A 4H-benzo[b]pyran derivative B78 and its application in resisting Chinese soft-shelled turtle iris virus

Abstract

The present invention belongs to the field of new drugs, and specifically relates to a 4H-benzo[b]pyran derivative B78 and its application in resisting Chinese soft-shelled turtle iris virus. The present invention provides the application of B78 or a pharmaceutically acceptable salt thereof in preparing a product for resisting Chinese soft-shelled turtle iris virus. B78 has an anti-Chinese soft-shelled turtle iris virus effect both in vivo and in vitro, and is a potential candidate drug for resisting Chinese soft-shelled turtle iris virus.

Description

Technical Field

[0001] The invention belongs to the field of new drugs, and specifically relates to a 4H-benzo[b]pyran derivative B78 and its application in resisting Chinese soft-shelled turtle iris virus. Background Art

[0002] Chinese soft-shelled turtle not only has high nutritional value, but also has good cultural value and research value. It is deeply favored by consumers and has a strong market demand, which has promoted the sustainable development of its breeding industry. Chinese soft-shelled turtle virus (Soft-shelled turtle iridvirus, STIV) is an important viral pathogen that harms Chinese soft-shelled turtle. The virus particles are spherical. Proliferation in carp epithelioma cell (EPC) strains causes cytopathic changes, forming many small plaques, which gradually expand until all cells fall off; no inclusion bodies are produced. The virus liquid amplified by cultured cells and the virus liquid crudely extracted from sick turtles were injected into the abdominal cavity of healthy turtles through the base of the hind legs. Bleeding spots began to appear after 3-4 days, and death began to occur after 4-5 days, and all died around 15 days.

[0003] Benzopyran compounds are widely found in natural products and bioactive drug molecules, such as coumarins, flavanoids, flavonoids, alkaloids and pterostilbene. They have good photochemical stability and pharmacological activity and are widely used in anticoagulants, antitumor drugs, antiallergic drugs and other preparations. Studies have shown that the coumarin derivative 7,9-dihydroxy-3-(4,5,7-trihydroxy-2-oxo-2H-benzopyran-3-yl)-4H-furo[3,2-c]benzopyran-4-one (1) has antiviral activity against human immunodeficiency virus type 1 (HIV-1), while 2-amino-4-(3-trifluoromethylphenyl)-3-cyano-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzopyran has an antiviral effect against Hantaan virus. The newly synthesized B78 of the present invention is a member of the 4H-benzo[b]pyran compounds. It has never been reported in the prior art that the newly synthesized B78 has an effect of resisting Chinese soft-shelled turtle iris virus. Summary of the invention

[0004] The object of the present invention is to provide a 4H-benzo[b]pyran derivative B78, the structural formula of B78 is:

[0005]

[0006] Another object of the present invention is to provide the use of 4H-benzo[b]pyran derivative B78 in the preparation of a drug for treating or preventing Chinese soft-shell turtle iris virus infection.

[0007] In order to achieve the above object, the present invention adopts the following technical measures:

[0008] A 4H-benzo[b]pyran derivative B78, with the molecular formula C 19 H 17 F3N2O2, molecular weight 362.35, structural formula:

[0009] The preparation method of the 4H-benzo[b]pyran derivative B78 comprises the following steps:

[0010] After mixing 5,5-dimethyl-1,3-cyclohexanedione, p-trifluoromethylbenzaldehyde and malononitrile, triethylamine is added as a catalyst and ethanol is used as a solvent, and the mixture is stirred at room temperature. After the reaction is completed, extraction is performed and silica gel column chromatography is used for separation to obtain the target product B78.

[0011] In the above-mentioned steps, preferably, 1.5-2.5 mmol of 5,5-dimethyl-1,3-cyclohexanedione, 1.5-2.5 mmol of p-trifluoromethylbenzaldehyde and 1.5-2.5 mmol of malononitrile are mixed, and then triethylamine (65-75 μL) is added as a catalyst and ethanol (8-12 ml) is added as a solvent. The mixture is stirred at room temperature (23-27° C.) for 22-26 hours. After the reaction is completed, the mixture is filtered and the filter cake is washed with a small amount of ethanol to obtain the target product.

[0012] The protection scope of the present invention also includes:

[0013] A combination preparation containing 4H-benzo[b]pyran derivative B78.

