COVID-19 treatment

Abstract

To develop a therapeutic method for COVID-19 caused by SARS-CoV-2 or a variant thereof, provided is a prophylactic and / or therapeutic agent for COVID-19 caused by SARS-CoV-2 or a variant thereof, said agent comprising a specific compound.

Description

[Technical Field]

[0001] This disclosure relates to COVID-19 treatment agents, etc. All references cited herein are incorporated herein by reference. [Background technology]

[0002] COVID-19, caused by infection with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), is currently raging worldwide, and providing treatment for it is an urgent issue.

[0003] Furthermore, viruses are generally known to mutate in order to maintain their survival more efficiently, and therefore, we also need to consider treatment methods for COVID-19 caused by SARS-CoV-2 or its variants. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2006-76989 [Patent Document 2] International Publication No. 2012 / 074120 [Patent Document 3] International Publication No. 2017 / 204166 [Patent Document 4] Japanese Patent Publication No. 10-147564 [Non-patent literature]

[0005] [Non-Patent Document 1] Jun Inoue et al., J.Med.Chem. 2003, 46, 868-871. [Overview of the project] [Problems that the invention aims to solve]

[0006] The inventors aimed mainly at developing a treatment method for COVID-19.

Means for Solving the Problems

[0007] The inventors found that a specific compound might be effective in the treatment of COVID-19 and further improved it.

[0008] This disclosure includes, for example, the subject matter described in the following items.

[0009] Item 1. Formula (I):

[0010]

Chemical formula

[0011]

Chemical formula

[0012] Item 2. The prophylactic and / or therapeutic agent according to Item 1, which is for an infected person with SARS-CoV-2 or its variant.

[0013] Item 3. The prophylactic and / or therapeutic agent according to Item 2, which is for an asymptomatic or symptomatic person infected with SARS-CoV-2 or its variant.

[0014] Item 4. The preventive and / or therapeutic agent according to any one of items 1 to 3, wherein the mutant of SARS-CoV-2 is at least one selected from the group consisting of alpha mutant, beta mutant, gamma mutant, delta mutant, lambda mutant, mu mutant, epsilon mutant, eta mutant, iota mutant, kappa mutant, zeta mutant, and theta mutant.

[0015] Item 5. Formula (I):

[0016]

Chemical formula

[0017]

Chemical formula

[0018] Item 6. The degeneration inhibitor according to item 5, wherein the mutant of SARS-CoV-2 is at least one selected from the group consisting of alpha mutant, beta mutant, gamma mutant, delta mutant, lambda mutant, mu mutant, epsilon mutant, eta mutant, iota mutant, kappa mutant, zeta mutant, and theta mutant.

[0019] Item 7. Formula (I):

[0020]

Chemical formula

[0021]

Chemical formula

[0022] Section 8. The antiviral agent according to item 7, wherein the SARS-CoV-2 variant is at least one selected from the group consisting of α-mutant, β-mutant, γ-mutant, δ-mutant, λ-mutant, μ-mutant, ε-mutant, η-mutant, ι-mutant, κ-mutant, ζ-mutant, and θ-mutant.

[0023] Section 1a. Equation (I):

[0024] [ka] Compounds represented by formula (II):

[0025] [ka] Compounds represented by A method for preventing and / or treating COVID-19 caused by SARS-CoV-2 or its variants, comprising administering a required amount of at least one compound selected from the group consisting of the following to a target.

[0026] Section 2a. The method according to item 1a, wherein the subject required is a person infected with SARS-CoV-2 or a variant thereof.

[0027] Section 3a. The method according to item 1a, wherein the required subjects are asymptomatic or symptomatic individuals infected with SARS-CoV-2 or its variants.

[0028] Section 4a. The method according to any one of items 1a to 3a, wherein the SARS-CoV-2 variant is selected from the group consisting of α mutant, β mutant, γ mutant, δ mutant, λ mutant, μ mutant, ε mutant, η mutant, ι mutant, κ mutant, ζ mutant, and θ mutant.

[0029] Section 5a. Equation (I):

[0030] [ka] Compounds represented by formula (II):

[0031] [ka] Compounds represented by This includes applying at least one compound selected from the group consisting of the following to cells infected with SARS-CoV-2 or its variants: A method for suppressing the degeneration of cells infected with SARS-CoV-2 or its variants.

[0032] Section 6a. The method according to item 5a, wherein the SARS-CoV-2 variant described above is at least one selected from the group consisting of α mutant, β mutant, γ mutant, δ mutant, λ mutant, μ mutant, ε mutant, η mutant, ι mutant, κ mutant, ζ mutant, and θ mutant.

