Novel retinoic acid compounds, pharmaceutical compositions containing the same, and their uses

Abstract

Provided is a compound or its pharmaceutically acceptable salt, and its pharmaceutical composition, comprising retinoic acid conjugated to carbohydrate.In addition, provided is the use of the compound or its pharmaceutically acceptable salt, or its pharmaceutical composition in the manufacture of a medicament for inhibiting viral infection or replication, or for treating cancer.

Description

[Technical Field]

[0001] This disclosure relates to novel retinoic acid compounds, pharmaceutical compositions containing the same, and their use in the manufacture of pharmaceuticals for inhibiting viral infection or replication or for treating cancer. [Background technology]

[0002] Coronaviruses are a group of single-stranded positive-chain RNA viruses belonging to the Coronaviridae family, which includes seven species / lineages that infect humans: Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Human Coronavirus 229E (HCoV-229E), Human Coronavirus OC43 (HCoV-OC43), Human Coronavirus NL63 (HCoV-NL63), and Human Coronavirus HKU (HCoV-HKU1). Notably, SARS-CoV-2 is identified as the viral lineage that caused the Coronavirus Disease 2019 (COVID-19) pandemic.

[0003] The SARS-CoV-2 genome shares high sequence identity with the SARS-CoV genome. Both SARS-CoV-2 and SARS-CoV rely critically on the activity of two viral proteases, namely 3C-like protease (3CLpro, also known as major protease (Mpro) or non-structural protein 5 (nsp5)) and papain-like protease (PLpro, the protease domain of non-structural protein 3 (nsp3)), to carry out the viral replication cycle and spread. Total trans retinoic acid (ATRA, also known as vitamin A acid or tretinoin) has been reported to be a potential therapeutic agent against SARS-CoV-2 by inhibiting 3CLpro activity.

[0004] Apart from 3CLpro, PLpro is also a potential target, and therefore such enzymes play a crucial role in the cleavage and maturation of viral polyproteins, the assembly of replicase-transcriptase complexes, and the disruption of the host response. While the primary function of Mpro and PLpro is the coordinated processing of viral polyproteins, PLpro has the additional function of removing ubiquitin and IFN-stimulated gene factor 15 (ISG15) from host cell proteins, allowing coronaviruses to evade the host's innate immune response. In other words, PLpro is involved not only in viral replication but also in the abnormal regulation of the signaling cascade in infected cells, which leads to cell death in surrounding uninfected cells. Therefore, drugs are designed to inhibit the function of PLpro, which may also have the potential to combat SARS-CoV-2. Recently, in addition to ATRA, some researchers have reported its derivatives, and 13-cis-retinoic acid (also known as isotretinoin, a potential PLpro inhibitor) is a potential PLpro inhibitor that may be used to treat COVID-19 caused by SARS-CoV-2.

[0005] Furthermore, retinoic acids, including ATRA and 13-cis-retinoic acid, are promising compounds for the treatment of various cancers due to their specific effects on cell proliferation, differentiation, and apoptosis, as well as their low toxicity. Retinoic acid receptors in human cell nuclei were discovered by biochemists and found not to mutate in cancer cells; therefore, retinoic acid has been able to exert its anticancer effects in many malignancies. For example, treatment with 13-cis-retinoic acid in children at high risk of neuroblastoma has been shown to reduce the risk of cancer recurrence after high-dose chemotherapy and stem cell transplantation. ATRA has been studied in combination with other drugs in various cancers and precancerous lesions. Numerous clinical trials using ATRA as part of combination therapy are currently underway. For example, ATRA with different interferons (IFNs) has been shown to enhance the effects of both drugs, leading to growth inhibition and cell death in tumor cell lines. Nevertheless, to unlock the therapeutic potential of retinoic acid, numerous studies highlight the need for a better understanding of the mechanisms blocking retinoic acid signaling and retinoic acid regulatory gene expression in cancers such as acute myeloid leukemia (AML). Combination therapies targeting multiple gene silencing mechanisms are clearly the most effective strategies for reactivating ATRA-sensitive gene expression and differentiation in AML cells, and for generally mediating the anti-cancer activity of ATRA. Currently, the identification of classes of proteins that regulate gene expression via histone and DNA modification is driving the development of novel therapeutic agents that alter chromatin structure, so-called epigenetic drugs. However, these epigenetic modifiers have been shown to be only partially effective against different cancers when used alone. [Overview of the project]

[0006] For the reasons stated above, the present invention provides novel retinoic acid compounds and pharmaceutical compositions containing the same that can effectively inhibit viral infection or replication or treat cancer.

[0007] In one aspect of the present invention, a compound or a pharmaceutically acceptable salt thereof is provided, comprising retinoic acid conjugated to a carbohydrate.

[0008] Preferably, the compound is of formula (I):

[0009] [ka] [In the formula, R 1 [This refers to a substituted or unsubstituted functional group of a carbohydrate.] It is represented by [this].

[0010] Preferably, the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

[0011] Preferably, the oligosaccharide is a monooligosaccharide or heterooligosaccharide, and the polysaccharide is a monopolysaccharide or heteropolysaccharide.

[0012] Preferably, the carbohydrates include glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3 / 1,6-glucan oligosaccharides, raffinose, stachyose, verbascose, fructooligosaccharides, starch, glycogen, cellulose, or any combination thereof.

[0013] Preferably, the compound or a pharmaceutically acceptable salt thereof is encapsulated by liposomes.

[0014] In another aspect of the present invention, a pharmaceutical composition is provided comprising the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0015] Preferably, the pharmaceutically acceptable carrier comprises liposomes, and the compound or a pharmaceutically acceptable salt thereof is encapsulated by the liposomes.

[0016] Preferably, the pharmaceutical composition further comprises a metal ion.

[0017] Preferably, the metal ion comprises a monovalent ion, a divalent ion, or a combination thereof. More preferably, the monovalent ion is K + , Na + , or a combination thereof, and the divalent ion is Zn 2+ , Mg 2+ , Cu 2+ , Mn 2+ , Ca 2+ , Fe 2+ , or any combination thereof.

[0018] Preferably, the pharmaceutically acceptable carrier comprises liposomes, and the metal ion is encapsulated by the liposomes.

[0019] Preferably, the compound or the pharmaceutically acceptable salt thereof and the metal ion are encapsulated individually or simultaneously by the liposomes.

[0020] In yet another aspect, the present invention provides the use of the compound, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the manufacture of a medicament for inhibiting viral infection or replication.

[0021] In yet a further aspect, the present invention provides the use of the compound, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the manufacture of a medicament for treating cancer.

