic amino acids at physiological pH, such as approximately pH 7.0, the protonated form predominates. In some embodiments, the basic amino acid is arginine (Arg;R), lysine (Lys;K), or histidine (His;H). The basic amino acid can be a D-isomer or an L-isomer. In some embodiments, the basic amino acid is arginine. In some embodiments, the basic amino acid is histidine. In some embodiments, the basic amino acid is lysine. In some embodiments, the basic amino acid is a derivative of arginine, such as L-2-amino-3-guanidinopropionic acid. In some embodiments, the basic amino acid is a derivative of lysine, such as 5-hydroxylysine, ornithine, N-acetyl-L-lysine, or 2,4-diaminobutyric acid. In some embodiments, the basic amino acid is a derivative of histidine, such as deaminohistidine, hydroxyhistidine, acetylhistidine, homohistidine, N-methylhistidine, α-methylhistidine, imidazole acetic acid, or α,α-dimethylimidazolium acetic acid (DMIA).

[0053] The term "amino acid derivatives with amino groups in their side chains" refers to derivative compounds in which an amino (-NH2) functional group is introduced or present on the side chain (R group) of an amino acid. Amino acids, as α-amino acids containing both amino (-NH2) and carboxyl (-COOH) functional groups, exhibit diverse side chain structures (R groups) depending on the type of amino acid, thus determining their physicochemical properties and biological functions. "Amino acid derivatives with amino groups in their side chains" typically refers to amino acids whose side chains are modified by introducing an additional amino functional group (-NH2) to the side chain, thereby altering their structural characteristics and biological activities. These derivatives are often used to regulate biochemical processes such as the polarity and hydrophilicity of amino acids. Examples include β-aminopropionic acid, N-aminoacetylglutamic acid, 2,3-diaminobutyric acid, 2,3-diaminopropionic acid, 3-amino-2-hydroxypropionic acid, N-amino-tyrosine, 2-amino-3-nitropropionic acid, and N-amino-tyrosine.

[0054] Example 1: Design of Antimicrobial Peptides

[0055] To address the existing problems in the development of antimicrobial peptides, a novel class of amphiphilic bicyclic antimicrobial peptides with a novel structure was designed. These antimicrobial peptides contain two cyclic polypeptide structures, which are constructed by forming thioether bonds between chloroacetyl groups and cysteine ​​thiol groups. The cyclic structures are linked by multiple Gly or PEG chains. Antimicrobial peptides P-01, P-02, and P-03 were prepared as examples, and their structural formulas are shown in Formulas 1, 2, and 3, respectively.

[0056]

[0057] Example 2 Synthesis of antimicrobial peptides

[0058] The bicyclic peptides in this invention are synthesized using solid-phase synthesis and liquid-phase synthesis. Taking bicyclic peptides P-01, P-02, and P-03 as examples, their synthesis steps are as follows:

[0059] P-01:

[0060] 1. The fragment Cl-CH2-CO-HN-K(Dde)RWRWRWC-NH2 was synthesized using a solid-state synthesis method.

[0061] (1) Swelling of resin: Weigh 0.47g (0.25mmol) of Rink-amide resin (loading capacity of 0.53mmol / g) and put it into the reactor. Add 5mL of N,N-dimethylformamide (DMF) and stir to swell for 3h. After the resin expands, turn on the vacuum pump and remove the solvent.

[0062] (2) Deprotection: Add 5 mL of deprotection reagent (20% piperidine / DMF solution) to the reactor and stir to react. After 5 min, dry the mixture and add the deprotection reagent again and stir to react for 25 min to remove the Fmoc protecting group on the resin, exposing the amino group. Wash the resin three times each with DMF and dichloromethane (DCM) alternately.

[0063] (3) Amino acid detection: Dip a small amount of resin into a centrifuge tube using a capillary tube, add one drop each of ninhydrin solution and pyridine solution, place in a 100℃ metal bath heating device and heat for 5 minutes, then remove and observe. If the resin turns blue, it indicates that deprotection was successful and proceed to the next step; if there is no color change, deprotection failed, and the deprotection reagent is added again to repeat the above steps.

