Moiety probably happens inside a stereochemistry-dependent manner, as only the downfield

Moiety probably occurs within a stereochemistry-dependent manner, as only the downfield 27.4 ppm peak decreased concurrently together with the appearance in the upfield 16 ppm peaks (Figure S7). To clarify whether phosphonoamidate prodrugs have been, in general, inefficiently cleaved on five or whether this was specific to bis-amidate and McGuigan techniques, we synthesized a mono-POM, isopropyl L-alaninate prodrug (20o, Table 2) and evaluated this compound in our three-cell line system. The synthesis of phosphonoamidate POM esters 20b-20o was described previously.32 Treatment with 20o for 6 days resulted in dose-dependent, selective toxicity against D423 cells, with an IC50 worth of 202 nM. Resulting from the ubiquitous expression of esterases, we reasoned that the order of promoiety removal would 1st be the POM group followed by the isopropyl Ldoi.org/10.1021/acs.jmedchem.2c01039 J. Med. Chem. 2022, 65, 13813-Journal of Medicinal Chemistry Table two. SAR of POM Phosphonoamidate Prodrugs of 5apubs.acs.org/jmcArticlea cData are presented as the mean of N two. Full structures are readily available inside the Supporting Information. bValues calculated from Chemicalize. Prodrug 20p includes only dodecylamine and no POM ester.alaninate. There is certainly extensive literature on the mechanism of amidate hydrolysis on phosphonate- and phosphate-containing nucleotide analogues. Whereas phosphoramidates like sofosbuvir and remdesivir are mainly cleaved by the histidine nucleotide triad-binding protein 1 (HINT1), phosphonoamidates including TAF and GS-9191 mainly undergo lysosomal/ acid hydrolysis (Figure 4). 31,33-37 By comparing the mechanisms of bioactivation for 20o and 17 and 18, we reasoned that the inefficient bioactivation of 17 and 18 probably stems in the requisite intramolecular cyclization step common to McGuigan prodrugs. Compared to other McGuigan phosphonoamidates for example TAF and GS-9131 which both contain a mono-substituted C phosphonate5 includes a di-substituted C phosphonate. Higher-order substitution at C on 5 most likely impedes each the rate of intramolecular cyclization required to displace the phenol as well as the subsequent ring-opening hydrolysis.B18R Protein MedChemExpress Beyond nucleotide analogues, efficient bioactivation of McGuigan phosphonoamidate prodrugs has also been observed on phospho(no)antigens containing the phosph(on)ates on mono-substituted C, supporting the influence of sterics around the bioactivation of McGuigan prodrugs.CD59 Protein Species 38,39 The proposed causes for inefficient bioactivation are additional substantiated by the uniformly poor development inhibition observed for both 17 and 18 inside the NCI-60 cell line panel, which suggests that part of their bioactivation is non-enzymatic and independent of cell line-specific expression of prospective phosphonoamidate-hydrolyzing enzymes (Figures 3, S6).PMID:23672196 The intracellular metabolism of 20o is distinct from McGuigan prodrugs 17 and 18, which most likely explains the moreefficient prodrug bioactivation as well as the resulting lower IC50 worth in D423 cells. The activity of 20o against D423 cells prompted our exploration in to the ability for amines beyond amino acids to serve as second promoieties. Inspired by SAR research toward the anti-hepatitis C nucleotide prodrug, IDX-184, we first examined the activity of a POM/benzylamine prodrug 20b in our three-cell line method (Table 2).32,40 The synthesis of 20b and this series of amine prodrugs was reported previously.32 Briefly, phosphonoamidate intermediates have been reliably generated utilizing a modified Mitsunobu coupling amongst benzylated.