3-Hydroxypyrimidine-2,4-dione derivatives as monkeypox virus resolvase (Mpr) inhibitors

Holliday junction resolvase plays an important role in poxvirus genome replication and viral maturation. Recently, we developed the first in vitro assay measuring the activity of orthopoxvirus resolvases, particularly monkeypox virus resolvase (Mpr). Screening of our in-house compound library identified metal-chelating chemotypes as resolvase inhibitors, including 3-hydroxypyrimidine-2,4-dione (HPD). Herein we report the synthesis and Mpr-based structure-activity relationship (SAR) of 37 new HPD analogs. The SAR focused on the effects of various arylamino groups at C-6 and N-1 methyl substitution. Importantly, potency is improved by an electron-withdrawing group at the meta-position, or incorporation of oxazole or phenyl at the para-position, of the phenyl substituent. Methylating the N-1 position largely abrogated Mpr inhibitory activity. These efforts identified multiple analogs potently inhibiting purified Mpr in the nM range. All newly synthesized HPD analogs were also tested in a cell-based antiviral assay against a Gaussia luciferase (GLuc) reporter vaccinia virus (VACV). Despite their nM potency in the in vitro assay, most new analogs produced a modest 2-5-fold of Gluc reduction at 10 μM. The lack of strong correlation between antiviral potency and in vitro activity is consistent with poor permeability as measured in the PAMPA.