CN114436923A - A small-molecule antibacterial gel that promotes wound healing in antibiotic-resistant bacterial infections - Google Patents

Abstract

The invention relates to a ROS scavenging small molecular gelator for promoting the healing of antibiotic drug-resistant bacterial infection wound, and also discloses a preparation method of the gelator, which comprises the following steps: adding a catalytic amount of acid into 9-fluorenone for activation, and then adding mercaptoalkyl acid for reaction to prepare a compound shown in a formula (1); dropping organic alkali into the phenylalanine methyl ester hydrochloride solution under ice bath stirring, and stirring; adding the phenylalanine methyl ester solution with hydrochloric acid removed into the solution of the formula (1) activated by a condensing agent for reaction to prepare a solution of the formula (2); and thirdly, adding a solvent and hydrazine hydrate into the formula (2), and reacting under the protection of nitrogen to obtain the small molecular gel compound (3). The invention also discloses a small molecule gel prepared by using the gelator and application of the small molecule gel as a biological material, in particular to a small molecule gel which can be used as a wound auxiliary material and can promote the healing of infected wounds.

Description

A small-molecule antibacterial gel that promotes wound healing of antibiotic-resistant bacterial infections

technical field

The present invention relates to a small-molecule gelling factor for preparing biological materials, and also relates to a preparation method of the gelling factor, and a small-molecule gel obtained by the gelling factor and its application.

Background technique

The skin is the body's first line of defense and plays an extremely important role in preventing water loss and isolating harmful substances and pathogenic microorganisms from invading. Once the skin is damaged, it provides an opportunity for harmful bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, etc.) to invade living tissue, and infection is the most common complication of wounds. Bacterial infection often leads to insufficient oxygenation of wounds and reduced nutrients, thereby accelerating and aggravating the pathological changes of various wounds, hindering wound closure, and bringing great pain and economic burden to patients (Acta Biomaterialia, 2018, 72, 35-44 .).) Bacterial infections are one of the major threats to human health worldwide, and antibiotics are the most common drugs used to treat wound infections. In recent years, due to the abuse of antibiotics, the treatment effect of antibiotics on infected wounds has become increasingly poor, and antibiotic-resistant bacteria (methicillin-resistant Staphylococcus aureus, etc.) have subsequently appeared (ACS Applied Materials & Interfaces, 2019, 11, 16320-16327.). Antibiotic-resistant bacteria can cause serious infections that are clinically difficult, expensive, and have high mortality rates. There is an urgent need to develop new antimicrobial materials to replace antibiotics to relieve the threat to human health from antibiotic-resistant bacteria.

Reactive oxygen species (ROS) are produced by damaged tissues and bacteria, resulting in the accumulation of large amounts of ROS in the wound. Excessive accumulation of live ROS in the wound not only induces a strong inflammatory response and makes the wound vulnerable, but also inhibits the function of endogenous stem cells and macrophages, hinders wound tissue regeneration, and thus inhibits wound closure (Biomaterials, 2020, p. 258, 120286.). In addition, ROS generated by bacterial infection can cause significant damage to blood vessels and endothelial cells, inhibit the formation of blood vessels, hinder wound healing, and form long-term non-healing wounds (Journal of investigative of dermatology, 2017, 137, 237-244; Trends in Biotechnology , 2019, 37, 505-517.). Therefore, the key to promoting the healing of bacterial infection wounds is to scavenge microorganisms and ROS from the wound site.

In order to find alternatives to antibiotics, researchers have developed a large number of nanomaterials with antibacterial activity, such as metal nanomaterials (gold nanoparticles, copper oxide nanoparticles, zinc oxide nanoparticles, etc.) and carbon nanomaterials (carbon nanotubes, graphene, etc.) et al) (Nano Letter, 2020, 20, 5149-5158.). Limited by their own degradation properties or biocompatibility, metal nanomaterials and carbon nanomaterials are difficult to be practically applied as antibacterial materials (Small, 2019, 15, 1900999.). Antibacterial polypeptides (ε-polylysine, etc.) can reduce bacterial activity by affecting the plasma membrane and physiological metabolism of pathogen cells, thereby killing bacteria. They are ideal antibacterial materials for killing antibiotic-resistant microorganisms (ACS Nano, 2018). , 12, 10772-10784; Advanced. Functional Materials, 2017, 27, 1604894.).

