Amino-thiocarbonyl compound, preparation method thereof and application of amino-thiocarbonyl compound in preparation of medicine for preventing or treating toxoplasma gondii infection

Abstract

The invention provides an aminothiocarbonyl compound, a preparation method thereof and application of the aminothiocarbonyl compound in preparation of drugs for preventing or treating toxoplasma gondii infection. In long-term research, the inventor finds that the aminothiocarbonyl compound shown in the formula has a remarkable inhibitory activity effect on intracellular parasitic protozoa toxoplasma gondii, has small toxic and side effects, and has the potential of preparing drugs for preventing or treating toxoplasma gondii infection. Meanwhile, the toxoplasma gondii resisting activity and cell safety of the compound TU-BO and the compound TU-BP are verified, and results show that the half effective inhibition concentrations of the TU-BO and the TU-BP on toxoplasma gondii are 1.478 mu M and 7.219 mu M respectively, the half cytotoxic concentrations of the TU-BO and the TU-BP on Vero cells are 61.73 mu M and 654.5 mu M respectively, and the safety indexes SI are 41.77 and 90.66 respectively. Compared with existing drugs for treating toxoplasma gondii infection, the effective inhibition concentration of TU-BO and TU-BP is far smaller than that of cytotoxicity, and the TU-BO and TU-BP can significantly inhibit lysis cells of toxoplasma gondii, inhibit invasion and proliferation of toxoplasma gondii, and have significant effects of preventing and treating toxoplasma gondii infection.

Description

Technical Field

[0001] This invention belongs to the field of parasitic disease prevention and control technology, specifically relating to an aminothiocarbonyl compound, its preparation method, and its application 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 infected with Toxoplasma gondii. The seropositivity rate varies by region; for example, the positivity rate is about 30% in the Americas, Europe, and Asia, while it is over 60% in Africa. Most human Toxoplasmosis infections are asymptomatic, but infection in infants and immunocompromised patients (such as those with HIV / AIDS, organ transplant recipients, and those with malignant tumors) can lead to severe or fatal illness. Toxoplasmosis infection is also a significant cause of miscarriage, stillbirth, and other reproductive disorders in pregnant animals and women, and is one of the mandatory prenatal screening tests in my country. Many animals (pigs, cattle, sheep, horses, dogs, cats, chickens, etc.) have high infection rates of Toxoplasma gondii. Serological surveys show that up to 30% of pigs worldwide are infected with Toxoplasma gondii, and in some parts of my country, the infection rate in pigs is even higher than 70%. When acute toxoplasmosis occurs in pig farms, 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 carriers of Toxoplasma gondii among many food animals, posing a significant public health and safety risk. my country is the world's largest producer of pork; an outbreak of swine toxoplasmosis would cause enormous economic losses to the livestock industry and could also become a serious food safety issue.

[0003] 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 side effects, and it is not a complete cure, has a high relapse rate, and a high treatment failure rate.

[0004] Therefore, developing a safe and effective anti-Toxoplasma gondii drug is an urgent problem to be solved and also has broad market prospects. In view of this, this invention is proposed. Summary of the Invention

[0005] To address the problems of existing technologies, such as drug resistance, significant toxic side effects, and poor efficacy despite large dosages in preventing or treating toxoplasmosis, this invention specifically provides an aminothiocarbonyl compound, its preparation method, and its application in the preparation of drugs for preventing or treating toxoplasmosis. Specifically, it includes:

[0006] One objective of this invention is to provide an aminothiocarbonyl compound, specifically comprising the formula... The general formula of the structure shown is:

[0007] Mode , where R represents any alkane with 4-7 carbon atoms.

[0008] In a preferred embodiment, R is butyl or heptyl;

[0009] When R is heptyl, the molecular formula of the compound is C1. 21 H 26 N2OS, its chemical structural formula is:

[0010] ;

[0011] When R is butyl, the molecular formula of the compound is C. 18 H 20 N2OS, its chemical structural formula is:

[0012] ;

[0013] Preferably, when R is heptyl, the compound is designated as TU-BO;

[0014] When R is butyl, the compound is designated as TU-BP.

[0015] The second objective of this invention is to provide a formula The method for preparing the aminothiocarbonyl compound shown includes the following steps:

[0016] Add a straight-chain saturated alkanoyl chloride with any number of carbons (5-8) dropwise to a dry acetone solution of potassium thiocyanate. After the addition is complete, heat to reflux under nitrogen protection and maintain reflux for 1.5-2.5 h. After cooling the resulting reaction solution to room temperature, add 2-aminobiphenyl, heat to reflux again, and maintain reflux for 8-10 h. After cooling to room temperature, pour into crushed ice, allow to stand to precipitate, filter, and dry to obtain the crude product. Recrystallize the crude product from ethanol to obtain aminothiocarbonyl compounds.

