Use of old drugs and compositions thereof, such as auranofin, in the treatment of positive-sense single-stranded rna viruses

Abstract

The present application relates to the application of 73 old drugs such as auranofin and their compositions in anti-single-stranded RNA virus. Specifically, the present application relates to the use of auranofin and other old drugs and their pharmaceutical compositions as 2019 novel coronavirus (SARS-CoV-2) 3CL protease inhibitors in the preparation of drugs for treating and / or preventing, relieving diseases such as respiratory tract infection, pneumonia and other related diseases caused by 2019 novel coronavirus infection.

Description

[0001] This application is a divisional application of the invention patent application filed on February 21, 2020, with application number 202010109153.5 and invention title "Application of old drugs such as aurinol and their compositions in the fight against single-stranded positive-sense RNA viruses". Technical Field

[0002] This invention relates to the pharmaceutical field, specifically to the application of several existing drugs, such as aurinolone, and their combinations in the fight against single-stranded positive-sense RNA viruses. Background Technology

[0003] The vast majority of acute infectious diseases are viral, which have high morbidity and mortality rates. Due to limited testing and diagnostic methods, outbreaks caused by new viruses are often characterized by suddenness, randomness, and unpredictability. Once an outbreak occurs, without effective prevention and control measures, it can easily lead to large-scale epidemics, seriously threatening people's health and lives.

[0004] The transmission routes of the SARS-CoV-2 virus are not fully understood. It is known to spread through droplets and contact, and there is a risk of human-to-human transmission, infection of medical personnel, and community transmission. The virus also has the potential to mutate. Currently, there are no specific preventative or treatment methods for the disease caused by the novel coronavirus.

[0005] SARS-CoV-2 coronavirus belongs to the genus Coronavirus in the family Coronaviridae. It is an enveloped, single-stranded, positive-sense RNA virus. Similar to other known coronaviruses, SARS-CoV-2 coronavirus proliferates through several processes, including adsorption, penetration, uncoating, biosynthesis, and the assembly and release of progeny viruses. Infection of host cells by SARS-CoV-2 coronavirus begins with the binding of the spike glycoprotein on the viral envelope to the ACE2 receptor on the host cell surface. Following membrane fusion, the virus enters the host cell and, under the action of organelles such as lysosomes, releases its genetic material, single-stranded positive-sense RNA. This RNA is then translated into a multiprotein by the host cell's mitochondria, ribosomes, and other protein synthesis elements, along with necessary raw materials. Subsequently, two essential cysteine ​​proteases of SARS-CoV-2 coronavirus—papain-like protease (PL)—are involved. pro ) and 3C-like protease (3CL) proThe virus cleaves and processes the precursor polyprotein at specific sites, generating multiple non-structural proteins crucial to the viral life cycle. Under the influence of these non-structural proteins, viral RNA replicates to produce progeny viral nucleic acid material, and a large number of the required structural proteins are translated, completing the assembly and release of the progeny virus. Any step in the life cycle of SARS-CoV-2 coronavirus-infected cells, or any key enzyme, can serve as a research target for antiviral drugs, such as the cysteine ​​protease PL, which hydrolyzes the precursor polyprotein. pro and 3CL pro RNA polymerase, etc., are responsible for replicating the genetic material of progeny viruses.

[0006] 3CL protease (3chymotrypsin-like protease, 3CL) pro ), also known as main protease (M pro 3CL is a key protease in the process of hydrolysis of the coronavirus RNA after translation into polyproteins pp1a and pp1ab, producing multiple non-structural proteins. It is crucial for viral replication and infection. Inhibiting the catalytic function of the 3CL protease can effectively inhibit the cleavage of viral polyprotein precursors, block viral replication, and suppress progeny virus generation. pro It belongs to the cysteine ​​protease family, and its sequence and structure are highly conserved in all coronaviruses. It is also very similar to the 3C protease in small RNA viruses, and no similar protease exists in humans. Therefore, 3CL... pro It is currently recognized as an ideal target for developing broad-spectrum drugs against single-stranded positive-sense RNA viruses.

[0007] Currently, there are no specific vaccines or antiviral drugs for the severe pneumonia disease (COVID-19) caused by the SARS-CoV-2 coronavirus. These infectious diseases seriously impact people's lives and health, making the development of effective antiviral drugs an urgent priority. [The text then abruptly shifts to a seemingly unrelated topic:] SARS-CoV-2 coronavirus 3CL... pro Developing low-toxicity and highly effective antiviral drugs to meet the clinical needs of SARS-CoV-2 coronavirus patients both domestically and internationally is of great social significance.

