Preparation method and application of hydantoin dimer and its derivatives
By synthesizing hydantoin dimer derivatives in liquid phase, the problems of insufficient stability and activity of antimicrobial peptides were solved, efficient bactericidal effect was achieved without the risk of drug resistance, and a new antimicrobial drug option was provided.
Patent Information
- Application Number
- CN202410350248.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-26
- Publication Date
- 2025-09-30
- Estimated Expiration
- 2044-03-26
AI Technical Summary
Existing antimicrobial peptides have poor metabolic stability, low activity, large toxic side effects and high production costs, and no effective candidate lead drugs have been found for antimicrobial peptide mimetics.
Hydantoin dimer derivatives were prepared by a liquid phase synthesis method, and their structures were optimized to improve stability and antibacterial activity. A simple and easy synthetic route was adopted, including reductive amination, reaction steps under alkaline and acidic conditions, to synthesize hydantoin dimer and its derivatives.
The bactericidal efficiency and stability of hydantoin dimer are significantly improved, and bacteria can be killed quickly within 1 hour without developing drug resistance.
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Figure CN118290345B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of organic chemistry, and particularly relates to a preparation method and application of a hydantoin dimer and derivatives thereof. Background Art
[0002] At present, bacterial resistance is on the rise. We need to develop drugs that are sensitive to drug-resistant bacteria and not prone to drug resistance, with clear mechanism of action,
[0003] Antimicrobial drugs with novel structures have become an urgent problem to be solved in order to maintain human health. Antimicrobial peptides are polypeptides that are widely present in nature and have broad application prospects in the treatment of infectious diseases. Antimicrobial peptides targeting cell membranes have unique mechanisms and a broad antimicrobial spectrum. However, natural antimicrobial peptides have some defects, such as poor metabolic stability in the body, generally low activity, toxic side effects, and high production costs, which limit their application. One of the more effective ways to solve these problems is to design and synthesize antimicrobial peptide mimics based on the net positive charge properties and overall hydrophilic and lipophilic conformational characteristics of antimicrobial peptides. In recent years, the research on non-peptide or small molecule mimics of antimicrobial peptides has developed rapidly.
[0004] Peptide or macromolecular mimics of antimicrobial peptides have the structural characteristics of peptides. Activity studies have found that these peptides have strong antimicrobial effects. Macromolecular or peptide mimics of antimicrobial peptides improve the stability and antimicrobial activity of antimicrobial peptides and reduce toxicity. However, no potential candidate lead drugs have been found so far. Summary of the Invention
[0005] Peptide compounds rely on solid-phase synthesis technology, which has low yields, long reaction routes, multiple steps, and high production costs. Antimicrobial cationic polymers have large molecular weights and poor membrane permeability, which can easily induce bacterial resistance.
[0006] In order to solve the above-mentioned technical problems, this application provides the following technical solutions:
[0007] The present invention obtains a series of hydantoin dimer derivatives through simple and easy liquid phase synthesis. The synthesis and structural optimization of such small molecule antimicrobial peptide mimetics are relatively easy to improve activity, and the compounds have good stability and are immune to protease degradation.
[0008] The present invention provides a method for preparing a hydantoin dimer derivative, comprising the following steps:
[0009] S11: Reductive amination reaction of the first mixed solution and a reducing agent at 20-30° C. for 6-12 hours to obtain a secondary amine A2; the first mixed solution is prepared by dissolving an aromatic dialdehyde and a protected amino acid hydrochloride in a first organic solvent and mixing them; the solution is alkaline during mixing;
[0010] S12: dissolving the secondary amine A2 and isocyanate in a first organic solvent, and reacting under alkaline conditions at 20-30° C. for 20-24 hours to obtain a secondary amine A4;
[0011] S13: dissolving the secondary amine A4 in a first organic solvent and performing a deprotection reaction to obtain a hydantoin dimer;
[0012] S14: dissolving the hydantoin dimer and 1,3-di(tert-butoxycarbonyl)-2-methyl-2-isothiourea (CAS No.: 107819-90-9) in a second organic solvent containing mercuric chloride or dimethylaminopyridine (0.2 equivalents), and reacting under alkaline conditions at 20-30° C. for 2-3 hours to obtain an intermediate containing an arginine residue;
[0013] S15: dissolving the intermediate containing the arginine residue in a first organic solvent and performing a deprotection reaction to obtain the hydantoin dimer derivative; in steps S13 and S15, the deprotection reaction is carried out under acidic conditions at 20-30° C. for 30-60 minutes.
