A sulfathiazole-trimethoprim drug crystal salt, its preparation method and application

Abstract

This invention provides a sulfathiazole-trimethoprim drug crystal salt, its preparation method, and its application, belonging to the field of drug crystal salt technology. The sulfathiazole-trimethoprim drug crystal salt provided by this invention is orthorhombic with a space group of [space group missing]. P bca; cell parameters are: a =17.3213(3)Å, b =12.2894(2)Å, c =23.7549(3)Å, α =90°, β =90°, c =90°; wherein the molar ratio of sulfathiazole to trimethoprim is 1:1, and there is no crystallizing solvent. The sulfathiazole-trimethoprim drug crystal salt provided by this invention has high solubility in water, which is beneficial to improving absorption and metabolism in humans and animals, and has excellent antibacterial effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical crystal salt technology, and in particular to a sulfathiazole-trimethoprim pharmaceutical crystal salt, its preparation method, and its application. Background Technology

[0002] Compound sulfamethoxazole is a common antibacterial drug, a combination of sulfamethoxazole and trimethoprim (TMP), in which TMP inhibits bacterial DHFR enzymes. This antibacterial drug has advantages such as ease of use, stability, and low cost, and is widely used clinically. However, with the widespread use of synthetic antibacterial drugs, bacterial resistance is becoming increasingly serious, making the development of more novel antibacterial drugs a pressing technical problem to be solved in this field.

[0003] Sulfathiazole (STZ) is a typical sulfonamide antibiotic that inhibits bacterial DHPS enzymes and belongs to the broad-spectrum antibacterial class. Combining STZ with TMP can block bacterial folic acid metabolism, thereby enhancing the antibacterial effect and reducing the emergence of drug-resistant strains. However, both drugs have low solubility, resulting in poor absorption and metabolism in humans and animals. Summary of the Invention

[0004] The purpose of this invention is to provide a sulfathiazole-trimethoprim drug crystal salt, its preparation method, and its application. The sulfathiazole-trimethoprim drug crystal salt provided by this invention has high solubility in water, which is beneficial to improving its absorption and metabolism in humans and animals, and has excellent antibacterial effects.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a sulfathiazole-trimethoprim drug crystal salt, which is orthorhombic and has a space group of [missing information]. P bca; 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 crystallizing solvent.

[0007] Preferably, Cu-K is used. α Radiation, at 2 θ The angle indicates that the characteristic diffraction peaks of the powder X-ray diffraction pattern of the sulfathiazole-trimethoprim drug crystal salt include 11.54°, 17.06°, 19.50°, 20.96°, 21.44°, 22.60°, 25.92° and 26.24°.

[0008] Preferably, the asymmetric unit of the sulfathiazole-trimethoprim drug crystal salt comprises a sulfathiazole cation and a trimethoprim anion, wherein the interaction between the sulfathiazole cation and the trimethoprim anion includes ionic bonding, hydrogen bonding, and π-π stacking.

[0009] Preferably, the melting point of the sulfathiazole-trimethoprim drug crystal salt is 169.5°C.

[0010] This invention provides a method for preparing the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution, comprising the following steps:

[0011] Sulfathiazole and trimethoprim are dissolved in a solvent to obtain a mixed solution; the solvent is acetone and ethanol.

[0012] The mixed solution was crystallized to obtain the sulfathiazole-trimethoprim drug crystal salt.

[0013] 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 volume ratio of sulfathiazole to solvent is 0.5~2.5 mmol:30 mL.

[0014] Preferably, the crystallization temperature is 15~40℃ and the time is 4~6 days.

[0015] This invention provides the application of the sulfathiazole-trimethoprim drug crystal salt described in the above-described technical solution or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in the above-described technical solution in the preparation of drugs for treating bacterial and / or fungal infectious diseases.

[0016] Preferably, the bacteria include one or more of the following: Gram-positive Staphylococcus aureus, Gram-negative Klebsiella pneumoniae, Shigella dysenteriae, and Pseudomonas aeruginosa; and the fungus includes Candida albicans.

[0017] This invention provides a medicament for treating bacterial and / or fungal infectious diseases, the active ingredient of which includes 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.

