A 4H-benzo[b]pyran derivative B77 and its application in anti-cyprinid herpesvirus type Ⅱ

Abstract

The present invention belongs to the field of new drugs and specifically relates to a 4H-benzo[b]pyran derivative B77 and its use in treating carp herpesvirus type II. The present invention provides the use of a substance comprising B77 or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a product for treating carp herpesvirus type II. B77 exhibits anti-carp herpesvirus type II activity both in vivo and in vitro, making it a potential candidate drug for treating carp herpesvirus type II.

Description

Technical Field

[0001] The present invention belongs to the field of new drugs, and specifically relates to a 4H-benzo[b]pyran derivative B77 and its application in resisting carp herpesvirus type II. Background Art

[0002] Hematopoietic necrosis of crucian carp (CR), caused by Cyprinid herpesvirus 2 (CyHV-2), is an epidemic disease of fish, causing significant economic losses to the crucian carp and goldfish aquaculture industries. CyHV-2 is highly contagious, with mortality rates reaching 100%. The virus can infect fish at all stages of growth, with juveniles being more susceptible and potentially fatal within one to two days of infection. CyHV-2 outbreaks typically occur in spring and autumn when water temperatures range from 15-25°C. CyHV-2 can be transmitted both vertically and horizontally: it can be detected in eggs or hatchlings of diseased fish, as well as in healthy fish co-housed with diseased fish. CyHV-2 shares the characteristics of latent infection with other herpes viruses, lacking obvious clinical symptoms during its incubation period but maintaining a certain level of infectiousness. Currently, severe CyHV-2 outbreaks have occurred in many countries, causing significant economic losses to the crucian carp aquaculture industry. However, there is currently no effective treatment, and prevention and control efforts primarily rely on prevention.

[0003] Benzopyran compounds are widely present in natural products and bioactive drug molecules and are important components of many natural substances, such as coumarins, flavans, flavonoids, alkaloids and pterostilbene. 4H-Benzopyran compounds have a variety of biological activities, such as antioxidant, antibacterial, antifungal, antiviral, and antitumor. 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); 2-amino-4-(3-trifluoromethylphenyl)-3-cyano-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzopyran has anti-Hantavirus activity. The newly synthesized B77 of the present invention is a member of the 4H-benzo[b]pyran compound class and has the function of resisting carp herpesvirus type II. Summary of the Invention

[0004] The object of the present invention is to provide a 4H-benzo[b]pyran derivative B77, the structural formula of B77 is:

[0005]

[0006] Another object of the present invention is to provide the use of 4H-benzo[b]pyran derivative B77 in the preparation of drugs for treating or preventing carp herpesvirus type II infection.

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

[0008] A 4H-benzo[b]pyran derivative B77, molecular formula C 19 H 20 N2O3, molecular weight 324.38, structural formula:

[0009] The preparation method of the 4H-benzo[b]pyran derivative B77 comprises the following steps:

[0010] After mixing 5,5-dimethyl-1,3-cyclohexanedione, m-methoxybenzaldehyde and malononitrile, triethylamine was added as a catalyst and ethanol was used as a solvent, and the mixture was stirred at room temperature. After the reaction was completed, the mixture was extracted and separated using silica gel column chromatography to obtain the target product B77.

[0011] Preferably, the above-described steps are performed by mixing 1.5-2.5 mmol of 5,5-dimethyl-1,3-cyclohexanedione, 1.5-2.5 mmol of m-anisaldehyde, and 1.5-2.5 mmol of malononitrile, then adding triethylamine (65-75 μL) as a catalyst and ethanol (8-12 ml) as a solvent. The mixture is stirred at room temperature (23-27°C) for 22-26 hours. After completion of the reaction, the mixture is filtered and the filter cake is washed with a small amount of ethanol to obtain the desired product.

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

[0013] A combination preparation containing 4H-benzo[b]pyran derivative B77.

[0014] Application of 4H-benzo[b]pyran derivative B77, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B77 in the preparation of drugs for treating or preventing hematopoietic organ necrosis in crucian carp.

