Broad-spectrum antiviral activity of Virazole: 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide.

Virazole is a synthetic nucleoside active in tissue culture against at least 16 DNA and RNA viruses. Applied topically, it inhibits herpetic keratitis in rabbits and tail lesions induced by herpes, vaccinia, and vesicular stomatitis viruses in mice. Injected intraperitoneally into mice, it inhibits splenomegaly and hepatomegaly induced by Friend leukemia virus and respiratory infections caused by influenza A(O), A(2), and B viruses and parainfluenza 1 virus. infections is also effective.

Imidazo[1,2-a]-s-triazine nucleosides. Synthesis and antiviral activity of the N-bridgehead guanine, guanosine, and guanosine monophosphate analogues of imidazo[1,2-a]-s-triazine.

The first chemical synthesis of 2-aminoimidazo[1,2-a]-s-triazin-4-one (8), the corresponding nucleoside and nucleotide, and certain related derivatives of a new class of purine analogues containing a bridgehead nitrogen atom is described. Condensation of 2-amino-4-chloro-6-hydroxy-s-triazine (2) with aminoacetaldehyde dimethyl acetal followed by the ring annulation gave the guanine analogue 8. A similar ring annulation of 4-(2,2-dimethoxyethylamino)-s-triazine-2,6-dione (5) gave imidazo[1,2-a]-s-triazine-4,6-dione (9). Direct glycosylation of the trimethylsilyl derivative of 8 with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in the presence of stannic chloride, followed by debenzoylation, gave the guanosine analogue 2-amino-8-(beta-D-ribofuranosyl)imidazo[1,2-a]-s-triazin-4-one (12b), which on deamination gave the xanthosine analogue 13. Phosphorylation of 12b gave 2-amino-8-(beta-D-ribofuranosyl)imidazo[1,2-a]-s-triazin-4-one 5'-monophosphate (II). The anomeric configuration has been determined unequivocally by using NMR of the 2',3'-O-isopropylidene derivate 10 and the site of ribosylation has been established by using 13C NMR spectroscopy. These compounds were tested against type 1 herpes, type 13 rhino, and type 3 parainfluenza viruses in tissue culture. Moderate rhinovirus activity was observed for several compounds at nontoxic dosage levels.

Synthesis and antiviral and antimicrobial activity of certain 1-beta-D-ribofuranosyl-4,5-disubstituted imidazoles.

Starting with AICA ribonucleoside the following nucleosides were prepared. Methyl 5-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carboxylate (5) was converted into methyl 5-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carboxylate (6) via diazotization in the presence of cuprous chloride. Similarly, 5-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuanosyl)imidazole-4-carbonitrile (9) was converted into 5-chloro-, 5-bromo-, and 5-iodo-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carbonitrile derivatives. These 5-halogenated imidazole nucleosides were treated with several nucleophiles such as ammonia, hydroxylamine, and hydrogen sulfide to provide, respectively, 5-haloimidazole-4-carboxamide, 5-haloimidazole-4-carboxamidoxime, and 5-haloimidazole-4-thiocarboxamide ribonucleosides. 5-Chloro- or 5-bromo-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)imidazole-4-carbonitrile was treated with potassium hydrosulfide to yield 5-mercapto-1-beta-D-ribofuranosylimidazole-4-thiocarboxamide (16). The catalytic reduction of 5-chloro- or 5-bromo-1-beta-D-ribofuranosylimidazole-4-carboxamidoxime provided 1-beta-D-ribofuranosylimidazole-4-carboxamidines as their hydrochloride and hydrobromide salts, respectively. These nucleosides were tested for in vitro antiviral, antifungal, and antibacterial activity. The 5-halo analogues of 1-beta-D-ribofuranosylimidazole-4-carboxamide showed significant antiviral activity whereas compound 16 was found inhibitory to fungi.

Synthesis, antitumor activity, and antiviral activity of 3-substituted 3-deazacytidines and 3-substituted 3-deazauridines.

