Synthesis, metabolic stability and antiviral evaluation of various alkoxyalkyl esters of cidofovir and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine.

Alkoxyalkyl esters of cidofovir (CDV) are orally active agents which inhibit the replication of a variety of double stranded DNA (dsDNA) viruses including variola, vaccinia, ectromelia, herpes simplex virus, cytomegalovirus, adenovirus and others. One of these compounds, hexadecyloxypropyl-CDV (HDP-CDV, CMX001) is in clinical development for prevention and treatment of poxvirus infection, vaccination complications, and for infections caused by cytomegalovirus, adenovirus, herpesviruses and other dsDNA viruses. This class of lipid analogs is potentially prone to undergo omega oxidation of the alkyl moiety which can lead to a short chain carboxylic acid lacking antiviral activity. To address this issue, we synthesized a series of alkoxyalkyl or alkyl glycerol esters of CDV and (S)-HPMPA having modifications in the structure of the alkyl residue. Antiviral activity was assessed in cells infected with vaccinia, cowpox or ectromelia viruses. Metabolic stability was determined in S9 membrane fractions from rat, guinea pig, monkey and human liver. All compounds had substantial antiviral activity in cells infected with vaccinia, cowpox or ectromelia. Metabolic stability was lowest in monkey liver S9 incubations where rapid disappearance of HDP-CDV and HDP-(S)-HPMPA was noted. Metabolic stability in monkey preparations increased substantially when a ω-1 methyl group (15-methyl-HDP-CDV) or a terminal cyclopropyl residue (14-cyclopropyl-tetradecyloxypropyl-CDV) was present in the alkyl chain. The most stable compound was 1-O-octadecyl-2-O-benzyl-sn-glycero-3-CDV (ODBG-CDV) which was not metabolized extensively by monkey liver S9. In rat, guinea pig or human liver S9 incubations, most of the modified antiviral compounds were considerably more stable.

Synthesis and antiviral evaluation of alkoxyalkyl-phosphate conjugates of cidofovir and adefovir.

Esterification of cidofovir (CDV), an antiviral nucleoside phosphonate, with alkyl or alkoxyalkyl groups increases antiviral activity by enhancing cell uptake and conversion to CDV diphosphate. Hexadecyloxypropyl-CDV (HDP-CDV) has been shown to be 40-100 times more active than CDV in vitro in cells infected with herpes group viruses, variola, cowpox, vaccinia or ectromelia viruses. Since the first phosphorylation of CDV may be rate limiting, we synthesized the hexadecyloxypropyl-phosphate (HDP-P-) and octadecyloxyethyl-phosphate (ODE-P-) conjugates of CDV and phosphonomethoxy-ethyl-adenine (PMEA, adefovir). We tested the CDV analogs in cells infected with human cytomegalovirus, herpes simplex virus, cowpox virus and vaccinia virus; the analogs of PMEA were tested in cells infected with the human immunodeficiency virus, type 1. In general, the alkoxyalkyl-phosphate conjugates of CDV were substantially more active than CDV. HDP-P-CDV and ODE-P-CDV were 4.6-40 times more active against HCMV and 7-30 times more active against cowpox and vaccinia in vitro. Although the compounds of this type were more cytotoxic than the unmodified bases, their selectivity for virally infected cells was generally greater than the parent nucleotides except that HDP-P-PMEA showed little or no selectivity in HIV-1 infected MT-2 cells. Although the new compounds with an interposed phosphate were generally less active than the corresponding alkoxyalkyl esters of CDV and PMEA, the present approach provides a possible alternative method for enhancing the antiviral activity of drugs of this class.

Synthesis and antiviral evaluation of alkoxyalkyl esters of phosphonopropoxymethyl-guanine and phosphonopropoxymethyl-diaminopurine.

Phosphonopropoxymethyl-guanine is the methylene phosphonate analogue of acyclovir. Although not highly active against HSV, 4-38 microM of phosphonopropoxymethyl-guanine has been reported to be active against human and murine cytomegalovirus. Recently we found that cidofovir, when esterified with alkoxyalkyl moieties, showed greatly increased antiviral activity against cytomegalovirus, herpes simplex virus and orthopoxviruses, in vitro. The alkoxyalkyl esters of cidofovir are orally active in murine models of human and murine cytomegalovirus and orthopoxviruses in vivo. To see if the antiviral activity of phosphonopropoxymethyl-guanine, phosphonopropoxymethyl-diaminopurine and phosphonopropoxymethyl-N6-cyclopropyl-diaminopurine could be increased by this approach, we synthesized their hexadecyloxypropyl- and octadecyloxyethyl- esters and evaluated antiviral activity and cytotoxicity in cells infected with HSV-1 and HCMV, in vitro. Marked increases in antiviral activity were noted in the alkoxyalkyl esters of phosphonopropoxymethyl-guanine. Alkoxyalkyl esters of diaminopurine and N6-cyclopropyl-diaminopurine showed slight increases in activity against HSV-1 and marked increases in activity against HCMV. The results suggest that esterification with alkoxyalkyl moieties may be a generally useful way to increase antiviral activity of nucleoside phosphonates.