[0033] Section 7a. Equation (I):

[0034] [ka] Compounds represented by formula (II):

[0035] [ka] Compounds represented by The method involves administering at least one compound selected from the group consisting of the following to a person infected with SARS-CoV-2 or a variant thereof. A method for suppressing the degeneration of SARS-CoV-2 or its variant-infected cells in individuals infected with SARS-CoV-2 or its variants.

[0036] Section 8a. The method according to item 7a, wherein the SARS-CoV-2 variant described above is at least one selected from the group consisting of α mutant, β mutant, γ mutant, δ mutant, λ mutant, μ mutant, ε mutant, η mutant, ι mutant, κ mutant, ζ mutant, and θ mutant.

[0037] Section 1b. For use in the prevention and / or treatment of COVID-19, Equation (I):

[0038] [ka] Compounds represented by formula (II):

[0039] [ka] Compounds represented by At least one compound selected from the group consisting of the following.

[0040] Section 2b. The compound described in item 1b for use in a person infected with SARS-CoV-2 or a variant thereof.

[0041] Section 3b. The compound described in item 2b for use in asymptomatic or symptomatic individuals infected with SARS-CoV-2 or its variants.

[0042] Section 4b. For use in suppressing the degeneration of cells infected with SARS-CoV-2 or its variants, Equation (I):

[0043] [ka] Compounds represented by formula (II):

[0044] [ka] Compounds represented by At least one compound selected from the group consisting of the following.

[0045] Section 5b. For use in suppressing the degeneration of SARS-CoV-2 or its variant-infected cells in individuals infected with SARS-CoV-2 or its variants, Equation (I):

[0046] [ka] Compounds represented by formula (II):

[0047] [ka] Compounds represented by At least one compound selected from the group consisting of the following.

[0048] Section 1c. For the manufacture of compositions (preferably pharmaceutical compositions) for the prevention and / or treatment of COVID-19 caused by SARS-CoV-2 or its variants, Equation (I):

[0049] [ka] Compounds represented by formula (II):

[0050] [ka] Compounds represented by The use of at least one compound selected from the group consisting of the following.

[0051] Section 2c. Formula (I): For the manufacture of a composition (preferably a pharmaceutical composition) for the treatment of patients infected with SARS-CoV-2 or its variants:

[0052] [ka] Compounds represented by formula (II):

[0053] [ka] Compounds represented by The use of at least one compound selected from the group consisting of the following.

[0054] Section 3c. Use as described in item 2c, where a person infected with SARS-CoV-2 or its variants is asymptomatic or symptomatic.

[0055] Section 4c. For the manufacture of a composition (preferably a pharmaceutical composition) for suppressing the degeneration of cells infected with SARS-CoV-2 or its variants, Equation (I):

[0056] [ka] Compounds represented by formula (II):

[0057] [ka] Compounds represented by The use of at least one compound selected from the group consisting of the following.

[0058] Section 5c. For the production of a composition (preferably a pharmaceutical composition) for suppressing the degeneration of SARS-CoV-2 or its variant-infected cells in individuals infected with SARS-CoV-2 or its variants, Equation (I):

[0059] [ka] Compounds represented by formula (II):

[0060] [ka] Compounds represented by The use of at least one compound selected from the group consisting of the following. [Effects of the Invention]

[0061] Novel preventive and / or therapeutic agents for COVID-19 caused by SARS-CoV-2 or its variants are provided. [Brief explanation of the drawing]

[0062] [Figure 1] This figure shows the effects of the compound of the present invention on the various mutants described in the examples. [Modes for carrying out the invention]

[0063] The embodiments included in this disclosure will be described in further detail below. This disclosure preferably includes, but is not limited to, agents for the prevention and / or treatment of COVID-19 caused by SARS-CoV-2 or its variants, and this disclosure includes everything disclosed herein and recognizable to those skilled in the art.

[0064] The preventive and / or therapeutic agents for COVID-19 caused by SARS-CoV-2 or its variants, as contained herein, have the effect of suppressing physiological phenomena caused by SARS-CoV-2 or its variants and contain certain compounds that can act as active ingredients for COVID-19 caused by SARS-CoV-2 or its variants. Such preventive and / or therapeutic agents for COVID-19 caused by SARS-CoV-2 or its variants may be referred to as therapeutic agents of this disclosure.

[0065] Two specific compounds are listed below. The first compound is given by formula (I):

[0066] [ka] This is a compound represented by [formula]. This compound is sometimes referred to as "Compound (I)" or "SNJ-1945".

[0067] Compound (I) is a known compound, as described, for example, in the above-mentioned Patent Documents 1 to 3. Furthermore, compound (I) can be produced according to the methods described in these documents.

[0068] The second compound is given by formula (II):

[0069] [ka] This is a compound represented by [formula]. This compound is sometimes referred to as "Compound (II)" or "SJA-6017".

