The Identification and Pharmacological Characterization of 6-(tert

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The identification and pharmacological characterization of 6(tert-butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK’583), a highly potent and selective inhibitor of RIP2 Kinase Pamela A Haile, Bartholomew J. Votta, Robert W Marquis, Michael J. Bury, John F. Mehlmann, Robert R. Singhaus, Adam K. Charnley, Ami S. Lakdawala, Maire A. Convery, David B. Lipshutz, Biva M. Desai, Barbara Swift, Carol A. Capriotti, Scott B. Berger, Mukesh K. Mahajan, Michael A. Reilly, Elizabeth J. Rivera, Helen H. Sun, Rakesh Nagilla, Allison M. Beal, Joshua N. Finger, Michael N. Cook, Bryan W. King, Michael T. Ouellette, Rachel D Totoritis, Maria Pierdomenico, Anna Negroni, Laura Stronati, Salvatore Cucchiara, Bartlomiej Ziolkowski, Anna Vossenkamper, Thomas T. MacDonald, Peter J. Gough, John J. Bertin, and Linda N. Casillas J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00211 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Journal of Medicinal Chemistry

MacDonald, Thomas; Blizard Institute, Queen Mary University of London Gough, Peter; GlaxoSmithKline Bertin, John; GlaxoSmithKline Casillas, Linda; GSK, ImmunoInflammation

ACS Paragon Plus Environment


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The identification and pharmacological characterization of 6-(tert-butylsulfonyl)-N-(5fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK’583), a highly potent and selective inhibitor of RIP2 Kinase

Pamela A. Haile,1‡ Bartholomew J. Votta,1‡ Robert W. Marquis,1 Michael J. Bury,1† John F. Mehlmann,1 Robert Singhaus, Jr.,1 Adam K. Charnley,1 Ami S. Lakdawala,2 Maire A. Convery,2 David B. Lipshutz,1 Biva M. Desai,1 Barbara Swift,1 Carol A. Capriotti,1 Scott B. Berger,1§ Mukesh K. Majahan,1 Michael A. Reilly,1 Elizabeth J. Rivera,1 Helen H. Sun,1 Rakesh Nagilla,1 Allison M. Beal,1 Joshua N. Finger,1 Michael N. Cook,1 Bryan W. King,2 Michael T. Ouellette,2 Rachel D. Totoritis, 2 Maria Pierdomenico,3 Anna Negroni,3 Laura Stronati,4 Salvatore Cucchiara,5 Bartłomiej Ziółkowski,6 Anna Vossenkämper,7 Thomas T. MacDonald,7 Peter J. Gough,1§ John Bertin,1 Linda N. Casillas1*

1

Pattern Recognition Receptor Discovery Performance Unit, Immuno-inflammation Therapy Area Unit; 2Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA &


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Stevenage, UK; 3 ENEA, Italian National Agency for new Technologies, Energy and Sustainable Economic Development, Rome, Italy; 4Department of Cellular Biotechnology and Hematology, Sapienza University Hospital Umberto I, Rome, Italy; 5Department of Pediatrics, Pediatric Gastroenterology and Liver Unit, Sapienza University Hospital Umberto I, Rome, Italy; 6 Royal London Hospital, Endoscopy Unit, Barts Health, London, UK. 7Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK



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Abstract RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2 leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, since they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK‘583, a next generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

Introduction The NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2) pathways have generated much interest in recent years. Through human and murine genetic evidence, as well as varied in vitro and in vivo investigations, this pathway has been implicated in a wide variety of inflammatory disorders, including inflammatory bowel disease (IBD),1,2 Blau Syndrome,3,4 rheumatoid arthritis,5,6 sarcoidosis,7 and asthma.8,9,10 NOD1 and NOD2 are well characterized cytoplasmic NLR’s (nucleotide-binding domain leucine-rich repeat family)11,12 that have been shown to play essential roles in innate immunity by recognizing peptidoglycan fragments from both gram negative and gram positive bacteria.13,14,15,16 Activation of either NOD1 by diaminopimelic acid (meso-DAP) or NOD2 by muramyldipeptide (MDP) initiates a signaling cascade culminating in synthesis of pro-inflammatory cytokines and


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chemokines. Under healthy physiological conditions, this pathway functions as a sentinel by recognizing and responding to invasive bacteria.

Dysregulation of this signaling pathway,

however, can lead to pathological levels of inflammatory cytokines, and ultimately disease.17 RIP2 kinase (also known as RIPK2, RICK, CARDIAK, and CARD3) is a multi-domain, dual specificity kinase that is expressed ubiquitously, and is the convergence point for NOD1 and NOD2 signaling.18,19,20,21,22 The C-terminal CARD domain (Caspase Activation and Recruitment Domain) of RIP2 is required for association with both NOD1 and NOD2, and functional activity of the N-terminal kinase domain is required for downstream signal transduction. The kinase domain of RIP2 is known to undergo auto-phosphorylation and subsequent ubiquitinylation, thus enabling downstream signaling through activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and mitogen-activated protein (MAP) kinases, resulting in the transcription of multiple inflammatory cytokine genes.23,24,25,26 In Blau syndrome patients, a gain-of-function mutation in NOD2 results in over-activation of this pathway, evidenced by a high level of phospho-RIP2.3

Genetically modified mice lacking the RIP2 protein are

unresponsive to either NOD1 or NOD2-activating ligands, highlighting the essential role of RIP2 kinase in NOD1 and NOD2 signaling.27 Additionally, it has been shown that the abundance of activated, phosphorylated RIP2 kinase is increased in intestinal biopsies obtained from pediatric Crohn’s and ulcerative colitis patients.28,29,30 Thus, pharmacologically targeting the NOD/RIP2 signaling pathway (or “nodosome”) via direct inhibition of RIP2 is expected to have broad therapeutic utility for auto-inflammatory diseases. In this paper we detail our efforts to identify and characterize a potent and highly selective inhibitor of RIP2 kinase, with the intent to further elucidate the biological effects of aberrant NOD1 and NOD2 signaling. Ultimately, we aim to discover a safe and effective inhibitor of



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RIP2 kinase for the treatment of chronic inflammatory diseases. Both endeavors require a rigorous commitment to achieving broad kinase selectivity. Until recently, kinase inhibitors have not been utilized for chronic non-oncology indications due to off-target toxicities associated with a lack of selectivity of many of these compounds. Several investigational and research compounds have been reported in the literature to have activity against RIP2 kinase;31 however, those with the highest affinity for RIP2 (e.g. Ponatinib; 4-chloro-7,10-dioxa-13,17,18,21tetraazatetracyclo[12.5.2.12,6.017,20]docosa-1(20),2(22),3,5,14(21),15,18-heptaene (OD36); and, SB-203580, 2) 32,33,34 suffer from poor overall kinome selectivities that could readily confound interpretation of their pharmacological effects in experimental systems. Other inhibitors such as Gefitinib and N-(2-(4-amino-3-(p-tolyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2methylpropyl)isonicotinamide (WEHI-345), although more selective across the kinome, have somewhat reduced activity in complex systems such as bone marrow-derived macrophage (BMDM) cultures and in vivo.30,35 To attain the highest quality molecule, we pursued a screening and triage strategy which allowed us to rapidly identify a broadly selective hit chemotype, that quickly evolved into the discovery of a precision tool molecule. In this report, we detail the identification and characterization of a highly differentiated RIP2 inhibitor tool compound distinguished from currently available tools by its combination of exquisite selectivity, robust potency, and demonstrated efficacy in both rodent and human models of inflammatory disease. Screening and Hit Identification We employed a broad screening strategy attempting to identify chemical diversity in a rapid fashion. This included screening the GSK kinase inhibitor set against the full RIP2 protein utilizing a fluorescence polarization (FP) based binding assay, as well as screening the kinase domain truncate protein (residues 1-310) in an affinity screen against our DNA Encoded Library


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(ELT) collection.36 Not unexpectedly, a variety of potent, ATP-competitive, Type I and Type II kinase inhibitor chemotypes were identified from this screening effort. Compounds 1, 2 and 3 illustrate screening hits from 3 different chemotypes and 2 different binding modes (Figure 1). Compound 1,37 is an example of a type II inhibitor that targets the DFG-out inactive conformation of the kinase. It had a modest binding affinity (pIC50 = 6.5) but relatively poor binding and ligand efficiencies typical of this class of inhibitor (BEI = 13.9, LipE = 1.2). The type I inhibitor 2,34 which targets the active DFG-in conformation, had a lower MW and clogP and improved binding affinity compared to 1, which led to superior BEI and LipE values.38,39 Compound 3, also a Type I inhibitor, had the best balance of binding affinity (pIC50 = 8.5) and properties, with BEI, and LipE far exceeding the values for the other hits. By extension, the physicochemical properties of 3, namely, the low molecular weight (MW = 328) and lipophilicity (clogP = 3.1), presented an excellent starting point for further optimization.

Interestingly,

although screening hits 2 and 3 were identified specifically from the FP binding screen, the ELT affinity screen also identified compounds related to these chemotypes.

Figure 1. Representative hits from the high throughput screen with binding affinity and efficiency measures. BEI = (pIC50/MW)x1000; LipE = pIC50 – clogP.



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To drive toward selection of a highly selective biological tool, a strategy to rapidly triage the screening hits by a cell-based RIP2-dependent selectivity assay was devised. To do this we evaluated inflammatory chemokine IL-8 secretion from HEK cells by two distinct but parallel cellular pathways. In a direct measure of RIP2-dependent cellular function, IL-8 production was measured in HEK cells over expressing NOD2 following stimulation with the NOD2-specific ligand MDP. In contrast, stimulation by the TLR2-specific ligand, Pam2CSK4, in HEK cells over expressing TLR2 resulted in IL-8 production by a RIP2-independent pathway. Evaluation of multiple compounds from each chemotype represented by compounds 1, 2 and 3 in the 2 orthogonal cell assays resulted in clear differentiation in their cell-based selectivities. Specifically, compound 3 exhibited an IC50 of 20 nM in the MDP stimulated assay and an IC50 of >5000 nM in the Pam2CSK4-stimulated TLR2-dependent assay, translating to >500-fold difference in activity between RIP2-dependent and RIP2-independent cellular pathways (Table 1). In fact, the 4-aminoquinoline chemotype emerged as the most compelling series by virtue of its superior selectivity in these cellular assays in comparison to all other chemotypes identified in the screen (Figure 2).

