Magnolol derivative B51 and application thereof in resisting white spot syndrome virus

Abstract

The application belongs to the field of new drugs, and particularly relates to a magnolol derivative B51 and application thereof in resisting white spot syndrome virus. The application provides application of B51 or a derivative thereof or a pharmaceutically acceptable salt thereof in resisting white spot syndrome. B51 has an effect of resisting white spot syndrome in vivo and in vitro, and is a potential candidate drug for resisting white spot syndrome.

Description

Technical Field

[0001] This invention belongs to the field of new drugs, specifically relating to a magnolol derivative B51 and its application in treating vitiligo syndrome virus. Background Technology

[0002] White spot syndrome virus (WSSV) is the most serious viral pathogen affecting the red swamp crayfish (Procambarus clarkii) aquaculture. WSSV-induced white spot syndrome is classified as a Class II animal disease. Infected shrimp typically exhibit reduced feeding, lethargy, and easy separation of the cephalothorax. Within 3-10 days of infection, the cumulative mortality rate can reach 100%. WSSV can be horizontally transmitted through crustaceans consuming WSSV-carrying aquatic organisms, and vertically transmitted to offspring via oocytes. It is characterized by rapid onset, high mortality, and strong infectivity. Currently, there is no effective treatment for white spot syndrome; prevention is the only way to control outbreaks and minimize economic losses.

[0003] Magnolol is an active substance extracted from the traditional Chinese medicine Magnolia officinalis, and is one of the main components responsible for its medicinal effects. It possesses various physiological functions, including anti-inflammatory, antioxidant, anticancer, and antibacterial properties, and is considered a natural, pollution-free, and non-toxic antibiotic alternative with promising applications in animal production. Magnolol derivatives refer to a class of compounds obtained by introducing different functional groups or structural units based on the structure of this natural compound through chemical synthesis or biotransformation. Modifying and derivatizing its structure can not only improve its pharmacological activity and efficacy but also expand its application scope and explore new therapeutic pathways. Summary of the Invention

[0004] The purpose of this invention is to provide a magnolol derivative B51, the structural formula of which is:

[0005]

[0006] Another object of the present invention is to provide the use of magnolol derivative B51 in the preparation of medicaments for the treatment or prevention of vitiligo syndrome.

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

[0008] A magnolol derivative B51, with the molecular formula C 34 H 32 N2O8S2, molecular weight 660.76, structural formula is:

[0009]

[0010] The preparation method of the above-mentioned magnolol derivative B51 includes the following steps:

[0011] Dichloromethane, magnolol, triethylamine and p-acetamidobenzenesulfonyl chloride were mixed and stirred at 48-52°C. After the reaction was completed, the mixture was extracted and separated by silica gel column chromatography to obtain the target product B51.

[0012] The preferred method described above is to mix 35-45 ml of dichloromethane, 1.5-2.5 mmol of magnolol, 5-7 mmol of triethylamine, and 7-9 mmol of p-acetamidobenzenesulfonyl chloride, stir at 47-53 °C, and after the reaction is complete, extract and separate the product B51 using silica gel column chromatography.

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

[0014] A compound preparation containing magnolol derivative B51.

[0015] The use of magnolol derivative B51, its pharmaceutically acceptable salt, or compound preparations containing magnolol derivative B51 in the preparation of drugs for the treatment or prevention of vitiligo syndrome;

[0016] The use of magnolol derivative B51, its pharmaceutically acceptable salt, or complex formulations containing magnolol derivative B51 in the preparation of drugs for the treatment or prevention of vitiligo syndrome viral infection.

[0017] Application of magnolol derivative B51, its pharmaceutically acceptable salt, or complex formulations containing magnolol derivative B51 in the preparation of vitiligo syndrome virus inhibitors.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] In the antiviral activity experiment of the magnolol derivative B51 provided by the present invention, it was found that this compound has a high anti-white spot syndrome effect and has little toxicity to Procambarus clarkii. B51 is a new compound with very few side effects and anti-white spot syndrome. Attached Figure Description

[0020] Figure 1 This is the synthetic route for magnolol derivative B51.

[0021] Figure 2 The synthetic route for the control compound magnolol derivative B44 is shown.

[0022] Figure 3 To assess the in vivo anti-WSSV activity of different drugs;

[0023] Results are expressed as viral load in the drug-treated group compared to the untreated group. Effects of B51 and B44 on viral load in *Procambarus clarkii* infection (*P<0.05; **P<0.01).

[0024] Figure 4 Experiments on the prevention of WSSV infection with different drugs. The figure shows the viral load results after drug treatment for different time periods (*P<0.05; **P<0.01).

[0025] Figure 5 The effects of different drugs on the direct killing of viruses (*P<0.05; **P<0.01).

