4-hydroxycoumarin derivatives x72 and their use in the preparation of a medicament for the treatment or prevention of ranavirus infections

Abstract

This invention belongs to the field of aquatic virus drugs, and discloses 4-hydroxycoumarin derivative X72 and its application in the preparation of drugs for treating or preventing frog virus infections. The applicant has synthesized a new compound, 4-hydroxycoumarin derivative X72, which exhibits anti-frog virus activity both in vitro and in vivo, particularly against largemouth bass frog virus and mandarin fish frog virus. It has minimal toxicity to normal cells, making it a novel compound with minimal side effects and anti-frog virus activity, laying the foundation for future frog virus drug development.

Description

Technical Field

[0001] This invention belongs to the field of aquatic virus drugs, specifically relating to 4-hydroxycoumarin derivative X72 and its application in the preparation of drugs for treating or preventing frog virus infection. Background Technology

[0002] Ranaviruses are a class of large DNA viruses, approximately 120-150 nm in diameter, belonging to the Iridoviridae family. They widely infect fish and amphibians, with high mortality rates. Representative viruses include largemouth bass ranavirus (LMBRaV) and mandarin fish ranavirus (MFRV). Ranaviruses are horizontally transmissible, highly infectious, and spread rapidly. Currently, the control of aquatic animal ranaviruses is still in its early stages, with prevention remaining the primary focus. Therefore, developing novel drugs for aquatic animal ranaviruses is of positive significance and offers significant advantages in preventing the recurring ranavirus diseases in aquaculture.

[0003] Coumarin compounds are widely found in natural products and bioactive drug molecules, exhibiting good photochemical stability and pharmacological activity. They are widely used in anticoagulant, antitumor, and antibacterial / anti-inflammatory formulations. Studies have shown that aescin, a coumarin derivative, has antibacterial activity against Escherichia coli and other bacteria, while another coumarin derivative, stigmosiderin, has antibacterial activity against Galanz-positive bacteria. The newly synthesized X72 in this invention is a member of the 4-hydroxycoumarin family and possesses antiviral activity against frog viruses in aquatic animals. Summary of the Invention

[0004] The purpose of this invention is to provide a 4-hydroxycoumarin derivative X72, the structural formula of which is:

[0005] .

[0006] Another object of the present invention is to provide the use of 4-hydroxycoumarin derivative X72 in the preparation of medicaments for treating or preventing frog virus infection.

[0007] To achieve the above objectives, the present invention adopts the following technical measures:

[0008] A 4-hydroxycoumarin derivative, X72, with the molecular formula C 17 H 11 N3O6S, molecular weight 385.35, structural formula is:

[0009] .

[0010] The preparation method of the above-mentioned 4-hydroxycoumarin derivative X72 includes the following steps:

[0011] 4-hydroxycoumarin, thiourea, and 3-nitrobenzaldehyde were mixed and reacted under alkaline catalyst with ethanol-water as solvent by stirring and reflux. The crude product was purified by silica gel column chromatography to obtain the final product.

[0012] Preferably, the steps described above involve placing 2.0 mmol of 4-hydroxycoumarin, 2.2 mmol of thiourea, and 2.0 mmol of 3-nitrobenzaldehyde in an ethanol-water mixture with a volume ratio of 8:2, adding 0.2 mmol of triethylamine catalyst, stirring and refluxing at 60°C for 5-7 hours, cooling to room temperature after the reaction is complete, adding water to precipitate the solid, filtering, washing with dilute ethanol and water sequentially, and purifying the crude product by a petroleum ether / ethyl acetate gradient elution.

[0013] The scope of protection of this invention also includes:

[0014] A compound preparation containing 4-hydroxycoumarin derivative X72.

[0015] The use of 4-hydroxycoumarin derivative X72, its pharmaceutically acceptable salt, or complex formulations containing 4-hydroxycoumarin derivative X72 in the preparation of medicaments for the treatment or prevention of frog virus infection.

[0016] The use of 4-hydroxycoumarin derivative X72, its pharmaceutically acceptable salt, or complex formulations containing 4-hydroxycoumarin derivative X72 in the in vitro non-therapeutic inhibition of frog viruses.

[0017] In the above-described applications, preferably, the frog viruses include largemouth bassranavirus (LMBRaV) and / or mandarin fish ranavirus (MFRV).