[0070] Compound (II) is a known compound, described, for example, in Patent Document 4 and Non-Patent Document 1. Furthermore, compound (II) can be produced according to the methods described in these documents.

[0071] Furthermore, unless otherwise specified, compound (I) or compound (II) also includes diastereomixtures of compound (I) or compound (II).

[0072] The therapeutic agent of this disclosure may consist solely of compound (I) and / or compound (II), or it may be a composition (particularly a pharmaceutical composition) of compound (I) and / or compound (II) with other components. For example, compound (I) and / or compound (II), which are the active ingredients, may be compounded with pharmaceutically acceptable bases, carriers, and additives (e.g., excipients, binders, disintegrants, lubricants, solvents, sweeteners, colorants, flavoring agents, odoring agents, surfactants, humectants, preservatives, pH adjusters, viscosity modifiers, etc.) as needed. Such bases, carriers, and additives can be appropriately selected from components known in the art. Furthermore, the formulation form is not particularly limited, and the active ingredient and other components can be mixed by conventional methods to prepare formulations such as tablets, coated tablets, powders, granules, fine granules, capsules, pills, liquids (e.g., injections, drips), suspensions, emulsions, jellies, chewables, soft tablets, etc.

[0073] The amounts of compound (I) and / or (II) in the therapeutic agent of this disclosure are not particularly limited as long as a therapeutic effect against COVID-19 is exerted, and can be set as appropriate. For example, 0.0005 to 100% by mass, more preferably 0.005 to 90% by mass, and even more preferably 0.05 to 80% by mass.

[0074] COVID-19 is an infectious disease characterized by respiratory symptoms caused by a newly identified coronavirus, formally named SARS-CoV-2. It was first reported in Wuhan, Hubei Province, China, in December 2019, and has since spread widely throughout China and the world. COVID-19 is thought to spread mainly from person to person through airborne droplets dispersed by the coughs and sneezes of infected individuals. Most infected individuals show no symptoms or only mild symptoms, but some develop severe symptoms and die. Symptoms include fever, cough, and shortness of breath. Symptoms usually appear approximately 1 to 14 days after infection.

[0075] SARS-CoV-2 is a coronavirus with a single-stranded positive-sense RNA genome, classified in the β-coronavirus genus of the Coronaviridae family, and possessing the nucleotide sequence (SEQ ID NO: 1) shown at NCBI accession number NC_045512. Among coronaviruses that infect humans, four types that routinely infect humans (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1) and two types of severe pneumonia viruses transmitted from animals (SARS-CoV, MERS-CoV) are known. SARS-CoV-2 is thought to have been transmitted from bats to humans.

[0076] Coronaviruses have a double membrane on the outermost part of the particle, called an "envelope," which contains lipids as a component. Coronaviruses cannot reproduce on their own, but they can attach to and enter host cells and multiply. The S protein expressed on the surface of SARS-CoV-2 binds to the ACE2 receptor on the surface of host cells, enabling infection of host cells. The use of the ACE2 receptor for infection of humans is similar to SARS-CoV, but differs from MERS-CoV, which uses the DPP-4 receptor. Furthermore, SARS-CoV-2 has structural proteins that differ from the other coronaviruses mentioned above. Preferred SARS-CoV-2 targeted by the therapeutic agent of this disclosure includes, for example, those having the following homology. The homology between this SARS-CoV-2 and SARS-CoV is approximately 76% for the S protein, approximately 90% for the N protein, approximately 90% for the M protein, and approximately 94% for the E protein. Furthermore, the homology between SARS-CoV-2 and MERS-CoV is approximately 35% for the S protein, 48% for the N protein, 42% for the M protein, and 36% for the E protein.

[0077] Numerous mutant strains of the S protein expressed on the surface of SARS-CoV-2 have been reported (Abdul Aleem, et al. Emerging Variants of SARS-CoV-2 And Novel Therapeutics Against Coronavirus (COVID-19). StatPearls [Internet]: 2021), and the reduced effectiveness of SARS-CoV-2 vaccines is considered a challenge in the treatment of COVID-19 (avindra K Gupta. Nat. Rev. Immunol. 21:340-341: 2021 and Matthew McCallum, et al. Science 373:648-654: 2021).

[0078] The therapeutic agents disclosed herein are effective against and / or preventive against all types of SARS-CoV-2 variants, regardless of their pathogenicity, and are not particularly limited. For example, variants having a nucleotide sequence with 70% or more homology to the nucleotide sequence shown in SEQ ID NO: 1 can be cited. More preferably, variants having a nucleotide sequence with 75% or more homology, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, and even more preferably 95% or more homology to the nucleotide sequence shown in SEQ ID NO: 1 can be cited.

