Novel retinoic acid compounds, pharmaceutical compositions containing same and uses thereof

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

[0002] Coronaviruses are a group of single-stranded, positive-stranded 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 causing the coronavirus disease 2019 (COVID-19) pandemic.

[0003] The SARS-CoV-2 genome shares high sequence identity with that of SARS-CoV. Both SARS-CoV-2 and SARS-CoV critically rely on the activity of two viral proteases, namely, 3C-like protease (3CL, also known as major protease (M) or nonstructural protein 5 (nsp5)) and papain-like protease (PL, the protease domain of nonstructural protein 3 (nsp3)), to achieve their viral multiplication cycle and spread. It has been reported that all-trans retinoic acid (ATRA, also known as vitamin A acid or tretinoin) could be considered a potential therapeutic agent against SARS-CoV-2 by inhibiting 3CL activity.

[0004] Aside from 3CL, PL is also a potential target; therefore, such enzymes play important roles in the cleavage and maturation of viral polyproteins, the assembly of replicase-transcriptase complexes, and the disruption of host responses. Although the primary function of M and PL is to process viral polyproteins in a coordinated manner, PL has an additional function of removing ubiquitin and IFN-stimulated gene factor 15 (ISG15) from host cellular proteins, enabling coronaviruses to evade the host's innate immune response. Thus, PL is not only involved in viral replication but also in the dysregulation of signaling cascades in infected cells that result in cell death in surrounding uninfected cells. Therefore, drugs designed to inhibit the function of PL could potentially combat SARS-CoV-2. In addition to ATRA, some researchers have recently reported its derivative, 13-cis-retinoic acid (also known as isotretinoin), as a potential PL inhibitor and 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. Biochemists discovered that retinoic acid receptors in the human cell nucleus are not mutated in cancer cells, thus potentially enabling retinoic acid to exert its anticancer effects in many malignancies. For example, treatment of children at high risk for neuroblastoma with 13-cis-retinoic acid 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 a different interferon (IFN) has been shown to potentiate 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 have emphasized the need for a better understanding of the mechanisms that block retinoic acid signaling and retinoic acid-regulated gene expression in cancers such as acute myeloid leukemia (AML). It is clear that combination therapy targeting multiple gene silencing mechanisms may be the most effective strategy for reactivating ATRA-sensitive gene expression and differentiation in AML cells, as well as mediating ATRA's overall anticancer activity. Currently, the identification of classes of proteins that control gene expression through histone and DNA modifications is driving the development of new therapeutic agents that alter chromatin structure, so-called epigenetic drugs. However, these epigenetic modifiers, when used alone, have been shown to be only partially effective against different cancers. Summary of the Invention

[0006] Based on the above reasons, the present invention provides novel retinoic acid compounds and pharmaceutical compositions containing the same, which 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 a retinoic acid conjugated to a carbohydrate.

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

[0009] [ka] [In the formula, R 1 is a substituted or unsubstituted functional group of the carbohydrate] is expressed by

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

[0011] Preferably, the oligosaccharide is a mono- or hetero-oligosaccharide, and the polysaccharide is a monopolysaccharide or heteropolysaccharide.

[0012] Preferably, 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.

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

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

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

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

[0017] Preferably, the metal ions include monovalent ions, divalent ions, or a combination thereof. More preferably, the monovalent ions are K + , Na + or a combination thereof, wherein 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 a liposome and the metal ion is encapsulated by the liposome.

