The application of phosphoramidate ProTide technology to the potent anti-HCV compound 4′-azidocytidine (R1479)

Article abstract of DOI:10.1016/j.bmcl.2009.05.099

We report the design, synthesis and evaluation of a family of ca 50 phosphoramidate ProTides of the potent anti-HCV compound 4′-azidocytidine (R1479), with variation on the ester, amino acid and aryl moiety of the ProTide. Sub-μM inhibitors of HCV emerge.

Full text of DOI:10.1016/j.bmcl.2009.05.099

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4250–4254
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The application of phosphoramidate ProTide technology to the potent
anti-HCV compound 40-azidocytidine (R1479)
a
a
a
a
Christopher McGuigan a, , Mary Rose Kelleher , Plinio Perrone , Sinead Mulready , Giovanna Luoni ,
Felice Daverio a, Sonal Rajyaguru b, Sophie Le Pogam b, Isabel Najera b, Joseph A. Martin b, Klaus Klumpp b,
David B. Smith b
*
a Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK
b Roche Palo Alto, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report the design, synthesis and evaluation of a family of ca 50 phosphoramidate ProTides of the
potent anti-HCV compound 40-azidocytidine (R1479), with variation on the ester, amino acid and aryl
Received 15 April 2009
Revised 21 May 2009
Accepted 22 May 2009
Available online 29 May 2009
moiety of the ProTide. Sub-
lM inhibitors of HCV emerge. The compounds are all non-cytotoxic in the rep-
licon assay. We herein report detailed SARs for each of the regions of the ProTide.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
HCV
Antiviral
R1479
ProTide
40-Azidocytidine
Prodrug
Nucleotide
The discovery of new inhibitors of the hepatitis C virus (HCV) is
a major current activity of both the pharmaceutical industry and
academia. The Roche group have previously reported that various
40-substituted ribonucleosides were endowed with potent and
selective inhibitory activity against HCV1 with 40-azidocytidine
(R1479) (1) emerging as a promising clinical candidate.2
the triphosphate of 2 is a similarly potent inhibitor of HCV RNA
polymerase as the triphosphate of 12,8 even though the parent
nucleoside (2) is inactive versus the virus; presumably due to poor
phosphorylation. Thus, the ProTide method boosted 2 from inac-
tive to sub-
l
M potency.8 We subsequently noted a similar effect
on the purine analogue 40-azidoadenosine (3).9
As with the majority of nucleoside therapeutics (1) requires
intracellular phosphorylation to its 50-triphosphate form to inter-
act at its viral (RNA polymerase) target.2 As HCV lacks any suitable
nucleoside kinase expression, the necessary phosphorylation steps
must be host kinase mediated. Since such phosphorylations may
be inefficient for modified nucleoside analogues a number of labo-
ratories have developed phosphate pro-drug methods to allow the
direct use of the masked monophosphate.3 The Cardiff group first
introduced phosphoramidate ProTides in 19964 and this motif
has been reviewed by us5 and now adopted for example by Gilead
for phosphonate delivery6 and Pharmasset for their 20-modified
anti-HCV family.7 In a collaboration between the Cardiff and Roche
laboratories we have previously reported the significant enhance-
ment in the potency of the uridine analogue of 1, compound 2 by
the application of phosphoramidate ProTide methods.8 Indeed,
NH2
N
O
N
NH2
N
N
N
NH
(1)
(2)
(3)
N
O
O
N
HO
N3
HO
N3
HO
N3
O
O
O
OH OH
OH OH
OH OH
Given the clinical progression of 1 as its ester pro-drug10 we were
interested to consider whether the antiviral activity of 1 might be
further enhanced by our ProTide methodology and we herein report
the early results of this study.
The synthesis of 1 was achieved entirely as has been recently
reported,11 involving the stereo- and regio-selective addition of
TMS-azide to the appropriate 40,50-olefin related to 1. We prepared
a family of 50-ProTides of 1 using phosphorochloridate chemistries
we have extensively reported.4,8,9
* Corresponding author. Tel./fax: +44 29 20874537.
