A 4H-benzo[b]pyran derivative B63 and its application in resisting largemouth bass frog iridescent virus

Abstract

This invention belongs to the field of new drugs, specifically relating to a pyran derivative, B63, and its use in the preparation of drugs for use against largemouth bass frog iridescence virus. The invention provides the use of B63, its derivatives, or pharmaceutically acceptable salts thereof, or substances containing B63, its derivatives, or pharmaceutically acceptable salts as active ingredients, in the preparation of products for use against largemouth bass frog iridescence virus. B63 exhibits both in vitro and in vivo anti-largemouth bass frog iridescence virus activity and is a potential drug candidate for use against largemouth bass frog iridescence virus.

Description

Technical Field

[0001] The present invention belongs to the field of new drugs, and specifically relates to a 4H-benzo[b]pyran derivative B63 and its application in resisting largemouth bass frog iridescent virus. Background Art

[0002] Largemouth bass (Micropterus salmoides), commonly known as California bass, is native to the Mississippi River basin in North America. A eurythermal fish, it is favored by consumers for its fast growth, low disease burden, cold tolerance, delicious meat, and ease of harvest. However, in recent years, with increasing stocking densities and deteriorating aquaculture environments, disease outbreaks have become increasingly severe, severely impacting the development of the aquaculture industry. Among the most damaging diseases during the aquaculture phase is ulcer syndrome, caused by largemouth bass ranavirus (LMBRaV). LMBRaV can cause massive mortality in largemouth bass during aquaculture, with mortality rates exceeding 70%. The prevalence of this disease poses a significant threat to largemouth bass aquaculture in my country. Currently, no effective medication, vaccination, or treatment exists to control the disease. Among them, drug prevention and control is a common and effective means in freshwater fish seed cultivation, but there are very few antiviral drugs available in fishery production, and human antiviral raw materials are prohibited from use in aquaculture. Therefore, it is of great significance to develop healthy, efficient and green drugs to prevent and control LMBRaV and other iridovirus infections in aquatic animals.

[0003] Pyran is a fully unsaturated six-membered heterocyclic compound containing one oxygen atom. It has two double bonds and can form two isomers, 2H-pyran and 4H-pyran, depending on the position of the double bonds. Pyran is not a closed conjugated system, but its quaternary pyridinium salt analog, the pyrylium salt, is a closed conjugated system with a certain degree of aromaticity and stability. Unsubstituted pyran has not been found in nature and is of little value on its own. However, pyran derivatives, particularly pyrone, are widely present in many natural substances. There are two types of saturated pyran derivatives: α-pyran, also known as 2H-pyran, and γ-pyran, also known as 4H-pyran. α-pyran has never been synthesized. γ-pyran can be obtained by cyclization of glutaraldehyde in a solution of hydrogen chloride and dichloromethane, followed by dehydrochlorination with N,N-diethylaniline at 90°C. Neither of these pyran nuclei is found in nature; all naturally occurring substances are derivatives of them.

[0004] 4H-benzo[b]pyran compounds are a very important class of organic heterocyclic compounds with significant biological activity and extensive medicinal value. For example, they have diuretic and antispasmodic effects, can be used as anticoagulants, and have certain therapeutic effects on cancer. In addition, they can also be used as cognitive enhancers to treat neurodegenerative diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Parkinson's disease, etc. Currently, the synthesis of these compounds is mainly through a three-component "one-pot" reaction synthesis of aldehydes, active methylene compounds, and 1,3-cyclohexanedione compounds. Over the years, people have synthesized many high-efficiency, low-toxic medicinal compounds through various methods. A study published in the journal Nature showed that a 4H-benzo[b]pyran compound called "ebselen" can effectively inhibit the replication of the new coronavirus. In addition, studies have shown that certain 4H-benzo[b]pyran compounds also have inhibitory effects on other viruses such as hepatitis B virus and HIV virus. At the same time, compounds containing 4H-benzo[b]pyrans generally exhibit high efficacy, low toxicity, safety against non-target organisms, easy degradation in the environment, and resistance to pest resistance. Furthermore, compounds with novel structures and excellent performance are constantly emerging. Therefore, in the research and development of antiviral drugs, compounds containing 4H-benzo[b]pyrans will receive more extensive attention and become a hot spot and frontier for new antiviral drug innovation.