[0022] Thus, the present invention provides at least the following advantages: 1. The claimed compound, which is a retinoic acid conjugated to a carbohydrate, and its pharmaceutical composition can enhance the ability to inhibit viral infection and / or replication as compared to retinoic acid alone. 2. The claimed compound and its pharmaceutical composition can be provided as potential drugs against coronaviruses, particularly SARS-CoV-2 that causes COVID-19. 3. The claimed compound and its pharmaceutical composition can inhibit cancer cells and thus enhance the ability to effectively treat various cancers such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, and liver cancer. [Brief explanation of the drawing]

[0023] [Figure 1] This figure illustrates the results of liquid chromatography-mass spectrometry (LC-MS) analysis of compound (Ia) according to an embodiment of the present invention. [Figure 2] This figure illustrates the absorbance versus time curve in a SARS-CoV-2 PLPro inhibition assay of a novel retinoic acid compound according to an embodiment of the present invention. [Figure 3] This figure illustrates the TCID50 chart showing the antiviral effect of a novel retinoic acid compound in a SARS-CoV-2 animal model according to embodiments of the present invention. [Figure 4] This figure illustrates a curve showing the cell viability of a novel retinoic acid compound on the AsPC-1 cancer cell line according to an embodiment of the present invention. [Figure 5] This figure illustrates a curve showing the cell viability of a novel retinoic acid compound against the MDA-MB-231 cancer cell line according to an embodiment of the present invention. [Figure 6] This figure illustrates a curve showing the cell viability of a novel retinoic acid compound against an HCT-116 cancer cell line according to an embodiment of the present invention. [Figure 7] This figure illustrates a curve showing the cell viability of a novel retinoic acid compound against a Huh-7 cancer cell line according to an embodiment of the present invention. [Figure 8] This figure illustrates a curve showing the cell viability of a novel retinoic acid compound on a SKOV-3 cancer cell line according to an embodiment of the present invention. [Figure 9] This figure illustrates a curve showing the cell viability of a novel retinoic acid compound on an A549 cancer cell line according to an embodiment of the present invention. [Figure 10] This figure illustrates a curve showing the cell viability of a novel retinoic acid compound on an H460 cancer cell line according to an embodiment of the present invention. [Figure 11] This figure illustrates the absorbance-to-time curve in a SARS-CoV-2 PLPro inhibition assay of a second pharmaceutical composition according to an embodiment of the present invention. [Figure 12] This figure illustrates TCID50 charts showing the antiviral effect of the second pharmaceutical composition in a SARS-CoV-2 animal model according to embodiments of the present invention. [Modes for carrying out the invention]

[0024] While the present invention is open to various modifications and alternative forms, specific embodiments are shown as examples in the drawings and described in detail. However, it should be understood that the description is not intended to limit the invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives that fall within the spirit and scope of the invention.

[0025] definition Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having the same meaning as their meaning in the context of the relevant art, and it will be further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0026] The terminology used herein is intended solely to describe specific embodiments and is not intended to be a limitation of the embodiments of the present invention. Where used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless the context clearly indicates otherwise. Where used herein, the terms “comprises,” “comprising,” “includes,” and / or “including,” specify the presence of the features, integers, steps, operations, elements, parts, and / or combinations thereof described herein, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts, and / or combinations thereof.

[0027] In this specification, the term “subject” refers to a mammal whose diagnosis, prognosis, or therapy is desired. Generally, mammals are humans. In certain embodiments, mammals may refer to non-human mammals such as non-human primates, cows, horses, goats, sheep, dogs, cats, rabbits, pigs, mice, or rats, used, for example, when screening, characterizing, and evaluating drugs, and in therapy.

[0028] In this specification, the terms “administer” or “to be administered” mean introducing, providing or delivering a given active ingredient to a subject by any preferred route for performing its intended function.

[0029] In this specification, the term “cancer” refers to leukemia, lymphoma, carcinoma, sarcoma, and other malignant tumors of potentially unrestrained growth that can spread locally by invasion and throughout the body by metastasis. Examples of cancer include, but are not limited to, cancers of the ovaries, adrenal glands, bones, brain, breasts, bronchi, colon and / or rectum, gallbladder, head and neck, kidneys, larynx, liver, lungs, nerve tissue, pancreas, prostate, parathyroid glands, skin, stomach, and thyroid gland. Certain other examples of cancer include cholangiocarcinoma, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, epidermal carcinoma, giant cell tumor, glioblastoma (multiforma), hairy cell tumor, enteric ganglion cell tumor, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, Marfanoid habitus tumor, medullary carcinoma, metastatic cutaneous carcinoma, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, pheochromocytoma, and polycythemia vera. This includes vera), primary brain tumors, small cell lung tumors, squamous cell carcinomas of both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumors, focal skin lesions, veticulum cell sarcoma, and Wilms' tumor.

[0030] In this specification, the term "oligosaccharide" refers to a carbohydrate composed of a small number of monosaccharides, usually about 3 to 10 monosaccharide units. Here, oligosaccharides with one type of monosaccharide subunit are called monooligosaccharides, and oligosaccharides with two or more types of monosaccharide subunits are called heterooligosaccharides.

[0031] In this specification, the term "polysaccharide" refers to a carbohydrate composed of numerous monosaccharide units. Here, a polysaccharide having one type of monosaccharide subunit is called a monopolysaccharide, and a polysaccharide having two or more types of monosaccharide subunits is called a heteropolysaccharide.

[0032] In this specification, the term "liposome" refers to a particle characterized by having an aqueous internal space isolated from an external medium by one or more bilayer membranes forming a vesicle. The main types of liposomes are multilayer vesicles (MLVs, having several lamellar-phase lipid bilayers), small monolayer liposome vesicles (SUVs, having a single lipid bilayer), and large monolayer vesicles (LUVs, having a single lipid bilayer). The bilayer membranes of monolayer or multilayer vesicles are typically formed by lipids, i.e., synthetic or naturally occurring amphiphilic molecules containing spatially separated hydrophobic and hydrophilic domains.

[0033] Explanatory Embodiment Hereafter, exemplary embodiments of the present invention will be described in more detail with reference to the attached drawings.

[0034] In one embodiment, a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound (hereinafter referred to as the “first” pharmaceutical composition) is provided, wherein the compound comprises retinoic acid conjugated to a carbohydrate.

[0035] In an exemplary embodiment, the compound is of formula (I):

[0036] [ka] [In the formula, R 1 [This refers to a substituted or unsubstituted functional group of a carbohydrate.] It is represented by [this].

[0037] In certain embodiments, the concentration of the compound containing retinoic acid conjugated to a carbohydrate may be, but is not limited to, 0.1 μM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 500 μM, 0.1 μM to 250 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0038] In another embodiment, a pharmaceutical composition comprising retinoic acid and a carbohydrate (hereinafter referred to as the “second” pharmaceutical composition) is provided. In a particular embodiment, the retinoic acid is 13-cis-retinoic acid (also known as isotretinoin).

[0039] In certain embodiments, the concentration of retinoic acid may be, but is not limited to, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0040] In certain embodiments, the carbohydrate may be a monosaccharide, disaccharide, oligosaccharide, or polysaccharide. The oligosaccharide may be a monooligosaccharide or a heterooligosaccharide. The polysaccharide may be a monopolysaccharide or a heteropolysaccharide. The monosaccharide may be selected from, but is not limited to, glucose, fructose, galactose, and mannose. The disaccharide may be selected from, but is not limited to, sucrose, lactose, and maltose. The oligosaccharide may be selected from, but is not limited to, β-1,3 / 1,6-glucan oligosaccharide, raffinose, stachyose, verbascose, and fructooligosaccharide. The polysaccharide may be selected from, but is not limited to, starch, glycogen, and cellulose.