[0017] In a preferred embodiment, the straight-chain saturated alkanoyl chloride with any number of carbons of 5-8 is octanoyl chloride or valerate chloride.

[0018] In a preferred embodiment, the molar ratio of potassium thiocyanate to 3,5-dinitrobenzoyl chloride is 6:(3-5); preferably, the molar ratio of potassium thiocyanate to 3,5-dinitrobenzoyl chloride is 3:2.

[0019] In a preferred embodiment, 8-10 mL of dry acetone is used for every 1 mmol of potassium thiocyanate; preferably, 9.1 mL of dry acetone is used for every 1 mmol of potassium thiocyanate.

[0020] In a preferred embodiment, the molar ratio of potassium thiocyanate to 2-aminobiphenyl is 6:(3-5); preferably, the molar ratio of potassium thiocyanate to 2-aminobiphenyl is 3:2.

[0021] In a preferred embodiment, the volume of the crushed ice is 2-4 times the volume of the reaction product; the standing time is 0.5-3 h; the drying can be carried out using conventional methods known to those skilled in the art, such as drying at 40-60℃ for 4-8 h; the ethanol recrystallization uses anhydrous ethanol, and the amount of anhydrous ethanol used is 3-8 times the mass of the crude product.

[0022] The third objective of this invention is to provide a formula The application of aminothiocarbonyl compounds shown is in the preparation of drugs for the prevention or treatment of Toxoplasma gondii infection.

[0023] 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.

[0024] In a preferred embodiment, the drug for preventing or treating toxoplasmosis infection is a drug that inhibits the invasion and proliferation of Toxoplasma gondii.

[0025] In a preferred embodiment, when R is butyl, the compound has an LD50 of 1.478 μM against Toxoplasma gondii and an LD50 of 61.73 μM against Vero cells.

[0026] In a preferred embodiment, when R is heptyl, the compound has an LD50 of 7.219 μM against Toxoplasma gondii and an LD50 of 654.5 μM against Vero cells.

[0027] A fourth objective of this invention is to provide a composition for the prevention or treatment of toxoplasmosis infection, wherein the active ingredient of the composition comprises the formula... The shown are aminothiocarbonyl compounds.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] In this invention, the dosage forms of the composition include, but are not limited to, oral dosage forms, topical dosage forms, suppositories, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays. It is understood that the dosage forms of the composition described in this invention are... The combinations of aminothiocarbonyl compounds shown can be administered in various ways depending on the different excipients and dosage forms.

[0032] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0033] 1. The inventors discovered the formula during long-term research. The aminothiocarbonyl compounds with the specific structure shown have significant inhibitory activity against the intracellular parasitic protozoan Toxoplasma gondii, and have few toxic side effects, showing potential for the preparation of drugs for the prevention or treatment of Toxoplasma gondii infection.

[0034] 2. The inventor has proven the formula through extensive experiments. The aminothiocarbonyl compounds (TU-BO and TU-BP) with specific structures shown exhibit significant preventive or therapeutic effects against Toxoplasma gondii infection: using Vero cells infected with Toxoplasma gondii as an experimental model, EC50 was measured... 50 The results showed that the application of compound TU-BO at a concentration of 1.478 μM had a 50% inhibitory effect on Toxoplasma gondii, and the application of compound TU-BP at a concentration of 7.219 μM had a 50% inhibitory effect on Toxoplasma gondii. Small amounts of the compound can achieve a highly effective anti-Toxoplasma gondii infection effect.

[0035] 3. Further experiments have confirmed that the formula... The aminothiocarbonyl compounds with the specific structures shown in this invention also exhibit low toxicity as anti-Toxoplasma gondii drugs: according to the CCK8 assay, the half-maximal cytotoxic concentration (ICC) of TU-BO against Vero cells was 61.73 μM, and that of TU-BP against Vero cells was 654.5 μM. Therefore, compared to existing drugs for treating Toxoplasma gondii infection, which suffer from significant side effects and poor efficacy, the aminothiocarbonyl compounds with the specific structures provided in this invention have an effective inhibitory concentration far lower than their cytotoxicity, offering significant advantages in the preparation of drugs for the prevention or treatment of Toxoplasma gondii infection: high efficiency, low toxicity, and safety.

