Activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) against guinea pig cytomegalovirus infection in cultured cells and in guinea pigs

(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC, and two HPMPC-related nucleoside analogs, (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine, HPMPA, and (2-phosphonylmethoxyethyl)guanine, PMEG, were evaluated for their antiviral activities against guinea pig cytomegalovirus (GPCMV) infection in guinea pig embryo (GPE) cells and human cytomegalovirus (HCMV) infection in human diploid fibroblast (MRC-5) cells. DHPG, 9-(1,3-dihydroxy-2-propoxymethyl)guanine, was used for comparison. The antiviral activity of HPMPC against GPCMV infection in vivo and its toxicity to Hartley guinea pigs were also evaluated. The 50% antiviral effective doses (ED50) of HPMPC, HPMPA, PMEG and DHPG against GPCMV infection in GPE cells were 0.22, 1.4, 0.07 and 62 microM, respectively; and against HCMV infection in MRC-5 cells, the ED50s were 0.51, 0.72, 0.01 and 17.5 microM, respectively. Their cytotoxic doses (CyD50) in GPE replicating cells were 84, 35, 1.4 and 700 microM, respectively and in MRC-5 cells were approximately 114, 31, 0.86 and 750 microM, respectively. Based on their calculated therapeutic indexes, HPMPC was the most potent and selective of the four compounds tested. In vivo, during acute infection, the spleen indexes of all infected animals that were treated with 1.25 to 5.0 mg/kg/day of HPMPC for 5 days were significantly reduced as compared with sham-treated animals. Virus infectivity titers in blood and various tissues of infected animals treated with HPMPC, 2.5 or 1.25 mg/kg/day were not significantly lower than those of the infected, sham-treated animals; with 5 mg/kg/day, infectivity titers in the blood, spleen, and salivary gland were significantly lower in HPMPC-treated than in sham-treated animals. However, HPMPC was toxic to guinea pigs especially at doses of 5 to 10 mg/kg/day. These data showed that HPMPC was highly active and selective in cultured guinea pig cells and human fibroblast cells against CMV infection but did not effectively inhibit GPCMV infection in guinea pigs at minimum toxic concentrations.     

A new class of acyclic phosphonate nucleotide analogues: phosphonate isosteres of acyclovir and ganciclovir monophosphates as antiviral agents.

Novel phosphonate isosteres of acyclovir (ACV) and ganciclovir (DHPG) monophosphates were found to be potent and selective antiherpesvirus agents. In the series of phosphonate analogues of ACV monophosphate, only the guanine analogue 20 exhibited activity against herpesviruses, similar to the structure-activity relationship observed for base modification of ACV analogues. The phosphonate isostere of ACV monophosphate (20) was more effective than ACV in the HSV-1 infected mouse model. The 3'-carba analogues of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]purines/pyrimidines (adenine, HPMPA; guanine, HPMPG; cytosine, HPMPC) are devoid of antiherpesvirus activity. This result confirms that the beta-oxygen atom of the phosphonomethyl ether functionality in HPMP-purines/pyrimidines plays a critical role for activity against herpesviruses.     

Particular characteristics of the anti-human cytomegalovirus activity of (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) in vitro.

The acyclic nucleoside phosphonate analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]cytosine (HPMPC) is a potent and selective inhibitor of human cytomegalovirus (HCMV) replication and DNA synthesis. Unlike 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), HPMPC inhibits HCMV replication in cell cultures which have been treated with the compound before infection. Upon short-pulse treatment of HCMV-infected cells with HPMPC, a long-lasting antiviral response is obtained. The antiviral activity of HPMPC, unlike the antiviral activity of other cytosine derivatives (i.e. Ara-C, FIAC), is not readily reversed by 2'-deoxycytidine or cytidine. Neutral and alkaline sucrose gradient analysis of HCMV DNA isolated from HPMPC-treated HCMV-infected cell cultures revealed that HPMPC does not cause (detectable) single- or double-strand breakage of HCMV DNA.     

