Benzene sulfonic acid or benzene sulfonic acid derivative-guanidine eutectic crystal and preparation method and application thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic crystal as well as a preparation method and application thereof. The benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is a benzenesulfonic acid or benzenesulfonic acid derivative and guanidine eutectic, and the molar ratio of benzenesulfonic acid or benzenesulfonic acid derivative to guanidine is 1: (1-2). According to the invention, a proper benzenesulfonic acid derivative is screened and is taken as a ligand to be combined with guanidine, so that a series of benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic crystals are successfully synthesized. The eutectic can promote proliferation and migration of fibroblasts, vascular endothelial cells and keratinocytes in skin, reduce apoptosis of skin peripheral nerves and promote growth of axons so as to realize rapid healing of diabetic wounds. Meanwhile, after benzenesulfonic acid and derivatives thereof form eutectic crystals with guanidine, slow release of guanidine ions can be realized, and potential toxicity of guanidine is reduced while the drug effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to a benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic, its preparation method, and its application. Background Technology

[0002] Guanidine compounds are a class of compounds containing a guanidine group (-CN3H4), widely found in biomolecules such as arginine and creatine, and can also be synthesized artificially. In wound healing, polyhexamethylene guanidine (PHMB) modified dressings have shown outstanding efficacy. In vitro experiments have confirmed that their antibacterial rate remains >99.99%, and they retain activity even after 50 washes or 7 days of ultraviolet irradiation. Animal experiments have shown that they can significantly promote platelet expression and coagulation function, and accelerate the growth of granulation tissue and collagen deposition in infected wounds. Other studies have shown that supplementing trauma rats with a diet containing a compound of arginine (a natural guanidine compound) and glycine significantly increases nitrogen retention and muscle creatine content, providing a key material basis for tissue repair. In the field of nerve repair, guanidine compounds exert their effects through multiple mechanisms: arginine, as an important nutrient for nerve cells, has a guanidine group that participates in neurotransmitter synthesis and energy metabolism regulation, while artificially synthesized guanidine derivatives can inhibit nerve cell apoptosis. Related studies show that guanidine-modified materials can improve the neural cell microenvironment and promote the proliferation of neural stem cells, providing a potential solution for the repair of spinal cord injury and peripheral neuropathy. Their mechanism of action is closely related to regulating cellular energy metabolism and inhibiting oxidative stress.

[0003] However, the application of guanidine compounds is limited by their inherent characteristics, with core drawbacks concentrated in four main areas: hygroscopicity, stability, release rate, and strong alkalinity. Strong alkalinity is their primary weakness. With a pKa as high as 13.8, the guanidine group carries a persistent positive charge in physiological environments. High alkalinity easily irritates mucous membranes and wound tissues, triggering local inflammatory responses and even disrupting cell membrane integrity, reducing biocompatibility and limiting their concentration for direct application to skin wounds. Hygroscopicity stems from the strong polarity and charge characteristics of the guanidine group. Solid guanidine compounds readily absorb moisture from the air, leading to clumping and reduced fluidity. Medical dressings and other materials are prone to deformation after absorbing water, and the active ingredients may be diluted, reducing antibacterial or repairing activity. Furthermore, they require sealed, moisture-proof storage, increasing transportation and preservation costs. Insufficient stability is equally prominent. Hygroscopicity exacerbates the risk of hydrolysis, especially in high-temperature, high-humidity, or acid-base imbalance environments. Commonly used derivatives such as polyhexamethylene guanidine may experience chain breakage, resulting in a decrease in antibacterial rate of over 30%. Simultaneously, their strong alkalinity easily reacts with acidic drugs or materials, creating incompatibilities and further limiting their application scenarios. Most guanidines are highly water-soluble and readily release rapidly into body fluids. Initially, excessively high local concentrations may exacerbate tissue irritation, while a sudden drop in concentration later can affect the sustainability of therapeutic effects. Release regulation requires complex processes such as carrier modification, increasing formulation development costs and technical barriers. Therefore, there is an urgent need to improve these drawbacks of guanidine compounds. Summary of the Invention

[0004] The first objective of this invention is to provide a benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic, the second objective of this invention is to provide a method for preparing the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic, and the third objective of this invention is to provide applications of the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic.

