Antiviral (RNA) activity of selected Amaryllidaceae isoquinoline constituents and synthesis of related substances.

A series of 23 Amaryllidaceae isoquinoline alkaloids and related synthetic analogues were isolated or synthesized and subsequently evaluated in cell culture against the RNA-containing flaviviruses (Japanese encephalitis, yellow fever, and dengue viruses), bunyaviruses (Punta Toro, sandfly fever, and Rift Valley fever viruses), alphavirus (Venezuelan equine encephalomyelitis virus), lentivirus (human immunodeficiency virus-type 1) and the DNA-containing vaccinia virus. Narciclasine [1], lycoricidine [2], pancratistatin [4], 7-deoxypancratistatin [5], and acetates 6-8, isonarciclasine [13a], cis-dihydronarciclasine [14a], trans-dihydronarciclasine [15a], their 7-deoxy analogues 13b-15b, lycorines 16 and 17, and pretazettine [18] exhibited consistent in vitro activity against all three flaviviruses and against the bunyaviruses, Punta Toro and Rift Valley fever virus. Activity against sandfly fever virus was only observed with 7-deoxy analogues. In most cases, however, selectivity of the active compounds was low, with toxicity in uninfected cells (TC50) occurring at concentrations within 10-fold that of the viral inhibitory concentrations (IC50). No activity was observed against human immunodeficiency virus-type 1, Venezuelan equine encephalomyelitis virus, or vaccinia viruses. Pancratistatin [4] and its 7-deoxy analogue 5 were evaluated in two murine Japanese encephalitis mouse models (differing in viral dose challenge, among other factors). In two experiments (low LD50 viral challenge, variant I), prophylactic administration of 4 at 4 and 6 mg/kg/day (2% EtOH/saline, sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 100% and 90%, respectively. In the same model, prophylactic administration of 5 at 40 mg/kg/day in hydroxypropylcellulose (sc, once daily for 7 days, day -1 to +5) increased survival of Japanese-encephalitis-virus-infected mice to 80%. In a second variant (high LD50 viral challenge), administration of 4 at 6 mg/kg/day (ip, twice daily for 9 days, day -1 to +7) resulted in a 50% survival rate. In all cases, there was no survival in the diluent-treated control mice. Thus, 4 and 5 demonstrated activity in mice infected with Japanese encephalitis virus but only at near toxic concentrations. To our knowledge, however, this represents a rare demonstration of chemotherapeutic efficacy (by a substance other than an interferon inducer) in a Japanese-encephalitis-virus-infected mouse model.

The importance of IMP dehydrogenase inhibition in the broad spectrum antiviral activity of ribavirin and selenazofurin.

The nucleosides ribavirin, selenazofurin, tiazofurin and bredinin all exhibit the lowering of guanylate pools in vitro and in vivo by the inhibition of IMP dehydrogenase. However, each of these nucleosides has a separate profile of antiviral and antitumor activity. The IMP dehydrogenase inhibition in the case of ribavirin and bredinin appears to be due to the nucleoside 5'-monophosphate and in the case of selenazofurin and tiazofurin to the NAD analogs formed intracellularly. With regard to the antiviral activity of these nucleosides, although selenazofurin was the most potent antiviral agent in vitro, its antiviral activity was also most readily reversed by exogenous guanosine. The antiviral effects of ribavirin were only partially reversed under the conditions studied. These and related studies show that each of these nucleosides form nucleotide metabolites which act as enzyme inhibitors at additional sites other than IMP dehydrogenase. As in the case of ribavirin such inhibition of IMP dehydrogenase may result in an increased "self potentiation" by the lowering of guanylate pools in those instances where guanylate analogs are involved as inhibitors of viral specific or viral induced enzymes. Further studies should more clearly elucidate the importance of the simultaneous inhibition of various enzyme sites by different metabolic nucleotide forms of the same nucleoside analog.

Broad-spectrum synergistic antiviral activity of selenazofurin and ribavirin.

The antiviral effects of selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide, selenazole), ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), and 3-deazaguanosine (6-amino-1-beta-D-ribofuranosylimidazo-[4.5-C]pyridin-4(5H)-one) were investigated separately and in various combinations in an in vitro study. The combination interactions were evaluated at seven drug concentrations, graphically (isobolograms) or by using fractional inhibitory concentration indices against mumps, measles, parainfluenza virus type 3, vaccinia and herpes simplex virus type 2 viruses in Vero and HeLa cells. Selenazofurin in combination with ribavirin produced the greatest synergistic antiviral activity. However, the degree of synergy depended on the virus and cell line used. In contrast, selenazofurin combined with 3-deazaguanosine consistently yielded an indifferent or an antagonistic response, or both, whereas the ribavirin-3-deazaguanosine interaction was additive against the same viruses. Single-drug cytotoxicity was minimal for the cytostatic agents selenazofurin and ribavirin but was markedly higher for cytocidal 3-deazaguanosine, as determined by relative plating efficiency after drug exposure. The drug combinations did not significantly increase cytotoxicity (they were only additive) when used on uninfected cells. Therefore, the enhanced antiviral activities of the drug combinations (shown to be synergistic) were due to specific effects against viral replication. These results indicated that in Vero and HeLa cells (i) the combination of selenazofurin and ribavirin produced an enhanced antiviral effect, thus requiring smaller amounts of drug to cause the same antiviral effect relative to a single compound; (ii) selenazofurin when compared with ribavirin and 3-deazaguanosine appeared to have a somewhat different mode of antiviral action; (iii) 3-deazaguanosine combined with selenazofurin was an unsuitable antiviral combination; and (iv) the antiviral activity of 3-deazaguanosine appeared to be due largely to its general overall cytotoxic effect.

Broad-spectrum antiviral activity of 2-beta-D-ribofuranosylselenazole-4-carboxamide, a new antiviral agent.

The relative in vitro antiviral activities of three related nucleoside carboxamides, ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), and selenazole (2-beta-D-ribofuranosylselenazole-4-carboxamide), were studied against selected DNA and RNA viruses. Although the activity of selenazole against different viruses varied, it was significantly more potent than ribavirin and tiazofurin against all tested representatives of the families Paramyxoviridae (parainfluenza virus type 3, mumps virus, measles virus), Reoviridae (reovirus type 3), Poxviridae (vaccinia virus), Herpes-viridae (herpes simplex virus types 1 and 2), Togaviridae (Venezuelan equine encephalomyelitis virus, yellow fever virus, Japanese encephalitis virus), Bunyaviridae (Rift Valley fever virus, sandfly fever virus [strain Sicilian], Korean hemorrhagic fever virus), Arenaviridae (Pichinde virus), Picornaviridae (coxsackieviruses B1 and B4, echovirus type 6, encephalomyocarditis virus), Adenoviridae (adenovirus type 2), and Rhabdoviridae (vesicular stomatitis virus). The antiviral activity of selenazole was also cell line dependent, being greatest in HeLa, Vero-76, and Vero E6 cells. Selenazole was relatively nontoxic for Vero, Vero-76, Vero E6, and HeLa cells at concentrations of up to 1,000 micrograms/ml. The relative plating efficiency at that concentration was over 90%. The effects of selenazole on viral replication were greatest when this agent was present at the time of viral infection. The removal of selenazole from the medium of infected cells did not reverse the antiviral effect against vaccinia virus, but there was a gradual resumption of viral replication in cells infected with parainfluenza type 3 or herpes simplex virus type 1 (strain KOS). However, the antiviral activity of ribavirin against the same viruses was reversible when the drug was removed.