Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase.

Cyclopropavir (CPV) is a promising antiviral drug against human cytomegalovirus (HCMV). As with ganciclovir (GCV), the current standard for HCMV treatment, activation of CPV requires multiple steps of phosphorylation and is enantioselective. We hypothesized that the resulting CPV triphosphate (CPV-TP) would stereoselectively target HCMV DNA polymerase and terminate DNA synthesis. To test this hypothesis, we synthesized both enantiomers of CPV-TP [(+) and (-)] and investigated their action on HCMV polymerase. Both enantiomers inhibited HCMV polymerase competitively with dGTP, with (+)-CPV-TP exhibiting a more than 20-fold lower apparent Ki than (-)-CPV-TP. Moreover, (+)-CPV-TP was a more potent inhibitor than GCV-TP. (+)-CPV-TP also exhibited substantially lower apparent Km and somewhat higher apparent kcat values than (-)-CPV-TP and GCV-TP for incorporation into DNA by the viral polymerase. As is the case for GCV-TP, both CPV-TP enantiomers behaved as nonobligate chain terminators, with the polymerase terminating DNA synthesis after incorporation of one additional nucleotide. These results elucidate how CPV-TP acts on HCMV DNA polymerase and help explain why CPV is more potent against HCMV replication than GCV.

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.

Synthesis and antiviral activity of certain second generation methylenecyclopropane nucleosides.

A second-generation series of substituted methylenecyclopropane nucleosides (MCPNs) has been synthesized and evaluated for antiviral activity against a panel of human herpesviruses, and for cytotoxicity. Although alkylated 2,6-diaminopurine analogs showed little antiviral activity, the compounds containing ether and thioether substituents at the 6-position of the purine did demonstrate potent and selective antiviral activity against several different human herpesviruses. In the 6-alkoxy series, antiviral activity depended on the length of the ether carbon chain, with the optimum chain length being about four carbon units long. For the corresponding thioethers, compounds containing secondary thioethers were more potent than those with primary thioethers.

Synthesis and antiviral activity of 6-deoxycyclopropavir, a new prodrug of cyclopropavir.

Synthesis of 6-deoxycyclopropavir (10), a prodrug of cyclopropavir (1) and its in vitro and in vivo antiviral activity is described. 2-Amino-6-chloropurine methylenecyclopropane 13 was transformed to its 6-iodo derivative 14 which was reduced to prodrug 10. It is converted to cyclopropavir (1) by the action of xanthine oxidase and this reaction can also occur in vivo. Compound 10 lacked significant in vitro activity against human cytomegalovirus (HCMV), human herpes virus 1 and 2 (HSV-1 and HSV-2), human immunodeficiency virus type 1 (HIV-1), human hepatitis B virus (HBV), Epstein-Barr virus (EBV), vaccinia virus and cowpox virus. In contrast, prodrug 10 given orally was as active as cyclopropavir (1) reported previously [Kern, E. R.; Bidanset, D. J.; Hartline, C. B.; Yan, Z.; Zemlicka, J.; Quenelle, D. C. et al. Antimicrob. Agents Chemother. 2004, 48, 4745] against murine cytomegalovirus (MCMV) infection in mice and against HCMV in severe combined immunodeficient (SCID) mice.

Phosphorylation of antiviral and endogenous nucleotides to di- and triphosphates by guanosine monophosphate kinase.

