Use of squaraine compounds in the preparation of anti-toxoplasmosis drugs

Abstract

The application discloses application of squaraine compounds in preparation of anti-toxoplasmosis drugs. The application provides a new application of squaraine compounds in anti-toxoplasma infection. Through synthesis of squaraine compounds and insect inhibiting experiments, two preferred squaraine compounds, F-squaraine and 3-F-squaraine, with better insect inhibiting effects are found. The two preferred squaraine compounds have higher in-vitro insect inhibiting rates on toxoplasma, can well inhibit in-vitro replication of toxoplasma, significantly inhibit formation of insect plaques, and can effectively inhibit growth of toxoplasma. The two preferred squaraine compounds have good safety and do not have cytotoxicity, and belong to squaraine compounds with low toxicity or no toxicity. Meanwhile, the squaraine compounds provided by the application can also inhibit reproduction of toxoplasma in mice, treat toxoplasmosis, have significant treatment effects, and will not affect growth of the mice. The squaraine compounds can be used for resisting toxoplasma infection, and can be used for preparing and developing more products for resisting toxoplasma infection and drugs for treating toxoplasmosis. The squaraine compounds have important significance for research and development of drugs for treating toxoplasmosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of squaric acid compounds in the preparation of drugs for treating toxoplasmosis. Background Technology

[0002] Toxoplasma gondii is an important zoonotic parasitic protozoan that can infect almost all warm-blooded animals, including humans, and invade all nucleated cells. Toxoplasma gondii has a wide host range and diverse transmission routes. Its life cycle is very complex; its definitive host is a cat or other felines, while humans, pigs, cattle, and sheep are intermediate hosts. Within the intermediate host, under immune pressure, Toxoplasma gondii can transform from rapidly proliferating tachyzoites to slowly proliferating bradyzoites, forming tissue cysts that can persist in muscles or the brain for extended periods. In most immunocompetent individuals, infection with Toxoplasma gondii can be effectively controlled by the immune system, thus not showing obvious clinical symptoms. However, in immunocompromised individuals, it can cause lymphadenopathy, central nervous system damage, and toxoplasmic eye disease. Toxoplasmosis infection can lead to miscarriage, stillbirth, and fetal malformations in pregnant women and livestock. Children and the elderly infected with Toxoplasma gondii are prone to meningitis and pneumonia. Infection with livestock such as pigs, cattle, and sheep can also cause reproductive disorders or miscarriages, seriously harming the development of animal husbandry and causing significant economic losses.

[0003] Traditional anti-Toxoplasma gondii drugs, such as pyrimethamine and sulfadiazine, while effective in treating acute infections, also have varying degrees of toxic side effects and are ineffective against chronic infections. Long-term use can easily lead to drug resistance in the parasites. In addition, some alternative therapies, such as clindamycin and atorvaquinone, while having some therapeutic effect on chronic infections, still cannot eradicate the infection. Therefore, finding more highly effective and low-toxicity anti-Toxoplasma gondii drugs is of great significance.

[0004] Anti-toxoplasmosis drugs can also be obtained through the development of natural products and the synthesis of new drugs targeting specific drug molecules. Repurposing existing drugs offers advantages over developing entirely new drugs, such as lower cost and significantly shorter development time. Imidazole is a promising anti-babesiosis drug with antiparasitic potential. However, because imidazole is not easily metabolized in animals, it tends to accumulate in the kidneys and liver, persisting in its original form for extended periods, causing hepatotoxicity and nephrotoxicity, which to some extent hinders its application. Therefore, improving the safety of anti-toxoplasmosis drugs and developing more safe, non-toxic, and side-effect-free new drugs for the treatment or prevention of toxoplasmosis is of great practical significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing anti-toxoplasmosis drugs, such as side effects, drug resistance, and low safety, and to provide the application of squaric acid compounds in the preparation of anti-toxoplasmosis drugs.

