Anti‐HIV Nucleoside Phosphonate GS‐9148 and Its Prodrug GS‐9131: Scale Up of a 2′‐F Modified Cyclic Nucleoside Phosphonate and Synthesis of Selected Amidate Prodrugs

Richard L. Mackman1

1 Gilead Sciences, Inc, Foster City, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 14.10
DOI:  10.1002/0471142700.nc1410s56
Online Posting Date:  March, 2014
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Nucleoside phosphonate analogs are an important class of antiviral drugs for the treatment of HIV and HBV. The most recent nucleoside phosphonate to progress to clinical development is GS‐9131, a cyclic nucleoside phosphonate (CNP). This unit contains procedures for the synthesis of the parent CNP 2′‐Fd4AP (GS‐9148) and selected monoamidate and bisamidate prodrugs, including the monoamidate clinical prodrug GS‐9131. The first basic protocol of this unit details improved procedures for the preparation of 2′‐Fd4AP and related phosphonate esters by introduction of a hydroxylmethyl phosphonate ester regioselectively and stereoselectively onto a furanose core via a glycal intermediate. The method described is believed to be robust and flexible, allowing for a variety of analogs with other nucleobases or furanose 2′‐ring substitutions to be prepared. The preparation of monoamidate and bisamidate prodrugs either on the phosphonate diacid or its monophenyl ester is then described in the second and third basic protocols of this unit. Curr. Protoc. Nucleic Acid Chem. 56:14.10.1‐14.10.21. © 2014 by John Wiley & Sons, Inc.

Keywords: antiviral; anti-HIV; nucleoside; phosphonate; prodrugs

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of 2′‐F Nucleoside Phosphonates via a Glycal Furanoid Intermediate
  • Basic Protocol 2: Preparation of Monoamidate Prodrugs from Mono and Diacid Phosphonates
  • Basic Protocol 3: Preparation of Bisamidate Prodrugs
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of 2′‐F Nucleoside Phosphonates via a Glycal Furanoid Intermediate

  • 2‐Fluoro‐1,3,5‐tri‐O‐benzoyl‐β‐D‐arabinofuranosyl (1; Davos Chemical)
  • Dichloromethane (CH 2Cl 2), anhydrous
  • 33% (w/v) hydrogen bromide (HBr) in acetic acid (Sigma‐Aldrich)
  • Sodium hydrogen carbonate (NaHCO 3)
  • Saturated aqueous sodium hydrogen carbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Sodium hydride (NaH; 60% dispersion in mineral oil)
  • Acetonitrile (CH 3CN), anhydrous
  • Nitrogen (N 2) gas
  • 6‐Chloropurine, 99% pure
  • Glacial acetic acid (AcOH), ACS reagent
  • Methanol (MeOH), analytical grade
  • Potassium carbonate (K 2CO 3), anhydrous powder
  • Hexanes, HPLC grade
  • Acetone, HPLC grade
  • Celite‐545
  • Jones reagent: 2.5 M CrO 3 in 25% aqueous H 2SO 4 (dissolve 25 g CrO 3 in 75 mL water and 25 mL of 98% H 2SO 4)
  • 2‐Propanol (i‐PrOH), HPLC grade
  • Ethyl acetate (EtOAc), HPLC grade
  • Tetrahydrofuran (THF), anhydrous
  • Tetrahydrofuran (THF), HPLC grade
  • Brine: saturated aqueous NaCl
  • Triphenylphosphine (Ph 3P), 99% pure
  • Diisopropylazodicarboxylate (DIAD), 98% pure
  • Diethyl (hydroxymethyl)phosphonate or diphenyl (hydroxymethyl)phosphonate, 97% pure (Epsilon Chimie)
  • Iodine monobromide solution (IBr), 1.0 M in methylene chloride
  • Sodium thiosulfate (Na 2S 2O 3), ACS reagent
  • Magnesium sulfate (MgSO 4), anhydrous
  • Silica gel: 0.040‐ to 0.063‐mm Macherey‐Nagel Kieselgel 60
  • Ethanol, ACS reagent, >99.5% (200 proof), absolute
  • Bleach: Clorox brand (10% to 13% NaOCl in H 2O)
  • Ammonium hydroxide (aq.), 28% to 30% concentrated
  • Octadecyl (C‐18), Bakerbond, 40 μm, prepLC packing
  • 2,6‐Lutidine, Reagent Plus, 98%
  • Bromotrimethylsilane (TMSBr), 97%
  • 12‐L glass three‐necked round‐bottom flask
  • Overhead mechanical stirrer
  • 4‐ and 2‐L separatory funnels
  • Rotary evaporator equipped with a vacuum pump
  • High‐vacuum oil vacuum pump
  • 5‐L reaction vessel
  • Büchner funnel with sealed‐in fritted‐glass disc
  • 7 × 50–cm chromatography column
  • 5‐L 3‐neck round‐bottom flask
  • Addition funnel
  • LC‐MS (see, e.g., unit 7.16) for reaction analysis
  • Parr reaction vessel, capable of heating and shaking, supporting pressures up to 1000 psi (Bench Top Reactor 1 L, Parr Instrument Co., model no. 4523)
  • Lyophilizer
  • Additional reagents and equipment for column chromatography ( appendix 3E) and liquid chromatography–mass spectroscopy (e.g., unit 7.16)