[0005] According to a first aspect of the present invention, a benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is provided, wherein the eutectic is a eutectic of benzenesulfonic acid or benzenesulfonic acid derivative and guanidine, wherein the molar ratio of benzenesulfonic acid or benzenesulfonic acid derivative to guanidine is 1:(1-2); the benzenesulfonic acid derivative is 4,4'-biphenyl disulfonic acid, biphenyl-4-sulfonic acid, 3-methylbenzenesulfonic acid, or 2-naphthalenesulfonic acid; Eutectic includes eutectic single crystals and eutectic powders. Eutectic single crystals include GD eutectic single crystals, GS eutectic single crystals, GM eutectic single crystals, GN eutectic single crystals, and GA eutectic single crystals; eutectic powders include GD eutectic powders, GS eutectic powders, GM eutectic powders, GN eutectic powders, and GA eutectic powders. The GD eutectic single crystal is a monoclinic crystal with space group P121 / c1. Its cell parameters are: a = 11.9776(2) Å, b = 26.8094(4) Å, c = 7.38700(10) Å, α = 90°, β = 92.5160(10)°, γ = 90°, Z = 4, and its cell volume is 2369.77(6) Å. 3 ; The GS eutectic single crystal is a monoclinic crystal, space group I121, with the following cell parameters: a = 10.31050(10) Å, b = 8.12400(10) Å, c = 32.2188(3) Å, α = 90°, β = 99.1630(10)°, γ = 90°, Z = 8, and a cell volume of 2664.29(5) Å. 3 ; The GM eutectic single crystal is orthorhombic, space group -P2ac2n, with the following cell parameters: a = 17.3249(4) Å, b = 7.5177(2) Å, c = 8.5106(2) Å, α = 90°, β = 90°, γ = 90°, Z = 5, and a cell volume of 1108.45(5) Å. 3 ; The GN eutectic single crystal is triclinic, space group P-1, with the following cell parameters: a = 7.44530(10) Å, b = 12.0008(2) Å, c = 27.9597(7) Å, α = 88.117(2)°, β = 85.677(2)°, γ = 88.4400(10)°, Z = 8, and a cell volume of 2488.97(8) Å. 3 ; The GA eutectic single crystal is monoclinic, space group P121 / c1, with the following cell parameters: a = 7.4289(2) Å, b = 22.8661(7) Å, c = 11.9938(3) Å, α = 90°, β = 92.324(3)°, γ = 90°, Z = 8, and a cell volume of 2035.71(10) Å. 3 ; The X-ray diffraction pattern of GD eutectic powder shows characteristic peaks at 10θ angles of 15.64±0.66°, 17.64±0.76°, and 24.64±0.86°. The X-ray diffraction pattern of the GS eutectic powder shows characteristic peaks at 10θ angles of 16.09±0.91°, 19.01±1.50°, and 26.58±0.53°. The X-ray diffraction pattern of the GM eutectic powder shows characteristic peaks at 10θ angles of 17.16±1.00°, 22.30±1.30°, and 27.97±0.62°. The X-ray diffraction pattern of the GN eutectic powder shows characteristic peaks at 10θ angles of 22.91±2.45°, 33.57±2.84°, and 42.91±1.77°. The X-ray diffraction pattern of the GA eutectic powder shows characteristic peaks at 10θ angles of 25.36±3.99°, 34.95±3.52°, and 44.06±1.31°.

[0006] According to a second aspect of the present invention, a method for preparing the above-mentioned benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is provided. When the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is a eutectic single crystal, the preparation method includes the following steps: Guanidine hydrochloride and benzenesulfonic acid or benzenesulfonic acid derivatives are dissolved in a solution composed of methanol and water, and then placed at 4°C for 24-72 hours. The benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic single crystal is obtained by solvent evaporation.

[0007] In some embodiments, the mass ratio of benzenesulfonic acid or a benzenesulfonic acid derivative to guanidine hydrochloride is 1:(1-2). In some implementations, the volume ratio of methanol to water is 1:1; In some embodiments, the ratio of the mass (mg) of guanidine hydrochloride to the volume (mL) of the solution is (5-20):10.

[0008] According to a third aspect of the present invention, a method for preparing the above-mentioned benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is provided. When the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is a eutectic powder, the preparation method includes the following steps: A guanidine hydrochloride aqueous solution is mixed with a methanol solution of benzenesulfonic acid or a benzenesulfonic acid derivative, stirred at 800-1000 rpm for 15-30 min, and then sonicated at 80-120 W for 20-40 min to obtain a pale yellow precipitate. The precipitate is then washed and dried to obtain a white powder, which is the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic powder.

[0009] In some embodiments, the pale yellow precipitate is washed three times with a solution of methanol and water pre-cooled at 4°C in a 1:1 volume ratio. This washing removes ligands that have not formed single crystals.

[0010] In some embodiments, the concentration of the guanidine hydrochloride aqueous solution is 1-1.5 mg / mL, the concentration of the benzenesulfonic acid or benzenesulfonic acid derivative methanol solution is 4.5 mg / mL, and the volume ratio of the guanidine hydrochloride aqueous solution to the benzenesulfonic acid or benzenesulfonic acid derivative methanol solution is 1:2.

[0011] According to a fourth aspect of the present invention, the use of the above-mentioned benzenesulfonic acid or benzenesulfonic acid derivative-guanidine cocrystal in the preparation of a medicament for treating diabetic foot or diabetic chronic ulcers is provided, wherein the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine cocrystal is a GD cocrystal single crystal or a GD cocrystal powder.

[0012] The beneficial effects of this invention include: (1) This invention addresses the problem of poor wound healing in diabetic patients by synthesizing a series of structurally stable binary ionic cocrystals with sustained-release guanidine properties using guanidine hydrochloride and benzenesulfonic acid and its derivatives. The cocrystals formed by benzenesulfonic acid and its derivatives and guanidine can synergistically improve the inherent defects of guanidine through intermolecular forces (such as hydrogen bonds and π-π stacking), while enhancing the therapeutic effect on diabetic wounds.

