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  • In the previous paper we described the


    In the previous paper, we described the design and synthesis of tricyclic thiazoles as FBPase inhibitors, and a series of SAR studies led to the identification of phosphate 3 and difluoromethylenephosphonate 4 exhibiting potent FBPase inhibitory activities (IC50=13, 47nM, respectively) (Fig. 2). In addition, in order to improve metabolic stability and enhance FBPase inhibitory activity, we further developed tricyclic-based FBPase inhibitors with the aid of structure-based drug design, which led to the discovery of phosphonate 5, exhibiting more potent FBPase inhibitory activity (IC50=1nM). The key feature of phosphonate 5 was that this compound possessed no amino group which might be the cause of metabolic instability. The X-ray co-crystal structure of 5 suggested that high affinity was achieved by hydrophobic interaction which compensated for the loss of the amino group, and also by a hydrogen-bonding network involving the amide side chain of 5 (Fig. 3). This paper describes our continuing efforts to explore the effect of the tricyclic phosphonates in GW 6471 and in vivo. Our phosphonate compounds seemed to have low membrane permeability due to the high negative charge of phosphonate moieties, which prompted us to convert the phosphonate compounds into corresponding prodrugs. There are many classes of phosphonate prodrugs, from which we selected phosphonic diamides as prodrugs in consideration of the advantage that the cleavage byproducts are nontoxic amino acids. In addition, the significant glucose-lowering effect of diamide prodrug CS-917 in animal models encouraged us to explore this type of prodrug. In order to investigate the cellular activity and in vivo efficacy of our tricyclic-based inhibitors, we focused our attention on developing diamide prodrugs of tricyclic phosphonates.
    Results and discussion The prodrugs of the tricyclic phosphonates were synthesized according to Scheme 1, Scheme 2. The prodrugs containing amide side chains (11a–c) were prepared from commercially available 4-bromophenol 6 (Scheme 1). Acylation of 6 followed by a Fries rearrangement and the introduction of a diethyl phosphonate unit resulted in diethyl phosphonate 7. CO insertion of 7 followed by a transesterification reaction afforded allyl ester 8, which was transformed into tricyclic thiazole 9 via bromination and cyclization with thioformamide (in situ generation). l-Alanine diamide 10 was obtained by cleaving the phosphonate ethyl groups of 9, dichlorination, and subsequent condensation with l-alanine ethyl esters. Cleaving an allyl group of 10 and amidation with corresponding amines led to prodrugs 11a–c. Prodrugs containing alkyl side chains were synthesized by methods similar to those described for prodrugs 11a–c, using side chains containing phenols 12a–c as starting materials (Scheme 2). The prodrugs 15a–e were obtained by condensation with the corresponding amino acid esters in the final step. The inhibitory effects on glucose production in monkey hepatocytes varied widely with the side chains of the parent phosphonates (Table 1). With the intent to rigorously evaluate the in vivo potential of prodrugs, this cell assay was performed under severe conditions wherein the hepatocytes were preincubated with prodrugs for only a short time (2min). Initially, phosphonate 5, which possesses an amide side chain and demonstrated potent inhibitory activities against human and monkey FBPase (IC50=1, 22nM, respectively), was converted to l-alanine diamide 11a. However, prodrug 11a showed only a modest inhibitory effect on glucose production, about ninefold less potent than CS-917. Similarly, prodrug 11b and 11c containing another amide side chain showed little effect. In contrast, our efforts to convert the phosphonates containing alkyl side chains to prodrugs resulted in a major increase in inhibitory activities in the cell assays. Prodrug 15a containing a methyl side chain exhibited high inhibitory activity (IC50=7.4μM), whereas prodrug 15b containing a ethyl side chain was about threefold less potent (IC50=23μM) than 15a. In addition, prodrug 15c containing two methyl side chains exhibited inhibitory activity (IC50=6.8μM) similar to that of prodrug 15a. Prodrugs derived from other amino acid esters such as l-alanine isopropyl esters (15d) and glycine ethyl esters (15e) were slightly less potent (IC50=9.0, 9.4μM, respectively) than l-alanine diamide 15c. Large differences in the inhibitory activity between prodrugs 11a–c and 15a–e suggested that a judicious combination of tricyclic-based phosphonate and prodrug moiety was required to exhibit high inhibitory activity in cell assays.