A 4H-benzo[b]pyran derivative B62 and its application in resisting largemouth bass rhabdovirus

Abstract

The present invention belongs to the field of new drugs, specifically relating to a 4H-benzo[b]pyran derivative, B62, and its use in the preparation of drugs for treating largemouth bass rhabdoviruses. The present invention provides the use of B62, its derivatives, or pharmaceutically acceptable salts thereof, or substances containing B62, its derivatives, or pharmaceutically acceptable salts thereof as active ingredients, in the preparation of products for treating largemouth bass rhabdoviruses. B62 exhibits both in vitro and in vivo anti-largemouth bass rhabdovirus effects and is a potential drug candidate for treating largemouth bass rhabdoviruses.

Description

Technical Field

[0001] The present invention belongs to the field of new drugs, and specifically relates to a 4H-benzo[b]pyran derivative B62 and its application in resisting largemouth bass rhabdovirus. Background Art

[0002] Largemouth bass rhabdovirus disease is an epidemic disease of fish caused by the largemouth bass rhabdovirus (Micropterus salmoidesrhabdovirus, MSRV). Largemouth bass rhabdovirus disease mainly infects largemouth bass in the early growth and development stages and is a major cause of low survival rate of fry. Largemouth bass rhabdovirus disease outbreaks have a distinct seasonal pattern. The disease spreads rapidly, has a short incubation period, and has a high mortality rate, especially for largemouth bass fry, with an infection rate as high as 80%, and a mortality rate of over 90% within a short period of time after infection. The main route of infection is horizontal transmission through water bodies, and it can also be transmitted vertically from broodstock to fry. At present, the prevention and control of largemouth bass rhabdovirus disease is still in its infancy, and prevention and control measures are still mainly based on prevention.

[0003] Benzopyran compounds are widely present in natural products and bioactive drug molecules, such as coumarins, flavans, flavonoids, alkaloids, and pterostilbene. They have good photochemical stability and pharmacological activity and are widely used in preparations such as anticoagulants, antitumor drugs, and antiallergic drugs. 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 B62 of the present invention is a member of the 4H-benzo[b]pyran compound class and has the function of resisting largemouth bass rhabdovirus. Summary of the Invention

[0004] The object of the present invention is to provide a 4H-benzo[b]pyran derivative B62, the structural formula of B62 is:

[0005]

[0006] Another object of the present invention is to provide the use of 4H-benzo[b]pyran derivative B62 in the preparation of a drug for treating or preventing largemouth bass rhabdovirus infection.

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

[0008] A 4H-benzo[b]pyran derivative B62, molecular formula C 18 H 17 FN2O2, molecular weight 312.34, structural formula:

[0009] The preparation method of the 4H-benzo[b]pyran derivative B62 comprises the following steps:

[0010] After mixing 5,5-dimethyl-1,3-cyclohexanedione, p-fluorobenzaldehyde 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 B62.

[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 p-fluorobenzaldehyde, 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 B62.

[0014] Application of 4H-benzo[b]pyran derivative B62, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B62 in the preparation of drugs for treating or preventing largemouth bass rhabdovirus disease.

[0015] Application of 4H-benzo[b]pyran derivative B62, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B62 in the preparation of drugs for treating or preventing largemouth bass rhabdovirus infection.

[0016] Application of 4H-benzo[b]pyran derivative B62, a pharmaceutically acceptable salt thereof or a composite preparation containing 4H-benzo[b]pyran derivative B62 in the preparation of a largemouth bass feed additive.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] In the antiviral activity experiment of the 4H-benzo[b]pyran derivative B62 provided by the present invention, it was found that the structural compound has a high anti-largemouth bass rhabdovirus effect and has little toxicity to normal cells. B62 is a new compound with very few side effects and has anti-largemouth bass rhabdovirus effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a synthetic route for 4H-benzo[b]pyran derivative B62.

[0020] Figure 2 This is the synthetic route of the reference compound 4H-benzo[b]pyran derivative B64.

[0021] Figure 3 To test the maximum safe concentration of different drugs;

[0022] In the figures, cell survival rate is expressed as the percentage of cell viability in the control group (*: P < 0.05; **: P < 0.01).

[0023] Figure 4 The in vitro anti-MSRV activities of different drugs are shown in Table 1 (*: P<0.05; **: P<0.01).