E-mail address: mcguigan@cardiff.ac.uk (C. McGuigan).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.099

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4250–4254
4251
NH2
N
In the first instance, the synthesis of phosphoramidates was
NH2
N
carried out in the presence of 1 with phosphorochloridate pre-
pared from phenyl phosphorochloridates and amino acid ester
hydrochlorides4 in THF in the presence of tBuMgCl followed by re-
peated column chromatography gave the initial family of phenyl
aminoacyl ProTides in low to moderate yield (Table 1, route
1).12 Thirty phosphoramidates were synthesized in this way with
problems of purification and solubility of the starting material (1)
leading to low yields. In order to overcome this problem and con-
sidering our previous work, the phosphoramidate synthesis was
performed with 20/30 protected nucleoside. In the first instance,
the 20,3-isopropylidine nucleoside was synthesized under stan-
dard conditions (Fig. 1, route 2). The synthesis of the phosphoram-
idates was then performed as above followed by deprotection in
the presence of acetic acid (Fig. 1). In this way 10 phosphorami-
dates were synthesized with improvements in the purification
and consequently in the overall yield. In order to further improve
the yields of this synthetic pathway, the cyclopentylidine group
was used as an alternative protecting group in 20- and 30-positions
(Fig. 1, route 3).
O
R
C
O
P
N
O
H
N
N
O
O
R''
O
HO
N3
R'
O
O
O
Ar
Route 3
N3
tBuMgBr
THF, RT, 17h
PTSA, 100 °C, 2h
O
O
O
O
O
O
Phosphorochloridate
NH2
N
80% aq HCOOH
NH2
N
Route 1
tBuMgBr
THF, RT, 17h
N
O
HO
N3
O
R
C
O
P
N
O
H
N
O
O
O
R
C
O
P
(4a-ah)
(5a-p)
(6-7)
R''
H
N
O
R'
O
O
Cl
OH OH
Ar
R''
O
R'
N3
O
Ar
OH OH
NH2
N
NH2
N
Route 2
60% Aq AcOH / 90°C
N
O
O
R
C
O
N
O
HO
N3
H
N
P
O
O
R''
O
R'
O
O
Ar
tBuMgBr
THF, RT, 17h
Phosphorochloridate
N3
O
O
O
O
The subsequent phosphoramidate synthesis was performed un-
der standard condition followed by deprotection reaction in the
presence of formic acid (80% v/v) at room temperature for 5 h. In
this way 10 phosphoramidates were synthesized with a purifica-
tion method that required only one column chromatography for
each step and with the consequent improvements in the overall
yield compared to routes 1 and 2.
Each of the phenyl ProTides 4a–at was tested for inhibition of
HCV in replicon as previously detailed with data being shown in
Table 1.
Figure 1.
Thus, the parent nucleoside (1) was active versus HCV replicon
with a mean IC50 value of 1.28 lM. We have often noted the benzyl
alanine phenyl ProTide to be a reasonable motif to embark with4,8,9
and we did so on this occasion, and with some variation of the ami-
no acid moiety.
Thus 4a–g represent the benzyl esters of
L
-Ala,
D-Ala, a,a-dim-
ethylglycine, Gly,
L
-Val,
L
-Leu and L-Phe, respectively. Compound
Table 1
Anti-HCV activity and cytotoxicity of 4a–ah
a
Route
Compd
Amino acid
Ala
R00
HCV replicon EC50
(l
M)
HCV replicon CC50 (lM)
1
1
1
2
1
1
1
2
2
1
1
1
1
1
1
1
2
1
3
2
1
1
1
3
3
2
1
1
1
1
1
3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4k
4l
4m
4n
4o
4p
4q
4r
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
iPr
iPr
iPr
iPr
iPr
iPr
Me
nBu
nBu
2-Bu
tBu
tBu
tBu
Ph
C12H25
—
6.0
2.2
9.2
2.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
—
D-Ala
Me2Gly
Gly
Val
Leu
Phe
5.6
88% (4.2)b
89% (7.7)b
0.38
Ala
D-Ala
2.3
11
>100
11
18
Leu
Ile
Phe
Met
EtAsp
EtGlu
CycloPntGly
Pro
N-MeGly
BetaAla
Ala
Me2Gly
Leu
Phe
88% (4.4)b
74% (44)b
69% (56)b
>100
72% (48)b
77% (47)b
0.99
4s
4t
4u
4v
3.1
>100
2.9
15
4.5
3.1
11
0.94
0.62
4w
4x
4y
4y0
4z
4aa
4ab
4ac
4ad
4ae
4af
4ag
4ah
1
Gly
Gly
Ala
Ala
D-Ala
Ala
Ala
0.72
D-Ala
>100
84% (6.7)b
2.9
30
1.28
Phe
Ala
3
1
D-Ala
R1479
—
a
HCV Con 1 (GT1b) replicon inhibition. All data are means from P2 experiments.
