4-hydroxycoumarin derivative X77 and application thereof in preparation of medicine for treating or preventing cyprinid herpesvirus infection
By synthesizing the 4-hydroxycoumarin derivative X77, the problem of prevention and control of carp herpesvirus was solved, and effective inhibition and killing of carp herpesvirus type II and III were achieved, providing a safe drug solution.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
- Filing Date
- 2026-01-27
- Publication Date
- 2026-05-12
AI Technical Summary
Current technologies have not yet effectively solved the prevention and control measures for carp herpesvirus, especially lacking effective drugs against carp herpesvirus types II and III, leading to the problem of rapid spread of infection and high mortality rate in aquaculture.
A 4-hydroxycoumarin derivative, X77, was synthesized and applied to the preparation of drugs for the treatment or prevention of cyprinid herpesvirus infection. Its inhibitory effect on cyprinid herpesvirus was verified through in vitro and in vivo experiments.
X77 showed significant inhibitory and killing effects on carp herpesvirus types II and III, with low toxicity to normal cells, providing a safe and effective prevention and control method.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of aquatic virus drugs, specifically relating to 4-hydroxycoumarin derivative X77 and its application in the preparation of drugs for treating or preventing carp herpesvirus infection. Background Technology
[0002] Cyprinid herpesvirus is a large DNA virus, approximately 80-100 nm in diameter, with an envelope, belonging to the Alloherpesvirus family, and primarily infects fish. It is classified into three types: Cyprinid herpesvirus type I (low mortality), Cyprinid herpesvirus type II (also known as CyHV-2) (primarily infecting crucian carp and goldfish with high mortality), and Cyprinid herpesvirus type III (also known as KHV) (primarily infecting carp with high mortality). Cyprinid herpesvirus is horizontally transmissible, highly contagious, and spreads rapidly. Currently, the control of cyprinid herpesvirus is still in its early stages, with prevention remaining the primary focus. Therefore, developing novel drugs for cyprinid herpesvirus is of positive significance and offers significant advantages in preventing the recurring cyprinid herpesvirus disease 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, while another coumarin derivative, stigmosiderin, has antibacterial activity against Galanz-positive bacteria. The newly synthesized X77 in this invention is a member of the 4-hydroxycoumarin family and possesses anti-carp herpesvirus function. Summary of the Invention
[0004] The purpose of this invention is to provide a 4-hydroxycoumarin derivative X77, the structural formula of which is:
[0005] .
[0006] Another object of the present invention is to provide the use of 4-hydroxycoumarin derivative X77 in the preparation of medicaments for the treatment or prevention of carp herpesvirus infection.
[0007] To achieve the above objectives, the present invention adopts the following technical measures:
[0008] A 4-hydroxycoumarin derivative, X77, has the molecular formula: C 20 H 17 N3O5S, molecular weight 423.44, structural formula is:
[0009] .
[0010] The preparation method of the above-mentioned 4-hydroxycoumarin derivative X77 includes the following steps:
[0011] 4-hydroxycoumarin, thiourea, and acetaminobenzaldehyde were reacted under reflux with ethanol-water as solvent in the presence of piperidine and glacial acetic acid as catalysts. The crude product was purified by silica gel column chromatography.
[0012] Preferably, the steps described above involve placing 2.0 mmol of 4-hydroxycoumarin, 2.4 mmol of thiourea, and 2.0 mmol of acetaminophen in an ethanol-water mixture with a volume ratio of 9:1, adding 0.1 mmol of piperidine and 0.05 mmol of glacial acetic acid as catalysts, stirring and refluxing at 60°C for 6 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 elution with a petroleum ether / ethyl acetate gradient.
[0013] The scope of protection of this invention also includes:
[0014] A compound preparation containing the 4-hydroxycoumarin derivative X77.
[0015] The use of 4-hydroxycoumarin derivative X77, its pharmaceutically acceptable salt, or a complex formulation containing 4-hydroxycoumarin derivative X77 in the preparation of medicaments for the treatment or prevention of carp herpesvirus infection.
