WO2019161495A1 - Ripk2 inhibitors - Google Patents

Ripk2 inhibitors Download PDF

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WO2019161495A1
WO2019161495A1 PCT/CA2019/050209 CA2019050209W WO2019161495A1 WO 2019161495 A1 WO2019161495 A1 WO 2019161495A1 CA 2019050209 W CA2019050209 W CA 2019050209W WO 2019161495 A1 WO2019161495 A1 WO 2019161495A1
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substituted
unsubstituted
ripk2
compound
inhibitor
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WO2019161495A9 (en
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Shairaz BAKSH
Carlos VELAZQUEZ-MARTINEZ
Rodrigo AGUAYO-ORTIZ
Ratmir Derda
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The Governors Of The University Of Alberta
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/439—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom the ring forming part of a bridged ring system, e.g. quinuclidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4406—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 3, e.g. zimeldine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/48—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine

Definitions

  • This disclosure relates generally to compounds, compositions, methods, and kits, for treating a subject with, or suspected of having, inflammation, an inflammatory disorder and/or cancer.
  • Inflammation is a complex defense mechanism against biological and chemical insults which is largely beneficial.
  • persistent inflammation can cause cellular damage resulting in many diseases including, for example, inflammatory bowel disease (IBD) and CRC (colorectal cancer).
  • IBD inflammatory bowel disease
  • CRC colonal cancer
  • Inflammation is characterized by the hyperactivation of transcription factors (such as NF-kB) and elevated production of cytokines (Baumgart and Carding, 2007; de Ridder et al. , 2007) to amplify the inflammatory response. (Xiao and Ghosh, 2005).
  • Activation of NF-kB proceeds through multiple pathways (both classical and non- classical) that includes TNF-R1 and the pathogen recognition pathway involving Toll like receptors (TLR) (Hayden and Ghosh, 2004; Madrid and Baldwin, 2003; Orlowski and Baldwin, 2002) and the NOD family of receptors (NOD1 and NOD2), intracellular pattern recognition receptors.
  • NOD2 is mainly stimulated by bacterial products containing muramyl dipeptide (MDP) and requires the obligate kinase, RIPK2, to promote an autophagic response or a non-classical NF-kB activation response (Tigno-Aranjuez et al., 2010).
  • Mice with genetic disruption ol Nod2/Ripk2 have a dysbiotic intestinal flora resulting in altered susceptibility to intestinal inflammation (Ermann et a!., 2014).
  • the loss of Ripk2 has been demonstrated to result in the inability of cells to carry out mitophagy leading to enhanced mitochondrial production of superoxide/reactive oxygen species and accumulation of damaged mitochondria that will trigger a capase-11 dependent
  • metastasis (Wu et a!., 2012) and offer alternate novel therapeutics for abnormal inflammation driven by the NOD2/R1PK2 pathway
  • RIPK2 inhibitors have been characterized over the past decade; however, most RIPK2 inhibitors were not designed to inhibit RIPK2 and, thus, RIPK2 inhibition was an off target effect of the use of these drugs.
  • Compounds known to inhibit R1 PK2 include Geftinib/lressa (ICso at 50 nJVl), DCAM-253 (at ICso at 70 nM), p38 MAPK inhibitor SB20358Q (at IC50 at 50 - 100 nM), Src kinase inhibitor 1 and 1 -NM-PP1 (at IC50 at 26 nM but will inhibit several other targets), Regorafenib, and other protein tyrosine kinases (Canning et al , 201 5) Tignc-Aranjuez et al.
  • WEHI-435 was demonstrated to delay RIPK2 ubiquitylation and NF-kB activation downstream of NOD2 activation and interfere with cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice (Nachbur et al., 2015).
  • GlaxoSmithKline has isolated a RIPK2 inhibitor, GSK583 based on using the full RIPK2 protein to screen a DNA-encoded library collection with a fluorescence polarization (FP) based binding assay as a readout.
  • FP fluorescence polarization
  • This inhibitor bound to the ATP binding pocket of the kinase domain and inhibited RIKP2 with an ICso of 5 - 50 nM depending on the assay.
  • An improved version was generated, GSK2983559, that is currently in a Phase 1 trial on healthy volunteers.
  • RIPK2 inhibitors described herein did not inhibit RIPK1 activity involved in ferroptosis or cell death nor effect on mitochondrial biology.
  • the RIPK2 inhibitors described herein appears to also inhibit cell proliferation as determined by MTT assay that does not appear to be promoting cell death.
  • RIPK2 has been demonstrated to be involved in the active growth of CD9G ⁇ +) intestinal stromal cells to suggest an “inflammatory” cross talk between intestinal stromal cells and the epithelial cells (Owens et al.
  • Inflammation signals from both will drive abnormal states to produce a cytokine storm that fuels malignant growth.
  • Inhibitors to RIPK2 may have promising therapeutic potential to uniquely interfere with NFuB-dependent biology and offer an alternative to existing anti-inflammatory therapies.
  • the invention comprises the novel compounds described herein, or the pharmaceutically acceptable salts thereof.
  • the invention relates to any of the compounds of Formulas I, II, III , IV or V, as defined herein and the pharmaceutically acceptable salts thereof
  • the invention comprises pharmaceutical preparations, containing as active substance one or more compounds described herein, or the pharmaceutically acceptable derivatives thereof, optionally combined with conventional excipients and/or carriers.
  • the invention comprises methods of treating inflammation, an inflammatory disorder, or a cancer in a subject comprising administering to said subject a therapeutically effective amount of a compound described or claimed herein, or a pharmaceutically acceptable salt thereof, or a composition comprising such a compound.
  • the invention may comprise a compound or composition described herein for use in the treatment of inflammation, an inflammatory disorder, or cancer in a subject.
  • the inflammation may be associated with inflammatory bowel disease, asthma, obesity, diabetes, cystic fibrosis, psoriasis, arthritis, Parkinson’s Disease, Alzheimer’s Disease or neuropathic pain.
  • the cancer is metastatic cancer, such as metastatic pancreatic or colorectal cancer
  • the invention may comprise the use of a compound, or a pharmaceutically acceptable salt thereof to treat inflammation, an inflammatory disorder or a cancer in a subject.
  • FIG. 1 Schematic of RIPK2 active site.
  • B Representation of pharmacophore points: hydrogen bond donor (HBD), hydrogen bond acceptor (H BA), hydrophobic (Hyd) and aromatic (Ar).
  • HBD hydrogen bond donor
  • H BA hydrogen bond acceptor
  • Hyd hydrogen bond acceptor
  • Ar aromatic
  • FIG. 1 Mass spectrometry and NMR confirmation of mass and structure of RIPK2 Inhibitor 1 .
  • A Direct-infusion measurements were carried out on an LTQ Orbifrap XL (Thermo Scientific) mass spectrometer using the Ion Max ESI source. Mass confirmation was obtained as indicated
  • B Two-dimensional 5 H J H ROESY spectrum of 3-benzamido-4-methyl-N-[3-(1 -methy!-1 H-imidazol-2-yl)phenyl]benzamide. Boxes (orange) represents through-space connectivities between protons in the compound. Blue boxes are signals due to 3-bond J-coupling between aromatic ring protons. 4-bond J- coupling signal is less intense.
  • FIG. 1 Kinase activity for c-ABL was tested in the presence of inhibitor 1 and 2, lCso for c-ABL for both inhibitors is > 100 mM. Analysis was carried out using purified c-ABL and the substrate peptide,
  • Y474 is shown inhibition was carried out on cells followed by lysis and
  • IP immu noprecipitation
  • FIG. 1 Figure 4. Characterization of RIPK2 kinase Inhibitors.
  • A Immunoblot illustration of use of RIPK2 phosphoantibodies in two Hodgkin’s lymphoma cells that have constitutive active RIPK2. A comparison with a known RIPK2 inhibitor is shown
  • KMH2 Hodgkin's lymphoma cells were inhibited in vivo for 33-36 hours using the indicated concentration of RIPK2 inhibitor 1 .
  • an in vitro kinase assay was carried out by IP overnight with 1 pg of the rabbit-anti RIPK2 antibody from ProteinTech and 1 ml of lysate form a confluent 6 well dish of KMH2 cells. Immune complexes were separated by SDS-PAGE and captured by
  • FIG. 1 NFkB gene reporter assay determination of inhibition of MDP stimulated NFkB activity with RIPK2 inhibitors (A) and IC50 determination for inhibition of MDP driven-NFkB activation using RIPK2
  • IC 50 determination for inhibition of LPS-NFkB driven inflammation using RIPK2 inhibitor 1 and 2 (B).
  • FIG. 6 In vivo inhibition of proliferation, intestinal and lung inflammation using RIPK2 inhibitors.
  • HCT116 (+ inhibitor 1 ) versus HCT1 16 (+ inhibitor 2) was ⁇ 0.03; DLD-1 (+ inhibitor 1) versus DLD-1 (+inhibitor 2) was ⁇ 0.02; DLD-1 (+ inhibitor 1 ) versus DLD-1 (+ Regorafenib) was ⁇ 0.006
  • RIPK2 inhibitor 1 treated DLD-1 or HCT116 vs RIPK2 inhibitor 1 treated ModeK or Rat-1 cells (normal cells), p ⁇ 0,002 (analysis in either cell type);
  • RIPK2 inhibitor 2 treated DLD-1 or HCT116 vs RIPK2 inhibitor 2 treated ModeK cells (normal cells), p ⁇ 0.02 and 0.098 (analysis in DLD-1 or HCT116 cells respectively); for
  • RIPK2 is activated via tyrosine (Y) (pY474) and serine (S) (pS176) phosphorylation and ubiquitination events to allow for associations with downstream components RIPK2 inhibitor 1 inhibits phosphorylation at serine 176 and tyrosine 474 and possible inhibition of ubiquitination of RIPK2.
  • HRE hypoxia response element
  • FIG. 10 Related to Figure 5. Effect of RIPK2 inhibitors on NFkB, cell growth and cell cycle control.
  • A EMSA was carried out using the NFkB binding site on the IL-6 promoter. HCT1 16 cells were pretreated with the indicated RIPK2 inhibitors for 2 days, followed by stimulation with MDP and nuclear/cytoplasmic extraction. 4 pg of nuclear extract was incubated with the IL-6 probe in an NFkB DNA binding assay
  • R1PK2 Inhibitor 1 can modulate the growth of several breast cancer cells.
  • MTT assay was carried out with the 100 nM of RIPK2 inhibitors in the indicated breast cancer cell lines with HCT1 143 and MDA-MB31 as triple negative breast cancer cells and JMT-1 is a Fler2 overexpressing cell line.
  • n 4 - 10.
  • P value -/+ RIPK2 inhibitor 1 for JMT-1 was 0.0001
  • for FICT 1143 was 0 0007
  • for MDA-MB231 was 0.05.
  • C Growth inhibition MTT curves for FICT1 16 (A) and BT-549 (B, breast cancer cells ⁇ were examined in the presence of RIPK2 inhibitor 1 at the concentrations indicated and ICso
  • FIG. 1 RIPK2 inhibitor 1 does not affect the cell cycle fraction distribution at 100 nM.
  • HCT-1 16 colon (A) or MCF-7 breast (B) cancer cells were treated with Vehicle or the indicated inhibitors (5 mM) for 36 hours. After an overnight ethanol fixation, celts were PI stained 15000 cells were analyzed on BD Accuri flow cytometer. Different cell cycle phases were quantitated for 3 independent experiments and plotted as percentage of mean ⁇ SD (far right A and B) No major difference was observed between treatments. The data for 5 mM of the drug is presented but similar results were observed at 100 nM RIPK2 inhibitor 1 drug treated cells.
  • R1PK1 activity was measured in FIT-29 cells by activation of ceil death using 20 mM z-VAD-fmk (Z), 100 nM Smac Mimetic-164 (S) and 10 ng/mL TNFcr (T) combination in the presence or absence of 100 nM Ripki inhibitor or 1 mM
  • FIG. 13 Related to Table 4 and Figure 6. Mice were exposed to 7 days of 3% DSS to induce inflammation injury and 7 days with water to promote recovery. RIPK2 inhibitors were intraperitoneal (IP) injected on day 5, 7 and 9 Serum was harvested 1 1 days after last IP injection and blood chemistry was carried out at the Prairie Diagnostics for numerous factors. The ones shown have some significant changes. For alkaline phosphatase, Two-way Anova reveals P value ⁇ 0.0001 for all measured marker [0031] Figure 14. Characterization of Identified RIPK2 Inhibitors.
  • Figure 15 RIPK2 Inhibition of NFKB, a key transcription factor driving inflammation.
  • Figure 17 Identified RIPK2 Inhibitors are effective in alleviating in vivo inflammation.
  • Active RIPK2 can be detected in tissue sections of DSS-treated pre-clinical models of IBD using or proprietary rabbit anti-phosphotyrosine 474 RIPK2 antibody to immunohistochemistry and is a strong driver of in vivo inflammation.
  • FIG. 20 Clinical characterization of active RIPK2.
  • A RIPK2 activity analysis in a pediatric patient with UC RIPK2 IFIC was carried out using he pY 474 RIPK2 antibody used in and the bottom panel, summary of clinical make up of pediatric case study patient with UC. Robust staining for phosphotyrosine R1PK2 was evident in
  • Figure 21 Pseudomonas aeruginosa model for lung inflammation.
  • FIG. 22 An immunoblot showing validation of the use of RIPK2 inhibitor
  • FIG. 23 RIPK2 Inhibitor 1 interfering with cell invasion in Hep3, a human squamous carcinoma cell line, and in MDA231 , a triple negative breast cancer ceil line. Similar results were obtained in the inflammatory breast cancer cell line IBC-3.
  • Figure 24 Empirical Testing of Potential off Target Effect on PKB/Akt.
  • Figure 28 NFKB gene reporter assay determination of inhibition of MDP stimulated NFKB activity with RIPK2 inhibitor 5 and 6 as in Fig 6A at the indicated concentrations.
  • FIG. 31 Model for intestinal Inflammation involving RASSF1 A
  • Figure 32 Biological characterization of RIPK2 Inhibitors. Test of RIPK2 inhibitor-1 modulation of RIPK2 autophosphorylation at Y474 in HL cells (HDMYZ and L428) and RIPK2 activity in an NHL (DOHH2) and primary AML (HL-60) cell lines Inhibition was carried out on cells followed bY lysis and IP with rabbit anti-RIPK2 overnight. Following protein G pull down and wash, 32-P-y-ATP is added and kinase reaction allowed to proceed for 30 minutes at 30 C. Following kinase reaction, SDS- PAGE was used to separate out the proteins, gel was then dried and exposed to film.
  • FIG. 33 Characterization of RIPK2 Inhibitors 6, 7 and gtiquidione was determined at the International Kinase Profiling Center in Dundee, Scotland. Substrate for RIPK2 was myelin basic protein and a peptide substrate (EAIYAAPFAKKK) for ABL in vitro kinase assay. RIPK2 utilized was truncated recombinant RIPK2 (2-31 1 )
  • Figure 35 TCGA analysis of RIPK2 expression in indicated cancers.
  • Inhibitor 1 was extracted from 0.1 mL alkalinized rat plasma using liquid-liquid extraction of (3 mL of tert-butyl methyl ether). Chromatography was accomplished with a 15 cm C18 analytical column with a mobile phase of 75% phosphate solution in acetonitrile and UV detection at 261 nm. The chromatogram above shows a sample with 1000 ng/ l inhibitor extracted from rat plasma vs unspiked inhibitor-free rat plasma.
  • the standard curve of detection in rat plasma was highly linear from 25 to 500 ng/mL.
  • FIG. 38 NFkB and growth inhibition of RIPK2 inhibitors.
  • MTT assay was carried out with the indicated concentrations of R1PK2 inhibitors in breast cancer cells.
  • RIPK2 inhibitor 1 does not appear to interfere with growth of MCF-7 cells, an early breast cancer luminal A cell.
  • MDA MB231 and BT549 are triple negative (ER-PR-Her2-) cells and SUM1 9 and IBC-3 are inflammatory breast cancer cells (ER-PR-Her2- and ER-PR- HER2+ cells, respectively).
  • n 4 - 6.
  • FIG. 41 Summary model of RASSF1A influences on inflammation and cancer signaling.
  • RASSF1 A as indicated in I BD and CRC.
  • Figure 43 Diagram depicting the pathogensis of IBD subtypes.
  • FIG 44 Summary of the characteristics of the RASSF1 gene family.
  • FIG. 45 Rassfl a animals are sensitive to dextran sodiumsulphate (DSS) treatment. Mice were subjected to 3% DSS solution followed by day 7 replacement with regular water to allow for recovery A Kaplan-Meier curve monitoring % survival following DSS treatment.
  • DSS dextran sodiumsulphate
  • FIG. 46 A representative picture of DSS treated colons is shown indicating how colon length was measured. DSS-treated Rassfl a+/ revealed a similar loss of colon length (data not shown).
  • Figure 47 Longitudinal cross-section of the descending colon stained with
  • H&E is shown for untreated and DSS-treated animals. All untreated colon sections samples were very similar to untreated colon sections from wild type mice
  • FIG. 48 Characterization of DSS induced inflammation injury in the epithelial and macrophage specific knock outs to RASSF1A. Sibling matches as also characterized that do not contain the macrophage specific knockout
  • mice were subjected to 3% DSS solution followed by day seven replacements with regular water to allow for recovery.
  • DAI Disease activity index
  • FIG. 50 Model for pathways driving colonic inflammation.
  • High levels of NFkB transcriptional activity can result in intestinal inflammation and abnormal activation of apoptosis leading to inflammation induced damage.
  • the presence of pathogens can also result in the activation of another pattern recognition receptor, NOD2 and its obligate kinase (R1PK2), to result in NFkB activation and initiation of the autophagic response (right side pathway).
  • NOD2 a pattern recognition receptor
  • R1PK2 obligate kinase
  • RASSF1A is epigenetically silenced in most of human cancers (a known) and in IBD (an unknown). As such, the lack of RASSF1 A in IBD and CRC patients would result in hyperactive RIPK2, TLR and TBK1 and, in turn, hyperactive NFkB and inflammation.
  • Figure 51 Survival curve in response to DSS-induced inflamamtion injury in the presence of the broad spectrum anti-inflammatory resveratrol.
  • FIG. 52 Expression of RASSF1 A and active YAP in IBD patients.
  • RASSF1 A is epigenetically silenced in IBD patients and as such low to no detection of RASSF1A is observed in IBD patients.
  • YAP is a co-transcriptional activator that can drive malignancy.
  • RIPK2 is the obligate kinase to the NOD2 pathogen receptor pathway and has been demonstrated to be involved in NFkB activation and metastatic behaviour in some cancers (Jun et al., 2013) and has a distinct activity versus RIPK1 , 3 or 4
  • CRC Colorectal cancer
  • IPK2 inhibitors reveal lower binding free energies that other RIPK2 inhibitors identified suggesting that the inhibitor may be more active
  • R1PK2 inhibitors appear to be have more affinity for the active site of RIPK2 than others, more effective at inhibition of proliferation and can effectively resolve lung inflammation and intestinal inflammation more robustly than gefitinib ( Figure 6B and Table 4).
  • inflammation may arise from the fact that it can have a small but significant effect on TNFa, IL-1 and LPS-driven NFkB activation as seen in Figure 5 and Figure 9B.
  • Most inflammatory diseases are complex diseases that have a multitude of inflammatory pathways targeting the area.
  • a broad spectrum inhibitor may be needed to target multiple TLRs and pathogen receptors (Murgueitio et al. , 2017)
  • RIPK2 has a unique requirement for NOD1 and NOD2 and functions in many pathways different from RIPK1 , 3 or 4.
  • Chirieleison et al. (2016) summarized the uniqueness of RIPK2 kinase domain within the RIPK family that could not be substituted for the kinase domain from RIPK1 or RIPK4.
  • RIPK2 inhibitors 1 and 2 did not inhibit RIPK1 biology nor ferroptosis, a form of cell death influenced by proteins of the RIPK family ( Figure 12A and B).
  • the identified RtPK2 inhibitor 1 and 2 can selectively modulate RIPK2 specific biology in agreement with the observations of Chirieleison et al. (2016).
  • inflammation damage RIPK2 may also be important in driving inflammation damage in other diseases involving inflammation such as obesity, diabetes, multiple sclerosis, cystic fibrosis, psoriasis (NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis Sci reports 2016), arthritis/osteoarthritis (Jurynec, M. J.
  • et ai A hyperactivating proinflammatory R1 PK2 allele associated with early-onset osteoarthritis, Hum Mol Genet 2018), Parkinson's (Ma et al, P268S in NOD2 associates with susceptibility to Parkinson’s disease in Chinese population, Behav Brain Funct 201 3 and cheng et al, NOD2 promotes dopaminergic degeneration regulated by NADPH oxidase 2 in d-hydroxydopamine model of Parkinson's disease, J Meuroinflamrmation 201 8) and Alzheimer’s Disease and neuropathic pain (Santa-Cecilia, F V. et al, The
  • IMOD2 signaling in peripheral macrophages contributes to neuropathic pain development, Pain 2018). It is expected that the RIPK2 inhibitors described herein may also alleviate the clinical symptoms associated with these diseases.
  • the present invention comprises novel compounds which inhibit the receptor-interacting serine/threonine protein kinase 2 (RIPK2) and are thus useful for treating a variety of diseases and disorders that are mediated or sustained through the activity of RI PK2, including numerous inflammatory and neurological diseases and cancers (including metastatic cancers).
  • This invention also relates to pharmaceutical compositions comprising these compounds, as well as methods of using RIPK2 inhibitor compounds, including in the treatment of various diseases and disorders.
  • the invention comprises a compound of Formula I,
  • Y is C or N
  • i is selected from the group consisting of: H, substituted or unsubstituted C -C straight alkyl, substituted or unsubstituted C -C branched alkyl, substituted or unsubstituted C - Ct straight alkenyl, substituted or unsubstituted C -C branched alkenyl, substituted or
  • Rz and R 4 are each independently selected from the group consisting of: H, substituted or unsubstituted C -C straight alkyl, substituted or unsubstituted C -C branched alkyl, substituted or unsubstituted C 3 -Ciz straight alkenyl, substituted or unsubstituted C -C branched alkenyl, substituted or unsubstituted C 8 -C 8 cycloalkyl, substituted or
  • R 3 and Rs are each independently selected from the group consisting of: H , substituted or unsubstituted C -C straight alkyl, substituted or unsubstituted C -Ciz branched alkyl, substituted or unsubstituted C 3 -Ci a straight alkenyl, substituted or unsubstituted C3-Ci 2 branched alkenyl, substituted or unsubstituted C -C cycloalkyl, substituted or
  • X is selected from the group consisting of; carbon, ortho- Oxygen, mefa-Oxygen, para-Oxygen, orfbo-Nitrogen, mefa-Nitrogen, para-Nitrogen, orfbo-Sulfur, mefa-Sulfur, and para-Sulfur; except that Formula 1 excludes 3-benzamido-4-methyl-N-[3-(1-methyl-1 H-imidazol-2-yl) phenyl]benzamide (RIPK2 Inhibitor 1 ).
  • Y is N and R5 is a substituted or unsubstituted heterocycle:
  • R is H or an substituted or unsubstituted aliphatic moeity, preferably Ci-C alkyl, more preferably C -C alkyl, or Z-R7, where Z is a linker and R7 is a functional group.
  • R7 is NH .
  • the compound is a compound of Formula II;
  • X is a divalent aliphatic or polymeric linker
  • X is a substituted or unsubstituted C -C alkylene, CID-C 2O alkylene, C -C alkenylene, C -C alkenylene, or C -C alkynylene, C -C alkynylene; a substituted or unsubstituted C3-C10 cycloalkylene, C -C cycloalkyfene, C4-C 10 cyc!oalkenylene, C -C cycloalkenylene, or Cio-C 2 o cycloalkynylene; a substituted or unsubstituted divalent ethylene glycol or a substituted or unsubstituted divalent polyethylene glycol; or, a substituted or unsubstituted divalent ether or a substituted or unsubstituted divalent polyether
  • the compound is a compound of Formula II selected from the group consisting of:
  • the compound is a compound of Formula III:
  • R6 is H (RIPK2 Inhibitor 1 C) or an substituted or unsubstituted aliphatic moeity, preferably C 1 -C 0 alkyl, more preferably Ci-Cs alkyl; or Z-R7, where Z is a linker and R7 is a functional group.
  • R7 is NH 2
  • Z is a divalent aliphatic or polymeric linker, such as a substituted or unsubstituted C 1 -C 10 alkylene, C 10 -C 20 a!kylene, C 2 -Ci 0 alkenylene, C10-C 20 alkeny!ene, or C2-C10 alkynylene, C 10 -C 20 alkynylene; a substituted or unsubstituted C3-C 10 cyc!oalkylene, C 10 -C 20 cycloalkylene, C 4 -C 10 cycloalkenylene, C 10 -C 20 cycloalkenylene, or C10-C 20 cycloaikynylene; a substituted or unsubstituted ethylene glycol or a substituted or unsubstituted divalent polyethylene glycol; or, a substituted or unsubstituted ether or a substituted or unsubstituted
  • R6 is the same linker amine in the derivatives of Formula I! shown above, and accordingly may be selected from the group consisting of:
  • the invention may comprise compounds of Formula IV or V:
  • any of the compounds described herein may comprise a tautomer, or a pharmaceutically acceptable salt, or a solvate, or a functional derivative thereof.
  • the term“functional derivative’ as used herein refers to a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original compound.
  • a functional derivative or equivalent may be a natural derivative or is prepared synthetically.
  • prodrug or a "physiologically functional derivative” of the compounds described herein.
  • physiologically functional derivative refers to compounds which are not pharmaceutically active themselves but which are transformed into their pharmaceutically active form in vivo, i.e. in the subject to which the compound is administered.
  • prodrug refers to a derivative of a substance that, following administration, is metabolized in vivo, e.g by hydrolysis or by processing through an enzyme, into an active metabolite.
  • the compounds described herein may be prepared by the methods and examples presented below and methods Known to those of ordinary skill in the art. In each of the examples below, the groups are as defined above for the various formulae. Optimum reaction conditions and reaction times may vary depending on the particular reactants used Unless otherwise specified, solvents, temperatures, pressures, and other reaction conditions may be readily selected by one of ordinary skill in the art. Specific procedures are provided below intermediates used in the syntheses below are either commercially available or easily prepared by methods known to those skilled in the art. Reaction progress may be monitored by conventional methods such as thin layer chromatography (TLC) or high pressure liquid chromatography-mass spec ⁇ HPLC-MS ⁇ . Intermediates and products may be purified by methods known in the art, including column chromatography, HPLC, preparative TLC or Preparatory HPLC
  • a method of synthesizing a compound comprises reacting
  • carboxylic acid such as 1 1 and amine, such as 2
  • a method of synthesizing the compound of Formula III comprises reacting
  • R1PK2 Inhibitor 1 may be synthesized as follows, and derivatives thereof may be synthesized in an analogous scheme:
  • EDC 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • Boc tert-butyioxycarbonyl
  • the compounds described herein are typically administered in the form of a pharmaceutical composition.
  • Such compositions can be prepared using procedures well known in the pharmaceutical art and comprise at least one compound described herein.
  • the compounds described herein may also be administered alone or in combination with adjuvants that enhance stability of the compounds, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increased antagonist activity, provide adjunct therapy, and the like.
  • the compounds may be used on their own or in conjunction with other active substances, optionally also in conjunction with other pharmacologically active substances.
  • the compounds described herein are administered in a therapeutically or pharmaceutically effective amount, but may be administered in lower amounts for diagnostic or other purposes
  • Administration of the compounds, in pure form or in an appropriate pharmaceutical composition can be carried out using any of the accepted modes of administration of pharmaceutical compositions.