Hydrogel has a porous structure, good biocompatibility, high oxygen permeability and excellent performance in maintaining a moist environment, and is an ideal wound excipient (Advanced. Science, 2019, 6, 1801664.). Through rational design, the hydrogel can also obtain responsive release properties for controllable local drug delivery. Antibacterial gels can be prepared by constructing hydrogels with compounds with their own antibacterial properties or loading antibacterial agents with hydrogels. Antibacterial gels are promising new antibacterial materials (Chemical Engineering Journal, 2021, 420, 127638.). The construction of antimicrobial gels loaded with antimicrobial peptides on stimuli-responsive gels is an effective way to treat wounds infected with antibiotic-resistant microorganisms. Therefore, developing antibacterial gels with ROS scavenging function and loaded with antimicrobial peptides is an effective way to promote the healing of wounds infected by antibiotic-resistant microorganisms.

Compared with polymer gels, small molecular gels (small molecular weight) degrade faster and can release drugs at the target site quickly, which has been shown in the fields of biomedicine such as drug delivery, tissue engineering, anticancer and antibacterial physiotherapy. Huge application prospects. Small-molecule gels are formed by small-molecule gelling factors in a solvent through non-covalent bond forces (hydrogen bonding, van der Waals force, π-π stacking force, hydrophobic force, electrostatic force, etc.). Small-molecule gelling factors have a single structure, small molecular weight, and can be introduced into corresponding functional units to achieve special functionalization as needed, and are widely used to construct stimuli-responsive small-molecule gels. Amino acids and peptides are widely used in the construction of small molecule gels due to their good biocompatibility, non-toxicity, and multiple hydrogen bond sites in their structures that are easy to self-assemble. Recent studies have shown that phenylalanine-based small-molecule gels can exhibit antibacterial activity through the combined action of membrane disruption and oxidative stress (Nature Communications, 2017, 8, 1365.). Many clinical drug molecules with antibacterial and anti-inflammatory activities contain hydrazide groups, and hydrazide compounds are potential antibacterial agents (ACS Applied Bio Materials, 2020, 3, 2295-2304.). Therefore, the present invention uses 9-fluorenone as a substrate to construct ROS-responsive thioketal ketals with different alkyl chain lengths, and introduces phenylalanine methyl ester fragments that are prone to self-assembly at both ends of the thioketal ketal. Then, it reacted with hydrophilic hydrazine hydrate to regulate the amphiphilicity to construct a ROS-responsive small molecule gelling factor. Small molecule antibacterial gel can be prepared by using the small molecule gel factor and antibacterial peptide in a biocompatible solvent. As a wound adjuvant, high concentration of ROS can be removed by ROS response in wounds infected with antibiotic-resistant microorganisms, and at the same time, a high concentration of ROS can be released. Antimicrobial peptides kill antimicrobial-resistant bacteria in the wound, thereby promoting the healing of infected wounds.

SUMMARY OF THE INVENTION

The first technical problem to be solved by the present invention is to provide a ROS scavenging type small molecule gelling factor that promotes the healing of antibiotic-resistant bacterial infection wounds in view of the above-mentioned technical status.

The second technical problem to be solved by the present invention is to provide a preparation method of a ROS scavenging type small molecule gelling factor that promotes the healing of antibiotic-resistant bacterial infection wounds in view of the above-mentioned technical status.

The third technical problem to be solved by the present invention is to provide a ROS-scavenging small-molecule antibacterial gel that promotes the healing of wounds infected with antibiotic-resistant bacteria in view of the above-mentioned technical status.

The fourth technical problem to be solved by the present invention is to provide a ROS scavenging small molecule antibacterial gel that promotes the healing of wounds infected with antibiotic-resistant bacteria as a wound adjuvant for promoting the healing of infected wounds.

The technical solution adopted by the present invention to solve the above-mentioned first technical problem is: a ROS scavenging type small molecule gel for promoting the healing of wounds infected with antibiotic-resistant bacteria, characterized in that the small molecule gelling factor has a structural formula (3) as follows:

n in the formula is an integer of 1-20.

Preferably, n in the structural formula (3) is 2-12.

The technical solution adopted by the present invention to solve the above-mentioned second technical problem is: a preparation method of a ROS scavenging type small molecule antibacterial gel factor that promotes the healing of wounds infected with antibiotic-resistant bacteria, which is characterized by comprising the following steps:

1. Add a catalytic amount of acid A to 9-fluorenone, add mercaptoalkyl acid after activation, and stir to obtain reaction solution a. After reaction solution a is treated, a product of structural formula (1) is obtained. The 9-fluorenone and The ratio of the amount of mercaptoalkanoic acid is 1:2 to 2.4.

