Discovery of Novel Orally Active Anti-Inflammatory N-Phenylpyrazolyl-N-Glycinyl-Hydrazone Derivatives That Inhibit TNF-a Production Renata B. Lacerda 1,2 , Leandro L. da Silva 1,3 , Cleverton K. F. de Lima 1,3 , Eduardo Miguez 4 , Ana Luisa P. Miranda 1,3 , Stefan A. Laufer 5 , Eliezer J. Barreiro 1,2,3 , Carlos A. M. Fraga 1,2,3 * 1 Laborato ´ rio de Avaliac ¸a ˜o e Sı ´ntese de Substa ˆncias Bioativas (LASSBio), Faculty of Pharmacy, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil, 2 Programa de Po ´ s-Graduac ¸a ˜o em Quı ´mica, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil, 3 Programa de Po ´ s-Graduac ¸a ˜o em Farmacologia e Quı ´mica Medicinal, Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil, 4 Institute of Macromolecules Professora Eloisa Mano, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil, 5 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Eberhard Karls University of Tu ¨ bingen, Tuebingen, Germany Abstract Herein, we describe the synthesis and pharmacological evaluation of novel N-phenylpyrazolyl-N-glycinyl-hydrazone derivatives that were designed as novel prototypes of p38 mitogen-activated protein kinase (MAPK) inhibitors. All of the novel synthesized compounds described in this study were evaluated for their in vitro capacity to inhibit tumor necrosis factor a (TNF-a production in cultured macrophages) and in vitro MAPK p38a inhibition. The two most active anti-TNF-a derivatives, (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-((4-(2-morpholinoethoxy)naphthalen-1-yl)methylene)ace- tohydrazide (4a) and (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-(4-chlorobenzylidene)acetohydrazide (4f), were evaluated to determine their in vivo anti-hyperalgesic profiles in carrageenan-induced thermal hypernociception model in rats. Both compounds showed anti-inflammatory and antinociceptive properties comparable to SB-203580 used as a standard drug, by oral route at a dose of 100 mmol/kg. This bioprofile is correlated with the ability of NAH derivatives (4a) and (4f) suppressing TNF-a levels in vivo by 57.3 and 55.8%, respectively. Citation: Lacerda RB, da Silva LL, de Lima CKF, Miguez E, Miranda ALP, et al. (2012) Discovery of Novel Orally Active Anti-Inflammatory N-Phenylpyrazolyl-N- Glycinyl-Hydrazone Derivatives That Inhibit TNF-a Production. PLoS ONE 7(10): e46925. doi:10.1371/journal.pone.0046925 Editor: Joao B. Calixto, Universidad Federal de Santa Catarina, Brazil Received April 14, 2012; Accepted September 6, 2012; Published October 8, 2012 Copyright: ß 2012 Lacerda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank Coordenac ¸a ˜o de Aperfeic ¸oamento de Pessoal de Nı ´vel Superior (CAPES, Brazil), Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´ gico (CNPq, Brazil), Fundac ¸a ˜o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ, Brazil), Programa de Apoio a Nu ´ cleos de Excele ˆncia (PRONEX, Brazil) and Instituto Nacional de Cie ˆncia e Tecnologia de Fa ´rmacos e Medicamentos (INCT-INOFAR, Brazil, Grant #573.564/2008-6) for financial support and fellowships (to RBL, CKFdL, LLdS, ALPM, EJB, CAMF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: cmfraga@ccsdecania.ufrj.br Introduction The production of proinflammatory cytokines, e.g., TNF-a, IL- 1b and IL-6, is a key factor in chronic inflammatory diseases, such as rheumatoid arthritis, Crohn’s disease, psoriasis and asthma [1,2]. Moreover, evidence exists that supports the involvement of cytokines in other diseases, including cardiac heart failure, ischemic retinopathy [3] and the development of insulin resistance in diabetes [4]. Due to the role of cytokines in various inflammatory diseases, many pharmaceutical companies have made efforts to develop new orally active substances that can modulate the production of proinflammatory cytokines. Tumor necrosis factor-alpha (TNF-a) is a pleiotropic cytokine that possesses proinflammatory and osmoregulator actions [5]. It is the major cytokine mediator of acute inflammation, it activates platelets, and it is also involved in the genesis of fever and anemia. TNF-a also mediates many inflammatory events in rheumatoid arthritis, including immune cell activation, proliferation, apoptosis and regulation of leukocyte movement [6], which has led to the development of strategies to block TNF-a-mediated effects. The currently available anti-TNF-a strategies involve either adminis- tration of anti-TNF-a antibodies or soluble TNF receptors to remove circulating TNF-a [7]. These inhibitors act by binding to TNF-a and preventing it from binding to its receptors on nearby cells, thus preventing the initiation of apoptosis or an inflamma- tory response [8]. Despite the approval of anti-TNF-a drugs, e.g., infliximab, etanercept and adalimumab, which demonstrated the effectiveness of therapeutic strategies based on the depletion of TNF-a, the appearance of side effects resulting from the debilitating actions of these drugs on the immune system highlights the necessity of identifying new alternative mechanisms to modulate the actions of pro-inflammatory cytokines [9,10]. One of the most promising targets involved in modulating the production of pro-inflammatory cytokines is the mitogen-activated protein kinase (MAPK) pathway, particularly p38 MAPK, a serine–threonine protein kinase that has been identified as a molecular target of the pyridinyl-imidazole derivatives SB-203580 (1) and SB-202190 (2) (Figure 1) [11,12]. These terphenyl- heterocyclic derivatives, which have been widely used to study p38 MAPK function, competitively bind at the ATP-binding pocket of p38 MAPK and inhibit TNF-a and IL-1b production. PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46925