Discovery of Clinical Candidate 1-{[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin-1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug Design.

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.

Identification and optimization of indolo[2,3-c]quinoline inhibitors of IRAK4.

IRAK4 is responsible for initiating signaling from Toll-like receptors (TLRs) and members of the IL-1/18 receptor family. Kinase-inactive knock-ins and targeted deletions of IRAK4 in mice cause reductions in TLR induced pro-inflammatory cytokines and these mice are resistant to various models of arthritis. Herein we report the identification and optimization of a series of potent IRAK4 inhibitors. Representative examples from this series showed excellent selectivity over a panel of kinases, including the kinases known to play a role in TLR-mediated signaling. The compounds exhibited low nM potency in LPS- and R848-induced cytokine assays indicating that they are blocking the TLR signaling pathway. A key compound (26) from this series was profiled in more detail and found to have an excellent pharmaceutical profile as measured by predictive assays such as microsomal stability, TPSA, solubility, and clogP. However, this compound was found to afford poor exposure in mouse upon IP or IV administration. We found that removal of the ionizable solubilizing group (32) led to increased exposure, presumably due to increased permeability. Compounds 26 and 32, when dosed to plasma levels corresponding to ex vivo whole blood potency, were shown to inhibit LPS-induced TNFα in an in vivo murine model. To our knowledge, this is the first published in vivo demonstration that inhibition of the IRAK4 pathway by a small molecule can recapitulate the phenotype of IRAK4 knockout mice.

The cPLA2α inhibitor efipladib decreases nociceptive responses without affecting PGE2 levels in the cerebral spinal fluid.

The contribution of central PGE(2) levels to the nociceptive response in rats was assessed and the effects of the selective cPLA(2)α inhibitor efipladib, and pain therapies of different classes on these responses was determined. An inflammatory pain model was optimized in rats so that PGE(2) levels in the cerebrospinal fluid (CSF) could be directly correlated to the nociceptive response. Since efipladib appears to have limited permeation of the blood-brain barrier, we used this compound to determine the extent of pain reversal resulting primarily from peripheral, but not central, inhibition of the arachidonic acid (AA) pathway. The nociceptive response was significantly inhibited by orally administered efipladib, yet spinal fluid levels of PGE(2) and temperature measurements were unaffected compared to vehicle-treated animals. Conversely, intrathecal (IT) administration of efipladib reduced PGE(2) levels in the CSF by 45-60%, yet there was no effect on the nociceptive response. With COX-2 selective inhibitors and ibuprofen, a return of the nociceptive response developed over time, despite complete inhibition of PGE(2) in the spinal fluid. The opposite was true with low doses of indomethacin: inhibition of the nociceptive response was observed despite the lack of effect on central PGE(2) levels. Our results demonstrate that levels of PGE(2) in the spinal fluid do not directly correlate with the nociceptive response and that blocking cPLA(2)α in the periphery significantly decreases inflammatory pain.

Benzhydrylquinazolinediones: novel cytosolic phospholipase A2alpha inhibitors with improved physicochemical properties.

The synthesis and optimization of a class of trisubstituted quinazoline-2,4(1H,3H)-dione cPLA(2)alpha inhibitors are described. Utilizing pharmacophores that were found to be important in our indole series, we discovered inhibitors with reduced lipophilicity and improved aqueous solubility. These compounds are active in whole blood assays, and cell-based assay results indicate that prevention of arachidonic acid release arises from selective cPLA(2)alpha inhibition.

Reactions of functionalized sulfonamides: application to lowering the lipophilicity of cytosolic phospholipase A2alpha inhibitors.

The cPLA(2)alpha inhibitors we reported earlier were potent in both isolated enzyme and rat whole blood assays but have high plogD(7.4). To address these issues, reactions of electrophilic sulfonamides 9-12 were employed to incorporate various heterocyclic or heteroatom-based reagents into cPLA(2)alpha inhibitors. For example, reactions of 9 with sulfur nucleophiles such as thiophenol allowed rapid assembly of thioether analogues that were converted into the corresponding sulfoxides to afford less lipophilic derivatives. Reactions of 10 and 11 with various nitrogen nucleophiles, including aromatic heterocycles and aliphatic amines, provided an efficient way to introduce polarity into cPLA(2)alpha inhibitors. Finally, we report the first application of (2-formylphenyl)methanesulfonyl chloride, 13. Reductive amination of 2-formylphenylmethane sulfonamides allowed the introduction of various nitrogen nucleophiles. Several inhibitors obtained herein have plogD(7.4) values 3-4 units lower than previously synthesized compounds and yet maintain in vitro potency.

Blockade of cytosolic phospholipase A2 alpha prevents experimental autoimmune encephalomyelitis and diminishes development of Th1 and Th17 responses.

