The Metabolism and Disposition of Brepocitinib in Humans and Characterization of the Formation Mechanism of an Aminopyridine Metabolite.

Brepocitinib is an oral once-daily Janus kinase 1 and Tyrosine kinase 2 selective inhibitor currently in development for the treatment of several autoimmune disorders. Mass balance and metabolic profiles were determined using accelerator mass spectrometry in six healthy male participants following a single oral 60 mg dose of 14C-brepocitinib (~300 nCi). The average mass balance recovery was 96.7% {plus minus} 6.3% with the majority of dose (88.0% {plus minus} 8.0%) recovered in urine and 8.7% {plus minus} 2.1% of the dose in recovered in feces. Absorption of brepocitinib was rapid, with maximal plasma concentrations of total radioactivity and brepocitinib achieved within 0.5 hours after dosing. Circulating radioactivity consisted primarily of brepocitinib (47.8%) and metabolite M1 (37.1%) derived from hydroxylation at the C5' position of the pyrazole ring. Fractional contributions to metabolism via cytochrome P450 (CYP) enzymes were determined to be 0.77 for CYP3A4/5 and 0.14 for CYP1A2 based on phenotyping studies in HLM. However, additional clinical studies are required to understand the potential contribution of CYP1A1. Approximately 83% of the dose was eliminated as N-methylpyrazolyl oxidative metabolites, with 52.1% of the dose excreted as M1 alone. Notably, M1 was not observed as a circulating metabolite in earlier metabolic profiling of human plasma from a multiple ascending study with unlabeled brepocitinib. Mechanistic studies revealed M1 was highly unstable in human plasma and phosphate buffer, undergoing chemical oxidation leading to loss of the 5-hydroxy-1-methylpyrazole moiety and formation of aminopyrimidine cleavage product M2. Time dependent inhibition and trapping studies with M1 yielded insights into the mechanism of this unusual and unexpected instability. Significance Statement This work describes the mass balance and metabolic profile of brepocitinib in human, a JAK1/TYK2 inhibitor being developed for treatment of autoimmune diseases.

In Retrospect: Root-Cause Analysis of Structure-Activity Relationships in IRAK4 Inhibitor Zimlovisertib (PF-06650833).

In this paper, we disclose insights on the root causes of three structure-activity relationship (SAR) observations encountered in the discovery of the IRAK4 inhibitor Zimlovisertib (PF-06650833). The first is a nonlinear potency SAR encountered with the isoquinoline ether substituent, the second is a potency enhancement introduced by fluorine substitution on the lactam, and the third is a slight potency preference for all-syn (2S,3S,4S) stereochemistry in the fluorine-substituted lactam. We present new data that help to inform us of the origins of these unexpected SAR trends.

Synthesis of Deuterated Enaminones with High Isotopic Fidelity.

The unexpected oxidation of nitrogenous heterocycles by aldehyde oxidase (AO) may be addressed by the substitution of deuterium for hydrogen adjacent to the heterocycle nitrogen atom. Enaminones are versatile intermediates in the synthesis of nitrogenous heterocycles. We report that heretofore inaccessible monodeuterated enaminones of the general structure RC(=O)CH=CDNMe2 may be synthesized from methyl ketones and DCO2Me with high isotopic fidelity and efficient utilization of deuterium.

Small Change, Big Impact: Reversal of Diastereoselection in Cuprate Conjugate Additions to α,ß-Unsaturated Lactams and Identification of a Competing Mechanism.

A seemingly minor change to a reactant is shown to cause a change in reaction mechanisms. Conjugate addition of organocopper reagents to bicyclic α,ß-unsaturated lactams derived from pyroglutaminol is determined by the nature of the aminal group. Aminals derived from aldehydes give anti addition; those from ketones give syn addition. Divergence in diastereoselection occurs because the substrates react by different mechanisms, ultimately due to a small but significant difference in pyramidalization of the aminal nitrogen.

Synthesis of Deuterated Heterocycles with Me2NCD(OMe)2 and Evaluation of the Products for Metabolism by Aldehyde Oxidase.

The metabolic oxidation of drug-like small molecules by aldehyde oxidase (AO) has commonly been mitigated through the incorporation of deuterium at the oxidation site. We report that dimethylformamide dimethyl acetal and related compounds undergo rapid CH to CD isotopic exchange upon exposure to methanol-d and similar deuterated alcohols. This isotopic exchange process can be used to synthesize Me2NCD(OMe)2 and has significant implications for the use of Me2NCD(OMe)2 in the synthesis of specifically deuterium-labeled compounds. The application of Me2NCD(OMe)2 to the synthesis of various heterocycles that have been associated with AO metabolism is described, and we report the impact of deuteration on the rate of in vitro AO-mediated metabolism.

