TW202434203A - Methods and compositions for inhibiting coronavirus infection - Google Patents

Abstract

Provided is a method for inhibiting an infection of a cell by a coronavirus, including contacting the cell with an effective amount of a coordination compound represented by formula (I). Also provided is a method for preventing or treating a coronavirus infection, including administering to a subject in need an effective amount of the coordination compound of formula (I). Also provided is a pharmaceutical composition for use in preventing or treating a coronavirus infection, including the coordination compound of formula (I) and a pharmaceutically acceptable carrier.

Description

Methods and compositions for inhibiting coronavirus infection

This application claims priority to U.S. Provisional Application No. 63/476,380, filed on December 21, 2022, the contents of which are incorporated herein by reference in their entirety.

The present invention relates to a coordination compound for use in fighting viral infection. Specifically, the present invention relates to a method for using the coordination compound to prevent or treat coronavirus infection. The present invention also relates to a pharmaceutical composition for preventing or treating coronavirus infection, which comprises the coordination compound.

Since the end of 2019, the 2019 novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly around the world, leading to a severe COVID-19 pandemic. According to statistics from the World Health Organization (WHO), as of February 2022, 220 countries have reported 403 million COVID-19 patients and more than 5.7 million deaths. SARS-CoV-2 is an enveloped positive-strand RNA virus that is about 80% similar to SARS-CoV, which caused the 2002 SARS pandemic, in terms of genome, but is more contagious.

The genomes of coronaviruses (CoVs) usually encode structural proteins (including envelope protein, nucleocapsid protein, membrane protein, and spike protein) as well as nonstructural 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) and transmembrane serine protease 2 (TMPRSS2) on the surface of host cells as receptors. The viral spike protein binds to the host's ACE2 receptor and is cleaved by TMPRSS2, resulting in membrane fusion of the virus and host cell. Therefore, inhibiting the expression or function of ACE2 or TMPRSS2 is a possible target to prevent 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 proteases and 3-chymotrypsin-like (3CL) proteases to form 16 non-structural proteins (acting in 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.

Dozens of vaccines have been approved as base vaccines or boosters to fight COVID-19 until 2022. However, the effectiveness of vaccines is affected by SARS-CoV-2 variants (such as alpha, beta, gamma, delta, and omicron variants). Therefore, there is an urgent need to develop universal and effective drugs to prevent this dangerous infectious disease. In addition to vaccines as preventive therapies, several small molecule compounds are 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 prevent viruses from developing drug resistance.

The present disclosure relates to a coordination compound that can be used as an antiviral agent to inhibit coronavirus infection of cells. The present disclosure further relates to the use of the coordination compound in preventing or treating coronavirus infection in an individual in need. The coordination compound is represented by formula (I): [XZ ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO ₃ . X and Z in formula (I) refer to different metal ions, 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).

One object of the present disclosure is to provide a method for inhibiting coronavirus infection of cells, comprising contacting the cells with an effective amount of a 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 zirconium. The coordination compound preferably comprises trivalent chromium ions and trivalent iron ions, and is represented by formula (II).

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

In some embodiments, the ligand compound inhibits infection of cells from the respiratory, digestive, urinary, or cardiovascular systems of a subject.

In some embodiments, the coordination compound inhibits infection by preventing the coronavirus from entering the cell. For example, the coordination compound can prevent the coronavirus from entering the cell by blocking the binding between a spike protein of the coronavirus and the 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 of the coronavirus and/or the replication of the coronavirus. For example, the coordination compound can inhibit the maturation of the coronavirus by inhibiting the activity of the 3CL protease, and further inhibit the replication of the coronavirus by inhibiting the activity of the RNA-dependent RNA polymerase (RdRp).

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

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 variant of SARS-CoV-2 can be selected from alpha, beta, gamma, delta, or omicron variants.

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

The present disclosure also relates to a pharmaceutical composition for preventing or treating the aforementioned coronavirus infection. The pharmaceutical composition comprises an effective amount of a 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 zirconium.

In some embodiments, the coordination compound contained in the pharmaceutical composition comprises trivalent chromium ions and trivalent iron ions and is represented by formula (II).

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

The coordination compound disclosed herein can effectively inhibit the entry, maturation and replication of coronavirus into cells. The coordination compound has also been shown to reduce the accumulation of coronavirus in individuals infected with coronavirus. Therefore, the compound can be used to prevent coronavirus infection, or to treat infected persons and reduce the severity of the disease.

