TWI777479B - A use of cytisine derivatives - Google Patents

Abstract

A cytisine derivative of following general formula (I), a synthesis method thereof, a pharmaceutical composition thereof and a use thereof are disclosed.

In formula (I), each symbol is as defined in the specification. The cytisine derivative can inhibit the cytopathic effect and viral infectivity of dengue virus. Therefore, it is useful as a prophylactic and therapeutic drug for treating dengue virus.

Description

Uses of cystein derivatives

The present invention relates to a cystein derivative, in particular to a cystein derivative that can prevent, treat and improve dengue virus, its synthesis method, its pharmaceutical composition and its use.

Dengue virus (DENV) belongs to the genus Flavivirus, which forms a cycle of viral transmission through vector mosquitoes and primates of the genus Aedes, and causes primates to be bitten by vector mosquitoes with dengue virus. Animals suffering from dengue fever, dengue hemorrhagic fever or dengue shock syndrome.

Dengue virus has a wide geographical distribution and has been found in all countries in the world. The most common areas are 35 degrees north latitude to 35 degrees south latitude and below 1,000 meters above sea level. In addition to dengue fever, dengue hemorrhagic fever or In addition to dengue shock syndrome, other neurological diseases may also be complicated, such as transverse myelitis and Guillain-Barre syndrome (Guillain-Barre Syndrome), floaters, and other rare complications include heart infection and acute liver failure.

Although the disease caused by dengue virus is quite serious, it can only be improved by rest and symptomatic treatment. There is no drug or vaccine. For the prevention or treatment of dengue virus, it is important to develop a novel drug to fight against this virus.

One object of the present invention is to provide a genus derivative, its synthesis method, its pharmaceutical composition and its use, and to apply the genus derivative to the purpose of resisting dengue virus. The base derivative has a virus-inhibiting effect to effectively treat dengue virus.

其中式(I)中，R₁為甲基、如式(MAI)所示之一基團或如式(MAII)所示之一基團，R₂為氫原子或硫原子，R₃為氫原子或硫原子，R₄為氫原子、硝基、如式(MBI)所示之一基團或如式(MBII)所示之一基團，R₅為氧原子或硫原子，

表示單鍵或雙鍵以便滿足所有價數：

One embodiment of the present invention provides the use of a cystein derivative, which is used to prepare a medicament for preventing, treating or ameliorating dengue virus infection, wherein the cyphine derivative has the formula (I ) shown in one of the structures:

Wherein in formula (I), R ₁ is a methyl group, a group shown in formula (MAI) or a group shown in formula (MAII), R ₂ is a hydrogen atom or a sulfur atom, and R ₃ is hydrogen atom or sulfur atom, R ₄ is a hydrogen atom, a nitro group, a group shown in formula (MBI) or a group shown in formula (MBII), R ₅ is an oxygen atom or a sulfur atom,

Represent single or double bonds to satisfy all valences:

Thereby, the cystein derivatives of the present invention have been proved to have anti-dengue virus efficacy through experimental data, can inhibit the cytopathic effect and virus infectivity of dengue virus in vitro, and can block the early and late replication of the virus A step of. Therefore, the cystein derivatives of the present invention are suitable as medicines for preventing, treating and ameliorating dengue virus infection, or can be used in combination with pharmaceutically acceptable excipients or carriers to prevent, treat and improve dengue virus. The pharmaceutical composition for diseases has the potential to be used in the biomedical market.

