Application of Salvia miltiorrhiza extract and quinone compounds in anti-coronavirus

Abstract

The present invention discloses an application of a salvia miltiorrhiza extract and a quinone compound in anti-coronavirus. One or more compounds selected from tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, β-lapachone, levorotatory shikonin, 2-methoxy-1,4-naphthoquinone, CPI-169, S3I-201 and plumbagin, their pharmaceutically acceptable salts, their solvates, their pharmaceutically acceptable salt solvates, or their crystal forms are used in the preparation of anti-coronavirus agents, in the preparation of drugs for treating and / or preventing diseases caused by coronaviruses, and / or in the preparation of inhibitors for inhibiting papain. These compounds can well inhibit the activity of papain in coronaviruses, and inhibit the ability of coronaviruses to replicate in cells and thus inhibit the activity of coronaviruses. When these compounds are used to treat diseases caused by coronaviruses, the toxic side effects are low, and oral administration is easy to implement.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to the use of salvia miltiorrhiza extracts and quinone compounds in anti-coronavirus, and in particular to the use of one or more compounds selected from tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, β-lapachone, levorotatory shikonin, 2-methoxy-1,4-naphthoquinone, CPI-169, S3I-201 and plumbagin, their pharmaceutically acceptable salts, their solvates, their pharmaceutically acceptable salt solvates, or their crystal forms in the preparation of anti-coronavirus agents, in the preparation of drugs for treating and / or preventing diseases caused by coronaviruses, and / or in the preparation of inhibitors for inhibiting papain. Background Art

[0002] Coronaviruses are a class of viruses that are closely related to humans and animals. Coronaviruses HCoV-229E and HCoV-OC43 cause the common cold 1 . The severe acute respiratory syndrome (SARS) caused by the SARS coronavirus in 2002 and 2003 caused a huge stir around the world. HCoVNL63, identified in 2004, can also cause respiratory diseases similar to the common cold. 1 . In 2012, the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) appeared. The new coronavirus (SARS-CoV-2) that has recently spread around the world can cause new coronavirus pneumonia (COVID-19), with clinical manifestations of fever, dry cough and difficulty breathing, and severe cases can cause death. 4 Coronaviruses also have a huge impact on animal husbandry: porcine epidemic diarrhea virus (PEDV), gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV, also known as deltavirus) can cause severe enteritis, diarrhea, vomiting and dehydration in pigs, causing huge losses to the pig industry. 5 Feline infectious peritonitis virus (FIPV) is a lethal disease in cats. Avian infectious bronchitis virus (IBV) infects birds and is a widespread poultry disease with a huge economic impact on the poultry industry.

[0003] The coronavirus genome is a single-stranded positive-sense RNA with a length of about 28kb. It mainly encodes the structural proteins required for virus packaging and non-structural proteins related to replication and transcription. The development of drugs and vaccines for the treatment of coronavirus-related diseases mainly targets the above two types of proteins. Two-thirds of the genes in the viral genome mainly encode non-structural proteins, which are used to participate in the replication process of the virus. Before the production of mature non-structural proteins, coronavirus encodes two replicase polypeptides pp1a and pp1ab. Pp1a and pp1ab are cut into 16 non-structural proteins (nsp1-16) by two proteases encoded by the virus itself, namely the main protease and papain-like protease. Only when these functional subunits are correctly cut into independent protein units by proteases and then assembled into replication and transcription complexes can the virus complete normal transcription and replication functions.

[0004] Papain is located in the non-structural protein nsp3 (non-structural protein 3). During the cleavage process, it is responsible for cleaving the three cleavage sites at the N-terminus of the replicase polypeptide, thereby releasing nsp1, nsp2 and nsp3. 6,7,8 . Since papain has important functions such as proteolysis, deubiquitination, and antagonism of host IFN, it plays a key regulatory role in the transcription and replication of the virus, and thus becomes an important coronavirus drug target. Therefore, it is essential to find inhibitors with strong specificity and high safety against the catalytic site of papain for drug development. Inhibitors against papain, especially small molecule compounds targeting its substrate binding pocket, may become potential drugs for the treatment of coronavirus.

[0005] At present, the first-line clinical treatment of coronavirus-induced infections mostly uses broad-spectrum antiviral drugs, such as the anti-HIV drugs lopinavir / ritonavir (Kaletra), Abidol, and the anti-Ebola virus drug remdesivir. However, all of them have more or less side effects and even cause bradycardia in some subjects, so they are not the best choice for treating coronavirus. Therefore, based on existing evidence and experience, the treatment of coronavirus has its own particularity, and it is still necessary to develop drugs that can effectively treat and / or prevent diseases caused by coronavirus.

[0006] Tanshinone I (Molecular formula: C 18 H 12 O3, CAS No.: 568-73-0) is a chemical molecule extracted from the Chinese herbal medicine Salvia miltiorrhiza Bunge. Its molecular formula is as follows:

[0007]

[0008] Tanshinone I has anti-inflammatory effects and can regulate or inhibit the metastasis of breast cancer by regulating adhesion molecules (Nizamutdinova, Irina Tsoy, et al. "Tanshinone I suppresses growth and invasion of human breast cancer cells, MDA-MB-231, through regulation of adhesion molecules." Carcinogenesis 29.10 (2008): 1885-1892). CN102552236A discloses the use of Tanshinone I for treating microglia-mediated diseases, and CN102988370A also discloses the use of Tanshinone I in the preparation of drugs for treating psoriasis.

[0009] β-lapachone (Beta-lapachone, molecular formula: C 15 H 14 O3; CAS number: 4707-32-8) is an o-naphthoquinone, and its molecular formula is as follows:

[0010]

[0011] Studies have shown that Beta-lapachone can cause the release of cytochrome C, thereby causing cell apoptosis and necrosis. Its lethal dose for a variety of cancer cell cultures is at the micromolar level (the IC50 values ​​for AD2780s, HT-29, DLD, G480 and MCF-7 cell killing are 2μM, 5μM, 5μM, 4μM and 2μM, respectively), indicating that it has the potential to inhibit the growth of cancer cells. In addition, Beta-lapachone has also obtained positive results in some anti-tumor experiments in animals. In addition, Beta-lapachone can also inhibit the repair of cellular DNA damage, thereby making tumor cells more sensitive to X-rays, alkylating agents, etc. (Li, You-Zhi, et al. "Release of mitochondrialcytochrome C in both apoptosis and necrosis induced byβ-lapachone in humancarcinoma cells." Molecular Medicine 5.4 (1999): 232-239.) (Pardee, Arthur B., Y. Li, and Chiang J. Li. "Cancer therapy withβ-lapachone." Current cancer drug targets 2.3 (2002): 227-242).

