Compounds for chronic disorders

Abstract

The present disclosure provides compounds and compositions useful for treating chronic disorders, including cancer and viral diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 005,730, filed on April 6, 2020, and Indian Application No. 202041001163, filed on January 10, 2020, each of which is incorporated herein by reference in its entirety. Background Art

[0003] A chronic condition is a human health condition or disease that persists or otherwise has effects that are long-lasting, or a disease that develops over time. The term chronic is usually applied when the condition lasts for more than three months. Common chronic diseases include arthritis, asthma, cancer, chronic obstructive pulmonary disease, diabetes, and some viral diseases such as hepatitis C and acquired immunodeficiency syndrome.

[0004] Cancer, also called a malignant tumor, is an abnormal growth of cells. Cancer develops when the body's normal control mechanisms stop working. Instead of dying, old cells grow uncontrollably, forming new abnormal cells. These extra cells may form a mass of tissue called a tumor. Some cancers, such as leukemia, do not form tumors.

[0005] There are more than 100 types of cancer, including breast cancer, skin cancer, lung cancer, colon cancer, prostate cancer, and lymphoma. Symptoms vary depending on the type.

[0006] Treatment options depend on the type of cancer and its stage. The goal of therapy is to kill as many cancer cells as possible while minimizing damage to nearby normal cells.

[0007] The three main treatments are:

[0008] Surgery: Direct removal of the tumor;

[0009] Chemotherapy: the use of chemicals to kill cancer cells;

[0010] Radiation therapy: Uses X-rays to kill cancer cells.

[0011] The major unsolved problems of metastatic cancer are recurrence after receiving an objective response to chemotherapy, drug-induced side effects of first-line chemotherapy, and delayed response to second-line treatment. Unfortunately, there are few options that can be used as third-line treatment. Therefore, there is a growing need to find new chemopreventive agents that can effectively prevent and / or manage chronic conditions such as cancer.

[0012] Natural products (such as flavonoids) can be used to prevent or treat chronic disorders. However, due to the solubility and bioavailability of reduction, natural products are not effective substitutes for current therapeutic agents. Therefore, there is a need to develop novel drugs in the prior art, and the novel drugs provide increased bioavailability, solubility and tissue distribution capabilities for targeted delivery. Such agents can be active for a variety of chronic conditions, and in some embodiments, can show anticancer or antiviral activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The foregoing and following information and other features of the present disclosure will become more apparent from the following description and the appended claims in conjunction with the accompanying drawings. It should be understood that these drawings depict only several embodiments according to the present disclosure and are therefore not to be considered limiting of its scope, and the present disclosure will be described with additional specificity and detail through the use of the accompanying drawings.

[0014] Figure 1 Depicted is the spectrum of compound I

[0015] a. Mass spectrometry

[0016] b. IR spectrum

[0017] c. 1 H NMR spectrum

[0018] d. 13 C NMR spectrum

[0019] Figure 2 The spectrum of compound II is depicted

[0020] a. Mass spectrometry

[0021] b. IR spectrum

[0022] c. 1 H NMR spectrum

[0023] d. 13 C NMR spectrum

[0024] Figure 3 Depicted is the spectrum of compound III

[0025] a. Mass spectrometry

[0026] b.IR spectrum

[0027] c. 1 H NMR spectrum

[0028] d. 13 C NMR spectrum

[0029] Figure 4 The spectrum of compound IV is depicted

[0030] a. Mass spectrometry

[0031] b.IR spectrum

[0032] c. 1 H NMR spectrum

[0033] d. 13 C NMR spectrum

[0034] Figure 5 Depicted is the spectrum of compound V

[0035] a. Mass spectrometry

[0036] b.IR spectrum

[0037] c. 1 H NMR spectrum

[0038] d. 13 C NMR spectrum

[0039] Figure 6 The spectrum of compound VI is depicted

[0040] a. Mass spectrometry

[0041] b.IR spectrum

[0042] c. 1 H NMR spectrum

[0043] d. 13 C NMR spectrum

[0044] Figure 7 The spectrum of compound VII is depicted

[0045] a. Mass spectrometry

[0046] b.IR spectrum

[0047] c. 1 H NMR spectrum

[0048] d. 13 C NMR spectrum

[0049] Figure 8 Depicted is the spectrum of compound VIII

[0050] a. Mass spectrometry

[0051] b.IR spectrum

[0052] c. 1 H NMR spectrum

[0053] d. 13 C NMR spectrum

[0054] Fig. 9 Depicted is the spectrum of compound IX

[0055] a. Mass spectrometry

[0056] b.IR spectrum

[0057] c. 1 H NMR spectrum

[0058] d. 13 C NMR spectrum

[0059] Fig. 10A The anticancer activity of Compound I on different cell lines is depicted.

[0060] A: Effects of molecules on different cancer cell lines;

[0061] B: Effects of molecules on normal cell lines;

[0062] C: Comparison with other standard drugs:

[0063] abbreviation

[0064] MCF-7 (human breast cancer cells);

[0065] MDAMB 231 (triple-negative breast cancer cell line);

[0066] PANC-1 (human pancreatic cancer cell line);

[0067] HT-29 (human colon cancer cell line);

[0068] T-ALL (T-cell acute lymphoblastic leukemia);

[0069] HDF (human dermal fibroblasts);

[0070] AC-16 (human cardiomyocytes);

[0071] HBMSC (human bone marrow mesenchymal stem cells);

[0072] MCF-12A (human normal breast cells);

[0073] CRL2989 (human normal pancreatic cells);

[0074] NCM 60 (normal colon epithelial cell line)

[0075] Fig. 10B Apoptosis induction and mitochondrial membrane potential of control and Compound I are depicted.

[0076] Fig.11 Depicted are a series of graphs illustrating the mechanism of action of compounds of the present disclosure (eg, Compound 1) that inhibit histone deacetylase (HDAC) activity.

[0077] Fig.12 A series of graphs depicting Compound I-induced formation of neuronal cell types.

[0078] a. Neuronal differentiation of human stem cells-molecules

[0079] b. Molecular analysis of tumor gene expression

[0080] Fig.13 Inhibition of cancer stem cell markers by Compound I is depicted.

[0081] Fig.14 Cancer cell kinetics and treatment with conventional drugs and compounds of the present invention are depicted.

[0082] Fig.15 The breakthroughs achieved by the compounds of the present disclosure are depicted.

[0083] Fig.16 Cancer stem cells and receptors are depicted.

[0084] Fig.17 Aberrant signaling pathways in cancer stem cells are delineated and cancer stem cells are shown as targets.

[0085] Fig.18 Genes targeted by the compounds of the present disclosure are depicted for various types of cancer.

[0086] Fig.19 Depicted is the evaluation of the cytotoxicity of anticancer agents by measuring IC50 against a panel of normal cell lines that have not been treated with compound 1 ( Fig.19 A) and ( Fig.19 B).

[0087] Fig. 20 Depicted is an in vivo study in a stage IV translocation tumor model comparing conventional drugs to Compound I of the present disclosure.

[0088] Fig.21 A PANC-1 mouse xenograft model is provided to evaluate the efficacy of Compound I compared to gemcitabine.

[0089] Fig. 22 Steps in the coronavirus replication pathway targeted by the compounds of the present disclosure are depicted.

[0090] Fig.23 Depicted is how the binding of ACE-2 inhibitors disrupts the interaction between the virus and the receptor.

[0091] Fig.24A The molecular binding site of Nspl5 is depicted.

[0092] Fig. 24B A model showing the binding of small molecules to Nsp15 is depicted.

[0093] Fig.24C A model showing binding site residues proposed to interact with the compounds of the present disclosure is depicted.

[0094] Fig.25 A depicts normal Vero cells and Vero cells infected with SARS-CoV-2 at an MOI of 0.1, treated with different doses of the indicated antiviral drugs for 48 h. Virus yields in cell supernatants were then quantified by qRT-PCR.

[0095] Fig.25 B depicts graphs representing the average % inhibition of viral yield and cytotoxicity of drugs (eg, Compound I), respectively. Experiments were performed in triplicate.

[0096] Fig.25 C depicts immunofluorescence microscopy of virus infection after treatment with compound I. At 48 h after infection, infected cells were fixed and then stained with rabbit serum against the nucleoprotein (NP) of SARS-related CoV as the primary antibody and goat anti-rabbit IgG labeled with Alexa 488 as the secondary antibody. Cell nuclei were stained with Hoechst dye. Scale bar, 20 μm.

[0097] Fig.25 D depicts Western blot analysis of nucleoprotein (NP) expression in cells infected with SARS-CoV-2 at an MOI of 0.1 at 24 h post-infection (pi).

[0098] Fig.25 E depicts a graph representing the expression of nuclear protein (NP) normalized to GAPDH. Virus yield in infected cell supernatants was quantified by qRT-PCR. Experiments were performed in triplicate.

[0099] Fig.25 F-25H depicts a graph showing that a reduction in viral RNA was found in supernatants and cell pellets from samples treated with 1 μM Compound I.

[0100] Fig.26 A depicts a cell viability assay of calu-3 cells treated with different concentrations of compound I.

[0101] Fig.26 B depicts the percentage plaque reduction assay in calu-3 cells treated with various concentrations of Compound I.

[0102] Fig. 27FIG. 8A depicts H&E staining analysis of liver and kidney samples from a tissue distribution study in nude mice administered a single dose of 10 mg / kg by weight of Compound 1.

[0103] Fig. 27 FIG. 8B depicts the results of a tissue distribution study from 0.5 h to 96 h in the heart, lung, muscle, spleen, tibia, and femur of nude mice administered a single dose of 10 mg / kg by weight of Compound I.

[0104] Fig.28 A- Fig.28 E is a graph showing the effects of oral administration of various amounts of Compound I on organ weights of male and female Wistar rats ( Fig.28 A- Fig.28 B), liver weight ( Fig.28 C) and weight ( Fig.28 D-2 Figure 8 E) The impact of .

[0105] Fig.28 F- Fig.28 FIG1 is a graph showing the effects on hematological parameters (urea, creatinine, etc.) when various amounts of Compound I are orally administered to male and female Wistar rats.

[0106] Fig.28 J- Fig.28 FIG. 4 is a graph showing the effects on electrolytes when various amounts of Compound I were orally administered to male and female Wistar rats.

[0107] Fig.28 L- Fig.28 FIG. 0 is a graph showing the effects of oral administration of various amounts of Compound I on liver function in male and female Wistar rats.

[0108] Fig.28 P- Fig.28 Q is a graph showing the effects of various amounts of Compound I on renal function in male and female mice after oral administration.

[0109] Fig.29 Depicts the interaction between males and Fig.29 A) and female rats ( Fig.29 B) Histopathological study of liver, kidney, skeletal muscle, heart and spleen organ samples analyzed. Tissue samples were collected on the last day of the treatment period. Summary of the invention

[0110] The present disclosure relates to compounds having anticancer and antiviral activity. The compounds can be used to treat subjects suffering from chronic disorders. The compounds have a structure of Formula 1A

[0111]

[0112] or any derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0113] in:

[0114] R 1 is H, OH or alkoxy;

[0115] R 2 is alkoxy or OH;

[0116] R 3 is alkoxy or OH;

[0117] X is C 1 -C 15 Alkyl, C 2 -C 15 Alkenyl, C 2 -C 15 an alkynyl or aralkyl chain, each independently substituted with at least one alkoxy, OH, =NH or oxo group;

[0118] Y is H or alkyl; and

[0119] X is located at R 2 The ortho position, or R 1 The counterpoint. DETAILED DESCRIPTION

[0120] definition

[0121] "Alkyl" or "alkyl group" refers to a fully saturated straight or branched hydrocarbon chain having from one to fifteen carbon atoms and which is attached to the rest of the molecule by a single bond. Unless otherwise specifically stated in the specification, an alkyl group may be optionally substituted.

[0122] "Alkenyl" or "alkenyl group" refers to a straight or branched hydrocarbon chain having two to fifteen carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Unless otherwise specifically stated in the specification, an alkylene chain may be optionally substituted.

[0123] "Alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain radical having from two to twelve carbon atoms and having one or more carbon-carbon double bonds. The alkenylene chain is attached to the rest of the molecule by a single bond and to the free radical by a single bond. Unless specifically stated otherwise in the specification, the alkenylene chain may be optionally substituted.

[0124] "Alkoxy" refers to a group having the formula -OR a A group in which Ra is an alkyl, alkenyl or alkynyl group as defined above containing from one to twelve carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted.

