Emergence of resistance to carbocyclic oxetanocin G in herpes simplex virus type 1 and genetic analysis of resistant mutants

AIM: To elucidate the potentiality of emergence of drug-resistance to carbocyclic oxetanocin G (C.OXT-G), a new effective antiviral drug for herpetic keratitis during treatment and the mechanism of this drug resistance.METHODS: A C.OXT-G resistant strain (C.OXT-Gr) was established by serially propagating the herpes simplex virus (HSV) -1 in African green monkey kidney (VERO) cells in the presence of C.OXT-G. After the drug sensitiv-ity assay and the thymidine kinase (TK) activity assay, the molecular basis for the drug resistance was studied using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and PCR direct se-quencing technology. RESULTS: After the 10th passage in 10 μm C.OXT-G, the ED50 of the C.OXT-G^r was 17.08-fold greater than that of the original strain on the average and the TK activities of these resistant strains were extremely reduced. PCR-SSCP analysis on TK gene of the wild HSV-1 and the C.OXT-Gr showed altered migration patterns in part 3 and part 4, while PCR-SSCP analysis on DNA polymerase gene showed no difference among the viruses. Sequence analysis revealed a deletion of G at position of 430 that caused frameshift, resulting in premature termination in the TK gene. CONCLUSION: The drug resistance to C.OXT-G may appear during the treatment due to the deficiency of TK activity caused by a single mutation in the TK gene of HSV-1.     

Synthesis and biological evaluation of carbocyclic analogues of lyxofuranosides of 2-amino-6-substituted-purines and 2-amino-6-substituted-8-azapurines

Carbocyclic analogues of lyxofuranosides of 2-amino-6-substituted-purines and 2-amino-6-substituted-8-azapurines were synthesized from (+/-)-(1 alpha, 2 alpha, 3 alpha, 5 alpha)-3-amino-5- (hydroxymethyl)-1,2-cyclopentanediol (2) and 2-amino-4,6-dichloropyrimidine (3). The 2-amino-6-chloropurine (8 and 11), the 2,6-diaminopurine (10 and 13), as well as the guanine (9) and 8-azaguanine (12) derivatives were all constructed from the key intermediate (+/-)-(1 alpha, 2 alpha, 3 alpha, 5 alpha)- 3-[(2,5-diamino-6-chloro-4-pyrimidinyl)amino]-5-(hydroxymethyl)-1,2- cyclopentanediol (7) by using established methodology. Compounds 8-13 were evaluated for both antitumor and antiviral activity. None of these materials exhibited appreciable activity against P-388 mouse leukemia cells in vitro. All of these analogues were investigated for activity versus herpes simplex virus type 1 (HSV-1) and influenza virus (IV-A), as well as the human immunodeficiency virus (HIV). Against HSV-1, only compound 9, the carbocyclic analogue of the lyxofuranoside of guanine, exhibited significant activity, yielding a virus rating (VR) of 2.1. The corresponding 2,6-diamino compound (10) demonstrated marginal activity, VR = 0.6, against that virus. The test compounds failed to exhibit inhibition of either IV-A or HIV. Additionally, 9 was tested against human cytomegalovirus (HCMV) and was found to display definite activity at concentrations as low as 32 microM.     

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.     

Antiviral activity of ganciclovir elaidic acid ester against herpesviruses

A fatty acid derivative of ganciclovir (GCV), elaidic acid ganciclovir (E-GCV), has been evaluated for its inhibitory activity against laboratory and clinical strains of herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) in human embryonic lung fibroblasts. GCV, cidofovir, acyclovir (ACV), brivudin (BVDU) and foscarnet (PFA) were included as reference compounds. The viruses studied were wild-type, thymidine kinase-deficient (TK(-)) and PFA-resistant (PFA(r)) HSV strains. The IC(50) values obtained for E-GCV were 5- to 30-fold lower than those observed for GCV, the IC(50) value of E-GCV for HSV-1 strain KOS being 0.07 nM. A similarly increased activity of E-GCV (as compared to GCV) was noted for TK(-) and PFA(r) HSV-1 or HSV-2 strains. However, E-GCV did not exhibit superior activity over GCV to VZV or HCMV in vitro. The antiviral efficacy of E-GCV was also evaluated in vivo against intracerebral HSV-2 infection in NMRI mice. Animals were treated intraperitoneally or perorally with E-GCV, GCV or placebo once daily for 10 days, starting the day of infection. E-GCV compared to GCV at equimolar doses, proved markedly more efficacious than GCV in terms of reduction of mortality rate and delay of mean time of death. The elaidic acid ester of GCV should therefore be considered as a novel approach towards the treatment of HSV infections.     

