Application of compound CTU-B2R in preparation of medicine for treating toxoplasmosis

Abstract

The invention provides an application of a compound CTU-B2R in preparation of a medicine for treating toxoplasmosis. The compound CTU-B2R comprises CTU-B2N (the molecular formula is C28H18O3BrN3S) and / or CTU-B2Br (the molecular formula is C28H18OBr2N2S). The inventor finds and verifies that CTU-B2R has a remarkable inhibitory activity effect on intracellular parasitic protozoa toxoplasma gondii through a large number of researches, Vero cells infected with the toxoplasma gondii are used as cells of an experimental model, and EC50 determination shows that the CTU-B2N has a half of inhibitory effect on the toxoplasma gondii when the CTU-B2N is 0.7 mu M and CTU-B2Br is 0.4 mu M, and the CTU-B2R has a remarkable inhibitory activity effect on the toxoplasma gondii when the CTU-B2N is 0.7 mu M and the CTU-B2Br is 0.4 mu M. And an efficient toxoplasma gondii infection resisting effect can be achieved by applying a small amount. Meanwhile, the cytotoxicity of the CTU-B2R to the Vero is detected by a CCK8 method, and the result shows that when the dosage of the CTU-B2N and the CTU-B2Br is maximally 500 mu M, the CTU-B2N and the CTU-B2Br still have no cytotoxicity to the Vero cells. Compared with the existing drugs for treating toxoplasma gondii infection, which have the problems of large side effect, poor effect and the like, the effective inhibition concentration of the CTU-B2R provided by the invention is far less than the cytotoxicity of the CTU-B2R, and the CTU-B2R has the remarkable advantages of high efficiency, low toxicity and safety when being used for preparing the drugs for preventing or treating toxoplasma gondii infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of parasitic disease prevention and treatment, and particularly relates to the use of the compound CTU-B2R in preparing a drug for treating toxoplasmosis. Background Art

[0002] Toxoplasmosis is an obligate intracellular parasitic protozoan disease caused by Toxoplasma gondii. Toxoplasma gondii infects almost all warm-blooded animals and humans, even some cold-blooded animals, and can parasitize all nucleated cells in the animal body. According to statistics, about one-third of the world's population is infected with Toxoplasma gondii, and the serum positivity rate of Toxoplasma gondii varies from region to region. For example, the positivity rate in the Americas, Europe and Asia is about 30%, while the positivity rate in Africa is over 60%. Most people have latent toxoplasmosis infection, but infants and immunosuppressed patients (such as AIDS patients, organ transplant patients and malignant tumor patients) infected with Toxoplasma gondii will develop severe or fatal diseases. Toxoplasmosis infection is also an important cause of miscarriage, stillbirth and other reproductive disorders in pregnant animals and pregnant women, and is one of the must-check items for prenatal examinations in my country.

[0003] In the livestock sector, a wide range of animals (pigs, cattle, sheep, horses, dogs, cats, chickens, etc.) have high infection rates for Toxoplasma gondii. Serological surveys indicate that up to 30% of pigs worldwide are infected with Toxoplasma gondii, and in parts of my country, infection rates in pigs reach over 70%. When acute toxoplasmosis occurs on pig farms, the morbidity rate can reach 100%, with mortality rates exceeding 60%. In the United States and China, pigs are considered the most important transmitters of Toxoplasma among food animals, posing a significant public health and safety risk. my country ranks first in the world in pork production, and an outbreak of Toxoplasmosis in pigs would result in significant economic losses to the livestock industry and potentially become a serious food safety issue.

[0004] Currently, clinical treatment for toxoplasmosis still relies on chemical drugs. Due to the complexity of the parasite's life cycle, diverse pathogenesis, and variability in its biological characteristics, there are currently no preventive or specific drug treatments. Although the combination of pyrimethamine and sulfadiazine is currently the gold standard for clinical treatment of toxoplasmosis, this treatment method is often accompanied by serious side effects, is incomplete, and is prone to relapse, resulting in a high failure rate. Therefore, screening for safe and effective anti-toxoplasmosis drugs is an urgent issue and has great market prospects.

