Synthesis and biological evaluation of acyclic phosphonic acid nucleoside derivatives

Article abstract of DOI:10.1080/15257770.2013.820833

As part of a project to generate a library of nucleosides as potential antiviral agents, a small subset of novel acyclic phosphonic acid nucleosides was prepared. Practical synthetic routes are described for three targets, which were then tested against H

Full text of DOI:10.1080/15257770.2013.820833

This article was downloaded by: [RMIT University]
On: 02 October 2013, At: 00:51
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic
Acids
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lncn20
Synthesis and Biological Evaluation of
Acyclic Phosphonic Acid Nucleoside
Derivatives
Philip Wainwright a , Adrian Maddaford a , Xiurong Zhang a , Helen
Billington a , David Leese a , Rebecca Glen a , David C. Pryde b ,
Donald S. Middleton b , Peter T. Stephenson b & Scott Sutton c
a Peakdale Molecular Ltd., Chapel-en-le-Frith , Derbyshire , United
Kingdom
b Worldwide Medicinal Chemistry, Pfizer Neusentis , Cambridge ,
United Kingdom
c Pfizer Global Research and Development , San Diego , California ,
USA
To cite this article: Philip Wainwright , Adrian Maddaford , Xiurong Zhang , Helen Billington ,
David Leese , Rebecca Glen , David C. Pryde , Donald S. Middleton , Peter T. Stephenson & Scott
Sutton (2013) Synthesis and Biological Evaluation of Acyclic Phosphonic Acid Nucleoside Derivatives,
Nucleosides, Nucleotides and Nucleic Acids, 32:9, 477-492, DOI: 10.1080/15257770.2013.820833
To link to this article: http://dx.doi.org/10.1080/15257770.2013.820833
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &

Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Nucleosides, Nucleotides and Nucleic Acids, 32:477–492, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.820833
SYNTHESIS AND BIOLOGICAL EVALUATION OF ACYCLIC
PHOSPHONIC ACID NUCLEOSIDE DERIVATIVES
Philip Wainwright,1 Adrian Maddaford,1 Xiurong Zhang,1 Helen Billington,1
David Leese,1 Rebecca Glen,1 David C. Pryde,2 Donald S. Middleton,2
Peter T. Stephenson,2 and Scott Sutton3
1Peakdale Molecular Ltd., Chapel-en-le-Frith, Derbyshire, United Kingdom
2Worldwide Medicinal Chemistry, Pfizer Neusentis, Cambridge, United Kingdom
3Pfizer Global Research and Development, San Diego, California, USA
As part of a project to generate a library of nucleosides as potential antiviral agents, a small
2
subset of novel acyclic phosphonic acid nucleosides was prepared. Practical synthetic routes are
described for three targets, which were then tested against HIV, hepatitis C virus (HCV), and Dengue
virus.
Keywords Antivirals; acyclic phosphonic acid nucleosides
INTRODUCTION
Acyclic phosphonic acid nucleosides, in which the 5’ hydroxyl group has
been replaced by a phosphonate group, have been shown to have promising
antiviral activity.[1] Among these analogues are bis(POC)PMPA 1 (Tenofovir
disoproxil) as an anti-HIV agent, HPMPC 2 (Cidofovir) that exhibits activity
against herpes viruses, and PMEA 3 (Adefovir, PMEA) used to treat infections
of the hepatitis B virus.
Received 31 May 2013; accepted 26 June 2013.
Address correspondence to Philip Wainwright, Ph.D., Peakdale Molecular Ltd., Peakdale Sci-
ence Park, Sheffield Road, Chapel-en-le-frith, Derbyshire SK23 0PG, United Kingdom. E-mail:
phil.wainwright@peakdale.co.uk
477

478
P. Wainwright et al.
The phosphorylation sequence for nucleosides is often a three-step pro-
cess and performed by more than one cellular kinase. The initial phos-
phorylation is often the most difficult step and strategies to facilitate or
bypass include prodrugs or inserting the initial monophosphate as part of
the nucleoside. Phosphonic acids are more chemically stable analogues of
the monophosphate, and in some cases still undergo the phosphorylation
and generate effective mimics of nucleoside triphosphates.
As part of a project to generate a library of nucleosides as potential
antiviral agents, we have been investigating synthetic procedures that allow
us to access a range of phosphonic acid nucleoside analogues.[2] A subset
of these is the novel phosphonic acid acyclic nucleosides 4–6 (adenine base
provided as an example).
As we wished to prepare an array of nucleosides 4–5, it was necessary to
prepare a synthon that could be coupled to a wide selection of purine and
pyrimidine bases. The synthetic strategy undertaken was to target the corre-
sponding bromide and introduce the base units by alkylation (Figure 1).
FIGURE 1 Retrosynthetic approach to phosphonic acid nucleosides 4 and 5.
Although they were relatively structurally simple, it was necessary to pre-
pare large (>10 g) quantities of the bromides (7, 8) to provide sufficient
material for the array of target nucleosides. This required the development
of a robust synthesis that was amenable to a multi-hundred gram scale.
Due to the more complex nature of 6, a longer synthesis was required
and a smaller set of nucleosides was therefore targeted (uracil and adenine).
We have previously reported[3] the synthesis of 10 and wished to utilize this
intermediate to obtain 6 via the phosphonate ester 9 (Figure 2).

