Synthesis and Biological Evaluation of ( E)-4-Hydroxy-3-methylbut-2-enyl Phosphate (HMBP) Aryloxy Triester Phosphoramidate Prodrugs as Activators of Vγ9/Vδ2 T-Cell Immune Responses

Abstract

The aryloxy triester phosphoramidate prodrug approach has been used with success in drug discovery. Herein, we describe the first application of this prodrug technology to the monophosphate derivative of the phosphoantigen HMBPP and one of its analogues. Some of these prodrugs exhibited specific and potent activation of Vγ9/Vδ2 T-cells, which were then able to lyse bladder cancer cells in vitro. This work highlights the promise of this prodrug technology in the discovery of novel immunotherapeutics.

Figure 1

Chemical structures of natural phosphoantigens HMBPP and IPP as well as synthetic molecules risedronate and zoledronate, which activate Vγ9/Vδ2 T-cells.

Scheme 1. (a) Synthesis of HMBP ProPAgens and (b) Synthesis of Aryl Phosphorochloridates

Part a, reagents and conditions: (a) TBSCl, imidazole, DCM, rt, yield 95%; (b) triethyl phosphonoacetate, NaH, THF, 0 °C, yield 50%; (c) LiAlH₄, THF, 0 °C, yield 40%; (d) 9a–d, TEA or NMI, DCM, yields 36–56%; (e) TBAF, THF; (f) HCl, MeOH, yields 20–74%. Part b, reagents and conditions: (g) Et₂O, TEA, −78 °C, yields 54–95%. Me, methyl; iPr, isopropyl; ^tBu, tert-butyl; Bn, benzyl.

Figure 2

Activation of human Vγ9/Vδ2⁺ T cells by HMBP ProPAgens. (a) Human peripheral blood mononuclear cells (PBMC) were incubated with media or indicated concentrations of HMB-PP or zoledronate for 18 h. TCR Vγ9/Vδ2⁺ T cells were then assessed for the upregulation of cell surface markers, CD69 and CD25. Data representative of n = 5. (b) As in (a), with PBMC incubated with indicated concentrations of HMBP ProPAgens. Data representative of n = 4. (c) As in (a) and (b), with data showing titrations of each of HMB-PP, zoledronate, and HMBP ProPAgens, alongside a medium control (M). Data from n = 4–5 donors. (d) EC₅₀, ED₅₀, and selectivity index (SI) values for each HMBP ProPAgens. Specific cell death, for ED₅₀ values, was calculated after adjusting for nonspecific cell death in medium controls. (e) CD69 and CD25 activation in CD3⁺ CD8⁺ αβ T-cells for each compound.

Figure 3

HMBP ProPAgens mediate the specific lysis of bladder cancer cells by Vγ9/Vδ2 T-cells. (a) Human T24 urinary bladder carcinoma cell lines (target) were pulsed for 4 h with 10 nM of the indicated ProPAgens, media, or 10 μM zoledronate, then cocultured for 18 h with expanded Vγ9/Vδ2 T cells (effector) at the indicated target/effector ratios. Specific killing of target cells was measured by amine-reactive dead cell marker staining. Data display the mean of n = 2. (b) As in (a) showing the mean ± SD specific killing of T24 cells at 1:10 target/effector ratio for each indicated treatment, n = 2: (∗) P < 0.05; (∗∗) P < 0.01; determined by one-way ANOVA.