100: Synthesis method of cystein derivatives

110: Precursor providing step

120: Chemical Reaction Steps

130: Product purification step

In order to make the above-mentioned and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows: Fig. 1 shows the synthesis of cystein derivatives according to another embodiment of the present invention. The flow chart of the steps of the method; Fig. 2A, Fig. 2B, Fig. 2C, Fig. 2D, Fig. 2E, Fig. 2F and Fig. 2G illustrate the cell viability test of Vero E6 cells according to an embodiment of the present invention result bar chart; Fig. 3A, Fig. 3B, Fig. 3C, Fig. 3D, Fig. 3E, Fig. 3F and Fig. 3G are bar graphs showing the results of the cell viability test on A549 cells according to the embodiments of the present invention; Fig. 4 Figure 1 is a microscopic image of the inhibitory ability of Example 1 of the present invention to DENV-2-induced Vero E6 cytopathies; Figure 5 is a microscopic image of the inhibitory ability of Example 1 of the present invention to DENV-2-induced A549 cytopathic changes ; Figure 6A, Figure 6B, Figure 6C, Figure 6D, Figure 6E, Figure 6F, and Figure 6G illustrate the infection inhibition of DENV-2-infected Vero E6 cells by embodiments of the present invention Test results; Fig. 7A, Fig. 7B, Fig. 7C, Fig. 7D, Fig. 7E, Fig. 7F and Fig. 7G show the infection inhibition of DENV-2 infected A549 cells by the examples of the present invention The results of the force test; Figure 8A, Figure 8B, Figure 8C, Figure 8D and Figure 8E show the results of the infection inhibition test results of the embodiments of the present invention on Vero E6 cells infected with DENV-1; Figure 9 The figure is a microscopic image of the inhibitory ability of DENV-2 to induce Vero E6 cytopathic changes in the pretreatment test of virus infection (without washing the drug) in Example 5 of the present invention; Figure 10A, Figure 10B and Figure 10C are series Figure 11 shows the results of the infection inhibition test on Vero E6 cells infected with DENV-2 in the pretreatment test of virus infection (without cleaning drugs) according to the embodiment of the present invention; In the pretreatment test (washing the drug with PBS), the inhibition of DENV-2-induced Vero E6 cytopathic changes Figure 12A, Figure 12B, and Figure 12C show the microscopic images of DENV-2-infected Vero E6 cells in a pre-treatment test for virus infection (drug washing with PBS) according to an example of the present invention. Infection inhibition test results; Figure 13 is a microscopic image of the inhibitory ability of DENV-2-induced Vero E6 cytopathies in the treatment test of Example 1 of the present invention during virus infection; Figure 14A, Figure 14B, Figure 14C Figure, Figure 14D, Figure 14E, Figure 14F and Figure 14G show the results of the infection inhibition test on DENV-2-infected Vero E6 cells in the treatment test of the embodiment of the present invention; Figure 15 is a microscopic image of the inhibitory ability of DENV-2 to induce Vero E6 cytopathic changes in the post-treatment test of virus infection in Example 1 of the present invention; and Figure 16A, Figure 16B, Figure 16C, Figure 16D, Figure 16E, Figure 16F and Figure 16G show the results of the infection inhibition test on DENV-2-infected Vero E6 cells in the virus infection post-treatment test according to the embodiment of the present invention.

Several embodiments of the present invention will be described below with reference to the drawings. For the sake of clarity, many practical details are set forth in the following description. It should be understood, however, that these practical details should not be used to limit the invention. That is, in some embodiments of the present invention, these practical details are unnecessary. In addition, in order to simplify the diagram, some conventional The structures and elements in the drawings will be shown in a simplified schematic manner; and repeated elements will possibly be designated by the same reference numerals.

<Cycylate Derivatives>

其中式(I)中，R₁為甲基、如式(MAI)所示之一基團或如式(MAII)所示之一基團，R₂為氫原子或硫原子，R₃為氫原子或硫原子，R₄為氫原子、硝基、如式(MBI)所示之一基團或如式(MBII)所示之一基團，R₅為氧原子或硫原子：

One embodiment of the present invention provides a cystein derivative, which has a structure as shown in formula (I):

Wherein in formula (I), R ₁ is a methyl group, a group shown in formula (MAI) or a group shown in formula (MAII), R ₂ is a hydrogen atom or a sulfur atom, and R ₃ is hydrogen atom or sulfur atom, R ₄ is a hydrogen atom, a nitro group, a group as shown in formula (MBI) or a group as shown in formula (MBII), R ₅ is an oxygen atom or a sulfur atom:

<Synthesis method of cystein derivatives>

Another embodiment of the present invention provides a gorse flower as described above Synthesis of base derivatives. Please refer to FIG. 1. FIG. 1 is a flow chart showing the steps of the method 100 for synthesizing the cystein derivatives of the present invention. The method 100 for synthesizing cystein derivatives includes step 110 , step 120 and step 130 .

