ACYCLIC/CARBOCYCLIC GUANOSINE ANALOGUES AS ANTI-HERPESVIRUS AGENTS

Several guanosine analogues, i.e. acyclovir (and its oral prodrug valaciclovir), penciclovir (in its oral prodrug form, famciclovir) and ganciclovir, are widely used for the treatment of herpesvirus [i.e. herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2),varicella-zoster virus (VZV) and/or human cytomegalovirus (HCMV)] infections. In recent years, several new guanosine analogues have been developed, including the 3-membered cyclopropylmethyl and-methenyl derivatives (A-5021 and synguanol) and the 6-membered D-and L-cyclohexenyl derivatives. The activity of the acyclic/carbocyclic guanosine analogues has been determined against a wide spectrum of viruses, including the HSV-1, HSV-2, VZV, HCMV, and also human herpesviruses type 6 (HHV-6), type 7 (HHV-7) and type 8 (HHV-8), and hepatitis B virus (HBV). The new guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) were found to be particularly active against those viruses (HSV-1, HSV-2, VZV) that encode for a specific thymidine kinase (TK), suggesting that their antiviral activity (at least partially) depends on phosphorylation by the virus-induced TK. Marked antiviral activity was also noted with A-5021 against HHV-6 and with D- and L-cyclohexenyl G against HCMV and HBV. The antiviral activity of the acyclic/carbocyclic nucleoside analogues could be markedly potentiated by mycophenolic acid, a potent inhibitor of inosine 5′-monophosphate (IMP) dehydrogenase. The new carbocyclic guanosine analogues (i.e. A-5021 and D- andL-cyclohexenyl G) hold great promise, not only as antiviral agents for the treatment of herpesvirus infections, but also an antitumor agents for the combined gene therapy/chemotherapy of cancer, provided that (part of) the tumor cells have been transduced by the viral (HSV-1, VZV) TK gene.     

Acyclic/carbocyclic guanosine analogues as anti-herpesvirus agents

Several guanosine analogues, i.e. acyclovir (and its oral prodrug valaciclovir), penciclovir (in its oral prodrug form, famciclovir) and ganciclovir, are widely used for the treatment of herpesvirus [i.e. herpes simplex virus type 1 (HSV-1), and type 2 (HSV-2), varicella-zoster virus (VZV) and/or human cytomegalovirus (HCMV)] infections. In recent years, several new guanosine analogues have been developed, including the 3-membered cyclopropylmethyl and -methenyl derivatives (A-5021 and synguanol) and the 6-membered D- and L-cyclohexenyl derivatives. The activity of the acyclic/carbocyclic guanosine analogues has been determined against a wide spectrum of viruses, including the HSV-1, HSV-2, VZV, HCMV, and also human herpesviruses type 6 (HHV-6), type 7 (HHV-7) and type 8 (HHV-8), and hepatitis B virus (HBV). The new guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) were found to be particularly active against those viruses (HSV-1, HSV-2, VZV) that encode for a specific thymidine kinase (TK), suggesting that their antiviral activity (at least partially) depends on phosphorylation by the virus-induced TK. Marked antiviral activity was also noted with A-5021 against HHV-6 and with D- and L-cyclohexenyl G against HCMV and HBV. The antiviral activity of the acyclic/carbocyclic nucleoside analogues could be markedly potentiated by mycophenolic acid, a potent inhibitor of inosine 5'-monophosphate (IMP) dehydrogenase. The new carbocyclic guanosine analogues (i.e. A-5021 and D- and L-cyclohexenyl G) hold great promise, not only as antiviral agents for the treatment of herpesvirus infections, but also an antitumor agents for the combined gene therapy/chemotherapy of cancer, provided that (part of) the tumor cells have been transduced by the viral (HSV-1, VZV) TK gene.     

John Montgomery's legacy: carbocyclic adenosine analogues as SAH hydrolase inhibitors with broad-spectrum antiviral activity

Ever since the S-adenosylhomocysteine (AdoHcy, SAH) hydrolase was recognized as a pharmacological target for antiviral agents (J. A. Montgomery et al., J. Med. Chem. 25:626-629, 1982), an increasing number of adenosine, acyclic adenosine, and carbocyclic adenosine analogues have been described as potent SAH hydrolase inhibitors endowed with broad-spectrum antiviral activity. The antiviral activity spectrum of the SAH hydrolase inhibitors include pox-, rhabdo-, filo-, arena-, paramyxo-, reo-, and retroviruses. Among the most potent SAH hydrolase inhibitors and antiviral agents rank carbocyclic 3-deazaadenosine (C-c3 Ado), neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A. These compounds are particularly active against poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus). The in vivo efficacy of C-c3 Ado and 3-deazaneplanocin A has been established in mouse models for vaccinia virus, vesicular stomatitis virus, and Ebola virus. SAH hydrolase inhibitors such as C-c3Ado and 3-deazaneplanocin A should in thefirst place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola.     