2. After dissolving the product of structural formula (1) in solvent B, add condensing agent C for activation; dissolve phenylalanine methyl ester hydrochloride in solvent B, add organic base D dropwise under ice-bath stirring; Under stirring, the phenylalanine methyl ester solution of the hydrochloric acid molecule is removed dropwise into the activated (1) solution to react, and the reaction solution b is processed to obtain a product with the structural formula (2), and the organic base D and benzene are The ratio of the amount of alanine methyl ester hydrochloride substance is 1-1.2:1; the ratio of the amount of the compound (1) to the substance of phenylalanine methyl ester hydrochloride is usually 1:2-2.4.

3. add solvent E and hydrazine hydrate to the compound whose structural formula is (2), react under nitrogen protection to obtain reaction solution c, and after-processing reaction solution c to obtain the product whose structural formula is (3), the consumption of described hydrazine hydrate is formula ( 2) 5 to 20 times the amount of the substance.

The structural formula involved in the preceding steps is as follows:

n in the formula is an integer of 1-20.

The reaction formula involved in the above reaction is as follows:

Preferably, the acid A described in step ① is at least one of trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid or sulfuric acid.

As preferably, the treatment method of reaction solution a described in step 1. is as follows: the solid is collected by glacial ether precipitation, dried to obtain the product (1) or placed at -50 ° C and left to stand until the solid is precipitated and then filtered, alternately with ice water and ice n-hexane The solid is washed and dried to obtain a product of formula (1).

Preferably, at least one of the solvent B in step ② is at least one of dichloromethane, chloroform or tetrahydrofuran.

As preferably, the volume consumption of solvent B described in step 2. is calculated as 1～5mL/mmol with the amount of substance of phenylalanine methyl ester hydrochloride.

Preferably, the condensing agent C described in step ② is N,N'-carbonyldiimidazole, dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide salt acid salt, 2-(1H-benzotriazo L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate, 2-(7-azabenzotriazole )-N,N,N',N'-tetramethylurea tetrafluoroborate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetrafluoroborate At least one of methyl urea hexafluorophosphate or benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate.

Preferably, the organic base D described in step ② is at least one of triethylamine, pyridine or N,N-diisopropylethylamine.

As preferably, the treatment mode of reaction solution b described in step 2. is as follows: after concentrating the organic phase, add tetrahydrofuran to stir, filter, collect the filtrate, and after concentrating the filtrate, use petroleum ether and ethyl acetate column chromatography to separate the product whose structural formula is (2) The volume ratio of petroleum ether and ethyl acetate used in column chromatography is generally 2:1 to 4:1.

Preferably, the solvent E described in step 3. is at least one of methanol, ethanol, isopropanol, dichloromethane or chloroform.

Preferably, the volume dosage of the solvent E described in step ③ is calculated as 1-5 mL/mmol based on the amount of the product of the structural formula (2).

Preferably, the reaction solution c described in step 3. is treated as follows: the reaction solution c is filtered, washed with water and dichloromethane for several times in turn, the solid is collected, and the product with the structural formula (3) is obtained after drying.

The technical solution adopted by the present invention to solve the above-mentioned third technical problem is: a ROS scavenging type small-molecule antibacterial gel for promoting the healing of wounds infected with antibiotic-resistant bacteria, the antibacterial gel is composed of compound (3) and antibacterial peptide in Prepared in a solvent, specifically prepared by the following steps:

The small molecule gelling factor and the antibacterial peptide are added into a sealable container, a solvent is added, and after sealing, the compound is heated until the compound is completely dissolved, and after standing, the small molecule antibacterial gel can be formed. The small molecule gelling factor adopts the substance represented by the structural formula (3).

Preferably, the concentration of the small molecule gel is 1-30 mg/mL.

Preferably, the solvent is at least one of water, methanol, ethanol, PEG200, PEG400 or a mixed solution of PEG200 (PEG400) and water.

The technical solution adopted by the present invention to solve the above-mentioned fourth technical problem is as follows: using compound (3) and antimicrobial peptides in biocompatible solvents such as water, PEG200, PEG400 or a mixed solution of PEG200 and water to prepare small-molecule antibacterial condensate Adhesive for wound dressings to promote the healing of wounds infected with antibiotic-resistant bacteria.

Compared with the prior art, the present invention has the advantages that: based on biocompatible phenylalanine, an easy-to-synthesize, ROS-responsive small-molecule gelling factor is designed, and the gelling factor itself has potential antibacterial properties. performance. This small-molecule gelling factor can prepare biocompatible small-molecule gels in biocompatible solvents. This kind of gel has good biocompatibility, shear-thinning behavior, damage recovery ability and high mechanical strength. It is an excellent class of biomedical materials. It has potential applications in drug controlled release and cancer physiotherapy. In particular, the antibacterial peptides loaded on the small molecule gel can prepare antibacterial gels, kill antibiotic-resistant bacteria, and have a significant therapeutic effect on wounds infected with drug-resistant bacteria as wound accessories.

