Methods and compositions for inhibiting coronavirus infection

Abstract

The invention provides a method for inhibiting coronavirus infected cells. The method comprises the step of contacting the cells with an effective amount of a coordination compound shown in a formula (I). The present invention also provides a method of preventing or treating coronavirus infection comprising administering to a subject in need thereof an effective amount of the coordination compound of formula (I). The invention also provides a pharmaceutical composition for preventing or treating coronavirus infection. The pharmaceutical composition comprises the coordination compound of formula (I) and a pharmaceutically acceptable carrier.

Description

Technical Field

[0001] This application claims priority to U.S. Provisional Application No. 63 / 476,380, filed on December 21, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to a coordination compound for combating viral infections. Specifically, the present invention relates to a method of using a coordination compound to prevent or treat coronavirus infections. The present invention also relates to a pharmaceutical composition for preventing or treating coronavirus infections, which comprises the coordination compound.

[0003] Prior Art

[0004] Since the end of 2019, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly worldwide, resulting in a severe COVID-19 pandemic. According to the statistics of the World Health Organization (WHO), as of February 2022, 403 million COVID-19 patients and more than 5.7 million deaths have been reported in 220 countries. SARS-CoV-2 is an enveloped positive-strand RNA virus, which has about 80% genomic similarity with SARS-CoV that caused the 2002 SARS pandemic, but is more infectious.

[0005] The genomes of coronaviruses (CoVs) generally encode structural proteins (including envelope protein, nucleocapsid protein, membrane protein, and spike protein) and non-structural proteins (such as proteases nsp3 and nsp5), and have similar infection mechanisms. In the case of SARS-CoV-2, the infection process involves angiotensin-converting enzyme 2 (ACE2) as a receptor on the surface of host cells and transmembrane serine protease 2 (TMPRSS2). The spike protein of the virus binds to the host's ACE2 receptor and is cleaved by TMPRSS2, resulting in membrane fusion between the virus and the host cell. Therefore, inhibiting the expression or function of ACE2 or TMPRSS2 is a potential target for preventing SARS-CoV-2 from entering host cells. After SARS-CoV-2 infects host cells, the RNA genome of SARS-CoV-2 is released, and polyproteins 1a and 1ab are translated in the cytoplasm. The polyprotein is further cleaved by papain-like protease and 3-chymotrypsin-like (3CL) protease to form 16 non-structural proteins (which function as replication-transcription complexes). The replication-transcription complex proteins include RNA-dependent RNA polymerase (RdRp), which is responsible for the replication of viral RNA. The structural proteins translated from the viral genome are inserted into the endoplasmic reticulum and secreted from the endoplasmic reticulum-Golgi junction in the form of vesicles to encapsulate the viral genome. Finally, mature SARS-CoV-2 is released from host cells through the constitutive exocytic pathway.

[0006] As of 2022, dozens of vaccines have been approved as primary vaccines or boosters to combat COVID-19. However, the effectiveness of vaccines can be affected by SARS-CoV-2 variants (such as alpha, beta, gamma, delta, and omicron variants). Therefore, there is an urgent need to develop a universal and effective drug to prevent this dangerous infectious disease. In addition to vaccines as preventive therapies, several small molecule compounds are also used in the treatment of COVID-19, including lopinavir, molnupiravir, nirmatrelvir, remdesivir, and ritonavir. Some of these compounds have been used in clinical applications to reduce the severity of COVID-19. However, new compounds are still needed to avoid the emergence of viral resistance. Summary of the Invention

[0007] The present invention relates to a coordination compound that can be used as an antiviral agent to inhibit the infection of cells by coronaviruses. The present invention further relates to the use of the coordination compound in preventing or treating coronavirus infection in an individual in need thereof. The coordination compound is represented by formula (I):

[0008] [XZ2(CH3CO2)6(H2O)4(OH)2]NO3. X and Z in formula (I) refer to different metal ions, where X is a trivalent ion of chromium (Cr) or molybdenum (Mo), and Z is a trivalent ion of iron (Fe), ruthenium (Ru), or osmium (Os).

[0009] An object of the present invention is to provide a method for inhibiting the infection of cells by coronaviruses, comprising contacting the cells with an effective amount of the coordination compound represented by formula (I), where X is a trivalent ion of chromium or molybdenum, and Z is a trivalent ion of iron, ruthenium, or osmium. The coordination compound preferably contains trivalent chromium ions and trivalent iron ions and is represented by formula (II).

[0010] In some embodiments, the coordination compound inhibits the infection of cells by severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or a variant of SARS-CoV or SARS-CoV-2. The aforementioned variants of SARS-CoV-2 can be selected from alpha, beta, gamma, delta, or omicron variants.

[0011] In some embodiments, the coordination compound inhibits the infection of cells from the respiratory system, digestive system, urinary system, or cardiovascular system of an individual.

[0012] In some embodiments, the coordination compound inhibits infection by preventing the coronavirus from entering cells. For example, the coordination compound can avoid the entry of the coronavirus into cells by blocking the binding between a spike protein of the coronavirus and angiotensin-converting enzyme 2 (ACE2) of the cell and / or inhibiting the expression of ACE2 or transmembrane serine protease 2 (TMPRSS2) in the cell. In some embodiments, the coordination compound inhibits infection by inhibiting the maturation and / or replication of the coronavirus. For example, the coordination compound can inhibit the maturation of the coronavirus by inhibiting the activity of 3CL protease and further inhibit the replication of the coronavirus by inhibiting the activity of RNA-dependent RNA polymerase (RdRp).