[0013] When benzenesulfonic acid and its derivatives form a cocrystal with guanidine, the benzenesulfonic acid and its derivatives can block the sites where guanidine is easily metabolized through steric hindrance or electronic effects. Simultaneously, guanidine may exhibit a slow release rate, high bioavailability, good therapeutic effect, and high safety due to the stable cocrystal structure. The cocrystal of guanidine with benzenesulfonic acid and its derivatives enhances structural stability, enabling the slow release of guanidine and promoting the healing of diabetic foot ulcers through both epidermal migration and nerve repair.

[0014] This invention screened suitable benzenesulfonic acid derivatives and used them as ligands to bind with guanidine, successfully synthesizing a series of benzenesulfonic acid or benzenesulfonic acid derivative-guanidine cocrystals. These cocrystals can promote the proliferation and migration of fibroblasts, vascular endothelial cells, and keratinocytes in the skin, reduce apoptosis of peripheral nerves, and promote axonal growth, thus achieving rapid healing of diabetic wounds. Simultaneously, the cocrystal formation of benzenesulfonic acid and its derivatives with guanidine enables the slow release of guanidine ions, improving efficacy while reducing the potential toxicity of guanidine. This invention provides a new approach for promoting the healing of diabetic foot ulcers using drug cocrystals.

[0015] (2) The acidity of benzenesulfonic acid and its derivatives can neutralize the strong alkalinity of guanidine, making the pH of the cocrystal tend to be neutral (about 6.5-7.2), thereby avoiding the irritation of mucous membranes or wounds when guanidine is used alone, significantly improving biocompatibility, and making it more suitable for the fragile wound microenvironment of diabetic patients. In addition, the cocrystal structure can shield the polar sites of the guanidine group, reducing hygroscopicity by 40%-60% compared with pure guanidine, effectively preventing dosage form clumping, and ensuring the storage stability of the preparation and the effective concentration during use.

[0016] (3) In terms of stability, the dense crystal structure formed by the co-crystal can inhibit the hydrolysis and chain breakage of guanidine, extending the shelf life to more than 24 months under normal temperature storage conditions, which is 50% higher than that of pure guanidine, and avoiding incompatibility with acidic excipients. In terms of release rate, the co-crystal achieves a sustained-release effect through crystal lattice regulation, extending the release cycle of the active ingredient to 72-96 hours, avoiding excessively high local concentrations caused by burst release, while maintaining healing-promoting activity, which meets the treatment needs of slow wound healing in diabetes. This invention solves the bottleneck of guanidine used alone, synergistically exerting anti-inflammatory and repair-promoting effects, providing a safer, more stable, and more efficient formulation for the treatment of diabetic chronic ulcers.

[0017] (4) This invention uses low-temperature reaction (low-temperature solution crystallization) and other methods to prevent the system from completing proton transfer in time, thereby kinetically preventing the formation of ionic salts and instead locking the molecular co-crystal assembly of free guanidine and benzenesulfonic acid or benzenesulfonic acid derivatives. Rapid low-temperature crystallization allows molecules to quickly arrange themselves into crystals in a non-ionic form, avoiding the time required for proton transfer.

[0018] Benzenesulfonic acid and its derivatives such as 4,4'-biphenyl disulfonic acid and biphenyl-4-sulfonic acid have a large conjugated structure and steric hindrance. Their sulfonic acid groups form a multi-hydrogen bond array with guanidine (rather than a simple ionic bond). The steric hindrance inhibits complete proton transfer and instead forms a eutectic supramolecular network dominated by hydrogen bonds and π-π stacking.

[0019] This invention strictly controls the stoichiometry during the preparation process, adjusting the non-equimolar feed ratio of guanidine to benzenesulfonic acid and benzenesulfonic acid derivatives. By adjusting the concentration effect, complete proton transfer is suppressed. Cl in guanidine hydrochloride... -It can also inhibit the protonation of guanidine and the deprotonation of sulfonic acid, thus stabilizing the eutectic phase. Attached Figure Description

[0020] Figure 1 This is a synthetic route diagram for the eutectic single crystal of guanidine and 4,4'-biphenyl disulfonic acid.

[0021] Figure 2 Characterization results of five benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic single crystals prepared in Examples 1-5.

[0022] Figure 3 The physicochemical characterization results are for the five benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic powders prepared in Examples 6-10.

[0023] Figure 4 The results show the stability and release rate of the five benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic powders prepared in Examples 6-10.

[0024] Figure 5 This is a result of cytotoxicity.

[0025] Figure 6 These are the results of a cell proliferation experiment.

[0026] Figure 7 The results are from a cell scratch assay.

[0027] Figure 8 The effect of SA, GD, BD and GH on promoting wound healing in diabetic mice. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. The experimental materials and reagents involved in the following embodiments are all commercially available. Experimental methods not specifically specified in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0029] I. Research and Development Approach A eutectic refers to a homogeneous crystalline mixture with a fixed melting point formed under certain conditions by two or more different substances through hydrogen bonding, π-π conjugation, or charge interactions. Guanidine molecules have abundant hydrogen bond donors and acceptors. Under physiological conditions, guanidine readily protonates to acquire a positive charge, further enhancing its ability to form hydrogen bonds. If guanidine forms a eutectic with a negatively charged ligand, the remaining empty hydrogen bond binding sites decrease, weakening its ability to bind with water and reducing its hygroscopicity. The positive and negative charge interactions further improve the stability of the eutectic, enabling the slow release of guanidine, thus improving bioavailability while reducing toxicity. Meanwhile, benzenesulfonic acid and its derivatives have been used in drug design to prolong half-life, improve bioavailability, enhance efficacy, and reduce metabolite toxicity. Currently, several drugs using benzenesulfonic acid are used to treat cancer, inflammation, and metabolic diseases; for example, calcium benzenesulfonate silicate can significantly improve symptoms of diabetic retinopathy and diabetic nephropathy. This invention attempts to prepare a eutectic using guanidine and benzenesulfonic acid and its derivatives.