Many fraudulent nucleosides including the antivirals acyclovir (ACV) and ganciclovir (GCV) must be metabolized to triphosphates to be active. Cyclopropavir (CPV) is a newer, related guanosine nucleoside analog that is active against human cytomegalovirus (HCMV) in vitro and in vivo. We have previously demonstrated that CPV is phosphorylated to its monophosphate (CPV-MP) by the HCMV pUL97 kinase. Consequently, like other nucleoside analogs phosphorylated by viral kinases, CPV most likely must be converted to a triphosphate (CPV-TP) in order to elicit antiviral activity. Once formed by pUL97, we hypothesized that guanosine monophosphate kinase (GMPK) is the enzyme responsible for the conversion of CPV-MP to CPV-DP. Incubation of CPV-MP with GMPK resulted in the formation of CPV-DP and, surprisingly, CPV-TP. When CPV-DP was incubated with GMPK, a time-dependent increase in CPV-TP occurred corresponding to a decrease in CPV-DP thereby demonstrating that CPV-DP is a substrate for GMPK. Substrate specificity experiments revealed that GMP, dGMP, GDP, and dGDP are substrates for GMPK. In contrast, GMPK recognized only acyclovir and ganciclovir monophosphates as substrates, not their diphosphates. Kinetic studies demonstrated that CPV-DP has a K(M) value of 45±15µM. We were, however, unable to determine the K(M) value for CPV-MP directly, but a mathematical model of experimental data gave a theoretical K(M) value for CPV-MP of 332±60µM. We conclude that unlike many other antivirals, cyclopropavir can be converted to its active triphosphate by a single cellular enzyme once the monophosphate is formed by a virally encoded kinase.

Stereoselective phosphorylation of cyclopropavir by pUL97 and competitive inhibition by maribavir.

Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Kinetic studies with CPV as the substrate for pUL97 demonstrated a Km of 1,750+/-210 microM. Introduction of 1.0 or 10 nM maribavir, a known pUL97 inhibitor, and subsequent Lineweaver-Burk analysis demonstrated competitive inhibition of CPV phosphorylation, with a Ki of 3.0+/-0.3 nM. Incubation of CPV with pUL97 combined with GMP kinase [known to preferentially phosphorylate the (+)-enantiomer of CPV-MP] established that pUL97 stereoselectively phosphorylates CPV to its (+)-monophosphate. These results elucidate the mechanism of CPV phosphorylation and help explain its selective antiviral action.

L-valine ester of cyclopropavir: a new antiviral prodrug.

BACKGROUND: Following the example of L-valine prodrugs of antiviral nucleoside analogues, L-valine ester of cyclopropavir (valcyclopropavir) was synthesized. METHODS: The known tetrahydropyranylcyclopropavir was transformed to N-(tert-butoxycarbonyl)-L-valine ester, which was deprotected to valcyclopropavir. RESULTS: Stability of valcyclopropavir towards hydrolysis at pH 7.0 roughly corresponded to that of valganciclovir. Valcyclopropavir inhibited replication of human cytomegalovirus (HCMV, Towne and AD169 strains) to approximately the same extent as the parent drug cyclopropavir. Pharmacokinetic studies in mice established that the oral bioavailability of valcyclopropavir was 95%. CONCLUSIONS: The prodrug valcyclopropavir offers some improved therapeutic parameters over the parent compound cyclopropavir.

Phosphonate analogues of cyclopropavir phosphates and their E-isomers. Synthesis and antiviral activity.

Z- and E-Phosphonate analogues 12 and 13 derived from cyclopropavir and the corresponding cyclic phosphonates 14 and 15 were synthesized and their antiviral activity was investigated. The 2,2-bis(hydroxymethylmethylenecyclopropane acetate (17) was transformed to tetrahydropyranyl acetate 18. Deacetylation gave intermediate 19 which was converted to bromide 20. Alkylation with diisopropyl methylphosphonate afforded after protecting group exchange (21 to 22) acetylated phosphonate intermediate 22. Addition of bromine gave the dibromo derivative 16 which was used in the alkylation-elimination procedure with 2-amino-6-chloropurine to give Z- and E-isomers 23 and 24. Hydrolytic dechlorination coupled with removal of all protecting groups gave the guanine phosphonates 12 and 13. Cyclization afforded the cyclic phosphonates 14 and 15. Z-Phosphonate 12 was a potent and non-cytotoxic inhibitor of human and murine cytomegalovirus (HCMV and MCMV) with EC(50) 2.2-2.7 and 0.13 microM, respectively. It was also an effective agent against Epstein-Barr virus (EBV, EC(50) 3.1 microM). The cyclic phosphonate 14 inhibited HCMV (EC(50) 2.4-11.5 microM) and MCMV (EC(50) 0.4 microM) but it was ineffective against EBV. Both phosphonates 12 and 14 were as active against two HCMV Towne strains with mutations in UL97 as they were against wild-type HCMV thereby circumventing resistance due to such mutations. Z-Phosphonate 12 was a moderate inhibitor of replication of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) but it was a potent agent against varicella zoster virus (VZV, EC(50) 2.9 microM). The cyclic phosphonate 14 lacked significant potency against these viruses. E-isomers 13 and 15 were devoid of antiviral activity.

