br Materials and methods Unless stated otherwise all chemica

Materials and methods Unless stated otherwise, all chemicals were from Sigma-Aldrich and were of the cell culture grade. HL-1 cardiomyocytes were presented by Dr. W.C. Claycomb (Louisiana State University Health Science Center, New Orleans, LA, USA) [8].
Results and discussion
Acknowledgements We wish to thank Professor J. Kubrakiewicz for kindly allowing us to use his electron microscope facilities. This work was supported by Polish Ministry of Science and Higher Education Grant No NN301 069139.
Fructose-1,6-bisphosphatase (FBPase) is one of the enzymes involved in the rate-limiting steps of hepatic gluconeogenesis, and catalyzes the hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate. FBPase inhibitors would lower blood glucose levels by reducing hepatic glucose output and are expected to be a novel class of drugs for the treatment of Type 2 diabetes mellitus. Several galeterone of small-molecule inhibitors of FBPase have been reported., , , , Among them, 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 (). In the previous Letter, we described the design and synthesis of tricyclic thiazoles as FBPase inhibitors, and a series of SAR studies led to the identification of phosphate exhibiting potent FBPase inhibitory activity (IC=13nM) (). In addition, we reported the finding of non-hydrolyzable difluoromethylenephosphonate which also showed high inhibitory activity (IC=47nM). However, subsequent attempts to evaluate the inhibitory effect on cellular glucose production of and corresponding prodrug compound were not successful. These results were rationalized by considering the metabolism of the amino group on the tricyclic scaffold. In fact, the amino groups of these compounds were readily metabolized by -acetyltransferases. In order to improve metabolic stability and to enhance FBPase inhibitory activity, we turned our attention to further modification of our tricyclic-based inhibitors with the aid of structure-based drug design. In order to address the problem of the metabolically unstable amino group on tricyclic scaffolds, we examined the conversion of the amino group. Our previous SAR studies led to the identification of compound which possessed the non-hydrolyzable oxymethylphosphonate moiety and showed moderate FBPase inhibitory activity (IC=124nM). Therefore, we selected compound for the modification of the amino group. An X-ray crystal structure of human liver FBPase in complex with suggested the possibility of removing the amino group (). The complex structure suggested that the amino group of interacted with a carbonyl oxygen of Val17 and a side chain oxygen (Oγ) of Thr31 through hydrogen-bonding interaction. On the other hand, the amino group was located near the hydrophobic surface area formed by hydrophobic residues of Val17, Leu34, and Met177. In addition, the carbonyl oxygen of Val17 and Oγ of Thr31 forms hydrogen bonds to backbone amide nitrogen of Gly21 and a water molecule, respectively, suggesting that the interaction to the amino group of compound might not be crucial in retaining the protein structure to the inhibited conformation. Based on these observations, we hypothesized that hydrophobic interaction instead of the interaction through the amino group might be beneficial to obtain high affinity. This led us to focus on replacing the amino group of compound with other hydrophobic substituents. Tricyclic thiazoles without amino group were synthesized according to . Commercially available 7-hydroxy-1-indanone was converted to diethyl phosphonate . Bromination of followed by cyclization with thioacetamide, thioformamide (in situ generation), and thiourea gave 2-methythiazole (), thiazole (), and 2-aminothiazole () intermediates, respectively. The hydrolysis of intermediates , afforded the desired compounds ,. In addition, 2-halogen thiazole analogues , were prepared from by a Sandmeyer reaction followed by hydrolysis.