[0064] (4) Condensation: Weigh three times the amount of the corresponding amino acid (0.75 mmol) into a 10 mL centrifuge tube along with 0.13 g of 1-hydroxy-7-azobenzotriazole (HOAT). Add 5 mL of DMF and 150 μL of N,N'-diisopropylcarbodiimide (DIC), mix well, and add to the reactor. Turn on the stirrer and react at room temperature for 3 h. After the reaction is complete, drain the reaction solution and wash the resin with DMF and DCM. Repeat the detection operation. If the resin shows no color reaction, the amino acid condensation is successful. If the resin shows blue, the condensation has failed or the reaction is incomplete. Add the material again and repeat the condensation operation until the condensation is successful. Then repeat the deprotection operation and couple the next cis amino acid. Connect the amino acids sequentially (C, W, R, W, R, W, R, K (Dde)).

[0065] (5) Deprotection with chloroacetic anhydride: Remove the Fmoc protecting group from the last amino acid, clean the resin with DMF and DCM, and repeat the detection process. After successful deprotection, weigh 0.43 g of chloroacetic anhydride, dissolve it in 8 mL of DCM, pour it into the reactor, and stir at room temperature for 0.5 h. Clean the resin with DMF and DCM, and repeat the detection process.

[0066] (6) Cleavage: Add 5 mL of anhydrous diethyl ether to the resin after the reaction is complete, wash three times, and dry under vacuum. Transfer the resin to a round-bottom flask, pour in 15 mL of lysis buffer (trifluoroacetic acid / m-methylphenol / anisole / ethylenedithiol / water = 17 / 1 / 1 / 1 / 0.5), stir at low speed, and lyse under ice-water bath conditions for 0.5 h, then lyse at room temperature for 3 h. After complete lysis, add 200 mL of diethyl ether to terminate the lysis, stir vigorously for 10 min, and let stand for 0.5 h. After the precipitate has fully separated, filter it using a G4 funnel, and wash the resin repeatedly with diethyl ether about 6 times. Then, dissolve the peptide in acetic acid / acetonitrile / water solution (v / v / v = 14 / 3 / 3) and filter to obtain the filtrate. Finally, freeze-dry the filtrate to obtain the crude linear peptide.

[0067] 2. Cyclic reaction under liquid phase conditions

[0068] Weigh 60.6 mg of the crude linear peptide and dissolve it in 20 mL of acetonitrile / water mixed solution (v / v = 1 / 1) to a concentration of 2 mM. Dissolve 66 μL of N,N-diisopropylethylamine (DIEA) in 20 mL of acetonitrile aqueous solution, bringing the DIEA concentration to 20 mM. Transfer this solution to a round-bottom flask and add the 2 mM linear peptide solution dropwise under stirring. After reacting for 0.5 h, freeze-dry the cyclized solution.

[0069] 3. Removal of Dde protecting groups from lysine side chains under liquid phase conditions

[0070] Weigh 59.2 mg of the cyclic peptide solid and dissolve it in 20 mL of 2% (v / v) hydrazine hydrate / ethanol solution to a concentration of 2 mM. After stirring the reaction for 0.5 h, remove the ethanol using a vacuum rotary evaporator, dissolve the peptide in water, and then freeze-dry it.

[0071] 4. Identification and purification

[0072] Identification: 0.1 mg of the cyclic peptide powder (with the Dde protecting group removed) was dissolved in 100 μL of ultrapure water and analyzed by analytical high performance liquid chromatography. The peak tip of the main peak was collected, and the molecular weight of the peptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After confirming the correct molecular weight, the peptide was purified.

[0073] Purification: Based on the physicochemical properties of each target peptide, appropriate analytical and purification conditions were selected. The target peptides were collected by gradient elution at room temperature using a preparative high-performance liquid chromatography (HPLC) instrument. Purity analysis was performed using an analytical HPLC instrument. Peptide solutions with a purity greater than 95% were collected, lyophilized, and pure peptide powder was obtained. The powder was collected, weighed, sealed in centrifuge tubes, and stored at -40°C.

[0074] Chromatographic column selection: analytical high performance liquid chromatography column (C18), preparative high performance liquid chromatography column (C18).

