A compound for treating and / or preventing a disease caused by a coronavirus and its application

Abstract

Compound YM155, tanshinone I or cryptotanshinone, or a pharmaceutically acceptable salt thereof, a solvate thereof, a solvate of a pharmaceutically acceptable salt thereof, or a crystal form thereof, is used for treating and / or preventing diseases caused by coronaviruses.

Description

[0001] This application claims the priority of Chinese Patent Application CN202010418069.1 with an application date of May 18, 2020, and the priority of PCT Application PCT / CN2020 / 090534 with an application date of May 15, 2020. This application incorporates the entire texts of the above two patent applications by reference. Technical Field

[0002] The present invention belongs to the technical field of biomedicine, and particularly relates to a compound for treating and / or preventing diseases caused by coronaviruses and its applications. Background Art

[0003] Coronaviruses are a group of viruses closely related to humans and animals. Coronaviruses HCoV-229E and HCoV-OC43 cause the common cold (van der Hoek, L., Pyrc, K., Jebbink, M. et al. Identification of a new human coronavirus. Nat Med 2004, 10, 368–373). The severe acute respiratory syndrome (SARS) caused by the SARS coronavirus during the period from 2002 to 2003 infected 8,098 people globally and 774 patients died, with a fatality rate of 10% (Stadler, K., Masignani, V., Eickmann, M. et al. SARS—beginning to understand a new virus. Nat Rev Microbiol 2003, 1, 209–218). HCoV-NL63 identified in 2004 can also cause cold-like respiratory diseases (van der Hoek, L., Pyrc, K., Jebbink, M. et al. Identification of a new human coronavirus. Nat Med 2004, 10, 368–373). The Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012. As of April 26, 2016, it caused 1,728 infections in 27 countries, of which 624 people died (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). The novel coronavirus (SARS-CoV-2) that has recently been prevalent globally causes coronavirus disease 2019 (COVID-19), with clinical manifestations such as fever, dry cough, and difficulty breathing, and severe cases can cause death (Jeannette Guarner, MD, Three Emerging Coronaviruses in Two Decades: The Story of SARS, MERS, and Now COVID-19, American Journal of Clinical Pathology,, aqaa029).Coronaviruses also have a huge impact on the livestock industry: Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and Porcine delta coronavirus (PDCoV, also known as delta virus) can cause severe enteritis, diarrhea, vomiting, and dehydration in pigs, bringing huge losses to the pig farming industry (Akimkin V, Beer M, Blome S, et al. New Chimeric Porcine Coronavirus in Swine Feces, Germany, 2012. Emerg Infect Dis. 2016, 22(7):1314–1315.). Feline infectious peritonitis virus (FIPV) can cause a lethal disease in felines. Avian infectious bronchitis virus (IBV) infects poultry and is a widespread poultry disease that has had a huge impact on the poultry industry.

[0004] The genome of coronaviruses is single-stranded positive-sense RNA, about 28 kb in length, mainly encoding the structural proteins required for virus packaging and the non-structural proteins related to replication and transcription. The development of drugs and vaccines for treating coronavirus-related diseases mainly targets these two types of proteins. Two-thirds of the genes in the virus genome mainly encode non-structural proteins, which are involved in the virus replication process. Before the mature non-structural proteins are produced, coronaviruses encode two replicase polypeptides, pp1a and pp1ab. pp1a and pp1ab are cleaved into 16 non-structural proteins (nsp1-16) by two proteases encoded by the virus itself, namely the main protease and the papain-like protease. Only when these functional subunits are correctly cleaved by proteases into independent protein units and then assembled into a replication-transcription complex can the virus complete its normal transcription and replication functions.

