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Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step of the NAD+ salvage pathway. Since NAD+ plays a pivotal role in many biological processes including metabolism and aging, activation of NAMPT is an attractive therapeutic target for treatment of diverse array of diseases. Herein, we report the continued optimization of novel urea-containing derivatives which were identified as potent NAMPT activators. Early optimization of HTS hits afforded compound 12, with a triazolopyridine core, as a lead compound. CYP direct inhibition (DI) was identified as an issue of concern, and was resolved through modulation of lipophilicity to culminate in 1-[2-(1-methyl-1H-pyrazol-5-yl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-3-(pyridin-4-ylmethyl)urea (21), which showed potent NAMPT activity accompanied with attenuated CYP DI towards multiple CYP isoforms.

Since elevated levels of gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] are associated with a poor prognosis in cancer patients, these enzymes are potential targets for tumor imaging. In the present study, a cyclic decapeptide, cCTTHWGFTLC (CTT), was selected as a mother compound because of its selective inhibitory activity toward gelatinases. For imaging gelatinase activity in tumors, we designed a CTT-based radiopharmaceutical taking into consideration that (1) the HWGF motif of the peptide is important for the activity, (2) hydrophilic radiolabeled peptides show low-level accumulation in the liver and (3) an increase in the negative charge of radiolabeled peptides is effective in reducing renal accumulation. Thus, a highly hydrophilic and negatively charged radiolabel, indiun-111-diethylenetriaminepentaacetic acid ( 111 In-DTPA), was attached to an N-terminal residue distant from the HWGF motif ( 111 In-DTPA-CTT). In MMP-2 inhibition assays, In-DTPA-CTT significantly inhibited the proteolytic activity in a concentration-dependent fashion. When injected into normal mice, 111 In-DTPA-CTT showed low levels of radioactivity in the liver and kidney. A comparison of the pharmacokinetic characteristics of 111 In-DTPA-CTT with those of other CTT derivatives having different physicochemical properties revealed that the increase in hydrophilicity and negative charge caused by the conjugation of 111 In-DTPA reduced levels of radioactivity in the liver and kidney. In tumor-bearing mice, a significant correlation was observed between the accumulation in the tumor as well as tumor-to-blood ratio of 111 In-DTPA-CTT and gelatinase activity. These findings support the validity of the chemical design of 111 In-DTPA-CTT for reducing accumulation in nontarget tissues and maintaining the inhibitory activity of the mother compound. Furthermore, 111 In-DTPA-CTT derivatives would be potential radiopharmaceuticals for the imaging of gelatinase activity in metastatic tumors in vivo.