[0075] Mobile phase preparation: Mobile phase A is a 1‰ trifluoroacetic acid (TFA) / water solution, which is mixed and then filtered through a 0.45μm aqueous phase filter membrane to remove impurities; Mobile phase B is a 1‰ TFA / (acetonitrile:water = 7:3) solution, which is mixed and then filtered through a 0.45μm organic phase filter membrane to remove impurities.

[0076] 5. The fragment Cl-CH2-CO-HN-RWRWRWC-GGG-COOH was synthesized using a solid-phase synthesis method.

[0077] (1) Swelling of resin: Weigh 0.64g (0.25mmol) of Fmoc-Gly-CTC resin (loading capacity of 0.39mmol / g) and put it into the reactor. Add 5mL of DMF and stir to swell for 3h. After the resin expands, turn on the vacuum pump and remove the solvent.

[0078] (2) Repeat steps 1 (2)-(4) (G, G, C, W, R, W, R, W, R).

[0079] (3) Repeat steps (5) and (6) in step 1 to obtain the crude peptide of the linear peptide.

[0080] 6. Cyclic reaction under liquid phase conditions

[0081] Weigh 55.8 mg of the crude linear peptide and dissolve it in 20 mL of acetonitrile / water (v / v = 1 / 1) mixed solution to a concentration of 2 mM. Dissolve 66 μL of N,N-diisopropylethylamine (DIEA) in 20 mL of acetonitrile aqueous solution, bringing the DIEA concentration to 20 mM. Transfer this solution to a round-bottom flask and add the 2 mM linear peptide solution dropwise under stirring. After reacting for 0.5 h, freeze-dry the cyclized solution to obtain cyclic peptide powder.

[0082] 7. Identification and purification

[0083] Dissolve 0.1 mg of the cyclic peptide powder obtained in step 6 in 100 μL of ultrapure water. The remaining operations are the same as the identification and purification methods in step 4.

[0084] 8. Fragmented amide condensation reaction under liquid phase conditions

[0085] Weigh 4.55 mg of the pure peptide obtained in step 4, 14.13 mg of the pure peptide obtained in step 7, 7.21 mg of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), and 3.38 mg of HOAT, dissolve them in 346 μL of anhydrous DMF, add 12 μL of DIEA, and stir the reaction for 5 h.

[0086] 9. Identification and purification of bicyclic peptides

[0087] Dissolve 0.1 mg of the cyclic peptide powder obtained in step 8 in 100 μL of ultrapure water. The remaining procedures are the same as in step 4. The results are as follows: Figure 1A , Figure 2A As shown.

[0088] P-02:

[0089] 1. The fragment Cl-CH2-CO-HN-K(Dde)RWRWC-NH2 was synthesized using a solid-phase synthesis method.

[0090] (1) Swelling of resin: Weigh 0.47g (0.25mmol) of Rink-amide resin (loading capacity of 0.53mmol / g) and put it into the reactor. Add 5mL of N,N-dimethylformamide (DMF) and stir to swell for 3h. After the resin expands, turn on the vacuum pump and remove the solvent.

[0091] (2) Deprotection: Add 5 mL of deprotection reagent (20% piperidine / DMF solution) to the reactor and stir to react. After 5 min, dry the mixture and add the deprotection reagent again and stir to react for 25 min to remove the Fmoc protecting group on the resin, exposing the amino group. Wash the resin three times each with DMF and dichloromethane (DCM) alternately.

[0092] (3) Amino acid detection: Dip a small amount of resin into a centrifuge tube using a capillary tube, add one drop each of ninhydrin solution and pyridine solution, place in a 100℃ metal bath heating device and heat for 5 minutes, then remove and observe. If the resin turns blue, it indicates that deprotection was successful and proceed to the next step; if there is no color change, deprotection failed, and the deprotection reagent is added again to repeat the above steps.

