A fatty cationic amphipathic peptide mimetic and its preparation method and application
Patent Information
- Application Number
- CN202510925347.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-05
- Publication Date
- 2026-08-28
- Estimated Expiration
- 2045-07-05
Smart Images
Figure CN120987817B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of antibacterial drug development technology, and particularly relates to an aliphatic cationic amphiphilic peptide mimic, its preparation method and application. Background Technology
[0002] Since the discovery of penicillin, traditional antibiotics have served as an effective weapon against infectious diseases for many years. However, the widespread and excessive use of antibiotics in daily life has led to a significant increase in bacterial resistance. Currently, resistant bacterial strains have been found for almost all antibiotics used clinically, foreshadowing the arrival of a post-antibiotic era. Therefore, there is an urgent need to develop new antimicrobial strategies to address the increasingly serious problem of bacterial resistance.
[0003] Developing novel antimicrobial agents with new structures and unique mechanisms of action is a key strategy for treating drug-resistant bacterial infections and effectively addressing bacterial resistance. However, these novel antimicrobial agents currently used in clinical practice are merely chemically modified versions of existing antibiotics, and the problem of cross-resistance among bacteria remains difficult to avoid.
[0004] Inspired by host defense peptides (HDPs), researchers have focused on developing antimicrobial peptide mimics, aiming to retain the key properties of HDPs while addressing their poor stability and high production costs. Unlike HDPs, peptide mimics possess more diverse chemical structures, better scalability, and lower production costs. They also retain the amphiphilicity of HDPs, enabling them to target bacterial cell membranes. Similar to HDPs, they exert their effects through physical interactions (such as electrostatic and hydrophobic interactions) rather than biochemical interactions, making them less likely to induce bacterial resistance. However, the antimicrobial activity of peptide mimics is closely related to their amphiphilic balance, and optimizing this balance is key to improving their efficacy. Summary of the Invention
[0005] To overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide an aliphatic cationic amphiphilic peptide mimic.
[0006] Another object of the present invention is to provide a method for preparing the above-mentioned fatty cationic amphiphilic peptide mimic.
[0007] Another object of the present invention is to provide the application of the above-mentioned fatty cationic amphiphilic peptide mimics.
[0008] This invention is achieved by providing an aliphatic cationic amphiphilic peptide mimic, the chemical structural formula of which is shown in (Ⅰ) below:
[0009]
[0010] In formula (Ⅰ), the R1 group is When the R2 group is selected from as well as Any one of them;
[0011] Alternatively, the R2 group is When the R1 group is selected from as well as Any one of them.
[0012] This invention further discloses a method for preparing aliphatic cationic amphiphilic peptide mimic, the method comprising the following steps:
[0013] (1) Fmoc-Cys(DPM)-OH (1 eq), HBTU (2.5 eq), HOBT (2.5 eq) and DIPEA (6 eq) were dissolved in DMF solution, and then N-Boc-ethylenediamine (1.5 eq) was added. The mixture was stirred at room temperature for 3 h.
[0014] (2) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the first intermediate product.
[0015] (3) Add piperidine and tetrahydrofuran (V:V=3:7) to the first intermediate product and stir for 1 hour to remove the Fmoc protecting group. After removing the solvent under reduced pressure, add ice-cold diethyl ether to precipitate and obtain the second intermediate product.
[0016] (4) Dissolve the second intermediate product in DMF solution, add 2.5 eq HBTU, HOBT, 6 eq DIPEA and 1.5 eq fatty acid (R2-OH) and react at room temperature for 3 h; wherein the fatty acid (R2-OH) is selected from any one of butyric acid, hexanoic acid, octanoic acid, n-decanoic acid, lauric acid, myristic acid, palmitic acid and stearic acid;
[0017] (5) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the third intermediate product.
[0018] (6) Trifluoroacetic acid (8 mL), dichloromethane (20 mL), and TIPS (200 μL) were added to the third intermediate and stirred for 3 h. The solvent was removed under reduced pressure, and ice-cold diethyl ether was added to precipitate the product, yielding an aliphatic cationic amphiphilic peptide mimic with the chemical structure shown in formula (II) below:
[0019]
[0020] In formula (II), the R2 group is selected from... as well as Any one of them.
[0021] Preferably, in steps (2) and (5), the washing, drying, and solvent removal specifically involve: washing the organic phase with 1N hydrochloric acid, sodium bicarbonate, and saturated brine, collecting EA, adding anhydrous sodium sulfate for drying, and removing the solvent using a rotary evaporator.
[0022] This invention further discloses a method for preparing aliphatic cationic amphiphilic peptide mimic, the method comprising the following steps:
[0023] (1) Fmoc-Cys(R1)-OH (1 eq), HBTU (2.5 eq), HOBT (2.5 eq) and DIPEA (6 eq) were dissolved in DMF solution, and then N-Boc-ethylenediamine (1.5 eq) was added. The mixture was stirred at room temperature for 3 h. The Fmoc-Cys(R1)-OH (1 eq) was selected from any one of Fmoc-Cys(Me)-OH, Fmoc-Cys(tBu)-OH, Fmoc-Cys(BZL)-OH, Fmoc-Cys(4-MBZL)-OH, Fmoc-Cys(4-methoxybenzyl)-OH and Fmoc-Cys(Trt)-OH.
[0024] (2) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the first intermediate product.
[0025] (3) Add piperidine and tetrahydrofuran (V:V=3:7) to the first intermediate product and stir for 1 hour to remove the Fmoc protecting group. After removing the solvent under reduced pressure, add ice-cold diethyl ether to precipitate and obtain the second intermediate product.
[0026] (4) Dissolve the second intermediate in DMF solution, add 2.5 eq HBTU, HOBT, 6 eq DIPEA and 1.5 eq lauric acid and react at room temperature for 3 h;
[0027] (5) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the third intermediate product.
[0028] (6) Trifluoroacetic acid (8 mL), dichloromethane (20 mL), and TIPS (200 μL) were added to the third intermediate and stirred for 3 h. The solvent was removed under reduced pressure, and ice-cold diethyl ether was added to precipitate the product, yielding an aliphatic cationic amphiphilic peptide mimic with the chemical structure shown in formula (III) below:
[0029]
[0030] In formula (III), the R1 group is selected from... as well as Any one of them.
[0031] Preferably, in steps (2) and (5), the washing, drying, and solvent removal specifically involve: washing the organic phase with 1N hydrochloric acid, sodium bicarbonate, and saturated brine, collecting EA, adding anhydrous sodium sulfate for drying, and removing the solvent using a rotary evaporator.
[0032] The present invention further discloses the above-mentioned cationic amphiphilic peptide mimicry, stereoisomers, tautomers or complexes of the cationic amphiphilic peptide mimicry, pharmaceutically acceptable salts of the cationic amphiphilic peptide mimicry, or solvates of the pharmaceutically acceptable salts, in the preparation of medicaments for treating, improving and / or preventing infections caused by drug-resistant bacteria and / or drug-resistant fungi.
[0033] Preferably, the drug-resistant bacteria are selected from at least one of Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, Gram-negative bacilli, Klebsiella pneumoniae, Enterobacter, Hafnia, Enterobacter schoenleinii, Proteus, Yersinia, and trophotropic Gram-negative bacilli; the drug-resistant fungi are selected from at least one of Candida, Cryptococcus neoformans, Aspergillus, Mucor, dermatophytes, Sporothrix, Histoplasma, and Pneumocystis carinii.
