WO2025217320A1 - Inhibitors of ripk3 and methods of use thereof - Google Patents

Inhibitors of ripk3 and methods of use thereof

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Publication number
WO2025217320A1
WO2025217320A1 PCT/US2025/023933 US2025023933W WO2025217320A1 WO 2025217320 A1 WO2025217320 A1 WO 2025217320A1 US 2025023933 W US2025023933 W US 2025023933W WO 2025217320 A1 WO2025217320 A1 WO 2025217320A1
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WO
WIPO (PCT)
Prior art keywords
ripk3
infection
iav
compound
necroptosis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023933
Other languages
French (fr)
Inventor
Gregory Cuny
Manu BALA
Raghavender BODA
Alexei Degterev
Siddharth Balachandran
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute for Cancer Research
Tufts University
University of Houston System
Original Assignee
Institute for Cancer Research
Tufts University
University of Houston System
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Filing date
Publication date
Application filed by Institute for Cancer Research, Tufts University, University of Houston System filed Critical Institute for Cancer Research
Publication of WO2025217320A1 publication Critical patent/WO2025217320A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • protein kinase inhibitors can be used as therapeutic agents for the treatment of various diseases.
  • 15 Human auto-immune, auto-inflammatory and degenerative conditions involve a continuous loop of cell death and inflammation, leading to tissue injury and dysfunction. This is seen in progressive chronic diseases with inflammatory etiology, including atherosclerosis and TNF-driven pathologies such as rheumatoid arthritis and ulcerative colitis. This also occurs upon deleterious acute inflammatory responses, such as upon infection by influenza virus.
  • IAV Influenza A virus
  • Influenza A virus is a negative-sense RNA virus of the family Orthomyxoviridae.
  • IAV Influenza A virus
  • the primary IAV reservoir virus replication occurs within the gastrointestinal tract and is typically asymptomatic.
  • mammalian IAV strains replicate in the respiratory tract and produce Page 1 QB ⁇ 166118.01517 ⁇ 95733313.1
  • IAV infects epithelial cells along the entire respiratory tract. Transmission is associated with infection of the upper tract, whereas severe disease is associated with infection in the lower lung, with the extent of lung involvement correlating with disease outcomes in humans and animal models. 5 As a lytic virus, IAV kills most lung cell types in which it replicates. But not all cell death by IAV is pathogenic. While programmed cell death is essential for early control of IAV replication and prevention of virus spread to the whole lung, the death of the lung epithelial layer is also one of the primary drivers of IAV infection-associated morbidity and mortality10.
  • Type I airway epithelial cells which are essential for gas exchange
  • AECs which are essential for gas exchange
  • a threshold of ⁇ 10% is strongly correlated with mortality in the mouse model of IAV infection.
  • cell death When cell death is well-controlled and apoptotic, it represents a host defense mechanism that limits both virus spread and immunopathology. But when cell death is uncontrolled or primarily necrotic (i.e., highly pro-inflammatory), it can lead to hyper-inflammatory responses, severe degradation of airway epithelia and consequent host mortality even if virus is cleared.
  • RIPK3 was recently uncovered as a targetable signaling pathway that accounts for almost all IAV-activated pathogenic death in infected cells. This pathway is initiated when the host sensor protein DAI detects IAV genomic RNA and activates RIPK3 kinase. RIPK3 then triggers a form of cell death called necroptosis (or programmed necrosis), responsible for much of the lung injury seen during IAV infection. 30 Fascinatingly, eliminating necroptosis not only drastically reduces lung damage and improves animal survival, but does so without impeding virus clearance. This is because RIPK3 also Page 2 QB ⁇ 166118.01517 ⁇ 95733313.1
  • apoptosis non-pathogenic cell death
  • apoptosis non-pathogenic cell death
  • RIPK3 kinase activity inhibitors of RIPK3 kinase function will be expected to ameliorate necrotic lung injury without affecting virus clearance, and potentially represent an entirely new strategy for treatment of IAV disease.
  • two separate 5 studies showed that animals deficient in Ripk3 gene show resistance in the development of atherosclerosis due to the inhibition of macrophage cell death and systemic inflammation. No selective RIPK3 kinase inhibitors are in clinical use or have been advanced into clinical trials.
  • R 1 is H or - –(CH 2 ) n –X 1 wherein X 1 is a heterocycloalkyl optionally substituted with alkyl and n is an integer between 0-3; and R 2 is H or - –(CH2)m–X 2 wherein X 2 is a heterocycloalkyl optionally substituted with alkyl and m is an integer between 0-3.
  • Page 3 QB ⁇ 166118.01517 ⁇ 95733313.1
  • the disclosed compounds may exhibit one or more biological activities.
  • the disclosed compounds may inhibit necroptosis.
  • the disclosed compounds may inhibit receptor interacting kinase 3 (RIPK3).
  • Another aspect of the invention provides for a pharmaceutical composition.
  • the 5 pharmaceutical composition comprises an effective amount of the compound as described herein, and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof.
  • Another aspect of the invention provides for a method of treating a protein kinase related disease or condition. The method comprises administering to the subject the compound as 10 described herein or the pharmaceutical composition as described herein.
  • Another aspect of the invention provides for a method of treating a subject for a respiratory virus infection. The method comprises administering to the subject a RIPK3 inhibitor two or more days after infection.
  • Influenza A viruses activate ZBP1 and trigger RIPK3-driven parallel pathways of MLKL-dependent necroptosis and caspase 8-driven apoptosis. Only necroptosis is reliant on RIPK3 kinase activity.
  • a IAV mRNA expression in lung cell types at 6 days after infection with PR8 (2500 EID50; i.n.).
  • b Distribution of IAV + primary lung cell types at 6 days after infection
  • c IAV replication and 15 ZBP1 expression in Type I AECs following IAV infection in vivo.
  • Cell viability was determined by Trypan Blue exclusion in panels d, f, and i.
  • Fig. 3 shows that UH15-38 prevents lethality in severe influenza.
  • b Survival analysis of mice challenged with PR8 (4500 EID50; ⁇ LD60) and treated i.p.
  • Fig.4 shows that UH15-38 prevents necroptosis, inflammation, and injury in IAV-infected lungs.
  • a, b Immunofluorescence staining of pMLKL (a) and quantification of pMLKL signal (b) in lung sections harvested on the indicated days after infection from mice infected with PR8 (6000 EID 50 ) and treated i.p. with either vehicle or UH15-38 (30 mg/kg once-daily), starting one day after infection, for up to four days.
  • mice were treated with either vehicle or UH15-38 (30 mg/kg once-daily) starting D1 post-infection.
  • d Lung images (d) and quantification (e) of neutrophil influx three days after infection with PR8 (6000 EID50), following treatment with either vehicle or UH15-38 (30 mg/kg, i.p., once-daily) starting one day after infection.
  • f H & E stained lung sections of vehicle- and UH15-38-treated mice nine days after 30 infection with PR8 (4500 EID 50 ). Left arrow shows hyaline membranes and right arrow depicts denuded bronchioles in vehicle-treated tissue.
  • g Histological scores of late lung injury Page 6 QB ⁇ 166118.01517 ⁇ 95733313.1
  • SpO2 Arterial oxygen saturation
  • a Immunoblot analysis of pMLKL and cleaved caspase 8 (CC8) in lysates obtained from Ripk3 -/- MEFs stably expressing 2xFv-RIPK3 and treated with dimerizer (AP20187, 100 nM) in the presence of the pan-caspase inhibitor (IDN-6556, 20 ⁇ M) and UH15-38 (500 nM) for 12 h.
  • b Table comparing UH15-38 to 15 GSK’872 in the indicated recombinant kinase assays.
  • c Overview of docking of UH15-38 into human RIPK3.
  • d Docking of GSK’872 into mouse RIPK3.
  • e Docking of GSK’843 into mouse RIPK3.
  • g Cell survival kinetics of iBMDMs treated with LPS (10 ng/ml) and TAK1 inhibitor 5z7 (200 nM) following exposure to the indicated concentrations of the RIPK3 inhibitors UH15- 38 and GSK’872 or the RIPK1 inhibitor GSK’547 for 6 h.
  • Ripk3 +/+ and Ripk3 -/- MEFs were treated with TNF/5z7/IDN6556 and TNF/5z7, respectively, in the presence of the indicated concentrations of UH15-38, and viability was determined after 24 h. Ripk3 -/- MEFs treated with 25 UH15-38 alone were used as controls. i, Viability of Ripk3 -/- MEFs treated with TNF/5z7 in the presence of DMSO or GSK’547 (10 ⁇ M).
  • GSDMD Gasdermin D
  • k Immunoblot analysis of Gasdermin D (GSDMD) cleavage in primary BMDMs pre-treated with LPS (10 ng/ml) for 3 h followed by Nigericin (10 ⁇ M) or ATP (5 mM), along with DMSO or UH15-38 (500 nM), for an additional 1 h.
  • k Viability of primary BMDMs pre-treated with LPS (10 ng/ml for 3 h) followed by treatment with Nigericin 30 (10 ⁇ M) and the indicated concentrations of UH15-38 for 1 h. Viability was determined by using CellTiter-Glo assay (as in g, h, k) or Trypan Blue exclusion assay (as in f). Bars from left to right: Page 7 QB ⁇ 166118.01517 ⁇ 95733313.1
  • Fig. 6 shows the pharmacological profiling of UH15-38. a, Half-life (T1/2) and peak 5 concentrations (Cmax) of UH15-38 in tissue samples collected from mice treated with four once- daily) doses of UH15-38 (30 mg/kg/day, i.p.).
  • PDPN Podoplanin staining demonstrates purity of primary Type I AECs.
  • CD140a was used as control for fibroblastic (Fibs) contamination.
  • b Cell survival kinetics of Type I AECs 20 treated with TCZ and exposed to the indicated concentrations of the RIPK3 kinase inhibitors UH15-38, GSK’843 or GSK’872).
  • e Cell survival analysis following infection of MEFs with a panel of IAV and IBV strains and exposure to the indicated concentrations of UH15-38 in the presence or absence of zVAD (50 ⁇ M).
  • HeLa- 10 RIPK3 cells treated with TCZ and the indicated concentrations of UH15-38 were examined for pMLKL, total MLKL, and RIPK3 by immunoblot analysis 18 h after treatment.
  • H1N1 influenza strains A/Puerto Rico/8/1934 (PR8), A/California/04/2009 (Cal/09); H3N2 influenza strains: A/Brisbane/10/2007 15 (Bri/07), A/Singapore/INFIMN-16-0019/2016 (Sin/16); Influenza B virus strains B/Colorado/06/2017 (Col/17) and B/Florida/04/2006 (Flo/06).
  • Fig.9 shows that UH15-38 prevents lethality in severe influenza.
  • d, e, Survival (d) and weight loss (e) curves of mice (n 7) infected with PR8 (4500 EID50) and treated i.p. with vehicle or GSK’872 (30mg/kg) as indicated above the graph.
  • a, b Immunofluorescence staining of cleaved caspase 3 (CC3) (a) and quantification of CC3 signal (b) in lung sections harvested on the indicated days post-infection 25 from mice infected with PR8 (6000 EID50) and treated i.p.
  • Fig. 12 shows the gating strategy for flow cytometric analyses.
  • a, b Gating strategy for the flow cytometric analyses presented in Fig.4, panel i and Fig.11, panel d, respectively.
  • 30 Fig 13 shows that MLKL binding requires the displacement of the RIPK3 ⁇ C helix.
  • a,b Structures of monomeric (a) and MLKL-bound (b) mRIPK3.
  • Fig. 14 shows viability of (a) HT29, (b) THP1, and (c) FADD-def Jurkat cells after treatment with selected compounds. Cells were treated with the compounds at indicated doses.
  • necroptosis was induced by adding 10 ng/ml human TNF ⁇ (a-c), 100 nM SM164 (a,b) and 20 ⁇ M IDN6556. Following 16-18 hr incubation, cell viability was determined using CellTiter-Glo assay. DMSO-treated unstimulated cells were used as a 100% viability control. IC50 10 values were determined using non-linear regression analysis in GraphPad Prism. DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to protein kinase inhibitors and uses thereof.
  • alkyl as contemplated herein includes a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C12 alkyl, C1-C10-alkyl, and C1-C6-alkyl, respectively.
  • alkylene refers to a diradical of an alkyl group (e.g., -(CH 2 ) n - where n is an 25 integer such as an integer between 1 and 20).
  • An exemplary alkylene group is -CH 2 CH 2 -.
  • haloalkyl refers to an alkyl group that is substituted with at least one halogen.
  • haloalkyl refers to an alkyl group that is substituted with at least one halogen.
  • heteroalkyl refers to an “alkyl” group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom).
  • a heteroatom e.g., an O, N, or S atom
  • One type of 30 heteroalkyl group is an “alkoxy” group. Page 12 QB ⁇ 166118.01517 ⁇ 95733313.1
  • alkenyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl, respectively.
  • alkynyl refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl, respectively.
  • cycloalkyl refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic 10 (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derived from a cycloalkane.
  • cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, 15 hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl.
  • the cycloalkyl group is not substituted, i.e., it is unsubstituted.
  • cycloheteroalkyl or “heterocycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least 20 one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S.
  • cycloalkylene refers to a cycloalkyl group that is unsaturated at one or more ring bonds.
  • partially unsaturated carbocyclyl refers to a monovalent cyclic hydrocarbon that contains at least one double bond between ring atoms where at least one ring of the carbocyclyl 25 is not aromatic.
  • the partially unsaturated carbocyclyl may be characterized according to the number oring carbon atoms.
  • the partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, and accordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl, respectively.
  • the partially unsaturated carbocyclyl may be in the form of a monocyclic carbocycle, bicyclic carbocycle, tricyclic carbocycle, bridged 30 carbocycle, spirocyclic carbocycle, or other carbocyclic ring system.
  • Exemplary partially unsaturated carbocyclyl groups include cycloalkenyl groups and bicyclic carbocyclyl groups that Page 13 QB ⁇ 166118.01517 ⁇ 95733313.1
  • partially unsaturated carbocyclyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, 5 hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl.
  • the partially unsaturated carbocyclyl is not substituted, i.e., it is unsubstituted.
  • aryl is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like.
  • aryl 10 includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls.
  • the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, 15 alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF 3 , -CN, or the like.
  • the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, 20 the aryl group is a 6-10 membered ring structure.
  • the terms “heterocyclyl” and “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3-to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur.
  • the number of ring atoms in the heterocyclyl group can be specified using 5 25 Cx-Cx nomenclature where x is an integer specifying the number of ring atoms.
  • a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur.
  • the designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines (e.g., mono-substituted amines or di-substituted amines), wherein substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.
  • substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl.
  • alkoxy or “alkoxyl” are art-recognized and refer to an alkyl group, as defined 5 above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tert-butoxy and the like.
  • O-alkylamine refers to –O– (CR 1 R 2 ) m –NR 3 R 4 , wherein m is an integer between 1-6, and R 1 , R 2 , R 3 and R 4 , for example, are each independently hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, and aryl. R 3 and R 4 , together with the nitrogen they are attached to, may also be optionally 10 joined to form a 4-8 membered cycloalkyl.
  • heterocycloalkyl refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S(O) n , wherein n is an integer of 0-2.
  • “Four to seven membered heterocycloalkyl” refers to a heterocycloalkyl 15 containing from four to seven atoms, including one or more heteroatoms, in the cyclic moiety of the heterocycloalkyl.
  • single-ring heterocycloalkyls examples include azetidinyl, oxetanyl, thietanyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, dihydropyranyl, piperidinyl, morpholinyl, piperazinyl, azepinyl, 20 oxepinyl, and diazepinyl.
  • the heterocycloalkyl described herein may be fused with a cycloalkyl, an aryl, or a heteroaryl, as described herein.
  • the heterocycloalkyl described herein may be , . lently linked by an oxygen. Accordingly, the 25 substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, and the like.
  • carbonyl refers to the radical -C(O)-.
  • oxo refers to a divalent oxygen atom –O-.
  • carboxymido refers to the radical -C(O)NRR', where R and R' may be the same or different. R and R', for example, may be independently alkyl, aryl, arylalkyl, 5 cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl.
  • carboxy as used herein refers to the radical -COOH or its corresponding salts, e.g. -COONa, etc.
  • amide or “amido” or “amidyl” as used herein refers to a radical of the form – R 1 C(O)N(R 2 )-, -R 1 C(O)N(R 2 )R 3 -, -C(O)NR 2 R 3 , or -C(O)NH 2 , wherein R 1 , R 2 and R 3 , for 10 example, are each independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro.
  • the compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or 15 diastereomers.
  • stereoisomers when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” or “+” or “-” depending on the configuration of substituents around the stereogenic carbon atom and or the optical rotation observed.
  • Stereoisomers include enantiomers and diastereomers.
  • Mixtures 20 of enantiomers or diastereomers may be designated ( ⁇ )” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise.
  • compositions comprising, consisting essentially of, or consisting of an enantiopure compound, 25 which composition may comprise, consist essential of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% of an R enantiomer of a given compound).
  • Compounds Disclosed herein includes a compound having a formula of Page 16 QB ⁇ 166118.01517 ⁇ 95733313.1
  • R 1 is H or - –(CH2)n–X 1 wherein X 1 is a heterocycloalkyl optionally substituted with alkyl and n is an integer between 0-3; and 5 R 2 is H or - –(CH2)m–X 2 wherein X 2 is a heterocycloalkyl optionally substituted with alkyl and m is an integer between 0-3.
  • n is 0 or 1 or m is 0 or 1.
  • X 1 is , 10 X 2 is In some embodiments, X 1 is 15 , In some embodiments, X 2 is Page 17 QB ⁇ 166118.01517 ⁇ 95733313.1
  • X is or 5 10 , , Page 18 QB ⁇ 166118.01517 ⁇ 95733313.1
  • the disclosed compounds may exhibit one or more biological activities. In some embodiments, the disclosed compounds inhibit necroptosis. In some embodiments, the disclosed compounds modulate the activity of receptor interacting kinase 3 (RIPK3). In some embodiments, the disclosed compounds inhibit the activity of RIPK3 in vivo or in vitro.
  • RIPK3 receptor interacting kinase 3
  • 5 the disclosed compounds inhibit the activity of RIPK3 by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 100 ⁇ M, 50 ⁇ M, 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 0.05 ⁇ M, 0.01 ⁇ M, 0.005 ⁇ M, 0.001 ⁇ M, 0.1 nM, 0.01 nM, or less.
  • Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.01 nM, 0.1 nM, 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, 10 ⁇ M, 10 and 100 ⁇ M.
  • the disclosed compounds inhibit necroptosis, such as RIPK3- dependent necroptosis of cells.
  • the necroptosis may be induced by IAV.
  • the necroptosis may be induced by lipopolysaccharide (LPS), tumor necrosis factor (TNF), pan-caspase inhibitor, or a combination thereof.
  • %Ctrl Percent Control
  • %Ctrl 100 x (TS – CPOS) / (CNEG – CPOS) (eqn 1) where TS is the test compound signal, CPOS is the positive control signal (0 %Ctrl), CNEG is the DMSO negative control (100 %Ctrl). 5 Selectivity Score (S ⁇ scores). Selectivity Score or S-score is a quantitative measure of compound selectivity.
  • the compound may have a %Ctrl less than 4%, 3%, 2%, 1%, 0.5%, or 0.1% and greater than 0% for RIPK3. 20
  • the disclosed compounds may have an S(35) value less than 0.50. In some instances, the disclosed compounds may have an S(35) value less than 0.40 or 0.30.
  • Pharmaceutical Compositions Another aspect of the present disclosure provides a pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt thereof disclosed herein and a 25 pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof.
  • the compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein.
  • compositions may take any physical form which is pharmaceutically acceptable; 30 illustratively, they can be orally administered pharmaceutical compositions.
  • Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is Page 22 QB ⁇ 166118.01517 ⁇ 95733313.1
  • Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose.
  • the amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and 5 other factors, such as the indication for which it is given.
  • the pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.
  • the compounds disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more 10 compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents.
  • the pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg).
  • the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg/kg body weight (preferably about 0.5 to about 500 mg/kg body weight, 15 more preferably about 50 to about 100 mg/kg body weight).
  • the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.01 nM, 0.1 nM, 0.001 ⁇ M, 0.005 ⁇ M, 0.01 ⁇ M, 0.5 ⁇ M, 0.1 ⁇ M, 1.0 ⁇ M, 10 ⁇ M, and 100 ⁇ M (e.g., 0.1 ⁇ M - 1.0 ⁇ M). 20 It is understood by those skilled in the art that dosage amount will vary with the activity of a particular inhibitor compound, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts.
  • a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect.
  • an effective dose of such a compound, pharmaceutically acceptable salt thereof, or related 25 composition may be administered in two or more sub-doses, administered separately over an appropriate period of time.
  • the pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include 30 the compound in a range of from about 1 to 100 mg.
  • the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg/kg body Page 23 QB ⁇ 166118.01517 ⁇ 95733313.1
  • the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.5 to about 500 mg/kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 50 to about 100 mg/kg body weight. 5
  • the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier.
  • the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.
  • the compounds utilized in the methods disclosed herein may be formulated as a 10 pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.
  • Filling agents may include lactose monohydrate, lactose anhydrous, and various starches;
  • binding agents are various celluloses and cross- linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® 15 PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCCTM).
  • Suitable lubricants may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • colloidal silicon dioxide such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.
  • sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. 20
  • flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like.
  • preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.
  • Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing.
  • diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and 30 glucose.
  • microcrystalline cellulose such as Avicel® PH101 and Avicel® PH102
  • lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21
  • dibasic calcium phosphate such as Emcompress®
  • mannitol starch
  • sorbitol sucrose
  • sucrose and 30 glucose.
  • Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.
  • effervescent agents are effervescent couples such as an organic acid and a 5 carbonate or bicarbonate.
  • Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts.
  • Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate.
  • sodium bicarbonate component of the effervescent 10 couple may be present.
  • the compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients 15 as appropriate to the desired preparation.
  • compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
  • Such formulations may be 20 prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
  • compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non- aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid 25 emulsions.
  • Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis.
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, Page 25 QB ⁇ 166118.01517 ⁇ 95733313.1
  • the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream.
  • the compound When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. 10
  • Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas.
  • Pharmaceutical compositions adapted for nasal administration where the carrier is a solid 15 include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose).
  • Suitable formulations where the carrier is a liquid for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.
  • Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.
  • Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed 30 ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Page 26 QB ⁇ 166118.01517 ⁇ 95733313.1
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, 5 sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate.
  • binding agents for example syrup, acacia, gelatin, 5 sorbitol, tragacanth, or polyvinylpyrrolidone
  • fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or g
  • Oral liquid preparations may be in the form of, for example, 10 aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan 15 monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents.
  • suspending agents for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan 15 monooleate,
  • the disclosed compounds or pharmaceutical compositions comprising the 20 disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders.
  • one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after 25 administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds.
  • the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders.
  • Methods of preparing pharmaceutical formulations or compositions include the step of bringing an inhibitor compound into association with a carrier and, optionally, one or more Page 27 QB ⁇ 166118.01517 ⁇ 95733313.1
  • compositions comprising the disclosed compounds may be administered in methods of treating a subject in need thereof.
  • methods of treatment a subject in need thereof may include a subject having a cell proliferative disease, disorder, or condition such as cancer.
  • Another aspect of the present disclosure provides a method of treating a protein kinase related disease or condition comprising administering the pharmaceutical composition disclosed herein to a subject in need thereof.
  • Another aspect of the present disclosure provides a method of treating a subject for a respiratory virus infection comprising administering the subject a RIPK3 inhibitor, such as a RIPK3 inhibitor disclosed herein, two or more days after infection.
  • a RIPK3 inhibitor such as a RIPK3 inhibitor disclosed herein
  • the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to 20 prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder.
  • the methods disclosed herein encompass both therapeutic and prophylactic administration.
  • a “subject in need thereof” as utilized herein refers to a subject in need of treatment for a disease or disorder associated with a protein kinase activity.
  • a “subject in 25 need thereof” as utilized herein refers to a subject in need of treatment for a disease or disorder associated with receptor interacting kinase 3 (RIPK3) activity.
  • the term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects.
  • the treated subject may be a mammalian subject.
  • mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs. Page 28 QB ⁇ 166118.01517 ⁇ 95733313.1
  • disorders refers to a condition in which there is a disturbance of normal functioning.
  • a “disease” is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant 5 behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms “disease”, “disorder”, “condition” and “illness”, are equally used herein. Diseases and disorders associated with RIPK3 activity may include, but are not limited to, human auto-immune, auto-inflammatory, and degenerative diseases and/or disorders.
  • the diseases 10 and/or disorders may include, but are not limited to, progressive chronic diseases with inflammatory etiology including atherosclerosis and TNF-driven pathologies such as rheumatoid arthritis and ulcerative colitis; deleterious acute inflammatory responses such as upon infection by influenza virus; auto-immune diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and lupus; lipid and lysosomal storage diseases such as Neiman-Pick and Gaucher 15 disease; ischemia-reperfusion injuries such as stroke, myocardial infarction, kidney, retinal and liver ischemia, immune checkpoint inhibitor-induced myocarditis, scleroderma (SSc), scleroderma-associated interstitial lung disease (SSc-ILD), psoriasis, sepsis, age-related macular degeneration (AMD), asthma, lichen planus (LP), Steven-Johnson syndrome (SJS), toxic epidermal necrolysis
  • the diseases and/or disorders may be influenza A virus (IAV) infection, influenza-induced lung damage, interstitial pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome (ARDS), viral pneumonia or bacterial pneumonia, necrotic lung injury, and/or RIPK3-mediated necroptosis.
  • the compounds and/or pharmaceutical compositions disclosed herein may be administered 25 in methods of treating a protein kinase related disease or condition, such as diseases or conditions involving RIPK3 activity, including inflammatory and degenerative diseases or conditions.
  • the diseases or conditions may be a respiratory virus, such as Influenza A virus (IAV) infection.