[0005] Papain-like protease is located in non-structural protein nsp3 and is responsible for cleaving three cleavage sites at the N-terminus of the replicase polypeptide during the cleavage process, thereby releasing nsp1, nsp2, and nsp3 (Ziebuhr, J.; Snijder, E. J.; Gorbalenya, A. E. Virus-encoded proteinases and proteolytic processing in the Nidovirales. J Gen Virol 2000, 81, 853-79.; Ziebuhr, J. Molecular biology of severe acute respiratory syndrome coronavirus. Curr Opin Microbiol 2004, 7, 412-9.; Ratia, Kiira, et al. Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proceedings of the National Academy of Sciences 103.15 (2006): 5717-5722.). Since papain-like protease has important functions such as enzymatic cleavage, deubiquitination, and antagonizing host IFN, and plays a key regulatory role in the transcription and replication processes of the virus, it has become an important drug target for coronaviruses. Therefore, it is essential to search for inhibitors with high specificity and safety for the catalytic site of papain-like protease for drug development. Inhibitors against the papain-like protease of coronaviruses, especially small molecule compounds targeting its substrate-binding pocket, may become potential drugs for treating coronaviruses. Sepantronium Bromide (YM155, molecular formula: C 20 H 19 BrN 4 O 3 , CAS number: 781661-94-7), the structural formula is as follows:

[0006]

[0007] YM155 is a novel small molecule survivin inhibitor.

[0008] Survivin is a member of the IAP (Inhibitors of apoptosis) family, which plays a role in inhibiting cell apoptosis and regulating the cell cycle. Under normal circumstances, survivin is mainly expressed during embryonic development and ceases to be expressed in terminally differentiated cells and tissues. However, in many tumors, the expression of survivin is upregulated, resulting in the inhibition of apoptosis in tumor cells, thereby reducing the death of tumor cells and conferring a certain degree of resistance to chemotherapy. Therefore, survivin has become an important target for the development of cancer therapeutic drugs (Garg, Himani, et al. "Survivin: a unique target for tumor therapy." Cancer cell international 16.1 (2016): 49.).

[0009] According to research, YM155 can inhibit the expression of survivin, thereby inhibiting the growth and proliferation of tumor cells and inducing apoptosis in tumor cells. At a concentration of 10 nM, YM155 can induce apoptosis in PC-3 and PPC-1 human HRPC cell lines (prostate cancer cell lines) (Nakahara, Takahito, et al. "YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts." Cancer research 67.17 (2007): 8014-8021.).

[0010] Although the killing effect of YM155 on tumor cells is relatively significant, YM155 is relatively safe for normal cell tissues. In the phase I clinical trial of YM155 (Satoh, Taroh, et al. "Phase I study of YM155, a novel survivin suppressant, in patients with advanced solid tumors." Clinical Cancer Research 15.11 (2009): 3872-3880.), the MTD (maximum tolerated dose) of YM155 was 8.0 mg / m 2 / d, indicating that YM155 has certain potential for clinical application.

[0011] Tanshinone I (molecular formula: C 18 H 12 O 3 , CAS No.: 568 - 73 - 0) is a chemical molecule extracted from the Chinese herbal medicine Salvia miltiorrhiza Bunge, and its molecular formula is shown as follows:

[0012]

[0013] Tanshinone I has an anti - inflammatory effect 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). The use of Tanshinone I for treating microglia - mediated diseases is disclosed in CN102552236A, and the application of Tanshinone I in the preparation of drugs for treating psoriasis is also disclosed in CN102988370A.

[0014] Cryptotanshinone (molecular formula: C 19 H 20 O 3 , CAS No.: 35825 - 57 - 1) is a quinone diterpene isolated from the Chinese herbal medicine Salvia miltiorrhiza Bunge, and its molecular formula is shown as follows:

[0015]

[0016] Studies have shown that the drug target of cryptotanshinone is STAT3, and it can effectively inhibit STAT3. Cryptotanshinone can rapidly inhibit the phosphorylation of Tyr at position 705 of STAT3 through a JAK2-independent mechanism. Cryptotanshinone binds to monomeric STAT3 to block STAT3 dimerization, 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). Additionally, there are also 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.)

[0017] In the prior art, there is no such drug available for treating diseases caused by coronaviruses. Summary of the Invention

[0018] The technical problem to be solved by the present invention is to provide a compound for treating and / or preventing diseases caused by coronaviruses and its application, aiming at the defect that there is no effective drug for treating and / or preventing diseases caused by coronaviruses in the prior art. The compound specifically relates to YM155.

[0019] The technical solution of the present invention is introduced in detail below.

