Use of a protac compound targeting m2-1 protein in the preparation of an anti-rsv drug

Abstract

The application relates to an application of a PROTAC compound targeting M2-1 protein in preparation of an anti-RSV drug and belongs to the technical field of biological medicines. The bifunctional small molecule capable of simultaneously combining M2-1 protein and CRBN E3 ubiquitin ligase is constructed, the selective degradation of the M2-1 protein is realized, the limitation that the prior art cannot effectively target the protein is broken through, and a new technical approach is provided for solving the problems of single target, high drug resistance risk and lack of specific drugs in the treatment of RSV infection, and obvious novelty and significant substantial progress are achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a PROTAC compound targeting the M2-1 protein in the preparation of anti-RSV drugs. Background Technology

[0002] Respiratory syncytial virus (RSV) is one of the leading pathogens causing respiratory infections in children under 5 years old worldwide, posing a serious threat to children's health. Therefore, effectively controlling RSV infection and reducing hospitalization and mortality rates in infants and young children has become a pressing clinical problem to be solved globally.

[0003] RSV belongs to the genus Pneumovirus in the family Paramyxoviridae. It is a single-stranded, negative-sense RNA virus with a genome length of 15.2 kb, encoding 11 functional proteins. Among these proteins, G and F proteins mediate viral attachment and fusion through interaction with host cells; N protein protects viral RNA from degradation; M protein regulates viral assembly; and M2-1 protein plays a crucial role in viral transcriptional elongation and replication. Studies have shown that M2-1 protein not only enhances mRNA synthesis but also promotes the formation of ribonucleoprotein complexes when interacting with other viral proteins (such as N, P, and L proteins), thereby supporting viral replication. Therefore, M2-1 protein is an important regulator in the RSV life cycle and an ideal potential therapeutic target.

[0004] Currently, treatment options for RSV remain very limited, with clinical practice mainly relying on supportive care. In recent years, some progress has been made in the development of RSV vaccines and antiviral drugs, but the F protein remains the core target. For example, nirsevimab has been used to prevent lower respiratory tract infections in infants and young children, and ziresovir inhibits viral replication by blocking the fusion of the virus with host cells, but it is still in phase III clinical trials. Although these strategies have brought hope for RSV prevention and control, they still have several limitations: (1) the F protein is prone to mutation, making it difficult for existing treatments to cover different circulating strains; (2) some vaccines have insufficient immunogenicity, unstable protective effects, and still pose safety risks such as enhanced respiratory disease (ERD); (3) there are currently no antiviral drugs specifically for treating RSV infection in children, and the risk of drug resistance cannot be ignored. Given the limitations of the F protein targeting strategy, it is increasingly important to develop new targets focusing on key proteins in the RSV life cycle. M2-1, as an RSV transcription elongation factor, is an indispensable core regulatory molecule for viral replication and has become a potential new target for anti-RSV. Currently, vaccines targeting M2-1 are mainly gene transcription-expression-based, such as PanAd3-RSV, MVA-RSV, MVA-BN-RSV, and ChAd155-RSV. However, their protective efficacy in infants and young children remains controversial, and there is still a lack of antiviral drugs that directly target M2-1. Although some studies have attempted to intervene around the structure and function of M2-1, such as inhibiting the zinc-binding domain (ZBD) in its RNA-binding domain with AT-2, or disrupting inclusion body structure and blocking the interaction between M2-1 and the P protein with cyclopamine, these molecules often suffer from insufficient targeting, limited inhibitory efficiency, or lack of drug-like properties. More importantly, there are few reports on the host regulatory mechanisms of M2-1 protein expression and stability, and there is still a lack of effective means to precisely regulate its protein levels. This gap severely limits the development of M2-1-targeted drugs and also provides a breakthrough opportunity for innovative treatment strategies. Against this backdrop, with the development of protein degradation technology, protein hydrolysis targeting chimeras (PROTAC) provide a new feasible pathway for the direct and effective removal of key viral proteins, bringing an important opportunity to target M2-1 and achieve its complete and selective degradation.

