Use of compound sb1 in the preparation of a medicament for preventing or treating toxoplasma infection

Abstract

The application provides application of a compound SB1 in preparation of a drug for preventing or treating toxoplasma infection. The compound SB1 is a Schiff base as a core skeleton, and the structure comprises a sulfonamide group, an imine bond, a thiophene ring and a 5-position bromine atom, and the molecular formula is C 11 H9BrN2O2S2. The inventors find through a large number of experiments that SB1 has a significant inhibitory activity effect on intracellular parasitic protozoa toxoplasma, has small toxic side effects, and can expand a new way for effective use of SB1. Vero cells infected with toxoplasma are used as an in vitro experimental model, and the results show that the half effective inhibitory concentration of SB1 on toxoplasma is 20.641 μM, can significantly inhibit the proliferation of toxoplasma in host cells, and the half cytotoxicity concentration of the Vero cells is 266.15 μM, and the safety index SI is 12.89, the effective inhibitory concentration of SB1 is far less than the cytotoxicity, has the effect of preventing and treating toxoplasma infection, and can be used for preparing a drug for preventing or treating toxoplasma infection.

Description

Technical Field

[0001] This invention belongs to the field of parasitic disease prevention and control technology, specifically relating to the application of compound SB1 in the preparation of drugs for the prevention or treatment of Toxoplasma gondii infection. Background Technology

[0002] Toxoplasmosis is an obligate intracellular parasitic protozoan disease caused by *Toxoplasma gondii*. *Toxoplasma gondii* infects almost all warm-blooded animals and humans, and even some cold-blooded animals, residing within all nucleated cells of the animal's body. It is estimated that about one-third of the global population is at risk of toxoplasmosis infection. Most infected individuals do not show obvious clinical symptoms, but infants, young children, and immunocompromised patients (such as those with HIV / AIDS, organ transplant recipients, and those with malignant tumors) are highly susceptible to developing severe illness and even death once infected. Furthermore, toxoplasmosis infection is a major cause of miscarriage, stillbirth, and other reproductive abnormalities in pregnant women and pregnant animals; therefore, toxoplasmosis screening has been included in routine prenatal checkups in my country.

[0003] The seropositivity rate of Toxoplasma gondii varies significantly by region: in many parts of the Americas, Europe, and Asia, the seropositivity rate is approximately 30%, while in Africa, the infection rate exceeds 60%. Pigs, cattle, sheep, poultry, and companion animals are all susceptible to Toxoplasma gondii infection, with pigs being particularly vulnerable. The global overall infection rate in pigs reaches 30%, and when an acute Toxoplasma gondii outbreak occurs in a pig farm, the morbidity rate can reach 100%, and the mortality rate can exceed 60%. In the United States and China, pigs are considered the most important vector for Toxoplasma gondii transmission among many food animals, posing a significant public health and safety risk.

[0004] Currently, clinical treatment of toxoplasmosis still relies on chemotherapy. Due to the complexity of the Toxoplasma gondii life cycle, the diversity of its pathogenesis, and the differences in its biological characteristics, there are currently no preventative or specific drug treatments. Although the combination of pyrimethamine and sulfadiazine is currently the gold standard for treating toxoplasmosis in clinical practice, this treatment method is often accompanied by serious adverse reactions and suffers from problems such as incomplete treatment, high relapse rate, and ineffectiveness against cysts, resulting in a high overall treatment failure rate.

[0005] Therefore, developing a safe and effective alternative drug to treat Toxoplasma gondii is an urgent problem that needs to be solved and has broad market prospects. In view of this, this invention is proposed. Summary of the Invention

[0006] To address the problems of existing drugs for preventing or treating toxoplasmosis infection, such as easy drug resistance, significant toxic side effects, and poor efficacy against toxoplasmosis cysts, this invention specifically provides compound SB1 and its preparation method, as well as its application in the preparation of drugs for preventing or treating toxoplasmosis infection, specifically including:

[0007] One of the objectives of this invention is to provide a compound SB1 with the molecular formula C 11 H9BrN2O2S2, with a molecular weight of 345.23, has the following chemical structural formula:

[0008] .

[0009] The compound SB1 provided by this invention has a Schiff base as its core skeleton and its structure includes a sulfonamide group (-SO2NH2), an imine bond (-CH=N-), a thiophene ring, and a bromine atom at the 5-position. It is a novel compound synthesized by the Chemistry Laboratory of Qued I Azzam University in Islamabad, Pakistan.