[0012] DNA topoisomerase I is the drug target of Beta-lapachone that has been discovered so far. Beta-lapachone can inhibit the activity of DNA topoisomerase I in unwinding DNA supercoils. And this inhibitory effect is specific. Studies have shown that Beta-lapachone has no inhibitory activity against DNA topoisomerase II (Li CJ, Averboukh L, Pardee AB. beta-Lapachone, a novel DNA topoisomerase I inhibitor with a mode of action different from camptothecin. J Biol Chem. 1993 Oct 25; 268 (30): 22463-8.). US20050187288A1 discloses a method for treating cancer or precancerous conditions or preventing cancer in a subject using β-lapachone or its derivatives or analogs or its pharmaceutically acceptable salts.

[0013] Tanshinone IIA-sulfonic sodium (Molecular formula: C 19 H 17 NaO6S, CAS No.: 69659-80-9) is a water-soluble derivative of Tanshinone IIA, which is a chemical molecule extracted from the Chinese herbal medicine Salvia miltiorrhiza Bunge. The molecular formula of Tanshinone IIA sodium sulfonate is as follows:

[0014]

[0015] Tanshinone IIA sodium sulfonate has a variety of pharmacological functions, including anti-oxidation, anti-inflammatory and anti-apoptosis, and has been approved by CFDA (China State Food and Drug Administration) for the treatment of cardiovascular diseases. In addition, in recent decades, many studies and clinical trials have also shown that tanshinone IIA sodium sulfonate has certain potential in the treatment of brain diseases, lung diseases, cancer, and sepsis (Zhou, Zhong-Yan, et al. "Sodium tanshinoneIIA sulfonate: A review of pharmacological activity and pharmacokinetics." Biomedicine & Pharmacotherapy 118 (2019): 109362). JP5247469B2 discloses that the use of tanshinone IIA sodium sulfonate can effectively prevent or reduce the physiological effects of endotoxin shock in mammals. CN101120940A discloses the application of tanshinone IIA sodium sulfonate as a blood-brain barrier protective agent in brain diseases.

[0016] Cryptotanshinone (Cryptotanshinone, molecular formula: C 19 H 20 O3, CAS No.: 35825-57-1) is a quinone diterpenoid isolated from the Chinese herbal medicine Salvia miltiorrhiza Bunge, and its molecular formula is as follows:

[0017]

[0018] Studies have shown that the drug target of cryptotanshinone is STAT3, which can effectively inhibit STAT3. Cryptotanshinone can quickly inhibit the phosphorylation of STAT3 Tyr 705 through a JAK2-independent mechanism. Cryptotanshinone blocks STAT3 dimerization by binding to monomeric STAT3, ultimately inhibiting the transcriptional regulatory activity of STAT3 (Shin, Dae-Seop, et al. "Cryptotanshinone inhibits constitutive signal transducer and activator of transcription 3 function through blocking the dimerization in DU145 prostate cancer cells." Cancer research 69.1 (2009): 193-202). In addition, there are studies reporting that cryptotanshinone can play an anti-inflammatory role by inhibiting the activity of cyclooxygenase II (Jin, Dao-Zhong, et al. "Cryptotanshinone inhibits cyclooxygenase-2 enzyme activity but not its expression." European journal of pharmacology 549.1-3 (2006): 166-172). CN106798737A also discloses the role of cryptotanshinone in preventing and treating pulmonary fibrosis. JP2004517939A also discloses the role of cryptotanshinone in preventing and treating early Alzheimer's disease.

[0019] Dihydrotanshinone I (Molecular formula: C 18 H 14 O3, CAS No.: 87205-99-0) is a natural compound extracted from the Chinese herbal medicine Salvia miltiorrhiza Bunge. Its molecular formula is as follows:

[0020]

[0021] Dihydrotanshinone I is cytotoxic to a variety of tumor cells. Studies have shown that dihydrotanshinone I can activate NOX5 by increasing calcium ion concentration. Further, NOX5 can induce the production of ROS (reactive oxygen species), and ultimately through the ROS / Stat3 signaling pathway, dihydrotanshinone I can inhibit the growth of breast cancer stem cells. (Kim, Su-Lim, et al. "Dihydrotanshinone-induced NOX5 activation inhibits breast cancer stem cellthrough the ROS / Stat3 signaling pathway." Oxidative medicine and cellular longevity 2019(2019).)

[0022] In addition, dihydrotanshinone I can produce strong cytotoxicity to umbilical vein endothelial cells (IC50 value is about 1.28μg / ml), indicating that dihydrotanshinone I has potential anti-angiogenesis ability (Bian, W; Chen, F; Bai, L; Zhang, P; Qin, W (2008). "Dihydrotanshinone I inhibits angiogenesis both in vitro and invivo". Acta Biochimica et Biophysica Sinica. 40 (1): 1-6). CN108379273A discloses the use of dihydrotanshinone I in drugs for treating multidrug-resistant tumors, and CN110613708A also discloses the use of dihydrotanshinone I in the preparation of P53 agonists and signal transduction and transcription activator 3 inhibitors.

[0023] Levorotatory shikonin (Alkannin, molecular formula: C 16 H 16 O5, CAS: 517-88-4) is a natural red naphthoquinone pigment, its molecular formula is as follows:

[0024]

[0025] Levorotatory shikonin has antibacterial, antitumor and anti-inflammatory effects; purified shikonin preparations are widely used in the production of medicines, cosmetics and certain foods; shikonin can also be incorporated into anti-inflammatory ointments and cream compositions for treating burns. It inhibits the viability, adhesion, invasion and migration of MGC-803 cells through TLR2 or NF-κB-mediated pathways (Takano-Ohmuro H, Yoshida LS, Yuda Y, et al. Shikonin inhibits IgE-mediated histamine release by human basophils and Syk kinase activity[J]. Inflammation Research, 2008, 57(10): 484-488.)(Liu JP, Liu D, Gu JF, et al. Shikonin inhibits the cell viability, adhesion, invasion and migration of the human gastric cancer cell line MGC-803via the Toll-like receptor 2 / nuclear factor-kappa B pathway[J]. Journal of Pharmacy & Pharmacology, 2015, 67(8): 1143-1155), and targets PI3Kα. CN104771384B discloses the use of shikonin for preparing antiviral drugs such as anti-herpes simplex virus and vesicular stomatitis virus.

[0026] CPI-169 (anti-tumor drug, Synonyms: CPI 169R-enantiomer, molecular formula: C 27 H 36 N4O5S, CAS: 1802175-07-0) is a novel and potent EZH2 inhibitor that can inhibit the activity of EZH2 WT, EZH2 Y641N and EZH1 with IC50 values ​​of 0.24nM, 0.51nM and 6.1nM, respectively. Its molecular formula is shown below:

[0027]

[0028] CPI-169 inhibits the catalytic activity of PRC2 with IC50 <1 nM, reduces the cellular level of H3K27me3 with EC50 of 70 nM, and triggers cell cycle arrest and apoptosis in multiple cell lines (Vidya Balasubramanian, et al. CPI-169, a novel and potent EZH2 inhibitor, synergizes with CHOP in vivo and achieves complete regression in lymphoma xenograft models. Cancer Res October 1, 201474; 1697).

