Compounds for suppressing inflammation

Abstract

To provide compounds that inhibit inflammasome activation.SOLUTION: The present application provides chemical compounds of formula (1) useful, for example, in inhibiting gasdermin pore formation in a cell, inhibiting inflammasome-mediated death of a cell (pyroptosis), inhibiting cytokine secretion from a cell, inhibiting an inflammatory caspase in a cell, and / or covalently reacting with a cysteine of a gasdermin protein in a cell. These compounds are also useful in treating or preventing diseases or conditions in which inflammasome activation is implicated in pathogenesis. One example of such disease or condition is sepsis. In the formula (1), R1, R2, R3 and R4 are each independently H, alkyl, haloalkyl, alkenyl etc.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Priority claims This application claims priority to U.S. Provisional Patent Application No. 62 / 690,788, filed June 27, 2018, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to chemical compounds, in particular compounds that inhibit inflammation and are useful in the treatment of conditions associated with inflammation. [Background technology]

[0003] Inflammasomes are multiprotein signaling scaffolds that assemble in response to invasive pathogens and sterile danger signals to activate inflammatory caspases (1 / 4 / 5 / 11), triggering inflammatory death (pyroptosis) and the processing and release of proinflammatory cytokines. Inflammasome activation contributes to many human diseases, including inflammatory bowel disease, gout, type II diabetes, cardiovascular disease, Alzheimer's disease, and sepsis (an often fatal response to systemic infection). Summary of the Invention

[0004] In a first general aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: Inhibiting gasdermin pore formation in cells, and / or Inhibiting inflammasome-mediated death of cells (pyroptosis), and / or Inhibits cytokine secretion from cells, and / or Inhibiting intracellular inflammatory caspases, and / or react covalently with the cysteines of the gasdermin protein within the cell, and / or covalently reacting with a cysteine ​​of an inflammatory signaling molecule selected from a sensor, an adaptor, and a transcription factor, or a regulator thereof; The method includes contacting the cell with an effective amount of any one of the compounds described herein, or a pharmaceutically acceptable salt thereof.

[0005] In a second general aspect, the disclosure provides a method for treating or preventing a disease or condition in which inflammasome activation and / or gasdermin-mediated inflammatory cell death is involved in the pathogenesis, the method comprising administering to a subject in need thereof a therapeutically effective amount of any one of the compounds described herein, or a pharmaceutically acceptable salt thereof.

[0006] In a third general aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: Inhibiting gasdermin pore formation in cells, and / or Inhibiting inflammasome-mediated death of cells (pyroptosis), and / or Inhibits cytokine secretion from cells, and / or Inhibiting intracellular inflammatory caspases, and / or react covalently with the cysteines of the gasdermin protein within the cell, and / or A method for identifying a compound that covalently reacts with a cysteine ​​of an inflammatory signaling molecule selected from a sensor, an adaptor, and a transcription factor, or a regulator thereof, is provided, The method comprises: a) providing a sample comprising liposomes containing a metal cation capable of complexing with a chelating ligand, the chelating ligand, a test compound, and gasdermin protein, or a fragment thereof; b) contacting the gasdermin protein in the sample with a protease enzyme; c) determining whether the test compound inhibits leakage of metal cations from the liposomes, wherein inhibition of leakage of metal cations from the liposomes indicates that the test compound: Inhibiting gasdermin pore formation in cells, and / or Inhibiting inflammasome-mediated death of cells (pyroptosis), and / or Inhibits cytokine secretion from cells, and / or Inhibiting intracellular inflammatory caspases, and / or react covalently with the cysteines of the gasdermin protein within the cell, and / or determining that the inflammatory signaling molecule covalently reacts with a cysteine ​​of the inflammatory signaling molecule selected from a sensor, an adaptor, and a transcription factor, or a regulator thereof.

[0007] In a fourth general aspect, the disclosure provides a pharmaceutical composition comprising any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0008] Specific implementations of the first, second, third, and fourth general aspects are described herein.

[0009] In some embodiments, the present disclosure provides a composition comprising any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, for treating or preventing any one of the diseases or conditions described herein.

[0010] In some embodiments, the present disclosure provides any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, for use as a medicament for treating or preventing any one of the diseases or conditions described herein.

[0011] In some embodiments, the present disclosure provides the use of any one of the compounds described herein, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prevention of any one of the diseases or conditions described herein.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application belongs. The methods and materials used in this application are described herein, and other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0013] Other features and advantages of the present application will become apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0014] [Figure 1] A diagrammatic representation of the terbium (Tb3+) / dipicolinic acid (DPA) fluorescent liposome leakage assay is included. [Figure 2] 1 includes a line graph showing the dose-response curve of disulfiram in a liposome leakage assay. [Figure 3] Included are line graphs showing MST measurements of the binding of Alexa 488-labeled His-MBP-GSDMD (80 nM) to C-22, C-23, or C-24. [Figure 4] 1 includes a bar graph showing cell viability after treatment with compounds C-22, C-23, and C-24 in the presence of nigericin or medium. [Figure 5] Included are bar graphs showing cell viability after pretreatment with each test compound (before electroporation with PBS or LPS). [Figure 6] 1 includes a line graph showing the IC50 for inhibition of canonical inflammasome activation by compound C-23. [Figure 7] 1 includes a line graph showing the IC50 for inhibition of non-canonical inflammasome activation by compound C-23. [Figure 8]Included is a bar graph showing the levels of IL-1β in culture supernatants treated with compound C-23, as assessed by ELISA (cells treated with LPS, or LPS and nigericin). [Figure 9] Included is a bar graph showing the levels of IL-1β in culture supernatants treated with compound C-23, as assessed by ELISA (PBS, or LPS-transfected cells). [Figure 10] Included are bar graphs showing cell viability after pretreatment with C-23 prior to transfection with PBS or poly(dA:dT). [Figure 11] Contains the chemical structures of compounds C-5, C-7, C-8, C-22, C-23, C-24, and C-25. [Figure 12] Included are dose-response curves for the inhibition of liposome leakage by disulfiram (C-23) or its metabolite DTC in the presence or absence of Cu(II). [Figure 13] Included are line graphs showing that LPS-primed THP-1 were pretreated with C-23 or DTC in the presence or absence of Cu(II) for 1 hour, followed by the addition of nigericin or medium for 2 hours. [Figure 14] Included is a line graph showing % mouse survival after challenge with 15 mg / kg LPS and treatment with C-23. [Figure 15] Included are bar graphs showing serum IL-1β measured by ELISA in mice pretreated with C-23 and challenged with 15 mg / kg LPS. [Figure 16] Included is a line graph showing % mouse survival after challenge with 25 mg / kg LPS and treatment with C-23. [Figure 17] Included is a line graph showing % mouse survival after challenge with 50 mg / kg LPS and treatment with C-23. [Figure 18]Contains line graphs showing % mouse survival after mice were treated with C-23 (50 mg / kg), C-23 (50 mg / kg) plus copper gluconate (0.15 mg / kg), or vehicle (control) by intraperitoneal injection at 0 and 12 hours after intraperitoneal LPS challenge (25 mg / kg). [Figure 19] Included is a chemical scheme showing the chemical reaction between DTC and Cu2+. [Figure 20] Contains MS / MS spectra of Cys191-containing human GSDMD peptides. [Figure 21] 1 contains the MS / MS spectrum of the GSDMD peptide after incubation with C-23 having covalent modification on Cys191 with a diethyldithiocarbamate moiety at C-23. [Figure 22] Included are images showing a model of full-length human GSDMD in its autoinhibited form, based on the corresponding structure of GSDMA3, and a model of the pore form of the GSDMDN terminal fragment (GSDMD-NT). [Figure 23] Included are dose-response curves for C-23 inhibition of liposome leakage induced by wild-type, C38A, or C191A GSDMD (0.3 μM) plus caspase-11 (0.15 μM). [Figure 24] Included are bar graphs showing C-23 inhibition of pyroptosis in LPS+nigericin-treated THP-1 cells after 1 hour preincubation with N-acetylcysteine ​​(NAC, 500 μM) or medium. [Figure 25] Included is a dose-response curve for compound C-23 on liposome leakage induced by human GSDMD-3C (0.3 μM) plus 3C protease (0.15 μM). [Figure 26] Included is a dose-response curve for compound C-23 on liposome leakage induced by human GSDMD-3C (0.3 μM) plus 3C protease (0.15 μM). [Figure 27] Contains the MS / MS spectrum of peptide FSLPGATCLQGEGQGHLSQK modified on cysteine ​​191 with carbamidomethyl. [Figure 28]Contains the MS / MS spectrum of peptide FSLPGATCLQGEGQGHLSQK modified on cysteine ​​191 by C-23. [Figure 29] Included is a sequence alignment of GSDMA3, hGSDMA, mGSDMD, and hGSDMD showing Cys residues. [Figure 30] Included are line graphs showing Tb3+ / DPA fluorescence of GSDMD (0.3 μM) preincubated for different periods (2–90 min) with the indicated concentrations of C-23 (0–50 μM) before the addition of caspase-11 (0.15 μM) in liposomes (50 μM). [Figure 31] 1 includes a line graph showing the time course of caspase-1 activity in the presence of the indicated concentrations of compound C-23. [Figure 32] 1 includes a line graph showing the time course of caspase-11 activity in the presence of the indicated concentrations of compound C-23. [Figure 33] 1 includes a dose-response curve for compound C-23 in a caspase-1 activity assay. [Figure 34] 1 includes a dose-response curve for compound C-23 in a caspase-11 activity assay. [Figure 35] 1 includes a line graph showing the time course of caspase-1 activity in the presence of the indicated concentrations of compound C-23 plus Cu(II). [Figure 36] 1 includes a line graph showing the time course of caspase-11 activity in the presence of the indicated concentrations of compound C-23 plus Cu(II). [Figure 37] 1 includes a dose-response curve for compound C-23 + Cu(II) in a caspase-1 activity assay. [Figure 38] 1 includes a dose-response curve for compound C-23 + Cu(II) in a caspase-11 activity assay. [Figure 39] Table 2 contains the chemical structures of the test compounds presented. [Figure 40] 39 includes a bar graph showing the results of cell viability assays for the compounds presented in Table 2 and FIG. [Figure 41]39. Contains bar graphs showing the results of cell viability assays for the compounds of Table 2 and FIG. 39 with and without nigericin. [Figure 42] 1 includes a bar graph showing the results of a cell viability assay for compounds C-23A1, C-23A2, C-23A9, and C-23A10 after addition of nigericin. [Figure 43] 1 includes a bar graph showing the results of a cell viability assay for compounds C-23, Bay11-7082, and C-23+Bay11-7082. [Figure 44] 1 includes a bar graph showing the results of a cell viability assay for compounds C-23 and Bay11-7082 after LPS transfection. [Figure 45] Contains images of immunoblots of THP-1 cells pretreated with C-23 and Bay11-7082. [Figure 46] Images of LPS-primed activated THP-1 cells pretreated with C-23, Bay11-7082, or z-VADfmk are included. [Figure 47] Included are bar graphs showing the % of cells with APS aggregates after treatment with C-23, Bay11-7082, or z-VADfmk. [Figure 48] Images of LPS-primed activated THP-1 cells pretreated with C-23 alone or together with Cu(II) are included. [Figure 49] Included are bar graphs showing the % of cells with APS aggregates after treatment with C-23 alone or together with Cu(II). [Figure 50] Included are images of immunoblots showing lysates of cells pretreated with C-23, Bay11-7082, or z-VADfmk and visualized with the indicated antibodies. [Figure 51] Included are images of immunoblots showing lysates of cells pretreated with C-23 alone or with Cu(II) and visualized with the indicated antibodies. [Figure 52] Included are bar graphs showing caspase-1 activity of C-23, Bay11-7082, and z-VADfmk. [Figure 53]Images of LPS-activated THP-1 cells pretreated with C-23, Bay11-7082, or z-VAD-fmk and stained with mouse anti-GSDMD monoclonal antibody are included. [Figure 54] Includes bar graphs showing quantification of the percentage of cells with GSDMD membrane staining and pyroptotic bubbles. [Figure 55] Response curves for Bay11-7082 inhibition of liposome leakage by wild-type, C38A, or C191A human GSDMD are included. [Figure 56] 1 includes a line graph showing thermophoresis measurements of the direct binding of Alexa 488-labeled His-MBP-GSDMD to Bay11-7082. [Figure 57] Included is a dose-response curve of the effect of Bay11-7082 on caspase-1 activity. [Figure 58] Included is a dose-response curve of the effect of Bay11-7082 on caspase-11 activity. [Figure 59] 1 includes the MS spectrum of the GSDMD peptide modified on Cys191 with carbamidomethyl. [Figure 60] 1 includes the MS spectrum of the GSDMD peptide modified at Cys191 and following GSDMD incubation with Bay11-7082. [Figure 61] 1 includes a dose-response curve of the effect of Bay11-7082 on liposome leakage induced by human GSDMD-3C. [Figure 62] 1 includes a dose-response curve of the effect of Bay11-7082 on liposome leakage induced by mouse GSDMD-3C. [Figure 63] 1 includes a bar graph showing the effect of pre-incubation of Bay11-7082 with N-acetylcysteine ​​(NAC) on the inhibition of pyroptosis. [Figure 64] Images of immunoblots of HEK293T cells transfected with the indicated plasmids and gels probed with the indicated antibodies are included. [Figure 65]Images of immunoblots of HCT116, 293T, and THP-1 cells transfected with the indicated plasmids and gels probed with the indicated antibodies are included. [Figure 66] Images of 293T and THP-1 cells immunostained with anti-GSDMD monoclonal antibody and co-stained with DAPI are included. [Figure 67] Included is a scheme showing the biochemical processes leading to the formation of gasdermin D pores and the subsequent release of inflammatory mediators. [Figure 68] Negative-stain EM images of PS-containing nanodiscs with and without incubation with GSDMD-3C+3C protease are included. In the third image from the left, C-23 was added to the GSDMD-3C+3C protease mixture, followed by addition of C-23 to the nanodiscs; in the fourth image, C-23 was added after the mixture was incubated with the nanodiscs when pores formed. Scale bar, 100 nm. Arrows point to empty nanodiscs and pores. [Figure 69] HT-29 cells were pretreated with or without disulfiram (C-23), 2 μM necrosulfonamide (NSA), or 10 μM necrostatin-1 (Nec) for 1 hour (10 μM and 50 μM) before the addition of 20 ng / ml TNFα (T), 100 nM SMAC mimetic (S), and 20 μM z-VAD-fmk (Z), and cell viability was analyzed 24 hours later by the CellTiter-Glo assay. Graphs show mean ± SD, and data are representative of three independent experiments. **P<0.01. [Figure 70] Included are line graphs showing the results of pyroptosis measured by SYTOX Green uptake in the absence of inhibitors or in the presence of 30 μM C-23 or z-VAD-fmk. [Figure 71]1 includes a bar graph showing the results of an experiment in which full-length (FL) human GSDMD and GSDMD C191S were co-expressed with caspase-11 in HEK293T cells. Cell death was determined by CytoTox96 cytotoxicity assay 20 hours after transfection. [Figure 72] FL includes a bar graph showing the results of an experiment in which human WT or C191S GSDMD was coexpressed with caspase-11 in HEK293T cells. Eight hours after transfection, the indicated amount of disulfiram was added, and cell death was determined by LDH release 12 hours later. Bar graphs show the mean ± sd of one representative experiment out of three independent experiments performed. *P<0.05, **P<0.01, and ns indicates not significant. [Figure 73] 1 includes a line graph showing the dose-response curve of disulfiram on liposome leakage induced by pre-cleaved human GSDMD (0.3 μM). [Figure 74] 1 includes a line graph showing the dose-response curve of disulfiram on liposome leakage induced by pre-cleaved murine GSDMA3-3C (0.3 μM). [Figure 75] Included are images showing LPS-primed activated THP-1 cells pretreated with or without 30 μM disulfiram or z-VAD-fmk for 1 hour and stimulated with nigericin or medium. [Figure 76] 1 includes a bar graph showing the results of an analysis of LPS-primed activated THP-1 cells for ASC specks. [Figure 77] Included are images showing the results of analyzing LPS-primarily activated THP-1 cells for NLRP3. [Figure 78] Included are images showing the analysis of LPS-primarily activated THP-1 cells for caspase-1, GSDMD, and pro-IL-1β cleavage, and IL-1 release by immunoblotting of whole cell lysates (WCL) or culture supernatants. [Figure 79]Images and bar graphs showing GSDMD redistribution to the plasma membrane are included. Cells were fixed 30 min after addition of nigericin and stained for GSDMD using a previously unreported monoclonal antibody generated in-house. Representative confocal microscopy images and quantification of the percentage of cells with GSDMD membrane staining and pyroptotic bubbles are shown. Arrows point to GSDMD staining of pyroptotic bubbles. Graphs show mean ± SD, and data are representative of three independent experiments. *P<0.05, **P<0.01. [Figure 80] Included are images showing a model of the inflammasome pathway steps and their inhibition by disulfiram, which have a major effect on GSDMD. [Figure 81] Included are plots showing the results of an experiment in which mice were pretreated with disulfiram (50 mg / kg) or vehicle (control) by intraperitoneal injection 24 and 4 hours before intraperitoneal challenge with 15 mg / kg LPS and followed for survival. TNFα was measured by ELISA (n=5 / group) 12 hours after LPS challenge. Mean values ​​± SD are shown. [Figure 82] Included are plots showing the results of an experiment in which mice were pretreated with disulfiram (50 mg / kg) or vehicle (control) by intraperitoneal injection 24 and 4 hours before intraperitoneal challenge with 15 mg / kg LPS and followed for survival. Serum IL-6 was measured by ELISA (n=5 / group) 12 hours after LPS challenge. Mean values ​​± SD are shown. [Figure 83] Included are line graphs showing the results of an experiment in which mice were pretreated with disulfiram (50 mg / kg) or vehicle (control) by intraperitoneal injection 4 hours before and daily after intraperitoneal LPS challenge (25 mg / kg) and followed for survival. [Figure 84] Contains images showing the results of experiments in which peritoneal macrophages from the four indicated mouse groups were analyzed for NLRP3, GSDMD, and HMGB1 by immunoblotting. [Figure 85]A line graph showing the results of a liposome leakage assay is included. GSDMD (2.5 μM) and caspase-11 (2.5 μM) were incubated in liposome solutions at various concentrations in 20 mM HEPES buffer (150 mM NaCl) for 1 hour. The concentration of liposomal lipids in the screen was set at 50 μM. [Figure 86] A line graph showing the results of a liposome leakage assay is included. Different concentrations of GSDMD and caspase-11 (1:1 ratio) were incubated in liposome (50 μM) solution for 1 hour. The concentration of GSDMD used in the screen was set to 0.3 μM. [Figure 87] A line graph showing the results of a liposome leakage assay is included. Different concentrations of caspase-11 and GSDMD (0.3 μM) were incubated in liposome (50 μM) solution for 1 hour. The concentration of caspase-11 used in the screen was set to 0.15 μM. Fluorescence intensity at 545 nm was measured after excitation at 276 nm. [Figure 88] Contains bar graphs showing the results of experiments in which mouse iBMDMs were pretreated with or without disulfiram (C-23) ranging from 5 to 40 μM for 1 hour before transfection with PBS or poly(dA:dT), and cell viability was analyzed by CellTiter-Glo assay 4 hours later. **P<0.01. [Figure 89] Included is an image showing a sequence alignment of GSDMA3, hGSDMA, mGSDMD, and hGSDMD, showing Cys residues. [Figure 90] Included are bar graphs showing the results of experiments in which FL mouse GSDMD or WT, C192S, or C39A GSDMD-NT was transiently expressed in HEK293T cells. Cell death was determined by CytoTox96 cytotoxicity assay 20 hours after transfection. c shows the mean ± SD of one representative experiment out of three independent experiments performed. **P<0.05. [Figure 91]1 includes a line graph (dose-response curve) showing the results of a GSDMD-mediated liposome leakage assay induced by 0.3 μM GSDMD+0.15 μM caspase-11 for the compound necrosulfonamide. [Figure 92] 1 includes a line graph (dose-response curve) showing the results of a GSDMD-mediated liposome leakage assay induced by 0.3 μM GSDMD+0.15 μM caspase-11 for the compound dimethyl fumarate. [Figure 93] 1 includes a line graph (dose-response curve) showing the results of a GSDMD-mediated liposome leakage assay induced by 0.3 μM GSDMD+0.15 μM caspase-11 for the compound afatinib. [Figure 94] 1 includes a line graph (dose-response curve) showing the results of a GSDMD-mediated liposome leakage assay induced by 0.3 μM GSDMD+0.15 μM caspase-11 for the compound ibrutinib. [Figure 95] 1 includes a line graph (dose-response curve) showing the results of a GSDMD-mediated liposome leakage assay induced by 0.3 μM GSDMD+0.15 μM caspase-11 for compound LDC7559. [Figure 96] Included are bar graphs showing the results of experiments in which LPS-primarily activated THP-1 cells, pretreated with or without 30 μM disulfiram or z-VAD-fmk for 1 hour and stimulated with nigericin or medium, were analyzed for caspase-1 activity 0.5 hours later with the cell-permeable fluorescent caspase activity probe FAM-YVAD-FMK. [Figure 97] Included are bar graphs showing the results of experiments in which, after removal of medium, LPS-activated THP-1 cells were incubated with the probe FAM-YVAD-FMK in FLICA assay buffer for an additional 0.5 h before fluorescence reading. iBMDMs were pretreated with disulfiram, Bay11-7082, necrosulfonamide (NSA), or z-VAD-fmk for 1 h and then treated with or without nigericin for 0.5 h. Whole cell lysates and culture supernatants were immunoblotted with the indicated antibodies. [Figure 98] Included are bar graphs showing the results of experiments in which, after removal of medium, LPS-activated THP-1 cells were incubated with the probe FAM-YVAD-FMK in FLICA assay buffer for an additional 0.5 h before fluorescence reading. iBMDMs were pretreated with disulfiram, Bay11-7082, necrosulfonamide (NSA), or z-VAD-fmk for 1 h, and then treated with or without nigericin for 1 h. Whole cell lysates and culture supernatants were immunoblotted with the indicated antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0015] As discussed in more detail below, the pore-forming protein gasdermin (such as gasdermin D) is the final pyroptosis initiator downstream of inflammasome activation. The compounds of the present application potently inhibit gasdermin pore formation and the subsequent secretion of inflammatory mediators such as IL-1β. Thus, the compounds of the present application are useful for treating diseases and conditions mediated by inflammation, such as sepsis. Pharmaceutical compositions containing the compounds of the present disclosure, as well as various methods for using and producing these compounds, are described below.