[0020] One of the technical solutions of the present invention is: Compound YM155, Tanshinone I or Cryptotanshinone, their pharmaceutically acceptable salts, their solvates, solvates of their pharmaceutically acceptable salts, or their crystal forms, which are used for the treatment and / or prevention of diseases caused by coronaviruses; the CAS number of Compound YM155 is 781661-94-7, the CAS number of Tanshinone I is 568-73-0, and the CAS number of Cryptotanshinone is 35825-57-1.

[0021] Another technical solution of the present invention is: Compound YM155, Tanshinone I or Cryptotanshinone, their pharmaceutically acceptable salts, their solvates, solvates of their pharmaceutically acceptable salts, or their crystal forms, which are used for preparing a drug or a papain-like protease inhibitor for the treatment and / or prevention of diseases caused by coronaviruses, and the drug is used for the treatment and / or prevention of diseases caused by coronaviruses; the CAS number of Compound YM155 is 781661-94-7, the CAS number of Tanshinone I is 568-73-0, and the CAS number of Cryptotanshinone is 35825-57-1.

[0022] A third technical solution of the present invention is: A pharmaceutical composition comprising Compound YM155, Tanshinone I or Cryptotanshinone described in one or two of the above technical solutions, their pharmaceutically acceptable salts, their solvates, solvates of their pharmaceutically acceptable salts, or their crystal forms.

[0023] In a preferred embodiment of the present invention, the pharmaceutical composition further comprises other drugs; wherein the other drugs are used for the treatment and / or prevention of diseases caused by coronaviruses.

[0024] A fourth technical solution of the present invention is: The application of Compound YM155, Tanshinone I or Cryptotanshinone, their pharmaceutically acceptable salts, their solvates, solvates of their pharmaceutically acceptable salts, or their crystal forms, or a pharmaceutical composition containing them in the preparation of a drug or a papain-like protease inhibitor for the treatment and / or prevention of diseases caused by coronaviruses, the CAS number of Compound YM155 is 781661-94-7, the CAS number of Tanshinone I is 568-73-0, and the CAS number of Cryptotanshinone is 35825-57-1.

[0025] The fifth technical solution of the present invention is: a method for treating a disease caused by a coronavirus, which includes administering to a patient in need a compound YM155, tanshinone I or cryptotanshinone, their pharmaceutically acceptable salts, their solvates, solvates of their pharmaceutically acceptable salts, or their crystal forms, or a pharmaceutical composition containing the same; the CAS number of the compound YM155 is 781661-94-7, the CAS number of tanshinone I is 568-73-0, and the CAS number of cryptotanshinone is 35825-57-1.

[0026] In the present invention, the disease is preferably a mammalian or avian disease.

[0027] In the present invention, the mammals preferably include humans, pigs and cats.

[0028] The coronavirus described in the present invention is defined as well-known in the art and belongs to the family Coronaviridae, subfamily Orthocoronavirinae, order Nidovirales in the system classification. Coronaviruses are RNA viruses with an envelope and a single-stranded positive-strand genome, and are a large class of viruses widely existing in nature.

[0029] The purpose of the present invention is to provide a potential treatment plan for diseases caused by coronavirus infection. The coronavirus described in the present invention preferably belongs to the subfamily Orthocoronavirinae, more preferably belongs to the genus Alpha coronavirus, Beta coronavirus, Gamma coronavirus or Delta coronavirus.

[0030] In a preferred embodiment of the present invention, the compound YM155, tanshinone I or cryptotanshinone can not only be used to treat diseases caused by SARS-CoV-2 (genus Betacoronavirus), but also be used to treat major infectious diseases caused by other coronaviruses such as SARS-CoV (genus Betacoronavirus) and MERS-CoV. It can also be used as a common cold medicine to treat diseases caused by coronaviruses such as HCoV-HKU1 (Human coronavirus HKU1; genus Betacoronavirus), HCoV-NL63 (Human coronavirus NL63; genus Alphacoronavirus), HCoV-OC43 (Human coronavirus OC43), and HCoV-229E (Human coronavirus 229E; genus Alphacoronavirus). It can also be used as a veterinary medicine to treat animal diseases such as transmissible gastroenteritis virus (TGEV; genus Alphacoronavirus), porcine epidemic diarrhea virus (PEDV; genus Alphacoronavirus), porcine delta coronavirus (PDCoV; genus Deltacoronavirus), feline infectious peritonitis virus (FIPV; genus Alphacoronavirus), and infectious bronchitis virus (IBV; genus Gammacoronavirus).