[0029] In KARPAS-422 cells, CPI-169, when used in combination with ABT-199, had a dose-dependent inhibitory effect on cell viability and produced synergistic antiproliferative activity. CPI-169 also inhibited cell growth in 16 of 25 NHL cell lines, with GI50 < 5 μM (Bradley WD, et al. EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation. ChemBiol. 2014 Nov 20; 21(11): 1463-75). US20190134025A1 also discloses the use of drugs such as CPI-169 in the treatment of cancer in human subjects with cancer whose cancer cells overexpress arginine methyltransferase CARM1.

[0030] S3I-201 (anti-tumor drug, Synonyms: NSC 74859, molecular formula: C 16 H 15 NO7S, CAS: 501919-59-1) is a selective Stat3 inhibitor with an IC50 of 86 μM. Its molecular formula is as follows:

[0031]

[0032] NSC 74859 (S3I-201) preferentially inhibits Stat3 DNA binding activity relative to Stat1 (IC50 values, Stat3·Stat3, 86±33μM; Stat1·Stat3, 160±43μM; Stat1·Stat1, >300μM) and inhibits Stat5 with an IC50 of 166±17μM). NSC 74859 significantly reduced the number of viable cells and inhibited the growth of transformed mouse fibroblasts NIH3T3 / v-Src and breast cancer cell lines (MDA-MB-231, MDA-MB-435, and MDA-MB-468). NSC 74859 induced significant apoptosis in representative human breast cancer cell lines MDA-MB-435 and NIH 3T3 / v-Src at concentrations of 30-100μM, both of which have constitutively active Stat3. The breast cancer MDA-MB-435 cell line was more sensitive to 30μM NSC74859. In contrast, human breast cancer MDA-MB-453 cells and normal mouse fibroblasts (NIH 3T3) do not contain abnormal Stat3 activity and are insensitive to NSC 74859 at 100 μM or less. At concentrations of 300 μM or higher, NSC 74859 causes general nonspecific cytotoxicity that is independent of the Stat3 activation state (Siddiquee K, et al. Selective chemical probe inhibitor of Stat3, identified through structure-based virtual screening, induces antitumor activity. Proc Natl Acad Sci US A. 2007 May 1; 104 (18): 7391-6.). Huh-7 cells do not express β2SP or TBGFR2 and are sensitive to STAT3 inhibition, with an IC50 of 100 μM for NSC 74859, regardless of the CD133+ state. The IC50 of NSC 74859 is 150 μM for Huh-7 and SNU-398 cells, 15 μM for SNU-475 cells, and 200 μM for SNU-182 cells.NSC 74859 inhibits breast cancer MDA-MB-435, MDA-MB-453 and MDA-MB-231 cell lines, with an IC50 close to 100 μM (Lin L, et al. The STAT3 inhibitor NSC 74859 is effective in hepatocellular cancers with disrupted TGF-β signaling. Oncogene. 2009 Feb 19; 28(7): 961-72.). US20140296270A1 also mentioned that S3I-201 (5 mg / kg every 2-3 days) can inhibit the growth of breast cancer cell lines.

[0033] 2-Methoxy-1,4-naphthoquinone (2-Methoxy-1,4-naphthoquinone, molecular formula: C 11 H8O3,CAS:2348-82-5) is an extract from Impatiens balsamina leaves, and its molecular formula is as follows:

[0034]

[0035] 2-Methoxy-1,4-naphthoquinone specifically inhibited the expression of PKCβI, PKCδ and PKCζ in Raji cells in a concentration-dependent manner. 2-Methoxy-1,4-naphthoquinone (MNQ) exhibited antifungal, antibacterial activities and showed potent killing effects against K562, HL-60, Raji, MCF-7, H-23, IMR-32, LA-174-T, HSC-2 and SK-MEL-28. MNQ was able to trigger the intrinsic apoptotic pathway in K562 cells and caused p53-dependent cell cycle arrest in p53 wild-type cell lines. MNQ inhibited TCF / β-catenin (TOP) transcriptional activity at an IC50 of 2.9μM and also reduced transcriptional activity in FOP (mutated TCF binding site) transfected cells. MNQ had an anti-metastatic effect on MDA-MB-231 cells. MNQ showed remarkable selectivity for PKCβI and ζ inhibition. MNQ moderately inhibits PKCδ, where MNQ inhibits half of PKCδ expression at a concentration of 10 μM. MNQ does not inhibit PKCβII (Wong Teck Yew, et al. Journal of Applied Pharmaceutical Science). MNQ can inhibit the invasion and migration properties of highly metastatic MDA-MB-231 cancer cells (Liew K, et al. Toxicol In Vitro. 2014, 28 (3): 335-9.). TW201726169A discloses the use of 2,3-dimethoxy-1,4-naphthoquinone as a ROS generator in the preparation of a pharmaceutical composition.

[0036] Plumbagin (molecular formula C11H8O3., CAS: 481-42-5) is a quinone component that has been shown to have anti-cancer, anti-atherosclerotic and antibacterial effects. Its molecular formula is shown below:

[0037]

[0038] In vitro experiments found that plumbagin exhibited potent cytostatic effects by inducing cancer cells to undergo G2 / M arrest and apoptosis. The blockade of the cell cycle was associated with increased levels of p21 and decreased levels of Cdc2, Cdc25C, and cyclinB1. Plumbagin treatment was also found to enhance the levels of inactivated phosphorylated Cdc2 and Cdc25C. Blocking p53 activity partially reduced plumbagin-induced apoptosis and G2 / M arrest, indicating that it may act through p53-dependent and independent pathways. In vivo experiments, to determine whether plumbagin inhibits tumor growth in vivo, A549 cells were injected into nude mice. Tumor growth inhibition was most pronounced in mice treated with 2 mg / kg / day of plumbagin, in which a reduction of approximately 80% in tumor size was observed compared to mice treated with vehicle. Judging by monitoring body weight, no signs of toxicity were observed in mice treated with plumbagin (Hsu YL, Cho CY, Kuo PL, Huang YT, Lin CC. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) induces apoptosis and cell cycle arrestin A549 cells through p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo. J Pharmacol Exp Ther. 2006 Aug; 318 (2): 484-94. Chemical Properties). US20140271923A1 also discloses the use of compounds such as plumbagin for patients with various chronic diseases of aggregation.