[0008] In summary, there is an urgent need in this field to develop inhibitors against the SARS-CoV-2 coronavirus 3CL protease for the treatment of pneumonia caused by novel coronavirus infection and other single-sense positive-sense RNA virus infections. Summary of the Invention

[0009] The purpose of this invention is to provide a drug molecule that can effectively inhibit coronavirus 3CL protease and its novel use in inhibiting single-stranded positive RNA virus infection.

[0010] Specifically, this invention provides the use of several existing drugs, such as aurinolone, and their compositions in the fight against single-stranded positive-sense RNA virus infection, especially in the fight against novel coronavirus disease (COVID-19).

[0011] In a first aspect of the invention, there is provided the use (or pharmaceutical use) of an active ingredient or a formulation containing said active ingredient, said active ingredient being selected from the group consisting of:

[0012] (Z1) is selected from any active ingredient (old drug) in A1 to A73, or a pharmaceutically acceptable salt thereof or an extract thereof;

[0013] (A1) Aurinophene;

[0014] (A2) Rabeprazole sodium;

[0015] (A3) Heparin sodium;

[0016] (A4) Disulfiram;

[0017] (A5) Iprazosin;

[0018] (A6) Boseraphim;

[0019] (A7) Timetprazole;

[0020] (A8) Ethacridine;

[0021] (A9) Ethacridine;

[0022] (A10) Bropol;

[0023] (A11) Fondaparin sodium;

[0024] (A12) Sucralfate;

[0025] (A13) Octenidine;

[0026] (A14) Hexachlorophenol;

[0027] (A15) Evans Blue;

[0028] (A16) Demethyldihydroguaiac acid;

[0029] (A17) Thorium bromide;

[0030] (A18) Tannic acid;

[0031] (A19) Omeprazole;

[0032] (A20) Benzelazid (Serrazid);

[0033] (A21) Felodipine;

[0034] (A22) Rilpivirine;

[0035] (A23) Phenoxyaniline;

[0036] (A24) Anthralin;

[0037] (A25) Dimercaptosuccinic acid;

[0038] (A26) Levothyroxine sodium;

[0039] (A27) Cefaclor;

[0040] (A28) Tilatreco;

[0041] (A29) Quinine;

[0042] (A30)TIBSOVO;

[0043] (A31) Iodine;

[0044] (A32) Benzbromarone;

[0045] (A33) Quinine;

[0046] (A34) Diiodoquinoline;

[0047] (A35) Granstron;

[0048] (A36) Mianselin;

[0049] (A37) Canagliflozin;

[0050] (A38) Bedaquiline;

[0051] (A39) Triclosan;

[0052] (A40) Ziprasidone;

[0053] (A41) Elbavir;

[0054] (A42) Bedaquiline;

[0055] (A43) Pentachlorosalicylate;

[0056] (A44) Hexadecylpyridine chloride;

[0057] (A45) Fenodolphine;

[0058] (A46) Triiodothyronine;

[0059] (A47) Anise trisulfite;

[0060] (A48) Djenadalon;

[0061] (A49) Tazarotene;

[0062] (A50)6-Mercaptopurine;

[0063] (A51) Malathion;

[0064] (A52) Butoconazole;

[0065] (A53) Percidatinib;

[0066] (A54) Otilonine bromide;

[0067] (A55) Nitrohydroxyquinoline;

[0068] (A56) Temopofen;

[0069] (A57) Caspofungin;

[0070] (A58) Menaquinone;

[0071] (A59) Diacerein;

[0072] (A60)(R)-Lansoprazole;

[0073] (A61) Enbecic acid;

[0074] (A62) Lometape;

[0075] (A63) Tiranavir;

[0076] (A64) Sofadone;

[0077] (A65) Zarust;

[0078] (A66) Lansoprazole;

[0079] (A67) Raloxifene;

[0080] (A68) Trapwe;

[0081] (A69) Riboflavin tetrabutyrate;

[0082] (A70) Levothyroxine;

[0083] (A71) Sodium docusate;

[0084] (A72) Djenadalon;

[0085] (A73) Econazole;

[0086] (Z2) Any combination of the above active ingredients A1 to A73;

[0087] The active ingredient or preparation containing the active ingredient is used to prepare (a) an inhibitor of coronavirus 3CL protease; and / or (b) a drug for treating and / or preventing or alleviating diseases caused by coronavirus (or single-stranded positive-sense RNA virus) infection.