[0034] The present invention further discloses a medicament for treating infections caused by drug-resistant bacteria or fungi, wherein the active ingredient of the medicament comprises the aforementioned aliphatic cationic amphiphilic peptide mimicry, a stereoisomer, tautomer, or complex of the cationic amphiphilic peptide mimicry, a pharmaceutically acceptable salt of the cationic amphiphilic peptide mimicry, or a solvate of the pharmaceutically acceptable salt.
[0035] Preferably, the drug-resistant bacteria are selected from at least one of Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, Gram-negative bacilli, Klebsiella pneumoniae, Enterobacter, Hafnia, Enterobacter schoenleinii, Proteus, Yersinia, and trophotropic Gram-negative bacilli; the drug-resistant fungi are selected from at least one of Candida, Cryptococcus neoformans, Aspergillus, Mucor, dermatophytes, Sporothrix, Histoplasma, and Pneumocystis carinii.
[0036] This invention overcomes the shortcomings of existing technologies and provides an aliphatic cationic amphiphilic peptide mimic, its preparation method, and its application. First, this invention develops a cysteine-based amphiphilic cationic peptide mimic system. Then, ethylenediamine is introduced as a cationic group by forming a peptide bond at the carboxyl terminus, while a hydrophobic group containing an aromatic ring is introduced into the thiol side chain. The cationic group provides positive charge and hydrophilicity, facilitating electrostatic interaction with negatively charged bacterial membranes, while the hydrophobic group facilitates membrane insertion into the phospholipid bilayer. Furthermore, this invention fine-tunes the amphiphilic balance of the cysteine-based cationic peptide mimic by modifying the N-terminus with fatty acids of different chain lengths, ultimately synthesizing an aliphatic cationic amphiphilic peptide mimic with the chemical structure shown in formula (Ⅰ).
[0037]
[0038] In formula (Ⅰ), the R1 group is When the R2 group is selected from as well as Any one of them;
[0039] Alternatively, the R2 group is When the R1 group is selected from as well as Any one of them.
[0040] In this invention, the aliphatic cationic amphiphilic peptide mimicry shown in formula (I) is composed of compounds shown in formulas (II) and (III).
[0041]
[0042] In formula (II), the R2 group is selected from... as well as Any one of them.
[0043]
[0044] In formula (III), the R1 group is selected from... as well as Any one of them.
[0045] The aliphatic cationic amphiphilic peptide mimicry shown in formula (II) was synthesized via scheme 1.
[0046] In Scheme 1, parental antimicrobial peptide mimics of the (I) series were synthesized using N-Boc-ethylenediamine and commercially available Fmoc-Cys(DPM)-OH; the coupling step was carried out in the liquid phase using dimethylformamide (DMF 30 ml) as the solvent. Specifically, Scheme 1 includes the following steps:
[0047] Fmoc-Cys(DPM)-OH, HBTU (2.5 eq), HOBT (2.5 eq), and DIPEA (6 eq) were dissolved in DMF solution, and then N-Boc-ethylenediamine (1.5 eq) was added. The mixture was stirred at room temperature for 3 h. After confirming the completion of the reaction by thin-layer chromatography (TLC), 60 mL of ethyl acetate and 150 mL of saturated saline were added, and the organic phase was retained. The organic phase was washed with 1N hydrochloric acid, sodium bicarbonate, and saturated saline, respectively. EA was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the first intermediate. Piperidine and tetrahydrofuran (V:V = 3:7) were added to the first intermediate and the mixture was stirred for 1 h to remove the Fmoc protecting group. After removing the solvent under reduced pressure, ice-cold diethyl ether was added to precipitate the second intermediate. The second intermediate was dissolved in DMF solution, and 2.5 eq of HBTU, HOBT, 6 eq of DIPEA, and 1.5 eq of fatty acid were added. The mixture was reacted at room temperature for 3 h. After confirming the reaction was complete by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, and the organic phase was retained. The organic phase was washed with 1N hydrochloric acid, sodium bicarbonate, and saturated saline, respectively. EA was collected and dried over anhydrous sodium sulfate. The solvent was removed using a rotary evaporator to obtain the third intermediate. Trifluoroacetic acid (8 mL), dichloromethane (20 mL), and TIPS (200 μL) were added to the third intermediate, and the mixture was stirred for 3 h. The solvent was removed under reduced pressure, and precipitate was obtained by adding ice-cold diethyl ether to obtain the aliphatic cationic amphiphilic peptide mimic of formula (II), namely peptide mimic 5a–5h.
[0048] The synthesis route for Scheme 1 is shown below:
[0049] Option 1
[0050]
[0051] The aliphatic cationic amphiphilic peptide mimic shown in Formula III was synthesized via Scheme 2. In Scheme 2, Fmoc-Cys(R1)-OH, HBTU (2.5 eq), HOBT (2.5 eq), and DIPEA (6 eq) were dissolved in DMF solution, and then N-Boc-ethylenediamine (1.5 eq) was added. The mixture was stirred at room temperature for 3 h. After confirming the completion of the reaction by thin-layer chromatography (TLC), 60 mL of ethyl acetate and 150 mL of saturated brine were added, and the organic phase was retained. The organic phase was washed with 1N hydrochloric acid, sodium bicarbonate, and saturated brine. EA was collected and dried over anhydrous sodium sulfate. The solvent was removed using a rotary evaporator to obtain the first intermediate. Piperidine and tetrahydrofuran (V:V = 3:7) were added to the first intermediate and the mixture was stirred for 1 h to remove the Fmoc protecting group. After solvent removal under reduced pressure, refractory ether was added to precipitate the product, yielding the second intermediate. The second intermediate was dissolved in DMF solution, and 2.5 eq HBTU, HOBT, 6 eq DIPEA, and 1.5 eq lauric acid were added. The reaction was carried out at room temperature for 3 h. After confirming the completion of the reaction by thin-layer chromatography (TLC), 60 mL ethyl acetate and 150 mL saturated saline were added. The organic phase was retained and washed with 1N hydrochloric acid, sodium bicarbonate, and saturated saline, respectively. EA was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation to obtain the third intermediate. Trifluoroacetic acid (8 mL), dichloromethane (20 mL), and TIPS (200 μL) were added to the third intermediate and the mixture was stirred for 3 h. The solvent was removed under reduced pressure, and icy diethyl ether was added to precipitate the aliphatic cationic amphiphilic peptide mimics shown in formula (III), namely peptide mimics 10a to 10f.
[0052] The synthesis route for Scheme 2 is shown below:
[0053] Option 2
[0054]
[0055] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects: The fatty acid-modified cationic amphiphilic peptide mimicry of the present invention has good antibacterial activity, which can induce cell membrane depolarization, disrupt cell membrane integrity, and rapidly kill bacteria, and is not prone to inducing bacterial resistance. Therefore, the stereoisomers, tautomers, or complexes of the cationic amphiphilic peptide mimicry, pharmaceutically acceptable salts of the cationic amphiphilic peptide mimicry, or solvates of pharmaceutically acceptable salts can be used to prepare treatments, improve and / or prevent drug-resistant bacteria. The drug is an antimicrobial agent for bacteria and / or drug-resistant fungi. In this invention, the drug-resistant bacteria are selected from at least one of Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, Gram-negative bacilli, Klebsiella pneumoniae, Enterobacter, Hafnia, Enterobacter schoenleinii, Proteus, Yersinia, and trophotropic Gram-negative bacilli; the drug-resistant fungi are selected from at least one of Candida, Cryptococcus neoformans, Aspergillus, Mucor, dermatophytes, Sporothrix, Histoplasma, and Pneumocystis carinii. This drug has a wide range of applications. Attached Figure Description
[0056] Figure 1 The hemolysis rate of rat erythrocytes was tested using peptide mimics 5a–10f at concentrations of 6.25–200 μg / mL.