Step 110 is a step of providing a precursor, which provides a precursor to perform a chemical reaction, and the used precursor is cytisine ((-)-Cytisine).

Step 120 is a chemical reaction step, which is to chemically react the precursor in a reaction manner to obtain a product, and the reaction manner is stirring at room temperature or refluxing at high temperature.

Step 130 is a product purification step, which is to purify the product to obtain a cysteine derivative. The purification method may be thin layer chromatography (TLC), but the present invention is not limited thereto.

The following specific test examples are hereby used to further demonstrate the present invention, so as to help those of ordinary knowledge in the technical field to which the present invention pertains to fully utilize and practice the present invention without excessive interpretation, and should not be regarded as a It is intended to limit the scope of the invention, but to illustrate how the materials and methods of the invention may be practiced.

<Test example>

1. Synthesis and structural identification of cystein derivatives of the present invention

Please refer to Table 1 below, which are the structural formulas of compound formula (IA) to compound formula (IJ) of Example 1 to Example 10 of the cystein derivatives of the present invention.

Please refer to the above Table 1, the various embodiments of the cystein derivatives of the present invention, the structural formulas are shown in Table 1.

The synthesis methods of Example 1 and Example 3 of the present invention are as follows: react with (-)-cytisine in acetone with methyl iodide and potassium carbonate at 56° C. After the reaction is completed, acetone is evaporated to obtain N-methylcytisine (hereinafter referred to as N-methylcytisine). Intermediate 1), the intermediate 1 was nitrated with sodium nitrate in sulfuric acid at room temperature, the product was poured on ice and neutralized with sodium carbonate, extracted with ethyl acetate, dried with anhydrous sodium sulfate and concentrated to obtain the implementation The cytidine derivative of Example 3 was dissolved in ethyl acetate, and the cytidine derivative of Example 3 was dissolved in ethyl acetate with a palladium-carbon catalyst under a hydrogen atmosphere. The nitro group is reduced to amine to obtain 9-amino-N-methylcytisine (hereinafter referred to as intermediate 2). After the intermediate 2 is reacted with 1,3-benzylformyluracil, it is dissolved in methanol for hydroboration Sodium is subjected to carbonyl reduction, and the product is extracted with chloroform and dried with anhydrous sodium sulfate to obtain the cysteine derivative of Example 1.

The synthesis method of Example 2 of the present invention is as follows: at room temperature, the (-)-cytisine dissolved in concentrated sulfuric acid is nitrified with sodium nitrate, neutralized with sodium carbonate, extracted with ethyl acetate, and then extracted with anhydrous sodium sulfate. Dry and concentrate to obtain 9-nitrocytisine (hereinafter referred to as intermediate 3), which is dissolved in benzene and reacted with phenyl isocyanate at room temperature and concentrated to obtain 9-Nitro-8-oxo-N -phenyl-1,5,6,8-tetrahydro-2H-1,5-methanopyrido[1,2-a][1,5]diazocine-3(4H)-carboxamide (hereinafter referred to as intermediate 4), the intermediate Compound 4 was dissolved in ethyl acetate, and the intermediate compound 2-2 was mixed with palladium-carbon catalyst under a hydrogen atmosphere. The nitro group is reduced to an amine to give 9-Amino-8-oxo-N-phenyl-1,5,6,8-tetrahydro-2H-1,5-methanopyrido[1,2-a][1,5]diazocine -3(4H)-carboxamide (hereinafter referred to as intermediate 5), the intermediate 5 and 1,3-dimethyl-5-formyluracil were refluxed in benzene at 80 °C, and the product was dissolved in methanol to The carbonyl group was reduced by sodium borohydride at 0° C., extracted with carbon trichloride, and dried with anhydrous sodium sulfate to obtain the cysteine derivative of Example 2.

