Discovery and biochemical characterization of selective ATP competitive inhibitors of the human mitotic kinesin KSP.

The kinesin spindle protein (KSP, also known as Eg5) is essential for the proper separation of spindle poles during mitosis, and inhibition results in mitotic arrest and the formation of characteristic monoaster spindles. Several distinct classes of KSP inhibitors have been described previously in the public and patent literature. However, most appear to share a common induced-fit allosteric binding site, suggesting a common mechanism of inhibition. In a high-throughput screen for inhibitors of KSP, a novel class of thiazole-containing inhibitors was identified. Unlike the previously described allosteric KSP inhibitors, the thiazoles described here show ATP competitive kinetic behavior, consistent with binding within the nucleotide binding pocket. Although they bind to a pocket that is highly conserved across kinesins, these molecules exhibit significant selectivity for KSP over other kinesins and other ATP-utilizing enzymes. Several of these compounds are active in cells and produce a phenotype similar to that observed with previously published allosteric inhibitors of KSP.

Stereospecific reduction of a potent kinesin spindle protein (KSP) inhibitor in human tissues.

Compound A, 1-{(3R,3aR)-3-[3-(4-acetylpiperazin-1-yl)propyl]-7-fluoro-3-phenyl-3a,4-dihydro-3H-pyrazolo[5,1-c][1,4]benzoxazin-2-yl}ethanone, is a novel and potent inhibitor of the mitotic kinesin spindle protein. Metabolism studies with human hepatocytes showed that Compound A underwent significant ketone reduction to its biologically active metabolite M1. Here, we describe the studies that characterized the metabolic interconversion between Compound A and M1 in vitro in human tissues. LC-MS/MS analysis showed that the ketone reduction was stereospecific for M1 with no diastereomer of M1 detected in incubations with human hepatocytes. Interestingly, such stereospecific ketone reduction was not observed with Compound B, the enantiomer of Compound A. No reductive products were observed when Compound B was incubated with human hepatocytes. Studies with human liver subcellular fractions showed that Compound A was reduced to M1 primarily by human liver cytosol with little contribution from human liver microsomes and mitochondria. NADPH was the preferred cofactor for the reduction reaction. Reverse oxidation of M1 back to Compound A was also observed, preferentially in human liver cytosol with NADP(+) as the cofactor. The interconversion between Compound A and M1 in human liver cytosol was inhibited significantly by flufenamic acid and phenolphthalein (potent inhibitors for aldo-keto reductase 1Cs, p<0.05), but not by menadione, a selective inhibitor for carbonyl reductase. In addition to the liver, S9 from human lung and kidney was also capable of catalyzing this interconversion. Collectively, the results implicated the aldo-keto reductase 1Cs as the most likely enzymes responsible for the metabolic interconversion of Compound A and its active metabolite M1.

Optimization of a pyrazoloquinolinone class of Chk1 kinase inhibitors.

The development of 2,5-dihydro-4H-pyrazolo[4,3-c]quinolin-4-ones as inhibitors of Chk1 kinase is described. Introduction of a fused ring at the C7/C8 positions of the pyrazoloquinolinone provided an increase in potency while guidance from overlapping inhibitor bound Chk1 X-ray crystal structures contributed to the discovery of a potent and solubilizing propyl amine moiety in compound 52 (Chk1 IC(50)=3.1 nM).

Synthesis and evaluation of substituted benzoisoquinolinones as potent inhibitors of Chk1 kinase.

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.

Kinesin spindle protein (KSP) inhibitors. Part 7: Design and synthesis of 3,3-disubstituted dihydropyrazolobenzoxazines as potent inhibitors of the mitotic kinesin KSP.

Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.

Kinesin spindle protein (KSP) inhibitors. Part 2: the design, synthesis, and characterization of 2,4-diaryl-2,5-dihydropyrrole inhibitors of the mitotic kinesin KSP.

The evolution of 2,4-diaryl-2,5-dihydropyrroles as inhibitors of KSP is described. Introduction of basic amide and urea moieties to the dihydropyrrole nucleus enhanced potency and aqueous solubility, simultaneously, and provided compounds that caused mitotic arrest of A2780 human ovarian carcinoma cells with EC(50)s<10nM. Ancillary hERG activity was evaluated for this series of inhibitors.

Kinesin spindle protein (KSP) inhibitors. Part 3: synthesis and evaluation of phenolic 2,4-diaryl-2,5-dihydropyrroles with reduced hERG binding and employment of a phosphate prodrug strategy for aqueous solubility.

2,4-Diaryl-2,5-dihydropyrroles have been discovered to be novel, potent and water-soluble inhibitors of KSP, an emerging therapeutic target for the treatment of cancer. A potential concern for these basic KSP inhibitors (1 and 2) was hERG binding that can be minimized by incorporation of a potency-enhancing C2 phenol combined with neutral N1 side chains. Aqueous solubility was restored to these, and other, non-basic inhibitors, through a phosphate prodrug strategy.

Design and synthesis of 3,7-diarylimidazopyridines as inhibitors of the VEGF-receptor KDR.

3,7-Diarylsubstituted imidazopyridines were designed and developed as a new class of KDR kinase inhibitors. A variety of imidazopyridines were synthesized and potent inhibitors of KDR kinase activity were identified with good aqueous solubility.