KR102145197B1 - Use of l-nucleosides for treating corona virus - Google Patents

Abstract

The present invention relates to a therapeutic agent for coronavirus, and more specifically, to use for the purpose of treating coronavirus infection by administering a drug or a pharmaceutical composition containing L-nucleoside of formula 1.

Description

Use of L-nucleosides to treat coronavirus {USE OF L-NUCLEOSIDES FOR TREATING CORONA VIRUS}

The present invention relates to a coronavirus therapeutic agent, and more particularly, to a use for the purpose of treating a coronavirus infection by administering a drug or a pharmaceutical composition comprising an L-nucleoside of Formula 1 below:

[Formula 1]

In Formula 1, R and R” are as defined herein.

Coronavirus disease 2019 (COVID-19) is a virus first discovered in Wuhan City, Hubei Province, China in 2019, belongs to the coronavirus (Coroviridae) group, and SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) ), is known as a virus similar to MERS coronavirus (MERS-CoV, Middle East respiratory syndrome coronavirus). Although no clear source of infection and path of infection have been identified, it has been reported that the possibility of infection through contact with bats in the Wuhan area is high and transmission through close contact between humans is possible.

COVID-19 coronavirus (CoV) is an RNA virus with a gene size of 27 to 32 kb, which can infect humans and various animals. Coronavirus disease 2019 (COVID-19) has an incubation period of about 2 weeks and mainly shows respiratory symptoms such as fever-associated cough, shortness of breath, shortness of breath, and sputum.In addition, headache, chills, runny nose, and muscle pain In addition, digestive symptoms such as loss of appetite, nausea, vomiting, abdominal pain, and diarrhea may also appear.

Until now, antiviral drugs for the treatment of coronavirus disease 2019 (COVID-19) have not been developed, and treatment for symptoms is mainly performed, and in severe cases, a respirator or artificial hemodialysis may be required.

The present invention is to provide a use of the L-nucleoside compound of Formula 1 or a pharmaceutically acceptable salt, ester or prodrug thereof for use in the treatment of coronavirus infection:

[Formula 1]

In Formula 1, R and R” are as defined herein.

The present invention provides a therapeutic agent for treating coronavirus infection, comprising a therapeutically effective amount of the L-nucleoside compound of Formula 1, or a pharmaceutically acceptable salt, ester or prodrug thereof:

[Formula 1]

In Formula 1,

R is a purine or pyrimidine base;

R″ is hydrogen, acyl, alkyl, monophosphate, diphosphate or triphosphate.

In addition, the present invention comprises a therapeutically effective amount of the L-nucleoside compound of Formula 1, or a pharmaceutically acceptable salt, ester or prodrug thereof, and additionally a pharmaceutically acceptable carrier, treating coronavirus infection It provides a pharmaceutical composition for.

In addition, the present invention provides a method for treating a coronavirus infection, comprising administering the L-nucleoside compound of Formula 1, or a pharmaceutically acceptable salt, ester, or prodrug thereof in a therapeutically effective amount to a subject to be treated. to provide.

In addition, the present invention provides a use of the L-nucleoside compound of Formula 1, or a pharmaceutically acceptable salt, ester, or prodrug thereof for treating coronavirus infection.

In one embodiment of the present invention, the coronavirus infection is human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 ( HCoV-NL63, New Heaven Coronavirus), human coronavirus HKU1, Middle East Respiratory Syndrome coronavirus (MERS-CoV) and coronavirus disease 2019 (COVID-19).

In another embodiment of the present invention, the term "alkyl" in Formula 1 is, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, s-butyl, t-butyl, isopentyl, amyl , t-pentyl, cyclopentyl and C ₁ -C ₁₀ alkyl groups including cyclohexyl, but are not limited thereto.

In another embodiment of the present invention, the term “acyl” in Formula 1 is, for example, acetyl, propionyl, butyryl, pentanoyl, 3-methylbutyryl, succinic acid hydrogen salt, 3-chlorobenzoate, benzoyl, acetyl , Pivaloyl, mesylate, propionyl, valeryl, caproic, caprylic, capric, lauric, myristic, palmitic, stearic and oleic.

In another embodiment of the present invention, unless otherwise defined herein, the term "aryl" in Formula 1 means phenyl.

In another embodiment of the present invention, the “base” of Formula 1 may be selected from the group consisting of 5-methyluracil (thymine), 5-iodouracil, cytosine, and 5-ethyluracil.