[0013] Another object of the present invention is to provide a method for preventing or treating coronavirus infection, comprising administering to an individual in need an effective amount of the coordination compound represented by formula (I), wherein X is a trivalent ion of chromium or molybdenum, and Z is a trivalent ion of iron, ruthenium, or osmium. The coordination compound preferably contains a trivalent chromium ion and a trivalent iron ion and is represented by formula (II).

[0014] In some embodiments, the coronavirus infection is caused by SARS-CoV, SARS-CoV-2, or a variant of SARS-CoV or SARS-CoV-2. The aforementioned variants of SARS-CoV-2 can be selected from alpha, beta, gamma, delta, or omicron variants.

[0015] In some embodiments, the coordination compound is administered to the individual in solid form or liquid form. In some embodiments, the coordination compound is administered orally, intranasally, topically, transmucosally, intravenously, or enterally.

[0016] The present invention also relates to a pharmaceutical composition for preventing or treating the aforementioned coronavirus infection. The pharmaceutical composition comprises an effective amount of the coordination compound represented by formula (I) and a pharmaceutically acceptable carrier, wherein X is a trivalent ion of chromium or molybdenum, and Z is a trivalent ion of iron, ruthenium, or osmium.

[0017] In some embodiments, the coordination compound contained in the pharmaceutical composition contains a trivalent chromium ion and a trivalent iron ion and is represented by formula (II).

[0018] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an antiviral agent, an immunomodulator, or an anti-infective agent.

[0019] The coordination compounds disclosed herein can effectively inhibit the entry, maturation, and replication of coronaviruses into cells. The coordination compounds have also been shown to reduce viral accumulation in individuals infected with coronaviruses. Accordingly, the compounds can be used to prevent coronavirus infection, or to treat infected individuals and reduce the severity of the disease. Brief Description of the Drawings

[0020] Those skilled in the art will clearly understand the present invention through the following detailed description of the preferred embodiments in conjunction with the accompanying drawings, in which:

[0021] Figure 1A is the 1 1H-NMR spectrum of the coordination compound of formula (II);

[0022] Figure 1B is the 13 13C-NMR spectrum of the coordination compound of formula (II);

[0023] Figure 1C is the Fourier transform infrared (FT-IR) spectrum of the coordination compound of formula (II);

[0024] Figure 2A shows the effect of the coordination compound of formula (II) on the interaction between recombinant human ACE2 and the SARS-CoV-2 spike protein; ** and *** indicate p < 0.01 and p < 0.001, respectively, compared to the compound-untreated (control) group;

[0025] Figure 2B shows that the coordination compound of formula (II) has no significant cytotoxicity to BEAS-2B cells;

[0026] Figure 3A shows the effect of the coordination compound of formula (II) on the expression of ACE2 protein in Calu-3 cells; ** and * ** indicate p < 0.01 and p < 0.001, respectively, compared to the compound-untreated (control) group;

[0027] Figure 3B shows the effect of the coordination compound of formula (II) on the expression of TMPRSS2 protein in Calu-3 cells; ** and * ** indicate p < 0.01 and p < 0.001, respectively, compared to the compound-untreated (control) group;

[0028] Figure 4A shows the effect of the coordination compound of formula (II) on the 3CL protease activity of SARS-CoV-2;

[0029] Figure 4BEffect of the coordination compound of formula (II) on the activity of viral RNA-dependent RNA polymerase; * indicates p < 0.05 compared with the untreated compound (control group);

[0030] Figure 5A Effect of the coordination compound of formula (II) on the infection of Calu-3 cells by SARS-CoV-2 alpha variant pseudovirus; ** and * ** respectively indicate p < 0.01 and p < 0.001 compared with the untreated compound (control group);

[0031] Figure 5B Effect of the coordination compound of formula (II) on the infection of Caco-2 cells by SARS-CoV-2 alpha variant pseudovirus; *, ** and * ** respectively indicate p < 0.05, p < 0.01 and p < 0.001 compared with the untreated compound (control group);

[0032] Figure 6A Effect of the coordination compound of formula (II) on the infection of Calu-3 cells by SARS-CoV-2 beta variant pseudovirus; ** and *** respectively indicate p < 0.01 and p < 0.001 compared with the untreated compound (control group);

[0033] Figure 6B Effect of the coordination compound of formula (II) on the infection of Calu-3 cells by SARS-CoV-2 gamma variant pseudovirus; ** and * ** respectively indicate p < 0.01 and p < 0.001 compared with the untreated compound (control group);

[0034] Figure 6C Effect of the coordination compound of formula (II) on the infection of Caco-2 cells by SARS-CoV-2 delta variant pseudovirus; ** and * ** respectively indicate p < 0.01 and p < 0.001 compared with the untreated compound (control group);

[0035] Figure 7A Schematic diagram of in vivo infection study using SKH-1 mice;

[0036] Figure 7B Effect of the coordination compound of formula (II) on the in vivo virus accumulation in SKH-1 mice infected with wild-type SARS-CoV-2 pseudovirus;

[0037] Figure 7CEffect of the coordination compound of formula (II) on in vivo viral accumulation in SKH-1 mice infected with SARS-CoV-2 delta variant pseudovirus;

[0038] Figure 7D Effect of the coordination compound of formula (II) on in vivo viral accumulation in SKH-1 mice infected with SARS-CoV-2 beta variant pseudovirus; and

[0039] Figure 7E Effect of the coordination compound of formula (II) on in vivo viral accumulation in SKH-1 mice infected with SARS-CoV-2 omicron variant pseudovirus. Detailed implementation mode

[0040] The following implementation modes and examples further illustrate the present invention. It should be understood that the following listed examples are not used to limit the scope of the present invention, and those skilled in the art can make adjustments and modifications without exceeding the scope of the appended claims.

