Synthesis and in vitro biological evaluation of valine-containing prodrugs derived from clinically used HIV-protease inhibitors

In an approach to improve the pharmacological properties and pharmacokinetic profiles of the current protease inhibitors (PIs) used in clinics, and consequently, their therapeutic potential, we performed the synthesis of PI–spacer–valine prodrugs (PI = saquinavir, nelfinavir and indinavir; spacer = –C(O)(CH2)5NH–), and evaluated their in vitro stability with respect to hydrolysis, anti-HIV activity, cytotoxicity, and permeation through a monolayer of Caco-2 cells (used as a model of the intestinal barrier), as compared with their parent PI and first generation of valine–PIs (wherein valine was directly connected through its carboxyl to the PIs). The PI–spacer–valine conjugates were prepared in two steps, in good yields, by condensing an acid derivative of the appropriate protected valine–spacer moiety with the PI, followed by deprotection of the valine protecting group. With respect to hydrolysis, we found that the PI–spacer–valine prodrugs were chemically more stable than the first generation of PI–Val prodrugs. Their stabilities correlated with the low to very low in vitro anti-HIV activity measured for those prodrugs wherein the coupling of valine–spacer residue to the PIs was performed onto the peptidomimetic PI's hydroxyl. Prodrugs wherein the coupling of the valine–spacer residue was performed onto the non-peptidomimetic PI hydroxyl displayed a higher antiviral activity, indicating that these prodrugs are also to some extent anti-HIV drugs by themselves. While the direct conjugation of l-valine to the PIs constituted a most appealing alternative, which improved their absorptive diffusion across Caco-2 cell monolayers and reduced their recognition by efflux carriers, its conjugation to the PIs through the –C(O)(CH2)5NH– spacer was found to inhibit their absorptive and secretory transepithelial transport. This was attributable to a drastic reduction of their passive permeation and/or active transport, indicating that the PI–spacer–valine conjugates are poor substrates of the aminoacid carrier system located at the brush border side of the Caco-2 cell monolayer.

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