Synthesis of uracil nucleotide analogs with a modified, acyclic ribose moiety as P2Y2 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2009.05.062

A series of new uracil nucleotide analogs (monophosphates, triphosphates, and phosphonates) was synthesized, in which the ribose moiety was replaced by acyclic chains, including branched or linear alkyl or dialkylether linkers. 1-ω-Bromoalkyluracil deriva

Full text of DOI:10.1016/j.bmc.2009.05.062

Bioorganic & Medicinal Chemistry 17 (2009) 5071–5079
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of uracil nucleotide analogs with a modified, acyclic ribose moiety
as P2Y2 receptor antagonists
*
Roland Sauer, Ali El-Tayeb , Marko Kaulich, Christa E. Müller
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new uracil nucleotide analogs (monophosphates, triphosphates, and phosphonates) was syn-
thesized, in which the ribose moiety was replaced by acyclic chains, including branched or linear alkyl or
dialkylether linkers. 1-x-Bromoalkyluracil derivatives (2) were converted to the corresponding alcohols
by treatment with sodium hydroxide and subsequently phosphorylated using phosphorus oxychloride
followed by hydrolysis to yield the monophosphates, or by coupling with diphosphate to form the tri-
phosphates. Reaction of 2 with triethyl phosphite followed by deprotection with trimethylsilyl bromide
Received 29 March 2009
Revised 16 May 2009
Accepted 23 May 2009
Available online 30 May 2009
Keywords:
P2Y2 receptors
led to the
x-phosphonylalkyluracil derivatives. These products could be further phosphorylated by con-
verting them into their imidazolides and subsequent treatment with diphosphate yielding the corre-
sponding UTP analogs. Nucleoside analogs with an oxygen atom in the 20-position, which are more
similar to the natural ribosides, were synthesized from silylated uracil and trimethylsilyl iodide-treated
1,3-dioxolane, or 1,3-dioxane, respectively, and subsequently phosphorylated by standard procedures.
The nucleotide analogs were investigated in a functional assay at NG108-15 cells, a neuroblastoma ꢀ gli-
oma hybrid cell line which expresses the UTP- and ATP-activated nucleotide receptor subtype P2Y2. The
acyclic nucleotide analogs were generally weaker ligands than UTP, and—in contrast to UTP—they were
antagonistic. The most potent compound was diphosphoric 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
UTP, UDP, UMP analogs
Nucleotide analogs
Acyclic ribose modification
Phosphonates
yl)pentylphosphonic anhydride (5c) with an IC50 value of 92
-phosphate by phosphonate, which leads to enhanced stability, was well tolerated.
Ó 2009 Elsevier Ltd. All rights reserved.
lM showing that the replacement of the
a
1. Introduction
further P2Y12 antagonists are currently evaluated.7 Another P2Y
receptor subtype which is of considerable interest as a new drug tar-
get, is the P2Y2 receptor subtype.3,8–11 P2Y2 agonists are being devel-
oped for the treatment of cystic fibrosis (INS37217, denufosol, III)
and dry eye syndrome (INS 365, diquafosol, IV), see Figure 1.12–14
However, III and IV are moderately potent and weakly selective
P2Y2 agonists. P2Y2 receptor antagonists possess therapeutic poten-
tial for the treatment of inflammatory conditions, pain, coronary
vasospastic disorders, and neurodegeneration.2,3 However, potent
and selective P2Y2 antagonists are currently not available.3
In previous studies, we modified the uracil base and the phos-
phate residue of UDP and UTP.15,16 The resulting compounds were
agonists at P2Y2 receptors. In the present study we modified the ri-
bose part of UTP with the goal to develop novel P2Y2 receptor li-
gands. We selected UTP over ATP, as a lead structure due to the
expected higher selectivity of pyrimidine nucleotides versus the
other P2 receptor subtypes that are activated by the adenine nucle-
otides ATP or ADP. Furthermore, the degradation products of ATP
analogs (the nucleoside adenosine and the nucleobase adenine)
may interact with adenosine (P1) or adenine (P0) receptors and
thus lead to unwanted side-effects.3 Our goal was to investigate
the consequences of replacing the ribose moiety in uracil nucleo-
tides by various acyclic ribose-mimetic structures.