Novel 3-substituted analogues of 4-amino-1-beta-D-ribofuranosyl-2(1H)-pyridinone (3-deazacytidine, 3) and 4-hydroxy-1-beta-D-ribofuranosyl-2(1H)-pyridinone (3-deazauridine, 4) have been synthesized and tested for antitumor and antiviral activity. Thus the 3-chloro (9a), 3-bromo (9b), and 3-nitro (9c) analogues of 3 and the 3-chloro (9d), 3-bromo (9e), and 3-nitro (9f) analogues of 4 were prepared by standard glycosylating procedures. Novel requisite heterocycles 4-amino-3-chloro-2(1H)-pyridinone (7a) and 4-amino-3-bromo-2(1H)-pyridinone (7b) were prepared by halogenating 4-amino-2(1H)-pyridinone (5). Requisite heterocycles 4-amino-3-nitro-2(1H)-pyridinone (7c), 3-chloro-4-hydroxy-2(1H)-pyridinone (7d), 3-bromo-4-hydroxy-2(1H)-pyridinone (7e), and 4-hydroxy-3-nitro-2(1H)-pyridinone (7f) were synthesized by known procedures from 4-hydroxy-2(1H)-pyridinone (6). Structure proof of target nucleosides was provided by independent synthesis, 1H NMR, and UV. Compounds 9a-f were devoid of activity against intraperitoneally implanted L1210 leukemia in mice. Compound 9f displayed significant activity against rhinovirus type 34 grown in WISH cells. 4-Amino-3-fluoro-1-beta-D-ribofuranosyl-2(1H)-pyridinone (1) displayed good activity against intraperitoneally implanted P388 leukemia in mice, but it was devoid of activity against M5076 sarcoma, amelanotic (LOX) melanoma xenograft, and subrenal capsule human mammary carcinoma MX-1 xenograft in mice. Compound 1 also displayed significant activity against rhinovirus type 34.

Synthesis and antiviral acticity of some phosphates of the broad-spectrum antiviral nucleoside, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin).

1-beta-D-Ribofuranosyl-1,2,4-triazole-3-carboxamide 5'-phosphate (2) was prepared and converted into the following derivatives: the 5'-phosphoramidate 3, the 5'-diphosphate 4, the 5'-triphosphate 5, and the cyclic 3',5'-phosphate 6. The cyclic 2',3'-phosphate 7 was prepared from the parent nucleoside, 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (1), and was opened to the 2'(3')-phosphate 8. These compounds were found to exhibit significant antiviral activity against several viruses in cell culture. Ribavirin 5'-phosphate (2) was shown to be effective when tested against lethal infections in mice caused by influenza A2, influenza B, and murine hepatitis viruses.

Synthesis and antitumor and antiviral activities of 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-iminopyrimidine and its derivatives.

1-beta-D-Arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine (10), 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-fluoropyrimidine 3'-phosphate (9), and 1-beta-D-arabinofuranosyl-2-amino-1,4(2H)-imino-5-chloropyrimidine (11) have been synthesized from 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine (5), 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-fluorocytosine 3'-phosphate (4), and 2,2'-anhydro-1-beta-D-arabinofuranosyl-5-chlorocytosine (6), respectively. 2,2'-Anhydro-1-beta-D-arabinofuranosylcytosine 3'-phosphate (7), 1-beta-D-arabinofuranosyl-2-amino-1,4-(2H)-iminopyrimidine (13), 1-beta-D-arabinofuranosyl-2-amino-1,3(2H)-iminopyrimidine 3'-phosphate (12), and compounds 4, 5, and 9 showed significant in vitro activity against a number of DNA viruses. Compounds 7 and 12 were also effective in vivo against type 1 herpes simplex virus. Compounds 7, 12, and 13 were extremely effective in the treatment of mice bearing leukemia L1210.

Synthesis and antiviral activity of certain 5'-monophosphates of 9-D-arabinofuranosyladenine and 9-D-arabinofuranosylhypoxanthine.

A number of 5'-phosphates of 9-D-arabinofuranosyladenine and 9-D-arabinofuranosylhypoxanthine were prepared and tested against a variety of DNA viruses in tissue culture. The syntheses of the antiviral agent 9-beta-D-arabinofuranosylhypoxanthine 5'-monophosphate (6) and a series of related nucleotides, 9-beta-D-arabinofuranosyladenine 5'-O-methylphosphate (3), 9-beta-D-arabinofuranosylhypoxanthine 5'-O-methylphosphate (7), 9-beta-D-arabinofuranosylhypaxanthine cyclic 3',5'-phosphate (13), and 9-alpha-D-arabinofuranosylhypoxanthine 5'-monophosphate (17), are described. The concepts underlying the development of these antiviral agents are discussed. Comparison of the anti-DNA viral activity is made with 9-beta-D-arabinofuranosyladenine (ara-A). Reproducible antiviral activity against three DNA viruses in vitro at nontoxic dosage levels is demonstrated by 3,6, and other related nucleotides.

