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.

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.

Elucidating the mechanism of cytochrome P450-mediated pyrimidine ring conversion to pyrazole metabolites with the BACE1 inhibitor GNE-892 in rats.

We investigated an uncommon biotransformation of pyrimidine during the metabolism of GNE-892 ((R)-2-amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one), a ß-secretase 1 inhibitor. Three novel metabolites, formed by conversion of pyrimidine to pyrazole, were observed in the (14)C-radiolabeled mass balance study in rats. Their structures were characterized by high-resolution mass spectrometry and nuclear magnetic resonance. Although these metabolites accounted for <5% of the administered dose, their unique nature prompted us to conduct further investigations. The pyrazole-containing metabolites were formed in vitro with rat hepatocytes and liver microsomes, which supported that they were formed during hepatic metabolism. Further, their generation was inhibited by 1-aminobenzotriazole, indicating involvement of cytochrome P450s. Studies with rat recombinant enzymes identified that CYP2D2 generated the N-hydroxypyrazole metabolite from GNE-892. This biotransformation proceeded through multiple steps from the likely precursor, pyrimidine N-oxide. On the basis of these data, we propose a mechanism in which the pyrimidine is activated via N-oxidation, followed by a second oxidative process that opens the pyrimidine ring to form a formamide intermediate. After hydrolysis of the formamide, a carbon is lost as formic acid, together with ring closure to form the pyrazole ring. This article highlights a mechanistic approach for determining the biotransformation of the pyrimidine to a pyrazole for GNE-892.

Spirocyclic ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors: from hit to lowering of cerebrospinal fluid (CSF) amyloid ß in a higher species.

A hallmark of Alzheimer's disease is the brain deposition of amyloid beta (Aß), a peptide of 36-43 amino acids that is likely a primary driver of neurodegeneration. Aß is produced by the sequential cleavage of APP by BACE1 and γ-secretase; therefore, inhibition of BACE1 represents an attractive therapeutic target to slow or prevent Alzheimer's disease. Herein we describe BACE1 inhibitors with limited molecular flexibility and molecular weight that decrease CSF Aß in vivo, despite efflux. Starting with spirocycle 1a, we explore structure-activity relationships of core changes, P3 moieties, and Asp binding functional groups in order to optimize BACE1 affinity, cathepsin D selectivity, and blood-brain barrier (BBB) penetration. Using wild type guinea pig and rat, we demonstrate a PK/PD relationship between free drug concentrations in the brain and CSF Aß lowering. Optimization of brain exposure led to the discovery of (R)-50 which reduced CSF Aß in rodents and in monkey.

Discovery and preclinical pharmacology of a selective ATP-competitive Akt inhibitor (GDC-0068) for the treatment of human tumors.

The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.

A potential therapeutic target for FLT3-ITD AML: PIM1 kinase.

Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) mutation have a poor prognosis, and FLT3 inhibitors are now under clinical investigation. PIM1, a serine/threonine kinase, is up-regulated in FLT3-ITD AML and may be involved in FLT3-mediated leukemogenesis. We employed a PIM1 inhibitor, AR00459339 (Array Biopharma Inc.), to investigate the effect of PIM1 inhibition in FLT3-mutant AML. Like FLT3 inhibitors, AR00459339 was preferentially cytotoxic to FLT3-ITD cells, as demonstrated in the MV4-11, Molm-14, and TF/ITD cell lines, as well as 12 FLT3-ITD primary samples. Unlike FLT3 inhibitors, AR00459339 did not suppress phosphorylation of FLT3, but did promote the de-phosphorylation of downstream FLT3 targets, STAT5, AKT, and BAD. Combining AR00459339 with a FLT3 inhibitor resulted in additive to mildly synergistic cytotoxic effects. AR00459339 was cytotoxic to FLT3-ITD samples from patients with secondary resistance to FLT3 inhibitors, suggesting a novel benefit to combining these agents. We conclude that PIM1 appears to be closely associated with FLT3 signaling, and that inhibition of PIM1 may hold therapeutic promise, either as monotherapy, or by overcoming resistance to FLT3 inhibitors.

Discovery of spirocyclic sulfonamides as potent Akt inhibitors with exquisite selectivity against PKA.

We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors. Through structure-based rational design, we have successfully improved PKA selectivity of previously disclosed spirochromanes. Representative compounds showed favorable Akt potency while exhibiting up to 1000-fold selectivity against PKA.

Discovery and SAR of spirochromane Akt inhibitors.

A novel series of spirochromane pan-Akt inhibitors is reported. SAR optimization furnished compounds with improved enzyme potencies and excellent selectivity over the related AGC kinase PKA. Attempted replacement of the phenol hinge binder provided compounds with excellent Akt enzyme and cell activities but greatly diminished selectivity over PKA.

Discovery of dihydrothieno- and dihydrofuropyrimidines as potent pan Akt inhibitors.

Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.

Discovery of pyrrolopyrimidine inhibitors of Akt.

The discovery and optimization of a series of pyrrolopyrimidine based protein kinase B (Pkb/Akt) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent inhibition of all three Akt isoforms and knockdown of phospho-PRAS40 levels in LNCaP cells and tumor xenografts.