Application of diaminodiketopiperazine compound

Abstract

The invention relates to the technical field of biological polysaccharide cross-linking agents, in particular to application of a diaminodiketopiperazine compound, and the diaminodiketopiperazine compound is used as a cross-linking agent in the preparation process of cross-linked biological polysaccharide gel. The diaminodiketopiperazine compound is a compound with a structure shown as a formula I or a pharmaceutically acceptable salt thereof: # imgabs0 #, wherein R is normal alkyl or isoalkyl with the carbon atom number smaller than or equal to 10; the preparation process of the cross-linked biological polysaccharide gel comprises the following steps: dissolving and uniformly mixing biological polysaccharide and the diaminodiketopiperazine compound in water or a buffer solution, then adding a condensing agent to form a reaction solution, and carrying out a cross-linking reaction to obtain the cross-linked biological polysaccharide gel, according to the invention, the diaminodiketopiperazine compound is used as a cross-linking agent to cross-link the biological polysaccharide, so that the biological polysaccharide gel has higher cell survival rate and better biological safety, and the prepared gel has better viscoelasticity and thermal stability and can tolerate moist heat sterilization.

Description

Technical Field

[0001] The invention relates to the technical field of biopolysaccharide crosslinking agents, and in particular to application of a diaminodiketopiperazine compound. Background Art

[0002] Hydrogel is a material with a three-dimensional network structure that can absorb and retain a large amount of water. They are widely used in biomedicine, environmental protection, food industry and other fields. Among them, biopolysaccharide hydrogel has become a hot topic of research due to its good biocompatibility, biodegradability and low toxicity. Sodium hyaluronate, sodium alginate and other commonly used biopolysaccharides form cross-linked hydrogels with specific properties and network structures through the auxiliary effect of cross-linking agents.

[0003] Cross-linking agents play a bridging role in the formation of hydrogels. They can form chemical bonds between polysaccharide molecular chains, thereby constructing a stable three-dimensional network structure. The type and properties of cross-linking agents directly affect the physical and chemical properties of hydrogels.

[0004] Commonly used cross-linking agents or cross-linking methods for biopolysaccharides are as follows: (1) Boric acid and its derivatives: They can cross-link with biopolysaccharides containing cis-diol structures, but may be toxic to cells, and the cross-linking speed and strength are greatly affected by pH. (2) Metal ions: For example, calcium ions (Ca 2+ ), aluminum ions (Al 3+ ) are commonly used to crosslink anionic polysaccharides such as sodium alginate, but the crosslinking process may be irreversible and high concentrations of metal ions may be toxic to cells. (3) Polyphenolic compounds: such as gallic acid and tannic acid, which form hydrogen bonds or covalent bonds with the hydroxyl groups of polysaccharides through phenolic hydroxyl groups, but the crosslinking conditions may be harsh and may affect the biocompatibility of hydrogels. (4) Enzymes: such as glutaminase, which are often used to catalyze the formation of covalent bonds between polysaccharide chains, but they usually have high costs and the crosslinking process may be difficult to control. (5) Ultraviolet irradiation: Free radical crosslinking is induced by photoinitiators, but it may have a destructive effect on biological molecules and the crosslinking depth is limited. (6) Chemical crosslinkers: such as glutaraldehyde, genipin, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), polyethylene glycol diglycidyl ether (PEGDE), etc. These crosslinkers form covalent bonds through chemical reactions. These crosslinkers contain chemically active structural units such as epoxy or double bonds, which can covalently bind to functional proteins and nucleic acids in the body, thus affecting their normal functions. They have high potential cytotoxicity and poor biocompatibility. In addition, the residues of these crosslinkers themselves or their degradation products can also cause inflammatory reactions in the body, have certain cytotoxicity, and the crosslinking process may release harmful byproducts.

[0005] Therefore, selecting a suitable cross-linker is a key step in preparing high-performance hydrogels. Summary of the invention

[0006] In order to solve the technical problem that the cross-linking agent for cross-linking biological polysaccharides in the prior art usually has certain cytotoxicity and poor biocompatibility, a use of a diaminodiketopiperazine compound is provided. The present invention synthesizes a diaminodiketopiperazine compound by a method of cyclization condensation of trifluoroacetyl-protected amino acids and then deprotection of the amino groups. The compound has the advantages of low cytotoxicity and high safety when used as a cross-linking agent for cross-linking biological polysaccharides, and the obtained cross-linked gel has a high modulus and can withstand wet heat sterilization, ensuring the technical requirements of terminal application products and application safety.

