Bifunctional pyran ring derivative as well as preparation method and application thereof

Abstract

The invention provides a bifunctional pyran ring derivative as well as a preparation method and application thereof, and relates to the field of biological medicines. The structure of the bifunctional pyran ring derivative is # imgabs0, hyaluronic acid is used as a raw material, sodium periodate, ethylene glycol and EDCI are sequentially added and stirred, then the mixture reacts with an amine compound or a hydrazide compound, and the bifunctional pyran ring derivative is obtained. The bifunctional pyran ring derivative can be used in the fields of biological medicine or medical cosmetology and the like. According to the invention, two functional groups which react with each other are creatively grafted on a 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. The preparation process is simple, the bifunctional pyran ring derivative can be obtained by adopting a one-pot two-step method, the use of an organic solvent is avoided, the problem of residual organic solvent in the product is fundamentally eliminated, the biocompatibility is better, and the occurrence probability of rejection reaction and inflammatory reaction when the bifunctional pyran ring derivative is implanted into a human body can be greatly reduced.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a bifunctionalized pyran ring derivative and a preparation method and application thereof. Background Art

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

[0003] The physicochemical properties of HA have been extensively studied since the 1940s, and its chemical structure was solved by Meyer and Weissmann in 1954. In the second half of the 20th century, the gradual understanding of the biological role of HA led to a strong interest in its production and development as a medical product. As a result, the process of extracting HA from animal tissues has been gradually optimized, but there are still some problems in removing unwanted contaminants (such as microorganisms and proteins). Before the 1970s, preliminary studies on the production of HA by bacterial fermentation and chemical synthesis had been carried out.

[0004] Pharmaceutical-grade HA was first produced by Balazs in 1979, who developed an efficient method for extracting and purifying the polymer from chicken combs and human umbilical cords. 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 the development of implantable artificial lenses and has become a major product in the field of ophthalmology due to its safety and protective effects on the corneal endothelium. In addition, HA has been found to be beneficial in the treatment of joint and skin diseases, wound healing, and soft tissue volume expansion.

[0005] HA has also been used to formulate drug delivery systems since the late 1980s, and efforts to develop HA-based carriers to improve therapeutic efficacy continue to this day. In the 1990s and 2000s, particular attention was paid to identifying and characterizing the 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 a variety of medical, pharmaceutical, nutritional, and cosmetic applications. Therefore, HA remains a hot topic of research to elucidate its biosynthetic pathways and molecular biology, optimize its biotechnological production, synthesize derivatives with improved properties, and optimize and implement its therapeutic and aesthetic uses.

[0006] Functionalized modified HA exhibits many unique advantages in the fields 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 the body and reduce the degradation of HA enzymes. This enhanced stability allows HA to maintain its efficacy for a longer period of time during application.

[0008] Stimulus responsiveness: Modified HA can be combined with different stimulus responsive groups to achieve response to specific external stimuli (such as pH, temperature, light, electric field or magnetic field). This property enables HA to release drugs under specific conditions, improving the targeting and effectiveness of drugs. For example, pH-responsive HA can trigger drug release under acidic conditions in the tumor microenvironment, while temperature-responsive HA can adjust the drug release rate when body temperature changes.

[0009] Enhanced biocompatibility: Modified HA is able to interact better with cells and tissues, providing excellent biocompatibility. This property makes it excellent in applications such as tissue engineering, wound healing, and soft tissue augmentation.

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

[0011] Improve drug delivery efficiency: Modified HA as a carrier of drug delivery system can improve the solubility and bioavailability of drugs and achieve controlled release. This smart drug delivery system has significant advantages in cancer treatment, inflammation control and chronic disease management.

