Sulfathiazole-trimethoprim medicine crystal salt as well as preparation method and application thereof

Abstract

The invention provides a sulfathiazole-trimethoprim medicine crystal salt as well as a preparation method and application thereof, and belongs to the technical field of medicine crystal salts. The sulfathiazole-trimethoprim drug crystal salt provided by the invention is an orthorhombic crystal system, and the space group is Pbca; the cell parameters are as follows: a is equal to 17.3213 (3), b is equal to 12.2894 (2), c is equal to 23.7549 (3), alpha is equal to 90 degrees, beta is equal to 90 degrees, and gamma is equal to 90 degrees; wherein the molar ratio of sulfathiazole to trimethoprim is 1: 1, and no crystalline solvent exists. The sulfathiazole-trimethoprim drug crystal salt provided by the invention has high solubility in water, is beneficial to improvement of absorption and metabolism conditions in human bodies and animal bodies, and has an excellent antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the technical field of drug crystal salts, and in particular to a sulfathiazole-trimethoprim drug crystal salt, a preparation method thereof, and an application thereof. Background Art

[0002] Co-trimoxazole is a common antibacterial drug, a combination of sulfamethoxazole and trimethoprim (TMP), which inhibits the bacterial DHFR enzyme. This antibacterial drug is widely used clinically due to its ease of use, stability, and affordability. However, with the widespread use of synthetic antibacterial drugs, bacterial resistance is becoming increasingly serious, necessitating the development of a wider range of new antibacterial drugs.

[0003] Sulfathiazole (STZ) is a typical sulfonamide antibiotic that inhibits bacterial DHPS enzymes and is a broad-spectrum antibacterial agent. The combined use of STZ and TMP can block bacterial folate metabolism, thereby enhancing antibacterial efficacy and reducing the emergence of drug-resistant strains. However, the low solubility of both drugs results in poor absorption and metabolism in humans and animals. Summary of the Invention

[0004] The present invention aims to provide a sulfathiazole-trimethoprim drug crystal salt, a preparation method and an application thereof. The sulfathiazole-trimethoprim drug crystal salt provided by the present invention has high solubility in water, is beneficial to improving absorption and metabolism in the human body and animals, and has excellent antibacterial effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a sulfathiazole-trimethoprim drug crystal salt, which is an orthorhombic crystal system with a space group of P bca; the unit cell parameters are: a =17.3213(3)Å, b =12.2894(2)Å, c =23.7549(3)Å, α =90°, β =90°, γ =90°; wherein the molar ratio of sulfathiazole to trimethoprim is 1:1, and there is no crystallization solvent.

[0006] Preferably, Cu-K is used α Radiation, with 2 θ Angles indicate that the characteristic diffraction peaks of the powder X-ray diffraction spectrum of the sulfathiazole-trimethoprim drug crystalline salt include 11.54°, 17.06°, 19.50°, 20.96°, 21.44°, 22.60°, 25.92° and 26.24°.

[0007] Preferably, the asymmetric unit of the sulfathiazole-trimethoprim drug crystal salt comprises a sulfathiazole cation and a trimethoprim anion, and the interaction between the sulfathiazole cation and the trimethoprim anion includes ionic bonding, hydrogen bonding and π-π stacking.

[0008] Preferably, the melting point of the sulfathiazole-trimethoprim drug crystal salt is 169.5°C.

[0009] The present invention provides a method for preparing the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution, comprising the following steps: Dissolving sulfathiazole and trimethoprim in a solvent to obtain a mixed solution; the solvent is acetone and ethanol; The mixed solution is crystallized to obtain the sulfathiazole-trimethoprim drug crystal salt.

[0010] Preferably, the molar ratio of sulfathiazole to trimethoprim is 1:1; the volume ratio of acetone to ethanol is 1:0.25-4; and the amount ratio of sulfathiazole to solvent is 0.5-2.5 mmol:30 mL.

[0011] Preferably, the crystallization temperature is 15-40° C., and the crystallization time is 4-6 days.

[0012] The present invention provides the use of the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in the above technical solution in the preparation of drugs for treating bacterial and / or fungal infectious diseases.

[0013] Preferably, the bacteria include one or more of the gram-positive bacteria Staphylococcus aureus, the gram-negative bacteria Klebsiella pneumoniae, Shigella dysenteriae and Pseudomonas aeruginosa; and the fungus includes Candida albicans.

[0014] The present invention provides a drug for treating bacterial and / or fungal infectious diseases, wherein the active ingredient comprises the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in the above technical solution.