Synthesis and anti-DNA -virus activity of the 5'-monophosphate and the cyclic 3',5'-monophosphate of 9-(beta-D-xylofuranosyl) guanine.

9-(beta-TD-xylofuranosyl)guanine (xylo-G) was converted chemically to the 9-(beta-D-xylofuranosyl)guanine 5'-monophosphate (xylo-GMP) and 9-(beta-D-xylofuranosyl)guanine cyclic 3',5'-monophosphate (c-xylo-GMP). These compounds were tested against a variety of DNA viruses in tissue culture in parallel with 9-(beta-D-arabinofuranosyl)adenine (ara-A). This evaluation revealed that xylo-G, xylo-GMP, and c-xylo-GMP were all moderately active but less effective than ara-A. When the four compounds were administered intracerebrally as a treatment for herpes virus, type 1 induced encephalitis in mice, c-xylo-GMP exhibited superior activity to that shown by the other three. When administered intraperitoneally, c-xylo-GMP was found to have a therapeutic index of about 4, which is less than that for ara-A (approximately 30) in the same system.

Synthesis and antiviral activity of certain thiazole C-nucleosides.

A general reaction of glycosyl cyanides with liquid hydrogen sulfide in the presence of 4-dimethylaminopyridine to provide the corresponding glycosylthiocarboxamides is described. These glycosylthiocarboxamides were utilized as the precursors for the synthesis of 2-D-ribofuranosylthiazole-4-carboxamide and 2-beta-D-ribofuranosylthiazole-5-carboxamide (23). The structural modification of 2-beta-D-ribofuranosylthiazole-4-carboxamide (12) into 2-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiazole-4-carboxamide (15), 2-beta-D-ribofuranosylthiazole-4-thiocarboxamide (17), and 2-(5-deoxy-beta-D-ribofuranosyl)thiazole-4-carboxamide (19) is also described. These thiazole nucleosides were tested for in vitro activity against type 1 herpes virus, type 3 parainfluenza virus, and type 13 rhinovirus and an in vivo experiment was run against parainfluenza virus. They were also evaluated as potential inhibitors of purine nucleotide biosynthesis. It was shown that the compounds (12 and 15) which possessed the most significant antiviral activity were also active inhibitors (40-70%) of guanine nucleotide biosynthesis.

Differential antiviral activity of two TIBO derivatives against the human immunodeficiency and murine leukemia viruses alone and in combination with other anti-HIV agents.

R82913 and R86183, two derivatives of tetrahydroimidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO), were found to potently and selectively inhibit the replication and cell killing effects of a panel of biologically diverse laboratory and clinical strains of HIV-1. The two compounds exhibited significant activity in all human cell lines tested, as well as in fresh human peripheral blood lymphocytes and macrophages. One of these two compounds (R82913) was found to significantly inhibit the replication of a murine retrovirus (Rauscher murine leukemia virus) in both UV-XC plaque formation and virus yield reduction assays. R86183, despite differing from R82913 only in the positioning of a single chlorine molecule, was not active against the murine retrovirus but was 10-fold more potent in inhibiting HIV-1 replication. Combination antiviral assays with other reverse transcriptase inhibitors, including AZT, ddC, and carbovir, yielded synergistic anti-HIV activity with both TIBO derivatives. Additive to slightly synergistic results were obtained in combinations with ddI and phosphonoformic acid whereas additive to antagonistic activity was detected in combination with dextran sulfate.

An ELISA system for evaluating antiretroviral activity against Rauscher murine leukemia virus.

A system for evaluating the activity of antiviral agents against Rauscher murine leukemia virus (R-MuLV) has been developed using an enzyme linked immunosorbent assay technique. The activity of various antiviral compounds demonstrated in this assay system has been compared to their activity in the UV-XC plaque reduction assay, which has been used historically for evaluating anti-R-MuLV compounds. The assay is based upon detection of R-MuLV encoded p30 protein production in virus infected murine cells. The assay reagents are readily available and the assay system is amenable to automated data collection systems. Cytotoxicity evaluations are conducted in parallel to the Rauscher MuLV ELISA assay in order to assess drug-induced reductions in cell viability. Cytotoxicity evaluations are important to interpretation of the ELISA results since reductions in cell viability reduce viral protein production which would indicate an antiviral drug effect. This system is less sensitive than the classical UV-XC plaque reduction assay; however, it does offer an alternative to the time-consuming and labor-intensive plaque assay.