[0007] In order to achieve the above objectives, the present invention is implemented by the following technical solutions:

[0008] A use of a diaminodiketopiperazine compound, wherein the diaminodiketopiperazine compound is used as a cross-linking agent in the preparation process of a cross-linked biopolysaccharide gel;

[0009] The diaminodiketopiperazine compound is a compound of the following formula I or a pharmaceutically acceptable salt thereof:

[0010]

[0011] wherein R is a normal alkyl group or an isomeric alkyl group having 10 or less carbon atoms.

[0012] Furthermore, R is a normal alkyl group or an isomeric alkyl group having 5 or less carbon atoms.

[0013] Furthermore, the diaminodiketopiperazine compound is selected from one or more of 3,6-di(4-aminobutyl)-2,5-diketopiperazine or its salt, 3,6-di(3-aminopropyl)-2,5-diketopiperazine or its salt, and the pharmaceutically acceptable salt is selected from one of hydrochloride, sulfate and nitrate.

[0014] Furthermore, the method for obtaining the diaminodiketopiperazine compound comprises the following steps:

[0015]

[0016] S1. Condensation cyclization: Two molecules of trifluoroacetyl-protected amino acid derivatives are dehydrated under the action of the condensate to form a molecule of cyclization intermediate;

[0017] S2, deprotection: deprotecting the cyclization intermediate by aminolysis to obtain a free diaminodiketopiperazine compound.

[0018] Furthermore, the method for obtaining the diaminodiketopiperazine compound specifically comprises the following steps:

[0019] S1, uniformly dissolving the trifluoroacetyl protected amino acid derivative in a solvent, adding phosphorus pentoxide condensate, under the action of which the trifluoroacetyl protected amino acid derivative undergoes dehydration, condensation and cyclization to form a cyclization intermediate, and precipitating with pure water to obtain the cyclization intermediate. The precipitation process may be to add the reaction system to water to precipitate, or to drop pure water into the reaction system to slowly precipitate the cyclization intermediate in the form of crystals. The latter precipitation process can improve the purity of the cyclization intermediate;

[0020] S2. Add the cyclized intermediate into aqueous ammonia, stir at 20°C to 50°C to carry out a deprotection reaction (react until the solution is almost clear), then filter while hot, and concentrate the filtrate under reduced pressure to obtain a yellow oil. Use an organic solvent to slurry the yellow oil at 10°C to 60°C for 1h to 2h, filter and dry to obtain a free diaminodiketopiperazine compound.

[0021] Preferably, the trifluoroacetyl-protected amino acid derivative is selected from trifluoroacetyl lysine or trifluoroacetyl ornithine;

[0022] The solvent is selected from N-methylpyrrolidone or dimethyl sulfoxide;

[0023] The reaction temperature of the condensation cyclization is in the range of 150°C-170°C, and the reaction time is in the range of 1h-3h;

[0024] The precipitation process is: adjusting the temperature of the reaction system to 80°C-90°C, adding pure water dropwise while maintaining the temperature, cooling to 20°C-30°C after the addition is complete for crystallization, and obtaining the cyclized intermediate through suction filtration, water rinsing, and reduced pressure drying.

[0025] Preferably, the trifluoroacetyl-protected amino acid derivative, the phosphorus pentoxide condensate, and the solvent are used in a ratio within the range of 0.4-0.6 mol: 0.11-0.15 mol: 180-220 mL.

[0026] Preferably, the mass percentage of the aqueous ammonia is in the range of 2%-25%; the time of the deprotection reaction is in the range of 18h-25h; the amount of the aqueous ammonia is 1.2 to 1.3 times the volume of the solvent; and the diaminodiketopiperazine compound is crystallized and purified using water and / or tetrahydrofuran as needed.