In another embodiment of the present invention, the L-nucleoside compound of Formula 1 is 2'-fluoro-5-methyl-β-L-arabinofuranosiluridine (INN: clevudine) of Formula 2 )to be:

[Formula 2]

In another embodiment of the present invention, the L-nucleoside compound of Formula 1 is N1-(2'-deoxy-2'-fluoro-β-L-arabinofuranosyl)-5-ethyluracil, N1 -(2'-deoxy-2'-fluoro-β-L-arabinofuranosyl)-5-iodocytosine) and N1-(2'-deoxy-2'-fluoro-β-L-ara Vinofuranosyl)-5-iodouracil can be selected from the group consisting of.

The L-nucleoside compounds of Formulas 1 and 2 can be prepared by using a known compound or a compound easily prepared therefrom if one has ordinary knowledge about the synthesis of the compound in the art. In particular, the preparation of the L-nucleoside compounds of Formulas 1 and 2 is described in detail in International Publication No. WO 1995/20595 (published date: August 3, 1995), and the document is incorporated herein by reference. do. However, the document is not intended to limit the scope of the invention, as only one exemplary method is presented, and the order of unit operations can be selectively changed as necessary.

In another embodiment of the present invention, the L-nucleoside compounds of Formulas 1 and 2 may include a prodrug form that is converted in vivo to exhibit the same effect.

In the pharmaceutical or pharmaceutical composition according to another embodiment of the present invention, the "therapeutically effective amount" for an individual subject means an amount sufficient to achieve the above-described pharmacological effect, that is, a therapeutic effect, The amount of the compound will vary depending on the condition of the subject and its severity, the mode of administration, and the age of the subject to be treated, but those skilled in the art can determine based on their own knowledge.

In the present invention, the "pharmaceutical composition" may include a pharmaceutically acceptable carrier in addition to the L-nucleoside compounds of Formulas 1 and 2 according to the present invention, and the "carrier" in the present invention is a cell Or it means a compound that facilitates the injection of the compound into the tissue, there is no particular limitation thereto.

In the present specification, “pharmaceutically acceptable” means a property that does not impair the biological activity and physical properties of the compound.

In the present invention, the compounds may be formulated in various pharmaceutical dosage forms depending on the purpose. When preparing the pharmaceutical composition according to the present invention, an active ingredient, specifically an L-nucleoside compound of Formula 1 or 2, or a pharmaceutically acceptable salt, ester or prodrug thereof, is prepared according to the formulation to be prepared. It is mixed with various pharmaceutically acceptable carriers that may be selected.

The term "treatment" as used herein is to eliminate all or part of a disease and/or its accompanying symptoms.

As used herein, the term “subject” refers to animals, preferably mammals (eg, primates) that are the subject of treatment, observation or experimentation.

The pharmaceutical or pharmaceutical composition according to the present invention can effectively treat coronavirus infection.

1 is a result of measuring the inhibitory effect on COVID-19 replication by a plaque reduction assay.
2 is a result of evaluation of antiviral efficacy based on real time qRT-PCR for COVID-19 replication.
3 is a real time qRT-PCR evaluation result for measuring the IC ₅₀ of clevudine for COVID-19 replication.
4 is a real time qRT-PCR evaluation result for measuring the IC ₅₀ of clevudine and remdesivir for COVID-19 replication in human lung epithelial cells (Calu-3 cells) (A: clevudine, B: remdesivir ).
5 is a result of measuring cell viability according to drug concentration at 24 and 48 hours (Rem: remdesivir, Hy-Ch: hydroxychloroquine, Cv: clevudine).

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the invention is not limited thereto.

Example 1: Plaque Reduction Assay

The virus was SARS-CoV-2 (COVID-19), the cell line was Vero, and the evaluation method was tested by plaque reduction assay, and the concentration of drugs (Lopinavir, Ritonavir, Chloroquine and Clevudine) Was set to 12.5 to 3.13 μM. In order to compare and analyze the number of plaques observed after treatment with the drug, a virus having an MOI of 0.1 was infected with the cells at 37°C for 1 hour, and then washed with phosphate-buffered saline (PBS). Thereafter, an agar medium containing the drug was overlaid on the cells and cultured at 37°C for 3 days. Three days after infection, the number of plaques that appeared by staining infected cells with crystal violet was counted and compared with the control group that was not treated with the drug.

The results are shown in FIG. 1, and when clevudine was used, the reduction rate was 26% at 12.5 μM and 36% at 3.13 μM, showing an excellent reduction rate.

Example 2: Real time qRT-PCR based antiviral efficacy evaluation

The virus was SARS-CoV-2 (COVID-19), the cell line was Vero cells (2 x 10 ⁴ /well), and the evaluation method was real time quantitative reverse transcription PCR, Real time qRT-PCR) (100 μl) was used, and the concentration of drug (Clevudine) was 100 μM to 0.39 μM (2-fold dilution, 2% FBS, DMEM). After culturing Vero cells in a 96 well plate (2 x 10 ⁴ cells/well), a virus of MOI 0.1 (2 x 10 ³ PFU/100 μl) was inoculated and infected in a 37°C incubator for 1 hour. . The infected cells were washed with PBS, and a cell culture solution containing drugs at different concentrations was added to the cells. Thereafter, cultures were obtained at 24 and 48 hours, and RNA levels in the SARS-CoV-2 virus particles were measured by real time qRT-PCR, and compared with the control group (viral infected group without drug treatment).

