Compound EA02, preparation method thereof and application of compound EA02 in preparation of antiviral drugs

Abstract

The invention discloses a compound EA02, a preparation method thereof and application of the compound EA02 in preparation of antiviral drugs. The invention identifies that a small molecule compound EA02 (7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy) phenyl)-N-(2-oxo-1, 2, 3, 4-tetrahydroisoquinoline-6-yl) pyrazolo [1, 5-a] pyrimidine-2-formamide (EA02)) is an activator for dimerization of EHD4, and proves that the treatment of the small molecule compound can activate non-classical autophagy and realize antiviral infection on the cell and animal level. The compound EA02 is expected to become a new drug and is applied to anti-infection treatment and health care product medicine industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a compound EA02, a preparation method thereof and application thereof in preparing antiviral drugs. BACKGROUND

[0002] Autophagy is a fundamental process that exists in eukaryotic organisms to maintain cellular homeostasis and life activities. As a dynamic recycling mechanism for redundant or misfolded proteins in cells, autophagy provides support for cell renewal and homeostasis by producing reusable small molecules and energy, and plays a key role in physiological processes including metabolic adaptation, intracellular quality control, and renewal during development and differentiation. The molecular mechanisms of autophagy-related proteins regulating classic macroautophagy have been relatively clear, however, more and more evidence shows that these proteins also play other functions beyond classic autophagy, and the molecular mechanisms and final target of regulating these functions are different from classic autophagy, these "autophagy-like" molecular pathways are called non-canonical autophagy. LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO) are representatives of non-canonical autophagy. The molecular mechanisms of LAP and LANDO are similar to some extent, but also different from classic autophagy. First, LAP and LANDO are not initiated by stress conditions such as starvation, and have no correlation with the upstream initiation signal AMPK-mTORC1 axis of classic autophagy. Second, non-canonical autophagy occurs on single-membrane vesicles, rather than forming double-membrane autophagosomes like classic autophagy. Third, the initiation of LAP and LANDO does not require the initiation complex of classic autophagy such as ULK1 complex and PI3KC3 complex I, but depends on the member VPS34 protein in PI3KC3 complex I. Fourth, the initiation of LAP and LANDO both depends on the regulator Rubicon protein, which as an inhibitor of classic autophagy, binds to Beclin-1, UVRAG and VPS34 protein to form PI3KC3 complex II to initiate non-canonical autophagy. Fifth, the WD40 domain of ATG16L1, an important molecule in the lipidation process of ATG8 family proteins, is essential for non-canonical autophagy, but not for classic autophagy. Functionally, LAP mediates the fusion of phagocytic vesicles with lysosomes to degrade phagocytic substrates, and there is evidence that it is involved in immune regulation related to macrophages. LANDO does not mediate the fusion of endocytic vesicles with lysosomes, but regulates the transport and recycling of endocytic vesicles, and is very important for the recycling of amyloid-β (Aβ) receptors, and its dysfunction is significantly associated with the occurrence of neurodegenerative diseases. Therefore, non-canonical autophagy has a key regulatory role in many physiological activities and is an important homeostatic mechanism in cells. However, current research in the field of cellular autophagy focuses on classic autophagy, and the diversity and specific mechanisms of non-canonical autophagy are not well understood. Moreover, few potential drugs targeting non-canonical autophagy have been developed, and the reason may be that the only known regulator of non-canonical autophagy, Rubicon, is an inhibitor of classic autophagy, and therefore is not suitable as a drug target for specifically initiating non-canonical autophagy.Therefore, finding a molecular pathway that specifically regulates non-canonical autophagy and designing small molecule drugs to activate non-canonical autophagy by targeting the pathway is a safe and efficient strategy.

[0003] Viruses are an important factor threatening human health and public safety. Given the high heterogeneity and rapid variation of viruses, it is important to design virus prevention and control strategies from the perspective of the host's pathogen defense system. There is some evidence that classical autophagy plays a crucial role in anti-infection, for example, in vitro infection of human neutrophils with Streptococcus pneumoniae can induce autophagy and enhance phagocytic activity, and exposure to autophagy inhibitors can significantly impair phagocytic activity. In Mycobacterium tuberculosis infection, autophagy can partially reduce the entry of mycobacteria into the cytosol through the ESX-1 secretion system and prevent necrosis to protect host macrophages. However, for many viruses that enter cells through endocytosis (such as VSV, HSV, IAV, DENV, etc.), it is still unclear whether the body can initiate endocytosis-related non-canonical autophagy to clear them. In addition, there is currently no anti-infective small molecule drug targeting non-canonical autophagy. SUMMARY

[0004] The purpose of the present application is to elucidate a new molecular initiation mechanism of non-canonical autophagy, develop small molecule activators targeting this pathway, and clear endocytosis viruses by specifically activating endocytosis-related non-canonical autophagy, thereby providing a safe and efficient new strategy for anti-infective drugs.

[0005] Autophagy is divided into canonical autophagy and non-canonical autophagy, and is a highly conserved physiological process in eukaryotic organisms, which plays an important role in maintaining cell homeostasis and a number of basic life activities. The molecular mechanism of canonical autophagy has been widely studied, however, the molecular mechanism and function of non-canonical autophagy are not clear, and few small molecule compounds have been confirmed to be able to regulate the non-canonical autophagy pathway. By combining bioinformatics and high-throughput mass spectrometry analysis, the present application finds that the small molecule compound 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) can activate non-canonical autophagy, and resist viral infection by starting non-canonical autophagy. We found that the RUN domain and FYVE domain containing protein 2 (RUFY2) of vacuolar sorting protein 34 (VPS34, encoded by the PI3KC3 gene) can promote the synthesis of phosphatidylinositol-3-phosphate (PI3P) on the endosome membrane, and recruit EH domain containing protein 4 (EHD4) in non-canonical autophagy. EHD4 binds to autophagy-related gene 7 (ATG7) protein, promotes the lipidation of autophagy-related gene 8 (ATG8) family protein, and acts on endocytosis-related non-canonical autophagy with RUFY2. In combination with the characteristic of EHD4 activation process that will occur polymerization, using molecular docking technology, we identify the small molecule compound EA02 as an activator of EHD4 dimerization, and prove that its treatment can activate non-canonical autophagy and achieve anti-viral infection at the cellular and animal levels. In summary, we identify a new non-canonical autophagy initiation mechanism, and for the first time find that the small molecule EA02 can activate non-canonical autophagy and help the body resist viral infection by activating the molecular pathway. The related application is expected to become a new drug, which can be used in anti-infection treatment and health care product medical industry, thereby achieving the purpose of the present application.

