TWI841929B - Novel retinoic acid compound, pharmaceutical composition including thereof and use thereof - Google Patents

Abstract

A compound or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof are provided, wherein the compound includes a retinoic acid conjugated with a carbohydrate. In addition, use of the compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in the manufacture of a medicament for inhibiting infection or replication of a virus or for treating a cancer is also provided.

Description

Novel retinoic acid compound, pharmaceutical composition containing the same and use thereof

The present invention relates to a novel retinoic acid compound, a pharmaceutical composition containing the same and the use thereof in the manufacture of a drug for inhibiting viral infection or replication or for treating cancer.

Coronaviruses are a group of positive-sense, single-stranded RNA viruses belonging to the Coronaviridae family, which include seven species/strains that infect humans, namely 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). It is worth noting that SARS-CoV-2 has been identified as the strain that caused the 2019 coronavirus disease (COVID-19) pandemic.

The genomes of SARS-CoV-2 and SARS-CoV have high sequence identity. Both SARS-CoV-2 and SARS-CoV are highly dependent on the activity of two viral proteases, namely 3C-like protease (3CLpro, also known as main protease (Mpro) or nonstructural protein 5 (nsp5)) and papain-like protease (PLpro, which is the protease domain of nonstructural protein 3 (nsp3)), to complete the viral proliferation cycle and viral transmission. It has been reported that all-trans retinoic acid (ATRA, also known as vitamin A acid or retinoin) can be considered as a potential therapeutic agent against SARS-CoV-2 by inhibiting the activity of 3CLpro.

In addition to 3CLpro, PLpro is also a potential target, as this type of enzyme plays an essential role in the cleavage and maturation of viral polyproteins, the assembly of the replicase-transferase complex, and the disruption of host responses. Although the main function of Mpro and PLpro is to process viral polyproteins in a synergistic manner, PLpro also has the additional function of stripping ubiquitin and interferon-stimulatory gene factor 15 (ISG15) from host cell proteins to allow coronaviruses to evade the host's innate immune response. In other words, PLpro is not only related to viral replication, but also to the dysregulation of the signaling cascade in infected cells, which increases cell death in surrounding uninfected cells. Therefore, drugs designed to inhibit the function of PLpro also have the potential to fight SARS-CoV-2. Recently, in addition to ATRA, some researchers have reported that its derivative 13-cis-retinoic acid (also known as isotretinoin) is a potential PLpro inhibitor and can be used to treat COVID-19 caused by SARS-CoV-2.

In addition, retinoic acid (including ATRA and 13-cis retinoic acid) has become a promising compound for the treatment of various cancers due to its specific effects on cell proliferation, differentiation and apoptosis and low toxicity. The retinoic acid receptor in the nucleus of human cells has been found by biochemists and found that it does not mutate in cancer cells, so retinoic acid may exert its anti-cancer effect in many malignant tumors. For example, it was found that in children with high-risk neuroblastoma, treatment with 13-cis retinoic acid can reduce the risk of cancer recurrence after high-dose therapy and stem cell transplantation. ATRA has been studied in combination with other drugs for various cancers and precancerous lesions. Several clinical trials using ATRA as part of combination therapy are currently underway. For example, ATRA combined with different interferons (IFNs) was shown to enhance the effects of both drugs and lead to growth inhibition and cell death of tumor cell lines. Nevertheless, in order to unlock the therapeutic potential of retinoic acid, many 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). Clearly, combination therapies targeting multiple gene silencing mechanisms may be the most effective strategy to reactivate ATRA-sensitive gene expression and differentiation of AML cells and mediate the general anticancer activity of ATRA. Currently, the identification of protein classes that control gene expression through histone and DNA modifications can promote the development of novel therapeutic agents, so-called epigenetic drugs that alter chromatin structure. However, these epigenetic modifying drugs have been shown to be only partially effective against different cancers when used alone.

Based on the above reasons, the present invention provides a novel retinoic acid compound and a pharmaceutical composition comprising the same, which can effectively inhibit viral infection or replication, or treat cancer.

In one aspect of the present invention, a compound or a pharmaceutically acceptable salt thereof is provided, wherein the compound comprises retinoic acid conjugated to a carbohydrate.

Preferably, the compound is represented by formula (I): (I), wherein R ¹ is a substituted or unsubstituted functional group of a carbohydrate.

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

Preferably, the oligosaccharide is a mono-oligosaccharide or a hetero-oligosaccharide; and the polysaccharide is a mono-polysaccharide or a hetero-polysaccharide.

Preferably, 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.

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

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

Preferably, the pharmaceutically acceptable carrier comprises liposomes, and the compound or its pharmaceutically acceptable salt is encapsulated in the liposomes.

Preferably, the pharmaceutical composition further comprises metal ions.

Preferably, the metal ions include monovalent ions, divalent ions or combinations thereof. More preferably, the monovalent ions include K ⁺ , Na ⁺ or combinations thereof; and the divalent ions include Zn ²⁺ , Mg ²⁺ , Cu ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ or any combination thereof.

Preferably, the pharmaceutically acceptable carrier comprises liposomes, and the metal ions are encapsulated by the liposomes.

Preferably, the compound or its pharmaceutically acceptable salt and the metal ion are encapsulated by liposomes alone or simultaneously.

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

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

Therefore, the present invention provides at least the following advantages: 1.        Compared with retinoic acid alone, the compounds and pharmaceutical compositions claimed in this case can enhance the ability to inhibit viral infection and/or replication, wherein the compound is retinoic acid conjugated to a carbohydrate. 2.        The compounds and pharmaceutical compositions claimed in this case can be used as potential anti-coronavirus drugs (especially SARS-CoV-2 that causes COVID-19). 3.        The compounds and pharmaceutical compositions claimed in this case can also enhance the ability to inhibit cancer cells, thereby effectively treating a variety of cancers, such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer and liver cancer.