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

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

[0021] In yet another aspect, the present invention provides the use of said compound or said pharmaceutically acceptable salt thereof, or said 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 compounds, which are retinoic acid conjugated to carbohydrates, and pharmaceutical compositions thereof, can enhance the ability to inhibit viral infection and / or replication compared to retinoic acid alone. 2. The claimed compounds and pharmaceutical compositions thereof may serve as potential drugs against coronaviruses, particularly SARS-CoV-2, which causes COVID-19. 3. The claimed compounds and pharmaceutical compositions thereof can also enhance the ability to inhibit cancer cells and thus effectively treat various cancers such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, and liver cancer. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 illustrates the results of liquid chromatography-mass spectrometry (LC-MS) analysis of compound (Ia) in accordance with an embodiment of the present invention. [Figure 2] FIG. 1 illustrates absorbance versus time curve plots in a SARS-CoV-2 PLPro inhibition assay for novel retinoic acid compounds according to an embodiment of the present invention. [Figure 3] FIG. 1 illustrates a TCID50 plot showing the antiviral efficacy of novel retinoic acid compounds in a SARS-CoV-2 animal model according to an embodiment of the present invention. [Figure 4] FIG. 1 illustrates a cell viability curve showing the cytotoxic effect of novel retinoic acid compounds on AsPC-1 cancer cell line in accordance with an embodiment of the present invention. [Figure 5] FIG. 1 illustrates a cell viability curve showing the cytotoxic effect of novel retinoic acid compounds on MDA-MB-231 cancer cell line in accordance with an embodiment of the present invention. [Figure 6] FIG. 1 illustrates a cell viability curve showing the cytotoxic effect of novel retinoic acid compounds on HCT-116 cancer cell line in accordance with an embodiment of the present invention. [Figure 7] FIG. 1 illustrates cell viability curves showing the cytotoxic effects of novel retinoic acid compounds on Huh-7 cancer cell line in accordance with an embodiment of the present invention. [Figure 8] FIG. 1 illustrates a cell viability curve showing the cytotoxic effect of novel retinoic acid compounds on SKOV-3 cancer cell line in accordance with an embodiment of the present invention. [Figure 9] FIG. 1 illustrates a cell viability curve showing the cytotoxic effect of novel retinoic acid compounds on A549 cancer cell line in accordance with an embodiment of the present invention. [Figure 10] FIG. 1 illustrates a cell viability curve showing the cytotoxic effect of novel retinoic acid compounds on H460 cancer cell line in accordance with an embodiment of the present invention. [Figure 11] FIG. 1 illustrates an absorbance versus time curve plot in a SARS-CoV-2 PLPro inhibition assay for a second pharmaceutical composition according to an embodiment of the present invention. [Figure 12] FIG. 1 illustrates a TCID50 plot showing the antiviral efficacy of a second pharmaceutical composition in a SARS-CoV-2 animal model according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] While the invention is susceptible to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and will be described in detail. It should be understood, however, that the description is not intended to limit the invention to the specific embodiments, but on the contrary, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

[0025] definition Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with 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 as such in this specification.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of embodiments of the present invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will be further understood that the terms "comprises," "comprising," "includes," and / or "including," when used herein, specify the presence of stated features, integers, steps, operations, elements, components, and / or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or combinations thereof.

[0027] The term "subject" as used herein refers to a mammal for which diagnosis, prognosis, or therapy is desired. Generally, the mammal is a human. In certain embodiments, the mammal may refer to a non-human mammal, such as a non-human primate, cow, horse, goat, sheep, dog, cat, rabbit, pig, mouse, or rat, used in, for example, screening, characterizing, and evaluating drugs, and in therapy.

[0028] As used herein, the term "administration" or "administered" refers to introducing, providing, or delivering a given active ingredient to a subject by any suitable route to perform its intended function.

[0029] The term "cancer" as used herein refers to leukemia, lymphoma, carcinoma, sarcoma, and other malignant tumors of potentially unlimited growth that can spread locally by invasion and throughout the body by metastasis. Examples of cancer include, but are not limited to, cancer of the ovary, adrenal gland, bone, brain, breast, bronchus, colon and / or rectum, gallbladder, head and neck, kidney, larynx, liver, lung, nervous tissue, pancreas, prostate, parathyroid gland, skin, stomach, and thyroid gland. Certain other examples of cancers include cholangiocarcinoma, acute and chronic lymphocytic and granulocytic tumors, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma (multiforma), hairy cell tumor, intestinal ganglioneuroma, hyperplastic corneal nerve tumor, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, Marfanoid habitus tumor, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, pheochromocytoma, polycythermia vera, and leiomyoma. vera), primary brain tumors, small cell lung tumors, squamous cell carcinoma, both ulcerative and papillary types, hyperplasia, seminoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, veticulum cell sarcoma, and Wilms' tumor.

[0030] As used herein, the term "oligosaccharide" refers to a carbohydrate composed of a small number of monosaccharide units, usually about 3 to 10 monosaccharide units, where oligosaccharides with one type of monosaccharide subunit are called mono-oligosaccharides and oligosaccharides with two or more types of monosaccharide subunits are called hetero-oligosaccharides.

[0031] As used herein, the term "polysaccharide" refers to a carbohydrate composed of multiple monosaccharide units, where polysaccharides with one type of monosaccharide subunit are called monopolysaccharides and polysaccharides with two or more types of monosaccharide subunits are called heteropolysaccharides.

