Methods and compositions for treating RNA virus-induced diseases

Abstract

The present invention provides methods and compositions for treating or lessening the symptoms of, or preventing, an RNA virus-induced disease in a subject by a cyclohexenone compound.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for treating or alleviating symptoms of, or preventing, an RNA virus-induced disease, and more particularly, to a method of administering a cyclohexenone compound. BACKGROUND

[0002] An RNA virus is a virus whose genetic material is RNA (ribonucleic acid). This nucleic acid is usually single-stranded RNA (ssRNA) but can be double-stranded RNA (dsRNA). Notable human diseases caused by RNA viruses include the common cold, influenza, SARS, MERS, COVID-19, dengue fever, hepatitis C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, and measles.

[0003] RNA virus-induced diseases, such as RNA virus-induced pneumonia, are a common cause of many deaths. There are approximately 450 million cases of pneumonia each year. Of these cases, viral pneumonia accounts for about 200 million cases, which includes about 100 million children and 100 million adults. Viral pneumonia is pneumonia caused by a virus. Pneumonia is an infection that causes inflammation in one or both lungs. The air sacs fill with fluid or pus, making it hard to breathe.

[0004] Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans, these viruses cause respiratory tract infections that can range from mild to lethal. Mild conditions include some cases of the common cold (which is also caused by some other viruses, mainly rhinoviruses), while more lethal variants are able to cause SARS, MERS, and COVID-19. SUMMARY

[0005] In one aspect, provided herein is a method for treating or alleviating symptoms of, and / or preventing, an RNA virus-induced disease, such as RNA virus-induced pneumonia, in a subject, comprising administering to the subject a therapeutically effective amount of a cyclohexenone compound having the structure:

[0006]

[0007] wherein each of X and Y is independently oxygen, NR5, or sulfur;

[0008] R is hydrogen or C(=O)C1-C8alkyl;

[0009] each of R1, R2, and R3is independently hydrogen, optionally substituted methyl, or (CH2) m -CH3;

[0010] R4is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0011] halogen, 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, glucosyl, wherein the 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from:

[0012] NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0013] C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8halo

[0014] alkyl;

[0015] each of R5and R6is independently hydrogen or C1-C8alkyl;

[0016] R7is C1-C8alkyl, OR5, or NR5R6;

[0017] m = 1-12; and

[0018] n = 1-12; or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof.

[0019] Incorporated by Reference

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF DRAWINGS

[0021] The novel features of the application are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative

[0022] Figures 1A / B Results from a study showing exemplary Compound 1 reduced HBeAg (1A) and HBsAg (1B) expression levels are shown.

[0023] Figures 2A / B Results from a study showing exemplary Compound 1 reduced HBV NDA expression levels (2A) and HCV RNA activity (2B) are shown.

[0024] Figure 3 The potential clinical progression of SARS-CoV-2 is illustrated.

[0025] Figure 4 The various pathways of antiviral, anti-inflammatory, and anti-fibrotic of exemplary Compound 1 are provided.

[0026] Figure 5 The results of Nrf-2 nuclear translocation studies of exemplary Compound 1 compared to silymarin are provided.

[0027] Figure 6 The results of oxidative stress studies of exemplary Compound 1 are provided.

[0028] Figure 7 provides the results of kidney inflammation studies of the NF-kB activation model of exemplary Compound 1.

[0029] Figure 8 provides the results of local kidney inflammation studies of exemplary Compound 1 in the context of MCP-1, IL-6, and CD3 markers.

[0030] Figures 9A / B The results of anti-fibrotic activity of exemplary Compound 1 by TGF-β1 inhibition (9A) and fibrosis-related proteins (9B) are provided.

[0031] Figure 10 The results of SARS inhibition by exemplary Compound 1 are provided.

[0032] Figure 11 The results of cell culture studies of exemplary Compound 1 compared to a control group (DMSO only) are provided.

[0033] Figures 12A-C The gene expression levels of CXCL10 (12A), IL6 (12B), and IL18 (12C) are provided, respectively.

[0034] Figures 13A-B provide the gene expression levels of TGFB1 (13A) and COL4A1 (13B), respectively. DETAILED DESCRIPTION

[0035] While a number of therapeutic agents have been developed for treating coronavirus-induced diseases such as SARS and MERS, the drugs developed to date have not found significant effects.

[0036] In some embodiments, the cyclohexenone compounds are obtained from extracts of natural products or prepared by synthesis or semi-synthesis. In some embodiments, the present application provides the therapeutic and prophylactic potential of exemplary cyclohexenone compounds (e.g., Compound 1) for treating or alleviating symptoms of or preventing an RNA virus-induced disease in a subject.

[0037] In some embodiments, there is provided a method for treating or alleviating symptoms of an RNA virus-induced disease, such as an RNA virus-induced pneumonia, and / or preventing an RNA virus-induced disease, such as an RNA virus-induced pneumonia, in a subject, comprising administering to said subject a therapeutically effective amount of a cyclohexenone compound having the structure:

[0038]

[0039] wherein each of X and Y is independently oxygen, NR5, or sulfur;

[0040] R is hydrogen or C(=O)C1-C8alkyl;

[0041] each of R1, R2, and R3is independently hydrogen, optionally substituted methyl, or

[0042] (CH2) m -CH3;

[0043] R4is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0044] halogen, 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, glucosyl, wherein the 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from the group consisting of NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl;

[0045] each of R5and R6is independently hydrogen or C1-C8alkyl;

[0046] R7is C1-C8alkyl, OR5, or NR5R6;

[0047] m = 1-12; and

[0048] n = 1-12; or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof.

[0049] In some embodiments, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a cyclohexenone compound having the structure:

[0050] wherein each of X and Y is independently oxygen, NR5, or sulfur;

[0051] R is hydrogen or C(=O)C1-C8alkyl;

[0052] each of R1, R2, and R3is independently hydrogen, optionally substituted methyl, or

[0053] (CH2) m -CH3;

[0054] R4is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0055] halogen, 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, glucosyl, wherein the 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from:

[0056] NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8

[0057] alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl;

[0058] each of R5and R6is independently hydrogen or C1-C8alkyl;

[0059] R7is C1-C8alkyl, OR5, or NR5R6;

[0060] m = 1-12; and

[0061] n = 1-12; or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof; for use in treating or reducing symptoms of an RNA virus-induced disease (such as a virus-induced pneumonia) in a subject and / or preventing an RNA virus-induced disease (such as a virus-induced pneumonia) in a subject.

[0062] In some embodiments, there is provided use of a therapeutically effective amount of a cyclohexenone compound having the structure or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof, in the manufacture of a medicament for treating or reducing symptoms of an RNA virus-induced disease (such as an RNA virus-induced pneumonia) in a subject and / or preventing an RNA virus-induced disease (such as an RNA virus-induced pneumonia) in a subject,

[0063] wherein each of X and Y is independently oxygen, NR5, or sulfur;

[0064] R is hydrogen or C(=O)C1-C8alkyl;

[0065] each of R1, R2, and R3is independently hydrogen, optionally substituted methyl, or (CH2) m -CH3;

[0066] R4is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0067] halogen, 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, glucosyl, wherein the 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl,

[0068] C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from the group consisting of NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5,

[0069] C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl

[0070] and C1-C8haloalkyl;

[0071] each of R5and R6is independently hydrogen or C1-C8alkyl;

[0072] R7is C1-C8alkyl, OR5, or NR5R6;

[0073] m = 1-12; and n = 1-12.

[0074] In some embodiments, the RNA virus-induced disease is an RNA virus-induced pneumonia, a coronavirus-induced pneumonia, or a SARS-CoV-2-induced pneumonia, among others. In certain embodiments, the RNA virus is a coronavirus. In some embodiments, the RNA virus-induced disease is caused or induced by a Coronaviridae infection. In some embodiments, in certain embodiments, the Coronaviridae infection is caused by or associated with an alphacoronavirus 229E (HCoV-229E), NL63 (HCoV-NL63, New Haven coronavirus), betacoronavirus OC43 (HCoV-OC43), HKU1, MERS-CoV (coronavirus that causes Middle East respiratory syndrome), SARS-CoV (coronavirus that causes severe acute respiratory syndrome), or SARS-CoV-2 (coronavirus that causes severe acute respiratory system syndrome, formerly known as the novel coronavirus, or 2019-nCoV), among others. In certain embodiments, the Coronaviridae infection is caused by or associated with severe acute respiratory system syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the RNA virus-induced disease is an RNA virus-induced pneumonia. In certain embodiments, the Coronaviridae infection is caused by or associated with severe acute respiratory system syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the cyclohexenone compound reduces RNA virus concentration or prevents RNA virus replication. In certain embodiments, the cyclohexenone compound reduces RNA virus concentration or prevents RNA virus replication of an alphacoronavirus 229E (HCoV-229E), NL63 (HCoV-NL63, New Haven coronavirus), betacoronavirus OC43 (HCoV-OC43), HKU1, MERS-CoV (coronavirus that causes Middle East respiratory syndrome), SARS-CoV (coronavirus that causes severe acute respiratory syndrome), or SARS-CoV-2 (coronavirus that causes severe acute respiratory system syndrome, formerly known as the novel coronavirus, or 2019-nCoV), among others. In some embodiments, the subject is a human.

[0075] In some embodiments, a method for treating, inhibiting, and / or preventing a coronavirus-induced pneumonia in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a cyclohexenone compound of the following formula (I).

[0076] In some embodiments, a method for treating, inhibiting, and / or preventing RNA virus replication (e.g., coronavirus replication) in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a cyclohexenone compound disclosed herein.

[0077] In some embodiments, a method for reducing the concentration of an RNA virus in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a cyclohexenone compound disclosed herein.

[0078] In some embodiments, a method for inhibiting and / or preventing an RNA virus infection in a subject in need thereof is provided, the method comprising administering to the subject an effective amount of a cyclohexenone compound disclosed herein.

[0079] In some embodiments, a cyclohexenone compound having the following structure is prepared from any suitable starting material by synthesis or semi-synthesis, In other embodiments, cyclohexenone compounds are prepared by fermentation or the like. For example, compounds 1 and 3-7 are isolated from organic solvent extracts. Non-limiting exemplary compounds are illustrated below.

[0080]

[0081]

[0082]

[0083] In other embodiments, a cyclohexenone compound having the following structure is isolated from an organic solvent extract of Antrodia cinnamomea In some embodiments, the organic solvent is selected from alcohols (e.g., methanol, ethanol, propanol, etc.), esters (e.g., methyl acetate, ethyl acetate, etc.), alkanes (e.g., pentane, hexane, heptane, etc.), haloalkanes (e.g., chloromethane, chloroethane, chloroform, dichloromethane, etc.), and the like. For example, exemplary compounds 1-7 are isolated from organic solvent extracts. In certain embodiments, the organic solvent is an alcohol. In certain embodiments, the alcohol is ethanol. In some embodiments, the cyclohexenone compounds are isolated from aqueous extracts of Antrodia cinnamomea. In certain embodiments, the cyclohexenone compounds disclosed herein are prepared by synthesis or semi-synthesis.

