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°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.

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Modes of action of five antirhinovirus agents were compared. Ro 09-0410, 4',6-dichloroflavan, and RMI-15,731 were active preferentially against human rhinovirus. Serotypes of the virus varied in their susceptibility to these three agents, whereas Ro 09-0179 and enviroxime showed activity against all the serotypes of the virus tested to date. Ro 09-0410, RMI-15,731, and 4',6-dichloroflavan inactivated the virus directly, although 4',6-dichloroflavan did so only slightly. Inactivation by 4',731 was associated with the binding of the agents to the virus, since the infectivity, reduced by exposure to the agents, was restored to the original level by extraction of the agents with chloroform. The binding of [3H]Ro 09-0410 to human rhinovirus type 2 was inhibited by unlabeled Ro 09-0410, 4',6-dichloroflavan, and RMI-15,731 but not by Ro 09-0179 or enviroxime. Furthermore, subtypes resistant to both 4',6-dichloroflavan and RMI-15,731 showed cross-resistance to Ro 09-0410 and vice versa. On the other hand, sublines resistant to these three agents were not cross-resistant to Ro 09-0179 or enviroxime. These results indicate (i) that Ro 09-0410, 4',6-dichloroflavan, and RMI-15,731 exert their activities through the same mode of action, namely, binding to or interaction with some specific site on the viral capsid protein, and (ii) that the binding or interaction sites for these three agents are either the same or very close to each other.

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N4-Trimethoxybenzoyl-5'-deoxy-5-fluorocytidine (Ro 09-1390), a prodrug of the cytostatic 5'-deoxy-5-fluorouridine (5'-DFUR), was synthesized with the aim of reducing of the dose-limiting toxicity of 5'-DFUR, which is diarrhea. In mice bearing Lewis lung carcinoma, 5'-DFUR given po produced a substantial amount of 5-fluorouracil (5-FU) in the intestinal tract as well as tumors, where the enzyme pyrimidine nucleoside phosphorylase, essential for conversion of 5'-DFUR to 5-FU, is predominantly located. With the oral administration of Ro 09-1390 only a small amount of 5-FU was formed in the intestine; however, the administration of Ro 09-1390 and 5'-DFUR at the same dose produced similar amounts of 5-FU in tumor tissues. These differences in metabolism were reflected in their toxicity and antitumor efficacy. The administration of 5'-DFUR resulted in damage to the intestinal mucosal membrane and diarrhea in normal mice, whereas Ro 09-1390 was much less toxic to the intestinal tract. As regards antitumor activity, Ro 09-1390 and 5'-DFUR at equivalent doses inhibited the growth of Lewis lung carcinoma to similar extents. Since Ro 09-1390 was much less toxic to the intestinal tract than 5'-DFUR, mice bearing Lewis lung carcinoma could be given Ro 09-1390 daily over a longer period and at a higher dose, resulting in a longer survival time.

The modes of action of antirhinovirus agents and mechanisms of resistance to the agents are compared. Chalcone Ro 09-0410, 4',6-dichloroflavan (DCF) and RMI-15,731, which were active against rhinoviruses, inactivated the virus directly. Inactivation was associated with the binding of the agents to the virus particles, since the infectivity, reduced by exposure to the compounds, was restored to the original level by extraction of the agents with chloroform. The binding of [3H]chalcone to rhinovirus type 2 was inhibited by any of the three unlabelled agents. Furthermore, virus sublines selected for resistance to both dichloroflavan and RMI-15,731 showed cross-resistance to chalcone and vice versa. These findings indicate that the three agents exert their activities through the same mode of action (namely binding to or interaction with a specific site on the viral capsid protein) and that the binding or interaction sites for these three agents are either the same or very close to each other. Since the sublines resistant to chalcone and to RMI-15,731 have little or no capability to bind to chalcone, the resistance to these agents is conferred by changes in the viral capsid protein. On the other hand, flavone Ro 09-0179 and enviroxime, which were active widely against picornaviruses, had no ability to inactivate the virus directly, and their antiviral activity was not associated with the capsid protein.

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