[0057] Figure 2 The bactericidal kinetics of peptide mimic 10c against Staphylococcus aureus were determined at concentrations of 2×MIC and 4×MIC.
[0058] Figure 3 To induce drug resistance in Staphylococcus aureus using peptide mimic 10c, ciprofloxacin was used as a positive control.
[0059] Figure 4 The cell membrane depolarization induced by peptide mimic 10c after treatment of Staphylococcus aureus with 1-8×MIC for 30 minutes was shown. PBS and Triton X-100 were used as negative and positive controls, respectively.
[0060] Figure 5 Fluorescent images of Staphylococcus aureus after treatment with peptide mimic 10c and incubation with PI staining solution for detection by laser confocal microscopy; PBS was used as a negative control and polymyxin B was used as a positive control.
[0061] Figure 6 Acute toxicity of peptide mimic 10c to mice at doses of 35, 50, 65, 80, and 95 mg / kg.
[0062] Figure 7 The results of lung tissue colony counts in mice with MRSA-infected pneumonia after treatment with peptide mimic 10c, with linezolid as a positive control.
[0063] Figure 8 The results of H&E staining of corneal colonies, ocular images, and corneal tissue sections were obtained after treatment of MRSA-infected mice with keratitis using peptide mimic 10c. The results of H&E staining of tissue sections from important organs (including heart, liver, spleen, lung, and kidney) were also obtained. Linezolid was used as a positive control. Detailed Implementation
[0064] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.
[0065] Example 1
[0066] In this embodiment, Fmoc-Cys(DPM)-OH, HBTU, HOBT, and DIPEA used in the synthesis of peptide mimics were purchased from Gil Biochemical Co., Ltd. (Shanghai, China). DMF, trifluoroacetic acid, piperidine, and triisopropylsilane were purchased from Maclean's Pharmaceuticals Ltd. (Shanghai, China).
[0067] I. Synthesis of peptide mimics 5a-5h
[0068] (1) Fmoc-Cys(DPM)-OH (1 eq), HBTU (2.5 eq), HOBT (2.5 eq) and DIPEA (6 eq) were dissolved in DMF solution, and then N-Boc-ethylenediamine (1.5 eq) was added. The mixture was stirred at room temperature for 3 h.
[0069] (2) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the organic phase was washed with 1N hydrochloric acid, sodium bicarbonate and saturated saline. EA was collected, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporator to obtain the first intermediate product.
[0070] (3) Add piperidine and tetrahydrofuran (V:V=3:7) to the first intermediate product and stir for 1 hour to remove the Fmoc protecting group. After removing the solvent under reduced pressure, add ice-cold diethyl ether to precipitate and obtain the second intermediate product.
[0071] (4) Dissolve the second intermediate in DMF solution, add 2.5 eq HBTU, HOBT, 6 eq DIPEA and 1.5 eq fatty acid (R2-OH) and react at room temperature for 3 h;
[0072] (5) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the organic phase was washed with 1N hydrochloric acid, sodium bicarbonate and saturated saline. EA was collected, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporator to obtain the third intermediate product.
[0073] (6) Trifluoroacetic acid (8 mL), dichloromethane (20 mL) and TIPS (200 μL) were added to the third intermediate and stirred for 3 h. The solvent was removed under reduced pressure and precipitated with ice-cold ether to obtain the following aliphatic cationic amphiphilic peptide mimics 5a-5h.
[0074]
[0075] In step (2), the relationship between the choice of fatty acid (R2-OH) and the resulting aliphatic cationic amphiphilic peptide mimicry is shown in Table 1 below:
[0076] Table 1. Correspondence between fatty acids (R2-OH) and peptide mimics 5a–5h
[0077] butyric acid Peptide mimic 5a hexanoic acid Peptide mimic 5b bitter Peptide mimic 5c n-decanoic acid Peptide mimic 5d Lauric acid peptide mimic 5e Myristic acid peptide mimic 5f Palmitic acid 5g peptide mimic stearic acid peptide mimic 5h
[0078] II. Synthesis of peptide mimics 10a-10f
[0079] (1) Fmoc-Cys(R1)-OH (1 eq), HBTU (2.5 eq), HOBT (2.5 eq) and DIPEA (6 eq) were dissolved in DMF solution, and then N-Boc-ethylenediamine (1.5 eq) was added. The mixture was stirred at room temperature for 3 h.
[0080] (2) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the organic phase was washed with 1N hydrochloric acid, sodium bicarbonate and saturated saline. EA was collected, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporator to obtain the first intermediate product.
[0081] (3) Add piperidine and tetrahydrofuran (V:V=3:7) to the first intermediate product and stir for 1 hour to remove the Fmoc protecting group. After removing the solvent under reduced pressure, add ice-cold diethyl ether to precipitate and obtain the second intermediate product.
[0082] (4) Dissolve the second intermediate in DMF solution, add 2.5 eq HBTU, HOBT, 6 eq DIPEA and 1.5 eq lauric acid and react at room temperature for 3 h;
[0083] (5) After confirming the completion of the reaction by thin-layer chromatography (TLC detection), 60 mL of ethyl acetate and 150 mL of saturated saline were added, the organic phase was retained, and the organic phase was washed with 1N hydrochloric acid, sodium bicarbonate and saturated saline. EA was collected, dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporator to obtain the third intermediate product.
[0084] (6) Trifluoroacetic acid (8 mL), dichloromethane (20 mL) and TIPS (200 μL) were added to the third intermediate and stirred for 3 h. The solvent was removed under reduced pressure and precipitated with ice-cold ether to obtain the following aliphatic cationic amphiphilic peptide mimics 10a-10f.
[0085]
[0086] In step (1), the relationship between the selection of Fmoc-Cys(R1)-OH and the resulting aliphatic cationic amphiphilic peptide mimics 10a to 10f is shown in Table 2 below:
[0087] Table 2. Correspondence between Fmoc-Cys(R1)-OH and peptide mimics 10a–10f
[0088] Fmoc-Cys(tBu)-OH Peptide mimic 10a Fmoc-Cys(4-MBZL)-OH Peptide mimic 10b Fmoc-Cys(BZL)-OH Peptide mimic 10c Fmoc-Cys(Me)-OH Peptide mimicry 10d Fmoc-Cys(4-methoxybenzyl)-OH Peptide mimic 10e Fmoc-Cys(Trt)-OH peptide mimic 10f
[0089] III. Purification and Identification of Peptide Mimics
[0090] Peptide mimics were purified using reversed-phase high-performance liquid chromatography (Waters Massachusetts, USA). The chromatography was performed on a C18 column (19 mm × 300 mm, 10 μm) at a flow rate of 8 mL / min using a linear gradient elution from ACN / H2O = 10:90 to ACN / H2O = 90:10.
[0091] The obtained peptide mimic 1 H and 13 3C NMR was performed using a Bruker AVANCE III 400 NMR spectrometer. All data processing was performed offline using Mestre Nova (version 14.2). Purity analysis of the peptide mimics was performed using standard analytical RT-HPLC (Waters Delta 600) with a Waters Deltapak C18 column (4.6 mm × 250 mm, 10 μm). Elution was performed using a linear gradient from 10% to 90% in acetonitrile / water solution containing 0.1% trifluoroacetic acid at a flow rate of 1 mL / min. Finally, the molecular weight (MW) of the peptide mimics was determined using ESI-MS (MaXis 4G, Bruker, Germany) for confirmation.
