Anti-inflammatory activity of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid in LPS-induced rat model.

INTRODUCTION: Salicylic acid derivate is very popular for its activity to suppress pain, fever, and inflammation. One of its derivatives is acetylsalicylic acid (ASA) which has been reported repeatedly that, as a non-steroidal anti-inflammatory drug (NSAID), it has a cardioprotective effect. Although ASA has various advantages, several studies have reported that it may induce severe peptic ulcer disease. We recently synthesized a new compound derived from salicylic acid, namely 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid (3-CH2Cl) which still has the benefit of acetylsalicylic acid as an analgesic and antiplatelet, but lacks its harmful side effects (Caroline et al., 2019). In addition, in silico studies of 3-CH2Cl showed a higher affinity towards protein receptor cyclooxygenase-2 (COX-2; PDB: 5F1A) than ASA. We hypothesized that 3-CH2Cl inhibits the COX-2 activity which could presumably decrease the inflammatory responses. However, no knowledge is available on the anti-inflammatory response and molecular signaling of this new compound. Hence, in this study, we investigated the potential functional relevance of 3-CH2Cl in regulating the inflammatory response in lipopolysaccharide (LPS)-induced rats. The results of this study show that this compound could significantly reduce the inflammatory parameter in LPS-induced rats. MATERIAL AND METHODS: Rats were induced with LPS of 0.5 mg/kg bw intravenously, prior oral administration with vehicle (3% Pulvis Gummi Arabicum / PGA), 500 mg/60 kg body weight (bw; rat dosage converted to human) of 3-CH2Cl and ASA. The inflammatory parameters such as changes in the temperature of septic shock, cardiac blood plasma concentrations of IL-1ß and TNF-α (ELISA), blood inflammation parameters, white blood cell concentrations, and lung histopathology were observed. Meanwhile, the stability of 3-CH2Cl powder was evaluated. RESULT: After the administration of 500 mg/60 kg bw of 3-CH2Cl (rat dosage converted to human) to LPS-induced rats, we observed a significant reduction of both TNF-α (5.70+/-1.04 × 103 pg/mL, p=<0.001) and IL-1ß (2.32+/-0.28 × 103 pg/mL, p=<0.001) cardiac blood plasma concentrations. Besides, we found a reduction of white blood cell concentration and the severity of lung injury in the 3-CH2Cl group compared to the LPS-induced rat group. Additionally, this compound maintained the rat body temperature within normal limits during inflammation, preventing the rats to undergo septic shock, characterized by hypothermic (t = 120 min.) or hyperthermic (t = 360 min) conditions. Furthermore, 3-CH2Cl was found to be stable until 3 years at 25°C with a relative humidity of 75 ± 5%. CONCLUSION: 3-CH2Cl compound inhibited inflammation in the LPS-induced inflammation response model in rats, hypothetically through binding to COX-2, and presumably inhibited LPS-induced NF-κß signaling pathways. This study could be used as a preliminary hint to investigate the target molecular pathways of 3-CH2Cl as a novel and less toxic therapeutical agent in alleviating the COX-related inflammatory diseases, and most importantly to support the planning and development of clinical trial.

Evaluation of analgesic and antiplatelet activity of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid.

Acetylsalicylic acid is used as a non-steroidal anti-inflammatory drugs (NSAID) and antiplatelet agents by inhibiting cyclooxygenases. However, therapy using acetylsalicylic acid could induce gastric bleeding and cause other gastrointestinal toxicity. The aim of this study was to demonstrate the synthesis of a new compound bearing salicylic acid residue namely 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid, to analyze its potential as a ligand for human cyclooxygenase-2 (COX-2) receptor, to evaluate its toxicity level and its effectiveness for analgesic and antiplatelet agent compared with acetylsalicylic acid. Synthesis of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid was conducted by microwave irradiation. The purity of this compound was evaluated with TLC, IR, NMR, and EDS spectroscopy. The chemical characterization and docking studies against human COX-2 (PDB:5F1A) was performed in-silico. The acute oral toxicity assay was performed under OECD guidelines. The analgesic activity study was performed by plantar and writhing test on animal model. For anti-platelet activity study, we performed tail-bleeding assay and flow cytometry based platelet aggregation assay. We could successfully synthesize a pure white crystalline 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid. In-Silico G-Score result of those compounds gives us preliminary hint of the potential affinity of this compound as a ligand for COX-2 receptor (PDB: 5F1A). Acute toxicity and microscopic gastrointestinal assessments indicated non-observable harmful toxicity parameters. The plantar response time of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid treated groups showed a significant increment (P < 0.01), and the nociceptive response in writhing test demonstrated a significant dose-dependent decrement. This indicated that its analgesic activity was better than acetylsalicylic acid. The platelet aggregation of 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid was lower than its controls, indicating an aggregation inhibition pattern. The animals treated with 2-((3-(chloromethyl)benzoyl)oxy)benzoic acid gave a longer bleeding time. Overall, this study demonstrated a successful synthesis of pure 2-((3-(chloromethyl)benzoyl)oxy) benzoic acid. We postulated that this compound was better than acetylsalicylic acid, exhibiting excellent analgesic and antiplatelet activity with no toxicity impact.