Generation of Stable Isopentenyl Monophosphate Aryloxy Triester Phosphoramidates as Activators of Vγ9Vδ2 T Cells

Abstract

Aryloxy triester phosphoramidate prodrugs of the monophosphate derivatives of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) were synthesized as lipophilic derivatives that can improve cell uptake. Despite the structural similarity of IPP and DMAPP, it was noted that their phosphoramidate prodrugs exhibited distinct stability profiles in aqueous environments, which we show is due to the position of the allyl bond in the backbones of the IPP and DMAPP monophosphates. As the IPP monophosphate aryloxy triester phosphoramidates showed favorable stability, they were subsequently investigated for their ability to activate Vγ9/Vδ2 T cells and they showed promising activation of this subset of T cells. Together, these findings represent the first report of IPP and DMAPP monophosphate prodrugs and the ability of IPP aryloxy triester phosphoramidate prodrugs to activate Vγ9/Vδ2 T cells highlighting their potential as possible immunotherapeutics.

Keywords: DMAPP; IPP; Phosphoramidates; Prodrugs; T-cells.

Figure 1

A. Chemical structures of Zoledronate (1), IPP (2) and DMAPP (3). B. Chemical structures and IPP and DMAPP monophosphate derivatives 4 and 5, which are used in this study.

Figure 2

Synthesis of IPP and DMAP monophosphate aryloxytriester phosphoramidates 8  a‐d and 9  a‐d. Reagents and conditions: (i) phenyl dichlorophosphate 6, TEA, DCM, −78 °C. 30 min then room temperature 3 h (ii) 3‐methylbut‐3‐en‐1‐ol, DCM, TEA, 16 h, 5–25 % over two steps; (iii) or 3‐methylbut‐2‐en‐1‐ol, DCM, TEA, 16 h, 5–25 % over two steps.

Figure 3

Stability of IPP and DMAPP monophosphate derivatives aryloxytriester phosphoramidates in acid. A. ³¹P NMR of compound 8  d in acidic buffer (pH=1) at 37 °C for 12 h. B. ³¹P NMR of compound 9  d in acidic buffer (pH=1) at 37 °C for 12 h.

Figure 4

A. Proposed mechanism of 9  d breakdown in aqueous acidic buffer. B. Scheme showing the possible mechanism by which strong nucleophiles mediate the breakdown of 9  d.

Figure 5

³¹P NMR of the reaction involving compound 9  d and the nucleophile O,O‐diethyl thiophosphate after 1 h.

Figure 6

IPP monophosphate aryloxytriester phosphoramidates‐mediated activation of Vγ9/Vδ2 T‐cells. Levels of activation are measured as the % of Vγ9/Vδ2 T‐cells expressing both CD69 and CD25 following overnight incubation with 100 μM (A) or 30 μM (B) of IPP monophosphate aryloxytriester phosphoramidates, Zoledronate and HMBPP. Data is shown as mean±SEM (n=3).