  • administration can be, for example, orally, buccaliy (e.g., sublingually), nasally, parenterally, topically, transdermally, vagtnally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.
  • the pharmaceutical compositions will generally include a conventional pharmaceutical carrier or excipient and a compound described herein as the/an active agent, and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, vehicles, or combinations thereof.
  • Such pharmaceutically acceptable excipients, carriers, or additives as well as methods of making pharmaceutical compositions for various modes or administration are well-known to those of skill in the art. The state of the art is evidenced, e.g , by Remington: The Science and Practice of Pharmacy, 20th Edition, A. Gennaro (ed.), Uppincott Williams & Wilkins, 2000; Handbook of Pharmaceutical Additives, Michael & Irene Ash (eds ), Gower, 1995; Handbook of Pharmaceutical Excipients, A.
  • the forms of the compounds described herein utilized in a particular pharmaceutical formulation will be selected (e.g., salts) that possess suitable physical characteristics (e.g., water solubility) that are required for the formulation to be efficacious.
  • compositions herein can be used for treating a subject with, or suspected of having, a cancer, especially with metastatic disease.
  • a RIPK2 inhibitor described herein can significantly interfere with the steps prior to malignant transformation and thus be a robust anti-cancer drug.
  • the invention may comprise a method for treating an inflammatory disorder, comprising administering one or more compounds or compositions described herein to a subject having or suspected of having an inflammatory disorder, an acute inflammatory disease or disorder, thereby treating the inflammatory disorder.
  • the inflammatory disorder is acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterative, parenchymatous, plastic, productive, proliferous, pseudomembranous, purulent, sclerosing, seropiastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and/or ulcerative inflammation
  • the inflammatory disorder is from gastrointestinal disorders (such as peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic) gastrointestinal disorders (such as, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea,
  • gastrointestinal disorders such as peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic
  • gastrointestinal disorders such as, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis
  • gastritis such as, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis
  • GORD gastroesophageal reflux disease
  • GERD gastroesophageal reflux disease
  • inflammatory bowel disease 1BD
  • Crohn's disease ulcerative colitis
  • collagenous colitis lymphocytic colitis
  • ischaemic colitis diversion colitis
  • Behcet's syndrome indeterminate colitis
  • IBS inflammatory bowel syndrome
  • the inflammatory disorder is a disorder of the lung selected from pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis,
  • bronchiectasis diffuse panbronchiolitis, hypersensitivity pneumonitis, asthma, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis.
  • the inflammatory disorder is multiple sclerosis (MS).
  • MS multiple sclerosis
  • multiple scleoris include, but are not limited to relapsing-remitting MS, secondary-progressive MS, primary-progressive MS, progressive-relapsing MS.
  • a subject with an inflammatory disorder can be treated to provide cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse of the patient from or as a result of the treatment.
  • a subject with Parkinson's disease or Alzheimer’s disease may be treated with an RIPK2 inhibitor, which may slow down the progression of these diseases by controlling the inflammation coming from the gut-brain-axis (Ma ef a!, Behavioral and Brain Function, P268S in NOD2 associates with susceptibility to
  • Parkinson's disease in a Chinese population Cheng L et al, NOD2 promotes dopaminergic degeneration regulated by NADPH oxidase 2 in 6-hydroxydopamine model of Parkinson’s Disease, N euro inflammation 2018).
  • a subject with neuropathic pain may be treated with an RIPK2 inhibitor to alleviate neuropathic pain, a condition resulting from inflammation activating the pain receptors in the brain and the NOD2/RIPK2 pathway has been shown to drive this processs (FV Santa-Ceci!ia et al Pain 2019).
  • cancer refers to a variety of conditions caused by abnormal, uncontrolled growth of cells.
  • Cells capable of causing cancer referred to as “cancer cells” possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain typical morphological features.
  • Cancer cells may be in the form of a tumour, but such cells may also exist alone within a subject, or may be a non-tumorigenic cancer ceil.
  • a cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e g.
  • a negative result in one or more of the above detection methods does not necessarily indicate the absence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse
  • the cancer is a lymphoma.
  • lymphoma refers to a malignant growth of B or T cells in the lymphatic system “Lymphoma” includes numerous types of malignant growths, including Hodgkin's Lymphoma and non-Hodgkin's lymphoma.
  • non-Hodgkin's Lymphoma refers to a malignant growth of B or T cells in the lymphatic system that is not a Hodgkin's Lymphoma (which is characterized, e.g.
  • Non-Hodgkin’s lymphomas encompass over 29 types of lymphoma, the distinctions between which are based on the type of cancer ceils.
  • the cancer is Hodgkin ' s lymphoma, relapsed non-Hodgkin’s lymphoma, or relapsed leukemia
  • the cancer is triple negative and inflammatory breast cancer, pancreatic cancer, or colorectal cancer
  • a subject with cancer can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent recurrence.
  • a compound or composition described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
  • a therapeutically effective amount may be any therapeutically effective amount.
  • the term“therapeutically effective amount” refers to an amount that is effective for preventing, ameliorating, or treating a disease or disorder (e g., inflammatory bowel disease, e.g., ulcerative colitis or Crohn's disease, e.g., cancer).
  • a disease or disorder e g., inflammatory bowel disease, e.g., ulcerative colitis or Crohn's disease, e.g., cancer.
  • Formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • the compounds and compositions may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g.
  • vaginal parenteral, for example, by injection, including subcutaneous, tntradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinai, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
  • compositions comprising compounds disclosed herein may be used in the methods described herein in combination with standard treatment regimes, as would be known to the skilled worker. Common drugs/combinations or treatments are well known to those skilled in the art.
  • therapeutic formulations comprising the compounds or compositions as described herein may be prepared by mixing compounds or compositions having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, in the form of aqueous solutions, iyophilized or other dried formulations.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
  • hexamethonium chloride benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyciohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino adds such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexe
  • the therapeutic formulation may also contain more than one active compound as necessary for the particular indication being treated, typically those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • kits preferably contains the composition.
  • Such a kit preferably contains instructions for the use thereof
  • the invention may comprise the use of a compound described herein as a ligand to bind to and purify proteins via affinity chromatography, wherein the ligand is coupled to a solid support.
  • derivatives of a RIPK2 inhibitor described herein may be coupled to an agarose or sepharose beaded matrix to form an affinity matrix for targeted proteins.
  • Cell lysates containing non-active or active RIPK2 (for example, generated using -/+ MDP treatment respectively) will be added to the RIPK2 inhibitor derivative- agarose or sepharose beads, incubated overnight at 4 ° C followed by SDS-PAGE separation of associated proteins.
  • Mass spectrometry may then be utilized to identify the protein target associated with RIPK2 inhibitor derivative-agarose. This analysis will allow for the possible identification of off target effects (mainly kinase off target effects) of a RIPK2 inhibitor.
  • any compound described herein having a reactive functional group may be coupled to a solid support, such as agarose for example, by a scheme such as:
  • Agarose-linked reagents are commercially available, and schemes to react the reagent to the functional group are well known to those skilled in the art.
  • the invention may comprise a method of identifying a polypeptide that binds to a RIPK2 inhibitor compound, comprising: (i) incubating a cell lysate comprising a RIPK2-inhibitor binding polypeptide with a support comprising the compound, to form a complex between said RIP K2 inhibitor binding polypeptide and the compound;
  • Example 1 Identification and Characterization of Novel Receptor Interacting Serine/threonine-Protein Kinase 2 (RIPK2) Inhibitors Using Structural Similarity Analysis
  • Receptor interacting protein kinase 2 (RIP2 or RICK herein referred to as RIPK2) is linked to the pathogen pathway that activates NFkB and autophagic activation.
  • RIPK2 Receptor interacting protein kinase 2
  • RIPK2 Receptor interacting protein kinase 2
  • Identified RIPK2 inhibitors potently inhibited proliferation of cancer cells by > 70% as well as inhibition of NFkB activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggest effectiveness to resolve inflammation with low toxicity to the animals.
  • the identified RIPK2 inhibitor may offer a possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cho
  • PC 57405602 -P .6 PC 58945669 -1 1.3 PC 58945635 1 1 6 PC_40780I 19 11 1
  • the residue was reconstituted in 0.15 mL of mobile phase and 0.05 mL injected into the HPLC.
  • the mobile phase was a mixture of [25 mM KFUPCh: 3 mM sulfuric acid: 3.6 mM triethyiamine]: acetonitrile in the proportion 64:36 v/v.
  • the mobile phase was passed (0.9 mL/min at room temperature) in series through a C1 8 2 cm> ⁇ 4.0mm, 5m guard column (Supelco, PA, USA) then a Symmetry C18 analytical column, 3.5m, 150mmx4.6mm (Waters, MA, USA).
  • Chromatographic data were collected and compiled by use of EZChrom software.
  • Detection was by ultraviolet absorption using a Waters 486 detector, with the Amax being set to 261 nm. Standard curves of peak area or height from 25 to 1000 ng/mL were highly linear (r 2 > 0.99).
  • Binding reactions were initiated by combining kinase, liganded affinity beads and test compounds in binding buffer.
  • Test compounds were prepared as 100 x stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Assay plates containing kinase/ligand and small molecule inhibitor were incubated at 25 °C with shaking for 1 h, washed extensively to remove unbound protein and eluted. Kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound /ids were determined using eleven serial threefold dilutions
  • ABL (5-20mU diluted in 50 mM Tris pF! 7.5, 0 1 mM EGTA, 1 mg/ml 8SA) was assayed against substrate peptide (EAIYAAPFAKKK) in a final volume of 25 5 pi containing 50mM Tris pH 7,5, 0 1 mM EGTA, 1 mM DTT, 300mM substrate peptide, 10 M magnesium acetate and 0.005 mM [33P-y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays were stopped by addition of 5 pi of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. The dried Unifilter plates are then sealed on
  • !VITT Growth Assay Cell proliferation assay was performed to evaluate cell viability after drug treatment. Cells were counted using hemocytometer and seeded in a 96-well plate at 1 -2 X 10 4 cells/well and incubated for 24 hours. Different concentrations of RIPK2 inhibitor was added to the cells and incubated for 48h. MTT [3- ⁇ 4,5- Dimethylthiazol-2-y[)-2,5-Diphenyltetrazolium Bromide] was then added and incubated for 4h. Absorbance was measured at 560 nm using vICToRTM Multilabel Plate Reader from PerkinElmer. For L428, cells were stained with Trypan Blue to determine cell viability after 1 - 6 days incubation with the drugs.
  • Membranes were then washed TBS-T (Tris-buffered saline containing 0, 1 % Tween 20) and incubated with appropriate secondary antibody (anti-rabbit or anti-mouse IgG) for one hour
  • Cell cycle analysis Cells were harvested by trypsin ization, washed with PBS and resuspended in 75% ethanol in PBS and kept at 48°C for at least 30 min. Prior to analysis, cells were washed again with PBS and resuspended and incubated for 30 min in a solution containing 0 05 mg/ml propidium iodide (Sigma), 1 mM EDTA, 0.1 % Triton-X-100 and 1 mg/ml RNAse A in PBS. The suspension was then passed through a nylon mesh ®lter and analyzed on a Becton Dickinson FACScan.
  • RIPK1 activity, HT-29, L929 and HaCaT cells were seeded in a 96-well plate and then treated with the indicated amounts of RIPK2 inhibitors. After 24 hours, the media was removed and replaced with fresh media containing 20 mM z-VAD-fmk (Z), 100 nM Smac Mimetic-164 (S) and 10 ng/mL TNFa (T) in the presence or absence of 100 nM Ripkl inhibitor or 1 uM necrosu!fanamide (NSA) or the indicated amounts of RIPK2 inhibitors for 24 hours (HaCaT and HT-29) or 8 h (L929). After the indicated time, the cells were double stained with hoechst and propidium iodide for 30 min at 37 °C and then cell death was measured using the Celigo cell cytometer.
  • Filtered compounds were then subjected to another filtering step based on Lipinski’s rule of five, (Lipinski et al , 2001 ) removing duplicates and inorganic molecules with the FAFDrugs3 server (Lagorce et al. , 2015) (22,274 compounds). Finally, a total of 3,000 compounds were selected and optimized using the obminimize tool of the OpenBabel toolbox (O'Boyie et al., 201 1 ) for the docking studies
  • NFKB Gene Reporter Luciferase Assay Dual-Luciferase Reporter Assay System (DLR assay system, Promega, E1910) was used to perform dual-reporter assays on NFkB Luciferase and Renil!a Luciferase (internal control). Briefly, HCT1 16 colon cancer cells were equally seeded at a density of 3 x 10 4 in 6-we!l plates and allowed to attach for 24 hrs. Prior to transfection, cells were washed with serum free media 3 times. Dual transfection was carried out using 12 pi of PEI to 3 pg of NFKB Luciferase construct
  • mice were challenged intranasally with 25 mI of saline containing 50 pg ovalbumin or saline alone as control on days 12 and 14 RIPK2 inhibitor -1 (1 pg/g body weight) or 30% DMSO (solvent control for drug) were injected intraperitoneaily on day 12, 13 and 14 (on day 12 and 1 the injection was performed 1 hr before intranasal challenge with ovalbumin).
  • mice were euthanized with an intraperitoneal injection of 2 mg sodium pentobarbital. Cardiac puncture was used to collect blood, followed by tracheal intubation with polyethylene tubing.
  • Mass Spectrometry Direct-infusion measurements were carried out on an LTQ Orbitrap XL (Thermo Scientific) mass spectrometer using an Ion Max ESI source. An on-board syringe pump was used with a 100uL Hamilton syringe and a flow rate of
  • NMR Spectroscopy NMR experiments were run on a Varian Inova 500 MHz spectrometer at 30°C. Proton chemical shifts were measured relative to residual proionated methyl (CHD2) signal of de-DMSO (2.50 ppm); coupling constants (J) are reported in Hertz (Hz). Standard notation was used to describe multiplicity of signals observed in ⁇ NMR spectra: singlet (s), doublet (d), triplet (t), broad (b) etc. Through- space and through-bond connectivities were observed using 2D 1 H- 1 H ROESY experiment (mixing time 200 ms). One-dimensional experiments were processed using VNMRJ (Varian Associates) and 2D 1 H- 1 H ROESY was processed using
  • R1PK2 inhibitor 1 was prepared by dissolving 0.3 mg in 500 pL d 6 -DMSO.
  • RIPK2 inhibitor 2 was prepared by dissolving 0.3 mg in 500 pL d s - DMSO (Delaglio et al. , 1995).
  • Figure 1 B shows the proposed binding mode of these ligands in the RIPK2 binding site. This figure also shows that all the compounds identified possible form hydrogen bonds between the Glu66 residue and the carboxamide group.
  • the initial virtual screening ranking, chemical structures and the Pan Assay Interference Compounds (PAINS) (Sterling and Irwin, 2015) check of the selected compounds are reported in the Table 2.
  • MDP is the molecular ligand for the NOD2 pathogen receptor whereby R1 PK2 can be activated. Similar results for RIPK2 inhibitor 1 inhibition of RIPK2 were obtained in breast cancer cells (BT549 and MDA-MB231 cells, data not shown).
  • RIPK2 inhibitor 1 was more effective inhibiting NFkB activity when compared to RIPK2 inhibitor 2, gefitinib or regorafenib (p value comparing RIPK2 inhibitor 1 vs RIPK2 inhibitor 2 or gefitinib or regorafenib inhibition of MDP-driven NFkB activation was 0.0015, 0.0006 and 0.004 respectively).
  • RIPK2 inhibitor 1 was as effective as ponatinib in NFkB inhibition (p value - 0.06 when comparing RIPK2 inhibitor 1 vs ponatinib inhibition of MDP-driven NFkB activation).
  • RIPK2 inhibitor GSK-583 did not inhibit MDP-dependent NFkB activity at 100 nM in HCT116 cells (data not shown) although known to in primary immune cells.
  • a small but significant reduction was observed in LPS (via TLR4 and TLR2), TNFa, and IL-i -dependent activation of NFkB ( Figure 5C and ICso in Figure 5D, Figure 9B). Although a reduction, the IC 5 o for RIPK2 inhibition of LPS-driven NFkB
  • activation is > 20 mM and it may be that it will also be much higher for inhibition of TNFa, and IL-1 p-dependent activation of NFkB.
  • RIPK2 inhibitor 1 can also inhibit the activation of hypoxia response element I response to chemical induction using 1 % H z 0 2 ( Figure 9C) to suggest either a link to inflammation or a direct modulation of HIFa function.
  • RIPK2 Inhibitors Can Inhibit the Proliferation of Several Cancer Cells but Not Promote Apoptosis or Cell Cycle Arrest. Inflammation is a strong driver of malignant transformation and abnormal proliferation, especially in the coion (Lasry et al.
  • RIPK2 Inhibitors Do Not Inhibit RIPK1 Directed Cell Death
  • RIPK2 inhibitor 1 did not interfere with these forms of cell death to support the cell cycle effects and to suggest no overlap with RI PK1 biology ( Figure 12A) Recently, RIPK3 was demonstrated to require Bax/Bak effect on the mitochondrial permeability transition pore (MPTP) in order to carry out
  • RIPK2 Inhibitors Can Efficiently Resolve Intestinal Inflammation in an Ulcerative Colitis Model.
  • DSS-induced intestinal inflammation is a model for ulcerative colitis (UC, a form of inflammatory bowel disease, I8D).
  • UC ulcerative colitis
  • I8D ulcerative colitis
  • DSS functions to irritate the colonic mucosa to promote localized inflammation, active cell death and localized destruction of the epithelial barrier to the lumen of the colon (Dieleman et al , 1998).
  • the Rassfl a knockout mice are very susceptible to DSS- induced inflammation injury (Gordon et al., 2013) mainly due to uncontrolled inflammation linked to the NOD2/RIPK2 pathway (Said et al, unpublished observation),
  • RIPK2 inhibitor can also inhibit intestinal inflammation injury but only promote a 41 % survival vs 73% survival with RIPK2 inhibitor 1 (Figure 6B and Table 4). Similar results were obtained for the protein tyrosine kinase inhibitor, regorafenib (data not shown). Since both gefitinib and the RIPK2 inhibitors described herein can inhibit RIPK2, either the inhibitors have more affinity for RIPK2 or off target effects of RIP 2 inhibitor 1 is beneficial to aid in recovery from inflammation injury using the DSS model. Table 4. Survival of Rassfla A animals during DSS-induced inflammation injury in the presence of RIPK2 inhibitors Acute intestinal inflammation injury was carried out using the dextran sodium sulfate (DSS) model.
  • DSS dextran sodium sulfate
  • RIPK2 inhibitor 1 may be efficiently metabolized within 2 hours and cleared from the blood (Figure 12D, bottom panel). Furthermore, toxicity analysis carried out 9 days after the last dose of intraperitoneal injection of 2 pg/g body weight revealed little of no changes is > 98% of the markers characterized in a complete blood count ( Figure 13). However, creatine kinase activity was significantly elevated in animals treated with RIPK2 inhibitor 1 as opposed to RIPK2 inhibitor 2. During treatment with RI PK2 inhibitor 1 (and post-treatment) phenotypic changes were not overtly observed in these animals, nor evidence of health conditions to support a creatine kinase abnormality (such as skeletal muscle defect and movement abnormalities or unexpected euthanasia
  • creatine/phosphocreatine pathway may play a central role in energy metabolism and nutritional creatine supplementation has been shown to impart beneficial effects in a number of diverse disease pathologies (Kitzenberg et al., 2016). It was speculated that this was the case as resetting of metabolic abnormalities is needed for recovery of IBD patients that have metabolic syndrome disorder ⁇ Goncalves et al., 2015).
  • RIPK2 Inhibitors Can Also Efficiently Resolve Lung Inflammation in an Asthma Model.
  • RIPK2 has also been implicated in allergic airway inflammation.
  • RIPK2 gene silencing in the airways decreased allergic airway inflammation in an ovalbumin-mediated mouse model of asthma (Jun et a)., 2013).
  • an association between a RIPK2 promoter polymorphism and childhood severe asthma has been shown in a Japanese population (Nakashima et al. , 2006).
  • RIPK2 Inhibitors Comparison with Recently Identified RIPK2 Inhibitors. Recently, five inhibitors to RIPK2 were identified as OD36/OD38 (Tigno-Aranjuez et al., 2014) WEHI- 435 (Nachbur et al , 2015), GSK-583/GSK-2983559 (Haile et al , 2016) and Novartis (He et al., 2017) OD36 and OD38 were obtained through a small molecule macrocylization process from Oncodesign.
  • IC $ o values of ⁇ 100 nM and the ability to interfere with MDP dependent RIPK2 activity WEHi-435 was obtained by analysis of the RIPK2/ponatinb structure and the necrostatin-1 /murine RIPK1 structure to obtain a structural face for the murine RIPK2 kinase domain (18-249) (Nachbur et al., 2015). Using this structural face, the utilized computational biology to obtain small molecules to associate with the RIPK2 ATP-binding pocket, GSK-583 was obtained using structural comparisons of RIPK2 with ponatinib. Lastly, the Novartis RIPK2 inhibitor was obtained in a similar manner to GSK583 and was based on a proprietary chemical library screen. Several hits were obtained and after structural optimization a RIPK2 inhibitor was obtained to inhibit RIPK2 kmase activity at 3 nM (He et al. : 2017).
  • toxicity analysis revealed little or no changes in > 98% of the markers characterized in a complete blood count.
  • creatine kinase activity was significantly elevated in animals treated with RIPK2 inhibitor 1 as opposed to RIPK2 inhibitor 2.
  • RIPK2 inhibitor 1 and post-treatment, phenotypic changes were not overtly observed in these animals nor was it noticed that there was evidence of health conditions to support a creatine kinase abnormality (such as skeletal muscle defect and movement abnormalities or unexpected euthanasia due to a cardiac abnormality).
  • RIPK2 inhibitor 1 can also inhibit the growth of Hodgkins’ lymphoma cells (A) but not the growth of non-Hodgkin's lymphoma cells (Pfeifer or BL2) nor an ALL cell line, Jurkat T cells (B). Curiously in (B), RIPK2 inhibitor 1 can inhibit the growth of relapse NHL cell lines, a condition that may promote a robust activation of R1 PK2. It was confirmed by immunoblot and in patient lymph node tissue sections that RIPK2 is not significantly elevated in ALL or primary NHL Thus, a RIPK2 inhibitor will not have an effect on growth rate in cell lines from ALL or primary NHL patients. All MTT assays were carried out with 100 nM of RIPK2 inhibitors with drug was added on day 1 and day 3. For blood cancer cells, all cells were counted using a hemocytometer on day 5 post-plating.
  • Rassf1a* A animals were also treated at 2 pg/g body weight with in vitro established FDA approved drugs, Gefiinib and Regorafenib, that can also inhibit RIPK2 with ECso values of 7.4 pM and 3.8 nM (Canning et al, 2015)
  • C A representative picture of the longitudinal H&E stained colon section from -/+ RIPK2 inhibitor treated animals (see A for survival curves). Inhibitor treated animals reveal an ordered structure for the crypts whereby the non-RIPK2 inhibitors reveal disrupted crypts and infiltration of immune cells (dense blue dots).
  • NFKB DNA binding assay was carried out using nuclear extracts derived from bone marrow derived macrophages and the l L-6 probe. In vivo injection of RIPK2 inhibitor 1 was efficient in eliminated NFKB DNA binding activity as shown.
  • n 4-6.
  • P values for DSS treated Rassf1 a+/- vs DSS treated Rassf1 a+/- + RIPK2 inhibitor 1 ranged from 0.005 to 0.01.
  • D Colon lysates from -/+ DSS treated animals and DSS treated/RIPK2 inhibitor 1 treated animals were used to immunostain for active RIPK1 using pY 474 RIPK2 phosphoantibody. See Figure 17 for details on the use
  • n 8-10 samples from independent Rassfta-/- animals is shown. Bottom is densitometric quantitation of the phospho-RIPK2 signal.
  • Bottom panel is the quantitation of the IHC result using a modified ImageJ software Y axis in graph denotes“Average density of DAB Areas”.
  • the H-10-/- is an established animal model for inflammatory bowel disease and the Rassffa _A mouse is very sensitive to developing colitis to suggest importance of RASSF1 A in preventing excessive inflammation. It is known that RASSF1 A can restrict pathogen activation of RIPK2 and other elements connected to innate immunity. Please see model in Fig. 31 .
  • Lung inflammation model for Cystic Fibrosis With reference to Figure 21 : Pseudomonas aeruginosa model for lung inflammation.
  • P. aeruginosa (PA) is a common pathogen associated with respiratory-tract infections in diverse clinical settings that induces oxidative stress, accentuates lung injury and elevated lung inflammation.
  • Mice received PBS or 1 X 3 10 6 CFU/mouse P. aeruginosa intratracheaily under anesthesia.
  • BAL was obtained using 1 ml of sterile Hanks’ Balanced Salt Buffer for the measurement of cell count, protein concentration (Panfeng Fu et al. ,
  • R1PK2 inhibitor 1 for P. aeruginosa challenge model of cystic fibrosis may be suitable.
  • RIPK2 has been demonstrated to be involved in the metastatic nature of epithelial cells.
  • Use of the RIPK2 inhibitor to prevent the spread of cancer cells in an extravasation assay or an assay of invasion was explored. This was carried out using the chick embryo chorioallantoic membrane (CAM) model, a low cost- and time-efficient model used in vivo for cancer research, especially for metastasis.
  • the CAM is immunodeficient early during hatching and therefore can tolerate the transplantation of human tumor cells (in a very similar way xenograft assays are carried out on immunocompromised mice).
  • cancer cell extravasation assay was carried out in two different cell types, Hep3, a human squamous carcinoma cell and in MDA-MB 231 , a triple negative breast cancer cell.
  • the left panels are 2D optical slices of chicken vasculature plus cancer cells. Vasculat re is grey with "black hole looking areas" being the outside of the vessels.
  • Right panels are 3D reconstructions of cells within the dashed squares. Red arrows point to the cell that are extravasated, white to the ones that are still in.
  • (B) is quantification of % cells extravasated as described in https.V/www. ncbi.nlm.nih gov/pubmed/25176655).
  • RIPK2 inhibitor 1 (Drug 1 ) at 1.5 yg/g body weight can robustly inhibit the movement of both of these cells and thus will interfere with metastasis
  • Beads were prepared according to manufacturer’s recommendation. Cell pellets (containing about 5 million cells) from various cancer cell lines were lysed with RIPA buffer and immunoprecipitated with 15 mI bead volume of RIPK2 inhibitor 1 F- Agarose Beads overnight. Following overnight incubation, beads were washed 2 times with 1 XPBS, followed by a 1 0% SDS-PAGE analysis for bound proteins. As shown in Figure 22, the gel was transferred to PVDF membrane and immunoblotted as indicated.
  • HCT1 16 is a colorectal cancer cell line that can be stimluated to promote the activation of R1 PK2 upon addition of the ligand of the NOD/RIPK2 receptor complex, muramyl dipeptide (MDP)
  • top panel of Figure 22 indicates what associated with R1PK2 Inhibitor
  • NFKB gene reporter assay determination of inhibition of MDP stimufated NFKB activity with RIPK2 inhibitor 1 analogs as stated in Table 5. All drugs in were utilized at 100 nM for 24 hours prior to adding MDP P values for RIPK inhibitor treated vs MDP was ⁇ 0.005 and n 4-10 for all inhibitors.
