In order to discover new ³¹P NMR markers for probing subtle pH changes (<0.2 pH unit) in biological environments, fifteen new conformationally constrained or sterically hindered α-aminophosphonates derived from diethyl(2-methylpyrrolidin-2-yl)phosphonate were synthesized and tested for their pH reporting and cytotoxic properties in vitro. All compounds showed near-neutral pK_as (ranging 6.28–6.97), chemical shifts not overlapping those of phosphorus metabolites, and spectroscopic sensitivities (i.e., chemical shifts variation Δδ_ab between the acidic and basic forms) ranging from 9.2–10.7 ppm, being fourfold larger than conventional endogenous markers such as inorganic phosphate. X-ray crystallographic studies combined with predictive empirical relationships and ab initio calculations addressed the inductive and stereochemical effects of substituents linked to the protonated amine function. Satisfactory correlations were established between pK_as and both the 2D structure and pyramidalization at phosphorus, showing that steric crowding around the phosphorus is crucial for modulating Δδ_ab. Finally, the hit ³¹P NMR pH probe 1b bearing an unsubstituted 1,3,2-dioxaphosphorinane ring, which is moderately lipophilic, nontoxic on A549 and NHLF cells, and showing pK_a = 6.45 with Δδ_ab = 10.64 ppm, allowed the first clear-cut evidence of trans-sarcolemmal pH gradients in normoxic Dictyostelium discoideum cells with an accuracy of <0.05 pH units.

Glioblastoma is the most common cerebral tumor in adults. The median survival of glioblastoma patients is 12 months.

Glioblastoma is one of the most highly aggressive types of brain tumor in adults. With limited treatment options, current therapies remain insufficient due to its invasiveness and immune evasion, highlighting the urgent need for new treatments. Bifunctional molecules targeting multiple aspects of the disease could be promising to overcome drug resistance and tumor heterogeneity. Metformin has demonstrated protective effects against brain tumors but requires high doses for efficacy, making it of great interest for molecular optimization. In this context, we synthesized a series of nine metformin–phenolic molecules, combining the metformin guanidine framework with phenolic acids, which have well-established properties in inhibiting cancer cell migration and adhesion. Their impact on cytotoxicity, reactive oxygen species inhibition, and signaling pathways was investigated for glioma cell lines and stem cells. Two of these hybrids, 5a and 5h, particularly enhanced cytotoxicity in glioblastoma cells, selectively targeting cancer cells while sparing healthy ones. Their mechanism of action differed significantly from metformin. Unlike metformin, which mainly triggers metabolic stress, the hybrids broadly inhibit RTK–MAPK–PI3K signaling, leading to cell cycle arrest and apoptosis. The results suggest that these compounds could offer a more effective and synergistic approach for glioblastoma treatment.