Synthesis and P2Y2 receptor agonist activities of uridine 5′-phosphonate analogues

Article abstract of DOI:10.1016/j.bmc.2012.02.012

We explored the influence of modifications of uridine 5′- methylenephosphonate on biological activity at the human P2Y₂ receptor. Key steps in the synthesis of a series of 5-substituted uridine 5′-methylenephosphonates were the reaction of a su

Full text of DOI:10.1016/j.bmc.2012.02.012

Bioorganic & Medicinal Chemistry 20 (2012) 2304–2315
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and P2Y2 receptor agonist activities of uridine
50-phosphonate analogues
Sara Van Poecke a, , Matthew O. Barrett b, , T. Santhosh Kumar c, Davy Sinnaeve d, José C. Martins d,
Kenneth A. Jacobson c, T. Kendall Harden b, Serge Van Calenbergh a,
⇑
a Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium
b Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599-7365, USA
c Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892, USA
d NMR and Structure Analysis Unit, Department of Organic Chemistry, Ghent University, Krijgslaan 281 S4, B-9000 Gent, Belgium
a r t i c l e i n f o
a b s t r a c t
We explored the influence of modifications of uridine 50-methylenephosphonate on biological activity at
the human P2Y2 receptor. Key steps in the synthesis of a series of 5-substituted uridine 50-meth-
ylenephosphonates were the reaction of a suitably protected uridine 50-aldehyde with [(diethoxyphos-
phinyl)methylidene]triphenylphosphorane, C-5 bromination and a Suzuki–Miyaura coupling. These
analogues behaved as selective agonists at the P2Y2 receptor, with three analogues exhibiting potencies
in the submicromolar range. Although maximal activities observed with the phosphonate analogues were
much less than observed with UTP, high concentrations of the phosphonates had no effect on the stim-
ulatory effect of UTP. These results suggest that these phosphonates bind to an allosteric site of the P2Y2
receptor.
Article history:
Received 29 October 2011
Revised 27 January 2012
Accepted 4 February 2012
Available online 15 February 2012
Keywords:
P2Y2 receptor
G protein-coupled receptor
Uracil nucleotides
Nucleoside phosphonates
Partial agonists
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
human P2Y4, P2Y6, and P2Y14 subtypes respond exclusively to var-
ious uracil nucleotides, and the human P2Y2 receptor is activated
Extracellular purine and pyrimidine nucleosides and nucleo-
tides act as messengers via specific receptors on the plasma mem-
brane. These receptors exist in two families: P1 receptors (also
termed adenosine receptors) activated by adenosine and P2 recep-
tors activated by adenosine 50-tri- or diphosphate (ATP or ADP)
and/or uridine 50-tri- or diphosphate (UTP or UDP). P2 receptors
are further divided as P2X and P2Y receptors, which are ligand-
gated ion channels and G protein-coupled receptors (GPCRs),
respectively.1–4 At least eight different subtypes of P2Y receptors
are known, that is P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13 and
P2Y14. The missing numbers belong to non-mammalian receptors,
which may be orthologs of mammalian subtypes, or receptors that
do not appear to be bona-fide P2Y receptor family members.5
The various human P2Y receptor subtypes are activated by
different physiological nucleotides. The human P2Y1, P2Y12, and
P2Y13 receptors are activated preferentially by ADP, while the
human P2Y11 receptor is activated preferentially by ATP. The
by UTP and ATP with similar potency. The P2Y receptors are pref-
erentially coupled to heterotrimeric G proteins of the Gq (P2Y1–
P2Y11) or Gi (P2Y12–P2Y14) families, to stimulate phospholipase C
(PLC) or to inhibit adenylyl cyclase (AC), respectively. The P2Y11
receptor is also coupled to Gs proteins.
The P2Y2 receptor is the most widely studied uracil nucleotide
receptor.5 It is broadly distributed throughout the body and is most
prominently expressed in the lung, heart, skeletal muscle, spleen,
kidney, and liver.6,7 The P2Y2 receptor is known to play important
physiological roles in epithelial cells of the lung, gastrointestinal
tract and the eye, and therefore, it is under investigation as a ther-
apeutic target. Agonists are promising for treatment of cystic fibro-
sis, cancer and dry eye syndrome,8,9 while P2Y2 antagonists might
have anti-inflammatory10 and neuroprotective effects.11
The major limitations associated with known agonists for the
P2Y2 receptor are (i) the lack of selectivity versus closely related
P2Y receptor subtypes and (ii) their fast degradation by nucleo-
tide-hydrolyzing ecto-enzymes, which results in a relative short
duration of action.5 In that context, we recently explored to what
Abbreviations: NBS, N-bromosuccinimide; TMSBr, trimethylsilyl bromide; TFA,
trifluoroacetic acid; NMO, N-methylmorpholine-N-oxide.
extent replacement of the a-phosphate group of UTP by an isoster-
ic phosphonate affected P2Y2 receptor activity.9 Since the carbon–
phosphorus bond cannot be hydrolyzed, this analogue was ex-
pected to exhibit prolonged metabolic stability. While we initially
⇑
Corresponding author. Tel.: +32 9 264 81 24; fax: +32 9 264 81 46.
E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2012.02.012

S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
2305
O
O
O
P
O
O
O
O
N
O
N
HO
HO
NH
NH
HO P O P O P X
OH OH OH
O
O
OH
HO
OH
HO
1: X = O (UTP)
2: X = CH2
3
Figure 1. Structure of UTP (1), diphosphophosphonate 2, and 50-methylenephosphonate 3.
focused on a diphosphophosphonate mimic of UTP (2), it was for-
tuitously discovered that its synthetic precursor 3 was also capable
of activating the P2Y2 receptor but was inactive at the P2Y4 recep-
tor (Fig. 1).9
often these involve a Wittig-type15 or an Arbuzov16 reaction. In
addition, Barton et al.17 published a radical approach for the intro-
duction of the carbon–phosphorous bond. We decided to follow
the method described by Xu et al.18 in which the isosteric analogue
was prepared by treatment of a suitably protected uridine 50-alde-
hyde with a stabilized [Ph3P@CHPO(OEt)2] ylide. Hydrogenation of
the obtained olefin in MeOH using a 10% palladium on carbon
catalyst gave access to the known compound 4.9 C-5 selective
NBS-mediated bromination of this intermediate followed by a
Suzuki–Miyaura coupling with a number of commercial aryl and
heteroaryl boronic acids gave access to a series of C-5 substituted
analogues. The latter transformation took place in a DMF–H2O
solution and was catalyzed by Pd(PPh3)4. Sodium carbonate was
used for the activation of the boronic acids.19 One-pot deprotection
of the phosphonate diester and the 20,30-O-isopropylidene group by
consecutive treatment with TMSBr and TFA afforded the desired
phosphonates 7a–h in variable yields (Scheme 1).
In this study, we explore the influence of further modifications
of the 50-methylenephosphonate 3 on activity at the P2Y2 receptor.
A patent application from Astra-Zeneca indicated that incorpora-
tion of a large semiplanar, hydrophobic aromatic ring at position
5 of thiouridine triphosphate may be accommodated by the P2Y2
receptor but tends to preclude the conformational change required
for receptor activation.12 Therefore, we introduced several smaller
(hetero)aromatic substituents at the 5-position of analogue 3 to
enhance binding but still allow for receptor activation. To sort
out the influence of replacement of the 20-OH group of 3, we envis-
aged the synthesis of a 20-chloro and a 20-amino analogue. Besides
establishing possible interactions with the target receptor, a 20-
chloro substituent may impact on the furanose ring conformation.
In the case of UTP, a 20-amino modification was associated with in-
creased P2Y2 selectivity while maintaining excellent potency.13 To
assess the influence of rigidifying the ribofuranose conformation of
3, an N-methanocarba analogue was synthesized. Finally, another
phosphonate bioisoster of uridine 50-monophosphate (UMP), ob-
tained by the inversion of the 40-CH2–O group of UMP, was also ex-
plored. Transforming a phosphate moiety ((RO)2P(O)–O–C–) to its
isomeric catabolically stable phosphonomethyl ether ((RO)2P(O)–
C–O–) has proven to be a successful strategy in the development
of antiviral drugs.14
The synthesis of 20-substituted 50-methylenephosphonates 12
and 14 started from intermediate 4 (Scheme 2). Following depro-
tection of the 20- and 30-hydroxyl groups of 4, 20,2-O-anhydro ana-
logue 9 was formed using thionyl chloride in CH3CN followed by
the addition of NaOAc.20 Introduction of an amino group at the
20-position was accomplished via opening of the anhydro-deriva-
tive 9 with NaN3 in DMF. Interestingly, these conditions caused
concomitant incomplete hydrolysis of the phosphonate ester to af-
ford 10 as reported previously by Holy.21 Staudinger reduction of
the azide followed by deprotection of mono ethylphosphonate 11
resulted in the desired 20-amino-20-deoxyuridine-50-phosphonate
analogue 12. Similarly, treatment of 9 with a 2 N HCl in diethyl
ether solution followed by the hydrolysis of the phosphonate dies-
ter resulted in 20-chloro-20-deoxy analogue 14.
2. Results and discussion
2.1. Chemistry
The synthetic procedure for N-methanocarba analogue 17 is de-
picted in Scheme 3. An initial Mitsunobu base coupling reaction of
previously reported alcohol 1522 using triphenylphosphine,
Different methods for the preparation of isosteric phosphonate
analogues of nucleoside phosphates have been reported. Most
Br
O
O
O
O
N
O
O
N
EtO
EtO
NH
P
a
b
EtO
EtO
NH
P
O
O
O
O
O
O
4
5
R
R
O
O
P
O
O
N
c
a: R = phenyl
EtO
EtO
O
P
NH
O
N
b: R = naphthalen-2-yl
c: R = 4- fluorophenyl
d: R = styryl
HO
HO
NH
O
O
O
O
e: R = p-tolyl
OH
HO
f : R = furan-2-yl
g: R = thien-2-yl
h: R = benzothiophen-2-yl
7a-h
6a-h
Scheme 1. Reagents and conditions: (a) NBS, DMF, rt, overnight, 79%; (b) R-B(OH)2, Na2CO3, Pd(PPh3)4, DMF, H2O, reflux, 4 h, 39–76%; (c) (i) TMSBr, CH2Cl2, rt, overnight; (ii)
TFA, H2O, rt, 4 h, 11–78%.