[0026] Figure 6 The efficacy of different drugs in treating WSSV-infected Procambarus clarkii (*P<0.05; **P<0.01). Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] The white spot syndrome virus used in this invention was obtained from the Yangtze River Fisheries Research Institute of the Chinese Academy of Fishery Sciences; the red swamp crayfish were tested for the virus to confirm that they did not have WSSV.

[0030] The purchased juvenile red swamp crayfish were stocked in a culture tank at 28°C and fed four times daily with commercial feed pellets. The virus was diluted to a series of concentrations (1.5 × 10⁻⁶). 2 1.5×10 3 1.5×10 4 1.5×10 5 1.5×10 6 and 1.5×10 7 The virus was administered separately at a concentration of 1.5 × 10⁻⁶ copies / μL. The concentration was set at which the shrimp mortality rate would be 100% within 3 days. 5 The drug was used as the infection concentration for subsequent experiments. A stock solution of the drug was prepared using DMSO organic solvent at a concentration of 50 mg / mL. 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.

[0031] Example 1:

[0032] The preparation method of magnolol derivative B51 includes the following steps:

[0033] Add 40 ml of dichloromethane (CAS No.: 75-09-2), 0.534 g (2 mmol) of honokiol (CAS No.: 528-43-8), and triethylamine (CAS No.: 528-43-8) to a 50 ml round-bottom flask in sequence. 121-44-8 835 μl (6 mmol), p-acetamidobenzenesulfonyl chloride (CAS No.: 121-60-8 1.402 g (8 mmol) was added and stirred at 50 °C for 8 h. The reaction progress was monitored by TLC (VL). 乙酸乙酯 V 石油醚 =1:1). After the reaction was complete, extraction was performed and silica gel column chromatography was used (V 石油醚 :V 乙酸乙酯 The target product was obtained by separation using a 1:1 elution system. The synthetic route and B51 structural diagram are shown below. Figure 1 .

[0034] The preparation method of magnolol derivative B44 (as a control group for magnolol derivative B51) includes the following steps:

[0035] Add 40 ml of dichloromethane (CAS No.: 75-09-2), 0.534 g (2 mmol) of honokiol (CAS No.: 528-43-8), and triethylamine (CAS No.: 528-43-8) to a 50 ml round-bottom flask in sequence. 121-44-8 835 μl (6 mmol) of cyclopropylformyl chloride (CAS No.: 4023-34-1) and 545 μl (8 mmol) of cyclopropylformyl chloride were added and stirred at 50 °C for 8 h. The reaction progress was monitored by TLC (VL). 乙酸乙酯 V 石油醚 =1:1). After the reaction was complete, extraction was performed and silica gel column chromatography was used (V 石油醚 :V 乙酸乙酯 The target product was obtained by separation using a 1:1 elution system. The synthetic route and B44 structural diagram are shown below. Figure 2 .

[0036] Example 2:

[0037] Safety tests of different drugs on red swamp crayfish

[0038] Red swamp crayfish were randomly separated into glass tanks, and then serially diluted B51, B44, or honokiol (10, 20, 30, 40, 50, 60 mg / L) were added. The control group was treated with 0.05% DMSO. The tanks were incubated at 28°C for 72 hours. Each tank contained 30 red swamp crayfish. Mortality rates were recorded every 12 hours. The maximum safe concentrations for B51, B44, and honokiol were all 40 mg / L.

[0039] Example 3:

[0040] Inhibitory effects of different drugs on WSSV in Procambarus clarkii

[0041] The experiment was conducted in glass tanks, with 30 shrimp per tank. Different drugs were added to the tanks to achieve a final concentration of 40 mg / L for B51, B44, or honokiol. The control group was treated with 0.04% DMSO (V). DMSO / V Water 12 hours later, WSSV (final concentration 1.5 × 10⁻⁶) was added to the tank. 5 (copies / μL), incubate red swamp crayfish for 72 hours.

[0042] After the experiment, the red swamp crayfish were euthanized using MS-222, and tissue DNA was extracted. WSSV load was then detected using qPCR (the qPCR method is described in Example 7). The results showed that B51 significantly inhibited WSSV replication in the red swamp crayfish, with an inhibition rate of 64.5%, while magnolol and B44 had no significant inhibitory effect on the virus. Figure 3 ).

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

[0044] Example 4:

[0045] Trials of different drugs for the prevention of WSSV infection

[0046] Each tank contained 30 shrimp. The red swamp crayfish were treated with B51 (40 mg / L), B44 (40 mg / L), or magnolol (40 mg / L), or DMSO (0.04% DMSO, V). DMSO / V Water Pre-incubate for 0, 3, 6, and 12 hours, then transfer to a glass tank and add WSSV (final concentration 1.5 × 10⁻⁶). 5 Infected with (copies / μL) shrimp for 72 hours. The experiment was repeated 3 times, and the survival rate of shrimp was recorded every 24 hours.