Stereoselective approach to the Z-isomers of methylenecyclopropane analogues of nucleosides: a new synthesis of antiviral synguanol.

Stereoselective synthesis of antiviral synguanol (1) is described. Reaction of 6-benzyloxy-2-(dimethylaminomethyleneamino)purine (10) with ethyl (cis,trans)-2-chloro-2-(chloromethyl) cyclopropane-1-carboxylate (2c) under the conditions of alkylation-elimination gave (Z)-6- benzyloxy-2-formylamino-9-[(2-carbethoxycyclopropylidene)methyl]purine (11) but no E,N(9)-isomer. Minor amounts of (Z)-6-benzyloxy-2-formylamino-7-[(2-carbethoxy-cyclopropylidene)methyl]purine (13) were also obtained. Hydrolysis of compounds 11 and 13 in 80% acetic acid afforded (Z)-9-[2-(carbethoxycyclopropylidene)methyl]guanine (14) and (Z)-7-[2-(carbethoxy- cyclopropylidene)methyl]guanine (15). Reduction of 14 furnished synguanol (1). Reaction of N(4)-acetylcytosine (7) with ester 2c led to (Z,E)-1-(2-carbethoxycyclopropropylidenemethyl)cytosine (8, Z/E ratio 6.1:1). Basicity of purine base, lower reactivity of alkylation intermediates as well as interaction of the purine N(3) or cytosine O(2) atoms with the carbonyl group of ester moiety seem to be essential for the observed high stereoselectivity of the alkylation-elimination. The Z-selectivity is interpreted in terms of E1cB mechanism leading to a transitory "cyclic" cyclopropenes which undergo a cyclopropene-methylenecyclopropane rearrangement.

(Z)- and (E)-2-(1,2-dihydroxyethyl)methylenecyclopropane analogues of 2'-deoxyadenosine and 2'-deoxyguanosine. Synthesis of all stereoisomers, absolute configuration, and antiviral activity.

Chiral Z- and E-stereoisomers of (1,2-dihydroxyethyl)methylenecyclopropane analogues of 2'-deoxyadenosine and 2'-deoxyguanosine were synthesized, and their antiviral activity was investigated. (S)-Methylenecyclopropylcarbinol (16) was converted in seven steps to reagents 26 and 27, which were used for alkylation-elimination of adenine and 2-amino-6-chloropurine to get ultimately analogues 12a, 12b, 13a, 13b, 14a, 14b, 15a, and 15b. The enantiomeric series ent-12a, ent-12b, ent-13a, ent-13b, ent-14a, ent-14b, ent-15a, and ent-15b was obtained by similar procedures starting from (R)-methylenecyclopropylcarbinol (ent-16). The Z-isomer ent-12b was an inhibitor of two strains of human cytomegalovirus (HCMV) with EC(50) of 6.8 and 7.5 microM and of murine cytomegalovirus (MCMV) with EC(50) of 11.3 microM. It was less active against HCMV with mutated gene UL97. It inhibited Epstein-Barr virus (EBV) with EC(50) of 8 microM. The E-isomers ent-15a, ent-13a, and 15b were less effective. All adenine analogues with the exception of the Z-isomers ent-12a and ent-14a were moderate substrates for adenosine deaminase.

Synthesis and enantioselectivity of cyclopropavir phosphates for cellular GMP kinase.