2306
S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
O
O
O
O
P
O
P
O
O
O
O
N
N
N
EtO
EtO
EtO
EtO
EtO
EtO
N
NH
NH
P
a
b
O
O
HO
O
O
OH
O
HO
4
9
8
O
O
O
c
O
P
O
P
O
P
e
d
O
O
O
N
N
N
H4NO
H4NO
EtO
HO
EtO
HO
NH
NH
NH
O
O
O
HO
NH2
HO
NH2
HO
N3
12
11
10
O
O
f
e
O
P
O
P
O
O
N
N
H4NO
H4NO
EtO
EtO
NH
NH
O
O
Cl
Cl
HO
HO
13
14
Scheme 2. Reagents and conditions: (a) 50% aq HCOOH, rt, 4.5 h, 57%; (b) (i) thionyl chloride, CH3CN; (ii) CH3COONa, DMF, 85 °C, 92%; (c) NaN3, DMF, 150 °C, overnight, 88%;
(d) PPh3, THF, H2O, rt, overnight, quant.; (e) TMSBr, CH2Cl2, rt, overnight, 78% for 20-amino and 58% for 20-chloro; (f) 2 N HCl in Et2O–dioxane (3:1), rt, overnight, 54%.
O
O
O
P
O
P
O
P
N
N
O
O
O
O
Et3HNO
Et3HNO
NH
NH
a
b
OH
O
O
O
O
OH
O
O
HO
15
16
17
Scheme 3. Reagents and conditions: (a) (i) Triphenylphosphine, 3-N-benzoyluracil, diisopropyl azodicarboxylate, anhyd THF; (ii) 6 N NH3/MeOH, 50 °C, 24% (over two steps);
(b) iodotrimethylsilane, anhyd CH2Cl2, 19%.
diisopropyl azodicarboxylate and 3-N-benzoyluracil23 followed by
a debenzoylation using 6 N NH3/MeOH gave phosphonate diester
16 in 24% overall yield. Simultaneous deprotection of the phospho-
nate diester and the 20,30-O-isopropylidene group using freshly
opened iodotrimethylsilane24 afforded target phosphonate 17.
The synthesis of 40-O–CH2 phosphonate analogue 22 started
phonate 19 in 35% overall yield. Assignment of the stereoarrange-
ment in 19 was based on mechanistic considerations.26
Phosphonate 19 was further transformed into olefin 20 via sodium
periodate oxidation of selenium and subsequent elimination.
OsO4-promoted dihydroxylation in the presence of N-methylmor-
pholine-N-oxide followed by TMSBr hydrolysis of the phosphonate
ester groups provided compound 22 in moderate yield. A positive
NOE was observed between the H-10 and H-40 of compound 20,
confirming the cis stereoarrangement of the uracil base and the
phosphonethoxy side chain.
from 20,30-dideoxy-30,40-didehydro-b-
D-erythrofuranosyl)uracil 18
(Scheme 4).25 Treatment of this glycal with phenylselenyl chloride
at ꢀ70 °C followed by the addition of silver perchlorate in the
presence of diethyl (hydroxymethyl)phosphonate afforded phos-
O
P
O
O
O
P
O
EtO
EtO
EtO
EtO
O
O
O
N
H
N
N
O
H
O
NH
NH
NH
b
a
O
O
O
NOE
PhSe
18
19
20
O
P
O
O
P
O
H4NO
H4NO
EtO
EtO
O
O
N
N
O
O
O
NH
NH
d
c
O
HO
OH
HO
OH
22
21
Scheme 4. Reagents and conditions: (a) (i) PhSeCl, CH2Cl2, ꢀ70 °C, 1 h; (ii) HOCH2PO(OEt)2, AgClO4, CH2Cl2, CH3CN, ꢀ70 to 0 °C, 15 min, 35%; (b) NaIO4, NaHCO3, MeOH, rt,
1 h then 80 °C, 75 min, 68%; (c) OsO4, NMO, acetone–H2O (5:1), rt, 48 h, 60%; (d) TMSBr, 2,6-lutidine, DMF, 0 °C to rt, overnight, 50%.

S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
2307
2.2. Pharmacological evaluation
effect observed with each of these molecules was only 40–55% of
that observed with UTP (Fig. 3 and Table 1). The most potent
agonists were the 5-(4-fluorophenyl) (7c), thien-2-yl (7g), and
5-(2-furanyl) (7f) analogues, which exhibited potencies in the
submicromolar range (Table 1).
Activities of analogues at the P2Y2 receptor were determined
measuring PLC-dependent phosphoinositide hydrolysis in
1321N1 human astrocytoma cells stably expressing the human
P2Y2 receptor (P2Y2-1321N1 cells).27,28 As previously shown for
UTP and UDP,27 none of the phosphonate analogues reproducibly
promoted inositol phosphate accumulation in wild-type 1321N1
cells (Fig. 2A).
In contrast, quantification of inositol phosphate accumulation
in P2Y2 receptor-expressing 1321N1 cells revealed that the newly
synthesized 50-methylenephosphonate analogues are P2Y2 recep-
tor agonists (Fig. 2B), although none of the analogues produced
Both 20-modified analogues (12 and 14) were essentially inac-
tive at the P2Y2 receptor. The negative impact of the replacement
of a 20-OH by a 20-NH2 group may indicate that these phosphonates
have a different mode of binding to the P2Y2 receptor, since a sim-
ilar modification on UTP was well tolerated.
Conformationally constrained compound 17 behaved as a low
efficacy P2Y2 agonist compared to UTP. This indicated that a (N)
conformation of a ribose-like moiety in this series of nucleoside
monophosphonates is compatible with the observed biological ef-
fect. Likewise, compound 22 was significantly less potent than the
lead phosphonate indicating that replacement of the 50-CH2 group
by an oxygen tends to reduce agonist activity.
The activities of two of the most potent new compounds 7c and
7f were directly compared in the same experiments to the 5-
unsubstituted parent compound 3 (Fig. 4). Both analogues pro-
duced maximal effects at the P2Y2 receptor that were similar to
that of 3, but were approximately 10-fold more potent than this
previously studied analogue.
maximal effects at 100 lM concentration as great as UTP. Given
the novel activity observed with these phosphonate analogues at
the P2Y2 receptor, we also determined whether they interacted
with the UTP-activated P2Y4 receptor or the UDP-activated P2Y6
receptor. Little or no reproducible stimulation was observed over
basal activity for any of these analogues in 1321N1 human astrocy-
toma cells stably expressing the human P2Y4 (Fig. 2C) or human
P2Y6 (Fig. 2D) receptors.
Full concentration effect curves were generated in P2Y2-
1321N1 cells with each of the analogues to more clearly assess
their maximal effects relative to UTP and to establish their relative
potencies as P2Y2 receptor agonists. To enhance clarity of compar-
ison of the relative activities of these analogues, the data are arbi-
trarily presented in four separate panels (Fig. 3).
The idea that these modified uridine 50-phosphonate analogues
are orthosteric ligands that exhibit less intrinsic efficacy than UTP
was examined by assessing their capacity at high concentrations to
inhibit the effect of a near-maximal concentration of UTP (Fig. 5A).
No inhibitory effect on the action of UTP was observed with any of
these phosphonate analogues. Concentration effect curves for UTP
The 5-modified uridine 50-phosphonate analogues activated the
P2Y2 receptor with a range of apparent potencies, and the maximal
120
120
B
A
P2Y2
wild-type
100
100
80
60
40
20
0
80
60
40
20
0
f
7
4
a
e
2
2
2
b
d
g
7
7f
er
er
7a
7c
7e
12
1
22
7
7c
7
1
14
7b
7d
7g
7h
7
7
7h
f
f
hol
f
UTP
Buf
Bu
Carbac
120
100
80
60
40
20
0
120
100
80
60
40
20
0
C
D
P2Y6
P2Y4
f
r
e
7
4
g
7
h
7
7
7f
er
f
e
7a
7c
12
1
22
7a
7c
7e
12
14
22
7b
7d
7g
7h
7b
7d
f
f
UTP
UDP
Buf
Bu
Figure 2. Inositol phosphate accumulation in (A) wild-type 1321N1 human astrocytoma cells or in 1321N1 cells stably expressing the human (B) P2Y2, (C) P2Y4, or (D) P2Y6
receptor. The concentration of all phosphonate analogues was 100 M, of carbachol was 100 M, of UTP was 100 nM, and UDP was 1
M. [3H]inositol phosphate
l
l
l
accumulation was quantified as described in Methods. The results are presented as the percent of response observed with carbachol (A), UTP (B and C), or UDP (D). Values are
presented as the mean SEM and are the average of results obtained in at least three separate experiments.

2308
S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
120
120
100
80
60
40
20
0
B
A
100
80
UTP
7e
7g
7h
UTP
7a
7b
60
7d
40
20
0
-9
-8
-7
-6
-5
-4
-9
-8
-7
-6
-5
-4
log [Compound], M
log [Compound], M
120
100
80
60
40
20
0
120
100
80
60
40
20
0
D
C
UTP
12
14
UTP
7c
7f
22
-9
-8
-7
-6
-5
-4
-9
-8
-7
-6
-5
-4
log [Compound], M
log [Compound], M
Figure 3. Concentration dependent activation of the P2Y2 receptor by phosphonate nucleotide derivatives. Accumulation of [3H]inositol phosphates was quantified as
described under Methods. The data are plotted as percent of the maximal response observed with UTP, are presented as the mean SEM, and are representative of results
obtained in three or more separate experiments.
Table 1
120
Relative potencies and maximal effects of UTP and phosphonate analogues at the
human P2Y2 receptor
Compound
EC50
(
l
M)
Max response
100
100
80
60
40
20
0
1
3
0.02 0.003
5.1 0.9
1.5 0.5
2.1 0.6
0.4 0.2
3.1 1.3
1.0 0.8
0.7 0.3
0.5 0.3
>20
32
53
54
43
9
7
7
5
7a
7b
7c
7d
7e
7f
7g
7h
12
14
17
22
41 12
UTP
7c
7f
3
41
45
35
45
9
7
5
5
5
>20
>20
4.0 0.7
>20
5
8
9
nd
19
9
Agonist potency (EC50 value) and maximal response relative to UTP was determined
for each analogue in P2Y2-1321N1 cells as described under Methods. The data are
presented as the mean SEM of values obtained from 3 to 6 separate experiments
with each analogue.
-9
-8
-7
-6
-5
-4
log [Compound], M
Figure 4. Comparison of the concentration dependence of the most potent
phosphonates for activation of the P2Y2 receptor. Accumulation of [3H]inositol
phosphates was quantified as described under Methods. The data are plotted as
percent of the maximal response observed with UTP, are presented as the
mean SEM, and are representative of results obtained in three separate
experiments.
also were carried out in the absence and presence of a high
(100 lM) concentration of 7c or 7f to determine whether the activ-
ity of these molecules was consistent with that of agonists acting at
the orthosteric or an allosteric site of the P2Y2 receptor. As
illustrated in Figure 5, neither 7c nor 7f affected the potency of

