Antiviral effect of aqueous extracts from species of the Lamiaceae family against Herpes simplex virus type 1 and type 2 in vitro

Aqueous extracts from species of the Lamiaceae family were examined for their antiviral activity against Herpes simplex virus (HSV). Extracts from lemon balm (Melissa officinalis), peppermint (Mentha x piperita), prunella (Prunella vulgaris), rosemary (Rosmarinus officinalis), sage (Salvia officinalis) and thyme (Thymus vulgaris) were screened. Their inhibitory activity against Herpes simplex virus type 1 (HSV-1), type 2 (HSV-2) and an acyclovir-resistant strain of HSV-1 (ACV (res)) was tested in vitro on RC-37 cells in a plaque reduction assay. The 50% inhibitory concentrations (IC (50)) of the extracts for HSV plaque formation were determined in dose-response studies. All test compounds showed a high antiviral activity against HSV-1, HSV-2 and ACV (res). In order to identify the mode of antiviral action, the extracts were added to the cells or viruses at different stages of infection. Both types of Herpes virus including ACV (res) were considerably neutralized after treatment with the extracts prior to infection. At maximum non-cytotoxic concentrations of the extracts, plaque formation was significantly reduced by > 90% for HSV-1 and HSV-2 and > 85% for ACV (res). In time-response studies over a period of 2 hours, a clearly time-dependent activity was demonstrated. These results indicate that the extracts affect HSV before adsorption, but have no effect on the intracellular virus replication. Therefore, the extracts exert their antiviral effect on free HSV and offer a chance to use them for topical therapeutic application against recurrent HERPES infections.     

Selective nonpeptidic inhibitors of herpes simplex virus type 1 and human cytomegalovirus proteases

The proteases encoded by herpesviruses including herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) are attractive targets for antiviral drug development because of their important roles in viral replication. We randomly screened a chemical compound library for inhibitory activity against HSV-1 protease. 1,4-Dihydroxynaphthalene and three naphthoquinones were found to be potent inhibitors of HSV-1 protease with IC50 values of 6.4 to 16.9 microM. Inhibitory mode analysis of the compounds against HSV-1 protease suggested that, in spite of structural similarities, only 1,4-dihydroxynaphthalene was a competitive inhibitor, whereas the three naphthoquinones were noncompetitive inhibitors. Among all assayed dihydroxynaphthalene derivatives in the chemical compound library, 1,4-dihydroxynaphthalene proved to be the most potent inhibitor of HSV-1 protease. Therefore, the two hydroxyl groups located at positions 1 and 4 on the naphthalene structure seemed essential for exertion of a potent inhibitory activity against HSV-1 protease. In addition, we have found that these compounds are also potent inhibitors of HCMV protease with extremely low micromolar IC50 values. This differed from the results of inhibitory mode analysis of HSV-1 protease, 1,4-dihydroxynaphthalene was a noncompetitive inhibitor of HCMV protease, and three naphthoquinones were competitive inhibitors. These compounds showed no effective inhibitory activity against several mammalian serine proteases (trypsin, chymotrypsin, kallikrein, plasmin, thrombin and Factor Xa) at 100 microM. These results suggest that 1,4-dihydroxynaphthalene and three naphthoquinones may be useful in the development of nonpeptidic antiherpesvirus agents.     

Improved synthesis and biological evaluation of an acyclic thiosangivamycin active against human cytomegalovirus.

We previously described the synthesis and in vitro antiviral activity of an acyclic thiosangivamycin analog (Gupta et al., 1989a). In order to extend these initial studies, a new, multi-gram synthesis of 4-amino-7-[(2-hydroxy- ethoxy)methyl]pyrrolo]2,3-d]pyrimidine-5-thiocarboxamide (compound 229) was achieved in 5 steps from the known 5-amino-2-bromo-3,4-dicyanopyrrole in good overall yield. In plaque reduction assays with HCMV, compound 229 had an IC50 of 7 microM; in yield reduction assays the IC90 was 25 microM. The compound was less active against MCMV, HSV-1, HSV-2, and least active against VZV. Concentrations of compound 229 up to 32 microM did not affect the growth of KB cells for incubation periods up to 72 h. At 100 microM, a prolongation in population doubling time from 21 h (untreated) to 35 h was noted. This inhibition, however, was reversible upon removal of the compound suggesting the inhibition was cytostatic rather than cytotoxic. Flow cytometric studies with compound 229 in HFF cells revealed an accumulation of cells in S phase and a concurrent loss of cells in G2/M phase, suggesting an early S phase blockage. We conclude there is adequate separation between antiviral activity and cytotoxicity to merit further work with this class of pyrrolopyrimidines.     