[0084] In some embodiments, each of X and Y is independently oxygen or sulfur. It is known in the art that compounds in which each of X and Y is independently sulfur can be prepared by similar or identical routes as compounds in which each of X and Y is independently oxygen, as oxygen and sulfur have similar chemical properties in structure. In some embodiments, compounds in which each of X and Y is independently NR5 can be prepared by similar routes as compounds in which each of X and Y is independently oxygen or sulfur, by way of appropriate protecting groups.

[0085] In some embodiments, R is hydrogen, C(=O)C3H8, C(=O)C2H5, or C(=O)CH3. In some embodiments, R1is hydrogen, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In certain embodiments, R1is hydrogen or methyl. In some embodiments, R2is hydrogen, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In certain embodiments, R2is hydrogen or methyl. In some embodiments, R3is hydrogen, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R4is halogen, NH2, NHCH3, N(CH3)2, OCH3, OC2H5, C(=O)CH3, C(=O)C2H5, C(=O)OCH3, C(=O)OC2H5, C(=O)NHCH3, C(=O)NHC2H5, C(=O)NH2, OC(=O)CH3, OC(=O)C2H5, OC(=O)OCH3, OC(=O)OC2H5, OC(=O)NHCH3, OC(=O)NHC2H5, or OC(=O)NH2. In some embodiments, R4is C2H5C(CH3)2OH, C2H5C(CH3)2OCH3, CH2COOH, C2H5COOH, CH2OH, C2H5OH, CH2Ph, C2H5Ph, CH2CH=C(CH3)(CHO), CH2CH=C(CH3)(C(=O)CH3), 5- or 6-membered lactone, C2-C8alkenyl, C2-C8alkynyl, aryl, or glucosyl, wherein the 5- or 6-membered lactone 、 C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from the group consisting of NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl. In certain embodiments, R4is 5- or 6-membered lactone, C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl optionally substituted with one or more substituents selected from the group consisting of NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl. In certain embodiments, R4is CH2CH=C(CH3)2. In certain embodiments, the compound is Certain Pharmaceutical and Medical Terms

[0086] Unless otherwise stated, the following terms used in this application (including the specification and claims) have the definitions given below. It must be noted that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” as used in the specification and appended claims include a plural referent. Unless otherwise stated, conventional methods such as mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology are employed. In this application, “or” or “and” is used to mean “and / or” unless otherwise stated. Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive. Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0087] The "alkyl" group refers to an aliphatic hydrocarbon group. An alkyl group can be a saturated alkyl group (meaning it does not contain any carbon-carbon double or triple bonds) or an unsaturated alkyl group (meaning it contains at least one carbon-carbon double or triple bond). The alkyl moiety, whether saturated or unsaturated, can be branched or straight-chain.

[0088] The "alkyl" group can have 1 to 12 carbon atoms (wherever it appears herein, the numerical range, such as "1 to 12," refers to each integer within a given range; for example, "1 to 12 carbon atoms" means that an alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to a maximum of 12 carbon atoms, but this definition also covers the occurrence of the term "alkyl" where no numerical range is specified). The alkyl group of the compounds described herein may be specified as "C1-C8 alkyl" or similar designations. By way of example only, "C1-C8 alkyl" means that the alkyl chain has 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. In one aspect, the alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl, allyl, but-2-enyl, but-3-enyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. In one aspect, the alkyl group is a C1-C8 alkyl group.

[0089] The term "alkylene" refers to a divalent alkyl group. Any monovalent alkyl group can become an alkylene group by abstracting a second hydrogen atom from the alkyl group. In one respect, alkylene groups are C1-C... 12Alkylene. In another aspect, alkylene is Ci-C8alkylene. Typical alkylene groups include, but are not limited to, -CH2-, -CH(CH3)-, -C(CH3)2-, -CH2CH2-, -CH2CH(CH3)-, -CH2C(CH3)2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2(CH2)3CH2-, and the like.

[0090] As used herein, the term "aryl" refers to an aromatic ring in which each atom forming the ring is a carbon atom. The aryl ring is formed of 5, 6, 7, 8, 9, or more than 9 carbon atoms. The aryl group is optionally substituted. In one aspect, the aryl group is phenyl or naphthyl. In one aspect, the aryl group is phenyl. In one aspect, the aryl group is C6-C 10 Aryl. Depending on the structure, the aryl group can be a monovalent group or a divalent group (i.e., an arylene group). In one aspect, the arylene group is C6-C 10 Arylene. Exemplary arylene groups include, but are not limited to, phenyl-1,2- ene, phenyl-1,3-ene, and phenyl-1,4-ene.

[0091] The term "arene" refers to a planar ring having a delocalized pi-electron system containing 4n+2 pi-electrons, where n is an integer. The aromatic ring can be formed of 5, 6, 7, 8, 9, 10, or more than 10 atoms. The arene is optionally substituted. The term "arene" includes carbocyclic aryl ("aryl," e.g., phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroarene") groups (e.g., pyridine). The term includes single ring or fused ring polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) groups.

[0092] The term "halo" or "halogen" or "halide" refers to fluorine, chlorine, bromine, or iodine.

[0093] The term "lactone" refers to a cyclic ester, which can be viewed as a condensation product of an alcohol group -OH and a carboxylic acid group -COOH in the same molecule. It is characterized by a closed ring composed of two or more carbon atoms and a single oxygen atom, with a keto group =0 in one of the carbons adjacent to the other oxygen.

[0094] The term "heterocycle" or "heterocyclyl" refers to heteroaromatic rings (also referred to as heteroaryl groups) and heterocycloalkyl rings (also referred to as heteroaliphatic groups) containing from 1 to 4 heteroatoms in the ring, wherein each heteroatom in the ring is selected from O, S, and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system, and provided that any ring does not contain two adjacent O or S atoms. Non-aromatic heterocyclyl groups (also referred to as heterocycloalkyl groups) include groups having only 3 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. Heterocyclic groups include benzo-fused ring systems. An example of a 3-membered heterocyclic group is aziridinyl. An example of a 4-membered heterocyclic group is azetidinyl. An example of a 5-membered heterocyclic group is thiazolyl. An example of a 6-membered heterocyclic group is pyridinyl, and an example of a 10-membered heterocyclic group is quinolinyl. Examples of non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, oxazolidinonyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, pyrrolin-2-yl, pyrrolin-3-yl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl, and quinolinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furanyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. If possible, the above groups can be C-linked or N-linked. For example, a group derived from pyrrole can be pyrrol-1-yl (N-linked) or pyrrol-3-yl (C-linked).Furthermore, the groups derived from imidazole can be imidazole-1-yl or imidazole-3-yl (both N-linked) or imidazole-2-yl, imidazole-4-yl or imidazole-5-yl (all C-linked). Heterocyclic groups include benzo[a]-fused ring systems. Non-aromatic heterocycles can be substituted with one or two oxo (=O) moieties, for example, pyrrolidone-2-one.

[0095] As used herein, the term "alkenyl" refers to a straight-chain, branched, or cyclic (in this case, also referred to as "cycloalkenyl") hydrocarbon containing 2-10 carbons and at least one carbon-carbon double chain formed by the removal of two hydrogens. In some embodiments, depending on the structure, the alkenyl group is a monovalent or divalent group (i.e., an alkenylene). In some embodiments, the alkenyl group is optionally substituted. Illustrative examples of alkenyl groups include, but are not limited to, vinyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl (cecenyl).

[0096] As used herein, the term "alkynyl" refers to a straight-chain, branched, or cyclic (in this case, also referred to as "cycloalkynyl") hydrocarbon containing 2-10 carbons and at least one carbon-carbon triple chain formed by the removal of four hydrogens. In some embodiments, depending on the structure, the alkynyl group is a monovalent or divalent group (i.e., an alkynylene). In some embodiments, the alkynyl group is optionally substituted. Illustrative examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentyynyl, hexynyl, heptyynyl, and so on.

[0097] As used herein, the term "alkoxy" refers to an alkyl group as defined herein, which is attached to a portion of the parent molecule via an oxygen atom. Illustrative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentoxy, and hexoxy.

[0098] As used herein, the term "cycloalkyl" refers to a monocyclic or polycyclic group containing only carbon and hydrogen, including those that are saturated, partially unsaturated, or fully unsaturated. Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative examples of cyclic groups include, but are not limited to, the following:

[0099]

[0100] In some implementations, depending on the structure, the cycloalkyl group is a monovalent or divalent group (i.e., a cycloalkylene group).

[0101] The terms "haloalkyl," "haloalkenyl," "haloalkynyl," and "haloalkoxy" as used herein include alkyl, alkenyl, alkynyl, and alkoxy structures in which at least one hydrogen is replaced with a halogen atom. In certain embodiments in which two or more hydrogen atoms are replaced with halogen atoms, the halogen atoms are all identical to one another. In other embodiments in which two or more hydrogen atoms are replaced with halogen atoms, the halogen atoms are not all identical to one another. The terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy, respectively, in which the halogen is fluorine. In certain embodiments, the haloalkyl is optionally substituted.

[0102] The term "glucosyl" as used herein includes D- or L-form glucosyl groups, in which the glucosyl group is attached through any of the hydroxyl groups on the glucose ring.

[0103] The term "acceptable" with respect to a formulation, composition, or ingredient as used herein means having no persistent detrimental effects on the general health of the subject being treated.

[0104] Antrodia is a genus of fungi in the family Antrodiaiaceae. The fruiting bodies of Antrodia species are usually flat or spread out on the growth surface with the hymenium exposed on the outside; the edges can turn up to form narrow brackets. Most species occur in temperate and boreal forests and cause brown-rot.

[0105] Antrodia cinnamomea, also known as the stout camphor fungus or Taiwan lingzhi, is a fungus endemic to Taiwan that only grows on the endemic camphor tree, causing brown heart rot. This unique mushroom of Taiwan has been used as a traditional medicine to protect against different disease conditions.

[0106] It is known in the art that the active ingredients isolated from different parts of Antrodia cinnamomea vary depending on the culture medium and method. For example, certain cyclohexenone compounds disclosed herein can only be isolated from a unique solid-state fermentation process used to culture Antrodia cinnamomea, which is different from other known methods.

[0107] The term "carrier" as used herein refers to a relatively nontoxic chemical compound or agent that facilitates the incorporation of a compound into a cell or tissue.

