Int. J. Mol. Sci. 2021, 22, 7079. https://doi.org/10.3390/ijms22137079 www.mdpi.com/journal/ijms Article Design, Synthesis, In Silico and In Vitro Studies for New Nitric Oxide‐Releasing Indomethacin Derivatives with 1,3,4‐oxadiazole‐2‐thiol Scaffold Alexandru Sava 1,2 , Frederic Buron 2 , Sylvain Routier 2, * ,† , Alina Panainte 1 , Nela Bibire 1 , Sandra Mădălina Constantin 3 , Florentina Geanina Lupașcu 3 , Alin Viorel Focșa 3 and Lenuţa Profire 3, * ,† 1 Department of Analytical Chemistry, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and Pharmacy of Iași, 16 University Street, 700115 Iasi, Romania; alexandru.i.sava@umfiasi.ro (A.S.); alina‐diana.panainte@umfiasi.ro (A.P.); nela.bibire@umfiasi.ro (N.B.) 2 Institut de Chimie Organique et Analytique ICOA, CNRS UMR 7311, Université d’Orléans, 45067 Orléans, France; frederic.buron@univ‐orleans.fr 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and Pharmacy of Iași, 16 University Street, 700115 Iasi, Romania; constantin.sandra@umfiasi.ro (S.M.C.); florentina‐geanina.lupascu@umfiasi.ro (F.G.L.); alin‐viorel‐v‐focsa@d.umfiasi.ro (A.V.F.); lenuta.profire@umfiasi.ro (L.P.) * Correspondence: sylvain.routier@univ‐orleans.fr (S.R.); lenuta.profire@umfiasi.ro (L.P.) † Both authors contributed equally as senior authors. Abstract: Starting from indomethacin (IND), one of the most prescribed non‐steroidal anti‐inflam‐ matory drugs (NSAIDs), new nitric oxide‐releasing indomethacin derivatives with 1,3,4‐oxadiazole‐ 2‐thiol scaffold (NO‐IND‐OXDs, 8a‐p) have been developed as a safer and more efficient multitarget therapeutic strategy. The successful synthesis of designed compounds (intermediaries and finals) was proved by complete spectroscopic analyses. In order to study the in silico interaction of NO‐ IND‐OXDs with cyclooxygenase isoenzymes, a molecular docking study, using AutoDock 4.2.6 software, was performed. Moreover, their biological characterization, based on in vitro assays, in terms of thermal denaturation of serum proteins, antioxidant effects and the NO releasing capacity, was also performed. Based on docking results, 8k, 8l and 8m proved to be the best interaction for the COX‐2 (cyclooxygense‐2) target site, with an improved docking score compared with celecoxib. Referring to the thermal denaturation of serum proteins and antioxidant effects, all the tested com‐ pounds were more active than IND and aspirin, used as references. In addition, the compounds 8c, 8h, 8i, 8m, 8n and 8o showed increased capacity to release NO, which means they are safer in terms of gastrointestinal side effects. Keywords: indomethacin; 1,3,4‐oxadiazole‐2‐thiol; nitric oxide; docking study; cyclooxygenase; inflammation 1. Introduction The non‐steroidal anti‐inflammatory drugs (NSAIDs) are the most prescribed drugs for management of different pathological conditions where inflammation is involved, based on their analgesic, anti‐inflammatory and antipyretic effects [1,2]. The inflammation is a defense reaction of the human body to various harmful agents in restoring the body homeostasis [3–6]. When inflammation persists for a long time, hold‐ ing the body in a constant state of alert, it may become chronic, with a negative impact on tissue and organs [7]. The clinical consequences of chronic inflammation‐driven damage can be severe and include increased risk of many chronic diseases such as: rheumatoid arthritis [8], inflammatory bowel disease [9], metabolic disorders (diabetes mellitus and obesity) [10–12], cardiovascular disorders (ischemic heart disease, atherosclerosis) [13,14], Citation: Sava, A.; Buron, F.; Routier, S.; Panainte, A.; Bibire, N.; Constantin, S.M.; Lupașcu, F.G.; Focșa, A.V.; Profire, L. Design, Synthesis, In Silico and In Vitro Studies for New Nitric Oxide‐Releasing Indomethacin Derivatives with 1,3,4‐oxadiazole‐2‐thiol Scaffold. Int. J. Mol. Sci. 2021, 22, 7079. https://doi.org/10.3390/ijms22137079 Academic Editor: Antonio Palumbo Piccionello Received: 25 May 2021 Accepted: 25 June 2021 Published: 30 June 2021 Publisher’s Note: MDPI stays neu‐ tral with regard to jurisdictional claims in published maps and insti‐ tutional affiliations. Copyright: © 2021 by the authors. Li‐ censee MDPI, Basel, Switzerland. This article is an open access article distrib‐ uted under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecom‐ mons.org/licenses/by/4.0/).