Inactivation of HSV-1 and HSV-2 and prevention of cell-to-cell virus spread by Santolina insularis essential oil

The essential oil obtained in toto from Santolina insularis was investigated for its antiviral activity on herpes simplex type 1 (HSV-1) and type 2 (HSV-2) in vitro. The IC(50) values, determined by plaque reduction assays, were 0.88 and 0.7 microg/ml for HSV-1 and HSV-2, respectively, while the CC(50) determined by the MTT test on Vero cells was 112 microg/ml, indicating a CC(50)/IC(50) ratio of 127 for HSV-1 and 160 for HSV-2. Results obtained by plaque reduction assays also indicated that the antiviral activity of S. insularis was principally due to direct virucidal effects. Antiviral activity against HSV-1 and HSV-2 was not observed in a post-attachment assay, and attachment assays indicated that virus adsorption was not inhibited. Up to 80% inhibition of HSV-1 was achieved at the concentration of 40 microg/ml by yield reduction assay. Furthermore, reduction of plaque formation assays also showed that S. insularis essential oil inhibits cell-to-cell transmission of both HSV-1 and HSV-2.     

Broad-spectrum antiviral activity of PNU-183792, a 4-oxo-dihydroquinoline, against human and animal herpesviruses

We identified a novel class of 4-oxo-dihydroquinolines represented by PNU-183792 which specifically inhibit herpesvirus polymerases. PNU-183792 was highly active against human cytomegalovirus (HCMV, IC(50) value 0.69 microM), varicella zoster virus (VZV, IC(50) value 0.37 microM) and herpes simplex virus (HSV, IC(50) value 0.58 microM) polymerases but was inactive (IC(50) value >40 microM) against human alpha (alpha), gamma (gamma), or delta (delta) polymerases. In vitro antiviral activity against HCMV was determined using cytopathic effect, plaque reduction and virus yield reduction assays (IC(50) ranging from 0.3 to 2.4 microM). PNU-183792 antiviral activity against both VZV (IC(50) value 0.1 microM) and HSV (IC(50) ranging from 3 to 5 microM) was analyzed using plaque reduction assays. PNU-183792 was also active (IC(50) ranging 0.1-0.7 microM) in cell culture assays against simian varicella virus (SVV), murine cytomegalovirus (MCMV) and rat cytomegalovirus (RCMV). Cell culture activity was compared with the appropriate licensed drugs ganciclovir (GCV), cidofovir (CDV) and acyclovir (ACV). PNU-183792 was also active against both GCV-resistant and CDV-resistant HCMV and against ACV-resistant HSV. Toxicity assays using four different species of proliferating mammalian cells indicated PNU-183792 was not cytotoxic at relevant drug concentrations (CC(50) value >100 microM). PNU-183792 was inactive against unrelated DNA and RNA viruses indicating specificity for herpesviruses. In animals, PNU-183792 was orally bioavailable and was efficacious in a model of lethal MCMV infection.     