[0010] A second objective of this invention is to provide a method for preparing compound SB1, comprising the following steps:

[0011] (1) Dissolve p-aminobenzenesulfonamide and 5-bromothiophene-2-carboxaldehyde in anhydrous methanol, and add glacial acetic acid as a catalyst. Heat the mixture under reflux to carry out a condensation reaction.

[0012] (2) After the reaction is complete, cool the reaction solution to room temperature, pour it into crushed ice, and let it stand to precipitate a yellow solid crude product;

[0013] (3) The crude solid was collected by vacuum filtration, washed with deionized water, and then transferred to boiling methanol and stirred until dissolved. The filtrate was cooled to room temperature, allowed to stand, recrystallized and purified, and the crystalline solid was collected and dried to obtain a yellow crystalline compound, denoted as SB1.

[0014] In a preferred embodiment, in step (1), the molar ratio of p-aminobenzenesulfonamide to 5-bromothiophene-2-carboxaldehyde is 1:(0.9-1.1); preferably, the molar ratio of p-aminobenzenesulfonamide to 5-bromothiophene-2-carboxaldehyde is 1:1.

[0015] In a preferred embodiment, in step (1), 4-6 mL of anhydrous methanol is added for every 1 mmol of p-aminobenzenesulfonamide; preferably, 5 mL of anhydrous methanol is added for every 1 mmol of p-aminobenzenesulfonamide.

[0016] In a preferred embodiment, in step (1), 0.2-0.8 mL of glacial acetic acid is added for every 1 mmol of p-aminobenzenesulfonamide; preferably, 0.5 mL of glacial acetic acid is added for every 1 mmol of p-aminobenzenesulfonamide.

[0017] In a preferred embodiment, in step (1), the heating reflux temperature is the reflux temperature of methanol (64-66°C), and the heating reflux time is 3.5-7.5 hours; preferably, the heating reflux temperature is 65°C, and the heating reflux time is 5 hours.

[0018] In a preferred embodiment, in step (2), the volume of the crushed ice is 2-4 times the volume of the reaction product; and the settling time is 0.5-1 hour.

[0019] In a preferred embodiment, in step (3), the number of times the deionized water is washed is 2-4 times, and the solid-liquid ratio of the crude solid product to the deionized water is 1g:(2-4)mL during each wash.

[0020] In a preferred embodiment, in step (3), the solid-liquid ratio of the crude solid product to boiling anhydrous methanol is 1 g: (8-16) mL.

[0021] In this step, vacuum filtration and washing with deionized water are used to thoroughly remove residual solvent and water-soluble impurities. Dissolving the impurities in boiling anhydrous methanol with stirring removes insoluble impurities while the liquid is hot, thus achieving purification.

[0022] In a preferred embodiment, in step (3), the settling recrystallization time is 3-7 hours; preferably, the settling time is 5 hours.

[0023] In a preferred embodiment, in step (3), the method for collecting the crystalline solid can be a conventional method in the art, such as using a sintered glass funnel or gravity filtration to filter the crystals that appear under vacuum; the drying can be a conventional method in the art, such as vacuum drying at 35-45°C to constant weight.

[0024] A third objective of this invention is to provide the use of compound SB1 in the preparation of drugs for the prevention or treatment of toxoplasmosis infection.

[0025] In a preferred embodiment, the drug for preventing or treating Toxoplasma gondii infection is a drug that inhibits the ability of Toxoplasma gondii to lyse cells.

[0026] In a preferred embodiment, the drug for preventing or treating toxoplasmosis infection is a drug that inhibits the invasion and proliferation of Toxoplasma gondii.

[0027] In a preferred embodiment, the SB1 has a 50% inhibitory effect on Toxoplasma gondii at 20.641 μM.

[0028] A fourth objective of the present invention is to provide a composition for the prevention or treatment of toxoplasmosis infection, wherein the active ingredient of the composition includes SB1.

[0029] In a preferred embodiment, the composition for preventing or treating Toxoplasma gondii infection is: a composition that inhibits the ability of Toxoplasma gondii to lyse cells, a composition that inhibits the invasion and proliferation of Toxoplasma gondii, or a composition that eliminates Toxoplasma gondii parasites from the body of humans or non-human animals; preferably, the non-human animals include, but are not limited to, pigs, cattle, sheep, horses, dogs, cats, chickens, etc., which are susceptible to Toxoplasma gondii.

