Design And Synthesis Of Potent And Multifunctional Aldose Reductase Inhibitors Based On Pyrido[2,3-b] Pyrazine And Benzothiazine

Diabetes mellitus?DM?,companied with cancer and cardiovascular disease,are recognized as the three most leading cause to human death.All form of DM,include type I and type II,are characterized by hyperglycaemia and the development of chronic diabetic complications including neuropathy,nephropathy,cataracts and retinopathy.More and more evidence has established that all forms of diabetes are vulnerable to chronic diabetic complications,and those diseases are the major menace to diabetic patients.In past several decades,increasing studies have shown that the polyol pathway of glucose plays a crucial role in the development of chronic diabetic complications.In this glucose metabolism pathway,the enzyme ALR2 firstly catalyses the reduction of glucose to sorbitol with NADPH as the rate-limiting step of this metabolism pathway,and then segmental sorbitol dehydrogenase converts sorbitol into fructose with reduction of NAD⁺.Under euglycemia circumstances,the ALR2 has little activity to the polyol pathway,and the most glucose is predominantly converted to glucose-6-phosphate by hexokinase and then implements the glycolytic pathway.However,in hyperglycemia,the activity of ALR2 is stimulated and about one third of the total glucose is metabolized through the polyol pathway in tissues demonstrating insulin-independent uptake of glucose,such as lens,kidney,retina,and peripheral nerves.Sorbitol is endowed with stronger polarity and hard to penetrate through cell membrane,therefore,the abnormal accumulation of sorbitol through the polyol pathway results in osmotic imbalance,cell swelling,and membrane permeability changes,and further grows into diabetic complications.Concurrently,the alteration in cellular redox potentials and the activity of enzymes such as nitric oxide synthase?NOS?and glutathione reductase caused by the decrease of NADPH and NAD⁺would further lead to the cellular oxidative stress,as a consequence of the imbalance between increased production of radical oxygen species?ROS?and reduced intracellular antioxidant defense.In addition,the increased AGEs caused by abnormal enhance of the levels of glycating agents could in return give rise to other pathological change in the functions of intracellular proteins and result in further accumulation of ROS.All those abnormal changes rooted in the polyol pathway regulated by ALR2 were responsible for multiple pathogenic mechanisms of the diabetic complications.Therefore,inhibiting the activity of ALR2 and further reducing the polyol pathway of glucose metabolism is supposed to be an efficient therapy to remedy and delay the development of diabetic complications.In recent decades,a number of structurally different ARIs have been developed and some of them are endowed with excellent inhibitory activity,but epalrestat is the only ARI available for the therapy in Japan and more recently in China and India.Most of those ARIs that appeared to be promising have not yet succeeded in the clinical trials mainly due to low in vivo efficacies or pharmacokinetic drawbacks.Although at present the underlying mechanism of the low efficacy is not clear,indeed,the pathogenesis of the chronic diabetes complications and oxidative stress always promote to each other.Thus,the design and synthesis of multifunctional ARIs to inhibit the activity of ALR2 and simultaneously to reduce the progress of oxidative stress process may be a new efficient therapy strategy.Based on this thinking,some different series of multifunctional ARIs were designed and synthesized.Firstly,aseriesofaldosereductaseinhibitorsbasedonpyrido[2,3-b]pyrazin-3?4H?-one was obtained.Most of these 28 derivatives,having a substituted C2 aromatic group and a N4 acetic acid group on the core structure,were found to be potent and selective aldose reductase inhibitors in vitro with submicromolar IC₅₀ values against ALR2 and no more than 40%to ALR1 at the concentration of 10?M.In particular,64c was proved to be the most active with IC₅₀ value 0.009?M.On the other hand,some of these endowed with not only good activity in the ALR2 inhibition but also potent antioxidant activity.Compound 67i,having excellent inhibitory activity of ALR2 was proved to be a better antioxidant compare to the well-known antioxidant Trolox at the concentration of 100?M.Structure-activity relationship and molecular docking studies highlighted the importance of substitution features of phenolic hydroxyl-substituted C2aromatic group and carbon-carbon double bond as spacer in the pyrido[2,3-b]-pyrazin-3?4H?-one scaffold to be potent multifunctional ALR2 inhibitors.Continue investigation have resulted in other series of 2H-benzothiazine 1,1-dioxide derivatives as novel,potent and multifunctional ALR2 inhibitors,of which the series 82a-f with N2 styryl side chain was potently active in the ALR2 inhibition with IC₅₀ values at submicromolar level,but less active in the ALR1 inhibition with inhibition percentages no more than 25.1%at concentration of 10?M,indicating good selectivity of the compounds for ALR2.82a bearing the phenolic 4-hydroxyl at the N2 styryl side chain was found to be the most potent ARI with IC₅₀ value of 0.094?M.In addition,DPPH radical scavenging potency was tested in order to verify the antioxidant activity of the designed compounds.All compounds of series 80-82 with the N2 styryl side chain displayed good antioxidant activity by the test of DPPH radical scavenging,and 82c containing 3,4-dihydroxyl groups in the N2-styryl was identified as a distinguished antioxidant even comparable with Trolox.82c was also excellent in the ALR2 inhibition.Structure-activity relationship studies on the compounds suggest that the phenolic hydroxyl-substituted N2 styryl besides the carboxylate head group is the key structure for the potent ARIs,and the vinyl spacer of the N2 side chain is essential for the antioxidant ability of ARIs.Lastly,in order to get more detailed information in the process of interaction between ALR2 and compounds,molecular docking studies were execute on compounds 64i and 82a,the result shows that the compound 64i fitted well to the active site of ALR2,in which the carboxylate group formed tight hydrogen bonds with Tyr48?2.62??,Trp111?3.12??and His110?2.49?and 2.87??.On the other hand,the 4-hydroxyl oxygen atom of the C2-side chain formed an additional hydrogen bond with the side chain of Thr113?3.09??explaining the key role of the hydroxyl in the enhancement of the ALR2 inhibition.Moreover,the phenyl ring of C2 side chain was well paralleled to the indole ring of Trp 111forming a stacking interaction,which well explain the experimental results,the similar results were obtained from the molecular docking studies on compounds 82a.The results from these studies have encouraged us to continue our investigations into the design of more potent,selective and multifunctional analogues by conducting appropriate modifications based on aromatic heterobicyclics.