[0032] The term "liposome" as used herein refers to a particle characterized by an aqueous interior space separated from the external medium by one or more bilayer membranes forming a vesicle. The main types of liposomes are multilamellar vesicles (MLVs, with several lamellar lipid bilayers), small unilamellar vesicles (SUVs, with a single lipid bilayer), and large unilamellar vesicles (LUVs, with a single lipid bilayer). The bilayer membrane of unilamellar or multilamellar vesicles is typically formed by lipids, i.e., amphiphilic molecules of synthetic or natural origin containing spatially separated hydrophobic and hydrophilic domains.

[0033] Illustrative Embodiments Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings.

[0034] In certain embodiments, 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 has Formula (I):

[0036] [ka] [In the formula, R 1 is a substituted or unsubstituted functional group of the carbohydrate] is expressed by

[0037] In certain embodiments, the concentration of a compound comprising a 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 (hereinafter referred to as the "second" pharmaceutical composition) is provided that includes retinoic acid and a carbohydrate. In certain embodiments, 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 mono-oligosaccharide or a hetero-oligosaccharide. The polysaccharide may be a mono-polysaccharide or a hetero-polysaccharide. 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 certain embodiments, the carbohydrate concentration is from 0.1 μM to 200 mM, from 0.1 μM to 150 mM, from 0.1 μM to 100 mM, from 0.1 μM to 10 mM, from 0.1 μM to 1 mM, from 0.1 μM to 500 μM, from 0.1 μM to 250 μM, from 0.1 μM to 100 μM, from 0.1 μM to 50 μM, from 1 μM to 200 mM, from 1 μM to 150 mM, from 1 μM to 10 ... The concentration may be, but is not limited to, from 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 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.

[0042] In certain embodiments, the molar ratio of retinoic acid to carbohydrate in the second pharmaceutical composition is about 1:10. -3 -100, 1:10 -3 -20, 1:1-20, or 1:1-10.

[0043] In certain embodiments, the first and second pharmaceutical compositions each optionally further comprise a metal ion. Preferably, the metal ion comprises a monovalent ion, a divalent ion, or a combination thereof. In certain embodiments, the monovalent ion is K + , Na + or a combination thereof, wherein the divalent ion is Zn 2+ , Mg 2+ , Cu 2+ , Mn 2+ , Ca 2+ , Fe 2+ , or any combination thereof.

[0044] In certain embodiments, the concentration of the metal ion is from 1 μM to 300 mM, from 1 μM to 250 mM, from 1 μM to 200 mM, from 1 μM to 150 mM, from 1 μM to 100 mM, from 1 μM to 10 mM, from 1 μM to 1 mM, from 1 μM to 500 μM, from 1 μM to 250 μM, from 1 μM to 100 μM, from 1 μM to 50 μM , 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 certain embodiments, the molar ratio of the compound and the metal ion in the first pharmaceutical composition is about 1:10 -3 -10 3 , 1:0.1-20, 1:0.1-10, or 1:1-10.

[0046] In certain embodiments, the molar ratio of retinoic acid, carbohydrate, and metal ion in the second pharmaceutical composition is about 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 -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 each optionally further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are widely used in the technical field of drug manufacturing. Examples of pharmaceutically acceptable carriers may include, but are not limited to, liposomes, excipients, adjuvants, solvents, buffer solutions, emulsifiers, suspending agents, disintegrating agents, disintegrating agents, dispersing agents, binders, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, etc. The selection and amount of pharmaceutically acceptable carriers are within the expertise of those skilled in the art.

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

[0049] Exemplary liposomes can be neutral, positively or negatively charged liposomes.In general, the lipids commonly used in liposomes typically include two-chain lipids such as phospholipids, diglycerides and dialiphatic glycolipids; single lipids such as sphingomyelin and glycosphingolipids; 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-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearo ... 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 soy phosphatidylcholine (HSPC), 1,2-dimyristoyl-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) (DSPG), 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-sn-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphatase 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 EPC), L-alpha-phosphatidylethanolamine (EPE), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.

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

[0051] The mole percentage of lipid in the bilayer may be about 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or less, or any value or range of values ​​therebetween (e.g., about 5-50%, about 5-45%, about 5-40%, about 5-35%, about 5-30%, about 5-25%, about 5-20%, about 5-15%, or about 5-10%).