[0108] The terms "co-administration" and the like as used herein refer to the inclusion of selected therapeutic agents in treatment protocols that are administered to a single patient and are intended to encompass administration of the agents by the same or different routes of administration or at the same or different times.

[0109] The term "diluent" refers to a compound used to dilute a compound of interest prior to delivery. Diluents can also be used to stabilize a compound as they can provide a more stable environment. Salts dissolved in buffered solutions, which can also provide pH control or maintenance, are used as diluents in the art, including but not limited to phosphate buffered saline solutions.

[0110] The term "effective amount" or "therapeutically effective amount" as used herein refers to a sufficient amount of a pharmaceutical agent or compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of a sign, a symptom, or a cause of disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of a composition comprising a compound as disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate "effective" amount in any individual case can be determined using techniques, such as a dose escalation study.

[0111] The term "enhance" or "enhancing" as used herein refers to increasing or prolonging the desired effect in potency or duration. Thus, with respect to enhancing the effect of a therapeutic agent, the term "enhance" refers to the ability to increase or prolong the effect of another therapeutic agent on a system in potency or duration. An "enhancing effective amount" as used herein refers to an amount sufficient to enhance the effect of another therapeutic agent in a desired system.

[0112] A "metabolite" of a compound disclosed herein is a derivative of the compound formed when the compound is metabolized. The term "active metabolite" refers to a biologically active derivative of a compound formed when the compound is metabolized. The term "metabolism" as used herein refers to the sum of the processes (including but not limited to hydrolysis reactions and reactions catalyzed by enzymes) by which an organism alters a particular substance. Thus, enzymes can make specific structural changes to a compound. For example, cytochrome P450 catalyzes a variety of oxidation and reduction reactions, while uridine diphosphate glucuronyl transferases catalyze the transfer of an activated glucuronic acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines, and free thiols. Metabolites of the compounds disclosed herein are optionally identified by administering a compound to a host and analyzing tissue samples from the host, or by incubating a compound with hepatocytes in vitro and analyzing the resulting compounds.

[0113] The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients (e.g., a compound (i.e., a cyclohexenone compound described herein)) and adjunct are both administered to the patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients (e.g., a compound (i.e., a cyclohexenone compound described herein)) and adjunct are administered to the patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limitations, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapies, that is, the administration of three or more active ingredients.

[0114] The term "pharmaceutical composition" means a mixture of a compound (i.e., a cyclohexenone compound described herein) with other chemical components such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

[0115] The term "subject" or "patient" includes mammals and birds. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates (such as chimpanzees and other apes and monkey species); farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one embodiment, the mammal is a human.

[0116] The terms "treat," "treating," or "treatment" as used herein include alleviating, reducing or abating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and / or therapeutically. In particular, the terms "treat," "treatment," or "treating" refer to reducing the frequency, degree, severity, and / or duration of symptoms experienced by a subject (e.g., a patient) of a coronavirus-induced disease.

[0117] The terms "prevent," "prevention," or "preventing" refer to inhibiting, reducing the risk of, reducing the onset of, or avoiding a symptom associated with a coronavirus-induced disease.

[0118] Routes of administration and dosage

[0119] Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transmucosal, transdermal, vaginal, aural, nasal, and topical administration. Moreover, parenteral delivery includes, by way of example only, intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.

[0120] In certain embodiments, the compounds as described herein are administered in a local rather than systemic manner, for example, via injection directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long acting formulations are administered by implantation (for example, subcutaneous or intramuscular implantation) or by intramuscular injection. Furthermore, in other embodiments, the drug is delivered in a targeted drug delivery system (for example, in a liposome coated with organ-specific antibody). In such embodiments, the liposome targets the organ and is selectively taken up by the organ. In yet other embodiments, the compounds as described herein are provided in a fast release formulation, in an extended release formulation, or in an intermediate release formulation. In yet other embodiments, the compounds described herein are administered topically.

[0121] In some embodiments, the cyclohexenone compound or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof is administered parenterally or intravenously. In other embodiments, the cyclohexenone compound or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof is administered by injection. In some embodiments, the cyclohexenone compound or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof is administered orally.

[0122] In the event that the patient's condition does not improve, the administration of the compound can be extended for prolonged periods of time (including for the duration of the patient's life), according to the judgment of the physician, to improve or otherwise control or limit the symptoms of the patient's disease or condition. In the event that the patient's status improves, the administration of the compound can be continued or temporarily suspended for a period of time (i.e., a "drug holiday"), according to the judgment of the physician.

[0123] The above-mentioned ranges are merely suggested, as there is a large number of variables with respect to individual treatment regimens, and considerable departures from these recommended values are often necessary depending on the needs of a particular patient, the severity of the disease or condition being treated, and the physician's judgment. The dosage can be altered on the basis of various factors, including but not limited to the activity of the compound used, the disease or condition being treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practicing physician.

[0124] Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determining the LD50 (50% of the population) and ED 50 (50% of the population). The ratio of the dose between the toxic and therapeutic effects is the therapeutic index and is expressed as the ratio LD 50 and ED 50 between them. Compounds exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0125] It is understood that in some embodiments, the dosage regimen for treating, preventing, or ameliorating the condition for which relief is sought is modified according to various factors. These factors include the disorder suffered by the subject, and the age, body weight, sex, diet, and medical condition of the subject. Thus, in other embodiments, the dosage regimen actually employed varies widely, and therefore deviates from the dosage regimens set forth herein.

[0126] Pharmaceutical Formulations

[0127] Some embodiments provide a pharmaceutical composition comprising: a therapeutically effective amount of a cyclohexenone compound having the structure:

[0128] wherein each of X and Y is independently oxygen, NR5, or sulfur;

[0129] R is hydrogen or C(=O)C1-C8alkyl;

[0130] each of R1, R2, and R3is independently hydrogen, optionally substituted methyl, or

[0131] (CH2) m -CH3;

[0132] R4is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0133] halogen, 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, glucosyl, wherein the 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from:

[0134] NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8

[0135] alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl;

[0136] each of R5and R6is independently hydrogen or C1-C8alkyl;

[0137] R7is C1-C8alkyl, OR5, or NR5R6;

[0138] m = 1-12; and n = 1-12; or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof; and a pharmaceutically acceptable excipient.

[0139] In some embodiments, the cyclohexenone compound of the pharmaceutical composition has the following structure:

[0140]

[0141] wherein each of X and Y is independently oxygen, NR5, or sulfur;

[0142] R is hydrogen or C(=O)C1-C8alkyl;

[0143] each of R1, R2, and R3is independently hydrogen, optionally substituted methyl, or (CH2) m -CH3;

[0144] R4is NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0145] halogen, 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, glucosyl, wherein the 5- or 6-membered lactone, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from:

[0146] NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6,

[0147] C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8halo

[0148] alkyl;

[0149] each of R5and R6is independently hydrogen or C1-C8alkyl;

[0150] R7is C1-C8alkyl, OR5, or NR5R6;

[0151] m = 1-12; and n = 1-12; or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof.

[0152] In some embodiments, R is hydrogen, C(=O)C3H8, C(=O)C2H5, or C(=O)CH3. In some embodiments, each of R1, R2, and R3is independently hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl. In certain embodiments, R1is hydrogen or methyl. In certain embodiments, R2is hydrogen, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In certain embodiments, R3is hydrogen, methyl, ethyl, propyl, butyl, pentyl, or hexyl. In some embodiments, R4is halogen, NH2, NHCH3, N(CH3)2, OCH3, OC2H5, C(=O)CH3, C(=O)C2H5, C(=O)OCH3, C(=O)OC2H5, C(=O)NHCH3, C(=O)NHC2H5, C(=O)NH2, OC(=O)CH3, OC(=O)C2H5, OC(=O)OCH3, OC(=O)OC2H5, OC(=O)NHCH3, OC(=O)NHC2H5, or OC(=O)NH2. In certain embodiments, R4is C2H5C(CH3)2OH, C2H5C(CH3)2OCH3, CH2COOH, C2H5COOH, CH2OH, C2H5OH, CH2Ph, C2H5Ph, CH2CH=C(CH3)(CHO), CH2CH=C(CH3)(C(=O)CH3), 5- or 6-membered lactone, aryl, or glucosyl, wherein the 5- or 6-membered lactone, aryl, and glucosyl are optionally substituted with one or more substituents selected from the group consisting of NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl. In certain embodiments, R4is CH2COOH, C2H5COOH, CH2OH, C2H5OH, CH2Ph, C2H5Ph, CH2CH=C(CH3)(CHO), CH2CH=C(CH3)(C(=O)CH3), 5- or 6-membered lactone, aryl, or glucosyl, wherein the 5- or 6-membered lactone, aryl, and glucosyl are optionally substituted with one or more substituents selected from the group consisting of NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl.In certain embodiments, R4is a 5 or 6 membered lactone optionally substituted with one or more substituents selected from NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl.

[0153] In certain embodiments, the compound is selected from:

[0154]

[0155]

[0156] In certain embodiments, the compound is selected from:

[0157]

[0158]

[0159] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. In particular embodiments, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the chosen route of administration. Any pharmaceutically acceptable techniques, carriers, and excipients are suitable for formulating the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0160] Provided herein are pharmaceutical compositions comprising a compound (i.e., a cyclohexenone compound described herein) and a pharmaceutically acceptable diluent, excipient, or carrier. In certain embodiments, as in combination therapy, the described compounds are administered as pharmaceutical compositions in which the compound (i.e., a cyclohexenone compound described herein) is mixed with other active ingredients. All combinations of active agents set forth in the Combination Therapy section below and throughout the present disclosure are contemplated herein. In specific embodiments, the pharmaceutical compositions include one or more compounds (i.e., a cyclohexenone compound described herein).

[0161] The term "pharmaceutical composition" as used herein refers to a mixture of a compound (i.e., a cyclohexenone compound described herein) with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. In certain embodiments, a pharmaceutical composition facilitates administration of the compound to an organism. In some embodiments, in practicing the therapeutic methods or uses provided herein, a therapeutically effective amount of a compound (i.e., a cyclohexenone compound described herein) is administered to a mammal suffering from a disease or condition to be treated in the form of a pharmaceutical composition. In particular embodiments, the mammal is a human. In certain embodiments, the therapeutically effective amount varies depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. The compounds described herein are used either alone or as a component of mixtures in combination with one or more therapeutic agents.

[0162] In an embodiment, a compound (i.e., a cyclohexenone compound described herein) is formulated into an aqueous solution. In particular embodiments, the aqueous solution is selected from, by way of example only, a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. In other embodiments, a compound (i.e., a cyclohexenone compound described herein) is formulated for transmucosal administration. In particular embodiments, transmucosal formulations include penetrants suitable for the barrier to be permeated. In yet other embodiments in which a compound described herein is formulated for other parenteral injection, suitable formulations include aqueous or non-aqueous solutions. In particular embodiments, such solutions contain physiologically compatible buffers and / or excipients.

