Synthesis of Cidofovir and (S)‐HPMPA Ether Lipid Prodrugs

James R. Beadle1

1 University of California, San Diego, La Jolla
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 15.2
DOI:  10.1002/0471142700.nc1502s29
Online Posting Date:  June, 2007
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Abstract

Cidofovir [(S)‐1‐(3‐hydroxy‐2‐phosphonomethoxypropyl)cytosine] and (S)‐HPMPA [(S)‐9‐(3‐hydroxy‐2‐phosphonomethoxypropyl)adenine] are potent nucleoside phosphonate antiviral agents that are not orally bioavailable unless one or both of their negative charges are masked. This unit describes the synthesis of hexadecyloxypropyl esters of cidofovir and (S)‐HPMPA. These prodrugs are readily absorbed after oral administration and are converted intracellularly to the corresponding diphosphates. The hexadecyloxypropyl esters of cidofovir and (S)‐HPMPA are orally active in animal models of viral infection. Two synthetic strategies are employed. In the first, cyclic cidofovir is coupled to 3‐hexadecyloxy‐1‐propanol using the Mitsunobu reaction (triphenylphosphine, DIAD), followed by basic hydrolysis of the cyclic ester. In the second, the lipid moiety is incorporated into a phosphonate synthon and a stepwise approach is used to assemble the (S)‐HPMPA analog.

Keywords: acyclic nucleoside phosphonates; ether lipid prodrugs; cidofovir; (S)‐HPMPA; antivirals

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of Hexadecyloxypropyl‐Cidofovir
  • Basic Protocol 2: Synthesis of Hexadecyloxypropyl‐(S)‐HPMPA
  • Support Protocol 1: Synthesis of 3‐Hexadecyloxy‐1‐Propanol
  • Support Protocol 2: Large‐Scale Synthesis of 3‐Hexadecyloxy‐1‐Propanol
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Hexadecyloxypropyl‐Cidofovir

  Materials
  • Cidofovir (CDV; WaterStone Technology; also see unit 14.2)
  • N,N‐Dimethylformamide, anhydrous (DMF; Aldrich)
  • N,N′‐Dicyclohexyl‐4‐morpholinecarboxamidine (Aldrich)
  • 1,3‐Dicyclohexylcarbodiimide (DCC; Aldrich)
  • Pyridine
  • Diethyl ether
  • Dichloromethane (CH 2Cl 2)
  • 1 N hydrochloric acid (HCl) in H 2O
  • Acetone
  • Toluene
  • 3‐Hexadecyloxy‐1‐propanol (S.2; see protocol 3 or protocol 42)
  • Triphenylphosphine (Aldrich)
  • Diisopropylazodicarboxylate (DIAD; Aldrich)
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • Concentrated ammonium hydroxide (NH 4OH)
  • Phospray TLC reagent (Supelco)
  • Ethanol (EtOH)
  • Silica gel 60 (230 to 400 mesh; EMD Chemicals)
  • p‐Dioxane
  • 0.5 N sodium hydroxide (NaOH) in H 2O
  • Nitrogen tank
  • 50% (v/v) acetic acid in H 2O
  • Isopropanol
  • 500‐, 250‐, and 100‐mL round‐bottom flasks, oven dried
  • 60‐mL addition funnel
  • 60° and 40°C oil baths
  • Rotary evaporator equipped with a vacuum pump
  • Frit funnel (medium porosity, 10 to 16 µm) with vacuum
  • Azeotropic distillation apparatus
  • Dean‐Stark trap
  • Vacuum oven (70°C)
  • Silica gel thin‐layer chromatography (TLC) plates (e.g., 250‐µm Silica Gel GF Uniplates; Analtech)
  • Filter funnel with vacuum
  • UV lamp
  • Muffle furnace set to 400°C
  • Glass flash chromatography columns: 50 × 457 mm
  • Vacuum oil pump

