DOI: 10.1002/cmdc.201000397 Nitrooxyacyl Derivatives of Salicylic Acid: Aspirin-Like Molecules that Covalently Inactivate Cyclooxygenase-1 Clara Cena, [a] Paolo Tosco, [a] Elisabetta Marini, [a] Loretta Lazzarato, [a] Marco Piccinini, [b] Cristina Ramondetti, [b] Elisa Lupino, [b] Roberta Fruttero, [a] and Alberto Gasco* [a] Introduction Acetylsalicylic acid (aspirin, ASA) is the most prominent repre- sentative of the nonsteroidal anti-inflammatory drugs (NSAIDs). It was introduced as a therapeutic agent at the end of the 19th century to treat a number of conditions associated with pain, inflammation, and fever. [1] It was later found to act as a potent antiplatelet agent as well, with proven benefits in the acute management of myocardial infarction, unstable angina, and embolic stroke. [2] More recently, a number of studies showed that the regular use of ASA decreases the risk of color- ectal adenoma or cancer, as well as the recurrence of colorec- tal adenoma in high risk patients. [3] ASA, and NSAIDs in general, inhibit cyclooxygenase (COX), an enzyme involved in the synthesis of prostanoids from arachidonic acid. [4] Reduced formation of prostanoids in differ- ent tissues is principally responsible for the pharmacological effects of these drugs. COX is present as two isoforms: COX-1 and COX-2. The former is constitutively expressed in platelets and a number of tissues, while the latter is primarily an induci- ble form which is present at sites of inflammation. ASA is cur- rently the sole NSAID able to irreversibly inactivate both COX isoforms, with a preference for COX-1. [5] It forms a covalent bond with the Ser 530 residue (residue numbering follows ovine COX-1 primary sequence throughout this paper), which is positioned at the entrance of the arachidonic acid binding channel and blocks access and subsequent oxygenation of the substrate. The formation of this covalent bond is the molecular basis for the use of ASA as an antithrombotic agent. Indeed, platelets are anuclear cells and are consequently devoid of the biosynthetic machinery necessary to synthesize new COX enzyme during their life span. [6] The prolonged use of ASA is problematic due to its high gastrotoxicity, which manifests as irritation, ulcer bleeding, per- foration, and death. [7] A number of factors may be responsible for this serious drawback, in particular the presence of a free carboxylic group in the compound structure and its ability to preferentially inhibit the COX-1 isoform, which is largely ex- pressed in gastric epithelial cells. [8] The free carboxylic group induces local irritant effects, while blockade of the COX-1 iso- form impairs the synthesis of prostaglandins, which display an important cytoprotective role for gastric mucosa. In view of the well known gastrosparing properties of nitric oxide (NO), [9] NO donor moieties were linked to the carboxylic group in an attempt to limit the gastrotoxicity of ASA. [10–12] Recently, we developed a new class of NO-donor aspirin-like molecules in which the acetyl group of ASA is replaced by acyl groups containing NO donor nitrooxy (-ONO 2 ) moieties (Scheme 1). [13] These products are generally stable over 3 h in acidic medium and at physiological pH. In human serum, some of the compounds have greater stability than aspirin, whereas others are less stable, depending on the structure of the nitro- oxyacyl moiety. Many of these compounds display anti-inflam- matory activity similar to that of ASA in the carrageenan-in- duced paw edema assay. They exhibit decreased or no gastro- toxicity when administered by intragastric route to conscious rats at a dose equimolar to that of ASA. Products 1–4, 7, and 8 (Scheme 1) are quite potent inhibitors of collagen-induced pla- A recently described series of nitrooxyacyl derivatives of salicyl- ic acid, displaying aspirin-like anti-inflammatory and platelet anti-aggregatory properties, were evaluated for their abilities to inhibit cyclooxygenase (COX). A number of these com- pounds irreversibly inhibited both COX-1 and COX-2 isoforms when tested in isolated human platelets and monocytes. Fur- ther studies using COX-1 expressed in human HEK293T cells showed that this inhibition mechanism is similar to that of as- pirin; namely, the products are able to covalently bind to the Ser 530 residue present in the active cleft of the enzyme. Mo- lecular modeling enabled us to rationalize this behavior. Be- cause these products were previously found to display NO-de- pendent properties in rat animal models, particularly as they decreased in vivo gastrotoxicity and induced in vitro vasodila- tion, they represent a new and interesting class of potential as- pirin-like antithrombotic agents worthy of further study. [a] Prof.Dr. C. Cena, Dr. P. Tosco, Dr. E. Marini, Dr. L. Lazzarato, Prof. R. Fruttero, Prof. A. Gasco Dipartimento di Scienza e Tecnologia del Farmaco Università degli Studi di Torino Via Pietro Giuria 9, 10125 Torino (Italy) Fax: (+ 39) 011-6707286 E-mail : alberto.gasco@unito.it [b] Prof. Dr. M. Piccinini, Dr. C. Ramondetti, Dr. E. Lupino Dipartimento di Medicina e Oncologia Sperimentale Sezione di Biochimica, Università degli Studi di Torino Via Michelangelo 27/B, 10126 Torino (Italy) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000397. ChemMedChem 2011, 6, 523 – 530  2011 Wiley-VCH Verlag GmbH & Co. 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