The pyrimidine nucleotides UTP and UDP have been recognized
as important signalling molecules activating G protein-coupled
membrane receptors (GPCRs) of the P2 (nucleotide) receptor fam-
ily.1–3 P2Y receptors are currently subdivided into eight members,
3
P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14
.
While the
human P2Y1, P2Y11, P2Y12, and P2Y13 receptors are activated exclu-
sively by adenine nucleotides, the human P2Y subtypes P2Y4, 6, 14 are
only activated by uracil nucleotides. The human P2Y2 receptor re-
sponds to ATP and UTP.2,3 Species differences exist, for example, in
contrast to the human P2Y4 receptor (a UTP receptor), the rat ortho-
logue is potently activated by UTP and ATP.2–4 P2Y receptors have
been found to be implicated in a variety of physiological functions
and pathophysiological conditions. Therefore P2Y receptors are of
interest as drug targets. Irreversible P2Y12 antagonists are already
in use as antithrombotic drugs, for example, clopidogrel (I).5 The
ATP analogue AR-C69931 MX (cangrelor, II) was studied in clinical
trials as a competitive P2Y12 antagonist for antithrombotic therapy;6
* Corresponding author. Tel.: +49 228 73 2301; fax: +49 228 73 2567.
E-mail address: christa.mueller@uni-bonn.de (C.E. Müller).
A.E.-T. is on leave from the University of Al-Azhar, Assiut, Egypt.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.062

5072
R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
OCH3
N
acyclic nucleotides to P2Y2 receptors and investigate the role of the
O
Cl
sugar moiety on the interaction of UTP with P2Y2 receptors. As a
replacement of the ribose moiety, short aliphatic chains with a ter-
minal hydroxyl group or acyclic ribose fragments were considered.
S
To stabilize the compounds, the
phosphonate in some derivatives.
a-phosphate was replaced by
I Clopidogrel
1-Phosphonylalkyluracil derivatives, 4b–d, were prepared as
metabolically stable nucleotide analogs according to the procedure
described in Scheme 1. The synthesis of 1-bromoalkyluracil and
1-bromoalkylthymine derivatives had previously been descri-
bed.28–31 Modification of the reported methods led to compounds
2a–d via silylation of uracil 1 by excess hexamethyldisilazane
under reflux condition. Due to steric effects the N1-position of
the uracil ring is more reactive than the N3-position. As shown
in Scheme 1, the N1-substituted 1-bromoalkyluracil derivatives
2a–d were obtained after reaction times of a few days in accept-
able yields. As described in the literature,32 by-products, such as
dimers, can be formed during the reaction. Therefore chromato-
graphic purification was required to obtain the pure products.
The 1-bromoalkyluracil derivatives 2b–d were refluxed with
triethyl phosphite yielding products 3b–d by Arbuzov reaction.
In order to hydrolyze the ethyl ester functions, compounds 3b–d
were dissolved in acetonitrile and reacted with trimethylsilyl
bromide under mild conditions to afford 4b–d in good yields.
Compounds 4b–d could be converted to their sodium salts via
ion exchange resin Dowex WX8 (Na+ form).
S
HN
N
CH3
N
N
O
P
O
P
O
P
Cl
CF3
N
S
HO
O
O
OH OH OH
O
Cl
OH OH
II ARC69931MX (Cangrelor)
O
N
NH2
H
O
N
N
O
P
O
P
O
P
O
P
O
N
O
O
O
O
O
HO
OH OH OH
O
O
OH OH
O
OH
III INS37217 (Denufosol)
O
The phosphorylation of 4b–d to obtain UTP analogs 5b–d was
achieved according to the method described by Moffat33 for the
preparation of nucleoside triphosphates. The phosphonic acids
4b–d were activated with 1,10-carbonyldiimidazole under argon
in the presence of tributylamine as a base to yield phosphonic acid
imidazolides which were not isolated but immediately reacted
with 5 equiv of tributylammonium diphosphate in DMF to give
the corresponding diphosphoryl phosphonates 5b–d (Scheme 1).
A number of by-products were formed during these reaction
steps. In Scheme 2 the main side products observed during the for-
mation of 5d are shown as an example. When the reaction time
was extended to more than 30 min, dimerization occurred and
the corresponding tetraphosphate derivative was formed. This
dimerization product was analyzed by 31P NMR spectroscopy and
identified by comparison with literature data for similar com-
pounds.34 The same dimerization reaction was also observed dur-
ing the synthesis of 5b and 5c. After anion exchange
chromatography of the reaction mixture, the desired triphosphate
analog 5d (see Scheme 2) was isolated as the main product. In
addition, the diphosphate analog 6d was formed after hydrolysis
of the dimeric tetraphosphate during the purification process. Both
products, 5d and 6d were obtained at a ratio of 4:1 (Scheme 2).
