Methods and compositions for the treatment of SARS-CoV-2

Abstract

This invention provides a useful method for treating patients with pathogenic infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). [Solution] The method involves administering a therapeutically effective amount of the following compounds to the target. TIFF2026094257000085.tif48150 The aim is to reduce the likelihood of a target becoming infected with a pathogen and to decrease the transmission of pathogens from one target to another. The method may be combined with additional drugs, such as anti-infective agents.

Description

[Technical Field]

[0001] This application is U.S. Provisional Patent Application No. 62 / 704,340, filed on May 5, 2020. , Patent No. 62 / 705,288 filed on June 19, 2020, and October 2020 Claiming priority rights to Patent No. 63 / 107,893 filed on the 30th, these The disclosure of the application is incorporated into this disclosure as if it were fully described herein. ru. [Background technology]

[0002] Coronavirus infection can result in substantial morbidity and mortality. Vaccination is one While vaccines may be effective against a variety of viral infections in general, they do not necessarily protect against all specific viruses. It is not always completely effective against Russ. Severe Acute Respiratory Syndrome Coronavirus 2 ( The long-term effectiveness of currently known vaccines against SARS-CoV-2 is particularly limited. Considering the emergence of SARS-CoV-2 strains that could reduce the overall impact of the virus, a decision has not yet been made. It has not been found. Therefore, the treatment of this virus and the prevention of its transmission to others are being addressed by some young people. This is particularly important among the elderly, the elderly, or those with an immune deficiency. [Overview of the Initiative] [Means for solving the problem]

[0003] Therefore, in one embodiment, the present disclosure relates to treating a subject having an infection caused by a pathogen. A method is provided. This method involves using apirimod, remdesivir, and hydroxychloroquine. Excluding therapeutically effective amounts of at least one compound selected from Table 1 or Table 4 described herein. This includes administering a substance to a target.

[0004] In various embodiments, the pathogen is a coronavirus. For example, in one embodiment, the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 ).

[0005] In some embodiments, the method further comprises administering an anti-infective agent to the subject. The anti-infective agent, in various aspects, is an antiviral agent, such as an entry inhibitor, an uncoating inhibitor, a reverse transcriptase inhibitor, an antisense agent, a ribozyme agent, a protease inhibitor, an assembly inhibitor, and an egress inhibitor, selected from the group consisting of. In some embodiments, the antiviral agent comprises remdesivir.

[0006] Another embodiment of the present disclosure is a compound according to formula RFM-011-200-5 or a pharmaceutically acceptable salt thereof:

Chemical formula

[0007] RFM-011-200-5.

[0008] Yet another embodiment of the present disclosure is a compound according to formula RFM-007-454-4 or a pharmaceutically acceptable salt thereof:

Chemical formula

[0009] RFM-007-454-4.

[0010] In a further embodiment, the present disclosure provides at least one compound selected from Table 1 or Table 4 described herein in a therapeutically effective amount, a therapeutically effective amount of an anti-infective agent described herein, and a pharmaceutically acceptable carrier. The present invention provides a pharmaceutical composition containing an acceptable carrier. [Brief explanation of the drawing]

