Design,Synthesis And Structure-Activity Relationship Of Maleimide And Nitrovinyl Ester FBPase Inhibitors

Diabetes is one of the most important non-communicable diseases currently threatening global human health.There are approximately 425 million people with diabetes worldwide,and the most common among these people with diabetes is type Ⅱdiabetes.Clinical studies have shown that excessive hepatic glucose production is responsible for the increase in fasting blood glucose in type 2 diabetic patients.Hepatic glucose is mainly derived from hepatic glycogenolysis and gluconeogenesis,and FBPase is a key regulatory enzyme in the gluconeogenesis pathway.Since FBPase is only involved in the gluconeogenesis process and does not participate in hepatic glycogenolysis,inhibition of FBPase can reduce blood sugar while avoiding the risk of hypoglycemia.Therefore,FBPase is considered to be a novel potential target for the treatment of type Ⅱ diabetes.According to the literature,FBPase usually has two key sites:fructose-1,6-diphosphate(FBP)substrate site and adenosine phosphate(AMP)allosteric site.At present,inhibitors designed with substrate sites are rarely reported,and most of them designed for allosteric sites.However,in the human body,many other enzymes(such as adenosine kinase,AMP deaminase,etc.)also have sites that bind to AMP.Therefore,so inhibitors designed for AMP sites may lead to inhibition of other enzymes while inhibiting FBPase and resulting in toxic side effects.In the previous study of our group,we were found that the small molecule of nitrostyrene had a good inhibitory effect on FBPase.And its mutant experiments showed that the compound form a covalent bond with the cysteine at position 128 near the substrate cavity to inhibit FBPase.The nitrostyrene has a molecular docking with FBPase,and its docking mode was analyzed.It was found that there was a small cavity near the binding site.In order to obtain a more active inhibitor,an extended nitrostyrene molecule is designed to occupy the cavity to better bind to FBPase.Therefore,we synthesized a nitrovinyl phenyl benzoate compound(SH series).However,in the study of the structure-activity relationship of SH series compounds,it is found that the change of substituents on the benzene ring has little effect on the inhibitory activity of SH series compounds.The activity of SH series compounds may be mainly derived from that nitrostyrene(electrophilic warhead)covalently bound to cysteine.It suggests that the electrophilic warhead is important for inhibitor’s inhibitory activity,so we tried to choose other electrophilic warheads to combine with FBPase.The combination of NEM and FBPase has been reported in the crystal.The structure of the crystal complex(pdb:4H46)shows that NEM forms a covalent bond with C128 of FBPase.Therefore,NEM can be used as an electrophilic warhead that is covalently bound to FBPase.Molecular docking of NEM and FBPase revealed that the NEM had no significant interaction with other surrounding amino acids except for covalent binding to cysteine.Therefore,it is considered to replace the ethyl moiety of NEM with an amide or urea which having more hydrogen bond acceptors,and at the same time,to extend the NEM molecule with a linking group,and it is expected that the extended portion can extend into the nearby cavity to be more compatible with FBPase.Based on this idea,an amide-substituted maleimide compound(Yr series)and a urea-substituted maleimide compound(Yrx series)were synthesized.The most potent inhibitory compound was Yr11,which had an inhibition constant of 0.19±0.02 μM,which was about 80-fold higher than that of NEM(IC50=16.85±1.63 μM).