Antiviral properties of a series of 1,6-naphthyridine and 7, 8-dihydroisoquinoline derivatives exhibiting potent activity against human cytomegalovirus

Abstract

A series of 1,6-naphthyridine (L. Chan, H. Jin, T. Stefanac, J. F. Lavallee, G. Falardeau, W. Wang, J. Bedard, S. May, and L. Yuen, J. Med. Chem. 42:3023-3025, 1999) and isoquinoline (L. Chan, H. Jin, T. Stefanac, W. Wang, J. F. Lavallee, J. Bedard, and S. May, Bioorg. Med. Chem. Lett. 9:2583-2586, 1999) analogues exhibiting a high level of anti-human cytomegalovirus (HCMV) activity were investigated in a series of studies aimed at better understanding the mechanism of action of some representatives of this class of compounds. In vitro antiviral profiling revealed that these compounds were active against a narrow spectrum of viruses, essentially the human herpesviruses and type 2 rhinovirus. In HCMV assays, a 39- to 223-fold lower 50% inhibitory concentration was obtained for compound A1 than for ganciclovir against strains AD 169 and Towne. In addition, ganciclovir, foscarnet, cidofovir, and BDCRB (2-bromo-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole)-resistant HCMV strains remained susceptible to 1,6-naphthyridines and 7, 8-dihydroisoquinolines tested in this study, supporting the view that a novel mechanism of action could be involved. Drug combination studies showed a small but significant synergistic antiviral effect between compound B2 and ganciclovir. Cytotoxicity profiling of representative compounds under various cell growth conditions indicated a generally similar cytotoxic effect, relative to ganciclovir, in log-phase growing cells. However, in stationary cells, a relatively higher level of toxicity was observed than that for control compound. Effect of time of drug addition showed that the anti-HCMV activity of compound A1, ganciclovir, and cidofovir was lost at approximately the same time (72 h postinfection), indicating that the compound was affecting events at the early and late stage of virus replication. This interpretation is also supported by reduction of de novo synthesis of pp65 tegument protein and lack of any effect of the compound on viral adsorption. A reduction of the HCMV enhancer-promoter-directed luciferase expression was also observed in a stably transfected cell line when compound A1 was present at relatively high concentrations.

FIG. 1

Chemical structures of a 1,6-naphthyridine derivative (A) and 7,8-dihydroisoquinoline derivatives (B).

FIG. 2

Effect of time of drug addition on anti-HCMV activity of compound A1. HFF cells were infected with HCMV strain Towne at an MOI of 0.5 PFU/cell and exposed to GCV (○), CDV (▴), TCRB (□), and compound A1 (■) at 100, 10, 100, and 10 μM, respectively. Drugs were added at 8, 16, 24, 32, 40, 48, 56, 64, 72, 80, and 88 h postinfection and were allowed to remain on the infected cultured cells for the 96-h duration of the experiment. Thereafter, infected cells were subjected to one cycle of freezing and thawing, and the titer of virus produced was determined as described previously (37). The 0-h time point represents the end of the viral adsorption step. The antiviral activity of the drugs is depicted as a percentage of the virus titer obtained with the infected untreated cells.

FIG. 3

Interaction of compound B2 and GCV. MRC-5 cells were infected with HCMV at an MOI of 0.001 and treated with combinations of the two compounds. The amount of HCMV replication was determined by an ELISA. Data analysis with MacSynergy II revealed a volume of synergy of 92.7 μM²% at 95% confidence level.

FIG. 4

Effect on HCMV ie1/ie2 promoter-enhancer-dependent luciferase activity. HCF cells were incubated in the presence of GCV (▵) and compounds A1 (○), B1 (▴), B2 (●), and B3 (■) at concentrations ranging from 1.8 to 39 μM for 40 h. Luciferase activity was measured as described in Materials and Methods. All points are averages of triplicate experiments. Standard deviation was less than 15%.

FIG. 5

Effect of compound A1 (5473) on the expression of IE 1 and the tegument proteins of HCMV strain AD 169 using indirect immunofluorescence assay. MRC-5 cells were infected at an MOI of 0.1 PFU/cell in the presence of either no compound (A and D), compound A1 (B and E), or GCV (C and F) at 0.62 and 10.2 μM, respectively. Cell cultures were sampled and examined at 72 h postinfection for the expression of the IE 1 (A to C) and tegument (D to F) proteins as described in Materials and Methods.