[0088] In another preferred embodiment, the drug further comprises one or more additional active ingredients selected from the group consisting of:

[0089] (Y1) Traditional Chinese medicine preparations or their active ingredients used to inhibit viruses;

[0090] (Y2) RNA replicase inhibitors (such as Remdesivir or GS-5734);

[0091] (Y3) Lopinavir, Ritonavir;

[0092] (Y4) Chloroquine (Sigma-C6628) or a pharmaceutically acceptable salt thereof;

[0093] (Y5) Hydroxychloroquine, or a pharmaceutically acceptable salt thereof.

[0094] In another preferred embodiment, the coronavirus 3CL protease is selected from the group consisting of: 2019 novel coronavirus (SARS-CoV-2) 3CL protease, SARS virus 3CL protease, MERS virus 3CL protease, or combinations thereof.

[0095] In another preferred embodiment, the active ingredient is used to prepare (a) a SARS-CoV-2 3CL protease inhibitor; and / or (b) a medicament for treating and / or preventing or alleviating diseases related to SARS-CoV-2 infection.

[0096] In another preferred embodiment, the coronavirus is selected from the group consisting of alpha coronaviruses, beta coronaviruses, or combinations thereof.

[0097] In another preferred embodiment, the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, SARS-CoV-2, or combinations thereof.

[0098] In another preferred embodiment, the coronavirus is selected from the group consisting of: 2019 novel coronavirus (SARS-CoV-2), SARS virus, MERS virus, or combinations thereof.

[0099] In another preferred embodiment, the related diseases caused by infection with the 2019 novel coronavirus are selected from the group consisting of respiratory infections, pneumonia and its complications, or combinations thereof.

[0100] In another preferred embodiment, the active ingredient is selected from the active compounds in Table 1.

[0101] In another preferred embodiment, the active ingredient is selected from the group consisting of any active ingredient (old drug) selected from A1 to A18, or a pharmaceutically acceptable salt thereof or an extract thereof.

[0102] In another preferred embodiment, the active ingredient is selected from the group consisting of: aurinophene, rabeprazole sodium, heparin sodium, disulfiram, ilaprazole, berberine, taliprazole, or pharmaceutically acceptable salts thereof.

[0103] In another preferred embodiment, the active ingredient (or active compound) is artificially synthesized and / or extracted from plants.

[0104] In another preferred embodiment, the plant or medicinal material includes roots, stems, leaves, flowers, fruits, or combinations thereof.

[0105] In another preferred embodiment, the drug further includes additional components selected from the group consisting of antiretroviral drugs or immune-enhancing drugs.

[0106] In another preferred embodiment, the composition or drug comprises: oral formulation and non-oral formulation.

[0107] In another preferred embodiment, the formulation includes: powder, granules, capsules, injections, tinctures, oral liquids, tablets, lozenges, or drops.

[0108] In a second aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0109] (a) A first active ingredient, wherein the first active ingredient is selected from the group consisting of any active ingredient (old drug) from A1 to A73, or a pharmaceutically acceptable salt thereof or an extract thereof; or a combination thereof;

[0110] and (b) pharmaceutically acceptable carriers.

[0111] In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for inhibiting coronavirus 3CL protease.

[0112] In another preferred embodiment, the pharmaceutical composition contains no other active ingredients (such as antiviral active ingredients) except for any active ingredient selected from A1 to A73 (old drugs), or a pharmaceutically acceptable salt thereof or an extract thereof.

[0113] In another preferred embodiment, the pharmaceutical composition comprises:

[0114] (a1) A first active ingredient, wherein the first active ingredient is selected from any active ingredient (preservative) from A1 to A73, or a pharmaceutically acceptable salt thereof or an extract thereof; or a combination thereof; and

[0115] (a2) A second active ingredient, wherein the second active ingredient is selected from the group consisting of: RNA replicase inhibitors (such as Remdesivir or GS-5734); Lopinavir, Ritonavir; Chloroquine (Sigma-C6628), hydroxychloroquine, or combinations thereof;

[0116] and (b) pharmaceutically acceptable carriers.

[0117] In another preferred embodiment, the dosage form of the drug is an oral or non-oral dosage form.

[0118] In another preferred embodiment, the oral dosage form is a tablet, powder, granule or capsule, or an emulsion or syrup.

[0119] In another preferred embodiment, the non-oral dosage form is an injection or syringe.

[0120] In a third aspect of the invention, the use of the pharmaceutical composition described in the second aspect of the invention is provided for the preparation of (a) an inhibitor of coronavirus 3CL protease; and / or (b) a medicament for the treatment and / or prevention and relief of diseases related to coronavirus infection.

[0121] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating and / or preventing or alleviating diseases related to infection with the 2019 novel coronavirus (SARS-CoV-2).

[0122] In another preferred embodiment, the related diseases caused by infection with the 2019 novel coronavirus are selected from the group consisting of respiratory infections, pneumonia and its complications, or combinations thereof.