[0015] Beneficial effect: The present invention provides a sulfathiazole-trimethoprim drug crystal salt, which is an orthorhombic crystal system with a space group of P bca; the unit cell parameters are: a =17.3213(3)Å, b =12.2894(2)Å, c =23.7549(3)Å, α =90°, β =90°, γ=90°; wherein the molar ratio of sulfathiazole to trimethoprim is 1:1, and no crystallization solvent is present. The present invention designed and successfully synthesized a pharmaceutical crystal salt of STZ and TMP, namely, STZ-TMP pharmaceutical crystal salt. This crystal salt has high solubility in water, which improves absorption and metabolism in humans and animals, and exhibits excellent antibacterial efficacy. The structure of the STZ-TMP pharmaceutical crystal salt was characterized in a test example of the present invention. Single crystal X-ray diffraction revealed that the molar ratio of the two components in the pharmaceutical crystal salt structure is 1:1, and that STZ and TMP are primarily bound together by electrostatic attraction (ionic bonds). Ionic bonds, hydrogen bonds, and π-π stacking interactions maintain the stability of the crystal structure. Notably, the STZ-TMP pharmaceutical crystal salt of the present invention improves the solubility of both active pharmaceutical ingredients. Furthermore, compared to a physical mixture of STZ and TMP, the STZ-TMP pharmaceutical crystal salt of the present invention exhibits enhanced antibacterial activity, a larger inhibition zone diameter, and enhanced inhibition of microbial DHPS and DHFR enzymes. It is shown that the present invention forms a drug crystal salt with STZ and TMP. Compared with the physical mixture of STZ-TMP, it can make STZ and TMP produce synergistic effects more efficiently in the same system, significantly improve the efficacy of combined medication, and bring new possibilities for conquering difficult diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the asymmetric unit structure of STZ-TMP drug crystal salt with atomic labels; Figure 2 The powder X-ray diffraction patterns of STZ, TMP and STZ-TMP drug crystal salts; Figure 3 Fourier transform infrared spectra of STZ, TMP and STZ-TMP drug crystal salts; Figure 4 DSC curves of STZ, TMP and STZ-TMP drug crystal salts; Figure 5 TGA curves of STZ, TMP and STZ-TMP drug crystal salts; Figure 6 This is a comparison chart of the solubility of STZ, TMP and STZ-TMP drug crystal salts under different pH conditions; Figure 7 This is a comparison chart of the inhibitory effects of STZ, TMP, STZ-TMP physical mixture and STZ-TMP drug crystal salt on DHPS enzyme and DHFR enzyme. DETAILED DESCRIPTION

[0017] The present invention provides a sulfathiazole-trimethoprim drug crystal salt, which is an orthorhombic crystal system with a space group of P bca; the unit cell parameters are: a=17.3213(3)Å, b =12.2894(2)Å, c =23.7549(3)Å, α =90°, β =90°, γ =90°; wherein the molar ratio of sulfathiazole to trimethoprim is 1:1, and there is no crystallization solvent.

[0018] The combined use of sulfathiazole (STZ) and trimethoprim (TMP) in related technologies does not alter the physical and chemical properties of the active pharmaceutical ingredients themselves. The present invention utilizes pharmaceutical crystal salt technology to form a pharmaceutical crystal salt from STZ and TMP. This significantly enhances the physical and chemical properties (such as solubility, stability, and antibacterial activity) while maintaining the chemical structure of the active pharmaceutical ingredients. This achieves synergistic treatment and enhances efficacy and reduces toxicity. The structural formulas of STZ (a) and TMP (b) in the present invention are shown below: .

[0019] As an embodiment of the present invention, the unit cell volume of the sulfathiazole-trimethoprim drug crystal salt is V =5055.82(14)Å 3 , Z= 8.

[0020] As an embodiment of the present invention, Cu-K α Radiation, with 2 θ Angles indicate that the characteristic diffraction peaks of the powder X-ray diffraction spectrum of the sulfathiazole-trimethoprim drug crystalline salt include 11.54°, 17.06°, 19.50°, 20.96°, 21.44°, 22.60°, 25.92° and 26.24°.

[0021] As one embodiment of the present invention, the asymmetric unit of the sulfathiazole-trimethoprim drug crystal salt contains a sulfathiazole cation and a trimethoprim anion, and the interaction between the sulfathiazole cation and the trimethoprim anion includes ionic bonds, hydrogen bonds and π-π stacking. Specifically, the sulfathiazole cation and the trimethoprim anion of the present invention are mainly combined together by ionic bonds. As one embodiment of the present invention, the sulfathiazole cation and the trimethoprim anion are connected by two pairs of intermolecular hydrogen bonds, and the bond lengths of the two pairs of intermolecular hydrogen bonds are 1.922Å and 2.123(2)Å respectively. As one embodiment of the present invention, of the two pairs of intermolecular hydrogen bonds, the first pair of hydrogen bonds N31-H31B···N13 is formed by N31-H31B of trimethoprim and N13 of sulfathiazole; the second pair of hydrogen bonds N25-H25···N10 is formed by N25-H25 of trimethoprim and N10 of sulfathiazole. At the same time, in terms of symmetry operation, N31-H31A of trimethoprim forms an intermolecular hydrogen bond with O9 of sulfathiazole (symmetry operation 1: -x+1, y-1 / 2, -z+1 / 2), with a hydrogen bond length of 2.895(2)Å; N30-H30B of trimethoprim forms an intermolecular hydrogen bond with O8 of sulfathiazole (symmetry operation 2: x+1 / 2, y, -z+1 / 2), with a hydrogen bond length of 2.869(2)Å. As an embodiment of the present invention, in the sulfathiazole-trimethoprim drug crystal salt, the face-to-face stacking of adjacent benzene rings in sulfathiazole forms a π-π stacking interaction force, and the distance between adjacent parallel planes is 0.35448nm. In the present invention, ionic bonds, hydrogen bond interactions, and π-π stacking interactions jointly maintain the stability of the sulfathiazole-trimethoprim drug crystal salt structure.

[0022] As an embodiment of the present invention, the melting point of the sulfathiazole-trimethoprim drug crystal salt is 169.5°C.

