Synthesis of 5′-methylene-phosphonate furanonucleoside prodrugs: Application to D-2′-deoxy-2′-α-fluoro-2′-β-C-methyl nucleosides

Article abstract of DOI:10.1021/ol301937v

A new and facile synthetic pathway to metabolically stable 5′-methylene-bis(pivaloyloxymethyl)(POM)phosphonate furanonucleoside prodrugs is reported. The key step involves a Horner-Wadsworth-Emmons reaction of a tetra(pivaloyloxymethyl) bisphosphonate salt with appropriately protected 5′-aldehydic nucleosides. This efficient approach was applied for the synthesis HCV related 2′-deoxy-2′-α-fluoro-2′-β-C- methyl nucleosides.

Full text of DOI:10.1021/ol301937v

ORGANIC
LETTERS
Synthesis of 50-Methylene-Phosphonate
Furanonucleoside Prodrugs: Application
to D‑20-Deoxy-20-r-fluoro-20-β‑C‑methyl
Nucleosides
2012
Vol. 14, No. 17
4426–4429
Ugo Pradere,† Franck Amblard,† Steven J. Coats,‡ and Raymond F. Schinazi*,†
Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of
Pediatrics, Emory University School of Medicine and Veterans Affairs Medical Center,
Decatur, Georgia 30033, United States, and RFS Pharma, LLC, 1860 Montreal Road,
Tucker, Georgia 30084, United States
rschina@emory.edu
Received July 12, 2012
ABSTRACT
A new and facile synthetic pathway to metabolically stable 50-methylene-bis(pivaloyloxymethyl)(POM)phosphonate furanonucleoside prodrugs is
reported. The key step involves a HornerꢀWadsworthꢀEmmons reaction of a tetra(pivaloyloxymethyl) bisphosphonate salt with appropriately
protected 50-aldehydic nucleosides. This efficient approach was applied for the synthesis HCV related 20-deoxy-20-R-fluoro-20-β-C-methyl nucleosides.
Over the past 40 years, nucleoside analogues have
become essential agents for the treatment of various infec-
tious diseases such as human immunodeficiency virus
(HIV), herpes simplex virus (HSV), hepatitis C virus
(HCV) and hepatitis B virus (HBV). Their mechanism of
action generally involves the direct or delayed chain termi-
nation of viral DNA or RNA elongation by incorporation
of nucleoside triphosphate analogues. The biotransforma-
tion of nucleosides into their corresponding triphosphates
involves three successive phosphorylation steps catalyzed
by viral and/or cellular kinases, and in many cases, the
limiting step in this process is the conversion to the
corresponding 50-monophosphate.1 Within the nucleoside
analogues family, nucleoside phosphonates have emerged
as an important class since the discovery of compounds
such as tenofovir, adefovir and cidofovir.2 Because a carbonꢀ
phosphorus bond replaces the 50-oxygenꢀphosphorus bond,
nucleoside phosphonates are more stable analogues of
nucleoside monophosphates and can, in theory, bypass
the first limiting phosphorylation step. However, as with
their phosphate counterparts, the ionic character of these
phosphonic acid derivatives is generally an obstacle for
absorption and/or cellular penetration, which often trans-
lates into a lack of in vivo activity for these compounds. In
order to mask the negative charges, phosphonate prodrugs
bearing biolabile protecting groups, such as pivaloyloxy-
methyl (POM) and isopropyloxymethyl groups (POC),
have been developed and incorporated into the FDA
approved adefovir dipivoxil 2 and tenofovir diisoproxil
fumarate 3, which are utilized clinically for the treatment
of HBV and HIV infections, respectively (Figure 1).2
The apparently simple replacement of the oxygen atom
by a carbon atom, to form 50-methylene nucleoside phos-
phonates prodrugs, is actually a low yielding multistep
sequence. Synthesis of such compounds involves (1)
introduction of a simple phosphonate moiety; (2) a tri-
methylsilyl bromide driven cleavage of the phosphonate
esters; (3) purification of highly polar phosphonic acids;
and (4) variable yields when coupling phosphonic acids
with the prodrug portion. A recently published exception
to this sequence is the approach developed by Agrofoglio
† Emory University School of Medicine
‡ RFS Pharma, LLC
(1) (a) De Clercq, E. Nat. Rev. Microbiol. 2004, 2, 704. (b) Cihlar, T.;
Ray, A. S. Antiviral Res. 2010, 85, 39.
