Novel Quinoline-based Derivatives:A New Class Of PDE4B Inhibitors For Adjuvant-induced Arthritis

Inflammation is a dynamic process in which the body’s immune system responds to external factors such as injury,organic toxins,hypersensitivity reactions and microbial invasion.When the body is stimulated by external factors,it sends chemical signals to the immune system,which checks and repairs the injured tissues to protect itself from the attack.Acute inflammation is more transient,but if the acute inflammatory response fails to eliminate the stimulus,it can evolve into chronic inflammation with continued stimulation.Chronic inflammation is thought to be associated with the physiological and pathological processes of a variety of diseases,including rheumatoid arthritis,parkinson’s disease,alzheimer’s disease,inflammatory bowel disease and systemic lupus erythematosus.The inflammatory response is more complex,and it is associated with a multifaceted biological and biochemical process of defense system cells and overactive mediators.During the inflammatory process,inflammatory cells in the body release specific substances that accelerate the inflammatory process.These substances include specific substances of vasoactive amines such as histamine and bradykinin,endothelialderived nitric oxide(NO)and pro-inflammatory cytokines such as interleukins,lymphokines,interferons,tumor necrosis factor(TNF-α),etc.The current classical antiinflammatory agents are represented by NSAIDs and glucocorticosteroids,which cause gastrointestinal,cardiovascular,and renal adverse effects,and therefore there is an urgent need to develop more effective and less toxic drugs for inflammatory applications.In this experiment,by reviewing the literature related to inflammation,it was found that many clinically applied anti-inflammatory drugs have the structure of quinoline ring,so the quinoline ring was chosen as the basic parent nucleus and 31 quinoline derivatives were designed and synthesized by introducing two commonly used sulfonamide fragments on it for the novelty and anti-inflammatory activity improvement.For in vitro studies,the commonly used mouse mononuclear macrophage RAW264.7 was chosen as the inflammatory cell model.When lipopolysaccharide(LPS)stimulated RAW264.7 cells,the cells over-secreted NO,IL-1β and TNF-α,while the inflammatory mediator inducible nitric oxide synthase(iNOS)and inducible cyclooxygenase(COX-2)were also overexpressed.Therefore,the anti-inflammatory activity of the synthetic compounds was assessed in vitro by inhibiting lipopolysaccharide(LPS)-induced NO production levels in RAW264.7 cells(100 μM)after evaluating their toxicity in vitro.Among them,SAR analysis revealed that the best anti-inflammatory activity was achieved when three identical groups CH3(f4)were introduced on the benzene ring.the IC50 value was 20.40± 0.94 μM.f4 was further analyzed using ELISA to inhibit the production of proinflammatory cytokines IL-1β and TNF-α,where f4 inhibited the release of IL-1β and TNF-α in a concentration-dependent manner.the IC50 values were 18.98 ± 0.21 and 23.48± 0.46 μM,respectively.western blot results showed that f4 inhibited the expression of inflammatory mediators iNOS and COX-2 in LPS-induced RAW264.7 cells.Therefore,f4 was selected as a representative compound for reverse virtual screening and subsequent biological testing.The results of the virtual screen revealed a potential target for f4,PDE4B.molecular docking(CDOCKER)analysis of its interaction pattern further confirmed that f4 can access the PDE4B receptor binding pocket and interact with some amino acids of the receptor protein by hydrogen bonding.Cellular thermal displacement assay(CETSA)was performed to analyze the interaction of f4 with PDE4B protein in RAW264.7 cells.The results showed that the stability of PDE4B protein bound to f4 was enhanced.Also,the compound inhibited PDE4B enzyme(IC50=0.94±0.36 μM)comparable to the positive drug rolipram(IC50=1.04 ± 0.28 μM).In vivo studies showed that f4 improved the degree of foot swelling and knee joint pathology in rats with adjuvant-induced arthritis and dose-dependently reduced serum inflammatory factors TNF-α and IL-1β levels.The above experiments suggest that this quinoline derivative f4 is a novel anti-inflammatory agent targeting PDE4B.Therefore,the development and design of quinoline-based derivatives for anti-inflammatory applications may be seen as both an opportunity and a challenge.