The following embodiments and examples are used to further illustrate the present invention. It should be understood that the following embodiments are not intended to limit the scope of the present invention, and that those skilled in the art may make modifications within the scope of the appended claims.

Unless otherwise defined, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Definition

Unless the context clearly indicates 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.

Data are usually presented as mean ± standard deviation. The values provided herein are approximate, and experimental values may vary within a range of 20%, preferably within a range of 10%, and more preferably within a range of 5%. Therefore, the terms "about" and "approximately" refer to within a range of 20%, preferably within a range of 10%, and more preferably within a range of 5% of a given value or range.

As used herein, the term "coordination compound" refers to a metal complex defined by formula (I) comprising three metal ion centers, each surrounded by six ligands.

Coordination compounds

The coordination compound disclosed herein is shown in formula (I): [XZ ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO _3Formula (I) 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 the coordination compound can contain Cr or Mo (both are Group 6 metals), and Fe, Ru or Os (all are Group 8 metals), the coordination compound can be represented by formula (II), (III), (IV), (V), (VI), or (VII) based on various combinations of central metal ions: _Formula (II): [CrFe ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO 3Formula (III): [CrRu ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO _3Formula (IV): [CrOs ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO _3Formula (V): [MoFe ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO ₃ Formula (VI): [MoRu ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO ₃ Formula (VII): [MoOs ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO ₃

In some embodiments, the coordination compound comprises trivalent chromium ions and trivalent iron ions and is represented by formula (II). The compound of formula (II) may have structure (a) as shown below or other structures representing different stereoisomers. That is, the compound of formula (II) means any compound of formula (II) in which the arrangement of the ligands around the metal ions may be different. Similarly, the compound of formula (III), (IV), (V), (VI), or (VII) may include various stereoisomers. Structure (a)

Use of coordination compounds to inhibit coronavirus infection

The present disclosure provides a method for inhibiting coronavirus infection of cells, comprising contacting the cells with an effective amount of a coordination compound represented by formula (I): [XZ ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO ₃ , wherein X is a trivalent ion of chromium or molybdenum, and Z is a trivalent ion of iron, ruthenium, or zirconium. The term "inhibiting infection" as used herein means that after using the coordination compound of formula (I), the number of cells infected with coronavirus can be reduced, or the number of coronaviruses produced by infected cells can be reduced to avoid further infection. In other words, the coordination compound can be used as an antiviral agent based on its preventive effect (e.g., preventing viruses from attaching to and/or entering host cells), or based on its therapeutic effect (e.g., inhibiting the replication of viral RNA in host cells).

Coronavirus infections that can be inhibited by contact of cells with the coordination compounds of formula (I) include any infection caused by a coronavirus having one or more of the following characteristics: (1) it has a spike protein that is structurally similar to the spike protein of SARS-CoV-2, and/or (2) it uses ACE2 or a membrane protein that is structurally similar to ACE2 as a receptor for entering host cells, and/or (3) it has a protease that is structurally similar to the 3CL protease of SARS-CoV-2, and/or (4) it has an RNA-dependent RNA polymerase (RdRp) that is structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2. The so-called "a spike protein that is structurally similar to the spike protein of SARS-CoV-2" refers to a spike protein that has 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 that is structurally similar to ACE2" refers to a membrane protein that has 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 that is structurally similar to the 3CL protease of SARS-CoV-2” refers to a protease that has 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 "RdRp that is structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2" refers to an RdRp that has 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. The term "variant" as used herein refers to a subtype of a coronavirus that has one or more mutations in its genome and is therefore genetically different from the main strain. For example, a variant of SARS-CoV-2 may be an alpha, beta, gamma, delta, or omicron variant published by WHO. Each SARS-CoV-2 variant includes a group of closely related viruses that have a common ancestry.

The cell (also referred to as host cell) in which the coordination compound of formula (I) can exert its antiviral effect can be any cell that can be infected by the coronavirus 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 a body, or a cell separated from a body (such as a human), or a derivative cell of the separated cell. In addition, the cell can have a variety of 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 bronchus. In other embodiments, the cell is an epithelial cell of pancreatic, small intestine, large intestine, kidney, heart, or vascular tissue. In some preferred embodiments, the cell is a cell expressing ACE2.