[0014] Application of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a composite preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of drugs for treating or preventing Chinese soft-shell turtle iris virus disease.

[0015] Application of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a composite preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of a drug for treating or preventing Chinese soft-shell turtle iris virus infection.

[0016] Application of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a composite preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of Chinese soft-shelled turtle iris virus inhibitor.

[0017] Application of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a composite preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of Chinese soft-shelled turtle feed additive.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] In the antiviral activity experiment of the 4H-benzo[b]pyran derivative B78 provided by the present invention, it was found that the structural compound has a high anti-Chinese soft-shelled turtle iris virus effect and has little toxicity to normal cells. B78 is a new compound with very little side effects and anti-Chinese soft-shelled turtle iris virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a synthetic route for 4H-benzo[b]pyran derivative B78.

[0021] Figure 2 This is the synthetic route of the reference compound 4H-benzo[b]pyran derivative B64.

[0022] Figure 3 To test the maximum safe concentration of different drugs;

[0023] In the figure, the cell survival rate is expressed as the percentage of cell viability in the control group (*: P < 0.05; **: P < 0.01).

[0024] Figure 4 In vitro anti-STIV activity of different drugs (*: P<0.05; **: P<0.01).

[0025] Figure 5 It is a time-of-addition assay experiment for the antiviral time course of different drugs;

[0026] The figure shows the viral load test results after drug treatment at different times (*: P<0.05; **: P<0.01).

[0027] Figure 6 This is an experiment on the inhibitory effect of different drugs on virus adsorption (*: P<0.05; **: P<0.01).

[0028] Figure 7 To test the inhibition of STIV replication by different drugs in Chinese soft-shell turtle;

[0029] Figures A and B show the effects of B78, B64 and 4H-benzo[b]pyran on the viral load in the kidney and spleen of Chinese soft-shelled turtle 4, 7 and 10 days after infection (*: P<0.05; **: P<0.01). DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0031] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0032] The Chinese soft-shell turtle iridovirus used in the present invention comes from the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences; the blackhead soft-shelled minnow epithelioma cell line (EPC) sensitive to the Chinese soft-shell turtle iridovirus is cultured in M199 supplemented with 10% fetal bovine serum. The culture medium used for cytotoxicity and antiviral tests contains 5% serum. The virus is propagated in EPC cells, and the TCID is calculated using the Reed-Muench method. 50 For 10 7.5 / mL and stored at -80°C until use. The drug was prepared with DMSO organic solvent to a stock solution concentration of 50 mg / mL. Chinese soft-shell turtles were purchased from a farm with no record of STIV detection in the past 5 years, and the fish were tested to confirm the absence of STIV. All animal experiments were conducted at the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, in full compliance with the guidelines of the Ethical Review Committee.

[0033] Embodiment 1:

[0034] The preparation method of 4H-benzo[b]pyran derivative B78 comprises the following steps:

[0035] 283 mg (2 mmol) 5,5-dimethyl-1,3-cyclohexanedione (CAS No.: 126-81-8), 348 mg (2 mmol) p-trifluoromethylbenzaldehyde (CAS No.: 455-19-6) and 133 mg (2 mmol) of malononitrile (CAS No.: 109-77-3) were added to a 25 mL round-bottom flask, and then 70 μL triethylamine (CAS No.: 121-44-8) was added as a catalyst and 10 ml ethanol (CAS No.: 64-17-5) was added as a solvent. The reaction was stirred at room temperature for 24 hours, and the reaction progress (V 丙酮 :V 石油醚 =1:1). After the reaction is completed, filter and wash the filter cake with a small amount of ethanol to obtain the target product. The synthetic route and B78 structure diagram are shown in Figure 1 .

[0036] The preparation method of 4H-benzo[b]pyran derivative B64 (as a control group of 4H-benzo[b]pyran derivative B78) comprises the following steps:

[0037] 283 mg (2 mmol) 5,5-dimethyl-1,3-cyclohexanedione (CAS No.: 126-81-8), 244 mg (2 mmol) p-hydroxybenzaldehyde (CAS No.: 123-08-0) and 133 mg (2 mmol) malononitrile (CAS No.: 109-77-3) were added to a 25 mL round-bottom flask, and then 70 μL triethylamine (CAS No.: 121-44-8) was added as a catalyst and 10 ml ethanol (CAS No.: 64-17-5) was added as a solvent. The reaction was stirred at room temperature for 24 hours, and the reaction progress was monitored by TLC (V 丙酮 :V 石油醚 =1:1). After the reaction is completed, filter and wash the filter cake with a small amount of ethanol to obtain the target product. The synthesis route and B64 structure diagram are shown in Figure 2 .