Description of drawings

FIG. 1 is a photograph of the appearance of the gel formed in Example 2. FIG.

FIG. 2 is a photograph of the appearance of the gel formed in Example 3. FIG.

FIG. 3 is the ¹ H nuclear magnetic resonance spectrum of compound (1) (n=2) in Example 4. FIG.

FIG. 4 is the ¹ H nuclear magnetic resonance spectrum of compound (2) (n=2) in Example 5. FIG.

FIG. 5 is the ¹ H nuclear magnetic resonance spectrum of compound (3) (n=2) in Example 6. FIG.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

Example 1, (Compound (3) (n=10))

(1) Add 10 mL of dichloromethane, 1.80 g (10 mmol) of 9-fluorenone and two drops of trifluoroacetic acid into a 50 mL branched-mouth reaction flask, and stir and activate at room temperature for 30 min. 4.37 g (20 mmol) of 11-mercaptoundecanoic acid was added to the activated 9-fluorenone, and the mixture was stirred under vacuum ice bath for 48 h. Subsequently, unreacted 9-fluorenone was removed by precipitation with glacial ether, and the precipitate was collected and dried to obtain the product (1) (n=10) as a white solid with a yield of 70.5%.

(2) 1. get the 100mL branched-mouth reaction flask, add 20mL methylene chloride, 4.74g (22mmol) phenylalanine methyl ester hydrochloride to it, slowly drip 5.9mL (44mmol) triethyl under ice bath conditions The amine, after the dropwise addition, was stirred for 10 minutes to remove the hydrochloric acid molecule of phenylalanine methyl ester hydrochloride. ② After 5.99 g (10 mmol) of compound formula (1) (n=10) was dissolved in 15 mL of dichloromethane, 3.57 g (22 mmol) of N,N-carbonyldiimidazole was added and activated for 30 minutes. ③ The activated solution was slowly added dropwise to ① in an ice bath, protected by nitrogen, and reacted at room temperature. The progress of the reaction was detected by a spot plate of petroleum ether and ethyl acetate in a volume ratio of 3:1. After the reaction is completed, the organic phase is concentrated and then tetrahydrofuran is added to stir, filter, discard the solid, collect the filtrate, concentrate the filtrate and carry out column chromatography. The volume ratio of petroleum ether and ethyl acetate used in the column chromatography is generally 6:1～2:1 , the product formula (2) (n=10) was obtained, and the yield was 78.1%.

(3) Take a 100 mL branched-mouth reaction flask, add 4.61 g (5 mmol) of compound formula (2) (n=10), 20 mL of methanol, 20 mL of dichloromethane, 3.2 mL (50 mmol) of 80% hydrazine hydrate to it , vacuumed and filled with nitrogen, reacted in an ice bath for 24 h, filtered, and the filter cake was washed with deionized water and dichloromethane in turn, and dried to obtain a white solid of formula (3) (n=10) with a yield of 90.2%.

Example 2, Small Molecule Gels

Add 10 mg of compound (3) obtained in Example 1 into a 4 mL screw bottle, then add 1 mL of chloroform, seal it, heat it to 100 °C to dissolve it, and let it stand to cool to room temperature to form a small molecule of 5 mg/mL. Hydrogel, as shown in Figure 1, the resulting material is an almost transparent gel.

Example 3, Small Molecule Gels

Add 10 mg of compound (1) obtained in Example 1 into a 4 mL screw bottle, then add 0.5 mL of water and 0.5 mL of PEG200, seal it, heat it to 90°C to dissolve it, and let it stand to cool to room temperature to form a mixed solvent. Gel, as shown in Figure 2, the obtained material is a translucent gel.

Example 4, compound (1) (n=2)

12mg of compound (1) (n=2) was put into an NMR tube, and then 0.5mL of deuterated dimethyl sulfoxide was added. After sealing, it was dissolved by heating with a hair dryer for ¹ H NMR test. NMR results As shown in FIG. 3, it was shown that compound (1) (n=2) was successfully synthesized.

Example 5, compound (2) (n=2)

Put 12 mg of compound (2) (n=2) into a NMR tube, then add 0.5 mL of deuterated chloroform, and after sealing, heat it with a hair dryer to dissolve it for ¹ H NMR test. The NMR results are shown in Figure 4 As shown, it was shown that compound (2) (n=2) was successfully synthesized.