  • the diseases or conditions may be inflammatory conditions driven by TNF, influenza-induced lung damage, interstitial pulmonary fibrosis, chronic 30 obstructive pulmonary disease, acute respiratory distress syndrome (ARDS), viral pneumonia or bacterial pneumonia, necrotic lung injury, and/or RIPK3-mediated necroptosis.
  • TNF inflammatory conditions driven by TNF, influenza-induced lung damage, interstitial pulmonary fibrosis, chronic 30 obstructive pulmonary disease, acute respiratory distress syndrome (ARDS), viral pneumonia or bacterial pneumonia, necrotic lung injury, and/or RIPK3-mediated necroptosis.
  • ARDS acute respiratory distress syndrome
  • the compounds and/or pharmaceutical compositions disclosed herein may be administered in methods of treating a subject for a respiratory virus infection.
  • the method may comprise administering the subject a RIPK3 inhibitor two or more days after infection.
  • the RIPK3 inhibitor may be administered five or more days after infection.
  • the RIPK3 inhibitor may be UH15-38 or any of the compounds disclosed herein.
  • the compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds.
  • a compound that modulates RIPK3 activity may be administered as a single compound or in combination with another compound that modulates RIPK3 activity or that has a different pharmacological activity.
  • the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 15 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject.
  • a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg,
  • the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 20 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject.
  • a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg
  • Minimal and/or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg – 200 mg). 25
  • a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject.
  • a 30 maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, Page 30 QB ⁇ 166118.01517 ⁇ 95733313.1
  • Minimal and/or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of 5 these disclosed dose levels (e.g., 500 – 2000 ng/kg body weight of the subject).
  • the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment.
  • the disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) 10 for treating a disease or disorder associated with RIPK3 activity.
  • An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the 15 subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
  • a typical daily dose may contain from about 0.01 mg/kg to about 100 mg/kg (such as from 20 about 0.05 mg/kg to about 50 mg/kg and/or from about 0.1 mg/kg to about 25 mg/kg) of each compound used in the present method of treatment.
  • Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and/or about 25 mg.
  • unit 25 dosage form refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.
  • Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein.
  • Other illustrative routes of administration include 30 transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes.
  • the route of administration may be varied in any way, limited Page 31 QB ⁇ 166118.01517 ⁇ 95733313.1
  • administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof.
  • miscellaneous 10 Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill 15 in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ⁇ 10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
  • the terms “comprise” and “comprising” should be interpreted as 20 being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
  • the terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims.
  • the term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not 25 fundamentally alter the nature of the claimed subject matter. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
  • IAV-activated necrotic cell death is potentially one such entry point.
  • IAV kills most lung cell types in which it replicates 12 .
  • it is an effective mechanism of virus clearance, but when death is unchecked, or primarily necrotic, then 25 lung injury and severe illness ensues, despite virus clearance 12,13 .
  • Such severe pathology is observed in mouse models, where destruction of airway epithelia is a hallmark of lethal IAV infection 4,14,15 , and in humans, where extensive death of distal pulmonary epithelia, marked by areas of bronchoalveolar necrosis, is a classic feature of IAV-induced ARDS, and of viral and secondary bacterial pneumonia 2,3,12 .
  • Page 33 QB ⁇ 166118.01517 ⁇ 95733313.1
  • Necroptosis drives influenza severity. Necroptosis accounts for most IAV-activated programmed necrotic death in infected lung epithelial cells 6-8,12,16,17 . Necroptosis is initiated by the host sensor protein Z-form nucleic acid Binding Protein 1 (ZBP1), which detects IAV-generated Z-RNA and activates RIPK3 18,19 . RIPK3 then phosphorylates Mixed Lineage Kinase Like protein 5 (MLKL), which induces necroptosis 6,8,12,13,18 (Fig. 1, panel a).
  • ZBP1 Z-form nucleic acid Binding Protein 1
  • MLKL Mixed Lineage Kinase Like protein 5
  • necroptosis triggers pulmonary tissue necrosis, pathogenic neutrophil recruitment and lung inflammation during severe IAV infections, but is dispensable for either CD8 + T cell-mediated antiviral responses or for virus clearance 8,13 .
  • RIPK3 also activates a parallel pathway of apoptosis, which is fully capable of restricting IAV in the absence of necroptosis 10 signaling 6 .
  • Inhibitors of RIPK3 kinase activity may be expected to ameliorate necrotic lung injury without affecting virus clearance, and, therefore, may represent an entirely new strategy for treatment of IAV-triggered lung inflammation and injury.
  • IAV H1N1 strain A/PuertoRico/8/1934; hereafter PR8
  • Mlkl -/- mice are selectively deficient in RIPK3 kinase-driven necroptosis, we reasoned that results from these mice will foreshadow the effects of RIPK3 kinase inhibition 20 during severe influenza in vivo.
  • RIPK3 represents a natural target for necroptosis blockade as it is the only kinase known to phosphorylate MLKL, and 5 as phosphorylation of MLKL is the only known function of its kinase activity. This therapeutic strategy is, however, currently hindered by a lack of efficient and selective RIPK3 inhibitors for in vivo use.
  • GlaxoSmithKline identified three molecules (GSK’872, GSK’843, and GSK’840) with potent activity against recombinant RIPK3 in vitro, but unexpectedly modest activity in cells 20 . Furthermore, even a two-fold increase in the concentration 10 of these inhibitors triggered RIPK3-mediated apoptosis and unleashed consequent on-target toxicity 20 . In agreement with these findings, knock-in mice harboring a mutation (D161N) in RIPK3 designed to nullify necroptosis by inactivating catalytic function succumbed instead to fulminant RIPK3-mediated apoptosis in utero 21 .
  • D161N mutation
  • UH15-38 prevented 30 phosphorylation of MLKL following necroptotic stimulation by TNF ⁇ (Fig.1e), or upon enforced Page 35 QB ⁇ 166118.01517 ⁇ 95733313.1
  • UH15-38 may display unique features in its RIPK3 binding mode that are distinct from GSK inhibitors and that translate strong RIPK3 binding into potent inhibition of necroptosis.
  • molecular docking studies showed that UH15-38 can interact with the ATP binding pocket of both 10 mouse and human RIPK3 in the active conformation, indicative of a Type I binding mode (Fig.1, panels g-i, Fig.5, panel c).
  • UH15-38 makes interactions with the backbone of the gatekeeper Met98 residue (Met97 in humans) in the hinge segment of the RIPK3 kinase domain (Fig.1, panels h, i; Fig.5, panel c).
  • Met98 residue Met97 in humans
  • Fig.1, panels h, i; Fig.5, panel c the hinge segment of the RIPK3 kinase domain
  • modeling and X-ray crystal structure data 26 for GSK’872 and GSK’843, 15 respectively displayed generally similar hinge binding poses to UH15-38 ( Fig. 5, panels d, e), which may explain why the in vitro affinities of the GSK molecules for RIPK3 are on par with that of UH15-38.
  • UH15-38 differed significantly from both GSK compounds in its interactions with the rear pocket of the RIPK3 active center. While GSK’872 and GSK’843 only made contacts with the backbone of the DFG motif of mouse RIPK3 ( Fig.5, panels d, e), UH15- 20 38 not only engaged in similar interactions with the DFG motif, but also participated (via the phenol) in a broader network of hydrogen bonds and ionic-dipole interactions with the charged side-chains of catalytic Lys51 and the central residue in the ⁇ C helix, Glu61 (Fig.1, panels h, i; Fig.5, panel c).
  • UH15-38 was able to effectively prevent MLKL phosphorylation in all tested organs (lung, liver, heart, kidney) in Casp8 -/- Mlkl FLAG/FLAG mice, demonstrating its potential for therapeutic 15 deployment in necroinflammatory diseases not just of the lung, but of these other organs as well ( Fig.6, panels b, c).
  • Comprehensive safety profiling of UH15-38 for off-target activity against a panel of 50 critical protein targets whose inhibition has been linked to potentially serious side effects in humans demonstrated that UH15-38 did not significantly inhibit any 20 of these key targets ( Fig. 6, panel d, Table 1A).
  • UH15-38 was exceptionally well tolerated in vivo, with no detectable signs in any of these organs of on-target apoptosis as measured by cleaved caspase 3 ( Fig.6, panels e, f), or of general toxicity (Tables 2A and 2B), even after extended dosing at 30 mg/kg/day (i.p) for seven consecutive days.
  • Tables 2A and 2B show whole animal and organ weight changes after treatment 5 with UH15-38 for 7 consecutive days at 30 mg/kg/day (i.p).
  • the formulation was prepared using 100 mg/ml DMSO stock diluted to 30 mg/kg of mice (C57BI/6) weight in 10/90 solutol/saline. Table 2A.
  • ALT Alanine Aminotransferase
  • BUN Blood Urea Nitrogen
  • the BUN is useful in the evaluation of kidney function and nutritional status. Increased levels will be seen with kidney disease, increased protein intake (high protein diet), and dehydration. Decreased levels will be seen with overhydration, severe primary liver disease and malnutrition.
  • CRE Creatinine
  • Elevations in creatinine generally suggest chronic kidney disease.
  • UH15-38 blocks IAV-activated necroptosis.
  • 15 small molecule blockade of RIPK3 kinase activity by UH15-38 will be efficacious in mitigating IAV-induced pathology, we first sought to identify the primary cell type(s) undergoing RIPK3- dependent necroptosis in infected lungs, as these cell types will represent the dominant targets for pharmacological blockade of RIPK3 activity in vivo.
  • Type I alveolar epithelial cells (AECs) as the primary replicative 20 niche for IAV in murine lungs; Type I AECs displayed significantly (84-fold) higher levels of IAV mRNA than any other cell type (Fig.2, panel a) and comprised over half of all IAV-positive cells in the infected lung (Fig.2, panel b). High levels of IAV mRNA were readily observed in Type I AECs by three days after infection, coincident with induction of Zbp1 mRNA (Fig. 2, panel c). Death of Type I AECs is also highly correlated with compromised lung function and mortality in 25 the mouse model 15 .
  • Type I AECs succumbed to IAV by 24 hours (h), similarly infected Type I AECs from littermate-matched Zbp1 -/- animals remained mostly viable for over 48 h (Fig.2, panel d).
  • Type I AECs strongly upregulated ZBP1 upon infection, possessed the entire ZBP1-dependent cell death machinery, and displayed markers of both necroptosis (pMLKL) and 5 apoptosis (cleaved caspases-8 and -3) activation following infection with IAV (Fig. 2, panel e).
  • UH15-38 15 also prevented activation of MLKL and selectively inhibited necroptosis in cells infected with a panel of clinically relevant IAV and IBV strains ( Fig.7, panels e, f). UH15-38 completely blocked IAV-induced necroptosis in Ripk1 -/- MEFs ( Fig.7, panel g).
  • UH15-38 was derived from a BCR-ABL inhibitor with inhibitory activity against RIPK1 and RIPK2, neither the ABL inhibitor imatinib (Gleevec) nor the selective RIPK2 inhibitor CSLP37 32 blocked IAV-triggered necroptosis 20 in cells ( Fig.7, panels h, i), demonstrating that while UH15-38 can inhibit RIPK1, RIPK2 and ABL kinase activity in vitro (Tables 1A-1B, Fig. 16, and Fig. 5, panel b), the ability to target RIPK3 is central to its necroptosis-inhibitory activity in cells.
  • TNF ⁇ -induced necroptosis in HeLa-RIPK3 cells ( FIG.7, panels l, m).
  • Most human cell lines (such as HeLa cells, as noted above) lack expression of one or more components of the necroptosis machinery, but we identified a human malignant mesothelioma-derived cell line (M29) 33 which expressed ZBP1, RIPK3, and MLKL and underwent necroptosis following infection with IAV 5 (Fig.2, panel i, Fig.7, panel n).
  • M29 human malignant mesothelioma-derived cell line
  • UH15-38 was able to prevent IAV-induced phosphorylation of MLKL at nanomolar concentrations ( Fig.
  • UH15-38 The toxicity manifested by UH15-38 at 100x IC 50 was indeed 20 RIPK3-dependent apoptosis, as it was accompanied by activation of caspases 8 and -3 ( Fig. 8, panels b, e), and was eliminated in Ripk3 -/- MEFs, or by addition of pan-caspase inhibitor zVAD.fmk (Fig. 8, panel c).
  • a compound with potent in cellulo efficacy in Type I AECs and other cell types as well as wide separation of necroptosis- inhibitory activity from the capacity to trigger on-target apoptosis. 25 UH15-38 prevents IAV-driven lethality.
  • mice with maximum protection observed at 30 mg/kg/day (Fig.3, panel a, Fig.9, panel a).80% of mice receiving the 30mg/kg/day dose of UH15-38 manifested significantly reduced (and delayed) weight loss and were fully protected from a lethal inoculum of IAV; these mice made full recoveries by three weeks after infection (Fig.3, panel a, Fig.9, panel a). Increasing the dose of 5 UH15-38 to 50 mg/kg/day did not provide any discernible additional benefit to survival outcomes of infected mice (Fig. 3, panel a, Fig. 9, panel a).
  • UH15-38 Evaluating UH15-38 for protection when administered over a shortened time course (30 mg/kg/day, once daily, for two days, starting one day after infection), or delaying administration by up to two days (i.e., starting UH15-38 at 30 mg/kg/day two or three days after infection) also reduced weight loss and prevented lethality in a 10 significant proportion of mice infected with this highly lethal dose of virus ( Fig.9, panels b, c).
  • UH15-38 was fully (100%) protective against a less lethal, but more patient- relevant inoculum of IAV (4500 EID50; ⁇ LD60), even at doses as low as 1 mg/kg/day (Fig.3, panel b).
  • UH15-38 was also able to afford full protection against lethality triggered by the H1N1 20 A/California/04/2009 strain of pandemic IAV (Fig.3, panel d, Fig.9, panel f), demonstrating its potential for therapeutic benefit against seasonal and pandemic strains of IAV.
  • UH15- 38 was able to protect almost all mice infected with IAV at the LD 60 dose even when administered up to five days after infection (Fig. 3, panel e, Fig. 9, panel g). This represents a significant advantage over the frontline antiviral agent oseltamivir (Tamiflu, Roche), which loses therapeutic 25 benefit if administered more than 48 h after infection in mice 36 and humans 37 .
  • UH15-38 failed to protect Ripk3 -/- or Mlkl -/- mice from IAV, confirming that its protective effects are mediated by blockade of RIPK LKL signaling (Fig. 3, panel f).
  • UH15-38 treatment was notably superior to germline MLKL loss in preventing IAV-induced lethality at all doses of virus tested ( Fig.8, panels h, i; also compare Fig.1, panel b to Fig.3, panel a).
  • We attribute this result to the 30 benefit some early necroptosis may provide in sparking the antiviral immune response, or to some other currently unknown protective function of MLKL in maintaining baseline immune Page 49 QB ⁇ 166118.01517 ⁇ 95733313.1
  • UH15-38 dampens IAV-induced lung injury.
  • PR8 6000 EID 50
  • UH15-38 (30 mg/kg) to these mice i.p. once-daily for a total of four days, starting 24 h after infection.
  • UH15-38 did not significantly alter the proportion of infected cells undergoing apoptosis (CC3 + ) ( Fig.10, panels a, b), demonstrating that UH15-38 is a selective inhibitor of IAV-triggered necroptosis in vivo.
  • 15 UH15-38 was able to prevent the release of inflammatory cytokines and chemokines (e.g., IL-1 ⁇ , IL-33, IL-6, TNF ⁇ , CXCL1) from infected Type I AECs in culture ( Fig.10, panel c), and this activity was recapitulated in vivo.
  • key inflammatory cytokines and neutrophil chemoattractants e.g., 20 IL-1 ⁇ , IL-6, IL-18, TNF ⁇ , CXCL1, GMCSF
  • lungs from infected animals treated with UH15-38 showed a decrease in diffuse alveolar damage 25 and the formation of hyaline membranes (Fig. 4, panel f, left arrow; Fig. 4, panel g), as well as notably reduced bronchiolar denudation (Fig. 4, panel f, right arrow; Fig. 4, panel g) and histological appearance of fibrosis (Fig.4, panel g).
  • Staining pulmonary tissues for Tenascin C a marker of fibrotic remodeling following injury 38 , confirmed that UH15-38 reduced the extent of fibrotic lung damage ( Fig. 10, panels d,e).
  • BALF from mice 30 receiving UH15-38 displayed significantly lower levels of the pro-fibrotic mediators (IL-1 ⁇ , IL-6, IL-18, IL-17, and CCL5), compared to controls, early (three days) after infection (Fig.4, panel c, Page 50 QB ⁇ 166118.01517 ⁇ 95733313.1
  • Fig. 10, panel f Lungs from UH15-38 treated mice also displayed dampened alveolar and interstitial infiltration, accompanied by decreases in septal thickening and epithelial metaplasia ( Fig. 10, panel g). In addition, UH15-38 completely blocked the release of IAV-triggered inflammatory cytokines IL-1 ⁇ and IL-18 from necroptotic BMDMs ( Fig. 10, panel h). 5 Importantly, UH15-38-treated animals maintained relatively normal lung function, as measured by evaluating arterial oxygen saturation levels three and six days after infection (Fig.4, panel h), and by measuring airway resistance ten days after infection ( Fig.10, panel i).
  • UH15-38 did not alter the extent of virus replication and spread, or the rate of virus clearance from infected lungs ( Fig.11, panels a-c). UH15-38 also did not negatively impact the 10 frequencies of IAV-specific CD8 + cytotoxic T cells (CTLs) in BALF ( Fig.11, panel d, Fig.12, panel a), while the functional profile of these cells, as measured by IFN ⁇ secretion, was improved (Fig. 4, panel i, Fig. 12, panel b). Together, these results demonstrate that UH15-38 dampens neutrophil influx into infected pulmonary tissues and significantly reduces the extent of virus- triggered lung injury, without negatively impacting either virus clearance or anti-IAV CD8 + T cell 15 responses.
  • CTLs cytotoxic T cells
  • necroptosis blockade may represent a therapeutic strategy for a range of lung pathologies, whether of viral etiology or otherwise.
  • RIPK3 kinase inhibitors such as UH15-38 will potentially have 10 benefit in a wider range of acute and chronic inflammatory conditions involving necroptosis than will anti-TNF approaches or RIPK1 inhibitors.
  • Materials and Methods Mice, cells, viruses, reagents 15 Zbp1 -/- 45 , Mlkl -/- 46 , Ripk3 -/- 47 , Ripk1 K45A 48 , Casp8 -/- Mlkl FLAG/FLAG 28 animals have been described previously. C57BL/6 mice were obtained from Taconic and were allowed at least 1 week to acclimate to housing conditions before use in experiments.
  • mice were randomly assigned into groups before each experiment. Mice were housed in SPF facilities at the Fox Chase Cancer Center, St. Jude Children’s Research Hospital, and all in vivo experiments were conducted under 20 protocols approved by the Committee on Use and Care of Animals at these institutions. No statistical methods were used to predetermine sample size. The investigators were not blinded to allocation during experiments. Primary MEFs were obtained from E14.5 embryos and were immortalized using the 3T3 protocol. Immortalized MEFs were routinely tested by qPCR and by immunoblot analyses for protein expression.
  • tandem inducible dimerization domains (hereafter 2xFv) derived from the protein FKBP were placed at the N-terminus of full length human RIPK3 or murine ZBP1.2xFv-tagged constructs were cloned into the Dox-inducible vector pRetroX-TRE3G (631188, Clontech).
  • Activation of RIPK3 or ZBP1 30 in cells expressing the 2xFv constructs was achieved by treatment with doxycycline (5 ⁇ g/ml) for 12 h, followed by exposure to B/B homodimerizer, AP20187 (100 nM).
  • HeLa ATCC, CCL-2
  • Page 53 QB ⁇ 166118.01517 ⁇ 95733313.1
  • H1N1 Human RIPK3 cells were retrovirally transduced with full-length human RIPK3 to generate HeLa-RIPK3 cells.
  • Mouse adapted strains of Influenza A/Puerto Rico/8/1934 (H1N1), A/California/04/2009 (H1N1), Influenza A/Brisbane/10/2007 (H3N2), A/Singapore/INFIMN-16-0019/2017 (H3N2), Influenza B/Florida/04/2006, and B/Colorado/06/2017 were propagated by allantoic cavity inoculation of 10 5 day-embryonated chicken egg.
  • Influenza virus titers were determined by plaque assay on MDCK cells obtained from the ATCC.
  • MEFs and Type I AECs were infected with virus or treated with 100 ng/mL TNF ⁇ (R&D Systems, 410-MT) in the presence of 250 ng/mL cycloheximide (Sigma, 01810) and 50 ⁇ M zVAD (Bachem, N-1510); RAW264.7 cells were treated with 100ng/ml LPS (Sigma, L2630), 20 ⁇ M IDN-6556 (Medkoo, 210530); 10 immortalized Bone marrow derived macrophages (iBMDM, gift of Dr.
  • iBMDM immortalized Bone marrow derived macrophages
  • All cell lines obtained from ATCC were maintained in DMEM 10% FBS, 1mM sodium pyruvate, 1x GlutaMAX and 1% penicillin/streptomycin at 37 o C and 5% CO 2 .
  • the human malignant mesothelial cell line M29 was a gift of Dr. J. Testa, and maintained in RPMI medium supplemented with 10% FBS, 1x GlutaMAX and 1% penicillin/streptomycin.
  • M29 cells were authenticated for 20 use by the Cell Culture Facility at Fox Chase Cancer Center. Immortalized BMDMs were authenticated by FACS for the presence of BMDM markers.
  • UH15-38 Pharmacokinetic and toxicological assessment of UH15-38. Pharmacokinetics of UH15-38 were evaluated in male (9-10 weeks old) C57Bl/6 mice following once-daily dosing at 30 mg/kg for four consecutive days. UH15-38 was administered by i.p. injection, and tissues were collected from three mice for each time point. Plasma was generated by standard centrifugation techniques and immediately frozen. Drug levels are determined by mass 20 spectrometry using an ABSciex 6500 mass spectrometer and multiple reaction monitoring analytical methods. Pharmacokinetic parameters were calculated using a non-compartmental model (Phoenix WinNonlin, Pharsight Inc.). UH15-38 concentrations in each tissue were modeled separately.
  • a polyethylene tube (PE50, BD, 427410) was inserted through the incision and fixed inside the trachea by suture. Lungs were lavaged three times with PBS containing 5 mM EGTA and 5 mM EDTA and instilled with 3ml of RPMI containing 25 mM 10 HEPES, 10% Dextran40, and 4.5 U/ml Elastase, immediately followed by 0.5 ml of 1% (w/v) low melting temperature agarose in water. The trachea was tied below the incision point and the tube was removed.
  • Single cells were centrifuged (250g for 8 min), rinsed in Wash Buffer, and incubated with 40 ⁇ g/ml rabbit polyclonal anti-Aqp5 antibody at 4 o C in a rotator for 40 min. After washing20 (3x) and resuspension in Wash Buffer, cells were incubated with 400 ⁇ l pre-washed goat anti- rabbit IgG BioMag ® beads and rotated at 4 o C for 40 min. Antibody-bound cells were precipitated using a magnetic field, and any unbound cells were removed by washing in Wash Buffer.
  • Antibody-bound cells were washed and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, 1 mM sodium pyruvate, 1x GlutaMAX (Thermo Fisher Scientific) and 1% 25 penicillin/streptomycin.
  • DMEM Modified Eagle Medium
  • Thermo Fisher Scientific 1% 25 penicillin/streptomycin.
  • the purity of Type I AEC isolated in this manner was assessed by staining for podoplanin with an anti-podoplanin antibody (1:500), and for the presence of contaminating fibroblasts using an antibody to CD140a (PDGFR ⁇ ) antibody (1:500).
  • PDGFR ⁇ CD140a
  • Single-cell gene expression analyses were performed on datasets of mouse lung cells isolated pre-infection and 1, 3, and 6 days after infection 51 . Data were processed as previously described 51 . Briefly, sequencing data were processed using CellRanger (v.3.0.2, 10x Genomics) with the mouse mm10 reference genome adapted to include the influenza A/Puerto Rico/8/1934 5 genome. Feature-barcode matrices were then loaded into Seurat (v.3.0.0.900) using a scaling factor of 1x10 4 for subsequent analysis and data visualization 52 . Log-normalization of gene-expression counts was performed, and variable genes were identified using the ‘vst’ method with default parameters. Next, gene expression was scaled to regress out any effects of total number of transcripts, cell cycle scores, and percent mitochondrial expression.
  • PCs principal components
  • Epcam epithelial
  • Pecam1 endothelial
  • Col1a2 mesenchymal
  • Ptprc immune
  • Data were then subset on non- immune cells and dimensionality reduction using t-SNE and clustering was performed.
  • Immunoblotting Cells were lysed in RIPA lysis buffer (Thermo Scientific, 89900) containing protease and phosphatase inhibitors (Thermo Scientific, 815-968-0747).
  • human FADD (1:1000), Mouse monoclonal anti-human caspase 8 (1:1000), Rabbit polyclonal anti-mouse caspase 8 (1:1000), Rabbit monoclonal anti-mouse cleaved caspase-8 (1:1000), Rabbit polyclonal anti-caspase-3 (1:1000), Rabbit polyclonal anti-influenza A virus NP (1:5000), Mouse monoclonal anti-influenza A virus NS1 (1:2000), Mouse monoclonal anti-influenza B 5 Nucleoprotein (1:2500), Rabbit polyclonal anti-GSDMD (1:1000), Mouse monoclonal anti- ⁇ - actin ((1:2000)), Mouse monoclonal GAPDH (1:2000), Rabbit polyclonal anti-alpha tubulin (1:5000).
  • anti-phosphorylated murine MLKL 53 (1:1000), rabbit polyclonal anti-cleaved caspase 3 (1:500) and anti-podoplanin (1:500), Donkey anti-Rabbit IgG (H+L) Highly 20 Cross-Adsorbed Secondary Antibody (1:500), Alexa FluorTM 594, Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (1:500), Alexa FluorTM 488, Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (1:500), Alexa FluorTM 488, Donkey anti- Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa FluorTM Plus 594 A32744 (1:500).