  • Inhibitor ? is also known as PP121 , a dual inhibitor of receptor tyrosine kinases (RTKs) (IC ⁇ 0.02 pM for Abl, Src, VEGFR-2 and PDGFR) and PI 3-K family kinases (IC S o ⁇
  • the RI PK2 assay was carried out using RIPK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0 1 mM EGTA, 0. 1 % b-mercaptoethanol, 1 mg/ml BSA) and assayed against MBP in a final volume of 25 5 pi containing 50m Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 1 0 mM magnesium acetate and 0 02 mM [33P-y-ATP] (50-1000 cpm/pmoie) and incubated for
  • Mammosphere is a 3-dtmensional formation that better represents how these cells exist in vivo They form spheroids that have established contacts with a substrate that can direct growth and signaling it is very clear that both RIPK2 inhibitor 1 and 7 are efficient in slowing down the growth rate of these spheroids and the limiting their size n for (A) and (C) is 4 and p values ⁇ 0,05.
  • RASSF1 A functions to restrict NFKB activity by interfering with Toll receptor 10 (TLR) activation of NFKB.
  • TLR Toll receptor 10
  • NOD2 another pattern recognition receptor
  • RIPK2 obligate kinase
  • % MDP (Muramyl dipeptide) is a stimulator of the NOD2/RIPK2 pathway that will drive activation of RIPK2.
  • ATP competition may determine how tightly the compound can bind to the ATP binding site This assay was carried out at two concentrations depending on which version of RIPK2 was utilized. Since RIKP2 inhibitor 1 , 1 B, 1 C did not inhibit the full length recombinant RIPK2 until > 200 mM, the ATP competition assay could not be done against full length recombinant RIPK2. For RIPK2 inhibitor 1 , at 200 mM, 85% activity is observed to indicate that ATP can successfully interfere with the inhibition of RSPK2 inhibitor 1 . At 400 mM, 88% activity is observed.
  • R1PK2 inhibitor 1 C at 200 mM, 60% activity is observed to indicate that ATP can not successfully interfere with the inhibition of RIPK2 inhibitor 1 C At 400 pM, 115% activity is observed.
  • RIPK2 inhibitor 7 and GSK298559 at 200 mM, 80% and 105% of the activity is observed to indicate that ATP can successfully interfere with the inhibition of RIPK2 inhibitor 7 and GSK298559.
  • subject refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc. , livestock (e.g, , cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g , mouse, rabbit, rat, guinea pig, etc ), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • livestock e.g, cattle, horses, pigs, sheep, goats, etc.
  • laboratory animals e.g , mouse, rabbit, rat, guinea pig, etc
  • mammals non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal.
  • the subject is a human.
  • treatment refers to obtaining beneficial or desired results, including clinical resuits.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating” and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • amelioration or “ameliorates” as used herein refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
  • symptom of a disease or disorder is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease.
  • a “treatment regimen” as used herein refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication
  • aliphatic refers to hydrocarbon moieties that are linear, branched or cyclic, may be alkyl, alkyiene, alkenyl, alkenylene, or alkynyl, alkynylene and may be substituted or unsubstituted.
  • Alkyl or alkyiene refers to a linear, branched or cyclic saturated hydrocarbon group
  • Alkenyl or “alkenylene” means a hydrocarbon moiety that is linear, branched or cyclic and contains at least one carbon to carbon double bond.
  • alkynyl or“alkynylene” means a hydrocarbon moiety that is linear, branched or cyclic and contains at least one carbon to carbon triple bond.
  • [inker” refers to a divalent moiety that bonds two molecular or atomic species by a covalent bond.
  • Compounds described herein may also include their isotopicallydabe!led forms.
  • An isotopica!ly-!abelled form of an active agent of a combination of the present invention is identical to said active agent but for the fact that one or more atoms of said active agent have been replaced by an atom or atoms having an atomic mass or mass number different from the atomic mass or mass number of said atom which is usually found in nature.
  • isotopes which are readily available commercially and which can be incorporated into an active agent of a combination of the present invention in accordance with well established procedures, include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, e.g., 2 H, 3 H, 13 C, 14 C, 15 N, 1 S 0, 17 0, 31 P, 32 P, 35 S, 1 B F, and 36 CI, respectively.
  • An active agent of a combination of the present invention, a prodrug thereof, or a pharmaceutically acceptable salt of either which contains one or more of the above-mentioned Isotopes and/or other isotopes of other atoms is contemplated to be within the scope of the present invention.
  • the invention includes the use of any compounds of described above containing one or more asymmetric carbon atoms may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Isomers shall be defined as being enantiomers and diastereomers. All such isomeric forms of these compounds are expressly included in the present invention.
  • Each stereogenic carbon may be in the R or S configuration, or a combination of
  • alkyl, aikenyl, and alkynyl groups shall be understood as being branched or unbranched where structurally possible and unless otherwise specified.
  • the term“alkyl” refers to both branched and unbranched alkyl groups. It should be understood that any combination term using an“aik” or“alkyl” prefix refers to analogs according to the above definition of“alkyl” For example, terms such as “alkoxy”,“alkythio" refer to
  • alkyl groups linked to a second group via an oxygen or sulfur atom “Alkanoyl” refers to an alkyl group linked to a carbonyl group (C— O).
  • N is not substituted then it Is NH.
  • nitrogen and sulfur include any oxidized form of nitrogen and sulfur and the quaternized form of any basic nitrogen.
  • a— S—C -s alkyl radical unless otherwise specified, shall be understood to include— S ⁇ 0)— Ci. s alkyl and— S(0) 2 — Ch-e alkyl
  • aryl refers to aromatic hydrocarbon rings containing from six to ten carbon ring atoms.
  • the term aryl includes monocyclic rings and bicyclic rings where at least one of the rings is aromatic.
  • Non-limiting examples of C 3 -io aryls include phenyl, indanyl, indenyl, benzocyclobutanyl, dihydronaphthyl, tetrahydronaphthyl, naphthyl, benzocycloheptanyl and benzocycloheptenyl.
  • heterocycle refers to a stable nonaromatic 4-8 membered monocyclic heterocyclic radical or a stable nonaromatic 6 to 1 1-membered fused bicyclic, bridged bicyclic or spirocyclic heterocyclic radical.
  • the 5 to 11-membered heterocycle consists of carbon atoms and one or more, preferably from one to four heteroatoms chosen from nitrogen, oxygen and sulfur.
  • the heterocycle may be either saturated or partially unsaturated.
  • Non-limiting examples of nonaromatic 4-8 membered monocyclic heterocyclic radicals include tetrahydrofuranyl, azetidinyl, pyrrolidinyl, pyranyl, tetrahydropyranyl, dioxanyl, thiomorpholinyl, 1 ,1-dioxo-1A 0 -thiomorpholinyl, morpholinyl, piperidinyl, piperazinyl, and azepinyl.
  • Non-!imiting examples of nonaromatic 6 to 1 1- membered fused bicyclic radicals include octahydroindolyl, octahydrobenzofuranyl, and octahydrobenzothiophenyl.
  • Non-limiting examples of nonaromatic 6 to 11-membered bridged bicyclic radicals include 2-azabicyclo[2.2.1]heptanyl, 3-azabicyclo[3.1 0]hexanyi, and 3-azabicyclo[3.2 1 ]octanyl.
  • Non-limiting examples of nonaromatic 6 to 1 1 -membered spirocyclic heterocyclic radicals include 7-aza-spiro[3,3]heptanyl, 7-$piro[3 4]octanyl, and 7-aza-spiro[3,4]octanyl.
  • heteroaryl shall be understood to mean an aromatic 5 to 6- membered monocyclic heteroaryl or an aromatic 7 to 1 1 -membered heteroaryl bicyclic ring where at least one of the rings is aromatic, wherein the heteroaryl ring contains 1 -4 heteroatoms such as N, O and S.
  • Non-limiting examples of 5 to 6-membered monocyclic heteroaryl rings include furanyi, oxazolyl, isoxazoly!, oxadiazolyl, thiazoiyl, pyrazolyl, pyrrolyl, imidazolyl, tetrazolyl, triazolyl, thienyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, and purinyl.
  • Non-limiting examples of 7 to 1 1-membered heteroaryl bicyclic heteroaryl rings include benzimidazolyl, quinolinyl, dihydro-2H- quinoliny!, isoquinolinyl, quinazolinyl, indazo!yl, thieno[2, 3-d]pyrim idinyl, indolyl, isoindolyl, benzofurany!, benzopyranyi, benzodioxolyf, benzoxazoiyl, benzothiazo!yl, dihydroindolyl, azaindolyl, benzothiazoly!, benzpyrrolyl, benzpyrazolyl, pyridopyrazolyl,
  • heteroatom as used herein shall be understood to mean atoms other than carbon such as O, N, and S.
  • haiogen as used in the present specification shall be understood to mean bromine, chlorine, fluorine or iodine.
  • alkyl a non-limiting example would be— CH2CHF2,— CF 3 etc.
  • the invention includes pharmaceutically acceptable derivatives of the compounds described herein
  • A“pharmaceutically acceptable derivative” refers to any pharmaceutically acceptable salt or ester, or any other compound which, upon administration to a patient, is capable of providing (directly or indirectly) a compound useful for the invention, or a pharmacologically active metabolite or pharmacologically active residue thereof
  • a pharmacologically active metabolite shall be understood to mean any compound of the invention capable of being metabolized enzymatically or chemically. This includes, for example, hydroxylated or oxidized derivative compounds described herein.
  • prodrugs of compounds described herein Prodrugs include those compounds that, upon simple chemical transformation, are modified to produce compounds described herein.
  • Simple chemical transformations include hydrolysis, oxidation and reduction. Specifically, when a prodrug is administered to a patient, the prodrug may be transformed into a compound disclosed hereinabove, thereby imparting the desired pharmacological effect.
  • Pharmaceutically acceptable salts of the compounds may include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
  • acids examples include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfuric, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfuric and benzenesuifonic acids.
  • Other acids such as oxalic acid, while not themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds and their pharmaceutically acceptable acid addition salts.
  • Salts derived from appropriate bases include alkali metal (e g , sodium), alkaline earth metal (e.g , magnesium), ammonium and N— (C1-C4 alkyl) ⁇ + salts.
  • references in the specification to "one embodiment”, “an embodiment”, etc , indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to combine, affect or connect such aspect, feature, structure, or characteristic with other embodiments, whether or not such connection or combination is explicitly described. In other words, any element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility between the two, or it is specifically excluded
  • ail ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values.
  • a recited range e.g., weight percents or carbon groups
  • Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • ChemmineR a compound mining framework for R Bioinformatics 24, 1733-1734. Cheng L et al, NOD2 promotes dopaminergic degeneration regulated by NADPH oxidase 2 in 6-hydroxydopamine model of Parkinson’s Disease, Neuroinflammation 2018
  • NMRPipe a multidimensional spectral processing system based on UNIX pipes. Journal of biomolecular NMR 6, 277-293.
  • Metabolic inflammation in inflammatory bowel disease crosstalk between adipose tissue and bowel
  • the tumor suppressor gene, RASSF1 A is essential for protection against inflammation - induced injury.
  • Kitzenberg, D , Colgan, S.P., and Glover, L. E. (2016). Creatine kinase in ischemic and inflammatory disorders. Clinical and translational medicine 5, 31.
  • FAF-Drugs3 a web server for compound property calculation and chemical library design. Nucleic Acids Res 43, W200-207.
  • a RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production Nature communications 6, 6442.
  • CD90(+) Stromal Cells are Non-Professional Innate Immune Effectors of the Human Colonic Mucosa. Frontiers in immunology 4, 307
  • NOD2/RIPK2 signaling regulates IL-17 axis and contributes to the development of experimental arthritis. Journal of immunology 188, 51 16-5122.

Abstract

Compounds which inhibits RIPK2, and compositions including such compounds, as well as uses, methods and kits for treating a subject with, or suspected of having, inflammation, an inflammatory disorder and/or a cancer. The invention relates to compounds of Formulas (I), (II), (III), (IV) or (V), as defined herein and the pharmaceutically acceptable salts thereof.

Description

RIPK2 INHIBITORS
FIELD
[0001] This disclosure relates generally to compounds, compositions, methods, and kits, for treating a subject with, or suspected of having, inflammation, an inflammatory disorder and/or cancer.
BACKGROUND
[0002] Inflammation is a complex defense mechanism against biological and chemical insults which is largely beneficial. However, persistent inflammation can cause cellular damage resulting in many diseases including, for example, inflammatory bowel disease (IBD) and CRC (colorectal cancer). About 15-20% of all cancer cases are preceded by chronic inflammation, including chronic IBD leading to CRC.
[0003] Inflammation is characterized by the hyperactivation of transcription factors (such as NF-kB) and elevated production of cytokines (Baumgart and Carding, 2007; de Ridder et al. , 2007) to amplify the inflammatory response. (Xiao and Ghosh, 2005). Activation of NF-kB proceeds through multiple pathways (both classical and non- classical) that includes TNF-R1 and the pathogen recognition pathway involving Toll like receptors (TLR) (Hayden and Ghosh, 2004; Madrid and Baldwin, 2003; Orlowski and Baldwin, 2002) and the NOD family of receptors (NOD1 and NOD2), intracellular pattern recognition receptors.
[0004] NOD2 is mainly stimulated by bacterial products containing muramyl dipeptide (MDP) and requires the obligate kinase, RIPK2, to promote an autophagic response or a non-classical NF-kB activation response (Tigno-Aranjuez et al., 2010). Mice with genetic disruption ol Nod2/Ripk2 have a dysbiotic intestinal flora resulting in altered susceptibility to intestinal inflammation (Ermann et a!., 2014). In addition, the loss of Ripk2 has been demonstrated to result in the inability of cells to carry out mitophagy leading to enhanced mitochondrial production of superoxide/reactive oxygen species and accumulation of damaged mitochondria that will trigger a capase-11 dependent
[nflammosome activation (Lupfer et al., 2013; Lupfer et al , 2014).
[0005) in the context of malignant transformation, knockdown of Ripk2 down- regulated RNA expression of E-cadherin and vimentin, proteins involved in epitheliai-to- mesenchymal transition (EMT) and the promotion of the metastatic phenotype (Wu et al., 201 2). RIPK2 might thus play an important role in cell migration (Wu et al., 2012) and
Figure imgf000003_0001
metastasis (Wu et a!., 2012) and offer alternate novel therapeutics for abnormal inflammation driven by the NOD2/R1PK2 pathway
[0006] Several RIPK2 inhibitors have been characterized over the past decade; however, most RIPK2 inhibitors were not designed to inhibit RIPK2 and, thus, RIPK2 inhibition was an off target effect of the use of these drugs. Compounds known to inhibit R1 PK2 include Geftinib/lressa (ICso at 50 nJVl), DCAM-253 (at ICso at 70 nM), p38 MAPK inhibitor SB20358Q (at IC50 at 50 - 100 nM), Src kinase inhibitor 1 and 1 -NM-PP1 (at IC50 at 26 nM but will inhibit several other targets), Regorafenib, and other protein tyrosine kinases (Canning et al , 201 5) Tignc-Aranjuez et al. (Tigno-Aranjuez et al. , 2014} identified a novel class of RIPK2 inhibitors (OD36 and OD38) that can inhibit 92% kinase activity of RIPK2 at 1 00 nM and can inhibit alleviate inflammation in a rodent model for inflammatory bowel disease. However, like most RIPK2 inhibitors, at 100 nM. > 80% inhibition of Fyn, TGFB2, ALK-2, and Lck was also observed. They concluded that OD36 and OD38 were as effective as Gefitinb for modulating RIPK2 and some forms of inflammation injury promoting colitis. WEHI-435 was demonstrated to delay RIPK2 ubiquitylation and NF-kB activation downstream of NOD2 activation and interfere with cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice (Nachbur et al., 2015).
GlaxoSmithKline has isolated a RIPK2 inhibitor, GSK583 based on using the full RIPK2 protein to screen a DNA-encoded library collection with a fluorescence polarization (FP) based binding assay as a readout. (Haile, Marquis, and Bury et al J Med Chem 2016). This inhibitor bound to the ATP binding pocket of the kinase domain and inhibited RIKP2 with an ICso of 5 - 50 nM depending on the assay. An improved version was generated, GSK2983559, that is currently in a Phase 1 trial on healthy volunteers.
[0007] Novartis has published an RIPK2 inhibitor (compound 8) obtained by virtual screening of their proprietary library that can inhibit RIPK2 with an IC50 of 3 nM (He et al., 2017). No in vivo applicability was analyzed but it was shown to selectively inhibit MDP-promoted cytokine production in peripheral blood mononuclear cells and bone marrow derived mouse macrophages. A kinome analysis was not carried out and thus it is unknown if significant off target effects exist.
[0008] Although RIPK2 inhibitors have been identified, most do not perform well when trying to alleviate DSS-induced inflammation injury or colitis-like symptoms in mice.
[0009] This background information is provided for the purpose of making known information believed to be of possible relevance to the present invention. No admission is
Figure imgf000004_0001
necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention,
SU MMARY
[0010] A similarity-based virtual screening and molecular docking analysis was utilized to identify potential RIPK2 inhibitors that can empirically inhibit the autophosphory!ation of RIPK2, and/or inhibit NFKB and alleviate lung and intestinal inflammation. RIPK2 inhibitors described herein did not inhibit RIPK1 activity involved in ferroptosis or cell death nor effect on mitochondrial biology. In addition, the RIPK2 inhibitors described herein appears to also inhibit cell proliferation as determined by MTT assay that does not appear to be promoting cell death. Interestingly, RIPK2 has been demonstrated to be involved in the active growth of CD9G{+) intestinal stromal cells to suggest an “inflammatory” cross talk between intestinal stromal cells and the epithelial cells (Owens et al. , 2013). Inflammation signals from both will drive abnormal states to produce a cytokine storm that fuels malignant growth. Inhibitors to RIPK2 may have promising therapeutic potential to uniquely interfere with NFuB-dependent biology and offer an alternative to existing anti-inflammatory therapies.
[0011] In one aspect, the invention comprises the novel compounds described herein, or the pharmaceutically acceptable salts thereof. In one embodiment, the invention relates to any of the compounds of Formulas I, II, III , IV or V, as defined herein and the pharmaceutically acceptable salts thereof
[0012] In another aspect, the invention comprises pharmaceutical preparations, containing as active substance one or more compounds described herein, or the pharmaceutically acceptable derivatives thereof, optionally combined with conventional excipients and/or carriers.
[0013] In yet another aspect, the invention comprises methods of treating inflammation, an inflammatory disorder, or a cancer in a subject comprising administering to said subject a therapeutically effective amount of a compound described or claimed herein, or a pharmaceutically acceptable salt thereof, or a composition comprising such a compound. Thus, the invention may comprise a compound or composition described herein for use in the treatment of inflammation, an inflammatory disorder, or cancer in a subject.
Figure imgf000005_0001
[0014·] In some embodiments, the inflammation may be associated with inflammatory bowel disease, asthma, obesity, diabetes, cystic fibrosis, psoriasis, arthritis, Parkinson’s Disease, Alzheimer’s Disease or neuropathic pain.
[0015] In some embodiments, the cancer is metastatic cancer, such as metastatic pancreatic or colorectal cancer
[0016] In yet another aspect, the invention may comprise the use of a compound, or a pharmaceutically acceptable salt thereof to treat inflammation, an inflammatory disorder or a cancer in a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Embodiments of the present invention may be described, by way of example only, with reference to the attached Figures.
[0018] Figure 1. (A) Schematic of RIPK2 active site. (B) Representation of pharmacophore points: hydrogen bond donor (HBD), hydrogen bond acceptor (H BA), hydrophobic (Hyd) and aromatic (Ar). Predicted binding modes of (i) ponatinib, (ii) MoiPort-016-359-762 and (iii) MolPort-016-412-727 in RIPK2 binding site (iv)
Comparison between binding position of ponatinib found within the crystal structure and binding mode predicted by AutoDock Vina. Predicted binding modes of (v) PC_57410628 (purple), PC_6834961 1 (brown), and (vi) PC_44716361 (MolPort-001 -746-327, orange) in RIPK2 binding site. All hydrogens were omitted for clarity. Dotted lines represent distances for the formation of hydrogen bonds.
[0019] Figure 2. Mass spectrometry and NMR confirmation of mass and structure of RIPK2 Inhibitor 1 . (A) Direct-infusion measurements were carried out on an LTQ Orbifrap XL (Thermo Scientific) mass spectrometer using the Ion Max ESI source. Mass confirmation was obtained as indicated (B) Two-dimensional 5HJ H ROESY spectrum of 3-benzamido-4-methyl-N-[3-(1 -methy!-1 H-imidazol-2-yl)phenyl]benzamide. Boxes (orange) represents through-space connectivities between protons in the compound. Blue boxes are signals due to 3-bond J-coupling between aromatic ring protons. 4-bond J- coupling signal is less intense.
[0020] Figure 3. Analysis of RIPK2 Kinase Activity. (A) Kinase activity for c-ABL was tested in the presence of inhibitor 1 and 2, lCso for c-ABL for both inhibitors is > 100 mM. Analysis was carried out using purified c-ABL and the substrate peptide,
EAIYAAPFAKKK, in association with the MRC Unit on Phosphorylation and
Ubiquitination, University of Dundee, Scotland. (B) RIPK2 autophosphorylation at site
Figure imgf000006_0001
Y474 is shown inhibition was carried out on cells followed by lysis and
immu noprecipitation (IP) with rabbit anti-RIPK2 overnight. Following protein G puli down and wash. 32P-y-ATP is added and kinase reaction allowed to proceed for 30 minutes at 30 °C. Following kinase reaction, SDS-PAGE was used to separate out the proteins, gel was then dried and exposed to film. (C) RIPK2 in HCT116 ceils were IP with an anti- RIPK2 antibody (A) and the supernatant after IP in (A) was IP in (B) with the same RIPK2 antibody and in vitro kinase (IVK) assay carried out on both samples. (D) Crispr/Cas9 knockout of RI PK2 in HCT1 16 colon cells was carried out followed by MDP activation and RIPK2 IVK analysis To illustrate use of RIPK2 Crispr/Cas9 reagents and specificity of our IVK assay (E) Left panel, Cells were stimulated with MDP for 3 hours and RIPK2 activation was monitored by a phosphotyrosine (pY) 474 R1PK2 antibody (in house antibody) and phosphoserine (pS)176 phosphorylation (Cell Signaling). Right panel, several colon cancer cells were investigated for constitutive activation of RIPK2 using phosphorylation specific antibodies to pY474 and pS176 as indicated. Bottom, constitutive activation of RIPK2 in numerous cell lines are explored.
[0021] Figure 4. Characterization of RIPK2 kinase Inhibitors. (A) Immunoblot illustration of use of RIPK2 phosphoantibodies in two Hodgkin’s lymphoma cells that have constitutive active RIPK2. A comparison with a known RIPK2 inhibitor is shown
(regorafenib). All inhibitor concentrations were 100 nM. Similar results were observed for a third Hodgkin's lymphoma cell line, KMH2. (B) KMH2 Hodgkin's lymphoma cells were inhibited in vivo for 33-36 hours using the indicated concentration of RIPK2 inhibitor 1 . Top, an in vitro kinase assay was carried out by IP overnight with 1 pg of the rabbit-anti RIPK2 antibody from ProteinTech and 1 ml of lysate form a confluent 6 well dish of KMH2 cells. Immune complexes were separated by SDS-PAGE and captured by
autoradiography. Bottom, quantitation of 3-5 independent experiments to reveal an IC50 of 5-10 nM for in vivo RIPK2 inhibition. (C) Analysis of effect of drug solvent (DMSO), salt concentration and detergents on the kinase activity of RIPK2. Following IP, the indicated reagents were added for 10 minutes followed by 32-g-ATR to initiate the kinase reaction as described in Example 5. (D) RIPK2 in vitro kinase assay carried out as in Figure 3B in KMH2 and HCT116 cells in presence of ponatinib or RIPK2 inhibitor 1 in order to carry out a comparison of RIPK2 inhibition.
[0022] Figure 5. RIPK2 Inhibition of NFkB Activity. (A-B) NFkB gene reporter assay determination of inhibition of MDP stimulated NFkB activity with RIPK2 inhibitors (A) and IC50 determination for inhibition of MDP driven-NFkB activation using RIPK2
Figure imgf000007_0001
inhibitors 1 and 2 (B). All drugs in (A) were utilized at 100 nM P values for RIPK inhibitor treated vs MDP (no drug) = < 0.0001 (inhibitor 1 ), 0.06 for inhibitor 2, < 0.006 (Gefitinib), 0.0002 (Regorafinib), and 0.0008 (Ponatinib). For all n = 4-1 0 and IC50 curves are shown in (B) (C) Left panel, NFkB gene reporter assay determination of inhibition of LPS stimulated NFkB activity with RIPK2 inhibitors (concentration as indicated). P values for RIPK2 inhibitor treated vs LPS (no drug) is < 0.02 (inhibitor 1 ) and < 0 268 (for inhibitor 2) (n = 4-6). Right panel, IC50 determination for inhibition of LPS-NFkB driven inflammation using RIPK2 inhibitor 1 and 2 (B).
[0023] Figure 6. In vivo inhibition of proliferation, intestinal and lung inflammation using RIPK2 inhibitors. (A) RI PK2 Inhibitors modulate proliferation of several colon cancers but not normal cells. MTT assay was carried out with indicated concentrations of RIPK2 inhibitors in indicated cell lines. HCT116 and DLD-1 are colon cancer cell lines while RAT-1 and MODEK are normal rat and mouse intestinal cell lines, respectively. RIPK2 inhibitor 1 does not appear to interfere with proliferation of normal intestinal cells (Rat-1 and ModeK) at 45 nM. For all experiments, n = 5 - 29. P value of HCT116 (+ inhibitor 1 ) versus HCT1 16 (+ inhibitor 2) was < 0.03; DLD-1 (+ inhibitor 1) versus DLD-1 (+inhibitor 2) was < 0.02; DLD-1 (+ inhibitor 1 ) versus DLD-1 (+ Regorafenib) was < 0.006 For RIPK2 inhibitor 1 treated DLD-1 or HCT116 vs RIPK2 inhibitor 1 treated ModeK or Rat-1 cells (normal cells), p < 0,002 (analysis in either cell type); For RIPK2 inhibitor 2 treated DLD-1 or HCT116 vs RIPK2 inhibitor 2 treated ModeK cells (normal cells), p < 0.02 and 0.098 (analysis in DLD-1 or HCT116 cells respectively); for
Regorafenib treated DLD-1 vs Regorafenib treated ModeK (normal cells), p < 0 24. (B) Intestinal inflammation injury was carried out using the dextran sodium sulfate (DSS) model. DSS is taken up in the drinking water and migrates to the colon to cause irritation and localized inflammation Rassfl a-/- mice are extremely sensitive to this model with < 25% survival following a 3% DSS insult for 7 days followed by water for 7 days. Most Rassfla-/- mice require euthanasia by day 7-9 Disease activity is scored based on rectal bleeding, piloerection, movement and body weight changes as described previously (Madsen et al , 2001 ). N = 10 -25 and p value of Rassfl a-/- (DSS) vs WT (DSS) was < 0.0001 ; Rassfl a-/- (DSS) vs 1 a-/- (DSS + inhibitor 1 or 2 or gefitinib) was < 0.0001. (C) In vivo inhibition of lung inflammation in an ovalbumin-induced asthma model in mice. Sensitization and challenge with ovalbumin in this model induces accumulation of inflammatory cells in the BAL that are primarily eosinophils Administration of RIPK2 inhibitor-1 significantly decreased both the number of total cells and the number of
Figure imgf000008_0001
eosinophils in the BAL fluid (n=8 for each group). P value for comparison between OVA alone and OVA+RIPK2 inhibitor-1 was 0.019 for total cell numbers and 0.003 for eosinophils Please note that RIPK2 inhibitor 2 was not utilized in this experiment due to its higher 1C50 for NFkB (see Fig 65B), (D) Model for disruption of NOD2/RIPK2 biology in the presence of RIPK2 inhibitor 1. RIPK2 is activated via tyrosine (Y) (pY474) and serine (S) (pS176) phosphorylation and ubiquitination events to allow for associations with downstream components RIPK2 inhibitor 1 inhibits phosphorylation at serine 176 and tyrosine 474 and possible inhibition of ubiquitination of RIPK2.
