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Using structure-guided design, several cell based assays, and microdosed positron emission tomography (PET) imaging, we identified a series of highly potent, selective, and brain-penetrant oxazole-4-carboxamide-based inhibitors of glycogen synthase kinase-3 (GSK-3). An isotopologue of our first-generation lead, [(3)H]PF-367, demonstrates selective and specific target engagement in vitro, irrespective of the activation state. We discovered substantial ubiquitous GSK-3-specific radioligand binding in Tg2576 Alzheimer’s disease (AD), suggesting application for these compounds in AD diagnosis and identified [(11)C]OCM-44 as our lead GSK-3 radiotracer, with optimized brain uptake by PET imaging in nonhuman primates. GSK-3β-isozyme selectivity was assessed to reveal OCM-51, the most potent (IC(50) = 0.030 nM) and selective (>10-fold GSK-3β/GSK-3α) GSK-3β inhibitor known to date. Inhibition of CRMP2(T514) and tau phosphorylation, as well as favorable therapeutic window against WNT/β-catenin signaling activation, was observed in cells.

Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1β1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of β1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1β1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.

Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.

Leucine rich repeat kinase 2 (LRRK2) has been genetically linked to Parkinson's disease (PD) by genome-wide association studies (GWAS). The most common LRRK2 mutation, G2019S, which is relatively rare in the total population, gives rise to increased kinase activity. As such, LRRK2 kinase inhibitors are potentially useful in the treatment of PD. We herein disclose the discovery and optimization of a novel series of potent LRRK2 inhibitors, focusing on improving kinome selectivity using a surrogate crystallography approach. This resulted in the identification of 14 (PF-06447475), a highly potent, brain penetrant and selective LRRK2 inhibitor which has been further profiled in in vivo safety and pharmacodynamic studies.

Several multistep syntheses of substituted benzenes are reported. The benzene analogues were designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa enzyme. A variety of chemical transformations including nucleophilic additions, reductive aminations, Stille couplings, and polymer-assisted solution-phase (PASP) techniques were used to prepare key intermediates and final products. The initial analogues identified some weakly active compounds which ultimately led to a 340 nM (IC(50)) tissue Factor VIIa inhibitor with selectivity over other related enzymes. The structure-activity relationship of these inhibitors and the synthetic progression from the discovery of the lead compound to the development of potent analogues will be discussed. The X-ray crystal structures of fluorobenzene 50c and benzoquinone 54 inhibitors complexed with the TF/VIIa enzyme will also be described.

High-throughput screening (HTS) of compound libraries is used to discover novel leads for drug development. When a structure is available for the target, computer-based screening using molecular docking may also be considered. The two techniques have rarely been used together on the same target. The opportunity to do so presented itself in a project to discover novel inhibitors for the enzyme protein tyrosine phosphatase-1B (PTP1B), a tyrosine phosphatase that has been implicated as a key target for type II diabetes. A corporate library of approximately 400 000 compounds was screened using high-throughput experimental techniques for compounds that inhibited PTP1B. Concurrently, molecular docking was used to screen approximately 235 000 commercially available compounds against the X-ray crystallographic structure of PTP1B, and 365 high-scoring molecules were tested as inhibitors of the enzyme. Of approximately 400 000 molecules tested in the high-throughput experimental assay, 85 (0.021%) inhibited the enzyme with IC50 values less than 100 microM; the most active had an IC50 value of 4.2 microM. Of the 365 molecules suggested by molecular docking, 127 (34.8%) inhibited PTP1B with IC50 values less than 100 microM; the most active of these had an IC50 of 1.7 microM. Structure-based docking therefore enriched the hit rate by 1700-fold over random screening. The hits from both the high-throughput and docking screens were dissimilar from phosphotyrosine, the canonical substrate group for PTP1B; the two hit lists were also very different from each other. Surprisingly, the docking hits were judged to be more druglike than the HTS hits. The diversity of both hit lists and their dissimilarity from each other suggest that docking and HTS may be complementary techniques for lead discovery.