[0041] In a particular embodiment, the carbohydrate concentration is 0.1 μM to 200 mM, 0.1 μM to 150 mM, 0.1 μM to 100 mM, 0.1 μM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 500 μM, 0.1 μM to 250 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 200 mM, 1 μM to 150 mM, 1 μM to 100 mM, 1 μM to 10 mM, 1 The range may be from μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 200 mM, 10 μM to 150 mM, 10 μM to 100 mM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM, but is not limited to these ranges.

[0042] In a particular embodiment, the molar ratio of retinoic acid to carbohydrates in the second pharmaceutical composition is approximately 1:10 -3 -100, 1:10 -3 It may also be -20, 1:1-20, or 1:1-10.

[0043] In certain embodiments, the first and second pharmaceutical compositions each optionally further contain metal ions. Preferably, the metal ions include monovalent ions, divalent ions, or combinations thereof. In certain embodiments, the monovalent ions are K + kaNa + , or a combination thereof, the divalent ions are Zn 2+ Mg 2+ Cu 2+ Mn 2+ Ca 2+ Fe 2+ , or any combination thereof.

[0044] In certain embodiments, the concentration of metal ions is 1 μM to 300 mM, 1 μM to 250 mM, 1 μM to 200 mM, 1 μM to 150 mM, 1 μM to 100 mM, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, and 1 μM to 50 μM. The concentration may be 10 μM to 300 mM, 10 μM to 250 mM, 10 μM to 200 mM, 10 μM to 150 mM, 10 μM to 100 mM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0045] In a particular embodiment, the molar ratio of the compound to the metal ion in the first pharmaceutical composition is approximately 1:10 -3 -10 3 It may also be 1:0.1-20, 1:0.1-10, or 1:1-10.

[0046] In a particular embodiment, the molar ratio of retinoic acid, carbohydrates, and metal ions in the second pharmaceutical composition is approximately 1:10. -4 -20:10 -4 -10 3 , 1:10 -4 -20:10 -3 -10 3 , 1:10 -4 -20:10-3 -20, 1:10 -4 -20:0.1-20, 1:10 -4 -20:1-10, 1:0.1-20:10 -4 -10 3 , 1:0.1-20:10 -3 -10 3 , 1:0.1-20:10 -3 -20, 1:0.1-20:0.1-20, 1:0.1-20:1-20, or 1:0.1-20:1-10, 1:0.1-1:10 -4 -10 3 , 1:0.1-1:10 -3 -10 3 , 1:0.1-1:10 -3 It may also be -20, 1:0.1-1:0.1-20, 1:0.1-1:1-20, or 1:0.1-1:1-10.

[0047] In certain embodiments, the first and second pharmaceutical compositions may further include, respectively, pharmaceutically acceptable carriers. pharmaceutically acceptable carriers are widely used in the art of drug manufacturing. Examples of pharmaceutically acceptable carriers may include, but are not limited to, liposomes, excipients, adjuvants, solvents, buffers, emulsifiers, suspending agents, degrading agents, disintegrants, dispersants, binders, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption retarders, etc. The selection and quantity of pharmaceutically acceptable carriers are within the scope of the expertise of those skilled in the art.

[0048] In certain embodiments, the pharmaceutically acceptable carrier is a liposome. The compound or a pharmaceutically acceptable salt thereof is encapsulated by the liposome. The compound or a pharmaceutically acceptable salt thereof and metal ions in the first pharmaceutical composition may be encapsulated individually or simultaneously by the liposome. The retinoic acid and carbohydrates in the second pharmaceutical composition may be encapsulated individually or simultaneously by the liposome. All or at least two of the retinoic acid, carbohydrates, and metal ions in the second pharmaceutical composition may be encapsulated individually or simultaneously by the liposome.

[0049] Exemplary liposomes may be neutral, positively, or negatively charged. Generally, lipids commonly used in liposomes typically include dialiphatic chain lipids such as phospholipids, diglycerides, and dialiphatic glycolipids; monolipids such as sphingomyelin and sphingoglycolipids; steroids such as cholesterol and its derivatives, and combinations thereof. Examples of phospholipids include phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine ( PSPC), 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soybean phosphatidylcholine (HSPC), 1,2-dimirystoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG), 1,2 -Dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol)(sodium salt)(DPPG), l-palmitoyl-2-stearoyl-sn-glycero-3-phospho(1'-rac-glycerol)(sodium salt)(PSPG), 1,2-distearoyl-sn-glycero-3-phospho(1'-rac-glycerol)(sodium salt)(DSPG), 1,2-dioleoyl-sn-glycero-3-phospho(1'-rac-glycerol)(sodium salt) c-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (seine) (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-Dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA), 1,2-Dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2-Distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA), 1,2-Dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), 1,2-Dipalmitoyl-9n-glycero-3-phosphoethanolamine (DPPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine Phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-myo-inositol) (ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (DSPI), 1,2-dioleoyl-sn-glycero-3-phospho(1'-myo-inositol) (ammonium salt) (DOPI), cardiolipin, L-alpha-phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 This includes, but is not limited to, EPC, L-alpha-phosphatidylethanolamine (EPE), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and 3-beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.

[0050] Suitable lipids may be a mixture of one or more of the aforementioned lipids, or a mixture of one or more of the aforementioned lipids with one or more other lipids not listed above, membrane stabilizers, or antioxidants.

[0051] The molar percentage of lipids in the bilayer may be approximately 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less, or any value or range in between (for example, approximately 5-50%, approximately 5-45%, approximately 5-40%, approximately 5-35%, approximately 5-30%, approximately 5-25%, approximately 5-20%, approximately 5-15%, or approximately 5-10%).

[0052] The lipids of the bilayer may be a mixture of a first phospholipid and a second phospholipid. The first phospholipid may be selected from the group essentially consisting of PC, HSPC, DOPC, POPC, DSPC, DPPC, DMPC, PSPC and combinations thereof, and the second phospholipid may be selected from the group essentially consisting of PE, PG, DOPE, PEG-DSPE, DPPG, DOPG, DOTAP, DOTMA, DDAB and combinations thereof. In other embodiments, the molar percentage of the first phospholipid in the bilayer is approximately 50, 45, 40, 35, 30, 25, 20, 15, 10 or any value or range in between (e.g., approximately 5-50%, approximately 5-45%, approximately 5-40%, approximately 5-35%, approximately 5-30%, approximately 5-25%, approximately 5-20%, approximately 5-15%, or approximately 5-10%), and the molar percentage of the second phospholipid in the bilayer is between 0.1 and approximately 15, 14, 13, 12, 11, 10, 9, 8, 7 or any value or range in between (e.g., approximately 0.1-15%, approximately 0.1-10%, approximately 0.5-15%, approximately 0.5-10%, or approximately 0.5-7%). In exemplary embodiments, the first phospholipid (DSPC) and the second phospholipid (DOPE, DOPG, or DDAB) may be in a molar ratio of 4:1 to 6:1.