[0024] Figure 7. (A) Ή-NMR chemical shift assignment of RIPK1 inhibitor 1 :
10.32 (s, 1 H), 10.09 (s, 1 H), 8.13 (t, J = 2 Hz, 1 H), 8.02 (m, 3H), 7.88 (dt, J = 8 Hz, 2 Hz, 1 H) 7.80 (dd, J = 8 Hz, 2Hz, 1 H), 7.60-7 64 (m, 1 H) 7.52-7.57 (m, 2H) 7.44-7,48 (m, 2H} 7.42 (dt, J = 8 Hz, 2 Hz, 1 H) 7.26 (d, J = 2 Hz, 1 H) 6.94 (d, J = 2 Hz, 1 H) 3.78 (s, 3H)
2.32 (s, 3H) ppm (B) Analysis of through-space connectivities suggested that RIPK2 inhibitor 1 has two major rotomeric forms in d6-DMSO. The second rotomer is slightly more favored, perhaps due to the formation of an intramolecular hydrogen bond
[0025] Figure 8. (A) 1 H-NMR chemical shift assignment of RIPK1 inhibitor 2: 9.6
(s, 1 H), 8.31 (s, 1 H), 8.00 (dd, J = 9 Hz, 2Hz, 1 H), 7.86-7.93 (m, 3H), 7.45-7.52 (m, 2H), 7.37 (d, J = 9 Hz, 2H), 7.09 (d, J = 9 Hz, 2H), 6.9 (s, 1 H), 4.32 (br, 1 H), 4.10 (br, J = 12 Hz, 5 Hz, 2H) 3.8 (s, 3H), 3.02-3.14 (br, 2H), 2.85-2.92 (br, 2H), 2.78 (sep, J = 7Hz, 1 H), 1 ,9-1 .97 (br, 1 H), 1 80-1.63 (br, 2H), 1.44-1 .54 (br, 2H), 1.40-1.29 (br, 2H), 1.20-1 .26 (br,
1 H), 1 .1 5 (d, J = 7 Hz, 6H) ppm. Top, enlarged aromatic region; Bottom, full 1 D. Note that 1 H signals attached to C17-C25 are broadened by conformational exchange, indicating conformational transitions within the fused piperidine ring structure (B) Twodimensional 1H-1H ROESY spectrum of [(2R,4S,5R)-5-[1 -methyl-3-(naphthalen-2-yl)-1 H-pyrazol-5-yl]- 1-azabicyclo[2.2.2]octan-2-yl]methy[ N-[4-(propan-2-yl)phenyl]carbamate showing through-bond and through-space connections between hydrogen atoms. Boxes (blue) indicate cross peak signals due to 3-bond and 4-bond J-coupling. Signals due to 4-bond J- coupling are less intense. Orange boxes denote through-space ROE connectivities.
[0026] Figure 9. Related to Figure 2. Mass spectrometry confirmation of mass for
RIPK2 Inhibitor 2 (A) Direct-infusion measurements were carried out on an LTQ Orbitrap XL (Thermo Scientific) mass spectrometer using the Ion Max ESI source. Mass confirmation was obtained as indicated (B) NFkB gene reporter assay of inhibition of NFkB activity with RIPK2 inhibitor 1 (concentration as indicated and several stimuli presented). P values for TNFct treatment was < 0 4; IL-1 b was < 0.0007 and IFN-y was <
Figure imgf000009_0001
0.07 (for inhibitor 1 treated vs non-treated). (C) RIPK2 inhibitor 1 effect on the activation of hypoxia response element (HRE) activation following 1 % H O addition overnight. P value for 1 % H O (treated with inhibitor 1 vs no drug) was < 0 02 (n = 3).
[0027] Figure 10. Related to Figure 5. Effect of RIPK2 inhibitors on NFkB, cell growth and cell cycle control. (A) EMSA was carried out using the NFkB binding site on the IL-6 promoter. HCT1 16 cells were pretreated with the indicated RIPK2 inhibitors for 2 days, followed by stimulation with MDP and nuclear/cytoplasmic extraction. 4 pg of nuclear extract was incubated with the IL-6 probe in an NFkB DNA binding assay (B) R1PK2 Inhibitor 1 can modulate the growth of several breast cancer cells. MTT assay was carried out with the 100 nM of RIPK2 inhibitors in the indicated breast cancer cell lines with HCT1 143 and MDA-MB31 as triple negative breast cancer cells and JMT-1 is a Fler2 overexpressing cell line. For all experiments, n = 4 - 10. P value -/+ RIPK2 inhibitor 1 for JMT-1 was 0.0001 , for FICT 1143 was 0 0007 and for MDA-MB231 was 0.05. (C) Growth inhibition MTT curves for FICT1 16 (A) and BT-549 (B, breast cancer cells} were examined in the presence of RIPK2 inhibitor 1 at the concentrations indicated and ICso
concentrations determined an indicated on top right portion of the graph.
[0028] Figure 1 1. RIPK2 inhibitor 1 does not affect the cell cycle fraction distribution at 100 nM. HCT-1 16 colon (A) or MCF-7 breast (B) cancer cells were treated with Vehicle or the indicated inhibitors (5 mM) for 36 hours. After an overnight ethanol fixation, celts were PI stained 15000 cells were analyzed on BD Accuri flow cytometer. Different cell cycle phases were quantitated for 3 independent experiments and plotted as percentage of mean ± SD (far right A and B) No major difference was observed between treatments. The data for 5 mM of the drug is presented but similar results were observed at 100 nM RIPK2 inhibitor 1 drug treated cells.
[0029] Figure 12. Related to Figure 3, 4 and 5 RIPK2 inhibitors do not inhibit
RIP1 or RIP3 Function. (A) Ferroptosis was measured in A549 lung cancer cells following treatment with Erastin. After 48 hours, cells were harvested, resuspended in PBS and stained with 0 04% trypan blue for 5-10 minutes af room temperature Cell death was quantified by flow cytometry using the FACSCalibur flow cytometer and CeilQuest software. P value for Erastin vs DMSO was < 0.0001 ; No significant differences were observed for Erastin vs. Erastin + inhibitor (1 or 2) (N=6). Similar results were obtained in the U87 cells. (B) R1PK1 activity was measured in FIT-29 cells by activation of ceil death using 20 mM z-VAD-fmk (Z), 100 nM Smac Mimetic-164 (S) and 10 ng/mL TNFcr (T) combination in the presence or absence of 100 nM Ripki inhibitor or 1 mM
Figure imgf000010_0001
necrosulfanamide (NSA) or the indicated amounts of RIPK2 inhibitors for 24 hours. After the indicated time, the cells were double stained with hoechst and propidiu iodide for 30 min at 37 °C and then cell death was measured using the Celigo cell cytometer. The effect of the RIPK2 inhibitors on RIPK2-directed cell death in L929 and HaCaT cells showed similar results P value for ZST vs DMSO was < 0.0001. No significant differences were observed for ZST vs. ZST + inhibitor (1 or 2). (C) Characterization of Sirtuin activity using the BIOMOL {Enzo Life Sciences} assay to monitor NAD activity. (D) Preliminary pharmacokenetics in mouse sera revealed a half-life of 1.89 hour and possible rapid metabolism of RIPK2 inhibitor 1 following 15 pg/g intraperitoneal injection. Upper panel, Chromatograms from untreated mouse serum (bottom trace) and from a mouse serum sample containing 500 ng/mL of inhibitor 1 (upper trace). The inhibitor eluted at 3.7 min. Lower panel, mean ± SD serum concentration vs. time measures of Inhibitor 1 in sera from mice.
[0030] Figure 13. Related to Table 4 and Figure 6. Mice were exposed to 7 days of 3% DSS to induce inflammation injury and 7 days with water to promote recovery. RIPK2 inhibitors were intraperitoneal (IP) injected on day 5, 7 and 9 Serum was harvested 1 1 days after last IP injection and blood chemistry was carried out at the Prairie Diagnostics for numerous factors. The ones shown have some significant changes. For alkaline phosphatase, Two-way Anova reveals P value < 0.0001 for all measured marker [0031] Figure 14. Characterization of Identified RIPK2 Inhibitors.
[0032] Figure 15. RIPK2 Inhibition of NFKB, a key transcription factor driving inflammation.
[0033] Figure 16. Characterization of Growth Suppressive Function of Identified
RIPK2 Inhibitors
[0034] Figure 17. Identified RIPK2 Inhibitors are effective in alleviating in vivo inflammation.
[0035] Figure 18 Further characterization of R1PK2 inhibitor 1.
[0036] Figure 19. Active RIPK2 can be detected in tissue sections of DSS-treated pre-clinical models of IBD using or proprietary rabbit anti-phosphotyrosine 474 RIPK2 antibody to immunohistochemistry and is a strong driver of in vivo inflammation.
[0037] Figure 20. Clinical characterization of active RIPK2. (A) RIPK2 activity analysis in a pediatric patient with UC RIPK2 IFIC was carried out using he pY 474 RIPK2 antibody used in and the bottom panel, summary of clinical make up of pediatric case study patient with UC. Robust staining for phosphotyrosine R1PK2 was evident in
Figure imgf000011_0001
biopsy 2 and 3 samples Top panel, IHC staining examples. Bottom panel is quantitation of top panel. (B) RIPK2 activity analysis in adult UC and CD patients using RIPK2 IHC was utilising the pY 474 R1PK2 antibody. Right panel is quantitation of the IHC signal.
[0038] Figure 21. Pseudomonas aeruginosa model for lung inflammation.
[0039] Figure 22. An immunoblot showing validation of the use of RIPK2 inhibitor
1 F-Agarose beads
[0040] Figure 23. RIPK2 Inhibitor 1 interfering with cell invasion in Hep3, a human squamous carcinoma cell line, and in MDA231 , a triple negative breast cancer ceil line. Similar results were obtained in the inflammatory breast cancer cell line IBC-3.
[0041] Figure 24. Empirical Testing of Potential off Target Effect on PKB/Akt.
[0042] Figure 25. Comparison of Analogs with Parent R1PK2 Inhibitor 1.
[0043] Figure 26 Kinase assay for RIPK2 -/+ RIPK2 inhibitor 7.
[0044] Figure 27. RIPK2 autophosphorylation analysis.
[0045] Figure 28. NFKB gene reporter assay determination of inhibition of MDP stimulated NFKB activity with RIPK2 inhibitor 5 and 6 as in Fig 6A at the indicated concentrations.
[0046] Figure 29. (A) NFKB gene reporter assay determination of inhibition of
MDP stimulated N FKB activity with R1PK2 inhibitor 7. (B) EMSA was carried out using the NFKB binding site on the IL-6 and IL-8 promoter. (C) Mammosphere formation in KPL-4 inflammatory breast cancer cell -/+ RIPK2 inhibitor 1 and 7.
[0047] Figure 30 Clinical characterization of RIPK2 Inhibitor 7.
[0048] Figure 31 . Model for intestinal Inflammation involving RASSF1 A
[0049] Figure 32. Biological characterization of RIPK2 Inhibitors. Test of RIPK2 inhibitor-1 modulation of RIPK2 autophosphorylation at Y474 in HL cells (HDMYZ and L428) and RIPK2 activity in an NHL (DOHH2) and primary AML (HL-60) cell lines Inhibition was carried out on cells followed bY lysis and IP with rabbit anti-RIPK2 overnight. Following protein G pull down and wash, 32-P-y-ATP is added and kinase reaction allowed to proceed for 30 minutes at 30 C. Following kinase reaction, SDS- PAGE was used to separate out the proteins, gel was then dried and exposed to film.
[0050] Figure 33. Characterization of RIPK2 Inhibitors 6, 7 and gtiquidione was determined at the International Kinase Profiling Center in Dundee, Scotland. Substrate for RIPK2 was myelin basic protein and a peptide substrate (EAIYAAPFAKKK) for ABL in vitro kinase assay. RIPK2 utilized was truncated recombinant RIPK2 (2-31 1 )
- I Q - [0051] Figure 34 NFkB and growth inhibition of RIPK2 Inhibitors Gene reporter analysis of MDP treated HCT116 cells treated with herein described RIPK2 inhibitors and compared to a recently characterized R1 PK2 inhibitor, regorafenib P values for most are < 0 05 (n = 5 for all).
[0052] Figure 35. TCGA analysis of RIPK2 expression in indicated cancers.
RIPK2 is overexpressed in cancers
[0053] Figure 36. Identification of RIPK2 inhibitors using Computational
Biology/Molecular Docking studies, and characterization of R1PK2 Inhibitors.
[0054] Figure 37. Extraction and quantitation of RIPK2 Inhibitor in rat plasma.
Extraction and quantition of RIPK2 inhibitor 1 in rat plasma. (A) Inhibitor 1 was extracted from 0.1 mL alkalinized rat plasma using liquid-liquid extraction of (3 mL of tert-butyl methyl ether). Chromatography was accomplished with a 15 cm C18 analytical column with a mobile phase of 75% phosphate solution in acetonitrile and UV detection at 261 nm. The chromatogram above shows a sample with 1000 ng/ l inhibitor extracted from rat plasma vs unspiked inhibitor-free rat plasma. (B) The standard curve of detection in rat plasma was highly linear from 25 to 500 ng/mL. A similar assay was developed for RIPK2 inhibitor 2 with chromatography using 15 cm C18 analytical column with a mobile phase of 35% phosphate solution in acetonitrile, UV detection at 290 nm and a standard curve that was highly linear from 50 to 1000 ng/mL.
[0055] Figure 38 NFkB and growth inhibition of RIPK2 inhibitors. MTT assay was carried out with the indicated concentrations of R1PK2 inhibitors in breast cancer cells. RIPK2 inhibitor 1 does not appear to interfere with growth of MCF-7 cells, an early breast cancer luminal A cell. MDA MB231 and BT549 are triple negative (ER-PR-Her2-) cells and SUM1 9 and IBC-3 are inflammatory breast cancer cells (ER-PR-Her2- and ER-PR- HER2+ cells, respectively). For all experiments, n = 4 - 6.
[0056] Figure 39. Identification of RIPK2 inhibitors using computational biology/molecular docking studies.
[0057] Figure 40. Characterization of Other RIPK2 inhibitors
[0058] Figure 41. Summary model of RASSF1A influences on inflammation and cancer signaling.
[0059] Figure 42. Methylation analysis of all 32 CpGs in the promoter region of
RASSF1 A as indicated in I BD and CRC.
[0060] Figure 43. Diagram depicting the pathogensis of IBD subtypes.
[0061] Figure 44 Summary of the characteristics of the RASSF1 gene family.
Figure imgf000013_0001
[0062] Figure 45. Rassfl a animals are sensitive to dextran sodiumsulphate (DSS) treatment. Mice were subjected to 3% DSS solution followed by day 7 replacement with regular water to allow for recovery A Kaplan-Meier curve monitoring % survival following DSS treatment. P value= 0 0001 (WT/Rassf5a vs Rassfl a) and 0.0023 (WT/Rassf5a vs Rassfl a+/); n=12-20 (B) Body weight changes following DSS treatment P value =0.003 (WT/ RassfSa vs Rassfl a); n=12 - 20 Rassfl a+/ animals revealed between 20-25% body weight loss (data not shown).
[0063] Figure 46. A representative picture of DSS treated colons is shown indicating how colon length was measured. DSS-treated Rassfl a+/ revealed a similar loss of colon length (data not shown).
[0064] Figure 47. Longitudinal cross-section of the descending colon stained with
H&E is shown for untreated and DSS-treated animals. All untreated colon sections samples were very similar to untreated colon sections from wild type mice
(doi: 10.1 371 /journal pone.0075483. g001 )
[0065] Figure 48. Characterization of DSS induced inflammation injury in the epithelial and macrophage specific knock outs to RASSF1A. Sibling matches as also characterized that do not contain the macrophage specific knockout
[0066] Figure 49. Rassfl a-'· Rod2 ' mice showed improve survival against DSS.
Mice were subjected to 3% DSS solution followed by day seven replacements with regular water to allow for recovery. Left panel, A Kaplan-Meier curve is monitoring % survival following DSS treatment n =14-43. p-value is < 0.0001 WT vs. Rassfl a ' and Rassfl a ' vs Nod2 ' and Rassfl a ' Nod 2J p-value = 0 0053 (**) Right panel, Disease activity index (DAI) of these mice were monitored following DSS treatment. P value <0.0001 (***), n = 11-25
[0067] Figure 50. Model for pathways driving colonic inflammation. High levels of NFkB transcriptional activity can result in intestinal inflammation and abnormal activation of apoptosis leading to inflammation induced damage. Furthermore, the presence of pathogens can also result in the activation of another pattern recognition receptor, NOD2 and its obligate kinase (R1PK2), to result in NFkB activation and initiation of the autophagic response (right side pathway). We have biochemical evidence that RASSF1 A, a tumor suppressor, can interefere with TLR, NOD2/RIPK2 and TBK1 biology (not shown) in order to restrict NFxB-dependent activation and hence inflammation. RASSF1A is epigenetically silenced in most of human cancers (a known) and in IBD (an unknown). As
Figure imgf000014_0001
such, the lack of RASSF1 A in IBD and CRC patients would result in hyperactive RIPK2, TLR and TBK1 and, in turn, hyperactive NFkB and inflammation.
[0068] Figure 51 . Survival curve in response to DSS-induced inflamamtion injury in the presence of the broad spectrum anti-inflammatory resveratrol.
[0069] Figure 52. Expression of RASSF1 A and active YAP in IBD patients.
RASSF1 A is epigenetically silenced in IBD patients and as such low to no detection of RASSF1A is observed in IBD patients. YAP is a co-transcriptional activator that can drive malignancy.
DETAILED DESCRIPTION
[0070] RIPK2 is the obligate kinase to the NOD2 pathogen receptor pathway and has been demonstrated to be involved in NFkB activation and metastatic behaviour in some cancers (Jun et al., 2013) and has a distinct activity versus RIPK1 , 3 or 4
(Chirieleison et al., 2016). In addition, several reports suggest involvement in the activation of the immune response upon viral infection (Lupfer et al., 2013) and the requirement for the NOD2/RIPK2 molecular pathway in several models on inflammatory diseases including experimental colitis (Branquinho et al., 2016) and arthritis (Vieira et al., 2012). Colorectal cancer (CRC) is only one example of a disease that can arise from a prior state of persistent or chronic inflammation. Individuals with IBD and primary sclerosing cholangitis (inflammation of the bile ducts) are at a much higher risk for progressing to CRC and require closer management of their debilitating disease (Dyson and Rutter, 2012; Williamson and Chapman, 2015). As such, therapies to modulate and control inflammation are robust cancer prevention strategies.
[0071] Pharmacophore and similarity-based search and molecular docking studies were performed toward the identification of novel RIPK2 inhibitors. In the pharmacophore search several molecules were identified that share the pharmacophore arrangement with ponatinib.
[0072] The identified IPK2 inhibitors reveal lower binding free energies that other RIPK2 inhibitors identified suggesting that the inhibitor may be more active
Furthermore, the identified R1PK2 inhibitors appear to be have more affinity for the active site of RIPK2 than others, more effective at inhibition of proliferation and can effectively resolve lung inflammation and intestinal inflammation more robustly than gefitinib (Figure 6B and Table 4). The effectiveness of RIPK2 inhibitor 1 in resolving intestinal
Figure imgf000015_0001
inflammation may arise from the fact that it can have a small but significant effect on TNFa, IL-1 and LPS-driven NFkB activation as seen in Figure 5 and Figure 9B. Most inflammatory diseases are complex diseases that have a multitude of inflammatory pathways targeting the area. Thus, to effectively resolve inflammation in these areas, a broad spectrum inhibitor may be needed to target multiple TLRs and pathogen receptors (Murgueitio et al. , 2017)
[0073] All of the in vivo inhibition was at 1 - 2 pg/g body weight, which is much lower than the level used for most small molecule inhibitors. Preliminary
pharmacokinetics suggest a 1 .89 hour half-life in the blood (Figure 12D) and chronic experiments using R1PK2 inhibitor 1 (data not shown) agree with the preliminary pharmacokinetics to suggest the effectiveness of this inhibitor can last for up to 5-6 days after a 1 -2 pg/g body weight intraperitoneal injection.
[0074] It is known that RIPK2 has a unique requirement for NOD1 and NOD2 and functions in many pathways different from RIPK1 , 3 or 4. Recently, Chirieleison et al. (2016) summarized the uniqueness of RIPK2 kinase domain within the RIPK family that could not be substituted for the kinase domain from RIPK1 or RIPK4. Indeed, RIPK2 inhibitors 1 and 2 did not inhibit RIPK1 biology nor ferroptosis, a form of cell death influenced by proteins of the RIPK family (Figure 12A and B). As such, it has been considered that the identified RtPK2 inhibitor 1 and 2 can selectively modulate RIPK2 specific biology in agreement with the observations of Chirieleison et al. (2016).
[0075] Although a robust inhibition of autophosphorylation of RIPK2, NFKB and alleviation of intestinal and airway inflammation injury in mouse models of colitis and asthma was observed, the kinome screen demonstrated in vitro inhibition of several other kinases including 20-30% inhibition of c-ABL, Aurora kinase B or ERBB2. There has been a demonstrated importance of c-ABL in driving inflammation injury in the acute DSS model in the publication of Gordon et a!. (2013). It is likely that RIPK2 inhibitor 1 may interfere with the kinase activity of these off targets indirectly by resolving the inflammation. Many of these kinases are involved in growth related pathways, tumorigenesis pathways and ceil cycle control such as Aurora Kinase B (Borisa and Bhatt, 2017) These off target effects may actually be beneficial in treating IBD, PSC, PSC/IBD-related CRC and asthma whereby a multitude of abnormal signaling involving inflammation, oxidative damage, DMA damage and apoptotic abnormalities exist to manifest itself into a diseased state. Thus, eliminating all of these abnormalities using a single small molecule may promote a return to homeostasis and recovery from
Figure imgf000016_0001
inflammation damage RIPK2 may also be important in driving inflammation damage in other diseases involving inflammation such as obesity, diabetes, multiple sclerosis, cystic fibrosis, psoriasis (NOD-like receptor signaling and inflammasome-related pathways are highlighted in psoriatic epidermis Sci reports 2016), arthritis/osteoarthritis (Jurynec, M. J. et ai, A hyperactivating proinflammatory R1 PK2 allele associated with early-onset osteoarthritis, Hum Mol Genet 2018), Parkinson's (Ma et al, P268S in NOD2 associates with susceptibility to Parkinson’s disease in Chinese population, Behav Brain Funct 201 3 and cheng et al, NOD2 promotes dopaminergic degeneration regulated by NADPH oxidase 2 in d-hydroxydopamine model of Parkinson's disease, J Meuroinflamrmation 201 8) and Alzheimer’s Disease and neuropathic pain (Santa-Cecilia, F V. et al, The
IMOD2 signaling in peripheral macrophages contributes to neuropathic pain development, Pain 2018). It is expected that the RIPK2 inhibitors described herein may also alleviate the clinical symptoms associated with these diseases. Compounds
[0076] I n some aspects, the present invention comprises novel compounds which inhibit the receptor-interacting serine/threonine protein kinase 2 (RIPK2) and are thus useful for treating a variety of diseases and disorders that are mediated or sustained through the activity of RI PK2, including numerous inflammatory and neurological diseases and cancers (including metastatic cancers). This invention also relates to pharmaceutical compositions comprising these compounds, as well as methods of using RIPK2 inhibitor compounds, including in the treatment of various diseases and disorders.
[0077] In one broad embodiment, the invention comprises a compound of Formula I,
Figure imgf000017_0001
wherein Y is C or N;
i is selected from the group consisting of: H, substituted or unsubstituted C -C straight alkyl, substituted or unsubstituted C -C branched alkyl, substituted or unsubstituted C - Ct straight alkenyl, substituted or unsubstituted C -C branched alkenyl, substituted or
Figure imgf000017_0002
unsubstituted C3-Cs cycloalkyl, substituted or unsubstituted Morpholino, Amino, substituted or unsubstituted Benzyl, substituted or unsubstituted Phenyl, substituted or unsubstituted C- -C aryl alkyl, substituted or unsubstituted Heteroaryl, substituted or unsubstituted Arylamino, substituted or unsubstituted Dialkylamino, substituted or unsubstituted Diarylamino, substituted or unsubstituted Carboxyalkylamino, substituted or unsubstituted Carboxydialky!amino, substituted or unsubstituted Tolyl, Xy!yl, Anisyl, Mesityl, Acetoxy, and Hydroxyl;
Rz and R4 are each independently selected from the group consisting of: H, substituted or unsubstituted C -C straight alkyl, substituted or unsubstituted C -C branched alkyl, substituted or unsubstituted C3-Ciz straight alkenyl, substituted or unsubstituted C -C
Figure imgf000018_0001
branched alkenyl, substituted or unsubstituted C8-C8 cycloalkyl, substituted or
unsubstituted Morpholino, substituted or unsubstituted Benzyl, substituted or
unsubstituted Phenyl, substituted or unsubstituted C -C aryl alkyl, and substituted or unsubstituted Heteroaryl;
R3 and Rs are each independently selected from the group consisting of: H , substituted or unsubstituted C -C straight alkyl, substituted or unsubstituted C -Ciz branched alkyl, substituted or unsubstituted C3-Cia straight alkenyl, substituted or unsubstituted C3-Ci2 branched alkenyl, substituted or unsubstituted C -C cycloalkyl, substituted or
unsubstituted Atkoxy, Nitrile, Halogen, substituted or unsubstituted Morpholino, Amino, substituted or unsubstituted Benzyl, substituted or unsubstituted Phenyl, substituted or unsubstituted C -C aryl alkyl, substituted or unsubstituted Heteroaryl, substituted or unsubstituted Arylamino, substituted or unsubstituted Dialkylamino, substituted or unsubstituted Diarylamino, substituted or unsubstituted Carboxyalkylamino, substituted or unsubstituted Carboxydialkylamino, substituted or unsubstituted Tolyl, Xylyl, Anisyl, Mesityl, Acetoxy, Carboxy, substituted or unsubstituted Carboxyethyl, substituted or unsubstituted Alkylcarbonyl, Thiol, substituted or unsubstituted Alkylthiol, substituted or unsubstituted Alkyloxy, substituted or unsubstituted Carboxamido, and Hydroxyl; and
X is selected from the group consisting of; carbon, ortho- Oxygen, mefa-Oxygen, para-Oxygen, orfbo-Nitrogen, mefa-Nitrogen, para-Nitrogen, orfbo-Sulfur, mefa-Sulfur, and para-Sulfur; except that Formula 1 excludes 3-benzamido-4-methyl-N-[3-(1-methyl-1 H-imidazol-2-yl) phenyl]benzamide (RIPK2 Inhibitor 1 ).
Figure imgf000018_0002
[0078] In some examples of Formula I, Y is N and R5 is a substituted or unsubstituted heterocycle:
Figure imgf000019_0001
or
wherein R is H or an substituted or unsubstituted aliphatic moeity, preferably Ci-C alkyl, more preferably C -C alkyl, or Z-R7, where Z is a linker and R7 is a functional group. In some embodiments, R7 is NH .