The synthesis method of Example 4 of the present invention is as follows: After the intermediate 2 is reacted with 4-hydroxybenzaldehyde in benzene at 80°C, the benzene is evaporated and the product is dissolved in methanol, and the hydroboration is carried out at 0°C. After carbonyl reduction with sodium, extraction with carbon trichloride and drying with anhydrous sodium sulfate, the cystein derivative of Example 4 was obtained.

The synthesis method of Example 5 of the present invention is as follows: react (-)-cytisine with allyl isothiocyanate in benzene at 35°C to 40°C to obtain the cytisine derivative of Example 5.

The synthesis method of Example 6 of the present invention is as follows: the intermediate 1 is dissolved in toluene and refluxed at 110° C. with Lawesson's Reagent, the solvent is evaporated after the reaction is completed, the product is alkalized with sodium carbonate, and the reaction is carried out with three After extraction with methyl chloride, drying over anhydrous sodium sulfate and concentration, the cystein derivative of Example 6 was obtained.

The synthesis methods of Example 7 and Example 10 of the present invention are as follows: the intermediate 1 and potassium permanganate are reacted in an aqueous acetonitrile solution (acetonitrile:water=10:1) at room temperature for 2 hours to obtain (1R,5R)-3-Methyl-3,4,5,6-tetrahydro-2H-1,5-methanopyrido[1,2-a][1,5]diazocine-2,8(1H)-dione( Hereinafter referred to as intermediate 6), the intermediate 6 was dissolved in toluene and refluxed at 110 ° C with Lawesson's reagent. After the reaction was completed, the solvent was evaporated, and the product was alkalized with sodium carbonate, extracted with chloroform and then extracted with anhydrous sodium sulfate. Drying and concentration gave the cystein derivatives of Example 7 and Example 10.

The synthesis methods of Example 8 and Example 9 of the present invention are as follows: Intermediate 1 and potassium permanganate are reacted in an aqueous acetonitrile solution (acetonitrile:water=10:1) at room temperature for 2 hours, then dissolved in toluene to 0.5 equivalent of Lawson's reagent was refluxed at 110°C to obtain the cystein derivatives of Example 8 and Example 9.

In each embodiment of the present invention, infrared spectroscopy and nuclear magnetic resonance spectroscopy are used to perform infrared spectroscopy and nuclear magnetic resonance spectroscopy analysis data as shown in Table 2 below.

2. Anti-dengue virus test of the genistein derivatives of the present invention

2-1: Cell Viability Test

The cell lines used for the cell viability test were African green monkey kidney epithelial cells Vero E6 (ATCC ^® No.CRL-1586, hereinafter referred to as Vero E6 cells) and human lung adenocarcinoma cells A549 (ATCC ^® No. CLL-185 ^TM , hereinafter referred to as referred to as A549 cells). First, Vero E6 cells or A549 cells were cultured in a 96-well plate at a cell volume of 5 × 10 ³ per well, and were prepared in DMEM (Dulbecco's modified eagle medium, Gibco ^® ) containing 10% FBS (Fetal bovine serum, Gibco ^® ). To a total volume of 160 μL/well, incubate overnight in an incubator at 37°C and 5% CO ₂ . On the next day, without removing the culture medium, the cystein derivatives of Example 1-Example 10 were added respectively, and the drug concentration of each example reached 0.1 μM, 1 μM, 10 μM and 50 μM, respectively. Four replicate experiments were performed. After 96 hours of drug treatment, add 10 μL of MTT solution to each well of cells, react in the dark for 4 hours, and then add 100 μL of Solution C (JT Baker ^® ) to disperse the crystal violet, and use an ELISA reader or high-brightness optical reading. The instrument measures the absorbance at the background wavelength of 670nm and the sample at the wavelength of 570nm respectively, and subtracts the absorbance of the background value at 670nm from the absorbance of the detected sample at the wavelength of 570nm, and evaluates the cells treated in each well. The absorbance (OD value) of the treated group was 100%, and the cell survival rate (%) and the half-toxic dose (CC ₅₀ ) of the derivatives to Vero E6 cells or A549 cells were calculated.