[0005] In summary, developing a drug that can effectively prevent or treat Toxoplasma gondii infection is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0006] In view of the problems in the prior art of drugs for preventing or treating Toxoplasma infection, such as drug resistance, severe toxic side effects, high drug dosage and poor efficacy in preventing and treating Toxoplasma infection, the present invention specifically provides the use of the compound CTU-B2R in the preparation of drugs for preventing or treating Toxoplasma infection, which specifically includes:

[0007] One of the objects of the present invention is to provide the use of compound CTU-B2R in the preparation of a drug for preventing or treating Toxoplasma gondii infection. The structural formula of the compound CTU-B2R is shown in Formula I:

[0008]

[0009] Wherein, R is Br or NO2.

[0010] In a preferred embodiment, R is NO2, the compound is CTU-B2N, and the molecular formula is C 28 H 18 O3BrN3S, molecular weight 556.4, C, 60.44; H, 3.26; N, 7.55; S, 5.76; the specific structural formula is shown in Formula II:

[0011]

[0012] In a preferred embodiment, R is Br, the compound is CTU-B2Br, and the molecular formula is C 28 H 18 OBr2N2S, molecular weight 590.3, C, 56.97; H, 3.07; N, 4.75; S, 5.43; the specific structural formula is shown in Formula III:

[0013]

[0014] In a preferred embodiment, the preparation method of the CTU-B2R (CTU-B2N and / or CTU-B2Br) comprises the following steps:

[0015] Step 1: Under nitrogen protection, reacting the compound 2-bromobenzoyl chloride represented by 1a or the compound 2-nitrobenzoyl chloride represented by 1b with potassium thiocyanate in anhydrous acetone at 40°C for 1 hour to obtain a compound represented by formula 2a / 2b: wherein the molar ratio of the compound represented by formula 1 (1a / 1b) to potassium thiocyanate is 1:(0.8-1.2).

[0016] Step 2: reflux the compound of formula 2a / 2b with an equimolar amount of 2-phenylaniline (3) in anhydrous acetone for 2-3 hours, continue stirring overnight, cool, filter, and collect the solid to obtain the compound of formula 4a / 4b;

[0017] Step 3: Under nitrogen protection, the compound represented by Formula 4a / 4b and an equimolar amount of triethylamine are dissolved in anhydrous dichloromethane, and a mixed solution of 2,4'-dibromoacetophenone (5) and anhydrous dichloromethane is slowly added dropwise to the mixed solution, and the mixture is refluxed for 2 hours. After the reaction is completed, the mixture is filtered and concentrated, and the crude product is recrystallized from ethanol to obtain the compound represented by Formula 6a / 6b CTU-B2N / CTU-B2Br; wherein the molar ratio of 2,4'-dibromoacetophenone to the compound represented by Formula 4 (4a / 4b) is (0.8-1.2):1.

[0018] The specific synthetic route of step 1 to step 4 is as follows:

[0019]

[0020] In a preferred embodiment, the drug for preventing or treating Toxoplasma gondii infection is a drug for preventing or treating toxoplasmosis.

[0021] In a preferred embodiment, the drug for preventing or treating Toxoplasma infection is a drug that inhibits the proliferation of Toxoplasma or inhibits the ability of Toxoplasma to lyse cells.

[0022] In a preferred embodiment, the drug for preventing or treating Toxoplasma infection is: a drug that eliminates Toxoplasma gondii parasites in humans or non-human animals; wherein, the non-human animals include but are not limited to pigs, cattle, sheep, horses, dogs, cats, chickens, etc. that are susceptible to Toxoplasma gondii.

[0023] In a preferred embodiment, the CTU-B2N has a half inhibitory effect on Toxoplasma gondii at 0.7 μM.

[0024] In a preferred embodiment, the CTU-B2Br has a half inhibitory effect on Toxoplasma gondii at 0.4 μM.

[0025] Another object of the present invention is to provide a composition for preventing or treating Toxoplasma gondii infection, wherein the active ingredient of the composition includes CTU-B2R.

[0026] In a preferred embodiment, the composition further comprises one or more of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an antioxidant, a metal complexing agent, a preservative, a pH regulator, a surfactant, an excipient, a filler and a synergist.