[0005] Proteolytic targeting chimeras (PROTACs) are a class of bifunctional small molecules composed of two specific ligands covalently linked by a linker chain. One ligand recruits an E3 ubiquitin ligase, while the other specifically recognizes the target protein. When a PROTAC binds simultaneously to both the E3 ubiquitin ligase and the target protein, it promotes polyubiquitination of the target protein and guides its degradation by the proteasome. Unlike traditional "occupation-driven" drugs, PROTACs have the advantage of a catalytic mechanism, enabling efficient clearance of target proteins at lower concentrations and significantly improving drug utilization. Furthermore, PROTAC technology overcomes the dependence on traditional "drugable targets," providing a new intervention pathway for proteins that are difficult to target.

[0006] Although PROTAC technology has seen rapid development in the fields of oncology and immune diseases in recent years, and has shown great application potential in antiviral applications, current published research mainly focuses on virus-related targets such as HBV, HIV, influenza virus, and coronavirus. Existing literature shows that CRBN, as one of the most mature druggable E3 ubiquitin ligases, has had its ligand pomalidomide used to construct various highly efficient PROTAC molecules, demonstrating the feasibility of the CRBN-PROTAC system. However, to date, no publicly reported PROTAC molecules have been constructed and effectively degraded targeting the key regulatory protein M2-1 of respiratory syncytial virus (RSV). Furthermore, research on M2-1 mainly focuses on structural analysis, functional domain characterization, and the exploration of small molecule inhibitors; a mature drug strategy capable of effectively clearing M2-1 protein at the cellular level or in animal models has not yet been developed. More importantly, existing publicly available technologies do not include a technical solution for linking an M2-1-specific ligand with the CRBN ligand pomalidomide to form a bifunctional molecule, thereby recruiting the CRBN E3 complex to achieve M2-1 ubiquitination and degradation. Therefore, the development of CRBN-PROTAC degradative agents for RSV M2-1 is still in a technological gap. Summary of the Invention

[0007] Therefore, the purpose of this invention is to overcome the problems of existing RSV treatments, such as single target, poor adaptability to viral variants, and lack of effective drugs that directly act on key RSV proteins, and to provide a novel PROTAC compound that can specifically target and degrade the RSV M2-1 protein to achieve efficient blocking of RSV replication.

[0008] To address the aforementioned technical problems, this invention provides the application of a PROTAC compound targeting the M2-1 protein in the preparation of anti-RSV drugs. By constructing a bifunctional small molecule capable of simultaneously binding to both the M2-1 protein and the CRBN E3 ubiquitin ligase, this invention achieves selective degradation of the M2-1 protein, overcoming the limitations of existing technologies that cannot effectively target this protein. This provides a new technical approach to solving problems such as single target, high risk of drug resistance, and lack of specific drugs in the treatment of RSV infection, demonstrating significant novelty and substantial progress.

[0009] This invention is achieved through the following technical solution:

[0010] The purpose of this invention is to provide the application of a PROTAC compound targeting the M2-1 protein in the preparation of an anti-RSV drug, wherein the structural formula of the PROTAC compound is as follows:

[0011] .

[0012] In one embodiment of the present invention, the method for preparing the PROTAC compound includes the following steps:

[0013] (1) 1,3-benzothiazol-6-methyl-carboxylic acid was subjected to an amino protecting group removal reaction to obtain 4-benzylpiperazine hydrochloride;

[0014] (2) The hydroxyl group of 1,3-benzothiazol-6-carboxylic acid is activated by acyl chloride, and then undergoes a nucleophilic substitution reaction with potassium tert-butoxide to achieve tert-butoxylation of the hydroxyl group, so as to obtain 1,3-benzothiazol-6-carboxylic acid ester;

[0015] (3) The obtained 1,3-benzothiazole-6-carboxylic acid ester was reacted with hydrazine monohydrate in an alcohol solvent to obtain tert-butyl-4-amino-mercaptobenzoate;

[0016] (4) tert-butyl 4-amino-mercaptobenzoate was reacted with chloroacetyl chloride in an organic solvent to obtain tert-butyl 2-(chloromethyl)benzothiazole-6-carboxylic acid ester;

