A pyrrolidine ketone compound and its application

Abstract

The present invention discloses a pyrrolidine ketone compound and its application. The compound can bind to E3 ubiquitin ligase triple motif 25 (TRIM25), promote TRIM25 to recognize pathogens, induce proteasome-dependent ubiquitination degradation of pathogen proteins, and is expected to be used as a ligand of TRIM25 to achieve a wider range of applications, such as for the preparation of PROTAC molecules. Therefore, the compound has very good research and development value and application prospects.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a pyrrolidine ketone compound and its application, for example, in the preparation of drugs for preventing and / or treating diseases, as a ligand of E3 ubiquitin ligase tripartite motif 25 (TRIM25), in regulating target ubiquitination levels, and in the preparation of targeted proteolysis chimeras (PROTACs). Background Art

[0002] In recent years, cell therapy, immunotherapy and gene editing technologies have developed rapidly. Protein degradation targeting chimeras (PROTACs) are an emerging direction in the field of drug development. They can directly induce the degradation of substrate proteins through the ubiquitin-proteasome protein degradation pathway in vivo and have attracted widespread attention from researchers.

[0003] The mechanism of PROTAC is to connect the small molecule inhibitor to the ligand of E3 ubiquitin ligase through a linker to form a consortium that targets and induces protein degradation. In vivo, the inhibitor part of this bifunctional molecule can recognize the target protein, and the ligand part of E3 can recognize the E3 ubiquitin ligase, thereby bringing the target protein and the E3 ubiquitin ligase closer in space, transferring the ubiquitin on the E3 ubiquitin conjugating enzyme to the target protein, ubiquitinizing the target protein, and then degrading the target protein through the ubiquitin-proteasome pathway.

[0004] In order to expand the application scope of PROTAC in drug development, finding small molecule ligands for E3 ubiquitin ligases is an important breakthrough. In 2008, Craig et al. reported a PROTAC that linked nutlin, a ligand of E3 ubiquitin ligase (MDM2), to an androgen receptor inhibitor (SCHNEEKLOTH AR, PUCHEAULT M, TAE HS, et al. Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics. Bioorganic & medicinal chemistry letters, 2008, 18 (22): 5904-5908). Crews et al. designed and modified the ligand of E3 ubiquitin ligase (VHL) and discovered a VHL ligand with high affinity (BUCKLEY DL, GUSTAFSON JL, VAN MOLLE I, et al. Small-molecule inhibitors of the interaction between the E3 ligase VHL and HIF1alpha. Angewandte Chemie, 2012, 51(46): 11463-11467). In 2015, Bradner's group and Crews' group reported methods for designing bifunctional molecules dBET1 and ARV-825 respectively using ligands of E3 ubiquitin ligase (CRBN) (diostatin drugs) and BRD4 inhibitor JQ1 (WINTER GE, BUCKLEY DL, PAULK J, et al. DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science, 2015, 348(6241): 1376-1381; LU J, QIAN Y, ALTIERI M, et al. Hijacking the E3ubiquitin ligase cereblon to efficiently target brD4. Chemistry & biology, 2015, 22(6): 755-763).

[0005] Although PROTAC technology has developed rapidly, it still faces many challenges in its application, for example, there are fewer ligands for E3 ubiquitin ligases.

[0006] E3 ubiquitin ligase tripartite motif 25 (TRIM25) is a member of the tripartite protein family among E3 ubiquitin ligases. Currently, there are few reports on the ligands of TRIM25, and there are no reports on PROTACs using TRIM25 ligands. Summary of the invention

[0007] The inventors of the present invention have discovered a class of pyrrolidine ketone compounds in their research, which can bind to E3 ubiquitin ligase triple motif 25 (TRIM25) in vitro, promote TRIM25 to recognize PA protein, and induce proteasome-dependent ubiquitination degradation of PA protein. They are expected to be used as ligands of E3 ubiquitin ligase TRIM25 and achieve wider applications, such as for the preparation of PROTAC molecules. Therefore, this class of compounds has very good application prospects and research and development value.

[0008] In a first aspect of the present invention, a compound is provided, which has the following structure:

[0009]

[0010] in,

[0011] Bonds at 1 and 2 represents a single bond or a double bond, and 1 and 2 are not double bonds at the same time;

[0012] R1 to R 18 are independently selected from: H, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, halogen, -CN, -NO2, -COR A 、-C(O)OR A 、-OCOR A 、-C(O)NR A R B 、-CH=NR A 、-OR A 、-OC(O)R A 、-S(O) t -R A 、-S(O) t -NR A R B 、-NR A R B 、-NR A C(O)R B ; Optionally, the H on each group may be substituted by one or more groups selected from the following: halogen, -CN, -CF3, -NO2, -CHO, -COOH, -C(O)NH2, -OH, -OC(O)H, -SH, -S(O)2H, -NH2.

[0013] R 19 and R 20 independently selected from: H, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl; optionally, the H on each group may be substituted by one or more groups selected from the following: substituted or unsubstituted heterocyclyl, halogen, -CN, -NO2, -COR A 、-C(O)OR A 、-C(O)NR A R B 、-CH=NR A 、-OR A 、-OC(O)R A 、-S(O) t -R A 、-S(O) t -NR A R B 、-NR A R B 、-NR A C(O)R B or, R 19 and R 20 Together with the carbon atom to which they are attached, they form a substituted or unsubstituted cycloalkyl or heterocyclic group; or, R 17 and R 19 Together with the carbon atom to which they are commonly attached, they form a substituted or unsubstituted cycloalkyl or heterocyclyl group;

[0014] t is selected from 0, 1 and 2;

[0015] Each R A and R B are independently selected from the group consisting of: H, alkyl, cycloalkyl, alkenyl, aryl, heterocyclyl and halogen.

[0016] Specifically, R1 to R 18independently selected from: H, C1-10 alkyl, C1-10 haloalkyl (such as fluorinated alkyl, for example trifluoromethyl), C1-10 alkenyl, C3-6 cycloalkyl, C4-10 cycloalkylalkyl, phenyl, four-membered to six-membered heterocycloalkyl, halogen, -CN, -NO2, -CHO, -CO(C1-10 alkyl), -COOH, -C(O)O(C1-10 alkyl), -C(O)NH2, -C(O)N(C1-10 -10 alkyl), -OH, -O(C1-10 alkyl), -OC(O)H, -OC(O)(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl), -NH2, -N(C1-10 alkyl)(C1-10 alkyl), -NHC(O)H, -N(C1-10 alkyl)C(O)(C1-10 alkyl).

[0017] More specifically, R1 is selected from: -OH, -O(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl); in some embodiments of the present invention, R1 is -OH.

[0018] More specifically, R2 is selected from: H, halogen, -CN, -CF3, -NO2, -CHO, -COOH, -C(O)NH2, -NH2; in some embodiments of the present invention, R2 is H.

[0019] More specifically, R3 is selected from: C1-10 alkyl, C1-10 haloalkyl (such as fluorinated alkyl, for example trifluoromethyl), C1-10 alkenyl, C3-6 cycloalkyl, C4-10 cycloalkylalkyl, in particular C1-10 alkyl, for example C1-6 alkyl, C1-3 alkyl (such as methyl, ethyl, n-propyl, isopropyl); in some embodiments of the present invention, R3 is methyl.

[0020] More specifically, R4 is selected from: -OH, -O(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl). In some embodiments of the present invention, R4 is -OH.

[0021] More specifically, R5 is selected from: H, C1-10 alkyl, C1-10 haloalkyl (such as fluoroalkyl, for example trifluoromethyl), C1-10 alkenyl, C3-6 cycloalkyl, C4-10 cycloalkylalkyl, in particular C1-10 alkyl, for example C1-6 alkyl, C1-3 alkyl (such as methyl, ethyl, n-propyl, isopropyl); in some embodiments of the present invention, R5 is methyl; in other embodiments of the present invention, R5 is H.

[0022] More specifically, R6 is selected from: -OH, -O(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl); in some embodiments of the present invention, R6 is -OH.

[0023] More specifically, R7 is selected from: -OH, -O(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl); in some embodiments of the present invention, R7 is -OH.

[0024] More specifically, R8, R9, R 11 , R 12 , R 13 , R 15 , R 16 are independently selected from: H, halogen, -CN, -CF3, -NO2, -CHO, -COOH, -C(O)NH2, -NH2; in some embodiments of the present invention, R8, R9, R 11 , R 12 , R 13 , R 15 , R 16 Both are H.

[0025] More specifically, R 10 and R 14 is independently selected from: C1-10 alkyl, C1-10 haloalkyl (such as fluoroalkyl, for example trifluoromethyl), C1-10 alkenyl, C3-6 cycloalkyl, C4-10 cycloalkylalkyl, in particular C1-6 alkyl, for example C1-3 alkyl (such as methyl, ethyl, n-propyl, isopropyl); in some embodiments of the present invention, R 10 and R 14 All are methyl.

[0026] More specifically, R 17 is selected from: H, -OH, -O(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl); in some embodiments of the present invention, R 17 is H. In other embodiments of the present invention, R 17 It is -OH.

[0027] More specifically, R 18Selected from: H, C1-10 alkyl, C1-10 haloalkyl (such as fluoroalkyl, for example trifluoromethyl), halogen, -CN, -CF3, -NO2, -CHO, -CO(C1-10 alkyl), -COOH, -C(O)O(C1-10 alkyl), -C(O)NH2, -C(O)N(C1-10 alkyl)(C1-10 alkyl), -OH, -O(C1-10 alkyl), -OC(O)H, -OC(O)(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl), -NH2, -N(C1-10 alkyl)(C1-10 alkyl), -NHC(O)H, -N(C1-10 alkyl)C(O)(C1-10 alkyl) ; Optionally, one or more H on each group may be substituted by a group selected from the group consisting of halogen, -CN, -CF3, -NO2, -CHO, -CO(C1-10 alkyl), -COOH, -C(O)O(C1-10 alkyl), -C(O)NH2, -C(O)N(C1-10 alkyl)(C1-10 alkyl), -OH, -O(C1-10 alkyl), -OC(O)H, -OC(O)(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl), -NH2, -N(C1-10 alkyl)(C1-10 alkyl), -NHC(O)H, -N(C1-10 alkyl)C(O)(C1-10 alkyl); More specifically, R 18 is selected from: H, C1-6 alkyl, C1-6 haloalkyl (such as fluoroalkyl, for example, trifluoromethyl), C1-6 hydroxy substituted alkyl (C1-6 alkyl substituted with one or more hydroxy groups, for example, hydroxymethyl), -CHO, -CO(C1-10 alkyl), -COOH, -C(O)O(C1-10 alkyl); in some embodiments of the present invention, R 18 Selected from: H, methyl, ethyl, n-propyl, isopropyl, -CH2OH, -COOH, -COOCH3, -CHO.