Cytosolic phospholipase A2 alpha (cPLA2 alpha) is the rate-limiting enzyme for release of arachidonic acid, which is converted primarily to prostaglandins via the cyclooxygenase (COX) 1/2 pathways, and leukotrienes via the 5-lipoxygenase (LO) pathway. We utilized inhibitors of cPLA2 alpha, COX-1/2 and 5-LO to determine the potential roles of these enzymes in development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Blocking cPLA2 alpha prevented EAE development and greatly reduced antigen-induced production of Th1-type cytokines and IL-17. Blocking COX-1/2 delayed onset and reduced severity of EAE, and reduced production of Th1-type cytokines, but not IL-17. Blocking 5-LO delayed onset and reduced cumulative severity of EAE, but did not reduce production of Th1-type cytokines or IL-17. Finally, blockade of cPLA2 alpha from the onset of clinical EAE reduced duration of EAE relapses. Therefore, cPLA2 alpha represents a potential therapeutic target for treatment of MS.

Thermodynamic characterization of cytosolic phospholipase A2 alpha inhibitors.

Cytosolic phospholipase A(2) alpha (cPLA(2)alpha, type IVA phospholipase) acts at the membrane surface to release free arachidonic acid, which is metabolized into inflammatory mediators, including leukotrienes and prostaglandins. Thus, specific cPLA(2)alpha inhibitors are predicted to have antiinflammatory properties. However, a key criterion in the identification and development of such inhibitors is to distinguish between compounds that bind stoichiometrically to cPLA(2)alpha and nonspecific membrane perturbants. In the current study, we developed a method employing isothermal titration calorimetry (ITC) to characterize the binding of several distinct classes of cPLA(2)alpha inhibitors. Thermodynamic parameters and the binding constants were obtained following titration of the inhibitor to the protein at 30 degrees C and pH 7.4. The compounds tested bound cPLA(2)alpha with a 1:1 stoichiometry, and the dissociation constant K(d) of the inhibitors calculated from the ITC experiments correlated well with the IC(50) values obtained from enzymatic assays. Interestingly, binding was observed only in the presence of a micellar surface, even for soluble compounds. The site of binding of these inhibitors within cPLA(2)alpha was analyzed by testing for binding in the presence of methyl arachidonyl fluorophosphonate (MAFP), an irreversible active site inhibitor of cPLA(2)alpha. Lack of binding of inhibitors in the presence of MAFP suggested that the compounds tested bound specifically at or near the active site of the protein. Furthermore, the effect of various detergents on the binding of certain inhibitors to cPLA(2)alpha was also tested. The results are discussed with reference to thermodynamic parameters such as changes in enthalpy (DeltaH), entropy (DeltaS), and free energy (DeltaG). The data obtained from these studies provide not only structure-activity relationships for compounds but also important information regarding mechanism of binding. This is the first example of ITC used for studying inhibitors of enzymes with interfacial kinetics.

Indole cytosolic phospholipase A2 alpha inhibitors: discovery and in vitro and in vivo characterization of 4-{3-[5-chloro-2-(2-{[(3,4-dichlorobenzyl)sulfonyl]amino}ethyl)-1-(diphenylmethyl)-1H-indol-3-yl]propyl}benzoic acid, efipladib.

The optimization of a class of indole cPLA 2 alpha inhibitors is described herein. The importance of the substituent at C3 and the substitution pattern of the phenylmethane sulfonamide region are highlighted. Optimization of these regions led to the discovery of 111 (efipladib) and 121 (WAY-196025), which are shown to be potent, selective inhibitors of cPLA 2 alpha in a variety of isolated enzyme assays, cell based assays, and rat and human whole blood assays. The binding of these compounds has been further examined using isothermal titration calorimetry. Finally, these compounds have shown efficacy when dosed orally in multiple acute and chronic prostaglandin and leukotriene dependent in vivo models.

Benzenesulfonamide indole inhibitors of cytosolic phospholipase A2alpha: optimization of in vitro potency and rat pharmacokinetics for oral efficacy.

The synthesis and structure-activity relationship of a series of benzenesulfonamide indole inhibitors of cPLA(2)alpha are described. Substitution of the benzenesulfonamide led to analogues with 50-fold improvement in potency versus the unsubstituted benzenesulfonamide lead compound. Rat pharmacokinetics in a minimal formulation was used to prioritize compounds, leading to the discovery of a potent inhibitor of cPLA(2)alpha with oral efficacy in models of rat carrageenan paw edema and Ascaris suum airway challenge in naturally sensitized sheep.

Discovery of Ecopladib, an indole inhibitor of cytosolic phospholipase A2alpha.

The synthesis and structure-activity relationship of a series of indole inhibitors of cytosolic phospholipase A2alpha (cPLA2alpha, type IVA phospholipase) are described. Inhibitors of cPLA2alpha are predicted to be efficacious in treating asthma as well as the signs and symptoms of osteoarthritis, rheumatoid arthritis, and pain. The introduction of a benzyl sulfonamide substituent at C2 was found to impart improved potency of these inhibitors, and the SAR of these sulfonamide analogues is disclosed. Compound 123 (Ecopladib) is a sub-micromolar inhibitor of cPLA2alpha in the GLU micelle and rat whole blood assays. Compound 123 displayed oral efficacy in the rat carrageenan air pouch and rat carrageenan-induced paw edema models.