Enantiomeric cyclopropavir phosphates (+)-9 and (-)-9 were synthesized and investigated as substrates for GMP kinase. N(2)-Isobutyryl-di-O-acetylcyclopropavir (11) was converted to (+)-monoacetate 12 using hydrolysis catalyzed by porcine liver esterase. Phosphorylation via phosphite 13 gave after deacylation, phosphate (+)-9. Acid-catalyzed tetrahydropyranylation of (+)-monoacetate 12 gave, after deacylation, tetrahydropyranyl derivative 14. Phosphorylation via phosphite 15 furnished, after deprotection, enantiomeric phosphate (-)-9. Racemic diphosphate 16 was also synthesized. The phosphate (+)-9 is a relatively good substrate for GMP kinase with a K(M) value of 57 microM that is similar to that of the natural substrates GMP (61 microM) and dGMP (82 microM). In contrast, the enantiomer (-)-9 is not a good substrate (K(M) 1200 microM) indicating a significant enantioselectivity for the GMP kinase catalyzed reaction of monophosphate to diphosphate.

Anti-BK virus activity of nucleoside analogs.

Polyomavirus BK is an important pathogen in transplant recipients with no effective therapy. This study demonstrates that alkoxyalkyl esters of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and fatty acid derivatives of 9-[2-(phosphonomethyoxy)ethyl]adenine (P393 and P405) are potent and selective inhibitors of BK virus replication in vitro, with a 50% effective concentration in the micromolar-to-nanomolar range.

Fluorinated methylenecyclopropane analogues of nucleosides. Synthesis and antiviral activity of (Z)- and (E)-9-{[(2-fluoromethyl-2-hydroxymethyl)-cyclopropylidene]methyl}adenine and -guanine.

Synthesis and antiviral activity of the title fluoromethylenecyclopropane analogues 15a, 15b, 16a, and 16b is described. Methylenecyclopropane carboxylate was first transformed to 2,2-bis-hydroxymethylmethylenecyclopropane. Selective monoacetylation followed by introduction of fluorine gave 2-acetoxymethyl-2-fluoromethylmethylenecyclopropane as the key intermediate. The synthesis of analogues 15a, 15b, 16a, and 16b then followed alkylation-elimination procedure as described previously for other methylenecyclopropane analogues [corrected] Compounds 15a, 15b, 16a and 16b were not active against Epstein-Barr virus (EBV) [corrected] Analogue 15a inhibited hepatitis C virus by virtue of its cytotoxicity and it moderately inhibited replication of the Towne strain of human cytomegalovirus (HCMV). The E-isomer 16a was a substrate for adenosine deaminase, whereas the Z-isomer 15a was not deaminated.

Synthesis of 2,2,3-tris(hydroxymethyl)methylenecyclopropane analogues of nucleosides.

Synthesis of 2,2,3-tris(hydroxymethyl)methylenecyclopropane analogues 16a, 16b, 17a, and 17b is described. Diethyl ester of Feist's acid 18b was hydroxymethylated via carbanion formation using formaldehyde under simultaneous isomerization to cis diester to give intermediate 19. Reduction followed by acetylation gave triacetate 22. Addition of bromine afforded reagent 23, which was used for alkylation-elimination of adenine and 2-amino-6-chloropurine to provide Z,E-isomeric mixtures of 24a and 24b. Deacetylation and separation furnished the Z-isomers 16a, 16c and E-isomers 17a, 17c. Hydrolytic dechlorination of 16c and 17c gave guanine analogues 16b and 17b. None of the analogues exhibited a significant antiviral activity. Adenosine deaminase is refractory toward adenine analogues 16a and 17a.

Fluoroanalogues of anti-cytomegalovirus agent cyclopropavir: synthesis and antiviral activity of (E)- and (Z)-9-{[2,2-bis(hydroxymethyl)-3-fluorocyclopropylidene]methyl}-adenines and guanines.