[0093] (4) Condensation: Weigh three times the amount of the corresponding amino acid (0.75 mmol) into a 10 mL centrifuge tube along with 0.13 g of 1-hydroxy-7-azobenzotriazole (HOAT). Add 5 mL of DMF and 150 μL of N,N'-diisopropylcarbodiimide (DIC), mix well, and add to the reactor. Turn on the stirrer and react at room temperature for 3 h. After the reaction is complete, drain the reaction solution and wash the resin with DMF and DCM. Repeat the detection operation. If the resin shows no color reaction, the amino acid condensation is successful. If the resin shows blue, the condensation has failed or the reaction is incomplete. Add the material again and repeat the condensation operation until the condensation is successful. Then repeat the deprotection operation and proceed with the coupling of the next cis amino acid. Connect the amino acids (C, W, R, W, R, K (Dde)) in sequence.

[0094] (5) Deprotection with chloroacetic anhydride: Remove the Fmoc protecting group from the last amino acid, clean the resin with DMF and DCM, and repeat the detection process. After successful deprotection, weigh 0.43 g of chloroacetic anhydride, dissolve it in 8 mL of DCM, pour it into the reactor, and stir at room temperature for 0.5 h. Clean the resin with DMF and DCM, and repeat the detection process.

[0095] (6) Cleavage: Add 5 mL of anhydrous diethyl ether to the resin after the reaction is complete, wash three times, and dry under vacuum. Transfer the resin to a round-bottom flask, pour in 15 mL of lysis buffer (trifluoroacetic acid / m-methylphenol / anisole / ethylenedithiol / water = 17 / 1 / 1 / 1 / 0.5), stir at low speed, and lyse under ice-water bath conditions for 0.5 h, then lyse at room temperature for 3 h. After complete lysis, add 200 mL of diethyl ether to terminate the lysis, stir vigorously for 10 min, and let stand for 0.5 h. After the precipitate has fully separated, filter it using a G4 funnel, and wash the resin repeatedly with diethyl ether about 6 times. Then, dissolve the peptide in acetic acid / acetonitrile / water solution (v / v / v = 14 / 3 / 3) and filter to obtain the filtrate. Finally, freeze-dry the filtrate to obtain the crude linear peptide.

[0096] 2. Cyclic reaction under liquid phase conditions

[0097] Weigh 47.5 mg of the crude linear peptide and dissolve it in 20 mL of acetonitrile / water mixed solution (v / v = 1 / 1) to a concentration of 2 mM. Dissolve 66 μL of N,N-diisopropylethylamine (DIEA) in 20 mL of acetonitrile aqueous solution, bringing the DIEA concentration to 20 mM. Transfer this solution to a round-bottom flask and add the 2 mM linear peptide solution dropwise under stirring. After reacting for 0.5 h, freeze-dry the cyclized solution.

[0098] 3. Removal of Dde protecting groups from lysine side chains under liquid phase conditions

[0099] Weigh 46.0 mg of the cyclic peptide solid and dissolve it in 20 mL of 2% (v / v) hydrazine hydrate / ethanol solution to a concentration of 2 mM. After stirring for 0.5 h, remove the ethanol using a vacuum rotary evaporator, dissolve the peptide in water, and then freeze-dry it.

[0100] 4. Identification and purification

[0101] Identification: 0.1 mg of the cyclic peptide powder (with the Dde protecting group removed) was dissolved in 100 μL of ultrapure water and analyzed by analytical high performance liquid chromatography. The peak tip of the main peak was collected, and the molecular weight of the peptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After confirming the correct molecular weight, the peptide was purified.

[0102] Purification: Based on the physicochemical properties of each target peptide, appropriate analytical and purification conditions were selected. The target peptides were collected by gradient elution at room temperature using a preparative high-performance liquid chromatography (HPLC) instrument. Purity analysis was performed using an analytical HPLC instrument. Peptide solutions with a purity greater than 95% were collected, lyophilized, and pure peptide powder was obtained. The powder was collected, weighed, sealed in centrifuge tubes, and stored at -40°C.

[0103] Chromatographic column selection: analytical high performance liquid chromatography column (C18), preparative high performance liquid chromatography column (C18).

[0104] Mobile phase preparation: Mobile phase A is a 1‰ trifluoroacetic acid (TFA) / water solution, which is mixed and then filtered through a 0.45μm aqueous phase filter membrane to remove impurities; Mobile phase B is a 1‰ TFA / (acetonitrile:water = 7:3) solution, which is mixed and then filtered through a 0.45μm organic phase filter membrane to remove impurities.

