A 4H-benzo[b]pyran derivative B86, preparation method and application thereof in resisting yellow eel rhabdovirus

Abstract

The present invention belongs to the field of new drugs, and specifically discloses a 4H-benzo[b]pyran derivative B86, a preparation method and its application in the preparation of anti-eel rhabdovirus. In the antiviral activity experiment of the 4H-benzo[b]pyran derivative B86 provided by the present invention, it is found that the structural compound has a high anti-eel rhabdovirus effect, and has a small toxic effect on normal cells. B86 is a new compound with very small side effects and anti-eel rhabdovirus.

Description

Technical Field

[0001] The invention belongs to the field of new drugs, and specifically relates to a 4H-benzo[b]pyran derivative B86, a preparation method and application thereof in resisting yellow eel rhabdovirus. Background Art

[0002] Chinese rice-field eel rhabdovirus (CrERV) is one of the main pathogens causing hemorrhagic disease in rice-field eels, which can cause more than 80% mortality in diseased rice-field eels, seriously affecting the yield and economic benefits of rice-field eel farming. CrERV belongs to the genus Perhabdovirus of the family Rhabdoviridae. It is a negative-sense single-stranded RNA virus with a genome length of 11.45 bp, encoding five major functional proteins, namely (from 3'-5') nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and large polymerase (L). The main symptoms are: swelling of the head, hemorrhages of varying sizes on the body surface or severe diffuse hemorrhage and congestion. Dissection revealed petechial hemorrhages inside the kidneys, and congestion of the spleen and liver. At present, there is no vaccine or marketed drug for CrERV. Therefore, it is urgent to develop new antiviral drugs to prevent and control rice-field eel rhabdovirus disease.

[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.

[0004] B86 is a 4H-benzo[b]pyran derivative synthesized by the applicant, and was first found to have the ability to inhibit the eel rhabdovirus. Summary of the invention

[0005] The object of the present invention is to provide a 4H-benzo[b]pyran derivative B86, the structural formula of B86 is:

[0006]

[0007] Another object of the present invention is to provide a method for preparing 4H-benzo[b]pyran derivative B86.

[0008] Another object of the present invention is to provide the use of 4H-benzo[b]pyran derivative B86 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing yellow eel rhabdovirus disease.

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

[0010] A 4H-benzo[b]pyran derivative B86, with the molecular formula C 19 H 20 N2O4, molecular weight 340.38, structural formula:

[0011] A method for preparing a 4H-benzo[b]pyran derivative B86 comprises:

[0012] After mixing 5,5-dimethyl-1,3-cyclohexanedione, 4-hydroxy-3-methoxybenzaldehyde and malononitrile, triethylamine is added as a catalyst and ethanol is used as a solvent, and 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 B86.

[0013] The above steps are preferably performed by mixing 1.5-2.5 mmol of 5,5-dimethyl-1,3-cyclohexanedione, 1.5-2.5 mmol of 4-hydroxy-3-methoxybenzaldehyde and 1.5-2.5 mmol of malononitrile, and then adding 65-75 μL of triethylamine as a catalyst and 8-12 ml of ethanol as a solvent. The mixture is stirred at room temperature (23-27° C.) for 22-26 hours, and 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.

[0014] The protection scope of the present invention also includes:

[0015] A combination preparation containing 4H-benzo[b]pyran derivative B86.

[0016] Application of 4H-benzo[b]pyran derivative B86, its pharmaceutically acceptable salt or a composite preparation containing 4H-benzo[b]pyran derivative B62 in the preparation of drugs for treating or preventing yellow eel rhabdovirus disease.

[0017] Application of 4H-benzo[b]pyran derivative B86, pharmaceutically acceptable salts thereof or composite preparations containing 4H-benzo[b]pyran derivative B62 in the preparation of drugs for treating or preventing yellow eel rhabdovirus infection.

