Discovery of 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine inhibitors of Erk2.

The discovery and optimization of a series of tetrahydropyridopyrimidine based extracellular signal-regulated kinase (Erks) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent and selective inhibition of Erk2 and knockdown of phospho-RSK levels in HepG2 cells and tumor xenografts.

Discovery of 5-Azaquinoxaline Derivatives as Potent and Orally Bioavailable Allosteric SHP2 Inhibitors.

SHP2 has emerged as an important target for oncology small-molecule drug discovery. As a nonreceptor tyrosine phosphatase within the MAPK pathway, it has been shown to control cell growth, differentiation, and oncogenic transformation. We used structure-based design to find a novel class of potent and orally bioavailable SHP2 inhibitors. Our efforts led to the discovery of the 5-azaquinoxaline as a new core for developing this class of compounds. Optimization of the potency and properties of this scaffold generated compound 30, that exhibited potent in vitro SHP2 inhibition and showed excellent in vivo efficacy and pharmacokinetic profile.

Anti-tumor efficacy of a potent and selective non-covalent KRASG12D inhibitor.

Recent progress in targeting KRASG12C has provided both insight and inspiration for targeting alternative KRAS mutants. In this study, we evaluated the mechanism of action and anti-tumor efficacy of MRTX1133, a potent, selective and non-covalent KRASG12D inhibitor. MRTX1133 demonstrated a high-affinity interaction with GDP-loaded KRASG12D with KD and IC50 values of ~0.2 pM and <2 nM, respectively, and ~700-fold selectivity for binding to KRASG12D as compared to KRASWT. MRTX1133 also demonstrated potent inhibition of activated KRASG12D based on biochemical and co-crystal structural analyses. MRTX1133 inhibited ERK1/2 phosphorylation and cell viability in KRASG12D-mutant cell lines, with median IC50 values of ~5 nM, and demonstrated >1,000-fold selectivity compared to KRASWT cell lines. MRTX1133 exhibited dose-dependent inhibition of KRAS-mediated signal transduction and marked tumor regression (≥30%) in a subset of KRASG12D-mutant cell-line-derived and patient-derived xenograft models, including eight of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models. Pharmacological and CRISPR-based screens demonstrated that co-targeting KRASG12D with putative feedback or bypass pathways, including EGFR or PI3Kα, led to enhanced anti-tumor activity. Together, these data indicate the feasibility of selectively targeting KRAS mutants with non-covalent, high-affinity small molecules and illustrate the therapeutic susceptibility and broad dependence of KRASG12D mutation-positive tumors on mutant KRAS for tumor cell growth and survival.

Non-oxime pyrazole based inhibitors of B-Raf kinase.

The synthesis and biological evaluation of non-oxime pyrazole based B-Raf inhibitors is reported. Several oxime replacements have been prepared and have shown excellent enzyme activity. Further optimization of fused pyrazole 2a led to compound 38, a selective and potent B-Raf inhibitor.

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.

Non-oxime inhibitors of B-Raf(V600E) kinase.

The development of inhibitors of B-Raf(V600E) serine-threonine kinase is described. Various head-groups were examined to optimize inhibitor activity and ADME properties. Several of the head-groups explored, including naphthol, phenol and hydroxyamidine, possessed good activity but had poor pharmacokinetic exposure in mice. Exposure was improved by incorporating more metabolically stable groups such as indazole and tricyclic pyrazole, while indazole could also be optimized for good cellular activity.

The discovery of furo[2,3-c]pyridine-based indanone oximes as potent and selective B-Raf inhibitors.

Virtual and high-throughput screening identified imidazo[1,2-a]pyrazines as inhibitors of B-Raf. We describe the rationale, SAR, and evolution of the initial hits to a series of furo[2,3-c]pyridine indanone oximes as highly potent and selective inhibitors of B-Raf.

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 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.

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.

Potent and selective pyrazole-based inhibitors of B-Raf kinase.

Herein we describe a novel pyrazole-based class of ATP competitive B-Raf inhibitors. These inhibitors exhibit both excellent cellular potency and striking B-Raf selectivity. A subset of these inhibitors has demonstrated the ability to inhibit downstream ERK phosphorylation in LOX tumors from mouse xenograft studies.

Discovery of (S)-1-(1-(4-Chloro-3-fluorophenyl)-2-hydroxyethyl)-4-(2-((1-methyl-1H-pyrazol-5-yl)amino)pyrimidin-4-yl)pyridin-2(1H)-one (GDC-0994), an Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) Inhibitor in Early Clinical Development.

The extracellular signal-regulated kinases ERK1/2 represent an essential node within the RAS/RAF/MEK/ERK signaling cascade that is commonly activated by oncogenic mutations in BRAF or RAS or by upstream oncogenic signaling. While targeting upstream nodes with RAF and MEK inhibitors has proven effective clinically, resistance frequently develops through reactivation of the pathway. Simultaneous targeting of multiple nodes in the pathway, such as MEK and ERK, offers the prospect of enhanced efficacy as well as reduced potential for acquired resistance. Described herein is the discovery and characterization of GDC-0994 (22), an orally bioavailable small molecule inhibitor selective for ERK kinase activity.

Discovery of highly potent, selective, and efficacious small molecule inhibitors of ERK1/2.

Using structure-based design, a novel series of pyridone ERK1/2 inhibitors was developed. Optimization led to the identification of (S)-14k, a potent, selective, and orally bioavailable agent that inhibited tumor growth in mouse xenograft models. On the basis of its in vivo efficacy and preliminary safety profiles, (S)-14k was selected for further preclinical evaluation.

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 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.