Discovery of potent and selective spiroindolinone MDM2 inhibitor, RO8994, for cancer therapy.

The field of small-molecule inhibitors of protein-protein interactions is rapidly advancing and the specific area of inhibitors of the p53/MDM2 interaction is a prime example. Several groups have published on this topic and multiple compounds are in various stages of clinical development. Building on the strength of the discovery of RG7112, a Nutlin imidazoline-based compound, and RG7388, a pyrrolidine-based compound, we have developed additional scaffolds that provide opportunities for future development. Here, we report the discovery and optimization of a highly potent and selective series of spiroindolinone small-molecule MDM2 inhibitors, culminating in RO8994.

Pyrido[2,3-d]pyrimidines: discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors.

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.

Pyrrolopyrazines as selective spleen tyrosine kinase inhibitors.

We describe the discovery of several pyrrolopyrazines as potent and selective Syk inhibitors and the efforts that eventually led to the desired improvements in physicochemical properties and human whole blood potencies. Ultimately, our mouse model revealed unexpected toxicity that precluded us from further advancing this series.

Discovery of a highly potent, orally active mitosis/angiogenesis inhibitor r1530 for the treatment of solid tumors.

A new series of 7,8-disubstituted pyrazolobenzodiazepines based on the lead compound 1 have been synthesized and evaluated for their effects on mitosis and angiogenesis. Described herein is the design, synthesis, SAR, and antitumor activity of these compounds leading to the identification of R1530, which was selected for clinical evaluation.

Rational design of highly selective spleen tyrosine kinase inhibitors.

A novel approach to design selective spleen tyrosine kinase (Syk) inhibitors is described. Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of autoimmune diseases such as asthma, rheumatoid arthritis, and SLE. Fostamatinib, a Syk inhibitor that successfully completed phase II clinical trials, also exhibits some undesirable side effects. More selective Syk inhibitors could offer safer, alternative treatments. Through a systematic evaluation of the kinome, we identified Pro455 and Asn457 in the Syk ATP binding site as a rare combination among sequence aligned kinases and hypothesized that optimizing the interaction between them and a Syk inhibitor molecule would impart high selectivity for Syk over other kinases. We report the structure-guided identification of three series of selective spleen tyrosine kinase inhibitors that support our hypothesis and offer useful guidance to other researchers in the field.

Identification of novel, potent and selective inhibitors of Polo-like kinase 1.

A series of pyrimidodiazepines was identified as potent Polo-like kinase 1 (PLK1) inhibitors. The synthesis and SAR are discussed. The lead compound 7 (RO3280) has potent inhibitory activity against PLK1, good selectivity against other kinases, and excellent in vitro cellular potency. It showed strong antitumor activity in xenograft mouse models.

Inactivation of mirk/dyrk1b kinase targets quiescent pancreatic cancer cells.

A major problem in the treatment of cancer arises from quiescent cancer cells that are relatively insensitive to most chemotherapeutic drugs and radiation. Such residual cancer cells can cause tumor regrowth or recurrence when they reenter the cell cycle. Earlier studies showed that levels of the serine/theronine kinase Mirk/dyrk1B are elevated up to 10-fold in quiescent G(0) tumor cells. Mirk uses several mechanisms to block cell cycling, and Mirk increases expression of antioxidant genes that decrease reactive oxygen species (ROS) levels and increase quiescent cell viability. We now show that a novel small molecule Mirk kinase inhibitor blocked tumor cells from undergoing reversible arrest in a quiescent G(0) state and enabled some cells to exit quiescence. The inhibitor increased cycling in Panc1, AsPc1, and SW620 cells that expressed Mirk, but not in HCT116 cells that did not. Mirk kinase inhibition elevated ROS levels and DNA damage detected by increased phosphorylation of the histone protein H2AX and by S-phase checkpoints. The Mirk kinase inhibitor increased cleavage of the apoptotic proteins PARP and caspase 3, and increased tumor cell kill several-fold by gemcitabine and cisplatin. A phenocopy of these effects occurred following Mirk depletion, showing drug specificity. In previous studies Mirk knockout or depletion had no detectable effect on normal tissue, suggesting that the Mirk kinase inhibitor could have a selective effect on cancer cells expressing elevated levels of Mirk kinase.

Biological evaluation of a multi-targeted small molecule inhibitor of tumor-induced angiogenesis.

RO4396686 is a small molecule KDR, FGFR, and PDGFR inhibitor with good pharmacokinetic properties in rodents. In a mouse corneal neovascularization assay, this compound inhibited VEGF-induced angiogenesis. Tested in a H460a xenograft tumor model this agent effected significant tumor growth inhibition at doses as low as 50mg/kg.

RO4383596, an orally active KDR, FGFR, and PDGFR inhibitor: synthesis and biological evaluation.

(+/-)-1-(anti-3-Hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido[4,5-d]pyrimidin-2-one (RO4383596) is a potent and selective inhibitor of the pro-angiogenic receptor tyrosine kinases KDR, FGFR, and PDGFR. This agent has an excellent pharmacokinetic profile and is highly efficacious in rodent models of angiogenesis upon oral administration.

A new series of potent oxindole inhibitors of CDK2.

A novel series of oxindole-type inhibitors of CDK2 that have heteroatom substituted alkynyl moieties at their C-4 position is described. These novel 4-alkynyl-substituted inhibitors have superior potency relative to their parent compound in free enzyme and in cell based assays. The crystal structure of CDK2 in complex with one of these analogues was determined and gives insight to their increased potency. The biochemical evaluation of a representative derivative is also described.

3,5,6-Trisubstituted naphthostyrils as CDK2 inhibitors.

A novel class of 3,5,6-trisubstituted naphthostyril analogues was designed and synthesized to study the structure-activity relationship for inhibition of cyclin-dependent kinase 2 (CDK2). These compounds, particularly molecules with side-chain modifications providing additional hydrogen bonding capability, were demonstrated to be potent CDK2 inhibitors with cellular activities consistent with CDK2 inhibition. These molecules inhibited tumor cell proliferation and G1-S and G2-M cell-cycle progression in vitro. The X-ray crystal structure of a 2-aminoethyleneamine derivative bound to CDK2, refined to 2.5A resolution, is presented.

Synthesis of potent oxindole CDK2 inhibitors.

A series of oxindole CDK2 inhibitors was synthesized. These novel analogues have a saturated monosubstituted cyclic moiety at their C-4 position that mimics the ribofuranoside of ATP. This substitution afforded agents with increased potency relative to the parent indolinone and nanomolar range IC(50) against the CDK2 enzyme and two cancer cell lines.