Application of a phenylurea compound as a Babesia growth inhibitor

Abstract

The present invention relates to the application of a phenylurea compound as a Babesia growth inhibitor, belonging to the field of pharmaceutical technology. The phenylurea compound is 1,3-bis(3-chlorophenyl)urea or 1,3-bis(3-cyanophenyl)urea. The two phenylurea compounds have a small molecular weight, a relatively simple structure, are soluble in a variety of solvents, and have unique structures and chemical properties. Experiments have shown that both phenylurea compounds can be used as Babesia growth inhibitors and can effectively inhibit the growth of Babesia. In addition, the two phenylurea compounds have good bioavailability and safety characteristics, have no obvious toxic side effects on host cells, and have broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to the use of a phenylurea compound as a Babesia growth inhibitor. Background Art

[0002] Babesiosis, a tick-borne blood-borne parasite, is widespread in humans and animals and poses a significant threat to global public health. Babesia parasites live within the host's red blood cells and can cause symptoms such as high fever, anemia, jaundice, hemoglobinuria, and, in severe cases, death. Currently, treatment for babesiosis relies primarily on chemical drugs. Long-term use can lead to the development of resistance to traditional drugs, posing new challenges to existing treatment options. Traditional drug resistance in Babesia is primarily due to the parasite's ability to modulate drug permeability and resistance mechanisms. Furthermore, resistance mechanisms also include genetic variation and modulation of drug metabolism pathways. These factors contribute to resistance to traditional drugs, further complicating the treatment of babesiosis. Therefore, the search for new Babesia inhibitors to overcome drug resistance is crucial. Phenylureas, a class of compounds with a broad range of biological activities, have garnered significant attention in recent years in drug development. Previous studies have shown that phenylureas have potential inhibitory activity in the field of parasites. However, their application in the inhibition of Babesia has not been thoroughly studied.

[0003] The present invention aims to provide novel phenylurea compounds for use as Babesia growth inhibitors. These compounds have unique chemical structures and biological activity characteristics and have certain insect-inhibiting effects. Summary of the Invention

[0004] The present invention aims to provide a phenylurea compound for use as a Babesia growth inhibitor. The phenylurea compound is 1,3-bis(3-chlorophenyl)urea or 1,3-bis(3-cyanophenyl)urea. The above two phenylurea compounds have unique structures and chemical properties. Experiments have shown that both prepared phenylurea compounds can be used as Babesia growth inhibitors and can effectively inhibit the growth of Babesia.

[0005] The technical problem to be solved by the present invention is that currently, the treatment of babesiosis relies primarily on chemical drugs. Long-term use can lead to parasite resistance to traditional drugs, posing new challenges to existing treatment options. Traditional Babesia drug resistance is primarily due to the parasite's ability to tolerate drugs and the modulation of its resistance mechanisms. Furthermore, Babesia drug resistance mechanisms also include genetic variation and modulation of drug metabolism pathways. These factors contribute to Babesia's resistance to traditional drugs, exacerbating the difficulty and challenges of treating babesiosis. Therefore, the search for new Babesia inhibitors to overcome this drug resistance is crucial.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A phenylurea compound as shown in formula I as a Babesia growth inhibitor;

[0008]

[0009] Wherein, the substituent R is selected from Cl or CN.

[0010] Furthermore, the Babesia is Babesia gibsoni.

[0011] Furthermore, when the substituent R is Cl, the phenylurea compound is 1,3-bis(3-chlorophenyl)urea, and its structural formula is shown below:

[0012]

[0013] Furthermore, when the substituent R is CN, the phenylurea compound is 1,3-bis(3-cyanophenyl)urea, and its structural formula is as follows:

[0014]

[0015] Furthermore, the synthesis method of the phenylurea compound is as follows: dissolving an aniline compound and di-tert-butyl dicarbonate in dichloromethane, then adding triethylenediamine, and reacting at room temperature to obtain the phenylurea compound.

[0016] Furthermore, the molar ratio of the aniline compound, di-tert-butyl dicarbonate and triethylenediamine is 2-2.5:1:0.2-0.25.

[0017] Furthermore, the aniline compound is m-chloroaniline or m-cyanoaniline.

[0018] Furthermore, the reaction time is 3 h.

