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

Abstract

Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum (t_1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC₅₀ ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro. Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.

Figure 1

Chemical structure of reported small molecule Vγ9/Vδ2 T-cell activators; HMBPP (EC₅₀: 60–500 pM),⁻ IPP (EC₅₀: 1–10 μM),^, risedronate (EC₅₀: 0.08–5 μM),^, and zoledronate (EC₅₀: 0.003–0.5 μM) [this work and Davey et al.(20)]

Figure 2

Chemical structures of HMBP ProPAgens (previous work) and the ProPAgens of their phosphonate derivatives (this work) as well as a general indication of their stability and Vγ9/Vδ2 T-cell activation.

Figure 3

pK_a values of phosphate and different phosphonate groups.

Scheme 1

Reagents and conditions: (i) TMSBr, DCM, rt, 2 h then (COCl)₂, DMF cat, DCM, rt, 18 h; (ii) a. Phenol, Et₃N, DCM, −78 °C for 30 min then rt, 3 h; b. Substituted l-alanine ester hydrochloride, Et₃N, DCM, rt, 12 h, yields: 38–61%; (iii) 2-methyl-2-propenol, 1,4-benzoquinone, Hoveyda–Grubbs catalyst 2nd generation, DCM, rt, yields: 57–64%; (iv) diethyl (bromodifluoromethyl)phosphonate, DMF, zinc powder, rt, N₂, 3 h then CuBr, allyl bromide, rt, 40 h; (v) (i) TMSBr, DCM, rt, 2 h then (COCl)₂, DMF cat, DCM, rt, 18 h; (ii) a. Phenol, Et₃N, DCM, −78 °C for 30 min then rt, 3 h; b. Substituted l-alanine ester hydrochloride, Et₃N, DCM, rt, 12 h, yields: 24–46%; and (iii) 2-methyl-2-propenol, 1,4-benzoquinone, Hoveyda–Grubbs Catalyst 2nd generation, DCM, rt, yields: 58–69%.

Figure 4

Stability of HMBP monophosphonate ProPAgen 9a in human serum at 37 °C for 12 h as monitored by ³¹P NMR.

Figure 5

In vitro carboxypeptidase-mediated breakdown of HMBP phosphonate ProPAgen 9b. (a) Postulated mechanism of aryloxy diester prodrugs metabolism. (b) ³¹P NMR spectrum of ProPAgen 9b alone and at different time points, following incubation with recombinant carboxypeptidase Y at 37 °C for 15 h.

Figure 6

In vitro ProPAgen-mediated activation of Vγ9/Vδ2 T-cells, following overnight incubation with HMBPP, zoledronate, and HMBPP ProPAgens. Levels of activation are represented as % of Vγ9/Vδ2 T-cells that are CD69+ CD25+. Data is shown as mean ± SE (n = 4). (A) Activation of Vγ9/Vδ2 stimulated by ProPAgens 4a–d. (B) Activation of Vγ9/Vδ2 stimulated by ProPAgens 9a–d. (C) Activation of Vγ9Vδ2 stimulated by HMBPP and zoledronate. (D) EC₅₀ values calculated based on the results of the activation assay. CLogP values were calculated using ChemDraw.

Figure 7

Cytotoxicity of Vγ9/Vδ2 T-cells toward ProPAgen-treated T24 cells. % killing represents % GFP + eFluor780+ cells (i.e. dead T24 cells). (A) Cytotoxicity of Vγ9/Vδ2 T-cells toward T24 cells treated with ProPAgens 4c, 4d, 9c, and 9d at different effector/target ratios. Ratio 0:1 refers to the basal effect of ProPAgens on T24 cells in the absence of effectors. (B,C) Cytotoxicity of ProPAgens 4c, 4d, 9c, and 9d (used at 10 pM) compared to the 10 pM HMBPP-induced effect after subtracting the basal cytotoxic effect of Vγ9/Vδ2 on untreated T24 cells. Data is shown as mean ± SE (n = 3). Statistical analysis was performed using one-way ANOVA and Tukey’s multiple comparisons test. *p < 0.05.