Min-Hwa Jasmine Lin

Discovery and optimization of potent and selective triazolopyridazine series of c-Met inhibitors

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.

Relationship between Passive Permeability, Efflux, and Predictability of Clearance from In Vitro Metabolic Intrinsic Clearance

In vitro intrinsic metabolic clearance (CLint) is used routinely for compound selection in drug discovery; however, in vitro CLint often underpredicts in vivo clearance (CL). Forty-one proprietary compounds and 16 marketed drugs were selected to determine whether permeability and efflux status could influence the predictability of CL from in vitro CLint obtained from liver microsomal and hepatocyte incubations. For many of the proprietary compounds examined, rat CL was significantly underpredicted using the well stirred model incorporating both fraction of unbound drug in blood and fraction of unbound drug in the microsomal or hepatocyte incubation. Further analysis revealed that the accuracy of the prediction was differentiated by permeability and P-glycoprotein- (P-gp) and mouse breast cancer resistance protein (mBcrp)-mediated efflux. For proprietary compounds with passive permeability greater than 5 × 10−6 cm/s and efflux ratios less than 5 in both P-gp- and mBcrp-expressing cells, CLint provided reasonable prediction. The average -fold error (AFE) was 1.8 for rat liver microsomes (RLMs) and 2.3 for rat hepatocytes. In contrast, CL was dramatically underpredicted for compounds with passive permeability less than 5 × 10−6 cm/s; AFEs of 54.4 and 29.2 were observed for RLM and rat hepatocytes, respectively. In vivo CL was also underpredicted for compounds that were good efflux substrates (permeability >5 × 10−6 cm/s). The AFEs were 7.4 and 8.1 for RLM and rat hepatocytes, respectively. A similar relationship between permeability, efflux status, and human CL prediction reported in the literature was observed for 16 marketed drugs. These data show that permeability and efflux status are determinants for the predictability of CL from in vitro metabolic CLint.

Prediction of Vss from In Vitro Tissue-Binding Studies

To predict volume of distribution at steady-state (Vss), empirical (e.g., allometry) and mechanistic (using physicochemical property data and plasma protein binding) methods have been used. None of these approaches has been able to predict Vss accurately for the total compliment of a wide range of drugs. Therefore, alternative approaches would be of value. This study evaluates the utility of in vitro nonspecific tissue-binding measurements in predicting Vss for a wide range of drugs in rats. Literature as well as proprietary compounds were studied. It was found that in vitro tissue-binding measurements combined with calculated effects of the pH partition hypothesis often predict Vss more accurately than other available mechanistic methods and that this approach can compliment existing methods. The Vss values for some compounds were not accurately predicted using either nonspecific tissue-binding experiments or other available mechanistic methods. The Vss for these drugs may not be describable by nonspecific tissue binding alone; there may be significant specific components to the mechanism of distribution for these drugs, such as pH-dependent uptake into lysosomes (primarily strongly basic drugs), active transport, and/or enterohepatic recirculation. A lack of prediction for certain drugs warrants further investigation into these mechanisms and their application to more accurate prediction of Vss by mechanistic means.

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.

Sulfonamides as Selective NaV1.7 Inhibitors: Optimizing Potency and Pharmacokinetics While Mitigating Metabolic Liabilities

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. The optimization of a series of internal NaV1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.

Sulfonamides as Selective NaV1.7 Inhibitors: Optimizing Potency, Pharmacokinetics, and Metabolic Properties to Obtain Atropisomeric Quinolinone (AM-0466) that Affords Robust in Vivo Activity

Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.

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.

Discovery of (R)-6-(1-(8-Fluoro-6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-a]pyridin-3-yl)ethyl)-3-(2-methoxyethoxy)-1,6-naphthyridin-5(6H)-one (AMG 337), a Potent and Selective Inhibitor of MET with High Unbound Target Coverage and Robust In Vivo Antitumor Activity.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

In vitro and in vivo pharmacokinetic characterizations of AMG 900, an orally bioavailable small molecule inhibitor of aurora kinases

AMG 900 is a small molecule being developed as an orally administered, highly potent, and selective pan-aurora kinase inhibitor. The aim of the investigations was to characterize in vitro and in vivo pharmacokinetic (PK) properties of AMG 900 in preclinical species. AMG 900 was rapidly metabolized in liver microsomes and highly bound to plasma proteins in the species tested. It was a weak Pgp substrate with good passive permeability. AMG 900 exhibited a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4 h. AMG 900 was well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. Food intake had an effect on rate (rats) or extent (dogs) of AMG 900 oral absorption. The clearance and volume of distribution at steady state in humans were predicted to be 27.3 mL/h/kg and 93.9 mL/kg, respectively. AMG 900 exhibited acceptable PK properties in preclinical species and was predicted to have low clearance in humans. AMG 900 is currently in Phase I clinical testing as a treatment for solid tumours. Preliminary human PK results appear to be consistent with the predictions.

Discovery of Potent and Selective 8-Fluorotriazolopyridine c-Met Inhibitors

The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.