[0092] IV. Nuclear Magnetic Resonance Characterization Results
[0093] Nuclear magnetic resonance spectroscopy data of peptide mimic 5a: 1 H NMR (400MHz, DMSO) δ8.29(t,J=5.8Hz,1H),8.17(d,J=8.0Hz,1H),7.84(s,3 H),7.45-7.38(m,4H),7.32(dtt,J=6.3,4.8,2.4Hz,4H),7.27-7.20(m,2H), 5.34(s,1H),4.44(td,J=8.0,6.1Hz,1H),3.29(q,J=6.5Hz,2H),3.21-2.54 (m,4H),2.11(t,J=7.3Hz,2H),1.50(q,J=7.3Hz,2H),0.84(t,J=7.4Hz,3H). 13 C10 NMR (101MHz, DMSO) δ 172.31, 170.89, 141.34 (d, J = 5.9Hz), 128.51 (d, J = 3.0Hz), 127.99 (d, J = 3.0Hz), 127.10, 52.73, 51.99, 38.32, 37.00, 36.42, 33.55, 18.53, 13.56. Compound molecular weight data: Theoretical molecular weight C10 22 H 29 N3O2S[M+H] + =400.21, and the molecular weight determined by mass spectrometry was 400.2641. The purity of this peptide mimic was determined to be 99.9% by reversed-phase high-performance liquid chromatography (RP-HPLC).
[0094] Nuclear magnetic resonance spectroscopy data of peptide mimic 5b: 1 H NMR (400MHz, DMSO) δ8.31(t,J=5.8Hz,1H),8.19(d,J=8.1Hz,1H),7.85(s,3H),7.42 (dd,J=12.2,7.6Hz,4H),7.33(q,J=7.1Hz,4H),7.24(dt,J=7.7,3.8Hz,2H),5.34(s ,1H),4.45(q,J=7.5Hz,1H),3.29(q,J=6.5Hz,2H),3.09-2.56(m,4H),2.13(t,J=7. 4Hz, 2H), 1.49 (t, J = 7.2Hz, 2H), 1.23 (td, J = 6.9, 4.4Hz, 4H), 0.82 (t, J = 6.8Hz, 3H). 13C10 NMR (101MHz, DMSO) δ 172.49, 170.89, 141.33 (d, J = 7.7Hz), 128.51 (d, J = 4.5Hz), 127.98 (d, J = 3.4Hz), 127.10 (d, J = 2.0Hz), 52.66, 51.90, 38.30, 36.40, 35.07, 33.54, 30.80, 24.82, 21.87, 13.81. Compound molecular weight data: Theoretical molecular weight C10 24 H 33 N3O2S[M+H] + =428.24, and the molecular weight determined by mass spectrometry was 428.2862. The purity of this peptide mimic was determined to be 99.9% by reversed-phase high-performance liquid chromatography (RP-HPLC).
[0095] Nuclear magnetic resonance spectroscopy data of peptide mimic 5c: 1 H NMR(400MHz,DMSO)δ8.30(t,J=5.8Hz,1H),8.19(d,J=8.1Hz,1H),7.90-7.8 0(m,3H),7.45-7.39(m,4H),7.36-7.30(m,4H),7.27-7.21(m,2H),5.34(s, 1H),4.52-4.42(m,1H),3.32-3.26(m,2H),3.14-2.57(m,4H),2.13(td,J=7 .4,2.8Hz,2H),1.52-1.45(m,2H),1.25-1.18(m,8H),0.85(d,J=6.8Hz,3H). 13 CNMR (101MHz, DMSO) δ 172.49, 170.89, 141.33 (d, J = 8.0Hz), 128.50 (d, J = 5.1Hz), 127.98 (d, J = 3.7Hz), 127.09 (d, J = 2.5Hz), 52.67, 51.89, 38.30, 36.40, 35.12, 33.56, 31.14, 28.53, 28.46, 25.15, 22.03, 13.91. Compound molecular weight data: theoretical molecular weight C 26 H 37 N3O2S[M+H] + =456.27, actual molecular weight 456.2681. RP-HPLC determined the purity of this peptide mimic to be 99.68%.
[0096] Nuclear magnetic resonance spectroscopy data of peptide mimics 5d: 1H NMR (400MHz, DMSO) δ8.29(t,J=5.8Hz,1H),8.17(d,J=8.2Hz,1H),7.89-7.79(m,3H ),7.41(dd,J=12.9,7.4Hz,4H),7.32(q,J=7.4Hz,4H),7.26-7.20(m,2H),5.33(s, 1H),4.44(td,J=8.2,5.8Hz,1H),3.29(q,J=6.4Hz,2H),2.95-2.61(m,4H),2.12(t d,J=7.3,3.1Hz,2H),1.47(q,J=6.6Hz,2H),1.24-1.19(m,12H),0.86-0.83(m,3H). 13 C NMR (101MHz, DMSO) δ 172.49, 170.89, 141.34 (d, J = 8.8Hz), 128.52, 128.46, 128.00, 127.96, 127.08 (d, J = 2.9Hz), 52.68, 51.89, 38.30, 36.40, 35.13, 31.25, 28.87, 28.82, 28.65, 28.58, 25.15, 22.07, 13.93. Compound molecular weight data: theoretical molecular weight C 28 H 40 N3O2S[M+H] + =484.30, actual molecular weight 484.3545. RP-HPLC determined the purity of this peptide mimic to be 98.76%.
[0097] Nuclear magnetic resonance spectroscopy data of compound 5e: 1 H NMR (400MHz, DMSO) δ8.25(t,J=5.8Hz,1H),8.17(d,J=8.1Hz,1H),7.64(s,3H),7.47-7.38(m,4H),7.36-7.30(m,4H),7.24(td,J=7.1,5.0Hz,2H),5. 34(s,1H),4.44(q,J=7.7Hz,1H),3.27(t,J=6.4Hz,2H),2.99-2.59(m,4H) ,2.13(h,J=7.0Hz,2H),1.48(s,2H),1.22(s,16H),0.86(t,J=6.7Hz,3H). 13C10 NMR (151MHz, DMSO) δ 172.96, 171.38, 141.84 (d, J = 13.6Hz), 129.02, 128.96, 128.51, 128.47, 127.58 (d, J = 4.9Hz), 53.17, 52.37, 38.80, 36.90, 31.78, 29.49, 29.27 (d, J = 20.6Hz), 25.67, 22.58, 14.44. Compound molecular weight data: theoretical molecular weight C10 30 H 45 N3O2S[M+H] + =512.23, actual molecular weight 512.3312. RP-HPLC determined the purity of this peptide mimic to be 99.9%.
[0098] Nuclear magnetic resonance spectroscopy data of peptide mimic 5f: 1 H NMR (400MHz, DMSO) δ8.26 (q, J=6.0Hz, 1H), 8.15 (d, J=8.2Hz, 1H), 7.82 (s, 3H), 7 .51-7.37(m,4H),7.32(q,J=7.5Hz,4H),7.26-7.19(m,2H),5.33(s,1H),4.45(qd ,J=8.0,5.4Hz,1H),3.32-3.26(m,2H),2.82(dq,J=9.9,4.6Hz,4H),2.12(td,J=7 .3,3.9Hz,2H),1.47(q,J=6.4Hz,2H),1.22(d,J=6.7Hz,20H),0.86-0.83(m,3H). 13 C10 NMR (101MHz, DMSO) δ 172.46, 170.89, 141.34 (d, J = 9.3Hz), 128.48 (d, J = 5.7Hz), 127.99 (d, J = 3.2Hz), 127.07 (d, J = 3.7Hz), 52.71, 51.90, 38.33, 36.42, 31.26, 29.03-28.99 (m), 28.92, 28.82, 28.68, 28.60, 25.16, 22.06, 13.91. Compound molecular weight data: theoretical molecular weight C10 32 H 49 N3O2S[M+H] + =540.36, measured molecular weight 540.3977. RP-HPLC determined the purity of this peptide mimic to be 99.9%.