Synthesis of Acyclic Phosphonic Acid Nucleosides
479
FIGURE 2 Retrosynthetic approach to phosphonic acid nucleosides 6a.
RESULTS AND DISCUSSION
For target 4, the strategy was to alkylate cyclopropyl acetic acid with 1,2-
dibromoethane to access the desired carbon skeleton (Scheme 1). From
there manipulation of the acid to the bromide should provide the precursor
to the nucleoside couplings.
SCHEME 1 Reagents and conditions: (a) P(OiPr3), reflux; (b) LDA, THF, 5◦C to 50◦C; (c) BH3, THF,
5◦C; (d) MsCl, NEt3, DCM, 5◦C; (e) LiBr, CH3CN, 75◦C; (f) LiH, DMF, adenine or N-acetyl cytosine; (g)
TMSBr, CH3CN.
Reaction of 1,2-dibromoethane (11) with triisopropyl phosphite[4] on a
150 g scale yielded 12 in a 71% yield. To form the dilithium salt of cyclopropyl
acetic acid, it was necessary to react the acid initially at 5◦C and then at 50◦C
for 1 hour. The salt was then reacted with 12 at 50◦C to yield, after workup,
13 in 63% yield, with no purification required. The acid was reduced with

480
P. Wainwright et al.
borane and the crude product immediately treated with methanesulfonyl
chloride to give 15 in 56% overall yield. Rather than treating 15 with the
adenine or cytosine direct, it was found that cleaner reactions were obtained
utilizing the bromide 7. Reaction of 15 with lithium bromide gave 48 g of
7 in 84% yield. This provided more than enough material for the array and
was coupled to a selection of pyrimidine and purine bases[5]; conditions are
provided for cytosine and adenine as representative examples. Reaction of
N-acetyl cytosine with 7 proceeded smoothly, although the N-acetyl group of
16b was hydrolyzed during the reaction.
For target 5, the carbon chain could be created in one step from α-
methyl-butyrolactone[6] (Scheme 2). The bromide could be used as a handle
to introduce the phosphonate group, and the acid to provide the second
bromide for the nucleoside couplings.
SCHEME 2 Reagents and conditions: (a) PBr3, 140◦C; (b) BH3, THF, 0◦C; (c) Ac2O, NEt3, DMAP,
DCM; (d) P(OiPr)3, reflux; (e) 20% NH3 in MeOH; (f) TsCl, NEt3, DCM; (g) LiBr, 2-butanone, reflux;
(h) LiH, DMF; (i) NaOMe, MeOH; (j) TMSBr, CH3CN.

Synthesis of Acyclic Phosphonic Acid Nucleosides
481
On a 130 g scale 17 was treated with phosphorous tribromide to yield
18 in 56% yield after purification by chromatography. The carboxylic acid
of 18 was reduced to the alcohol with borane; rather than losing material in
the aqueous phase of a traditional workup, the reaction mixture was instead
evaporated to dryness followed by evaporation from methanol to remove
any volatile borates. The crude product was then immediately acetylated
with acetic anhydride to 20 and isolated without the need for chromatog-
raphy in 31% yield. The low yield was presumably due to the presence of
SCHEME 3 Reagents and conditions: (a) LiH, DMSO, 90◦C; (b) TBAF, THF; (c) I2, imidazole, PPh3,
PhCH3, reflux; (d) P(OEt)3, reflux; (e); aq 0.1M HCl; (f) NaIO4, NaBH4, dioxane, water; (g) TMSBr,
CH3CN.

482
P. Wainwright et al.
alcohol/borate complexes that were retained in the aqueous phase. The Ar-
buzov reaction on 20 gave 21 in 74% yield after purification by distillation.
This was followed by deacetylation with 20% ammonia in methanol to give
74 g of crude 22, and then directly reacted to the tosylate to yield 50 g of
23. Reaction of the tosylate with lithium bromide yielded a total of 32 g
of 8 providing ample of material for the array. Again this was treated with
a selection of pyrimidine and purine bases,[5] conditions for cytosine and
adenine are provided as representative samples.
For acyclic phosphonic acid nucleoside 6, the base unit was introduced
at the beginning of the synthesis to allow us to shorten the route by omitting
the need for unnecessary protecting groups (Scheme 3).
Adenine was treated with lithium hydride and reacted with tosylate
10 to give 25 in 55% yield. The silyl group was removed with tetra-n-
butylammonium fluoride (TBAF), and liberated hydroxyl transformed to
the iodide 27 with iodine/triphenylphosphine. The Arbuzov reaction was
carried out in 48% yield, and the acetonide of 9 was removed with aqueous
0.1M HCl. The diol 28 was cleaved with sodium periodate, which also
unexpectedly deprotected one of the phosphonate esters. The remaining
ester was removed with trimethylsilylbromide (TMSBr) to provide the final
target 6a in 52% yield. The same approach was used to access the uracil
analogue (Scheme 4).
SCHEME 4
All prepared phosphonic acid nucleosides were tested in a cell-based
hepatitis C virus (HCV) replicon assay, a cell-based HIV replication screen,
and a Dengue Virus genotype 2 antiviral cell-based screen. None were shown
to have any appreciable activity.
CONCLUSION
We have developed efficient reliable synthetic routes to three new classes
of acyclic phosphonic acid nucleosides. Although none of the nucleosides
showed antiviral activity in the three assays screened, they may be of interest