Although the present invention is susceptible to various modifications and alternative forms, specific embodiments are shown in the drawings and described in detail by way of example. However, it should be understood that this description is not intended to limit the present invention to specific embodiments, but on the contrary, the present invention will cover all modifications, equivalents and alternative forms that fall within the spirit and scope of the present invention.

Definition

Unless otherwise defined, all terms (including technical and scientific terms) used in this document have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It will be further understood that, unless expressly defined as such herein, terms defined in commonly used dictionaries should be interpreted as having the same meaning as in the context of the relevant technology and will not be interpreted in an idealized or overly formal sense.

The terms used herein are used for the purpose of describing specific embodiments only and are not intended to limit the embodiments of the present invention. Unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" used herein are intended to include the plural forms as well. It will be further understood that the terms "comprising", "including", "including" and/or "comprising", when used herein, specify the presence of the described features, integers, steps, operations, elements, parts and/or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, parts and/or combinations thereof.

The term "subject" herein refers to a mammal for whom diagnosis, prognosis or treatment 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, for example, to screen, characterize and evaluate drugs and therapies.

The term "administering" or "administered" herein means introducing, providing or delivering the intended active ingredient into a subject by any suitable route to perform its intended function.

The term "cancer" as used herein refers to leukemia, lymphoma, carcinoma, sarcoma and other malignant tumors that may grow indefinitely and may spread locally by invasion and systemically by metastasis. Examples of cancer include, but are not limited to, ovarian cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colon cancer and/or rectal cancer, gallbladder cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, cancer of nervous tissue, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer and thyroid cancer. Some other examples of cancer include cholangiocarcinoma, acute and chronic lymphocytic and granulocytic neoplasms, adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforma, trichomema, intestinal ganglionic cell tumor, hyperplastic corneal neuroma, pancreatic 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, primary brain tumors, small cell lung tumors, ulcerative and papillary squamous cell carcinomas, cell hyperplasia, spermatoma, soft histosarcoma, retinoblastoma, rhabdomyosarcoma, nephrocytoma, localized skin lesions, veticulum cell sarcoma, and Wilm's tumor.

The term "oligosaccharide" herein refers to a carbohydrate composed of a small number of monosaccharides, usually about three to ten monosaccharide units. Among them, oligosaccharides with one type of monosaccharide subunit are called mono-oligosaccharides; oligosaccharides with more than one type of monosaccharide subunits are called hetero-oligosaccharides.

The term "polysaccharide" herein refers to a carbohydrate composed of a large number of monosaccharide units. Among them, polysaccharides with one monosaccharide subunit are called monopolysaccharides; polysaccharides with more than one monosaccharide subunit are called heteropolysaccharides.

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

Description of Embodiments

Hereinafter, exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

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 with a carbohydrate.

In an exemplary embodiment, the compound is represented by formula (I): , wherein R ¹ is a substituted or unsubstituted functional group of a carbohydrate.

In certain embodiments, the concentration of the compound comprising 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.

In another embodiment, a pharmaceutical composition is provided, which includes retinoic acid and a carbohydrate (hereinafter referred to as the "second" pharmaceutical composition). In certain embodiments, the retinoic acid is 13-cis-retinoic acid (also known as isoretinic acid).

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.

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

In certain embodiments, the concentration of the carbohydrate may be, but is not limited to, 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 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.

In certain embodiments, the molar ratio of retinoic acid to carbohydrate in the second pharmaceutical composition may be about 1:10 ^-3 ~100, 1:10 ^-3 ~20, 1:1~20, or 1:1~10.

In certain embodiments, the first pharmaceutical composition and the second pharmaceutical composition each optionally further include metal ions. Preferably, the metal ions include monovalent ions, divalent ions, or combinations thereof. In a specific embodiment, the monovalent ions include K ⁺ , Na ⁺ , or combinations thereof; and the divalent ions include Zn ²⁺ , Mg ²⁺ , Cu ²⁺ , Mn ²⁺ , Ca ²⁺ , Fe ²⁺ , or any combination thereof.

In certain embodiments, the concentration of the metal ion can be 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, 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.

In certain embodiments, the molar ratio of the compound to the metal ion in the first pharmaceutical composition may be about 1:10 ^-3 to 10 ³ , 1:0.1 to 20, 1:0.1 to 10, or 1:1 to 10.

In certain embodiments, the molar ratio of retinoic acid, carbohydrate and metal ion in the second pharmaceutical composition may be about 1: ^10-4 ~20: ^10-4 ~ ¹⁰³ , 1:10-4~20:10-3~103, 1: ^10-4 ~20:10-3 ^{~ 20} , 1: ^10-4 ~20:0.1 ^~ 20, 1: ^10-4 ~20:1~10, 1: ^0.1 ~20:10-4~103, 1:0.1~20:10-3 ^{~ 103} , 1:0.1~20:10-3~ ²⁰ , 1: ^0.1 ~20:0.1 ^~ 20, 1:0.1~20:1~20, 1:0.1~20:1~20, 1:0.1~20:1~10, 1:0.1~1: ^10-4 ~ ¹⁰³ , 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.

In certain embodiments, the first pharmaceutical composition and the second pharmaceutical composition each optionally further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is widely used in the field of drug manufacturing. Examples of pharmaceutically acceptable carriers may include (but are not limited to) liposomes, excipients, adjuvants, solvents, buffers, emulsifiers, suspending agents, decomposing agents, disintegrants, dispersants, adhesives, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents and the like. The selection and dosage of pharmaceutically acceptable carriers are within the expertise of those skilled in the art.