[0018] Beneficial effects: This invention provides a sulfathiazole-trimethoprim drug crystal salt, which is orthorhombic and has a space group of [missing information]. P bca; 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. This invention designed and successfully synthesized a drug crystal salt of STZ and TMP, namely STZ-TMP drug crystal salt, which has high solubility in water, beneficial for improving absorption and metabolism in humans and animals, and exhibits excellent antibacterial effects. The structure of the STZ-TMP drug crystal salt was characterized in the test examples of this invention. Single-crystal X-ray diffraction showed that the molar ratio of the two components in the drug crystal salt structure is 1:1, and STZ and TMP are mainly bonded together by electrostatic attraction (ionic bonds). Ionic bonds, hydrogen bonds, and π-π stacking interactions jointly maintain the stability of the crystal structure. It is worth mentioning that the STZ-TMP drug crystal salt of this invention can improve the solubility of the two active pharmaceutical ingredients. Furthermore, compared with a physical mixture of STZ-TMP, the STZ-TMP drug crystal salt of this invention exhibits enhanced antibacterial activity, a larger inhibition zone diameter, and enhanced inhibitory ability against microbial DHPS and DHFR enzymes. This invention demonstrates that by forming drug crystal salts from STZ and TMP, compared to physical mixtures of STZ and TMP, STZ and TMP can produce synergistic effects more efficiently within the same system, significantly improving the efficacy of combined drug therapy and bringing new possibilities for overcoming intractable diseases. Attached Figure Description

[0019] Figure 1 A schematic diagram of the asymmetric unit structure of STZ-TMP drug crystal salt with atomic labels;

[0020] Figure 2 Powder X-ray diffraction patterns of STZ, TMP, and STZ-TMP drug crystal salts;

[0021] Figure 3 Fourier transform infrared spectra of STZ, TMP, and STZ-TMP drug crystal salts;

[0022] Figure 4 DSC curves for STZ, TMP, and STZ-TMP drug crystal salts;

[0023] Figure 5 TGA curves for STZ, TMP, and STZ-TMP drug crystal salts;

[0024] Figure 6 A comparison graph showing the solubility of STZ, TMP, and STZ-TMP drug crystal salts under different pH conditions;

[0025] Figure 7 A comparative diagram showing the inhibitory effects of STZ, TMP, STZ-TMP physical mixtures, and STZ-TMP drug crystal salt on DHPS and DHFR enzymes. Detailed Implementation

[0026] This invention provides a sulfathiazole-trimethoprim drug crystal salt, which is orthorhombic and has a space group of [missing information]. P bca; 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 crystallizing solvent.

[0027] In related technologies, sulfathiazole (STZ) and trimethoprim (TMP) are used in combination, but this cannot alter the physicochemical properties of the active pharmaceutical ingredients themselves. This invention utilizes a drug crystallization technique to form drug crystals from STZ and TMP, which significantly enhances the physicochemical properties (such as solubility, stability, and antibacterial activity) while maintaining the basic chemical structure of the active pharmaceutical ingredients, thereby achieving synergistic therapy and enhanced efficacy with reduced toxicity. The structural formulas of STZ (a) and TMP (b) in this invention are shown below:

[0028] .

[0029] As one embodiment of the present invention, the cell volume of the sulfathiazole-trimethoprim drug crystal salt V =5055.82(14)Å 3 , Z= 8.

[0030] As one embodiment of the present invention, Cu-K is used. α Radiation, at 2 θ The angle indicates that the characteristic diffraction peaks of the powder X-ray diffraction pattern of the sulfathiazole-trimethoprim drug crystal salt include 11.54°, 17.06°, 19.50°, 20.96°, 21.44°, 22.60°, 25.92° and 26.24°.

[0031] In 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 linked together by ionic bonds. In 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 1.922 Å and 2.123(2) Å, respectively. In 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 the N31-H31B of trimethoprim and the N13 of sulfathiazole; the second pair of hydrogen bonds N25-H25···N10 is formed by the N25-H25 of trimethoprim and the N10 of sulfathiazole. Simultaneously, in terms of symmetry operations, 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 one embodiment of the present invention, in the sulfathiazole-trimethoprim drug crystal salt, the face-to-face stacking of benzene rings in adjacent sulfathiazoles forms a π-π stacking interaction force, with a distance of 0.35448 nm between adjacent parallel planes. In the present invention, ionic bonds, hydrogen bond interactions, and π-π stacking interactions collectively maintain the stability of the sulfathiazole-trimethoprim drug crystal salt structure.

[0032] In one embodiment of the present invention, the melting point of the sulfathiazole-trimethoprim drug crystal salt is 169.5°C.

[0033] This invention provides a method for preparing the sulfathiazole-trimethoprim drug crystal salt described in the above technical solution, comprising the following steps:

[0034] Sulfathiazole and trimethoprim are dissolved in a solvent to obtain a mixed solution; the solvent is acetone and ethanol.

[0035] The mixed solution was crystallized to obtain the sulfathiazole-trimethoprim drug crystal salt.