[0028] In one embodiment of the present invention, R 19 Has the following structure: in,

[0029] represents a single bond or a double bond;

[0030] R 21 To R 24independently selected from: H, C1-10 alkyl, C1-10 haloalkyl (such as fluorinated alkyl, for example trifluoromethyl), C1-10 alkenyl, C3-6 cycloalkyl, C4-10 cycloalkylalkyl, phenyl, four-membered to six-membered heterocycloalkyl, halogen, -CN, -NO2, -CHO, -CO(C1-10 alkyl), -COOH, -C(O)O(C1-10 alkyl), -C(O)NH2, -C(O)N(C1-10 alkyl) alkyl), -OH, -O(C1-10 alkyl), -OC(O)H, -OC(O)(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl), -NH2, -N(C1-10 alkyl)(C1-10 alkyl), -NHC(O)H, -N(C1-10 alkyl)C(O)(C1-10 alkyl); or, R 21 To R 24 The two of them together with the carbon atom in between form a substituted or unsubstituted cycloalkyl or heterocyclic group; When it represents a double bond, R 24 does not exist;

[0031] R 25 and R 26 Independently selected from: C1-10 alkyl, C1-10 haloalkyl (such as fluorinated alkyl, for example trifluoromethyl), C1-10 alkenyl, C3-6 cycloalkyl, C4-10 cycloalkylalkyl, phenyl, four-membered to six-membered heterocycloalkyl.

[0032] Specifically, R 25 and R 26 are independently selected from C1-10 alkyl, such as C1-6 alkyl, C1-3 alkyl; in some embodiments of the present invention, R 25 and R 26 All are methyl.

[0033] In some embodiments of the present invention, R 21 For H.

[0034] In some embodiments of the present invention, R 19 for

[0035] Specifically, R 24 is selected from -OH, -O(C1-10 alkyl), or, R 23 and R 24 Together with the carbon atom in the middle thereof, they form a substituted or unsubstituted cycloalkyl or heterocyclic group.

[0036] Specifically, R 22 Selected from: H, -OC(O)H, -OC(O)(C1-10 alkyl), for example -OC(O)CH3.

[0037] Specifically, R 23 Selected from: H, -OH, -O(C1-10 alkyl), -SH, -S(C1-10 alkyl), for example H or -OH.

[0038] In other embodiments of the present invention, R 20 and R 22 Together with the carbon atom in the middle, it forms a heterocyclic group.

[0039] In other embodiments of the present invention, R 20 and R 23 Together with the carbon atom in the middle, it forms a heterocyclic group.

[0040] Specifically, the above R 19 In the definition of , heterocyclyl is heterocycloalkyl, for example, four-membered to six-membered heterocycloalkyl, in particular oxygen-containing five-membered to six-membered heterocycloalkyl, for example Among them, R C is one or more independent substituents on the ring selected from: C1-10 alkyl, C1-10 haloalkyl, C1-10 hydroxy-substituted alkyl, C1-10 alkenyl, halogen, -CN, -NO2, -CHO, -CO(C1-10 alkyl), -COOH, -C(O)O(C1-10 alkyl), -C(O)NH2, -C(O)N(C1-10 alkyl)(C1-10 alkyl), -OH, -O(C1-10 alkyl), -OC(O)H, -OC(O)(C1-10 alkyl), -SH, -S(C1-10 alkyl), -S(O)2H, -S(O)2(C1-10 alkyl), -NH2, -N(C1-10 alkyl)(C1-10 alkyl), -NHC(O)H, -N(C1-10 alkyl)C(O)(C1-10 alkyl).

[0041] Specifically, R C is one or more independent substituents on the ring selected from: C1-6 alkyl, C1-6 haloalkyl, C1-6 hydroxy-substituted alkyl, C1-6 alkenyl, halogen, -CN, -NO2, -CHO, -COOH, -C(O)NH2, -OH, -OC(O)H, -SH; In some embodiments of the present invention, R C is one or more independent substituents on the ring selected from: methyl, ethyl, n-propyl, isopropyl.

[0042] In some embodiments of the present invention, R 19 Selected from:

[0043] In some embodiments of the present invention, R 20 For H.

[0044] In some embodiments of the present invention, R 19 and R 20 Together with the carbon atom to which they are attached, they form the following group:

[0045] In some embodiments of the present invention, one bond is a double bond and two bonds are single bonds.

[0046] In other embodiments of the present invention, one bond is a single bond and two bonds are double bonds.

[0047] In other embodiments of the present invention, one valence bond is a single bond, and two valence bonds are single bonds.

[0048] Specifically, the compound may have the following structure:

[0049]

[0050] Among them, R1, R4, R5, R6, R7, R 17 , R 18 , R 19 , R 20 It has the corresponding definitions of the present invention as mentioned above.

[0051] More specifically, the compound may have the following structure:

[0052]

[0053] Among them, R1, R4, R5, R6, R 17 , R 18 , R 19 , R 20 It has the corresponding definitions of the present invention as mentioned above.

[0054] More specifically, the compound may have the following structure:

[0055]

[0056] Among them, R5, R 17 , R 18 , R 19 , R 20 It has the corresponding definitions of the present invention as mentioned above.

[0057] More specifically, the compound can be selected from the following structures:

[0058]

[0059] In an embodiment of the present invention, the compound has the following structure:

[0060]

[0061] Preferably, the following compounds are not included:

[0062]

[0063] In the second aspect of the present invention, provided are pharmaceutically acceptable salts, esters, stereoisomers, prodrugs and solvates of the compound described in the first aspect.

[0064] Specifically, the stereoisomers of the compound may have the following structures:

[0065]

[0066] More specifically, the stereoisomers of the compound may have the following structures:

[0067]

[0068] In some embodiments of the present invention, the stereoisomers of the compound have the following structures:

[0069]

[0070] Specifically, the compounds described in the first aspect of the present invention and the pharmaceutically acceptable salts, esters, stereoisomers, prodrugs, solvates described in the second aspect of the present invention can be prepared by any suitable method known in the art, such as chemical synthesis, semi-synthesis, microbial fermentation or plant and animal extraction. For example, they can be obtained by extracting and separating the fermentation products of microorganisms (such as Aspergillus iizukae CPCC401321, whose preservation number is CGMCC No. 22467), and can also be obtained by chemically modifying the compounds obtained by extraction and separation (and physical treatment process) (semi-synthesis), or can be obtained from chemical raw materials with simpler chemical structures through a series of chemical synthesis and physical treatment processes (total synthesis).

[0071] In some embodiments of the present invention, the method for preparing the compound described in the first aspect of the present invention may include the step of extracting and separating the fermentation product of a microorganism (e.g., Aspergillus iizukae CPCC 401321, whose preservation number is CGMCC No. 22467); further, the preparation method may also include the step of chemically modifying the compound obtained by extraction and separation.

[0072] In other embodiments of the present invention, the method for preparing the compound described in the first aspect of the present invention may include the step of preparing the compound (total synthesis) from chemical raw materials with relatively simple chemical structures through a series of chemical synthesis and physical treatment processes.

[0073] In the third aspect of the present invention, a pharmaceutical composition is provided, comprising the compound described in the first aspect of the present invention or the pharmaceutically acceptable salt, ester, stereoisomer, prodrug, solvate described in the second aspect, and one or more pharmaceutically acceptable excipients.

[0074] Specifically, the pharmaceutical composition can be in any suitable dosage form, such as, but not limited to, tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, granules, pills, powders, pastes, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, and patches; oral dosage forms are preferred, such as: capsules, tablets, oral liquids, granules, pills, powders, pills, pastes, etc.

[0075] Specifically, pharmaceutically acceptable excipients may be, for example, but not limited to, binders, fillers, diluents, tableting agents, lubricants, disintegrants, colorants, flavoring agents and wetting agents, etc.; suitable fillers may be, for example, cellulose, mannitol, lactose and other similar fillers; suitable disintegrants may be, for example, starch, polyvinyl pyrrolidone and starch derivatives may be, for example, sodium starch glycolate; suitable lubricants may be, for example, magnesium stearate; suitable wetting agents may be, for example, sodium lauryl sulfate.

[0076] Specifically, the pharmaceutical composition may further comprise one or more other ingredients selected from the following: inosine monophosphate dehydrogenase (IMPDH) inhibitors, interferon inducers, M2 ion channel protein inhibitors and neuraminidase inhibitors.

[0077] Specifically, the inosine monophosphate dehydrogenase inhibitor may be, for example, ribavirin.

[0078] Specifically, the interferon inducer may be, for example, arbidol hydrochloride.

[0079] Specifically, the M2 ion channel protein inhibitor may be, for example, amantadine hydrochloride or rimantadine hydrochloride.

[0080] Specifically, the neuraminidase inhibitor may be, for example, osehamivir phosphate, oseltamivir, zanamivir or peramivir.

[0081] In the fourth aspect of the present invention, provided is the use of the compound described in the first aspect of the present invention or the pharmaceutically acceptable salt, ester, stereoisomer, prodrug, solvate described in the second aspect, and the pharmaceutical composition described in the third aspect in the preparation of a pharmaceutical composition for preventing and / or treating a disease.

[0082] In one embodiment of the present invention, the above-mentioned disease is a disease caused by infection with pathogens.

[0083] Specifically, the above-mentioned pathogens can be viruses, for example, but not limited to, Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (external genital herpes), VZV (varicella), EBV (Epstein-Barr virus), CMV (cytomegalovirus)), Poxviridae (such as smallpox virus, vaccinia virus), Papovaviridae (such as papillomavirus), Parvoviridae (such as B19 virus), Hepadnaviridae (such as hepatitis B virus), Polyomaviridae (such as polyomavirus), Reoviridae (such as reovirus, rotavirus), Picornaviridae (such as enterovirus, foot-and-mouth disease virus), Caliciviridae (such as Norwalk virus, hepatitis E virus), Togaviridae (such as rubella virus), Arenaviridae (such as lymphocytic choriomeningitis virus), Retroviridae (HIV-1, HIV-2, HTLV-1), Flaviviridae (such as dengue virus , Zika virus, Japanese encephalitis virus, Chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.), Orthomyxoviridae (such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.)), Paramyxoviridae (such as human parainfluenza virus (HPV) type 1, HPV type 2, HPV type 3, HPV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.), Bunyaviridae (such as California encephalitis virus, Hantavirus), Rhabdoviridae (such as rabies virus), Filoviridae (such as Ebola virus, Marburg virus), Coronaviridae (such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.), Astroviridae (such as astrovirus), Bornaviridae (such as Borna virus).

[0084] In some embodiments of the present invention, the virus is an influenza virus, such as one or more of influenza A virus, influenza B virus and influenza C virus, in particular influenza A virus.

[0085] Specifically, the influenza A virus may be an influenza A virus of the H1N1 subtype, H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.