[0123] In another preferred embodiment, the coronavirus is selected from the group consisting of HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1, MERS-CoV, SARS-CoV-2, or combinations thereof.

[0124] In a fourth aspect of the invention, a method for inhibiting coronavirus 3CL protease is provided, comprising the steps of: contacting a first active ingredient or an preparation containing the first active ingredient with coronavirus 3CL protease, thereby inhibiting the activity of said 3CL protease;

[0125] The first active ingredient is selected from the following group:

[0126] (Z1) is selected from any active ingredient (old drug) in A1 to A73, or a pharmaceutically acceptable salt thereof or an extract thereof;

[0127] (Z2) Any combination of the above active ingredients A1 to A73.

[0128] In another preferred embodiment, the inhibition method is an in vitro method and is a non-therapeutic and non-diagnostic method.

[0129] In another preferred embodiment, the inhibition method is an in vivo method and a therapeutic method.

[0130] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0131] In another preferred embodiment, the method is performed in vitro.

[0132] In another preferred embodiment, the 3CL protease is a recombinant or coronavirus-expressed 3CL protease.

[0133] In another preferred embodiment, the SARS-CoV-2 3CL protease is recombinant or SARS-CoV-2 expressed 3CL protease.

[0134] In another preferred embodiment, the first active ingredient is selected from the group consisting of any active ingredient from A1 to A18, or a pharmaceutically acceptable salt thereof or an extract thereof; or a combination thereof.

[0135] In another preferred embodiment, the first active ingredient is selected from the group consisting of any active ingredient from A1 to A10, or a pharmaceutically acceptable salt thereof or an extract thereof; or a combination thereof.

[0136] In another preferred embodiment, the first active ingredient is selected from the group consisting of: aurinophene, rabeprazole sodium, heparin sodium, disulfiram, ilaprazole, berberine, taliprazole, or pharmaceutically acceptable salts thereof; or combinations thereof.

[0137] In a fifth aspect of the invention, a method for treating, preventing, and / or alleviating diseases related to coronavirus infection is provided, comprising the steps of: administering to a desired subject a safe and effective amount of a first active ingredient or a formulation containing the first active ingredient, wherein the first active ingredient is selected from the group consisting of:

[0138] (Z1) is selected from any active ingredient (old drug) in A1 to A73, or a pharmaceutically acceptable salt thereof or an extract thereof;

[0139] (Z2) Any combination of the above active ingredients A1 to A73.

[0140] In another preferred embodiment, the preparation is a non-traditional Chinese medicine preparation.

[0141] In another preferred embodiment, the method further includes: administering a safe and effective amount of a second active ingredient and, optionally, a third active ingredient to the desired object, wherein...

[0142] The second active ingredient is selected from the group consisting of: RNA replicase inhibitors (such as Remdesivir or GS-5734); Lopinavir, Ritonavir; Chloroquine (Sigma-C6628), hydroxychloroquine, or combinations thereof;

[0143] In another preferred embodiment, the object is a mammal, preferably a primate mammal, and more preferably a human.

[0144] In another preferred embodiment, the first active ingredient is selected from the group consisting of any active ingredient from A1 to A18, or a pharmaceutically acceptable salt thereof or an extract thereof; or a combination thereof.

[0145] In another preferred embodiment, the first active ingredient is selected from the group consisting of any active ingredient from A1 to A10, or a pharmaceutically acceptable salt thereof or an extract thereof; or a combination thereof.

[0146] In another preferred embodiment, the first active ingredient is selected from the group consisting of: aurinophene, rabeprazole sodium, heparin sodium, disulfiram, ilaprazole, berberine, taliprazole, or pharmaceutically acceptable salts thereof; or combinations thereof.

[0147] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0148] Figure 1 The curves showing the concentration-dependent inhibition of SARS-CoV-2 3CL protease activity by auronoxine are presented.

[0149] Figure 2 The curves showing the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by rabeprazole sodium are displayed.

[0150] Figure 3 The curves showing the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by heparin sodium are displayed.

[0151] Figure 4 The curves showing the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by disulfiram are displayed.

[0152] Figure 5 The curves showing the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by ipramazole are displayed.

[0153] Figure 6 The curves showing the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by β-prevectin are displayed.