[0027] Furthermore, the method for obtaining the diaminodiketopiperazine compound further comprises the following step S3: subjecting the diaminodiketopiperazine compound to a salt-forming reaction to obtain a diaminodiketopiperazine compound hydrochloride:

[0028]

[0029] Furthermore, the S3 specifically includes the following operation process:

[0030] The diaminodiketopiperazine compound is dissolved in a dilute hydrochloric acid solution and filtered, tetrahydrofuran is added dropwise to the obtained filtrate at 40°C to 50°C, and after the addition is completed, the temperature is lowered for crystallization, and the mixture is filtered, washed, and dried to obtain the diaminodiketopiperazine compound hydrochloride.

[0031] Preferably, the molar concentration of the dilute hydrochloric acid solution is 1 M, wherein the amount of dilute hydrochloric acid is 0.10 to 0.15 times the molar amount of the trifluoroacetyl-protected amino acid derivative; the temperature of the cooling crystallization is 20° C. to 25° C., and the volume amount of the tetrahydrofuran added dropwise to the filtrate is 10 to 20 times the volume amount of the dilute hydrochloric acid solution.

[0032] Furthermore, the preparation process of the cross-linked biopolysaccharide gel includes: after the biopolysaccharide and the diaminodiketopiperazine compound are dissolved and mixed evenly in water or a buffer solution, one of them can be dissolved first and then the other can be added, or both can be dissolved evenly and then combined, the purpose is to dissolve and mix both evenly, the process does not matter, and then a condensation agent is added to form a reaction solution, and a cross-linking reaction is carried out to obtain a cross-linked biopolysaccharide gel.

[0033] Furthermore, the dosage of the diaminodiketopiperazine compound is 1%-20% of the molar amount of the biopolysaccharide; the temperature of the cross-linking reaction is in the range of 0°C-30°C, and the time is in the range of 10h-80h.

[0034] Furthermore, the biopolysaccharide is selected from one or more of hyaluronic acid or its salts, alginate or its salts, carboxymethyl chitin or its salts, carboxymethyl chitosan or its salts, carboxymethyl cellulose or its salts, and chondroitin compounds, and the mass fraction of the biopolysaccharide in the reaction solution is 0.5%-10%.

[0035] In addition, the buffer is selected from one of a phosphate buffer system and a phosphate buffer system / sodium chloride solution, and the pH of the buffer is in the range of 6.0-8.5; the condensing agent is a conventional condensing agent, such as 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine tetrafluoroborate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, 2-chloro-1-methylpyridinium iodide, etc., and the amount of the condensing agent is the conventional technical amount. For related cross-linked biopolysaccharide gels, please refer to the relevant patent of the applicant with application number 202411477353.0.

[0036] Beneficial technical effects:

[0037] The present invention is an invention of the use of diaminodiketopiperazine compounds, which are used as crosslinking agents to prepare crosslinked biopolysaccharide gels. The diaminodiketopiperazine compounds of the present invention have extremely low cytotoxicity as crosslinking agents and are highly safe. The obtained gel has high viscoelasticity, low impurity content, uniform particles, improved thermal stability and resistance to enzyme degradation, and can withstand wet heat sterilization. The product has high safety and can reduce the frequency of injections in clinical use.

[0038] The present invention uses trifluoroacetyl protected amino acid derivatives as starting materials and obtains diaminodiketopiperazine compounds through condensation cyclization and deprotection. The obtaining method is simple and efficient and suitable for process scale-up production.

[0039] The present invention proves for the first time the biological safety of using diaminodiketopiperazine compounds as crosslinking agents, so that they can be used in the preparation of biomedical gels such as medical filling, surgical adhesion prevention and bone joint cavity injection;

[0040] The diaminodiketopiperazine compound crosslinking agent of the present invention is covalently bonded to the carboxyl group of the biopolysaccharide through an amide bond. The reactivity of the amino group in the crosslinking agent is much higher than the reactivity of the hydroxyl group of the biopolysaccharide itself, which can improve the crosslinking efficiency and avoid self-crosslinking within or between biopolysaccharide molecular chains.

[0041] The diaminodiketopiperazine compound or a pharmaceutically acceptable salt thereof provided by the present invention has excellent water solubility, can ensure sufficient dissolution and distribution in water during the cross-linking reaction, and can completely remove the unreacted cross-linking agent from the product through a rinsing process during post-treatment, and has better biological safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a diagram of the intradermal reaction test after intradermal injection of compound a. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. The technology and method known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology and method should be considered as a part of the specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values.