S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
2309
Table 2
1H NMR derived mole fraction of the (N)-type conformer of 3 and 7a at different
temperatures
T (°C)
XN
3
7a
20
27
35
42
50
55.5
54.8
54.4
54.1
54.0
52.9
53.1
54.1
53.8
53.0
To assess the impact of the presence of a substituent at the C-5
position of the uracil base on the relative proportions of (N) and (S)
conformers, these scalar couplings were measured at five different
temperatures for the 5-phenyl substituted uridine phosphonate 7a,
while the same was done for the non-C-5 substituted uridine phos-
phonate 3. The fraction of the (N)-type conformer was obtained by
minimizing the sum of square difference between the experimen-
tal couplings and those calculated from the above equations. The
results are shown in Table 2.
For compound 3, the (N) and (S) conformers are nearly equally
present, with a small preference for the (N). From Table 1, it ap-
pears that compound 7a has similar (N)/(S) populations as 3. It
can therefore be concluded that the incorporation of a phenyl
group at the C-5 position appears to have no effect on the confor-
mational state of the ribofuranose ring. Interestingly, this is in con-
trast to the effect of implanting a phenyl ring at C-6 of the base as
was published recently by Nencka et al.,31 that clearly shows an
enhanced preference for the (N) conformation. A second aspect of
the conformation is the orientation of the base relative to the ring
structure, which is here assumed to be either syn (with the substi-
tuent pointing away from the ring) or anti (with the substituent
pointing towards the ring). The NOESY spectrum of 3 confirmed
that the anti rotamer could be adopted. However, when a bulky
group is implanted at the C-5 position, it orients away from the ri-
bose ring, meaning that in this case only the syn rotamer will be
available.
Figure 5. Effect of high concentrations of phosphonate analogues on the P2Y2
receptor-mediated activity of UTP. (A) The effects of 100 M concentrations of the
3. Conclusions
l
indicated phosphonates were tested in the presence of 100 nM UTP in P2Y2-1321N1
cells as described in Methods. (B) Concentration response curves for UTP were
In this study we explored the influence of modifications of uri-
dine 50-methylenephosphonate (3) on activity at the human P2Y2
receptor. A Suzuki–Miyaura coupling of a suitable 5-bromo precur-
sor with aryl and heteroaryl boronic acids allowed generation of a
small series of C-5 substituted analogues of 3. All 5-modified
analogues caused P2Y2 receptor-dependent inositol phosphate
accumulation. Within this series, 5-(2-furanyl) (7f), 5-(4-fluoro-
phenyl) (7c), and thien-2-yl (7g) substitutions afforded the highest
potencies. Interestingly, none of the phosphonate analogues exhib-
ited the same maximal activity as UTP, and these analogues failed
to inhibit activity of UTP down to the level of activity observed
with the 5-modified analogues alone as would be expected for
classical partial agonists acting at the orthosteric binding pocket.
Indeed, no change in the concentration effect curve to UTP oc-
curred in the presence of supramaximal concentrations of 7c or
7f. These results are consistent with the concept that these ana-
logues allosterically activate the P2Y2 receptor in a manner that
has no obvious influence on the effects of UTP mediated through
its orthosteric binding site. Although our observations strongly
suggest allosteric regulation by the phosphonate analogues, they
do not prove it, and the lack of availability of a radioligand for di-
rect assessment of the orthosteric binding site prevents more con-
fident conclusions about their mechanism of action. By altering the
sugar part of 3, we showed that the SAR in the phosphonate series
does not fully parallel that observed in the triphosphate class, fur-
generated in P2Y2-1321N1 cells in the absence or presence of 100 lM 7c or 7f.
[3H]inositol phosphate accumulation was quantified as described in Methods. The
data are presented as the mean SEM and are representative of results obtained in
three separate experiments.
UTP. These results are consistent with the idea that the uridine 50-
phosphonate analogues act at an allosteric site rather than the
orthosteric binding pocket to produce their effect on P2Y2 receptor
activity.
2.3. Conformational analysis
The furanose ring of nucleotides displays a characteristic equi-
librium between two favored puckering conformations, that is
the (N) and South (S) state.29 Davies et al.30 derived the following
equations that relate the fraction of the (N) conformer XN with
the values of the vicinal three scalar couplings that can be mea-
sured between the protons of ring:
0
1;H02
JH
JH
JH
¼ 9:3ð1 ꢀ XNÞ
¼ 4:6XN þ 5:3ð1 ꢀ XNÞ
¼ 9:3XN
0
2;H03
0
3;H04

2310
S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
ther suggesting that the current phosphonates may bind at a site
that differs from the cognate agonist binding pocket.
3.91–4.02 (m, 5H, OCH2CH3, H-40), 4.67–4.71 (m, H-30), 5.14 (dd,
J = 2.4, 6.6 Hz, H-20), 5.85 (d, J = 2.1 Hz, H-10), 7.29–7.41 (m, 3H,
Ph), 7.52–7.55 (m, 2H, Ph), 7.91 (s, H-6), 11.62 (s, 3-NH). 31P
NMR (DMSO-d6): d 31.55. 13C NMR (75 MHz, DMSO-d6): d 16.22,
16.30 (OCH2CH3), 20.81 (d, J = 140 Hz, C-60), 25.26, 27.03
(C(CH3)2), 25.80 (C-50), 60.94, 60.98 (d, J = 6.3 Hz, OCH2CH3),
82.76 (C-30), 83.42 (C-20), 85.55 (d, J = 17.9 Hz, C-40), 91.96 (C-10),
113.44, 113.85 (C-5, (C(CH3)2)), 127.39, 128.05, 128.27, 132.69,
140.40 (Ph,C-6), 149.70 (C-2), 162.21 (C-4). HRMS (ESI) for
4. Experimental section
4.1. Chemical synthesis
All reagents were from standard commercial sources and of
analytic grade. Precoated Merck Silica Gel F254 plates were used
for TLC, spots were examined under ultraviolet light at 254 nm
and further visualized by sulfuric acid-anisaldehyde spray. Column
C
23H32N2O8P [M+H]+ found, 495.1891; calcd, 495.1891.
4.2.2. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b-D-ribo-hexofuranosyl]-5-(naphthalen-2-yl)uracil
(6b)
chromatography was performed on silica gel (63–200 lm, 60 Å,
