Evaluation of PNU-159682 antibody drug conjugates (ADCs).

PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.

Discovery of a class of highly potent Janus Kinase 1/2 (JAK1/2) inhibitors demonstrating effective cell-based blockade of IL-13 signaling.

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).

Optimization of Pan-Pim Kinase Activity and Oral Bioavailability Leading to Diaminopyrazole (GDC-0339) for the Treatment of Multiple Myeloma.

Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.

Discovery of a Potent (4 R,5 S)-4-Fluoro-5-methylproline Sulfonamide Transient Receptor Potential Ankyrin 1 Antagonist and Its Methylene Phosphate Prodrug Guided by Molecular Modeling.

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.

Selective Inhibitors of Dual Leucine Zipper Kinase (DLK, MAP3K12) with Activity in a Model of Alzheimer's Disease.

Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.

Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.

α-Aryl pyrrolidine sulfonamides as TRPA1 antagonists.

A series of α-aryl pyrrolidine sulfonamide TRPA1 antagonists were advanced from an HTS hit to compounds that were stable in liver microsomes with retention of TRPA1 potency. Metabolite identification studies and physicochemical properties were utilized as a strategy for compound design. These compounds serve as starting points for further compound optimization.

Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety.

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

Novel antibody-antibiotic conjugate eliminates intracellular S. aureus.

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.

Discovery of 3,5-substituted 6-azaindazoles as potent pan-Pim inhibitors.

Pim kinase inhibitors are promising cancer therapeutics. Pim-2, among the three Pim isoforms, plays a critical role in multiple myeloma yet inhibition of Pim-2 is challenging due to its high affinity for ATP. A co-crystal structure of a screening hit 1 bound to Pim-1 kinase revealed the key binding interactions of its indazole core within the ATP binding site. Screening of analogous core fragments afforded 1H-pyrazolo[3,4-c]pyridine (6-azaindazole) as a core for the development of pan-Pim inhibitors. Fragment and structure based drug design led to identification of the series with picomolar biochemical potency against all three Pim isoforms. Desirable cellular potency was also achieved.

Probing Mechanisms of CYP3A Time-Dependent Inhibition Using a Truncated Model System.

Time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes may incur serious undesirable drug-drug interactions and in rare cases drug-induced idiosyncratic toxicity. The reactive metabolites are often generated through multiple sequential biotransformations and form adducts with CYP enzymes to inactivate their function. The complexity of these processes makes addressing TDI liability very challenging. Strategies to mitigate TDI are therefore highly valuable in discovering safe therapies to benefit patients. In this Letter, we disclose our simplified approach toward addressing CYP3A TDI liabilities, guided by metabolic mechanism hypotheses. By adding a methyl group onto the α carbon of a basic amine, TDI activities of both the truncated and full molecules (7a and 11) were completely eliminated. We propose that truncated molecules, albeit with caveats, may be used as surrogates for full molecules to investigate TDI.

Effect of selective LRRK2 kinase inhibition on nonhuman primate lung.

Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.

Discovery of dual leucine zipper kinase (DLK, MAP3K12) inhibitors with activity in neurodegeneration models.

Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.

Dose-dependent exposure and metabolism of GNE-892, a ß-secretase inhibitor, in monkeys: contributions by P450, AO, and P-gp.

(R)-2-Amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one (GNE-892) is an orally administered inhibitor of ß-secretase 1 (ß-site amyloid precursor protein cleaving enzyme 1, BACE1) that was developed as an intervention therapy against Alzheimer's disease. A clinical microdosing strategy was being considered for de-risking the potential pharmacokinetic liabilities of GNE-892. We tested whether dose-proportionality was observed in cynomolgus monkey as proof-of-concept for a human microdosing study. With cryopreserved monkey hepatocytes, concentration-dependency for substrate turnover and the relative contribution of P450- versus AO-mediated metabolism were observed. Characterization of the kinetics of these metabolic pathways demonstrated differences in the affinities of P450 and AO for GNE-892, which supported the metabolic profiles that had been obtained. To test if this metabolic shift occurred in vivo, mass balance studies in monkeys were conducted at doses of 0.085 and 15 mg/kg. Plasma exposure of GNE-892 following oral administration was more than 20-fold greater than dose proportional at the high-dose. P-gp-mediated efflux was unable to explain the discrepancy. The profiles of metabolites in circulation and excreta were indicative that oxidative metabolism limited the exposure to unchanged GNE-892 at the low dose. Further, the in vivo data supported the concentration-dependent metabolic shift between P450 and AO. In conclusion, microdosing of GNE-892 was not predictive of pharmacokinetics at a more pharmacologically relevant dose due to saturable absorption and metabolism. Therefore, it is important to consider ADME liabilities and their potential concentration-dependency when deciding upon a clinical microdosing strategy.

8-Tetrahydropyran-2-yl chromans: highly selective beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors.