[0154] Figure 7 The curves showing the concentration-dependent inhibition of SARS-CoV-2-3CLpro enzyme activity by tetoraphite are displayed. Detailed Implementation

[0155] Through extensive and in-depth research, and by screening 2000 approved drug molecules, the inventors unexpectedly developed, for the first time, several existing drugs, including aurinolone, that effectively inhibit the activity of the 3CL protease of coronaviruses such as SARS-CoV-2. Experiments show that the active molecules of this invention (such as aurinolone and other existing drugs, or their pharmaceutically acceptable salts) can efficiently inhibit the activity of the 3CL protease of coronaviruses such as SARS-CoV-2, thereby inhibiting the replication and activity of SARS-CoV-2 coronavirus. This invention was completed based on this.

[0156] Specifically, this invention discloses the use of several existing drugs, including aurinolone, and their combinations in combating single-sense positive-sense RNA viruses, particularly in the treatment of COVID-19. These existing drugs, including aurinolone, and their combinations exhibit excellent inhibitory activity against the highly conserved 3CL hydrolase, which is essential for coronavirus replication, and show promising clinical application prospects.

[0157] the term

[0158] As used herein, "active compound of the present invention" and "active compound of the present invention for inhibiting 3CL protease" are used interchangeably, referring to compounds with excellent 3CL protease inhibitory activity, especially known pharmaceutical active ingredients (old drugs) such as aurinophene or combinations thereof.

[0159] As used herein, "medicinal materials of the present invention" refers to Chinese medicinal materials containing the active compounds of the present invention.

[0160] As used herein, "medicinal extract of the present invention" or "extract of the present invention" refers to an extract obtained from Chinese medicinal herbs or corresponding plants and containing one or more active compounds of the present invention.

[0161] As used herein, "the formulation of the present invention" refers to a formulation containing the active compounds of the present invention, including traditional Chinese medicine formulations and non-traditional Chinese medicine formulations.

[0162] As used herein, the term "comprising" or its variations such as "including" or "comprises" will be understood to include the stated elements or components without excluding other elements or other components.

[0163] Coronavirus and 3CL protease

[0164] Coronavirus (CoV) belongs to the order Nidovirales and the family Coronaviridae. It is an enveloped positive-sense RNA virus, and its subfamily includes four genera: α, β, δ, and γ.

[0165] Of the coronaviruses currently known to infect humans, HCoV-229E and HCoV-NL63 belong to the alpha coronavirus genus, while HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV, and SARS-CoV-2 belong to the beta coronavirus genus.

[0166] The highly pathogenic coronaviruses SARS-CoV (2003) and MERS-CoV (2012), which broke out in 2003 and 2012 respectively, both belong to the β-coronavirus genus. The novel coronavirus (SARS-CoV-2) that broke out at the end of 2019 has about 80% similarity to SARS-CoV and 40% similarity to MERS-CoV, and also belongs to the β-coronavirus genus.

[0167] The genome of this type of virus is a single-stranded positive-sense RNA, making it one of the largest RNA viruses in terms of genome size. It encodes replicase, spike protein, envelope proteins, and nucleocapsid proteins. In the initial stage of viral replication, the genome is translated into two polypeptide chains of several thousand amino acids each, known as precursor polyproteins. Subsequently, the precursor proteins are cleaved by proteases to generate non-structural proteins (such as RNA polymerase and helicase), structural proteins (such as spike protein), and accessory proteins.

[0168] 3CL protease (3Chymotrypsin-like protease, 3CLpro) is the main protease in coronaviruses responsible for cleaving precursor proteins (hence also known as M). pro ( ), which is indispensable for the replication of the virus.

[0169] 3CLpro is a cysteine ​​hydrolase that is highly conserved among various coronaviruses and is similar to 3C protease in small RNA viruses. However, there is no protease similar to it in the human body, making it an ideal target for developing broad-spectrum anti-single-strand RNA virus drugs.

[0170] The active compounds and active ingredients of the present invention

[0171] This invention provides an active ingredient that can effectively inhibit the replication of coronaviruses such as the 2019 novel coronavirus (SARS-CoV-2). This active ingredient is selected from the following group:

[0172] (Z1) is selected from any active ingredient in A1 to A73, or a pharmaceutically acceptable salt thereof or an extract thereof;

[0173] (Z2) Any combination of the above active ingredients A1 to A73 (including any combination of two or more of the above active ingredients Z1).

[0174] Experiments have shown that the active compounds of the present invention can effectively inhibit the 3CL protease of the 2019 novel coronavirus (SARS-CoV-2), thereby inhibiting the replication of the 2019 novel coronavirus (SARS-CoV-2) and thus preventing, treating and / or alleviating SARS-CoV-2 related diseases.

[0175] As used herein, "active compound of the present invention" and "active compound of the present invention for inhibiting 3CL protease" are used interchangeably, referring to compounds with excellent 3CL protease inhibitory activity, especially any active ingredient in A1 to A73, or a combination thereof, represented by aurinol.

