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Bis-amidate derivatives have been viewed as attractive phosphonate prodrug forms because of their straightforward synthesis, lack of phosphorus stereochemistry, plasma stability and nontoxic amino acid metabolites. However, the efficiency of bis-amidate prodrug forms is unclear, as prior studies on this class of prodrugs have not evaluated their activation kinetics. Here, we synthetized a small panel of bis-amidate prodrugs of butyrophilin ligands as potential immunotherapy agents. These compounds were examined relative to other prodrug forms delivering the same payload for their stability in plasma and cell lysate, their ability to stimulate T cell proliferation in human PBMCs, and their activation kinetics in a leukemia co-culture model of T cell cytokine production. The bis-amidate prodrugs demonstrate high plasma stability and improved cellular phosphoantigen activity relative to the free phosphonic acid. However, the efficiency of bis-amidate activation is low relative to other prodrugs that contain at least one ester such as aryl-amidate, aryl-acyloxyalkyl ester, and bis-acyloxyalkyl ester forms. Therefore, bis-amidate prodrugs do not drive rapid cellular payload accumulation and they would be more useful for payloads in which slower, sustained-release kinetics are preferred., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Human Vγ9Vδ2 T cells respond to small phosphorus-containing compounds, often called phosphoantigens, which are now known to be intracellular ligands of the immune receptor butyrophilin 3A1 (BTN3A1). In order to compare the efficiency of butyrophilin ligands, we developed a luciferase-based lysis assay that measures the direct cytolysis by Vγ9Vδ2 T cells of luciferase-expressing K562 leukemia cells sensitized by phosphoantigen prodrugs. Our results show that the luciferase-based lysis assay allows in vitro and in vivo assessment of phosphoantigen activity in a way that does not require the extensive processing of flow cytometry or ELISA based approaches. In cellular assays, the structure activity relationships of phosphoantigen prodrugs correlate with ELISA-based activation assays, though phosphoantigen induced target cell lysis occurs at lower concentrations relative to T cell interferon γ production measured by ELISA. In mice dosed with phosphoantigens, a racemic aryl phosphonamidate prodrug, methyl 2-[[[(E)-5-hydroxy-4-methyl-pent-3-enyl]-(1-naphthyloxy)phosphoryl]amino]acetate (1-Nap/GlyOMe C-HMBP, 5), sensitized subcutaneous K562 tumors within minutes, and this effect was maintained at least four hours after treatment. In vivo activity of compound 5 was stronger than that of an equivalent dose of zoledronate. This luciferase lysis assay can be used for evaluation of phosphoantigens due to its time efficiency, high sensitivity, and in vivo compatibility and demonstrates rapid in vitro and in vivo sensitization of tumor cells by phosphoantigen prodrugs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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 50 = 340 pM) and interferon γ production in response to loaded K562 cells (9e, EC 50 = 62 nM). Importantly, all compounds of this class display extended plasma stability ( t 1/2 > 24 h). These findings increase our understanding of metabolism of butyrophilin ligands and the structure-activity relationships of phosphonamidate prodrugs.