Synthesis of Acyclic Nucleoside Phosphonates

Antonin Holý1

1 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 14.2
DOI:  10.1002/0471142700.nc1402s22
Online Posting Date:  October, 2005
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Abstract

Acyclic nucleoside phosphonates (ANPs) are important biologically active nucleotide analogs. They contain an isopolar phosphonomethyl function linked to the hydroxyl group of an acyclic side‐chain via an undegradable ether group. Though their most important activity is antiviral, some exhibit cytostatic or antiprotozoic effects. The three most important groups of ANP are presented here as synthetic procedures for a large laboratory scale. Synthesis follows three principles: (1) introduction of a protected phosphonomethyl group to the hydroxyl on an appropriate alkyl side‐chain of an acyclic nucleoside, (2) alkylation of the heterocyclic base by a synthon with all characteristic features of the future phoshonate‐bearing side‐chain, or (3) transformation of a reactive group at the heterocyclic base. The last step in all these cases is removal of the phosphonate esters. Preparation methods are described in detail for PMEA, PMEG, PMEDAP and its N6‐cyclopropyl derivative, (R)‐PMPA, and (S)‐HPMPA, as well as all intermediates and synthons.

Keywords: Acyclic nucleoside phosphonates; purine nucleotide analogs; tenofovir; adefovir; antiviral

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 9‐[2‐(Phosphonomethoxy)Ethyl]Adenine (Adefovir, PMEA)
  • Basic Protocol 2: Synthesis of 2,6‐Diamino‐9‐[2‐(Phosphonomethoxy)Ethyl]Purine (PMEDAP)
  • Basic Protocol 3: Synthesis of 9‐[2‐(Phosphonomethoxy)Ethyl]Guanine (PMEG)
  • Basic Protocol 4: Synthesis of 2‐Amino‐N6‐(Cyclopropylamino)‐9‐[2‐(Phosphonomethoxy)Ethyl]Purine
  • Support Protocol 1: Preparation of the PME Synthon: Diisopropyl 2‐Chloroethoxymethylphosphonate
  • Basic Protocol 5: Synthon Method for Synthesis of 9‐(R)‐[2‐(Phosphonomethoxy)Propyl]Adenine [Tenofovir, (R)‐PMPA]
  • Support Protocol 2: Preparation of (R)‐2‐O‐Tetrahydropyranylpropane‐1,2‐Diol
  • Support Protocol 3: Preparation of the (R)‐PMP Synthon: (R)‐2‐O‐(Diisopropylphosphonylmethoxy)Propyl p‐Tolylsulfonate
  • Support Protocol 4: Preparation of 1‐O‐Benzyl‐(R)‐2‐O‐(Diisopropylphosphonylmethyl)Propane‐1,2‐Diol Via Direct Etherification
  • Support Protocol 5: Preparation of the C1 Synthon: Diisopropyl p‐Tolylsulfonyloxymethylphosphonate
  • Support Protocol 6: Preparation of the (R)‐PMP Synthon
  • Alternate Protocol 1: Stepwise Method for Tenofovir Synthesis
  • Basic Protocol 6: Synthesis of 9‐(S)‐[3‐Hydroxy‐2‐(Phosphonomethoxy)Propyl]Adenine (HPMPA)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 9‐[2‐(Phosphonomethoxy)Ethyl]Adenine (Adefovir, PMEA)

  Materials
  • Sodium hydride, 60% dispersion in paraffin oil
  • Dimethylformamide (DMF)
  • Adenine ( S.1)
  • Diisopropyl 2‐chloroethoxymethylphosphonate ( S.16; see protocol 5)
  • Toluene
  • Chloroform
  • Celite
  • Silica gel
  • Methanol
  • Ethyl acetate
  • Hexane
  • Bromotrimethylsilane
  • Acetonitrile
  • Conc. aqueous ammonia
  • Dowex 50 × 8, acid form (see recipe)
  • 1:1 (v/v) diluted HCl (from conc. hydrochloric acid and water)
  • Ethanol
  • Diethyl ether
  • 500‐mL and 1‐L round‐bottom flasks
  • Magnetic stirrer with heating plate and silicon oil bath
  • 250‐mL dropping funnel
  • Rotary evaporator with diaphragm pump
  • Glass filter funnels with 1‐L filter flasks
  • Reflux condenser
  • 6 × 40–cm and 5 × 40–cm chromatography columns
  • Fraction collector (optional)
  • UV detector (optional)
  • 2‐L thick‐wall beaker
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of 2,6‐Diamino‐9‐[2‐(Phosphonomethoxy)Ethyl]Purine (PMEDAP)