[0005] So far, there has been no report on the research of the newly synthesized 4H-benzo[b]pyran compounds of the present invention as aquatic antiviral drugs. Summary of the Invention

[0006] The present invention provides a 4H-benzo[b]pyran derivative B63, which has significant protective and therapeutic effects on sea bass infected with largemouth bass frog iridescence virus. The structural formula of B63 is:

[0007]

[0008] Another object of the present invention is to provide the use of 4H-benzo[b]pyran derivative B63 in the preparation of a drug for treating or preventing largemouth bass frog iridescent virus infection.

[0009] In order to achieve the above object, the present invention adopts the following technical measures:

[0010] A 4H-benzo[b]pyran derivative B63, molecular formula C 18 H 17 N3O4, molecular weight 339.35, structural formula:

[0011]

[0012] The preparation method of the 4H-benzo[b]pyran derivative B62 comprises the following steps:

[0013] After mixing 5,5-dimethyl-1,3-cyclohexanedione, p-nitrobenzaldehyde 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 by silica gel column chromatography to obtain the target product B62.

[0014] Preferably, the above-described step is performed by mixing 1.5-2.5 mmol of 5,5-dimethyl-1,3-cyclohexanedione, 1.5-2.5 mmol of p-nitrobenzaldehyde, and 1.5-2.5 mmol of malononitrile, then adding 65-75 μL of triethylamine as a catalyst and 8-12 ml of ethanol as a solvent. The reaction 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.

[0015] The protection scope of the present invention also includes:

[0016] A combination preparation containing 4H-benzo[b]pyran derivative B63.

[0017] Application of 4H-benzo[b]pyran derivative B63, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B63 in the preparation of drugs for treating or preventing largemouth bass frog iridovirus disease.

[0018] Application of 4H-benzo[b]pyran derivative B63, a pharmaceutically acceptable salt thereof, or a compound preparation containing 4H-benzo[b]pyran derivative B63 in the preparation of a drug for treating or preventing largemouth bass frog iridescent virus infection.

[0019] Application of 4H-benzo[b]pyran derivative B63, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B63 in the preparation of largemouth bass frog iridescent virus inhibitors.

[0020] Application of 4H-benzo[b]pyran derivative B63, a pharmaceutically acceptable salt thereof or a composite preparation containing 4H-benzo[b]pyran derivative B63 in the preparation of a largemouth bass feed additive.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] In the antiviral activity experiment of the 4H-benzo[b]pyran derivative B63 provided by the present invention, it was found that the structural compound has a high anti-largemouth bass frog iridescent virus effect and has little toxicity to normal cells. B63 is a new compound with very few side effects and has anti-largemouth bass frog iridescent virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a synthetic route for 4H-benzo[b]pyran derivative B63.

[0024] Figure 2 This is the synthetic route of the reference compound 4H-benzo[b]pyran derivative B64.

[0025] Figure 3 To test the maximum safe concentration of different drugs;

[0026] In the figures, cell survival rate is expressed as the percentage of cell viability in the control group (*: P < 0.05; **: P < 0.01).

[0027] Figure 4 In vitro anti-LMBRaV activity of different drugs (*: P<0.05; **: P<0.01).

[0028] Figure 5 This is a time-of-addition assay experiment for the antiviral effects of different drugs;

[0029] The figure shows the viral load test results after drug treatment at different times (*: P < 0.05; **: P < 0.01).

[0030] Figure 6 This is a test on the direct killing of viruses by different drugs (*: P<0.05; **: P<0.01).

[0031] Figure 7 The effects of different drugs in inhibiting LMBRaV replication in largemouth bass;

[0032] Figures A and B show the effects of B63, B64, and 4H-benzo[b]pyran on viral loads in the spleen and kidney of largemouth bass 3, 6, and 9 days after infection (*: P < 0.05; **: P < 0.01). DETAILED DESCRIPTION

[0033] 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.