[0036] 4. Through extensive experimental exploration, this invention has also discovered that formula... The aminothiocarbonyl compounds with the specific structures shown exhibit anti-Toxoplasma gondii activity mainly by inhibiting its invasion and intracellular proliferation. Furthermore, the effect of these compounds on Toxoplasma gondii is dose-dependent, indicating that higher concentrations result in better anti-parasitic effects. Attached Figure Description

[0037] 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.

[0038] Figure 1 This is a graph showing the toxicity test results of TU-BO on Vero cells in Example 1 of the present invention;

[0039] Figure 2This is a graph showing the toxicity test results of TU-BP on Vero cells in Example 1 of the present invention;

[0040] Figure 3 This is a graph showing the inhibitory effect of TU-BO on Toxoplasma gondii in Example 2 of the present invention.

[0041] Figure 4 This is a graph showing the inhibitory effect of TU-BP on Toxoplasma gondii in Example 2 of the present invention.

[0042] Figure 5 The figures show the plaque phagocytic results of TU-BO and the control group in Example 3 of this invention;

[0043] Figure 6 The figures show the plaque phagocytic results of TU-BP and the control group in Example 3 of this invention;

[0044] Figure 7 The graph shows the anti-proliferative effect of TU-BO and the control group (left figure), and TU-BP and the control group (right figure) on intracellular RH in Example 4 of the present invention.

[0045] Figure 8 The images show the results of the anti-invasion effects of TU-BO and the control group (left image), and TU-BP and the control group (right image) on extracellular RH-type Toxoplasma gondii in Example 4 of this invention. Detailed Implementation

[0046] 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.

[0047] 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 ℃. 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 10% fetal bovine serum, supplemented with 100 U / mL penicillin and 100 μg / mL streptomycin. In this invention, the evaluation of the anti-Toxoplasma gondii effects of compounds TU-BO and TU-BP was conducted independently, and both were tested according to the methods in Examples 1-4. Both groups of experiments used the same control and blank group settings, with only the experimental groups receiving either compound TU-BO or compound TU-BP individually; the two were not administered in combination.

[0048] In this embodiment of the invention, the preparation method of compound TU-BO includes the following steps:

[0049] Freshly prepared octanoyl chloride (1.1 mmol) was added dropwise to a freshly prepared solution of potassium thiocyanate (1.65 mmol) and dry acetone (15 mL). After the addition was complete, the solution was heated to 56 °C under nitrogen protection and refluxed for 2 h to obtain a milky white solution, which was then cooled to room temperature. 2-Aminobiphenyl (1.1 mmol) was added to the reaction solution, and the solution was heated again to 56 °C and refluxed for 10 h. After cooling to room temperature, the solution was poured into crushed ice twice the volume of the reaction product. The solution was allowed to stand for 1 h to precipitate the solid, which was then collected, filtered, and dried to constant weight to obtain the crude product. Anhydrous ethanol three times the mass of the crude product was added, and the solution was heated to reflux (approximately 78 °C) to completely dissolve the solid. The solution was then allowed to cool naturally to room temperature to precipitate crystals, yielding the product N-([1,1'-biphenyl]-2-ylaminothiocarbonyl)octanoamide, denoted as TU-BO. The molecular formula of TU-BO is C2. 21 H 26 N2OS, chemical structural formula is:

[0050] .

[0051] In this embodiment of the invention, the preparation method of compound TU-BP includes the following steps:

[0052] Freshly prepared pentanoyl chloride (1.1 mmol) was added dropwise to a freshly prepared potassium thiocyanate (1.65 mmol) and dry acetone (15 mL) solution. After the addition was complete, the mixture was heated to 56 °C under nitrogen protection and refluxed for 2 h to obtain a milky white solution, which was then cooled to room temperature. 2-aminobiphenyl (1.1 mmol) was added to the reaction solution, and the mixture was heated to 56 °C again and refluxed for 8-10 h. TLC monitoring (hexane and ethyl acetate, volume ratio 4:1) was performed until the starting material spots disappeared. The mixture was then cooled to room temperature and poured into crushed ice with a volume twice that of the reaction product. After standing for 1 h, a precipitate was formed. The solid was collected, filtered, and dried to constant weight to obtain the crude product. Anhydrous ethanol with a mass three times that of the crude product was added, and the mixture was heated to reflux (approximately 78 °C) to completely dissolve the solid. The mixture was then allowed to cool naturally to room temperature for recrystallization to obtain the product N-([1,1'-biphenyl]-2-ylaminothiocarbonyl)pentanoamide, denoted as TU-BP. The molecular formula of TU-BP is C 18 H 20 N2OS, chemical structural formula is:

[0053] .

