Compounds and methods for ameliorating or treating coronavirus infection

Abstract

The present invention relates to anti-coronavirus compounds. The present invention includes a method for preventing and / or treating coronavirus infection by inhibiting cysteine proteases in viruses, in particular SARS-COV-2. Also provided are compositions / pharmaceutical compositions for preventing and / or treating coronavirus infection comprising any of the compounds, pharmaceutically acceptable salts thereof or mixtures thereof, and uses of the compounds.

Description

[0001] Cross-references to related applications

[0002] This application claims the interest and priority of U.S. Provisional Application No. 63 / 018,690, filed May 1, 2020, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention provides certain anti-coronavirus compounds and methods and compositions / pharmaceutical compositions for the prevention and treatment of coronavirus infection, specifically disease caused by SARS-CoV-2. Background Technology

[0004] SARS is an enveloped beta-coronavirus containing positive-stranded single-stranded RNA with a genome of approximately 30 kb. Open reading frames (ORFs) 1a and 1b encode two complex large proteins (pps), pp1a and pp1ab, respectively (Hegyi et al., 2002; Needle et al., 2015). Successful replication and proteolytic processes are essential for completing its life cycle (Herold et al., 1998). In fact, the common functions of these virally encoded proteolytic proteins are found in all coronaviruses, particularly papain-like protease (PLpro) and chymotrypsin-like protease (3CLpro) (Herold et al., 1998). During the proteolysis of pp1a and pp1ab, PLpro and 3CLpro cleave the first three sites and the remaining 11 sites, respectively, generating a total of 16 nonstructural proteins (nsp1-16) (Hegyi et al., 2002; Needle et al., 2015). Therefore, 3CLpro inhibition has been considered a molecular approach for exploring and developing anti-SARS drugs (Chen et al., 2005; Jo et al., 2020).

[0005] SARS-COV-2 is a novel coronavirus that has spread rapidly since its discovery and there is currently no drug or vaccine approved for the treatment of human coronavirus (Li & De Clercq, 2020). In light of the current SARS-CoV-2 outbreak and the treatment experience with SARS and MERS (another beta coronavirus), many studies have extensively investigated the possibility of using existing antiviral drugs for the treatment or intervention of SARS-COV-2 that are used to treat HIV, hepatitis B virus, hepatitis C virus, and influenza virus infections (De Clercq & Li, 2016; Li & De Clercq, 2020). At the same time, the characteristics of SARS-CoV-2 have been identified as an enveloped, positive-stranded, single-stranded RNA beta coronavirus similar to SARS and MERS (Li & De Clercq, 2020). In line with the characteristics of coronaviruses, the SARS-CoV-2 genome encodes structural proteins (e.g., spike glycoprotein), non-structural proteins (e.g., 3CLpro, PLpro, helicase, RNA-dependent RNA polymerase), and accessory proteins. With the available genome sequences of SARS-COV-2, SARS, and MERS, proteolytic sites and proteolytic enzymes were found to be highly conserved, so it is worth considering the reuse of SARS and MERS protease inhibitors to treat SARS-COV-2 (Liu et al., 2020). Since 3CLpro plays a key role in SARS, it is reasonable to inhibit proteases by targeting 3CLpro rather than its PLpro of SARS-COV-2 to intercept its life cycle (Chen et al., 2005; Jo et al., 2020; Liu et al., 2020).

[0006] Currently, disulfiram is an approved drug for the treatment of alcohol dependence and has been reported to inhibit PLpro of MERS and SARS in cell cultures, but has not been clinically evaluated (Li & De Clercq, 2020). In addition, clinical trials of HIV protease inhibitors (lopinavir and ritonavir) for SARS-CoV-2 patients have also been initiated, but it is uncertain whether they can effectively inhibit SARS-CoV-2, because HIV and beta coronavirus proteases belong to the aspartic acid protease family and the cysteine protease family, respectively (Li & De Clercq, 2020; Zumla et al., 2016). On the other hand, remdesivir is a nucleotide analog of an RNA-dependent RNA polymerase inhibitor approved for HIV treatment, which is currently undergoing clinical trials in SARS-CoV-2 patients, with an expected completion date of April 2020; galidesivir is another nucleotide analog of an RNA-dependent RNA polymerase inhibitor used in early clinical studies for HCV treatment, which showed broad-spectrum 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 analogs can be considered to cause unknown toxicity (Feng, 2018).