[0041] Unless otherwise defined, the meanings of all technical and scientific terms and abbreviations used herein are the same as those commonly understood by those skilled in the art to which the present invention belongs.

[0042] Definition

[0043] Unless the context clearly defines otherwise, the singular forms "a", "an" and "the" used herein include plural references. For example, "a pharmaceutically acceptable carrier" includes a mixture of pharmaceutically acceptable carriers, and "an additional pharmaceutically active agent" includes more than one pharmaceutically active agent.

[0044] Data are usually expressed as mean ± standard deviation. The numerical values provided herein are approximate values, and experimental numerical values can vary within a range of 20%, preferably within a range of 10%, more preferably within a range of 5%. Therefore, terms such as "about" and "approximate" refer to within a range of 20% of a given numerical value or range, preferably within a range of 10%, more preferably within a range of 5%.

[0045] The term "coordination compound" as used herein refers to a metal complex defined by formula (I), which contains three metal ion centers, each center being surrounded by six ligands.

[0046] Coordination compound

[0047] The coordination compounds disclosed herein are shown as formula (I):

[0048] [XZ2(CH3CO2)6(H2O)4(OH)2]NO3 Formula (I).

[0049] X can be a trivalent ion of chromium (Cr) or molybdenum (Mo), and Z can be a trivalent ion of iron (Fe), ruthenium (Ru), or osmium (Os). Since this coordination compound can contain Cr or Mo (both Group 6 metals), and Fe, Ru, or Os (all Group 8 metals), this coordination compound can be represented by Formula (II), (III), (IV), (V), (VI), or (VII) based on various combinations of central metal ions:

[0050] Formula (II): [CrFe2(CH3CO2)6(H2O)4(OH)2]NO3

[0051] Formula (III): [CrRu2(CH3CO2)6(H2O)4(OH)2]NO3

[0052] Formula (IV): [CrOs2(CH3CO2)6(H2O)4(OH)2]NO3

[0053] Formula (V): [MoFe2(CH3CO2)6(H2O)4(OH)2]NO3

[0054] Formula (VI): [MoRu2(CH3CO2)6(H2O)4(OH)2]NO3

[0055] Formula (VII): [MoOs2(CH3CO2)6(H2O)4(OH)2]NO3

[0056] In some embodiments, this coordination compound contains trivalent chromium ions and trivalent iron ions and is represented by Formula (II). The compound of Formula (II) can have the structure (a) shown in the following figure or other structures representing different stereoisomers. That is, the compound of Formula (II) means any compound of Formula (II) in which the arrangement of ligands around the metal ions may be different. Similarly, the compounds of Formula (III), (IV), (V), (VI), or (VII) can include various stereoisomers.

[0057]

[0058] Use of the coordination compound for inhibiting coronavirus infection

[0059] The present invention provides a method for inhibiting the infection of cells by a coronavirus, which comprises contacting the cells with an effective amount of a coordination compound represented by formula (I): [XZ2(CH3CO2)6(H2O)4(OH)2]NO3, wherein X is a trivalent ion of chromium or molybdenum, and Z is a trivalent ion of iron, ruthenium, or osmium. As used herein, "inhibiting infection" means that after using the coordination compound of formula (I), the number of cells infected with a coronavirus can be reduced, or the number of coronaviruses produced by the infected cells can be reduced to avoid further infection. In other words, the coordination compound, as an antiviral agent, can be based on its prophylactic utility (e.g., preventing virus attachment and / or entry into host cells), or on its therapeutic utility (e.g., inhibiting the replication of viral RNA in host cells).

[0060] Coronavirus infections that can be inhibited by contacting cells with the coordination compound of formula (I) include any infection caused by a coronavirus having one or more of the following characteristics: (1) having a spike protein structurally similar to that of SARS-CoV-2, and / or (2) using ACE2 or a membrane protein structurally similar to ACE2 as a receptor for entering host cells, and / or (3) having a protease structurally similar to the 3CL protease of SARS-CoV-2, and / or (4) having an RNA-dependent RNA polymerase (RdRp) structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2. The so-called "a spike protein structurally similar to that of SARS-CoV-2" refers to a spike protein having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the protein sequence of the spike protein of SARS-CoV-2. The so-called "a membrane protein structurally similar to ACE2" refers to a membrane protein having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the protein sequence of human ACE2. The so-called "a protease structurally similar to the 3CL protease of SARS-CoV-2" refers to a protease having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the protein sequence of the 3CL protease of SARS-CoV-2. The so-called "an RdRp structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2" refers to an RdRp having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the protein sequence of the RNA-dependent RNA polymerase of SARS-CoV-2. Examples of such coronaviruses include, but are not limited to, SARS-CoV, variants of SARS-CoV, SARS-CoV-2, and variants of SARS-CoV-2. As used herein, the term "variant" refers to a subtype of coronavirus that has one or more mutations in its genome and is thus genetically distinct from the main strain.For example, the variants of SARS-CoV-2 can be the alpha, beta, gamma, delta, or omicron variants released by the WHO. Each SARS-CoV-2 variant includes a group of closely related viruses with a common ancestor.