An effective amount of a coordination compound administered to cells to inhibit coronavirus infection refers to an amount sufficient to reduce coronavirus infection of host cells or an amount sufficient to reduce coronavirus production by host cells. As those skilled in the art will appreciate, the effective amount will vary depending on a variety of 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.

The step of contacting the cells with the coordination compound can be performed 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.

In some embodiments, the ligands inhibit infection by preventing coronaviruses from entering cells. For example, the ligands disclosed herein can prevent coronaviruses (including but not limited to SARS-CoV-2) from entering cells by one of the following actions: (1) blocking the binding between the coronavirus spike protein and ACE2, which is a protein expressed on the surface of host cells and is a receptor for coronaviruses when the virus attaches; (2) inhibiting the expression of ACE2 in cells; (3) inhibiting the expression of transmembrane serine protease 2 (TMPRSS2), which is a protease expressed on the cell surface and is essential for the cleavage of the spike protein and the fusion of the coronavirus membrane with the cell membrane.

In addition, in some embodiments, the coordination compounds inhibit 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 polyproteins translated from viral RNA to form non-structural proteins and is therefore extremely important for viral maturation.

Use of coordination compounds to prevent or treat coronavirus infection

The coordination compound exhibits antiviral activity in an individual. Therefore, the present disclosure further provides a method for preventing or treating coronavirus infection, comprising administering to an individual in need thereof an effective amount of a 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 zirconium.

As used herein, the term "subject" refers to a mammal. The subject may be human or non-human, including but not limited to primates, rodents, dogs, cats, cows, goats, sheep, horses, rabbits, pigs, etc. Therefore, the present invention contemplates antiviral methods and compositions for veterinary and medical use.

Coronavirus infections in individuals that can be prevented or treated by the ligand compounds of formula (I) include any infection caused by a coronavirus having one or more of the following characteristics: (1) it has a spike protein that is structurally similar to the spike protein of SARS-CoV-2, and/or (2) it utilizes ACE2 or a membrane protein that is structurally similar to ACE2 as a receptor for entering host cells, and/or (3) it has a protease that is structurally similar to the 3CL protease of SARS-CoV-2, and/or (4) it has an RNA-dependent RNA polymerase (RdRp) that is structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2. The so-called "a spike protein that is structurally similar to the spike protein of SARS-CoV-2" refers to a spike protein that has 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 that is structurally similar to ACE2" refers to a membrane protein that has 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 that is structurally similar to the 3CL protease of SARS-CoV-2” refers to a protease that has 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 "RdRp that is structurally similar to the RNA-dependent RNA polymerase of SARS-CoV-2" refers to an RdRp that has 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. The term "variant" as used herein refers to a subtype of a coronavirus that has one or more mutations in its genome and is therefore genetically different from the main strain. For example, a variant of SARS-CoV-2 may be an alpha, beta, gamma, delta, or omicron variant published by WHO. Each SARS-CoV-2 variant includes a group of closely related viruses that have a common ancestry.

The effective amount of the coordination compound administered to an individual in need can be a preventive effective amount or a therapeutic effective amount. A "preventive effective amount" refers to an amount sufficient to prevent or delay the occurrence of coronavirus infection or the appearance of its symptoms or signs in an individual. A "therapeutically effective amount" refers to an amount sufficient to stop or delay the development of coronavirus infection or at least one symptom or sign of an individual, or to alleviate the coronavirus infection state or at least one symptom or sign of an individual. As understood by those skilled in the art in this technical field, the effective amount will vary with a variety of factors, such as the type of coronavirus infection, the age, weight, physical condition and responsiveness of the individual being treated, the specific coordination compound administered, the route of administration, the use of formulations, and the co-use with other pharmaceutically active agents.

The coordination compound of formula (I) can be administered to a subject by any suitable route, preferably in the form of a pharmaceutical composition suitable for such route. In some embodiments, the coordination compound is administered orally, intranasally, topically, transmucosally, intravenously, or enterally.

In some embodiments, the coordination compound of formula (I) is administered to an individual before or at the beginning of an infection with a coronavirus. In some embodiments, the coordination compound is administered to an individual who has been infected with a coronavirus. In some embodiments, the coordination compound is administered at least once, twice, three times or more per day. In some embodiments, the coordination compound is administered every day, every other day, several times per week, weekly or at a lower frequency to maintain effective dose levels and patient compliance. In some embodiments, the administration of the coordination compound will continue for two days, three days, four days, five days, six days, seven days or longer. The frequency and duration of treatment may vary depending on the response of an individual to treatment.

