A novel series of beta-amino amides incorporating fused heterocycles, i.e., triazolopiperazines, were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (1) is a potent, orally active DPP-IV inhibitor (IC(50) = 18 nM) with excellent selectivity over other proline-selective peptidases, oral bioavailability in preclinical species, and in vivo efficacy in animal models. MK-0431, the phosphate salt of compound 1, was selected for development as a potential new treatment for type 2 diabetes.

Dipeptidyl peptidase (DPP)-IV inhibitors are a new approach to the treatment of type 2 diabetes. DPP-IV is a member of a family of serine peptidases that includes quiescent cell proline dipeptidase (QPP), DPP8, and DPP9; DPP-IV is a key regulator of incretin hormones, but the functions of other family members are unknown. To determine the importance of selective DPP-IV inhibition for the treatment of diabetes, we tested selective inhibitors of DPP-IV, DPP8/DPP9, or QPP in 2-week rat toxicity studies and in acute dog tolerability studies. In rats, the DPP8/9 inhibitor produced alopecia, thrombocytopenia, reticulocytopenia, enlarged spleen, multiorgan histopathological changes, and mortality. In dogs, the DPP8/9 inhibitor produced gastrointestinal toxicity. The QPP inhibitor produced reticulocytopenia in rats only, and no toxicities were noted in either species for the selective DPP-IV inhibitor. The DPP8/9 inhibitor was also shown to attenuate T-cell activation in human in vitro models; a selective DPP-IV inhibitor was inactive in these assays. Moreover, we found DPP-IV inhibitors that were previously reported to be active in models of immune function to be more potent inhibitors of DPP8/9. These results suggest that assessment of selectivity of potential clinical candidates may be important to an optimal safety profile for this new class of antihyperglycemic agents. Diabetes

BPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-beta-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.

There is currently intense interest in the emerging group of proline-specific dipeptidases, and their roles in the regulation of biological processes. Dipeptidyl peptidase IV (DPP-IV) is involved in glucose metabolism by contributing to the regulation of glucagon family peptides and has emerged as a potential target for the treatment of metabolic diseases. Two other proline-specific dipeptidases, DPP-VII (also known as quiescent cell proline dipeptidase) and DPP-II, have unknown functions and have recently been suggested to be identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) [Araki, Li, Yamamoto, Haneda, Nishi, Kikkawa and Ohkubo (2001) J. Biochem. 129, 279-288; Fukasawa, Fukasawa, Higaki, Shiina, Ohno, Ito, Otogoto and Ota (2001) Biochem. J. 353, 283-290]. To facilitate the identification of selective substrates and inhibitors for these enzymes, a complete biochemical profile of these enzymes was obtained. The pH profiles, substrate specificities as determined by positional scanning, Michaelis-Menten constants and inhibition profiles for DPP-VII and DPP-II were shown to be virtually identical, strongly supporting the hypothesis that they are the same protease. In addition, substrate specificities, catalytic constants and IC(50) values were shown to be markedly different from those of DPP-IV. Selective DPP-IV and DPP-VII substrates were identified and they can be used to design selective inhibitors and probe further into the biology of these enzymes.

As part of a structure-aided effort to design clinically useful inhibitors of metallo-beta-lactamases, the X-ray crystal structure of a complex between the metallo-beta-lactamase from Bacteroides fragilis and 4-morpholinoethanesulfonic acid (MES) has been determined and a model for the structure has been refined to a crystallographic R-factor of 0.151 for data between 10.0- and 1.85-A resolution. Although the binding of MES was an adventitious result of the use of MES as a buffer in the crystallization mixture, MES was subsequently shown to be a competitive inhibitor of the enzyme, with a Ki of 23 +/- 5 mM. MES binds in the same fashion to both of the molecules in the crystallographic asymmetric unit; both direct and solvent-mediated hydrogen bonds to the protein and to the binuclear zinc cluster are observed, involving the oxygens of the sulfonic acid group and the nitrogen of the morpholino ring. In addition, there are hydrophobic interactions between the morpholino ring and residues in the flexible beta-strand of the enzyme between residues 26 and 36. Comparison of this structure with the previously reported unliganded structures of the same enzyme [Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. -M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] reveals that although the overall conservation of structure in the three different crystal lattices is very high, binding of MES is correlated with a significant change in the conformation of this beta-strand. The flexibility of this beta-strand will be an important consideration in the design of inhibitors of the metallo-beta-lactamases.