[0053] In exemplary embodiments, the liposome bilayer contains a steroid, preferably cholesterol, at a molar percentage of less than about 55 moles. The molar percentage of steroids (such as cholesterol) in the bilayer may be about 15-55%, about 20-55%, about 25-55%, about 15-50%, about 20-50%, about 25-50%, about 15-45%, about 20-45%, about 25-45%, about 15-40%, about 20-40%, or about 25-40%. The molar percentages of phospholipids and cholesterol in the bilayer may be about 25-50%:15-55%, 25-50%:20-55%, or 25-50%:15-50%. The molar ratio of phospholipids and cholesterol may be 1:1 to 3:1. The molar percentage ratios of the first phospholipid, the second phospholipid, and cholesterol in the bilayer may be approximately 25-50%:0.1-15%:15-55%, 5-50%:0.1%-15%:10-40%, or 25-50%:0.5-10%:5-20%.

[0054] Liposomes encapsulating the scavenger can be prepared by any currently known or subsequently developed technology. For example, MLV liposomes can be formed directly from a hydrated lipid film, spray-dried powder, or freeze-dried cake of a selected lipid composition with a scavenger, while SUV liposomes and LUV liposomes can be sized from MLV liposomes by ultrasonic treatment, homogenization, microfluidization, or extrusion.

[0055] In yet another embodiment, a compound containing retinoic acid conjugated to a carbohydrate or a pharmaceutically acceptable salt thereof, as well as the first and second pharmaceutical compositions, can be used to inhibit viral infection or replication. The virus may be an RNA virus. The RNA virus may include coronavirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, or any combination thereof. The coronavirus may include severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU (HCoV-HKU1), or any combination thereof. In an exemplary embodiment, the coronavirus is SARS-CoV-2, which causes COVID-19.

[0056] In yet another embodiment, cancer can be treated using compounds containing retinoic acid conjugated to a carbohydrate, pharmaceutically acceptable salts thereof, and the first and second pharmaceutical compositions. Cancer may include leukemia, lymphoma, carcinoma, or sarcoma. In exemplary embodiments, cancer may be lung cancer, ovarian cancer, breast cancer, liver cancer, pancreatic cancer, or cholangiocarcinoma.

[0057] In certain embodiments, the compounds and metal ions in the first pharmaceutical composition may be administered separately, simultaneously, or sequentially. The retinoic acid and carbohydrates in the second pharmaceutical composition may be administered separately, simultaneously, or sequentially. All or at least two of the retinoic acid, carbohydrates, and metal ions in the second pharmaceutical composition may be administered separately, simultaneously, or sequentially. In exemplary embodiments, the metal ions and carbohydrates are administered simultaneously, followed by the retinoic acid.

[0058] In exemplary embodiments, the time interval between administrations may be, in order, 1 to 30 minutes, 30 to 60 minutes, 60 to 90 minutes, 90 to 120 minutes, 2 to 3 hours, 3 to 12 hours, or 12 to 24 hours.

[0059] In certain embodiments, compounds containing retinoic acid conjugated to a carbohydrate, pharmaceutically acceptable salts thereof, and the first and second pharmaceutical compositions may be prepared for administration in dimethyl sulfoxide (DMSO), ethanol, buffer, or water, respectively. The dosage and frequency of administration may vary depending on the following factors: the severity of the viral infection (e.g., coronavirus infection) or disease (e.g., cancer) being treated, as well as the weight, age, physical condition, and response of the subject being treated. The daily dosage of the treatment agent may be administered as a single dose or in multiple doses.

[0060] In certain embodiments, compounds containing retinoic acid conjugated to a carbohydrate, pharmaceutically acceptable salts thereof, and the first and second pharmaceutical compositions are administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, or topically. [Examples]

[0061] The present invention will be further illustrated by the following examples. However, it should be understood that these examples are intended for illustrative purposes only and should not be construed as actually limiting the present invention.

[0062] material and method A. Preparation of galactose-modified isotretinoin, a novel retinoic acid compound represented by formula (Ia) based on the following synthetic scheme:

[0063] [ka]

[0064] I. General experimental methods All reagents were commercially available and used without further purification. Yields refer to purified and spectroscopically pure compounds. Thin-layer chromatography (TLC) was performed using Merck TLC aluminum sheet silica gel 60F 254 The procedure was performed using plates and visualized by fluorescence quenching under UV light. Flash chromatography was performed using silica gel (Chromatrex, MB70-40 / 75, 40-75 μm) purchased from Fuji Silicia Chemical Co., Ltd. The NMR spectrum was obtained as follows: 1 For H, the data was recorded using a Varian-400MR operating at 400 MHz. Chemical shifts are reported in ppm using solvent resonance as an internal standard. Data are reported as follows: s = single line, br = wide, d = double line, t = triple line, q = quadruple line, m = multiple line, dd = double line of double line; coupling constant in Hz; integral. Purity was recorded using a Waters e2695 separation module / 2998 PDA detector HPLC system (column: crossbridge C18, 5 μM, 4.6 mm (ID) × 150 mm (L), eluent: mixture of mobile phases A and B, mobile phase A: 100% acetonitrile; mobile phase B: pure water containing 0.1% formic acid and 10 mM NH4OAc, flow rate: 0.5 mL / min, detection: UV, 254 nm).

[0065] II. Synthesis of compound (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(3-bromopropoxy)tetrahydro-2H-pyran-3,4,5-triyltriacetate (2)

[0066] [ka] To a solution of galactose pentaacetate (compound (1), 5.0 g, 13 mmol), 3-bromo-1-propanol (1.73 mL, 19.0 mmol), and fresh, dried molecular sieve in anhydrous dichloromethane (50 mL), boron trifluoride-diethyl etherate (8.04 mL, 64.0 mmol) was added at 0°C. The mixture was stirred overnight at room temperature. The solution was neutralized with triethylamine, the molecular sieve was removed by passing it through Celite, and the reaction mixture was washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The crude product was purified by silica gel column chromatography (Â:hexane = 1:2) to obtain the desired product (2) (0.625 g, 10%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 5.40 (dd, J = 3.4, 1.2 Hz, 1H), 5.22-5.16 (m, 1H), 5.04-5.01 (m, 1H), 4.48 (d, J = 8 Hz, 1H), 4.21-4.11 (m, LCMS (ESI) m / z C 17 H 25 BrO 10 Calculated value: 469.28; Measured value: 491.04 [M + Na] + .

[0067] III. Synthesis of compound (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(3-azidopropoxy)tetrahydro-2H-pyran 3,4,5-triyltriacetate (3)

[0068] [ka] Sodium azide (0.43 g, 6.6 mmol) was added to a solution of compound (2) (0.62 g, 1.3 mmol) in DMF (4 mL). The mixture was stirred at 100°C for 12 hours. The solution was concentrated to dryness. The crude product was purified by silica gel column chromatography (siRNA:hexane = 1:2) to obtain the desired product (3) (458 mg, 80%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 5.39 (dd, J = 3.4, 0.8 Hz, 1H), 5.22-5.18 (m, 1H), 5.03-5.00 (m, 1H), 4.48 (d, J = 8 Hz, 1H), 4.21-4.10 (m, 2H), 3.99-3.89 (m, 1H), 3.63-3.58 (m, 1H), 3.39-3.35 (m, 2H), 2.15 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H), 1.92-1.77 (m, 2H); LCMS (ESI) m / z C 17 H 25 N3O 10 Calculated value: 431.40; Measured value: 454.2 [M + Na] + .