[0006] The present application provides 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide and its synthesis steps, and experimentally verifies its functions of activating non-canonical autophagy and resisting viruses.

[0007] The compound EA02 of the present application, i.e. 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02), has the following chemical structure:

[0008]

[0009] 7-(3-(Cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) can interact with EHD4.

[0010] Treatment of cells with appropriate concentrations of 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) can increase the level of multimerization activation of EHD4. Treatment of cells with appropriate concentrations of 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) can initiate EHD4-RUFY2 axis-mediated non-canonical autophagy.

[0011] Treatment of cells or mice with appropriate concentrations of 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) can effectively resist viral infection in a manner dependent on the EHD4-RUFY2 axis.

[0012] Therefore, the present application also provides the use of the above-mentioned compound EA02 or its pharmaceutically acceptable salt, solvate or derivative thereof in the preparation of a non-canonical autophagy agonist or an antiviral drug.

[0013] Preferably, the virus can be various viruses, such as Vesicular Stomatitis Virus (VSV).

[0014] The present application also provides a non-canonical autophagy agonist or an antiviral drug containing the above-mentioned compound EA02 or its pharmaceutically acceptable salt, solvate or derivative thereof as an active ingredient.

[0015] Preferably, the drug can be an injection, oral, inhalation, transdermal drug.

[0016] The present application also provides a preparation method of the compound EA02, which comprises the following steps:

[0017] The reaction formula is as follows:

[0018]

[0019] Compound 1 undergoes a nucleophilic substitution reaction with ethyl 2-chloro-2,2-difluoroacetate in the presence of sodium carbonate in DMF solvent to generate compound 2. Compound 2 then undergoes a reaction with bromomethylcyclopropane in the presence of potassium carbonate in DMF solvent to generate compound 3. Compound 3 reacts with N,N-dimethylformamide dimethyl acetal (DMFDMA) in DMF solvent to generate key intermediate compound 4. Compound 4 undergoes a cyclization reaction with methyl 5-amino-1H-pyrazole-3-carboxylate in the presence of acetic acid to generate compound 5. Compound 5 is hydrolyzed with sodium hydroxide in a mixture of ethanol / water solvent to convert its methyl ester to carboxylic acid to generate compound 6. Finally, compound 6 undergoes an amide condensation reaction with 6-amino-3,4-dihydro-1H-quinolin-2-one in DMF solvent with the addition of HATU and DIPEA to generate the target compound EA02.

[0020] 1. The present application first discovers that the small molecule compound 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) specifically initiates non-canonical autophagy by targeting the EHD4-RUFY2 axis.

[0021] 2. The present application first discovers that the small molecule compound 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) can initiate non-canonical autophagy by targeting the EHD4-RUFY2 axis to reduce the level of viral infection in the body.

[0022] 3. The present application first describes the specific molecular mechanism of the small molecule compound 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02) in initiating non-canonical autophagy by targeting the EHD4-RUFY2 axis and mediating viral clearance. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1Figure A is the analysis result of searching for proteins with RUN domain in human genome. Figure B is the sequence alignment result of RUFY family proteins and RUN domain sequence of RUBCN. Figure C is the immunoblotting result of detecting the protein level change of endogenous LC3B type and type I in HeLa cells transfected with Flag-RUFY1-4 for 48 h, treated with Baf Al (250 nM) for 3 h, and collected protein samples. Figure D is the quantitative analysis result of similar samples as in Figure C. Figure E is the immunofluorescence result of staining LC3B aggregates with LC3B antibody and green fluorescent secondary antibody in HeLa cells transfected with siRNA-Scr or siRNA-RUFY2, treated with Torin-1 (2 μM, 3 h) or monensin (50 μM, 1 h) for different groups, and fixed after 60 h of transfection. Figure F is the quantitative analysis result of similar samples as in Figure E. Figure G is the immunofluorescence result of HeLa cell lines of wild type or RUFY2 knockout transfected with GFP-LC3B for 24 h, treated with dextran (200 μg / mL) and monensin (50 μM) for 1 h. Figure H is the quantitative analysis result of similar samples as in Figure G. Figure I is the immunofluorescence result of HeLa cell lines of RUBCN knockout transfected with siRNA-Scr or siRNA-RUFY2, treated with monensin (50 μM, 1 h) for different groups, and fixed after 60 h of transfection. Figure J is the quantitative analysis result of similar samples as in Figure I.

[0024] Figure 2Experimental results of RUFY2 regulating the level of PI3P on endosome membrane by interacting with VPS34 in the embodiments of the present application: Figure A is the immunofluorescence result of transfected GFP-FYVE (used to indicate PI3P) for 24h in wild type or RUFY2 knock-out HeLa cell lines, dextran (200 μg / mL) and monensin (50 μM) treatment for 1h. Figure B and Figure C are the quantitative analysis results of similar samples as Figure A. Figure D is the MTM1-inducible-HeLa cells transfected with siRNA-Scr or siRNA-RUFY2, treated with monensin (50 μM, 1h). Fixed after 60h of transfection, the immunofluorescence result of LC3B puncta staining using LC3B antibody and green fluorescent secondary antibody. Figure E is the quantitative analysis result of similar samples as Figure D. Figure F is the experimental result of HeLa cells treated with monensin (50 μM, 1h), after sample collection, the whole cell lysate (WCL) of HeLa cells was immunoprecipitated by protein A / G agarose beads and anti-VPS34 antibody, then the expression level of each molecule in WCL was analyzed by immunoblotting reaction and the level of RUFY2 protein after immunoprecipitation was analyzed using RUFY2 endogenous antibody. Figure G is the quantitative analysis result of similar samples as Figure F. Figure H is the immunofluorescence result of PI3KC3 knock-out HeLa cell lines transfected with siRNA-Scr or siRNA-RUFY2, treated with monensin (50 μM, 1h) for different groups, fixed after 60h of transfection, LC3B puncta staining using LC3B antibody and green fluorescent secondary antibody. Figure I is the quantitative analysis result of similar samples as Figure H.