Level of inhibition at top concentration (100 lM) in % (approximate IC50).
b

4252
C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4250–4254
4a is slightly less potent than 1 in this assay, implying a slightly
lower eventual level of the triphosphate of (1) being delivered by
(4a) as compared to (1) under the assay conditions. This is in con-
trast to the corresponding ProTide of (2), where we noted a >200-
fold boost in potency.8 This most likely relates to the relative effi-
ciency of phosphorylation of (1) and (2) in the replicon system,
a chain extended beta amino acid; beta alanine (4t), which was
poorly active. Alpha amino acids have been considered essential
by us for the putative activation mechanism of ProTides14 and in-
deed early chain-extended examples were inactive when applied
to the anti-HIV agent d4T.15 Thus, it appears here too that an alpha
amino acid with a free NH and a small amino acid motif is essential
for potent activity.
Gilead have found success in applying our approach to acyclic
nucleoside phosphonates6 and have found particular advantage
from alanines with branched aliphatic esters such as iPr. For com-
pound 2 we found that iPr esters were roughly equiactive with Et
examples.8 Thus, here we prepared iPr examples of Ala (4u), dim-
ethylglycine (4v), Leu (4w), Phe (4x), and Gly (4y). In general activ-
ities fell within the range observed with the ethyl and benzyl series
above. Notably, the Phe compound which showed 29-fold reduced
with (1) being
a
good substrate for deoxycytidine kinase.20
-amino acids, such as
-Ala, were poorly effective as ProTide motifs13 we did observe that
Although we have previously noted that
D
D
the benzyl
D
-Alanine derivative of (2) showed similar potency with
-analogue.8 In
-Ala analogue (4b) was
slightly (2.7-fold) more active as compared to the -Ala analog
only a twofold increased IC50 as compared to the
the case of the cytidine analog (1), the
L
D
L
(4a). Also, replacement of both glycine hydrogen atoms by methyl
groups only led to a rather small loss of potency for the dimethyl-
glycine compound (4c), with 4.2-fold as compared to the
D-Ala
potency as compared to L-Ala in the Et ester series had less of a det-
analog (4b), similar to what had been seen with analogous pro-
rimental effect in the benzyl and iPr series. In the case of the iPr
glycine compound we observed a separation of the two diastereo-
isomers arising from mixed stereochemistry at the phosphate cen-
tre. Such an isomeric pair was present in every case, and roughly
1:1 as judged by 31P NMR. In most cases the isomers were tested
as mixtures. However, the fortuitous separation of the isomers in
the case of 4y lead to two fractions being evaluated separately.
Although not fully resolved, 4y was primarily the more polar iso-
mer, based on reverse phase HPLC (9:1 ‘fast’/’slow’) while 4y0
was primarily the less polar isomer (3.5:6.5 ‘fast’/’slow’). The 3.3-
fold difference in potency noted between 4y and 4y0 suggests a
ca. sevenfold difference for the pure separate isomers, with the
more lipophilic compound being more potent. In the case of two
diastereomers of a ProTide of 2 we previously noted no significant
difference in activity,8 although we16 and others6 have seen more
substantial isomers differences in some cases.
drugs of (2).8
The glycine analogue (4d) showed similar potency as compared
to the
D-Ala analog (4b). The Valine analogue (4e) was similarly po-
tent as compared to
L-Ala (4a). Longer amino acids such as Leu (4f)
and Phe (4g) could also be introduced without a large impact on
apparent EC50, although these compounds did not reach 90% inhi-
bition levels under the assay conditions.