[0016] therapeutic compounds In one general aspect, the disclosure provides a compound of formula (I): [ka] or a pharmaceutically acceptable salt thereof, wherein: R 1 , R 2 , R 3 , and R 4 are independently H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, Cy 1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)ORa1 , S(O)2R b1 , and S(O)NR c1 R d1 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1 , Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)NR c1 R d1 , S(O)2R b1 and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Alternatively, R 1 and R 2 together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Cy2 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Alternatively, R 3 and R 4 together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Cy3 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Each Cy 1 independently, C 6~10 Aryl, C 3~10cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from R Cy1 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Each R Cy1 , R Cy2 , and R Cy3 independently, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~6 Haloalkyl, Halo, CN, NO2, OR a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , N.R. c2 C(O)OR a2 , N.R. c2 C(O)NR c2 R d2 , S(O)2R b2 and S(O)NR c2 R d2 is selected from R a1 , R a2 , R c1 , R c2 , R d1 , and R d2 are independently H, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~4 Haloalkyl, Cy 1 , C(O)R b3 , C(O)NR c3 R d3 , C(O)OR a3 , S(O)2R b3 , and S(O)NR c3 R d3 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1, Halo, CN, NO2, OR a3 , S.R. a3 , C(O)R b3 , C(O)NR c3 R d3 , C(O)OR a3 , N.R. c3 R d3 , N.R. c3 C(O)R b3 , N.R. c3 C(O)OR a3 , N.R. c3 C(O)NR c3 R d3 , N.R. c3 S(O)2R b3 , N.R. c3 S(O)NR c3 R d3 , S(O)2R b3 and S(O)NR c3 R d3 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R b1 and R b2 are each independently 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~4 Haloalkyl, and Cy 1 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1 , Halo, CN, NO2, OR a3 , S.R. a3 , C(O)R b3 , C(O)NR c3 R d3 , C(O)OR a3 , N.R. c3 R d3 , N.R. c3 C(O)R b3 , N.R. c3 C(O)OR a3 , N.R. c3 C(O)NR c3 R d3 , N.R. c3 S(O)2R b3 , N.R. c3S(O)NR c3 R d3 , S(O)2R b3 and S(O)NR c3 R d3 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R a3 , R c3 , and R d3 are independently H, C 1~6 Alkyl, C 1~4 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, (4-12 membered heterocycloalkyl)-C 1~4 Alkylene, C(O)R b4 , C(O)NR c4 R d4 , C(O)OR a4 , N.R. c4 R d4 , S(O)2R b4 , and S(O)NR c4 R d4 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, and (4- to 12-membered heterocycloalkyl)-C 1~4 Alkylene is oxo, C 1~6 Alkyl, C1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~6 Cyanoalkyl, halo, CN, NO2, OR a4 , S.R. a4 , C(O)R b4 , C(O)NR c4 R d4 , C(O)OR a4 , N.R. c4 R d4 , N.R. c4 C(O)R b4 , N.R. c4 C(O)OR a4 , N.R. c4 C(O)NR c4 R d4 , N.R. c4 S(O)2R b4 , N.R. c4 S(O)NR c4 R d4 , S(O)2R b4 , and S(O)NR c4 R d4 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Each R b3 independently, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~4 Haloalkyl, C 6~10 Aryl, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, and (4- to 12-membered heterocycloalkyl)-C 1~4 alkylene, 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, and (4- to 12-membered heterocycloalkyl)-C 1~4 The alkylene is, respectively, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~6 Cyanoalkyl, halo, CN, NO2, OR a4 , S.R. a4 , C(O)R b4 , C(O)NR c4 R d4 , C(O)OR a4 , N.R. c4 R d4 , N.R. c4 C(O)R b4 , N.R. c4 C(O)OR a4 , N.R. c4 C(O)NR c4 R d4 , N.R. c4 S(O)2R b4 , N.R. c4 S(O)NR c4 R d4 , S(O)2R b4 , and S(O)NR c4 R d4 and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from R a4 , R c4 , and R d4 are independently H, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Cyanoalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, (4-12 membered heterocycloalkyl)-C 1~4 Alkylene and R g wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, and (4- to 12-membered heterocycloalkyl)-C 1~4 Each alkylene is represented by R g and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Each R b4 independently, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Cyanoalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, (4-12 membered heterocycloalkyl)-C 1~4 Alkylene and R g wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 6~10 Aryl, C 3~10Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, and (4- to 12-membered heterocycloalkyl)-C 1~4 Alkylene is R g and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Each R g are independently OH, NO2, CN, halo, C 1~6 Alkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, C 1~4 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, Cyano-C 1~3 Alkylene, HO-C 1~3 Alkylene, C 6~10 Aryl, C 6~10 Aryloxy, C 3~10 Cycloalkyl, 5-10 membered heteroaryl, 4-12 membered heterocycloalkyl, C 6~10 Aryl-C 1~4 Alkylene, C 3~10 Cycloalkyl-C 1~4 Alkylene, (5-10 membered heteroaryl)-C 1~4 Alkylene, (4-12 membered heterocycloalkyl)-C 1~4 Alkylene, Amino, C 1~6 Alkylamino, di(C 1~6 Alkyl)amino, thio, C 1~6 Alkylthio, C 1~6 Alkylsulfinyl, C 1~6 Alkyl sulfonyl, carbamyl, C 1~6 Alkylcarbamyl, di(C 1~6 Alkyl) carbamyl, carboxy, C 1~6 Alkyl carbonyl, C 1~6 Alkoxycarbonyl, C 1~6 Alkylcarbonylamino, C 1~6Alkyl sulfonyl amino, amino sulfonyl, C 1~6 Alkylaminosulfonyl, di(C 1~6 Alkyl)aminosulfonyl, aminosulfonylamino, C 1~6 Alkylaminosulfonylamino, di(C 1~6 Alkyl)aminosulfonylamino, aminocarbonylamino, C 1~6 Alkylaminocarbonylamino, and di(C 1~6 alkyl)aminocarbonylamino.

[0017] In some embodiments, R 1 is H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, and Cy 1 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1 , Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)NR c1 R d1 , S(O)2R b1 and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0018] In some embodiments, R 1is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and Cy 1 wherein C is selected from 1~6 Alkyl is Cy 1 , Halo, CN, NO2, OR a1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and N.R. c1 S(O)2R b1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0019] In some embodiments, R 1 Cy 1 C optionally substituted with 1~6 In some aspects of these embodiments, R 1 is selected from methyl, ethyl, propyl, isopropyl, n-butyl, and t-butyl, each of which is selected from Cy 1 In other aspects of these embodiments, R 1 Cy 1 In some embodiments, R 1 Cy 1 In some embodiments, R 1 Cy 1 and Cy 1 C optionally substituted with 1~6 alkyl.

[0020] In some embodiments, R 2 is H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, and Cy 1 wherein C is selected from 1~6 Alkyl, C 2~6Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1 , Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)NR c1 R d1 , S(O)2R b1 and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0021] In some embodiments, R 2 is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and Cy 1 wherein C is selected from 1~6 Alkyl is Cy 1 , Halo, CN, NO2, OR a1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and N.R. c1 S(O)2R b1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0022] In some embodiments, R 2 Cy 1C optionally substituted with 1~6 In some aspects of these embodiments, R 2 is selected from methyl, ethyl, propyl, isopropyl, n-butyl, and t-butyl, each of which is selected from Cy 1 In other aspects of these embodiments, R 2 Cy 1 In some embodiments, R 2 Cy 1 In some embodiments, R 2 Cy 1 and Cy 1 C optionally substituted with 1~6 alkyl.

[0023] In some embodiments, R 3 is H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, and Cy 1 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1 , Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)NR c1 R d1 , S(O)2R b1 and S(O)NR c1 Rd1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0024] In some embodiments, R 3 is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and Cy 1 wherein C is selected from 1~6 Alkyl is Cy 1 , Halo, CN, NO2, OR a1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and N.R. c1 S(O)2R b1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0025] In some embodiments, R 3 Cy 1 C optionally substituted with 1~6 In some aspects of these embodiments, R 3 is selected from methyl, ethyl, propyl, isopropyl, n-butyl, and t-butyl, each of which is selected from Cy 1 In other aspects of these embodiments, R 3 Cy 1 In some embodiments, R 3 Cy 1 In some embodiments, R 3 Cy 1 and Cy 1 C optionally substituted with 1~6 alkyl.

[0026] In some embodiments, R 4 is H, C 1~6 Alkyl, C 1~6Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, and Cy 1 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1 , Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)NR c1 R d1 , S(O)2R b1 and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0027] In some embodiments, R 4 is H, C 1~6 Alkyl, C 1~6 Haloalkyl, and Cy 1 wherein C is selected from 1~6 Alkyl is Cy 1 , Halo, CN, NO2, OR a1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , and N.R. c1 S(O)2R b1and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0028] In some embodiments, R 4 Cy 1 C optionally substituted with 1~6 In some aspects of these embodiments, R 4 is selected from methyl, ethyl, propyl, isopropyl, n-butyl, and t-butyl, each of which is selected from Cy 1 In other aspects of these embodiments, R 4 Cy 1 In some embodiments, R 4 Cy 1 In some embodiments, R 4 Cy 1 and Cy 1 C optionally substituted with 1~6 alkyl.

[0029] In some embodiments, R 1 and R 2 are respectively Cy 1 C optionally substituted with 1~6 In some embodiments, R 1 and R 2 are respectively Cy 1 In some embodiments, R 1 Cy 1 C optionally substituted with 1~6 alkyl, and R 2 Cy 1 In some embodiments, R 1 Cy 1 and R 2 Cy 1 C optionally substituted with 1~6 It is alkyl.

[0030] In some embodiments, R 3 and R 4 are respectively Cy 1 C optionally substituted with1~6 In some embodiments, R 3 and R 4 are respectively Cy 1 In some embodiments, R 3 Cy 1 C optionally substituted with 1~6 alkyl, and R 4 Cy 1 In some embodiments, R 3 Cy 1 and R 4 Cy 1 C optionally substituted with 1~6 It is alkyl.

[0031] In some embodiments, R 1 and R 2 together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Cy2 In some aspects of the foregoing embodiments, the 4- to 12-membered heterocycloalkyl is selected from any one of the following groups: [ka]

[0032] In some embodiments, R 3 and R 4 together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Cy3 In some aspects of the foregoing embodiments, the 4- to 12-membered heterocycloalkyl is selected from any one of the following groups: [ka]

[0033] In some embodiments, Cy 1 is C 6~10 aryl, and RCy1 In some aspects of these embodiments, C is optionally substituted with 1, 2, or 3 substituents independently selected from 6~10 Aryl is phenyl or naphthyl.

[0034] In some embodiments, each Cy 1 independently, C 6~10 aryl and 5- to 10-membered heteroaryl, each of which is selected from R Cy1 is optionally substituted with 1, 2, or 3 substituents independently selected from

[0035] In some embodiments, Cy 1 is C 3~10 is cycloalkyl, and R Cy1 In some aspects of these embodiments, C is optionally substituted with 1, 2, or 3 substituents independently selected from 3~10 Cycloalkyl is selected from cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0036] In some embodiments, Cy 1 is a 5- to 10-membered heteroaryl, and R Cy1 and optionally substituted with 1, 2, or 3 substituents independently selected from: In some aspects of these embodiments, the 5- to 10-membered heteroaryl is selected from thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, 1,3,4-oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl. In other aspects of these embodiments, the 5- to 10-membered heteroaryl is selected from pyridin-2-yl, pyridin-3-yl, and pyridin-4-yl.

[0037] In some embodiments, Cy 1 is a 4- to 12-membered heterocycloalkyl; R Cy1 In some aspects of these embodiments, the 4- to 12-membered heterocycloalkyl is selected from tetrahydropuranyl, oxetanyl, azetidinyl, morpholinyl, thiomorpholinyl, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, and benzazapenyl.

[0038] In some embodiments, each R Cy1 independently, C 1~6 Alkyl, C 1~6 Haloalkyl, Halo, CN, NO2, OR a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , and N.R. c2 C(O)OR a2 In some embodiments, each R Cy1 is C 1~6 It is alkyl.

[0039] In some embodiments, each R Cy2 independently, C 1~6 Alkyl, C 1~6 Haloalkyl, Halo, CN, NO2, OR a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , and N.R. c2 C(O)OR a2In some embodiments, each R Cy2 is C 1~6 It is alkyl.

[0040] In some embodiments, each R Cy3 independently, C 1~6 Alkyl, C 1~6 Haloalkyl, Halo, CN, NO2, OR a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , and N.R. c2 C(O)OR a2 In some embodiments, each R Cy3 is C 1~6 It is alkyl.

[0041] In some embodiments, R a1 , R a2 , R c1 , R c2 , R d1 , and R d2 are independently H, C 1~6 Alkyl, Cy 1 , C(O)R b3 , C(O)NR c3 R d3 , C(O)OR a3 , S(O)2R b3 , S(O)NR c3 R d3 wherein C is selected from 1~6 Alkyl is Cy 1 , Halo, CN, NO2, OR a3 , N.R. c3 R d3 , N.R. c3 C(O)R b3 , N.R. c3 C(O)OR a3 , and N.R. c3 S(O)2R b3 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0042] In some embodiments, R b1 and R b2 are each independently 1~6 Alkyl and Cy 1 wherein C is selected from 1~6 Alkyl, halo, Cy 1 , CN, NO2, OR a3 , N.R. c3 R d3 , N.R. c3 C(O)R b3 , N.R. c3 C(O)OR a3 , and N.R. c3 S(O)2R b3 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0043] In some embodiments, R a3 , R c3 , and R d3 are independently H, C 1~6 Alkyl, C 1~4 Haloalkyl, C 6~10 Aryl, C 3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~6 Cyanoalkyl, halo, CN, NO2, OR a4 , N.R. c4 R d4 , N.R. c4 C(O)R b4 , N.R. c4 C(O)OR a4 , and N.R. c4 S(O)2R b4 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0044] In some embodiments, each R b3 independently, C 1~6 Alkyl, C 1~4 Haloalkyl, C 6~10 Aryl, C3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~6 Cyanoalkyl, halo, CN, NO2, OR a4 , N.R. c4 R d4 , N.R. c4 C(O)R b4 , N.R. c4 C(O)OR a4 , and N.R. c4 S(O)2R b4 and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0045] In some embodiments, R a4 , R c4 , and R d4 are independently H, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Cyanoalkyl, C 6~10 Aryl, C 3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from R g and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0046] In some embodiments, each R b4 independently, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Cyanoalkyl, C 6~10 Aryl, C 3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from R g and optionally substituted with 1, 2, or 3 substituents independently selected from:

[0047] In some embodiments, each R g are independently OH, NO2, CN, halo, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, Cyano-C 1~3 Alkylene, and HO-C 1~3 alkylene.