Basic Protocol 2: Preparation of Monoamidate Prodrugs from Mono and Diacid Phosphonates

  • Disodium salt 9 or mono phenyl ester 8b ( protocol 1)
  • L‐alanine ethyl ester hydrochloride, 99% (Sigma‐Aldrich, cat. no. 855669)
  • Phenol, ACS reagent, loose crystals, >99.0%
  • Pyridine, anhydrous, 99.8%
  • Triethylamine
  • Nitrogen (N 2) gas
  • Aldrithiol‐2, 98% (Sigma‐Aldrich)
  • Triphenylphosphine (Ph 3P), 99% pure
  • Pyridine, anhydrous, 99.8%
  • Ethyl acetate (EtOAc), HPLC grade
  • Saturated aqueous sodium hydrogen carbonate (NaHCO 3)
  • Brine: saturated aqueous NaCl
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Dichloromethane (DCM)
  • Silica gel: 0.040‐ to 0.063‐mm Macherey‐Nagel Kieselgel 60
  • Methanol (MeOH), analytical grade
  • Acetonitrile, HPLC grade
  • Diphenyl chlorophosphate, 99% (Sigma‐Aldrich, cat. no. D206555)
  • Buffer A: 5% (v/v) ethanol in acetonitrile
  • Buffer B: 505 (v/v) methanol in acetonitrile
  • 200‐mL round‐bottom flask
  • Rotary evaporator
  • 125‐mL separatory funnel
  • 4 × 13–cm chromatography column
  • Gilson Liquid Handler 215 equipped with 322 pumps
  • UV/Vis‐156 detector
  • Phenomenex Synergi Combi‐HTS 4 micron hydro‐RP C18 column (150 × 30 mm)
  • Lyophilizer
  • Diacel Chiralpak AS‐H column (20 × 250 mm)
  • Additional reagents and equipment for HPLC (unit 10.5) and column chromatography ( appendix 3E)

Basic Protocol 3: Preparation of Bisamidate Prodrugs

  • Disodium salt 9 ( protocol 1)
  • L‐Alanine isopropyl ester hydrochloride
  • L‐Alanine propyl ester hydrochloride
  • Cyclobutyl (2S)‐2‐aminopropanoate hydrochloride
  • Glycine isopropyl ester hydrochloride
  • L‐Phenylalanine isopropyl ester hydrochloride
  • Additional reagents and equipment for preparation of 2′‐F nucleoside phosphonates ( protocol 1) and monoamidite prodrugs ( protocol 2)
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Literature Cited

Literature Cited
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