Table 1. Activity of representative hits across biochemical and cell assays Cmpd

RIP2 FP IC50 (nM)

MDP/NOD2 IL-8 IC50 (nM)

Pam2CSK4 / TLR2 IL-8 IC50 (nM)

Fold difference in cell assays

1

790

320

2000

6.2x

2

250

250

6310

25x

3

3

20

>10000

>500x


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8

TLR2(HEK) Pam2CSK4-IL8 pIC50

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Non 4AQ

7.5

4AQ

7 6.5 6 5.5 5 4.5 4 4

6

8

10

NOD2(HEK) MDP-IL8 pIC50

Figure 2. Correlation of inhibition of IL-8 production (pIC50) in NOD2(HEK) cells stimulated by MDP (x-axis) and in TRL2(HEK) cells stimulated by Pam2CSK4 (y-axis) for the 4aminoquinoline (4AQ) series compared to other inhibitor series (Non 4AQ).

Encouraged by the excellent cell-based selectivity demonstrated by 3, the compound was further evaluated against a panel of 337 kinases in a kinase activity assay at Reaction Biology Corporation (RBC).40 At 1 micromolar concentration, compound 3 inhibited 38 kinases by ≥90% and 17 kinases by 70 – 90% (Supporting Information, Table S2). We presumed that the lack of broader kinase selectivity of 3 was driven by the promiscuous binding nature of the 3-amino-4methyl-phenol back-pocket moiety, and as such, this emerged as the first area of exploration for optimization of this template.

Chemistry



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The synthesis of 4-aminoquinoline analogs including hit compound 3 was conducted as described in the literature (Scheme 1).41 Condensation of 4-(methylthio)aniline with diethyl 2(ethoxymethylene)malonate by cyclization in inert solvent at high temperature provided the quinoline 5. Decarboxylation in two steps afforded quinoline 6. Oxidation of the sulfide to the sulfone utilizing potassium peroxymonosulfate followed by chlorination at the 4-position of the quinoline with thionyl chloride provided the intermediate required for diversification with multiple aromatic back-pocket groups.

Heating of chloroquinoline 7 under microwave

conditions in the presence of the appropriate amines afforded compounds 3 and 8 - 10.

Scheme 1. Synthesis of analogs containing varied back-pocket moieties.

Reagents and conditions: (a) diethyl 2-(ethoxymethylene)malonate, 160 °C, 83%; (b) Ph2O, 250 °C, 100%; (c) NaOH, EtOH/H2O, 130 °C, 97%; (d) Ph2O, 250 °C, 60%; (e) potassium peroxymonosulfate, MeOH/ H2O; (f) SOCl2, DMF, 100 °C, 91% over two steps; (g) EtOH, 150 °C,

microwave

with

3-amino-4-methylphenol,

1H-benzo[d][1,2,3]triazol-5-amine,


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benzo[d]thiazol-5-amine or 5-fluoro-1H-indazol-3-amine [3 (39%), 8 (64%), 9 (67%), 10 (14%)].

4-Chloro-quinolines with sulfone alternatives at the C6 position such as hydrogen, trifluoromethyl, and methoxy were purchased from commercial sources (Scheme 2). Addition of 3-amino-4-methylphenol under microwave conditions provided analogs 15, 19 and 20. From the 6-iodoquinoine 16, the meta-pyridyl quinoline 17 was formed via a Suzuki reaction while the tetrahydropyranylmethylamide 18 was installed by a one pot carbonylation–amidation sequence.42

Scheme 2. Synthesis of analogs containing sulfone alternatives.

Reagents and conditions: (a) 3-amino-4-methylphenol, EtOH, 150 °C, microwave [15 (55%), 16 (92%), 19, 20 (15%)]; (b) 3-(1,3,2-dioxaborinan-2-yl)pyridine, Pd(dppf[Cl2]), K2CO3, 150 °C, microwave, 41%; (c) (tetrahydro-2H-pyran-4-yl)methanamine; DBU, Mo(CO)6, t-Bu3PBF4, trans-di(µ-acetato)bis[0-(di-o-tolyl-phosphino)benzyl]dipalladium(II), THF, 150 °C, microwave, 11%.



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Diversity on the sulfone substituent was facilitated by utilizing a C6 halogenated quinoline core as in Scheme 3. Condensation of 4-iodoaniline with Meldrum’s acid afforded the malonic acid ester 22. Cyclization to the 4-hydroxy-6-iodoquinoline under heated conditions and subsequent treatment with POCl3 provided the chlorinated quinoline core 12 setting the stage for eventual introduction of the aniline moiety.

Alternatively, commercially available 6-bromo-4-

chloroquinoline was used as in 25. Palladium catalyzed coupling at C6 of either iodide 12 or bromide 25 was used to install the desired thioether. Oxidation to sulfones 23 and 26 was followed by introduction of the back-pocket moiety as in Scheme 1 leading to 24 and 27.

Scheme 3. Synthesis of analogs containing varied alkyl sulfone moieties.

Reagents and conditions: (a) triethoxymethane, Meldrum’s acid, 90 °C to 70 °C, 75%; (b) Ph2O, 240 °C, 82%; (c) POCl3, 92%; (d) i-PrSH, Pd(PPh3)4, KOt-Bu, toluene, 50 °C, 45%; (e) potassium peroxymonosulfate, THF/ H2O, 80%; (f) EtOH with benzo[d]thiazol-5-amine or 5fluoro-1H-indazol-3-amine, 47%; (g) i-PrSH, Pd(PPh3)4, Et3N, acetonitrile, 90° C, 91%; (h)


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potassium peroxymonosulfate, THF/ H2O, 41%; (i) HCl, EtOH, 5-fluoro-1H-indazol-3-amine, 94%.

Results and Discussion Following identification of 4-aminoquinoline 3, we established unambiguous biophysical evidence that compound 3 binds within the ATP binding site of the kinase domain of RIP2. We recently published the first X-ray crystal structure of apo-RIP2 at 2.4Å resolution and the cocrystal structures of several RIP2/inhibitor complexes with different binding motifs within the ATP pocket of RIP2.43

Utilizing this in-house RIP2 crystallization methodology, we

subsequently solved the co-crystal structure of aminoquinoline 3 bound to the kinase domain of RIP2 (Figure 3 and Supporting Information Table S1). The co-crystal structure confirms that 3 binds within the ATP binding pocket of RIP2 in a traditional Type I DFG-in inhibitor binding motif with a single hinge interaction between the backbone N-H of Met98 and the quinoline nitrogen of 3. The glycine-rich loop of RIP2 possesses a polar Ser25 residue, which is rare among protein kinases with only three other kinases having a serine at this position. Interestingly, the crystal structure revealed a 2.7Å hydrogen bonding interaction between the sulfone oxygen of 3 and Ser25. The methyl group of the C6 methyl sulfone projects into a small pocket on the floor of the active site, and is also solvent accessible suggesting an attractive point for inhibitor optimization. The quinoline core of 3 occupies the adenine binding pocket and projects the C4 aniline around the Thr95 gatekeeper and into the hydrophobic back-pocket of RIP2. The phenol group forms hydrogen bonds with 2 of the 3 conserved catalytic residues, Glu66 and Asp164, and the methyl group is oriented toward a hydrophobic patch in the ceiling of the back-pocket of RIP2. Unlike the Ser25 interaction, these interactions within the back-pocket



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are not predicted to be unique to RIP2 and are likely the basis for the broad kinase inhibition seen with 3.

Figure 3. Co-Crystal structure of 4-anilinoquinoline 3 bound within the ATP binding pocket of RIP2 kinase (2.7Å).

By leveraging our diverse collection of co-crystal structures of ligand-bounded RIP2, we postulated that the large and flexible hydrophobic back-pocket provided an opportunity to drive optimization of inhibitor selectivity. Interpretation of the ELT affinity screening results from a 4substituted quinoline containing library of >400 anilines led to the identification of two amines, 5-aminobenzothiazole and 5-aminobenzotriazole, that presented unique structural features when compared to the SAR from the FP screen. Drawing on the observation of complementary SAR between hits identified by both ELT and FP based screens, we incorporated these amines as part of an array of >200 aniline backpocket replacements on the methylsulfone quinoline core using the method described in Scheme 1. This combined approach led to the identification of several


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analogs with dramatically improved kinase selectivity over the screening lead 3. Exemplars 8 (5-aminobenzotriazole), 9 (5-aminobenzothiazole) and 10 (3-amino-5-fluoroindazole) stood out in terms of activity and selectivity as shown in Table 2. Kinase targets p38α and VEGFR2 were chosen as surrogate kinases because they represent different branches of the kinome and cross reactivity between RIP2 with p38α and VEGFR2 has been observed with earlier inhibitors. Inhibition data for these kinases provided surrogate kinase selectivity data in a high through-put fashion, thus providing a filter preceding the more extensive kinome profiling applied to advanced analogs.