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

Exemplary liposomes may be neutral, positively charged or negatively charged. In general, lipids commonly used in liposomes typically include dialiphatic chain lipids such as phospholipids, diglycerides and diglycerides; monolipids such as sphingomyelin and glycosphingolipids; steroids such as cholesterol and its derivatives, and combinations thereof. Examples of phospholipids include, but are not limited to, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), 1,2-dilauryl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HPC), 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-dioleyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-distearyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine DMPA, 1,2-dipalmitoyl-sn-glycerol-3-phosphate (sodium salt) (DPPA), 1,2-distearyl-sn-glycerol-3-phosphate (sodium salt) (DSPA), 1,2-dioleoyl-sn-glycerol-3-phosphate (sodium salt) (DOPA), 1,2-dipalmitoyl-sn-glycerol-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolamine (POPE), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), 1 ,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium salt) (DPPI), 1,2-distearyl-sn-glycero-3-phosphoinositol (ammonium salt) (DSPI), 1,2-dioleyl-sn-glycero-3-phospho-(1-myo-inositol) (ammonium salt) (DOPI), cardiolipin, L-α-phosphatidylcholine (EPC), 1,2-dioleyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), L-α-phosphatidylethanolamine (EPE), dimethyldioctadecylammonium (DDAB), 1,2-dioleyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), and 3β-[N-(N',N'-dimethylaminoethane)aminomethyl]cholesterol hydrochloride.

The lipids suitable for use may be a lipid mixture of one or more of the aforementioned lipids, or a mixture of one or more of the aforementioned lipids and one or more other lipids, membrane stabilizers or antioxidants not listed above.

The molar percentage of lipid in the bilayer membrane can be equal to or less than about 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or any value or range 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%).

The lipid of the bilayer membrane can be a mixture of a first phospholipid and a second phospholipid. The first phospholipid can be selected from the group consisting of PC, HSPC, DOPC, POPC, DSPC, DPPC, DMPC, PSPC and combinations thereof, and the second phospholipid is selected from the group 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 membrane 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 membrane 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 molar ratio of the first phospholipid (DSPC) to the second phospholipid (DOPE, DOPG or DDAB) may be 4:1 to 6:1.

In an exemplary embodiment, the bilayer membrane of the liposome includes less than about 55 molar percentage of a steroid, preferably cholesterol. The molar percentage of the steroid (e.g., cholesterol) in the bilayer membrane 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 percentage of phospholipids and cholesterol in the bilayer membrane may be about 25-50%, 15-55%, 25-50%, 20-55%, or 25-50%, 15-50%. The molar ratio of phospholipids to cholesterol may be 1:1 to 3:1. The molar percentage of the first phospholipid, the second phospholipid, and cholesterol in the bilayer membrane may be about 25-50%, 0.1-15%, 15-55%, 5-50%, 0.1-15%, 10-40%, or 25-50%, 0.5-10%, 5-20%.

Liposomes coated with the capture agent can be prepared by any technique currently known or later developed. For example, MLV liposomes can be formed directly by hydrated lipid films, spray-dried powders or freeze-dried cakes of the selected lipid composition and the capture agent; SUV liposomes and LUV liposomes can be sized from MLV liposomes by ultrasonic vibration, homogenization, microfluidization or extrusion.

In yet another embodiment, the compound comprising retinoic acid conjugated to a carbohydrate or a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition and the second pharmaceutical composition can be used to inhibit viral infection or replication. The virus can be an RNA virus. The RNA virus can include a coronavirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus, or any combination thereof. The coronavirus can 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.

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

In a specific embodiment, the compound and the metal ion in the first pharmaceutical composition can be administered separately, simultaneously or sequentially. The retinoic acid and the carbohydrate in the second pharmaceutical composition can be administered separately, simultaneously or sequentially. All or at least two of the retinoic acid, carbohydrate and metal ion in the second pharmaceutical composition can be administered separately, simultaneously or sequentially. In an exemplary embodiment, the metal ion and the carbohydrate are administered simultaneously, followed by the retinoic acid.

In exemplary embodiments, the time interval between sequential administrations may be 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.

In certain embodiments, the compound comprising retinoic acid conjugated to a carbohydrate, its pharmaceutically acceptable salt, and each of the first pharmaceutical composition and the second pharmaceutical composition can be prepared in dimethyl sulfoxide (DMSO), ethanol, buffer or water for administration. The dosage and frequency of administration can vary according to the following factors: the severity of the viral infection (e.g., coronavirus infection) or the disease to be treated (e.g., cancer) and the weight, age, physical condition and response of the individual to be treated. The daily dosage of the above-mentioned therapeutic agent can be administered as a single dose or several doses.

In certain embodiments, the compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition and the second pharmaceutical composition are each administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally or topically.

Examples

The present invention will be further described by the following examples. However, it should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the present invention in practice.

Materials and methods

A. A novel retinoic acid compound represented by formula ( Ia ), namely galactose-modified isoretinic acid, was prepared according to the following synthetic flow chart:

I. General Experimental Methods All reagents were commercially available and used without further purification. Yields refer to compounds that were purified and spectrally identified as high purity. Thin layer chromatography (TLC) was performed using Merck TLC aluminum silica 60 F ₂₅₄ plates and visualized by fluorescence quenching under UV light. Flash chromatography was performed using silica gel (Chromatorex, MB 70-40/75, 40~75 μm) purchased from Fuji Silysia Chemicals. NMR spectra were recorded on a Varian-400MR operating at 400 MHz for ¹ H. Chemical shifts are reported in ppm and the solvent resonance is used as an 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 are in Hz; integration was performed. Purity was recorded on a Waters e2695 separation module/2998 PDA detector HPLC system (column: XBridge C18, 5 μm, 4.6 mm (ID) x 150 mm (L), eluent: a 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 NH ₄ OAc, flow rate: 0.5 mL/min. Detection: UV, 254 nm).