[0099] Nuclear magnetic resonance spectroscopy analysis data of 5g of peptide mimic: 1H NMR (400MHz, DMSO) δ8.27(t,J=5.7Hz,1H),8.15(d,J=8.1Hz,1H),7.81(s,3H),7. 44-7.38(m,4H),7.32(d,J=7.5Hz,4H),7.25-7.20(m,2H),5.33(s,1H),4.44(td, J=8.2,5.9Hz,1H),3.29(d,J=6.3Hz,2H),2.82(h,J=5.1Hz,4H),2.12(td,J=7.3, 4.3Hz, 2H), 1.47 (q, J = 6.8Hz, 2H), 1.22 (d, J = 7.2Hz, 24H), 0.84 (d, J = 7.0Hz, 3H). 13 C10 NMR (101MHz, DMSO) δ 172.46, 170.89, 141.34 (d, J = 9.3Hz), 128.50, 128.45, 128.00, 127.97, 127.06 (d, J = 3.7Hz), 52.71, 51.90, 38.33, 36.42, 35.14, 33.59, 31.26, 29.02, 28.92, 28.83, 28.68, 28.60, 25.16, 22.06, 13.91. Compound molecular weight data: theoretical molecular weight C10 34 H 53 N3O2S[M+H] + =568.39, actual molecular weight 568.3935. RP-HPLC determined the purity of this peptide mimic to be 99.9%.
[0100] Nuclear magnetic resonance spectroscopy data of peptide mimics after 5 hours: 1 H NMR(400MHz,DMSO)δ8.27(t,J=5.8Hz,1H),8.15(d,J=8.1Hz,1H),7.81(s,3H), 7.43-7.38(m,4H),7.31(d,J=7.5Hz,4H),7.23(dd,J=7.7,5.8Hz,2H),5.33(s,1 H),4.47-4.42(m,1H),3.31-3.26(m,2H),2.83(p,J=6.3Hz,4H),2.12(td,J=7. 3,4.4Hz,2H),1.47(p,J=6.6Hz,2H),1.22(d,J=9.8Hz,28H),0.86-0.83(m,3H). 13C10 NMR (101MHz, DMSO) δ 172.46, 170.89, 141.34 (d, J = 9.4Hz), 128.50, 128.44, 128.00, 127.97, 127.06 (d, J = 4.0Hz), 52.71, 51.89, 38.33, 36.42, 35.14, 33.60, 31.26, 29.01, 28.93, 28.83, 28.67, 28.61, 22.06, 13.91. Compound molecular weight data: Theoretical molecular weight C10 36 H 57 N3O2S[M+H] + =596.42, actual molecular weight 596.4549. RP-HPLC determined the purity of this peptide mimic to be 95.27%.
[0101] Nuclear magnetic resonance spectroscopy data of peptide mimic 10a: 1 H NMR (400MHz, DMSO) δ8.22(t,J=5.7Hz,1H),8.11(d,J=8.0Hz,1H),7.80(s,3H),4.30(td,J=8.0,6.0Hz,1H),3.28(t,J =6.5Hz,2H),2.87-2.64(m,4H),2.12(h,J=7.0Hz,2H),1.48(s,2H),1.26(s,9H),1.24(s,16H),0.86(t,J=6.7Hz,3H). 13 C10 NMR (101MHz, DMSO) δ 172.37, 170.94, 53.04, 41.93, 38.38, 36.42, 35.07, 31.26, 29.91, 29.01-28.52 (m), 25.12, 22.07, 13.93. Compound molecular weight data: theoretical molecular weight C10 21 H 43 N3O2S[M+H] + =402.31, measured molecular weight 402.3164. RP-HPLC determined the purity of this peptide mimic to be 89.33%.
[0102] Nuclear magnetic resonance spectroscopy data of peptide mimic 10b: 1H NMR(400MHz,DMSO)δ8.30(t,J=5.7Hz,1H),8.14(d,J=8.1Hz,1H),7.78(s,3H), 7.19(d,J=8.0Hz,2H),7.11(d,J=7.8Hz,2H),4.44(td,J=8.1,6.0Hz,1H),3.70 (d,J=3.1Hz,2H),3.30(hept,J=6.9Hz,2H),2.88-2.71(m,4H),2.27(s,3H),2. 12(hept,J=7.0Hz,2H),1.49(d,J=6.9Hz,2H),1.23(s,16H),0.90-0.81(m,3H). 13 C10 NMR (151MHz, CDCl3) δ 177.70, 176.29, 141.15, 140.35, 134.07 (d, J = 14.5Hz), 57.24, 43.58, 41.66, 40.36, 40.09, 38.07, 36.53, 34.25, 34.08, 33.95, 33.84, 30.42, 27.32, 25.88, 19.18. Compound molecular weight data: theoretical molecular weight C10 25 H 43 N3O2S[M+H] + =450.31, actual molecular weight 450.3170. RP-HPLC determined the purity of this peptide mimic to be 99.9%.
[0103] Nuclear magnetic resonance spectroscopy data of peptide mimic 10c: 1 H NMR(400MHz,DMSO)δ8.28(t,J=5.7Hz,1H),8.16(d,J=8.0Hz,1H),7.67(s,3H ),7.32(d,J=0.9Hz,2H),7.31(s,2H),7.24(ddd,J=8.7,5.2,3.5Hz,1H),4.55 -4.37(m,1H),3.75(d,J=2.8Hz,2H),3.31-3.23(m,2H),3.12-2.59(m,4H),2 .13(hept,J=7.0Hz,2H),1.55-1.43(m,2H),1.23(s,16H),0.89-0.82(m,3H). 13C10 NMR (151MHz, CDCl3) δ 177.71, 176.26, 143.50, 134.11, 133.56, 132.05, 57.25, 43.57, 41.67, 40.39, 36.53, 34.25 (d, J = 2.7Hz), 34.20, 34.06, 33.95, 33.85, 30.42, 27.33, 19.17. Compound molecular weight data: Theoretical molecular weight C10 24 H 41 N3O2S[M+H] + =436.30, actual molecular weight 436.2992. RP-HPLC determined the purity of this peptide mimic to be 99.13%.
[0104] Nuclear magnetic resonance spectroscopy data of peptide mimics at 10 days: 1 H NMR (400MHz, DMSO) δ8.25(t,J=5.6Hz,1H),8.11(d,J=8.0Hz,1H),7.68(s,3H),4.38(td,J=8.4,5.6Hz,1H),3.31 -3.22(m,2H),2.86-2.56(m,4H),2.19-2.08(m,2H),2.06(s,3H),1.48(s,2H),1.24(s,16H),0.88-0.82(m,3H). 13 C10 NMR (151MHz, CDCl3) δ 177.66, 176.37, 57.09, 43.59, 41.70, 40.75, 40.37, 36.52, 34.24, 34.19, 34.03, 33.95, 33.85, 30.39, 27.33, 20.25, 19.17. Compound molecular weight data: Theoretical molecular weight C10 18 H 37 N3O2S[M+H] + =360.27, actual molecular weight 360.3391. RP-HPLC determined the purity of this peptide mimic to be 99.9%.
