Serine peptide phosphoester prodrugs of cyclic cidofovir: synthesis, transport, and antiviral activity

Abstract

Cidofovir (HPMPC, 1), a broad-spectrum antiviral agent, is currently used to treat AIDS-related human cytomegalovirus (HCMV) retinitis and has recognized therapeutic potential for orthopox virus infections, but is limited by its low oral bioavailability. Cyclic cidofovir (2) displays decreased nephrotoxicity compared to 1, while also exhibiting potent antiviral activity. Here we describe in detail the synthesis and evaluation as prodrugs of four cHPMPC dipeptide conjugates in which the free POH of 2 is esterified by the Ser side chain alcohol group of an X-L-Ser(OMe) dipeptide: 3 (X=L-Ala), 4 (X=L-Val), 5 (X=L-Leu), and 6 (X=L-Phe). Perfusion studies in the rat establish that the mesenteric permeability to 4 is more than 20-fold greater than to 1, and the bioavailability of 4 is increased 6-fold relative to 1 in an in vivo murine model. In gastrointestinal and liver homogenates, the cHPMPC prodrugs are rapidly hydrolyzed to 2. Prodrugs 3, 4, and 5 are nontoxic at 100 microM in HFF and KB cells and in cell-based plaque reduction assays had IC 50 values of 0.1-0.5 microM for HCMV and 10 microM for two orthopox viruses (vaccinia and cowpox). The enhanced transport properties of 3-6, conferred by incorporation of a biologically benign dipeptide moiety, and the facile cleavage of the Ser-O-P linkage suggest that these prodrugs represent a promising new approach to enhancing the bioavailability of 2.

Figure 1

Electrostatic potential maps of diastereomer S (left) and diastereomer F (right) as calculated on Spartan ’02.

Figure 2

Prodrugs 3–5 show increased permeability over 1 or 2 in an in situ single pass perfusion assay. Plasma was sampled at the indicated times, and the total cidofovir content (prodrug and 1 or 2) was determined using LC–MS/MS analysis. The plasma level of drug at 90 min was used to determine the permeability shown in Table 3.

Figure 3

Dose corrected total cidofovir found in plasma after intestinal dosing of 3–5. Dosing of the prodrug boosts the plasma levels of total cidofovir over 1 and 2 controls. Rats were dosed by direct injection of 3 mg (~10 mg/kg) of material into the intestine, and plasma was sampled over a time course of 8 h. Prodrug 4 showed a greater than 8-fold increase in total cidofovir levels over 2. Prodrugs 3 and 5 showed a greater than 5-fold increase in absorption.

Figure 4

The left panel shows the drug species found in the plasma after intestinal dosing of 4. The right panel shows plasma concentration time data for an intravenous dose of 1 and 2. Both compounds have similar kinetics of disappearance. The AUC values from the IV dose of 2 were used to calculate the bioavailability of the prodrug compounds.

Figure 5

Drug plasma profile in mice after dosing with 2 or 4. Mice were dosed by oral gavage. Prodrug 4 (■) showed an approximate 2-fold increase in AUC of total cidofovir compared with that seen after dosing with 2 (○).

Figure 6

Plasma profiles of 2 after oral dosing of 4 to mice with and without protease inhibitors (left panel) and plasma profiles of 4 after oral dosing of 4 to mice with and without protease inhibitors (right panel). Mice were dosed with 0.3 mg of 4 with and without the protease inhibitors, which were dissolved in water. For each dosing cohort, groups of 3 mice were sacrificed at the indicated time points and their plasma was analyzed for 2 and prodrug by LC–MS/MS. These figures show a comparison of the plasma profiles of 2 or 4 after dosing with 4 alone (▲), 4 with 100 μM bestatin (□), or 4 with the mixture of inhibitors (■).

Scheme 1

Synthesis of Prodrugs 3, 4, 5, and 6

Chart 1

Structures of HPMPC (1) and cHPMPC (2)

Chart 2

Structures of 3–6