[0079] In some examples, the compound is a compound of Formula II;
Figure imgf000019_0002
wherein X is a divalent aliphatic or polymeric linker
[0080] In some examples, X is a substituted or unsubstituted C -C alkylene, CID-C2O alkylene, C -C alkenylene, C -C alkenylene, or C -C alkynylene, C -C alkynylene; a substituted or unsubstituted C3-C10 cycloalkylene, C -C cycloalkyfene, C4-C 10 cyc!oalkenylene, C -C cycloalkenylene, or Cio-C2o cycloalkynylene; a substituted or unsubstituted divalent ethylene glycol or a substituted or unsubstituted divalent polyethylene glycol; or, a substituted or unsubstituted divalent ether or a substituted or unsubstituted divalent polyether
[0081] In some examples, the compound is a compound of Formula II selected from the group consisting of:
Figure imgf000019_0003
Figure imgf000020_0001
Figure imgf000021_0001
5
[0082] In some examples, the compound is a compound of Formula III:
Figure imgf000021_0002
Figure imgf000022_0001
{li t)
wherein R6 is H (RIPK2 Inhibitor 1 C) or an substituted or unsubstituted aliphatic moeity, preferably C1-C 0 alkyl, more preferably Ci-Cs alkyl; or Z-R7, where Z is a linker and R7 is a functional group. In some embodiments, R7 is NH2 In some embodiments, Z is a divalent aliphatic or polymeric linker, such as a substituted or unsubstituted C1-C10 alkylene, C10-C20 a!kylene, C2-Ci0 alkenylene, C10-C20 alkeny!ene, or C2-C10 alkynylene, C10-C20 alkynylene; a substituted or unsubstituted C3-C10 cyc!oalkylene, C10-C20 cycloalkylene, C4-C10 cycloalkenylene, C10-C20 cycloalkenylene, or C10-C20 cycloaikynylene; a substituted or unsubstituted ethylene glycol or a substituted or unsubstituted divalent polyethylene glycol; or, a substituted or unsubstituted ether or a substituted or unsubstituted divalent polyether.
[0083] In some examples, R6 is the same linker amine in the derivatives of Formula I! shown above, and accordingly may be selected from the group consisting of:
Figure imgf000022_0002
Figure imgf000023_0001
[0084] In another aspect, the invention may comprise compounds of Formula IV or V:
Figure imgf000023_0002
Figure imgf000024_0001
[RIPK2-inhibitor 1 D] (V)
[0085] Any of the compounds described herein may comprise a tautomer, or a pharmaceutically acceptable salt, or a solvate, or a functional derivative thereof. The term“functional derivative’ as used herein refers to a molecule that retains a biological activity (either function or structural) that is substantially similar to that of the original compound. A functional derivative or equivalent may be a natural derivative or is prepared synthetically.
[0086] Also encompassed is a prodrug or a "physiologically functional derivative" of the compounds described herein. The term "physiologically functional derivative” as used herein refers to compounds which are not pharmaceutically active themselves but which are transformed into their pharmaceutically active form in vivo, i.e. in the subject to which the compound is administered. The term "prodrug" as used herein, refers to a derivative of a substance that, following administration, is metabolized in vivo, e.g by hydrolysis or by processing through an enzyme, into an active metabolite.
General Synthetic Methods
[0087] The compounds described herein may be prepared by the methods and examples presented below and methods Known to those of ordinary skill in the art. In each of the examples below, the groups are as defined above for the various formulae. Optimum reaction conditions and reaction times may vary depending on the particular reactants used Unless otherwise specified, solvents, temperatures, pressures, and other reaction conditions may be readily selected by one of ordinary skill in the art. Specific procedures are provided below intermediates used in the syntheses below are either commercially available or easily prepared by methods known to those skilled in the art. Reaction progress may be monitored by conventional methods such as thin layer chromatography
Figure imgf000024_0002
(TLC) or high pressure liquid chromatography-mass spec {HPLC-MS}. Intermediates and products may be purified by methods known in the art, including column chromatography, HPLC, preparative TLC or Preparatory HPLC
[0088] In one example, a method of synthesizing a compound (such as RIPK2 Inhibitor 1 ), comprises reacting
Figure imgf000025_0001
carboxylic acid, such as 1 1 and amine, such as 2
Figure imgf000025_0002
s in the presence of a coupling reagent
[0089] In another example, a method of synthesizing the compound of Formula III comprises reacting
Figure imgf000025_0003
carboxylic acid 1 and tert-butyloxycarbonyl-protected amine 3
Figure imgf000025_0004
in the presence of a coupling reagent, wherein X is a linker as defined above.
[0090] For example, R1PK2 Inhibitor 1 may be synthesized as follows, and derivatives thereof may be synthesized in an analogous scheme:
Figure imgf000025_0005
Figure imgf000026_0001
EDC = 1 -(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
[0091] In another example, compounds with a linker X may be synthesized as follows, or in an analogous scheme:
Figure imgf000026_0002
Boc = tert-butyioxycarbonyl
General Administration and Pharmaceutical Compositions
[0092] When used as pharmaceuticals, the compounds described herein are typically administered in the form of a pharmaceutical composition. Such compositions can be prepared using procedures well known in the pharmaceutical art and comprise at least one compound described herein. The compounds described herein may also be administered alone or in combination with adjuvants that enhance stability of the compounds, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increased antagonist activity, provide adjunct therapy, and the like. The compounds may be used on their own or in conjunction with other active substances, optionally also in conjunction with other pharmacologically active substances. In general, the compounds described herein are
Figure imgf000026_0003
administered in a therapeutically or pharmaceutically effective amount, but may be administered in lower amounts for diagnostic or other purposes
[0093] Administration of the compounds, in pure form or in an appropriate pharmaceutical composition, can be carried out using any of the accepted modes of administration of pharmaceutical compositions. Thus, administration can be, for example, orally, buccaliy (e.g., sublingually), nasally, parenterally, topically, transdermally, vagtnally, or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages. The pharmaceutical compositions will generally include a conventional pharmaceutical carrier or excipient and a compound described herein as the/an active agent, and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, vehicles, or combinations thereof. Such pharmaceutically acceptable excipients, carriers, or additives as well as methods of making pharmaceutical compositions for various modes or administration are well-known to those of skill in the art. The state of the art is evidenced, e.g , by Remington: The Science and Practice of Pharmacy, 20th Edition, A. Gennaro (ed.), Uppincott Williams & Wilkins, 2000; Handbook of Pharmaceutical Additives, Michael & Irene Ash (eds ), Gower, 1995; Handbook of Pharmaceutical Excipients, A. H. Kibbe (ed.), American Pharmaceutical Ass'n, 2000; H C. Ansel and N. G. Popovish, Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th ed., Lea and Febiger, 1990; each of which is incorporated herein by reference in their entireties to better describe the state of the art
[0094] As one of skill in the art would expect, the forms of the compounds described herein utilized in a particular pharmaceutical formulation will be selected (e.g., salts) that possess suitable physical characteristics (e.g., water solubility) that are required for the formulation to be efficacious.
Use of the Compounds or Compositions
[0095] The compounds described above and also the compounds of Formula l-A, VI , VII, VIII, or IX:
Figure imgf000027_0001
Figure imgf000028_0001
[RIPK2-inbibitor 7] (IX)
may be used for treating a subject with, or suspected of having, inflammation or an inflammatory disorder, a neurological disorder or neuropathic pain. In addition, the
Figure imgf000028_0002
compounds and compositions herein can be used for treating a subject with, or suspected of having, a cancer, especially with metastatic disease.
[0096] In some examples, a RIPK2 inhibitor described herein can significantly interfere with the steps prior to malignant transformation and thus be a robust anti-cancer drug.
[0097] In one aspect, the invention may comprise a method for treating an inflammatory disorder, comprising administering one or more compounds or compositions described herein to a subject having or suspected of having an inflammatory disorder, an acute inflammatory disease or disorder, thereby treating the inflammatory disorder.
[0098] In some examples, the inflammatory disorder is acute, adhesive, atrophic, catarrhal, chronic, cirrhotic, diffuse, disseminated, exudative, fibrinous, fibrosing, focal, granulomatous, hyperplastic, hypertrophic, interstitial, metastatic, necrotic, obliterative, parenchymatous, plastic, productive, proliferous, pseudomembranous, purulent, sclerosing, seropiastic, serous, simple, specific, subacute, suppurative, toxic, traumatic, and/or ulcerative inflammation
[0099] In some examples, the inflammatory disorder is from gastrointestinal disorders (such as peptic ulcers, regional enteritis, diverticulitis, gastrointestinal bleeding, eosinophilic) gastrointestinal disorders (such as, eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis), gastritis, diarrhea,
gastroesophageal reflux disease (GORD, or GERD), inflammatory bowel disease (1BD) (such as Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminate colitis) and inflammatory bowel syndrome (IBS)}.
[00100] In some examples, the inflammatory disorder is a disorder of the lung selected from pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis,
bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, asthma, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis.
[00101] In one example, the inflammatory disorder is multiple sclerosis (MS). Examples of multiple scleoris include, but are not limited to relapsing-remitting MS, secondary-progressive MS, primary-progressive MS, progressive-relapsing MS.
[00102] In another example, a subject with an inflammatory disorder can be treated to provide cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse of the patient from or as a result of the treatment.
Figure imgf000029_0001
[00103] In another example, a subject with Parkinson's disease or Alzheimer’s disease may be treated with an RIPK2 inhibitor, which may slow down the progression of these diseases by controlling the inflammation coming from the gut-brain-axis (Ma ef a!, Behavioral and Brain Function, P268S in NOD2 associates with susceptibility to
Parkinson's disease in a Chinese population; Cheng L et al, NOD2 promotes dopaminergic degeneration regulated by NADPH oxidase 2 in 6-hydroxydopamine model of Parkinson’s Disease, N euro inflammation 2018).
[00104] In another example, a subject with neuropathic pain may be treated with an RIPK2 inhibitor to alleviate neuropathic pain, a condition resulting from inflammation activating the pain receptors in the brain and the NOD2/RIPK2 pathway has been shown to drive this processs (FV Santa-Ceci!ia et al Pain 2019).
[00105] The term“cancer", as used herein, refers to a variety of conditions caused by abnormal, uncontrolled growth of cells. Cells capable of causing cancer, referred to as “cancer cells", possess characteristic properties such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain typical morphological features. Cancer cells may be in the form of a tumour, but such cells may also exist alone within a subject, or may be a non-tumorigenic cancer ceil. A cancer can be detected in any of a number of ways, including, but not limited to, detecting the presence of a tumor or tumors (e g., by clinical or radiological means), examining cells within a tumor or from another biological sample (e g. , from a tissue biopsy), measuring blood markers indicative of cancer, and detecting a genotype indicative of a cancer. However, a negative result in one or more of the above detection methods does not necessarily indicate the absence of cancer, e.g., a patient who has exhibited a complete response to a cancer treatment may still have a cancer, as evidenced by a subsequent relapse
[00106] In some examples, the cancer is a lymphoma.The term lymphoma” as used herein refers to a malignant growth of B or T cells in the lymphatic system “Lymphoma” includes numerous types of malignant growths, including Hodgkin's Lymphoma and non-Hodgkin's lymphoma. The term“non-Hodgkin's Lymphoma” as used herein, refers to a malignant growth of B or T cells in the lymphatic system that is not a Hodgkin's Lymphoma (which is characterized, e.g. , by the presence of Reed-Stern berg cells in the cancerous area) Non-Hodgkin’s lymphomas encompass over 29 types of lymphoma, the distinctions between which are based on the type of cancer ceils. In some
Figure imgf000030_0001
examples, the cancer is Hodgkin's lymphoma, relapsed non-Hodgkin’s lymphoma, or relapsed leukemia
[00107] In some examples, the cancer is triple negative and inflammatory breast cancer, pancreatic cancer, or colorectal cancer
[00108] A subject with cancer can be treated to prevent progression or alternatively a subject in remission can be treated with a compound or composition described herein to prevent recurrence.
[00109] A compound or composition described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
[00110] In treating a subject, a therapeutically effective amount may be
administered to the subject. As used herein, the term“therapeutically effective amount” refers to an amount that is effective for preventing, ameliorating, or treating a disease or disorder (e g., inflammatory bowel disease, e.g., ulcerative colitis or Crohn's disease, e.g., cancer).
[00111] Formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
[001 12] The compounds and compositions may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, tntradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinai, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
[00113] Compounds and/or compositions comprising compounds disclosed herein may be used in the methods described herein in combination with standard treatment regimes, as would be known to the skilled worker. Common drugs/combinations or treatments are well known to those skilled in the art.
Figure imgf000031_0001
[00114] In some examples, therapeutic formulations comprising the compounds or compositions as described herein may be prepared by mixing compounds or compositions having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, in the form of aqueous solutions, iyophilized or other dried formulations. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, histidine and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyciohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino adds such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
[001 15] The therapeutic formulation may also contain more than one active compound as necessary for the particular indication being treated, typically those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
[00116] A skilled worker will be able to determine the appropriate dose for the individual subject by following the instructions on the label. Preparation and dosing schedules for commercially available second therapeutic and other compounds administered in combination with or concomitantly with compounds or compositions described herein may be used according to manufacturers' instructions or determined empirically by the skilled practitioner.
[00117] Methods as described herein are conveniently practiced by providing the compounds and/or compositions used in such methods in the form of a kit. Such a kit preferably contains the composition. Such a kit preferably contains instructions for the use thereof
Use for Purification of Polypeptides or Identification of Binding Polypeptides
Figure imgf000032_0001
[001 18] In another aspect, the invention may comprise the use of a compound described herein as a ligand to bind to and purify proteins via affinity chromatography, wherein the ligand is coupled to a solid support.
[00119] For example, derivatives of a RIPK2 inhibitor described herein may be coupled to an agarose or sepharose beaded matrix to form an affinity matrix for targeted proteins. Cell lysates containing non-active or active RIPK2 (for example, generated using -/+ MDP treatment respectively) will be added to the RIPK2 inhibitor derivative- agarose or sepharose beads, incubated overnight at 4 ° C followed by SDS-PAGE separation of associated proteins. Mass spectrometry may then be utilized to identify the protein target associated with RIPK2 inhibitor derivative-agarose. This analysis will allow for the possible identification of off target effects (mainly kinase off target effects) of a RIPK2 inhibitor.
[00120] Thus, any compound described herein having a reactive functional group, and particularly those comprising a linker arm and a reactive functional group, may be coupled to a solid support, such as agarose for example, by a scheme such as:
Figure imgf000033_0001
Agarose-linked reagents are commercially available, and schemes to react the reagent to the functional group are well known to those skilled in the art.
[00121] In another aspect, the invention may comprise a method of identifying a polypeptide that binds to a RIPK2 inhibitor compound, comprising:
Figure imgf000033_0002
(i) incubating a cell lysate comprising a RIPK2-inhibitor binding polypeptide with a support comprising the compound, to form a complex between said RIP K2 inhibitor binding polypeptide and the compound;
(ii) separating said RIPK2-inhibitor binding polypeptide from the complex, for example by using SDS-PAGE analysis; and
(iii) identifying said RIPK2-inhibitor binding polypeptide, for example by using mass spectrometry.
EXAMPLES
[00122] To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these example are for illustrative purposes only, Therefore, they should not limit the scope of this invention in any way.
[00123] Example 1 : Identification and Characterization of Novel Receptor Interacting Serine/threonine-Protein Kinase 2 (RIPK2) Inhibitors Using Structural Similarity Analysis
[00124] Receptor interacting protein kinase 2 (RIP2 or RICK herein referred to as RIPK2) is linked to the pathogen pathway that activates NFkB and autophagic activation. Using molecular modeling (docking) and chemoinformatics analyses the RIPK2/ponatinib crystal structure was utilized and searched in chemical databases for small molecules exerting binding interactions similar to those exerted by ponatinib. Identified RIPK2 inhibitors potently inhibited proliferation of cancer cells by > 70% as well as inhibition of NFkB activity. More importantly, in vivo inhibition of intestinal and lung inflammation rodent models suggest effectiveness to resolve inflammation with low toxicity to the animals. Thus, the identified RIPK2 inhibitor may offer a possible therapeutic control of inflammation in diseases such as inflammatory bowel disease, asthma, cystic fibrosis, primary sclerosing cholangitis and pancreatitis
[00125] Most RIPK2 inhibitors were not designed to inhibit RIPK2 and, thus, RiPK2 inhibition was an off target effect of the use of these drugs. Because of robust off target effects of most R1PK2 inhibitors, molecular modeling (docking) and cheminformatics analyses was carried out by carefully analyzing the only known crystal structure of RIPK2 in association with the c-ABL kinase inhibitor, ponatinibl , (Canning and Bullock). Utilizing this structure, chemical databases were searched for scaffolds exerting significant binding interactions similar (or stronger) to those exerted by ponatinib, determined by comparing
Figure imgf000034_0001
their free energies when docked inside of the RIPK2 binding site. In addition to R1PK2 binding as part of this computer-based (initial) screening, compounds were selected that inhibited EGFR (ICSO’s > 000 nM; weak inhibitory activity) along with weak binding interactions in the EGFR and c-ABL binding sites, two common off-targets for previously identified RIPK2 inhibitors. Several hits are shown in Figure 1 and Table 1 and these compounds have unknown biological features. Since RIPK2 signals downstream of NOD2 and is linked to NFkB and autophagy activation, we believe, without restriction to a theory, that in vivo use of the selective RIPK2 inhibitors described herein will target NFkB activation and resolve abnormal inflammation states.
Table 1. Calculated binding free energies of potential R1PK2 inhibitors. ZINC, MolPort and PubChem (PC) molecule identification numbers are shown. iVtoJPort-016-359-762 is RIPK2 inhibitor 1 and PC_44716361 (MolPort-001 -746-327) is RIPK2 inhibitor 2. What is indicated are ZINC, MolPort and PubChem (PC) molecule identification numbers
Figure imgf000035_0001
_ _
PC_44716361 1 1.8 PC 58945685 -11.5
Structure similarity PC_2482680 I - 1 1.8 PC_6834961 ) - 1 1.5
PC 57405602 -P .6 PC 58945669 -1 1.3 PC 58945635 1 1 6 PC_40780I 19 11 1
Materials and Methods
[00126] Pharmacokenetics. Intraperitoneal injection of 15 pg/g body weight of RIPK2 inhibitor 1 was carried out on wild type C56BL/6 mice. At indicated times, cardiac puncture was performed and mouse serum obtained. Inhibitor 1 was measured in mouse serum injected using a high performance liquid chromatographic (FiPLC) assay. To 0.1 mL of mouse serum in glass tubes was added 0 05 ml_ each of 1 M NaOH and methanol. This was followed by addition of 3 mL of t-butyl methyl ether, vortex mixing for 30 sec at high speed, and centrifugation at high speed for 3 min. Supernatant was transferred to clean glass tubes and solvent evaporated in vacuo. The residue was reconstituted in 0.15
Figure imgf000035_0002
mL of mobile phase and 0.05 mL injected into the HPLC. The mobile phase was a mixture of [25 mM KFUPCh: 3 mM sulfuric acid: 3.6 mM triethyiamine]: acetonitrile in the proportion 64:36 v/v. The mobile phase was passed (0.9 mL/min at room temperature) in series through a C1 8 2 cm><4.0mm, 5m guard column (Supelco, PA, USA) then a Symmetry C18 analytical column, 3.5m, 150mmx4.6mm (Waters, MA, USA).
Chromatographic data were collected and compiled by use of EZChrom software.
Detection was by ultraviolet absorption using a Waters 486 detector, with the Amax being set to 261 nm. Standard curves of peak area or height from 25 to 1000 ng/mL were highly linear (r2 > 0.99).
[00127] Kinase Profiling. All assays are carried out in 97 well plate format as service at DiscoverX using their 97 target KinomeScan Profiling Service. For the 97 KinomeScan profiling assay, competition binding assays were carried as described previously (Karaman et al., 2008). Briefly, kinases were generated either as full length fusions to T7 phage6 or expressed in HEK-293 ceils and subsequently tagged with DNA for PCR detection. For the binding assays, biotinylated affinity resins were coupled to streptavidin-coated magnetic beads, blocked with excess biotin and washed with blocking buffer (SeaB!ock, Pierce) to remove unbound ligand and to reduce nonspecific binding. Binding reactions were initiated by combining kinase, liganded affinity beads and test compounds in binding buffer. Test compounds were prepared as 100 x stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Assay plates containing kinase/ligand and small molecule inhibitor were incubated at 25 °C with shaking for 1 h, washed extensively to remove unbound protein and eluted. Kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound /ids were determined using eleven serial threefold dilutions
[00128] Kinase Activity Assay for c-Abl. C-Abl kinase assay (Figure 4D) was carried out by MRC Protein Phosphorylation and Ubiquitylation Unit at
http://www.ppu.mrc.ac.uk/. ABL (5-20mU diluted in 50 mM Tris pF! 7.5, 0 1 mM EGTA, 1 mg/ml 8SA) was assayed against substrate peptide (EAIYAAPFAKKK) in a final volume of 25 5 pi containing 50mM Tris pH 7,5, 0 1 mM EGTA, 1 mM DTT, 300mM substrate peptide, 10 M magnesium acetate and 0.005 mM [33P-y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays were stopped by addition of 5 pi of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. The dried Unifilter plates are then sealed on
Figure imgf000036_0001
addition of MicroScint O and were counted in Packard Topcount NXT scintillation counters
[00129] !VITT Growth Assay. Cell proliferation assay was performed to evaluate cell viability after drug treatment. Cells were counted using hemocytometer and seeded in a 96-well plate at 1 -2 X 104 cells/well and incubated for 24 hours. Different concentrations of RIPK2 inhibitor was added to the cells and incubated for 48h. MTT [3-{4,5- Dimethylthiazol-2-y[)-2,5-Diphenyltetrazolium Bromide] was then added and incubated for 4h. Absorbance was measured at 560 nm using vICToR™ Multilabel Plate Reader from PerkinElmer. For L428, cells were stained with Trypan Blue to determine cell viability after 1 - 6 days incubation with the drugs.
[00130] Immunoblot and Immunoprecipitation Analysis. Cells were co llected and lysed using RIPA lysis buffer. Samples were denatured in SDS sample buffer and run on 7.5% gel and blotted onto PVDF membrane. Blotted membranes were blocked with 10% milk for 30 min and then incubated with primary antibodies overnight at 4°C
Membranes were then washed TBS-T (Tris-buffered saline containing 0, 1 % Tween 20) and incubated with appropriate secondary antibody (anti-rabbit or anti-mouse IgG) for one hour
[00131] Cell cycle analysis. Cells were harvested by trypsin ization, washed with PBS and resuspended in 75% ethanol in PBS and kept at 48°C for at least 30 min. Prior to analysis, cells were washed again with PBS and resuspended and incubated for 30 min in a solution containing 0 05 mg/ml propidium iodide (Sigma), 1 mM EDTA, 0.1 % Triton-X-100 and 1 mg/ml RNAse A in PBS. The suspension was then passed through a nylon mesh ®lter and analyzed on a Becton Dickinson FACScan.
[00132] RIPK1 activity, HT-29, L929 and HaCaT cells were seeded in a 96-well plate and then treated with the indicated amounts of RIPK2 inhibitors. After 24 hours, the media was removed and replaced with fresh media containing 20 mM z-VAD-fmk (Z), 100 nM Smac Mimetic-164 (S) and 10 ng/mL TNFa (T) in the presence or absence of 100 nM Ripkl inhibitor or 1 uM necrosu!fanamide (NSA) or the indicated amounts of RIPK2 inhibitors for 24 hours (HaCaT and HT-29) or 8 h (L929). After the indicated time, the cells were double stained with hoechst and propidium iodide for 30 min at 37 °C and then cell death was measured using the Celigo cell cytometer.
[00133] Ferroptosis Analysis. A549 and U87 cells plated in 12 well dishes were pretreated with the inhibitors for one hour before cells were treated with Erastin (a compound known to induce ferroptosis). After 48 hours, cells were harvested, re-
Figure imgf000037_0001
suspended in PBS and stained with 0 04% trypan blue for 5-10 minutes at room temperature. Cell death was quantified by flow cytometry using the FACSCalibur flow cytometer and CellQuest software Ferrostatin-1 (Fer-1 ) and Deferoxamine (DFO) were used as positive controls for inhibition of ferroptosis.
[00134] Assay of SIRT1 activity. BiOMOL (Enzo Life Sciences) assay was performed as previously described {Hubbard et al., 2013). 2 pg of recombinant SIRT1 was used for each reaction, and (3-NAD and FdL-p53 peptide concentrations used were 200 mM and 20 mM, respectively 0,5 pL of vehicle or test compound was added to each 50 pL reaction Fluorescence values corresponding to SIRT1 activity were calculated by subtracting parallel reactions in the absence of b-NAD from those in the presence of b- NAD (F corrected = F+NAD - F -MAD)· Fold activation was calculated by dividing fluorescence values in the presence of drug by those in the presence of DMSO only.
[00135] Pharmacophore Search. Commercially available subsets of ZINC (15,868,179 compounds) and MolPort (7,241 ,662 compounds) databases were screened using lead drug ponatinib as a reference. ZINCPharmer pharmacophore search server was used (Baksh et al , 2005) and pharmacophore features were identified directly from RIPK2 structure (PDB ID: 4C8B) and used for screening. In this regard, a total of 74 compounds were selected from both databases. The selected compounds were then docked into the RIPK2 binding site.
[00136] Structure Similarity Search. PubChem (Kim et al., 2016) compound library (more than 80 million molecules) was compared to Ponatinib, to filter all molecules which were at least 60% similar to this compound (10,655, 787 molecules). Most (> 90%; 803 compounds) and less (< 62%; 50,000 compounds randomly selected from a total of 2,607,266) similar structures were selected employing PubChem Fingerprint (PCFP) implemented in the ChemmtneR package (Cao et al., 2008) of the R program (Baksh et at , 2002) (R Development Group, 2008). Filtered compounds were then subjected to another filtering step based on Lipinski’s rule of five, (Lipinski et al , 2001 ) removing duplicates and inorganic molecules with the FAFDrugs3 server (Lagorce et al. , 2015) (22,274 compounds). Finally, a total of 3,000 compounds were selected and optimized using the obminimize tool of the OpenBabel toolbox (O'Boyie et al., 201 1 ) for the docking studies
[00137] Molecular Docking. Crystal structure of human RIPK2 (PDB: 4C8B) (Canning and Bullock) was retrieved from Protein Data Bank (PDB) (Bernstein et al.,
1 977). Some residues in the three-dimensional structures were missing, and
Figure imgf000038_0001
consequently, these side chain atoms were added using WHAT IF Web Interface. (Vriend, 1990) The protein was subjected to an energy minimization employing AMBER99SB force field by 1000-step steepesl-descent minimization followed by 100-conjugate gradient minimization using UCSF CHIMERA v1.9 software (Pettersen et al., 2004). Polar hydrogens, Gasteiger-Marsili empirical atomic partial charges and AutoDock atom types were computed employing MGLTools v1.5.4 package (Morris et al., 2009) Torsional root and branches of the ligands were chosen with the same program, allowing flexibility for all rotational bonds (except for the amide bond). In addition, the software was used to assign Gasteiger-Marsili atomic charges to all ligands (Gasteiger et al 1978). Docking calculations were performed using AutoDock Vina software (Trott and Olson, 2010). A grid box of 28 A3 and centered at the binding site was used to calculate atom types needed for the calculation A total of 9 runs were performed with an exhaustiveness of 50. Best binding mode of each molecule was selected based on lowest binding free energy score. 3D figures were generated using PyMOL Molecular Graphics System (Delano Scientific LLC, Palo Alto, CA, 2007) (Baksh et al. , 1992).