Biosolve, Valkenswaard, The Netherlands). 1H, 13C, and 31P NMR
spectra were recorded in CDCl3, DMSO-d6, CD3OD or D2O on a Var-
ian Mercury 300 MHz and a Bruker 700 MHz spectrometer. Chem-
ical shifts are given in parts per million (ppm), d relative to residual
solvent peak for 1H and 13C and to external D3PO4 for 31P. Struc-
tural assignment was confirmed with COSY and DEPT. All signals
assigned to hydroxyl groups were exchangeable with D2O. Exact
mass measurements were performed on a Waters LCT Premier
XETM Time of flight (TOF) mass spectrometer equipped with a
standard electrospray ionization (ESI) and modular LockSpray TM
interface. Samples were infused in a CH3CN–H2O (1:1) mixture at
Reaction of compound 5 (63 mg, 0.13 mmol) with naphthalene-
1-boronic acid (45 mg, 0.25 mmol), Pd(PPh3)4 (15 mg, 0.013 mmol)
and Na2CO3 (44 mg, 0.42 mmol) in DMF (3 mL) and degassed H2O
(0.4 mL) was performed as described in the general procedure for
the synthesis of 5-modified phosphonates. Purification of the crude
mixture on a silica gel column (CH2Cl2/MeOH 96:4) afforded com-
pound 6b as a colorless solid (42 mg, 61%). 1H NMR (300 MHz,
DMSO-d6): d 1.19 (dt, 6H, J = 1.5, 7.2 Hz, OCH2CH3), 1.31 (s, 3H,
CH3), 1.50 (s, 3H, CH3), 1.74–1.91 (m, 4H, H-50, H-60), 3.90–4.04
(m, 5H, OCH2CH3, H-40), 4.71 (dd, J = 4.5 Hz, J = 6.3 Hz, H-30), 5.18
(dd, J = 2.4, 6.6 Hz, H-20), 5.87 (d, J = 2.1 Hz, H-10), 7.49–7.55 (m,
2H, naphthyl), 7.69 (dd, J = 1.8, 8.4 Hz, naphthyl), 7.90–7.95 (m,
3H, naphthyl), 8.06 (s, H-6), 8.12 (d, J = 1.2 Hz, naphthyl), 11.69
(s, 3-NH). 31P NMR (DMSO-d6): d 16.21, 16.28 (OCH2CH3), 20.81
(d, J = 140 Hz, C-60), 25.30, 27.05 (C(CH3)2), 25.88 (C-50), 61.01
(OCH2CH3), 82.80 (C-30), 83.47 (C-20), 85.56 (C-40), 92.28 (C-10),
113.40, 113.66 (C-5, (C(CH3)2)), 126.09, 126.19, 126.49, 126.82,
127.28, 127.42, 127.94, 130.42, 132.12, 132.78, 140.89 (naphthyl,
C-6), 149.80 (C-2), 162.51 (C-4). HRMS (ESI) for C27H34N2O8P
[M+H]+ found, 545.2076; calcd, 545.2047.
10 l .
L minꢀ1
4.1.1. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b- -ribo-hexofuranosyl]-5-bromouracil (5)
D
To a solution of compound 49 (399 mg, 0.95 mmol) in DMF
(8 mL) was added N-bromosuccinimide (NBS, 187 mg, 1.05 mmol)
under N2. The reaction mixture was stirred at room temperature
for 16 h. DMF was removed in vacuo and the residue was purified
by column chromatography (CH2Cl2/MeOH 95:5) to afford com-
pound 5 (376 mg, 79%) as a colorless oil. 1H NMR (300 MHz,
DMSO-d6): d 1.20–1.28 (m, 9H, OCH2CH3, CH3), 1.47 (s, 3H, CH3),
1.69–1.92 (m, 4H, H-50, H-60), 3.90–4.04 (m, 5H, OCH2CH3, H-40),
4.64–4.67 (m, H-30), 5.04 (dd, J = 2.1, 6.6 Hz, H-20), 5.73 (d,
J = 2.1 Hz, H-10), 8.23 (s, H-6), 11.91 (s, 3-NH). 31P NMR (DMSO-
4.2.3. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b-D-ribo-hexofuranosyl]-5-(4-fluorophenyl)uracil (6c)
Reaction of compound 5 (78 mg, 0.16 mmol) with 4-fluoro-
phenyl boronic acid (44 mg, 0.31 mmol), Pd(PPh3)4 (18 mg,
0.016 mmol) and Na2CO3 (54 mg, 0.51 mmol) in DMF (4 mL) and
degassed H2O (0.5 mL) was performed as described in the general
procedure for the synthesis of 5-modified phosphonates. Purifica-
tion of the crude mixture on a silica gel column (CH2Cl2/MeOH
95:5) afforded compound 6c as a colorless solid (56 mg, 70%). 1H
NMR (300 MHz, DMSO-d6): d 1.18 (dt, 6H, J = 0.9, 7.2 Hz, OCH2CH3),
1.27 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.72–1.89 (m, 4H, H-50, H-60),
3.92–4.02 (m, 5H, OCH2CH3, H-40), 4.69 (dd, J = 4.8, 6.6 Hz, H-30),
5.14 (dd, J = 2.4, 6.9 Hz, H-20), 5.83 (d, J = 2.4 Hz, H-10), 7.19–7.25
(m, 2H, Ph), 7.57–7.63 (m, 2H, Ph), 7.92 (s, H-6), 11.64 (s, 3-NH).
31P NMR (DMSO-d6): d 31.56. 13C NMR (75 MHz, DMSO-d6): d
16.22, 16.29 (OCH2CH3), 20.82 (d, J = 139 Hz, C-60), 25.27, 27.03
(C(CH3)2), 25.85 (C-50), 60.95, 60.99 (d, J = 6.0 Hz, OCH2CH3),
82.77 (C-30), 83.40 (C-20), 85.56 (d, J = 17.9 Hz, C-40), 92.01 (C-10),
112.89, 113.46, 114.74, 115.02 (Ph, C-5, (C(CH3)2)), 129.08,
130.24, 130.35 (Ph), 140.45 (C-6), 149.71 (C-2), 159.94 (Ph),
d6):
d d 16.25, 16.32
31.53. 13C NMR (75 MHz, DMSO-d6):
(OCH2CH3), 20.83 (d, J = 140 Hz, C-60), 25.24, 27.00 (C(CH3)2),
25.70 (C-50), 60.91, 61.00 (OCH2CH3), 82.61 (C-30), 83.38 (C-20),
85.65 (d, J = 17.9 Hz, C-40), 91.77 (C-10), 96.12 (C-5), 113.49
(C(CH3)2), 142.53 (C-6), 149.56 (C-2), 159.23 (C-4). HRMS (ESI)
for C17H27BrN2O8P [M+H]+ found, 497.0697; calcd, 497.0683.
4.2. General procedure for the synthesis of 5-modified nucleo-
side phosphonates via Suzuki–Miyaura coupling (6a–h)
A mixture of compound 5 (1 equiv), aryl boronic acid (2 equiv),
Pd(PPh3)4 (0.1 equiv) and Na2CO3 (3.3 equiv) in DMF and degassed
H2O was heated ( 130 °C, oil bath) under argon for 6 h or until TLC
indicated consumption of all starting material. The mixture was
then concentrated and co-distilled with toluene. The residue was
purified by column chromatography affording 5-modified ana-
logues 6a–h in moderate yield.
162.26 (C-4). HRMS (ESI) for
513.1815; calcd, 513.1797.
C
23H31FN2O8P [M+H]+ found,
4.2.1. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b-D-ribo-hexofuranosyl]-5-phenyluracil (6a)
4.2.4. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b- -ribo-hexofuranosyl]-5-styryluracil (6d)
Reaction of compound 5 (78 mg, 0.16 mmol) with phenylbo-
ronic acid (39 mg, 0.32 mmol), Pd(PPh3)4 (18 mg, 0.016 mmol)
and Na2CO3 (55 mg, 0.52 mmol) in DMF (4 mL) and degassed H2O
(0.5 mL) was performed as described in the general procedure.
Purification of the crude mixture on a silica gel column (CH2Cl2/
MeOH 95:5) afforded compound 6a as a colorless oil (33 mg,
43%). 1H NMR (300 MHz, DMSO-d6): d 1.18–1.23 (m, 6H, OCH2CH3),
1.30 (s, 3H, CH3), 1.49 (s, 3H, CH3), 1.74–1.89 (m, 4H, H-50, H-60),
D
Reaction of compound 5 (91 mg, 0.18 mmol) with trans-2-phe-
nylvinyl boronic acid (56 mg, 0.37 mmol), Pd(PPh3)4 (21 mg,
0.018 mmol) and Na2CO3 (64 mg, 0.60 mmol) in DMF (4 mL) and
degassed H2O (0.5 mL) was performed as described in the general
procedure for the synthesis of 5-modified phosphonates. Purifica-
tion of the crude mixture on a silica gel column (CH2Cl2/MeOH

