Discovery of SARxxxx92, a pan-PIM kinase inhibitor, efficacious in a KG1 tumor model.

N-substituted azaindoles were discovered as potent pan-PIM inhibitors. Lead optimization, guided by structure and focused on physico-chemical properties allowed us to solve inherent hERG and permeability liabilities, and provided compound 27, which subsequently impacted KG-1 tumor growth in a mouse model.

Discovery of N-(4-(3-(2-aminopyrimidin-4-yl)pyridin-2-yloxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine (AMG 900), a highly selective, orally bioavailable inhibitor of aurora kinases with activity against multidrug-resistant cancer cell lines.

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

An Orally Available BACE1 Inhibitor That Affords Robust CNS Aß Reduction without Cardiovascular Liabilities.

BACE1 inhibition to prevent Aß peptide formation is considered to be a potential route to a disease-modifying treatment for Alzheimer's disease. Previous efforts in our laboratory using a combined structure- and property-based approach have resulted in the identification of aminooxazoline xanthenes as potent BACE1 inhibitors. Herein, we report further optimization leading to the discovery of inhibitor 15 as an orally available and highly efficacious BACE1 inhibitor that robustly reduces CSF and brain Aß levels in both rats and nonhuman primates. In addition, compound 15 exhibited low activity on the hERG ion channel and was well tolerated in an integrated cardiovascular safety model.

Development of 2-aminooxazoline 3-azaxanthenes as orally efficacious ß-secretase inhibitors for the potential treatment of Alzheimer's disease.

The ß-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is one of the most hotly pursued targets for the treatment of Alzheimer's disease. We used a structure- and property-based drug design approach to identify 2-aminooxazoline 3-azaxanthenes as potent BACE1 inhibitors which significantly reduced CSF and brain Aß levels in a rat pharmacodynamic model. Compared to the initial lead 2, compound 28 exhibited reduced potential for QTc prolongation in a non-human primate cardiovascular safety model.

Inhibitors of ß-site amyloid precursor protein cleaving enzyme (BACE1): identification of (S)-7-(2-fluoropyridin-3-yl)-3-((3-methyloxetan-3-yl)ethynyl)-5'H-spiro[chromeno[2,3-b]pyridine-5,4'-oxazol]-2'-amine (AMG-8718).

We have previously shown that the aminooxazoline xanthene scaffold can generate potent and orally efficacious BACE1 inhibitors although certain of these compounds exhibited potential hERG liabilities. In this article, we describe 4-aza substitution on the xanthene core as a means to increase BACE1 potency while reducing hERG binding affinity. Further optimization of the P3 and P2' side chains resulted in the identification of 42 (AMG-8718), a compound with a balanced profile of BACE1 potency, hERG binding affinity, and Pgp recognition. This compound produced robust and sustained reductions of CSF and brain Aß levels in a rat pharmacodynamic model and exhibited significantly reduced potential for QTc elongation in a cardiovascular safety model.

Lead optimization and modulation of hERG activity in a series of aminooxazoline xanthene ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

The optimization of a series of aminooxazoline xanthene inhibitors of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) is described. An early lead compound showed robust Aß lowering activity in a rat pharmacodynamic model, but advancement was precluded by a low therapeutic window to QTc prolongation in cardiovascular models consistent with in vitro activity on the hERG ion channel. While the introduction of polar groups was effective in reducing hERG binding affinity, this came at the expense of higher than desired Pgp-mediated efflux. A balance of low Pgp efflux and hERG activity was achieved by lowering the polar surface area of the P3 substituent while retaining polarity in the P2' side chain. The introduction of a fluorine in position 4 of the xanthene ring improved BACE1 potency (5-10-fold). The combination of these optimized fragments resulted in identification of compound 40, which showed robust Aß reduction in a rat pharmacodynamic model (78% Aß reduction in CSF at 10 mg/kg po) and also showed acceptable cardiovascular safety in vivo.

