Compounds and methods for preventing and treating viral infections

Abstract

The present invention relates to antiviral compounds. The present disclosure includes methods for preventing and / or treating viral infections through the inhibition of viral cysteine ​​proteases and / or intracellular sodium-taurocholate cotransporting polypeptides, particularly SARS-COV-2 and Hepatitis B virus (HBV). Also provided are compositions / pharmaceutical compositions containing any of the compounds, their pharmaceutically acceptable salts, or mixtures thereof, and uses of the compounds for preventing and / or treating viral infections.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority to U.S. Provisional Patent Application 63 / 071,564, filed August 28, 2020.

[0002] (Field of the Invention) The present invention relates to anti - viral compounds, methods, and combinations / compositions / pharmaceutical compositions for preventing and treating viral infections, particularly diseases caused by coronaviruses or hepatitis viruses.

Background Art

[0003] Viruses are entities in which genetic material is enclosed within a protein coat, invade living normal cells, and use those cells to proliferate and produce other viruses like themselves, thereby causing common infectious diseases such as influenza and warts, or severe diseases such as smallpox and acquired immunodeficiency syndrome (AIDS).

[0004] For example, hepatitis viruses include five different types: A, B, C, D, and E, as well as types X and G. Hepatitis A and E viruses are induced by ingesting contaminated water or food. However, hepatitis B, C, and D viruses are caused by parenteral contact with infected body fluids. Also, infections with hepatitis C and D viruses are increasing, and effective treatments are being sought.

[0005] Hepatitis B virus (HBV) causes acute and chronic viral hepatitis in humans. HBV infection is often associated with severe liver diseases including cirrhosis and hepatocellular carcinoma (HCC) [1]. The prevalence of HBV infection in the world is very high. Despite the availability of effective vaccines for over 25 years, approximately 350 million people are chronically infected. The relative risk of HCC in HBV carriers increases by about 100 - fold compared to non - carriers [2].

[0006] Currently approved anti-HBV drugs, such as interferon alpha or nucleoside (nucleotide) analogs that inhibit the viral reverse transcriptase, are unsuitable for the growing number of HBV-infected patients due to side effects and the development of drug resistance [3].

[0007] Therefore, in order to improve treatment effectiveness, it is necessary to search for effective, safe, and reasonably priced anti-HBV drugs that aim to disrupt other stages in the viral life cycle.

[0008] HBV is a small DNA virus consisting of a nucleocapsid protecting a 3.2 kb viral genome [4]. The HBV nucleocapsid is enclosed in an envelope consisting of the hepatitis B surface antigen (HBsAg). HBsAg is encoded within a single read frame having three in-phase start codons. MHBsAg has a 55 amino acid (aa) extension from the S domain, which is called the pre-S2 domain. LHBsAg has a further 108-aa region extending from the pre-S2 domain, constituting the pre-S1 domain. Recently, sodium-taurocholic acid cotransport polypeptide (NTCP) has been identified as an HBV receptor [5, 6]. HBV entry into uninfected hepatocytes has long been proposed as a potential target for antiviral interventions [7]. On the other hand, HepG2.2.15 cells contain the entire HBV genome and have been widely used in studies of HBV replication, assembly, and secretion.

[0009] The attachment of HBV to hepatocytes during the infection phase has long been proposed as a potential target for antiviral interventions. Molecules that specifically bind to HBV particles are thought to interfere with viral attachment, thereby reducing or preventing subsequent infection.[8]

[0010] Our understanding of the initial infection event of human HBV is limited due to the lack of cell culture systems that support a complete replication cycle. To date, two cell types have been shown to be susceptible to HBV infection. One is the human hepatocellular carcinoma cell line HepaRG, which becomes infectious after differentiation induced by dimethyl sulfoxide (DMSO) [7,9], and the other is normal human primary hepatocytes, which are readily infected with HBV [10,11], but their limited in vitro cell lifespan and lack of a stable supply source are considered to have significant limitations for future applications.

[0011] Furthermore, herpes simplex virus (HSV) also consists of a DNA genome enclosed within a protein coat. Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2) are causative agents of human diseases including gingivostomatitis, pharyngitis, oral herpes, encephalitis, and infections of the eyes and genitals

[12] . Herpesvirus infections generally have a mild or asymptomatic initial stage, after which the virus persists in a non-replicating latent state or at clinically undetectable levels of replication

[13] . Primary HSV-1 infections are most associated with the mouth and / or throat, causing gingivostomatitis and pharyngitis. Even after recovering from a primary oropharyngeal infection, individuals may retain HSV DNA in the trigeminal ganglion for life, leading to recurrent outbreaks of oral herpes. Studies have also revealed a possible link between some members of the herpesvirus family and periodontal disease

[14] . Human herpesviruses may be present at a relatively high rate in periodontal lesions

[15] . HSVs are associated with the severity of periodontal disease in terms of clinical attachment loss

[16] . Viral gingival infections can impair the host's defense mechanisms, thereby leading to a stage of abnormal proliferation of pathogenic oral bacteria [15, 17].

[0012] HSV generally attacks mucous membranes, skin, eyes, and the nervous system and can infect a wide variety of cells

[18] . Organ cultures of human gingival mucosa can be infected with HSV-1 and HSV-2

[19] . In addition, human gingival keratinocytes and gingival fibroblasts cultured in vitro can sustain HSV proliferation [20,21]. HSV-1 encodes a viral thymidine kinase, which indirectly metabolizes acyclovir to acyclovir triphosphate, a chain terminator substrate of HSV DNA polymerase, thereby halting viral DNA replication

[22] . However, resistance to acyclovir has been reported in 5–30% of cases

[23] ). Acyclovir-resistant HSV-1 strains frequently occur in immunocompromised patients and can cause serious complications

[24] . As no vaccine exists, topical fungicides are considered an important strategy for preventing HSV transmission.

