The design and study of two classes of noncompetitive acetylcholinesterase inhibitors (AChEIs) which also function as NSAID prodrugs are reported. The most potent AChEIs disclosed contain an aromatic alkyl-aryl linker between an NSAID and a lipophilic choline mimic and they inhibit acetylcholinesterase (AChE) in the submicromolar range. These agents have the therapeutic potential to dually target inflammation by releasing an NSAID in vivo and activating the cholinergic antiinflammatory pathway via cholinergic up-regulation.Pro-inflammatory cytokine up-regulation plays a role in the pathogenesis of a wide range of disorders including osteoarthritis, 1 psoriasis, 2 multiple sclerosis, 3 and other autoimmune disorders. 4 Despite decades of research, non-steroidal anti-inflammatory drugs (NSAIDs) are still one of the most commonly used, highly effective treatments for such disorders. However, since chronic NSAID-use often leads to gastrointestinal (GI) side effects, NSAID ester prodrugs have been explored to mask the acidic GI-irritating portion of the NSAID. 5 For conditions such as arthritis, topical NSAID prodrugs are particularly useful as their therapeutic action is localized, resulting in minimal systemic side effects. 6 Alternately, acetylcholinesterase inhibitors (AChEIs) have implications in the treatment of severe inflammation resulting from sepsis, 7 endotoxemia, 8 and rheumatoid arthritis, 9 as well as in the treatment of neuroinflammation associated with Alzheimer's disease [10][11][12] and Myasthenia Gravis. 13 The administration of CNS-active AChEIs such as galanthamine depletes systemic pro-inflammatory cytokines and ameliorates both central and peripheral inflammation. 8 AChEIs seem to suppress inflammation via the cholinergic anti-inflammatory pathway, a mechanism by which the vagus nerve of the CNS regulates the production and release of tumor necrosis factor and other cytokines. 14,15 *Corresponding author. Tel.: 1-610-758-3464; fax.: 1-610-758-3461; ndh0@lehigh.edu. † Current address: PTC Therapeutics, Inc., South Plainfield, NJ 07080-2449, USA ‡ Current address: Digestive Care, Inc., Bethlehem, PA 18017-7059, USA Supplementary data Synthetic procedures, physical characterization of compounds, in vitro assay methods and Lineweaver-Burk plots can be found in the online version of this article.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Figure 1). 18 The synthesis and evaluation of these compounds as anti-inflammatory and anticholinesterase agents for topical or oral administration are presented herein. NIH Public AccessAuthor Manuscript Bioor...

As part of a continuous effort to develop efficient countermeasures against sulfur mustard injuries, several unique NSAID prodrugs have been developed and screened for anti-inflammatory properties. Presented herein are three classes of prodrugs which dually target inflammation and cholinergic dysfunction. Compounds 1–28 contain common NSAIDs linked either to choline bioisosteres or to structural analogs of acetylcholinesterase (AChE) inhibitors. These agents have shown utility as anti-vesicants and anti-inflammatory agents when screened in a mouse ear vesicant model (MEVM) against both 2-chloroethyl ethyl sulfide (CEES), a blistering agent, and 12-O-tetradecanoylphorbol-13-acetate (TPA), a common topical irritant. Many of the prodrugs have activity against CEES, with 5, 18, 22 and 27 reducing inflammation by more than 75 % compared to a control. Compounds 12, 13, 15 and 22 show comparable activity against TPA. Promising activity in the MEVM is related to half-lives of NSAID-release in plasma, moderate to high lipophilicity, and some degree of inhibition of AChE, a potential contributor to sulfur mustard-mediated tissue damage.

Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 hr post-SM exposure. After 96 hr, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermalepidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure.

PurposeSulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial–stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial–stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing.MethodsRabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3–100 nmol in 20 μL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays.ResultsNitrogen mustard–induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial–stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial–stromal attachment.ConclusionsMustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial–stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial–stromal separation.

Nuclear magnetic resonance (NMR) spectroscopy is an integral part of the undergraduate chemistry curriculum. In addition to structure determination, NMR spectroscopy is used to analyze chemical reactions and equilibria in situ. Determination of the position of equilibria is well-suited to NMR analysis in the undergraduate laboratory as an extension of peak identification and signal integration, and the determination of keto−enol equilibrium remains a popular undergraduate laboratory experiment. Several factors affect the position of keto−enol equilibrium, defined here as K e/k = [enol]/[keto], including structure (steric bulk, conjugation, electron-withdrawing/donating groups, resonance), temperature, and solvent. A judiciously selected set of compounds that have a common 1,3-dicarbonyl moiety with progressively changing ligands at the 1 and 3 positions is presented. This array allows students to investigate structure−function relationships that affect keto−enol equilibrium in a cumulative fashion and affords instructors a broad selection of compounds for study in both introductory and advanced laboratory courses using a variety of pedagogic approaches.