The results are shown in FIG. 2, and antiviral efficacy according to the concentration of the drug was observed at 48 hours when clevudine was used, and the reduction rate was 63% at 50 μM, 44% at 12.5 μM, and 38% at 3.13 μM. Showed.

Example 3: Real time qRT-PCR based IC ₅₀ Evaluation (Vero Cell)

The virus was SARS-CoV-2 (COVID-19), the cell line was Vero cells (2 x 10 ⁴ /well), and the evaluation method was tested with real time qRT-PCR (100 μl). The concentrations of (Lopinavir, Chloroquine and Clevudine) were 0.05 μM, 0.1 μM, 0.2 μM, 0.39 μM, 0.79 μM, 1.57 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM. After culturing Vero cells in a 96-well plate (2 x 10 ⁴ cells/well), a virus of MOI 0.1 (2 x 10 ³ PFU/100 μl) was inoculated and infected in a 37°C incubator for 1 hour. The infected cells were washed with PBS, and a cell culture solution containing drugs at different concentrations was added to the cells. Thereafter, cultures were obtained at 24 and 48 hours, and RNA levels in the SARS-CoV-2 virus particles were measured by real time qRT-PCR, and compared with the control group (viral infected group without drug treatment).

The results are shown in Table 1 and Fig. 3, and the IC ₅₀ of 24 hours and 48 hours when clevudine was used was 0.53 μM and 32.78 μM, respectively.

[Table 1]

Example 4: Real time qRT-PCR based IC ₅₀ Assessment (human lung epithelial cell)

The virus was SARS-CoV-2 (COVID-19), the cell line was human lung epithelial cells, Calu-3 cells, and the evaluation method was tested with real time qRT-PCR (100 μl), and the drug ( Remdesivir and Clevudine) concentrations were 0.05 μM, 0.1 μM, 0.2 μM, 0.39 μM, 0.79 μM, 1.57 μM, 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM and 100 μM. For antiviral efficacy, the IC ₅₀ was derived by measuring real-time RT-PCR by extracting RNA from cell culture solution 48 hours after virus infection.

The results are shown in Tables 2 and 4, and the IC ₅₀ of 48 hours when clevudine was used was 0.2 μM.

[Table 2]

Example 5: Cell Cytotoxicity Assay

In order to analyze the toxicity of clevudine to vero cells, which are monkey kidney cells, it was diluted from 800 μM to 0.0000975 μM by 2 steps. PBS was used as a negative control, and remdesivir, hydroxychloroquine, and DMSO at the same concentration were used as controls. Cell viability and CC ₅₀ (50% cytotoxic concentration) values according to the drug concentration were measured at 24 hours and 48 hours after each drug was treated with cells by concentration, and are shown in FIGS. 5 and 3, respectively. As can be seen from FIG. 5 and Table 3, clevudine exhibited drug-induced cytotoxicity at concentrations exceeding 400 μM and 200 μM, respectively, at 24 and 48 hours, and thus lower cells than remdesivir and hydroxychloroquine used as controls. Showed toxicity.

[Table 3]

As can be seen from the results of Examples 1 to 4, it was confirmed that clevudine inhibits the replication of COVID-19, thereby inhibiting the infection caused by COVID-19. In addition, human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus NL63 (HCoV-NL63, New Heaven Coronavirus), human coronavirus HKU1, and Middle East Respiratory Syndrome coronavirus (MERS-CoV) are expected to inhibit infection.

In addition, from the results of Example 5, it was found that the toxicity of clevudine was significantly lower than that of other drugs having an antiviral effect against coronavirus.

Claims (12)

Coronavirus disease 19 (coronavirus disease 2019, comprising a therapeutically effective amount of 2'-fluoro-5-methyl-β-L-arabinofuranosiluridine or a pharmaceutically acceptable salt thereof of Formula 2 below) COVID-19) treatment drugs:
[Formula 2]

. delete delete delete delete delete Corona comprising a therapeutically effective amount of 2'-fluoro-5-methyl-β-L-arabinofuranosiluridine or a pharmaceutically acceptable salt thereof of Formula 2, together with a pharmaceutically acceptable carrier Pharmaceutical composition for the treatment of coronavirus disease 2019 (COVID-19):
[Formula 2]

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