[0176] It should be understood that the active ingredients of the present invention include the active compound of the present invention that inhibits 3CL protease, or a pharmaceutically acceptable salt thereof, enantiomer, diastereomer or racemate thereof, or a prodrug thereof. It should be understood that the active ingredients of the present invention also include the active compound of the present invention in crystal form, amorphous form, deuterated form, solvate, hydrate, or other forms.

[0177] The "pharmaceutically acceptable salt" refers to a conventional, non-toxic salt formed by reacting the active compound of the present invention with an inorganic or organic acid. For example, conventional non-toxic salts can be prepared by reacting the active compound of the present invention with an inorganic or organic acid, wherein the inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, and phosphoric acid, etc., and the organic acid includes citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pyric acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid, and hydroxyethanesulfonic acid, etc. Alternatively, the active compound of the present invention can form an ester with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, aspartic acid, or glutamic acid, and then form a sodium salt, potassium salt, calcium salt, aluminum salt, or ammonium salt with an inorganic base; or the active compound of the present invention can form a methylamine salt, ethylamine salt, or ethanolamine salt with an organic base; or the active compound of the present invention can form an ester with lysine, arginine, or ornithine, and then form a corresponding inorganic acid salt with hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, or phosphoric acid, or form a corresponding organic acid salt with formic acid, acetic acid, picric acid, methanesulfonic acid, or ethanesulfonic acid.

[0178] Furthermore, the active ingredient of this invention is particularly suitable for use in combination with other antiviral drugs. Other representative antiviral drugs include (but are not limited to): reverse transcriptase inhibitors, protease inhibitors, co-receptor antagonists, retroviral integrase inhibitors, viral adsorption inhibitors, specific viral transcription inhibitors, antibodies, or combinations thereof.

[0179] The active ingredient of this invention can inhibit the infectivity of novel coronaviruses such as SARS-CoV-2. Therefore, when the active ingredient of this invention is applied or administered therapeutically, it can inhibit the activity of 3CL protease, thereby inhibiting the infection of 2019 novel coronavirus (SARS-CoV-2) and thus achieving an antiviral effect.

[0180] Pharmaceutical Compositions and Applications

[0181] The present invention also provides the use of one or more of the active compounds of the present invention that inhibit 3CL protease, or pharmaceutically acceptable salts thereof, or prodrugs thereof, or extracts thereof, or medicinal materials thereof, as active ingredients in the preparation of medicaments for the treatment and / or prevention and relief of respiratory infections, pneumonia and other related diseases caused by 2019 novel coronavirus infection.

[0182] The pharmaceutical composition provided by the present invention preferably contains 0.001-99 wt% of an active ingredient, and the preferred proportion is that the active compound of the present invention accounts for 0.1 wt% to 90 wt% or 1 wt% to 50 wt% of the total weight, with the remainder being a pharmaceutically acceptable carrier, diluent, solution or salt solution.

[0183] When necessary, one or more pharmaceutically acceptable carriers may be added to the drug of this invention. These carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., which are conventional in the pharmaceutical field.

[0184] The compounds and pharmaceutical compositions provided by this invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions, and aerosols, and can be present in suitable solid or liquid carriers or diluents and in suitable sterilization devices for injection or infusion.

[0185] Various dosage forms of the pharmaceutical compositions of the present invention can be prepared according to conventional pharmaceutical preparation methods. The dosage form typically contains 0.05-400 mg of the active compound of the present invention per unit volume, preferably 1-500 mg per unit volume.

[0186] The compounds and pharmaceutical compositions of the present invention are suitable for clinical use in mammals, including humans and animals, and can be administered via oral, nasal, skin, lung, or gastrointestinal routes. Oral administration is preferred. The most preferred daily dose is 0.01-400 mg / kg body weight, administered as a single dose, or 0.01-200 mg / kg body weight, administered in divided doses. Regardless of the method of administration, the optimal dose for an individual should be determined based on the specific treatment. Generally, a low dose is started, and the dose is gradually increased until the most suitable dose is found.

[0187] The drugs or inhibitors of the present invention can be administered in various ways, such as by injection, spray, nasal drops, eye drops, penetration, absorption, physical or chemical mediated methods, into the body such as muscles, intradermal, subcutaneous, veins, and mucous membranes; or by being mixed with or encapsulated by other substances and introduced into the body.

[0188] The main advantages of this invention include:

[0189] (a) The active compounds of this invention can efficiently inhibit SARS-CoV-2 3CL protease, and the IC50 of some active compounds is [missing information]. 50 The value is below 5 μM or lower.