ꢀ
(2) De Clercq, E.; Holy, A. Nat. Rev. Drug Discovery 2005, 4, 928.
r
10.1021/ol301937v
Published on Web 08/23/2012
2012 American Chemical Society

We sought to extend the approach of Montgomery and
Hewson9 by preparing a dialkyl bisphosphonate where the
alkyl portion was the desired final prodrug substituent.
Utilization of such a reagent in a modified HWE reaction
followed by reduction would eliminate the traditional4,12
and often problematic phosphate ester cleavage and also
make the overall synthesis more convergent by shifting the
coupling of the phosphonic acid with the prodrug portion
to the HWE reagent synthesis.
Herein, we report a general method to prepare nucleo-
side 50-methylene bis(POM)-phosphonate prodrugs that
allows (1) direct introduction of the phosphonate moiety
bearing the biolabile POM leaving group and (2) apparent
compatibility with most 50-aldehydic nucleosides, indepen-
dent of the nucleobase by condensation of tetra(POM)-
bisphosphonate B via a modified HWE olefination
(Figure 2). As part of our HCV research program, we
applied this strategy to the 20-deoxy-20-R-fluoro-20-β-C-
methyl sugar derivatives whose most advanced member,
PSI-7977 (GS-7977),13,14 is currently in phase III clinical
trials as a safe and effective anti-HCV agent (Figure 1).15
The required tetra(POM)-bisphosphonate (TPBP, 5)
was prepared by a reported method from tetramethyl
bisphosphonate 4 and chloromethyl pivalate in presence
of sodium iodide (Scheme 1).16 Previous studies involving
5 have been limited to its use as a methylenebisphospho-
nate prodrug for bone resorption disorders16 and the
closely related mimics of farnesyl diphosphate.17 The use
of tetra substituted bisphosphonates in modified HWE
reactions have, to date, been limited to CH3-, CH3CH2-,
CF3CH2-, i-Bu-, Ph-, and Bn-substitutions.
Figure 1. Biologically active phosphonate and phosphorami-
date nucleosides.
in which butenyl acylic nucleoside phosphonate prodrugs
are prepared using an olefin cross metathesis reaction with
POM/POC protected allylphosphonates.3
Simple 50-methylene-alkylphosphonates are most often
preparedvia aninitial Wittigreaction between 50-aldehydic
nucleosides and dialkyl ((triphenylphosphoranylidene)-
methyl)phosphonate reagents.4,5 Alternative synthetic path-
ways include (1) Arbuzov condensation on a 50-deoxy-60-
halo-sugar, subsequent glycosylation and deprotection;6 (2)
30,40-β-oxetane ring-opening with alkylphosphonate anions
followed by 30-hydroxy inversion;7 and (3) 40-C-radical gen-
eration by photolysis of the 40-(2-thiopyridone) ester (Barton
reaction) via protection and 50-oxidation to the carboxylate.8
An attractive but surprisingly underexploited synthetic path-
way for the synthesis of 50-methylene-nucleoside phospho-
nates reported by Montgomery, involves a modified Hornerꢀ
WadsworthꢀEmmons (HWE) olefination by addition of
dialkyl bisphosphonate salt on a 50-aldehydic sugar and its
subsequent glycosylation.9 A similar approach was used later
by Blackburn and Rashid for the synthesis of 3-phospho-
D-glyceric acid analogues starting from a β-D-ribopentodialde-
hydo-l,4-furanoside10 and by Van Calenbergh for the synth-
esis of 6-substituted uridine phosphonic acid analogues.11
Figure 2. Retrosynthetic analysis.