[0011] [Figure 1A] This figure illustrates a primary cell-based HCI assay for identifying compounds active against SARS-CoV-2 infection. A) A simplified assay workflow. B) Representative images from dimethyl sulfoxide (DMSO), remdesivir, or appilimod-treated wells. For each treatment, the entire imaging area per well (captured with a 10x objective lens and combined into four fields) is shown, as well as an 8x magnified segment delimited by a white box. DNA signals [4',6-diamidino-2-phenylindole (DAPI)] are colored green, and immunofluorescence-visualized viruses are colored magenta. Infected cells (arrows) and uninfected cells (arrowheads) are shown. Scale bars of 500 μm and 50 μm are shown in the combined and magnified images, respectively. Values ​​calculated from raw and normalized (Norm.) images are shown. C) Box plot of SARS-CoV-2 assay control EC50 obtained from independent biological experiments, showing the mean, bars, and all data points. Whiskers indicate minimum and maximum values. D) Heatmap image of normalized data from 1.9 μM ReFRAME screening plates. Normalized activity values ​​for infected cell percentage and total cell count are shown according to the scale bars, and density plots for compound and control wells are shown. DMSO-treated wells are in column 24, and positive control-treated wells (blocks of wells containing 2.5 μM remdesivir, 2.5 μM apirimod, or 9.6 μM puromycin) are in column 23. Density plots representing the frequency of values ​​associated with each well type are shown on the right. E) Figure of the distribution of 1.9 μM ReFRAME screening data for compound wells and control wells. F) Figure of the screen hit selection threshold. [Figure 1B]This figure illustrates a primary cell-based HCI assay for identifying compounds active against SARS-CoV-2 infection. A) A simplified assay workflow. B) Representative images from dimethyl sulfoxide (DMSO), remdesivir, or appilimod-treated wells. For each treatment, the entire imaging area per well (captured with a 10x objective lens and combined into four fields) is shown, as well as an 8x magnified segment delimited by a white box. DNA signals [4',6-diamidino-2-phenylindole (DAPI)] are colored green, and immunofluorescence-visualized viruses are colored magenta. Infected cells (arrows) and uninfected cells (arrowheads) are shown. Scale bars of 500 μm and 50 μm are shown in the combined and magnified images, respectively. Values ​​calculated from raw and normalized (Norm.) images are shown. C) Box plot of SARS-CoV-2 assay control EC50 obtained from independent biological experiments, showing the mean, bars, and all data points. Whiskers indicate minimum and maximum values. D) Heatmap image of normalized data from 1.9 μM ReFRAME screening plates. Normalized activity values ​​for infected cell percentage and total cell count are shown according to the scale bars, and density plots for compound and control wells are shown. DMSO-treated wells are in column 24, and positive control-treated wells (blocks of wells containing 2.5 μM remdesivir, 2.5 μM apirimod, or 9.6 μM puromycin) are in column 23. Density plots representing the frequency of values ​​associated with each well type are shown on the right. E) Figure of the distribution of 1.9 μM ReFRAME screening data for compound wells and control wells. F) Figure of the screen hit selection threshold. [Figure 1C]This figure illustrates a primary cell-based HCI assay for identifying compounds active against SARS-CoV-2 infection. A) A simplified assay workflow. B) Representative images from dimethyl sulfoxide (DMSO), remdesivir, or appilimod-treated wells. For each treatment, the entire imaging area per well (captured with a 10x objective lens and combined into four fields) is shown, as well as an 8x magnified segment delimited by a white box. DNA signals [4',6-diamidino-2-phenylindole (DAPI)] are colored green, and immunofluorescence-visualized viruses are colored magenta. Infected cells (arrows) and uninfected cells (arrowheads) are shown. Scale bars of 500 μm and 50 μm are shown in the combined and magnified images, respectively. Values ​​calculated from raw and normalized (Norm.) images are shown. C) Box plot of SARS-CoV-2 assay control EC50 obtained from independent biological experiments, showing the mean, bars, and all data points. Whiskers indicate minimum and maximum values. D) Heatmap image of normalized data from 1.9 μM ReFRAME screening plates. Normalized activity values ​​for infected cell percentage and total cell count are shown according to the scale bars, and density plots for compound and control wells are shown. DMSO-treated wells are in column 24, and positive control-treated wells (blocks of wells containing 2.5 μM remdesivir, 2.5 μM apirimod, or 9.6 μM puromycin) are in column 23. Density plots representing the frequency of values ​​associated with each well type are shown on the right. E) Figure of the distribution of 1.9 μM ReFRAME screening data for compound wells and control wells. F) Figure of the screen hit selection threshold. [Figure 1D-1]This figure illustrates a primary cell-based HCI assay for identifying compounds active against SARS-CoV-2 infection. A) A simplified assay workflow. B) Representative images from dimethyl sulfoxide (DMSO), remdesivir, or appilimod-treated wells. For each treatment, the entire imaging area per well (captured with a 10x objective lens and combined into four fields) is shown, as well as an 8x magnified segment delimited by a white box. DNA signals [4',6-diamidino-2-phenylindole (DAPI)] are colored green, and immunofluorescence-visualized viruses are colored magenta. Infected cells (arrows) and uninfected cells (arrowheads) are shown. Scale bars of 500 μm and 50 μm are shown in the combined and magnified images, respectively. Values ​​calculated from raw and normalized (Norm.) images are shown. C) Box plot of SARS-CoV-2 assay control EC50 obtained from independent biological experiments, showing the mean, bars, and all data points. Whiskers indicate minimum and maximum values. D) Heatmap image of normalized data from 1.9 μM ReFRAME screening plates. Normalized activity values ​​for infected cell percentage and total cell count are shown according to the scale bars, and density plots for compound and control wells are shown. DMSO-treated wells are in column 24, and positive control-treated wells (blocks of wells containing 2.5 μM remdesivir, 2.5 μM apirimod, or 9.6 μM puromycin) are in column 23. Density plots representing the frequency of values ​​associated with each well type are shown on the right. E) Figure of the distribution of 1.9 μM ReFRAME screening data for compound wells and control wells. F) Figure of the screen hit selection threshold. [Figure 1D-2]This figure illustrates a primary cell-based HCI assay for identifying compounds active against SARS-CoV-2 infection. A) A simplified assay workflow. B) Representative images from dimethyl sulfoxide (DMSO), remdesivir, or appilimod-treated wells. For each treatment, the entire imaging area per well (captured with a 10x objective lens and combined into four fields) is shown, as well as an 8x magnified segment delimited by a white box. DNA signals [4',6-diamidino-2-phenylindole (DAPI)] are colored green, and immunofluorescence-visualized viruses are colored magenta. Infected cells (arrows) and uninfected cells (arrowheads) are shown. Scale bars of 500 μm and 50 μm are shown in the combined and magnified images, respectively. Values ​​calculated from raw and normalized (Norm.) images are shown. C) Box plot of SARS-CoV-2 assay control EC50 obtained from independent biological experiments, showing the mean, bars, and all data points. Whiskers indicate minimum and maximum values. D) Heatmap image of normalized data from 1.9 μM ReFRAME screening plates. Normalized activity values ​​for infected cell percentage and total cell count are shown according to the scale bars, and density plots for compound and control wells are shown. DMSO-treated wells are in column 24, and positive control-treated wells (blocks of wells containing 2.5 μM remdesivir, 2.5 μM apirimod, or 9.6 μM puromycin) are in column 23. Density plots representing the frequency of values ​​associated with each well type are shown on the right. E) Figure of the distribution of 1.9 μM ReFRAME screening data for compound wells and control wells. F) Figure of the screen hit selection threshold. [Figure 1E]This figure illustrates a primary cell-based HCI assay for identifying compounds active against SARS-CoV-2 infection. A) A simplified assay workflow. B) Representative images from dimethyl sulfoxide (DMSO), remdesivir, or appilimod-treated wells. For each treatment, the entire imaging area per well (captured with a 10x objective lens and combined into four fields) is shown, as well as an 8x magnified segment delimited by a white box. DNA signals [4',6-diamidino-2-phenylindole (DAPI)] are colored green, and immunofluorescence-visualized viruses are colored magenta. Infected cells (arrows) and uninfected cells (arrowheads) are shown. Scale bars of 500 μm and 50 μm are shown in the combined and magnified images, respectively. Values ​​calculated from raw and normalized (Norm.) images are shown. C) Box plot of SARS-CoV-2 assay control EC50 obtained from independent biological experiments, showing the mean, bars, and all data points. Whiskers indicate minimum and maximum values. D) Heatmap image of normalized data from 1.9 μM ReFRAME screening plates. Normalized activity values ​​for infected cell percentage and total cell count are shown according to the scale bars, and density plots for compound and control wells are shown. DMSO-treated wells are in column 24, and positive control-treated wells (blocks of wells containing 2.5 μM remdesivir, 2.5 μM apirimod, or 9.6 μM puromycin) are in column 23. Density plots representing the frequency of values ​​associated with each well type are shown on the right. E) Figure of the distribution of 1.9 μM ReFRAME screening data for compound wells and control wells. F) Figure of the screen hit selection threshold. [Figure 1F]This figure illustrates a primary cell-based HCI assay for identifying compounds active against SARS-CoV-2 infection. A) A simplified assay workflow. B) Representative images from dimethyl sulfoxide (DMSO), remdesivir, or appilimod-treated wells. For each treatment, the entire imaging area per well (captured with a 10x objective lens and combined into four fields) is shown, as well as an 8x magnified segment delimited by a white box. DNA signals [4',6-diamidino-2-phenylindole (DAPI)] are colored green, and immunofluorescence-visualized viruses are colored magenta. Infected cells (arrows) and uninfected cells (arrowheads) are shown. Scale bars of 500 μm and 50 μm are shown in the combined and magnified images, respectively. Values ​​calculated from raw and normalized (Norm.) images are shown. C) Box plot of SARS-CoV-2 assay control EC50 obtained from independent biological experiments, showing the mean, bars, and all data points. Whiskers indicate minimum and maximum values. D) Heatmap image of normalized data from 1.9 μM ReFRAME screening plates. Normalized activity values ​​for infected cell percentage and total cell count are shown according to the scale bars, and density plots for compound and control wells are shown. DMSO-treated wells are in column 24, and positive control-treated wells (blocks of wells containing 2.5 μM remdesivir, 2.5 μM apirimod, or 9.6 μM puromycin) are in column 23. Density plots representing the frequency of values ​​associated with each well type are shown on the right. E) Figure of the distribution of 1.9 μM ReFRAME screening data for compound wells and control wells. F) Figure of the screen hit selection threshold. [Figure 2A]This figure shows the identification of potent and selective compounds with anti-SARS-CoV-2 activity in the ReFRAME library. A) Figure of the composition of the ReFRAME reuse library in relation to the clinical stage of the outbreak and disease indications. B) Figure plotting the SARS-CoV-2 EC50 of each compound against its host cytotoxicity CC50 when evaluated in uninfected HeLa-ACE2 cells, based on dose-response reconfirmation results. The dotted line represents the maximum concentration tested in the dose-response study for the assay compound (40 μM) and the control apirimodo and remdesivir (10 μM). The activity of the control (black diamond) and the assay compound (pink diamond) is shown. The activity of the ReFRAME library copy of puromycin, which was screened as part of this hit reconfirmation, is also shown (red diamond). C) Dose-response curves for SARS-CoV-2 EC50 (blue), infected HeLa-ACE2 EC50 (orange), and uninfected HeLa-ACE2 CC50 for remdesivir, apirimodo, and puromycin control compounds, performed as part of ReFRAME hit reconfirmation. D) Classification of 75 potent and selective compounds and 135 weakly active or non-selective compounds by functional annotation. E) Representative output (single replica) of the synergistic analysis of the apirimodo + remdesivir drug synergy landscape, showing the overall synergistic effect (-10 < δ < 10). F) Addition reaction between remdesivir and two fixed concentrations of apirimodo (approximately EC30 and EC65 during the synergistic experiment). Technical triplicate median + / - sem is shown. [Figure 2B]This figure shows the identification of potent and selective compounds with anti-SARS-CoV-2 activity in the ReFRAME library. A) Figure of the composition of the ReFRAME reuse library in relation to the clinical stage of the outbreak and disease indications. B) Figure plotting the SARS-CoV-2 EC50 of each compound against its host cytotoxicity CC50 when evaluated in uninfected HeLa-ACE2 cells, based on dose-response reconfirmation results. The dotted line represents the maximum concentration tested in the dose-response study for the assay compound (40 μM) and the control apirimodo and remdesivir (10 μM). The activity of the control (black diamond) and the assay compound (pink diamond) is shown. The activity of the ReFRAME library copy of puromycin, which was screened as part of this hit reconfirmation, is also shown (red diamond). C) Dose-response curves for SARS-CoV-2 EC50 (blue), infected HeLa-ACE2 EC50 (orange), and uninfected HeLa-ACE2 CC50 for remdesivir, apirimodo, and puromycin control compounds, performed as part of ReFRAME hit reconfirmation. D) Classification of 75 potent and selective compounds and 135 weakly active or non-selective compounds by functional annotation. E) Representative output (single replica) of the synergistic analysis of the apirimodo + remdesivir drug synergy landscape, showing the overall synergistic effect (-10 < δ < 10). F) Addition reaction between remdesivir and two fixed concentrations of apirimodo (approximately EC30 and EC65 during the synergistic experiment). Technical triplicate median + / - sem is shown. [Figure 2C]This figure shows the identification of potent and selective compounds with anti-SARS-CoV-2 activity in the ReFRAME library. A) Figure of the composition of the ReFRAME reuse library in relation to the clinical stage of the outbreak and disease indications. B) Figure plotting the SARS-CoV-2 EC50 of each compound against its host cytotoxicity CC50 when evaluated in uninfected HeLa-ACE2 cells, based on dose-response reconfirmation results. The dotted line represents the maximum concentration tested in the dose-response study for the assay compound (40 μM) and the control apirimodo and remdesivir (10 μM). The activity of the control (black diamond) and the assay compound (pink diamond) is shown. The activity of the ReFRAME library copy of puromycin, which was screened as part of this hit reconfirmation, is also shown (red diamond). C) Dose-response curves for SARS-CoV-2 EC50 (blue), infected HeLa-ACE2 EC50 (orange), and uninfected HeLa-ACE2 CC50 for remdesivir, apirimodo, and puromycin control compounds, performed as part of ReFRAME hit reconfirmation. D) Classification of 75 potent and selective compounds and 135 weakly active or non-selective compounds by functional annotation. E) Representative output (single replica) of the synergistic analysis of the apirimodo + remdesivir drug synergy landscape, showing the overall synergistic effect (-10 < δ < 10). F) Addition reaction between remdesivir and two fixed concentrations of apirimodo (approximately EC30 and EC65 during the synergistic experiment). Technical triplicate median + / - sem is shown. [Figure 2D]This figure shows the identification of potent and selective compounds with anti-SARS-CoV-2 activity in the ReFRAME library. A) Figure of the composition of the ReFRAME reuse library in relation to the clinical stage of the outbreak and disease indications. B) Figure plotting the SARS-CoV-2 EC50 of each compound against its host cytotoxicity CC50 when evaluated in uninfected HeLa-ACE2 cells, based on dose-response reconfirmation results. The dotted line represents the maximum concentration tested in the dose-response study for the assay compound (40 μM) and the control apirimodo and remdesivir (10 μM). The activity of the control (black diamond) and the assay compound (pink diamond) is shown. The activity of the ReFRAME library copy of puromycin, which was screened as part of this hit reconfirmation, is also shown (red diamond). C) Dose-response curves for SARS-CoV-2 EC50 (blue), infected HeLa-ACE2 EC50 (orange), and uninfected HeLa-ACE2 CC50 for remdesivir, apirimodo, and puromycin control compounds, performed as part of ReFRAME hit reconfirmation. D) Classification of 75 potent and selective compounds and 135 weakly active or non-selective compounds by functional annotation. E) Representative output (single replica) of the synergistic analysis of the apirimodo + remdesivir drug synergy landscape, showing the overall synergistic effect (-10 < δ < 10). F) Addition reaction between remdesivir and two fixed concentrations of apirimodo (approximately EC30 and EC65 during the synergistic experiment). Technical triplicate median + / - sem is shown. [Figure 2E]This figure shows the identification of potent and selective compounds with anti-SARS-CoV-2 activity in the ReFRAME library. A) Figure of the composition of the ReFRAME reuse library in relation to the clinical stage of the outbreak and disease indications. B) Figure plotting the SARS-CoV-2 EC50 of each compound against its host cytotoxicity CC50 when evaluated in uninfected HeLa-ACE2 cells, based on dose-response reconfirmation results. The dotted line represents the maximum concentration tested in the dose-response study for the assay compound (40 μM) and the control apirimodo and remdesivir (10 μM). The activity of the control (black diamond) and the assay compound (pink diamond) is shown. The activity of the ReFRAME library copy of puromycin, which was screened as part of this hit reconfirmation, is also shown (red diamond). C) Dose-response curves for SARS-CoV-2 EC50 (blue), infected HeLa-ACE2 EC50 (orange), and uninfected HeLa-ACE2 CC50 for remdesivir, apirimodo, and puromycin control compounds, performed as part of ReFRAME hit reconfirmation. D) Classification of 75 potent and selective compounds and 135 weakly active or non-selective compounds by functional annotation. E) Representative output (single replica) of the synergistic analysis of the apirimodo + remdesivir drug synergy landscape, showing the overall synergistic effect (-10 < δ < 10). F) Addition reaction between remdesivir and two fixed concentrations of apirimodo (approximately EC30 and EC65 during the synergistic experiment). Technical triplicate median + / - sem is shown. [Figure 2F]This figure shows the identification of potent and selective compounds with anti-SARS-CoV-2 activity in the ReFRAME library. A) Figure of the composition of the ReFRAME reuse library in relation to the clinical stage of the outbreak and disease indications. B) Figure plotting the SARS-CoV-2 EC50 of each compound against its host cytotoxicity CC50 when evaluated in uninfected HeLa-ACE2 cells, based on dose-response reconfirmation results. The dotted line represents the maximum concentration tested in the dose-response study for the assay compound (40 μM) and the control apirimodo and remdesivir (10 μM). The activity of the control (black diamond) and the assay compound (pink diamond) is shown. The activity of the ReFRAME library copy of puromycin, which was screened as part of this hit reconfirmation, is also shown (red diamond). C) Dose-response curves for SARS-CoV-2 EC50 (blue), infected HeLa-ACE2 EC50 (orange), and uninfected HeLa-ACE2 CC50 for remdesivir, apirimodo, and puromycin control compounds, performed as part of ReFRAME hit reconfirmation. D) Classification of 75 potent and selective compounds and 135 weakly active or non-selective compounds by functional annotation. E) Representative output (single replica) of the synergistic analysis of the apirimodo + remdesivir drug synergy landscape, showing the overall synergistic effect (-10 < δ < 10). F) Addition reaction between remdesivir and two fixed concentrations of apirimodo (approximately EC30 and EC65 during the synergistic experiment). Technical triplicate median + / - sem is shown. [Figure 3] This figure shows the effect of MOI on the EC50 of control compounds. The activity of remdesivir, apirimod, and puromycin controls in the SARS-CoV-2 / HeLa-ACE2 assay was evaluated using MOIs ranging from 1 to 26. The EC50 of each compound at the indicated MOIs is shown. [Modes for carrying out the invention]