50μM)。 Fig. 2A, Fig. 2B, Fig. 2C, Fig. 2D, Fig. 2E, Fig. 2F, and Fig. 2G respectively illustrate Embodiment 1, Embodiment 2, Embodiment 3, Embodiment 4, and Embodiment of the present invention Bar graphs of cell viability assay results of Example 5, Example 6 and Example 7 on Vero E6 cells; Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, Figure 3F, and Figure 3G The figure shows a bar graph of the cell viability test results of Example 1, Example 2, Example 3, Example 4, Example 5, Example 6 and Example 7 of the present invention on A549 cells, respectively. It can be seen from the test results that even if the added drug concentration of Example 1-Example 7 reaches 50 μM, it does not obviously lead to the death of Vero E6 cells or A549 cells, which shows that the cytidine derivatives of the present invention are effective against Vero E6 cells and on Vero E6 cells. A549 cells had no toxic effects (CC ₅₀

50 μM).

2-2: Cytopathic inhibition test and immunofluorescence staining analysis

The cell lines for cytopathic inhibition test and immunofluorescence staining analysis were Vero E6 cells and A549 cells, and the virus strains were Dengue virus type 1 (DENV-1) and Dengue virus type 2 (Dengue virus type 1, DENV-1). virus type 2, DENV-2) of the 16681 strain (Strain 16681).

First, Vero E6 cells or A549 cells were cultured in a 6-well dish at 2×10 ⁵ cells per well, prepared in DMEM containing 10% FBS to a total volume of 2 mL/well, and incubated at 37°C and 5% CO ₂ Incubator overnight. On the next day, the culture medium was removed and the 6-well plate was washed twice with Phosphate buffered saline (PBS) and once with DMEM without FBS, and then DENV-1 or DENV-2 was added. Vero E6 cells and A549 cells were infected with DENV-1 with a multiplicity of infection (MOI) of 0.1, Vero E6 cells were infected with DENV-2 with an MOI of 0.005, and A549 cells were infected with DENV-2 with an MOI of 0.05. The genistein derivatives of Example 1-Example 10 were added to their drug concentrations of 0.1 μM, 1 μM, and 10 μM, respectively, while the most effective derivatives were increased to 0.001 μM and 0.01 μM to treat cells. deal with. After 96 hours of culture, the results of the cytopathic inhibition test can be obtained by observing the cytopathic state and taking pictures.

Please refer to Figure 4 and Figure 5. Figure 4 is a microscopic image of the inhibitory ability of Example 1 of the present invention to DENV-2-induced Vero E6 cell lesions, and Figure 5 is a microscopic image of Example 1 of the present invention to DENV-2 2 Microscopic images of the inhibitory ability to induce pathological changes in A549 cells. As can be seen from the figure, Example 1 inhibited DENV-2-induced Vero E6 cytopathic and A549 cytopathic in a concentration-dependent manner, and showed a significant inhibitory effect when the drug concentration was 10 μM. Taking the inhibitory effect shown in Figure 4 as a significant inhibitory effect (+++), the inhibitory effects of the derivatives of each example under this test on the cytopathic effects of infected Vero E6 cells are listed in Table 3 below. The results of the graphs show that the genistein derivatives of the present invention have a significant inhibitory effect on DENV-2-induced cytopathies.