Synthesis of Acyclic Phosphonic Acid Nucleosides
483
to other areas of medicinal research where such classes of nucleosides have
shown promise.[7]
EXPERIMENTAL
General
All purchased materials and reagents were used without further pu-
1
rification unless noted. H NMR spectral data were recorded on a JEOL
ECX300 MHz spectrometer. Chemical shifts are expressed in parts per mil-
lion (ppm). Splitting patterns describe apparent multiplicities and are des-
ignated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
or br s (broad singlet). Liquid chromatography–mass spectra (LCMS) was
recorded on a Waters 2795-ZQ instrument (Waters Limited, 730-740 Cen-
tennial Court, Centennial Park, Elstree, Hertfordshire, WD6 3SZ, United
Kingdom). Purification was performed by flash chromatography using Flu-
orochem silicagel LC60A 40–63 micron (Fluorochem, Unit 14, Graphite
Way, Hadfield, Derbyshire, SK13 1QH, United Kingdom), or where noted
by reverse phase high performance liquid chromatography (HPLC) on a
Waters 2525 Binary Gradient module with a Waters 2487 Dual λ absorbance
detector (set at 254 and 210 nm) with a X-Terra C18 column.
Dipropyl (2-bromoethyl)phosphonate (12)
A mixture of 1,2-dibromoethane (400 g, 2.1 mol) and triisopropylphos-
phite (146.2 g, 0.70 mol) was heated at reflux for 4 hours, cooled, and the
product was distilled at 80◦C/1 mmHg to give 12 (135.5 g, 71%) as a clear
colorless oil. 1H NMR (300 MHz, CDCl3) δ 1.29 (d, 12H), 2.24–2.36 (m, 2H),
3.40–3.51 (m, 2H), 4.60–4.71 (m, 2H).
2-Cyclopropyl-4-(dipropoxyphosphoryl)butanoic acid (13)
To a solution of cyclopropylacetic acid (47.3 g, 0.473 mol) in anhydrous
tetrahydrofuran (1.4 L) was added 2.0M lithium diisopropylamine in tetrahy-
drofuran (496 mL, 0.993 mol) at 5◦C over 25 minutes. The reaction mixture
was then heated at 50◦C for 1 hour. The reaction then cooled to 5◦C and 12
(135.5 g, 0.496 mol) was added as a solution in tetrahydrofuran (200 mL)
over 15 minutes. The reaction was allowed to warm to ambient temperature
and then heated at 50◦C for 3 hours. The reaction was cooled and diluted
with water (1 L). The aqueous phase was washed with ethyl acetate (1 L),
made acidic with 3M HCl, and extracted with ethyl acetate (3×1 L). This
was dried over magnesium sulphate, filtered, and concentrated to give 13
1
(91.9 g, 63%) as a yellow oil. H NMR (300 MHz, CDCl3) δ 0.07–0.16 (m,
1H), 0.24–0.32 (m, 1H), 0.40–0.58 (m, 2H), 0.84–0.94 (m, 1H), 1.28 (d,
12H), 1.48–1.88 (m, 5H), 4.60–4.71 (m, 2H).

484
P. Wainwright et al.
2-Cyclopropyl-4-(dipropoxyphosphoryl)butyl methanesulfonate (15)
To a solution of 13 (91.9 g, 0.300 mol) in anhydrous tetrahydrofuran
(900 mL) at 5◦C was added a 1.0M solution of borane/tetrahydrofuran com-
plex (300 mL, 0.315 mol). The reaction was stirred for 30 minutes and then
warmed to ambient temperature and stirred for 2 hours. The reaction was
carefully diluted with methanol (100 mL) and then concentrated in vacuo.
The residue was dissolved in dichloromethane (1.0 L) and washed with satu-
rated sodium hydrogen carbonate. The organics were dried over magnesium
sulphate, filtered, and evaporated to give a yellow oil (66 g). This was redis-
solved in dichloromethane (600 mL), and triethylamine (94 mL, 0.900 mol)
was added followed by methanesulfonyl chloride (26 mL, 0.450 mol) at
5◦C. The reaction was allowed to warm to ambient temperature and stirred
overnight. The reaction was diluted with 1M hydrochloric acid (700 mL)
and the organic phase was separated. The aqueous phase was extracted with
dichloromethane (2 × 500 mL) and the organic phases were combined,
dried over magnesium sulphate, filtered, and evaporated. The residue was
purified by flash chromatography (19:1 dichloromethane:methanol) to give
15 (60 g, 56%) as a yellow oil. Rf 0.42 (ethyl acetate). 1H NMR (CDCl3)
0.10–0.21 (m, 2H), 0.42–0.61 (m, 3H), 0.97–1.04 (m, 1H), 1.29 (d, 12H),
1.66–1.94 (m, 4H), 3.13 (s, 3H), 4.12 (dd, 1H), 4.26 (dd, 1H), 4.60–4.71 (m,
2H).
Diisopropyl (4-bromo-3-cyclopropylbutyl)phosphonate (7)
A solution of 15 (60.0 g, 0.169 mol) and lithium bromide (29.3 g,
0.337 mol) in acetonitrile (500 mL) was heated at 70◦C for 2.5 hours. The
mixture was cooled and concentrated in vacuo. The residue was dissolved
in dichloromethane (200 mL) and washed with sodium hydrogen carbon-
ate solution (200 mL), then dried over magnesium sulphate, filtered, and
evaporated. The residue was purified by flash chromatography (1:1 ethy-
lacetate:hexane) to give 7 (48.1 g, 84%) as a clear yellow oil. Rf 0.38 (1:1
1
hexane:ethyl acetate). H NMR (CDCl3) 0.06–0.13 (m, 1H), 0.14–0.24 (m,
1H), 0.42–0.71 (m, 3H), 0.84–0.97 (m, 1H), 1.31 (d, 12H), 1.67–1.96 (m,
4H), 3.43 (dd, 1H), 3.54 (dd, 1H), 4.61–4.74 (m, 2H).
Diisopropyl (4-(6-amino-9H-purin-9-yl)-3-cyclopropylbutyl)phosphonate (16a)
To a suspension of adenine (277 mg, 2.05 mmol) in N,N-
dimethylformamide (10 mL) was added lithium hydride (20 mg, 2.46 mmol).
This was stirred under argon at 120◦C for 1 hour, and then 7 (0.7 g,
2.05 mmol) was added. The reaction was then stirred under argon at 120◦C
for further 14 hours. After cooling, the solution was concentrated in vacuo
and purified by flash chromatography (20:1 dichloromethane:methanol) to
give 16a (400 mg, 50%) as an oil. Rf 0.30 (9:1 dichloromethane:methanol).
1H NMR (CDCl3) δ −0.70 (m, 1H), 0.10 (m, 1H), 0.25 (m, 1H), 0.5 (m, 2H),

