Bifunctionalized pyrylium ring derivatives, processes for their preparation and use thereof

Abstract

The application provides a bifunctional pyran ring derivative and a preparation method and application thereof, and relates to the field of biological medicines.The structure of the bifunctional pyran ring derivative is: the derivative is prepared from hyaluronic acid, sodium periodate, ethylene glycol and EDCI are sequentially added and stirred, and then reacted with amine compounds or hydrazine compounds respectively.The bifunctional pyran ring derivative can be used in the fields of biological medicines, medical cosmetology and the like.The bifunctional pyran ring derivative is prepared by grafting two kinds of functional groups which react with each other on the hyaluronic acid main chain at the same time, so that the obtained pyran ring derivative can spontaneously form a crosslinked hydrogel structure after being dissolved.The preparation process is simple, the bifunctional pyran ring derivative can be obtained by using a one-pot two-step method, the use of organic solvents is avoided, the problem of residual organic solvents in the product is fundamentally eliminated, the biological compatibility is better, and the probability of rejection reaction and inflammatory reaction of the implant in the human body can be greatly reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a bifunctional pyrylium derivative, a preparation method and application thereof. BACKGROUND

[0002] The first studies on HYALURONIC ACID (HA) date back to 1880: the French scientist Portes observed that the vitreous humor mucin was different from other mucopolysaccharides in the cornea and cartilage and named it "hyaline mucin". However, it was not until 1934 that Meyer and Palmer isolated a new polysaccharide containing amino sugars and uronic acids from bovine vitreous humor and named it HA, meaning "hyaloid" (vitreous body) and "uronic acid". During the 1930s and 1950s, HA was also isolated from human umbilical cord, chicken comb and streptococci.

[0003] Since the 1940s, the physicochemical properties of HA have been extensively studied and its chemical structure was elucidated in 1954 by Meyer and Weissmann. During the second half of the 20th century, the progressive understanding of the biological role of HA led to a strong interest in its production and development as a medical product. Therefore, the process of extracting HA from animal tissues was gradually optimized, but there were still problems in removing unwanted contaminants such as microorganisms, proteins. Preliminary studies on the production of HA by bacterial fermentation and chemical synthesis were carried out before the 1970s.

[0004] In 1979, Balazs first produced pharmaceutical-grade HA, he developed an effective method for extracting and purifying the polymer from chicken combs and human umbilical cord. Balazs' method laid the foundation for the industrial production of HA. Since the early 1980s, HA has been extensively studied as a raw material for developing implantable intraocular lenses, and has become a major product in the field of ophthalmology due to its safety and protective effect on the corneal endothelium. In addition, HA has also been found to be beneficial for the treatment of joint and skin diseases, wound healing and soft tissue augmentation.

[0005] Since the late 1980s, HA has also been used to formulate drug delivery systems, and efforts are still being made to develop HA-based carriers to improve therapeutic efficacy. In the 1990s and 2000s, particular attention was paid to identifying and characterizing enzymes involved in HA metabolism, and developing bacterial fermentation techniques to produce HA with controlled size and polydispersity. Today, HA occupies a key position in various medical, pharmaceutical, nutritional and cosmetic applications. Therefore, HA remains a hot topic for research to elucidate its biosynthetic pathway and molecular biology, optimize its biotechnological production, synthesize derivatives with improved properties, and optimize and implement its therapeutic and aesthetic uses.

[0006] Functionalized HA exhibits many unique advantages in the field of medicine and bioengineering, making it an ideal biomaterial.

[0007] Enhanced stability and durability: Through chemical modification and cross-linking, modified HA can significantly improve its stability in vivo, reducing the degradation effect of HA enzymes. This enhanced stability enables HA to maintain longer-term therapeutic effects in applications.

[0008] Stimulus responsiveness: Modified HA can incorporate different stimulus-responsive groups to respond to specific external stimuli such as pH, temperature, light, electric field, or magnetic field. This feature enables HA to release drugs under specific conditions, improving drug targeting and effectiveness. For example, pH-responsive HA can trigger drug release under the acidic conditions of the tumor microenvironment, while temperature-responsive HA can adjust drug release rates when body temperature changes.

[0009] Enhanced biocompatibility: Modified HA can better interact with cells and tissues, providing excellent biocompatibility. This feature makes it excel in applications such as tissue engineering, wound healing, and soft tissue filling.