For the synthesis of uracil-1-alkyl mono- and triphosphates 1-
H
H
O
P
HO
N
O
P
O
P
O
P
N
O
O
N
O
O
O
N
O
O
O
OH OH OH
O
OH OH
OH OH
IV INS365 (Diquafosol)
Figure 1. Selection of biologically active P2Y receptor ligands.
Several acyclic analogs of adenine nucleotides have been de-
scribed and evaluated;17,18 some have been reported to be moder-
ately potent P2Y1 receptor antagonists,19,20 while a series of acyclic
adenine nucleotide analogs was found to inhibit platelet aggrega-
tion by blocking P2Y12 receptors.21 Furthermore, acyclic nucleotide
analogs have been investigated as antiviral, cytostatic, antiparasitic
and immunomodulatory agents.22,23 Three compounds bearing a
phosphonate residue, namely cidofovir, adefovir, and tenofovir,
are therapeutically used as antiviral drugs for the treatment of
hepatitis B, AIDS, and various diseases caused by DNA viruses.23
A large number of acyclic nucleoside analogs has been developed,
especially adenosine and guanosine derivatives, such as the antivi-
ral drug acyclovir (9-(2-hydroxyethoxymethyl)guanine).24 Because
of their antiviral effects the interest in such compounds has been
high.25,26 Due to the recent discovery of the important roles of
small interfering ribonucleic acids (siRNAs), a novel area of drug
development focuses on modified small RNAs.27 In this respect,
the newly synthesized acyclic nucleotide analogs may be useful
for the preparation of artificial, stabilized siRNAs and RNA chain
terminators.
(
x
-bromoalkyl)uracils 2a,c–d were reacted with 1 N aqueous so-
dium hydroxide solution to obtain the corresponding 1-(
x
-
hydroxyalkyl)uracils 7a,c–d as previously described (see Scheme
3).35 If a higher concentration of sodium hydroxide or higher tem-
peratures than 50 °C were applied, the alkyl residues were split off
and the formation of uracil was observed as a side reaction.
Furthermore, we introduced oxygen-containing dialkyl ether
linkers as more ribose-like replacements of the sugar moiety. Sily-
lation of uracil was performed by treatment with bis(trimethyl-
silyl)acetamide in acetonitrile followed by reaction with the
alkylating reagent formed in situ by treatment of 1,3-dioxolane,
or 1,3-dioxane, respectively, with trimethylsilyl iodide. 1-(2-
Hydroxyethoxymethyl)uracil (8a) and 1-(3-hydroxypropoxymeth-
yl)uracil (8b) were obtained in 25% and 35% yields, respectively
(Scheme 3).
2. Results and discussion
2.1. Chemistry
A series of uridine analogs in which the ribose moiety was re-
placed by a ‘spacer’ was synthesized and converted to analogs of
uracil nucleotides. Our goal was to investigate the binding of such
The synthesized nucleoside analogs 7a,c–d and 8a,b were
susceptible to phosphorylation yielding the corresponding

R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
5073
O
N
O
H
O
N
H
N
H
O
4
N3
5
6
O
O
P
O
b
a
2
1
N
O
N
O
(
)
Br
n
H
( )
n
1
2a-d
3a-d
a n = 1
b n = 2
c n = 3
d n = 4
c
O
N
O
N
H
O
O
N
H
O
N
N
H
O
N
O
P
O
O
P
O
O
P
O
d
O
P
e
-
O
P
O
O
-
O
-
O
-
( )
OH
OH
( )
n
N
-
n
(
)
5b-d
4a-d
N
n
Scheme 1. Synthesis of 1-phosphonylalkyluracil and 1-(x-phosphonylalkyl)uracil diphosphate derivatives 5b–d. Reagents and conditions: (a) (1) HMDS, Me3SiCl, 12 h,
110 °C; (2) Br(CH2)nBr, I2, 4–7 d, 60 °C, 21–46%; (b) P(OEt)3, 3 h, 150 °C; (c) Me3SiBr, acetonitrile, 12 h, rt, 82–85%; (d) Bu3N, 1,10-carbonyldiimidazole, 12 h, rt; (e) 5 equiv
(Bu3NH+)4P2O7, Bu3N, DMF, 30 min, rt.