[0079] Specifically, the mutants mentioned above include α mutants, β mutants, γ mutants, δ mutants, λ mutants, μ mutants, ε mutants, η mutants, ι mutants, κ mutants, ζ mutants, θ mutants, and others. These mutants are named by the WHO.

[0080] Among the above SARS-CoV-2 mutants, α mutants, β mutants, γ mutants, ε mutants, δ mutants, μ mutants, or κ mutants are preferred, and α mutants, β mutants, γ mutants, or ε mutants are even more preferred.

[0081] The α-mutant is a mutant strain that is believed to have been first discovered in the UK, and is designated as lineage B.1.1.7. It is mainly known for having mutations such as N501Y.

[0082] The β-mutant is a mutant strain that was first discovered in South Africa, and is classified as lineage B.1.351. It is mainly known for having mutations such as N501Y, E484K, and K417N.

[0083] The γ mutant strain is a mutant strain that was first discovered in Brazil, and is classified as lineage P.1. It is mainly known for having mutations such as N501Y, E484K, and K417T.

[0084] The δ mutant strain is a mutant strain that was first discovered in India, and is classified as lineage B.1.617.2. It is mainly known for having mutations such as L452R, T478K, D614G, and P681R.

[0085] The λ mutant strain is a mutant strain that is believed to have been first discovered in Peru, and is classified as lineage C.37. It is mainly known for having mutations such as L452Q, F490S, D614G, and T859N.

[0086] The μ mutant is a mutant strain that was first discovered in Colombia, and is classified as lineage B.1.621.1. It is mainly known for having mutations such as E484K, N501Y, D614G, and P681H.

[0087] The ε-mutant strain is a mutant strain that is believed to have been first discovered in the United States, and is known as lineage B.1.427 / B.1.429, mainly possessing mutations such as S13L, W152C, L452R, and D614G.

[0088] The η mutant strain is a mutant strain that was first discovered in the UK and Nigeria, and is classified as lineage B.1.525. It is mainly known for having mutations such as E484K, D614G, and F888L.

[0089] The ι mutant is a mutant strain that is believed to have been first discovered in the United States, and is classified as lineage B.1.526. It is mainly known for having mutations such as E484K, D614G, and A701L.

[0090] The κ mutant is a mutant strain that was first discovered in India, and is classified as lineage B.1.617.1. It is mainly known for having mutations such as L452R, E484Q, D614G, and P681R.

[0091] The ζ mutant strain is a mutant strain that was first discovered in Brazil, and is classified as lineage P2 or B.1.1.28.2. It is mainly known for having mutations such as E484K, D614G, and V1176F.

[0092] The θ mutant strain is a mutant strain that was first discovered in the Philippines and is classified as lineage P.3 or B.1.1.28.3. It is mainly known for having mutations such as E484K, N501Y, D614G, P681H, E1092K, H1101Y, and V1176F.

[0093] The subjects to whom the therapeutic agents of this disclosure are administered are not particularly limited, but are preferably, for example, infected with SARS-CoV-2 or its variants, and are particularly preferred to be subjects who show a positive reaction to SARS-CoV-2 or its variants. Such a positive reaction is, for example, a positive reaction obtained by a method capable of detecting SARS-CoV-2 or its variants (preferably specifically), and such methods include, for example, PCR tests and antigen / antibody tests (e.g., ELISA) capable of detecting SARS-CoV-2 or its variants.

[0094] Furthermore, the severity of COVID-19 is not particularly limited, and the treatment can be used for asymptomatic, mild, and moderate to severe cases. The therapeutic agent disclosed herein is useful not only for suppressing symptoms in symptomatic individuals (mild, moderate to severe cases) but also for suppressing (reducing) SARS-CoV-2 or its variants present in the bodies of asymptomatic and symptomatic individuals. The symptoms of symptomatic individuals are not particularly limited and include, for example, fever, dry cough, fatigue, sputum, shortness of breath, sore throat, headache, diarrhea, pneumonia, and difficulty breathing. Moreover, it can also be used prophylactically in subjects who have not yet been infected with SARS-CoV-2 or its variants.

[0095] Furthermore, the therapeutic agents disclosed herein are applicable not only to humans but also to non-human mammals (particularly non-human mammals infected with SARS-CoV-2 or its variants). Such non-human mammals are especially preferred if they are kept as pets or livestock. Examples include dogs, cats, monkeys, cattle, horses, sheep, goats, pigs, rabbits, mice, rats, camels, and llamas. In the case of mammals, as in humans, the therapeutic agents disclosed herein can also be used prophylactically.