[0013] 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 more than 29 countries and 8,465 possible cases worldwide.

[25] SARS is an enveloped β-coronavirus containing positive-sense single-stranded RNA with a genome size of approximately 30 kb, where read frames (ORFs) 1a and 1b encode two respective polyproteins (pps), pp1a and pp1ab.[26, 27] Successful replication and proteolysis are required to complete its life cycle.

[28] In fact, consensus functions of proteolytic proteins encoded by these viruses are found in all coronaviruses, particularly papline-like protease (PLpro) and chymotrypsin-like protease (3CLpro).

[28] In the proteolytic treatment of pp1a and pp1ab, PLpro and 3CLpro cleave the first three sites and the remaining 11 sites, respectively, yielding a total of 16 non-structural proteins (nsp1-16) [26, 27]. Therefore, inhibition of 3CLpro is considered a molecular approach in the discovery and development of anti-SARS drugs [25, 29].

[0014] SARS-CoV-2 is a novel coronavirus (named COVID-19) that has spread rapidly since its initial identification in a patient with severe pneumonia in Wuhan, China. As of February 17, 2020, it has been reported in 25 countries worldwide, with approximately 72,000 laboratory-confirmed cases and 1,775 deaths.

[30] Tragically, there are still no approved treatments or vaccines for human coronaviruses.

[31] Given the current SARS-CoV-2 outbreak and our experience treating SARS and MERS (another beta-coronavirus), many studies have broadly investigated the potential use of existing antiviral agents used for HIV, hepatitis B virus, hepatitis C virus, and influenza infections to treat or intervene in SARS-CoV-2.[31, 32] Meanwhile, SARS-CoV-2 is characterized by being an enveloped, positive-sense, single-stranded RNA beta-coronavirus, similar to SARS and MERS.

[31] Consistent with the characteristics of coronaviruses, the SARS-CoV-2 genome encodes structural proteins (e.g., spike glycoprotein), non-structural proteins (e.g., 3CLpro, PLpro, helicases, RNA-dependent RNA polymerases), and accessory proteins. In the available genome sequences of SARS-CoV-2, SARS, and MERS, high levels of conserved proteolytic sites and proteolytic enzymes have been found, making it worthwhile to explore the repurposing of SARS and MERS protease inhibitors for the treatment of SARS-CoV-2

[33] . 3CLpro plays a crucial role in SARS, and it is reasonable to approach protease inhibition by targeting SARS-CoV-2's 3CLpro instead of SARS-CoV-2's PLpro and disrupting its life cycle [25, 29, 33].

[0015] Disulfiram, currently approved as a drug to treat alcohol dependence, has been reported to inhibit MERS and SARS PLpro in cell cultures, but has not yet undergone clinical evaluation

[31] . Furthermore, clinical trials of HIV protease inhibitors (lopinavir and ritonavir) have been initiated in SARS-CoV-2 patients, but it is unclear whether they can effectively inhibit SARS-CoV-2 proteases, as HIV and β-coronavirus proteases belong to the asparagine protease family and cysteine ​​protease family, respectively [31, 34]. On the other hand, remdesivir (RDV), a nucleotide analog of an RNA-dependent RNA polymerase inhibitor approved for the treatment of HIV, is currently undergoing clinical trials in SARS-CoV-2 patients, scheduled for completion in April 2020; galidesivir, another nucleotide analog of an RNA-dependent RNA polymerase inhibitor in early-stage clinical studies for the treatment of HCV, has shown broad-spectrum antiviral activity against severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) in preclinical studies [34, 35]. However, nucleoside analogs are still expected to induce toxicity beyond human comprehension

[36] .

[0016] No antiviral drugs have yet been found to prevent or treat human coronavirus infection. The search for and development of safe anti-coronavirus therapies, particularly against SARS-CoV-2, is urgently needed.

[0017] The development of new antiviral therapies and medications remains desirable. [Overview of the Initiative]

[0018] In the present invention, it was unexpectedly discovered that several triterpenes are effective in inhibiting viral infections, particularly hepatitis B virus (HBV) infection and / or herpes simplex virus (HSV) infection and / or coronavirus infection, especially SARS-CoV-2 infection.

[0019] In one aspect, the present invention is a method of inhibiting viral 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 Formula I:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0020] In some specific examples of the present invention, the compounds are selected from the group consisting of ugonin J, ugonin N, (6-(3,4-dihydroxyphenyl)-4-hydroxyhexa-3,5-dien-2-one), (2-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-6-hydroxypyran-4-one), dehydroebric acid, 3-O-methylkaempferol, kaempferol-3-O-(3,4-diacetyl-alpha-L-rhamnopyranoside, kaempferol-3-O-(2,4-diacetyl-alpha-L-rhamnopyranoside), dehydrosulfurenic acid, sulfurenic acid, percisponate D, and trans-p-ment-6-en-2,8-diol, and anthocine K.