Continue on (after 96 hours of culture), add 1 mL of 4% formalin to each well of cells to fix the cells, then react on a shaker for 30 minutes at room temperature, then wash twice with PBS, and then add to each well of cells 1mL of 50mM NH ₄ Cl, then reacted on a shaker for 30 minutes at room temperature to remove autofluorescence, then washed once with PBS, and then added BSA:Triton-100 as a 1000:1 blocking solution, and then in The cells were reacted with shaking in a cold room at 4°C for 4 hours, washed with PBS, and then stained with antibodies. Specifically, Rabbit anti-DENV2-NS4B (GeneTex, Inc.) was used as the primary antibody, and then the cells were shaken in a refrigerated room at 4°C overnight. After washing three times with PBS every other day, Goat anti-rabbit IgG H & L (Alexa Fluor ^® 555, ThermoFisher) antibody was used as the secondary antibody for staining. Nuclei were stained with DAPI (4'6-diamidino-2-phenylindole) fluorescent dye after antibody removal to count the number of cells, and Image J software was used to determine the percentage of NS4B positive cells in the infected cells. Infection inhibition was calculated as: (percent NS4B positive in infected cells not treated with derivative - percent NS4B positive in infected cells treated with derivative)/percent NS4B positive in infected cells treated with derivative. According to the results of the infection inhibition, the half-maximum inhibitory concentration (IC ₅₀ ) was calculated, which is the antiviral activity of the genistein derivatives of the present invention against DENV-2.

Fig. 6A, Fig. 6B, Fig. 6C, Fig. 6D, Fig. 6E, Fig. 6F, and Fig. 6G respectively illustrate Embodiment 1, Embodiment 2, Embodiment 3, Embodiment 4, and Embodiment of the present invention Infection inhibition test results of Example 5, Example 6 and Example 7 on Vero E6 cells infected with DENV-2, Figure 7A, Figure 7B, Figure 7C, Figure 7D, Figure 7E, Figure 7F and FIG. 7G respectively illustrate Embodiment 1, Embodiment 2, Embodiment 3, Embodiment 4, Embodiment 5, Embodiment 6 and Embodiment of the present invention. 7. The results of the infection inhibition test on A549 cells infected with DENV-2. Figure 8A, Figure 8B, Figure 8C, Figure 8D and Figure 8E respectively depict Example 1, Example 2, and Figure 8E of the present invention. Infection inhibition test results of Example 3, Example 4 and Example 7 on Vero E6 cells infected with DENV-1. Please also refer to Table 4 below. Table 4 shows the data on the infection inhibition and antiviral activity of DENV-2 infected cells in Examples 1 to 10 of the present invention. The results of the graph show that each embodiment of the present invention significantly reduces the percentage of NS4B positive cells in the infected cells in a concentration-dependent manner, that is, each embodiment of the present invention can significantly reduce the effect of DENV-2 on Vero E6 cells. and the infectivity of A549 cells, which indicates that the cystein derivatives of the present invention can significantly reduce the infectivity of dengue virus to cells and have a significant inhibitory effect on DENV-2-induced cytopathic effects.

2-3 Virus infection pretreatment test

The cell line used for virus infection pretreatment test was Vero E6 cell, the 16681 strain of Dengue virus type 2 (DENV-2) (Strain 16681).

First, Vero E6 cells were cultured at 2×10 ⁵ cells per well in a 6-well dish, prepared in DMEM containing 10% FBS to a total volume of 2 mL/well, and incubated at 37°C in an incubator with 5% CO ₂ . Incubate overnight. The next day, the culture medium was removed and the 6-well plate was washed twice with Phosphate buffered saline (PBS), and then washed once with DMEM without FBS, and then the drugs of Example 1-Example 10 were added to make the drug concentration. At 0.1 μM, 1 μM and 10 μM, Vero E6 cells were infected with DENV-2 at an MOI of 0.005 after culturing for 24 hours in an incubator at 37°C and 5% CO ₂ without washing the drug/after washing the drug with PBS, Finally, DMEM without FBS was added to bring the total volume to 2 mL, and then placed in an incubator at 37°C and 5% CO ₂ for 96 hours to observe the inhibitory effect of cytopathic changes, and immunofluorescence staining was performed. The process of immunofluorescence staining has been completed. As mentioned in the previous paragraph, I will not repeat them here. The fluorescence quantity of NS4B viral protein and DAPI was observed under a fluorescence microscope and photographed, and then Image J software was used to calculate the quantity of NS4B viral protein and DAPI to calculate the ratio of virus infection.