Synthesis of fluorinated cyclopropavir analogues 13a, 13b, 14a, and 14b is described starting from alkene 15. Addition of carbene derived from dibromofluoromethane gave bromofluoro cyclopropane 16. Reduction (compound 17) followed by desilylation gave intermediate 18, which was transformed to 2-nitrophenylselenenyl derivative 19. Oxidation to selenoxide 20 was followed by beta-elimination to afford methylenecyclopropane 21. Addition of bromine provided compound 22 for alkylation-elimination of adenine and 2-amino-6-chloropurine. The resultant E,Z isomeric mixtures of methylenecyclopropanes 23a + 24a and 23c + 24c were resolved and the individual isomers were deprotected to give adenine analogues 13a and 14a as well as compounds 13c and 14c. Hydrolytic dechlorination of 13c and 14c furnished guanine analogues 13b and 14b. The only significant antiviral effects were observed with analogue 13a against HCMV and 14a against VZV in cytopathic inhibition assays.

Synthesis of "reversed" methylenecyclopropane analogues of antiviral phosphonates.

Synthesis of "reversed" methylenecyclopropane analogues of nucleoside phosphonates 6a,7a, 6b, and 7b is described. 1-Bromo-1-bromomethylcyclopropane 8 was converted to the bromocyclopropyl phosphonate 9 by Michaelis-Arbuzov reaction with triisopropyl phosphite. Base-catalyzed beta-elimination and deacetylation gave the key Z- and E-hydroxymethylcyclopropyl phosphonates 10 and 11 separated by chromatography. The Mitsunobu type of alkylation of 10 or 11 with adenine or 2-amino-6-chloropurine afforded phosphonates 12a, 12b, 13a, and 13b. Acid hydrolysis furnished the adenine and guanine analogues 6a, 7a, 6b, and 7b. The E and Z configuration was assigned on the basis of NOE experiments with phosphonates 6b and 7b. All Z- and E-isomers were also distinguished by different chemical shifts of CH2O or CH2N (H4 or H4'). Significant differences of the chemical shifts of the cyclopropane C3(3') carbons and coupling constants 3JP,C2(2') or 3JP,C3(3') selective for the Z- or E-isomers were also noted. Phosphonates 6a, 7a, 6b, and 7b are devoid of significant antiviral activity.

9-{[3-fluoro-2-(hydroxymethyl)cyclopropylidene]methyl}adenines and -guanines. Synthesis and antiviral activity of all stereoisomers1.

All stereoisomers of adenine and guanine methylene-3-fluoromethylenecyclopropane analogues of nucleosides 9a, 9b, 10a, 10b, 11a, 11b, 12a, and 12b were synthesized and their antiviral activities were evaluated. A highly convergent approach permitted the synthesis of all these analogues using a single intermediate 15. Reaction of aldehyde 13 with fluorotrichloromethane and tri-n-butylphosphine gave fluoroalkenes 14a+14b (83:17). Addition of carbene derived from ethyl diazoacetate gave cyclopropane 15 as the major product. Reduction (19), bromination (20), and phenylselenenylation (21), followed by Se oxidation and beta-elimination gave cis-methylenecyclopropane 22. Addition of bromine provided the reagent 23 for alkylation-elimination. Reaction of 23 with adenine led to an isomeric mixture 25a+26a that after deprotection afforded analogues 9a and 10a. The 2-amino-6-chloropurine furnished 25e+26e and after deblocking (9e and 10e) and hydrolysis gave targets 9b and 10b. Intermediate 15 provided, after debenzylation (27), 2-nitrophenylselenenylation (28), reduction (29), benzylation (30), and oxidation-elimination trans-methylenecyclopropane 31. Addition of bromine gave reagent 32. Further transformations followed the sequence outlined for analogues 9a, 9b, 10a, and 10b. Analogue 9b was effective against human cytomegalovirus (HCMV; Towne) with EC50 2.9 microM. The trans-isomer 10b inhibited AD169 strain of HCMV (EC50 15 microM) and the murine virus MCMV (EC50 2.5 microM). Compound 12a was effective against Epstein-Barr virus (EC50<0.03 microM). Analogue 9a inhibited varicella zoster virus (EC50 5.9 microM) and human immunodeficiency virus type 1 (EC50 5.2 microM). Analogues 9a, 10a, and 11a are moderate substrates for adenosine deaminase. The structure-activity relationships will be discussed in context with other methylenecyclopropane analogues.