[0027] In a preferred embodiment, the diluent includes starch, sucrose, cellulose, inorganic salts, etc.; the wetting agent includes water, ethanol, etc.; the binder includes starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; the disintegrant includes starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium dicarboxymethyl cellulose, surfactants, etc.; the lubricant includes talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, polyethylene glycol, etc.; the color, flavor and flavoring agent includes pigment, sweetener, flavor, mucilage, etc.; the solvent includes water, glycerol, ethanol, etc.; the solubilizer includes Tweens, sorbits, sulfates, sulfonates, etc.; the cosolvents include organic acids (such as citric acid) and their salts, inorganic salts, polyethylene glycol, etc.; the emulsifiers include spans, glycerol fatty acid esters, gum arabic, gelatin, agar, sodium alginate, etc.; the antioxidants include sulfites, ascorbic acid, gallic acid and its salts, etc.; the metal chelating agents include disodium edetate, 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.

[0028] In the present invention, the composition dosage forms include, but are not limited to, powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, and other oral dosage forms, topical preparations, suppositories, and sterile injectable solutions. It will be appreciated that the CTU-B2R-containing compositions of the present invention can be administered in a variety of ways, depending on the different excipients and dosage forms.

[0029] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0030] 1. During the research process, the inventors found that CTU-B2R (CTU-B2N, CTU-B2Br) has a significant inhibitory effect on the intracellular parasite Toxoplasma gondii, and its toxic side effects are small, which can expand new avenues for the effective utilization of CTU-B2R.

[0031] 2. The inventors have verified through a large number of experiments that CTU-B2R has a significant effect in preventing or treating Toxoplasma gondii infection. Specifically, using Vero cells infected with Toxoplasma gondii as the experimental model, the EC 50 The results showed that CTU-B2R had a 50% inhibitory effect on Toxoplasma gondii at 0.7 μM (CTU-B2N) and 0.4 μM (CTU-B2Br). Therefore, a small amount of administration can achieve a highly effective anti-Toxoplasma infection effect.

[0032] 3. Furthermore, experiments have confirmed that CTU-B2R, as an anti-Toxoplasma drug, exhibits low toxicity. Specifically, the cytotoxicity of CTU-B2R against green monkey kidney (Vero) cells was tested using the CCK8 assay. The results showed that CTU-B2N and CTU-B2Br remained non-cytotoxic to Vero cells at a maximum dose of 500 μM. Therefore, compared to existing drugs for the treatment of Toxoplasma infection, which suffer from significant side effects and poor efficacy, the CTU-B2R provided in the present invention has an effective inhibitory concentration far below its cytotoxicity, offering significant advantages for the preparation of drugs for the prevention or treatment of Toxoplasma infection, including high efficacy, low toxicity, and safety.

[0033] 4. Through a large number of experimental explorations, the present invention also found that the anti-Toxoplasma effect of CTU-B2R is mainly through inhibiting its intracellular proliferation, and the effect of CTU-B2R on Toxoplasma is dose-dependent, indicating that the higher the concentration, the better the anti-insect effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The advantages of CTU-B2N and CTU-B2Br, such as good anti-Toxoplasma efficacy and low toxicity and low side effects, will become more apparent and easier to understand from the following detailed description of the embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0035] Figure 1 This is a graph showing the results of a toxicity test on Vero cells of CTU-B2N and CTU-B2Br in Example 1 of the present invention;

[0036] Figure 2 This is a graph showing the inhibitory effect of CTU-B2N and CTU-B2Br on Toxoplasma gondii in Example 2 of the present invention;

[0037] Figure 3 This is a diagram of a plaque assay of different concentrations of CTU-B2N, CTU-B2Br, and a control group in Example 3 of the present invention;

[0038] Figure 4 This is a statistical graph showing the anti-proliferative effects of different concentrations of CTU-B2N, CTU-B2Br and a control group on intracellular RH tachyzoites in Example 4 of the present invention;

[0039] Figure 5 This is a graph showing the anti-invasion effects of different concentrations of CTU-B2N, CTU-B2Br and the control group on extracellular RH-type Toxoplasma gondii in Example 4 of the present invention. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that the protection scope of the present invention is not limited to the specific embodiments.