[0105] 5. The fragment Cl-CH2-CO-HN-RWRWC-GGG-COOH was synthesized using a solid-phase synthesis method.

[0106] (1) Swelling of resin: Weigh 0.64g (0.25mmol) of Fmoc-Gly-CTC resin (loading capacity of 0.39mmol / g) and put it into the reactor. Add 5mL of DMF and stir to swell for 3h. After the resin expands, turn on the vacuum pump and remove the solvent.

[0107] (2) Repeat steps (2)-(4) in step 1 (G, G, C, W, R, W, R).

[0108] (3) Repeat steps (5) and (6) in step 1 to obtain the crude peptide of the linear peptide.

[0109] 6. Cyclic reaction under liquid phase conditions

[0110] Weigh 42.2 mg of the crude linear peptide and dissolve it in 20 mL of acetonitrile / water (v / v = 1 / 1) mixed solution to a concentration of 2 mM. Dissolve 66 μL of N,N-diisopropylethylamine (DIEA) in 20 mL of acetonitrile aqueous solution, bringing the DIEA concentration to 20 mM. Transfer this solution to a round-bottom flask and add the 2 mM linear peptide solution dropwise under stirring. After reacting for 0.5 h, freeze-dry the cyclized solution to obtain cyclic peptide powder.

[0111] 7. Identification and purification

[0112] Dissolve 0.1 mg of the cyclic peptide powder obtained in step 6 in 100 μL of ultrapure water. The remaining operations are the same as the identification and purification methods in step 4.

[0113] 8. Fragmented amide condensation reaction under liquid phase conditions

[0114] Weigh 3.36 mg of the pure peptide obtained in step 4, 10.57 mg of the pure peptide obtained in step 7, 7.21 mg of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), and 3.38 mg of HOAT, dissolve them in 346 μL of anhydrous DMF, add 12 μL of DIEA, and stir the reaction for 5 h.

[0115] 9. Identification and purification of bicyclic peptides

[0116] Dissolve 0.1 mg of the cyclic peptide powder obtained in step 8 in 100 μL of ultrapure water. The remaining procedures are the same as in step 4. The results are as follows: Figure 1B , Figure 2B As shown.

[0117] P-03:

[0118] 1. The fragment Cl-CH2-CO-HN-K(Dde)RWRWRWRWC-NH2 was synthesized using a solid-state synthesis method.

[0119] (1) Swelling of resin: Weigh 0.47g (0.25mmol) of Rink-amide resin (loading capacity of 0.53mmol / g) and put it into the reactor. Add 5mL of N,N-dimethylformamide (DMF) and stir to swell for 3h. After the resin expands, turn on the vacuum pump and remove the solvent.

[0120] (2) Deprotection: Add 5 mL of deprotection reagent (20% piperidine / DMF solution) to the reactor and stir to react. After 5 min, dry the mixture and add the deprotection reagent again and stir to react for 25 min to remove the Fmoc protecting group on the resin, exposing the amino group. Wash the resin three times each with DMF and dichloromethane (DCM) alternately.

[0121] (3) Amino acid detection: Dip a small amount of resin into a centrifuge tube using a capillary tube, add one drop each of ninhydrin solution and pyridine solution, place in a 100℃ metal bath heating device and heat for 5 minutes, then remove and observe. If the resin turns blue, it indicates that deprotection was successful and proceed to the next step; if there is no color change, deprotection failed, and the deprotection reagent is added again to repeat the above steps.

[0122] (4) Condensation: Weigh three times the amount of the corresponding amino acid (0.75 mmol) into a 10 mL centrifuge tube along with 0.13 g of 1-hydroxy-7-azobenzotriazole (HOAT). Add 5 mL of DMF and 150 μL of N,N'-diisopropylcarbodiimide (DIC), mix well, and add to the reactor. Turn on the stirrer and react at room temperature for 3 h. After the reaction is complete, drain the reaction solution and wash the resin with DMF and DCM. Repeat the detection operation. If the resin shows no color reaction, the amino acid condensation is successful. If the resin shows blue, the condensation has failed or the reaction is incomplete. Add the material again and repeat the condensation operation until the condensation is successful. Then repeat the deprotection operation and couple the next cis amino acid. Connect the amino acids sequentially (C, W, R, W, R, W, R, W, R, K (Dde)).