[0061] The cells (also referred to as host cells) in which the coordination compound of formula (I) can exert its antiviral effect can be any cells that can be infected by the coronaviruses described herein in the absence of the coordination compound. The cell can be a single cell or a group of homogeneous or heterogeneous cells. The cell can be a cell present in an individual's body, or a cell isolated from an individual (such as a human) body, or a derivative cell of the isolated cell. In addition, the cell can have various sources. For example, the cell is from the respiratory system, digestive system, urinary system, or cardiovascular system of an individual. In some embodiments, the cell is an epithelial cell of the lung, trachea, bronchus, or bronchiole. In other embodiments, the cell is an epithelial cell of the pancreas, small intestine, large intestine, kidney, heart, or vascular tissue. In some preferred embodiments, the cell is a cell expressing ACE2.

[0062] The effective amount of the coordination compound administered to the cells to inhibit coronavirus infection refers to an amount sufficient to reduce the amount of host cells infected by the coronavirus or sufficient to reduce the amount of coronavirus produced by the host cells. As understood by those skilled in the art, the effective amount will vary with various factors, such as the type of coronavirus, the source of the host cells, the specific coordination compound administered, and the manner in which the coordination compound is delivered to the host cells.

[0063] The step of contacting the cells with the coordination compound can be carried out according to methods known in the art. In some embodiments, the cells are contacted with the coordination compound by being cultured in a cell culture medium supplemented with the coordination compound. In some embodiments, the cells are contacted with the coordination compound by being exposed to a composition containing the coordination compound and a pharmaceutically acceptable carrier in vitro or in vivo.

[0064] In some embodiments, the coordination compound inhibits infection by preventing the coronavirus from entering the cells. For example, the coordination compounds disclosed herein can prevent coronaviruses (including but not limited to SARS-CoV-2) from entering the cells through one of the following actions: (1) blocking the binding between the spike protein of the coronavirus and ACE2, which is a protein expressed on the surface of host cells and serves as the receptor for the coronavirus during virus attachment; (2) inhibiting the expression of ACE2 in the cells; (3) inhibiting the expression of transmembrane serine protease 2 (TMPRSS2), which is a protease expressed on the surface of cells and is crucial for the cleavage of the spike protein and the fusion of the coronavirus membrane with the cell membrane.

[0065] In addition, in some embodiments, the coordination compound inhibits infection by inhibiting the maturation and / or replication of coronaviruses (including but not limited to SARS-CoV-2). For example, the coordination compounds disclosed herein can inhibit 3CL protease activity and / or RNA-dependent RNA polymerase (RdRp) activity, both of which are important drug targets for treating viral infections. RdRp is required for viral RNA replication. 3CL protease is a major protease in coronaviruses that cleaves the polyprotein translated from viral RNA to form non-structural proteins and is thus essential for viral maturation.

[0066] Use of a coordination compound for preventing or treating coronavirus infection

[0067] The coordination compound exhibits antiviral activity in a subject. Accordingly, the present invention further provides a method for preventing or treating coronavirus infection, comprising administering to a subject in need thereof an effective amount of the coordination compound of formula (I), wherein X is a trivalent ion of chromium or molybdenum and Z is a trivalent ion of iron, ruthenium, or osmium.

[0068] As used herein, the term "subject" refers to a mammal. The subject can be human or non-human and includes but is not limited to primates, rodents, dogs, cats, cows, goats, sheep, horses, rabbits, pigs, etc. Accordingly, antiviral methods and compositions for veterinary and medical uses are contemplated herein.

[0069] Coronavirus infections in an individual that can be prevented or treated by the coordination compound of formula (I) include any infection caused by a coronavirus having one or more of the following characteristics: (1) having a spike protein structurally similar to the spike protein of SARS-CoV-2, and / or (2) using ACE2 or a membrane protein structurally similar to ACE2 as a receptor for entering host cells, and / or (3) having a protease structurally similar to the 3CL protease of SARS-CoV-2, and / or (4) having an RNA-dependent RNA polymerase (RdRp) structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2. The so-called "a spike protein structurally similar to the spike protein of SARS-CoV-2" refers to a spike protein having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein sequence of the spike protein of SARS-CoV-2. The so-called "a membrane protein structurally similar to ACE2" refers to a membrane protein having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein sequence of human ACE2. The so-called "a protease structurally similar to the 3CL protease of SARS-CoV-2" refers to a protease having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein sequence of the 3CL protease of SARS-CoV-2. The so-called "an RdRp structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2" refers to an RdRp having at least 75%, preferably at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein sequence of the RNA-dependent RNA polymerase of SARS-CoV-2. Examples of such coronaviruses include, but are not limited to, SARS-CoV, variants of SARS-CoV, SARS-CoV-2, and variants of SARS-CoV-2. As used herein, the term "variant" refers to a subtype of coronavirus that has one or more mutations in its genome and is thus genetically distinct from the main strain.For example, the variant strains of SARS-CoV-2 can be the alpha, beta, gamma, delta, or omicron variant strains released by the WHO. Each SARS-CoV-2 variant strain includes a group of closely related viruses with a common ancestor.

[0070] An effective amount of the coordination compound administered to an individual in need can be a prophylactically effective amount or a therapeutically effective amount. A "prophylactically effective amount" means an amount sufficient to prevent or delay the occurrence of a coronavirus infection or the appearance of its symptoms or signs in an individual. A "therapeutically effective amount" means an amount sufficient to halt or delay the progression of a coronavirus infection or at least one of its symptoms or signs in an individual, or to alleviate the state of a coronavirus infection or at least one of its symptoms or signs in an individual. As understood by those skilled in the art, the effective amount will vary with various factors, such as the type of coronavirus infection, the age, weight, physical condition, and reactivity of the individual being treated, the particular coordination compound being administered, the route of administration, the use of excipients, and the co-use with other pharmaceutically active agents, etc.