[0086] Specifically, the above-mentioned diseases caused by viral infection include, but are not limited to, influenza, SARS, COVID-19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), AIDS, rabies, dengue fever, Ebola virus disease, etc.

[0087] In another embodiment of the present invention, the above-mentioned disease is a tumor.

[0088] Specifically, the above-mentioned tumors are malignant tumors, which include but are not limited to: breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, gastric cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, bladder cancer, anal cancer, gallbladder cancer, bile duct cancer, teratoma and heart tumor, etc.; especially lung cancer (such as non-small cell lung cancer), prostate cancer, liver cancer, breast cancer, gastric cancer, colorectal cancer.

[0089] Specifically, the subject of the drug can be a mammal (eg, human, ape, monkey, pig, cattle or sheep) or poultry (domestic poultry such as chicken, duck or goose, or wild poultry).

[0090] In a fifth aspect of the present invention, there is provided a compound as shown below and its pharmaceutically acceptable salts, esters, stereoisomers, prodrugs and solvates thereof as a ligand of E3 ubiquitin ligase TRIM25, in regulating target ubiquitination levels, and in the preparation of targeted proteolysis chimeras (PROTACs),

[0091]

[0092] in,

[0093] Bonds at 1 and 2 represents a single bond or a double bond, and 1 and 2 are not double bonds at the same time;

[0094] R1 to R 18 are independently selected from: H, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, halogen, -CN, -NO2, -COR A 、-C(O)OR A 、-OCOR A 、-C(O)NR A R B 、-CH=NR A 、-OR A 、-OC(O)R A 、-S(O) t -R A 、-S(O) t -NR A RB 、-NR A R B 、-NR A C(O)R B ; Optionally, the H on each group may be substituted by one or more groups selected from the following: halogen, -CN, -CF3, -NO2, -CHO, -COOH, -C(O)NH2, -OH, -OC(O)H, -SH, -S(O)2H, -NH2.

[0095] R 19 and R 20 independently selected from: H, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl; optionally, the H on each group may be substituted by one or more groups selected from the following: substituted or unsubstituted heterocyclyl, halogen, -CN, -NO2, -COR A 、-C(O)OR A 、-C(O)NR A R B 、-CH=NR A 、-OR A 、-OC(O)R A 、-S(O) t -R A 、-S(O) t -NR A R B 、-NR A R B 、-NR A C(O)R B or, R 19 and R 20 Together with the carbon atom to which they are attached, they form a substituted or unsubstituted cycloalkyl or heterocyclic group; or, R 17 and R 19 Together with the carbon atom to which they are commonly attached, they form a substituted or unsubstituted cycloalkyl or heterocyclyl group;

[0096] t is selected from 0, 1 and 2;

[0097] Each R A and R B are independently selected from the group consisting of: H, alkyl, cycloalkyl, alkenyl, aryl, heterocyclyl and halogen.

[0098] Specifically, each group has the corresponding definition described in the first aspect of the present invention.

[0099] Specifically, the target is a target protein to be degraded, which can be an endogenous protein in the body or a foreign protein, such as a viral protein.

[0100] In one embodiment of the present invention, the regulating ubiquitination level includes promoting the ubiquitination of PA protein, promoting the binding of PA protein to E3 ubiquitin ligase TRIM25, acting as a ligand of E3 ubiquitin ligase TRIM25, and the like.

[0101] In some embodiments of the present invention, in the above application, the compound has the following structure:

[0102]

[0103] in particular

[0104] In some embodiments of the present invention, in the above application, the stereoisomer of the compound has the following structure:

[0105]

[0106] in particular

[0107] Specifically, in the above application, PROTAC may have the following structure: SMI-L-E3L, wherein SMI is a small molecule inhibitor portion (which may be formed by any suitable small molecule inhibitor of the target known in the prior art), E3L is a ligand portion of the E3 ubiquitin ligase (for example, a structural portion formed by the above compound or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, or solvate thereof), and L is a connecting bond or linker between SMI and E3L.

[0108] In a sixth aspect of the present invention, a PROTAC is provided, which has the following structure:

[0109] SMI-L-E3L

[0110] (Ⅻ)

[0111] Among them, SMI is the small molecule inhibitor part;

[0112] E3L is the ligand portion of E3 ubiquitin ligase, which is formed by the compound described in the sixth aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate thereof;

[0113] L is a connecting bond or a connecting group between SMI and E3L.

[0114] Specifically, SMIs can be formed by small molecule inhibitors of any suitable target known in the art.

[0115] In the seventh aspect of the present invention, a method for preparing PROTAC is provided, comprising the step of using the compound described in the sixth aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, or solvate thereof.

[0116] In the eighth aspect of the present invention, an Aspergillus fungus is provided, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on July 8, 2021 (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with a deposit number of CGMCC No. 22467, and a classification name of Aspergillus iizukae. In the ninth aspect of the present invention, a method for preventing and / or treating a disease is provided, comprising administering to a subject in need thereof an effective amount of a compound described in the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate thereof, a pharmaceutical composition described in the third aspect of the present invention, and a PROTAC described in the sixth aspect of the present invention.

[0117] Specifically, the above-mentioned diseases can be diseases caused by pathogen infection, such as influenza, SARS, COVID-19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), AIDS, rabies, dengue fever, Ebola virus disease, etc.; tumors, such as breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, gastric cancer, gastroesophageal adenocarcinoma, esophageal cancer, small intestine cancer, cardia cancer, bladder cancer, anal cancer, gallbladder cancer, bile duct cancer, teratoma and heart tumor, etc.

[0118] Specifically, the subject may be a mammal (eg, human, ape, monkey, pig, cattle or sheep) or poultry (domestic poultry such as chicken, duck or goose, or wild poultry).

[0119] In the tenth aspect of the present invention, a method for regulating target ubiquitination levels is provided, comprising the step of administering to a subject in need thereof an effective amount of the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt, stereoisomer, ester, prodrug, solvate thereof, the pharmaceutical composition described in the third aspect of the present invention, or the PROTAC described in the sixth aspect of the present invention.

[0120] The present invention provides a class of pyrrolidine compounds, which can bind to TRIM25, promote TRIM25 to recognize pathogen proteins (such as viruses), and induce proteasome-dependent ubiquitination degradation of pathogens. They are expected to be used as ligands of E3 ubiquitin ligase TRIM25 to achieve wider applications, such as for the preparation of PROTAC molecules. Therefore, the compounds have very good application prospects and research and development value.

[0121] The deposit information of the biological material of the present invention is as follows:

[0122] An Aspergillus fungus, Aspergillus iizukae CPCC 401321, was deposited in the General Microbiology Center of China Culture Collection Administration (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on July 8, 2021, with the deposit number CGMCC No. 22467 and the classification name Aspergillus iizukae. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 Shown is compound C1 1 H-NMR spectrum.

[0124] Figure 2 Shown is compound C1 13 C-NMR spectrum.

[0125] Figure 3 Shown is compound C2 1 H-NMR spectrum.

[0126] Figure 4 Shown is compound C2 13 C-NMR spectrum

[0127] Figure 5 Shown is compound C3 1 H-NMR spectrum.

[0128] Figure 6 Shown is compound C3 13 C-NMR spectrum

[0129] Figure 7 Shown is the HSQC spectrum of compound C3.

[0130] Figure 8 Shown is the HMBC spectrum of compound C3.

[0131] Fig. 9 Shown is compound C3 1 H- 1 H COSY spectrum.

[0132] Fig.10 Shown is the NOESY spectrum of compound C3.

[0133] Fig.11 Shown is compound C4 1 H-NMR spectrum.

[0134] Fig.12 Shown is compound C4 13C-NMR spectrum.

[0135] Fig.13 Shown is the HSQC spectrum of compound C4.

[0136] Fig.14 Shown is the HMBC spectrum of compound C4.

[0137] Fig.15 Shown is compound C4 1 H- 1 H COSY spectrum.

[0138] Fig.16 Shown is the NOESY spectrum of compound C4.

[0139] Fig.17 Shown is compound C5 1 H-NMR spectrum.

[0140] Fig.18 Shown is compound C5 13 C-NMR spectrum.

[0141] Fig.19 Shown is the HSQC spectrum of compound C5.

[0142] Fig. 20 Shown is the HMBC spectrum of compound C5.

[0143] Fig.21 Shown is compound C5 1 H- 1 H COSY spectrum.

[0144] Fig. 22 Shown is the NOESY spectrum of compound C5.

[0145] Fig.23 Shown is compound C6 1 H-NMR spectrum.

[0146] Fig.24 Shown is compound C6 13 C-NMR spectrum.

[0147] Fig.25 Shown is the HSQC spectrum of compound C6.

[0148] Fig.26 Shown is the HMBC spectrum of compound C6.

[0149] Fig. 27 Shown is compound C6 1 H- 1 H COSY spectrum.

[0150] Fig.28 Shown is the NOESY spectrum of compound C6.

[0151] Fig.29 Shown is compound C7 1 H-NMR spectrum.

[0152] Fig.30 Shown is compound C7 13 C-NMR spectrum.

[0153] Fig.31 Shown is the HSQC spectrum of compound C7.

[0154] Fig.32 Shown is the HMBC spectrum of compound C7.

[0155] Fig.33 Shown is compound C7 1 H- 1 H COSY spectrum.

[0156] Fig.34 Shown is the NOESY spectrum of compound C7.

[0157] Fig.35 Shown is compound C8 1 H-NMR spectrum.

[0158] Fig.36 Shown is compound C8 13 C-NMR spectrum.

[0159] Fig.37 Shown is the HSQC spectrum of compound C8.

[0160] Fig.38 Shown is the HMBC spectrum of compound C8.

[0161] Fig.39 Shown is compound C8 1 H- 1 H COSY spectrum.

[0162] Fig.40 Shown is the NOESY spectrum of compound C8.

[0163] Fig.41 Shown is compound C9 1 H-NMR spectrum.

[0164] Fig.42 Shown is compound C9 13 C-NMR spectrum.

[0165] Fig.43 Shown is the HSQC spectrum of compound C9.

[0166] Fig.44 Shown is the HMBC spectrum of compound C9.

[0167] Fig.45 Shown is compound C9 1 H- 1 H COSY spectrum.

[0168] Fig.46 Shown is the NOESY spectrum of compound C9.

[0169] Fig.47 Shown is compound C10 1 H-NMR spectrum.

[0170] Fig.48 Shown is compound C10 13 C-NMR spectrum.

[0171] Fig.49 Shown is the HSQC spectrum of compound C10.

[0172] Fig.50 Shown is the HMBC spectrum of compound C10.

[0173] Fig.51 Shown is compound C10 1 H- 1 H COSY spectrum.

[0174] Fig.52 Shown is compound C11 1 H-NMR spectrum.

