In addition, a human plasma protein binding (PPB) assay was performed to determine the fraction unbound (Fu) of 9. This allows an assessment of the free drug concentration available to distribute into the tissues, and interact with the target. The extent to which a compound binds to the constituents of blood plasma can influence drug distribution. This assay was performed after six hours incubation time, after which 98.3 % of 9 was recovered (Table † ). At this time, 99.7 % of 9 was bound to the proteins present in the plasma, with 0.3 % of this agent remaining free, corresponding to a very highly bound compound (Table † ).