[00138] RIPK2 In Vitro Kinase Assay. For this protocol, samples were lysed in 1 X RIPK2 lysis buffer (50 mM Tris, pH 7.5, 10mM MgCE, 1 % Triton X-100, 1 mM DTT, 1 mM EDTA, 1 mM EG A, with freshly added 1 mM b-glycerophosphate and protease inhibitors [PMSF and aprotinin]) and immunoprecipitated overnight using 1.5 pg rabbit anti-RIPK2 antibody. The next day, protein G sepharose was used to immunoprecipitate the RIPK2 protein complex IP for 1 .5 hours Following this incubation, samples were washed once in 1 X kinase wash buffer (50 mM Tris, pH 7.5, 150 mM NaCI, 1 % Trition X-100, 1 mM EDTA) followed by 2 washes with 1 X Kinase buffer (30 mM Hepes, pH 7 5, 10 mM MgCI2, 2 mM MnCh) After last wash, 20 pi water was added to beads, followed by 1 X Kinase buffer and 3zP-y-ATP and kinase reaction was allowed to proceed for 45-60 minutes at 30 °C. Protein loading dye was added to the mix and samples were boiled and separated on an SDS-PAGE gel The gel was dried and then exposed to X-ray film to capture the autophosphorylation of RIPK2.
[00139] NFKB Gene Reporter Luciferase Assay. Dual-Luciferase Reporter Assay System (DLR assay system, Promega, E1910) was used to perform dual-reporter assays on NFkB Luciferase and Renil!a Luciferase (internal control). Briefly, HCT1 16 colon cancer cells were equally seeded at a density of 3 x 104 in 6-we!l plates and allowed to attach for 24 hrs. Prior to transfection, cells were washed with serum free media 3 times. Dual transfection was carried out using 12 pi of PEI to 3 pg of NFKB Luciferase construct
Figure imgf000039_0001
and 60 ng of Renilla Luciferase construct. After 24 hours after transfection, cells were treated with the different drugs for 24-36 hrs. Cells were then lysed using the passive lysis buffer provided by the kit for 30 minutes on ice. Lysate was spun down for 8 minutes at 10,000 rpm and 20 pi of cell lysate were transferred in 96-well plate. Luciferase assays were analyzed based on ratio of Firefly/Ren ilia to normalize cell number and transfection efficiency
[00140] Intestinal Inflammation Injury Model. Animals were administered 3% w/v DSS (molecular weight of 40 000 - 50 000, MP Biomedicals) in the drinking water for 7 days followed by recovery for 7 days. They were monitored for: piloerection,
bioatednesss, tremors, lack of movement, rectal bleeding and weight loss (all on a scale of 0-5 with 5 being very severe, adapted from Madsen et al, 2001 ). Animals were euthanized once rectal bleeding became grossly apparent. For weight loss, a score of 0 for no weight loss, 1 if 5% loss, 2 for 5- 10% loss, 3 for 10 - 15% loss, 4 for 15 - 20% loss and a score of 5 for > 20% loss in initial body weight. Disease activity indices (DAI) were the sum of all individual scores. All animals were male of the C57BL/6 background and at 10 - 12 weeks of age or 25 g in body weight at the beginning of the experiment.
[00141] Lung Inflammation Model for Asthma. Male Balb/c mice (8-8 weeks) were sensitized on days 1 and 6 via intraperitoneal injection of 0.9% sterile saline (0.5 mL) or 0.9% saline containing 10 pg ovalbumin and 2 mg AI(OH)3. After light anesthesia with ketamine (75 mg/kg) and acepromazine (2.5 mg/kg), mice were challenged intranasally with 25 mI of saline containing 50 pg ovalbumin or saline alone as control on days 12 and 14 RIPK2 inhibitor -1 (1 pg/g body weight) or 30% DMSO (solvent control for drug) were injected intraperitoneaily on day 12, 13 and 14 (on day 12 and 1 the injection was performed 1 hr before intranasal challenge with ovalbumin). On day 15, mice were euthanized with an intraperitoneal injection of 2 mg sodium pentobarbital. Cardiac puncture was used to collect blood, followed by tracheal intubation with polyethylene tubing. Lungs were washed twice, with 1 mL of phosphate buffered saline (PBS), pH 7.4, and 2 mL of broncho-alveolar lavage (BAL) fluid was collected. BAL fluid was spun at 300 g for 5 min. Ceil pellets were re-suspended in PBS, total cell count was performed and then cytospins were prepared with 5000 cells. Slides were stained with Diff Quick and differential cells counts were done to assess allergic airway inflammation.
[00142] Mass Spectrometry. Direct-infusion measurements were carried out on an LTQ Orbitrap XL (Thermo Scientific) mass spectrometer using an Ion Max ESI source. An on-board syringe pump was used with a 100uL Hamilton syringe and a flow rate of
Figure imgf000040_0001
5uL/mtn. Ffollowing parameters were used: sheath gas flow of 15 arbitrary units, auxiliary gas flow of 5 arbitrary units, spray voltage of 3.5 kV, tube lens voltage of 1 10 V, capillary temperature of 275C, capillary voltage of 35V, capillary temperature of 275 °C. AGC target was set to 1e6 and the maximum injection time to 200 ms. Resolution was set at 100000 and two microscans were recorded.
[00143] NMR Spectroscopy. NMR experiments were run on a Varian Inova 500 MHz spectrometer at 30°C. Proton chemical shifts were measured relative to residual proionated methyl (CHD2) signal of de-DMSO (2.50 ppm); coupling constants (J) are reported in Hertz (Hz). Standard notation was used to describe multiplicity of signals observed in Ή NMR spectra: singlet (s), doublet (d), triplet (t), broad (b) etc. Through- space and through-bond connectivities were observed using 2D 1 H-1H ROESY experiment (mixing time 200 ms). One-dimensional experiments were processed using VNMRJ (Varian Associates) and 2D 1H-1H ROESY was processed using
NMRPipe/NMRDraw software. R1PK2 inhibitor 1 was prepared by dissolving 0.3 mg in 500 pL d6-DMSO. RIPK2 inhibitor 2 was prepared by dissolving 0.3 mg in 500 pL ds- DMSO (Delaglio et al. , 1995).
Results
[00144] Pharmacophore search. Ponatinib/RiPK2 complex (PDB: 4C8B) has revealed significant binding interactions of this inhibitor in the RIPK2 active site. Based on this crystal structure, the active site is localized in a binding pocket formed by at least eight different amino acid residues including Val32, Lys47, Glu66, Ile69, Leu70, Thr95, Met98, and Asp164 (Figure 1A), ZINC and MolPort databases were screened with the ZINCPharmer pharmacophore search server using the lead drug ponatinib as query. Several pharmacophoric features were identified directly from the RIPK2 structure and used for the screening. In this regard, a total of 74 compounds were identified from both databases with similar pharmacophoric points to ponatinib (Figure 1 B). The compounds were then docked into the same RIPK2 binding site that in which ponatinib is located. Interestingly, 54 compounds shared the pharmacophore distribution of ponatinib. Table 1 summarizes the docking scores of the compounds with the lowest binding free energies. Figure 1 B shows the location of the pharmacophoric points identified for the interaction of ponatinib with the RIPK2 active site and the proposed binding modes of the ligands with the lowest binding free energies, respectively. It is noteworthy that the compounds with the lowest calculated binding free energies exhibit a high structural similarity between
Figure imgf000041_0001
them (MolPort-016-359-762 [labelled as RIPK2 inhibitor 1 in subsequent Figures, 3- benzamido-4-methyl-N-[3-(1 -methyl- 1H-imidazol-2yl)phenyl] benzamide], MolPort-015- 752-252, MolPort-015-604-588, and Mo!Port-016-412-727) and a high correlation with pharmacophoric points of ponatinib. These results suggest that compound MolPort-016- 359-762 and its derivatives have high probability to inhibit RIPK2
[00145] Structure similarity search A total of 10,655,787 compounds with at least 60% of similarity to ponatinib were obtained from the PubChem database to dock them into this binding site and identify new potential inhibitors. The most {³ 90%; 803 compounds) and less (< 62%; 50,000 compounds randomly selected from a total of 2,607,266) similar structures were selected in order to identify ponatinib-like molecules and novel scaffolds, respectively. After filtering with Lipinski’s rule and removing duplicates and inorganic molecules, molecular docking studies in the RIPK2 active site were performed using AutoDock Vina, from which more than 500 compounds with a calculated AGbind < -10.0 kcal/mol were identified. The docking scores of ponatinib and the compounds with the lowest binding free energies are summarized in Table 1. The compounds with the highest similarity values showed similar binding modes compared to ponatinib (PC_24826801 , PC_57405602, PC_58945635, PC_58945682, PC_58945685), However, compound PC_57410628 was the only molecule with a lower binding free energy than the lead molecule. Moreover, compound PC_44716361 (MolPort-001 -746- 327, labelled as RIPK2 inhibitor 2), which present a lower similarity to ponatinib, represents a new scaffold with unique structural features offering alternatives from a Medicinal Chemistry perspective. Figure 1 B shows the proposed binding mode of these ligands in the RIPK2 binding site. This figure also shows that all the compounds identified possible form hydrogen bonds between the Glu66 residue and the carboxamide group. The initial virtual screening ranking, chemical structures and the Pan Assay Interference Compounds (PAINS) (Sterling and Irwin, 2015) check of the selected compounds are reported in the Table 2.
Table 2. Related to Table 1 . Pharmacophore (Pharm) and similarity (Sim) compound ranking, PCFP similarity, chemical structure and SMILES of molecules retrieved from the pharmacophore and similarity-based virtual screenings The compounds were also filtered for PAINS and aggregators (http://zinc15.docking.org/patterns/home/).
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000043_0002
Figure imgf000044_0001
Figure imgf000045_0001
[00146] Utilizing mass spectrometry and 1 H-MMR (Figure 2 and 7, 8, 9A), > 95% purity was confirm of the two lead compounds and confirmed structure of the identified R1PK2 inhibitors 1 and 2. ROESY (rotating-frame Overhauser enhancement spectroscopy) spectra provided through-bond and through-space connectivities that were
Figure imgf000045_0002
essential for obtaining complete chemical shift assignments of both molecules (Figures 7 and 8). In addition, through-space ROE (rotating-frame nuclear Overhauser
enhancement) connectivities provided valuable information about the conformational preferences of RIPK2 inhibitor 1 in solution (Figure 2B). The lack of ROEs between HN22 and either HC17 or HC25 indicates that the central methyl-containing aromatic ring is roughly perpendicular to the amide plane of HN22, as would be expected based on steric considerations in contrast, HN13 shows ROEs to HC8, HC17, and HC21 , indicating that the two central benzene rings are more planar with respect to the central amide bond of HN13, similar to the conformation seen in ponatinib (Figure 1 B). Thus, the arrangement of benzene rings found in RIPK2 inhibitor 1 resembles the geometry of the corresponding aromatic rings in ponatinib bound to RIPK2 The presence of both HN13-HC17 and HN13-HC21 ROEs suggest rotation about the C14-C16 bond, with rotomer i (Figure 7B) matching the conformation found in ponatinib-RIPK2. However, the presence of an HN13- HC8 ROE coupled with the absence of an HN 13-CH10 ROE suggests that rotation about the C9-N13 bond is more restricted, which may force the imidazole ring of RIPK2 inhibitor 1 into a position opposite to the corresponding CF3 group of ponatinib-RIPK2 (see Fig. 1 Biii), which would place it in a more solvent-exposed position than the buried position of the CF3 group.
[00147] Validation of RIPK2 Inhibitors using Kinome Analysis. The specificity of the identified RIPK2 inhibitors to inhibit numerous kinases was explored utilizing the Kinome Scan profiling service at DiscoverX. This utilizes an in vitro kinase approach with purified recombinant kinases/peptide substrates to determine activity of kinases in the panel. It was explored if lead compounds, RIPK2 inhibitor 1 and 2, can inhibit other targets at 1 00 n in the 97 kinome panel (Table 3). This concentration was utilized due to robust inhibition of NFkB and RIPK2 kinase assay in assays outlined in Figures 2-6 The kinome inhibition analysis revealed robust selectivity towards R1PK2 for RIPK2 inhibitor 1 with 20-27 % inhibition of c-ABL, Aurora kinase B or ERBB2 (Table 3). RIPK2 inhibitor 2 appears to inhibit 38% of the activity of SNARK and 27% of the activity of FGFR2, GSK3- b, JNK1 , CSNK1 G2 (Caesin kinase 1), and MET tyrosine kinase. These off target inhibitions are not surprising as it is difficult to obtain a specific small molecule inhibitor. It would suggest possible activities beyond inhibiting RIPK2 that will need to be empirically tested Initial empirical testing for c-ABL was carried out via an in vitro kinase approach with purified recombinant kinases at the MRC Protein Phosphorylation and Ubiquitination Unit in Dundee, Scotland. Both of the R1PK2 small molecule lead compounds inhibited c-
Figure imgf000046_0001
ABL but with 1C50 of > 100 mM (Figure 3A). Given this observation, it is considered that the off target effects on Aurora kinase B and ERBB2 (if any) will also be in a vastly different concentration range of 100 nM. Table 3. Related to Figure 3. Kinome inhibition in the presence of 100 nM RIPK2 Inhibitor 1 or 2 Kinase activity assays of 97 kinases were carried using peptides or defined substrate as carried out by a "Kinome Scan” analysis by DiscoverX
(httpsY/www.discoverx.com/sen/ices/drug-discovery-development- services/kinaseprofiling/
kinomescan/)
Figure imgf000047_0002
Figure imgf000047_0001
Figure imgf000048_0002
Figure imgf000048_0001
Figure imgf000049_0002
Figure imgf000049_0003
[00148] Cell based Confirmation of RIPK2 Inhibition and Inhibition of NFkB Activation. Cell based in vivo inhibition of RIPK2 was explored. Clearly observed was
Figure imgf000049_0001
RIPK2 inhibitor 1 and 2 inhibition of MDP-dependent activation of R1PK2
autophosphorylation (on tyrosine 474) using an in vitro kinase assay in HCT116 cells (Figure 3B). MDP is the molecular ligand for the NOD2 pathogen receptor whereby R1 PK2 can be activated. Similar results for RIPK2 inhibitor 1 inhibition of RIPK2 were obtained in breast cancer cells (BT549 and MDA-MB231 cells, data not shown).
Immunodepeletion using an anti-RIPK2 antibody (Figure 3C, condition B) or Crispr/Cas9 knockout of RIPK2 (Figure 3D) confirmed specificity of detection of the
autophosphorylated RIPK2 band in Figure 3B. Furthermore, using a phospho-tyrosine [Y] 474 (in-house generated) and a phospho-serine [S] 176 (Cell Signaling, Inc.) RIPK2 antibodies, it was confirmed that active RIPK2 is recognized by these antibodies upon activation with MDP treatment in 293HEK fibroblast and HCT1 16 colon cancer epithelial cells (Figure 3E, left panel) and detection of constitutively active RIPK2 in the colon cancer cel! line, SW480 (Figure 3E, right panel). Interestingly, one can observe constitutively active RIPK2 in Hodgkin’s Lymphoma cell lines HDMYZ, L428 (Figure 4A) and KMH2 cells (Figure 4B). This signal can effectively be inhibited using RIPK2 inhibitor J , 2 and regorafenib (a known RIPK2 kinase inhibitors) at 100 nM as determined using RIPK2 phospho-specific antibodies (Figure 4A and B). Furthermore, it was determined that RIPK2 inhibitor 1 inhibited KHM2 cells at 5-10 nM and kinase activity is somewhat sensitive to detergent conditions (Figure 4B and C). When compared to inhibition with ponatinib, RI PK2 inhibitor 1 performed equally to inhibit RIPK2 kinase activity in an in vitro kinase assay in two different cell lines (Figure 4D).
[00149] The functional consequence of inhibiting MDP-dependent activation of RI PK2 is the loss of NFkB activity (Figure 5A-C and Figure 9B) and DNA binding ability, especially for the IL-8 promoter (Figure 10A). RIPK2 inhibitor 1 was more effective inhibiting NFkB activity when compared to RIPK2 inhibitor 2, gefitinib or regorafenib (p value comparing RIPK2 inhibitor 1 vs RIPK2 inhibitor 2 or gefitinib or regorafenib inhibition of MDP-driven NFkB activation was 0.0015, 0.0006 and 0.004 respectively). However, RIPK2 inhibitor 1 was as effective as ponatinib in NFkB inhibition (p value - 0.06 when comparing RIPK2 inhibitor 1 vs ponatinib inhibition of MDP-driven NFkB activation). RIPK2 inhibitor GSK-583 (Haile et a!., 2016) did not inhibit MDP-dependent NFkB activity at 100 nM in HCT116 cells (data not shown) although known to in primary immune cells. A small but significant reduction was observed in LPS (via TLR4 and TLR2), TNFa, and IL-i -dependent activation of NFkB (Figure 5C and ICso in Figure 5D, Figure 9B). Although a reduction, the IC5o for RIPK2 inhibition of LPS-driven NFkB
Figure imgf000050_0001
activation is > 20 mM and it may be that it will also be much higher for inhibition of TNFa, and IL-1 p-dependent activation of NFkB.
[00150] RIPK2 inhibitor 1 can also inhibit the activation of hypoxia response element I response to chemical induction using 1 % Hz02 (Figure 9C) to suggest either a link to inflammation or a direct modulation of HIFa function. These observations validate the specificity of the RIPK2 inhibitors described herein for NOD/R1PK2 biology and usefulness as an in vim inhibitor of DP-driven inflammation
[00151] RIPK2 Inhibitors Can Inhibit the Proliferation of Several Cancer Cells but Not Promote Apoptosis or Cell Cycle Arrest. Inflammation is a strong driver of malignant transformation and abnormal proliferation, especially in the coion (Lasry et al. , 2016) and breast (Suman et al., 2016) The ability of RIPK2 inhibitors to reduce the proliferative rate of highly metastatic cancer cells was explored For most of the cancer cells, 45-100 nM of RIPK2 inhibitor 1 inhibited > 70% of the proliferation of colon and blood cancer cells while preserving the proliferation of normal Rat-1 and ModeK intestinal epithelial cells (Figure 6A) These data would suggest strong suppressive properties of RIPK2 inhibitor 1 and to some extent inhibitor 2 on cell proliferation. Regorafenib, a recently characterized RI PK2 inhibitor, did not significantly inhibit the proliferation of these ceils in an MTT assay (Figure 6A). Inhibition of triple negative breast cancer cells, HCT1 143 and MDA-MB231 , with RIPK2 inhibitor 1 (Figure 10B) was also observed to suggest effective anti-proliferative properties. The ICsofor inhibition was around 30-60 nM for most of the cells tested (two are shown in Figure 10C). This effect on growth inhibition was not due to alterations in cell cycle (Figure 1 1 ) nor a significant increase in the apoptotic cell populations to suggest that RIPK2 inhibition of proliferation is independent of cell cycle or apoptotic changes (Figure 1 1).
[00152] RIPK2 Inhibitors Do Not Inhibit RIPK1 Directed Cell Death,
Ferroptosis or Modulation of Mitochondrial Physiology. The RIPK family of proteins are actively involved in numerous cell death processes beyond death receptor dependent cell death (Vanden Berghe et al. , 2016). Two forms of cell death were characterized involving erastin stimulated ferrroptosis (an iron dependent form of cell death, Figure 12A) and VAD-fmk (Z), Smac Mimetic-164 (S) and TNFa (T) (ZST) stimulation of RIPK1- directed cell death (Figure 1 B). in both cases, RIPK2 inhibitor 1 did not interfere with these forms of cell death to support the cell cycle effects and to suggest no overlap with RI PK1 biology (Figure 12A) Recently, RIPK3 was demonstrated to require Bax/Bak effect on the mitochondrial permeability transition pore (MPTP) in order to carry out
Figure imgf000051_0001
necroptosis and the importance of both R1PK1 and RIPK3 in relation to necroptosis- directed degenerative, inflammatory and infectious diseases has been published (Moriwaki et el., 2015). It was thus explored how the identified RIPK2 inhibitors can perturb mitochondrial physiology by evaluating the activity of the sirtuins, a class of deacetylases that use NAD4 to remove acetyl groups from proteins. (Tang, 2016) Indeed, the putative S1RT1 activator, resveratrol, can stimulate mitochondrial biogenesis to promote increased wellness in the individual (Ostojic, 2017) Therefore, the effect on Sirtuin activity using the BIOMOL assay was explored as described previously.(Dai et al., 2016) No statistically significant increase in S1RT1 activation was observed when using the compounds at 0.1 or 10 mM, indicating that phenotypes observed for the use of R1PK2 inhibitors at these doses in cells are likely through SIRT1 -independent mechanisms when compared to the effect of resveratrol (Figure 12C). The identified RIPK2 inhibitors are thus selective inhibitors of RIPK2-dependent NFkB pathways if the data in Figure 5 and Figures 9B, 10 and 1 1 are considered.
[00153] RIPK2 Inhibitors Can Efficiently Resolve Intestinal Inflammation in an Ulcerative Colitis Model. DSS-induced intestinal inflammation is a model for ulcerative colitis (UC, a form of inflammatory bowel disease, I8D). DSS functions to irritate the colonic mucosa to promote localized inflammation, active cell death and localized destruction of the epithelial barrier to the lumen of the colon (Dieleman et al , 1998).
These events subsequently drive inflammation damage indicative of what IBD patients encounter. NOD2 mutations have been observed in IBD patients lending support for dysregulated NOD2/RIPK2 signaling driving inflammation in IBD patients (Branquinho et al. , 2016). The importance was demonstrated of Ras association domain family protein 1A, RASSF1A (or 1A), in the pathogenesis of colitis in a rodent model (Gordon et al. , 2013). RASSF1 A is a tumor suppressor involved in TNF-R1 dependent apoptosis, cell cycle control and restriction of NFkB activation (Gordon et al., 2013). The Rassfl a knockout mice (both heterozygous and homozygous forms) are very susceptible to DSS- induced inflammation injury (Gordon et al., 2013) mainly due to uncontrolled inflammation linked to the NOD2/RIPK2 pathway (Said et al, unpublished observation),
[00154] Therefore, intraperitoneal injection of 1 pg/g body weight of RI PK2 inhibitor 1 or 2 on day 5, 7, and 9 was carried out to offset the pre-inflammation damage stage, peak inflammation damage stage and post-inflammation damage/resfitution phase. Using this treatment scheme, a robust difference was observed on day 9 in the disease activity indices of wild type and Rassfla knockout mice that was significantly inhibited with the
Figure imgf000052_0001
use of the RIPK2 inhibitors, especially R1PK2 inhibitor 1 (Figure 6B and Table 4).
Interestingly, a newly characterized RIPK2 inhibitor, gefitinib, can also inhibit intestinal inflammation injury but only promote a 41 % survival vs 73% survival with RIPK2 inhibitor 1 (Figure 6B and Table 4). Similar results were obtained for the protein tyrosine kinase inhibitor, regorafenib (data not shown). Since both gefitinib and the RIPK2 inhibitors described herein can inhibit RIPK2, either the inhibitors have more affinity for RIPK2 or off target effects of RIP 2 inhibitor 1 is beneficial to aid in recovery from inflammation injury using the DSS model. Table 4. Survival of Rassfla A animals during DSS-induced inflammation injury in the presence of RIPK2 inhibitors Acute intestinal inflammation injury was carried out using the dextran sodium sulfate (DSS) model.
Figure imgf000053_0002
[00155] Pharmacokinetic properties of RIPK2 inhibitor 1 in the sera of mice was explored. Analysis revealed that RIPK2 Inhibitor 1 can be eluted at ~3.7 minutes in a region of the chromatogram free of interfering substances (Figure 12D, top panel). After a dose of 1 -2 mg/kg IP of inhibitor, the compound was not quantifiable in mouse serum. However, after dosing mice with 15 mg/kg IP, the R1PK2 inhibitor was of measurable concentrations in most of the serum samples assayed. The mean C max was 114 ng/mL occurring at 1 h after dosing, and the area under the serum concentration versus time curve was 573 ngxh/mL, with a terminal phase half-life of 1 ,9 h being observed.
Therefore, it appears that RIPK2 inhibitor 1 may be efficiently metabolized within 2 hours and cleared from the blood (Figure 12D, bottom panel). Furthermore, toxicity analysis carried out 9 days after the last dose of intraperitoneal injection of 2 pg/g body weight revealed little of no changes is > 98% of the markers characterized in a complete blood count (Figure 13). However, creatine kinase activity was significantly elevated in animals treated with RIPK2 inhibitor 1 as opposed to RIPK2 inhibitor 2. During treatment with RI PK2 inhibitor 1 (and post-treatment) phenotypic changes were not overtly observed in these animals, nor evidence of health conditions to support a creatine kinase abnormality (such as skeletal muscle defect and movement abnormalities or unexpected euthanasia
Figure imgf000053_0001
due to a cardiac abnormality) Interestingly, it has been shown that the
creatine/phosphocreatine pathway may play a central role in energy metabolism and nutritional creatine supplementation has been shown to impart beneficial effects in a number of diverse disease pathologies (Kitzenberg et al., 2016). It was speculated that this was the case as resetting of metabolic abnormalities is needed for recovery of IBD patients that have metabolic syndrome disorder {Goncalves et al., 2015).
[00156] RIPK2 Inhibitors Can Also Efficiently Resolve Lung Inflammation in an Asthma Model. RIPK2 has also been implicated in allergic airway inflammation. RIPK2 gene silencing in the airways decreased allergic airway inflammation in an ovalbumin-mediated mouse model of asthma (Jun et a)., 2013). Furthermore, an association between a RIPK2 promoter polymorphism and childhood severe asthma has been shown in a Japanese population (Nakashima et al. , 2006). Since one can observe robust inhibition of NFKB in the presence of RIPK2 inhibitor 1 and a > 70% recovery of animals from DSS-induced inflammation injury (Figure 4 and 6B), the ability to resolve lung inflammation using the ovalbumin challenge model was explored (Kumar et al., 2008) Ovalbumin elicits a robust inflammatory reaction in the airways characterized by increased number of total cells and eosinophils in the bronchoalveolar lavage (BAL) of ovalbumin sensitized mice (Figure 6B). In the presence of RIPK2 inhibitor 1 (and to some extent with RIPK2 inhibitor 2), a significant reduction was observed in both total cell numbers and eosinophil numbers in BAL fluid. This data suggest effective inhibition of lung inflammation
[00157] Comparison with Recently Identified RIPK2 Inhibitors. Recently, five inhibitors to RIPK2 were identified as OD36/OD38 (Tigno-Aranjuez et al., 2014) WEHI- 435 (Nachbur et al , 2015), GSK-583/GSK-2983559 (Haile et al , 2016) and Novartis (He et al., 2017) OD36 and OD38 were obtained through a small molecule macrocylization process from Oncodesign. IC$o values of < 100 nM and the ability to interfere with MDP dependent RIPK2 activity WEHi-435 was obtained by analysis of the RIPK2/ponatinb structure and the necrostatin-1 /murine RIPK1 structure to obtain a structural face for the murine RIPK2 kinase domain (18-249) (Nachbur et al., 2015). Using this structural face, the utilized computational biology to obtain small molecules to associate with the RIPK2 ATP-binding pocket, GSK-583 was obtained using structural comparisons of RIPK2 with ponatinib. Lastly, the Novartis RIPK2 inhibitor was obtained in a similar manner to GSK583 and was based on a proprietary chemical library screen. Several hits were
Figure imgf000054_0001
obtained and after structural optimization a RIPK2 inhibitor was obtained to inhibit RIPK2 kmase activity at 3 nM (He et al.: 2017).