[0021] In some specific examples of the present invention, the above combination is a combination of two or more compounds selected from the group consisting of ugonin J, ugonin N, (6-(3,4-dihydroxyphenyl)-4-hydroxyhexa-3,5-dien-2-one), (2-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-6-hydroxypyran-4-one), dehydroebric acid, 3-O-methylkaempferol, kaempferol-3-O-(3,4-diacetyl-alpha-L-rhamnopyranoside, kaempferol-3-O-(2,4-diacetyl-alpha-L-rhamnopyranoside), ovatodiol, dehydrosulfurenic acid, sulfurenic acid, percisponate D, and trans-p-ment-6-en-2,8-diol, and anthocine K.

[0022] In a further embodiment, the present invention provides a combination / composition / pharmaceutical composition for preventing or treating viral infections, particularly coronaviruses, such as SARS-CoV-2, comprising a therapeutically effective amount of any of the compounds described in the present invention, or a pharmaceutically acceptable salt thereof, or a mixture thereof, in combination with a pharmaceutically acceptable carrier.

[0023] In a further embodiment, the present invention provides a composition / pharmaceutical composition for preventing and / or treating hepatitis virus infection, particularly HBV, comprising a therapeutically effective amount of any of the compounds disclosed herein or a pharmaceutically acceptable salt thereof, or a mixture thereof, in combination with a pharmaceutically acceptable carrier.

[0024] Depending on the circumstances, the composition / pharmaceutical composition according to the present invention may contain at least one additional antiviral therapeutic substance.

[0025] In a further embodiment, the present invention provides the use of compounds described in the present invention or pharmaceutically acceptable salts thereof, or mixtures thereof, for the manufacture of pharmaceuticals for the prevention or treatment of viral infections, particularly coronaviruses, such as SARS-CoV-2.

[0026] In one example of the present invention, the virus is a hepatitis virus, particularly hepatitis B virus (HBV).

[0027] In one example of the present invention, the virus is herpes simplex virus (HSV).

[0028] It should be understood that both the general description above and the detailed description below are illustrative and descriptive only, and do not limit the invention. [Brief explanation of the drawing]

[0029] The above summary and 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 present invention, the drawings show currently preferred embodiments.

[0030] In the drawing,

[0031] [Figure 1] Figure 1 shows the relative activity (%) of 3CLpro and IC50 = 3.065 μM for AR100-DS1+ARH 101-DS2(0.25p / 0.6FP).

[0032] [Figure 2] Figure 2 shows the relative activity (%) of 3CLpro and IC50 = 2.934 μM for AR100-DS1+ARH 101-DS3 (0.25 p / 0.6 FP).

[0033] [Figure 3] Figure 3 shows the IC50 of ARH 020-DS1-SARS-CoV-2 = 14.93 μM.

[0034] [Figure 4] Figure 4 shows the IC50 of ARH 020-DS2-SARS-CoV-2 = 6.329 μM.

[0035] [Figure 5] Figure 5 shows the IC50 of ARH 019-DS1-SARS-CoV-2 = 19.21 μM.

[0036] [Figure 6] Figure 6 shows the IC50 of ARH 019-DS2-SARS-CoV-2 = 2.487 μM.

[0037] [Figure 7] Figure 7 shows the IC50 of ARH 007-DS3-SARS-CoV-2 = 11.61 μM.

[0038] [Figure 8] Figure 8 shows the IC50 of ARH 007-DS4-SARS-CoV-2 = 18.85 μM.

[0039] [Figure 9] Figure 9 shows the IC50 of ARH 007-DS5-SARS-CoV-2 = 48.22 μM.

[0040] [Figure 10]Figure 10 shows the relative activity (%) of 3CLpro for AR101-DS2+ARH013-DS1 and IC50 = 2.409 μM.

[0041] [Figure 11] Figure 11 shows the relative activity (%) of 3CLpro for ARH007-DS3 + ARH013-DS1 and the IC50 = 18.2 μM.

[0042] [Figure 12] Figure 12 shows the relative activity (%) of 3CLpro for AR100-DS1+ARH007-DS3 and the IC50 = 8.646 μM.

[0043] [Figure 13] Figure 13 shows ARH013-DS1-SARS-Cov-2 = 32.89 μM.

[0044] [Figure 14] Figure 14 shows the effects of 0 μM, 20 μM, and 40 μM of AR101-DS2 on HBsAg secretion from HepG2.2.15 cells (*, P<0.05; **, P<0.01; ***, P<0.001).

[0045] [Figure 15] Figure 15 shows the effects of 0 μM, 20 μM, and 40 μM AR101-DS2 on HBV DNA levels in the culture medium of HepG2.2.15 cells (*, P<0.05; **, P<0.01; ***, P<0.001).

[0046] [Figure 16] Figure 16 shows the effects of 0 μM, 20 μM, and 40 μM of AR101-DS2 on HBsAg secretion from HuS-E / 2 cells (*, P<0.05; **, P<0.01; ***, P<0.001).

[0047] [Figure 17]Figure 17 shows the effects of 0 μM, 20 μM, and 40 μM of AR101-DS2 on HBV mRNA expression levels in HuS-E / 2 cells.

[0048] [Figure 18] Figure 18 shows the inhibitory effects of AR101-DS3 at 0 μM, 10 μM, 20 μM, and 100 μM on NTCP (*, P<0.05; **, P<0.01; ***, P<0.001).

[0049] [Figure 19] Figure 19 shows the inhibitory effects of AR101-DS4 at 0 μM, 10 μM, 20 μM, and 100 μM on NTCP (*, P<0.05; **, P<0.01; ***, P<0.001).

[0050] [Figure 20] Figure 20 shows the effects of 0 μM, 40 μM, and 80 μM of AR101-DS1+AR101-DS3 on HBsAg secretion from Hus-E / 2 cells (*, P<0.05; **, P<0.01; ***, P<0.001).

