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We describe a new type of barbiturate-based matrix metalloproteinase (MMP) inhibitor incorporating a nitric oxide (NO) donor/mimetic group (series 1). The compounds were designed to inhibit MMP at enzyme level and to attenuate MMP-9 secretion arising from inflammatory signaling. To detect effects related to the nitrate, we prepared and studied an analogous series of barbiturate C5-alkyl alcohols that were unable to release NO (series 2). Both series inhibited recombinant human MMP-2/9 activity with nanomolar potency. Series 1 consistently inhibited the secretion of MMP-9 from TNFα/IL1β stimulated Caco-2 cells at 10 μM, which could be attributed to NO related effects because the non-nitrate panel did not affect enzyme levels. Several compounds from series 1 (10 μM) inhibited tumor cell invasion but none from the non-nitrate panel did. The work shows that MMP-inhibitory barbiturates are suitable scaffolds for hybrid design, targeting additional facets of MMP pathophysiology, with potential to improve risk-benefit ratios.

Ursodeoxycholic acid (UDCA) is used for the treatment of hepatic inflammatory diseases. Recent studies have shown that UDCA's biological effects are partly glucocorticoid receptor (GR) mediated. UDCA derivatives were synthesized and screened for ability to induce GR translocation in a high content analysis assay using the esophageal cancer SKGT-4 cell line. UDCA derivatives induced GR translocation in a time dependent manner with equal efficacy to that of dexamethasone (Dex) and with greatly increased potency relative to UDCA. The cyclopropylamide 1a suppressed TNF-α induced NF-κB activity and it induced GRE transactivation. 1a was unable to displace Dex from the GR ligand binding domain (LBD) in a competition experiment but was capable of coactivator recruitment in a time-resolved fluorescence energy transfer assay (TR-FRET). This represents a novel mechanism of action for a GR modulator. These derivatives could result in a new class of GR modulators.

Isosorbide-2-benzyl carbamate-5-benzoate is a highly potent and selective BuChE inhibitor. Meanwhile, isosorbide-2-aspirinate-5-salicylate is a highly effective aspirin prodrug that relies on the salicylate portion to interact productively with human BuChE. By integrating the salicylate group into the carbamate design, we have produced isosorbide-2-benzyl carbamate-5-salicylate, an inhibitor of high potency (150 pM) and selectivity for human BuChE over AChE (666000) and CES2 (23000). Modeling and mutant studies indicate that it achieves its exceptional potency because of an interaction with the polar D70/Y332 cluster in the PAS of BuChE in addition to pseudosubstrate interactions with the active site.

Aspirin prodrugs and related nitric oxide releasing compounds hold significant therapeutic promise, but they are hard to design because aspirin esterification renders its acetate group very susceptible to plasma esterase mediated hydrolysis. Isosorbide-2-aspirinate-5-salicylate is a true aspirin prodrug in human blood because it can be effectively hydrolyzed to aspirin upon interaction with plasma BuChE. We show that the identity of the remote 5-ester dictates whether aspirin is among the products of plasma-mediated hydrolysis. By observing the requirements for aspirin release from an initial panel of isosorbide-based esters, we were able to introduce nitroxymethyl groups at the 5-position while maintaining ability to release aspirin. Several of these compounds are potent inhibitors of platelet aggregation. The design of these compounds will allow better exploration of cross-talk between COX inhibition and nitric oxide release and potentially lead to the development of selective COX-1 acetylating drugs without gastric toxicity.

Glucocorticoids are used in the treatment of inflammatory bowel disease. A limitation to their use is that they undergo absorption from the GIT before reaching the colon causing severe systemic side effects. We report here on a novel prodrug approach to targeting corticosteroids to the colon. The design involves attaching a 21-ester group that suppresses absorption during transit to the colon. The prodrug is designed to be primed by colonic microflora liberating an amino ester that cyclizes releasing the steroid. One of the prodrugs 5b was as efficacious as prednisolone in the murine DSS model but did not cause thymic atrophy, a marker for systemic steroid effects.

Aspirin prodrugs formed by derivatization at the benzoic acid group are very difficult to obtain because the promoiety accelerates the rate of hydrolysis by plasma esterases at the neighboring acetyl group, generating salicylic acid derivatives. By tracing the hydrolysis pattern of the aspirin prodrug isosorbide-2,5-diaspirinate (ISDA) in human plasma solution, we were able to identify a metabolite, isosorbide-2-aspirinate-5-salicylate, that undergoes almost complete conversion to aspirin by human plasma butyrylcholinesterase, making it the most successful aspirin prodrug discovered to date.

In this study, we report the SAR and characterization of two groups of isosorbide-based cholinesterase inhibitors. The first was based directly on the clinically used nitrate isosorbide mononitrate (ISMN) retention of the 5-nitrate group and introduction of a series of 2-carbamate functionalities. The compounds proved to be potent and selective inhibitors of human plasma butyrylcholinesterase ( huBuChE). In the second group, the nitrate ester was removed and replaced with a variety of alkyl and aryl esters. These generally exhibited nanomolar potency with high selectivity for BuChE over acetylcholinesterase (AChE). The most potent and selective compound was isosorbide-2-benzyl carbamate-5-benzoate with an IC 50 of 4.3 nM for BuChE and >50000 fold selectivity over human erythrocyte AChE. Inhibition with these compounds is time-dependent, competitive, and slowly reversible, indicating active site carbamylation.