  • peptide:MHC tetramer staining was performed on 1x10 6 total BAL cells per sample. Cells were incubated with PE-PB1 (residues 703 – 711) and APC-PA (residues 224-233) tetramers at a dilution of 1:750 for each for 1 h on ice.
  • cytokine staining was performed 20 following stimulation with IAV peptides. For each condition, 5x10 5 cells from each sample were plated in a 96-well U-bottom plate. Cells were incubated with either PB1 (residues 703 – 711) or PA (residues 224-233) peptides at a concentration of 1 ⁇ M in complete RPMI in the presence of Protein Transport Inhibitor Cocktail, containing Monensin and Brefeldin A, (eBioscience, 1:500) for 4 h at 37 degrees C.
  • PB1 PB1
  • PA protein Transport Inhibitor Cocktail
  • Immune complexes were detected using the Ventana OmniMap anti-Rabbit detection kit (760-4311) and developed using the Ventana ChromMap DAB detection kit (760-159) per to the manufacturer’s instructions. Slides were then counterstained with hematoxylin II (790-2208) for 8 min, followed by Bluing reagent (760-2037) for 4 min. The slides were then dehydrated with ethanol series, cleared in xylene, and mounted. As a negative control, 20 the primary antibody was replaced with normal rabbit IgG to confirm absence of specific staining. Immunostained slides were scanned using a Leica Aperio ScanScope CS 5 slide scanner (Aperio, Vista, CA, USA).
  • the percentage of each lung field with infection/lesion was calculated using the Aperio ImageScope software (version 12.3.3). 10
  • lungs were first embedded in paraffin, sectioned at 4 ⁇ m, mounted on positively charged glass slides (Superfrost Plus; Thermo Fisher Scientific, Waltham, MA), and dried at 60°C for 20 min.
  • Antigen retrieval at 100°C for 20 min in Epitope Retrieval solution 2 (ER2) was performed on a Bond Max immunostainer (Leica Biosystems, Buffalo Grove, IL).
  • a rat monoclonal primary antibody to Ly- 15 6B.2 (1:30,000) was applied for 15 minutes followed by biotinylated secondary rabbit anti-rat antibody 1:400 (Vector Laboratories) for 10 minutes and then ready-to-use BOND DAB Enhancer (AR9432; Leica Biosystems) and a hematoxylin counterstain.
  • Slides were scanned and digital whole lung images were evaluated using the DenseNet algorithm of the image analysis platform HALO (version 3.1; Albuquerque, NM). Alveoli in the lung images were classified as being either 20 normal or infiltrated with neutrophil exudates and subjected to deep learning (DL) until convergence to a cross-entropy that was ⁇ 0.1.
  • Lungs were then analyzed by the trained model, and pathologists visually confirmed the results.
  • the regions were corrected or appropriately annotated, and then the DL was repeated to construct the trained model for the automated recognition. To improve the recognition 25 accuracy, the cycle was repeated until the number of misrecognized regions was reduced to an absolute minimum, or until the analysis results did not change even when further DL was performed.
  • the fraction of lung volume that was occupied by neutrophil infiltrates was determined by quantitative morphometry using the HALOTM Area Quantification v2.1.11 algorithm (IndicaLabs, NM, USA).
  • Concentrations of analytes were determined using the 15 ProcartaPlex Mouse Immune Monitoring Panel 48-Plex kit (ThermoFisher, EPX480-20834-901). Briefly, samples were centrifuged at 1400 rpm for 10 min at 4 o C. Prepared standards, high and low controls, diluent only controls, and clarified supernatants were added to plates containing capture beads, according to manufacturer’s instructions. Plates were shaken at 600 rpm overnight at 4 o C, then warmed to room temperature for an additional 30 min with shaking. The remaining 20 steps were performed according to manufacturer’s instructions.
  • Cellular inhibitor of apoptosis protein cIAP2 protects against pulmonary tissue necrosis during influenza virus infection to promote host survival.
  • Compromised respiratory function in lethal influenza infection 15 is characterized by the depletion of type I alveolar epithelial cells beyond threshold levels.
  • 16 Shubina, M. et al. Necroptosis restricts influenza A virus as a stand-alone cell death mechanism. J Exp Med 217, e20191259 (2020). 17 Upton, J. W., Shubina, M. & Balachandran, S. RIPK3-driven cell death during virus 20 infections. Immunol Rev 277, 90-101 (2017). 18 Thapa, R. J. et al.
  • Ethyl 6-Chloro-4-(methylamino) nicotinate (2) Compound 1 (0.03 mol) was dissolved in acetonitrile (80 mL). The solution was cooled to 0 °C, and methylamine (40% water solution, 18.00 mL) was slowly added. The reaction was stirred at 0 °C for 20 min and then warmed to room temperature for 3 h. The solvent was removed under reduced pressure, and the crude product was 5 purified by silica gel chromatography (20-100% EtOAc in hexane) to provide 2 (96% yield) as a white powder.
  • 6-Chloro-4-(methylamino)pyridine-3-carbaldehyde (4) The crude alcohol was 15 dissolved in DCM (100 mL), and MnO2 (200 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. After completion, the reaction mixture was filtered through Celite to remove the MnO 2 . The collected filtrate was concentrated under reduced pressure, and the crude mixture was purified by silica gel chromatography (30-100% EtOAc in hexane) to provide 4 (75% yield) as a white solid powder.

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Abstract

Disclosed herein are compounds and derivatives thereof for use as inhibitors of receptor interacting kinase 3 (RIPK3). The disclosed compounds and pharmaceutical compositions thereof may be used in methods for treating a disease or disorder associated with RIPK3 activity, including inflammatory and degenerative diseases and disorders.

Description

INHIBITORS OF RIPK3 AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority of United States Provisional Patent 5 Application No.63/631,965, filed April 09, 2024, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under AI144400 awarded by the National Institutes of Health. The government has certain rights in the invention. 10 BACKGROUND OF THE INVENTION This disclosure pertains to compounds that demonstrate protein kinase inhibitory activity. Protein kinases are important enzymes in cellular signal transduction. In many pathological conditions aberrant signal transduction occurs. Therefore, protein kinase inhibitors can be used as therapeutic agents for the treatment of various diseases. 15 Human auto-immune, auto-inflammatory and degenerative conditions involve a continuous loop of cell death and inflammation, leading to tissue injury and dysfunction. This is seen in progressive chronic diseases with inflammatory etiology, including atherosclerosis and TNF-driven pathologies such as rheumatoid arthritis and ulcerative colitis. This also occurs upon deleterious acute inflammatory responses, such as upon infection by influenza virus. Although 20 treatments for some of these conditions exist, many inflammatory diseases remain poorly controlled and in significant need of new therapeutic strategies. Influenza A virus (IAV) infections account for up to 700,000 hospitalizations and 50,000 annual deaths in the US alone. Worryingly, even though highly pathogenic H5 and H7 strains of avian IAV are thus far limited in their spread between humans, they require only a small number 25 of mutations to become transmissible. The lung epithelium is a primary site of mammalian Influenza A virus pathology. Influenza A virus (IAV) is a negative-sense RNA virus of the family Orthomyxoviridae. In aquatic birds, the primary IAV reservoir, virus replication occurs within the gastrointestinal tract and is typically asymptomatic. In contrast, mammalian IAV strains replicate in the respiratory tract and produce Page 1 QB\166118.01517\95733313.1
symptoms ranging from mild cases of ‘the flu’ to severe, sometimes lethal disease. IAV infects epithelial cells along the entire respiratory tract. Transmission is associated with infection of the upper tract, whereas severe disease is associated with infection in the lower lung, with the extent of lung involvement correlating with disease outcomes in humans and animal models. 5 As a lytic virus, IAV kills most lung cell types in which it replicates. But not all cell death by IAV is pathogenic. While programmed cell death is essential for early control of IAV replication and prevention of virus spread to the whole lung, the death of the lung epithelial layer is also one of the primary drivers of IAV infection-associated morbidity and mortality10. In particular, the loss of Type I airway epithelial cells (AECs, which are essential for gas exchange) 10 above a threshold of ~10% is strongly correlated with mortality in the mouse model of IAV infection. When cell death is well-controlled and apoptotic, it represents a host defense mechanism that limits both virus spread and immunopathology. But when cell death is uncontrolled or primarily necrotic (i.e., highly pro-inflammatory), it can lead to hyper-inflammatory responses, severe degradation of airway epithelia and consequent host mortality even if virus is cleared. Such 15 severe pathology is observed in mouse models, where destruction of airway epithelia is a common feature of lethal IAV infection, and in humans, where death of distal pulmonary epithelia, marked by areas of bronchioalveolar necrosis, is a classic feature of IAV-induced Acute Respiratory Distress Syndrome (ARDS). Hence, the ideal treatment for IAV disease should encourage clearance-promoting death while inhibiting degradation-inducing inflammatory death, as well as 20 of viral and secondary bacterial pneumonia. Receptor-interacting protein kinase 3 (RIPK3) is a multi-functional protein involved in cell death pathways in various tissues. Prior work has demonstrated that loss of RIPK3 kinase activity provides a protection in a variety of animal models of inflammatory and degenerative conditions, including sepsis, brain trauma, acute kidney injuries, lung injury associated with influenza (IAV) 25 infection, atherosclerosis, and many others. For example, RIPK3 was recently uncovered as a targetable signaling pathway that accounts for almost all IAV-activated pathogenic death in infected cells. This pathway is initiated when the host sensor protein DAI detects IAV genomic RNA and activates RIPK3 kinase. RIPK3 then triggers a form of cell death called necroptosis (or programmed necrosis), responsible for much of the lung injury seen during IAV infection. 30 Fascinatingly, eliminating necroptosis not only drastically reduces lung damage and improves animal survival, but does so without impeding virus clearance. This is because RIPK3 also Page 2 QB\166118.01517\95733313.1
activates a parallel pathway of non-pathogenic cell death (apoptosis) that mediates virus clearance. As only necroptosis, and not apoptosis, relies on RIPK3 kinase activity, inhibitors of RIPK3 kinase function will be expected to ameliorate necrotic lung injury without affecting virus clearance, and potentially represent an entirely new strategy for treatment of IAV disease. Similarly, two separate 5 studies showed that animals deficient in Ripk3 gene show resistance in the development of atherosclerosis due to the inhibition of macrophage cell death and systemic inflammation. No selective RIPK3 kinase inhibitors are in clinical use or have been advanced into clinical trials. In 2014, a panel of RIPK3-selective inhibitors was reported, but these compounds displayed only modest activity in cells, and, unfortunately, induced an unanticipated conformational change 10 in the structure of RIPK3 that resulted in toxicity. As current vaccines and antiviral strategies are either limited in their efficacy or susceptible to viral resistance and evasion, identifying new therapeutic entry-points for seasonal and virulent IAV pulmonary disease, preferably those that target pathogenic, host signaling pathways, is an urgent imperative. Clinically-viable inhibitors of RIPK3 kinase activity are a major unmet need. 15 BRIEF SUMMARY OF THE INVENTION Disclosed herein are compounds that demonstrate protein kinase inhibitory activity and methods for using the same. One aspect of the invention provides for compounds having a formula of: , 20 or a pharmaceutically accept , R1 is H or - –(CH2)n–X1 wherein X1 is a heterocycloalkyl optionally substituted with alkyl and n is an integer between 0-3; and R2 is H or - –(CH2)m–X2 wherein X2 is a heterocycloalkyl optionally substituted with alkyl and m is an integer between 0-3. Page 3 QB\166118.01517\95733313.1
The disclosed compounds may exhibit one or more biological activities. The disclosed compounds may inhibit necroptosis. The disclosed compounds may inhibit receptor interacting kinase 3 (RIPK3). Another aspect of the invention provides for a pharmaceutical composition. The 5 pharmaceutical composition comprises an effective amount of the compound as described herein, and a pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof. Another aspect of the invention provides for a method of treating a protein kinase related disease or condition. The method comprises administering to the subject the compound as 10 described herein or the pharmaceutical composition as described herein. Another aspect of the invention provides for a method of treating a subject for a respiratory virus infection. The method comprises administering to the subject a RIPK3 inhibitor two or more days after infection. BRIEF DESCRIPTION OF THE DRAWINGS 15 Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is 20 not necessary to allow those of ordinary skill in the art to understand the invention. Fig. 1 shows that UH15-38 is a potent RIPK3 kinase inhibitor. a, Influenza A viruses activate ZBP1 and trigger RIPK3-driven parallel pathways of MLKL-dependent necroptosis and caspase 8-driven apoptosis. Only necroptosis is reliant on RIPK3 kinase activity. b, Survival analysis of mice of indicated genotypes (Wild-type [WT], n=12; Mlkl-/-, n=15; Ripk1kinase-dead, 25 n=13; Ripk3-/-, n=12; Zbp1-/-, n=11) following intranasal challen h IAV H1N1 strain PR8 (6000 Egg Infectious Dose [EID]50). c, Structure of UH15-38. d, Cell viability of primary wild- type MEFs exposed to the combination of murine TNF^ (100 ng/ml), cycloheximide (250 ng/ml), and zVAD (50 ^M) (TCZ) in the presence of UH15-38, GSK’872 or GSK’843. Viability was assessed 12 h after treatment. Data are mean ± SD, n = 3, examined over three biologically 30 independent experiments. e, MEFs treated with TCZ in the presence of UH15-38 were examined Page 4 QB\166118.01517\95733313.1
for phosphorylated MLKL (pMLKL), total MLKL, ZBP1 and RIPK3 12 h after treatment. f, Comparative efficacy of UH15-38, GSK’872 and GSK’843 in human and mouse cells. g, Molecular docking of UH15-38 into mouse RIPK3. N-terminal lobe, ^C helix, activation loop, hinge region, and C-terminal lobe are shown. The inhibitor is shown in ball-and stick 5 representation with a mesh surface. h, Detailed active site view of UH15-38 docked into mouse RIPK3. Hydrogen bonds (2.0 - 3.1 Å) to the hinge residue M98 as well as the K51, E61 of the ^C helix and D161 and F162 of the DFG motif are shown as dashes. i, LigPlot analysis showing interactions of UH15-38 with amino acids in mouse RIPK3. Hydrogen bonds are shown with dashes and labeled with the bond length in Angstroms. Cell viability in d was determined using 10 the Trypan Blue exclusion assay, and in f by the CellTiter-Glo assay. Groups were compared using the Log-rank (Mantel Cox) test (as in b). Fig. 2 shows that UH15-38 selectively blocks IAV-induced necroptosis in Type I AECs. a, IAV mRNA expression in lung cell types at 6 days after infection with PR8 (2500 EID50; i.n.). b, Distribution of IAV+ primary lung cell types at 6 days after infection c, IAV replication and 15 ZBP1 expression in Type I AECs following IAV infection in vivo. d, Viability of primary Type I AECs from Zbp1+/+ and Zbp1-/- mice following infection with PR8 (MOI = 2). e, Expression of the indicated proteins in primary WT (Zbp1+/+) Type I AECs following PR8 (MOI=2) infection. f, Viability of Type I AECs after infection with PR8 (MOI = 2) and treatment with UH15-38, GSK’872 and GSK’843 with or without zVAD (50 ^M). g, Photomicrographs of primary Type I 20 AECs infected with PR8 (MOI = 2) and treated with UH15-38 (1 ^M) in presence or absence of zVAD (50 ^M). Images are representative of three independent experiments. h, Immunoblots of lysates from primary Type I AECs infected with PR8 (MOI = 2, 12h) in the presence of UH15-38. Data are representative of at least two independent experiments. i, Viability of M29 cells infected with PR8 (MOI =10, 24h) and treated with or without zVAD (50 ^M) or UH15-38 (1 ^M). j-l, 25 Live human lung sections (n=3/group) infected with PR8 (5 x 106 pfu/ml) and treated with either DMSO or UH15-38 (1^M) for 24 h were stained for pMLKL (j, k) and IAV H1N1 antigen (j, l). Cell viability was determined by Trypan Blue exclusion in panels d, f, and i. Data are average of n=2 as in f and mean ± SD (n=3) as in d, i examined over three independent experiments with similar outcomes. Groups were compared using the unpaired two-sided Student’s t test (as in d), 30 or by ordinary one-way ANOVA (as in i, k, l). Page 5 QB\166118.01517\95733313.1
Fig. 3 shows that UH15-38 prevents lethality in severe influenza. a, Survival analysis of mice infected with PR8 (6000 EID50) followed by i.p. administration of vehicle (n=20) or the following doses of UH15-38: 50 mg/kg (n = 20), 30 mg/kg (n = 21), 15 mg/kg (n = 15), 7.5 mg/kg (n = 15). Vehicle or UH15-38 was administered once-daily per the dosing schedule shown above 5 the graph. b, Survival analysis of mice challenged with PR8 (4500 EID50; ~LD60) and treated i.p. with vehicle (n = 15) or the following doses of UH15-38: 30 mg/kg, (n = 15); 15 mg/kg (n = 9), 7.5 mg/kg (n = 10), 3 mg/kg (n = 9), 1 mg/kg (n = 9). Vehicle or UH15-38 was administered once- daily per the dosing schedule shown above the graph. c, Weight loss analysis of PR8 (4500 EID50)- infected mice (n = 15/group) following four once-daily i.p. doses of vehicle or UH15-38 (30 10 mg/kg). d, Survival analysis of mice infected with IAV H1N1 strain A/California/04/09 (600 EID50; ~LD60) and treated once-daily with vehicle (n = 10) or UH15-38 (30 mg/kg, i.p., n =13) per the dosing schedule shown above the graph. e, Survival analysis of PR8 (4500 EID50)-infected mice treated with UH15-38 (30 mg/kg, i.p.) starting either two (n = 10), three (n = 9), four (n = 10), or five (n = 10) days after infection. f, Survival analysis of PR8 (LD60)-infected Mlkl-/- (dashed 15 lines) mice (vehicle group, n = 11; UH15-38 group, n = 10) or Ripk3-/- (solid lines) mice (vehicle group, n = 10; UH15-38 group, n = 9) following once-daily treatment with vehicle or UH15-38 (30 mg/kg, i.p.) administered per the dosing schedule shown above the graph. The LD60 for PR8 in Mlkl-/- mice was 6500 EID50, and for Ripk3-/- mice was 2500 EID50. Groups were compared using the Log-rank (Mantel-Cox) test. 20 Fig.4 shows that UH15-38 prevents necroptosis, inflammation, and injury in IAV-infected lungs. a, b, Immunofluorescence staining of pMLKL (a) and quantification of pMLKL signal (b) in lung sections harvested on the indicated days after infection from mice infected with PR8 (6000 EID50) and treated i.p. with either vehicle or UH15-38 (30 mg/kg once-daily), starting one day after infection, for up to four days. c, Levels of inflammatory mediators in BALF (n = 4/group) 25 from mice infected with PR8 (6000 EID50) three days after infection. Mice were treated with either vehicle or UH15-38 (30 mg/kg once-daily) starting D1 post-infection. d, e, Lung images (d) and quantification (e) of neutrophil influx three days after infection with PR8 (6000 EID50), following treatment with either vehicle or UH15-38 (30 mg/kg, i.p., once-daily) starting one day after infection. f, H & E stained lung sections of vehicle- and UH15-38-treated mice nine days after 30 infection with PR8 (4500 EID50). Left arrow shows hyaline membranes and right arrow depicts denuded bronchioles in vehicle-treated tissue. g, Histological scores of late lung injury Page 6 QB\166118.01517\95733313.1
characterized by hyaline membrane formation, denuded bronchioles, and fibrosis nine days after infection. h. Arterial oxygen saturation (SpO2) measured in uninfected (n = 4/group) or infected mice (PR8, 4500 EID50; n = 6/group) 3 and 6 days after infection following treatment with vehicle or UH15-38 (30 mg/kg, i.p.) once daily for four days starting 24 h after infection. Graphs from top 5 to bottom: Mock + vehicle, Mock + UH15-38, PR8 + UH15-38, PR8 + vehicle. i, Frequencies of IFN^-producing CD8+ T cells nine days after infection with PR8 in BALF of vehicle- and UH15- 38-treated mice following in vitro stimulation with peptides corresponding to PB1 residues 703- 711 or PA residues 224-233 (n = 10/group). Data are mean ± SD. n = 6 in b or n = 5 mice/group unless stated otherwise. Groups were compared using the two-sided Mann-Whitney U test (as in 10 b, c, e, g, i, j), or by two-way ANOVA (as in h). Fig.5 shows that UH15-38 is a potent RIPK3 kinase inhibitor. a, Immunoblot analysis of pMLKL and cleaved caspase 8 (CC8) in lysates obtained from Ripk3-/- MEFs stably expressing 2xFv-RIPK3 and treated with dimerizer (AP20187, 100 nM) in the presence of the pan-caspase inhibitor (IDN-6556, 20 ^M) and UH15-38 (500 nM) for 12 h. b, Table comparing UH15-38 to 15 GSK’872 in the indicated recombinant kinase assays. Kinase assays were performed using ADPGlo (Promega) and on the DiscoverX platform (Eurofins DiscoverX). ND = Not determined. c, Overview of docking of UH15-38 into human RIPK3. d, Docking of GSK’872 into mouse RIPK3. e, Docking of GSK’843 into mouse RIPK3. f, MEFs infected with PR8 (MOI =2) were treated with indicated drugs in the presence of zVAD (50 ^M) and cell viability was determined 20 after 24 h. g, Cell survival kinetics of iBMDMs treated with LPS (10 ng/ml) and TAK1 inhibitor 5z7 (200 nM) following exposure to the indicated concentrations of the RIPK3 inhibitors UH15- 38 and GSK’872 or the RIPK1 inhibitor GSK’547 for 6 h. h, Ripk3+/+ and Ripk3-/- MEFs were treated with TNF/5z7/IDN6556 and TNF/5z7, respectively, in the presence of the indicated concentrations of UH15-38, and viability was determined after 24 h. Ripk3-/- MEFs treated with 25 UH15-38 alone were used as controls. i, Viability of Ripk3-/- MEFs treated with TNF/5z7 in the presence of DMSO or GSK’547 (10 ^M). j, Immunoblot analysis of Gasdermin D (GSDMD) cleavage in primary BMDMs pre-treated with LPS (10 ng/ml) for 3 h followed by Nigericin (10 ^M) or ATP (5 mM), along with DMSO or UH15-38 (500 nM), for an additional 1 h. k, Viability of primary BMDMs pre-treated with LPS (10 ng/ml for 3 h) followed by treatment with Nigericin 30 (10 ^M) and the indicated concentrations of UH15-38 for 1 h. Viability was determined by using CellTiter-Glo assay (as in g, h, k) or Trypan Blue exclusion assay (as in f). Bars from left to right: Page 7 QB\166118.01517\95733313.1
Mock, LPS, and LPS + Nigericin. Error bars represent mean ± SD (n=3). Data are from one of the at least two independent experiments. Groups were compared using ordinary one-way ANOVA (as in f, k) or the unpaired two-sided Student’s t test (as in i). Fig. 6 shows the pharmacological profiling of UH15-38. a, Half-life (T1/2) and peak 5 concentrations (Cmax) of UH15-38 in tissue samples collected from mice treated with four once- daily) doses of UH15-38 (30 mg/kg/day, i.p.). b, c, Immunofluorescence staining for phosphorylated-MLKL (pMLKL) (b) and quantification of pMLKL signal in lung, heart kidney and liver sections (c) obtained from Casp8-/-MlklFLAG/FLAG mice following i.p administration of either vehicle (n = 3 mice) or UH15-38 (30 mg/kg, once-daily, n = 4 mice) for four days. Six fields 10 of each tissue section per mice were quantified. d, InVEST Safety Panel profile of UH15-38 showing percent inhibition of each target in the presence of 1^M compound. e, f, Immunohistochemistry for cleaved caspase 3 (CC3) (e) and quantification of cleaved caspase 3 signal (f) in lung, heart, kidney and liver sections (n = 4 mice) following i.p. administration of either vehicle or UH15-38 (30 mg/kg, once-daily) for seven days. IAV PR8-infected lung tissue 15 (n = 4 mice) is shown as positive control in e and f. Groups were compared using the two-sided Mann-Whitney U test. Fig 7 shows that UH15-38 is a potent and specific inhibitor of necroptosis in murine and human cells. a, Podoplanin (PDPN) staining demonstrates purity of primary Type I AECs. CD140a was used as control for fibroblastic (Fibs) contamination. b, Cell survival kinetics of Type I AECs 20 treated with TCZ and exposed to the indicated concentrations of the RIPK3 kinase inhibitors UH15-38, GSK’843 or GSK’872). c, Cell survival kinetics of primary wild-type MEFs infected with PR8 (MOI = 2) and treated with the indicated RIPK3 kinase inhibitors in the presence or absence (DMSO) of zVAD. d, Immunoblot analysis of the indicated proteins in lysates prepared from primary MEFs infected with PR8 (MOI = 2) and treated with the indicated concentrations of 25 UH15-38. Cell lysates were prepared 12 h after infection. e, Cell survival analysis following infection of MEFs with a panel of IAV and IBV strains and exposure to the indicated concentrations of UH15-38 in the presence or absence of zVAD (50 ^M). f, Immunoblot analysis of the indicated proteins in cell lysates prepared from primary MEFs infected with a panel of IAV or IBV strains (MOI = 5) and treated with DMSO or UH15-38 (1 ^M). Cell lysates were prepared 30 12 h after infection. g, Viability of primary Ripk1-/- MEFs infected with PR8 (MOI = 2) and treated with DMSO or UH15-38 (1^M). h, Viability of primary MEFs after infection with PR8 (MOI = Page 8 QB\166118.01517\95733313.1
