Several classes of small molecule

Several Gedunin synthesis of small-molecule inhibitors of FBPase have been reported. These inhibitors can be structurally classified into two groups; non-phosphorus-based inhibitors and phosphorus-based inhibitors. In the former group, several chemotypes including anilinoquinazoline, indole dicarboxylic acid, piperazinedione, and benzoxazole benzenesulfonamide were reported, although they showed only modest inhibitory potency against FBPase. Also in this group, bissulfonylurea was described as a potent FBPase inhibitor with novel interaction. In the latter group, in contrast, AMP mimetic MB05032 () exhibited high inhibitory activity. A prodrug of MB05032 (CS-917, ) lowered blood glucose levels in animal models and was entered into clinical development (). With the intention of developing a novel class of FBPase inhibitors, we turned our attention to phosphorus-based AMP mimetics in recognition of their in vitro and in vivo potencies. From the binding mode of AMP and FBPase, the hydrogen bonding interactions of the NH and POH groups of AMP seemed to play an important role in realizing high affinity. Based on this structural information, we focused our attention on placing the two groups at the appropriate position to obtain such high affinity. AMP mimetic MB05032 has a rotatable biheteroaryl moiety; we expected that increasing the structural rigidity of the compound would be useful to fix the NH and POH groups in the appropriate binding orientation. This led us to design rigid tricyclic scaffolds, and the three kinds of the scaffolds were designed by varying the central ring size from a five- to a seven-membered ring (). In order to facilitate changing the positions of the POH group, a benzene ring instead of a furan ring was introduced to the tricyclic scaffolds. A series of tricyclic derivatives was efficiently synthesized according to . The bicyclic phenols were converted to diethyl phosphonate via triflation and Pd-catalyzed phosphonylation. Diethyl phosphate were obtained by direct phosphorylation of , and diethyl phosphonate were acquired by condensation of with diethoxyphosphorylmethyl tosylate. Bromination of followed by cyclization with thiourea and subsequent hydrolysis afforded the final compounds . The inhibitory activities against human FBPase of the tricyclic derivatives are summarized in . Initially, to optimize the position of the POH group, a variety of benzene phosphonic acid derivatives were prepared (). Most of these compounds showed low activity, but only , possessing a relatively flexible scaffold with a seven-membered central ring, showed moderate activity (IC=0.169μM). This result suggested that some degree of structural flexibility was beneficial in improving inhibitory activity and prompted us to introduce a spacer group of 1–2 atoms in length connecting the POH group with the tricyclic scaffolds. Due to synthetic feasibility, linkers such as –O– and –CHO– were selected for further investigation (). Introducing the spacer groups showed a tendency to increase activity, and several compounds bearing –O– linker (i.e., phosphate compounds) exhibited enhanced activity (, , and , IC=0.070, 0.013, and 0.022μM, respectively). In particular, exhibited the highest potency, almost equal to that of MB05032. In order to obtain insights into the binding mode of this novel series of tricyclic inhibitors, an X-ray crystal structure of human liver FBPase in complex with was determined (). As expected, binds at four AMP binding sites of FBPase tetramer and the -bound FBPase exists in the inactive T-state. The phosphate group of occupies a similar position to that of the phosphate group of AMP. Similarly, the amino group of is placed in the same position as the 6NH of AMP. The phosphate group interacts with the backbone nitrogens (NH) of Gly26, Thr27, Gly28, Glu29, Leu30, Thr31 and the side chains of Thr27, Lys112, Tyr113 and Arg140, and some of these interactions are formed via water molecules. A water molecule placed between phosphate and the aromatic N atom also contributes to the hydrogen-bonding network among the phosphate group and FBPase. The amino group of makes hydrogen bonds with the carbonyl oxygen of Val17 (Val17CO) and Thr31Oγ. These hydrogen bonds may contribute to causing the quaternary conformational change of FBPase towards the inactive T-state., This complex structure suggests that the tricyclic scaffold of is beneficial in retaining the proper distance and directions between the phosphate group and the amino group. In addition, this planar tricyclic scaffold can fit well to the pocket of the AMP-binding site, resulting in relatively high affinity and therefore high inhibitory activity of .