Mutagenicity of methyl 2-benzimidazolecarbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister-chromatid exchanges, C-mitoses, polyploidies and micronuclei

Methyl 2-benzimidazolecarbamate (MBC), diethylstilbestrol (DES) and estradiol were tested with regard to their ability to induce C-mitoses, polyploidies, micronuclei, structural chromosomal aberrations and sister-chromatid exchanges (SCE) in human peripheral lymphocytes in vitro. The compounds did not induce structural chromosomal aberrations either in the presence or absence of metabolic activation. MBC and estradiol were negative in the SCE test. DES induced SCE rates which were not even twice the control level and which were independent of dose and of metabolic activation. All compounds induced C-mitoses, polyploidies and micronuclei. The micronuclei are interpreted as resulting from errors in the anaphase distribution of chromosomes by spindle disturbances rather than from structural chromosomal aberrations.     

John Montgomery's Legacy: Carbocyclic Adenosine Analogues as Sah Hydrolase Inhibitors with Broad-Spectrum Antiviral Activity

Ever since the S-adenosylhomocysteine (AdoHcy, SAH) hydrolase was recognized as a pharmacological target for antiviral agents (J. A. Montgomery et al., J. Med. Chem. 25:626–629, 1982), an increasing number of adenosine, acyclic adenosine, and carbocyclic adenosine analogues have been described as potent SAH hydrolase inhibitors endowed with broad-spectrum antiviral activity. The antiviral activity spectrum of the SAH hydrolase inhibitors include pox-, rhabdo-, filo-, arena-, paramyxo-, reo-, and retroviruses. Among the most potent SAH hydrolase inhibitors and antiviral agents rank carbocyclic 3-deazaadenosine (C-c³Ado), neplanocin A, 3-deazaneplanocin A, the 5′-nor derivatives of carbocyclic adenosine (C-Ado, aristeromycin), and the 2-halo (i.e., 2-fluoro) and 6′-R-alkyl (i.e., 6′-R-methyl) derivatives of neplanocin A. These compounds are particularly active against poxviruses (i.e., vaccinia virus), and rhabdoviruses (i.e., vesicular stomatitis virus). The in vivo efficacy of C-c³Ado and 3-deazaneplanocin A has been established in mouse models for vaccinia virus, vesicular stomatitis virus, and Ebola virus. SAH hydrolase inhibitors such as C-c³Ado and 3-deazaneplanocin A should in the first place be considered for therapeutic (or prophylactic) use against poxvirus infections, including smallpox, and hemorrhagic fever virus infections such as Ebola.     

Influence of diethylenetriamine (DETA) and sodium nitroprusside (NaNP) on sister chromatid exchange frequency and cell kinetics in cultured human lymphocytes

Diethylenetriamine (DETA) and Sodium Nitroprusside (NaNP), the exogenous NO-generating compounds, were tested for their genotoxicity in human lymphocytes in vitro using the sister chromatid exchange (SCE) technique. Both compounds were found to be inactive in inducing SCE in concentrations from 0.3 to 30 pM. However both compounds displayed an inhibiting effect on cell kinetics.     

Synthesis and antiviral activity of carbocyclic nucleosides incorporating a modified cyclopentane ring. Part 3: Adenosine and uridine analogues

Six new carbocyclic nucleosides were prepared by mounting a purine (compounds 4-6), 8-azapurine (7 and 8) or uridine (9) base on the amino group of (1S,3R)-3-amino-2,2,3-trimethylcyclopentylmethanol (10). At subtoxic concentrations, compounds 5-9 showed at best marginal antiviral activity.     

Carbocyclic Adenosine Analogues as S-Adenosylhomocysteine Hydrolase Inhibitors and Antiviral Agents: Recent Advances

Abstract

Various carbocyclic analogues of adenosine, including aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A, 3-deazaneplanocin A, the 5′-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, neplanocin A and 3-deazaneplanocin A, and the 2-halo (i.e., 2-fluoro) and 6′-R-alkyl (i.e., 6′-R-methyl) derivatives of neplanocin A have been recognized as potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. This enzyme plays a key role in methylation reactions depending on S-adenosylmethionine (AdoMet) as methyl donor. AdoHcy hydrolase inhibitors have been shown to exert broad-spectrum antiviral activity against pox-, paramyxo-, rhabdo-, filo-, bunya-, arena-, and reoviruses. They also interfere with the replication of human immunodeficiency virus through inhibition of the Tat transactivation process.     