Antiviral activity of hop constituents against a series of DNA and RNA viruses

We investigated whether crude hop extracts and purified hop components representing every major chemical class of hop compound have antiviral activity. These hop constituents were tested for antiviral activity against bovine viral diarrhea virus (BVDV) as a surrogate model of hepatitis C virus (HCV), human immunodeficiency virus (HIV), influenza A virus (FLU-A), influenza B virus (FLU-B), rhinovirus (Rhino), respiratory syncytial virus (RSV), yellow fever virus (YFV), cytomegalovirus (CMV), hepatitis B virus (HBV), and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The extracts all failed to prevent the replication of HIV, FLU-A, FLU-B, RSV and YFV. A xanthohumol-enriched hop extract displayed a weak to moderate antiviral activity against BVDV (therapeutic index (TI)=6.0), HSV-2 (TI=>5.3), Rhino (TI=4.0) and HSV-1 (TI=>1.9) with IC(50) values in the low microg/ml range. Pure iso-alpha-acids demonstrated low to moderate antiviral activity against both BVDV (TI=9.1) and CMV (TI=4.2) with IC(50) values in the low microg/ml range. No antiviral activity was detected using beta-acids or a hop oil extract. Ultra-pure preparations (>99% pure) were used to show that xanthohumol accounted for the antiviral activity observed in the xanthohumol-enriched hop extract against BVDV, HSV-1 and HSV-2. Xanthohumol was found to be a more potent antiviral agent against these viruses than the isomer iso-xanthohumol. With Rhino, the opposite trend was observed with iso-xanthohumol showing superior antiviral activity to that observed with xanthohumol. Xanthohumol also showed antiviral activity against CMV, suggesting that it might have a generalized anti-herpesvirus antiviral activity. Again, superior antiviral activity was observed with the xanthohumol isomer against CMV. In summary, iso-alpha-acids and xanthohumol were shown to have a low-to-moderate antiviral activity against several viruses. These hop constituents might serve as interesting lead compounds from which more active anti-HCV, anti-Rhino and anti-herpesvirus antiviral agents could be synthesized.     

Antiherpes simplex virus type 2 activity of casuarinin from the bark of Terminalia arjuna Linn

Casuarinin, a hydrolyzable tannin isolated from the bark of Terminalia arjuna Linn. (Combretaceae), was investigated for its antiviral activity on herpes simplex type 2 (HSV-2) in vitro. Results showed that the IC(50) of casuarinin in XTT and plaque reduction assays were 3.6+/-0.9 and 1.5+/-0.2 microM, respectively. The 50% cytotoxic concentration for cell growth (CC(50)) was 89+/-1 microM. Thus, the selectivity index (SI) (ratio of CC(50) to IC(50)) of casuarinin was 25 and 59 for XTT and plaque reduction assays, respectively. Casuarinin continued to exhibit antiviral activity even added 12 h after infection. During the attachment assay, casuarinin was shown to prevent the attachment of HSV-2 to cells. Furthermore, casuarinin also exhibited an activity in inhibiting the viral penetration. Interestingly, casuarinin was virucidal at a concentration of 25 microM, reducing viral titers up to 100,000-fold. This study concludes that casuarinin possesses anti-herpesvirus activity in inhibiting viral attachment and penetration, and also disturbing the late event(s) of infection.     

Antiviral activity of Australian tea tree oil and eucalyptus oil against herpes simplex virus in cell culture

The antiviral effect of Australian tea tree oil (TTO) and eucalyptus oil (EUO) against herpes simplex virus was examined. Cytotoxicity of TTO and EUO was evaluated in a standard neutral red dye uptake assay. Toxicity of TTO and EUO was moderate for RC-37 cells and approached 50% (TC50) at concentrations of 0.006% and 0.03%, respectively. Antiviral activity of TTO and EUO against herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) was tested in vitro on RC-37 cells using a plaque reduction assay. The 50% inhibitory concentration (IC50) of TTO for herpes simplex virus plaque formation was 0.0009% and 0.0008% and the IC50 of EUO was determined at 0.009% and 0.008% for HSV-1 and HSV-2, respectively. Australian tea tree oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. At noncytotoxic concentrations of TTO plaque formation was reduced by 98.2% and 93.0% for HSV-1 and HSV-2, respectively. Noncytotoxic concentrations of EUO reduced virus titers by 57.9% for HSV-1 and 75.4% for HSV-2. Virus titers were reduced significantly with TTO, whereas EUO exhibited distinct but less antiviral activity. In order to determine the mode of antiviral action of both essential oils, either cells were pretreated before viral infection or viruses were incubated with TTO or EUO before infection, during adsorption or after penetration into the host cells. Plaque formation was clearly reduced, when herpes simplex virus was pretreated with the essential oils prior to adsorption. These results indicate that TTO and EUO affect the virus before or during adsorption, but not after penetration into the host cell. Thus TTO and EUO are capable to exert a direct antiviral effect on HSV. Although the active antiherpes components of Australian tea tree and eucalyptus oil are not yet known, their possible application as antiviral agents in recurrent herpes infection is promising.     

