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Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is an important kinase of the innate immune system. Herein, we describe the optimization of a series of RIPK2 PROTACs which recruit members of the inhibitor of apoptosis (IAP) family of E3 ligases. Our PROTAC optimization strategy focused on reducing the lipophilicity of the early lead which resulted in the identification of analogues with improved solubility and increased human and rat microsomal stability. We identified a range of IAP binders that were successfully incorporated into potent RIPK2 PROTACs with attractive pharmacokinetic profiles. Compound 20 possessed the best overall profile with good solubility, potent degradation of RIPK2, and associated inhibition of TNFα release. A proof-of-concept study utilizing a slow release matrix demonstrated the feasibility of a long-acting parenteral formulation with >1 month duration. This represents an attractive alternative dosing paradigm to oral delivery, especially for chronic diseases where compliance can be challenging.

Receptor Interacting Protein 2 (RIP2) kinase inhibitors have been reported for therapeutic opportunities in inflammatory bowel diseases such as Ulcerative Colitis and Crohn's disease. During lead optimization, team identified 4-aminoquinoline series and several compounds from this series were investigated in rat and dog pharmacokinetic studies. While compounds such as GSKA and GSKB demonstrated acceptable pharmacokinetics in rat and dog, further progression of these compounds was halted due to adverse findings in advanced safety studies. Structurally similar analogues incorporating polarity at C-7 position of 4-aminoquinoline resulted in identification of GSKC - GSKF. Interestingly, following oral administration to rat at similar low dose, GSKC - GSKF demonstrated significantly low systemic drug exposure compared to GSKA and GSKB (3-17-fold difference). However, in dog, dose normalized oral systemic exposure for GSKC - GSKF was comparable to GSKA and GSKB (within 2-fold). A series of studies were conducted to understand the disconnect which highlighted that an intrinsic reduction in permeability and high P-glycoprotein (P-gp) efflux ratio for C-7 substituted analogues were driving pharmacokinetic disconnect between rat and dog. Oral absorption was minimally impacted in dog by P-gp mediated efflux compared to rat because the leakier gastrointestinal tract in dog likely overcomes this effect.

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines., Mares et al. develop Proteolysis-Targeting Chimeras (PROTACs) that degrade its target RIPK2 in vivo at low doses for a prolonged period of time. This study suggests that PROTAC has a therapeutic potential that is superior to traditional RIPK2 small-molecule inhibitors.

[Image: see text] Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.

[Image: see text] RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC(50) = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC(50) = 10 nM) with reduced hERG activity (14 μM).

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

Brevetoxin A is a decacyclic ladder toxin that possesses five-,six-, seven-, eight-, and nine-membered oxacycles, as well as 22 tetrahedral stereocenters. Herein, we describe a unified approach to the B, E, G, and J rings predicated upon a ring-closing metathesis strategy from the corresponding dienes. The enolate technologies developed in our laboratory allowed access to the precursor acyclic dienes for the B, E, and G medium ring ethers. The strategies developed for the syntheses of these four monocycles ultimately provided multigram quantities of each of the rings, supporting our efforts toward the convergent completion of brevetoxin A.

Molecular clusters of BBr3 were subjected to electron ionization and mass analysis in a reflectron time-of-flight mass spectrometer. Five series of cluster ions were observed, with formulas corresponding to each of the possible fragment ions of BBr3 being solvated by neutral BBr3 molecules. Geometry optimizations on the observed cluster ions using density functional theory (B3LYP/6-31G*) predict that fragment ions smaller than BBr3+ undergo reactions with neutral BBr3 molecules to form covalently bound adduct species that function as core ions within the clusters. Once all boron atoms are saturated, the reactions cease, and larger cluster ions consist of BBr3 molecules loosely bound to the core ions. Divalent bromine atoms are present in at least three of the cluster ions, and most of the intermolecular contact within the clusters is between Br atoms. Enthalpies of formation, addition reactions, and BBr3 elimination from the cluster ions were derived from B3LYP and MP2 calculations at the B3LYP/6-31G* geometries using both the 6-31G* and the 6-311++G(2df,2p) basis sets. The results are compared to limiting expectations based on known bulk thermochemistry.

