Synthesis of Peptidomimetic Conjugates of Cyclic Nucleoside Phosphonates

Michaela Serpi1, Ivan S. Krylov1, Valeria M. Zakharova1, Charles E. McKenna1

1 University of Southern California, Los Angeles, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 15.4
DOI:  10.1002/0471142700.nc1504s43
Online Posting Date:  December, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Cyclic nucleoside phosphonates connected through a P‐O‐C linkage to a promoiety represent a class of prodrugs designed to overcome the low oral bioavailability of parent antiviral acyclic nucleoside phosphonates. In our prodrug approach, a nontoxic promoiety, such as an amino acid or dipeptide, is conjugated to the cyclic form of the parent drug by esterification of the phosphonic acid moiety with an alcoholic amino acid side chain (Ser, Tyr, and Thr) or a glycol linker. For the biological evaluation and investigation of the pharmacokinetic profiles of these modified nucleoside phosphonates, a reliable synthetic procedure that allows preparation of sufficient amount of potential prodrugs is needed. This unit provides a procedure for synthesizing peptidomimetic conjugates of two broad‐spectrum antiviral acyclic nucleoside phosphonates: (S)‐HPMPC and (S)‐HPMPA. Two alternate strategies allowing synthesizing selected amino acid, dipeptide, or ethylene glycol‐linked amino acid prodrugs of (S)‐HPMPC and (S)‐HPMPA in solution and using a solid‐phase approach are presented. Curr. Protoc. Nucleic Acid Chem. 43:15.4.1‐15.4.13. © 2010 by John Wiley & Sons, Inc.

Keywords: (S)‐HPMPC; (S)‐HPMPA; peptidomimetic prodrugs; acyclic nucleoside phosphonates

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Amino Acid, Dipeptide, and Ethylene Glycol‐Linked Amino Acid Cyclic (S)‐HPMPC and (S)‐HPMPA Conjugates
  • Alternate Protocol 1: Solid‐Phase Synthesis of Amino Acid Cyclic (S)‐HPMPA Conjugates
  • Support Protocol 1: Synthesis of the Peptidomimetic Promoiety
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Synthesis of Amino Acid, Dipeptide, and Ethylene Glycol‐Linked Amino Acid Cyclic (S)‐HPMPC and (S)‐HPMPA Conjugates

  • Dry nitrogen (N 2) or Argon (Ar)
  • 1‐[(2S)‐3‐Hydroxy‐2‐phosphonomethoxypropyl]cytosine: Cidofovir, CDV, (S)‐HPMPC; Rasayan Inc. (Holý, ; Eriksson et al., ) or 9‐[(2S)‐3‐Hydroxy‐2‐phosphonomethoxypropyl]adenine: (S)‐HPMPA; Rasayan Inc. (Holý, )
  • N,N‐Dimethylformamide (DMF; EMD), anhydrous
  • N‐Ethyldiisopropylamine, (DIEA; Alfa Aesar), freshly distilled over KOH
  • Amino acids or dipeptide:
    • Methyl N‐(tert‐butoxycarbonyl)‐(L)‐serinate ( S.3a)
    • Methyl N‐(tert‐butoxycarbonyl)‐(L)‐tyrosinate ( S.3b)
    • Methyl N‐(tert‐butoxycarbonyl)‐(L)‐valyl‐L‐serinate ( S.3c; see protocol 3)
    • 2‐Hydroxyethyl‐N‐(tert‐butoxycarbonyl)‐(L)‐valinate ( S.3d; see protocol 3)
  • Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; Aldrich)
  • Diethylether (Et 2O), ACS reagent grade
  • Dichloromethane (CH 2Cl 2), ACS reagent grade
  • Acetone, ACS reagent grade
  • Methanol (MeOH), ACS reagent grade
  • Silica gel (60 to 200 mesh; EMD)
  • KMnO 4 spray solution, optional
  • Trifluoroacetic acid (TFA; EMD), anhydrous
  • Acetonitrile (CH 3CN), ACS reagent grade
  • 500‐, 100‐, 50‐, 25‐mL round‐bottom flasks
  • Magnetic stir bar
  • Heat gun (Master Appliance Corporation, model no. HG‐201A)
  • Rotary evaporator equipped with a vacuum pump
  • 40°C oil bath
  • Glass flash chromatography columns: 2.5 × 30–cm and 1.5 × 25–cm
  • 254‐nm UV lamp
  • C18 HPLC column (10 µM, 80 Å pore size, 21.2 × 150–mm; Beckman Coulter)
  • Silica gel thin‐layer chromatography (TLC) plates (Merck)
  • Preparative TLC chamber
  • Glass filter funnels
  • Additional reagents and equipment for TLC ( appendix 3D)