[0007] Currently, there is no vaccine or antiviral drug to prevent or treat human coronavirus infection. There is an urgent need to explore and develop safe anti-coronavirus therapies, specifically for SARS-COV-2. SUMMARY

[0008] The present invention surprisingly found that some triterpenoids are effective in inhibiting coronavirus infection, particularly SARS-COV-2.

[0009] In one aspect, the present invention provides a method of preventing and / or treating coronavirus infection, comprising administering to a subject in need thereof a compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of:

[0010]

[0011]

[0012] wherein R1is O, a-OH or b-H; R2is H or OH; R3is O, a-H, b-OH, b-OAc or H2; R4is H or OH; R5is H, OH or ORx; R6is COORx or COO(CH2) n -CH3; n is an integer from 0 to 3; R7is H, OH, ORxor OAc; R8is CH3or COORx; R 21 is CH3, COORxor COO(CH2) n -CH3; n is an integer from 0 to 3; the dotted line represents a single bond or a double bond, Rxis H or a C 1-8 alkyl group; and

[0013]

[0014] wherein R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , X1and X2are each H, OH, C 1-8 alkyl, NRx, SRx, ORx, pyrazoline, cysteine, glutathione, halogen, COORxor COO(CH2) n -CH3; n is an integer from 0 to 3; Y1, Y2, Z1, Z2, W1and W2are each H, OH, C 1-8 alkyl, or X1and X2together form -O-, or Y1and Y2, Z1and Z2, or W1and W2together form a cyclic epoxy resin.

[0015] In some specific embodiments of the present application, the compound is selected from the group consisting of:

[0016] (dehydroeburicoic acid),

[0017] (variisponic acid D),

[0018] (dehydroeburicoic acid),

[0019] (dehydroeburicoic acid), ​

[0020] antcin K), and

[0021] ovatodiolide.

[0022] In another aspect, the present application provides a composition / pharmaceutical composition for preventing and / or treating a coronavirus infection, in particular SARS-COV-2, comprising a therapeutically effective amount of any of the compounds disclosed herein, or pharmaceutically acceptable salts thereof, or mixtures thereof, in combination with a pharmaceutically acceptable carrier.

[0023] Optionally, the composition / pharmaceutical composition according to the present application can comprise at least one additional antiviral therapeutic agent.

[0024] In yet another aspect, the present application provides the use of any of the compounds disclosed herein, or pharmaceutically acceptable salts thereof, or mixtures thereof, in the manufacture of a medicament for preventing and / or treating a coronavirus infection, in particular SARS-COV-2.

[0025] 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 application. BRIEF DESCRIPTION OF DRAWINGS

[0026] The foregoing summary, as well as the following detailed description of the application, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the application, there is shown in the drawings a presently preferred embodiment. It should be understood, however, that the application is not limited to the embodiment shown in the drawings.

[0027] In the drawings:

[0028] Figure 1 AR100-DS1, AR101 DS1, AR101 DS2, AR101 DS3 and AR101 DS4 at 20 mM concentration. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0029] Figure 2A Relative 3CLpro activity (%) of AR101 DS2 (0.5 p / 5 FP) is shown. IC50= 39 mM.

[0030] Figure 2B Relative 3CLpro activity (%) of AR101 DS2 (0.125 p / 1.25 FP) is shown. IC50= 15.82 mM.

[0031] Figure 2CRelative 3CLpro activity (%) for AR101 DS3 (0.5p / 5FP) is shown, IC50= 14.61 mM.

[0032] Figure 2D Relative 3CLpro activity (%) for AR101 DS3 (0.5p / 5FP) is shown, IC50= 11.06 mM.