[0018] Application of 4H-benzo[b]pyran derivative B86, pharmaceutically acceptable salts thereof or composite preparations containing 4H-benzo[b]pyran derivative B86 in the preparation of feed additives for rice field eels.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The antiviral activity experiment of the 4H-benzo[b]pyran derivative B86 provided by the present invention found that the structural compound has a high anti-eel rhabdovirus effect and has a small toxic effect on normal cells. B86 is a new compound with very small side effects and anti-eel rhabdovirus effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a synthetic route for 4H-benzo[b]pyran derivative B86.

[0022] Figure 2 This is a comparative synthetic route for 4H-benzo[b]pyran derivative B64.

[0023] Figure 3 To test the toxicity of different drugs on GiCB cells;

[0024] 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).

[0025] Figure 4 Schematic diagram of the in vitro anti-CrERV activity of different drugs (*: P<0.05; **: P<0.01).

[0026] Figure 5 This is a time-of-addition assay experiment of the anti-CrERV effects of different drugs (*: P<0.05; **: P<0.01).

[0027] Figure 6 The direct killing effect of different drugs on CrERV virus (*: P<0.05; **: P<0.01).

[0028] Figure 7 The effects of different drugs on CrERV infection in yellow eel;

[0029] Figures A and B show the effects of drugs including B86 and 4H-benzo[b]pyran on the viral load in the kidney and spleen of the eel 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 eel rhabdovirus used in the present invention comes from the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences; the crucian carp brain tissue cell line (GiCB) sensitive to the eel rhabdovirus 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 GiCB cells, and the TCID is calculated using the Reed-Muench method. 50 For 10 6.5 / 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. Yellow eel was purchased from a farm with no record of CrERV detection in the past 5 years, and the fish were tested to confirm the absence of CrERV. 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] Preparation of 4H-benzo[b]pyran derivative B86

[0035] 283 mg (2 mmol) 5,5-dimethyl-1,3-cyclohexanedione (CAS No.: 126-81-8), 304 mg (2 mmol) 4-hydroxy-3-methoxybenzaldehyde (CAS No.: 50773-56-3) 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 B86 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 B86):

[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 GiCB cells

[0040] The GiCB cells in good growth state were taken and digested with trypsin. Then, the GiCB cells were inoculated 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 28°C incubator for 24 hours, fresh culture medium containing B86, 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. Three parallels were set in each group, and a control group without drug was set up at the same time. After culturing at 28°C for 48 hours, cytotoxicity detection was performed according to the steps recommended 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 the maximum safe concentration of the drug was selected for subsequent experiments. The maximum safe concentrations of B86, B64 and 4H-benzo[b]pyran were determined by CCK-8 method to be 50 mg / L, 50 mg / L and 40 mg / L, respectively ( Figure 3 ).

[0041] Embodiment 3:

[0042] Inhibitory effects of different drugs on CrERV in GiCB cells

[0043] 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. 3 TCID 50 ) After infection at 28°C for 2h, cells were treated with B86 (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 48h, cells were collected and the intracellular viral load of CrERV after drug treatment was detected (see Example 7). B86 can significantly inhibit CrERV infection in GiCB cells, with a maximum inhibition rate of 63.90±3.93%, while B64 and 4H-benzo[b]pyran have less inhibitory effects on the virus than B86 ( 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] GiCB 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 infection with CrERV, cells were treated with B86 (50 mg / L), B64 (50 mg / L), and 4H-benzo[b]pyran (40 mg / L). 3 TCID 50 Virus infection was performed. The CrERV 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 up in 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 B86 can significantly inhibit CrERV infection, especially at 2 hours and 4 hours after infection. The viral load was reduced by 61.5% at 4 hours. B64 and 4H-benzo[b]pyran had no obvious inhibitory effect on the virus ( Figure 5 ).

