Rapid Diversity-Oriented Synthesis in Microtiter Plates for in situ Screening: Discovery of Potent and Selective α-Fucosidase Inhibitors

Polyhydroxypiperidines and pyrrolidines (also called iminocyclitols or aza-sugars) have attracted increasing attention because of their inhibitory activity against various glycosidases. The glucose-type iminocyclitol deoxynojirimycin (DNJ), for example, has been used for the treatment of noninsulin-dependent diabetes (miglitol, targeting the intestinal disaccharidases). Recent studies further indicate that DNJ and its derivatives are effective against hepatitis B and C, as well as glycosphingolipid storage disorders (such as Gaucher disease). The efficacy of iminocyclitols is attributed to their mimicry of the transition state of enzymatic glycosidic cleavage. Of the members of the glycosidase family, a-fucosidase is involved in the hydrolytic degradation of numerous fucosecontaining glycoconjugates. The existence of this enzyme is associated with a variety of essential functions. For example, the abnormal accumulation of fuco-glycoconjugates, which results from the absence or deficiency of a-fucosidase, leads to the genetic neurovisceral storge disease fucosidosis. An aberrant distribution of intracellular and extracellular afucosidase is also found in cystic fibrosis 11] and colon–rectal cancer. 13] Though the physiological functions of a-fucosidase are not completely understood, potent fucosidase inhibitors may be used as probes for the study of fucosidases with regard to their functions and for the development of potential therapeutic agents. Recently we have established a novel combinatorial approach to five-membered iminocyclitols based on reductive amination and the Strecker reaction, and a potent inhibitor has been identified from the library. The process, that is, synthesis and isolation of individual compounds for testing, is however very slow. To further facilitate the discovery of new glycosidase inhibitors, we aimed to develop a new method for the rapid derivatization of an iminocyclitol core designed for a specific glycosidase family (for example, fuconojirimycin derivatives for fucosidases in this study) without protecting group manipulation and under such conditions that the product could be used directly for screening in situ without isolation. This concept was first demonstrated by the development of new HIV protease inhibitors. Herein we report the generation of a library based on fuconojirimycin (FNJ) in a microtiter plate, followed by the direct in situ evaluation of these reaction mixtures as fucosidase inhibitorse without product isolation. This approach has led to the discovery of the most potent inhibitors of a-fucosidases from bovine kidney and Corynebacterium sp. Previous work on the development of glycosidase inhibitors indicated the existence of an additional binding component in the inhibitor. 16–25] This study was thus aimed at generating an FNJ derivative 1 (Scheme 1) as a core structure to mimic the transition state of the fucose moiety. A baminomethyl group was attached to the C1-position for identification of a new binding component throught the subsequent in situ amide-bond formation and screening. Since 1-deoxy-FNJ has been shown to be a potent inhibitor against a-fucosidases (Ki= 9.8 nm for the a-fucosidase from human neutrophil and 6.2 nm from bovine epididymal)