Effect of the estrous cycle on susceptibility of female mice to intravaginal inoculation of herpes simplex virus type 2 (HSV-2).

In order to explore the effect of sexual maturity on the susceptibility of mice to genital herpesvirus infections, mice were separated into the four stages of the estrous cycle and inoculated intravaginally with varying doses of HSV-2, strain 186. Deaths were observed as indicators of susceptibility and were recorded as follows: proestrous, 33%; estrous, 16%; metestrous, 9% and diestrous, 75%. To determine the course of infection in animals inoculated at different stages of estrous, cotton swabs were used to collect vaginal specimens at various times post-virus inoculation for virus titration. All mice inoculated during diestrous were positive for virus as early as 6 hours post-virus inoculation and had titers that increased over a 3 day period. Mice inoculated in other stages of estrus were positive only briefly (at 6 h) or had no detectable virus. In order to verify the susceptibility associated with diestrous, mice were ovariectomized to produce a continuous diestrous (pseudodiestrous) and when inoculated greater than or equal to 66.7% died. In contrast, none of the mice which had been ovariectomized and treated with estrogen to simulate the estrus stage died. We postulate that in stages other than diestrous virus may adsorb to epithelial cells in the lumen of the vagina and/or be expelled from the body by nonspecific resistance functions, thus reducing the likelihood of vaginal infection.

Evaluation of the anti-herpesvirus drug combinations: virazole plus arabinofuranosylhypoxanthine and virazole plus arabinofuranosyladenine.

Combinations of Virazole plus arabinofuranosylhypoxanthine (ara-Hx) and Virazole plus arabinofuranosyladenine (ara-A) were investigated in KB or BHK cells infected with types 1 or 2 herpes viruses. Combinations of Virazole and ara-Hx exhibited significant synergy as evaluated graphically (isobolograms) or by fractional inhibitory concentration (FIC) indices. Optimal ratios for the combination were 1:1 to 1:10 for Virazole to ara-Hx. At these ratios, FIC indices in the range of 0.5-0.2 were commonly observed. Combinations of Virazole and ara-A were antagonistic when observed in the presence of pentostatin, an adenosine deaminase inhibitor. In the absence of pentostatin, the minimum inhibitory concentration (MIC) of ara-A and degree of synergy with Virazole were variable.

Antiviral and cytotoxicity evaluation of 3-nitro-3-deazauridine.

3-Nitro-3-deazauridine (3N-3DU) is a new synthetic nucleoside having activity against members of 5 RNA virus families including: paramyxoviruses (parainfluenza, PIV), picornaviruses (rhino-, RV), rhabdoviruses (vesicular stomatitis, VSV), togaviruses (Semliki Forest, SFV) and bunyaviruses (Punta Toro, PTV). In this report, we evaluate and compare its activity with the parent nucleoside, 3-deazauridine (3DU) and ribavirin as drug standards. Comparison of drug activities utilizes observations of antiviral indices, which are determined by the following formula: maximum tolerated dose (MTD)/minimum inhibitory concentration (MIC). The antiviral index (AI) of 3N-3DU (AI 15.3) was comparable to ribavirin and much higher than 3DU when evaluated against PIV. The 3N-3DU was the most active of the three when tested against RV (AI 24.1), SFV (AI 76.9) or VSV (AI 50). In contrast to the RV activity, 3N-3DU (AI 0.5) and 3DU (AI less than 0.1) were less active than ribavirin (AI 1.3) when evaluated against poliovirus, type 1 (PoV). Ribavirin (AI 10.0) was more active than 3N-3DU (AI 2.4) and 3DU (AI less than 0.1) against PTV. 3N-3DU exhibited comparable toxicity to ribavirin in KB cells, was 4-fold less toxic in WISH cells and 4-fold more toxic in LLC-MK2 cells. Overall, 3N-3DU is markedly less toxic than its parent nucleoside, 3DU. It appears from this study that the structural modification of 3DU resulting from the addition of the nitro group in the 3 position of the base reduces toxicity and enhances the antiviral activity.

Evaluation of anti-AIDS drugs in conventional mice implanted with a permeable membrane device containing human T cells infected with HIV.