Clearly, selectivity increased dramatically for compounds 8, 9, and 10

relative to p38α and VEGFR2 kinases in comparison to 3. While it is unclear precisely what is driving the selectivity for RIP2 over VEGFR2 and p38α, we have surmised that subtle electrostatic potential differences in the back-pocket region of each protein may play a role, in conjunction with the higher degree of flexibility in the back-pocket of RIP2. This hypothesis is based upon SAR observations of selectivity differences between back-pocket groups and correlative electrostatic potential calculations using ab initio DFT/B3LYP/6-31G* methods.44,45 While all three backpocket groups exhibited desirable selectivity profiles combined with potent RIP2 inhibition and favorable LipE, benzotriazole 8 was not progressed further due to poor oral rat PK (data not shown). Representative inhibitors containing both benzothiazole 9 and indazole 10 backpocket groups were evaluated in a kinase panel screen against >280 kinases at Reaction Biology Corporation (RBC). Selectivity of the 3-aminoidazole was generally superior to that of the benzothiazole. Despite the slightly lower potency and ligand efficiency, the 3-aminoindazole was chosen as the preferred back-pocket substitution based on the kinase selectivity profile. Table 2. Back-pocket analogs



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Cmpd

R

RIP2 FP IC50 (nM)

LipE

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p38α VEGFR2 IC50 (nM) IC50 (nM)

3

3

5.5

502

186

8

50

4.7

>5000

>5000

9

6.3

4.7

>5000

2385

10

50

4.0

>5000

>5000

Next we set out to determine whether sterics or electostatic interactions were driving other aspects of the protein/ligand interaction for this chemical series. To assess the importance of the sulfone / Ser25 interaction observed in the co-crystal structure, a set of C6-substituted compounds with varying abilities to hydrogen bond with serine were tested against RIP2 as well as p38α and VEGFR2 (Table 3). Because we expected many of these substitutions to have varying electronic effects on the quinoline ring system, thus affecting the N1 interaction with the NH of Met98 at the hinge of the kinase, analogs were also designed to determine the effect of the electron donating and electron withdrawing groups on RIP2 activity as a measure of hinge binding affinity. As a baseline comparison, the C6 unsubstituted quinoline 15 was prepared, which should have no interaction with Ser25 and a stronger N1 – Met98(NH) interaction relative to sulfone 3. A >200 fold shift in potency was observed between the hit sulfone 3 and the


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unsubstituted quinoline 15 suggesting that the Ser25 electrostatic interaction was indeed important for binding affinity. The pyridine-containing quinoline 17 and amide 18 both retained activities of 10 and 20 nM, respectively at RIP2, as expected, based on the predicted interactions of these substituents with Ser25 from modelling exercises.

Methoxyquinoline 19 and

trifluoromethylquinoline 20 were designed as analogs that would have negligible interactions with Ser25, and so would allow an isolated evaluation of the electron withdrawing and donating substituent effect upon hinge binding.

It is generally accepted that increasing the H-bond

strength at the hinge binding interaction should lead to higher affinity inhibitors.46 Indeed, ab initio calculations47 showed that the quinoline N1 hydrogen bonding strength was predicted to increase with increasing electron donating abilities of the C6 substituents [OMe (19) > H (15) > CF3 (20) > SO2Me (3)]. Thus, it was anticipated that 19 would retain RIP2 activity despite no Ser25 interaction due to the increased hinge binding strength, but that analog 20 would lose activity. It was therefore surprising that 20 was very active at RIP2 (IC50 = 8 nM) despite the predicted weaker hinge binding affinity. Interestingly, while fluorine generally forms weaker hydrogen bonds than oxygen (2.4 kcal/mol vs. 5-10 kcal/mol), they have been reported in the literature.48,49 Modelling of trifluoromethylquinoline 20 in the RIP2 active site revealed a very close interaction between the fluorine and the Ser25 hydroxyl of RIP2 with an F-H distance of 1.7Å. There are very few documented examples in which a fluorine and hydroxyl are in such close proximity suggesting that this fluorine hydrogen bond is particularly strong. 50,51 We believe that this putative H-F bonding interaction is compensating for the reduced hinge binding affinity for this compound. In addition, most of these C6 substitutions had a beneficial impact on kinase selectivity compared to the methyl sulfone as indicated by the activity at the surrogate kinases p38α and VEGFR2. In the end, the methyl sulfone was chosen as the preferred C6



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substitution because it provided the best balance of potency, physicochemical properties, permeability, and lipophilic ligand efficiency (LipE).

Table 3. Changes at C6 to Assess the Importance of the Ser25 Interaction

Compound

3

15

RIP2 FP

p38α

VEGFR2

Permeability

IC50 (nM)

IC50 (nM)

IC50 (nM)

(nm/sec)

3

500

190

79

5.5

630

>5000

>5000

250

1.9

10

3740

460

230

3.5

20

>5000

>5000

15

3.8

R

H

17

LipE

18

19

CH3O

250

>5000

3000

230

2.0

20

CF3

8

1080

100

490

3.0

Combining the highly selective 3-aminoindazole back-pocket binder with more sterically demanding C6 sulfone substituents led to compounds 24 (GSK’214)3 and 27 (GSK’583) (Table


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4). Although these changes did not provide any additional benefits in terms of kinase selectivity or rat oral pharmacokinetics, increasing the bulk and lipophilicity at the sulfone functionality translated to moderate improvements in the binding affinity while maintaining reasonable lipophilic ligand efficiency (LipE). In addition to robust selectivity against p38α and VEGFR2, 27 exhibited excellent selectivity in a panel of 300 kinases at 1 µM compound concentration (Figure 4, and Supporting Information Table S3). In this panel, only RIP2 was inhibited >50% by 27, and only two additional kinases were inhibited by approximately 30% (BRK, Aurora A). This level of broad kinome selectivity is a significant improvement over most published kinase inhibitors.21,31-34 Inhibitor 27 possesses comparable binding affinity for RIP3 kinase (not included in RBC panel) as demonstrated by an in-house FP binding assay configured similarly to the RIP2 FP assay (RIP2 FP IC50 = 5 nM; RIP3 FP IC50 = 16 nM). Despite this potent biochemical activity against RIP3 kinase, 27 showed little or no inhibition of RIP3-dependent necroptotic cell death in a cellular assay up to 10 µM concentration (Supporting Information Figure S3). The reason for this shift in potency between biochemical and cell-based formats is not understood completely. However, we have experimentally determined that the KM,ATP for RIP3 is 5 µM, a relatively low KM,ATP among kinases. By comparison, the KM,ATP for RIP2 is 100 µM (data not shown). Given the relationship between KM,ATP and IC50 from the ChengPrusoff equation,52,53 and the expectation that the intracellular concentration of ATP is in the 1 – 10 mM range, it is not surprising that for RIP3 in particular, there would be a large shift in cellular potency.54

Table 4. Small Modifications of the Methylsulfone



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Compound

Structure

RIP2 FP IC50 (nM)

Page 20 of 57

Rat Oral PK

p38α

VEGFR2

IC50 (nM)

IC50 (nM)

LipE

a

Cmax (ng/mL) / AUC (ng*h/mL)

a

10

50

4.0

>5000

>5000

500 / 1700

24

10

3.9

>5000

>5000

370 / 1100

27

5

3.8

5119

>5000

260 / 980

Cassette data, 2 mg/kg oral dose; female Sprague-Dawley rat

Figure 4. Kinome plot for 27, 1 µM compound, RBC panel of 300 kinases. Green = 1000

n.d.

9

n.d.

2

Human whole blood, MDP-stimulated TNFα release

80 237

44

6

Rat whole blood, MDP-stimulated TNFα release

133

65

5

MDP-stimulated IL8 release (HEK/NOD2) Pam2CSK4-stimulated IL8 release (HEK/TLR2) Human monocytes, MDP-stimulated TNFα release*

*Individual values: 7 nM and 9 nM in two separate experiments.

The cellular specificity of 27-mediated inhibition of pro-inflammatory cytokine production in human monocytes was further established using ligands specific to other innate immune receptors constitutively expressed in monocytes, which are known to signal primarily via RIP2independent pathways.

Following treatment with 27 at 1 µM, little inhibition of pro-

inflammatory signaling was observed upon activation of Toll-like receptors (TLR2, TLR4, TLR7) or cytokine receptors (IL-1R, TNFR) (Figure 6). In contrast, complete inhibition was


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observed upon activation of both NOD1 and NOD2 receptors, which signal in a RIP2-dependent manner. These data demonstrate that the potency and selectivity of 27 demonstrated in biochemical assays translates to cellular systems.

Figure 6. Cellular selectivity of RIP2 kinase inhibitor 27 was evaluated in primary human monocytes. In the presence of 1 µM 27, NOD1- or NOD2-mediated cytokine production was completely inhibited. In contrast, only weak inhibition was observed upon stimulation with ligands which selectively activate various TLR or cytokine receptors. The targeted receptors were stimulated with the following ligands: NOD1 (300 µg/mL, ieDAP), NOD2 (1 µg/mL, MDP); TLR2 (10 ng/mL Pam2Csk4), TLR4 (10 ng/mL, ultrapure LPS), TLR7 (10 µg/mL, gardiquimod), IL-1R (10 ng/mL, IL-1β), and TNFR (100 ng/mL, TNFα). Release of proinflammatory cytokines, either TNFα (NOD2, TLR2, TLR4, IL1R) or IL-8 (NOD1, TLR7, TNFR) were measured by immunoassay. % control indicates the mean ± SD (N=3) of the cytokine response in the absence of inhibitor.

Compound 27 was further profiled in discrete oral and i.v. crossover experiments in both rat and mouse. The pharmacokinetic parameters are consistent across rodent species exemplifying low clearance, moderate volumes of distribution, and moderate oral bioavailability (Table 6).



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Unfortunately the PK/PD relationship incorporating rodent PK with the concentration-response data from the human whole blood assay suggests 27 would not produce a human phamacodynamic response within an acceptable dose range, thus precluding this molecule from further development as a drug candidate. However, the oral PK in rat and mouse provides sufficient systemic exposure for use as a preclinical in vivo tool molecule in an acute inflammation challenge model.