It was also of interest to explore other esters. Firstly, we pre-
pared a series of ethyl esters of varying amino acids (4h–t). The
ethyl alanine compound (4h) was the most potent of the series
with a mean EC50 of 380 nM, thus 3.4-fold more potent than
R1479 (1) and 16-fold more potent than the benzyl equivalent
(4a). In the case of Et esters, the
D-Ala analogue (4i) was sixfold less
potent as compared to the -Ala analogue. The introduction of
L
bulky amino acid side chains significantly reduced potency in the
Et-ester subseries; Leu (4k), and Phe (4m) showed 29-fold reduced
Shortening (4z) and linear extension (4aa–ac) of the ester chain
was also explored. The activities and SARs noted were rather sim-
ilar to those captured above, although the n-butyl case was anom-
potency as compared to the
inactive. These results are in contrast to the case of nucleoside 2
where the Et-Phe analogue, was similarly potent as the Et- -Ala
L-Ala analogue, whereas Ile (4l) was
L
alous, with the
D-Ala example (4ab) being unusually more active
analogue.8 Several other amino acids were also explored as their
ethyl esters. Thus, the Met (4n), ethyl aspartate (4o), and particu-
larly ethyl glutamate (4p) analogues were relatively poorly active.
The unusual spiro fused cyclopentyl glycine motif in (4q) has been
found to be effective in other ProTide programmes in our labora-
tory, but was poorly active here, as was the Proline compound
(4r). The poor activity of the latter could correspond to its unique
cyclic structure amongst natural amino acids, or could arise from
the need for a free NH. The poor activity of the N-methylglycine
compound (4s) supports the latter point at least, although the Pro-
line ring may also be detrimental. Lastly in this series we prepared
than the -compound (4aa).
L
The t-Butyl case was even more intriguing. Historically, we have
generally observed a reduction, and in some cases a loss, of activity
for t-Bu esters,17 this being attributed to poor hydrolysis of these
systems in vitro. In the case of ProTides of (2) we did see activity
for a t-Bu ester,8 although it was a log less active than the benzyl
lead. In the case of (1), the tBuAla compound (4ad) was surpris-
ingly similarly potent as compared to the Et-Ala lead, while the
tBu-D-Ala analogue (4ae) was inactive, as was the Phe compound
(4af), thus differing from the SAR in the Et ester series. This is
amongst the most striking and specific example of the apparent
Table 2
Anti-HCV activity and cytotoxicity of 5a–p
Route
Compd
Ph subst
Amino acid
R00
HCV replicon EC50
(lM)
HCV replicon CC50 (lM)
3
2
1
3
3
1
2
3
3
2
2
3
1
1
1
1
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
o-Cl
m-Cl
p-Cl
o-OMe
m-OMe
p-OMe
o-Me
p-Me
p-Br
3,4-Cl2
p-Me
2,6-(OMe)
p-Cl
p-OMe
p-Me
3,4-Cl2
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Ala
Leu
Leu
Leu
Leu
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Et
3.5
3.7
6.2
2.3
8.8
>100
1.3
0.51
6.5
9.2
2.9
4.1
2.5
9.3
4.3
2.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Et
Et
Et
Et
Et

C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4250–4254
4253
Table 3
tential of such ProTides is only likely to become apparent with fur-
ther in vitro and in vivo study. For example, it is notable that the
lead agent 5h has a calculated lipophilicity (Clog P, ChemDraw Ul-
tra 11.0) of 1.2 versus ꢁ1.9 for parent nucleoside 1; the almost 3-
log lipophilicity boost may well lead to more efficient passive dif-
fusion into cells and independence from nucleoside transporters,
which could convey significant in vivo advantage. Nucleoside
deaminase resistance is also likely for these ProTides, based on
prior examples.21 Also, notably two cytidine analogues for HCV,
NM 283 and R1626, have recently shown efficacy in HIV infected
subjects, but have been withdrawn due to safety reasons.22 Poten-
tial targeting of these ProTides to liver is quite likely based on re-
cent data on some of our 20-modified nucleoside ProTides,23 and
this could well lead to enhanced clinical safety profiles for such
Anti-HCV activity and cytotoxicity of 6–7
Route
Compd
Nap
Isomer
HCV replicon
HCV replicon
CC50 (lM)
EC50
(lM)
3
3
6
60
1-Nap
1-Nap
Polar
Non-
polar
Polar
Non-
polar
0.95
1.2
>100
>100
1
1
7
70
2-Nap
2-Nap
4.3
2.9
>100
>100
interplay between ester and amino acid motifs in ProTide SAR that
we have observed.