[0071] The coordination compound of formula (I) can be administered to an individual through any suitable route, preferably in the form of a pharmaceutical composition suitable for that route. In some embodiments, the coordination compound is administered orally, intranasally, topically, transmucosally, intravenously, or enterally.

[0072] In some embodiments, the coordination compound of formula (I) is administered to an individual before or at the beginning of a coronavirus infection. In some embodiments, the coordination compound is administered to an individual who has already been infected with a coronavirus. In some embodiments, the coordination compound is administered at least once, twice, three times, or more times per day. In some embodiments, the coordination compound is administered daily, every other day, several times a week, weekly, or at a lower frequency to maintain an effective dose level and patient compliance. In some embodiments, the administration of the coordination compound continues for up to two days, three days, four days, five days, six days, seven days, or longer. The frequency and duration of treatment can be changed according to an individual's response to the treatment.

[0073] Pharmaceutical composition

[0074] The present invention further provides a pharmaceutical composition for preventing or treating coronavirus infection, comprising an effective amount of the coordination compound represented by formula (I) and a pharmaceutically acceptable carrier, wherein X is a trivalent ion of chromium or molybdenum, and Z is a trivalent ion of iron, ruthenium, or osmium. In some preferred embodiments, the pharmaceutical composition comprises the coordination compound of formula (II) as an active ingredient.

[0075] The pharmaceutical composition can be in any suitable form, such as tablets, powders, solutions, suspensions, emulsions, liposomes, nanoparticles, or other formulations.

[0076] As used herein, the term "pharmaceutically acceptable carrier" refers to any carrier or excipient that is compatible with the coordination compound and other active ingredients, if any, and is preferably capable of stabilizing the active ingredient and being harmless to the individual to be treated. Pharmaceutically acceptable carriers can be excipients, diluents, antioxidants, and preservatives known in the art. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, physiological saline, buffers, organic solvents, hydrophilic polymers, carbohydrates, peptides, amino acids, and surfactants.

[0077] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent. The term "pharmaceutically active agent" refers to a small molecule compound or a macromolecule (such as an antibody or a fragment thereof) having the desired pharmacological action and therapeutic effect. The pharmaceutically active agent can be an antiviral agent, an immunomodulator, an anti-infective agent, or any combination thereof. Examples of antiviral agents include, but are not limited to, protease inhibitors, viral protease inhibitors, viral polymerase inhibitors, antisense nucleic acids, viral entry inhibitors, viral replication inhibitors (such as RNA polymerase inhibitors), viral assembly inhibitors, and interferons. Examples of immunomodulators include immunosuppressants (such as cytokine production inhibitors) and immunostimulants. Examples of anti-infective agents include antifungal agents, antibacterial agents, and antiparasitic agents.

[0078] Examples

[0079] Example 1: Preparation of the coordination compound

[0080] The preparation process of the coordination compound of formula (II) (hereinafter referred to as "Compound (II)") described below is an example for illustrating the preparation method of the coordination compounds invented herein. The preparation process of the coordination compound of formula (II) (hereinafter referred to as "Compound (II)") described below is an example for illustrating the preparation method of the coordination compounds invented herein. Mix chromium(III) nitrate and iron(III) nitrate in a flask, with a molar ratio of about 1:1 to 1:3. Inject alcohol with an ethanol content of 60 - 95% (the rest is water) into the flask at room temperature, such that the weight-to-volume ratio (g / ml) of iron(III) nitrate to alcohol is about 1:1 to 1:3, and stir the mixture until all solids dissolve. Thereafter, add acetic anhydride to the flask such that the volume ratio of acetic anhydride to alcohol is between about 4:1 and 7:1, then stir the reaction mixture for about 2 to 6 hours. The reaction with acetic anhydride is carried out below 70 °C. Subsequently, stir the reaction mixture for 20 to 28 hours, and then collect the solid precipitate of Compound (II) by filtration. Dry the solid precipitate in an oven to a constant weight.

[0081] By mass spectrometry, the molecular weight of Compound (II) is about 686.11. Elemental analysis shows that Compound (II) contains about 7.36% chromium, about 19.31% iron, about 2.21% nitrogen, about 21.73% carbon, about 3.86% hydrogen, and about 44.21% oxygen (by mass). The structural characteristics of Compound (II) are verified using 1 1H nuclear magnetic resonance (NMR) spectroscopy, 13 13C-NMR spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. Figure 1A Showing the 1 1H-NMR spectrum of Compound (II), where the signals at about 3.3 ± 0.2 and 1.9 ± 0.2 ppm correspond to the hydrogens of H2O and the acetate group, respectively. In addition, 13 the signals in the 13C-NMR spectrum at about 21.2 ± 0.5, 39.7 ± 0.5, and 171.9 ± 0.5 ppm indicate the presence of the acetate group ( Figure 1B ). In addition, the infrared spectrum shows absorption peaks at about 3400 ± 10, 3200 ± 10, 3000 ± 10, 1683 ± 10, 1585 ± 10, 1428 ± 10, 1349 ± 10, 1292 ± 10, and 1035 ± 10 cm -1 -1 ( Figure 1C ). All data support the structure of Compound (II) invented herein. Compound (II) is used in the efficacy studies in Examples 2 - 6.

[0082] The coordination compounds of formulas (III) to (VII) can be prepared according to the above method but using appropriate metal salts in place of chromium(III) nitrate and / or iron(III) nitrate.