[0012] This disclosure, in part, describes the treatment of subjects with pathogenic infections such as SARS-CoV-2. Regarding the method of treatment: In early December 2019, severe acute respiratory syndrome was diagnosed with coronavirus 2. SARS-CoV-2) has caused a rapid increase in the number of people with severe pneumonia-like symptoms, known as COVID-19. It was identified as the cause of the increase (Huang, C. et al. Clinical f eatures of patients infected with 2019 n ovel coronavirus in Wuhan,China.Lancet 3 95,497-506,doi:10.1016 / S0140-6736(20)301 83-5 (2020). Since then, SARS-CoV-2 has been identified by the World Health Organization (WHO) The pandemic situation has been officially recognized. As of February 10, 2021, SARS -CoV-2 has spread worldwide, infecting 106,555,200 people in 223 different countries. It has caused more than 2,333,440 confirmed infections and has resulted in over 2,333,440 reported deaths. (Organization, WH Vol.2020 (ed World H) health organization)(World Health Organization) (ation, 2020). Development of several effective anti-SARS-CoV-2 vaccines While it will undoubtedly contribute to controlling the pandemic, it will render some vaccines less effective. The emergence of SARS-CoV-2 strains with escape mutations and overall restricted C The global supply of OVID-19 vaccines is the foundation for ongoing efforts to identify therapeutic interventions. This is the basis. However, despite the tremendous efforts of research groups, the antiviral activity against COVID-19 has not been found. Treatment options remain very limited. These include severe or critical COVID-19 cases. This includes corticosteroids such as dexamethasone for the treatment of patients with D-19. (Group,RCet al.Dexamethasone in Hospital alized Patients with Covid-N Engl J Med, doi:10.1056 / NEJMoa2021436(2020)and the i ntravenously-delivered antiviral remdesi vir(de Wit,E.et al.Prophylactic and ther apeutic remdesivir(GS-5734) treatment in the rhesus macaque model of MERS-CoV inf ection.Proc Natl Acad Sci USA 117,6771 -6776,doi:10.1073 / pnas.1922083117(2020); Sheahan,TPet al.Broad-spectrum antivir al GS-5734 inhibits both epidemic and zo onotic coronaviruses.Sci Transl Med 9,do i:10.1126 / scitranslmed.aal3653(2017);Lo, MKet al.GS-5734 and its parent nucleos ide analog inhibit Filo-,Pneumo-,and Par amyxoviruses.Sci Rep 7,43395,doi:10.1038 / srep43395(2017)). Nucleotide analogues with broad antiviral activity. The somatic prodrugs remdesivir and RdRp inhibitors are Phase III approved for COVID-19. 19 treatment trials demonstrated a positive clinical endpoint (median time to recovery was 15 to 15 days). The period was shortened to 11 days (Health, NIo (2020)), U.S. Food and Drug Administration The bureau justified its emergency use authorization for the treatment of hospitalized COVID-19 patients. ministration,USFD(ed USFood&Drug A dministration)(USFood&Drug Administration) (ion, 2020). However, hydroxychloroquine, lopinavir and interf In conjunction with the effler regimen, the large-scale, multicenter WHO Solidarity trial was conducted. Recently, it has not been possible to reduce the mortality rate of COVID-19 patients in hospitals (Pan, He t al.Repurposed antiviral drugs for COVI D-19-interim WHO SOLIDARITY trial result s.medRxiv,2020.2010.2015.20209817,doi:10 .1101 / 2020.10.15.20209817(2020)). This disclosure also includes, Partially, two different cell-based SARS-CoV-2 infection assays and remdesivir Screening of a large drug library (ReFRAME) in a re-enhanced format This relates to the profiling of identified hits in the quadrature assay. The screening cascade and subsequent hit prioritization are based on SARS-CoV-2 We identified and validated MK-4482 as a promising hit as a potent inhibitor, and in vitro findings were This was converted into an in vivo hamster model of SARS-CoV-2 infection. Other identified hits are being used to develop treatments and tools to elucidate the coronavirus replication pathway. Useful for practical purposes.

[0013] definition As used herein, the term “compound” shall not apply unless otherwise specified. This includes compounds or their pharmaceutically acceptable salts, stereoisomers, and / or tautomers. It is comprehensive in that it is comprehensive. Therefore, for example, the compounds described herein are comprehensive in their chemical It contains pharmaceutically acceptable salts of tautomers of the compound.

[0014] In this disclosure, “pharmaceutically acceptable salt” means the pharmaceutically acceptable salt of the compound described herein. These are salts of organic or inorganic acids or bases that are acceptable in pharmaceutically. Representative pharmaceutically acceptable salts include... For example, alkali metal salts, alkaline earth salts, ammonium salts, water-soluble and water-insoluble Salt, for example, acetate, amsonate (4,4-diaminostilbene-2,2-disulfide). Honate, benzenesulfonate, benzoate, bicarbonate, bisulfate, Vitaltrate, volat, bromide, butyrate, calcium, calcium edetate, ca Musilate, carbonate, chloride, citrate, clavularate, dihydrochloride, edetate Ejisirato, Estrato, Esirato, Fiunarato, Gruceptato, G Luconate, glutamate, glycolyl arsanilate, hexafluorophosphate, Hexylresorcinate, hydravamin, hydrobromide, hydrochloride, hydroxynaphthol Iodide, isothionate, lactate, lactetobionate, laurate, marathon To, Maleat, Mandelat, Mesylate, Methylbromide, Methylnitrate, Meth Lusulfate, muquet, napsilate, nitrate, N-methylglucamine ammonia Umium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate Pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate, ionbo Naat, pantothenat, phosphate / diphosphate, picrate, polygalactus Lonato, propionate, p-toluenesulfonate, salicylate, stealthart, Suacetato, Suxinato, Sulfato, Sulfosalitzlat, Slamet, Tanna Salt, tartrato, teocrato, tosilato, triechojido, and barrerato salt Examples include: A pharmaceutically acceptable salt is one that has two or more charged atoms in its structure. This is possible. In this case, a pharmaceutically acceptable salt may have multiple counterions. Therefore, Therefore, a pharmaceutically acceptable salt is one or more charged atoms and / or one or more counterions. It is possible.

[0015] The terms “to treat,” “to treat,” and “treatment” refer to a disease or disease-related This refers to the improvement or eradication of symptoms. In various embodiments, this term refers to having such a disease. Diseases resulting from the administration of one or more prophylactic or therapeutic compounds described herein to a patient. This refers to minimizing the spread or worsening of the condition.

[0016] The terms "prevent," "preventing," and "prevention" are used in the chemicals described herein. This refers to the prevention of the onset, recurrence, or transmission of disease in patients caused by the administration of a substance.

[0017] The term "effective dose" refers to the therapeutic or preventive benefit in the treatment or prevention of a disease. This specification is sufficient to provide benefits or to delay or minimize disease-related symptoms. This refers to the amount of the compound or other active ingredient described in this document. Furthermore, it relates to the compound described herein. The therapeutically effective dose is the amount of a therapeutic agent that provides a therapeutic benefit in the treatment or prevention of a disease, when used alone. , or in combination with other therapies. Used in connection with the compounds described herein. In some cases, this term means improving the overall treatment, reducing or avoiding the symptoms or causes of the disease. or contains an amount that enhances the therapeutic effect of another therapeutic agent or is synergistic with another therapeutic agent. It is possible.

[0018] "Patient" or "subject" can include humans, cattle, horses, sheep, lambs, pigs, chickens, and venison. Animals such as finches, quail, cats, dogs, mice, rats, rabbits, or guinea pigs are included. In some embodiments, the animals are non-primates and primates (e.g., monkeys and The patient is a mammal such as a human. In one embodiment, the patient is a human, for example, a human infant, child, or adolescent. or being an adult. In this disclosure, the terms “patient” and “subject” are used interchangeably.

[0019] Screening assay and method ReFRAME(Repurposing,Focused Rescue,and The Accelerated Medchem drug collection is suitable for direct use in humans. A wide range of drug reuse studies have shown that nearly 12,000 small molecule drugs are being carried out. It is a lari (5), and its effectiveness can be further enhanced as a monotherapy or in combination with remdesivir. A wealth of resources to discover new therapies that may be useful in improving and reducing the potential for drug resistance It provides a source. This can inhibit the entry or replication of SARS-CoV-2 in human cells. In order to identify compounds in the library, the inventors have identified human SARS-CoV-2 receptors He expresses angiotensin-converting enzyme 2 or ACE2 (HeLa-ACE2). High-content imaging (HCI) 384-well format using La cells We developed an assay. In this assay, HeLa-ACE2 cells are subjected to the presence of the target compound. The subjects were then infected with the SARS-CoV-2 virus, and the viral infection was quantified after 24 hours (Figure). 1. Panel A). The assay uses serum purified from patients exposed to the virus. This relies on immunofluorescence detection of the ARS-CoV-2 protein, which is performed in conjunction with host cell nuclear staining. This allows for the quantification of the percentage of infected cells in each well (Figure 1, Panel B).

[0020] Activity against Ebola has been reported, and activity against SARS-CoV-2 is suspected or Previously examined compound: remdesivir (GS-5734) (6) (EC 50 =194± 20 nM, mean ± sem of 5 independent experiments and PIKfyve inhibitor apirimod ( EC 50 =50±11nM (mean ± sem of 4 independent experiments) (Figure 1, Panel B) The assay was validated using this method. High concentrations of remdesivir almost completely eliminate infected cells. This can be achieved (Figure 1, Panel C), and the inventors used this as a positive control at a concentration of 2.5 μM. The data was used and normalized to this and the neutral DMSO control well. Apilimodo is It was more potent than remdesivir, but had a slightly lower maximum efficacy compared to remdesivir. It contained (85-90% uninfected cells at the highest effective concentration). Furthermore, the inventors have found that positive cells Using the cytotoxic protein synthesis inhibitor puromycin as a saturation agent, per well Compound toxicity was evaluated in relation to infection by quantifying the total number of cells (average EC2). 50 = 547 ± 27 nM, mean ± sem of 5 independent experiments, HeLa-ACE2 CC 50 = 2.45 ± 0.23 μM, mean ± sem of 5 independent experiments). In particular, the increase in the number of associated cells is likely due to the decrease in the growth of infected cells, which is consistent with the antiviral activities of remdesivir and apilimod (Figure 1, panels B - E). Changing the multiplicity of infection had a moderate effect on the potency of the control compounds in the same experiment, and the EC 50 of remdesivir increased 2.7-fold from MOI = 1 to M OI = 26, and the EC 50 of apilimod increased 3.7-fold, but the EC of puromycin 50 did not increase (Figure 3).

[0021] Using the developed assay, we performed a pilot screening to evaluate the activities of 148 small molecules suspected of having therapeutic potential against coronavirus infection (7) (RZ’ = 0.84). We identified 19 compounds with an EC 50 < 9.6 μM, and based on the data obtained from the 24-hour viability / cytotoxicity assay of non-infected HeLa-ACE2, 10 of these were selected (non-infected HeLa-ACE2 CC / SARS-CoV-2 EC 50 / SARS-CoV-2 EC 50 > 10 or non-infected HeLa-ACE2 CC 50 > 40 μM) (Table 1). This included a library / screening of multiple apilimod (EC 50 = 184 nM, CC 50 > 40 μM) and remdesivir (EC 50 = 300 nM CC 50 > 40 μM) that were'rediscovered' in the assay. The Higher e-commerce 50 This was compared to a newly prepared control powder stock in the screening dataset. This is most likely due to slight decomposition within the material over time. [Table 1] TIFF2026094257000004.tif223158TIFF2026094257000005.tif189156TIFF2026094257000006.tif205157TIFF20260942570 00007.tif221156TIFF2026094257000008.tif232156TIFF2026094257000009.tif222157TIFF2026094257000010.tif133156 TIFF2026094257000011.tif200156TIFF2026094257000012.tif215156TIFF2026094257000013.tif204155TIFF20260942570 00014.tif219157TIFF2026094257000015.tif212156TIFF2026094257000016.tif203157TIFF2026094257000017.tif221155 TIFF2026094257000018.tif232156TIFF2026094257000019.tif213157TIFF2026094257000020.tif198156TIFF20260942570 00021.tif218156TIFF2026094257000022.tif173156TIFF2026094257000023.tif187156TIFF2026094257000024.tif205157 TIFF2026094257000025.tif213156TIFF2026094257000026.tif212155TIFF2026094257000027.tif217156TIFF20260942570 00028.tif195155TIFF2026094257000029.tif236158TIFF2026094257000030.tif223156TIFF2026094257000031.tif207157

[0022] The inventors have found that 12,000 compounds ReFRA at final concentrations of 1.9 μM and 9.6 μM are used. ME reuse libraries were screened. Assay quality is as shown in Table 2 below. , maintained throughout both screenings (RZ' of 0.87 and 0.72, respectively) . [Table 2]

[0023] Furthermore, DMSO vehicle (neutral control), remdesivir (positive control), apirimod And a clear distinction in the activity profile of the puromycin-(toxicity control) treated well. This was evident (Figure 1, panels D and E). Hit selection was based on the number of infected cells per well. >50% reduction (normalized <-50% activity compared to neutral control negative inhibitors) and <40% toxicity based on total cell count per well (including 10 μM puromycin) This is based on demonstration of compound activity (normalized to >-40% activity) (Figure 1, panel) E and F), 61 primary hits at 1.9 μM and a screening concentration of 9.6 μM. We identified 266 primary hits (with hit rates of 0.51% and 2.24% respectively), totaling 3 There were 11 hits.