[0125] "Aralkyl" or "arylalkyl" refers to a group having the formula -R b -R c A group in which R b is an alkylene or alkenylene group as defined above, and R c is one or more aryl groups as defined above, for example, benzyl, diphenylmethyl, etc. Unless stated otherwise specifically in the specification, an aralkyl group may be optionally substituted.

[0126] "Oxo" refers to the following groups having a double bond with an oxygen atom.

[0127] The term "substituted" as used herein refers to any of the above groups, wherein at least one hydrogen atom is replaced by a bond to a non-hydrogen atom, including but not limited to: halogen atoms, such as F, Cl, Br and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups and triarylsilyl groups; and other heteroatoms in various other atoms. "Substituted" also refers to any of the above groups, wherein one or more hydrogen atoms are replaced by oxygen in heteroatoms (such as oxo, carbonyl, carboxyl and ester groups); and higher-order bonds (such as double bonds or triple bonds) to nitrogen in groups such as imines, oximes, hydrazones and nitrile groups. For example, "substituted" includes any of the above groups in which one or more hydrogen atoms are replaced by: -NR g R h 、-NR g C(=O)R h 、-NR g C(=O)NR g R h 、-NR g C(=O)OR h 、-NR g SO 2 R h 、-OC(=O)NR g R h 、-OR g 、-SR g 、-SOR g 、-SO 2 Rg 、-OSO 2 R g 、-SO 2 OR g , =NSO 2 R g and-SO 2 NR g R h "Substituted" also refers to any of the above groups in which one or more hydrogen atoms are replaced by: -C(=O)R g 、-C(=O)OR g 、-C(=O)NR g R h 、-CH 2 SO 2 R g 、-CH 2 SO 2 NR g R h In the above text, R g and R h "substituted" further refers to any one of the above groups, wherein one or more hydrogen atoms and amino, cyano, hydroxyl, imino, nitro, oxo, sulfo, halogen, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclic radical, N-heterocyclic radical, heterocyclic radical alkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl are replaced by the key of alkyl, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, alkylthio, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclic radical, N-heterocyclic radical, heterocyclic radical alkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl groups. In addition, each of the aforementioned substituents can also be optionally substituted by one or more of the above substituents.

[0128] As used herein, the symbol (hereinafter may be referred to as "point of attachment") represents a point of attachment between two chemical entities, one of which is depicted as being attached to the point of attachment and the other is not depicted as being attached to the point of attachment. For example, Indicates that the chemical entity "XY" is bound to another chemical entity via a bonding point of attachment. In addition, the specific point of attachment to an undescribed chemical entity may be specified by inference. For example, the compound CH 3 -R 3 , where R 3 is H or It means when R 3 When "XY", the attachment point is the same as R3 Described as CH 3 The bonds that bind are identical bonds.

[0129] "Pharmaceutically acceptable salts" include pharmaceutically acceptable base addition salts and acid addition salts, such as metal salts (such as alkali metal and alkaline earth metal salts), ammonium salts, organic amine addition salts and amino acid addition salts and sulfonates, where appropriate. Acid addition salts include inorganic acid addition salts, such as hydrochlorides, sulfates and phosphates; and organic acid addition salts, such as alkyl sulfonates, aryl sulfonates, acetates, maleates, fumarates, tartrates, citrates and lactates. Other examples of acid addition salts include acetates, benzenesulfonics (benzenesulfonic, besylate), benzoates, camphorsulfonates, citrates, ethylenesulfonates, fumarates, gluconates, glutamates, hydrobromides, hydrochlorides, isethionates, lactates, maleates, malates, mandelates, methanesulfonates, mucates, nitrates, palmitates, pantothenates, phosphates, succinates, sulfates, tartrates, p-toluenesulfonates, etc. Examples of metal salts are alkali metal salts such as lithium, sodium and potassium salts; alkaline earth metal salts such as magnesium and calcium, aluminum and zinc salts. Examples of ammonium salts are ammonium and tetramethylammonium salts. Examples of organic amine addition salts are salts containing morpholine and piperidine. Examples of amino acid addition salts are salts containing glycine, phenylalanine, glutamic acid and lysine. Sulfonates include methanesulfonates, toluenesulfonates and benzenesulfonates. When the compound contains an acidic side chain, suitable pharmaceutically acceptable base addition salts include metal salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc, or organic salts made from lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.

[0130] The term "therapeutically effective" as applied to dosage or amount refers to that amount of a compound or pharmaceutical formulation sufficient to produce the desired clinical benefit upon administration to a patient in need thereof.

[0131] In the following detailed description, reference is made to the accompanying drawings, which form a part of the description. In the drawings, similar symbols generally identify similar parts unless the context indicates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as generally described herein and shown in the drawings, can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0132] In general, the present invention discloses novel therapeutic compounds for the treatment of chronic disorders, including cancer.

[0133] In some embodiments, the present invention discloses a compound having a structure of Formula 1A,

[0134]

[0135] or any derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0136] in:

[0137] R 1 is H, OH or alkoxy;

[0138] R 2 is alkoxy or OH;

[0139] R 3 is alkoxy or OH;

[0140] X is C 1 -C 15 Alkyl, C 2 -C 15 an alkenyl or aralkyl chain, each independently substituted with at least one alkoxy, OH, =NH, or oxo group;

[0141] Y is H or alkyl; and

[0142] X is located at R 2 The ortho position, or R 1 The counterpoint.

[0143] In some embodiments of the compound having Formula 1A,

[0144] R 1 It's H.

[0145] R 2 is -OH;

[0146] R 3 It is C 1 -C 3 alkoxy; and

[0147] X is C substituted with 2 oxo groups 4 -C 8 Alkenyl.

[0148] Y is for C 1 -C 3 alkyl.

[0149] In some embodiments, the compound of Formula 1A has a structure of Formula 1

[0150]

[0151] or any derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0152] in:

[0153] R 1 is H, alkoxy or OH;

[0154] R 2 is alkoxy or OH;

[0155] R 3 is alkoxy or OH;

[0156] X is C 1 -C 15 Alkyl, C 2 -C 15 Alkenyl, or aralkyl chains, each independently substituted with at least one alkoxy, OH, =NH, or oxo group.

[0157] In some embodiments of Formula 1, X is

[0158] (i) C substituted with four substituents 6 -C 10 The substituents are independently selected from the group consisting of: oxo, -OH and C 1 -C 3 Alkoxy;

[0159] (ii) Oxygenated and C 1 -C 3 Alkoxy substituted C 8 -C 12 alkenyl;

[0160] (iii) C substituted with 2 oxo groups 4 -C 8 alkenyl;

[0161] (iv) Oxygenated and C 1 -C 3 Alkoxy substituted C 2 -C 6 alkenyl;

[0162] (v) C substituted with =NH 1 -C 6 alkyl;

[0163] (vi) C substituted by -OH 2 -C 3 alkenyl;

[0164] (vii) Oxygenated and 2 C 1 -C 3 Alkoxy-substituted C 8 -C12 alkenyl; or

[0165] (viii) Contains C 6 -C 8 Alkyl and C 6 Aralkyl of aryl, wherein the alkyl is substituted with oxo and the aryl is substituted with two alkoxy groups.

[0166] In some embodiments of Formula 1,

[0167] R 1 It is H;

[0168] R 2 It is C 1 -C 3 Alkoxy;

[0169] R 3 It is C 1 -C 3 alkoxy; and

[0170] X is C 6 -C 1o Alkenyl, substituted with oxo, -OH and 2 C 1 -C 3 Alkoxy group substitution;

[0171] In some embodiments of Formula 1,

[0172] R 1 It is H;

[0173] R 2 It is C 1 -C 3 Alkoxy;

[0174] R 3 It is C 1 -C 3 alkoxy; and

[0175] X is oxidized and C 1 -C 3 Alkoxy substituted C 8 -C 12 alkenyl;

[0176] In some embodiments of Formula 1,

[0177] R 1 It is OH;

[0178] R 2 It is C 1 -C 3 Alkoxy;

[0179] R 3 It is C1 -C 3 alkoxy; and

[0180] X is C substituted with two oxo groups 4 -C 8 alkenyl;

[0181] In some embodiments of Formula 1,

[0182] R 1 It is C 1 -C 3 Alkoxy;

[0183] R 2 It is OH;

[0184] R 3 It is C 1 -C 3 alkoxy; and

[0185] X is C substituted with 2 oxo groups 4 -C 8 alkenyl;

[0186] In some embodiments of Formula 1,

[0187] R 1 It is H;

[0188] R 2 It is OH;

[0189] R 3 is OH; and

[0190] X is C substituted with =NH 1 -C 6 alkyl;

[0191] In some embodiments of Formula 1,

[0192] R 1 It is OH;

[0193] R 2 It is C 1 -C 3 Alkoxy;

[0194] R 3 It is C 1 -C 3 alkoxy; and

[0195] X is C substituted with OH 2 -C 3 Alkenyl.

[0196] In some embodiments of Formula 1,

[0197] R 1 It is H;

[0198] R 2 It is C 1 -C 3 Alkoxy;

[0199] R 3 It is C 1 -C 3 alkoxy; and

[0200] X is oxidized and two C 1 -C 3 Alkoxy-substituted C 8 -C 12 Alkenyl.

[0201] In some embodiments of Formula 1,

[0202] R 1 It is OH;

[0203] R 2 It is C 1 -C 3 Alkoxy;

[0204] R 3 It is C 1 -C 3 alkoxy; and

[0205] X is the number containing C 6 -C 8 Alkyl and C 6 Arylalkyl of an aryl group, wherein the alkyl group is substituted with oxo and the aryl group is substituted with two C 1 -C 3 Alkoxy groups are substituted.

[0206] In some embodiments, the compound having Formula 1A has a structure of Formula 2

[0207]

[0208] or any derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0209] in

[0210] R 1 is H or OH;

[0211] R 2 is alkoxy or OH;

[0212] R 3 is alkoxy or OH; and

[0213] R4 It is C 1 -C 15 Alkyl, C 2 -C 15 Alkenyl, aralkyl, each of which is substituted by at least one alkoxy group, -OH or oxo.

[0214] In some embodiments of Formula 2, R 4 yes:

[0215]

[0216] In some embodiments of Formula 2,

[0217] R 1 It is H;

[0218] R 2 OCH 3 ;

[0219] R 3 OCH 3 ;and

[0220] R 4 yes

[0221] In some embodiments of Formula 2,

[0222] R 1 It is H;

[0223] R 2 OCH 3 ;

[0224] R 3 OCH 3 ;and

[0225] R 4 yes.

[0226] In some embodiments of Formula 2,

[0227] R 1 It is OH;

[0228] R 2 OCH 3 ;

[0229] R 3 OCH 3 ;and

[0230] R 4 yes

[0231] In some embodiments of Formula 2,

[0232] R 1 It is OH;

[0233] R 2 OCH 3 ;

[0234] R 3 OCH 3 ;and

[0235] R 4 yes

[0236] In some embodiments of Formula 2,

[0237] R 1 It is H;

[0238] R 2 OCH 3 ;

[0239] R 3 OCH 3 ;and

[0240] R 4 yes

[0241] In some embodiments, Formula 1A has a structure of Formula 3

[0242]

[0243] or any derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0244] in:

[0245] R 1 is H, alkoxy or OH;

[0246] R 2 is alkoxy or OH;

[0247] R 3 is alkoxy or OH; and

[0248] R 5 It is C 1 -C 12 Alkyl or C 2 -C 12 Alkenyl independently substituted with 1 or 2 substituents selected from the group consisting of: =NH or oxo.

[0249] In some embodiments of Formula 3,

[0250] R 1 It is H;

[0251] R 2 It is OH;

[0252] R 3 is OH; and

[0253] R 5 Yes = NH.

[0254] In some embodiments, R 5 Located in R 2 The adjacent position.

[0255] In some embodiments of Formula 3,

[0256] R 1 Yes-OC 2 H 5 ;

[0257] R 2 It is OH;

[0258] R 3 Yes-OCH 3 ;and

[0259] R 5 yes

[0260] In some embodiments of Formula 3, R 5 Located in R 1 The counterpoint.

[0261] In some embodiments, the compound of Formula 1A has a structure of Formula 4

[0262]

[0263] or any derivative thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0264] in:

[0265] R 1 It is OH;

[0266] R 2 It is C 1 -C 3 Alkoxy;

[0267] R 3 It is C 1 -C 3 alkoxy; and

[0268] R 6 It is C(CH 2 )OH.

[0269] In some embodiments of Formula 4,

[0270] R 1 It is OH;

[0271] R 2 OCH 3 ;

[0272] R 3 OCH 3 ;and

[0273] R 6 It is C(CH 2 )OH.