[0031] Therefore, the coronaviruses of the present invention are preferably selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, HCoV-NL63, HCoV-OC43, HCoV-229E, TGEV, PEDV, PDCoV, FIPV or IBV.

[0032] The pharmaceutical compositions containing various compounds of the present invention as active ingredients involved in the present invention can all be prepared according to methods well 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 their pharmaceutical compositions is usually 0.1-99.0%.

[0033] The pharmaceutically acceptable carrier described above 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 pharmaceutically acceptable excipients, fillers, diluents, etc. More preferably, the pharmaceutical composition comprises 0.01-99.99% of the above-mentioned protein and / or the above-mentioned antibody-drug conjugate, and 0.01-99.99% of a pharmaceutical carrier, and the percentages are by mass of the pharmaceutical composition.

[0034] The compounds of the present invention or the pharmaceutical compositions containing the same can be administered in unit dosage forms, and the administration routes can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0035] The papain inhibitor described above may exist in the form of a coronavirus inhibitor, for example: a conventional drug for preventing and treating coronavirus.

[0036] Term Explanation

[0037] 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.

[0038] The term "pharmaceutically acceptable salt" refers to salts prepared from the compounds of the present invention, and the drugs, pharmaceutical compositions containing them, and relatively non-toxic, pharmaceutically acceptable acids or bases. When the compounds of the present invention, and the drugs, pharmaceutical compositions containing them, contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such drugs 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, diethanolamine salts. When the drugs of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such drugs with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acids include inorganic acids, which include, but are not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, phosphoric acid, phosphorous acid, sulfuric acid, etc. The pharmaceutically acceptable acids include organic acids, which include, but are 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, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citrate, oleic acid, tannic acid, pantothenic acid, acid tartrate, ascorbic acid, gentisic acid, fumaric acid, gluconic acid, saccharic acid, formic acid, ethanesulfonic acid, pamoic acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (such as glutamic acid, arginine), etc. When the drugs 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).

[0039] The "multiple" in the term "one or more" can refer to 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0040] The compounds of the present invention, the drugs or pharmaceutical compositions containing them can be administered in unit dosage forms, and the administration routes can be enteral or parenteral, such as oral administration, external application, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc., preferably by oral administration or external application.

[0041] The pharmaceutical dosage form can be a liquid dosage form, a solid dosage form or a semi-solid dosage form. The liquid dosage form can be a solution (including true solution and colloidal solution), an emulsion (including o / w type, w / o type and multiple emulsion), a suspension, an injection (including aqueous injection, powder injection and infusion), an eye drop, a nasal drop, a lotion, a liniment, etc.; the solid dosage form can be a tablet (including ordinary tablet, enteric-coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), a capsule (including hard capsule, soft capsule, enteric-coated capsule), a granule, a powder, a pellet, a dropping pill, a suppository, a film, a patch, an aerosol (powder aerosol), a spray, etc.; the semi-solid dosage form can be an ointment, a gel, a paste, etc.

[0042] The drug or pharmaceutical composition of the present invention can be made into an ordinary preparation, or can also be made into a sustained-release preparation, a controlled-release preparation, a targeted preparation and various particulate drug delivery systems.

[0043] "Pharmaceutical composition" refers to the mixing of one or more of the compounds in the present invention or their pharmaceutically acceptable salts, solvates, hydrates or prodrugs with other chemical components, such as pharmaceutically acceptable carriers. The purpose of the pharmaceutical composition is to facilitate the process of administration to an animal.

[0044] "Pharmaceutically acceptable carrier" refers to the inactive ingredient in a pharmaceutical composition that does not cause significant irritation to the 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 polymer or methacrylic polymer, gel, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil, etc.

[0045] In the aforementioned pharmaceutical composition, in addition to including pharmaceutically acceptable carriers, it can also include adjuvants commonly used in pharmacy, such as: antibacterial agents, antifungal agents, antimicrobials, preservatives, colorants, solubilizers, thickeners, surfactants, complexing agents, proteins, amino acids, fats, sugars, vitamins, minerals, trace elements, sweeteners, pigments, flavors or their combinations, etc.