[0018] Application of 4-hydroxycoumarin derivative X72, its pharmaceutically acceptable salt, or compound formulations containing 4-hydroxycoumarin derivative X72 in the preparation of feed additives for largemouth bass or mandarin fish.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] This invention is the first synthesis of a 4-hydroxycoumarin derivative, X72. Verification has shown that this derivative has a good inhibitory effect on frog viruses, with minimal toxicity to normal cells. It is a novel compound with minimal side effects and anti-frog virus activity, laying the foundation for future drug development against frog viruses. Attached Figure Description

[0021] Figure 1 The synthetic route for the 4-hydroxycoumarin derivative X72 is as follows.

[0022] Figure 2 The in vitro anti-LMBRaV activity of different drugs was measured (*: P<0.05; **: P<0.01).

[0023] Figure 3 The in vitro anti-MFRV activity of different drugs was compared (*: P<0.05; **: P<0.01).

[0024] Figure 4 For the time-of-addition assay of different drugs against LMBRBraV (*: P<0.05; **: P<0.01).

[0025] Figure 5 For the time-of-addition assay of different drugs against MFRV (*: P<0.05; **: P<0.01).

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

[0027] Figure 7 The effects of different drugs on the direct killing of MFRV (*: P<0.05; **: P<0.01).

[0028] Figure 8 The effects of different drugs on LMBRaV replication in largemouth bass;

[0029] The figure shows the effect of X72, X70 and 4-hydroxycoumarin on the viral copy number in the spleen of largemouth bass on day 7 of challenge (*: P<0.05; **: P<0.01).

[0030] Figure 9 The effects of different drugs on MFRV replication in mandarin fish;

[0031] The figure shows the effects of X72, X70 and 4-hydroxycoumarin on the viral copy number in the spleen of mandarin fish on day 7 of challenge (*: P<0.05; **: P<0.01). Detailed Implementation

[0032] To make the present invention easier to understand, embodiments of the present invention will be further described below. The present invention will be further described and demonstrated in conjunction with these embodiments. However, these embodiments are not intended to limit the present invention. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all derived from commercial sources.

[0033] The largemouth bass frog virus and mandarin fish frog virus used in this invention were both obtained from the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. Carp epithelial cell lines (EPC) sensitive to both viruses were cultured in M199 culture medium supplemented with 10% fetal bovine serum. The culture medium used for cytotoxicity and antiviral assays contained 5% serum. Both viruses were passaged in EPC cells, and the TCID50 of both viruses was calculated to be 10 using the Reed-Muench method. 8 / mL, stored at -80℃ until use. The drug was prepared as a stock solution with a concentration of 50 mg / mL using DMSO organic solvent. Largemouth bass were purchased from farms with no record of largemouth bass frog virus detection in the past 5 years, and the fish were tested to confirm the absence of largemouth bass frog virus. Mandarin fish were purchased from farms with no record of mandarin fish frog virus detection in the past 5 years, and the mandarin fish were tested to confirm the absence of mandarin fish frog virus. The mandarin fish were domesticated and edible. 4-Hydroxycoumarin was purchased from Yuanye Company with a purity of 98%. All animal experiments were conducted at the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences, in strict accordance with the guidelines of the Ethics Review Committee.

[0034] Example 1:

[0035] The preparation method of 4-hydroxycoumarin derivative X72 includes the following steps:

[0036] 390 mg (2 mmol) of 4-hydroxycoumarin (CAS No.: 1076-38-6), 168 mg (2.2 mmol) of thiourea (CAS No.: 62-56-6), and 363 mg (2 mmol) of 3-nitrobenzaldehyde (CAS No.: 99-61-6) were added to a 25 mL round-bottom flask. 12 mL of ethanol (CAS No.: 64-17-5)-water (8:2) was added as a solvent, followed by 28 µL (0.2 mmol) of triethylamine (CAS No.: 121-44-8) as a catalyst. The reaction was stirred at 60 °C for 6 h, and the reaction progress was monitored by TLC (VL). 丙酮 V 石油醚 =1:1). After the reaction was complete, the mixture was cooled to room temperature, water was added to precipitate the solid, and the solid was filtered and washed successively with dilute ethanol and water. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate gradient elution) to obtain the target product. The synthetic route and X72 structure diagram are shown in [reference needed]. Figure 1 The structural formula of X72 is as follows:

[0037] .