[0045] The experimental methods in the following examples without specifying specific conditions are usually measured in accordance with national standards; if there is no corresponding national standard, they are carried out in accordance with general standard requirements or general methods.

[0046] The following abbreviations h or hr all represent the time unit - hour.

[0047] Example 1

[0048] This case is the preparation of the cross-linking agent 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride:

[0049] S1, take 2-amino-6-(trifluoroacetylamino) hexanoic acid (i.e. trifluoroacetyl lysine, CAS10009-20-8) 100g, phosphorus pentoxide 17.5g, N-methyl pyrrolidone 206mL and place them in a reaction vessel, carry out condensation cyclization reaction at 165°C for 2 hours, adjust the temperature of the reaction system to 80°C and add pure water, after the water is added, cool to 20°C for crystallization, filter, rinse with water, and dry under reduced pressure to obtain a cyclized intermediate;

[0050] S2, then placing the cyclized intermediate in 270 mL of aqueous ammonia (mass fraction 19%), stirring at 50° C. for detrifluoroacetylating reaction for 22 hours, then filtering while hot, and concentrating the filtrate under reduced pressure to obtain a yellow oily substance, and beating the yellow oily substance with tetrahydrofuran at 50° C. for 1 hour, filtering with suction, and drying to obtain a crude free 3,6-di(4-aminobutyl)-2,5-diketopiperazine;

[0051] S3, the crude product was dissolved in 60 mL of 1M dilute hydrochloric acid at 30°C, the filtrate was filtered and 600 mL of tetrahydrofuran was added dropwise at 42°C, after the addition was completed, the temperature was lowered to 20°C for crystallization, suction filtered, tetrahydrofuran rinsed, and dried to obtain 30 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride (molecular weight 329.27 g / mol), HPLC purity 99%. The product was tested by carbon and hydrogen nuclear magnetic resonance spectra, and the results were as follows:

[0052] 13 CNMR(101MHz,D2O)δ:170.28,53.98,39.16,31.76,26.41,20.17;

[0053] 1 HNMR(400MHz,D2O)δ:4.25(t,2H),3.01(t,4H),1.99-1.77(m,4H),1.74-1.62(m,4H),1.54-1.28(m,4H).

[0054] Example 2

[0055] This case is the preparation of the cross-linking agent 3,6-bis(3-aminopropyl)-2,5-diketopiperazine hydrochloride:

[0056] S1, take 2-amino-5-(trifluoroacetylamino) valeric acid (i.e. trifluoroacetyl ornithine, CAS 5123-49-9) 100g, phosphorus pentoxide 17.2g, N-methylpyrrolidone 200mL and place them in a reaction container, carry out condensation cyclization reaction at 165°C for 2 hours, adjust the temperature of the reaction system to 90°C and add pure water, after the water is added, cool to 30°C for crystallization, filter, rinse with water, and dry under reduced pressure to obtain a cyclized intermediate;

[0057] S2, then placing the cyclized intermediate in 250 mL of aqueous ammonia (mass fraction 19%), stirring at 50° C. for 20 hours to remove the trifluoroacetyl protecting group, then filtering while hot, and concentrating the filtrate under reduced pressure to obtain a yellow oily substance, and beating the yellow oily substance with tetrahydrofuran at 30° C. for 2 hours, filtering with suction, and drying to obtain a crude product of 3,6-bis(3-aminopropyl)-2,5-diketopiperazine;

[0058] S3 The crude product was dissolved in 60 mL of 1M dilute hydrochloric acid at 30°C, the filtrate was filtered, and 600 mL of tetrahydrofuran was added dropwise at 48°C. After the addition was completed, the temperature was lowered to 25°C for crystallization, and suction filtration, tetrahydrofuran elution, and drying were performed to obtain 25 g of 3,6-bis(3-aminopropyl)-2,5-diketopiperazine hydrochloride (molecular weight 301.21 g / mol), with an HPLC purity of 99%. The product was tested by nuclear magnetic resonance hydrogen spectrum, and the results were as follows:

[0059] 1 HNMR(400MHz,D2O)δ:4.17(t,2H),3.05(t,4H),1.96-1.71(m,4H),1.78-1.65(m,4H).

[0060] Example 3

[0061] This case is a cytotoxicity test on the products of Example 1 and Example 2.