[0025] Figure 3The following are experimental results from this invention regarding RUFY2 recruiting EHD4 and co-regulating non-canonical autophagy: Figures A and B show the GO-BP analysis of the RUFY2 protein-protein interactionome (A) and the changes in the abundance of proteins related to endocytosis or possessing ATP / GTPase activity before monensin treatment (B). Data were obtained from two independent replicate experiments. Figure C shows the results of treating HeLa cells with monensin (50 μM, 1 h), followed by immunoprecipitation of whole-cell lysate (WCL) of HeLa cells using protein A / G agarose beads and anti-RUFY2 antibody. The expression levels of various molecules in WCL were then analyzed by Western blotting, and the level of EHD4 protein after immunoprecipitation was analyzed using an endogenous EHD4 antibody. Figure D shows the quantitative analysis results for samples similar to those in Figure C. Figure E shows the immunofluorescence results of different groups of wild-type or EHD4 knockout HeLa cells treated with Torin-1 (2 μM, 3 h) or monensin (50 μM, 1 h), and the staining of LC3B aggregates with LC3B antibody and green fluorescent secondary antibody. Figure F shows the quantitative analysis results of samples similar to those in Figure E. Figure G shows the immunoblotting results of wild-type or EHD4 knockout HeLa cells transfected with HA-EHD4 wild-type, G68R mutant, or K330D mutant, and the detection of changes in endogenous LC3B and type I protein levels in the cells. Figure H shows the immunoblotting results of wild-type or RUFY2 knockout HeLa cells transfected with HA-EHD4, with or without Flag-RUFY2 replenishment in RUFY2 knockout HeLa cells, treated with monensin (50 μM, 1 h), and the detection of changes in endogenous LC3B and type I protein levels in the cells. Figure I shows the results of an immunoblotting experiment to detect changes in the protein levels of endogenous LC3B and I proteins in wild-type or RUBCN knockout HeLa cells after transfection with HA-EHD4 and treatment with monensin (50 μM, 1 h).

[0026] Figure 4The following are experimental results illustrating how EHD4 and ATG7 binding promotes the interaction between ATG7 and LC3 in this embodiment of the invention: Figures A and B show the GO-BP analysis of the EHD4 protein interactome (A) and the abundance of autophagy-related proteins (B), with data from two independent replicate experiments. Figure C shows the results of treating HeLa cells with monensin (50 μM, 1 h), followed by immunoprecipitation of whole-cell lysate (WCL) of HeLa cells using protein A / G agarose beads and anti-ATG7 antibody. The expression levels of various molecules in WCL were then analyzed by Western blotting, and the level of EHD4 protein after immunoprecipitation was analyzed using an endogenous EHD4 antibody. Figure D shows the quantitative analysis results for samples similar to those in Figure C. Figure E shows the experimental results of HeLa cells transfected with siRNA-Scr or siRNA-RUFY2, treated with torin-1 (2 μM, 3 h) or monensin (50 μM, 1 h), and then subjected to immunoprecipitation of whole-cell lysates (WCL) with protein A / G agarose beads and anti-ATG7 antibody. The expression levels of various molecules in WCL were analyzed by Western blotting, and the level of LC3B protein after immunoprecipitation was analyzed using an endogenous LC3B antibody. Figure F shows the quantitative analysis results for samples similar to Figure E. Figure G shows the experimental results of HeLa cells transfected with HA-EHD4 wild-type or G68R mutant, and then subjected to immunoprecipitation of whole-cell lysates (WCL) with protein A / G agarose beads and anti-ATG7 antibody. The expression levels of various molecules in WCL were analyzed by Western blotting, and the level of LC3B protein after immunoprecipitation was analyzed using an endogenous LC3B antibody. Figure H shows the quantitative analysis results for samples similar to Figure G.

[0027] Figure 5The following are the experimental results for screening and validating EHD4 activators in this invention: Figure A shows the results of immunoblotting experiments on the dimerization and oligomerization levels of endogenous EHD4 after HeLa cells were treated with monensin (50 μM, 1 h) or Torin-1 (2 μM, 3 h) and then subjected to protein cross-linking using DSS. Figure B shows the molecular docking analysis results of EHD4. Figure C shows the screening results of the top ten small molecule drugs in (B) on the dimerization and oligomerization of EHD4. The drug treatment concentration and time were both 5 μM for 24 h, and the results were obtained from three biological replicate experiments. Figure D shows the structural formula of 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinoline-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide (EA02). Figure E is a schematic diagram of the three-dimensional structure of EA02. Figures F and G illustrate the interaction between EA02 and EHD4. Figure H shows the results of an experiment where HeLa cells were transfected with Flag-EHD4, treated with EA02 (5 μM, 24 h) for 12 h, then collected, and treated at the temperatures shown in the figure for 3 min before lysing for immunoblotting analysis. Figure I shows the results of an immunoblotting experiment on wild-type and EHD4 knockout HeLa cells treated with the concentration of EA02 shown in the figure for 24 h, collecting protein samples, and detecting changes in the protein levels of endogenous LC3B and I types in the cells.

[0028] Figure 6 Experimental results related to the antiviral function of the EHD4-RUFY2 axis and EA02 in the embodiments of the present invention: Figure A shows the results of A549 cells transfected with EGFP-EHD4 for 24 hours, after which pre-chilled VSV-red (MOI=1000) was added on ice (via VSV and Alexa Fluor). TM The sample was obtained by co-incubation with 568 succinimide ester, placed on ice for 30 min, then cultured at 37℃ for 1 h, and observed using a confocal microscope after fixation. Figure B shows the quantitative analysis results of a sample similar to Figure A. Figure C shows the results of A549 cells transfected with EGFP-EHD4 for 24 h, with the medium changed and pre-chilled VSV-red (MOI = 1000) added (via VSV and Alexa Fluor). TMThe sample was obtained by co-incubation with 568 succinimide ester, placed on ice for 30 min, and then cultured at 37℃ for 1 h. After fixation, the LC3B aggregates were stained with LC3B antibody and far-infrared fluorescent secondary antibody and observed using a confocal microscope. Figure D shows the quantitative analysis results of a sample similar to Figure C. Figure E shows the results of Western blotting experiments on VSV-G protein levels in A549 cells transfected with siRNA-Scr, siRNA-EHD4 (#1 or #2) or siRNA-RUFY2 (#1 or #2) for 60 h, and then infected with VSV (MOI = 0.1) for 18 h. Figures I to K show the results of mouse tail vein injection of AAV-shScr or AAV-shEhd4 for two weeks, followed by oral administration of EA02 (40 mg / mL) for 15 days, and tail vein injection of VSV virus (pfu = 5 × 10⁻⁶). 7 Results of experiments at 6 days (survival curve, F) or 3 days (HE and IHC staining, G and H). Detailed Implementation

[0029] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0030] Example 1:

[0031] 1. RUFY2 can initiate non-classical autophagy related to endocytosis.