[0048] In some embodiments, Each R 1 , R 2 , R 3 , and R 4 are independently H, C 1~6 Alkyl, C 1~6 Haloalkyl, C 2~6 Alkenyl, C 2~6 Alkynyl, and Cy 1 wherein C is selected from 1~6 Alkyl, C 2~6 Alkenyl, and C 2~6 The alkynyl is, respectively, Cy 1 , Halo, CN, NO2, OR a1 , S.R. a1 , C(O)R b1 , C(O)NR c1 R d1 , C(O)OR a1 , N.R. c1 R d1 , N.R. c1 C(O)R b1 , N.R. c1 C(O)OR a1 , N.R. c1 C(O)NR c1 R d1 , N.R. c1 S(O)2R b1 , N.R. c1 S(O)NR c1 R d1 , S(O)2R b1 and S(O)NR c1 R d1 and optionally substituted with 1, 2, or 3 substituents independently selected from Alternatively, R1 and R 2 together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Cy2 and optionally substituted with 1, 2, or 3 substituents independently selected from Alternatively, R 3 and R 4 together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Cy3 and optionally substituted with 1, 2, or 3 substituents independently selected from Each Cy 1 independently, C 6~10 aryl and 5- to 10-membered heteroaryl, each of which is selected from R Cy1 and optionally substituted with 1, 2, or 3 substituents independently selected from Each R Cy1 , R Cy2 , and R Cy3 independently, C 1~6 Alkyl, C 1~6 Haloalkyl, Halo, CN, NO2, OR a2 , C(O)R b2 , C(O)NR c2 R d2 , C(O)OR a2 , N.R. c2 R d2 , N.R. c2 C(O)R b2 , and N.R. c2 C(O)OR a2 is selected from R a1 , R a2 , R c1 , R c2 , R d1 , and R d2 are independently H, C 1~6 Alkyl, Cy 1 , C(O)R b3 , C(O)NR c3 R d3 , C(O)OR a3 , S(O)2R b3 , S(O)NR c3 R d3 wherein C is selected from1~6 Alkyl is Cy 1 , Halo, CN, NO2, OR a3 , N.R. c3 R d3 , N.R. c3 C(O)R b3 , N.R. c3 C(O)OR a3 , and N.R. c3 S(O)2R b3 and optionally substituted with 1, 2, or 3 substituents independently selected from R b1 and R b2 are each independently 1~6 Alkyl and Cy 1 wherein C is selected from 1~6 Alkyl, halo, Cy 1 , CN, NO2, OR a3 , N.R. c3 R d3 , N.R. c3 C(O)R b3 , N.R. c3 C(O)OR a3 , and N.R. c3 S(O)2R b3 and optionally substituted with 1, 2, or 3 substituents independently selected from R a3 , R c3 , and R d3 are independently H, C 1~6 Alkyl, C 1~4 Haloalkyl, C 6~10 Aryl, C 3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~6 Cyanoalkyl, halo, CN, NO2, OR a4 , N.R. c4 R d4 , N.R. c4 C(O)R b4 , N.R. c4 C(O)OR a4 , and N.R. c4 S(O)2R b4and optionally substituted with 1, 2, or 3 substituents independently selected from Each R b3 independently, C 1~6 Alkyl, C 1~4 Haloalkyl, C 6~10 Aryl, C 3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~6 Cyanoalkyl, halo, CN, NO2, OR a4 , N.R. c4 R d4 , N.R. c4 C(O)R b4 , N.R. c4 C(O)OR a4 , and N.R. c4 S(O)2R b4 and optionally substituted with 1, 2, or 3 substituents independently selected from R a4 , R c4 , and R d4 are independently H, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Cyanoalkyl, C 6~10 Aryl, C 3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from R g and optionally substituted with 1, 2, or 3 substituents independently selected from Each R b4 independently, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~4 Hydroxyalkyl, C 1~4 Cyanoalkyl, C 6~10 Aryl, C 3~10 cycloalkyl, 5- to 10-membered heteroaryl, and 4- to 12-membered heterocycloalkyl, each of which is selected from R gand optionally substituted with 1, 2, or 3 substituents independently selected from Each R g are independently OH, NO2, CN, halo, C 1~6 Alkyl, C 1~4 Haloalkyl, C 1~6 Alkoxy, C 1~6 Haloalkoxy, Cyano-C 1~3 Alkylene, and HO-C 1~3 alkylene.

[0049] In some aspects of the foregoing embodiments, R 1 , R 2 , R 3 , and R 4 are each independently 1 and Cy 1 C optionally substituted with 1~6 alkyl.

[0050] In some embodiments, the compound of formula (I) is a compound listed in Table A below: [Table 1] TIFF0007787250000005.tif106170, or a pharmaceutically acceptable salt thereof.

[0051] In some embodiments, the compound of formula (I) is not any one of the compounds listed in Table A.

[0052] In some embodiments, the present application provides the following compound: [Table 2] TIFF0007787250000007.tif227170TIFF0007787250000008.tif108170, or a pharmaceutically acceptable salt thereof.

[0053] In some embodiments, the compound of the present application is not C-5, C-7, C-8, C-22, C-24, C-25, Bay11-7082, ASN-08966899, LDC7559, ibrutinib, afatinib, dimethyl fumarate, or necrosulfonamide.

[0054] In some embodiments, the present application provides the following compound: [Table 3] TIFF0007787250000010.tif159170, or a pharmaceutically acceptable salt thereof.

[0055] In some embodiments, the compound of the present application is not C-5, C-7, C-8, C-22, C-24, C-25, Bay11-7082, or ASN-08966899.

[0056] pharmaceutically acceptable salts In some embodiments, salts of the compounds disclosed herein are formed between an acidic and a basic group of the compound, such as an amino functional group, or between a basic and an acidic group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.

[0057] In some embodiments, acids commonly used to form pharmaceutically acceptable salts of compounds of the present disclosure include inorganic acids such as hydrodisulfic acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid, and organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, parabromophenylsulfonic acid, carbonic acid, succinic acid, cutic acid, citric acid, benzoic acid, and acetic acid, and related inorganic and organic acids. Thus, such pharmaceutically acceptable salts include gluconate, sulfate, pyrosulfate, bisulfate, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprylate, heptanoate, propionate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyrate, hexylate, hexylate-1,4-dione, hexylate-2, hexylate-3, hexylate-4, hexylate-5, hexylate-6, hexylate-7, hexylate-8, hexylate-9, hexylate-10, hexylate-11, hexylate-12, hexylate-13, hexylate-14, hexylate-15, hexylate-16, hexylate-17, hexylate-18, hexylate-19, hexylate-19, hexylate-19, hexylate-19, hexylate-19, hexylate-19, hexylate-20, hexylate-21, hexylate-22, hexylate-23, hexylate-24, hexylate-25, hexylate-26, hexylate-27, hexylate-28, hexylate-29 ... 6-dicarboxylic acid, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

[0058] In some embodiments, bases commonly used to form pharmaceutically acceptable salts of compounds of the present disclosure include alkali metal hydroxides, including sodium, potassium, and lithium; alkaline earth metal hydroxides, such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; organic amines, such as ammonia, unsubstituted or hydroxyl-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids, such as arginine and lysine.

[0059] In some embodiments, the compounds disclosed herein, or pharmaceutically acceptable salts thereof, are substantially isolated.

[0060] Production method The compounds disclosed herein (including salts thereof) can be prepared using known organic synthesis techniques and can be synthesized according to any of many possible synthetic routes. Those skilled in the art know how to select and implement appropriate synthetic protocols and understand that a wide repertoire of synthetic organic reactions is available for potential use in synthesizing the compounds provided herein.

[0061] Suitable methods for synthesizing starting materials, intermediates, and products can be identified by reference to the literature, including the following cited sources: Advances in Heterocyclic Chemistry,Vols.1-107(Elsevier,1963-2012);Journal of Heterocyclic Chemistry Vols.1-49(Journal of Heterocyclic Chemistry,1964-2012);Carreira,et al.(Ed.)Science of Synthesis,Vols.1-48(2001-2010)and Knowledge Updates KU2010 / 1-4;2011 / 1-4;2012 / 1-2 (Thieme,2001-2012);Katritzky, et al.(Ed.)Comprehensive Organic Functional Group Transformations,(Pergamon Press,1996);Katritzky et al.(Ed.);Comprehensive Organic Functional Group Transformations II (Elsevier,2 nd Edition,2004);Katritzky et al.(Ed.),Comprehensive Heterocyclic Chemistry(Pergamon Press,1984);Katritzky et al.,Comprehensive Heterocyclic Chemistry II,(Pergamon Press,1996);Smith et al.,March's Advanced Organic Chemistry:Reactions,Mechanisms,and Structure,6 th Ed. (Wiley, 2007); Trost et al. (Ed.), Comprehensive Organic Synthesis (Pergamon Press, 1991).

[0062] The reaction for preparing the compounds provided herein can be carried out in a suitable solvent that can be easily selected by those skilled in the art of organic synthesis.A suitable solvent can be substantially non-reactive with the starting material (reactant), intermediate, or product at the temperature at which the reaction is carried out, for example, a temperature that can range from the freezing temperature of the solvent to the boiling temperature of the solvent.A given reaction can be carried out in one solvent or a mixture of two or more solvents.Depending on the specific reaction step, a suitable solvent for a specific reaction step can be selected by those skilled in the art.

[0063] The preparation of the compounds provided herein can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. Protecting group chemistry is described, for example, in P.G.W. Muts and T.W. Greene, Protective Groups in Organic Synthesis, 4 th Ed., Wiley & Sons, Inc., New York (2006).

[0064] How to use Referring to Figure 67, the inflammatory cascade begins when pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), also known as alarmins, are sensed by cell surface and endosomal pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), as well as cytoplasmic sensors. Examples of PAMPs and DAMPs include LPS, bacterial toxins, bacterial proteins and nucleic acids, particles (such as uric acid and cholesterol crystals and amyloid-β fibrils), hyaluronan, and extracellular ATP. In response, the cellular machinery activates procaspase canonical or noncanonical inflammasomes, resulting in the release of active inflammatory caspases. Examples of inflammatory caspases include caspase-1, caspase-11, and caspase-4 and caspase-5. Activation of caspases in the inflammasome leads to caspase cleavage of the cytoplasmic protein gasdermin, producing the gasdermin N-terminal fragment (gasdermin-NT). In some cases, the caspase-cleavable gasdermin protein is selected from the following members of the gasdermin family: GSDMA, GSMDB, GSDMC, GSDMD, DFNA5, and DFNB59. Gasdermin-NT then binds to the plasma membrane from the cytoplasmic side, forming a pore that permeabilizes the plasma membrane, leading to cytokine secretion and pyroptosis. DFNA5 is activated by caspase-3 during classical apoptosis. Other gasdermin-activating proteases are currently unknown, but they may be activated independently of inflammasomes and not caspases. Typically, gasdermin binds to acidic lipids restricted to the inner leaflet of mammalian membranes, such as phosphatidylinositol phosphate (PIP), phosphatidylserine (PS), and phosphatidic acid (PA), as well as the bacterial and mitochondrial lipid cardiolipin. Gasdermin genes are typically expressed in epithelial and immune cells of various tissues, and all are capable of forming pores upon cleavage by inflammatory caspases.In one example, canonical inflammasome activation activates caspase-1, which cleaves pro-IL-1β, pro-IL-18, and gasdermin D, which forms the pore necessary to release the pro-inflammatory cytokine IL-1β.

[0065] The compounds of the present disclosure effectively block gasdermin pore formation and, therefore, any of the individual downstream mediators. Therefore, these compounds are more effective at inhibiting inflammation than anti-inflammatory agents that inhibit individual upstream or downstream inflammatory pathways, such as those clinically tested (IL-1 receptor antagonists, TNFα antibodies). The compounds are also more effective at mediating multiple, difficult-to-control dysregulated events that lead to patient death, such as disseminated intravascular coagulation (inhibited with activated protein C infusion). Inhibition of gasdermin (e.g., gasdermin D) by the compounds of the present application prevents cytokine storm, which is more effective than conventional anti-inflammatory treatments that attempt to reduce the complications of cytokine storm. Similarly, the compounds of the present application are also more effective than agents that neutralize LPS or its extracellular receptors (TLR4, CD14). Gram bacteria produce many PAMPs (toxins, flagella, and bacillus proteins), but not all are known. Therefore, neutralizing LPS may not prevent Gram sepsis if LPS inhibition is incomplete, especially in LPS-hypersensitive humans. TLR4 may be a less important LPS sensor than the noncanonical inflammasome, which is constitutively expressed in humans, not only on immune antigen-presenting cells but also on mucosal epithelia. LPS is a crucial trigger, and inhibiting it, or if its initial detection is unsuccessful, then inhibiting one of the other PAMP or DAMP sensors, may be effective, for example, in pleiotropically triggered sepsis in humans, where the triggering PAMP is generally unknown at the time of treatment. Furthermore, the compounds of the present application are more effective than individual inhibitors of inflammatory caspases. This is because the potential cross-reactivity of these inhibitors with apoptotic caspases and other cysteine ​​proteases may result in undesirable toxicity (e.g., liver fibrosis). Unwanted inhibition of caspase-8 can also lead to necroptosis. In some embodiments, inhibition of gasdermin pore formation occurs as a result of the compounds of the present application reacting with cysteines in the gasdermin protein.In some embodiments, the cysteine ​​is Cys191. In some embodiments, the compound also reacts with cysteines of inflammatory signaling molecules selected from sensors, adaptors, and transcription factors or regulators thereof. In some embodiments, the compound's diverse reactivity with protein cysteine ​​residues does not result in any undesirable toxicity or adversely affect the efficacy of the compound.

[0066] In some cases, the compounds of the present application are useful in treating or preventing inflammatory disorders or ameliorating symptoms associated with these disorders. Such disorders typically result in the immune system attacking one's own cells or tissues and can lead to conditions such as sepsis (e.g., acute sepsis), alopecia, hearing loss syndrome, gout, arthritis, rheumatoid arthritis, sclerosis, inflammatory bowel disease, ankylosing spondylitis (AS), antiphospholipid syndrome (APS), myositis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, vasculitis, familial Mediterranean fever, neonatal-onset multisystem inflammatory disease, Behcet's disease, cutaneous inflammatory disease, and the like. Inflammatory diseases include dermatosis, type 1 diabetes, autoimmune diseases, psoriasis, psoriatic arthritis, multiple sclerosis, Addison's disease, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, celiac disease, chronic inflammation, rheumatoid arthritis, encephalomyelitis, post-infectious cerebellitis, neuromyelitis optica (e.g., Devic's disease), encephalitis, metabolic encephalitis, asthma, periodontitis, ulcerative colitis, Crohn's disease, sinusitis, atherosclerosis, hypercholesterolemia, and peptic ulcer.In some cases, inflammatory diseases include eye diseases such as glaucoma, dry eye, and retinal ischemia-reperfusion.In some cases, inflammatory diseases include chronic lung disease and injury, as well as NASH and other inflammatory liver diseases.In some cases, inflammatory diseases are genetic autoinflammatory conditions.

[0067] Symptoms associated with inflammatory disorders typically include chronic pain, redness, swelling of joints and other tissues, stiffness, fever, buildup of blood proteins in organs, hair loss, fatigue, and damage to normal tissues. The compounds of the present application are useful in ameliorating these symptoms.

[0068] In some cases, the compounds of the present application are useful in treating sepsis or ameliorating symptoms associated with this condition. Examples of symptoms associated with sepsis include vascular leakage, circulatory collapse, coagulation activation, and multiple organ failure. Without appropriate treatment, sepsis is fatal in approximately one-third of cases. It is the leading cause of death among newborns and young children worldwide and contributes to one in two or three deaths among hospitalized adults in the United States. Current treatments for sepsis are limited to antibiotics and supportive care, and over 100 clinical trials designed to quiesce the immune response to infection have failed to produce a single new, effective therapy. Advantageously, the compounds of the present application reduce the innate immune response to disseminated and uncontrolled infection, successfully treating sepsis.

[0069] In some cases, the compounds of the present application may be used to prevent sepsis, for example, in patients at high risk of developing sepsis, suitable examples of such patients include neutropenic patients undergoing bone marrow transplantation.

[0070] In some cases, the compounds of the present disclosure are useful in treating or preventing cardiovascular diseases, including stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysm, peripheral arterial disease, thromboembolic disease, coronary artery disease, myocardial infarction, and venous thrombosis.

[0071] In some cases, the compounds of the present disclosure are useful in treating or preventing metabolic disorders, such as metabolic syndrome, type II diabetes, cystinosis, cystinuria, Fabry disease, galactosemia, Gaucher disease (type I), Hartnup disease, homocystinuria, Hunter syndrome, Hurler syndrome, Lesch-Nyhan syndrome, maple syrup urine disease, Maroteaux-Lamy syndrome, Morquio syndrome, Niemann-Pick disease (type A), phenylketonuria, Pompe disease, porphyria, Scheie syndrome, Tay-Sachs disease, tyrosinemia (hepatorenal), and von Gierke disease.

[0072] In some cases, the compounds of the present application are useful in treating or preventing neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, dementia, frontotemporal dementia, Huntington's disease, amyotrophic lateral sclerosis (ALS), motor neuron disease, and schizophrenia.

[0073] In particular, any disease may have an inflammatory component when infection or cell death is involved in the disease.Therefore, the compounds of the present application are useful in treating or preventing such diseases.Suitable examples of such diseases include infections caused by gram-positive bacteria, polymicrobial infections, infections caused by parasites (e.g., malaria, toxoplasmosis, trypanosomiasis, leishmaniasis), transplant rejection, eye inflammation (e.g., retinitis, uveitis), and cancer.

[0074] Combination therapy In some cases, the method of using the compounds described herein, or pharmaceutically acceptable salts thereof, includes administering the compound to a subject in combination with at least one additional therapeutic agent. In this method, the compound and the additional therapeutic agent may be administered to the subject simultaneously (e.g., in the same dosage form or in separate dosage forms) or sequentially (e.g., the additional therapeutic agent may be administered before or after the compound of the present disclosure, or a pharmaceutically acceptable salt thereof).

[0075] In some cases, the additional therapeutic agent includes an anti-inflammatory agent. Suitable examples include non-steroidal anti-inflammatory drugs such as celecoxib, rofecoxib, ibuprofen, naproxen, aspirin, diclofenac, sulindac, oxaprozin, piroxicam, indomethacin, meloxicam, fenoprofen, diflunisal, BAY11-7082, or pharmaceutically acceptable salts thereof. Suitable examples of steroid (e.g., corticosteroid) anti-inflammatory agents include cortisol, corticosterone, hydrocortisone, aldosterone, deoxycorticosterone, triamcinolone, bardoxolone, bardoxolone methyl, triamcinolone, cortisone, prednisone, and methylprednisolone, or pharmaceutically acceptable salts thereof. Other suitable examples of anti-inflammatory agents include anti-inflammatory antibodies (e.g., anti-IL-1, anti-TNF), and proteins such as integrins.

[0076] In some cases, the additional therapeutic agent is an antibiotic, which may be selected from quinolones, beta-lactams, cephalosporins, penicillins, carbapenems, lipopeptides, aminoglycosides, glycopeptides, macrolides, ansamycins, sulfonamides, monobactams, oxazolidinones, lipopeptides, macrolides, and cationic antimicrobial peptides (CAMPs).

[0077] Suitable examples of cationic antimicrobial peptides include defensin peptides (e.g., defensin 1, such as beta-defensin 1 or alpha-defensin 1), or cecropin, andropin, moricin, ceratototoxin, melittin, magainin, dermaseptin, bombinin, brevinin (e.g., brevinin-1), esculentin, buforin II (e.g., from amphibians), CAP18 (e.g., from rabbits), LL37 (e.g., from humans), abaecin, apidaecin (e.g., from honeybees), prophenin (e.g., from pigs), indolisin (e.g., from cattle), brevinin, protegrin (e.g., from pigs), tachyplesin (e.g., from horseshoe crabs), and drosomycin (e.g., from fruit flies).

[0078] Suitable examples of quinoline antibiotics include levofloxacin, norfloxacin, ofloxacin, ciprofloxacin, perfloxacin, lomefloxacin, fleroxacin, sparfloxacin, grepafloxacin, trovafloxacin, clinafloxacin, gemifloxacin, enoxacin, sitafloxacin, nadifloxacin, tosulfloxacin, cinnoxacin, rosoxacin, These include miroxacin, moxifloxacin, gatifloxacin, cinnoxacin, enoxacin, fleroxacin, romafloxacin, lomefloxacin, miroxacin, nalidixic acid, nadifloxacin, oxolinic acid, pefloxacin, pyrimidic acid, pipemidic acid, losoxacin, rufloxacin, temafloxacin, tosufloxacin, trovafloxacin, and besifloxacin.

[0079] Suitable examples of cephalosporin antibiotics include cefazolin, cefuroxime, ceftazidime, cephalexin, cephaloridine, cefamandole, cefsulodin, cefonicid, cefoperazine, cefopromisol, and ceftriaxone.