We now report the confirmation of the work of Hollingshead et al. (1995) on development of a cell based hollow fiber (HF) system for evaluating potential anti-AIDS drugs in vivo using conventional mice rather than SCID mice. CD4 +, CEM-SS cells infected with HIV/1, strain RF, at a multiplicity of infection of 0.1 were placed into HFs. The fibers were implanted into the peritoneal cavity of outbred Swiss mice. Using this model, the antiviral activity of azidothymidine (AZT) at doses of approximately 150, 75 and 37.5 mg/kg/day was evaluated by administering AZT to the mice in drinking water. Upon fiber removal on day 6, AZT treatment was shown to significantly increase CEM cell viability over the untreated, virus control group and significantly reduced the levels of HIV p24 and HIV RT activity.

In vitro and in vivo enhancement of ddI activity against Rauscher murine leukemia virus by ribavirin.

Ribavirin has been reported to enhance the activity of ddI against HIV. We explored this enhancement of antiviral activity in Rauscher murine leukemia virus (RMuLV) models in vitro and in vivo. The significant finding in these studies was that combinations of the drugs exhibited virus titer reductions that were greater than would be expected if the drug interactions were simply additive. These effects were designated synergistic by the method of Prichard and Shipman (Prichard, M.N. and Shipman, C., Jr. (1990). A three-dimensional model to analyze drug-drug interaction, Antiviral Res. 14, 181-206). In addition to the antiviral synergy, we also observed some synergistic toxicity in the animal model.

Thiazolobenzimidazole: biological and biochemical anti-retroviral activity of a new nonnucleoside reverse transcriptase inhibitor.

Thiazolobenzimidazole (NSC 625487) was a highly potent inhibitor of human immunodeficiency virus-induced cell killing and viral replication in a variety of human cell lines, as well as fresh human peripheral blood lymphocytes and macrophages. The compound was active against a panel of biologically diverse laboratory and clinical strains of HIV-1, including the AZT-resistant strain G910-6. However, the agent was inactive against HIV-2 and a pyridinone-resistant strain (A17) of HIV-1, a strain which is cross-resistant to several structurally diverse members of a common pharmacologic class of nonnucleoside reverse transcriptase inhibitors. The compound selectively inhibited HIV-1 reverse transcriptase but not HIV-2 reverse transcriptase. Combinations of thiazolobenzimidazole with either AZT or ddI synergistically inhibited HIV-1 induced cell killing in vitro. Thiazolobenzimidazole also inhibited the replication of the Rauscher murine leukemia retrovirus. Thus, thiazolobenzimidazole is a new active anti-HIV-1 chemotype and may represent a subclass of nonnucleoside reverse transcriptase inhibitors with an enhanced range of anti-retroviral activity.

Acceleration of scrapie in mice by target-organ treatment with interferon inducers.

Interferon inducers were used in the target-organ treatment of scrapie in mice. Intracerebral treatments began 24 hr prior to intracerebral inoculation of 10(4.8) LD50 of the Chandler strain of scrapie agent. The treatments included 30 and 0.3 microgram poly(I:C) given weekly 9 times, 45 microgram statolon given biweekly 7 times, or 1.5 HA units of Sendai virus given biweekly 6 times. All treatments except the lower dose of poly(I:C) accelerated death in scrapie-affected mice. Compared to saline-treated control groups, 30 microgram poly(I:C), given weekly, shortened the mean survival time 13.5 days. Groups treated with statolon or Sendai virus had their mean survival times shortened 18.5 and 21.7 days, respectively. Infected mice were also evaluated for signs of disease at approximately weekly intervals using a numerical scoring method. Acceleration was also apparent using this parameter of disease. When treatment occurred only once, Sendai virus was the only inducer to significantly shorten the survival of mice.

Comparison of the development of resistant strains of Type 1 herpes simplex virus to in vitro antiviral activity of 5-iodo-2'-deoxyuridine or ribavirin.

Exposure of HSV/1 to low concentrations of ribavirin during 4--5 passages does not produce ribavirin-resistant virus. IDU resistance was developed by HSV/1 while it was being passed simultaneously. This resistance was seen to develop in both KB and Vero cells. The IDU-resistant virus is also resistant to ribavirin in KB cells.

Effect of ribavirin on Type 2 Herpesvirus hominis (HVH/2) in vitro and in vivo.

Ribavirin was found to inhibit five strains of HVH/2 tested in KB cells with the M1C range being 10-100 microgram/ml. In a mouse tail animal model, it effectively reduced HVH/2-induced lesions particularly at the peak lesion periods. Ribavirin was remarkably effective when intravaginal treatment was initiated late in the infection, which suggests that it may have potential for treatment of human cutaneous infections.