[0175] Fig.53 Shown is compound C11 13 C-NMR spectrum.

[0176] Fig.54 Shown is the HSQC spectrum of compound C11.

[0177] Fig.55 Shown is the HMBC spectrum of compound C11.

[0178] Fig.56 Shown is compound C11 1 H- 1 H COSY spectrum.

[0179] Fig.57 Shown is compound C12 1 H-NMR spectrum.

[0180] Fig.58 Shown is compound C12 13 C-NMR spectrum.

[0181] Fig.59 Shown is the HSQC spectrum of compound C12.

[0182] Fig.60 Shown is the HMBC spectrum of compound C12.

[0183] Fig.61 Shown is compound C12 1 H- 1 H COSY spectrum.

[0184] Fig.62 Shown is the NOESY spectrum of compound C12.

[0185] Fig.63 Shown is compound C13 1 H-NMR spectrum.

[0186] Fig.64 Shown is compound C13 13 C-NMR spectrum.

[0187] Fig.65 Shown is the HSQC spectrum of compound C13.

[0188] Fig.66 Shown is the HMBC spectrum of compound C13.

[0189] Fig.67 Shown is compound C13 1 H- 1 H COSY spectrum.

[0190] Fig.68 Shown is the NOESY spectrum of compound C13.

[0191] Fig.69 Shown is compound C14 1 H-NMR spectrum.

[0192] Fig.70 Shown is compound C14 13 C-NMR spectrum.

[0193] Fig.71 Shown is the HSQC spectrum of compound C14.

[0194] Fig.72 Shown is the HMBC spectrum of compound C14.

[0195] Fig.73 Shown is compound C14 1 H- 1 H COSY spectrum.

[0196] Fig.74Shown is the NOESY spectrum of compound C14.

[0197] Fig.75 Shown are experimental results showing the effects of compounds C1-C14 on influenza virus PA protein expression.

[0198] Fig.76 Shown are experimental results showing that compound C1 downregulates the PA protein degradation pathway.

[0199] Fig.77 Shown are the results of an experiment showing that compound C1 induces PA polyubiquitination.

[0200] Fig.78 Shown are experimental results showing that compound C1 induces PA degradation by recognizing the E3 ligase TRIM25.

[0201] Fig.79 Shown are experimental results showing that compound C1 promotes the interaction of TRIM25 and PA.

[0202] Fig.80 Shown are the results of an experiment showing that the compounds bind to TRIM25 in vitro.

[0203] Fig.81 Shown are the results of an experiment demonstrating the ability of compounds to bind PA in vitro.

[0204] Fig.82 Shown are experimental results showing that compound C1 promotes the polyubiquitination level of PA protein in vitro. DETAILED DESCRIPTION

[0205] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0206] The term "alkyl" refers to a straight or branched hydrocarbon group that does not contain unsaturated bonds, and the hydrocarbon group is connected to the other parts of the molecule with a single bond. The alkyl group used in this article usually contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkyl), preferably contains 1 to 6 carbon atoms (i.e., C1-6 alkyl). The example of alkyl includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. If the alkyl group is replaced by a cycloalkyl group, it is correspondingly a "cycloalkylalkyl group", such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. If the alkyl group is replaced by an aryl group, it is correspondingly a "aralkyl group", such as benzyl, diphenylmethyl or phenethyl. If the alkyl group is replaced by a heterocyclic group, it is correspondingly a "heterocyclic alkyl group".

[0207] The term "alkenyl" refers to a straight or branched hydrocarbon group containing at least two carbon atoms, at least one unsaturated bond, and the hydrocarbon group is connected to the rest of the molecule by a single bond. The alkenyl group used in this article generally contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkenyl), preferably contains 1 to 6 carbon atoms (i.e., C1-6 alkenyl). Examples of alkenyl include, but are not limited to, vinyl, 1-methyl-vinyl, 1-propenyl, 2-propenyl or butenyl, etc.

[0208] The term "cycloalkyl" refers to alicyclic hydrocarbons. The cycloalkyl group used in this article usually contains 1 to 4 single rings and / or condensed rings, 3-18 carbon atoms, preferably 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (e.g., C3-10 cycloalkyl, C3-6 cycloalkyl), such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or adamantyl, etc.

[0209] The term "aryl" refers to any functional group or substituent derived from a simple aromatic ring, including monocyclic aryl groups and / or fused ring aryl groups, such as those containing 1-3 rings, monocyclic or fused rings and having 6-18 (e.g., 6, 8, 10, 12, 14, 16, 18) carbon ring atoms. The aryl used herein is generally an aryl (i.e., C6-12 aryl) containing 1-2 rings, monocyclic or fused rings and having 6-12 carbon ring atoms, wherein the H on the carbon atom can be substituted, for example, by groups such as alkyl, halogen, etc. Examples of the aryl include, but are not limited to, phenyl, p-methylphenyl, naphthyl, biphenyl, indenyl, etc.

[0210] The term "halogen" refers to bromine, chlorine, iodine or fluorine.

[0211] The term "heterocyclyl" refers to a 3- to 18-membered non-aromatic ring group containing 2 to 17 carbon atoms and 1 to 10 heteroatoms. The heterocyclyl group can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can contain fused, spirocyclic or bridged ring systems. The heterocyclyl group can be partially saturated (heteroaryl) or fully saturated (heterocycloalkyl). Suitable heteroaryl groups in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms, and include, for example, coumarin, including 8-coumarin, quinolyl, including 8-quinolyl, isoquinolyl, pyridinyl, pyrazinyl, pyrazolyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridinyl. Suitable heterocycloalkyl groups in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms, and include, for example, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathiolanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxirane, thiirane, azepine, oxazepine, diazepine, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothiolanyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl and quinolizinyl.

[0212] The pharmaceutically acceptable salts of the present invention include acid addition salts and base addition salts.

[0213] The acid addition salts include, but are not limited to, salts from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid and phosphonic acid, and salts from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids and aliphatic and aromatic sulfonic acids. Therefore, these salts include but are not limited to sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, iodate, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate and mesylate, also comprising amino acid salts such as arginate, gluconate, galacturonate etc. Acid addition salts can be prepared by contacting the free alkali form with a sufficient amount of the required acid to form a salt in a conventional manner. The free alkali form can be regenerated by contacting the salt form with an alkali, and the free alkali is separated in a conventional manner.

[0214] Base addition salts of the present invention refer to salts formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of suitable amines include, but are not limited to, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N-methylglucamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form a salt. The free acid form can be regenerated by contacting the salt form with an acid, and the free acid is separated in a conventional manner.

[0215] Stereoisomers of the present invention include enantiomers, diastereomers and geometric isomers. Some compounds of the present invention have cycloalkyl groups, which can be substituted on more than one carbon atom, in which case all geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention. The cycloalkyl groups include alicyclic hydrocarbon groups and aryl groups, wherein the alicyclic hydrocarbon groups can be non-aromatic monocyclic, condensed rings, bridged rings or spirocyclic saturated or unsaturated cyclic hydrocarbon groups, aryl groups such as phenyl, naphthyl, phenanthrenyl, biphenyl etc.

[0216] Solvates of the present invention refer to the physical combination of a compound of the present invention and one or more solvent molecules. The physical combination includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate can be separated, for example, when one or more solvent molecules are incorporated into the lattice of a crystalline solid. Solvates include solution phases and separable solvates. Representative solvates include ethanolates, methanolates, etc.

[0217] The prodrugs of the present invention refer to forms of the compounds of formula I which are suitable for administration to patients without excessive toxicity, irritation, allergic reactions, etc. and are effective for their intended use, including acetal, ester and zwitterion forms. The prodrugs are transformed in vivo, such as by hydrolysis in the blood, to obtain the parent compound.

[0218] The "patient" or "subject" and the like described in the present invention are used interchangeably herein and refer to any animal or cell thereof treated according to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animal includes mammals, for example, rats, mice, guinea pigs, rabbits, dogs, monkeys or humans, especially humans.

[0219] The term "treatment" as used herein refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, stopping and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0220] The term "prevention" as used in the present invention refers to preventing, minimizing or making it difficult for a disease to occur or develop by treatment before the disease occurs.

[0221]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0222] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0223] Example 1: Preparation of Aspergillus iizukae CPCC 401321 fermentation culture

[0224] Aspergillus iizukae CPCC 401321 is a high-yield strain of pyrrolidone compounds (its deposit number is CGMCC No. 22467), which is cultured on a PDA slant at 28° C. for 7 days, and then a mycelium block is picked and inoculated in a 500 ml triangular flask containing 100 ml of PDB seed culture medium, and shake-cultured at 28-30° C. for 5 days as a seed solution. Then, 10 ml of the seed solution is inoculated in a 500 ml triangular flask containing rice culture medium (a 500 ml triangular flask is filled with 100 g of rice, 100 ml of deionized water is added to soak, and the rice culture medium is obtained by sterilization at 121° C. for 20 minutes), and cultured at 28° C. for 20 days to obtain a solid fermentation culture of Aspergillus iizukae CPCC401321.

[0225] Example 2: Preparation of ethyl acetate extract from fermentation culture of Aspergillus iizukae CPCC 401321

[0226] Take 10 kg of the solid fermentation culture of Aspergillus iizukae CPCC 401321 obtained in Example 1, crush it with a glass rod, add 20 L of ethyl acetate, and perform ultrasonic extraction at room temperature for 3 times, each time for 30 min. The ethyl acetate extracts are combined and the ethyl acetate in the extracts is removed by rotary evaporation using a rotary evaporator (temperature: 40° C.). The obtained substance is the ethyl acetate extract of the fermentation culture of Aspergillusiizukae CPCC 401321.

[0227] Example 3: Separation of ethyl acetate extract

[0228] (1) The ethyl acetate extract (250 g) of Example 2 was dissolved in an ethyl acetate-methanol mixed solution and then separated by silica gel column chromatography. The silica gel used in the silica gel column chromatography was silica gel H, the size of the silica gel column used was 12×40 cm, and the column volume was 4522 mL. The elution procedure used in the silica gel column chromatography was as follows: linear gradient elution: the mobile phase used was a mixture of petroleum ether and acetone, and the volume ratio of petroleum ether to acetone in the linear gradient elution mobile phase was linearly reduced from 4:1 to 1:1. The eluted liquid was collected continuously from the beginning of the elution process, 500 ml of each fraction (200 ml / fraction) was collected, and 275 fractions were collected continuously, which were recorded as: Fr.1, Fr.2, Fr.3, Fr.4, ..., Fr.275. The same fractions were combined according to TLC detection, and finally 19 combined components were obtained: Fr.1-5 (mixed from F.1 to Fr.5 fractions, the same below), Fr.6, Fr.7-11, Fr. 12-23, Fr.24-26, Fr.27-29, Fr.30-44, Fr.45-51, Fr.52-67, Fr.68-77, Fr.78-91, Fr.92-99 , Fr.100-115, Fr.116-127, Fr.128-137, Fr.138-196, Fr.197-227, Fr.228-258, Fr.259-275.