[0190] (b) Due to the high conservation of 3CLpro and its substrate binding pocket in various coronaviruses, the active compounds of the present invention are expected to exert broad-spectrum antiviral activity by inhibiting 3CLpro of other coronaviruses.

[0191] (c) The active compounds of this invention are all old drugs with low toxicity and side effects and good drug properties.

[0192] (d) The technical platform of the present invention can quickly and effectively discover inhibitors of coronavirus 3CL hydrolase, and the method was used to discover the inhibitory effects of several old drugs such as aurinol and their combinations on SARS-CoV-2 virus 3CL hydrolase.

[0193] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0194] The analytical data of the samples were determined by the following instruments: nuclear magnetic resonance (NMR) was measured by a GEMINI-300, Bruker AMX-400, and INVOA-600 NMR spectrometer, with TMS (tetramethylsilane) as an internal standard. Chemical shifts were measured in ppm, and coupling constants were measured in Hz. Mass spectrometry (MS) was measured by a Finnigan MAT-711, MAT-95, and LCQ-DECA mass spectrometer, as well as an IonSpec 4.7 Tesla mass spectrometer.

[0195] Column chromatography used 200-300 mesh silica gel (produced by Qingdao Ocean Chemical Plant); TLC silica gel plates were HSGF-254 type thin-layer chromatography pre-prepared plates produced by Yantai Chemical Plant; petroleum ether had a boiling range of 60-90℃; ultraviolet light and an iodine tank were used for color development. Unless otherwise specified, all conventional reagents and chemicals used in the following examples were purchased from Sinopharm Group. Reagents and solvents used in the experiments were handled according to the specific reaction conditions.

[0196] Example 1: Establishment of a method for discovering SARS-CoV-2-3CLpro inhibitors

[0197] The inhibitory activity of single compounds and mixtures against SARS-CoV-23CLpro enzyme activity was evaluated using fluorescence resonance energy transfer (FRET). The total volume of the enzymatic reaction system was 120 μL, with a final protease concentration of 30 nM and a final substrate concentration of 20 μM. The reaction buffer consisted of 50 mM Tris (pH 7.3) and 1 mM EDTA. SARS-CoV-23CLpro protease and samples of different concentrations of compounds or mixtures were added to 96-well plates and incubated at 30 °C for 10 min. The substrate was then added, and the plates were quickly transferred to a microplate reader for reading. The excitation and emission wavelengths were 340 nm and 405 nm, respectively. The assay lasted 10 min, with fluorescence readings taken every 30 s. The final result was calculated by fitting the reaction rate to the readings from the first 2 mins and comparing it with the control group (DMSO). The IC50 was obtained using GraphPad Prism 8 software. 50 value.

[0198] This method is simple, sensitive, and can perform high-throughput screening of inhibitors of SARS-CoV-2-3CLpro (including various solid samples such as monomers and mixtures). Furthermore, due to the high conservation of 3CLpro and its substrate binding pockets among various coronaviruses, this method is also applicable to the discovery of inhibitors of other coronavirus 3CLpro, such as SARS-nCoV-3CLpro.

[0199] Example 2: Determination of the inhibitory effects of several existing drugs, including aurinolone, on SARS-CoV-2-3CLpro

[0200] In this embodiment, the 2000 pre-existing drugs tested were obtained through purchase, and their structure and purity (>90%) were confirmed. The compounds were first dissolved in DMSO to prepare a stock solution, and the DMSO stock solution was diluted with enzyme activity test buffer to prepare compound samples of various concentrations.

[0201] Using the SARS-CoV-2-3CLpro inhibitor discovery method established in Example 1, the inhibitory effects of compounds (including several existing drugs such as aurinolone) from a library of 2000 existing drugs on SARS-CoV-2-3CLpro were determined. The inhibitory rates and IC50 values ​​of different concentrations of the compounds on SARS-CoV-2-3CLpro were also measured. 50 As shown in Table 1.

[0202] The experimental results are shown in Table 1.

[0203] Table 1. Inhibitory activity of several existing drugs, including aurinophene, against the 3CL protease of the 2019 novel coronavirus.

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] The results showed that several existing drugs, including auronoxine (A1–A73), exhibited highly significant inhibitory effects against SARS-CoV-2-3CLpro (Table 1). These existing drugs, including auronoxine, are newly discovered inhibitors of SARS-CoV-2-3CLpro derived from existing drug sources, with IC50 values ​​of [missing information]. 50 The optimal IC50 values ​​for compounds such as aurinofen, rabeprazole sodium, heparin sodium, disulfiram, esomeprazole, berberine, and teloprazole are at the single-digit micromolar level. 50 The concentrations were 1.1 μM, 1.5 μM, 1.5 μM, 1.6 μM, 2.6 μM, 4.8 μM, and 6.5 μM, respectively. Figure 1-7 ).

