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4340 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 18 PerspectiveThis is in clear distinction from classical prodrugs,which involve a biologically inactive compound beingconverted to a biologically active one by chemical orenzymatic means (usually via hydrolytic biotransformation).The concept of active metabolite as defined hereis also distinct from reactive metabolites, which arechemically reactive intermediates formed during metabolismthat may elicit toxicological activity throughcovalent binding to biomolecules. The importance of anactive metabolite is measured by the degree of itscontribution to the total pharmacological activity of agiven dose of the parent compound and by its intrinsicactivity and the relative concentration at the site ofaction. 13,14Generation of Chemical Diversity throughBiological TransformationFigure 1. Examples of common biotransformation reactionsthat led to pharmacologically active or inactive metabolites.sulfation of acetaminophen leads to bioinactivation(Figure 1). The 6-O-glucuronide metabolite of morphineis pharmacologically active, whereas morphine 3-Oglucuronideis not (Figure 1). 12 In silico methods havethe potential to predict and assess the formation ofmetabolites from a precursor compound and whetherthese metabolites are active against known pharmacologicaltargets. However, a robust in silico method thatcould fulfill these objectives across a wide variety ofchemical space has yet to be developed. Therefore,experimental methods are needed to generate, detect,and characterize important active metabolites. In thispaper, we will discuss the importance of embracing theconcept of active metabolites as part of the drug designprocess and will review experimental observations thatcan trigger the search for active metabolites. We willalso discuss methodological advances in the detection,generation, and structural characterization of activemetabolites.Definition of an Active MetaboliteHere, we define an active metabolite as a pharmacologicallyactive metabolic product with activity againstthe same pharmacological target as the parent molecule.Current drug discovery paradigms involve (1) randomor directed screening of natural or synthetic productlibraries, (2) combinatorial chemistry and molecularmodification of pharmacologically active synthetic ornatural products, and (3) a de novo rational drug design.As described above, discovering drugs by methods ofbiological transformation may also be a viable approachto drug design. It is therefore beneficial to have methodsin place to allow screening of compounds for therapeuticallyuseful biotransformation products or active metabolitesduring the drug discovery phase.During lead optimization, drug candidates are routinelyscreened for metabolic stability or in vivo systemicexposure and rank-ordered according to the rate andextent of metabolism or systemic exposure level. 15 Inthe case of metabolic screening, this is usually performedin vitro after incubations of the drug candidateswith subcellular fractions such as liver microsomes orintact cellular systems (e.g., hepatocytes) containing afull complement of drug-metabolizing enzymes. Compoundswith low metabolic stability are then excludedfrom further consideration because most therapeutictargets require compounds with an extended pharmacokinetichalf-life. The same is true with in vivoexposure studies, where high-clearance compounds arediscarded. In these early screens, the concentration ofthe parent compound is typically the only measurementmade. Consequently, there is no information on thenumber, identity, and pharmacological significance ofmetabolites that may have been formed. Even whenmetabolic profiling is completed and metabolites areidentified, the information is typically used to directsynthesis of analogues with improved metabolic stabilitythrough the modification of metabolic soft or hot spots. 16,17Thus, the information is rarely used for the purpose ofsearching for pharmacologically active products as newanalogues. However, rapid metabolism of parent compoundscan lead to the formation of pharmacologicallyactive metabolites that may have comparatively superior“developability” characteristics. As a result, metabolicinstability, which otherwise may be considered aliability, can be used as an advantage during leadoptimization.