Although exhibited high inhibitory activity was found to be

Although exhibited high inhibitory activity, was found to be metabolically unstable due to the hydrolysis of the phosphate group (data not shown), which prompted us to modify the phosphate moiety of . Comparison of the activities between three compounds bearing no or different spacer groups (, , and , IC=9.32, 0.013, and 19.1μM, respectively) and the X-ray structure of bound to FBPase suggested that a spacer group of 1 atom in length was favorable for potent inhibitory activity. Therefore, we decided to evaluate phosphate mimics such as methylenephosphonate and difluoromethylenephosphonate. The syntheses of these phosphate mimics are illustrated in . Methylene analog and difluoromethylene analog were prepared from 7-hydroxy-1-tetralone . Triflation of followed by ketalization and Pd-catalyzed coupling with vinyl bromide gave olefin . Olefin was transformed into alcohol through dihydroxylation, oxidative cleavage, and subsequent reduction. Diethyl phosphonate was obtained by bromination of and subsequent Arbuzov reaction. Deketalization of provided ketone , and difluorination of followed by deketalization led to ketone . Bromination of , followed by cyclization with thiourea and subsequent hydrolysis afforded the final compounds . The inhibitory activities against human FBPase of these analogs are summarized in . Methylene analog displayed reduced activity (IC=0.840μM) relative to . In contrast, difluoromethylene analog showed similar potency (IC=0.047μM) to . Interestingly, the second p of benzylic α,α-difluorophosphonic nutlin (5.71) is more like that of phenylphosphate (6.22) than that of benzylic phosphonic acid (7.72), which suggests that the high charge density on the POH group is favorable to the enzyme-inhibitor interaction. Contrary to the phenyl phosphate structure in , the difluoromethylenephosphonate structure in is chemically stable to hydrolysis. This finding provided us with the possibility for further development of this tricyclic scaffold as an FBPase inhibitor. In summary, we designed and synthesized a series of tricyclic compounds as FBPase inhibitors. Compound exhibited high inhibitory activity, and an X-ray crystallographic study revealed that binds in the AMP binding site with hydrogen bonding interactions similar to those of AMP. In order to enhance metabolic stability, the structure of was modified to difluoromethylene analog which showed inhibitory activity similar to . Further studies on enhancing the inhibitory activity and developing prodrug compounds are being intensively conducted at this time. Acknowledgements
Introduction In vertebrate tissues, two distinct isozymes of fructose 1,6-bisphosphatase (FBPase; EC 3.1.3.11) are present [1]. The liver isozyme is a regulator of glucose synthesis from carbohydrate precursors. Muscle FBPase is expressed in all vertebrate cells and a growing body of research suggests that its physiological function goes beyond participation in the synthesis of glucose/glycogen from non-carbohydrates. Previously we showed that, muscle FBPase is found inside the nuclei and we suggested that this localization accompanies the cell’s potential to divide [2], [3]. We showed that, in HL-1 cardiomyocytes, FBPase disappearance from the nuclei coincided with a significant increase of the cells mortality [3]. Proteomic studies to date imply that FBPase might be involved in apoptosis [4], [5]. However, the data are still few and inconsistent, and the precise role of FBPase in the process remains unknown. Glycogen synthase kinase-3 (GSK3) has been recognized as a multi-functional kinase and a key element of a range of cellular processes, including glycogen biosynthesis, apoptosis or necrosis. In the heart, the β isoform of GSK3 acts as an inhibitor of hypertrophic signaling [6], where suppression of the enzyme reduces the ischemia/reperfusion injury through a mechanism dependent on the mitochondria [7]. This suggests a crucial role of GSK3β in cardioprotection.