[0033] Figure 2E Relative 3CLpro activity (%) for AR101 DS3 (0.125p / 1.25FP) is shown, IC50= 11.7 mM.

[0034] Figure 3 Relative 3CLpro activity (%) for AR100 DS1 (0.125p / 1.25FP) is shown, IC50= 21.31 mM. DETAILED DESCRIPTION

[0035] The above summary of the present application will be further described with reference to the following examples of embodiments. However, it should not be understood that the content of the present application is limited only to the following embodiments, but all the inventions based on the above content of the present application are within the scope of the present application.

[0036] 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 application belongs.

[0037] As used herein, the singular forms "a", "an" and "the" include plural referents 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.

[0038] In the present application, to assess the impact of prospecting drugs on high-throughput proteolytic processing inhibition, synthetic peptides using a labeled fluorescence resonance energy transfer (FRET) pair were used, as those used in previous studies in which the quenched fluorophore was released immediately after 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). The present application demonstrates that any of the compounds or mixtures thereof disclosed herein are effective in inhibiting cysteine proteases, in particular 3CLpro of SARS-CoV-2.

[0039] The present disclosure provides a method for preventing and / or treating a coronavirus infection, in particular 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 selected from the group consisting of: (1)

[0041]

[0042]

[0043] wherein R1 is O, a-OH or b-H; R2 is H or OH; R3 is O, a-H, b-OH, b-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 bond or a double bond, Rx is H or a C 1-8 alkyl group; and (2)

[0045]

[0046] wherein R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , X1 and X2 are each H, OH, C 1-8 alkyl, NRx, SRx, ORx, pyrazoline, cysteamine, glutathione, halogen, COORx, or COO(CH2) n -CH3; n is an integer from 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 a cyclic epoxy.

[0047] The present disclosure also provides a composition / pharmaceutical composition for preventing and / or treating a coronavirus infection, in particular SARS-COV-2, comprising a therapeutically effective amount of a compound disclosed herein, or a mixture thereof, and a pharmaceutically acceptable carrier.

[0048] In one embodiment, the compound is (1)

[0050]

[0051]

[0052] wherein R1is O, a-OH or β-H; R2is H or OH; R3is O, a-H, β-OH, β-OAc or H2; R4is H or OH; R5is H, OH or ORx; R6is COORxor COO(CH2) n -CH3; n is an integer from 0 to 3; R7is H, OH, ORxor OAc; R8is CH3or COORx; R9is H, OH, ORxor OAc; and Rxis H or a C 21 -CH3; n is an integer from 0 to 3; R7is H, OH, ORxor OAc; R8is CH3or COORx; R9is H, OH, ORxor OAc; and Rxis H or a C n -CH3; n is an integer from 0 to 3; R7is H, OH, ORxor OAc; R8is CH3or COORx; R9is H, OH, ORxor OAc; and Rxis H or a C 1-8 -CH3; n is an integer from 0 to 3; R7is H, OH, ORxor OAc; R8is CH3or COORx; R9is H, OH, ORxor OAc; and Rxis H or a C (2)

[0054]

[0055] wherein R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , X1and X2are each H, OH, C 1-8 alkyl, NRx, SRx, ORx, pyrazoline, cysteamine, glutathione, halogen, COORxor COO(CH2) n -CH3; n is an integer from 0 to 3; R7is H, OH, ORxor OAc; R8is CH3or COORx; R9is H, OH, ORxor OAc; and Rxis H or a C 1-8 alkyl; Y1, Y2, Z1, Z2, W1and W2are each H, OH, C 1-8 alkyl, or X1and X2together form -O-, or Y1and Y2, Z1and Z2, or W1and W2together form a cyclic epoxy.