Synthesis of Methylenecyclopropane Analogues of Antiviral Nucleoside Phosphonates.

Synthesis of methylenecyclopropane analogues of nucleoside phosphonates 6a, 6b, 7a and 7b is described. Cyclopropyl phosphonate 8 was transformed in four steps to methylenecyclopropane phosphonate 16. The latter intermediate was converted in seven steps to the key Z- and E-methylenecyclopropane alcohols 23 and 24 separated by chromatography. Selenoxide eliminations (15 --> 16 and 22 --> 23 + 24) were instrumental in the synthesis. The Z- and E-isomers 23 and 24 were transformed to bromides 25a and 25b which were used for alkylation of adenine and 2-amino-6-chloropurine to give intermediates 26a, 26b, 26c and 26d. Acid hydrolysis provided the adenine and guanine analogues 6a, 6b, 7a and 7b. Phosphonates 6b and 7b are potent inhibitors of replication of Epstein-Barr virus (EBV).

Synthesis, antiviral, and antitumor activity of 2-substituted purine methylenecyclopropane analogues of nucleosides.

The Z- and E-2-fluoro- and 2-chloropurine methylenecyclopropanes 9a,b and 10a,b as well as enantiomeric Z-isoguanine methylenecyclopropanes 11a,b and their phenyl phosphoralaninate pronucleotides 11c,d were synthesized and their antiviral activity against several viruses was evaluated. Fluoro analogues 9a and 10a were active against human cytomegalovirus but they were cytotoxic at approximately the same concentrations. Chloro derivatives 9b and 10b were non-cytotoxic and effective against Epstein-Barr virus in Daudi cells. Isoguanine analogues 11a-d lacked antiviral activity but pronucleotides 11c,d were substrates for porcine liver esterase. From the group of 9a,b and 10a,b, the fluoro analogues 9a and 10a exhibited antitumor activity but only the Z-isomer 9a had a selective effect.

In vitro activity and mechanism of action of methylenecyclopropane analogs of nucleosides against herpesvirus replication.

We have reported previously that methylenecyclopropane analogs of nucleosides have excellent activity against certain members of the herpesvirus family. A second generation, the 2,2-bis-hydroxymethyl derivatives, were synthesized, and 18 compounds were tested for activity in vitro against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), human and murine cytomegalovirus (HCMV and MCMV), varicella-zoster virus (VZV), and Epstein-Barr virus (EBV). Selected analogs were also evaluated against human herpesvirus type 6 (HHV-6) and HHV-8. None of the 18 compounds had activity against HSV-1 or HSV-2, but four were active against VZV by plaque reduction (PR) assay at 50% effective concentration (EC(50)) levels of < or =50 microM. Six of the 18 compounds were active against HCMV by cytopathic effect or PR assays with EC(50)s of 0.5 to 44 microM, and all were active against MCMV by PR (0.3 to 54 microM). Four of the compounds were active against EBV by enzyme-linked immunosorbent assay (<0.3 to 4.4 microM). Four compounds with CMV activity were also active against HHV-6A and HHV-6B (0.7 to 28 microM), and three compounds were active against HHV-8 (5.5 to 16 microM). One of these, ZSM-I-62, had particularly good activity against CMV, HHV-6, and HHV-8, with EC(50)s of 0.7 to 8 microM. Toxicity was evaluated in adherent and nonadherent cells, and minimal cytotoxicity was observed. Mechanism of action studies with HCMV suggested that these compounds are phosphorylated by the ppUL97 phosphotransferase and are potent inhibitors of viral DNA synthesis. These results indicate that at least one of these compounds may have potential for use in treating CMV and other herpesvirus infections in humans.