[0014] Preferably, the protected amino acid hydrochloride is selected from Nε-tert-butyloxycarbonyl-L-lysine tert-butyl ester hydrochloride (CAS No.: 13288-57-8); the aromatic dialdehyde is selected from 4,4'-biphenyldicarboxaldehyde, 3,3'-biphenyldicarboxaldehyde, terephthalaldehyde, isophthalaldehyde, naphthalene-1,4-dicarboxaldehyde, naphthalene-2,6-dicarboxaldehyde, 1,4-bis(4-formaldehydephenyl)benzene, 1,2-bis(4'-formaldehydephenyl)acetylene, (E)-4,4'-dibenzaldehyde ethylene, 4,4'-(ethane-1,2-diyl)dibenzaldehyde or 4,4'-oxybenzaldehyde; the reducing agent is selected from sodium triacetoxyborohydride or sodium cyanoborohydride.
[0015] Preferably, the molar ratio of the aromatic dialdehyde, the protected amino acid hydrochloride and the reducing agent is 1:2-2.2:4-6.
[0016] Preferably, the isocyanate is selected from 1-adamantyl isocyanate, dodecyl isocyanate, butyl isocyanate, amyl isocyanate, hexyl isocyanate, heptyl isocyanate or octyl isocyanate.
[0017] Preferably, in step S12, the molar ratio of the secondary amine A2 to the isocyanate is 1-1.1:2.5.
[0018] Preferably, the first organic solvent is dichloromethane, and the second organic solvent is N,N-dimethylformamide.
[0019] Preferably, in step S14, the molar ratio of hydantoin dimer, 1,3-di(tert-butoxycarbonyl)-2-methyl-2-isothiourea and mercuric chloride is 1:2-2.2:2.
[0020] Preferably, in steps S11, S12 and S14, the alkaline condition is obtained by adding N,N-diisopropylethylamine or triethylamine; and in steps S13 and S15, the acidic condition is obtained by adding trifluoroacetic acid or hydrochloric acid.
[0021] Specifically, the reaction steps are as follows Figure 1 As shown, the following steps are included:
[0022] a) dissolving an aromatic dialdehyde and a protected amino acid hydrochloride in a solvent and stirring under alkaline conditions, adding a reducing agent, and reducing the resulting imine to a secondary amine through a reductive amination reaction;
[0023] b) dissolving the isocyanate and the secondary amine obtained in step a) in a solvent and reacting them under alkaline conditions to obtain a new secondary amine;
[0024] c) dissolving the secondary amine obtained in step b) in a solvent and removing the protecting group under acidic conditions to obtain a hydantoin dimer derivative;
[0025] d) dissolving the hydantoin dimer obtained in step c) and 1,3-di(tert-butoxycarbonyl)-2-methyl-2-isothiourea in a solvent and reacting them under alkaline conditions in the presence of mercuric chloride to obtain an intermediate containing an arginine residue.
[0026] e) dissolving the intermediate obtained in step d) in a solvent and removing the protecting group under acidic conditions to obtain a new hydantoin dimer derivative.
[0027] The present invention also provides a hydantoin dimer and a hydantoin dimer derivative prepared by the above preparation method. The chemical formula of the hydantoin dimer is shown in Compound 1-17, and the chemical formula of the hydantoin dimer derivative is shown in Compound 18-20:
[0028]
[0029]
[0030]
[0031] The present invention also provides use of the hydantoin dimer and hydantoin dimer derivatives in treating bacterial infections.
[0032] The technical solution of the present invention has the following advantages over the prior art:
[0033] The antibacterial activity and mechanism of action of the successfully synthesized hydantoin dimer compound were determined. The results showed that the dimer had significantly improved bactericidal efficiency and stability, rapidly killing bacterial strains within an hour. The mechanism of action, similar to previous research, is through direct interaction with the bacterial cell membrane, damaging it and leading to bacterial cell death. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 is the general reaction formula of the present invention; wherein, the structure of R is as follows:
[0035] DETAILED DESCRIPTION
[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.
