Ing electron-withdrawing groups in the C or C could increase its susceptibility to hydrolysis. In contrast to 25, 4-fluorophenol phosphonoamidate 26 was inert and didn’t exhibit meaningful cell killing in our three-cell line system for the duration on the experiment (Table 1). Ultimately, the irinotecan-like [1,4-bipiperidine]-1-carboxyl phosphonoamidate 27 did not exhibit meaningful activity against D423 cells below 15 M (Table 1). We also synthesized and evaluated non-phosphonoamidatebased prodrugs of five. These included a lipid prodrug 28, a biscyanoethyl prodrug 29, and salicylic alcohol (“cycloSal”) 30 prodrugs (Table 1, entries 19-21). The proposed mechanisms of action for these prodrugs are as follows: phospholipase and alkalinity.52-55 Lipid prodrug 28 was synthesized by N,Ndicyclohexylcarbodiimide coupling amongst intermediate 7 and 3-(hexadecyloxy)propan-1-ol, followed by the palladiumcatalyzed hydrogenation from the solution precursor. Lipid prodrug 28 was obtained as an off-white waxy solid in 36 yield. Bis-cyanoethyl prodrug 29 was synthesized by reacting 2cyanoethanol with dichlorinated 7, followed by the palladiumcatalyzed hydrogenation (72 yield over 3 measures). Finally, cycloSal prodrug 30 was synthesized by isobutyrylation of 5, followed by thionyl chloride-mediated dichlorination of intermediate 9 and reaction with salicylic alcohol (39-65 yield more than three steps). Treatment with 28 in our three-cell line panel resulted in dose-dependent, selective killing of Ddoi.org/10.1021/acs.jmedchem.2c01039 J. Med. Chem. 2022, 65, 13813-Journal of Medicinal Chemistrypubs.acs.org/jmcArticleFigure 5. Activity and stability of a cycloSal prodrug of 5. A cycloSal prodrug of 5 is hydrolyzable under biological conditions. (A) Proposed mechanism of bioactivation of 30; the 2-(hydroxymethyl)phenol (cycloSal) prodrug is indicated in blue. (B) Activity of 30 in D423 cells at different oxygen levels. Whilst 30 exhibits a slight reduce in potency below hypoxic circumstances (red), it really is roughly 40-fold additional potent below hyperoxic situations (blue).EGF, Mouse Across all three conditions, cells have been treated with 30 for 6 days.FLT3LG, Human (HEK293, His) Information are plotted relative to untreated controls and are the imply SD of N two. (C) Stability of 30 (two mM, 1:1 ratio of Sp and Rp isomers) in 80 human plasma, 20 D2O as measured by 31P NMR spectroscopy. Peaks have been integrated and normalized relative towards the phosphate peak at 0 ppm. (D) Time course 31P NMR traces of 30 in human plasma. Intact 30 seems as a broad peak at 21.1 ppm inside the human plasma/D2O resolution and is completely hydrolyzed to five (13.four ppm) soon after 3 h.PMID:35954127 (E) 1H-31P HSQC spectra of intact 30 at time 0 (major) and at 72 h (bottom) with proton resonances on the structure indicated in addition to a focused view from the resonances in the boxed region (major). Top: protons highlighted in light blue correspond to the downfield benzylic protons at five.three and five.five ppm. The two x-axis proton resonances correspond to the broad y-axis peak at 21.1 ppm due to the presence of Sp and Rp isomers. Highlighted in dark blue around the structure will be the C proton, which corresponds to the upfield proton resonance at two.2 ppm. Bottom: hydrolysis of 30 to 5 is total soon after three h. The 72 h spectrum shows that five could be the only species detectable in human plasma, as indicated by the C proton resonance at two.six ppm, which can be highlighted in blue around the structure.cells, with an IC50 worth of 207 nM. Lipid prodrug 28 was submitted for additional evaluation within the NCI-60 cell line panel.
