Kenneth L. Arrington

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.

An Inhibitor of the Kinesin Spindle Protein Activates the Intrinsic Apoptotic Pathway Independently of p53 and De Novo Protein Synthesis

ABSTRACT The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.

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.

Novel inhibitors of checkpoint kinase 1.

To date, DNA-damaging chemotherapeutic agents constitute a basic tool set in the treatment of cancer. Whereas a number of such drugs have achieved widespread use, their shortcomings are well documented and include toxicity associated with lack of selectivity for tumors over normal proliferating cells, as well as efficacy limitations. Thus, widening the therapeutic window of DNA-damaging chemotherapies would lead to significant improvement in cancer care. Ideally, a dividing tumor cell would progress into mitosis after sustaining DNA damage inflicted by chemotherapy and subsequently undergo mitotic catastrophe and apoptosis. However, cells have the ability to halt the cell cycle in G1, G2, or S phases and pause to allow for DNA repair. Temporary arrest of the progression of the cell cycle therefore provides a survival mechanism for tumor cells and can be mediated through both p53 and Chk1 pathway activation. Whereas normal proliferating cells have an intact p53 pathway, it is compromised in many tumors, and for these Chk1-mediated G2/S arrest becomes a dominant defense mechanism from DNA-damaging chemotherapy. Inhibition of Chk1 in such p53-deficient tumor cells with damaged DNA would abrogate the cell-cycle arrest and force the progression into mitosis resulting in cell death, thus selectively sensitizing these tumor types to chemotherapy. Accordingly, combination therapy comprising a DNA-damaging agent with a Chk1 inhibitor will potentially have a significantly higher therapeutic index than chemotherapy alone. Such realizations have driven great interest in the discovery of selective inhibitors of Chk1 kinase with a number of pharmaceutical companies and academic research labs contributing to the effort. This minireview will focus on the challenges and recent progress achieved in this area from a medicinal chemistry perspective. In a majority of publications, inhibition of Chk1 enzymatic activity serves as a primary assay for compound screening. This can be followed by a number of experiments designed to evaluate functional activity in a cellular context, which typically measure effects of compound administration on either checkpoint abrogation or antiproliferative activity of a DNA-damaging agent. Cell cycle profile (FACS) analysis has been employed to observe changes in cell distribution from abrogation of checkpoint arrest and apoptosis following administration of a DNA damaging agent and Chk1 inhibitor. A similar observation is made in the checkpoint escape assay which utilizes an immunohistochemistry approach to measure the extent of mitotic marker expression. Antiproliferative effects of Chk1 inhibitors have been investigated with inhibition of colony formation or cell viability (for example, MTS) assays demonstrating sensitization of tumor cell lines to a DNA-damaging agent. Finally, measurement of Chk1 autophosphorylation provides a direct readout of a compound’s inhibitory activity at the cellular level. In recent years (2005–2007) both enzymatic and functional activity data have become available for novel Chk1 inhibitors belonging to a number of structurally distinct series. Among these promising series, the diaryl ureas, fusedpyrazoles, quinolinones, aminopyrimidines, and others, are described below.

Pyridyl aminothiazoles as potent inhibitors of Chk1 with slow dissociation rates.

Pyridyl aminothiazoles comprise a novel class of ATP-competitive Chk1 inhibitors with excellent inhibitory potential. Modification of the core with ethylenediamine amides provides compounds with low picomolar potency and very high residence times. Investigation of binding parameters of such compounds using X-ray crystallography and molecular dynamics simulations revealed multiple hydrogen bonds to the enzyme backbone as well as stabilization of the conserved water molecules network in the hydrophobic binding region.

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.

Pyridyl aminothiazoles as potent Chk1 inhibitors: Optimization of cellular activity

Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.