[0274] In some embodiments, the present invention provides a pharmaceutical composition comprising one or more of the compounds described above and a pharmaceutically acceptable carrier.

[0275] In some embodiments of the present disclosure, pharmaceutically acceptable carriers include gum arabic, animal oil, benzyl alcohol, benzyl benzoate, calcium stearate, carbomer, cetearyl alcohol, cetyl alcohol, cholesterol, cyclodextrin, dextrose, diethanolamine, emulsifying wax, ethylene glycol palmitostearate, glycerol, glyceryl monostearate, glyceryl stearate, glyceryl monooleate, glyceryl monostearate, hydrate, histidine, hydrochloric acid, hydroxypropyl cellulose, hydroxypropyl-β-cyclodextrin (HPBCD), hydroxypropyl methylcellulose (hydroxypropyl methylcellulose (HPMC)), lanolin, lanolin alcohol, lecithin, medium chain triglycerides, metal soaps, methylcellulose, mineral oil, sodium dihydrogen phosphate, monoethanolamine, oleic acid, polyethylene glycol (PEG 3350, PEG 4000, PEG6000), polyoxyethylene-polyoxypropylene copolymer (poloxamers), polyoxyethylene alkyl ethers, polyoxyethylene castor oil, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polysorbates, polyoxyethylene (20) sorbitan monolaurate (Tween 20, polysorbate 20), polyoxyethylene (20) sorbitan monooleate (Tween 80, polysorbate 80), povidone, propylene glycol alginate, saline, sodium chloride, sodium citrate, sodium citrate dihydrate, sodium hydroxide, sodium lauryl sulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sorbitan esters, stearic acid, stearyl alcohol, sunflower oil, tragacanth gum, triethanolamine, vegetable oil, water, xanthan gum, or a combination thereof.

[0276] Any chemical structure of Formula 1A-4 can be prepared according to the exemplary synthetic routes disclosed herein.

[0277] In one embodiment, exemplary compounds I having Formula 1A can be prepared by Scheme I provided below.

[0278]

[0279] Chemical synthesis: In a dry three-necked flask, acetylacetone (5mmol, 0.51ml) and boric oxide (3.5mmol, 0.244g) were dissolved in anhydrous ethyl acetate and stirred at 40°C for 30min. Then the corresponding aldehyde (10mmol) and tributyl borate (10mmol, 2.4ml) were added and stirred for another 30min. n-Butylamine (7.5mmol) was dissolved in anhydrous ethyl acetate and then added over a period of 15min. The mixture was heated to 40°C for 24h. Then 5ml HCl (10%) was added and heated to 60°C for another hour. The aqueous phase was extracted several times with ethyl acetate and the organic layer was purified by Na 2 SO 4 Dry and distill off the solvent. Filter off the insoluble precipitate (part of the product) and recrystallize the residue from various solvents. Purify by column chromatography (eluent toluene / ethyl acetate 8:2) and crystallize from ethanol (80%).

[0280] The synthesized compound having Formula 1A-4 was then subjected to molecular characterization to determine the structure of the compound.

[0281] Characterization:

[0282] The purity of the synthesized product was verified by melting point, thin layer chromatography, HPLC, IR, mass spectrometry and NMR analysis. Figure 1-9 In the above, the structures of the synthesized compounds having Formula 1A-4 are illustrated as Compounds I to IX in Table 1 below.

[0283] Table 1

[0284]

[0285]

[0286]

[0287]

[0288] In some embodiments, compounds of formula 1A-4 and compounds I-IX can encompass cis isomers, trans isomers, or cis isomers and trans isomers. In some embodiments, compounds of formula 1A-4 and compounds I-IX can be mixtures of cis isomers and trans isomers. In some embodiments, compounds of formula 1A-4 and compounds I-IX can be cis isomers (i.e., Z-isomers). In some embodiments, compounds of formula 1A-4 and compounds I-IX can be trans-isomers (i.e., E-isomers).

[0289] In some embodiments, compounds of Formula 1A-4 and compounds I-IX can encompass R or S stereoisomers and mixtures of stereoisomers (e.g., mixtures of diastereomers). In some embodiments, compounds of Formula 1A-4 can be racemic mixtures or enantiomerically pure.

[0290] In some embodiments, compounds of Formula 1A-4 and compounds I-IX are enantiomerically pure (e.g., containing the R or S enantiomer), diastereomerically pure, or contain a mixture of stereoisomers (e.g., a racemic mixture or a mixture of diastereomers). In some embodiments, compounds of Formula 1A-4 are racemic mixtures. In some embodiments, compounds of Formula 1A-4 are enantiomerically pure. In some embodiments, compounds of Formula 1A-4 are diastereomerically pure.

[0291] In some embodiments, an enantiomerically pure compound is one having an enantiomeric excess (ee) greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99%.

[0292] In some embodiments, a diastereomerically pure compound is one having a diastereomeric excess (de) greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99%.

[0293] The compounds of the invention can be used to perform or provide any of the biological functions described herein.

[0294] Pharmaceutical composition

[0295] The present disclosure also includes pharmaceutical compositions comprising a therapeutically effective amount of one or more compounds disclosed herein. In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more compounds having Formula 1A, 2, 3 and / or 4, or a pharmaceutically acceptable salt thereof. In other embodiments, the pharmaceutical composition comprises a therapeutically effective amount of one or more compounds selected from Table 1, or a pharmaceutically acceptable salt thereof.

[0296] In various aspects, a compound having Formula 1A-4 (including compounds in Table 1) or a pharmaceutically acceptable salt thereof can be administered at about 0.001 mg / kg to about 100 mg / kg body weight (e.g., about 0.01 mg / kg to about 10 mg / kg or about 0.1 mg / kg to about 5 mg / kg).

[0297] The concentration of the disclosed compounds in the pharmaceutically acceptable mixture will vary according to several factors, including the dose of the compound to be administered, the pharmacokinetic characteristics of the compound or compounds used, and the route of administration. The medicament may be administered in single doses or repeated doses. The dosage regimen for using the compounds of the invention is selected according to a variety of factors, including the type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; the patient's renal and liver function; and the specific compound or salt thereof used. Treatment may be administered once a day or more frequently, depending on many factors, including the patient's overall health, and the formulation and route of administration of the selected compound or compounds.

[0298] The compounds or pharmaceutical compositions of the present disclosure may be manufactured and / or administered in single or multiple unit dosage forms.

[0299] In some embodiments, the compounds of the present disclosure (compounds of Formula 1-4, and Table 1) are administered to patients suffering from chronic conditions. In the context of some embodiments of the present invention, the term "chronic disorder" refers to, but is not limited to, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, basal cell carcinoma, bladder cancer, brain cancer, brain stem glioma, breast cancer, bronchial adenoma / carcinoid tumor, Burkitt's lymphoma, carcinoid tumor, cerebellar or brain astrocytoma, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic or chronic lymphocytic leukemia, chronic myelocytic leukemia or chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, cytokine production ... Desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma of the brain stem, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip and oral cancer, liposarcoma, lymphoma, male breast cancer, malignant mesothelioma, medulloblastoma, melanoma, Merkel cell skin cancer, mesothelioma, metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasmacytoma, mycosis fungoides, myelodysplastic / myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, ovarian cancer, ovarian germ cell tumor, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian low malignant potential tumor, pancreatic cancer, parathyroid cancer, penile cancer, nasopharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasmacytoma, pleuropulmonary blastoma, primary carcinoma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, cancer of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Wilm's tumor, Parkinson's disease and Parkinson's disorder, Huntington's disease, Alzheimer's disease, multiple sclerosis,Amyotrophic lateral sclerosis, Shy-Drager syndrome, progressive supranuclear palsy, Lewy body dementia, spinal cord ischemia, spinal cord injury, ischemic stroke, cerebral infarction, spinal cord injury, and cancer-related brain and spinal cord injury, multi-infarct dementia, Alzheimer's disease, other cognitive impairment, depression, onychomycosis (fungal infection of the nails), gingivitis and periodontal disease (gum disease), obesity and diabetes. The compounds disclosed herein are also good candidates for severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19) and different cancers with KRAS oncogene mutations.

[0300] In some embodiments, the chronic condition is cancer. In some embodiments, the cancer is colon cancer, prostate cancer, breast cancer, or leukemia. In some embodiments, the cancer is stage 4 cancer. In some embodiments, the colon cancer, prostate cancer, breast cancer, or leukemia is stage 4. In some embodiments, the chronic condition is a KRAS oncogene mutation in various cancers.

[0301] In some embodiments, the chronic condition is a viral infection, such as SARS or COVID-19.

[0302] In certain embodiments, the methods, compounds, and compositions described herein are administered in combination with one or more of the following: other antibody molecules, chemotherapy, other anti-cancer therapies (e.g., targeted anti-cancer therapies, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, or oncolytic drugs), cytotoxic agents, immune-based therapies (e.g., cytokines or cell-based immunotherapy), surgery (e.g., lumpectomy or mastectomy), or radiation therapy, or a combination of any of the foregoing.

[0303] Alternatively, or in combination with the foregoing combinations, the methods and compositions described herein can be administered in combination with one or more of the following: a vaccine (eg, a therapeutic cancer vaccine); or other forms of cellular immunotherapy.

[0304] In another embodiment, the methods, compounds and compositions described herein are used in combination with one, two or all of oxaliplatin, leucovorin or 5-FU (e.g., FOLFOX co-treatment). Alternatively or in combination, the combination further comprises a VEGF inhibitor (e.g., a VEGF inhibitor as disclosed herein).

[0305] Non-limiting examples of additional therapeutic agents that can be combined with the methods disclosed herein include: taxol, imatinib, doxorubicin, paclitaxel, fluorouracil (5-FU), and vinblastine.

[0306] In some embodiments, the methods and compositions described herein can be administered in combination with one or more antiviral agents.

[0307] Non-limiting examples of additional therapeutic agents (e.g., antiviral agents) that can be used in combination with the methods disclosed herein include: remdesivir, lopinavir / ritonavir, favilavir, chloroquine, hydroxychloroquine, azithromycin, or a combination thereof.

[0308] Numbered implementations:

[0309] 1. A compound having formula 1A,

[0310]

[0311] or a pharmaceutically acceptable salt thereof,

[0312] in:

[0313] R 1 is H, OH or alkoxy;

[0314] R 2 is alkoxy or OH;

[0315] R 3 is alkoxy or OH;

[0316] X is C 1 -C 15 Alkyl, C 2 -C 15 Alkenyl, C 2 -C 15 an alkynyl or aralkyl chain, each independently substituted with at least one alkoxy, OH, =NH, or oxo group;

[0317] Y is H or alkyl; and

[0318] X is located at R 2 The ortho position, or R 1 The counterpoint.

[0319] 2. A compound as described in embodiment 1, wherein:

[0320] R 2 It is C 1 -C 3 Alkoxy.

[0321] 3. A compound as described in embodiment 1 or 2, wherein:

[0322] R 3 It is C 1 -C 3 Alkoxy.

[0323] 4. A compound as described in embodiments 1-3, wherein:

[0324] X is C 1 -C 15 Alkyl or C 2 -C 15 Alkenyl groups, each independently substituted with 1, 2, 3 or 4 substituents selected from the group consisting of alkoxy, OH, =NH or oxo groups.

[0325] 5. A compound as described in embodiments 1-4, wherein:

[0326] R 1 It is H or OH.

[0327] 6. A compound as described in embodiments 1-5, wherein:

[0328] Y is H.

[0329] 7. A compound as described in embodiments 1-5, wherein:

[0330] R 1 It is H;

[0331] R 2 is -OH;

[0332] R 3 It is C 1 -C 3 Alkoxyl;

[0333] X is C substituted with two oxo groups 4 -C 8 alkenyl; and

[0334] Y is for C 1 -C 3 alkyl.

[0335] 8. The compound according to embodiment 1, which has a structure of formula 1

[0336]

[0337] or a pharmaceutically acceptable salt thereof,

[0338] in:

[0339] R 1 is H, alkoxy or OH;

[0340] R 2 is alkoxy or OH;

[0341] R 3 is alkoxy or OH; and

[0342] X is C 1 -C 15 Alkyl, C 2 -C 15 Alkenyl, C 2 -C 15 an alkynyl or aralkyl chain, each independently substituted with at least one alkoxy, OH, =NH or oxo group;

[0343] 9. The compound according to embodiment 8, wherein:

[0344] R 1 is H or OH;

[0345] R 2 It is C 1 -C 3 Alkoxy or OH;

[0346] R 3 It is C 1 -C 3 alkoxy or OH; and

[0347] X is C 1 -C 12 Alkyl, C 2 -C 12 an alkenyl group, or an aralkyl group, each independently substituted with 1, 2, 3 or 4 substituents independently selected from the group consisting of an alkoxy group, OH, =NH and an oxo group.

