Single Diastereomers of the Clinical Anticancer ProTide Agents NUC-1031 and NUC-3373 Preferentially Target Cancer Stem Cells In Vitro

Abstract

A 3′-protected route toward the synthesis of the diastereomers of clinically active ProTides, NUC-1031 and NUC-3373, is described. The in vitro cytotoxic activities of the individual diastereomers were found to be similar to their diastereomeric mixtures. In the KG1a cell line, NUC-1031 and NUC-3373 have preferential cytotoxic effects on leukemic stem cells (LSCs). These effects were not diastereomer-specific and were not observed with the parental nucleoside analogues gemcitabine and FUDR, respectively. In addition, NUC-1031 preferentially targeted LSCs in primary AML samples and cancer stem cells in the prostate cancer cell line, LNCaP. Although the mechanism for this remains incompletely resolved, NUC-1031-treated cells showed increased levels of triphosphate in both LSC and bulk tumor fractions. As ProTides are not dependent on nucleoside transporters, it seems possible that the LSC targeting observed with ProTides may be caused, at least in part, by preferential accumulation of metabolized nucleos(t)ide analogues.

Synthesis and Characterization of NUC-7738, an Aryloxy Phosphoramidate of 3′-Deoxyadenosine, as a Potential Anticancer Agent

“…Our attempts to separate the diastereoisomers of ProTides 7a (NUC-7738) (selected compound from this study) via direct phase silica gel column chromatography were ineffective due to the very close retention times of both isomers on silica. Attempts to separate 3′-dA phosphoramidate diastereoisomers bearing different silane protecting groups at the 2′ position, as previously reported for ProTides of different nucleosides, were also unsuccessful. This protection strategy, although ineffective for the separation of the two diastereoisomers, proved to be a more efficient way to prepare 7a , with an overall yield of 42% .…”

“…We have recently reported on the preferential targeting of leukemic stem cells (LSCs) by the gemcitabine ProTide, NUC-1031, in the acute myeloid leukemia cell line KG1a and in primary AML blasts . Therefore, once we evaluated the LC 50 of ProTide 7a and 7b in KG1a cells (Table S2), we went on to assess the relative ability of ProTides 7a and 7b to preferentially kill the LSCs compared to the bulk tumor within the KG1a cell line (LSCs were defined by the phenotype Lin – /CD34 + /CD38 – /CD123 + ) .…”

“…We have recently reported on the preferential targeting of leukemic stem cells (LSCs) by the gemcitabine ProTide, NUC-1031, in the acute myeloid leukemia cell line KG1a and in primary AML blasts. 62 Therefore, once we evaluated the LC 50 of ProTide 7a and 7b in KG1a cells ( Table S2 ), we went on to assess the relative ability of ProTides 7a and 7b to preferentially kill the LSCs compared to the bulk tumor within the KG1a cell line (LSCs were defined by the phenotype Lin – /CD34 + /CD38 – /CD123 + ). 67 While ProTide 7b did not lead to any significant alteration of the proportion of LSCs across the entire range of concentrations, 3′-dA and ProTide 7a showed increased selectivity and potency against the putative leukemic stem cells with 7a significantly reducing the LSC population to a greater extent than the parent nucleoside at concentrations above 1 μM ( Figure 8 ).…”

Advances in the Synthesis and Analysis of Biologically Active Phosphometabolites

“…The phosphoramidate prodrug approach, which was named as ProTide approach, was developed by the McGuigan group and uses the masking of the monophosphate or monophosphonates of nucleoside analogues for efficiently delivering into cells where, after cleavage by intracellular enzymes, the bioactive free nucleoside monophosphates and monophosphonates are released [ 55 ]. The application of this approach continues to be of interest not only for antiviral and anti-cancer nucleoside analogues [ 56 ], but also for further extensions to non-nucleoside analogues [ 57 ].…”

Design, Synthesis, and Anti-Cancer Evaluation of Novel Cyclic Phosphate Prodrug of Gemcitabine

“…16 Recent studies demonstrated that NUC-1031 has preferential cytotoxic effects on leukemic and prostate cancer stem cells. 22 However, a phase III study of NUC1031 as a monotherapy versus gemcitabine for patients with metastatic pancreatic cancer is recently discontinued for efficacy-related futility (NCT03610100). 26 Cyclic phosphate ester prodrug is another widely used strategy for designing nucleoside analogue prodrugs, and several cyclic phosphate ester prodrugs have been reported, such as PSI352938, SATE-cMP prodrug 18, and so on (Figure 2).…”