Table 6. PK parameters for 27 in mouse and rat, discrete iv/po studies Oral / IV Oral Dose Cmax (mg/kg) (ng/mL)

Species

Strain

Rat

SpragueDawley

2.0 / 1.0

Mouse

C57BL/6

2.0 / 1.0

Oral AUC (ng*h/mL)

Cl (mL/min /kg)

Vdss (L/kg)

T½ (h)

%F

Free Fraction (%)

260

980

15

2.0

3.1

39

2.6

340

860

13

1.2

2.1

34

--

To demonstrate the utility of 27 in this context, we employed a combination of models which measured local and systemic inflammatory responses to intra-peritoneal injection of L18-MDP in mice and intra-venous injection of L18-MDP in rats with oral doses of 27 of 0.1, 1, and 10 mg/kg. Compound 27 inhibited serum KC (the rodent ortholog of IL-8) levels in rats in a dosedependent manner with an IC50 derived from rat blood concentrations of 50 nM (or 20 ng/mL). Similarly, 27 inhibited serum KC levels (data not shown) and recruitment of neutrophils into the peritoneal cavity in mice in a dose-dependent manner with an IC50 of 37 nM (15 ng/mL) derived from mouse blood concentrations (Figure 7). As expected, a single oral dose of 10 mg/kg of 27 also inhibited the increase in serum levels of KC following administration of the NOD1 ligand, FK156 in mice, but had no effect on cytokine production in response to stimulation with the


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TLR4 ligand, LPS at the same dose (Figure 7). These data demonstrate that 27 is a potent and selective RIP2 kinase inhibitor in vivo as well as in vitro.

Figure 7. 27 inhibited MDP-induced inflammatory responses in vivo. Oral administration of 27 (0.1, 1, 10 mg/kg) produced a dose-dependent inhibition of serum KC in rats following intravenous challenge with L18-MDP (left panel) and inhibition of neutrophil levels from peritoneal lavage in mice following intraperitoneal challenge with L18-MDP (center panel). The RIP2 specificity in vivo was confirmed by demonstrating that an oral dose (10 mg/kg) of 27 inhibited the increase in serum KC levels in mice following an intravenous challenge with the NOD1 ligand, FK156, but not with TLR4 ligand, LPS.

Recent publications have shown that inhibitors of RIP2 kinase are effective in alleviating inflammation in animal models of experimental colitis.32,34,55 As it is well established that these animal models of colitis and inflammation have limited translatability, we decided to employ a human translational model using intestinal mucosal tissue from IBD patients.

Human intestine mucosal biopsy experiments



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Page 26 of 57

Previous studies have provided evidence of increased activation of the NOD2/RIP2 signaling pathway in intestinal biopsies taken from pediatric UC and CD patients with active disease, including increased expression of the activated form of RIP2 (pS176).16-18 We examined RIP2 expression and phosphorylation in biopsies from adult UC (n=14) and CD (n=14) patients (Supporting Information Figure S5). Total levels of RIP2 protein were similar to those seen in biopsies from normal healthy volunteers, but the proportion of phosphorylated RIP2 (anti-pS176) was significantly increased in both CD and UC biopsies suggesting that the RIP2 pathway was similarly activated in adult disease. To explore the significance of RIP2 kinase pathway activation on inflammation in adult CD and UC patient, we employed an ex vivo culture system to assess the effect of RIP2 kinase inhibition on spontaneous pro-inflammatory cytokine release by intestinal explants.

As previously

described, intestinal mucosal tissue obtained from inflamed areas of the gut spontaneously release significantly elevated levels of inflammatory cytokines compared to non-diseased mucosa, recapitulating the inflammatory phenotype found in vivo.56 In this human biopsy assay, 27 significantly inhibited both TNFα and IL-6 production in a concentration-dependent fashion in both CD (n=12) and UC (n=5) samples, and this inhibition was comparable in magnitude to that seen with the steroid prednisolone (Figure 8). The observed potency of 27 in these explant cultures (IC50 ~ 200 nM) was comparable to the potency observed in MDP-stimulated human whole blood cultures (IC50 = 237 nM). This agreement in apparent IC50 values combined with the exquisite selectivity of 27 strongly supports the hypothesis that inhibition of this inflammatory signal is primarily mediated through selective inhibition of RIP2 kinase activity.


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Figure 8. Spontaneous release of pro-inflammatory cytokines by intestinal mucosal biopsies from Crohn’s Disease (left, n = 12 patients) and Ulcerative Colitis (right, n = 5 patients) patients is inhibited by 27. Explants were cultured overnight in the presence of medium alone (Ctrl) or prednisolone (1 µM) or the indicated concentration of 27. Cytokine concentration was assessed by immunoassay. Bars represent the mean ± SEM.

Given the number of different PRRs that are likely activated by penetration of commensal luminal bacteria following breakdown of the intestinal epithelial barrier in the context of diseases such as UC and CD, the magnitude of inhibition provided by the RIP2 inhibitor in these models is somewhat surprising. Recent data have suggested that there is significant synergy in the inflammatory response following simultaneous stimulation of multiple PRRs.57,58,59,60,61 Together, these data suggest that RIP2 kinase may be a key node in the signaling network connecting multiple PRRs.

Conclusion



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Herein, we have reported the identification and characterization of 27, an exceptional RIP2 tool inhibitor.

The potency, selectivity, and PK/PD properties of 27 make it an ideal tool for

elucidating the pharmacology of RIP2 inhibition in a variety of in vitro and in vivo settings. Pursuing a screening strategy designed to identify potent, efficient, and selective inhibitors of RIP2 kinase by first intent has set the stage for rapid identification of 27. By focusing an SAR strategy around the co-optimization of RIP2 kinase potency and broad kinome selectivity, we were able to develop an exquisitely selective pharmacological tool compound. Although 27 has excellent kinase selectivity, it does inhibit both the hERG channel and Cyp3A4, which precludes it from further progression as a drug candidate. Nonetheless, by employing a chemically-driven biology approach to specifically target inhibition of RIP2 kinase activity, we have unambiguously demonstrated the role for RIP2 kinase activity in modulating intestinal inflammation in both murine and human systems. These new data suggest that RIP2 kinase is a key regulator of the inflammatory response in intestinal models of inflammation, and adds to the growing body of evidence supporting RIP2 kinase as an attractive therapeutic target for the treatment of IBD.

Further, the exquisite selectivity of 27 lends high confidence to the

interpretation of these data and the conclusions herein. The added cellular, ex vivo and in vivo characterization of potency and selectivity, and the robust evidence of consistent pharmacology across species and assay systems, provides a comprehensive data package supporting the use of 27 in future RIP2 pharmacological investigations.

Experimental Section


Page 29 of 57

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All animal studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents. Chemistry: Without specific statement, all solvents and reagents were available from SigmaAldrich, VWR, or Fisher Scientific and used as supplied or purified by standard laboratory methods as required.

1

H NMR spectra were recorded at 600, 500, 400, or 250 MHz at 25 °C.

13

C NMR spectra were recorded at 151 or 126 MHz at 25 °C. Deuterated solvents are noted with

the NMR data. High resolution mass spectrometry were performed by either a time of flight mass spectrometer or Fourier transform mass spectrometer using electrospray (ES) techniques. Melting points were obtained on a digital OptiMelt system by Stanford Research Systems. Purifications were accomplished via flash chromatography on ISCO or Biotage systems with normal phase silica gel column or via reverse phase chromatography on a Gilson or Waters preparatory HPLC using a TFA and acetonitrile mixture in water.

Purity of tested compounds

was assessed to be at least 95% by UV and ELSD using LCMS was carried out using a Waters Acquity fitted with Waters SQD mass spectrometer (column temp 45 oC, UV = 210 – 350 nm, MS = ESI with pos/neg switching) and a Waters BEH C18 (50 mm × 2.1 mm, 1.7 μm) at a flow rate of 1 mL/min using a solvent system of 3% water with 0.1% formic acid to 100% CH3CN with 0.1% formic acid over 2.0 min with a 0.4 min hold. Purity of key compound 27 was further assessed by elemental analysis.



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Page 30 of 57

Ethyl 4-hydroxy-6-(methylthio)quinoline-3-carboxylate (5). 4-(methylthio)aniline (42 mL, 340 mmol) and diethyl [(ethyloxy)methylidene]propanedioate (110 mL, 540 mmol) were combined and heated to 160 °C for 2 hours in a round bottom flask with a reflux condenser attached. The condenser was then removed, and the mixture was heated for an additional hour. The reaction mixture was then cooled to room temperature where it solidified overnight. The solid mass was broken up, suspended in hexanes, and filtered. The cake was rinsed with hexanes three times. Intermediate diethyl ({[4-(methylthio)phenyl]amino}methylidene) propanedioate was obtained asa pale yellow solid (92 g, 83 %).

1

H NMR (400 MHz, DMSO-d6)  10.70 (d,

J=14.1 Hz, 1H), 8.37 (d, J=14.1 Hz, 1H), 7.32-7.39 (m, 2H), 7.25-7.31 (m, 2H), 4.21 (q, J=7.2 Hz, 2H), 4.12 (q, J=7.0 Hz, 2H), 2.48 (s, 3H), 1.20-1.30 (m, 6H); LC/MS (m/z) exact mass C15H19NO4S 309.3807, observed 310.1 (M+H+). To diphenyl ether (62 mL, 390 mmol) at 250 °C

was

added

the

intermediate

diethyl

({[4-(methylthio)phenyl]amino}methylidene)

propanedioate (10 g, 32 mmol) in a steady addition. The reaction was vigorously stirred and heated for the next 3 hours before cooling to room temperature overnight. The resulting solid mass was then transferred to a beaker, broken up, and suspended in 500 mL of hexanes before being filtered. The solid was rinsed with hexanes to afford the title product (8.5 g, 100 %). 1H NMR (400 MHz, DMSO-d6)  12.34 (br. s., 1H), 8.52 (s, 1H), 7.93 (d, J=2.0 Hz, 1H), 7.52-7.68 (m, 2H), 4.21 (q, J=7.2 Hz, 2H), 2.55 (s, 3H), 1.28 (t, J=7.1 Hz, 3H); LC/MS (m/z) exact mass C13H13NO3S 263.0616, observed 264.1 (M+H+). 6-(Methylthio)quinolin-4-ol

(6).

