Kathleen E. Coll

Synthesis and initial SAR studies of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines: A new class of KDR kinase inhibitors

We have synthesized and evaluated the activity of 3,6-disubstituted pyrazolo[1,5-a]pyrimidines as a new class of KDR kinase inhibitors. Starting with screening lead 1, potency against isolated KDR was fully optimized with 3-thienyl and 4-methoxyphenyl substituents at the 6- and 3-positions (3g, KDR IC(50)=19 nM), respectively. The synthesis and SAR of these compounds are described.

Optimization of a pyrazolo[1,5-a]pyrimidine class of KDR kinase inhibitors: improvements in physical properties enhance cellular activity and pharmacokinetics.

We have introduced solubilizing functionality to a 3,6-disubstituted pyrazolo[1,5-a]pyrimidine series of KDR kinase inhibitors to improve the physical properties of these compounds. The addition of a basic side-chain to the 6-aryl ring, introduction of 3-pyridyl groups, and most significantly, incorporation of a 4-pyridinonyl substituent at the 6-position of the core are modifications that maintain and often enhance the intrinsic potency of this class of inhibitors. Moreover, the improvements in physical properties result in marked increases in cellular activity and more favorable pharmacokinetics in rats. The synthesis and SAR of these compounds are described.

Dissociation of Phospholamban Regulation of Cardiac Sarcoplasmic Reticulum Ca2+ATPase by Quercetin

Quercetin had a biphasic effect on Ca2+ uptake and calcium-stimulated ATP hydrolysis in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was observed at low quercetin concentrations (<25 μM) followed by inhibition at higher concentrations. The effects were dependent upon the SR protein concentration, the MgATP concentration, and intact phospholamban regulation of cardiac Ca2+ATPase. Only the inhibitory effects at higher quercetin concentrations were observed in skeletal muscle SR which lacks phospholamban and in cardiac SR treated to remove phospholamban regulation. Stimulation was additive with monoclonal antibody 1D11 (directed against phospholamban) at submaximal antibody concentrations; however, the maximal antibody and quercetin stimulation were identical. Quercetin increased the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. In addition, low concentrations of quercetin increased the steady-state formation of phosphoenzyme from ATP or Pi, but higher quercetin decreased phosphoenzyme levels. Quercetin, even under stimulatory conditions, was a competitive inhibitor of ATP, but appears to relieve the Ca2+ATPase from phospholamban inhibition, thereby, producing an activation. The subsequent inhibitory action of higher quercetin concentrations results from competition of quercetin with the nucleotide binding site of the Ca2+ATPase. The data suggest that quercetin interacts with the nucleotide binding site to mask phospholambans inhibition of the SR Ca2+ATPase and suggests that phospholamban may interact at or near the nucleotide binding site.

A novel orally bioavailable inhibitor of kinase insert domain-containing receptor induces antiangiogenic effects and prevents tumor growth in vivo.

A strategy for antagonizing vascular endothelial growth factor (VEGF) -induced angiogenesis is to inhibit the kinase activity of its receptor, kinase insert domain-containing receptor (KDR), the first committed and perhaps the last unique step in the VEGF signaling cascade. We synthesized a novel ATP-competitive KDR tyrosine kinase inhibitor that potently suppresses human and mouse KDR activity in enzyme (IC50 = 7.8–19.5 nm) and cell-based assays (IC50 = 8 nm). The compound was bioavailable in vivo, leading to a dose-dependent decrease in basal- and VEGF-stimulated KDR tyrosine phosphorylation in lungs from naïve and tumor-bearing mice (IC50 = 23 nm). Pharmacokinetics and pharmacodynamics guided drug dose selection for antitumor efficacy studies. HT1080 nude mice xenografts were treated orally twice daily with vehicle, or 33 or 133 mg/kg of compound. These doses afforded trough plasma concentrations approximately equal to the IC50 for inhibition of KDR autophosphorylation in vivo for the 33 mg/kg group, and higher than the IC99 for the 133 mg/kg group. Chronic treatment at these doses was well-tolerated and resulted in dose-dependent inhibition of tumor growth, decreased tumor vascularization, decreased proliferation, and enhanced cell death. Antitumor efficacy correlated with inhibition of KDR tyrosine phosphorylation in the tumor, as well as in a surrogate tissue (lung). Pharmacokinetics and pharmacodynamics assessment indicated that the degree of tumor growth inhibition correlated directly with the extent of inhibition of KDR tyrosine phosphorylation in tumor or lung at trough. These observations highlight the need to design antiangiogenic drug regimens to ensure constant target suppression and to take advantage of PD end points to guide dose selection.

Property-based design of KDR kinase inhibitors.

Small molecule inhibitors of KDR kinase activity have typically possessed poor intrinsic physical properties including low aqueous solubility and high lipophilicity. These features have often conferred limited cell permeability manifested in low levels of cell-based KDR inhibitory activity and oral bioavailability. Thus, the design of inhibitors with appropriate physical properties has played a critical role in the development of clinical candidates. We present a variety of structural modifications that have afforded improvements in physical properties and thereby have addressed suboptimal cellular potency and pharmacokinetics for three unique classes of KDR kinase inhibitors.

2,4-disubstituted pyrimidines: a novel class of KDR kinase inhibitors.

2,4-Disubstituted pyrimidines were synthesized as a novel class of KDR kinase inhibitors. Evaluation of the SAR of the screening lead compound 1 (KDR IC(50)=105 nM, Cell IC(50)=8% inhibition at 500 nM) led to the potent 3,5-dimethylaniline derivative 2d (KDR IC(50)=6 nM, cell IC(50)=19 nM).

Design and synthesis of 1,5-diarylbenzimidazoles as inhibitors of the VEGF-receptor KDR

1,5-Diarylbenzimidazoles have been identified as potent inhibitors of KDR kinase activity. The series was developed with a goal of finding compounds with optimal drug-like properties. This communication describes structural modifications in the series that enhance solubility, lower protein binding, and provide compounds with excellent potency and pharmacokinetic profiles.

Discovery and evaluation of 3-(5-Thien-3-ylpyridin-3-yl)-1H-indoles as a novel class of KDR kinase inhibitors

We have discovered 3-(5-thien-3-ylpyridin-3-yl)-1H-indoles as potent inhibitors of KDR kinase activity. This communication details the evolution of this novel class from a potent screening lead of vastly different structure with an emphasis on structural modifications that retained activity and provided improvements in key physical properties. The synthesis and in-depth evaluation of these inhibitors are described.