Studies of various analogs related to the antipicornavirus agent, 4',5-dihydroxy-3,3',7-trimethoxyflavone (Ro 09-0179), led to the identification of 4'-ethoxy-2'-hydroxy4,6'-dimethoxychalcone (Ro 09-0410), a new and different type of antiviral agent. Ro 09-0410 had a high activity against rhinoviruses but no activity against other picorraviruses. Of 53 rhinovirus serotypes so far tested, 46 were susceptible to Ro 09-0410 in HeLa cell cultures. The concentration of Ro 09-0410 inhibiting 50% of the types of rhinovirus was about 0.03 ,ug/ml, whereas the 50%o cytotoxic concentration was 30 ,Ig/thl. Ro 09-0410 inactivated rhinoviruses in direct dose-, time-, and temperture-dependent fashion. Since infectivity, reduced by exposure to the agent, completely regained the original level by extraction of the agent with chloroform, the inactivation may be associated with the binding of the agent to some specific site of the rhinovirus capsid.Ro 09-0179 (4',5-dihydroxy-3,3',7-trimethoxyflavone), a potent antipicornavirus agent, was isolated from a Chinese medicinal herb, Agastache Folium (Agastache rugosa Kuntze) (3). The discovery of this agent led to the synthesis of a series of derivatives and testing of their antiviral activities in tissue culture. Among hundreds of compounds prepared and evaluated, Ro 09-0410 (4'-ethoxy-2'-hydroxy-4,6'-dimethoxychalcone) (Fig. 1) emerged as an agent exclusively active against rhinoviruses.The mode of action of Ro 09-0410 differed significantly from that of Ro 09-0179. The latter appeared to inhibit the replication of picornavirus at a stage between uncoating and the initiation of RNA synthesis in infected cells (3). On the other hand, Ro 09-0410 directly inactivated rhinoviruses. Subsequent studies showed that the infectivity of rhinoviruses was reduced by exposure to Ro 09-0410 but was regained completely by the extraction of the agent with chloroform, to which rhinovirus is resistant (1). While bound to the agent, rhinovirus was inactive. In this report, we describe the results of tissue culture studies on the antirhinovirus activity of ( All rhinoviruses used in this study were purchased from the American Type Culture Collection, Rockville, Md., except type 2 (HGP), which was kindly supplied by R. Kohno, the National Institute of Health of Japan. Viruses were propagated in HeLa and WI-38 cells at 33掳C. Poliovirus, echoviruses, coxsackieviruses, mengovirus, vesicular stomatitis virus, respiratory syncytial virus, herpes simplex virus, vacciia virus, and influenza virus used in this study were propagated as described in the accompanying paper (3).Asy of rhino titer and ePt"mation of MIC5..The rhinovirus titer was assayed, and the 509o minimal inhibitory concentration (MIC50) was estimated as described in the accompanying paper (3). The virus titer was expressed as plaque-forming units per milliliter. The MIC50 was expressed as the concentration at which the viral cytopathic effect was inhibited by about 50%6 as compared with the control.