A second generation of benzothiadiazine dioxide (BTD) derivatives was synthesized employing benzylation reactions mainly. The chlorophenylmethyl BTD derivatives showed activity against human cytomegalovirus (HCMV) with IC(50) values ranging from 3 to 10 microM. Their 50% cytotoxic concentrations were often >200 microM to lung fibroblast HEL cell proliferation and between 20 and 35 microM for lymphocyte CME cell growth. When cytotoxicity for cell morphology was considered, the minimum cytotoxic concentration for the different BTD derivatives varied between 5 and 200 microM. Some of the anti-HCMV compounds also showed activity against HIV-1 and HIV-2. The chlorophenylmethyl derivative 21 was active against a variety of HCMV clinical isolates from patients with different clinical manifestations and fully maintained its activity against a ganciclovir-resistant HCMV strain. The dibenzyl BTD derivatives did not inhibit HCMV protease, and preliminary pharmacological experiments revealed that their anti-HCMV action stems from interference with an early stage of the viral replicative cycle.