Phosphonamidate Prodrugs of a Butyrophilin Ligand Display Plasma Stability and Potent Vγ9 Vδ2 T Cell Stimulation

Abstract

Small organophosphorus compounds stimulate Vγ9 Vδ2 T cells if they serve as ligands of butyrophilin 3A1. Because the most potent natural ligand is ( E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), which is the last intermediate in bacterial biosynthesis of isoprenoids that is not found in mammalian metabolism, activation of these T cells represents an important component of the immune response to bacterial infections. To identify butyrophilin ligands that may have greater plasma stability, and clinical potential, we have prepared a set of aryl phosphonamidate derivatives (9a-i) of the natural ligand. Testing of these new compounds in assays of T cell response has revealed that this strategy can provide compounds with high potency for expansion of Vγ9 Vδ2 T cells (9f, EC = 340 pM) and interferon γ production in response to loaded K562 cells (9e, EC = 62 nM). Importantly, all compounds of this class display extended plasma stability ( t > 24 h). These findings increase our understanding of metabolism of butyrophilin ligands and the structure-activity relationships of phosphonamidate prodrugs.

Aryloxy Diester Phosphonamidate Prodrugs of Phosphoantigens (ProPAgens) as Potent Activators of Vγ9/Vδ2 T-Cell Immune Responses

“… 19 Some of these reactions are low yielding such as the oxidation of the side chain to introduce HMBP’s side chain, the hydroxyl group. 33 Notably, to date, no synthesis of HMBP difluoromethylene monophosphonate prodrugs has been reported.…”

“…An example of this is the aryloxy triester phosphoramidate prodrugs of the anticancer agent gemcitabine, where the benzyl ester phosphoramidates exhibit potent sub-micromolar potencies across four cancer cell lines compared to the isopropyl ester prodrugs, which had an IC 50 > 5 μM. 43 Additionally, HMBP methylene phosphoramidate prodrugs using glycine as an amino acid have been shown to exhibit sub-nanomolar activation of Vγ9/Vδ2 T-cells, 33 and this was the same with mixed aryl prodrugs of HMBP. 44 , 45 Notably, glycine phosphoramidates often generate less potent prodrugs compared to the l -alanine counterparts.…”

Stability and Efficiency of Mixed Aryl Phosphonate Prodrugs

“…The three compounds containing unmodified naphthyl substituents (compounds 5 , 12 , and 9 ) did show some toxicity at 100 μ m following a 72‐hour treatment (Figure ). This mild cytotoxicity is unlikely to be due to the free 1‐ or 2‐naphthol, as prior studies have shown no impact of those naphthols at 100 μ m …”

“…Other bis‐POM compounds, while used clinically, have limitations themselves with respect to plasma stability, so further investigations into other prodrug forms are important. While compound 2 remains the most efficient known phosphoantigen prodrug based on both speed and potency of triggering a T cell response, it does undergo rapid plasma metabolism that could limit in vivo applications …”

“…We also have applied the phosphonamidate strategy to produce phosphoantigen prodrugs such as compound 3 . Indeed, this prodrug form did improve plasma stability, resulting in potent compounds.…”