Pharmaceutical compositions

The present disclosure further provides a pharmaceutical composition for preventing or treating coronavirus infection, comprising an effective amount of a 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 zirconium. In some preferred embodiments, the pharmaceutical composition comprises the coordination compound represented by formula (II) as an active ingredient.

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

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

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 a 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 immunostimulators. Examples of anti-infective agents include antifungal agents, antibacterial agents, and antiparasitic agents.

Embodiment

Example 1: Preparation of coordination compound

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

The molecular weight of compound (II) was determined to be about 686.11 by mass spectrometry. Elemental analysis showed that compound (II) contained 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) were verified by ¹ H nuclear magnetic resonance (NMR) spectroscopy, ¹³ C-NMR spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. FIG1A shows the ¹ H-NMR spectrum of compound (II), in which the signals at about 3.3 ± 0.2 and 1.9 ± 0.2 ppm correspond to H ₂ O and hydrogen of the acetate group, respectively. In addition, the signals at about 21.2 ± 0.5, 39.7 ± 0.5 and 171.9 ± 0.5 ppm in the ¹³ C-NMR spectrum indicate the presence of an acetate group ( FIG. 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 ( FIG. 1C ). All data support the structure of the compound (II) disclosed herein. Compound (II) was used in Examples 2-6 for efficacy studies.

The coordination compounds of formula (III) to formula (VII) can be prepared according to the above method but using appropriate metal salts instead of chromium (III) nitrate and/or iron (III) nitrate.

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

To evaluate whether the ligands can inhibit the interaction between coronavirus spike protein and host cell receptors, an enzyme-linked immunosorbent assay (ELISA) was performed using the COVID-19 Spike-ACE2 Binding Assay Kit from RayBiotech (Peachtree Corners, Georgia, USA) to measure the binding of recombinant human ACE2 to SARS-CoV-2 spike protein in the presence or absence of compound (II). The receptor binding domain (RBD) of the SARS-CoV-2 spike protein was pre-coated on a clear 96-well flat-bottom plate according to the manufacturer's instructions. Compound (II) at different concentrations (0, 100, 200, 300, 400, or 500 μg/ml) was mixed with ACE2 protein and then reacted in a 96-well plate 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 the primary antibody (goat anti-human ACE2 antibody) was added to the 96-well plate. After that, the washing step was repeated, and the anti-goat secondary antibody coupled with horseradish peroxidase was added to the 96-well plate and reacted at 22°C and 200 rpm for 60 minutes. After that, 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 allowed to react at 22°C and 200 rpm in the dark for 60 minutes. The reaction was stopped with 50 µl of stop solution, and the absorbance of each well at 450 nm (OD450) was measured with a plate reader.

As shown in Figure 2A, compared with the control group (without compound (II)), about 100 µg/ml of compound (II) can block the binding of SARS-CoV-2 spike protein to ACE2 by more than 50%, and the blocking is dose-dependent. This result shows that compound (II) has an inhibitory effect on the interaction between SARS-CoV-2 spike protein and host cell ACE2.

In addition, non-tumorous 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 ^™ , Thermo Fisher Scientific, Waltham, MA, USA) at 37°C in an incubator supplied with 5% carbon dioxide. After BEAS-2B cells were treated with different doses (0, 200, 400, 600 or 800 µg/ml) of compound (II) for 24 hours, the cell viability was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. As shown in FIG2B , compound (II) did not significantly inhibit the survival of BEAS-2B cells, indicating that the ligand disclosed herein does not inhibit the growth of human lung cells.

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

Both ACE2 and TMPRSS2 are transmembrane proteins required for SARS-CoV-2 to enter cells. To evaluate the effects of the ligands 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) under the catalog number HTB-55) were used as a cell model in the expression assay. Calu-3 cells were cultured in Eagle's Minimum Essential Medium supplemented with 20% fetal bovine serum (Gibco ^TM , Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin in an incubator at 37°C with 5% CO2. Cells were treated with different concentrations (0, 100, 200, 300, or 600 µg/ml) of compound (II). 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 frozen and lysed 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. The polyvinylidene fluoride (PVDF) membrane for Western blot was blocked with 5% skimmed milk and then reacted with primary antibodies against ACE2 (sc-390851; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TMPRSS2 (ab92323; Abcam, Cambridge, UK), and β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for more than 12 hours. Then, the PVDF membrane was washed and reacted with secondary antibodies. The transfer results were detected using a chemiluminescent reagent (WesternBright® ECL) and observed under an iBright 1500 chemiluminescent image analyzer.