Hydroxyethylamine-based inhibitors of BACE1: P1-P3 macrocyclization can improve potency, selectivity, and cell activity.

We describe a systematic study of how macrocyclization in the P1-P3 region of hydroxyethylamine-based inhibitors of ß-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid ß-peptide (Aß)-lowering activity in HEK293T cells stably expressing APPSW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.

Structure- and property-based design of aminooxazoline xanthenes as selective, orally efficacious, and CNS penetrable BACE inhibitors for the treatment of Alzheimer's disease.

A structure- and property-based drug design approach was employed to identify aminooxazoline xanthenes as potent and selective human ß-secretase inhibitors. These compounds exhibited good isolated enzyme, cell potency, and selectivity against the structurally related aspartyl protease cathepsin D. Our efforts resulted in the identification of a potent, orally bioavailable CNS penetrant compound that exhibited in vivo efficacy. A single oral dose of compound 11a resulted in a significant reduction of CNS Aß40 in naive rats.

2-Phenylamino-6-cyano-1H-benzimidazole-based isoform selective casein kinase 1 gamma (CK1γ) inhibitors.

Screening of the Amgen compound library led to the identification of 2-phenylamino-6-cyano-1H-benzimidazole 1a as a potent CK1 gamma inhibitor with excellent kinase selectivity and unprecedented CK1 isoform selectivity. Further structure-based optimization of this series resulted in the discovery of 1h which possessed good enzymatic and cellular potency, excellent CK1 isoform and kinase selectivity, and acceptable pharmacokinetic properties.

Structure guided P1' modifications of HEA derived ß-secretase inhibitors for the treatment of Alzheimer's disease.

The synthesis and SAR of a series of BACE-1 hydroxyethyl amine inhibitors containing substitutions on a spirocyclobutyl moiety is described. Selectivity against cathepsin D, a related aspartyl protease with potential off target toxicity, and improved microsomal stability is exemplified.

Design and preparation of a potent series of hydroxyethylamine containing ß-secretase inhibitors that demonstrate robust reduction of central ß-amyloid.

A series of potent hydroxyethyl amine (HEA) derived inhibitors of ß-site APP cleaving enzyme (BACE1) was optimized to address suboptimal pharmacokinetics and poor CNS partitioning. This work identified a series of benzodioxolane analogues that possessed improved metabolic stability and increased oral bioavailability. Subsequent efforts focused on improving CNS exposure by limiting susceptibility to Pgp-mediated efflux and identified an inhibitor which demonstrated robust and sustained reduction of CNS ß-amyloid (Aß) in Sprague-Dawley rats following oral administration.

Design and synthesis of potent, orally efficacious hydroxyethylamine derived ß-site amyloid precursor protein cleaving enzyme (BACE1) inhibitors.

We have previously shown that hydroxyethylamines can be potent inhibitors of the BACE1 enzyme and that the generation of BACE1 inhibitors with CYP 3A4 inhibitory activities in this scaffold affords compounds (e.g., 1) with sufficient bioavailability and pharmacokinetic profiles to reduce central amyloid-ß peptide (Aß) levels in wild-type rats following oral dosing. In this article, we describe further modifications of the P1-phenyl ring of the hydroxyethylamine series to afford potent, dual BACE1/CYP 3A4 inhibitors which demonstrate improved penetration into the CNS. Several of these compounds caused robust reduction of Aß levels in rat CSF and brain following oral dosing, and compound 37 exhibited an improved cardiovascular safety profile relative to 1.

Synthesis of 4-substituted chlorophthalazines, dihydrobenzoazepinediones, 2-pyrazolylbenzoic acid, and 2-pyrazolylbenzohydrazide via 3-substituted 3-hydroxyisoindolin-1-ones.