[0123] (5) Deprotection with chloroacetic anhydride: Remove the Fmoc protecting group from the last amino acid, clean the resin with DMF and DCM, and repeat the detection process. After successful deprotection, weigh 0.43 g of chloroacetic anhydride, dissolve it in 8 mL of DCM, pour it into the reactor, and stir at room temperature for 0.5 h. Clean the resin with DMF and DCM, and repeat the detection process.

[0124] (6) Cleavage: Add 5 mL of anhydrous diethyl ether to the resin after the reaction is complete, wash three times, and dry under vacuum. Transfer the resin to a round-bottom flask, pour in 15 mL of lysis buffer (trifluoroacetic acid / m-methylphenol / anisole / ethylenedithiol / water = 17 / 1 / 1 / 1 / 0.5), stir at low speed, and lyse under ice-water bath conditions for 0.5 h, then lyse at room temperature for 3 h. After complete lysis, add 200 mL of diethyl ether to terminate the lysis, stir vigorously for 10 min, and let stand for 0.5 h. After the precipitate has fully separated, filter it using a G4 funnel, and wash the resin repeatedly with diethyl ether about 6 times. Then, dissolve the peptide in acetic acid / acetonitrile / water solution (v / v / v = 14 / 3 / 3) and filter to obtain the filtrate. Finally, freeze-dry the filtrate to obtain the crude linear peptide.

[0125] 2. Cyclic reaction under liquid phase conditions

[0126] Weigh 74.9 mg of the crude linear peptide and dissolve it in 20 mL of acetonitrile / water mixed solution (v / v = 1 / 1) to a concentration of 2 mM. Dissolve 66 μL of N,N-diisopropylethylamine (DIEA) in 20 mL of acetonitrile aqueous solution, bringing the DIEA concentration to 20 mM. Transfer this solution to a round-bottom flask and add the 2 mM linear peptide solution dropwise under stirring. After reacting for 0.5 h, freeze-dry the cyclized solution.

[0127] 3. Removal of Dde protecting groups from lysine side chains under liquid phase conditions

[0128] Weigh 73.4 mg of the cyclic peptide solid and dissolve it in 20 mL of 2% (v / v) hydrazine hydrate / ethanol solution to a concentration of 2 mM. After stirring the reaction for 0.5 h, remove the ethanol using a vacuum rotary evaporator, dissolve the peptide in water, and then freeze-dry it.

[0129] 4. Identification and purification

[0130] Identification: 0.1 mg of the cyclic peptide powder (with the Dde protecting group removed) was dissolved in 100 μL of ultrapure water and analyzed by analytical high performance liquid chromatography. The peak tip of the main peak was collected, and the molecular weight of the peptide was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). After confirming the correct molecular weight, the peptide was purified.

[0131] Purification: Based on the physicochemical properties of each target peptide, appropriate analytical and purification conditions were selected. The target peptides were collected by gradient elution at room temperature using a preparative high-performance liquid chromatography (HPLC) instrument. Purity analysis was performed using an analytical HPLC instrument. Peptide solutions with a purity greater than 95% were collected, lyophilized, and pure peptide powder was obtained. The powder was collected, weighed, sealed in centrifuge tubes, and stored at -40°C.

[0132] Chromatographic column selection: analytical high performance liquid chromatography column (C18), preparative high performance liquid chromatography column (C18).

[0133] Mobile phase preparation: Mobile phase A is a 1‰ trifluoroacetic acid (TFA) / water solution, which is mixed and then filtered through a 0.45μm aqueous phase filter membrane to remove impurities; Mobile phase B is a 1‰ TFA / (acetonitrile:water = 7:3) solution, which is mixed and then filtered through a 0.45μm organic phase filter membrane to remove impurities.

[0134] 5. The fragment Cl-CH2-CO-HN-RWRWRWRWC-GGG-COOH was synthesized using a solid-phase synthesis method.

[0135] (1) Swelling of resin: Weigh 0.64g (0.25mmol) of Fmoc-Gly-CTC resin (loading capacity of 0.39mmol / g) and put it into the reactor. Add 5mL of DMF and stir to swell for 3h. After the resin expands, turn on the vacuum pump and remove the solvent.

