Evaluation of a 7‐Methoxycoumarin‐3‐carboxylic Acid Ester Derivative as a Fluorescent, Cell‐Cleavable, Phosphonate Protecting Group

Abstract

Cell-cleavable protecting groups often enhance cellular delivery of species that are charged at physiological pH. Although several phosphonate protecting groups have achieved clinical success, it remains difficult to use these prodrugs in live cells to clarify biological mechanisms. Here we present a strategy that uses a 7-methoxycoumarin-3-carboxylic acid ester as a fluorescent protecting group. This strategy is applied to synthesis of an HMBPP analogue to assess cellular uptake and human Vγ9Vδ2 T cell activation. The fluorescent ester displays low cell toxicity (IC50 >100 μM) and strong T cell activation (EC50 = 0.018 μM) relative to the unprotected anion (EC50 = 23 μM). The coumarin-derived analogue allows no-wash analysis of biological deprotection, which reveals rapid internalization of the prodrug. These results demonstrate that fluorescent groups can be applied both as functional drug delivery tools and useful biological probes of drug uptake.

Phosphinophosphonates and Their Tris-pivaloyloxymethyl Prodrugs Reveal a Negatively Cooperative Butyrophilin Activation Mechanism

“…It should be noted this value is higher than other values reported for HMBPP because the two hour exposure does not allow for maximum uptake of charged phosphoantigens. 21, 28 In this assay, compound 10 triggered lysis with an EC 50 of 41 µM, while its tris-POM analog 4 triggered lysis with a 0.28 µM EC 50 , representing a 150-fold increase in cellular potency. Compound 11 also demonstrated activity in this assay, with an EC 50 of 7.3 µM.…”

“…compound 2 ) potently trigger cellular activation of Vγ9Vδ2 T cells. 10, 21 However, when drugs are designed to interact with phosphate binding sites, it is often desirable to deliver a diphosphate analog rather than a monophosphate analog to avoid the need for cellular phosphorylation (i.e. kinase bypass).…”

The Role of the Phosphorus Atom in Drug Design

“…[127] Therefore, evaluation of cellular uptake and human Vg9Vd2T cell activation by compounds 140-143 was very remarkable. In fact, 140 was found to be am etabolically stable analogueo ft he natural agonist 139.I nterestingly,t he prodrug 140 exhibited improved cellular potencyt han the free monophosphonate 141, [128,130,131] whereas the tris-POMp rodrug 142,w hich is am etabolically stable and close structural relative to 139,s howedi ncreased stimulatory effects on Tcell proliferation, being two orders of magnitude more potent than the free salt 143. [131] Mixed aryl phosphonate prodrugs such as 144-149 were found to be potent agonists of butyrophilin,a cting in the sub-nanomolar range.…”

Synthesis of phosphoantigens and chiral trisphosphonates

“…Figure 23. Half-life of CCOM ester cleavage 40,44 Once CCOM phosphonate esters were shown to be cleavable by non-specific esterases, these new compounds also were examined for their ability to stimulate Vγ9Vδ2…”

“…Expansion of Vγ9Vδ2 T-cells 31 Table 5. Stimulation Vγ9Vδ2 T-cells of novel phosphonates 40,44 Table 7. Vγ9Vδ2 T-cells proliferation and lysis assays 19,31 Phosphorus is a vital element required for life that is used for numerous processes in the body, along with carbon, hydrogen, nitrogen, oxygen, and sulfur.…”