Compounds and methods for preventing and treating coronavirus infections

Abstract

The present invention relates to anti-coronavirus compounds. The present disclosure includes methods for preventing and / or treating coronavirus infections through the inhibition of cysteine ​​proteases in viruses, particularly SARS-COV-2. Also provided are compositions / pharmaceutical compositions for preventing and / or treating coronavirus infections, comprising the compounds, their pharmaceutically acceptable salts, or mixtures thereof, and the use of the compounds.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 018,690, filed May 1, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention provides several anti-coronavirus compounds and methods and compositions / pharmaceutical compositions for preventing and treating coronavirus infections, particularly diseases caused by SARS-COV-2. [Background technology]

[0003] According to the World Health Organization (WHO), the severe acute respiratory syndrome (SARS) outbreak from November 1, 2002, to June 18, 2003, resulted in 801 deaths in over 29 countries and 8,465 probable cases reported worldwide (Chen et al., 2005). SARS, an enveloped beta-coronavirus containing positive-sense single-stranded RNA, has a genome size of approximately 30 kb, and its open reading frames (ORFs) 1a and 1b encode two polyproteins (pps), pp1a and pp1ab, respectively (Hegyi et al., 2002; Needle et al., 2015). Successful replication and proteolytic processing are essential for the completion of its life cycle (Herold et al., 1998). In fact, the common function of these virus-encoded proteolytic proteins is found in all coronaviruses, particularly the papulin-like protease (PL) and chymotrypsin-like protease (3CL) (Herold et al., 1998). During proteolytic processing of pp1a and pp1ab, PL and 3CL cleave the first three and the remaining 11 positions, respectively, to give a total of 16 nonstructural proteins (nsp1-16) (Hegyi et al., 2002; Needle et al., 2015). Thus, 3CL inhibition is considered to provide a molecular approach for the discovery and development of anti-SARS drugs (Chen et al., 2005; Jo et al., 2020).

[0004] SARS-COV-2 is a novel coronavirus (designated COVID-19) that has spread rapidly since its identification in patients with severe pneumonia in Wuhan, China. It has been reported in 25 countries, with approximately 72,000 laboratory-confirmed cases and 1,775 deaths worldwide as of February 17, 2020 (Coronavirus disease 2019 (Covid-19) Situation Report - 28, 2020; Li & De Clercq, 2020). It is depressing that no drugs or vaccines have yet been approved to treat human coronaviruses (Li & De Clercq, 2020). Regarding the current SARS-CoV-2 outbreak and the therapeutic experience with SARS and MERS (another beta coronavirus), many studies have extensively examined the potential use of existing antiviral drugs used for HIV, hepatitis B virus, hepatitis C virus, and influenza infections for the treatment or intervention of SARS-CoV-2 (De Clercq & Li, 2016; Li & De Clercq, 2020). Meanwhile, SARS-CoV-2 has been characterized as an enveloped, positively-oriented, single-stranded RNA beta coronavirus, similar to SARS and MERS (Li & De Clercq, 2020). Consistent with coronavirus characteristics, the SARS-CoV-2 genome encodes structural proteins (e.g., spike glycoprotein), nonstructural proteins (e.g., 3CLpro, PLpro, helicase, RNA-dependent RNA polymerase), and accessory proteins. High levels of conservation of proteolytic sites and proteolytic enzymes were found in the available genome sequences of SARS-CoV-2, SARS, and MERS, making it worth considering repurposing SARS and MERS protease inhibitors for the treatment of SARS-CoV-2 (Liu et al., 2020). Because 3CL plays a crucial role in SARS, it is reasonable to target 3CL rather than PL of SARS-CoV-2 to inhibit the protease by blocking its life cycle (Chen et al., 2005; Jo et al., 2020; Liu et al., 2020).