[0036] In this invention, unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0037] This invention involves dissolving sulfathiazole and trimethoprim in a solvent to obtain a mixed solution. In this invention, the solvent is acetone and ethanol, specifically anhydrous ethanol; the volume ratio of acetone to ethanol can be 1:0.25~4, more preferably 1:0.5~2, and specifically 1:1. As one embodiment of this invention, the molar ratio of sulfathiazole to trimethoprim can be 1:1; the volume ratio of sulfathiazole to solvent can be 0.5~2.5 mmol:30 mL, more preferably 0.5~1 mmol:30 mL.

[0038] After obtaining the mixed solution, the present invention crystallizes the mixed solution to obtain the sulfathiazole-trimethoprim drug crystal salt. In one embodiment of the present invention, the crystallization temperature can be 15~40℃, specifically room temperature (25℃), and the time can be 4~6 days, specifically 5 days. In a specific embodiment of the present invention, the mixed solution is placed in a beaker, the beaker containing the mixed solution is sealed with a semi-permeable membrane, and then holes are punched in the semi-permeable membrane, and crystallization is carried out under static conditions.

[0039] This invention provides the application of the sulfathiazole-trimethoprim drug crystal salt described in the above-described technical solution or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in the above-described technical solution in the preparation of drugs for treating bacterial and / or fungal infectious diseases.

[0040] In 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 fungi may include Candida albicans. In another embodiment of the present invention, the content of sulfathiazole-trimethoprim drug crystal salt in the drug may be 50-99.9 wt%, more preferably 80-95 wt%. In the present invention, the drug includes pharmaceutically acceptable excipients, which 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.

[0041] This invention provides a medicament for treating bacterial and / or fungal infections, the active ingredient of which includes the sulfathiazole-trimethoprim drug crystal salt described in the above-described technical solution or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in the above-described technical solution. The content of the sulfathiazole-trimethoprim drug crystal salt, the types of pharmaceutically acceptable excipients, and the dosage form of the medicament of this invention are consistent with the above-described technical solution and will not be repeated here.

[0042] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0043] The drugs and reagents used in the following experiments are shown in Table 1.

[0044] Table 1. Drugs and Reagents

[0045]

[0046] The instruments used in the following experiments are shown in Table 2.

[0047] Table 2 Instruments

[0048]

[0049] Example 1

[0050] The steps for preparing sulfathiazole (STZ)-trimethoprim (TMP) drug crystal salts are as follows:

[0051] STZ (0.1275 g, 0.5 mmol) and TMP (0.1450 g, 0.5 mmol) were dissolved in 30 mL of a mixed solvent of acetone and anhydrous ethanol (the volume ratio of acetone to anhydrous ethanol was 1:1). The resulting mixed solution was placed in a beaker, and the beaker containing the mixed solution was sealed with a semi-permeable membrane. Then, holes were punched in the semi-permeable membrane, and crystallization was carried out for 5 days at room temperature (25 °C). White, transparent, fine needle-like crystals were obtained, which were the STZ-TMP drug crystal salts, with a yield of 70%.

[0052] Test Example 1: Physicochemical Characterization of Sulfathiazole-Trimethoprim Drug Crystal Salts

[0053] 1. Single-crystal X-ray diffraction (SCXRD)

[0054] The single-crystal X-ray diffraction experiment used a single-crystal X-ray diffractometer (Xtal AB) from Rigaku Corporation, Japan, employing a sealed X-ray tube to generate Cu-K... αRadiation. All data were integrated using the CrysAlisPro software suite, and surface-indexed absorption correction was applied to each acquired data set. Crystal structures were resolved using the SHELXT program and refined using SHELXT-2018 / 3 within the Olex2 graphical user interface. All non-hydrogen atoms were anisotropically refined, with most hydrogen atoms generated based on ideal positions of C or N atoms, and some hydrogen atoms calculated using Fourier electron cloud density. The asymmetric unit cell structure of the STZ-TMP drug crystal salt was plotted using Diamond 3.2 software.

[0055] Single-crystal structure data of STZ-TMP drug crystal salts were collected at room temperature, and their crystal data and structural refinement parameters are summarized in Table 3. Hydrogen bonds within STZ-TMP drug crystal salts are summarized in Table 4. Figure 1 This is a schematic diagram of the asymmetric unit structure of STZ-TMP drug crystal salt with atomic labels (specifically, a schematic diagram of ORTEP).

[0056] Table 3 Crystal data and structural refinement parameters of STZ-TMP drug crystals at room temperature.

[0057]

[0058] Table 4 Hydrogen bonds within STZ-TMP drug crystal salts

[0059]

[0060] Note: Symmetric operation 1: -x+1, y-1 / 2, -z+1 / 2; Symmetric operation 2: x+1 / 2, y, -z+1 / 2.