O
N
O
N
H
O
H
O
N
N
a
O
P
O
P
O
N
OH
N
OH
-
4d
b
b
O
N
O
N
O
N
O
N
H
O
H
O
H
O
H
O
N
N
N
N
c
c
O
P
O
O
P
O
O
P
O
O
P
O
O
P
O
O
P
O
O
P
O
O
P
O
O
P
O
-
-
O
-
O
-
O
-
O
-
O
-
O
-
O
-
O
-
-
5d
6d
Scheme 2. Observed side reactions during the synthesis of 1-(
carbonyldiimidazole, 12 h, rt; (b) 1.5 equiv (Bu3NH+)4P2O7, Bu3N, DMF, 2 h, rt; (c) hydrolysis, DEAE-A25 Sephadex chromatography.
x
-phosphonylalkyl)uracil diphosphates (example 6d). Reagents and conditions: (a) Bu3N, DMF, 1,10-
triphosphates. The acyclic nucleoside analogs 8a,b were dissolved
in dry trimethylphosphate under an argon atmosphere, and ‘proton
sponge’ (1,8-bis(dimethylamino)naphthaline) was added in order
to prevent the lowering of the pH value of the reaction mixture
as described by Ludwig.36 After stirring the solution at 0–4 °C for
some minutes, phosphorus oxychloride was added. After several
hours of stirring the ‘activated’ monophosphate was formed, which
was either hydrolyzed with triethylammonium hydrogencarbon-
ate (pH 7.5) to yield the monophosphates 9a,b, or further reacted
with the tributylammonium salt of diphosphate (dissolved in
DMF) to afford, after hydrolysis, the triphosphates 10a,b (Scheme
3). The same synthetic procedure was also used for the phosphor-
As an alternative to the phosphorylation using POCl3, the Lud-
wig–Eckstein phosphorylation method37 was applied to phosphor-
ylate compound 7c. This was done in order to investigate whether
higher yields could be achieved with that procedure, which was,
however, not the case. As shown in Scheme 4, the first reaction
step was the formation of phosphate using 2-chloro-4H-1,3,2-ben-
zodioxaphosphorine-4-one as a phosphitylating agent. The result-
ing nucleoside phosphite was immediately coupled with
tributylammonium diphosphate in DMF in the presence of tribu-
tylamine leading to the displacement of the salicylate resulting
in the formation of a cyclic intermediate (Scheme 4). This reaction
was monitored by 31P NMR spectroscopy. The signal for the triva-
ylation of 1-(
x
-hydroxyalkyl)uracil derivatives 7a,c–d (Scheme 3).
lent a-phosphorus atom in the cyclic intermediate showed a typi-
The monophosphates 11a,c–d were obtained due to the hydrolysis
of the phosphorodichloridate intermediate during the reaction and
isolated as side products during the synthesis of the triphosphates
12a,c–d. The yield ratio between the triphosphates 12a,c–d and
the corresponding monophosphates 11a,c–d was about 4:1. After
chromatographic purification, the total yield of all described tri-
phosphates synthesized by the Ludwig method ranged between
30% and 40%.
cal shift of +104 ppm and appeared as a triplet with a coupling
constant of 43 Hz as a result of the coupling with the two phos-
phate groups in the cyclic intermediate (Scheme 4). As it is an achi-
ral molecule, the two cyclic phosphate groups showed a single
signal and appeared as a doublet (J = 43 Hz) with a characteristic
shift of ꢁ20 ppm. The addition of iodine in aqueous solution led
to oxidation of the
a-phosphorus atom and ring opening to the lin-
ear triphosphate 12c. As in the other phosphorylation methods,

5074
R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
O
N
O
O
O
H
H
O
H
O
O
P
Cl
H
O
N
N
N
c
c
a
N
b
Cl
O
O
OH
N
N
( )
N
( )
N
H
1
R
(
)
O
n
(
)
Br
OH
n
n
n
2a,c-d
7a,c-d
8a,b
O
N
H
O
e
d
11a,c-d
12a,c-d
a n = 1
b n = 2
c n = 3
d n = 4
=
=
R =
or
O
O
P
O
O
P
O
P
O
O
P
O
-
H
O
-
O
N
9a,b
O
O
-
O
-
O
-
O
-
R =
O
R
10a,b
R
(
)
(
)
n
n
N
O
9a,b
11a,c-d
10a,b
12a,c-d
Scheme 3. Synthesis of acyclic nucleoside analogs 7a,c–d and 8a,b and acyclic nucleotide analogs 9a,b, 11a,c–d (monophosphates) and 10a,b, 12a,c–d (triphosphates).