[0037] Example 1 Synthesis of Intermediate A2
[0038]
[0039] Nε-tert-Butyloxycarbonyl-L-lysine tert-butyl ester hydrochloride (2.2 equiv) was dissolved in dichloromethane, and DIPEA (2.2 equiv) was added. The mixture was stirred at room temperature (25±5°C) for 10 min. Aromatic aldehyde A1 (1.0 equiv) was added and allowed to react for 3 h. Sodium triacetoxyborohydride (6.0 equiv) was then added and stirred for another 6 h until the reaction was complete. The reaction solution was quenched with aqueous sodium bicarbonate and extracted three times with dichloromethane (DCM). The organic phases were combined and back-extracted once with water and saturated brine, dried over anhydrous sodium sulfate, and the solvent evaporated to dryness. The mixture was then purified by flash silica gel chromatography (PE / EA = 5:1 to 2:1) to obtain Intermediate A2 (94% yield, colorless oil).
[0040] Table 1 Selection of aromatic aldehydes A1
[0041]
[0042] Example 2 Synthesis of Intermediate A4
[0043]
[0044] Intermediate A2 (1.0 equiv) was dissolved in dichloromethane, and N,N-diisopropylethylamine (DIPEA) (2.5 equiv) and isocyanate A3 (2.5 equiv) were added, followed by overnight reaction at room temperature. The reaction solution was quenched with dilute hydrochloric acid (1 M), extracted three times with dichloromethane (DCM), and then dried over anhydrous sodium sulfate. The solvent was then evaporated to dryness. The product was then purified by flash silica gel chromatography (PE / EA = 5:1 to 2:1) to afford Intermediate A4 as a colorless oil in a 77% yield.
[0045] Table 2 Selection of various compounds of isocyanate A3
[0046]
[0047] Example 3 Synthesis of Final Product 1-17 and Intermediate A5
[0048]
[0049] Compound A4 was dissolved in a mixture of dichloromethane and trifluoroacetic acid (1:1 volume ratio) and stirred at room temperature for 0.5-1 hour. The reaction progress was monitored by TLC. After completion of the reaction, the solvent was evaporated to afford the final product 1-17 and intermediate A5 (100% yield, yellow oil). This completes the synthesis of compound 1-17.
[0050] Example 4 Synthesis of Intermediate A6 and Final Products 18-20
[0051]
[0052] Intermediate A5 (1 equivalent) of compound 18-20 and 1,3-di(tert-butoxycarbonyl)-2-methyl-2-isothiourea (2 equivalents) were dissolved in N,N-dimethylformamide. Triethylamine (6 equivalents) was added and stirred at 0°C for 10 minutes. Mercuric chloride (2 equivalents) was added and the reaction continued for 2 hours. The reaction progress was monitored by TLC. The reaction solution was added with water and extracted three times with ethyl acetate. The organic phases were combined and washed three times with water and once with saturated sodium chloride solution. After dehydration with anhydrous sodium sulfate, the solvent was evaporated and the product was purified by flash silica gel chromatography (PE / EA = 4:1 to 2:1) to obtain Intermediate A6 (67% yield, colorless oil).
[0053] Intermediate A6 was dissolved in a mixture of dichloromethane and trifluoroacetic acid (1:1 volume ratio) and stirred at room temperature for 0.5-1 hour. The reaction progress was monitored by thin-layer chromatography (TLC). After completion of the reaction, the solvent was evaporated to afford the final product A7 (100% yield, yellow oil).
[0054] Application Example 1
[0055] Compounds 1 to 20 were dissolved in dimethyl sulfoxide (DMSO) to prepare 5 mg mL -1 The mother solution was diluted to 1 mg mL with TSB liquid medium. -1 , used for minimum inhibitory concentration (MIC) testing, and ciprofloxacin was used as the positive control drug.
[0056] The antibacterial effects of all compounds were determined against eight different bacterial strains: ESKAPE and standard strains of MRSA and Bacillus subtilis (E: Enterococcus faecalis (ATCC 29212), S: Staphylococcus aureus (ATCC 29213), K: Klebsiella pneumoniae (ATCC 10031), A: Acinetobacter baumannii (ATCC 19606), P: Pseudomonas aeruginosa (CMCC 10104), E: Enterobacter (ATCC 25922), MRSA (ATCC 43300), and Bacillus subtilis (CMCC 63501).