(3) (a) Topalis, D.; Pradere, U.; Roy, V.; Caillat, C.; Azzouzi, A.; Broggi,
J.; Snoeck, R.; Andrei, G.; Lin, J.; Ericksson, S.; Alexandre, J. A. C.;
El-Amri, C.; Deville-Bonne, D.; Meyer, P.; Balzarini, J.; Agrofoglio, L. A.
J. Med. Chem. 2011, 54, 222. (b) Pradere, U.; Clavier, H.; Roy, V.; Nolan,
S. P.; Agrofoglio, L. A. Eur. J. Org. Chem. 2011, 36, 1712.
(4) For recent reports using this procedure see: (a) Gallier, F.;
Alexandre, J. A. C.; El Amri, C.; Deville-Bonne, D.; Peyrottes, S.;
Perigaud, C. ChemMedChem. 2011, 6, 1094. (b) Kim, B.-S.; Kim, B.-T.;
Hwang, K.-J. Bull. Korean Chem. Soc. 2010, 31, 1643. (c) Cosyn, L.; Van
Calenbergh, S.; Joshi, B. V.; Ko, H.; Carter, R. L.; Harden, T. K.;
Jacobson, K. A. Bioorg. Med. Chem. Lett. 2009, 19, 3002. (d) Suk, D.-H.;
Bonnac, L.; Dykstra, C. C.; Pankiewicz, K. W.; Patterson, S. E. Bioorg.
Med. Chem. Lett. 2007, 17, 2064. (e) Koh, Y.-H.; Shim, J. H.; Wu, J. Z.;
Zhong, W.; Hong, Z.; Girardet, J.-L. J. Med. Chem. 2005, 48, 2867.
(5) (a) Jones, G. H.; Moffatt, J. G. J. Am. Chem. Soc. 1968, 90, 5337.
(b) Jones, G. H.; Hamamura, E. K.; Moffatt, J. G. Tetrahedron Lett.
1968, 9, 5731.
(7) (a) Tanaka, H.; Fukui, M.; Haraguchi, K.; Masaki, M.; Miyasaka,
T. Tetrahedron Lett. 1989, 30, 2567. (b) Hutter, D.; Blaettler, M. O.;
Benner, S. A. Helv. Chim. Acta 2002, 85, 2777. (c) Barral, K.; Priet, S.; De
Michelis, C.; Sire, J.; Neyts, J.; Balzarini, J.; Canard, B.; Alvarez, K. Eur.
J. Med. Chem. 2010, 45, 849.
(8) Barton, D. H. R.; Gero, S. D.; Quiclet-Sire, B.; Samadi, M. J. Chem.
Soc., Chem. Commun. 1989, 1000. (b) Barton, D. H. R.; Gero, S. D.; Quiclet-
Sire, B.; Samadi, M. Tetrahedron Lett. 1989, 30, 4969. (c) Barton, D. H. R.;
Gero, S. D.; Quiclet-Sire, B.; Samadi, M. Tetrahedron 1992, 48, 1627.
(9) Montgomery, J. A.; Hewson, K. Chem. Commun. 1969, 15.
(10) Blackburn, G. M.; Rashid, A. J. Chem. Soc., Chem. Commun.
1989, 40.
(11) Nencka, R.; Sinnaeve, D.; Karalic, I.; Martins, J. C.; Van
Calenbergh, S. Org. Biomol. Chem. 2010, 8, 5234.
(12) Mackman, R. L.; Ray, A. S.; Hui, H. C.; Zhang, L.; Birkus, G.;
Boojamra, C. G.; Desai, M. C.; Douglas, J. L.; Gao, Y.; Grant, D.;
Laflamme, G.; Lin, K.-Y.; Markevitch, D. Y.; Mishra, R.; McDermott,
M.; Pakdaman, R.; Petrakovsky, O. V.; Vela, J. E.; Cihlar, T. Bioorg.
