William M. Shannon

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.

A TIBO derivative, R82913, is a potent inhibitor of HIV-1 reverse transcriptase with heteropolymer templates.

R82913, (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (a TIBO derivative), inhibited the replication of thirteen different strains of HIV-1 in CEM cells with a median IC50 of 0.15 microM. The concentration of compound that killed 50% of the cells was much higher (46 microM), indicating that R82913 has a high selectivity index. R82913 was 20-fold more potent than AZT-TP in the inhibition of HIV-1 reverse transcriptase in an assay using a naturally occurring template (ribosomal RNA) that more accurately resembles native viral RNA than a synthetic homopolymer. With this template, R82913 inhibited HIV-1 reverse transcriptase with an ID50 (0.01 microM) that is equal to, or lower than, the IC50 for this compound in all of our cell culture assays (0.01-0.65 microM). R82913 has no effect on the replication of HIV-2 in CEM cells and does not inhibit the reverse transcriptase from this virus.

Adenosylhomocysteine hydrolase inhibitors: synthesis of 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine and its effect on Rous sarcoma virus and Gross murine leukemia virus.

Abstract The synthesis of a new analog of adenosylhomocysteine (AdoHcy), 5′-deoxy-5′-(isobutylthio)-3-deazaadenosine, is described. The compound is a non-competitive inhibitor of AdoHcy hydrolase, with a K i of 0.4 mM, as compared to a K i of 3 μM for 3-deazaadenosine, a competitive inhibitor of the enzyme. 5′-Deoxy-5′-(isobutylthio)-3-deazaadenosine is not hydrolyzed by AdoHcy hydrolase. 5′-Deoxy-5′-(isobutylthio)-3-deazaadenosine when tested has a selective antiviral activity against Rous sarcoma virus in chick embryo cells, and against Gross murine leukemia virus in mouse embryo cells. Possible mechanisms of this anti-viral activity are discussed.

SELECTIVE INHIBITION OF RNA TUMOR VIRUS REPLICATION IN VITRO AND EVALUATION OF CANDIDATE ANTIVIRAL AGENTS IN VIVO

A limited number of biologically active materials were examined for their relative ability to selectively inhibit the replication of Gross or Rauscher murine leukemia virus (MLV) in Swiss mouse embryo cells by means of the UV-XC plaque-reduction assay. Among the compounds demonstrating significant antiviral activity against Gross MLV in vitro were 1-(4-fluorobenzyloxy) adenosine (FBAR), polyadenylic acid [poly(A)], the carbocyclic analogue of 6-methylthiopurine ribonucleoside (C-MeMPR), 3-(2,4-dinitrophenylhydrazonemethyl)rifamycin SV (AF/DNFI), and phosphonoacetic acid (PAA). Five compounds that exhibited significant antiviral activity against MLV in vitro were tested for similar activity against Rauscher MLV in vivo. Three of these selected compounds, pyrazofurin (pyrazomycin), ribavirin (Virazole), and 9-beta-D-arabinofuranosyladenine (ara-A), produced a significant (50%-100%) inhibition of virus-induced splenomegaly development in mice, whereas the other two candidate inhibitors, 3-deazauridine (deazaUR) and rifamycin SV, the other two candidate inhibitors, 3-deazauridine (deazaUR) and rifamycin SV, failed to demonstrate any in vivo activity in this 21-day leukemogenesis assay. The administration of an inhibitor of adenosine deaminase (Co-vidarabine) in combination with ara-A resulted in an enhanced antiviral response in both infected cell cultures and animals. Co-vidarabine also increased the potency of ara-AMP against Gross MLV in vitro, indicating the probable dephosphorylation of the compound to ara-A and its subsequent deamination to ara-H in this system.

Cell-Based and Biochemical Analysis of the anti-HIV Activity of Combinations of 3′-azido-3′-deoxythymidine and Analogues of TIBO

The toxicity of 3′-azido-3′-deoxythymidine (AZT) and the appearance of drug-resistant mutants in patients treated with AZT emphasizes the critical importance of the development of alternative strategies for the therapy of AIDS patients. Combination antiviral chemotherapy provides an attractive therapeutic strategy since the dose of the individual agents may be lowered to reduce toxicity and the use of two potent antiviral agents may limit the development of drug resistance. Two analogues of tetrahydro-imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione (TIBO) potently and selectively inhibit the replication of HIV-1 in cell culture. In combination with AZT, either of the two TIBO compounds, R82913 and R86183, was highly synergistic in cell culture against HIV-1. However, in biochemical enzyme inhibition assays, utilizing recombinant HIV-1 reverse transcriptase, synergy was not detected at the enzymatic level. These results suggest that one of these two known inhibitors of HIV-1 reverse transcriptase may have a secondary mechanism of action distinct from inhibition of the reverse transcriptase.