[0034] 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.

[0035] The largemouth bass frog iridovirus used in this study was obtained from the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. A carp epithelial cell line (EPC) sensitive to largemouth bass frog iridovirus 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 EPC cells, and the TCID was calculated using the Reed-Muench method. 50 is 10 7.5 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. Largemouth bass were purchased from a farm with no record of LMBRaV detection in the past five years, and the fish were tested to confirm the absence of LMBRaV. 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.

[0036] Example 1:

[0037] The preparation method of 4H-benzo[b]pyran derivative B63 comprises the following steps:

[0038] To a 25 mL round-bottom flask, 283 mg (2 mmol) of 5,5-dimethyl-1,3-cyclohexanedione (CAS No.: 126-81-8), 302 mg (2 mmol) of p-nitrobenzaldehyde (CAS No.: 555-16-8), and 133 mg (2 mmol) of malononitrile (CAS No.: 109-77-3) were added. 70 μL of triethylamine (CAS No.: 121-44-8) was added as a catalyst, and 10 ml of ethanol (CAS No.: 64-17-5) was added 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 B63 structure diagram are shown in Figure 1 .

[0039] Preparation method of 4H-benzo[b]pyran derivative B64 (as a control group of 4H-benzo[b]pyran derivative B63):

[0040] 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 .

[0041] Example 2:

[0042] Detection of the maximum safe concentration of different drugs on cells:

[0043] EPC cells in good growth condition were taken and digested with trypsin. Then, EPC cells were seeded into 96-well cell culture plates with M199 cell culture medium containing 10% fetal bovine serum. Each well had 1×10 cells. 4 After culturing the 96-well plate in a 25°C incubator for 24 hours, fresh culture medium containing B63, B64 or 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. 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, EPC cytotoxicity test was performed according to the recommended steps in the instructions of the Cellcountingkit-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 was > 80%, which was selected as the maximum safe concentration of the drug for subsequent experiments. The maximum safe concentrations of B63, B64 and 4H-benzo[b]pyran were determined by CCK-8 method to be 80 mg / L, 80 mg / L and 60 mg / L, respectively. Figure 3 ).

[0044] Example 3:

[0045] Inhibitory effects of different drugs on LMBRaV in EPC cells

[0046] 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 28°C for 2 hours, cells were treated with B63 (80 mg / L), B64 (80 mg / L) or 4H-benzo[b]pyran (60 mg / L). Three replicates were set up in each group. After 48 hours of culture in a 28°C incubator, cells were collected and the intracellular viral load of LMBRaV after B63 treatment was detected (see Example 7). B63 can significantly inhibit LMBRaV replication in EPC cells within a safe concentration range, with a maximum inhibition rate of 70.52±0.32%, respectively, while B64 and 4H-benzo[b]pyran had no significant inhibitory effect ( Figure 4 ).

[0047] Inhibition rate = (viral load of the control group - viral load of the experimental group) / viral load of the control group, the same below.

[0048] Example 4:

[0049] Time-of-addition assay of different antiviral drugs

[0050] EPC 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, during, or after infection with LMBRaV, cells were treated with different drugs. 10 3 TCLD 50 Virus infection was performed. The LMBRaV infection time was set to 0h, and the cells were treated with B63 (80mg / L), B64 (80mg / L) or 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 RNA 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 B63 can significantly inhibit LMBRaV infection, and the viral load was reduced by 81.96% at -12 hours and by 50.85% at -6 hours ( Figure 5 ). B63 infection and post-treatment also significantly inhibited viral load ( Figure 5 ), particularly at 2, 4, 6, and 8 hours after infection. Thus, B63 inhibited LMBRaV infection before, during, and after infection. B64 and 4H-benzo[b]pyran had no significant inhibitory effect.

[0051] Example 5:

[0052] Test on direct virus killing by different drugs:

[0053] Well-grown EPC cells were seeded into 12-well plates, and LMBRaV (10 3 TCLD 50 ) were incubated with B63 (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 the cells for 2 h. After washing with PBS, the cells were replaced with cell maintenance medium. Three parallels were set up in each group, and the 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 was incubated with B63 for 30, 60, and 90 min, the viral load was significantly reduced ( Figure 6 ), the highest inhibition rate reached 53.84%. B64 and 4H-benzo[b]pyran had no obvious inhibitory effect.