2) and treatment with UH15-38 or CSLP37 (a RIPK2 inhibitor) in the presence of either DMSO or zVAD (50 ^M). (zVAD is included in the assays to block apoptosis so that effects of the test compounds on necroptosis can be evaluated). i, Viability of primary MEFs after infection with PR8 (MOI = 2) and treatment with UH15-38 or imatinib (an ABL inhibitor) in the presence of 5 either DMSO or zVAD (50 ^M). j, k, Zbp1-/- MEFs stably expressing 2xFv-tagged murine ZBP1 were exposed to dimerizer (AP20187, 100 nM) in the presence of the indicated concentrations of UH15-38. Cell viability (j) and immunoblot analyses (k) of the indicated proteins (right) are shown. l, Viability of HeLa-RIPK3 cells treated with human TNF^ (100 ng/mL) + cycloheximide (2.5 ^g/mL) + zVAD (50 ^M) (TCZ) in the presence or absence of UH15-38 (1 ^M). m, HeLa- 10 RIPK3 cells treated with TCZ and the indicated concentrations of UH15-38 were examined for pMLKL, total MLKL, and RIPK3 by immunoblot analysis 18 h after treatment. n, M29 cells infected with PR8 (MOI=10) were treated with increasing concentrations of UH15-38 for 24 h and examined for the indicated proteins by immunoblot analysis. H1N1 influenza strains: A/Puerto Rico/8/1934 (PR8), A/California/04/2009 (Cal/09); H3N2 influenza strains: A/Brisbane/10/2007 15 (Bri/07), A/Singapore/INFIMN-16-0019/2016 (Sin/16); Influenza B virus strains B/Colorado/06/2017 (Col/17) and B/Florida/04/2006 (Flo/06). zVAD (50 ^M) was used to prevent apoptosis in this experiment. Cell viability in all panels was determined at 24 h after infection, using the Trypan Blue exclusion assay. Error bars represent mean ± SD of n =3 samples. All data are from one of at least two independent experiments with similar outcomes. Groups were 20 compared using ordinary one-way ANOVA. Fig.8 shows that UH15-38 is a potent inhibitor of RIPK3 in cellulo. a, Viability of MEFs following treatment with indicated concentrations of UH15-38. b, Immunoblot analysis of the lysates of MEFs treated with indicated concentrations of UH15-38 for the markers of apoptosis. CC8 = cleaved caspase 8, CC3 = cleaved caspase 3. c, Viability of wild-type MEFs treated with 25 100 x IC50 (5 ^M) of UH15-38 in the presence of DMSO or zVAD (50 ^M), as compared to the viability of Ripk3 -/- MEFs treated with 100x IC50 UH15-38. d, Viability of Type I AECs following treatment with indicated concentrations of UH15-38. e, Immunoblot analysis of the lysates of Type I AECs treated with indicated concentrations of UH15-38 for markers of apoptosis. Cell viability in all panels was determined using the Trypan Blue exclusion assay. Groups were compared using 30 ordinary one-way ANOVA. Error bars are mean ± SD of n = 3 samples. Figures are representative of two (b, e) or three (a, c, d) independent experiments with similar outcomes. Page 9 QB\166118.01517\95733313.1
Fig.9 shows that UH15-38 prevents lethality in severe influenza. a, Weight loss curves of mice infected with PR8 (6000 EID50; ~LD100) followed by i.p. administration of vehicle (n=20) or the following doses of UH15-38: 50 mg/kg (n = 20), 30 mg/kg (n = 21), 15 mg/kg (n = 15), 7.5 mg/kg (n = 15). Vehicle or UH15-38 was administered once-daily per the dosing schedule shown 5 above the graph. b, c, Survival graphs (b) and weight loss curves (c) of mice (n=10) infected with PR8 (6000 EID50) and treated with vehicle (n=12) or with UH15-38 (30 mg/kg, i.p.) for a shortened-, or delayed-dosing regimens, as indicated above panel b. d, e, Survival (d) and weight loss (e) curves of mice (n=7) infected with PR8 (4500 EID50) and treated i.p. with vehicle or GSK’872 (30mg/kg) as indicated above the graph. f, Weight loss analysis of mice infected with 10 IAV H1N1 strain A/California/04/09 (600 EID50; ~LD60) and treated once-daily with either vehicle (n = 10) or UH15-38 (30 mg/kg, i.p., n =13) per the dosing schedule shown above the graph. g, Weight loss curves of mice infected with PR8 (4500 EID50; ~LD60) and treated i.p. with UH15-38 (30 mg/kg, i.p.), per dosing regimens shown above the graph (drug treatment beginning at day 3 after infection, n = 9; all other groups, n = 10/group). h, Wild type mice or Mlkl -/- mice (n = 10) 15 were infected with PR8 (2500 EID50). Wild type mice were treated with either vehicle (n = 10) or 30 mg/kg UH15-38 (n=12) once daily for four days starting 24 h after infection. i, Wild type mice or Mlkl -/- mice (n = 15) were infected with PR8 (4500 EID50). Wild type mice were treated with either vehicle (n = 14) or 30 mg/kg UH15-38 (n=13) once daily for four days starting 24 h after infection. Mice were observed until 21 days and survival curves were plotted. Wild-type (Mlkl+/+) 20 mice in panels h and i were generated by intercrossing Mlkl+/- mice. Groups were compared using the Log-rank (Mantel-Cox) test. Fig. 10 shows that UH15-38 prevents necroptosis, inflammation, and injury in IAV- infected lungs. a, b, Immunofluorescence staining of cleaved caspase 3 (CC3) (a) and quantification of CC3 signal (b) in lung sections harvested on the indicated days post-infection 25 from mice infected with PR8 (6000 EID50) and treated i.p. with either vehicle or UH15-38 (30 mg/kg once-daily), starting one day after infection, for up to four days. c, Primary Type I AECs were infected with PR8 (MOI = 2) and treated with DMSO or UH15-38 (1 ^M). Bars from left to right: Mock, PR8, and PR8 + UH15-38. Supernatants were collected 12 h after infection and the indicated chemokines were analyzed on the Luminex platform. d, e, Morphometric images of 30 Tenascin C stained lung sections showing Tenascin C positive area, inflamed tenascin C negative lung area, larger airways and normal lung area (d) and proportion of Tenascin C positive area (e) Page 10 QB\166118.01517\95733313.1
in infected lungs (n = 5/group) nine days after infection, following i.p. administration of either vehicle or UH15-38 (30 mg/kg once-daily), starting one day after infection, for four days. f, Levels of IL-17 and CCL5 in BALF from infected mice (n=4/group) measured on the Luminex platform three days after infection, following i.p. treatment with either vehicle or UH15-38 (30 mg/kg once- 5 daily), starting one day after infection, for two days. g, Histological scores of lung sections obtained from mice (n = 5/group) three days (alveolar inflammation and interstitial inflammation) or nine days (septal thickening and epithelial metaplasia) following infection with PR8 (6000 EID50) and treated i.p. with either vehicle or UH15-38 (30 mg/kg once-daily), starting one day after infection, for up to four days. h, Primary BMDMs were infected with PR8 (MOI = 5) and 10 treated with UH15-38 (1 ^M) in the presence or absence of zVAD (50 ^M). Supernatants were collected 24 h after infection and IL-1^ and IL-18 levels were measured by ELISA. i, Airway resistance (RI) was measured in mice uninfected (Mock / Vehicle, n = 5; Mock / UH15-38, n = 4) or infected (PR8, 2500 EID50, n = 4/group) 10 days after infection following treatment with either vehicle or UH15-38 (30 mg/kg) intraperitoneally one dose daily for four days starting 24 h post 15 infection. Graphs from top to bottom: PR8 / vehicle, Mock / UH15-38, Mock / Vehicle, and PR8 / UH15-38. Data are presented as mean ± SD. (n = 6/group in b or n = 3/treatment condition in c, h). Comparison between groups was carried out by two sided Mann-Whitney U test (as in b, e, f, g) or by ordinary one-way ANOVA (as in c, h) or two-way ANOVA (as in i). Fig. 11 shows that UH15-38 does not impede virus clearance or anti-IAV CD8+ T cell 20 responses. a, b, Lung morphometry showing virus spread (indicated by arrows) in lungs (a) and quantitation of percentage of viral antigen-positive lung area (b) at 6 days after infection. n = 5/group. c, Virus titers in lungs (day 3 and day 6, n = 10/group; day 9 and day 12, n = 5/group) at indicated time points as determined by plaque assay. d, Frequencies of IAV-specific CD8+ T cells nine days after infection with PR8 in BALF of vehicle- and UH15-38-treated mice using 25 peptide:MHC tetramers incorporating PB1 residues 703-711 or PA residues 224-233 (n = 10/group). Comparison between groups was carried out by the two-sided Mann-Whitney U test (as in b, c, d), nd = not detected. Fig. 12 shows the gating strategy for flow cytometric analyses. a, b, Gating strategy for the flow cytometric analyses presented in Fig.4, panel i and Fig.11, panel d, respectively. 30 Fig 13 shows that MLKL binding requires the displacement of the RIPK3 ^C helix. a,b, Structures of monomeric (a) and MLKL-bound (b) mRIPK3. The side chains of key active site Page 11 QB\166118.01517\95733313.1
residues are shown as sticks and labeled. c. Overlaying the monomeric and MLKL-bound conformations of mRIPK3 shows that the ^C helix swivels (arrow) outwards in the MLKL-bound conformation, with the E61 residue now facing away from the active site. The dashed lines indicate axes of the ^C helices in inactive and active conformations. 5 Fig. 14 shows viability of (a) HT29, (b) THP1, and (c) FADD-def Jurkat cells after treatment with selected compounds. Cells were treated with the compounds at indicated doses. After 15 min, necroptosis was induced by adding 10 ng/ml human TNF^ (a-c), 100 nM SM164 (a,b) and 20 ^M IDN6556. Following 16-18 hr incubation, cell viability was determined using CellTiter-Glo assay. DMSO-treated unstimulated cells were used as a 100% viability control. IC50 10 values were determined using non-linear regression analysis in GraphPad Prism. DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates to protein kinase inhibitors and uses thereof. Compounds that demonstrate inhibitory activity of receptor interacting kinase 3 (RIPK3) and the uses thereof have been previously reported in International Patent Publication WO 2020232190, which is 15 incorporated by reference in its entirety. Chemical Entities New chemical entities and uses for chemical entities are disclosed herein. The chemical entities may be described using terminology known in the art and further discussed below. As used herein, an asterisk “*” or a plus sign “+” may be used to designate the point of 20 attachment for any radical group or substituent group. The term “alkyl” as contemplated herein includes a straight-chain or branched alkyl radical in all of its isomeric forms, such as a straight or branched group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C1-C12 alkyl, C1-C10-alkyl, and C1-C6-alkyl, respectively. The term “alkylene” refers to a diradical of an alkyl group (e.g., -(CH2)n- where n is an 25 integer such as an integer between 1 and 20). An exemplary alkylene group is -CH2CH2-. The term “haloalkyl” refers to an alkyl group that is substituted with at least one halogen. For example, -CH2F, -CHF2, -CF3, -CH2CF3, -CF2CF3, and the like. The term “heteroalkyl” as used herein refers to an “alkyl” group in which at least one carbon atom has been replaced with a heteroatom (e.g., an O, N, or S atom). One type of 30 heteroalkyl group is an “alkoxy” group. Page 12 QB\166118.01517\95733313.1
The term “alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkenyl, C2-C10-alkenyl, and C2-C6-alkenyl, respectively. 5 The term “alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond, such as a straight or branched group of 2-12, 2-10, or 2-6 carbon atoms, referred to herein as C2-C12-alkynyl, C2-C10-alkynyl, and C2-C6-alkynyl, respectively. The term “cycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic 10 (e.g., adamantyl) hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons, referred to herein, e.g., as “C4-8-cycloalkyl,” derived from a cycloalkane. Unless specified otherwise, cycloalkyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halo, haloalkyl, heteroaryl, heterocyclyl, 15 hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In certain embodiments, the cycloalkyl group is not substituted, i.e., it is unsubstituted. The term “cycloheteroalkyl” or “heterocycloalkyl” refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least 20 one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S. The term “cycloalkylene” refers to a cycloalkyl group that is unsaturated at one or more ring bonds. The term “partially unsaturated carbocyclyl” refers to a monovalent cyclic hydrocarbon that contains at least one double bond between ring atoms where at least one ring of the carbocyclyl 25 is not aromatic. The partially unsaturated carbocyclyl may be characterized according to the number oring carbon atoms. For example, the partially unsaturated carbocyclyl may contain 5-14, 5-12, 5-8, or 5-6 ring carbon atoms, and accordingly be referred to as a 5-14, 5-12, 5-8, or 5-6 membered partially unsaturated carbocyclyl, respectively. The partially unsaturated carbocyclyl may be in the form of a monocyclic carbocycle, bicyclic carbocycle, tricyclic carbocycle, bridged 30 carbocycle, spirocyclic carbocycle, or other carbocyclic ring system. Exemplary partially unsaturated carbocyclyl groups include cycloalkenyl groups and bicyclic carbocyclyl groups that Page 13 QB\166118.01517\95733313.1
are partially unsaturated. Unless specified otherwise, partially unsaturated carbocyclyl groups are optionally substituted at one or more ring positions with, for example, alkanoyl, alkoxy, alkyl, haloalkyl, alkenyl, alkynyl, amido, amidino, amino, aryl, arylalkyl, azido, carbamate, carbonate, carboxy, cyano, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, 5 hydroxyl, imino, ketone, nitro, phosphate, phosphonato, phosphinato, sulfate, sulfide, sulfonamido, sulfonyl or thiocarbonyl. In certain embodiments, the partially unsaturated carbocyclyl is not substituted, i.e., it is unsubstituted. The term “aryl” is art-recognized and refers to a carbocyclic aromatic group. Representative aryl groups include phenyl, naphthyl, anthracenyl, and the like. The term “aryl” 10 includes polycyclic ring systems having two or more carbocyclic rings in which two or more carbons are common to two adjoining rings (the rings are “fused rings”) wherein at least one of the rings is aromatic and, e.g., the other ring(s) may be cycloalkyls, cycloalkenyls, cycloalkynyls, and/or aryls. Unless specified otherwise, the aromatic ring may be substituted at one or more ring positions with, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, 15 alkoxyl, amino, nitro, sulfhydryl, imino, amido, carboxylic acid, -C(O)alkyl, -CO2alkyl, carbonyl, carboxyl, alkylthio, sulfonyl, sulfonamido, sulfonamide, ketone, aldehyde, ester, heterocyclyl, aryl or heteroaryl moieties, -CF3, -CN, or the like. In certain embodiments, the aromatic ring is substituted at one or more ring positions with halogen, alkyl, hydroxyl, or alkoxyl. In certain other embodiments, the aromatic ring is not substituted, i.e., it is unsubstituted. In certain embodiments, 20 the aryl group is a 6-10 membered ring structure. The terms “heterocyclyl” and “heterocyclic group” are art-recognized and refer to saturated, partially unsaturated, or aromatic 3- to 10-membered ring structures, alternatively 3-to 7-membered rings, whose ring structures include one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The number of ring atoms in the heterocyclyl group can be specified using 5 25 Cx-Cx nomenclature where x is an integer specifying the number of ring atoms. For example, a C3-C7 heterocyclyl group refers to a saturated or partially unsaturated 3- to 7-membered ring structure containing one to four heteroatoms, such as nitrogen, oxygen, and sulfur. The designation “C3-C7” indicates that the heterocyclic ring contains a total of from 3 to 7 ring atoms, inclusive of any heteroatoms that occupy a ring atom position. Page 14 QB\166118.01517\95733313.1
The terms “amine” and “amino” are art-recognized and refer to both unsubstituted and substituted amines (e.g., mono-substituted amines or di-substituted amines), wherein substituents may include, for example, alkyl, cycloalkyl, heterocyclyl, alkenyl, and aryl. The terms “alkoxy” or “alkoxyl” are art-recognized and refer to an alkyl group, as defined 5 above, having an oxygen radical attached thereto. Representative alkoxy groups include methoxy, ethoxy, tert-butoxy and the like. The term “O-alkylamine” refers to –O– (CR1R2)m–NR3R4, wherein m is an integer between 1-6, and R1, R2, R3 and R4, for example, are each independently hydrogen, alkyl, haloalkyl, alkenyl, alkynyl, and aryl. R3 and R4, together with the nitrogen they are attached to, may also be optionally 10 joined to form a 4-8 membered cycloalkyl. The term "heterocycloalkyl" refers to a monovalent saturated cyclic, bicyclic, or bridged cyclic hydrocarbon group of 3-12, 3-8, 4-8, or 4-6 carbons in which at least one carbon of the cycloalkane is replaced with a heteroatom such as, for example, N, O, and/or S(O)n, wherein n is an integer of 0-2. "Four to seven membered heterocycloalkyl" refers to a heterocycloalkyl 15 containing from four to seven atoms, including one or more heteroatoms, in the cyclic moiety of the heterocycloalkyl. Examples of single-ring heterocycloalkyls include azetidinyl, oxetanyl, thietanyl, dihydrofuranyl, tetrahydrofuranyl, dihydrothiophenyl, tetrahydrothiophenyl, pyrrolinyl, pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl, isothiazolinyl, thiazolidinyl, isothiazolidinyl, dihydropyranyl, piperidinyl, morpholinyl, piperazinyl, azepinyl, 20 oxepinyl, and diazepinyl. In some embodiments, the heterocycloalkyl described herein may be fused with a cycloalkyl, an aryl, or a heteroaryl, as described herein. In some embodiments, the heterocycloalkyl described herein may be , . lently linked by an oxygen. Accordingly, the 25 substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, and the like. Page 15 QB\166118.01517\95733313.1
The term “carbonyl” as used herein refers to the radical -C(O)-. The term “oxo” refers to a divalent oxygen atom –O-. The term “carboxamido” as used herein refers to the radical -C(O)NRR', where R and R' may be the same or different. R and R', for example, may be independently alkyl, aryl, arylalkyl, 5 cycloalkyl, formyl, haloalkyl, heteroaryl, or heterocyclyl. The term “carboxy” as used herein refers to the radical -COOH or its corresponding salts, e.g. -COONa, etc. The term “amide” or “amido” or “amidyl” as used herein refers to a radical of the form – R1C(O)N(R2)-, -R1C(O)N(R2)R3-, -C(O)NR2R3, or -C(O)NH2, wherein R1, R2 and R3, for 10 example, are each independently alkoxy, alkyl, alkenyl, alkynyl, amide, amino, aryl, arylalkyl, carbamate, cycloalkyl, ester, ether, formyl, halogen, haloalkyl, heteroaryl, heterocyclyl, hydrogen, hydroxyl, ketone, or nitro. The compounds of the disclosure may contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as geometric isomers, enantiomers or 15 diastereomers. The term “stereoisomers” when used herein consist of all geometric isomers, enantiomers or diastereomers. These compounds may be designated by the symbols “R” or “S,” or “+” or “-” depending on the configuration of substituents around the stereogenic carbon atom and or the optical rotation observed. The present invention encompasses various stereo isomers of these compounds and mixtures thereof. Stereoisomers include enantiomers and diastereomers. Mixtures 20 of enantiomers or diastereomers may be designated (±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. It is understood that graphical depictions of chemical structures, e.g., generic chemical structures, encompass all stereoisomeric forms of the specified compounds, unless indicated otherwise. Also contemplated herein are compositions comprising, consisting essentially of, or consisting of an enantiopure compound, 25 which composition may comprise, consist essential of, or consist of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of a single enantiomer of a given compound (e.g., at least about 99% of an R enantiomer of a given compound). Compounds Disclosed herein includes a compound having a formula of Page 16 QB\166118.01517\95733313.1
, or a pharmaceutically accept R1 is H or - –(CH2)n–X1 wherein X1 is a heterocycloalkyl optionally substituted with alkyl and n is an integer between 0-3; and 5 R2 is H or - –(CH2)m–X2 wherein X2 is a heterocycloalkyl optionally substituted with alkyl and m is an integer between 0-3. In some embodiments, n is 0 or 1 or m is 0 or 1. In some embodiments, X1 is , 10 X2 is In some embodiments, X1 is 15 , In some embodiments, X2 is Page 17 QB\166118.01517\95733313.1
, rogen. In some embodiments, X is or 5 10 , , Page 18 QB\166118.01517\95733313.1
, S 5 , Page 19 QB\166118.01517\95733313.1
or 5 . e scose co pou s ay e o uae as parmaceutical compositions further comprising a pharmaceutically acceptable excipient, carrier, or diluent. Page 20 QB\166118.01517\95733313.1
The disclosed compounds may exhibit one or more biological activities. In some embodiments, the disclosed compounds inhibit necroptosis. In some embodiments, the disclosed compounds modulate the activity of receptor interacting kinase 3 (RIPK3). In some embodiments, the disclosed compounds inhibit the activity of RIPK3 in vivo or in vitro. In some embodiments, 5 the disclosed compounds inhibit the activity of RIPK3 by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 100 µM, 50 µM, 10 µM, 1 µM, 0.1 µM, 0.05 µM, 0.01 µM, 0.005 µM, 0.001 µM, 0.1 nM, 0.01 nM, or less. Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.01 nM, 0.1 nM, 0.001 µM, 0.005 µM, 0.01 µM, 0.5 µM, 0.1 µM, 1.0 µM, 10 µM, 10 and 100 µM. In some embodiments, the disclosed compounds inhibit necroptosis, such as RIPK3- dependent necroptosis of cells. In some embodiments, the necroptosis may be induced by IAV. In some embodiments, the necroptosis may be induced by lipopolysaccharide (LPS), tumor necrosis factor (TNF), pan-caspase inhibitor, or a combination thereof. In some embodiments, the disclosed 15 compounds inhibit necroptosis by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than 100 µM, 50 µM, 10 µM, 1 µM, 0.1 µM, 0.05 µM, 0.01 µM, 0.005 µM, 0.001 µM, 0.1 nM, 0.01 nM, or less. In some embodiments, the disclosed compounds are selective RIPK3 inhibitors. In some embodiments, kinase selectivity may be assessed through a kinase binding assay 20 in a kinase panel and selectivity scores (S^scores). In short, compounds that bind the kinase active site and directly (sterically) or indirectly (allosterically) prevent kinase binding to the immobilized ligand, will reduce the amount of kinase captured on the solid support. Conversely, test molecules that do not bind the kinase have no effect on the amount of kinase captured on the solid support. Screening "hits" are identified by measuring the amount of kinase captured in test versus control 25 samples. In some embodiments, selective RIPK3 inhibitors exhibit the strongest inhibition of RIPK3 among all the kinases screened. Percent Control (%Ctrl). The compounds may be screened at any suitable concentration. Results for primary screen binding interactions are reported as “% Ctrl” or “POC”, where lower 30 numbers indicate stronger hits in the matrix. %Ctrl is defined as (eqn 1): Page 21 QB\166118.01517\95733313.1
%Ctrl = 100 x (TS – CPOS) / (CNEG – CPOS) (eqn 1) where TS is the test compound signal, CPOS is the positive control signal (0 %Ctrl), CNEG is the DMSO negative control (100 %Ctrl). 5 Selectivity Score (S^scores). Selectivity Score or S-score is a quantitative measure of compound selectivity. The S-score is calculated by dividing the number of kinases that compounds bind to by the total number of distinct kinases tested, excluding mutant variants (eqn 2): S = Number of hits / Number of assays (eqn 2) 10 This value can be calculated using %Ctrl as a potency threshold and provides a quantitative method of describing compound selectivity to facilitate comparison of different compounds: S(35) = (# of non-mutant kinases with %Ctrl <35)/(# of non-mutant kinases tested) (eqn 3) 15 S(10) = (# of non-mutant kinases with %Ctrl <10)/(# of non-mutant kinases tested) (eqn 4) S(1) = (# of non-mutant kinases with %Ctrl <1)/(# of non-mutant kinases tested) (eqn 5) The disclosed compounds may have a %Ctrl less than 5% for RIPK3. In some instances, the compound may have a %Ctrl less than 4%, 3%, 2%, 1%, 0.5%, or 0.1% and greater than 0% for RIPK3. 20 The disclosed compounds may have an S(35) value less than 0.50. In some instances, the disclosed compounds may have an S(35) value less than 0.40 or 0.30. Pharmaceutical Compositions Another aspect of the present disclosure provides a pharmaceutical composition comprising the compound or the pharmaceutically acceptable salt thereof disclosed herein and a 25 pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof. The compounds employed in the compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein. Such compositions may take any physical form which is pharmaceutically acceptable; 30 illustratively, they can be orally administered pharmaceutical compositions. Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is Page 22 QB\166118.01517\95733313.1