[0057] Experimental steps: A single colony of each strain was inoculated into 4 ml TSB liquid medium and incubated at 37 °C for 6 h. The bacteria grew to the logarithmic growth phase and then diluted 1000 times with tryptic soy broth (TSB) (the bacterial solution concentration was 1 × 10 6 CFU·mL -1 50 μL of TSB medium was added to each well of a 96-well plate. After the compound was serially diluted in a two-fold gradient, 50 μL of the diluted bacterial solution was added to each well. After incubation at 37°C for 16-20 h, the absorbance at a wavelength of 600 nm (OD600) was recorded on a microplate reader.
[0058] The in vitro antibacterial activities of compounds 1-20 against Gram-positive and Gram-negative bacteria are shown in Table 3 below:
[0059] Table 3 Antibacterial performance test
[0060]
[0061] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.
Claims
1. A method for preparing a hydantoin dimer and its derivatives, characterized in that: The following steps are involved: S11: Reductive amination reaction of the first mixed solution and the reducing agent at room temperature for 6-12 hours to obtain secondary amine A2; the first mixed solution is prepared by dissolving an aromatic dialdehyde and a protected amino acid hydrochloride in dichloromethane and mixing them in an alkaline solution; the protected amino acid hydrochloride is selected from Nε-tert-butyloxycarbonyl-L-lysine tert-butyl ester hydrochloride; the aromatic dialdehyde is selected from 4,4'-biphenyldicarboxaldehyde, 3,3'-biphenyldicarboxaldehyde, terephthalaldehyde, m-phthalaldehyde, naphthalene-1,4-dicarboxaldehyde, naphthalene-2,6-dicarboxaldehyde, 1,4-bis(4-formylphenyl)benzene, 1,2-bis(4'-formylphenyl)acetylene, (E)-4,4'-dibenzaldehyde ethylene, 4,4'-(ethane-1,2-diyl)dibenzaldehyde or 4,4'-oxybenzaldehyde; the reducing agent is selected from sodium triacetoxyborohydride; S12: dissolving the secondary amine A2 and the isocyanate in dichloromethane, and reacting them under alkaline conditions at room temperature for 20-24 hours to obtain the secondary amine A4; in the steps S11 and S12, the alkaline conditions are obtained by adding N,N-diisopropylethylamine; the isocyanate is selected from 1-adamantyl isocyanate, dodecyl isocyanate, butyl isocyanate, amyl isocyanate, hexyl isocyanate, heptyl isocyanate or octyl isocyanate; S13: dissolving the secondary amine A4 in dichloromethane, and removing the protecting group under acidic conditions with the addition of trifluoroacetic acid to obtain a hydantoin dimer; S14: dissolving the hydantoin dimer and 1,3-di(tert-butoxycarbonyl)-2-methyl-2-isothiourea in N,N-dimethylformamide containing mercuric chloride, and reacting at 0°C under alkaline conditions for 2 h to obtain an intermediate containing an arginine residue; in step S14, the alkaline conditions are obtained by adding triethylamine; S15: dissolving the intermediate containing the arginine residue in dichloromethane and performing a deprotection reaction to obtain the hydantoin dimer derivative; in steps S13 and S15, the deprotection reaction is carried out under acidic conditions at room temperature for 30 minutes; the acidic conditions are obtained by adding trifluoroacetic acid, and the volume ratio of dichloromethane to trifluoroacetic acid is 1:1; The preparation process of the hydantoin dimer and its derivatives is as follows: ; wherein RT represents room temperature, Overnight represents overnight, A1 is an aromatic dialdehyde, A3 is an isocyanate, A5 is a hydantoin dimer, A6 is an intermediate containing an arginine residue, and A7 is a hydantoin dimer derivative.
2. The preparation method according to claim 1, wherein The molar ratio of the aromatic dialdehyde, the protected amino acid hydrochloride and the reducing agent is 1:2-2.2:4-6.
3. The preparation method according to claim 1, wherein In step S12, the molar ratio of the secondary amine A2 to the isocyanate is 1-1.1:2.
5.
4. The preparation method according to claim 1, wherein In step S14, the molar ratio of hydantoin dimer, 1,3-di(tert-butoxycarbonyl)-2-methyl-2-isothiourea and mercuric chloride is 1:2-2.2:2.