[0023] The present invention provides a method for preparing the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution, comprising the following steps: Dissolving sulfathiazole and trimethoprim in a solvent to obtain a mixed solution; the solvent is acetone and ethanol; The mixed solution is crystallized to obtain the sulfathiazole-trimethoprim drug crystal salt.

[0024] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known to those skilled in the art or are prepared using methods well known to those skilled in the art.

[0025] The present invention dissolves sulfathiazole and trimethoprim in a solvent to obtain a mixed solution. In the present invention, the solvent is acetone and ethanol, and the ethanol is specifically anhydrous ethanol. The volume ratio of the acetone to the ethanol can be 1:0.25-4, further can be 1:0.5-2, and specifically can be 1:1. As one embodiment of the present invention, the molar ratio of the sulfathiazole to the trimethoprim can be 1:1. The amount ratio of the sulfathiazole to the solvent can be 0.5-2.5 mmol:30 mL, further can be 0.5-1 mmol:30 mL.

[0026] After obtaining the mixed solution, the present invention crystallizes the mixed solution to obtain the sulfathiazole-trimethoprim drug crystal salt. As one embodiment of the present invention, the crystallization temperature can be 15-40°C, specifically room temperature (25°C), and the crystallization time can be 4-6 days, specifically 5 days. In the practice of the present invention, the mixed solution is placed in a beaker, the beaker containing the mixed solution is sealed with a semipermeable membrane, and then holes are punctured in the semipermeable membrane. The crystallization is then performed under static conditions.

[0027] The present invention provides the use of the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in the above technical solution in the preparation of drugs for treating bacterial and / or fungal infectious diseases.

[0028] As one embodiment of the present invention, the bacteria include one or more of the Gram-positive bacteria Staphylococcus aureus, the Gram-negative bacteria Klebsiella pneumoniae, Shigella dysenteriae, and Pseudomonas aeruginosa; the fungus may include Candida albicans. As one embodiment of the present invention, the content of sulfathiazole-trimethoprim drug crystal salt in the drug may be 50-99.9wt%, and further may be 80-95wt%. In the present invention, the drug includes pharmaceutically acceptable excipients, and the pharmaceutically acceptable excipients may include one or more of starch, sodium carboxymethyl cellulose, and magnesium stearate; the dosage form of the drug may be tablets, capsules, or eye drops.

[0029] The present invention provides a drug for treating bacterial and / or fungal infections, the active ingredient of which comprises the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in the above technical solution. The content of the sulfathiazole-trimethoprim drug crystal salt, the types of pharmaceutically acceptable excipients, and the dosage form of the drug are consistent with those in the above technical solution and are not further described here.

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] Some of the drugs and reagents used in the following experiments are shown in Table 1.

[0032] Table 1 Drugs and reagents

[0033] The instruments used in the following experiments are shown in Table 2.

[0034] Table 2 Instruments

[0035] Example 1 The steps for preparing sulfathiazole (STZ)-trimethoprim (TMP) drug crystal salt are as follows: STZ (0.1275 g, 0.5 mmol) and TMP (0.1450 g, 0.5 mmol) were dissolved in 30 mL of an acetone-anhydrous ethanol mixed solvent (the volume ratio of acetone to anhydrous ethanol was 1:1 by volume). The resulting mixed solution was placed in a beaker, and the beaker containing the mixed solution was sealed with a semipermeable membrane. The semipermeable membrane was then punctured and crystallized at room temperature (25°C) for 5 days to obtain white, transparent, fine needle-shaped crystals, i.e., STZ-TMP drug crystalline salt, with a yield of 70%.

[0036] Test Example 1 Characterization of the Physical and Chemical Properties of Sulfathiazole-Trimethoprim Drug Crystalline Salt 1. Single Crystal X-ray Diffraction (SCXRD) Single crystal X-ray diffraction experiments were performed using a single crystal X-ray diffractometer (Xtal AB) produced by Rigaku Corporation, Japan. α All data were integrated in the CrysAlisPro software suite, and surface-indexed absorption correction was applied to each acquired data set. The crystal structure was solved using the SHELXT program, and structure refinement was performed using SHELXT-2018 / 3 within the Olex2 graphical user interface. All non-hydrogen atoms were anisotropically refined, and most hydrogen atoms were generated based on the ideal positions of carbon or nitrogen atoms. Some hydrogen atoms were calculated using Fourier electron cloud density. The asymmetric unit structure diagram of the STZ-TMP drug crystalline salt was drawn using Diamond 3.2 software.

[0037] The single crystal structure data of the STZ-TMP drug crystalline salt were collected at room temperature, and its crystal data and structure refinement parameters are summarized in Table 3. The hydrogen bonds within the STZ-TMP drug crystalline salt are summarized in Table 4. Figure 1 Schematic diagram of the asymmetric unit structure of the STZ-TMP drug crystal salt with atomic labels (specifically, the ORTEP schematic diagram).

[0038] Table 3 Crystallographic data and structure refinement parameters of STZ-TMP drug crystal salt at room temperature

[0039] Table 4 Hydrogen bonds in STZ-TMP drug crystal salt

[0040] Note: Symmetry operation 1: -x+1, y-1 / 2, -z+1 / 2; Symmetry operation 2: x+1 / 2, y, -z+1 / 2.

