molecules Article Synthesis, Characterization, and Anti-Inflammatory Activities of Methyl Salicylate Derivatives Bearing Piperazine Moiety Jingfen Li 1,†, Yong Yin 2,†, Lisheng Wang 2, Pengyun Liang 2, Menghua Li 2, Xu Liu 2, Lichuan Wu 2,* and Hua Yang 2,* 1 Department of Life Science, Huzhou Teachers’ College, Huzhou 313000, China; ljfl[email protected] 2 School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China; [email protected] (Y.Y.); [email protected] (L.W.); [email protected] (P.L.); [email protected] (M.L.); [email protected] (X.L.) * Correspondence: [email protected] (L.W.); [email protected] (H.Y.); Tel.: +86-771-3270-732 (L.W.) † These authors contributed equally to this work. Academic Editor: Diego Muñoz-Torrero Received: 11 October 2016; Accepted: 11 November 2016; Published: 23 November 2016 Abstract: In this study, a new series of 16 methyl salicylate derivatives bearing a piperazine moiety were synthesized and characterized. The in vivo anti-inflammatory activities of target compounds were investigated against xylol-induced ear edema and carrageenan-induced paw edema in mice. The results showed that all synthesized compounds exhibited potent anti-inflammatory activities. Especially, the anti-inflammatory activities of compounds M15 and M16 were higher than that of aspirin and even equal to that of indomethacin at the same dose. In addition, the in vitro cytotoxicity activities and anti-inflammatory activities of four target compounds were performed in RAW264.7 macrophages, and compound M16 was found to significantly inhibit the release of lipopolysaccharide (LPS)-induced interleukin (IL)-6 and tumor necrosis factor (TNF)-α in a dose-dependent manner. In addition, compound M16 was found to attenuate LPS induced cyclooxygenase (COX)-2 up-regulation. The current preliminary study may provide information for the development of new and safe anti-inflammatory agents. Keywords: inflammation; salicylate; piperazine; derivatives; anti-inflammatory activity 1. Introduction Inflammation is a complex biological process that occurs when body tissues are exposed to hazardous stimuli, such as irritants and pathogens [1]. Inflammation seriously threatens human health, and exaggerated and prolonged inflammation may cause various diseases, including arthritis, sepsis, and even cancer [2]. Presently, the most widely used drugs in inflammation treatment are non-steroidal anti-inflammatory drugs (NSAIDs), accounting for 35% of the global market of prescription of pain medications [3,4]. The common NSAIDs, such as aspirin and indomethacin, can inhibit cyclooxygenases (COX-1 and COX-2) [5,6], which are involved in the catalyzation of arachidonic acid to prostaglandins (PGs). Considering the significant toxicity of NSAIDs to the gastrointestinal tract and kidney [7], it is of great importance and urgent need to develop new anti-inflammation drugs [8]. Pharmaceuticals bearing a piperazine moiety are widely used in medicinal and pesticide chemistry [9]. Piperazine can effectively change the physicochemical properties of drugs and improve their pharmacokinetic properties and biological activity. Piperazine derivatives can act as histamine and serotonin receptor antagonists in the control of inflammation [10–12]. Molecules 2016, 21, 1544; doi:10.3390/molecules21111544 www.mdpi.com/journal/molecules Molecules 2016, 21, 1544 2 of 11 Salicylates are a class of chemicals with favorable anti-inflammatory effects. A large number of studies have indicated that methyl salicylate derivatives exhibited great anti-inflammatory effectsMolecules [13–15 2016]., In 21, the1544 current study, methyl salicylate and piperazine were combined to synthesize2 of 10 a new series of methyl salicylate derivatives, and their anti-inflammation activities were screened [13–15]. In the current study, methyl salicylate and piperazine were combined to synthesize a new in vitroseries and of in methyl vivo. salicylate derivatives, and their anti-inflammation activities were screened in vitro and in vivo. 2. Results 2. Results 2.1. Synthesis of Salicylate Derivatives Bearing Piperazine Moiety The2.1. Synthesis synthesis of ofSalic targetylate compoundsDerivatives Bearing is outlined Piperazine in Scheme Moiety1 . Methyl salicylate (a) firstly reacted with 3-chloro-1,2-epoxypropaneThe synthesis of target (b) compounds to generate is methyl-2-(oxiran-2-ylmethoxy)outlined in Scheme 1. Methyl salicylate benzoate (a) firstly (c) (Schemereacted 1A, upper).with Meanwhile,3-chloro-1,2-epoxypropane various aryl (b) carboxylic to generate meth acidsyl-2-(oxiran-2-ylmethoxy) (d) reacted with thionyl benzoate chloride (c) (Scheme to obtain 1A, the intermediateupper). Meanwhile, (e) which subsequently various aryl reactedcarboxylic with acids anhydrous (d) reacted piperazine with thionyl (f) in chloride the presence to obtain of acetic the acid at roomintermediate temperature, (e) which generating subsequently intermediate reacted with (g) anhydrous (Scheme 1piperazineA, bottom). (f) in Then, the presence based onof acetic combination acid principles,at room intermediate temperature,c generating reacted with intermediate g to generate (g) (Scheme products 1A,M1 bottom).