Antibacterial amide compounds and methods for preparing hydrogels thereof
The hydrogel was prepared by mixing N,N’-bis(3-pyridyl)pyrimidine-4,6-dicarboxamide with silver nitrate, dimethyl sulfoxide and water. The problems of poor mechanical properties and instability of existing silver ion hydrogels were solved, and the preparation of small-molecular gels with good antibacterial effects and greater application prospects were achieved.
Patent Information
- Application Number
- CN202411975973.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-08
AI Technical Summary
The mechanical properties of existing silver ion hydrogels are poor and unstable, which limits the expansion of their application fields.
The hydrogel was prepared by mixing N,N’-bis(3-pyridyl)pyrimidine-4,6-dicarboxamide with silver nitrate, dimethyl sulfoxide and water. Through specific process steps and reaction conditions, a small molecule gel with good antibacterial effect was prepared.
The prepared hydrogel not only has good antibacterial effect, but also has good mechanical properties and stability, expanding its application prospects in various application fields.
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Figure CN119775258A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of hydrogels, and in particular to an antibacterial amide compound and a method for preparing a hydrogel thereof. Background Art
[0002] Silver ions have broad-spectrum antibacterial activity and have antibacterial effects on Gram-negative bacteria, Gram-positive bacteria, fungi and some viruses. Therefore, they have a long history as antibacterial materials. Silver ion hydrogel is a new type of gel antibacterial material. It can continuously and effectively release silver ions to kill wounds and surrounding bacteria, thereby improving the wound healing environment. Hydrogel is a polymer compound with a three-dimensional network structure that can absorb and slowly release a large amount of water without changing its own structural morphology. Due to its excellent water retention and adjustable mechanical properties, it is widely used in many fields. The unique three-dimensional network structure of hydrogel can contain a large number of free water molecules, providing a moist microenvironment for the wound surface, which is conducive to the healing of the wound surface. At the same time, the evaporation of water reduces the temperature of the wound surface, relieves the patient's pain, and does not adhere to the wound surface. Due to its good biocompatibility and no skin sensitization and irritation, it is widely used in wound care.
[0003] However, most silver ion hydrogels currently on the market are prepared using traditional chemical cross-linking methods, but their poor mechanical properties and instability limit the expansion of their application areas. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide an antibacterial amide compound and a method for preparing a hydrogel thereof in view of the deficiencies of the above-mentioned prior art. The antibacterial amide compound is N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide. The compound is mixed with silver nitrate, dimethyl sulfoxide and water to prepare a hydrogel. The hydrogel has good antibacterial effect and is a small molecule gel, which has great application prospects.
[0005] The invention provides an antibacterial amide compound, which is N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide.
[0006] The present invention also provides a method for preparing a hydrogel using the above-mentioned antibacterial amide compounds, comprising the following steps:
[0007] S1, adding thionyl chloride and DMF to pyrimidine-4,6-dicarboxylic acid, fully reflux, and then distill under reduced pressure to obtain pyrimidine-4,6-dicarbonyl chloride;
[0008] S2, adding anhydrous dichloroethane and triethylamine to 3-aminopyridine, stirring evenly to obtain a mixed solution;
[0009] S3, dissolving the pyrimidine-4,6-dicarbonyl chloride prepared in S1 in anhydrous dichloromethane, and adding it dropwise to the mixed solution prepared in S2 at 0°C. After sufficient reaction, the mixture is filtered and dried to obtain a primary product;
[0010] S4, purifying the initial product prepared in S3 by recrystallization with DMSO and water, and drying to obtain a pure product of antibacterial amide compounds, namely N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide;
[0011] S5. Take the N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide and silver nitrate prepared in S4, dissolve them in a mixed solvent of dimethyl sulfoxide and water, mix them evenly and let them stand to obtain a hydrogel.
[0012] According to the method for preparing hydrogel from antibacterial amide compounds provided by the present invention, the molar volume ratio of pyrimidine-4,6-dicarboxylic acid, thionyl chloride and DMF in S1 is 20 mmol:25 mL:0.2 mL, the reflux temperature is 75° C., and the reflux time is 4 h.
