Sharadha Subramanian

Design and synthesis of 5,6-fused heterocyclic amides as Raf kinase inhibitors.

Two scaffolds based on 5,6-fused heterocyclic backbones were designed and synthesized as Raf kinase inhibitors. The scaffolds were assessed for in vitro pan-Raf inhibition, activity in cell proliferation and target modulation assays, and pharmacokinetic parameters.

2-Amino-7-substituted benzoxazole analogs as potent RSK2 inhibitors.

2-Amino-7-substituted benzoxazole analogs were identified by HTS as inhibitors of RSK2. Molecular modeling and medicinal chemistry techniques were employed to explore the SAR for this series with a focus of improving in vitro and target modulation potency and physicochemical properties.

Design and Discovery of N-(2-Methyl-5′-morpholino-6′-((tetrahydro-2H-pyran-4-yl)oxy)-[3,3′-bipyridin]-5-yl)-3-(trifluoromethyl)benzamide (RAF709): A Potent, Selective, and Efficacious RAF Inhibitor Targeting RAS Mutant Cancers

RAS oncogenes have been implicated in >30% of human cancers, all representing high unmet medical need. The exquisite dependency on CRAF kinase in KRAS mutant tumors has been established in genetically engineered mouse models and human tumor cells. To date, many small molecule approaches are under investigation to target CRAF, yet kinase-selective and cellular potent inhibitors remain challenging to identify. Herein, we describe 14 (RAF709) [ Aversa , Biaryl amide compounds as kinase inhibitors and their preparation . WO 2014151616, 2014 ], a selective B/C RAF inhibitor, which was developed through a hypothesis-driven approach focusing on drug-like properties. A key challenge encountered in the medicinal chemistry campaign was maintaining a balance between good solubility and potent cellular activity (suppression of pMEK and proliferation) in KRAS mutant tumor cell lines. We investigated the small molecule crystal structure of lead molecule 7 and hypothesized that disruption of the crystal packing would improve solubility, which led to a change from N-methylpyridone to a tetrahydropyranyl oxy-pyridine derivative. 14 proved to be soluble, kinase selective, and efficacious in a KRAS mutant xenograft model.

Design and synthesis of potent RSK inhibitors

Utilizing the already described 3,4-bi-aryl pyridine series as a starting point, incorporation of a second ring system with a hydrogen bond donor and additional hydrophobic contacts yielded the azaindole series which exhibited potent, picomolar RSK2 inhibition and the most potent in vitro target modulation seen thus far for a RSK inhibitor. In the context of the more potent core, several changes at the phenol moiety were assessed to potentially find a tool molecule appropriate for in vivo evaluation.

Abstract B38: Inhibiting mutated KRAS, a broken switch of effector pathways

Mutated forms of KRAS are no longer able to switch effectors between “on” and “off” states. It is known that the function of KRAS is controlled by key parts in the C-terminus, including six consecutive lysines, a terminal prenyl moiety and a terminal carboxymethyl functional group. We set out to discover compounds which would inhibit the function of mutated KRAS as an activator for effectors. This campaign yielded several compounds that blocked biochemical and cellular functions of KRAS with low micromolar activity while not affecting markers outside of KRAS pathways in cells. In order to understand the mode of binding of these compounds to KRAS, we generated different forms of the protein, including unprenylated truncated and fully processed full-length protein. NMR studies with truncated protein (amino acids 1-169) identified a site at which compound binding stabilized the inactive conformation of KRAS. This site is located adjacent to switch-II and is similar to sites described by others. The Kd determined for this binding event is almost 3 orders of magnitude higher than the IC50 and EC50 values measured in biochemical and cellular assays. In order to understand this difference, we developed a biophysical assay using the Fortebio system which enabled binding studies in a system with full-length prenylated protein in the presence of lipids, to match the context of the biochemical and cellular assays. Micromolar binding to the full-length prenylated KRAS protein was observed in the Fortebio assay and binding was not observed in the absence of prenylation, consistent with the near millimolar Kd observed by NMR for truncated KRAS. Curiously, similar micromolar binding was seen to a peptide derived from the C-terminus of KRAS (amino acids 168-185) with and without prenyl modification while related compounds that do not bind to the full-length prenylated KRAS also do not bind to these peptides. It is still unclear whether binding to the terminal peptide in lipid context is related to the binding site adjacent to switch-II. From a drug discovery perspective, it remains to be confirmed whether current inhibitors can be optimized. Citation Format: Johanna Jansen, Wolfgang Jahnke, Susan Fong, Laura Tandeske, Charles Wartchow, Keith Pfister, Tatiana Zavorotinskaya, Anke Blechschmidt, Dirksen Bussiere, Yumin Dai, Jeff Dove, Eric Fang, David Farley, Jean-Michel Florent, John Fuller, Simona Gokhin, Alvar Gossert, Mohammad Hekmat-Nejad, Chrystele Henry, Julia Klopp, Bill Lenahan, Andreas Lingel, Arndt Meyer, Jamie Narberes, Gwynn Pardee, C Gregory Paris, Savithri Ramurthy, Paul Renhowe, Sebastien Rieffel, Kevin Shoemaker, Sharadha Subramanian, Tiffany Tsang, Stephania Widger, Armin Widmer, Isabel Zaror, Stephen Hardy. Inhibiting mutated KRAS, a broken switch of effector pathways. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B38. doi: 10.1158/1557-3125.RASONC14-B38