[0024] The hit rate for the initial screening of the ReFRAME library was high (2.51%). ), this collection of bioactive small molecules is not unexpected, and many of them are approved drugs. or are in the clinical development stage and are used for a wide range of indications (Figure 2, Panel A). In order to reconfirm and evaluate the effectiveness and selectivity of the hit, the inventors have used a 9.6 μM solution. 325 available compounds were tested in a high-concentration 10-point 1:3 dilution dose-response format. Of these, 233 (71.7%) were EC-19 in response to SARS-CoV-2. 50 <9. It showed activity at 6 μM, and further 42 (12.9%) showed weak activity (EC 50 >9.6μ M). However, many of the primary screening hits are also cytotoxic and insensitive. Unacceptably low selectivity ratios (non-infected CC) as determined by stained HeLa-ACE2 cells 50 / EC 50 <10) accompanied by (Table 3, Figure 2, Panel B). [Table 3] TIFF2026094257000034.tif232159

[0025] Because viruses depend on host mechanisms for replication, many compounds possess antiviral activity. It was not unexpected that this would also affect important host processes. Interestingly, Protein synthesis inhibitors such as puromycin and even compounds like hydroxychloroquine. The reduction in viral infections provided benefits to cell health in the infected environment, but not to uninfected cells. Since it was not provided, this toxicity could be shielded in infected cells (Figure 2, panel) (Lu C).

[0026] Between the small-scale pilot and the ReFRAME screening, the inventors obtained 76 ( (75 unique units identified as GW-803430 from two different lots) Na (EC 50 <9.6μM) and selective (CC 50 / EC 50 >10 or CC 50 >3 A 9.8 μM compound and 135 compounds with weak activity (EC 50 >9.6μM ) or strong but not sufficiently selective (EC 50 <9.6μM, CC 50 / EC 50 <10) Compounds were identified (Figure 2 Panels B and D, Table 1). Top 4 classes of potency Furthermore, selective compounds include tumor-disintegrating compounds, ion channel modulators, antipsychotics, and receptors. It was a conjugated compound (Figure 2, Panel D). In the case of weak activity or non-selective hits, the top 4 The two categories are similarly tumor-disintegrating compounds, ion channel modulators, antipsychotics, and It contained signaling modulators. The fifth of the potent and selective hits was a tumor-clearing agent. It can be classified as a virus that exists in rapidly proliferating cells and is a virus that interferes with host cell processes. This further reflects the dependence on the disease. Antipsychotic, cardiovascular, and even antiparasitic (neglected tropical diseases) The identification of compounds belonging to the class involves the cationic and amphiphilic properties of some of these molecules. , and their ability to accumulate in and affect acidic intracellular compartments may be reflected (for example) (Late endosome / lysosome). The resulting dysregulation of intralysosomal pathways and Lipid homeostasis has been suggested to impair viral entry and / or replication. 8) Both of these modes of action were found in the inventors' screening of SARS-CoV Amiodarone and hydroxy, identified here as potent and selective hits against -2 The inventors have also considered cycloloquine (Tables 1 and 3). Two selective estrogens, a class of compounds previously found to inhibit infection. EC receptor modulators (bazedoxifene, EC) 50 =3.47 μM and raloxifene EC5 We identified 0 (4.13 μM) (9).

[0027] Further embodiments for use in any of the methods or compositions described herein are provided below. This includes further confirmed hits from FRAME screening. These are specified in this document. SARS-CoV-2 / HeLa-ACE2 high-content screening app described above SARS-CoV-2 / Calu-3 high-content screening assay ( The corresponding data from the examples are presented in Table 4. [Table 4] TIFF2026094257000036.tif226158TIFF2026094257000037.tif220157TIFF2026094257 000038.tif215156TIFF2026094257000039.tif230157TIFF2026094257000040.tif21315 5TIFF2026094257000041.tif207157TIFF2026094257000042.tif220154TIFF2026094257 000043.tif202157TIFF2026094257000044.tif233156TIFF2026094257000045.tif52157

[0028] Among the identified hits, according to various embodiments, relatively high exposure as a therapeutic agent Or, due to a long history of use, halophanthrin HCl, amiodarone, mesylate nelfi New oral medications, navir, simperevir, manidipine, and ozanimod, have been identified and approved. Therefore, after further efficacy testing in animal models, it will be considered as a treatment for COVID-19. It has the potential to be reused quickly. For example, the viral protease inhibitor Nelphy Navir and simeprevir show excellent exposure.

[0029] In another embodiment, the compound is a selective sphingosine-1-phosphate (S1P1). The receptor modulator is ozanimod. Selective S1P1 agonists reduce inflammation at the site of infection. (This reduces the release of cytokines by pulmonary endothelial cells and the infiltration of lymphocytes into the lungs.) This provides significant protection against influenza virus infection in a mouse model. It has been shown that (10) ozanimod is a direct-acting antiviral drug This could serve as an excellent combination partner.

[0030] According to another embodiment, the compound administered by the method described herein provides excellent exposure ( C max Approved drugs containing approximately 684 μM of amiodarone, or low exposure to hypertension. Approved calcium channel blockers that may improve the outcomes of patients with COVID-19 It is nidipine. Amiodarone has broad-spectrum antiviral activity in in vitro screening. They have been further identified as having sex (11).

[0031] Apilimodo (found in filoviruses, due to disruption of endolysosome transport) Assay controls that can inhibit viral entry (12), protease inhibitors NCO 19 different development stages of 700 (cathepsin B) and dutacatib (cathepsin K), etc. Other compounds in the species may also affect viral entry, and all of these may have an effect on their efficacy or Efficacy can be demonstrated due to the pharmacokinetic profile (Table 3). Most of these Except for the extremely potent appilimod, the potency of remdesivir does not exceed 1 μM. Medium-sized e-commerce 50 He possessed it.

[0032] This disclosure also describes, in some embodiments, the likelihood of pathogen infection occurring in the subject. This provides a method for reducing or decreasing the transmission of pathogens from infected targets to other targets. The method may be applied to the subject in combination with at least one anti-infective agent described herein. i. This includes administering at least one compound listed in Table 1 or Table 4.

[0033] Combination therapy In various embodiments, the method of the present disclosure further includes administering an anti-infective agent. The dye contains at least one compound listed in this specification (Table 1 and Table 2) in the same formulation or dosage form. 4) can be administered simultaneously. Alternatively, the anti-infective agent can be administered before or after the compound. It is possible.

[0034] In some embodiments, the antiinfectant is an entry inhibitor (including enfuvirtide), a decongestant, or Reverse transcriptase inhibitors (including amantadine, rimantadine, and preconalil), reverse transcriptase Inhibitors (including acyclovir, zidovudine, and lamivudine), antisense drugs (fomid) (including Pyrrhizin), lipozymes, protease inhibitors, aggregation inhibitors (rifampicin) Selected from the group consisting of (including), and release inhibitors.

[0035] In some embodiments, further agents include dexamethasone and amodiaquine. [ka] [ka]

[0036] In some embodiments, the additional anti-infective agent is an antiviral agent. So, the antiviral drugs are abacavir, acyclovir, and adefovir. , amantadine, amprigen, amprenavir (agenase), arbidol, atta Zanavir, Atripura, Baravir, Baloxavir marbocil (Xofluza), Victor Ruby, boceprevir (victrelis), cidofovir, cobicistat (Tybost) Combivir, daclatasvir (daclinza), darunavir, delavirdin, descobi Didanosine, docosanol, dolutegravir, doravirine (Pifertro), Ecoliva - Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfvir Tide, entecavir, etravirine (Intelence), fanciclovir, favi Pyravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) Fomivirsen, fosaprenavir, foscanet, phosphonet, fusion inhibitor Ganciclovir (Cytovene), ivacitabine, ibalizumab (Trogarz) o) Idoxuridine, imiquimod, immunovir, indinavir, inosine, integr Lase inhibitors, interferon type I, interferon type II, interferon II Type I, interferon, lamivudine, letermovir (Prevymis), lopinavir , robilide, maraviloc, methisazone, moloxidine, nelfinavir, nevirapine, Nexavir, nitazoxanide, norvir, nucleoside analogs, oseltamivir (tamif (L), Peginterferon alpha-2a, Peginterferon alpha-2b, Nonciclovir, peramivir (rapivab), preconalil, podophyllotoxin, protea Reverse transcriptase inhibitors (pharmacology), pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors , ribavirin, rilpivirine (edurant), rimantadine, ritonavir, saquinavir Simeprevir (Olysio), sofosbuvir, stabuzin, synergistic enhancer (anti-retino) Rovirus), telaprevir, terbivudine (Taizeca), tenofovir alafenamide, Tenofovir disoproxil, tenofovir, tipranavir, trifluridine, tridivir, Tromantadine, Tolvada, Valacyclovir (Valtrex), Valganciclovir, Bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza) The group is selected from the group consisting of and zidovudine.

[0037] Due to the need for intravenous administration and the potentially limited efficacy of remdesivir, alternative therapies or Further investigation of replacement therapy was prompted. Therefore, according to various embodiments, this disclosure is also The compounds disclosed herein are provided as partners with remdesivir in therapeutic use. .

[0038] The combination therapies disclosed herein reduce the drug dose of one or both combination partners. This can increase the effectiveness of the treatment, and therefore, regarding the administration of higher doses It can prevent potential side effects. Drug combinations also delay the development of drug tolerance. It can be made to increase the activity of combination therapies beyond the expectations of additive interactions. Synergistic drug effects are rare, and furthermore, additive effects themselves can improve treatment regimens. Conversely, antagonistic effects occur, where the combination of compounds exceeds what would be expected if they acted independently. Inhibition of overall activity is an undesirable characteristic.

[0039] Therefore, between the FDA-approved remdesivir hit and ReFRAME hit To identify synergistic, additive, and antagonistic interactions, the inventors used a checkerboard. In experiments, synergistic interaction studies were conducted to determine the complete use of remdesivir in the dose response of 11 hits. Dose response, attractive safety and pharmacokinetic profiles in a 10x10 matrix Compared with (Figure 2, Panel E). The inventors proposed the zero interaction efficacy model (ZIP) ( 14) To evaluate the interactions between compounds tested using the synergy finder of R -Using package (13), a δ score > 10 indicates the possibility of synergy, and δ < -10 The results show antagonistic activity, and the δ between -10 and 10 suggests additive interaction. We demonstrated that several exemplary combinations are additive (Figure 2, Panels E and F, Table 1). .