  Materials
  • 2,6‐Diaminopurine ( S.5; TCI America)
  • Cesium carbonate
  • Dimethylformamide (DMF)
  • Diisopropyl 2‐chloroethoxymethylphosphonate ( S.16; see protocol 5)
  • Celite
  • Toluene
  • Chloroform
  • Silica gel
  • Methanol
  • Ethanol
  • Hexane
  • Acetonitrile
  • Bromotrimethylsilane
  • Conc. aqueous ammonia
  • 1:1 (v/v) diluted HCl (from conc. hydrochloric acid and water)
  • Diethyl ether
  • 1‐L round‐bottom flasks
  • Magnetic stirrer with heating plate and silicon oil bath
  • Glass filter funnels with 1‐L filter flasks
  • Rotary evaporator with diaphragm pump
  • Reflux condenser
  • 6 × 40–cm chromatography column
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Synthesis of 9‐[2‐(Phosphonomethoxy)Ethyl]Guanine (PMEG)

  Materials
  • 2‐Amino‐6‐chloropurine ( S.8)
  • Cesium carbonate
  • Diisopropyl 2‐chloroethoxymethylphosphonate ( S.16; see protocol 5)
  • Dimethylformamide (DMF)
  • Toluene
  • Chloroform
  • Celite
  • Silica gel
  • Ethyl acetate
  • Hexane
  • Diethyl ether
  • 1 M HCl
  • Conc. aqueous ammonia
  • Acetone
  • Bromotrimethylsilane
  • Acetonitrile
  • 0.25 M sulfuric acid
  • 500‐mL and 1‐L round‐bottom flasks
  • Magnetic stirrer with heating plate and silicon oil bath
  • Rotary evaporator with diaphragm pump
  • Glass filter funnels with 1‐L filter flasks
  • 6 × 40–cm chromatography column
  • Reflux condenser
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Synthesis of 2‐Amino‐N6‐(Cyclopropylamino)‐9‐[2‐(Phosphonomethoxy)Ethyl]Purine

  Materials
  • Cyclopropylamine
  • 2‐Amino‐6‐chloro‐9‐[2‐(diisopropylphosphonylmethoxy)ethyl]purine ( S.9; see protocol 3)
  • Dioxane
  • Chloroform
  • Methanol
  • Ethanol
  • Dowex 50 × 8, acid form (see recipe)
  • 1:10 (v/v) aqueous ammonia in 20% aqueous methanol
  • Acetonitrile
  • Bromotrimethylsilane
  • Dowex 1 × 2, acetate form (see recipe)
  • 0.2 and 1 M acetic acid
  • Acetone
  • Diethyl ether
  • 250‐mL and 1‐L round‐bottom flasks
  • Reflux condenser
  • Soda lime protecting tube
  • Magnetic stirrer with heating plate and silicon oil bath
  • 4 × 40–cm glass chromatography column
  • Peristaltic pump with column adapter
  • UV detector
  • Fraction collector (optional)
  • Rotary evaporator with diaphragm pump
  • Vacuum desiccator with P 2O 5
  • 1‐L cylinder‐shaped bottles
  • Plastic tubing
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Preparation of the PME Synthon: Diisopropyl 2‐Chloroethoxymethylphosphonate

  Materials
  • 1,3,5‐Trioxane
  • 2‐Chloroethanol ( S.14)
  • Dry ice/ethanol bath
  • Hydrogen chloride (gas)
  • Dry nitrogen (gas)
  • Calcium chloride
  • Celite
  • Dichloromethane
  • Conc. sulfuric acid
  • Triisopropyl phosphite
  • 3‐L reaction flask with inlet/outlet system
  • Thermometer
  • Plastic tubing
  • Calcium chloride protecting tube
  • Magnetic stirrer with heating plate
  • Hydrogen chloride drying system (composed of washing bottle with conc. sulfuric acid, protective flasks before and after, inlet tube with sinter, with all parts interconnected in‐line by polypropylene tubing)
  • 2‐L separatory funnel
  • Rotary evaporator with diaphragm pump
  • 2‐L distillation flask
  • Sintered glass filter funnels with 2‐L filter flasks
  • 40‐cm distillation columns with head and thermometer
  • 1‐L two‐neck round‐bottom flask
  • 250‐mL dropping funnel
  • Diaphragm vacuum pump
  • Oil vacuum pump
  • 250‐mL and 1‐L round‐bottom receiving flasks
CAUTION: This procedure makes use of gaseous hydrogen chloride and must be performed in a well‐ventilated fume hood. The product itself or the side products of the reaction may be carcinogens. All precautions must be taken to avoid inhalation of the vapors or contact with skin or eyes.