[0038] Embodiment 2:

[0039] Detection of the maximum safe concentration of different drugs on cells:

[0040] EPC cells in good growth state were taken, digested with trypsin, and then inoculated into 96-well cell culture plates with M199 cell culture medium containing 10% fetal bovine serum, with 1×10 cells per well. 4 After culturing the 96-well plate in a 25°C incubator for 24 hours, fresh culture medium containing B78 and 4H-benzo[b]pyran was added, and 6 concentration gradients were set, namely 100 mg / L, 80 mg / L, 60 mg / L, 40 mg / L, 20 mg / L, and 10 mg / L. A control group without drug was set up at the same time, and 3 parallels were set up in each group. After culturing at 28°C for 48 hours, EPC cytotoxicity detection was performed according to the recommended steps in the instructions of the Cell counting kit-8 kit (CCK-8, C0038, Beyotime, China). The cell survival rate was calculated as [(OD 450 -Blank control OD 450 ) / (control cell OD 450 -Blank control OD 450 )] × 100%, cell survival rate> 80%, then it was selected as the maximum safe concentration of the drug for subsequent experiments. The maximum safe concentrations of B78, B64 and 4H-benzo[b]pyran were determined by CCK-8 method to be 80 mg / L, 80 mg / L and 60 mg / L ( Figure 3 ).

[0041] Embodiment 3:

[0042] Inhibitory effects of different drugs on STIV in EPC cells

[0043] EPC cells were seeded into 12-well plates at a density of 1 × 10 5 / well, culture to a density of about 90% per well. 3 TCLD 50 ) After infection at 25°C for 2h, cells were treated with B78 (80 mg / L), B64 (80 mg / L) and 4H-benzo[b]pyran (60 mg / L), with 3 parallels per group. After culturing in a 28°C incubator for 48h, cells were collected and the intracellular viral load of STIV after B78 treatment was detected (see Example 7). B78 can significantly inhibit STIV infection in EPC cells, with the maximum inhibition rates of 70.43±3.48%, respectively, while B64 and 4H-benzo[b]pyran were not as effective as B78 ( Figure 4 ).

[0044] Inhibition rate = (viral load of the control group - viral load of the experimental group) / viral load of the control group, the same below.

[0045] Embodiment 4:

[0046] Time-of-addition assay experiment of different drugs' antiviral effects

[0047] EPC cells were seeded into 12-well plates at a density of 1 × 10 5 / well, and cultured to a density of about 90% per well. Before, during, or after STIV infection, cells were treated with B78 (80 mg / L), B64 (80 mg / L), and 4H-benzo[b]pyran (60 mg / L). 10% of STIV was used. 3 TCLD 50 Virus infection was performed. The STIV infection time was set at 0h, and the cells were treated with B78 (80mg / L), B64 (80mg / L) and 4H-benzo[b]pyran (60mg / L) at -12, -6, 0, 2, 4, 6, 8, 10, and 12h, respectively. Three parallels were set up in each group. The cells were collected after 48h, and the total cell DNA was extracted to detect the viral load. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the viral load (see Example 7). At the same time, cells cultured in ordinary culture medium were set as the control group. Pretreatment with B78 can significantly inhibit STIV infection, and the viral load was reduced by 35.76% (-12 hours). Figure 4 ). B78 infection and post-treatment also significantly inhibited viral load ( Figure 5 ), especially at 2 hours and 4 hours after infection. Therefore, B78 can inhibit STIV infection before, during and after STIV infection. B64 and 4H-benzo[b]pyran are not as effective as B78 in inhibiting the virus.

[0048] Embodiment 5:

[0049] Experiment on direct virus killing by different drugs

[0050] Well-growing EPC cells were seeded into 12-well plates and STIV (10 3 TCLD 50 ) were incubated with B78 (80 mg / L), B64 (80 mg / L) or 4H-benzo[b]pyran (60 mg / L) at room temperature for 0, 30, 60, 90 min, and then incubated in cells for 2 h. After washing with PBS, the cells were replaced with cell maintenance medium. Three parallels were set for each group, and cells were collected after 48 h. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the viral load (see Example 7). After the virus and drug were incubated for 30, 60, and 90 min, the viral load was significantly reduced ( Figure 6 ), the highest inhibition rate reached 54.54%. B64 and 4H-benzo[b]pyran had no obvious inhibitory effect.