Efficiency and selectivity of (E)-5-(2-bromovinyl)-2'-deoxyuridine and some other 5-substituted 2'-deoxypyrimidine nucleosides as anti-herpes agents

A number of novel 5-substituted 2'deoxypyrimidine nucleosides exhibited antiviral activity against herpes simplex virus type 1 strain V3 (HSV-1-V3) when assayed under one-step conditions in primary human lung fibroblast j(PHLF) cell cultures, and compared with the reference compounds cytosine arabinoside (ara-C), 5-iodo-2'-deoxyuridine (IUdR), and 5-iodo-5'amino-2',5'-dideoxyuridine (AIU). The most effective of these were (in order of decreasing activity): (E)-5-(2-bromovinyl)-UdR (BrVUdR) greater than ara-C greater than IUdR greater than 5-azidomethyl-UdR (AMeUdR) greater than 5-formyl-UdR (fUdR) greater than 5-hydroxymethyl-UdR (HMeUdR) greater than AIU greater than 5-mercaptomethyl-UdR (MMeUdR) = 5-hydroxymethyl-2'-deoxy-cytidine (HMeCdR) greater than 5-benzyloxymethyl-UdR (BOMeUdR). In a multistep virus replication experiment (plaque reduction assay on Vero cells) the order of decreasing activity was as follows: BrVUdR = ara-C greater than HMeUdR greater than fUdR IUdR greater than HMeCdR greater than BOMeUdR greater than AMeUdR greater than AIU greater than MMeUdR. BrVUdR effected a 50% reduction in plaque formation of different strains of HSV-1 at a concentration of 0.06-0.22 microM, of pseudorabies virus (PRV) at 0.02-0.23 microM, and of herpes simplex virus type 2 (HSV-2) at 8 microM, whereas the ID50 values for adenovirus type 2 and type 5 were 100 and 50-100 microM, respectively. The growth of synchronied baby hamster kidney cells in suspension cultures was inhibited by 50% at concentrations of 100, 70, 20, 4, 8, and 0.2 microM for BrVUdR, HMeCdR, IUdR, fUdR, BOMeUdR, and HMeUdR, respectively.     

Design, synthesis, and antiviral activity of certain 3-substituted 2,5,6-trichloroindole nucleosides

A series of trichlorinated indole nucleosides has been synthesized and tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1) and for cytotoxicity. Modifications of the previously reported 2,5,6-trichloro-1-(beta-d-ribofuranosyl)indole at the 3-position of the heterocycle were designed in part to test our hypothesis that hydrogen bonding is required at that position for antiviral activity. Analogues were synthesized using electrophilic addition at the 3-position or by synthesis of modified indole heterocycles followed by glycosylation and modification of the sugar. Among the modifications at the 3-position, only those analogues with hydrogen-bond-accepting character were active against HCMV (e.g., 3-formyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole, FTCRI, IC50 = 0.23 microM). Conversely, analogues with non-hydrogen-bonding substituents at the 3-position (e.g., 3-methyl-2,5,6-trichloro-1-(beta-D-ribofuranosyl)indole) were much less active (IC50 = 32 microM) than those with the requisite hydrogen-bonding capacity. The 5'-O-acyl analogue of FTCRI was obtained as an intermediate and also found to be a potent inhibitor of HCMV (IC50 < 0.1 microM). The synthesis of some additional 5'-O-acylated analogues did not provide a compound with increased antiviral activity. None of the indole nucleosides had significant activity against HSV-1, and none were cytotoxic to uninfected cells in their antiviral dose range. Results obtained from the antiviral evaluations have validated our hypothesis that hydrogen bonding at the 3-position is required for antiviral activity in this series of chlorinated indole nucleosides.     

Oligomeric proanthocyanidins from Rumex acetosa L. inhibit the attachment of herpes simplex virus type-1