[0038] Preparation method of 4-hydroxycoumarin derivative X70 (as X72 control group):

[0039] 390 mg (2 mmol) of 4-hydroxycoumarin (CAS No.: 1076-38-6), 168 mg (2.2 mmol) of thiourea (CAS No.: 62-56-6), and 248 mg (2 mmol) of p-fluorobenzaldehyde (CAS No.: 459-57-4) were added to a 25 mL round-bottom flask. 12 mL of ethanol (CAS No.: 64-17-5)-water (8:2) was added as a solvent, followed by 28 µL (0.2 mmol) of triethylamine (CAS No.: 121-44-8) as a catalyst. The reaction was stirred at 60 °C for 6 h, and the reaction progress was monitored by TLC (VL). 丙酮 V 石油醚 =1:1). After the reaction was complete, the mixture was cooled to room temperature, water was added to precipitate the solid, and the solid was filtered and washed successively with dilute ethanol and water. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate gradient elution) to obtain the target product.

[0040] The structural formula of X70 is as follows:

[0041] .

[0042] Example 2:

[0043] Maximum safe concentration detection of different drugs in cells:

[0044] Healthy EPC cells were collected, digested with trypsin, and then seeded into 96-well cell culture plates using M199 cell culture medium containing 10% fetal bovine serum, at a density of 1 × 10⁶ cells per well. 4 After culturing 96-well plates at 25°C for 24 hours, fresh culture medium containing X72, X70, or 4-hydroxycoumarin was added at six concentration gradients: 100 mg / L, 80 mg / L, 60 mg / L, 40 mg / L, 20 mg / L, and 10 mg / L. A control group without drug was also included. Each group had three replicates. After culturing at 28°C for 48 hours, EPC cytotoxicity was performed according to the recommended procedures in the Cellcounting kit-8 (CCK-8, C0038, Beyotime, China) instructions. Cell viability was calculated as [(OD450 of drug-treated group - OD450 of blank control) / (OD450 of control cells - OD450 of blank control)] × 100%. Cell viability > 80% was selected as the maximum safe concentration of the drug for subsequent experiments. The maximum safe concentrations of X72, X70, and 4-hydroxycoumarin were determined using the CCK-8 assay to be 60 mg / L, 60 mg / L, and 60 mg / L, respectively.

[0045] Example 3:

[0046] Inhibitory effects of different drugs on LMBRBraV and MFRV in EPC cells

[0047] EPC cells were seeded into 12-well plates at a density of 1×10⁶ cells / well. 5 / well, cultured to approximately 90% density per well. In LMBRBraV (10 4 Cells were infected at 28°C for 2 hours with TCLD50 / mL and then treated with X72 (60 mg / L), X70 (60 mg / L), or 4-hydroxycoumarin (60 mg / L), with three replicates per group. After culturing at 28°C for 48 hours, cells were collected, and the LMBRBraV viral load after X72 treatment was measured.

[0048] Viral DNA was extracted using a viral DNA extraction kit (OMEGA, USA) and stored at -20 °C until use. Primers used for digital PCR are shown in Table 1. Viral load was determined using established digital PCR methods for LMBRBraV and MFRV.

[0049] Table 1 Primers used in digital PCR

[0050]

[0051] The results showed that X72 significantly inhibited LMBRaV infection in EPC cells, with a maximum inhibition rate of 79.32 ± 0.97%, while X70 and 4-hydroxycoumarin were less effective than X72 in inhibiting LMBRaV. Figure 2 ).

[0052] Also in MFRV (10 4 Cells were infected with TCLD50 / mL at 28℃ for 2 h, and then treated with X72 (60 mg / L), X70 (60 mg / L), or 4-hydroxycoumarin (60 mg / L), respectively. Each group had three replicates. After culturing at 28℃ for 48 h, cells were collected, and the MFRV viral load after X72 treatment was measured. Results showed that X72 significantly inhibited MFRV infection in EPC cells, with a maximum inhibition rate of 74.59 ± 0.81%. X70 and 4-hydroxycoumarin showed less inhibitory effect on MFRV than X72 (60 mg / L). Figure 3 ).

[0053] Inhibition rate = (viral load in control group - viral load in experimental group) / viral load in control group, the same applies below.

[0054] Example 4:

[0055] Time-of-addition assay for antiviral effects of different drugs

[0056] EPC cells were seeded into 12-well plates at a density of 1×10⁶ cells / well. 5 / well, cultured to approximately 90% density per well. Cells were treated with the test drug (X72, X70, or 4-hydroxycoumarin, with DMSO as a control) before, concurrently with, or after LMBRaV infection, and then treated with 10% LMBRaV. 3 TCLD50 cells were used for viral infection. The LMBRaV infection time was set at 0 h. Cells were treated with X72 (60 mg / L), X70 (60 mg / L), or 4-hydroxycoumarin (60 mg / L) at -12, -6, 0, 2, 6, and 12 h, respectively. Cells were collected after 48 h, and viral DNA was extracted to detect viral load. The viral load was detected by digital PCR (see Example 3). Each group was divided into 3 replicates, and cells cultured in ordinary culture medium were set up as a control group.