A convenient synthesis of an isotopically-labelled version (13C,2H3) of the long-acting β2-adrenergic agonist, salmeterol, is described, starting from methyl salicylate, 1,6-dibromohexane and 4-phenyl-1-butanol. This compound was designed as an analytical internal standard for mass spectrometric determination of salmeterol in pharmacokinetic studies. Copyright © 2000 John Wiley & Sons, Ltd.

(-)-Isopulegol derivatives undergo a ring contraction under silylene-mediated conditions to provide cyclopentane products. Silylene transfer to other homoallylic ethers did not provide the ring contraction products. Allylic silane products were elaborated to determine the stereochemical course of the ring contraction reaction. A mechanism for the transformation is proposed.

Boron trifluoride clusters are formed in a supersonic expansion and ionized by electron impact. The resulting ion distribution is analyzed by time-of-flight mass spectrometry. The only consistently observed, extended series of peaks in EI of BF3 clusters is BnF3n-1+. Ab initio modeling of these ions informs our speculation regarding their internal structure and ion−molecule chemistry involved in their formation. Formation of B2F5δ+ is computationally predicted to occur in all of the cluster ions we observe (n = 2−8) except B3F8+. BF4δ- units are evident in the predicted structure of B8F23+.

NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.

A series of biarylsulfonamides was identified as hCCR2 receptor antagonist but suffered from high plasma protein binding resulting in a >100 fold shift in activity in a functional GTPγS assay run in tandem in the presence and absence of human serum albumin. Introduction of an aryl amide with ethylenediamine linker led to compounds with reduced shifts and improved activity in whole blood.

Brevetoxin A is a decacyclic ladder toxin that possesses 5-, 6-, 7-, 8-, and 9-membered oxacycles, as well as 22 tetrahedral stereocenters. Herein, we describe a unified approach to the B, E, G, and J rings based upon a ring-closing metathesis strategy from the corresponding dienes. The enolate technologies developed in our laboratory allowed access to the precursor acyclic dienes for the B, E, and G medium-ring ethers. The strategies developed for the syntheses of these four monocycles ultimately provided multigram quantities of each of the rings, supporting our efforts toward the completion of a convergent synthesis of brevetoxin A.

Allylic ethers undergo insertions of silylenes into C–O bonds to form allylic silanes. Silylene insertion into C–O acetal bonds was also observed. Formation of silylene ylide intermediates led to [1,2]-Stevens rearrangement products as well as [2,3]-sigmatropic products depending upon the steric environment of the starting allylic ether.

Allylic ethers undergo insertions of silylenes into C–O bonds to form allylic silanes. Silylene insertion into C–O acetal bonds was also observed. Formation of silylene ylide intermediates led to [1,2]-Stevens rearrangement products as well as [2,3]-sigmatropic products depending upon the steric environment of the starting allylic ether.

A total synthesis of brevetoxin A is reported. Two tetracyclic coupling partners, prepared from previously reported advanced fragments, were effectively united via a Horner—Wittig olefination. The resulting octacycle was progressed to substrates that were explored for reductive etherification, the success of which led to a penultimate tetraol intermediate. The tetraol was converted to the natural product through an expeditious selective oxidative process, followed by methylenation.

NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.

Blau Syndrome (Blau) is a granulomatous auto-inflammatory disease caused by mutations in NOD2 that have been proposed to result in phosphorylation of RIP2 kinase and the production of pro-inflammatory cytokines. Such monogenic diseases can bring to light pathways that are also likely to be involved in more genetically complex diseases. For example, increased RIP2 phosphorylation has been observed in inflammatory bowel disease (IBD), although the role of RIP2 in IBD has not been determined. We are developing a first-in-class, highly potent and selective inhibitor of RIP2 kinase, which may provide therapeutic benefit in both Blau and IBD.