Second-generation lymphocyte function-associated antigen-1 inhibitors: 1H-imidazo[1,2-alpha]imidazol-2-one derivatives.

A novel class of lymphocyte function-associated antigen-1 (LFA-1) inhibitors is described. Discovered during the process to improve the physicochemical and metabolic properties of BIRT377 (1, Figure 1), a previously reported hydantoin-based LFA-1 inhibitor, these compounds are characterized by an imidazole-based 5,5-bicyclic scaffold, the 1,3,3-trisubstituted 1H-imidazo[1,2-alpha]imidazol-2-one (i.e. structure 3). The structure-activity relationship (SAR) shows that electron-withdrawing groups at C5 on the imidazole ring benefit potency and that oxygen-containing functional groups attached to a C5-sulfonyl or sulfonamide group further improve potency. This latter gain in potency is attributed to the interaction(s) of the functionalized sulfonyl/sulfonamide groups with the protein, likely polar-polar in nature, as suggested by SAR data. X-ray studies revealed that these bicyclic inhibitors bind to the I-domain of LFA-1 in a pattern similar to that of compound 1.

Small molecule LFA-1 antagonists compete with an anti-LFA-1 monoclonal antibody for binding to the CD11a I domain: development of a flow-cytometry-based receptor occupancy assay.

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.