Peptide carbocyclic derivatives as inhibitors of the viroporin function of RNA-containing viruses

New carbocyclic derivatives of amino acids, peptides, and other compounds have been synthesized, and their antiviral activity toward the influenza A/H5N1 and hepatitis C viruses has been studied in vitro. It has been shown that the aminoacyl derivatives of aminoadamantane are capable of inhibiting the replication of the highly virulent strain of the avian influenza virus (H5N1), which is resistant to aminoadamantanes amantadine and rimantadine. The effect of the configuration of the carbocyclic moiety of the dipeptide H-Pro-Trp-OH on the antiviral properties toward the hepatitis C virus has been studied. The cyclohexyloxycarbonyl derivative of the H-Pro-Trp-OH dipeptide strongly inhibited the replication of HCV in vitro. Some compounds have been found to exhibit a high virucidal activity toward influenza A/H5N1 and HCV virions.     

Chemically-induced sister-chromatid exchange in vivo in bone marrow of Chinese hamsters. An evaluation of 24 compounds

Chemically-induced sister-chromatid exchange (SCE) was measured in vivo in bone marrow of Chinese hamsters. Chemicals were administered either intraperitoneally or orally and increased SCE frequencies were noted with 6 of 6 direct-acting genotoxins and with 9 of 14 activation-dependent genotoxins. Metronidazole, o-toluidine, 4-nitro-o-phenylenediamine and 2-nitro-p-phenylenediamine, compounds which have shown either mutagenic or carcinogenic activity, did not induce SCE in vivo, 4 non-genotoxins and 4 different control treatments did not induce SCE. The results show that the in vivo SCE method may be useful for the identification of genotoxins and that the outcome of the test is, for certain chemicals, dependent upon the route of exposure.     

A comparison of sister-chromatid exchange in mouse peripheral blood lymphocytes exposed in vitro and in vivo to phosphoramide mustard and 4-hydroxycyclophosphamide

Cyclophosphamide (CP) and two of its known metabolites, 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), were analyzed for their ability to induce sister-chromatid exchanges (SCEs) in mouse peripheral blood lymphocytes (PBLs) in vitro and in vivo. At equimolar concentrations, CP is a more potent SCE inducer in vivo than PAM and PAM and 4-OHCP induce equal numbers of SCEs in a dose-dependent manner. The present study also shows that these metabolites of CP are more potent SCE inducers than CP itself in vitro. This relationship might be explained by the differences in pharmacokinetics of these compounds.     

Cinnamic esters of acyclovir-synthesis and biological activity

In the present study we have synthesized esters of acyclovir with cinnamic acids (p-coumaric, ferulic, and sinapic acids) and evaluated them for their antiviral and antioxidant potential. The antiviral activity of the newly synthesized compounds has been tested against human herpes virus 1 (HSV-1) in vitro. The results indicate that none of the synthesized compounds inhibits the tested virus strain. The antioxidant properties have been studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH)* test.     

Synthesis,Antiviral and Cytostatic Activities of Carbocyclic Nucleosides Incorporating a Modified Cyclopentane Ring. Part 2:1 Adenosine and Uridine Analogues

Abstract

Six new carbocyclic nucleosides were prepared by mounting a purine (compounds 5–7), 8-azapurine (compounds 9 and 10) or pyrimidine (compound 13) base on the amino group of (1R,cis)-3-(aminornethyl)-1,2,2-trimethylcyclopentylmethanol (2). The antiviral activity of compounds 5–7, 10 and 13, and their cytostatic activity, were evaluated. At subtoxic concentrations, the compounds showed no or marginal antiviral activity. Compound 5 showed moderate inhibition on tumor cell proliferation.     

Chemically-induced sister-chromatid exchange in vivo in bone marrow of Chinese hamsters An evaluation of 24 compounds

Chemically-induced sister-chromatid exchange (SCE) was measured in vivo in bone marrow of Chinese hamsters. Chemicals were administered either intraperitoneally or orally and increased SCE frequencies were noted with 6 of 6 direct-acting genotoxins and with 9 of 14 activation-dependent genotoxins. Metronidazole, O-toluidine, 4-nitro-O-phenylenediamine and 2-nitro-p-phenylenediamine, compounds which have shown either mutagenic or carcinogenic activity, did not induce SCE in vivo. 4 non-genotoxins and 4 different control treatments did not induce SCE. The results show that the in vivo SCE method may be useful for the identification of genotoxins and that the outcome of the test is, for certain chemicals, dependent upon the route of exposure.     