[0046] The term "treatment" refers to therapeutic treatment. When referring to a specific disease or disorder, treatment means: (1) alleviating one or more biological manifestations of the disease or disorder, (2) interfering with (a) one or more points in the biological cascade that causes or gives rise to the disorder or (b) one or more biological manifestations of the disorder, (3) improving one or more symptoms, effects or side effects associated with the disorder, or one or more symptoms, effects or side effects associated with the disorder or its treatment, or (4) slowing down the development of the disorder or one or more biological manifestations of the disorder.

[0047] The term "solvate" refers to a substance formed by the combination of a compound of the present invention with a stoichiometric or non-stoichiometric amount of a solvent. The solvent molecules in the solvate can exist in an ordered or disordered arrangement. The solvents include, but are not limited to, water, methanol, ethanol, etc.

[0048] In the term "solvate of a pharmaceutically acceptable salt", "pharmaceutically acceptable salt" and "solvate" are as described above, and refer to a substance formed by the combination of a compound of the present invention with 1. a relatively non-toxic, pharmaceutically acceptable acid or base, and 2. a stoichiometric or non-stoichiometric amount of a solvent. The "solvate of a pharmaceutically acceptable salt" includes, but is not limited to, the monohydrate hydrochloride of the compound of the present invention.

[0049] The terms "compound", "pharmaceutically acceptable salt", "solvate" and "solvate of a pharmaceutically acceptable salt" can exist in crystalline or amorphous forms. The term "crystalline form" means that the ions or molecules therein are arranged in a strictly periodic manner in three-dimensional space according to a certain pattern and have a regular periodic repetition at a certain interval; due to the different periodic arrangements, there can be multiple crystalline forms, that is, the phenomenon of polymorphism. The term "amorphous" means that the ions or molecules therein are in a disorderly distribution state, that is, there is no periodic arrangement rule between ions and molecules.

[0050] In the present invention, the term "comprising, containing or including" can mean that in addition to the components listed later, there are other components; it can also mean "consisting of...", that is, only the components listed later are included and there are no other components.

[0051] The positive and progressive effects of the present invention are as follows:

[0052] Through in vitro enzyme activity experiments and in vitro cell virus experiments, it was unexpectedly found that YM155 can be used to treat diseases caused by coronaviruses. Currently, there is no approved specific drug for human coronaviruses. The YM155 treatment regimen can fill the deficiencies of the existing technology, has strong antiviral activity and low toxicity and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 Showing that YM155 has strong inhibitory activity against the papain-like protease of the novel coronavirus (IC 50 = 2.47 μM).

[0054] Figure 2 Showing that YM155 can inhibit the replication of SARS-CoV-2 in cells (EC 50 = 0.17 μM).

[0055] Figure 3It is shown that YM155 has low cytotoxicity (CC 50 > 133 μM).

[0056] Figure 4 It is shown the inhibitory activity of tanshinone I against the papain-like protease of the novel coronavirus; 1: DMSO (negative control), 2: compound molecule, 3: positive control (GRL-0617).

[0057] Figure 5 It is shown the inhibitory activity of cryptotanshinone against the papain-like protease of the novel coronavirus; 1: DMSO (negative control), 2: compound molecule, 3: positive control (GRL-0617).

[0058] Figure 6 It is shown that tanshinone I has strong inhibitory activity against the papain-like protease of the novel coronavirus (IC50 = 2.21 μM).

[0059] Figure 7 It is shown that tanshinone I can inhibit the replication of SARS-CoV-2 in cells (EC50 = 2.26 μM), and has low cytotoxicity (CC50 > 300 μM).

[0060] Figure 8 It is shown that cryptotanshinone has strong inhibitory activity against the papain-like protease of the novel coronavirus (IC50 = 5.63 μM).

[0061] Figure 9 It is shown that cryptotanshinone can inhibit the replication of SARS-CoV-2 in cells (EC50 = 0.7 μM), and has low cytotoxicity (CC50 > 300 μM). Detailed implementation manners

[0062] The present invention will be further described below in conjunction with embodiments.