[0061] Table 3 shows that the single-crystal X-ray structure determination indicates that the STZ-TMP drug crystal salt belongs to the orthorhombic crystal system with space group [space group number missing]. 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 methoxybenzidine anion [C 14 H 19 N4O3] -It should be noted here that the proton is transferred from the N10 atom of the secondary amine ring of sulfathiazole to the N25 atom of the pyrimidine ring of methoxybenzyl. Therefore, the sulfathiazole cation is [C9H8N3O2S2]. + With trimethoprim anion [C 14 H 19 N4O3] - They are mainly bonded together by electrostatic attraction (ionic bonds). Meanwhile, the STZ cation is linked to the TMP anion via hydrogen bonds, with bond lengths of 2.123(2) Å and 1.922 Å, respectively (Table 4). The first pair of hydrogen bonds, N31-H31B···N13, is formed by the N31-H31B of TMP and the N13 of STZ. The second pair of hydrogen bonds, N25-H25···N10, is formed by the N25-H25 of TMP and the N10 of STZ. Figure 1 Meanwhile, in terms of symmetry operations, N31-H31A of TMP forms intermolecular hydrogen bonds with O9 atoms of STZ (symmetry operation 1: -x+1, y-1 / 2, -z+1 / 2), with a bond length of 2.895(2) Å; N30-H30B of TMP forms intermolecular hydrogen bonds with O8 atoms of STZ (symmetry operation 2: x+1 / 2, y, -z+1 / 2), with a bond length of 2.869(2) Å (Table 4). Furthermore, in the STZ-TMP drug crystal salt, the face-to-face stacking of adjacent benzene rings in STZ forms π-π stacking forces, with a distance of 0.35448 nm between adjacent parallel planes. Ionic bonds, hydrogen bond interactions, and π-π stacking interactions collectively maintain the stability of the STZ-TMP drug crystal salt structure.

[0062] 2. Powder X-ray diffraction (PXRD)

[0063] Powder X-ray diffraction (XRD) was performed using a TD-3500 XRD powder diffractometer. Small amounts of STZ, TMP, and STZ-TMP drug crystals were dried, ground into fine powder, and then evenly spread in the sample cell. Cu-K... α ray( λ The X-ray source was a diode (1.54060 Å), with 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 was acquired every 0.5 s. Powder X-ray diffraction results for STZ, TMP, and STZ-TMP drug crystal salts were obtained. The simulated powder X-ray diffraction pattern of STZ-TMP drug crystal salt was calculated using Mercury 2023 software (Cambridge Crystallography Data Centre, UK).

[0064] Powder X-ray diffraction is a rapid, convenient, and reliable analytical method used to verify the formation of new compounds and the purity of samples. Figure 2Powder X-ray diffraction (PXRD) spectra of STZ, TMP, and STZ-TMP drug crystal salts were obtained. The results showed that the spectra of STZ and TMP were consistent with the corresponding literature (Wang LY, Bu FZ, Li YT, et al. A sulfathiazole-amantadine hydrochloride cocrystal: The first codrug simultaneously comprising antiviral and antibacterial components [J]. Cryst Growth Des, 2020, 20(5): 3236-3246; Maddileti D, Swapna B, Nangia A K. Tetramorphs of the antibiotic drug trimethoprim: Characterization and stability [J]. CrystGrowth Des, 2015, 15(4): The characteristic peaks of the STZ-TMP drug crystal salt (1745-1756) 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 crystal salt shows new peaks and the disappearance of old peaks, indicating the formation of a new compound. The PXRD powder diffraction pattern of the STZ-TMP drug crystal salt also matches the SCXRD simulation data, indicating that the prepared STZ-TMP drug crystal salt has high purity, which is beneficial for subsequent tests.

[0065] 3. Fourier Transform Infrared Spectroscopy (FTIR)

[0066] Appropriate amounts of STZ, TMP, and STZ-TMP drug crystal salt samples were weighed separately, and each sample was ground and mixed with potassium bromide at a mass ratio of 1:100. After removing static electricity, the mixture was pressed into tablets for analysis. Infrared spectral data were obtained by scanning the 4000–400 cm⁻¹ range on a PerkinElmer Spectrum 2 Fourier transform infrared spectrometer. -1 Range obtained.