Herein we describe a general three-step synthesis of 4-substituted chlorophthalazines in good overall yields. In the key step, N,N-dimethylaminophthalimide (8a) directs the selective monoaddition of alkyl, aryl, and heteroaryl organometallic reagents to afford 3-substituted 3-hydroxyisoindolinones 9b, 9i-9am. Many of these hydroxyisoindolinones are converted to chlorophthalazines 1b-1v via reaction with hydrazine, followed by chlorination with POCl(3). We have also discovered two novel transformations of 3-vinyl- and 3-alkynyl-3-hydroxyisoindolinones. Addition of vinyl organometallic reagents to N,N-dimethylaminophthalimide (8a) provided dihydrobenzoazepinediones 15a-15c via the proposed ring expansion of 3-vinyl-3-hydroxyisoindolinone intermediates. 3-Alkynyl-3-hydroxyisoindolinones react with hydrazine and substituted hydrazines to afford 2-pyrazolyl benzoic acids 16a-16d and 2-pyrazolyl benzohydrazides 17a-17g rather than the expected alkynyl phthalazinones.

Discovery and optimization of aminopyrimidinones as potent and state-dependent Nav1.7 antagonists.

Clinical genetic data have shown that the product of the SCN9A gene, voltage-gated sodium ion channel Nav1.7, is a key control point for pain perception and a possible target for a next generation of analgesics. Sodium channels, however, historically have been difficult drug targets, and many of the existing structure-activity relationships (SAR) have been defined on pharmacologically modified channels with indirect reporter assays. Herein we describe the discovery, optimization, and SAR of potent aminopyrimidinone Nav1.7 antagonists using electrophysiology-based assays that measure the ligand-receptor interaction directly. Within this series, rapid functionalization at the polysubstituted aminopyrimidinone head group enabled exploration of SAR and of pharmacokinetic properties. Lead optimized N-Me-aminopyrimidinone 9 exhibited improved Nav1.7 potency, minimal off-target hERG liability, and improved rat PK properties.

Structure-Based Design of Potent and Selective CK1γ Inhibitors.

Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.

Identification of a potent, state-dependent inhibitor of Nav1.7 with oral efficacy in the formalin model of persistent pain.

Clinical human genetic studies have recently identified the tetrodotoxin (TTX) sensitive neuronal voltage gated sodium channel Nav1.7 (SCN9A) as a critical mediator of pain sensitization. Herein, we report structure-activity relationships for a novel series of 2,4-diaminotriazines that inhibit hNav1.7. Optimization efforts culminated in compound 52, which demonstrated pharmacokinetic properties appropriate for in vivo testing in rats. The binding site of compound 52 on Nav1.7 was determined to be distinct from that of local anesthetics. Compound 52 inhibited tetrodotoxin-sensitive sodium channels recorded from rat sensory neurons and exhibited modest selectivity against the hERG potassium channel and against cloned and native tetrodotoxin-resistant sodium channels. Upon oral administration to rats, compound 52 produced dose- and exposure-dependent efficacy in the formalin model of pain.

Preclinical evaluation of AMG 900, a novel potent and highly selective pan-aurora kinase inhibitor with activity in taxane-resistant tumor cell lines.

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.

Discovery of a potent, selective, and orally bioavailable pyridinyl-pyrimidine phthalazine aurora kinase inhibitor.

The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.

Discovery of alpha-amidosulfones as potent and selective agonists of CB2: synthesis, SAR, and pharmacokinetic properties.

A series of alpha-amidosulfones were found to be potent and selective agonists of CB(2). The discovery, synthesis, and structure-activity relationships of this series of agonists are reported. In addition, the pharmacokinetic properties of the most promising compounds are profiled.

Pyridyl-pyrimidine benzimidazole derivatives as potent, selective, and orally bioavailable inhibitors of Tie-2 kinase.

Selective small molecule inhibitors of Tie-2 kinase are important tools for the validation of Tie-2 signaling in pathological angiogenesis. Reported herein is the optimization of a nonselective scaffold into a potent and highly selective inhibitor of Tie-2 kinase.