Reagents and conditions: (a) 1 N NaOH, 48 h, 50 °C, 45–70%; (b) (1) bis(trimethylsilyl)acetamide, acetonitrile, 3 h, rt; (2) KI, Me3SiI, 1,3-dioxolane or 1,3-dioxane, 24 h, rt; 8a
(25%), 8b (35%); (c) POCl3, OP(OMe)3, 1 h, 0–4 °C; (d) (1) (Bu3NH+)4P2O7, Bu3N, DMF, 1 h, 0–4 °C; (2) (Et3NH)HCO3, 25–30%; (e) (Et3NH)HCO3 ; 9a,b (50–60%), 11a,c–d (10–
15%).
O
N
O
N
O
O
H
O
H
O
N
N
O
P
a
OH
O
7c
b
O
N
O
H
O
O
P
H
O
-
-
N
N
O
P
O
O
P
O
O
P
O
O
O
O
c
-
O
P
O
O
-
O
-
N
O
O
-
O
P
O
12c
Scheme 4. Ludwig–Eckstein phosphorylation procedure for the synthesis of nucleoside triphosphate 12c. Reagents and conditions: (a) 2-chloro-4H-1,3,2-benzodioxaphosph-
orine-4-one, dioxane/DMF, 0–4 °C; (b) (Bu3NH+)4P2O7, Bu3N, DMF; (c) I2, H2O, 25%.
this reaction is characterized by formation of numerous, mostly
unidentified by-products, so that the total yield after chromato-
graphic purification was even lower in comparison with the Lud-
wig method.
toma ꢀ rat glioma hybrid cell line) was determined by a fluores-
cence method using the calcium-chelating fluorophor Oregon
GreenÒ as previously described.8–10 NG108-15 cells express the
mouse P2Y2 receptor, which is coupled to phospholipase C activa-
tion and subsequent intracellular calcium release.38 They also ex-
press UDP-activated P2Y6 receptors, but not UTP-sensitive P2Y4
receptors as shown by reverse transcriptase polymerase chain
reaction.39 Therefore the addition of UTP will only activate the
P2Y2 receptor subtype in NG108-15 cells.
P2Y2 agonists as well as antagonists have been shown to inhibit
a subsequent receptor stimulation, agonists by inducing receptor
desensitization, and antagonists by a blockade of the receptors.40
The mouse P2Y2 receptor is very similar to the human subtype
due to high sequence homology (89% identity in amino acid se-
quence).41 Therefore data from mouse P2Y2 receptors are highly
predictive of activity at human P2Y2 receptors.9 As an agonist,
UTP induced a concentration-dependent increase in [Ca2+]i with
Since the phosphorylation reactions are usually complicated by
the formation of side products, chromatographic purification of all
synthesized mono- and triphosphates (and phosphonates) was
performed in two steps: anion exchange chromatography was ap-
plied in order to separate the formed mono- from the triphos-
phates; in a second step, preparative HPLC was applied, in order
to remove inorganic impurities. The purity of the final products
was controlled by 31P NMR spectroscopy, capillary electrophoresis,
and HPLC–ESI-MS analysis (see Supplementary data) and found to
be >95% in all cases.
3. Biological investigations
3.1. P2Y2-antagonistic effect
an EC50 value of 1.26 0.45 lM (n = 10). This relatively high EC50
value is probably due to the moderate receptor expression level
in the NG108-15 cells. In contrast, UTP usually shows a much low-
er EC50 value in artificial cell lines with high receptor expression
The potency of the test compounds to inhibit UTP-induced
intracellular calcium release in NG108-15 cells (mouse neuroblas-

R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
5075
levels.8,16,18 In antagonist assays, a UTP concentration of 1
responding to the EC50 value was used for stimulation. Initial
screening of the nucleotide analogs for inhibition of UTP- (1 M)
induced calcium mobilization was carried out at concentrations
of 10, 100, and 500 M. For selected compounds full concentra-
l
M cor-
anhydride, with an IC50 value of 92
less potent than UTP (preincubation) in preventing UTP-induced
P2Y2 activation.
l
M. The compound was 61-fold
l
l
3.2. P2Y2-agonistic effect
tion–response curves were determined and IC50 values were calcu-
lated. The results are depicted in Table 1. Typical concentration–
inhibition curves are shown in Figure 2.