[0041] The single crystal X-ray structure determination in Table 3 shows that the STZ-TMP drug crystal salt belongs to the orthorhombic system with a space group of P bca. Its unit cell parameters are: a =17.3213(3)Å, b =12.2894(2)Å, c =23.7549(3)Å, α =90°, β =90°, γ =90°, V =5055.82(14)Å 3 , Z= 8. The asymmetric unit of the STZ-TMP drug crystal salt contains a sulfathiazole cation [C9H8N3O2S2] + With a trimethoprim anion [C 14 H 19 N4O3] - It should be noted that the proton is transferred from the secondary amine N10 atom of sulfathiazole to the N25 atom of the pyrimidine ring of trimethoprim. Therefore, the sulfathiazole cation [C9H8N3O2S2] + With trimethoprim anion [C 14 H 19 N4O3] -They are mainly bound together by electrostatic attraction (ionic bond). At the same time, the STZ cation is connected to the TMP anion by hydrogen bonds, and the hydrogen bond lengths are 2.123(2)Å and 1.922Å, respectively (Table 4). The first pair of hydrogen bonds N31-H31B···N13 is formed by N31-H31B of TMP and N13 of STZ. The second pair of hydrogen bonds N25-H25···N10 is formed by N25-H25 of TMP and N10 of STZ ( Figure 1 ). At the same time, in terms of symmetry operation, N31-H31A of TMP forms an intermolecular hydrogen bond with O9 of STZ (symmetry operation 1: -x+1, y-1 / 2, -z+1 / 2) atom, with a hydrogen bond length of 2.895(2)Å; N30-H30B of TMP forms an intermolecular hydrogen bond with O8 of STZ (symmetry operation 2: x+1 / 2, y, -z+1 / 2), with a hydrogen bond length of 2.869(2)Å (Table 4). In addition, in the STZ-TMP drug crystal salt, the face-to-face stacking of adjacent benzene rings in STZ forms a π-π stacking force, and the distance between adjacent parallel planes is 0.35448nm. Ionic bonds, hydrogen bond interactions and π-π stacking interactions jointly maintain the stability of the STZ-TMP drug crystal salt structure.

[0042] 2. Powder X-ray Diffraction (PXRD) Powder X-ray diffraction test was performed using TD-3500 X-ray powder diffractometer. A small amount of STZ, TMP, and STZ-TMP drug crystal salt was taken, dried, ground into fine powder, and then evenly spread in the sample tank. α ray( λ =1.54060Å) as the X-ray source, with the tube voltage and current set to 35 kV and 20 mA, respectively. The scanning range was 5°–50°, with a step size of 0.02° / s. Data were collected every 0.5 s. Powder X-ray diffraction patterns of STZ, TMP, and STZ-TMP drug crystal salts were obtained. Simulated powder X-ray diffraction patterns of STZ-TMP drug crystal salts were calculated using Mercury 2023 software (Center for Crystallographic Data, Cambridge, UK).

[0043] Powder X-ray diffraction is a fast, convenient, and reliable analytical method for verifying the formation of new compounds and the purity of samples. Figure 2The powder X-ray diffraction spectra of STZ, TMP and STZ-TMP drug crystal salts are consistent with the corresponding literature (Wang LY, Bu FZ, Li YT, et al. A sulfathiazole-amantadine hydrochloride cocrystal: The first codrug simultaneouslycomprising antiviral and antibacterial components [J]. Cryst Growth Des,2020, 20(5): 3236-3246;Maddileti D, Swapna B, Nangia A K. Tetramorphs of theantibiotic drug trimethoprim: Characterization and stability [J]. CrystGrowth Des, 2015, 15(4): 1745-1756), the characteristic peaks of the STZ-TMP drug crystalline salt are 11.54°, 17.06°, 19.50°, 20.96°, 21.44°, 22.60°, 25.92°, and 26.24°. Compared with the two raw materials, the STZ-TMP drug crystalline salt exhibits new peaks and disappears old peaks, indicating the formation of a new compound. The powder diffraction pattern of the STZ-TMP drug crystalline salt tested by PXRD also matches the SCXRD simulation data, indicating that the prepared STZ-TMP drug crystalline salt is of high purity, which is beneficial for subsequent testing.

[0044] 3. Fourier Transform Infrared Spectroscopy (FTIR) Appropriate amounts of STZ, TMP, and STZ-TMP drug crystal salt samples were weighed and ground with potassium bromide at a mass ratio of 1:100. After removing static electricity, the tablets were pressed for measurement. Infrared spectral data were obtained by scanning the 4000-400 cm-1 region on a PerkinElmer Spectrum 2 Fourier transform infrared spectrometer. -1 Range obtained.