[0229] (2) The supernatants of Fr.12-23 obtained in step (1) were combined and subjected to Sephadex LH-20 gel column chromatography. The size of the gel column used was 3×120 cm. The elution procedure used was to use methanol as the mobile phase for elution. The eluted liquid was collected continuously from the beginning of the elution procedure. 30 ml (30 ml / fraction) was collected in each tube. 25 fractions were collected continuously and recorded as: tube.1, tube.2, tube.3, ..., tube.25. Tubes 5-9 (the fractions from Tube.5 to Tube.9 were mixed to obtain a component named tubes 5-9) were subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation). The filler used in the preparative high performance liquid chromatography separation was octadecylsilane bonded silica filler with a particle size of 5 μm. The chromatographic column used in the preparative high performance liquid chromatography separation had a diameter of 8 mm and a height of 250 mm. The mobile phase was a mixture of methanol and water (containing 0.05% formic acid), with a volume ratio of methanol to water of 90:10; the flow rate was 4.0 mL / min. R The elution peaks with (retention time) of 16.0 min, 18.0 min and 19.9 min were removed by rotary evaporation using a rotary evaporator (temperature: 50°C) to obtain 80.5 mg of compound C3, 39.1 mg of compound C4 and 4.1 mg of compound C5, respectively.

[0230] (3) Fr.92-99 obtained in step (1) was subjected to MCI column chromatography. The specification of the MCI column used was 2×30 cm. The elution procedure used was to use a mixed liquid consisting of methanol and water in a volume ratio of 90:10 as the mobile phase for elution. The eluted liquid was collected continuously from the beginning of the elution procedure. 40 ml was collected in each tube (40 ml / fraction). 20 fractions were collected continuously and recorded as: tube.1, tube.2, tube.3, ..., tube.20. Tubes 5-9 (obtained by mixing the fractions from tube.5 to tube.9) were subjected to Sephadex LH-20 gel column chromatography. The size of the gel column used was 2×80 cm. The elution procedure used was to use methanol as the mobile phase for elution. The eluted liquid was collected continuously from the beginning of the elution procedure. 20 ml (20 ml / fraction) was collected in each tube. 25 fractions were collected continuously and recorded as: tube.1′, tube.2′, tube.3′, ..., tube.25′. Tubes 2′-5′ (obtained by mixing the fractions from Tube.2′ to Tube.5′) were subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation). The filler used in the preparative high performance liquid chromatography separation was octadecylsilane bonded silica filler with a particle size of 5 μm. The chromatographic column used in the preparative high performance liquid chromatography separation had a diameter of 8 mm and a height of 250 mm. The mobile phase was a mixture of methanol and water (containing 0.05% formic acid), with a volume ratio of methanol to water of 85:15; the flow rate was 4.0 mL / min. RThe elution peaks with retention times of 11.8min, 12.5min and 17.7min were removed by rotary evaporation with a rotary evaporator (temperature: 50°C) to obtain 21.6mg of compound C9, 10.6mg of compound C10 and 102.9mg of compound C6, respectively. Tubes 11-15 (mixed from tube.11 to tube.15 fractions) were further subjected to Sephadex LH-20 gel column chromatography. The size of the gel column used was 2×80cm. The elution procedure used was to use methanol as the mobile phase for elution. The eluted liquid was continuously collected from the beginning of the elution procedure. 20ml was collected in each tube (20ml / fraction). 15 fractions were collected continuously and recorded as: tube.1″, tube.2″, tube.3″, ..., tube.15″. tubes 2″-8″ (mixed from tube.2″ to tube.8″) were subjected to preparative HPLC separation (RP-C18 liquid phase preparative separation). The filler used in the preparative HPLC separation was octadecylsilane bonded silica gel filler, the particle size of the filler was 5μm, the chromatographic column used in the preparative HPLC separation was 8mm in diameter and 250mm in height. The mobile phase was a mixed liquid of methanol and water (containing 0.05% formic acid), the volume ratio of methanol to water in the mobile phase was 87:13; the flow rate was 4.0mL / min. The collected t R The elution peaks with (retention times) of 17.2 min, 19.4 min and 21.1 min were removed by rotary evaporation using a rotary evaporator (temperature: 50°C) to obtain 40.5 mg of compound C6, 28.2 mg of compound C8 and 425.6 mg of compound C7, respectively.

[0231] Fr.228-258 obtained in step (1) was subjected to MCI column chromatography. The specification of the MCI column used was 6×18 cm. The elution procedure used was to use a mixed liquid consisting of methanol and water in a volume ratio of 80:20 as the mobile phase for elution. The eluted liquid was collected continuously from the beginning of the elution procedure. 100 ml was collected in each tube (100 ml / fraction). 39 fractions were collected continuously and recorded as: tube.1, tube.2, tube.3, ..., tube.39. Tubes 16-39 (mixed from tube.16 to tube.39) were subjected to ODS column chromatography. The ODS column used was 5.5×28 cm in size. The elution procedure used was to use a mixed liquid consisting of methanol and water in a volume ratio of 75:25 as the mobile phase for elution. The eluted liquid was collected continuously from the beginning of the elution procedure. 100 ml was collected in each tube (100 ml / fraction). 50 fractions were collected continuously and recorded as: tube.1′, tube.2′, tube.3′, ..., tube.50′. Tubes 34-45 (mixed from tube.34′ to tube.55′) were subjected to rotary evaporation (temperature: 50°C) to remove methanol and water to obtain 1.25 g of compound C1. Tubes 31-33 (mixed from tube.31′ to tube.33′) were subjected to preparative high performance liquid chromatography separation (RP-C18 liquid phase preparative separation). The filler used in the preparative HPLC separation is octadecylsilane bonded silica filler, the particle size of the filler is 5 μm, the diameter of the chromatographic column used in the preparative HPLC separation is 8 mm, and the height is 250 mm. The mobile phase is a mixed liquid composed of methanol and water (containing 0.05% formic acid), and the volume ratio of methanol to water in the mobile phase is 73:27; the flow rate is 4.5 mL / min. Collect t R The elution peak with a retention time of 14.0 min was evaporated by rotary evaporation of methanol and water using a rotary evaporator (temperature: 50° C.) to obtain 14.4 mg of compound C11.

[0232] Fr.30-44 obtained in step (1) was subjected to MCI column chromatography. The size of the MCI column used was 3×23 cm. The elution procedure used was to use a mixed liquid consisting of methanol and water in a volume ratio of 90:10 as the mobile phase for elution. The eluted liquid was collected continuously from the beginning of the elution procedure. 50 ml (50 ml / fraction) was collected in each tube. 20 fractions were collected continuously and recorded as: tube.1, tube.2, tube.3, ..., tube.20. Tubes 7-20 (mixed from tube.7 to tube.20 fractions) were then subjected to Sephadex LH-20 gel column chromatography. The size of the gel column used was 2×80 cm. The elution procedure used was to use methanol as the mobile phase for elution. The eluted liquid was collected continuously from the beginning of the elution procedure. 20 ml (20 ml / fraction) was collected in each tube. 18 fractions were collected continuously and recorded as: tube.1′, tube.2′, tube.3′, ..., tube.18′. tubes 2′-6′ (mixed from tube.2′ to tube.6′) were subjected to preparative HPLC separation (RP-C18 liquid phase preparative separation). The filler used in the preparative HPLC separation was octadecylsilane bonded silica gel filler, the particle size of the filler was 5 μm, the chromatographic column used in the preparative HPLC separation was 8 mm in diameter and 250 mm in height. The mobile phase was a mixed liquid of methanol and water (containing 0.05% formic acid), the volume ratio of methanol to water in the mobile phase was 84:16; the flow rate was 4.5 mL / min. Collect t R The elution peaks with (retention time) of 20.0min, 23.7min, 30.4min and 33.7min were removed by rotary evaporation using a rotary evaporator (temperature: 50°C) to obtain 3.2mg of compound C14, 33.5mg of compound C13, 152.3mg of compound C12 and 206.1mg of compound C2, respectively.

[0233] Compounds C1 to C14 are all brown colloidal solids, and are easily soluble in solvents such as methanol, ethanol, and DMSO, but poorly soluble in water. Compounds C1 to C14 are developed by silica gel thin layer chromatography using a solvent system of chloroform-methanol-water (volume ratio 70:15:2) with Rf values ​​of 0.4-0.8, and have obvious fluorescence at 254nm and 365nm, and vanillin sulfuric acid is brown-red.

[0234] Example 4: Structural Identification

[0235] (1) Compounds C1 and C2

[0236] The spectral data of compounds C1 and C2 are shown in Table 1. 1 H NMR and 13C NMR spectra are shown in Figure 1-4 The structures of compounds C1 and C2 are shown in Table 6.

[0237] Table 1. NMR data of compounds C1 and C2 ( 1 H NMR 600 MHz; 13 C NMR 150MHz; DMSO-d6)

[0238]

[0239]

[0240] (2) Compound C3

[0241] HRESIMS (negative ion) peak of compound C3: m / z 573.2825 [MH] - The molecular formula is C 35 H 42 O7. Comprehensive analysis 1 H NMR ( Figure 5 ), 13 C NMR ( Figure 6 ) and HSQC spectra ( Figure 7 ), it is speculated that the compound should contain 2 keto carbonyl groups, 1 aldehyde carbonyl group, 18 olefinic carbons and 14 sp 3 Hybridized carbon. Characteristic signals can be seen in the hydrogen spectrum: δ14.33 (1H, s), 13.11 (1H, s), 9.29 (1H, s), 6.83 (1H, s), 6.50 (1H, t, J = 7.3 Hz), 5.05 (1H, t, J = 7.2 Hz), 4.94 (1H, t, J = 6.8 Hz), 4.81 (1H, t, J = 7.8 Hz), 2.83 (3H, s), 2.56 (2H, d, J = 7.9 Hz), 2.14 (3H, s), 1.57 (3H, s), 1.49 (3H, s), 1.44 (3H, s), 1.28 (3H, s). The above characteristic signals are similar to those of compound C2. A detailed comparison of the NMR signals of compounds C3 and C2 revealed that they both have the same three-membered ring structure of naphthylenediol, and the only difference is in the diterpene side chain. Figure 8) can be found in the following related signals: δ1.28 (H-35) is related to δ115.4 (C-17), 139.4 (C-18), 39.2 (C-19), δ1.44 (H-34) is related to 124.4 (C-21), 133.3 (C-22), 37.4 (C-23), δ9.29 (H-33) is related to δ155.2 (C-25), 142.1 (C-26), 23.5 (C-27), δ5.05 (H-29 ) is correlated with δ23.5 (C-27), 25.3 (C-31), 17.3 (C-32), δ6.50 (H-25) is correlated with δ26.9 (C-24), 23.5 (C-27), 194.9 (C-33), δ4.94 (H-21) is correlated with δ38.2 (C-19), 37.4 (C-23), 15.3 (C-34), δ4.81 (H-17) is correlated with δ39.2 (C-19), 15.5 (C-35). 1 H- 1 H COSY spectrum ( Fig. 9 ) can be found in the following related signals: δ4.81 (H-17) is related to δ2.56 (H-16), δ4.94 (H-21) is related to δ1.56 (H-20), δ1.56 (H-20) is related to δ1.64 (H-19), δ6.50 (H-25) is related to δ2.36 (H-24), δ2.36 (H-24) is related to δ2.02 (H-23), δ5.05 (H-29) is related to δ1.93 (H-28), and δ1.93 (H-28) is related to δ2.14 (H-27). The above HMBC and 1 H- 1 The H COSY related signals confirmed the planar structure of the diterpene side chains. Fig.10 ) can be found in the following related signals: δ4.81 (H-17) is related to δ1.64 (H-19), δ4.94 (H-21) is related to δ2.02 (H-23), and δ6.50 (H-25) is related to δ9.29 (H-33), which proves that the configurations of 17 (18)-ene, 21 (22)-ene and 25 (26)-ene are E-, E- and E-, respectively. In view of the same biosynthetic pathway as compounds C1 and C2, it is judged that the absolute configuration of the C-1 position of compound C3 should also be S configuration. Finally, compound C3 was identified as the structure shown in Table 6, and its NMR signal attribution is specifically shown in Table 2.