Example 6, compound (3) (n=2)

12mg of compound (3) (n=2) was put into an NMR tube, and then 0.5mL of deuterated dimethyl sulfoxide was added. After sealing, it was dissolved by heating with a hair dryer for ¹ H NMR test. NMR results As shown in Figure 5, it was shown that the small molecule gelling factors were successfully prepared.

Claims (10)

1. a ROS scavenging type small-molecule antibacterial gel factor that promotes the healing of anti-biodrug-resistant bacteria infection wounds is characterized in that the structural formula (3) of this small-molecule gel factor is as follows:

n in the formula is an integer of 1-20. 2. a preparation method of the ROS scavenging type small molecule antibacterial gel factor that promotes antibiotic-resistant bacterial infection wound healing, is characterized in that comprising the steps: ①Add catalytic amount of acid A to 9-fluorenone, add mercaptoalkyl acid after activation, obtain reaction solution a after stirring, and process reaction solution a to obtain a product with structural formula (1), the cinnamaldehyde and mercaptoalkyl The ratio of the amount of acid substances is 1:2 to 2.4; ② Dissolve compound formula (1) in solvent B and add condensing agent C for activation; dissolve phenylalanine methyl ester hydrochloride in solvent B, add dropwise organic base D under ice bath stirring; The alanine methyl ester solution is added dropwise to the activated product (1) solution, a reaction solution b is obtained after stirring, and a product whose structural formula is (2) is obtained after processing the reaction solution b. The ratio of the amount of amino acid methyl ester hydrochloride is usually 1:2 to 2.4; the ratio of the amount of the organic base D to the amount of phenylalanine methyl ester hydrochloride is 1 to 1.2:1; the The ratio of the amount of compound formula (1) to the amount of the condensing agent C is usually 1:2 to 2.2; 3. after adding solvent E and hydrazine hydrate to compound formula (2), react in ice bath under nitrogen protection to obtain reaction solution c, and processing reaction solution c to obtain a product whose structural formula is (3), the compound formula (2) and hydration The ratio of the amount of hydrazine is 1:5 to 20; The structural formula involved in the preceding steps is as follows:

n in the formula is an integer of 1-20. 3. preparation method according to claim 2 is characterized in that the acid A described in step 1. is at least one in trifluoroacetic acid, p-toluenesulfonic acid, hydrochloric acid or sulfuric acid; The reaction solution a described in step 1. is processed The method is as follows: directly filter or place at -50°C and stand for the solid to separate out and then filter, alternately wash the solid with ice water and ice n-hexane, and obtain the product of formula (1) after drying; the condensing agent C described in step 2 is: N,N'-Carbonyldiimidazole, Dicyclohexylcarbodiimide, 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(1H-Benzodiimide Nitrogen L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate, 2-(7-azabenzotriazole)-N,N,N',N'- Tetramethylurea tetrafluoroborate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate or benzotriazepine At least one of azole-N,N,N',N'-tetramethylurea hexafluorophosphate. 4. preparation method according to claim 2 is characterized in that reaction solution b described in step 2. is treated as follows: after concentrating the organic phase, add tetrahydrofuran and stir, filter, collect filtrate, use petroleum ether and ethyl acetate after concentrating the filtrate The product of structural formula (2) is obtained by column chromatography, and the volume ratio of petroleum ether and ethyl acetate used in column chromatography is generally 2:1 to 4:1. 5. a small molecule gel prepared by the compound of claim 1, the small molecule gel is prepared by adopting the following steps: adding a certain amount of compound into a sealable container, adding a solvent, and heating until the compound is completely dissolved after sealing , small molecule gels can be formed after standing. 6 . The small molecule gel according to claim 5 , wherein the concentration of the small molecule gel is 1-50 mg/mL. 7 . 7. a small molecule gel compound according to claim 1 prepares biomedical small molecule gel, it is characterized in that solvent is water, methanol, ethanol, PEG200, PEG400, water and ethanol mixed solution, water and PEG200 mixed solution, At least one of water and PEG400 mixed solution. 8. A biomedical small molecule gel according to claim 7, used for cell culture, tissue engineering, drug delivery, and anti-tumor therapy. 9 . The biomedical small molecule gel loaded with antibiotics according to claim 7 is used as an antibacterial gel and dressing for promoting the healing of bacterial infection wounds. 10 . 10. A biomedical small molecule gel loaded with antimicrobial peptides according to claim 7 is used as an antimicrobial gel and dressing for promoting wound healing of antibiotic-resistant bacterial infection.

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