[0069] IV. Synthesis of the compound (2R,3R,4S,5R,6R)-2-(3-azidopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (4)

[0070] [ka] Sodium methoxide (28 mg, 0.5 mmol) was added to a solution of compound (3) (0.45 g, 1 mmol) in dichloromethane (1.0 mL) and methanol (4.0 mL). The mixture was stirred at room temperature for 10 hours. The solution was neutralized with Dowex 50WX8 and evaporated to dryness to obtain the desired product (4), which was used in the next step without further purification. LCMS(ESI)m / z C9H 17Calculated value of N3O6: 263.25; measured value: 286.1 [M+Na] + .

[0071] V. Synthesis of compound (2R,3R,4S,5R,6R)-2-(3-aminopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (5)

[0072] [ka] Crude compound (4) in methanol (10.5 mL) was treated with Pd / C (11 mg) and acetic acid (6.3 mg) under an H2 atmosphere. The solution was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure to yield (2R,3R,4S,5R,6R)-2-(3-aminopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (5), which was used in the next step without further purification. LCMS(ESI)m / z C9H 19 Calculated value for NO6: 237.25; measured value: 237.8 [M] + .

[0073] VI. Synthesis of the compound perfluorophenyl(2Z,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexa-1-en-1-yl)nonano-2,4,6,8-tetraenoatee (6)

[0074] [ka] To a solution of retinoic acid (compound (7), 0.10 g, 0.33 mmol) and triethylamine (0.093 mL, 0.67 mmol) in anhydrous DMF (1.0 mL), pentafluorophenyl trifluoroacetate (compound (8), 0.081 mL, 0.47 mmol) was added at 0°C. The solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with RINKAN and washed with 0.1 N aqueous HCl, followed by aqueous NaHCO3 and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated to dryness to obtain the desired product (6) without further purification.

[0075] VII. Synthesis of the target compound (2Z,4E,6E,8E)-3,7-dimethyl-N-(3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)-9-(2,6,6-trimethylcyclohexa-1-en-1-yl)nona-2,4,6,8-tetraenamide (Ia)

[0076] [ka] Triethylamine (0.049 mL, 0.33 mmol) was added at room temperature to a solution of crude compound (5) (30 mg, 0.13 mmol) and activated ester (6) (150 mg, 0.33 mmol) in DMF (1.0 mL). The mixture was stirred overnight. The solution was concentrated to dryness. The crude product was purified by silica gel column chromatography (methanol:dichloromethane = 1:9). LCMS(ESI) m / z C 29 H 45 Calculated value for NO7: 519.68; Measured value: 520.5 [M+H] + Please refer to Figure 1.

[0077] B. Preparation of SARS-CoV-2 PLPro To determine whether the claimed compounds and pharmaceutical compositions can inhibit SARS-CoV-2, their in vitro inhibitory effects on the activity of SARS-CoV-2 papain-like protease (PLpro) were first evaluated.

[0078] The codon-optimized gene sequence encoding wild-type SARS-CoV-2 PLpro was synthesized by Biotools Co., Ltd. (New Taipei City, Taiwan), subcloned into the pET-21a (Novagen) vector using NdeI and XhoI restriction sites, while retaining the His tag coding region (-LEHHHHHH-) at the C-terminus.

[0079] A vector containing the SARS-CoV-2 PLPro gene was converted to E. coli BL21(DE3) strain (Yeastern Biotech Co., Ltd., New Taipei City, Taiwan) for overexpression of PLPro. Culturing was performed in LB medium (containing 1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with ampicillin (100 μg / mL), which serves as an antibiotic marker. The resulting culture was initially incubated at 37°C with shaking at 200 rpm. Optical density (OD) at 600 nm between 0.6 and 0.8 was measured. 600PLpro expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. Incubation was continued at 18°C ​​and 200 rpm for 20 hours. Cells were harvested by centrifugation (5,000 × g) and destroyed by sonication in a lysis buffer containing 50 mM sodium phosphate (pH 7.4), 1.0 mM DTT, 5% glycerol, and 100 mM NaCl. Cell debris was then removed by centrifugation at 20,000 × g for 50 minutes. The supernatant was loaded into a 5 mL His-Trap HP column (GE Healthcare Life Sciences), and the proteins were eluted using a gradient of 0–500 mM imidazole in 50 mM sodium phosphate (pH 7.4) and 100 mM NaCl. The fraction containing His-tagged SARS-CoV-2 PLpro was pooled and concentrated using a Sentricon membrane (10K cutoff, GE Healthcare Life Sciences). The His-tagged SARS-CoV-2 PLpro was further purified by gel filtration chromatography using a Superdex 75 gel filtration column (GE Healthcare Life Sciences) in 50 mM sodium phosphate buffer (pH 7.4). The SARS-CoV-2 PLpro concentration was 45270M. -1 cm -1 The extinction coefficient (ε280) was used and determined by measuring the ultraviolet absorbance at 280 nm.

[0080] C. SARS-CoV-2 PLPro activity assay The enzymatic activity of SARS-CoV-2 PLpro obtained above was measured by a colorimetric-based peptide cleavage assay using a 6-mer peptide substrate, FRLKGG-para-nitroanilide (FG6-pNA) (purity 97% by HPLC; GL Biochem Ltd., Shanghai, China). In the cleavage assay, the 6-mer peptide substrate was cleaved at the Gly-pNA bond, releasing free pNA, which changed the color of the solution to yellow. Enzymatic activity was measured using a 96-well microplate spectrophotometer (Epoch® 2, Biotek) by measuring the absorbance (A) at 405 nm. 405 This was determined by continuously monitoring the temperature at 30°C.

[0081] Specifically, the cleavage assay was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4), and FG6-pNA was added to each well to prepare substrate solutions with various concentrations of FG6-pNA (0.1875 mM, 0.375 mM, 0.75 mM, 1.5 mM, 3.0 mM, 6.0 mM). The assay mixture (180 μL in each well) was pre-incubated for 10 minutes for precise temperature control, and the reaction was initiated by adding 20 μL of SARS-CoV-2 PLpro solution (1.75 μM) to the assay mixture. The SARS-CoV-2 PLpro solution was prepared by mixing the SARS-CoV-2 PLpro obtained above with 50 mM sodium phosphate buffer (pH 7.4). The concentration of pNA released by proteolysis was measured at 9800 M. -1 cm -1 Using the extinction coefficient (ε405) of A 405 (A 405 9.8 at 1 mM.

[0082] Steady-state enzyme reaction kinetic parameters were obtained by fitting initial velocity (V0) data based on the Michaelis-Menten equation using OriginPro 8.0 software (OriginLab Corporation, USA). All measurements were performed in triplicate. The obtained data are expressed as mean ± standard deviation.