II. Synthesis of (2R,3S,4S,5R,6R)-2-( acetoxymethyl ) -6-(3 - bromopropoxy ) tetrahydro - 2H -pyran- 3,4,5 - triyl triacetate ( 2 ) Boron trifluoride-diethyl etherate (8.04 mL, 64.0 mmol) was added 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) at 0°C. The resulting 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, CDCl ₃ ): 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, 2H), 4.03-3.98 (m, 1H), 3.92 (td, J = 5.8, 0.8 Hz, 1H), 3.72-3.66 (m, 1H), 3.50-3.47 (m, 2H), 2.25-1.99 (m, 14H); LCMS (ESI) m/z calcd for C ₁₇ H ₂₅ BrO ₁₀ 469.28; found 491.04 [M + Na] ⁺ .

III. Synthesis of the compound (2R,3S,4S,5R,6R)-2-( acetoxymethyl ) -6-(3 - azidopropoxy ) tetrahydro - 2H -pyran 3,4,5 - triyl triacetate ( 3 ) 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 resulting mixture was stirred at 100°C for 2 hours. The solution was concentrated to dryness. The crude product was purified by silica gel column chromatography (EtOAc: hexane = 1:2) to obtain the desired product ( 3 ) (458 mg, 80%) as a colorless oil. ¹ H NMR (400 MHz, CDCl ₃ ): 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 calculated for C ₁₇ H ₂₅ N ₃ O ₁₀ 431.40; found 454.2 [M + Na] ⁺ .

IV. Synthesis of compound ( 2R,3R,4S,5R,6R)-2-(3- azidopropoxy )-6-( hydroxymethyl ) tetrahydro - 2H -pyran- 3,4,5 - triol ( 4 ) 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 resulting 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 calculated for C9H17N3O6 263.25 ; found} 286.1 [M+Na] ⁺ .

V. Synthesis of compound ( 2R,3R,4S,5R,6R)-2-(3 - aminopropoxy )-6-( hydroxymethyl ) tetrahydro - 2H-pyran- 3,4,5 - triol ( 5 ) The crude compound ( 4 ) in methanol (10.5 mL) was treated with Pd/C (11 mg) and acetic acid (6.3 mg) under _H2 atmosphere. The solution was filtered through a pad of diatomaceous earth 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 calculated for _{C9H19NO6 237.25} ; found 237.8 [M] ⁺ .

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 ) Pentafluorophenyl trifluoroacetate (compound ( 8 ), 0.081 mL, 0.47 mmol) was added 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) at 0°C. The solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with EtOAc and washed with 0.1N aqueous HCl solution, followed by aqueous _NaHCO3 solution and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated to dryness to obtain the desired product ( 6 ) without further purification.

VII. 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 ) Synthesis Triethylamine (0.049 mL, 0.33 mmol) was added to a solution of crude compound ( 5 ) (30 mg, 0.13 mmol) and active ester ( 6 ) (150 mg, 0.33 mmol) in DMF (1.0 mL) at room temperature. The resulting 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 calculated for C29H45NO7 519.68 ;} found 520.5 [M+H] ⁺ . See Figure 1 .

B , SARS-CoV-2 PLPro Preparation

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.

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

The vector containing the SARS-CoV-2 PLPro gene was transformed into the E. coli BL21 (DE3) strain (Yeastern Biotech Co., Ltd., New Taipei City, Taiwan) to express PLPro in large quantities. The culture was cultured in LB medium (containing 1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with ampicillin (100 μg/mL) as an antibiotic marker. The resulting culture was initially cultured at 37°C while shaking at 200 rpm. At an optical density at 600 nm (OD ₆₀₀ ) between 0.6 and 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce the expression of PLpro. The culture was continued at 18°C and 200 rpm for 20 hours. The cells were harvested by centrifugation (5,000 x g) and disrupted by ultrasonic vibration in a lysis buffer containing 50 mM sodium phosphate (pH 7.4), 1.0 mM DTT, 5% glycerol, and 100 mM NaCl. Subsequently, the cell debris was removed by centrifugation at 20,000 x 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 50 mM sodium phosphate (pH 7.4) and 100 mM NaCl containing 0-500 mM imidazole. Fractions containing His-tagged SARS-CoV-2 PLpro were pooled and concentrated using Centricon membrane (10 K cutoff, GE Healthcare Life Sciences). His-tagged SARS-CoV-2 PLpro was further purified by gel filtration using a Superdex 75 gel filter column (GE Healthcare Life Sciences) in 50 mM sodium phosphate buffer (pH 7.4). The concentration of SARS-CoV-2 PLpro was determined by measuring UV absorbance at 280 nm (using an extinction coefficient (ε280) of 45270 M ^-1 cm ^-1 ).

C , SARS-CoV-2 PLPro Activity test

The enzymatic activity of the obtained SARS-CoV-2 PLpro was measured by a colorimetry-based peptide cleavage assay using a 6-mer peptide substrate, FRLKGG-p-nitroanilide (FG6-pNA) (HPLC purity 97%; 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 turned the solution yellow. Enzyme activity was determined by continuously monitoring the absorbance at 405 nm (A ₄₀₅ ) at 30°C using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek).

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, so that a substrate solution with various FG6-pNA concentrations (0.1875 mM, 0.375 mM, 0.75 mM, 1.5 mM, 3.0 mM, 6.0 mM) was prepared. The assay mixture (180 μL per well) was pre-incubated for 10 minutes to precisely control the temperature, 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 calculated by measuring A ₄₀₅ and using the extinction coefficient (ε405) of 9800 M ^-1 cm ^-1 (A ₄₀₅ = 9.8 at 1 mM).