[0045] Infrared technology is a powerful tool for identifying hydrogen bonds, which can be used to verify the interactions between the components in crystal salts. Figure 3 The following are Fourier transform infrared spectra of STZ, TMP, and STZ-TMP drug crystal salts. In the infrared spectrum of TMP, the symmetric and asymmetric stretching vibrations of N-H appear at 3318 cm-1, respectively. -1 and 3469cm -1In the infrared spectrum of STZ-TMP drug crystal salt, the symmetric and asymmetric stretching vibrations of N−H are 3340 cm -1 and 3410cm -1 This indicates that the peak shift is caused by the formation of N31−H31B···N13 hydrogen bonds between TMP and STZ. In the infrared spectrum of STZ, the symmetric and asymmetric stretching vibration shifts of −NH2 are 3280 cm -1 and 3320cm -1 . The characteristic peak intensity of −NH− in the infrared spectrum is weaker than that of −NH2 in this region, and will be masked by −NH2. Therefore, the formation of hydrogen bond N25−H25···N10 cannot be directly determined by infrared shift alone. However, since the STZ-TMP drug crystal salt is an ionic compound, it can be indirectly judged by comparing the differences in the symmetric and asymmetric stretching vibration shifts of −SO2 in STZ and STZ-TMP drug crystal salts. In STZ, the symmetric and asymmetric stretching vibrations of −SO2 in the sulfonamide group are 1137 cm -1 and 1323cm -1 , while in STZ-TMP drug crystal salt, they become 1128 cm -1 and 1310cm -1 In addition, the bending vibration of the C=N of the thiazole ring should be 1531cm in STZ. -1 The single peak is 1527 cm in the STZ-TMP drug crystal salt. -1 and 1504cm -1 This indicates that these positions may be affected by the formation of ionic bonds.

[0046] 4. Thermogravimetric analysis and differential scanning calorimetry (TGA / DSC) Differential scanning calorimetry (DSC) analysis was performed on a PerkinElmer DSC 4000. 3–6 mg of STZ, TMP, and STZ-TMP drug salt crystals were weighed into an alumina crucible. The aluminum pans were pressed into pellets, and the instrument scanned the sample between 30 and 250°C at a heating rate of 10°C / min. Thermogravimetric analysis (TGA) studies were performed on a PerkinElmer TGA 8000, with the sample placed in an alumina crucible. The tests were performed in a nitrogen atmosphere over a temperature range of 30–500°C at a heating rate of 10°C / min.

[0047] Figure 4Figure 3 is the DSC curve of STZ, TMP and STZ-TMP drug crystal salt. The results show that the melting point of TMP is 202.0℃; STZ may undergo crystal transformation at 172.5℃, and the final melting point is 202.5℃; the melting point of STZ-TMP drug crystal salt is different from the other two, and its melting point is significantly lower, at 169.5℃, which also indicates that it has formed a new compound and there is no crystal transformation before the melting point.

[0048] Figure 5 The TGA curves for STZ, TMP, and STZ-TMP drug crystal salts show that they all decompose after reaching their melting point, which is reflected by a decrease in mass on the thermogravimetric analysis curve. It is also important to note that no mass loss was observed for the STZ-TMP drug crystal salt before its melting point, further confirming the absence of solvent molecules within the molecule, a conclusion confirmed by SCXRD.

[0049] 5. Equilibrium Solubility Drug solubility is a key physical parameter of a drug, significantly impacting its absorption, oral bioavailability, and drugability. In this experiment, solubility was assessed using an excess powder dissolution method and analyzed by spectrophotometry to generate a standard curve. Accurately weighed STZ, TMP, and STZ-TMP drug crystals were dissolved in 10 mL of dilute hydrochloric acid (pH 1.2), phosphate buffer (pH 6.8), and pure water (pH 7.0), respectively. The mixtures were shaken in a waterbath (37°C, 200 rpm) for 24 hours and then filtered through a 0.22 μm microporous filter to obtain saturated solutions. The saturated solutions were diluted to the appropriate concentrations and substituted into the corresponding standard curve equations to determine the concentrations of the saturated solutions. Absorbance was measured at different detection wavelengths using a UV9100 UV-Vis spectrophotometer. Measurements were repeated three times.

[0050] Figure 6The following figure compares the solubility of STZ, TMP, and STZ-TMP drug crystal salts at different pH values. The results show that the solubility of TMP, STZ, and STZ-TMP drug crystal salts exhibits a consistent trend with pH: pH 1.2 > pH 6.8 > pH 7.0. At pH 1.2 (gastric acid environment), the solubility of STZ released from STZ-TMP drug crystal salt is 10.96 g / L, while the solubility of STZ alone is 4.39 g / L, a 2.50-fold increase. The solubility of TMP released from STZ-TMP drug crystal salt is 11.03 g / L, while the solubility of TMP alone is 3.02 g / L, a 3.65-fold increase. At pH 6.8 (small intestine), the solubility of STZ released from the STZ-TMP crystal salt was 2.29 g / L, while the solubility of STZ alone was 0.56 g / L, a 4.09-fold increase. The solubility of TMP released from the STZ-TMP crystal salt was 2.31 g / L, while the solubility of TMP alone was 0.46 g / L, a 5.02-fold increase. At pH 7.0, the solubility of STZ released from the STZ-TMP crystal salt was 0.98 g / L, while the solubility of STZ alone was 0.32 g / L, a 3.06-fold increase. The solubility of TMP released from the STZ-TMP crystal salt was 0.94 g / L, while the solubility of TMP alone was 0.29 g / L, a 3.24-fold increase. In summary, STZ-TMP drug crystal salt significantly increased the solubility of STZ and TMP under all pH conditions, particularly in the acidic environment of the stomach (pH 1.2) and the small intestine (pH 6.8). This suggests that STZ-TMP drug crystal salt can effectively increase the solubility of STZ and TMP, potentially promoting absorption and enhancing drug therapeutic efficacy, reducing drug dosage, and improving patient compliance.