[0052] The lipids of the bilayer membrane may be a mixture of a first phospholipid and a second phospholipid, wherein the first phospholipid is selected from the group consisting essentially of PC, HSPC, DOPC, POPC, DSPC, DPPC, DMPC, PSPC, and combinations thereof, and the second phospholipid is selected from the group consisting essentially 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 about 50, 45, 40, 35, 30, 25, 20, 15, 10, or any value or range of values ​​therebetween (e.g., about 5-50%, about 5-45%, about 5-40%, about 5-35%, about 5-30%, about 5-25%, about 5-20%, about 5-15%, or about 5-10%), and the molar percentage of the second phospholipid in the bilayer is between 0.1 and about 15, 14, 13, 12, 11, 10, 9, 8, 7, or any value or range of values ​​therebetween (e.g., about 0.1-15%, about 0.1-10%, about 0.5-15%, about 0.5-10%, or about 0.5-7%). In an exemplary embodiment, 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 less than about 55 molar percent of a steroid, preferably cholesterol. The molar percentage of steroid (e.g., 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 phospholipid and cholesterol in the bilayer may be about 25-50%:15-55%, 25-50%:20-55%, or 25-50%:15-50%. The phospholipid and cholesterol may be in a molar ratio of 1:1 to 3:1. The molar percentage ratio of the first phospholipid, the second phospholipid, and the cholesterol in the bilayer membrane 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 entrapment agent can be prepared by any of the currently known or later developed techniques. For example, MLV liposomes can be formed directly by hydrated lipid films, spray-dried powders, or lyophilized cakes of selected lipid compositions with the entrapment agent, and SUV and LUV liposomes can be sized from MLV liposomes by sonication, homogenization, microfluidization, or extrusion.

[0055] In yet another embodiment, a compound comprising retinoic acid conjugated to a carbohydrate or a pharmaceutically acceptable salt thereof, and 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, a compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and the first and second pharmaceutical compositions can be used to treat cancer.Cancer may include leukemia, lymphoma, carcinoma, or sarcoma.In an exemplary embodiment, cancer may be lung cancer, ovarian cancer, breast cancer, liver cancer, pancreatic cancer, or bile duct cancer.

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

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

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

[0060] In certain embodiments, the compound comprising retinoic acid conjugated to a carbohydrate, the pharmaceutically acceptable salt thereof, and the first and second pharmaceutical compositions are each administered by oral, intravenous, intramuscular, subcutaneous, intraperitoneal, intranasal, or topical routes. [Example]

[0061] The present invention is further illustrated by the following examples, however, it should be understood that the following examples are intended for illustrative purposes only and should not be construed as limiting the invention in any way.

[0062] material and method A. Preparation of Galactose-Modified Isotretinoin, a Novel Retinoic Acid Compound Represented by Formula (Ia) Based on the Following Synthesis 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 on Merck TLC aluminum sheets silica gel 60F. 254 The NMR spectra were visualized by fluorescence quenching under UV light. Flash chromatography was performed using silica gel (Chromatorex, MB70-40 / 75, 40-75 μm) purchased from Fuji Silysia Chemical Co., Ltd. 1 H were recorded on a Varian-400MR operating at 400 MHz. Chemical shifts are reported in ppm using the solvent resonance as the internal standard. Data are reported as follows: s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets; coupling constants in Hz; integrals. Purity data were recorded on 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-triyl triacetate (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 freshly dried molecular sieves in anhydrous dichloromethane (50 mL) was added boron trifluoride-diethyl etherate (8.04 mL, 64.0 mmol) at 0 °C. The mixture was stirred at room temperature overnight. The solution was neutralized with triethylamine, the molecular sieves were removed by passing 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 (EtOAc:hexane = 1:2) to give 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; Found 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-triyl triacetate (3)

[0068] [ka] To a solution of compound 2 (0.62 g, 1.3 mmol) in DMF (4 mL) was added sodium azide (0.43 g, 6.6 mmol). 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 (EtOAc:hexane=1:2) to give 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; Found value, 454.2 [M + Na] + .

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

[0070] [ka] To a solution of compound (3) (0.45 g, 1 mmol) in dichloromethane (1.0 mL) and methanol (4.0 mL) was added sodium methoxide (28 mg, 0.5 mmol). The mixture was stirred at room temperature for 10 hours. The solution was neutralized with Dowex 50WX8 and evaporated to dryness to give the desired product (4), which was used in the next step without further purification. LCMS (ESI) m / z C9H 17Calculated N3O6 value 263.25; Found 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 H atmosphere. The solution was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to give (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 NO6 value: 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-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenoate (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) was added pentafluorophenyl trifluoroacetate (compound (8), 0.081 mL, 0.47 mmol) at 0° C. The solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with EtOAc 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 give the desired product (6) without further purification.

[0075] VII. Synthesis of 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-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide (Ia)

[0076] [ka] 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) was added triethylamine (0.049 mL, 0.33 mmol) at room temperature. 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; Found value: 520.5 [M+H] + See 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 assessed.