[0013] According to the method for preparing hydrogel from antibacterial amide compounds provided by the present invention, the molar volume ratio of 3-aminopyridine, anhydrous dichloroethane and triethylamine in S2 is 20mmol:100mL:3mL.
[0014] According to the method for preparing hydrogel from antibacterial amide compounds provided by the present invention, the molar volume ratio of pyrimidine-4,6-dicarbonyl chloride and anhydrous dichloromethane in S3 is 10 mmol:50 mL, and the sufficient reaction time is 12 h.
[0015] According to the method for preparing hydrogel from antibacterial amide compounds provided by the present invention, the molar volume ratio of N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide, silver nitrate, dimethyl sulfoxide and water in S5 is 1:3:2:1, and the standing time is 30 minutes.
[0016] Compared with the prior art, the present invention has the following advantages:
[0017] The invention provides an antibacterial amide compound N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide. The compound is mixed with silver nitrate, dimethyl sulfoxide and water to prepare a hydrogel. The hydrogel has good antibacterial effect and is a small molecule gel with good mechanical properties and strong stability, thus having great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0019] Figure 1 It is a flow chart of the method for preparing the bacteramide hydrogel factor;
[0020] Figure 2 It is a flow chart of the inversion test;
[0021] Figure 3 is the SEM image of the antimicrobial amide hydrogel;
[0022] Figure 4 This is the rheological test graph of antibacterial amide hydrogel;
[0023] Figure 5 is the FT-IR image of antimicrobial amide hydrogel;
[0024] Figure 6 is the XRD spectrum of antimicrobial amide hydrogel;
[0025] Figure 7 This is the result of in vitro antibacterial experiment;
[0026] Figure 8 This is a graph of cytotoxicity test results;
[0027] Fig. 9 This is a comparison of histological damage of in vivo experimental wounds;
[0028] Fig.10 This is a comparison of H&E staining results from in vivo experiments. DETAILED DESCRIPTION
[0029] Example 1
[0030] This embodiment provides a method for preparing an antibacterial amide compound, and the specific steps are as follows:
[0031] S1. Take 20 mmol of pyrimidine-4,6-dicarboxylic acid and place it in a 50 mL round-bottom flask, add 25 mL of thionyl chloride and 0.2 mL (4 drops) of DMF (dimethylformamide), reflux at 75 ° C for 4 h, and then distill under reduced pressure to obtain pyrimidine-4,6-dicarboxylic acid chloride;
[0032] S2, take 20mmol 3-aminopyridine and put it in a 250mL round-bottom flask, add 100mL anhydrous dichloroethane and 3mL triethylamine, stir and dissolve evenly to obtain a mixed solution;
[0033] S3, take 10mmol of pyrimidine-4,6-dicarbonyl chloride prepared in S1 and dissolve it in 50mL of anhydrous dichloromethane, slowly add it dropwise to the round-bottom flask containing the mixed solution prepared in S2 at 0°C, react for 12h, and then filter and dry to obtain the initial product;
[0034] S4. The initial product prepared in S3 is purified by recrystallization with DMSO (dimethyl sulfoxide) and water, and dried to obtain a pure product of antibacterial amide compounds, namely N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide (denoted as MDA-N3).
[0035] The chemical structure of the product N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide is:
[0036]
[0037] The flow chart of the preparation method of antibacterial amide compounds is as follows Figure 1 shown.
[0038] Example 2
[0039] This embodiment provides a method for preparing a hydrogel from the antibacterial amide compound MDA-N3 prepared in Example 1, and the specific steps are as follows:
[0040] The gel-forming ability of metal supramolecular gel was evaluated by a typical “inversion test”.
[0041] First, a certain amount of N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide and silver nitrate prepared in this example are dissolved in a certain volume of dimethyl sulfoxide and water, respectively, and the specific ratios are shown in Table 1 below. Subsequently, the solution is immediately mixed in a scintillation vial, and the system is shaken to make the mixture uniform. After mixing, the system is left to stand for 30 minutes. Finally, the scintillation vial is turned upside down to observe whether the solution inside can still flow. If no flow occurs, gelation is formed, which is recorded as "G". The process of the inversion test is as follows: Figure 2 shown.