[0051] Test Example 2 Study on the biological activity of sulfathiazole-trimethoprim drug crystal salt 1. In vitro antibacterial activity test In order to evaluate the antibacterial activity of STZ, TMP, STZ-TMP physical mixture and STZ-TMP drug crystal salt, the minimum inhibition test was conducted on Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Klebsiella pneumoniae, Shigella dysenteriae and Pseudomonas aeruginosa) and fungi (Candida albicans), as follows: (1) Preparation of culture medium and sample solution Prepared broth (LB) and nutrient agar (NA) media were autoclaved at 121°C for 30 min and set aside. STZ (0.0255 g, 0.1 mmol), TMP (0.0290 g, 0.1 mmol), a physical mixture of STZ and TMP (0.0545 g, 0.1 mmol each), and a crystalline STZ-TMP salt (0.0545 g, 0.1 mmol) were weighed and dissolved in 5 mL of dimethyl sulfoxide (DMSO). Ultrapure water was then added to 100 mL, giving a final concentration of 1 μmol / mL for each of the four samples. A mixed solution of DMSO and water (5:95 volume ratio) was used as a control solution. Finally, each solution was sterilized by filtering once through a 0.22 μm sterile filter.

[0052] (2) Preparation of bacterial solution Five bacteria (from the laboratory of Jiangxi University of Traditional Chinese Medicine, School of Basic Medical Sciences), namely Staphylococcus aureus ( S. aureus ), Klebsiella pneumoniae ( K. Pneumoniae ), Shigella dysenteriae ( S. dysenteriae ), Pseudomonas aeruginosa ( P. aeruginosa ) and Candida albicans ( C. albicans ) into a sterile test tube containing 6 mL of LB medium and incubate in a constant temperature shaking incubator at 37°C and 200 rpm for 24 hours. Dilute the bacterial solution to a desired multiple and then spread it onto a prepared culture dish containing NA medium. After incubation in a 37°C incubator for 24 hours, calculate the concentration of each bacterial solution by colony count and store at 2-8°C until needed.

[0053] (3) Determination of antibacterial rate by microbroth dilution method The microbroth dilution method was used to determine the effects of STZ, TMP, STZ-TMP physical mixture and STZ-TMP drug crystal salt on the tested strains ( S. aureus 、 K. Pneumoniae 、 S. dysenteriae 、 P. aeruginosa and C. albicans ) inhibition rate. 100 μL of different sample solutions at a concentration of 1 μmol / mL were added to the wells of the first column of a 96-well plate and then serially diluted to obtain six different drug concentrations. The positive control group consisted of a bacterial solution in LB medium, the negative control group consisted of a drug solution in LB medium, and the blank control group consisted of pure LB medium. After incubating the plates in a 37°C incubator for 24 hours, the absorbance (OD) at 600 nm was measured using a microplate reader to determine the inhibition rate of the four samples. All tests were repeated three times.

[0054] (4) Determination of antibacterial properties by Oxford cup method Take 100 μL of the solution with a concentration of 1×10 7 A bacterial suspension of 100 CFU / mL was evenly spread on a sterile culture dish containing NA medium. Five Oxford cups were evenly placed in each culture dish. 100 μL of 2 μmol / mL STZ, TMP, STZ-TMP physical mixture, and STZ-TMP drug crystal salt solution were added to four of the Oxford cups respectively. At the same time, the prepared control solution was added to the remaining Oxford cup as a control group. The culture dish with the Oxford cup was pre-diffused at room temperature for 30 minutes, then placed in a 37°C incubator for 24 hours, and then the size of the inhibition zone was measured. Each group was repeated 3 times in parallel.

[0055] The minimum inhibitory concentrations (MIC, μmol / mL) of STZ, TMP, STZ-TMP physical mixture and STZ-TMP drug crystal salt against Staphylococcus aureus, Candida albicans, Klebsiella pneumoniae, Shigella dysenteriae and Pseudomonas aeruginosa are shown in Tables 5 to 9, respectively. The inhibition zone diameters of STZ, TMP, STZ-TMP physical mixture and STZ-TMP drug crystal salt are shown in Table 10.

[0056] Table 5 Minimum inhibitory concentration of each drug against Staphylococcus aureus

[0057] Table 6 Minimum inhibitory concentration of each drug against Candida albicans

[0058] Table 7 Minimum inhibitory concentration of each drug against Klebsiella pneumoniae

[0059] Table 8 Minimum inhibitory concentration of each drug against Shigella dysenteriae

[0060] Table 9 Minimum inhibitory concentration of each drug against Pseudomonas aeruginosa

[0061] Tables 5-9 show that the four drugs exhibited significantly greater inhibition against Gram-positive bacteria than against Gram-negative bacteria and fungi. This is because the rigid cell walls of Gram-positive bacteria cannot prevent the entry of small-molecule antimicrobial drugs, while the complex outer membrane structure and composition of Gram-negative bacteria reduce the effectiveness of small-molecule antimicrobial drugs. For Gram-negative bacteria (such as Klebsiella pneumoniae, Shigella dysenteriae, and Pseudomonas aeruginosa) and fungi (such as Candida albicans), at high sample concentrations, the inhibition rates of both STZ-TMP crystal salt and the STZ-TMP physical mixture were higher than those of STZ and TMP alone; the order was STZ-TMP crystal salt > STZ-TMP physical mixture > STZ > TMP. Furthermore, for Gram-negative bacteria (such as Klebsiella pneumoniae) and fungi (such as Candida albicans), STZ-TMP crystal salt reached the MIC (i.e., 90% inhibition) earlier than the STZ-TMP physical mixture. The MIC of STZ-TMP drug crystal salt against Klebsiella pneumoniae and Candida albicans was 0.25 μmol / mL, while the MIC of STZ-TMP physical mixture against Klebsiella pneumoniae and Candida albicans was greater than 0.25 μmol / mL; in addition, for Pseudomonas aeruginosa, the inhibition rate of STZ-TMP drug crystal salt and STZ-TMP physical mixture at a concentration of 0.5 μmol / mL was greater than 90%, while the inhibition rate of STZ at a concentration of 1 μmol / mL was greater than 90%, and the inhibition rate of TMP at a concentration of 1 μmol / mL was only about 25.9%.