S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
2311
95:5) afforded compound 6d as a colorless solid (72 mg, 76%). 1H
NMR (300 MHz, DMSO-d6): d 1.18 (app dt, 6H, J = 1.2, 6.9 Hz,
OCH2CH3), 1.28 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.71–1.88 (m, 4H,
H-50, H-60), 3.90–4.01 (m, 5H, OCH2CH3, H-40), 4.68 (dd, J = 4.8,
6.6 Hz, H-30), 5.06 (dd, J = 2.4, 6.6 Hz, H-20), 5.80 (d, J = 2.4 Hz, H-
10), 6.88 (d, J = 16.2 Hz, styryl), 7.20–7.25 (m, styryl), 7.33 (d, 2H,
J = 7.8 Hz, styryl), 7.43–7.48 (m, 3H, styryl), 7.94 (H-6). 31P NMR
(DMSO-d6): d 31.51. 13C NMR (75 MHz, DMSO-d6): d 16.22, 16.30
(OCH2CH3), 20.83 (d, J = 140 Hz, C-60), 25.29, 27.02 (C(CH3)2),
25.89 (C-50), 60.97, 61.01 (d, J = 6.3 Hz, OCH2CH3), 82.64 (C-30),
83.49 (C-20), 85.41 (d, J = 17.6 Hz, C-40), 91.34 (C-10), 111.22 (C-5),
113.54 (C(CH3)2), 120.83, 125.99, 127.42, 128.32, 128.74, 137.43
(styryl), 140.09 (C-6), 149.26 (C-2), 162.21 (C-4). HRMS (ESI) for
8.20 (s, H-6). 31P NMR (DMSO-d6): d 31.64. 13C NMR (75 MHz,
DMSO-d6): d 16.25, 16.33 (OCH2CH3), 20.85 (d, J = 140 Hz, C-60),
25.31, 27.07 (C(CH3)2), 60.99 (OCH2CH3), 82.87 (C-30), 83.57 (C-
20), 85.86 (C-40), 93.79 (C-10), 108.56 (C-5), 113.40 (C(CH3)2),
128.73–138.33 (thienyl). HRMS (ESI) for C21H30N2O8PS [M+H]+
found, 501.1453; calcd, 501.1455.
4.2.8. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b-D-ribo-hexofuranosyl]-5-(benzothiophen-3-yl)uracil
(6h)
Reaction of compound 5 (105 mg, 0.21 mmol) with 1-benzo-
thiophen-3-ylboronic acid (77 mg, 0.42 mmol), Pd(PPh3)4 (24 mg,
0.021 mmol) and Na2CO3 (74 mg, 0.70 mmol) in DMF (5 mL) and
degassed H2O (0.7 mL) was performed as described in the general
procedure for the synthesis of 5-modified phosphonates. Purifica-
tion of the crude mixture on a silica gel column (CH2Cl2/MeOH
95:5) afforded compound 6h as a brown solid (49 mg, 42%). 1H
NMR (300 MHz, CDCl3): d 1.25–1.32 (m, 9H, OCH2CH3, CH3), 1.55
(s, 3H, CH3), 1.77–1.93 (m, 2H, H-60), 1.98–2.08 (m, 2H, H-50),
4.02–4.12 (m, 5H, OCH2CH3, H-40), 4.61 (t, J = 5.7 Hz, H-30), 5.00
(app d, J = 6.6 Hz, H-20), 5.70 (app s, H-10), 7.32–7.41 (m, 2H, benzo-
thiophenyl), 7.46–7.48 (m, benzothiophenyl), 7.54 (s, H-6), 7.67 (d,
J = 7.8 Hz, benzothiophenyl), 7.86 (d, J = 7.8 Hz, benzothiophenyl),
9.53 (br s, 3-NH). 31P NMR (CDCl3): d 31.01. 13C NMR (75 MHz,
CDCl3): d 16.52, 16.60 (OCH2CH3), 21.99 (d, J = 142 Hz, C-60),
25.48, 27.31 (C(CH3)2), 26.41 (C-50), 61.81 (OCH2CH3), 83.43 (C-
30), 84.41 (C-20), 86.64 (d, J = 17.9 Hz, C-40), 93.79 (C-10), 110.69,
115.11 (C-5, (C(CH3)2)), 122.48, 123.08, 124.65, 124.71, 126.86,
126.94, 137.70 (benzothiophenyl), 140.15, 140.39 (C-6, benzothi-
ophenyl), 149.54 (C-2), 162.12 (C-4). HRMS (ESI) for C25H32N2O8PS
[M+H]+ found, 551.1620; calcd, 551.1612.
C
25H34N2O8P [M+H]+ found, 521.2049; calcd, 521.2047.
4.2.5. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b- -ribo-hexofuranosyl]-5-(p-tolyl)uracil (6e)
D
Reaction of compound 5 (63 mg, 0.13 mmol) with 4-meth-
ylphenylboronic acid (36 mg, 0.25 mmol), Pd(PPh3)4 (15 mg,
0.013 mmol) and Na2CO3 (45 mg, 0.42 mmol) in DMF (3 mL) and
degassed H2O (0.4 mL) was performed as described in the general
procedure for the synthesis of 5-modified phosphonates. Purifica-
tion of the crude mixture on a silica gel column (CH2Cl2/MeOH
97:3) afforded compound 6e as a colorless solid (29 mg, 46%). 1H
NMR (300 MHz, CDCl3): d 1.24–1.33 (m, 9H, OCH2CH3, CH3), 1.54
(s, 3H, CH3), 1.72–2.07 (m, 4H, H-50, H-60), 2.71 (s, 3H, p-tolyl),
4.01–4.13 (m, 5H, OCH2CH3, H-40), 4.64 (dd, J = 4.5, 6.3 Hz, H-30),
5.04 (dd, J = 2.1, 6.3 Hz, H-20), 5.67 (d, J = 2.4 Hz, H-10), 7.16–7.79
(m, 4H, p-tolyl), 8.00 (H-6). 31P NMR (CDCl3): d 31.27. HRMS (ESI)
for C24H34N2O8P [M+H]+ found, 509.2076; calcd, 509.2047.
4.2.6. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b-
D
-ribo-hexofuranosyl]-5-(furan-2-yl)uracil (6f)
4.2.9. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
D-ribo-hexo-
Reaction of compound 5 (93 mg, 0.19 mmol) with furan-2-boro-
nic acid (42 mg, 0.37 mmol), Pd(PPh3)4 (22 mg, 0.019 mmol) and
Na2CO3 (65 mg, 0.62 mmol) in DMF (4.5 mL) and degassed H2O
(0.6 mL) was performed as described in the general procedure for
the synthesis of 5-modified phosphonates. Purification of the crude
mixture on a silica gel column (CH2Cl2/MeOH 95:5) afforded com-
pound 6f as a brown solid (36 mg, 40%). 1H NMR (300 MHz, DMSO-
d6): d 1.16–1.28 (m, 6H, OCH2CH3) 1.28 (s, 3H, CH3), 1.48 (s, 3H,
CH3), 1.76–1.81 (m, 4H, H-50, H-60), 3.82–4.07 (m, 5H, OCH2CH3,
H-40), 4.58–4.68 (m, H-30), 5.03–5.13 (m, H-20), 5.84–5.86 (m, H-
10), 6.50–6.54 (m, furanyl), 6.83–6.88 (m, furanyl), 7.61–7.66 (m,
furanyl), 8.02 (app d, J = 4.8 Hz, H-6). 31P NMR (DMSO-d6): d
31.63. 13C NMR (75 MHz, DMSO-d6): d 16.29, 16.37 (OCH2CH3),
20.99 (d, J = 148 Hz, C-60), 25.31, 27.07 (C(CH3)2), 60.99, 61.11
(OCH2CH3), 82.83 (C-30), 83.74 (C-20), 85.95 (d, J = 7.5 Hz, C-40),
92.37 (C-10), 105.72, 108.40 (furanyl), 111.73 (C-5), 113.46
((C(CH3)2)), 128.83, 131.55 (furanyl), 141.79 (C-6), 149.24 (C-2),
furanosyl]-5-phenyluracil (7a)
Phosphonic ester 6a (32 mg, 0.068 mmol) was dissolved in
1.4 mL of CH2Cl2 under argon and treated with TMSBr (18 L,
l
1.35 mmol) and the solution was stirred overnight. The solvents
were evaporated and the residue was co-distilled with toluene.
Then, 0.7 mL of H2O was added followed by 1.4 mL of a 50% aque-
ous TFA solution and the mixture was stirred for 4 h. The solvent
was evaporated and subsequently portioned between EtOAc/Et2O
(1:1) and H2O. The organic phase was washed with H2O and the
aqueous layers were combined and lyophilized. Purification of
the crude mixture using RP high-performance liquid chromatogra-
phy (HPLC, Phenomenex Luna C-18, H2O/0.1% HCOOH in CH3CN,
90:10?0:100 in 23 min, flow 17.5 mL/min) afforded compound
7a as a white powder (14.1 mg, 55%). 1H NMR (300 MHz, DMSO-
d6): d 1.40–1.43 (m, 2H, H-60), 1.80–1.84 (m, 2H, H-50), 3.82–3.83
(m, 2H, H-30, H-40), 4.19 (t, J = 5.1 Hz, H-20), 5.72 (d, J = 4.5 Hz, H-
10), 7.26–7.40 (m, 3H, Ph), 7.51–7.54 (m, 2H, Ph), 7.64 (s, H-6).
31P NMR (DMSO-d6): d 25.89. 13C NMR (75 MHz, DMSO-d6): d
26.89 (C-50), 72.46 (C-30), 72.52 (C-20), 83.59 (d, J = 15.9 Hz, C-40),
89.54 (C-10), 113.97 (C-5), 127.36, 128.16, 128.22, 132.80 (Ph),
138.34 (C-6), 150.11 (C-2), 162.07 (C-4). HRMS (ESI) for
160.37 (C-4). HRMS (ESI) for
485.1708; calcd, 485.1683.
C
21H30N2O9P [M+H]+ found,
4.2.7. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-20,30-O-isopro-
pylidene-b- -ribo-hexofuranosyl]-5-(thien-2-yl)uracil (6g)
D
C
16H18N2O8P [MꢀH]ꢀ found, 397.0815; calcd, 397.0806.
Reaction of compound 5 (81 mg, 0.16 mmol) with thiophene-2-
boronic acid (42 mg, 0.33 mmol), Pd(PPh3)4 (19 mg, 0.016 mmol)
and Na2CO3 (57 mg, 0.54 mmol) in DMF (4 mL) and degassed H2O
(0.5 mL) was performed as described in the general procedure for
the synthesis of 5-modified phosphonates. Purification of the crude
mixture on a silica gel column (CH2Cl2/MeOH 95:5) afforded com-
pound 6g as a colorless solid (32 mg, 39%). 1H NMR (300 MHz,
DMSO-d6): d 1.1–1.22 (m, 6H, OCH2CH3) 1.28 (s, 3H, CH3), 1.48
(s, 3H, CH3), 1.68–1.91 (m, 4H, H-50, H-60), 3.90–3.97 (m, 5H,
OCH2CH3, H-40), 4.67–4.69 (m, H-30), 5.13–5.15 (m, H-20), 5.82
(m, H-10), 7.05–7.07 (m, thienyl), 7.46–7.47 (m, 2H, m, thienyl),
4.2.10. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
D-ribo-
hexofuranosyl]-5-(naphthalen-2-yl)uracil (7b)
Reaction of compound 6b (70 mg, 0.073 mmol) with TMSBr
L, 1.46 mmol), work-up and purification as described for 7a,
(19
l
afforded compound 7b as a white powder (22.6 mg, 39%). 1H
NMR (300 MHz, DMSO-d6): d 1.60 (br s, 2H, H-60), 1.78–1.84 (m,
2H, H-50), 3.77–3.85 (m, 2H, H-30, H-40), 4.25 (t, J = 5.4 Hz, H-20),
5.73 (d, J = 4.8 Hz, H-10), 7.29–7.41 (m, 3H, naphthyl), 7.52–7.69
(m, 2H, naphthyl), 7.69 (s, H-6), 11.57 (s, 3-NH). 31P NMR
13
(DMSO-d6): d 25.71. C NMR (75 MHz, DMSO-d6): d 23.80 (C-60),