[0030] II. Preparation of benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic single crystals In all the following examples, the hydrochloride form of guanidine (guanidine hydrochloride GH) is used, but hydrochloric acid does not participate in the formation of the final eutectic structure.

[0031] Example 1 The preparation method of 4,4'-biphenyl disulfonic acid-guanidine eutectic single crystal in this embodiment includes the following steps: 10 mg of guanidine hydrochloride (GH) and 7.5 mg of 4,4'-biphenyl disulfonic acid (BD) were dissolved in 10 mL of a solution composed of methanol and water in a 1:1 volume ratio. The container containing the above materials was sealed with tin foil and a small hole was punched. The container was then placed at 4 °C for 24 h. The 4,4'-biphenyl disulfonic acid-guanidine (GD) eutectic single crystal was obtained by solvent evaporation.

[0032] Example 2 The method for preparing biphenyl-4-sulfonic acid-guanidine eutectic single crystals in this embodiment includes the following steps: 10 mg of guanidine hydrochloride (GH) and 10 mg of biphenyl-4-sulfonic acid (BS) were dissolved in 10 mL of a solution composed of methanol and water in a 1:1 volume ratio. The container containing the above materials was sealed with tin foil and a small hole was punched. The container was then placed at 4 °C for 24 h. Biphenyl-4-sulfonic acid-guanidine (GS) eutectic single crystals were obtained by solvent evaporation.

[0033] Example 3 The method for preparing 3-methylbenzenesulfonic acid-guanidine eutectic single crystals in this embodiment includes the following steps: 5 mg of guanidine hydrochloride (GH) and 5 mg of 3-methylbenzenesulfonic acid (BM) were dissolved in 10 mL of a solution composed of methanol and water in a 1:1 volume ratio. The container containing the above materials was sealed with tin foil and a small hole was punched. The container was then placed at 4 °C for 24 h. The 3-methylbenzenesulfonic acid-guanidine (GM) eutectic single crystal was obtained by solvent evaporation.

[0034] Example 4 The preparation method of 2-naphthalenesulfonic acid-guanidine eutectic single crystal in this embodiment includes the following steps: 20 mg of guanidine hydrochloride (GH) and 20 mg of 2-naphthalenesulfonic acid (BN) were dissolved in 10 mL of a solution composed of methanol and water in a 1:1 volume ratio. The container containing the above materials was sealed with tin foil and a small hole was punched. The container was then placed at 4 °C for 24 h. The 2-naphthalenesulfonic acid-guanidine (GN) eutectic single crystal was obtained by solvent evaporation.

[0035] Example 5 The method for preparing benzenesulfonic acid-guanidine eutectic single crystals in this embodiment includes the following steps: 10 mg of guanidine hydrochloride (GH) and 10 mg of benzenesulfonic acid (BA) were dissolved in 10 mL of a solution composed of methanol and water in a 1:1 volume ratio. The container containing the above materials was sealed with tin foil and a small hole was punched. The container was then placed at 4 °C for 24 h. The benzenesulfonic acid-guanidine (GA) eutectic single crystal was obtained by solvent evaporation.

[0036] III. Preparation of benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic powder In all the following examples, the hydrochloride form of guanidine (guanidine hydrochloride GH) is used, but hydrochloric acid does not participate in the formation of the final eutectic structure.

[0037] Example 6 The preparation method of the 4,4'-biphenyl disulfonic acid-guanidine eutectic powder in this embodiment includes the following steps: 50 mL of guanidine hydrochloride (GH) aqueous solution with a concentration of 1 mg / mL was slowly added to 100 mL of 4,4'-biphenyl disulfonic acid (BD) methanol solution with a concentration of 4.5 mg / mL. The mixture was stirred at 800 rpm for 30 min and then sonicated at 100 W for 30 min to obtain a pale yellow precipitate. The precipitate was then washed three times with a solution of methanol and water pre-cooled at 4 °C in a 1:1 volume ratio. Finally, the precipitate was dried under vacuum at 26 °C for 24 h to obtain a white powder, which is the 4,4'-biphenyl disulfonic acid-guanidine (GD) eutectic powder.

[0038] Example 7 The preparation method of biphenyl-4-sulfonic acid-guanidine eutectic powder in this embodiment includes the following steps: 50 mL of guanidine hydrochloride (GH) aqueous solution with a concentration of 1 mg / mL was slowly added to 100 mL of biphenyl-4-sulfonic acid (BS) methanol solution with a concentration of 4.5 mg / mL. The mixture was stirred at 800 rpm for 15 min and then sonicated at 100 W for 30 min to obtain a pale yellow precipitate. The precipitate was then washed three times with a solution of methanol and water pre-cooled at 4 °C in a 1:1 volume ratio. The precipitate was then dried under vacuum at 26 °C for 24 h to obtain a white powder, which is the biphenyl-4-sulfonic acid-guanidine (GS) eutectic powder.

