A synthetic library of cell-permeable molecules

Small molecules that induce or stabilize the association of macromolecules have proven to be useful effecters of a wide variety of biological processe s. To date, all examples of such chemical inducers of dimerization have inv olved known ligands to well-characterized proteins. The generality of this approach could be broadened by enabling the discovery of heterodimerizers t hat target known macromolecules having no established ligand, or heterodime rizers that produce a novel biologic response in screens having no predeter mined macromolecular target. Toward this end, we report the construction of a diversified library of synthetic heterodimerizers consisting of an invar iant ligand that targets the FK506-binding protein (AP1867) attached to 320 substituted tetrahydrooxazepines (THOXs). The THOX components were generat ed by a combination of liquid- and solid-phase procedures employing sequent ial Mitsonobu displacements to join two structurally diversified olefin-con taining monomers, followed by ruthenium-mediated olefin metathesis to effec t closure of the seven-membered ring. The 320 resin-bound THOX ligands were coupled in parallel to AP1867, and the products were released from the res in to yield candidate heterodimerizers in sufficient yield and purity to be used directly in biologic testing. A representative panel of 25 candidate heterodimerizers were tested for their ability to pass through the membrane of human fibrosarcoma cells, and all were found to possess activity in thi s tissue culture system. These studies pave the way for further studies aim ed at using small-molecule inducers of heterodimerization to effect novel b iological responses in intact cells.