[0062] 3,6-bis(4-aminobutyl)-2,5-diketopiperazine hydrochloride (hereinafter referred to as compound a) and 3,6-bis(3-aminopropyl)-2,5-diketopiperazine hydrochloride (hereinafter referred to as compound b) were respectively prepared into samples with different crosslinker concentrations as shown in Table 1 below (the solvent used was phosphate buffer solution), and incubated with different cells for a certain period of time, and then cell viability was detected. Specifically: When the cells grow to a good exponential growth phase, live cells are counted using a cell counter. The cell suspension is adjusted to an appropriate cell concentration with the corresponding culture medium. 100 μL of cell suspension was added to each well of a 96-well cell culture plate, 5000 cells / well, 3 replicates, and cultured overnight (about 24 hours) in a 37°C, 5% CO2 incubator. After the cells adhered to the wall the next day, the supernatant of the culture medium was gently aspirated, and 120 μL of different concentrations of the test compound prepared with the culture medium was added to each group. After incubation for a certain period of time, cell viability was detected.

[0063] Cell viability was determined by chemiluminescence to measure the ATP content to reflect the number of living cells. According to the operating instructions of the kit, each well was added with a reagent that had been melted and equilibrated to room temperature (equilibrium time was about 30 minutes), and the mixture was shaken at room temperature for 2 minutes to fully induce cell lysis. The fluorescence signal value was measured using a multifunctional microplate reader.

[0064] Calculation of cell survival rate: Cell survival rate = each experimental well (RLU value) / blank control group (average RLU value) × 100%.

[0065] The results are shown in Table 1.

[0066] Table 1 Cytotoxicity results of cross-linking agents

[0067]

[0068] As can be seen from Table 1, under experimental conditions far exceeding the crosslinker concentration used in gel preparations, both crosslinkers of the present invention did not show significant inhibitory effects on the proliferation of the four cell types, that is, the diaminodiketopiperazine compounds of the present invention have the advantages of low cytotoxicity and high safety as crosslinkers.

[0069] Example 4

[0070] In this case, New Zealand rabbits were used for intradermal reaction test, and normal saline was used as control to investigate the non-specific acute irritation of the skin to the local area of ​​the skin and the biocompatibility of the material after injection of the cross-linking agent compound a at concentrations of 0.8 mg / mL and 1.6 mg / mL. Figure 1 As shown. Figure 1 The experiment showed that no erythema or other irritation was observed after intradermal injection of compound a at 0 hr (i.e., immediately after injection), 1 hr, and 24 hr after injection, and there was no significant difference from the area injected with normal saline. This indicates that the two diaminodiketopiperazine compounds of the present invention are highly safe and can be used as cross-linking agents to prepare cross-linked polysaccharide gels, thereby preparing medical injection materials.

[0071] Application Example 1

[0072] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0073] S1. Weigh 8.0172 g of sodium hyaluronate (molecular weight 0.8 MDa), add 171.74 g of phosphate buffer system / sodium chloride solution (pH=6.74), and stir and mix for 24 h;

[0074] Weigh 0.6598 g of 3,6-bis(3-aminopropyl)-2,5-diketopiperazine hydrochloride of Example 2 as a crosslinking agent, add phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.0056 g, and dissolve evenly;

[0075] The two were stirred and mixed for 30 min to form a mixed solution;

[0076] S2, weighing 1.4864 g of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, adding a phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.0539 g, and dissolving evenly to form a condensing agent solution; filtering the condensing agent solution, adding it to the mixed solution, and stirring for about 30 minutes to form a reaction solution, and then stirring at 25° C. for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0077] S3, take 100.80 g of the reaction product after the cross-linking reaction of S2, add phosphate buffer system / sodium chloride solution (pH=6.74) to the reaction product to make the total weight 611.42 g, stir and wash for 24 hours; filter through a mesh, drain and crush the retained gel to obtain the product, and detect the elastic modulus G' and viscous modulus G" of the product at a frequency of 2.5 Hz, and calculate the swelling ratio and the final concentration of the product;

[0078] The obtained product was packed into multiple vials, stoppered and capped, and sterilized by wet heat sterilization at 121°C for 15 min, 20 min, and 30 min, respectively. The elastic modulus G' and viscous modulus G" of the product after sterilization at a frequency of 2.5 Hz were tested.