[0042] Table 1
[0043]
[0044] In Table 1 above, the molar ratio of MDA-N3 to silver nitrate and the volume ratio of dimethyl sulfoxide to water are shown;
[0045] Therefore, the MDA-N3 prepared in Example 1 and 3 times the molar amount of silver nitrate were dissolved together in dimethyl sulfoxide and water in a volume ratio of 2:1, the solution was immediately mixed in a scintillation vial and the system was shaken to mix the mixture evenly, and then the system was allowed to stand for 30 minutes to obtain an antibacterial amide hydrogel.
[0046] At the same time, this example characterizes the antibacterial amide hydrogel prepared as above.
[0047] ②Surface microstructure detection of hydrogel
[0048] The antibacterial amide hydrogel was subjected to SEM detection, and the results obtained were as follows: Figure 3 As shown, it can be seen that the hydrogel is a small molecule gel.
[0049] ③Metallogel rheological characterization
[0050] Figure 4 The metallogel rheological test graphs of the antibacterial amide hydrogel prepared in this example are oscillation angular frequency sweep test, dynamic strain sweep test and temperature strain test.
[0051] The oscillation angular frequency sweep test was carried out at 25±0.1°C and a constant strain of 10%. The storage modulus (G') and loss modulus (G") of the hydrogel were measured at an angular frequency of 0.1 to 100 rad / s. The sample was a hydrogel with a molar ratio of MDA-N3 and AgNO3 of 1:3.
[0052] The dynamic strain sweep measurement was performed at a constant frequency of 10 rad / s in the strain range of 1 to 1000%, and the sample was a hydrogel with a molar ratio of MDA-N3 to AgNO3 of 1:3.
[0053] Rheological testing provides a rapid and sensitive method to evaluate the mechanical properties of hydrogels.
[0054] It is generally believed that when the elastic modulus (G') is greater than the viscous modulus (G"), a hydrogel is formed.
[0055] The test results of this embodiment are as follows Figure 4 As shown, it can be seen that the hydrogel is very stable.
[0056] ④FT-IR spectrum characterization
[0057] Figure 5The FT-IR spectrum of the antibacterial amide hydrogel was obtained by vacuum freeze-drying the hydrogels of MDA-N3, AgNO3 and MDA-N3:AgNO3=1:3 and then pressing them with KBr powder. The infrared spectra of the samples were measured by Fourier transform infrared spectrometer with the scanning wave number range of 4000-400 cm -1 , resolution 4cm -1 .
[0058] As shown in the figure, the hydrogel formed by MDA-N3:AgNO3=1:3 lacks the 3326 peak and the 1377 peak compared with MDA-N3 and AgNO3, indicating that MDA-N3:AgNO3=1:3 is formed by the -NH2 bond of MDA-N3 and the -NO2 bond of AgNO3.
[0059] ⑤XRD spectrum characterization
[0060] Figure 6 The XRD spectrum of the antibacterial amide hydrogel, the X-ray diffraction pattern of dried silver nitrate, MDA-N3 and hydrogel samples were measured under the following test conditions: the scanning rate was 4° / min, the scanning angle was within the range of 5 to 60°, and Cu target radiation was used, with λ=0.154nm.
[0061] The compatibility and crystal structure of the material were further verified by XRD. Figure 6 As shown, compared with MDA-N3 and AgNO3, the hydrogel has no sharp diffraction peaks, and a diffuse peak appears at 10-20°. The results show that the hydrogel has an amorphous structure and is non-crystalline.