[0005] Disulfiram, currently approved as a drug for treating alcoholism, has been reported to inhibit the PLpro of MERS and SARS in cell culture, but has not yet been clinically evaluated (Li & De Clercq, 2020). Clinical trials of HIV protease inhibitors (lopinavir and ritonavir) in patients with SARS-CoV-2 have also begun, but it is unclear whether they can effectively inhibit SARS-CoV-2 proteases, as the proteases of HIV and betacoronaviruses belong to the aspartic and cysteine ​​protease families, respectively (Li & De Clercq, 2020; Zumla et al., 2016). On the other hand, remdesivir, a nucleotide analogue of an RNA polymerase inhibitor approved for the treatment of HIV, is currently undergoing clinical trials in SARS-CoV-2 patients with a planned completion date of April 2020; galidesivir, another nucleotide analogue of an RNA-dependent RNA polymerase inhibitor in early clinical trials for the treatment of HCV, has demonstrated broad antiviral activity against severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) in preclinical studies (Wang et al., 2020; Zumla et al., 2016). However, nucleoside analogues are expected to induce toxicities beyond our current understanding (Feng, 2018).

[0006] No vaccines or antiviral drugs have yet been discovered to prevent or treat human coronavirus infections, and there is an urgent need to discover and develop safe anticoronavirus therapies, especially for SARS-COV-2. Summary of the Invention

[0007] The present invention unexpectedly discovered that some triterpenes are effective in inhibiting coronavirus infection, particularly SARS-COV-2.

[0008] In one aspect, the present invention provides a method for preventing and / or treating a coronavirus infection, comprising administering to a subject in need thereof a compound or a pharmaceutically acceptable salt thereof, wherein the compound is formula: [ka] [ka] [ka] [ka] [ka] [Wherein R1 is O, α-OH or β-H; R2 is H or OH; R3 is O, α-H, β-OH, β-OAc or H2; R4 is H or OH; R5 is H, OH or ORx; R6 is COORx or COO(CH2)n-CH3; n is an integer from 0 to 3; R7 is H, Oh, ORx or OAc; R8 is CH3 or COORx; R 21 is CH3, COORx, or COO(CH2)n-CH3; n is an integer from 0 to 3; the dotted line represents a single or double bond, and Rx is H or C 1-8 alkyl] a compound represented by the formula: formula: [ka] [In the formula, R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , X1 and X2 are H, OH, C, respectively 1-8alkyl, NRx, SRx, ORx, pyrazoline, cysteine, glutathione, halogen, COORx, or COO(CH2)n-CH3; n is an integer of 0 to 3; Y1, Y2, Z1, Z2, W1, and W2 are each H, OH, C 1-8 alkyl, or X1 and X2 together form -O-, or Y1 and Y2, Z1 and Z2, or W1 and W2 together form an epoxy." a compound represented by The method is selected from the group consisting of:

[0009] In some specific examples of the present invention, the compound is formula: [ka] [ka] [ka] [ka] [ka] [ka] The compound is selected from the group consisting of:

[0010] In a further aspect, the present invention provides a composition / pharmaceutical composition for treating and / or preventing coronavirus infection, particularly SARS-COV-2, comprising a therapeutically effective amount of any of the compounds disclosed herein or a pharmaceutically acceptable form thereof, or a mixture thereof, in combination with a pharmaceutically acceptable carrier.

[0011] Optionally, the composition / pharmaceutical composition according to the present invention may comprise at least one additional antiviral therapeutic agent.

[0012] In a further aspect, the present invention provides the use of any compound disclosed herein or a pharmaceutically acceptable salt thereof, or a mixture thereof, for the manufacture of a medicament for the prevention and / or treatment of a coronavirus infection, in particular SARS-COV-2.

[0013] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention. [Brief explanation of the drawings]

[0014] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred.

[0015] In the figure,

[0016] [Figure 1] Figure 1 shows the inhibition profiles of AR100-DS1, AR101 DS1, AR101 DS2, AR101 DS3, and AR101 DS4 at a concentration of 20 μM. *, P<0.05; **, P<0.01; ***, P<0.001.

[0017] [Figure 2] Figure 2A shows the relative 3CLpro activity (%) of AR101 DS2 (0.5p / 5FP), IC50 = 39 μM.

[0018] [Figure 2] Figure 2B shows the relative 3CLpro activity (%) of AR101 DS2 (0.125p / 1.25FP), IC50 = 15.82 μM.

[0019] [Figure 2] Figure 2C shows the relative 3CLpro activity (%) of AR101 DS3 (0.5p / 5FP), IC50 = 14.61 μM.