[0054] Example 1: Study on the toxicity of TU-BO and TU-BP to Vero cells

[0055] 1. Experimental Procedure

[0056] Cytotoxicity assay: The cytotoxicity of TU-BO or TU-BP to Vero cells was determined using the CCK-8 assay.

[0057] African green monkey kidney (Vero) cell suspension was seeded into 96-well cell culture plates, approximately 100 μL (5000 cells) per well. The culture plates were then pre-cultured in an incubator for 18 h (37℃, 5% CO2, saturated humidity) to allow the cells to adhere.

[0058] The study included a blank control group, a control group, and an experimental group. The blank control group was not seeded with cells, but only received complete culture medium containing 0.1% DMSO. Both the control and experimental groups were seeded with Vero cells, with the control group receiving complete culture medium containing 0.1% DMSO. The experimental groups were prepared with nine concentration gradients of TU-BO or TU-BP: 2 μM, 4 μM, 8 μM, 16 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, and 500 μM. The final DMSO concentration for all three groups was 0.1%. The original culture medium in each well was gently aspirated, and the corresponding culture medium or drug-containing medium was added to each group, with a final volume of 100 μL per well. Each group was divided into three replicates.

[0059] The culture plates were 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 plates were incubated in the dark for 2 h. The absorbance (OD) at 450 nm was measured using a microplate reader. Cell viability was calculated using the following formula. The independent experiments were repeated three times, and the half-maximal cytotoxic concentration (MCC) was obtained by fitting a dose-response curve. 50 .

[0060] Cell viability (%) = (OD) 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组） ×100%

[0061] 2. Experimental Results

[0062] Figure 1 and Figure 2 The graphs show the toxicity test results of TU-BO and TU-BP on Vero cells, with the vertical axis representing cell viability (with the 0.1% DMSO control group as 100%) and the horizontal axis representing the concentration of compound TU-BO or TU-BP administered.

[0063] It can be seen that cell viability decreased in a dose-dependent manner with increasing TU-BO or TU-BP concentrations: when the TU-BO concentration reached 61.73 μM or the TU-BP concentration reached 654.5 μM, the cell viability dropped to 50%, which is the half-maximal cytotoxic concentration (CC50) of compounds TU-BO and TU-BP on Vero cells. 50 The effective concentrations were 61.73 μM and 654.5 μM, respectively, indicating that the two compounds have advantages such as low toxicity and safety to host cells within the effective concentration range.

[0064] Example 2: Inhibitory effects of TU-BO and TU-BP on Toxoplasma gondii

[0065] 1. Experimental Procedure

[0066] This experiment included a control group (DMSO group) and an experimental group (TU-BO or TU-BP treatment group), both inoculated with Vero cells and Toxoplasma gondii tachyzoites. Specific procedures included:

[0067] African green monkey kidney (Vero) cell suspension was seeded into 96-well cell culture plates, approximately 100 μL (5000 cells) per well. The culture plates were then pre-cultured in an incubator for 18 h (37℃, 5% CO2, saturated humidity) to allow the cells to adhere.

[0068] Fresh, viable Luciferase tachyzoites were harvested from Vero cells, counted using a hemocytometer, and 3 × 10⁻⁶ cells were collected. 5One 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.

[0069] The control group was given complete culture medium containing 0.1% DMSO; the experimental groups were prepared with nine concentration gradients of compound TU-BO or TU-BP: 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, 50 μM, 75 μM, and 100 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.

[0070] 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 11000 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. After mixing, the luminescence value (RLU) was detected using a chemiluminescence analyzer. The inhibition rate of Toxoplasma gondii proliferation by compound TU-BO or TU-BP 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 .

[0071] Inhibition rate % = (RLU) DMSO -RLU 处理组 ) / RLU DMSO ×100%.

[0072] 2. Experimental Results

[0073] Figure 3 and Figure 4 The graphs show the inhibitory effects of TU-BO and TU-BP on Toxoplasma gondii, with the vertical axis representing the Toxoplasma gondii proliferation inhibition rate and the horizontal axis representing the dosage concentration of compound TU-BO or TU-BP.