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

Example 4: Inhibitory effect of the coordination compound of formula (II) on 3CL protease and RNA-dependent RNA polymerase activity

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

In the SARS-CoV-2 3CL protease activity assay, 3CL protease cleavage of a 3CL protease fluorescent substrate was measured using the SensoLyte ^® 520 SARS-CoV-2 3CL Protease Activity Assay Kit (SARS-CoV-2 3CL Protease Activity Assay Kit, AS-72262; Kaneka Eurogentec SA, Serran, Belgium). SARS-CoV-2 3CL protease solution (40 µl/well) was mixed with compound (II) (10 µl/well) or control buffer in PBS in a 96-well plate according to the manufacturer's instructions. Afterwards, 50 µl of 3CL protease substrate solution was added to each well, the reaction mixture was reacted at 37°C for 30 minutes, and the fluorescence intensity at 490 nm (excitation)/520 nm (radiation) was measured using a plate reader to determine the protease activity. As shown in Figure 4A, compound (II) at a concentration of 100 µg/ml or more significantly inhibited the activity of 3CL protease, and when the amount of compound (II) was between 100 µg/ml and 400 µg/ml, the inhibitory effect was dose-dependent.

The RNA molecules synthesized by RNA-dependent RNA polymerase of positive-stranded single-stranded RNA viruses were detected using the Virus (Flavivirus) RNA-dependent RNA polymerase Assay Kit (ProFoldin; Hudson, MA, USA). According to the manufacturer's instructions, the reaction mixture (30 µl) containing RNA template, RNA polymerase, nucleoside triphosphates (NTPs), and MnCl ₂ was reacted at 37°C for 60 minutes with or without the addition 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 shown in Figure 4B, compound (II) at a concentration of about 800 µg/ml or higher could significantly inhibit RNA synthesis. In summary, compound (II) can effectively inhibit the activity of 3CL protease and RdRp, thereby reducing viral maturation and replication.

Example 5: Use of the coordination compound of formula (II) to inhibit the infection of SARS-CoV-2 pseudovirus to lung cells and colon cells

The inhibitory effect of the coordination compounds on coronavirus infection was studied using multiple SARS-CoV-2 spike protein-pseudolentiviruses (hereinafter referred to as SARS-CoV-2 pseudoviruses) in ACE2-enriched Calu-3 cells (ATCC HTB-55) and human colon epithelial cells CaCo-2 (available from ATCC, No. HTB-37). Briefly, each SARS-CoV-2 pseudovirus was co-transfected into human 293T cells (available from ATCC, CRL-3216) with a lentivirus vector, an expression plasmid of the recombinant spike protein of a SARS-CoV-2 variant (selected from alpha, beta, gamma or delta variants), and an expression plasmid of a fluorescent reporter protein (e.g., green fluorescent protein (GFP)) to generate fluorescent virus particles. Calu-3 cells and CaCo-2 cells were cultured in Eagle's Minimum Essential Medium supplemented with 20% fetal bovine serum (Gibco ^™ , Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin. Cells were cultured at 37°C and 5% CO2.

In the infection assay, cells were first inoculated into cell culture chamber slides (2 × 10 ³ cells/slide) and cultured overnight. Cells were then 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 slides and cultured for 12 hours. Cells were then washed with PBS-T buffer and the slides were sealed with mounting medium. Changes in green fluorescence intensity were observed using an Axio Observer 207 A1 digital fluorescence microscope (Olympus, Tokyo, Japan).

In Figures 5A and 5B, 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 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), it was observed that the green fluorescence intensity of both cells decreased, indicating that the compound can inhibit the infection of SARS-CoV-2 alpha variant pseudovirus. Similarly, when cells were infected with pseudoviruses carrying SARS-CoV-2 beta (N501Y.V2), gamma (P1), or delta (B.1.617.2) variant spike proteins, the cells emitted bright green fluorescence, but after pre-treatment of the cells with 100 to 600 µg/ml of compound (II), the fluorescence intensity decreased significantly (Figures 6A-6C). This result shows that compound (II) can effectively inhibit the entry of multiple SARS-CoV-2 pseudoviruses into lung cells and colon cells and cause infection to them; this result also indicates that the compound can be used to prevent coronavirus infection of multiple cells.

