Design,Synthesis And Biological Activities Of Human Urate-anion Transporter-1(hURAT1)Inhibitors

Uric acid comes mianly from the degradation of purine and the biological transformation of nucleic acids.High uric acid level is easily to lead gout.Gout is a metabolic disease caused by the deposition of sodium urate crystals in joints and soft tissue that results from high serum uric acid level.Current uric-acid-lowering drugs such as allopurinol and benzbromarone inevitably have side effects,for example,they may cause bone marrow suppression,allergy,liver damage and other adverse reactions.So the development of a new class of low-toxic drugs is very important.Promoting uric acid excretion is one of the main ways to reduce the uric acid level,and uric acid excretion is dependent on a variety of urate transporters which locate in renal tubular epithelial cells.The human urate-anion transporter-1(hURAT1)is an important transporter that situate in the apical membranes of the epithelial cells,and it can exchange anion with uric acid and promote uric acid reabsorption.So,inhibit the activity of hURAT1 can reduce the reabsorption of uric acid and lower the serum uric acid level effectively.HURAT1 can only be transport uric acid and its analogs or the structures with an aromatic carbon contains a pyrimidine or imidazole groups although it is an anion transporter,and the substrate binding specificity making hURATl a good target for drug therapy.Currently,hURAT1 inhibitors have become a hotspot in the treatment of gout.Benzbromarone inhibits multiple urate transporters including hURATl,the non-specificity may be one of the reasons to produce adverse reactions;and the study of benzbromarone structure-activity relationship exhibits that the the stronger the acidity is,the better the activity of reducing uric acid level is.So we take benzbromarone as the lead compound,introduce the carboxyl groups which have higher acidic to replace the hydroxyl group to design 3 target compounds of B series,and expect the introduction of carboxyl groups can improve the ability of lowering serum uric acid level and the hURAT1 receptor selectively.The p-/m-methyl terephthalate or 1,3,5-Benzenetricarboxylicacid as raw material,after acylation,Friedel-Crafts acylation and hydrolysis acidification reaction to obtain the target compounds.Lesinurad is the first specificity hURAT1 inhibitor approves by FDA,we take it as the lead compound,design me-too drug by the use of biological electronic principle.Since the imidazole group can be specificity combined with hURPAT1,we design L-2 that introduce imidole to alternative triazole and hope the introduction of imidazole could improve the activity and selectivity.The 1-Cyclopropyl-4-isothiocyanato-napht-halene as raw material,after the reaction with thiophosgene,annulation reaction,bromination,nucleophilic substitution reaction and hydrolytic reaction,we get compound L-2.We adopt a new method to synthesis Lesinurad,use formylhydrazine complete cyclization reaction,and explore the cyclization reaction and nucleophilic substitution reaction comditions to improve the yield of the synthesis.Various potent hURATl inhibitors have following characteristics:(1)They all have a planar aromatic ring;(2)Aromatic ring combine with an atom with unshared pair electrons such as O,S,N;(3)Acetoacethl group connect with O,S,N.Based on these characteristics,we design 4 compounds of Py series and 6 compounds of In series.Since URAT1 is a membrane protein,its crystal structure has not been reported,we use protein homology modeling constructs its crystal structure,and the structures of the two series are molecular docking with hURAT1,docking results show that the compounds of Py-3 and In-1 have excellent binding capacity to hURAT1,their scores are 4.3 and 6.7,and the docking score of In-1 higher than Lesinurad(5.4).The hydroxyl or mercapto-substituted pyridine as raw material,after nucleophilic substitution reaction and hydrolytic reaction we get Py series;the In series get by amidation reaction,Boc protective reaction,nucleophilic substitution reaction and so on.All the target compounds are characterized by ESI-MS,1H-NMR,13C-NMR.We test the in vitro hURAT1 and hOAT1 inhibitory activity of these target compounds with benzbromarone or Lesinurad as a positive control and in vivo hURAT1 inhibitory activity of activity compounds in vitro.Test results are as follows:(1)Compound B-1 retains a certain inhibition of hURAT1,and exhibits better hURATl selectivity than Benzbromarone;(2)Compound L-2 shows better inhibition and specificity than Lesinurad,its IC50(3.22 ± 0.42 μM)is lower than the positive drug(65.47 ± 6.87 μM)an order of magnitude;and its IC50(9.09 ± 2.67 μM)of hOAT1 is also lower than Lesinurad(43.94 ± 4.06 μM);(3)In vivo tests of compound L-2 in rats shows stronger capacity to reduce uric acid than Lesinurad;(4)The compounds of Py series have weak inhibitory on hURAT1 and certain stimulative effect on hOAT1,which can also promote the excretion of uric acid;(5)The compounds of In series can specifically inhibit hURAT1,compounds In-4 is the most potent hURATl inhibitor.The results also presents some simple structure-activity relationships,which are summarized as follows:(1)The introduction of a single carboxyl group in the structure skeleton of B series can improve the specificity of hURAT1 and retain the ability of anti-hURATl;the compound containing double carboxyl groups will lose its inhibitory to hURATl;(2)The introduction of imidazole group in the structure skeleton of Lesinurad may improve the inhibitory activity and selectivity of the compound to hURAT1;(3)The introduction of electron withdrawing groups to benzene ring and steric hindrance group to side chain in the structure skeleton of In series will enhance the hURATl inhibitory.We proved the importance of carboxylic group in B series compound and imidazole group in compound L-2 on the activity and specificity to hURAT1 inhibitors in this paper,and successfully synthesized compound L-2 which shows better activity and selectivity than the positive control in vitro test and in vivo test,also we get the structure skeleton of In series which exhibit potent hURATI inhibitory.We will continue to design other compounds on the basis of compound In-4 to screen out the compounds with better activity and specificity.