[0017] (5) 4-Benzylpiperazine hydrochloride was reacted with CS2 and NaOMe in an organic solvent, and tert-butyl 2-(chloromethyl)benzothiazole-6-carboxylic acid ester was added and reacted to obtain tert-butyl 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid ester;

[0018] (6) The tert-butyl 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid ester was subjected to a tert-butoxy protecting group removal reaction to obtain 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid;

[0019] (7) 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid was mixed with HATU, DIEA, pomalidomide-PEG3-C2-amino (trifluoroacetate) in an organic solvent to obtain a PROTAC compound targeting the M2-1 protein.

[0020] In one embodiment of the present invention, the concentration of the PROTAC compound is 10 mg / kg to 30 mg / kg.

[0021] In one embodiment of the present invention, the dosage form of the drug is selected from tablets, capsules, granules, oral liquids, emulsions, dry suspensions, dry extracts, or injections.

[0022] In one embodiment of the invention, the drug further includes a pharmaceutically or pharmacologically acceptable carrier and / or salt.

[0023] In one embodiment of the present invention, the carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, filler, suspending agent, surfactant, and preservative.

[0024] In one embodiment of the invention, the filler is selected from one or more of starch, sucrose, and lactose; the wetting agent includes glycerin; and the surfactant includes hexadecyl alcohol.

[0025] In one embodiment of the present invention, the adhesive is selected from one or more of cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone.

[0026] In one embodiment of the present invention, the disintegrant is selected from one or more of agar, calcium carbonate, and sodium bicarbonate.

[0027] In one embodiment of the invention, the pharmaceutically or pharmacologically acceptable salt is selected from inorganic acid salts and / or organic acid salts; the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

[0028] This invention provides a PROTAC compound that targets the M2-1 protein. This PROTAC compound can target the E3 ubiquitin ligase CRBN and the target protein RSV M2-1 and promote their degradation through a proteolytic targeting mechanism (PROTAC), thereby effectively inhibiting RSV replication.

[0029] Selection of CRBN-binding ligand for E3 ligase: This invention is based on CRBN as a recruitment element for E3 ubiquitin ligase, and pomalidomide is selected as a high-affinity, commercially available CRBN-binding ligand. The introduction of pomalidomide ensures that the PROTAC molecule can stably and efficiently recruit CRBN, thereby triggering ubiquitination of the target protein and subsequent proteasome degradation.

[0030] Specific binding ligand for RSV core transcriptional regulatory protein M2-1: This invention uses 10e as a small molecule ligand to recognize and bind to the RSV M2-1 protein. This ligand can specifically bind to the functional domain of M2-1, blocking the integrity of the viral transcription complex. The use of 10e as a targeting ligand to induce M2-1 degradation is unique and pioneering.

[0031] This invention uses polyethylene glycol (PEG3) as the linker arm connecting pomalidomide and 10e. PEG3 has moderate flexibility and spatial length, which ensures that the PROTAC molecule maintains a reasonable conformation in vivo, enabling CRBN and M2-1 to form a stable ternary complex and improving degradation efficiency.

[0032] This invention constructs a PROTAC molecule with a complete dual-ligand-connector arm structure, capable of simultaneously recruiting CRBN and targeting RSV M2-1, ultimately inducing ubiquitination of the M2-1 protein. This PROTAC molecule is not disclosed in existing technologies and is the first CRBN-based RSV antiviral degrader, which can be applied to reduce RSV infection.