[0010] Multifunctionality and controllability: Through different chemical modifications, modified HA can have multiple functions such as increasing its mechanical strength, adjusting its degradation rate, and improving its drug carrier performance. This multifunctionality enables HA to adapt to various biomedical needs, providing personalized and customized treatment plans.

[0011] Improved drug delivery efficiency: As a carrier for drug delivery systems, modified HA can improve drug solubility and bioavailability, and achieve controlled release. This intelligent drug delivery system has significant advantages in cancer treatment, inflammation control, and chronic disease management.

[0012] These advantages of modified HA make it an important part of modern biomedicine and drive its widespread application in the fields of treatment and cosmetics. With further research and technological progress, the application prospects of modified HA will be even broader. SUMMARY

[0013] The present application successfully prepares a bifunctional modified pyran ring derivative with a novel structure through a one-pot two-step process. The bifunctional groups are aldehyde and amine groups or aldehyde and hydrazine groups. The bifunctional pyran ring derivative can form in-situ self-crosslinking hydrogel through Schiff base condensation reaction in aqueous solution, and due to the reversibility of Schiff base condensation reaction, the gel has self-healing properties, which is a good new biocompatible polymer biomaterial. It can be used as a raw material for hydrogel and developed into wound dressings, healing powder, gel drug carriers, gel cell carriers, and other biomedical products.

[0014] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0015] The present application provides a kind of bifunctional pyran ring derivative, its structure is as shown in formula I:

[0016]

[0017] In formula I, x, y, z are percentage of three structural units respectively, x+y+z=100%;

[0018] R is amido or hydrazine group.

[0019] Further, R is any of the following groups:

[0020] Further, the molecular weight of the bifunctional pyran ring derivative is 10-2500kDa, preferably 200-1000kDa, more preferably 400-600kDa.

[0021] Further, the degree of substitution of aldehyde group in the compound of formula I is 10-90%, preferably 30-60%, more preferably 40-50%; the degree of substitution of amido or hydrazine group is 20-80%, preferably 30-60%, more preferably 45-55%.

[0022] In the present application, the degree of substitution represents the percentage of substituted pyran ring units in all pyran ring units.

[0023] The present application also provides a preparation method of the above-mentioned bifunctional pyran ring derivative, comprising the following steps:

[0024] (1) dissolve hyaluronic acid, add sodium periodate, stir for a certain time;

[0025] (2) add ethylene glycol, stir for a certain time;

[0026] (3) adjust the pH value of the solution to 4.7-4.8, add EDCI (1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole hydrochloride) and stir for a certain time;

[0027] (4) add amine compound or hydrazine compound, stir for a certain time;

[0028] (5) the reaction is purified by dialysis, and the obtained purified liquid is freeze-dried to obtain the bifunctional pyran ring derivative.

[0029] The specific reaction route of the above reaction is as follows:

[0030]

[0031] Further, the hyaluronic acid in step (1) has a molecular weight of 10-2500 kDa, preferably 200-1000 kDa, and more preferably 400-600 kDa.

[0032] Further, the solvent for dissolving the hyaluronic acid in step (1) is water, physiological saline or PBS buffer.

[0033] Further, the hyaluronic acid in step (1) has a molecular weight of 10-2500 kDa, preferably 200-1000 kDa, and more preferably 400-600 kDa.

[0034] Further, the molar ratio of sodium periodate to hyaluronic acid in step (1) is 0.1-100:1, preferably 1-10:1.

[0035] Further, the stirring time in step (1) is 1-24 hours.

[0036] Further, the molar ratio of sodium periodate to ethylene glycol is 1:10-1000, preferably 1:20-200.

[0037] Further, the stirring time in step (2) is 1-24 hours.

[0038] Further, the molar ratio of EDCI to hyaluronic acid in step (3) is 0.1-100:1, preferably 1-10:1.

[0039] Further, the stirring time in step (3) is 1-24 hours.

[0040] Further, the amine compound in step (4) is an organic compound containing two or more amine groups.

[0041] Further, the amine compound in step (4) is ethylenediamine, propylenediamine, butylenediamine, p-phenylenediamine or m-phenyl triamine.

[0042] Further, the hydrazine compound in step (4) is an organic compound containing two or more hydrazine groups.

[0043] Further, the hydrazine compound in step (4) is ethanedioic acid dihydrazide, propanedioic acid dihydrazide, butanedioic acid dihydrazide, p-phenylenedioic acid dihydrazide, m-phenylenedioic acid dihydrazide or m-phenyl triacid trihydrazide.