[0080] Suitable examples of penicillin antibiotics include penicillin G, penicillin V, procaine penicillin, and benzathine penicillin, ampicillin, and amoxicillin, benzylpenicillin, phenoxymethylpenicillin, oxacillin, methicillin, dicloxacillin, flucloxacillin, temocillin, azlocillin, carbenicillin, licacillin, mezlocillin, piperacillin, apalcillin, hetacillin, bacampicillin, sulbenicillin, mecicilam, pevmecillinam, cyclacillin, talapicillin, aspoxicillin, cloxacillin, nafcillin, and pivampicillin.

[0081] Suitable examples of carbapenem antibiotics include thienamycin, tomopenem, lenapenem, tebipenem, razupenem, imipenem, meropenem, ertapenem, doripenem, panipenem (betamipron), and biapenem.

[0082] Suitable examples of lipopeptide antibiotics include polymyxin B, colistin (polymyxin E), and daptomycin.

[0083] Suitable examples of aminoglycoside antibiotics include gentamicin, amikacin, tobramycin, debekacin, kanamycin, neomycin, netilmicin, paromomycin, sisomicin, spectinomycin, and streptomycin.

[0084] Suitable examples of glycopeptide antibiotics include vancomycin, teicoplanin, telavancin, ramoplanin, daptomycin, decaplanin, and bleomycin.

[0085] Suitable examples of macrolide antibiotics include azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midokamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin / tylosin, roxithromycin, dirithromycin, troleandomycin, spectinomycin, methymycin, neomethymycin, erythronolide, megalomycin, pikromycin, narbomycin, oleandomycin, triacetyl-oleandomycin, laucamycin, kuzimycin A, albocyclin, and cineromycin B.

[0086] Suitable examples of ansamycin antibiotics include streptovaricin, geldanamycin, herbimycin, rifamycin, rifampin, rifabutin, rifapentine, and rifamixin.

[0087] Suitable examples of sulfonamide antibiotics include sulfanilamide, sulfacetarnide, sulfapyridine, sulfathiazole, sulfadiazine, sulfamerazine, sulfadimidine, sulfasomidine, sulfasalazine, mafenide, sulfamethoxazole, sulfamethoxypyridazine, sulfadimethoxine, sulfasimazine, sulfadoxine, sulfamethpyrazine, sulfaguanidine, succinylsulfathiazole, and phthalylsulfathiazole.

[0088] Pharmaceutical Compositions The present application also provides a pharmaceutical composition comprising an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition may also comprise any one of the additional therapeutic agents described herein. In certain embodiments, the present application also provides pharmaceutical compositions and dosage forms comprising any one of the additional therapeutic agents described herein. The carrier(s) are "acceptable" in the sense of being compatible with the other ingredients of the formulation, and, in the case of a pharmaceutically acceptable carrier, not harmful to the recipient thereof in the amounts used in the pharmaceutical.

[0089] Pharmaceutically acceptable carriers, adjuvants, and vehicles that may be used in the pharmaceutical compositions of the present application include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or electrolytes such as protamine sulfate, disodium monohydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0090] The composition or dosage form may contain in the range of 0.005%-100% of any one of the compounds and therapeutic agents described herein, with the remainder consisting of suitable pharmaceutically acceptable excipients. Contemplated compositions may contain 0.001%-100% of any one of the compounds and therapeutic agents provided herein, and in one embodiment 0.1-95%, in another embodiment 75-85%, and in a further embodiment 20-80% of any one of the compounds and therapeutic agents provided herein, with the remainder consisting of any pharmaceutically acceptable excipient described herein, or any combination of these excipients.

[0091] Route of administration and dosage form Pharmaceutical compositions of the present application include those suitable for any accepted route of administration. Acceptable routes of administration include, but are not limited to, buccal, intradermal, intracervical, intranasal, intrasinus, intratracheal, enteral, epidural, intrainterstitial, intraabdominal, intra-arterial, intrabronchial, oral, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepithelial, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intraspinal, intrameningeal, intramuscular, intranasal, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intranasal, intrathecal, intrasynovial, intratesticular, intrathecal, intratubular, intratumor, intrauterine, intravascular, intravenous, intranasal, nasogastric, oral, parenteral, transdermal, epidural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral, ​​and intravaginal.

[0092] The compositions and formulations described herein can be conveniently provided in unit dosage form, for example, tablets, sustained-release capsules, and liposomes, and can be prepared by any method well known in the field of pharmacy.See, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, Baltimore, MD (20th ed.2000).Such preparation methods include the step of bringing the molecule to be administered into association with ingredients such as a carrier, which constitutes one or more accessory ingredients.In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier, liposomes, or finely divided solid carrier, or both, and then, if necessary, forming the product.

[0093] In some embodiments, any one of the compounds and therapeutic agents disclosed herein is orally administered.The compositions of the present application suitable for oral administration can be provided as separate units, such as capsules, sachets, granules or tablets, powders or granules, solutions or suspensions in aqueous or non-aqueous liquids, oil-in-water emulsions, water-in-oil emulsions, filled in liposomes, or as boluses, each containing a predetermined amount (e.g., effective amount) of active ingredient.Soft gelatin capsules can be useful for containing such suspensions, which can advantageously increase the absorption rate of the compound.For tablets for oral use, commonly used carriers include lactose, sucrose, glucose, mannitol, and silicic acid and starch. Other acceptable excipients include: a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retardants such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) humectants such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspension is administered orally, active ingredient is combined with emulsifier and suspending agent.If desired, certain sweetener and / or flavoring agent and / or coloring agent can be added.The composition suitable for oral administration includes the lozenge that comprises flavor base, usually with sucrose and acacia or tragacanth, and the pastille that comprises active ingredient in the inactive base such as gelatin and glycerin or sucrose and acacia.

[0094] Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection or infusion solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The preparations may be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a lyophilized (lyophilized) state, requiring only the addition of a sterile liquid carrier, such as water for injection, saline (e.g., 0.9% saline), or 5% dextrose solution, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Injectable solutions may be in the form of, for example, sterile injectable aqueous or oleaginous suspensions. The suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, as solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may contain long-chain alcohol diluents or dispersants.

[0095] The pharmaceutical composition of the present application can be administered in the form of suppositories for rectal administration.These compositions can be prepared by mixing the compound of the present application with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, so that it melts in the rectum and releases the active ingredient.Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0096] The pharmaceutical compositions of the present application can be administered by nasal aerosol or inhalation. Such compositions can be prepared according to techniques well known in the art of pharmaceutical formulation, and can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. See, for example, U.S. Patent No. 6,803,031. Additional formulations and methods for intranasal administration can be found in Ilium, L., J Pharm Pharmacol, 56:3-17, 2004 and Ilium, L., Eur J Pharm Sci 11:1-18, 2000.

[0097] The topical compositions of the present disclosure can be prepared and used in the form of aerosol sprays, creams, emulsions, solids, liquids, dispersions, foams, oils, gels, hydrogels, lotions, mousses, ointments, powders, patches, pomades, solutions, pump sprays, sticks, towelettes, soaps, or other forms commonly used in the field of topical administration and / or cosmetics and skin care formulations. The topical composition can be in emulsion form. Topical administration of the pharmaceutical compositions of the present application is particularly useful when the desired treatment involves areas or organs that are easily accessible by topical application. In some embodiments, a topical composition comprises any one of the compounds and therapeutic agents disclosed herein in combination with one or more additional ingredients, carriers, excipients, or diluents, including, but not limited to, absorbents, anti-irritants, anti-wheezing agents, preservatives, antioxidants, colorants / pigments, emollients (moisturizers), emulsifiers, film-forming / retentive agents, fragrances, foliar exfoliants, prescription medications, preservatives, exfoliants, silicones, skin-toning / repairing agents, slip agents, sunscreen actives, surfactants / detergent cleansers, penetration enhancers, and thickeners.

[0098] The compounds and therapeutic agents of the present application may be incorporated into compositions for coating implantable medical devices, such as prostheses, artificial valves, vascular grafts, stents, or catheters. Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in U.S. Patent Nos. 6,099,562, 5,886,026, and 5,304,121. The coating is typically a biocompatible polymeric material, such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate, and mixtures thereof. The coating may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids, or combinations thereof to impart controlled-release properties to the composition. Coatings for invasive devices are included within the definition of pharmaceutically acceptable carrier, adjuvant, or vehicle, as those terms are used herein.

[0099] According to another embodiment, the present application provides an implantable drug release device that contains or contains a compound or therapeutic agent, or a composition that includes a compound or therapeutic agent of the present application, such that the compound or therapeutic agent is released from the device and is therapeutically active.

[0100] Dosage and Regimen In the pharmaceutical compositions of the present application, the compounds described herein are present in an effective amount (eg, a therapeutically effective amount).

[0101] The effective amount may vary depending on the condition being treated, the severity of the condition, the route of administration, the sex, age, and general health of the subject, the use of excipients, the possibility of co-administration with other therapeutic treatments such as the use of other drugs, and the judgment of the treating physician.

[0102] In some embodiments, the compounds of the present application are used at concentrations that are easily and safely achieved in human blood and tissues.

[0103] In some embodiments, an effective amount of a compound described herein can be, for example, about 0.001 mg / kg to about 500 mg / kg (e.g., about 0.001 mg / kg to about 200 mg / kg, about 0.01 mg / kg to about 200 mg / kg, about 0.01 mg / kg to about 150 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0 The dose may be in the range of 0.01 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 200 mg / kg, about 0.1 mg / kg to about 150 mg / kg, about 0.1 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 2 mg / kg, about 0.1 mg / kg to about 1 mg / kg, or about 0.1 mg / kg to about 0.5 mg / kg).

[0104] In some embodiments, an effective amount of a compound described herein is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, or about 150 mg / kg.

[0105] The aforementioned dosages can be administered on a daily basis (e.g., as a single dose or as two or more divided doses, e.g., once daily, twice daily, three times daily) or non-daily (e.g., every other day, every two days, every three days, once a week, twice a week, every two weeks, once a month).

[0106] kit The present disclosure also provides pharmaceutical kits useful for treating the disorders, diseases, and conditions mentioned herein, for example, comprising one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the present disclosure.Such kits can optionally further comprise one or more of various conventional pharmaceutical kit components, such as a container with one or more pharmaceutically acceptable carriers, additional containers, etc.The kit can also include instructions, either as a package insert or label, indicating the amount of components to be administered, directions for administration, and / or directions for mixing the components.The kit can optionally include any one of the additional therapeutic agents described herein, or a pharmaceutically acceptable salt thereof, in any one of the amounts and dosage forms described herein.

[0107] Screening assays In some cases, the present application provides screening assays for identifying inhibitors of gasdermin pore formation, inflammasome-mediated cell death (pyroptosis), cellular cytokine secretion, and / or inflammatory caspases. Referring to FIG. 1 , in such assays, a sample may contain liposomes formed such that metal cations are trapped inside the liposomes. The sample may also contain a full-length gasdermin protein containing a protease cleavage site, a test compound, and a ligand capable of complexing with the metal cation trapped within the liposome. To determine whether a compound inhibits pore formation, a protease enzyme is added to the sample. The protease enzyme cleaves N-terminal gasdermin fragments from the full-length gasdermin protein. In the absence of the test compound, or if the test compound is inactive in the assay, these N-terminal fragments then bind to the liposomal lipids, forming pores within the liposomes through which the metal cations leak from the liposomes into the external buffer. In the external buffer, the metal cations bind to and complex with the chelating ligand. This complex has higher fluorescence than the metal cation or chelating ligand when the cation and ligand are not bound to each other. The increased fluorescence of the sample can be detected using appropriate instrumentation, indicating leakage of the metal cation from the liposome. For example, in the presence of an active test compound that chemically reacts with gasdermin, the NT gasdermin fragment chemically modified by the test compound does not form pores in the liposome. Therefore, the metal cation remains encapsulated in the liposome and does not bind to the chelating ligand in the external buffer. Therefore, there is no liposome leakage in the sample, and no increase in fluorescence is detected. Active compounds can be identified in an assay by comparing the fluorescence of a sample containing the test compound with the fluorescence of a control sample that does not contain any test compound. If a compound is considered active in the assay, the fluorescence of the sample will be lower than the fluorescence of the control. In some embodiments, if a compound is considered active, the fluorescence of the sample will be at least about 10%, 20%, 30%, 40%, 50%, or 60% lower than the fluorescence of the control.

[0108] In some cases, the metal cation is Ce 3+ , Fe 2+ , Fe 3+ , Zn 2+ , Cu 2+ , Mg 2+ , and Tb 3+ In some embodiments, the metal cation is selected from Tb 3+ In some cases, the chelating ligand is selected from ethylenediaminetetraacetic acid (EDTA), dipicolinic acid (DPA), ethylenediamine, porphyrin, and dimercaptol. In some embodiments, the chelating ligand is dipicolinic acid (DPA).

[0109] In some cases, the gasdermin protein in the sample is selected from GSDMA, GSMDB, GSDMC, GSDMD, DFNA5, and DFNB59. In some cases, the gasdermin protein contains a rhinovirus 3C protease cleavage site (GSDM-3C). For example, the gasdermin protein in the sample is a gasdermin D protein having a 3C protease cleavage site (GSDMD-3C).

[0110] In some cases, the protease enzyme is selected from an inflammatory caspase and a rhinovirus 3C protease. The inflammatory caspase can be caspase 1 or caspase 11. In some embodiments, the gasdermin protein is GSDM-3C and the protease enzyme is 3C protease. In other embodiments, the gasdermin protein is GSDMD-3C and the protease enzyme is 3C protease.

[0111] In yet another general aspect, the application provides: Inhibiting gasdermin pore formation in cells, and / or Inhibiting inflammasome-mediated death of cells (pyroptosis), and / or Inhibits cytokine secretion from cells, and / or Inhibiting intracellular inflammatory caspases, and / or react covalently with the cysteines of the gasdermin protein within the cell, and / or A method for identifying a compound that covalently reacts with a cysteine ​​of an inflammatory signaling molecule selected from a sensor, an adaptor, and a transcription factor, or a regulator thereof, is provided, The method comprises: d) providing a sample comprising a liposome comprising a metal cation capable of complexing with a chelating ligand, the chelating ligand, and a test compound; e) contacting the test compound with the N-terminal gasdermin protein fragment; f) determining whether the test compound inhibits leakage of metal cations from the liposomes, wherein inhibition of leakage of metal cations from the liposomes indicates that the test compound: Inhibiting gasdermin pore formation in cells, and / or Inhibiting inflammasome-mediated death of cells (pyroptosis), and / or Inhibits cytokine secretion from cells, and / or Inhibiting intracellular inflammatory caspases, and / or react covalently with the cysteines of the gasdermin protein within the cell, and / or determining that the inflammatory signaling molecule covalently reacts with a cysteine ​​of the inflammatory signaling molecule selected from a sensor, an adaptor, and a transcription factor, or a regulator thereof.

[0112] definition As used herein, the term "about" means "approximately" (eg, plus or minus about 10% of the stated value).

[0113] At various places in the present specification, substituents of compounds of the invention are disclosed in groups or in ranges. It is specifically intended that the invention include each and every individual subcombination of the members of such groups and ranges. For example, "C 1~6The term "alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0114] Various aryl, heteroaryl, cycloalkyl, and heterocycloalkyl rings are described in various places herein. Unless otherwise specified, these rings can be attached to the rest of the molecule at any ring member, as permitted by valence. For example, the term "pyridine ring" or "pyridinyl" can refer to a pyridin-2-yl, pyridin-3-yl, or pyridin-4-yl ring.

[0115] It will be further understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0116] The term "aromatic" refers to a carbocyclic or heterocyclic ring having one or more polyunsaturated rings that have aromatic character (i.e., have (4n+2) delocalized π (pi) electrons, where n is an integer).

[0117] The term "n-membered," where n is an integer, typically describes the number of ring-forming atoms in a moiety where n is the number of ring-forming atoms. For example, piperidinyl is an example of a 6-membered heterocycloalkyl ring, pyrazolyl is an example of a 5-membered heteroaryl ring, pyridyl is an example of a 6-membered heteroaryl ring, and 1,2,3,4-tetrahydro-naphthalene is an example of a 10-membered cycloalkyl group.

[0118] As used herein, the phrase "optionally substituted" means unsubstituted or substituted. The substituents are independently selected, and the substitution can be at any chemically accessible position. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced with a substituent. A single divalent substituent, for example, oxo, can replace two hydrogen atoms. It is understood that substitution at a given atom is limited by valency.

[0119] Throughout the definition, "C n~m " denotes a range inclusive of the endpoints, where n and m are integers and indicate the number of carbons. Examples include C 1~4 , C 1~6 Examples include:

[0120] As used herein, "C" when used alone or in combination with other terms n~m The term "alkyl" refers to a saturated hydrocarbon group that may be straight or branched, having n to m carbons. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, and higher homologs such as 2-methyl-1-butyl, n-pentyl, 3-pentyl, n-hexyl, and 1,2,2-trimethylpropyl. In some embodiments, alkyl groups contain 1 to 6 carbon atoms, 1 to 4 carbon atoms, 1 to 3 carbon atoms, or 1 to 2 carbon atoms.

[0121] As used herein, "C" when used alone or in combination with other terms n~m The term "haloalkyl" refers to an alkyl group having 1 to 2s+1 halogen atoms, which may be the same or different, where "s" is the number of carbon atoms in the alkyl group, and the alkyl group has n to m carbon atoms. In some embodiments, the haloalkyl group is exclusively fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0122] As used herein, "C n~m "Alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds and having n to m carbons. Exemplary alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like. In some embodiments, the alkenyl moiety has 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0123] As used herein, "C n~m "Alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds and having n to m carbons. Exemplary alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some embodiments, the alkynyl moiety has 2 to 6, 2 to 4, or 2 to 3 carbon atoms.

[0124] As used herein, "C" when used alone or in combination with other terms n~m The term "alkylene" refers to a divalent alkyl linker having n to m carbons. Examples of alkylene groups include, but are not limited to, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,1-diyl, propane-1,3-diyl, propane-1,2-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, 2-methyl-propane-1,3-diyl, and the like. In some embodiments, the alkylene moiety has 2 to 6, 2 to 4, 2 to 3, 1 to 6, 1 to 4, or 1 to 2 carbon atoms.

[0125] As used herein, "C" when used alone or in combination with other terms n~mThe term "alkoxy" refers to a group of formula -O-alkyl, where the alkyl group has n to m carbons. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), butoxy (e.g., n-butoxy and tert-butoxy), and the like. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0126] As used herein, "C n~m "Haloalkoxy" refers to a group of the formula -O-haloalkyl having n to m carbon atoms. An exemplary haloalkoxy group is OCF. In some embodiments, the haloalkoxy group is exclusively fluorinated. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0127] As used herein, the term "amino" refers to a group of formula -NH2.

[0128] As used herein, "C" when used alone or in combination with other terms n~m The term "alkylamino" refers to a group of formula -NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylamino groups include, but are not limited to, N-methylamino, N-ethylamino, N-propylamino (e.g., N-(n-propyl)amino and N-isopropylamino), N-butylamino (e.g., N-(n-butyl)amino and N-(tert-butyl)amino), and the like.

[0129] As used herein, "di(C n~m The term "N(alkyl)amino" refers to a group of formula -N(alkyl)2, where the two alkyl groups each independently have n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0130] As used herein, "C n~m The term "alkoxycarbonyl" refers to a group of formula -C(O)O-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl (e.g., n-propoxycarbonyl and isopropoxycarbonyl), butoxycarbonyl (e.g., n-butoxycarbonyl and tert-butoxycarbonyl), and the like.