EXAMPLE 2:
[00158] As depicted in Figure 13, toxicity analysis revealed little or no changes in > 98% of the markers characterized in a complete blood count. However, creatine kinase activity was significantly elevated in animals treated with RIPK2 inhibitor 1 as opposed to RIPK2 inhibitor 2. During treatment with RIPK2 inhibitor 1 (and post-treatment), phenotypic changes were not overtly observed in these animals nor was it noticed that there was evidence of health conditions to support a creatine kinase abnormality (such as skeletal muscle defect and movement abnormalities or unexpected euthanasia due to a cardiac abnormality). Interestingly, it has been shown that the creatine/phosphocreatine pathway may play a central role in energy metabolism and nutritional creatine supplementation has been shown to impart beneficial effects in a number of diverse disease pathologies (Kitzenberg et al., 2016). It was considered that this may be the case as resetting of metabolic abnormalities is needed for recovery of IBD patients that have metabolic syndrome disorder (Goncalves et al., 2015).
[00159] With reference to Figure 14: Similar to Figure 3B, RIPK2 kinase assay was determined in triple negative breast cancer cells. MDP (muramyl dipeptide) is the ligand found in pathogens to activate RIPK2 It has been considered that the RIPK2 inhibitor 1 ICso for RIPK2 is much lower than d mM.
[00160] With reference to Figure 15: RIPK2 Inhibition of NFKB Activity. N FKB gene reporter assay of inhibition of NFKB activity with the recently isolated GSK-583 inhibitor (concentration as indicated and several stimuli presented). P values for LPS or MDP treatment vs drug treatment was > 0.05, n = 3.
[00161] With reference to Figure 16: RIPK2 inhibitor 1 can also inhibit the growth of Hodgkins’ lymphoma cells (A) but not the growth of non-Hodgkin's lymphoma cells (Pfeifer or BL2) nor an ALL cell line, Jurkat T cells (B). Curiously in (B), RIPK2 inhibitor 1 can inhibit the growth of relapse NHL cell lines, a condition that may promote a robust activation of R1 PK2. It was confirmed by immunoblot and in patient lymph node tissue sections that RIPK2 is not significantly elevated in ALL or primary NHL Thus, a RIPK2 inhibitor will not have an effect on growth rate in cell lines from ALL or primary NHL patients. All MTT assays were carried out with 100 nM of RIPK2 inhibitors with drug was
Figure imgf000055_0001
added on day 1 and day 3. For blood cancer cells, all cells were counted using a hemocytometer on day 5 post-plating.
[00162] With reference to Figure 17: Pre-clinical characterization of RIPK2 Inhibitor 1 . (A) The use of dextran sodium sulphate in the drinking water of animals will promote acute inflammation injury that closely resembles ulcerative colitis. DSS-induced inflammation can be inhibited by the use of RIPK2 inhibitor 1 at 1 pg/g body weight injected intraperitonea I at day 3, 5 and 7. The Kapian-Meir survival curve is plotted for both Rassfla-'- and Rassf1a* animals. Rassf1a+J animals. (B) Rassf1a*A animals were also treated at 2 pg/g body weight with in vitro established FDA approved drugs, Gefiinib and Regorafenib, that can also inhibit RIPK2 with ECso values of 7.4 pM and 3.8 nM (Canning et al, 2015) (C) A representative picture of the longitudinal H&E stained colon section from -/+ RIPK2 inhibitor treated animals (see A for survival curves). Inhibitor treated animals reveal an ordered structure for the crypts whereby the non-RIPK2 inhibitors reveal disrupted crypts and infiltration of immune cells (dense blue dots). (D) Inflammation scores were obtained by blinded scoring by a gastrointestinal pathologist based on a combined analysis of infiltration of enterocytes, neutrophils, lamina propria cellularity, crypt structure and epithelial hyperplasia (scored as 0-2 where 2 = maximal injury as described, ref. 60), For A and B, n = 12-15 for non-FDA drug treated animals and n = 8 for FDA drug treated animals.
[00163] With reference to Figure 18: Further characterization of RIPK2 Inhibitor 1. (A) Serum cytokine production from DSS and RIPK2 inhibitor treated animals harvested on day 9. in addition to the above cytokines being inhibited, several chemokine production was also down by > 70% to include MCP-1 , MIG, MIRΊ b, MIP-2 and
RANTES/IP-10/CXCL-10 data not shown). (B) NFKB DNA binding assay was carried out using nuclear extracts derived from bone marrow derived macrophages and the l L-6 probe. In vivo injection of RIPK2 inhibitor 1 was efficient in eliminated NFKB DNA binding activity as shown. (C) Tissue yleoperoxidase was measured in colon tissues after in vivo DSS treatment and treatment with the RIPK2 inhibitor 1 MPO was analyzed using the o-dianisidine peroxidase substrate/PEOs method and colometric reading at 450 nm. 1 unit of MPO = 1 mM of HJ02 split per minute = 1 .13 x 10-2 change in absorbance. For all experiments, n = 4-6. P values for DSS treated Rassf1 a+/- vs DSS treated Rassf1 a+/- + RIPK2 inhibitor 1 ranged from 0.005 to 0.01. (D) Colon lysates from -/+ DSS treated animals and DSS treated/RIPK2 inhibitor 1 treated animals were used to immunostain for active RIPK1 using pY 474 RIPK2 phosphoantibody. See Figure 17 for details on the use
Figure imgf000056_0001
of the RIPK2 inhibitor 1 at 1 pg/g body weight. For all experiments, n = 8-10 samples from independent Rassfta-/- animals is shown. Bottom is densitometric quantitation of the phospho-RIPK2 signal.
[00164] With reference to Figure 19: (A} Pre-clinical characterization of RIPK2 I nhibitor 2. DSS-induced inflammation can be inhibited by the use of RIPK2 inhibitor 2 at 1 pg/g body weight injected intraperitoneal at day 3, 5 and 7 The Kaplan-Meir survival curve is plotted for la+/- animals -/+ RIPK2 inhibitor 2. (B) Analysis of active RI PK2 in mouse tissues via immunohistochemicaf analysis using the pY474 RIPK2 antibody #125. Analysis was carried in untreated and DSS treated wild type, Rassffa A and ii-ICr^ mice. Bottom panel is the quantitation of the IHC result using a modified ImageJ software Y axis in graph denotes“Average density of DAB Areas". The H-10-/- is an established animal model for inflammatory bowel disease and the Rassffa_A mouse is very sensitive to developing colitis to suggest importance of RASSF1 A in preventing excessive inflammation. It is known that RASSF1 A can restrict pathogen activation of RIPK2 and other elements connected to innate immunity. Please see model in Fig. 31 .
[00165] With reference to Figure 20: Clinical characterization of active RIPK2. Analysis of active RIPK2 in human tissues via immunohistochemical analysis using the pY474 RIPK2 antibody #126. Analysis was carried in FFPE unstained sections from (A) a pediatric patient from early diagnosis to 2015 and (B) in several UC and CD patients as well as non-IBD patients. Active RIPK2 is indicated by the brown stained areas that appear to be absent in the control non-IBD patient sections. Graph is quantitation of n = 25 patients in each category CRC patients also have a > 0.15 staining index for active RIPK2 as seen in the panel A graph.
[00166] Lung inflammation model for Cystic Fibrosis: With reference to Figure 21 : Pseudomonas aeruginosa model for lung inflammation. P. aeruginosa (PA) is a common pathogen associated with respiratory-tract infections in diverse clinical settings that induces oxidative stress, accentuates lung injury and elevated lung inflammation. Mice received PBS or 1 X 3 106 CFU/mouse P. aeruginosa intratracheaily under anesthesia.
24 or 48 hours after treatment, BAL was obtained using 1 ml of sterile Hanks’ Balanced Salt Buffer for the measurement of cell count, protein concentration (Panfeng Fu et al. ,
Am J Respir Cell Mol Biol Vol 48 (4), 477-488, (2013). P value PA -/+ RIPK2 inhibitor 1 = 0 021 , n = 4 and for Gefitinib, p value = 0 1 29, n = 4. Increased BAL total cellular content represents leakiness of the lung due to inflammation induced damage. RIPK2 inhibitor was given 24 hours prior to PA infection and BAL harvested 24 post-PA infection. A range
Figure imgf000057_0001
of 5-15 g/g body weight use of R1PK2 inhibitor 1 for P. aeruginosa challenge model of cystic fibrosis may be suitable.
[00167] With reference to Figure 23; RIPK2 has been demonstrated to be involved in the metastatic nature of epithelial cells. Use of the RIPK2 inhibitor to prevent the spread of cancer cells in an extravasation assay or an assay of invasion was explored. This was carried out using the chick embryo chorioallantoic membrane (CAM) model, a low cost- and time-efficient model used in vivo for cancer research, especially for metastasis. The CAM is immunodeficient early during hatching and therefore can tolerate the transplantation of human tumor cells (in a very similar way xenograft assays are carried out on immunocompromised mice).
[00168] To model the growth of primary tumor, cells were implanted into the CAM of ex ovo embryos using the glass microcapillary vasculature labelled with lectin- rhodamine or lectin-AF647 via injection into the feeding arteriole of the CAM. Cancer cells were allowed to form primary tumors and metastatic lesions for 5 days after which embryos were used for intravital imaging and reconstruction of cellular movement (reference for methodology can be found at
https://www ncbi.nlm.nih gov/pubmed/251766551.
[00169] As depicted in Figure 23, cancer cell extravasation assay was carried out in two different cell types, Hep3, a human squamous carcinoma cell and in MDA-MB 231 , a triple negative breast cancer cell. (A) In Figure 23, the left panels are 2D optical slices of chicken vasculature plus cancer cells. Vasculat re is grey with "black hole looking areas" being the outside of the vessels. Right panels are 3D reconstructions of cells within the dashed squares. Red arrows point to the cell that are extravasated, white to the ones that are still in. (B) is quantification of % cells extravasated as described in https.V/www. ncbi.nlm.nih gov/pubmed/25176655). RIPK2 inhibitor 1 (Drug 1 ) at 1.5 yg/g body weight can robustly inhibit the movement of both of these cells and thus will interfere with metastasis
[001 0] With reference to Table 5, there is provided structural analogs of RIPK2 Inhibitor 1 and creation of RIPK2 bait molecules.
Table 5. Chemical information on RIPK2 inhibitors
Figure imgf000058_0001
Figure imgf000059_0002
Figure imgf000059_0001
Figure imgf000060_0002
Figure imgf000060_0001
Figure imgf000061_0002
Figure imgf000061_0001
Figure imgf000062_0003
[00171] With reference to Figure 24: Empirical Testing of Potential off Target Effect on PKB/Akt. RIPK2 inhibitor 1 does not appear to promote cell death nor does it interfere with the Akt/PI3K pathway or downstream effectors of Akt (p4eBP1 ) that may control cell growth and translation. 4E-BP1 (also known as PHAS-1 ) inhibits cap-dependent translation and can be regulated by Akt and mTOR.
[00172] Creation of a RIPK2 Inhibitor 1 Screening Tool (Detailed Scheme) (RIPK2 Bait Molecule 1 E and 1 F in Table 5.
[00173] Original scheme for FCG806791773 (RIPK2 Inhibitor 1 ):
Figure imgf000062_0001
1 2 FCG80S791773
Synthetic procedure for FCG806791773: To a solution of acid 1 (1 mmol) in DMF was added DIPEA (3 mmol). The mixture was cooled to 0"C and treated stepwise with EDC- HCI (2 mmol), HOBt (2 mmoi) and amine 2 (1.5 mmol). The reaction mixture was stirred at room temperature for 1 h, diluted with brine, and extracted with EtOAc. The organic layer was dried with Mg2S04 and concentrated in vacuo to give a crude product. The crude was purified using reverse phase HPLC (C18, mobile phase: H20-MeOH) Yield: 167 mg: 28%; 1 H NMR (400 MHz. DMSO-d6): 5 = 2.35 (m: 3H), 3.82 (s, 3H), 6.92 (s, 1 H), 7.09 (s, 1 H), 7.49 (m, 6H), 8.03 (m, 6H), 9.95 (s, 1 H), 10.19 (s, 1 H); MS(APSI) m/z
[M+H]+ calculated for C25H22N4Q2: 411 2; found: 411.0.
Figure imgf000062_0002
[00174] Scheme for FCG30033793xx molecules with linker (creation of A RIPK2 inhibitor 1 tool for off target search):
Figure imgf000063_0001
[00175] Table 6. Example derivatives of RIPK2 Inhibitor 1 with different linkers
Figure imgf000063_0003
Figure imgf000063_0002
Figure imgf000064_0002
Figure imgf000064_0001
[00176] Validation of the use of RIPK2 Inhibitor 1 F-Agarose Beads
[00177] Beads were prepared according to manufacturer’s recommendation. Cell pellets (containing about 5 million cells) from various cancer cell lines were lysed with RIPA buffer and immunoprecipitated with 15 mI bead volume of RIPK2 inhibitor 1 F- Agarose Beads overnight. Following overnight incubation, beads were washed 2 times with 1 XPBS, followed by a 1 0% SDS-PAGE analysis for bound proteins. As shown in Figure 22, the gel was transferred to PVDF membrane and immunoblotted as indicated.
[00178] Evidence thus far would suggest constitutively active RIPK2 in primary
Hodgkin’s lymphoma cells, while little to no detection of active R1PK2 in most non- Hodgkin’s lymhoma (NHL) but possible detection in relapsed NHL. HCT1 16 is a colorectal cancer cell line that can be stimluated to promote the activation of R1 PK2 upon addition of the ligand of the NOD/RIPK2 receptor complex, muramyl dipeptide (MDP)
[00179] Then top panel of Figure 22 indicates what associated with R1PK2 Inhibitor
1 F-Agarose Beads Bottom panel is an rmmuno-blot for total RIPK2 in lysates from the input samples used to immunoprecipitate This was important to show what was the pool level of RIPK2 was before immunoprecipitation.
[00180] With reference to Figure 25: NFKB gene reporter assay determination of inhibition of MDP stimufated NFKB activity with RIPK2 inhibitor 1 analogs as stated in Table 5. All drugs in were utilized at 100 nM for 24 hours prior to adding MDP P values for RIPK inhibitor treated vs MDP was < 0.005 and n = 4-10 for all inhibitors.
[00181] With reference to Figure 26: RIPK2 Inhibitor 7 was isolated using a second screen of molecular docking analysis of which inhibitor 5 and 6 were also identified.
Inhibitor ? is also known as PP121 , a dual inhibitor of receptor tyrosine kinases (RTKs) (IC < 0.02 pM for Abl, Src, VEGFR-2 and PDGFR) and PI 3-K family kinases (ICSo <
0.06 mM for p1 10a, DNA-PK and mTOR). Exhibits no significant effect on receptor serine/threonine kinases (RSTKs). Blocks the proliferation of tumor cells by direct inhibition of PI 3-K, mTOR, Src and the VEGF receptor. No known activities or use has been found for inhibitor 5 and 6
[00182] Kinase assay for RIPK2 -/+ RIPK2 inhibitor 7 was carried out at the MRC
Protein Phosphorylation and Ubiquityiation Unit at http://www.ppu.mrc ac.uk/. The RI PK2 assay was carried out using RIPK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0 1 mM EGTA, 0. 1 % b-mercaptoethanol, 1 mg/ml BSA) and assayed against MBP in a final volume of 25 5 pi containing 50m Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 1 0 mM magnesium acetate and 0 02 mM [33P-y-ATP] (50-1000 cpm/pmoie) and incubated for
Figure imgf000065_0001
30 min at room temperature Assays are stopped by addition of 5 m! of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.
[00183] With reference to Figure 27: (A) RIPK2 autophosphorylation at site Y474 is shown and Inhibition was carried out as in Figure 3B. (B) Analysis of RIPK2
autophosphorylation using the pS176- phosphospecific RIPK2 antibody is shown for an inflammatory breast cancer cell line. Comparison to two known RIPK2 inhibitors is shown, Regorafenib and GSK-583. Bottom is quantitation of results from 4 independent experiments (C) MTT assay to explore effects on growth of 3 inflammatory breast cancer cells with RIPK2 inhibitor 7 at the indicated concentrations. Inhibition of HCT1 16 colon cancer cells was also tested and ICso of < 250 nM (data not shown). *P< 0.01 **P< 0.05 and n=4. RIPK2 inhibitor 5 and 6 do not appear to significantly affect growth rates of cancer cells (data not shown).
[00184] With reference 1o Figure 28: NFKB gene reporter assay determination of inhibition of MDP stimulated NFKB activity with RIPK2 inhibitor 5 and 6 as in Figure 34 at the indicated concentrations.
[00185] With reference to Figure 29: (A) NFKB gene reporter assay determination of inhibition of MDP stimulated N FKB activity with RIPK2 inhibitor 7 as in Figure 34 at the indicated concentrations. (B) EMSA was carried out using the NFKB binding site on the I L-6 and IL-8 promoter as in Figure 10A. A loss of signal would indicate inhibition of DNA binding abilities. 6 and 5 refer to failed RIPK2 inhibitors isolated in the screen while G (GSK-583) and R (Regorafenib) are known RIPK2 inhibitors used for comparison (C) Mammosphere formation in KPL-4 inflammatory breast cancer cell -/+ RIPK2 inhibitor 1 and 7. Mammosphere is a 3-dtmensional formation that better represents how these cells exist in vivo They form spheroids that have established contacts with a substrate that can direct growth and signaling it is very clear that both RIPK2 inhibitor 1 and 7 are efficient in slowing down the growth rate of these spheroids and the limiting their size n for (A) and (C) is 4 and p values < 0,05.
[00186] With reference to Figure 30: Clinical characterization of RI PK2 Inhibitor 7. (A) DSS-induced inflammation can be inhibited by the use of RIPK2 inhibitor 7 at 2 ng/g body weight injected intraperitoneal at day 5 and 7 The Kaplan-Meir survival curve is plotted for the 1 a-/- animals (B) DAI is plotted for disease scoring and compared to Gefiinib, a known RI PK2 inhibitor with an EC50 values of 7.4 mM and with another RIPK2 inhibitor identified in our screen, inhibitor 6. Based on this and other results, figwe
Figure imgf000066_0001
focused on inhibitor 7. Please note that both 1 a-/- and the 1 a+/- animals respond similarly to DSS induced inflammation injury and can be used interchangeably. (C) Ex vivo analysis of bone marrow derived macrophages in response to LPS stimulation. BMDM was obtained from the Rassf1a/ mice. These mice were utilized as they have a 5 robust activation of NFKB aided by the loss of RASSF1A. RIPK2 inhibitor 7 can efficiently reduce the secreted cytokine production in response to LPS, a trigger of NFKB activation and production of cytokines.
[00187] With reference to Figure 31 : Model for intestinal inflammation involving RASSF1 A. RASSF1 A functions to restrict NFKB activity by interfering with Toll receptor 10 (TLR) activation of NFKB. High levels of NFKB transcriptional activity can result in
intestinal inflammation and abnormal activation of apoptosis leading to inflammation induced damage. Furthermore, the presence of pathogens can also result in the activation of another pattern recognition receptor, NOD2 and its obligate kinase (RIPK2), to result in NFKB activation and initiation of the autophagic response (right side pathway). 15 Once NOD2 is stimulated, it recruits R1PK2 which undergoes autophosphorylation on tyrosine 474 (Y474) and polyubiqu itinated at lysine (K) 209. Activation of RI PK2 promotes the recruitment and activation of the TAK1 and /or Atg 16 L 1 resulting in activation of NFKB and autophagy respectively. There is biochemical evidence that RASSF1 A can interfere with NOD2/RIPK2 association to restrict NOD2 signaling and activation of both 0 NF-kB and autophagy. Thus the loss of RASSF1A would result in activation of RIPK2 and uncontrolled inflammation and autophagic signaling Inhibiting RIPK2 would thus be a novel therapeutic angle to restrict NF-kB driven inflammation. Prolonged intestinal inflammation will lead to hyperplasia and tumorigenesis. 5 Table 7A; Summary of Functional Properties of RIPK2 Inhibitors
Figure imgf000067_0002
Figure imgf000067_0001
Figure imgf000068_0002
*ND = Not determined
L In vitro kinase assay (an immunoprecipitation for RIPK2 followed by a kinase assay with 32P-y-ATP.
% MDP (Muramyl dipeptide) is a stimulator of the NOD2/RIPK2 pathway that will drive activation of RIPK2.
# Derivative of GSK-583 currently in phase 1 toxicity trial
& In comparison, lapatinb {a drug in human use) is predicted to 57 2% Bioavailable RIPK2 inhibitor 1 ICso for inhibition of recombinant truncated RIPK2 is 6.4 p and that IPK2 inhibitor 1 C IC5D for inhibition of recombinant truncated RIPK2 is 4.2 mM
Table 7B: Inhibition of Truncated vs Full length RIPK2 and ATP competition
Figure imgf000068_0003
Figure imgf000068_0001
Figure imgf000069_0002
[00188] Two versions of the recombinant protein was utilized to carry out the kinase assay as described in Salla et al, JPET 2018. Inhibition of c-ABL is a common off target effect of RIPK2 inhibitors. As such, determination of c-ABL inhibition is a good test for specificity of a new RIPK2 inhibitor.
[00189] ATP competition may determine how tightly the compound can bind to the ATP binding site This assay was carried out at two concentrations depending on which version of RIPK2 was utilized. Since RIKP2 inhibitor 1 , 1 B, 1 C did not inhibit the full length recombinant RIPK2 until > 200 mM, the ATP competition assay could not be done against full length recombinant RIPK2. For RIPK2 inhibitor 1 , at 200 mM, 85% activity is observed to indicate that ATP can successfully interfere with the inhibition of RSPK2 inhibitor 1 . At 400 mM, 88% activity is observed. For R1PK2 inhibitor 1 C, at 200 mM, 60% activity is observed to indicate that ATP can not successfully interfere with the inhibition of RIPK2 inhibitor 1 C At 400 pM, 115% activity is observed. For RIPK2 inhibitor 7 and GSK298559, at 200 mM, 80% and 105% of the activity is observed to indicate that ATP can successfully interfere with the inhibition of RIPK2 inhibitor 7 and GSK298559.
DEFINITIONS AND INTERPRETATION
[00190] The description of the present invention has been presented for purposes of illustration and description, but it is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Embodiments were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various
Figure imgf000069_0001
modifications as are suited to the particular use contemplated. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention.
[00191] The term "subject", as used herein, refers to an animal, and can include, for example, domesticated animals, such as cats, dogs, etc. , livestock (e.g, , cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g , mouse, rabbit, rat, guinea pig, etc ), mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. In a specific example, the subject is a human.
[00192] The term’'treatment”, "treat", or“treating” as used herein, refers to obtaining beneficial or desired results, including clinical resuits. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. "Treating" and "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.
[00193] The term "amelioration" or "ameliorates" as used herein refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
[00194] The term "symptom" of a disease or disorder (e g , inflammatory bowel disease, e.g , ulcerative colitis or Crohn’s disease, e.g. cancers) is any morbid phenomenon or departure from the normal in structure, function, or sensation, experienced by a subject and indicative of disease.
[00195] A "treatment regimen” as used herein refers to a combination of dosage, frequency of administration, or duration of treatment, with or without addition of a second medication
[00196] As used herein, "aliphatic" refers to hydrocarbon moieties that are linear, branched or cyclic, may be alkyl, alkyiene, alkenyl, alkenylene, or alkynyl, alkynylene and may be substituted or unsubstituted. "Alkyl" or "alkyiene” refers to a linear, branched or cyclic saturated hydrocarbon group "Alkenyl” or "alkenylene" means a hydrocarbon moiety that is linear, branched or cyclic and contains at least one carbon to carbon double
Figure imgf000070_0001
bond.“Alkynyl" or“alkynylene” means a hydrocarbon moiety that is linear, branched or cyclic and contains at least one carbon to carbon triple bond.
[00197] As used herein, the term "[inker" refers to a divalent moiety that bonds two molecular or atomic species by a covalent bond.
[00198] For all compounds disclosed in this application, in the event the nomenclature is in conflict with the structure, it shall be understood that the compound is defined by the structure. For compounds with stereogenic centers, the structures show the absolute stereochemistry.
[00199] Compounds described herein may also include their isotopicallydabe!led forms. An isotopica!ly-!abelled form of an active agent of a combination of the present invention is identical to said active agent but for the fact that one or more atoms of said active agent have been replaced by an atom or atoms having an atomic mass or mass number different from the atomic mass or mass number of said atom which is usually found in nature. Examples of isotopes which are readily available commercially and which can be incorporated into an active agent of a combination of the present invention in accordance with well established procedures, include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine, e.g., 2H, 3H, 13C, 14C, 15N, 1 S0, 170, 31 P, 32P, 35S, 1 BF, and 36CI, respectively. An active agent of a combination of the present invention, a prodrug thereof, or a pharmaceutically acceptable salt of either which contains one or more of the above-mentioned Isotopes and/or other isotopes of other atoms is contemplated to be within the scope of the present invention.
[00200] The invention includes the use of any compounds of described above containing one or more asymmetric carbon atoms may occur as racemates and racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Isomers shall be defined as being enantiomers and diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. Each stereogenic carbon may be in the R or S configuration, or a combination of
configurations. Some of the compounds described herein can exist in more than one tautomeric form. The invention includes methods using all such tautomers.
[00201] All alkyl, aikenyl, and alkynyl groups shall be understood as being branched or unbranched where structurally possible and unless otherwise specified. The term“alkyl" refers to both branched and unbranched alkyl groups. It should be understood that any combination term using an“aik" or“alkyl" prefix refers to analogs according to the above definition of“alkyl” For example, terms such as "alkoxy”,“alkythio" refer to
Figure imgf000071_0001
alkyl groups linked to a second group via an oxygen or sulfur atom.“Alkanoyl" refers to an alkyl group linked to a carbonyl group (C— O).
[00202] It shall be understood that if N is not substituted then it Is NH. As used herein,“nitrogen” and "sulfur” include any oxidized form of nitrogen and sulfur and the quaternized form of any basic nitrogen. For example, for a— S—C -s alkyl radical, unless otherwise specified, shall be understood to include— S{0)— Ci.s alkyl and— S(0)2— Ch-e alkyl
[00203] The term“aryl” refers to aromatic hydrocarbon rings containing from six to ten carbon ring atoms. The term aryl includes monocyclic rings and bicyclic rings where at least one of the rings is aromatic. Non-limiting examples of C3-io aryls include phenyl, indanyl, indenyl, benzocyclobutanyl, dihydronaphthyl, tetrahydronaphthyl, naphthyl, benzocycloheptanyl and benzocycloheptenyl.
[00204] The term "heterocycle" refers to a stable nonaromatic 4-8 membered monocyclic heterocyclic radical or a stable nonaromatic 6 to 1 1-membered fused bicyclic, bridged bicyclic or spirocyclic heterocyclic radical. The 5 to 11-membered heterocycle consists of carbon atoms and one or more, preferably from one to four heteroatoms chosen from nitrogen, oxygen and sulfur. The heterocycle may be either saturated or partially unsaturated. Non-limiting examples of nonaromatic 4-8 membered monocyclic heterocyclic radicals include tetrahydrofuranyl, azetidinyl, pyrrolidinyl, pyranyl, tetrahydropyranyl, dioxanyl, thiomorpholinyl, 1 ,1-dioxo-1A0-thiomorpholinyl, morpholinyl, piperidinyl, piperazinyl, and azepinyl. Non-!imiting examples of nonaromatic 6 to 1 1- membered fused bicyclic radicals include octahydroindolyl, octahydrobenzofuranyl, and octahydrobenzothiophenyl. Non-limiting examples of nonaromatic 6 to 11-membered bridged bicyclic radicals include 2-azabicyclo[2.2.1]heptanyl, 3-azabicyclo[3.1 0]hexanyi, and 3-azabicyclo[3.2 1 ]octanyl. Non-limiting examples of nonaromatic 6 to 1 1 -membered spirocyclic heterocyclic radicals include 7-aza-spiro[3,3]heptanyl, 7-$piro[3 4]octanyl, and 7-aza-spiro[3,4]octanyl.