2312
S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
27.04 (C-50), 72.56 (C-30), 72.62 (C-20), 83.52 (C-40), 89.83 (C-10),
113.94 (C-5), 126.19, 126.27, 126.58, 126.78, 127.50, 128.22,
130.51, 132.23, 132.91 (naphthyl), 138.95 (C-6), 150.21 (C-2),
4.2.15. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
D-ribo-
hexofuranosyl]-5-(thien-2-yl)uracil (7g)
Reaction of compound 6g (32 mg, 0.063 mmol) with TMSBr
(17 L, 1.26 mmol), work-up and purification as described for 7a,
162.32 (C-4). HRMS (ESI) for
447.0969; calcd, 447.0963.
C
20H20N2O8P [MꢀH]ꢀ found,
l
afforded compound 7g as a white powder (9.3 mg, 37%). 1H NMR
(300 MHz, DMSO-d6): d 1.61 (br s, 2H, H-60), 1.75–1.91 (m, 2H,
H-50), 3.80–3.85 (m, 2H, m, H-30, H-40), 4.22–4.28 (m, H-20), 5.71
(d, J = 4.5 Hz, H-10), 7.02–7.10 (m, thienyl), 7.46 (d, J = 5.1 Hz, thie-
nyl), 7.50 (s, thienyl), 7.96 (s, H-6). 31P NMR (DMSO-d6): d 25.42.
13C NMR (175 MHz, DMSO-d6): d 24.21 (C-60), 26.99 (C-50), 72.36
(C-30), 72.73 (C-20), 83.68 (C-40),89.80 (C-10), 108.63 (C-5), 123.20
(C-3’’), 125.80 (C-5’’), 126.54 (C-4’’), 133.39 (C-2’’), 135.82 (C-6),
149.48 (C-2), 161.30 (C-4). HRMS (ESI) for C14H16N2O8PS [MꢀH]ꢀ
found, 403.0343; calcd, 403.0371.
4.2.11. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
D-ribo-
hexofuranosyl]-5-(4-fluorophenyl)uracil (7c)
Reaction of compound 6c (56 mg, 0.11 mmol) with TMSBr
(29 L, 2.20 mmol), work-up and purification as described for 7a,
l
afforded compound 7c as a white powder (26.1 mg, 57%). 1H
NMR (300 MHz, DMSO-d6): d 1.62 (br s, 2H, H-60), 1.79 (br s, 2H,
H-50), 3.77–3.84 (m, 2H, H-30, H-40), 4.26 (t, J = 5.1 Hz, H-20), 5.73
(d, J = 4.8 Hz, H-10), 7.21 (t, 2H, J = 8.7 Hz, Ph), 7.56–7.60 (m, 2H,
Ph), 7.70 (s, H-6), 11.59 (s, 3-NH). 31P NMR (DMSO-d6): d 26.17.
13C NMR (75 MHz, DMSO-d6): d 27.05 (C-50), 72.57 (C-30), 72.60
(C-20), 83.65 (C-40), 89.67 (C-10), 113.11 (C-5), 114.92, 115.20,
129.21, 129.25, 130.29, 130.40 (Ph), 138.44 (C-6), 150.18 (C-2),
160.01 (Ph), 162.17 (C-4). HRMS (ESI) for C16H17FN2O8P [MꢀH]ꢀ
found, 415.0722; calcd, 415.0712.
4.2.16. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
D-ribo-
hexofuranosyl]-5-(benzothiophen-3-yl)uracil (7h)
Reaction of compound 6h (49 mg, 0.088 mmol) with TMSBr
(23 L, 1.76 mmol), work-up and purification as described for 7a,
l
afforded compound 7h as a white powder (31.5 mg, 78%). 1H
NMR (300 MHz, DMSO-d6): d 1.59 (br s, 2H, H-60), 1.76–1.80 (m,
2H, H-50), 3.76–3.83 (m, 2H, H-30, H-40), 4.21–4.4.24 (m, H-20),
5.77 (d, J = 4.8 Hz, H-10), 7.36–7.44 (m, 2H, benzothiophenyl),
7.63–7.66 (m, benzothiophenyl), 7.78 (app d, 2H, J = 7.2 Hz, benzo-
thiophenyl, H-6), 7.98–8.02 (m, benzothiophenyl), 11.66 (s, 3-NH).
31P NMR (DMSO-d6): d 26.13. 13C NMR (75 MHz, DMSO-d6): d 26.65
(C-50), 72.49 (C-30), 72.44 (C-20), 83.47 (C-40), 89.37 (C-10), 109.30
(C-5), 123.05, 124.28, 124.39, 126.65, 128.23, 138.02, 139.14,
139.68 (benzothiophenyl, C-6), 150.29 (C-2), 162.00 (C-4). HRMS
(ESI) for C18H18N2O8PS [MꢀH]ꢀ found, 453.0531; calcd, 453.0527.
4.2.12. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
D-ribo-
hexofuranosyl]-5-styryluracil (7d)
Reaction of compound 6d (72 mg, 0.14 mmol) with TMSBr
(36 L, 2.75 mmol), work-up and purification as described for
l
7a, afforded compound 7d as a white powder (6.5 mg, 11%). 1H
NMR (300 MHz, DMSO-d6): d 1.60 (br s, 2H, H-60), 1.87 (br s,
2H, H-50), 3.78–3.86 (m, 2H, H-30, H-40), 4.15 (app s, H-20),
5.75–5.76 (m, H-10), 6.98 (d, J = 16.2 Hz, styryl), 7.15–7.57 (m,
6H, styryl), 7.78 (s, H-6). 31P NMR (DMSO-d6): d 24.42. 13C
NMR (175 MHz, DMSO-d6): d 24.35 (C-60), 27.07 (C-50), 72.35
(C-30), 72.72 (C-20), 83.48 (C-40), 88.68 (C-10), 111.27 (C-5),
121.28 (5-CH@CH–), 125.97 (Cortho), 127.27 (Cpara), 128.20 (5-
CH@CH–), 128.57 (Cmeta), 137.57 (Cipso), 138.78 (C-6), 149.62
(C-2), 162.04 (C-4). HRMS (ESI) for C18H20N2O8P [MꢀH]ꢀ found,
423.0954; calcd, 423.0963.
4.2.17. 1-[50,60-Dideoxy-60-(diethoxyphosphinyl)-b-
D-ribo-
hexofuranosyl]uracil (8)
A solution of compound 4 (428 mg, 1.02 mmol) and 50% aq
HCOOH (10 mL) was stirred for 4.5 h at room temperature. The
mixture was evaporated in vacuo and purified on a silica column
using CH2Cl2/MeOH (90:10) as a solvent. Compound 8 was ob-
tained as a colorless solid (220 mg, 57%). 1H NMR (300 MHz,
DMSO-d6): d 1.23 (t, 6H, J = 6.9 Hz, OCH2CH3), 1.72–1.86 (m, 4H,
H-50, H-60), 3.32–3.83 (m, 2H, H-30, H-40), 3.93–4.11 (m, 5H,
OCH2CH3, H-20), 5.12 (d, J = 5.7 Hz, 30-OH), 5.35 (d, J = 5.7 Hz, 20-
OH), 5.63 (d, J = 8.1 Hz, CH@CH), 5.68 (d, J = 5.1 Hz, H-10), 7.59 (d,
J = 8.1 Hz, CH@CH), 11.34 (s, 3-NH). 31P NMR (DMSO-d6): d 31.86.
13C NMR (75 MHz, DMSO-d6): d 16.26, 16.33 (OCH2CH3), 20.92 (d,
J = 140 Hz, C-60), 25.93 (C-50), 60.93, 60.96 (d, J = 6 Hz, OCH2CH3),
72.48, 72.57 (C-20, C-30), 82.65 (d, J = 17 Hz, C-40), 88.93 (C-10),
102.06 (C-5), 141.42 (C-6), 150.61 (C-2), 163.03 (C-4). HRMS (ESI)
for C14H24N2O8P [M+H]+ found, 379.1262; calcd, 379.1265.
4.2.13. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
hexofuranosyl]-5-(p-tolyl)uracil (7e)
D-ribo-
Reaction of compound 6e (29 mg, 0.058 mmol) with TMSBr
(15 L, 1.15 mmol) work-up and purification as described for 7a,
l
afforded compound 7e as a white powder (4.3 mg, 18%). 1H NMR
(300 MHz, DMSO-d6): d 1.40–1.99 (m, 4H, H-50, H-60), 3.77–3.82
(m, 2H, H-30, H-40), 4.22 (m, H-20), 5.73 (d, J = 4.5 Hz, H-10), 7.19
(d, 2H, J = 7.5 Hz, p-tolyl), 7.42 (d, 2H, J = 7.8 Hz, p-tolyl), 7.62 (s,
H-6). 31P NMR (DMSO-d6): d 25.55. 13C NMR (75 MHz, DMSO-d6):
d 20.61 (CH3), 27.00 (C-60), 72.33 (C-30), 72.42 (C-20), 83.64 (C-40),
89.28 (C-10), 113.74 (C-5), 127.85, 128.58, 129.71, 136.47 (p-tolyl),
137.58 (C-6), 149.92 (C-2), 161.95 (C-4). HRMS (ESI) for
4.2.18. 1-[2,20-O-Anhydro-50,60-dideoxy-60-(diethoxyphosphi-
nyl)-b-D-arabinofuranosyl]uracil (9)
C
17H20N2O8P [MꢀH]ꢀ found, 411.0970; calcd, 411.0963.
Compound 8 (205 mg, 0.54 mmol) was dissolved into a mixture
of thionyl chloride (0.17 mL) and CH3CN (1.4 mL) with vigorously
stirring and then the reaction was maintained at room tempera-
ture. After 2 h, the mixture was poured into H2O and extracted
with CH2Cl2. The combined organic layers were dried over MgSO4,
filtered and concentrated in vacuo. Without further purification,
the 20,30-sulfonyl derivative of 8 and sodium acetate (223 mg,
2.72 mmol) were heated in DMF at about 85 °C. After stirring for
3 h, solvents were removed in vacuo and the crude mixture puri-
fied on a silica gel column (CH2Cl2/MeOH (95:5?80:20)) yielding
180 mg (92%) of 9 as a white foam. 1H NMR (300 MHz, DMSO-
d6): d 1.14–1.23 (m, 7H, OCH2CH3, H-50a), 1.59–1.68 (m, 3H, H-
50b, H-60), 3.84–3.94 (m, 4H, OCH2CH3), 4.00–4.10 (m, H-40), 4.32
(app s, H-30), 5.19 (d, J = 5.7 Hz, H-20), 5.87 (d, J = 7.5 Hz, CH@CH),
6.30 (d, J = 5.7 Hz, H-10), 7.91 (d, J = 7.5 Hz, CH@CH). 31P NMR
4.2.14. 1-[50,60-Dideoxy-60-(dihydroxyphosphinyl)-b-
D-ribo-
hexofuranosyl]-5-(furan-2-yl)uracil (7f)
Reaction of compound 6f (32 mg, 0.067 mmol) with TMSBr
(18 L, 1.34 mmol), work-up and purification as described for 7a,
l
afforded compound 7f as a white powder (5.8 mg, 22%). 1H NMR
(300 MHz, DMSO-d6): d 1.61 (br s, 2H, H-60), 1.83–1.91 (m, 2H,
H-50), 3.80–3.82 (m, 2H, H-30, H-40), 4.12 (s, H-20), 5.73 (d,
J = 3.9 Hz, H-10), 6.51 (d, J = 1.8 Hz, furanyl), 6.87 (d, J = 3.3 Hz, fura-
nyl), 7.66 (s, furanyl), 7.82 (s, H-6). 31P NMR (DMSO-d6): d 25.08.
13C NMR (75 MHz, DMSO-d6): d 27.12 (C-50), 72.58, 73.34 (C-20,
C-30), 83.56 (C-40), 89.56 (C-10), 105.69, 108.10, 111.56 (C-5, fura-
nyl), 134.25 (C-6), 141.81, 146.08 (furanyl), 149.42 (C-2), 160.07
(C-4). HRMS (ESI) for C14H16N2O9P [MꢀH]ꢀ found, 387.0604; calcd,
387.0599.