[0020] [Figure 2] Figure 2D shows the relative 3CLpro activity (%) of AR101 DS3 (0.5p / 5FP), IC50 = 11.06 μM.

[0021] [Figure 2] Figure 2E shows the relative 3CLpro activity (%) of AR101 DS3 (0.125p / 1.25FP), IC50 = 11.7 μM.

[0022] [Figure 3] Figure 3 shows the relative 3CLpro activity (%) of AR100 DS1 (0.125p / 1.25FP), IC50 = 21.31 μM.

[0023] Detailed Description of the Invention The above-mentioned summary of the present invention will be further explained with reference to the following examples. However, the content of the present invention should not be construed as being limited to the following examples, and all inventions based on the content of the present invention described above belong to the scope of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0025] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "sample" includes a plurality of such samples and equivalents thereof known to those skilled in the art.

[0026] In the present invention, to evaluate the efficacy of discovery drugs in inhibiting proteolytic processing in a high-throughput manner, synthetic peptides labeled with fluorescence resonance energy transfer (FRET) pairs are used, similar to those used in previous studies, where the fluorophore is released upon cleavage of the FRET-labeled peptide, generating a fluorescent signal that can be monitored in real time (Chen et al., 2005; Jean et al., 1995; Jo et al., 2020). In the present invention, any compound or mixture thereof disclosed herein is confirmed to be effective in inhibiting cysteine ​​proteases, particularly 3CL of SARS-CoV-2.

[0027] The present invention relates to a method for preventing and / or treating a coronavirus infection, particularly SARS-COV-2, comprising administering to a subject in need thereof a compound or a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the compound is (1) Formula: [ka] [ka] [ka] [ka] [ka] [Wherein R1 is O, α-OH or β-H; R2 is H or OH; R3 is O, α-H, β-OH, β-OAc or H2; R4 is H or OH; R5 is H, OH or ORx; R6 is COORx or COO(CH2)n-CH3; n is an integer from 0 to 3; R7 is H, Oh, ORx or OAc; R8 is CH3 or COORx; R 21is CH3, COORx, or COO(CH2)n-CH3; n is an integer from 0 to 3; the dotted line represents a single or double bond, and Rx is H or C 1-8 alkyl] a compound represented by the formula: (2) Formula: [ka] [In the formula, R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , X1 and X2 are H, OH, C, respectively 1-8 alkyl, NRx, SRx, ORx, pyrazoline, cysteine, glutathione, halogen, COORx, or COO(CH2)n-CH3; n is an integer of 0 to 3; Y1, Y2, Z1, Z2, W1, and W2 are each H, OH, C 1-8 alkyl, or X1 and X2 together form -O-, or Y1 and Y2, Z1 and Z2, or W1 and W2 together form an epoxy. a compound represented by The method is selected from the group consisting of:

[0028] The present invention also provides a composition / pharmaceutical composition for preventing and / or treating a coronavirus infection, particularly a SARS-COV-2 infection, comprising a therapeutically effective amount of a compound disclosed herein or a mixture thereof and a pharmaceutically acceptable carrier.

[0029] In one embodiment, the compound is (1) Formula: [ka] [ka] [ka] [ka] [ka] [Wherein R1 is O, α-OH or β-H; R2 is H or OH; R3 is O, α-H, β-OH, β-OAc or H2; R4 is H or OH; R5 is H, OH or ORx; R6 is COORx or COO(CH2)n-CH3; n is an integer from 0 to 3; R7 is H, Oh, ORx or OAc; R8 is CH3 or COORx; R 21 is CH3, COORx, or COO(CH2)n-CH3; n is an integer from 0 to 3; the dotted line represents a single or double bond, and Rx is H or C 1-8 alkyl] a compound represented by the formula: (2) Formula: [ka] [In the formula, R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , X1 and X2 are H, OH, C, respectively 1-8 alkyl, NRx, SRx, ORx, pyrazoline, cysteine, glutathione, halogen, COORx, or COO(CH2)n-CH3; n is an integer from 0 to 3, and Rx is H or C 1-8 alkyl; Y1, Y2, Z1, Z2, W1 and W2 are each H, OH, C 1-8 alkyl, and X1 and X2 together form -O-, or Y1 and Y2, Z1 and Z2, or W1 and W2 together form an epoxy. a compound represented by is selected from the group consisting of:

[0030] In an embodiment of the present invention, the compound of formula (I) is [ka] [Table 1] It could be.