[00205] The term“heteroaryl” shall be understood to mean an aromatic 5 to 6- membered monocyclic heteroaryl or an aromatic 7 to 1 1 -membered heteroaryl bicyclic ring where at least one of the rings is aromatic, wherein the heteroaryl ring contains 1 -4 heteroatoms such as N, O and S. Non-limiting examples of 5 to 6-membered monocyclic heteroaryl rings include furanyi, oxazolyl, isoxazoly!, oxadiazolyl, thiazoiyl, pyrazolyl, pyrrolyl, imidazolyl, tetrazolyl, triazolyl, thienyl, thiadiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, and purinyl. Non-limiting examples of 7 to 1 1-membered heteroaryl bicyclic heteroaryl rings include benzimidazolyl, quinolinyl, dihydro-2H- quinoliny!, isoquinolinyl, quinazolinyl, indazo!yl, thieno[2, 3-d]pyrim idinyl, indolyl, isoindolyl, benzofurany!, benzopyranyi, benzodioxolyf, benzoxazoiyl, benzothiazo!yl, dihydroindolyl, azaindolyl, benzothiazoly!, benzpyrrolyl, benzpyrazolyl, pyridopyrazolyl,
dihydrobenzofuranyl, benzothienyl, benzodioxany!, dihydrobenzo[1 ,4]dioxanyl and benzo[1 ,3]dioxolyl.
[00206] The term“heteroatom” as used herein shall be understood to mean atoms other than carbon such as O, N, and S.
[00207] The term“haiogen” as used in the present specification shall be understood to mean bromine, chlorine, fluorine or iodine. The definitions“halogenated", “partially or fully halogenated"; partially or fully fluorinated;“substituted by one or more halogen atoms", includes for example, mono, di or tri halo derivatives on one or more carbon atoms. For alkyl, a non-limiting example would be— CH2CHF2,— CF3etc. Each alkyl, aryl, cycloalky!/carbocycle, heterocycle or heteroaryl, or the analogs thereof, described herein shall be understood to be optionally partially or fully halogenated.
[00208] The compounds described herein are only those which are contemplated to be 'chemically stable’ as will be appreciated by those skilled in the art. For example, a compound which would have a‘dangling valency’, or a‘carbanion’ are not compounds contemplated by the inventive methods disclosed herein.
[00209] The invention includes pharmaceutically acceptable derivatives of the compounds described herein A“pharmaceutically acceptable derivative” refers to any pharmaceutically acceptable salt or ester, or any other compound which, upon administration to a patient, is capable of providing (directly or indirectly) a compound useful for the invention, or a pharmacologically active metabolite or pharmacologically active residue thereof A pharmacologically active metabolite shall be understood to mean any compound of the invention capable of being metabolized enzymatically or chemically. This includes, for example, hydroxylated or oxidized derivative compounds described herein. In addition, within the scope of the invention is use of prodrugs of compounds described herein. Prodrugs include those compounds that, upon simple chemical transformation, are modified to produce compounds described herein. Simple chemical transformations include hydrolysis, oxidation and reduction. Specifically, when a prodrug is administered to a patient, the prodrug may be transformed into a compound disclosed hereinabove, thereby imparting the desired pharmacological effect. [00210] Pharmaceutically acceptable salts of the compounds may include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfuric, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfuric and benzenesuifonic acids. Other acids, such as oxalic acid, while not themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds and their pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e g , sodium), alkaline earth metal (e.g , magnesium), ammonium and N— (C1-C4 alkyl)^ + salts.
[00211] References in the specification to "one embodiment", "an embodiment", etc , indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to combine, affect or connect such aspect, feature, structure, or characteristic with other embodiments, whether or not such connection or combination is explicitly described. In other words, any element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility between the two, or it is specifically excluded
[00212] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or use of a "negative" limitation. The terms "preferably,’ “preferred,” "prefer,”“optionally,” "may,” and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention.
[00213] The singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated.
[00214] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, ail ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range and bounding the range Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
[00215] As will also be understood by one skilled in the art, all ranges described herein, and all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number(s) recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above.
REFERENCES
[00216] All publications, patents and patent applications mentioned in this specification, and/or listed beiow, are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference
Bernstein, F.C., Koetzle, T.F., Williams, G J., Meyer, E.E., Brice, M.D., Rodgers, J.R , Kennard, O., Shimanouchi, T., and Tasumi, M. (1977). The Protein Data Bank: a computer-based archival file for macromolecular structures. Journal of molecular biology 122, 535.
Borisa, A.C , and Bhatt, H G (2017). A comprehensive review on Aurora kinase: Small molecule inhibitors and clinical trial studies. European journal of medicinal chemistry 140, 1 -19.
Branquinho, D., Freire, P. , and Sofia, C. (2016). NOD2 mutations and colorectal cancer - Where do we stand? World journal of gastrointestinal surgery 8, 284-293.
Canning, P., and Bullock, A. Structure of the kinase domain of human RIPK2 in complex with ponatinib. To be Published.
Canning, P., Ruan, Q., Schwerd, T., Hrdinka, M. , Maki, J.L , Saleh, D.,
Suebsuwong, C. , Ray, S., Brennan, P.E , Cuny, G.D., et al (2015). Inflammatory Signaling by NOD-RIPK2 Is Inhibited by Clinically Relevant Type II Kinase
Inhibitors. Chemistry & biology 22, 1 174-1 184
Cao, Y., Charisi, A., Cheng, L.C. , Jiang, T. , and Girke, T. (2008) ChemmineR: a compound mining framework for R Bioinformatics 24, 1733-1734. Cheng L et al, NOD2 promotes dopaminergic degeneration regulated by NADPH oxidase 2 in 6-hydroxydopamine model of Parkinson’s Disease, Neuroinflammation 2018
Chirieleison, S.M. , Kertesy, S. B., and Abbott, D.W. (2016). Synthetic Biology Reveals the Uniqueness of the RIP Kinase Domain Journal of immunology 196, 4291 -4297
Dai, H. , Eilis, J. L., Sinclair, D A., and Hubbard, B.P (2016). Synthesis and Assay of SIRT1 -Activating Compounds Methods Enzymol 574, 213-244
Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. Journal of biomolecular NMR 6, 277-293.
Dieleman, L.A., Palmen, M.J., Akol, H., Bioemena, E., Pena, A.S., Meuwtssen,
S.G. , and Van Rees, E P (1998). Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clinical and experimental immunology 1 14, 385-391.
D.R Koes, C J Camacho. ZINCPharmer: pharmacophore search of the ZINC database. Nucleic Acids Research 40 (2012): W409-414
Dyson, J. K., and Rutter, M.D. (2012). Colorectal cancer in inflammatory bowel disease: what is the real magnitude of the risk? World journal of gastroenterology 18, 3839-3848.
Ermann, J., Staton, T., Glickman, J.N., de Waal Malefyt, R., and Glimcher, L.H. (2014). Nod/Ripk2 signaling in dendritic celts activates IL-17A-secreting innate lymphoid cells and drives colitis in T-bet-/-.Rag2-/- (TRUC) mice. Proceedings of the National Academy of Sciences of the United States of America 1 1 1 , E2559- 2566.
Goncalves, P., Magro, F., and Martel, F. (2015). Metabolic inflammation in inflammatory bowel disease: crosstalk between adipose tissue and bowel
I nflammatory bowel diseases 21 , 453-467.
Gordon, M. , E!-Kalla, M., Zhao, Y. , Fiteih, Y., Law, J,, Volodko, N , Anwar- Mohamed, A., El-Kadi, A.O., Liu, L., Odenbach, J., et al. (2013). The tumor suppressor gene, RASSF1 A, is essential for protection against inflammation - induced injury. PloS one 8, e75483.
Haile, P.A , Votta, B J. , Marquis, R.W., Bury, M.J., Mehlmann, J.F., Singhaus, R. , Jr., Charnley, A.K. , Lakdawala, A.S., Convery, M.A , Lipshutz, D.B., et al, (2016). The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N- (5-fluoro-1 H-indazol-3-yl)quinoiin-4-amine (G5K583), a Highly Potent and Selective Inhibitor of RIP2 Kinase. Journal of medicinal chemistry 59, 4867-4880.
Hayden, M.S., and Ghosh, S. (2004). Signaling to NF-kappaB. Genes &
development 18, 2195-2224.
He, X., Da Ros, S , Nelson, J , Zhu, X., Jiang, T. , Okram, B., Jiang, S., Michel lys, P Y. , Iskandar, M., Espinola, S., et al. (2017). Identification of Potent and Selective RIPK2 Inhibitors for the Treatment of Inflammatory Diseases ACS medicinal chemistry letters 8, 1048-1053. J. Gasteiger, M. Marsilli. A new model for calculating atomic charges in molecules, Tetrahedron Letters 19 (1978): 3181 - 3184
Jun, J.C., Cominelli, F. , and Abbott, D.W. (2013). RIP2 activity in inflammatory disease and implications for novel therapeutics Journal of leukocyte biology 94, 927-932.
Kim, S , Thiessen, P.A., Bolton, E.E., Chen, J. , Fu, G., Ginduiyte, A. , Han, L, He, J., He, S. , Shoemaker, B.A., et al. (2016). PubChem Substance and Compound databases. Nucleic Acids Res 44, D1202-1213.
Kitzenberg, D , Colgan, S.P., and Glover, L. E. (2016). Creatine kinase in ischemic and inflammatory disorders. Clinical and translational medicine 5, 31.
Kuhn, R., Lohler, J., Rennick, D , Rajewsky, K., and Muller, W (1993). Inter!eukin- 10-deficient mice develop chronic enterocolitis Cell 75, 263-274
Kumar, R.K , Herbert, C , and Foster, P.S. (2008). The "classical" ovalbumin challenge model of asthma in mice. Current drug targets 9, 485-494.
Lagorce, D., Sperandio, O., Baell, J.B , Miteva, M.A., and Villoutreix, B.O. (2015). FAF-Drugs3: a web server for compound property calculation and chemical library design. Nucleic Acids Res 43, W200-207.
Lasry, A., Zinger, A., and Ben-Neriah, Y. (2016) Inflammatory networks underlying colorectal cancer. Nature immunology 17, 230-240.
Lipinski, C.A., Lombardo, F. , Dominy, B.W., and Feeney, P.J. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced drug delivery reviews 46, 3-26
Lupfer, C., Thomas, P.G., Anand, P.K., Vogel, P., Milasta, S., Martinez, J., Huang, G., Green, M., Kundu, M , Chi, H. et ai. (2013) Receptor interacting protein kinase 2-mediated mitophagy regulates inf!ammasome activation during virus infection. Nature immunology 14, 480-488.
Lupfer, C. R. , Anand, P.K. , Liu, Z. , Stokes, K. L., Vogel, P , Lamkanfi, M., and Kanneganti, T.D (2014). Reactive oxygen species regulate caspase-1 1 expression and activation of the non-canonical NLRP3 inflammasome during enteric pathogen infection PLoS pathogens 10, e1004410.
Ma et a!, Behavioral and Brain Function, P268S in NOD2 associates with susceptibility to Parkinson’s disease in a Chinese population;
Madrid, L.V., and Baldwin, A.S., Jr. (2003). Regulation of NF-kappaB by oncoproteins and tumor suppressor proteins. Methods in molecular biology 223, 523-532.
Madsen, K , Cornish, A., Soper, P., McKaigney, C , Jijon, H. , Yachimec, C., Doyle, J., Jewell, L., and De Simone, C (2001 ). Probiotic bacteria enhance murine and human intestinal epithelial barrier function. Gastroenterology 121 , 580-591.
Moriwaki, K., Berlin, J , Gough, P.J , Orlowski, G.M. , and Chan, F K. (2015).
Differential roles of RIPK1 and RIPK3 in TNF-induced necroptosis and
chemotherapeutic agent-induced cell death Cell death & disease 6, e1636. Morris, G.M. , Huey, R. , Lindstrom, W., Sanner, M. F., Belew, R.K., Goodsell, D.S., and Olson, A.J. (2009). AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility J Comput Chem 30, 2785-2791.
Murgueitio, M.S. , Rakers, C. , Frank, A., and Wolber, G. (2017). Balancing
Inflammation: Computational Design of Small-Molecule Toll-like Receptor
Modulators. Trends in pharmacological sciences 38, 155-168.
Nachbur, U , Stafford, C.A., Bankovacki, A., Zhan, V., Lindqvist, L.M., Fill, B.K. , Khakham, Y., Ko, H.J., Sandow, J.J., Falk, H., et al. (2015). A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production Nature communications 6, 6442.
akashima, K , Hirota, T , Suzuki, Y., Akahoshi, M. , Shimizu, M., Jodo, A., Doi, $., Fujita, K., Ebisawa, M. , Yoshihara, S., et al. (2006) Association of the RIP2 gene with childhood atopic asthma. Allergology international : official journal of the Japanese Society of Allergology 55, 77-83.
O'Boyle, N M., Banck, M. , James, C A., Morley, C., Vandermeersch, T., and Hutchison, G R. (201 1). Open Babel: An open chemical toolbox. Journal of cheminformatics 3, 33.
Orlowski, R.Z., and Baldwin, A.S., Jr. (2002). NF-kappaB as a therapeutic target in cancer. Trends in molecular medicine 8, 385-389
Ostojic, S. (2017). Mitochondria-targeted nutraceuticals In sports medicine: a new perspective. Research in sports medicine 25, 91-100
Owens, B.M. , Steevels, T.A., Dudek, M., Walcott, D. , Sun, M Y., Mayer, A., Allan,
P. , and Simmons, A. (2013). CD90(+) Stromal Cells are Non-Professional Innate Immune Effectors of the Human Colonic Mucosa. Frontiers in immunology 4, 307
Pettersen, E.F., Goddard, T.D., Huang, C.C , Couch, G.S , Greenbiatt, D.M., Meng, E.C., and Ferrin, T.E (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612.
R Development Group (2008). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria
Sal!a et al, Identification and Characterization of Novel Receptor-Interacting Serine/Threonine-Protein Kinase 2 Inhibitors Using Structural Similarity Analysis, J Pharmacol Exp Ther 365:354-367, May 2018
V. Santa-Cecilia, Flavia & W. Ferreira, David & M. Guimaraes, Rafaeia & Cecilio, Nerry & Fonseca, Miriam & H. Lopes, Alexandre & Ferreira, Marcela & Kusuda, Ricardo & Souza, Guilherme & Nachbur, Ueli & Alves-Filho, Jose C. & Teixeira, Mauro & Zamboni, Dario & Cunha, Fernando & Cunha, Thiago. (2018). The NOD2 signaling in peripheral macrophages contributes to neuropathic pain development. PAIN. 160. 1. 10.1097/j. pain 0000000000001383.
Sterling, T , and Irwin, J.J. (2015). ZINC 15-Ligand Discovery for Everyone. J Chem Inf Model 55, 2324-2337.
Suman, S , Sharma, P. K., Rai, G., Mishra, S. , Arora, D., Gupta, P., and Shukta, Y. (2016) Current perspectives of molecular pathways involved in chronic inflammation-mediated breast cancer. Biochemical and biophysical research communications 472, 401-409.
Tang, B. L (2016). Sirtl and the Mitochondria. Molecules and cells 39, 87-95.
Tigno-Aranjuez, J.T., Asara, J.M., and Abbott, D.W. (2010). Inhibition of RIP2’s tyrosine kinase activity limits NOD2-driven cytokine responses Genes & development 24, 2666-2677.
Tigno-Aranjuez, J.T , Benderitter, P., Rombouts, F., Deroose, F , Bai, X. , Mattiofi,
B., Cominelli, F., Pizarro, T.T , Hoflack, J., and Abbott, D.W. (2014). In vivo inhibition of RIPK2 kinase alleviates inflammatory disease. The Journal of biological chemistry 289, 29651 -29664.
Trott, O., and Olson, A.J. (2010). AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31 , 455-461.
Vanden Berghe, T., Hassannia, B., and Vandenabeeie, P. (2016). An outline of necrosome triggers. Cellular and molecular life sciences: CMLS 73, 2137-2152.
Vieira, S.M , Cunha, T. M., Franca, R.F., Pinto, L.G., Talbot, J., Turato, W.M., Lemos, H P., Lima, J, B. , Verri, W A , Jr., Almeida, S.C., et al. (2012) Joint
NOD2/RIPK2 signaling regulates IL-17 axis and contributes to the development of experimental arthritis. Journal of immunology 188, 51 16-5122.
Vriend, G. (1990). WHAT IF: a molecular modeling and drug design program. J Mol Graph 8, 52-56, 29.
W.L. DeLano, The PyMOL Molecular Graphics System, DeLano Scientific LLC,
Palo Alto, CA (2007)
Williamson, K.D., and Chapman, R.W. (2015). Primary sclerosing cholangitis: a clinical update. British medical bulletin 1 14, 53-64.
Wu, S. , Kanda, T., Nakamoto, S. , Imazeki, F., and Yokosuka, O. (2012) Knockdown of receptor-interacting serine/threonine protein kinase-2 (RIPK2) affects EMT- associated gene expression in human hepatoma cells. Anticancer research 32, 3775- 3783.

Claims

1. A compound of Formula I
Figure imgf000080_0001
wherein Y is C or N,
Ri is selected from the group consisting of: H, substituted or unsubstituted C1-C12 straight alkyl, substituted or unsubstituted C3-C12 branched alkyl, substituted or unsubstituted C3- C12 straight alkenyl, substituted or unsubstituted C3-C12 branched alkenyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted Morpholino, Amino, substituted or unsubstituted Benzyl, substituted or unsubstituted Phenyl, substituted or unsubstituted C1-C4 aryl alkyl, substituted or unsubstituted Heteroaryl, substituted or unsubstituted Arylamino, substituted or unsubstituted Dialkylamino, substituted or unsubstituted Diarylamino, substituted or unsubstituted Carboxyalkylamino, substituted or unsubstituted Carboxydialkylamino, substituted or unsubstituted Tolyl, Xylyl, Anisyl, Mesityl, Acetoxy, and Hydroxyl;
R2 and R4 are each independently selected from the group consisting of: H, substituted or unsubstituted C1-C12 straight alkyl, substituted or unsubstituted C3-C12 branched alkyl, substituted or unsubstituted C3-C12 straight alkenyl, substituted or unsubstituted C3-C12 branched alkenyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted Morpholino, substituted or unsubstituted Benzyl, substituted or
unsubstituted Phenyl, substituted or unsubstituted C1-C4 aryl alkyl, and substituted or unsubstituted Heteroaryl;
R3 and R5 are each independently selected from the group consisting of: H, substituted or unsubstituted C1-C12 straight alkyl, substituted or unsubstituted C3-C12 branched alkyl, substituted or unsubstituted C3-C12 straight alkenyl, substituted or unsubstituted C3-C12 branched alkenyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted Alkoxy, Nitrile, Halogen, substituted or unsubstituted Morpholino, Amino, substituted or unsubstituted Benzyl, substituted or unsubstituted Phenyl, substituted or unsubstituted C1-C4 aryl alkyl, substituted or unsubstituted Heteroaryl, substituted or unsubstituted Arylamino, substituted or unsubstituted Dialkylamino, substituted or unsubstituted Diarylamino, substituted or unsubstituted Carboxyalkylamino, substituted or unsubstituted Carboxydialkylamino, substituted or unsubstituted Tolyl, Xylyl, Anisyl, Mesityl, Acetoxy, Carboxy, substituted or unsubstituted Carboxyethyl, substituted or unsubstituted Alkylcarbonyl, Thiol, substituted or unsubstituted Alkylthiol, substituted or unsubstituted Alkyloxy, substituted or unsubstituted Carboxamido, and Hydroxyl; and
X is selected from the group consisting of: carbon, ortho- Oxygen, meta- Oxygen, para-Oxygen, ortho- Nitrogen, mefa-Nitrogen, para-Nitrogen, o/ΐRo-Sulfur, mefa-Sulfur, and para-Sulfur; and excluding the compound 3-benzamido-4-methyl-N-[3-(1 -methyl-1 H-imidazol-2-yl) phenyl]benzamide.
2. The compound of claim 1 wherein Y is N and R5 is a substituted or unsubstituted heterocyclyl which is either:
Figure imgf000081_0001
wherein R6 is H or an substituted or unsubstituted aliphatic moeity, preferably C1-C10 alkyl, more preferably C1 -C5 alkyl, or Z-R7, where Z is a linker and R7 is a functional group.
3. The compound of claim 2 wherein R7 is NH2
4. The compound of claim 3 which is a compound of Formula II:
Figure imgf000081_0002
wherein X is a divalent aliphatic or polymeric linker.
5. The compound of claim 4 wherein X is a substituted or unsubstituted C1-C10 alkylene, C10-C20 alkylene, C2-C10 alkenylene, C10-C20 alkenylene, or C2-C10 alkynylene, C10-C20 alkynylene; a substituted or unsubstituted C3-C10 cycloalkylene, C10-C20 cycloalkylene, C4-C10 cycloalkenylene, C10-C20 cycloalkenylene, or C10-C20 cycloalkynylene; a substituted or unsubstituted divalent polyethylene glycol; or, a substituted or unsubstituted divalent polyether.
6. The compound of claim 4 or 5 wherein the compound is selected from the group consisting of:
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
82
7. A compound of Formula III:
Figure imgf000085_0001
wherein R6 is H; an substituted or unsubstituted aliphatic moeity, preferably C1-C10 alkyl, more preferably C1-C5 alkyl; or Z-R7, where Z is a linker and R7 is a functional group.
8. The compound of claim 7 wherein Z is a divalent aliphatic or polymeric linker and R7 is NH2.
9. The compound of claim 8 wherein Z is a substituted or unsubstituted C1-C10 alkylene, C10-C20 alkylene, C2-C10 alkenylene, C10-C20 alkenylene, or C2-C10 alkynylene, C10-C20 alkynylene; a substituted or unsubstituted C3-C10 cycloalkylene, C10-C20 cycloalkylene, C4-C10 cycloalkenylene, C10-C20 cycloalkenylene, or C10-C20 cycloalkynylene; a substituted or unsubstituted divalent (poly)ethylene glycol; or, a substituted or unsubstituted divalent (poly)ether.
10. The compound of claim 7 wherein R6 is selected from the group consisting of:
Figure imgf000085_0002
Figure imgf000086_0002
Figure imgf000086_0001
11. A compound of Formula IV or V:
Figure imgf000087_0002
12. A pharmaceutical composition comprising a compound of any one of claims 1-11, or a compound of one of Formula l-A, VI, VII, VIII, or IX:
Figure imgf000087_0001
Figure imgf000088_0001
(IX) or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.
13. A method for treating inflammation, an inflammatory disorder, or a cancer in a subject comprising administering to said subject a therapeutically effective amount of a compound of any one of claims 1-11 , or a compound of Formula l-A, VI, VII, VIII, or IX, or a pharmaceutically acceptable salt thereof, or a composition of claim 12.
Figure imgf000088_0002
14. The method of claim 13 wherein the inflammation is associated with inflammatory bowel disease, asthma, obesity, diabetes, cystic fibrosis, psoriasis, arthritis, Parkinson’s Disease, Alzheimer’s Disease or neuropathic pain.
15. The method of claim 13 wherein the cancer is metastatic cancer.
16. The method of claim 15 wherein the cancer is metastatic pancreatic or colorectal cancer.
17. Use of a compound of any one of claims 1-13 or a compound of any one of Formula l-A, VI, VII, VIII, or IX, to treat inflammation, an inflammatory disorder or a cancer in a subject.
18. The use of claim 17 wherein the inflammation is associated with inflammatory bowel disease, asthma, obesity, diabetes, cystic fibrosis, psoriasis, arthritis, Parkinson’s Disease, Alzheimer’s Disease or neuropathic pain.
19. The use of claim 17 wherein the cancer is a metastic cancer, such as metastatic pancreatic or colorectal cancer.
20. A ligand for purifying or identifying a polypeptide which binds to a RIPK2 inhibitor, comprising a compound of any one of claims 1-11 or a compound of any one of Formula I- A, VI, VII, VIII, or IX, coupled to a solid support such as agarose.
21. The ligand of claim 20 which comprises a compound of claim 6.
22. A method of purifying or identifying a polypeptide which binds to a RIPK2 inhibitor, comprising the steps of providing a ligand comprising a compound of any one of claims 1- 11 or a compound of any one of Formula l-A, VI, VII, VIII, or IX, coupled to a solid support, such as agarose, and contacting the ligand with a mixture comprising the polypeptide.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112877332A (en) * 2021-03-16 2021-06-01 扬州大学 Method for detecting activity of chicken RIPK2 promoter by using dual-luciferase reporter gene
WO2022066917A1 (en) * 2020-09-23 2022-03-31 Takeda Pharmaceutical Company Limited 3-(6-aminopyridin-3-yl)benzamide derivatives as ripk2 inhibitors
CN115429803A (en) * 2022-08-08 2022-12-06 中南民族大学 Application of PP121 in preparation of medicine for relieving asthma symptoms
CN115611767A (en) * 2021-07-12 2023-01-17 沈阳化工大学 Amide compound and application thereof
CN116637108A (en) * 2023-03-21 2023-08-25 杭州师范大学 Application of salweione derivative in preparation of medicines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015179436A1 (en) * 2014-05-19 2015-11-26 Sanford-Burnham Medical Research Institute Inflammation therapy using mekk3 inhibitors or blocking peptides

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015179436A1 (en) * 2014-05-19 2015-11-26 Sanford-Burnham Medical Research Institute Inflammation therapy using mekk3 inhibitors or blocking peptides

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
APSEL, BETH ET AL.: "Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases", NATURE CHEM. BIOL., vol. 4, no. 11, 2008, pages 691 - 699, XP007909493 *
CHE, HUAN-YONG ET AL.: "PP121, a dual inhibitor of tyrosine and phosphoinositide kinases, inhibits anaplastic thyroid carcinoma cell proliferation and migration", TUMOR BIOL., vol. 35, no. 9, 2014, pages 8659 - 8664, XP035405604 *
MARIJANA HRANJEC ET AL.: "Synthesis, crystal structure determination and antiproliferative activity of novel2-amino-4-aryl-4,10-dihydro[1,3,5]triazino[1,2-a]benzimidazoles", J. MOL. STRUCT., vol. 1007, 2012, pages 242 - 251, XP028343019 *
YU JIAO ET AL.: "Design, synthesis and evaluation of novel 2-(1H-imidazol-2-yl)pyridine Sorafenib derivatives as potential BRAF inhibitors andanti-tumor agents", EUR. J. MED. CHEM., vol. 90, 2015, pages 170 - 183, XP029125945 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022066917A1 (en) * 2020-09-23 2022-03-31 Takeda Pharmaceutical Company Limited 3-(6-aminopyridin-3-yl)benzamide derivatives as ripk2 inhibitors
CN112877332A (en) * 2021-03-16 2021-06-01 扬州大学 Method for detecting activity of chicken RIPK2 promoter by using dual-luciferase reporter gene
CN115611767A (en) * 2021-07-12 2023-01-17 沈阳化工大学 Amide compound and application thereof
CN115429803A (en) * 2022-08-08 2022-12-06 中南民族大学 Application of PP121 in preparation of medicine for relieving asthma symptoms
CN116637108A (en) * 2023-03-21 2023-08-25 杭州师范大学 Application of salweione derivative in preparation of medicines

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