In the case of ProTides of the anti-HIV agent d4T we have pre-
viously studied the effect of substitutions in the phenyl moiety,18
and noted the beneficial effect of a p-Cl group for example. Thus
we pursued Ph substitution in the present case. The same synthetic
access was used as for the parent Ph systems 4a–ah but it was now
necessary to prepare the appropriate substituted phenyl phospho-
rodichloridates by reaction of the corresponding phenol and POCl3
in inert solvent. The dichloridates were assayed by 31P NMR and
used without purification in most cases. Thus, as noted in Table
2, we explored a number of o-, m-, and p-mono- and di- substitu-
tions on the Ph ring, with electron-withdrawing and donating
groups, and examples in the BnAla, EtAla and EtLeu series.
Overall, phenyl substitution did not have a great affect on po-
tency in this series, although 5h did emerge as slightly more active
than the parent nucleoside 1 (ca threefold) and its parent ProTide
4a (ca 10-fold).
We previously noted a boost in efficacy for anti-cancer ProTides
of BVDU19 and also for the anti-HCV activity of ProTides of (2) on
substitution of the phenyl moiety entirely by a 1-naphthyl unit.
Thus, we prepared the 1-naphthyl analogue of (4a), using the phos-
phorochloridate prepared from naphthyl phosphorodichloridate
and benzylalanine hydrochloride. The product was separated into
its polar (6) and non-polar (60) diastereomers by reverse phase
HPLC. The compounds were essentially pure by 31P NMR, with 6
resonating upfield of 60 (dP 3.75, 3.87). We also prepared the corre-
sponding 2-naphthyl analogue and also resolved it into its separate
isomers 7 and 70. Data on the naphthyl family are presented in Ta-
ble 3.
agents. As a sub-lM lead, the p-MePh analogue 5h may represent
a useful point of departure.
References and notes
1. Smith, D. B.; Martin, J. A.; Klumpp, K.; Baker, S. J.; Blomgren, P. A.; Devos, R.;
Granycome, C.; Hang, J.; Hobbs, C. J.; Jiang, W.-R.; Laxton, C.; Le Pogam, S.;
Leveque, V.; Ma, H.; Maile, G.; Merrett, J. H.; Pichota, A.; Sarma, K.; Smith, M.;
Swallow, S.; Symons, J.; Vesey, D.; Najera, I.; Cammack, N. Bioorg. Med. Chem.
Lett. 2007, 17, 2570.
2. Klumpp, K.; Levenque, V.; Le Pogam, S.; Ma, H.; Jiang, W.-R.; Kang, H.;
Granycome, C.; Singer, M. L.; Laxton, C.; Hang, J. Q.; Sarma, K.; Smith, D. B.;
Heindl, D.; Hobbs, C. J.; Merrett, J. H.; Symons, J.; Cammack, N.; Martin, J. A.;
Devos, R.; Najera, I. J. Biol. Chem 2006, 281, 3739.
3. Arizo, M. E. Drug Design Rev. 2005, 2, 373.
4. McGuigan, C.; Cahard, D.; Sheeka, H. M.; De Clercq, E.; Balzarini, J. J. Med. Chem.
1996, 39, 1748.
5. Cahard, D.; McGuigan, C.; Balzarini, J. Mini-Rev. Med. Chem. 2004, 4,
371.
6. Birkus, G.; Kutty, N.; He, G.-X.; Mulato, A.; Lee, W.; McDermott, M.; Cihlar, T.
Antiviral Res. 2007, 74, A57.
7. Furman, P.A.; Wang, P.; Niu, C.; Bao, D.; Symonds, W.; Nagarathnam, D.; H.M.
Steuer.; Rachakonda, S.; Ross, B.S.; Otto, M.J.; Sofia, M.J. The 59th Annual
Meeting of the American Association for the Study of Liver Diseases (AASLD),
San Francisco, California, 2008.