1,2,4-Oxadiazolidin-3,5-diones and 1,3,5-triazin-2,4,6-triones as cytosolic phospholipase A2alpha inhibitors.

1,2,4-Oxadiazolidin-3,5-dione and 1,3,5-triazin-2,4,6-trione scaffolds were employed as templates to incorporate the pharmacophore requirements of cytosolic phospholipase A2alpha substrate mimetics. Inhibitors that are active in both enzyme, and cell-based assays were identified from both classes. From the SAR work carried out and modeling efforts around these templates, the triazinetrione scaffold with an additional substitution point was found to be more favorable.

Inhibition of cytosolic phospholipase A2alpha: hit to lead optimization.

Compound 1 was previously reported to be a potent inhibitor of cPLA(2)alpha in both artificial monomeric substrate and cell-based assays. However, 1 was inactive in whole blood assays previously used to characterize cyclooxygenase and lipoxygenase inhibitors. The IC(50) of 1 increased dramatically with cell number or lipid/detergent concentration. In an attempt to insert an electrophilic ketone between the indole and benzoic acid moieties, we discovered that increasing the distance between the two moieties gave a compound with activity in the GLU (7-hydroxycoumarinyl-gamma-linolenate) micelle assay, which contains lipid and detergent. Extensive structure-activity relationship work around this lead identified a potent pharmacophore for cPLA(2)alpha inhibition. The IC(50)s between the GLU micelle and rat whole blood assays correlated highly. No correlation was found for other parameters, including lipophilicity or acidity of the required acid functionality. Compounds 25, 39, and 94 emerged as potent, selective inhibitors of cPLA(2)alpha and represent well-validated starting points for further optimization.

Cytosolic phospholipase A2 alpha-deficient mice are resistant to experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), a Th1-mediated inflammatory disease of the central nervous system (CNS), is a model of human multiple sclerosis. Cytosolic phospholipase A2alpha (cPLA2alpha), which initiates production of prostaglandins, leukotrienes, and platelet-activating factor, is present in EAE lesions. Using myelin oligodendrocyte glycoprotein (MOG) immunization, as well as an adoptive transfer model, we showed that cPLA2alpha-/- mice are resistant to EAE. Histologic examination of the CNS from MOG-immunized mice revealed extensive inflammatory lesions in the cPLA2alpha+/- mice, whereas the lesions in cPLA2alpha-/- mice were reduced greatly or completely absent. MOG-specific T cells generated from WT mice induced less severe EAE in cPLA2alpha-/- mice compared with cPLA2alpha+/- mice, which indicates that cPLA2alpha plays a role in the effector phase of EAE. Additionally, MOG-specific T cells from cPLA2alpha-/- mice, transferred into WT mice, induced EAE with delayed onset and lower severity compared with EAE that was induced by control cells; this indicates that cPLA2alpha also plays a role in the induction phase of EAE. MOG-specific T cells from cPLA2alpha-/- mice were deficient in production of Th1-type cytokines. Consistent with this deficiency, in vivo administration of IL-12 rendered cPLA2alpha-/- mice susceptible to EAE. Our data indicate that cPLA2alpha plays an important role in EAE development and facilitates differentiation of T cells toward the Th1 phenotype.

A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening.

A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for FAAH, was designed and synthesized. FAAH catalyzes the hydrolysis of AAMCA to generate arachidonic acid and a highly fluorescent 7-amino, 4-methyl coumarin (AMC). The assay was done at 25 degrees C by incubating whole cell or microsomal preparations from FAAH-expressing cells with AAMCA. Release of AMC was monitored continuously using a fluorometer. Microsomal FAAH catalyzed the hydrolysis of AAMCA with an apparent K(m) of 0.48muM and V(max) of 58pmolmin(-1)mgprotein(-1). The assay is specific for FAAH given that microsomes prepared from cells expressing FAAH-S241A or vector alone had no significant activity against AAMCA. Furthermore, the activity was inhibited by URB-597, an FAAH-specific inhibitor, in a concentration-dependent manner with an IC(50) of 33.5nM. The assay was optimized for HTS and had a Z' value ranging from 0.7 to 0.9. The assay is also compatible with ex vivo analysis of FAAH activity.

Structure-activity relationships of indole cytosolic phospholipase A(2)alpha inhibitors: substrate mimetics.

An SAR effort focused on generating cPLA(2)alpha inhibitors using a substrate mimetic approach is reported. Indole inhibitors of cPLA(2)alpha with promising pharmacokinetic parameters that were active in both an isolated enzyme assay and in cell-based assays were discovered. Modeling these compounds into the cPLA(2)alpha structure validated the assumptions made at the start of the SAR effort.