related to the daily dose of the compound to be administered. Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one-third of the dose. The amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and 5 other factors, such as the indication for which it is given. The pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures. In some embodiments, the compounds disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more 10 compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg/kg body weight (preferably about 0.5 to about 500 mg/kg body weight, 15 more preferably about 50 to about 100 mg/kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.01 nM, 0.1 nM, 0.001 µM, 0.005 µM, 0.01 µM, 0.5 µM, 0.1 µM, 1.0 µM, 10 µM, and 100 µM (e.g., 0.1 µM - 1.0 µM). 20 It is understood by those skilled in the art that dosage amount will vary with the activity of a particular inhibitor compound, disease state, route of administration, duration of treatment, and like factors well-known in the medical and pharmaceutical arts. In general, a suitable dose will be an amount which is the lowest dose effective to produce a therapeutic or prophylactic effect. If desired, an effective dose of such a compound, pharmaceutically acceptable salt thereof, or related 25 composition may be administered in two or more sub-doses, administered separately over an appropriate period of time. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg. In some embodiments, the pharmaceutical composition may include the compound in a range of from about 0.5 to 500 mg. In some embodiments, the pharmaceutical composition may include 30 the compound in a range of from about 1 to 100 mg. The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to about 1000 mg/kg body Page 23 QB\166118.01517\95733313.1
weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.5 to about 500 mg/kg body weight. In some embodiments, the pharmaceutical composition may be administered to provide the compound at a daily dose of about 50 to about 100 mg/kg body weight. 5 The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste. The compounds utilized in the methods disclosed herein may be formulated as a 10 pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross- linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® 15 PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. 20 Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride. 25 Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and 30 glucose. Page 24 QB\166118.01517\95733313.1
Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof. Examples of effervescent agents are effervescent couples such as an organic acid and a 5 carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent 10 couple may be present. The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients 15 as appropriate to the desired preparation. Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be 20 prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non- aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid 25 emulsions. Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis. 30 Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated dressings, Page 25 QB\166118.01517\95733313.1
sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams. For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are in some embodiments applied as a topical ointment or cream. 5 When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. 10 Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for nasal administration where the carrier is a solid 15 include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. 20 Pharmaceutical compositions adapted for administration by inhalation include fine particle dusts or mists which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations. 25 Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed 30 ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Page 26 QB\166118.01517\95733313.1
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, 5 sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tableting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, 10 aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan 15 monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents. Optionally, the disclosed compounds or pharmaceutical compositions comprising the 20 disclosed compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the disclosed compounds or with pharmaceutical compositions comprising the disclosed compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after 25 administering the disclosed compounds or the pharmaceutical compositions comprising the disclosed compounds. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the disclosed compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders. 30 Methods of preparing pharmaceutical formulations or compositions include the step of bringing an inhibitor compound into association with a carrier and, optionally, one or more Page 27 QB\166118.01517\95733313.1
additional adjuvants or ingredients. For example, standard pharmaceutical formulation techniques can be employed, such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. Regardless of composition or formulation, those skilled in the art will recognize various 5 avenues for medicament administration, together with corresponding factors and parameters to be considered in rendering such a medicament suitable for administration. Methods The disclosed compounds and pharmaceutical compositions comprising the disclosed compounds may be administered in methods of treating a subject in need thereof. For example, in 10 the methods of treatment a subject in need thereof may include a subject having a cell proliferative disease, disorder, or condition such as cancer. Another aspect of the present disclosure provides a method of treating a protein kinase related disease or condition comprising administering the pharmaceutical composition disclosed herein to a subject in need thereof. 15 Another aspect of the present disclosure provides a method of treating a subject for a respiratory virus infection comprising administering the subject a RIPK3 inhibitor, such as a RIPK3 inhibitor disclosed herein, two or more days after infection. As used herein, the terms “treating” or “to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to 20 prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder. As such, the methods disclosed herein encompass both therapeutic and prophylactic administration. A “subject in need thereof” as utilized herein refers to a subject in need of treatment for a disease or disorder associated with a protein kinase activity. In some embodiments, a “subject in 25 need thereof” as utilized herein refers to a subject in need of treatment for a disease or disorder associated with receptor interacting kinase 3 (RIPK3) activity. The term “subject” may be used interchangeably with the terms “individual” and “patient” and includes human and non-human mammalian subjects. In some embodiments, the treated subject may be a mammalian subject. Although the methods disclosed herein are particularly intended for the treatment of proliferative 30 disorders in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, mice, rats and pigs. Page 28 QB\166118.01517\95733313.1
As used herein, the term "disorder" refers to a condition in which there is a disturbance of normal functioning. A "disease" is any abnormal condition of the body or mind that causes discomfort, dysfunction, or distress to the person affected or those in contact with the person. Sometimes the term is used broadly to include injuries, disabilities, syndromes, symptoms, deviant 5 behaviors, and atypical variations of structure and function, while in other contexts these may be considered distinguishable categories. It should be noted that the terms "disease", "disorder", "condition" and "illness", are equally used herein. Diseases and disorders associated with RIPK3 activity may include, but are not limited to, human auto-immune, auto-inflammatory, and degenerative diseases and/or disorders. The diseases 10 and/or disorders may include, but are not limited to, progressive chronic diseases with inflammatory etiology including atherosclerosis and TNF-driven pathologies such as rheumatoid arthritis and ulcerative colitis; deleterious acute inflammatory responses such as upon infection by influenza virus; auto-immune diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and lupus; lipid and lysosomal storage diseases such as Neiman-Pick and Gaucher 15 disease; ischemia-reperfusion injuries such as stroke, myocardial infarction, kidney, retinal and liver ischemia, immune checkpoint inhibitor-induced myocarditis, scleroderma (SSc), scleroderma-associated interstitial lung disease (SSc-ILD), psoriasis, sepsis, age-related macular degeneration (AMD), asthma, lichen planus (LP), Steven-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and fibrotic diseases of different organs (including lung, liver, 20 kidney). In some embodiments, the diseases and/or disorders may be influenza A virus (IAV) infection, influenza-induced lung damage, interstitial pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress syndrome (ARDS), viral pneumonia or bacterial pneumonia, necrotic lung injury, and/or RIPK3-mediated necroptosis. The compounds and/or pharmaceutical compositions disclosed herein may be administered 25 in methods of treating a protein kinase related disease or condition, such as diseases or conditions involving RIPK3 activity, including inflammatory and degenerative diseases or conditions. In some embodiments, the diseases or conditions may be a respiratory virus, such as Influenza A virus (IAV) infection. In some embodiments, the diseases or conditions may be inflammatory conditions driven by TNF, influenza-induced lung damage, interstitial pulmonary fibrosis, chronic 30 obstructive pulmonary disease, acute respiratory distress syndrome (ARDS), viral pneumonia or bacterial pneumonia, necrotic lung injury, and/or RIPK3-mediated necroptosis. Page 29 QB\166118.01517\95733313.1
The compounds and/or pharmaceutical compositions disclosed herein may be administered in methods of treating a subject for a respiratory virus infection. The method may comprise administering the subject a RIPK3 inhibitor two or more days after infection. In some embodiments, the RIPK3 inhibitor may be administered five or more days after infection. In some 5 embodiments, the RIPK3 inhibitor may be UH15-38 or any of the compounds disclosed herein. The compounds for use according to the methods of disclosed herein may be administered as a single compound or a combination of compounds. For example, a compound that modulates RIPK3 activity may be administered as a single compound or in combination with another compound that modulates RIPK3 activity or that has a different pharmacological activity. 10 In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 15 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 20 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and/or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg – 200 mg). 25 In some embodiments of the disclosed treatment methods, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject. In some embodiments, a 30 maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, Page 30 QB\166118.01517\95733313.1
500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng/kg body weight of the subject. Minimal and/or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end-points any of 5 these disclosed dose levels (e.g., 500 – 2000 ng/kg body weight of the subject). As used herein the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g., as present in a pharmaceutical composition) 10 for treating a disease or disorder associated with RIPK3 activity. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the 15 subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. A typical daily dose may contain from about 0.01 mg/kg to about 100 mg/kg (such as from 20 about 0.05 mg/kg to about 50 mg/kg and/or from about 0.1 mg/kg to about 25 mg/kg) of each compound used in the present method of treatment. Compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and/or about 25 mg. The term “unit 25 dosage form” refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient. Oral administration is an illustrative route of administering the compounds employed in the compositions and methods disclosed herein. Other illustrative routes of administration include 30 transdermal, percutaneous, intravenous, intramuscular, intranasal, buccal, intrathecal, intracerebral, or intrarectal routes. The route of administration may be varied in any way, limited Page 31 QB\166118.01517\95733313.1
by the physical properties of the compounds being employed and the convenience of the subject and the caregiver. The compounds and compositions disclosed herein may be administered in methods of treatment as known in the art. Accordingly, various such compounds and compositions can be 5 administered in conjunction with such a method in any suitable way. For example, administration may comprise oral, intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral, transdermal, intravaginal, intranasal, mucosal, sublingual, topical, rectal or subcutaneous administration, or any combination thereof. Miscellaneous 10 Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill 15 in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as 20 being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not 25 fundamentally alter the nature of the claimed subject matter. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. 30 No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Page 32 QB\166118.01517\95733313.1
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Preferred aspects of this invention are described herein, including the best mode known to 5 the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited 10 in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. EXAMPLES Example 1 15 Seasonal influenza A virus (IAV) infections cause up to 5 million cases of severe illness, resulting in up to 650,000 deaths globally each year. Worryingly, while highly pathogenic H3, H5 and H7 strains of avian IAV are thus far limited in their spread between humans, they may require only a small number of mutations to become transmissible and achieve pandemic potential9-11. As current vaccines and antiviral strategies are either limited in their efficacy or susceptible to viral 20 resistance and evasion, identifying new therapeutic approaches for virulent IAV-triggered pulmonary disease is imperative. IAV-activated necrotic cell death is potentially one such entry point. As a lytic virus, IAV kills most lung cell types in which it replicates12. When such death is well-controlled, it is an effective mechanism of virus clearance, but when death is unchecked, or primarily necrotic, then 25 lung injury and severe illness ensues, despite virus clearance12,13. Such severe pathology is observed in mouse models, where destruction of airway epithelia is a hallmark of lethal IAV infection4,14,15, and in humans, where extensive death of distal pulmonary epithelia, marked by areas of bronchoalveolar necrosis, is a classic feature of IAV-induced ARDS, and of viral and secondary bacterial pneumonia2,3,12. Page 33 QB\166118.01517\95733313.1
Necroptosis drives influenza severity. Necroptosis accounts for most IAV-activated programmed necrotic death in infected lung epithelial cells6-8,12,16,17. Necroptosis is initiated by the host sensor protein Z-form nucleic acid Binding Protein 1 (ZBP1), which detects IAV-generated Z-RNA and activates RIPK318,19. RIPK3 then phosphorylates Mixed Lineage Kinase Like protein 5 (MLKL), which induces necroptosis6,8,12,13,18 (Fig. 1, panel a). We have previously shown that necroptosis triggers pulmonary tissue necrosis, pathogenic neutrophil recruitment and lung inflammation during severe IAV infections, but is dispensable for either CD8+ T cell-mediated antiviral responses or for virus clearance8,13. This is because RIPK3 also activates a parallel pathway of apoptosis, which is fully capable of restricting IAV in the absence of necroptosis 10 signaling6. These observations position necroptosis as an attractive node for therapeutic intervention, as only necroptosis, and not apoptosis, relies on RIPK3 kinase function6,20 (Fig. 1, panel a). Inhibitors of RIPK3 kinase activity may be expected to ameliorate necrotic lung injury without affecting virus clearance, and, therefore, may represent an entirely new strategy for treatment of IAV-triggered lung inflammation and injury. 15 To examine this possibility, we infected wild-type and Mlkl-/- mice with a lethal dose of IAV (H1N1 strain A/PuertoRico/8/1934; hereafter PR8) and assessed animal survival over a three- week time course. As Mlkl-/- mice are selectively deficient in RIPK3 kinase-driven necroptosis, we reasoned that results from these mice will foreshadow the effects of RIPK3 kinase inhibition 20 during severe influenza in vivo. Whereas wild-type (WT) mice succumbed to IAV by 12 days after infection, ~70% of the necroptosis-deficient Mlkl-/- mice made complete recoveries, in line with our previous findings8 (Fig. 1, panel b). Mice lacking Zbp1 or Ripk3 succumbed to this dose of IAV, as these animals lack both necroptosis and apoptosis signaling and therefore cannot control virus spread within the infected lung87,18 (Fig. 1, panel b). Mice harboring a kinase-inactivating 25 point-mutation in RIPK1 also succumbed to virus with kinetics and magnitude indistinguishable from wild-type animals, demonstrating that targeting RIPK1 kinase activity does not prevent IAV- induced lethality in the mouse model (Fig. 1, panel b); these findings are consistent with our previous observations that RIPK3-driven necroptosis in IAV-infected murine cells does not necessitate the kinase activity of RIPK16. Thus, RIPK3 kinase-dependent necroptosis is not only 30 unnecessary for animal survival or virus clearance but is actually deleterious to survival at lethal doses of IAV. Page 34 QB\166118.01517\95733313.1
Based on these findings, we hypothesized that a pharmacological agent which selectively blocks RIPK3 kinase function, without interfering with RIPK3-induced apoptosis, will be able to protect mice from IAV-triggered necroinflammatory injury and lethality. RIPK3 represents a natural target for necroptosis blockade as it is the only kinase known to phosphorylate MLKL, and 5 as phosphorylation of MLKL is the only known function of its kinase activity. This therapeutic strategy is, however, currently hindered by a lack of efficient and selective RIPK3 inhibitors for in vivo use. For example, GlaxoSmithKline (GSK) identified three molecules (GSK’872, GSK’843, and GSK’840) with potent activity against recombinant RIPK3 in vitro, but unexpectedly modest activity in cells20. Furthermore, even a two-fold increase in the concentration 10 of these inhibitors triggered RIPK3-mediated apoptosis and unleashed consequent on-target toxicity20. In agreement with these findings, knock-in mice harboring a mutation (D161N) in RIPK3 designed to nullify necroptosis by inactivating catalytic function succumbed instead to fulminant RIPK3-mediated apoptosis in utero21. These observations raise the question of whether targeting necroptosis by blocking RIPK3 kinase function in vivo is even feasible or will inevitably15 produce on-target toxicity. Other groups, including ours, have since identified a subset of anti- cancer kinase inhibitors (e.g., ponatinib22,23 and dabrafenib24) capable of blocking RIPK3 without triggering on-target apoptosis, but these molecules are poorly-suited for non-oncological settings. UH15-38 is a potent RIPK3 inhibitor. To address the absence of a clinically viable 20 RIPK3 inhibitor, we undertook a fresh approach to targeting this kinase. We focused on the molecule PD180970, originally developed as an inhibitor of the oncoprotein BCR-ABL, but shown in a proteomics analysis25 as capable of inhibiting RIPK2, a close mammalian homolog of RIPK3. We discovered that a PD180970 analog (PD166285) inhibited RIPK3 in vitro (IC50=1.08 ^M) and was moderately potent at blocking necroptosis in cells (IC50 = 2.88 ^M). We generated >40 25 PD166285 analogs, one of which showed potent anti-necroptosis activity in cells. We named this molecule UH15-38 (Fig. 1, panel c). UH15-38 blocked TNF^-induced necroptosis in primary murine embryonic fibroblasts (MEFs) at concentrations (IC50 = 98 nM) that were ~8.5-fold lower than GSK’843 (IC50 = 843 nM) and ~6-fold lower than GSK’872 (IC50 = 582 nM), the two best- performing GSK inhibitors of mouse RIPK320 (Fig. 1, panel d). UH15-38 prevented 30 phosphorylation of MLKL following necroptotic stimulation by TNF^ (Fig.1e), or upon enforced Page 35 QB\166118.01517\95733313.1
dimerization of RIPK3 (Fig.5, panel a) in MEFs. UH15-38 also robustly inhibited necroptosis in an additional panel of human and mouse cell lines (Fig.1, panel f). Notably, the previously developed GSK compounds showed reduced anti-necroptotic potency in cells, compared to UH15-38 (Fig. 1, panels d, f), despite robust inhibition of RIPK3 5 kinase function in vitro (Fig.5, panel b), and in a RIPK3 target engagement assay in cellulo (Fig. 1, panel f). These observations suggested that UH15-38 may display unique features in its RIPK3 binding mode that are distinct from GSK inhibitors and that translate strong RIPK3 binding into potent inhibition of necroptosis. To explore this possibility, we carried out molecular docking studies. These studies showed that UH15-38 can interact with the ATP binding pocket of both 10 mouse and human RIPK3 in the active conformation, indicative of a Type I binding mode (Fig.1, panels g-i, Fig.5, panel c). Consistent with the binding mode of other Type I kinase inhibitors, UH15-38 makes interactions with the backbone of the gatekeeper Met98 residue (Met97 in humans) in the hinge segment of the RIPK3 kinase domain (Fig.1, panels h, i; Fig.5, panel c). Interestingly, modeling and X-ray crystal structure data26 for GSK’872 and GSK’843, 15 respectively, displayed generally similar hinge binding poses to UH15-38 ( Fig. 5, panels d, e), which may explain why the in vitro affinities of the GSK molecules for RIPK3 are on par with that of UH15-38. However, UH15-38 differed significantly from both GSK compounds in its interactions with the rear pocket of the RIPK3 active center. While GSK’872 and GSK’843 only made contacts with the backbone of the DFG motif of mouse RIPK3 ( Fig.5, panels d, e), UH15- 20 38 not only engaged in similar interactions with the DFG motif, but also participated (via the phenol) in a broader network of hydrogen bonds and ionic-dipole interactions with the charged side-chains of catalytic Lys51 and the central residue in the ^C helix, Glu61 (Fig.1, panels h, i; Fig.5, panel c). Methylation of the phenol (e.g., as in the analog UH15-38 Me) resulted in loss of activity ( Fig.5, panel f). The elaborate interaction network unique to UH15-38 may account for 25 the increased cellular potency of UH15-38 over the GSK molecules. Although we found that UH15-38 binds RIPK1 in vitro ( Fig.5, panel b), it displayed no significant activity against RIPK1 kinase-driven caspase-8-dependent cell death27 induced in macrophages by the combination of LPS and a TAK1 inhibitor ( Fig. 5, panel g), or in Ripk3-/- MEFs stimulated with TNF^ and a TAK1 inhibitor ( Fig.5, panel h), whereas the RIPK1 inhibitor 30 GSK’547 was able to block such death in Ripk3-/- MEFs ( Fig.5, panel i). UH15-38 also did not inhibit either Gasdermin D cleavage ( Fig.5, panel j) or pyroptosis ( Fig.5, panel k) upon canonical Page 36 QB\166118.01517\95733313.1
activation of the NLRP3 inflammasome by Nigericin, demonstrating selectivity of this molecule for RIPK3-driven necroptotic cell death. To assess if UH15-38 will be efficacious when administered systemically in vivo, we dosed wild type C57BL/6 mice with this compound at 30 mg/kg/day for four consecutive days via the 5 intraperitoneal (i.p) route, and found that it achieved excellent accrual in lung tissues (Cmax = 42 ^M) following this dosing regimen, which is ~8-fold higher than its levels in plasma ( Fig.6, panel a). UH15-38 also accumulated to significant levels in the liver, heart, kidney, and colon ( Fig.6, panel a). Next, we administered UH15-38 at 30 mg/kg/day for four consecutive days (i.p) to Casp8- /-Mlkl FLAG/FLAG mice28. In these mice, germline loss of caspase 8 triggers spontaneous multi-organ 10 RIPK3 activation and phosphorylation of FLAG-tagged MLKL, but the N-terminal FLAG tag prevents MLKL from executing necroptosis28. This mouse model thus allows one to identify all cells harbouring phosphorylated MLKL without losing these cells to eventual necrotic demise. UH15-38 was able to effectively prevent MLKL phosphorylation in all tested organs (lung, liver, heart, kidney) in Casp8-/-Mlkl FLAG/FLAG mice, demonstrating its potential for therapeutic 15 deployment in necroinflammatory diseases not just of the lung, but of these other organs as well ( Fig.6, panels b, c). Comprehensive safety profiling of UH15-38 for off-target activity against a panel of 50 critical protein targets whose inhibition has been linked to potentially serious side effects in humans (InVEST, Reaction Biology) demonstrated that UH15-38 did not significantly inhibit any 20 of these key targets ( Fig. 6, panel d, Table 1A). When profiled for inhibitory activity against a panel of 90 non-mutant human kinases (DiscoverX KinomeScan), UH15-38 displayed an S(35) selectivity score of ~0.25 (Table 1B), in line with other pre-clinical stage Type I kinase inhibitors, and on par with some of the current FDA-approved TKIs, such as Sorafenib [S(35) = 0.22] and Dasatinib [S(35) = 0.28]. 25 Table 1A. InVEST Safety Panel Profiling of UH15-38 (1 µM) Remainin activit (% control) Page 37 QB\166118.01517\95733313.1
alpha2A adrenergic 92 95 beta 1 adrenergic 89 82 Page 38 QB\166118.01517\95733313.1
CYP2D6 98 95 CYP3A4 70 72 No . A value ≥ 100 indicates no effect of test compound. Table 1B. Profiling of UH15-38 (10 x IC50, 500 nM) against 90 non-mutant kinases on the 5 DiscoverX KinomeScan platform. Remaining activity (% 0 Page 39 QB\166118.01517\95733313.1
ALK 64 AURKA 76 Page 40 QB\166118.01517\95733313.1
IKK-beta 70 INSR 88 Page 41 QB\166118.01517\95733313.1
PIK3CA 70 PIK3CG 71 Page 42 QB\166118.01517\95733313.1