[0039] Example 8 The preparation method of the 3-methylbenzenesulfonic acid-guanidine eutectic powder in this embodiment includes the following steps: 50 mL of guanidine hydrochloride (GH) aqueous solution with a concentration of 1 mg / mL was slowly added to 100 mL of 3-methylbenzenesulfonic acid (BM) methanol solution with a concentration of 4.5 mg / mL. The mixture was stirred at 800 rpm for 15 min and then sonicated at 100 W for 30 min to obtain a pale yellow precipitate. The precipitate was then washed three times with a solution of methanol and water pre-cooled at 4 °C in a 1:1 volume ratio. Finally, the precipitate was dried under vacuum at 26 °C for 24 h to obtain a white powder, which is the 3-methylbenzenesulfonic acid-guanidine (GM) eutectic powder.

[0040] Example 9 The preparation method of the 2-naphthalenesulfonic acid-guanidine eutectic powder in this embodiment includes the following steps: 50 mL of guanidine hydrochloride (GH) aqueous solution with a concentration of 1 mg / mL was slowly added to 100 mL of 2-naphthalenesulfonic acid (BN) methanol solution with a concentration of 4.5 mg / mL. The mixture was stirred at 800 rpm for 25 min and then sonicated at 100 W for 30 min to obtain a pale yellow precipitate. The precipitate was then washed three times with a solution of methanol and water pre-cooled at 4 °C in a 1:1 volume ratio. Finally, the precipitate was dried under vacuum at 26 °C for 24 h to obtain a white powder, which is the 2-naphthalenesulfonic acid-guanidine (GN) eutectic powder.

[0041] Example 10 The preparation method of benzenesulfonic acid-guanidine eutectic powder in this embodiment includes the following steps: 50 mL of guanidine hydrochloride (GH) aqueous solution with a concentration of 1 mg / mL was slowly added to 100 mL of benzenesulfonic acid (BA) methanol solution with a concentration of 4.5 mg / mL. The mixture was stirred at 800 rpm for 25 min and then sonicated at 100 W for 30 min to obtain a pale yellow precipitate. The precipitate was then washed three times with a solution of methanol and water pre-cooled at 4 °C in a 1:1 volume ratio. Finally, the precipitate was dried under vacuum at 26 °C for 24 h to obtain a white powder, which is the benzenesulfonic acid-guanidine (GA) eutectic powder.

[0042] IV. Characterization of benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic 1. Characterization of eutectic single crystals X-ray single-crystal diffraction was used to determine the eutectic single crystal formed by the combination of benzenesulfonic acid derivative and guanidine, and to study detailed data on the binding mode, binding ratio and crystal structure of the ligands.

[0043] The characterization results of the five benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic single crystals prepared in Examples 1-5 are as follows: Figure 2 As shown in the figure, (A) is a scanning electron microscope (SEM) image, a polarized light microscope (PLM) image, and a 3D model of the crystal morphology; (B) is a diagram of the single crystal chemical structure, hydrogen bond length, and planar packing interaction.

[0044] from Figure 2 (A) It can be seen that the five crystals GD, GS, GM, GN, and GA all exhibit crystalline morphology, and the observation results from SEM (scanning electron microscopy) and PLM (polarizing light microscopy) are consistent. From Figure 2 (B) It can be seen that the chemical structure, hydrogen bond length, and planar packing of the five single crystals (GD, GS, GM, GN, and GA) were all determined by single-crystal XRD, indicating that the above-mentioned eutectic single crystals were successfully synthesized. Figure 2 (B) It can also be found that the molar ratio of 3-methylbenzenesulfonic acid to guanidine in the GM eutectic single crystal is 1:1, the molar ratio of biphenyl-4-sulfonic acid to guanidine in the GS eutectic single crystal is 1:1, the molar ratio of 2-naphthalenesulfonic acid to guanidine in the GN eutectic single crystal is 1:1, the molar ratio of 4,4'-biphenyl disulfonic acid to guanidine in the GD eutectic single crystal is 1:2, and the molar ratio of benzenesulfonic acid to guanidine in the GA eutectic single crystal is 1:2.

[0045] The crystal data and structural details of GD, GS, GM, GN, and GA are shown in Table 1-5.

[0046] Table 1. Crystal data and structural details of GD

[0047] Table 2 shows the crystal data and structural details of GS.

[0048] Table 3 shows the crystal data and structural details of GM.

[0049] Table 4. Crystal data and structural details of GN

[0050] Table 5 shows the crystal data and structural details of GA.

[0051] 2. Characterization of eutectic powder (1) Powder X-ray diffraction (P-XRD) After vacuum drying, the characteristic peaks of the eutectic powders of GD, GS, GM, GN and GA were determined by P-XRD and compared with the predicted XRD patterns.

[0052] (2) Nuclear magnetic resonance hydrogen spectrum ( 1 H-NMR) Vacuum-dried GD, GS, GM, GN and GA eutectic powders were dissolved in deuterated DMSO, and the samples were tested using a proton nuclear magnetic resonance spectrometer.

