Design,Synthesis,and Biological Activity Evaluation Of Novel Fused Pyrimidines As Potent URAT1 Inhibitors

Gout is an inflammatory metabolic disease,caused by the chronic deposition of monosodium urate(MSU)crystals due to elevated uric acid concentrations in the body.Currently,hundreds of millions of people worldwide suffer from gout,and men are the most commonly affected.At present,the types of drugs used clinically to treat gout are very limited,mainly drugs that inhibit uric acid production and promote uric acid excretion from the body,but these drugs have many shortcomings in terms of efficacy and safety.Therefore,there is an urgent need to develop novel potent and safe anti-gout drugs.Urate transporter 1(URAT1)is a critical protein involved in the reabsorption of uric acid,which controls over 90%of uric acid reabsorption after glomerular filtration.Inhibition of URAT1 reduces the uric acid reabsorption and increases uric acid excretion,ultimately reducing the serum uric acid levels and the risk of gout attacks.Therefore,URAT1 is a crucial target for developing safe and highly effective uricosuric agents to manage hyperuricemia and gout.Unfortunately,the crystal structure of the URAT1 protein remains unresolved.As a result,conducting target-based rational drug design is currently impossible.Lesinurad is the first approved URAT1 inhibitor,but it was found to have severe nephrotoxicity and poor curative efficacy during use.It was listed as a black box warning drug by the FDA and was withdrawn from the market in the United States in 2019.In light of the insufficient efficacy and safety of lesinurad,and the increasing demand for more effective and safe anti-gout drugs,this study focused on URAT1 as the target and utilized a combination of scaffold hopping and molecular hybridization strategies to modify various structural regions of the lead compound lesinurad based on pharmacophore and 3D-QSAR models.The fused pyrimidine backbones containing carboxylic acid side chains were spliced with the privileged cyanobenzene backbone containing amino groups,resulting in the design and synthesis of 54 novel URAT1 inhibitors containing fused pyrimidines.Through evaluation via the mouse acute hyperuricemia model(subcutaneous injection of 400 mg/kg potassium oxonate and intragastric administration of 600 mg/kg hypoxanthine),40 compounds were found to be superior to the positive drug lesinurad(36.5%)in terms of the in vivo serum uric acid lowering activity.Among them,the fluoroquinazolines YJ-20(74.2%)and YJ-21(71.9%),thienopyrimidine YJ-26(75.4%),dihydrocyclopentapyrimidines YJ-49(89.9%)and YJ-50(78.3%)had a serum uric acid reduction rate of more than 70%,being about two-fold more potent than that of lesinurad.In addition,the in vitro URAT1 target validation assay was performed on these representative compounds in HEK293T cells stably expressing hURAT1,and the changes in the radioactive intensity of 14C-labeled uric acid uptake by cells at different concentrations were measured.Among them,the thienopyrimidine YJ-26(IC50=6.90 μM)exhibited URAT1 inhibitory activity comparable to that of lesinurad(IC50=7.17 μM).It is worth noting that YJ-26 exhibited good safety in the acute toxicity experiment,as no acute toxicity was observed even at a single dose of 1000 mg/kg.In conclusion,we designed and synthesized 54 URAT1 inhibitors with fused pyrimidine cores using a ligand-based drug design strategy.Several derivatives exhibited significant in vivo serum uric acid lowering activity in an acute hyperuricemia model.In vitro URAT1 target validation assays and in vivo acute toxicity experiments demonstrated that YJ-26 had excellent activity and safety profiles.All these results suggest that thienopyrimidines are promising scaffolds for the development of novel URAT1 inhibitors with potential for further optimization.Meanwhile,YJ-20 and YJ-49,which showed weak inhibitory activity against URAT1 but strong uric acid-lowering activity,are currently being evaluated for their activity against other targets such as XO and GLUT9,which are related to uric acid metabolism and transport.