[0056] In embodiments of the present application, the compound of formula (I) can be:

[0057]

[0058] [R1] [R2] [R3] [R4] Delta Tumulosic acid A O H [H2] H Tumulosic acid B O H O H Tumulosic acid C O H Beta-OH H Tumulosic acid D O H O OH Tumulosic acid E O H [H2] 14 Tumulosic acid F O H Beta-OH 14 Tumulosic acid K Alpha-OH OH Beta-OH H

[0059] In another embodiment of the application, the compound of formula (I) can be:

[0060]

[0061] R7= H; R8= CH3

[0062]

[0063] In another embodiment of the application, the compound of formula (I) can be:

[0064]

[0065]

[0066] In another embodiment of the application, the compound of formula (I) can be:

[0067]

[0068] [R7] [R8] Delta Dehydroeburicoic acid H COOH 7.9(11) Buricinol H CH3 8 15 Alpha-acetyldihydroeburicoic acid OAc COOH 7.9(11) Dihydroeburicoic acid OH COOH 7.9(11) Eburoic acid H COOH 8 Variecoic acid OAc COOH 8 Sulphureburicoic acid OH COOH 8

[0069] In a particular embodiment of the application, the compound of formula (I) can be a lanostane:

[0070]

[0071] In addition, the compound of formula (II) can be Ovatodiolide,

[0072] Ovatodiolide acid, Anisomelic acid,

[0073]

[0074] Modification 1,

[0075]

[0076] Modification 2,

[0077]

[0078] Modification 3,

[0079]

[0080]

[0081] or a derivative thereof, for example

[0082]

[0083] Thus, preferred compounds are selected from the group consisting of: (Dehydrotumolosaeure),

[0084] (Dehydrotumulosic acid),

[0085] (3-Epi-dehydrotumulosic acid),

[0086] (Dehydrosulphurenic acid),

[0087] (Sulphurenic acid),

[0088] (Dehydrotumolosaeure-methylester),

[0089]

[0090] ((20ξ)-3β,15α,16α-trihydroxy-24-methyllanosta-7,9(11),24(241)-trien-21-oic acid; 15α-hydroxydehydrotumulosic acid),

[0091]

[0092] (Methyl 25-hydroxy-3-epidehydrotumulosate (methyl)),

[0093] (Dehydropachymic acid),

[0094] (15α-acetyldehydrosulfuric acid),

[0095] (15α-acetyldehydrosulphurenic acid)

[0096] (variable hole) Porcini acid D, versisponic acid D)

[0097]

[0098] (29-hydroxydehydropachymic acid; (3β,16α)-3-(acetyloxy)-16,29-dihydroxy-24-methylidenelanosta-7,9(11)-dien-21-oic acid),

[0099] (dehydroeburicoic acid)

[0100] (Antropicillin A, antcin A)

[0101] (Antrocin B)

[0102] (Ganoderic acid C, antcin C)

[0103] (Antrothocarboxylic acid H, antcin H), and

[0104] (Antrothocarboxylic acid K, antcin K), and

[0105] Ovatodiolide.

[0106] According to the present invention, the preferred compounds are selected from the group consisting of:

[0107] (Gachollic acid K, AR101DS01);

[0108] (Dehydroerythrocinic acid, AR101 DS2),

[0109]

[0110] (Erythrocinic acid, AR101 DS2),

[0111] (Variccinic acid D, AR101 DS3), and (Erythrocinic acid, AR101 DS2),

[0112] (Dehydroerythrocinic acid, AR101 DS2).

[0113] In one embodiment of the present application, the compound of Formula (II) is

[0114] (Fishhookin, AR100 DS1).

[0115] As used herein, the term “coronavirus” refers to a coronavirus in the order Nidovirales, family Coronaviridae, subfamily Orthocoronavirinae, having a positive-sense single-stranded RNA genome and an enveloped virus with a helical symmetry of the nucleocapsid. They have characteristic club-shaped spikes protruding from their surface, which create a crown-like appearance that is reminiscent of the solar corona when viewed under an electron microscope, from which they derive their name. Coronaviruses cause diseases in mammals and birds, including humans. In humans, coronaviruses cause respiratory tract infections, including the common cold, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and SARS-CoV-2.

[0116] As used herein, the term “cysteine protease” refers to a thiol protease, an enzyme that degrades proteins, having a common catalytic mechanism involving a nucleophilic cysteine thiol in a catalytic triad or diad. One example of a cysteine protease in a virus is 3CLpro in SARS-CoV-2.