[0006] The purpose of this invention is to provide a new application of squaric acid compounds in the treatment of toxoplasmosis.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] This invention provides the use of squaric acid compounds in the preparation of drugs for treating toxoplasmosis, wherein the structural formula of the squaric acid compounds is shown below:

[0009] Among them, R1~R4 are selected from hydrogen or halogen F.

[0010] This invention demonstrates that specific structurally derived squaric acid compounds inhibit the growth of Toxoplasma gondii, exhibiting a high inhibitory rate. These compounds not only effectively inhibit in vitro replication and growth of Toxoplasma gondii but also significantly suppress plaque formation and proliferation. Furthermore, the compound's EC50... 50 The squaric acid compounds provided by this invention exhibit good efficacy and promising anti-toxoplasmosis potential. Furthermore, they demonstrate good safety profiles, exhibiting low cytotoxicity to HFF cells, classifying them as low-toxicity or non-toxic squaric acid compounds. In vivo experiments further demonstrate that these compounds can inhibit the reproduction of Toxoplasma gondii in mice, exhibiting a certain therapeutic effect on toxoplasmosis infection in mice without affecting their normal growth. This invention provides a novel application of specific squaric acid compounds in the treatment of toxoplasmosis, offering new ideas and methods for the preparation and development of more anti-toxoplasmosis drugs, and showing promising application prospects in the research and development of drugs for the treatment or prevention of toxoplasmosis.

[0011] Therefore, the present invention provides the use of succinic acid compounds in the preparation of products that inhibit the growth or reproduction of Toxoplasma gondii.

[0012] This invention provides the application of succinic acid compounds in the preparation of products for treating Toxoplasma gondii infection.

[0013] Preferably, the squaric acid compound is:

[0014] or .

[0015] Furthermore, the product or drug can inhibit the growth of Toxoplasma gondii.

[0016] Furthermore, the product or drug can inhibit the proliferation of Toxoplasma gondii.

[0017] Furthermore, the product or drug can inhibit the replication of Toxoplasma gondii tachyzoites.

[0018] Furthermore, the product or drug can inhibit the formation of Toxoplasma gondii plaques.

[0019] Preferably, the product further comprises a squaric acid compound or a pharmaceutically acceptable salt, hydrate, or combination thereof, or excipients.

[0020] Preferably, the dosage form of the drug is a tablet, capsule, oral liquid preparation, spray, or injection.

[0021] The present invention has the following beneficial effects:

[0022] This invention provides a novel application of squaric acid compounds in the preparation of anti-toxoplasmosis drugs. Through the synthesis and insecticidal experiments of squaric acid compounds, two preferred squaric acid compounds, F-squaric acid and 3-F-squaric acid, with good insecticidal effects were identified. These compounds exhibit high in vitro inhibition rates against Toxoplasma gondii, effectively inhibiting its in vitro replication, significantly suppressing plaque formation, and effectively inhibiting its in vitro growth. Furthermore, they demonstrate good safety, lacking cytotoxicity, and are classified as low-toxicity or non-toxic squaric acid compounds. The EC50 of these compounds... 50 The drug has a low efficacy and good anti-Toxoplasma gondii potential. Furthermore, the squaric acid compounds provided by this invention can also inhibit the reproduction of Toxoplasma gondii in mice, treating toxoplasmosis without affecting mouse growth, demonstrating significant therapeutic effects and resistance to Toxoplasma gondii infection. These compounds can be used to prepare and develop more anti-Toxoplasma gondii products and drugs for treating toxoplasmosis, showing promising application prospects in the research and development of drugs for the treatment or prevention of toxoplasmosis. Attached Figure Description

[0023] Figure 1 The figure shows the preliminary screening results of the insect inhibition rate of different styrax compounds.

[0024] Figure 2 This is a diagram showing the in vitro plaque formation of Toxoplasma gondii caused by squaric acid compounds.

[0025] Figure 3 This is a graph showing the statistical results of the effects of squaric acid compounds on the phagocytic plaque area of ​​Toxoplasma gondii.

