Background: Influenza is still a serious threat to human health with significant morbidity and mortality, so it is desirable to develop novel anti-flu drug agents with novel structures. Objective: The main purpose of this research was to explore broad-spectrum anti-flu agents and provide antiviral stockpiles in response to potential future influenza pandemics. Methods: Fifteen novel 12N-substituted tricyclic matrinic derivatives were synthesized and evaluated for their anti-influenza activities against H1N1 subtype taking 12N-p-cyanobenzenesulfonyl matrinane (1) as the lead. All prepared compounds were characterized by H-1 NMR, C-13 NMR and ESI-HRMS. The pharmacokinetics (PK) profile of the key compound was also examined in this study. Result: The structure-activity relationship study indicated that suitable benzyl groups on 12N atom might be beneficial for the activity. Among them, 12N-p-carboxybenzyl matrinic butane (17g) exhibited the most promising activity with an IC50 value of 16.2 mu M and a selective index (SI) value of over 33.4. In addition, compound 17g displayed a good in vivo pharmacokinetic profile with an area under the curve (AUC(0-infinity)) value of 9.89 mu M.h. Conclusion: We consider tricyclic matrinic butane derivatives to be a new class of anti-influenza agents and this study provided useful information on further optimization.