Design, synthesis, and antiviral activity of alpha-nucleosides: D- and L-isomers of lyxofuranosyl- and (5-deoxylyxofuranosyl)benzimidazoles.

Several 2-substituted alpha-D- and alpha-L-lyxofuranosyl and 5-deoxylyxofuranosyl derivatives of 5,6-dicholro-2-(isopropylamino)-1-(beta-L-ribofuranosyl) benzimidazole (1263W94) and 2,5,6-trichloro-1(beta-D-ribofuranosyl)benzimidazole (TCRB) were synthesized and evaluated for activity against two herpesviruses (HSV-1 and HCMV) and for their cytotoxicity against HFF and KB cells. Condensation of 1,2,3,5-tetra-O-acetyl-L-lyxofuranose (2a) with 2,5,6-trichlorobenzimidazole (1) yielded the alpha-nucleoside 3a. The 2-bromo derivative and 2-methylamino derivative were prepared by treatment of 3a with HBr followed by deprotection or from methylamine, respectively. Compound 3a was deprotected and the resultant nucleoside used to prepare the 2-cyclopropylamino and 2-isopropylamino derivatives. The 2-alkylthio nucleosides were prepared by condensing 2a with 5,6-dichlorobenzimidazole-2-thione followed by deprotection. Alkylation of this adduct gave the 2-methylthio and 2-benzylthio derivatives. Condensation of 5-deoxy-1,2,3-tri-O-acetyl-L-lyxofuranosyl, prepared from L-lyxose, with 1 or 2-bromo-5,6-dichlorobenzimidazole (15), followed by deprotection, gave the 2-chloro or 2-bromo-5'-deoxylyxo-furanosyl derivative, respectively. The cyclopropylamino derivative was prepared from the 2-chloro derivative. All D-isomers were prepared in an analogous fashion from D-lyxose. Either compounds were inactive against HSV-1 or weak activity was poorly separated from cytotoxicity. In contrast, the 2-halogen derivatives in both the alpha-lyxose and 5-deoxy-alpha-lyxose series were active against the Towne strain of HCMV. The 5-deoxy alpha-L analogues were the most active, IC50's = 0.2-0.4 microM, plaque assay; IC90's = 0.2-2 microM, yield reduction assay. All of the 2-isopropylamino or 2-cyclopropylamino derivatives were less active (IC50's = 60-100 microM, plaque assay; IC90's = 17-100 microM, yield reduction assay) and were not cytotoxic. The methylamino, thio, and methylthio derivatives were neither active nor cytotoxic. The benzylthio derivatives were weakly active, but this activity was poorly separated from cytotoxicity. The alpha-lyxose L-isomers were more active in a plaque assay against the AD169 strain of HCMV compared to the Towne strain, thereby providing additional evidence of antiviral specificity.

Synthesis and antiviral evaluation of certain novel pyrazinoic acid C-nucleosides.

Pyrazine (1,4-diazine) C-nucleosides constitute a rare class of nucleic acid analogues that has only recently been reported in the literature. As part of our ongoing investigation into the synthesis and reactivity of these compounds, we have developed an electrophilic esterification of a lithiated pyrazine C-nucleoside (1) to give, following deprotection, the versatile intermediate ethyl 3,5-dichloro-6-(beta-d-ribofuranosyl)pyrazine-2-carboxylate (4). This intermediate was subjected to a variety of reaction conditions to generate a series of pyrazinoic acid C-nucleosides. These compounds, along with 3, 5-dichloro-2-(beta-d-ribofuranosyl)pyrazine (2) and 4, were evaluated for antiviral activity and cytotoxicity. No significant activity was observed for compounds 2 and 5-9, but 4 was active against two herpes viruses and cytotoxic in the micromolar range.

Design, synthesis, and antiviral evaluations of 1-(substituted benzyl)-2-substituted-5,6-dichlorobenzimidazoles as nonnucleoside analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole.

We have recently reported that certain ribosylated polyhalogenated benzimidazoles are potent and selective inhibitors of HCMV replication at noncytotoxic concentrations. To extend the structure-activity relationship beyond these first-generation compounds, we alkylated 5,6-dichloro-2-substituted-benzimidazoles with either a series of substituted benzyl halides or (2-bromoethyl)benzene to obtain five series of nonnucleoside analogues. Evaluation of these compounds for activity against herpes viruses revealed that the new compounds were less active than the benzimidazole ribonucleosides against human cytomegalovirus (HCMV) and inactive against herpes simplex virus type 1 (HSV-1). However, as part of our broader antiviral testing, we found that some of these compounds were active against HIV. Comparisons of the biological data revealed that a chloro or bromo group was required at the 2-position for the best separation of activity against HIV and cytotoxicity. Evaluation of the most active compounds against drug-resistant HIV suggested that they act by a mechanism other than inhibition of reverse transcriptase.

Thermal inactivation as a means of inhibiting the serum-associated deamination of 9-beta-D Arabinofuranosyladenine in tissue culture media.

9-beta-d-Arabinofuranosyladenine (ara-A) was deaminated to 9-beta-d-arabinofuranosylhypoxanthine by adenosine deaminase present in fetal bovine serum, newborn calf serum, and calf serum used to supplement tissue culture media. Heating newborn calf serum or calf serum for 12 h at 56 C completely eliminated the enzymatic deamination of ara-A. The deaminase activity associated with fetal bovine serum was more refractory to heating, requiring 24 h for complete inactivation. The nutritive value of heat-inactivated calf serum did not differ significantly from that of unheated serum based on considerations of population doubling times, deoxyribonucleic acid synthesis, and relative cloning efficiencies of KB cells.