A 4H-benzo[b]pyran derivative B95, its preparation method and its application in resisting carp spring viremia virus

Abstract

The present invention belongs to the field of new drugs and specifically relates to a 4H-benzo[b]pyran derivative B95, a preparation method, and its use in combating carp spring viremia virus. Antiviral activity experiments of the 4H-benzo[b]pyran derivative B95 provided by the present invention revealed that the structural compound has a high anti-carp spring viremia virus activity and has minimal toxicity to normal cells. B95 is a new compound with minimal side effects that can combat carp spring viremia 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 B95, a preparation method and application thereof in resisting carp spring viremia virus. Background Art

[0002] Spring viremia of carp (SVC), a viral disease of fish, is an acute hemorrhagic viral disease caused by the spring viremia of carp virus (SVCV), a serious threat to carp fish. Current research indicates that SVCV is an enveloped virus with typical rhabdovirus morphology, exhibiting a rod or bullet shape, 80-180 nm in length, and 60-90 nm in diameter. Genomically, it is a single-stranded, negative-sense RNA virus with an approximately 11 kb genome encoding five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L). Juvenile fish mortality can reach up to 90% at temperatures between 11 and 17°C, making it a notifiable pathogen by the World Organization for Animal Health (WOAH). Despite the widespread spread of SVCV, which threatens the carp aquaculture industry, comprehensive prevention and treatment strategies are still in their infancy.

[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.

[0004] B95 is a 4H-benzo[b]pyran derivative synthesized by the applicant, and it was first discovered that it has the effect of inhibiting carp spring viremia virus. Summary of the Invention

[0005] The present invention aims to provide a 4H-benzo[b]pyran derivative B95, the structural formula of which is:

[0006]

[0007] Another object of the present invention is to provide a method for preparing B95.

[0008] Another object of the present invention is to provide the use of 4H-benzo[b]pyran derivative B95 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating or preventing carp spring viremia.

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

[0010] A 4H-benzo[b]pyran derivative B95, molecular formula C 16 H 16 N2O3, molecular weight 284.32, structural formula:

[0011]

[0012] A method for preparing a 4H-benzo[b]pyran derivative B95 comprises:

[0013] Mix 5,5-dimethyl-1,3-cyclohexanedione, furan-2-carboxaldehyde and malononitrile, use triethylamine as catalyst and ethanol as solvent, stir and react at room temperature, filter after the reaction is completed, and wash the filter cake with ethanol to obtain the product.

[0014] In the above-described steps, preferably, 1.5-2.5 mmol of 5,5-dimethyl-1,3-cyclohexanedione, 1.5-2.5 mmol of furan-2-carboxaldehyde and 1.5-2.5 mmol of malononitrile are mixed, and then 65-75 μL of triethylamine is added as a catalyst and 8-12 mL of ethanol 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 ethanol to obtain the target product.

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

[0016] A combination preparation containing 4H-benzo[b]pyran derivative B95.

[0017] Application of 4H-benzo[b]pyran derivative B95, pharmaceutically acceptable salts thereof or compound preparations containing 4H-benzo[b]pyran derivative B95 in preparing drugs for treating or preventing carp spring viremia.

[0018] Application of 4H-benzo[b]pyran derivative B95, a pharmaceutically acceptable salt thereof or a compound preparation containing 4H-benzo[b]pyran derivative B95 in the preparation of a drug for treating or preventing carp spring viremia virus infection.

[0019] Application of 4H-benzo[b]pyran derivative B95, a pharmaceutically acceptable salt thereof or a composite preparation containing 4H-benzo[b]pyran derivative B95 in the preparation of a carp feed additive.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] In the antiviral activity experiment of the 4H-benzo[b]pyran derivative B95 provided by the present invention, it was found that the structural compound has a high anti-carp spring viremia virus effect and has little toxicity to normal cells. B95 is a new compound with very few side effects that can resist carp spring viremia virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Synthetic route of 4H-benzo[b]pyran derivative B95.

[0023] Figure 2 The synthetic route of the comparative 4H-benzo[b]pyran derivative B64 is shown.

[0024] Figure 3 To test the toxicity of different drugs on EPC cells;

[0025] In the figures, cell survival rate is expressed as the percentage of cell viability in the control group (*: P < 0.05; **: P < 0.01).