[0083] Example 2: Inhibitory effect of the coordination compound of formula (II) on the interaction between SARS-CoV-2 spike protein and ACE2

[0084] To evaluate whether the coordination compound can inhibit the interaction between the spike protein of coronaviruses and the host cell receptor, an enzyme-linked immunosorbent assay (ELISA) was performed using a COVID-19 Spike-ACE2 Binding Assay Kit from RayBiotech (Peachtree Corners, Georgia, USA) to determine the binding of recombinant human ACE2 to the SARS-CoV-2 spike protein in the presence or absence of compound (II). According to the manufacturer's instructions, the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was pre-coated on a transparent 96-well flat-bottom plate. Different concentrations (0, 100, 200, 300, 400, or 500 μg / ml) of compound (II) were mixed with the ACE2 protein, and then the mixture was reacted with the 96-well plate by shaking at 22 °C and 200 rpm for 2.5 hours. After the reaction, the reaction mixture was removed, and the 96-well plate was washed four times with 300 μl / well of washing buffer (washing step), and then a primary antibody (goat anti-human ACE2 antibody) was added to the 96-well plate. Subsequently, the washing step was repeated, and an anti-goat secondary antibody conjugated with horseradish peroxidase was added to the 96-well plate and reacted by shaking at 22 °C and 200 rpm for 60 minutes. Subsequently, the washing step was repeated, and 100 μl of 3,3',5,5'-tetramethylbenzidine substrate was added to each well of the 96-well plate, and then the reaction was carried out by shaking at 22 °C and 200 rpm in the dark for 60 minutes. The reaction was blocked with 50 μl of termination solution, and the absorbance value (OD450) of each well at a wavelength of 450 nm was measured with a plate reader.

[0085] As Figure 2A shown, compared with the control group (without using compound (II)), compound (II) at about 100 μg / ml could block the binding of the SARS-CoV-2 spike protein to ACE2 by more than 50%, and this blocking showed a dose-dependence. This result indicates that compound (II) has an inhibitory effect on the interaction between the SARS-CoV-2 spike protein and the host cell ACE2.

[0086] In addition, non-tumorigenic human bronchial epithelial cells BEAS-2B (95102433, Sigma-Aldrich) were used as a cell model to study the cytotoxicity of compound (II). BEAS-2B cells were cultured in LHC-9 medium (Gibco TM , ThermoFisher Scientific, Waltham, Massachusetts, USA) in an incubator at 37 °C with 5% carbon dioxide supply. After treating BEAS-2B cells with different doses (0, 200, 400, 600 or 800 μg / ml) of compound (II) for 24 hours, cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. As Figure 2B shown, compound (II) did not significantly inhibit the survival of BEAS-2B cells, indicating that the coordination compounds of the present invention do not hinder the growth of human lung cells.

[0087] Example 3: Inhibitory effect of the coordination compound of formula (II) on the expression of ACE2 and TMPRSS2 proteins

[0088] Both ACE2 and TMPRSS2 are transmembrane proteins required for SARS-CoV-2 entry into cells. To evaluate the effect of the coordination compound on the expression of ACE2 and TMPRSS2 proteins in host cells, human lung epithelial cells Calu-3 (available from the American Type Culture Collection (ATCC), catalog number HTB-55) were used as a cell model in the expression assay. Calu-3 cells were cultured in medium supplemented with 20% fetal bovine serum (Gibco TM, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and Eagle's Minimum Essential Medium supplemented with 1% penicillin / streptomycin, and cultured in an incubator at 37 °C with 5% carbon dioxide supply. Cells were treated with compound (II) at different concentrations (0, 100, 200, 300, or 600 μg / ml). After 24 hours, the cells were washed twice with phosphate buffered saline (PBS), and then centrifuged at 5000 rpm for 10 minutes to collect the cells. The cell pellet was lysed by freezing in RIPA buffer containing protease inhibitors and phosphatase inhibitors for 1 hour, and then centrifuged at 12,500 rpm for 30 minutes at 4 °C. The supernatant containing the target protein was collected for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. After blocking the polyvinylidene difluoride (PVDF) membrane for western blot with 5% skim milk, it was reacted with primary antibodies against ACE2 (sc-390851; Santa Cruz Biotechnology, Santa Cruz, California, USA), TMPRSS2 (ab92323; Abcam, Cambridge, UK), and β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, California, USA) at 4 °C for more than 12 hours. Then, the PVDF membrane was washed and reacted with secondary antibodies. The transfer result was detected using a chemiluminescent reagent ( ECL), and observed under an iBright 1500 chemiluminescent imaging analyzer.

[0089] As Figure 3A and Figure 3B shown, compared with the control group (without using compound (II)), compound (II) inhibited the protein expression of ACE2 and TMPRSS2 in Calu-3 cells in a dose-dependent manner. This result also showed that compound (II) at about 300 to 600 μg / ml could significantly inhibit the protein expression of TMPRSS2 and ACE2 in host cells.

[0090] Example 4: Inhibitory effects of the coordination compound of formula (II) on 3CL protease and RNA-dependent RNA polymerase activities

[0091] 3CL protease and RNA-dependent RNA polymerase (RdRp) are viral proteins required for the maturation and replication of SARS-CoV-2. To evaluate whether coordination compounds would affect the activities of these enzymes, the 3CL protease activity of SARS-CoV-2 and the viral RdRp activity were measured in the presence or absence of compound (II).