[0039] There is no drug available in the prior art that can be used to treat diseases related to coronavirus. Summary of the invention

[0040] The technical problem to be solved by the present invention is to address the defect that there is no effective drug for treating and / or preventing diseases caused by coronavirus in the prior art, and provide one or more compounds selected from tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, β-lapachone, levorotatory shikonin, 2-methoxy-1,4-naphthoquinone, CPI-169, S3I-201 and plumbagin, their pharmaceutically acceptable salts, their solvates, their pharmaceutically acceptable salt solvates, or their crystal forms, and pharmaceutical compositions and kits containing them for use in anti-coronavirus and / or in inhibiting papain. The anti-coronavirus may be the use of the compounds described in the present invention as anti-coronavirus agents (for example, they can be used to prevent and / or treat patients carrying coronavirus who do not show a disease state), or the use of the compounds described in the present invention to treat and / or prevent diseases caused by coronavirus. These compounds of the present invention can well inhibit the activity of papain in coronavirus, and can well inhibit the replication ability of coronavirus in cells and thus inhibit the activity of coronavirus, and can be used to treat and / or prevent related diseases caused by coronavirus, or be prepared into anti-coronavirus agents, filling the gap of the prior art in lacking treatment and / or prevention of diseases caused by coronavirus. When the compounds of the present invention are used to treat and / or prevent diseases caused by coronavirus, the toxic side effects are low, and oral administration is easy to implement. The compound can also be further studied and modified for application.

[0041] After a large number of experiments, the inventors unexpectedly discovered that certain compounds of Salvia miltiorrhiza extract, certain compounds of quinones (such as naphthoquinones), certain EZH2 inhibitors, certain STAT3 inhibitors and other compounds can significantly inhibit the activity of papain (especially coronavirus papain) and can be used to treat related diseases caused by coronavirus.

[0042] In order to solve the above technical problems, the first aspect of the present invention provides a method selected from tanshinone I (CAS No.: 568-73-0), tanshinone IIA, cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), β-lapachone (CAS No.: 4707-32-8), levorotatory shikonin (CAS No.: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5), CPI-169 (CAS No.: 1802175-07-0), S3I-201 (CAS No.: 501919-59-1) and plumbagin (CAS: 481-42-5), their pharmaceutically acceptable salts, their solvates, their pharmaceutically acceptable salt solvates, or their crystal forms in the preparation of anti-coronavirus agents, in the preparation of drugs for treating and / or preventing diseases caused by coronaviruses, and / or in the preparation of inhibitors for inhibiting papain.

[0043] Preferably, the pharmaceutically acceptable salt is a sulfonate and / or a sodium salt.

[0044] Preferably, the pharmaceutically acceptable salt of Tanshinone IIA is Tanshinone IIA sodium sulfonate (CAS No.: 69659-80-9).

[0045] Preferably, the papain is a coronavirus papain.

[0046] Preferably, the compound, its pharmaceutically acceptable salt, its solvate, its pharmaceutically acceptable salt solvate, or its crystalline form inhibits the papain (especially the papain of coronavirus, for example, inhibits the activity of the enzyme).

[0047] Preferably, the disease is a mammalian or avian disease, and the mammal is preferably a human, a pig and / or a cat.

[0048] The coronavirus described in the present invention is defined as being well known in the art and belonging to the subfamily Coronaviridae of the order Nidovirales in terms of systematic classification. Coronavirus is an RNA virus with an envelope and a single-stranded positive-strand genome, and is a large class of viruses that are widely present in nature.

[0049] The object of the present invention is to provide a potential treatment for a disease caused by coronavirus infection. The coronavirus of the present invention preferably belongs to the subfamily Orthocoronavirinae, more preferably to the genus Alphacoronavirus, genus Betacoronavirus, genus Gammacoronavirus and / or genus Deltacoronavirus.

[0050] Preferably, the coronavirus is selected from one or more of coronaviruses that cause upper respiratory tract infection, viruses that cause acute respiratory syndrome, such as SARS-related coronaviruses, and Middle East respiratory syndrome coronavirus (MERS-CoV). The coronavirus that causes upper respiratory tract infection is preferably human coronavirus 229E, human coronavirus HKU1, human coronavirus OC43, human coronavirus NL63 and / or mouse hepatitis virus A59. The SARS-related coronavirus is preferably SARS-CoV or SARS-CoV-2.

[0051] In the present invention, the compounds can be used to treat diseases caused by SARS-CoV-2 (Beta coronavirus), and can also be used to treat major infectious diseases caused by other coronaviruses such as SARS-CoV (Beta coronavirus) and MERS-CoV. They can also be used as common cold medicines to treat diseases caused by coronaviruses such as HCoV-HKU1 (Human coronavirus HKU1; Beta coronavirus), HCoV-NL63 (Human coronavirus NL63; Alpha coronavirus), HCoV-OC43 (Human coronavirus OC43) and HCoV-229E (Human coronavirus 22E; Alpha coronavirus); they can also be used as veterinary drugs to treat porcine transmissible gastroenteritis virus (TGEV; Alpha coronavirus), porcine epidemic diarrhea virus (PEDV; Alpha coronavirus), porcine delta coronavirus (PDCoV; Delta coronavirus), and feline infectious peritonitis virus (Feline infectious peritonitis virus). virus, FIPV; Alpha coronavirus genus), avian infectious bronchitis virus (Infectious bronchitis virus, IBV; Gamma coronavirus genus) and other animal diseases.

[0052] In the present invention, the SARS-related coronavirus is the coronavirus that causes severe acute respiratory syndrome (SARS). The disease caused by the SARS-related coronavirus is a symptom or disease caused by viral infection. The disease is mainly reflected in respiratory diseases in the early stage, and clinically manifested as fever, fatigue, dry cough, cough, accelerated breathing or acute respiratory distress syndrome, shortness of breath, etc. A small number of patients are accompanied by symptoms such as nasal congestion, runny nose, and sore throat. Imaging shows varying degrees of changes in the lungs such as multiple spots and ground-glass shadows. The main mode of virus transmission is close-range droplet transmission or contact with the patient's respiratory secretions. In addition, there may be digestive tract symptoms such as diarrhea, acute gastroenteritis in infants and neonates, and in rare cases, neurological syndrome. Many complications may occur in the later stage, including difficult-to-correct metabolic acidosis and coagulation dysfunction, respiratory failure, fulminant myocarditis to heart failure, liver and kidney failure, and multiple organ failure and septic shock. Severe patients often experience dyspnea and / or hypoxemia one week after onset, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis and coagulopathy, and multiple organ failure. The above diseases are all expected to be treated with the PARP inhibitors of the present invention. In addition, there are also some virus carriers who do not show the above symptoms, and the PARP inhibitors of the present invention can also be used to inhibit viral replication and spread, thereby preventing the virus carriers from becoming ill or spreading the virus to produce new cases. In a certain embodiment, the virus of the genus Betacoronavirus is a virus that causes acute respiratory syndrome, such as a SARS-related coronavirus; preferably, the SARS-related coronavirus is SARS-CoV (severe acute respiratory syndrome coronavirus) and / or SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2).