[0015] Application of 4H-benzo[b]pyran derivative B77, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B77 in the preparation of drugs for treating or preventing cyprinid herpesvirus type II infection.

[0016] Application of 4H-benzo[b]pyran derivative B77, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B77 in the preparation of cyprinid herpesvirus type II inhibitors.

[0017] Application of 4H-benzo[b]pyran derivative B77, pharmaceutically acceptable salts thereof or composite preparations containing 4H-benzo[b]pyran derivative B77 in the preparation of crucian carp feed additives.

[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 B77 provided by the present invention, it was found that the structural compound has a high anti-cyprinid herpesvirus type II effect and has little toxicity to normal cells. B77 is a new compound with very little side effects and anti-cyprinid herpesvirus type II. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a synthetic route to 4H-benzo[b]pyran derivative B77.

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

[0024] Figure 4 The in vitro anti-CyHV-2 activity of different drugs;

[0025] Effect of B77 on the expression rate of viral proteins in GiCB cells infected with CyHV-2 (*P<0.05; **P<0.01).

[0026] Figure 5 This is a time-of-addition assay for the antiviral effects of different drugs;

[0027] The figure shows the viral load test results after drug treatment at different times (*P<0.05; **P<0.01).

[0028] Figure 6 The direct virus killing effects of different drugs (*P<0.05; **: P<0.01).

[0029] Figure 7 This is an experiment on the inhibition of CyHV-2 replication in crucian carp by different drugs (*P<0.05; **P<0.01). DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0031] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0032] The cyprinid herpesvirus type II used in this study was obtained from the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The Gibel carp brain cell line (GiCB), which is sensitive to cyprinid herpesvirus type II, was cultured in M199 supplemented with 10% fetal bovine serum. The culture medium used for cytotoxicity and antiviral testing contained 5% serum. The virus was propagated in GiCB cells, and the TCID was calculated using the Reed-Muench method. 50 is 10 7.2 The drug was prepared in DMSO (DMSO) as a stock solution at a concentration of 50 mg / mL and stored at -80°C until use. Crucian carp were purchased from a farm with no record of CyHV-2 detection in the past five years, and the fish were tested to confirm the absence of CyHV-2. 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 institutional review committee.

[0033] Example 1:

[0034] The preparation method of 4H-benzo[b]pyran derivative B77 comprises the following steps:

[0035] To a 25 mL round-bottom flask, 283 mg (2 mmol) of 5,5-dimethyl-1,3-cyclohexanedione (CAS No.: 126-81-8), 272 mg (2 mmol) of m-anisaldehyde (CAS No.: 591-31-1), and 133 mg (2 mmol) of malononitrile (CAS No.: 109-77-3) were added, followed by 70 μL of triethylamine (CAS No.: 121-44-8) as a catalyst and 10 ml of ethanol (CAS No.: 64-17-5) as a solvent. The reaction was stirred at room temperature for 24 h, 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 synthetic route and B77 structure diagram are shown in Figure 1 .

[0036] Preparation method of 4H-benzo[b]pyran derivative B64 (as a control group of 4H-benzo[b]pyran derivative B77):

[0037] To a 25 mL round-bottom flask, 283 mg (2 mmol) of 5,5-dimethyl-1,3-cyclohexanedione (CAS No. 126-81-8), 244 mg (2 mmol) of p-hydroxybenzaldehyde (CAS No. 123-08-0), and 133 mg (2 mmol) of malononitrile (CAS No. 109-77-3) were added, followed by 70 μL of triethylamine (CAS No. 121-44-8) as a catalyst and 10 ml of ethanol (CAS No. 64-17-5) as a solvent. The reaction was stirred at room temperature for 24 h, 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 synthetic route and B64 structure diagram are shown in Figure 2 .