Basic Protocol 5: Synthon Method for Synthesis of 9‐(R)‐[2‐(Phosphonomethoxy)Propyl]Adenine [Tenofovir, (R)‐PMPA]

  Materials
  • Sodium hydride, 60% dispersion in paraffin oil
  • Dimethylformamide (DMF)
  • Adenine
  • (R)‐PMP synthon ( S.32; see protocol 8 or protocol 116)
  • Toluene
  • Chloroform
  • Celite
  • Methanol
  • Silica gel
  • Acetonitrile
  • Bromotrimethylsilane
  • Conc. aqueous ammonia
  • Dowex 50 × 8, acid form (see recipe)
  • Dowex 1 × 2, acetate form (see recipe)
  • 1 M acetic acid
  • Ethanol
  • Diethyl ether
  • 250‐mL round‐bottom flasks
  • Magnetic stirrer with heating plate and silicon oil bath
  • Rotary evaporator with diaphragm pump
  • Glass filter funnel with filter flask
  • 4 × 40–cm and 3 × 40–cm chromatography columns
  • Fraction collector (optional)
  • Gradient maker (see protocol 4)
  • Calcium chloride protecting tube
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 2: Preparation of (R)‐2‐O‐Tetrahydropyranylpropane‐1,2‐Diol

  Materials
  • Isobutyl (R)‐lactate ( S.20)
  • Dihydropyran
  • 6 M HCl in DMF (see recipe)
  • Silver oxide
  • Celite
  • Triethylamine
  • Red‐Al (Aldrich, Fluka)
  • Diethyl ether
  • Ethyl acetate
  • Dioxane
  • Magnesium sulfate, anhydrous
  • 500‐mL round‐bottom flask
  • Calcium chloride protecting tube
  • Glass filter funnels (≥12 cm diameter) with 2‐L filter flasks
  • Distillation apparatus with 250‐mL flask
  • 500‐mL three‐neck flask
  • Mechanical KPG stirrer
  • Reflux condenser with calcium chloride protecting tube
  • 200‐mL dropping funnel
  • Thermometer
  • 600‐mL glass beaker
  • Broad glass funnel

Support Protocol 3: Preparation of the (R)‐PMP Synthon: (R)‐2‐O‐(Diisopropylphosphonylmethoxy)Propyl p‐Tolylsulfonate

  Materials
  • Sodium hydride, 60% dispersion in paraffin oil
  • Hexane
  • Dimethylformamide (DMF), reagent grade
  • (R)‐2‐O‐Tetrahydropyranyl‐1,2‐propanediol ( S.22; see protocol 7)
  • Benzyl bromide
  • Saturated ammonia in methanol
  • Ethyl acetate
  • 80% (v/v) aqueous methanol
  • Dowex 50 × 8, acid form (see recipe)
  • Methanol
  • Diethyl ether
  • Magnesium sulfate, anhydrous
  • 1,2‐Dichloroethane
  • 1,3,5‐Trioxane
  • Calcium chloride, anhydrous
  • Toluene
  • Triisopropyl phosphite
  • Aluminum oxide, neutral
  • Conc. aqueous HCl
  • Argon
  • Palladium‐on‐charcoal catalyst
  • Celite
  • Hydrogen
  • Triethylamine
  • Pyridine
  • 4‐Dimethylaminopyridine
  • p‐Tolylsulfonyl chloride
  • Chloroform
  • Glass receiver with glass filter (see Fig. )
  • Diaphragm vacuum pump
  • 2‐L round‐bottom reaction flasks
  • Mechanical KPG stirrer
  • 200‐mL dropping funnel
  • Magnetic stirrer
  • Rotary evaporator with diaphragm pump
  • Reflux condensers
  • Glass filter funnels with 2‐L filter flasks
  • 250‐mL distillation flask in distillation apparatus
  • 500‐mL three‐neck round‐bottom flask with inlet and outlet tubes
  • HCl drying system with dry HCl from a cylinder and an inlet tube
  • 8 × 40–cm chromatography columns
  • Vacuum desiccator with P 2O 5
  • Extraction funnel
  • 2‐L Erlenmeyer flask
  • 1‐L two‐neck round‐bottom flask
  • Calcium chloride protecting tube at the end of polypropylene tubing reaching to the vent
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 4: Preparation of 1‐O‐Benzyl‐(R)‐2‐O‐(Diisopropylphosphonylmethyl)Propane‐1,2‐Diol Via Direct Etherification

  Materials
  • Sodium hydride, 60% dispersion in paraffin oil
  • Hexane
  • Tetrahydrofuran, freshly distilled from NaH
  • (R)‐1‐O‐Benzylpropane‐1,2‐diol ( S.28; see protocol 8)
  • Diisopropyl p‐tolylsulfonyloxymethylphosphonate ( S.19; see protocol 10)
  • Celite
  • Ethyl acetate
  • Toluene
  • Silica gel
  • Glass receiver with glass filter (Fig. )
  • Rotary evaporator with diaphragm pump
  • 4‐L three‐neck round‐bottom flask
  • Magnetic stirrer
  • 250‐mL dropping funnel
  • Calcium chloride protecting tube
  • Thermometer
  • Glass filter funnel with filter flask
  • 8 × 40–cm glass chromatography column
  • Vacuum desiccator with P 2O 5
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 5: Preparation of the C1 Synthon: Diisopropyl p‐Tolylsulfonyloxymethylphosphonate