[0074] The results showed that TU-BO had a half-maximal effective concentration (EC50) against Toxoplasma gondii proliferation. 50 The half-maximal effective concentration (EC50) of TU-BP against Toxoplasma gondii proliferation is 1.478 μM. 50 The concentration was 7.219 μM. Furthermore, the inhibitory effects of both TU-BO and TU-BP on Toxoplasma gondii were dose-dependent, with higher concentrations resulting in stronger inhibitory effects, indicating that TU-BO and TU-BP have good inhibitory effects on Toxoplasma gondii. Meanwhile, the safety index SI of TU-BO (SI=CC) was [not specified in the original text]. 50 / EC50 The safety index (SI) of TU-BO and TU-BP was 41.77, and the safety index (SI) of TU-BP was 90.66, indicating that TU-BO and TU-BP have strong selectivity for parasites and low host toxicity, and have excellent potential for the development of anti-Toxoplasma gondii drugs.

[0075] Example 3: The inhibitory effect of TU-BO and TU-BP on intracellular Toxoplasma gondii plaque formation.

[0076] 1. Experimental Procedure

[0077] 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, 5% CO2, and saturated humidity until the cells were 100% confluent. The cells were then cultured statically for another 24 hours to form a monolayer of cells with complete contact inhibition.

[0078] Tachyzoites of freshly lysed HFF monolayers of Toxoplasma gondii type I (RH) were collected, and 150 live tachyzoites were inoculated into each well of HFF monolayers (MOI approximately 1:600). The culture plates were divided into control and experimental groups, with 6 replicates in each group. The control group was given complete medium containing 0.1% DMSO. The experimental group of TU-BO was given 7 μM of compound TU-BO and 0.1% DMSO in complete medium per well, or the experimental group of TU-BP was given 35 μM of compound TU-BP and 0.1% DMSO in complete medium per well. The final volume of medium per well was fixed at 1 mL, and the final concentration of DMSO in both groups was 0.1%.

[0079] After incubating the culture plate at 37℃, 5% CO2, and saturated humidity for 7 days, the culture medium in the wells was gently aspirated, and the plate was washed twice with sterile PBS. 1 mL of 4% paraformaldehyde was added to each well, and the plate was fixed 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, and the plate was stained 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.

[0080] 2. Experimental Results

[0081] like Figure 5 and Figure 6 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 cells with added TU-BO or TU-BP, indicating that TU-BO and TU-BP can significantly inhibit the growth of Toxoplasma gondii in host cells and plaque formation.

[0082] Example 4: Antiproliferative effects of TU-BO and TU-BP on intracellular Toxoplasma gondii type I strain (RH)

[0083] 1. Experimental Procedure

[0084] Proliferation experiment: Collect 1×10 5 One RH tachyzoite was inoculated into a 12-well plate 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 7 μM of TU-BO (5 times EC50) per well. 50 The experimental groups using complete medium of 0.1% DMSO or TU-BP were prepared by adding 35 μM of compound TU-BP (5 times EC50) to each well. 50 The two groups were prepared with complete culture medium containing 0.1% DMSO, with the final concentration of DMSO being 0.1%.

[0085] After culturing 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 aspirated, the cells were washed three times with PBS, and 0.1% Triton X-100 was added for permeation at room temperature for 10 min. The permeation solution was aspirated, the cells were washed three times with PBS, and 1% BSA / PBS was added for blocking at room temperature for 30 min.

[0086] 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 DAPI working solution (1 μg / mL) was added and stained at room temperature in the dark for 5 min to label the host cell nuclei.

[0087] 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.

[0088] 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 used, and the tachyzoites were resuspended in the control group and the experimental group, respectively. The control group received complete medium of 0.1% DMSO, while the TU-BO experimental group received 7 μM TU-BO (5 times EC50) per well. 50The experimental groups using complete medium of 0.1% DMSO or TU-BP were prepared by adding 35 μM of compound TU-BP (5 times EC50) to each well. 50 Complete culture medium containing 0.1% DMSO was added simultaneously to the corresponding wells, with a final concentration of 0.1% DMSO in both groups.

[0089] After culturing in an incubator at 37℃, 5% CO2, and saturated humidity for 30 min, immediately remove the culture medium and gently wash three times with sterile PBS to thoroughly remove any uninvaded tachyzoites (observe under a microscope to ensure all extracellular tachyzoites are clean). Replace with fresh complete culture medium and continue culturing for 20 h. Then, proceed with the fixation, permeabilization, blocking, and IFA staining steps described above for the proliferation experiment (nucleus staining with primary antibody GAP45, secondary antibody FITC, and DAPI).

[0090] During fluorescence microscopy, 8-10 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.