An in vitro nucleoside analog screening method for cancer gene therapy

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene therapy research. We have developed an in vitro screening assay to assess the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). The thymidine kinase gene enhances nucleoside phosphorylation to nucleotides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transfected 3T3-NIH). Two types of analysis are performed: a cytotoxicity assay, the neutral red uptake assay to assess the IC₅₀ on the two cell lines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic profiles after incubation of cells with tritiated analogs. Results show that cells expressing the HSV-TK gene are more sensitive than the parent cells to the effect of acyclovir or ganciclovir, the reference purine analog drugs, and also to the effect of pyrimidine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxyuridine. Promising nucleoside analogs for gene therapy that can be achieved by HSV-TK could be evaluated using this model.Abbreviations ACV acyclovir - ACV-MP acyclovir monophosphate - ACV-DP acyclovir diphosphate - ACV-TP acyclovir triphosphate - BDU bromodeoxyuridine - BVDU bromovinyldeoxyuridine - EDU ethyldeoxyuridine - FDU fluorodeoxyuridine - GCV ganciclovir - HSV-TK herpes simplex virus thymidine kinase gene - IDU iododeoxyuridine - NA nucleoside analog     

V79-hCYP2E1-hSULT1A1, a cell line for the sensitive detection of genotoxic effects induced by carbohydrate pyrolysis products and other food-borne chemicals

We recently constructed a Chinese hamster V79-derived cell line that stably expresses human cytochrome P450 (CYP) 2E1 and human sulphotransferase (SULT) 1A1. These enzymes are involved in the bioactivation of numerous promutagens/procarcinogens, but are not taken into account in standard in vitro mutagenicity assays. Various carbohydrate pyrolysis products and other food contaminants that induce tumours or preneoplastic lesions in laboratory animals are inactive or only weakly active in standard in vitro genotoxicity assays. This is the case for acrylamide, furan, 5-hydroxymethylfurfural, nitrofen and N-nitrosodimethylamine. These compounds were investigated for induction of sister chromatid exchange (SCE) in V79-hCYP2E1-hSULT1A1 cells. All test compounds showed positive results over a wide concentration range, starting at 0.01 microM for N-nitrosodimethylamine, 3 microM for furan, 12.5 microM for nitrofen, 20 microM for 5-hydroxymethylfurfural, and 200 microM for acrylamide. The concentration-response curve of furan was unusual, as this compound induced a statistically significant, but rather constant and weak increase in SCE over an extremely wide concentration range (3-16,000 microM). Furan was slightly less active, whereas the remaining compounds were much less active in the parental V79 cell line than in V79-hCYP2E1-hSULT1A1 cells. Compared to many other genotoxic effects, the study of SCE only requires small numbers of cells (and incubation volumes) and usually is detected even at low concentrations of the genotoxicant. Therefore, induction of SCE in V79-hCYP2E1-hSULT1A1 cells may be useful in the genotoxicity testing of preparations of heated food and in their bioassay-directed fractionation.     

Antiviral compounds in extracts of Korean seaweeds: Evidence for multiple activities

Extracts of 13 Korean seaweeds, previously shown to contain antiviral activity, were investigated in more detail in order to learn the nature of the antiviral compounds and their mechanisms of action. One extract, from Codium fragile, was active against all three test viruses (herpes simplex, HSV; Sindbis, SINV; polio), whereas the others were more selective. Thus four species, Enteromorpha linza, Colpomenia bullosa, Scytosiphon lomentaria, and Undaria pinnatifida, were active against HSV and SINV, but not poliovirus. The other eight were active against either HSV or SINV. In all cases there was evidence for photosensitizers, since the antiviral activities required or were enhanced substantially by light. In general UVA (long wave ultraviolet) was much more effective than visible light in promoting activity, although the extract of Sargassum sagamianum could be activated equally by either. In experiments to determine the site of action of these antiviral extracts, the predominant activity was virucidal (i.e. direct inactivation of virus particles), rather than inhibition of virus replication, although Sargassum sagamianum also could protect cells against subsequent virus infection. These results imply that different antiviral compounds are present among the extracts, and furthermore the activities cannot be explained in terms of common ingredients such as polysaccharides or tannins. We suggest that seaweeds may be a source of potentially useful and interesting antiviral compounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro studies of biological effects of cigarette smoke condensate. II. Induction of sister-chromatid exchanges in human lymphocytes by weakly acidic, semivolatile constituents

