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 30-fold greater than to 1, and the bioavailability of 4 is increased 8-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 μM in HFF and KB cells and in cell-based plaque reduction assays had IC 50 values of 0.1-0.5 μM for HCMV and 10 μM for two orthopox viruses (vaccinia and cowpox). The enhanced transport properties of 3-6, conferred by incorporation of a toxicologically 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.

The design of smart nonviral vectors for gene delivery is of prime importance for the successful implementation of gene therapies. In particular, degradable analogues of macromolecules represent promising targets as they would combine the multivalent presentation of multiple binding units that is necessary for achieving effective complexation of therapeutic oligonucleotides with the controlled degradation of the vector that would in turn trigger drug release. Toward this end, we have designed and synthesized hybrid polyacylhydrazone-based dynamic materials that combine bis-functionalized cationic monomers with ethylene oxide containing monomers. Polymer formation was characterized by (1) H and DOSY NMR spectroscopy and was found to take place at high concentration, whereas macrocycles were predominantly formed at low concentration. HPLC monitoring of solutions of these materials in aqueous buffers at pH values ranging from 5.0 to 7.0 revealed their acid-catalyzed degradation. An ethidium bromide displacement assay and gel electrophoresis clearly demonstrated that, despite being dynamic, these materials are capable of effectively complexing dsDNA in aqueous buffer and biological serum at N/P ratios comparable to polyethyleneimine polymers. The self-assembly of dynamic covalent polymers through the incorporation of a reversible covalent bond within their main chain is therefore a promising strategy for generating degradable materials that are capable of establishing multivalent interactions and effectively complexing dsDNA in biological media.

An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but with benzene added, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands, and were transfected into E. coli. Although yielding fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture.

Nucleotide analogues have been well accepted as therapeutic agents active against a number of viruses. However, their use as antiviral agents is limited by the need for phosphorylation by endogenous enzymes, and if the analogue is orally administered, by low bioavailability due to the presence of an ionizable diacid group. To circumvent these limitations, a number of prodrug approaches have been proposed. The ideal prodrug achieves delivery of a parent drug by attachment of a non-toxic moiety that is stable during transport and delivery, but is readily cleaved to release the parent drug once at the target. Here, a brief overview of several promising prodrug strategies currently under development is given.