Discovery Of Novel 3-Hydroxyandrosta-5,7-Diene-17-Carboxylic Acid Derivatives As Anti-Inflammatory Bowel Diseases Agents

Inflammatory bowel diseases(IBD),including ulcerative colitis(UC)and Crohn’s disease(Crohn’s disease,CD),is a type of autoimmune disease occurring frequently in human.The diseases are mainly characterized by uncontrolled inflammation of the intestinal mucosa,accompanied by abdominal pain,diarrhea,mucus and blood stool,intestinal obstruction,and weight loss.With the global morbidity rising year by year,IBD has been considered as a global disease affecting human health.The current treatment of IBD is limited to controlling symptoms,maintaining remission and preventing recurrence,usually with aminosalicylate preparations,glucocorticoids,immunomodulators and biological preparations.Among them,glucocorticoids are still the most effective treatment for patients with an acute disease flare,despite the severe side effects of long-term medication.New steroid drugs with less side effects but better curative effect is of great significance for the treatment of IBD.Based on the structure of 3-hydroxyandrostane-5,7-diene-17-carboxylic acid(17COOH-7DA),a series of new steroids were designed and synthesized.RAW 264.7 macrophages and DSS-induced colitis mouse model were used to evaluate in vivo and in vitro anti-inflammation activities and explore their possible mechanisms of action.The specific experiments are as follows:(1)Based on the structure of introduction of 17-COOH-7DA,side chains with different lengths were introduced;further,the core structure was changed to pregnenolone,and side chains with different heteroatoms were introduced to improve the anti-inflammatory activity.With pregnenolone acetate as the raw material,the intermediate 3-hydroxyandrostan-5-ene-17-carboxylic acid is obtained by oxidation of acetyl group to the carboxyl group.The intermediate is esterified in the side chain,and dehydrogenated at the 7 position of the steroid nucleus to obtain 3-hydroxy androstane5,7-diene-17-carboxylate series compounds(5a-d).Compounds 6a-s were obtained by amidation of the side chain of the intermediate.Compound 8a-c were synthesized through dehydrogenation of the steroidal nucleus of pregnenolone and then alkylation by Grignard reagent;(2)The nitrite measurement was used to evaluate the anti-inflammatory activity of the synthesized compounds.The experimental results showed that in the LPSinduced RAW 264.7 macrophages,9 compounds significantly inhibited the generation of NO at 20 μM(inhibition rate>50%);qPCR,Western Blot and Elisa assays have further verified the inhibitory effect of compound 6q on inflammatory factors at the translation and protein levels respectively,and this anti-inflammatory effect may be achieved through the NF-κB signal transduction pathway;(3)After one-week acclimation,mice were given DSS aqueous solution to induce acute colitis,and treated with 6q solution through intraperitoneally injection for one week.The mouse colon was taken for histological,immunohistochemical and protein content analysis.The experimental results showed that 6q could effectively alleviate the inflammatory response such as colon shortening and crypt abscess induced by DSS,probably by blocking the NF-κB signaling pathway.However,the inflammatory response did not improve significantly when the dose was too high.In summary,a series of 17-COOH-7DA derivatives were designed,synthesized,and biologically evaluated.Compound 6q was found to have a significant antiinflammatory effect in vitro.In addition,in the DSS-induced colitis model,6q significantly improved the inflammatory response in mice,indicating that the novel compound is expected to be used in the treatment of IBD and is worthy of further study.