[0117] As used herein, the terms “treat,” “treating,” or “treatment” and the like refer to the application of or administration of a composition comprising one or more active agents to or on a body having a disease, a symptom or condition of the disease, or a progression of the disease, with the purpose of treating, curing, mitigating, relieving, altering, remedying, improving, enhancing, or affecting the disease, the symptom or condition of the disease, a disability caused by the disease, or the progression of the disease.

[0118] As used herein, the terms “prevent,” “prevention,” or “preventing,” and the like, refer to preventing the recurrence, onset, or development of a viral infection, one or more symptoms thereof, or a respiratory condition associated with, exacerbated by, or exacerbated by a coronavirus infection in an individual.

[0119] As used herein, the term “individual” includes a human or non-human animal, such as a companion animal (e.g., a dog, cat, and the like), a farm animal (e.g., a cow, sheep, pig, horse, and the like), or a laboratory animal (e.g., a rat, mouse, guinea pig, and the like).

[0120] As used herein, the term “therapeutically effective amount” refers to the amount of an agent that, compared to a corresponding individual who has not received such amount, has an effect in treating, curing, preventing or ameliorating a disease, disorder, or side effect, or reducing the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

[0121] For use in therapy, the therapeutically effective amount of the compound is formulated into a pharmaceutical composition for administration. Accordingly, the present application further provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the compounds disclosed herein, or a mixture thereof, and one or more pharmaceutically acceptable carriers.

[0122] For delivery and absorption, a therapeutically effective amount of the active ingredient according to the present application can be formulated with a pharmaceutically acceptable carrier into a pharmaceutical composition in a suitable form. Based on the route of administration, the pharmaceutical composition of the present application is preferably 0.1% to 100% by weight of the total weight of the active ingredient.

[0123] The term “pharmaceutically acceptable carrier” as used herein refers to an acceptable carrier, diluent, or excipient with which the formulation is compounded and which is compatible with the other ingredients of the formulation and not deleterious to the individual to whom the pharmaceutical composition will be administered. Any carrier, diluent, or excipient commonly known or used in the art, depending on the requirements of the pharmaceutical formulation, can be used in the present application. The carrier can be a diluent, vehicle, excipient, or matrix of the active ingredient. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The composition can additionally contain lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents and suspending agents; preservatives such as methyl and propylhydroxybenzoates; sweetening agents; and flavoring agents.

[0124] The compositions of the present application can provide the effect of rapid, sustained or delayed release of the active ingredients after administration to a patient. In accordance with the present application, 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.

[0125] For parenteral administration, it is preferred to use a sterile aqueous solution which can contain other substances, such as salts or glucose, sufficient to make the solution isotonic with the blood. The aqueous solution can be appropriately buffered (preferably at a pH of 3 to 9) as needed. The preparation of suitable parenteral compositions under sterile conditions can be accomplished by standard pharmacological techniques known to those skilled in the art.

[0126] In one embodiment of the present application, the pharmaceutical compositions are formulated for oral administration. Such formulations can be prepared by any method known in the pharmaceutical art. In accordance with the present application, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, dragees, elixirs, suspensions, lotions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and powders in a hermetically sealed container.

[0127] In the present application, the methods and compositions / pharmaceutical compositions are effective in treating viral infections by inhibiting cysteine proteases in a virus, in particular, an RNA-dependent virus. Accordingly, the present application also provides methods and compositions / pharmaceutical compositions for treating and / or preventing viral infections by inhibiting cysteine proteases in a virus, comprising the use of the compounds disclosed herein or pharmaceutically acceptable salts thereof.

[0128] Exemplary viruses that are responsive include, but are not limited to, coronaviruses and HIV. Preferably, the viral infection is a coronavirus. More preferably, the viral infection is SARS, MERS and SARS-COV-2.

[0129] In another aspect, the present application provides a method for treating or preventing an RNA-dependent viral infection by inhibiting cysteine proteases in a virus. An example of the virus is an RNA-dependent virus, such as SARS, MERS and SARS-COV-2; in particular, SARS-COV-2.