[0062] Example 3
[0063] This embodiment provides a product prepared by Example 2, namely, an antibacterial amide hydrogel, Ag + -Gel was used to conduct an in vitro antibacterial experiment. The specific steps are as follows:
[0064] Inhibition of antibacterial amide hydrogels on Gram-negative Escherichia coli (E.coli) and Gram-positive Staphylococcus aureus (S.aureus): The antibacterial amide hydrogels were cut into discs with a diameter of about 1 cm for antibacterial experiments. After the samples were sterilized by ultraviolet irradiation for 15 minutes, E. coli were spread on LB solid agar flat culture medium; Staphylococcus aureus was spread on Baird-Parker agar plates, where LB solid agar and Baird-Parker agar plates were purchased from Haibo Biotechnology and cultured in a constant temperature incubator at 37°C for 24 hours. The size of the inhibition zone around the antibacterial amide hydrogel in the bacterial plate reflects the antibacterial activity of the antibacterial amide hydrogel.
[0065] like Figure 7 As shown, Ag + -Gel group was higher than that of Ag alone + The group had better antibacterial activity against Escherichia coli and Staphylococcus aureus, and with the increase of Ag + The higher the concentration, the better the antibacterial effect.
[0066] Example 4
[0067] This embodiment provides a product prepared by Example 2, namely, an antibacterial amide hydrogel, Ag + -Gel was used to evaluate cytotoxicity. The specific steps are as follows:
[0068] 200 μL of Ag with a mass concentration of 0.02 g / L + -Gel was evenly coated in a six-well plate and allowed to stand at 37°C for about 30 min. After the gel solidified, the six-well plate was sterilized by ultraviolet irradiation for 30 min, and then 4×10 4 fibroblasts / mL.
[0069] After culturing for 4 hours, 2 days, 4 days, and 6 days, the fibroblasts were stained with calcein. Figure 8 Shown: 0.02g / L Ag + There was no significant difference in cell density and state between the -Gel and Control groups. The results showed that antimicrobial amide hydrogels were non-toxic to fibroblasts.
[0070] Example 5
[0071] This embodiment provides a product prepared by Example 2, namely, an antibacterial amide hydrogel, Ag + -Gel in vivo experiment, the specific steps are as follows:
[0072] (1) Establishment of rat skin defect and wound infection model:
[0073] After one week of adaptive feeding, male rats were used for formal experiments. The full-thickness skin defect infection model was used to evaluate the antimicrobial amide hydrogel Ag. + -Gel's effect on wound healing.
[0074] After the rats were anesthetized, the hair on their backs was shaved with a shaver, and an appropriate amount of depilatory cream was evenly applied to the exposed skin on their backs. After about 2 minutes, the remaining hair was removed with gauze, and the depilatory area of the rats was disinfected with alcohol. A circular hole puncher with a diameter of 1 cm was used to form a circular wound with a diameter of 1 cm, and surgical scissors were used to trim and remove the fascia. Four wounds were formed on each rat, with symmetrical positions and a certain distance between them.
[0075] 50 μL of Staphylococcus aureus was injected into each wound surface. After 8 hours of bacterial treatment, a rat full-thickness skin defect infection model was formed for subsequent experiments.
[0076] (2) All animals were randomly divided into three groups, with 5 animals in each group: a blank group with 200 μL sterile PBS solution, a control group with 200 μL sulfadiazine silver latex, and a control group with 200 μL 0.02 g / L Ag + -Gel hydrogel was the treatment group.
[0077] The recovery of the rats was recorded by taking photos on days 0, 1, 5 and 10. All rats were housed in separate cages, once in the morning and once in the evening, and the dressing was changed every three days until they were killed on the 14th day.
[0078] (3) Collection, embedding and preparation of skin tissue
[0079] In this experiment, the animals were killed on the 14th day after surgery, and the tissues near the wound were collected and immersed in 4% paraformaldehyde solution for 24 hours.
[0080] Then, skin tissue samples were taken for embedding. First, they were rinsed with distilled water for 2 hours to remove the paraformaldehyde solution, and the tissues were trimmed with scissors and placed in embedding boxes for gradient alcohol continuous dehydration. The dehydration sequence was first 75% ethanol immersion for 1 hour, 85% ethanol treatment for 1 hour, 95% ethanol immersion for 1 hour, and finally 2 times of anhydrous ethanol immersion for 2 hours respectively; then the tissue was transparentized and immersed in xylene twice, each time for 15 minutes; finally, the tissue was immersed in wax for 3 hours and embedded in paraffin.