[0031] In another example of the present invention, the compound of formula (I) is [ka] [Table 2] It could be.

[0032] In a further example of the present invention, the compound of formula (I) is [ka] [Table 3] It could be.

[0033] In a further embodiment of the present invention, the compound of formula (I) is [ka] [Table 4] It could be.

[0034] In one particular example of the present invention, the compound of formula (I) is lanostane: [ka] It could be.

[0035] Furthermore, the compound represented by formula (II) [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or derivatives thereof, e.g. [ka] It could be.

[0036] Thus, preferred compounds are [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0037] According to the present invention, the most preferred compounds are [ka] [ka] [ka] [ka] [ka] is selected from the group consisting of:

[0038] In one particular example of the present invention, the compound of formula (II) is [ka] is.

[0039] As used herein, the term "coronavirus" refers to a coronavirus in the Orthocoronavirus subfamily, Coronaviridae family, Nidovirales order, Riboviridae domain, which is an enveloped virus with a positively oriented single-stranded RNA genome and a nucleocapsid with helical symmetry. They have characteristic club-shaped spikes protruding from their surface, producing an image in electron micrographs reminiscent of the solar corona from which they are named. Coronaviruses cause illness in mammals, including humans, and birds. In humans, they cause respiratory infections such as the common cold, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and SARS-CoV-2.

[0040] As used herein, the term "cysteine ​​protease" refers to a thiol protease, a protein-degrading enzyme that shares a common catalytic mechanism in which a catalytic triad or duad contains a nucleophilic cysteine ​​thiol. An example of a viral cysteine ​​protease is 3CLpro in SARS-COV-2.

[0041] As used herein, the terms "treat," "treating," or "treatment" refer to the application or administration of a composition containing one or more active substances to a subject afflicted with a disease, a symptom or condition of a disease, or the progression of a disease, for the purpose of curing, healing, alleviating, mitigating, altering, ameliorating, or affecting the disease, the symptom or condition of a disease, the disorder caused by the disease, or the progression of the disease.

[0042] As used herein, the terms "prevent," "prevention," or "preventing" refer to preventing the recurrence, onset, or progression of a viral infection, one or more symptoms thereof, or a respiratory condition associated with, enhanced by, or that enhances a coronavirus infection in a subject.

[0043] As used herein, the term "subject" includes a human or non-human animal, such as a companion animal (e.g., dog, cat, etc.), a farm animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, etc.).

[0044] As used herein, the term "therapeutically effective amount" refers to an amount of a pharmaceutical agent that is effective in treating, curing, preventing, or ameliorating a disease, disorder, or side effect, or reducing the rate of progression of a disease or disorder, compared to a corresponding subject not receiving such amount. The term also encompasses amounts effective to enhance normal physiological function.

[0045] For use in therapy, a therapeutically effective amount of the compound is formulated as a pharmaceutical composition for administration. Accordingly, the present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of any compound disclosed herein or a mixture thereof, and one or more pharmaceutically acceptable carriers.

[0046] For the purpose of delivery and absorption, a therapeutically effective amount of the active ingredient according to the present invention can be formulated into a pharmaceutical composition in a suitable form together with a pharmaceutically acceptable carrier. Depending on the route of administration, the pharmaceutical composition of the present invention preferably comprises 0.1% to 100% by weight of the total weight of the active ingredient.

[0047] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier(s), diluent(s), or excipient(s) that is / are acceptable in the sense that it is compatible with other ingredients of the formulation and does not dramatically alter the subject to which the pharmaceutical composition is administered. Any carrier, diluent, or excipient commonly known or used in the art can be used in the present invention, depending on the requirements of the pharmaceutical formulation. The carrier may be a diluent, vehicle, excipient, or matrix for the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbose, mannose, starch, gum arabic, calcium phosphate, alginates, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The compositions can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents.

[0048] The compositions of the present invention can provide rapid, sustained, or delayed release of the active ingredient after administration to a patient. According to the present invention, the pharmaceutical compositions can be adapted for administration by any suitable route, including, but not limited to, oral, rectal, nasal, topical, vaginal, or parenteral routes (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods.

