Synthesis and Deployment of an Elusive Fluorovinyl Cation Equivalent: Access to Quaternary α-(1'-Fluoro)vinyl Amino Acids as Potential PLP Enzyme Inactivators.

Developing specific chemical functionalities to deploy in biological environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor development but also is central to other protein-tagging methods in modern chemical biology including activity-based protein profiling and proteolysis-targeting chimeras. We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1'-fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with ß,ß-difluorovinyl phenyl sulfone, a new (1'-fluoro)vinyl cation equivalent, and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biologically relevant AA side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine, and tryptophan. Following addition/elimination, the resulting transoid α-(1'-fluoro)-ß-(phenylsulfonyl)vinyl AA-esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1'-fluoro)-ß-(tributylstannyl)vinyl AA-esters. Protodestannylation and global deprotection then yield these sterically encumbered and densely functionalized quaternary amino acids. The α-(1'-fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1'-fluoro)vinyllysine is seen to act as a time-dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved, and each is seen to give time-dependent inactivation with this enzyme. Kitz-Wilson analysis reveals similar inactivation parameters for the two antipodes, L-α-(1'-fluoro)vinyllysine (Ki = 630 ± 20 µM; t1/2 = 2.8 min) and D-α-(1'-fluoro)vinyllysine (Ki = 470 ± 30 µM; t1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.

Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle glaucoma.

Inhibition of Rho kinase (ROCK) to improve fluid outflow through the trabecular meshwork and lower intraocular pressure is a strategy for the development of new anti-glaucoma agents. Alpha-aryl-beta-amino isoquinoline analogs were identified as potent ROCK inhibitors. Compounds that provided a longer duration of intraocular pressure reduction in Dutch Belted rabbits also inhibited norepinephrine transporter. Ester 60 improved bioavailability of its parent ROCK inhibitor, 29 (Ki=0.2nM) and demonstrated an effective and sustained IOP reduction for 24h after dosing. From these studies, netarsudil (a.k.a. AR-13324) was discovered and is currently in clinical trials for the treatment of glaucoma and ocular hypertension.

Eyes on Topical Ocular Disposition: The Considered Design of a Lead Janus Kinase (JAK) Inhibitor That Utilizes a Unique Azetidin-3-Amino Bridging Scaffold to Attenuate Off-Target Kinase Activity, While Driving Potency and Aqueous Solubility.

An unmet medical need remains for patients suffering from dry eye disease (DED). A fast-acting, better-tolerated noncorticosteroid anti-inflammatory eye drop could improve patient outcomes and quality of life. Herein, we describe a small-molecule drug discovery effort to identify novel, potent, and water-soluble JAK inhibitors as immunomodulating agents for topical ocular disposition. A focused library of known 3-(4-(2-(arylamino)pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitriles was evaluated as a molecular starting point. Structure-activity relationships (SARs) revealed a ligand-efficient (LE) JAK inhibitor series, amenable to aqueous solubility. Subsequent in vitro analysis indicated the potential for off-target toxicity. A KINOMEscan selectivity profile of 5 substantiated the likelihood of widespread series affinity across the human kinome. An sp2-to-sp3 drug design strategy was undertaken to attenuate off-target kinase activity while driving JAK-STAT potency and aqueous solubility. Tactics to reduce aromatic character, increase fraction sp3 (Fsp3), and bolster molecular complexity led to the azetidin-3-amino bridging scaffold in 31.