[0038] Example 2:

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

[0040] The GiCB cells that were in good growth condition were taken and digested with trypsin. Then, the GiCB cells were seeded into 96-well cell culture plates with M199 cell culture medium containing 10% fetal bovine serum. The number of cells per well was 1×10 4 After culturing the 96-well plate in a 28°C incubator for 24 hours, fresh culture medium containing B77, B64 and 4H-benzo[b]pyran was added, and 6 concentration gradients were set, namely 60 mg / L, 50 mg / L, 40 mg / L, 30 mg / L, 20 mg / L, and 10 mg / L. At the same time, a control group without drug was set up, with 3 parallels in each group. After culturing at 28°C for 48 hours, cytotoxicity detection was performed according to the steps recommended in the instructions of the Cellcounting 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%, and the cell survival rate>80%, it was selected as the maximum safe concentration of the drug for subsequent experiments.

[0041] The maximum safe concentrations of B77, B64 and 4H-benzo[b]pyran were determined to be 50 mg / L, 50 mg / L and 40 mg / L, respectively, using the CCK-8 method. Figure 3 ).

[0042] Example 3:

[0043] Inhibitory effects of different drugs on CyHV-2 in GiCB cells

[0044] GiCB 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 28 ° C for 2 hours, cells were treated with B77 (50 mg / L), B64 (50 mg / L) and 4H-benzo [b] pyran (40 mg / L), with 3 parallels per group. After culturing in a 28 ° C incubator for 48 hours, cells were collected and the intracellular viral load of CyHV-2 after drug treatment was detected (see Example 7). B77 can significantly inhibit the CyHV-2 load in GiCB cells within a safe concentration range, with a maximum inhibition rate of 63.98%, while B64 and 4H-benzo [b] pyran had no significant inhibitory effect on the virus ( Figure 4 ).

[0045] Inhibition rate = (viral load of the control group - viral load of the experimental group) / viral load of the control group, the same below.

[0046] Example 4:

[0047] Time-of-addition assay experiment of viral inhibition by different drugs

[0048] GiCB cells were seeded into 12-well plates at a density of 1 × 10 5 / well, cultured to a density of about 90% per well. Before infection with CyHV-2 or while infected with CyHV-2 or after infection with CyHV-2, cells were treated with B77 (50 mg / L), B64 (50 mg / L) and 4H-benzo[b]pyran (40 mg / L). 3 TCLD 50 Virus infection was performed. The CyHV-2 infection time was set to 0h, and the cells were treated with drugs at -12, -6, 0, 2, 4, 6, 8, 10, and 12h, respectively. Three parallels were set for each group, and the cells were collected at 48h. The viral load was detected by fluorescent quantitative PCR (see Example 7). At the same time, cells cultured in ordinary culture medium were set as the control group. Pretreatment with B77 significantly inhibited CyHV-2 infection, with the viral load reduced by 57.76% at -12 hours and by 33.95% at -6 hours ( Figure 5 ). B77 infection and post-treatment also significantly inhibited viral load ( Figure 5), especially 2 hours, 4 hours, 6 hours and 8 hours after infection. Therefore, B77 can inhibit CyHV-2 infection before, during and after CyHV-2 infection. B64 and 4H-benzo [b] pyrans have no obvious inhibitory effect on the virus.

[0049] Example 5

[0050] Different drugs directly kill viruses:

[0051] Well-grown GiCB cells were seeded into 12-well plates, and CyHV-2 (10 3 TCLD 50 ) were incubated with B77 (50 mg / L), B64 (50 mg / L) and 4H-benzo[b]pyran (40 mg / L) at room temperature for 0, 30, 60, and 90 min, and then incubated in cells for 2 h. After washing with PBS and changing to cell maintenance medium, 3 parallels were set for each group, and cells were collected after 48 h. Fluorescence quantitative PCR was used to detect viral load (see Example 7). After incubation of the virus with the drug for 30, 60, and 90 min, the viral load was significantly inhibited ( Figure 6 ), the highest inhibition rate reached 78.56%. B64 and 4H-benzo[b]pyran had no obvious killing effect on the virus.