[0051] [Figure 21] Figure 21 shows the effects of 0 μM, 40 μM, and 80 μM of AR101-DS1+AR101-DS3 on HBV mRNA expression levels in Hus-E / 2 cells (*, P<0.05;**, P<0.01;***, P<0.001).

[0052] [Figure 22] Figure 22 shows the effects of 0 μM, 40 μM, and 80 μM of AR101-DS1+AR101-DS4 on HBsAg secretion from Hus-E / 2 cells (*, P<0.05; **, P<0.01; ***, P<0.001).

[0053] [Figure 23]Figure 23 shows the effects of 0 μM, 40 μM, and 80 μM of AR101-DS1+AR101-DS4 on HBV mRNA expression levels in Hus-E / 2 cells (*, P<0.05; **, P<0.01; ***, P<0.001).

[0054] Detailed description of the present invention The above-described outline of the present invention will be further explained with reference to the embodiments of the following examples. However, the content of the present invention is limited to the following embodiments, and it should be understood that all inventions based on the above-described content of the present invention fall within the scope of the present invention.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0056] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to "sample" includes multiple such samples and their equivalents known to those skilled in the art.

[0057] In this invention, to evaluate the effect of a predicted drug on high-throughput inhibition of proteolytic degradation, a synthetic peptide of labeled fluorescence resonance energy transfer (FRET) pairs is employed, similar to those used in prior studies. Here, when the FRET-labeled peptide is cleaved, the quenched phosphor is released, generating a fluorescence signal that can be monitored in real time (Chen et al., 2005; Jean et al., 1995; Jo et al., 2020). In this invention, it has been confirmed that any of the compounds disclosed herein, or mixtures thereof, are effective in inhibiting cysteine ​​proteases, particularly 3CLpro of SARS-CoV-2.

[0058] The present invention relates to a method for preventing and / or treating a viral infection, comprising administering a compound or a pharmaceutically acceptable salt thereof, or a mixture thereof, to a subject in need thereof, wherein the compound is Formula I: [ka] Ugonin J having the structure and its derivatives, Formula II: [ka] Ugonin N having the structure and its derivatives, Formula III: [ka] 6-(3,4-dihydroxyphenyl)-4-hydroxyhexa-3,5-dien-2-one and its derivatives having the structure, Formula IV: [ka] 2-[(E)-2-(3,4-dihydroxyphenyl)ethenyl]-6-hydroxypyran-4-one and its derivatives having the structure, Formula V: [ka] Dehydroebrycoic acid having the structure and its derivatives, Equation VI: [ka] 3-O-methylkaempferol having the structure and its derivatives, Formula VII: [ka] Kaempferol-3-O-(3,4-O-diacetyl-alpha-L-rhamnopyranoside) and its derivatives having the structure, Formula VIII: [ka] Kaempferol-3-O-(2,4-O-diacetyl-alpha-L-rhamnopyranoside) and its derivatives having the structure, Formula IX: [ka] Dehydrosulfurenic acid having the structure and its derivatives, Formula X: [ka] Sulfurenic acid having the structure and its derivatives, Formula XI: [ka] Percisponate D having the structure and its derivatives, Formula XII: [ka] Trans-p-ment-6-ene-2,8-diol having the structure and its derivatives, Formula XIII: [ka] Anthosin K having the structure and its derivatives, The present invention provides a method selected from a group consisting of these combinations.

[0059] The present invention also provides combinations / compositions / pharmaceutical compositions for preventing and / or treating viral infections, particularly coronaviruses, such as SARS-CoV-2, comprising a therapeutically effective amount of a compound described in the present invention and a pharmaceutically acceptable carrier.

[0060] As used herein, the term "virus" means any small infectious substance that replicates only within the living cells of an organism and can infect all types of living organisms, from animals and plants to microorganisms, including bacteria and archaea. Exemplary viruses include, but are not limited to, hepatitis viruses, influenza viruses, herpes simplex viruses (HSV), enteroviruses, rotaviruses, dengue viruses, poxviruses, human immunodeficiency viruses, adenoviruses, measles viruses, retroviruses, coronaviruses, or noroviruses.

[0061] As used herein, the term "hepatitis virus" means the virus that causes hepatitis, in particular hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis D virus (HDV).

[0062] As used herein, the term "coronavirus" refers to coronaviruses belonging to the subfamily Orthocoronavirinae, family Coronaviridae, order Nidovirales, and realm Riboviria. These are enveloped viruses with a positive-sense single-stranded RNA genome and a helical nucleocapsid. They possess characteristic club-shaped spikes protruding from their surface, and images created using electron microscopy resemble the sun's corona, hence the name. Coronaviruses cause disease in mammals, including humans, and birds. In humans, coronaviruses cause respiratory infections, including the common cold, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and SARS-CoV-2.

[0063] As used herein, the term "cysteine ​​protease" refers to a thiol protease, an enzyme that breaks down proteins and shares a common catalytic mechanism involving three or two catalytic cysteine ​​thiols (duad). An example of a viral cysteine ​​protease is SARS-CoV-2 3CLpro.

[0064] As used herein, the terms “to treat” or “to treat” mean applying or administering a composition comprising one or more active substances to a subject suffering from a disease, symptoms or conditions of a disease, or the progression of a disease, for the purpose of treating, curing, reducing, mitigating, altering, restoring, improving, or influencing the disease, symptoms or conditions of a disease, physical impairments caused by the disease, or the progression of the disease.