5-(Haloalkyl)-2'-deoxyuridines: a novel type of potent antiviral nucleoside analogue

Syntheses of 5-(2-haloethyl)-2'-deoxyuridines, 5-(3-chloropropyl)-2'-deoxyuridines, and 5-(2-chloroethyl)-2'-deoxycytidine are described. The antiviral activities of these compounds were determined in cell culture against herpes simplex virus types 1 and 2. All compounds were shown to possess significant and selective antiviral activity. The most potent derivative, 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), inhibited HSV-1 at concentrations below 0.1 microgram/mL. It exerted measurable inhibitory effects on cell proliferation only at concentrations higher than 100 micrograms/mL. In vivo CEDU reduced the mortality rate of HSV-1-infected mice at concentrations lower than 5 mg/kg per day when given intraperitoneally and orally. Thus, it proved to be more effective in this in vivo model than the reference compounds (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-[(2-hydroxyethoxy)methyl]guanine (ACV).     

Evaluation of anti-herpesvirus activity of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]- guanine (A-5021) in mice.

The anti-herpesvirus activity of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guani ne (A-5021) was evaluated in murine cells and in several murine models of herpes simplex virus (HSV) infection. Against HSV type 1 (HSV-1), A-5021 was 15-30- and 30-60-fold more active, and against HSV type 2 (HSV-2), it was 2- and 8-fold more active than acyclovir and penciclovir in Balb/3T3 cells, respectively. When antiviral compounds were administered orally (once daily) to mice infected intraperitoneally with HSV-1 (Tomioka), A-5021 was more active than acyclovir or famciclovir in spite of its relatively low oral bioavailability. A-5021 was as active as penciclovir when the antiviral compounds were given intravenously (three times daily) to mice infected intraperitoneally with HSV-2 (186). In mice with a cutaneous HSV-1 (KOS) infection, three times daily oral therapy with A-5021 at 25 mg/kg per day produced more significant reduction in severity of skin lesions than equivalent treatment with acyclovir or famciclovir. In mice infected intracerebrally with HSV-1 (Tomioka), complete survival was observed in the group treated intravenously with A-5021 at 25 mg/kg per day (three times daily), while more than 50% of mice died in the groups treated intravenously with acyclovir of up to 100 mg/kg per day (three times daily). Moreover, A-5021 was more effective than acyclovir in clearing infectious virus from the brain. These findings demonstrate that A-5021 has potent anti-HSV activity in several murine models.     

The activity of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC) against equine herpesvirus-1 (EHV-1) in cell cultures, mice and horses.

The activity of the nucleotide analogue, (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC), against equine herpesvirus-1 (EHV-1) was tested in cell culture, mice and foals. The ED50 for plaque reduction was found to be 0.07 and 0.03 microgram/ml in RK-13 and EEL cells respectively. In mice, a single administration of HPMPC (20 mg/kg, s.c.) was very effective at reducing clinical signs and virus replication if given on the day before intranasal inoculation with EHV-1. Treatment on the day of infection or day 1 p.i. was less effective, but still significantly reduced clinical signs and virus titres in the target organs (lungs and nasal tissue). Furthermore, HPMPC was found to protect mice partially from an intracerebral inoculation with EHV-1. Experiments in the horse suggested that HPMPC was also very active against EHV-1 in the natural host. Thus a single administration of HPMPC at 20 mg/kg, s.c., on the day of infection, markedly reduced clinical signs and nasal excretion of virus following intranasal inoculation with EHV-1. HPMPC given as a divided dose of 1 mg/kg on day 0 and day 3 p.i. had no effect on clinical signs but did reduce nasal excretion of virus. The significance of these results is discussed.     