[0026] Figure 4 Schematic diagram of the in vitro anti-SVCV activity of different drugs;

[0027] Figure 2 shows the effect of B95 on the expression of viral proteins in EPC cells infected with SVCV: SVCV (10 3 TCID 50 ) were infected at 28°C for 2 hours. Equal volumes of B95 or 4H-benzo[b]pyran (40 mg / L) were added and incubated for 48 hours. RT-qPCR analysis was performed, and the results are expressed as the viral expression level in the drug-treated group compared with the untreated group. Effects of B95 on the expression of viral proteins in EPC cells infected with SVCV (*: P < 0.05; **: P < 0.01).

[0028] Figure 5 This is a time-of-addition assay experiment of the anti-SVCV effects of different drugs (*: P<0.05; **: P<0.01).

[0029] Figure 6 The direct killing effects of different drugs on SVCV virus (*: P < 0.05; **: P < 0.01).

[0030] Figure 7 The effects of different drugs on carp after SVCV infection;

[0031] Figures A and B show the effects of B95 and 4H-benzo[b]pyran on the relative expression levels of viral mRNA in the kidney and spleen of carp 4, 7, and 10 days after infection (*: P < 0.05; **: P < 0.01). DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] The carp spring viremia virus used in this study was obtained from the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The epithelial carp tumor cell line (EPC), which is sensitive to carp spring viremia, was cultured in M199 medium 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 7.2 / mL and stored at -80°C until use. B95 was prepared in DMSO to a stock concentration of 50 mg / mL. Common carp were purchased from a farm with no record of SVCV detection in the past five years, and the fish were tested to confirm the absence of SVCV. 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.

[0035] Example 1:

[0036] Preparation method of 4H-benzo[b]pyran derivative B95, Figure 1 As shown, the following steps are included:

[0037] 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 furan-2-carbaldehyde (CAS No. 98-01-1), 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, the filter cake is filtered and washed with a small amount of ethanol to obtain the target product. The obtained compound is: 4H-benzo[b]pyran derivative B95, referred to as B95 in the present invention. Its synthesis route and structure are shown in Figure 1 .

[0038] Preparation method of 4H-benzo[b]pyran derivative B64 (as a control group of 4H-benzo[b]pyran derivative B95):

[0039] 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, the filter cake is filtered and washed with a small amount of ethanol to obtain the target product. The obtained compound is: 4H-benzo[b]pyran derivative B64, referred to as B64 in the present invention. Its synthesis route and structure are shown in Figure 2 .

[0040] Example 2:

[0041] Detection of cytotoxicity of different drugs on EPC cells:

[0042] 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. 4After culturing the 96-well plate in a 28°C incubator for 24 hours, fresh culture medium containing B95, 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 for 48 hours at 28°C, EPC cytotoxicity test 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%, 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 B95, 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 ).

[0043] Example 3:

[0044] Inhibitory effects of different drugs on SVCV in EPC cells:

[0045] 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 TCID 50 ) After infection at 28°C for 2 h, the cells were treated with B95 (80 mg / L), B64 (80 mg / L), or 4H-benzo[b]pyran (60 mg / L). Each group had three replicates. After 48 h of incubation in a 28°C incubator, the cells were harvested and the expression of intracellular viral glycoproteins of SVCV after B95 treatment was detected (see Example 7).

[0046] The results showed that B95 could significantly inhibit SVCV in EPC cells within a safe concentration range, with a maximum inhibition rate of 66.21±3.96%, while B64 and 4H-benzo[b]pyran had less inhibitory effects on the virus than B95 ( 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 drugs' antiviral effects

[0050] 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 B95 before, during, or after SVCV infection. Use 10 3 TCID 50 Viral infection was performed. The SVCV infection time was set to 0 h, and the cells were treated with B95 (80 mg / L), B64 (80 mg / L), or 4H-benzo[b]pyran (60 mg / L) at -12, -6, 0, 2, 4, 6, 8, 10, and 12 h, respectively. Three parallels were set for each group. Cells were collected after 48 h, and total cell RNA was extracted to detect the viral load. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the viral RNA expression level (see Example 7). Cells cultured in ordinary medium were also set as a control group.