Synthesis and biological evaluation of novel 2',3',4'-triply branched carbocyclic nucleosides as potential antiviral agents

Novel 2',3',4'-triply branched carbocyclic nucleosides were synthesized in this study. The introduction of two methyl groups in the 2'- and 3'-position was accomplished by a Horner-Wadsworth-Emmons reaction and isopropenyl magnesium bromide addition, respectively. The construction of the required 4'-quaternary carbon was carried out using a [3,3]-sigmatropic rearrangement. Bis-vinyls were successfully cyclized using a Grubbs' catalyst II. The natural bases (adenine, cytosine) were efficiently coupled using a Pd(0) catalyst. The antiviral activities of the synthesized compounds were evaluated against HIV-1, HSV-1, HSV-2 and HCMV. Compound 30 displayed moderate anti-HCMV activity (EC50 = 30.1 microg/mL), without exhibiting any cytotoxicity at up to 100 microM.     

Highly selective cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine derivatives for murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 thymidine kinase gene

(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVDU) and various structurally related analogues thereof, i.e., (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC), and the carbocyclic analogues of BVDU, IVDU, and BVDC, were evaluated for their inhibitory effects on the growth of murine mammary carcinoma FM3A cells, deficient in thymidine kinase (TK) activity but transformed with the herpes simplex virus type 1 (HSV-1) TK gene (designated FM3A/TK-/HSV-1 TK+). BVDU and its congeners were much more inhibitory to the growth of FM3A/TK-/HSV-1 TK+ than to the growth of the wild type (FM3A/0) cells. For BVDU, for example, the 50% inhibitory dose for the FM3A/TK-/HSV-1 TK+ cells was 0.5 ng/ml, as compared to 11 micrograms/ml for the FM3A/0 cells. Evidently, BVDU and its congeners required phosphorylation by the HSV-1 TK to exert their cytostatic action. In attempts to evaluate further the mechanism of this cytostatic action, BVDU, IVDU, and their carbocyclic analogues were evaluated for their inhibitory effects on thymidylate synthetase (TS) and their incorporation into DNA. TS was identified as one, but not the sole, target in the cytostatic activity of BVDU and its derivatives. With [125I]IVDU and its carbocyclic analogue C-[125I]IVDU, clear evidence was obtained for the incorporation of these radiolabeled analogues into DNA of the FM3A/TK-/HSV-1 TK+ cell line and a TS-deficient mutant thereof, FM3A/TK-/HSV-1 TK+/TS-. No incorporation was detected with [125I]IVDU or C-[125I]IVDU into DNA of FM3A/0 and FM3A/TS- cells. To what extent the incorporation of [125I]IVDU and C-[125I]IVDU contributed to their cytostatic action against FM3A/TK-/HSV-1 TK+ cells remains the subject of further study.     