Combined antiviral effects of paired nucleosides against guinea pig cytomegalovirus replication in vitro

Several promising antiviral nucleosides have been tested in paired combinations against guinea pig cytomegalovirus (GPCMV) replication in guinea pig embryo (GPE) cells by plaque reduction assay; these are [9-(2-hydroxy-1-3-2-dioxaphosphorinan-5-yl)oxymethyl]-guanin e P-oxide (2'nor-cGMP, compound 164), [4-amino-5-bromo-7-(2-hydroxyethoxymethyl)-pyrrolo(2,3-d)pyrimidine] (compound 102), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), 9-(2-hydroxyethoxymethyl)-guanine (acyclovir, ACV) and 3'-azido-3'-deoxythymidine (zidovudine, AZT). Various degrees of interactions were observed; i.e. synergistic reactions were noted in the presence of compound 164/compound 102 and compound 164/DHPG combinations at all concentrations tested. HPMPC/DHPG combinations were synergistic at relatively lower concentrations of DHPG, but became antagonistic as the concentration of DHPG increased. Combinations of compound 164/ACV and DHPG/AZT were antagonistic.     

Treatment of experimental herpes simplex virus type 1 encephalitis in mice with (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil: comparison with bromovinyl-deoxyuridine and acyclovir

The efficiency of (E)-5-(2-bromovinyl)- and 5-vinyl-1-beta-D-arabinofuranosyluracil (BrVaraU, VaraU) as inhibitors of three herpes simplex virus type 1 (HSV-1) strains was assessed in comparison to (E)-5-(2-bromovinyl)-2'-deoxyuridine (BrVUdR), 9-(2-hydroxyethoxymethyl)guanine (ACV), and trisodium phosphonoformate (Na3PFA) using a plaque assay in human embryonic lung fibroblast (HELF) cell cultures. The following order of decreasing activity was found: BrVaraU greater than VaraU greater than BrVU-dR greater than ACV much greater than Na3PFA. In HELF cell cultures, the selectivity indexes of VaraU and BrVaraU were 10 times higher than those of BrVUdR and ACV. Protection of mice from encephalitis and death due to intracerebral (i.c.) infection with a clinical HSV-1 isolate was nearly complete if mice were treated intraperitoneally (i.p.) with two daily doses of VaraU and BrVaraU (100 or 200 mg/kg per day) over a period of 5 or 10 days. The efficacy was similar to ACV, but, using a treatment schedule of three daily i.p. doses over 10 days, with equimolar amounts of the nucleoside analogs, VaraU and BrVaraU (140 and 180 mg/kg per day) were superior to ACV (130 mg/kg per day) (P less than 0.05).     

Antiviral evaluation of octadecyloxyethyl esters of (S)-3-hydroxy-2-(phosphonomethoxy)propyl nucleosides against herpesviruses and orthopoxviruses

Our previous studies showed that esterification of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) or 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP and ODE esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine (HPMPT), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC₅₀ values were observed with ODE–HPMPA and ODE–HPMPC while ODE–HPMPG had intermediate antiviral activity with an EC₅₀ of 40 nM. In HFF cells infected with HCMV, the lowest EC₅₀ values were observed with ODE–HPMPC, 0.9 nM. ODE–HPMPA was highly active with an EC₅₀ of 3 nM, while ODE–HPMPG and ODE–HPMPDAP were also highly active with EC₅₀s of 22 and 77 nM, respectively. Against vaccinia and cowpox viruses, ODE–HPMPG and ODE–HPMPDAP were the most active and selective compounds with EC₅₀ values of 20–60 nM and selectivity index values of 600–3500. ODE–HPMPG was also active against ectromelia virus with an EC₅₀ value of 410 nM and a selectivity index value of 166. ODE–HPMPG and ODE–HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases.     