[0130] In another aspect, the present application provides a composition / pharmaceutical composition for treating and / or preventing viral infections by inhibiting cysteine proteases in a virus, comprising any of the compounds disclosed herein, pharmaceutically acceptable salts thereof or mixtures thereof. Optionally, the composition / pharmaceutical composition can comprise at least one additional antiviral therapeutic agent.

[0131] In another aspect, the present application provides the use of any of the compounds disclosed herein for the manufacture of a medicament for the treatment or prevention of viral infection by inhibiting cysteine proteases in a virus.

[0132] The present application is further illustrated by the following examples which are provided by way of illustration and not by way of limitation.

[0133] Example

[0134] Materials and Methods

[0135] I. FRET protease assay of SARS-CoV-2 3CLpro

[0136] The ED-FRET platform was set up according to the protocol proposed by Jo et al. (2020). Briefly, a custom-made proteolytic fluorogenic peptide with DABCYL and EDANS at both ends, DABCYL-TSAVLQSGFRKMG-EDANS (KIRGEN BIOTECH, Taiwan), contains a consensus nsp4 / nsp5 cleavage sequence that can be recognized by SARS-CoV-2 3CLpro. The peptide was dissolved in distilled water and incubated with SARS-CoV-2 3CLpro. Spectrofluorometric measurements were determined by a Multifunction Microplate Reader (TECAN). The fluorescence intensity of EDANS upon proteolysis of the peptide as a function of time was determined at 37°C with an excitation wavelength of 340 nm and an emission wavelength of 490 nm with a bandwidth of 9, 15 nm, respectively. Prior to the assay, the test drug was checked for emission wavelength at 340 nm excitation to ensure that it did not overlap with the emission spectrum of EDANS.

[0137] Three replicates were performed in black 96-well microplates (Greiner) in 100 μL assay buffer containing SARS-CoV-2 3CLpro and custom-made peptide. In the SARS 3CLpro assay, 1 μM SARS-CoV-2 3CLpro containing 50 mM Tris pH 6.5 was incubated with 5 μM fluorogenic substrate at 37°C for 3 hours prior to measuring the Relative Fluorescence Unit (RFU).

[0138] II. Inhibition assay in the presence of a compound according to the present application

[0139] Five example compounds (AR100-DS1, AR101-DS1, AR101-DS2, AR101-DS3 and AR101-DS4) were screened for the first time at 20 μM (n = 3). ​

[0140] The following table provides the five compounds.

[0141]

[0142]

[0143]

[0144] First, SARS-CoV-2 3CLpro and Arjil drugs were mixed and pre- incubated for 1 hour at 37 °C. Using GraphPad Prism 7.03 (GraphPad Software, Inc., San Diego, CA, USA), those drugs that showed inhibitory activity against SARS-CoV-2 3CLpro at different concentrations were further investigated to characterize their IC50 values.

[0145] Based on the knowledge and sequence-based SARS-CoV-2 3CLpro, the efficacy of 3CLpro inhibitors according to the present application was evaluated in vitro to determine their therapeutic potential in the treatment of SARS-CoV-2. Given that no drug or vaccine has been approved for the treatment of human SARS-CoV-2 infection, it is extremely important and urgent to develop a broad-spectrum antiviral drug to combat SARS-CoV-2. The implementation of the ED-FRET technique and its workflow provides a powerful high-throughput drug discovery for the laboratory. At the same time, the identification of SARS-CoV-2 3CLpro inhibitors from the five example compounds according to the present application can serve as a guide for possible therapeutic doses in clinical evaluation and promote future patent applications, which helps to construct an antiviral library.