[0348] 10. A compound as described in embodiment 8 or 9, wherein X is:

[0349] (i) C substituted with four substituents 6 -C 10 The substituents are independently selected from the group consisting of: oxo, -OH and C 1 -C 3 Alkoxy;

[0350] (ii) Oxygenated and C 1 -C 3 Alkoxy substituted C 8 -C 12 alkenyl;

[0351] (iii) C substituted with 2 oxo groups 4 -C 8 alkenyl;

[0352] (iv) Oxygenated and C 1 -C 3 Alkoxy substituted C 2 -C 6 alkenyl;

[0353] (v) C substituted with =NH 1 -C 6 alkyl;

[0354] (vi) C substituted by -OH 2 -C 3 alkenyl;

[0355] (vii) is oxidized and has two C 1 -C 3 Alkoxy-substituted C 8 -C 12 alkenyl; and

[0356] (viii) Contains C 6 -C 8 Alkyl and C 6 Aralkyl of aryl, wherein the alkyl is substituted with oxo and the aryl is substituted with two alkoxy groups.

[0357] 11. The compound according to embodiments 8-10, wherein R 2 It is an alkoxy group.

[0358] 12. The compound according to embodiment 11, wherein R 2 It is C 1 -C 3 Alkoxy.

[0359] 13. The compound as described in embodiment 12, wherein R 2 Yes-OCH 3 .

[0360] 14. The compound according to embodiment 8-13, wherein R 3 It is an alkoxy group.

[0361] 15. The compound as described in embodiment 14, wherein R 3 It is C 1 -C 3 Alkoxy.

[0362] 16. The compound according to embodiment 15, wherein R 3 Yes-OCH 3 .

[0363] 17. A compound as described in embodiment 8 or 9, wherein:

[0364] R 1 It is H;

[0365] R 2 It is C 1 -C 3 Alkoxy;

[0366] R 3 It is C 1 -C 3 alkoxy; and

[0367] X is substituted with oxo, -OH or two C 1 -C 3 Alkoxy-substituted C 6 -C 10 alkenyl;

[0368] 18. A compound as described in embodiment 8 or 9, wherein

[0369] R 1 It is H;

[0370] R 2 It is C 1 -C 3 Alkoxy;

[0371] R 3 It is C 1 -C 3 alkoxy; and

[0372] X is oxidized and C 1 -C 3 Alkoxy substituted C 8 -C 12 alkenyl;

[0373] 19. The compound according to embodiment 8 or 9, wherein

[0374] R 1 It is OH;

[0375] R 2 It is C 1 -C 3 Alkoxy;

[0376] R 3 It is C 1 -C 3 alkoxy; and

[0377] X is C substituted with two oxo groups 4 -C 8 Alkenyl.

[0378] 20. The compound according to embodiment 8 or 9, wherein

[0379] R 1 It is C 1 -C 3 Alkoxy;

[0380] R 2 It is OH;

[0381] R 3 It is C 1 -C 3 alkoxy; and

[0382] X is C substituted with two oxo groups 4 -C 8 Alkenyl.

[0383] 21. A compound as described in embodiment 8 or 9, wherein

[0384] R 1 It is H;

[0385] R 2 It is OH;

[0386] R 3 is OH; and

[0387] X is C substituted with =NH 1 -C 6 alkyl.

[0388] 22. The compound of embodiment 8 or 9, wherein

[0389] R 1 It is OH;

[0390] R 2 It is C 1 -C 3 Alkoxy;

[0391] R 3 It is C 1 -C 3 alkoxy; and

[0392] X is C substituted with OH 2 -C 3 Alkenyl.

[0393] 23. A compound as described in embodiment 8 or 9, wherein

[0394] R 1 It is H;

[0395] R 2 It is C 1 -C 3 Alkoxy;

[0396] R 3 It is C 1 -C 3 alkoxy; and

[0397] X is oxidized and two C 1 -C 3 Alkoxy-substituted C8 -C 12 Alkenyl.

[0398] 24. The compound according to embodiment 8 or 9, wherein

[0399] R 1 It is OH;

[0400] R 2 It is C 1 -C 3 Alkoxy;

[0401] R 3 It is C 1 -C 3 alkoxy; and

[0402] X is the number containing C 6 -C 8 Alkyl and C 6 Aralkyl of aryl, wherein the alkyl is substituted with oxo and the aryl is substituted with two alkoxy groups.

[0403] 25. The compound according to embodiment 1, which has a structure of formula 2

[0404]

[0405]

[0406] or a pharmaceutically acceptable salt thereof;

[0407] in:

[0408] R 1 is H or OH;

[0409] R 2 is alkoxy or OH;

[0410] R 3 is alkoxy or OH; and

[0411] R 4 It is C 1 -C 15 Alkyl, C 2 -C 15 Alkenyl, C 2 -C 15 Alkynyl or aralkyl, each of which is substituted with at least one alkoxy, -OH or oxo.

[0412] 26. The compound as described in embodiment 25, wherein R 4 Selected from the group consisting of:

[0413]

[0414] 27. The compound as described in embodiment 25 or 26, wherein R 2 It is an alkoxy group.

[0415] 28. The compound as described in embodiment 27, wherein R 2 It is C 1 -C 3 Alkoxy.

[0416] 29. The compound as described in embodiment 28, wherein R 2 Yes-OCH 3 .

[0417] 30. A compound as described in embodiments 25-29, wherein R 3 It is an alkoxy group.

[0418] 31. The compound as described in embodiment 30, wherein R 3 It is C 1 -C 3 Alkoxy.

[0419] 32. The compound as described in embodiment 31, wherein R 3 Yes-OCH 3 .

[0420] 33. A compound as described in embodiment 25 or 26, wherein

[0421] R 1 It is H;

[0422] R 2 is an alkoxy group;

[0423] R 3 is an alkoxy group; and

[0424] R 4 yes

[0425] 34. The compound of embodiment 25 or 26, wherein

[0426] R 1 It is H;

[0427] R 2 is an alkoxy group;

[0428] R 3 is an alkoxy group; and

[0429] R 4 yes

[0430] 35. The compound of embodiment 25 or 26, wherein

[0431] R 1 It is OH;

[0432] R 2 is an alkoxy group;

[0433] R 3 is an alkoxy group; and

[0434] R 4 yes

[0435] 36. The compound of embodiment 25 or 26, wherein

[0436] R 1 It is OH;

[0437] R 2 is an alkoxy group;

[0438] R 3 is an alkoxy group; and

[0439] R 4 yes

[0440] 37. The compound of embodiment 25 or 26, wherein

[0441] R 1 It is H;

[0442] R 2 is an alkoxy group;

[0443] R 3 is an alkoxy group; and

[0444] R 4 yes

[0445] 38. The compound according to embodiment 1, which has a structure of formula 3

[0446]

[0447] or a pharmaceutically acceptable salt thereof,

[0448] in:

[0449] R 1 is H, alkoxy or OH;

[0450] R 2 is alkoxy or OH;

[0451] R 3 is alkoxy or OH; and

[0452] R 5 It is C1 -C 12 Alkyl or C 2 -C 12 Alkenyl independently substituted with 1 or 2 substituents selected from the group consisting of =NH and oxo.

[0453] 39. The compound as described in embodiment 38, wherein R 5 Yes = NH.

[0454] 40. The compound of embodiment 38 or 39, wherein R 5 Located in R 2 The adjacent position.

[0455] 41. A compound as described in any one of embodiments 38-40, wherein R 2 It's OH.

[0456] 42. A compound as described in any one of embodiments 38-41, wherein R 3 It's OH.

[0457] 43. A compound as described in any one of embodiments 38-42, wherein R 1 It's H.

[0458] 44. The compound as described in embodiment 38, wherein R 5 yes:

[0459]

[0460] 45. The compound of embodiment 38 or 44, wherein R 3 It is an alkoxy group.

[0461] 46. ​​A compound as described in any one of embodiments 38, 44 or 45, wherein R 3 It is C 1 -C 3 Alkoxy.

[0462] 47. A compound as described in any one of embodiments 38 or 44-46, wherein R 3 Yes-OCH 3 .

[0463] 48. A compound as described in any one of embodiments 38 or 44-47, wherein R 1 It is an alkoxy group.

[0464] 49. A compound as described in any one of embodiments 38 or 44-48, wherein R 1 It is C 1 -C 3 Alkoxy.

[0465] 50. A compound as described in any one of embodiments 38 or 44-49, wherein R 1 Yes-OC 2 H 5 .

[0466] 51. A compound as described in any one of embodiments 38 or 44-50, wherein R 2 It is -OH.

[0467] 52. A compound as described in any one of embodiments 44-51, wherein R 5 Located in R 1 The counterpoint.

[0468] 53. The compound of embodiment 1, which has the structure of formula 4;

[0469]

[0470] or a pharmaceutically acceptable salt thereof;

[0471] in:

[0472] R 1 It is OH;

[0473] R 2 It is C 1 -C 3 Alkoxy;

[0474] R 3 It is C 1 -C 3 alkoxy; and

[0475] R 6 It is C(CH 2 )OH.

[0476] 54. The compound of embodiment 53, wherein

[0477] R 1 It is OH;

[0478] R 2 OCH 3 ;

[0479] R 3 OCH 3 ;and

[0480] R 6 It is C(CH 2 )OH.

[0481] 55. A pharmaceutical composition comprising a compound as described in any one of embodiments 1-54 and a pharmaceutically acceptable carrier.

[0482] 56. A method of treating a chronic disorder in a patient in need thereof, the method comprising administering a compound of embodiments 1-54 or a pharmaceutical composition of embodiment 55.

[0483] 57. The method of claim 56, wherein the chronic disorder is acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, basal cell carcinoma, bladder cancer, brain cancer, brain stem glioma, breast cancer, bronchial adenoma / carcinoid tumor, Burkitt's lymphoma, carcinoid tumor, cerebellar or brain astrocytoma, cervical cancer, bile duct cancer, chondrosarcoma, chronic lymphocytic or chronic lymphocytic leukemia, chronic myeloid leukemia or chronic myelocytic leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumors, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), gestational trophoblastic tumors, brainstem gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway gliomas, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, laryngeal cancer, leukemia, lip and oral cancer, liposarcoma, lymphoma, male breast cancer, malignant mesothelioma, medulloblastoma, melanoma, Merkel cell skin cancer, mesothelioma, metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple Myeloma, Multiple Myeloma / Plasmacytoma, Mycosis Fungoides, Myelodysplasia / Myeloproliferative Disorders, Nasal and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Melanoma Skin Cancer, Non-Small Cell Lung Cancer, Oligodendroglioma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma, Ovarian Cancer, Ovarian Germ Cell Tumor, Ovarian Epithelial Cancer (Surface Epithelial-Mesenchymal Tumor), Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Parathyroid Cancer, Penile Cancer, Nasopharyngeal Cancer, Pheochromocytoma, Pineal Astrocytoma, Pineal Germ Cell Tumor, Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumor, Pituitary Adenoma, Plasma Cell Tumor, Pleuropulmonary Blastoblastoma, Primary Cancer, Primary Central Nervous System Nervous system lymphoma, primary liver cancer, prostate cancer, colorectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, Wilm's tumor, Parkinson's disease and Parkinson's disorder, Huntington's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, Shy-Drager syndromesyndrome), progressive supranuclear palsy, dementia with Lewy bodies, spinal cord ischemia, spinal cord injury, ischemic stroke, cerebral infarction, spinal cord injury, and brain and spinal cord injury related to cancer, multi-infarct dementia, Alzheimer's disease, other cognitive impairment, depression, onychomycosis (nail inflammation), Fungal infections https: / / en.wikipedia.org / wiki / Nail_(anatomy)), gingivitis and periodontal disease (gum disease), obesity, diabetes, SARS, COVID-19, or cancer with mutations in the KRAS oncogene.

[0484] 58. The method of embodiment 56 or 57, wherein the chronic disorder is cancer.

[0485] 59. The method of embodiment 58, wherein the cancer is colon cancer, prostate cancer, breast cancer, or leukemia.

[0486] 60. The method of embodiment 58 or 59, wherein the cancer has a KRAS oncogene mutation.

[0487] 61. The method of embodiment 56 or 57, wherein the chronic disorder is SARS or COVID-19.