[0048] Embodiment 5:

[0049] Effects of different drugs on inhibiting virus adsorption

[0050] Well-growing GiCB cells were seeded into T25 cm 2 In the culture flask, cells were inoculated with CrERV (10 3 TCID 50 ) were incubated with B86 (50 mg / L), B64 (50 mg / L) and 4H-benzo[b]pyran (40 mg / L) at 4°C for 30 min, with 3 parallels in each group. The cells were washed with PBS and collected. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression level of viral RNA. During the virus attachment and internalization stage, the viral load was significantly inhibited ( Figure 6 ), the inhibition rate reached more than 60%. B64 and 4H-benzo[b]pyran had no significant inhibitory effect on the virus.

[0051] Embodiment 6:

[0052] Effects of different drugs on inhibiting viral replication in yellow eel

[0053] To determine the antiviral effect of B86 in rice field eels, 150 healthy rice field eels with an average body length of 12±3 cm were acclimatized to the laboratory environment at 25°C for 2 weeks and fed with dry pelleted feed ad libitum before the experiment.

[0054] 150 healthy eels were randomly divided into 5 groups, 30 eels in each group:

[0055] CrERV DMSO Group, CrERV+B86 group (corresponding to Figure 7 B86 group in CrERV+B64 group (corresponding to Figure 7 B64 group in CrERV) and 4H-benzo[b]pyran group (corresponding to Figure 7 The 4H-benzo[b]pyran group) were injected intraperitoneally with 50 μL of CrERV virus solution, and the control group DMSO The same volume of PBS was injected in each group.

[0056] After 12 hours, the eels in the CrERV+B86, CrERV+B64, and CrERV+4H-benzo[b]pyran groups were fed with feeds containing B86 (50 mg / kg), B64 (50 mg / kg), and 4H-benzo[b]pyran (40 mg / kg), respectively. DMSO Group and CrERV DMSO The groups were fed with the same volume of feed containing 0.12% DMSO.

[0057] To evaluate the viral load of rice field eels, three rice field eels were collected from each group at 4, 7, and 10 days after treatment with B86, 4H-benzo[b]pyran, or 0.12% DMSO, and the kidneys and spleens were dissected and RNA was extracted. CrERV load was detected by RT-qPCR (see Example 7). B86 significantly reduced the CrERV load in the spleen at 4, 7, and 10 days after CrERV infection ( Figure 7 Middle A and Figure 7 In addition, B64 and 4H-benzo[b]pyran had no significant effect on the decrease of viral load in kidney and spleen. The above results indicate that B86 can inhibit the replication of CrERV in yellow eel.

[0058] Embodiment 7:

[0059] Viral load determination

[0060] Total RNA was extracted using a total RNA extraction kit (Yisheng, Shanghai, China). III 1stStrand cDNA Synthesis SuperMix qPCR reverse transcribed RNA (+gDNA digester plus) (Yisheng, Shanghai, China), and all cDNAs were stored at -20°C until use. The obtained cDNA was then used as a template and purified using Hieff 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.

[0061] Table 1. Primers for CrERV RNA assay

[0062]

[0063] Note: β-actin and EF1α are the internal reference genes of GiCB cells and rice field eel, respectively. CrERV-Fq / Rq are the primers used to detect the relative expression of CrERV virus glycoprotein 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 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 B86, its pharmaceutically acceptable salt or a compound preparation containing H-benzo[b]pyran derivative B86 in the preparation of a drug for treating or preventing yellow eel rhabdovirus disease, wherein the molecular formula of the 4H-benzo[b]pyran derivative B86 is C 19 H 20 N2O4, molecular weight 340.38, structural formula: 。 2. Use of 4H-benzo[b]pyran derivative B86, its pharmaceutically acceptable salt or a composite preparation containing H-benzo[b]pyran derivative B86 in the preparation of a feed additive for rice field eels, wherein the molecular formula of the 4H-benzo[b]pyran derivative B86 is C 19 H 20 N2O4, molecular weight 340.38, structural formula: 。

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