[0033] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0034] This invention provides the application of a PROTAC compound targeting the M2-1 protein in the preparation of an anti-RSV drug. The PROTAC compound molecule provided by this invention can target the active pocket of M2-1, causing M2-1 degradation, and inhibiting RSV infection by recruiting CRBN protein. The PROTAC compound targeting the M2-1 protein provided by this invention has been shown to have low cytotoxicity through CCK-8 assays; Western blot experiments have demonstrated the degradation effect of this PROTAC compound; RT-qPCR, Western blot, and immunofluorescence experiments have demonstrated that the PROTAC compound targeting the M2-1 protein can inhibit RSV infection in vitro; and mouse RSV infection model experiments have demonstrated the in vivo anti-RSV infection effect of this PROTAC compound. It shows promise for development into a drug targeting the M2-1 protein to treat RSV infection. Attached Figure Description

[0035] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0036] Figure 1 This is a graph showing the degradation results of the PROTAC compound of this invention at five concentrations;

[0037] Figure 2 This is a diagram showing the experimental results of Hep2 cell proliferation inhibition in this invention;

[0038] Figure 3 The results of RT-qPCR after treating RSV-infected cells with DMSO and PROTAC compounds in this invention;

[0039] Figure 4 This invention utilizes DMSO and PROTAC compounds to treat RSV-infected cells and perform Western blot analysis.

[0040] Figure 5 This is the result of viral immunofluorescence after treating RSV-infected cells with DMSO and PROTAC compounds according to the present invention;

[0041] Figure 6 This is the result of virus titer detection after RSV-infected cells were treated with DMSO and PROTAC compounds according to the present invention;

[0042] Figure 7 This invention provides RT-qPCR detection results of the antiviral activity of PROTAC compounds in vivo.

[0043] Figure 8 This invention provides Western blot detection results of the antiviral activity of PROTAC compounds in vivo.

[0044] Figure 9 This invention uses HE staining to detect pathological changes in the lungs of RSV-infected mice after treatment with PROTAC compounds;

[0045] Figure 10 This invention uses a Co-IP experiment to detect the ubiquitination level of M2-1 mediated by PROTAC compounds. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0048] Example 1:

[0049] This embodiment provides a PROTAC compound targeting the M2-1 protein, with the following structural formula:

[0050] .

[0051] The specific steps for preparing the aforementioned PROTAC compound targeting the M2-1 protein are as follows:

[0052] Step 1:

[0053]

[0054] 1,3-benzothiazol-6-methyl-carboxylic acid (HM-2972) _ 1. CAS No. 57260-70-5, 5.0 g, 18.1 mmol, 1.0 eq.) was suspended in HCl / 1,4-dioxane (4.0 M) (100 mL), and the mixture was reacted at 20-30 °C for 2 hours. LCMS analysis showed that the reactants had largely reacted. MTBE (80 mL) was added to the mixture, and the mixture was filtered. The solid was collected and dried using a vacuum oil pump to obtain the target product, 4-benzylpiperazine hydrochloride (HM-2972). _ 2. CAS No. 110475-31-5, 3.56 g, yield: 85%, white solid. LCMS: SY48365-009-02-1 (ESI), rt=0.31 min, m / z: 177.2 [M+H] + P=95.1%.

[0055] Step Two:

[0056]

[0057] 1,3-benzothiazole-6-carboxylic acid (HM-2972) _3. CAS No. 3622-35-3, 4.9 g, 27.3 mmol, 1.0 eq.) was suspended in anhydrous toluene (200 mL), and N,N-dimethylformamide (200 mg, 2.73 mmol, 0.1 eq.) and thionyl chloride (3.90 g, 32.8 mmol, 1.2 eq.) were added. The mixture was stirred at 70-80°C for 2 hours. A small amount of the reaction solution was taken, quenched with methanol, and LCMS analysis confirmed the completion of the acyl chloride reaction. The mixture was evaporated to dryness under vacuum to obtain a yellow solid, dissolved in tetrahydrofuran (200 mL), and the system was cooled to -20°C to -15°C. Potassium tert-butoxide (dissolved in tetrahydrofuran, 1M) (35.5 mL, 35.5 mmol, 1.3 eq.) was added dropwise. After the addition was complete, the system was slowly heated to 20-30°C and stirred for 2 hours. LCMS analysis confirmed that the reaction of the raw materials was essentially complete. The mixture was then purified by column chromatography [silica gel, EA / PE = 0:100~70:30] to obtain the target product, 1,3-benzothiazol-6-carboxylic acid ester (HM-2972_4, CAS No. 1443543-00-7, 4.7 g, yield: 73%, yellow solid). LCMS: SY48365-003-01-1 (ESI), rt = 1.603 min, m / z: 236.0 [M+H] + P=99.54%.