[0016] The use of 4-hydroxycoumarin derivative X77, its pharmaceutically acceptable salt, or complex formulations containing 4-hydroxycoumarin derivative X77 in the in vitro non-therapeutic inhibition of chevroherpesvirus.
[0017] In the above-described applications, preferably, the carp herpesvirus includes Cyprinidherpesvirus II (CyHV-2) and / or Koi herpesvirus (KHV).
[0018] Application of 4-hydroxycoumarin derivative X77, its pharmaceutically acceptable salt, or compound formulations containing 4-hydroxycoumarin derivative X77 in the preparation of feed additives for carp or crucian carp.
[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, X77. Verification has shown that this derivative has a good inhibitory effect on carp herpesvirus, with minimal toxicity to normal cells. It is a novel compound with minimal side effects and anti-carp herpesvirus activity, laying the foundation for future drug development for carp herpesvirus. Attached Figure Description
[0021] Figure 1 The synthetic route for the 4-hydroxycoumarin derivative X77 is as follows.
[0022] Figure 2 The in vitro anti-CyHV-2 activity of different drugs was measured (*: P<0.05; **: P<0.01).
[0023] Figure 3 The in vitro anti-KHV 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 CyHV-2 (*: P<0.05; **: P<0.01).
[0025] Figure 5 For the time-of-addition assay of different drugs against KHV (*: P<0.05; **: P<0.01).
[0026] Figure 6 The effects of different drugs on the direct killing of CyHV-2 (*: P<0.05; **: P<0.01).
[0027] Figure 7 The effects of different drugs on the direct killing of KHV (*: P<0.05; **: P<0.01).
[0028] Figure 8 To investigate the effects of different drugs on CyHV-2 replication in crucian carp, the effects of X72, X70 and 4-hydroxycoumarin on viral copy number in crucian carp kidneys on day 7 of challenge (*: P<0.05; **: P<0.01).
[0029] Figure 9 To investigate the effects of different drugs on KHV replication in carp, the effects of X72, X70 and 4-hydroxycoumarin on viral copy number in carp kidneys on day 7 of challenge (*: P<0.05; **: P<0.01). Detailed Implementation
[0030] 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.
[0031] The CyHV-2 and KHV viruses used in this invention were both derived from the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. The CyHV-2-sensitive crucian carp brain cell line (GiCB) and the KHV-sensitive carp fin stripe cell line (KF-1) 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 the cells, and the TCID50 of both was calculated using the Reed-Muench method to be 10. 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. Crucian carp were purchased from farms with no CyHV-2 detection records in the past 5 years, and the fish were tested to confirm the absence of CyHV-2. Carp were purchased from farms with no KHV detection records in the past 5 years, and the carp were tested to confirm the absence of KHV. 4-Hydroxycoumarin was purchased from Yuan Ye Company with a purity of 98%. All animal experiments were conducted at the Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, in strict accordance with the guidelines of the Ethics Review Committee.
[0032] Example 1:
[0033] The preparation method of 4-hydroxycoumarin derivative X77 includes the following steps:
[0034] 390 mg (2 mmol) of 4-hydroxycoumarin (CAS No.: 1076-38-6), 183 mg (2.4 mmol) of thiourea (CAS No.: 62-56-6), and 392 mg (2 mmol) of p-acetaminobenzaldehyde (CAS No.: 122-85-0) were added to a 25 mL round-bottom flask. 12 mL of ethanol (CAS No.: 64-17-5)-water (9:1) was added as a solvent, followed by 11.8 µL (0.1 mmol) of piperidine (CAS No.: 110-89-4) and 2.9 µL (0.05 mmol) of glacial acetic acid (CAS No.: 64-19-7) as catalysts. The reaction was stirred at 60 °C for 6 h, and the reaction progress was monitored by TLC (V acetone:V petroleum ether = 1:1). After the reaction was complete, the mixture was cooled to room temperature, and ice water was added to precipitate the solid. The solid was filtered, washed successively with dilute ethanol and water, and 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 X77 structural diagram are shown below. Figure 1 .