Carbocyclic analogues of 5-halocytosine nucleosides

Carbocyclic analogues of 5-halocytosine nucleosides were prepared by direct halogenation of the carbocyclic analogues of cytidine, 2'-deoxycytidine, 3'-deoxycytidine, or ara-C. The 5-chloro and 5-bromo derivatives of the cytidine (carbodine) and of the 2'-deoxycytidine analogues and the 5-iodo derivatives of all four of the cytosine nucleoside analogues were prepared. All of the C-5-halocytosine nucleosides, as well as the parent C-cytosine nucleosides, were tested against a strain of herpes simplex virus type 1 (HSV-1) that induces thymidine kinase in host cells. Carbodine, 5-bromocarbodine, C-2'-deoxycytidine, C-5-bromo-2'-deoxycytidine, the four C-5-iodocytosine nucleosides, and C-ara-C inhibited replication of this strain of HSV-1 in cultured cells. Most of these compounds were tested also against the type 2 virus (HSV-2) in vitro and were active. The greatest activity observed was exerted by C-5-iodo-2'-deoxycytidine in inhibiting replication of HSV-1 in L929 cells. In tests against these DNA viruses, carbodine, a ribofuranoside analogue that had been shown previously to be highly active against human influenza A virus in vitro, was the most active compound against HSV-2 and one of the most active compounds against HSV-1 in Vero cells. 5-Bromocarbodine was active against influenza virus, but it was less active than carbodine.     

Synthesis and antiviral evaluation of carbocyclic analogues of 2-amino-6-substituted-purine 3'-deoxyribofuranosides

Carbocyclic analogues of 2-amino-6-substituted-purine 3'-deoxyribofuranosides were synthesized by beginning with (+/-)-(1 alpha,3 alpha,4 beta)-3-amino-4-hydroxycyclopentanemethanol and 2-amino-4,6-dichloropyrimidine. The route parallels the earlier syntheses of the corresponding ribofuranoside and 2'-deoxyribofuranoside analogues. The 2-amino-6-chloropurine, guanine, and 2,6-diaminopurine derivatives and the analogous 8-azapurines were prepared. The analogue (3'-CDG) of 3'-deoxyguanosine is active in vitro against a strain of type 1 herpes simplex virus (HSV-1) that induces thymidine kinase and is modestly active against a thymidine kinase inducing strain of type 2 HSV. 3'-CDG is not active against a strain of HSV-1 that lacks the thymidine kinase inducing capacity, whereas the carbocyclic analogue of 2-amino-6-chloropurine 3'-deoxyribofuranoside is active against that strain. The carbocyclic analogue of 2,6-diaminopurine 3'-deoxyribofuranoside displayed modest activity in vitro against influenza virus.     

Synthesis and antiherpes simplex virus activity of 9-[(1,3-dihydroxy-2-propylthio)methyl]guanine

The synthesis of the thio analogue (thio-DHPG, 2) of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) is described. The synthesis of 2 proceeded via the condensation of acetoxymethyl sulfide 9 with diacetylguanine 10 to give the protected nucleoside analogue 11. Although catalytic hydrogenolysis failed, the benzyl ether functionalities of 11 were successfully cleaved by an acetolysis reaction to furnish 14. Ammonolysis of 14 gave 2, which was also transformed to sulfoxide 15 and sulfone 16. Preliminary in vitro screening indicated that 2 exhibited comparable activity to DHPG against herpes simplex virus type 1 (HSV-1) but was less active against the type 2 virus (HSV-2) and human cytomegalovirus (HCMV). In a mouse encephalitis model (HSV-2), subcutaneous treatment with 2 led to a 53% reduction in mortality at a dose of 100 mg/kg per day.     

二羟丙氧甲基乌嘌呤抗单纯疱疹病毒Ⅰ型的实验研究

二羟丙氧甲基鸟嘌岭(DHPG),在组织培养中对单纯疱疹病毒Ⅰ型(HSV-1)KOS株抑制50%空斑形成的浓度(IC_(50))为0.1μg/ml。DHPG与环胞苷联合应用的抗HSV活性呈协同作用;DHPG分别与无环鸟苷、酞丁安合用有相加作用。0.1和0.05%DHPG溶液滴眼对家兔实验性浅层单纯疱疹病毒角膜炎有显著治疗作用,浓度降低至0.025和0.0l%无明显疗效。对实质层型单疱角膜炎,0.1%DHPG溶液滴眼治疗有效。     

Distinct thymidine kinases encoded by cowpox virus and herpes simplex virus contribute significantly to the differential antiviral activity of nucleoside analogs