[0030] In a preferred embodiment, the composition further includes one or more pharmaceutically acceptable carriers, the carriers comprising one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, flavor and color modifiers, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, preservatives, pH adjusters, surfactants, excipients, fillers, and synergists.

[0031] In a preferred embodiment, the diluent includes substances such as starch, sucrose, cellulose, and inorganic salts; the wetting agent includes substances such as water and ethanol; the binder includes substances such as starch paste, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol; the disintegrant includes substances such as starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium bicarboxymethyl cellulose, and surfactants; the lubricant includes substances such as talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, and polyethylene glycol; the flavoring and coloring agents include substances such as pigments, sweeteners, flavorings, and gelling agents; the solvent includes substances such as water, glycerin, and ethanol; and the solubilizer includes substances such as... The ingredients include: Tween compounds, maltose compounds, sulfates, sulfonates, etc.; the cosolvents include organic acids (such as citric acid) and their salts, inorganic salts, polyethylene glycol, etc.; the emulsifiers include Span compounds, glycerol fatty acid esters, gum arabic, gelatin, agar, sodium alginate, etc.; the antioxidants include sulfites, ascorbic acid, gallic acid and their salts, etc.; the metal complexing agents include disodium ethylenediaminetetraacetate, polycarboxylic acid compounds, etc.; the preservatives include parabens, quaternary ammonium compounds, chlorhexidine acetate, etc.; the pH adjusters include hydrochloric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, citrates, etc.

[0032] In this invention, the dosage forms of the composition include, but are not limited to, oral, topical, suppository, and sterile injectable solutions in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays. It is understood that the SB1-containing compositions of this invention can be administered in various ways depending on the different excipients and dosage forms.

[0033] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0034] 1. Through long-term research, the inventors have discovered that compound SB1 has a significant inhibitory effect on the intracellular parasitic protozoan Toxoplasma gondii, and has few toxic side effects, which can expand new avenues for the effective utilization of SB1.

[0035] 2. The inventors have demonstrated through extensive experiments that compound SB1 has a significant preventive or therapeutic effect against Toxoplasma gondii infection. Specifically, using Vero cells infected with Toxoplasma gondii as an experimental model, the EC50 was measured... 50 This indicates that SB1 has a 50% inhibitory effect on Toxoplasma gondii at 20.641 μM. Therefore, a small amount of application can achieve a highly effective anti-Toxoplasma gondii infection effect.

[0036] 3. Further experiments have confirmed that compound SB1, as an anti-Toxoplasma gondii drug, also has the advantage of low toxicity. Specifically, the cytotoxicity of SB1 against green monkey kidney cells (Vero) was detected by the CCK8 method, and the results showed that its CCK8 activity was low. 50 The value is 266.15 μM, obtained by CC. 50 With EC 50 The calculated selectivity index (SI) was 12.89. In the art, an SI value greater than 10 is generally used as a standard for determining whether a drug has a good safety window. Therefore, compound SB1 not only significantly inhibits the proliferation of Toxoplasma gondii but also effectively maintains the activity of host cells, achieving a balance between in vitro safety and selective inhibitory activity. Compared to the significant side effects of existing conventional Toxoplasma gondii treatments, compound SB1 provided by this invention exerts its antiparasitic efficacy while having minimal impact on host cells, exhibiting good safety and representing a significant improvement in high efficiency, low toxicity, and safety.

[0037] 4. Through extensive experimental research, this invention has found that the anti-Toxoplasma gondii effect of SB1 is mainly achieved by inhibiting its intracellular proliferation, and the effect of SB1 on Toxoplasma gondii is dose-dependent, indicating that the higher the concentration, the better the anti-parasitic effect. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 This is a synthetic route diagram for compound SB1 of the present invention;

[0040] Figure 2 This is a graph showing the toxicity test results of compound SB1 in Example 1 of the present invention on Vero cells;

[0041] Figure 3 This is a graph showing the inhibitory effect of compound SB1 from Example 2 of the present invention on Toxoplasma gondii.

[0042] Figure 4 These are plaque phagocytic experiments of compound SB1 and the control group in Example 3 of this invention;

[0043] Figure 5 This is a statistical graph showing the antiproliferative effect of compound SB1 and the control group against intracellular RH in Example 4 of the present invention.