Example 6: Use of the coordination compound of formula (II) to prevent and treat SARS-CoV-2 pseudovirus infection in mice

To evaluate the antiviral effect of the coordination compound in vivo, SKH-1 mice were infected with the SARS-CoV-2 spike protein-pseudolentivirus described in Example 5. Eight-week-old female SKH-1 mice were orally administered with compound (II) (10 mg/kg/day or 100 mg/kg/day; dissolved in PBS) or PBS (control group) for 6 days (Figure 7A). On days 4 to 6, mice were intranasally injected with wild-type SARS-CoV-2 pseudovirus or SARS-CoV-2 variant pseudovirus (1.2×10 ⁶ virus particles in 500 µl saline) every day using a nebulizer (Aeroneb USB controller; Kent Scientific Corporation Torrington, CT, USA). On the seventh day, the viral load in mice was determined by detecting the fluorescence of multiple SARS-CoV-2 pseudoviruses using an in vivo imaging system (IVIS; PerkinElmer, UK).

Three days after infection of mice with wild-type SARS-CoV-2 pseudovirus, we observed virus accumulation in the nasopharynx, chest, and abdomen of 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, the high-dose group (100 mg/kg/day) had a better therapeutic effect than the low-dose group (10 mg/kg/day). The high-dose group reduced the viral load in the nasopharynx/chest by about 85% and the viral load in the abdomen by about 90% compared with the control group. In contrast, the low-dose group reduced the viral load in the nasopharynx/chest by about 80% and the viral load in the abdomen by about 56% compared with the control group. Similar antiviral effects were also observed 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) show that compound (II) can reduce the spread and accumulation of coronaviruses throughout the body, which is helpful for preventing and treating various coronavirus infections in individuals.

Table 1 Inhibitory effect on viral particles Compared with the control group, the amount of virus accumulation decreased (%) Nasopharynx / Chest abdomen Low dose High dose Low dose High dose Wild-type SARS-CoV-2 80 ± 3% 85 ± 2% 56 ± 5% 90 ± 8% Delta variant 50 ± 5% 86 ± 4% 30 ± 7% 70 ± 6% Beta variant 60 ± 6% 66 ± 2% 25 ± 5% 80 ± 7% Omicron variant 75 ± 3% 80 ± 2% 70 ± 5% 80 ± 4% Note: The low dose refers to 10 mg/kg body weight/day for mice, and the equivalent dose for adults is 61.4 mg/70 kg/day. The high dose refers to 100 mg/kg body weight/day for mice, and the equivalent dose for adults is 614.8 mg/70 kg/day.

In summary, the coordination compound disclosed herein effectively inhibits coronavirus infection by preventing coronavirus from entering host cells or inhibiting the maturation and replication of coronavirus. 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 shown to reduce the invasion and accumulation of multiple coronaviruses in multiple body parts (including the chest and abdomen) of an individual. Therefore, the coordination compound can be used to prepare a drug for preventing or treating coronavirus infection in an individual in need, such as an elderly or young person who is susceptible to coronavirus infection, or a coronavirus infected person.

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Those skilled in the art will be able to clearly understand the present invention through the following detailed description of the preferred embodiment in conjunction with the attached drawings, in which:

FIG1A is a ¹ H-NMR spectrum of the coordination compound of formula (II);

FIG1B is the ¹³ C-NMR spectrum of the coordination compound of formula (II);

FIG1C is a Fourier transform infrared (FT-IR) spectrum of the coordination compound of formula (II);

FIG2A shows the effect of the coordination compound of formula (II) on the interaction between recombinant human ACE2 and SARS-CoV-2 spike protein; ^** and ^*** respectively indicate p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG2B shows that the coordination compound of formula (II) has no obvious cytotoxicity to BEAS-2B cells;

FIG3A shows the effect of the coordination compound of formula (II) on the expression of ACE2 protein in Calu-3 cells; ^** and ^*** respectively indicate p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG3B shows the effect of the coordination compound of formula (II) on the expression of TMPRSS2 protein in Calu-3 cells; ^** and ^*** respectively indicate p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG4A shows the effect of the coordination compound of formula (II) on the 3CL protease activity of SARS-CoV-2;