Orthopoxviruses and herpesviruses are both large enveloped DNA viruses, yet these virus families exhibit very different susceptibilities to antiviral drugs. We investigated the activation of nucleoside analogs by the types I and II thymidine kinase (TK) homologs expressed by herpes simplex virus type 1 (HSV-1) and cowpox virus (CV). Antiviral activity against TK(-) and TK(+) strains of HSV-1 and CV was determined, and the ratio of the EC(50) values was used as a measurement of TK dependence. As to HSV-1, most of the selected compounds were markedly less effective against the TK(-) strains, suggesting that this enzyme was required for the activation of these nucleoside analogs. This differs from the results for CV where only idoxuridine and bromodeoxyuridine appeared to be activated, putatively by the type II TK expressed by this virus. These data confirm that the type II TK encoded by CV exhibits a more limited substrate specificity than the type I TK encoded by HSV-1. These data suggest that the inefficient activation of nucleoside analogs by the orthopoxvirus TK significantly limits their activity. Additional screening against orthopoxviruses will be required to identify nucleoside analogs that are efficiently activated by their type II TK.     

Synthesis and antiviral evaluation of carbocyclic analogues of ribofuranosides of 2-amino-6-substituted-purines and of 2-amino-6-substituted-8-azapurines

Carbocyclic analogues of ribofuranosides of 2-amino-6-substituted-purines and of 2-amino-6-substituted-8- azapurines were prepared from the 2-amino-6-chloropurine ribofuranoside analogue (2) and the 2-amino-6-chloro-8- azapurine ribofuranoside analogue (9), respectively. Analogues of purine ribofuranosides with the chloro, amino, methylamino, or methylthio group at position 6, the thioguanosine analogue, and the previously reported guanosine analogue were evaluated in vitro against herpes simplex virus, type 1 (HSV-1). 8- Azapurine ribofuranoside analogues with the chloro, amino, or methylthio group at position 6 and the previously reported 8- azaguanosine analogue were also evaluated against HSV-1. The carbocyclic analogue (6) of 2,6-diaminopurine ribofuranoside is highly active against HSV-1 and, also, against vaccinia virus. The 2-amino-6-chloropurine, 2-amino-6-(methylamino)purine, and the 2,6-diamino-8- azapurine derivatives also demonstrated significant activity against HSV-1.     

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

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

Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides

A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.     

Inhibition of herpes simplex virus thymidine kinases by 2-phenylamino-6-oxopurines and related compounds: structure-activity relationships and antiherpetic activity in vivo

Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.     

Cyclopentenylcytosine. A carbocyclic nucleoside with antitumor and antiviral properties

Cyclopentenylcytosine (CPE-C, 2), a pyrimidine analogue of the fermentation derived carbocyclic nucleoside neplanocin A, has been synthesized from the optically active cyclopentenylamine 3b by two synthetic routes. CPE-C demonstrates significant antitumor activity against both the sensitive and ara-C resistant lines of L1210 leukemia in vivo. Multiple long term survivors are produced in both tumor models. The compound also gives 100% growth inhibition of the solid human A549 lung and MX-1 mammary tumor xenografts grown in athymic mice. Good activity is also observed against a third human tumor xenograft model, metastatic LOX melanoma. CPE-C has significant activity against both DNA and RNA viruses in vitro. Potent activity is observed against HSV-1 (TK+ and TK-), HSV-2, vaccinia, cytomegalovirus, and varicella-zoster virus. Good activity is also found against a strain of influenza virus (Hong Kong flu), vesicular stomatitis virus, Japanese encephalitis virus, and Punta Toro virus.     

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.     

Phenoxazine derivatives inactivate human cytomegalovirus, herpes simplex virus-1, and herpes simplex virus-2 in vitro.