[0163] In another embodiment, a compound described herein is formulated for oral administration. A compound described herein, including a compound (i.e., a cyclohexenone compound described herein), is formulated by combining the active compound with, for example, a pharmaceutically acceptable carrier or excipient. In various embodiments, a compound described herein is formulated into oral dosage forms including, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

[0164] In certain embodiments, pharmaceutical formulations for oral use are obtained by mixing one or more solid excipient with one or more of the compounds described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or sugar-coated tablets. Suitable excipients, in particular fillers, are, for example, sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, microcrystalline cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose; or others, such as, for example, polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. In particular embodiments, disintegrating agents are optionally added. Disintegrating agents include, by way of example only, the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0165] In an embodiment, the dosage forms such as sugar-coated tablet cores and tablets have one or more suitable coatings. In particular embodiments, a concentrated sugar solution is used to coat the dosage forms. The sugar solution optionally contains additional components such as, by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbomer gels, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes and / or pigments are optionally added to the coatings for identification purposes. Furthermore, dyes and / or pigments are optionally used to distinguish different combinations of active compound doses.

[0166] In certain embodiments, therapeutically effective amounts of at least one of the compounds described herein are formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In particular embodiments, the push-fit capsules contain the active ingredients in admixture with one or more filler. Fillers include, by way of example only, lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and optional stabilizers. In other embodiments, the soft capsules contain one or more active compounds dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers are optionally added.

[0167] In other embodiments, a therapeutically effective amount of at least one compound described herein is formulated for buccal or sublingual administration. Formulations suitable for buccal or sublingual administration include, only as examples, tablets, lozenges, or gels. In yet other embodiments, a compound described herein is formulated for parenteral injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, formulations for injection are in unit dosage form (e.g., in ampoules) or in multi-dose containers. Preservatives are, optionally, added to the injection formulations. In yet other embodiments, a pharmaceutical composition of a compound (i.e., a cyclohexenone compound described herein) is formulated in a form suitable for parenteral injection as a sterile suspension, solution or emulsion in an oily or aqueous vehicle. The parenteral injection formulations optionally contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In specific embodiments, pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In additional embodiments, suspensions of the active compounds are prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles for pharmaceutical compositions described herein include, only as examples, fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate or triglycerides, or liposomes. In certain specific embodiments, aqueous injection suspensions contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension includes suitable stabilizers or agents which increase the solubility of the compounds to allow for a

[0168] In one aspect, a compound (i.e., a cyclohexenone compound described herein) is prepared as a solution for parenteral injection as described herein or known in the art and administered with an auto-injector. Auto-injectors, such as those disclosed in U.S. Patent Nos. 4,031,893; 5,358,489; 5,540,664; 5,665,071; 5,695,472; and WO / 2005 / 087297 (each of which is incorporated herein by reference), are known. Generally, all auto-injectors contain a volume of solution that includes a compound (i.e., a cyclohexenone compound described herein) to be injected. Typically, an auto-injector includes a reservoir for holding the solution in fluid communication with a needle for delivering the drug, and a mechanism for automatically deploying the needle, inserting the needle into a patient, and delivering the dose into the patient. Exemplary injectors provide about 0.3 mL, 0.6 mL, 1.0 mL, or other suitable volume of solution at a concentration of about 0.5 mg to 50 mg of compound (i.e., a cyclohexenone compound described herein) per 1 mL of solution. Each injector can only deliver one dose of the compound.

[0169] In yet other embodiments, the compounds (i.e., the cyclohexenone compounds described herein) are administered topically. The compounds described herein are formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, sticks, balms, creams, or ointments. Such pharmaceutical compositions optionally include solubilizing agents, stabilizing agents, tonicity enhancing agents, buffering agents, and preservatives.

[0170] In yet other embodiments, the compounds (i.e., the cyclohexenone compounds described herein) are formulated for transdermal administration. In particular embodiments, transdermal formulations employ transdermal delivery devices and transdermal delivery patches and can be either lipophilic emulsions or buffered aqueous solutions dissolved and / or dispersed in a polymer or adhesive. In various embodiments, such patches are configured for continuous, pulsatile, or on demand delivery of a medicament. In further embodiments, transdermal delivery of the compounds (i.e., the cyclohexenone compounds described herein) is achieved by iontophoresis patches and the like. In certain embodiments, transdermal patches provide controlled delivery of the compounds (i.e., the cyclohexenone compounds described herein). In particular embodiments, the rate of absorption is slowed by the use of rate-controlling membranes or by trapping the compounds within a polymer matrix or gel. In alternative embodiments, absorption enhancers are used to increase absorption. Absorption enhancers or vehicles include absorbable, pharmaceutically acceptable solvents that aid passage through the skin. For example, in one embodiment, the transdermal device is in the form of a bandage that includes a backing member; a reservoir containing the compounds, optionally with a vehicle; an optional rate-controlling barrier for delivering the compounds to the skin of the host at a controlled and predetermined rate over an extended period of time; and means for securing the device to the skin.

[0171] Various devices already described in the art can be used to administer the transdermal formulations described herein. For example, such devices include, but are not limited to, U.S. Patent Nos. 3,598,122; 3,598,123; 3,710,795; 3,731,683; 3,742,951; 3,814,097; 3,921,636; 3,972,995; 3,993,072; 3,993,073; 3,996,934; 4,031,894; 4,060,084; 4,069,307; 4,077,407; 4,201,211; 4,230,105; 4,292,299; 4,292,303; 5,336,168; 5,665,378; 5,837,280; 5,869,090; 6,923,983; 6,929,801; and 6,946,144.

[0172] The transdermal dosage forms described herein can include certain pharmaceutically acceptable excipients that are routine in the art. In one embodiment, the transdermal formulations described herein include at least three components: (1) a formulation of a compound (i.e., a cyclohexenone compound described herein); (2) a penetration enhancer; (3) an aqueous adjuvant. In addition, the transdermal formulations can include additional components such as, but not limited to, gelling agents, cream and ointment bases, and the like. In some embodiments, the transdermal formulations further include a woven or non-woven backing material to enhance absorption and prevent removal of the transdermal formulation from the skin. In other embodiments, the transdermal formulations described herein are maintained in a saturated or supersaturated state to promote diffusion into the skin.

[0173] In other embodiments, the compound (i.e., a cyclohexenone compound described herein) is formulated for administration by inhalation. Various forms suitable for administration by inhalation include, but are not limited to, aerosols, mists, or powders. The pharmaceutical compositions of the compound (i.e., a cyclohexenone compound described herein) are conveniently in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In particular embodiments, the dosage unit for a pressurized aerosol is determined by providing a valve to deliver a metered amount. In certain embodiments, for example, gelatin capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated to contain a powder mix of the compound and a suitable powder base such as, for example, lactose or starch.

[0174] Intranasal formulations are known in the art and are described, for example, in U.S. Patent Nos. 4,476,116; 5,116,817; and 6,391,452, each of which is specifically incorporated herein by reference. Formulations including a compound (i.e., a cyclohexenone compound described herein) are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents known in the art, according to these and other techniques known in the art. See, e.g., Ansel, H.C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably, these compositions and formulations are prepared with suitable nontoxic pharmaceutically-acceptable ingredients. These ingredients are found in standard references such as Remington: The Science and Practice of Pharmacy, 21st edition, 2005. The choice of suitable carriers depends highly on the exact nature of the nasal dosage form desired (e.g., solution, suspension, ointment, or gel). In addition to the active ingredient, nasal dosage forms typically contain a large amount of water. Small amounts of other ingredients can also be present, such as pH adjusters, emulsifiers or dispersants, preservatives, surfactants, gelling agents, or buffers and other stabilizers and solubilizers. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[0175] For inhalation administration, the compounds described herein can be in the form of an aerosol, mist, or powder. The pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example only, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of a compound described herein and a suitable powder base such as lactose or starch.

[0176] In other embodiments, the compounds (i.e., cyclohexenone compounds described herein) are formulated into rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository wages such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In the suppository form of the composition, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides is first melted and optionally combined with cocoa butter.

[0177] In certain embodiments, the pharmaceutical compositions are formulated in any conventional way using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are optionally used, as appropriate and as understood in the art. Pharmaceutical compositions comprising a compound (i.e., a cyclohexenone compound described herein) can be manufactured in a conventional manner, such as, by way of example only, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0178] The pharmaceutical compositions include at least one pharmaceutically acceptable carrier, diluent or excipient and as active ingredient at least one compound described herein (i.e., a cyclohexenone compound described herein). The active ingredient is in a free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and pharmaceutical compositions described herein include the use of crystalline forms (also known as polymorphs), as well as active metabolites of these compounds having the same type activity. All tautomers of the compounds described herein are included within the scope of the compounds described herein. The term "pharmaceutically acceptable salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malc acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.

[0179] In addition, the compounds described herein include unsolvated as well as solvated forms, with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms presented herein are also considered to be disclosed herein. In addition, the pharmaceutical compositions optionally include other pharmaceutically acceptable formulations or agents, carriers, adjuvants (such as preservatives, stabilizers, wetting or emulsifying agents), solution promoters, salts for

[0180] Methods for preparing compositions including the compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid, or liquid. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles, or nanoparticles including the compounds disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions, and creams. The forms of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for dissolving or suspending in liquid prior to use, or as emulsions. These compositions also optionally contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0181] In some embodiments, the pharmaceutical compositions comprising at least a compound (i.e., the cyclohexenone compounds described herein) illustratively take the form of a liquid, wherein the medicament is present in solution, suspension, or both. Typically, when the composition is administered as a solution or suspension, a first portion of the medicament is present in solution, and a second portion of the medicament is present in suspension in particulate form in a liquid matrix. In some embodiments, the liquid composition includes a gel formulation. In other embodiments, the liquid composition is aqueous.

[0182] In certain embodiments, the aqueous pharmaceutical suspensions include one or more polymers as suspending agents. Polymers include water-soluble polymers (such as cellulosic polymers, e.g., hydroxypropyl methylcellulose) and water-insoluble polymers (such as cross-linked carboxy-containing polymers). Certain pharmaceutical compositions described herein include mucoadhesive polymers selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymers), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymers, sodium alginate, and dextran.

[0183] The pharmaceutical compositions also optionally include a solubilizing agent to facilitate the solubility of the compound (i.e., the cyclohexenone compounds described herein). The term “solubilizing agent” generally includes an agent that results in the formation of a micellar solution or true solution of the agent. Certain acceptable non-ionic surfactants (e.g., polysorbate 80) can be used as solubilizing agents, and ophthalmically acceptable glycols, polyglycols (e.g., polyethylene glycol 400 and glycol ethers) can also serve as solubilizing agents.