[0212] Due to the high conservation of 3CLpro and its substrate binding pockets among various coronaviruses, these newly discovered inhibitors hold promise for inhibiting 3CLpro in other coronaviruses and exerting broad-spectrum antiviral activity.

[0213] Example 3: Evaluation of the inhibitory activity of compound against 2019 novel coronavirus replication

[0214] Determining the inhibitory activity of active compounds (A1–A73) against the replication of each 2019 novel coronavirus (SARS-CoV-2): Cells were cultured at a density of 5 × 10⁻⁶ cells / year. 4 Cells were cultured overnight in 48-well cell culture dishes. Cells were pretreated for 1 hour with different concentrations of active compounds (such as aurinolone and other older drugs), followed by infection with virus (multiple infection multiplicity MOI of 0.05) for 2 hours. The virus-compound mixture was then removed, and cells were further cultured in fresh medium containing the active compounds. At 48 hours post-infection (pi), cell supernatant was collected and lysed in lysis buffer. The viral copy number in the cell supernatant was quantitatively assessed by quantitative real-time RT-PCR (qRT-PCR), and the EC50 of the compounds inhibiting viral activity was calculated. 50 .

[0215] The results show that the active compounds of this invention (such as aurinophene and other existing drugs) can effectively inhibit the replication of the 2019 novel coronavirus and have a certain inhibitory effect on different isolated virus strains.

[0216] discuss

[0217] The sequence identity of SARS-CoV-2 3CLpro is as high as 96% with that of SARS-CoV 3CLpro, and the substrate binding pocket region is 100% conserved. Following the SARS-CoV outbreak in 2003, several inhibitors targeting SARS-CoV 3CLpro were designed and discovered, most of which are covalently acting peptide and polypeptide inhibitors, with a small number of heterocyclic esters, pyrazoles, and macrocyclic non-covalent inhibitors also reported. The bioactivity of these known inhibitors is mainly detected at the in vitro enzyme level, with a few inhibitors showing some inhibitory efficiency at the cellular level (EC50). 50 (Mostly at the micromolar level).

[0218] Searching for compounds with anti-SARS-CoV-2 activity from already approved drugs is currently the best strategy for rapidly finding clinical treatments for COVID-19.

[0219] Prior to this invention, the inhibitory effects of several existing drugs, such as aurinophene, on coronavirus 3CL hydrolase had not been reported.

[0220] In this invention, the inventors disclose for the first time that several existing drugs, including aurinolone, have significant inhibitory effects on SARS-CoV-2 3CLpro (Table 1), representing novel SARS-CoV-2 3CLpro inhibitors derived from existing drugs, with an IC50 value of [missing information]. 50 The levels reached single-digit micromolar levels. Due to the high conservation of 3CLpro and its substrate binding pocket among various coronaviruses, the SARS-CoV-2 3CLpro inhibitor of this invention is expected to exert broad-spectrum antiviral activity by inhibiting 3CLpro of other coronaviruses.

[0221] Therefore, several existing drugs, such as aurinofen, have provided important candidate drugs for the development of broad-spectrum anti-single-strand RNA virus drugs and have good prospects for clinical application.

[0222] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of an active ingredient or a formulation containing said active ingredient in the preparation of (a) a 2019 novel coronavirus 3CL protease inhibitor; and / or (b) a medicament for treating and / or preventing or alleviating diseases related to infection with the 2019 novel coronavirus, wherein said active ingredient is selected from the group consisting of tannic acid or a pharmaceutically acceptable salt thereof.

2. The use as described in claim 1, characterized in that, The diseases related to infection with the 2019 novel coronavirus are selected from the following group: respiratory infections, pneumonia and its complications, or combinations thereof.

3. The use of a pharmaceutical composition in the preparation of (a) an inhibitor of the 2019 novel coronavirus 3CL protease; and / or (b) a medicament for treating and / or preventing or alleviating diseases related to infection with the 2019 novel coronavirus, characterized in that, The pharmaceutical composition contains: (a) A first active ingredient, wherein the first active ingredient is selected from: tannic acid or a pharmaceutically acceptable salt thereof; and (b) pharmaceutically acceptable carriers.

4. The use as described in claim 3, characterized in that, The pharmaceutical composition contains: (a1) A first active ingredient, wherein the first active ingredient is selected from the group consisting of tannic acid or a pharmaceutically acceptable salt thereof; (a2) The second active ingredient, wherein the second active ingredient is remdesivir; and (b) pharmaceutically acceptable carriers.

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