[0131] As used herein, "C n~m The term "alkylcarbonyl" refers to a group of formula -C(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkylcarbonyl groups include, but are not limited to, methylcarbonyl, ethylcarbonyl, propylcarbonyl (e.g., n-propylcarbonyl and isopropylcarbonyl), butylcarbonyl (e.g., n-butylcarbonyl and tert-butylcarbonyl), and the like.

[0132] As used herein, "C n~m The term "alkylcarbonylamino" refers to a group of formula -NHC(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0133] As used herein, "C n~m The term "alkylsulfonylamino" refers to a group of formula -NHS(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0134] As used herein, the term "aminosulfonyl" refers to a group of formula -S(O)2NH2.

[0135] As used herein, "C n~m The term "alkylaminosulfonyl" refers to a group of formula -S(O)NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0136] As used herein, "di(C n~m The term "(alkyl)aminosulfonyl" refers to a group of formula -S(O)N(alkyl), where each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0137] As used herein, the term "aminosulfonylamino" refers to a group of formula -NHS(O)2NH2.

[0138] As used herein, "C n~m The term "alkylaminosulfonylamino" refers to a group of formula -NHS(O)NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0139] As used herein, "di(C n~m The term "NHS(O)N(alkyl)aminosulfonylamino" refers to a group of formula -NHS(O)N(alkyl), where each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0140] As used herein, the term "aminocarbonylamino," employed alone or in combination with other terms, refers to a group of formula -NHC(O)NH2.

[0141] As used herein, "C n~m The term "alkylaminocarbonylamino" refers to a group of formula -NHC(O)NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0142] As used herein, "di(C n~m The term "NHC(O)N(alkyl)aminocarbonylamino" refers to a group of formula -NHC(O)N(alkyl)2, where each alkyl group independently has n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0143] As used herein, the term "carbamyl" refers to a group of formula -C(O)NH2.

[0144] As used herein, "C n~m The term "alkylcarbamyl" refers to a group of formula -C(O)-NH(alkyl), where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0145] As used herein, "di(C n~m The term "-C(O)N(alkyl)carbamyl" refers to a group of formula -C(O)N(alkyl)2, where the two alkyl groups each independently have n to m carbon atoms. In some embodiments, each alkyl group independently has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0146] As used herein, the term "thio" refers to a group of formula -SH.

[0147] As used herein, "C n~m The term "alkylthio" refers to a group of formula -S-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0148] As used herein, "C n~m The term "alkylsulfinyl" refers to a group of formula -S(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0149] As used herein, "C n~m The term "alkylsulfonyl" refers to a group of formula -S(O)-alkyl, where the alkyl group has n to m carbons. In some embodiments, the alkyl group has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.

[0150] As used herein, the term "carbonyl," employed alone or in combination with other terms, refers to a -C(=O)- group, which may also be written as C(O).

[0151] As used herein, the term "carboxy" refers to the group --C(O)OH.

[0152] As used herein, "cyano-C" 1~3 The term "alkyl" refers to a group of the formula -(C 1~3 refers to the group alkylene-CN.

[0153] As used herein, "HO-C 1~3 The term "alkyl" refers to a group of the formula -(C 1~3 refers to the group alkylene-OH.

[0154] As used herein, "halo" refers to F, Cl, Br, or I. In some embodiments, halo is F, Cl, or Br.

[0155] As used herein, the term "aryl," used alone or in combination with other terms, refers to an aromatic hydrocarbon group that can be monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings). n~m "Aryl" refers to an aryl group having n to m ring carbon atoms. Examples of aryl groups include phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, an aryl group has 6 to 10 carbon atoms. In some embodiments, an aryl group is phenyl or naphthyl.

[0156] As used herein, "cycloalkyl" refers to a non-aromatic cyclic hydrocarbon, including cyclized alkyl and / or alkenyl groups. Cycloalkyl groups can include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocyclic rings. The ring-forming carbon atoms of a cycloalkyl group can be optionally substituted with one or two independently selected oxo or sulfido groups (e.g., C(O) or C(S)). The definition of cycloalkyl also includes moieties having one or more aromatic rings fused with (i.e., having a common bond with) a benzo or thienyl derivative, such as cyclopentane, cyclohexane, etc. Cycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including the ring-forming atoms of the fused aromatic ring. Cycloalkyl groups can have 3, 4, 5, 6, 7, 8, 9, or 10 ring-forming carbon atoms (C 3~10 In some embodiments, cycloalkyl can have C 3~10 In some embodiments, the cycloalkyl is C 3~7Monocyclic cycloalkyl. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcarnyl, adamantyl, and the like. In some embodiments, the cycloalkyl is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0157] As used herein, "heteroaryl" refers to a monocyclic or polycyclic aromatic heterocycle having at least one heteroatom ring member selected from sulfur, oxygen, and nitrogen. In some embodiments, the heteroaryl ring has 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, any ring-forming N in the heteroaryl moiety can be an N-oxide. In some embodiments, the heteroaryl is a 5- to 10-membered monocyclic or bicyclic heteroaryl having 1, 2, 3, or 4 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5- to 6-membered monocyclic heteroaryl having 1 or 2 heteroatom ring members independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heteroaryl is a 5- or 6-membered heteroaryl ring. A 5-membered heteroaryl ring is a heteroaryl with a ring having 5 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 5-membered heteroaryls are thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl, 1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl. A 6-membered heteroaryl ring is a heteroaryl with a ring having 6 ring atoms, wherein one or more (e.g., 1, 2, or 3) ring atoms are independently selected from N, O, and S. Exemplary 6-membered heteroaryls are pyridyl, pyrazinyl, pyrimidinyl, triazinyl, and pyridazinyl.

[0158] As used herein, "heterocycloalkyl" refers to a non-aromatic monocyclic or polycyclic heterocycle having one or more ring-forming heteroatoms selected from O, N, or S. Included within heterocycloalkyl are monocyclic 4-, 5-, 6-, 7-, 8-, 9-, or 10-membered heterocycloalkyl groups. Heterocycloalkyl groups can also include spirocycles. Exemplary heterocycloalkyl groups include pyrrolidin-2-one, 1,3-isoxazolidin-2-one, pyranyl, tetrahydropyran, oxetanyl, azetidinyl, morpholino, thiomorpholino, piperazinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, pyrrolidinyl, isoxazolidinyl, isothiazolidinyl, pyrazolidinyl, oxazolidinyl, thiazolidinyl, imidazolidinyl, azepanyl, benzazepene, and the like. The ring-forming carbon atoms and heteroatoms of a heterocycloalkyl group can be optionally substituted with one or two independently selected oxo or sulfido groups (e.g., C(O), S(O), C(S), or S(O)). The heterocycloalkyl group can be bonded through a ring-forming carbon atom or ring-forming heteroatom. In some embodiments, a heterocycloalkyl group contains zero to three double bonds. In some embodiments, a heterocycloalkyl group contains zero to two double bonds. Also included within the definition of heterocycloalkyl are moieties that have one or more aromatic rings fused (i.e., sharing a common bond with) the cycloalkyl ring, e.g., benzo or thienyl derivatives such as piperidine, morpholine, azepine, etc. Heterocycloalkyl groups containing fused aromatic rings can be bonded through any ring-forming atom, including a ring-forming atom of the fused aromatic ring. In some embodiments, a heterocycloalkyl is a monocyclic 4- to 6-membered heterocycloalkyl having one or two heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members. In some embodiments, the heterocycloalkyl is a monocyclic or bicyclic 4-10 membered heterocycloalkyl having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur and having one or more oxidized ring members.

[0159] In certain places, definitions or embodiments refer to specific rings (for example, azetidine rings, pyridine rings, etc.). Unless otherwise specified, these rings can be bonded to any ring member as long as the valence of the atom is not exceeded. For example, the azetidine ring can be bonded to any position on the ring, while the pyridin-3-yl ring is bonded to the 3-position.

[0160] As used herein, the term "oxo" refers to an oxygen atom as a divalent substituent that forms a carbonyl group when attached to carbon (e.g., C=O) or to a heteroatom that forms a sulfoxide or sulfone group.

[0161] The term "compound," as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the depicted structure. A compound identified herein by name or structure as one particular tautomeric form is intended to include other tautomeric forms unless otherwise specified.

[0162] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). Unless otherwise specified, all stereoisomers, such as enantiomers and diastereomers, are intended. Compounds of the present invention containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials, such as by resolution of racemic mixtures or stereoselective synthesis, are known in the art. Many geometric isomers of olefins, C=N double bonds, N=N double bonds, and the like, can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and can be isolated as a mixture of isomers or as separated isomeric forms. In some embodiments, the compounds have the (R)-configuration. In some embodiments, the compounds have the (S)-configuration.

[0163] The compounds provided herein also include tautomeric forms. Tautomeric forms result from the exchange of a single bond with an adjacent double bond, accompanied by the simultaneous migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states with the same empirical formula and total charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and cyclic forms, in which protons can occupy two or more positions in the heterocyclic ring system, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0164] As used herein, the term "cell" is intended to refer to a cell that is in vitro, ex vivo, or in vivo. In some embodiments, an ex vivo cell may be part of a tissue sample excised from an organism, such as a mammal. In some embodiments, an in vitro cell may be a cell in cell culture. In some embodiments, an in vivo cell is a cell that lives within an organism, such as a mammal.

[0165] As used herein, the term "contacting" refers to bringing together the indicated moieties in an in vitro system or an in vivo system. For example, "contacting" a compound of the present invention with gasdermin includes administering a compound of the present invention to an individual or patient, such as a human, who has gasdermin, as well as introducing a compound of the present invention into a sample containing, for example, a cell preparation or purified preparation containing gasdermin.

[0166] As used herein, the terms "individual," "patient," or "subject" refer to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse, or primate, and most preferably a human.

[0167] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to an amount of an active compound or pharmaceutical agent that elicits the biological or medical response in a tissue, system, animal, individual or human that is desired by a researcher, veterinarian, physician or other clinician.

[0168] As used herein, the term "treating" or "treatment" refers to 1) inhibiting a disease, e.g., inhibiting a disease, condition, or disorder in an individual experiencing or displaying the pathology or symptoms of the disease, condition, or disorder (i.e., arresting further development of the pathology and / or symptoms), or 2) ameliorating a disease, e.g., ameliorating a disease, condition, or disorder in an individual experiencing or displaying the pathology or symptoms of the disease, condition, or disorder (i.e., reversing the pathology and / or symptoms).

[0169] As used herein, the term "preventing" or "prevention" of a disease, condition, or disorder refers to reducing the risk of development of the disease, condition, or disorder in a subject or group of subjects (e.g., a subject or group of subjects susceptible to or susceptible to the disease, condition, or disorder). In some embodiments, preventing a disease, condition, or disorder refers to reducing the likelihood of acquiring the disease, condition, or disorder and / or its associated symptoms. In some embodiments, preventing a disease, condition, or disorder refers to completely or nearly completely halting the development of the disease, condition, or disorder. [Example]

[0170] Cytoplasmic sensing of pathogens and danger by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to induce cytokine maturation and inflammatory cell death (pyroptosis). Inflammation recruits immune cells to orchestrate a protective immune response but can also cause pathology. Pore formation by the inflammatory caspase substrate gasdermin D (GSDMD) has recently been identified as the mechanism responsible for pyroptosis and the release of inflammatory mediators. Inhibiting GSDMD is an attractive strategy for suppressing inflammation. The experimental results described below demonstrate that disulfiram, a drug used to treat chronic alcoholism, is an inhibitor of pore formation by GSDMD but not other members of the GSDMD family. Disulfiram blocks inflammasome-mediated pyroptosis and cytokine release in cells and inhibits LPS-induced septic death in mice. At nanomolar concentrations, disulfiram covalently modifies human Cys191 (mouse Cys192) in GSDMD, blocking pore formation and pyroptosis.

[0171] General method Mice. Eight-week-old female C57BL / 6 wild-type mice were purchased from The Jackson Laboratory and maintained in the SPF facility at Harvard Medical School. All mouse experiments were performed using protocols approved by the Animal Care and Use Committees of Boston Children's Hospital and Harvard Medical School.

[0172] Drug administration and LPS-induced sepsis in mice. Mice were treated with disulfiram (C-23, DSF, 50 mg / kg) formulated in sesame oil or vehicle (control) by intraperitoneal injection at the indicated times. In the group of mice shown in Figure 5h, copper gluconate (0.15 mg / kg) was administered intraperitoneally 6 h before the first injection of DSF. Sepsis was induced in C57BL / 6 mice (8–10 weeks old) by intraperitoneal injection of LPS (E. coli O111:B4) at the indicated concentrations. In some experiments, mice were treated with copper gluconate (0.15 mg / kg) or vehicle by intraperitoneal injection 5 h before LPS challenge, and then administered DSF (50 mg / kg) dissolved in sesame oil or vehicle intraperitoneally 4 h before and immediately before LPS challenge (15 mg / kg intraperitoneally). Six hours after LPS challenge, peritoneal cells were harvested by rinsing the peritoneal cavity with ice-cold PBS containing 3% FBS. To measure cytokines, blood samples were collected by tail vein bleeding 12 hours after LPS challenge and allowed to clot at room temperature. Serum obtained after centrifugation at 2,000 × g for 10 minutes was analyzed for inflammatory cytokines by ELISA.

[0173] Reagents. β-Mercaptoethanol (2ME), dithiothreitol (DTT), terbium(III) chloride (TbCl), dipicolinic acid (DPA), and copper gluconate were obtained from Sigma-Aldrich. Compound C-23 and its analogs: tetraethylthiuram disulfide (C-23), tetramethylthiuram disulfide (C-23A1), tetrabutylthiuram disulfide (C-23A3), 4-methylpiperazine-1-carbothioic acid dithioperoxyanhydride (C-23A4), tetraphenylthiuram disulfide (C-23A5), N,N'-dimethyl-N,N'-(4,4'-dimethyldiphenyl)-2-methyl-4-methyl-1-methyl-2-methyl-4-methyl-1-methyl-2-methyl-1 ... N,N'-dimethyl-N,N'-di(4-pyridinyl)thiuram disulfide (C-23A6), di(4-morpholinyl)dithioperoxyanhydride (C-23A7), N,N'-dimethyl-N,N'-di(4-pyridinyl)thiuram disulfide (C-23A8), pyrrolidine-1-carbothioic acid dithioperoxyanhydride (C-23A10), and dimethyldiphenylthiuram disulfide (C-23A11) were obtained from Sigma-Aldrich. Tetraisopropylthiuram disulfide (C-23A2) and dicyclopentamethylenethiuram disulfide (C-23A9) were obtained from Oakwood Chemicals. Tetrabenzylthiuram disulfide (C-23A12) was obtained from AK Scientific. Phorbol 12-myristate 13-acetate (PMA) and DMSO were obtained from Sigma-Aldrich. Ultra LPS and nigericin were obtained from InvivoGen. The pan-caspase inhibitor z-VAD-fmk was obtained from BD Bioscience. Complete protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail were obtained from Roche. Necrosulfonamide, necrostatin-1, dimethyl fumarate, ibrutinib, and afatinib were obtained from Sigma-Aldrich. LDC7559 was synthesized by Information Research Labs.

[0174] Biomolecule: Monoclonal antibodies against GSDMD were generated in-house by immunizing 6-week-old BALB / c mice with recombinant human GSDMD and boosting them with recombinant human GSDMD-NT according to standard protocols. Serum samples were collected to assess the titer of reactive antibodies, and spleen cells were fused with SP2 / 0 myeloma cells. Hybridomas were selected, and supernatants from the resulting clones were screened by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunofluorescence microscopy. Tubulin antibodies were obtained from Sigma-Aldrich. Phospho-IκBα, IκBα, phospho-NF-κBp65, cleaved human caspase-1 (Asp297), and NLRP3 antibodies were obtained from Cell Signaling Technology. ASC antibody (AL177) and mouse caspase-1p20 antibodies were obtained from AdipoGen. Human and mouse IL-1β antibodies were obtained from R&D Systems. HMGB1 and mouse GSDMD antibodies were obtained from Abcam.

[0175] Liposome leakage assay: A fluorogenic liposome leakage assay was performed using Tb incubated with GSDMD and caspase-11. 3+ Tb from loaded liposomes 3+ (See References 7 and 9). See Figure 1. 3+ Liposome leakage was detected by an increase in fluorescence when GSDMD bound to dipicolinic acid (DPA) in buffer C. Human GSDMD (0.3 μM) was dispensed into wells (Corning 3820) containing PC / PE / CL liposomes (50 μM liposomal lipid) and incubated with test compounds for 1 h before adding caspase-11 (0.15 μM) to each well. Fluorescence intensity of the wells was measured at 545 nm with excitation at 276 nm 1 h after addition of caspase-11 using a Perkin Elmer EnVision plate reader. Final percent inhibition was calculated as [(fluorescence 試験化合物 -fluorescence 陰性対照 ) / (fluorescence 陽性対照 -fluorescence 陰性対照The IC of the test compound was calculated as (IC = IC = 1 / 2 × 100), where wells with GSDMD without the test compound were used as positive controls and wells without caspase-11 were used as negative controls. 50 was determined in concentration-response experiments over the dose range of 0.008 to 50 μM.

[0176] Protein expression and purification: The full-length human GSDMD sequence was cloned into the pDB.His.MBP vector with a tobacco etch virus (TEV)-cleavable N-terminal His6-MBP tag using NdeI and XhoI restriction sites. Human GSDMD-3C and mouse GSDMA3-3C mutants were constructed by QuikChange Mutagenesis (Agilent Technologies). For expression of full-length GSDMD, GSDMD-3C, GSDMA3, and GSDMA3-3C, E. coli BL21(DE3) cells harboring the indicated plasmids were cultured at OD . 600 After induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the β-glucan reached 0.8, 50 μg ml -1 Cells were grown overnight at 18°C ​​in LB medium supplemented with 100 mM kanamycin. Cells were sonicated in lysis buffer containing 25 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, and 5 mM 2ME at pH 8.0. The lysate was clarified by centrifugation at 40,000 x g for 1 hour at 4°C. The supernatant containing the target protein was incubated with Ni-NTA resin (Qiagen) for 30 minutes at 4°C. After incubation, the resin-supernatant mixture was poured into a column, and the resin was washed with lysis buffer. The protein was eluted using lysis buffer supplemented with 100 mM imidazole. The His6-MBP tag was removed by TEV protease digestion overnight at 16°C. The cleaved protein was purified using a HiTrap Q ion exchange and Superdex 200 gel filtration column (GE Healthcare Life Sciences).

[0177] The caspase-11 sequence was cloned into the pFastBac-HTa vector with a TEV-cleavable N-terminal His6-tag using EcoRI and XhoI restriction sites. Baculovirus was prepared using the Bac-to-Bac system (Invitrogen), and the protein was expressed in Sf9 cells according to the manufacturer's instructions. 10 ml of His-caspase-11 baculovirus was used to infect 1 L of Sf9 cells. Cells were harvested 48 hours postinfection, and His6-caspase-11 was purified according to the same protocol as for His6-MBP-GSDMD. The eluate from the Ni-NTA resin was collected for subsequent assays.