[0044] Figure 6 The figure shows the results of the anti-invasion effect of compound SB1 and the control group on extracellular RH-type Toxoplasma gondii in Example 4 of this invention. Detailed Implementation

[0045] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0046] Unless otherwise specified, the technical means used in this invention are conventional means well known to those skilled in the art. All raw materials, reagents, instruments, and equipment used in this invention can be purchased commercially or prepared using existing methods. Unless otherwise specified, all reagents used in this invention are of analytical grade. In the embodiments of this invention, the room temperature is 20-30 °C. The Vero cells and HFF cells used in this invention were purchased from the ATCC cell bank. In the embodiments of this invention, the complete culture medium used is: high-glucose DMEM medium containing 8% fetal bovine serum, supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin.

[0047] In this embodiment of the invention, the preparation method of compound SB1 includes the following steps:

[0048] like Figure 1The synthetic route shown involves adding equimolar amounts (1.0 mmol each) of p-aminobenzenesulfonamide and 5-bromothiophene-2-carboxaldehyde to 4 mL of anhydrous methanol and stirring until completely dissolved. Then, 0.5 mL of glacial acetic acid is added as a catalyst for the condensation reaction. The resulting mixture is heated to a stable reflux state and maintained at reflux for 5 hours. After the reaction is complete, the reaction solution is naturally cooled to room temperature and poured into three times its volume of crushed ice. After standing for 30 minutes, a yellow crude solid precipitates. The crude solid is collected by vacuum filtration, washed three times with deionized water (the solid-liquid ratio of crude product to deionized water is 1 g: 3 mL for each wash), and then dissolved in boiling methanol (the solid-liquid ratio of crude product to boiling methanol is 1 g: 12 mL). The mixture is naturally cooled to room temperature and allowed to stand for 5 hours to complete recrystallization. After separating the solid, it is dried to obtain a yellow crystalline compound, denoted as SB1, with an overall reaction yield of 78%.

[0049] Example 1: Toxicity study of SB1 on Vero cells

[0050] 1. Experimental Procedure

[0051] Cytotoxicity assay: The cytotoxicity of SB1 to Vero cells was determined using the CCK-8 assay.

[0052] (1) Seed Vero cells in the logarithmic growth phase into 96-well culture plates, about 100 μL (5000 cells) per well, and cultured in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere fully and form a stable monolayer.

[0053] (2) After the cells stabilized, the original culture medium was discarded, and the cells were gently rinsed twice with sterile PBS buffer to remove residual serum components. Then, different concentrations of drug working solutions (1 μM, 4 μM, 10 μM, 100 μM, 200 μM, 500 μM) prepared with complete culture medium were added to the experimental group, and DMSO culture medium was added to the control group. Cell-free blank wells were also set up. The final concentration of DMSO in the blank group, control group and experimental group was 0.1%, and the final volume of each well was 100 μL. Three replicates were set up for each group.

[0054] (3) The culture plate was incubated at 37℃, 5% CO2, and saturated humidity for 24 h. After incubation, 10 μL of CCK-8 reagent was added to each well, and the plate was incubated in the dark for another 2 h. After incubation, the absorbance (OD value) of each well was measured at 450 nm using a Tecan M200 Pro multi-mode microplate reader. Cell viability was calculated according to the following formula based on the OD results. The independent experiment was repeated 3 times, and the half-maximal cytotoxic concentration (MCC) was obtained by fitting the dose-response curve. 50 .

[0055] Cell viability (%) = (OD)实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组） ×100%

[0056] 2. Experimental Results

[0057] like Figure 2 The graph shows the toxicity test results of compound SB1 on Vero cells. The vertical axis represents cell viability, with the 0.1% DMSO control group as the baseline (100%), and the horizontal axis represents the administered concentration of compound SB1. It can be seen that in the low concentration range of 1 μM-10 μM, cell viability is close to 100%, and compound SB1 has no significant effect on cell growth and survival. As the SB1 concentration increases, cell viability decreases in a dose-dependent manner. When the concentration reaches 266.15 μM, cell viability drops to 50%, which is the half-maximal cytotoxic concentration (MCC) of compound SB1 on Vero cells. 50 The value was 266.15 μM.