[0041] Unless otherwise specified, the technical means used in the present invention are conventional means well known to those skilled in the art. The various raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods. All reagents used in the present invention were of analytical grade unless otherwise specified. Vero cells and HFF cells used in the present invention were purchased from the ATCC cell bank.

[0042] According to the synthetic route of CTU-B2R, the preparation method of CTU-B2Br comprises the following steps:

[0043] Step 1: Under nitrogen, dissolve 0.05 mol of 2-bromobenzoyl chloride (compound 1a) in 20 mL of anhydrous acetone and slowly add the resulting mixture dropwise to a solution of 0.04 mol of potassium thiocyanate in 100 mL of anhydrous acetone. Reflux the mixture and monitor by TLC (n-hexane:ethyl acetate = 4:6) until the starting material spots disappear. Cool the reaction mixture and pour it into ice water to precipitate a yellow precipitate. Filter, wash with ethanol, and vacuum dry to obtain the compound of Formula 2a.

[0044] Step 2: 0.04 mol of the compound represented by Formula 2a and 0.04 mol of 2-phenylaniline were added to 80 mL of anhydrous acetone. The mixture was refluxed for 3 hours and then stirred at room temperature overnight. TLC was monitored until the intermediate spot disappeared. The reaction solution was poured into ice water to precipitate a white precipitate. The precipitate was filtered, recrystallized from ethanol, and dried in vacuo to obtain the compound represented by Formula 4a.

[0045] Step 3: Under nitrogen protection, 0.01 mol of the compound represented by formula 4a was dissolved in 30 mL of anhydrous dichloromethane. 0.01 mol of triethylamine was added, and a solution of 0.01 mol of 2,4'-dibromoacetophenone (5) in 7 mL of anhydrous dichloromethane was slowly added dropwise. The reaction was continued until the raw material spot disappeared as monitored by TLC. The reaction solution was filtered and concentrated under reduced pressure. The crude product was recrystallized from ethanol and dried in vacuo to obtain CTU-B2Br.

[0046] The preparation method of CTU-B2N comprises the following steps:

[0047] Step 1: Under nitrogen, dissolve 0.05 mol of 2-nitrobenzoyl chloride (compound 1b) in 20 mL of anhydrous acetone and slowly add dropwise to a solution of 0.04 mol of potassium thiocyanate in 100 mL of anhydrous acetone. Reflux the mixture and monitor by TLC (n-hexane:ethyl acetate = 4:6) until the starting material spots disappear. Cool the reaction solution and pour it into ice water to precipitate a yellow precipitate. Filter, wash with ethanol, and vacuum dry to obtain the compound of Formula 2b.

[0048] Step 2: 0.04 mol of the compound represented by Formula 2b and 0.04 mol of 2-phenylaniline were added to 80 mL of anhydrous acetone. The mixture was refluxed for 3 hours and then stirred at room temperature overnight. TLC was monitored until the intermediate spot disappeared. The reaction solution was poured into ice water to precipitate a white precipitate. The precipitate was filtered, recrystallized from ethanol, and dried in vacuo to obtain the compound represented by Formula 4b.

[0049] Step 3: Under nitrogen protection, 0.01 mol of the compound represented by Formula 4b was dissolved in 30 mL of anhydrous dichloromethane. 0.01 mol of triethylamine was added, and a solution of 0.01 mol of 2,4'-dibromoacetophenone (5) in 7 mL of anhydrous dichloromethane was slowly added dropwise. The reaction was continued until the raw material spot disappeared as monitored by TLC. The reaction solution was filtered and concentrated under reduced pressure. The crude product was recrystallized from ethanol and dried under vacuum to obtain CTU-B2N.