Ethyl

4-hydroxy-6-(methylthio)-3-quinoline-3-

carboxylate (5) (8.5 g, 19 mmol) was dissolved in ethanol (16 mL) before NaOH (3.9 g, 97 mmol) and water (32 mL) were added. The suspension was heated to 130 °C for 2 hours. The reaction was cooled to room temperature and stirred for 72 hours. The residual ethanol was


Page 31 of 57

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removed by rotary evaporation and the aqueous solution was acidified using concentrated HCl. The solid that formed was filtered and washed with water and diethyl ether and then air dried. The solid was then triturated with acetone and filtered to give intermediate 4-hydroxy-6(methylthio)quinoline-3-carboxylic acid as a tan solid (4.4 g, 97 %).

1

H NMR (400 MHz,

DMSO-d6)  15.34 (br. s., 1H), 13.47 (br. s., 1H), 8.87 (s, 1H), 7.99 (d, J=1.8 Hz, 1H), 7.72-7.83 (m, 2H), 2.60 (s, 3H); LC/MS (m/z) exact mass C11H9NO3S 235.2591, observed 236.0 (M+H+). The intermediate carboxylic acid (49.8 g, 212 mmol) was steadily added to diphenyl ether (500 mL, 3.2 mol) at 250 °C. Once the reaction was complete, the homogeneous solution was cooled to room temperature. Hexanes (500 mL) was added to the reaction which was then filtered and the cake was rinsed with hexanes and dried. Title compound was obtained as a brown solid (25.8 g, 60 %). 1H NMR (400 MHz, DMSO-d6)  11.81 (br. s., 1H), 7.83-7.91 (m, 1H), 7.67-7.73 (m, 1H), 7.54-7.60 (m, 1H), 7.46-7.53 (m, 1H), 6.05 (d, J=6.5 Hz, 1H), 2.53 (s, 3H); LC/MS (m/z) exact mass C10H9NOS 191.0405, observed 192.1 (M+H+). 4-chloro-6-(methylsulfonyl)quinoline (7).

6-(Methylthio)-4-quinolinol (6) (1.6 g, 8.6

mmol) and potassium peroxymonosulfate (5.8 g, 9.4 mmol) were suspended in methanol (34 mL) and water (34 mL), and the mixture was stirred at room temperature for 4 hours. Upon filtration the cake was washed with methanol, and the filtrate was concentrated.

The residue

was suspended in methanol, filtered, and concentrated. The residue was triturated with acetone and concentrated to a yellow solid to provide 6-(methylsulfonyl)quinolin-4-ol.

1

H NMR (400

MHz, DMSO-d6)  12.14 (br. s., 1H), 8.58 (d, J=2.0 Hz, 1H), 8.11 (dd, J=2.2, 8.7 Hz, 1H), 8.03 (d, J=7.3 Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 6.17 (d, J=7.6 Hz, 1H), 3.25 (s, 3H); LC/MS (m/z) exact mass C10H9NO3S 223.0303, observed 224.1 (M+H+). The sulfone (3.4 g, 9.1 mmol) was suspended in thionyl chloride (26 mL, 360 mmol) before DMF (0.035 mL, 0.45 mmol) was



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Page 32 of 57

added and the reaction was heated to 100 °C for 1 hour. After cooling to room temperature the mixture was concentrated.

The reaction was incomplete and was resubjected to reaction

conditions. Complete conversion was observed after 1 hour. The reaction mixture was cooled and concentrated to a yellow solid (2.0 g, 91 % over two steps). 1H NMR (400 MHz, DMSO-d6)  9.06 (d, J=4.5 Hz, 1H), 8.75 (d, J=1.3 Hz, 1H), 8.35 (dd, J=1.4, 3.9 Hz, 2H), 7.99 (d, J=4.8 Hz, 1H), 3.40 (s, 3H); LC/MS (m/z) exact mass C10H8ClNO2S 240.9964, observed 242.0, 244.0 (M+H). 4-methyl-3-((6-(methylsulfonyl)quinolin-4-yl)amino)phenol

(3).

4-chloro-6-

(methylsulfonyl)quinoline (7) (0.75 g, 3.10 mmol), 3-amino-4-methylphenol (0.38 g, 3.1 mmol), and ethanol (12.4 mL) were added to a vial which was capped and heated to 150 °C for 10 minutes in a microwave reactor. The reaction was diluted with 1 mL of DMSO, filtered through a syringe filter, and the solution was purified by reverse phase prep HPLC (0.1 % TFA) using a gradient of 10 % to 50 % MeCN in water. The desired fractions were neutralized with saturated aqueous NaHCO3, combined, and partially concentrated to remove any organics. The solid that formed was filtered and dried overnight to provide the title compound (396 mg, 39%). 1H NMR (400 MHz, DMSO-d6)  9.44 (s, 1H), 9.26 (s, 1H), 9.11 (s, 1H), 8.49 (d, J=5.5 Hz, 1H), 8.078.14 (m, 1H), 8.00-8.06 (m, 1H), 7.18 (d, J=8.3 Hz, 1H), 6.64-6.74 (m, 2H), 6.21 (d, J=5.3 Hz, 1H), 3.34 (s, 3H), 2.04 (s, 3H); HRMS (TOFMS ES+, m/z) exact mass C17H16N2O3S 328.3860, found 329.0960; LC/MS observed 329.1 (M+H), purity 99%. Compounds 8 – 10 were made in an analogous fashion to 3 using the appropriate commercially available amine in the last step: N-(1H-benzo[d][1,2,3]triazol-5-yl)-6-(methylsulfonyl)quinolin-4-amine (TFA salt) (8): 1

H NMR (400 MHz, DMSO-d6)  9.58 (br. s., 1H), 9.13 (s, 1H), 8.52 (d, J = 5.0 Hz, 1H), 8.07 -


Page 33 of 57

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8.13 (m, 1H), 8.00 - 8.06 (m, 1H), 7.83 (d, J = 8.8 Hz, 1H), 7.65 (s, 1H), 7.15 (dd, J = 8.6, 1.5 Hz, 1H), 6.88 (d, J = 5.3 Hz, 1H), 3.33 (s, 3H); HRMS (TOFMS ES+) exact mass C16H13N5O2S 339.0790, found 340.0868; LC/MS observed 340.0 (M+H), purity 100%, 64% yield. N-(6-(methylsulfonyl)quinolin-4-yl)benzo[d]thiazol-5-amine (TFA salt) (9):

1

H NMR

(400 MHz, CHLOROFORM-d)  9.11 (s, 1H), 8.88 (s, 1H), 8.67 (d, J = 5.6 Hz, 1H), 8.24 (d, J = 8.8 Hz, 1H), 8.09 - 8.18 (m, 2H), 8.05 (d, J = 8.6 Hz, 1H), 7.48 (dd, J = 8.6, 2.0 Hz, 1H), 7.09 (d, J = 5.6 Hz, 1H), 3.20 (s, 3H); HRMS (TOFMS ES+) exact mass C17H13N3O2S2 355.0449, found 356.0527; LC/MS observed 356.1 (M+H), purity 99%, 67% yield. N-(5-fluoro-1H-indazol-3-yl)-6-(methylsulfonyl)quinolin-4-amine (10):

1

H NMR (400

MHz, DMSO-d6)  12.94 (br. s., 1H), 9.85 (br. s., 1H), 9.27 (s, 1H), 8.64 (m, 1H), 8.06 - 8.17 (m, 2H), 7.56 - 7.64 (m, 2H), 7.44 (d, J = 5.3 Hz, 1H), 7.33 (td, J = 9.1, 2.5 Hz, 1H), 3.36 (s, 3H); HRMS (TOFMS ES+) exact mass C17H13FN4O2S 356.0743, found 357.0822; LC/MS observed 357.1 (M+H), purity 95%, 14% yield. 4-Chloro-6-iodoquinoline (12). To diphenyl ether (1.3 L, 8.0 mol) at 240 °C was added 5{[(4-iodophenyl)amino]methylidene}-2,2-dimethyl-1,3-dioxane-4,6-dione (120 g, 322 mmol) portion-wise. The reaction was heated for 1.5 hours before cooling to room temperature and pouring into 1.5 L of hexanes. The resulting suspension was then filtered. The cake was broken up and rinsed with hexanes (2 x 500 mL). The solid was dried under vacuum to afford 6iodoquinolin-4-ol as a brown solid (80 g, 82 %). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.89 (d, J = 4.0 Hz, 1H), 8.36 (d, J = 2.3 Hz, 1H), 7.89 - 7.98 (m, 2H), 7.37 (d, J = 8.6 Hz, 1H), 6.07 (dd, J = 7.5, 1.1 Hz, 1H); LC/MS (m/z) exact mass C9H6INO 271.0545, observed 272.0 (M+H+). 6-Iodoquinolin-4-ol (100 g, 3.7 mmol) was suspended in POCl3 (340 mL, 3.7 mol) at room temperature. After 1 hour, the reaction mixture was concentrated and the resulting residue was



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Page 34 of 57

placed in an ice water bath and carefully neutralized using saturated aqueous Na2CO3. The resulting brown suspension was filtered and the solid was rinsed with water (2 x 500 mL) and dried under vacuum overnight. 4-Chloro-6-iodoquinoline was obtained as a brown solid (103 g, 92 %). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.88 (d, J = 4.8 Hz, 1H), 8.54 (d, J = 1.8 Hz, 1H), 8.15 (dd, J = 8.8, 2.0 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 4.8 Hz, 1H); LC/MS (m/z) exact mass C9H5ClIN 288.9155, observed 289.9 (M+H+). 4-Methyl-3-(quinolin-4-ylamino)phenol

(15).