UH15-38 was exceptionally well tolerated in vivo, with no detectable signs in any of these organs of on-target apoptosis as measured by cleaved caspase 3 ( Fig.6, panels e, f), or of general toxicity (Tables 2A and 2B), even after extended dosing at 30 mg/kg/day (i.p) for seven consecutive days. Tables 2A and 2B show whole animal and organ weight changes after treatment 5 with UH15-38 for 7 consecutive days at 30 mg/kg/day (i.p). The formulation was prepared using 100 mg/ml DMSO stock diluted to 30 mg/kg of mice (C57BI/6) weight in 10/90 solutol/saline. Table 2A. Whole animal weight (in grams) over the 7-day treatment period Subjects Pretreatment D1 D2 D3 D4 D5 D6 D7 V hi l 0 0 0 0 0 0 Page 43 QB\166118.01517\95733313.1
Table 2B. Key organ weight after 7 days Subjects BW (g) Heart (g) liver (g) kidneys (g) spleen (g) Vehicle male-1 19.780 0.131 1.308 0.313 0.069 ere were no s gns o ver tox c ty (e.g., ncrease n serum a an ne amn notransferase [ALT] or albumin levels), even though the compound reached peak concentrations >100 ^M in 5 this organ following dosing at 30 mg/kg/day (i.p) for four consecutive days ( Fig.6, panel a, Tables 3A and 3B). Collectively, these data indicate that UH15-38 possesses a pharmacological profile that is suitable for safe and effective in vivo deployment. Table 3A. Multi-day dose PK study in male C57Bl\6J mice of UH15-38 (MW: 441.2) Organ Dose T1/2 Tmax Cmax Cmax AUClast AUClast ) 1 Page 44 QB\166118.01517\95733313.1
AUC_% AUCINF Organ Extrap Cl_F_obs Cmin Tmin C24 nts w a eas wo va va ues were use n p armaco ne c ca cu a ons. *** Does not converge N/A - Cannot be curve-fit 5 Table 3B. Serum biomarker profiling after dosing with UH15-3830mg/kg/day for 4 days (IP) Sample ID ALB ALT BUN CRE NA+ TP 3 *https:// services/private/serum_chemistry_reference_ranges_mice.pdf 10 Albumin (ALB)- Albumin is approximately 60% of the total protein. It is formed in the liver and transports important blood constituents such as drugs, hormones and enzymes. Decreased levels are associated with liver disease and malnutrition. Increased levels are seen with dehydration. Alanine Aminotransferase (ALT)- Usually measured to evaluate liver disease. Elevation 15 of ALT is rarely observed in conditions other than liver disease. Page 45 QB\166118.01517\95733313.1
Blood Urea Nitrogen (BUN)- The BUN is useful in the evaluation of kidney function and nutritional status. Increased levels will be seen with kidney disease, increased protein intake (high protein diet), and dehydration. Decreased levels will be seen with overhydration, severe primary liver disease and malnutrition. 5 Creatinine (CRE)- Used in the evaluation of kidney function. Elevations in creatinine generally suggest chronic kidney disease. Electrolytes- Sodium (Na)- there are almost no metabolic processes that are not dependent on or affected by electrolytes. Thus, abnormal levels of electrolytes may be either the cause or the consequence of a variety of disorders. 10 Total Protein (TP)-The sum of the circulating proteins in the blood. Proteins are found in muscle, enzymes, hormones, and several other key functional and structural entities within the body. UH15-38 blocks IAV-activated necroptosis. Before attempting to determine whether 15 small molecule blockade of RIPK3 kinase activity by UH15-38 will be efficacious in mitigating IAV-induced pathology, we first sought to identify the primary cell type(s) undergoing RIPK3- dependent necroptosis in infected lungs, as these cell types will represent the dominant targets for pharmacological blockade of RIPK3 activity in vivo. We carried out scRNA-Seq analyses of IAV- infected lung cells and identified Type I alveolar epithelial cells (AECs) as the primary replicative 20 niche for IAV in murine lungs; Type I AECs displayed significantly (84-fold) higher levels of IAV mRNA than any other cell type (Fig.2, panel a) and comprised over half of all IAV-positive cells in the infected lung (Fig.2, panel b). High levels of IAV mRNA were readily observed in Type I AECs by three days after infection, coincident with induction of Zbp1 mRNA (Fig. 2, panel c). Death of Type I AECs is also highly correlated with compromised lung function and mortality in 25 the mouse model15. Thus, although other lung cell types (most notably Type II AECs and ciliated cells) also represent important replication niches for IAV (Fig. 2, panels a, b), we focused on examining the efficacy of UH15-38 in preventing necroptosis of IAV-infected Type I AECs. We isolated primary Type I AECs from mouse lungs by immunomagnetic selection with anti-aquaporin 5 (Aqp5) antibodies29,30. Staining these cells with the Type I AEC marker 30 podoplanin31 showed them to be >95% pure ( Fig.7, panel a). Using these cells, we first established that they underwent ZBP1-dependent cell death upon infection with IAV (Fig.2, panel d). Whereas Page 46 QB\166118.01517\95733313.1
~80% of infected wild type (Zbp1+/+) Type I AECs succumbed to IAV by 24 hours (h), similarly infected Type I AECs from littermate-matched Zbp1-/- animals remained mostly viable for over 48 h (Fig.2, panel d). Type I AECs strongly upregulated ZBP1 upon infection, possessed the entire ZBP1-dependent cell death machinery, and displayed markers of both necroptosis (pMLKL) and 5 apoptosis (cleaved caspases-8 and -3) activation following infection with IAV (Fig. 2, panel e). After confirming that UH15-38 was able to protect Type I AECs from canonical TNF^-induced necroptosis (IC50 = 114 nM, Fig.7, panel b), we tested if UH15-38 could selectively prevent IAV- induced necroptosis, without impeding apoptosis, in this cell type. We found that UH15-38 was a highly potent inhibitor of IAV-induced necroptosis in primary Type I AECs (Fig. 2, panel f, 10 IC50=39.5 nM) and primary MEFs ( Fig.7, panel c, IC50=51.9 nM). Inhibition was selective for necroptosis, as UH15-38 did not prevent apoptosis (i.e., cell death in the absence of pan-caspase inhibitor zVAD) to any significant extent in these cells (Fig.2, panels f, g, Fig. 7, panels c, d). UH15-38 blocked IAV-induced phosphorylation of MLKL in Type I AECs (Fig.2, panel h) and MEFs ( Fig. 7, panel d), without inhibiting cleavage of either caspase-8 or caspase-3. UH15-38 15 also prevented activation of MLKL and selectively inhibited necroptosis in cells infected with a panel of clinically relevant IAV and IBV strains ( Fig.7, panels e, f). UH15-38 completely blocked IAV-induced necroptosis in Ripk1-/- MEFs ( Fig.7, panel g). Although UH15-38 was derived from a BCR-ABL inhibitor with inhibitory activity against RIPK1 and RIPK2, neither the ABL inhibitor imatinib (Gleevec) nor the selective RIPK2 inhibitor CSLP3732 blocked IAV-triggered necroptosis 20 in cells ( Fig.7, panels h, i), demonstrating that while UH15-38 can inhibit RIPK1, RIPK2 and ABL kinase activity in vitro (Tables 1A-1B, Fig. 16, and Fig. 5, panel b), the ability to target RIPK3 is central to its necroptosis-inhibitory activity in cells. To examine if UH15-38 could block necroptosis in a virus-independent system of ZBP1 activation, we appended two Fv domains to the N-terminus of full-length murine ZBP1 (2xFv- 25 ZBP1) and reconstituted Zbp1-/- MEFs with this chimeric construct. Chemical oligomerization of these Fv domains leads to ZBP1 activation, RIPK-mediated phosphorylation of MLKL, and necroptosis ( Fig.7, panels j, k). UH15-38 fully inhibited ZBP1-dependent RIPK3 activation and phosphorylation of MLKL and consequent necroptosis in this system ( Fig.7, panels j, k). To test the efficacy of UH15-38 in human cells, we first reconstituted HeLa cells (which 30 normally do not express RIPK3) with full-length human RIPK3 (HeLa-RIPK3) and exposing them to TNF^, in the presence of zVAD. We found that UH15-38 was again able to effectively prevent Page 47 QB\166118.01517\95733313.1
TNF^-induced necroptosis in HeLa-RIPK3 cells ( Fig.7, panels l, m). Most human cell lines (such as HeLa cells, as noted above) lack expression of one or more components of the necroptosis machinery, but we identified a human malignant mesothelioma-derived cell line (M29)33 which expressed ZBP1, RIPK3, and MLKL and underwent necroptosis following infection with IAV 5 (Fig.2, panel i, Fig.7, panel n). UH15-38 was able to prevent IAV-induced phosphorylation of MLKL at nanomolar concentrations ( Fig. 7, panel n), resulting in full protection from IAV- induced necroptosis at 1 ^M (Fig.2, panel i) in these cells. We observed that M29 cells manifested basally elevated levels of pRIPK3 (Thr224/Ser227), which were reduced upon infection, and further dampened by UH15-38 at concentrations ≥500 nM ( Fig. 7, panel n). It should be noted 10 that the relationship between RIPK3 autophosphorylation and catalytic activity remains incompletely understood, as the best-characterized sites of RIPK3 autophosphorylation (Thr231/Ser232 in mouse RIPK3 and Thr224/Ser227 in human RIPK3) are dispensable for RIPK3 catalytic activity34,35. Of particular clinical relevance, UH15-38 completely inhibited MLKL phosphorylation in human donor lung tissue, following infection of these tissues with IAV ex vivo 15 (Fig. 2, panels j-l). Together, these results demonstrate that UH15-38 is a potent and specific inhibitor of IAV-triggered necroptosis in murine and human cells. UH15-38 did not induce apoptosis even at fifty times the IC50 dose for necroptosis blockade in type I AECs (IC50 = 39.5 nM) and MEFs (IC50 = 50 nM) and only modest toxicity was seen at 100x IC50 (Fig. 8, panels a, d). The toxicity manifested by UH15-38 at 100x IC50 was indeed 20 RIPK3-dependent apoptosis, as it was accompanied by activation of caspases 8 and -3 ( Fig. 8, panels b, e), and was eliminated in Ripk3-/- MEFs, or by addition of pan-caspase inhibitor zVAD.fmk (Fig. 8, panel c). Thus, we have achieved in UH15-38 a compound with potent in cellulo efficacy in Type I AECs and other cell types, as well as wide separation of necroptosis- inhibitory activity from the capacity to trigger on-target apoptosis. 25 UH15-38 prevents IAV-driven lethality. To test if UH15-38 can prevent IAV-triggered lethality when delivered as a systemic therapeutic, we next infected WT mice with a lethal dose of IAV (6000 EID50), treated these mice starting one day after infection with a four intraperitoneal (i.p.) once-daily doses of UH15-38 ranging from 7.5 mg/kg/day to 50 mg/kg/day, and compared 30 their overall survival rates, as well as their rates of weight loss, to mice receiving vehicle alone. We found that doses of UH15-38 as low as 7.5 mg/kg/day prevented lethality in 40% of infected Page 48 QB\166118.01517\95733313.1
mice, with maximum protection observed at 30 mg/kg/day (Fig.3, panel a, Fig.9, panel a).80% of mice receiving the 30mg/kg/day dose of UH15-38 manifested significantly reduced (and delayed) weight loss and were fully protected from a lethal inoculum of IAV; these mice made full recoveries by three weeks after infection (Fig.3, panel a, Fig.9, panel a). Increasing the dose of 5 UH15-38 to 50 mg/kg/day did not provide any discernible additional benefit to survival outcomes of infected mice (Fig. 3, panel a, Fig. 9, panel a). Evaluating UH15-38 for protection when administered over a shortened time course (30 mg/kg/day, once daily, for two days, starting one day after infection), or delaying administration by up to two days (i.e., starting UH15-38 at 30 mg/kg/day two or three days after infection) also reduced weight loss and prevented lethality in a 10 significant proportion of mice infected with this highly lethal dose of virus ( Fig.9, panels b, c). Remarkably, UH15-38 was fully (100%) protective against a less lethal, but more patient- relevant inoculum of IAV (4500 EID50; ~LD60), even at doses as low as 1 mg/kg/day (Fig.3, panel b). UH15-38 administered at 30 mg/kg/day not only delayed the onset of weight loss, but markedly mitigated the extent of such weight loss, compared to vehicle-treated controls, in mice infected 15 with IAV at this dose (Fig. 3, panel c). In contrast, mice receiving four once-daily doses of GSK’872 (30 mg/kg, i.p.) were not protected from IAV (PR8, LD60); in fact, GSK’872-treated mice fared worse than vehicle-treated controls over the three-week time course of this experiment ( Fig.9, panels d, e), possibly reflecting the on-target toxicity associated with this class of RIPK3 inhibitor18. UH15-38 was also able to afford full protection against lethality triggered by the H1N1 20 A/California/04/2009 strain of pandemic IAV (Fig.3, panel d, Fig.9, panel f), demonstrating its potential for therapeutic benefit against seasonal and pandemic strains of IAV. Importantly, UH15- 38 was able to protect almost all mice infected with IAV at the LD60 dose even when administered up to five days after infection (Fig. 3, panel e, Fig. 9, panel g). This represents a significant advantage over the frontline antiviral agent oseltamivir (Tamiflu, Roche), which loses therapeutic 25 benefit if administered more than 48 h after infection in mice36 and humans37. UH15-38 failed to protect Ripk3-/- or Mlkl-/- mice from IAV, confirming that its protective effects are mediated by blockade of RIPK LKL signaling (Fig. 3, panel f). In fact, UH15-38 treatment was notably superior to germline MLKL loss in preventing IAV-induced lethality at all doses of virus tested ( Fig.8, panels h, i; also compare Fig.1, panel b to Fig.3, panel a). We attribute this result to the 30 benefit some early necroptosis may provide in sparking the antiviral immune response, or to some other currently unknown protective function of MLKL in maintaining baseline immune Page 49 QB\166118.01517\95733313.1
homeostasis or limiting early disease, both of which may be lost when MLKL is permanently ablated. UH15-38 dampens IAV-induced lung injury. To examine if UH15-38 prevented IAV- 5 induced mortality by blocking necroptosis and consequent lung injury in vivo, we infected WT mice with a lethal dose of PR8 (6000 EID50), and administered UH15-38 (30 mg/kg) to these mice i.p. once-daily for a total of four days, starting 24 h after infection. Lungs from vehicle-treated mice showed significant evidence of necroptosis (i.e., pMLKL+) as early as day 3 post infection; by day 6, necroptosis was seen in a substantial fraction of Type I AECs (PDPN+) (Fig.4, panels a, 10 b). Mice receiving UH15-38, however, showed markedly reduced numbers of pMLKL+ necroptotic cells at both these time points (Fig.4, panels a, b). As we have previously shown, the majority of pMLKL+ cells are also IAV+ 16. Importantly, UH15-38 did not significantly alter the proportion of infected cells undergoing apoptosis (CC3+) ( Fig.10, panels a, b), demonstrating that UH15-38 is a selective inhibitor of IAV-triggered necroptosis in vivo. 15 UH15-38 was able to prevent the release of inflammatory cytokines and chemokines (e.g., IL-1^, IL-33, IL-6, TNF^, CXCL1) from infected Type I AECs in culture ( Fig.10, panel c), and this activity was recapitulated in vivo. Bronchoalveolar lavage fluid (BALF) from UH15-38 treated mice three days after infection, and following just two doses of UH15-38, also showed significantly reduced levels of key inflammatory cytokines and neutrophil chemoattractants (e.g., 20 IL-1^, IL-6, IL-18, TNF^, CXCL1, GMCSF) (Fig. 4, panel c), accompanied by a marked diminishment in the magnitude and extent of infiltrating neutrophils into pulmonary tissues (Fig. 4, panels d, e), compared to animals receiving vehicle. By nine days after infection, following the full four-dose cycle of vehicle or UH15-38, lungs from infected animals treated with UH15-38 showed a decrease in diffuse alveolar damage 25 and the formation of hyaline membranes (Fig. 4, panel f, left arrow; Fig. 4, panel g), as well as notably reduced bronchiolar denudation (Fig. 4, panel f, right arrow; Fig. 4, panel g) and histological appearance of fibrosis (Fig.4, panel g). Staining pulmonary tissues for Tenascin C, a marker of fibrotic remodeling following injury38, confirmed that UH15-38 reduced the extent of fibrotic lung damage ( Fig. 10, panels d,e). Consistent with this observation, BALF from mice 30 receiving UH15-38 displayed significantly lower levels of the pro-fibrotic mediators (IL-1^, IL-6, IL-18, IL-17, and CCL5), compared to controls, early (three days) after infection (Fig.4, panel c, Page 50 QB\166118.01517\95733313.1
Fig. 10, panel f). Lungs from UH15-38 treated mice also displayed dampened alveolar and interstitial infiltration, accompanied by decreases in septal thickening and epithelial metaplasia ( Fig. 10, panel g). In addition, UH15-38 completely blocked the release of IAV-triggered inflammatory cytokines IL-1^ and IL-18 from necroptotic BMDMs ( Fig. 10, panel h). 5 Importantly, UH15-38-treated animals maintained relatively normal lung function, as measured by evaluating arterial oxygen saturation levels three and six days after infection (Fig.4, panel h), and by measuring airway resistance ten days after infection ( Fig.10, panel i). UH15-38 did not alter the extent of virus replication and spread, or the rate of virus clearance from infected lungs ( Fig.11, panels a-c). UH15-38 also did not negatively impact the 10 frequencies of IAV-specific CD8+ cytotoxic T cells (CTLs) in BALF ( Fig.11, panel d, Fig.12, panel a), while the functional profile of these cells, as measured by IFN^ secretion, was improved (Fig. 4, panel i, Fig. 12, panel b). Together, these results demonstrate that UH15-38 dampens neutrophil influx into infected pulmonary tissues and significantly reduces the extent of virus- triggered lung injury, without negatively impacting either virus clearance or anti-IAV CD8+ T cell 15 responses. Discussion In this study, we report that RIPK3 kinase activity, the key enzymatic driver of necroptosis, is a new host target for the prevention of lung inflammation and injury during severe influenza 20 virus infections. We have developed a potent new RIPK3 kinase inhibitor, UH15-38, which effectively and selectively blocked activation of necroptosis in IAV-infected alveolar epithelial cells in vivo, preventing both the aberrantly high neutrophil recruitment that characterizes severe influenza, as well as the histological sequelae, such as the presence of hyaline membranes and widespread bronchiolar denudation, which are hallmarks of IAV-triggered lung injury and ARDS. 25 Our data also demonstrate that the production and release of a set of important inflammatory mediators capable of promoting secondary inflammation, fibrosis and cycles of necroinflammation, is almost completely dependent on necroptosis, highlighting the uniquely inflammatory nature of this form of cell death. Our findings support a model in which UH15-38, by preventing necroptosis of infected 30 type I AECs and other lung cell types, blocks the release of DAMPs, alarmins, and other inflammatory mediators from these cells. This reduces neutrophil influx into the lung, dampens Page 51 QB\166118.01517\95733313.1
necroinflammation, and prevents consequent loss of alveolar integrity and gas exchange function, without affecting either beneficial anti-IAV adaptive immune responses or virus clearance rates, both of which are fully enabled via the more immunologically silent pathway of apoptosis. Neither virus spread, progeny virion titers, or eventual virus clearance were significantly impacted by 5 necroptosis blockade with UH15-38. In fact, we noted a small, but significant, increase in PA- specific CTLs in the BAL fluid of UH15-38 treated mice, compared to controls, suggesting that the anti-IAV T cell response may proceed more efficiently when the infected lung is not clogged by neutrophils (which by themselves can dampen CTL-driven antiviral responses39,40) or by other inflammatory mediators. 10 Strikingly, UH15-38 was efficacious even when first administered as late as five days after infection. To our knowledge, no other host-targeted therapeutic has been shown to prevent lethality in the mouse model when dosed this late after infection and suggests that blocking feed-forward cycles of neutrophil-driven necro-inflammation affords strong therapeutic benefit even during the peak of virus replication, which is a finding of significant clinical importance. 15 While the in vitro affinity of UH15-38 towards RIPK3 was on par with previous RIPK3 inhibitors GSK’843 and GSK’872, its activity in cells is markedly greater than either GSK compound, whose unexpected loss of cellular potency had been previously noted20. As GSK’872 showed comparable in cellulo target engagement to UH15-38, the difference in cellular potency between these compounds is unlikely to result from differential cell penetration. Rather, we 20 propose that the ability of the phenol of UH15-38 to engage an interaction network in the back pocket of RIPK3 may define a binding mode that translates more efficiently into cellular potency. We have previously observed similar discrepancies between in vitro and cell-based inhibitors for another RIPK family member, RIPK232,41,42. In that case, a subset of RIPK2 inhibitors capable of specifically occluding the deep pocket along the ^C helix were effective at blocking RIPK2- 25 mediated cellular signaling, whereas other structurally similar RIPK2 inhibitors lacking the ability to occlude this pocket, were equally potent in vitro, but manifested poor cellular activity against RIPK2. Mechanistically, the ‘deep pocket occluders’, were able to allosterically interfere with RIPK2 binding to the E3 ligase XIAP, which is important for RIPK2 signaling in cells. Our modeling suggests that similar engagement of the deep pocket of RIPK3 by UH15-38 may produce 30 allosteric effects on RIPK3 kinase function beyond simple catalytic blockade. A comparison of the published structures of monomeric RIPK3 to the MLKL-bound form of RIPK3 showed that Page 52 QB\166118.01517\95733313.1
MLKL binding requires the displacement of the RIPK3 ^C helix34 ( Fig. 13, panels a, b); this transition is likely to be impeded by UH15-38 engagement of Glu61 in the ^C helix, but whether this occurs in cells remains to be verified. As several other respiratory viruses, including RSV and SARS-family CoVs have been 5 shown to trigger necroptosis in infected lungs5,43, and as necroptosis has also been implicated in the pathogenesis of several chronic inflammatory and fibrotic lung diseases, such as COPD and IPF44, our findings suggest that necroptosis blockade may represent a therapeutic strategy for a range of lung pathologies, whether of viral etiology or otherwise. Because RIPK3 is essential for all known pathways of necroptosis, RIPK3 kinase inhibitors such as UH15-38 will potentially have 10 benefit in a wider range of acute and chronic inflammatory conditions involving necroptosis than will anti-TNF approaches or RIPK1 inhibitors. Materials and Methods. Mice, cells, viruses, reagents 15 Zbp1-/- 45, Mlkl-/- 46, Ripk3-/- 47, Ripk1K45A 48, Casp8-/-MlklFLAG/FLAG 28 animals have been described previously. C57BL/6 mice were obtained from Taconic and were allowed at least 1 week to acclimate to housing conditions before use in experiments. Mice were randomly assigned into groups before each experiment. Mice were housed in SPF facilities at the Fox Chase Cancer Center, St. Jude Children’s Research Hospital, and all in vivo experiments were conducted under 20 protocols approved by the Committee on Use and Care of Animals at these institutions. No statistical methods were used to predetermine sample size. The investigators were not blinded to allocation during experiments. Primary MEFs were obtained from E14.5 embryos and were immortalized using the 3T3 protocol. Immortalized MEFs were routinely tested by qPCR and by immunoblot analyses for protein expression. To create the 2xFv-RIPK3 and 2xFv-ZBP1 25 constructs, tandem inducible dimerization domains (hereafter 2xFv) derived from the protein FKBP were placed at the N-terminus of full length human RIPK3 or murine ZBP1.2xFv-tagged constructs were cloned into the Dox-inducible vector pRetroX-TRE3G (631188, Clontech). Immortalized Ripk3-/- or Zbp1−/− MEFs inducibly expressing 2xFv-RIPK3 or 2xFv-ZBP1, respectively, were produced using the retroviral Tet-On 3G System. Activation of RIPK3 or ZBP1 30 in cells expressing the 2xFv constructs was achieved by treatment with doxycycline (5 ^g/ml) for 12 h, followed by exposure to B/B homodimerizer, AP20187 (100 nM). HeLa (ATCC, CCL-2) Page 53 QB\166118.01517\95733313.1
cells were retrovirally transduced with full-length human RIPK3 to generate HeLa-RIPK3 cells. Mouse adapted strains of Influenza A/Puerto Rico/8/1934 (H1N1), A/California/04/2009 (H1N1), Influenza A/Brisbane/10/2007 (H3N2), A/Singapore/INFIMN-16-0019/2016 (H3N2), Influenza B/Florida/04/2006, and B/Colorado/06/2017 were propagated by allantoic cavity inoculation of 10 5 day-embryonated chicken egg. Influenza virus titers were determined by plaque assay on MDCK cells obtained from the ATCC. For necroptosis induction, MEFs and Type I AECs were infected with virus or treated with 100 ng/mL TNF^ (R&D Systems, 410-MT) in the presence of 250 ng/mL cycloheximide (Sigma, 01810) and 50 ^M zVAD (Bachem, N-1510); RAW264.7 cells were treated with 100ng/ml LPS (Sigma, L2630), 20 ^M IDN-6556 (Medkoo, 210530); 10 immortalized Bone marrow derived macrophages (iBMDM, gift of Dr. Kate Fitzgerald) were treated with 10ng/ml LPS, 20 ^M IDN-6556, 100nM 5z-7-oxozaenol (MedChemExpress, HY- 12686); HeLa RIPK3 and U937 cells were treated with 20 ng/ml human TNF^ (R&D Systems, 210-TA), 20 ^M IDN-6556, 100 nM Smac Mimetic SM164 (MedChemExpress, HY-15989); FADD-deficient Jurkat cells were treated with 20ng/ml human TNF^. RIPK1 dependent apoptosis 15 in iBMDM was induced by treatment with 10ng/ml LPS and 100 nM 5z-7-oxozaenol. All cell lines obtained from ATCC were maintained in DMEM 10% FBS, 1mM sodium pyruvate, 1x GlutaMAX and 1% penicillin/streptomycin at 37oC and 5% CO2. The human malignant mesothelial cell line M29 was a gift of Dr. J. Testa, and maintained in RPMI medium supplemented with 10% FBS, 1x GlutaMAX and 1% penicillin/streptomycin. M29 cells were authenticated for 20 use by the Cell Culture Facility at Fox Chase Cancer Center. Immortalized BMDMs were authenticated by FACS for the presence of BMDM markers. All other cell lines were authenticated by ATCC.All cell linese were routinely tested for Mycoplasma contamination and only used when negative for Mycoplasma sp. Synthesis of UH15-38 and analogs 25 UH15-38 was prepared in a two-step procedure, as described below, and used as the hydrochloride salt (UH15-38.HCl) in all experiments. UH15-38.HCl is commercially available. Page 54 QB\166118.01517\95733313.1
