Human cytomegalovirus resistance to deoxyribosylindole nucleosides maps to a transversion mutation in the terminase subunit-encoding gene UL89.

Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 µM) than ganciclovir (EC50 = 7.4 µM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50 = 3.1 ± 0.7 µM) compared to that of wild-type virus (EC50 = 0.17 ± 0.04 µM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50 for wild-type HCMV = 0.25 ± 0.04 µM, EC50 for HCMV pUL89 E256Q = 0.23 ± 0.04 µM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).

Resistance of human cytomegalovirus to cyclopropavir maps to a base pair deletion in the open reading frame of UL97.

Human cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC50s] = 0.46 and 4.1 µM, respectively). We hypothesize that the mechanism of action of CPV is similar to that of GCV: phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation to a triphosphate by endogenous kinases, resulting in inhibition of viral DNA synthesis. To test this hypothesis, we isolated a CPV-resistant virus, sequenced its genome, and discovered that bp 498 of UL97 was deleted. This mutation caused a frameshift in UL97 resulting in a truncated protein that lacks a kinase domain. To determine if this base pair deletion was responsible for drug resistance, the mutation was engineered into the wild-type viral genome, which was then exposed to increasing concentrations of CPV. The results demonstrate that the engineered virus was approximately 72-fold more resistant to CPV (EC50 = 25.8 ± 3.1 µM) than the wild-type virus (EC50 = 0.36 ± 0.11 µM). We conclude, therefore, that this mutation is sufficient for drug resistance and that pUL97 is involved in the mechanism of action of CPV.

Benzimidazole analogs inhibit human herpesvirus 6.

Several benzimidazole nucleoside analogs, including 1H-ß-D-ribofuranosyl-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and 1H-ß-L-ribofuranosyl-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir [MBV]), inhibit the replication of human cytomegalovirus. Neither analog inhibited the related betaherpesvirus human herpesvirus 6 (HHV-6). Additional analogs of these compounds were evaluated against both variants of HHV-6, and two L-analogs of BDCRB had good antiviral activity against HHV-6A, as well as more modest inhibition of HHV-6B replication.

Synthesis and antiviral activity of (Z)- and (E)-2,2-[bis(hydroxymethyl)cyclopropylidene]methylpurines and -pyrimidines: second-generation methylenecyclopropane analogues of nucleosides.

The second generation of methylenecyclopropane analogues of nucleosides 5a-5i and 6a-6i was synthesized and evaluated for antiviral activity. The 2,2-bis(hydroxymethyl)methylenecyclopropane (11) was converted to dibromo derivative 7 via acetate 12. Alkylation-elimination of adenine (16) with 7 afforded the Z/E mixture of acetates 17 + 18, which was deacetylated to give analogues 5a and 6a separated by chromatography. A similar reaction with 2-amino-6-chloropurine (19) afforded acetates 20 + 21 and, after deprotection and separation, isomers 5f and 6f. The latter served as starting materials for synthesis of analogues 5b, 5e, 5g-5i and 6b, 6e, 6g-6i. Alkylation-elimination of N(4)-acetylcytosine (22) with 7 afforded a mixture of isomers 5c + 6c which were separated via N(4)-benzoyl derivatives 23 and 24. Deprotection furnished analogues 5c and 6c. Alkylation of 2,4-bis(trimethylsilyloxy)-5-methylpyrimidine (25) with 7 led to bromo derivative 26. Elimination of HBr followed by deacetylation and separation gave thymine analogues 5d and 6d. The guanine Z-isomer 5b was the most effective against human and murine cytomegalovirus (HCMV and MCMV) with EC(50) = 0.27-0.49 microM and no cytotoxicity. The 6-methoxy analogue 5g was also active (EC(50) = 2.0-3.5 microM) whereas adenine Z-isomer 5a was less potent (EC(50) = 3.6-11.7 microM). Cytosine analogue 5c was moderately effective, but 2-amino-6-cyclopropylamino derivative 5e was inactive. All E-isomers were devoid of anti-CMV activity, and none of the analogues was significantly active against herpes simplex viruses (HSV-1 or HSV-2). The potency against Epstein-Barr virus (EBV) was assay-dependent. In Daudi cells, the E-isomers of 2-amino-6-cyclopropylamino- and 2,6-diaminopurine derivatives 6e and 6h were the most potent (EC(50) approximately 0.3 microM), whereas only the thymine Z-isomer 5d was active (EC(50) = 4.6 microM). Guanine Z-derivative 5b was the most effective compound in H-1 cells (EC(50) = 7 microM). In the Z-series, the 2-amino-6-methoxypurine analogue 5g was the most effective against varicella zoster virus (VZV, EC(50) = 3.3 microM) and 2,6-diaminopurine 5h against hepatitis B virus (HBV, EC(50) = 4 microM). Adenine analogues 5a and 6a were moderately active as substrates for adenosine deaminase.