We examined whether phenoxazine derivatives, 2-amino-4,4alpha-dihydro-4alpha-7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-amino-1,4alpha-dihydro-4alpha-8-dimethyl-2H-phenoxazine-2-one (Phx-2), and 2-amino-phenoxazine-3-one (Phx-3) may have antiviral activity against herpes family viruses: human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2). The antiviral activity was evaluated by the selectivity index (SI), which is the ratio of 50% cytotoxic concentration (CC(50)) and 50% antiviral concentration (IC(50)). Among these phenoxazines, Phx-2 exerted strong antiviral activity to HCMV with the SI of 200, while Phx-1 and Phx-3 exerted no marked anti-HCMV activity. Phx-2 also showed moderate inhibition of HSV-1 and HSV-2, with the SI of 6.7 and 17, respectively. In the time-of-addition experiments, inhibitory effect of Phx-2 against HCMV was active even when applied to cells at 100 h after HCMV infection, while ganciclovir (GCV) showed potent inhibition when applied to cells before 42-h post-infection, but its inhibitory effects disappeared thereafter. Attachment and penetration of HCMV was not affected by the presence of Phx-2. When HCMV was pretreated with Phx-2, concentration-dependent virucidal action was observed, suggesting that Phx-2 inactivates HCMV directly. From these data, it was found that Phx-2 might have a different anti-HCMV target from GCV.     

Antiviral activity of Plantago major extracts and related compounds in vitro

Plantago major L., a popular traditional Chinese medicine, has long been used for treating various diseases varying from cold to viral hepatitis. The aim of present study was to examine the antiviral activity of aqueous extract and pure compounds of P. major. Studies were conducted on a series of viruses, namely herpesviruses (HSV-1, HSV-2) and adenoviruses (ADV-3, ADV-8, ADV-11). The antiviral activity of EC50 was defined as the concentration achieved 50% cyto-protection against virus infection and the selectivity index (SI) was determined by the ratio of CC50 (concentration of 50% cellular cytotoxicity) to EC50. Results showed that aqueous extract of P. major possessed only a slight anti-herpes virus activity. In contrast, certain pure compounds belonging to the five different classes of chemicals found in extracts of this plant exhibited potent antiviral activity. Among them, caffeic acid exhibited the strongest activity against HSV-1 (EC50=15.3 microg/ml, SI=671), HSV-2 (EC50=87.3 microg/ml, SI=118) and ADV-3 (EC50=14.2 microg/ml, SI=727), whereas chlorogenic acid possessed the strongest anti-ADV-11 (EC50=13.3 microg/ml, SI=301) activity. The present study concludes that pure compounds of P. major, which possess antiviral activities are mainly derived from the phenolic compounds, especially caffeic acid. Its mode of action against HSV-2 and ADV-3 was found to be at multiplication stages (postinfection of HSV-1: 0-12 h; ADV-3: 0-2 h), and with SI values greater than 400, suggesting the potential use of this compound for treatment of the infection by these two viruses.     

Patent Evaluation: Aryl Macrocyclic Compositions for Treating Viral Infections

Summary

Novelty: New compostions consisting of aryl macrocyclic derivatives and suitable carriers are disclosed. They are potentially useful for the treatment of viral infections, such as herpes, HSV-1, HSV-2, DNA viruses (Roizman), HIV etc.

Biology: Fourteen biological assays are described; these include cytotoxicity in proliferating human cells, inhibition of HSV activity for cytopathic effects, plaque reduction in Vero cells, viral yield reduction in HeLa cells and activity against drug-resistant strains of HSV-1 and HSV-2 using KOS and KOS(PFA). Typically for the specified compound cytotoxicity was IC₅₀= 100 mg/ml or greater and inhibtion of HSV-1 and -2 showed IC₅₀ values of 1.25 and 1.8 μg/ml. Generally the data demonstrate that aryl macrocyclic derivatives are effective against drug resistant HSV strains at concentrations which are comparable to those effective against the wild-type virus. By contrast, and with the exception of DHPG as an inhibitor of HSV-1 strains, both drug-resistant strains showed a significant resistance to ACV, DHPG, PFA and PMEA.

Chemistry: The composition consists of phenyl or naphthyl macrocyclic derivatives with protamine. Antiviral nucleoside analogues may be included in a suitable formulation for topical, oral or intravenous adminsitration.