Basic Protocol 2: Synthesis of Hexadecyloxypropyl‐(S)‐HPMPA

  Materials
  • Adenine (S.5; Aldrich)
  • (S)‐Trityl glycidyl ether (Daiso)
  • N,N‐Dimethylformamide (DMF), anhydrous
  • 1,8‐Diazabicyclo[5,4,0]undec‐7‐ene (DBU; Aldrich)
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • Toluene, dry
  • Trityl chloride (Aldrich)
  • N,N‐Dimethylaminopyridine (DMAP; Aldrich)
  • Pyridine (anhydrous, Aldrich)
  • Ethyl acetate (EtOAc)
  • Saturated sodium bicarbonate (NaHCO 3), cold
  • Anhydrous magnesium sulfate (MgSO 4)
  • Silica gel 60 (230 to 400 mesh; EMD Chemicals)
  • Hexanes
  • Diethyl toluenesulfonyloxymethylphosphonate (Biofine International)
  • Dichloromethane (CH 2Cl 2), anhydrous
  • Bromotrimethylsilane (Aldrich)
  • Oxalyl chloride (Acros Organics)
  • Diethyl ether
  • Triethylamine, dry
  • Sodium t‐butoxide (Aldrich)
  • NaCl, cold saturated solution
  • Ethanol (EtOH)
  • 80% acetic acid in water
  • Phospray TLC reagent (Supelco)
  • Concentrated ammonium hydroxide (NH 4OH)
  • 250‐, 100‐, and 50‐mL round‐bottom flasks, oven dried
  • Reflux condensers
  • 50°, 60°, and 100°C oil baths
  • Silica gel thin‐layer chromatography (TLC) plates (e.g., 250‐µm Silica Gel GF Uniplates, Analtech)
  • UV lamp
  • Rotary evaporator and vacuum pump
  • Filter funnels with vacuum
  • Vacuum oil pump
  • 250‐mL and 1‐L separatory funnels
  • Glass flash chromatography columns: 50 × 457 mm and 64 × 457 mm
  • 150‐mL dropping funnel
  • Muffle furnace set to 400°C

Support Protocol 1: Synthesis of 3‐Hexadecyloxy‐1‐Propanol

  Materials
  • 1‐Hexadecanol (Aldrich)
  • Triethylamine (Aldrich), dry
  • Methanesulfonyl chloride (Aldrich)
  • Dichloromethane (CH 2Cl 2), dry
  • 0.1 N HCl, cold
  • NaCl, saturated solution
  • MgSO 4, anhydrous
  • 95% ethanol (EtOH)
  • Sodium hydride, 60% dispersion in paraffin oil (Aldrich)
  • N,N‐Dimethylformamide (DMF), dry
  • 1,3‐Propanediol (Shell Chemical or Aldrich)
  • Hexanes
  • 1‐L round‐bottom flasks, oven dried
  • 250‐mL dropping funnels
  • 2‐L beaker
  • Glass filter funnels
  • Separatory funnel
  • Rotary evaporator with vacuum pump
  • Vacuum oil pump
  • 500‐mL Erlenmeyer flask

Support Protocol 2: Large‐Scale Synthesis of 3‐Hexadecyloxy‐1‐Propanol

  Materials
  • 1,3‐Propanediol (Shell Chemical or Aldrich)
  • 85% potassium hydroxide (KOH; Fisher Scientific)
  • 1‐Bromohexadecane (Aldrich)
  • Hexanes
  • 1‐L round‐bottom flask, oven dried
  • Heating mantle
  • Vacuum distillation apparatus (distillation head)
  • Reflux condenser
  • Bent adapter
  • Thermometer
  • Vacuum pump
  • Nitrogen tank
  • 2‐L three‐neck flask
  • Overhead mechanical stirrer
  • 250‐mL dropping funnel
  • 3‐L beaker
  • Glass filter funnel with 2‐L filter flask
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Figures

Videos

Literature Cited

Literature Cited
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