[0019] The phenylurea compounds provided by the present invention have a low molecular weight, a relatively simple structure, are easily soluble in various solvents, and possess unique structural and chemical properties. Experimental studies have demonstrated that these phenylurea compounds exhibit significant Babesia inhibitory activity, effectively inhibiting the growth of Babesia and ultimately killing it. Furthermore, these compounds have excellent bioavailability and safety profiles, with no significant toxic side effects on host cells.

[0020] Beneficial effects of the present invention:

[0021] (1) In the technical solution of the present invention, the phenylurea compound provided has a small molecular weight, a relatively simple structure, and is easily soluble in a variety of solvents.

[0022] (2) The phenylurea compounds provided in the technical solution of the present invention have good specificity, effectively inhibiting the growth of Babesia and ultimately killing it. Furthermore, they have good bioavailability and safety characteristics, with no significant toxic side effects on host cells, providing a new therapeutic strategy and option for the treatment of babesiosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The erythrocyte infection rate of each treatment sample was observed under a 100x oil microscope. From left to right, they are the control group without the inhibitor 1,3-bis(3-chlorophenyl)urea and the treatment groups with different concentrations of the inhibitor.

[0024] Figure 2 The growth of Babesia gibsoni at various inhibitor concentrations in the half-maximal inhibitory concentration assay; from left to right, the concentrations of the phenylurea compound, i.e., the inhibitor 1,3-bis(3-chlorophenyl)urea, are: 0, 10, 20, 40, 60, 80 μM;

[0025] Figure 3 The erythrocyte infection rate of each treatment sample was observed under a 100x oil microscope. From left to right, they are the control group without the addition of the inhibitor 1,3-bis(3-cyanophenyl)urea, and the treatment groups with different concentrations of the inhibitor 1,3-bis(3-cyanophenyl)urea.

[0026] Figure 4 This is the growth of Babesia gibsoni at various inhibitor concentrations in the half-maximal inhibitory concentration assay; from left to right, the concentrations of the phenylurea compound, i.e., the inhibitor 1,3-bis(3-cyanophenyl)urea, are: 0, 2.5, 5, 10, 20, 40, 80, 160 μM;

[0027] Figure 5 The invention relates to a method for synthesizing phenylurea compounds. DETAILED DESCRIPTION

[0028] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0029] Example 1

[0030] The cultivation of Babesia strains includes the following steps:

[0031] A1. Preparation of Babesia strains for testing:

[0032] The parasite strain tested in this example relates to Babesia gibsoni.

[0033] A2. Preparation of Babesia culture medium:

[0034] Preparation of Babesia culture medium: Prepare 1000 ml of SFM basal medium. Upon use, transfer 47.5 ml of SFM to a 50 ml centrifuge tube and add 2.5 ml of self-collected canine serum (from beagle dogs). This liquid culture medium is a typical liquid culture medium for Babesia gibsoni.

[0035] The components and proportions of SFM basal medium are as follows:

[0036]

[0037] A3. Babesia culture method:

[0038] A conventional Babesia gibbsii culture system (50 μL of red blood cells, 450 μL of Babesia gibbsii culture medium (SFM culture medium containing canine serum)) was added to a 48-well culture plate and cultured in a 5% CO2 incubator at 37°C. The culture medium was changed every 24 hours (400 μL of culture medium was aspirated and replaced with 400 μL of new culture medium) and thoroughly mixed with the red blood cells at the bottom. The cells were passaged every 72 hours (after thoroughly mixing the old well, 50 μL was aspirated and added to a new well, followed by 45 μL of fresh canine red blood cells and 405 μL of Babesia gibbsii culture medium).

[0039] All the above operations were performed in the sterile environment of a conventional cell room clean bench.

[0040] Example 2

[0041] Preparation of candidate inhibitors:

[0042] The inhibitor 1,3-bis(3-chlorophenyl)urea in this example was used as a solution. The inhibitor powder was dissolved in dimethyl sulfoxide (DMSO), 10× phosphate buffered saline (10× PBS), and SFM culture medium for subsequent experimental use. The specific preparation method was as follows: First, a concentrated inhibitor solution with an initial concentration of 100 mM was prepared for storage, and then diluted with 10× PBS to an intermediate concentration (4 mM). When determining the half-maximal inhibitory concentration of the inhibitor, 1,3-bis(3-chlorophenyl)urea, against Babesia gibbsii, the prepared inhibitor concentrate was diluted with SFM culture medium to conduct a concentration gradient experiment. The concentration gradient was designed to be: 80, 60, 40, 20, and 10 μM.