3-Deazauridine: Inhibition of Ribonucleic Acid Virus-Induced Cytopathogenic Effects In Vitro

3-Deazauridine, a synthetic analogue of uridine and a potential antitumor agent, was found to possess antiviral activity against a number of ribonucleic acid-containing animal viruses in mammalian cell culture. Inhibition of virus-induced cytopathogenic effects by 3-deazauridine was observed in cells infected with rhinovirus types 1A and 13, coxsackievirus type A21, and PR-8 influenza virus, but not in cells infected with poliovirus type 2 or echovirus type 12. The extracellular yield of progeny influenza virus was found to be significantly reduced in titer in 3-deazauridine-treated Madin-Darby canine kidney cell cultures at 12 to 24 hr after infection. Although the precise biochemical mechanism of action of this antimetabolites antiviral activity is not known, the compound does not appear to exert a direct virucidal effect on the influenza virion itself.

Synthesis, Antiproliferative, and Antiviral Evaluation of Certain Acyclic 6-Substituted Pyrrolo[2,3-D]-pyrimidine Nucleoside Analogs Related to Sangivamycin and Toyocamycin

Abstract A number of 6-substituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine and 7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyrimidine derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their biological activity. Treatment of 2-amino-5-bromo-3,4-dicyanopyrrole (2) with triethylorthoformate, followed by alkylation via the sodium salt method with either 2-(acetoxyethoxy)methyl bromide or (1,3-diacetoxy-2-propoxy)methyl bromide, furnished the corresponding N-substituted pyrroles 3a and 3b. These compounds were then smoothly converted to the requisite deprotected 4-amino-6-bromopyrrolo[2,3-d]-pyrimidine-5-carbonitriles 5a and 5b (toyocamycin analogs) by methanolic ammonia. The 6-amino-derivatives were obtained by a displacement of the bromo group with liquid ammonia. Conventional functional group transformations involving the 5-cyano group furnished the 5-carboxamide (sangivamycin) and 5-thioamide analogs. Compounds substituted at the 7-...

SYNTHESIS AND ANTIVIRAL EVALUATION OF ANALOGS OF ADENOSINE-N1-OXIDE AND 1-(BENZYLOXY)ADENOSINE

The activity of a series of compounds related to adenosine-N1-oxide (1) and 1-(benzyloxy)adenosine (42) against vaccinia virus has been determined both in vitro and in a vaccinia mouse tailpox model. Significant activities have been found both in vitro and in vivo for a number of the synthetic compounds.

In vitro and in vivo antiviral (RNA) evaluation of orotidine 5'-monophosphate decarboxylase inhibitors and analogues including 6-azauridine-5'-(ethyl methoxyalaninyl) phosphate (a 5'-monophosphate prodrug)

A series of 29 pyrimidines comprising analogues of 6-azauridine (e.g. 2- and 4-thio-6-azauridine), 6-substituted uridines (including several known inhibitors of orotidine 5′-monophosphate decarboxylase, ODCase, e.g. pyrazofurin), and 6-azauridine-5′-(ethyl methoxyalaninyl) phosphate (a potential prodrug of 6-AU-5′-MP) were synthesized and evaluated in vitro and in vivo against five RNA viruses: Japanese encephalitis (JE), yellow fever (YF), sandfly fever (SF), Punta Tora (PT) and Venezuelan equine encephalomyelitis (VEE) viruses. 2-Thio-6-azauridine demonstrated the best In vitro activity against all five viruses. However, in vivo activity was not observed in JE-, PT- and VEE-infected mice. The phosphate prodrug of 6-azauridine was significantly more effective than the parent compound in the PT virus mouse model. Optimum in vivo dose/route/schedule was determined for pyrazofurin in PT-virus-infected mice.

Antiviral activity of 9-beta-D-arabinofuranosyladenine (ara-A) against Gross murine leukemia virus in vitro.

Summary Ara-A, an antiviral agent with broad-spectrum activity against DNA viruses, was observed to inhibit Gross murine leukemia virus (an RNA tumor virus) in cell culture. Its activity was selective and was observed at concentrations well below cytotoxic levels.