[0032] By searching the human genome, we found that RUFY family proteins carry a similar RUN domain to the non-classical autophagy regulator Rubicon. Figure 1 A and Figure 1 B), among which RUFY2 overexpression significantly increased autophagy levels (B). Figure 1 C and Figure 1 D). Treatment of cells with the classical autophagy activator torin-1 and the non-classical autophagy activator monensin, respectively, showed that knocking down RUFY2 reduced the number of monensin-induced LC3B aggregates but had no effect on the number of torin-1-induced LC3B aggregates. Figure 1 E and Figure 1 F. Immunofluorescence results using the endocytosis substrate dextran to label endosomes showed that a large number of endosomes were encapsulated by LC3B after monensin treatment, but the proportion of endosomes encapsulated by LC3B was significantly reduced in RUFY2 knockout cells. Figure 1 G and Figure 1 H). Meanwhile, in RUBCN knockout cells, the function of RUFY2 in regulating non-classical autophagy is unaffected (H). Figure 1 I and Figure 1 J). The above experiments demonstrate that RUFY2 can independently mediate endocytosis-related nonclassical autophagy.

[0033] 2. RUFY2 regulates nonclassical autophagy by modulating PI3P levels on the endosomal membrane via VPS34.

[0034] We used GFP-FYVE to indicate the level and localization of PI3P in cells and dextran to indicate endosomes. We found that in RUFY2 knockout cells, PI3P levels were significantly reduced, the area of ​​PI3P aggregates was smaller, and the co-localization level of dextran with PI3P aggregates was significantly reduced. Figure 2 (A to 2C). We constructed an MTM1 (Myotubularin 1) gene-inducible cell line to create a PI3P-depleted cell model. When PI3P in the cells was depleted, knocking down RUFY2 no longer affected LC3B aggregation (…). Figure 2 D and 2E). RUFY2 binds to VPS34 (gene name PI3KC3), and their interaction level increases after monensin treatment. Figure 2 F and 2G). In PI3KC3 knockout cells, knockdown of RUFY2 no longer significantly inhibited monensin-induced noncanonical autophagy levels (F and 2G). Figure 2 H and Figure 2 I). Therefore, the function of RUFY2 in regulating intracellular PI3P and promoting non-canonical autophagy depends on VPS34. These results indicate that RUFY2 interacts with VPS34 to promote the production of PI3P on the endosome membrane, thereby initiating endocytosis-related non-canonical autophagy.

[0035] 3. RUFY2 recruits EHD4 in nonclassical autophagy

[0036] We enriched Flag-RUFY2 protein and its interacting proteins using anti-Flag agarose beads and analyzed the RUFY2 protein-interaction genome using LC-MS / MS mass spectrometry. We found that RUFY2 has a strong interaction with EHD4, a member of the EH domain-containing protein family, and that the interaction level was significantly upregulated after monensin treatment. Figure 3 A and Figure 3 B). Immunoprecipitation experiments further confirmed the existence of the interaction between RUFY2 and EHD4 proteins and their upregulation in non-classical autophagy. Figure 3 C and Figure 3 D).

[0037] In EHD4 knockout HeLa cells, the number and area of ​​LC3B aggregates induced by momenisn were significantly reduced, while the baseline level and the number of LC3B aggregates induced by torin-1 remained unchanged. Figure 3 E and Figure 3F). The function of EHD4 in promoting autophagy depends on its membrane binding site (K330) and ATPase active site (G68). Figure 3 G), and this function depends on RUFY2 ( Figure 3 H) but independent of Rubicon ( Figure 4 I). The above experimental results indicate that EHD4 is recruited by RUFY2 in nonclassical autophagy, and together they initiate nonclassical autophagy.

[0038] 4. EHD4 promotes the function of ATG7 in nonclassical autophagy, thereby promoting LC3 lipidation.

[0039] We enriched Flag-EHD4 protein and its interacting proteins using anti-Flag agarose beads and analyzed the EHD4 interactionome using LC-MS / MS mass spectrometry. We found that EHD4 interacts with various autophagy-related proteins, with the strongest interaction being with ATG7, an E1 enzyme involved in the lipidation of ATG8 family proteins (ubiquitin-like proteins). Figure 4 A and Figure 4 B). Immunoprecipitation experiments further confirmed the existence of the interaction between EHD4 and ATG7 proteins, and this interaction was significantly upregulated in non-classical autophagy. Figure 4 C and Figure 4 D). Knockdown of EHD4 specifically downregulates monensin-induced ATG7-LC3B interaction, but has no significant effect on baseline levels or torin-1-induced ATG7-LC3B interaction. Figure 4 E and Figure 4 F). Overexpression of EHD4 directly promotes the interaction between ATG7 and LC3B, and this function depends on the ATPase activity of EHD4. Figure 4 G and Figure 5 H). In summary, EHD4 binds to ATG7 in nonclassical autophagy, promotes the interaction between ATG7 and LC3, and ultimately promotes ATG7-mediated LC3 lipidation.

[0040] 5. Screening of EHD4 activators

[0041] The degree of polymerization reflects the activation level of the EHD4 protein. Our DSS crosslinking experiment confirmed that the dimerization and oligomerization levels of EHD4 were significantly increased after monensin treatment, while torin-1 treatment had no significant effect. Figure 5 A). We screened a batch of small molecule compounds that interact with EHD4 and promote the dimerization and oligomerization of EHD4 through molecular docking. Figure 5B), of which compound 7-(3-(cyclopropylmethoxy)-4-(difluoromethoxy)phenyl)-N-(2-oxo-1,2,3,4-tetrahydroisoquinoline-6-yl)pyrazolo[1,5-a]pyrimidine-2-carboxamide showed the most significant effect in promoting the polymerization of EHD4. Figure 5 C to Figure 5 We named this EHD4 activator #02 (hereinafter referred to as EA02). Molecular docking simulation results show that EA02 binds to EHD4 through the ATP-binding pocket of EHD4. Figure 5 F and Figure 5 G). Cell thermal migration assay results showed that EA02 treatment significantly improved the thermal stability of intracellular EHD4 protein. Figure 5 The results indicate that EA02 interacts with EHD4. Furthermore, in wild-type cells, but not in EHD4 knockout cells, EA02 treatment significantly increased LC3B-II levels. Figure 6 I). The above results indicate that EA02 can act as a small molecule compound targeting EHD4 to promote autophagy.