To 4-chloroquinoline (11) (50 mg, 0.31

mmol) in a microwave vial was added 3-amino-4-methylphenol (76 mg, 0.62 mmol) and DMF (0.5 mL). The microwave vial was then placed in a microwave reactor at 200 °C for 15 minutes. The crude mixture was then concentrated at atmospheric pressure by allowing a stream of nitrogen to flow into the flask. The compound was purified by reverse phase HPLC to provide 42.6 mg (55%) of the title compound. 1H NMR (600 MHz, DMSO-d6) δ 10.72 (br. s., 1H), 9.85 (br. s., 1H), 8.70 (d, J=8.3 Hz, 1H), 8.49 (d, J=6.8 Hz, 1H), 8.02 (d, J=3.8 Hz, 2H), 7.80 (td, J=4.0, 8.2 Hz, 1H), 7.26 (d, J=8.3 Hz, 1H), 6.84 (dd, J=1.9, 8.3 Hz, 1H), 6.77 (d, J=1.9 Hz, 1H), 6.30 (d, J=7.2 Hz, 1H), 2.06 (s, 3H); HRMS (FTMS ESI+, m/z) exact mass C16H14N2O 250.1106, found 251.1179; LC/MS observed 250.1 (M+H), purity 100%. 3-((6-Iodoquinolin-4-yl)amino)-4-methylphenol (16). To 4-chloro-6-iodoquinoline (12) (1.3 g, 4.5 mmol) in a microwave vial was added 3-amino-4-methylphenol (550 mg, 4.5 mmol) and EtOH (6.9 mL). The microwave vial was then placed in a microwave reactor at 150 °C for 20 minutes. The crude mixture was then filtered and used without further purification (1.6 g, 92% crude yield). LC/MS (m/z) exact mass C16H13IN2O 376.0073, observed 377.0 (M+H). 4-Methyl-3-((6-(pyridin-3-yl)quinolin-4-yl)amino)phenol (17).

To 3-((6-Iodoquinolin-4-

yl)amino)-4-methylphenol (16) (100 mg, 0.27 mmol) in dioxane (1.1 mL) and water (0.2 mL) in


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a microwave vial was added 3-(1,3,2-dioxaborinan-2-yl)pyridine (43 mg, 0.27 mmol), potassium carbonate (180 mg, 1.3 mmol) and 1,1′-bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex (43 mg, 0.05 mmol). The microwave vial was then placed in a microwave reactor at 150 °C for 20 minutes. The crude mixture was then filtered and purified by reverse phase HPLC to afford the title compound (36 mg, 41%). 1H NMR (500 MHz, DMSOd6)  9.37 (s, 1H), 9.15 (m, 1H), 8.83 (d, J=11.2 Hz, 2H), 8.61 (d, J=4.4 Hz, 1H), 8.37 (d, J=4.9 Hz, 1H), 8.29 (d, J=8.3 Hz, 1H), 8.08 (d, J=8.8 Hz, 1H), 7.95 (d, J=8.8 Hz, 1H), 7.56 (dd, J=4.9, 7.8 Hz, 1H), 7.17 (d, J=7.8 Hz, 1H), 6.66-6.72 (m, 2H), 6.17 (d, J=4.9 Hz, 1H), 2.06 (s, 3H); HRMS (FTMS ESI+, m/z) exact mass C21H17N3O 327.1372, found 328.1445; LC/MS observed 328.1 (M+H), purity 95%. 4-((5-Hydroxy-2-methylphenyl)amino)-N-((tetrahydro-2H-pyran-4-yl)methyl)quinoline6-carboxamide (TFA salt) (18).

To a microwave vial containing 3-[(6-iodo-4-

quinolinyl)amino]-4-methylphenol (16) (20 mg, 0.053 mmol), trans-di(µ-acetato)bis[0-(di-otolyl-phosphino)benzyl]dipalladium (II) (1.2 mg, 1.3 µmol), tri-tert-butylphosphinonium tetrafluoroborate (0.77 mg, 2.7 µmol), and molybdenum hexacarbonyl (14 mg, 0.053 mmol) was added NMP (0.5 mL), 1,8-diazabicyclo[5.4.0]undec-7-ene (24 µL, 0.16 mmol), and (tetrahydro2H-pyran-4-yl)methanamine (46 µL, 0.16 mmol). The resulting mixture was sealed in a microwave vial and heated to 170 ºC for 20 minutes in a microwave reactor. After cooling to room temperature, the mixture was filtered through a PTFE syringe filter and purified by reverse phase HPLC to afford the title compound (2.3 mg, 11%). 1H NMR (400 MHz, DMSO-d6)  8.91 (d, J=1.5 Hz, 1H), 8.85 (s, 1H), 8.52 (t, J=5.7 Hz, 1H), 8.40 (d, J=5.3 Hz, 1H), 8.06 (dd, J=1.8, 8.8 Hz, 1H), 7.87 (d, J=8.8 Hz, 1H), 7.16 (d, J=8.8 Hz, 1H), 6.64-6.72 (m, 2H), 6.13 (d, J=5.3 Hz, 1H), 3.87 (dd, J=2.5, 11.4 Hz, 2H), 3.20-3.30 (m, 4H), 2.04 (s, 3H), 1.86 (ddd, J=3.9, 7.4,



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11.2 Hz, 1H), 1.61-1.70 (m, 2H), 1.17-1.31 (m, 2H); HRMS (FTMS ESI+, m/z) exact mass C23H25N3O3 391.1896, found 392.1968; LC/MS observed 392.3 (M+H), purity 96%.

3-((6-Methoxyquinolin-4-yl)amino)-4-methylphenol

(TFA

salt)

(19).

3-((6-

Methoxyquinolin-4-yl)amino)-4-methylphenol was synthesized by a procedure analogous to compound 20. 1H NMR (250 MHz, DMSO-d6) δ 8.29 (d, J=5.7 Hz, 1H), 8.16 (s, 1H), 7.79-7.91 (m, 2H), 7.38-7.48 (m, 1H), 7.20 (d, J=7.5 Hz, 1H), 6.65-6.77 (m, 2H), 6.16 (d, J=5.7 Hz, 1H), 3.94 (s, 3H), 2.05 (s, 3H); HRMS (FTMS ESI+, m/z) exact mass C17H16N2O2 280.1212, found 281.1285; LC/MS observed 280.8 (M+H), purity 100%. 4-Methyl-3-((6-(trifluoromethyl)quinolin-4-yl)amino)phenol (20). A mixture of 3-amino4-methylphenol (8.1 g, 65 mmol) and 4-chloro-6-trifluoromethylquinoline (14) (15 g, 65 mmol) in n-butanol (250 mL) was gently refluxed in an oil bath for 20 hours. The mixture was allowed to cool to room temperature and was then partitioned between saturated sodium bicarbonate solution and ethyl acetate. The aqueous phase was separated and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and concentrated. The residue was chromatographed on silica gel (50% - 66% ethyl acetate:hexanes) to give 3.1 g (15%) of the desired compound.

1

H NMR (400 MHz, DMSO-d6) δ 9.45 (br. s., 1H), 9.10 (s,

1H), 8.93 (s, 1H), 8.47 (d, J=5.0 Hz, 1H), 7.98-8.06 (m, 1H), 7.92 (dd, J=1.8, 8.8 Hz, 1H), 7.18 (d, J=8.0 Hz, 1H), 6.65-6.73 (m, 2H), 6.20 (d, J=5.5 Hz, 1H), 2.04 (s, 3H); HRMS (FTMS ESI+, m/z) exact mass C17H13F3N2O 318.0980, found 319.1052; LC/MS observed 319.0 (M+H), purity 100%. 5-(((4-Iodophenyl)amino)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione (22). A mixture of Meldrum's acid (227 g, 1.58 mol) and triethyl orthoformate (262 mL, 1.58 mol) were heated


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to 90 °C for 1.5 hours before being cooled to 70 ºC where 4-iodoaniline (21) (300 g, 1.37 mol) was added in portions. In order for the reaction to be continually stirred via mechanical stirrer, MeOH was added (500 mL). Once the addition was complete, the reaction was stirred at 70 ºC for another 1 hour before it was diluted with MeOH (1.5 L). The suspension was filtered. The cake was broken up and washed with MeOH (2 x 1 L) and dried under vacuum overnight to afford the title compound as a tan solid (389 g, 75 %). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.22 (d, J = 14.4 Hz, 1H), 8.55 (d, J = 14.7 Hz, 1H), 7.76 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 8.8 Hz, 2H), 1.67 (s, 6H). 4-Chloro-6-(isopropylsulfonyl)quinoline (23).

