John C. Martin

Synthesis and in vitro evaluation of a phosphonate prodrug: bis(pivaloyloxymethyl) 9-(2-phosphonylmethoxyethyl)adenine

9-(2-Phosphonylmethoxyethyl)adenine (PMEA; 1) was acylated with chloromethyl pivalate to afford bis(pivaloyloxymethyl) PMEA (2). The ester prodrug demonstrated enhanced in vitro potency against HSV-2 greater than 150-fold higher than the parent compound. The antiviral activity of 2 was 50-fold better than PMEA against HSV-1, and equipotent against HIV and HCMV. The toxicity of 2 was studied in both resting and growing cells.

Preparation of the geometric isomers of DDC, DDA, D4C and D4T as potential anti-HIV agents

Abstract β-L-ddC ( 5 ), β-L-ddA ( 7 ), β-L-d4T ( 7 ) and β-L-d4C ( 8 ) (enantiomers of natural dideoxynucleoside analogues with known potent HIV activity) were prepared as potential anti-HIV agents.

Treatment of latent rabbit and human papillomavirus infections with 9-(2-phosphonylmethoxy)ethylguanine (PMEG)

The acyclic nucleotide PMEG was studied for effectiveness against Shope papillomavirus (CRPV) infection of rabbits and human papillomavirus type 11 (HPV-11) infections of human foreskin xenografts in athymic mice. PMEG given in the latent period strongly suppressed the subsequent growth rates of Shope papillomas. PMEG starting in the latent period and continuing for the duration of the experiment, inhibited HPV-11 infections of human skin, including condyloma growth, and synthesis of viral DNA and capsid antigen. Drug toxicity paralleled the therapeutic effects in rabbits but there was much less toxicity in athymic mice.

Antiherpes Simplex Virus Activity of 9-[4-Hydroxy-3-(hydroxymethyl)-1-butyl] guanine

Abstract The carba analogue 2 of DHPG (1) was found to be highly inhibitory to herpes simplex virus type 1 replication but less active against the type 2 virus.

Biochemical Pharmacology of Acyclic Nucleotide Analogues

Our studies have shown that the acyclic nucleotide analogues PMEA and HPMPC are able to penetrate into cells and are then activated to mono- and diphosphate derivatives. The latter correspond to triphosphate analogues and presumably serve an important role in the biological activity exerted by these antiviral agents. In support of this idea, the inhibitory effect of PMEApp on HIV reverse transcriptase has been demonstrated with both RNA and DNA template-primer systems. Further studies will be undertaken to determine the effect of HPMPCpp on viral DNA polymerases. Whereas the metabolism of PMEA in CEM cells gives rise to only PMEAp and PMEApp, additional metabolites were obtained in MRC-5 cells; the identity of these metabolites remains to be determined. In the case of HPMPC, a third metabolite was obtained in addition to HPMPCp and HPMPCpp, which has been tentatively assigned as a phosphate-choline adduct by analogy with activation of cytosine-based nucleoside derivatives. The metabolism of HPMPC was unchanged between uninfected and infected cells, indicating that viral enzymes are not necessary for the activation of HPMPC. The long intracellular half-lives of the HPMPC metabolites may have implications for the antiviral efficacy of this compound. The persistence of activated metabolites suggests that infrequent dosing may be possible due to a prolonged antiviral effect. Our results on the effectiveness of infrequent dosing schedules with HPMPC in the treatment of HSV 2 infections in mice support this hypothesis. It is also possible that HPMPCp-choline may serve as a reservoir for HPMPC and therefore for the presumed active metabolite HPMPCpp.

(S)-1-(3-hydroxy-2-(phosphonylmethoxy)propyl)cytosine (HPMPC): a potent antiherpesvirus agent.

(S)-1-(3-Hydroxy-2-(phosphonylmethoxy)propyl)cytosine (HPMPC; 1, fig. 1, left) has been identified from a series of phosphonate nucleotide analogues as having the greatest therapeutic index against lethal herpes simplex virus (HSV) infections in mice (1, 2). The prototype compound of the series is the adenine analogue 2 (HPMPA; 2, fig. 1, right), which was first described by De Clercq et al. in 1986 as a broad-spectrum antiviral agent (3). HPMPA was shown to have a mechanism of action that involves, at least in part, two intracellular phosphorylations to give a diphosphate, which then selectively inhibits the viral polymerase. Since the discovery of HPMPA, additional studies have shown that many related phosphonylmethoxy nucleotide analogues have broad-spectrum in vitro activity (4, 5). Of this class of nucleotide analogues, HPMPC has been described as having the best in vitro therapeutic index against cytomegalovirus (CMV) (2, 6).

Synthesis and Antiviral Evaluation of 3′-Branched Nucixoside Analogues

Abstract A common structural feature shared by the representative nucleoside analogues active against HIV is the lack of hydroxyl substituents at the positions C -2′ and C-3′ of the furanose ring.1 These analogues which include AZT, ddI, D4T, ddC, 3′Fddt, are first converted to their 5′-0- triphosphates, which then exert their biological effect either as reverse transcriptase (RT) inhibitors or chain tetminators or both.2

A Radioimmunoassay Procedure for the Determination of the Antiviral Nucleoside DHPG (9-[(l,3-Dihydroxy-2-propoxy)-methyl]guanine) in Plasma or Serum

A procedure is described that is suitable for the radioimmunoassay (RIA) of 9-[(l,3-dihydroxy-2-propoxy)-methyl]guanine (DHPG) in plasma or serum at concentrations as low as 0.7 ng/ml (2.75 × 10−9M). Antiserum was prepared by coupling DHPG monohemisuccinate to bovine serum albumin and immunizing rabbits with the resulting conjugate. The antibodies did not show significant cross-reactivities with structurally related endogenous compounds. For RIA, tritium-labeled DHPG was used as the tracer and charcoal–dextran was used to separate the free and bound fractions. No purification of samples was required prior to RIA. The accuracy of the method was assessed by adding known quantities of DHPG to DHPG-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.0007 to 15.0 µg/ml yielded the following equation; y = 0.90 x + 0.033 (r = 0.999). Additional validation was obtained from studies in which DHPG was administered to a monkey, mice, dogs, and rats, and plasma-clearance profiles were determined by RIA and high-performance liquid chromatography (HPLC). The results obtained by RIA were in good agreement with those obtained by HPLC.

The Use of Acetyl Bromide for the Multigram Synthesis of the Anti-HIV Agent 2′,3′-Didehydro-2′,3′-Dideoxycytidine (d4C)

Abstract Treatment of uridine (1) with acetyl bromide produced bromoacetate 2 which was reduced with Zn/Cu to give the 2′,3′-unsaturated uridine (d4U; 3). Conversion of the uracil moiety of 3 to thioamide 7 with Lawessons reagent, followed by amination and deprotection with methanolic ammonia, afforded d4C (9). This multigram scale process for the synthesis of d4C proceeded in 20% yield from uridine.

Synthesis and HIV activity of phosphonate isosteres of D4T monophosphate

Abstract Two new phosphonate isosteres of d4T monophosphate have been prepared and tested against HIV.