OriginPro 8.0 software (OriginLab Corporation, USA) was used to fit the initial velocity (V ₀ ) data based on the Michaelis-Menten equation to obtain steady-state enzyme kinetic parameters. All measurements were performed in triplicate. The data obtained are expressed as mean ± standard deviation.

Results: The K _M and k _cat values were 2.50±0.03 mM and 0.85±0.01 s ^-1 , respectively. Therefore, it was verified that the SARS-CoV-2 PLPro with protease activity was successfully prepared according to the above-mentioned manufacturing method, which can be used for subsequent SARS-CoV-2 PLPro inhibition tests.

D. SARS-CoV-2 Establishing the Animal Model

Golden Syrian hamsters (5–6 weeks old, average weight approximately 100 g) were obtained from the National Laboratory Animal Center, National Research Institutes of Taiwan (Taipei, Taiwan). The hamsters were housed in an animal room under specific pathogen-free (SPF) conditions commonly used in the field. In addition, all hamsters were provided with water and feed ad libitum. All experiments involving hamsters were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Academia Sinica (Taiwan).

Hamsters were intranasally inoculated with ^1x104 plaque-forming units (PFU) and infected at 12:00 PM with phosphate-buffered saline (PBS) containing SARS-CoV-2 (obtained from P3 Laboratory, Genomic Research Center, Academia Sinica; Wuhan wild-type) to establish the SARS-CoV-2 animal model. The established SARS-CoV-2 animal model was confirmed (data not shown).

Examples 1

For in vitro evaluation of novel retinoic acid compounds (Galactose-modified isoretinal acid) SARS-CoV-2 Antiviral effect SARS-CoV-2 PLPro Inhibition test

The enzyme inhibition test 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 solution in each well was divided into a control group (abbreviated as "Control"), a comparison group (abbreviated as "Comparison Example_1") and an experimental group (abbreviated as "Experimental Example_1"). The corresponding inhibitor shown in Table 1 below was added to each group of enzyme solutions to form a test mixture (total volume of 180 μL). Pre-incubation was performed for 30 minutes.

surface 1 Group Components Comparison PLpro Comparison example_1 PLpro + 50 μM isoretinic acid Experimental example_1 PLpro + 50 μM galactose-modified isoretinal acid

20 μL of the above FG6-pNA (1.2 mM) was added to each test mixture to start the enzyme reaction. The enzyme reaction was allowed to proceed for 300 seconds at 30°C. The enzyme activity was determined by continuously monitoring the absorbance at 405 nm (A ₄₀₅ ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). The reaction rate was calculated based on this, where the reaction rate was the slope of the change in absorbance A ₄₀₅ with time (seconds), and the total reaction time was 300 seconds. The data are presented in Figure 2 .

The inhibition percentage was calculated using the following formula: A = [1-(B/C)]x100 ( I ) where A = inhibition percentage, B = reaction rate of each group, and C = reaction rate of the control group. The data obtained were expressed as mean ± standard deviation.

Results: The inhibition percentages of different treatment groups are shown in Table 2 below.

surface 2 Group Inhibition percentage ( % ） Comparison: PLpro 0 Comparison Example 1: PLpro + isoretinal acid 20.90 ± 1.24 Experimental Example 1: PLpro + galactose-modified isoretinal acid 34.21 ± 0.17

As shown in Figure 2 and Table 2 , the inhibition rate of Experimental Example 1 is significantly higher than that of Comparative Example 1, which indicates that galactose-modified isoretinoic acid is more effective than isoretinoic acid alone in inhibiting the activity of PLpro enzyme. This result indicates that galactose-modified isoretinoic acid has a better inhibitory effect on the activity of papain-like protease.

Furthermore, it provides important insights into the biochemical properties of the coronavirus PLpro family and leads the way to promising therapeutic strategies against SARS-CoV-2.

Examples 2

Novel Retinoic Acid Compounds (Galactose-modified isoretinal acid) SARS-CoV-2 Animal Mode SARS-CoV-2 In vivo evaluation of the antiviral effect of

Since the novel retinoic acid compound galactose-modified isoretinic acid has been shown to have an inhibitory effect on SARS-CoV-2 in vitro, the antiviral effect of the novel retinoic acid compound on SARS-CoV-2 was further evaluated in an in vivo animal test.

The infected hamsters obtained above were divided into the following four groups (n=5 in each group): a control group (abbreviated as "control"), two comparison groups (abbreviated as "comparative example_1" and "comparative example_2") and an experimental group (abbreviated as "experimental example_1"). The treatment agents used in these groups are listed in Table 3 below.

surface 3 Group Therapeutic agents Comparison Buffer containing 40% ethanol, 40% Span ^® 80 (sorbitan monooleate) (Sigma-Aldrich), and 20% peanut oil Comparison example_1 Isoretinoic acid Comparison example_2 Liposome-encapsulated isoretinal acid Experimental example_1 Galactose-modified isoretinal acid

Specifically, for each hamster in Experimental Example 1, a novel retinoic acid compound was intranasally administered at 0.35 mg/kg at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, Day 1) and at 0.35 mg/kg at 8:00 PM on the day of infection. On the next two days (Day 2 and Day 3), the novel retinoic acid compound was administered nasally twice a day at 8:00 AM and 8:00 PM at a dose of 0.35 mg/kg each.