[0062] For Gram-positive bacteria (Staphylococcus aureus), TMP has a positive effect on Staphylococcus aureus ( S. aureus) has a greater degree of inhibition than STZ, which may be due to the different permeability of the two drugs. TMP belongs to BCS class II (Piccirillo G, Aroso R, Baptista JA, et al. Trimethoprim-based multicomponent solidsystems: Mechanochemical screening, characterization and antibacterialactivity assessment [J]. Int J Pharmaceut, 2024, 661: 124416) (low solubility and high permeability), while STZ belongs to BCS class IV (de Moura Oliveira CH, C. de Melo C, Doriguetto A C.Sulfamethoxazole salts: Crystal structures, conformations and solubility [J]. New J Chem, 2019, 43(26): 10250-10258) (low solubility and low permeability), which makes TMP easier to penetrate the cell wall of Staphylococcus aureus than STZ, thereby producing a better antibacterial effect.

[0063] Table 10 Inhibition zone diameters of various drugs (all concentrations are 2 μmol / mL, unit: mm)

[0064] Note: In the determination of antibacterial effect: the inhibition zone diameter > 20 mm is extremely sensitive (++), 15-20 mm is highly sensitive (++), 10-15 mm is moderately sensitive (+), and the inhibition zone diameter < 10 mm is low sensitive (Zhou J, Li N, Liu P, et al. Preparation of fluorescently labeled chitosan-quercetin drug-loaded nanoparticles with excellent antibacterial properties [J]. J Funct Biomater, 2022, 13(3): 141).

[0065] From the above results, it can be seen that TMP has a strong antibacterial effect on Staphylococcus aureus, and the inhibition rate has reached 99% at a concentration of 0.0039μmol / mL. Therefore, in the Oxford Cup experiment, only STZ, TMP, STZ-TMP physical mixture and STZ-TMP drug crystal salt were tested for inhibition zone against Gram-negative bacteria and fungi. As can be seen from Table 10, at a concentration of 2μmol / mL, the two raw materials had poor inhibition effects on the four bacteria. In contrast, due to the synergistic effect of the STZ-TMP physical mixture, its inhibition zone was expanded on the four strains. The diameters of the inhibition zones for Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa and Candida albicans were approximately 14.3mm, 11.8mm, 12.2mm and 12.6mm, respectively, but they were still in the moderately sensitive range. It is worth noting that STZ-TMP drug crystal salt showed a stronger inhibitory effect, with the inhibition zone diameters of Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa and Candida albicans being approximately 17.1mm, 14.8mm, 15.0mm and 15.9mm, respectively, all reaching high sensitivity. Among them, the inhibition zone diameter for Klebsiella pneumoniae was approximately 2.1 times and 2.4 times that of STZ and TMP, respectively, and 1.2 times that of the STZ-TMP physical mixture. Taking Candida albicans as an example, the inhibition zone diameter of the STZ-TMP physical mixture for Candida albicans was approximately 12.6mm, and the inhibition zone diameter of the STZ-TMP drug crystal salt for Candida albicans was approximately 15.9mm, which was an increase of approximately 26.19% compared with the STZ-TMP physical mixture. Compared to physical mixtures, after the STZ-TMP drug crystal salt is dissolved in the medium, STZ-TMP may appear in a whole form, acting synergistically on bacteria; however, after the STZ-TMP physical mixture is dissolved in the medium, the two active ingredients STZ and TMP may not form a whole, which may be the reason why the antibacterial effect of the STZ-TMP drug crystal salt is stronger than that of the STZ-TMP physical mixture. This shows that the STZ-TMP drug crystal salt can improve the drug's physical and chemical properties while also improving its biological activity.

[0066] 2. Inhibition of DHFS and DHFR Enzyme Activity Test DHFS and DHFR enzymes are ubiquitous in microorganisms and are essential for nucleic acid synthesis. STZ and TMP achieve their antimicrobial effects by inhibiting the activity of these two enzymes, respectively. In this experiment, DHFS and DHFR enzyme-linked immunosorbent assay kits (Jiangxi Apid Biotechnology Co., Ltd.) were used to test the inhibitory effects of STZ, TMP, a physical mixture of STZ and TMP, and a crystal salt of STZ and TMP on these enzymes. A 450 U / L standard was diluted with standard diluent to 300 U / L, 200 U / L, 100 U / L, 50 U / L, and 25 U / L to establish a standard curve. The 450 U / L standard was diluted to 250 U / L for later use. 40 μL of the 250 U / L standard was added to the experimental wells, followed by 10 μL of a 2 μmol / mL solution of STZ, TMP, a physical mixture of STZ and TMP, and a crystal salt of STZ and TMP. The standard concentration in each well was 200 U / L. Therefore, 50 μL of a 200 U / L standard was used as a control. The OD values at a wavelength of 450 nm were measured using a SpectraMax M2 microplate reader. These values were then inserted into the standard curve to calculate the degree of inhibition of DHFS and DHFR activity by the four samples. Three parallel groups were set up for each sample.