[0054] Example 6:

[0055] Experiment on the inhibition of LMBRaV replication in largemouth bass by different drugs

[0056] To determine the antiviral effect of the drug in largemouth bass, 150 healthy largemouth bass with an average body length of 8.5±0.6 cm were acclimated to the laboratory environment at 25℃ for 2 weeks and fed dry pellet feed ad libitum before the experiment. The 150 healthy largemouth bass were randomly divided into 5 groups, 30 in each group: DMSO Group, B63+LMBRaV group (corresponding Figure 7 B63 in), B64+LMBRaV group (corresponding to Figure 7 B64) and 4H-benzo[b]pyran + LMBRaV group (corresponding to Figure 7 4H-benzo[b]pyran) was injected intraperitoneally with 15 μL (10 3 TCLD 50 )LMBRaV virus solution, to the control group DMSO The same volume of PBS was injected into the control group. 12 hours later, the largemouth bass in the B63+LMBRaV group, the B64+LMBRaV group, and the 4H-benzo[b]pyran+LMBRaV group were fed with feed containing B63 (80 mg / kg), B64 (80 mg / kg), and 4H-benzo[b]pyran (60 mg / kg). DMSO Group and LMBRaV DMSO The groups were fed with the same volume of DMSO-containing feed.

[0057] To evaluate the viral load of largemouth bass, three largemouth bass were collected from each group 3, 6, and 9 days after treatment with B63, B64, 4H-benzo[b]pyran, or 0.02% DMSO, and their spleens and kidneys were dissected and DNA was extracted. The LMBRaV viral load was detected by RT-qPCR (see Example 7). On the 3rd day after LMBRaV infection, B63 significantly reduced the LMBRaV load in the kidneys. On the 6th day after LMBRaV infection, B63 significantly reduced the LMBRaV load in the spleen and kidneys. On the 9th day after infection, B63 significantly reduced the LMBRaV load in the spleen and kidneys ( Figure 7 China A and Figure 7 Middle B). Control DMSO No LMBRaV was detected in the spleen and kidney of largemouth bass in the control group. Furthermore, B64 and 4H-benzo[b]pyran had no significant inhibitory effect on the virus in the kidney and spleen. These results suggest that B63 can inhibit the proliferation of LMBRaV in largemouth bass.

[0058] Example 7:

[0059] LMBRaV viral load determination

[0060] 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 The relative expression levels of target genes were calculated.

[0061] Table 1 Primer sequences

[0062]

[0063] Note: β-actin is the internal reference gene, and LMBRaV-MCP-F / R are primers used to detect the relative expression level of the LMBRaV virus MCP gene by RT-qPCR.

[0064] 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 B63, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B63 in the preparation of a drug for treating or preventing largemouth bass frog iridovirus disease, wherein the 4H-benzo[b]pyran derivative B63 has a molecular formula of C 18 H 17 N3O4, molecular weight 339.35, structural formula: 。 2. Use of 4H-benzo[b]pyran derivative B63, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B63 in the preparation of a drug for treating or preventing largemouth bass frog iridescent virus infection, wherein the 4H-benzo[b]pyran derivative B63 has a molecular formula of C 18 H 17 N3O4, molecular weight 339.35, structural formula: 。 3. Use of 4H-benzo[b]pyran derivative B63, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B63 in the preparation of largemouth bass frog iridescent virus inhibitors, wherein the 4H-benzo[b]pyran derivative B63 has a molecular formula of C 18 H 17 N3O4, molecular weight 339.35, structural formula: 。 4. Use of 4H-benzo[b]pyran derivative B63, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B63 in the preparation of a feed additive for largemouth bass, wherein the 4H-benzo[b]pyran derivative B63 has a molecular formula of C 18 H 17 N3O4, molecular weight 339.35, structural formula: 。

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