[0049] For parenteral administration, the compositions are preferably used in the form of a sterile aqueous solution, which may contain sufficient salts or other substances, such as glucose, to render the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of 3 to 9), if desired. The preparation of suitable parenteral compositions under sterile conditions can be accomplished by standard pharmacological techniques well known to those skilled in the art.

[0050] In a specific example of the present invention, the pharmaceutical composition is formulated for oral administration.Such formulations can be prepared by any method known in the art of pharmacy.According to the present invention, the composition may be in the form of tablets, pills, powders, lozenges, packets, troches, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injections, or packaged powders.

[0051] The methods and compositions / pharmaceutical compositions of the present invention are effective in treating viral infections through the inhibition of cysteine ​​proteases in viruses, particularly RNA-dependent viruses. Accordingly, the present invention also provides methods and compositions / pharmaceutical compositions for treating and / or preventing viral infections through the inhibition of cysteine ​​proteases in viruses, comprising using the compounds disclosed herein or pharmaceutically acceptable salts thereof.

[0052] Reactive exemplary viruses include but are not limited to coronavirus and HIV.Preferably, viral infection is coronavirus.More preferably, viral infection is SARS, MERS and SARS-COV-2.

[0053] In another aspect, the present invention provides a method for treating or preventing RNA-dependent viral infection by inhibiting cysteine ​​protease in the virus.Examples of viruses include RNA-dependent viruses, such as SARS, MERS and SARS-COV-2; particularly SARS-COV-2.

[0054] In a further aspect, the present invention provides a composition / pharmaceutical composition for treating and / or preventing viral infections through inhibiting cysteine ​​proteases in the viruses, the composition / pharmaceutical composition comprising any of the compounds disclosed herein, a pharmaceutically acceptable salt thereof, or a mixture thereof. Optionally, the composition / pharmaceutical composition may also comprise at least one additional antiviral therapeutic agent.

[0055] In a further aspect, the present invention provides the use of any of the compounds disclosed herein for the manufacture of a medicament for treating or preventing a viral infection through inhibiting a cysteine ​​protease in the virus.

[0056] The present invention is further described by the following examples, which are offered by way of illustration and not by way of limitation. [Example]

[0057] material and method

[0058] I. FRET Protease Assay Using SARS-CoV-2 3CLpro

[0059] The construction of the ED-FRET platform follows the protocol described by Jo et al. (2020). Briefly, a custom proteolytic fluorescent peptide with DABCYL and EDANS at its termini, DABCYL-TSAVLQSGFRKMG-EDANS (Genomics, Taiwan), contains the consensus nsp4 / nsp5 cleavage sequence that can be recognized by SARS-CoV-2 3CL. This peptide is dissolved in distilled water and incubated with SARS-CoV-2 3CL. Spectral-based fluorescence measurements are performed using a SPARK® multimode microplate reader provided by TECAN. Proteolytic activity is measured at 37°C by measuring the fluorescence intensity of EDANS as a function of time during peptide hydrolysis, λ. 励起 = 340 nm, λ 発光 Prior to the assay, the emission wavelength of the test drug at 340 nm excitation was checked to ensure that it did not overlap with the emission spectrum of EDANS.

[0060] Assays are performed in triplicate in black 96-well microplates (Greiner) in 100 μL of assay buffer containing SARS-CoV-2 3CLpro and customized peptides. The SARS 3CLpro assay involves incubating 1 μM SARS-CoV-2 3CLpro in 50 mM Tris pH 6.5 with 5 μM fluorescent substrate for 3 hours at 37°C before measuring relative fluorescence units (RFU).

[0061] II. Inhibition assay in the presence of compounds according to the invention

[0062] In the first screening at 20 μM (n=3), there were five exemplary compounds (AR100-DS1, AR101-DS1, AR101-DS2, AR101-DS3, and AR101-DS4). These five compounds are shown in the table below. [Table 5-1] [Table 5-2]

[0063] First, SARS-CoV-2 3CLpro and Arjil drugs were mixed and pre-incubated at 37°C for 1 hour. Those that showed inhibitory activity against SARS-CoV-2 3CLpro were further examined at different concentrations, and their IC50 values ​​were characterized using GraphPad Prism 7.03 (GraphPad Software, San Diego, CA, USA).