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

[0079] The vector with the inserted SARS-CoV-2 PLPro gene was transformed into Escherichia coli (E. coli) BL21(DE3) strain (Yeastern Biotech Co., Ltd., New Taipei City, Taiwan) for overexpression of PLPro therein. Cultivation was performed in LB medium (containing 1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with ampicillin (100 μg / mL), which served as an antibiotic marker. The resulting culture was initially incubated at 37°C with shaking at 200 rpm. The optical density at 600 nm (OD ) was between 0.6 and 0.8. 600At 4°C, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce PLpro expression. Incubation continued for 20 hours at 18°C ​​and 200 rpm. Cells were harvested by centrifugation (5,000 × g) and disrupted 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 onto a 5 mL His-Trap HP column (GE Healthcare Life Sciences), and the protein was eluted using a gradient of 0 to 500 mM imidazole in 50 mM sodium phosphate (pH 7.4) and 100 mM NaCl. Fractions containing His-tagged SARS-CoV-2 PLpro were pooled and concentrated using a Centricon membrane (10K cutoff, GE Healthcare Life Sciences). 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 45270 M. -1 cm -1 The extinction coefficient (ε280) was determined by measuring the UV absorbance at 280 nm.

[0080] C. SARS-CoV-2 PLPro Activity Assay The enzymatic activity of the SARS-CoV-2 PLpro obtained above was measured by a colorimetric peptide cleavage assay using a 6-mer peptide substrate, FRLKGG-para-nitroanilide (FG6-pNA) (97% pure by HPLC; GL Biochem Ltd., Shanghai, China). In the cleavage assay, the 6-mer peptide substrate was cleaved at the Gly-pNA bond to release free pNA, which changed the color of the solution to yellow. The enzymatic activity was measured by measuring the absorbance at 405 nm (A ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). 405 ) was determined by continuous monitoring 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, and 6.0 mM). The assay mixture (180 μL per well) was preincubated 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 using a 9800 mM phosphate buffer. -1 cm -1 Using the extinction coefficient (ε405) of A 405 was calculated by measuring 405 = 9.8 at 1 mM).

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

[0083] Result:K M and k cat The values ​​were 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 was successfully prepared according to the above manufacturing method and can be used to perform the following SARS-CoV-2 PLPro inhibition assay.

[0084] D. Establishment of SARS-CoV-2 Animal Models Golden Syrian hamsters (5-6 weeks old, with an average weight of approximately 100 g) were obtained from the National Laboratory Animal Center (Taipei, Taiwan). Hamsters were housed in an animal room under specific pathogen-free (SPF) conditions, as commonly applied in the art. Furthermore, all hamsters were provided with water and food ad libitum. All experiments involving hamsters were submitted to, reviewed, and approved by the Institutional Animal Care and Use Committee (IACUC) of Academia Sinica (Taiwan).

[0085] To establish a SARS-CoV-2 animal model, hamsters were infected with SARS-CoV-2 (obtained from P3Lab, Genomics Research Center, Academia Sinica; Wuhan wild type) in phosphate-buffered saline (PBS) at 1 × 10 4 Infection was initiated via intranasal inoculation of plaque-forming units (PFU) at 12:00 PM. Establishment of the SARS-CoV-2 animal model was confirmed (data not shown).

[0086] [Example 1] SARS-CoV-2 PLPro Inhibition Assay to Evaluate the Antiviral Effect of Galactose-Modified Isotretinoin, a Novel Retinoic Acid Compound, against SARS-CoV-2 in Vitro The enzyme inhibition assay was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4). The 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 comparative group (abbreviated as "Comparative_1"), and one experimental group (abbreviated as "Experiment_1"). The corresponding inhibitor shown in Table 1 below was added to the enzyme solution of each group to form a test mixture (in a total volume of 180 μL). Pre-incubation was performed for 30 minutes.

[0087] [Table 1]

[0088] 20 μL of the above-mentioned FG6-pNA (1.2 mM) was added to each test mixture to initiate the enzyme reaction. The enzyme reaction was allowed to proceed for 300 seconds at 30°C. The enzymatic activity was measured by measuring the absorbance at 405 nm (A ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). 405 The reaction rate was determined by continuously monitoring the absorbance A over a total reaction time of 300 seconds. 405 The data are presented in Figure 2.

[0089] Percent inhibition was calculated using the following equation: A = [1-(B / C)] × 100 (I) Where A = percent inhibition B = reaction rate of each group C = control response rate The data obtained are expressed as the mean ± standard deviation.

[0090] Results: The percentage of inhibition for the different treatment groups is shown in Table 2 below.