Discovery of a Series of Pyrimidine Carboxamides as Inhibitors of Vanin-1.

A diaryl ketone series was identified as vanin-1 inhibitors from a high-throughput screening campaign. While this novel scaffold provided valuable probe 2 that was used to build target confidence, concerns over the ketone moiety led to the replacement of this group. The successful replacement of this moiety was achieved with pyrimidine carboxamides derived from cyclic secondary amines that were extensively characterized using biophysical and crystallographic methods as competitive inhibitors of vanin-1. Through optimization of potency and physicochemical and ADME properties, and guided by co-crystal structures with vanin-1, 3 was identified with a suitable profile for advancement into preclinical development.

Prevention and partial reversion of the lupus phenotype in ABIN1[D485N] mice by an IRAK4 inhibitor.

OBJECTIVE: We have reported previously that the IRAK4 inhibitor PF06426779 given to ubiquitin-binding-defective ABIN1[D485N] mice at 6 weeks of age prevents the major facets of lupus that develop 10 weeks later. The present study was undertaken to investigate whether PF06426779 could reverse the lupus phenotype when administered to 13-week-old ABIN1[D485N] mice that had already developed symptoms of lupus. METHODS: Splenomegaly, the number of splenic neutrophils, TFH and Germinal Centre B (GCB) cells, serum levels of immunoglobulins, the extent of kidney, liver and lung pathology, and glomerular IgA and IgM were measured after feeding 13-week-old ABIN1[D485N] and wild-type mice for another 10 weeks with R&M3 diet with and without PF06426779 (4 g/kg). RESULTS: Following drug treatment, spleen size and weight, splenic neutrophil numbers, and serum IgA and glomerular IgA levels of ABIN1[D485N] mice returned to those seen in wild-type mice. The rise in splenic TFH and GCB numbers, the increase in kidney and liver pathology, and the concentrations of serum IgG1, IgG2A and IgE between 13 and 23 weeks were suppressed. There was no reduction in the level of anti-self double-stranded DNA, anti-self nuclear antigens or IgM during the drug treatment. CONCLUSIONS: The results demonstrate the therapeutic potential of IRAK4 inhibitors for the treatment of lupus and raise the possibility of monitoring efficacy by measuring decreases in the serum levels of IgA. Our results support the view that there may be a closer connection between lupus and IgA nephropathy than realised previously.

Elucidation of a Sequential Iminium Ion Cascade Reaction Triggered by a Silica Gel-Promoted Aza-Peterson Reaction.

In a recent methodological study investigating the synthesis of N-alkoxyazomethine ylides, an unexpected aminal byproduct was generated during our attempt to isolate O-benzyl-N-((trimethylsilyl)methyl)hydroxylamine. After a strategic investigation, silica gel was discovered to be the cause of the byproduct formation. Through the mechanistic insight from control and trapping experiments, we propose the formation of a methaniminium ion via a novel aza-Peterson reaction, which ultimately triggers a sequential iminium ion cascade sequence. Herein, we discuss the elucidation of this cascade reaction mechanism and the constraints for the byproduct formation.

Discovery of Tyrosine Kinase 2 (TYK2) Inhibitor (PF-06826647) for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).

Distinct signals and immune cells drive liver pathology and glomerulonephritis in ABIN1[D485N] mice.

We report that TLR7, IL-6, and the adaptive immune system are essential for autoimmunity and glomerulonephritis but not for liver pathology in mice expressing the ubiquitin-binding-defective ABIN1[D485N] mutant. The blood and organs of ABIN1[D485N] mice have exceptionally high numbers of patrolling monocytes (pMo), which develop independently of IL-6 and the adaptive immune system. They are detectable in the blood months before autoimmunity and organ pathology are seen and may have diagnostic potential. The splenic pMo, inflammatory monocytes (iMo), and neutrophils of ABIN1[D485N] mice expressed high levels of mRNAs encoding proteins released during NETosis, which together with the high numbers of monocyte-derived dendritic cells (MoDCs) may drive the liver pathology in ABIN1[D485N] mice, and contribute to the pathology of other organs. The splenic iMo of ABIN1[D485N] mice displayed high expression of mRNAs encoding proteins controlling cell division and were actively dividing; this may underlie the increased pMo and MoDC numbers, which are derived from iMo. An orally active IRAK4 inhibitor suppressed all facets of the disease phenotype and prevented the increase in pMo numbers.

Identification of Morpholino-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-ones as Nonsteroidal Mineralocorticoid Antagonists.