[0146] III. Results

[0147] 1. Identification of SARS-CoV-2 3CLpro inhibitors

[0148] To determine the efficacy of the five example compounds according to the present application, SARS-CoV-2 3CLpro, IQF peptide substrate, and example compounds were incubated at concentrations of 0.5 μM, 5 μM, and 20 μM, respectively, in the constructed ED-FRET platform. As shown in Table 2, the five example compounds according to the present application showed inhibitory activity against SARS-CoV-2 3CLpro, with IC50 values ranging from 0.5 μM to 20 μM. Figure 1As shown, all five example compounds significantly inhibited the peptidolytic activity of SARS-CoV-2 3CLpro. Among the five example compounds, AR101 DS2 and AR101 DS3 exhibited the greatest inhibition at 20 μΜ. Specifically, about 50% inhibition of SARS-CoV-2 3CLpro was found after 3 hours of AR101 DS3 treatment, while AR101 DS2 exhibited 40% inhibition under the same conditions, indicating their potential in countering the multi-protein processing cycle of SARS-CoV-2.

[0149] 2. Characterization of the half maximal inhibitory concentration of the inhibitors

[0150] The inhibition curves of AR100-DS1, AR101 DS1, AR101 DS2, AR101 DS3, and AR101 DS4 at a concentration of 20 μΜ are shown in Figure 1 As shown in Figure 1 , the half maximal inhibitory concentration (IC) of SARS-CoV-2 3CLpro was characterized by treating the compounds at a specified concentration ranging from 0 μΜ to 200 μΜ. The IC50values of AR101 DS2 and AR101 DS3 are shown in Figures 2A-2E As shown in Figure 2A , the IC50value of AR101 DS2 in the presence of 0.5 μΜ SARS-CoV-2 3CLpro and 5 μΜ IQF peptidic substrate (FP) was 39 μΜ. At the same time, the inhibition of 0.125 μΜ AR101 DS2 on SARS-CoV-2 3CLpro and 1.25 μΜ IQF peptidic substrate was determined (see Figure 2B ), the IC50value of AR101 DS2 on SARS-CoV-2 was 15.82 μΜ. As for the IC50value of AR101 DS3, 14.61 μΜ of AR101 DS3 achieved 50% inhibition of SARS-CoV-2 3CLpro in the presence of 0.5 μΜ SARS-CoV-2 3CLpro and 5 μΜ IQF peptidic substrate (see Figure 2C ), the IC50value of AR101 DS3 was 11.06 μΜ in the presence of 0.25 μΜ SARS-CoV-2 3CLpro and 2.5 μΜ IQF peptidic substrate (see Figure 2D ), or the IC50value of AR101 DS3 was 11.7 μΜ in the presence of 0.125 μΜ SARS-CoV-2 3CLpro and 1.25 μΜ IQF peptidic substrate (see Figure 2E ).

[0151] The IC50value of AR100 DS1 is shown in Figure 3AR100 DS1 was 21.31 μΜ. The IC50value of AR100 DS1 in the presence of 0.125 μΜ SARS-CoV-2 3CLpro and 1.25 μΜ IQF peptidomimetic substrate was 21.31 μΜ.

[0152] In view of the above, all the exemplified compounds including AR100 DS1, AR101 DS1, AR101 DS2, AR101 DS3 and AR101 DS4 were confirmed to have inhibitory effect on SARS-CoV-2 3CLpro, highlighting their therapeutic potential against SARS-CoV-2, especially AR100 DS1, AR101 DS2 and AR101 DS3.

[0153] All publications, patents, and patent documents cited herein are incorporated by reference herein in their entirety as if each were individually incorporated by reference.

[0154] The application has been described with reference to various specific and preferred embodiments and techniques. However, one skilled in the art will understand that many variations and modifications can be made while remaining within the spirit and scope of the application.

[0155] References

[0156] 1. Chan, J. F.-W., Kok, K.-H., Zhu, Z., Chu, H., To, K. K.-W., Yuan, S., & Yuen, K.-Y. (2020). Emerging Microbes & Infections, 9(1), 221-236.

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Claims

1. Use of a compound, a pharmaceutically acceptable salt thereof, or a mixture thereof in the preparation of a medicament for inhibiting SARS-CoV-2 infection, wherein said compound is selected from the group consisting of: (dehydroeburicoic acid); and (variable hole) Verisponic acid D).

2. The use as described in claim 1 further includes the administration of at least one additional antiviral therapeutic agent.

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