[0043] Example 3

[0044] Preparation of candidate inhibitors:

[0045] The inhibitor 1,3-bis(3-cyanophenyl)urea in this example was used as a solution. The inhibitor powder was dissolved in dimethyl sulfoxide (DMSO), 10× phosphate buffered saline (10× PBS), and SFM culture medium for subsequent experimental use. The specific preparation method was as follows: First, a concentrated inhibitor solution with an initial concentration of 200 mM was prepared for storage, and then diluted with 10× PBS to an intermediate concentration (4 mM). When determining the 50% inhibitory concentration of the inhibitor, 1,3-bis(3-cyanophenyl)urea, against Babesia gibbsii, the prepared inhibitor concentrate was diluted with SFM culture medium to conduct a concentration gradient experiment. The concentration gradient was designed to be: 160, 80, 40, 20, 10, 5, and 2.5 μM.

[0046] Example 4

[0047] In vitro growth inhibition test of Babesia gibsoni

[0048] Inhibition test on Babesia gibbsii: According to the aseptic operation procedure of Example 1, 0.3 μL of red blood cell mud was aspirated in the 48-well culture plate of Babesia gibbsii in Example 1, and a blood smear was made. After Giemsa staining, the infection rate was observed under a microscope. The infection rate was diluted to 0.2% with red blood cells of a healthy dog. Mix thoroughly with SFM culture medium and divide equally into new 96-well culture plates according to a volume of 50 μL per well. Take the candidate inhibitor 1,3-bis(3-chlorophenyl)urea prepared in Example 2, and perform three repeated experiments for each concentration. Mix the mixture in each well thoroughly, culture it at 37°C for 72 hours, and observe the effect of 1,3-bis(3-chlorophenyl)urea on the in vitro growth of Babesia gibbsii by counting the infection rate, and calculate the half maximal inhibitory concentration (IC50). 50 The entire operation was carried out under the aseptic operation procedure of the commonly used cell-internal ultra-clean workbench. Figure 1 and Figure 2 shown.

[0049] from Figure 1 and Figure 2 It can be seen that 1,3-bis(3-chlorophenyl)urea can effectively inhibit the growth of Babesia gibbsii in vitro, IC 50 It is 11.44μM.

[0050] Example 5

[0051] In vitro growth inhibition test of Babesia gibsoni

[0052] Inhibition test on Babesia gibbsii: According to the aseptic operation procedure of Example 1, 0.3 μL of red blood cell mud was aspirated in the 48-well culture plate of Babesia gibbsii in Example 1, and a blood smear was made. After Giemsa staining, the infection rate was observed under a microscope. The infection rate was diluted to 0.2% with red blood cells from a healthy dog. Mix thoroughly with SFM culture medium and divide equally into new 96-well culture plates according to a volume of 50 μL per well. Take the candidate inhibitor 1,3-bis(3-cyanophenyl)urea prepared in Example 3, and perform three repeated experiments for each concentration. The mixture in each well was thoroughly mixed and cultured at 37°C for 72 hours. The infection rate was counted to observe the effect of 1,3-bis(3-cyanophenyl)urea on the in vitro growth of Babesia gibbsii, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The entire operation was carried out under the aseptic operation procedure of the commonly used cell-internal ultra-clean workbench. Figure 3 and Figure 4 shown.

[0053] from Figure 3 and Figure 4 It can be seen that 1,3-bis(3-cyanophenyl)urea can effectively inhibit the in vitro growth of Babesia gibbsii, IC 50 It is 9.32μM.

[0054] Throughout the specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0055] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in similar ways. As long as they do not deviate from the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. Use of a phenylurea compound of formula I in the preparation of a medicament for inhibiting the growth of Babesia; Formula I; In the formula I, the substituent R is selected from Cl or CN.

2. The use according to claim 1, characterized in that: The Babesia is Babesia gibsoni.

3. The use according to claim 1, characterized in that: The synthesis method of the phenylurea compound comprises the following steps: dissolving an aniline compound and di-tert-butyl dicarbonate in dichloromethane, adding triethylenediamine, and reacting at room temperature to obtain the phenylurea compound.

4. The use according to claim 3, characterized in that: The molar ratio of the aniline compound, di-tert-butyl dicarbonate and triethylenediamine is 2-2.5:1:0.2-0.

25.

5. The use according to claim 3, characterized in that: The aniline compound is m-chloroaniline or m-cyanoaniline.

6. The use according to claim 3, characterized in that: The reaction time is 3 hours.

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