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 antiinflammatory 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 cyclooxygenase2 (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-1β 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-1β
(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-κβ 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.