[0067] Infrared technology is a powerful tool for identifying hydrogen bonds, which can be used to verify the interactions between components in a crystalline salt. Figure 3 Fourier transform infrared spectra of STZ, TMP, and STZ-TMP drug crystal salts are shown. In the infrared spectrum of TMP, the symmetric and asymmetric stretching vibrations of N−H appear at 3318 cm⁻¹, respectively. -1 and 3469cm -1At the location; in the infrared spectrum of STZ-TMP drug crystal salt, the symmetric and asymmetric stretching vibrations of N−H are 3340 cm⁻¹. -1 and 3410cm -1 This indicates that the peak shift is due to 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. -1 and 3320cm -1 In the infrared spectrum, the characteristic peak intensity of −NH− is weaker than that of −NH2 in this region and is thus masked by −NH2. Therefore, the formation of hydrogen bonds N25−H25···N10 cannot be directly determined by infrared shift alone. However, since STZ-TMP drug crystal salts are ionic compounds, the difference in the symmetric and asymmetric stretching vibration shifts of −SO2 in STZ and STZ-TMP drug crystal salts can be used to indirectly determine this. In STZ, the symmetric and asymmetric stretching vibrations of −SO2 in the sulfonamide group are 1137 cm⁻¹. -1 and 1323cm -1 In STZ-TMP drug crystals, they become 1128 cm⁻¹ -1 and 1310cm -1 Furthermore, the bending vibration of the thiazole ring C=N should be 1531 cm in the STZ. -1 The single peak in STZ-TMP drug crystals is 1527 cm⁻¹. -1 and 1504cm -1 The double peaks suggest that these positions may have been affected by the formation of ionic bonds.

[0068] 4. Thermogravimetric analysis and differential scanning calorimetry (TGA / DSC)

[0069] Differential scanning calorimetry (DSC) analysis was performed on a PerkinElmer DSC 4000. 3–6 mg of STZ, TMP, and STZ-TMP drug crystals were weighed into an alumina crucible. The aluminum disc was pressed into a sheet, and the instrument scanning range was 30–250 °C with a heating rate of 10 °C / min. Thermogravimetric analysis (TGA) was performed using a PerkinElmer TGA 8000. The samples were placed in an alumina crucible for thermogravimetric testing; the tests were conducted under a nitrogen atmosphere, with a temperature range of 30–500 °C and a heating rate of 10 °C / min.

[0070] Figure 4DSC curves of STZ, TMP, and STZ-TMP drug crystal salts were obtained. The results showed that TMP had a melting point of 202.0℃; STZ may undergo a crystal transformation at 172.5℃, with a final melting point of 202.5℃; while the melting point of STZ-TMP drug crystal salt was different from the other two, being significantly lower at 169.5℃. This indicates that it formed a new compound and did not undergo a crystal transformation before reaching its melting point.

[0071] Figure 5 The TGA curves for STZ, TMP, and STZ-TMP drug crystal salts are shown. The results indicate that all three drug crystal salts undergo decomposition after reaching their melting points, which is reflected as a decrease in mass on the thermogravimetric analysis curves. It is worth noting that no mass loss was observed in STZ-TMP drug crystal salt before its melting point, further confirming the absence of solvent molecules within its molecule. This conclusion has been confirmed by SCXRD.

[0072] 5. Equilibrium solubility

[0073] Drug solubility is one of the important physical parameters of a drug, significantly affecting its absorption, oral bioavailability, and drug-likeness. In this experiment, solubility was assessed using an excess powder dissolution method, and analyzed by spectrophotometry to plot a standard curve. STZ, TMP, and STZ-TMP crystalline salts were accurately weighed and dissolved in 10 mL of dilute hydrochloric acid solution (pH 1.2), phosphate buffer solution (pH 6.8), and pure water (pH 7.0), respectively. The solutions were then shaken in a water bath at 37°C and 200 rpm for 24 h, followed by filtration through a 0.22 μm microporous membrane to obtain saturated solutions. The saturated solutions were diluted to appropriate concentrations, and the concentrations were calculated by substituting the values ​​into the corresponding standard curve equation. The absorbance was measured at different detection wavelengths using a UV9100 UV-Vis spectrophotometer. Measurements were repeated three times.

[0074] Figure 6A comparison graph shows the solubility of STZ, TMP, and STZ-TMP drug crystals under different pH conditions. The results show that the solubility of TMP, STZ, and STZ-TMP drug crystals exhibits a consistent trend with pH: pH 1.2 > pH 6.8 > pH 7.0. At pH 1.2 (acidic gastric environment), the solubility of STZ released from STZ-TMP drug crystals is 10.96 g / L, while the solubility of STZ alone is 4.39 g / L, representing a 2.50-fold increase in solubility. The solubility of TMP released from STZ-TMP drug crystals is 11.03 g / L, while the solubility of TMP alone is 3.02 g / L, representing a 3.65-fold increase in solubility. At pH 6.8 (small intestine environment), the solubility of STZ released from STZ-TMP drug crystals was 2.29 g / L, while the solubility of STZ alone was 0.56 g / L, representing a 4.09-fold increase in solubility. The solubility of TMP released from STZ-TMP drug crystals was 2.31 g / L, while the solubility of TMP alone was 0.46 g / L, representing a 5.02-fold increase in solubility. At pH 7.0, the solubility of STZ released from STZ-TMP drug crystals was 0.98 g / L, while the solubility of STZ alone was 0.32 g / L, representing a 3.06-fold increase in solubility. The solubility of TMP released from STZ-TMP drug crystals was 0.94 g / L, while the solubility of TMP alone was 0.29 g / L, representing a 3.24-fold increase in solubility. In summary, STZ-TMP crystal salt significantly improved the solubility of both 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 indicates that STZ-TMP crystal salt can effectively improve the solubility of STZ and TMP, potentially promoting absorption and enhancing therapeutic efficacy, reducing dosage, and improving patient compliance.