Synthesis and antiherpetic activity of (S)-, (R)-, and (+/-)-9-[(2,3-dihydroxy-1-propoxy)methyl]guanine, linear isomers of 2'-nor-2'-deoxyguanosine

Racemic 9-[(2,3-dihydroxy-1-propoxy)methyl]guanine [(+/-)-iNDG], a new analogue of acyclovir (ACV) and a structural analogue of 2'-nor-2'-deoxyguanosine (2'NDG), was synthesized and found to inhibit the replication of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2). Subsequently, its optical isomers, (R)- and (S)-iNDG, were prepared from chiral intermediates. The chloromethyl ethers of 1,2-di-O-benzyl-D- and -L-glycerol were made and reacted with tris(trimethylsilyl)guanine to give the 9-alkylated guanines, which were deprotected by catalytic hydrogenolysis. Against HSV-1 and HSV-2 in cell culture, (S)-iNDG was approximately 10- to 25-fold more active than the R enantiomer and had an ED50 comparable to those for ACV and 2'NDG. The inferior activity of (R)-iNDG paralleled the poor inhibition of viral DNA polymerase by its phosphorylation products. In mice infected intraperitoneally or orofacially with HSV-1 or intravaginally with HSV-2, (S)-9-[(2,3-dihydroxy-1-propoxy)methyl]guanine [(S)-iNDG] was less efficacious than 2'NDG but comparable to or more active than ACV.     

A novel class of phosphonate nucleosides. 9-[(1-phosphonomethoxycyclopropyl)methyl]guanine as a potent and selective anti-HBV agent

9-[1-(Phosphonomethoxycyclopropyl)methyl]guanine (PMCG, 1), representative of a novel class of phosphonate nucleosides, blocks HBV replication with excellent potency (EC(50) = 0.5 microM) in a primary culture of HepG2 2.2.15 cells. It exhibits no significant cytotoxicity in several human cell lines up to 1.0 mM. It does not inhibit replication of human immunodeficiency virus (HIV-1) or herpes simplex virus (HSV-1) at 30 microM. Many purine base analogues of 1 also exhibit inhibitory activity against HBV, but at 30 microM, pyrimidine analogues do not. 1 is 4 times more potent than 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), which was used as a positive control (EC(50) = 2.0 microM). The characteristic cyclopropyl moiety at the 2'-position of 1 was prepared by titanium-mediated Kulinkovich cyclopropanation. 1 was modified to give the orally available drug candidate, PMCDG Dipivoxil (2). Compound 2 exhibited excellent efficacy when administered at 5 mg per kg per day in a study with woodchucks infected with woodchuck hepatitis B virus (WHBV). Drug candidate 2 has successfully completed phase I clinical trials and is currently undergoing phase II clinical studies for evaluation of efficacy.     

Synthesis and antiviral activity of (S)-9-[4-hydroxy-3-(phosphonomethoxy)butyl]guanine

The synthesis of (S)-9-[4-hydroxy-3-(phosphonomethoxy)butyl]guanine (3), starting from (S)-4-(2-hydroxyethyl)-2,2-dimethyl-1,3-dioxolane (4), is described. Alkylation of trityl derivative 7 with (diethylphosphono)methyl triflate provided phosphonate 8, which was readily converted to mesylate 12 in three steps. Nucleophilic substitution of the mesylate group of 12 by 2-amino-6-chloropurine sodium salt led to (S)-2-amino-6-chloro-9-[3-[(diethyl-phosphono)methoxy]-4-(tetrahydro- 2H-pyran-2-yloxy)butyl]purine (13). Sequential treatment of 13 with trimethylsilyl bromide and then with 2 N HCl furnished 3. Preliminary in vitro screening indicated that 3 exhibited a potent activity against human cytomegalovirus (HCMV) but was not active against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The adenine and cytosine derivatives (14 and 15) did not exhibit activity against HSV-1 and -2 and HCMV.     

Anti-herpesvirus activity of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)-cycloprop-1'-yl]methyl] x guanine (A-5021) in vitro and in vivo

