Identification of novel 2-(benzo[ d ]isoxazol-3-yl)-2-oxo- N -phenylacetohydrazonoyl cyanide analoguesas potent EPAC antagonists

Abstract

Two series of novel EPAC antagonists are designed, synthesized and evaluated in an effort to develop diversified analogues based on the scaffold of the previously identified high-throughput (HTS) hit 1 (ESI-09). Further SAR studies reveal that the isoxazole ring A of 1 can tolerate chemical modifications with either introduction of flexible electron-donating substitutions or structurally restrictedly fusing with a phenyl ring, leading to identification of several more potent and diversified EPAC antagonists (e.g., 10 (NY0617), 14 (NY0460), 26 (NY0725), 32 (NY0561), and 33 (NY0562)) with low micromolar inhibitory activities. Molecular docking studies on compounds 10 and 33 indicate that these two series of compounds bind at a similar site with substantially different interactions with the EPAC proteins. The findings may serve as good starting points for the development of more potent EPAC antagonists as valuable pharmacological probes or potential drug candidates.

The Potential of a Novel Class of EPAC-Selective Agonists to Combat Cardiovascular Inflammation

“…Subsequently, concerns were raised over the specificity of ESI-09, which is thought to display non-specific protein denaturing properties [ 104 ]. Despite this, several more potent EPAC antagonistic ESI-09 and ESI-05 analogues ( Figure 4 ) have been developed [ 105 , 106 , 107 ].…”

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Not surprisingly, the majority of hit compounds from the HTS perform as pan competitive EPAC antagonists inhibiting cAMP-induced activation of both EPAC1 and EPAC2 (17,1043). Improved chemical synthesis and structure-activity relationship (SAR) analysis have resulted in the identification of more potent dual EPAC antagonists (165,167,1162,1163,1210). In particular, structural optimization of hit ESI-09 reveals that the 3-, 4-, and 5-positions of the phenyl scaffold and the 5-position of the isoxazole moiety are highly tunable for improving compound potency.…”

“…A simple addition of a Cl atom at the 5-position of the phenyl ring leads to a fivefold improvement in potency, and another Cl addition at the 4-position generates the potent compound NY0123 with a relative binding affinity more than 35-fold higher than cAMP (1162,1210). On the other hand, the isoxazole ring can tolerate chemical modifications with either introduction of flexible electron-donating substitutions or structurally restrictive fusing with a phenyl ring to generate potent dual inhibitors with apparent IC 50 values in the low micromolar range (1163). These SAR data are consistent with the molecular modeling results illustrating that while the isoxazole moiety docks into a hydrophobic pocket and interacts with residues Phe367, Leu406, Ala407, and Ala415, the phenyl ring forms hydrophobic interactions with Val386 and Leu397 within the CNB-B of EPAC2 (1162).…”

The Epac1 Protein: Pharmacological Modulators, Cardiac Signalosome and Pathophysiology

“…However, these compounds, particularly NY0123, were even better inhibitors of 8-NBD-cAMP binding to Epac2 than to Epac1. Recently, further chemical optimizations involving modifications of the isoxazole and phenyl rings of ESI-09 have resulted in the discovery of several novel Epac antagonists, among which NY0460 and NY0562 (Table 1) show low micromolar inhibitory activities (IC50 = 2.4 µM and 2.7 µM, respectively), but without specificity for Epac1 with respect to Epac2 [39]. In another optimization study, ZL0524 (Table 1) compound has been discovered as a potent Epac inhibitor with IC50 values of 3.6 µM and 1.2 µM against Epac1 and Epac2, respectively [40], and therefore without specificity in favour of Epac1.…”