Discovery Of Novel Fused Heterocycle-containing Lead Compounds And Drug Candidates Targeting URAT1 For Treating Gout

Gout is caused by the disorder of purine metabolism in human body,and hyperuricemia(HUA)is the pathological basis.Both of gout and HUA are metabolic diseases that seriously endanger human health.In the past half century,incidence of gout has explosively increased and showed the increasing trend in younger people.Moreover,the clinically used anti-gout drugs possess disadvantages such as low activity and severe side effects,and long-term usage also leads to drug resistance.It makes clinical treatment facing the dilemma of shortage of effective and new drugs.Therefore,the novel safe and effective drugs are urgently needed for treating gout and hyperuricemia.The studies about pathogenesis of gout showed that abnormal reabsorption of uric acid by anion transporters in the glomerulus resulted in excretion disorder,which existed in more than 90%of hyperuricemia patients.Among the anion transporters,uric acid transporter 1(URAT1)plays an important role in the process of uric acid reabsorption,making it an attractive target for the design of novel hypouricemic drugs.However,the approved uricosuric drugs have the disadvantages of insufficient activity and high hepatorenal toxicity.Considering disadvantages of the clinically used anti-gout drugs and the urgent clinical needs,systematic structural modifications were carried out via the strategies of pharmacophore-based scaffold hopping and bioisosterism with lesinurad and candidate T7 as lead compounds in this study.Meanwhile,a stable mouse hyperuricemia model was well established,with which the activity and druggability of the novel compounds were evaluated.In addition,the virtual screening of URAT1 inhibitors was displayed by establishing URAT1 homology modules.This dissertation is described as the following five parts.Establishment of an in vivo evaluation method for URAT1 inhibitors and druggability evaluation of pyridoimidazole or acylsulfonamide compounds T7 and S3.In order to evaluate the in vivo activity of anti-hyperuricemic compounds more effectively and accurately,a stable acute hyperuricemia model in mice was firstly optimized and evaluated by orthogonal test method.At the same time,a stable and efficient hyperuricemia model in rat and a screening method of target inhibitory activity in vitro for further evaluation of the SUA-reducing activity of the compounds were as well established.Then,the in vitro and in vivo activities of the Lead compounds T7 and S3 which were discovered previously were studied via the models mentioned above.T7 and S3 showed excellent in vivo activity,with decrease ratio(DR)of 2.5 times higher than that of lesinurad.Furthermore,the lowest effective dose of T7 in mice was finally determined to be 1 mg/kg by gradient dose administration,and meanwhile both compounds exhibited excellent SUA-lowering activity in rat models.The in vitro activity results showed that inhibitory activity of T7(IC50=1.57μM)was 4.6-fold stronger than that of lesinurad(IC50=7.21 μM).In this chapter,we also systematically evaluated the druggability of T7 and S3.The bioavailability of T7 in rats was 76.3%,and the maximum tolerated dose was 500 mg/kg.All the results indicated that T7 and S3 were promising anti-gout drug candidates.Meanwhile,T7 and S3 showed several shortcomings through the above evaluations,which compelled us to undertake in-depth optimization.According to the structural characteristics of lesinurad derivatives,the key pharmacophores in different components of the structures were modified to obtain URAT1-targeting drug candidates with high efficiency and low toxicity.The work was described in the following three chapters successively.Discovery of novel ligand-based pyridoimidazole mercaptoacetic acid URAT1 inhibitor TD-3 as a hypouricemic drug candidate.In order to carry out ligand-based compound design more efficiently,the lead compounds obtained in the previous work and listed drugs were firstly used to construct the pharmacophore of lesinurad derivatives.Considering the "three-point" pharmacophore and the disadvantages of insufficient efficacy and poor safety of the approved URAT1 inhibitors,the hydrophobic regions of the structures of lesinurad and T7 were modified via scaffold hopping and bioisosterism strategies.Finally,a total of 60 novel URAT1 inhibitors were designed and synthesized via Buchwald Hartwig cross coupling,cyclization and nucleophilic substitution reactions successively.The in vivo activity results in mice showed that 26 compounds possessed robust SUA-lowering activity compared to that of lesinurad.Among them,compound TD-3 showed a decrease rate of 96.1%in the in vivo activity assay,which was the most potent compound in this section.Meanwhile,the in vitro activity results suggested that TD-3 could significantly inhibit URAT1 with IC50 of 1.36 μM,which was 4.1 times higher than that of lesinurad(IC50=5.54 μM).Further activity results show that TD-3 also possessed outstanding SUA-lowering activity in rats,and the minimum effective dose in mice was 0.5 mg/kg,which was significantly better than those of T7 and lesinurad.Finally,in the druggability evaluation experiment,TD-3 achieved excellent pharmacokinetic properties with oral bioavailability of 59.3%.Additionally,TD-3 