Med. Chem. 2010, 18, 3606.
(6) (a) Padyukova, N. S.; Karpeisky, M. Y.; Kolobushkina, L. I.;
Mikhailov, S. N. Tetrahedron Lett. 1987, 28, 3623. (b) Mikhailov, S. N.;
Paoyukova, N. S.; Karpeiskii, M. Y.; Kolobushkina, L. I.; Beigelman,
L. N. Collect. Czech. Chem. Commun. 1989, 54, 1055. (c) Ioannidis, P.;
Classon, B.; Samuelsson, B.; Kvarnstrom, I. Acta Chem. Scand. 1991,
45, 746.
Org. Lett., Vol. 14, No. 17, 2012
4427

trans-bis(POM)-vinylphosphonate 8 was obtained as the
major isomer (E/Z ratio of 95:5 as determined by 1H NMR).
Inordertogeneralizeour strategy, thisroutewas applied
to the preparation of synthetically more challenging cyto-
sine (C), adenine (A), guanine (G), and 2,6-diamino purine
20-F-20-C-Me nucleoside bis(POM)-phosphonate analogues
22ꢀ25. For each nucleoside base, selective protections were
utilized to be compatible with the key oxidation/HWE
reaction sequence. Thus, cytosine derivative 9 was first
treated with TBSCl and then with 2 equiv of Boc2O to give
the fully protected compound 10. Finally, selective deprotec-
tion using trichloroacetic acid (TCA) afforded the 30-OTBS-
N4-Boc nucleoside 11 in 81% overall yield with no observed
Boc cleavage. A similar sequence was applied to prepare
30-OTBS-N6-diBoc adenine nucleoside 14. The exocyclic
amine was bis-Boc protected in high yield by treatment with
excess Boc2O in presence of N,N-dimethylaminopyridine
(DMAP).21 The TCA mediated deprotection gave the corre-
sponding 50-hydroxyl product 14 in an overall 74% yield with
both Boc groups intact. In order to prepare guanosine and
2,6-diaminopurine phosphonate derivatives 19 and 20, we
started from the 6-chloro precursor 16, which was synthesized
from 1522 in 89% yield by persilylation of the sugar hydroxy
groups using 5 equiv of TBSCl in presence of imidazole.
Substitution of the 6-chloro of nucleoside analogue 16 with
either sodium benzyloxide or sodium azide followed by
2-amino protection with Boc2O led to compounds 17 and
18. Selective 50-deprotection with TCA afforded the desired
protected intermediates 19 and 20 readily available for con-
version to bis(POM)-phosphonate prodrugs (Scheme 3).
Scheme 1. Synthesis of Tetra(POM)-bisphosphonate (TPBP) 5
In consideration of the chemical stability of POM
groups, the selection of appropriate protective groups for
our strategy was crucial and required investigation. We sought
a protecting group that (1) can be selectively introduced on
the 30-position of the sugar in high yield; (2) is stable under
oxidative and bisphosphonate salt addition conditions; and
(3) can easily be removed without affecting the POM groups.
