Full Paper Synthesis of New Tricyclic and Tetracyclic Fused Coumarin Sulfonate Derivatives and Their Inhibitory Effects on LPS-Induced Nitric Oxide and PGE 2 Productions in RAW 264.7 Macrophages: Part 2 Mohammed I. El-Gamal 1,2,3 , Woo-Seok Lee 4,5 , Ji-Sun Shin 4,5 , Chang-Hyun Oh 6,7 , Kyung-Tae Lee 4,5 , Jungseung Choi 8 , Nohsun Myoung 8 , and Daejin Baek 8 1 Department of Medicinal Chemistry, College of Pharmacy, University of Sharjah, Sharjah, United Arab Emirates 2 Sharjah Institute for Medical Research, University of Sharjah, Sharjah, United Arab Emirates 3 Faculty of Pharmacy, Department of Medicinal Chemistry, University of Mansoura, Mansoura, Egypt 4 Department of Pharmaceutical Biochemistry, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea 5 Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University, Seoul, Republic of Korea 6 Center for Biomaterials, Korea Institute of Science and Technology (KIST), Seoul, Republic of Korea 7 Department of Biomolecular Science, University of Science and Technology (UST), Daejeon, Republic of Korea 8 Department of Chemistry, Hanseo University, Seosan, Republic of Korea The synthesis of a new series of 21 fused coumarin derivatives is described, and the biological evaluation of their in vitro antiinflammatory effects as inhibitors of lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production in RAW 264.7 macrophages. The target compounds 1a–u were first tested for cytotoxicity to determine a non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE 2 production would not be caused by cytotoxicity. Compounds 1f and 1p were the most active PGE 2 inhibitors with IC 50 values of 0.89 and 0.95 mM, respectively. Western blot and cell-free COX-2 screening showed that their effects were due to inhibition of both COX-2 protein expression and COX-2 enzyme activity. Their IC 50 values against the COX-2 enzyme were 0.67 and 0.85 mM, respectively, which is more potent than etoricoxib. The selectivity indexes of compounds 1f and 1p against COX-2 compared to COX-1 were 41.1 and 42.5, respectively. Compound 1f showed strong inhibitory effects at 5 mM concentration on COX-2 mRNA expression in LPS-induced RAW 264.7 macrophages. Moreover, the tricyclic compounds 1l and 1n as well as the tetracyclic analog 1u were the most potent NO inhibitors, with one-digit micromolar IC 50 values. They showed dose-dependent inhibition of inducible nitric oxide synthase (iNOS) protein expression. The tetracyclic derivative 1u was the most potent inhibitor of NO production. It also exhibited a strong inhibitory effect on iNOS mRNA expression in LPS-induced RAW 264.7 macrophages. Keywords: Antiinflammatory / Coumarin sulfonate / COX-2 / NO / PGE 2 Received: August 23, 2016; Revised: October 9, 2016; Accepted: October 11, 2016 DOI 10.1002/ardp.201600243 : Additional supporting information may be found in the online version of this article at the publisher’s web-site. Introduction Inflammation is a biological symptom that occurs as a normal response to some external factors such as edema or microbial infection [1]. Mild and short-term inflammation is not Correspondence: Dr. Daejin Baek, Department of Chemistry, Hanseo University, Seosan 356-706, Republic of Korea. E-mail: djbaek@hanseo.ac.kr Fax: þ82-41-660-1119  Additional correspondence: Dr. Kyung-Tae Lee, E-mail: ktlee@khu.ac.kr Arch. Pharm. Chem. Life Sci. 2016, 349,1–11 Archiv der Pharmazie ARCH RCH PHARM HARM ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.archpharm.com 1