[0184] In addition, the pharmaceutical compositions optionally comprise one or more pH adjusting agents or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. These acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition within acceptable ranges.

[0185] In addition, the pharmaceutical compositions optionally comprise one or more salts, included in amounts necessary to bring the osmolality of the composition within acceptable ranges. Such salts include those having a sodium cation, potassium cation, or ammonium cation and a chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anion; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0186] Other pharmaceutical compositions optionally comprise one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0187] Still other pharmaceutical compositions comprise one or more surfactants to enhance physical stability or for other purposes. Suitable non-ionic surfactants include polyoxyethylene fatty acid glycerol esters and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

[0188] Still other pharmaceutical compositions can comprise one or more antioxidants to enhance chemical stability, if needed. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0189] In certain embodiments, the pharmaceutical aqueous suspension compositions are packaged in single-dose, non-reclosable containers. Alternatively, multi-dose, reclosable containers are used, in which case a preservative is typically included in the composition.

[0190] In alternative embodiments, other delivery systems for hydrophobic pharmaceutical compounds are employed. Liposomes and emulsions are examples of delivery vehicles or carriers herein. In certain embodiments, organic solvents such as N-methylpyrrolidinone are also employed. In additional embodiments, the compounds described herein are delivered using a slow release system, for example, a semipermeable matrix containing a therapeutic agent in a solid hydrophobic polymer. A variety of slow release materials can be used herein. In some embodiments, slow release capsules release the compound for a few hours to more than 24 hours. Additional protein stabilization strategies can be employed depending on the chemical nature and biological stability of the therapeutic agent.

[0191] In certain embodiments, the formulations described herein comprise one or more antioxidants, metal chelators, thiol-containing compounds, and / or other general stabilizers. Examples of such stabilizers include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0192] Examples

[0193] Example 1: Preparation of exemplary cyclohexenone compounds

[0194] One hundred grams of mycelium from T. cerifera was placed in a flask. An appropriate amount of water and alcohol (70-100% ethanol solution) was added to the flask and stirred at 20-25 °C for at least 1 hour. The solution was filtered through a filter and a 0.45 μιη membrane, and the filtrate was collected as an extract. In one example, for example, the extract was prepared by the solid state fermentation mycelium conditions and compositions disclosed in Lee, T-H. et al., Planta Med 2007; 73: 1412-1415.

[0195] The filtrate of T. cerifera was subjected to high performance liquid chromatography (HPLC) analysis. Separation was performed on a RP18 column with a mobile phase consisting of methanol (A) and 0.3% acetic acid (B) with gradient conditions of 95%-20% B for 0-10 min, 20%-10% B for 10-20 min, 10%-10% B for 20-35 min, 10%-95% B for 35-40 min at a flow rate of 1 ml / min. The column effluent was monitored with a UV-visible detector.

[0196] Fractions collected between 21.2 and 21.4 min were collected and concentrated to yield compound 5 as a pale yellow liquid product. Compound 5 was analyzed as 4-hydroxy-5-(11-hydroxy-3,7,11- trimethyldodeca-2,6-dienyl)-2,3-dimethoxy-6-methylcyclohex-2-enone with a molecular weight of 408 (formula: C 24 H 40 O5). 1 H-NMR (CDC13) δ (ppm) = 1.21, 1.36, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.68, 4.05, 5.71 and 5.56. 13 C-NMR (CDC13) δ (ppm): 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 30.10, 40.27, 43.34, 59.22, 60.59, 71.8, 120.97, 123.84, 124.30, 131.32, 134.61, 135.92, 138.05, 160.45 and 197.11.

[0197]

[0198] Compound 5: 4-hydroxy-5-(11-hydroxy-3,7,11-trimethyldodeca-2,6-dienyl)-2,3- dimethoxy-6-methylcyclohex-2-enone

[0199] Fractions collected between 23.7 and 24.0 min were collected and concentrated to yield compound 7 as a pale yellow liquid product. Compound 7 was analyzed as 4-hydroxy-2,3- dimethoxy-5-(11-methoxy-3,7,11-trimethyldodeca-2,6-dienyl)-6-methylcyclohex-2-enone with a molecular weight of 422 (formula: C 25 H 42 O5). 1 H-NMR (CDC13) δ (ppm) = 1.21, 1.36, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.24, 3.68, 4.05, 5.12, 5.50 and 5.61. 13C-NMR (CDC13) δ (ppm) = 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 40.27, 43.34, 59.22, 60.59, 120.97, 123.84, 124.30, 131.32, 135.35, 135.92, 138.05, 160.45 and 197.12.

[0200]

[0201] Compound 7: 4-Hydroxy-2,3-dimethoxy-5-(11-methoxy-3,7,11- trimethyldodeca-2,6-dienyl)-6-methylcyclohex-2-enone

[0202] Fractions collected between 25 and 30 min were collected and concentrated to give 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11-trimethyldodeca-2,6,10- trienyl)cyclohex-2-enone (Compound 1, also known as andrographolide) as a light yellow-brown liquid product. Analysis of Compound 1 showed a C 24 H 38 O4 having a molecular weight of 390 and a melting point of 48 to 52 °C. Nuclear magnetic resonance spectra indicated, 1 H-NMR (CDC13) δ (ppm) = 1.51, 1.67, 1.71, 1.75, 1.94, 2.03, 2.07, 2.22, 2.25, 3.68, 4.05, 5.07 and 5.14; 13 C-NMR (CDC13) δ (ppm) = 12.31, 16.1, 16.12, 17.67, 25.67, 26.44, 26.74, 27.00, 39.71, 39.81, 40.27, 43.34, 59.22, 60.59, 120.97, 123.84, 124.30, 131.32, 135.35, 135.92, 138.05, 160.45 and 197.12.

[0203]

[0204] Compound 1: 4-Hydroxy-2,3-dimethoxy-6-methyl-5-(3,7,11- trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone

[0205] Compound 27 (a metabolite of Compound 1) was obtained from urine samples of rats that were fed Compound 1 in animal studies. Compound 27 was determined to be 4-hydroxy-2,3-dimethoxy-6-methyl-5-(3-methyl-2-hexenoic acid)cyclohex-2-enone, with a molecular weight of 312 (C 16 H 24 O6). Compound 25, determined to be 2,3-dimethoxy-5-methyl-6-((2E,6E)-3,7,11- trimethyldodeca-2,6,10-trienyl)cyclohexa-2,5-diene-1,4-dione (molecular weight of 386.52, C 24 H 34 Compound 25 was obtained from the purification process.

[0206]

[0207] Compound 26, 4-hydroxy-2-methoxy-6-methyl-5-((2E,6E)-3,7,11- trimethyldodeca-2,6,10-trienyl)cyclohex-2-enone, was also prepared by the purification process, with a molecular weight of 350.53 (C 23 H 36 Compound 28 was also prepared.

[0208]

[0209] Alternatively, exemplary compounds can be prepared from 4-hydroxy-2,3- dimethoxy-6-methylcyclohexa-2,5-dienone, and the like. See, for example, the examples in U.S. Patent No. 9,365,481 and U.S. Patent Publication No. 2016-0237012.

[0210] Similarly, other cyclohexenone compounds having the structure were isolated from T. ceriferum or prepared synthetically or semi-synthetically from suitable starting materials. One of ordinary skill in the art would readily utilize suitable conditions for such synthesis.

[0211] Example 2: Study of antiviral, anti-inflammatory, anti-fibrotic activity of Compound 1 (Andrographolide)

[0212] Exemplary Compound (Compound 1) was subjected to antiviral, anti-inflammatory, and anti-fibrotic activity studies.

[0213] Materials and Methods

[0214] Cell CultureHepG2.2.15 cell line was cultured at 37°C in a 5% CO2 incubator in MEM medium supplemented with 10% fetal bovine serum, penicillin (100 IU / ml; Gibco, USA) and streptomycin (100 ug / ml; Gibco, USA). This is a cell line derived from the human hepatoblastoma cell line HepG2 characterized by having stable HBV expression. Qs5 is a rat hepatoma cell line that produces HBV.

[0215] Lamivudine (3TC) and adefovir (Adv) were purchased as positive controls for HBV treatment. Gossypium quinoa (G4) and lamivudine (3TC) were dissolved in dimethyl sulfoxide (DMSO) only for stock solutions and dilution in culture medium. The final concentration of DMSO in cells was lower than 0.1%. Drugs were treated with 2 x 104cells in 96-well plates for 72 hours.

[0216] HBsAg and HBeAg levels in HepG2.2.15 supernatants were measured with their commercial enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer's instructions.

[0217] MTT Assay . 1.25 x 10 5 Cells / well were seeded in 24-well plates. Cells were incubated with different concentrations of G4 and 3TC (1, 5, 25, 50, 100 and 200 uM) for 72 hours. 1 mg / ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well and incubated at 37°C for 2 hours to form colored crystals. The medium was replaced by DMSO and the plates were incubated at room temperature for 15 minutes with shaking to solubilize the crystals. The absorbance was measured by a microplate reader. The optical density was measured at 490 nm.

[0218] Southern Blot Analysis of Viral DNA . DNA associated with HBV core particles was resolved on 1.2% native agarose gels and detected by Southern blot analysis using a specific HBV DNA probe. The total amount of virus DNA associated with core particles extracted from each treated culture dish was loaded on each lane of the Southern blot gel, plated at the same cell density the night before transfection.

[0219] Results

[0220] To examine the effect of Andrographolide on HBV protein expression, including surface antigen (HBsAg) and e antigen (HBeAg), ELISA assays were used to quantify HBV replication. The results of the study showed that Compound 1 (i.e., Andrographolide) reduced the expression level of HBeAg (see FIG. 1A) and HBsAg (see FIG. IB). Compared to Lamivudine, Compound 1 showed a 50% and 40% reduction in the expression level of two major HBV biomarkers, HBeAg and HBsAg, respectively. Thus, in short, exemplary Compound 1 showed significant inhibitory effects on not only HBeAg but also HBsAg at different doses.