[0042] 6. Synthesis of compound EA02

[0043] (1) Synthesis of compound 2

[0044]

[0045] 7.0 g of compound 1 (46.0 mmol, 1.0 equiv) was weighed, and 5.85 g of sodium carbonate (55.2 mmol, 1.2 equiv) was added. DMF (50 mL) was then added, followed by 7.3 g of ethyl difluorochloroacetate (46.0 mmol, 1.2 equiv). The mixture was reacted at 80 °C for 12 hours. After the reaction was complete, the mixture was extracted three times with ethyl acetate, and the organic phase was washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give 3.5 g of compound 2, with a yield of 37%.

[0046] Compound 2 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results were as follows: 1 ¹H NMR (600MHz, CDCl₃) δ 7.63 (d, J = 1.8 Hz, 1H), 7.51 (dd, J = 9.0, 2.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.64 (t, J = 73.2 Hz, 1H), 6.33 (br, 1H), 2.58 (s, 3H). ESI-HRMS m / z: Calculated value is C₁₈H₈O₃F₂Na. + [M+Na] +,225.0334; the measured value is 225.0311.

[0047] (2) Synthesis of compound 3

[0048]

[0049] 3.1 g of compound 2 (15 mmol, 1.0 equiv) was weighed, 4.2 g of potassium carbonate (30.0 mmol, 2.0 equiv) was added, followed by 5 mL of DMF, and finally 2.49 g of bromomethylcyclopropane (18.0 mmol, 1.2 equiv). The reaction was carried out at 80 °C for 5 hours until complete. The reaction was extracted three times with ethyl acetate and washed with saturated sodium chloride solution. The organic phase was concentrated and subjected to rapid column chromatography to give 2.2 g of compound 3 in 57% yield.

[0050] Compound 3 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results were as follows: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.87 (dd, J = 8.4, 2.0Hz, 1H), 7.69 (d, J = 2.0Hz, 1H), 7.24 (d, J = 8.8Hz, 1H), 7.17 (t, J = 74.4Hz, 1H), 3.99 (d, J = 7.2Hz, 2H), 2.53 (s, 3H), 1.30–1.23 (m, 1H), 0.65–0.52 (m, 2H), 0.43–0.27 (m, 2H). ESI-HRMS m / z: calculated value is C 13 H 14 O3F2Na + [M+Na] + ,279.0803; measured value is 279.0808.

[0051] (3) Synthesis of compound 4

[0052]

[0053] 2 g of compound 3 (7.81 mmol, 1.0 equiv.) and 1.13 g of N,N-dimethylformamide dimethyl acetal (9.37 mmol, 1.2 equiv.) were weighed and added to a reaction flask. 2 mL of N,N-dimethylformamide was added, and the mixture was stirred overnight at 120 °C. The mixture was monitored by TLC. The mixture was extracted three times with ethyl acetate, washed once with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The organic phase was concentrated and subjected to rapid column chromatography to give 1.8 g of compound 4 in 74% yield.

[0054] Compound 4 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results were as follows:1 ¹H NMR (400MHz, DMSO-d⁶) δ 7.70 (d, J = 12.4Hz, 1H), 7.57–7.47 (m, 2H), 7.19 (d, J = 8.0Hz, 1H), 7.16 (t, J = 74.4Hz, 1H), 5.81 (d, J = 12.4Hz, 1H), 3.94 (d, J = 6.8Hz, 2H), 3.14 (s, 3H), 2.92 (s, 3H), 1.27 (d, J = 8.1Hz, 1H), 0.61–0.49 (m, 2H), 0.42–0.30 (m, 2H). ESI-HRMS m / z: calculated value is C 16 H 19 O3NF2Na + [M+Na] + ,334.1225; the measured value is 334.1225.

[0055] (4) Synthesis of compound 5

[0056]

[0057] 2.48 g of compound 4 (7.97 mmol, 1.0 equiv.) and 1.46 g of methyl 5-amino-1H-pyrazole-3-carboxylic acid (23.91 mmol, 3.0 equiv.) were weighed and added to a reaction flask. 10 mL of acetic acid was added, and the mixture was stirred overnight at 80 °C. The reaction mixture was monitored by TLC. The organic phase was extracted three times with ethyl acetate, washed once with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The organic phase was concentrated and subjected to rapid column chromatography to give 2.6 g of compound 5 in 84% yield.

[0058] Compound 5 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results were as follows: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.72 (d, J = 4.4Hz, 1H), 7.89 (d, J = 2.0Hz, 1H), 7.71 (dd, J = 8.4, 2.0Hz, 1H), 7.45–7.39 (m, 2H), 7.28 (s, 1H), 7.27 (t, J = 74.0Hz, 1H), 3.99 (d, J = 6.8Hz, 2H), 3.88 (s, 3H), 1.42–1.26 (m, 1H), 0.66–0.51 (m, 2H), 0.43–0.24 (m, 2H). ESI-HRMS m / z: calculated value is C 19 H 17 O4N3F2Na + [M+Na] + ,412.1079; measured value is 412.1083.

[0059] (5) Synthesis of compound 6

[0060]

[0061] 1.0 g of compound 5 (2.6 mmol, 1.0 equiv.) was dissolved in a mixture of ethanol and water (1:1), and 510 mg of sodium hydroxide (13.0 mmol, 5.0 equiv.) was added. The mixture was stirred at 80 °C and monitored by TLC. After the reaction was complete, the ethanol was removed by rotary evaporation, and an appropriate amount of water was added. The pH was adjusted to 6 with dilute hydrochloric acid. The solid was obtained by filtration and dried to give 1.2 g of compound 6, with a yield of 98%.