[0063] Tanshinone I (CAS No.: 568-73-0) was purchased from Approved Drug Library (Target Mol), and cryptotanshinone (CAS No.: 35825-57-1) was purchased from Natural Product Library (Selleck).

[0064] The positive control used was the GRL-0617 inhibitor (purchased from Guangyuan Biotech), which is known to be able to effectively inhibit the papain-like protease of coronaviruses (see specifically 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)). Its structural formula is shown below:

[0065]

[0066] Example 1

[0067] Through in vitro enzyme activity experiments, it was found that YM155 could significantly inhibit the papain-like protease activity of the novel coronavirus (SARS-CoV-2) ( Figure 1 ).

[0068] The steps of the in vitro enzyme activity experiment were as follows: 200 nM final concentration of SARS-CoV-2 papain-like protease, YM155 solutions at different concentration gradients (0.1 nM - 5 μM), and 20 μM of the fluorescent substrate (Arg-Leu-Arg-Gly-Gly-AMC) were added to a 60 μl reaction system (50 mM HEPES, pH = 7.5, 0.1 mg ml-1 BSA), incubated at room temperature for 10 min, and the initial reaction rate of the fluorescent substrate was detected using an emission wavelength of 340 nm and an excitation wavelength of 460 nm. By comparing with the initial rate of the control group without the drug, an inhibition rate curve was obtained. The experiment was repeated 3 times biologically.

[0069] Example 2

[0070] Through in vitro cell virus experiments, it was found that YM155 could significantly inhibit the replication ability of SARS-CoV-2 in cells ( Figure 2 ). At the same time, in vitro cytotoxicity tests ( Figure 3 ) and the reported clinical phase I trial (Satoh, Taroh, et al. "Phase I study of YM155, a novel survivin suppressant, in patients with advanced solid tumors." Clinical Cancer Research 15.11 (2009): 3872 - 3880.) confirmed that YM155 had high safety. Therefore, YM155 can be used to treat diseases caused by coronaviruses.

[0071] The detailed experimental methods for in vitro cell virus experiments are as follows:

[0072] 1) In the plaque assay antiviral test, after pretreating Vero E6 cells (purchased from ATCC) seeded in 24-well culture dishes with different concentrations (0.1 μM - 10 μM) of YM155 for 1 hour, the clinically isolated virus strain SARS-CoV-2 (nCoV-2019 BetaCoV / Wuhan / WIV04 / 2019) (MOI = 0.05) was added and infected for 1 hour. Then, the virus-drug mixture was removed, and after washing twice with DMEM medium, the cells were re-added to a new medium containing the corresponding concentration of the pretreated drug and 0.9% agarose. After continued culture for 4 days, the cells were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet, and observed and counted. All virus tests were completed in a biosafety level 3 laboratory and had 3 biological replicates.

[0073] 2) In the cell viability experiment, Vero E6 cells were cultured in 96-well plates. After 1 day, different concentrations (0.06 μM - 400 μM) of YM155 were added to the DMEM medium and continued for 1 day, and then the relative number of viable cells was measured by the CCK8 assay to obtain cell viability data. All experiments had 3 biological replicates.

[0074] For the detailed experimental methods of in vitro enzyme activity experiments and in vitro cell virus experiments, see 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).

[0075] Example 3

[0076] The specific conditions for the in vitro enzyme activity experiment are as follows:

[0077] Fluorescent substrate for the enzyme activity experiment: Z-Arg-Leu-Arg-Gly-Gly-AMC (from Gil Biochemical), and the excitation wavelength and emission wavelength of this fluorescent substrate are 340 nm and 460 nm, respectively.

[0078] The enzyme activity reaction buffer used in the experimental procedure is 50 mM HEPES, pH 7.5, 0.1 mg / ml BSA, 2 mM DTT; the enzyme activity reaction temperature: 30 °C.

[0079] The specific experimental steps are as follows: At the start of the reaction, papain-like protease was added, and the final concentration of papain-like protease was 0.2 μM. A 50 μM inhibitor (tanshinone I, cryptotanshinone) solution and an enzyme activity assay fluorescent substrate (Z-Arg-Leu-Arg-Gly-Gly-AMC, concentration 20 μM) were added to a 60 μl reaction system (50 mM HEPES, pH = 7.5, 0.1 mg / ml BSA), and incubated at room temperature for 10 min. The initial reaction rate of the fluorescent substrate was detected using an emission wavelength of 340 nm and an excitation wavelength of 460 nm. By comparing with the initial rate of the control group without drugs, the inhibition rate curve was obtained. The experiment was performed with 3 biological replicates (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)).