For each hamster in the comparison group, isotretinoic acid in Comparative Example 1 or liposome-encapsulated isotretinoic acid in Comparative Example 2 was intranasally administered at 0.35 mg/kg at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection and at 0.35 mg/kg at 8:00 PM on the day of infection. On the next two days after the infection day, isotretinoic acid in Comparative Example 1 or liposome-encapsulated isotretinoic acid in Comparative Example 2 was intranasally administered at 0.35 mg/kg twice a day at 8:00 AM and 8:00 PM. The liposome-encapsulated isoretinoid in Comparative Example 2 was prepared by Taipei Medical University (Taiwan) using the commonly used technology in this field and is described in detail above. Since the main technical feature of the present invention is the novel retinoic acid compound galactose-modified isoretinoid, for the sake of brevity, the details of the liposome are omitted here.

For each hamster in the control group, 100 μL of buffer solution was administered through the nose at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection and 100 μL of buffer solution was administered through the nose at 8:00 PM on the day of infection. For two days after the infection day, 100 μL of buffer solution was administered through the nose twice a day at 8:00 AM and 8:00 PM.

After 3 days of treatment, hamsters were sacrificed and their lungs were collected for measurement of live viral load in Vero E6 cells by TCID ₅₀ assay. Virus titers were determined based on 50% tissue culture infectious dose (TCID ₅₀ ) using the Reed-Muench method. All experiments with SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by Academia Sinica (Taipei, Taiwan).

The experimental data were analyzed using Tukey's test to assess the differences between the groups. Statistical significance was indicated at p < 0.05.

Results: See Figure 3 . The TCID ₅₀ of Experimental Example ₁ is significantly lower than that of Comparative Example 1 and Comparative Example 2, indicating that the novel retinoic acid compound galactose-modified isoretinoic acid has a higher in vivo efficacy against SARS-CoV-2 compared to isoretinoic acid alone or isoretinoic acid encapsulated in liposomes.

Examples 3

Novel Retinoic Acid Compounds Evaluation of the in vitro cytotoxic effects of galactose-modified isoretinoic acid on different cancer cell lines

In this example, seven cell lines were used, namely, AsPC-1, MDA-MB-231, HCT-116, Huh-7, SKOV-3, A549 and H460 cancer cell lines. Each cell line was cultured in a designated growth medium containing 10% FBS in a humidified incubator at 37°C containing 5% CO ₂ .

One day before treatment, cells in logarithmic growth phase were harvested, counted and seeded in a 96-well plate at a density of 1x10 ⁴ cells/100 μL/well. After overnight culture, the medium in each well was gently removed and fresh medium (200 μL/well) was added.

The test article (TA) is a novel retinoic acid compound (abbreviated as "novel compound" in this example), which is freshly prepared as a 96 mM stock solution in 100% DMSO on the day of treatment. Isoretinoic acid is serially diluted 2-fold using 100% DMSO to obtain different concentrations of isoretinoic acid from 7.5 mM to 96 mM. Cells are treated by adding 2.02 μL of the specified concentration of isoretinoic acid to each well to obtain a final concentration range of 7.5 μM to 960 μM of isoretinoic acid, and all wells are maintained with 1% DMSO, including DMSO controls. All components are mixed gently and incubated for 24 hours.

Cells were treated by adding 2.02 μL of the indicated concentration of novel compounds or DMSO to each well, followed by gentle mixing and incubation for 24 hours.

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

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

Results: See Figures 4 to 10 , which show that the novel compound can significantly reduce the cell survival rate of all seven cancer cell lines, indicating that the novel retinoic acid compound galactose-modified isoretinic acid can be a potential anticancer drug for the effective treatment of various cancers (such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer and liver cancer). IC ₅₀ is shown in Table 4 .

surface 4 Tumor type Cell lines IC ₅₀ (μM) Non-small cell lung cancer NCI-H460 22.95 Non-small cell lung cancer A549 21.88 Liver cancer Huh-7 77.46 Pancreatic cancer AsPC-1 23.00 Colorectal cancer HCT-116 123.44 Ovarian cancer SK-OV-3 13.82 Breast cancer MDA-MB-231 115.88

Examples 4

For in vitro evaluation of the effect of a second pharmaceutical composition on SARS-CoV-2 Antiviral effect SARS-CoV-2 PLPro Inhibition test

The enzyme inhibition test 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 obtained above was added to each well to form an enzyme solution with a final concentration of 0.9 μM. The enzyme solution in each well was divided into a control group (abbreviated as "Control"), four comparison groups (abbreviated as "Comparison Example 1", "Comparison Example 2", "Comparison Example 3" and "Comparison Example 4") and two experimental groups (abbreviated as "Experimental Example 1" and "Experimental Example 2"). The corresponding inhibitor shown in Table 5 below was added to each group of enzyme solutions to form a test mixture (total volume of 180 μL). Pre-incubate for 30 minutes.

surface 5 Group Components Comparison PLpro Comparison example_1 PLpro + oligosaccharide mixture [(β-1,3/1,6-glucan oligosaccharide) (25 μM) + raffinose (25 μM) + stachyose (25 μM) + verbascose (25 μM)] Comparison example_2 PLpro + Mg ²⁺ (0.9 μM) Comparison example_3 PLpro + Zn ²⁺ （0.09 μM） Comparison example_4 PLpro + isoretinic acid (50 μM) Experimental example_1 PLpro + isoretinic acid (50 μM) + oligosaccharide mixture [(β-1,3/1,6-glucan oligosaccharide) (25 μM) + raffinose (25 μM) + stachyose (25 μM) + verbascose (25 μM)] Experimental example_2 PLpro + isoretinic acid (50 μM) + oligosaccharide mixture [(β-1,3/1,6-glucan oligosaccharide) (25 μM) + raffinose (25 μM) + stachyose (25 μM) + verbascose (25 μM)] + Mg ²⁺ (0.9 μM) + Zn ²⁺ (0.09 μM)

20 μL of the above FG6-pNA (1.2 mM) was added to each test mixture to start the enzyme reaction. The enzyme reaction was allowed to proceed for 300 seconds at 30°C. The enzyme activity was determined by continuously monitoring the absorbance at 405 nm (A ₄₀₅ ) using a 96-well microplate spectrophotometer (Epoch™ 2, Biotek). The reaction rate was calculated based on this, where the reaction rate is the slope of the change in absorbance A ₄₀₅ with time (seconds), and the total reaction time is 300 seconds. The data are presented in Figure 11. The calculation of the inhibition percentage is as described in Example 3.