[0044] Further, the molar ratio of the amine compound or hydrazine compound to hyaluronic acid in step (4) is 2-1000:1, preferably 5-40:1.

[0045] Further, the stirring time in step (4) is 1-24 hours.

[0046] The application further provides a hydrogel prepared from the bifunctionalized pyran ring derivative.

[0047] Further, the preparation method of the hydrogel comprises: crushing the bifunctionalized pyran ring derivative of the application, dissolving it in water, and stirring to obtain the hydrogel.

[0048] The application further provides the use of the bifunctionalized pyran ring derivative or the hydrogel in the fields of biological medicine, medical cosmetology, etc., and the bifunctionalized pyran ring derivative or the hydrogel can be specifically used for preparing wound dressings, healing powders, gel drug carriers, gel cell carriers, tumor radioactive isolation gels, vascular embolism gels, aneurysm blocking agents, ophthalmic viscoelastic agents, knee joint injection gels, etc.

[0049] Compared with the prior art, the application has the following beneficial effects:

[0050] 1. The preparation process of the application is simple, the bifunctionalized pyran ring derivative can be obtained by one-pot two-step method, the intermediate product does not need to be purified, and the substitution degree of aldehyde groups, amine groups or hydrazine groups can be adjusted.

[0051] 2. The use of organic solvents is avoided, the problem of residual organic solvents in the product is fundamentally eliminated, the biological compatibility is better, and the probability of rejection reaction and inflammatory reaction of the human body after implantation can be greatly reduced.

[0052] 3. Two kinds of functional groups that react with each other are grafted on the hyaluronic acid main chain at the same time, so that the obtained pyran ring derivative can spontaneously form a cross-linked hydrogel structure after being dissolved. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 The H-NMR spectrum of HA-CHO-EDA prepared for the embodiment 1 of the application. 1

[0054] Figure 2 The H-NMR spectrum of HA-CHO-SDH prepared for the embodiment 2 of the application. 1

[0055] Figure 3 The MTT cell compatibility test results of the bifunctionalized pyran ring derivative in the embodiment 5 of the application. DETAILED DESCRIPTION

[0056] ​​The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application. In addition, it is worth mentioning that the raw materials involved in the present application are all common commercially available products unless otherwise specified.

[0057] Example 1, synthesis of aldehyde-amine-bifunctional pyran ring derivative (HA-CHO-EDA)

[0058] Dissolve 10 g of hyaluronic acid with a molecular weight of 500 kDa in 1 L of pure water, add 1 equivalent of sodium periodate, stir at room temperature for 24 hours, add 50 equivalents of ethylene glycol, and stir at room temperature for 2 hours. Adjust the pH of the solution to 4.7-4.8 using 1 M hydrochloric acid, add 2 equivalents of condensing agent EDCI and stir for 15 minutes, add 5 equivalents of ethylenediamine (EDA) and stir for 4 hours. Transfer the reaction system to a dialysis bag with a molecular weight cut-off of 1 kDa for dialysis purification. The purified solution is freeze-dried to obtain 8.2 g of white fibrous HA-CHO-EDA product. The product has a molecular weight of 500 kDa, and the degree of substitution of the aldehyde group is 16%, and the degree of substitution of the amine group is 47.5%. 1 The H-NMR spectrum is shown in the following figure. Figure 1

[0059] The degree of substitution of the aldehyde group in the product is 16%, and the degree of substitution of the amine group is 47.5%.

[0060] The structural formula of HA-CHO-EDA is as follows:

[0061]

[0062] Example 2, synthesis of aldehyde-hydrazine-bifunctional pyran ring derivative (HA-CHO-SDH)

[0063] Dissolve 10 g of hyaluronic acid with a molecular weight of 1000 kDa in 1 L of pure water, add 2 equivalents of sodium periodate, stir at room temperature for 24 hours, add 100 equivalents of ethylene glycol, and stir at room temperature for 2 hours. Adjust the pH of the solution to 4.7-4.8 using 1 M hydrochloric acid, add 2 equivalents of condensing agent EDCI and stir for 15 minutes, add 8 equivalents of succinic acid dihydrazide (SDH) and stir for 4 hours. Transfer the reaction system to a dialysis bag with a molecular weight cut-off of 1 kDa for dialysis purification. The purified solution is freeze-dried to obtain 8.6 g of white fibrous HA-CHO-SDH product. The product has a molecular weight of 1000 kDa, and the degree of substitution of the aldehyde group is 20%, and the degree of substitution of the hydrazine group is 48%. 1 The H-NMR spectrum is shown in the following figure. Figure 2