[0012] These advantages of modified HA make it occupy an important position in modern biomedicine and promote its wide application in the fields of treatment and beauty. With the deepening of research and the advancement of technology, the application prospects of modified HA will be broader. Summary of the invention

[0013] The present invention successfully prepares a bifunctional modified pyran ring derivative with a novel structure through a one-pot two-step process, wherein the bifunctionalized groups are aldehyde and amine or aldehyde and hydrazide groups. The bifunctionalized pyran ring derivative can form an in-situ self-crosslinking hydrogel in an aqueous solution through a Schiff base condensation reaction, and since the Schiff base condensation reaction is reversible, the gel has a self-healing property and is a good new biocompatible polymer biomaterial. It can be used as a hydrogel raw material and developed into biomedical products such as wound dressings, healing powders, gel drug carriers, and gel cell carriers.

[0014] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0015] The present invention provides a bifunctional pyran ring derivative, the structure of which is shown in the following formula I:

[0016]

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

[0018] R is an amine group or a hydrazide group.

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

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

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

[0022] In the present invention, the degree of substitution refers to the percentage of substituted pyran ring units to all pyran ring units.

[0023] The present invention also provides a method for preparing the above-mentioned bifunctionalized pyran ring derivative, comprising the following steps:

[0024] (1) Dissolve hyaluronic acid, add sodium periodate, and stir for a certain period of time;

[0025] (2) Add ethylene glycol and stir for a certain period of time;

[0026] (3) adjusting the pH value of the solution to 4.7-4.8, adding EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and stirring for a certain period of time;

[0027] (4) adding an amine compound or a hydrazide compound and stirring for a certain period of time;

[0028] (5) The reactant is dialyzed for purification, and the obtained purified solution is freeze-dried to obtain the bifunctionalized pyran ring derivative.

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

[0030]

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

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

[0033] Furthermore, the mass concentration of the hyaluronic acid after dissolution in step (1) is 0.1-20%, preferably 0.5-4%.

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

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

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

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

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

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

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

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

[0042] Furthermore, the hydrazide compound in step (4) is an organic compound containing two or more hydrazines.

[0043] Furthermore, the hydrazide compound in step (4) is oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, terephthalic acid dihydrazide, isophthalic acid dihydrazide or trimesophthalic acid trihydrazide.

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

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

[0046] The invention also provides a hydrogel prepared from the bifunctionalized pyran ring derivative.

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

[0048] The present invention also provides the use of the above-mentioned bifunctionalized pyran ring derivative or the above-mentioned hydrogel in the fields of biomedicine or medical cosmetology, and can be specifically used to prepare wound dressings, healing powders, gel drug carriers, gel cell carriers, tumor radioisolation gels, vascular embolization gels, aneurysm occluding agents, ophthalmic viscoelastics, knee joint injection gels, etc.

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

[0050] 1. The preparation process of the present invention is simple, and a bifunctionalized pyran ring derivative can be obtained by a one-pot two-step method without purifying the intermediate product, and the degree of substitution of the aldehyde group, the amine group or the hydrazide group can be adjusted.

[0051] 2. It avoids the use of organic solvents and fundamentally eliminates the problem of residual organic solvents in the product. It has better biocompatibility and can greatly reduce the probability of rejection and inflammatory reactions after implantation in the human body.

[0052] 3. In a pioneering way, two mutually reactive functional groups are simultaneously grafted onto the main chain of hyaluronic acid, so that the resulting pyran ring derivative can spontaneously form a cross-linked hydrogel structure after dissolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 HA-CHO-EDA prepared in Example 1 of the present invention 1 H-NMR spectrum.

[0054] Figure 2 HA-CHO-SDH prepared in Example 2 of the present invention 1 H-NMR spectrum.

[0055] Figure 3 The results of the MTT cell compatibility experiment of the bifunctional pyran ring derivative in Example 5 of the present invention. DETAILED DESCRIPTION

[0056] The technical scheme in the embodiment of the present invention will be clearly and completely described below. Obviously, the described embodiment is only a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In addition, it is worth noting that the raw materials involved in the present invention are all common commercial products unless otherwise specified.