[0065] As used herein, the terms “prevent,” “prevention,” or “preventing” mean the prevention of a viral infection, one or more of its symptoms, or a recurrence, onset, or progression of a respiratory condition associated with, enhanced by, or enhancing a coronavirus infection in a subject.

[0066] As used herein, the term “subject” includes human or non-human animals, such as companion animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, sheep, pigs, horses, etc.), or laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0067] As used herein, the term “therapeutic dose” means the amount of a drug that, compared to a corresponding subject not receiving such an amount, has the effect of treating, curing, preventing or improving a disease, disorder or side effect, or reducing the rate of progression of the disease or disorder. The term also includes amounts that are effective in improving normal physiological function.

[0068] For therapeutic use, a therapeutically effective amount of a compound is formulated as a pharmaceutical composition for administration. Therefore, the present invention further provides a pharmaceutical composition comprising a therapeutically effective amount of any of the compounds disclosed herein or a mixture thereof, and one or more pharmaceutically acceptable carriers.

[0069] For the purpose of delivery and absorption, a therapeutically effective amount of the active ingredient according to the present invention can be incorporated into a pharmaceutical composition in a suitable form together with a pharmaceutically acceptable carrier. Based on the route of administration, the pharmaceutical composition of the present invention preferably contains 0.1% to 100% by weight of the active ingredient relative to the total weight.

[0070] As used herein, the term “pharmaceutically acceptable carrier” means a carrier(s), diluent(s), or excipient(s) that is compatible with the other components of the formulation and is acceptable in the sense that it does not dramatically alter the pharmaceutical composition to the recipient. In the present invention, any carrier, diluent, or excipient that is commonly known or used in the art may be used, 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 composition may further contain lubricants, such as talc, magnesium stearate, and mineral oil; humectants; emulsifiers and suspending agents; preservatives, such as methyl and propyl hydroxybenzoates; sweeteners; and flavoring agents.

[0071] The compositions of the present invention can provide the effect of rapidly, continuously, or delayedly releasing the active ingredient after administration to a patient. According to the present invention, the pharmaceutical compositions may be adapted for administration by any suitable route, including but not limited to oral, rectal, nasal, topical, vaginal, or non-enteral routes (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal), perdermal, suppository, and intranasal methods.

[0072] For non-enteral administration, the product is preferably used in the form of a sterile aqueous solution, which may contain sufficient salts or other substances such as glucose to make the solution isotonic with respect to blood. The aqueous solution may be appropriately buffered (preferably to a pH of 3-9) as needed. The preparation of a suitable non-enteral composition under sterile conditions can be achieved by standard pharmaceutical techniques well known to those skilled in the art.

[0073] In one 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 pharmaceuticals. According to the present invention, the form of the composition may be tablets, pills, powders, lozenges, packets, oral tablets, elixirs, suspensions, lotions, solutions, syrups, soft gelatin capsules and hard gelatin capsules, suppositories, sterile injections, and packaged powders.

[0074] In the present invention, the methods and compositions / pharmaceutical compositions described above are effective in treating viral infections by inhibiting the cysteine ​​protease of viruses, particularly RNA-dependent viruses. Accordingly, the present invention also provides methods and compositions / pharmaceutical compositions for treating and / or preventing viral infections by inhibiting the cysteine ​​protease of viruses, comprising using the compounds disclosed herein or pharmaceutically acceptable salts thereof.

[0075] Exemplary responsive viruses include, but are not limited to, hepatitis viruses, influenza viruses, herpes simplex viruses, enteroviruses, rotaviruses, dengue viruses, poxviruses, human immunodeficiency viruses, adenoviruses, coronavirus infections, arenavirus infections, measles viruses, coronaviruses, or noroviruses. Preferably, the virus is a hepatitis virus, including hepatitis B virus, hepatitis C virus, hepatitis D virus, or SARS-CoV-2.

[0076] In another aspect, the present invention provides a method for treating or preventing RNA-dependent viral infections by inhibiting viral cysteine ​​proteases. Examples of viruses include RNA-dependent viruses such as SARS, MERS, and SARS-CoV-2; in particular, SARS-CoV-2.

[0077] In a further embodiment, the present invention provides a composition / pharmaceutical composition for treating and / or preventing viral infection by inhibiting viral cysteine ​​proteases, comprising any of the compounds disclosed herein, pharmaceutically acceptable salts thereof, or mixtures thereof. Optionally, the composition / pharmaceutical composition may comprise at least one additional antiviral therapeutic agent.

[0078] In a further embodiment, the present invention provides a composition / pharmaceutical composition for treating and / or preventing viral infection by inhibiting intracellular sodium-taurocholic acid cotransport polypeptides (NTCPs), comprising a compound disclosed herein, a pharmaceutically acceptable salt thereof, or a mixture thereof. The composition / pharmaceutical composition may optionally comprise at least one additional antiviral therapeutic agent.

[0079] In a further embodiment, the present invention provides the use of any of the compounds disclosed herein for producing a pharmacopoeia for treating or preventing a viral infection by inhibiting the cysteine ​​protease of a virus.

[0080] In a further embodiment, the present invention provides the use of any of the compounds disclosed herein for producing a pharmacopoeci for treating or preventing viral infections by inhibiting intracellular sodium-taurocholic acid cotransport polypeptides (NTCPs).

[0081] In another embodiment, the present invention provides a method for treating or preventing DNA-dependent viral infections by inhibiting intracellular sodium-taurocholic acid cotransport polypeptides (NTCPs). Examples of viruses include DNA-dependent viruses such as hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis D virus (HDV).