Synthesis and antiviral activity of 2-substituted analogs of triciribine.

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore the effect of substitutions at the 2-position of triciribine. 2-Methyl- (2-Me-TCN), 2-ethyl- (2-Et-TCN), 2-phenyl- (2-Ph-TCN), 2-chloro- (2-Cl-TCN), and 2-aminotriciribine (2-NH2-TCN) were designed and synthesized to determine the effects of substitutions at the 2-position which change the steric, electronic, and hydrophobic properties of TCN, while maintaining the integrity of the tricyclic ring system. These compounds were evaluated for activity against human immunodeficiency virus (HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV) and were found to be either less active than TCN and TCN-P or inactive at the highest concentrations tested, 100 microM. We conclude that substitutions at the 2-position of triciribine adversely affect the antiviral activity most likely because these analogs are not phosphorylated to active metabolites.

Tyrosine-based 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine and -adenine ((S)-HPMPC and (S)-HPMPA) prodrugs: synthesis, stability, antiviral activity, and in vivo transport studies.

Eight novel single amino acid (6-11) and dipeptide (12, 13) tyrosine P-O esters of cyclic cidofovir ((S)-cHPMPC, 4) and its cyclic adenine analogue ((S)-cHPMPA, 3) were synthesized and evaluated as prodrugs. In vitro IC(50) values for the prodrugs (<0.1-50 µM) vs vaccinia, cowpox, human cytomegalovirus, and herpes simplex type 1 virus were compared to those for the parent drugs ((S)-HPMPC, 2; (S)-HPMPA, 1; IC(50) 0.3-35 µM); there was no cytoxicity with KB or HFF cells at ≤100 µM. The prodrugs exhibited a wide range of half-lives in rat intestinal homogenate at pH 6.5 (<30-1732 min) with differences of 3-10× between phostonate diastereomers. The tyrosine alkylamide derivatives of 3 and 4 were the most stable. (l)-Tyr-NH-i-Bu cHPMPA (11) was converted in rat or mouse plasma solely to two active metabolites and had significantly enhanced oral bioavailability vs parent drug 1 in a mouse model (39% vs <5%).

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

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.

Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir.

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH-dependent stability (3 and 4), potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver (t(1/2) = 3.7 min), intestinal (t(1/2) = 12.5 min), and Caco-2 cell homogenates (t(1/2) = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC50 value, 0.68 microM vs 3.0 microM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.

Amino acid ester prodrugs of 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole enhance metabolic stability in vitro and in vivo.

2-Bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV), but it lacks clinical utility due to rapid in vivo metabolism. We hypothesized that amino acid ester prodrugs of BDCRB may enhance both in vitro potency and systemic exposure of BDCRB through evasion of BDCRB-metabolizing enzymes. To this end, eight different amino acid prodrugs of BDCRB were tested for N-glycosidic bond stability, ester bond stability, Caco-2 cell uptake, antiviral activity, and cytotoxicity. The prodrugs were resistant to metabolism by BDCRB-metabolizing enzymes, and ester bond cleavage was rate-limiting in metabolite formation from prodrug. Thus, BDCRB metabolism could be controlled by the selection of promoiety. In HCMV plaque-formation assays, l-Asp-BDCRB exhibited 3-fold greater selectivity than BDCRB for inhibition of HCMV replication. This potent and selective antiviral activity in addition to favorable stability profile made l-Asp-BDCRB an excellent candidate for in vivo assessment and pharmacokinetic comparison with BDCRB. In addition to rapid absorption and sufficient prodrug activation after oral administration to mice, l-Asp-BDCRB exhibited a 5-fold greater half-life than BDCRB. Furthermore, the sum of area under the concentration-time profile (AUC)(BDCRB) and AUC(prodrug) after l-Asp-BDCRB administration was roughly 3-fold greater than AUC(BDCRB) after BDCRB administration, suggesting that a reservoir of prodrug was delivered in addition to parent drug. Overall, these findings demonstrate that amino acid prodrugs of BDCRB exhibit evasion of metabolizing enzymes (i.e., bioevasion) in vitro and provide a modular approach for translating this in vitro stability into enhanced in vivo delivery of BDCRB.