[0064] Based on the knowledge and sequence of SARS-CoV-2 3CL, we evaluated the 3CL inhibitory effects of compounds according to the present invention in vitro to determine their therapeutic potential in treating SARS-CoV-2. Because no drugs or vaccines have yet been approved to treat SARS-CoV-2 infection, the development of broad-spectrum antivirals against SARS-CoV-2 is of utmost importance and urgency. The introduction of ED-FRET technology and its workflow provides robust, high-throughput drug discovery in the laboratory. Meanwhile, the identification of SARS-CoV-2 3CL inhibitors from the five exemplary compounds according to the present invention will serve as a guideline for possible therapeutic doses in clinical evaluation and potentially encourage patent applications, contributing to the construction of antiviral libraries.

[0065] III. Results

[0066] 1. Identification of SARS-CoV-2 3CLpro inhibitors

[0067] To measure the efficacy of five exemplary compounds according to the present invention, SARS-CoV-2 3CL, the IQF peptide substrate, and the exemplary compounds were incubated in the constructed ED-FRET platform at 0.5 μM, 5 μM, and 20 μM, respectively. As shown in Figure 1, all five exemplary compounds substantially inhibited the peptide cleavage activity of SARS-CoV-2 3CL. Among these five exemplary compounds, AR101 DS2 and AR101 DS3 showed the highest inhibitory effect at 20 μM. In particular, AR101 DS3 inhibited SARS-CoV-2 3CL by approximately 50% after 3 hours of treatment, while AR101 DS2 inhibited it by 40% under the same conditions, suggesting their potential to counteract the SARS-CoV-2 polyprotein processing cycle.

[0068] 2. Characterization of half-maximal inhibitory concentrations of inhibitors

[0069] The inhibition profiles of AR100-DS1, AR101 DS1, AR101 DS2, AR101 DS3, and AR101 DS4 at a concentration of 20 μM were measured and are shown in Figure 1. As shown in Figure 1, the half-maximal inhibitory concentrations (IC) against SARS-CoV-2 3CL were characterized by treating the compounds at the indicated concentrations ranging from 0 μM to 200 μM. The IC50 values ​​of AR101 DS2 and AR101 DS3 are shown in Figures 2A–2E. As shown in Figure 2A, AR101 DS2 had an IC50 value of 39 μM in the presence of 0.5 μM SARS-CoV-2 3CL and 5 μM IQF peptide substrate (FP). Meanwhile, when the inhibitory rate of 0.125 μM AR101 DS2 against SARS-CoV-2 3CLpro and 1.25 μM IQF peptide substrate (FP) was measured (see Figure 2B), the IC50 value of AR101 DS2 against SARS-CoV-2 was 15.82 μM. The IC50 values ​​for AR101 DS3 reached 50% SARS-CoV-2 3CL inhibition at 14.61 μM with 0.5 μM SARS-CoV-2 3CL and 5 μM IQF peptide substrate (see Figure 2C), 11.06 μM with 0.25 μM SARS-CoV-2 3CL and 2.5 μM IQF peptide substrate (see Figure 2D), or 11.7 μM with 125 μM SARS-CoV-2 3CL and 1.25 μM IQF peptide substrate (see Figure 2E).

[0070] The IC50 value of AR100 DS1 is shown in Figure 3. AR100 DS1 exhibited an IC50 value of 21.31 μM in the presence of 0.125 μM SARS-CoV-2 3CLpro and 1.25 μM IQF peptide substrate.

[0071] From the above, it was confirmed that all the exemplified compounds, including AR100 DS1, AR101 DS1, AR101 DS2, AR101 DS3 and AR101 DS4, have inhibitory effects against SARS-CoV-2 3CLpro, and in particular, AR100 DS1, AR101 DS2 and AR101 DS3 highlighted their therapeutic potential against SARS-CoV-2.

[0072] All publications, patents, and patent documents cited above are herein incorporated by reference in their entirety.