[0091] [Table 2]

[0092] As shown in Figure 2 and Table 2, the inhibition rate of Experiment 1 was significantly higher than that of 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] Additionally, this provides important insights into the biochemical properties of the coronavirus PLpro family, paving the way for promising therapeutic strategies against SARS-CoV-2.

[0094] [Example 2] Evaluation of the in vivo antiviral effect of a novel retinoic acid compound, galactose-modified isotretinoin, against SARS-CoV-2 in a SARS-CoV-2 animal model Because the novel retinoic acid compound, galactose-modified isotretinoin, was demonstrated to have in vitro inhibitory effects against SARS-CoV-2, we further evaluated in vivo animal studies to assess the antiviral effects of the novel retinoic acid compound against SARS-CoV-2.

[0095] The infected hamsters obtained as mentioned above were divided into four groups (n=5 per group): a control group (abbreviated as "Control"), two comparison groups (abbreviated as "Comp_1" and "Comp_2", respectively), and one experimental group (abbreviated as "Experiment_1"). The therapeutic agents used for each of these groups are listed in Table 3 below.

[0096] [Table 3]

[0097] Specifically, for each hamster in Experiment 1, a new retinoic acid compound was administered intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, Day 1) at a dose of 0.35 mg / kg and at 8:00 PM on the day of infection at a dose of 0.35 mg / kg. For the next two days (Days 2 and 3), the new retinoic acid compound was administered intranasally twice daily at 8:00 AM and 8:00 PM, each at a dose of 0.35 mg / kg.

[0098] For each hamster in the comparison group, isotretinoin (Comparative_1) or liposomal isotretinoin (Comparative_2) was administered intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) at a dose of 0.35 mg / kg and at 8:00 PM on the day of infection at a dose of 0.35 mg / kg. Isotretinoin (Comparative_1) or liposomal isotretinoin (Comparative_2) was administered intranasally at 8:00 AM and 8:00 PM twice daily for the next two days after the day of infection at a dose of 0.35 mg / kg. The liposomal isotretinoin (Comparative_2) was prepared by Taipei Medical University (Taiwan) using techniques commonly used in the art and described in detail above. Since the main technical feature of this disclosure is the novel retinoic acid compound, galactose-modified isotretinoin, details of the liposome will be omitted here for the sake of brevity.

[0099] For each hamster in the control group, buffer was administered intranasally in 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 8:00 PM on the day of infection. Buffer was administered intranasally in a volume of 100 μL twice daily at 8:00 AM and 8:00 PM for two days after the day of infection.

[0100] After 3 days of treatment, the hamsters were sacrificed and their lungs were cultured at TCID 200 in Vero E6 cells. 50 The virus was collected for live virus assay. The 50% tissue culture infectious dose (TCID) was determined using the Reed-Muench method. 50 Virus titers were determined in terms of . All experiments using SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by Academia Sinica (Taipei, Taiwan).

[0101] The experimental data were analyzed by Tukey's test to assess differences between groups, with statistical significance indicated by p<0.05.

[0102] Results: Referring to Figure 3, the TCID of Experiment 1 50 was significantly lower than those of Comparison 1 and Comparison 2, respectively, demonstrating that the novel retinoic acid compound, galactose-modified isotretinoin, has higher in vivo efficacy against SARS-CoV-2 compared to isotretinoin alone or liposome-encapsulated isotretinoin alone.

[0103] [Example 3] Evaluation of the in vitro cytotoxic effects of galactose-modified isotretinoin, a novel retinoic acid compound, against different cancer cell lines Seven cell lines were used in the present examples: AsPC-1, MDA-MB-231, HCT-116, Huh-7, SKOV-3, A549, and H460 cancer cell lines. 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 treatment, cells in logarithmic growth phase were harvested, counted, and diluted to 1 × 10 4 The cells were seeded into a 96-well plate at a density of 100 μL / well. After overnight culture, the culture medium in each well was gently removed, and then fresh medium was added (200 μL / well).

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

[0106] Cells were treated by adding 2.02 μL of fresh Compound A or DMSO at the indicated concentration to each well, which was then mixed gently and incubated for 24 hours.

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

[0108] The percentage of cell viability was calculated using the following formula: Viability % = (TA [A 570 / A 600 ratio] / DMSO [A 570 / A 600 ratio])×100% Where TA = test article A 570 = absorbance at 570 nm A 600 = absorbance at 600 nm

[0109] Results: Referring to Figures 4-10, they show that the new compound can significantly reduce the cell viability of all seven cancer cell lines, revealing that the novel retinoic acid compound, galactose-modified isotretinoin, can be a potential anticancer drug for effectively treating various cancers, such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colon cancer, and liver cancer. Table 4 shows the IC 50 showed.