[0178] Liposome preparation: PC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 25 mg / mL in chloroform; 80 μL), PE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 25 mg / mL in chloroform; 128 μL), and CL (1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (sodium salt), 25 mg / mL in chloroform; 64 μL) were mixed, and the solvent was evaporated under a stream of N gas. The lipid mixture was suspended in 1 mL of buffer A (20 mM HEPES, 150 mM NaCl, 50 mM sodium citrate, and 15 mM TbCl) for 3 min. The suspension was pushed 30 times through a 100 nm Whatman® Nuclepore™ track-etched membrane to obtain homogeneous liposomes. The filtered suspension was purified by a size-exclusion column (Superose 6, 10 / 300GL) in buffer B (20 mM HEPES, 150 mM NaCl) to remove TbCl3 from the outer liposomes. The void fractions were pooled to create a stock of PC / PE / CL liposomes (1.6 mM). The liposomes were diluted to 50 μM in buffer C (20 mM HEPES, 150 mM NaCl, and 50 μM DPA) for use in high-throughput screening.

[0179] Fluorescent protein labeling and microscale thermophoresis binding assay: His6-MBP-GSDMD was labeled with AlexaFluor-488 using a Molecular Probes protein labeling kit. Inhibitor binding to GSDMD was assessed using microscale thermophoresis (MST). Ligands (49 nM–150 μM) were incubated with purified AlexaFluor-488-labeled protein (80 nM) in assay buffer (20 mM HEPES, 150 mM NaCl, 0.05% Tween 20) for 30 min. Samples were loaded into NanoTemper Monolith NT.115 glass capillaries, and MST was performed using 20% ​​LED power and 40% MST power. The mass action equation and NanoTemper software were used to calculate the K d values ​​were calculated.

[0180] Caspase-1 and caspase-11 inhibition assay: The fluorogenic assay for caspase-1 and caspase-11 activity is based on the release of 7-amino-4-methylcoumarin (AMC) from the caspase substrate Ac-YVAD-AMC. Compounds (8 nM–50 μM) were incubated with 0.5 U of caspase-1 and caspase-11 in assay buffer (20 mM HEPES, 150 mM NaCl) in a 384-well plate (Corning 3820) for 30 min before adding Ac-YVAD-AMC (40 μM) to initiate the reaction. The reaction was monitored in a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, California, USA) with excitation / emission wavelengths of 350 / 460 nm. The fluorescence intensity of each reaction was recorded every 2 min for 2 h.

[0181] Cell viability assay. THP-1 cells seeded at a density of 4000 cells per well in 96-well plates (Corning 3610) were differentiated by exposure to 50 nM PMA for 36 hours before priming with 100 ng / mL LPS. Initially activated THP-1 cells were pretreated with each test compound for 1 hour, followed by the addition of 20 μM nigericin or medium as a control. The number of viable cells was determined by CellTiter-Glo assay after 1.5 hours. The final percent cell viability was calculated using the formula [(luminescence 試験化合物 - Luminous 陰性対照 ) / (Light 陽性対照 - Luminous 陰性対照 The IC of each test compound in the cell viability assay was calculated using the formula: (IC value) × 100, where wells with LPS only were used as positive controls and wells treated with LPS and nigericin were used as negative controls. 50 was determined by concentration-response experiments over the dose range of 0.39 to 50 μM.

[0182] Mass Spectrometry and Sample Preparation. Gel bands were cut into 1 mm slices and placed in separate 1.5 mL polypropylene tubes. 100 μL of 50% acetonitrile in 50 mM ammonium bicarbonate buffer was added to each tube, and the samples were then incubated at room temperature for 20 minutes. This step was repeated, if necessary, to destain the gel. The gel slices were then incubated with 55 mM iodoacetamide (in 50 mM ammonium bicarbonate) for 45 minutes at room temperature in the dark, after which the gel was washed sequentially with 50 mM ammonium bicarbonate, water, and acetonitrile. The samples were then dried in a Speedvac for 20 minutes. Trypsin (Promega Corp.) (10 ng / μL in 25 mM ammonium bicarbonate, pH 8.0) was added to each sample tube to cover the gel, and the samples were then incubated at 37°C for 6 hours or overnight.

[0183] After digestion, samples were acidified with 0.1% formic acid (FA), and 3 μl of the tryptic peptide solution was injected. Nano-LC / MS / MS was performed on a Thermo Scientific Orbitrap Fusion system coupled to a Dionex Ultimat 3000 nano-HPLC with a 40-well standard tray and autosampler. Samples were injected onto a trap column (300 μM, 1 d. × 5 mm, C18 PepMap 100) and then onto a C18 reversed-phase nano-LC column (Acclaim PepMap 100, 75 μm × 25 cm) heated to 50 °C. Mobile phases A (99.9% water, 0.1% FA) and B (99.9% acetonitrile, 0.1% FA) were used with a 60-minute LC gradient at a flow rate of 400 nL / min. The eluted peptides were sprayed into a mass spectrometer through a charged emitter tip (PicoTip Emitter, New Objective, 10 ± 1 μm) using the following parameters: tip voltage, +2.2 kV; Fourier transform mass spectrometry (FTMS) mode (resolution 120,000) for MS acquisition of precursor ions; and ion trap mass spectrometry (ITMS) mode (3 s) for subsequent MS / MS via higher energy collisional dissociation (HCD).

[0184] Proteome Discoverer 1.4 was used for protein identification and modification analysis. Raw data were analyzed using the UniPort Human Database. Other parameters included: enzyme selection as trypsin; maximum missed cleavage count = 2; dynamic modifications were carbamidomethyl (control), diethyldithiocarbamate (from C-23), and Bay11-7082 on cysteine; oxidized methionine; deaminated asparagine; and glutamine; precursor tolerance set to 10 ppm; MS / MS fragment tolerance set to 0.6 Da; and +2 to +4 charged peptides were considered. For significant matches, the peptide false discovery rate (FDR) was set to less than 1%.

[0185] Cell lines and treatments: THP-1 and HEK293T cells (obtained from ATCC) were grown in RPMI with 10% heat-inactivated fetal bovine serum supplemented with 100 U / ml penicillin G, 100 μg / ml streptomycin sulfate, 6 mM HEPES, 1.6 mM L-glutamine, and 50 μM 2ME. iBMDM cells from C57BL / 6 mice were kindly provided by J. Kagan (Boston Children's Hospital) and cultured in DMEM with the same supplements. Cells were confirmed to be free of mycoplasma contamination. Transient transfection of HEK293T cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. iBMDM cells were transfected by nucleofection using the Amaxa Nucleofector kit (VPA-1009). Generally, THP-1 cells were differentiated by incubation with 50 nM PMA for 36 hours, followed by initial activation with LPS (1 μg / ml) for 4 hours and treatment with nigericin (20 μM). To examine IκBα phosphorylation and degradation and IL-1β induction, PMA-differentiated THP-1 cells were stimulated with LPS (1 μg / ml) for 0.5, 1, and 4 hours, respectively. For noncanonical inflammasome activation, 1 million iBMDM cells were electroporated with 1 μg of ultraLPS.

[0186] Cytotoxicity and cell viability assays: Cell death and cell viability were determined by lactate dehydrogenase release assay using the CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega) and by measuring ATP levels using the CellTiter-Glo Luminescent Cell Viability Assay (Promega), respectively, according to the manufacturer's instructions. Luminescence and absorbance were measured on a BioTek Synergy2 plate reader.

[0187] Nanodiscs and pore reconstitution in negative-stain electron microscopy: The coding sequence for the membrane skeletal protein NW50 was cloned into the pET-28a vector, and the protein was expressed in E. coli BL21(DE3), purified by a refolding procedure, and covalently cyclized with sortase according to a previously described protocol. A lipid mixture containing phosphatidylserine (PS) and phosphatidylcholine (PC) (3:7 molar ratio) was solubilized in 60 mM sodium cholate and incubated with cyclized NW50 on ice for 1 h to assemble nanodiscs. The sodium cholate was then removed by overnight incubation with BioBeads SM-2 (Bio-Rad) at 4 °C. The biobeads were then removed using a 0.22 μm filter, and the assembled nanodiscs were further purified using a Superose6 10 / 300 gel filtration column (GE Healthcare Life Sciences) equilibrated with buffer D (50 mM Tris-HCl, 150 mM NaCl at pH 8.0) to remove excess lipids. To form GSDMD pores on nanodiscs, purified human GSDMD-3C was incubated with 3C protease in the presence of nanodiscs for 6 hours on ice. The pores were further purified on a Superose 6 column equilibrated with buffer D. To evaluate the effect of C-23, either human GSDMD-3C + 3C protease was incubated with C-23 (1:1 molar ratio) on ice for 30 minutes before addition to nanodiscs (pretreatment), or C-23 was added to pre-assembled pores for 30 minutes on ice (posttreatment). For negative stain electron microscopy, 5 μl of sample was placed on glow-discharged carbon-coated copper grids (Electron Microscopy Sciences), washed twice with buffer A, stained with 1% uranyl formate for 1 minute, and air-dried. Grids were prepared using Tecnai G 2 Images were taken on a Spirit BioTWIN electron microscope and recorded with an AMT 2k CCD camera (Harvard Medical School Electron Microscopy Facility).

[0188] Immunoblot analysis: Cell extracts were prepared using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with Complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor cocktail (Roche). Samples were subjected to SDS-PAGE, and resolved proteins were then transferred to PVDF membranes (Millipore). Immunoblots were probed with the indicated antibodies and visualized using the SuperSignal West Pico Chemiluminescence ECL kit (Pierce).

[0189] Caspase-1 activity assay in cells: To measure caspase-1 activation, THP-1 cells were seeded in 96-well plates and differentiated with PMA. After the indicated treatments, cells were incubated with the fluorescently activated caspase-1 substrate FAM-YVAD-FMK (Immunochemistry Technologies). Samples were read on a BioTek Synergy2 plate reader.

[0190] Cytokine measurement: The concentration of IL-1β in the culture supernatant or mouse serum was measured by ELISA kit (R&D Systems) according to the manufacturer's instructions.

[0191] Immunostaining and confocal microscopy: Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 minutes, permeabilized in 0.1% Triton X-100 in PBS for 5 minutes, and blocked with 5% BSA for 1 hour. Cells were then stained with the indicated primary antibodies followed by incubation with fluorescently conjugated secondary antibodies (Jackson ImmunoResearch). Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole) (Sigma-Aldrich). Slides were mounted using Aqua-Poly / Mount (Dako). Images were captured using a laser scanning confocal microscope (Olympus Fluoview FV1000 Confocal System) equipped with a 63x water immersion objective and Olympus Fluoview software (Olympus). All confocal images are representative of three independent experiments.

[0192] Statistics: Statistical analysis of two independent treatments was performed using Student's t-test. Survival curves and statistics of mice were analyzed using the Mantel-Cox log-rank test.

[0193] Example 1 - Inhibition of GSDMD pore formation by test compounds C-23 is a symmetric molecule known as disulfiram, a drug used to treat alcoholism (see reference 12). [ka]

[0194] IC of test compound 50 The values ​​and GSDMD binding results (assessed by microscale thermophoresis (MST)) are shown in Table 1. The chemical structures of the test compounds are shown in FIG. [Table 4] TIFF0007787250000013.tif36170

[0195] Test compounds were evaluated for GSDMD binding by microscale thermophoresis (MST). Figure 3 shows MST measurements of the binding of Alexa 488-labeled His-MBP-GSDMD (80 nM) to C-22, C-23, or C-24.

[0196] To assess whether test compounds inhibit pyroptosis, test compounds were added to PMA-differentiated and LPS-activated human THP-1 cells or mouse immortalized bone marrow-derived macrophages (iBMDMs) prior to activating canonical inflammasomes with nigericin or noncanonical inflammasomes by LPS electroporation. As discussed in the next paragraph, C-23 had IC values ​​of 7.67 ± 0.29 μM and 10.33 ± 0.50 μM for canonical and noncanonical inflammasome-dependent pyroptosis, respectively. 50 Disulfiram blocked pyroptosis at low levels and attenuated AIM2 inflammasome-induced cell death in poly(dA:dT)-transfected mouse iBMDMs (see Figure 10). Disulfiram also inhibited nigericin- or LPS-transfection-induced IL-1β secretion with potency comparable to that of the pan-caspase inhibitor z-VAD-fmk.

[0197] Experimental Results: The response curve of the compound disulfiram (C-23) in the liposome leakage assay is shown in Figure 2. In Figures 4, 6, and 8, PMA-differentiated LPS-activated human THP-1 cells were pretreated with the indicated concentrations of each compound for 1 hour before adding nigericin or medium. The number of viable cells was determined by CellTiter-Glo assay (Figures 4 and 6), and IL-1β in the culture supernatant was assessed by ELISA 2 hours later (Figure 8). In Figures 5, 7, and 9, mouse iBMDMs were pretreated with each test compound for 1 hour before electroporation with PBS or LPS. The number of viable cells was determined by CellTiter-Glo assay (Figures 5 and 7), and IL-1β in the culture supernatant was assessed by ELISA 2.5 hours later (Figure 9). In Figures 8 and 9, the test compound was added at a concentration of 40 μM. In Figure 10, mouse iBMDMs were pretreated with or without 30 μM C-23 for 1 hour before transfection with PBS or poly(dA:dT), and cell viability was analyzed 4 hours later using the CellTiter-Glo assay. The graph shows the mean ± SD, and the data are representative of three independent experiments. ** P<0.01.

[0198] To confirm that C-23 inhibits pore formation, we reconstituted human GSDMD-NT pores on covalently cyclized lipid nanodiscs constructed with the acidic lipids phosphatidylserine (PS) and phosphatidylcholine. Full-length GSDMD was engineered to replace the caspase cleavage site with a rhinovirus 3C protease cleavage site (GSDMD-3C) as previously described. 3C protease cleavage of the engineered GSDMD-3C liberates the active NT fragment. Addition of GSDMD-3C+3C protease to assembled nanodiscs reconstituted pores that were visible by negative staining electron microscopy (EM). Pretreatment with C-23 before addition to the nanodiscs completely blocked pore formation by GSDMD-3C+3C protease. However, addition of C-23 after pore formation did not disrupt the already assembled pores. Thus, disulfiram inhibits pore formation but does not disassemble already formed pores.

[0199] To assess whether C-22, -23, and -24 inhibit pyroptosis, these compounds were added to PMA-differentiated and LPS-primed human THP-1 cells before activating the canonical NLRP3 inflammasome with nigericin, or to murine immortalized bone marrow-derived macrophages (iBMDMs) before activating the noncanonical inflammasome by LPS electroporation (see figure). Only C-23 exhibited similar IC values ​​of 7.7 ± 0.3 μM and 10.3 ± 0.5 μM for inflammation-dependent pyroptosis in canonical human and noncanonical mouse models, respectively. 50 Disulfiram blocked pyroptosis at 1000-fold higher than that of the control group. It also attenuated cell death induced by the AIM2 inflammasome in poly(dA:dT)-transfected mouse iBMDMs in a dose-dependent manner, supporting its inhibition of a common downstream component of the inflammasome pathway. Inhibition was demonstrated by cell viability, as assessed by CellTiter-Glo ATP luminescence, and membrane permeability, as assessed by uptake of the membrane-impermeable dye SYTOX Green. Furthermore, disulfiram inhibited nigericin-induced IL-1β secretion in THP-1 cells and LPS-transfection-induced IL-1β secretion in iBMDM cells with potency comparable to that of the pan-caspase inhibitor z-VAD-fmk. In contrast, disulfiram had no effect on necroptosis induced in HT-29 cells by treatment with TNFα, SMAC mimetics, and z-VAD-fmk, which was blocked by either necrosulfonamide (NSA) or necrostatin-1 (Nec). These data indicate that disulfiram inhibits pyroptosis induced by canonical and noncanonical inflammasomes in both human and mouse cells, but not necroptosis.

[0200] Example 2 - Disulfiram protects against LPS-induced sepsis Disulfiram is being investigated as an anticancer drug because epidemiological studies have shown that individuals taking disulfiram for alcoholism are less likely to die from cancer (see Reference 24). In cells, disulfiram is rapidly metabolized to diethyldithiocarbamate (DTC) (see References 25 and 26). [ka] The in vivo anticancer activity of DTC is significantly enhanced by complexation with copper, presumably due to the enhanced electrophilicity of the DTC thiol (see, e.g., Reference 24). In liposome leakage assays, copper gluconate (Cu 2+ ) was found to only slightly increase the inhibition of disulfiram or DTC, likely due to the high reactivity of the Cys residues of the GSDMD involved (see Examples below). However, Cu 2+ strongly enhanced the ability of either disulfiram or DTC to protect LPS-activated THP-1 cells from pyroptosis (Figure 13). 2+ With the IC of C-23 to inhibit pyroptosis 50 was reduced 24-fold to 0.41 ± 0.02 μM, which was similar to its efficacy in preventing liposome leakage. 2+ In the presence of α-tocopherol, the activity in cells was almost equivalent to that of C-23.

[0201] Because C-23 inhibited intracellular pyroptosis and IL-1β release, its ability to protect C57BL / 6 mice from LPS-induced sepsis was also tested. Prior to LPS challenge, mice were treated intraperitoneally with vehicle or disulfiram. A low concentration of LPS (15 mg / kg) killed three of eight control mice after 96 hours, whereas all disulfiram-treated mice survived (P<0.05) (Figure 14). When all mice survived, C-23 strongly reduced serum IL-1β concentrations 12 hours after LPS challenge (281 ± 149 ng / mL in disulfiram-pretreated mice and 910 ± 140 ng / mL in control mice (P<0.0001)) (Figure 15). After LPS challenge at the intermediate concentration (25 mg / kg), all control mice died within 72 hours, whereas 5 of 8 disulfiram-treated mice survived (P<0.01) (Figure 16). At the highest LPS challenge (50 mg / kg), all control mice died within 1 day, but disulfiram treatment significantly delayed death, with 1 of 8 mice surviving (P<0.0001) (Figure 17). To determine whether treatment could be delayed until after LPS challenge and whether adding copper could improve protection, mice were challenged intraperitoneally with 25 mg / kg LPS and administered C-23 immediately and 24 hours later with or without copper gluconate. Post-LPS treatment still improved survival (P=0.041 without copper, P=0.024 with copper). All control mice and mice treated without copper died, whereas 2 of 8 mice given copper-complexed disulfiram survived (Figure 18). Thus, disulfiram given before or after LPS partially protected mice from septic death and reduced IL-1β secretion.

[0202] Experimental Results: Figure 12 shows the dose-response curves for the inhibition of liposome leakage by C-23 or its metabolite DTC in the presence or absence of Cu(II). In Figure 13, LPS-activated THP-1 cells were pretreated with C-23 or DTC for 1 hour in the presence or absence of Cu(II), followed by the addition of nigericin or medium for 2 hours. Cell death was determined by CytoTox96 assay. In Figures 14-17, mice were pretreated with C-23 (50 mg / kg) or vehicle (control) by intraperitoneal injection 24 and 4 hours before intraperitoneal LPS challenge (Figures 14 and 15: 15 mg / kg, Figure 16: 25 mg / kg, Figure 17: 50 mg / kg), and survival was followed. Statistical analysis was performed using the log-rank test (Figures 14, 16, and 17: mice / group). In Figure 15, serum IL-1β was measured by ELISA in mice (n=5 / group) pretreated with C-23 as described above and challenged with 15 mg / kg LPS. Serum was obtained 12 hours after LPS challenge. Mean values ​​± SD are shown. In Figure 18, mice were treated with C-23 (50 mg / kg), C-23 (50 mg / kg) plus copper gluconate (0.15 mg / kg), or vehicle (control) by intraperitoneal injection 0 and 12 hours after intraperitoneal LPS challenge (25 mg / kg). Statistical analysis was performed using the log-rank test (8 mice / group).