[0058] Example 2: Inhibitory effect of SB1 on Toxoplasma gondii

[0059] 1. Experimental Procedure

[0060] This experiment included a control group (DMSO group) and an experimental group (SB1 treatment group), both of which were inoculated with Vero cells and Toxoplasma gondii tachyzoites. Specific procedures included:

[0061] Vero cells in the logarithmic growth phase were seeded into 96-well culture plates, approximately 100 μL (5000 cells) per well, and cultured at 37°C in a 5% CO2 incubator for 24 h to allow the cells to adhere fully and form a stable monolayer.

[0062] Fresh, viable Luciferase tachyzoites were harvested from Vero cells, counted using a hemocytometer, and 3 × 10⁻⁶ cells were collected. 5 One tachyzoite was inoculated into Vero cells (multiple of infection MOI = 60:1) and incubated at 37°C, 5% CO2, and saturated humidity for 2 hours to allow the tachyzoites to complete their invasion. The original culture medium in the wells was gently aspirated and the cells were gently washed twice with sterile PBS to remove any uninvaded free tachyzoites.

[0063] The control group was given complete culture medium containing 0.1% DMSO; the experimental group was prepared with compound SB1 at nine concentration gradients: 0.5 μM, 1 μM, 2 μM, 3.2 μM, 5 μM, 10 μM, 16 μM, 40 μM, and 63 μM. The final concentration of DMSO in both the control and experimental groups was 0.1%, with a final volume of 200 μL per well and three replicates per group.

[0064] After incubating the culture plates at 37℃, 5% CO2, and saturated humidity for 24 h, the culture medium was gently aspirated from each well, and 100 μL of cell lysis buffer was added to each well. After lysis at room temperature for 5 min, the lysis buffer was aspirated and transferred to a 1 mL EP tube. The plates were centrifuged at 12000 r / min for 5 min, and 100 μL of the supernatant was added to a 96-well microplate. Then, under dark conditions, 100 μL of luciferase substrate working solution was added to each well, and the mixture was stirred. The luminescence intensity (RLU) was measured using a chemiluminescence analyzer. The inhibition rate of Toxoplasma gondii proliferation by compound SB1 was calculated using the following formula. The independent experiments were repeated three times, and the half-maximal effective concentration (EC50) was obtained by fitting a dose-response curve. 50 .

[0065] Inhibition rate % = (RLU) DMSO -RLU 处理组 ) / RLU DMSO ×100%

[0066] 2. Experimental Results

[0067] Figure 3 The graph shows the inhibitory effect of compound SB1 on Toxoplasma gondii, with the vertical axis representing the Toxoplasma gondii proliferation inhibition rate and the horizontal axis representing the administered concentration of compound SB1. The results show that SB1 has a half-maximal effective concentration (EC50) against Toxoplasma gondii proliferation. 50 The concentration of SB1 was 20.641 μM, and its inhibitory effect on Toxoplasma gondii was dose-dependent, with higher concentrations showing stronger inhibitory effects, indicating that SB1 has a good inhibitory effect on Toxoplasma gondii. Meanwhile, the safety index SI (SI=CC) was [not specified in the original text]. 50 / EC 50 The value reached 12.89, indicating that SB1 has strong selectivity for parasites and low host toxicity, and has excellent potential for the development of anti-Toxoplasma gondii drugs.

[0068] Example 3: The effect of SB1 on inhibiting intracellular Toxoplasma gondii plaque formation.

[0069] 1. Experimental Procedure

[0070] Human foreskin fibroblasts (HFFs), a classic cell model for Toxoplasma gondii research, were seeded into 12-well cell culture plates and cultured at 37°C with 5% CO2 for 24 hours to form a monolayer of cells.

[0071] Fresh Toxoplasma gondii type I strain (RH) tachyzoites were collected, and 150 live tachyzoites were inoculated into each well of a monolayer of HFF cells (MOI approximately 1:200). The culture plates were divided into a control group and an experimental group, with 6 replicates in each group. The control group was given complete medium containing 0.1% DMSO, while the experimental group was given complete medium containing 40 μM compound SB1 and 0.1% DMSO. The final volume of medium in each well was fixed at 2 mL, and the final concentration of DMSO in both groups was 0.1%.