[0062] Compound 6 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results were as follows: 1 ¹H NMR (400MHz, DMSO-d⁶) δ 8.58 (d, J = 4.4Hz, 1H), 7.85 (d, J = 1.6Hz, 1H), 7.78 (dd, J = 8.4, 2.0Hz, 1H), 7.38 (d, J = 8.4Hz, 1H), 7.27 (d, J = 4.0Hz, 1H), 7.25 (t, J = 74.0Hz, 1H), 6.95 (s, 1H), 3.99 (d, J = 6.8Hz, 2H), 1.37–1.30 (m, 1H), 0.65–0.54 (m, 2H), 0.39–0.26 (m, 2H), OH (not observed). ESI-HRMS m / z: calculated value is C 18 H 15 O4N3F2Na + [M+Na] + ,398.0923; the measured value is 398.0909.

[0063] (6) Synthesis of compound EA02

[0064]

[0065] 50 mg of compound 6 (0.13 mmol, 1.0 equiv.) was dissolved in N,N-dimethylformamide, and 51 mg of HATU (0.13 mmol, 1.0 equiv.) and 52 mg of DIPEA (0.39 mmol, 3.0 equiv.) were added. The mixture was stirred at room temperature for 20 min, and then 29 mg of 6-amino-3,4-dihydro-2(1H)-quinolinone (0.13 mmol, 1.0 equiv.) was added. The reaction was carried out at room temperature for 6 h. The reaction was monitored by TLC. After the reaction of the starting material was completely quenched with water, the mixture was extracted three times with ethyl acetate, washed once with saturated sodium chloride solution, and the ethyl acetate layer was dried over anhydrous sodium sulfate. The solution was concentrated and purified by column chromatography to give 16 mg of compound EA02, with a yield of 20%.

[0066] The obtained compound EA02 was identified using nuclear magnetic resonance spectroscopy and mass spectrometry. The identification results are as follows: 1 H NMR (600MHz, DMSO-d6) δ10.09(s,1H),10.07(s,1H),8.71(d,J=4.2Hz,1H),8.09(s,1H),7.88(d ,J=9.0Hz,1H),7.61(s,1H),7.55(dd,J=8.4,2.4Hz,1H),7.47(d,J=4.2Hz,1H),7.42(d,J=8.4H z,1H),7.28(s,1H),7.28(t,J=74.4Hz,1H),6.84(d,J=8.4Hz,1H),4.06(d,J=6.6Hz,2H),2.88( t,J=7.2Hz,2H),2.46(t,J=7.2Hz,2H),1.38–1.31(m,1H),0.59–0.52(m,2H),0.37–0.30(m,2H). 13 C NMR (151MHz, DMSO-d6) δ 170.0, 159.5, 155.4, 151.1, 150.1, 148.2, 142.1, 136.4, 134.6, 134.3, 132.8, 123.7, 120.9, 120.3, 120.2, 119.5, 116.6 (t, J = 256.5Hz), 115.0, 112.9, 107.7, 96.9, 73.3, 30.4, 25.1, 10.0, 3.1 (2×C). ESI-HRMS m / z: Calculated value is C 27 H 23 O4N5F2Na + [M+Na] + ,542.1610; measured value is 542.1624.

[0067] 7. EA02 initiates non-classical autophagy virus removal via the EHD4-RUFY2 axis.

[0068] I. Materials and Reagents

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional biochemical methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0070] 1. Cell lines

[0071] The human non-small cell lung cancer cell line A549 and the African green monkey kidney cell line Vero E6 are held in our laboratory. They can also be obtained commercially available (such as from the Cell Bank of the Chinese Academy of Sciences).

[0072] 2. Virus

[0073] The vesicular stomatitis virus (VSV) was kept and donated by Professor Li Yiping's laboratory at Sun Yat-sen University School of Medicine.

[0074] 3. Plasmids

[0075] The eGFP-EHD4 plasmid was purchased from Shenzhen Hermetic Biotechnology Co., Ltd.

[0076] 4. Animals

[0077] C57BL / 6 wild-type mice were purchased from Jicui Pharmaceutical Co., Ltd.

[0078] 5. Molecular biology reagents and antibodies

[0079] Superluminal transfection reagent was purchased from MIKX; Lipofectamine RNAiMAX transfection reagent and RUFY2 antibody were purchased from Thermo Fisher; MAP1LC3B antibody, β-actin antibody, and DAPI were purchased from Sigma-Aldrich; EHD4 antibody was purchased from Proteintech; goat anti-rabbit IgG (H+L)-HRP, goat anti-rabbit IgG (H+L) cross-adsorption Alexa Fluor 568 fluorescent secondary antibody, and Alexa Fluor... TM 568NHS ester was purchased from Invitrogen; PBS was purchased from Gibco; methanol was purchased from Guangzhou Chemical Reagent Factory; tissue fixative was purchased from Meilunbio; and concentrated normal goat serum was purchased from Boster.

[0080] Both the qPCR primers and siRNA were purchased from Sangon Biotech.

[0081] AAV-shScr and AAV-shEhd4 adenoviruses were purchased from Heyuan Biotechnology Co., Ltd. The specific sequence information of the shRNA is as follows:

[0082] Table 1. shRNA sequences

[0083]

[0084] II. Methods

[0085] 1. Immunofluorescence detection

[0086] Immunofluorescence was used to detect changes in the amount of VSV-568 in cells transfected with eGFP-EHD4 and the co-localization relationship among EHD4, VSV-568, and LC3B. The specific steps are as follows:

[0087] 1.1 Construction of vesicular stomatitis virus (VSV)-568 with Alexa Fluor 568 fluorescent group: Wild-type VSV virus particles were precipitated by ultracentrifugation (10,000×g, 4 hours, 4℃). After discarding the supernatant, the virus was dialyzed overnight in 0.1M NaHCO3 buffer (pH 8.3) at 4℃. After quantifying the protein concentration, NHS Alexa Fluor was added at a viral protein:dye molar ratio of 1:4 under continuous vortexing. TM 568NHS ester dye. The reaction system was incubated by inversion and rotation at room temperature for 2 hours. Then, it was purified by sucrose gradient centrifugation, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain VSV-568 virus particles;

[0088] 1.2 Cell Seeding: A549 cells were seeded at a rate of 5 × 10⁻⁶ cells / year. 3 Seeds were placed at a density of 10 cells / mL into a confocal microplate and cultured for 24 hours.

[0089] 1.3 Transfection of eGFP-EHD4 plasmid: The eGFP-EHD4 plasmid was transfected using Superluminal transfection reagent (refer to the instruction manual for specific methods) at a transfection amount of 400 ng / plate, and cultured for another 24 h.