[0058] Step 3:

[0059]

[0060] 1,3-benzothiazole-6-carboxylic acid ester (HM-2972_4, CAS No. 1443543-00-7, 4.6 g, 19.5 mmol, 1.0 eq.) was dissolved in anhydrous 95% EtOH (35.0 mL), the system was cooled to 0℃-5℃, and hydrazine monohydrate (85% w / w) (29.3 g) was added dropwise. After the addition was complete, the mixture was slowly heated to 20℃-30℃ and stirred for 16 h. LCMS analysis indicated that the reaction of the raw materials was essentially complete. The mixture was adjusted to pH 6.0 with glacial acetic acid, diluted with 200 mL of water, and the product was extracted with DCM (100 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness and purified by column chromatography [silica gel, EA:PE = 0:100~50:50] to obtain the target product, tert-butyl-4-aminothiobenzoate (HM-2972_5, 3.1 g, yield: 70%, yellow solid). LCMS: SY48365-007-02-1 (ESI), rt = 1.548 min, m / z: 226.0 [M+H] +P=83.32%.

[0061] Step Four:

[0062]

[0063] tert-butyl 4-aminothiobenzoate (HM-2972_5, 3.0 g, 13.3 mmol, 1.0 eq.) was dissolved in anhydrous DCM (100 mL). The system was cooled to 0℃-5℃, and chloroacetyl chloride (1.80 g, 16.0 mmol, 1.2 eq.) was added dropwise. After the addition was complete, the mixture was slowly heated to 20℃~30℃ and stirred for 16 hours. LCMS analysis showed that the reaction of the starting material was basically complete. Silica gel (100 g) was added to the mixture, and DCM was removed by rotary evaporation. The mixture was then purified by column chromatography [silica gel, EA / PE = 0:100~70:30] to obtain the target product, tert-butyl 2-(chloromethyl)benzothiazole-6-carboxylic acid ester (HM-2972_6, 2.0 g, yield: 53%, yellow solid). LCMS: SY48365-013-02-1 (ESI), rt=1.817 min, m / z:284.0 [Μ+H] + P=97.26%.

[0064] Step 5:

[0065]

[0066] 4-Benzylpiperazine hydrochloride (HM-2972_2, 3.34 g, 13.4 mmol, 2.0 eq.) was dissolved in DMF (80 mL). CS2 (1.02 g, 13.4 mmol, 2.0 eq.) and NaOMe (1.09 g, 20.1 mmol, 3.0 eq.) were added with stirring at 0-5°C. The mixture was stirred at 20-30°C for 1 hour. Then, tert-butyl-2-(chloromethyl)benzothiazole-6-carboxylic acid ester (HM-2972_6, 1.9 g, 6.70 mmol, 1.0 eq.) was added. The mixture was stirred at 20-30°C for 16 hours. LCMS analysis confirmed that the reaction of the raw materials was essentially complete. The mixture was then purified by Prep-Rp-HPLC to obtain the target product, tert-butyl 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid ester (HM-2972_7, 2.1 g, yield: 63%, yellow solid). LCMS: SY48365-017-P1 (ESI), rt=2.275 min, m / z: 500.1 [M+H] + P=96.36%.

[0067] Step Six:

[0068]

[0069] tert-butyl 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid ester (HM-2972_7, 2.0 g, 4.0 mmol, 1.0 eq.) was dissolved in DCM (18 mL), and TFA (15 mL) was added with stirring. The mixture was stirred at 20-30°C for 1 hour. LCMS analysis showed that the reaction of the starting material was basically complete. The mixture was rotary evaporated to remove DCM, and the residue was purified by column chromatography [C18, CH3CN: 0.1% TFA / water = 0:100-40:60]. After lyophilization, the target product 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid (HM-2972_8, 1.5 g, yield: 85%, yellow solid) was obtained. LCMS: SY48365-021-P1 (ESI), rt=0.841 min, m / z:444.1 [Μ+H] + P=93.78%.