[0026] Figure 4 The graph shows the survival curve results of the toxicity of succinic acid compounds against Toxoplasma gondii in mice. Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] Example 1 Synthesis of Squamous Acid Compounds

[0030] Referring to existing technology (Pfeifer, Lukas, et al., Hydrogen-Bonded Homoleptic Fluoride-Diarylurea Complexes: Structure, Reactivity, and Coordinating Power; Journal of the American Chemical Society (2016), 138(40), 13314-13325.), five squaric acid compounds were synthesized by chemical synthesis using 3,4-diethoxycyclobutene-3-en-1,2-dione, amine compounds with different substituents, and zinc trifluoromethanesulfonate in a mixed solution of appropriate amounts of toluene and N,N-dimethylformamide. The synthetic route is as follows:

[0031]

[0032] By introducing trifluoromethyl, cyano, hydroxyl, or halogen (F) groups into the R group of amine compounds containing different substituents, squaric acid compounds with different substituents can be synthesized. For example, the specific synthetic steps of compound 5 are as follows: Anhydrous toluene (38 mL), anhydrous DMF (2 mL), 3,4-diethoxycyclobut-3-ene-1,2-dione (5.11 g, 30.0 mmol), and zinc trifluoromethanesulfonate (2.18 g, 6.0 mmol) are added to a dry 100 mL round-bottom flask. The resulting suspension is stirred at room temperature, and then 4-fluoroaniline (6.67 g, 60.0 mmol) is added. The solution is heated to 100 °C and stirred for 18 hours, at which point a yellow precipitate is observed. The reaction mixture is cooled to room temperature, filtered, and washed successively with methanol (3 x 50 mL) and diethyl ether (50 mL). The mixture is dried under high vacuum to obtain the desired product, a powdery, pale yellow solid (8.65 g, 96% yield).

[0033] The following compounds were all prepared by the above method and then... 1 The target compound was fully characterized by H-NMR, and the characterization results were consistent with the synthesized structure.

[0034] Compound 1: ,3,4-bis{[3-(trifluoromethyl)phenyl]amino}cyclobut-3-ene-1,2-dione. 1 H NMR (400 MHz, DMSO- d6) δ 10.17 (s, 2H), 7.90 (s, 2H), 7.61 (d, J = 8.0 Hz, 4H), 7.42 (d, J = 6.8 Hz, 2H).

[0035] Compound 2: ,4-{[4-(ethylamino)-2,3-dioxane-cyclobut-4-enyl]amino}benzene-1-carboxynitrile. 1 H NMR (400 MHz, DMSO- d 6) δ 11.07 (s, 1H), 7.84 – 7.79 (m, 2H), 7.59 – 7.54 (m, 2H), 4.80 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 6.8 Hz, 3H).

[0036] Compound 3: ,3,4-bis[(4-fluorophenyl)amino]cyclobut-3-ene-1,2-dione. 1 H NMR (400 MHz, DMSO- d 6) δ 9.86 (s, 2H), 7.53 – 7.43 (m, 4H), 7.22 (t, J =8.8 Hz, 4H).

[0037] Compound 4: ,3,4-bis[(3-hydroxyphenyl)amino]cyclobut-3-ene-1,2-dione. 1 H NMR (400 MHz, DMSO- d 6) δ 9.72 (s, 2H), 9.60 (s, 2H), 7.15 (t, J = 8.0Hz, 2H), 6.97 – 6.91 (m, 4H), 6.53 – 6.47 (m, 2H).

[0038] Compound 5: ,3,4-bis[(3-fluorophenyl)amino]cyclobut-3-ene-1,2-dione. 1 H NMR (400 MHz, DMSO- d 6) δ 10.06 (s, 2H), 7.50 (dt, J = 11.2, 2.4 Hz, 2H), 7.42 (td, J= 8.2, 6.4 Hz, 2H), 7.19 (dd, J = 8.0, 2.4 Hz, 2H), 6.92 (td, J = 8.4, 2.5 Hz, 2H).