Synthesis of Acyclic Phosphonic Acid Nucleosides
485
1.25 (m, 13H), 1.8 (m, 4H), 4.15 (d, 2H), 4.75 (m, 2H), 5.8 (s, 2H), 7.80 (s,
1H), 8.35 (s, 1H).
Diisopropyl (4-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-cyclopropylbutyl)
phosphonate (16b)
Lithium hydride (47 mg, 5.9 mmol) was added to a solution of N-acetyl
cytosine (655 mg, 5.9 mmol) in N,N-dimethylformamide (10 mL) and stirred
for 30 minutes under argon at ambient temperature. 7 (2 g, 5.9 mmol) was
then added and the reaction mixture was heated to 120◦C for 16 hours. After
cooling, the reaction mixture was concentrated in vacuo and the resultant
residue purified by flash chromatography (9:1 dichloromethane:methanol)
to give 16b (1.01 g, 46%) as an oil. 1H NMR (DMSO) δ −0.20 (m, 1H), 0.05
(m, 1H), 0.25 (m, 1H), 0.40 (m, 2H), 1.0 (m, 1H), 1.20 (m, 12H), 1.45 (m,
2H), 1.60–1.90 (m, 2H), 3.60 (m, 2H), 4.50 (m, 2H), 5.60 (d, 1H), 7.0 (br
d, 2H), 7.50 (d, 2H).
(4-(6-Amino-9H-purin-9-yl)-3-cyclopropylbutyl)phosphonic acid (4a)
To a solution of 16a (400 mg, 1.01 mmol) in acetonitrile (10 mL)
was added TMSBr (0.43 mL, 3.2 mmol). The reaction mixture was stirred
overnight under argon and then the solvent was removed by rotary evapo-
ration. The residue was redissolved in acetonitrile (20 mL) and then con-
centrated by rotary evaporation again. Water (10 mL) was added to give a
suspension that was then heated at reflux. The solid was filtered while hot
and washed with water (2×10 mL). This was dried at high vacuum over
potassium hydroxide to give 4a (150 mg, 48%) as a white solid. 1H NMR
(50:50 D2O:TFA) δ −1.90–2.0 (m, 1H), −1.30–1.40 (m, 1H), −1.20–1.30
(m, 1H), −0.90–1.10 (m, 2H), −0.20 (1H), 0.25–0.80 (m, 3H), 2.05–2.2 (m,
1H), 2.90–3.10 (m, 2H), 6.95 (s, 1H), 7.20 (s, 1H). MS (ESI) m/z 312.11 [M
+ H]+. Chemical purity by HPLC, 100% (260 nm), Synergy Polar RP 4.6 ×
150 mm 4 μm, 0–40% methanol over 20 minutes then held for 5 minutes,
aqueous phase 2% trifluoroacetic acid (TFA) in water.
(4-(4-Amino-2-oxopyrimidin-1(2H)-yl)-3-cyclopropylbutyl)phosphonic
acid (4b)
Bromotrimethylsilane (10.2 mL, 8.6 mmol, 3.2 eq) was added to a solu-
tion of 16b (1.01 g, 2.7 mmol) in acetonitrile (20 mL) and stirred overnight
at ambient temperature under argon. The solvent was removed in vacuo and
the residue was added to acetonitrile (10 mL) and once again evaporated
to dryness. Water (10 mL) was added and the pH was adjusted to 4.5 with
1M sodium hydroxide solution. The solvent was removed in vacuo and the
resultant residue was purified by preparative HPLC (0–10% acetonitrile over
10 minutes, X-Terra C18). Subsequent freeze drying gave 771 mg (99%) of
4b as a white solid. Rf 0.15 (1:1:1:1:1 toluene:acetone:butanol:water:acetic

486
P. Wainwright et al.
acid). 1H NMR (DMSO-d6) δ −0.10–0.30 (m, 1H), 0.0–0.1 (m, 1H), 0.25–0.35
(m, 1H), 0.35–0.50 (m, 2H), 0.90–1.10 (m, 1H), 1.40–1.80 (m, 4H), 3.80 (m,
2H), 6.05 (d, 1H, J = 7.8 Hz), 8.0 (d, 1H, J = 7.3 Hz), 8.90 (s, 1H), 9.50 (s,
1H). MS (ESI), m/z 288.13 [M + H]+. Chemical purity by HPLC, 99.57%
(242 nm), Synergi Hydro 4.6×150 mm, 0–10% acetonitrile over 20 min-
utes then held for 5 minutes, aqueous phase 20 mM sodium dihydrogen
orthophosphate pH 2.
4-Bromo-2-methylbutanoic acid (18)
To α-methyl-γ -butyrolactone (130 g, 1.3 mol) was added phosphorous
tribromide (101 mL, 1.13 mol) and this was then heated at reflux for 8 hours.
The reaction was slowly poured onto iced water (500 mL). Dichloromethane
(500 mL) was added and this was stirred overnight at ambient temperature.
The layers were separated, dried over magnesium sulphate, filtered, and
evaporated to dryness. The resultant yellow oil was passed through a pad of
1
silica (3:1 hexane:ethyl acetate). This gave 18 (158.5 g, 67%) as an oil. H
NMR (CDCl3) δ 1.20 (d, 3 H); 1.90 (m, 1 H); 2.22 (m, 1 H); 2.75 (m, 1 H);
3.45 (t, 2 H); 11.35 (br s, 1 H).
4-Bromo-2-methylbutyl acetate (20)
To a solution of 18 (100 g, 0.55 mol) in tetrahydrofuran (420 mL) at 0◦C
and under argon was added dropwise 1M BH3-THF (770 mL, 0.77 mol). The
reaction mixture was stirred at 0◦C for 3 hours and then carefully quenched
with methanol. After stirring for 15 minutes, the mixture was rotary evapo-
rated to remove the solvent. The residue was dissolved in methanol and the
solvent was removed by rotary evaporation; this was repeated a further two
times. This gave crude 19 (80 g, 87%), which was used without further purifi-
cation. To a mixture of 4-dimethylaminopyridine (DMAP) (5.9 g, 48 mmol)
and triethylamine (133 mL, 96 mmol) in dichloromethane (200 mL) under
argon was added dropwise acetic anhydride (68 mL, 72 mmol). After stir-
ring for 5 minutes, 19 (80 g crude, 48 mmol) dissolved in dichloromethane
(100 mL) was added dropwise over 10 minutes. The reaction mixture was
stirred for 18 hours. To the reaction mixture, 10% citric acid (200 mL) was
added, this was stirred for 10 minutes, the layers were separated, and the
organic phase was dried over magnesium sulphate, and filtered. Removal of
solvent by rotary evaporation gave 20 (31 g, 31%). Rf 0.6 (1:1 ethyl acetate
in hexane). 1H NMR (CDCl3) δ 0.95 (d, 3 H), 1.72 (m, 1 H), 1.95 (m, 2 H),
2.03 (s, 3 H), 3.45 (m, 2 H), 3.92 (m, 2 H).
4-(Diisopropoxyphosphoryl)-2-methylbutyl acetate (21)
A mixture of 20 (84 g, 0.4 mol) and triisopropylphosphite (160 mL,
0.7 mol) was heated at reflux for 4 days. The remaining triisopropy-
lphosphite was removed by distillation at 26◦C, 0.1 mmHg. The product

