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Synthesis of Peptidomimetic Conjugates of Cyclic Nucleoside Phosphonates

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Abstract
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Abstract

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

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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

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

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.3 a )
- Methyl N ‐(tert ‐butoxycarbonyl)‐(L)‐tyrosinate ( S.3 b )
- Methyl N ‐(tert ‐butoxycarbonyl)‐(L)‐valyl‐L‐serinate ( S.3 c ; see protocol 3 )
- 2‐Hydroxyethyl‐N ‐(tert ‐butoxycarbonyl)‐(L)‐valinate ( S.3 d ; see protocol 3 )
Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; Aldrich)
Diethylether (Et 2 O), ACS reagent grade
Dichloromethane (CH 2 Cl 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 3 CN), 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.6 a , see protocol 3 )
Methyl O‐[tert‐butyl(dimethyl)silyl]‐(D )‐serinate ( S.6 b , see protocol 3 )
Isopropyl O‐[tert‐butyl(dimethyl)silyl]‐(L )‐serinate ( S.6 c , 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

N ‐tert‐ Butoxycarbonyl‐(L )‐valine hydrochloride (Fluka)
(L )‐Serine methyl ester hydrochloride (Aldrich)
Ethan‐1,2‐diol (ethylene glycol)
1‐Hydroxybenzotriazole hydrate (HOBt; Aldrich)
Triethylamine (Et 3 N; 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 2 SO 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

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Figure 15.4.1 Synthesis of peptidomimetic cHPMPC and cHPMPA conjugates (S.5 a‐d ) from ( S )‐HPMPC (S.1 ) or ( S )‐HPMPA (S.2 ) and amino acid (S.3 a,b ), dipeptide (S.3 c ), or ethylene glycol–linked amino acid (S.3 d ).

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Figure 15.4.2 Synthesis of peptidomimetic cyclic ( S )‐HPMPA conjugates (S.7 a‐c ) from ( S )‐HPMPA (S .2) and amino acid (S.6 a‐c ) on solid support.

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Figure 15.4.3 Synthesis of the peptidomimetics promoieties S.3 c,d and S.6 a‐c .

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Literature Cited

Literature Cited
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