Rea c)2 , Xantphos, Cs2CO3, dioxane, 80 oC, 24 h; (ii) 2.0 M HCl in diethylether, diethylether: MeOH, 0 °C – rt, 16 h. Synthesis of 7-Chloro-3-(3-hydroxyphenyl)-1-methyl-1,6-naphthyridin-2(1H)-one (3a) 5 To a stirred solution of 1 (1.0 g, 5.71 mmol) and 2a (1.60 g, 5.71 mmol) in dry DMA (15 mL), KF/Al2O3 (6.0 g, 40 wt%) was added at 0 °C portion wise under argon. The reaction mixture was stirred at room temperature (rt) for 2 h. After completion, the reaction mixture was filtered through Celite and the residual solid was washed with MeOH and DCM several times. The filtrate was concentrated, and the residue was purified by column chromatography over silica gel using10 25% EtOAc/hexane to give 3a (1.30 g, 80%) as a light-yellow solid.1H NMR (600 MHz, DMSO- d6) ^ 9.56 (s, 1H), 8.78 (s, 1H), 8.15 (s, 1H), 7.64 (s, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.13-7.05 (m, 2H), 6.80 (d, J = 7.4 Hz, 1H), 3.64 (s, 3H); 13C-NMR (151 MHz, DMSO-d6) ^ 160.5, 157.0, 150.5, 150.4, 150.3, 145.9, 137.0, 134.2, 132.5, 129.2, 119.4, 115.8, 115.4, 108.5, 29.8. 15 Synthesis of UH15-38 To a stirred solution of 3a (1.0 g, 3.50 mmol) and 4 (668 mg, 3.50 mmol) in dioxane (20 mL), were added Xantphos (405 mg, 0.70 mmol) and cesium carbonate (2.28 g, 7.0 mmol). The reaction mixture was degassed with argon for 10 min. Pd(OAc)2 (79 mg, 0.35 mmol) was added to the mixture and the flask flushed with argon for another 10 min. Next, the reaction mixture was 20 heated at 80 oC for 24 h. The reaction mixture was then partitioned between ethyl acetate and water, dried over anhydrous Na2SO4, filtered, concentrated and purified by column chromatography using silica gel (3 % MeOH/DCM) to give UH-15-38 (540 mg, 35 %) as a white Page 55 QB\166118.01517\95733313.1
solid. mp.206-208 oC. 1H NMR (600 MHz, CDCl3) ^ 8.47 (s, 1H), 7.72 (s, 1H), 7.26-7.23 (m, 4H), 7.13 (t, J = 7.5 Hz, 2H), 6.89 (s, 1H), 6.81 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 6 Hz, 1H), 6.66 (s, 1H), 3.50 (s, 3H), 3.26 (t, J = 4.5 Hz, 4H), 2.64 (t, J = 4.5 Hz, 4H), 2.39 (s, 3H).13C NMR (150 MHz, CDCl3) ^162.2, 157.0, 156.4, 152.5, 150.5, 146.6, 140.3, 137.8, 135.2, 130.3, 129.6, 128.5, 5 120.3, 116.2, 115.5, 112.9, 112.3, 111.4, 109.0, 89.0, 55.0, 48.7, 46.0, 29.6; HRMS (ESI): m/z [M + H]+ calcd for C26H28N5O2: 442.2238; found: 442.2241. Synthesis of UH-15-38•HCl UH-15-38 (500 mg, 1.13 mmol) was dissolved in 10 mL diethyl ether and then 2 mL methanol was added to speed up dissolution. 2.0 M HCl in diethyl ether (2.82 mL, 5.65 mmol) 10 was added to the mixture dropwise with constant and vigorous stirring at 0 ֯C. Stirring then continued at rt for 16 h. The precipitate was collected by filtration, washed with diethyl ether, and dried under vacuum to furnish UH15-38•HCl (450 mg, 83%) as a white solid; mp.245-250 °C; 1H-NMR (600 MHz, DMSO-d6) ^ 11.01 (bs, 1H), 10.10 (bs, 1H), 8.63 (s, 1H), 8.04 (s, 1H), 7.29 – 7.26 (m, 1H), 7.23 – 7.21 (m, 2H), 7.10 (s, 1H), 7.05 – 7.03 (m, 2H), 6.83 (s, 1H), 6.78 (d, J = 15 1.8 Hz, 2H), 3.84 – 3.81 (m, 2H), 3.54 (s, 3H), 3.49 – 3.46 (m, 2H), 3.19 – 3.12 (m, 4H), 2.81 (s, 3H); 13C-NMR (100 MHz, DMSO-d6) ^ 160.8, 157.0, 152.1, 150.6, 147.7, 143.0, 138.9, 137.0, 134.3, 130.2, 129.5, 129.0, 119.3, 115.7, 115.2, 113.1, 112.4, 110.6, 108.9, 92.5, 51.8, 45.3, 41.9, 29.5; HRMS (ESI): m/z [M + H]+ calcd for C26H28N5O2: 442.2238; found: 442.2236. Purity was determined to be 98% by analytical high-performance liquid chromatography (WATERS 2545 20 HPLC) using binary gradient pump Kinetex 5 ^m C18 100 Å column (250 x 4.6 mm). UV absorption was monitored at ^ = 254 nm. The injection volume was 100 ^L. The gradient of acetonitrile/water (water containing 0.1% formic acid) was 2:98 to 98:2 over a total run time of 30 min and a flow rate of 1.2 mL/min. tR = 14.44 min. 25 Synthesis of UH15-38Me Page 56 QB\166118.01517\95733313.1
Re hos, Cs2CO3 , dioxane, MW, 90 °C, 20 h. Synthesis of 7-Chloro-3-(3-methoxyphenyl)-1-methyl-1,6-naphthyridin-2(1H)-one (3b) 5 To a stirred solution of compound 1 (70 mg, 0.41 mmol), 2b (88 mg, 0.50 mmol) in DMA (2 mL) was added KF/Al2O3 (420 mg) at rt and the reaction mixture was stirred at rt for 2 h. After completion of the reaction, filter the reaction mixture through a small Celite pad and water (5 mL) was added to the filtrate. The organic layer was extracted with ethyl acetate (2 x 15 mL). The combined organic layers washed with brine solution, dried over anhydrous Na2SO4, and filtered. 10 The solvents were removed under reduced pressure and purification of the residue on combiflash chromatography (0-10% ethyl acetate in hexane) furnished the product 3b as a white solid. mp. 165-167 °C; yield: 83 mg (67%); 1H-NMR (600 MHz, CDCl3) ^ 8.61 (s, 1H), 7.81 (s, 1H), 7.37 (t, J = 8.3 Hz, 1H), 7.26 - 7.23 (m, 3H), 6.98 - 6.96 (m, 1H), 3.86 (s, 3H), 3.72 (s, 3H); 13C-NMR (151 MHz, CDCl3) ^ 161.1, 159.4, 151.8, 149.8, 145.9, 136.8, 133.9, 133.7, 129.3, 121.2, 115.8, 15 114.5, 114.3, 107.8, 55.3, 29.8. Synthesis of UH15-38Me To a stirred solution of compound 3b (75 mg, 0.25 mmol), 3-(4-methylpiperazin-1-yl) aniline 4 (48 mg, 0.25 mmol), Cs2CO3 (162 mg, 0.5 mmol), Xantphos (29 mg, 0.05 mmol), Pd2(dba)3 (23 mg, 0.03 mmol) in dioxane (2 mL) and flushed argon gas for 10 minutes and refluxed 20 the reaction mixture at 90 °C for 20 h. After completion of the reaction, the reaction mixture was filtered through a small Celite pad and water (2 mL) was added to the filtrate. The organic layer was extracted with ethyl acetate (2 x 15 mL). The combined organic layers washed with brine Page 57 QB\166118.01517\95733313.1
solution, dried over anhydrous Na2SO4, and filtered. The solvents were removed under reduced pressure and purification of the residue on combiflash chromatography (0 - 10% methanol in CH2Cl2) furnished UH15-38Me as a pale-yellow solid. mp. 70-72 °C; yield: 22 mg (19%). 1H- NMR (600 MHz, CDCl3) ^ 8.44 (s, 1H), 7.71 (s, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.31 - 7.23 (m, 2H), 5 6.93 - 6.90 (m, 2H), 6.88 - 6.84 (m, 2H), 6.77 - 6.74 (m, 1H), 6.69 (s, 1H), 3.86 (s, 3H), 3.58 (s, 3H), 3.26 (t, J = 4.7 Hz, 4H), 2.60 (t, J = 4.8 Hz, 4H), 2.37 (s, 3H); 13C-NMR (151 MHz, CDCl3) ^ 162.0, 159.3, 157.0, 152.5, 150.4, 146.5, 140.3, 137.9, 135.0, 130.2, 129.1, 128.7, 121.2, 114.2, 113.8, 112.5, 111.9, 111.2, 108.9, 88.9, 55.3, 55.0, 48.7, 46.1, 29.5. HRMS (ESI): m/z [M + H]+ calcd for C27H30N5O2: 456.2394, found: 456.2385. Purity was determined to be 98% by analytical 10 high-performance liquid chromatography (WATERS 1525 HPLC) using binary pump Kinetex 5 ^m C18100 Å column (250 x 4.6 mm). UV absorption was monitored at ^ = 254 nm. The injection volume was 15 ^L. The gradient of acetonitrile/water (both containing 0.1% trifluoroacetic acid) was 2:98 to 90:10 over a total run time of 30 min and a flow rate of 1 mL/min. tR = 17.365 min. 15 Pharmacokinetic and toxicological assessment of UH15-38. Pharmacokinetics of UH15-38 were evaluated in male (9-10 weeks old) C57Bl/6 mice following once-daily dosing at 30 mg/kg for four consecutive days. UH15-38 was administered by i.p. injection, and tissues were collected from three mice for each time point. Plasma was generated by standard centrifugation techniques and immediately frozen. Drug levels are determined by mass 20 spectrometry using an ABSciex 6500 mass spectrometer and multiple reaction monitoring analytical methods. Pharmacokinetic parameters were calculated using a non-compartmental model (Phoenix WinNonlin, Pharsight Inc.). UH15-38 concentrations in each tissue were modeled separately. All procedures were approved by the IACUC at the UF Scripps Institute for Biomedical Innovation & Technology. For toxicological assessment of UH15-38, three male and three female 25 C57Bl/6 mice were dosed i.p. for 7 days with UH15-38 at 30 mg/kg/day, and potential toxicity of the drug was assessed 24 h after the last dose. Comprehensive blood chemistry and blood cell composition analyses were carried out by IDEXX Bioanalytics. Effects of UH15-38 on whole- animal and organ weights were assessed by Tufts University Comparative Medicine Services (TUCMS), and histological examination of tissues was carried out by a certified veterinary Page 58 QB\166118.01517\95733313.1
pathologist (Dr. Lauren Richey, TUCMS). IHC assessment of cleaved caspase 3 was carried out by the Histology Facility at the Fox Chase Cancer Center. In vitro RIPK3 and RIPK1 kinase and in cellulo RIPK3 target engagement assays ADPGlo RIPK3 kinase assays utilizing hRIPK3 (1-313 aa) construct expressed in Sf9 cells 5 were performed as previously described22. NanoBRET target engagement assays were performed as described earlier32. Briefly, NanoLuc-RIPK3 (Promega) was transfected into HEK293T cells. After 24-48 hr, cell density was adjusted to 2 × 105 cells/ml, and cells were incubated with PBI- 6948 nanoBRET tracer (Promega) and various inhibitor concentrations for 2 h at 37°C. Next, NanoLuc substrate mix (Promega) was added, and BRET ratios were determined using a Victor3V 10 plate reader (PerkinElmer). Determination of the affinities of the drugs towards recombinant RIPK1 and RIPK3 kinase domains was performed by DiscoverX using the KinomeScan platform, which is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. An 11-point 3-fold serial dilution of UH15-38 compound was tested in duplicate. Binding constants (Kd) were calculated 15 with a standard dose-response curve using the Hill equation: Response = Background + (Signal – Background)/ (1 + (KdHill Slope / DoseHill Slope). The Hill Slope was set to -1. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm. Molecular docking Docking of UH15-38 into the RIPK3 kinase domain was performed using standard 20 protocol of AutoDock Tools1.5.6 (http://autodock.scripps.edu), which is computed with MGLTools-1.5.4 (http://mgltools.scripps.edu). UH15-38 was created through ChemDrawBio and subjected to energy minimization using MM2 force field. The coordinates of mouse and human RIPK3 were extracted from crystal structures of mouse RIPK3 (PDB: 4M66) and human RIPK3 (PDB: 7MX3) using BIOVIA Discovery Studio Visualizer 2016 software 25 (http://www.3dsbiovia.com). Autodock Tools were used and grid boxes for docking simulations were selected based on each binding site. The docking results were analyzed using PyMOL visualization software (version PyMOL™ 2.5.4) and UCSF Chimera 1.1549. When needed, hydrogens and charges were assigned to docking models in Chimera prior to assessment of contacts between drug and RIPK3. 2D analysis was performed using LigPlot+ Page 59 QB\166118.01517\95733313.1
(https://www.ebi.ac.uk/thornton-srv/software/LigPlus/)50 and identified hydrogen bonds and salt bridges were confirmed in Chimera. High resolution rendering of models was done with Chimera. Isolation of Type I AECs C57BL/6 mice were anesthetized with 3% isoflurane and cardiac perfusion carried out by 5 inserting a 23 G needle attached to a 10 ml syringe containing RPMI (supplemented with 25 mM HEPES, at 37oC) into the right ventricle. The trachea was then exposed, and a small incision was made on the ventral surface. A polyethylene tube (PE50, BD, 427410) was inserted through the incision and fixed inside the trachea by suture. Lungs were lavaged three times with PBS containing 5 mM EGTA and 5 mM EDTA and instilled with 3ml of RPMI containing 25 mM 10 HEPES, 10% Dextran40, and 4.5 U/ml Elastase, immediately followed by 0.5 ml of 1% (w/v) low melting temperature agarose in water. The trachea was tied below the incision point and the tube was removed. Mouse carcasses were allowed to cool for 5 minutes at 4oC, lungs were then excised and placed into 15 ml tubes with 2 ml of Wash Buffer (RPMI containing 25 mM HEPES, 10% Dextran40, and 4.5U/ml Elastase) and rocked for 45 min at 25oC (Roto-Therm Plus, Benchmark 15 Scientific, G0520075). After digestion, lungs were disintegrated in 6 cm plates containing 8 ml RPMI supplemented with 25 mM HEPES, 20% FBS, and 100 ^g/ml DNAse I for 10 min. Crude single cell fractions were then serially passed through 100 ^m, and then through 40 ^m cell strainers. Single cells were centrifuged (250g for 8 min), rinsed in Wash Buffer, and incubated with 40 ^g/ml rabbit polyclonal anti-Aqp5 antibody at 4oC in a rotator for 40 min. After washing20 (3x) and resuspension in Wash Buffer, cells were incubated with 400 ^l pre-washed goat anti- rabbit IgG BioMag® beads and rotated at 4oC for 40 min. Antibody-bound cells were precipitated using a magnetic field, and any unbound cells were removed by washing in Wash Buffer. Antibody-bound cells were washed and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, 1 mM sodium pyruvate, 1x GlutaMAX (Thermo Fisher Scientific) and 1% 25 penicillin/streptomycin. The purity of Type I AEC isolated in this manner was assessed by staining for podoplanin with an anti-podoplanin antibody (1:500), and for the presence of contaminating fibroblasts using an antibody to CD140a (PDGFR^) antibody (1:500). Single-cell gene expression data and analysis Page 60 QB\166118.01517\95733313.1
Single-cell gene expression analyses were performed on datasets of mouse lung cells isolated pre-infection and 1, 3, and 6 days after infection51. Data were processed as previously described51. Briefly, sequencing data were processed using CellRanger (v.3.0.2, 10x Genomics) with the mouse mm10 reference genome adapted to include the influenza A/Puerto Rico/8/1934 5 genome. Feature-barcode matrices were then loaded into Seurat (v.3.0.0.900) using a scaling factor of 1x104 for subsequent analysis and data visualization52. Log-normalization of gene-expression counts was performed, and variable genes were identified using the ‘vst’ method with default parameters. Next, gene expression was scaled to regress out any effects of total number of transcripts, cell cycle scores, and percent mitochondrial expression. Based on an elbow plot of PC 10 standard deviations, 25 principal components (PCs) were selected for t-SNE dimensionality reduction and clustering using shared-nearest-neighbor modularity algorithm in Seurat. Marker gene expression was analyzed to identify major lung cell types, including epithelial (Epcam), endothelial (Pecam1), mesenchymal (Col1a2), and immune (Ptprc). Data were then subset on non- immune cells and dimensionality reduction using t-SNE and clustering was performed. 15 Immunoblotting Cells were lysed in RIPA lysis buffer (Thermo Scientific, 89900) containing protease and phosphatase inhibitors (Thermo Scientific, 815-968-0747). SDS sample buffer was added, and the samples were boiled for 10 min. Protein samples were then loaded onto 12-15% SDS-PAGE gels and proteins transferred overnight at 4oC onto PVDF membranes. The membranes were then 20 blocked in PBS containing 0.1% Tween-20 and 3% BSA, and treated with primary antibodies overnight at 4oC, followed by HRP-conjugated goat secondary antibodies at room temperature for 4 h. Membranes were washed and visualized following brief exposure to ECL substrate (Thermo Fisher, 34578). The following primary antibodies were used for immunoblotting: Mouse monoclonal anti-ZBP1 (1:2000), Rabbit polyclonal anti-ZBP1 (1:1000), Rabbit polyclonal anti- 25 RIPK3 (1:2000), Rabbit monoclonal anti-mouse (phospho T231/S232) RIPK3 (1:1000), Rabbit monoclonal anti-human RIPK3 (1:2000), Rabbit monoclonal anti-human (phospho S227) RIPK3 (1:1000), Rabbit monoclonal anti-mouse (phospho-S345) MLKL (1:2000),Rabbit monoclonal anti-human (phospho-S358) MLKL (1:2000), Rat monoclonal anti-MLKL (1:1000), Rabbit polyclonal anti-mouse MLKL (1;1000), Rabbit monoclonal anti-human MLKL (1:1000), Mouse30 monoclonal anti-RIP1 (1:1000 ) Rabbit monoclonal anti-FADD (1:1000), Rabbit polyclonal anti- Page 61 QB\166118.01517\95733313.1
human FADD (1:1000), Mouse monoclonal anti-human caspase 8 (1:1000), Rabbit polyclonal anti-mouse caspase 8 (1:1000), Rabbit monoclonal anti-mouse cleaved caspase-8 (1:1000), Rabbit polyclonal anti-caspase-3 (1:1000), Rabbit polyclonal anti-influenza A virus NP (1:5000), Mouse monoclonal anti-influenza A virus NS1 (1:2000), Mouse monoclonal anti-influenza B 5 Nucleoprotein (1:2500), Rabbit polyclonal anti-GSDMD (1:1000), Mouse monoclonal anti-^- actin ((1:2000)), Mouse monoclonal GAPDH (1:2000), Rabbit polyclonal anti-alpha tubulin (1:5000). Following secondary antibodies were used: Peroxidase-conjugated Affinity purified Goat anti-mouse IgG (H+L) (1:5000), Peroxidase-conjugated Affinity purified Goat anti-rat IgG (H+L) (1:2500), Peroxidase-conjugated Affinity purified Goat anti-rabbit IgG (H+L) (1:10000). 10 Immunofluorescence microscopy Cells were fixed with freshly prepared 4% (w/v) paraformaldehyde, permeabilized in 0.2% (v/v) Triton X-100, blocked with MAXblock™ Blocking Medium (Active Motif), and incubated overnight with primary antibodies at 4°C. After three washes in PBS, slides were incubated with fluorophore-conjugated secondary antibodies for 1 h at room temperature. Following an additional 15 three washes in PBS, slides were mounted in ProLong Gold antifade reagent (Thermo Fisher Scientific) and imaged by confocal microscopy on a Leica SP8 instrument. Fluorescence intensity was quantified using Leica LAS X software. The following antibodies were used for immunofluorescence studies: anti-phosphorylated murine MLKL53 (1:1000), rabbit polyclonal anti-cleaved caspase 3 (1:500) and anti-podoplanin (1:500), Donkey anti-Rabbit IgG (H+L) Highly 20 Cross-Adsorbed Secondary Antibody (1:500), Alexa Fluor™ 594, Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (1:500), Alexa Fluor™ 488, Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody (1:500), Alexa Fluor™ 488, Donkey anti- Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 594 A32744 (1:500). 25 In vivo IAV studies Age- (8-12 week-old) and sex-matched mice were anesthetized with Avertin (2,2,2- tribromoethanol) or 3% isoflurane and infected intranasally with virus inoculum diluted in endotoxin-free saline. UH15-38 dissolved in DMSO was diluted to the desired concentration in carrier solvent containing 10% Solutol HS (MedChemExpress, HY-Y1893) in normal saline for 30 in vivo use. Mice were treated i.p. with UH15-38 or vehicle (DMSO plus an equivalent volume of Page 62 QB\166118.01517\95733313.1
carrier solvent) in 400 ^L. Treatment groups that did not require drug treatment on a particular day(s), were administered vehicle. Mice were either monitored for survival over a period of 21 days or sacrificed at defined time points for analysis of histology and virus replication. Mice losing >30% body weight were considered moribund and euthanized by gradual CO2 asphyxiation. 5 Titration of virus was conducted by standard plaque assay of diluted lung homogenates on monolayers of MDCK cells, and plaques were scored after three days of incubation. IAV-specific CD8+ T cell responses were assessed on cells isolated from bronchoalveolar lavage (BAL) fluid collected 9 days after infection. BAL washes were performed using 2 mL of sterile 1x DPBS. BAL washes were centrifuged at 500 x g for 5 min before performing red blood cell lysis on cell pellets. 10 To analyze the frequency of IAV-specific CD8+ T cells, peptide:MHC tetramer staining was performed on 1x106 total BAL cells per sample. Cells were incubated with PE-PB1 (residues 703 – 711) and APC-PA (residues 224-233) tetramers at a dilution of 1:750 for each for 1 h on ice. Following incubation with tetramers, cells were washed with FACS buffer (1% FBS and 1 mM EDTA in 1x DPBS) and then incubated with Trustain FcX anti-mouse CD16/CD32 (Biolegend) 15 (1:100) for 10 min at room temperature. Cells were then stained for surface markers with a cocktail of the following antibodies and viability dye for 30 min on ice: Ghost Dye Violet 510 (1:100), APCFire750 anti-mouse CD45 (1:100), PE-Cy7 anti-mouse CD19 (1:100), BV421 anti-mouse CD3 (1:100), BV650 anti-mouse CD8a (1:200), and FITC anti-mouse CD4 (1:200). To analyze the frequency of cytokine producing CD8+ T cells, intracellular cytokine staining was performed 20 following stimulation with IAV peptides. For each condition, 5x105 cells from each sample were plated in a 96-well U-bottom plate. Cells were incubated with either PB1 (residues 703 – 711) or PA (residues 224-233) peptides at a concentration of 1 ^M in complete RPMI in the presence of Protein Transport Inhibitor Cocktail, containing Monensin and Brefeldin A, (eBioscience, 1:500) for 4 h at 37 degrees C. As positive control, cells were incubated with Cell Stimulation Cocktail 25 plus protein transport inhibitors, containing phorbol 12-myristate 13-acetate (PMA), Monensin, and Brefeldin A (eBioscience, 1:500). Following stimulation, cells were washed with FACS buffer and then incubated with Trustain FcX anti-mouse CD16/CD32 (Biolegend) (1:100) for 10 min at room temperature. Surface staining was then performed by incubating with a cocktail of the following antibodies for 30 min at room temperature: Ghost Dye Violet 510 (1:100), APCFire750 30 anti-mouse CD45 (1:100), PE anti-mouse CD3 (1:100), APC anti-mouse CD8a (1:200), and FITC anti-mouse CD4 (1:200). Intracellular staining was then performed by fixing and permeabilizing Page 63 QB\166118.01517\95733313.1
cells in 100 ^L of fix-perm solution (BD Biosciences) for 20 min on ice. The cells were then washed with permeabilization buffer and incubated with BV650 anti-mouse IFN^ (1:100) for 30 min on ice. For flow cytometry, all samples were analyzed on an LSR Fortessa (BD Biosciences) using BD FACSDIVA Software version 8.0.1. 5 Histological assessment of IAV-infected tissues For assessment of phosphorylated MLKL (pMLKL) in human pulmonary tissues, frozen human lung slices were purchased from the Institute for In Vitro Sciences Inc, and cultured per instructions provided by the supplier. Following infection of these sections with IAV, immunohistochemical staining of pMLKL was performed on a Ventana Discovery XT automated 10 staining instrument (Ventana Medical Systems) using Ventana reagents, according to the manufacturer's instructions. Slides were de-paraffinized using EZ Prep solution (950–102) for 16 min at 72 °C. Epitope retrieval was accomplished with CC1 solution (950–224) at 95–100 °C for 30 min. Rabbit primary antibody to human pMLKL (1:20) dilution) and goat polyclonal antibody to Influenza A (1:1000) were titered with a TBS antibody diluent into user fillable 15 dispensers for use on the automated stainer. Immune complexes were detected using the Ventana OmniMap anti-Rabbit detection kit (760-4311) and developed using the Ventana ChromMap DAB detection kit (760-159) per to the manufacturer’s instructions. Slides were then counterstained with hematoxylin II (790-2208) for 8 min, followed by Bluing reagent (760-2037) for 4 min. The slides were then dehydrated with ethanol series, cleared in xylene, and mounted. As a negative control, 20 the primary antibody was replaced with normal rabbit IgG to confirm absence of specific staining. Immunostained slides were scanned using a Leica Aperio ScanScope CS 5 slide scanner (Aperio, Vista, CA, USA). Scanned images were then viewed and captured with Leica Aperio's image viewer software (ImageScope, version 11.1.2.760, Leica Aperio). The pMLKL signal was quantified using the Aperio Positive Pixel Count algorithm. 25 To examine IAV spread and pathology in murine pulmonary tissues, lungs were first inflated with 1 ml of 10% neutral-buffered formalin, removed, and placed in 15 ml conical tube containing 3 ml of formalin and incubated at room temperature for 24 h to ensure complete fixation. Then lungs were embedded in paraffin blocks and sectioned onto glass slides. These lung sections were stained with hematoxylin and eosin for histological analyses. Immunohistochemical 30 labelling of viral antigen was performed with goat polyclonal antibody to influenza A (1:1,000) Page 64 QB\166118.01517\95733313.1
and a secondary biotinylated donkey anti-goat antibody (Santa Cruz Biotechnology; 1:200) on tissue sections subjected to antigen retrieval for 30 min at 98°C. The extent of virus spread was quantified by first capturing digital images of whole-lung sections stained for viral antigen by using an Aperio ScanScope XT Slide Scanner (Aperio Technologies), then manually outlining 5 fields with the alveolar areas containing virus antigen–positive pneumocytes highlighted in red (defined as “active” infection), whereas lesioned areas containing no antigen-positive cells and no/minimal antigen-positive debris were highlighted in yellow (defined as “inactive” infection). The percentage of each lung field with infection/lesion was calculated using the Aperio ImageScope software (version 12.3.3). 10 For the measure of extent of neutrophil infiltration, lungs were first embedded in paraffin, sectioned at 4 μm, mounted on positively charged glass slides (Superfrost Plus; Thermo Fisher Scientific, Waltham, MA), and dried at 60°C for 20 min. For detection of neutrophils, antigen retrieval at 100°C for 20 min in Epitope Retrieval solution 2 (ER2) was performed on a Bond Max immunostainer (Leica Biosystems, Buffalo Grove, IL). A rat monoclonal primary antibody to Ly- 15 6B.2 (1:30,000) was applied for 15 minutes followed by biotinylated secondary rabbit anti-rat antibody 1:400 (Vector Laboratories) for 10 minutes and then ready-to-use BOND DAB Enhancer (AR9432; Leica Biosystems) and a hematoxylin counterstain. Slides were scanned and digital whole lung images were evaluated using the DenseNet algorithm of the image analysis platform HALO (version 3.1; Albuquerque, NM). Alveoli in the lung images were classified as being either 20 normal or infiltrated with neutrophil exudates and subjected to deep learning (DL) until convergence to a cross-entropy that was <0.1. Lungs were then analyzed by the trained model, and pathologists visually confirmed the results. In the cases in which the auto-recognized regions were misrecognized, the regions were corrected or appropriately annotated, and then the DL was repeated to construct the trained model for the automated recognition. To improve the recognition 25 accuracy, the cycle was repeated until the number of misrecognized regions was reduced to an absolute minimum, or until the analysis results did not change even when further DL was performed. The fraction of lung volume that was occupied by neutrophil infiltrates was determined by quantitative morphometry using the HALO™ Area Quantification v2.1.11 algorithm (IndicaLabs, NM, USA). 30 For detection of Tenascin C, antigen retrieval at 100°C for 32 min was completed in Cell Conditioning 1 (CC1) buffer (Ventana Medical Systems, 950-500) on a Discovery Ultra Page 65 QB\166118.01517\95733313.1