[0081] The sample wax block was taken and made into a 5 μm thick sample in a microtome. After slicing, spreading and baking, it was used for subsequent related staining analysis.
[0082] (4) Histopathological examination and immunohistochemical testing
[0083] The above sections were dewaxed and stained with H&E according to the standard protocol. The fixed specimens were observed under an upright microscope to observe the differences in pathological changes in different sample tissues. Microscopic imaging system was used to take pictures and record the changes in wound histological damage. The results are as follows: Fig. 9 shown.
[0084] (5) Experimental results
[0085] (5.1) Wound healing analysis
[0086] The antibacterial effect and wound healing effect of the hydrogel were evaluated using a rat skin wound bacterial infection model.
[0087] Intuitively, 0.02g / L Ag +-Gel's wound healing effect was significantly better than the other two groups at all stages:
[0088] On the first day, it was found that the wound surface in the 0.02 g / L Ag+-Gel group was drier than that in the other two groups;
[0089] On the 5th day, it can be found that the wound healed faster than the other two groups;
[0090] Day 10, 0.02 g / L Ag + -The wounds in the Gel group were nearly completely healed, and the wound healing effect was significantly better than that in the control group and the sulfadiazine silver group;
[0091] These results clearly demonstrate that materials with antimicrobial and microcirculatory-regulating properties can aid wound recovery.
[0092] (5.2) Histopathological analysis
[0093] H&E staining results Fig.10 As shown, it can be clearly seen that on the 10th day, 0.02g / L Ag + The dermis in the -Gel group was completely regenerated, with fewer infiltrating inflammatory cells.
[0094] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.
Claims
1. An antibacterial amide compound, characterized in that: The antibacterial amide compound is N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide.
2. A method for preparing a hydrogel using the antibacterial amide compound as claimed in claim 1, characterized in that: The following steps are involved: S1, adding thionyl chloride and DMF to pyrimidine-4,6-dicarboxylic acid, fully reflux, and then distill under reduced pressure to obtain pyrimidine-4,6-dicarbonyl chloride; S2, adding anhydrous dichloroethane and triethylamine to 3-aminopyridine, stirring evenly to obtain a mixed solution; S3, dissolving the pyrimidine-4,6-dicarbonyl chloride prepared in S1 in anhydrous dichloromethane, and adding it dropwise to the mixed solution prepared in S2 at 0°C. After sufficient reaction, the mixture is filtered and dried to obtain a primary product; S4, purifying the initial product prepared in S3 by recrystallization with DMSO and water, and drying to obtain a pure product of antibacterial amide compounds, namely N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide; S5. Take the N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide and silver nitrate prepared in S4, dissolve them in a mixed solvent of dimethyl sulfoxide and water, mix them evenly and let them stand to obtain a hydrogel.
3. The method for preparing hydrogel using antibacterial amide compounds according to claim 2, characterized in that: The molar volume ratio of pyrimidine-4,6-dicarboxylic acid, thionyl chloride and DMF in S1 is 20 mmol:25 mL:0.2 mL, the reflux temperature is 75° C., and the reflux time is 4 h.
4. The method for preparing hydrogel using antibacterial amide compounds according to claim 3, characterized in that: The molar volume ratio of 3-aminopyridine, anhydrous dichloroethane and triethylamine in S2 is 20mmol:100mL:3mL.
5. The method for preparing hydrogel using antibacterial amide compounds according to claim 2, characterized in that: The molar volume ratio of pyrimidine-4,6-dicarbonyl chloride and anhydrous dichloromethane in S3 is 10 mmol:50 mL, and the sufficient reaction time is 12 h.
6. The method for preparing hydrogel using antibacterial amide compounds according to claim 2, characterized in that: The molar volume ratio of N,N'-di(3-pyridyl)pyrimidine-4,6-dicarboxamide, silver nitrate, dimethyl sulfoxide and water in S5 is 1:3:2:1, and the standing time is 30 minutes.