[0083] Result:K M and k cat The values ​​are 2.50 ± 0.03 mM and 0.85 ± 0.01 s, respectively. -1 Therefore, it has been verified that SARS-CoV-2 PLPro with protease activity can be successfully prepared according to the above manufacturing method and used to perform the following SARS-CoV-2 PLPro inhibition assay.

[0084] D. Establishment of an animal model for SARS-CoV-2 Golden Syrian hamsters (5-6 weeks old, with an average weight of approximately 100g) were obtained from the National Center for Experimental Animals (Taipei, Taiwan). The hamsters were housed in an animal room under specific pathogen-free (SPF) conditions, which are generally applicable in this field. Furthermore, all hamsters were provided with free access to water and feed. All experiments involving hamsters were commissioned to the Animal Experimentation Committee (IACUC) of Academia Sinica (Taiwan), reviewed, and approved.

[0085] To establish an animal model of SARS-CoV-2, hamsters were given SARS-CoV-2 (P3Lab, Genomics Research Center, Academia Sinica; Wuhan wild type) in phosphate-buffered saline (PBS) at a rate of 1 × 10⁻¹⁴. 4 Infection was induced at 12:00 PM via intranasal inoculation of plaque-forming units (PFUs). The establishment of a SARS-CoV-2 animal model was confirmed (data not shown).

[0086] [Example 1] SARS-CoV-2 PLPro Inhibition Assay for In vitro Evaluation of the Antiviral Effect of Galactose-Modified Isotretinoin, a Novel Retinoic Acid Compound, Against SARS-CoV-2 The enzyme inhibition assay was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4). SARS-CoV-2 PLPro (0.9 μM) obtained above was added to each well to form an enzyme solution. The enzyme solutions in the wells were divided into one control group (abbreviated as "control"), one comparison group (abbreviated as "comparison_1"), and one experimental group (abbreviated as "experiment_1"). The corresponding inhibitors shown in Table 1 below were added to the enzyme solution of each group to form test mixtures (total volume 180 μL). Pre-incubation was performed for 30 minutes.

[0087] [Table 1]

[0088] The enzymatic reaction was initiated by adding 20 μL of the aforementioned FG6-pNA (1.2 mM) to the test mixture of each group. The enzymatic reaction was allowed to proceed at 30°C for 300 seconds. Enzymatic activity was measured using a 96-well microplate spectrophotometer (Epoch® 2, Biotek) by measuring the absorbance at 405 nm (A). 405 The reaction rate was determined by continuously monitoring the absorbance A over a total reaction time of 300 seconds. The reaction rate was calculated as appropriate, where the reaction rate is defined as the absorbance A over a total reaction time of 300 seconds. 405 This is the slope relative to time (seconds). The data is shown in Figure 2.

[0089] The inhibition percentage was calculated using the following equation: A = [1 - (B / C)] × 100 (I) In the formula, A = Inhibition percentage B = reaction rate of each group C = reaction rate of the control group The acquired data is expressed as mean ± standard deviation.

[0090] Results: The inhibition percentages for the different treatment groups are shown in Table 2 below.

[0091] [Table 2]

[0092] As shown in Figure 2 and Table 2, the inhibition rate in Experiment 1 was significantly higher than that in Comparison 1, indicating that galactose-modified isotretinoin was more effective than isotretinoin alone in inhibiting the enzymatic activity of PLpro. This result suggests that galactose-modified isotretinoin has a better inhibitory effect on the activity of papain-like proteases.

[0093] In addition, this provides important insights into the biochemical characteristics of the PLpro family of coronaviruses, paving the way for promising therapeutic strategies against SARS-CoV-2.

[0094] [Example 2] Evaluation of the in vivo antiviral effect of galactose-modified isotretinoin, a novel retinoic acid compound, against SARS-CoV-2 in an animal model of SARS-CoV-2. Since galactose-modified isotretinoin, a novel retinoic acid compound, has been demonstrated to have an in vitro inhibitory effect against SARS-CoV-2, further in vivo animal studies were conducted to evaluate the antiviral effect of the novel retinoic acid compound against SARS-CoV-2.

[0095] As mentioned above, the infected hamsters were divided into the following four groups (n=5 per group): a control group (abbreviated as "control"), two comparison groups (abbreviated as "comparison_1" and "comparison_2," respectively), and one experimental group (abbreviated as "experiment_1"). The therapeutic agents used in each of these groups are listed in Table 3 below.

[0096] [Table 3]

[0097] Specifically, in Experiment 1, each hamster was administered a novel retinoic acid compound intranasally at a dose of 0.35 mg / kg at 8:00 a.m. on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, day 1) and again at 8:00 p.m. on the day of infection. For the next two days (days 2 and 3), the novel retinoic acid compound was administered twice daily, at 8:00 a.m. and 8:00 p.m., via intranasal administration at a dose of 0.35 mg / kg each time.

[0098] In the control group, each hamster received intranasal administration of isotretinoin in Control 1 or liposomal-encapsulated isotretinoin in Control 2 at a dose of 0.35 mg / kg at 8:00 a.m. on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and at 8:00 p.m. on the day of infection. In the control group, isotretinoin in Control 1 or liposomal-encapsulated isotretinoin in Control 2 was administered intranasally at a dose of 0.35 mg / kg twice daily at 8:00 a.m. and 8:00 p.m. for the following two days after the day of infection. The liposomal-encapsulated isotretinoin in Control 2 was prepared by Taipei Medical University (Taiwan) using a technique commonly used in the art and described in detail above. The main technical feature of this disclosure lies in the novel retinoic acid compound, galactose-modified isotretinoin; therefore, for brevity, details of the liposome are omitted here.

[0099] In the control group, each hamster was administered 100 μL of buffer via nasal infusion at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and again at 8:00 PM on the day of infection. The buffer was also administered 100 μL twice daily at 8:00 AM and 8:00 PM via nasal infusion for two days following the day of infection.

[0100] After 3 days of treatment, the hamsters were euthanized, and their lungs were examined for TCID in Vero E6 cells. 50 The viral load was collected for assay-based measurement of live viral load. The Reed-Muench method was used to determine the 50% tissue culture infectious dose (TCID). 50 The viral titer was determined from the perspective of [the specified criteria]. All experiments using SARS-CoV-2 were conducted in a biosafety level 3 (BSL-3) laboratory and were approved by Academia Sinica (Taipei, Taiwan).

[0101] Experimental data were analyzed using Tukey's test to evaluate differences between groups. Statistical significance is indicated by p<0.05.

[0102] Results: Refer to Figure 3, the TCID of Experiment 1. 50 This study revealed that galactose-modified isotretinoin, a novel retinoic acid compound, exhibits significantly lower in vivo efficacy against SARS-CoV-2 compared to isotretinoin alone or liposomal-encapsulated isotretinoin alone, both of which were significantly lower than in comparisons 1 and 2.

[0103] [Example 3] Evaluation of the in vitro cytotoxic effects of galactose-modified isotretinoin, a novel retinoic acid compound, on different cancer cell lines. In the embodiments of the present invention, seven cell lines were used, which are AsPC-1, MDA-MB-231, HCT-116, Huh-7, SKOV-3, A549, and H460 cancer cell lines, respectively. Each cell line was cultured in the indicated growth medium supplemented with 10% FBS and maintained in a humidified incubator at 37°C containing 5% CO2.