4-Chloro-6-iodoquinoline (12) (5 g, 17

mmol), Pd(Ph3P)4 (1.0 g, 0.99 mmol), and KOtBu (4.9 g, 43 mmol) were added to a flask. The flask was then purged with nitrogen for 10 min. Toluene (85 mL) was then added followed by 2propanethiol (1.7 mL, 18 mmol). The reaction mixture was then heated to 50 °C overnight. After cooling to room temperature, the mixture was diluted with EtOAc (100 mL) before being washed with aqueous 5:1 Na2S2O3:NaHCO3. The aqueous layer was backextracted with EtOAc (2 x 100 mL) and the combined organics were dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash chromatography (10 to 50 % EtOAc in hexanes). Desired fractions were combined and concentrated to give 2.3 g (45%) of 4-chloro-6(isopropylthio)quinoline. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.78 (d, J = 4.5 Hz, 1H), 8.048.00 (m, 2H), 7.82 (dd, J = 9.0, 2.1 Hz, 1H), 7.77 (d, J = 4.8 Hz, 1H), 3.79-3.73 (m, 1H), 1.33 (d, J = 6.6 Hz, 6H); LC/MS (m/z) exact mass C12H12ClNS 237.7484, observed 238 (M+H+). The intermediate sulfide (7.3 g, 31 mmol) was dissolved in tetrahydrofuran (THF) (100 mL) before water (80 mL) and then potassium peroxymonosulfate (19.8 g, 32.2 mmol) were added. The reaction was stirred at room temperature for 2 hours. The reaction mixture was then partitioned



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between water (100 mL) and EtOAc (100 mL). The organics were collected and the aqueous layer was backextracted with EtOAc (2 x 100 mL). Combined organics were dried over sodium sulfate, filtered, and concentrated. Crude material was purified by flash chromatography (0 to 50 % EtOAc in hexanes). Desired fractions were combined and concentrated to a pale pink solid (7.0 g, 25 mmol, 80%). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.06 (d, J = 4.8 Hz, 1H), 8.67 (d, J = 1.8 Hz, 1H), 8.36 (d, J = 8.8 Hz, 1H), 8.24 (dd, J = 8.6, 2.0 Hz, 1H), 7.99 (d, J = 4.5 Hz, 1H), 3.65 (sep, J = 6.8 Hz, 1H), 1.22 (d, J = 6.8 Hz, 6H); LC/MS (m/z) exact mass C12H12ClNO2S 269.0277, observed 270 (M+H+). N-(5-fluoro-1H-indazol-3-yl)-6-(isopropylsulfonyl)quinolin-4-amine (24). A solution of 4-chloro-6-(isopropylsulfonyl)quinoline (23) (4.0 g, 14.8 mmol) and 5-fluoro-1H-indazol-3amine (2.2 g, 14.8 mmol) in ethanol was stirred at room temperature for 3 days. The reaction was concentrated to dryness and purified via reverse phase HPLC (Waters Sunfire 30x150mm Acetonitrile:Water

TFA

26-60%)

to

obtain

N-(5-fluoro-1H-indazol-3-yl)-6-

(isopropylsulfonyl)quinolin-4-amine (2.7 g, 6.9 mmol, 46.5 % yield).

1

H NMR (400 MHz,

DMSO-d6) δ ppm 13.13 (d, J=1.8 Hz, 1 H), 10.09 - 10.77 (m, 1 H), 9.30 (s, 1 H), 8.66 (d, J=5.8 Hz, 1 H), 8.07 - 8.23 (m, 2 H), 7.62 (ddd, J=12.6, 9.2, 3.3 Hz, 2 H), 7.23 - 7.40 (m, 2 H), 3.59 (quin, J=6.8 Hz, 1 H), 1.25 (d, J=6.8 Hz, 6 H);

13

C NMR (126 MHz, DMSO-d6) δ 157.3, 154.6,

151.4, 149.3, 142.5, 138.9, 133.5, 131.4, 127.9, 126.3, 119.4, 117.2, 116.7, 113.1, 105.2, 104.9, 55.2, 16.1. HRMS (TOFMS ES+) exact mass C19H18FN4O2S 384.1056, found 385.1138; LC/MS observed 385.1 (M+H), purity 100%. Melting point 269 - 270 ºC. 6-(tert-Butylthio)-4-chloroquinoline (26). To a flask was added 6-bromo-4-chloroquinoline (25) (40 g, 170 mmol) and Pd(Ph3P)4 (4.8 g, 4.1 mmol). The flask was then evacuated and backfilled with nitrogen three times. Acetonitrile (180 mL) was then added followed by TEA


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(34.5 mL, 247 mmol) and 2-methyl-2-propanethiol (18.6 mL, 165 mmol). The reaction was then heated to 90 °C overnight followed by cooling to room temperature and partitioning between a 2M 5:1 Na2S2O3:NaHCO3 aqueous solution and EtOAc (500 mL each). The organics were collected and the aqueous layer was back extracted with EtOAc (2 x 500 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The crude product was dissolved in DCM and passed through a pad of silica gel eluting with EtOAc. After 2 L of solvent was collected,the organics were concentrated to an oil that solidified upon standing overnight to afford 6-(tert-butylthio)-4-chloroquinoline as a yellow solid (45 g, 91%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.89 (d, J=4.8 Hz, 1 H), 8.29 (d, J=1.8, 1 H), 8.10 (d, J=8.6, 1 H), 7.92 (dd, J=8.8, 2.0 Hz, 1 H), 7.83 (d, J=4.8 Hz, 1 H), 1.32 (s, 9 H); LC/MS (m/z) exact mass C13H14ClNS 251.0535, observed 252 (M+H+). The intermediate sulfide (43.8 g, 174 mmol) was dissolved in tetrahydrofuran (THF) (400 mL) before water (400 mL) and then potassium peroxymonosulfate (107 g, 174 mmol) were added.

The reaction was stirred at room

temperature for 2 hours. The mixture was diluted with water (200 mL) and extracted with EtOAc (3 x 500 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. They were then dissolved in DCM and purified by flash chromatography (30 to 70 % EtOAc in hexanes). Fractions containing product were combined and concentrated to provide the title compound as a pale yellow solid (20.6 g, 41%). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.08 (d, J=4.5 Hz, 1 H), 8.63 (d, J=2.0, 1 H), 8.36 (d, J=8.8, 1 H), 8.20 (dd, J=8.8, 2.0 Hz, 1 H), 8.00 (d, J=4.8 Hz, 1 H), 1.31 (s, 9 H); LC/MS (m/z) exact mass C13H14ClNO2S 283.0434, observed 284 (M+H+). 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (27). To a flask was added 6-(tert-butylsulfonyl)-4-chloroquinoline (26) (23 g, 81 mmol), 5-fluoro-1H-indazol-3-



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amine (12 g, 81 mmol), and ethanol (150 mL). Three drops of concentrated HCl were then added and the reaction was stirred at room temperature for 3 days.

The reaction was

concentrated and the residue was taken up in DCM. The resulting suspension was filtered. The solid cake was then partitioned between saturated aqueous NaHCO3 and DCM (750 mL each). The organic layer was separated and the aqueous layer was back extracted with DCM (2 x 500 mL). The combined organics were dried over sodium sulfate, filtered, and concentrated. The material was then suspended in MeCN (200 mg/mL), sonicated, and filtered. The cake was washed with MeCN. This solid was then boiled in MeOH (100 mg/mL) and subsequently allowed to cool to room temperature where it was filtered and rinsed with MeOH (50 mL). This process was repeated and the solid was dried in a vacuum oven for two days at 50 ºC to provide the desired product as a yellow solid (31 g, 94%). 1H NMR (400MHz, DMSO-d6)  ppm 13.00 (s, 1H), 9.96 (s, 1H), 9.19 (d, J=1.5 Hz, 1H), 8.64 (d, J=5.3 Hz, 1H), 8.07-8.11 (m, 1H), 7.988.04 (m, 1H), 7.60 (dd, J=9.1, 4.0 Hz, 1H), 7.54 (dd, J=9.2, 2.4 Hz, 1H), 7.33 (td, J=9.0, 2.4 Hz, 1H), 7.28 (d, J=5.6 Hz, 1H), 1.34 (s, 9H);

13

C NMR (151 MHz, DMSO-d6) δ 155.7, 153.8,

150.5, 148.7, 141.4, 138.1, 130.6, 130.1, 128.5, 127.1, 118.3, 116.4, 116.0, 112.3, 104.2, 103.9, 59.3, 23.2.

HRMS (TOFMS ES+) exact mass C20H19FN4O2S

398.1231, found 399.1292;

LC/MS observed 399.2 (M+H), purity 100%. Anal Calcd for C20H19FN4O2S: C 60.29; H 4.81; N 14.06. Found: C 59.94; H 4.48; N 13.82. Melting point 241 - 242 ºC.

Biological Assays. RIP2 FP binding Assay: A fluorescent polarization based binding assay was developed to quantitate interaction of

novel test compounds at the ATP binding pocket of RIP2K, by

competition with a fluorescently labeled ATP competitive ligand. Full length FLAG His tagged


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RIP2K was purified from a Baculovirus expression system and was used at a final assay concentration of twice the KD apparent. A fluorescent labeled ligand that is reversible and competitive with the inhibitors was used at a final assay concentration of 5 nM. Both the enzyme and ligand were prepared in solutions in 50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 1 mM CHAPS. Test compounds were prepared in 100% DMSO and 100 nL was dispensed to individual wells of a multi-well plate. Next, 5 μL RIP2K was added to the test compounds at twice the final assay concentration, and incubated at room temperature for 10 minutes. Following the incubation, 5 μL of the fluorescent labeled ligand solution, was added to each reaction, at twice the final assay concentration, and incubated at room temperature for at least 10 minutes. Finally, samples were read on an instrument capable of measuring fluorescent polarization. Test compound inhibition was expressed as percent (%) inhibition of internal assay controls. For concentration response experiments, normalized data were fit using the following four parameter logistic equation: y = A + ((B-C))/(1+(10x)/(10C)D), where: y is the % activity (% inhibition) at a specified compound concentration; A is the minimum % activity; B is the maximum % activity; C = log10(IC50); D= Hill slope; x = log10 (compound concentration [M]); and pIC50 = (-C).62 Preparations of FLAG His tagged RIP2 and the fluorescent ligand used in the assay have been published.63

MDP-Stimulated Human Whole Blood Assay: Heparinized blood was dispensed into 96 well polystyrene plates (160 microliters/well). Test compounds (0.001 – 10 µM, final concentration) were added to duplicate wells (20 µL/well). Plates were placed at 37 °C for 30 minutes on a plate shaker (~500 rotations per minute). Plates were removed and 20 µL of MDP (final concentration = 0.1 µg/mL) or sterile PBS was added to



