Synthesis and Biological Evaluation of Phosphoester and Phosphorothioate Prodrugs of STING Agonist 3',3'-c-Di(2'F,2'dAMP)

J Med Chem. 2021 Jun 10;64(11):7596-7616. doi: 10.1021/acs.jmedchem.1c00301. Epub 2021 May 21.

Abstract

Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3',3'-c-di(2'F,2'dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2' position of 3',3'-c-di(2'F,2'dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Crystallography, X-Ray
  • Density Functional Theory
  • Esters / chemistry*
  • Esters / pharmacology
  • Esters / therapeutic use
  • HEK293 Cells
  • Humans
  • Interferon-gamma / metabolism
  • Leukocytes, Mononuclear / cytology
  • Leukocytes, Mononuclear / drug effects
  • Leukocytes, Mononuclear / metabolism
  • Magnetic Resonance Spectroscopy
  • Membrane Proteins / agonists*
  • Membrane Proteins / metabolism
  • Phosphates / chemistry*
  • Phosphates / metabolism
  • Phosphates / pharmacology
  • Phosphates / therapeutic use
  • Prodrugs / chemical synthesis*
  • Prodrugs / chemistry
  • Prodrugs / metabolism
  • Prodrugs / pharmacology
  • Tumor Necrosis Factor-alpha / metabolism

Substances

  • Esters
  • Membrane Proteins
  • Phosphates
  • Prodrugs
  • STING1 protein, human
  • Tumor Necrosis Factor-alpha
  • Interferon-gamma
  • thiophosphoric acid