[0090] 1.4 Viral infection: VSV-568 virus (MOI=1000) was added to the cell supernatant and placed at 4℃ for 30 min to allow the virus to settle and attach to the cell surface. Then the cells were transferred to a 37℃ incubator and cultured for 1 h.

[0091] 1.5 Fixation: Aspirate the culture medium from the stem cell culture dish, add 1 mL of 4% paraformaldehyde solution to each dish, incubate on ice for 15–30 min, then wash three times with 1 mL of PBS in a shaker for 5 min each time;

[0092] 1.6 Permeabilization: Add 1 mL of pre-cooled methanol at -20℃, let stand at -20℃ for 15-20 min, then wash three times with 1 mL PBS on a shaker for 5 min each time;

[0093] 1.7 Blocking: Add 1 mL of 6% goat serum (diluted with PBS), block at room temperature for 1 h, then wash three times with 1 mL of PBS on a shaker for 5 min each time;

[0094] 1.8 Primary antibody incubation: Add 100 μL of LC3B antibody diluted 1:200 with 6% goat serum, incubate overnight at 4°C, and then wash three times with 1 mL PBS on a shaker for 5 min each time;

[0095] 1.9 Incubation with fluorescent secondary antibody: Add fluorescent secondary antibody diluted 1:500 with 1 mL of 6% goat serum, incubate at room temperature in the dark for 1 h, then wash three times with 1 mL of PBS on a shaker for 5 min each time;

[0096] 1.10 DAPI staining: Add 100 μL of DAPI diluted 1:5000 with PBS, incubate at room temperature in the dark for 15 min, then wash three times with 1 mL PBS on a shaker for 5 min each time, and finally add 1 mL PBS to keep the sample moist;

[0097] 1.11 The sample was photographed using an SP8 Lightning confocal microscope, and the images were analyzed using ImageJ software.

[0098] The results showed that 1 hour after viral invasion, the number of viral particles in cells overexpressing EGFP-EHD4 was significantly reduced. Figure 6 A and 6B). Confocal microscopy revealed the triphasic colocalization of VSV viral particles, LC3B, and EHD4, and overexpression of EGFP-EHD4 significantly increased the proportion of VSV viral particles colocalizing with LC3B. Figure 6 C and Figure 6 D).

[0099] 2. The impact of knocking down EHD4 or RUFY2 on VSV virus replication levels

[0100] 2.1 Cell Seeding: A549 cells were seeded at a rate of 1×10⁻⁶ cells / year. 5 The cells were seeded at a density of 10 cells / mL into 24-well plates and cultured for 24 hours.

[0101] 2.2 Transfection of siRNA: EHD4 or RUFY2 siRNA (specific sequences are shown in Table 2) was transfected into cells using Lipofectamine RNAiMAX transfection reagent (refer to the instruction manual for specific methods) and cultured for 60 h;

[0102] Table 2. siRNA sequences

[0103]

[0104] 2.3 Viral infection: Cells were infected with VSV virus (MOI = 0.1) 18 hours before sample collection;

[0105] 2.4 Immunoblotting: Aspirate the culture medium from the stem cell culture plate, add approximately 120 μL of low-salt lysis buffer (LSB) to each well (for a 24-well plate), and lyse on ice at 170 rpm for 30 min. Take 16 μL of LSB protein lysis buffer and add 4 μL of 5×SDS loading solution. The buffer was prepared into an electrophoresis system and boiled at 100°C for 5 min. SDS-PAGE electrophoresis was performed at 80V for 40 min, then switched to 120V for 60 min. Proteins on the gel were transferred to a PVDF membrane using a wet transfer method, with a constant current of 280 mA for 100 min. The PVDF membrane was blocked by treating it with 5% skim milk at room temperature for 1 h. β-actin antibody was diluted 1:1:3000 with antibody dilution buffer and incubated at room temperature for 1 h, followed by washing the membrane three times with TBST for 5–10 min each time. The endogenous primary antibody was diluted 1:1000 with antibody dilution buffer and incubated overnight at 4°C. After washing the membrane three times with TBST, the secondary antibody (diluted 1:3000 with 5% skim milk) was incubated at room temperature for 1 h, followed by washing the membrane three times with TBST for 5–10 min each time. ECL chromogenic reagent was evenly spread on the PVDF membrane, and the results were obtained by developing the gel chemistry on a Bio-rad gel chemistry imaging system.

[0106] The results showed that knockdown of both EHD4 and RUFY2 significantly increased intracellular VSV infection levels. Figure 6 E).

[0107] 2.5 Real-time quantitative PCR:

[0108] 2.5.1 Extraction of total intracellular RNA (Trizol method): Aspirate the supernatant culture medium from A549 cells using a pipette or vacuum aspirator. Add 0.5 mL Trizol to each well (using a 24-well plate as an example), thoroughly pipette, and transfer to a 1.5 mL EP tube. Incubate at room temperature for 5 min. Add 0.1 mL chloroform, vortex vigorously for 15 s, incubate at room temperature for 2 min, centrifuge at 12000 × g for 15 min at 4°C, and collect the upper aqueous phase in a new EP tube. Add 0.25 mL isopropanol, incubate at room temperature for 10 min, centrifuge at 12000 × g for 15 min at 4°C, and discard the supernatant. Add 0.5 mL ethanol, mix thoroughly, centrifuge at 7500 × g for 15 min at 4°C, and discard the supernatant. Open the EP tube and allow the precipitate to air dry at room temperature. Dissolve the RNA precipitate with DEPC H2O and determine the RNA concentration using NanoDrop.

[0109] 2.5.2 Reverse transcription: Using the HiScript III RT SuperMix for qPCR (+gDNAwiper) kit, 800 ng of RNA was added to a PCR tube, along with 4 μL of 4×gDNAwiper Mix. The total volume was brought to 16 μL with DEPC H2O. The tube was vortexed, centrifuged, and incubated at 42°C for 2 min. Then, 4 μL of 5×HiScript III qRT SuperMix was added to each tube. The tubes were vortexed, centrifuged, and incubated at 37°C for 15 min, followed by incubation at 85°C for 5 s. The mixture was then diluted 4-fold with ddH2O and set aside for later use.