Synthesis of Acyclic Phosphonic Acid Nucleosides
487
21 (86.5 g, 74%) was then collected at 122◦C at 0.25 mmHg. Rf 0.44 (20:1
dichloromethane:methanol). 1H NMR (CDCl3) δ 0.90 (d, 3 H), 1.27 (d, 12
H), 1.5–1.8 (m, 5 H), 2.02 (s, 3 H), 3.88 (d, 2 H), 4.64 (m, 2 H).
4-(Diisopropoxyphosphoryl)-2-methylbutyl 4-methylbenzenesulfonate (23)
A solution of 21 (86.5 g, 0.29 mol) in 20% methanol/ammonia solution
(600 mL) was stirred at ambient temperature and under argon for 48 hours.
The solvent was removed using rotary evaporation. The resultant residue was
redissolved in methanol/ammonia solution (500 mL) and left stirring at am-
bient temperature and under argon for 72 hours. The solvent was removed
using rotary evaporation to give crude 22 (74 g). This was used without
further purification. 22 (74 g, 0.293 mol) was dissolved in dichloromethane
(1.5 L) and cooled under argon to 0◦C. Triethylamine (43 mL, 0.32 mol) was
added, followed by portionwise addition of tosyl chloride (61 g, 0.32 mol).
After stirring for 18 hours at ambient temperature, a further 1.1 equivalents
of both triethylamine and tosyl chloride were added at 0◦C. After stirring
for a further 24 hours, water (1 L) was added and the organic layer was
separated and then washed twice with 2M hydrochloric acid (1 L), twice with
sodium hydrogen carbonate solution (1 L), and twice with water (1 L). After
drying, the solvent was removed by rotary evaporation. Purification by flash
chromatography (98:2 dichloromethane:methanol) gave 23 (50.4 g, 42%).
1
Rf 0.23 (20:1 dichloromethane:methanol). H NMR (CDCl3) δ 0.88 (d, 3
H), 1.25 (d, 12 H), 1.5–1.8 (m, 5 H), 2.43 (s, 3 H), 3.82 (d, 2 H), 4.65 (m, 2
H), 7.31 (d, 2 H), 7.75 (d, 2 H).
Diisopropyl (4-bromo-3-methylbutyl)phosphonate (8)
To a solution of 23 (50.4 g, 0.124 mol) in 2-butanone (700 mL) was added
lithium bromide (53.8 g, 0.62 mol). After stirring for 7 hours at reflux, the
solvent was removed by rotary evaporation. The resultant residue was diluted
with water (500 mL), extracted with dichloromethane (2 × 500 mL), dried
with magnesium sulphate, and filtered. The solvent was then removed by
rotary evaporation. Purification was achieved by gradient elution flash chro-
matography (100% hexane, 15% ethyl acetate in hexane, then 100% ethyl
acetate) to give 8 (32g, 91%). Rf 0.47 (19:1 dichloromethane:methanol). 1H
NMR (CDCl3) δ 1.01 (d, 3 H), 1.29 (d, 12 H), 1.55–1.85 (m, 5 H), 3.35 (d,
2 H), 4.67 (m, 2 H).
Diisopropyl (4-(6-amino-9H-purin-9-yl)-3-methylbutyl)phosphonate (24a)
To a suspension of adenine (500 mg, 3.7 mmol) in N,N-
dimethylformamide (20 mL) was added lithium hydride (31 mg, 3.9 mmol).
This was stirred under argon at 80◦C for 1 hour and then 8 (1.26 g, 4.4 mmol)
was added. The reaction was then stirred under argon at 120◦C for a further