[0092] In the SARS-CoV-2 3CL protease activity assay, the 520 SARS-CoV-2 3CL Protease Activity Assay Kit (SARS-CoV-2 3CL Protease Activity Assay Kit, AS-72262; Kaneka Eurogentec S.A., Seraing, Belgium) was used to measure the cleavage of a 3CL protease fluorogenic substrate by 3CL protease. According to the manufacturer's instructions, the SARS-CoV-2 3CL protease solution (40 μl / well) was mixed with compound (II) in PBS (10 μl / well) or the control buffer in a 96-well plate. Subsequently, 50 μl of the 3CL protease substrate solution was added to each well, and the reaction mixture was allowed to react at 37 °C for 30 minutes. The fluorescence intensity at 490 nm (excitation) / 520 nm (emission) was measured using a plate reader to determine the protease activity. As Figure 4A shown, compound (II) at a concentration of 100 μg / ml or higher significantly inhibited the activity of 3CL protease, and the inhibitory effect was dose-dependent when the amount of compound (II) was between 100 μg / ml and 400 μg / ml.

[0093] The ProFoldin (Hudson, MA, USA), a viral (Flavivirus) RNA-dependent RNA polymerase assay kit, was used to detect RNA molecules synthesized by the RNA-dependent RNA polymerase of positive-strand single-stranded RNA viruses. According to the manufacturer's instructions, the reaction mixture (30 μl) containing the RNA template, RNA polymerase, nucleoside triphosphates (NTPs), and MnCl2 was reacted at 37 °C for 60 minutes in the presence or absence of compound (II). After the reaction, the reaction mixture was mixed with a fluorescent dye, and the fluorescence intensity at 485 nm (excitation) / 535 nm (emission) was measured using a plate reader. As Figure 4B shown, compound (II) at a concentration of approximately 800 μg / ml or higher significantly inhibited RNA synthesis. In summary, compound (II) can effectively inhibit the activities of 3CL protease and RdRp, thereby reducing virus maturation and replication.

[0094] Example 5: Inhibition of SARS-CoV-2 pseudovirus infection of lung cells and colon cells using the coordination compound of formula (II)

[0095] In Calu-3 cells (ATCC HTB-55) rich in ACE2 and human colon epithelial cells CaCo-2 (available from ATCC, catalog number HTB-37), the inhibitory effect of the coordination compound on coronavirus infection was studied using a variety of SARS-CoV-2 spike protein-pseudolentiviruses (hereinafter referred to as SARS-CoV-2 pseudoviruses). Briefly, each of these SARS-CoV-2 pseudoviruses was co-transfected into human 293T cells (available from ATCC, catalog number CRL-3216) with a lentivirus vector, a plasmid expressing a recombinant spike protein of a SARS-CoV-2 variant (selected from the alpha, beta, gamma, or delta variant), and a plasmid expressing a fluorescent reporter protein (such as green fluorescent protein (GFP)) to generate fluorescent virus particles. Calu-3 cells and CaCo-2 cells were cultured in Minimum Essential Medium (Eagle’s Minimum Essential Medium) supplemented with 20% fetal bovine serum (Gibco TM , Thermo Fisher Scientific, Waltham, Massachusetts, USA) and 1% penicillin / streptomycin. Cell culture was carried out at 37 °C under a 5% carbon dioxide supply.

[0096] In the infection experiment, cells were first seeded on chamber slides (2 × 10 3 cells / slide) and cultured overnight. Then the cells were treated with different concentrations (0, 100, 200, 300, 400, 500, or 600 μg / ml) of compound (II). After 12 hours, different types of SARS-CoV-2 pseudoviruses were added to the slide and cultured for 12 hours. Then the cells were washed with PBS-T buffer, and the slide was sealed with a mounting medium. The change in green fluorescence intensity was observed using an Axio Observer 207A1 digital fluorescence microscope (Olympus, Tokyo, Japan).

[0097] In Figure 5A and Figure 5BAmong them, Calu-3 cells and Caco-2 cells infected with SARS-CoV-2 alpha (B.1.1.7) variant pseudovirus showed obvious green fluorescence, indicating that the SARS-CoV-2 pseudovirus has the ability to attach to and enter host cells. However, after pretreatment with at least 100 μg / ml of compound (II), a decrease in the green fluorescence intensity of these two types of cells was observed, indicating that this compound can inhibit the infection of SARS-CoV-2 alpha variant pseudovirus. Similarly, when cells were infected with pseudoviruses carrying the spike proteins of SARS-CoV-2 beta (N501Y.V2), gamma (P1), or delta (B.1.617.2) variants, the cells emitted bright green fluorescence, but after pretreatment of the cells with 100 to 600 μg / ml of compound (II), the fluorescence intensity decreased significantly ( Figure 6A-6C ). This result shows that compound (II) can effectively inhibit the entry of various SARS-CoV-2 pseudoviruses into lung cells and colon cells and cause infection to them; this result also indicates that this compound can be used to prevent the infection of coronavirus to various cells.

[0098] Example 6: Prevention and treatment of SARS-CoV-2 pseudovirus infection in mice using the coordination compound of formula (II)

[0099] To evaluate the antiviral effect of the coordination compound in vivo, SKH-1 mice were infected with SARS-CoV-2 spike protein-pseudolenti virus described in Example 5. Compound (II) (10 mg / kg / day or 100 mg / kg / day; dissolved in PBS) or PBS (control group) was administered orally to eight-week-old female SKH-1 mice for 6 days ( Figure 7A ). From day 4 to day 6, wild-type SARS-CoV-2 pseudovirus or SARS-CoV-2 variant pseudovirus (1.2×10 6 virus particles in 500 μl of physiological saline) was intranasally injected into the mice daily using a nebulizer (Aeroneb USB controller; Kent Scientific Corporation, Torrington, Connecticut, USA). On the seventh day, a live imaging system (IVIS; PerkinElmer, UK) was used to detect the fluorescence of various SARS-CoV-2 pseudoviruses to determine the viral load in the mice.