[0039] Example 2 Insect inhibition rate of squaric acid compounds

[0040] Five squaric acid compounds (prepared in Example 1) were subjected to in vitro insecticidal activity tests at an effective concentration of 10 μM. The specific method was as follows:

[0041] (1) Human foreskin fibroblasts (HFF) (ATCC, Manassas, VA, USA) were used to culture Toxoplasma gondii RH strain (from Huazhong Agricultural University) in vitro. When the parasite had a large vacuole, the parasite was lysed and released.

[0042] (2) The insect strain was inoculated onto a new 24-well plate with well-grown HFF cells and invaded the cells for 1 h at 37°C and 5% CO2.

[0043] (3) Wash away the uninvaded parasites with PBS, add culture medium containing 10 μM compound to each well of 24 plate, and continue to culture the parasites at 37℃ and 5% CO2 for 16 h.

[0044] (4) After washing away the free parasites with PBS, fix the crawling cells with 4% paraformaldehyde at 37°C for 20 min, and wash with PBS 3 times.

[0045] (5) Add rabbit anti-Toxoplasma gondii Tg Incubate with ALD (fructo-1,6-bisphosphate aldolase, prepared and preserved in our laboratory) polyclonal antibody for 30 min, then wash 3 times with PBS;

[0046] (6) Cells were permeabilized with 0.1% Triton X-100 for 20 min, washed 3 times with PBS, and then blocked with 10% FBS at 37℃ for 2 h.

[0047] (7) Add mouse anti-Toxoplasma gondii positive serum (prepared in our laboratory), place the 24-well plate in a 37°C incubator for 30 min, and wash 3 times with PBS;

[0048] (8) Add goat anti-rabbit 594 secondary antibody (Alexa Fluor 594-conjugated goat anti-rabbit (Cell Signalling Technology)), goat anti-mouse 488 secondary antibody (Alexa Fluor 488-conjugated goat anti-mouse (Cell Signalling Technology)) and Hoechst (labeling cell nuclei, Hoechst AG, Germany) diluted 1:1000, then incubate in the dark for 30 min and wash 3 times with PBS;

[0049] (9) Mount the slides, observe and record the number of vesicles containing 1, 2, 4, 8 or more 16 worms under an inverted fluorescence microscope, and count the number of vesicles in each group. At least 100 vesicles should be counted in each group. The experiment should be repeated at least 3 times. The inhibition rate of worms should be obtained by comparing with the negative control (using DMEM medium containing 2% FBS as the negative control). The inhibition rate of different compounds against Toxoplasma gondii should be calculated by Graphpad Prism 8.

[0050] Statistical results of insect inhibition rates of different succinic acid compounds are as follows: Figure 1 As shown in Table 1, among the five compounds, p-F-squamous acid and 3-F-squamous acid have a better inhibitory effect on Toxoplasma gondii, with 3-F-squamous acid having an inhibition rate of more than 65%. Meanwhile, the other three squamous acid compounds have a poor inhibitory effect on Toxoplasma gondii, and 3-OH-squamous acid cannot even inhibit Toxoplasma gondii.

[0051] Table 1. Structural formulas of squaric acid compounds and their insecticidal rates

[0052]

[0053] Example 3: In vitro insecticidal effect of squaric acid compounds

[0054] 1. Effects of squaric acid compounds on in vitro replication of tachyzoites

[0055] EC values ​​of p-F-squamous acid and 3-F-squamous acid compounds with good inhibition effects were determined in Example 2. 50 The specific steps are as follows:

[0056] (1) Use HFF cells to culture Toxoplasma gondii RH strain in vitro. When the parasite has a large vacuole, lyse the parasite and release it.

[0057] (2) The insect strain was inoculated onto a 24-well plate with HFF cells that had been grown, and invaded the cells for 1 h at 37°C and 5% CO2.