Alternate Protocol 1: Solid‐Phase Synthesis of Amino Acid Cyclic (S)‐HPMPA Conjugates

  • Trityl chloride polystyrene (TCP) resin (100 to 200 mesh, 1% cross‐linked, typical loading 1.0 to 1.8 mmol/g; Aldrich)
  • Methyl O‐[tert‐butyl(dimethyl)silyl]‐(L)‐serinate ( S.6a, see protocol 3)
  • Methyl O‐[tert‐butyl(dimethyl)silyl]‐(D)‐serinate ( S.6b, see protocol 3)
  • Isopropyl O‐[tert‐butyl(dimethyl)silyl]‐(L)‐serinate ( S.6c, see protocol 3)
  • Tetrahydrofurane (THF), ACS reagent grade
  • Tetrabutylammonium fluoride trihydrate (TBAF; Aldrich)
  • 0.1 N HCl in methanol
  • Desiccator

Support Protocol 1: Synthesis of the Peptidomimetic Promoiety

  • Ntert‐Butoxycarbonyl‐(L)‐valine hydrochloride (Fluka)
  • (L)‐Serine methyl ester hydrochloride (Aldrich)
  • Ethan‐1,2‐diol (ethylene glycol)
  • 1‐Hydroxybenzotriazole hydrate (HOBt; Aldrich)
  • Triethylamine (Et 3N; Aldrich), freshly distilled over KOH
  • 3‐(Ethyliminomethyleneamino)‐N,N‐dimethyl‐propan‐1‐amine hydrochloride (EDC⋅HCl; Aldrich)
  • Citric acid (Baker Analyzed)
  • Sodium bicarbonate (NaHCO 3; Mallinckrodt Chemicals)
  • Sodium chloride (NaCl; EMD)
  • Sodium sulfate (Na 2SO 4; EMD), anhydrous
  • tert‐Butyldimethylsilyl chloride (TBDMSCl; Aldrich)
  • (D)‐Serine methyl ester hydrochloride (Aldrich)
  • (L)‐Serine isopropyl ester hydrochloride (Aurora Building Blocks)
  • Imidazole (Aldrich)
  • Hexane
  • 250‐mL separatory funnel
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Ariza, M.E. 2005. Current prodrug strategies for the delivery of nucleotides into cells. Drug Des. Rev. Online 2:373‐387.
   De Clercq, E. 2007. The acyclic nucleoside phosphonates from inception to clinical use: Historical perspective. Antiviral Res. 75:1‐13.
   De Clercq, E. and Holý, A. 2005. Acyclic nucleoside phosphonates: A key class of antiviral drugs. Nat. Rev. Drug Discov. 4:928‐940.
   Eriksson, U., Hilfinger, J.M. Kim, J.‐S., Mitchell, S., Kijek, P., Borysko, K. Z., Breitenbach, J.M., Drach, J.C., Kashemirov, B.A., and McKenna, C.E. 2007. Synthesis and biological activation of an ethylene glycol‐linked amino acid conjugate of cyclic cidofovir. Bioorg. Med. Chem. Lett. 17:583‐586.
   Eriksson, U., Peterson, L.W., Kashemirov, B.A., Hilfinger, J.M., Drach, J.C., Borysko, K.Z., Breitenbach, J.M., Kim, J.S., Mitchell, S., Kijek, P., and McKenna, C.E. 2008. Serine peptide phosphoester prodrugs of cyclic cidofovir: Synthesis, transport, and antiviral activity. Mol. Pharmaceutics 5:598‐609.
   Holý, A. 2003. Phosphonomethoxyalkyl analogs of nucleotides. Curr. Pharm. Des. 9:2567‐2592.