[0024] Figure 5 A time-of-addition assay for the antiviral effects of different drugs;

[0025] *: P < 0.05; **: P < 0.01.

[0026] Figure 6 The direct virus killing effects of different drugs (*: P < 0.05; **: P < 0.01).

[0027] Figure 7 To investigate the effect of MSRV replication in largemouth bass with different drugs;

[0028] Figures A and B show the effects of B62, B64, and 4H-benzo[b]pyran on the relative expression levels of viral mRNA in the spleen and kidney of largemouth bass 3, 6, and 9 days after infection (*: P < 0.05; **: P < 0.01). DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] The largemouth bass rhabdovirus used in this study was obtained from the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. A common carp epithelial cell line (EPC) sensitive to largemouth bass rhabdovirus 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 MSRV detection in the past five years, and the fish were tested to confirm the absence of MSRV. 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.

[0032] Example 1:

[0033] The preparation method of 4H-benzo[b]pyran derivative B62 comprises the following steps:

[0034] To a 25 mL round-bottom flask, 283 mg (2 mmol) of 5,5-dimethyl-1,3-cyclohexanedione (CAS No.: 126-81-8), 248 mg (2 mmol) of p-fluorobenzaldehyde (CAS No.: 459-57-4), 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 B62 structure diagram are shown in Figure 1 .

[0035] Preparation method of 4H-benzo[b]pyran derivative B64 (as a control group of 4H-benzo[b]pyran derivative B62):

[0036] 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 .

[0037] Example 2:

[0038] Detection of the maximum safe concentration of different drugs on cells:

[0039] 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 B62, 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 of the Cellcountingkit-8 kit (CCK-8, C0038, Beyotime, China) manual. 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 B62, B64 and 4H-benzo[b]pyran were determined by CCK-8 method to be 60 mg / L, 80 mg / L and 60 mg / L, respectively. Figure 3 ).

[0040] Example 3:

[0041] Inhibitory effects of different drugs on MSRV in EPC cells

[0042] 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 B62 (60 mg / L), B64 (80 mg / L) or 4H-benzo[b]pyran (60 mg / L), with 3 replicates per group. After culturing in a 28°C incubator for 48h, cells were collected and the MSRV viral load after B62 treatment was detected (see Example 7). The results showed that B62 could significantly inhibit MSRV infection in EPC cells, with a maximum inhibition rate of 68.54±0.92%, respectively. However, the inhibitory effects of B64 and 4H-benzo[b]pyran on MSRV were not as good as those of B62 ( Figure 4 ).

[0043] Inhibition rate = (viral load of the control group - viral load of the experimental group) / viral load of the control group, the same below.

[0044] Example 4:

[0045] Time-of-addition assay of different antiviral drugs

[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. Treat cells with B62 before, during, or after infection with MSRV. Use 10 3 TCLD 50 Virus infection was performed. The MSRV infection time was set to 0h, and cells were treated with B62 (60mg / L), B64 (80mg / L) or 4H-benzo[b]pyran (60mg / L) at -12, -6, 0, 2, 4, 6, 8, 10, and 12h, respectively. Cells were collected at 48h, and total cell RNA was extracted to detect viral load. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect viral load (see Example 7 for RT-qPCR method). Three parallels were set for each group, and cells cultured in ordinary culture medium were set as a control group. Pretreatment with B62 significantly inhibited MSRV infection, with viral load reduced by 34.43% at -12 hours and by 28.62% at -6 hours. Figure 4 ). B62 infection and post-treatment also significantly inhibited viral load ( Figure 5 ), especially after infection 2 hours, 4 hours, 6 hours and 8 hours.Therefore, B62 can suppress MSRV infection before MSRV infection, when infecting and after infection.B64 and 4H-benzo [b] pyrans are worse than B62 to the inhibitory effect of MSRV.

[0047] Example 5:

[0048] Direct virus killing test of different drugs

[0049] Well-grown EPC cells were seeded into 12-well plates and MSRV (10 3 TCLD 50 ) were incubated with 60 mg / L B62 at room temperature for 0, 30, 60, and 90 min, and then incubated in the cells for 2 h. After washing with PBS, the cells were replaced with cell maintenance medium. After 48 h, the cells were collected, and 3 parallels were set up in each group. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the viral load (see Example 7 for RT-qPCR method). After incubation of the virus with the drug for 30, 60, and 90 min, the viral load was significantly reduced ( Figure 6 ), the highest inhibition rate reached 68.47%. B64 and 4H-benzo[b]pyran had a poorer direct killing effect on MSRV than B62.