[0488] Examples

[0489] Example 1. Anticancer activity study (Figure 10)

[0490] The synthesized compounds of formula 1-4 were evaluated for cell proliferation, apoptosis, cell cycle arrest, using MTT assay and flow cytometry to measure the generation of reactive oxygen species and calcium, respectively. The expression of apoptosis and proliferation-related proteins was determined by Western blotting. The effects of the molecules on the expression of apoptosis-related mRNA in cancer cells were detected by RT-PCR.

[0491] Molecule induces apoptosis and cell cycle arrest in pancreatic cancer cell line (PANC)

[0492] When pancreatic cancer cells (PANC) were treated with compound I at different doses (1-100 μg / mL) for 24 and 48 h, cell viability was significantly reduced in a time- and dose-dependent manner. Compared with standard 5-fluorouracil (10 μM), which showed a 30.21 ± 0.21% reduction in viable cells at 3 h, exposure of PANC cells to 10 μg / mL resulted in a reduction of approximately 65.5 ± 0.88% in viable cells. After 24 h of exposure, IC 50The values ​​were 5.74 ± 0.02 μg / ml and 5.21 ± 0.19 μM for 5-FU. Most importantly, these molecules showed an increase of 85.29% ± 0.98 in the protection of normal pancreatic cells, with IC 50 The toxicity of 5-FU was 109.24 μg / ml, while 5-FU showed 40.32±0.98% toxicity at 6 h. The changes in cell morphology were detected by cell membrane blebbing, chromatin condensation and the formation of apoptotic bodies ( Fig. 10A ).

[0493] In vitro studies have shown that the disclosed compounds potentially target proteins involved in the G2 / M checkpoint ( Fig. 10A and 10B ), as it has been shown to induce arrest in this checkpoint in breast, pancreatic, and other cancers, which prevents cells from passing the checkpoint and entering mitosis.

[0494] Cell growth inhibition was demonstrated by induction of apoptosis via the intrinsic pathway (caspases 3 and 9) in a caspase-dependent manner, which resulted in a subsequent loss of mitochondrial potential (ΔΨm) ( Fig. 10B ), increased ROS production and DNA damage, leading to cell cycle arrest at the G0 / G1 phase in a dose- and time-dependent manner.

[0495] The novel molecules are thought to induce cell cycle arrest and senescent changes, possibly by targeting STAT-3, β-catenin / Wnt signaling, MAPK, and / or JAK-1 / 2 / 3 Aurora kinase A, thereby inducing mitotic arrest, altering the expression of cell cycle-related proteins, and disrupting microtubules.

[0496] Example 2. Molecules that inhibit histone deacetylase (HDAC) activity

[0497] Next, the expression of Class I HDAC during pancreatic tumorigenesis was detected. Simply, different concentrations of compound I were added and incubated for 24 hours. Western blot analysis confirmed that the protein expression of Class I HDAC decreased in a dose-dependent manner compared with the control (p<0.05). Compound I significantly inhibited HDAC1, HDAC2, HDAC3 and HDAC8 from Class I. In general, these data show that the inhibition of Class I HDAC by molecules is sufficient to induce cell death in PANC cell lines. These results show that the molecule is an effective HDAC inhibitor. The inhibition of Class I HDAC is associated with the upregulation of histone H3 acetylation and p21 mRNA and protein expression, which is associated with antiproliferative activity. HDAC6 function is also reported as α-tubulin deacetylase, regulating tubulin stability. Because α-tubulin and histone H3 are common downstream targets of HDAC, the relationship between protein expression and molecular function is further studied. The effect of compound I on histone H3 acetylation in PANC cells was studied using Western blot analysis. Compound I induced stronger hyperacetylation of histone H3 compared to the control, consistent with its potent inhibitory effect on class I HDAC1, but no acetyl-α-tubulin was detected.

[0498] Mechanism of action Fig.11 )

[0499] Without being bound by any particular theory, the compounds disclosed herein may exhibit one or more of the following mechanisms of action:

[0500] ■ May induce cell cycle arrest and senescence changes by targeting STAT-3, β-catenin / Wnt signaling, MAPK, JAK-1 / 2 / 3 Aurora kinase A, induce mitotic arrest, and alter the expression of cell cycle-related proteins and disrupt microtubules.

[0501] ■Results in loss of mitochondrial membrane potential, cytochrome c release, Bax upregulation, Bcl-2 downregulation, and caspase-3 cleavage, indicating activation of mitochondrial-mediated apoptosis.

[0502] ■Multi-targeted kinase inhibitors inhibit the proliferation of various human cancer cells and cancer stem cells in vitro and in vivo through multiple pathways.

[0503] ■Histone deacetylase (HDAC) inhibitors and histone methyltransferase (HMT) inhibitors.

[0504] ■ Downregulates the levels of HDAC2 and HDAC3 at both the mRNA and protein levels, and has the potential to also downregulate the levels of KLF4, which plays an important role in stem cell formation.

[0505] ■Downregulate hTERT levels compared to the standard drug suberoylanilide hydroxamic acid (SAHA).

[0506] To elucidate the mechanisms underlying the antiproliferative effect, cell cycle distribution and apoptosis of PANC cells were investigated. Early apoptosis was observed as early as 6 h by binding of annexin V-fluorescein to the cell membrane. Compounds I-IX activated the intrinsic pathway of apoptosis by inducing caspase-3 and caspase-9. Involvement of the intrinsic pathway in mitochondria was seen, with a significant increase in mitochondrial permeability and cytochrome c release, while mitochondrial membrane potential decreased. Apoptosis was confirmed at the protein level, including Bax, Bcl-2, and survivin, while cell cycle disruption was used for the final validation of apoptosis. Propidium iodide / Annexin V double staining revealed a pro-apoptotic cell population in cells treated with PANC at 3 h. In addition, we observed molecular-induced cell cycle arrest associated with the important checkpoint control protein p21 as determined by fluorescence-activated cell sorting (FACS) analysis. WAF1 / KIP1 、p27 KIP1 , p53, and cyclin A, leading to G0 / G1 arrest in the PANC cell line, downregulating the expression of cyclin D3, cyclin E1, CDK2, CDK4, and CDK6, and upregulating the expression of p21, p27, and p53 via HDAC inhibition.

[0507] Example 3. Neurogenesis and Neuronal Development Molecule-Molecule-Induced Neuronal Cell Type Formation in Human Umbilical Cord Stem Cells ( Fig.12 )

[0508] At the beginning of induction, HUMSC cultures phenotypically showed large, thin, flat cell bodies with large nuclei. MSCs treated with molecules showed morphological changes within 48-72 hours. At this time, the MSC population showed spherical, refractive cell bodies with dendritic processes and slender axon-like processes extending from the cell body, which are typical characteristics of neurons. After 72 hours of treatment, the proportion of neuron-like cells reached a peak, in which the cell body shrank and the processes were further extended. MSCs maintained this morphology during induction.

[0509] Example 4. KRAS degradation

[0510] In short, the present disclosure provides compounds that can regulate G12C mutant KRAS proteins. In some cases, the compound, as an electrophilic agent, can form a covalent bond with a cysteine ​​residue at the position of the mutant protein K-Ras4BG12C / G12D / G12V-GTP / GDP, K-Ras4BG13D-GTP / GDP, K-Ras4BQ61H-GTP / GDP. A method for using such compounds to treat various diseases or conditions (such as cancer) is also provided. Additional mechanism studies have shown that compound AB-REV-001 can block the formation of guanosine triphosphate (GTP) and KRAS complexes in vitro. In addition, AB-REV-001 inhibits KRAS downstream signaling pathways RAF / MEK / ERK and RAF / P13K / AKT.

[0511] Example 5. Cancer stem cell inhibition

[0512] Cancer stem cells (CSCs) are believed to exhibit unique self-renewal, proliferation, and differentiation capabilities and therefore play important roles in various aspects of cancer. CSCs have important impacts on tumor progression, drug resistance, recurrence, and metastasis of different types of malignancies. Conventional cancer chemotherapy often fails because most anticancer drugs are ineffective against drug-resistant CSCs. These surviving CSCs lead to recurrence and metastasis. Most studies have reported that conventional therapeutic agents have limited effects on CSCs due to their hypoxic microenvironment and location away from the vasculature, which hinders the efficacy of anti-CSC drugs ( Fig.14 ). However, the disclosed compounds can overcome this limitation and can penetrate deeper and destroy CSCs. Therefore, in an embodiment, the present invention provides a method for inhibiting cancer stem cell survival and / or self-renewal, comprising administering to the cancer stem cells an effective amount of a compound disclosed herein, such as Compound I ( Fig.13 ).

[0513] Fig.17Abnormal signal transduction pathways in CSCs and strategies to target CSCs are described. Signal transduction pathways in CSCs play important roles in self-renewal, drug resistance, tumor recurrence, and distant metastasis. Signal transduction pathways Notch, Wnt, and Hedgehog signaling and downstream effectors, including transcription factors -catenin (-cat), signal transducer and activator of transcription 3 (STAT3), and Nanog play key roles in CSC characteristics. After interaction with xCT, CD44 variant (CD44v) enhances glutathione synthesis and defense against reactive oxygen species (ROS). Due to this abnormal state, CSCs acquire a unique phenotype. The best way to eradicate CSCs is to identify molecules responsible for the specific properties of CSCs, rather than those of normal cells. Target CSC phenotypes include: Delta-like ligand (DLL), Frizzled (FZD), Janus kinase (JAK), lipoprotein receptor-related protein (LRP), Patched (Ptch), Sonic Hedgehog (Shh), and Smoothened (Smo).

[0514] It has been demonstrated that the compounds of the present disclosure selectively target cancer stem cells in the tumor microenvironment and destroy CSCs by regulating genes for self-renewal and differentiation. The compounds of the present disclosure inhibit the expression of genes in the following cancers: Fig.18 ):

[0515] Breast cancer: CD44+CD24- / lineage-low, ALDH-1 high

[0516] Liver cancer: CD133+, CD49f+, CD90+

[0517] Colon cancer: CD133+, CD44+, CD166+, EpCAM+, CD24+

[0518] Pancreatic cancer: CD133+, CD44+, EpCAM+, CD24+

[0519] Leukemia: CD34+CD38-

[0520] Lung cancer: CD133+, ABCG2 高

[0521] Leukemia: CD34+, CD38-, HLA-, DR-, CD71-, CD90-, CD117-, CD123+

[0522] The reduction in stem cell surface marker expression was visible within one hour and up to 15 hours. In contrast to other standard drugs, no cancer stem cell markers were observed after 24 hours of treatment.

[0523] Example 6: In vitro cancer studies with Compound I

[0524] Chemotherapy is a drug therapy that uses powerful chemicals to kill fast-growing cells in the body. However, treatment shows adverse side effects, which may be simple gastritis and alopecia to severe bone marrow suppression, cardiotoxicity, etc. In terms of cytotoxicity, the effect of the combination of Compound I of the present invention and chemotherapeutic drugs has been studied. As described in detail below, if cells are treated with Compound I of the present invention before treating chemotherapeutic drugs, the cytotoxicity of chemotherapeutic drugs (e.g., docetaxel, paclitaxel, pazobanib, Endoxon, etoposide, adriamycin, dacromycin, Avastin, gemcitabine, cisplatin and oxaliplatin) is significantly reduced.

[0525] The cytotoxicity of anticancer agents was studied using a group of individual normal human cell lines and in combination with Compound I of the present invention. Cytotoxic effects were determined by MTT assay. The following cell lines were used: human epidermal keratinocytes (HaCaT), human dermal fibroblasts (HDF), human bone marrow mesenchymal stem cells (HBMSC), human normal hepatocytes (THLE2), human cardiomyocytes (AC-16), human intestinal epithelial cells (HIEC-6), human neuronal cells (SHSY-5Y), human vascular endothelial cells (HuVEC), human lung epithelial cells (Calu-3), human lung fibroblasts (MRC 5).

[0526] According to the data, after treatment with different concentrations (1-500 μM) of anticancer agents, normal cells were significantly reduced (p>0.05) compared with the control, and morphological changes were observed. It was found that the IC50 values ​​of doxorubicin, gemicitanib, 5-FU, cisplatin, lenalidomide, irinotecan, chloroquine, hydroxychloroquine, vincristine and vinblastine ranged from 12.90 μM to 40.50 μM ( Fig.19 A). Significant levels of apoptosis were observed in all treated normal cell lines compared to the negative control, showing their unique signature characteristics.

[0527] Next, the cells were treated with an anticancer agent in combination with Compound I of the present invention. After the combined treatment, an increase in cell viability was observed, and no signs of cell rounding, granulation, and cell shrinkage were observed, indicating that treatment of normal cells with Compound I reduced the toxic effects induced by the anticancer agent. An increase in IC50 was found compared to treatment with the anticancer agent alone. The average treatment in all cell lines showed IC50>100 μM, which shows the cytoprotective effect of Compound I ( Fig.19B). No significant apoptotic effect was observed in the combination study, indicating that compound I reduced the cytotoxicity produced by the chemotherapeutic agent. Even at the highest concentration, no significant cytotoxic effect was observed in normal cell lines.