[0242] (3) Compound C4

[0243] HRESIMS (negative ion) ion peak of compound C4: m / z 573.2838 [MH]- The molecular formula is C 35 H 42 O7. Comprehensive analysis 1 H NMR ( Fig.11 ), 13 C NMR ( Fig.12 ) and HSQC spectra ( Fig.13 ), it is speculated that the compound should contain 2 keto carbonyl groups, 18 olefinic carbons and 15 sp 3 Hybridized carbon. The NMR signal of compound C4 is basically the same as that of compound C2, with the only difference being the C-25 to C-28 structural fragments. 1 H- 1 H COSY spectrum ( Fig.15 ) shows that H-29 and H-28 are related, and combined with the HSQC related signal, signal δ H 4.43 and δ C 75.1 was assigned to C-28, and based on the signal chemical shift value, it was determined that C-28 was oxidized. Fig.14 ) can be found in the following related signals: H-25 (δ4.26, 4.06) is related to C-25 (δ119.0), C-26 (δ138.8), H-25 (δ4.26, 4.06) is related to C-28 (δ75.1), suggesting that C-28 and C-33 are connected by an ether bond. In the NOESY spectrum ( Fig.16 ) in which δ5.17 (H-25) is correlated with δ2.50 (H-27), proving that the configuration of 25(26)-ene is E-. In view of the same biosynthetic pathway as compounds C1 and C2, it is judged that the C-1 configuration of compound C4 is S. Finally, compound C4 was identified as the structure shown in Table 6, and the specific attribution of its NMR signals is shown in Table 2.

[0244] (4) Compound C5

[0245] HRESIMS (negative ion) ion peak of compound C5: m / z 617.3372 [MH] - The molecular formula is C 37 H 46 O8. Comprehensive analysis 1 H NMR ( Fig.17 ), 13 C NMR ( Fig.18 ) and HSQC spectra ( Fig.19 ), it is speculated that the compound should contain 2 keto carbonyl groups, 1 ester carbonyl group, 18 olefin carbons and 16 sp 3 Hybridized carbon. The NMR signals of compound C5 are similar to those of compound C2, except for the C-25 to C-30 structural fragments. Fig. 20 ) shows that δ1.54 (H-33) is related to 106.0 (C-5), 130.1 (C-26), 44.6 (C-27), δ1.62 (H-31) is related to 123.7 (C-29), 135.9 (C-30), 25.2 (C-31), δ1.65 (H-32) is related to δ123.7 (C-29), 135.9 (C-30), 18.0 (C-32), and 1 H- 1 HCOSY spectrum ( Fig.21 ) shows that δ5.50 (H-28) is related to δ2.2, 2.06 (H-27), and 5.07 (H-29), respectively. Based on the above evidence, the skeleton structure of C-25 to C-30 can be determined. In the HMBC spectrum, it can be seen that δ5.50 (H-28) is related to δ169.3 (- CO CH3) related, δ1.91(-CO CH3 ) and δ169.3(- CO CH3), proving that an acetyl group is connected to the C-28 position. Fig. 22 ) can be found in the following related signals: δ4.82 (H-17) is related to δ1.64 (H-19), δ4.87 (H-21) is related to δ1.83 (H-23), and δ5.05 (H-25) is related to δ2.20 (H-27), which proves that the configurations of 17 (18)-ene, 21 (22)-ene and 25 (26)-ene are E-, E- and E-, respectively. In view of the same biosynthetic pathway as compounds C1 and C2, the C-1 configuration of compound C5 should also be S configuration. Finally, compound C5 was identified as the structure shown in Table 6, and its NMR signal attribution is specifically shown in Table 2.

[0246] Table 2. NMR data of compounds C3 to C5 ( 1 H NMR 600 MHz; 13 C NMR 150MHz; DMSO-d6)

[0247]

[0248] (5) Compound C6

[0249] HRESIMS (negative ion) peak of compound C6: m / z 593.3115 [MH] - The molecular formula is C 35 H 46 O8. Comprehensive analysis 1 H NMR ( Fig.23 ), 13 C NMR ( Fig.24) and HSQC spectra ( Fig.25 ), it is speculated that the compound should contain 2 keto carbonyl groups, 16 olefinic carbons and 17 sp 3 Hybridized carbon. The NMR signals of compound C6 are basically the same as those of compound C1, with the only difference being at the C-26 position. Fig.26 ) can be found in the following related signals: δ5.04 (H-25) is related to δ36.5 (C-27), 15.9 (C-33), δ1.53 (H-33) is related to δ123.2 (C-25), 135.0 (C-26), 36.5 (C-27), suggesting that C-26 is a methyl substitution. In the NOESY spectrum ( Fig.28 ) in which δ5.04 (H-25) is correlated with δ1.85 (H-27), proving that the configuration of 25(26)-ene is E-. In view of the same biosynthetic pathway as compounds C1 and C2, it is judged that the C-1 and C-29 configurations of compound C6 are S and R, respectively. Finally, compound C6 was identified as shown in Table 6, and its NMR signal attribution is shown in Table 3.

[0250] (6) Compound C7

[0251] HRESIMS (negative ion) ion peak of compound C7: m / z 649.3383 [MH] - The molecular formula is C 38 H 50 O9. Comprehensive analysis 1 H NMR ( Fig.29 ), 13 C NMR ( Fig.30 ) and HSQC spectra ( Fig.31 ), it is speculated that the compound should contain 2 keto carbonyl groups, 16 olefinic carbons and 20 sp 3 Hybridized carbon. The NMR signals of compound C7 are basically the same as those of compound C1, with the only difference being the C-28 to C-32 structural fragments on the diterpene side chain. Compound C7 has three more carbon atoms than compound C1, including an oxidized quaternary carbon δ105.6 (C-1′) and two methyl carbon signals δ28.4 (C-2′) [δ1.29 (3H, s, H-2′)] and δ26.7 (C-3′) [δ1.21 (3H, s, H-3′)]. In the HMBC spectrum (Appendix Fig.46) δ1.29 (H-2′) is correlated with δ105.6 (C-1′), 26.7 (C-3′), δ1.21 (H-3′) is correlated with δ105.6 (C-1′), 28.4 (C-2′), indicating that C-1′ is substituted by two methyl groups. Moreover, the larger chemical shift of C-1′ suggests that it should be replaced by two oxygen atoms. HMBC related signals: δ1.15 (H-31) is correlated with δ79.4 (C-30), 82.1 (C-29), δ0.98 (H-32) is correlated with 79.4 (C-30) and 82.1 (C-29), combined with 1 H- 1 H COSY related signal δ3.61 (H-29) is related to δ2.04 (H-28), and δ2.04 (H-28) is related to δ2.19 (H-27a), 2.00 (H-27b), further confirming the structure of the C-28 to C-32 fragment. The larger chemical shifts of C-29 and C-30 and the unsaturation of compound C7 indicate that C-1′ is connected to C-29 and C-30 through an ether bond, respectively, to eventually form a 5-membered ring structure fragment. In view of the same biosynthetic pathway as compounds C1 and C2, it is judged that the absolute configuration of the C-1 position of compound C7 should also be S configuration. Finally, compound C7 was identified as the structure shown in Table 6, and the specific attribution of its NMR signals is shown in Table 3.

[0252] (7) Compound C8

[0253] HRESIMS (negative ion) peak of compound C8: m / z 663.3114 [MH] - The molecular formula is C 38 H 48 O 10 Comprehensive analysis 1 H NMR ( Fig.35 ), 13 C NMR ( Fig.36 ) and HSQC spectra ( Fig.37 ), it is speculated that the compound should contain 2 keto carbonyl groups, 1 ester carbonyl group, 16 olefin carbons and 19 sp 3 Hybridized carbon. The NMR signal of compound C8 is basically the same as that of compound C7, with the only difference being the C-26 position on the diterpene side chain. Fig.38 ) in which δ6.60 (H-25) correlates with δ23.6 (C-27), 37.8 (C-23), and 168.2 (C-33), suggesting that the C-26 position is substituted by a carboxyl group (-COOH). In the NOESY spectrum ( Fig.40) in which δ2.34 (H-27) is correlated with δ2.20 (H-24), proving that the configuration of 25(26)-ene is E-. Given that it has the same biosynthetic pathway as compounds C1 and C2, it is judged that the absolute configuration of C-1 of compound C8 should also be S-configuration. Finally, compound C8 was identified as shown in Table 6, and its NMR signal attribution is shown in Table 3.