8. Perrone, P.; Luoni, G. M.; Kelleher, M.-R.; Daverio, F.; Angell, A.; Mulready, S.;
Congiatu, C.; Rajyaguru, S.; Martin, J. A.; Lévêue, V.; Pogam, S. Le.; Najera, I. I.;
Klumpp, K.; Smith, D. B.; McGuigan, C. J. Med. Chem. 2007, 50,
1840.
9. Perrone, P.; Daverio, F.; Valente, R.; Rajyaguru, S.; Martin, J.; Leveque, V.;
LePogam, S.; Najera, I.; Klumpp, K.; Smith, D.; McGuigan, C. J. Med. Chem. 2007,
50, 5463.
10. Leveque, V.; Pogam, S.L.; Ao-Ieong, G.; Harris, S.; Kang, H.; Kosaka, A.;
Seshaadri, A.; Chiu, S.; Symons, J.; Cammack, N.; Klumpp K.; Najera, I. 43rd
Annual Meeting of the European Association for the Study of the Liver (EASL),
2008, Italy.
As we note in Table 2, the 1-naphthyl compound showed ꢀ6-
fold improved potency as compared to the parent Phenyl BnAla
(4a) compound, with no significant difference in potency between
the two diastereomers 6 and 60. By contrast, the 2-naphthyl com-
pounds (7 and 70) were slightly less potent (3–5-fold).
In conclusion, we herein report the application of the phosphor-
amidate ProTide method to the potent anti-HCV agent 40-azi-
docytidine (1). Approximately 50 new compounds were
prepared, with variations in the ester, amino acid, and aryl moiety.
11. Smith, D. B.; Kalayanov, G.; Sund, C.; Winqvist, A.; Pinho, P.; Maltseva, T.;
Morisson, V.; Leveque, V.; Rajyaguru, S.; Pogam, S. L.; Najera, I.; Benkestock, K.;
Zhou, X.-X.; Maag, H.; Cammack, N.; Martin, J. A.; Swallow, S.; Johansson, N. G.;
Klumpp, K.; Smith, M. J. Med. Chem. 2009, 52, 219.
12. Synthesis of 40-Azidocytidine-50-O- [phenyl-(benzyloxy-
L-alaninyl)]-phosphate
[4a] tBuMgCl (4.11 mL, 1 M solution in THF, 4.11 mmol) and 40-azido-
cytidine (500 mg, 1.643 mmol) were dissolved in dry THF (15 mL) and stirred
for
15 min.
Then
phenyl(benzyloxy-L-alaninyl)-phosphorochloridate
(4.11 mmol, 4.11 mL, 1 M in THF) was added dropwise at RT and stirred at
room temperature overnight. Then a solution of sat. NH4Cl was added to
quench the reaction. The solvent was removed under reduced pressure to yield
a yellow solid which was purified by repeat column chromatography, using
10–20% MeOH/DCM eluent gradients each time, then a preparative TLC using a
DCM/MeOH (9:1) solvent mixture. The obtained pure product was a white
solid (49 mg, 5%). 31P NMR (121.5 MHz, MeOH-d4): dP 4.70, 4.49; 1H NMR
(300 MHz, MeOH-d4): dH 7.65–7.58 (1H, m, H-6), 7.36–7.34 (7H, m, Ph-CH),
7.26–7.19 (3H, m, Ph-CH), 6.20–6.13 (1H, dd, J = 4.7 and 14.3 Hz, H-10), 5.92–
5.85 (1H, m, H-5), 5.20 (2H, s, Ph–CH2), 4.37–4.29 (2H, m, H-20 and H-30), 4.23–
4.11 (2H, m, H-50), 4.01 (1H, m, Ala-CH), 1.41–1.25 (3H, m, Ala-CH3). 13C NMR
(75.5 MHz, MeOH-d4): dC 174.94, 174.88, 174.62, 174.56 (C@O), 167.62 (C-4),
158.34 (C-2), 152.05, 151.97 (Ph-C), 143.12, 142.93 (C-6), 137.41, 137.25 (Ar-
C), 130.96, 130.17, 130.31, 130.171 (Ar-C), 129.66, 129.59, 129.41, 129.36,
129.29 (Ar-C), 126.44 (Ar-C), 124.34 (Ar-C), 123.92 (Ar-C) 121.66, 121.60,
121.47, 121.41 (Ar-C), 121.28, 121.22 (Ar-C), 98.83, 98.72, 98.60 (C-5), 97.04
(C-40), 93.87, 93.42 (C-10), 74.57, 74.37 (C-30), 73.50 (C-20), 68.82, 68.75 (Bn-
CH2), 68.12, 67.74 (Ala-CH), 20.52, 20.43, 20.30, 20.20 (Ala-CH3).
