The Discovery and Hit-to-Lead Optimization of Tricyclic Sulfonamides as Potent and Efficacious Potentiators of Glycine Receptors.

Current pain therapeutics suffer from undesirable psychotropic and sedative side effects, as well as abuse potential. Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels expressed in nerves of the spinal dorsal horn, where their activation is believed to reduce transmission of painful stimuli. Herein, we describe the identification and hit-to-lead optimization of a novel class of tricyclic sulfonamides as allosteric GlyR potentiators. Initial optimization of high-throughput screening (HTS) hit 1 led to the identification of 3, which demonstrated ex vivo potentiation of glycine-activated current in mouse dorsal horn neurons from spinal cord slices. Further improvement of potency and pharmacokinetics produced in vivo proof-of-concept tool molecule 20 (AM-1488), which reversed tactile allodynia in a mouse spared-nerve injury (SNI) model. Additional structural optimization provided highly potent potentiator 32 (AM-3607), which was cocrystallized with human GlyRα3cryst to afford the first described potentiator-bound X-ray cocrystal structure within this class of ligand-gated ion channels (LGICs).

Synthesis of 4-substituted chlorophthalazines, dihydrobenzoazepinediones, 2-pyrazolylbenzoic acid, and 2-pyrazolylbenzohydrazide via 3-substituted 3-hydroxyisoindolin-1-ones.

Herein we describe a general three-step synthesis of 4-substituted chlorophthalazines in good overall yields. In the key step, N,N-dimethylaminophthalimide (8a) directs the selective monoaddition of alkyl, aryl, and heteroaryl organometallic reagents to afford 3-substituted 3-hydroxyisoindolinones 9b, 9i-9am. Many of these hydroxyisoindolinones are converted to chlorophthalazines 1b-1v via reaction with hydrazine, followed by chlorination with POCl(3). We have also discovered two novel transformations of 3-vinyl- and 3-alkynyl-3-hydroxyisoindolinones. Addition of vinyl organometallic reagents to N,N-dimethylaminophthalimide (8a) provided dihydrobenzoazepinediones 15a-15c via the proposed ring expansion of 3-vinyl-3-hydroxyisoindolinone intermediates. 3-Alkynyl-3-hydroxyisoindolinones react with hydrazine and substituted hydrazines to afford 2-pyrazolyl benzoic acids 16a-16d and 2-pyrazolyl benzohydrazides 17a-17g rather than the expected alkynyl phthalazinones.

Negative ion tandem mass spectrometry for the detection of glutathione conjugates.

Characterization of S-linked conjugates of the endogenous tripeptide glutathione (gamma-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approach for the identification of chemically reactive, electrophilic intermediates formed during the metabolism of both foreign compounds and endogenous substances. In most cases, GSH adducts generated in vitro or excreted in the bile of animals are detected by the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing survey scans based on characteristic fragmentations of this class of conjugates. However, a limitation of current LC-MS/MS approaches, which typically employ electrospray ionization with analysis of positive ions, is that no single survey scan exhibits broad utility in the detection of unknown GSH adducts, since different structural classes of conjugate (aromatic, benzylic, aliphatic, thioester, etc.) behave differently upon collision-induced dissociation (CID) of the respective [M + H]+ parent ions. In the present study, we evaluated MS/MS in the negative ion mode as an alternative approach and report herein that the spectra obtained by CID of the [M - H]- ions of a number of representative GSH adducts, as well as GSH itself, are dominated by fragments originating from the glutathionyl moiety of the tripeptide. In particular, the anion at m/z 272, corresponding nominally to deprotonated gamma-glutamyl-dehydroalanyl-glycine, was abundant in the negative ion spectra of free GSH and all GSH conjugates examined, suggesting that scanning for precursors of this ion may provide a generally applicable technique for the detection of adducts of unknown structure. The utility of this novel detection strategy was demonstrated in a series of in vitro and in vivo experiments where compounds known to undergo metabolic activation were examined for their propensity to form conjugates with GSH. In all cases, scanning for precursors of m/z 272 in the negative ion mode revealed the presence of the expected adducts and in some instances revealed additional conjugates that had not been reported previously. Positive ion MS/MS, on the other hand, was more useful than the corresponding negative ion scans in providing information on the molecular structure of GSH conjugates.