[0528] Example 7: In vivo cancer studies with Compound I

[0529] Metastasis Research

[0530] In vivo studies have shown that tumor growth can be suppressed in a xenograft nude mouse model with an improved therapeutic window compared to standard drug treatment. Notably, no recurrence or relapse of tumors was observed in a follow-up study after 6 months.

[0531] The compounds of the present disclosure showed in vivo efficacy with 90% TGI (tumor growth inhibition) in triple negative breast cancer, pancreatic cancer model, liver cancer model and colon cancer model, without any mortality growth inhibition ( Fig. 20 ).

[0532] Furthermore, the compounds of the present disclosure significantly disrupted the surrounding ECM organization, resulting in increased quiescence, apoptosis, improved chemosensitivity, reduced invasiveness, metastatic spread, and a 6-fold reduction in tumor volume and cancer progression in vivo compared to gemcitabine and 5-fluorouracil treatment.

[0533] Further anticancer effects were demonstrated when single administration of the disclosed compounds was combined with standard drug regimens, showing significant inhibition of tumor recurrence in vivo.

[0534] Pancreatic cancer xenograft model (PANC-1)

[0535] Research design: Based on Fig.21 The PANC-1 xenograft mouse model was performed using the experimental design described in A. Tumor volume was monitored over 30 days following administration of PBS alone (vehicle control), 25 mg / kg gemcitabine, and 10 mg / kg Compound I.

[0536] Results: Fig.21 As shown in Figure B, from day 5 to day 30, tumor volume increased steadily in mice treated with 25 mg / kg gemcitabine (n=12). In contrast, mice treated with 10 mg / kg compound I had reduced tumor volume over the same period. This result was confirmed by imaging studies showing that no tumors were detected in mice treated with compound I ( Fig.21 C).

[0537] The reduction in tumor volume translated into improved survival of mice treated with Compound I ( Fig.21 D).

[0538] Comparison with standard drugs

[0539] Compared to current standard of care drugs (Table 1 below): Revlimid, Avastin, Herceptin, 5-fluorouracil and gemcitabine, the compounds of the present disclosure are 50 times more effective in reducing the number of tumor spheres and 100 times more effective in reducing the CSC population and preventing tumor recurrence.

[0540] No significant toxicity and mortality were observed in vital organs compared with standard drugs for treating stage IV cancer such as Revlimid (100% mortality), Avastin (60% mortality), Herceptin (60% mortality), 5-fluorouracil (70% mortality), and gemcitabine (50% mortality).

[0541] Furthermore, when administered in combination with standard drugs, the compounds of the present disclosure reduce mortality and enhance the sensitivity of tumor cells, thereby improving the therapeutic effect.

[0542] Table 1: 9 breakthrough small molecules and standard drugs

[0543]

[0544] Downregulation of migration genes and signaling pathways

[0545] Compared to standard drugs that exhibit limited access to CSCs due to the hypoxic environment, the compounds disclosed herein can prevent metastasis to the lung and lymph nodes by inhibiting lymphangiogenesis and angiogenesis (VRGFR) in pre-metastatic organs.

[0546] Without being bound by any particular theory, multi-tyrosine kinase inhibitors can induce apoptosis and inhibit cancer cell invasion by inhibiting activated NF-κB, Akt, ERK2, Tyk2 and PKC. Without being bound by theory, these compounds also reduce migration and invasion by inhibiting the PI3K / Akt / mTOR signaling pathway. Without being bound by any particular theory, the compounds effectively inhibit metastasis, angiogenesis and cancer cell invasion via ERK1 / 2-, Akt / NF-κB / mTOR- and p38 MAPK-dependent NF-KB signaling pathways.

[0547] Regulation of epithelial-mesenchymal transition (EMT)

[0548] All 9 compounds disclosed herein inhibit tumor growth in xenograft mouse models and modulate the expression of mesenchymal and epithelial markers. They suppress the expression of mesenchymal genes (such as fibronectin, vimentin, N-cadherin, TWIST and SNAIL) and increase the expression of epithelial genes (such as occlusion and E-cadherin) by specifically targeting the canonical WNT / p-catenin / Hedgehog, TGFβ / BMP-SMAD pathways.

[0549] Immunomodulation

[0550] The compounds of the present disclosure increase the number and activity of cytotoxic T cells and promote the activation of macrophages, NK cells and DCs, which promote the conversion of APCs to CD4+ and CD8+ by capturing, internalizing, processing and presenting tumor antigens via class I and class II MHC molecules.

[0551] In vivo tumors treated with the molecules further showed reduced ECM deposition and impaired CD31 + Endothelial cells, α-SMA + Infiltration of cancer-associated fibroblasts and F4 / 80+ macrophages indicated that the treatment generated a suppressive tumor microenvironment, which in turn suppressed tumor growth, invasion, and metastasis.

[0552] Example 8: Targeting lung cancer stem cells

[0553] Compound I has a significant effect on targeting specific lung cancer stem cells, and induces apoptosis and inhibits metastasis without damaging normal cells, which is helpful for the treatment of cancer therapy. Compound I is an orally bioavailable multi-tyrosine-kinase inhibitor with 120 target proteins, which induces apoptosis and suppresses the invasion of lung cancer stem cells without toxicity to normal cells. No signs of discomfort or any cardiovascular or respiratory disorders were observed in the animals after administration throughout the study.

[0554] Example 9: Compounds of the Disclosure as Inhibitors of ACE-2 and Nsp15

[0555] Objective: To determine whether compounds of the present disclosure (e.g., Compound 1) are potential inhibitors of ACE-2 and Nsp15, proteins responsible for the entry and replication of viruses such as SARS-CoV-2 in human cells.

[0556] Background: To infect a human host, viruses must be able to enter individual human cells. Viruses exploit the host cell machinery to produce copies of themselves, which then spill over and spread to new cells. Studies have shown that SARS-CoV-2 attaches to target cells through an interaction between its spike protein (S) and the host cell protein angiotensin-converting enzyme-2 (ACE-2), a transmembrane enzyme found on the cell surface. This interaction on the host cell surface is important because it initiates the infection process ( Fig. 22 ). Therefore, drugs that modulate the biological activity of ACE-2 are considered potential candidates for the treatment of this viral infection.

[0557] Another protein identified from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that may play a role in viral progression is Nsp15 ( Fig. 22 ). Nsp15 is 89% identical to an early outbreak protein from SARS-CoV. Analysis of SARS-CoV showed that inhibition of Nsp15 could slow viral replication. The newly mapped protein is conserved among coronaviruses and is essential in their life cycle and virulence. Initially, Nsp15 was thought to be directly involved in viral replication, but more recently it has been proposed that it may aid viral replication by interfering with the host's immune response.

[0558] Results: To evaluate the potential of the disclosed compounds to target ACE-2 and Nsp15, computer simulation methods and molecular docking were used to study. From these analyses, it was found that compound I had high binding affinity to all tested viral proteins ( Figure 22-2 4). Specifically, Fig.21 Compound I was shown to have affinity for the ACE-2 binding site, which resulted in disruption of the interaction between the virus and the receptor. Compound I was also found to have affinity for the Nsp15 binding site, as Fig.24A -C.

[0559] Summary: The present disclosure provides small molecule inhibitors targeting S-ACE-2 that mediates SARS-CoV-2 entry into human cells. In addition, the compounds disclosed herein also target Nsp15, which can slow viral replication. Therefore, compounds having formula 1-4 have the potential to be developed as effective drugs against COVID-19.

[0560] Example 10: In vitro antiviral activity of compound I in Vero cells

[0561] Study Design: Standard assays were performed to measure the effects of Compound I on SARS-CoV-2 cytotoxicity, viral yield, and infectivity ( Fig.25First, the cytotoxicity of compound I was tested on Vero cells. Then, SARS-CoV-2 ( Fig.25 A) Vero cells were infected with a multiplicity of infection (MOI) of 0.1 PFU / cell, and DMSO was used as a carrier. Efficacy was assessed by quantifying viral copy number in cell supernatants via quantitative real-time RT-PCR (qRT-PCR), and visualization of viral nucleoprotein (NP) expression was confirmed by immunofluorescence analysis at 48 h post-infection (pi) ( Fig.25 C). In addition, after infection, cells were exposed to test compounds to further evaluate their potential as prophylactic and therapeutic agents against coronaviruses. The protective effect of compound I was also evaluated in a lung epithelial (Calu-3) cell line.

[0562] Antiviral activity and cytotoxicity: To test the antiviral activity of compound I, Vero cells were infected with SARS-CoV-2 isolates at an MOI of 0.1 for 2 h ( Fig.25 B), followed by the addition of different concentrations of compound I (0.001 μM, 0.01 μM, 1 μM, 3 μM, 5 μM, respectively). According to the evaluation of the data obtained from the dose-response curve, it was found that compound I effectively exhibited antiviral activity, with an IC50 of 1 μM ( Fig.25 B, blue line, represents the average % inhibition of the virus), and the CC50 is >15 μM ( Fig.25 B, red line, represents the cytotoxicity of the drug).

[0563] To further determine the effectiveness of Compound I, cells infected with SARS-CoV-2 were treated with serial dilutions of Compound I, and supernatants and cell pellets were collected 2 h after infection for real-time RT-PCR ( Fig.25 F- Fig.25 H).

[0564] At 24 h, there was a 100% reduction in viral RNA (indicative of released virions) present in the supernatant of samples treated with Compound I compared to vehicle DMSO. Similarly, a 99% reduction in cell-associated viral RNA (indicative of unreleased and unpackaged virions) was observed with Compound I treatment. At 36 h, this effect was added to the reduction in viral RNA in Compound I treatment, which showed statistical significance when compared to control samples, indicating that Compound I treatment resulted in an effective loss of essentially all viral material at 36 h.

[0565] At 48 h, a reduction in viral RNA was observed in both the supernatant and cell pellet of samples treated with 1 μM Compound I, equivalent to a 99% reduction in viral RNA in these samples compared to control samples. Likewise, no toxicity was observed with Compound I at any concentration tested.

[0566] After treatment with compound I, immunofluorescence microscopy of virus-infected cells was performed. Specifically, Vero cells were infected with SARS-CoV-2 at an MOI of 0.1 and treated with 1 μM and 1 μM of compound I. After 48h pi, the infected cells were fixed and then probed with rabbit serum against NP of SARS-related CoV as the first antibody and Alexa 488-labeled goat anti-rabbit IgG as the second antibody. The nuclei were stained with Hoechst dye. Scale bar, 20 μm. The staining results showed that in Vero cells treated with either dose of compound I, the viral load measured by nucleoprotein expression was significantly reduced ( Fig.25 C). These results were confirmed by Western blot analysis of infected cells 24 h pi ( Fig.25 D) and quantified by normalization to GAPDH ( Fig.25 E).

[0567] Example 11: In vitro activity of Compound I in lung epithelial cells

[0568] The antiviral activity of compound I against SARS-CoV-2 was again evaluated in Calu-3 cultures. Dose-dependent reductions in replication proximity were observed at 0.1-5 μM, with an average IC50 value of 0.01 μM (SARS-CoV-2) compared to untreated controls. To determine whether compound I could inhibit replication, we first evaluated antiviral activity and cytotoxicity in a continuous human lung epithelial cell line, Calu-3 cells. Compound I inhibited SARS-CoV-2 replication in cells, with an average half-maximal effective concentration (IC50) value of 1.5 μM ( Fig.25 B). Importantly, we did not observe any cytotoxicity at concentrations up to 100 μM, thus demonstrating that the 50% cytotoxic concentration (i.e., CC50) of Compound I in Calu-3 cells exceeds 100 μM. Taken together, these results suggest that substantial inhibition of SARS-CoV-2 replication will be achieved at low micromolar concentrations in lung epithelial cells.

