Synthesis and Identification of Novel Arylnaphthalene V-ATPase Inhibitors as Selective Anti-Filoviral Agents

2020-01-16T19:06:32Z (GMT) by Aaron R. Lindstrom
Ebolavirus, a genus of filoviruses, are responsible for outbreaks that cause up to 90% fatality, including the recent outbreak in West Africa that has resulted in over 28,603 reported cases and 11,301 deaths according to the WHO. Inhibitors of Vacuolar-ATPase (V-ATPase), a key protein complex that is responsible for endosomal acidification and represents a unique method to block this common viral pathway. V-ATPase inhibitors have previously been explored as therapies for many diseases but have failed due to high toxicity. Diphyllin is a natural, arylnaphthalene lignan that represents a novel structural class of V-ATPase inhibitors with a greater selectivity index than previous V-ATPase inhibitors. Diphyllin has shown promising anti-tumor and anti-osteoclast activity, as well as strong anti-viral activity against Influenza and Dengue viruses.
Herein, novel modifications of the lactone and phenol functional groups of diphyllin were explored for the ability to enhance the potency or therapeutic selectivity of the diphyllin core. Four initial sets of derivatives were synthesized and assayed for activity against ebolavirus infection, inhibition of cellular endosomal acidification, cytotoxicity and biochemical inhibition of isolated V-ATPase. Modification of diphyllin’s lactone functional group reduced both activity and selectivity, while alkylation of the phenol groups significantly enhanced activity. The incorporation of basic heterocycles to the alkyl group created an alkylamino series of derivatives that exhibited significantly improved therapeutic selectivity compared to diphyllin. Further investigation of the alkylamino class indicated that they retained activity against Marburgvirus infection, a filovirus related to Ebolavirus. Alkylamino derivatives inhibited ebolavirus infection of human macrophages at low micromolar levels with no apparent cytotoxicity.
Further investigation of the alkylamino class of diphyllin derivatives was conducted to determine if potency and/or therapeutic selectivity could be optimized. The addition of a 1-methylpiperazine moiety to the end of the alkyl chain improved potency 1260-fold over diphyllin, though therapeutic selectivity was not improved. The modification of the alkylamino linker to an acetamide eliminated cytotoxicity but decreased derivative activity against V-ATPase activity. To evaluate if the cytotoxicity evidenced by the alkylamino derivatives was evidenced in organisms, the derivative toxicity was assessed in zebrafish and mouse models. Derivatives displayed toxicity in a zebrafish developmental model but were all at least 10-fold less toxic than the known V-ATPase inhibitor bafilomycin A1. Three derivatives were well tolerated in CD-1 mice when administered at therapeutically relevant concentrations and caused no abnormal changes in their blood chemistry. Overall, these results demonstrate that the alkylamino and acetamide diphyllin phenol derivatives should be further studied as therapies for ebolavirus infection in addition to other V-ATPase mediated diseases.