The novel nucleoside analog (1S',2R')-9-[[1',2'-bis(hydroxymethyl)cycloprop-1-yl]methyl]guanine (A-5021) was previously shown to be a potent inhibitor of the replication of herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) and varicella zoster virus (VZV), both in vitro and in vivo (J. Med. Chem. 41, 1284-1298; Antimicrob. Agents Chemother. 42, 1666-1670). Here we demonstrate that A-5021 is also a potent inhibitor of Epstein-Barr virus (EBV) and human herpes virus 6 (HHV-6A and HHV-6B), but that the compound lacks activity against HHV-8. A-5021, in comparison to acyclovir, was also assessed for protective activity against HSV-1-induced mortality in SCID mice. The compounds were administered at 50 mg/kg per day by subcutaneous injection for four consecutive days and treatment was initiated at either 2 h, 1 or 2 days post infection (p.i.). When administered from day 0 to 4 p.i., A-5021 conferred complete protection against the infection (as assessed at 22 days p.i.), whereas acyclovir delayed virus induced mortality by only 5 days. When treatment was begun on day 1 or 2, A-5021 still afforded marked protection against the infection, whereas acyclovir was virtually devoid of any activity under these conditions. Our data underline that A-5021 may offer great promise for the treatment of herpesvirus infections.     

Effect of acyclic pyrimidines related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine on herpesviruses

A series of pyrimidines related to the potent antiherpetic agent 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (1, BW B759U), all containing the same acyclic chain, have been synthesized. Some of the compounds were derivatives of the naturally occurring bases, cytosine, uracil, and thymine; others included compounds in which the 5-position of the cytosine and uracil moieties were substituted by bromo, iodo, fluoro, methyl, and amino groups. Other variations of the cytosine derivatives were the 5-aza, 2-mercapto, 4-methylamino, 4-dimethylamino, and isocytosine congeners. A 4-aminopyrimidine adduct was also made. Antiviral testing showed that 1-[(1,3-dihydroxy-2-propoxy)methyl]cytosine (18, BW A1117U) was equivalent to the guanine analogue in potency against human cytomegalovirus and Epstein Barr virus. Other compounds in the series were largely inactive in antiviral screening against the herpesviruses.     

Synthesis and antiviral activity of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine.

A number of methyl derivatives of 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG, 1) have been synthesized and tested in vitro for anti-herpes and anti-human immunodeficiency virus (HIV) activity. Among these analogues, (R)-2'-methyl-PMEG [(R)-3] and 2',2'-dimethyl-PMEG (7) demonstrated potent anti-HIV activity in the XTT assay with EC50 values of 1.0 and 2.6 microM, respectively. The corresponding (S)-2'-methyl-PMEG [(S)-3] was found to be less potent against HIV. In addition, the (R) and (S) enantiomers of 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG, 8) were prepared for comparison of biological activity, and shown to be active and equipotent against herpesviruses, but inactive against HIV.     

Purine acyclic nucleosides. Nitrogen isosteres of 9-[(2-hydroxyethoxy)methyl]guanine as candidate antivirals

A number of nitrogen analogues of 9-[(2-hydroxyethoxy)methyl]guanine [acylovir, Zovirax] containing amine functions in the side chain were synthesized and tested for antiviral activity. These purine acyclic nucleosides were prepared by reaction of tris(trimethylsilyl)guanine or 2,6-diaminopurine sodium salt with the chloromethyl ethers prepared from N-(2-hydroxyethyl)phthalimide, N-[2-(2-hydroxyethoxy)ethyl]phthalimide, or N-(2-hydroxyethyl)oxazolidin-2-one to give the N-blocked intermediates 5-8. Deprotection with hydrazine or by alkaline hydrolysis gave 9-[(2-aminoethoxy)methyl]guanine (9), 9-[(2-aminoethoxy)methyl]-2,6-diaminopurine (10), 9-[2(2-aminoethoxy]ethoxymethyl]guanine (11), and 9-[[2-[(2-hydroxyethyl)amino]ethoxy]methyl]guanine (12). When tested against herpes simplex virus type 1, only 9 was active with an IC50 = 8 microM. Little or no activity was observed against a range of other DNA and RNA viruses.     

Intracellular metabolism of the antiherpes agent (S)-1-[3-hydroxy-2-(phosphonylmethoxy)propyl]cytosine.