[0073] Although the invention has been described with reference to various specific and preferred embodiments and techniques, those skilled in the art will recognize that many variations and modifications can be made while remaining within the spirit and scope of the invention. Furthermore, the present invention includes the following aspects. [Aspect 1] 1. A method of inhibiting coronavirus infection, comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the compound is (1) Formula: [ka] [ka] [ka] [ka] [ka] [Wherein R1 is O, α-OH or β-H; R2 is H or OH; R3 is O, α-H, β-OH, β-OAc or H2; R4 is H or OH; R5 is H, OH or ORx; R6 is COORx or COO(CH2)n-CH3; n is an integer from 0 to 3; R7 is H, Oh, ORx or OAc; R8 is CH3 or COORx; R 21 is CH3, COORx, or COO(CH2)n-CH3; n is an integer from 0 to 3; the dotted line represents a single or double bond, and Rx is H or C 1-8 It is alkyl. a compound represented by the formula: (2) Formula: [ka] [In the formula, R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , X1 and X2 are H, OH, C, respectively 1-8 alkyl, NRx, SRx, ORx, pyrazoline, cysteine, glutathione, halogen, COORx, or COO(CH2)n-CH3; n is an integer of 0 to 3; Y1, Y2, Z1, Z2, W1, and W2 are each H, OH, C 1-8 alkyl, or X1 and X2 together form -O-, or Y1 and Y2, Z1 and Z2, or W1 and W2 together form an epoxy. a compound represented by The method is selected from the group consisting of: [Aspect 2] The compound of formula (I) may be of the formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and wherein the compound is selected from the group consisting of Aspects The method described in 1. [Aspect 3] The compound of formula (II) is of the formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] and wherein the compound is selected from the group consisting of Aspects The method described in 1. [Aspect 4] 1. The compound of formula: [ka] [ka] [ka] [ka] [ka] and wherein the compound is selected from the group consisting of Aspects The method described in 1. [Aspect 5] The coronavirus is selected from the group consisting of Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and SARS-COV-2; Aspects The method described in 1. [Aspect 6] The coronavirus is SARS-COV-2. Aspects The method described in 1. [Aspect 7] The compound is effective in inhibiting a cysteine ​​protease in a virus, Aspects The method described in 1. [Aspect 8] further comprising administering at least one additional antiviral therapeutic agent. Aspects Any of 1 to 7 crab The method described. [Aspect 9] 1. A method for treating or preventing a viral infection through inhibiting a cysteine ​​protease in the virus, comprising administering to a subject in need thereof a composition / pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt, or a mixture thereof, wherein the compound is present in an amount effective to inhibit the cysteine ​​protease in the virus: Aspects Any of 1 to 4 crab or a pharmaceutically acceptable salt thereof, or a mixture thereof. [Aspect 10] A composition / pharmaceutical composition for treating or preventing a viral infection, comprising an amount effective to inhibit a cysteine ​​protease in the virus, Aspects Any of 1 to 4 crabA composition / pharmaceutical composition comprising the compound described or a pharmaceutically acceptable salt thereof or a mixture thereof. [Aspect 11] The virus is an RNA-dependent virus, Aspects 11. The composition / pharmaceutical composition according to claim 10. [Aspect 12] The RNA-dependent virus is a coronavirus. Aspects 11. The composition / pharmaceutical composition according to claim 11. [Aspect 13] The coronavirus is SARS, MERS, or SARS-CoV-2; Aspects 13. The composition / pharmaceutical composition according to claim 12. [Aspect 14] The coronavirus is SARS-CoV-2. Aspects 13. The composition / pharmaceutical composition according to claim 12. [Aspect 15] For the manufacture of a medicament for preventing or treating a viral infection by inhibiting a cysteine ​​protease in the virus, Aspects Any of 1 to 4 crab Use of the compounds described or their pharmaceutically acceptable salts or mixtures thereof. [Aspect 16] The virus is an RNA-dependent virus, Aspects 15. Use according to claim 15. [Aspect 17] The RNA-dependent virus is a coronavirus. Aspects 16. Use according to claim 16. [Aspect 18] The coronavirus is SARS, MERS, or SARS-CoV-2; Aspects 17. Use according to claim 17. [Aspect 19] The coronavirus is SARS-CoV-2. Aspects 17. Use according to claim 17.

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Claims

[Claim 1] A compound for inhibiting 3-chymotrypsin-like protease (3CLpro) in viruses, 【Chemistry 1】 10. A pharmaceutical composition comprising:

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