   Hostetler, K.Y. 2009. Alkoxyalkyl prodrugs of acyclic nucleoside phosphonates enhance oral antiviral activity and reduce toxicity: Current state of the art. Antiviral Res. 82:A84‐A98.
   Krečmerová, M., Holý, A., Andrei, G., Pomeisl, K., Tichý, T., Břehová, P., Masojídková, M., Dračínský, M., Pohl, R., Laflamme, G., Naesens, L., Hui, H., Cihlar, T., Neyts, J., De Clercq, E., Balzarini, J., and Snoeck, R. 2010. Synthesis of ester prodrugs of 9‐(S)‐[3‐hydroxy‐2‐(phosphonomethoxy)propyl]‐2,6‐diaminopurine (HPMPDAP) as anti‐poxvirus agents. J. Med. Chem. 53:6825‐6837.
   Krylov, I.S., Peterson, L.W., Kashemirov, B.A., Breitenbach, J.M., Borysko, K.Z., Drach, J.C., Kim, J.S., Hilfinger, J.M., and McKenna, C.E. 2009. In vitro transport, activation and antiviral evaluation of new HPMPA prodrugs synthesized on a solid support. Antiviral Res. 82:A75.
   McKenna, C.E., Peterson, L.W., Kashemirov, B.A., Serpi, M., Mitchell, S., Kim, J.S., Hilfinger, J.M., and Drach, J.C. 2009. New peptidomimetic prodrugs of acyclic and cyclic cidofovir: SAR studies of chemical and enzymatic activation mechanisms. Antiviral Res. 82:A75.
   Mendel, D.B., Cihlar, T., Moon, K., and Chen, M.S. 1997. Conversion of 1‐[((S)‐2‐hydroxy‐2‐oxo‐1,4,2‐dioxaphosphorinan‐5‐yl)methyl]cytosine to cidofovir by an intracellular cyclic CMP phosphodiesterase. Antimicrob. Agents Chemother. 41:641‐646.
   Novachek, K.A. and Myers, A.I. 1996. A convenient procedure for the reduction of S‐(+)silyl serine methyl ester to chiral serinol derivatives. Tetrahedron Lett. 37:1743‐1746.
   Peterson, L.W. and McKenna, C.E. 2009. Prodrug approaches to improving the oral absorption of antiviral nucleotide analogues. Expert Opin. Drug Deliv. 6:405‐420.
   Peterson, L.W., Sala‐Rabanal, M., Krylov, I.S., Serpi, M., Kashemirov, B.A., and McKenna, C.E. 2010 Serine side chain‐linked peptidomimetic conjugates of cyclic HPMPC and HPMPA: Synthesis and interaction with hPEPT1. Mol. Pharm. In press.
   Soul‐Lawton, J., Seaber, E., On, N., Wootton, R., Rolan, P., and Posner, J. 1995. Absolute bioavailability and metabolic disposition of valaciclovir, the L‐valyl ester of acyclovir, following oral administration to humans. Antimicrob. Agents Chemother. 39:2759‐2764.
   Zakharova, V.M., Krylov, I.S., Serpi, M., Kashemirov, B.A., and McKenna, C.E. 2010. Approaches to tyrosine‐linked peptidomimetic prodrugs of (S)‐HPMP‐based acyclic nucleoside phosphonates. Phosphorus, Sulfur, Silicon and the Rel. Elem. Accepted.
PDF or HTML at Wiley Online Library