[0075] Test Example 2: Study on the bioactivity of sulfathiazole-trimethoprim drug crystal salts

[0076] 1. In vitro antibacterial activity test

[0077] To evaluate the antibacterial activity of STZ, TMP, STZ-TMP physical mixtures, and STZ-TMP crystalline salts, this experiment conducted minimum inhibitory activity (MIC) tests on Gram-positive bacteria (Staphylococcus aureus), Gram-negative bacteria (Klebsiella pneumoniae, Shigella dysenteriae, and Pseudomonas aeruginosa), and fungi (Candida albicans), as detailed below:

[0078] (1) Preparation of culture medium and sample solution

[0079] The 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), STZ-TMP physical mixture (0.0545 g, containing 0.1 mmol each of STZ and TMP), and STZ-TMP drug crystal salt (0.0545 g, 0.1 mmol) were weighed and dissolved in 5 mL of dimethyl sulfoxide (DMSO). Ultrapure water was then added to bring the volume to 100 mL, ensuring a final concentration of 1 μmol / mL for all four samples. A DMSO / water mixture (5:95 volume ratio) was used as a control solution. Finally, all solutions were filtered once through a 0.22 μm sterile filter for sterilization.

[0080] (2) Preparation of bacterial culture

[0081] Five types of bacteria (sourced from the Laboratory of Basic Medical College, Jiangxi University of Traditional Chinese Medicine), namely Staphylococcus aureus (…) S. aureus ), Klebsiella pneumoniae ( K. Pneumoniae ), Shigella dysenteriae ( S. dysenteriae ), Pseudomonas aeruginosa ( P. aeruginosa ) and Candida albicans ( C. albicans The bacterial culture was inoculated into sterile test tubes containing 6 mL of LB medium and incubated for 24 h at 37 °C and 200 rpm in a constant temperature shaking incubator. The bacterial culture was then diluted several times and spread onto prepared culture dishes containing NA medium. After incubation at 37 °C for 24 h, the concentration of each bacterial culture was calculated by colony counting, and the culture was stored at 2–8 °C for later use.

[0082] (3) Determination of antibacterial rate by micro-broth dilution method

[0083] The effects of STZ, TMP, STZ-TMP physical mixture, and STZ-TMP crystal salt on the tested bacterial strains were determined using the micro-broth dilution method. S. aureus , K. Pneumoniae , S. dysenteriae , P. aeruginosa and C. albicansThe antibacterial rate of the drug was determined. 100 μL of different sample solutions with a concentration of 1 μmol / mL were added to the wells in the first column of a 96-well plate, and then serially diluted to obtain six different drug concentrations. Simultaneously, the positive control group consisted of bacterial culture containing LB medium, the negative control group consisted of drug solution containing LB medium, and the blank control group consisted of pure LB medium. After incubating the plates at 37℃ for 24 h, the absorbance (OD) value at 600 nm was measured using a microplate reader to determine the antibacterial rate of the four samples. All tests were repeated three times.

[0084] (4) Determination of antibacterial properties using the Oxford cup method

[0085] Take 100 μL of a concentration of 1×10 7 A CFU / mL bacterial suspension was evenly spread onto sterile Petri dishes containing NA medium. Five Oxford cups were placed evenly in each Petri dish. 100 μL of 2 μmol / mL STZ, TMP, a physical mixture of STZ-TMP, and STZ-TMP crystalline salt solution were added to four of the Oxford cups, respectively. A prepared control solution was added to the remaining Oxford cup as a control group. The Petri dishes containing the Oxford cups were pre-diffused at room temperature for 30 min, then incubated at 37°C for 24 h. The size of the inhibition zone was then measured. Each group was repeated in triplicate.

[0086] The minimum inhibitory concentrations (MICs, μmol / mL) of STZ, TMP, STZ-TMP physical mixture, and STZ-TMP crystal salt against Staphylococcus aureus, Candida albicans, Klebsiella pneumoniae, Shigella dysenteriae, and Pseudomonas aeruginosa are shown in Tables 5 to 9, respectively. The diameters of the inhibition zones of STZ, TMP, STZ-TMP physical mixture, and STZ-TMP crystal salt are shown in Table 10.