[0105] Nuclear magnetic resonance spectroscopy data of peptide mimic 10e: 1H NMR (400MHz, DMSO) δ8.29(t,J=5.8Hz,1H),8.14(d,J=8.0Hz,1H),7.77(s,3 H),7.24-7.19(m,2H),6.88-6.84(m,2H),4.43(td,J=8.1,6.1Hz,1H),3.73 (s,3H),3.69(d,J=2.9Hz,2H),3.30(q,J=6.3Hz,2H),2.87-2.72(m,4H),2. 13(q,J=7.0Hz,2H),1.49(s,2H),1.23(d,J=3.7Hz,16H),0.87-0.83(m,3H). 13 C10 NMR (151MHz, CDCl3) δ 177.71, 176.30, 163.39, 135.23, 118.94, 60.22, 57.27, 43.57, 41.66, 40.36, 39.78, 38.03, 36.52, 34.27, 34.21, 34.06, 33.95, 33.85, 30.42, 27.32, 19.17. Compound molecular weight data: Theoretical molecular weight C10 25 H 43 N3O2S[M+H] + =466.30, actual molecular weight 466.3691. RP-HPLC determined the purity of this peptide mimic to be 98.40%.
[0106] Nuclear magnetic resonance spectroscopy data of peptide mimic 10f: 1 H NMR (400MHz, DMSO) δ8.17(d,J=8.2Hz,1H),8.10(t,J=5.7Hz,1H),7.71(s,3H),7.34-7.31(m,4H),7.29-7.26(m,4H),7.26-7.21(m,2H),4.37-4.1 8(m,1H),3.24(q,J=6.6Hz,2H),2.35(qd,J=11.9,7.0Hz,4H),2.19-2.03 (m,2H),1.60-1.41(m,2H),1.22(d,J=8.1Hz,16H),0.85(t,J=6.7Hz,3H). 13C10 NMR (151MHz, CDCl3) δ 177.56, 175.71, 149.51, 134.28, 133.25, 131.97, 71.01, 56.74, 43.51, 41.64, 40.34, 38.94, 36.53, 34.29, 34.23, 34.22, 34.11, 33.93, 33.80, 30.41, 27.32, 19.18. Compound molecular weight data: Theoretical molecular weight C10 36 H 49 N3O2S[M+H] + =588.39, measured molecular weight 588.4199. RP-HPLC determined the purity of this peptide mimic to be 99.9%.
[0107] Example 2
[0108] In this embodiment, the strains used to test the antimicrobial activity of the peptide mimics were standard strains (all strains below are from the U.S. Culture Collection): Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Cronobacter sakazakii ATCC 29544, Bacillus subtilis ATCC 23857, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Enterococcus quaternaria ATCC 49573, and Klebsiella pneumoniae ATCC 700603.
[0109] The Kunming mice used in this example weighed approximately 20 grams and were purchased from the Laboratory Animal Center of Lanzhou University.
[0110] I. Experimental Section
[0111] 1. Determination of minimum inhibitory concentration (MIC)
[0112] The minimum inhibitory concentration (MIC) values were determined using the standard two-fold dilution method recommended by the National Clinical and Laboratory Standards Institute (NCCLS). The procedure is briefly as follows: First, dilute the test bacteria in the logarithmic growth phase to 1×10⁻⁶ mmol / L using MH medium. 6 After CFU / mL, the sample was seeded into 96-well plates; then, an equal volume of the test peptide mimic was added to each well using a 2-fold dilution method, and the plates were incubated at 37°C for 18 hours. Finally, the turbidity of the 96-well plates was observed visually and the results were recorded. The drug concentration of the first well with no visible turbidity was recorded as the MIC. Each peptide mimic was subjected to at least three independent MIC experiments, with each experiment consisting of three replicates.
[0113] 2. Hemolysis test
[0114] This invention quantitatively analyzes the hemolytic activity of a synthetic peptide mimic against human erythrocytes using optical absorption spectroscopy. The experimental steps are as follows: First, fresh rat blood was collected into blood collection tubes containing heparin sodium anticoagulant. Blood cells were separated by centrifugation at 800g for 5 min, followed by washing three times with phosphate-buffered saline (PBS). The erythrocyte suspension was then diluted with PBS to an 8% (v / v) concentration and added to a 96-well plate. Next, an equal volume of the peptide mimic, diluted twofold, was added sequentially to the 96-well plate. PBS and 2% Triton X-100 were used as negative and positive controls, respectively. After incubation for 1 hour, the mixture was centrifuged at 1200g for 15 min, and the supernatant was carefully transferred to a new 96-well plate. The absorbance was measured at 490 nm. The hemolysis rate was calculated using the following formula:
[0115]
[0116] Each experiment was repeated three times independently, with three replicates for each experiment.
[0117] 3. Membrane depolarization experiment
[0118] This invention uses the membrane potential-sensitive fluorescent dye 3,3′-dipropylthiobiscarbocyanine iodide (Di SC2(3)) to study the degree of change in cytoplasmic membrane potential. Staphylococcus aureus was grown overnight in MH broth at 37°C, centrifuged at 1500 rpm for 10 min, washed three times with PBS, and resuspended in 0.1% glucose to OD200. 600 =0.5. 10c was serially diluted from 1×MIC to 8×MIC for later use, and DiSC2(3) dye (3mM) was diluted to 60μM for later use. An equal volume of bacterial suspension and dye was mixed and incubated at 37℃ for 15 min. After incubation, 50 μL of the co-incubation solution was added to a 96-well plate, and 50 μL of 10c dilution was added. After incubation at 37℃ for 30 min, fluorescence was measured (excitation wavelength, 530 nm; emission wavelength, 630 nm). The percentage of depolarized cells was calculated by comparing the fluorescence of treated and untreated bacteria.
[0119] 4. Propranolol Iodide (PI) Uptake Test
[0120] This invention uses a PI uptake assay to determine the effect of peptide mimics on the membrane integrity of *Escherichia coli* (ATCC 25922) and *Staphylococcus aureus* (ATCC 25923). Log-phase bacterial suspensions were mixed with 10c of peptide mimics at a concentration of 4×MIC and incubated for 30 minutes. Then, 1 mg / mL of PI was added and incubated for another 15 minutes at 37°C in the dark. The bacterial suspension was then washed three times with PBS to remove the PI dye, and the suspension was resuspended in PBS. Finally, the bacterial suspension was placed on a glass slide and observed and photographed under a laser confocal scanning microscope.
[0121] 5. Sterilization kinetics
[0122] The in vitro bactericidal time (bactericidal kinetics) of peptide mimic 10c against Staphylococcus aureus was determined using a colony forming unit (CFU) assay. In short, a concentration of 1.0 × 10⁻⁶ CFU was used during the logarithmic growth phase. 6 CFU / mL bacteria were treated with peptide mimicry 10c (concentrations of 2×MIC and 4×MIC) and incubated on a shaker at 37°C and 120 rpm. MH medium was used as a negative control, and vancomycin as a positive control. Co-incubation solutions were collected at 0, 10, 30, 60, 120, 180, 360, 720, and 1440 minutes, appropriately diluted, and plated onto MH agar plates. After incubation at 37°C for 24 hours, colonies were counted. This experiment was independently repeated three times, with two parallel samples per experiment.
[0123] 6. Assay for inducing bacterial resistance tendency
[0124] This invention utilizes the continuous measurement of the minimum inhibitory concentration (MIC) of peptide mimic 10c against Staphylococcus aureus (ATCC 25923) co-incubated with sublethal concentrations of peptide mimic 10c for 21 consecutive generations. The MIC values are recorded and the fold change in MIC is calculated. A coordinate axis is constructed with the generation number of MIC measurements as the x-axis and the fold change in MIC as the y-axis to represent the dynamic change in MIC values. Amoxicillin is used as a control in this invention.