Fig. 9 is a microscopic image of the inhibitory ability of DENV-2 to induce Vero E6 cytopathic changes in the pretreatment test of virus infection (without cleaning drugs) according to Example 5 of the present invention, Fig. 10A, Fig. 10B and Fig. 10C The figure shows the results of the infection inhibition test of DENV-2-infected Vero E6 cells in the pretreatment test of virus infection (without washing the drug) in Example 5, Example 6 and Example 7 of the present invention, the 11th The figure is a microscopic image of the inhibitory ability of DENV-2 to induce Vero E6 cytopathies in the pretreatment test of virus infection (washing the drug with PBS) in Example 5 of the present invention. Figure 12A, Figure 12B and Figure 12C are the series The results of the infection inhibition test of DENV-2-infected Vero E6 cells in the pretreatment test of virus infection (washing the drug with PBS) of Example 5, Example 6 and Example 7 of the present invention are shown. From the results of Figure 9, Figure 10A to Figure 10C, Figure 11, and Figure 12A to Figure 12C, Example 5 of the present invention significantly reduced the percentage of NS4B positive cells in the infected cells, and its IC ₅₀ is 0.45 μM in the state of not washing the drug, and its IC ₅₀ is 5.42 μM in the state of washing the drug with PBS, that is, the cytidine derivative of Example 5 of the present invention can significantly prevent DENV-2 for Vero E6 Infectivity of cells.

2-4 Virus infection and post-virus treatment test

First, two groups of Vero E6 cells were cultured in a 6-well dish in the aforementioned manner, and infected with DENV-2 in the aforementioned manner, and one group of cells was infected with the virus while adding the cysteine derivative of the embodiment (virus infection). Time treatment test), another group of cells was added with the cytidine derivative of the example 1 hour after infection (virus infection post treatment test). The examples used are Example 1, Example 2, Example 3, Example 4, Example 5, Example 6 or Example 7, and the drug concentration of each example reached 0.1 μM, 1 μM and 10 μM, respectively. After culturing in an incubator at 37°C and 5% CO ₂ for 1 hour, wash twice with PBS to remove the drug, and finally add FBS-free DMEM to make the total volume 2 mL, and then put it into an incubator at 37° C. and 5% CO ₂ . Incubator for 96 hours to observe the cytopathic inhibition effect, and perform immunofluorescence staining. The process of immunofluorescence staining has been mentioned in the previous paragraph and will not be repeated here. NS4B virus protein and DAPI fluorescence were observed under a fluorescence microscope. Quantity and taking pictures, and then use Image J software to calculate the amount of NS4B virus protein and DAPI, and calculate the ratio of virus infection.

Figure 13 is a microscopic image of the inhibitory ability of DENV-2 to induce Vero E6 cytopathic changes in the treatment test of Example 1 of the present invention during virus infection, Figure 14A, Figure 14B, Figure 14C, Figure 14D, Fig. 14E, Fig. 14F and Fig. 14G respectively illustrate the treatment test of the present invention in Example 1, Example 2, Example 3, Example 4, Example 5, Example 6 and Example 7 of the present invention Infection inhibition test results on Vero E6 cells infected with DENV-2. Please refer to the following table 5 at the same time, table 5 is the antiviral activity data of the embodiments 1-7 of the present invention to DENV-2 in the cell entry stage. The results of the graph show that each embodiment of the present invention significantly reduces the percentage of NS4B positive cells in the infected cells in a concentration-dependent manner, that is, each embodiment of the present invention can significantly reduce the effect of DENV-2 on Vero E6 cells. The infectivity of the present invention shows that the genistein derivatives of the present invention can significantly reduce the infectivity of dengue virus to cells and have a significant inhibitory effect on the early replication of DENV-2.