488
P. Wainwright et al.
14 hours. After cooling, the solution was concentrated in vacuo and puri-
fied by flash chromatography (20:1 dichloromethane:methanol progressing
to 10:1 dichloromethane:methanol) to give 24a (609 mg, 45%) as a yellow
oil. Rf 0.5 (4:1 dichloromethane:methanol). 1H NMR (CDCl3) δ 0.9 (d, 3H,
J = 6.7 Hz), 1.3 (m, 12H), 1.4 (m, 1H), 1.7 (m, 3H), 2.0 (m, 1H), 3.5 (m,
1H), 3.6 (m, 1H), 4.7 (m, 2H), 7.80 (s, 1H), 8.35 (s, 1H). MS (ESI), m/z
370.12 [M+H]+. Chemical purity by HPLC, 96.20% (273 nm), X-Terra RP18
3×150 mm, 10–80% acetonitrile over 20 minutes then held for 5 minutes,
aqueous phase 10 mM ammonium bicarbonate pH 10.
Diisopropyl (4-(4-acetamido-2-oxopyrimidin-1(2H)-yl)-3-methylbutyl)
phosphonate (24b)
To a suspension of N-acetyl cytosine (490 mg, 3.2 mmol, Avocado)
in N,N-dimethylformamide (10 mL) was added lithium hydride (28 mg,
3.5 mmol). This was stirred under argon at 80◦C for 1 hour and then 8 (1 g,
3.5 mmol) was added. The reaction was then stirred under argon at 120◦C
for a further 4 hours. The reaction mixture was allowed to cool overnight
with continuous stirring. The filtrate was diluted with water (50 mL) and
extracted three times with dichloromethane (50 mL). The organic layers
were combined, dried over magnesium sulphate, filtered, and then concen-
trated in vacuo. The residue was further purified by flash chromatography
(20:1 dichloromethane:methanol), followed by preparative HPLC (X-terra,
acetonitrile, water). Isolation and drying gave 24b (250 mg, 22%) as a white
solid. Rf 0.54 (9:1 dichloromethane:methanol). 1H NMR (CDCl3) δ 0.9 (d,
3H), 1.29 (d, 12H), 1.45 (m, 1H), 1.6–1.9 (m, 3H), 2.1 (m, 1H), 2.26 (s,
3H), 3.61 (dd, 1H), 3.81 (dd, 1H), 4.65 (m, 2H), 7.38 (d, 1H), 7.56 (d, 1H).
Diisopropyl (4-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-methylbutyl)
phosphonate (24c)
To a solution of 24b (250 mg, 0.7 mmol) in methanol (3 mL) was added
a solution of sodium methoxide (72 mg, 1.33 mmol) in methanol (2.7 mL).
The reaction mixture was stirred under argon at room temperature for
2 days. The reaction mixture was added to a small volume of water (10 mL)
and extracted with dichloromethane (3 × 20 mL). The combined organic
layers were dried over magnesium sulphate and filtered. The solvent was
then removed in vacuo to give 24c (160 mg, 66%) as a white solid. Mp
1
183–185◦C. Rf 0.4 (9:1 dichloromethane:methanol). H NMR (CDCl3) δ
0.80 (d, 3H, J = 6.7 Hz), 1.27 (d, 12H, J = 6.2 Hz), 1.5–1.9 (m, 4H), 1.9–2.1
(m, br, 1H), 3.48 (dd, 1H, J = 8, 13.4 Hz), 3.67 (dd, 1H, J = 6.3, 13.2 Hz),
4.55–4.65 (m, 2H), 5.75 (d, 1H, J = 7.2 Hz), 7.14 (d, 1H, J = 7.2 Hz). MS
(ESI), m/z 346.18 [M+H]+. Chemical purity by HPLC, 96.36% (276 nm),
X-Terra RP18 3×150 mm, 5–95% acetonitrile over 20 minutes then held for
5 minutes, aqueous phase 10 mM ammonium bicarbonate pH 10.

Synthesis of Acyclic Phosphonic Acid Nucleosides
489
(4-(6-Amino-9H-purin-9-yl)-3-methylbutyl)phosphonic acid (5a)
To a solution of 24a (570 mg, 1.54 mmol) in acetonitrile (6 mL) in an ice-
bath was added bromotrimethylsilane (0.65 mL, 4.93 mmol). This caused
precipitation of a white solid. This suspension was stirred under argon at
ambient temperature overnight. The reaction mixture was concentrated in
vacuo. The residue was dissolved in a further 10 mL of acetonitrile, which
was again removed in vacuo. The residue was then taken up in water and
basified to pH 4–5. This was passed through a pre-conditioned Isolute SAX
cartridge (0.3M acetic acid 100 mL followed by water 100 mL) and then
washed with water (5 × 5 mL) and 10% TFA in water (5 × 5 mL). The
fractions containing product were then concentrated by rotary evaporation.
This gave 5a (255 mg, 58%) as an orange oil. 1H NMR (DMSO-d6) δ 0.79 (d,
3H, J = 6.6 Hz), 1.2–1.7 (m, 4H), 2.0–2.1 (m, 1H), 4.05–4.15 (m, 2H), 4.89
(br s, 2H), 8.45 (s, 1H), 8.50 (s, 1H), 8.72 (br s, 1H), 9.62 (br s, 1H). MS (ESI),
m/z 286.09 [M+H]+. Chemical purity by HPLC, 98.50% (254 nm), Synergi
Hydro RP 18 3×150 mm, 0–100% 20% methanol in 20 mM ammonium
acetate at pH 3.65 over 20 minutes then held for 5 minutes, aqueous phase
20 mM ammonium acetate pH 3.65.
(4-(4-Amino-2-oxopyrimidin-1(2H)-yl)-3-methylbutyl)phosphonic acid (5b)
Bromotrimethylsilane (0.18 mL, 1.53 mmol) was added to a solution of
24c (165 mg, 0.48 mmol) in acetonitrile (5 mL). The reaction mixture was
stirred overnight at room temperature under argon. After concentration in
vacuo, the residue was dissolved in water (10 mL) and the pH was adjusted to
4.5 with 1M sodium hydroxide. The solvent was removed in vacuo. The resul-
tant residue was purified by preparative HPLC (Synergy-hydro RP 80A, 0.1%
TFA in water/acetonitrile). Subsequent freeze drying gave 5b (106 mg, 85%)
as a pale green solid. Rf 0.25 (1:1:1:1:1 toluene:acetone:butanol:water:acetic
1
acid). H NMR (DMSO-d6) δ 0.15 (d, 3H, J = 6.3 Hz), 0.3–1.2 (m, 5H),
2.85–2.95 (m, 1H), 3.0–3.1 (m, 1H), 5.35 (d, 1H, J = 7.4 Hz), 7.02 (d, 1H,
J = 7.5 Hz). MS (ESI), m/z 262.16 [M+H]+. Chemical purity by HPLC,
96.44% (284 nm), Synergy-hydro RP 18 4.6 × 150 mm, 4 μm, 0–15% ace-
tonitrile over 20 minutes then held for 5 minutes, aqueous phase 20 mM
sodium dihydrogen phosphate pH 2.
9-(((3aS,4S,6R,6aR)-6-(((Tert-butyldiphenylsilyl)oxy)methyl)-2,
2-dimethyltetrahydrofuro [3,4-d][1,3]dioxol-4-yl)methyl)-9H-purin-6-
amine (25)
To a suspension of adenine (7.34 g, 54.4 mmol) in dimethylsulfoxide
(30 mL) was added lithium hydride (399 mg, 49.8 mmol). This was stirred
under argon at 90◦C for 1/2 hour and then 10 (27.0 g, 45.3 mmol) was added.
The reaction was then stirred under argon at 90◦C for a further 7 hours. The
solution was diluted with water (1 L), and washed with dichloromethane
(3×1.5 L). The combined dichloromethane solution was then washed with