A series of 2,3,4,4a,10,10a-hexahydropyrano[3,2-b]chromene analogs was developed that demonstrated high selectivity (>2000-fold) for BACE1 vs Cathepsin D (CatD). Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Guided by structure based design, changes to P2' and P3 moieties were explored. A conformationally restricted P2' methyl group provided inhibitors with excellent cell potency (37-137 nM) and selectivity (435 to >2000-fold) for BACE1 vs CatD. These efforts lead to compound 59, which demonstrated a 69% reduction in rat CSF Aß1-40 at 60 mg/kg (PO).

Discovery of a Novel Class of Imidazo[1,2-a]Pyridines with Potent PDGFR Activity and Oral Bioavailability.

The in silico construction of a PDGFRß kinase homology model and ensuing medicinal chemistry guided by molecular modeling, led to the identification of potent, small molecule inhibitors of PDGFR. Subsequent exploration of structure-activity relationships (SAR) led to the incorporation of a constrained secondary amine to enhance selectivity. Further refinements led to the integration of a fluorine substituted piperidine, which resulted in significant reduction of P-glycoprotein (Pgp) mediated efflux and improved bioavailability. Compound 28 displayed oral exposure in rodents and had a pronounced effect in a pharmacokinetic-pharmacodynamic (PKPD) assay.

Synthesis, characterization, and PK/PD studies of a series of spirocyclic pyranochromene BACE1 inhibitors.

The development of 1,3,4,4a,5,10a-hexahydropyrano[3,4-b]chromene analogs as BACE1 inhibitors is described. Introduction of the spirocyclic pyranochromene scaffold yielded several advantages over previous generation cores, including increased potency, reduced efflux, and reduced CYP2D6 inhibition. Compound 13 (BACE1 IC50=110 nM) demonstrated a reduction in CSF Aß in wild type rats after a single dose.

Discovery of 7-tetrahydropyran-2-yl chromans: ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors that reduce amyloid ß-protein (Aß) in the central nervous system.

In an attempt to increase selectivity vs Cathepsin D (CatD) in our BACE1 program, a series of 1,3,4,4a,10,10a-hexahydropyrano[4,3-b]chromene analogues was developed. Three different Asp-binding moieties were examined: spirocyclic acyl guanidines, aminooxazolines, and aminothiazolines in order to modulate potency, selectivity, efflux, and permeability. Using structure-based design, substitutions to improve binding to both the S3 and S2' sites of BACE1 were explored. An acyl guanidine moiety provided the most potent analogues. These compounds demonstrated 10-420 fold selectivity for BACE1 vs CatD, and were highly potent in a cell assay measuring Aß1-40 production (5-99 nM). They also suffered from high efflux. Despite this undesirable property, two of the acyl guanidines achieved free brain concentrations (Cfree,brain) in a guinea pig PD model sufficient to cover their cell IC50s. Moreover, a significant reduction of Aß1-40 in guinea pig, rat, and cyno CSF (58%, 53%, and 63%, respectively) was observed for compound 62.

Discovery of highly potent, selective, and brain-penetrant aminopyrazole leucine-rich repeat kinase 2 (LRRK2) small molecule inhibitors.

Leucine-rich repeat kinase 2 (LRRK2) has drawn significant interest in the neuroscience research community because it is one of the most compelling targets for a potential disease-modifying Parkinson's disease therapy. Herein, we disclose structurally diverse small molecule inhibitors suitable for assessing the implications of sustained in vivo LRRK2 inhibition. Using previously reported aminopyrazole 2 as a lead molecule, we were able to engineer structural modifications in the solvent-exposed region of the ATP-binding site that significantly improve human hepatocyte stability, rat free brain exposure, and CYP inhibition and induction liabilities. Disciplined application of established optimal CNS design parameters culminated in the rapid identification of GNE-0877 (11) and GNE-9605 (20) as highly potent and selective LRRK2 inhibitors. The demonstrated metabolic stability, brain penetration across multiple species, and selectivity of these inhibitors support their use in preclinical efficacy and safety studies.

A hit to lead discovery of novel N-methylated imidazolo-, pyrrolo-, and pyrazolo-pyrimidines as potent and selective mTOR inhibitors.

A series of N-7-methyl-imidazolopyrimidine inhibitors of the mTOR kinase have been designed and prepared, based on the hypothesis that the N-7-methyl substituent on imidazolopyrimidine would impart selectivity for mTOR over the related PI3Kα and δ kinases. The corresponding N-Me substituted pyrrolo[3,2-d]pyrimidines and pyrazolo[4,3-d]pyrimidines also show potent mTOR inhibition with selectivity toward both PI3α and δ kinases. The most potent compound synthesized is pyrazolo[4,3-d]pyrimidine 21c. Compound 21c shows a Ki of 2 nM against mTOR inhibition, remarkable selectivity (>2900×) over PI3 kinases, and excellent potency in cell-based assays.