[0125] 7. Acute toxicity test
[0126] In this invention, mice were acclimatized in the laboratory for one week. First, mice weighing 20g ± 1g were randomly divided into 5 groups of 8 mice each. Then, the peptide mimic was administered to the mice intraperitoneally at doses of 35, 50, 65, 80, and 90 mg / kg (body weight), respectively. The mice's growth was observed for 7 days, and the number of surviving mice was recorded. Finally, the median lethal dose (LD50) of the peptide mimic was calculated using the Kohl's method. 50 To determine its acute toxicity in vivo.
[0127] 8. Treatment of methicillin-resistant Staphylococcus aureus (MRSA)-induced keratitis in mice with peptide mimicry.
[0128] This invention used male Kunming mice aged 6–8 weeks and weighing 20±2g as research subjects, and experiments began after one week of acclimatization. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (60mg / kg), followed by topical instillation of 5μL (0.3%) procaine hydrochloride into the right eye. The corneal epithelium of the mice was scraped with a sterile needle, followed by instillation of 5μL of a 1×10⁻⁶ solution. 8A CFU / mL MRSA bacterial solution was used. Corneal abscesses and opacities were observed 12 hours later to confirm successful establishment of the keratitis model. Mice were then randomly divided into four groups of seven each, treated with 1 mg / mL and 2 mg / mL peptide mimicry, vancomycin (a positive control), and saline, respectively, for 8 hours. Thirty minutes after the last administration, mice were euthanized, and their eyeballs were enucleated and homogenized. The homogenate was diluted and inoculated onto MH agar plates and incubated at 37°C for 16 hours. The number of bacteria on each eyeball was determined by colony counting.
[0129] 9. Treatment of horn pneumonia in mice infected with methicillin-resistant Staphylococcus aureus (MRSA) using peptide mimics.
[0130] In this experiment, 24 female Kunming mice were randomly divided into 4 groups of 6 mice each: a model group, a linezolid group, a peptide mimicry treatment group, and a blank control group (0.9% NaCl). Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg / kg) and then injected with 25 μL of 1×10... 8 A bacterial suspension of CFU / mL was nebulized and injected into the trachea of mice. Treatment was administered 2 hours and 12 hours post-infection. Mice were euthanized 24 hours after infection, and lung tissue homogenates were collected for bacterial count.
[0131] II. Results Analysis
[0132] 1. Antibacterial activity of peptide mimics
[0133] This invention used the classic two-fold dilution method to determine the minimum inhibitory concentrations (MICs) of the synthesized peptide mimics against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Enterobacter sakazakii ATCC 29544, Bacillus subtilis ATCC 23857, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Enterococcus quaternaria ATCC 49573, and Klebsiella pneumoniae ATCC 700603. Furthermore, this invention also determined the MICs of these peptide mimics against Candida albicans ATCC 14053, with the MICs ranging from 3.125 μg / mL to 200 μg / mL.
[0134] 2. Hemolytic activity of peptide mimics
[0135] This invention detects the hemolytic effect of peptide mimics 5a-10e on rat erythrocytes at concentrations ranging from 6.25 μg / mL to 200 μg / mL. The hemolysis rate is as follows: Figure 1 As shown.
[0136] 3. Time-kill kinetics of peptide mimics
[0137] To investigate the antibacterial mechanism and effects of peptide mimics, this invention conducted time-killing kinetic experiments using peptide mimic 10c as a representative, tracking the speed and extent of bacterial elimination over time. Results showed that 10c exhibits rapid bactericidal activity, significantly reducing bacterial count in a short period and maintaining high efficiency throughout the process. Figure 2 As shown, at a concentration of 4×MIC, 10c completely killed all bacteria within 2 hours; at a concentration of 2×MIC, complete bacterial elimination was achieved within 6 hours. In contrast, vancomycin, a commercial antibiotic used as a positive control, required 12 hours to eliminate bacteria even at concentrations of 4×MIC and 2×MIC. This indicates that 10c has a superior bactericidal rate compared to vancomycin, possibly due to its stronger membrane-disrupting ability. The rapid bactericidal mechanism of 10c is similar to that of natural antimicrobial peptides (HDPs), which is associated with a lower risk of antibiotic resistance.
[0138] 4. Peptide mimics tend to induce bacterial resistance.
[0139] Given that bacterial resistance to traditional antibiotics drives the demand for novel antimicrobial agents, the potential for resistance development has become a key criterion for evaluating the value of new drug candidates. In this embodiment, an experiment was conducted using multiple consecutive passages to detect the MIC of peptide mimics, dynamically monitoring changes in the MIC of Staphylococcus aureus cultured at sublethal concentrations against a representative peptide mimic, 10c. Figure 3 As shown, during 21 passages, the MIC of 10c remained stable within a narrow range (0.5–2×MIC), with the final MIC being the same as the initial value, significantly below the resistance threshold (≥8-fold increase). In contrast, the MIC of the clinical antibiotic ciprofloxacin increased significantly from the 3rd generation onwards, reaching a 16-fold increase by the 12th generation, consistent with the high risk of resistance associated with fluoroquinolone-induced gyrA mutations. Furthermore, the rapid bactericidal activity of 10c (>99.9% of bacteria were eliminated within 2 hours at a concentration of 4×MIC) minimized the exposure time window for resistant mutants, thereby suppressing their selective pressure.
[0140] 5. Effects of peptide mimics on bacterial membrane potential
[0141] Given the extremely low tendency of 10c to induce resistance, we hypothesize that its antibacterial activity may originate from a membrane-active mechanism. To verify this hypothesis, this invention used the 3,3′-dipropylthiodicarbocyanine iodide (DiSC2(3)) probe to evaluate the effect of 10c on bacterial membrane potential. Figure 4As shown, at 1×MIC, 10c significantly reduced the membrane potential of Staphylococcus aureus. At 2×MIC, membrane rupture comparable to that of the positive control Triton X-100 was achieved within 30 minutes. This rapid, concentration-dependent membrane depolarization indicates that 10c disrupts membrane permeability, leading to dysregulation of energy metabolism (e.g., loss of proton kinetics) and impaired substance transport.
[0142] 6. Effects of peptide mimic 10c on bacterial membrane integrity
[0143] To investigate the effect of peptide mimics on bacterial cell membrane integrity, this invention employs laser confocal scanning microscopy (LCSM) to observe bacterial uptake of propidium iodide (PI) and assess bacterial membrane integrity. PI is a fluorescent dye that can only penetrate damaged membranes and emit red light after binding to intracellular DNA. Figure 5 As shown, Staphylococcus aureus and Escherichia coli exhibited obvious red fluorescence in LCSM after co-incubation with 10c or the positive control polymyxin B (PMB), while no fluorescence signal was observed in the negative control group. This confirms that 10c, like PMB, disrupts the integrity of the bacterial membrane, promoting PI influx and binding to DNA.
[0144] 7. Acute toxicity of peptide mimics
[0145] To comprehensively evaluate the clinical safety of peptide mimics, this invention uses the Karber method to determine their median lethal dose (LD50) in female Kunming mice. 50 Mice were administered different doses of 10c (30–100 mg / kg body weight) via intraperitoneal injection and observed for 7 days. Figure 6 As shown, the survival rate of mice in the low-dose group (30 mg / kg bw) was 100%, indicating extremely low acute toxicity of 10c. The survival rate in the medium-dose group (40–70 mg / kg) decreased with increasing dose, exhibiting dose-dependent toxicity. The survival rate in the high-dose group (80–100 mg / kg) decreased significantly, but did not reach 100% mortality, suggesting the existence of a dose-dependent toxicity threshold. The LD50 was calculated... 50 The effective dose was 43.65 mg / kg (95% confidence interval: 37.5–68.3), indicating that it has a good safety window.