[0070] Step Seven:

[0071]

[0072] 2-((4-phenylmethylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid (HM-2972_8, 1.3 g, 2.93 mmol, 1.0 eq.) was dissolved in DMF (20 mL). HATU (1.34 g, 3.52 mmol, 1.2 eq.), DIEA (1.14 g, 8.79 mmol, 3.0 eq.), and HM-2972_9 (1.98 g, 3.52 mmol, 1.2 eq.) were added with stirring. The mixture was stirred at 20-30°C for 1 hour. LCMS analysis showed that the reaction of the starting materials was essentially complete. The mixture was purified by Prep-Rp-HPLC to obtain the target product HM-2972_10 (1.2 g, yield: 47%, yellow solid). LCMS: SY48365-025-P1(ESI), rt=1.782 min, m / z:874.2 [Μ+H] + P=99.70%.

[0073] Example 2: Verification of the effect of PROTAC compound on degrading viral protein M2-1.

[0074] M2-1 viral protein particles were transfected into hep2 cells. Twenty-four hours later, cells were treated with different concentrations (0, 10 nM, 100 nM, 1000 nM, 10000 nM) of PROTAC compound. Western blot analysis of M2-1 viral protein expression was performed 48 hours later. The experimental results are as follows: Figure 1 As shown, Figure 1 This indicates that the PROTAC compound successfully degraded the M2-1 protein, and the degradation effect was best at a concentration of 10 μM.

[0075] PROTAC was then subjected to cytotoxicity experiments. Figure 2 Experimental results showed that PROTAC did not affect cell viability within the concentration range of 0-10 μM, demonstrating its good biocompatibility and suitability for further functional studies. Therefore, a PROTAC concentration of 10 μM was selected as the optimal concentration for subsequent in vitro experiments.

[0076] Example 3: In vitro evaluation of the antiviral activity of PROTAC compounds.

[0077] Hep2 cells infected with RSV at different time points were treated with PROTAC compound and DMSO, respectively. After 48 hours of treatment, the viral load in Hep2 cells was analyzed by real-time quantitative PCR (RT-qPCR) and Western blot. The experimental results are as follows: Figure 3 , Figure 4 The results showed that the PROTAC compound effectively reduced viral mRNA and protein levels, and Figure 5 The expression level of RSV fluorescent protein decreased after administration of the PROTAC compound; furthermore, enzyme-linked immunospot assay showed that... Figure 6 The addition of PROTAC compounds resulted in a decrease in viral titers in hep2 cells. These experiments demonstrate that PROTAC compounds exert their antiviral effect by degrading viral components.

[0078] Example 4: In vivo evaluation of the antiviral ability of PROTAC compounds to degrade M2-1 viral proteins.

[0079] To further verify the antiviral activity of PROTAC compounds, 15 C57 mice (purchased from Shanghai Nanmo Biotechnology Co., Ltd.) weighing 20 g were selected as experimental animals. The mice were randomly divided into groups of 3 mice each. PBS was used as a control. The other mice were infected with RSV via nasal drip for 24 hours, and then administered the drug intraperitoneally daily at different concentrations (0, 10 mg / kg, 20 mg / kg, and 30 mg / kg) for 4 consecutive days. On the fifth day, lung tissue was collected from the mice, homogenized, and the viral load in the lungs was detected by RT-qPCR and Western blot. Lung damage was also assessed by HE staining. The results showed that the PROTAC compounds possess antiviral function in vivo. Figure 7 The study showed that the lowest RSV mRNA level in mice was observed at a PROTAC compound concentration of 20 mg / kg. Figure 8 , Figure 9 The results showed that when the concentration of PROTAC compound was 30 mg / kg, the RSV protein level in mice was the lowest, and the lung damage was the least, even approaching that of the PBS group.

[0080] Example 5: Co-IP assay showed that the PROTAC compound could enhance the ubiquitination level of M2-1 viral proteins.