[0082] The present invention is further illustrated by the following examples, which are provided for demonstrative purposes only and are not limiting. [Examples]

[0083] material and method

[0084] I. FRET protease assay with SARS-CoV-2 3CLpro

[0085] The establishment of the ED-FRET platform follows the protocol provided by Jo et al. (2020). Briefly, DABCYL-TSAVLQSGFRKMG-EDANS (Genomics, Taiwan), a custom proteolytic fluorescent peptide with DABCYL and EDANS at its termini, contains a consensus nsp4 / nsp5 cleavage sequence recognizable by SARS-CoV-2 3CLpro. This peptide is dissolved in distilled water and incubated with SARS-CoV-2 3CLpro. Spectroscopic fluorescence measurements are determined using a SPARK® multimode microplate reader provided by TECAN. Proteolytic activity is measured by the fluorescence intensity of EDANS as a function of time during peptide hydrolysis at 37°C (λ, respectively). 励起 =340nm, λ 放出 (=490nm, bandwidth=9, 15nm). Before the assay, the emission wavelength of the test drug is examined with 340nm excitation to ensure that it does not overlap with the emission spectrum of EDANS.

[0086] The assay was performed in a black 96-well microplate (Greiner) with 100 μL of assay buffer (50 mM Tris pH 6.5) containing 0.25 μM SARS-CoV-2 3CLpro and 0.6 μM customized IQF substrate peptide, and measurements were taken three times.

[0087] II. Real-time FRET protease assay and dose-response curve analysis of SARS-CoV-2 in 3CLpro.

[0088] Prior to the addition of the IQF peptide substrate, 0.25 μM SARS-CoV-2 3CLpro was incubated with the target compound at the indicated concentration (0–100 μM) in assay buffer at 37°C for 1 hour (SC-HM100, Sheng Ching Enterprise Co. Ltd). Subsequently, 6 μM IQF peptide substrate was added to the mixture in a black 96-well microtiter plate immediately before detection of RFU using a TECAN SPARK® multimode microplate reader. Small modifications were made to the measurement parameters, differing from those used in the protein activity assay. During execution, 10 detection cycles were performed at 1.5-minute intervals with a gain value of 80. The change in fluorescence intensity was calculated by subtracting the initial value of the condition from its final value. Subsequently, the change in fluorescence intensity for each condition was normalized against the change in the negative control (vehicle only) in each assay plate. For each drug, the dose-response point from 0 to 100 μM was fitted to the normalized dose-response model provided by GraphPad Prism 7.03 (GraphPad software). Here,

number

[0089] III. Inhibition assay in the presence of Arjil drug

[0090] Thirteen predicted Arjil drugs were pre-incubated with SARS-CoV-2 3CLpro at 37°C for 1 hour. The evident inhibitory activity against SARS-CoV-2 3CLpro was further investigated at various concentrations, and the IC50 values ​​were characterized using GraphPad Prism 7.03 (GraphPad Software, San Diego, CA, USA). The thirteen predicted Arjil drugs are shown in the table below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0091] Predicted results

[0092] Based on knowledge and sequencing of SARS-CoV-2 3CLpro, the efficacy of 3CLpro inhibitors provided by Arjil was evaluated in vitro to determine their therapeutic potential in the treatment of SARS-CoV-2. Since no drugs or vaccines to treat human SARS-CoV-2 infection have yet been approved, the development of broad-spectrum antiviral materials to combat SARS-CoV-2 is a critical and urgent issue. The introduction of ED-FRET technology and its workflow enables reliable and high-throughput drug discovery in the laboratory. Furthermore, the identification of SARS-CoV-2 3CLpro inhibitors from 13 tests proposed and provided by Arjil serves as a guideline for estimated therapeutic doses in clinical evaluation, facilitates future patent applications, and contributes to the construction of an antiviral library. The 13 tests are as follows: 1. AR100-DS1 + AR101-DS2; 2. AR100-DS1 + AR101-DS3; 3. ARH 020-DS1 (Real-time); 4. ARH 020-DS2 (Real-time); 5. ARH 019-DS1 (Real-time); 6. ARH 019-DS2 (Real-time); 7. ARH 007-DS3 (Real-time); 8. ARH 007-DS4 (Real-time); 9. ARH 007-DS5 (Real-time); 10. AR101-DS2+ARH013-DS1; 11. ARH007-DS3+ARH013-DS1 12. AR100-DS1+ARH007-DS3; and 13. ARH 013-DS1 (Real-time).

[0093] IV. HepG2.2.15 cells

[0094] Continuous HBV proliferation can be achieved in HepG2.2.15 cells (RRID:CVCL_L855) stably transfected with the adw2 subtype of the HBV genome. HepG2.2.15 cells were used for unlimited supply and consistent quality and were maintained in Dulbecco's modified Eagle medium (DMEM;Invitrogen) supplemented with 10% thermo-inactivated fetal bovine serum (FBS;Thermo) + 100 units of penicillin and 100 X g of streptomycin / ml (both from Invitrogen).

[0095] V. HuS-E / 2 cells

[0096] For HBV infection, we utilize HuS-E / 2 cells, which retain the characteristics of primary hepatocytes even after long-term culture. As described in our previous study

[38] , for HBV infection, we differentiate HuS-E / 2 cells in 2% DMSO for 7 days, collect viral particles, and infect and replicate them in HuS-E / 2 cells. These cells are useful for assays of HBV strain infectivity and for screening anti-HBV substances.