[0053] In a preferred embodiment of the present invention, the coronavirus includes severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

[0054] In a preferred embodiment of the present invention, the coronavirus is severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

[0055] In the present invention, the inhibitor of papain that inhibits coronavirus can be in the form of an inhibitor that inhibits coronavirus, such as a conventional drug used to fight coronavirus.

[0056] In order to solve the above technical problems, the second aspect of the present invention provides a pharmaceutical composition for use in the preparation of an anti-coronavirus agent, in the preparation of a drug for treating and / or preventing a disease caused by a coronavirus, and / or in the preparation of an inhibitor of papain, wherein the pharmaceutical composition comprises a compound selected from tanshinone I (CAS No.: 568-73-0), tanshinone IIA, cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), β-lapachone (CAS No.: 4707-32-8), levorotatory shikonin (CAS No.: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5), CPI-169 (CAS No.: 1802175-07-0), S3I-201 (CAS No.: 501919-59-1) and plumbagin (CAS: 481-42-5), a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystalline form thereof.

[0057] Preferably, the pharmaceutically acceptable salt is a sulfonate and / or a sodium salt.

[0058] Preferably, the pharmaceutically acceptable salt of Tanshinone IIA is Tanshinone IIA sodium sulfonate (CAS No.: 69659-80-9).

[0059] Preferably, the papain is a coronavirus papain.

[0060] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such as a pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical composition is usually selected from tanshinone I (CAS No.: 568-73-0), tanshinone IIA, cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), β-lapachone (CAS No.: 4707-32-8), levorotatory shikonin (CAS No.: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5), CPI-169 (CAS No.: 1802175-0 7-0), S3I-201 (CAS No.: 501919-59-1) and plumbagin (CAS: 481-42-5), their pharmaceutically acceptable salts, their solvates, their pharmaceutically acceptable salt solvates, or their crystalline forms as the sole active ingredient of the pharmaceutical composition, and other active ingredients may also be present, and the other active ingredients may be active ingredients for treating and / or preventing diseases related to coronavirus, or active ingredients for treating and / or preventing diseases caused by other viruses.

[0061] Preferably, the coronavirus is as described in the first aspect of the present invention. Specifically, it can be:

[0062] The coronavirus may be a coronavirus belonging to the subfamily Orthocoronavirinae. The subfamily Orthocoronavirinae virus is preferably a virus belonging to the genus Alphacoronavirus, genus Betacoronavirus, genus Gammacoronavirus and / or genus Deltacoronavirus.

[0063] The coronavirus may be one or more selected from coronaviruses that cause upper respiratory tract infections, viruses that cause acute respiratory syndrome, such as SARS-associated coronaviruses, and Middle East respiratory syndrome coronaviruses; the coronavirus that causes upper respiratory tract infections is preferably human coronavirus 229E, human coronavirus HKU1, human coronavirus OC43, human coronavirus NL63 and / or mouse hepatitis virus A59; the SARS-associated coronavirus is preferably SARS-CoV or SARS-CoV-2;

[0064] The coronavirus may also be porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine delta coronavirus, feline infectious peritonitis virus and / or avian infectious bronchitis virus.

[0065] In order to solve the above technical problems, the third aspect of the present invention provides a kit for use in the preparation of anti-coronavirus agents, in the preparation of drugs for treating and / or preventing diseases caused by coronaviruses, and / or in the preparation of inhibitors for inhibiting papain, wherein the kit comprises one or more compounds selected from tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, β-lapachone, levorotatory shikonin, 2-methoxy-1,4-naphthoquinone, CPI-169, S3I-201 and plumbagin, pharmaceutically acceptable salts thereof, solvates thereof, solvates of pharmaceutically acceptable salts thereof, or crystalline forms thereof.

[0066] Preferably, the pharmaceutically acceptable salt is a sulfonate and / or a sodium salt.

[0067] Preferably, the pharmaceutically acceptable salt of Tanshinone IIA is Tanshinone IIA sodium sulfonate.

[0068] Preferably, the papain is a coronavirus papain.

[0069] Preferably, the medicine kit further comprises drugs for treating and / or preventing diseases associated with coronavirus, and / or drugs for treating and / or preventing diseases caused by other viruses.

[0070] Preferably, the coronavirus is as described in the first aspect of the present invention. Specifically, it can be:

[0071] The coronavirus may be a coronavirus belonging to the subfamily Orthocoronavirinae. The subfamily Orthocoronavirinae virus is preferably a virus belonging to the genus Alphacoronavirus, Betacoronavirus, Gammacoronavirus and / or Deltacoronavirus.

[0072] The coronavirus may be one or more selected from coronaviruses that cause upper respiratory tract infections, viruses that cause acute respiratory syndrome, such as SARS-associated coronaviruses, and Middle East respiratory syndrome coronaviruses; the coronavirus that causes upper respiratory tract infections is preferably human coronavirus 229E, human coronavirus HKU1, human coronavirus OC43, human coronavirus NL63 and / or mouse hepatitis virus A59; the SARS-associated coronavirus is preferably SARS-CoV or SARS-CoV-2;

[0073] The coronavirus may also be porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine delta coronavirus, feline infectious peritonitis virus and / or avian infectious bronchitis virus.

[0074] In order to solve the above technical problems, the present invention also provides a kind of selected from tanshinone I (CAS number: 568-73-0), tanshinone IIA, cryptotanshinone (CAS number: 35825-57-1), dihydrotanshinone I (CAS number: 87205-99-0), β-lapachone (CAS number: 4707-32-8), levorotatory shikonin (CAS number: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS number: 2348-82-5), CPI-169 (CAS number: 1802175-07-0), S3I-201 (CAS number: 501919-59-1) and One or more compounds of plumbagin (CAS: 481-42-5), pharmaceutically acceptable salts thereof, solvates thereof, solvates of pharmaceutically acceptable salts thereof, or crystal forms thereof are used as anti-coronavirus agents, or for treating and / or preventing diseases caused by coronavirus, or for preparing anti-coronavirus agents or in preparing drugs for treating and / or preventing diseases caused by coronavirus, or for inhibiting papain (e.g., papain of coronavirus, for example, inhibiting the activity of papain), or for preparing inhibitors for inhibiting papain (e.g., papain of coronavirus, for example, inhibiting the activity of papain).

[0075] Preferably, one or more compounds selected from tanshinone I (CAS No.: 568-73-0), tanshinone IIA, cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), β-lapachone (CAS No.: 4707-32-8), levorotatory shikonin (CAS No.: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5), CPI-169 (CAS No.: 1802175-07-0), S3I-201 (CAS No.: 501919-59-1) and plumbagin (CAS: 481-42-5), their pharmaceutically acceptable salts, their solvates, their pharmaceutically acceptable salt solvates, or their crystalline forms as described in the first aspect or the second aspect of the present invention.