[0040] This screening also detects nucleoside analog ribopurine (nausea and surgical site infection). N6-isopentenyladenosine, previously studied as an antineoplastic agent for the treatment of the disease, Also prescribed prenatal / postnatal multivitamin / mineral tablets CitraNat al 90 (components of DHA) and folic acid antagonist 10 deazaaminopterin (currently under development) An antineoplastic compound in Phase I has activity that synergistically interacts with the activity of remdesivir. This was identified. The synergistic effect of both compounds was observed over specific concentrations, and a three-dimensional synergistic effect was observed. It was shown as a peak within the score landscape.

[0041] Ribopurine achieved maximum (100%) efficacy across the tested concentration range, but The addition of mudesivir's EC2 shifts its EC50 from 12 μM to 3.6 μM, and rem The addition of EC24 to desivir further increased its potency to EC50 = 1.6 μM. 10 - Deazaaminopterin showed a maximum efficacy of only 40% across the tested concentration range. However, the addition of remdesivir to EC2 increased the maximum efficacy from 40% to nearly 65%. A 2% shift is expected, and the addition of remdesivir to EC24 is a combination. Maximum effectiveness was increased from 40% to >80%.

[0042] Pharmaceutical composition In various embodiments, this disclosure provides therapeutically effective amounts from Table 1 or Table 4 described herein. At least one selected compound, a therapeutically effective amount of the anti-infective agent described herein, and pharmaceutical The present invention provides a pharmaceutical composition comprising a suitably permissible carrier. In some embodiments, the composition is In accordance with acceptable pharmaceutical formulation practices, one or more additional pharmaceutically acceptable excipients Diluents, adjuvants, stabilizers, emulsifiers, preservatives, colorants, buffers, and flavoring agents are used. It also includes.

[0043] The pharmaceutical compositions disclosed herein are formulated in a manner consistent with good medical practice and divided into appropriate amounts. It is administered. Factors to be considered in this regard include the specific disorder being treated, the specific disorder being treated. Target subjects, clinical condition of subjects, cause of impairment, site of drug delivery, method of administration, and administration schedule. This includes other factors known to healthcare professionals.

[0044] The "therapeutic effective dose" of the compound administered, including all the active ingredients of the combination therapy, is such It depends on the factors, and the minimum required to induce antiinfectiveness, such as an antiviral effect. It is a quantity. Such a quantity may be less than the amount that is toxic to normal cells or the entire subject. Generally, the initial therapeutic effective dose of the compound of this disclosure administered is approximately 0.01 to approximately 200 mg / The range is approximately 0.1 to 20 mg / kg patient body weight / day, with a typical initial range being approximately The dosage ranges from 0.3 to approximately 15 mg / kg / day. Oral dosage forms, such as tablets and capsules, are approximately It may contain 1 mg to about 1000 mg of the compound of this disclosure. In another embodiment, such The dosage form contains approximately 50 mg to approximately 500 mg of the compound disclosed herein. In yet another embodiment, Such dosage forms contain approximately 25 mg to approximately 200 mg of the compound of this disclosure. In embodiments, such dosage forms contain about 10 mg to about 100 mg of the compound of the present disclosure. In further embodiments, such a dosage form may contain about 5 mg to about 50 mg of the compound of the present disclosure. Contains.

[0045] The composition is administered in dose units by oral, topical, parenteral, inhalation, spray, or rectal administration. It is possible. As used herein, the term parenteral refers to subcutaneous injection, intravenous injection, intramuscular injection. , including intrasternal injection or infusion techniques.

[0046] Suitable oral compositions according to this disclosure include tablets, lozenges, medicinal drops, aqueous or oily suspensions. Turbidity, dispersible powder or granules, emulsion, hard or soft capsule, syrup or elixir This includes, but is not limited to, silicants.

[0047] The compounds disclosed herein or their pharmaceutically acceptable stereoisomers, salts, or tautomers and drugs The scope of this disclosure includes pharmaceutical compositions suitable for single-unit doses containing scientifically acceptable carriers. It can be done.

[0048] A composition suitable for oral use may be manufactured by any method known in the art for the production of pharmaceutical compositions. It can be prepared according to the following. For example, a liquid formulation of a compound is an arginase inhibitor drug. To provide a scientifically refined and palatable preparation, sweeteners, flavorings, colorings and It contains one or more drugs selected from the group consisting of preservatives.

[0049] The tablet composition is a mixture of the compound of this disclosure with a non-toxic, pharmaceutically acceptable excipient. These are used in the manufacture of tablets. Examples of such excipients include calcium carbonate and sodium carbonate. Inert diluents such as um, lactose, calcium phosphate, or sodium phosphate; granulation collapse Agents, such as corn starch or alginic acid; binders, such as starch, gelatin or red Shear; and lubricants, such as magnesium stearate, stearic acid, or talc. However, this is not limited to these. The tablets do not need to be coated, or they may be in the gastrointestinal tract. It delays the breakdown and absorption of the substance, thereby providing a sustained therapeutic effect over the desired period. For this purpose, it may be coated by a known coating technique. For example, By using time-delaying materials such as glyceryl nostearate or glyceryl distearate... It is possible.

[0050] Formulations for oral use may also contain an active ingredient with an inactive solid diluent, such as calcium carbonate. As hard gelatin capsules mixed with calcium phosphate or kaolin, or as active ingredients The mixture is mixed with water or an oily medium, such as peanut oil, liquid paraffin, or olive oil. It can be provided as a soft gelatin capsule.

[0051] In the case of an aqueous suspension, the compounds of this disclosure may be mixed with an excipient suitable for maintaining a stable suspension. They are combined. Examples of such excipients include sodium carboxymethylcellulose, Chill cellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinyl Examples include, but are not limited to, pyrrolidone, tragacanth gum, and gum arabic. .

[0052] Oral suspensions may also contain dispersants or wetting agents, such as natural phosphatides, such as lecithin, or These are condensation products of alkylene oxides and fatty acids, such as polyoxyethylene stearate. , or condensation products of ethylene oxide and long-chain aliphatic alcohols, such as heptadecaeth Lenoxycetanol, or a portion derived from ethylene oxide, fatty acids, and hexitol. Condensation products with esters, such as polyoxyethylene sorbitol monooleate, or Condensation formation of ethylene oxide with partial esters derived from fatty acids and hexitol anhydride. It may contain substances such as polyethylene sorbitan monooleate. The aqueous suspension is also 1 One or more preservatives, for example, ethyl or n-propyl p-hydroxybenzoic acid. Coloring agents, one or more flavoring agents, and one or more sweeteners, such as sucrose or saccharin. It may contain.

[0053] The oily suspension contains the compounds of this disclosure in vegetable oils, such as peanut oil, olive oil, sesame oil, or It can be formulated by suspending it in coconut oil or mineral oil such as liquid paraffin. The oily suspension contains a thickening agent, such as beeswax, hard paraffin, or cetyl alcohol. That's good too.

[0054] By adding the above-mentioned sweeteners and flavorings, we can provide an oral preparation with a pleasant taste. Yes, these compositions can be preserved by adding antioxidants such as ascorbic acid. .

[0055] Dispersible powders and granules suitable for preparing aqueous suspensions by adding water include dispersants or wetting agents. The present disclosure provides compounds of the present disclosure mixed with a suspending agent and one or more preservatives. A suitable dispersant or Wetting agents and suspending agents are exemplified by those already described above. Additional excipients, such as sweeteners, are also used. Flavoring agents, aromatherapy agents, and coloring agents may also be present.

[0056] The pharmaceutical compositions disclosed herein may be in the form of oil-in-water emulsions. The oily phase is plant Oil, for example olive oil or peanut oil, or mineral oil, for example liquid paraffin or similar It may be a mixture of these. Suitable emulsifiers are naturally occurring gums, such as gum arabic or Tragacanth gum, naturally occurring phosphatides, such as soy, lecithin, and fats. Esters or partial esters derived from acids and hexitol, anhydrides, such as sorbitan mo Nooleates, and condensation dione products of the partial ester and ethylene oxide, for example It may be polyoxyethylene sorbitan monooleate. The emulsion is also a sweetener. It may also contain flavoring agents.

[0057] The syrup and elixir contain sweeteners such as glycerol, propylene glycol, and It can be formulated with rubitol or sucrose. Such formulations can also be used as a lubricant and preservative. It may contain agents, as well as flavoring agents and coloring agents. The pharmaceutical composition may be a sterile injection, an aqueous suspension or It may be in the form of an oily suspension. This suspension may contain the above-mentioned suitable dispersant or wetting agent and suspension The agent can be used to formulate a drug according to known techniques. Sterile injectable preparations can also be prepared. A sterile injection solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, 1 It may also be a solution in 3-butanediol. Acceptable vehicles and The solvents include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, the sterilization fixing oil is , has been conventionally used as a solvent or suspension medium. For this purpose, synthetic monoglycerides Any non-irritating fixing oil containing lidocaine or diglycerides can be used. Furthermore, Fatty acids such as ic acid are used in the preparation of injectable drugs.

[0058] The compounds of this disclosure may also be administered in the form of suppositories for rectal administration of the compounds. The composition of the drug is solid at room temperature but liquid at rectal temperature, and therefore in the rectum Prepared by melting and releasing the drug, and by mixing with appropriate non-irritating excipients. This can be done. Such materials include cocoa butter and polyethylene glycol.

[0059] Compositions for parenteral administration are administered in sterile culture medium. Vehicles and formulations used. Depending on the concentration of the drug, parenteral preparations are either a suspension or a solution containing the dissolved drug. It could be any of the following: Adding an adjuvant such as a local anesthetic, preservative, or buffer to the parenteral composition. It is also possible.

[0060] [Examples] The following examples are illustrative and not limited to the scope of compositions, methods, and formulations described herein. It will not be done.

[0061] Virus generation. Vero E6 cells (ATCC CRL-1586) were used in 10% FBS. , 1×PenStrep(Corning20-002-CL), 2mM L-glutamic acid Complete DMEM containing Corning 25-005-CL Seeds were sown overnight in a T225 flask containing (15-013-CV) at 37°C with 5% CO2. Remove the culture medium from the lasco and add 2 mL of SARS-CoV-2 strain USA-W in complete DMEM. A1 / 2020 (BEI Resources NR-52281) with a MOI of 0.5 The solution was added to a flask and incubated at 34°C with 5% CO2 for 30 minutes. After the initial heating, 30 mL of complete DMEM was added to the flask. Then, the flask was heated to 34°C. The samples were placed in an incubator with 5% CO2 for 5 days. On the 5th day after infection, the supernatant was collected and 1,000 ml was used. Centrifuged at ×g for 5 minutes. Filter the supernatant through a 0.22 μM filter and store at -80°C. Ta.

[0062] ReFRAME Library: Compound management, drug annotation, and screen data Access. The ReFRAME Library Collection contains high-quality dimethyl sulfoxide (DM). It consists of approximately 12,000 high-purity compounds (>95%) dissolved in SO. Quality control is carried out as needed, including liquid chromatography-mass spectrometry and / or 1 By H-NMR That's what they said. Low concentration (2-10 μM) and high concentration (10-50 μM) screening type. To support the formula, libraries were prepared at two concentrations: 2 mM and 10 mM. Co-certified 384-well low dead volume + microplate (LP-0200- Using BC (Labcyte Inc.) as the library source plate, Ech Supports acoustic transmission via o 555 liquid handler (Labcyte Inc.) Compounds that do not dissolve in DMSO were plated in water (129 compounds). Compounds lacking long-term solubility were suspended immediately before dispensing to avoid precipitation (71 compounds). ).