[0110] [Table 4]

[0111] [Example 4] SARS-CoV-2 PLPro Inhibition Assay for Evaluating the Antiviral Effect of a Second Pharmaceutical Composition Against SARS-CoV-2 In Vitro 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 mentioned above was added to each well to form an enzyme solution with a final concentration of 0.9 μM. The enzyme solution in the wells was divided into one control group (abbreviated as "Control"), four comparison groups (abbreviated as "Comparative_1", "Comparative_2", "Comparative_3", and "Comparative_4", respectively), and two experimental groups (abbreviated as "Experiment_1" and "Experiment_2", respectively). The corresponding inhibitor shown in Table 5 below was added to the enzyme solution of each group to form a test mixture (in a total volume of 180 μL). Preincubation was performed for 30 minutes.

[0112] [Table 5]

[0113] 20 μL of the above-mentioned FG6-pNA (1.2 mM) was added to each test mixture to initiate the enzyme reaction. The enzyme reaction was allowed to proceed for 300 seconds at 30°C. The enzymatic activity was measured by measuring the absorbance at 405 nm (A ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). 405 The reaction rate was determined by continuously monitoring the absorbance A over a total reaction time of 300 seconds. 405 (Slope of curve versus time (seconds)). Data are presented in Figure 11. Percent inhibition was calculated as described in Example 3.

[0114] Results: The percentage of inhibition for the different treatment groups is shown in Table 6 below.

[0115] [Table 6]

[0116] As shown in Figure 11 and Table 6, the inhibitory effect of each group in the experimental group was significantly higher than that of each group in the comparative group. The two experimental groups: (1) isotretinoin and oligosaccharides, and (2) isotretinoin, oligosaccharides, and Zn 2+ and Mg 2+ However, isotretinoin, oligosaccharides, Zn 2+ or Mg 2+ These results indicate that the combined effect of isotretinoin, oligosaccharides, and divalent metal ions on the activity of papain-like proteases is more effective than the individual components of each compound alone. These results suggest that isotretinoin, oligosaccharides, and divalent metal ions have a synergistic inhibitory effect on the activity of papain-like proteases. Additionally, this provides important insights into the biochemical properties of the PLpro family of coronaviruses, paving the way for promising therapeutic strategies against SARS-CoV-2.

[0117] [Example 5] Evaluation of the in vivo treatment effects of a combination of isotretinoin, oligosaccharides, 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 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 agent was prepared by Taipei Medical University using liposomes according to the techniques commonly used in the art and described in detail above. Since the main technical feature of the present disclosure is the combination of retinoic acid and oligosaccharides with or without metal ions, the details of liposomes are omitted here for the sake of brevity.

[0120] Specifically, for each hamster in the experimental group, liposomal isotretinoin was administered intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, day 1) at a dose of 0.35 mg / kg and at 8:00 PM on the day of infection at a dose of 0.35 mg / kg. On the following two days (days 2 and 3), liposomal isotretinoin was administered intranasally twice daily at 8:00 AM and 8:00 PM, each at a dose of 0.35 mg / kg.

[0121] In addition, liposomal isotretinoin was administered intranasally at a dose of 0.35 mg / kg twice daily at 8:00 AM and 8:00 PM, and either a liposomal oligosaccharide mixture in a volume of 15 μL (i.e., Experiment_1) or a liposomal oligosaccharide mixture (15 μL) and Zn, both of which were encapsulated in liposomes. 2+ (100 μM), Mg 2+ (200 μM) and K +The combination (15 μL) of (200 μM) in a total volume of 30 μL (i.e., experiment_2) was given via intranasal administration once daily between 7:30 PM and 8:00 PM on the day of infection (i.e., 0.5 to 1 hour before the nighttime administration of liposomal isotretinoin), and two days later in a volume of 15 μL in experiment_1 or 30 μL in experiment_2, respectively.

[0122] For each hamster in the comparison group, isotretinoin in comparison_1 or liposomal isotretinoin in comparison_2 was administered intranasally at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection) at a dose of 0.35 mg / kg and at 8:00 PM on the day of infection at a dose of 0.35 mg / kg, and isotretinoin in comparison_1 or liposomal isotretinoin in comparison_2 was administered intranasally at a dose of 0.35 mg / kg twice daily at 8:00 AM and 8:00 PM for the next two days after the day of infection.