t-Butyldimethylsilyl (TBS) groups appeared to fit these
criteria. Therefore, 30-OTBS-20-C-Me-20F-uridine derivative
7 was prepared as outlined in Scheme 2, by persilylation
of 618 with TBSCl followed by selective 50-desilylation in
presence of aqueous trichloroacetic acid (TCA).19
Scheme 2. Introduction of 30-TBS Group and Oxidation Suitability
Scheme 3. Synthesis of Protected Nucleosides 11, 14, 19, and 20
Oxidation of the 30-OTBS derivative 7 was conducted
with both DessꢀMartin periodinane (DMP) at room
temperature and 2-iodoxybenzoic acid (IBX)20 at 80 °C
(Scheme 2). The IBX oxidation of 7 afforded clean alde-
hyde in almost quantitative yield after simple filtration,
while aldehyde resulting from DMP oxidation was mixed
with several DMP byproducts. Finally, reaction between
the crude aldehyde and 2.2 equiv of TPBP 5 led to the
formation of desired bis(POM)-vinylphosphonate 8. In-
terestingly, the use of a cleaner crude intermediate alde-
hyde obtained from IBX oxidation had an impact on the
efficiency of the overall reaction sequence (60% compared
to 84%). As expected with a HWE reaction mechanism,
(13) Sofia, M. J.; Bao, D.; Chang, W.; Du, J.; Nagarathnam, D.;
Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang, H.-R;
Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Micolochick Steuer,
H. M.; Niu, C.; Otto, M. J.; Furman, P. A. J. Med. Chem. 2010, 53, 7202.
(14) This compound can now be found under the name GS-7977 as
Gilead Sciences, Inc. acquired Pharmasset, Inc. in January 2012.
(15) For a review of NS5B anti-HCV agents, see: Sofia, M. J.; Chang,
W.; Furman, P. A.; Mosley, R. T.; Ross, B. S. J. Med. Chem. 2012, 55,
2481.
€ €
(16) Vepsalainen, J. J. Tetrahedron Lett. 1999, 40, 8491.
4428
Org. Lett., Vol. 14, No. 17, 2012

gel column chromatography (except in the case of the U
analogue; entry 1) and thus had to achieve subsequent
deprotection with an 80% HCOOH/water solution to
facilitate isolation. All attempts to use more traditional
Table 1. Conversion of Protected 50-Hydroxyl Nucleosides to
50-Methylene-bis(POM)-phosphonate Prodrugs 21ꢀ25
desilylation conditions (TBAF, TBAF/HOAc, HF Et3N
3
or 1 M HCl in dioxane) led to significant or complete
degradation of the substrates. Final hydrogenation using H2
and Pd/C24 provided the desired 50-methylene bis(POM)-
phosphonate nucleosides 21ꢀ25 in yields that ranged from
60 to 71% over 4 steps and 44 to 60% from the starting
nucleosides. All five final phosphonate prodrugs were de-
void of anti-HCV,25 anti-HIV, or cytotoxic activity.26,27
In conclusion, we report herein the first highly efficient
synthesis of 50-methylene bis(POM)-phosphonate pro-
drugs through a modified HWE reaction. The key HWE
reaction of the sequence involves the addition of a tetra-
(POM) bisphosphonate sodium salt to Boc/TBDMS
protected 50-aldehydic nucleosides. The 20-deoxy-20-R-
fluoro-20-β-C-methyl-50-methylene bis(POM)-phospho-
nate nucleoside prodrugs 21ꢀ25 were obtained with
excellent overall yields that ranged from 60 to 71% over
4 steps from the corresponding protected starting materials.
Through this work we have extended the scope of tetra-
substituted bisphosphonates that take part in a modified
HWE reaction. The versatility of this approach could be
utilized for the rapid synthesis of new and diverse phos-
phonate prodrugs with superior biological activity.
Acknowledgment. This work was supported in part by
NIH Grant 5P30-AI-50409 (CFAR) and by the Depart-
ment of Veterans Affairs. We thank Tami McBrayer and
Mervi Detorio for the biological testing.
a Isolated yield over 4 steps.
Supporting Information Available. Experimental pro-
cedures and full spectroscopic data for all new com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
The IBX oxidation/TPBP sodium salt addition sequence
was successfully applied to the protected 50-OH nucleosides
11, 14, 19 and 20 (Table 1). In the oxidation of the purine
analogues 14, 19 and 20, the N,N-bis(Boc) protection
provides enough steric and/or electron-withdrawing
effectstoavoidpotentialdecomposition.23 Itisnoteworthy
that after the modified HWE reaction, we were unable
to separate the bisPOM-vinylphosphonate nucleosides
from excess tetra(POM)-bisphosphonate 5 by flash silica
(23) A reviewer has pointed out to us that generation of the 50-
aldehyde with purine nucleobases is problematic becauseof cyclonucleo-
side formation with the N-3 amino group, which leads presumably to
deglycosylation and other degradation products.
