Purpose: Rho-associated protein kinase (ROCK) inhibitors lower intraocular pressure (IOP) by increasing aqueous outflow through the trabecular meshwork (TM). The preclinical characterization of netarsudil, a new ROCK/norepinephrine transporter (NET) inhibitor currently in clinical development, is presented herein.Methods: The kinase inhibitory activity of netarsudil was compared to its esterase metabolite, netarsudil-M1, and 3 other ROCK inhibitors using a commercially available kinase assay kit. Disruption of actin stress fibers was measured in primary porcine TM cells and disruption of focal adhesions in transformed human TM (HTM) cells. Induction of fibrosis markers after exposure to transforming growth factor-β2 (TGF-β2) was conducted in primary HTM cells. Ocular hypotensive activity and tolerability of topical formulations were evaluated in normotensive Dutch Belted rabbits and Formosan Rock monkeys. In vitro corneal metabolism assays were conducted using dog, pig, rabbit, monkey, and human corneas. In vivo ocular pharmacokinetics was studied in Dutch Belted rabbits.Results: Netarsudil inhibited kinases ROCK1 and ROCK2 with a Ki of 1 nM each, disrupted actin stress fibers and focal adhesions in TM cells with IC50s of 79 and 16 nM, respectively, and blocked the profibrotic effects of TGF-β2 in HTM cells. Netarsudil produced large reductions in IOP in rabbits and monkeys that were sustained for at least 24 h after once daily dosing, with transient, mild hyperemia observed as the only adverse effect.Conclusion: Netarsudil is a novel ROCK/NET inhibitor with high potency in biochemical and cell-based assays, an ability to produce large and durable IOP reductions in animal models, and favorable pharmacokinetic and ocular tolerability profiles.

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Starting from phenethanolamine aniline leads 3a and 3b, we have identified a series of functionally potent and selective beta(3) adrenergic receptor (AR) agonists containing acylsulfonamide, sulfonylsulfonamide, or sulfonylurea groups within the aniline phenethanolamine series. In beta(3), beta(2), and beta(1) AR cAMP functional assays, 3a and other right-hand side (RHS) carboxylate analogues were found to be full agonists that were modestly selective against beta(1) or beta(2) ARs, while analogues lacking RHS acid functionality were active at beta(3) AR but not selective. Replacement of the carboxylate with acylthiazole and acylmethylsulfone gave potent, but only modestly selective, compounds. Increasing the size of the RHS sulfonamide substituent with phenyl or p-toluene afforded compounds with good potency and functional selectivity (beta(3) AR pEC(50) greater than 8; beta(1) and beta(2) AR selectivity greater than 40- and 500-fold, respectively). Our SAR studies suggest that the potency and selectivity profile of the best analogues reported here is a result of both the steric bulk and acidity of the RHS sulfonamide NH group. Although all of the analogues had a pharmacokinetic half-life of less than 2 h, acylsulfonamides 43 and 44 did show moderately low clearance in dogs. These two compounds were further evaluated by thermographic imaging in mice and were found to produce a robust thermogenic response via oral administration.

The synthesis of a series of phenethanolamine aniline agonists that contain an aniline ring on the right-hand side of the molecule substituted at the meta position with a benzoic acid or a pyridyl carboxylate is described. Several of the analogues (e.g., 34, 36-38, 40, and 44) have high beta(3) adrenergic receptor (AR) potency and selectivity against beta(1) and beta(2) ARs in Chinese hamster ovary (CHO) cells expressing beta ARs. The dog pharmacokinetic profile of some of these analogues showed >25% oral bioavailability and po half-lives of at least 1.5 h. Among the compounds described herein, the 3,3'-biarylaniline carboxylate derivatives 36, 38 and the phenylpyridyl derivative 44 demonstrated outstanding in vitro properties and reasonable dog pharmacokinetic profiles. These three analogues also showed dose dependent beta(3) AR mediated responses in mice. The ease of synthesis and superior dog pharmacokinetics of compound 38 relative to that of 44 in combination with its in vitro profile led us to choose this compound as a development candidate for the treatment of type 2 diabetes.