[0096] The timing of administration of the therapeutic agent disclosed herein is not particularly limited, and the timing of administration can be appropriately selected considering, for example, the form of formulation, the age of the recipient, the severity of the symptoms of the recipient, etc. The form of administration is also not particularly limited, but oral administration, intravascular administration (especially intravenous administration), subcutaneous administration, etc., are preferred.

[0097] The dosage of the therapeutic agent disclosed herein may be appropriately selected depending on the age of the recipient, the severity of the symptoms, and other conditions. For example, it is preferable to use an amount of compound (I) in the therapeutic agent disclosed which is preferably in the range of 10 to 3000 mg per day for adults, more preferably in the range of 20 to 2000 mg. Also, for example, it is preferable to use an amount of compound (II) in the therapeutic agent disclosed which is preferably in the range of 10 to 3000 mg per day for adults, more preferably in the range of 20 to 2000 mg. It can be administered once a day or divided into multiple doses (preferably 2 to 3 times). In the case of mammals, the dosage can be appropriately set with reference to the case for humans. The therapeutic agent disclosed herein can preferably be used as a pharmaceutical composition.

[0098] Furthermore, as shown in the examples described below, the therapeutic agent of this disclosure has the effect of inhibiting the entry of SARS-CoV-2 or its variants into cells and suppressing the degeneration of infected cells. Therefore, it is considered useful for preventing infection by administering it before infection with SARS-CoV-2 or its variants. For this reason, the therapeutic agent of this disclosure can also be preferably used as a preventive agent. In other words, this disclosure preferably encompasses COVID-19 preventive and / or therapeutic agents. Cellular degeneration refers to physiological and / or morphological changes that occur after a cell is infected with a virus. Also, since the degeneration of SARS-CoV-2 infected cells is thought to be due to the proliferation of SARS-CoV-2, the therapeutic agent of this disclosure can be considered to have an inhibitory effect on the proliferation of SARS-CoV-2.

[0099] Furthermore, in this specification, the term "comprising" includes "consisting essentially of" and "consisting of." This disclosure also encompasses all any combination of the constituent elements described herein.

[0100] Furthermore, the various characteristics (properties, structure, function, etc.) described in each embodiment of this disclosure above may be combined in any way to identify the subject matter covered by this disclosure. In other words, this disclosure covers all subject matter consisting of any combination of the combinable characteristics described herein.

[0101] The contents of the above-mentioned documents may be incorporated herein by reference. [Examples]

[0102] The embodiments of this disclosure will be described in more detail below with examples, but the embodiments of this disclosure are not limited to the examples below.

[0103] Preparation of the compound (sample for investigation) As shown in Table 1 below, compound stock solutions (80 μL of dimethyl sulfoxide (DMSO) solution containing each compound) were placed in plates, and 40 μL of each stock solution was serially diluted twofold to prepare sample solutions. 30 nL of each sample solution was dispensed into a 384-well assay plate using the ECHO555 acoustic liquid handling system (Labcyte Inc.). In addition to SNJ-1945 and SJA-6017, chloroquine, hydroxychloroquine, E64d, and remdesivir were used as compounds. DMSO was dispensed into the control wells so that the DMSO concentration in all wells was 0.1%.

[0104] Furthermore, Chloroquine and Hydroxychloroquine are compounds that are thought to potentially be effective in treating COVID-19, Remdesivir is a drug approved in the United States in 2020 as a treatment for COVID-19, and E64d is a protease inhibitor that exerts antiviral activity by inhibiting the function of host proteases. Also, as mentioned above, SNJ-1945 is compound (I) and SJA-6017 is compound (II).

[0105] [Table 1]

[0106] Measurement of the antiviral effect of compounds (CPE assay) Vero E6 cells (a cell line derived from kidney epithelial cells of African green monkeys) were cultured in Minimum Essential Media (MEM) medium supplemented with 10% fetal bovine serum (FBS), and on the day of the assay, the medium was changed to MEM medium supplemented with 1% penicillin-streptomycin and 2% FBS. SARS-CoV-2 virus (USA_WA1 / 2020, MOI~0.002) was added to the Vero E6 cells so that the cell viability 72 hours after inoculation was 5%. Assay plates were prepared with 30 nL of the test compound and 5 μL of medium, and 25 μL of virus-inoculated Vero E6 cells (4000 cells / well) were added and incubated for 72 hours under conditions of 37°C / 5% CO2 and 90% humidity. As controls, only Vero E6 cells before virus inoculation (100% CPE suppression group) or only virus-inoculated Vero E6 cells (0% CPE suppression group) were set up. After 72 hours, Cell-Titer-Glo (Promega) was added to each well and incubated at room temperature for 10 minutes, after which luminescence was measured using a CLARIOstar plate reader (BMG Labtech).