[0104] One day before the procedure, harvest and count the cells in the logarithmic growth phase, and measure 1 × 10⁻⁶. 4 Cells were seeded in a 96-well plate at a density of 100 μL / well. After overnight incubation, the culture medium was gently removed from each well, and then fresh medium was added (200 μL / well).

[0105] The test material (TA) was a novel retinoic acid compound (abbreviated as "novel compound" in the examples of this invention) freshly prepared in a 96 mM stock solution using 100% DMSO on the day of treatment. Isotretinoin was serially diluted 2-fold using 100% DMSO to obtain different concentrations of isotretinoin from 7.5 mM to 96 mM. Cells were treated by adding 2.02 μL of isotretinoin at the indicated concentration to each well to obtain isotretinoin in a final concentration range from 7.5 μM to 960 μM, with all wells containing 1% DMSO, including the DMSO control. All components were gently mixed and incubated for 24 hours.

[0106] The cells were treated by adding 2.02 μL of the indicated concentration of new compound A or DMSO to each well, then gently mixed and incubated for 24 hours.

[0107] On the cell viability day, the culture medium in each well was replaced with freshly prepared Alamar blue dye (10% v / v) and incubated at 37°C for 2–3 hours. Spectrophotometric absorbance was recorded at wavelengths of 570 nm and 600 nm.

[0108] The percentage of cell viability was calculated using the following formula: Survival rate % = (TA's [A 570 / A 600 [A ratio] / DMSO 570 / A 600 ratio])×100% In the formula, TA = test item A 570 Absorbance at 570 nm A 600 = Absorbance at 600 nm

[0109] Results: As shown in Figures 4-10, the novel compounds significantly reduced cell viability in all seven cancer cell lines, demonstrating that galactose-modified isotretinoin, a novel retinoic acid compound, could be a potential anticancer drug for effectively treating various cancers, including lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, and liver cancer. See Table 4 for IC. 50 This was shown.

[0110] [Table 4]

[0111] [Example 4] SARS-CoV-2 PLPro Inhibition Assay for In vitro Evaluation of the Antiviral Efficacy of a Second Pharmaceutical Composition Against SARS-CoV-2 The enzyme inhibition assay was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4). SARS-CoV-2 PLPro, obtained as described above, was added to each well to form an enzyme solution with a final concentration of 0.9 μM. The enzyme solutions in the wells were divided into one control group (abbreviated as "control"), four comparison groups (abbreviated as "comparison_1", "comparison_2", "comparison_3", and "comparison_4", respectively), and two experimental groups (abbreviated as "experiment_1" and "experiment_2", respectively). The corresponding inhibitors shown in Table 5 below were added to the enzyme solution of each group to form test mixtures (total volume of 180 μL). Pre-incubation was performed for 30 minutes.

[0112] [Table 5]

[0113] 20 μL of the above-described FG6-pNA (1.2 mM) was added to the test mixtures of each group to initiate the enzyme reaction. The enzyme reaction was allowed to proceed at 30 °C for 300 seconds. The enzymatic activity was determined by continuously monitoring the absorbance (A 405 ) at 405 nm using a 96-well microplate spectrophotometer (Epoch (trademark) 2, Biotek). The reaction rate was calculated as appropriate. Here, the reaction rate is the slope of absorbance A 405 versus time (seconds) over the total reaction time of 300 seconds). The data is presented in Figure 11. The inhibition percentage was calculated as described in Example 3.

[0114] Results: The inhibition percentages of the different treatment groups are shown in Table 6 below.

[0115]

Table 6

[0116] As shown in Figure 11 and Table 6, the inhibitory effects of each group in the experimental groups were significantly higher than those of each group in the control group, and the two experimental groups: (1) isotretinoin and oligosaccharide, and (2) isotretinoin, oligosaccharide, Zn 2+ and Mg 2+ were more effective than the individual components of isotretinoin, oligosaccharide, Zn 2+ or Mg 2+ alone. These results suggest that isotretinoin, oligosaccharide and divalent metal ions have a synergistic inhibitory effect on the activity of papain-like protease. In addition, this provides important insights into the biochemical properties of the PLpro family of coronaviruses and paves the way for promising treatment strategies against SARS-CoV-2.

[0117] [Example 5] Evaluation of the in vivo treatment effect of a combination of isotretinoin, oligosaccharide or divalent metal ions against SARS-CoV-2 in a SARS-CoV-2 animal model The infected hamsters obtained in Part A of this case were divided into the following six groups (n=5 per group): a control group, three experimental groups (i.e., experimental groups 1, 2, and 3), and four comparison groups (i.e., comparison groups 1, 2, 3, and 4). The treatments used for each of these groups are listed in Table 7 below.

[0118] [Table 7]

[0119] The liposome-encapsulated therapeutic agents were prepared by Taipei Medical University using liposomes and techniques commonly used in the art and described in detail above. Since the main technical feature of this disclosure lies in the combination of retinoic acid and oligosaccharides with / without metal ions, details of the liposomes are omitted here for brevity.

[0120] Specifically, each hamster in the experimental group received liposomal-encapsulated isotretinoin intranasally at a dose of 0.35 mg / kg at 8:00 a.m. on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, day 1) and at 8:00 p.m. on the day of infection. For the next two days (days 2 and 3), liposomal-encapsulated isotretinoin was administered twice daily via intranasal administration at 8:00 a.m. and 8:00 p.m., each time at a dose of 0.35 mg / kg.

[0121] In addition, liposomal-encapsulated isotretinoin was administered twice daily, at 8:00 AM and 8:00 PM, via nasal administration at a dose of 0.35 mg / kg, along with a 15 μL volume of liposomal-encapsulated oligosaccharide mixture (i.e., Experiment 1), or a 15 μL volume of liposomal-encapsulated oligosaccharide mixture and Zn. 2+ (100 μM), Mg 2+ (200 μM) and K +(200 μM) combination (15 μL) in a total volume of 30 μL (i.e., Experiment_2) was administered once a day via nasal administration from 7:30 pm to 8:00 pm on the day of infection (i.e., 0.5 to 1 hour before the administration of liposome-encapsulated isotretinoin at night), and 15 μL in volume in Experiment_1 and 30 μL in volume in Experiment_2, respectively, 2 days later.

[0122] For each hamster in the comparison group, isotretinoin in Comparison_1 or liposome-encapsulated isotretinoin in Comparison_2 was administered intranasally at a dose of 0.35 mg / kg at 8:00 am on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and at a dose of 0.35 mg / kg at 8:00 pm on the day of infection, and isotretinoin in Comparison_1 or liposome-encapsulated isotretinoin in Comparison_2 was administered intranasally at a dose of 0.35 mg / kg twice a day at 8:00 am and 8:00 pm for the next 2 days after the day of infection.

[0123] For each hamster, Zn 2+ (100 μM), Mg 2+ (200 μM) and K + (200 μM) combination and a mixture of oligosaccharides encapsulated in liposomes in Comparison_4 were administered once a day via nasal administration from 7:30 pm to 8:00 pm on the day of infection and in a volume of 15 μL each 2 days later.