A novel series of morpholine-based nonsteroidal mineralocorticoid receptor antagonists is reported. Starting from a pyrrolidine HTS hit 9 that possessed modest potency but excellect selectivity versus related nuclear hormone receptors, a series of libraries led to identification of morpholine lead 10. After further optimization, cis disubstituted morpholine 22 was discovered, which showed a 45-fold boost in binding affinity and corresponding functional potency compared to 13. While 22 had high clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy (increased urinary Na+/K+ ratio) and safety. In contrast to rat, the dog and human MetID and PK profiles of 22 were adequate, suggesting that it could be suitable as a potential clinical asset.

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.

Synthesis of Chiral Azabicycles from Pyroglutaminols.

The stereocontrolled synthesis of a range of substituted bicyclic morpholine and piperazine derivatives is reported from substituted pyroglutaminols via an intramolecular SN2 cyclization as the key step. This enantiospecific approach toward chiral bicyclic morpholines and piperazines offers new opportunities to access these challenging ring systems, which are becoming increasingly common motifs in drug discovery.

Binding site elucidation and structure guided design of macrocyclic IL-17A antagonists.

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.

Lithium Enolates Derived from Pyroglutaminol: Mechanism and Stereoselectivity of an Azaaldol Addition.

A lithium enolate derived from an acetonide-protected pyroglutaminol undergoes a highly selective azaaldol addition with (E)-N-phenyl-1-[2-(trifluoromethyl)phenyl]methanimine. The selectivity is sensitive to tetrahydrofuran (THF) concentration, temperature, and the presence of excess lithium diisopropylamide base. Rate studies show that the observable tetrasolvated dimeric enolate undergoes reversible deaggregation, with the reaction proceeding via a disolvated-monomer-based transition structure. Limited stereochemical erosion stems from the intervention of a trisolvated-monomer-based pathway, which is suppressed at low THF concentrations and elevated temperature. Endofacial selectivity observed with excess lithium diisopropylamide (LDA) is traced to an intermediate dianion formed by subsequent lithiation of the monomeric azaaldol adduct, which is characterized as both a dilithio form and a trilithio dianion-LDA mixed aggregate.

Lithium Enolates Derived from Pyroglutaminol: Aggregation, Solvation, and Atropisomerism.

Lithium enolates derived from protected pyroglutaminols were characterized by using (6)Li, (13)C, and (19)F NMR spectroscopies in conjunction with the method of continuous variations. Mixtures of tetrasolvated dimers and tetrasolvated tetramers in different proportions depend on the steric demands of the hemiaminal protecting group, tetrahydrofuran concentration, and the presence or absence of an α-fluoro moiety. The high steric demands of the substituted bicyclo[3.3.0] ring system promote dimers to an unusual extent and allow solvents and atropisomers in cubic tetramers to be observed in the slow-exchange limit. Pyridine used as a (6)Li chemical shift reagent proved useful in assigning solvation numbers.

Reversal of Diastereoselection in the Conjugate Addition of Cuprates to Unsaturated Lactams.

We report that the stereochemical outcome of the conjugate addition of organocopper reagents to bicyclic α,ß-unsaturated lactams derived from pyroglutaminol is determined by the nature of the aminal group. Bicyclic α,ß-unsaturated lactams in which the aminal is derived from a ketone have been found to afford products of syn conjugate addition. By contrast, bicyclic α,ß-unsaturated lactams in which the aminal is derived from an aldehyde afford products of anti conjugate addition. These remarkably different results obtained from very similar starting materials are unexpected.

Aminocarbonylation of aryl tosylates to carboxamides.

The palladium - catalyzed aminocarbonylation of aryl tosylates with amines is reported. Suitable conditions were identified by high throughput reaction screening and then further optimized. The substrate scope of the reaction with respect to the aryl tosylate component and the amine component are reported. Competitive aminolysis of the aryl tosylates to afford the amine toluenesulfonamides and the phenol was not observed.

Identification of a novel conformationally constrained glucagon receptor antagonist.

Identification of orally active, small molecule antagonists of the glucagon receptor represents a novel treatment paradigm for the management of type 2 diabetes mellitus. The present work discloses novel glucagon receptor antagonists, identified via conformational constraint of current existing literature antagonists. Optimization of lipophilic ligand efficiency (LLE or LipE) culminated in enantiomers (+)-trans-26 and (-)-trans-27 which exhibit good physicochemical and in vitro drug metabolism profiles. In vivo, significant pharmacokinetic differences were noted with the two enantiomers, which were primarily driven through differences in clearance rates. Enantioselective oxidation by cytochrome P450 was ruled out as a causative factor for pharmacokinetic differences.

From partial to full agonism: identification of a novel 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole as a full agonist of the human GPR119 receptor.

A novel GPR119 agonist based on the 2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole scaffold was designed through lead optimization starting from pyrazole-based GPR119 agonist 1. The design is centered on the conformational restriction of the core scaffold, while minimizing the change in spatial relationships of two key pharmacophoric elements (piperidine-carbamate and aryl sulfone).