Discovery of 3-Cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide: A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C2 Inverse Agonist.

The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.

Discovery of novel cyanodihydropyridines as potent mineralocorticoid receptor antagonists.

A new 1,4-dihydropyridine 5a, containing a cyano group at the C3 position, was recently reported to possess excellent mineralocorticoid receptor (MR) antagonist in vitro potency and no calcium channel-blocker (CCB) activity. In the present study, we report the structure-activity relationships of this novel series of cyano ester dihydropyridines that resulted in R6 substituted analogues with improved metabolic stability while maintaining excellent MR antagonist activity and selectivity against other nuclear receptors. Further structure optimization with the introduction of five-membered ring heterocycles at R6 resulted in compounds with excellent MR antagonist potency and a suitable pharmacokinetic profile. In vivo studies of a promising tool compound in the Dahl salt-sensitive rat model of hypertension showed similar blood pressure (BP) reduction as the steroidal MR antagonist eplerenone, providing proof-of-concept (POC) for a nonsteroidal, orally efficacious MR antagonist.

Discovery of (3S,3aR)-2-(3-chloro-4-cyanophenyl)-3-cyclopentyl-3,3a,4,5-tetrahydro-2H-benzo[g]indazole-7-carboxylic acid (PF-3882845), an orally efficacious mineralocorticoid receptor (MR) antagonist for hypertension and nephropathy.

We have discovered a novel class of nonsteroidal pyrazoline antagonists of the mineralocorticoid receptor (MR) that show excellent potency and selectivity against other nuclear receptors. Early analogues were poorly soluble and had a propensity to inhibit the hERG channel. Remarkably, both of these challenges were overcome by incorporation of a single carboxylate moiety. Structural modification of carboxylate-containing lead R-4g with a wide range of substituents at each position of the pyrazoline ring resulted in R-12o, which shows excellent activity against MR and reasonable pharmacokinetic profile. Introduction of conformational restriction led to a novel series characterized by exquisite potency and favorable steroid receptor selectivity and pharmacokinetic profile. Oral dosing of 3S,3aR-27d (PF-3882845) in the Dahl salt sensitive preclinical model of salt-induced hypertension and nephropathy showed blood pressure attenuation significantly greater than that with eplerenone, reduction in urinary albumin, and renal protection. As a result of these findings, 3S,3aR-27d was advanced to clinical studies.

Stereochemical requirements for the mineralocorticoid receptor antagonist activity of dihydropyridines.

A number of known 1,4-dihydropyridine CCBs were identified as having comparable potency to the steroidal MR antagonist eplerenone. Chiral resolution of mebudipine revealed that MR and CCB activity reside in opposite enantiomers. Small molecule X-ray crystal structures showed that the C4 stereochemistry of optimized selective MR analogues, e.g. 5, is consistent with MR-active mebudipine. Molecular modeling supports a binding pose consistent with that previously proposed for DHP diesters.

A number of marketed dihydropyridine calcium channel blockers have mineralocorticoid receptor antagonist activity.

Calcium channel blockers are widely used antihypertensives. Mineralocorticoid receptor antagonists are also used to treat hypertension and heart failure. We report here that a number of widely used dihydropyridine class calcium channel blockers are able to inhibit aldosterone-induced activation of mineralocorticoid receptor. These dihydropyridines varied in the extent of their effect on mineralocorticoid receptor, with nimodipine and felodipine the most potent and amlodipine the least. In contrast, both diltiazem and verapamil, nondihydropyridine calcium channel blockers, had no effect on mineralocorticoid receptor. These dihydropyridines compete with aldosterone for binding and block aldosterone-induced coactivator recruitment to mineralocorticoid receptor. The mineralocorticoid receptor S810L mutant, which is activated by steroidal mineralocorticoid receptor antagonist such as eplerenone, is inhibited by these drugs. Furthermore, nimodipine decreased aldosterone-induced expression of the mineralocorticoid receptor target gene epithelial sodium channel gamma subunit in adrenalectomized rats, demonstrating that dihydropyridine calcium channel blockers can function as mineralocorticoid receptor antagonists in vivo. Molecular modeling indicates that dihydropyridines dock into the ligand binding domain of mineralocorticoid receptor in a consensus pose that partially overlaps with steroidal mineralocorticoid receptor antagonists. Together, our data suggest that, in addition to their calcium channel blocking activity, a number of dihydropyridine calcium channel blockers also have mineralocorticoid receptor antagonist activity at high doses, a finding which may thus prove useful for the design of novel antihypertensive drugs in the future.

The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria.

The oxazolidinones are one of the newest classes of antibiotics. They inhibit bacterial growth by interfering with protein synthesis. The mechanism of oxazolidinone action and the precise location of the drug binding site in the ribosome are unknown. We used a panel of photoreactive derivatives to identify the site of action of oxazolidinones in the ribosomes of bacterial and human cells. The in vivo crosslinking data were used to model the position of the oxazolidinone molecule within its binding site in the peptidyl transferase center (PTC). Oxazolidinones interact with the A site of the bacterial ribosome where they should interfere with the placement of the aminoacyl-tRNA. In human cells, oxazolidinones were crosslinked to rRNA in the PTC of mitochondrial, but not cytoplasmic, ribosomes. Interaction of oxazolidinones with the mitochondrial ribosomes provides a structural basis for the inhibition of mitochondrial protein synthesis, which is linked to clinical side effects associated with oxazolidinone therapy.