[0203] In cells, Cu(II) strongly promoted the ability of either disulfiram or DTC to protect LPS-activated THP-1 cells from pyroptosis, likely because Cu(II) promoted the activity of the major cellular metabolite DTC. The IC value of disulfiram for inhibiting pyroptosis with Cu(II) was significantly higher. 50 was reduced 24-fold to 0.41 ± 0.02 μM, similar to its potency in preventing liposome leakage. DTC became nearly as active as disulfiram in cells in the presence of Cu(II). The similar potency of disulfiram (when its major cellular metabolite is stabilized) in inhibiting GSDMD pore formation in liposomes and pyroptosis in cells supports GSDMD as a primary target in disulfiram's mechanism of action.

[0204] Example 3 - Disulfiram covalently modifies GSDMD Cys191 Disulfiram has been shown to inactivate reactive Cys residues by covalent modification (see Reference 27). To probe the mechanism of GSDMD inhibition by disulfiram, we analyzed disulfiram-treated human GSDMD using nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS / MS). Tryptic fragments revealed a dithiodiethylcarbamoyl adduct of Cys191, in which half of the symmetric disulfiram molecule is bound to a thiol (Figures 20, 21, 27, and 28). Indeed, Cys191 is required for GSDMD pore formation in cells, because oligomerization was blocked by an Ala mutation of the corresponding Cys192 in mouse GSDMD (see Reference 8). This Cys residue is conserved in GSDMD but not in other GSDM family members and is accessible in both the full-length autoinhibitor structural model and the N-terminal pore-forming model generated based on the mouse GSDMA3 structure (Refs. 7 and 14) (Figures 22 and 29). Corresponding to Leu183 in GSDMA3, Cys191 is located at the distal tip of the membrane-spanning region at the beginning of the β8 strand within the β7-β8 hairpin, a key component of the pore-forming β-barrel (Ref. 14). Analysis of Cys reactivity using PROPKA (Ref. 28) suggests that Cys191 is the most reactive of all Cys residues in GSDMD. Consistent with its high reactivity, time course analysis showed that disulfiram inhibited liposome leakage within 2 min of incubation (Figure 30). To confirm that disulfiram acts on Cys191, we generated Ala mutations at Cys191 and Cys38 as controls. Disulfiram IC for WT and C38A in liposome leakage assay 50 The IC values ​​for both were approximately 0.3 μM, but the IC 50The reduction in GSDMD pore formation was approximately 8-fold (Figure 23). Disulfiram was also incubated with N-acetylcysteine ​​(NAC), which contains a reactive Cys that can inactivate Cys-reactive drugs, to assess whether disulfiram protects THP-1 cells from nigericin-mediated pyroptosis. As expected, NAC abrogated the activity of disulfiram (Figure 24). Together, these data suggest that disulfiram inhibits GSDMD pore formation by selectively and covalently modifying Cys191.

[0205] Experimental Results: Figures 20 and 21 show the MS / MS spectrum of the Cys191-containing human GSDMD peptide FSLPGATCLQGEGQGHLSQK (aa 184-103; 2057.00 Da) modified on Cys191 by carbamidomethyl (a 57.0214 Da increase) [LC retention time, 22.85 min; triplet-charged precursor ion m / z 705.6827 (mass: 2114.0481 Da; delta M 2.27 ppm) was observed] (a), or the MS / MS spectrum of the corresponding GSDMD peptide after GSDMD incubation with disulfiram (C-23) modified on Cys191 by a diethyldithiocarbamate moiety (a 147.0255 Da increase). [LC retention time: 28.93 min; triplet-charged precursor ion m / z 735.6802 (mass: 2204.0406 Da; delta M 0.53 ppm) was observed.] (b) Figure 22 shows a model of full-length human GSDMD in its autoinhibited form based on the corresponding structure of GSDMA3, indicating the location of Cys191 cleaved by compound C-23, and a model of the pore form of the GSDMD DNA-terminal fragment (GSDMD-NT) (References 7 and 14). GSDMD-NT is shown in draft form, and GSDMD-CT is shown in gray. Figure 23 shows the dose-response curves for C-23's inhibition of liposome leakage induced by wild-type, C38A, or C191A GSDMD (0.3 μM) plus caspase-11 (0.15 μM). Figure 24 shows C-23 inhibition of pyroptosis in LPS + nigericin-treated THP-1 cells after 1 hour of C-23 preincubation with N-acetylcysteine ​​(NAC, 500 μM) or medium. Two-fold dilutions of C-23 ranging from 5 to 40 μM were used. Graphs show mean ± SD, and data are representative of three independent experiments. ** ***P<0.01. Figures 25 and 26 show the dose-response curves of compound C-23 on liposome leakage induced by human GSDMD-3C (0.3 μM) + 3C protease (0.15 μM) (Figure 25) or mouse GSDMA3-3C (0.3 μM) + 3C protease (0.15 μM) (Figure 26).

[0206] Figures 27 and 28 show MS / MS spectra for peptides containing Cys191 in human GSDMD. Figure 27 shows the MS / MS spectrum of peptide FSLPGATCLQGEGQGHLSQK modified on cysteine ​​by carbamidomethyl. Protein coverage is 73%. Figure 28 shows the MS / MS spectrum of peptide FSLPGATCLQGEGQGHLSQK modified on cysteine ​​by C-23. Protein coverage is 72%.

[0207] Figures 29 and 30 show that disulfiram covalently modifies Cys191 of GSDMD. In Figure 29, a sequence alignment of mouse GSDMA3, human GSDMA (hGSDMA), mouse GSDMD (mGSDMD), and human GSDMD (hGSDMD) shows the Cys residue. In Figure 30, GSDMD (0.3 μM) was preincubated with the indicated concentrations of C-23 (0-50 μM) for different times (2-90 min) before the addition of caspase-11 (0.15 μM) in liposomes (50 μM).

[0208] To confirm that disulfiram acts on Cys191, disulfiram IC 50 Values ​​were compared for pore formation in liposomes treated with WT, C38A control, or C191A human GSDMD + caspase-11. IC of disulfiram acting on C191A GSDMD 50The activity of C38A was approximately eightfold higher than that of WT GSDMD, whereas the activity of C38A was similar to that of WT GSDMD, confirming the importance of Cys191 for disulfiram activity. The residual inhibition of the Cys191 mutant may be due to disulfiram modification of other Cys residues within the mutant GSDMD. To confirm the importance of Cys191 in pore formation, we measured cell death by LDH release in HEK293T cells ectopically expressing full-length human WT or C191S mutant GSDMD with or without caspase-11. WT or C191S GSDMD alone did not impair cell survival, but both WT GSDMD and caspase-11 caused substantial cell death, and C191S GSDMD and caspase-11 reduced cell viability. Similarly, cell death caused by ectopic expression of mouse GSDMD-NT (mGSDMD-NT) was significantly reduced in HEK293T cells expressing the analogous C192S mutant but only modestly reduced in cells expressing C39A mGSDMD-NT. These results, consistent with previous results, confirm the role of Cys191 and Cys192 in GSDMD-NT pore formation in humans and mice, respectively.

[0209] To further confirm that disulfiram acts on Cys191, we evaluated disulfiram inhibition of LDH release in HEK293T cells expressing caspase-11 and either WT or C191S GSDMD. As expected, WT GSDMD-induced cell death was strongly inhibited by disulfiram in a dose-dependent manner starting from the lowest concentration tested (10 μM), whereas the reduced cell death caused by expression of caspase-11 and C191S GSDMD was only inhibited when fourfold more disulfiram was added. Collectively, these data indicate that disulfiram inhibits GSDMD pore formation by covalently modifying Cys191. Additionally, the data suggest that disulfiram inhibits cell death primarily through its effect on GSDMD-NT pore formation, since disulfiram would have provided better protection from death in cells expressing caspase-11 and C191S GSDMD if it had strongly inhibited caspase-11.

[0210] Example 4 - Disulfiram (C-23) inhibits caspase-1 and caspase-11 Disulfiram has been reported to inhibit caspases by binding to the catalytic Cys responsible for proteolysis (see Reference 29). Therefore, disulfiram likely inhibits both caspases and GSDMD. Using a fluorogenic caspase activity assay measuring the release of 7-amino-4-methylcoumarin (AMC) from the substrate Ac-YVAD-AMC, disulfiram exhibited IC values ​​of 0.15 ± 0.04 μM and 0.73 ± 0.07 μM, respectively. 50It was found that disulfiram indeed inhibited caspase-1 and caspase-11 (Figures 31-38). Addition of Cu(II) did not significantly alter disulfiram caspase inhibition in vitro. To determine the relative contribution of caspase-11 inhibition versus GSDMD inhibition by disulfiram in pore formation, the caspase cleavage site in GSDMD was replaced with the rhinovirus 3C protease site (GSDMD-3C) and the 3C protease was used instead of caspase-11 in liposome leakage assays. The resulting IC 50 The IC50 for murine GSDMA3 was 0.52 ± 0.03 μM, comparable to 0.30 ± 0.01 μM for caspase-11-induced liposome leakage (Figures 2 and 25). In contrast, disulfiram had a much weaker IC50 of 12.14 ± 2.10 μM, due to the lack of the conserved Cys191 residue in murine GSDMA3. 50 Disulfiram inhibited liposome leakage induced by 3C-cleaved GSDMA3 containing a 3C protease site (GSDMA3-3C) (Figure 26). Thus, the inhibitory effect of disulfiram in the liposome leakage assay is mediated by direct inhibition of GSDMD.

[0211] Experimental Results: Figures 31 and 32 show the time course of caspase-1 and caspase-11 activity in the presence of the indicated concentrations of Compound C-23. Caspase (0.5 U) was incubated with Compound C-23 at the indicated concentrations for 1 hour before adding Ac-YVAD-AMC (40 μM). Figures 33 and 34 show the dose-response curves of Compound C-23 in the caspase-1 and caspase-11 activity assays. Figures 35 and 36 show the time course of caspase-1 and caspase-11 activity in the presence of the indicated concentrations of Compound C-23 and Cu(II). Caspase (0.5 U) was incubated with Compound C-23 and Cu(II) at the indicated concentrations for 1 hour before adding Ac-YVAD-AMC (40 μM). Figures 37 and 38 show the dose-response curves of compound C-23 + Cu(II) in caspase-1 and caspase-11 activity assays. Fluorescence intensity at 460 nm was measured after excitation at 350 nm.

[0212] Example 5 - Test compounds inhibit GSDMD pore formation IC of test compounds shown in Figure 39 in the liposome leakage assay 50 The values ​​are shown in Figures 40-42. The data show that the tested compounds protected against nigericin-induced pyroptosis in THP-1. The results of the leakage assay are shown in Table 2. The chemical structures of the compounds listed in Table 2 are shown in Figure 39. [Table 5] TIFF0007787250000016.tif49170

[0213] Experimental Results: In Figure 40, PMA-differentiated, LPS-activated THP-1 cells were treated with the indicated compound (40 μM) for 3 hours and tested for viability by the CellTiter-Glo assay. In Figure 41, PMA-differentiated, LPS-activated THP-1 cells were pretreated with 40 μM disulfiram, the indicated test compound, or z-VAD-fmk for 1 hour before treatment, or were not treated with nigericin. Cells were assessed for cell viability by the CellTiter-Glo assay 2 hours after the addition of nigericin. In Figure 42, PMA-differentiated, LPS-activated THP-1 cells were pretreated with 40 μM disulfiram or z-VAD-fmk, or with two-fold serial dilutions of the indicated test compound (concentration range, 0.39-50 μM), for 1 hour, followed by the addition of nigericin. Cells were assessed for cell viability by the CellTiter-Glo assay 2 hours after the addition of nigericin. The graph shows the mean value±sd and the data are representative of three independent experiments. ** P<0.01. None of the tested compounds was significantly toxic to THP-1 cells (see figure). The tested compounds also significantly protected against nigericin-induced pyroptosis in THP-1 cells.

[0214] Example 6a - Disulfiram and Bay11-7082 inhibit multiple steps in the inflammasome activation cascade Pan-caspase inhibitor z-VAD-fmk (CAS Registry Number 187389-52-2): [ka] was found to inhibit the canonical inflammasome pathway in THP-1 cells.

[0215] Also, Bay11-7082 (CAS Registry Number 19542-67-7), [ka] Previously known inhibitors of NF-κB activation (13) and the NLRP3 pathway (30) (Figure 43) were found to also inhibit the canonical inflammasome pathway in THP-1 cells. As discussed below, Bay11-7082, for example, inhibits GSDMD, caspase-1, and caspase-11.

[0216] MST revealed that Bay11-7082 bound to GSDMD (see Figures 55 and 56 and Figure 2). Bay11-7082 inhibited caspase-1 and, to a lesser extent, caspase-11 (see Figures 55-58). Surprisingly, similar to disulfiram, Bay11-7082 functions by inactivating reactive Cys residues (see References 31 and 32), and Cys191 in GSDMD was covalently modified by Bay11-7082 (see Figures 59 and 60). In a liposome leakage assay, replacing C191A GSDMD with WT GSDMD reduced Bay11-7082's inhibition of liposome leakage by twofold (Figure 55). Much of the inhibition of liposome leakage by Bay11-7082 can be attributed to caspase-11 inhibition, as Bay11-7082 was less able to inhibit leakage by GSDMD-3C+3C protease than by GSDMD+caspase-11, and its activity toward mouse GSDMA3-3C, which lacks the equivalent reactive cysteine, as well as toward 3C protease, was similar to its activity toward GSDMD-3C (see Figures 61 and 62).

[0217] Bay11-7082 inhibited pyroptosis induced by both canonical and noncanonical inflammasomes in THP-1 cells, but was more active in nigericin-treated than in LPS-transfected cells (Figures 43 and 44). Bay11-7082 was more effective than disulfiram in inhibiting canonical inflammasome-dependent pyroptosis in the absence of copper, and the two drugs together had an additive protective effect but were cytotoxic at the highest concentrations tested (Figure 43). Bay11-7082 was less active than disulfiram in inhibiting pyroptosis induced by noncanonical inflammasome activation (Figure 44).

[0218] Because both disulfiram and Bay11-7082 nonspecifically modify reactive Cys residues, we next analyzed their effects on the processes leading to pyroptosis and inflammatory caspase activation. Several genes involved in the canonical inflammasome pathway are not expressed in unstimulated cells, and their expression must be induced by pathogen binding to cell surface sensors and danger-associated molecular patterns, such as Toll-like receptors (TLRs), in a process called initial activation (priming). Bay11-7082 is known to inhibit NF-κB activation, a key transcription factor in priming. We first examined the effects of disulfiram and Bay11-7082 on priming (Figure 45). NF-κB activation was assessed by examining IκBα phosphorylation and degradation and RelA (p65) phosphorylation. Induction of pro-IL-1β was assessed by immunoblotting for pro-IL-1β protein. In the absence of disulfiram or Bay11-7082, p65 phosphorylation was first detected 30 minutes after LPS addition and persisted for 4 hours. Phosphorylated and reduced IκBα was detected 1 hour after LPS addition, and increased pro-IL-1β was detected 4 hours after LPS addition. Both test compounds, added at a concentration of 30 μM, inhibited NF-κB activation, but Bay11-7082 had a stronger effect, and both blocked the induction of pro-IL-1β. Thus, both disulfiram and Bay11-7082 inhibit priming.

[0219] Nigericin activates the assembly of the NLRP3 canonical inflammasome using an adaptor called apoptosis-associated speck-like protein, which contains a caspase recruitment domain (ASC), and these can be visualized as specks by immunofluorescence microscopy. When LPS-activated THP-1 cells were treated with nigericin in the absence of inhibitors, ASC specks were detected in 30% of the cells (Figure 36). As expected, because caspase activation occurs downstream of inflammasome assembly, speck formation was not inhibited by z-VAD-fmk. However, both test compounds added after priming but 1 hour before nigericin inhibited ASC speck formation, but not completely. Bay11-7082 was more potent than disulfiram when used at the same concentrations. While 1 μM disulfiram was completely inactive in blocking pyroptosis induced by nigericin or transfected LPS (Figures 6 and 7), the same concentration of disulfiram in combination with copper gluconate completely blocked pyroptosis and also reduced ASC puncta (Figures 48 and 49).

[0220] To assess which steps in NLRP3-mediated inflammation were inhibited after ASC speck formation, LPS-activated THP-1 cells were treated with vehicle or 30 μM z-VAD-fmk, disulfiram, or Bay11-7082 for 1 h followed by the addition of nigericin. The cleavage and activation of caspase-1, GSDMD, and pro-IL-1β were analyzed by immunoblotting of whole cell lysates 30 min later (Figure 50). Secretion of treated IL-1β was also assessed by immunoblotting of culture supernatants. While cleavage of caspase-1, GSDMD, and pro-IL-1β into their active forms was clearly detected in the absence of inhibitors, it was dramatically reduced in cells treated with any of the three inhibitors. Furthermore, treated IL-1β was only detected in the culture supernatant in the absence of any inhibitors. When the same experiment was repeated by treating cells with 1 μM disulfiram alone in PBS or copper gluconate, copper-complexed disulfiram completely blocked caspase-1, GSDMD, and pro-IL-1β processing and IL-1β secretion, whereas copper-free disulfiram had no effect (Figure 51). Because immunoblots are not quantitative, caspase-1 activity 30 min after addition of nigericin was also assessed using a fluorescent substrate in intact cells. Caspase-1 activity was completely inhibited by z-VAD-fmk but only partially reduced by either disulfiram or Bay11-7082, and again strongly reduced by Bay11-7082 (Figure 52). Next, the effects of z-VAD-fmk, disulfiram, and Bay11-7082 on LPS + nigericin-induced GSDMD pore formation were assessed by immunofluorescence microscopy using monoclonal antibodies that recognize both uncleaved GSDMD and its pore form (Figures 53, 54, and 64-66). In the absence of any inhibitor, GSDMD antibodies stained both the cytosol and plasma membrane of LPS + nigericin-treated cells, which formed the characteristic pyroptotic bubbles (see reference 10). All three inhibitors completely blocked GSDMD membrane staining and the appearance of pyroptotic bubbles.Thus, disulfiram and Bay11-7082 inhibit multiple steps leading to canonical inflammasome-induced pyroptosis and proinflammatory cytokine release, including priming, inflammasome assembly, inflammatory caspase activation, proinflammatory cytokine processing, and GSDMD pore formation.