[0254] Table 3. NMR data of compounds C6 to C8 ( 1 H NMR 600 MHz; 13 C NMR 150MHz; DMSO-d6)

[0255]

[0256]

[0257] (8) Compound C9

[0258] HRESIMS (negative ion) peak of compound C9: m / z 609.3343 [MH] - The molecular formula is C 35 H 46 O9. Comprehensive analysis 1 H NMR ( Fig.41 ), 13 C NMR ( Fig.42 ) and HSQC spectra ( Fig.43 ), it is speculated that the compound should contain 2 keto carbonyl groups, 16 olefinic carbons and 17 sp 3 Hybridized carbon. The NMR signals of compound C9 are similar to those of compound C1, except for the C-24 to C-29 structural fragments. Fig.44 ) shows that δ1.00 (H-31) is correlated with δ85.9 (C-29), 69.8 (C-30), δ0.99 (H-33) is correlated with δ74.5 (C-25), 84.9 (C-26), 34.3 (C-27), δ3.13 (H-25) is correlated with δ35.9 (C-23), 84.9 (C-26), 34.3 (C-27), and 1 H- 1 H COSY spectrum ( Fig.45) shows that δ3.57 (H-29) is related to δ1.71 (H-28), δ1.71 (H-28) is related to δ1.91, 1.45 (H-27), δ3.13 (H-25) is related to δ1.56, 1.19 (H-24), and δ1.56, 1.19 (H-24) is related to δ2.07, 1.85 (H-24). The above evidence can determine the structure of C-24 to C-29 in compound C9. In the NOESY spectrum ( Fig.46 ) can be found that δ0.99 (H-33) is related to δ3.57 (H-29), and the relative configuration of the C-26 and C-29 substituent groups can be inferred. The final compound C9 was identified as shown in Table 6, and its NMR signal attribution is shown in Table 4.

[0259] (9) Compound C10

[0260] HRESIMS (negative ion) peak of compound C10: m / z 609.3343 [MH] - The molecular formula is C 35 H 46 O9. The NMR signals of compounds C10 and C9 are basically consistent, and it is inferred that they have the same planar structure. 1 HNMR Fig.47 ), 13 C NMR ( Fig.48 ) and HSQC spectra ( Fig.49 ), 1 H- 1 H COSY( Fig.50 ) and HMBC spectra ( Fig.51 ), proving the above conclusion. Carefully comparing the NMR signals of compounds C10 and C9, the structures of the two are slightly different at C-26 to C-29, and the relative configuration of the C-26 and C-29 substituent groups in compound C10 (different from compound C9) is inferred. Finally, compound C10 was identified as the structure shown in Table 6, and the specific attribution of its NMR signals is shown in Table 4.

[0261] (10) Compound C11

[0262] HRESIMS (negative ion) peak of compound C11: m / z 595.2946 [MH] - The molecular formula is C 34 H 44 O9. Comprehensive analysis 1 H NMR ( Fig.52 ), 13 C NMR ( Fig.53 ) and HSQC spectra ( Fig.54), it is speculated that the compound should contain 2 keto carbonyl groups, 16 olefinic carbons and 16 sp 3 Hybridized carbon. The NMR signals of compounds C11 and C1 are highly similar. After detailed comparison, it was found that the diterpene branched chain structures of the two are the same, with only slight differences in the three-membered ring structure of naphthoxybenzene. The hydrogen spectrum of compound C11 lacks a C-14 methyl hydrogen signal (approximately at δ2.13) compared with the hydrogen spectrum of compound C1, but has an additional single-peak aromatic hydrogen signal (δ6.31), indicating that there is no methyl substitution at the C-12 position in the structure of compound C11. In the HMBC spectrum ( Fig.55 ) in which δ13.78 (13-OH) correlates with δ101.7 (C-3), 99.1 (C-12), 167.0 (C-13), and δ6.32 (H-12) correlates with δ101.7 (C-3), 112.6 (C-10), further confirming the above conclusion. In view of the same biosynthetic pathway as compounds C1 and C2, it is inferred that the C-1 and C-29 configurations in the structure of compound C11 are S and R configurations, respectively, and the configurations of 17 (18)-ene, 21 (22)-ene and 25 (26)-ene are E-, E- and Z-, respectively. Finally, compound C11 was identified as the structure shown in Table 6, and the specific attribution of its NMR signals is shown in Table 4.

[0263] Table 4. NMR data of compounds C9 to C11 ( 1 H NMR 600 MHz; 13 C NMR 150MHz; DMSO-d6)

[0264]

[0265]

[0266] (11) Compound C12

[0267] HRESIMS (negative ion) peak of compound C12: m / z 589.2827 [MH] - The molecular formula is C 35 H 42 O8. Comprehensive analysis 1 H NMR ( Fig.57 ), 13 C NMR ( Fig.58 ) and HSQC spectra ( Fig.59 ), it is speculated that the compound should contain 2 keto carbonyl groups, 1 ester carbonyl group, 18 olefin carbons and 14 sp 3 Hybridized carbon. The NMR signals of compounds C12 and C2 are highly similar. After detailed comparison, it is found that the only difference between the two is the different substituents at the C-26 position. In the HMBC spectrum ( Fig.60) in which δ5.70 (H-25) correlates with δ34.3 (C-27), 38.4 (C-23), and 168.7 (C-33), confirming that the C-26 position of compound C12 is substituted by a carboxyl group (-COOH). In the NOESY spectrum ( Fig.62 ) in which δ5.70 (H-25) is related to δ2.11 (H-27), proving that the configuration of 25(26)-ene is E-. The configurations of other positions in the structure of compound C12 are the same as those of compound C2. Finally, compound C12 was identified as having the structure shown in Table 6, and the attribution of its NMR signals is shown in Table 5.

[0268] (12) Compound C13

[0269] HRESIMS (negative ion) peak of compound C13: m / z 591.2969 [MH] - The molecular formula is C 35 H 42 O7. Comprehensive analysis 1 H NMR ( Fig.63 ), 13 C NMR ( Fig.64 ) and HSQC spectra ( Fig.65 ), it is speculated that the compound should contain 2 keto carbonyl groups, 16 olefinic carbons and 17 sp 3 Hybridized carbon. The NMR signals of compounds C13 and C1 are similar. After detailed comparison, it is found that the difference between the two lies in the C-25 to C-29 structural fragments. In the HMBC spectrum ( Fig.66 ) in which H-33 (δ4.26, 4.06) correlates with C-25 (δ119.0), C-26 (δ138.8), H-33 (δ4.49, 3.63) correlates with C-28 (δ84.1), and H-33 (δ3.06) correlates with C-28 (δ66.1), confirming that C-29 and C-30 are connected by an ether bond to form a six-membered ring structure. In the NOESY spectrum ( Fig.68 ), δ5.12 (H-25) is related to δ2.50 (H-27), and δ4.49 (H-33) is related to δ1.99 (H-24), proving that the configuration of 25(26)-ene is Z-. The configurations of other positions in the structure are the same as those of compound C1. Finally, compound C13 was identified as shown in Table 6, and the specific attribution of its NMR signals is shown in Table 5.

[0270] (13) Compound C14

[0271] HRESIMS (negative ion) peak of compound C14: m / z 591.3248 [MH] - The molecular formula is C 35 H 44 O8. Comprehensive analysis1 H NMR ( Fig.69 ), 13 C NMR ( Fig.70 ) and HSQC spectra ( Fig.71 ), it is speculated that the compound should contain 2 keto carbonyl groups, 16 olefinic carbons and 17 sp 3 Hybridized carbon. The NMR signals of compound C14 are mostly similar to those of compound C1, with the difference being the C-24 to C-29 structural fragments. Fig.72 ) shows that δ1.03 (H-31) is related to δ84.6 (C-29), 70.0 (C-30), δ1.20 (H-33) is related to δ137.2 (C-25), 81.9 (C-26), 37.0 (C-27). 1 H- 1 HCOSY spectrum ( Fig.73 ) shows that δ3.60 (H-29) is correlated with δ1.78, 1.70 (H-28), δ1.78, 1.70 (H-28) is correlated with δ1.76, 1.75 (H-27), and δ5.39 (H-24) is correlated with δ5.41 (H-25) and δ2.53 (H-23). ​​The above evidence can determine the structure of C-24 to C-29 in compound C14. In the NOESY spectrum ( Fig.74 ) can be found that δ5.41 (H-25) is related to δ2.53 (H-23), which proves that the configuration of 24(25)-ene is E-. Finally, compound C14 was identified as the structure shown in Table 6, and the specific attribution of its NMR signals is shown in Table 5.

[0272] Table 5. NMR data of compounds C12 to C14 ( 1 H NMR 600 MHz; 13 C NMR 150MHz; DMSO-d6)

[0273]

[0274] Table 6. Structures of compounds C1 to C14

[0275]

[0276]

[0277] Example 5: Effects of Compounds C1 to C14 on PA Protein

[0278] 2.5×10 5HEK 293T cell suspension of 100 μg / mL was inoculated into a 6-well plate at 2 ml per well. When the cells grew to 80%, 500 ng pHW2000-PA plasmid was transfected into each well of the HEK 293T cell group. Four hours after transfection, the culture medium was replaced with DMEM culture medium containing 10% fetal bovine serum (FBS). One group added 2 μL of 5.00 mM test compound to each well, and the other group used DMSO (dimethyl sulfoxide) as a negative control, and then cultured for 24 hours. Discard the culture medium, add 80 μL of RIPA lysis buffer to each well, transfer the lysate to a 1.5 ml EP tube and lyse on ice for 20 minutes, add 20 μL of 5X protein loading buffer to each tube, and boil in a 100°C metal bath for 30 minutes. Wait for Western Blot to detect the expression of PA protein. See the test results. Fig.75 .

[0279] In addition, pHW2000-PA-Luc plasmid was transfected into HEK293T cells, and different concentrations of compounds were added for treatment. After 24 h, the expression level of Luc protein was detected, and the EC of the compound to degrade PA protein was calculated. 50 Values ​​(Table 7).

[0280] Table 7. EC of compounds for degradation of influenza virus PA protein 50 result

[0281]

[0282]

[0283] Example 6: Protease inhibitor MG132 can effectively block the degradation of PA protein by compound C1

[0284] pHW183-PA plasmid was transfected into HEK239T cells, and different concentrations of compound C1 (2μM and 10μM) were added for treatment. Lysosomal inhibitor ConA (Concanavalin A) or proteasome inhibitor MG-132 were also added to observe the expression of PA protein. The results showed that the expression level of PA protein was almost completely restored after treatment with proteasome inhibitor MG-132 ( Fig.76 A), but the recovery of PA protein was hardly affected by treatment with the lysosomal inhibitor ConA ( Fig.76 B), indicating that compound C1 degradation of PA protein is mainly achieved through the proteasome pathway.

[0285] Example 7: Compound C1 promotes the ubiquitination level of PA protein

[0286] HEK293T cells were transfected with PA and myc-CW7 ubiquitin plasmids. Compound C1 of different concentrations was added 3.5 hours after transfection, and MG-132 was added for incubation 8 hours before harvesting the cells. Samples were collected 24 hours after cell transfection, and the expression level of ubiquitinated PA protein was detected by Western Blotting. The experimental results showed that compound C1 could promote the polyubiquitination level of PA protein ( Fig.77 ).

