Peptidomimetic prodrugs of cidofovir: design, synthesis, transport, mechanism of activation, and antiviral activity

UnrestrictedCidofovir and its equivalently potent cyclic form are possible therapies for a broad spectrum of viral infections, but are limited in this role by low oral bioavailability, prompting efforts to create prodrugs incorporating a promoiety conjugated to the phosphoryl group via the side chain hydroxyl group of a single amino acid, di- or tripeptide promoiety. Sixteen novel cyclic cidofovir and four acyclic cidofovir prodrugs were synthesized utilizing PyBOP as a condensing agent. The promoiety was varied to explore the influence of the amino acid stereochemistry, the N-terminal amino acid (Val vs. Ala), the C-terminal group (methyl vs. isopropyl ester, carboxylic acid or amide), the size of promoiety (single amino acid, di- or tripeptide), and the linking amino acid (Ser, Thr or Tyr) on their biological activities. The products were characterized by 1H and 31P NMR, HR-MS, and HPLC.; The cidofovir and cyclic cidofovir conjugates’ stabilities in phosphate buffer pH 6.5 and in rat intestinal homogenate were studied using LC-MS. In general, the single amino acid conjugates had shorter half-lives than the di- and tripeptide prodrugs. These studies provided evidence for two pathways to release cyclic cidofovir from the prodrugs, where the predominant mechanism of activation is structure dependent. Enhanced levels of total cidofovir species in the plasma were observed after oral dosing of some of the prodrugs in a rat model. To elucidate the role of the active transporter, PEPT1, in the uptake of these compounds, electrophysiology experiments on Xenopus laevis oocytes over-expressing hPEPT1 were performed. In this model, the prodrugs did not evoke a current, indicating that their observed enhanced oral bioavailability is unlikely to be due to this particular active transporter. The conjugates were also evaluated for in vitro antiviral activity against vaccinia virus, cowpox virus, herpes simplex virus type 1, and human cytomegalovirus infections. The conjugates exhibited EC50 values in the micromolar to submicromolar range in the human cytomegalovirus assay. None of the prodrugs showed cytotoxicity in KB and HFF cells at concentrations up to 100 μM