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

surface 6 Group Inhibition percentage ( % ） Comparison: PLpro 0 Comparison Example 1: PLpro + Oligosaccharides 12.43 ± 0.37 Comparative Example 2: PLpro + Mg ²⁺ 14.21 ± 0.15 Comparative Example 3: PLpro + Zn ²⁺ 19.22 ± 0.21 Comparison example 4: PLpro + isoretinal acid 20.90 ± 1.24 Experimental Example 1: PLpro + isoretinic acid + oligosaccharides 26.61 ± 0.49 Experimental Example 2: PLpro + isoretinic acid + oligosaccharides + Mg ²⁺ + Zn ²⁺ 72.16 ± 0.92

As shown in Figure 11 and Table 6 , the inhibitory effects of each group in the experimental group were significantly higher than those of each group in the comparative group, indicating that the two experimental groups: (1) isoretinoic acid and oligosaccharides, and (2) isoretinoic acid, oligosaccharides, Zn ²⁺ , and Mg ²⁺ , were more effective than the individual components of isoretinoic acid, oligosaccharides, Zn ²⁺ , or Mg ²⁺ . These results suggest that isoretinoic acid, oligosaccharides, and divalent metal ions have a synergistic inhibitory effect on papain-like protease activity. In addition, it provides important insights into the biochemical properties of the coronavirus PLpro family and leads the way for promising therapeutic strategies against SARS-CoV-2.

Examples 5

Combinations of isoretinic acid, oligosaccharides or divalent metal ions in SARS-CoV-2 Animal Mode SARS-CoV-2 Evaluation of in vivo treatment effects

The infected hamsters obtained in Part A of this experiment were divided into the following six groups (n=5 in each group): one control group, two experimental groups (i.e., Experimental Example_1 and Experimental Example_2) and four comparative groups (i.e., Comparative Example_1, Comparative Example_2, Comparative Example_3 and Comparative Example_4). The treatment agents used in these groups are listed in Table 7 below.

surface 7 Group Therapeutic agents Control group Buffer containing 40% ethanol, 40% Span® 80 (sorbitan monooleate) (Sigma-Aldrich), and 20% peanut oil Comparison example_1 Isoretinoic acid Comparison example_2 Liposome-encapsulated isoretinal acid Comparison example_3 Liposome-encapsulated combination of Zn ²⁺ , Mg ²⁺ and K ⁺ Comparison example_4 Liposome-encapsulated oligosaccharide mixture Experimental Example_1 Liposome-encapsulated isoretinoic acid + Liposome-encapsulated oligosaccharide mixture Experimental example_2 Liposome-encapsulated isotretinoic acid + Liposome-encapsulated oligosaccharide mixture + Liposome-encapsulated Zn ²⁺ , Mg ²⁺ and K ⁺ combination

The liposome-encapsulated therapeutic agent was prepared by Taipei Medical University using the commonly used technology in the field and is described in detail above. Since the main technical feature of the present invention 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.

Specifically, for each hamster in the experimental group, liposome-encapsulated isoretinoids were intranasally administered at 0.35 mg/kg at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, day 1) and at 0.35 mg/kg at 8:00 PM on the day of infection. On the next two days (days 2 and 3), liposome-encapsulated isoretinoids were administered intranasally twice a day at 8:00 AM and 8:00 PM at a dose of 0.35 mg/kg each.

In addition to intranasal administration of 0.35 mg/kg of liposome-encapsulated isotretinoic acid twice a day at 8:00 AM and 8:00 PM, a 15 μL volume of liposome-encapsulated oligosaccharide mixture (i.e., Experimental Example_1) or a combination of oligosaccharide mixture (15 μL) and Zn ²⁺ (100 μM), Mg 2+ (200 μM), and K ⁺ (200 μM) (15 μL), both encapsulated in liposomes, in a total volume of 30 μL (i.e., Experimental Example_2) was intranasally administered once a day from 7:30 PM to 8:00 PM on the day of infection (i.e., 0.5 to 1 hour before the evening administration of liposome-encapsulated ^isotretinoic acid), and on the next two days, a 15 μL volume of Experimental Example_1 or 30 μL volume experimental example_2.

For each hamster in the comparison group, isotretinoic acid in Comparative Example 1 or liposome-encapsulated isotretinoic acid in Comparative Example 2 was intranasally administered at 0.35 mg/kg at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection and at 0.35 mg/kg at 8:00 PM on the day of infection. On the next two days after the infection day, isotretinoic acid in Comparative Example 1 or liposome-encapsulated isotretinoic acid in Comparative Example 2 was intranasally administered at 0.35 mg/kg twice a day at 8:00 AM and 8:00 PM.

To each hamster, the combination of liposome-encapsulated Zn ²⁺ (100 μM), Mg ²⁺ (200 μM) and K ⁺ (200 μM) in Comparative Example 3 and the liposome-encapsulated oligosaccharide mixture in Comparative Example 4 were administered intranasally once a day from 7:30 PM to 8:00 PM on the day of infection, and 15 μL of each was administered on the next two days.

For each hamster in the control group, buffer solution was administered intranasally at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection and at 8:00 PM on the day of infection. For the two days after the infection day, buffer solution was administered intranasally at 8:00 AM and 8:00 PM twice a day at 100 μL.