[0035] The structural formula of X77 is as follows:
[0036] .
[0037] Preparation method of 4-hydroxycoumarin derivative X80 (as X77 control group):
[0038] 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 244.2 mg (2 mmol) of p-hydroxybenzaldehyde (CAS No.: 123-08-0) were added to a 25 mL round-bottom flask. 12 mL of ethanol (CAS No.: 64-17-5)-water (9:1) was added as a solvent, followed by 11.8 µL (0.1 mmol) of piperidine (CAS No.: 110-89-4) and 2.9 µL (0.05 mmol) of glacial acetic acid (CAS No.: 64-19-7) as catalysts. The reaction was stirred at 60 °C for 6 h, and the reaction progress was monitored by TLC (V acetone:V petroleum ether = 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.
[0039] The structural formula of X80 is as follows:
[0040]
[0041] Example 2:
[0042] Maximum safe concentration detection of different drugs in cells:
[0043] GiCB cells in good growth condition were collected, digested with trypsin, and then EPC cells were seeded into 96-well cell culture plates using M199 cell culture medium containing 10% fetal bovine serum, with 1 × 10⁶ cells per well. 4 After culturing the 96-well plates at 25°C for 24 hours, fresh culture medium containing X77, X80, 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 the drug was also included. Each group had three replicates. After culturing at 25°C for 48 hours, GiCB cytotoxicity was assessed according to the instructions of the Cellcounting kit-8 (CCK-8, C0038, Beyotime, China). Cell viability was calculated as [(OD450 of drug-treated group - OD450 of blank control) / (OD450 of control cells - OD450 of blank control)] × 100%. A cell viability > 80% was selected as the maximum safe concentration of the drug for subsequent experiments. The maximum safe concentrations of X77, X80, and 4-hydroxycoumarin were determined using the CCK-8 assay to be 60 mg / L, 60 mg / L, and 60 mg / L, respectively.
[0044] KF-1 cells in good growth condition 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 the 96-well plates at 25°C for 24 hours, fresh culture medium containing X77, X80, 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 25°C for 48 hours, KF-1 cytotoxicity was assessed according to the instructions of the Cellcounting kit-8 (CCK-8, C0038, Beyotime, China). Cell viability was calculated as [(OD450 of drug-treated group - OD450 of blank control) / (OD450 of control cells - OD450 of blank control)] × 100%. A cell viability > 80% was selected as the maximum safe concentration of the drug for subsequent experiments. The maximum safe concentrations of X77, X80, 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 CyHV-2 and KHV in GiCB cells or KF-1 cells
[0047] GiCB 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 CyHV-2 (10 4 Cells were infected at 25°C for 2 hours with TCLD50 / mL and then treated with X77 (60 mg / L), X80 (60 mg / L), or 4-hydroxycoumarin (60 mg / L), with three replicates per group. After culturing at 25°C for 48 hours, cells were collected, and the CyHV-2 viral load after X77 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 CyHV-2 and KHV digital PCR methods.
[0049] Table 1. Primers used in digital PCR
[0050]
[0051] The results showed that X77 significantly inhibited CyHV-2 infection in GiCB cells, with a maximum inhibition rate of 75.89 ± 0.72%, while X80 and 4-hydroxycoumarin were less effective than X77 in inhibiting CyHV-2. Figure 2 ).