FIG4B shows the effect of the coordination compound of formula (II) on the activity of viral RNA-dependent RNA polymerase; ^* indicates p < 0.05 compared with the control group without compound treatment;

FIG5A shows the effect of the coordination compound of formula (II) on SARS-CoV-2 alpha variant pseudovirus-infected Calu-3 cells; ^** and ^*** respectively indicate p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG5B shows the effect of the coordination compound of formula (II) on SARS-CoV-2 alpha variant pseudovirus-infected Caco-2 cells; ^* , ^** and ^*** respectively indicate p < 0.05, p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG6A shows the effect of the coordination compound of formula (II) on SARS-CoV-2 beta variant pseudovirus-infected Calu-3 cells; ^** and ^*** respectively indicate p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG6B shows the effect of the coordination compound of formula (II) on SARS-CoV-2 gamma variant pseudovirus-infected Calu-3 cells; ^** and ^*** respectively indicate p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG6C shows the effect of the coordination compound of formula (II) on SARS-CoV-2 delta variant pseudovirus-infected Caco-2 cells; ^** and ^*** respectively indicate p < 0.01 and p < 0.001 compared with the control group without compound treatment;

FIG7A is a schematic diagram of in vivo infection studies using SKH-1 mice;

FIG7B shows the effect of the coordination compound of formula (II) on the in vivo viral accumulation of SKH-1 mice infected with wild-type SARS-CoV-2 pseudovirus;

FIG7C shows the effect of the coordination compound of formula (II) on the in vivo viral accumulation of SKH-1 mice infected with SARS-CoV-2 delta variant pseudovirus;

FIG7D shows the effect of the coordination compound of formula (II) on the in vivo viral accumulation of SKH-1 mice infected with SARS-CoV-2 beta variant pseudovirus; and

Figure 7E shows the effect of the coordination compound of formula (II) on the in vivo viral accumulation of SKH-1 mice infected with SARS-CoV-2 omicron variant pseudovirus.

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Claims (15)

A method for inhibiting coronavirus infection of cells comprises contacting the cells with an effective amount of a coordination compound represented by formula (I): [XZ ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO ₃ 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 niobium (Os). The method as claimed in claim 1, wherein the coordination compound is represented by formula (II): [CrFe ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO _3Formula (II). The method of claim 1, wherein the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), or a variant of SARS-CoV or SARS-CoV-2. A method as described in claim 1, wherein the coronavirus is SARS-CoV-2 or a variant thereof, and the variant is selected from alpha, beta, gamma, delta, or omicron variants. A method as described in any one of claims 1 to 4, wherein the cell is from the respiratory system, digestive system, urinary system, or cardiovascular system of a subject. A method as described in any one of claims 1 to 5, wherein the coordination compound prevents the coronavirus from entering the cell and/or inhibits the maturation of the coronavirus and/or inhibits the replication of the coronavirus. A method as described in any one of claims 1 to 6, wherein the ligand blocks the binding between a spike protein of the coronavirus and angiotensin-converting enzyme 2 (ACE2) of the cell, and/or inhibits the expression of ACE2 or transmembrane serine protease 2 (TMPRSS2) in the cell. A method as described in 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. A method for preventing or treating coronavirus infection comprises administering to a subject in need thereof an effective amount of a coordination compound represented by formula (I): [XZ ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO _3Formula (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 niobium (Os). The method as described in claim 9, wherein the coordination compound is represented by formula (II): [CrFe ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO _3Formula (II). The method of claim 9, wherein the coronavirus is severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), or a variant of SARS-CoV or SARS-CoV-2. A method as described in claim 9, wherein the coronavirus is SARS-CoV-2 or a variant thereof, and the variant is selected from alpha, beta, gamma, delta, or omicron variants. A pharmaceutical composition for preventing or treating coronavirus infection comprises an effective amount of a coordination compound represented by formula (I): [XZ ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO ₃ 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 niobium (Os). The pharmaceutical composition of claim 13, wherein the coordination compound is represented by formula (II): [CrFe ₂ (CH ₃ CO ₂ ) ₆ (H ₂ O) ₄ (OH) ₂ ]NO _3Formula (II). The pharmaceutical composition of claim 13 further comprises an additional pharmaceutically active agent.