[0051] Pretreatment with B95 significantly inhibited SVCV infection, with the viral load reduced by 41.23% at -12 hours ( Figure 5 B95 post-treatment also significantly suppressed viral load, especially at 2, 4, and 6 hours after infection ( Figure 5 ). Therefore, B95 can inhibit SVCV infection both before and after SVCV infection, while B64 and 4H-benzo[b]pyran have no significant inhibitory effect ( Figure 5 ).

[0052] Example 5:

[0053] Test on direct virus killing by different drugs:

[0054] Well-grown EPC cells were seeded into 12-well plates, and SVCV (10 3 TCID 50 ) were incubated with B95 (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. Three parallels were set up in each group. After washing with PBS, the cells were replaced with cell maintenance medium and collected after 48 h. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression level of viral RNA (see Example 7). After the virus was incubated with B95 for 0, 30, 60, 90 min, the viral load was significantly reduced ( Figure 6 ), the highest inhibition rate reached 49.65% (90min group). B64 and 4H-benzo[b]pyran had no significant inhibitory effect ( Figure 6 ).

[0055] Example 6:

[0056] Determination of viral load inhibition in carp by different drugs:

[0057] To determine the antiviral effect of B95 in common carp, 150 common carp with an average body length of 4.28 ± 0.15 cm were acclimatized to the laboratory environment at 25 °C for 2 weeks and fed dry pelleted feed ad libitum before the experiment.

[0058] 150 healthy carps were randomly divided into 5 groups, with 30 carps in each group:

[0059] To SVCV DMSO Group, SVCV+B95 group (corresponding Figure 7 B95 group), SVCV+B64 group (corresponding to Figure 7 B64 group in) and SVCV+4H-benzo[b]pyran group (corresponding to Figure 7 4H-benzo[b]pyran group) were injected intraperitoneally with 10 μL of SVCV virus solution, and the control group DMSO The same volume of PBS was injected into the other groups.

[0060] After 12 hours, the carp in the SVCV+B95 group and the SVCV+4H-benzo[b]pyran group were fed with feed containing B95 (80 mg / kg), B64 (80 mg / kg) and 4H-benzo[b]pyran (60 mg / kg). DMSO Group and SVCV DMSO The groups were fed with the same volume of feed containing 0.12% DMSO.

[0061] To evaluate the viral load of carp, 3 carp were collected from each group 4, 7, and 10 days after treatment with B95, 4H-benzo[b]pyran, or 0.12% DMSO, homogenized, and RNA was extracted. The SVCV viral load was detected by RT-qPCR (see Example 7). On the 4th day after SVCV infection, the SVCV+B95 group significantly reduced the SVCV viral load in the spleen; on the 7th day after SVCV infection, the SVCV+B95 group significantly reduced the SVCV viral load in the spleen and kidney; and 10 days after infection, the SVCV+B95 group significantly reduced the SVCV viral load in the kidney and significantly reduced the SVCV viral load in the spleen ( Figure 7 China A and Figure 7 In addition, B64 and 4H-benzo[b]pyran did not significantly reduce viral load in the kidney and spleen. These results indicate that B95 can inhibit the proliferation of SVCV in carp.

[0062] Example 7:

[0063] Viral load determination

[0064] 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.

[0065] Table 1. SVCV RNA assay primers

[0066]

[0067] Note: β-actin is an internal reference gene, and SVCV-Fq / Rq are primers used to detect the relative expression of SVCV viral glycoprotein by RT-qPCR.

[0068] 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 experiments, 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 of the present application and are made using conventional techniques known in the art.

Claims

1. Use of 4H-benzo[b]pyran derivative B95, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B95 in the preparation of a drug for treating or preventing carp spring viremia, wherein the 4H-benzo[b]pyran derivative B95 has a molecular formula of C 16 H 16 N2O3, molecular weight 284.32, structural formula: 。 2. Use of 4H-benzo[b]pyran derivative B95, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B95 in the preparation of a drug for treating or preventing carp spring viremia virus infection, wherein the 4H-benzo[b]pyran derivative B95 has a molecular formula of C 16 H 16 N2O3, molecular weight 284.32, structural formula: 。 3. Use of 4H-benzo[b]pyran derivative B95, its pharmaceutically acceptable salt or a compound preparation containing 4H-benzo[b]pyran derivative B95 in the preparation of carp feed additives, wherein the 4H-benzo[b]pyran derivative B95 has a molecular formula of C 16 H 16 N2O3, molecular weight 284.32, structural formula: 。

Images