[0107] Cytopathic effect (CPE) refers to the cytopathic effect of a virus on host cells, and the Cell-Titer-Glo (Promega) assay kit is used to measure intracellular ATP as a surrogate measure of host cell viability. When CPE occurs due to viral infection, ATP depletion is measured and is proportional to the viral load. Therefore, the detected amount of ATP is directly proportional to the number of living host cells in culture, allowing for the quantification of the viral cytopathic effect.

[0108] Evaluation of the cytotoxicity of compounds The cytotoxicity of the compounds was evaluated in parallel with the measurement of antiviral efficacy. Assay plates were prepared with 30 nL of the test compound and 5 μL of culture medium, 25 μL of Vero E6 cells (4000 cells / well) were added, and incubated for 72 hours at 37°C, 5% CO2, and 90% humidity. Vero E6 cells alone (100% viability) and cells with 100 μM hyamine added (0% viability) were set as high-signal and low-signal controls. After 72 hours, Cell-Titer-Glo (Promega) was added to each well and incubated at room temperature for 10 minutes, after which luminescence was measured using a PHERAstar plate reader (BMG Labtech).

[0109] Data Analysis For all assays, raw data from the plate reader was imported into Activity Base, a plate assay data management system.

[0110] In measuring the antiviral effect, the signal value was converted to the CPE suppression rate using the following formula. CPE reduction rate (%) = 100 × (Each sample value - Mean value of the 0% CPE reduction group) / (Mean value of the 100% CPE reduction group - Mean value of the 0% CPE reduction group)

[0111] The survival rate in the cytotoxicity assessment was calculated using the following formula. Survival rate (%) = 100 × (Each sample value - Mean value of low-signal control) / (Mean value of high-signal control - Mean value of low-signal control) 50% inhibitory concentration (IC 50 ) and 50% cytotoxic concentration (CC 50 The values ​​of were calculated from a 4-parameter logistic fit of the data using the Activity Base Xlfit module.

[0112] result The results are shown in Table 2 below. ICs of Remdesivir, Chloroquine, and Hydroxychloroquine, which have already been shown to be effective in suppressing SARS-CoV-2 virus replication. 50 These values ​​were 10.56, 2.06, and 6.49 μM, respectively. Also, the IC of E64d 50 The IC was 8.23 ​​μM. In contrast, the IC of SNJ-1945 and SJA-6017 50 These concentrations were 1.52 μM and 0.21 μM, respectively.

[0113] The results above are summarized in the table below.

[0114] [Table 2]

[0115] Furthermore, no cytotoxicity was observed in any of the compounds at the concentrations used in the tests.

[0116] Based on these results, it was found that SNJ-1945 and SJA-6017 have excellent inhibitory effects on the degeneration of SARS-CoV-2 virus-infected cells.

[0117] Effects on various mutants <1 Manufacturing of various coronavirus variants> Kits sold by Funakoshi Co., Ltd. System Biosciences, LLC's "pPACK-SPIKE SARS-CoV-2 "S" Pseudotype Lentivector Packaging Mix" (Wild Type: WT), "pPACK-SPIKE N501Y, SARS-CoV-2 "S" Pseudotype-N501Y Mutant-Lentivector Packaging Mix" (N501Y mutant), and "pPACK-SPIKE B.1.429, SARS-CoV-2 "S" Pseudotype-B.1.429 (CAL.20C) Variant-Lentivector Packaging Mix" Using "Mix" (B.1.429 mutant strain: ε strain), we expressed the spike proteins of coronavirus variants including WT, N501Y mutant strain, and S13I / W152C / L452R / D614G (B.1.429) mutant strain (ε strain), and created lentiviruses containing GFP expression plasmids (hereinafter sometimes referred to as "pseudotype viruses") to confirm the effects of the compounds of the present invention. These pseudotype viruses were concentrated and used for SARS-CoV-2 infection evaluation.

[0118] <2. Evaluation of the inhibitory effect on infection (invasion) of various coronavirus variants> The inhibitory effect of compounds on viral entry against infection by pseudotyped viral particles containing GFP-expressing plasmids was evaluated using ACE2-expressing HEK293T cells (Human ACE2 293T cells, Takara). SNJ-1945 (compound 1) was used as the test compound. A DMSO solution of each test compound was added to a dish seeded with ACE2-expressing HEK293T cells at the highest concentration (100 μM) that did not exhibit cytotoxicity, and incubated for 30 minutes. Subsequently, the entry inhibitory activity was examined by adding the pseudotyped viruses (WT, N501Y, and ε mutant strains). Infection by these pseudotyped viral particles was observed by confocal laser microscopy to measure GFP fluorescence, and the inhibitory activity was calculated.