[0063] Related compound annotations (Table 1) are three widely used commercial drug competitive intelligence days. Tabes: Clarivate Integrity, GVK Excelra GoS Supported by tar and Citeline Pharmaprojects Where available, annotation data should reflect the status of clinical development and the best development achieved. This may include stage, mechanism of action, one or more drug indications, and route of administration.

[0064] HeLa-ACE2 stable cell line. HeLa-ACE2 cells are converted to human ACE2 lentils. It was produced by transduction of Russ. The lentivirus is Lipofectamine20 Use 00 (Thermo Fisher Scientific, 11668019) Then, the pBOB-hACE2 construct and lentiviral packaging plasmid pMD Simultaneous tractor stimulation of HEK293T cells with L, pREV, and pVSV-G (Addgene) It was prepared by transfection. The supernatant was collected 48 hours after transfection. Next, it was used to transduce pre-sown HeLa cells. Transduction at 12:00 After a period of time, a stable cell line was recovered, scaled up, and stored. The cells were then treated with 10% FBS (G IBCO, 10438026) and 1x sodium pyruvate (Gibco, 11360) In DMEM (Gibco, 11965-092) containing 070), at 37°C and 5% CO2 It was maintained.

[0065] SARS-CoV-2 / HeLa-ACE2 high content screening assay. Compound 384-well μm transparent bottom plate (Greiner, Part. No. 781090-2) The cells were acoustically transferred to B). HeLa-ACE2 cells were placed in a well of 1.0 × 10³ cells. Cells were seeded at a density in 13 μL of DMEM containing 2% FBS. Plated cells were then placed in BSL3. The sample was transported to the facility, where it was adjusted to 0.65 for primary screening to reconfirm the powder. For the assay, with a MOI (Member of Infection) of 2.2, 13 μL of S diluted in assay medium was used. ARS-CoV-2 was added to each well. The plates were kept at 34°C and 5% CO2 for 24 hours. Incubate, then sterilize at 34°C with a final concentration of 4% formaldehyde and 5% CO2. Time fixation was performed. Between plate fixation and subsequent primary and secondary antibody staining, 1×PBS 0 was applied. Washed with 0.5% Tween 20. Perm / Wash buffer (BD Bioscien Human polyclonal plasma, diluted 1:500 with CES554723, was added to the plate. The sample was incubated at room temperature for 2 hours in SuperBlock T20 (PBS) buffer. 8 μg / mL (1 (Diluted 250 times) Goat anti-human H+L conjugate Alexa488 (Thermo Fisher Scientific A11013) 8 μM antifade-4 6-Diamidino-2-phenylindole (DAPI; Thermo Fisher S Add to the plate along with scientific D1306 and leave in the dark at room temperature for 1 hour. I modified the plate with an ImageXpress microphone with a 10x objective lens. ro Confocal High-Content Imaging System( Imaged using Molecular Devices, with four fields of view per well. The images were created. The images were stained with DAPI to identify the host cell nuclei (total number of cells in the image) and then processed. Using SARS-CoV-2 immunofluorescence signals to identify stained cells, multi-wavelength cell imaging is performed. The analysis was performed using the Coring Application Module (MetaXpress).

[0066] Time-of-Adsorption (TOA) assay. HeLa-ACE2 cells were added to assay medium (2% FBS). SARS-CoV-2 suspended in DMEM (containing ) was subjected to a 1.5 MOI at 34°C and 5% Infect with CO2 for 1 hour, then thoroughly wash with PBS and standard HeLa-ACE2 Similar to infection assays, the 384 wells of the assay are pre-spotted with the compound. Seeds were sown in a tray. For the time course experiment, cells were divided into groups 4, 5, 6, 7, 8, 10, 11, and 12. Fixation was performed with 4% formaldehyde at the final concentration of 24 hpi, in the case of a standard infection assay. Similarly, the cells were stained and imaged to determine the optimal time for the TOA assay. The sample was fixed to i, and the TOA assay was performed in the same manner.

[0067] Calu-3 high-content screening assay. The assay is performed with the following exceptions: The HeLa-ACE2 assay is then performed as outlined in the Calu-3 cell assay. (ATCC HTB-55) (Dr. and Scripps Res. of NCATS / NIH) A generous gift from Dr. Juan Carlos de la Torre of earch. ) in assay medium (MEM containing 2% FBS) at 20 μL / well for 5,000 cells. Seeds were seeded at a density, and SARS-CoV-2 diluted in assay medium was added at a MOI of 0.75-1. After adding the solution, approximately 30-60% of the cells were infected. The plates were then heated at 34°C in 5% CO2 for 4 minutes. The samples were incubated for 8 hours and then fixed with a final concentration of 4% formaldehyde. The cells were stained and imaged in the same manner as in the HeLa-ACE2 assay.

[0068] Non-infected host cytotoxicity counter screening. In the case of HeLa-ACE2 cells, Mixture 1,536 well μ clear plate (Greiner Part, No. 7890) 91) Acoustically transferred. The HeLa-ACE2 cells will be described in relation to the infection assay. Maintain the cells and prepare them for assay in DMEM containing 2% FBS at a well of 400 cells / well. The cells were seeded and incubated on plates at 37°C with 5% CO2 for 24 hours. Cell viability was evaluated. To evaluate, Image-iT DEAD Green Reagent (Thermo Fisher ) was used according to the manufacturer's instructions. Cells were fixed with 4% paraformaldehyde and DA Counterstaining was performed with PI. The image was taken using an ImageXpress micro confocal with a 10× objective lens. Using high-content imaging systems (Molecular Devices) The fixed cells are then imaged using the Live Dead application module (MetaXp Total viable cells per well were quantified in images obtained using (ress).

[0069] For Calu-3 cells, the assay medium (MEM containing 2% FBS) contains Calu-3 cells. Before seeding the cells at a density of 600 cells per 5 μL per well, the compound was added to 1,536 units. The sound was acoustically transferred to a cell plate (Corning No. 9006BC). The plate was moved to 3 The cells were incubated at 7°C and 5% CO2 for 48 hours. To assess cell viability, dilute the cells with water. 2 μL of 50% Cell-Titer Glo(Promega No. G757) 3) Add to the cells and use EnVision Plate Reader (Perkin E The luminescence was measured using an IMER (lmer).

[0070] HepG2 (ATCC HB-8065) ​​and HEK293T (ATCC CRL-3 216) Mammalian cell lines were subjected to 10% thermal inactivation in a humidified tissue culture incubator. yClone FBS (GE Healthcare Life Sciences), A 24-hour ointment containing 100 IU penicillin and 100 mg / mL streptomycin (Gibco) The samples were maintained in Rubecco's modified Eagle medium (DMEM, Gibco) at 37°C and 5% CO2. To assay the mammalian toxicity of the hit compound, 750 HepG2 and 375 HEK293T cells / well were acoustically transferred in 3-fold serial dilutions starting at 40 μM. 1536-well white tissue culture treated solid bottom plate containing the compound (Corning, 90 Assay medium in 06BC (DMEM, 2% FBS, 100 IU penicillin and 100 Seeds were seeded in mg / mL streptomycin. After 72 hours of incubation... , CellTiter-Glo Luminescent Cell Viabilit Using the y Assay (Promega NoG7573), in the case of Calu-3 cells... Similarly, cell viability was quantified.

[0071] SARS-CoV-2 primary ALI HBEC model. Normal primary human bronchial epithelial cells ( HBEC (Lonza) with a 1μm PET filter (Sigma) In a licell-96 cell culture insert plate, at the gas-liquid interface, PneumaCul Using t(trademark)-ALI medium (Stemcell Technologies), a small amount They were cultured for at least 4 weeks. To put it simply, HBEC was initially grown in a cell culture flask. After that, immerse in PneumaCult(trademark)-Ex Plus medium and 10 per well 0,000 cells were seeded. After one week, the cells were processed using Pneuma Cult™-AL. Switched to medium I and removed the medium from the apical surface. Maintained the gas-liquid interface and changed the medium every 2-3 days. The cells were replaced for at least 4 weeks to allow for cell differentiation. Before infection, the apical surface was treated with DPBS. After one rinse, the compound was added to the basal outer chamber. 20,000 PFU SARS- CoV-2 strain USA-WA1 / 2020 was added to the apical surface in 50 μL of PBS and left for 2 hours. The cells were incubated. Then, the inoculum was removed and the cells were rinsed once with DPBS. The solution was replaced, and fresh compound was added 24 and 48 hours after infection. 72 hours after infection Then, the apical wash solution is collected by adding 100 μL of DPBS to the apical surface for 15 minutes. I did it. I used PureLink(trademark) Pro 96 Viral RNA / DNA. Top-end cleaning using a Purification Kit (Thermo Fisher) Isolated from the liquid, SuperScript(trademark) III Platinum(trademark) On e-Step qRT-PCR Kit (Thermo Fisher) and 2019- nCoV N1 CDC Primer and Probe Set (Integrated DNA) Viral RNA level measured by RT-qPCR using Technologies) We analyzed the following: By isolating RNA from serial dilutions of stock viruses, we identified the target A quasi-curve was created and used to determine the PFU equivalent / mL of each sample. Then, a virus was generated. The reduction in volume was determined for each experimental compound treatment compared to a neutral DMSO control, and the logarithmic scale was used. The plot was made using a cytotoxicity detection kit (LDH) (Sigma). The LDH activity in the bottom culture medium was evaluated by measuring it according to the manufacturer's instructions. The average value for the experimental samples was taken and expressed as a percentage of the positive control puromycin. We performed a technical triplicate on both antiviral and cytotoxic readout.

[0072] A SARS-CoV-2 efficacy model in Golden Syrian hamsters. 8-week-old Golden Syrian hamsters. This will be shown to the Syrian hamster (Charles River) (5 per group). The drug was administered orally (PO). Four hours after the first dose, the animal was measured in 100 μL of DMEM. The hamsters were infected by placing a total of 106 PFUs into their nasal cavities. The mixture was administered twice daily (BID), and body weight was measured during the study period. On day 5 after infection, hams The tar was slaughtered and lung tissue was isolated for analysis. The research protocol was approved and Scrip ps Research is conducted in accordance with IACUC Protocol #20-0003. It was done.

[0073] Lung virus titer measurement. 100 μm cell strainer (Myriad 2825-8367) SARS- CoV2 titer was measured. Homogenized organs were titrated at a 1:10 ratio in six steps, and Ve It was overlaid onto ro cells. After incubation at 37°C for 1 hour, it was placed in a DMEM overlay. 1% methylcellulose was added, and the cells were incubated at 37°C for 3 days. The plaques were fixed with PFA and counted by crystal violet staining.

[0074] Pharmacokinetic studies. Pharmacokinetic studies follow the IACUC guidelines (IACUC protocol). According to #09-0004-5), Scripps Research Institute The experiment was conducted using te's Animal Models Core. It involved an 8-week-old male Syrian hares. Mustards (Charles River) (3 per group) were given each compound and formulation. As indicated, PO was administered. Plasma concentrations of each test substance were monitored for up to 48 hours. For both pharmacokinetic and efficacy studies, nelfinavir was administered in 10% DMSO / 90% tonic. Formulated in sorghum oil, MK-4482 is mixed with 10% PEG400 / 2.5% Cremaph or formulated with RH40.