  Materials
  • Triethylamine
  • Diisopropyl phosphite
  • Paraformadehyde, dried at atmospheric pressure over sulfuric acid in vacuum desiccator for ≥48 hr
  • Ethyl acetate
  • Ethanol
  • p‐Nitrobenzylpyridine reagent (see recipe)
  • Acetonitrile
  • p‐Tolylsulfonyl chloride
  • 4‐Dimethylaminopyridine (optional)
  • Diethyl ether
  • Magnesium sulfate, anhydrous
  • Toluene
  • Silica gel (40‐60 µm)
  • Chloroform
  • Methanol
  • Cyclohexane
  • Hexane
  • 500 ml round‐bottomed flask
  • Reflux condenser with calcium chloride protecting tube
  • Magnetic stirrer with heating plate and silicon oil bath
  • 1‐L Erlenmeyer flask
  • Glass filter funnel with filter flask
  • Rotary evaporator with diaphragm pump
  • 8 × 20– to 8 × 22–cm glass chromatography column
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
NOTE: All solvents and chemicals should be reagent grade.

Support Protocol 6: Preparation of the (R)‐PMP Synthon

  Materials
  • (R)‐1‐O‐Benzyl‐2‐O‐(diisopropylphosphonylmethyl)propane‐1,2‐diol ( S.29; see protocol 9)
  • Methanol, spectroscopically pure or HPLC pure
  • Argon
  • 10% palladium‐on‐charcoal catalyst
  • Celite
  • Toluene
  • Pyridine
  • 4‐Dimethylaminopyridine
  • p‐Tolylsulfonyl chloride
  • Ethyl acetate
  • Diluted HCl: 1:20 (v/v) in water
  • Saturated potassium hydrogencarbonate
  • Magnesium sulfate, anhydrous
  • Silica gel
  • 2‐L round‐bottom flask
  • Rotary evaporator with diaphragm pump
  • Bladder as hydrogen reservoir
  • Glass filter funnel with filter flask
  • 6 × 40–cm glass chromatography column

Alternate Protocol 1: Stepwise Method for Tenofovir Synthesis

  Materials
  • Adenine
  • Cesium carbonate
  • Dimethylformamide (DMF)
  • (R)‐Propanediol‐1,2‐carbonate ( S.26; Fig. )
  • Toluene
  • Ethanol
  • Diethyl ether
  • Sodium hydride, 60% dispersion in paraffin oil
  • Diisopropyl p‐tolylsulfonyloxymethylphosphonate ( S.19; see protocol 10)
  • Acetic acid
  • Chloroform
  • Dowex 50 × 8, acid form (see recipe)
  • Conc. aqueous ammonia
  • Methanol
  • Silica gel
  • 250‐mL round‐bottom flask
  • Magnetic stirrer with heating plate and silicon oil bath
  • Rotary evaporator with diaphragm pump
  • Glass filter funnel with filter flask
  • 3 × 40–cm and 4 × 40–cm glass chromatography columns
  • UV detector
  • Fraction collector (optional)
  • Calcium chloride protecting tube
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 6: Synthesis of 9‐(S)‐[3‐Hydroxy‐2‐(Phosphonomethoxy)Propyl]Adenine (HPMPA)

  Materials
  • Trityl‐(S)‐glycidol
  • Adenine
  • Dimethylformamide (DMF)
  • Cesium carbonate
  • Chloroform
  • Silica gel
  • Pyridine
  • Chlorotrimethylsilane
  • Benzoyl chloride
  • Conc. aqueous ammonia
  • Magnesium sulfate, anhydrous
  • Tetrahydrofuran
  • Sodium hydride, 60% dispersion in paraffin oil
  • Diisopropyl p‐tolylsulfonyloxymethylphosphonate ( S.19; see protocol 10)
  • Acetic acid
  • Ethyl acetate
  • Methanol
  • Sodium methoxide
  • Dowex 50 × 8, acid form (see recipe)
  • Triethylamine
  • Diethyl ether
  • Ethanol
  • Toluene
  • Bromotrimethylsilane
  • Acetonitrile
  • Dowex 1 × 2, acetate form (see recipe)
  • 500‐mL round‐bottom flask
  • Magnetic stirrer with heating plate and silicon oil bath
  • Rotary evaporator with a diaphragm pump
  • 5 × 50–cm glass chromatography column
  • 2‐L Erlenmeyer flask
  • Glass filter funnel with filter flask
  • Reflux condenser
  • Calcium chloride protecting tube
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
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Literature Cited

Literature Cited
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