[0221] To evaluate the effect of exemplary Compound 1 on viral replication, DNA associated with intracellular HBV core particles was isolated and analyzed. By Southern blot, exemplary Compound 1 (Andrographolide) showed significant inhibition of HBV replication intermediates (relaxed circular, linear, and single-stranded DNA). As shown in FIG. 2A, Compound 1 (Andrographolide) reduced the expression level of HBV DNA, and reduced HCV RNA activity (2B). In particular, compared to Lamivudine and Adefovir, Compound 1 showed more significant results in reducing the expression level of HBV DNA. Compound 1 also showed a significant reduction of 95% in HCV RNA activity. Figures 2A / B

[0222] Andrographolide inhibition of HBV production can be a result of its cytotoxicity, and this possibility was examined by using MTT assay. No significant cytotoxicity was detected upon exposure to up to 5 uM of Andrographolide, indicating that the inhibitory effect of Andrographolide on supernatant viral protein and DNA levels was not caused by its cytotoxicity. Notably, more than 25 uM of Andrographolide had cytotoxic effects on HepG2.2.15 cells.

[0223] To treat, alleviate symptoms of, or prevent the course of a coronavirus infection (e.g., SARS-CoV-2) in a subject, it is understood that in addition to antiviral activity, a variety of approaches are needed, such as Figure 3 As shown. Upon viral infection, the virus replicates and the number of viruses increases, inflammation occurs causing a cytokine storm, leading to lump fibrosis. Thus, a compound with antiviral, anti-inflammatory, and even anti-fibrosis capabilities, such as exemplary Compound 1, is a suitable drug against coronavirus infection, such as Figure 4 As shown. For example, this indicates that Compound 1 inhibits the function of mTOR and inhibits endocytosis.

[0224] In particular, the results of the study (see Figure 5 ​It is provided that Compound 1 shows effective increase in Nrf-2 nuclear translocation at lower concentration of administration as compared to silymarin. Figure 6 It is provided that Compound 1 significantly reduces ethanol-induced ALT and AST elevation and inhibits oxidative stress. Other anti-inflammatory results of Compound 1 include inhibition of NF-kB expression by 36% and enhancement of nuclear Nrf-2 expression by 2-fold, as shown in Figure 7. In Figure 8, Compound 1 also shows effective inhibition of MCP-1, IL-6 and CD3 expression by about 50%, 57% and 66%, respectively. All of the above study results indicate the effectiveness of Compound 1 in anti-inflammatory activity.

[0225] It is also found that exemplary Compound 1 provides anti-fibrotic activity. Figure 9A illustrates that exemplary Compound 1 effectively inhibits TGF-betal expression by about 64%. In studies utilizing fibrosis-related proteins (Col 1 and Col III), Compound 1 also shows anti-fibrotic properties as shown in Figure 9B. Thus, the data clearly indicate that Compound 1 eliminates viral activity, inflammatory effector protein expression and TGFb1 signal-mediated fibrosis.

[0226] Example 3: Study of the effect of exemplary Compound 1 on the progression of COVID-19

[0227] The study aims to evaluate the impact of exemplary Compound 1 on COVID-19 (i.e., SARS-CoV-2) progression through anti-SARS-CoV-2 (Specific Objective 1), anti-SARS-CoV-2 induced cytokine storm and anti-SARS-CoV-2 induced fibrosis (Specific Objective 2). The overall goal is to confirm whether exemplary Compound 1 (i.e., Andrographolide) provides a potential triple action for COVID-19 treatment and offers a new treatment option for SARS-CoV-2 patients.

[0228] Specific Objective 1. Study the functional effect of Andrographolide against SARS-CoV-2

[0229] The productivity reduction assay is used to determine the inhibitory rate (EC 50). Briefly, Vero E6 cells were seeded into 24-well plates containing DMEM with 2% FBS and treated with Compound 1 (i.e., Andrographolide, 10 or 20 mM) for 1 hour. Plates without any treatment in DMSO were used as controls. Then, the resulting cells were infected with SARS-CoV-2 (multiplicity of infection, MOI = 0.1) for 1 hour. After removing Andrographolide and virus, cells were washed once with PBS and overlaid with overlay medium containing different concentrations of Andrographolide for 24 hours. Cell culture medium was collected for viral plaque assay to determine the number of plaque forming units. One day before infection, Vero E6 cells were seeded into 24-well plates containing DMEM with 10% FBS and antibiotics. Cell culture medium was added to the cell monolayer and incubated at 37 °C for 1 hour. Subsequently, cell culture medium was removed and the cell monolayer was washed once with PBS and then overlaid with medium containing 1% methylcellulose for 5 days. Cells were fixed with 10% formaldehyde for 1 hour. After removing the overlay medium, cells were stained with 0.5% crystal violet and plaques were counted. Cells were collected for protein and RNA extraction by RIPA cell lysis buffer and NucleoSpin RNA kit (Macherey-Nagel), respectively, by AMRESCO. Then, the expression levels of nucleocapsid protein and E gene were detected by Western blot (antibody catalog number 40143-R019) and quantitative real-time PCR (qRT-PCR), respectively. In addition, isolated RNA was used for specific target 2. In addition, the cytotoxicity (i.e., IC 50 ) will be measured by acid phosphatase assay. Herein, remdesivir (1 mM) treatment will be used as a control. All results will be shown as mean ± s.d. from at least three independent experiments.

[0230] The results of the study showed that both 20 and 10 mM of Compound 1 significantly reduced the concentration of SARS-CoV-2 (99.93% for 20 mM and 91.20% for 10 mM). See Figure 10 . Figure 11 Cell culture results are also provided showing cell culture plates from Andrographolide and control (DMSO plate) treatment.

[0231] Specific objective 2. Explore the effect of Andrographolide on SARS-CoV-2-induced cytokine storm and on SARS-CoV-2-induced fibrosis

[0232] Current studies have shown that multiple cytokines / chemokines are significantly associated with COVID-19 disease. For example, plasma IP-10 (also known as CXCL10) is highly correlated with disease severity and can predict the progression of COVID-19. IL-6 can also serve as a predictor of progression to severe COVID-19, suggesting that targeting cytokines as a treatment option for COVID-19 patients. Regarding the long-term effects of COVID-19, TGF-b-mediated collagen deposition can be an important factor in causing irreversible pulmonary fibrosis.

[0233] To reveal the effects of andrographolide on SARS-CoV-2-induced cytokine storm and SARS-CoV-2-induced fibrosis, the RNA described in Specific Objective 1 was used to detect the gene expression of cytokines / chemokines (such as CXCL10, IL6, and IL18, see Figures 12A-C ), pro-fibrotic growth factors (such as TGFB1, see Figure 13A), and collagens (such as COL1A1, COL3A1, and COL4A1). Briefly, 5.4 pg of RNA was reverse transcribed into cDNA by M-MLV Reverse Transcriptase Kit. Real-time PCR analysis was established using SYBR TM Green Master Mix Kit and performed in a QuantStudio TM 5 Real-Time PCR System. The relative levels of target mRNA were determined by normalizing to actin rRNA.

[0234] Results of the study:

[0235] Figures 12A-C The gene expression levels of CXCL10 (12A), IL6 (12B), and IL18 (12C) are provided, respectively. Using 20 mM of andrographolide, CXCL10 expression changed by 1.01-fold, while 3.40-fold and 9.04-fold changes were observed using 10 mM of andrographolide and DMSO, respectively. Using 10 mM of andrographolide, IL6 expression changed by 11.88-fold, while 47.81-fold change was observed using DMSO. Using 20 mM of andrographolide, IL18 expression changed by 0.89-fold, while 1.36-fold change was observed using DMSO.

[0236] Figures 13A-B provide the gene expression levels of TGFB1 (13A) and COL4A1 (13B), respectively. Using 20 mM of andrographolide, TGFB1 expression changed by 0.99-fold, while 2.59-fold change was observed using DMSO. Using 20 mM of andrographolide, COL4A1 expression changed by 0.65-fold, while 2.37-fold change was observed using DMSO.

[0237] Thus, it is clear that the exemplary compound Andrographolide provides superior effects against SARS-CoV-2 induced cytokine storm and against SARS-CoV-2 induced fibrosis.

[0238] Example 4: Phase 2 study clinical trial for evaluating the safety and efficacy of Compound 1 in hospitalized patients with mild to moderate pneumonia due to COVID19 Primary efficacy analysis

[0239] The primary objectives of this study are:

[0240] • To evaluate the efficacy of Andrographolide treatment in patients with mild to moderate pneumonia due to COVID-19, as measured by:

[0241] o Time to clinical improvement

[0242] o Progression of disease.

[0243] • To evaluate the safety of Andrographolide treatment in patients with mild to moderate pneumonia due to COVID-19.

[0244] The secondary objectives are:

[0245] • To further evaluate the efficacy of Andrographolide compared to placebo in this patient population,

[0246] by the following measures:

[0247] o Duration of hospitalization

[0248] o Viral clearance

[0249] o Life status (death)

[0250] • To assess the pharmacokinetic (PK) plasma concentrations of Andrographolide in this patient population.

[0251] • To assess the safety of Andrographolide in this patient population.

[0252] Study design:

[0253] This is a Phase 2 clinical trial designed to evaluate the safety and efficacy of Andrographolide in hospitalized patients with mild to moderate pneumonia due to COVID-19.

[0254] The main characteristics of the hospitalized patients included in this study are: adult patients with onset of fever within 5 days prior to screening and respiratory rate > 24 / minute. Symptoms of mild to moderate pneumonia due to COVID 19 must be present (results confirmed by chest X-ray or computed tomography [CT] scan). The planned duration of treatment is 10 days of Andrographolide or placebo administration in addition to standard of care (SoC) therapy in line with local SoC policies.

[0255] A total of 166 patients are planned to be enrolled and randomized in a 1 : 1 ratio of Andrographolide to placebo.

[0256] As Andrographolide has shown antiviral and anti-inflammatory activity in earlier clinical studies, it is planned to be used to treat patients with COVID-19 infection. Therefore, the initial sentinel cohort of patients will be treated to assess the safety of Andrographolide. This sentinel cohort will include the first 20 patients (10 patients assigned to Andrographolide and 10 patients assigned to placebo). Recruitment will be paused once the first 20 patients have started treatment.

[0257] Once the first 20 patients have completed at least 10 days of therapy, the Data Monitoring Committee (DMC) will assess the safety and tolerability of Andrographolide in this sentinel cohort. The DMC can unblind the data from this assessment. Recruitment will resume once the 20 patients in the sentinel cohort have been treated for at least 10 days and the study has been assessed by the DMC as safe to continue.

[0258] All enrolled patients in the study, including the sentinel cohort, will be included in the primary analysis of the efficacy and safety of the study treatment. The DMC will continue to review safety and assess the risk / benefit profile.

[0259] The primary efficacy analysis will be performed once all patients have achieved clinical improvement, or have been followed up for 28 days from the start of therapy.

[0260] Number of patients:

[0261] The study plans to enroll a total of 166 patients (83 patients in the Andrographolide group and 83 patients in the placebo group). This enrollment level ensures approximately 135 improvement events. Enrollment will be based on the following assumptions:

[0262] A 1 : 1 randomization ratio of Andrographolide to placebo group;

[0263] • Clinical improvement is defined as a median change from 7 days to 4 days.