(S)-1-[3-Hydroxy-2-(phosphonylmethoxy)propyl]cytosine (HPMPC) is an antiviral phosphonate nucleotide analogue that displays activity against a range of herpesviruses. Anion exchange high performance liquid chromatography analysis of the 60% methanol extract from [14C]HPMPC-treated cells reveals the formation of three major metabolites. Two of these were identified as phosphorylated forms of HPMPC, HPMPC phosphate, and HPMPC diphosphate, by liberation of HPMPC upon acid digestion and coelution with synthetic standards on high performance liquid chromatography. The third metabolite, which is resistant to alkaline phosphatase cleavage but sensitive to phosphodiesterase, is proposed to be an HPMPC phosphate adduct. In herpes simplex virus-1-infected cells the same three metabolites are detected, at concentrations comparable to those in uninfected cells. When HPMPC is removed from the medium, the concentrations of the metabolites in cells decrease slowly, with half-lives of approximately 6, 17, and 48 hr for HPMPC phosphate, HPMPC diphosphate, and the HPMPC phosphate adduct, respectively. HPMPC diphosphate inhibits herpes simplex virus-1 and -2 DNA polymerases with a lower Ki than that for DNA polymerase alpha, and enzyme inhibition is competitive in each case. The formation and the persistence of HPMPC phosphates in cells and the selective inhibition of viral DNA polymerases by HPMPC diphosphate can explain why cells pretreated with HPMPC remain refractory to viral infection even long after HPMPC is removed from the medium.     

Effect of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine on the replication and morphogenesis of herpes simplex virus type 1.

Treatment of African green monkey kidney cells with 1 microgram/ml of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC) inhibited the release of infectious herpes simplex virus type 1 (HSV-1) by more than 90%. Electron microscopic observations of HPMPC-treated monkey kidney cells demonstrated few intracellular or extracellular viral particles. The viral particles seen were without dense cores. In addition, HPMPC blocked cell fusion induced by HSV-1 in monkey kidney cells. Immunoblot analysis showed that HPMPC significantly blocked the expression of HSV-1-specific proteins. Furthermore, HPMPC inhibited the synthesis of viral DNA as determined by in situ hybridization. These results indicate that HPMPC inhibits the replication of HSV by blocking one of the events involved in DNA synthesis.     

Antiviral activity of anti-cytomegalovirus agents (HPMPC, HPMPA) assessed by a flow cytometric method and DNA hybridization technique.

Phosphonylmethoxyalkylpurines and -pyrimidines, particularly (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) and (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and their cyclic forms (cHPMPC, cHPMPA) and the 3-deaza analogue of HPMPA (HPMPc3A) are selective and potent inhibitors of human cytomegalovirus (CMV) replication in vitro. Their anti-CMV activity has been monitored by flow cytometry and DNA hybridization. The anti-CMV agent ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG)] was included as a reference compound. From the flow cytometric assays, HPMPC and HPMPA were the most active compounds, with an EC50 (50% effective concentration) of approximately 0.6 microM. The EC50 values obtained by DNA hybridization ranged from 0.05 microM (HPMPC) to 0.74 microM (DHPG). Selectivity indexes, calculated as the ratio of the 50% inhibitory concentration (CC50) for cell growth or [methyl-3H]-thymidine incorporation to the EC50 for virus replication were highest for HPMPC and its cyclic derivative (cHPMPC). The flow cytometry and DNA hybridization assays thus confirm the results obtained by the classic plaque assay. They allow a quantitative estimation of the anti-CMV potency of the test compounds.     

Synthesis and antiviral properties of 9-[(2-methyleneaminoxyethoxy)methyl]guanine derivatives as novel Acyclovir analogues

This paper reports the synthesis and the antiviral properties of a series of 9-[(2-methyleneaminoxyethoxy)methyl]guanine derivatives, which can be viewed as analogues of the antiherpes drug Acyclovir (ACV) from which they differ in the replacement of its hydroxy group with variously substituted methyleneaminoxy moieties. Some of the newly synthesized compounds proved to possess a certain activity against HSV-1, albeit lower than that of ACV.