[0097] VI. Collection of HBV particles

[0098] The culture medium of drug-treated HepG2.2.15 cells was clarified by centrifugation at 1,000 X g for 10 minutes at 4°C. The supernatant was then placed on a 20% sucrose cushion (20% sucrose, 20 mM HEPES, pH 7.4, 0.1% bovine serum albumin [BSA]) and centrifuged at 197,000 X g for 3 hours at 4°C to pellet HBV particles. The mixture was then concentrated 100-fold to detect HBV DNA.

[0099] VII. Isolation, reverse transcription, and real-time PCR of DNA and RNA

[0100] Total DNA is extracted using a genomic DNA isolation kit (Nexttec Biotechnologie, Germany). Total RNA is isolated from cultured cells using TRIzol® reagent (Invitrogen). Reverse transcription is performed using an RNA template, AMV reverse transcriptase (Roche), and oligo-dT primers. The products are subjected to real-time PCR using primer sets for specific genes and SYBR Green PCR Master Mix (Bio-Rad). The primer sets used for HBV core, HBsAg, cccDNA, and GAPDH are described [3]. The results are analyzed using the iCycler iQ real-time PCR detection system (Bio-Rad). Plasmid p1.3HBcl is prepared at a 10-fold dilution (2*10⁴–2*10⁹ copies / ml) and a standard curve is created in parallel PCR reactions.

[0101] VIII. Enzyme-linked immunosorbent assay (ELISA)

[0102] The HBsAg ELISA kit (General Biologicals Corp.) is used to detect hepatitis B surface antigen (HBsAg) according to the proposed protocol.

[0103] IX. PreS1-NTCP Pulldown Assay

[0104] Recombinant fusion protein expression and purification, and GST pull-down assay were performed as described above

[40] . Briefly, GST fusion protein expression in E. coli BL21(DE3) was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside, and the bacterial cells were then lysed by sonication at 4°C in PBS containing 1% Triton X-100 (PBST), and separated into soluble and insoluble fractions by centrifugation at 13,800 g for 10 minutes at 4°C. To perform the GST pull-down assay, the soluble fraction of bacterial lysate containing the GST fusion protein was incubated with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences) at 4°C for 3 hours. The beads were then washed three times with PBST and incubated overnight at 4°C with overexpression HA-NTCP prepared by dissolving Huh7 cell lysate and a protease inhibitor cocktail (1 mM PMSF, 10 μg / ml aprotinin, 1 μg / ml pepstatin A, 1 μg / ml leupeptin) in PBST. The beads were then washed with PBST, resuspended in a buffer sample (12.5 mM Tris-HCl, pH 6.8, 2% SDS, 20% glycerol, 0.25% bromophenol blue, 5% β-mercaptoethanol), subjected to SDS-polyacrylamide gel electrophoresis, and examined by Western blot analysis.

[0105] X. Statistical analysis

[0106] All values ​​are shown as mean ± SE. Each value is the mean of at least three experiments in in vitro studies for each drug. Student's t-test is used for statistical comparisons. * indicates that the value is significantly different from the control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0107] III. Results

[0108] 1. Characterization of the median inhibitory concentration of inhibitory substances

[0109] The median inhibitory concentrations of SARS-CoV-2 3CLpro were characterized by treatment with Arjil drugs at indicated concentrations ranging from 0 μM to 100 μM. The IC50 values ​​of Arjil drugs / tests against SARS-CoV-2 are shown in Table 1. The IC50 values ​​for each Arjil drug / test are shown in the figure below. In summary, the inhibitory effects of the combinations AR100-DS1+AR101-DS2, AR101-DS2+ARH013-DS1, AR100-DS1+ARH007-DS3, and AR100-DS1+AR101-DS3 against SARS-CoV-2 3CLpro highlight their therapeutic potential against COVID-19. Furthermore, ARH 020-DS2, ARH 019-DS2, and ARH 007-DS3 are the most promising compounds for inhibiting SARS-CoV-2 3CLpro.

[0110] [Table 2]

[0111] Ten example compounds / tests (AR100-DS1+AR101-DS2, AR100-DS1+AR101-DS3, ARH 020-DS1, ARH 020-DS2, ARH 019-DS1, ARH 019-DS2, ARH 007-DS3, ARH 007-DS4, ARH 007-DS5, ARH101-DS2+ARH013-DS1, ARH007-DS3+ARH013-DS1, AR100-DS1+ARH007-DS3, ARH013-DS1) are shown in Figure 1-13.

[0112] As shown in Figure 1, AR100-DS1+AR101-DS2 exhibited an IC50 value of 3.065 μM in the presence of 0.25 μM SARS-CoV-2 3CLpro and 0.6 μM IQF peptide substrate (FP). On the other hand, when the inhibitory effect of 0.25 μM AR100-DS1+AR101-DS3 on SARS-CoV-2 3CLpro and 0.6 μM IQF peptide substrate was measured (see Figure 2), the IC50 value of AR100-DS1+AR101-DS3 against SARS-CoV-2 was located at 2.934 μM.

[0113] The IC50 values ​​for ARH 020-DS2, ARH 019-DS2, and ARH 007-DS3 were 6.329 μM, 2.487 μM, and 11.61 μM, respectively.

[0114] Based on the above, the combinations of AR100-DS1+AR101-DS2, AR101-DS2+ARH013-DS1, AR100-DS1+ARH007-DS3, and AR100-DS1+AR101-DS3 against SARS-CoV-2's 3CLpro highlight their therapeutic potential against COVID-19. Furthermore, ARH 020-DS2, ARH 019-DS2, and ARH 007-DS3 are the most promising compounds for inhibiting SARS-CoV-2's 3CLpro.