[0221] Experimental Results: In Figure 43, PMA-differentiated, LPS-primed THP-1 cells were pretreated with two-fold serial dilutions (ranging from 0.3125 to 40 μM) of C-23 and / or Bay11-7082 for 1 hour before treatment with nigericin. Cell death was determined by CytoTox96 assay. In Figure 44, mouse iBMDMs were pretreated with two-fold serial dilutions of C-23 or Bay11-7082 (ranging from 0.3125 to 40 μM) for 1 hour before electroporation with PBS or LPS. Cell death was determined by CytoTox96 assay. In Figure 45, THP-1 cells were pretreated with 30 μM of C-23 or Bay11-7082 for 1 hour, followed by the addition of LPS. Immunoblots of whole-cell lysates harvested 0.5 hours later are shown. In Figures 46, 47, 50, and 52, LPS-activated THP-1 cells were pretreated with 30 μM C-23, Bay11-7082, or z-VAD-fmk for 1 hour, followed by the addition of nigericin or medium. Representative images of ASC specks (arrows) and the mean ± SD percentage of cells with ASC specks were analyzed 20 minutes later (Figure 47). Thirty minutes after the addition of nigericin, whole cell lysates (WCL) and culture supernatants (Sup) were collected and immunoblotted with the indicated antibodies (Figure 50). Caspase-1 activity was assayed 30 minutes after the addition of nigericin using the cell-permeable fluorescent dye FAM-YVAD-FMK (Figure 52). In Figures 48, 49, and 51, LPS-activated THP-1 cells were pretreated with 1 μM C-23 in the absence of Cu(II) for 1 hour, followed by the addition of nigericin or medium. Representative images of ASC specks (arrows) and the mean ± SD percentage of cells with ASC specks were analyzed after 20 min (Figures 48 and 49). Thirty minutes after nigericin addition, whole cell lysates (WCL) and culture supernatants (Sup) were collected and analyzed by immunoblotting (Figure 51). In Figures 53 and 54, LPS-activated THP-1 cells were pretreated with 30 μM C-23, Bay11-7082, or z-VAD-fmk for 1 hour, followed by addition of nigericin or medium, and then stained with mouse anti-GSDMD monoclonal antibody 30 min later (see Figures 55-63).Representative confocal microscopy images and quantification of the percentage of cells with GSDMD membrane staining and pyroptotic bubbles are shown. Arrows point to GSDMD staining of pyroptotic bubbles. Figure 55 shows Bay11-7082 dose-response curves for inhibition of liposome leakage by wild-type, C38A, or C191A GSDMD (0.3 μM) plus caspase-11 (0.15 μM). Figure 56 shows MST measurements of direct binding of Alexa 488-labeled His-MBP-GSDMD (80 nM) to Bay11-7082 using NanoTemper. Figures 57 and 58 show dose-response curves for the effect of Bay11-7082 on caspase-1 (Figure 57) and caspase-11 (Figure 58) activity toward fluorescent peptide substrates. Figures 59 and 60 show the MS / MS spectrum of the Cys191-containing GSDMD peptide FSLPGATCLQGEGQGHLSQK (aa 184-103; 2057.00 Da) modified on Cys191 with carbamidomethyl (an increase of 57.0214 Da) [LC retention time, 22.85 min; triplet-charged precursor ion m / z 705.6827 (mass: 2114.0481 Da; delta M 2.27 ppm) was observed] (Figure 59) (a), or the MS / MS spectrum of the corresponding GSDMD peptide after GSDMD incubation with Bay11-7082 modified on Cys191 (an increase of 207.0354 Da) [LC retention time 17.20 min; triplet-charged precursor ion m / z 756.0229 (mass: 2264.0688 Da; delta M 11.7 ppm) was observed] (Figure 60). Figures 61 and 62 show dose-response curves for the effect of Bay11-7082 on liposome leakage induced by 0.3 μM human GSDMD-3C (Figure 61) or mouse GSDMA3-3C (Figure 62) plus 0.15 μM 3C protease. Figure 63 shows the effect of 1 hour pre-incubation of Bay11-7082 with N-acetylcysteine ​​(NAC, 500 μM) on inhibition of pyroptosis in THP-1 cells treated with LPS plus nigericin. Two-fold dilutions of Bay11-7082 from 5 to 40 μM were used. The graph shows the mean ± SD, and the data are representative of three independent experiments. * P<0.05, **P<0.01.

[0222] Compared to disulfiram, Bay11-7082 bound to GSDMD with lower affinity and was 23-fold less active in inhibiting liposome leakage (IC 50 Bay11-7082 also inhibited caspase-1 but was approximately threefold less active against caspase-11 than disulfiram. Like disulfiram, Bay11-7082 functions by inactivating reactive Cys residues 29 and 30. By nano-LC-MS / MS, Bay11-7082 was found to covalently modify Cys191 in GSDMD. However, replacing C191A GSDMD with WT GSDMD in the assay reduced Bay11-7082's inhibition of liposome leakage by only twofold. Thus, much of the inhibition of liposome leakage by Bay11-7082 can be attributed to caspase-11 inhibition, because Bay11-7082 was substantially less able to inhibit leakage by GSDMD-3C + 3C protease than by GSDMD + caspase-11, and its activity toward mouse GSDMA3-3C, which lacks the equivalent reactive cysteine, as well as the 3C protease, was similar to its activity toward GSDMD-3C. Thus, unlike disulfiram, Bay11-7082 is a caspase inhibitor rather than a GSDMD inhibitor in the liposome leakage assay.

[0223] Example 6b - Inhibitors of the inflammasome activation cascade Recently, the Cys-reactive necrosis factor inhibitor NSA was shown to also inhibit GSDMD-mediated pyroptosis. The potency of disulfiram in inhibiting GSDMD and caspase-11-mediated liposome leakage was compared to that of NSA and other Cys-reactive compounds, including dimethyl fumarate (DMF, a drug for psoriasis and multiple sclerosis), afatinib (a drug that inhibits epidermal growth factor receptor tyrosine kinase), ibrutinib (a drug that inhibits Bruton's tyrosine kinase), and LDC7559. NSA moderately inhibited liposome leakage but was approximately 30-fold less potent than disulfiram (IC of 9.50 ± 0.43 μM). 50 ).

[0224] Example 7 - Mouse monoclonal antibodies recognize full-length human GSDMD and GSDMD-NT pore forms on immunoblots and by immunofluorescence microscopy. Monoclonal antibodies against GSDMD were generated by immunizing mice with recombinant human GSDMD and boosting them with recombinant human GSDMD-NT according to the methods. In Figure 64, HEK293T cells were transfected with the indicated plasmids, and cell lysates were analyzed by immunoblotting of reduced gels probed with the indicated antibodies. In Figure 65, cell lysates from HCT116, 293T, and THP-1 cells treated with or without nigericin were immunoblotted with the indicated antibodies. 293T cells do not express endogenous GSDMD. In Figure 66, 293T and THP-1 cells were immunostained with anti-GSDMD monoclonal antibodies and co-stained with DAPI (blue). 293T cells, which do not express GSDMD, show no background staining.

[0225] Example 8 - Mechanical Studies To elucidate the cellular mechanism of pyroptosis inhibition by disulfiram, we analyzed its effect on the entire inflammasome activation pathway. Several genes involved in the canonical inflammasome pathway are not expressed in unstimulated cells, and their expression must be induced by pathogen binding to cell surface sensors and danger-associated molecular patterns, such as Toll-like receptors (TLRs), in a process called priming (initial activation). In previous experiments, disulfiram was added 4 h after LPS priming and 1 h before stimulation with nigericin, so the effect of disulfiram on inflammasome priming was not examined. To explicitly examine priming, THP-1 cells were pretreated with disulfiram for 1 h followed by LPS addition for up to 4 h. NF-κB activation, a key transcription factor in priming, was assessed by examining IκBα phosphorylation and degradation and RelA (p65) phosphorylation. Induction of NLRP3 and pro-IL-1β expression was assessed by immunoblotting. Bay11-7082 was used as a positive control due to its known inhibitory effect on NF-κB activation. In the absence of disulfiram or Bay11-7082, p65 phosphorylation was first detected 30 minutes after LPS addition and persisted for 4 hours. Phosphorylated and reduced IκBα was detected 1 hour after LPS addition, and increased NLRP3 and pro-IL-1β proteins were detected 4 hours after LPS addition. Both drugs inhibited NF-κB activation, but Bay11-7082 had a stronger effect, blocking the induction of NLRP3 and pro-IL-1β.

[0226] Nigericin activates the assembly of the NLRP3 canonical inflammasome using an adaptor called apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which can be visualized as specks by immunofluorescence microscopy. When LPS-activated THP-1 cells were treated with nigericin in the absence of inhibitors, ASC specks were detected in approximately 30% of cells. As expected, because caspase activation occurs downstream of inflammasome assembly, speck formation was not inhibited by z-VAD-fmk. Disulfiram added after priming but 1 h before nigericin moderately inhibited ASC speck formation, resulting in approximately 20% of cells. The modest decrease in speck formation is due to the subtle inhibition of priming by disulfiram, even though it was added 4 h after LPS priming. Indeed, immunoblotting showed that NLRP3 levels were reduced by disulfiram added after priming compared to cells incubated in medium.

[0227] Assembly of the canonical inflammasome activates caspase-1, which cleaves pro-IL-1β and GSDMD, the latter required for releasing processed IL-1β and inducing pyroptosis. To assess which steps in ASC-mediated inflammation were inhibited after NLRP3-induced inflammation, LPS-activated THP-1 cells were treated with vehicle, 30 μM z-VAD-fmk, or disulfiram for 1 hour before the addition of nigericin. The cleavage and activation of caspase-1, GSDMD, and pro-IL-1β were analyzed by immunoblotting of whole cell lysates 30 minutes and 1 hour later. IL-1β secretion was also assessed by immunoblotting of culture supernatants. Cleavage of caspase-1, GSDMD, and pro-IL-1β into their active forms was clearly detected in the absence of inhibitors, and their processing was reduced in cells treated with disulfiram or z-VAD-fmk 30 min after nigericin. However, consistent with disulfiram's weaker effect on caspases, by 60 min, processing of caspase-1, GSDMD, and pro-IL-1β in disulfiram-treated samples caught up with that detected in the absence of inhibitors, while samples treated with z-VAD-fmk still showed little cleavage of these proteins. The 1-h time point is relevant for cell death and IL-1β release measurements using cells stimulated with nigericin for 1 and 2 h, respectively. These data suggest that disulfiram delayed, but did not inhibit, caspase-1 activation. However, processed IL-1β was only detected in the culture supernatant in the absence of either inhibitor, suggesting that disulfiram completely inhibited cytokine release by blocking GSDMD pore formation despite limited caspase-1 inhibition.A similar preferential effect of disulfiram on IL-1β release (but not processing) was found in mouse iBMDMs, while NSA, Bay11-7082, and z-VAD-fmk still inhibited caspase-1, GSDMD, and IL-1β processing at 1 h.

[0228] Next, we evaluated the effects of z-VAD-fmk and disulfiram on LPS- and nigericin-induced GSDMD pore formation by immunofluorescence microscopy using a monoclonal antibody generated in the previous study that recognizes both uncleaved GSDMD and its pore form. In the absence of any inhibitor, the GSDMD antibody stained both the cytosol and plasma membrane of LPS + nigericin-treated cells, which formed the characteristic pyroptotic bubble. Both inhibitors completely blocked GSDMD membrane staining and the appearance of pyroptotic bubbles. Thus, disulfiram inhibits priming and delays caspase-1 activation, but its effects culminate at the bottleneck stage of GSDMD pore formation, suppressing both pyroptosis and inflammatory cytokine release in both THP-1 and iBMDM cells. In contrast, the control inhibitor z-VAD-fmk exclusively blocks caspase-1 activity.

[0229] To examine the in vivo effects of disulfiram, LPS-induced sepsis was examined in C57BL / 6 mice. After allometric scaling for body surface area, mice were treated intraperitoneally with vehicle or disulfiram before LPS challenge using a drug dose (50 mg / kg) equivalent to 284 mg / day in humans, within the clinically approved dose range of 125–500 mg / day for treating alcoholism. 32A low concentration of LPS (15 mg / kg) killed three of eight control mice after 96 hours, whereas all disulfiram-treated mice survived (P = 0.045). Serum IL-1β, TNFα, and IL-6 concentrations were strongly reduced 12 hours after LPS challenge, when all mice survived (P < 0.0003). After an intermediate LPS challenge (25 mg / kg), all control mice died within 72 hours, whereas five of eight disulfiram-treated mice survived (P < 0.008). At the highest LPS challenge (50 mg / kg), all control mice died within 1 day, but disulfiram treatment significantly delayed death, resulting in one of eight mice surviving (P = 0.007). LPS-induced sepsis in mice depends on cleavage of GSDMD by caspase-11 in the noncanonical inflammasome. Consistent with previous studies, Casp11 - / - Mouse and Gsdmd - / - Mice were resistant to death from LPS-induced sepsis, but Casp1 - / - As expected, disulfiram prevented Casp1 from a lethal LPS challenge. - / - Casp11 protected mice, but all but one mouse in each non-drug control group survived. - / - Mouse and Gsdmd - / - There was no significant effect on mouse survival.

[0230] To determine whether complexation with Cu(II) could improve protection from sepsis in vivo, we compared the efficacy of disulfiram administered with or without Cu(II) with the survival rate of mice challenged intraperitoneally with 25 mg / kg LPS. To better mimic the clinical situation in which sepsis is typically diagnosed only after the inflammatory cascade has been initiated, disulfiram administration was delayed until immediately after LPS injection and 12 h later. Disulfiram treatment after LPS significantly delayed death (p = 0.041 without Cu(II); p = 0.024 with copper). All control mice and mice treated with disulfiram alone died, whereas two of eight mice given Cu(II)-complexed disulfiram survived. However, the difference in survival rate between disulfiram treatment with or without Cu(II) did not reach significance (p = 0.064). Thus, mice given disulfiram after LPS partial protection, with administration of Cu(II), were able to improve its activity.

[0231] LPS not only induces noncanonical inflammasome activation in cells, which does not require priming, but also primes NLRP3 inflammasome activation, which amplifies septic shock. Previous studies have shown that genetic deficiency of NLRP3, ASC, caspase-1, or IL-1 receptor did not provide a substantial survival advantage in mice challenged with LPS, whereas caspase-11 or GSDMD deficiency protected mice from septic death. Therefore, it is inferred that protection from LPS-induced sepsis likely depends on inhibition of GSDMD cleavage or pore formation, but is independent of NLRP3 inflammasome priming. This inference supports the idea that disulfiram inhibits Casp1. - / - This is supported by our independent finding that IFN-γ protected WT and WT mice as well.

[0232] To determine whether disulfiram primarily inhibits GSDMD processing by caspase-11 or pore formation, four groups of mice were pretreated with disulfiram or vehicle 4 hours and immediately before intraperitoneal challenge with LPS or vehicle. Peritoneal macrophages were harvested 6 hours later and analyzed for NLRP3, GSDMD, and HMGB1 by immunoblotting. GSDMD was similarly expressed in LPS-challenged mice with or without disulfiram treatment, indicating that inhibition of GSDMD pore formation, rather than GSDMD cleavage, led to reduced mortality. Surprisingly, NLRP3 levels were also similar in LPS-challenged mice with or without disulfiram treatment, suggesting that disulfiram, although impairing NLRP3 priming in cells, did not inhibit NLRP3 priming in mice. These results strongly suggest that inhibiting GSDMD pore formation to block LPS-induced pyroptosis and the release of inflammatory mediators is the primary target of disulfiram in our model.

[0233] Disulfiram's inhibition of GSDMD pore formation in mouse and human cells complements its activity in blocking inflammasome priming and caspase activity, suppressing pyroptosis and inflammatory cytokine release induced by both canonical and noncanonical pathways. Simultaneous targeting of three steps in the inflammasome pathway means that disulfiram is a particularly potent inhibitor of inflammation, especially when administered with Cu(II) to stabilize its intermediates. The results presented herein demonstrate that inhibition of pore formation, a common and essential final step in both pyroptosis and inflammatory mediator release, governs disulfiram's anti-inflammatory activity. Its relatively weak activity in priming and caspase inhibition allows disulfiram to be nontoxic to humans, whereas more potent NF-κB inhibitors, such as Bay11-7082 and caspase inhibitors, are both associated with toxicity. Furthermore, noncanonical inflammasomes do not require priming, and in disease settings, priming of relevant immune and epithelial cells may already have occurred by the time signs and symptoms of inflammation are clinically recognized, suggesting that inhibiting GSDMD to halt pyroptosis and the most downstream steps in inflammatory mediator release would be particularly useful. Finally, the relative selectivity of disulfiram is supported by the lack of activity against GSDMD of several other covalent Cys-reactive compounds, including the highly reactive DMF.

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[0235] Other embodiments While the present application has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, but not limiting, of the scope of the application, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The following is one embodiment of the present invention. (1) Inhibiting gasdermin pore formation in cells, and / or Inhibiting inflammasome-mediated death of cells (pyroptosis), and / or Inhibits cytokine secretion from cells, and / or Inhibiting intracellular inflammatory caspases, and / or react covalently with the cysteines of the gasdermin protein within the cell, and / or covalently reacting with a cysteine ​​of an inflammatory signaling molecule selected from a sensor, an adaptor, and a transcription factor, or a regulator thereof, The method comprises treating the cells with an effective amount of a compound of formula (I),

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Table 8

Claims

1. an effective amount of a compound of formula (I), 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, wherein: R １ , R ２ , R ３ , and R ４ are independently H, C １～６ Alkyl, C １～６ Haloalkyl, and Cy １ and C is selected from １～６ The alkyl is Cy １ , Halo, CN, and NO ２ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R １ and R ２ together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Ｃｙ２ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Or, R ３ and R ４ together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Ｃｙ３ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Each Cy １ But independently, C ６～１０ aryl, and 5- to 10-membered heteroaryl, each of which is selected from R Ｃｙ１ and optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from Each R Ｃｙ１ , R Ｃｙ２ , and R Ｃｙ３ But independently, C １～６ Alkyl, C １～６ Haloalkyl, halo, CN, and NO ２ is selected from The composition is for use in a method of treating an inflammatory disease that is sepsis.

2. R １ , R ２ , R ３ , and R ４ are each independently Cy １ and Cy １ C optionally substituted with １～６ alkyl, Or, R １ and R ２ together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Ｃｙ２ and optionally substituted with 1, 2, or 3 substituents independently selected from Or, R ３ and R ４ together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Ｃｙ３ and optionally substituted with 1, 2, or 3 substituents independently selected from Each Cy １ But independently, C ６～１０ aryl, and 5- to 10-membered heteroaryl, each of which is selected from R Ｃｙ１ and optionally substituted with 1, 2, or 3 substituents independently selected from Each R Ｃｙ１ , R Ｃｙ２ , and R Ｃｙ３ But independently, C １～６ selected from alkyl, A composition for use according to claim 1.

3. R １ , R ２ , R ３ , and R ４ are each independently Cy １ and Cy １ C optionally substituted with １～６ The composition for use according to claim 1, wherein the alkyl is selected from the group consisting of aryl, arylsulfonyl ...

4. R １ and R ２ together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Ｃｙ２ 2. The composition for use according to claim 1, optionally substituted with 1, 2, or 3 substituents independently selected from:

5. R ３ and R ４ together with the N atom to which they are attached form a 4- to 12-membered heterocycloalkyl, which is Ｃｙ３ 2. The composition for use according to claim 1, optionally substituted with 1, 2, or 3 substituents independently selected from:

6. The 4- to 12-membered heterocycloalkyl is: 【Chemistry 2】 6. The composition for use according to claim 4 or claim 5, selected from any one of:

7. The compounds of formula (I) are those listed in Table A: Table 1 2. The composition for use according to claim 1, wherein the compound is selected from any one of the following:

8. The composition for use according to claim 1, wherein the inflammatory disease is acute sepsis.

9. 2. The composition for use of claim 1, wherein the method comprises administering to a subject the compound of formula (I), or a pharmaceutically acceptable salt thereof, in combination with at least one additional anti-inflammatory agent, or a pharmaceutically acceptable salt thereof, optionally wherein the additional anti-inflammatory agent is selected from an anti-IL1 antibody, an anti-TNF antibody, an NSAID, and a steroid anti-inflammatory agent.

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