[0064] The degree of substitution of the aldehyde group in the product is 20%, and the degree of substitution of the hydrazine group is 48%.​​

[0065] The structural formula of HA-CHO-SDH is as follows:

[0066]

[0067] Example 3, synthesis of HA-CHO-EDA gel

[0068] The HA-CHO-EDA prepared in Example 1 was crushed and 0.1 g, 0.2 g, 0.4 g, 0.6 g, and 0.8 g of the powder were respectively dissolved in 10 mL of pure water. The solution viscosity gradually increased during the powder dissolution process, and continuous stirring was performed until a uniform gel was formed, i.e., Hydrogel CHO-EDA1％ , Hydrogel CHO-EDA2％ , Hydrogel CHO-EDA4％ , Hydrogel CHO-EDA6％ , Hydrogel CHO-EDA8％ .

[0069] Example 4, synthesis of HA-CHO-SDH gel

[0070] The HA-CHO-SDH prepared in Example 2 was crushed and 0.1 g, 0.2 g, 0.4 g, 0.6 g, and 0.8 g of the powder were respectively dissolved in 10 mL of pure water. The solution viscosity gradually increased during the powder dissolution process, and continuous stirring was performed until a uniform gel was formed, i.e., Hydrogel CHO-SDH1％ , Hydrogel CHO-SDH2％ , Hydrogel CHO-SDH4％ , Hydrogel CHO-SDH6％ , Hydrogel CHO-SDH8％ .

[0071] Example 5, cell compatibility test

[0072] The pyran ring derivatives prepared in Examples 1-2 were respectively formulated into a series of samples (0, 500, 1000, 2500 g / mL) using DMEM cell formulation solution, and were filtered to remove bacteria. The L929 fibroblast cells were subjected to sample cytotoxicity test by MTT method. The group with a concentration of 0 was a blank control group, and the others were experimental groups. The L929 cells in the logarithmic growth phase were inoculated into a 96-well cell culture plate (cell concentration 8.0×10 3 cells / well), and were cultured overnight after cell adhesion. The above blank control group and experimental group solutions were respectively added to each well at 100 μL, and three replicate wells were set. After 24 h of culture, the absorbance value at 570 nm was determined by MTT method, and the relative survival rate of cells was calculated.

[0073] The calculation formula is: cell survival rate (%) = (experimental group OD average value / blank control group OD average value) x 100%.

[0074] Results are shown in the attached Figure 3 The cell survival rate of each experimental group was more than 90%, indicating that the pyran ring derivatives prepared in Examples 1-2 had no significant toxicity.

[0075] Example 6, detection of hydrogel viscoelasticity

[0076] The rheological properties of the hydrogels obtained in Examples 3 and 4 were detected using a TA-RH-2 rheometer with an 8mm probe, and 200,000 molecular weight hyaluronic acid was dissolved in water of the same concentration as the control group for viscoelasticity detection.

[0077] The results are shown in Table 1:

[0078] Table 1:

[0079]

[0080]

[0081] As shown in Table 1, the bifunctional HA derivative after dissolution, except for Hydrogel CHO-EDA1％ Except for the sample group that did not form a hydrogel, the remaining sample groups all formed a uniform cross-linked structure hydrogel (elastic modulus greater than viscous modulus), and the hydrogel strength range increased with increasing derivative concentration. 200,000 molecular weight hyaluronic acid as a control group was dissolved in water of the same concentration, as shown in Table 1, a viscous solution was obtained at 1%, 2%, and 4% concentrations, and no cross-linking reaction occurred (viscosity modulus greater than elastic modulus), and the 6% and 8% concentration samples did not dissolve to a uniform state.

[0082] Example 7, hemostatic effect experiment of bifunctional pyran ring derivative

[0083] 7.1 Preparation of materials

[0084] Pentobarbital anesthetic, 1ml sterile syringe, gauze, balance, box holder, forceps, scissors, surgical knife, alcohol disinfectant, paper towel.