The polyphenole-enriched acetone-water extract R2 from the aerial parts of Rumex acetosa L. containing high amounts of oligomeric and polymeric proanthocyanidins and flavonoids was tested for antiviral activity. R2 exhibited strong antiviral activity against herpes simplex virus type-1 (HSV-1) while the replication of adenovirus 3 was not affected. By plaque reduction test and MTT assay on Vero cells, the HSV-1-specific inhibitory concentration (IC(50)) and cytotoxic concentration (CC(50)) were determined. R2 exibited an IC(50) of 0.8 μg/mL and a selectivity index (SI) (ratio of IC(50) to CC(50)) of approximately 100 when added to the virus inoculum for 1h at 37°C prior to infection. The antiviral activity was due to the presence of flavan-3-ols and oligomeric proanthocyanidins in the extract. Structure-activity analyses indicated that flavan-3-ols and proanthocyanidins with galloylation at position O-3 are highly potent compounds (SI>40), while ungalloylated compounds did not exhibit antiviral effects (SI<1). R2 and a major proanthocyanidin from R2, epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate abolished virus entry into the host cell by blocking attachment to the cell surface. When added after attachment at a concentration of ≥ 12.5 μg/mL, R2 inhibited also penetration of HSV-1 into the host cell. R2 and epicatechin-3-O-gallate-(4β→8)-epicatechin-3-O-gallate were shown to directly interact with viral particles leading to the oligomerisation of envelope proteins as demonstrated for the essential viral glycoprotein gD. Using raft cultures with three-dimensional organotypic human skin equivalents it was shown that treatment of cultures with R2 after infection with HSV-1 resulted in a reduced viral spread.     

Inhibition of herpes simplex virus type 2 replication in vitro by 1-N-pentadecanoyl-3'-N-trifluoroacetyl kanamycin A

The in vitro antiviral activity as well as the mechanism of action of a new antiviral agent, a kanamycin analogue, 1-N-pentadecanoyl-3'-N-trifluoroacetyl kanamycin A (PTKA) against herpes simplex virus type 2 (HSV-2) was investigated. The drug showed excellent antiviral action with negligible cytotoxic effect on the culture cells. Based on plaque reduction assays the 50% inhibitory dose (ID50) of the drug was 1 microgram/ml, and at 20 micrograms/ml plaque formation was totally suppressed. The compound inhibited viral protein synthesis in infected cells without affecting RNA and DNA synthesis, when added to the cultures after virus adsorption. Moreover, pretreatment of the cells with PTKA before HSV-2 infection, increased the antiviral activity significantly. Dot-blot hybridization analysis revealed that the drug reduced the level of immediate early viral mRNA if applied before infection. There was no detectable action at the level of virus adsorption, penetration or uncoating. These results indicate that PTKA exerted its antiviral action at the early stage of viral replication as well as at the level of viral protein synthesis.     

Synthesis and antiviral evaluation of carbocyclic analogues of 2'-azido- and 2'-amino-2'-deoxycytidine

Carbocyclic analogues of 2'-azido- and 2'-amino-2'-deoxycytidine, compounds 8 and 9, were synthesized by an eight-step synthesis from (+/-)-(1 alpha,2 alpha,3 beta,5 beta)-3-amino-5-(hydroxymethyl)-1,2- cyclopentanediol (1), which was prepared from cyclopentadiene via an eight-step route. These compounds were tested in vitro against herpes simplex virus type 1 (HSV-1). The 2'-amino analogue was found to show moderate antiviral activity, with an ED50 of 50 microM. However, the 2'-azido analogue was not active at a concentration up to 400 microM.     

6-N-Acyltriciribine analogues: structure-activity relationship between acyl carbon chain length and activity against HIV-1

Triciribine (TCN) and triciribine-5'-monophosphate (TCN-P) are active against HIV-1 at submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore the tolerance of TCN to structural modifications at the 6-position. A number of 6-N-acyltriciribine analogues were synthesized and evaluated for antiviral activity and cytotoxicity. The cytotoxicity of these compounds was minimal in three human cell lines (KB, CEM-SS cells, and human foreskin fibroblasts (HFF)). The compounds were marginally active or inactive against herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). In contrast, most of the compounds exhibited moderate to high activity against human immunodeficiency virus type 1 (HIV-1), IC(50)'s = 0.03 to 1 microM. This structure-activity relationship study identified the N-heptanoyl group as having the optimal carbon chain length. This compound was as active against HIV-1 as TCN and TCN-P. Reverse phase HPLC of extracts from uninfected cells treated with 6-N-acyltriciribines detected sufficient TCN-P to account for anti-HIV activity thereby suggesting a prodrug effect. Studies in an adenosine kinase deficient cell line showed that the 6-N-acyl derivative was not phosphorylated directly but first was metabolized to triciribine which then was converted to TCN-P.     