[0076] Preferably, the coronavirus is as described in the first aspect or the second aspect of the present invention.

[0077] In order to solve the above technical problems, the present invention also provides a method selected from tanshinone I (CAS No.: 568-73-0), tanshinone IIA, cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), β-lapachone (CAS No.: 4707-32-8), levorotatory shikonin (CAS No.: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5), CPI-169 (CAS No.: 1 802175-07-0), S3I-201 (CAS No.: 501919-59-1) and plumbagin (CAS: 481-42-5), their pharmaceutically acceptable salts, their solvates, their pharmaceutically acceptable salt solvates, or their crystalline forms, for use in anti-coronavirus, or in treating and / or preventing diseases caused by coronavirus, or in inhibiting papain (e.g., papain of coronavirus, for example, inhibiting the activity of papain).

[0078] Preferably, the coronavirus is as described in the first aspect or the second aspect of the present invention.

[0079] In order to solve the above technical problems, the present invention also provides an anti-coronavirus method, or a method for treating and / or preventing a disease caused by a coronavirus, or a method for inhibiting papain (e.g., papain of coronavirus, such as inhibiting the activity of papain), which comprises administering a compound selected from tanshinone I (CAS No.: 568-73-0), tanshinone IIA, cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), β-lapachone (C The invention relates to a method for preparing one or more compounds of the group consisting of scutellaria baicalensis (CAS No.: 4707-32-8), levorotatory shikonin (CAS No.: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5), CPI-169 (CAS No.: 1802175-07-0), S3I-201 (CAS No.: 501919-59-1) and plumbagin (CAS: 481-42-5), pharmaceutically acceptable salts thereof, solvates thereof, solvates of pharmaceutically acceptable salts thereof, or crystalline forms thereof.

[0080] Preferably, the coronavirus is as described in the first aspect or the second aspect of the present invention.

[0081] The present invention also provides a kit for use in preparing an anti-coronavirus agent, in preparing a drug for treating and / or preventing a disease caused by a coronavirus, and / or in preparing an inhibitor for inhibiting papain (e.g., papain of coronavirus), wherein the kit comprises a compound selected from tanshinone I (CAS No.: 568-73-0), tanshinone IIA, cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), β-lapachone (CAS No.: 4 707-32-8), levorotatory shikonin (CAS No.: 517-88-4), 2-methoxy-1,4-naphthoquinone (CAS No.: 2348-82-5), CPI-169 (CAS No.: 1802175-07-0), S3I-201 (CAS No.: 501919-59-1) and plumbagin (CAS: 481-42-5), a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystalline form thereof.

[0082] Preferably, the medicine kit further comprises drugs for treating and / or preventing diseases associated with coronavirus, and / or drugs for treating and / or preventing diseases caused by other viruses.

[0083] Preferably, the coronavirus is as described in the first aspect or the second aspect of the present invention.

[0084] The pharmaceutical compositions involving the present invention and using the various compounds of the present invention as active ingredients can be prepared according to methods known in the art. The compounds of the present invention can be made into any dosage form suitable for human or animal use. The weight content of the compounds of the present invention in the pharmaceutical compositions is usually 0.1-99.0%.

[0085] The pharmaceutically acceptable carrier may be a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above protein and / or the above antibody-drug conjugate, and 0.01 to 99.99% of a pharmaceutical carrier, and the percentage is the mass percentage of the pharmaceutical composition.

[0086] The compound of the present invention or the pharmaceutical composition containing the compound can be prepared into a common preparation, or a sustained-release preparation, a controlled-release preparation, a targeted preparation and various microparticle drug delivery systems.

[0087] "Pharmaceutical composition" refers to a mixture of one or more of the compounds of the present invention or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof with another chemical component, such as a pharmaceutically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate the process of administration to an animal.

[0088] The aforementioned pharmaceutical composition, in addition to the pharmaceutically acceptable carrier, may also include adjuvants commonly used in pharmacy, such as: antibacterial agents, antifungal agents, antimicrobial agents, preservatives, colorants, solubilizers, thickeners, surfactants, chelating agents, proteins, amino acids, fats, carbohydrates, vitamins, minerals, trace elements, sweeteners, pigments, flavors or combinations thereof.

[0089] "Pharmaceutically acceptable carrier" refers to an inactive ingredient in a pharmaceutical composition that does not cause significant irritation to an organism and does not interfere with the biological activity and properties of the administered compound, such as but not limited to: calcium carbonate, calcium phosphate, various sugars (such as lactose, mannitol, etc.), starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic acid polymer or methacrylic acid polymer, gelatin, water, polyethylene glycol, propylene glycol, ethylene glycol, EZ sesame oil or hydrogenated EZ sesame oil or polyethoxylated hydrogenated EZ sesame oil, sesame oil, corn oil, peanut oil, etc.

[0090] The term "treat" refers to therapeutic treatment. When referring to a specific condition, treatment means: (1) ameliorating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade leading to or causing the condition or (b) one or more biological manifestations of the condition, (3) ameliorating one or more symptoms, effects, or side effects associated with the condition or one or more symptoms, effects, or side effects associated with the condition or its treatment, or (4) slowing the progression of the condition or one or more biological manifestations of the condition.

[0091] The compounds of the present invention or pharmaceutical compositions containing the same can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0092] The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w types, w / o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0093] The term "pharmaceutically acceptable" means that salts, solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

[0094] The term "pharmaceutically acceptable salt" refers to a salt prepared from a compound of the present invention and a relatively nontoxic, pharmaceutically acceptable acid or base. When the compound of the present invention contains a relatively acidic functional group, a base addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compound of the present invention contains a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acid includes an inorganic acid, and the inorganic acid includes, but is not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, and the like. The pharmaceutically acceptable acid includes organic acids, including but not limited to acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, sulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid, tannic acid, pantothenic acid, bitartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (e.g., glutamic acid, arginine), etc. When the compounds of the present invention contain relatively acidic and relatively basic functional groups, they can be converted into base addition salts or acid addition salts. For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0095] The term "solvate" refers to a substance formed by the combination of a compound of the present invention and a stoichiometric or non-stoichiometric solvent. The solvent molecules in the solvate may exist in an ordered or non-ordered arrangement. The solvent includes, but is not limited to, water, methanol, ethanol, etc.

[0096] The "pharmaceutically acceptable salt" and "solvate" in the term "pharmaceutically acceptable salt solvate" as described above refer to a substance formed by combining the compound of the present invention with 1. a relatively nontoxic, pharmaceutically acceptable acid or base prepared 2. a stoichiometric or non-stoichiometric solvent. The "pharmaceutically acceptable salt solvate" includes but is not limited to the hydrochloric acid monohydrate of the compound of the present invention.