Figure 15 is a microscopic image of the inhibitory ability of DENV-2 to induce Vero E6 cytopathic changes in the virus infection post-treatment test of Example 1 of the present invention, Figure 16A, Figure 16B, Figure 16C, Figure 16D, Fig. 16E, Fig. 16F and Fig. 16G illustrate the post-virus treatment test of Example 1, Example 2, Example 3, Example 4, Example 5, Example 6 and Example 7 of the present invention Infection inhibition test results on Vero E6 cells infected with DENV-2. Please refer to the following table 6 at the same time. Table 6 is the antiviral activity data of DENV-2 after infecting cells in Examples 1-7 of the present invention. The results of the graphs show that each embodiment of the present invention significantly reduces the percentage of NS4B positive cells in the infected cells in a concentration-dependent manner, that is, each embodiment of the present invention can significantly reduce the entry of DENV-2 into Vero E6 cells This shows that the genistein derivatives of the present invention can significantly reduce the replication ability of dengue virus after entering cells and have a significant inhibitory effect on the late replication of DENV-2.

To sum up, the genistein derivatives represented by the structure of the present invention as shown in formula (I) can inhibit the cytopathic effect and virus infectivity of DENV-1 and DENV-2 in vitro, and can also block the early stage of the virus and late replication steps, and its half-maximal inhibitory concentration on DENV-1 and DENV-2 are both less than 10 μM, so in addition to reducing the half-maximal inhibitory concentration, it can also improve Therapeutic Index (TI). Incidentally, the therapeutic index is the ratio of the half-toxic dose to the half-maximal inhibitory concentration. Therefore, the cystein derivatives of the present invention have the effect of preventing, treating and improving dengue virus infection, and are suitable as medicines for preventing, treating and improving dengue virus, or together with pharmaceutically acceptable excipients Or the carrier can be used as a pharmaceutical composition that can prevent, treat and improve dengue virus disease, and can further expand the use as an anti-dengue virus drug, which has deep potential in the biomedical market.

The present invention has been disclosed as above in embodiments, but it is not intended to limit the present invention. Anyone who is familiar with the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is The scope of the patent application attached shall prevail.

Claims (8)

A use of a cyphine derivative, which is used to prepare a medicine for preventing, treating or improving dengue virus infection; wherein the cyphine derivative has a structure as shown in formula (I):

Wherein in the formula (I), R ₁ is a methyl group, a group shown in formula (MAI) or a group shown in formula (MAII), R ₂ is a hydrogen atom or a sulfur atom, and R ₃ is A hydrogen atom or a sulfur atom, R ₄ is a hydrogen atom, a nitro group, a group shown in formula (MBI) or a group shown in formula (MBII), R ₅ is an oxygen atom or a sulfur atom,

Represent single or double bonds to satisfy all valences:

The use of the cystein derivative according to claim 1, wherein the cystein derivative has formula (IA), formula (IB), formula (IC), formula (ID), formula (IE) , one of the structures shown in formula (IF), formula (IG), formula (IH), formula (II) or formula (IJ):

The use of cystein derivatives according to claim 1, wherein an effective dose of the drugs for preventing, treating or ameliorating dengue virus infection is 0.1 μM to 10 μM. The use of cystein derivatives according to claim 3, wherein the effective dose is 10 μM. The use of cystein derivatives according to claim 1, wherein the medicine for preventing, treating or ameliorating dengue virus infection further comprises a pharmaceutically acceptable excipient or carrier. The use of a cystein derivative according to claim 1, wherein the dengue virus infection is caused by type 1 dengue virus or type 2 dengue virus. The use of the cystein derivatives according to claim 1, wherein the drug for preventing, treating or improving dengue virus infection is a drug for reducing the infectivity of dengue virus to cells. The use of cystein derivatives as claimed in claim 1, wherein the drug for preventing, treating or improving dengue virus infection is a drug for inhibiting early and late replication of dengue virus.

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