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achieve a final volume of 200 microliters/well. Plates were placed at 37 °C for six hours on a plate shaker (~500 rotations per minute). Plates were removed and 100 microliters PBS was added to each well. Plates were shaken gently (~100 rotations per minute) at room temperature for 10 minutes and then centrifuged (3000 rpm for 10 minutes at room temperature). The supernatant layer was removed (~100 microliters/well), transferred to a separate plate and frozen. Samples were assayed for TNFα by immunoassay using the Meso-Scale Discovery technology platform. Basal TNFα levels were generally < 10 pg/mL. Maximal MDP-stimulated TNFα levels range from 50 – 500 pg/mL, depending on the blood donor. The percentage inhibition was calculated as % Inhibiton = [(Test sample – Basal)/Maximal response – Basal) x 100]. The IC50 value was defined as the inhibitor concentration eliciting a 50% reduction in the magnitude of the maximal MDP response. The IC50 value for each test compound was determined by plotting the inhibitor concentration vs. the % inhibition and applying a sigmoidal 4-parameter curve fit. Data reported in the text are the mean values from 6 independent experiments. MDP-stimulated Human Monocyte Assay: Monocytes were isolated from heparinized blood using the two-step Ficoll-Paque and Percoll density gradient centrifugation method. Cells were washed using PBS, resuspended in RPMI-1640 supplemented with 10% FCS, 2 mM Lglutamine, 2.5 mM HEPES + Penicillin/Streptomycin), then seeded in 96-well plates at a final density of 50,000 cells/well. Following overnight incubation (5% CO2, 37 °C), media was replaced. Cells were treated with test compound (0.001 – 10 µM) for 30 minutes followed by addition of MDP (final concentration = 1 µg/mL). Six hours later, media was removed, transferred to a separate plate and stored at -80 °C. Levels of TNFα in the media were measured by immunoassay using the Meso-Scale Discovery technology platform. Percentage inhibition and IC50 values were calculated as described above.


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Cellular Selectivity: To assess cellular selectivity monocytes were pretreated with inhibitor for 30 min, then stimulated for 6 h with ligands which selectively agonize NLRs: NOD1 (300 µg/mL, ieDAP), NOD2 (1 µg/mL, MDP); Toll-like receptors: TLR2 (10 ng/mL Pam2Csk4), TLR4 (10 ng/mL, ultrapure LPS), TLR7 (10 µg/mL, gardiquimod), or cytokine receptors: IL-1R (10 ng/mL, IL-1β), TNFR (100 ng/mL, TNFα). Release of pro-inflammatory cytokines, either TNFα (NOD2, TLR2, TLR4, IL1R) or IL-8 (NOD1, TLR7, TNFR) was measured by immunoassay. % inhibition and/or IC50 values were calculated as described above.

Rodent Pharmacokinetics: The preliminary oral pharmacokinetic studies were carried out in cassette fashion in female Sprague-Dawley rats (n=3; 2 mg/kg). The follow up pharmacokinetic studies in mouse (male C57BL/6) and rat (female Sprague-Dawley) were conducted using non-crossover design on two study days where animals (n=3) were dosed at 1 and 2 mg/kg, iv and po, respectively. Blood samples (25 µL) were collected at various time points and diluted with equal volume of water and stored frozen at approximately -80 °C until analysis. Blood sample analysis was performed using liquid chromatography/tandem mass spectrometry (LC/MS/MS); blood concentrations were determined by standard calibration curve analysis. Pharmacokinetic parameters were calculated using non-compartmental methods with Phoenix WinNonlin and bioavailability was calculated in a non-cross over fashion using the average intravenous AUC and dose.

MDP-induced rat challenge model:

Female Crl:CD(SD) rats (n-8/treatment group) were

dosed orally with vehicle or 27 15 minutes prior to MDP challenge (150 µg/rat, IV). At 2 hours post MDP challenge rats were sacrificed, and terminal serum was prepared from blood collected



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via cardiac stick. Serum cytokine levels (IL-6, IL-8 or KC, IL-1β, and TNFα) were quantified by the MSD platform. MDP-induced mouse peritonitis model:

Female C57Bl/6 mice (for cytokine analyses) or

male Balb/c mice (for peritoneal neutrophil analyses) (n=10/treatment group) were dosed orally 15 minutes prior to MDP challenge with vehicle or 27 (0.1, 1, or 10 mg/kg). For peritoneal neutrophil analysis, mice were sacrificed at 4 hr post-MDP challenge (30 µg, ip) and peritoneal fluid was collected by lavage. Peritoneal neutrophils were quantified by FACS analysis. NOD1 (FK156) and TLR4 (LPS) challenge models in mouse: FK156: Female C57Bl/6 mice (n=10/treatment group) were dosed orally with either vehicle or 27 at 0.1, 1, 10, 100 mg/kg 15 minutes prior to FK156 injection (10 µg/mouse, IP). At 2.5 hours post FK156 administration the mice were sacrificed. Serum was prepared from terminal blood (via cardiac stick) and peritoneal lavage was harvested with 4 mL of cold PBS. IL-8 levels were quantified by the MSD platform in the serum and peritoneal lavage. Peritoneal neutrophils were quantified by FACS analysis. LPS: Female C57Bl/6 mice (n=8/treatment group) were dosed orally with either vehicle or 27at 10 mg/kg 15 minutes prior to LPS injection (1µg/200 µL/mouse, IP). At 2 hours post LPS administration the mice were sacrificed. IL-8 and IL-6 levels were quantified by the MSD platform in serum prepared from terminal blood collected via cardiac stick. All in vivo data are shown as mean ± SEM and were analyzed using one-way ANOVA, followed by Bonferroni post hoc analysis. Dose–response curves and IC50 values for the assays were generated using GraphPad Prism 6 software using non-linear regression log (inhibitor) vs. response (three parameters) with a standard hill slope and the top constrained at 100. Organ Culture of Human Intestine Mucosal Explants


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Tissue was obtained during routine endoscopy of patients with IBD. All patients took part in this study after providing informed written consent. The study was approved by the local ethics committee. Explant cultures were performed as previously described. 56 Biopsies were cultured in 24-well plates in 300 mL serum-free HL1 medium (Lonza, Cambridge, England) containing glutamine, Pen/Strep, and 50 mg/mL gentamicin. The RIP2 kinase inhibitor was added to the culture medium. Mucosal samples were emersed in liquid, and culture was performed for 24 hours at 37°C and 5% CO2. Supernatants and biopsies were subsequently snap-frozen and stored at -70 ˚C. Cytokine concentrations of IL-1β and TNFα were determined by Sandwich ELISA (R&D Systems) according to the manufacturer’s instructions.

Crystallography. Complex Crystallization of compounds 3 and 27 with RIP2: Flag-tagged RIP2 kinase domain (1-310) was expressed in the baculovirus expression vector system with Spodoptera frugiperda cells and purified in two steps by anti-flag and size exclusion chromatography.4 The protein (10 mgs/mL in 20 mM Tris pH 8, 0.15 M NaCl, 1 mM DTT and 5% glycerol) was incubated with 2 mM compound 3 prior to co-crystallization. Crystals grew from 1.5 M Ammonium Sulfate and 25% glycerol. Apo crystals were grown with seeding in 100 mM Mes pH 7, 12% PEG 400, 250 mM CaCl2. An apo crystal was soaked with 7.5 mM of 27 for 2 hours in 100 mM Mes pH 7, 24% PEG 400 and 100 mM CaCl2. Crystals in both cases were frozen without additional cryoprotectant. Data was collected at a synchrotron source. The structures were solved by molecular replacement using PDB:5AR4 as starting model. See supplemental information for data collection and refinement statistics and exemplars of the density.



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Associated Content: Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Data collection and refinement statistics and exemplars of the density for compounds 3 and 27; Kinome selectivity data for compounds 3, 24 and 27; Small molecule and determination and refinement methods and information, RIP3 cell data for 27; Adult ID biopsy showing RIP2 activation

Accession Codes: The PDB codes are as follows: 5j79 for RIP2 / 3 co-crystal structure and 5j7b for RIP2 / 27 cocrystal structure. Authors will release the atomic coordinates and experimental data upon article publication. Author Information Corresponding Author: *

L.N.C.: phone 610-917-7808; email, [email protected]

Author Contributions: ‡

P.A.H. and B.J.V. contributed equally to this work.

Current Addresses: †

Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA

§

Host Defense Discovery Performance Unit, Infectious Disease Therapy Area Unit,

GlaxoSmithKline, Collegeville, PA, USA,


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All authors have given approval for the final version of this manuscript.

Financial Disclosure: T.M. and A.V. have been funded by GSK in part. All other non-GSK authors declare no competing financial interest.

All GSK authors are/were employees and stockholders of

GlaxoSmithKline when this work was completed.

Acknowledgements The authors would like to express appreciation to the members of Platform Technology and Science at GlaxoSmithKline: Yong Jiang for protein production, Emma Jones for protein purification, Julie Cox for screening support, Minghui Wang for NMR support, and Ashley Leister and Amy Gibble for purification support. Also, many thanks are due to Rob Copely at GSK and Amy Sarjeant at Northwestern University for X-ray analysis.

Abbreviations NOD, nucleotide-binding oligomerization domain protein; PRR, pattern recognition receptors; RIP2 and RIP2K, receptor interacting protein 2 kinase; MDP, muramyl di-peptide; IBD, inflammatory bowel disease; FP, fluorescence polarization; clogP, calculated partition coefficient; MW, molecular weight; BEI, binding efficiency index; LipE, lipophilic ligand efficiency; UC, ulcerative colitis; CD, Crohn’s Disease; NF-B, nuclear factor kappa-lightchain-enhancer of activated B cells; CARD, caspase activation and recruitment domain; BMDM, bone marrow-derived macrophages.



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The Identification and Pharmacological Characterization of 6-(tert

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