[0072] After incubating the culture plate at 37℃, 5% CO2, and saturated humidity for 8 days, the culture medium in the wells was gently aspirated. Each well was washed twice with 1 mL of sterile PBS, and 1 mL of 4% paraformaldehyde was added to each well for fixation at room temperature for 30 min. The fixative was then aspirated, and the plate was washed three times with PBS. 1 mL of 0.5% crystal violet staining solution was added to each well for staining at room temperature for 20 min. The staining solution was then aspirated, and the plate was gently washed with sterile water until no excess staining solution remained. After air drying, the plaque morphology and plaque area were recorded by microscopic photography.

[0073] 2. Experimental Results

[0074] like Figure 4 As shown, under the same magnification microscope, larger plaques were observed in the DMSO negative control group, which were significantly different from the plaques formed in the SB1 group, indicating that SB1 can significantly inhibit the growth of Toxoplasma gondii in host cells and plaque formation.

[0075] Example 4: Antiproliferative effect of SB1 on intracellular Toxoplasma gondii type I strain (RH)

[0076] 1. Experimental Procedure

[0077] Proliferation experiment: Collect 1×10 5 One RH tachyzoite was inoculated onto a 12-well cell culture slide covered with a monolayer of HFF cells. After 4 hours of parasite invasion, the culture medium was changed, and the cells were divided into two groups: the control group was given complete medium containing 0.1% DMSO, and the experimental group was given complete medium containing 40 μM (2 times EC). 50 The compound SB1 and 0.1% DMSO were used in complete culture medium, with the final concentration of DMSO in both groups being 0.1%.

[0078] After incubation at 37℃, 5% CO2, and saturated humidity for 24 h, the original culture medium was gently aspirated, and the cells were gently washed twice with sterile PBS. 1 mL of 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 15 min. The fixative was then aspirated, and the cells were washed twice with PBS, each time for 5 min. 0.25% Triton X-100 permeabilization buffer prepared with PBS was added to the wells, and the cells were permeated at room temperature for 30 min. The permeabilization buffer was then aspirated, and the cells were washed twice with PBS. Finally, 1% BSA / PBS was added to block the cells at room temperature for 30 min.

[0079] After blocking, rabbit-derived anti-Toxoplasma gondii surface protein GAP45 primary antibody (1:300 diluted in 1% BSA / PBS) was added and incubated overnight at 4°C. The next day, the primary antibody was discarded, the cells were washed three times with PBS, and FITC-labeled goat anti-rabbit fluorescent secondary antibody (1:100 diluted in 1% BSA / PBS) was added and incubated at room temperature in the dark for 1 h. The secondary antibody was discarded, the cells were washed three times with PBS, and Hoechst 33342 dye (1 μg / mL) was added and stained at room temperature in the dark for 5 min to label the host cell nuclei.

[0080] Observation under a fluorescence microscope: multiple fields of view were randomly selected, covering the upper, lower, left, right, and center positions, and the number of Toxoplasma gondii tachyzoites in 100 vacuoles with worms was counted. The independent experiment was repeated 3 times.

[0081] Invasion experiment: Invasion was performed using 12-well culture plates filled with HFF cells, and 1×10⁶ cells with uniform viability were collected. 5 One RH tachyzoite was resuspended in complete medium containing 0.1% DMSO (control group) and complete medium containing 40 μM compound SB1 and 0.1% DMSO (experimental group), respectively, and added to the corresponding wells simultaneously. The final concentration of DMSO in both groups was 0.1%.

[0082] After culturing in an incubator at 37℃, 5% CO2, and saturated humidity for 30 min, the culture medium was immediately removed, and the cells were gently washed three times with sterile PBS to thoroughly remove any uninvaded tachyzoites (observation under a microscope was performed to ensure the extracellular tachyzoites were completely removed). Fresh complete culture medium was then replaced, and the cells were cultured for another 20 h. Following the same fixation, permeabilization, blocking, and IFA staining procedures as described in the proliferation experiment (primary antibody GAP45, secondary antibody FITC, and Hoechst 33342 for nuclei staining), the cells were then observed under a fluorescence microscope. Twenty fields of view were randomly selected (covering the upper, lower, left, right, and center positions) to acquire images of Toxoplasma gondii tachyzoites and host cell nuclei.

[0083] The number of vacuoles containing parasites and the number of host cells containing tachyzoites in each image were counted. The antiproliferative effects of SB1 expression and the control group on intracellular RH tachyzoites were investigated. Figure 5 As shown.