[0287] Example 8: Discovery of E3 ubiquitin ligase TRIM25 using surface plasmon resonance (SPR) technology

[0288] SPR technology is a classic method for detecting the binding of small molecules to proteins. Its advantage is that it does not require molecular labeling of the sample, that is, it does not change the properties of the small molecules, and it has high sensitivity. The basic principle is to first fix the small molecules to the surface of the chip, and continuously flow the cell lysate over the surface of the chip in the form of a solution. The changes in the molecular concentration on the surface of the sensor chip during the binding and dissociation of the small molecules and proteins are recorded by LC-MS coupling, thereby monitoring the interaction between small molecules and proteins in real time. The SPR technology was used to detect proteins involved in the polyubiquitination of PA proteins. The experimental results showed that the experiment captured a total of seven host proteins involved in protein polyubiquitination, including KEAP1, HERC5, RBP2, UBA7, TRIM25, ISG15 and UB2E2. Only the knockdown of TRIM25 significantly affected the anti-influenza activity of compound C1 ( Fig.78 A); Overexpression of TRIM25 in TRIM25 knockout cell lines can restore the anti-influenza activity of compound C1 to the level of normal cells ( Fig.78 B).

[0289] Example 9: Compound C1 promotes the interaction between TRIM25 and PA

[0290] HEK293T cells were transfected with PA and TRIM25 plasmids, and different concentrations of compound C1 were added for treatment 3.5 hours after transfection. MG-132 was added for incubation 8 hours before harvesting the cells. Samples were collected 24 hours after cell transfection, and TRIM25 protein antibody was used to capture, and Western Blotting was used to detect the expression level of ubiquitinated PA protein. The experimental results showed that compound C1 can promote the interaction between TRIM25 and PA protein ( Fig.79 ).

[0291] Example 10: Compounds C1-C14 directly bind to TRIM25 and PA proteins

[0292] In order to further study the mechanism of compound C1 in degrading PA protein, the inventors used biofilm layer interferometry (BLI) to detect the binding ability of the compound to TRIM25 protein and PA protein. The test results showed that compounds C1 to C14 can bind to TRIM25 protein, and the KD value is between 12-43μM ( Fig.80 ). This result indicates that the ability of the compound to bind to TRIM25 is directly related to its function of inducing PA degradation. At the same time, the above compounds also have the ability to bind to PA in vitro. For example, compounds C1 and C2 can bind to PA protein, and their KD values ​​are 11μM and 58μM, respectively ( Fig.81 ). The above results suggest that this class of compounds can recruit TRIM25 to PA by binding to TRIM25 and PA, thereby inducing its ubiquitination and degradation.

[0293] Example 11: Compound C1 promotes the ubiquitination level of PA protein in vitro

[0294] The results of in vitro ubiquitination assays showed that compound C1 could promote the polyubiquitination level of PA protein in vitro ( Fig.82 ).

[0295] Example 12: Anti-influenza virus activity

[0296] (1) Cell culture

[0297] Human embryonic kidney epithelial cells 293T and 293T-Gluc derived cell line were cultured in DMEM medium containing 10% fetal bovine serum (FBS).

[0298] (2) Preparation of recombinant influenza A virus

[0299] 1.8×10 cells were seeded in a 10 cm cell culture dish at a ratio of 3:1. 6 293T cells and 0.6×10 6MDCK cells. After 24 hours of culture, 8 plasmids (pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA, pHW185-NP, pHW186-NA, pHW187-M, pHW188-NS) of influenza A virus (IAV) A / WSN / 33 (H1N1) were transfected, the transfection amount was 1.2 μg, the transfection reagent was Lipofectamine2000, according to the instruction manual, 40 μl was used per dish. After 6 hours of transfection, fresh DMEM medium was replaced. TPCK-trypsin was added at a final concentration of 1 μg / mL 24 hours after transfection. After 48 hours, the supernatant was collected, centrifuged at 1000 rpm for 5 minutes to remove cell debris, filtered with a 0.45 μm filter membrane, and divided into small portions to obtain A / WSN / 33 (H1N1) recombinant influenza virus, which was stored in a -80°C refrigerator.

[0300] Among them, the influenza A virus (IAV) 8 plasmid reverse genetics system was donated by Dr. Robert G. Webster, namely: pHW181-PB2, pHW182-PB1, pHW183-PA, pHW184-HA, pHW185-NP, pHW186-NA, pHW187-M, pHW188-NS (Hoffmann, E., G. Neumann, et al. A DNA transfection system for generation of influenza A virus from eight plasmids [J]. Proc Natl Acad Sci USA, 2000, 97: 6108-6113).

[0301] (3) EC based on 293T-Gluc cells 50 Determination

[0302] 293T-Gluc cells (Gao Q, Wang Z, Liu Z, et al. A cell-based high-throughput approach to identify inhibitors of influenza A virus [J]. Acta Pharmaceutica Sinica B, 2014, 4 (4): 301-306) were plated in 96-well plates, and 2.5 × 10 4Cells were cultured in 100 μl of DMEM containing 10% FBS. 24 hours after cell plating, 1 μl of gradient dilution of the test compound was added to each well (the test compound was dissolved in DMSO (dimethyl sulfoxide) and diluted with DMSO). One hour after the test compound was added, the virus was infected at an MOI of 0.25. After 24 hours, 10 μl of the supernatant was taken to detect the Gluc protein content for calculating the EC 50 (Concentration required to inhibit the virus by 50%). The experiment was repeated three times.

[0303] Gasussia luciferase activity assay

[0304] Dissolve 250 μg of the substrate Coelenterazine-h lyophilized powder in 600 μL of anhydrous ethanol to prepare a substrate mother solution with a concentration of 1.022 mM, and store it at -20°C; before measurement, dilute the mother solution in PBS at a ratio of 1:60 to prepare a substrate working solution. Let it stand at room temperature for 30 minutes to stabilize the working solution. Since the substrate is unstable when exposed to light, it needs to be protected from light throughout the process; take 10 μL of cell culture supernatant (the cell supernatant after 24 hours of culture after transfection in the above Western Blot experiment) into a white opaque 96-well plate and use the microplate reader Centro XS 3 The LB 960 automatic sampler added the substrate working solution incubated in the dark to each well at a volume of 60 μL per well, and the signal was collected for 0.5 seconds. The measurement results were expressed in relative light units (RLU). Three groups of parallel experiments were set up. The experimental data are shown in GraphPad Prism 5.0 was used for graphing and statistical analysis.

[0305] (4) Cell viability assay

[0306] CCK-8 (Cell Counting Kit-8) is a rapid and highly sensitive detection kit based on WST-8 (water-soluble tetrazolium salt, chemical name: 2-(2-methyloxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazolyl monosodium salt) and widely used in cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT. In the presence of an electron coupling reagent, it can be reduced by some dehydrogenases in the mitochondria to generate orange-yellow formazan. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of color is linearly related to the number of cells. The light absorption value is measured at a wavelength of 450nm by an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells.

[0307] 293T-Gluc cells were seeded in 96-well plates at 2.5×10 4 cells, cultured in 100 μl DMEM culture medium containing 10% FBS. 24 hours after cell plating, add 1 μl of gradient dilution of the test compound to each well (the test compound is dissolved in DMSO and diluted with DMSO), set up blank control (only 100 μl DMEM culture medium), positive control (add 1 μl Ribavirin) and negative control (add 1 μl DMSO), and incubate at 37°C for 48 hours. Take out the 96-well plate, add 10 μl CCK-8 to each well, continue incubation at 37°C for 1-2 hours, and use Enspire2300 multifunctional microplate reader to detect the light absorption value of each well at a wavelength of 450nm to calculate the half cytotoxic concentration CC 50 (Refers to the drug concentration that causes 50% cell death.) The experiment was repeated three times.

[0308] Table 8. Anti-IAV activity results of compounds

[0309]

[0310] It can be seen that compounds C1-C14 have good anti-influenza A virus activity to varying degrees. 50 The values ​​were between 0.45 and 2.22 μM. In addition, these compounds had an 50 The values ​​were all greater than 100 μM. Therefore, this class of compounds exhibited strong antiviral ability and low cytotoxicity.

[0311] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0312] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0313] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A compound, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: The compound has the following structure: in, Bonds at 1 and 2 represents a single bond or a double bond, and 1 and 2 are not double bonds at the same time; R5 is selected from: H, methyl; R 17 Selected from: H, -OH; R 18 Selected from: H, methyl, ethyl, n-propyl, isopropyl, -CH2OH, -COOH, -COOCH3, -CHO; R 19 Selected from: R 20 is H; or, R 19 and R 20 Together with the carbon atom to which they are attached, they form a group selected from: The following compounds are not included:

2. The compound according to claim 1, characterized in that The compound is selected from the following structures:

3. The compound according to claim 1, characterized in that The stereoisomers are selected from the following structures:

4. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt, stereoisomer thereof, and one or more pharmaceutically acceptable excipients.

5. Use of the compound according to any one of claims 1 to 3 or its pharmaceutically acceptable salt or stereoisomer in the preparation of a pharmaceutical composition for preventing and / or treating a disease; The disease is a disease caused by infection with pathogens.

6. The use according to claim 5, characterized in that The disease caused by pathogen infection is selected from: influenza, SARS, COVID-19, viral hepatitis, AIDS, rabies, dengue fever, and Ebola virus disease.

7. Use of a compound or a pharmaceutically acceptable salt or stereoisomer thereof as a ligand of E3 ubiquitin ligase TRIM25 or in the preparation of a protein degradation targeting chimera PROTAC, wherein: The compound has the following structure: in, Bonds at 1 and 2 represents a single bond or a double bond, and 1 and 2 are not double bonds at the same time; R5 is selected from: H, methyl; R 17 Selected from: H, -OH; R 18 Selected from: H, methyl, ethyl, n-propyl, isopropyl, -CH2OH, -COOH, -COOCH3, -CHO; R 19 Selected from: R 20 is H; or, R 19 and R 20 Together with the carbon atom to which they are attached, they form a group selected from:

8. The use according to claim 7, characterized in that The compound is selected from the following structures:

9. The use according to claim 7, characterized in that The stereoisomers are selected from the following structures:

10. The use according to claim 7, characterized in that The PROTAC has the following structure: SMI-L-E3L (Ⅻ) Among them, SMI is the small molecule inhibitor part; E3L is the ligand portion of the E3 ubiquitin ligase; L is a connecting bond or a connecting group between SMI and E3L; Wherein, E3L is formed by the compound or its pharmaceutically acceptable salt or stereoisomer.

11. A PROTAC, characterized in that The PROTAC has the following structure: SMI-L-E3L (Ⅻ) Among them, SMI is the small molecule inhibitor part; E3L is the ligand portion of the E3 ubiquitin ligase; L is a connecting bond or a connecting group between SMI and E3L; Wherein, E3L is formed by the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt or stereoisomer thereof.

12. An Aspergillus fungus, whose strain number is CPCC 401321 and whose preservation number is CGMCC No.22467.

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