[0097] The terms "compound", "pharmaceutically acceptable salt", "solvate" and "solvate of a pharmaceutically acceptable salt" may exist in a crystalline or amorphous form. The term "crystalline form" means that the ions or molecules therein are arranged in a strict periodic manner in three-dimensional space in a certain manner and have a regularity of periodic repetition at a certain distance; due to the different periodic arrangements mentioned above, there may be multiple crystalline forms, i.e., polymorphism. The term "amorphous form" means that the ions or molecules therein are in a disorderly distribution state, i.e., there is no periodic arrangement between the ions and molecules.

[0098] The "plurality" in the term "one or more" may mean 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0099] In the present invention, the term “include, include or contain” may mean that in addition to the components listed below, other components are present; or it may mean “consisting of…”, that is, only the components listed below are present without other components.

[0100] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0101] The reagents and raw materials used in the present invention are commercially available.

[0102] The positive and progressive effects of the present invention are that the compounds of the present invention can well inhibit the activity of papain in coronavirus, and can well inhibit the replication ability of coronavirus in cells and thus inhibit the activity of coronavirus, and have low cytotoxicity, and can be used to treat and / or prevent related diseases caused by coronavirus, or be prepared into anti-coronavirus agents, filling the gap in the prior art in the lack of treatment and / or prevention of diseases caused by coronavirus. When the compounds of the present invention are used to treat and / or prevent diseases caused by coronavirus, the toxic side effects are low, and oral administration is easy to implement. The compound can also be further studied and modified for application. BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Figure 1 : Inhibitory activity of Tanshinone I on the papain-like protease of the new coronavirus; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0104] Figure 2 :Inhibitory activity of Beta-Lapachone on the papain-like protease of the new coronavirus; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0105] Figure 3 : Inhibitory activity of sodium tanshinone IIA sulfonate on the papain-like protease of the new coronavirus; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0106] Figure 4 : Inhibitory activity of cryptotanshinone on the papain-like protease of the new coronavirus; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0107] Figure 5 : Inhibitory activity of dihydrotanshinone I on the novel coronavirus papain; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0108] Figure 6 : Inhibitory activity of levorotatory shikonin on papain-like protease of novel coronavirus; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0109] Figure 7 : Inhibitory activity of CPI-169 against the novel coronavirus papain; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0110] Figure 8 :Inhibitory activity of S3I-201 on the novel coronavirus papain; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0111] Fig. 9 : Inhibitory activity of 2-methoxy-1,4-naphthoquinone (Impatiens balsamina leaf extract) on the novel coronavirus papain-like protease; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0112] Fig.10 : Inhibitory activity of plumbagin on papain-like protease of novel coronavirus; 1: DMSO (negative control), 2: compound molecule; 3: positive control (GRL-0617).

[0113] Fig.11Tanshinone I has a strong inhibitory activity against papain-like protease of novel coronavirus (IC 50 =2.21 μM).

[0114] Fig.12 Tanshinone I can inhibit the replication of SARS-CoV-2 in cells (EC 50 =2.26μM) and had lower cytotoxicity (CC 50 >300μM).

[0115] Fig.13 Cryptotanshinone has a strong inhibitory activity against papain-like protease of novel coronavirus (IC 50 =5.63 μM).

[0116] Fig.14 Cryptotanshinone can inhibit the replication of SARS-CoV-2 in cells (EC 50 =0.7 μM) and had low cytotoxicity (CC 50 >300μM). DETAILED DESCRIPTION

[0117] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0118] Source of compound

[0119] Tanshinone I (CAS No.: 568-73-0) and Tanshinone IIA sodium sulfonate (CAS No.: 69659-80-9) were purchased from Approved Drug Library (Target Mol).

[0120] Beta-lapachone (CAS No.: 4707-32-8) was purchased from Clinic Compound Library (Target Mol).

[0121] Cryptotanshinone (CAS No.: 35825-57-1), dihydrotanshinone I (CAS No.: 87205-99-0), L-shikonin (CAS: 517-88-4), 2-methoxy-1,4-naphthoquinone (Impatiens balsamina leaf extract, CAS: 2348-82-5), and plumbagin (CAS: 481-42-5) were purchased from Natural Product Library (Selleck).

[0122] CPI-169 (CAS: 1802175-07-0) and S3I-201 (CAS: 501919-59-1) were purchased from Selleck.

[0123] The positive control used was the GRL-0617 inhibitor (purchased from Guangyuan Biotechnology), which is known to effectively inhibit coronavirus papain-like protease (see Ratia, K. et al. A noncovalent class of papain-like protease / deubiquitinase inhibitors blocks SARS virus replication. Proceedings of the National Academy of Sciences 105, 16119-16124 (2008)), and its structural formula is shown below:

[0124]

[0125] Example 1 Through multiple in vitro enzyme activity experiments, it was found that 10 compounds including Tanshinone I can significantly inhibit the papain activity of the new coronavirus (SARS-CoV-2)

[0126] The specific conditions for in vitro enzyme activity experiments are as follows:

[0127] Enzyme activity experiment fluorescent substrate: Z-Arg-Leu-Arg-Gly-Gly-AMC (from Gill Biochemical), the excitation wavelength and emission wavelength of this fluorescent substrate are 340nm and 460nm, respectively.

[0128] The enzyme activity reaction buffer used in the experimental steps was 50mM HEPES, pH 7.5, 0.1mg / ml BSA, 2mM DTT; the enzyme activity reaction temperature was 30°C.

[0129] The specific experimental steps are as follows: the reaction is initiated by adding papain, the final concentration of papain is 0.2 μM, 50 μM inhibitor (10 small molecule compounds: tanshinone I, tanshinone IIA sulfonic acid, β-lapachone, cryptotanshinone, dihydrotanshinone I, levorotatory shikonin, 2-methoxy-1,4-naphthoquinone, plumbagin, CPI-169, S3I-201) solution and enzyme activity experiment fluorescent substrate (Z-Arg-Leu-Arg-Gly-Gly-AMC, concentration is 20 μM) are added to 60 μl reaction system (50 mM HEPES, pH = 7.5, 0.1 mg / ml BSA), incubated at room temperature for 10 min, and the initial reaction rate of the fluorescent substrate is detected by emission light 340 nm and excitation light 460 nm. The inhibition rate curve is obtained by comparing with the initial rate of the control group without drug addition. The experiment was repeated three times (for specific steps, refer to Lee, H., et al. (2019). "Identification and design of novel small molecule inhibitors against MERS-CoV papain-like protease via high-throughput screening and molecular modeling." Bioorganic & medicinal chemistry 27(10): 1981-1989; Jin, Z., Du, X., Xu, Y. et al. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature, doi: 10.1038 / s41586-020-2223-y (2020)).