[0100] Three days after infecting the mice with wild-type SARS-CoV-2 pseudovirus, we observed the accumulation of the virus in the nasopharynx, chest, and abdomen of the mice, but administration of compound (II) at a dose of 10 mg / kg / day or 100 mg / kg / day significantly reduced the viral load in the nasopharynx / chest and abdomen (Figure 7B ). In addition, compared with the low-dose group (10 mg / kg / day), the high-dose group (100 mg / kg / day) had a better therapeutic effect. The high-dose group had a reduction of approximately 85% in viral load in the nasopharynx / thorax and approximately 90% in viral load in the abdomen compared with the control group. In contrast, the low-dose group had a reduction of approximately 80% in viral load in the nasopharynx / thorax and approximately 56% in viral load in the abdomen compared with the control group. Similar antiviral effects were also seen in mice infected with SARS-CoV-2 delta variant pseudovirus ( Figure 7C ), SARS-CoV-2 beta variant pseudovirus ( Figure 7D ), and SARS-CoV-2 omicron variant pseudovirus ( Figure 7E ). All results (summarized in Table 1 below) showed that compound (II) could reduce the systemic spread and accumulation of coronaviruses, which helps to prevent and treat multiple coronavirus infections in individuals.

[0101] Table 1

[0102]

[0103] In summary, the coordination compound of the present invention effectively inhibits coronavirus infection by preventing the entry of coronaviruses into host cells or inhibiting the maturation and replication of coronaviruses. Therefore, the coordination compound can be used as an antiviral agent that targets one or more viral infection mechanisms, including viral attachment, entry, maturation, and replication. In addition, the coordination compound has been demonstrated to reduce the invasion and accumulation of multiple coronaviruses in multiple body parts (including the thorax and abdomen) of an individual. Therefore, the coordination compound can be used to prepare a medicament for preventing or treating coronavirus infection in individuals in need, such as the elderly or young people susceptible to coronavirus infection, or individuals infected with coronaviruses.

Claims

1. A method for inhibiting coronavirus from infecting cells, comprising contacting the cells with an effective amount of a coordination compound represented by formula (I): [XZ2(CH3CO2)6(H2O)4(OH)2]NO3 Formula (I), wherein X is a trivalent ion of chromium (Cr) or molybdenum (Mo), and Z is a trivalent ion of iron (Fe), ruthenium (Ru), or osmium (Os).

2. The method according to claim 1, wherein the coordination compound is represented by formula (II): [CrFe2(CH3CO2)6(H2O)4(OH)2]NO3 Formula (II).

3. The method according to claim 1, wherein the coronavirus is Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), or a variant of SARS-CoV or SARS-CoV-2.

4. The method according to claim 1, wherein the coronavirus is SARS-CoV-2 or a variant thereof, and the variant is selected from the alpha, beta, gamma, delta, or omicron variant.

5. The method according to any one of claims 1 to 4, wherein the cells are from the respiratory system, digestive system, urinary system, or cardiovascular system of an individual.

6. The method according to any one of claims 1 to 5, wherein the coordination compound prevents the coronavirus from entering the cells and / or inhibits the maturation of the coronavirus and / or inhibits the replication of the coronavirus.

7. The method according to any one of claims 1 to 6, wherein the coordination compound blocks the binding between a spike protein of the coronavirus and angiotensin-converting enzyme 2 (ACE2) of the cells, and / or inhibits the expression of ACE2 or transmembrane serine protease 2 (TMPRSS2) in the cells.

8. The method according to any one of claims 1 to 6, wherein the coordination compound inhibits the activity of 3CL protease and / or the activity of RNA-dependent RNA polymerase.

9. A method for preventing or treating coronavirus infection, comprising administering to an individual in need an effective amount of a coordination compound represented by formula (I): [XZ2(CH3CO2)6(H2O)4(OH)2]NO3 Formula (I), wherein X is a trivalent ion of chromium (Cr) or molybdenum (Mo), and Z is a trivalent ion of iron (Fe), ruthenium (Ru), or osmium (Os).

10. The method according to claim 9, wherein the coordination compound is represented by formula (II): [CrFe2(CH3CO2)6(H2O)4(OH)2]NO3 Formula (II).

11. The method according to claim 9, wherein the coronavirus is Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), or a variant of SARS-CoV or SARS-CoV-2.

12. The method according to claim 9, wherein the coronavirus is SARS-CoV-2 or a variant thereof, and the variant is selected from the alpha, beta, gamma, delta, or omicron variant.

13. A pharmaceutical composition for preventing or treating coronavirus infection, comprising an effective amount of the coordination compound represented by formula (I): [XZ2(CH3CO2)6(H2O)4(OH)2]NO3 Formula (I) and a pharmaceutically acceptable carrier, wherein X is a trivalent ion of chromium (Cr) or molybdenum (Mo), and Z is a trivalent ion of iron (Fe), ruthenium (Ru), or osmium (Os).

14. The pharmaceutical composition according to claim 13, wherein the coordination compound is represented by formula (II): [CrFe2(CH3CO2)6(H2O)4(OH)2]NO3 Formula (II).

15. The pharmaceutical composition according to claim 13, further comprising an additional pharmaceutically active agent.

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