–M13 inThen, the based presence on combination of toluene, with principles, intermediate c reacted with g to generate products M1–M13 in the presence of toluene, yields ranging from 41% to 62% (Scheme1A). For products M14–M16, raw material (h) reacted with with yields ranging from 41% to 62% (Scheme 1A). For products M14–M16, raw material (h) reacted methyl-2-(oxiran-2-ylmethoxy)with methyl-2-(oxiran-2-ylmethoxy) benzoate benzoate (c) in the(c) in presence the presence of toluene of toluene to generate to generate the the target target methyl salicylatemethyl derivatives salicylate derivatives (M14–M16 ()M14 (Scheme–M16)1 B),(Scheme with yields1B), with ranging yields fromranging 34% from to 67%. 34% Allto 67%. compounds All werecompounds characterized were by characterized microanalytical by microanalytical and spectral and data. spectral data. Scheme 1. Synthetic routes of methyl salicylate derivatives. (A) Synthetic routes of compounds M1~M13. Scheme 1. Synthetic routes of methyl salicylate derivatives. (A) Synthetic routes of compounds M1~M13. (B) Synthetic routes of compounds M14~M16. Reagents and conditions: (i) K2CO3, acetone, reflux, 30 h, (B) Synthetic routes of compounds M14~M16. Reagents and conditions: (i) K2CO3, acetone, reflux, 30 h, yield = 85%; (ii) SOCl2, reflux, 2 h, yield = 71%–87%; (iii) Piperazine, AcOH, r.t., 3 h, yield = 64%–79%; yield = 85%; (ii) SOCl , reflux, 2 h, yield = 71%–87%; (iii) Piperazine, AcOH, r.t., 3 h, yield = 64%–79%; (iv) toluene, reflux,2 12 h, yield = 34%–67%. (iv) toluene, reflux, 12 h, yield = 34%–67%. Molecules 2016, 21, 1544 3 of 11 2.2. In Vivo Anti-Inflammatory Activities of Salicylate Derivatives Bearing Piperazine Moiety The in vivo anti-inflammatory activities of target compounds were evaluated against xylol-induced ear edema and carrageenan-induced paw edema in mice. All of the target compounds were administeredMolecules 2016, 21,at 1544 a dose of 100 mg/kg, and aspirin (100 mg/kg) and indomethacin3 of (5 10 mg/kg) were used as standard controls. The results indicated that all compounds exhibited favorable anti-inflammatory2.2. In Vivo Anti-Inflammatory activity compared Activities to of aspirin, Salicylate exceptDerivatives for Bearing compound PiperazineM10 Moiety(Table 1). Among the tested compounds,The in vivoM2 anti-inflammatory, M14, M15, and activitiesM16 were of target much compounds more potent were thanevaluated others, against and xylol- compound M16 exhibitedinduced theear edema strongest and carrageenan-induced anti-inflammatory paw activity. edema To in further mice. All depict of the target the dose-effect compounds relationships were of the fouradministered compounds at a dose (M2 of 100, M14 mg/kg,, M15 and, aspirin and M16 (100), mg/kg) various and indomethacin doses (5, 10, (5 andmg/kg) 20 were mg/kg) used of the four compounds,as standard controls. aspirin, The and results indomethacin indicated that were all compounds intragastrically exhibited administered. favorable anti-inflammatory The results showed activity compared to aspirin, except for compound M10 (Table 1). Among the tested compounds, M2, that theM14 anti-inflammatory, M15, and M16 were activities much more of potent the four than others, compounds and compound were increased M16 exhibited in the a dose-dependent strongest manneranti-inflammatory (Figure1). Especially, activity. To the further anti-inflammatory depict the dose-effect activities relationships of compounds of the four compoundsM15 and (M2M16, were better thanM14, that M15 of, and aspirin, M16), and various even doses equal (5, to 10, that and of 20 indomethacin mg/kg) of the atfour the compounds, same dose. aspirin, and indomethacin were intragastrically administered. The results showed that the anti-inflammatory activities Tableof the 1. Anti-inflammatoryfour compounds were (in increased vivo) activity in a ofdose-dependent the target compounds manner (Figure against 1). xylol-induced Especially, the ear anti- edema andinflammatory carrageenan-induced activities of paw compounds edema in M15 mice. and M16 were better than that of aspirin, and even equal to that of indomethacin at the same dose. Dose Ear Edema Paw Edema Dose CompoundTable 1. Anti-inflammatory (in vivo) activity of the target compounds against xylol-induced ear (mg/kg) Swelling Degree (mg) Inhibition (%) Swelling Degree (mg) Inhibition (%) (mmol/kg)