[0119] Confocal laser microscopy (OLYMPUS: FV1000) observation was performed by fixing infected cells with 4% PFA / PBS, mounting them, and observing them at the excitation and fluorescence wavelengths for EGFP. In this experiment, a strong fluorescence intensity for GFP indicates that the pseudotyped virus infects ACE2-expressing HEK293T cells. The results are shown in Figure 1.

[0120] As shown in Figure 1, the results clearly demonstrate that compound 1 inhibits cell entry in all strains: WT, N501Y mutant, and ε mutant. Since the N501Y mutation is found in α, β, and γ mutants, compound 1 is expected to inhibit cell entry by these strains. Furthermore, since the ε mutant has mutations such as S13L, W152C, L452R, and D614G, compound 1 is also expected to inhibit cell entry by mutant strains such as the ε strain that exhibit mutations in any of these amino acid sequences.

[0121] Pharmacokinetic (PK) measurements of SNJ-1945 and SJA-6017 Test animals Eight-week-old male SD rats were used to study SNJ-1945. Each animal was housed in a room set to a temperature of 21.6°C to 22.3°C, humidity of 47.7% to 60.3%, 12 hours of lighting per day, and ventilation of 6 to 20 times per hour. The rats were fasted from the evening before administration, and feeding was resumed 8 hours after administration, once blood sampling was completed. Water was provided as an ad libitum.

[0122] For the study of SJA-6017, male SD rats weighing 160-210g were used. The animals were housed in a room set to a temperature of 23±2℃, humidity of 55±10%, 12-hour lighting, and ventilation of 15 / hr or more. The rats were fasted from the night before administration and feeding was resumed immediately after administration. Water was provided as an ad libitum.

[0123] Test method 1) Group composition For the study of SNJ-1945, four groups were set up at 1, 3, 10, and 30 mg / kg (n = 3 at each time point). For the study of SJA-6017, two groups were set up at 100 mg / kg (n = 3 at each time point) and 500 mg / kg (n = 8 at each time point).

[0124] 2) Preparation of the test specimens SNJ-1945 was prepared by dissolving methylcellulose in water for injection to a concentration of 1 w / v%. The test substance was suspended in this solution and adjusted to concentrations of 0.25 (for 1 mg / kg), 0.75 (for 3 mg / kg), 2.5 (for 10 mg / kg), and 7.5 mg / mL (for 30 mg / kg). The dosing solution was dispensed into brown glass bottles.

[0125] SJA-6017 was prepared by dissolving sodium carboxymethylcellulose in distilled water to a concentration of 0.5% (w / v). The test substance was suspended in this solution and adjusted to concentrations of 10% (for 500 g / kg) and 2% (for 100 mg / kg).

[0126] 3) Administration SNJ-1945 was administered by forced single oral administration using a gastric tube at doses of 1, 3, 10, and 30 mg / 4 mL / kg. SJA-6017 was administered by forced single oral administration using a gastric tube at a dose of 5 mL / kg.

[0127] 4) Method for collecting plasma For the SNJ-1945 administration group, blood was collected from the subclavian vein at 30 minutes, 1, 2, 4, and 8 hours after administration. The obtained blood was immediately centrifuged at 10,000 × g for 3 minutes to collect plasma.

[0128] For the SJA-6017 administration group, blood was collected from the abdominal aorta under ether anesthesia at 30 minutes, 1, 2, 4, and 8 hours after administration. The obtained blood was immediately centrifuged at 5,000 rpm for 10 minutes to collect plasma.

[0129] 5) Measurement of plasma concentration and calculation of pharmacokinetic parameters <SNJ-1945 administration group> Plasma (20 μL) obtained from the SNJ-1945 administration group was placed in a polypropylene tube, methanol (20 μL) was added, and internal standard solution (SNJ-1945-d7; 100 μL) was added and mixed for approximately 5 minutes. Then, it was centrifuged for 5 minutes (10,000 × g), and 100 μL of the supernatant was transferred to another polypropylene tube. 1 mmol / L EDTA·2K (100 μL) was added and mixed for approximately 10 seconds. This solution was placed in an autosampler and measured within 72 hours.

[0130] (LC / MS / MS conditions) The measuring instrument used was the Shimadzu 20AD LC System 2 (Prominence UFLC). XR A system (Shimadzu Corporation) and a mass spectrometer (API 5000; AB SCIEX) were used. High-performance liquid chromatography was performed under the following conditions: column: CAPCELL CORE AQ, 2.7 μm, 2.1 × 50 mm (Shiseido), column temperature: 60°C, mobile phase A: 5 mmol / L ammonium bicarbonate / 1 mmol / LEDTA·2K (500:1, v / v), mobile phase B: methanol, flow rate: 0.8 mL / min. The mobile phase gradient conditions were set as shown in Table 3 below.