490
P. Wainwright et al.
water (2×1 L), and dried over magnesium sulphate and filtered. The solvent
was removed and the product was purified by flash chromatography (20:1
dichloromethane:methanol) to give 25 (13.86 g, 55%) as a white solid. Rf
0.4 (2% methanol in ethylacetate); 1H NMR (CDCl3) δ 1.06 (s, 9H), 1.32 (s,
3H), 1.51 (s, 3H), 3.79 (m, 2H), 4.10 (q, 1H), 4.26 (m, 2H), 4.45 (m, 1H),
4.53 (m, 1H), 4.63 (m, 1H), 5.68 (br s, 2H), 7.41 (m, 6H), 7.65 (m, 4H),
7.78 (s, 1H), 8.37 (s, 1H).
((3aR,4R,6S,6aS)-6-((6-Amino-9H-purin-9-yl)methyl)-2,
2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methanol (26)
Tetra-n-butylammonium fluoride (2 mL, 1.0M in THF) was added to
a solution of 25 (1.0 g, 1.79 mmol) in THF (55 mL) at ambient tem-
perature and stirred for 20 minutes. The reaction mixture was concen-
trated in vacuo and the product was purified by flash chromatography (9:1
dichloromethane:methanol) to give 26 (563 mg, 98%) as a white solid. Rf
1
0.33 (9:1 dichloromethane:methanol). H NMR (CD3OD) δ 1.30 (s, 3H),
1.46 (s, 3H), 3.62 (m, 2H), 4.02 (m, 1H), 4.38 (m, 1H), 4.42 (m, 2H), 4.57
(m, 1H), 4.64 (m, 1H), 8.15 (s, 1H), 8.19 (s, 1H).
9-(((3aS,4S,6S,6aS)-6-(Iodomethyl)-2,2-dimethyltetrahydrofuro
[3,4-d][1,3]dioxol-4-yl)methyl)-9H-purin-6-amine (27)
To a solution of 26 (560 mg, 1.74 mmol) in toluene (40 mL) was added
imidazole (416 mg, 6.11 mmol), triphenylphosphine (503 mg, 1.92 mmol),
and iodine (553 mg, 2.18 mmol). The mixture was heated at reflux for
2 hours. More imidazole (208 mg, 3.05 mmol), triphenylphosphine (252 mg,
0.96 mmol), and iodine (277 mg, 1.04 mmol) were added and heated for
another half hour. After cooling to ambient temperature and removing the
solvent by evaporation, the product was purified by flash chromatography
(20:1 dichloromethane:methanol). The product was collected in two parts:
part 1 27 (330 mg, 44%, 95% pure); part 2, impure 27, 830 mg. Rf 0.4 (9:1
dichloromethane:methanol). 1H NMR (CD3OD) δ 1.28 (s, 3H), 1.46 (s, 3H),
3.35 (m, 2H), 3.93 (m, 1H), 4.31 (m, 1H), 4.45 (m, 3H), 4.59 (m, 1H), 8.13
(s, 1H), 8.20 (s, 1H).
Diethyl (((3aS,4S,6S,6aS)-6-((6-amino-9H-purin-9-yl)methyl)-2,
2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)methyl)phosphonate (9)
27 (325 mg, 0.745 mmol) in triethylphosphite (15 mL) was heated at
reflux for 7 hours. After cooling to ambient temperature, the solvent was
removed by reduced pressure. The product was purified by flash chromatog-
raphy (9:1 dichloromethane:methanol) to give 9 as a clear gum (160 mg,
1
48%). Rf 0.35 (9:1 dichloromethane:methanol). H NMR (CD3OD) δ 1.27
(m, 9H), 1.46 (s, 3H), 2.17 (m, 2H), 4.05 (m, 4H), 4.26 (m, 1H), 4.40 (m,
2H), 4.59 (m, 1H), 8.15 (s, 1H), 8.20 (s, 1H).