[0085] 7.2 Experimental steps

[0086] 1. 8 SD rats were weighed, anesthetized intraperitoneally, and marked;

[0087] 2. Use surgical cutting tools to cut 40% of the length of the rat tail, place the proximal tail on 10 layers of 5cm*5cm gauze pre-weighed, let the tail stay in the air for 15 seconds to ensure normal blood loss, and then proceed with the subsequent experiment;

[0088] 3. Treatment group: After the rats were castrated and bled normally, hyaluronic acid raw material and pyran ring derivative powder were applied to the bleeding site, 0.2 grams of powder was used for each group, and the amount of powder needed to cover the wound site. The time to stop bleeding was observed and recorded, and the weight of the gauze was recorded after stopping bleeding;

[0089] 4. Blank group: After the rats were castrated and bled normally, the time to stop bleeding was observed and recorded, and the weight of the gauze was recorded after stopping bleeding;

[0090] 5. After hemostasis, antibiotic ointment was applied to the wound site for protection (erythromycin eye ointment).

[0091] 6. After the rats woke up, the animal state was observed, and the crawling action behavior was recorded.

[0092] 7. The results are recorded in Table 2.

[0093] Table 2:

[0094]

[0095]

[0096] Finally, it should be noted that the above content is only used to illustrate the technical solutions of the present application, and is not a limitation on the protection scope of the present application. Simple modifications or equivalent replacements of the technical solutions of the present application made by those skilled in the art do not deviate from the essence and scope of the technical solutions of the present application.

Claims

1. A bifunctionalized hyaluronic acid, the structure of which is shown in Formula I below: Formula I In Equation I, x, y, and z represent the percentages of the three structural units, respectively, and x + y + z = 100%. R is any one of the following groups: , , , , , , , , , , ; The degree of substitution of the aldehyde group in the compound of formula I is 10-90%, and the degree of substitution of R is 30-60%.

2. The bifunctionalized hyaluronic acid according to claim 1, characterized in that, The molecular weight of the bifunctionalized hyaluronic acid is 10-2500 kDa.

3. The bifunctionalized hyaluronic acid according to claim 2, characterized in that, The molecular weight of the bifunctionalized hyaluronic acid is 200-1000 kDa.

4. The bifunctionalized hyaluronic acid according to claim 2, characterized in that, The molecular weight of the bifunctionalized hyaluronic acid is 400-600 kDa.

5. A method for preparing bifunctionalized hyaluronic acid according to any one of claims 1-4, comprising the following steps: (1) Dissolve hyaluronic acid, add sodium periodate, and stir for a certain period of time; (2) Add ethylene glycol and stir for a certain period of time; (3) Adjust the pH of the solution to 4.7-4.8, add EDCI and stir for a certain period of time; (4) Add amine compounds or acyl hydrazides and stir for a certain period of time; the amine compounds are ethylenediamine, propylenediamine, butanediamine, p-phenylenediamine or m-phenyltriamine, and the acyl hydrazides are oxalic acid dihydrazides, malonic acid dihydrazides, succinic acid dihydrazides, terephthalic acid dihydrazides, isophthalic acid dihydrazides or isophthalic trihydrazides; (5) The reactants were purified by dialysis, and the purified solution was freeze-dried to obtain the bifunctionalized hyaluronic acid. The mass concentration of the dissolved hyaluronic acid in step (1) is 0.5-4%.

6. The preparation method according to claim 5, characterized in that, In step (1), the solvent used to dissolve hyaluronic acid is water, physiological saline, or PBS buffer. The molar ratio of sodium periodate to hyaluronic acid in step (1) is 0.1-100:

1.

7. The preparation method according to claim 5, characterized in that, The molar ratio of sodium periodate to hyaluronic acid in step (1) is 1-10:

1.

8. The preparation method according to claim 5, characterized in that, The molar ratio of sodium periodate to ethylene glycol is 1:10-1000, the molar ratio of EDCI to hyaluronic acid is 0.1-100:1, and the molar ratio of amine compound or hydrazide compound to hyaluronic acid is 2-1000:

1.

9. The preparation method according to claim 8, characterized in that, The molar ratio of sodium periodate to ethylene glycol is 1:20-200, the molar ratio of EDCI to hyaluronic acid is 1-10:1, and the molar ratio of amine compound or hydrazide compound to hyaluronic acid is 5-40:

1.

10. A hydrogel prepared from the bifunctionalized hyaluronic acid according to any one of claims 1-4.

11. The method for preparing the hydrogel according to claim 10, the method comprising: The bifunctionalized hyaluronic acid described in any one of claims 1-4 is pulverized and dissolved in water, and the hydrogel is obtained by stirring.

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