John A. Mccauley

Orally Efficacious NR2B-Selective NMDA Receptor Antagonists

A novel series of benzamidines was synthesized and shown to exhibit NR2B-subtype selective NMDA antagonist activity. Compound 31 is orally active in a carrageenan-induced rat hyperalgesia model of pain and shows no motor coordination side effects.

NR2B subtype-selective NMDA receptor antagonists: 2001 – 2004

Discovery of the NR2A – D subtypes of the N-methyl-D-aspartate (NMDA) receptor, and of an allosteric binding site on the NR2B receptor subunit, has renewed interest in the therapeutic potential of NMDA receptor antagonists. Many previous clinical efforts with non-selective NMDA antagonists have failed due to unacceptable CNS side effects. NMDA antagonists that selectively bind to the NR2B subunit, however, have demonstrated a larger therapeutic window in animal models of a number of disease states. In the last few years, there has been a significant increase in the structural diversity of small-molecule NR2B-selective NMDA antagonists. This review will focus on patents and patent applications published from 2001 to 2004 and will divide compounds into the three general classes of phenols, phenol replacements and non-phenols, with further subdivisions within these classes.

In vitro characterization of novel NR2B selective NMDA receptor antagonists

N-Methyl-D-aspartate (NMDA) subunit specific receptor antagonism has potential therapeutic application for multiple CNS pathologies. MERCK 1, MERCK 2, and MERCK 3 are novel NR2B subtype selective NMDA receptor antagonists. The affinity and the kinetic mechanism of inhibition by these antagonists and ifenprodil were investigated using the whole-cell configuration of the patch clamp technique, calcium flux, and radioligand binding on a mouse cell line L(tk-) expressing recombinant human heteromeric NMDA receptors consisting of NR1a/NR2B subunit combinations. The rank order of potency, as determined by electrophysiology, was ifenprodil<MERCK 2<MERCK 1<MERCK 3 with K(D)s 79+/-8, 2.4+/-1.1, 1.3+/-0.9, and approximately 0.16+/-0.02 nM, respectively. The apparent dissociation rate constants among these compounds differed by as much as 394-fold whereas the apparent association constants varied less than 3-fold. Higher affinities were a result of slower drug dissociation kinetics of receptor unbinding. Maximal inhibition was not voltage-dependent and was not statistically different at saturating concentrations by these compounds. These results provide the first detailed functional analysis of the kinetic mechanism of MERCK 1, MERCK 2, and MERCK 3 inhibition of NMDA receptors.

P2‐Quinazolinones and Bis‐Macrocycles as New Templates for Next‐Generation Hepatitis C Virus NS3/4a Protease Inhibitors: Discovery of MK‐2748 and MK‐6325

With the goal of identifying inhibitors of hepatitis C virus (HCV) NS3/4a protease that are potent against a wide range of genotypes and clinically relevant mutant viruses, several subseries of macrocycles were investigated based on observations made during the discovery of MK‐5172. Quinazolinone‐containing macrocycles were identified as promising leads, and optimization for superior cross‐genotype and mutant enzyme potency as well as rat liver and plasma concentrations following oral dosing, led to the development of MK‐2748. Additional investigation of a series of bis‐macrocycles containing a fused 18‐ and 15‐membered ring system were also optimized for the same properties, leading to the discovery of MK‐6325. Both compounds display the broad genotype and mutant potency necessary for clinical development as next‐generation HCV NS3/4a protease inhibitors.

Kinetic characterization of novel NR2B antagonists using fluorescence detection of calcium flux.

To facilitate the discovery of novel N-methyl-d-aspartate (NMDA) receptor antagonists, we have developed a high-throughput functional assay based on fluorescence detection of free intracellular calcium concentrations. Mouse fibroblast L(tk-) cells expressing human NR1a/NR2B NMDA receptors were plated in 96-well plates and loaded with fluorescence calcium indicator fluo-3 AM. NR2B antagonists were added after stimulation of NMDA receptors with 10 microM glutamate and 10 microM glycine. Changes in fluorescence after the addition of the antagonists were fitted by a single exponential equation providing k(obs). The concentration dependence of k(obs) was linear for all NR2B antagonists at concentrations where k(obs) < 0.2 s(-1). The values of k(obs) for six structurally distinct NR2B antagonists were in the range of 1.1 to 7.5 x 10(5) M(-1)s(-1). These values were several orders of magnitude slower than that obtained for diffusion limited Mg(2+) channel block. The rate constants k(off) provided the values of t(1/2) for dissociation of NR2B antagonists in the range of 1.8 min for ifenprodil to 240 min for the slowest novel antagonist. The IC(50) values obtained from the end-point fluorescence measurements agree with K(d) values calculated from kinetic measurements. All kinetic constants, obtained using our fluorescence method, correlate well with data measured by voltage clamp.

Potent, selective and orally bioavailable leucine-rich repeat kinase 2 (LRRK2) inhibitors

Familial Parkinsons disease cases have recently been associated with the leucine rich repeat kinase 2 (LRRK2) gene. It has been hypothesized that inhibition of the LRRK2 protein may have the potential to alter disease pathogenesis. A dihydrobenzothiophene series of potent, selective, orally bioavailable LRRK2 inhibitors were identified from a high-throughput screen of the internal Merck sample collection. Initial SAR studies around the core established the series as a tractable small molecule lead series of LRRK2 inhibitors for potential treatment of Parkinsons disease. It was also found that incorporation of a lactam into the core drastically improved the CNS and DMPK properties of these small molecules.

Amide-containing NR2B/NMDA receptor antagonists

Discovery of the NR2A-D subtypes of the NMDA receptor and of an allosteric binding site on the NR2B receptor subunit has renewed interest in the therapeutic potential of NMDA receptor antagonists for a variety of CNS disease states. Recently, there has been a significant interest in increasing the selectivity of NR2B compounds versus hERG activity. This article will focus on a recent series of NR2B antagonists from Pfizer that show good selectivity over dofetilide binding.

Development of macrocyclic inhibitors of HCV NS3/4A protease with cyclic constrained P2–P4 linkers

A series of macrocyclic compounds containing a cyclic constraint in the P2-P4 linker region have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K, A156T, A156V, and D168V mutant activity while maintaining high rat liver exposure. The effect of the constraint is most dramatic against gt 1b A156 mutants where ~20-fold improvements in potency are achieved by introduction of a variety of ring systems into the P2-P4 linker.

Development of potent macrocyclic inhibitors of genotype 3a HCV NS3/4A protease.

A series of macrocyclic compounds containing 2-substituted-quinoline moieties have been discovered and shown to exhibit excellent HCV NS3/4a genotype 3a and genotype 1b R155K mutant activity while maintaining the high rat liver exposure. Cyclization of the 2-substituted quinoline substituent led to a series of tricyclic P2 compounds which also display superb gt3a potency.

Synthesis and evaluation of novel tricyclic benzo[4.5]cyclohepta[1.2]pyridine derivatives as NMDA/NR2B antagonists

A novel series of annulated tricyclic compounds was synthesized and evaluated as NMDA/NR2B antagonists. Structure-activity development was directed towards in vitro optimization of NR2B activity and selectivity over the hERG K(+) channel. Preferred compounds were subsequently evaluated for selectivity in an alpha(1)-adrenergic receptor binding counter-screen and a cell-based assay of NR2B activity.