Efficiency of bis-amidate phosphonate prodrugs

Abstract

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.

Keywords: Butyrophilin; Isoprenoid; Ligand; Phosphoantigen; Phosphonamidate prodrug.

Figure 1.. Natural phosphoantigens, a synthetic analog, and various prodrug forms.

HMBPP (1) is the prototypical natural phosphoantigen (pAg). IPP (2) and DMAPP (3) are ubiquitous but weaker natural phosphoantigens. C-HMBP (4) is a synthetic analog of HMBPP (1). Various prodrug forms of C-HMBP (5-8) designed to improve its potency, including bis-amidates (8, this work).

Figure 2.. Stimulation of human γδ T cell cytokine production by compound-loaded K562 cells.

K562 cells were loaded with test compounds 16 and 17, washed, then exposed to γδ T cells. The amount of interferon γ produced by the T cells was quantified by ELISA (n=3).

Scheme 1.. Synthesis of bis-amidates 11-17.

Reagents and conditions: a) TMSBr, pyridine, 0 °C to RT, overnight; b) amino acid ester hydrochloride salt, PPh₃, 2,2′-dithiodipyridine, pyridine, 60 °C, overnight; c) SeO₂, 70% aqueous t-BuOOH, pyridine, methanol, 0 °C to RT, overnight.