Synthesis of Acyclic Phosphonic Acid Nucleosides
491
Monoethyl ((S)-2-(((S)-1-(6-amino-9H-purin-9-yl)-3-hydroxypropan-2-yl)oxy)-
3-hydroxy propyl) phosphonate (29)
9 (156 mg, 0.35 mmol) was dissolved in water (10 mL). Hydrochloride
acid (1.0M, 1.77 mL, 1.77 mmol) was added and the reaction mixture was
stirred for 3 hours at ambient temperature. The pH was adjusted to 7 us-
ing 1M sodium hydroxide. The solvent was removed by reduced pressure.
This gave the crude product 28 that contained sodium chloride. This was
dissolved in the mixture of dioxane (4 mL) and water (20 mL). Sodium peri-
odate (150 mg, 0.70 mmol) was added and stirred for 1 hour at ambient tem-
perature. Sodium borohydride (34 mg, 0.89 mmol) was added and stirred
for 0.5 hour at ambient temperature. The solvent was removed by reduced
pressure, and the product was purified by preparative HPLC (30–60% ace-
tonitrile over 10 minutes, X-Terra C18). This gave 29 as a clear gum (68 mg,
1
52%). Rf 0.62 (1:1:1:1:1 toluene:acetone:butan-1-ol:water:acetic acid). H
NMR (CD3OD) δ 1.14 (t, 3H), 1.29 (m, 2H), 3.29 (m, 1H), 3.50 (m, 2H),
3.64–3.85 (m, 4H), 3.88 (m, 1H), 4.38 (m, 2H), 8.19 (s, 2H).
((S)-2-(((S)-1-(6-Amino-9H-purin-9-yl)-3-hydroxypropan-2-yl)oxy)-
3-hydroxypropyl)phosphonic acid (6a)
TMSBr (0.22 mL, 1.7 mmol) was added to the solution of 29 (65 mg,
0.17 mmol) in acetonitrile (5 mL) and stirred for 22 hours at ambient tem-
perature. The solvent was removed by reduced pressure and co-evaporated
with dichloromethane (2×10 mL) and methanol (5 mL). The residue was
dissolved in water (5 mL), and the pH was adjusted to 9 using 1M sodium
hydroxide and then to 3 by adding TFA. The solvent was removed and
evaporated again from methanol. The product was purified by preparative
HPLC (0–30% acetonitrile over 10 minutes, X-Terra C18). This gave 6a
as a white solid (46 mg, 78%). Rf 0.30 (1:1:1:1:1 toluene:acetone:butan-1-
ol:water:acetic acid). 1H NMR (DMSO) δ 1.30–1.45 (m, 1H), 1.50–1.60 (m,
1H), 3.25–3.40 (m, 3H), 3.48 (dd, 1H, J = 3.6, 11.5 Hz), 3.60–3.80 (m, 1H),
3.85–3.95 (m, 1H), 4.20 (dd, 1H, J = 6.3, 14.1 Hz), 4.35 (dd, 1H, J = 3.6,
11.5 Hz), 8.32 (s, 1H), 8.35 (s, 1H). MS (ESI) m/z 348.03 [M+H]+. Chemical
purity by HPLC, 98.72% (258 nm), Atlantis dC18 3 × 150 mm, 3 μM, 0–5%
acetonitrile over 20 minutes, then 5–95% acetonitrile over 5 minutes then
held for 5 minutes (aqueous phase containing 5% of 2% TFA in water).
((S)-2-(((S)-1-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
3-hydroxypropan-2-yl)oxy)-3-hydroxypropyl)phosphonic acid (6b)
Using the same procedure as 6a, 6b (140 mg) was isolated as a white
foam. 1H NMR (D2O) δ 1.60–1.78 (m, 2H), 3.36–3.50 (m, 2H), 3.57–3.77
(m, 5H), 3.90 (dd, 1H, J = 3.4, 13.8 Hz), 5.67 (d, 1H, J = 7.8 Hz), 7.50
(d, 1H, J = 7.8 Hz). MS (ESI) m/z (relative intensity) 325.06 [M+H]+.
Chemical purity by HPLC, 96.08% (266 nm), Atlantis dC18 3 × 150 mm,

492
P. Wainwright et al.
3 μM, 5%(2%TFA/water)/95%water held for 10 minutes, then ramped to
50%acetonitrile/5%(2%TFA/water)/45%water over 20 minutes.
REFERENCES
1. (a) Chu, C.K.; Cutler, S.J. Chemistry and antiviral activities of acyclonucleosides. J. Heterocycl. Chem.
1986, 23, 289–319. (b) De Clercq, E.; Holy´, A.; Rosenberg, I.; Sakuma, T.; Balzarini, J.; Maudgal, P.C.
A novel selective broad-spectrum anti-DNA virus agent. Nature 1986, 323, 464–467. (c) Casara, P.J.;
Altenburger, J.-M.; Taylor, D.J.; Tyms, A.S.; Kenny, M.; Nave, J.-F. Synthesis and antiviral activity of
rigid acyclonucleostide analogs. Bioorg. Med. Chem. Lett. 1995, 5, 1275–1280.
2. Wainwright, P.; Maddaford, A.; Bissell, R.; Fisher, R.; Leese, D.; Lund, A.; Runcie, K.; Dragovich,
P.S.; Gonzalez, J.; Kung, P.-P.; Middleton, D.S.; Pryde, D.C.; Stephenson, P.T.; Sutton, S.C. A flexible,
efficient synthesis of carbocyclic phosphonic acid nucleoside derivatives. Synlett 2005, 5, 765–768.
3. Pryde, D.C.; Middleton, D.S.; Stephenson, P.T.; Wainwright, P.; Maddaford, A.; Zhang, X.; Leese, D.;
Glen, R.; Hart, J.; Forrest, N.; Guyot, T. Practical synthetic routes to carbon-substituted nucleosides.
Tet. Lett. 2011, 52, 6415–6419.
4. Bhattacharya, A.K.; Thyagarajan, G. Michaelis-Arbuzov rearrangement. Chem. Rev. 1981, 81, 415–430.
5. Non natural bases used in the library include purine, 2,6-diaminopurine, 6-chloropurine, benzimida-
zole, 2-aminopurine, 1H-Benzoimidazol-4-ol, 5-fluorocytosine, 2-amino-6-aminocyclopropylpurine,
xanthine, 5-cyclopropyluracil, Imidazo[1,2-c]pyrimidin-5(1H)-one, 1,3a,4,6,7,8a-hexahydroimidazo
[4,5-e][1,4]diazepine-5,8-dione see Bridson, P.K.; Weirich, T.P. Cyclic homologs of xanthines. J. Het.
Chem. 1988, 25, 1179–1182.
6. Ziegler, F.E.; Sobolov, S.B. Synthesis of a highly functionalized carbon ring skeleton for the tri-
chothecene anguidine. J. Am. Chem. Soc. 1990, 112, 2749–2758.
7. (a) cˇesnek, M.; Hockova´, D.; Holy´, A.; Dracˇinsky´, M.; Baszczynˇski, O.; de Jersey, J.; Keough,
D.T.; Guddat L.W. Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: Evalu-
ation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and hu-
man hypoxanthine–guanine–(xanthine) phosphoribosyltransferases. Bioorg. Med. Chem. 2012, 20,
1076–1089. (b) Volpini, R.; Mishra, R.C.; Kachare, D.D.; Dal Ben, D.; Lambertucci, C.; Antonini,
I.; Vittori, S.; Marucci, G.; Sokolova, E.; Nistri, A.; Cristalli, G. Adenine-based acyclic nucleotides as
novel P2×3 receptor ligands. J. Med. Chem. 2009, 52, 4596–4603. (c) Sauer, R.; El-Tayeb, A.; Kaulich,
M.; Mu¨ller, C.E. Synthesis of uracil nucleotide analogs with a modified, acyclic ribose moiety as P2Y2
receptor antagonists. Bioorg. Med. Chem. 2009, 5071–5079.