[0067] Figure 7 The following chart compares the inhibitory effects of STZ, TMP, a physical mixture of STZ and TMP, and a crystal salt of STZ and TMP on DHPS and DHFR enzymes. The results show that in the wells containing STZ, the measured DHPS enzyme activity was 152.23 U / L, while the DHFR enzyme activity was 190.35 U / L. This indicates that STZ primarily acts on the DHPS enzyme and has little effect on the DHFR enzyme. In contrast, the data measured in the wells containing TMP were opposite to those of STZ, with the DHPS and DHFR enzyme activities measured at 192.73 U / L and 157.26 U / L, respectively. In the wells containing the physical mixture of STZ and TMP, the measured DHPS and DHFR enzyme activities were 151.79 U / L and 159.04 U / L, respectively, reflecting the nearly identical inhibition levels of STZ and TMP on these two enzymes. In the experimental wells where STZ-TMP drug crystal salt was added, the measured DHPS enzyme and DHFR enzyme activities were 124.69 U / L and 139.67 U / L, respectively. Compared with the two APIs, the degree of inhibition of these two enzymes was increased by 28 U / L and 18 U / L, respectively. This shows that the STZ-TMP drug crystal salt is not a simple combination of two APIs and has a "1+1=2" effect. On the contrary, the STZ-TMP drug crystal salt of the present invention can improve the physicochemical properties of the drug, enabling it to better exert its inhibitory effect on these two enzymes, thereby achieving a "1+1>2" effect.

[0068] Comparative Example 1 The method of Example 1 was used, except that different APIs and solvents were used, as shown in Tables 11 to 13. The molar ratio of API 1 to API 2 in Tables 11 to 13 was 1:1. As shown in Tables 11 to 13, no cocrystal or crystalline salt was formed using the APIs and solvents used in Comparative Example 1.

[0069] Table 11 API (API 1 is sulfathiazole) and solvent used in Comparative Example 1

[0070] Table 12 APIs (API 1 is trimethoprim) and solvents used in Comparative Example 1

[0071] Table 13 API (trimethoprim + sulfadiazine / mafenamide) and solvent used in Comparative Example 1

[0072] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A sulfathiazole-trimethoprim drug crystal salt, characterized in that: It is an orthorhombic crystal system with a space group of P bca; the unit cell parameters are: a =17.3213(3)Å, b =12.2894(2)Å, c =23.7549(3)Å, α =90°, β =90°, γ =90°; wherein the molar ratio of sulfathiazole to trimethoprim is 1:1, and there is no crystallization solvent.

2. The sulfathiazole-trimethoprim drug crystal salt according to claim 1, characterized in that Using Cu-K α Radiation, with 2 θ Angles indicate that the characteristic diffraction peaks of the powder X-ray diffraction spectrum of the sulfathiazole-trimethoprim drug crystalline salt include 11.54°, 17.06°, 19.50°, 20.96°, 21.44°, 22.60°, 25.92° and 26.24°.

3. The sulfathiazole-trimethoprim drug crystal salt according to claim 1 or 2, characterized in that The asymmetric unit of the sulfathiazole-trimethoprim drug crystal salt comprises a sulfathiazole cation and a trimethoprim anion, and the interaction modes between the sulfathiazole cation and the trimethoprim anion include ionic bond, hydrogen bond and π-π stacking.

4. The sulfathiazole-trimethoprim drug crystal salt according to claim 3, characterized in that The melting point of the sulfathiazole-trimethoprim drug crystal salt is 169.5°C.

5. The method for preparing the sulfathiazole-trimethoprim drug crystal salt according to any one of claims 1 to 4, characterized in that: The following steps are involved: Dissolving sulfathiazole and trimethoprim in a solvent to obtain a mixed solution; the solvent is acetone and ethanol; The mixed solution is crystallized to obtain the sulfathiazole-trimethoprim drug crystal salt.

6. The preparation method according to claim 5, characterized in that The molar ratio of sulfathiazole to trimethoprim is 1:1; the volume ratio of acetone to ethanol is 1:0.25-4; and the dosage ratio of sulfathiazole to solvent is 0.5-2.5 mmol:30 mL.

7. The preparation method according to claim 5 or 6, characterized in that: The crystallization temperature is 15-40° C. and the crystallization time is 4-6 days.

8. Use of the sulfathiazole-trimethoprim drug crystal salt according to any one of claims 1 to 4 or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a medicament for treating bacterial and / or fungal infectious diseases.

9. The use according to claim 8, characterized in that The bacteria include one or more of the gram-positive bacteria Staphylococcus aureus, the gram-negative bacteria Klebsiella pneumoniae, Shigella dysenteriae and Pseudomonas aeruginosa; the fungi include Candida albicans.

10. A drug for treating bacterial and / or fungal infectious diseases, characterized in that: The active ingredient comprises the sulfathiazole-trimethoprim drug crystal salt according to any one of claims 1 to 4 or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method according to any one of claims 5 to 7.

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