[0080] The results obtained are as Figure 4 , Figure 5 shown. The figure shows the enzyme activity assay curves of tanshinone I and cryptotanshinone. The experiments indicate that tanshinone I and cryptotanshinone can significantly inhibit the papain-like protease activity of the novel coronavirus (SARS-CoV-2), which is significantly different from the negative control. Both compounds have obvious inhibitory activity against the coronavirus papain-like protease.

[0081] Example 4

[0082] Through in vitro enzyme activity assays, it was found that tanshinone I and cryptotanshinone can significantly inhibit the papain-like protease activity of the novel coronavirus (SARS-CoV-2) ( Figure 4 ).

[0083] The in vitro enzyme activity assay steps are as follows: SARS-CoV-2 papain-like protease at a final concentration of 200 nM, inhibitor solutions at different concentration gradients, and a 20 μM fluorescent substrate (Arg-Leu-Arg-Gly-Gly-AMC) are added to a 60 μl reaction system (50 mM HEPES, pH = 7.5, 0.1 mg ml-1 BSA), incubated at room temperature for 10 min, and the initial reaction rate of the fluorescent substrate is detected using an emission wavelength of 340 nm and an excitation wavelength of 460 nm. The inhibition rate curve is obtained by comparing with the initial rate of the control group without the drug. The experiment was performed with 3 biological replicates.

[0084] The results obtained are as Figure 6 and Figure 8 shown. Tanshinone I and cryptotanshinone have strong inhibitory activities against SARS-CoV-2 papain-like protease, and the IC 50 values are 2.21 μM and 5.63 μM, respectively.

[0085] Example 5

[0086] Through in vitro cell virus experiments, it was found that tanshinone I and cryptotanshinone can significantly inhibit the replication ability of SARS-CoV-2 in cells and have low cytotoxicity. The EC 50 values are 2.26 μM and 0.7 μM, respectively (see Figure 7 and Figure 9 ). The CC50 values are all greater than 300 μM.

[0087] The detailed experimental methods for the in vitro cell virus experiments are as follows:

[0088] (1) In the plaque assay for antiviral testing, after pretreating Vero E6 cells (purchased from ATCC) seeded in 24-well culture plates with different concentrations of the inhibitor for 1 hour, the clinically isolated virus strain SARS-CoV-2 (nCoV-2019 BetaCoV / Wuhan / WIV04 / 2019) (MOI = 0.05) was added and incubated for 1 hour. Then, the virus-drug mixture was removed, and the cells were washed twice with DMEM medium. The cells were then re-added to a new medium containing the corresponding concentration of the pretreated drug and 0.9% agarose. After continued culture for 4 days, the cells were fixed with 4% paraformaldehyde for 30 minutes, stained with crystal violet, and observed and counted. All virus tests were performed in a biosafety level 3 laboratory and had 3 biological replicates.

[0089] (2) In the cell viability experiment, Vero E6 cells were cultured in 96-well plates. After 1 day, different concentrations of the inhibitor were added to the DMEM medium and incubated for another 1 day. Then, the relative number of viable cells was measured using the CCK8 assay to obtain cell viability data. All experiments had 3 biological replicates.

[0090] For the detailed experimental methods of in vitro enzyme activity assay and in vitro cell virus assay, 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).

[0091] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples. Without departing from the principle and essence of the present invention, various changes or modifications can be made to these embodiments. Therefore, the protection scope of the present invention is defined by the appended claims.

Claims

1. Use of compound YM155 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, in the preparation of a medicament for treating and / or preventing diseases caused by SARS-CoV-2; the CAS number of the compound YM155 is 781661-94-7.

2. The use according to claim 1, wherein, the disease is a mammalian or avian disease.

3. The use according to claim 2, wherein, the mammals are humans, pigs and cats.

4. The use according to claim 1, wherein, the disease caused by SARS-CoV-2 is coronavirus disease 2019 (COVID-19).

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