Our previous high throughput screening HTS campaign

Our previous high-throughput screening (HTS) campaign using automated, robust, and sensitive fluorescence based PP242 sale assay [10], [11] led to the identification of several EPAC specific inhibitors (ESIs), and was subsequently followed by extensive hit-to-lead optimizations [20], [21], [22], [23], [24]. Among these identified inhibitor hits, ESI-09 (1, Fig. 1) has been shown to selectively inhibit EPAC functions in vitro[12] and in vivo[13], [14]. With the aid of molecular docking studies of 1 into the cAMP binding domain B of active EPAC2 proteins, we hypothesized that binding interactions of inhibitors to EPAC2 proteins may primarily occur through two terminal hydrophobic pockets (P1 and P2) and the unique linker [7]. Later, systematic structure−activity relationships (SARs) studies were performed, leading to the discovery of several more active EPAC antagonists (e.g., 2 (NY0123)) with low micromolar inhibitory activity and improved solubility [24]. In a continuing effort to develop novel diversified analogues based on the scaffold of hit 1, we focus on our further chemical optimizations involving modifications of 5-tert-butyl group on the isoxazole ring A, meanwhile retaining favorable hydrophobic fragments of EPAC antagonists including fluorine-substitutions on the B-ring identified from our previous studies [24]. In order to explore the depth of the aforementioned hydrophobic pocket P2, as depicted in Fig. 1, series I was designed by inserting a rigid phenyl ring between the isoxazole A ring and its tert-butyl substitution at the 5-position. For comparison, we also attempted to make the molecular skeleton more compacted by fusing a phenyl ring with the isoxazole A (as depicted in series II, Fig. 1). Herein, we report such structural modifications of compound 1 with a focus on improving EPAC inhibitory activities and structural diversity of EPAC antagonists. The studies have resulted in the discovery of several novel potent EPAC antagonists such as 14 (NY0460), 26 (NY0725), 32 (NY0561), and 33 (NY0562), with low micromolar inhibitory activities for preclinical development.
Results and discussion
Conclusions Two series of novel EPAC antagonists based on the scaffold of the previously identified high-throughput hit 1 (ESI-09) have been designed, synthesized, and biologically evaluated for their EPAC1 and EPAC2 inhibitory activities. The SAR results based on EPAC2 activity comply with our docking studies in general, indicating that the isoxazole ring B of 1 can tolerate chemical modifications with either introduction of flexible electron-donating substitutions or structurally restrictedly fusing with a phenyl ring. The new scaffold of series II, as in compound 33 interacting with EPAC2 in a novel binding mode, may offer a good starting point for further drug design and structural optimizations. All these modification efforts 2allow us to further tune the original hit 1 to achieve more potent and structurally diverse EPAC1 and EPAC2 inhibitors, such as 10 (NY0617), 14 (NY0460), 26 (NY0725), 32 (NY0561), and 33 (NY0562) with IC50 values in the low micromolar range. These compounds may hold promise as potential drug candidates toward novel therapeutics against human diseases, and serve as valuable pharmacological probes to elucidate the physiological functions of EPAC proteins. Currently, the in vitro and in vivo activities of these selected compounds in infectious disease models (e.g. rickettsiosis) are being investigated. Further systematic optimizations based upon identified new scaffolds of these two series toward EPAC subtype selectivity are also under way and the findings will be reported in due course.
Experimental section
Notes
Acknowledgment This work was supported by grants R01 GM106218, R01 AI111464, and R01 GM066170 from the National Institutes of Health. We want to thank Drs. Lawrence C. Sowers and Cheryl F. Lichti at the Department of Pharmacology as well as Dr. Tianzhi Wang at the NMR core facility of UTMB for the NMR spectroscopy assistance.