[0053] (3) Fourier transform infrared spectroscopy (FTIR) Vacuum-dried GD, GS, GM, GN, and GA eutectic powders were mixed with KBr, ground uniformly, and then pressed into tablets. Fourier transform infrared spectroscopy was used for testing, with a scanning range of 500-4000 cm⁻¹. -1 The resolution is 4cm. -1 .

[0054] (4) Stability and release rate determination Vacuum-dried GD, GS, GM, GN, and GA eutectic powders were placed in 10% BSA solution (pH=5.5 or 7.4, 10 mL). Powders were collected at 24h, 96h, and 168h and vacuum-dried. Material stability was determined using P-XRD. The release of guanidine from the GD, GS, GM, GN, and GA eutectic powders at pH=5.5 and pH=7.4 was determined using dialysis at thirteen time points: 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h.

[0055] (5) Characterization results The physicochemical characterization results of the five benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic powders prepared in Examples 6-10 are as follows: Figure 3 As shown, where, Figure 3 (A) shows the P-XRD results of the eutectic powder. Figure 3 (B) is a eutectic powder. 1 H-NMR results, Figure 3 (C) shows the FTIR results of the eutectic powder.

[0056] from Figure 3 The P-XRD results in (A) show that the XRD peak positions of the five eutectic powders are consistent with the predicted XRD peak positions, indicating that the five eutectic materials were successfully synthesized and that the five eutectic materials have specific crystal structures.

[0057] from Figure 3 (B) 1 The H-NMR results show that all five eutectic powders simultaneously possess characteristic peaks belonging to guanidine and benzenesulfonic acid derivatives, indicating that the five eutectic powders were successfully synthesized.

[0058] from Figure 3 The FTIR results in (C) show that in the range of 500-4000 cm⁻¹ -1 Within the specified range, all five eutectic powders exhibited characteristic peaks of benzenesulfonic acid derivatives and guanidine, indicating that the five eutectic powders were successfully synthesized.

[0059] The stability and release rate of the five benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic powders prepared in Examples 6-10 are as follows: Figure 4 As shown, where, Figure 4 (A) is the stability result of the eutectic powder. Figure 4 (B) is the release rate result of the eutectic powder.

[0060] from Figure 4 The stability results of the eutectic powders in (A) show that the five eutectic powders can maintain their XRD patterns unchanged for 7 days under acidic and physiological pH conditions, indicating that the eutectic powders have good stability. Among them, GD has the best stability and no new peaks appeared.

[0061] from Figure 4 The release results of the eutectic powder in (B) show that GS, GM, GN and GA all released all guanidine molecules at 168h, while GD could still maintain a slow and continuous release of guanidine, indicating that GD has the slowest rate of guanidine release and is suitable for drug administration.

[0062] In summary, the above results demonstrate that the five eutectic powders—GD, GS, GM, GN, and GA—possess crystalline structures and exhibit characteristic peaks associated with guanidine, confirming the successful synthesis of these five eutectic powders. Furthermore, the results demonstrate that these five eutectic powders are stable under physiological conditions and exhibit slow-release properties.

[0063] V. Cell Experiments 1. Cytotoxicity The cytotoxicity of GD powder in Example 6 was qualitatively determined using the Calcein-AM / PI liveness-death assay. HaCaTs, HDF-as, HUVECs, and PC12 cells were co-cultured with complete culture medium containing 1 mg / mL GD powder sample for 48 h, with an equal volume of physiological saline added as a control. The cells were then incubated with Calcein-AM / PI dye for 30 min and photographed using a high-content fluorescence microscope.

[0064] The cytotoxicity of GD, BD, and GH powders in Example 6 was quantitatively determined using the CCK-8 assay. HaCaTs, HDF-as, HUVECs, and PC12 cells were co-cultured for 48 h with complete culture media containing 0.01, 0.1, 0.5, 1, and 10 mg / mL of GD, BD, and GH powder samples, respectively. Cytotoxicity was then measured using the CCK-8 reagent. An equal volume of physiological saline was added as a control group (SA). Cell viability was calculated using the following formula: Cell viability = (Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance) × 100%.

[0065] Cytotoxicity results such as Figure 5 As shown, where, Figure 5 (A) shows the results of GD staining experiments on four cell types (HUVEC, HaCaT, PC-12, and HDF) for both live and dead cells. Figure 5 (B) shows the results of the CCK-8 experiment, where * indicates P < 0.05 between the two groups, ** indicates P < 0.01 between the two groups, and *** indicates P < 0.001 between the two groups.

[0066] from Figure 5 (A) It can be seen that, compared with the control group, no large amount of red fluorescence appeared in the GD administration group, indicating that the GD co-crystal powder has low toxicity to all four types of cells.

[0067] from Figure 5 (B) It can be seen that the GD eutectic powder at a concentration of 1 mg / mL has no obvious toxicity to any of the four cell types.

[0068] The above results indicate that GD co-crystal powder has low toxicity to all four cell types, followed by GH. Although BD monomer has some toxicity, it is still within the safe range. The synthesis of GD co-crystal effectively reduces the toxicity of BD monomer.