S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
2313
(DMSO-d6): d 31.02. 13C NMR (75 MHz, CDCl3): d 16.45, 16.53
(OCH2CH3), 22.04 (d, J = 143 Hz, C-60), 26.42 (C-50), 62.14, 62.17
(d, J = 6.6 Hz, OCH2CH3), 87.72 (d, J = 16.7 Hz, C-40), 89.49 (C-20),
90.31 (C-10), 109.94 (C-5), 136.16 (C-6), 160.19 (C-2), 172.90 (C-
4). HRMS (ESI) for C14H22N2O7P [M+H]+ found, 361.1157; calcd,
361.1159.
(d, J = 8.4 Hz, CH@CH), 11.45 (s, 3-NH). 31P NMR (DMSO-d6): d
32.03. 13C NMR (75 MHz, DMSO-d6): d 16.43, 16.50 (OCH2CH3),
20.99 (d, J = 140 Hz, C-60), 25.82 (d, J = 3.9 Hz, C-50), 61.12 (C-20),
61.20 (OCH2CH3), 71.93 (C-30), 83.57 (d, J = 17 Hz, C-40), 88.83 (C-
10), 102.62 (C-5), 140.71 (C-6), 150.69 (C-2), 163.07 (C-4). HRMS
(ESI) for C14H23ClN2O7P [M+H]+ found, 397.0942; calcd, 397.0926.
4.2.19. 1-[20-Azido-20-deoxy-50,60-dideoxy-60-(ethoxyhydroxy-
4.2.23. 1-[20-Chloro-20-deoxy-50,60-dideoxy-60-(dihydroxyphos-
phosphinyl)-b-
D
-ribo-hexofuranosyl]uracil (10)
phinyl)-b-
To a solution of compound 13 (41 mg, 0.10 mmol) in CH2Cl2
(1.5 mL) was added TMSBr (27 L, 0.20 mmol). After stirring over-
D-ribo-hexofuranosyl]uracil (14)
A suspension of compound 9 (33 mg, 0.092 mmol) and NaN3
(37 mg, 0.56 mmol) in 0.7 mL DMF was heated overnight at
150 °C. The mixture was cooled to room temperature and evapo-
rated in vacuo. The residue was dissolved in CH2Cl2/MeOH (8:2)
and filtrated. Solvents were removed to afford compound 10 which
was used in the next reaction without further purification (30 mg,
88%, yellow oil). 1H NMR (300 MHz, DMSO-d6): d 1.08–1.20 (m, 3H,
OCH2CH3), 1.48–1.70 (m, 2H, H-60), 1.76–1.91 (m, 2H, H-50), 3.75–
3.89 (m, 3H, OCH2CH3, H-20), 4.13–4.17 (m, H-40), 4.24–4.26 (m, H-
30), 5.66–5.74 (m, 2H, H-10, CH@CH), 7.94–8.02 (m, CH@CH). 31P
NMR (DMSO-d6): d 25.36. HRMS (ESI) for C12H19N5O7P [M+H]+
found, 376.0997; calcd, 376.1017.
l
night, the volatiles were removed in vacuo. The residue was dis-
solved in H2O, washed with EtOAc/Et2O (1:1) and lyophilized.
The residue was purified using flash chromatography (iPrOH/
NH4OH/H2O 6:3:1) yielding 58% of compound 14 (21.8 mg, white
powder). Compound 14 was isolated in the ammonium salt form.
1H NMR (300 MHz, D2O): d 1.60–1.82 (m, 2H, H-60), 1.94–2.05
(m, 2H, H-50), 4.13–4.19 (m, H-40), 4.27 (app t, J = 5.4 Hz, H-30),
4.66 (app t, J = 5.1 Hz, H-20), 5.92 (d, J = 8.4 Hz, CH@CH), 6.08 (d,
J = 4.8 Hz, H-10), 7.71 (d, J = 8.1 Hz, CH@CH). 31P NMR (D2O): d
13
24.24. C NMR (75 MHz, D2O): d 24.17 (d, J = 134 Hz, C-60), 27.10
(d, J = 3.8 Hz C-50), 61.39 (C-20), 72.56 (C-30), 84.25 (d, J = 17.9 Hz
4.2.20. 1-[20-Amino-20-deoxy-50,60-dideoxy-60-(ethoxyhydroxy-
phosphinyl)-b-
C-40), 90.27 (C-10), 102.73 (C-5), 141.52 (C-6), 151.69 (C-2),
D
-ribo-hexofuranosyl]uracil (11)
166.25 (C-4). HRMS (ESI) for
339.0154; calcd, 339.0154.
C
10H13ClN2O7P [MꢀH]ꢀ found,
Compound 10 (24 mg, 0.065 mmol) and PPh3 (34 mg,
0.13 mmol) were dissolved in THF (1 mL). After stirring for 10 min-
utes, H2O was added (17
lL). After stirring overnight, the volatiles
4.2.24. (10S,20R,30S,40R,50S)-10-(Diisopropyl-phosphonoethenyl)-
20,30-O-(isopropylidine)-40-(uracil-1-yl)-bicyclo[3.1.0]hexane
(16)
were removed in vacuo. The residue dissolved in H2O, washed with
EtOAc/Et2O (1:1) and lyophilized to yield 24 mg of crude 11 as a
light yellow solid. 1H NMR (300 MHz, DMSO-d6): d 1.02–1.15 (m,
3H, OCH2CH3), 1.21–1.52 (m, 2H, H-60), 1.76 (br s, 2H, H-50),
3.56–3.77 (m, 3H, OCH2CH3, H-20), 4.04–4.10 (m, H-40), 4.22 (app
s, H-30), 5.83–5.88 (m, CH@CH), 6.27 (app d, J = 5.4 Hz, H-10),
7.91–7.93 (d, J = 7.5 Hz, CH@CH). 31P NMR (D2O-d6): d 26.76. HRMS
(ESI) for C12H19N3O7P [MꢀH]ꢀ found, 348.0954; calcd, 348.0966.
Diisopropyl azodicarboxylate (93 lL, 0.47 mmol) was added at
rt to a mixture of triphenylphosphine (123 mg, 0.47 mmol) and
3-N-benzoyluracil (101 mg, 0.47 mmol) in anhydrous THF (3 mL).
After stirring for 40 min, a solution of compound 1522 (85 mg,
0.23 mmol) in anhydrous THF (3 mL) was added to the mixture.
After stirring for 36 h, the reaction mixture was evaporated to dry-
ness. The resulting residue was purified by silica gel column chro-
matography (0–6% MeOH in EtOAc) to afford N-3-benzoyl
protected uracil nucleoside as a white solid material (Rf = 0.4,
EtOAc/MeOH 94:6), which was treated with 6 N NH3/MeOH
(6 mL) and heated up to 50 °C. After stirring for 19 h, the reaction
mixture was evaporated to dryness. The resulting residue was
purified by silica gel column chromatography (0–8% MeOH in
CH2Cl2) to afford phosphonate diester 16 (25 mg, 24%) as a white
solid material. Rf = 0.3 (CH2Cl2/MeOH 92:8). 1H NMR (CDCl3): d
0.72–0.80 (m), 0.89–0.95 (m), 1.24 (s, 3H), 1.28–1.33 (m, 12 H),
1.35–1.39 (m), 1.48 (s, 3H), 1.60–2.04 (m, 4H), 4.33 (s), 4.64–4.72
(m, 3H), 4.91 (d, J = 7.1 Hz), 5.70 (d, J = 7.69 Hz), 7.14 (d,
J = 7.69 Hz), 8.25 (s, NH). HRMS (ESI) for C21H34N2O7P [M+H]+
found, 457.2121; calcd, 457.2104.
4.2.21. 1-[20-Amino-20-deoxy-50,60-dideoxy-60-
(dihydroxyphosphinyl)-b-
To a solution of compound 11 (23.6 mg, 0.067 mmol) in CH2Cl2
(2.0 mL) was added TMSBr (90 L, 0.68 mmol). After stirring over-
D-ribo-hexofuranosyl]uracil (12)
l
night, the volatiles were removed in vacuo. The residue was dis-
solved in H2O, washed with EtOAc/Et2O (1:1) and lyophilized.
The residue was purified using flash chromatography (iPrOH/
NH4OH/H2O 6:3:1) yielding compound 12 (18.6 mg, 78%) as a light
yellow powder. Compound 12 was isolated in the ammonium salt
1
form. H NMR (300 MHz, D2O): d 1.61–1.78 (m, 2H, H-60), 1.89–
1.95 (m, 2H, H-50), 4.00–4.01 (m, H-20), 4.12–4.18 (m, H-40),
4.34–4.37 (m, H-30), 5.93 (d, J = 7.8 Hz, CH@CH), 6.06 (app d,
J = 6.6 Hz, H-10), 7.73 (d, J = 8.4 Hz, CH@CH). 31P NMR (D2O-d6): d
13
24.03. C NMR (75 MHz, D2O): d 24.10 (d, J = 134 Hz, C-60), 27.20
(d, J = 3.5 Hz, C-50), 55.79 (C-20), 71.65 (C-30), 86.20 (d, J = 17.3 Hz,
4.2.25. (10S,20R,30S,40R,50S)-20,30-(Dihydroxy)-10-(phosphono-
ethenyl)-40-(uracil-1-yl)-bicyclo[3.1.0]hexane (17)
C-40), 87.27 (C-10), 102.90 (C-5), 141.67 (C-6), 151.88 (C-2),
166.21 (C-4). HRMS (ESI) for
320.0646; calcd, 320.0653.
C
10H15N3O7P [MꢀH]ꢀ found,
Nucleoside 16 (22 mg, 0.048 mmol) was co-evaporated with
anhydrous toluene (3 ꢁ 2 mL) and dissolved in anhydrous CH2Cl2
(2 mL). Iodotrimethylsilane (96 lL, 0.48 mmol) was added. After
4.2.22. 1-[20-Chloro-20-deoxy-50,60-dideoxy-60-(diethoxyhydr-
oxyphosphinyl)-b- -ribo-hexofuranosyl]uracil (13)
stirring for 15 h, the reaction mixture was cooled to 0 °C followed
by the addition of ice-cold H2O (15 mL) and CH2Cl2 (15 mL). The
phases were separated, and the aqueous phase was washed with
CH2Cl2 (25 mL) and diethylether (3 ꢁ 35 mL). The resulting aque-
ous phase was evaporated to dryness and purified by RP-HPLC
(Phenomenex Luna C-18, 10 mM triethylammonium acetate
(TEAA)–CH3CN, 100:0?70:30 in 30 min, flow 3 mL/min) to afford
compound 17 (2.89 mg, 19%) as a white solid material. Compound
17 was isolated in the triethylammonium salt form. 1H NMR (D2O,
300 MHz): d 0.72–0.80 (m, 1H), 1.12–1.17 (m), 1.36–1.46 (m),
1.54–2.03 (m, 4H), 4.00 (d, J = 6.60 Hz), 4.46 (d, J = 6.60 Hz), 4.62
D
To a solution of compound 9 (76 mg, 0.21 mmol) in dry dioxane
(0.5 mL) was added 2 N HCl in diethylether (1.4 mL). After stirring
overnight, the mixture was concentrated in vacuo and the residue
purified on a silica gel column (CH2Cl2/MeOH 90:10) affording
compound 13 as
a
colorless solid (45 mg, 54%). 1H NMR
(300 MHz, DMSO-d6): d 1.23 (t, 6H, J = 7.2 Hz, OCH2CH3), 1.65–
1.98 (m, 4H, H-50, H-60), 3.87–3.89 (m, H-40), 3.94–4.02 (m, 5H,
OCH2CH3, H-30), 4.73 (app t, J = 6.0 Hz, H-20), 5.69 (d, J = 8.1 Hz,
CH@CH), 5.89 (d, J = 5.1 Hz, 30-OH), 5.96 (d, J = 6 Hz, H-10), 7.63