[0264] • Each patient will be followed up for up to 28 days

[0265] • 90% performance

[0266] • Two-sided alpha of 0.05.

[0267] Diagnosis, and primary inclusion and exclusion criteria:

[0268] Unless otherwise stated, patients must meet all of the following inclusion criteria at the screening visit: • Age 18 years or older • Diagnosis of COVID-19 infection

[0269] 1. Willing and able to provide informed consent.

[0270] 2. Male or female patients >18 years and <80 years of age.

[0271] 3. Hospitalized due to fever (defined as oral temperature > 38.6°C) and respiratory rate > 24 / minute. Fever (axillary > 36.6°C, or oral > 37.2°C, or rectal or ear > 37.8°C…Gilead)

[0272] Note: Hospitalized patients can also include patients admitted to hospital-based centers for treatment of COVID-19 patients.

[0273] 4. Chest X-ray or CT scan consistent with pneumonia.

[0274] Status: Unilateral and bilateral pneumonia (infiltrates / interstitial)

[0275] 5. Fever within 5 days prior to screening.

[0276] 6. SARS CoV 2 infection confirmed by PCR testing of nasopharyngeal sample (non-serological testing).

[0277] 7. Male and female patients of childbearing potential must agree to use a protocol-specified method of contraception.

[0278] 8. Female patients of childbearing potential must have a negative pregnancy test prior to screening and treatment on Day 1.

[0279] 9. Male patients must agree not to donate sperm from the first dose of study drug until 90 days after the last dose.

[0280] 10. Patient willing and able to comply with the study drug regimen and all other study requirements, as deemed by the Investigator.

[0281] 11. Hospitalized < 48 hours and randomized within 48 hours of meeting inclusion criteria.

[0282] Patients will be excluded from the study if they meet any of the following exclusion criteria at the screening visit unless otherwise specified:

[0283] 1. Female patients who are pregnant or breastfeeding.

[0284] 2. Any patient with a concomitant life-threatening condition, including but not limited to: need for mechanical ventilation, acute respiratory distress syndrome (ARDS), shock, or heart failure.

[0285] Patient requires invasive mechanical ventilation; or other organ failure requiring ICU monitoring

[0286] Is the patient able to receive mask oxygen therapy (O2 inhalation)?

[0287] 3. Evidence of lobar or sub-lobar consolidation on chest X-ray.

[0288] 4. Room air oxygen saturation (SpO2) < 90% or arterial partial pressure of oxygen (PaO2) / fraction of inspired oxygen (FiO2) < 200 mm Hg, severe dyspnea, or need for positive pressure ventilation (with or without intubation)

[0289] 5. Use of drugs or alcohol abuse that, in the investigator’s judgment, can interfere with compliance with study requirements.

[0290] 6. Treatment with other drugs considered likely to be active against COVID 19 within 7 days prior to enrollment.

[0291] 7. Use of other study drugs within 30 days prior to dosing, or planned enrollment in another study drug clinical trial concurrent with participation in this study.

[0292] 8. Clinically significant abnormal ECG at screening, as determined by the investigator.

[0293] 9. Patient requires frequent or long-term use of systemic corticosteroids or other immunosuppressive drugs (e.g., for organ transplant or autoimmune conditions).

[0294] 10. Abnormal laboratory values at screening:

[0295] a. Estimated glomerular filtration rate (GFR) < 50 mL / min.

[0296] b. Alanine aminotransferase (ALT) or aspartate aminotransferase (AST)

[0297] > 5 x upper limit of normal (ULN), or ALT / AST > 3 x ULN plus total bilirubin

[0298] > 2 x ULN.

[0299] c. Platelet count < 100 x 109 / L.

[0300] d. Total bilirubin > 1.5 x ULN, unless patient has known Gilbert’s syndrome.

[0301] e. Hemoglobin < 9 g / dL for females or < 11 g / dL for males.

[0302] f. Total white blood cell (WBC) count < 3,500 / mm3 or absolute neutrophil count (ANC) < 1,500 / mm3.

[0303] 11. Treatment with any antiviral medication or any medication known to be a strong inducer or inhibitor of CYP2C19, CYP3A4, CYP2C8, and CYP2E1 within 14 days prior to study treatment initiation.

[0304] 12. Any other clinically significant medical condition or laboratory abnormality that, in the investigator's opinion, can have compromised the patient's safety or potentially affected the patient's compliance or safety / efficacy observations in the study.

[0305] 13. Viral pneumonia due to other viruses than 2019-nCoV

[0306] 14. Patients unable to take oral medication

[0307] 15. Patients who were intubated or required immediate intubation at randomization

[0308] 16. Severe cognitive and psychiatric disorders

[0309] Test article, dose, and mode of administration:

[0310] Anudolutinol (100 mg capsules) will be administered orally at a dose of 200 mg (2 capsules) twice daily (BID) for 10 days.

[0311] Reference therapy, dose, dosage form, and mode of administration: placebo (capsules) will be administered orally BID for 10 days.

[0312] Duration of patient participation in the study:

[0313] The total study duration is planned to be up to 28 ± 2 days.

[0314] The screening period is planned to be up to 2 days. The planned treatment duration is 10 days. Follow-up safety assessments will be performed on Day 14 and Day 28 (± 2 days).

[0315] Study population:

[0316] Full Analysis Set (FAS): All randomized patients who received at least one dose of study medication. Patients will be analyzed according to the treatment to which they were randomized.

[0317] Per Protocol Set (PPS): All patients from the FAS who did not have major protocol deviations during the study. Patients with any major protocol deviations should be excluded from the PPS prior to database lock.

[0318] Safety Set (SS): All patients who received at least one dose of study medication. Patients will be analyzed according to the study treatment actually received by the patient.

[0319] Pharmacokinetic Set (PKS): All patients who have received at least 1 dose of study drug and have at least 1 evaluable plasma concentration without major protocol deviations or events considered to significantly affect PK.

[0320] Endpoints:

[0321] The primary efficacy endpoint is: Time to clinical improvement [time frame: within 28 days from start of medication]

[0322] Clinical improvement is defined as the time (in days) from start of study treatment to normalization of fever < 37.2°C, respiratory rate < 24 / minute in room air, and oxygen saturation (SpO2) > 94% in room air. (individual or total)

[0323] Resolution of hypoxia (defined as SpO2 > 93% in room air or PaO2 / FiO2 > 300 mmHg).

[0324] • Rate of disease resolution [time frame: days 14 and 28 from start of medication]

[0325] • Progression of disease

[0326] Progression of disease is defined as the need for positive pressure ventilation (intubated or not) or the need for ICU care. For the subgroup of patients who remain hospitalized and have an arterial blood gas (ABG) test as part of SoC, PaO2 / FiO2 < 200 mmHg will also be used as a measure of disease progression.

[0327] Rate of invasive mechanical ventilation when respiratory failure occurs [time frame: 10 days]

[0328] Secondary efficacy endpoints are:

[0329] • Length of hospital stay (in days).

[0330] • Viral clearance from nasopharyngeal or respiratory tract samples

[0331] o Time to viral clearance, measured in study days from start of treatment to first negative SARS-CoV-2 PCR test

[0332] o Rate of change in viral load will be evaluated depending on the validity of the quantitative analysis.

[0333] (real-time RT-PCR test)

[0334] • Vital status (death) will be collected up to days 14 and 28

[0335] • Time to improvement in pulmonary imaging [time frame: within 10 days post-dose]

[0336] Safety endpoints include the following variables:

[0337] • Adverse events (AEs).

[0338] • Chest imaging (X-ray or CT scan) results.

[0339] • Vital signs: blood pressure, pulse rate.

[0340] • Physical examination: general appearance, HEENT, lymphatic, cardiovascular, respiratory, gastrointestinal, skeletal muscle, neurological, dermatological.

[0341] • 12-lead electrocardiogram (ECG).

[0342] • Standard safety laboratory tests (hematology, chemistry, and urinalysis).

[0343] Pharmacokinetic evaluation

[0344] The PK parameters assessed from plasma samples are:

[0345] • Trough (initial dose) plasma concentration (Ctrough)

[0346] • Maximum plasma concentration (Cmax).

[0347] Statistical methods:

[0348] General principles:

[0349] Continuous variables will be summarized by standard descriptive statistics: number of patients (n), mean, standard deviation (SD), median, minimum (min), and maximum (max). Frequencies and percentages will be summarized for categorical variables.

[0350] Results will be considered statistically significant at the one-sided alpha of 0.025, and results will be considered to indicate a promising trend at the one-sided alpha of 0.2.

[0351] Efficacy analysis:

[0352] Secondary efficacy analysis

[0353] The hazard ratio (HR) and its 95% confidence interval (CI) for time to clinical recovery will be estimated by a Cox proportional hazards model, with data censored on death, on the provision of any non-study anti-viral treatment to the patient, or on Day 28 if the patient has not recovered. The median time to clinical improvement will be estimated by the Kaplan-Meier (KM) method, and KM curves will be provided. P-values for comparisons between groups will be obtained according to the log-rank test.

[0354] For disease progression, logistic regression will be used to calculate the proportion of patients requiring positive pressure ventilation and requiring ICU care in the two groups and the difference between groups and 95% CI. P-values will be based on the Chi-square test. For the patient subgroups with ABG data collected as part of SoC, PaO2 / FiO2 will also be evaluated.

[0355] Example 5: Oral formulations

[0356] • Length of stay, virologic clearance (time to virologic clearance, rate of change in viral load), and vital status will be analyzed using similar statistical methods as for the primary efficacy endpoint.

[0357] Safety analyses:

[0358] Adverse events will be coded according to the Medical Dictionary for Regulatory Activities (MedDRA).

[0359] The number and percentage of patients with treatment-emergent AEs (TEAEs), serious AEs (SAEs), TEAEs related to study treatment, SAEs related to study treatment TEAEs leading to discontinuation of study treatment, TEAEs leading to discontinuation of the study, and TEAEs leading to death will be summarized by system organ class (SOC), preferred term (PT), and treatment group. In addition, the severity of TEAEs and relationship to study treatment will be tabulated by SOC, PT, and treatment group.

[0360] The following standardized MedDRA queries (SMQs) identify AEs of special interest (AESI) and will be reported:

[0361] • Respiratory failure

[0362] • Opportunistic infections

[0363] Test values and changes from baseline for specific laboratory test results, vital signs, SpO2, physical examinations, and EGG results will be summarized descriptively. Transposed tables by treatment group will be presented where applicable.

[0364] Pharmacokinetic analyses:

[0365] Descriptive statistics for andorukirn plasma concentrations and / or PK parameters will be provided.