[0110] 2.5.3 Prepare the real-time quantitative PCR system. The primer sequences used are shown in Table 3, and the specific reagents and ratios for each well are shown in Table 4.

[0111] Table 3. Primer sequences for real-time quantitative PCR

[0112]

[0113] Table 4. Real-time quantitative PCR reaction system

[0114]

[0115] 2.5.4 passed Real-time quantitative PCR was performed using a 480 Instrument, and the obtained CT values ​​were calculated using the 2^-ΔΔCt method to obtain the final relative quantitative value.

[0116] The results showed that transfection of RUFY2 (Accession: AF461266.1) or / and EHD4 (Accession: NM_139265.4) plasmids fused with the Flag tag into A549 cells using Superluminal transfection reagent (see 1.3 for details) revealed that overexpression of EHD4 and RUFY2, alone or in combination, significantly reduced the intracellular VSV viral RNA (vRNA) level. Figure 6 F).

[0117] 3. The impact of EA02 treatment on VSV virus replication level

[0118] 3.1 Cell Seeding: A549 cells were seeded at a rate of 1×10⁻⁶ cells / year. 5 The cells were seeded at a density of 10 cells / mL into 24-well plates and cultured for 24 hours.

[0119] 3.2 Transfection of siRNA: EHD4 or RUFY2 siRNA (Table 2) was transfected into cells using Lipofectamine RNAiMAX transfection reagent and cultured for 60 h;

[0120] 3.3 Drug treatment: 24 hours before sample collection, add the concentration of EA02 shown in the figure (for...) to the cell supernatant. Figure 6 H (concentration of 2.5 μM);

[0121] 3.4 Viral infection: Cells were infected with VSV virus (MOI = 0.1) 18 hours before sample collection;

[0122] 3.5 Real-time quantitative PCR: The effect of EA02 treatment on intracellular VSV replication levels was detected using real-time quantitative PCR, with the specific experimental method being the same as in 2.5. The results showed that EA02 treatment significantly reduced VSV infection levels, and the best antiviral effect was observed at a concentration of 2.5 μM. Figure 6 G);

[0123] 3.6 Immunoblotting: The effect of EHD4 or RUFY2 knockdown on the antiviral function of EA02 was detected using immunoblotting, with the specific experimental method being the same as in 2.4. The results showed that the antiviral function of EA02 almost disappeared after EHD4 or RUFY2 knockdown. Figure 6 H).

[0124] 4. Verify the antiviral function of EA02 in animal models.

[0125] 4.1 Adenovirus injection: Dilute AAV-shScr and AAV-shEhd4 adenovirus to 2×10⁻⁶ using PBS. 12 VG / mL (Vector Genomes per mL) was administered to mice via tail vein injection, with 50 μL injected into each mouse, followed by a two-week wait.

[0126] 4.2 Administration: EA02 was prepared into a suspension using a 0.5% sodium carboxymethyl cellulose solution and administered to mice by gavage at a dose of 40 mg / kg once a day for 15 days.

[0127] 4.3 Viral Infection: VSV (10) was administered via tail vein injection on day 10 of drug administration. 7 PFU virus solution was injected into mice;

[0128] 4.4 Immunohistochemistry (IHC) and Hematoxylin-Einstein (H&E) Staining: Five days after viral infection, mice were sacrificed, and lung tissue was removed and fixed by immersion in 4% paraformaldehyde solution. Preparation of paraffin sections, IHC staining with VSV-G antibody, and H&E staining were performed by Savill Biotechnology Co., Ltd. IHC and H&E stained sections were observed and photographed using a microscope.

[0129] 4.5 Survival curve plotting: For mice whose survival was observed, they were observed for 6 days after viral infection, and their mortality was recorded. Survival curves were plotted in GraphPad software and significance difference analysis was performed.

[0130] The specific steps and grouping are as follows:

[0131] Table 5. Mouse experimental grouping information

[0132]

[0133] The results showed that VSV infection significantly increased the mortality rate of AAV-shEhd4 mice, while all mice in the EA02-only group survived the experimental period. However, administering the drug to AAV-shEhd4 mice did not reverse their high mortality rate. Figure 6 I). In control mice, EA02 administration significantly reduced viral load in the lungs and alleviated lung damage, while in AAV-shEhd4 mice, both of these indicators worsened significantly, and the effect of EA02 administration was not obvious. ​ J and 6K).

[0134] The results above indicate that the EHD4-RUFY2 axis inhibits viral infection by initiating non-classical autophagy, while the small molecule compound EA02 exerts its antiviral effect by targeting the EHD4-RUFY2 axis.

Claims

1. Compound EA02 or its pharmaceutical salt, the chemical structure of which is shown in the following formula:

2. The use of the compound EA02 of claim 1 or its pharmaceutical salt, solvate or derivative thereof in the preparation of nonclassical autophagy agonists or antiviral drugs.

3. The application according to claim 1, characterized in that, The viruses mentioned refer to various viruses.

4. The application according to claim 3, characterized in that, The virus in question is vesicular stomatitis virus.

5. A non-classical autophagy agonist or antiviral drug, characterized in that it contains the compound EA02 of claim 1 or its pharmaceutical salt, solvate or derivative thereof as an active ingredient.

6. The drug according to claim 5, characterized in that, The drug is a drug that can be administered by injection, oral administration, inhalation, or transdermal application.

7. A method for preparing the compound EA02 according to claim 1, characterized in that, Includes the following steps: The reaction formula is as follows: Compound 1 undergoes a nucleophilic substitution reaction with ethyl 2-chloro-2,2-difluoroacetate in DMF solvent in the presence of sodium carbonate to generate compound 2. Compound 2 then reacts with bromomethylcyclopropane in DMF solvent in the presence of potassium carbonate to give compound 3. Compound 3 reacts with N,N-dimethylformamide dimethyl acetal (DMFDMA) in DMF solvent to generate the key intermediate compound 4. Compound 4 undergoes a cyclization reaction with methyl 5-amino-1H-pyrazole-3-carboxylate under acetic acid catalysis to give compound 5. Compound 5 is hydrolyzed by sodium hydroxide in an ethanol / water mixed solvent to convert its methyl ester into a carboxylic acid to generate compound 6. Finally, compound 6 undergoes an amide condensation reaction with 6-amino-3,4-dihydro-1H-quinoline-2-one in DMF solvent with the addition of HATU and DIPEA to generate the target compound EA02.

Images