Nonnucleoside pyrrolopyrimidines with a unique mechanism of action againsthuman cytomegalovirus

Based upon a prior study which evaluated a series of nonnucleoside pyrrolo[ 2,3-d]pyrimidines as inhibitors of human cytomegalovirus (HCMV), we have se lected three active analogs for detailed study. In an HCMV plaque-reduction assay, compounds 828, 951, and 1028 had 50% inhibitory concentrations (IC( 50)s) of 0.4 to 1.0 PLM, Similar results were obtained when 828 and 951 wer e examined by HCMV enzyme-linked immunosorbent assay (IC(50)s = 1.9 and 0.4 mu M, respectively) and when 828 was tested in a viral DNA-DNA hybridizati on assay (IC50 = 1.3 mu M). In yield-reduction assays with a low multiplici ty of infection (MOI), all three compounds caused multiple log,, reductions in virus titer, and the activities of these compounds were comparable to t he activity of ganciclovir (GCV; IC90 = 0.2 mu M), In contrast to the reduc tion of viral titers by GCV, the reduction of viral titers by 828, 951, and 1028 decreased with increasing MOI. Cytotoxicity in human foreskin fibrobl asts and KB cells ranged from 32 to >100 mu M. In addition, 828 (the only c ompound tested) was less toxic against human bone marrow progenitor cells t han GCV, Time-of-addition and time-of-removal studies established that the three pyrrolopyrimidines inhibited HCMV replication before GCV had an effec t on viral DNA synthesis but after viral adsorption. Compound 828 was equal ly effective against GCV-sensitive and GCV-resistant HCMV clinical isolates . Combination studies with 828 and GCV showed that the effects of the two c ompounds on HCMV were additive but not synergistic. Taken together, the dat a indicate that these pyrrolopyrimidines target a viral protein that is req uired in an MOI-dependent manner and that is expressed early in the HCMV re plication cycle.