[0366] Table 1: Schedule of assessments

[0367]

[0368]

[0369] Abbreviations: AE = adverse event; C max = maximum plasma concentration; C trough = trough (pre dose) plasma concentration; CT = computed tomography; DMC = data monitoring committee; ECG = electrocardiogram; EOS = end of study; EOT = end of treatment; FiO2 = percentage of inspired oxygen; HEENT = head, eye, ear, nose, and throat; ICU = intensive care unit; PaO2 = arterial partial pressure of oxygen; PCR = polymerase chain reaction; PD = pharmacodynamics; PK = pharmacokinetics; SpO2 = blood oxygen saturation / pulse oximetry.

[0370] Footnotes:

[0371] a Patients can be discharged at any time during days 2-10 after reaching clinical recovery, defined as the time (in days) from start of study treatment to normalization of fever, respiratory rate, and oxygenation. Patients will then be required to receive treatment at home (per prescribed) and will be followed up by phone at day 14 and day 28 to assess symptoms. Discharged patients will need to come to the hospital / site to complete day 10 assessments / EOT visit.

[0372] b An initial cohort of 20 patients will be enrolled to assess safety and tolerability. Once the DMC confirms there are no safety issues, the study will resume enrollment of the remaining patients.

[0373] c Body temperature (oral, forehead, axillary, tympanic) > 38.6°C and respiratory rate > 24 / minute within 5 days prior to screening. A complete physical examination (general appearance, HEENT, lymphatic, cardiovascular, respiratory, gastrointestinal, skeletal muscle, neurological, and skin systems) will be performed at screening. Targeted physical examination for COVID-19 symptoms will be performed during hospitalization. Vital signs (respiratory rate, blood pressure, and pulse rate) will be assessed daily during hospitalization. Height and weight will be measured only at screening.d Chest x-ray or CT scan should show results consistent with COVID-19 pneumonia and will be performed at screening and at discharge.

[0374] e Female patients of childbearing potential will have a urine pregnancy test performed at the local (site) laboratory.

[0375] fEfficacy parameter assessments: [1] PCR testing for COVID-19 at local labs (until negative result) at screening and follow-up visits. This testing can be done at central labs for outpatients for their follow-up testing; [2] Patients can be assessed as clinically worsened status if they require prolonged hospitalization or disease progression (defined as the need for positive pressure ventilation (intubated or not), or the need for ICU care); for a subset of patients who remain hospitalized and have arterial blood gas (ABG) testing as part of SoC, PaO2 / FiO2 will also be used as a measure of disease progression; [3] Daily SpO2 monitoring on Day 1: average of 3 consecutive readings in 5 minutes; [4] Arterial blood gas evaluation of PaO2 / FiO2 at screening and during hospitalization (until discharge).

[0376] gSafety parameter assessments: [1] AEs will be assessed from treatment initiation (during hospitalization and at home after discharge) until EOS; [5] Standard safety laboratory tests will include all parameters of hematology, chemistry, and urinalysis and will be performed on Days 1, 5, and 10, prior to dosing; [6] 12-lead EGG will be performed at screening when the patient is in supine position; [7]

[0377] Concomitant medications will be recorded from screening until EOS.

[0378] hPharmacokinetic parameters include Ctrough and Cmax. Blood samples for these parameters will be assessed on Days 5 and 10 (if the patient is still hospitalized), prior to dosing and 2 hours post-dosing.

[0379] Example 6: Sublingual (hard pastille) formulations .

[0380] To prepare a pharmaceutical composition for oral delivery, an equal weight of the exemplary Compound 1 is mixed with an equal weight of corn oil (e.g., 25 mg, 50 mg, 100 mg, 200 mg). The mixture is incorporated into an oral dosage unit in a gelatin capsule suitable for oral administration.

[0381] In some cases, 100 mg of a compound described herein is mixed with 750 mg of starch. The mixture is incorporated into an oral dosage unit in a hard gelatin capsule suitable for oral administration.

[0382] Example 7: Inhalation compositions

[0383] To prepare a pharmaceutical composition for oral delivery, an equal weight of the exemplary Compound 1 is mixed with an equal weight of corn oil (e.g., 25 mg, 50 mg, 100 mg, 200 mg). The mixture is incorporated into an oral dosage unit in a gelatin capsule suitable for oral administration.

[0384] ​

[0385] For preparing a pharmaceutical composition for inhalation delivery, 20 mg of a compound described herein is mixed with 50 mg of anhydrous citric acid and 100 mL of a 0.9% sodium chloride solution. The mixture is incorporated into an inhalation delivery unit, such as a nebulizer, which is suitable for inhalation administration.

[0386] While the preferred embodiments of the application have been shown and described herein, it will be apparent to those skilled in the art that many changes, modifications, and substitutions can be made thereto without departing from the application. It is to be understood that various alternatives to the embodiments of the application described herein can be employed in practicing the application. The appended claims are intended to cover all methods and structures falling within the scope of the invention and their equivalents.

[0387] The present application also provides the following items:

[0388] 1. A method for treating or alleviating a symptom of an RNA virus-induced disease or preventing an RNA virus-induced disease in a subject, comprising administering to the subject a therapeutically effective amount of a cyclohexenone compound having the structure:

[0389]

[0390] wherein each of X and Y is independently oxygen, NR5, or sulfur;

[0391] R is hydrogen or C(=0)Ci-C8alkyl;

[0392] each of R1, R2, and R3is independently hydrogen, optionally substituted methyl, or (CH2) m -CH3;

[0393] R4is NR5R6, OR5, OC(=0)R7, C(=0)OR5, C(=0)R5, C(=0)NR5R6,

[0394] halogen, 5- or 6-membered lactone, Ci-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, glucosyl,

[0395] wherein the 5- or 6-membered lactone, Ci-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, aryl, and glucosyl are optionally substituted with one or more substituents selected from NR5R6, OR5, OC(=0)R7, C(=0)OR5, C(=0)R5, C(=0)NR5R6, Ci-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and Ci-C8haloalkyl;

[0396] each of R5and R6is independently hydrogen or Ci-C8alkyl;

[0397] R7is Ci-C8alkyl, OR5, or NR5R6;

[0398] m = 1-12; and

[0399] n = 1-12; or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof.

[0400] 2. The method of item 1, wherein the RNA virus is a coronavirus.

[0401] 3. The method of item 1, wherein the RNA virus-induced disease is caused or induced by a Coronaviridae infection.

[0402] 4. The method of item 1, wherein the cyclohexenone compound reduces RNA virus concentration or prevents RNA virus infection.

[0403] 5. The method of item 1, wherein the RNA virus-induced disease is coronavirus-induced pneumonia, or SARS-CoV-2-induced pneumonia.

[0404] 6. The method of item 1, wherein the RNA virus-induced disease is RNA virus-induced pneumonia.

[0405] 7. The method of item 3, wherein the Coronaviridae infection is caused by or associated with alpha coronavirus 229E (HCoV-229E), NL63 (HCoV-NL63, New Haven coronavirus), beta coronavirus OC43 (HCoV-OC43), HKU1, MERS-CoV (coronavirus that causes Middle East respiratory syndrome), SARS-CoV, or SARS-CoV-2 (2019-nCoV).

[0406] 8. The method of item 7, wherein the Coronaviridae infection is caused by or associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0407] 9. The method of any one of items 1-8, wherein the cyclohexenone compound or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof is administered orally, parenterally, or intravenously.

[0408] 10. The method of any one of items 1-8, wherein the cyclohexenone compound or a pharmaceutically acceptable salt, metabolite, solvate, or prodrug thereof is administered by injection.

[0409] 11. The method of any of items 1-10, wherein the subject is a human.

[0410] 12. The method of any of items 1-11, wherein R is hydrogen, C(=0)C3H8, C(=0)C2H5, or C(=0)CH3.

[0411] 13. The method of any of items 1-11, wherein each of R1, R2, and R3 is independently hydrogen, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl.

[0412] 14. The method of item 13, wherein R1 is hydrogen or methyl.

[0413] 15. The method of item 13, wherein R2 is hydrogen or methyl.

[0414] 16. The method of any of items 1-15, wherein R4 is halogen, NH2, NHCH3, N(CH3)2, OCH3, OC2H5, C(=0)CH3, C(=0)C2H5, C(=0)OCH3, C(=0)OC2H5, C(=0)NHCH3, C(=0)NHC2H5, C(=0)NH2, OC(=0)CH3, OC(=0)C2H5, OC(=0)OCH3, OC(=0)OC2H5, OC(=0)NHCH3, OC(=0)NHC2H5, or OC(=0)NH2.

[0415] 17. The method of any of items 1-15, wherein R4 is C2H5C(CH3)2OH, C2H5C(CH3)2OCH3, CH2COOH, C2H5COOH, CH2OH, C2H5OH, CH2Ph, C2H5Ph, CH2CH=C(CH3)(CHO), CH2CH=C(CH3)(C(=0)CH3), a 5- or 6-membered lactone, an aryl group, or a glucosyl group, wherein the 5- or 6-membered lactone, aryl group, and glucosyl group are optionally substituted with one or more substituents selected from NR5R6, OR5, OC(=0)R7, C(=0)OR5, C(=0)R5, C(=0)NR5R6, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, and C1-C8 haloalkyl.

[0416] 18. The method of any one of items 1-15, wherein R4is C1-C8alkyl optionally substituted with one or more substituents selected from NR5R6, OR5, OC(=O)R7, C(=O)OR5, C(=O)R5, C(=O)NR5R6, C1-C8alkyl, C2-C8alkenyl, C2-C8alkynyl, C3-C8cycloalkyl, and C1-C8haloalkyl.

[0417] 19. The method of item 18, wherein R4is CH2CH=C(CH3)2.

[0418] 20. The method of any one of items 1-19, wherein the cyclohexenone compound is

[0419]

Claims

1. The therapeutically effective amount has the following structure: The use of cyclohexenone compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating or alleviating symptoms of an RNA virus-induced disease in a subject and / or preventing an RNA virus-induced disease in a subject, wherein the RNA virus-induced disease is a SARS-CoV-2-induced disease.

2. The use as described in claim 1, wherein, The cyclohexenone compound reduces RNA virus concentration or prevents RNA virus infection.

3. The use as described in claim 1, wherein, The RNA virus-induced disease is SARS-CoV-2-induced pneumonia.

4. The use as described in any one of claims 1-3, wherein, The cyclohexenone compound or a pharmaceutically acceptable salt thereof is administered orally, parenterally, or intravenously.

5. The use as described in any one of claims 1-3, wherein, The cyclohexenone compound or a pharmaceutically acceptable salt thereof is administered by injection.

6. The use as described in any one of claims 1-3, wherein, The subjects were humans.

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