Synthesis, Hydrolysis, and Protonation-Promoted Intramolecular Reductive Breakdown of Potential NRTIs: Stavudine α-P-Borano-γ-P-N-L-tryptophanyltriphosphates.

Zhihong Xu,Barbara Ramsay Shaw

doi:10.3390/molecules201018808

Zhihong Xu, Barbara Ramsay Shaw

Open Access

https://doi.org/10.3390/molecules201018808

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Journal: Molecules (Basel, Switzerland)	Publication Date: Oct 16, 2015
Citations: 33	License type: CC BY 4.0

Affiliation: Duke University

Abstract

Phosphorus-modified prodrugs of dideoxynucleoside triphosphates (ddNTPs) have shown promise as pronucleotide strategies for improving antiviral activity compared to their parent dideoxynucleosides. Borane modified NTPs offer a promising choice as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, the availability of α-P-borano-γ-P-substituted NTP analogs remains limited due to challenges with synthesis and purification. Here, we report the chemical synthesis and stability of a new potential class of NRTI prodrugs: stavudine (d4T) 5′-α-P-borano-γ-P-N-l-tryptophanyltriphosphates. One-pot synthesis of these compounds was achieved via a modified cyclic trimetaphosphate approach. Pure Rp and Sp diastereomers were obtained after HPLC separation. Based on LC-MS analysis, we report degradation pathways, half-lives (5–36 days) and mechanisms arising from structural differences to generate the corresponding borano tri- and di-phosphates, and H-phosphonate, via several parallel routes in buffer at physiologically relevant pH and temperature. Here, the major hydrolysis products, d4T α-P-boranotriphosphate Rp and Sp isomers, were isolated by HPLC and identified with spectral data. We first propose that one of the major degradation products, d4T H-phosphonate, is generated from the d4T pronucleotides via a protonation-promoted intramolecular reduction followed by a second step nucleophilic attack. This report could provide valuable information for pronucleotide-based drug design in terms of selective release of target nucleotides.

Highlights

Occurring or chemically synthesized nucleoside analogs have been used as anti-viral and anti-tumor drugs for decades
Mechanistic studies show that a dideoxynucleoside must be activated in a series of phosphorylation reactions to the corresponding 5′-triphosphate active form, which is incorporated as a chain terminator by a viral reverse transcriptase into the viral
By contrast, when water was directly added, most compounds 5 decomposed, probably due to the increased acidity. This was in agreement with the reports [29,40] that the P–N bond of nucleoside N-alkylphosphoramidates was chemically cleaved in acidic solutions

Summary

Introduction

Occurring or chemically synthesized nucleoside analogs have been used as anti-viral and anti-tumor drugs for decades. Several nucleoside analogs such as AZT and d4T are widely used in the treatment of AIDS patients, but major concerns such as drug toxicity, ineffectiveness and resistance resulting from inefficient phosphorylation by intrinsic kinases after cell penetration are often associated. To overcome such disadvantages, various nucleotides and other modified analogs have been studied as potential nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). In our first synthesis [35] of α-P-borano- and thio- modified nucleoside γ-P-N-substituted triphosphate mimics via cyclic trimetaphosphate intermediates, the isolation of the respective Rp and Sp diastereomers in their triethylammonium salt (TEA salt) form was successfully achieved after HPLC purification. The prodrug degradation pathways and mechanisms imparted by the structural differences are proposed for the first time

Synthesis and Isolation

Stability Studies

General Information

LC-MS Analysis of Degradation Products from d4T Triphosphate Mimics

Spectroscopic Data

Conclusions