[0057] Pretreatment with X72 significantly inhibited LMBRBraV infection, reducing viral load by 47.22% at -12 hours and by 51.34% at -6 hours. Figure 4 X72 infection and post-treatment also significantly suppressed viral load ( ). Figure 4 X72 showed inhibitory effects on LMBRaV infection, particularly at 2 and 6 hours post-infection. Therefore, X72 inhibited LMBRaV infection before, during, and after infection. X70 and 4-hydroxycoumarin showed weaker inhibitory effects on LMBRaV than X72.

[0058] Similarly, cells were treated with the test drug (X72, X70, or 4-hydroxycoumarin, with DMSO as a control) before, during, or after MFRV infection, and then treated with MFRV 10. 3 TCLD50 cells were used for MFRV infection. The infection time was defined as 0 h. Cells were treated with X72 (60 mg / L), X70 (60 mg / L), or 4-hydroxycoumarin (60 mg / L) at -12, -6, 0, 2, 6, and 12 h. Cells were collected after 48 h, and viral DNA was extracted to detect viral load. Digital PCR was used to detect viral load (see Example 3). Each group had three replicates, and cells cultured in ordinary culture medium served as a control group. Pretreatment with X72 significantly inhibited MFRV infection, reducing viral load by 40.17% at -12 h and by 55.25% at -6 h. Figure 5 X72 infection and post-treatment also significantly suppressed viral load ( ). Figure 5 X72 showed inhibitory effects, particularly at 2 and 6 hours post-infection. Therefore, X72 inhibited MFRV infection before, during, and after infection. X70 and 4-hydroxycoumarin showed weaker inhibitory effects than X72.

[0059] Example 5:

[0060] Experiments on the direct killing of viruses by different drugs

[0061] Well-grown EPC cells were seeded into 12-well plates, and LMBRBraV (10) were added. 3 TCLD50 / mL and 60 mg / L of the test drug (X72, X70, or 4-hydroxycoumarin, with DMSO as a control) were incubated at room temperature for 0, 30, 60, and 90 min, followed by incubation in cells for 2 h. After washing with PBS, the cells were replaced with cell maintenance medium, and cells were collected after 48 h. Each group was divided into three replicates. Viral load was detected by digital PCR (see Example 3). After incubation of the virus with the drug for 30, 60, and 90 min, the viral load decreased significantly ( Figure 6 The inhibition rate reached a maximum of 85.34%. X70 and 4-hydroxycoumarin were less effective than X72 in directly killing LMBRBraV.

[0062] Similarly, MFRV (10 3 TCLD50 / mL and 60 mg / L of the test drug (X72, X70, or 4-hydroxycoumarin, with DMSO as a control) were incubated at room temperature for 0, 30, 60, and 90 min, followed by incubation in cells for 2 h. After washing with PBS, the cells were replaced with cell maintenance medium, and cells were collected after 48 h. Each group was divided into three replicates. Viral load was detected by digital PCR (see Example 3). After incubation of the virus with the drug for 30, 60, and 90 min, the viral load decreased significantly ( Figure 7 The inhibition rate reached a maximum of 72.51%. X70 and 4-hydroxycoumarin were less effective than X72 in directly killing LMBRBaV.

[0063] Example 6:

[0064] Experiments on the inhibition of LMBRaV replication in largemouth bass by different drugs

[0065] To determine the antiviral effect of X72 in largemouth bass, 150 healthy largemouth bass with an average body length of 10 ± 0.5 cm were acclimatized to the laboratory environment at 25°C for 2 weeks and fed dry pellet feed before the experiment. The 150 healthy largemouth bass were randomly divided into 5 groups of 30 each: the LMBRaV DMSO group, the LMBRaV + X72 group, the LMBRaV + X70 group, and the LMBRaV + 4-hydroxycoumarin group were intraperitoneally injected with 20 μL of LMBRaV virus solution (10 μL / 100 μL). 3The same volume of PBS was injected into the control group (Control DMSO group). Twelve h later, the LMBRaV + X72 group, LMBRaV + X70 group, and LMBRaV + 4-hydroxycoumarin group were fed diets containing X72 (60 mg / kg), X70 (60 mg / kg), and 4-hydroxycoumarin (60 mg / kg), respectively. Simultaneously, the Control DMSO group and the LMBRaV DMSO group were fed diets containing the same volume of DMSO.