(24) While 1 h completely reduced the vinylphosphonate, several
hours were required for one pot removal of the benzyl group and the
reduction of the azido group.
(25) Stuyver, L. J.; Whitaker, T.; McBrayer, T. R.; Hernandez-
Santiago, B. I.; Lostia, S.; Tharnish, P. M.; Ramesh, M.; Chu, C. K.;
Jordan, R.; Shi., J.; Rachakonda, S.; Watanabe, K. A.; Otto, M. J.;
Schinazi, R. F. Antimicrob. Agents Chemother. 2003, 47, 244.
(26) (a) Schinazi, R. F.; Sommadossi, J. P.; Saalmann, V.; Cannon,
D. L.; Xie, M.-W.; Hart, G. C.; Smith, G. A.; Hahn, E. F. Antimicrob.
Agents Chemother. 1990, 34, 1061. (b) Stuyver, L. J.; Lostia, S.; Adams,
M.; Mathew, J.; Pai, B. S.; Grier, J.; Tharnish, P.; Choi, Y.; Chong, Y.;
Choo, H.; Chu, C. K.; Otto, M. J.; Schinazi, R. F. Antimicrob. Agents
Chemother. 2002, 46, 3854.
(27) No significant antiviral activities were found for compounds
21ꢀ25 when tested up to 100 μM against HIV in peripheral blood
mononuclear cells and up to 10 μM against HCV in a subgenomic RNA
replicon system. No cytotoxicity was observed in Huh7 cells up to 10 μM
or in PBM, human lymphoblastoid CEM, or African Green monkey
Vero cells up to 100 μM.
(17) Troutman, J. M.; Chehade, K. A. H.; Kiegiel, K.; Andres, D. A.;
Spielmann, H. P. Bioorg. Med. Chem. Lett. 2004, 14, 4979.
(18) Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharnish,
P. M.; Lostia, S.; McBrayer, T. R.; Schinazi, R.; Watanabe, K. A.; Otto,
M. J.; Furman, P. A.; Stec, W. J.; Patterson, S. E.; Pankiewicz, K. W.
J. Med. Chem. 2005, 48, 5504.
(19) Zhu, X.-F.; Williams, H. J.; Scott, A. I. Synth. Commun. 2003,
33, 2011.
(20) More, J. D.; Finney, N. S. Org. Lett. 2002, 4, 3001.
(21) Ikeuchi, H.; Meyer, M.; E.; Ding, Y.; Hiratake, J.; Richards, N.;
G.; J. Bioorg. Med. Chem. 2009, 17, 6641.
(22) (a) Du, J.; Nagarathnam, D.; PamulapatiG. R.; Ross B. S.; Sofia
M. J. WO2009152095. (b) Pamulapati, G. R.; Chun, B.-K.; Zhang,
H.-R.; Rachakonda, S.; Ross, B. S.; Sofia, M. J. J. Org. Chem. 2011, 76,
3782. (c) Clark, J. L.; Mason, J. C.; Hollecker, L.; Stuyver, L. J.;
Tharnish, P. M.; McBrayer, T. R.; Otto, M. J.; Furman, P. A.; Schinazi,
R. F.; Watanabe, K. A. Bioorg. Med. Chem. Lett. 2006, 16, 1712.
The authors declare the following competing financial interest(s): Dr.
Schinazi is the founder and a major shareholder of RFS Pharma, LLC.
Emory received no funding from RFS Pharma, LLC to perform this
work and vice versa.
Org. Lett., Vol. 14, No. 17, 2012
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