[0052] KF-1 cells were seeded into 12-well plates at a density of 1 × 10⁶ cells / well. 5 / well, cultured to approximately 90% density per well. At KHV (10 4 Cells were infected at 25°C for 2 hours with TCLD50 / mL and then treated with X77 (60 mg / L), X80 (60 mg / L), or 4-hydroxycoumarin (60 mg / L), respectively. Each group had three replicates. After culturing at 25°C for 48 hours, cells were collected, and the KHV viral load after X77 treatment was measured (see Example 3). Results showed that X77 significantly inhibited KHV infection in KF-1 cells, with a maximum inhibition rate of 78.29 ± 1.23%. X80 and 4-hydroxycoumarin showed less inhibitory effects on KHV than X77 (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] GiCB cells were seeded into 12-well plates at a density of 1 × 10⁶ cells / well. 5 / well, cultured to approximately 90% density per well. GiCB cells were treated with X77 before, concurrently with, or after CyHV-2 infection, using 10 CyHV-2... 3 TCLD50 cells were used for viral infection. CyHV-2 infection was defined as 0 h. Cells were treated with X77 (60 mg / L), X80 (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 X77 significantly inhibited CyHV-2 infection, reducing viral load by 43.21% at -12 h and by 56.77% at -6 h. Figure 4 X77 infection and post-treatment also significantly suppressed viral load ( ). Figure 4X77 showed particularly strong inhibitory effects on CyHV-2 infection, especially at 2 and 6 hours post-infection. Therefore, X77 inhibited CyHV-2 infection before, during, and after infection. X80 and 4-hydroxycoumarin showed weaker inhibitory effects on CyHV-2 than X77.
[0057] Similarly, KF-1 cells were treated with X77 before, during, or after KHV infection, and KHV was used in 10... 3 TCLD50 cells were used for viral infection. The KHV infection time was defined as 0 h. Cells were treated with X77 (60 mg / L), X80 (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 medium served as a control group. Pretreatment with X77 significantly inhibited KHV infection, reducing viral load by 49.21% at -12 h and by 56.37% at -6 h. Figure 5 X77 infection and post-treatment also significantly suppressed viral load ( ). Figure 5 X77 showed particularly strong inhibitory effects at 2 and 6 hours post-infection. Therefore, X77 inhibited KHV infection before, during, and after infection. X80 and 4-hydroxycoumarin showed weaker inhibitory effects than X77.
[0058] Example 5:
[0059] Experiments on the direct killing of viruses by different drugs
[0060] Well-grown GiCB cells were seeded into 12-well plates, along with CyHV-2 (102) cells. 3 TCLD50 / mL) was incubated with 60 mg / L X77 at room temperature for 0, 30, 60, and 90 min, respectively, 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 80.66%. X80 and 4-hydroxycoumarin showed a weaker direct killing effect on CyHV-2 than X77.
[0061] Similarly, KHV(10) 3TCLD50 / mL) was incubated with 60 mg / L X77 at room temperature for 0, 30, 60, and 90 min, respectively, and then incubated in KF-1 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 83.24%. X80 and 4-hydroxycoumarin were less effective at directly killing KHV than X77.
[0062] Example 6:
[0063] Experiments on the inhibition of CyHV-2 replication in crucian carp by different drugs
[0064] To determine the antiviral effect of X77 in crucian carp, 150 healthy crucian carp with an average body length of 13 ± 0.5 cm were acclimatized to the laboratory environment at 25℃ for 2 weeks and fed dry pellet feed before the experiment. The 150 healthy crucian carp were randomly divided into 5 groups of 30 each: CyHV-2 DMSO group, CyHV-2+ X77 group (corresponding to...) Figure 8 X77), CyHV-2 + X80 group (corresponding to Figure 8 X80) and CyHV-2+4-hydroxycoumarin group (corresponding to Figure 8 4-hydroxycoumarin) Intraperitoneal injection of 20 μL CyHV-2 virus solution (10 3 The same volume of PBS was injected into the control group (TCLD50 / mL). Twelve h later, the crucian carp in the CyHV-2+ X77 group, CyHV-2+ X80 group, and CyHV-2+ 4-hydroxycoumarin group were fed diets containing X77 (60 mg / kg), X80 (60 mg / kg), and 4-hydroxycoumarin (60 mg / kg), respectively. Simultaneously, the Control DMSO group and the CyHV-2 DMSO group were fed diets containing the same volume of DMSO.