showed favourable safety profiles.The maximum tolerated dose of TD-3 was 500 mg/kg,and no obvious subacute toxicity was observed in Kunming mice.Overall,all the results indicated that TD-3 could be considered as a promising drug candidate for the treatment of hyperuricemia and gout.Bioisosterism-based discovery of novel pyridzimidazolyl sulfonamide-typed URAT1 inhibitor TS-2 as hypouricemic drug candidate.In this chapter,with drug candidate S3 as starting point,a variety of structural optimization were carried out on the peripheral substituents through bioisosterism and molecular hybridization strategies.Finally,two series of 40 novel URAT1 inhibitors were designed and synthesized.The in vivo activity results showed that 23 compounds in the two series had remarkable in vivo SUA-lowering activity,which were better than that of the control drug lesinurad.Among them,TS-2 showed the best activity.After administration,the blood uric acid level in disease model(SUA=1162.00 μM)was reduced to healthy level(SUA=143.20 μM)rapidly,with the DR of 96.8%.This was more than two times higher than that of lesinurad.In the in vitro activity assay,the URAT1 inhibitory activity of TS-2(IC50=0.19 μM)was 29.2 times higher than that of lesinurad.A more detailed activity study showed that TS-2 reduced the SUA level in the rat model(SUA=917.80μM)to the blank SUA level(SUA=108.75μM)within four hours,with DR of 91.9%.Through the dose gradient setting,it was found that the lowest effective dose of TS-2 in mice was 0.25 mg/kg.The activity of TS-2 was significantly improved compared with that of the lead compounds.Further druggability evaluation showed that TS-2 possessed acceptable pharmacokinetic properties and significantly improved safety properties.The maximum tolerated dose of TS-2 in mice was greater than 1000 mg/kg,which was significantly better than that of lesinurad.In conclusion,TS-2 is suitable for further investigations towards hypouricemic clinical candidates.Scaffold hopping-based discovery of novel fused pyrimidine URAT1 inhibitor F-5 as lead compound.In this chapter,the 3D-QSAR model of lesinurad derivatives was established based on our previous study.Here,combined with the tolerated region predicted by three-dimensional field of this model,multiple modifications were conducted for the core component of lesinurad and T7.The volume of the core ring and the spatial arrangement of the pharmacophore were modified through scaffold hopping and other strategies.And 9 series of URAT1 inhibitors,which contained 54 novel compounds,were finally designed and synthesized.The in vivo activity results showed that most compounds in this chapter exhibited potent SUA-lowering activity.Especially,compound F-5 displayed the best activity whether in vivo or in vitro,with DR of 93.2%and URAT1 inhibitory activity IC50 of 2.01 μM.The in vivo and in vitro activities of F-5 were both more than two times higher than those of lesinurad.Additionally,the lowest effective dose of F-5 was 0.5 mg/kg.At the end of this chapter,the preliminary druggability evaluation showed that F-5 possessed favorable safety and pharmacokinetic properties,and it could be treated as a lead compound for further optimization.AlphdFold2-based establishment of URAT1 homology model and virtual screening of URAT1 inhibitors.Since URAT1 is a transmembrane protein with complex structure,its crystal structure has not been analyzed yet,which restricts the target-based rational drug design.Therefore,in this chapter,the homology model establishment and potential binding pocket prediction of URAT1 were carried out through Alphdfold2.Furthermore,this model was used to predict the binding mode and pharmacophore characteristics of candidate drugs obtained above,which provided a theoretical basis for the rational drug design in subsequent research.A high-throughput virtual screening for different commercial compound libraries was as well conducted via well-established model and predicted binding sites,and several potential URAT1 inhibitors were finally obtained through extensive evaluation.Then the in vivo activity results showed that three small molecules showed obvious SUA-lowering activity in animals,which provided a basis for the follow-up exploration of new URAT1 inhibitors.It is expected that the drug candidates with novel structure,high efficiency and low toxicity would be obtained through further structural optimization.In summary,considering insufficient bioactivity and poor safety of existing URAT1 inhibitors,a total of 154 novel compounds were designed with the combined medicinal chemistry campaign,involving the strategies of molecular hybridization,scaffold hopping,bioisosterism and computer-aided drug design.Subsequently,in vivo activity screening in disease models,in vitro inhibitory activity assay and systematic druggability evaluation were performed,and several candidates were discovered.Among them,TD-3,TS-2,F-5 and T7 showed the outstanding properties.Moreover,the structure-activity relationship,binding mode and pharmacophore characteristics of lesinurad derivatives obtained here were predicted and analyzed through URAT1 homology model,pharmacophore model and 3D-QSAR model.Overall,this study established a solid foundation for the further structure-based design of URAT1 inhibitors with higher efficiency and lower toxicity.