[0136] (2) Repeat steps 1 (2)-(4) (G, G, C, W, R, W, R, W, R, W, R).

[0137] (3) Repeat steps (5) and (6) in step 1 to obtain the crude peptide of the linear peptide.

[0138] 6. Cyclic reaction under liquid phase conditions

[0139] Weigh 69.6 mg of the crude linear peptide and dissolve it in 20 mL of acetonitrile / water (v / v = 1 / 1) mixed solution to a concentration of 2 mM. Dissolve 66 μL of N,N-diisopropylethylamine (DIEA) in 20 mL of acetonitrile aqueous solution, bringing the DIEA concentration to 20 mM. Transfer this solution to a round-bottom flask and add the 2 mM linear peptide solution dropwise under stirring. After reacting for 0.5 h, freeze-dry the cyclized solution to obtain cyclic peptide powder.

[0140] 7. Identification and purification

[0141] Dissolve 0.1 mg of the cyclic peptide powder obtained in step 6 in 100 μL of ultrapure water. The remaining operations are the same as the identification and purification methods in step 4.

[0142] 8. Fragmented amide condensation reaction under liquid phase conditions

[0143] Weigh 5.73 mg of the pure peptide obtained in step 4, 17.68 mg of the pure peptide obtained in step 7, 7.21 mg of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), and 3.38 mg of HOAT, dissolve them in 346 μL of anhydrous DMF, add 12 μL of DIEA, and stir the reaction for 5 h.

[0144] 9. Identification and purification of bicyclic peptides

[0145] Dissolve 0.1 mg of the cyclic peptide powder obtained in step 8 in 100 μL of ultrapure water. The remaining procedures are the same as in step 4. The results are as follows: Figure 1C , Figure 2C As shown.

[0146] Example 3: Activity of antimicrobial peptides

[0147] To evaluate the in vitro antibacterial activity of peptides against multidrug-resistant (MDR) strains, the minimum inhibitory concentrations (MICs) of P-01, P-02, and P-03 in Staphylococcus aureus (MDR), Staphylococcus epidermidis (MDR), and Escherichia coli (MDR) were determined using the dilution method.

[0148] Using a pipette, add 50 μL of broth sequentially to rows 1-8 of a 96-well plate. Take 50 μL of each pre-prepared 400 μM antimicrobial peptide solution and add it to each well in row 1. Then, using a dilution method, pipette 50 μL of the peptide solution from row 1 to row 2, mix well, and then pipette the peptide solution from row 2 to row 3. Repeat this operation until row 8. Finally, take a new sterile sample loading tank and add the diluted bacterial solution (concentration 5 × 10⁻⁶). 5 (CFU / mL) 50 μL of the drug solution was pipetted into rows 1-8 of a 96-well plate. The drug solution and bacterial culture were thoroughly mixed, and the plate was incubated at 37°C with shaking at 180 rpm for 16 h. The MIC value was defined as the lowest concentration of peptide at which no visible bacterial growth was observed in the well when the plate was placed under fluorescent light. The experimental results are shown in Table 1.

[0149] Table 1. MIC values ​​of bicyclic peptides P-01, P-02, and P-03

[0150]

[0151] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An amphiphilic bicyclic peptide or a pharmaceutically acceptable salt thereof, characterized in that: The amphiphilic bicyclic peptide has any of the structures shown in Formulas 1-3: Formula 1 Formula 2 Formula 3.

2. A pharmaceutical composition, characterized in that: It includes the amphiphilic bicyclic peptide of claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

3. A method for preparing the amphiphilic bicyclic peptide or its pharmaceutically acceptable salt according to claim 1, the method comprising preparing a linear polypeptide fragment by solid-phase synthesis, performing a cyclization reaction under liquid-phase conditions to obtain a cyclic peptide fragment, and achieving the linkage of the cyclic peptide fragment by liquid-phase amide condensation.

4. The use of the amphiphilic bicyclic peptide of claim 1 or its pharmaceutically acceptable salt in the preparation of drugs against drug-resistant bacteria; in, The drug can be used to inhibit and / or kill drug-resistant strains of Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli.

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