[0058] (3) Wash away the uninvaded insect strains with PBS, add culture medium containing different concentrations of squaric acid compounds to 24-well plates, and continue to culture the insects at 37°C and 5% CO2 for 16 h.

[0059] (4) After washing away the free parasites with PBS, fix the crawling cells with 4% paraformaldehyde at 37°C for 20 min, and wash with PBS 3 times.

[0060] (5) Add rabbit anti-Toxoplasma gondii Tg Incubate with ALD polyclonal antibody for 30 min, then wash 3 times with PBS;

[0061] (6) Permeabilize cells with 0.1% Triton X-100 for 20 min, wash 3 times with PBS, and then block with 10% FBS at 37℃ for 2 h;

[0062] (7) After washing 3 times with PBS, mouse anti-Toxoplasma gondii positive serum was added to the 24-well plate and incubated in a 37°C incubator for 30 min. Then, the plate was washed 3 times with PBS.

[0063] (8) Add goat anti-rabbit 594 secondary antibody, goat anti-mouse 488 secondary antibody and Hoechst (1:1000 dilution), then incubate in the dark for 30 min, and wash 3 times with PBS;

[0064] (9) Mount the slides and observe them under an inverted fluorescence microscope. Record and count the number of vesicles containing 1, 2, 4, 8, or more than or equal to 16 vesicles. Count at least 100 vesicles in each group. Repeat the experiment at least 3 times. Calculate the inhibition rate of vesicles at different concentration gradients by comparing with the negative control. Calculate the EC50 of the compound using Graphpad Prism 8. 50 .

[0065] The results are shown in Table 2, indicating that the two squaric acid compounds have a significant effect on F-squaric acid and 3-F-squaric acid EC. 50 The concentrations were 5.69 μM and 0.997 μM, respectively, EC. 50 The lower the concentration, the better the efficacy, and it can effectively inhibit the growth of Toxoplasma gondii.

[0066] Table 2. Anti-Toxoplasma gondii styracifolium compounds EC 50

[0067]

[0068] 2. Cytotoxicity test

[0069] The above assays showed that 3-F-squamous acid had a better inhibitory effect. Further assays were performed to determine the cytotoxicity of this compound. The effect of 3-F-squamous acid at different concentrations on HFF cell growth was determined using a CCK8 assay kit (Beyotime, catalog number: C0038). The specific steps were as follows:

[0070] (1) According to 10 4 HFF cells were seeded into 96-well culture plates and cultured at 37°C and 5% CO2 for 24 h.

[0071] (2) Dilute the squaric acid compounds to different concentrations with DMEM medium containing 2% FBS, add 100 μL of the corresponding compound medium to each well, and make 3 replicate wells; use the same volume of DMSO 2% FBS DMEM medium with the same compound added and the blank group without cells (only DMEM medium containing 2% FBS added) as the control group (containing only cells and medium), and make 3 replicate wells in the same way;

[0072] (3) After incubation for 48 h, wash three times with PBS, add 100 μL of 10% CCK8 solution prepared with serum-free medium to each well, incubate for 1 h, and then use a multi-functional microplate reader to detect the absorbance at a wavelength of 450 nm.

[0073] (4) Using [1 - (experimental group - blank group) / (control group - blank group)] * 100% as the toxicity ratio of the corresponding concentration of the compound to the cells, calculate the relative toxicity ratio, and import the results into Graphpad Prism 8 to calculate the TC of the compound. 50 .

[0074] The results are shown in Table 3 below, which illustrates the TC of 3-F-squaric acid, a squaric acid compound. 50 The value was 146.7 μM, which showed low cytotoxicity and a selectivity of more than 100-fold for inhibiting parasite growth, indicating that the inhibitory effect of the squaric acid compound 3-F-squaric acid on Toxoplasma gondii growth was not due to significant host cytotoxicity.