2314
S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
(s), 5.86 (d, J = 8.25 Hz), 7.61 (d, J = 8.25 Hz). 31P NMR (D2O): d
26.82. HRMS (ESI) for C12H16N2O7P [MꢀH]ꢀ found, 331.0682;
calcd, 331.0695.
(d, J = 6.9 Hz, H-10), 7.49 (d, J = 8.1 Hz, CH@CH). 31P NMR (DMSO-
d6):
d d 16.44, 16.51
20.55. 13C NMR (75 MHz, DMSO-d6):
(OCH2CH3), 59.50 (C-50), 62.35, 62.43 (OCH2CH3), 73.07 (C-20),
73.53 (C-30), 87.78 (C-10), 103.17 (C-5), 107.87, 108.03 (C-40),
140.47 (C-6), 151.13 (C-2), 163.12 (C-4). HRMS (ESI) for
4.2.26. 20,30-Dideoxy-30-phenylselenyl-50-[(diethoxyphosphi-
nyl)-methoxy]-b-
D
-uridine (19)
C
13H22N2O9P [M+H]+ found, 381.1035; calcd, 381.1057.
A solution of phenylselenyl chloride (44 mg, 0.23 mmol) in
CH2Cl2 (0.1 mL) was added dropwise to a solution of glycal 1825
(41 mg, 0.23 mmol) in CH2Cl2 (0.7 mL). After the mixture was
stirred at ꢀ70 °C for 1 h, the solvent was removed under reduced
pressure and the residue redissolved in CH2Cl2 (0.5 mL).
4.2.29. 50-[(Dihydroxyphosphinyl)methoxy]-b-
D-uridine (22)
Compound 18 (23 mg, 0.061 mmol) was dissolved in dry DMF
(3.8 mL) and cooled to 0 °C. The solution was then treated with
2,6-lutidine (71
lL, 0.61 mmol) followed by the dropwise addition
Dimethyl(hydroxylmethyl)phosphonate (74
lL, 0.50 mmol) was
of TMSBr (41 L, 0.30 mmol). The mixture was allowed to warm to
l
added and the solution was cooled to ꢀ70 °C. Over 3 min a solution
of silver perchlorate (52 mg, 0.25 mmol) in CH3CN (0.1 mL) was
added and the mixture was allowed to warm to 0 °C and poured
into an aq NaHCO3 solution. The organic phase was separated,
dried over MgSO4 and evaporated in vacuo. The residual oil was
chromatographed on silica (CH2Cl2/MeOH 96:4) yielding com-
pound 19 (40 mg, 35%) as a colorless solid. 1H NMR (300 MHz,
CDCl3): d 1.26 (app dt, 6H, J = 1.5, 7.2 Hz, OCH2CH3), 2.41–2.45
(m, 2H, H-20), 3.59–3.67 (m, H-50a), 3.79–3.90 (m, 2H, H-50b, H-
30), 4.03–4.13 (m, 4H, OCH2CH3), 5.09 (app s, H-40), 5.74 (app dd,
J = 2.4, 8.4 Hz, CH@CH), 6.48 (app t, J = 7.2 Hz, H-10), 7.23–7.33
(m, 3H, Ph), 7.49–7.55 (m, 2H, Ph), 7.61 (d, J = 8.4 Hz, CH@CH),
8.51 (s, 3-NH). 31P NMR (CDCl3): d 20.12. 13C NMR (75 MHz, CDCl3):
d 16.65, 16.72 (OCH2CH3), 36.27 (C-20), 43.79 (C-30), 60.47 (C-50),
62.82, 62.91 (OCH2CH3), 86.53 (C-10), 103.84 (C-40), 110.04 (C-5),
127.36, 128.89, 129.73, 135.12 (Ph), 140.41 (C-6), 150.51 (C-2),
rt and stirred for 16 h. The solvent was evaporated and subse-
quently portioned between EtOAc/Et2O (1:1) and H2O. The organic
phase was washed with H2O and the aqueous layers were com-
bined and lyophilized. The residue was purified using flash chro-
matography (iPrOH/NH4OH/H2O 6:3:1) yielding compound 22
(9.0 mg, 50%) as a white powder. 1H NMR (300 MHz, CD3OD): d
3.47–3.55 (m, H-50a), 3.80–3.87 (m, H-50b), 4.09 (app d, J = 4.2 Hz,
H-30), 4.40–4.44 (m, H-20), 4.96 (H-40), 5.84 (d, J = 8.1 Hz, CH@CH),
6.25 (d, J = 6.6 Hz, H-10), 7.91 (d, J = 7.8 Hz, CH@CH). 31P NMR
13
(CD3OD): d 14.09. C NMR (75 MHz, CD3OD): d 64.88 (C-50),
66.99 (C-20), 73.62 (C-30), 89.86 (C-10), 104.15 (C-5), 110.31,
110.49 (C-40), 143.28 (C-6), 152.96 (C-2), 166.08 (C-4). HRMS
(ESI) for C9H12N2O9P [MꢀH]ꢀ found, 323.0296; calcd, 323.0286.
4.3. Assay of PLC activity
162.78 (C-4). HRMS (ESI) for
505.0630; calcd, 505.0637.
C
19H26N2O7PSe [M+H]+ found,
Stable cell lines for study of the human P2Y2, P2Y4, and P2Y6
receptors were generated by retroviral expression of the receptor
in 1321N1 human astrocytoma cells, which do not natively express
P2Y receptors.24 Agonist-induced [3H]inositol phosphate produc-
tion was measured in cells plated at 20,000 cells/well on 96-well
plates two days prior to assay. Sixteen hours before the assay,
the inositol lipid pool of the cells was radiolabeled by incubation
4.2.27. 20,30-Dihydro-50-[(diethoxyphosphinyl)methoxy]-b-
D-
uridine (20)
To a solution of compound 19 (41 mg, 0.082 mmol) in MeOH
(0.7 mL) was added dropwise a suspended solution of sodium
bicarbonate (12 mg, 0.14 mmol) and sodium periodate (22 mg,
0.10 mmol) in H2O (0.7 mL). After being stirred at rt for 1 h, the
mixture was heated at 80 °C for 75 min. Volatiles were removed
in vacuo and the residue was suspended in CH2Cl2, filtered through
celite and dried over MgSO4. After evaporation under reduced pres-
sure, the residue was purified on a silica gel column (CH2Cl2/MeOH
96:4) to give compound 20 (19 mg, 68%) as a yellow oil. 1H NMR
(300 MHz, CDCl3): d 1.31–1.36 (m, 6H, OCH2CH3), 3.87–3.94 (m,
H-50a), 4.03–4.22 (m, 5H, H-50b, OCH2CH3), 5.71 (d, J = 7.8 Hz,
CH@CH), 5.80 (app s, H-40), 6.10 (app d, J = 6.0 Hz, H-20), 6.28–
6.31 (m, H-30), 6.97 (app s, H-10), 7.39 (d, J = 8.1 Hz, CH@CH),
9.21 (s, 3-NH). 31P NMR (CDCl3): d 20.21. 13C NMR (75 MHz, CDCl3):
d 16.18, 16.25 (OCH2CH3), 61.17, 62.36, 63.41 (OCH2CH3, C-50),
87.77 (C-10), 102.92 (C-5), 108.30, 108.46 (C-40), 130.11 (C-20),
131.73 (C-30), 139.77 (C-6), 150.21 (C-2), 162.78 (C-4). HRMS
(ESI) for C13H20N2O7P [M+H]+ found, 347.0999; calcd, 347.1003.
in 100
medium, containing 1.0
medium were made subsequent to the addition of [3H]inositol.
On the day of the assay, cells were challenged with 25 L of a
l
L of serum-free inositol-free Dulbecco’s modified Eagle’s
l
Ci of [3H]myo-inositol. No changes of
l
five-fold concentrated solution of receptor agonists in 100 mM
Hepes (N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid),
pH 7.3 in HBSS, containing 50 mM LiCl for 30 min at 37 °C. Incuba-
tions were terminated by aspiration of the drug-containing med-
ium and addition of 90
lL of ice-cold 50 mM formic acid. After
30 min, supernatants were neutralized with 30
lL of 150 mM
NH4OH and applied to Dowex AG1-X8 anion exchange columns.
Total inositol phosphates were eluted and radioactivity was mea-
sured using a liquid scintillation counter.27
4.4. Data analysis
Agonist potencies (EC50 values) were determined from concen-
tration-response curves by non-linear regression analysis using the
GraphPad software package Prism (GraphPad, San Diego, CA). Each
concentration of drug was tested in triplicate assays, and concen-
tration effect curves for each test drug were repeated in at least
three separate experiments with freshly diluted molecule. The re-
sults are presented as mean SEM from multiple experiments or in
the case of concentration effect curves from a single experiment
carried out with triplicate assays that were representative of re-
sults from multiple experiments.
4.2.28. 50-[(Diethoxyphosphinyl)methoxy]-b-
D-uridine (21)
Compound 20 (37 mg, 0.11 mmol) was dissolved in a 5:1 ace-
tone–H2O mixture (4 mL) and treated with osmium tetraoxide
(0.19 mL [4 wt % solution], 0.032 mmol) and N-methylmorpho-
line-N-oxide (32 mg, 0.26 mmol). After stirring for 48 h, the mix-
ture was quenched with a saturated solution of Na2S2O3 (4 mL)
and stirred for 30 min. The black mixture was extracted with EtOAc
three times and the combined organic layers were dried over
MgSO4 and evaporated. Chromatography of the residue on silica
gel (CH2Cl2/MeOH 90:10) yielded diol 21 as a colorless solid
(24 mg, 60%). 1H NMR (300 MHz, DMSO-d6):
d
1.23 (t, 6H,
Acknowledgments
J = 6.9 Hz, OCH2CH3), 3.88–3.91 (m, 3H, H-30, H-50a, H-50b), 4.02–
4.10 (m, 4H, OCH2CH3), 4.93 (app s, H-40), 5.52 (d, J = 6.0 Hz, 20-
OH), 5.60 (d, J = 4.2 Hz, 30-OH), 5.65 (d, J = 8.1 Hz, CH@CH), 6.06
We highly appreciate the technical assistance of Izet Karalic.
This research was supported in part by the Intramural Research

S. Van Poecke et al. / Bioorg. Med. Chem. 20 (2012) 2304–2315
2315
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