[0079] Application Example 2

[0080] This case is a method for preparing a highly viscoelastic cross-linked biopolysaccharide gel, which specifically includes the following steps:

[0081] S1. Weigh 10.0011 g of sodium hyaluronate (molecular weight 1.2 MDa), add 169.21 g of phosphate buffer system / sodium chloride solution (pH=6.74), and stir and mix for 24 h;

[0082] Weigh 0.6598 g of 3,6-di(4-aminobutyl)-2,5-diketopiperazine hydrochloride of Example 1 as a crosslinking agent, add phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.0080 g, and dissolve evenly;

[0083] The two were stirred and mixed for 30 min to form a mixed solution;

[0084] S2, weighing 1.2452 g of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride monohydrate, adding a phosphate buffer system / sodium chloride solution (pH=6.74) to a total weight of 10.1350 g, and dissolving evenly to form a condensing agent solution; filtering the condensing agent solution, adding it to the mixed solution, and stirring for about 30 minutes to mix to form a reaction solution, and then stirring at 10° C. for 24 hours to perform a cross-linking reaction to obtain a reaction product, a highly viscoelastic cross-linked biopolysaccharide gel;

[0085] S3, taking 100.86 g of the reaction product after the cross-linking reaction of S2, adding phosphate buffer system / sodium chloride solution (pH=6.74) to the total weight of 707.25 g, stirring and washing for 96 hours; filtering through a mesh, draining and crushing the trapped gel to obtain a product, and detecting the elastic modulus G' and viscous modulus G" of the product at a frequency of 2.5 Hz, calculating the swelling ratio and the final concentration of the product;

[0086] The obtained product was packed into multiple vials, stoppered and capped, and sterilized by wet heat sterilization at 121°C for 15 min, 20 min, and 30 min, respectively. The elastic modulus G' and viscous modulus G" of the product after sterilization at a frequency of 2.5 Hz were tested.

[0087] The properties of the cross-linked biopolysaccharide gel obtained in the application example are shown in Table 2 below.

[0088] Table 2 Properties of cross-linked biopolysaccharide gels prepared in application examples

[0089]

[0090] (Note: Commercially available I is Hyruan ONE, cross-linked sodium hyaluronate with butylene glycol, manufactured by LG Chem, Ltd., with batch number SIH22068; commercially available II is Synvisc, manufactured by Genzyme Corporation, with batch number CRSP006B; commercially available III is Haimei, manufactured by Shanghai Qisheng Biological Products Co., Ltd., with batch number Q2305QS11; sodium hyaluronate concentration refers to the mass fraction of sodium hyaluronate in the reaction solution; the amount of cross-linking agent refers to the percentage of the molar amount of the cross-linking agent to the molar amount of the sodium hyaluronate repeating unit; the swelling ratio is the ratio of the increased weight of the gel after swelling to the original weight; the final concentration of the product is calculated based on the sodium hyaluronate content)

[0091] It can be seen from the above application cases that the cross-linked biopolysaccharide gel prepared by using the two diaminodiketopiperazine compounds prepared in Example 1 and Example 2 as cross-linking agents has an elastic modulus before sterilization that is three times that of commercially available products, and a viscous modulus that is four times that of commercially available products, and still has a high elastic modulus and viscous modulus after wet heat sterilization. After wet heat sterilization, the elastic modulus exceeds 300Pa, and the viscous modulus exceeds 50Pa. The cross-linked biopolysaccharide gel prepared by using the two diaminodiketopiperazine compounds as cross-linking agents in the present invention can withstand wet heat sterilization at 121°C for 15min or even longer while ensuring the viscoelastic properties of the product, thereby ensuring the application safety of the terminal product.

[0092] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A use of a diaminodiketopiperazine compound, characterized in that: The diaminodiketopiperazine compound is used as a cross-linking agent in the preparation process of cross-linked biopolysaccharide gel; The diaminodiketopiperazine compound is a compound of the following formula I or a pharmaceutically acceptable salt thereof: wherein R is a normal alkyl group or an isomeric alkyl group having 10 or less carbon atoms.

2. The use of a diaminodiketopiperazine compound according to claim 1, characterized in that: R is a normal alkyl group or an isomeric alkyl group having 5 or less carbon atoms.

