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Late sodium current (late INa) is enhanced during ischemia by reactive oxygen species (ROS) modifying the Nav 1.5 channel, resulting in incomplete inactivation. Compound 4 (GS-6615, eleclazine) a novel, potent, and selective inhibitor of late INa, is currently in clinical development for treatment of long QT-3 syndrome (LQT-3), hypertrophic cardiomyopathy (HCM), and ventricular tachycardia-ventricular fibrillation (VT-VF). We will describe structure-activity relationship (SAR) leading to the discovery of 4 that is vastly improved from the first generation late INa inhibitor 1 (ranolazine). Compound 4 was 42 times more potent than 1 in reducing ischemic burden in vivo (S-T segment elevation, 15 min left anteriorior descending, LAD, occlusion in rabbits) with EC50 values of 190 and 8000 nM, respectively. Compound 4 represents a new class of potent late INa inhibitors that will be useful in delineating the role of inhibitors of this current in the treatment of patients.

We started with a medium throughput screen of heterocyclic compounds without basic amine groups to avoid hERG and β-blocker activity and identified [1,2,4]triazolo[4,3-a]pyridine as an early lead. Optimization of substituents for Late INa current inhibition and lack of Peak INa inhibition led to the discovery of 4h (GS-458967) with improved anti-arrhythmic activity relative to ranolazine. Unfortunately, 4h demonstrated use dependent block across the sodium isoforms including the central and peripheral nervous system isoforms that is consistent with its low therapeutic index (approximately 5-fold in rat, 3-fold in dog). Compound 4h represents our initial foray into a 2nd generation Late INa inhibitor program and is an important proof-of-concept compound. We will provide additional reports on addressing the CNS challenge in a follow-up communication.

We hereby disclose the discovery of inhibitors of CaMKII (7h and 7i) that are highly potent in rat ventricular myocytes, selective against hERG and other off-target kinases, while possessing good CaMKII tissue isoform selectivity (cardiac γ/δ vs. neuronal α/β). In vitro and in vivo ADME/PK studies demonstrated the suitability of these CaMKII inhibitors for PO (7h rat F = 73%) and IV pharmacological studies.

We identified a series of structurally novel SCD (Delta9 desaturase) inhibitors via high-throughput screening and follow-up SAR studies. Modification of the central bicyclic scaffold has proven key to our potency optimization effort. The most potent analog (8g) had IC(50) value of 50 pM in a HEPG2 SCD assay and has been shown to be metabolically stable and selective against Delta5 and Delta6 desaturases.

We discovered a structurally novel SCD (Delta9 desaturase) inhibitor 4a (CVT-11,563) that has 119 nM potency in a human cell-based (HEPG2) SCD assay and selectivity against Delta5 and Delta6 desaturases. This compound has 90% oral bioavailability (rat) and excellent plasma exposure (dAUC 935 ng h/mL). Additionally, 4a shows moderately selective liver distribution (three times vs plasma and adipose tissue) and relatively low brain penetration. In a five-day study (high sucrose diet, rat) compound 4a significantly reduced SCD activity as determined by GC analysis of fatty acid composition in plasma and liver. We describe the discovery of 4a from HTS hit 1 followed by scaffold replacement and SAR studies focused on DMPK properties.

The Nav1.5 channel is responsible for the upstroke of the action potential in cardiac myocytes. Congenital mutations in hNav1.5 have been shown to impair fast inactivation, leading to an increase in persistent sodium current (late INa) and LQT3. Growing evidence suggests that an increase in late INa can contribute to the pathogenesis of several cardiac diseases. With the goal of finding small molecules that selectively target cardiac late INa over peak INa and have rapid unbinding kinetics, we developed three novel assays on PatchXpress using HEK-293 cells stably expressing hNav1.5. The first assay measures block of late INa generated by tefluthrin, a pyrethroid that impairs fast inactivation. We chose tefluthrin over another late INa activator, the sea anemone toxin ATX-II, because with tefluthrin late INa reaches steady state faster and more reliably. Importantly, block obtained with tefluthrin and ATX-II is usually similar. The second assay measures tonic and use-dependent block of peak INa stimulated at 0.1 Hz and 3 Hz, respectively. To reduce voltage clamp errors on PatchXpress due to large inward peak currents and access resistance, we used 50% series resistance compensation and reduced Na+ in the extracellular solution to 20 mM. The third assay is designed to determine the rate at which compounds dissociate from the channel and is calculated from the time-course of the decrease in peak INa when the stimulation frequency is reduced from 10 to 1 Hz. All three assays on PatchXpress were validated by comparing IC50s or off-rates for several standard Nav1.5 blockers (eg. flecainide, mexiletine, quinidine and lidocaine) to those obtained from manual patch clamp. IC50s agree within two-fold and off-rate rankings are identical.