[0131] [Table 3]

[0132] The mass spectrometer was set to the following conditions: Ionization mode: Turboionspray, Measurement mode: Multiple reaction monitoring, Detection mode: Negative, Collision gas: N2, 6 (Number of valves opened), Curtain gas: N2, 15 psi, Ion source gas 1: Air, 80 psi, Ion source gas 2: Air, 80 psi, Ionspray voltage: -4500V, Temperature: 500℃, Channel electron multiplier: 1800V or 2000V. The monitoring ions and dwell time were set as shown in Table 4 below.

[0133] [Table 4]

[0134] Using the plasma concentration measurement results, the pharmacokinetic analysis was performed using the non-compartmental analysis function of the pharmacokinetic analysis software Phoenix WinNonlin Ver.6.3 (Pharsight Corporation as part of Certara), and each pharmacokinetic parameter was calculated using the method shown in Table 5 below.

[0135] [Table 5]

[0136] [Experimental Results] Pharmacokinetic parameters (C) at 1 mg / kg administration max AUC 0-8 The levels were 186 ng / mL and 167.5 ng·h / mL respectively when administered at 3 mg / kg, 391 ng / mL and 379.8 ng·h / mL respectively when administered at 10 mg / kg, 553 ng / mL and 691.1 ng·h / mL respectively when administered at 30 mg / kg, and 821 ng / mL and 1165.9 ng·h / mL respectively when administered at 30 mg / kg.

[0137] <SJA-6017 Administration Group> Also, plasma (200 μL) obtained from the SJA-6017 administration group was taken into a brown spitz roll, 0.01 M hydrochloric acid (0.5 mL) was added, and further diethyl ether (3 mL) was added and shaken for 10 minutes. Then, it was centrifuged for 10 minutes (3,000 rpm) to separate the aqueous layer and the diethyl ether phase. Only the lower aqueous layer was frozen with dry ice-acetone, and the diethyl ether layer was separated into another brown spitz roll. Thereafter, it was dried under reduced pressure, and acetonitrile (100 μL) and a 0.01 M hydrochloric acid solution of 5 mM phenylhydrazine hydrochloride (200 μL) were added and shaken to dissolve. This solution was transferred to an HPLC vial and reacted at 40 °C for 1 hour using a water bath. 150 μL of this sample solution was analyzed by HPLC.

[0138] (HPLC Conditions) Detector: Ultraviolet absorption photometer (measurement wavelength: 272 nm) Column: Capcell-pak SG-120 (250 mm) Column temperature: 40 °C Mobile phase: 0.02 M phosphate buffer (pH 7.0) / acetonitrile mixture (45:55) Flow rate: 1.0 mL / min

[0139] C was calculated from the average plasma concentration at each time point. max and AUC 0-8 were calculated.

[0140] [Experimental Results] The pharmacokinetic parameters (C max , AUC 0-8 ) at a dose of 100 mg / kg were 284 ng / mL and 678.9 ng·h / mL respectively, and at a dose of 500 mg / kg they were 348 ng / mL and 1837.3 ng·h / mL respectively.

[0141] The above results are summarized in Table 6 below.

[0142] [Table 6]

[0143] The blood concentration of SNJ-1945 at a low dose (30 mg / kg) was higher than that of SJA-6017 at a high dose (100 mg / kg). Assuming that blood concentration increases proportionally with the dose, the difference in blood concentration was approximately 5 to 10 times higher for SNJ-1945.

[0144] Based on these findings, SJA-6017 exhibits stronger inhibitory activity against the degeneration of SARS-CoV-2 infected cells compared to SNJ-1945 (IC 50 (SNJ-1945: 1.52 μM, SJA-6017: 0.21 μM) Since SNJ-1945 has a blood concentration approximately 5 to 10 times higher, it is predicted that it will have higher penetration into target tissues and therefore exhibit stronger SARS-CoV-2 inhibitory activity in tissues than SJA-6017.

Claims

1. Equation (I): 【Chemistry 1】 A prophylactic and / or therapeutic agent for COVID-19 containing the compound represented by [formula].

2. The preventive and / or therapeutic agent according to claim 1, for use in persons infected with SARS-CoV-2 or its variants.

3. The preventive and / or therapeutic agent according to claim 2, for use by asymptomatic or symptomatic individuals infected with SARS-CoV-2 or its variants.

4. The preventive and / or therapeutic agent according to claim 2 or 3, wherein the SARS-CoV-2 mutant is at least one selected from the group consisting of α mutant, β mutant, γ mutant, δ mutant, λ mutant, μ mutant, ε mutant, η mutant, ι mutant, κ mutant, ζ mutant, and θ mutant.

Images