[0069] 2. Cell proliferation Cell proliferation experiments were conducted using the GD, BD, and GH powders from Example 6. HUVECs and PC12 cells were co-cultured for 24 h with 500 μL of complete culture medium containing 0.5 mg / mL of GD, BD, or GH powder samples, respectively. The control group (SA) received an equal volume of physiological saline. Each group was incubated with EdU working solution for 4 h, washed, and then incubated with Apollp staining solution for 30 min. Fluorescence microscopy was used for observation and photography, and fluorescence intensity was quantified using a microplate reader.

[0070] Cell proliferation experiment results as follows Figure 6 As shown, where, Figure 6 (A) and (B) show the cell proliferation results of PC12 cells. Figure 6 (C) and (D) show the cell proliferation results of HUVECs cells.

[0071] from Figure 6 (A) and (B) show that, compared with the control group (SA), GD can effectively promote the proliferation of PC-12 cells.

[0072] from Figure 6 (C) and (D) show that, compared with the control group (SA), GD can effectively promote the proliferation of HUVEC cells.

[0073] The above results indicate that GD eutectic powder can promote the proliferation of related cells.

[0074] 3. Cell scratch Cell scratch assays were performed using GD, BD, and GH powders as described in Example 6. HaCaTs and HDF-as cells were seeded in 48-well plates. After the cells had uniformly covered the bottom of the plate, a uniform vertical line was drawn along the center of each well using a 200µL pipette tip. The cells were washed three times with PBS solution to remove cell debris. The cells were then observed under an inverted microscope and bright-field images were taken. 500µL of GH, BD, or GD culture medium containing 0.5mg / mL of the drug (1% serum concentration) was added to each well, while the control group (SA) received an equal volume of physiological saline. After incubation for 24 hours, the cells were washed three times with PBS solution and observed under an inverted microscope and bright-field images were taken. The rate of change of scratch area over time was statistically analyzed using ImageJ software. Cell migration rate was calculated using the following formula: Cell migration rate (%) = (A0 - At) / A0 * 100%. A0: The central scratch-free area at 0h; At: The central scratch-free area after 24h.

[0075] Cell scratch assay results are as follows Figure 7 As shown, where, Figure 7 (A) is an image showing the effects of SA, GD, BD, and GH on HDF cell migration. Figure 7 (B) is a quantitative bar graph of the scratch healing rate of HDF cells. Figure 7 (C) shows images of the effects of SA, GD, BD, and GH on HaCaT cell migration. Figure 7 (D) is a quantitative bar graph of the scratch healing rate of HaCaT cells. ** indicates that the difference between the two groups is P < 0.01, and *** indicates that the difference between the two groups is P < 0.001.

[0076] from Figure 7 As can be seen from (A) and (B), compared with the control group (SA), the scratch area of ​​HDF cells in the GD group was significantly reduced after 24 hours.

[0077] from Figure 7(C) and (D) show that, compared with the control group (SA), the scratch area of ​​HaCaT cells in the GD group was significantly reduced after 24 hours.

[0078] The above results indicate that GD eutectic powder can significantly promote cell migration.

[0079] VI. Animal Experiments Wound healing experiments were conducted in type 2 diabetic mice using GD, BD, and GH powders as described in Example 6. Six-week-old male C57BL / 6 mice, weighing 20-30g, were acclimatized to a high-fat diet for one month before a diabetic model was established using streptozotocin (STZ). Experimental groups included a control group (SA) and experimental groups (GD, BD, and GH), with three mice in each group. After anesthesia and hair removal, circular full-skin excision wounds were made on both sides of the mouse's back. Surgical sutures were used to suture the skin around the adhesive band around the wound. Then, 40 μL of physiological saline suspension containing GD, BD, and GH powders (1 mg / mL) was evenly dripped into the wounds of mice in the GD, BD, and GH groups, respectively. An equal volume of physiological saline was evenly dripped into the wounds of mice in the control group. A sterile dressing was then applied to the adhesive band to seal the medication. Finally, two layers of self-adhesive bandages were wrapped around the wounds on the mouse's back. The wound condition was observed and photographed periodically. ImageJ software was used for statistical analysis of the photographs to calculate the wound healing rate.

[0080] The effects of SA, GD, BD, and GH on promoting wound healing in diabetic mice are as follows: Figure 8 As shown, where, Figure 8 (A) Images of wound healing in each group of mice. Figure 8 (B) shows the wound healing rate of mice in each group. Figure 8 (C) is a graph showing the changes in blood glucose levels in each group of mice. ☆ indicates that the wound is completely closed, * indicates that the difference between the GD group and the SA group is P < 0.05, and ** indicates that the difference between the GD group and the SA group is P < 0.01.

[0081] from Figure 8 (A) It can be seen that, compared with the control group, the wound in the GD group was closed at 21 days and covered with hair at 28 days.

[0082] from Figure 8 (B) It can be seen that the wound closure rate within the 28-day cycle is: GD > GH > BD > SA. Compared with the control group, the GD group significantly improved the wound healing rate.

[0083] from Figure 8 (C) It can be seen that the blood glucose levels of mice in each group were greater than 18 mmol / L, indicating that the diabetes model was successfully established.

[0084] The above results indicate that GD can effectively promote wound healing in diabetic mice.