Increased efficacy of acyclovir-loaded microparticles against herpes simplex virus type 1 in cell culture.

Acyclovir is one of the most effective and selective agents against viruses of the herpes group. In order to increase its antiviral activity, acyclovir loaded microparticles, prepared by an O/W solvent evaporation method were developed. Their antiviral activity against herpes simplex virus type 1 (HSV-1) and toxicity were evaluated on Vero cells and then compared with those presented by a drug solution. The 50% inhibitory concentration (IC(50)) values for acyclovir loaded microspheres determined by plaque reduction assays at 48 and 96 h, were found to be 1.06 +/- 0.01 microM and 0.15 +/- 0.03 microM, respectively, while the equivalent values obtained for an acyclovir solution were 1.28 +/- 0.04 microM at 48 h and 0.27 +/- 0.02 microM at 96 h. These results indicate that acyclovir shows a higher antiviral activity, against herpes simplex virus type 1, when this drug was loaded in microparticles rather than as a drug solution, especially after 96 h of incubation. The toxicity of these microparticles was determined by the MTT test at 48 and 96 h. At 48 h only a small toxicity was found (cell viability ranged from 72 to 82%, with the higher concentration tested) and it could not be attributed to the microparticles, since the acyclovir control solution showed similar toxicity values. However, after 96 h a higher toxicity was observed with acyclovir microparticles as well as with the unloaded ones (cell viability located between 60 and 70%). In summary, acyclovir-loaded microparticles have shown to be promising carriers for the effective delivery of acyclovir in the treatment of HSV-1 infections in cells so they can have a potential use in vivo.     

In vitro antiviral activity of prodelphinidin B-2 3,3'-di-O-gallate from Myrica rubra

In this study, the in vitro antiviral properties of prodelphinidin B-2 3,3'-di- O-gallate (PB233'OG) isolated from the bark of Myrica rubra (Myricaceae) was investigated. Results showed that PB233'OG exhibited anti-herpes simplex virus type 2 (HSV-2) activity with IC (50) values of 5.3 +/- 0.1 and 0.4 +/- 0.04 microM for XTT and plaque reduction assays, respectively. The IC (50) value increased with increasing MOI (multiplicity of infection). PB233'OG did not show a cellular cytotoxic effect at concentrations that possessed antiviral activity. Mechanistic studies demonstrated that PB233'OG inhibited HSV-2 attachment to the Vero cell, interfered with the penetration of HSV-2 into the Vero cell, affected the late stage(s) of the HSV-2 infection cycle, and also reduced the viral infectivity at high concentrations. It is concluded that PB233'OG exhibits various modes of action in its anti-HSV-2 effects.     

Design, synthesis, and structure-activity relationship of pyridyl imidazolidinones: a novel class of potent and selective human enterovirus 71 inhibitors

When skeletons of Win compounds were used as templates, computer-assisted drug design led to the identification of a novel series of imidazolidinone derivatives with significant antiviral activity against enterovirus 71 (EV 71), the infection of which had resulted in about 80 fatalities during the 1998 epidemic outbreak in Taiwan. In addition to inhibiting all the genotypes (A, B, and C) of EV 71 in the submicromolar to low micromolar range, compounds 1 and 8 were extensively evaluated against a variety of viruses, showing potent activity against coxsackievirus A9 (IC(50) = 0.47-0.55 microM) and coxsackievirus A24 (IC(50) = 0.47-0.55 microM) as well as moderate activity against enterovirus 68 (IC(50) = 2.13 microM) and echovirus 9 (IC(50) = 2.6 microM). Our SAR studies revealed that imidazolidinone analogues with an aryl substituent at the para position of the phenoxyl ring, such as compounds 20, 21, 27, 57, 58, and 61, in general exhibited the highest activity against EV 71. Among them, compound 20 and its corresponding hydrochloride salt 57, in terms of potency and selectivity index, appear to be the most promising candidates in this series for further development of anti-EV-71 agents. Preliminary results of the study on the mode of action by a time-course experiment suggest that test compounds 1 and 8 can effectively inhibit the virus replication at the early stages, referring to virus attachment or uncoating. This indicates that the surface protein may be the target for this type of compounds.     