[0091] The number of vacuoles containing parasites and the number of host cells containing tachyzoites in each image were counted. The anti-proliferative effect of TU-BO and control groups or TU-BP and control groups on intracellular RH tachyzoites was investigated. Figure 7 As shown.

[0092] 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 TU-BO and the control group, or TU-BP and the control group, on extracellular RH-type Toxoplasma gondii were investigated. Figure 8 As shown.

[0093] 2. Experimental Results

[0094] After Toxoplasma gondii invades a host cell, it forms a vacuolar vacuolar, in which tachyzoites multiply by internal binary fission. Therefore, within the same time frame after cell invasion, the number of tachyzoites in the vacuolar vacuolar can reflect the proliferative capacity of the parasite.

[0095] The results of the proliferation experiment showed that, compared with the control group (containing 0.1% DMSO), the number of tachyzoites in the vacuoles of the worm-carrying groups containing 7 μM compound TU-BO or 35 μM compound TU-BP was significantly reduced; the results of the invasion experiment showed that the invasion rate of the TU-BO experimental group or the TU-BP experimental group was significantly lower than that of the control group.

[0096] In summary, the present invention provides formula The aminothiocarbonyl compounds shown have great potential in the preparation of drugs for the prevention or treatment of Toxoplasma gondii infection, among which, compound TU-BO, has a high C64 content. 50=61.73 μM, EC 50 =1.478 μM, safety index SI=41.77, TU-BP's CC 50 =654.5 μM, EC 50 With a concentration of 7.219 μM and a safety index (SI) of 90.66, the 7 μM compound TU-BO and 35 μM compound TU-BP were within the safe range of no significant toxicity to host cells in experiments inhibiting intracellular plaque formation of Toxoplasma gondii (Example 3) and anti-proliferative effects against Toxoplasma gondii type I strains (Example 4). This indicates that TU-BO or TU-BP can significantly inhibit both Toxoplasma gondii cell lysis and the invasion and intracellular proliferation of RH strain Toxoplasma gondii, further verifying their low-toxicity and highly effective anti-Toxoplasma gondii activity.

[0097] 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. An aminothiocarbonyl compound, characterized in that, The compound includes the formula: The general formula of the structure shown is: Mode , where R represents any alkane with 4-7 carbon atoms.

2. The aminothiocarbonyl compound as described in claim 1, characterized in that, R is butyl or heptyl.

3. The method for preparing aminothiocarbonyl compounds as described in claim 1 or 2, characterized in that, Includes the following steps: Add a straight-chain saturated alkanoyl chloride with any number of carbons (5-8) dropwise to a dry acetone solution of potassium thiocyanate. After the addition is complete, heat to reflux under nitrogen protection and maintain reflux for 1.5-2.5 h. After cooling the resulting reaction solution to room temperature, add 2-aminobiphenyl, heat to reflux again, and maintain reflux for 8-10 h. After cooling to room temperature, pour into crushed ice, allow to stand to precipitate, filter, and dry to obtain the crude product. Recrystallize the crude product from ethanol to obtain aminothiocarbonyl compounds.

4. The method for preparing aminothiocarbonyl compounds as described in claim 3, characterized in that, The molar ratio of potassium thiocyanate to any straight-chain saturated alkanoyl chloride with 5-8 carbon atoms is 6:(3-5); 9.1 mL of dry acetone is used for every 1 mmol of potassium thiocyanate; the molar ratio of potassium thiocyanate to 2-aminobiphenyl is 6:(3-5).

5. The use of the aminothiocarbonyl compounds as described in claim 1 or 2 in the preparation of medicaments for the prevention or treatment of toxoplasmosis infection.

6. The use of the aminothiocarbonyl compound as described in claim 5 in the preparation of a medicament for the prevention or treatment of Toxoplasma gondii 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 the aminothiocarbonyl compound as described in claim 5 in the preparation of a medicament for the prevention or treatment of Toxoplasma gondii 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 the aminothiocarbonyl compound as described in claim 5 in the preparation of a medicament for the prevention or treatment of Toxoplasma gondii infection, characterized in that, When R is butyl, the compound has an LD50 of 1.478 μM against Toxoplasma gondii and an LD50 of 61.73 μM against Vero cells. When R is heptyl, the compound has an LD50 of 7.219 μM against Toxoplasma gondii and an LD50 of 654.5 μM against Vero cells.

9. A composition for the prevention or treatment of toxoplasmosis infection, characterized in that, The active ingredient of the composition includes the aminothiocarbonyl compound 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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