[0057] Example 1. Synthesis of Aldehyde-Amine-Bifunctionalized Pyran Ring Derivative (HA-CHO-EDA)

[0058] Dissolve 10 grams of 500 kDa molecular weight hyaluronic acid in 1L 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. Use 1M hydrochloric acid to adjust the solution pH to 4.7-4.8, 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 cutoff of 1 kDa for dialysis purification, and the purified solution is freeze-dried to obtain 8.2 grams of white fibrous HA-CHO-EDA product, the product 1 The H-NMR spectrum is attached. Figure 1 shown.

[0059] The results showed that the substitution degree of aldehyde group in the product was 16%, and the substitution degree of amine group was 47.5%.

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

[0061]

[0062] Example 2: Synthesis of Aldehyde-Hydrazide-Bifunctionalized Pyran Ring Derivative (HA-CHO-SDH)

[0063] Dissolve 10 grams of 1000 kDa molecular weight hyaluronic acid in 1L of pure water, add 2 times the equivalent of sodium periodate, stir at room temperature for 24 hours, add 100 times the equivalent of ethylene glycol, and stir at room temperature for 2 hours. Use 1M hydrochloric acid to adjust the solution pH to 4.7-4.8, add 2 times the equivalent of condensing agent EDCI and stir for 15 minutes, add 8 times the equivalent of succinic acid dihydrazide (SDH) and stir for 4 hours, transfer the reaction system to a dialysis bag with a molecular weight cutoff of 1 kDa for dialysis purification, and the purified solution is freeze-dried to obtain 8.6 grams of white fibrous HA-CHO-SDH product, the product 1 The H-NMR spectrum is attached. Figure 2 shown.

[0064] The results showed that the degree of substitution of aldehyde groups in the product was 20%, and the degree of substitution of hydrazide groups was 48%.

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

[0066]

[0067] Example 3: Synthesis of HA-CHO-EDA gel

[0068] 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 were respectively dissolved in 10 ml of pure water. The viscosity of the solution gradually increased during the dissolution of the powder. The solution was stirred continuously until a uniform gel was formed, thus obtaining 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 were dissolved in 10 ml of pure water. The viscosity of the solution gradually increased during the dissolution of the powder. The solution was stirred continuously until a uniform gel was formed, thus obtaining Hydrogel. CHO-SDH1％ 、Hydrogel CHO-SDH2％ 、Hydrogel CHO-SDH4％ 、Hydrogel CHO-SDH6％ 、Hydrogel CHO-SDH8％ .

[0071] Example 5: Cytocompatibility test

[0072] The pyran ring derivatives prepared in Example 1-2 were prepared into a series of samples with different concentrations (0, 500, 1000, 2500 g / mL) using DMEM cell sample preparation solution and sterilized by filtration. The cytotoxicity test of the samples was performed on L929 fibroblasts using the MTT method. The group with a concentration of 0 was the blank control group, and the others were the experimental groups. The L929 cells growing in the logarithmic phase were inoculated into a 96-well cell culture plate (cell concentration 8.0×10 3 After overnight culture, the blank control group and experimental group solutions were added, 100 μL per well, and three replicates were set. After 24 h of culture, the absorbance 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 (%) = (mean OD value of experimental group / mean OD value of blank control group) × 100%.

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

[0075] Example 6: Hydrogel viscoelasticity test

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

[0077] The results are shown in Table 1:

[0078] Table 1:

[0079]

[0080]

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

[0082] Example 7: Experiment on the hemostatic effect of bifunctional pyran ring derivatives

[0083] 7.1 Prepare items

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

[0085] 7.2 Experimental procedures

[0086] 1.8 SD rats, weighed, intraperitoneally anesthetized, and labeled;

[0087] 2. Use surgical cutting tools to cut 40% of the length of the mouse tail, place the proximal end of the mouse tail on a pre-weighed 10-layer 5cm*5cm gauze, and let the tail stay in the air for 15 seconds to ensure normal blood loss before conducting subsequent experiments;

[0088] 3. Treatment group: After the mice were tail amputated and bleeding was normal, the hyaluronic acid raw material and pyran ring derivative powder were applied to the bleeding site. The amount of powder used for each group was 0.2 g, and the amount was required to cover the wound site. The time for bleeding to stop was observed and recorded. After the bleeding stopped, the gauze was weighed and the amount of bleeding was recorded;

[0089] 4. Blank group: After the mice were tail amputated and bleeding occurred normally, the time of bleeding cessation was observed and recorded. After bleeding stopped, the gauze was weighed and the amount of bleeding was recorded.