[0081] Overexpression of CRBN, M2-1, and Ub plasmids was performed in 293T cells. After 24 hours, cells were treated with either DMSO (control) or PROTAC compounds. Following drug treatment, cells were lysed using IP lysis buffer and centrifuged at 12,000 rpm for 20 minutes. The supernatant was collected for Western blot or co-immunoprecipitation (Co-IP) analysis of whole-cell lysates. For Co-IP, the lysate was incubated overnight at 4°C with anti-Myc magnetic beads. The next day, the beads were washed three times with pre-cooled PBS, followed by elution with SDS-PAGE loading buffer to remove binding proteins, and then boiled for 10 minutes for complete denaturation. For Western blot analysis, the immunoprecipitate or whole-cell lysate was separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, and then immunoblotted using anti-HA, anti-Myc, anti-CRBN, and anti-GAPDH antibodies, respectively. The effects of PROTAC-mediated CRBN on the ubiquitination level of M2-1 protein were evaluated using the Co-IP experiment described above. Figure 10 As shown, the PROTAC compound significantly enhanced the level of CRBN-mediated M2-1 protein ubiquitination.

[0082] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of a PROTAC compound targeting the M2-1 protein in the preparation of an anti-RSV drug, characterized in that, The structural formula of the PROTAC compound is: 。 2. The application according to claim 1, characterized in that, The preparation method of the PROTAC compound includes the following steps: (1) 1-Benzyl-4-Boc-piperazine was subjected to an amino protecting group removal reaction to obtain 4-benzylpiperazine hydrochloride; (2) The hydroxyl group of 1,3-benzothiazol-6-carboxylic acid is activated by acyl chloride, and then undergoes a nucleophilic substitution reaction with potassium tert-butoxide to achieve tert-butoxylation of the hydroxyl group, so as to obtain 1,3-benzothiazol-6-carboxylic acid ester; (3) The obtained 1,3-benzothiazole-6-carboxylic acid ester was reacted with hydrazine monohydrate in an alcohol solvent to obtain tert-butyl-4-amino-mercaptobenzoate; (4) tert-butyl 4-amino-mercaptobenzoate was reacted with chloroacetyl chloride in an organic solvent to obtain tert-butyl 2-(chloromethyl)benzothiazole-6-carboxylic acid ester; (5) 4-Benzylpiperazine hydrochloride was reacted with CS2 and NaOMe in an organic solvent, and tert-butyl 2-(chloromethyl)benzothiazole-6-carboxylic acid ester was added and reacted to obtain tert-butyl 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid ester; (6) The tert-butyl 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid ester was subjected to a tert-butoxy protecting group removal reaction to obtain 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid; (7) 2-((4-benzylpiperazine-1-carbonylthio)thiomethyl)benzothiazole-6-carboxylic acid was mixed with HATU, DIEA, and pomalidomide-PEG3-C2-aminotrifluoroacetate in an organic solvent to obtain the PROTAC compound.

3. The application according to claim 1, characterized in that, The dosage form of the drug is selected from tablets, capsules, granules, oral liquids, emulsions, dry suspensions, dry extracts, or injections.

4. The application according to claim 1, characterized in that, The drug also includes pharmaceutically or pharmacologically acceptable carriers and / or salts.

5. The application according to claim 4, characterized in that, The carrier is selected from one or more of the following: disintegrant, diluent, lubricant, adhesive, humectant, flavoring agent, filler, suspending agent, surfactant, and preservative.

6. The application according to claim 5, characterized in that, The filler is selected from one or more of starch, sucrose, and lactose; the wetting agent includes glycerin; and the surfactant includes hexadecyl alcohol.

7. The application according to claim 5, characterized in that, The adhesive is selected from one or more of cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone.

8. The application according to claim 5, characterized in that, The disintegrant is selected from one or more of agar, calcium carbonate, and sodium bicarbonate.

9. The application according to claim 4, characterized in that, The pharmaceutically or pharmacologically acceptable salt is selected from inorganic acid salts and / or organic acid salts; the organic acid salt is selected from alkyl sulfonates and / or aryl sulfonates.

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