3. The use of a diaminodiketopiperazine compound according to claim 2, characterized in that: The diaminodiketopiperazine compound is selected from one or more of 3,6-di(4-aminobutyl)-2,5-diketopiperazine or its salt, 3,6-di(3-aminopropyl)-2,5-diketopiperazine or its salt, and the pharmaceutically acceptable salt is selected from one of hydrochloride, sulfate and nitrate.

4. The use of a diaminodiketopiperazine compound according to claim 3, characterized in that: The method for obtaining the diaminodiketopiperazine compound comprises the following steps: S1. Condensation cyclization: Two molecules of trifluoroacetyl-protected amino acid derivatives are dehydrated under the action of the condensate to form a molecule of cyclization intermediate; S2, deprotection: deprotecting the cyclization intermediate by aminolysis to obtain a free diaminodiketopiperazine compound.

5. The use of a diaminodiketopiperazine compound according to claim 4, characterized in that: The method for obtaining the diaminodiketopiperazine compound specifically comprises the following steps: S1, uniformly dissolving the trifluoroacetyl protected amino acid derivative in a solvent, adding phosphorus pentoxide condensate, under the action of which the trifluoroacetyl protected amino acid derivative undergoes dehydration, condensation and cyclization to form a cyclization intermediate, and precipitating with pure water to obtain the cyclization intermediate; S2. Add the cyclized intermediate into aqueous ammonia, stir at 20°C to 50°C to carry out a deprotection reaction, then filter while hot, and concentrate the filtrate under reduced pressure to obtain a yellow oily substance. Use an organic solvent to slurry the yellow oily substance at 10°C to 60°C for 1h to 2h, filter and dry to obtain a free diaminodiketopiperazine compound.

6. The use of a diaminodiketopiperazine compound according to claim 5, characterized in that: The trifluoroacetyl-protected amino acid derivative is selected from trifluoroacetyl lysine or trifluoroacetyl ornithine; The solvent is selected from N-methylpyrrolidone or dimethyl sulfoxide; The reaction temperature of the condensation cyclization is in the range of 150°C-170°C, and the reaction time is in the range of 1h-3h; The trifluoroacetyl protected amino acid derivative, the phosphorus pentoxide condensate, and the solvent are used in a ratio of 0.4-0.6 mol: 0.11-0.15 mol: 180-220 mL; The mass percentage of the ammonia water is in the range of 2%-25%; the time of the deprotection reaction is in the range of 18h-25h; the amount of the ammonia water used is 1.2 to 1.3 times the volume of the solvent.

7. The use of a diaminodiketopiperazine compound according to claim 5, characterized in that: The obtaining method specifically further comprises the following step S3: subjecting the diaminodiketopiperazine compound to a salt-forming reaction to obtain a diaminodiketopiperazine compound hydrochloride: Specifically, the diaminodiketopiperazine compound is dissolved in a dilute hydrochloric acid solution and filtered, tetrahydrofuran is added dropwise to the obtained filtrate at 40° C. to 50° C., the temperature is lowered after the addition is completed, crystallization is performed, the mixture is filtered, washed, and dried to obtain the diaminodiketopiperazine compound hydrochloride.

8. The use of a diaminodiketopiperazine compound according to any one of claims 1 to 7, characterized in that: The preparation process of the cross-linked biopolysaccharide gel comprises: dissolving and mixing the biopolysaccharide and the diaminodiketopiperazine compound in water or a buffer solution, adding a condensing agent to form a reaction solution, and performing a cross-linking reaction to obtain the cross-linked biopolysaccharide gel.

9. The use of a diaminodiketopiperazine compound according to claim 8, characterized in that: The dosage of the diaminodiketopiperazine compound is 1%-20% of the molar amount of the biopolysaccharide; the temperature of the cross-linking reaction is in the range of 0°C-30°C, and the time is in the range of 10h-80h.

10. The use of a diaminodiketopiperazine compound according to claim 8, characterized in that: The biopolysaccharide is selected from one or more of hyaluronic acid or its salt, alginic acid or its salt, carboxymethyl chitin or its salt, carboxymethyl chitosan or its salt, carboxymethyl cellulose or its salt, and chondroitin compounds, and the mass fraction of the biopolysaccharide in the reaction solution is 0.5%-10%.

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