Symmetrical Diamidate Prodrugs of Nucleotide Analogues for Drug Delivery

Fabrizio Pertusati1, Christopher McGuigan1, Michaela Serpi1

1 School of Pharmacy and Pharmaceutical Sciences, Cardiff University, King Edward VII Avenue, Cardiff
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 15.6
DOI:  10.1002/0471142700.nc1506s60
Online Posting Date:  March, 2015
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The use of pronucleotides to circumvent the well‐known drawbacks of nucleotide analogs has played a significant role in the area of antiviral and anticancer drug delivery. Several motifs have been designed to mask the negative charges on the phosphorus moiety of either nucleoside monophosphates or nucleoside phosphonates, in order to increase their hydrophobicity and allow entry of the compound into the cell. Among them the bis‐amidate analogs, having two identical amino acids as masking groups through a P–N bond, represent a more recent approach for the delivery of nucleotide analogs, endowed with antiviral or anticancer activity. Different synthetic strategies are commonly used for preparing phosphorodiamidates of nucleosides. In this protocol, we would like to focus on the description of the synthetic methodology that in our hand gave the best results using 2′‐3′‐didehydro‐2′‐3′‐dideoxythymidine (d4T, Stavudine) as model nucleoside. A second strategy for preparing diamidates of nucleoside phosphonates will be reported using {[2‐(6‐amino‐9 H‐purin‐9‐yl)ethoxy]methyl}phosphonic acid (PMEA, adefovir) as model substrate. © 2015 by John Wiley & Sons, Inc.

Keywords: phosphorodiamidates; nucleoside; phosphonate; acyclic nucleoside phosphonates (ANP); antiviral; bis‐amidate

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Bis‐Amidate Prodrugs of 2′,3′‐Didehydro‐2′,3′‐Dideoxythymidine
  • Basic Protocol 2: Synthesis of Bis‐Amidate Prodrugs of {[2‐(6‐Amino‐9 H‐Purin‐9‐YL)Ethoxy]Methyl}Phosphonic Acid
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of Bis‐Amidate Prodrugs of 2′,3′‐Didehydro‐2′,3′‐Dideoxythymidine

  • 2‐3‐didehydro‐2‐3‐dideoxythymidine (d4T, Stavudine), 1
  • Anhydrous tetrahydrofuran (THF; Sigma‐Aldrich)
  • Argon (Ar)
  • Triethylamine (Et 3N; Sigma‐Aldrich)
  • Phosphoryl chloride (POCl 3; Sigma‐Aldrich)
  • CDCl 3
  • Anhydrous dichloromethane (CH 2Cl 2) (Sigma‐Aldrich)
  • (L)‐Alanine cyclohexyl ester p‐toluensulfonate (see Support Protocol in unit 15.5; Serpi et al., )
  • (L)‐Alanine 2,2‐dimethylpropylester p‐toluensulfonate (see Support Protocol in unit 15.5; Serpi et al., )
  • Water HPLC grade (Fisher)
  • Brine
  • Anhydrous MgSO 4
  • Silica gel (35 to 70 μm, 60 A; Fluka/Fisher)
  • Dichloromethane (CH 2Cl 2; Fluka)
  • Sand (Sigma‐Aldrich)
  • Methanol (Fluka)
  • Anhydrous MgSO 4 (Sigma‐Aldrich)
  • MeOH‐d 4 (Goss)
  • HPLC grade Acetonitrile
  • 25‐, 50‐, 100‐, and 500‐mL round‐bottom flasks
  • Magnetic stirrer plate
  • 250‐mL separating funnel
  • Glass funnel
  • Filter paper
  • Rotary evaporator equipped with vacuum pump (BUCHI)
  • Vacuum desiccator
  • Chromatography columns: 2.5 × 30–cm
  • Analytical TLC plate (aluminum‐backed TLC plates, precoated with silica gel 60 F254, 0.2 mm; Merck Kieselgel)
  • UV light source
  • Dry ice/Acetone bath
  • Varian Pursuit XRs 5 C18, 150 × 4.6 mm

Basic Protocol 2: Synthesis of Bis‐Amidate Prodrugs of {[2‐(6‐Amino‐9 H‐Purin‐9‐YL)Ethoxy]Methyl}Phosphonic Acid

  • Adefovir (Hubei Maxsource Chemical)
  • Anhydrous acetonitrile (Sigma‐Aldrich)
  • Argon
  • Trimethylsilyl bromide (TMSBr; Sigma‐Aldrich)
  • Triethylamine (Et 3N)
  • Anhydrous pyridine (Sigma‐Aldrich)
  • (L)‐Alanine 2,2‐dimethylpropylester p‐toluensulfonate (see the Support Protocol in unit 15.5; Serpi et al., )
  • Aldrithiol‐2 (Sigma‐Aldrich)
  • Triphenylphosphine (Sigma‐Aldrich)
  • Methanol (Fluka)
  • HPLC grade water (Fisher)
  • Toluene (Fluka)
  • Hexane (Fluka)
  • Ethyl acetate (Fluka)
  • MgSO 4
  • Dichloromethane (CH 2Cl 2; Fluka)
  • Silica gel (35 to 70 μm, 60 A; Fluka/Fisher)
  • Sand (Sigma‐Aldrich)
  • Anhydrous MgSO 4 (Sigma‐Aldrich)
  • Dry Argon (Ar)
  • 25‐, 50‐, 100‐, and 500‐mL round‐bottom flasks (Fisher)
  • Magnetic stirrer plate
  • Rotary evaporator equipped with vacuum pump (BUCHI)
  • 250‐mL separatory funnel
  • Glass funnel
  • Filter paper
  • Chromatography columns: 5 × 20–cm and 4.5 × 23–cm
  • Analytical TLC plate (aluminum‐backed TLC plates, precoated with silica gel 60 F254, 0.2 mm; Merck Kieselgel)
  • UV light source
  • Vacuum desiccator
  • Varian Pursuit XRs 5 C18, 150 × 4.6 mm
  • Oil bath
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Literature Cited

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