immunostainer (Ventana Medical Systems). A rabbit monoclonal primary antibody to Tenascin C diluted at 1:100 (Abcam, ab108930) was applied for 32 min at 37°C. Secondary goat anti-rabbit polyclonal antibodies labeled with a proprietary hapten nitropyrazole (Ventana Medical Systems, 760-4311) were applied for 16 min at RT and then developed with ChromoMap DAB (3,3′- 5 Diaminobenzidine tetrahydrochloride) chromogen, and then counterstained with hematoxylin (all from Ventana Medical Systems). The extent and severity of pulmonary inflammation were determined from sections examined in a blinded fashion by a pathologist and scored on a severity scale as follows: 0 = no lesions; 1 = minimal, focal to multifocal, barely detectable; 15 = mild, multifocal, small but conspicuous; 40 = moderate, multifocal, prominent; 80 = marked, multifocal 10 coalescing, lobar; 100 = severe, diffuse, with extensive disruption of normal architecture and function. These scores were converted to a semiquantitative scale as described previously54. Cytokine and chemokine assessment in BALF For cytokine profile studies, BALF was collected using 1 ml of sterile phosphate-buffered saline (calcium-magnesium free). Concentrations of analytes were determined using the 15 ProcartaPlex Mouse Immune Monitoring Panel 48-Plex kit (ThermoFisher, EPX480-20834-901). Briefly, samples were centrifuged at 1400 rpm for 10 min at 4oC. Prepared standards, high and low controls, diluent only controls, and clarified supernatants were added to plates containing capture beads, according to manufacturer’s instructions. Plates were shaken at 600 rpm overnight at 4oC, then warmed to room temperature for an additional 30 min with shaking. The remaining 20 steps were performed according to manufacturer’s instructions. Plates were run on a Luminex 200 instrument (Luminex Corp), and data were analyzed using 5PL logistic fit in ProcartaPlex Analyst 1.0 software (ThermoFisher). Pulse oximetry and lung function studies For pulse oximetry studies, mice were infected with PR8 (4,500 EID50, i.n.). At day 3 and 25 day 6 after infection, the fur around the neck of the mouse was removed and arterial oxygen saturation (SpO2) was measured with a throat collar sensor using the MouseOx system (STARR Life Sciences). To assess airway resistance, mice were infected with PR8 (2,500 EID50, i.n.). At day 10 post infection, a tracheal tube was inserted into anesthetized mice, and airway resistance was measured on a Buxco Finepointe Resistance and Compliance instrument. Data were collected 30 using Buxco Finepointe software version 2.6 (Data Sciences International). Two methacholine Page 66 QB\166118.01517\95733313.1
challenges were performed at doses of 1.5625 and 3.125 mg/ml, administering the methacholine in 0.010 ml PBS. Following each methacholine challenge, airway resistance was measured for 3 min, allowing the mice to recover for 1 min after each measurement. Measurements for airway resistance were averaged over the 3 min measurement period for each methacholine challenge. 5 Statistics Statistical significance was determined by use of multiple unpaired Student’s t tests or the Mann-Whitney U test for comparison between two groups, or one-way ANOVA followed by Tukey's test or the two-way ANOVA, for comparisons between multiple (>2) groups. Significance of in vivo survival data was determined by the log-rank (Mantel-Cox) test. P-values of 0.05 or 10 lower were considered significant. Graphs were generated using Graph Pad Prism 9.5.0 software. References 1 Kalil, A. C. & Thomas, P. G. Influenza virus-related critical illness: pathophysiology and epidemiology. Crit Care 23, 258 (2019). 2 Korteweg, C. & Gu, J. Pathology, molecular biology, and pathogenesis of avian 15 influenza A (H5N1) infection in humans. Am J Pathol 172, 1155-1170 (2008). 3 Mauad, T. et al. Lung pathology in fatal novel human influenza A (H1N1) infection. Am J Respir Crit Care Med 181, 72-79 (2010). 4 Kash, J. C. et al. Genomic analysis of increased host immune and cell death responses induced by 1918 influenza virus. Nature 443, 578-581 (2006). 20 5 Flerlage, T., Boyd, D. F., Meliopoulos, V., Thomas, P. G. & Schultz-Cherry, S. Influenza virus and SARS-CoV-2: pathogenesis and host responses in the respiratory tract. Nat Rev Microbiol (2021). 6 Nogusa, S. et al. RIPK3 Activates Parallel Pathways of MLKL-Driven Necroptosis and FADD-Mediated Apoptosis to Protect against Influenza A Virus. Cell Host Microbe 20, 13- 25 24 (2016). 7 Rodrigue-Gervais, I. G. et al. Cellular inhibitor of apoptosis protein cIAP2 protects against pulmonary tissue necrosis during influenza virus infection to promote host survival. Cell Host Microbe 15, 23-35 (2014). 8 Zhang, T. et al. Influenza Virus Z-RNAs Induce ZBP1-Mediated Necroptosis. Cell 30 180, 1115-1129 e1113 (2020). Page 67 QB\166118.01517\95733313.1
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23 Fauster, A. et al. A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis. Cell Death Dis 6, e1767 (2015). 24 Li, J. X. et al. The B-Raf(V600E) inhibitor dabrafenib selectively inhibits RIP3 and alleviates acetaminophen-induced liver injury. Cell Death Dis 5, e1278 (2014). 5 25 Wissing, J. et al. Chemical proteomic analysis reveals alternative modes of action for pyrido[2,3-d]pyrimidine kinase inhibitors. Mol Cell Proteomics 3, 1181-1193 (2004). 26 Meng, Y. et al. Human RIPK3 maintains MLKL in an inactive conformation prior to cell death by necroptosis. Nat Commun 12, 6783 (2021). 27 Sarhan, J. et al. Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis 10 during Yersinia infection. Proc Natl Acad Sci U S A 115, E10888-E10897 (2018). 28 Rodriguez, D. A. et al. Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis. Proc Natl Acad Sci U S A 119, e2207240119 (2022). 29 Chen, J., Chen, Z., Narasaraju, T., Jin, N. & Liu, L. Isolation of highly pure alveolar epithelial type I and type II cells from rat lungs. Lab Invest 84, 727-735 (2004). 15 30 Wang, S. & Hubmayr, R. D. Type I alveolar epithelial phenotype in primary culture. Am J Respir Cell Mol Biol 44, 692-699 (2011). 31 Rishi, A. K. et al. Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal and neural derivatives. Dev Biol 167, 294- 306 (1995). 20 32 Hrdinka, M. et al. Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling. EMBO J 37 (2018). 33 Tan, Y. et al. Somatic Epigenetic Silencing of RIPK3 Inactivates Necroptosis and Contributes to Chemoresistance in Malignant Mesothelioma. Clin Cancer Res 27, 1200-1213 (2021). 25 34 Xie, T. et al. Structural insights into RIP3-mediated necroptotic signaling. Cell Rep 5, 70-78 (2013). 35 Chen, W. et al. Diverse sequence determinants control human and mouse receptor interacting protein 3 (RIP3) and mixed lineage kinase domain-like (MLKL) interaction in necroptotic signaling. J Biol Chem 288, 16247-16261 (2013). 30 36 Bantia, S. et al. Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir. Antimicrob Agents Chemother 45, 1162-1167 (2001). Page 69 QB\166118.01517\95733313.1
37 Groeneveld, G. H. et al. Effectiveness of oseltamivir in reduction of complications and 30-day mortality in severe seasonal influenza infection. Int J Antimicrob Agents 56, 106155 (2020). 38 Zuliani-Alvarez, L. & Piccinini, A. M. A virological view of tenascin-C in 5 infection. Am J Physiol Cell Physiol 324, C1-C9 (2023). 39 Pillay, J. et al. A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. J Clin Invest 122, 327-336 (2012). 40 Tak, T. et al. Neutrophil-mediated Suppression of Influenza-induced Pathology Requires CD11b/CD18 (MAC-1). Am J Respir Cell Mol Biol 58, 492-499 (2018). 10 41 Nikhar, S. et al. Design of pyrido[2,3-d]pyrimidin-7-one inhibitors of receptor interacting protein kinase-2 (RIPK2) and nucleotide-binding oligomerization domain (NOD) cell signaling. Eur J Med Chem 215, 113252 (2021). 42 Suebsuwong, C. et al. Activation loop targeting strategy for design of receptor- interacting protein kinase 2 (RIPK2) inhibitors. Bioorg Med Chem Lett 28, 577-583 (2018).15 43 Muraro, S. P. et al. Respiratory Syncytial Virus induces the classical ROS- dependent NETosis through PAD-4 and necroptosis pathways activation. Sci Rep 8, 14166 (2018). 44 Wang, L. et al. Necroptosis in Pulmonary Diseases: A New Therapeutic Target. Front Pharmacol 12, 737129 (2021). 45 Ishii, K. J. et al. TANK-binding kinase-1 delineates innate and adaptive immune 20 responses to DNA vaccines. Nature 451, 725-729 (2008). 46 Murphy, J. M. et al. The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism. Immunity 39, 443-453 (2013). 47 Newton, K., Sun, X. & Dixit, V. M. Kinase RIP3 is dispensable for normal NF- kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll- 25 like receptors 2 and 4. Mol Cell Biol 24, 1464-1469 (2004). 48 Berger, S. B. et al. Cutting Edge: RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. J Immunol 192, 5476-5480 (2014). 49 Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory 30 research and analysis. J Comput Chem 25, 1605-1612 (2004). Page 70 QB\166118.01517\95733313.1
50 Laskowski, R. A. & Swindells, M. B. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model 51, 2778-2786 (2011). 51 Boyd, D. F. et al. Exuberant fibroblast activity compromises lung function via ADAMTS4. Nature 587, 466-471 (2020). 5 52 Stuart, T. et al. Comprehensive Integration of Single-Cell Data. Cell 177, 1888- 1902 e1821 (2019). 53 Rodriguez, D. A. et al. Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis. Cell Death Differ 23, 76-88 (2016). 54 Matute-Bello, G. et al. An official American Thoracic Society workshop report: 10 features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol 44, 725-738 (2011). Example 2 15 Experimental Procedures 1. General Information All reactions involving air-sensitive reagents were carried out with magnetic stirring and oven-dried glassware with rubber septa under argon unless otherwise stated. All commercially available chemicals and reagent-grade solvents were used directly without further purification 20 unless otherwise specified. Reactions were monitored by thin-layer chromatography (TLC) on Baker-flex® silica gel plates (IB2-F) using UV-light (254 and 365 nm) detection or visualizing agents (e.g. iodine, ninhydrin, or phosphomolybdic acid stain). Flash chromatography was conducted on a silica gel (230-400 mesh) using a Teledyne ISCO CombiFalsh® Rf. NMR spectra were recorded at room temperature using a JEOL ECA-600 instrument (1H NMR at 600 MHz and 25 13C NMR at 151 MHz) with tetramethylsilane (TMS) as an internal standard. Chemical shifts (δ) are given in parts per million (ppm) with reference to solvent signals [1H-NMR: CDCl3 (7.26 ppm), DMSO-d6 (2.50 ppm), CD3CN (1.94 ppm); 13C-NMR: CDCl3 (77.0 ppm), DMSO-d6 (39.5 ppm), CD3CN (1.32 ppm)]. Signal patterns are reported as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad). Coupling constants (J) are given in Hz. High-resolution mass spectra 30 (HRMS) were obtained by the University of Texas, Austin mass spectral facility using a Qstar Elite-ESI and reported as m/z (relative intensity) for the molecular ion [M]. Page 71 QB\166118.01517\95733313.1
Ethyl 6-Chloro-4-(methylamino) nicotinate (2): Compound 1 (0.03 mol) was dissolved in acetonitrile (80 mL). The solution was cooled to 0 °C, and methylamine (40% water solution, 18.00 mL) was slowly added. The reaction was stirred at 0 °C for 20 min and then warmed to room temperature for 3 h. The solvent was removed under reduced pressure, and the crude product was 5 purified by silica gel chromatography (20-100% EtOAc in hexane) to provide 2 (96% yield) as a white powder. (6-chloro-4-(methylamino) pyridin-3-yl) methanol (3): A mixture of LAH (51.5 mmol) in anhydrous THF (50 mL) at –78°C was added slowly dropwise compound 1 (24 mmol) dissolved in anhydrous THF (45 mL) at -78 oC. The reaction mixture was stirred for 3h and then gradually 10 warmed to room temperature. A saturated solution of ammonium chloride was slowly added to quench LAH. The resulting mixture was filtered through Celite, and the filtrate was evaporated to dryness to get compound 3 as a white solid powder. The crude mixture was used for further step reaction. 6-Chloro-4-(methylamino)pyridine-3-carbaldehyde (4): The crude alcohol was 15 dissolved in DCM (100 mL), and MnO2 (200 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. After completion, the reaction mixture was filtered through Celite to remove the MnO2. The collected filtrate was concentrated under reduced pressure, and the crude mixture was purified by silica gel chromatography (30-100% EtOAc in hexane) to provide 4 (75% yield) as a white solid powder. 20 7-Chloro-1-methyl-2-oxo-1,2-dihydro-1,6-naphthyridine-3-carboxylic acid (5): To a stirred solution of compound 4 (1.17 mmol) in EtOH (5mL) were added Meldrum’s acid (1.3 mmol), piperidine (0.2 mmol), and acetic acid (0.4 mmol). The reaction mixture was stirred at room temperature for 20 mins and then heated at reflux for 2.5h. After completion, the reaction mixture was cooled to room temperature, and the precipitates formed were filtered, washed with 25 EtOH (10ml), and dried in vacuo to give 5 (90%) as a yellow solid powder. 7-chloro-3-iodo-1-methyl-1,6-naphthyridin-2(1H)-one (6): In a microwave vial (30mL), was added a solution compound 5(0.45 mmol) in a mixture of DMF/water (9:1, 6 mL) followed by addition of NIS (1.45 mmol) and LiOAc (0.55 mmol). The vial was sealed and heated in a microwave at a temperature of 110°C for 15 minutes. Once the reaction was completed, the 30 reaction mixture was cooled down, water was added, and the mixture was extracted with DCM. The organic layer was washed with brine, dried with Na2SO4, filtered, and then concentrated under Page 72 QB\166118.01517\95733313.1
reduced pressure. The crude mixture was purified using combi flash chromatography (30-100% EtOAc in hexane) to provide compound 6 as a light-yellow solid powder. 5-(4,4,5-trimethyl-1,3,2-dioxaborolan-2-yl) benzo[d]thiazole (9): To a sealed tube (20ml) was added compound 7 (1.41 mmol) in dioxane, followed by the addition of compound 8 5 (1.5 mmol), potassium acetate (4.98 mmol), and Pd (dppf)Cl2.DCM (0.14 mmol). The mixture was purged with argon, tube was sealed and heated at 110 oC for 16h. After completion, water was added, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by combiflash chromatography (10−100% EtOAc in hexane) to provide compound 10 as a brown 10 solid powder. Procedure for preparation of compound 10: To a vessel (25 mL) was added compound 6 (0.46 mmol) in 1,4-dioxane/water (9:1, 10 mL), the boron reagent 9 (0.46 mmol), Na2CO3 (1.17 mmol), and Pd (PPh3)4 (0.02 mmol). The tube was sealed, and the reaction mixture was heated at 90 °C for 16 h. After cooling, water was 15 added, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by combiflash chromatography (50−100% EtOAc in hexane) to provide compound 10 as a white solid powder. Procedure for preparation of compound 12a-12f: 20 To a vessel (10 mL) were added compound 10 (0.13 mmol) in 1,4-dioxane (4 mL), compound 11 (0.12 mmol), Cs2CO3 (0.27 mmol), Xanthphos (0.02 mmol) and Pd2(dba)3 (0.01 mmol). The tube was sealed, and the reaction mixture was heated under microwave irradiation at 110 °C for 40 min. After cooling, water was added, and the mixture was extracted with DCM. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated under reduced 25 pressure. The crude product was purified by combiflash chromatography (0−10% methanol in CH2Cl2) to provide compounds 12a-12f. Scheme 1 shows the overall synthetic scheme for the preparation of 12a-12f. Page 73 QB\166118.01517\95733313.1
Scheme 1. Reagents and conditions: (a) MeNH2, ACN, rt, 3h; (b) LAH, THF, -78 oC 3h; (c) MnO2, DCM: THF, rt, 24h; (d) Meldrum’s acid, Piperdine, Acetic acid, Ethanol, 80 oC, 2h; (e) NIS, LiOAc, DMF: water, MW, 110 oC, 15mins; (f) Pd(dpff)Cl2.DCM, AcOK, Dioxane, 100 oC, 16h; (g) Pd(PPh3)4, Na2CO3, Dioxane: water, 90 °C, 16 h; (h) Pd2(dba)3, Cs2CO3, Xantphos, 5 Dioxane, MW, 110 °C, 60 mins. O O O N O a N O b N OH c N H d S N     Compound R1 R2 Page 74 QB\166118.01517\95733313.1
3-(Benzo[d]thiazol-5-yl)-1-methyl-7-((3-(4-methylpiperazin-1-yl)phenyl)amino)-1,6- naphthyridin-2(1H)-one (BR-2-39F; 12a): 5 (s, Hz,
1H), 6.92 (s, 1H), 6.87 (d, J = 7.9 Hz, 1H), 6.83 (s, 1H), 6.76 (dd, J = 8.3, 1.7 Hz, 1H), 6.70 (s, 1H), 3.61 (s, 3H), 3.27 (t, J = 4.6 Hz, 4H), 2.60 (s, 4H), 2.37 (s, 3H); 13C-NMR (151 MHz, CDCl3- D) 13C-NMR (151 MHz, CDCl3) δ 162.1, 157.1, 154.3, 153.5, 152.5, 150.6, 146.7, 140.2, 135.5, 135.2, 133.2, 130.2, 128.3, 126.7, 123.6, 121.3, 112.6, 112.0, 111.3, 109.0, 88.9, 55.0, 48.7, 46.1, 5 29.5; HRMS (+ESI) m/z: [M + H]+ calcd for C27H26N6OS: 483.1962, found: 483.1958. 3-(benzo[d]thiazol-5-yl)-1-methyl-7-((3-(1-methylpiperidin-4-yl)phenyl)amino)-1,6- naphthyridin-2(1H)-one (MB-2-03; 12b): S N 10 Light yellow solid; mp.193-195 oC; yield: 10 mg (20%); 1H-NMR (600 MHz, DMSO-D6) δ 9.44 (d, J = 4.8 Hz, 2H), 8.63 (s, 1H), 8.42 (s, 1H), 8.21-8.17 (m, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.55- 7.51 (m, 2H), 7.23 (t, J = 7.8 Hz, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.70 (s, 1H), 3.58 (s, 3H), 2.88 (d, J = 10.7 Hz, 2H), 2.43 (s, 1H), 2.22 (s, 3H), 2.01-1.98 (m, 2H), 1.76 (d, J = 12.1 Hz, 2H), 1.69- 15 1.66 (m, 2H); 13C-NMR (151 MHz, DMSO-D6) δ 161.7, 157.3, 157.1, 153.7, 150.8, 147.4, 146.1, 141.5, 136.6, 135.5, 133.2, 129.3, 126.8, 123.4, 122.3, 120.6, 117.8, 117.3, 111.2, 91.6, 56.3, 46.7, 41.9, 33.5, 29.6; HRMS (+ESI) m/z: [M + H]+ calcd for C28H27N5OS: 482.2009, found: 482.2006. 20 3-(benzo[d]thiazol-5-yl)-1-methyl-7-((4-(1-methylpiperidin-4-yl)phenyl)amino)-1,6- naphthyridin-2(1H)-one (MB-1-158; 12f): 25 76
Light yellow solid; mp.193-195 oC; yield: 8 mg (18%); 1H-NMR (600 MHz, DMSO-D6) δ 9.42 (d, J = 6.0 Hz, 2H), 8.60 (s, 1H), 8.42 (s, 1H), 8.19 (t, J = 8.2 Hz, 2H), 7.81 (d, J = 8.4 Hz, 1H), 7.58 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 6.67 (s, 1H), 3.58 (s, 3H), 2.86 (d, J = 10.8 Hz, 2H), 2.41-2.39 (m, 1H), 2.19 (s, 3H), 1.96-1.93 (m, 2H), 1.73-1.63 (m, 4H); 13C NMR (151 MHz, 5 DMSO-D6) δ 161.7, 157.3, 157.1, 153.7, 150.8, 146.1, 140.0, 139.4, 136.7, 135.5, 133.2, 127.3, 126.6, 123.5, 123.3, 122.4, 119.8, 111.1, 91.4, 56.8, 46.8, 41.2, 33.7, 29.6. HRMS (+ESI) m/z: [M + H]+ calcd for C28H27N5OS: 482.2009, found: 482.2002. 3-(benzo[d]thiazol-5-yl)-1-methyl-7-((3-((4-methylpiperazin-1-yl)methyl)phenyl)amino)- 10 1,6-naphthyridin-2(1H)-one (MB-2-09; 12d): Light yellow solid; mp.190-192 oC ; yield: 5 mg (10%); 1H-NMR (600 MHz, MeOH-D4) δ 9.27 15 (s, 1H), 8.54 (s, 1H), 8.38 (s, 1H), 8.10 (d, J = 8.4 Hz, 1H), 8.05 (s, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.31-7.28 (m, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.71 (s, 1H), 3.66 (s, 3H), 3.57 (s, 2H), 2.88-2.61 (m, 8H), 2.37 (s, 3H); 13C-NMR (151 MHz, MeOH-D4) δ 162.5, 157.3,156.2, 152.8, 150.2, 150.1, 146.2, 140.8, 140.73, 137.7, 135.4, 133.0, 129.4, 127.3, 126.7, 122.9, 122.6, 118.0, 116.0, 111.4, 90.7, 62.6, 54.8, 53.0, 46.1, 29.6. 20 3-(benzo[d]thiazol-5-yl)-7-((3-(4-isopropylpiperazin-1-yl)phenyl)amino)-1-methyl-1,6- naphthyridin-2(1H)-one (MB-2-10; 12c): 25 77
Off white solid; mp.190-192 oC; yield: 6 mg (13%); 1H-NMR (600 MHz, MeOH-D4) δ 9.28 (s, 1H), 8.54 (s, 1H), 8.39 (s, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.05 (s, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.23-7.21 (m, 2H), 7.04 (d, J = 7.9 Hz, 1H), 6.72 (t, J = 8.3 Hz, 2H), 3.66 (s, 3H), 3.35 (s, 2H), 3.24 (s, 4H), 2.75-2.70 (m, 5H), 1.14 (d, J = 6.4 Hz, 6H); 13C-NMR (151 MHz, MeOH-D4) δ 161.5, 5 159.1, 157.7, 154.4, 153.7, 151.7, 147.7, 142.7, 138.0, 137.0, 134.4, 130.5, 128.5, 128.0, 124.3, 122.6, 113.5, 112.7, 112.3, 109.7, 92.8, 56.1, 50.5, 50.1, 30.1, 18.9. 3-(benzo[d]thiazol-5-yl)-1-methyl-7-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1,6- naphthyridin-2(1H)-one (MB-2-11; 12e): 10 Light yellow solid; mp. 190-192 °C; yield: 5mg (15%); 1H-NMR (600 MHz, CDCl3) δ 9.02 (s, 1H), 8.44 (s, 1H), 8.38 (d, J = 1.4 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.82 (dd, J = 8.3, 1.5 Hz, 1H), 15 7.80 (s, 1H), 7.27 (s, 1H), 7.00 (d, J = 8.8 Hz, 2H), 6.69 (s, 1H), 6.44 (s, 1H), 3.57 (s, 3H), 3.25 (t, J = 4.9 Hz, 4H), 2.61 (t, J = 4.9 Hz, 4H), 2.38 (s, 3H); 13C-NMR (151 MHz, CDCl3) δ 162.4, 158.6, 154.5, 153.7, 150.9, 149.1, 147.0, 135.5, 133.3, 131.1, 128.0, 127.0, 124.8, 123.8, 121.5, 117.3, 111.1, 88.0, 55.3, 49.5, 46.4, 29.7. 20 Necroptosis Inhibition Biochemical inhibition of human RIPK1 and RIPK3 was assessed using KINOMEscan™ and BROMOscan™ assay technology (see Fabian, M.A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol. 2005, 23, 329-336). Select compound of the invention and control compound UH15-38 inhibit necroptosis in various cells. Necroptosis was 25 induced by the combination of LPS (10 ng/ml) and pan-caspase inhibitor IDN6556 (20 µM). Fig. 14 shows viability of (a) HT29, (b) THP1, and (c) FADD-def Jurkat cells after treatment with selected compounds. Table 4 shows inhibition of RIPK1, RIPK3 and Necroptosis by selected compounds. Page 78 QB\166118.01517\95733313.1
Table 4. RIPK1, RIPK3 and Necroptosis Inhibition IC50 IC50, IC50 IC50 IC50 IC50 (nM) IC50 ) 1, Page 79 QB\166118.01517\95733313.1

Claims

CLAIMS We claim: 1. A compound having a formula of , 5 or a pharmaceutically acc R1 is H or - –
(CH2)n–X1 wherein X1 is a heterocycloalkyl optionally substituted with alkyl and n is an integer between 0-3; and R2 is H or - –(CH2)m–X2 wherein X2 is a heterocycloalkyl optionally substituted with alkyl and m is an integer between 0-3. 10 2. The compound of claim 1, wherein n is 0 or 1 or m is 0 or 1.
3. The compound of any one of claims 1-2, wherein— X1 is , 15 X2 is a 20 Page 80 QB\166118.01517\95733313.1
4. The compound of any one of claims 1-2, wherein X1 is ,
5. The compound of any one of claims 1-2, wherein X2 is 5 , rogen.
6. The compound of any one of claims 1-3, wherein— X1 is or 10
7. 15 81 QB\16611
8. The compound of any one of claims 1-3, wherein R1 is , 9. 5 , QB\16611
N S , 5 or QB\166118
, 10. A pharmaceutical composition comprising a therapeutically effective amount of the compound or the pharmaceutically acceptable salt of any one of claims 1-9, and a 5 pharmaceutically acceptable excipient, adjuvant, carrier, buffer, stabilizer, or mixture thereof.
11. A method of treating a protein kinase related disease or condition comprising administering the pharmaceutical composition of claim 10 to a subject in need thereof.
12. The method of claim 11, wherein the disease or condition involves receptor interacting kinase 3 (RIPK3) activity. 10
13. The method of claim 12, wherein the disease or condition is an inflammatory or degenerative disease.
14. The method of claim 13, wherein the disease or condition is a respiratory virus. 15. The method of claim 14, wherein the disease or condition is Influenza A virus (IAV) infection.
15
16. A method of treating a subject for a respiratory virus infection comprising administering the subject a RIPK3 inhibitor two or more days after infection.
17. The method of claim 16, wherein the RIPK3 inhibitor is administered five or more days after infection.
18. The method of any one of claims 16-17, wherein the RIPK3 inhibitor is 20 , or a pharmaceutically acceptable salt thereof. Page 84 QB\166118.01517\95733313.1
19. The method of any one of claims 16-17, wherein the RIPK3 inhibitor is one or more of the compounds according to any one of claims 1-9 or the pharmaceutical composition according to claim 10. Page 85 QB\166118.01517\95733313.1
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5095025A (en) * 1989-02-01 1992-03-10 Hodogaya Chemical Co., Ltd. Benzothiazole derivative
US20040224958A1 (en) * 2000-01-27 2004-11-11 Booth Richard John Pyridopyrimidinone derivatives for treatment of neurodegenerative disease

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5095025A (en) * 1989-02-01 1992-03-10 Hodogaya Chemical Co., Ltd. Benzothiazole derivative
US20040224958A1 (en) * 2000-01-27 2004-11-11 Booth Richard John Pyridopyrimidinone derivatives for treatment of neurodegenerative disease

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PUBCHEM: "6-(1,3-benzothiazol-6-ylamino)-4-(cyclopentylamino)-Nmethylpyridine-3-carboxamide, Database accession no. 90155375", DATABASE PUBCHEM 13 FEBRUARY 2015 (2015-02-13), DATABASE ACCESSION NO. 90155375, 13 February 2015 (2015-02-13), pages 1 - 6, XP093367723 *
PUBCHEM: "8-Ethyl-2-phenylamino-8H-pyrido[2,3-d]pyrimidin-7-one, Database accession no. 5330199", DATABASE PUBCHEM 30 JANUARY 2006 (2006-01-30), DATABASE ACCESSION NO. 5330199, 30 January 2006 (2006-01-30), pages 1 - 8, XP093367730 *

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