[0124] For each hamster in the control group, buffer was administered via nasal administration at a volume of 100 μL at 8:00 am on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) and at a volume of 100 μL at 8:00 pm on the day of infection. Buffer was administered via nasal administration twice a day at 8:00 am and 8:00 pm for the next 2 days after the day of infection at a volume of 100 μL.

[0125] After 3 days of treatment, the hamsters were sacrificed and their lungs were used for TCID in Vero E6 cells [[ID=The viral load was collected for assay-based measurement of live viral load. The Reed-Muench method was used to determine the 50% tissue culture infectious dose (TCID). 50 The viral titer was determined from the perspective of [the specified criteria]. All experiments using SARS-CoV-2 were conducted in a biosafety level 3 (BSL-3) laboratory and were approved by Academia Sinica (Taipei, Taiwan).

[0126] Experimental data were analyzed using Tukey's test to evaluate differences between groups. Statistical significance is indicated by p<0.05.

[0127] Results: Refer to Figure 12, and the respective TCIDs for Experiment 1, as well as Experiments 2 and 3. 50 This study revealed that the combination of isotretinoin with / without metal ions and oligosaccharides had significantly lower in vivo efficacy against SARS-CoV-2 compared to isotretinoin alone, metal ions alone, or oligosaccharides alone, with significantly lower values ​​than those in Comparatives 1, 2, 3, and 4.

[0128] conclusion Considering the results of Examples 1-5, it has been verified that the claimed novel retinoic acid compound or a pharmaceutically acceptable salt thereof, or the claimed pharmaceutical composition comprising the compound (i.e., the first pharmaceutical composition), or the claimed pharmaceutical composition comprising retinoic acid with / without metal ions and a carbohydrate (i.e., the second pharmaceutical composition), can provide improved and / or synergistic effects in inhibiting SARS-CoV-2 infection and replication, and in treating diseases associated with SARS-CoV-2 infection or cancer. As a result, the compounds comprising retinoic acid conjugated to a carbohydrate of the present invention, pharmaceutically acceptable salts thereof, and the first and second pharmaceutical compositions can, in fact, be supplied as drug repurposing agents.

[0129] The present invention has been described broadly and comprehensively herein. Each of the narrower species and subgroup classifications within the comprehensive disclosure also forms part of the present invention. In addition, where features or aspects of the present invention are described in terms of the Markush group, those skilled in the art will recognize that the present invention is also described in terms of any individual member of the Markush group or a subgroup of a member thereof. The present invention includes the following embodiments. [Claim 1] Compounds or pharmaceutically acceptable salts thereof containing retinoic acid conjugated to carbohydrates. [Claim 2] The aforementioned compound is of formula (I): [ka] [In the formula, R 1 [This refers to a substituted or unsubstituted functional group of a carbohydrate.] The compound or a pharmaceutically acceptable salt thereof as represented by claim 1. [Claim 3] The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides. [Claim 4] The compound or pharmaceutically acceptable salt thereof according to claim 3, wherein the oligosaccharide is a monooligosaccharide or heterooligosaccharide, and the polysaccharide is a monopolysaccharide or heteropolysaccharide. [Claim 5] The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the carbohydrate comprises glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3 / 1,6-glucan oligosaccharide, raffinose, stachyose, verbascose, fructooligosaccharide, starch, glycogen, cellulose, or any combination thereof. [Claim 6] A pharmaceutical composition comprising a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. [Claim 7] The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier comprises liposomes, and the compound or the pharmaceutically acceptable salt thereof is encapsulated by the liposomes. [Claim 8] The pharmaceutical composition according to claim 6, further comprising metal ions. [Claim 9] The pharmaceutical composition according to claim 8, wherein the metal ion comprises a monovalent ion, a divalent ion, or a combination thereof. [Claim 10] The aforementioned monovalent ion, K + kaNa + , or a combination thereof, wherein the divalent ion is Zn 2+ Mg 2+ Cu 2+ Mn2+ Ca 2+ Fe 2+ The pharmaceutical composition according to claim 9, comprising, or any combination thereof. [Claim 11] The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable carrier comprises liposomes, and the metal ions are encapsulated by the liposomes. [Claim 12] The pharmaceutical composition according to claim 11, wherein the compound or a pharmaceutically acceptable salt thereof and the metal ion are encapsulated individually or simultaneously by the liposome. [Claim 13] Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 6 to 12, in the manufacture of a pharmaceutical for inhibiting viral infection or replication. [Claim 14] The use according to claim 13, wherein the virus is an RNA virus. [Claim 15] The use according to claim 14, wherein the RNA virus includes coronavirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, or any combination thereof. [Claim 16] The use according to claim 15, wherein the coronavirus includes severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU (HCoV-HKU1), or any combination thereof. [Claim 17] Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 6 to 12, in the manufacture of a pharmaceutical for treating cancer. [Claim 18] The use according to claim 17, wherein the cancer includes leukemia, lymphoma, carcinoma, or sarcoma.

Claims

1. A compound or pharmaceutically acceptable salt thereof that is isotretinoin conjugated to a carbohydrate via a -C3H6NH- linker, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

2. The compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the oligosaccharide is a monooligosaccharide or heterooligosaccharide, and the polysaccharide is a monopolysaccharide or heteropolysaccharide.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the carbohydrate comprises glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3 / 1,6-glucan oligosaccharides, raffinose, stachyose, verbascose, fructooligosaccharides, starch, glycogen, cellulose, or any combination thereof.

4. The compound is of formula (Ia): 【Chemistry 10】 The compound according to claim 1 or a pharmaceutically acceptable salt thereof, represented by [formula].

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutically acceptable carrier comprises liposomes, and the compound or the pharmaceutically acceptable salt thereof is encapsulated by the liposomes.

7. The pharmaceutical composition according to claim 5, further comprising metal ions.

8. The pharmaceutical composition according to claim 7, wherein the metal ion comprises a monovalent ion, a divalent ion, or a combination thereof.

9. The aforementioned monovalent ion, K ＋ Na ＋ , or a combination thereof, wherein the divalent ion is Zn ２＋ Mg ２＋ ,Cd ２＋ Mn ２＋ Ca ２＋ Fe ２＋ The pharmaceutical composition according to claim 8, comprising, or any combination thereof.

10. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable carrier comprises liposomes, and the metal ions are encapsulated by the liposomes.

11. The pharmaceutical composition according to claim 10, wherein the compound or a pharmaceutically acceptable salt thereof and the metal ion are encapsulated individually or simultaneously by the liposome.

12. Use of a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 5 to 11, in the manufacture of a pharmaceutical for inhibiting viral infection or replication.

13. The use according to claim 12, wherein the virus is an RNA virus.

14. The use according to claim 13, wherein the RNA virus includes coronavirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, or any combination thereof.

15. The use according to claim 14, wherein the coronavirus includes severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU (HCoV-HKU1), or any combination thereof.

16. Use of a compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 5 to 11, in the manufacture of a pharmaceutical for treating cancer.

17. The use according to claim 16, wherein the cancer includes leukemia, lymphoma, carcinoma, or sarcoma.

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