[0065] To assess viral load in crucian carp, three crucian carp were collected from each group 7 days after treatment with X77, X80, and 4-hydroxycoumarin or DMSO. Their kidneys were dissected, and viral DNA was extracted. CyHV-2 load was detected by digital PCR (see Example 3). At day 7 post-CyHV-2 infection, X77 significantly reduced the viral load in the kidneys ( Figure 8CyHV-2 was not detected in the kidneys of crucian carp in the Control DMSO group. These results indicate that X77 can inhibit CyHV-2 replication in crucian carp. Furthermore, X80 and 4-hydroxycoumarin showed weaker inhibitory effects on CyHV-2 in fish than X77.
[0066] To determine the antiviral effect of X77 in carp, 150 healthy carp with an average body length of 15 ± 1 cm were acclimatized to the laboratory environment at 25℃ for 2 weeks and fed a diet before the experiment. The 150 healthy carp were randomly divided into 5 groups of 30 each: the KHV DMSO group, the KHV + X77 group (corresponding to...). Figure 9 X77), KHV+ X80 group (corresponding to Figure 9 X80) and KHV + 4-hydroxycoumarin group (corresponding to Figure 9 50 μL of KHV virus solution (containing 4-hydroxycoumarin) was injected intraperitoneally. 3 The same volume of PBS was injected into the control group (TCLD50 / mL). Twelve h later, the carp in the KHV + X77 group, KHV + X80 group, and KHV + 4-hydroxycoumarin group were fed diets containing X77 (60 mg / kg), X80 (60 mg / kg), and 4-hydroxycoumarin (60 mg / kg), respectively. Simultaneously, the Control DMSO group and the KHV DMSO group were fed diets containing the same volume of DMSO.
[0067] To assess viral load in carp, three carp were collected from each group 7 days after treatment with X77, X80, and 4-hydroxycoumarin or DMSO. Their kidneys were dissected, and viral DNA was extracted. KHV load was detected by digital PCR (see Example 3). At day 7 post-KHV infection, X77 significantly reduced the viral load in the kidneys ( Figure 9 KHV was not detected in the kidneys of carp in the Control DMSO group. These results indicate that X77 can inhibit KHV replication in carp. Furthermore, X80 and 4-hydroxycoumarin showed weaker inhibitory effects on KHV in carp than X77.
Claims
1. A synthetically produced 4-hydroxycoumarin derivative, X77, with the following structural formula: 。 2. The method for preparing the 4-hydroxycoumarin derivative X77 according to claim 1, comprising the following steps: 4-hydroxycoumarin, thiourea, and acetaminobenzaldehyde were reacted under reflux with ethanol-water as solvent in the presence of piperidine and glacial acetic acid as catalysts. The crude product was purified by silica gel column chromatography.
3. The preparation method according to claim 2, characterized in that: 2.0 mmol of 4-hydroxycoumarin, 2.4 mmol of thiourea, and 2.0 mmol of acetaminobenzaldehyde were placed in an ethanol-water mixture with a volume ratio of 9:
1. 0.1 mmol of piperidine and 0.05 mmol of glacial acetic acid were added as catalysts, and the mixture was stirred and refluxed at 60 °C for 6 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 preparation containing the 4-hydroxycoumarin derivative X77.
5. The use of the 4-hydroxycoumarin derivative X77 of claim 1, its pharmaceutically acceptable salt, or the complex formulation of claim 4 in the preparation of a medicament for treating or preventing carp herpesvirus infection.
6. The use of the 4-hydroxycoumarin derivative X77 of claim 1, its pharmaceutically acceptable salt, or the complex formulation of claim 4 in the in vitro non-therapeutic inhibition of chevroherpesvirus.
7. The application according to claim 5 or 6, wherein the carp herpesvirus includes carp herpesvirus type II (… Cyprinid herpesvirus II) or / and koi herpesvirus ( Koi herpesvirus ).
8. The use of the 4-hydroxycoumarin derivative X77 of claim 1, its pharmaceutically acceptable salt, or the compound formulation of claim 4 in the preparation of a feed additive for carp or crucian carp.