[0087] Table 5. Minimum inhibitory concentrations of each drug against Staphylococcus aureus

[0088]

[0089] Table 6. Minimum inhibitory concentrations of each drug against Candida albicans

[0090]

[0091] Table 7. Minimum inhibitory concentrations of each drug against Klebsiella pneumoniae

[0092]

[0093] Table 8. Minimum inhibitory concentrations of various drugs against Shigella dysenteriae

[0094]

[0095] Table 9. Minimum inhibitory concentrations of each drug against Pseudomonas aeruginosa

[0096]

[0097] As shown in Tables 5-9, the four drugs exhibited significantly better 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 small-molecule antibacterial drugs from entering the bacteria, while the complex structure and composition of the outer membrane of Gram-negative bacteria reduce the effectiveness of small-molecule antibacterial drugs. Specifically, for Gram-negative bacteria (such as Klebsiella pneumoniae, Shigella dysenteriae, and Pseudomonas aeruginosa) and fungi (such as Candida albicans), at higher sample concentrations, the inhibition rates of 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., an inhibition rate of 90%) earlier than the STZ-TMP physical mixture. The MIC of STZ-TMP crystal salt against Klebsiella pneumoniae and Candida albicans is 0.25 μmol / mL, while the MIC of STZ-TMP physical mixture against Klebsiella pneumoniae and Candida albicans is greater than 0.25 μmol / mL. In addition, for Pseudomonas aeruginosa, the inhibition rate of STZ-TMP crystal salt and STZ-TMP physical mixture at a concentration of 0.5 μmol / mL is greater than 90%, while the inhibition rate of STZ at a concentration of 1 μmol / mL is greater than 90%, and the inhibition rate of TMP at a concentration of 1 μmol / mL is only about 25.9%.

[0098] For Gram-positive bacteria (Staphylococcus aureus), TMP is effective against Staphylococcus aureus. S. aureusThe inhibitory effect of TMP is greater than that of STZ, which may be due to the difference in permeability between 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). This makes TMP more likely to penetrate the cell wall of Staphylococcus aureus than STZ, thus producing a better antibacterial effect.

[0099] Table 10. Diameter of inhibition zone for each drug (concentration is 2 μmol / mL, unit of measurement: mm)

[0100]

[0101] Note: In the determination of antibacterial effect: the diameter of the inhibition zone > 20 mm is extremely sensitive (++), 15~20 mm is highly sensitive (++), 10~15 mm is moderately sensitive (+), and the diameter of the inhibition zone < 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).

[0102] The aforementioned results indicate that TMP exhibits strong antibacterial activity against Staphylococcus aureus, achieving a 99% inhibition rate at a concentration of 0.0039 μmol / mL. Therefore, in the Oxford cup experiment, only the inhibition zone experiments of STZ, TMP, STZ-TMP physical mixture, and STZ-TMP crystal salt against Gram-negative bacteria and fungi were conducted. Table 10 shows that at a concentration of 2 μmol / mL, both raw materials showed poor inhibitory effects against the four bacteria. In contrast, due to synergistic effects, the STZ-TMP physical mixture exhibited expanded inhibition zones against all four strains, with inhibition zone diameters of approximately 14.3 mm, 11.8 mm, 12.2 mm, and 12.6 mm against Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, and Candida albicans, respectively, but still within the range of moderate sensitivity. Notably, STZ-TMP crystalline salt exhibited stronger inhibitory activity, with inhibition zone diameters of approximately 17.1 mm, 14.8 mm, 15.0 mm, and 15.9 mm against Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, and Candida albicans, respectively, all demonstrating high sensitivity. Specifically, the inhibition zone diameter against 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 was approximately 12.6 mm, while that of STZ-TMP crystalline salt was approximately 15.9 mm, representing an improvement of approximately 26.19% compared to the STZ-TMP physical mixture. Compared to physical mixtures, when STZ-TMP crystal salts dissolve in a medium, STZ-TMP may exist as a whole, acting synergistically against bacteria. However, when STZ-TMP physical mixtures dissolve in a medium, the two active pharmaceutical ingredients, STZ and TMP, may not form a cohesive whole. This may explain why STZ-TMP crystal salts exhibit stronger antibacterial effects than STZ-TMP physical mixtures. This indicates that STZ-TMP crystal salts can improve both the physicochemical properties and the biological activity of the drug.