[0066] To assess the viral load in largemouth bass, three largemouth bass were collected from each group 7 days after treatment with X72, X70, and 4-hydroxycoumarin or DMSO. Their spleens were dissected, and viral DNA was extracted. LMBRaV load was detected by digital PCR (see Example 3). At day 7 post-LMBRaV infection, X72 significantly reduced the viral load in the spleen ( Figure 8 LMBRaV was not detected in the spleen of largemouth bass in the ControlDMSO group. Figure 8 (Not shown in the image). These results indicate that X72 can inhibit LMBRaV replication in largemouth bass. Furthermore, X70 and 4-hydroxycoumarin showed weaker inhibitory effects on LMBRaV in fish than X72.

[0067] To determine the antiviral effect of X72 in mandarin fish, 150 healthy mandarin fish with an average body length of 8 ± 1 cm were acclimatized to the laboratory environment at 25°C for 2 weeks and fed a diet before the experiment. The 150 healthy mandarin fish were randomly divided into 5 groups of 30 fish each: the MFRV DMSO group, the MFRV + X72 group, the MFRV + X70 group, and the MFRV + 4-hydroxycoumarin group were intraperitoneally injected with 50 μL of MFRV virus solution (10... 3 The same volume of PBS was injected into the control group (Control DMSO group). Twelve h later, the MFRV + X72 group, MFRV + X70 group, and MFRV + 4-hydroxycoumarin group were fed diets containing X72 (60 mg / kg), X70 (60 mg / kg), and 4-hydroxycoumarin (60 mg / kg), respectively. Simultaneously, the Control DMSO group and the MFRV DMSO group were fed diets containing the same volume of DMSO.

[0068] To assess the viral load in mandarin fish, three mandarin fish were collected from each group seven days after treatment with X72, X70, and 4-hydroxycoumarin or DMSO. Their spleens were dissected, and viral DNA was extracted. MFRV load was detected by digital PCR (see Example 3). On day 7 post-MFRV infection, X72 significantly reduced the viral load in the spleen (…). Figure 9 MFRV was not detected in the spleen of mandarin fish in the Control DMSO group. These results indicate that X72 can inhibit MFRV replication in mandarin fish. Furthermore, X70 and 4-hydroxycoumarin showed weaker inhibitory effects on MFRV in mandarin fish than X72.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A synthetically produced 4-hydroxycoumarin derivative, X72, with the following structural formula: 。 2. The method for preparing the 4-hydroxycoumarin derivative X72 according to claim 1, comprising the following steps: 4-hydroxycoumarin, thiourea, and 3-nitrobenzaldehyde were mixed and reacted under alkaline catalyst with ethanol-water as solvent by stirring and reflux. The crude product was purified by silica gel column chromatography to obtain the final product.

3. The preparation method according to claim 2, characterized in that: 2.0 mmol of 4-hydroxycoumarin, 2.2 mmol of thiourea, and 2.0 mmol of 3-nitrobenzaldehyde were placed in an ethanol-water mixture with a volume ratio of 8:

2. 0.2 mmol of triethylamine catalyst was added, and the mixture was stirred and refluxed at 60 °C for 5-7 hours. After the reaction was completed, the mixture was cooled to room temperature, and water was added to precipitate the solid. The solid was filtered and washed successively with dilute ethanol and water. The crude product was purified by elution with a petroleum ether / ethyl acetate gradient.

4. A compound formulation containing the 4-hydroxycoumarin derivative X72 as described in claim 1.

5. The use of the 4-hydroxycoumarin derivative X72 of claim 1, its pharmaceutically acceptable salt, or the complex formulation of claim 4 in the preparation of a medicament for treating or preventing frog virus infection.

6. The use of the 4-hydroxycoumarin derivative X72 of claim 1, its pharmaceutically acceptable salt, or the complex formulation of claim 4 in the in vitro non-therapeutic inhibition of frog virus.

7. The application according to claim 5 or 6, wherein the frog virus is largemouthbass ranavirus and / or mandarin fish ranavirus.

8. The use of the 4-hydroxycoumarin derivative X72 of claim 1, its pharmaceutically acceptable salt, or the compound formulation of claim 4 in the preparation of feed additives for largemouth bass or mandarin fish.

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