[0146] 8. Intra-body therapeutic effects of peptides
[0147] To evaluate the in vivo therapeutic effect of the peptide mimic, this invention established mouse models of MRSA-infected pneumonia and keratitis, and then treated them with the representative peptide mimic 10c. In the mouse pneumonia model, 10c treatment significantly reduced the bacterial load in the lungs, decreasing it from 2.3 × 10⁻⁶ in the untreated group. 7 CFU / g decreased to 5.3×10 6The CFU / g level decreased by 0.639 log units, and its efficacy was comparable to that of the positive control drug linezolid (2.6 × 10⁻⁶ in the linezolid group). 6 CFU / g decreased by 0.69 log units; Figure 7 A) can significantly relieve tissue congestion and swelling caused by bacterial infection. Figure 7 B), and histopathological analysis showed that, compared with the model group, 10c significantly alleviated alveolar collapse, alveolar wall thickening, inflammatory cell infiltration, and pulmonary congestion (e.g., Figure 7 (As shown in C). In the keratitis model of MRSA infection, as... Figure 8 As shown in Figure A, topical ophthalmic administration for 10 days reduced the corneal bacterial load from 6.6 × 10⁻⁶. 6 CFU / mL decreased to 5.9 × 10⁻⁶ 6 CFU / mL (decreased by 0.049 log units), although slightly lower than vancomycin (4.9 × 10⁻⁶). 6 CFU / mL, decreased by 0.130 log (but visual observation showed reduced corneal opacity in the 10c treatment group, and histopathological examination confirmed reduced inflammatory infiltration, maintenance of corneal structural integrity, and reduced inflammatory response (e.g., CFU / mL, decreased by 0.130 log). Figure 8 (As shown in B and C). Importantly, histological examination of other organs revealed no pathological changes. Figure 8 D), highlighting the good in vivo safety of 10c. Overall, the amphiphilic peptide mimics of this invention are a promising new antimicrobial agent, and their balanced efficacy and safety support further preclinical development and potential clinical translation.
[0148] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A fatty cationic amphiphilic peptide mimic, characterized in that, The chemical structural formula of the fatty cationic amphiphilic peptide mimic is shown below (Ⅰ): （Ⅰ） In formula (Ⅰ), the R1 group is When the R2 group is selected from , , , , , , as well as Any one of them; Alternatively, the R2 group is When the R1 group is selected from , , , , as well as Any one of them.
2. A method for preparing an aliphatic cationic amphiphilic peptide mimic, characterized in that, The method includes the following steps: (1) Dissolve Fmoc-Cys(DPM)-OH, HBTU, HOBT and DIPEA in DMF solution, then add N-Boc-ethylenediamine and stir the reaction at room temperature for 3 h; (2) After confirming the completion of the reaction by TLC, ethyl acetate and saturated brine were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the first intermediate product. (3) Add piperidine and tetrahydrofuran to the first intermediate and stir for 1 hour to remove the Fmoc protecting group. After removing the solvent under reduced pressure, add ice-cold diethyl ether to precipitate and obtain the second intermediate. (4) Dissolve the second intermediate product in DMF solution, add HBTU, HOBT, DIPEA and fatty acids and react at room temperature for 3 hours; (5) After confirming the completion of the reaction by TLC, ethyl acetate and saturated brine were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the third intermediate product. (6) Trifluoroacetic acid, dichloromethane, and TIPS were added to the third intermediate and stirred for 3 h. The solvent was removed under reduced pressure, and ice-cold diethyl ether was added to precipitate the product, yielding an aliphatic cationic amphiphilic peptide mimic with the chemical structure shown in formula (II) below: (II) In formula (II), the R2 group is selected from... , , , , , , as well as Any one of them.
3. The method as described in claim 2, characterized in that, In steps (2) and (5), the washing, drying, and solvent removal specifically involve washing the organic phase with 1N hydrochloric acid, sodium bicarbonate, and saturated brine, collecting EA, drying with anhydrous sodium sulfate, and removing the solvent using a rotary evaporator.
4. A method for preparing an aliphatic cationic amphiphilic peptide mimic, characterized in that, The method includes the following steps: (1) Dissolve Fmoc-Cys(R1)-OH, HBTU, HOBT and DIPEA in DMF solution, then add N-Boc-ethylenediamine and stir the reaction at room temperature for 3 h; (2) After confirming the completion of the reaction by TLC, ethyl acetate and saturated brine were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the first intermediate product. (3) Add piperidine and tetrahydrofuran to the first intermediate and stir for 1 hour to remove the Fmoc protecting group. After removing the solvent under reduced pressure, add ice-cold diethyl ether to precipitate and obtain the second intermediate. (4) Dissolve the second intermediate in DMF solution, add HBTU, HOBT, DIPEA and lauric acid and react at room temperature for 3 h; (5) After confirming the completion of the reaction by TLC, ethyl acetate and saturated brine were added, the organic phase was retained, and the product was washed, dried and desolventized to obtain the third intermediate product. (6) Trifluoroacetic acid, dichloromethane, and TIPS were added to the third intermediate and stirred for 3 h. The solvent was removed under reduced pressure, and ice-cold diethyl ether was added to precipitate the product, yielding an aliphatic cationic amphiphilic peptide mimic with the chemical structure shown in formula (III) below: (III) In formula (III), the R1 group is selected from... , , , , as well as Any one of them.
5. The method as described in claim 4, characterized in that, In steps (2) and (5), the washing, drying, and solvent removal specifically involve washing the organic phase with 1N hydrochloric acid, sodium bicarbonate, and saturated brine, collecting EA, drying with anhydrous sodium sulfate, and removing the solvent using a rotary evaporator.
6. The cationic amphiphilic peptide mimicry of claim 1, a stereoisomer or tautomer of the cationic amphiphilic peptide mimicry, a pharmaceutically acceptable salt of the cationic amphiphilic peptide mimicry, and its use in the preparation of medicaments for treating, improving, and / or preventing infections caused by drug-resistant bacteria and / or drug-resistant fungi.
7. The application as described in claim 6, characterized in that, The drug-resistant bacteria are selected from at least one of Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, Gram-negative bacilli, Klebsiella pneumoniae, Enterobacter, Hafnia, Enterobacter schoenleinii, Proteus, Yersinia, and trophotropic Gram-negative bacilli; the drug-resistant fungi are selected from at least one of Candida, Cryptococcus neoformans, Aspergillus, Mucor, dermatophytes, Sporothrix, Histoplasma, and Pneumocystis carinii.
8. A drug for treating infections caused by drug-resistant bacteria or fungi, characterized in that, The active ingredient of the drug includes the fatty cationic amphiphilic peptide mimicry of claim 1, a stereoisomer or tautomer of the cationic amphiphilic peptide mimicry, and a pharmaceutically acceptable salt of the cationic amphiphilic peptide mimicry.
9. The drug as described in claim 8, characterized in that, The drug-resistant bacteria are selected from at least one of Gram-positive cocci, Gram-positive bacilli, Gram-negative cocci, Gram-negative bacilli, Klebsiella pneumoniae, Enterobacter, Hafnia, Enterobacter schoenleinii, Proteus, Yersinia, and trophotropic Gram-negative bacilli; the drug-resistant fungi are selected from at least one of Candida, Cryptococcus neoformans, Aspergillus, Mucor, dermatophytes, Sporothrix, Histoplasma, and Pneumocystis carinii.
Citation Information
Patent Citations
Adamantane modification-based antibacterial peptide simulant and application thereof
CN120208814A