[0085] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic, characterized in that, The eutectic is a eutectic of benzenesulfonic acid or a benzenesulfonic acid derivative and guanidine, wherein the molar ratio of benzenesulfonic acid or a benzenesulfonic acid derivative to guanidine is 1:(1-2); the benzenesulfonic acid derivative is 4,4'-biphenyl disulfonic acid, biphenyl-4-sulfonic acid, 3-methylbenzenesulfonic acid, or 2-naphthalenesulfonic acid. The eutectic includes eutectic single crystals and eutectic powders, wherein the eutectic single crystals include GD eutectic single crystals, GS eutectic single crystals, GM eutectic single crystals, GN eutectic single crystals, and GA eutectic single crystals; and the eutectic powders include GD eutectic powders, GS eutectic powders, GM eutectic powders, GN eutectic powders, and GA eutectic powders. The GD eutectic single crystal is a monoclinic crystal with space group P121 / c1. Its cell parameters are: a = 11.9776(2) Å, b = 26.8094(4) Å, c = 7.38700(10) Å, α = 90°, β = 92.5160(10)°, γ = 90°, Z = 4, and its cell volume is 2369.77(6) Å. 3 ; The GS eutectic single crystal is a monoclinic crystal, space group I121, with the following cell parameters: a = 10.31050(10) Å, b = 8.12400(10) Å, c = 32.2188(3) Å, α = 90°, β = 99.1630(10)°, γ = 90°, Z = 8, and a cell volume of 2664.29(5) Å. 3 ; The GM eutectic single crystal is orthorhombic, space group -P2ac2n, with the following cell parameters: a=17.3249(4)Å, b=7.5177(2)Å, c=8.5106(2)Å, α=90°, β=90°, γ=90°, Z=5, and a cell volume of 1108.45(5) Å. 3 ; The GN eutectic single crystal is triclinic, space group P-1, with the following cell parameters: a = 7.44530(10) Å, b = 12.0008(2) Å, c = 27.9597(7) Å, α = 88.117(2)°, β = 85.677(2)°, γ = 88.4400(10)°, Z = 8, and a cell volume of 2488.97(8) Å. 3 ; The GA eutectic single crystal is monoclinic, space group P121 / c1, with the following cell parameters: a = 7.4289(2) Å, b = 22.8661(7) Å, c = 11.9938(3) Å, α = 90°, β = 92.324(3)°, γ = 90°, Z = 8, and a cell volume of 2035.71(10) Å. 3 ; The X-ray diffraction pattern of the GD eutectic powder shows characteristic peaks at 10θ angles of 15.64±0.66°, 17.64±0.76°, and 24.64±0.86°. The X-ray diffraction pattern of the GS eutectic powder shows characteristic peaks at 10θ angles of 16.09±0.91°, 19.01±1.50°, and 26.58±0.53°. The X-ray diffraction pattern of the GM eutectic powder shows characteristic peaks at 10θ angles of 17.16±1.00°, 22.30±1.30°, and 27.97±0.62°. The X-ray diffraction pattern of the GN eutectic powder shows characteristic peaks at 10θ angles of 22.91±2.45°, 33.57±2.84°, and 42.91±1.77°. The X-ray diffraction pattern of the GA eutectic powder shows characteristic peaks at 10θ angles of 25.36±3.99°, 34.95±3.52°, and 44.06±1.31°.

2. The method for preparing benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic according to claim 1, characterized in that, When the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is a eutectic single crystal, its preparation method includes the following steps: Guanidine hydrochloride and benzenesulfonic acid or benzenesulfonic acid derivatives are dissolved in a solution composed of methanol and water, and then placed at 4°C for 24-72 hours. The benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic single crystal is obtained by solvent evaporation.

3. The preparation method according to claim 2, characterized in that, The mass ratio of benzenesulfonic acid or benzenesulfonic acid derivative to guanidine hydrochloride is 1:(1-2). The volume ratio of methanol to water is 1:1; The mass ratio of guanidine hydrochloride to the volume of the solution is (5-20):

10.

4. The method for preparing benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic according to claim 1, characterized in that, When benzenesulfonic acid or its derivative-guanidine eutectic is a eutectic powder, its preparation method includes the following steps: A guanidine hydrochloride aqueous solution is mixed with a methanol solution of benzenesulfonic acid or a benzenesulfonic acid derivative, stirred at 800-1000 rpm for 15-30 min, and then sonicated at 80-120 W for 20-40 min to obtain a pale yellow precipitate. The precipitate is then washed and dried to obtain a white powder, which is the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic powder.

5. The preparation method according to claim 4, characterized in that, The concentration of the guanidine hydrochloride aqueous solution is 1-1.5 mg / mL, the concentration of the benzenesulfonic acid or benzenesulfonic acid derivative methanol solution is 4.5 mg / mL, and the volume ratio of the guanidine hydrochloride aqueous solution to the benzenesulfonic acid or benzenesulfonic acid derivative methanol solution is 1:

2.

6. The use of the benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic according to claim 1 in the preparation of a medicament for treating diabetic foot or diabetic chronic ulcers, wherein, The benzenesulfonic acid or benzenesulfonic acid derivative-guanidine eutectic is a GD eutectic single crystal or a GD eutectic powder.

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