Inhibition of cyclin-dependent kinase 1 by purines and pyrrolo[2,3-d]pyrimidines does not correlate with antiviral activity.

We have previously shown that a series of nonnucleoside pyrrolo[2,3-d]pyrimidines selectively inhibit the replication of herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). These compounds act at the immediate-early or early stage of HCMV replication and have antiviral properties somewhat similar to those of roscovitine and olomoucine, specific inhibitors of cyclin-dependent kinases (cdks). In the present study we examine the hypothesis that pyrrolo[2,3-d]pyrimidines exert their antiviral effects by inhibition of cellular cdks. Much higher concentrations of a panel of pyrrolo[2,3-d]pyrimidine nucleoside analogs with antiviral activity were required to inhibit recombinant cdk1/cyclin B compared to the submicromolar concentrations required to inhibit HCMV and HSV-1 replication. 4,6-Diamino-5-cyano-7-(2-phenylethyl)pyrrolo[2,3-d]pyrimidine (compound 1369) was the best inhibitor of cdk1 and cyclin B, with a 50% inhibitory concentration (IC(50); 14 microM) similar to that of roscovitine; it was competitive with respect to ATP (K(i) = 14 microM). The potency of compound 1369 against cdk1 and cyclin B was similar to its cytotoxicity (IC(50)s, 32 to 100 microM) but not its antiviral efficacy (IC(50)s, 0.02 to 0.3 microM). Thus, our results indicated the null hypothesis. In contrast, roscovitine was only weakly active against HSV-1 (IC(50), 38 microM) and HCMV (IC(50), 40 microM). These values were similar to those derived by cytotoxicity and cell growth inhibition assays, thereby suggesting that roscovitine is not a selective antiviral. Therefore, we propose that inhibition of cdk1 and cyclin B is not responsible for selective antiviral activity and that pyrrolo[2,3-d]pyrimidines constitute novel pharmacophores which compete with ATP to inhibit cdk1 and cyclin B.

Phosphorylation of beta-D-ribosylbenzimidazoles is not required for activity against human cytomegalovirus.

We have previously reported that 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analog (2-bromo-5,6-dichloro-1-(beta-D-ribofuranosy)benzimidazole [BDCRB]) are potent and selective inhibitors of human cytomegalovirus (HCMV) replication that block viral DNA maturation via HCMV gene products UL89 and UL56. To determine if phosphorylation is required for antiviral activity, the in vitro metabolism of BDCRB was examined and the antiviral activities of nonphosphorylatable 5'-deoxy analogs were determined. Reverse-phase high-performance liquid chromatography (HPLC) analysis of extracts from uninfected and HCMV-infected cells incubated with [(3)H]BDCRB revealed two major metabolites. Both were less polar than naturally occurring nucleoside monophosphates, but one peak coeluted with a BDCRB-5'-monophosphate (BDCRB-5'-MP) standard. Further analysis revealed, however, that neither metabolite partitioned with BDCRB-5'-MP on anion-exchange HPLC. Their retention patterns were not affected by incubation with alkaline phosphatase, thereby establishing that the compounds were not nucleoside 5'-monophosphates. Both compounds were detected in uninfected and HCMV-infected cells and in mouse live extracts, but neither has been identified. Like TCRB and BDCRB, the nonphosphorylatable 5'-deoxy analogs were potent and selective inhibitors of HCMV replication. The 5'-deoxy analogs maintained inhibition of HCMV replication upon removal of BDCRB, whereas an inhibitor of DNA synthesis did not. Similar to TCRB, its 5'-deoxy analog (5'-dTCRB) did not affect viral DNA synthesis, but 5'-dTCRB did inhibit viral DNA maturation to genome-length units. Additionally, virus isolates resistant to TCRB were also resistant to 5'-dTCRB and the 5'-deoxy analog of BDCRB. Taken together, these results confirm that TCRB, BDCRB, and their 5'-deoxy analogs have common mechanisms of action and establish that these benzimidazole ribonucleosides, unlike other antiviral nucleosides, do not require phosphorylation at the 5' position for antiviral activity.