[0051] Embodiment 6:

[0052] Experiment on the inhibition of STIV replication in Chinese soft-shelled turtle by different drugs

[0053] To determine the antiviral effect of B78 in Chinese soft-shelled turtles, 150 healthy Chinese soft-shelled turtles with an average body weight of 50 ± 2.5 g were acclimatized to the laboratory environment at 25 °C for 2 weeks. The 250 healthy Chinese soft-shelled turtles were randomly divided into 5 groups, 50 in each group: DMSO Group, STIV+B78 group (corresponding to Figure 7 B78 group), STIV+B64 group (corresponding to Figure 7 B64 group) and STIV+4H-benzo[b]pyran group (corresponding to Figure 7 4H-benzo[b]pyran group) were intraperitoneally injected with 15 μL STIV virus solution, and the control group DMSO The same volume of PBS was injected into the groups. 12 h later, the STIV+B78, STIV+B64, and STIV+4H-benzo[b]pyran groups were fed feeds containing B78 (80 mg / kg), B64 (80 mg / kg), and 4H-benzo[b]pyran (60 mg / kg). DMSO Group and STIV DMSO The mice in each group were fed with a diet containing the same amount of DMSO.

[0054] To evaluate the viral load of Chinese soft-shelled turtles, 3 Chinese soft-shelled turtles were collected from each group 4, 7, and 10 days after treatment with B78, B64\4H-benzo[b]pyran, or 0.12% DMSO, and their kidneys and spleens were dissected and DNA was extracted. The STIV load was detected by RT-qPCR (see Example 7). On the 4th day after STIV infection, B78 significantly reduced the STIV load in the spleen and kidneys. On the 7th day after STIV infection, B78 significantly reduced the STIV load in the spleen and kidneys. On the 10th day after infection, B78 significantly reduced the STIV load in the spleen and kidneys ( Figure 7 Middle A and Figure 7 B) Control DMSO No STIV was detected in the spleen and kidney of the Chinese soft-shelled turtle in the group. The above results indicate that B78 can inhibit the proliferation of STIV in Chinese soft-shelled turtle. The inhibitory effects of B64 and 4H-benzo[b]pyran on the viral load in the kidney and spleen are not as good as those of B78.

[0055] Embodiment 7:

[0056] STIV viral load determination

[0057] Total DNA was extracted using a total DNA extraction kit (Yisheng, Shanghai, China). The obtained DNA was then used as a template and purified using Hieff UNICON qPCR was performed using the Universal Blue qPCR SYBR Green Master Mix Kit (RR420A) according to the instructions. The primers used for RT-qPCR are shown in Table 1. -ΔΔCT Methods The relative expression of target genes was calculated.

[0058] Table 1. Primers used in RT-qPCR

[0059]

[0060] Note: β-actin is the internal reference gene, and STIV-Fq / Rq are primers used to detect the relative expression of STIV virus MCP protein by RT-qPCR.

[0061] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of a drug for treating or preventing Chinese soft-shell turtle iris virus disease, wherein the molecular formula of the 4H-benzo[b]pyran derivative B78 is C 19 H 17 F3N2O2, molecular weight 362.35, structural formula: 。 2. Use of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of a drug for treating or preventing Chinese soft-shell turtle iris virus infection, wherein the molecular formula of the 4H-benzo[b]pyran derivative B78 is C 19 H 17 F3N2O2, molecular weight 362.35, structural formula: 。 3. Use of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of a Chinese soft-shell turtle iris virus inhibitor, wherein the molecular formula of the 4H-benzo[b]pyran derivative B78 is C 19 H 17 F3N2O2, molecular weight 362.35, structural formula: 。 4. Use of 4H-benzo[b]pyran derivative B78, its pharmaceutically acceptable salt or a composite preparation containing 4H-benzo[b]pyran derivative B78 in the preparation of a Chinese soft-shell turtle feed additive, wherein the molecular formula of the 4H-benzo[b]pyran derivative B78 is C 19 H 17 F3N2O2, molecular weight 362.35, structural formula: 。

Images