[0050] By providing the use of the compound CTU-B2R in the preparation of a drug for treating toxoplasmosis, the present invention effectively addresses several issues with existing drugs for preventing or treating toxoplasmosis, including their tendency to induce drug resistance, significant toxic side effects, and high dosages, all while being ineffective in preventing and treating toxoplasmosis. The mechanism behind this is believed to be that CTU-B2R, due to its specific structure containing a sulfur heterocycle, nitrogen atoms, and a benzene ring, may bind to the active sites of key Toxoplasma proteins, interfering with protein function and thereby inhibiting Toxoplasma proliferation, thereby preventing or treating toxoplasmosis. Furthermore, the introduction of bromine atoms increases the molecule's lipophilicity and electronic effects, potentially enhancing its selective binding to Toxoplasma through hydrophobic and electrostatic interactions.

[0051] Existing anti-toxoplasma drugs (such as pyrimethamine and sulfadiazine) can easily lead to drug resistance with long-term use. However, the novel structure of the present invention can reduce the risk of drug resistance in Toxoplasma gondii, broadening treatment strategies. Furthermore, compared to existing Toxoplasma gondii treatments that suffer from significant side effects and poor efficacy, the CTU-B2R provided by the present invention has an effective inhibitory concentration far lower than its cytotoxicity, making it highly effective, low-toxic, and safe for use in the preparation of drugs to prevent or treat Toxoplasma infection.

[0052] The technical solution of this application is described in detail below through specific embodiments:

[0053] Example 1

[0054] Study on the toxicity of CTU-B2N and CTU-B2Br to Vero cells

[0055] 1. Experimental process

[0056] Cytotoxicity experiment: The CCK-8 method was used to determine the toxicity of CTU-B2N and CTU-B2Br to Vero cells. African green monkey kidney (Vero) cell suspension was inoculated in a 96-well cell culture plate, with approximately 100 μL (5000 Vero cells) per well. The culture plate was placed in an incubator for pre-culture for a period of time (37°C, 5% CO2) to allow the cells to adhere to the wall for about four hours. CTU-B2N was set at a concentration gradient of 0.4 μM-500 μM for a total of ten times, and CTU-B2Br was set at a concentration gradient of 0.1 μM-500 μM for a total of twelve times. Vero cells were treated with different concentrations to observe whether their cytotoxicity was significantly different from that of the 0.1% DMSO control group. The absorbance at 450 nm was measured using a microplate reader. The cell viability was calculated according to the following formula. The independent experiment was repeated 3 times.

[0057] Cell viability (%) = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%

[0058] 2. Experimental results

[0059] like Figure 1 The absorbance values of African green monkey kidney cells treated with different concentrations of CTU-B2N and CTU-B2Br at 450nm were statistically analyzed. The results showed that there was no significant difference in absorbance between the groups treated with different concentrations of CTU-B2N and CTU-B2Br and the DMSO control group, indicating that CTU-B2N and CTU-B2Br have low toxicity and are safe for cells.

[0060] Example 2

[0061] Inhibitory effects of CTU-B2N and CTU-B2Br on Toxoplasma gondii

[0062] 1. Experimental process

[0063] Vero cells were spread over 96-well cell culture plates, and fresh and vigorous Luciferase tachyzoites were harvested from the Vero cells. The tachyzoites were counted with a hemocytometer. 3×10 5Tachyzoites were inoculated into Vero cells. DMSO was used as the control group, and eight concentration gradients of CTU-B2N and CTU-B2Br were added, ranging from 0.1μM to 10μM. The culture medium in each well was controlled at 150-300μL, and the independent experiment was repeated 3 times. After culturing for 24 hours, the DMEM in the well was gently aspirated, and 100μL of cell lysis solution was added to each well. After lysis for 5 minutes, the lysate was aspirated, placed in a 1mL EP tube, centrifuged at 12000r / min for 5 minutes, 100μL of supernatant was aspirated and added to a 96-well enzyme-labeled plate, and then 100μL of luciferase was added to each well under light-proof conditions. After mixing, the luminescence value was detected in a chemiluminescence instrument, and the survival rate of Luciferase was calculated. Inhibition rate % = (RLU DMSO -RLU 处理组 ) / RLU DMSO ×100

[0064] 2. Experimental results

[0065] like Figure 2 The results showed that the half maximum effective concentration (Effective Concentration 50%, EC 50 ) were 0.7μM and 0.4μM, respectively. Moreover, the inhibitory effect on Toxoplasma gondii was dose-dependent, with higher concentrations leading to stronger inhibitory effects. This indicates that CTU-B2N and CTU-B2Br have good inhibitory effects on Toxoplasma gondii.