[0090] 5. After the bleeding stops, apply antibiotic ointment to the wound for protection (erythromycin eye ointment).

[0091] 6. After the rat wakes up, observe the animal's condition and record its crawling behavior.

[0092] 7.3 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 solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A bifunctional pyran ring derivative, the structure of which is shown in the following formula I: In formula I, x, y, and z are the percentages of the three structural units, respectively, and x+y+z=100%; R is an amine group or a hydrazide group; preferably, R is any one of the following groups:

2. The bifunctionalized pyran ring derivative according to claim 1, characterized in that: The molecular weight of the bifunctionalized pyran ring derivative is 10-2500 kDa, preferably 200-1000 kDa, and more preferably 400-600 kDa.

3. The bifunctionalized pyran ring derivative according to claim 1, characterized in that: The degree of substitution of the aldehyde group in the compound of formula I is 10-90%, preferably 30-60%, more preferably 40-50%; the degree of substitution of the amine group or hydrazide group is 20-80%, preferably 30-60%, more preferably 45-55%.

4. The method for preparing the bifunctionalized pyran ring derivative according to any one of claims 1 to 3, 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 value of the solution to 4.7-4.8, add EDCI and stir for a certain period of time; (4) adding an amine compound or a hydrazide compound and stirring for a certain period of time; (5) The reactant is dialyzed for purification, and the obtained purified solution is freeze-dried to obtain the bifunctionalized pyran ring derivative.

5. The preparation method according to claim 4, characterized in that: The molecular weight of the hyaluronic acid in step (1) is 10-2500 kDa, preferably 200-1000 kDa, more preferably 400-600 kDa; Preferably, the solvent for dissolving hyaluronic acid in step (1) is water, physiological saline or PBS buffer; Preferably, the mass concentration of the hyaluronic acid after dissolution in step (1) is 0.1-20%, preferably 0.5-4%; Preferably, the molar ratio of sodium periodate to hyaluronic acid in step (1) is 0.1-100:1, preferably 1-10:

1.

6. The preparation method according to claim 4, characterized in that: The molar ratio of sodium periodate to ethylene glycol is 1:10-1000, preferably 1:20-200; preferably, the molar ratio of EDCI to hyaluronic acid is 0.1-100:1, preferably 1-10:1; preferably, the molar ratio of the amine compound or hydrazide compound to hyaluronic acid is 2-1000:1, preferably 5-40:

1.

7. The preparation method according to claim 4, characterized in that: In step (4), the amine compound is an organic compound containing two or more amine groups, and the hydrazide compound is an organic compound containing two or more hydrazines; Preferably, the amine compound is ethylenediamine, propylenediamine, butylenediamine, p-phenylenediamine or m-phenylenetriamine, and the hydrazide compound is oxalic acid dihydrazide, malonic acid dihydrazide, succinic acid dihydrazide, terephthalic acid dihydrazide, isophthalic acid dihydrazide or isophthalic acid trihydrazide.

8. A hydrogel prepared from the bifunctionalized pyran ring derivative according to any one of claims 1 to 3.

9. The method for preparing the hydrogel according to claim 8, comprising: The bifunctionalized pyran ring derivative according to any one of claims 1 to 3 is crushed, dissolved in water, and stirred to obtain the hydrogel.

10. Use of the bifunctionalized pyran ring derivative according to any one of claims 1 to 3 or the hydrogel according to claim 8 or 9 in the fields of biomedicine, medical cosmetology, etc.

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