Cigarette smoke condensate is known to enhance the frequency of sister-chromatid exchanges (SCE) in human lymphocytes in vitro and some of the activity has been found in the most volatile part of the particulate phase, the semivolatile fraction. In this study we have investigated the chemical composition and the SCE-inducing activity of the weakly acidic, semivolatile fraction of a cigarette smoke condensate. A number of individual weakly acidic compounds were also tested for their SCE-inducing effects. The weakly acidic fraction was separated by preparative gel chromatography into 11 subfractions (F1-F11). The chemical composition was determined by gas chromatography and gas chromatography-mass spectrometry. Measurements of the effects on SCE in human lymphocytes were used to evaluate the genotoxic effects. All fractions except F11 induced SCE in a dose-dependent way. The most active fraction was F4 which contained mainly alkyl-2-hydroxy-2-cyclopenten-1-ones. The individual compounds to be tested for induction of SCE were selected on the basis of their abundance in the weakly acidic subfractions and on the basis of their occurrence in the environment. Of 23 tested compounds, most of which were alkylphenols, 7 induced SCE, i.e., catechol, 2-(1-propenyl)phenol, cyclotene, maltol, isoeugenol, 2-methoxyphenol (guaiacol) and vanillin. Many of these are important flavor components that occur not only in tobacco and tobacco smoke but also in food, candies, beverages and perfumes.     

In vitro studies of biological effects of cigarette smoke condensate: II. Induction of sister-chromatid exchanges in human lymphocytes by weakly acidic,semivolatile constituents

Cigarette smoke condensate is known to enhance the frequency of sister-chromatid exchanges (SCE) in human lymphocytes in vitro and some of the activity has been found in the most volatile part of the particulate phase, the semivolatile fraction. In this study we have investigated the chemical composition and the SCE-inducing activity of the weakly acidic, semivolatile fraction of a cigarette smoke condensate. A number of individual weakly acidic compounds were also tested for their SCE-inducing effects.The weakly acidic fraction was separated by preparative gel chromatography into 11 subfractions (F1–F11). The chemical composition was determined by gas chromatography and gas chromatography-mass spectrometry. Measurements of the effects on SCE in human lymphocytes were used to evaluate the genotoxic effects. All fractions except F11 induced SCE in a dose-dependent way. The most active fraction was F4 which contained mainly alkyl-2-hydroxy-2-cyclopenten-1-ones.The individual compounds to be tested for induction of SCE were selected on the basis of their abundance in the weakly acidic subfractions and on the basis of their occurrence in the environment. Of 23 tested compounds, most of which were alkylphenols, 7 induced SCE, i.e., catechol, 2-(1-propenyl)phenol, cyclotene, maltol, isoeugenol, 2-methoxyphenol (guaiacol) and vanillin. Many of these are important flavor components that occur not only in tobacco and tobacco smoke but also in food, candies, beverages and perfumes.     

Pentacyclic triterpenes in birch bark extract inhibit early step of herpes simplex virus type 1 replication

Antiviral agents frequently applied for treatment of herpesvirus infections include acyclovir and its derivatives. The antiviral effect of a triterpene extract of birch bark and its major pentacyclic triterpenes, i.e. betulin, lupeol and betulinic acid against acyclovir-sensitive and acyclovir-resistant HSV type 1 strains was examined. The cytotoxic effect of a phytochemically defined birch bark triterpene extract (TE) as well as different pentacyclic triterpenes was analyzed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. TE, betulin, lupeol and betulinic acid exhibited high levels of antiviral activity against HSV-1 in viral suspension tests with IC₅₀ values ranging between 0.2 and 0.5 μg/ml. Infectivity of acyclovir-sensitive and clinical isolates of acyclovir-resistant HSV-1 strains was significantly reduced by all tested compounds and a direct concentration- and time-dependent antiherpetic activity could be demonstrated. In order to determine the mode of antiviral action, TE and the compounds were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had low effect on virus multiplication. Minor virucidal activity of triterpenes was observed, however both TE and tested compounds exhibited high anti-herpetic activity when viruses were pretreated with these drugs prior to infection. Pentacyclic triterpenes inhibit acyclovir-sensitive and acyclovir-resistant clinical isolates of HSV-1 in the early phase of infection.