Structure activity relationships support the notion that an a-amino
acid is essential for significant activity and a free NH on the amino
acid also seems important. The ester moiety can be varied signifi-
cantly. Interestingly, in this study t-butyl is an acceptable ester in
the case of
were best tolerated;
Larger and branched amino acids were generally less or poorly
effective. Substitution in the phenyl ring was tolerated by a variety
of substituents at various positions, though no substantial
enhancements in potency were seen. 1-Naphthyl provided a small
benefit over phenyl, whereas 2-naphthyl could substitute with no
loss of potency. Lastly, in three cases the phosphate diastereoiso-
mers were resolved and tested separately in replicon assay, and lit-
tle difference in potency noted between the stereoisomers.
L-alanine, but not other amino acids. Small amino acids
L
and (in most cases) -alanine, and glycine.
D
13. McGuigan, C.; Salgado, A.; Yarnold, C.; Harries, T. Y.; De Clercq, E.; Balzarini, J.
Antivir. Chem. Chemother. 1996, 7, 184.
14. Balzarini, J.; Karlsson, A.; Aquaro, S.; Perno, C.-F.; Cahard, D.; Naesens, L.; De
Clercq, E.; McGuigan, C. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 7295.
Many of the ProTides herein described retained or slightly (up
to 3–4 fold) enhanced the potency of 1 in replicon, but the true po-

4254
C. McGuigan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4250–4254
15. McGuigan, C.; Tsang, H.-W.; Sutton, P. W.; De Clercq, E.; Balzarini, J. Antivir.
Chem. Chemother. 1998, 9, 109.
16. McGuigan, C.; Turner, S.; Nicholls, S. R.; O’Connor, T. J.; Kinchington, D. Antivir.
Chem. Chemother. 1994, 5, 162.
17. McGuigan, C.; Sutton, P. W.; Cahard, D.; Turner, K.; O’Leary, G.; Wang, Y.;
Gumbleton, M.; De Clercq, E.; Balzarini, J. Antivir. Chem. Chemother. 1998, 9,
473.
18. Siddiqui, A. Q.; Ballatore, C.; McGuigan, C.; De Clercq, E.; Balzarini, J. J. Med.
Chem. 1999, 42, 393.
20. Klumpp, K.; Kalayanov, G.; Ma, H.; Le Pogam, S.; Leveque, V.; Jiang, W.-R.;
Inocencio, N.; De Witte, A.; Rajyaguru, S.; Tai, E.; Chanda, S.; Irwin, M. R.; Sund,
C.; Winqist, A.; Maltseva, T.; Eriksson, S.; Usova, E.; Smith, M.; Alker, A.; Najera,
I.; Cammack, N.; Martin, J. A.; Johansson, N. G.; Smith, D. J. Biol. Chem. 2008,
283, 2167.
[21]. McGuigan, C.; Tollerfield, S. M.; Riley, P. A. Nucleic Acids res. 1989, 17, 6065.
[22]. Brown NA. Expert Opin. Investig. Drugs. 2009. [Epub ahead of print].
[23]. Hutchins, J.; Chamberlain, S.; Chang, C.; Ganguly, B.; Gorovits, E.; Hall, A.;
Henson, G.; Kolykhalov, A.; Liu, Y.; Muhammad, J.; Perrone, P.; Gilles, A.; Holl,
S.; Madela, K.; McGuigan, C.; Patti, J., 22nd ICAR. Miami Beach, FL, 2009; p 42.
19. Congiatu, C.; McGuigan, C.; Jiang, W. G.; Davies, G.; Mason, M. D. Nucleosides
Nucleotides Nucleic Acids 2005, 24, 485.