[0084] The Toxoplasma gondii invasion rate was calculated as the number of vacuoles carrying the parasite divided by the number of host cells. The average value of all images was taken as the invasion rate for that group. The anti-invasion effects of SB1 and the control group against extracellular RH-type Toxoplasma gondii were investigated. Figure 6 As shown.

[0085] 2. Experimental Results

[0086] After Toxoplasma gondii invades a host cell, it forms vacuolar vesicles within which tachyzoites multiply by internal binary fission. Therefore, the number of tachyzoites in these vacuolar vesicles within the same timeframe after cell invasion reflects the proliferative capacity of the parasite. Statistical results from proliferation experiments showed that, compared to the control group (DMSO group), the number of tachyzoites in the vacuolar vesicles of the experimental group containing 40 μM compound SB1 was significantly reduced. Invasion experiments showed that the invasion rate of the SB1 experimental group was lower than that of the control group. Combined with the CC of SB1... 50 =266.15 μM, EC 50 With a concentration of 20.641 μM, a safety index (SI) of 12.89, and a dosage of 40 μM within the safe range where there is no significant toxicity to host cells, SB1 can significantly inhibit the intracellular proliferation of RH strain Toxoplasma gondii, further verifying its low-toxicity and highly effective anti-Toxoplasma gondii activity.

[0087] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A compound SB1, characterized in that, The molecular formula is C 11 H9BrN2O2S2, with a molecular weight of 345.23, has the following chemical structural formula: 。 2. The method for preparing compound SB1 as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve p-aminobenzenesulfonamide and 5-bromothiophene-2-carboxaldehyde in anhydrous methanol, and add glacial acetic acid as a catalyst. Heat the mixture under reflux to carry out a condensation reaction. (2) After the reaction is complete, cool the reaction solution to room temperature, pour it into crushed ice, and let it stand to precipitate a yellow solid crude product; (3) The crude solid was collected by vacuum filtration, washed with deionized water, and then transferred to boiling methanol and stirred until dissolved. The filtrate was cooled to room temperature, allowed to stand, recrystallized and purified, and the crystalline solid was collected and dried to obtain a yellow crystalline compound, denoted as SB1.

3. The method for preparing compound SB1 as described in claim 2, characterized in that, In step (1), the molar ratio of p-aminobenzenesulfonamide to 5-bromothiophene-2-carboxaldehyde is 1:(0.9-1.1); 4-6 mL of anhydrous methanol is added for every 1 mmol of p-aminobenzenesulfonamide; 0.2-0.8 mL of glacial acetic acid is added for every 1 mmol of p-aminobenzenesulfonamide; the heating reflux temperature is 64-66℃, and the heating reflux time is 3.5-7.5 hours.

4. The method for preparing compound SB1 as described in claim 2, characterized in that, In step (3), the solid-liquid ratio of the crude solid product to boiling anhydrous methanol is 1 g: (8-16) mL; the recrystallization time is 3-7 hours.

5. The use of compound SB1 as described in claim 1 in the preparation of a medicament for the prevention or treatment of toxoplasmosis infection.

6. The use of compound SB1 as described in claim 5 in the preparation of a medicament for the prevention or treatment of toxoplasmosis infection, characterized in that, The drugs mentioned for preventing or treating Toxoplasma gondii infection are drugs that inhibit the ability of Toxoplasma gondii to lyse cells.

7. The use of compound SB1 as described in claim 5 in the preparation of a medicament for the prevention or treatment of toxoplasmosis infection, characterized in that, The drugs mentioned for preventing or treating toxoplasmosis infection are drugs that inhibit the invasion and proliferation of Toxoplasma gondii.

8. The use of compound SB1 as described in claim 5 in the preparation of a medicament for the prevention or treatment of toxoplasmosis infection, characterized in that, The SB1 showed a 50% inhibitory effect on Toxoplasma gondii at 20.641 μM.

9. A composition for the prevention or treatment of toxoplasmosis infection, characterized in that, The active ingredient of the composition includes compound SB1 as described in claim 1.

10. The composition for preventing or treating toxoplasmosis infection as described in claim 9, characterized in that, The composition further includes one or more pharmaceutically acceptable carriers, said carriers comprising one or more of the following: diluents, wetting agents, binders, disintegrants, lubricants, flavor and color modifiers, solvents, solubilizers, cosolvents, emulsifiers, antioxidants, metal complexing agents, preservatives, pH adjusters, surfactants, excipients, fillers, and synergists.

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