4-Oxo-4,7-dihydrothieno[2,3-b]pyridines as non-nucleoside inhibitors of human cytomegalovirus and related herpesvirus polymerases

A novel series of 4-oxo-4,7-dihydrothieno[2,3-b]pyridine-5-carboxamides have been identified as potential antivirals against human herpesvirus infections resulting from human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and varicella-zoster virus (VZV). Compounds 10c and 14 demonstrated broad-spectrum inhibition of the herpesvirus polymerases HCMV, HSV-1, and VZV. High specificity for the viral polymerases was observed compared to human alpha polymerase. The antiviral activity of 10c and 14, as determined by plaque reduction assay, was comparable or superior to that of existing antiherpes drugs, ganciclovir (for HCMV) and acyclovir (for HSV-1 and VZV). Drug resistance to compound 14 correlated to point mutations in conserved domain III of the herpesvirus DNA polymerase, but these mutations do not confer resistance to existing nucleoside therapy. In addition, compound 14 maintained potent antiviral activity against acyclovir-resistant HSV-1 strains. Substitution to the pyridone nitrogen (N7) was found to be critical for enhanced in vitro antiviral activity.     

Antiviral activity of cyclosaligenyl prodrugs of the nucleoside analogue bromovinyldeoxyuridine against herpes viruses

A series of 42 lipophilic bromovinyldeoxyuridine monophosphates (BVDUMPs) are presented as potential prodrugs of the antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU). The 5'-cycloSal-masking group technique has been applied to this cyclic nucleoside analogue to achieve delivery of the monophosphate of BVDU inside the target cells. The new substances have been tested for their antiviral activity against herpes simplex virus types 1 and 2 (HSV-1 and -2), thymidine kinase-deficient (TK(-)) HSV-1, varicella-zoster virus (VZV), human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). The XTT-based tetrazolium reduction assay EZ4U (for HSV), the plaque inhibition test (for VZV and HCMV) and a DNA hybridisation assay (for EBV) were used to assess antiviral activity. The results indicate that cycloSal-BVDUMP triesters proved to be potent and selective inhibitors of HSV-1 comparable with aciclovir. VZV replication was inhibited by very low concentrations, and two substances had a slightly better anti-VZV activity than the parent compound BVDU. No antiviral effect could be demonstrated against TK(-)-HSV-1, HSV-2 and HCMV, most likely owing to the lack of phosphorylation to BVDU diphosphate. Most remarkably, several cycloSal-BVDUMP triesters yielded promising anti-EBV activity whereas the parent compound BVDU was entirely inactive.     

Broad-spectrum antiviral activity of the acyclic guanosine phosphonate (R,S)-HPMPG

(R,S)-9-(3-hydroxy-2-phosphonomethoxypropyl)guanine [(R,S)-HPMPG] exhibits broad spectrum antiviral activity with an ED50 of less than 1 microM against herpes simplex virus (HSV) types 1 and 2, varicella zoster virus, human cytomegalovirus (HCMV) and vaccinia in plaque reduction assays. Wild type HSV-2 and its thymidine kinase deficient variant are equally sensitive to (R,S)-HPMPG. (R,S)-HPMPG is 100-fold more potent than acyclovir (ED50 = 0.45 microM vs. 44 microM, respectively) against HCMV in cell culture, and 10-fold more active than acyclovir in extending survival time in mice intraperitoneally infected with 70 LD50 HSV-1. However, (R,S)-HPMPG is toxic when administered repeatedly at 44 mg/kg/day in uninfected adult mice. The diphosphoryl derivative of HPMPG was enzymatically synthesized and is a competitive inhibitor of HSV-1 DNA polymerase relative to dGTP (K1 = 0.03 microM). HPMPG-PP is 70-fold less active at inhibiting HeLa DNA polymerase alpha than HSV-1 DNA polymerase. At concentrations between 0.3 and 1.5 microM (R,S)-HPMPG inhibited HSV-1 DNA replication greater than or equal to 50% in infected cells as measured by nucleic acid hybridization. Consistent with inhibition of viral DNA synthesis, 6 to 30 microM (R,S)-HPMPG reduces late viral polypeptide synthesis in HSV-1 infected cells. These data indicate that (R,S)-HPMPG is a thymidine kinase independent broad spectrum antiviral drug which is capable of inhibiting viral DNA polymerase.