[0066] Example 3

[0067] Effects of CTU-B2N and CTU-B2Br on the inhibition of intracellular plaque formation of Toxoplasma gondii

[0068] 1. Experimental process

[0069] Experiment A: Human foreskin fibroblasts (HFFs), a classic cell model for Toxoplasma research, were spread over 12-well cell culture plates. Tachyzoites of Toxoplasma type I strain (RH) were collected, and then 150 tachyzoites were inoculated into a monolayer of HFF cells (the ratio of the number of inoculated Toxoplasma to the number of host cells was 1:2000). The cells were divided into three groups, each with 3 wells. Group (1) was the 1.4 μM CTU-B2N group, group (2) was the 6 μM CTU-B2N group, and group (3) was the 0.1% DMSO control group. After incubation for 7 days (the culture medium in each well was controlled at 2-3 mL), the cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes, washed with PBS, and stained with crystal violet to observe the size of the plaques.

[0070] Experiment B: Compared with Experiment A, the only difference is that group (1') is the group with 0.8 μM CTU-B2Br, group (2') is the group with 6 μM CTU-B2Br, and group (3) is the control group with 0.1% DMSO. The rest of the method steps are exactly the same as Experiment A.

[0071] 2. Experimental results

[0072] like Figure 3 At the same magnification, larger plaques were observed in the DMSO negative control group, which were significantly different from the plaque areas formed in the cells of the CTU-B2N and CTU-B2Br groups, indicating that CTU-B2N and CTU-B2Br significantly inhibited the growth of Toxoplasma gondii.

[0073] Example 4

[0074] Antiproliferative Effects of CTU-B2N and CTU-B2Br on Intracellular Toxoplasma gondii Type I Strain (RH)

[0075] 1. Experimental process

[0076] Proliferation assay A: HFF cells in good growth condition were seeded on a 12-well flyer plate, and 1×10 5 Fresh RH tachyzoites were inoculated with a ratio of 1:10 between the number of Toxoplasma and the number of host cells. After 2 hours of invasion, the cells were divided into three groups: (1) group with 1.4 μM CTU-B2N, (2) group with 6 μM CTU-B2N, and (3) group with 0.1% DMSO control. After 24 hours of culture, the cells were fixed, permeabilized, blocked, and stained. The primary antibody used was rabbit-derived Toxoplasma surface protein GAP45, with a dilution of 1:300, and the secondary antibody used was goat anti-rabbit fluorescent secondary antibody labeled with fluorescein isothiocyanate (FITC), with a dilution of 1:100. Under a fluorescence microscope, multiple fields of view were randomly selected, including the top, bottom, left, right, and middle positions of each well. The number of Toxoplasma tachyzoites in 100 parasite-carrying vacuoles was counted, and the independent experiment was repeated 3 times.

[0077] Invasion experiment A: HFF cells were used to invade a 12-well culture plate filled with cells. 1×10 5RH tachyzoites were divided into three groups: (1) group with 1.4μM CTU-B2N, (2) group with 6μM CTU-B2N, and (3) group with 0.1% DMSO control group. After culturing in a 37°C, 5% CO2 incubator for 30 minutes, the culture medium was immediately removed and the cells were washed three times with PBS to thoroughly wash away the uninvaded tachyzoites. The extracellular tachyzoites could be observed under a microscope to see if they were clean. The cells were cultured for 20 hours and an indirect immunofluorescence assay (IFA) experiment was performed. The cells were placed under a fluorescence microscope for observation. 8-10 fields of view were randomly selected from each well, including the top, bottom, left, right, and middle positions of each well. Images of Toxoplasma tachyzoites and host cell nuclei were collected using a fluorescence microscope. The number of parasite-carrying vacuoles and the number of tachyzoite host cells in each picture were counted to express the anti-proliferative effects of different concentrations of CTU-B2N and the control group on intracellular RH tachyzoites, as shown in the following figure: Figure 4 The invasion efficiency of each image is calculated as follows: the number of parasite-carrying vacuoles per image / the number of host cells per image. The average invasion efficiency of all collected images is the invasion efficiency of the parasite strain. The anti-invasion effect of different concentrations of CTU-B2N and the control group on extracellular RH-type Toxoplasma is shown in the figure. Figure 5 shown.