[0075] Hamster lung RNA analysis. Uninfected (U, n=2), vehicle-treated (V, n=4), and M Hamster lungs from K-4482 treated (T, n=4) samples were analyzed using an RNASeq platform. The analysis was performed using the Python package scipy.stats.median_a. Using bsolute_deviation, the mean absolute deviation for all genes ( Calculates the MAD (Magnetic Algorithm). StepMiner algorithm (Sahoo, D., Dill, D.) L., Tibshirani, R. & Plevritis, SKExtracti ng binary signals from microarray time-c ourse data.Nucleic Acids Res 35,3705-371 Applying 2,doi:10.1093 / nar / gkm284(2007), 22,2 A high MAD value was selected to filter the 84 genes down to 14,939. The epMiner algorithm was applied again, reducing the number of genes from 14,939 to 8,617. I performed the retarding. Using the Python seaborn cluster map library function, Hierarchical clustering analysis was performed on these 8,617 genes. The current analysis was performed using DESeq227 (MK-4482 treated vs. vehicle treated sample). Applying the adjusted p-value < 0.1 and the multiple change |log2| > 1, the upregulator Identify downregulated / recombined genes. Recombination pathway analysis (Fabr egat,A.et al.The Reactome Pathway Knowle dgebase.Nucleic Acids Res 46,D649-D655,d Perform the oi:10.1093 / nar / gkx1132(2018)) gene set We identified high levels of biological processes concentrated in the x-axis. We demonstrate the significance of the concentrated biological processes using a bar graph with r).

[0076] RNASeq. An RNA sequencing library, as accurately described 25 years ago. TruSeq Unique Dual Indexes (Illumina, San Illumina TruSeq Stranded To It was prepared using tal RNA Library Prep Gold. In short, Process the sample according to the manufacturer's instructions, except for changing the RNA shear time to 5 minutes. Done. The resulting library was multiplexed and sequenced on an Illumina NovaSeq 6000 by the Institute of Genomic Medicine (IGM) at the University of California, San Diego to a depth of approximately 25 to 40 million reads per sample with 100 base pair (bp) paired-end (PE100). Samples were demultiplexed using bcl2fastq v2.20 Conversion Software (Illumina, San Diego, CA). RNAseq data was processed using kallisto (version 0.45.0) and the Mesocricetus auratus genome (MesAur1.0). Gene-level TPM values and gene annotations were calculated using the tximport and biomaRt R packages. Custom Python scripts were used to format the data, and log2(TPM) was used if TPM > 1, and TPM - 1 was used to reduce the log if TPM <= 1. For the hamster experiments, the kallisto index was prepared with Mesocricetus_auratus.MesAur1.0.ncrna.fa.gz + Mesocricetus_auratus MesAur1.0cdna.all.fa.gz. Raw and processed data will be deposited in the Gene Expression Omnibus (GSEID pending from NCBI GEO). at the Institute of Genomic Med icine (IGM) at the University of California, San Diego on an Illumina NovaSeq 6000 to a depth of approximately 25 to 40 million reads per sample with 100 base pair (bp) paired-end (PE100 ) sequencing. Samples were demultiplexed using bcl2fastq v2.20 Conversion Soft ware (Illumina, San Diego, CA). RNAseq data was processed using kallisto (version 0.45.0), Mes ocricetus auratus genome (MesAur1.0). Gene-level TPM values and gene annotations were calculated using the tximport and biomaRt R packages. Custom Python scripts were used to format the data, and log2(TPM) was used if TPM > 1, and TPM - 1 was used to reduce the log if TPM <= 1. For the hamster experiments, the kallisto index was prepared with Mes ocricetus_auratus.MesAur1.0.ncrna.fa.gz + Mesocricetus_auratus MesAur1.0cdna.all. fa.gz. Raw and processed data will be deposited in the Gene Expressi on Omnibus (NCBI GEO). - 1 was used to reduce the log if TPM <= 1. For the hamster experiments, the kallisto index was prepared with Mes socricetus_auratus.MesAur1.0.ncrna.fa.gz + Mesocricetus_auratus MesAur1.0cdna.all. fa.gz. Raw and processed data will be deposited in the Gene Expressi on Omnibus (NCBI GEO).

[0077] Quantification of hamster lung histopathology / filtrate. ImageJ software was used to quantify H &E stained slide images (20x magnification). Images were first converted to 8-bit (image Image > Type > 8-bit), adjust their thresholds (Image > Adjust > Threshold), and select only the dark stained nuclei. To ensure reliable detection, select a threshold of 70-80%. After thresholding, convert the image into a mask. (Process > Binary > Convert to Mask), then analyze with default settings (Analyze > Analyze Particles) Add the display results to show the outline, and output to GraphPad Prism (V9 Export to .0.0) and use the nonparametric two-sided Mann-Whitney test. The significance level was calculated using this method.

[0078] Data analysis. Using MetaXpress (version 6.5.4.532), high cost Content image analysis was performed. Primary in vitro screening and host cytotoxicity counter were also performed. - Screening data from Genedata Screener, version 16.0 Uploaded to .3-Standard. HeLaACE2 data, neutral (DM SO) Negative inhibitor control (Regarding antiviral effect in HeLa-ACE2 cells) For 2.4 μM remdesivir and infection host cytotoxicity, use 10 μM puromycin. In contrast, the data was normalized. Calu-3 infection assay data was normalized using neutral (DMSO) negative inhibitory assay. The data was normalized to the drug control (10 μM remdesivir) and the Calu-3 cell count was read out. Then, all cells were subjected to a stimulating factor (10 μM remdesivir) minus a neutral control (DMSO). It was normalized. For screening non-infected host cytotoxicity counters, 40 μM pure Romycin (Sigma) is used in HeLa-ACE2, HepG2, and HEK293T cells. As a positive (inhibitory) control in [the following scenario], 30 μM puromycin (Sigma) was used. It was used as a positive (inhibitory) control against Calu-3 cells. For dose-response experiments, The compound was tested in a technical triplicate on different assay plates, and the dose curve was analyzed using four parameters. The model was fitted using the Tahir formula. Technically replicated data was analyzed using central condensation. Multiple Regarding the compound activity (EC50 and CC50) obtained from independent biological experiments, geometrically... Reports the mean and geometric standard deviation. R's Gynamic Finder package (version 3.6) .3) was used for synergistic effect analysis ((Ianevski, A., He, L., Aitto kallio,T.&Tang,J.SynergyFinder:a web app lication for analyzing drug combinations dose-response matrix data.Bioinformatics 33,2413-2415,doi:10.1093 / bioinformatics / btx162(2017)). The geometric mean is calculated by taking the logarithm (base 10) of all values ​​and... The geometric standard deviation was calculated by calculating the mean of the logarithms and then taking the inverse logarithm of that mean. This is done by taking the standard deviation of each logarithmically transformed value and then taking the inverse logarithm of that standard deviation. I calculated it. The geometric standard deviation is a unitless ratio and is reported as ×÷ instead of + / -. Therefore, for the reported 0.123 μM × ÷ 1.276, the standard deviation range is 0.096 μM to 0.157 μM (i.e., 0.123 μM ÷ 1.276 to 0.123 It is μM × 1.276.

[0079] High-throughput Calu-3 phenotype ReFRAME screening for SARS-CoV-2 Leaning. Complements a relatively rapid 24-hour HeLa-ACE2 assay, prioritizing hits. In order to rank them, the inventors relied on the same antibody detection and similar assay workflow. Using the existing Calu-3 cells, a second more physiologically relevant infection assay was developed and read out at 48 hours post SARS-CoV-2 infection (hpi) (as described above). Calu- 3 is a human lung epithelial cell that endogenously expresses both the ACE2 receptor and the host serine protease TMPRSS2, which is necessary for the processing of the SARS-CoV-2 spike protein and viral entry into host cells (Hoffmann, M. et al. S ARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell 181, 271-2 80 e278, doi:10.1016 / j.cell.2020.02.052 (2 020)). In contrast, strong infection in HeLa-ACE2 cells lacking TMPRSS2 expression is likely to depend on the cathepsin-mediated viral entry pathway of endosomes, a generally recognized mechanism of coronaviruses (Yang, N. & Shen, H. M. Ta rgeting the Endocytic Pathway and Autoph agy Process as a Novel Therapeutic Strat egy in COVID-19. Int J Biol Sci 16, 1724-1 731, doi:10.7150 / ijbs.45498 (2020)). Remdesivir was active in Calu-3 cells (EC50 = 444 nM ± 1 .514 (n = 4)) and was similar to the TMPRSS2 inhibitor nafamostat mesylate .

[0080] ​​(EC50 = 24 nM × ÷ 1.55 (n = 3)). HeLa-ACE2 screening and In contrast, the Calu-3 assay showed a more pronounced cytopathic effect (MOI was higher). (This is likely due to the higher cost and longer incubation time used.) As a result, Viral compounds also protect cells from virus-induced cell death and exhibit antiviral activity. A second metric related to this was provided. Notably, 52 HeLa-ACE2 Most of the ReFRAME hits were not active in Calu-3 cell-based assays. They either selected (58%, 30 / 52) or were not selective.

[0081] By limiting the overlap of activity between HeLa-ACE2 and Calu-3 cells, Calu -3 cells prompted re-screening of the ReFRAME library. The test was performed at a final concentration of 2.5 μM, and RZ' = 0.744, indicating inhibition of >50% of infections. Harm, <80% cytotoxicity or >40% inhibition of infection, and >40% increase in cell number (wi 235 major hits were identified that demonstrated protection from Ruth-induced cell death. Of these, 145 was moderately active when tested in a dose-response manner (EC50 < 10 μM). ), however, only 42 was also selective (CC50 / EC50>10 or CC50> (30 μM). 88 estimated hit compounds in fresh powder stock (CC50 / EC50 > 5 Alternatively, CC50 > 30 μM, CC50 / EC50 < 5, but not for non-infected cytotoxic assays. There was a decrease of less than 50%, and three additional compounds with EC50 < 1 μM increased the number of infected cells. Selected for testing as (showing read protection), 87 were reconfirmed to be strong. 41 of them were reconfirmed to be selective in Calu-3 cells. Retesting individual Calu-3ReFRAME hits similarly with the HeLa-ACE2 infection assay. The experiment was conducted. Of these, 63% (26 / 41) showed SAR in HeLa-ACE2 cells. Although inactive against S-CoV-2, 34% (14 / 41) were active, but the results were strong and fine. It was cytotoxic, and CC50 levels were less than 3 μM in uninfected HeLa-ACE2 cells. Identification of endosomal cathepsin-mediated entry inhibitors. HeL in Calu-3 assay. One possible source of the limited activity of a-ACE2 ReFRAME hit is Since this is an entry mechanism used by viruses in each cell line, the inventors of the present invention have found that He We established a time-of-adsorption (TOA) assay in La-ACE2 cells and used ReFRAME. Among them, cathepsin-mediated activity is less likely to be active in the context of TMPRSS2 entry. We identified a viral entry inhibitor. To first determine the dynamics of the infection, we used HeLa-ACE. Two cells were infected with SARS-CoV-2 for one hour, and then the unattached viruses were washed away. Wash the cells in the presence of DMSO, hydroxychloroquine, apirimod, or remdesivir. The cells were seeded in a 384-well plate at a final concentration of 10 μM. The cells in the wells are shown. Cells were fixed at 4 hpi to 24 hpi, and the percentage of infected cells was quantified at each time point.