[0075] 3. Effects of squaric acid compounds on in vitro plaque formation in Toxoplasma gondii

[0076] (1) Use HFF cells to culture RH parasites in vitro. When the parasites have large vesicles, lyse and release the parasites. Use a 3 μm filter membrane to filter and purify the freshly spilled Toxoplasma gondii tachyzoites.

[0077] (2) Inoculate RH strains (100 Tg / well, 3 replicates per strain) into 6-well plates containing HFF cells, and use EC. 50Six-well plates inoculated with 3-F-squamous acid at a concentration of 3-F-squamous acid were incubated; 5 μM pyrimethamine was set as a positive control; and an equal volume of 2% FBS DMEM medium without the compound was used as a negative control. The plates were cultured at 37°C and 5% CO2 for 7 days.

[0078] (3) After 7 days, wash the 6-well plate with PBS 1-2 times, fix the cells in the 6-well plate with 4% paraformaldehyde at 37℃ for 20 min, and wash with PBS 1-2 times.

[0079] (4) Stain with 0.1% crystal violet for 20 min, wash with PBS and air dry at room temperature;

[0080] (5) Scan the blank spot with a scanner and measure the area of ​​the blank spot.

[0081] The statistical diagram of plaque formation and plaque area in vitro of Toxoplasma gondii is shown below. Figure 2 and Figure 3 As shown, compared with the control group, the RH strain cultured with 3-F-squamous acid could not form visible plaques, affecting the in vitro growth of Toxoplasma gondii, indicating that 3-F-squamous acid not only has low cytotoxicity but also significantly inhibits the in vitro growth of Toxoplasma gondii.

[0082] Example 4: In vivo insecticidal effect of squaric acid compounds

[0083] The in vivo antiparasitic effect was determined using 3-F-squamous acid. ICR mice infected with Toxoplasma gondii (purchased from Ruige Biotechnology Co., Ltd.) were treated with ME49 strain (from Anhui Agricultural University) at a concentration of 10... 4 Mice were inoculated, and the first treatment was administered 4 hours later (50 mg / kg 3-F-squamous acid, with pyrimethamine as a positive control, 20 mg / kg, the compound was administered using 5% anhydrous ethanol + corn oil as a solvent; the negative control was given an equal volume of corn oil). The administration was repeated daily for 7 days. Afterwards, toxicity tests were conducted to observe mortality and weight changes in the mice. The specific steps were as follows:

[0084] (1) Using serum-free DMEM solution as the diluent, at 10 4 Each worm / mouse was used to inoculate mice with an infectious dose. Seven-week-old female ICR mice were infected by intraperitoneal injection of ME49 tachyzoites, with eight mice in each group.

[0085] (2) Seven days after infection, the mortality and weight changes of mice were observed for 23 consecutive days.

[0086] Test results as follows Figure 4As shown, the squaric acid compound 3-F-squaric acid can increase the survival rate of mice to 50% without affecting mouse growth, exhibiting significant antiparasitic effects and resistance to Toxoplasma gondii infection. Although its in vivo therapeutic effect is not as good as pyrimethamine, its in vitro effects are basically consistent with pyrimethamine. Considering it as a promising compound, further optimization is possible.

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The use of squaric acid compounds or pharmaceutically acceptable salts thereof in the preparation of products that inhibit the growth or reproduction of Toxoplasma gondii, characterized in that, The structural formula of the squaric acid compounds is shown below: or .

2. The use of squaric acid compounds or pharmaceutically acceptable salts thereof in the preparation of products for treating Toxoplasma gondii infection, characterized in that, The structural formula of the squaric acid compounds is shown below: or .

3. The application of squaric acid compounds in the preparation of drugs for treating toxoplasmosis, characterized in that, The structural formula of the squaric acid compounds is shown below: or .

4. The application according to claim 1 or 2, characterized in that, The product also includes auxiliary materials.

5. The application according to claim 3, characterized in that, The dosage form of the drug is tablets, capsules, oral liquid preparations, sprays, or injections.

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