[0052] Example 6

[0053] Experiment on the inhibition of viral replication by different drugs in crucian carp

[0054] In order to determine the antiviral effect of the drug in crucian carp, 250 healthy crucian carp with an average body length of 9.38±0.5cm were used, and they were acclimated to the laboratory environment at 25℃ for 2 weeks and fed dry pellet feed ad libitum before the experiment. The 250 healthy crucian carp were randomly divided into 5 groups, with 50 in each group: DMSO group, CyHV-2+B77 group (corresponding to Figure 7 B77 group), CyHV-2+B64 group (corresponding Figure 7 B64 group) and CyHV-2+4H-benzo[b]pyran group (corresponding to Figure 7 4H-benzo[b]pyran group) were injected intraperitoneally with 20 μL CyHV-2 virus solution, and the control group DMSO The same volume of PBS was injected into the control group. 12 hours later, the carp in the CyHV-2+B77 group, CyHV-2+B64 group, and CyHV-2+4H-benzo[b]pyran group were fed with feed containing B77 (50 mg / kg), B64 (50 mg / kg), and 4H-benzo[b]pyran (40 mg / kg). DMSO group and CyHV-2 DMSOThe rats in the two groups were fed with feed containing the same amount of DMSO.

[0055] In order to evaluate the viral load of crucian carp, 4, 7 and 10 days after using B77, B64 and 4H-benzo [b] pyran or DMSO treatment, 3 crucian carp were collected in each group, and their kidneys and spleens were dissected and DNA was extracted. CyHV-2 load was detected by RT-qPCR (see Example 7). At the 4th day after CyHV-2 infection, B77 significantly reduced the CyHV-2 load in spleen and kidney. At the 7th day after CyHV-2 infection, B77 significantly reduced the CyHV-2 load in spleen and kidney. And 10 days after infection, B77 significantly reduced the CyHV-2 load in spleen and kidney ( Figure 7 China A and Figure 7 In addition, B64 and 4H-benzo[b]pyran had no significant inhibitory effect on viral load in the kidney and spleen. These results indicate that B77 can inhibit the proliferation of CyHV-2 in crucian carp.

[0056] Example 7:

[0057] CyHV-2 viral load determination

[0058] Total DNA was extracted using a total DNA extraction kit (Yisheng, Shanghai, China). The obtained DNA was then used as a template and analyzed using Hieff UNICON qPCR was performed using the Universal Blue qPCR SYBR Green Master Mix Kit (RR420A) according to the manufacturer's instructions. The primers used for RT-qPCR are shown in Table 1. -ΔΔCT Methods Calculate the relative expression of target genes

[0059] Table 1. Primers used in RT-qPCR

[0060]

[0061] Note: β-actin is the internal reference gene, and CyHV-2-Fq / Rq are primers used to detect the relative expression of CyHV-2 viral proteins by RT-qPCR.

[0062] 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Use of 4H-benzo[b]pyran derivative B77, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B77 in the preparation of a drug for treating or preventing hematopoietic organ necrosis in crucian carp, wherein the 4H-benzo[b]pyran derivative B77 has a molecular formula of C 19 H 20 N2O3, molecular weight 324.38, structural formula: 。 2. Use of 4H-benzo[b]pyran derivative B77, its pharmaceutically acceptable salt or compound preparation containing 4H-benzo[b]pyran derivative B77 in the preparation of a drug for treating or preventing herpesvirus type II infection in cyprinids, wherein the 4H-benzo[b]pyran derivative B77 has a molecular formula of C 19 H 20 N2O3, molecular weight 324.38, structural formula: 。 3. Use of 4H-benzo[b]pyran derivative B77, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B77 in the preparation of cyprinid herpesvirus type II inhibitors, wherein the 4H-benzo[b]pyran derivative B77 has a molecular formula of C 19 H 20 N2O3, molecular weight 324.38, structural formula: 。 4. Use of 4H-benzo[b]pyran derivative B77, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B77 in the preparation of crucian carp feed additives, wherein the 4H-benzo[b]pyran derivative B77 has a molecular formula of C 19 H 20 N2O3, molecular weight 324.38, structural formula: 。

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