We wish to report a strategy that targets interleukin-2 inducible T cell kinase (Itk) with covalent inhibitors. Thus far, covalent inhibition of Itk has not been disclosed in the literature. Structure-based drug design was utilized to achieve low nanomolar potency of the disclosed series even at high ATP concentrations. Kinetic measurements confirmed an irreversible binding mode with off-rate half-lives exceeding 24 h and moderate on-rates. The analogues are highly potent in a cellular IP1 assay as well as in a human whole-blood (hWB) assay. Despite a half-life of approximately 2 h in resting primary T cells, the covalent inhibition of Itk resulted in functional silencing of the TCR pathway for more than 24 h. This prolonged effect indicates that covalent inhibition is a viable strategy to target the inactivation of Itk.

A series of pyrazole inhibitors of p38 mitogen-activated protein (MAP) kinase were designed using a binding model based on the crystal structure of 1 (SC-102) bound to p38 enzyme. New chemistry using dithietanes was developed to assemble nitrogen-linked substituents at the 5-position of pyrazoles. Calculated log D was used in tandem with structure-based design to guide medicinal chemistry strategy and improve the in vivo activity of a series of molecules. The crystal structure of an optimized inhibitor, 4 (SC-806), in complex with p38 enzyme was obtained to confirm the hypothesis that the addition of a basic nitrogen to the molecule induces an interaction with Asp112 of p38 alpha. A compound identified from this series was efficacious in an animal model of rheumatic disease.

A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

Targeted 2-pyridones were selected as tissue Factor VIIa inhibitors and prepared from 2,6-dibromopyridine via a multistep synthesis. A variety of chemical transformations, including regioselective nucleophilic addition, selective nitrogen alkylation, and a Suzuki coupling, afforded the targeted tissue Factor VIIa inhibitors. The pyridone core was selected as a replacement for the pyrazinone core of noncovalent tissue Factor VIIa inhibitors and designed such that their substitution pattern would occupy and interact with the S(1), S(2), and S(3) pockets of the tissue Factor VIIa enzyme. These compounds were tested in several serine protease enzyme assays involved in the coagulation cascade exhibiting modest activity on tissue Factor VIIa with excellent selectivity over thrombin and Factor Xa. Finally, an X-ray crystal structure of inhibitor 14a bound to tissue Factor VIIa was obtained and will be described.

A solution-phase synthesis of an alpha-ketothiazole library of the general form D-Phe-L-AA-Arg-alpha-ketothiazole is described. The five-step synthesis is accomplished using a combination of polymeric reagents and polymer-assisted solution-phase purification concepts, including reactant-sequestering resins, reagent-sequestering resins, and tagged reagents. The multistep synthesis affords desired alpha-ketothiazole products in excellent purities and yields. A variety of L-amino acid inputs were used to probe the S2 pocket of tissue Factor VIIa enzyme to influence both potency and selectivity. An X-ray crystal structure of compound 10k bound to the TF/VIIa complex was obtained that explains the observed selectivity. The alpha-ketothiazoles were found to be potent, reversible-covalent inhibitors of tissue Factor VIIa, with some analogues demonstrating selectivity over thrombin.

Structure-based drug design (SBDD) and polymer-assisted solution-phase (PASP) library synthesis were used to develop a series of pyrazinone inhibitors of the Tissue Factor/Factor VIIa (TF/VIIa) complex. The crystal structure of a tripeptide-alpha-ketothiazole complexed with TF/VIIa was utilized in a docking experiment to identify the pyrazinone core as a starting scaffold. The pyrazinone core could orient the substituents in the correct spatial arrangement to probe the S1, S2, and S3 pockets of the enzyme. A multistep PASP library synthesis was designed to prepare the substituted pyrazinones varying the P1, P2, and P3 moieties. Hundreds of pyrazinone TF/VIIa inhibitors were prepared and tested in several serine protease enzyme assays involved in the coagulation cascade. The inhibitors exhibited modest activity on TF/VIIa with excellent selectivity over thrombin (IIa) and Factor Xa. The structure-activity relationship of the pyrazinone inhibitors will be discussed and X-ray crystal structures of selected compounds complexed with the TF/VIIa enzyme will be described. This study ultimately led to the synthesis of compound 34, which exhibited 16 nM (IC50) activity on TF/VIIa with >6250 x selectivity vs Factor Xa and thrombin. This potent and highly selective inhibitor of TF/VIIa was chosen for preclinical, intravenous proof-of-concept studies to demonstrate the separation between antithrombotic efficacy and bleeding side effects in a nonhuman primate model of electrolytic-induced arterial thrombosis.