[0078] Proliferation assay B: Compared with proliferation assay A, the only difference is that group (1') is the group with 0.8 μM CTU-B2Br, group (2') is the group with 6 μM CTU-B2Br, and group (3') is the control group with 0.1% DMSO. The rest of the procedures are exactly the same as those in proliferation assay A.

[0079] Invasion assay B: Compared with invasion assay A, the only difference is that group (1') is the group with 0.8 μM CTU-B2Br, group (2') is the group with 6 μM CTU-B2Br, and group (3') is the control group with 0.1% DMSO. The rest of the procedures are exactly the same as those of invasion assay A.

[0080] 2. Experimental results

[0081] After Toxoplasma gondii invades host cells, it forms parasitic vacuoles, in which tachyzoites multiply by binary fission. Therefore, the number of tachyzoites in the parasitic vacuoles within the same time after invasion can reflect the proliferation ability of the parasite. The statistical results of the proliferation experiment showed that compared with the control group (DMSO group), the invasion and proliferation ability of the parasite in the CTU-B2N and CTU-B2Br groups was significantly reduced ( Figure 4 、 5 ). This showed that CTU-B2N and CTU-B2Br significantly inhibited the invasion and proliferation of RH Toxoplasma gondii.

[0082] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. Use of the compound CTU-B2R in the preparation of a drug for preventing or treating Toxoplasma gondii infection, characterized in that: The structural formula of the compound CTU-B2R is shown in Formula I: Wherein, R is Br or NO2.

2. Use of the compound CTU-B2R according to claim 1 in the preparation of a drug for preventing or treating Toxoplasma gondii infection, characterized in that: R is NO2, the compound is CTU-B2N, and the molecular formula is C 28 H 18 O3BrN3S, the specific structural formula is shown in Formula II:

3. Use of the compound CTU-B2R according to claim 1 in the preparation of a drug for preventing or treating Toxoplasma gondii infection, characterized in that: R is Br, the compound is CTU-B2Br, and the molecular formula is C 28 H 18 OBr2N2S, the specific structural formula is shown in Formula III:

4. Use of the compound CTU-B2R according to claim 1 in the preparation of a drug for preventing or treating Toxoplasma gondii infection, characterized in that: The drug for preventing or treating toxoplasmosis is a drug for preventing or treating toxoplasmosis.

5. Use of the compound CTU-B2R according to claim 1 in the preparation of a drug for preventing or treating Toxoplasma gondii infection, characterized in that: The drug for preventing or treating Toxoplasma infection is a drug that inhibits the proliferation of Toxoplasma or inhibits the ability of Toxoplasma to lyse cells.

6. Use of the compound CTU-B2R according to claim 1 in preparing a drug for preventing or treating Toxoplasma gondii infection, characterized in that: The drug for preventing or treating Toxoplasma infection is a drug that eliminates Toxoplasma gondii in humans or non-human animals.

7. Use of the compound CTU-B2R according to claim 2 in the preparation of a drug for preventing or treating Toxoplasma gondii infection, characterized in that: The CTU-B2N had a half inhibitory effect on Toxoplasma gondii at 0.7 μM.

8. Use of the compound CTU-B2R according to claim 3 in the preparation of a drug for preventing or treating Toxoplasma gondii infection, characterized in that: The CTU-B2Br has a half inhibitory effect on Toxoplasma gondii at 0.4 μM.

9. A composition for preventing or treating Toxoplasma gondii infection, characterized in that: The active ingredient of the composition includes CTU-B2R.

10. The composition for preventing or treating Toxoplasma gondii infection according to claim 9, wherein The composition further comprises one or more of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an antioxidant, a metal complexing agent, a preservative, a pH regulator, a surfactant, an excipient, a filler and a synergist.

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