[0050] Example 6:

[0051] Experiment on the inhibition of MSRV replication in largemouth bass by different drugs

[0052] To determine the antiviral effect of B62 in largemouth bass, 150 healthy largemouth bass with an average body length of 3.5 ± 0.5 cm were acclimated to the laboratory environment at 25 °C for 2 weeks and fed dry pelleted feed ad libitum before the experiment. DMSO Group, MSRV+B62 group (corresponding Figure 7 B62 in), MSRV+B64 group (corresponding to Figure 7 B64) and MSRV+4H-benzo[b]pyran group (corresponding to Figure 7 4H-benzo[b]pyran) was injected intraperitoneally with 15 μL of MSRV virus solution, and the control group was DMSO The same volume of PBS was injected into the two groups. 12 hours later, the largemouth bass in the MSRV+B62 group and the MSRV+4H-benzo[b]pyran group were fed with feed containing B62 (60 mg / kg), B64 (80 mg / kg) and 4H-benzo[b]pyran (60 mg / kg). DMSO Group and MSRV DMSO The rats in each group were fed with a diet containing the same volume of DMSO.

[0053] To evaluate the viral load of largemouth bass, 3 largemouth bass were collected from each group 4, 7, and 10 days after treatment with B62, B64, and 4H-benzo[b]pyran or DMSO, and their kidneys and spleens were dissected and RNA was extracted. The MSRV load was detected by RT-qPCR (see Example 7). At 4 days after MSRV infection, B62 significantly reduced the MSRV load in the spleen and kidneys. At 7 days after MSRV infection, B62 significantly reduced the MSRV load in the spleen and kidneys. At 10 days after infection, B62 significantly reduced the MSRV load in the spleen and kidneys. Figure 7 China A and Figure 7 Middle B) Control DMSO No MSRV was detected in the spleen and kidney of the largemouth bass in the control group. The above results indicate that B62 can inhibit the replication of MSRV in largemouth bass. In addition, the inhibitory effects of B64 and 4H-benzo[b]pyran on MRSV in fish were worse than those of B62. DMSO No MSRV was detected in the spleen and kidney of largemouth bass in the group.

[0054] Example 7:

[0055] MSRV viral load determination

[0056] Total RNA was extracted using a total RNA extraction kit (Yisheng, Shanghai, China). III 1stStrand cDNA Synthesis SuperMix qPCR reverse transcription RNA (+gDNAdigester plus) (Yisheng, Shanghai, China), all cDNAs were stored at -20 °C until use. The obtained cDNA was then used as a template and the cDNA was 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.

[0057] Table 1. Primers used in RT-qPCR

[0058]

[0059] Note: β-actin is the internal reference gene, and MSRV-Fq / Rq are primers used to detect the relative expression of MSRV viral glycoprotein by RT-qPCR.

[0060] 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 B62, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B62 in the preparation of a drug for treating or preventing largemouth bass rhabdovirus disease, wherein the 4H-benzo[b]pyran derivative B62 has a molecular formula of C 18 H 17 FN2O2, molecular weight 312.34, structural formula: 。 2. Use of 4H-benzo[b]pyran derivative B62, its pharmaceutically acceptable salt or compound preparation containing 4H-benzo[b]pyran derivative B62 in the preparation of a drug for treating or preventing largemouth bass rhabdovirus infection, wherein the 4H-benzo[b]pyran derivative B62 has a molecular formula of C 18 H 17 FN2O2, molecular weight 312.34, structural formula: 。 3. Use of 4H-benzo[b]pyran derivative B62, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B62 in the preparation of largemouth bass rhabdovirus inhibitors, wherein the 4H-benzo[b]pyran derivative B62 has a molecular formula of C 18 H 17 FN2O2, molecular weight 312.34, structural formula: 。 4. Use of 4H-benzo[b]pyran derivative B62, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B62 in the preparation of a feed additive for largemouth bass, wherein the 4H-benzo[b]pyran derivative B62 has a molecular formula of C 18 H 17 FN2O2, molecular weight 312.34, structural formula: 。

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