[0115] All publications, patents, and patent documents cited above are incorporated herein by their individual citations.

[0116] The present invention has been described with reference to various specific and preferred embodiments and techniques. However, those skilled in the art will understand that many variations and modifications can be made while remaining within the spirit and scope of the invention.

[0117] 2. Inhibitory effect on HBV secretion by HepG2.2.15 cells

[0118] To investigate whether the above compounds affect the replication, assembly, or secretion of the HBV genome, HepG2.2.15 cells stably transfected with the HBV genome were incubated with AR101-DS2 for 48 hours, and then HBsAg and HBV DNA collected from the culture medium were measured by ELISA and real-time PCR. The results are shown in Figures 14 and 15.

[0119] The effects of AR101-DS2 on HBsAg secretion from HepG2.2.15 cells are shown in Figure 14 (0 μM, 20 μM, and 40 μM of AR101-DS2). HBsAg secretion was significantly inhibited by treatment with AR101-DS2.

[0120] The effects of AR101-DS2 on HBV DNA levels in the culture medium are shown in Figure 15 (0 μM, 20 μM, and 40 μM of AR101-DS2). We found a significant decrease in DNA levels after treatment with either 20 μM or 40 μM AR101-DS2. These results indicate that AR101-DS2 suppresses HBV secretion from HepG2.2.15 cells.

[0121] 3. Inhibitory effect on the HBV infectivity of HuS-E / 2 cells.

[0122] To evaluate the effects of AR101-DS2 on HBV infectivity and replication, HuS-E / 2 cells were infected with any subtype of HBV derived from HepG2.2.15 cells. AR101-DS2 was added to the culture medium during HBV infection and incubated for 18 hours. The infected cells were then washed, incubated in fresh medium for 48 hours, and HBsAg in the medium was detected by ELISA. HBV mRNA was detected by real-time PCR, and these were used as indicators of HBV infection efficiency in HuS-E / 2 cells. The results are shown in Figures 16 and 17.

[0123] The effects of AR101-DS2 on HBV entry into HuS-E / 2 cells are shown in Figures 16 and 17. Neither the secretion level of HBsAg nor the expression level of HBV mRNA in the culture medium showed a dose-dependent decrease. Therefore, AR101-DS2 was unable to prevent HBV entry into HuS-E / 2 cells.

[0124] 4. Inhibitory effect on intracellular sodium-taurocholic acid cotransport polypeptide (NTCP)

[0125] To evaluate the effect of inhibitors on inhibiting HBV infection, cell lysates were extracted from cells treated with AR101-DS3 or AR101-DS4. As the amount of AR101-DS3 increased (0 μM, 10 μM, 20 μM, and 100 μM; see Figure 18), the amount of NTCP increased in a dose-dependent manner from the pull-down assay.

[0126] Similarly, AR101-DS4 also caused a significant decrease in the amount of NTCP from the pull-down assay, as shown in Figure 19. These results indicate that inhibitors can inhibit hepatitis virus infection by inhibiting NTCP.

[0127] 5. Effects of inhibitory combinations on the HBV infectivity of HuS-E / 2 cells.

[0128] To evaluate whether combining multiple inhibitors improves the ability to inhibit HBV infectivity and replication, HuS-E / 2 cells were infected with any subtype of HBV derived from HepG2.2.15 cells. During 18 hours of HBV infection, AR101-DS1+AR101-DS3 or AR101-DS1+AR101-DS4 was added to the culture medium. The infected cells were then washed and incubated in fresh medium for 48 hours. HBsAg in the medium was detected by ELISA, and HBV mRNA was detected by real-time PCR, serving as indicators of the HBV infection efficiency of HuS-E / 2 cells.

[0129] The effects of AR101-DS1+AR101-DS3 on HBV entry into HuS-E / 2 cells are shown in Figures 20 and 21. We found that both HBsAg secretion levels and HBV mRNA expression levels in the culture medium were dose-dependently reduced. Therefore, AR101-DS1+AR101-DS3 was able to prevent HBV infection of HuS-E / 2 cells.

[0130] Similarly, the effects of AR101-DS1+AR101-DS4 on HBV entry into HuS-E / 2 cells are shown in Figures 22 and 23. We found that both HBsAg secretion levels and HBV mRNA expression levels in the culture medium were reduced in a dose-dependent manner. Therefore, AR101-DS1+AR101-DS4 was able to prevent HBV infection of HuS-E / 2 cells.

[0131] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided for illustrative purposes only and can be implemented in combination. Numerous modifications, alterations, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to embodiments of the present invention may be adopted when implementing this disclosure. The following claims define the scope of the present invention, and methods and structures within these claims, as well as their equivalents, are intended to be covered thereby. Furthermore, the present invention includes the following embodiments. [Aspect 1] A method for inhibiting a viral 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 Formula I: [ka] Ugonin J having the structure and its derivatives, Formula II: [ka] Ugonin N having the structure and its derivatives, Formula III:

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Claims

[Claim 1] A pharmaceutical composition for inhibiting cysteine ​​protease in SARS-CoV-2, comprising an effective amount of a compound or a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the compound is Formula VI: 【Chemistry 1】 Compounds having the structure, Formula VII: 【Chemistry 2】 Compounds having the structure, and Formula VIII: 【Transformation 3】 A pharmaceutical composition, which is one selected from the group consisting of compounds having the structure.

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