On the basis of previous structure-activity relationship (SAR) and antiviral mechanism studies, antofine analogues with different substituent groups at the C-6 position targeting tobacco mosaic virus (TMV) RNA were synthesized for the first time. The antofine analogues 1a-8a and 1b-9b were evaluated for their antiviral activity against TMV. The SAR study of antofine analogues is discussed. Most of the compounds were found to exhibit higher antiviral activity than commercial Ningnanmycin in vitro and in vivo. The groups with hydrogen donor or electron-withdrawing groups at the C-6 position were found to be favorable for antiviral activity.