Bioreducible Phosphonoamidate Pro-drug Inhibitor of Enolase: Proof of Concept Study

Abstract

Glycolysis inhibition remains aspirational in cancer therapy. We recently described a promising phosphonate inhibitor of enolase for cancers harboring homozygous deletions of ENO1. Here, we describe the application of a nitroheterocycle phosphonoamidate pro-drug pair to capitalize on tumor hypoxia. This bioreducible prodrug exhibits greater-than 2-fold potency under hypoxic conditions compared to normoxia and exhibits robust stability in biological fluids. Our work provides strong in vitro proof-of-concept for using bioreduction as a pro-drug delivery strategy in the context of enolase inhibition.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers

“…Compared to the POM phosphonoamidate counterparts (Table ), the higher potency of these mono-SATE prodrugs may suggest slightly more efficient intracellular cleavage of the first ester, perhaps due to the ability for SATE to be cleaved by multiple types of esterases beyond carboxylesterases, such as thioesterases . For nitroheterocycle prodrug 24 , we also observed dose-dependent selective toxicity in D423 cells, with an IC 50 value of 299 nM (Table , compound 24 , 21% O 2 ) . Consistent with previously reported nitroheterocycle prodrugs, imaging agents, and histological stains, − the activity of 24 improved under hypoxic conditions (1% O 2 ), yielding an IC 50 value of 136 nM in D423 cells and lower IC 50 values in non-target D423 ENO1 and LN319 cell lines (Table , compound 24 : 21% vs 1% O 2 ).…”

“…To phosphonoamidate intermediates 21a or 21b , we evaluated the efficacies of SATE (IDX-184-like), nitroheterocycle, cyanoethanol, 4-fluorophenol, and [1,4′-bipiperidine]-1′-carboxyl strategies (Table , compounds 22–27 ). The putative mechanisms of the initial mechanism of bioactivation for these prodrugs are, respectively, as follows: esterase (exact identity unknown), hypoxia, alkalinity (based on synthesis approaches for 2-cyanoethanol and 4-fluorophenol), and butyrylcholinesterase. − …”

“…After purification, the desired products were obtained in approximately 10–15% yield. The synthesis of nitroheterocycle prodrug 24 was previously reported . Alkaline-labile prodrugs 24 and 25 were prepared by monochlorination of intermediate 20b in neat phosphorous oxychloride for 30 min.…”

An enolase inhibitor for the targeted treatment of ENO1-deleted cancers

“…Pro-drug moieties with alternate mechanisms of bioactivation have been synthesized 13 and is an ongoing area of research in our laboratory. One promising pro-drug strategy capitalizes on elevated expression of nitroreductases under hypoxic conditions 54 , 55 , a defining characteristic of many tumors such as GBM 56 . Such application to HEX could result in a more favorable distribution of Enolase inhibition in tumor versus surrounding tissues.…”

“…Such application to HEX could result in a more favorable distribution of Enolase inhibition in tumor versus surrounding tissues. Future work will be directed towards evaluating this strategy and other bioactivation strategies as alternative pro-drug approaches 55 , 57 , 58 .…”