After 3 days of treatment, hamsters were sacrificed and their lungs were collected for measurement of live viral load in Vero E6 cells by TCID ₅₀ assay. Virus titers were determined based on 50% tissue culture infectious dose (TCID ₅₀ ) using the Reed-Muench method. All experiments with SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by Academia Sinica (Taipei, Taiwan).

The experimental data were analyzed using Tukey's test to assess the differences between the groups. Statistical significance was indicated at p < 0.05.

Results: Referring to Figure 12 , the _TCID50 of each of Experimental Example 1 and Experimental Example 2 is significantly lower than the _TCID50 of each of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4, indicating that the combination of isotretinoic acid and oligosaccharides with/without metal ions has a higher in vivo efficacy against SARS-CoV-2 than isotretinoic acid alone, metal ions alone or oligosaccharides alone.

Conclusion

In view of the results of Examples 1 to 5, it is verified that the claimed novel retinoic acid compound or its pharmaceutically acceptable salt, 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 carbohydrates (i.e., the second pharmaceutical composition) can provide improved and/or synergistic effects in inhibiting the infection and replication of SARS-CoV-2 and treating diseases associated with SARS-CoV-2 infection or cancer. Therefore, the compound comprising retinoic acid conjugated with carbohydrates, its pharmaceutically acceptable salt, and the first pharmaceutical composition and the second pharmaceutical composition of the present invention can indeed be used as drug-repurposing agents.

The present invention has been described broadly and throughout this document. Each narrower form and subgroup falling within the overall disclosure also constitutes a part of the present invention. In addition, where features or aspects disclosed herein are described in terms of Markush groups, a person skilled in the art will understand that the present invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

without

FIG1 illustrates the results of liquid chromatography-mass spectrometry (LC-MS) analysis of compound ( Ia ) according to an example of the present invention.

FIG2 is a graph showing the absorbance variation over time in a SARS-CoV-2 PLPro inhibition assay of a novel retinoic acid compound according to an embodiment of the present invention.

FIG3 illustrates _{a graph of TCID 50} showing the antiviral effects of novel retinoic acid compounds according to embodiments of the present invention in a SARS-CoV-2 animal model.

FIG. 4 is a graph illustrating cell survival rate, which shows the cytotoxic effect of the novel retinoic acid compound according to an embodiment of the present invention on the AsPC-1 cancer cell line.

FIG. 5 is a graph illustrating cell viability, showing the cytotoxic effect of the novel retinoic acid compounds according to embodiments of the present invention on the MDA-MB-231 cancer cell line.

FIG. 6 illustrates a graph of cell viability, showing the cytotoxic effect of the novel retinoic acid compounds according to embodiments of the present invention on the HCT-116 cancer cell line.

FIG. 7 is a graph illustrating cell survival rate, which shows the cytotoxic effect of the novel retinoic acid compounds according to embodiments of the present invention on Huh-7 cancer cell lines.

FIG. 8 illustrates a graph of cell viability, which shows the cytotoxic effect of the novel retinoic acid compounds according to embodiments of the present invention on the SKOV-3 cancer cell line.

FIG. 9 is a graph illustrating cell viability, showing the cytotoxic effect of the novel retinoic acid compounds according to embodiments of the present invention on A549 cancer cell lines.

FIG. 10 is a graph illustrating cell survival rate, which shows the cytotoxic effect of the novel retinoic acid compound according to an embodiment of the present invention on the H460 cancer cell line.

Figure 11 illustrates a curve graph of absorbance variation over time in a SARS-CoV-2 PLPro inhibition test of a second pharmaceutical composition according to an embodiment of the present invention.

Figure 12 illustrates a graph of TCID ₅₀ , which shows the antiviral effect of the second pharmaceutical composition according to an embodiment of the present invention in the SARS-CoV-2 animal model.

Claims (10)

Use of a compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the manufacture of a drug for inhibiting viral infection or replication, wherein the compound comprises retinoic acid conjugated to a carbohydrate, wherein the retinoic acid is isoretinic acid, and the carbohydrate is selected from the group consisting of: monosaccharides, disaccharides, oligosaccharides and polysaccharides, wherein the pharmaceutical composition comprises the compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, wherein the virus is a coronavirus, and 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. The use of claim 1, wherein the compound is represented by formula (I):

wherein R ¹ is a substituted or unsubstituted carbohydrate. The use as claimed in claim 1, wherein the isoretinic acid is conjugated to the carbohydrate via a linker. The use according to claim 3, wherein the linker is -C ₃ H ₆ NH-. The use as claimed in claim 1, wherein the oligosaccharide is a mono-oligosaccharide or a hetero-oligosaccharide; and the polysaccharide is a mono-polysaccharide or a hetero-polysaccharide. For use as claimed in claim 1, 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. The use as claimed in claim 1, wherein the pharmaceutically acceptable carrier comprises liposomes, and the compound or its pharmaceutically acceptable salt is encapsulated by the liposomes. The use as claimed in claim 1, wherein the pharmaceutical composition further comprises metal ions, and the metal ions comprise monovalent ions, divalent ions or a combination thereof, and wherein the monovalent ions comprise K ⁺ , Na ⁺ or a combination thereof, and the divalent ions comprise Zn2 ⁺ , Mg2 ⁺ , Cu2 ⁺ , Mn2 ⁺ , Ca2 ⁺ , Fe2 ⁺ or any combination thereof. The use as claimed in claim 8, wherein the pharmaceutically acceptable carrier comprises liposomes, and the metal ions are encapsulated by the liposomes. The use as claimed in claim 9, wherein the compound or its pharmaceutically acceptable salt and the metal ion are encapsulated by the liposome alone or simultaneously.