[0123] For each hamster, Zn encapsulated in liposomes in Comparative_3 2+ (100 μM), Mg 2+ (200 μM) and K + The combination of 200 μM of guanosine monophosphate (200 μM) and the mixture of liposome-encapsulated oligosaccharides in Comparative_4 were given via intranasal administration once daily from 7:30 pm to 8:00 pm on the day of infection and two days later in a volume of 15 μL.

[0124] For each hamster in the control group, buffer was administered intranasally in 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 8:00 PM on the day of infection in a volume of 100 μL. Buffer was administered intranasally in a volume of 100 μL twice daily at 8:00 AM and 8:00 PM for the next two days after the day of infection.

[0125] After 3 days of treatment, the hamsters were sacrificed and their lungs were cultured at TCID 200 in Vero E6 cells. 50The virus was collected for live virus assay. The 50% tissue culture infectious dose (TCID) was determined using the Reed-Muench method. 50 Virus titers were determined in terms of . All experiments using SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by Academia Sinica (Taipei, Taiwan).

[0126] The experimental data were analyzed by Tukey's test to assess differences between groups, with statistical significance indicated by p<0.05.

[0127] Results: Referring to Figure 12, the TCID of Experiment_1 and Experiment_2 and 3, respectively, 50 was significantly lower than that of each of Comparison_1, Comparison_2, Comparison_3, and Comparison_4, revealing that the combination of isotretinoin and oligosaccharides with or without added metal ions has higher in vivo efficacy against SARS-CoV-2 compared to isotretinoin alone, metal ions alone, or oligosaccharides alone.

[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 and a carbohydrate with or without added metal ions (i.e., the second pharmaceutical composition) can provide improved and / or synergistic effects on inhibiting SARS-CoV-2 infection and replication and treating diseases associated with SARS-CoV-2 infection or cancer. As a result, the compound comprising retinoic acid conjugated to a carbohydrate of the present invention, the pharmaceutically acceptable salt thereof, and the first and second pharmaceutical compositions can actually serve as drug repurposing agents.

[0129] The invention has been described broadly and generically herein. Each of the narrower species and subgroup groupings falling within the generic disclosure also form part of the invention. In addition, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A compound, or a pharma- ceutically acceptable salt thereof, comprising a retinoic acid conjugated to a carbohydrate.

2. The compound has the formula (I): 【Chemistry 1】 [In the formula, R １ is a substituted or unsubstituted functional group of a carbohydrate.

2. The compound of claim 1, represented by: or a pharma- ceutically acceptable salt thereof.

3. 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, and polysaccharides.

4. 4. The compound of claim 3, wherein the oligosaccharide is a mono- or hetero-oligosaccharide and the polysaccharide is a mono- or hetero-polysaccharide, or a pharma- ceutically acceptable salt thereof.

5. 4. The compound of claim 3, or a pharma- ceutically 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.

6. 6. A pharmaceutical composition comprising a compound according to any one of claims 1 to 5, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

7. 7. The pharmaceutical composition of claim 6, wherein the pharma- ceutically acceptable carrier comprises a liposome and the compound or the pharma- ceutically acceptable salt thereof is encapsulated by the liposome.

8. The pharmaceutical composition of claim 6 further comprising a metal ion.

9. 9. The pharmaceutical composition of claim 8, wherein the metal ion comprises a monovalent ion, a divalent ion, or a combination thereof.

10. The monovalent ion is K ＋ , Na ＋ or a combination thereof, wherein the divalent ion is Zn ２＋ , Mg ２＋ , Cu ２＋ , Mn ２＋ , Ca ２＋ , Fe ２＋ or any combination thereof.

11. 9. The pharmaceutical composition of claim 8, wherein the pharma- ceutically acceptable carrier comprises a liposome and the metal ion is encapsulated by the liposome.

12. 12. The pharmaceutical composition of claim 11, wherein the compound or the pharma- ceutically acceptable salt thereof and the metal ion are individually or simultaneously encapsulated by the liposome.

13. 17. Use of a compound according to any one of claims 1 to 5 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 6 to 12, in the manufacture of a medicament for inhibiting viral infection or replication.

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

15. 15. The use of claim 14, wherein the RNA virus comprises a coronavirus, a human immunodeficiency virus (HIV), a hepatitis C virus (HCV), an influenza virus, or any combination thereof.

16. 16. The use of claim 15, wherein the coronavirus comprises 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.

17. 17. Use of a compound according to any one of claims 1 to 5 or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 6 to 12, in the manufacture of a medicament for treating cancer.

18. 18. The use of claim 17, wherein the cancer comprises leukemia, lymphoma, carcinoma, or sarcoma.

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