[0569] Example 12: Effect of Compound I on the Stage of Viral Infection

[0570] It was hypothesized that compound I would inhibit early replicating SARS-CoV-2 by inhibiting viral RNA synthesis. To test this hypothesis and determine at which stage of the viral replication cycle compound I inhibits SARS-CoV-2, Vero cells were infected with a multiplicity of infection (MOI) of 0.1 PFU / cell, resulting in a single-cycle infection, and treated with 1 μM compound I at 2h intervals from 2h before infection to 10h after infection. The greatest inhibition was observed when compound I was added between 2h before infection and 2h after infection. Less inhibition was detected when compound I was added between 6 and 8h after infection, and no inhibition was observed when compound I was added after 10h after infection. These results demonstrate that compound I inhibits SARS-CoV-2 in the early stages of infection. Because viral RNA is synthesized early in infection and compound I is associated with inhibiting viral RNA synthesis, after treatment with compound I, the cellular levels of viral RNA were next determined by real-time quantitative PCR (qPCR). Treatment with increasing concentrations of compound I resulted in reduced viral RNA levels, which were observed to be associated with reduced titers. These results suggest that compound I inhibits SARS-CoV-2 early after infection by interfering with viral RNA replication.

[0571] Compound I was studied in a normal epithelial cell SARS-CoV-2 infection model of human lung epithelial cells (calu-3 cell line) to see if it could also inhibit SARS-CoV-2 replication in human cells. These results show that compound I is able to inhibit SARS-CoV-2 replication at the same concentration as in VERO cells (IC 50 10 μM compound I treated cells = <102 TCID50 / m1), indicating that the antiviral activity of compound I is independent of the cell type ( Fig.26 A). As for other coronaviruses, expression of the SARS-CoV genome is mediated by translation of genomic RNA and a set of "nested" subgenomic messenger RNAs, which are generated by a unique mechanism involving discontinuous transcription during RNA synthesis. To determine whether compound I acts by blocking viral RNA synthesis in the case of SARS COV-2, Calu-3 cells were infected with SARS-CoV-2 shortly after the viral adsorption period and treated with different concentrations (1.5 and 10 μM) of compound I. Total RNA was extracted 24h after pi and analyzed by RT-PCR. Fig.26As shown in Figure B, compound I treatment resulted in a dose-dependent reduction in intracellular SARS-CoV-2 RNA levels, reaching more than 95% inhibition of the control at a concentration of compound I that did not affect RNA synthesis in uninfected cells. Viral genomic RNA was quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the clarified supernatant. Like the effect on infectious titers, a dose-dependent reduction was found in viral genomic RNA, and the IC50 was similarly calculated to be 5 μM. Collectively, these data demonstrate that compound I has a potent antiviral effect against two genetically distinct emerging CoVs.

[0572] Example 13: Immune Response to Compounds of the Disclosure

[0573] Compound I has the ability to activate CD4+ helper T cells and CD8+ cytotoxic T cells, and generate immune responses in vivo to defend against viral infection. Specifically, Compound I increases the number and activity of cytotoxic T cells, and promotes the activation of macrophages, NK cells, and DCs, which promote the conversion of APCs to CD4+ and CD8+ by capturing, internalizing, processing, and presenting antigens via class I and II MHC molecules.

[0574] Example 14: Proposed Mechanism of Action in the Lung

[0575] In a xenograft stage 4 metastatic breast cancer animal model, Compound I reduced the production of cytokines (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokines (CXCL10, CCL2, CCL3, CCL5) and correlated with the migration of natural killer cells and macrophages and observed in the lungs. From data collected from QPCR gene expression studies, at day 7, histopathological evidence showed normal lung pathology, without any pneumonitis observed, and reduced expression of cytokines (TNF-α, IL-6, gamma interferon [IFN-γ], IL-2, and IL-5), chemokines (CXCL9, CXCL10, CCL2, CCL3, and CCL5), and receptors (CXCR3, CCR2, and CCR5) was detected in the lungs, which correlated with the influx of T lymphocytes. No signs of clinical disease and histopathological evidence of disease characterized by bronchiolitis, interstitial pneumonia, diffuse alveolar damage, and fibrotic scarring were observed.

[0576] Example 15: Potentiating Hydroxychloroquine by Reducing Side Effects in COVID-19 Treatment

[0577] Coronavirus disease (COVID-19) is an infectious disease caused by the virus SARS-CoV-2. The disease causes respiratory illness, where symptoms include coughing, fever, and in more severe cases, difficulty breathing. Currently, one drug that may be effective in treating COVID-19 is hydroxychloroquine. However, the drug hydroxychloroquine can cause serious side effects, including cardiovascular disease, eye damage, mild or severe bronchospasm, and can also affect mental health. The effect of Compound I of the present invention in reducing the side effects associated with hydroxychloroquine was studied according to methods known in the art. It was found that Compound I of the present invention helps to enhance the efficacy of hydroxychloroquine ( Fig. 20 and 21 ).

[0578] Example 16: Pharmacokinetics (PK), tissue distribution and toxicity

[0579] The in vivo pharmacokinetics and toxicity of compound I were studied in a nude mouse model

[0580] PK studies :

[0581] Data analysis showed that the mean plasma concentration of Compound I was significantly higher than that of the control group throughout the 6 h sampling period. After intravenous administration, Compound I was widely distributed in several tissues, including hippocampus, heart, lung, stomach, liver, breast, kidney, spleen, femur and tibia ( Fig. 27 A- Fig. 27 B). As compared with the control group, the maximum plasma concentration (Cmax) of the treatment group increased 29-fold, and the area under the curve (AUC) increased 28-fold (p>0.05).

[0582] Notably, Compound I is widely distributed in several organs, and especially those with high porosity, which is consistent with its pharmacodynamic activity in these organs. In addition, when compared to the control, the distribution volume of Compound I after IV administration was found to be significant, which means good tissue distribution. Tissue distribution in the current study showed that Compound I achieved appropriate pharmacodynamic activity levels in the hippocampus, femur, tibia, and mammary gland at 10 mg / kg IV, which is relevant to the prevention and treatment goals of neurodegenerative diseases and other chronic diseases ( Fig. 27 B). No toxicity was observed.

[0583] At 1h, 2h, 4h, 24h after administration, the ratio of the tissue and plasma of compound I was recorded in the animals. At 60min, the highest level of compound I was found in the lungs, brain, stomach, liver, mammary gland and small intestine, all of which were highly perfused organs, followed by spleen and heart. Until 72h, compound I was detected in most organs, but not in femur and kidney. The ratio in most organs continued to increase to 4h. Interestingly, from 1, 2 and 24 hours after administration, the tissue and plasma ratio of compound I in the hippocampus and brain increased significantly and continuously. After administration and during the entire study period, no signs of discomfort or any cardiovascular or respiratory disorders were observed in the animals. In the 7 days after drug administration, the monitored body weight did not change.

[0584] PK Results: Pharmacokinetic studies conducted with the compounds disclosed herein revealed prolonged persistence in systemic blood circulation and no nephrotoxicity, cardiotoxicity and hepatotoxicity were observed compared to other standard drugs that showed severe cardiotoxicity, hepatotoxicity, gastrointestinal toxicity and respiratory disturbances.

[0585] Summary: The novel molecules of the present disclosure show enhanced pharmacokinetics, biodistribution and tolerability when compared to standard drug administration. In vivo biodistribution studies showed that the accumulation of the novel molecules in tumors of animal models was significantly higher than other organs analyzed (P<0.01).

[0586] Acute oral toxicity studies :

[0587] Experimental design: Acute toxicity determination was performed according to the Organization for Economic Cooperation and Development (OECD) Guide 423 (OECD, 2001a). A total of 60 mice weighing between 27 and 37 g were randomly divided into six experimental groups, 10 mice per group (5 males and 5 females per group). After fasting overnight, compound I was administered by oral gavage at a single dose of 200 mg / kg, 500 mg / kg, 1000 mg / kg, and 2000 mg / kg to each treatment group, respectively. The control group was treated with the same volume of distilled water. After administration, the mortality rate and general behavioral changes of all animals were observed within the first 30 min, and then at 2h, 4h, 6h, 10h and 24h after treatment, respectively. After administration of different doses, toxic symptoms such as hypoactivity, hair erection, dyspnea, tremor and convulsion were evaluated. The LD 50 value was determined according to the method described by OECD Guide 423 (OECD, 2001a). During the remaining experimental period, animal observations were performed at least once a day during the 14-day post-dose period. Body weights were measured at the beginning of treatment and on the 4th, 7th, 11th, and 14th days after administration. On the 14th day, mice were sacrificed under anesthesia and vital organs (heart, kidney, lung, spleen, and liver) were removed for macroscopic examination.

[0588] Subtoxicity study :

[0589] Experimental design: Subchronic toxicity studies were conducted according to OECD Test Guidelines 408 (OECD, 2008) for testing chemicals. A total of 48 male and female Wister rats weighing between 170 and 240 g were randomly divided into four groups (n=6 males and 6 females / group). Rats in the treatment group received compound I orally at doses of 200 mg / kg / day, 500 mg / kg / day, 1000 mg / kg / day and 2000 mg / kg / day. Compound I was administered by oral gavage at 10 mL / kg body weight, based on daily use, for 28 days. Rats in the control group were orally administered with the same volume of distilled water (vehicle). During the experiment, the body weight of all groups was measured once a week. Animals were also visually observed for mortality, changes in behavioral patterns, changes in physical appearance, and disease symptoms. At the end of the treatment period, all rats were fasted overnight (12h-16h) and then anesthetized with urethane by intraperitoneal injection (1mL / 100g body weight). Blood samples were collected for measurement of hematological (EDTA-2K coated tubes) and biochemical (dried tubes) parameters.After euthanasia, rats were sacrificed and organs were removed for necropsy, organ weight measurement and histopathological examination.

[0590] Urinalysis: Urinalysis was performed in all rat groups during the last week of the treatment period. Fresh urine was collected from all animals overnight to determine the levels of specific gravity, pH, leukocytes, nitrites, protein, glucose, ketones, blood, urobilinogen, and bilirubin. Urine samples were analyzed using an automated urine analyzer and test strips.

[0591] Hematology and serum biochemistry: For hematology studies, all animals were fasted overnight, but allowed to drink water ad libitum. The rats were then anesthetized, and blood samples were collected from the abdominal aorta. Whole blood was collected in EDTA tubes (containing the potassium salt of ethylenediaminetetraacetic acid) and immediately processed for hematological analysis. The parameters measured were red blood cell count (RBC), hematocrit (HCT), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), white blood cell count (WBC), neutrophil (NEU), eosinophil (EOS), basophil (BASO), lymphocyte (LYM) and monocyte (MONO). Hematological analysis was performed using an automated hematology analyzer. To measure biochemical parameters, a dry test tube containing collected blood was centrifuged at 3000rpm for 15min at 5°C to obtain serum. Serum samples were analyzed using an automated biochemical analyzer. Clinical biochemical parameters included: serum total protein (TP), albumin (ALB), total bilirubin (T-BIL), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (URIC), urea (UREA), creatinine (CREA), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), total cholesterol (TC), triglycerides (TG), and glucose (GLU). Serum electrolytes such as calcium (Ca2+), sodium (Na + ), potassium (K + ) and chlorine (Cl - ).

[0592] Necropsy and organ weights: A gross necropsy was performed on all rat groups, including examination of the chest organs, external surface organs, and all internal organs. A careful macroscopic examination of the vital organs was performed to detect any type of abnormality. Thereafter, various organs (including heart, liver, kidney, stomach, lung, spleen, adrenal gland, thymus, epididymis, testis, uterus, and ovary) were surgically removed, washed with ice-cold saline solution, placed on absorbent paper, and then weighed (absolute organ weight in grams). The relative organ weight (ROW) of each animal was then calculated as follows: ROW = [absolute organ weight (g) ÷ rat body weight on the day of sacrifice (g)] × 100.

[0593] Histopathology: Major organs (lungs, heart, liver, kidneys) and reproductive organs (testes and ovaries) were removed for histopathological examination. After measuring the weight, the organs were quickly fixed in 10% buffered formalin (pH 7.4). After fixation, the tissue specimens were dehydrated in a graded series of ethanol (70%-100%), cleaned in toluene, and finally wrapped in paraffin. Thereafter, 5 μm thin sections were prepared using a microtome (Leica) and stained with hematoxylin and eosin (H&E) before microscopic examination. The microscopic features of the organs of the treated groups were compared with those of the control group, and micrographs were recorded.

[0594] Statistical analysis: All data are expressed as mean ± standard deviation (SD). Statistical significance between control and treated groups was determined by one-way analysis of variance (ANOVA), followed by Dunnett's post hoc test. Graph Pad Prism version 6.0 for Windows was used for statistical analysis. Data from male and female groups were analyzed separately, and differences were considered statistically significant at p < 0.05.

[0595] Toxicity Results:

[0596] General signs and behavior analysis

[0597] ■Clinical signs and symptoms are important observation indicators for monitoring the toxic effects of drugs on organs (Jothy et al., 2011). In our current study, no treatment-related deaths were observed in animals of both sexes in acute and subacute toxicity studies at specific oral doses of Compound I.