[0130] The results are as follows Figure 1-Figure 10 As shown in the figure, the enzyme activity experimental curves of 10 compounds including Tanshinone I are shown. The experiment shows that 10 compounds including Tanshinone I can significantly inhibit the papain activity of the new coronavirus (SARS-CoV-2), which is significantly different from the negative control. These 10 compounds all have significant inhibitory activity against coronavirus papain.

[0131] Example 2

[0132] Through multiple in vitro enzyme activity experiments, it was found that tanshinone I and cryptotanshinone can significantly inhibit the papain activity of the new coronavirus (SARS-CoV-2) Fig.11 and 13 ).

[0133] The in vitro enzyme activity experiment steps are as follows: 200nM final concentration of SARS-CoV-2 papain (refer to Zhao Y, DuX, Duan Y, et al. High-throughput screening identifies established drugs as SARS-CoV-2PLpro inhibitors [J]. Protein & Cell, 2021: 1-12.), different concentration gradients of inhibitor solutions and 20μM fluorescent substrate (Arg-Leu-Arg-Gly-Gly-AMC) were added to a 60μl reaction system (50mM HEPES, pH = 7.5, 0.1mg ml-1BSA), incubated at room temperature for 10min, and the initial reaction rate of the fluorescent substrate was detected by emission light 340nm and excitation light 460nm. The inhibition rate curve was obtained by comparing the initial rate of the control group without drug addition. The experiment was repeated three times.

[0134] The results are as follows Fig.11 and Fig.13 As shown in Figure 2, tanshinone I and cryptotanshinone have strong inhibitory activity against the papain-like protease of the new coronavirus, IC 50 2.21 μM and 5.63 μM respectively.

[0135] Example 3

[0136] Through in vitro cell virus experiments, it was found that tanshinone I and cryptotanshinone can significantly inhibit the replication of SARS-CoV-2 in cells and have low cytotoxicity. 50 The values ​​were 2.26 μM and 0.7 μM, respectively. 50 The values ​​were all greater than 300 μM. Fig.12 and Fig.14 .

[0137] The detailed experimental method of in vitro cell virus experiment is as follows:

[0138] (1) In the plaque antiviral test, Vero E6 cells (purchased from ATCC) were pretreated with different concentrations of inhibitors in 24-well culture dishes for 1 hour, and then the clinically isolated virus strain SARS-CoV-2 (nCoV-2019BetaCoV / WIV04 / 2019) (MOI=0.05) (see Zhao Y, Du X, Duan Y, et al. High-throughput screening identifies established drugs as SARS-CoV-2PLpro inhibitors [J]. Protein & Cell, 2021: 1-12.) was added and infected for 1 hour. Then, the virus-drug mixture was removed, washed twice with DMEM medium, and the cells were re-added with new medium containing the corresponding concentration of drugs for pretreatment and 0.9% agarose. After 4 days of continuous culture, the cells were fixed with 4% polyformaldehyde for 30 minutes, stained with crystal violet, observed and counted. All virus experiments were completed in a biosafety level 3 laboratory with 3 biological replicates.

[0139] (2) In the cell viability experiment, Vero E6 cells were cultured in 96-well plates. After 1 day, different concentrations of inhibitors were added to DMEM medium for another day, and then the relative number of surviving cells was measured by CCK8 assay to obtain cell viability data. All experiments were repeated 3 times.

[0140] For detailed experimental methods of in vitro enzyme activity experiments and in vitro cell virus experiments, please refer to Jin, Z., Du, X., Xu, Y. et al. Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors. Nature, doi: 10.1038 / s41586-020-2223-y (2020).

[0141] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

[0142] References

[0143] (1)van der Hoek, L., Pyrc, K., Jebbink, M. et al.

[0144] (2)Stadler,K.,Masignani,V.,Eickmann,M.et al.SARS—beginning tounderstand a new virus.Nat Rev Microbiol 2003,1,209–218.

[0145] (3)de Wit,E.,van Doremalen,N.,Falzarano,D.et al.SARS and MERS:recent insights into emerging coronaviruses.Nat Rev Microbiol 2016,14,523–534.

[0146] (4)Jeannette Guarner, MD, Three Emerging Coronaviruses in Two Decades: The Story of SARS, MERS, and Now COVID-19, American Journal of Clinical Pathology, No

[0147] (5)Akimkin V,Beer M,Blome S,et al.New Chimeric Porcine Coronavirus inSwine Feces,Germany,2012.Emerg Infect Dis.2016,22(7):1314–1315.

[0148] (6)Ziebuhr,J.Snijder,EJ;Gorbalenya,AEProteolytic processing of virus-encoded proteins in the Nidovirales.J Gen Virol 2000,81,853-79.

[0149] (7)Ziebuhr,J.Molecular biology of severe acute respiratory syndromecoronavirus.Curr Opin Microbiol 2004,7,412-9.

[0150] (8)Ratia,Kiira,et al.Severe acute respiratory syndrome coronaviruspapain-like protease:structure of a viral deubiquitinating enzyme.Proceedingsof the National Academy of Sciences103.15(2006):5717-5722.

Claims

1. Use of one or more compounds selected from tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, or pharmaceutically acceptable salts thereof in the preparation of drugs for treating and / or preventing diseases caused by SARS-Cov-2.

2. The use according to claim 1, characterized in that The pharmaceutically acceptable salt is sulfonate and / or sodium salt.

3. The use according to claim 2, characterized in that The pharmaceutically acceptable salt of Tanshinone IIA is Tanshinone IIA sodium sulfonate.

4. The use according to any one of claims 1 to 3, characterized in that: The compound or a pharmaceutically acceptable salt thereof inhibits the papain of SARS-Cov-2.

5. Use of a pharmaceutical composition in the preparation of a drug for treating and / or preventing diseases caused by SARS-Cov-2, the pharmaceutical composition comprising one or more compounds selected from tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, or a pharmaceutically acceptable salt thereof.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutically acceptable salt is a sulfonate and / or a sodium salt; And / or, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutically acceptable salt of Tanshinone IIA is Tanshinone IIA sodium sulfonate.

8. Use of a medicine kit in the preparation of a drug for treating and / or preventing diseases caused by SARS-Cov-2, the medicine kit comprising one or more compounds selected from tanshinone I, tanshinone IIA, cryptotanshinone, dihydrotanshinone I, or a pharmaceutically acceptable salt thereof.

9. The medicine kit according to claim 8, characterized in that: The pharmaceutically acceptable salt is sulfonate and / or sodium salt.

10. The medicine kit according to claim 9, characterized in that: The pharmaceutically acceptable salt of Tanshinone IIA is Tanshinone IIA sodium sulfonate.

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