Synthesis of Phosphoramidate Prodrugs: ProTide Approach

Michaela Serpi1, Karolina Madela1, Fabrizio Pertusati1, Magdalena Slusarczyk1

1 School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, United Kingdom
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 15.5
DOI:  10.1002/0471142700.nc1505s53
Online Posting Date:  June, 2013
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Abstract

The ProTide (pronucleotide) approach is a prodrug strategy elaborated to deliver nucleoside monophosphate into the cell, circumventing the first and inefficient rate‐limiting phosphorylation step of nucleosides and improving the cellular penetration of nucleotides. The ProTide of a nucleoside phosphate is a phosphoramidate prodrug consisting of an amino acid ester promoiety linked via P‐N bond to a nucleoside aryl phosphate. Such prodrugs have increased lipophilicity and thus are capable of altering cell and tissue distribution. The ProTide technology was successfully and extensively applied to a wide variety of nucleoside phosphates, endowed with antiviral and anticancer activity. This unit describes two different synthetic strategies allowing the preparation of phosphoramidates of 6‐O‐methyl‐2′−β‐C‐methylguanosine as model compounds for nucleosides having only the 5′‐OH as reactive hydroxyl group, and phosphoramidates of 2′−β‐D‐arabinouridine (AraU) as model compounds for nucleosides containing two or more reactive hydroxyl groups. Curr. Protoc. Nucleic Acid Chem. 53:15.5.1‐15.5.15. © 2013 by John Wiley & Sons, Inc.

Keywords: ProTide; phosphoramidate nucleoside; nucleotide; antiviral and anticancer pro‐drugs

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of 6‐O‐Methyl‐2′−β‐C‐Methylguanosine 5′‐Protides
  • Basic Protocol 2: Preparation of 1‐(β‐D‐Arabinofuranosyl)Uracil‐5′‐Protides
  • Support Protocol 1: Synthesis of the Phosphorochloridate Intermediates
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 6‐O‐Methyl‐2′−β‐C‐Methylguanosine 5′‐Protides

  Materials
  • 1,2,3,5‐Tetra‐O‐benzoyl‐2‐C‐methyl‐β‐D‐ribofuranose ( 1) (Carbosynth Limited)
  • 2‐Amino‐6‐chloropurine (Carbosynth Limited)
  • Dry nitrogen (N 2) or Argon (Ar)
  • Anhydrous acetonitrile (Sigma‐Aldrich)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Sigma‐Aldrich)
  • Trimethylsilyl trifluoromethanesulfonate (TMS triflate; Sigma‐Aldrich)
  • Methanol (Fluka)
  • Chloroform (Fluka)
  • Sodium bicarbonate (Sigma‐Aldrich)
  • Dichloromethane (Fluka)
  • Anhydrous Na 2SO 4 (Sigma‐Aldrich)
  • Anhydrous methanol (MeOH) (Sigma‐Aldrich and Fluka)
  • Sodium methoxide (Sigma‐Aldrich)
  • Amberlite hydrogen form (Sigma‐Aldrich)
  • Silica gel (35‐70 µ, 60A; Fluka/Fisher)
  • Sand
  • Anhydrous tetrahydrofuran (THF; Sigma‐Aldrich)
  • Tert‐butylmagnesium chloride (tBuMgCl 1M in THF; Sigma‐Aldrich)
  • 12aor 12b as prepared in protocol 3
  • 50‐, 100‐, and 500‐mL round‐bottom flasks
  • Magnetic plate
  • Ice/H 2O bath
  • Oil bath
  • Analytical TLC plate (aluminum‐backed TLC plates, precoated with silica gel 60 F 254, 0.2 mm; Merck Kieselgel)
  • 1‐L separating funnel
  • Glass funnel
  • Filter paper
  • Rotary evaporator equipped with vacuum pump
  • Sintered glass funnel
  • Vacuum desiccator
  • pH paper (Fisher Brand)
  • Chromatography columns: 5 × 20–cm and 4.5 × 23–cm
  • UV light source

Basic Protocol 2: Preparation of 1‐(β‐D‐Arabinofuranosyl)Uracil‐5′‐Protides

  Materials
  • 1‐(β‐D‐Arabinofuranosyl)uracil ( 5) (Carbosynth Limited)
  • Anhydrous tetrahydrofuran (THF; Sigma‐Aldrich)
  • Anhydrous pyridine (Py)
  • N‐Methylimidazole (NMI) (Sigma‐Aldrich)
  • Argon
  • 12c or 12d as prepared in protocol 3
  • Dichloromethane (Fluka)
  • 0.5 M HCl
  • Anhydrous Na 2SO 4 (Sigma‐Aldrich)
  • Silica gel (35 to 70 µM)
  • Sand
  • Methanol (Fluka)
  • 50‐ and 500‐mL one‐neck round‐bottom flasks
  • Magnetic stirrer
  • 3 × 25–cm Chromatography columns
  • Analytical TLC plates (Aluminium backed TLC plates, precoated with silica gel 60 F 254, 0.2 mm; Merck Kieselgel)
  • UV light source
  • Rotary evaporator equipped with vacuum pump
  • 100‐mL separating funnel

Support Protocol 1: Synthesis of the Phosphorochloridate Intermediates

  Materials
  • L‐Alanine (Merck)
  • 2,2‐Dimethyl‐1‐propanol
  • p‐Toluenesulfonic acid (pTsOH; Sigma‐Aldrich)
  • Anhydrous toluene (Sigma‐Aldrich)
  • Anhydrous diethyl ether (Et 2O)
  • 2‐Propanol (Fluka)
  • Thionyl chloride (SOCl 2; Sigma‐Aldrich)
  • Dry nitrogen (N 2) or Argon (Ar)
  • Naphthol, ACS reagent grade (Sigma‐Aldrich)
  • Phosphorus oxychloride (POCl 3; Sigma‐Aldrich)
  • Anhydrous triethylamine (Et 3N; Sigma‐Aldrich)
  • Drierite or anhydrous calcium chloride (ACS reagent)
  • Phenyl dichlorophosphate ( 11) (Sigma‐Aldrich)
  • L‐Alanine benzyl ester p‐tosylate salt ( 8b) (Merck)
  • Anhydrous dichloromethane (CH 2Cl 2)
  • Silica gel (60 to 200 mesh; EMD)
  • Ethyl acetate (Fluka)
  • Hexane (Fluka)
  • 500‐, 250‐, and 100‐mL round‐bottom flasks
  • Dean‐Stark apparatus (Sigma‐Aldrich)
  • Glass‐sintered filter funnel (max pore size 16‐40 µm)
  • Magnetic stirring and heating plate
  • Vacuum pump
  • 500‐mL round‐bottom, two‐necked flasks
  • Condenser/airflux condenser
  • Ice/water bath
  • Rotary evaporator equipped with a vacuum pump
  • −78°C dry‐ice/acetone bath
  • UV light source
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Figures

Videos

Literature Cited

Literature Cited
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