[0598] ■ During the 14-day (acute) and 28-day (subacute) observation periods, animals did not show any adverse changes in physical behavior, food, and water consumption.

[0599] ■ No obvious or macroscopic abnormalities were observed in all animals in both groups (acute and subacute toxicity). Therefore, the median lethal dose (LD50) of the drug can be considered to be greater than 2,000 mg / kg.

[0600] ■ According to the Globally Harmonized Classification System (GHS), substances with LD50>2,000mg are considered relatively safe (Miyagawa, 2010). Therefore, according to GHS, Compound I can be classified as Class 5.

[0601] Effects of Compound I on body weight and organ weights ( Fig.28 A- Fig.28 E)

[0602] ■Exposure to potentially toxic drugs will cause a sharp decrease in weight gain in rats (Teo et al., 2002). Changes in body weight and relative organ weights are indicators of toxicity and health assessment in experimental animals (Piao et al., 2013).

[0603] ■The body weight of rats in the control group and the treatment group Fig.28 D- Fig.28 In this study, all rats in each dose group showed sustained weight gain during the experimental period, indicating that compound I did not cause any adverse effects on body weight in the acute and subacute toxicity groups.

[0604] ■ In addition, there was no significant difference in the percentage of weight gain compared between the treatment and control groups.

[0605] Relative organ weight (ROW)

[0606] ■ Relative organ weights (ROW) of liver, brain, kidney, heart, spleen for both tests are shown. The difference in ROW between the control and treated groups was statistically significant when evaluated against the control group.

[0607] ■ No significant changes were observed in all organs, so it was confirmed that the administration of Compound I did not produce any adverse effects on vital organs.

[0608] Effects of Compound I on food and water intake.

[0609] ■ Graph depicts the effect of Compound I on food and water intake during subacute treatment.

[0610] ■ Single daily administration of Compound I at the study dose for 28 days resulted in no significant changes in food and water intake when compared to the control group (P>0.05).

[0611] Effects of Compound I on hematological parameters ( Fig.28 F- Fig.28 I).

[0612] ■Hematological parameters play a crucial role in determining the toxicity induced by drugs (Petterino and Argentino-Storino, 2006). When the data come from experiments on laboratory animals, changes in blood parameters have a better predictive value for human safety assessment (Olson et al., 2000). The assessment of hematological parameters is very important in determining the health status of an individual.

[0613] ■The reference values ​​of RBC, WBC, PCV, MCH, and MCHC were 7-10×10^6 / μl, 6-18×10^3 / μl, 35%-64%, 14.3-19.5 pg, and 26.2-40 g / dl, respectively. In the current study, the mean values ​​of hematological parameters such as WBC, RBC, PCV, and hemoglobin in the treatment group did not change significantly in both groups (acute and subacute toxicity studies) when compared with the control group (Loha et al., 2019).

[0614] ■This result suggests that Compound I may not have any toxic substances that could cause symptoms like anemia or other abnormalities.

[0615] ■ Increased WBC release is a clear biomarker of stress and also helps protect the body against some inflammatory conditions such as bacterial infections, leukemia, and major bleeding.

[0616] ■The results obtained from this study showed that Compound I did not cause any significant changes in WBC count levels or their subtypes (including neutrophils, lymphocytes, monocytes, and eosinophils) at any dose relative to the control group. This suggests that Compound I is non-toxic.

[0617] Effects of compound I on biochemical parameters ( Fig.28 L- Fig.28 Q)

[0618] ■ Biochemical evaluation is essential for evaluating the safety of drugs on liver and kidney function. Data on biochemical parameters of the control and treatment groups are presented in Fig.28 L- Fig.28 Q. In the present study, all measured biochemical parameters did not show any significant changes in the acute and subacute study groups.

[0619] ■ In the present work, the hepatotoxic potential of compound I was evaluated by measuring the enzymatic activities of aminotransferases (ALAT and ASAT) - see Fig.28 L- Fig.28 M.

[0620] ■ Abnormal elevations in aminotransferase activity (ALAT and ASAT) may often be involved in hepatotoxicity [Fortson et al., 1985].

[0621] ■ The results showed that the biochemical parameters of the treatment group with the highest dose (2000 mg / kg body weight) were not directly affected when compared to the control group (p>0.05).

[0622] ■ These findings are consistent with those of acute toxicity, which showed no clinical signs or behavioral changes in mice treated with similar doses.

[0623] ■However, there was no effect on aminotransferase activity in the treated group when compared to the control group (p>0.05).

[0624] ■ In both sexes of animals, at doses of 200 mg / kg, 500 mg / kg, 1000 mg / kg and 2000 mg / kg, the plasma levels of AST, ALT, ALP activities did not change significantly, which clearly indicated that Compound I did not cause damage to the liver.

[0625] Kidney function tests ( Fig.28 J- Fig.28 K)

[0626] ■ Potential drug-induced toxic effects on renal function were also assessed by measuring urea and creatinine concentrations, as any significant changes in these parameters may involve induced nephrotoxicity [Mukinda et al., 2010; Gnanamani, A et al., 2008].

[0627] ■Retention of creatinine, electrolytes, urea, and uric acid in the body is an indicator of kidney damage. Changes in the levels of some electrolytes (such as Na+, K+, Cl-, and Mg2+) can also be a sign of kidney damage.

[0628] ■ Our findings showed no significant differences in the levels of creatinine, electrolytes, urea, or uric acid at all doses in both sexes of rats when compared to the control group, and compound I had no effect on serum electrolytes (Na+, Ca2+, Mg2+, and Cl-). Fig.28 J- Fig.28 of K).

[0629] ■In addition, there were no significant differences in the levels of total protein, albumin, conjugated bilirubin, and bilirubin when compared with the control group.

[0630] ■ This provides further support for the safety of Compound I at these doses as renal function was not altered.

[0631] Histopathological study ( Fig.29 A- Fig.29 B)

[0632] ■ On the last day of the treatment period, organ samples of liver, kidney, pancreas, heart, lung, stomach and reproductive organs of male and female rats were subjected to histopathological analysis and the results of some of these tissues are tabulated in Fig.29 A- Fig.29 B.

[0633] Liver cancer:

[0634] ■ Multiple sections of liver from males in the Compound I treated group showed normal hepatocytes as well as normal portal triad, sinusoids and central venous system.

[0635] ■ Sections of liver from female rats in the treatment group showed almost normal cellularity and normal hepatocytes. There was also a normal-appearing portal triad, including the hepatic portal vein, interlobular bile ducts, and hepatic artery branches. Sections of liver from male and female rats in the control group showed normal liver architecture.

[0636] kidney:

[0637] ■Multiple sections taken from renal biopsies of male and female rats in the treatment group showed almost normal size and shape of glomeruli, tubules, intestines and blood vesicles.

[0638] ■ There was no strong evidence of acute tubular necrosis and glomerular changes in the group treated with Compound I. Renal biopsy sections from male and female rats in the control group showed normal findings.

[0639] Pancreas (not shown):

[0640] ■ In the control treated group, sections of the pancreas of male and female rats showed normal architecture, whereas in the Compound I treated group, almost no abnormalities were observed in the architecture of pancreatic acini and islets.

[0641] heart:

[0642] ■ Heart sections taken from male and female rats appeared normal in control treated rats as well as Compound I treated rats, however no significant changes were observed in male rats.

[0643] Lungs (not shown):

[0644] ■ In both male and female rats, multiple sections of the lungs showed normal cellularity, alveoli, and lymphatic vessels in the control treatment group. In the Compound I treatment group, lymphocytic infiltration was observed in both male and female rats.

[0645] Stomach (not shown)

[0646] ■ In the control treated group, gastric sections from rats of both sexes showed normal findings. In the Compound I treated group, female rats showed normal cellularity with normal mucosa, submucosa, muscularis externa and serosa, while male rats showed normal cellularity with no polyp formation or hyperplastic changes observed.

[0647] Reproductive organs (not shown)

[0648] For both the control and Compound I treated groups, sections of reproductive organs (ie, testes in males and ovaries in females) showed normal pathology.

[0649] Example 17: Expected recovery of COVID-19 patients after administration of Compound I

[0650] Dose: 250 mg Compound I PO, BID, over 7 days

[0651] Day 01 :

[0652] The orally bioavailable drug Compound I is absorbed in the intestine and distributed throughout the body. It targets the SARS-CoV-2 viral entry point, inhibits replication and proteolytic processes, and shuts down the protein production machinery that the virus uses to enter host cells.

[0653] Day 02 :

[0654] Compound I stimulates the body's own immune system by acting as an immunomodulator, increasing the production of anti-inflammatory cytokines and interferons, and inhibiting lysosomal activity in host cells that targets viral infections. Elevated levels of circulating IL-6 will decrease, which is associated with controlled levels of lung elasticity and protection against more severe bronchoalveolar inflammation.

[0655] Day 03 :

[0656] The induced immune response inhibits further viral replication, promotes viral clearance from the respiratory tract, induces tissue repair, and triggers a prolonged adaptive immune response against the virus, thereby slowing disease progression. After treatment, peripheral lymphocytes will increase, C-reactive protein will decrease, and overactivated cytokine-secreting immune cells (CXCR3+CD4+T cells, CXCR3+CD8+T cells, CXCR3+NK cells) will decrease within 3-5 days, thereby reducing the cytokine storm induced by SARS-CoV-2.

[0657] Day 04-06 :

[0658] In general, treatment with compound I inhibits SARS-CoV-2 virus particles from invading the respiratory mucosa and infecting other cells, which in turn triggers a series of immune responses and the generation of cytokine storms in the body, which may be associated with the critical condition of COVID-19 patients. After administration of compound I, the patient's lung function and symptoms will improve within 4-5 days, and lung damage caused by excessive immune response to SARS-CoV-2 can be alleviated.

[0659] Summarize

[0660] Compound I has shown significant inhibitory effects on many key proteins of similar coronaviruses (such as SARS-CoV) ( Fig. 22). The compound inhibits viral enzymes (including protease ACE-2 receptor and viral replication protein Nsp15), thereby greatly reducing viral infection and replication in host cells. In vivo studies on mice further showed no toxicity to normal cells, and the animals were healthy and functioning normally with no recurrence of disease after 9 months of administration. Without being bound by theory, it is believed that the molecules of the present invention have the ability to activate CD4+ helper T cells and CD8+ cytotoxic T cells and generate an immune response in vivo to defend against viral infection.

[0661] In addition, dose-dependent inactivation of SARS-CoV-2 was observed upon direct exposure to compound I, and a 50% reduction (IC50) was achieved at 1 μM. In summary, these data support the further development of compound I for the treatment of CoV and propose a new mechanism by which compound I interacts with the CoV replication complex, which may elucidate key aspects of replication. As explained above, pharmacokinetic studies investigated the concentrations of compound I at different time intervals after oral administration or intravenous injection. In tissue distribution, compound I was found to be mainly concentrated in the heart, lungs, liver, and other organs. In the gastrointestinal tract, a large amount of compound I accumulated in the stomach, which means that compound I can be absorbed by gastric tissue and distributed throughout the body. In addition, less than 2% of compound I was excreted in urine or feces, indicating that approximately 98% of compound I was absorbed or distributed to important organs. In summary, our current findings will provide a more complete understanding of the biological effects of compound I in vivo.

[0662] From the above, it can be understood that various embodiments of the present disclosure have been described herein for illustrative purposes, and various modifications may be made without departing from the scope and spirit of the present disclosure. Therefore, the various embodiments disclosed herein are not intended to be limiting, with the true scope and spirit being indicated by the appended claims.

Claims

1. A compound of formula 2, or a pharmaceutically acceptable salt thereof, or a combination thereof, wherein: R1 is OH; R2 is C1-C3 alkoxy; R3 is C1-C3 alkoxy; and R4 is:

2. The compound of claim 1, wherein R1 is OH; R2 is OCH3; R3 is OCH3; and R4 is 3. The compound according to any one of claims 1 to 2, wherein the compound is:

4. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition of claim 4, wherein the compound is:

6. Use of the compound according to any one of claims 1 to 3 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating cancer, wherein the cancer is pancreatic cancer, liver cancer, colon cancer, lung cancer, breast cancer or leukemia.

7. The use of claim 6, wherein the cancer has a KRAS oncogene mutation.

8. The use according to any one of claims 6 to 7, wherein the compound is:

9. Use of a compound as described in any one of claims 1 to 3 or a pharmaceutical composition as described in claim 4 in the preparation of a medicament for treating a viral infection, wherein the viral infection is SARS or COVID-19.

10. The use according to claim 9, wherein the compound is:

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