[0082] In all treatments except remdesivir, viral infection is detected by antibody staining with 6 hpi. This was the first thing that became clear, and it reached almost the maximum level between 10 hpi and 12 hpi. Loss of activity of both apirimod and hydroxychloroquine when treatment is initiated with i, These compounds block viral entry into HeLa-ACE2 cells, while rem Desivir therapy is initiated with 1 hpi, consistent with its direct antiviral mechanism of action. Nevertheless, it shows that the infection was effectively blocked.

[0083] Based on these results, the inventors have determined that all HeLa-ACE2 in dose response In the TOA assay (above) which evaluated the activity of ReFRAME hits, the replication cycle A time point of 10 hpi was used to limit the number of cells. The inventors used the TOA assay. This is a decrease in their activity, i.e., >10 E between the standard 24 hours and the TOA assay. Based on the C50 ratio, 33% (10 / ) of the compounds were inactive in Calu-3 cells. 30) was found to be an entry inhibitor in HeLa-ACE2. In contrast, Ca The compound that was also active in lu-3 cells (EC50 < 10 μM) was found to be active at its threshold. It could not be clearly classified as an entry inhibitor. Osimertinib and MK-22 Each of the 06s has a ratio of >8, and these are related to viral entry in HeLa-ACE2 cells. Although it was suggested that they may be involved, their antiviral activity in Calu-3 cells was not It was specific (SI < 2).

[0084] ReFRAME hit prioritization and validation. The ReFRAME library is bioactive. They are aggregates of small molecules, many of which are approved drugs or in the clinical development stage, and are diverse in nature. It is used for the following indications. It has been reconfirmed as a powder in HeLa-ACE2 screening. Potent and selective compounds of class 5 are tumor-disintegrating compounds (9) and ion channel modulators. These were agents (7), anti-inflammatory agents (5), antiviral agents (5), and signal transduction modulators (5). However, in the Calu-3 screening, the top 5 classes were signaling modulators (14), tumor Cerebral erosion compounds (11), protease inhibitors (7), antibiotics (3), and ion channels It was a regulator (3). The fifth point reflects the virus's dependence on host cell processes present in rapidly proliferating cells. Therefore, it can be classified as a tumor-lysis agent. It exclusively inhibits psychiatric activity in HeLa-ACE2 cells. The identification of compounds belonging to the disease and antiparasitic (neglected tropical diseases) class of these molecules Some cationic amphiphilic properties, and accumulation in acidic intracellular compartments, affect them. This can reflect the capabilities of those endosomes (e.g., late endosomes / lysosomes). Dysregulation and lipid homeostasis resulting from somal pathways are associated with viral entry and / or it has been suggested that it may impair reproduction (Salata, C., Calistri, A ., Parolin, C., Baritussio, A. & Palu, G. Antivi ral activity of cationic amphiphilic dru gs.Expert Rev Anti Infect Ther 15,483-49 2,doi:10.1080 / 14787210.2017.1305888(2017 )), these modes of action are all SARS- Amiodarone and hydro It is speculated that hydroxychloroquine is the only one. It was identified as an entry inhibitor in the inventors' assay.

[0085] From the compounds identified as hits in the initial screening by the present inventors, we selected those of high interest. The compound exhibited activity and selectivity in both the HeLa-ACE2 and Calu-3 assays. Furthermore, remdesivir, which was not classified as an entry inhibitor in HeLa-ACE2 cells, It was a compound with a profile similar to that of a profile. Prodrug MK-448 N-hydroxycytidine, the parent of compound 2, is not active in vitro in MK-4482 itself. However, perhaps due to a lack of metabolism to convert MK-4482 into its active form, its p It matched the rofile.

[0086] Furthermore, while it is active in Calu-3 cells, it is not active in HeLa-ACE2 cells. Compounds such as nafamostat mesylate and TMPRSS2 inhibitors do not affect the progression of infection. It had the potential to be active in Calu-3 cells. Conversely, it was not active in Calu-3 cells. Invasion inhibitors in HeLa-ACE2 cells (e.g., apyrimod, hydroxychloroquine) The priority of (azithromycin) was lowered. Based on the inventors' prioritization, the invention The researchers used an orthogonal gas-liquid interface primary human bronchial epithelial cell (ALI-HBEC) model of infection. The activity of representative hits against SARS-CoV-2 was tested. These differentiated substances The tract cells express high levels of both ACE2 and TMPRSS2. As expected, remde Syvir and nafamostat mesylate inhibit viral replication in ALI-HBEC. However, apirimod did not inhibit it. Furthermore, nelfinavir mesylate, MK-4482 And all of its parent N-hydroxycytidines have an apical viral load greater than 1 log at 72 hpi. This resulted in a decrease. These results are consistent with the inventors' model of hit prioritization. did.

[0087] Overall, the present inventors have identified the approved oral drugs halophanthrin HCl and mesylate nel Finavir, simeprevir, and manidipine were tested for their activity in both assays. Furthermore, due to their relatively high exposure levels or long history of use as therapeutic agents, they are of the greatest interest. Identified as a hit, and therefore after further efficacy evaluation in animal models, COVID-19 19. The potential for rapid reuse as a treatment was identified. Viral protease inhibitors Rufinavir and simeprevir have reported good plasma exposure, and their described Based on their mode of action, they may directly inhibit SARS-CoV-2. The sium channel blocker manidipine has low plasma exposure, but in patients with COVID-19 disease... It may be possible to improve the outcome. Nine other compounds at various stages of development are also available. Potential to demonstrate efficacy for efficacy or pharmacokinetic profile in pharmacokinetic assays High (see table). Both TO-195 and RWJ-56423 are trypsin inhibitors. A harmful agent, avoralstat can inhibit viral entry and is active in Calu-3 cells. It is a sexual kallikrein inhibitor. It is p38 mitogen-activated protein kinase (MAP). K) Inhibitor, LY222820 / larimetinib mesylate, is a HeLa-ACE2 screen It was active in both the ning and Calu-3 screening, and inhibited p38 MAPK23. It has been previously shown that it inhibits the replication of other coronaviruses through harm. Therefore, p 38MAPK may be an important host target for inhibiting coronavirus replication. (Note) What should be done is to use N-hydroxycytidine (Morn), which is the parent of the prodrug MK-4482. Pyramid (EIDD-2801) is used in HeLa-ACE2 assays and Calu-3 assays. It was a powerful and selective hit in both aspects. MK-4482 is Ridgeb ack Biotherapeutics and Merck have identified COVID-19 patients. This is an oral antiviral nucleoside analog currently being evaluated for the treatment of [condition].

[0088] Oral administration of MK-4482 provides complete protection against SARS-CoV-2 infection. Demonstrated in vitro efficacy in the ALI-HBEC primary cell model and Nerfinavi Sufficient exposure to nelfinavir and MK-4482 / N-hydroxycytidine (single dose of 5 At a PO dose of 00 mg / kg, the EC50 of Calu-3 SARS-CoV-2 lasts approximately 3 hours. For the duration of HeLa- SARS-CoV-2 infection is progressing due to the time of ACE2 and Calu-3EC50 Efficacy of nelfinavir and MK-4482 in a Rudenshirean hamster animal model Sex was investigated. To evaluate dose-dependent protection, nelfinavir was administered at 500 mg / kg MK-4482 is delivered via BID (twice daily) at 500 mg / kg and 150 mg / kg. g and 50 mg / kg BID were delivered via PO. A suspension of only the matched vehicle was used as a control. It was used as follows: Four hours after the initial treatment, the animals were given 1 × 10⁶ PFU of S by intranasal administration. We took on the challenge with ARS-CoV-2 (USA-WA1 / 2020). We used a measure of disease progression and The animals' weight was measured daily, and lung tissue was isolated on the fifth day of infection to determine viral titer and lung histology. And the gene expression profile was determined. Nelfinavir is probably in hamsters Insufficient plasma exposure can lead to weight loss and may prevent animals from viral replication. It didn't work. However, MK-4482, at 500 mg / kg, can treat animals from severe weight loss. Protected and, on average, 97% of the initial body weight at 5 days after infection. 150 mg / kg group and The 50 mg / kg group and the vehicle control group showed the same developmental progress on day 5 of infection compared to 85% of the vehicle control group. Partial protection was observed with average weight reductions of 89% and 90% from the starting weight.

[0089] To analyze the correlation with weight loss, plaque ascent based on crystal violet Using (I), the relative viral titer was determined from lung samples taken on day 5. (500 mg / kg and A dose of 150 mg / kg results in an undetectable viable viral titer in the lungs, and the virus It demonstrated complete protection from replication. The 50 mg / kg group had an average of 4.5 × 10³ PFU / lung. Compared to the vehicle control group, the average efficacy was moderately good at 4.5 × 10⁵ PFU / lung. It showed a 99% reduction in the virus.

[0090] Protection from weight loss and viremia in the 500 mg / kg treatment group is observed in hamsters. RNA Seq analysis of lung tissue, followed by unsupervised hierarchical clustering, was used for determination. As such, it was associated with almost complete protection from the host immune response; MK-4482 treatment (500 mg / kg) samples were clustered together with non-infected samples. DESeq2 analysis was performed. Therefore, the infected vehicle-treated lungs undergo interferon signaling and interferon Regarding pathways that have been reported to be upregulated in COVID-19, including stimulating genes. It was confirmed that it induces the expression of 66 related genes (Tindle, C. et a l.Adult Stem Cell-derived Complete Lung Organoid Models Emulate Lung Disease in COVID-19.bioRxiv,doi:10.1101 / 2020.10.17. 344002(2020);Sahoo,D.et al.AI-guided dis coverage of the invariant host response to viral pandemics.bioRxiv,doi:10.1101 / 202 0.09.21.305698(2020)). Finally, histological examination revealed MK-4 The lungs of 482 treated hamsters were found to be protected and more similar to those of non-infected animals. In stark contrast, examination of lung tissue in the vehicle-treated control group showed alveolar cavity obstruction and overwhelming This revealed the infiltration of immune cells.

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Claims

1. A method for treating a subject infected with a pathogen, wherein the effective therapeutic dose is selected from the following table. Infection by a pathogen, comprising administering at least one selected compound to the subject. A method of treating the subject that possesses it. Table 1

2. A method for treating a subject infected with a pathogen, wherein the effective therapeutic dose is selected from the following table. Infection by a pathogen, comprising administering at least one selected compound to the subject. A method of treating the subject that possesses it. Table 2

3. The method according to claim 1 or 2, wherein the pathogen is a coronavirus.

4. The aforementioned pathogen is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The method according to any one of claims 1 to 3.

5. The method according to any one of claims 1 to 4, further comprising administering an anti-infective agent.

6. The method according to claim 5, wherein the anti-infective agent includes an antiviral agent.

7. The aforementioned antiviral agents include entry inhibitors, decoating inhibitors, reverse transcriptase inhibitors, and antiviral agents. A group consisting of sense drugs, lipozymes, protease inhibitors, aggregation inhibitors, and release inhibitors. The method according to claim 6, selected from the following.

8. The aforementioned antiviral agents include remdesivir, hydroxychloroquine, pyronarizine, and azithromycin. The method according to claim 5 or 6, selected from the group consisting of mycin and favipiravir. 。

9. The aforementioned antiviral agent may be amodiaquine, which may be combined with dexamethasone. 【Chemistry 1】 【Chemistry 2】 The method described in invoice 5 or 6, selected from the group consisting of (PF-835231).

10. Compounds according to formula RFM-011-200-5 or pharmaceutically acceptable salts thereof: 【Transformation 3】 RFM-011-200-5.

11. Compounds according to formula RFM-007-454-4 or pharmaceutically acceptable salts thereof: 【Chemistry 4】

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