[0103] 2. Assay for DHFS and DHFR enzyme activity inhibition

[0104] DHFS and DHFR enzymes are ubiquitous in microorganisms and are essential enzymes for nucleic acid synthesis. STZ and TMP achieve their antibacterial effects by inhibiting the activity of these two enzymes, respectively. In this experiment, an enzyme-linked immunosorbent assay kit (Jiangxi Aipude Biotechnology Co., Ltd.) was used to detect the inhibitory effects of STZ, TMP, STZ-TMP physical mixture, and STZ-TMP drug crystal salt on these two 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 each well, followed by 10 μL of a 2 μmol / mL solution of STZ, TMP, the STZ-TMP physical mixture, and STZ-TMP drug crystal salt, respectively. At this point, the concentration of the standard in each well was 200 U / L. Therefore, 50 μL of a standard with a concentration of 200 U / L was used as a control group. The OD value at 450 nm was measured using a SpectraMax M2 microplate reader. The OD values ​​were then substituted into the standard curve to calculate the inhibitory effect of the four samples on the activities of DHFS and DHFR enzymes. Three parallel groups were set up for each sample for testing.

[0105] Figure 7 This chart compares the inhibitory effects of STZ, TMP, a physical mixture of STZ and TMP, and STZ-TMP crystal salt on DHPS and DHFR enzymes. The results show that in the wells with 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 DHPS enzymes and has almost no effect on DHFR enzymes. In contrast, the data measured in the wells with TMP are the opposite of those with STZ, with DHPS and DHFR enzyme activities of 192.73 U / L and 157.26 U / L, respectively. In the wells with the STZ-TMP physical mixture, the measured DHPS and DHFR enzyme activities were 151.79 U / L and 159.04 U / L, respectively, which is almost the same inhibitory effect of STZ and TMP on these two enzymes. In the experimental wells containing STZ-TMP drug crystal salt, the measured activities of DHPS and DHFR enzymes were 124.69 U / L and 139.67 U / L, respectively. Compared with the two active pharmaceutical ingredients, the inhibition levels of these two enzymes increased by 28 U / L and 18 U / L, respectively. This indicates that STZ-TMP drug crystal salt is not a simple combination of two active pharmaceutical ingredients with a "1+1=2" effect. Rather, the STZ-TMP drug crystal salt described in this 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.

[0106] Comparative Example 1

[0107] The procedure was performed according to Example 1, except that the active pharmaceutical ingredient and solvent used were different, as shown in Tables 11-13. In Tables 11-13, the molar ratio of active pharmaceutical ingredient 1 to active pharmaceutical ingredient 2 was 1:1. As can be seen from Tables 11-13, neither the active pharmaceutical ingredient nor the solvent used in Comparative Example 1 formed a eutectic or crystalline salt.

[0108] Table 11. Active pharmaceutical ingredient (API 1 is sulfathiazole) and solvent used in Comparative Example 1

[0109]

[0110] Table 12. Active pharmaceutical ingredient (API 1 is trimethoprim) and solvent used in Comparative Example 1

[0111]

[0112] Table 13. Drug substances (trimethoprim + sulfadiazine / sulfamidone) and solvents used in Comparative Example 1

[0113]

[0114] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered 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 space group . P bca; 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; Using Cu-K α Radiation, at 2 θ The angle indicates that the characteristic diffraction peaks of the powder X-ray diffraction pattern of the sulfathiazole-trimethoprim drug crystal salt include 11.54°, 17.06°, 19.50°, 20.96°, 21.44°, 22.60°, 25.92° and 26.24°; The melting point of the sulfathiazole-trimethoprim drug crystal salt is 169.5℃.

2. The sulfathiazole-trimethoprim drug crystal salt according to claim 1, characterized in that, 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 bonding, hydrogen bonding and π-π stacking.

3. The method for preparing the sulfathiazole-trimethoprim drug crystal salt according to claim 1 or 2, characterized in that, Includes the following steps: Sulfathiazole and trimethoprim are dissolved in a solvent to obtain a mixed solution; the solvent is acetone and ethanol. The mixed solution was crystallized to obtain the sulfathiazole-trimethoprim drug crystal salt.

4. The preparation method according to claim 3, 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 volume ratio of sulfathiazole to solvent is 0.5~2.5 mmol:30 mL.

5. The preparation method according to claim 3 or 4, characterized in that, The crystallization temperature is 15~40℃, and the time is 4~6 days.

6. The use of the sulfathiazole-trimethoprim drug crystal salt according to claim 1 or 2, or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method according to any one of claims 3 to 5, in the preparation of medicaments for treating bacterial and / or fungal infectious diseases.

7. The application according to claim 6, 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.

8. A medicament for treating bacterial and / or fungal infections, characterized in that, The active ingredient includes the sulfathiazole-trimethoprim drug crystal salt as described in claim 1 or 2, or the sulfathiazole-trimethoprim drug crystal salt prepared by the preparation method described in any one of claims 3 to 5.

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