[0047] After the experiment, surviving *Procambarus clarkii* were euthanized using MS-222, and WSSV viral load was detected by qPCR after tissue DNA extraction (see Example 7). The results showed that pretreatment with B51 significantly inhibited WSSV infection in *Procambarus clarkii*. Infection with *Procambarus clarkii* 6 hours after pretreatment reduced the viral load by 46.84%, while infection with *Procambarus clarkii* 12 hours after pretreatment reduced the viral load by 63.28%. Figure 4 Therefore, B51 has a preventive effect against WSSV infection in Procambarus clarkii, while B44 and magnolol have no preventive effect against WSSV infection in Procambarus clarkii.

[0048] Example 5:

[0049] Experiments on the direct killing of WSSV by different drugs

[0050] WSSV (final concentration 7.5 × 10⁻⁶) 7 The virus-drug mixture (copies / μL) was incubated with B51 (final concentration 40 mg / L), B44 (final concentration 40 mg / L), or magnolol (final concentration 40 mg / L), or 0.04% DMSO at 28°C for 1, 2, and 4 h. Subsequently, the virus-drug mixture was diluted 100-fold in water to achieve a final WSSV concentration of 1.6 × 10⁻⁶. 5 The virus-drug mixture was diluted to 0.4 mg / L, far below the biosafety concentrations of these drugs. The diluted virus-drug mixture was then incubated with 30 red swamp crayfish per tank for 72 hours.

[0051] After the experiment, surviving shrimp were euthanized using MS-222, and tissue DNA was extracted. WSSV viral load was then detected using qPCR (see Example 7). Viral load significantly decreased after 1, 2, and 4 hours of incubation with B51. Figure 5 The inhibition rate reached a maximum of 71.32%. B44 and magnolol had no significant killing effect.

[0052] Example 6:

[0053] Trials of different drugs for treating WSSV infection

[0054] Add the red swamp crayfish to the glass tank and mix with WSSV (final concentration 1.5 × 10⁻⁶). 5 Pre-incubate with B51 (final concentration 40 mg / L), B44 (final concentration 40 mg / L), or magnolol (final concentration 40 mg / L), or 0.04% DMSO for 72 hours, and record survival rates. Each tank contains 30 red swamp crayfish. Three replicates are performed each time.

[0055] After the experiment, tissue DNA was extracted, and WSSV load was detected using qPCR (see Example 7). Treatment with B51 24 hours post-WSSV infection significantly reduced WSSV load (P<0.01), and post-treatment at 48 hours post-WSSV infection significantly reduced WSSV load (P<0.05). Figure 6 The results showed that B51 could inhibit WSSV replication in *Procambarus clarkii*. Magnolol and B44 had no significant inhibitory effect.

[0056] Example 7:

[0057] WSSV Virus Load Detection Methods

[0058] DNA was extracted from *Procambarus clarkii* (WSSV) using a rapid genomic DNA extraction kit (OMEGA, USA). DNA concentration and purity were determined using a micro-volume nucleic acid analyzer. WSSV genome copy number was then determined by qPCR. The WSSV envelope protein VP28 (141 bp) was amplified using primers (VP28-F: 5'-AAACCTCCGCATTCCTGTGA-3', VP28-R: 5'-TCCGCATCTTCTTCCTTCAT-3') and inserted into the pMD19-T vector (Takara, China). The vector was then transferred to *E. coli* (DH5α) for large-scale production. The copy number of the target amplicon was quantified using a spectrophotometer (Thermo, USA), and the plasmid was serially diluted 10 times as a template for qPCR. qPCR conditions were as follows: 95℃ for 2 min, 95℃ for 15 sec for 40 cycles, and 56℃ for 30 sec. Melting curve analysis (65-95℃, 5 sec per step) was performed at the end of each PCR thermal curve to verify the amplification of individual products. The cycle threshold (Ct) and WSSV copy number are used to determine the standard curve for viral load.

[0059] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A magnolol derivative B51, with the molecular formula C 34 H 32 N2O8S2, molecular weight 660.76, structural formula is: 。 2. The method for preparing magnolol derivative B51 according to claim 1, comprising the following steps: Dichloromethane, magnolol, triethylamine and p-acetamidobenzenesulfonyl chloride were mixed and stirred at 48-52°C. After the reaction was completed, the mixture was extracted and separated by silica gel column chromatography to obtain the target product B51.

3. A compound formulation containing the magnolol derivative B51 as described in claim 1.

4. The use of the magnolol derivative B51 of claim 1, its pharmaceutically acceptable salt, or the compound formulation of claim 3 in the preparation of a medicament for the treatment or prevention of vitiligo syndrome.

5. The use of the magnolol derivative B51 of claim 1, its pharmaceutically acceptable salt, or the compound formulation of claim 3 in the preparation of a medicament for the treatment or prevention of vitiligo syndrome viral infection.

6. The use of the magnolol derivative B51 of claim 1, its pharmaceutically acceptable salt, or the compound formulation of claim 3 in the preparation of a vitiligo syndrome virus inhibitor.

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