The next step was to investigate whether the new compounds
were agonists or antagonists at P2Y2 receptors. All compounds
UTP preincubation led to a dose-dependent inhibition of UTP-
stimulated intracellular calcium release due to receptor desensiti-
were investigated in concentrations of up to 100 lM. Cells were
treated with UTP (agonist) or test compound and changes in intra-
cellular calcium concentrations were recorded. While UTP showed
a concentration-dependent increase in [Ca2+]i with an EC50 value of
1.26 0.45 lM, the acyclic nucleotide analogs—with the exception
of 9b—did not show any effect indicating that they were antago-
zation. UTP showed an IC50 value of 1.51 0.60
inhibitor of UTP- (1 M) induced calcium mobilization (Table 1),
a value that was very close to the EC50 value determined for its
P2Y2-agonistic effect (1.26 M).
lM (n = 4) as an
l
l
All acyclic nucleotide analogs were less potent than UTP. Struc-
ture II (Table 1) is most similar to the natural nucleotides. In this
nists rather than agonists at P2Y2 receptors (data not shown). Com-
pound 9b showed an increase in fluorescence at 100 lM (ca. 40% of
set of compounds a chain length of n = 2 (chain of 6 atoms to P )
the maximal effect observed with UTP, data not shown), however
the increase in fluorescence was untypically slow and may have
been caused by a P2Y2 receptor-independent effect.
a
was superior to n = 1 (5 atom chain), and monophosphates were
similarly potent as triphosphates (compare 9b/10b). Activity of
nucleoside monophosphates had previously also been found for 2-
substituted adenosine 50-monophosphate derivatives at the P2Y1
and the P2Y12 receptor subtypes.11,42 The best compound in this ser-
ies was 10b, which was 175-fold less potent than UTP
4. Conclusions
In conclusion, we have synthesized a series of uracil nucleotide
analogs, in which the ribose moiety was replaced by acyclic struc-
(IC50 = 264 lM) in inhibiting or preventing P2Y2 stimulation by
UTP. In series III, where the oxygen atom corresponding to position
20 of ribose was lacking, a similar trend could be observed. The opti-
mal chain length of six carbon atoms (n = 4) was best tolerated by
the receptor in combination with a monophosphate substitution
(11d). This compound had a similar potency as the corresponding
oxygen-containing analog 9b. The best compounds were found
among the phosphonates (structure I). In this series a shorter chain
length was favorable (4 carbon atoms, n = 3, 5c), while 5 atoms (5d)
were also well tolerated. Three phosph(on)ate groups (UTP analogs)
were superior to only two (UDP analog; compare 5d/6d). The most
potent compound of the present study was 5c, diphosphoric
5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)pentylphosphonic
tures. In some of the compounds, the a-phosphate was replaced by
a phosphonate to enhance chemical and metabolic stability. Most
of the compounds were new derivatives not previously described
in the literature. Evaluation at P2Y2 receptors showed that—in con-
trast to the parent UTP—the acyclic nucleotide analogs were antag-
onists. The
a-phosphonate modification was well tolerated.
Despite their moderate potency they might serve as a starting
point for further optimization. Furthermore, some of the new com-
pounds may be of interest as building blocks for the preparation of
modified RNAs.
5. Experimental
5.1. General information
Table 1
Inhibitory potency of nucleotide analogs at P2Y2 receptors expressed in NG108-15
cells (UTP = 1 lM)
The 1H and 13C NMR spectra were measured on a Bruker AC-
200, or an AMX-300 spectrometer. Coupled 1H–31P NMR spectra
were measured on a Bruker Avance 500 spectrometer. Tetrameth-
ylsilane was used as an internal standard for the spectra measured
in CDCl3. 31P NMR spectra were determined on a Bruker AMX300
or an AMX-400 spectrometer. Phosphoric acid (85%) was used as
an external standard. The chemical shifts are given in ppm relative
to the external standard (31P NMR) or relative to the remaining
protons of the deuterated solvents used as internal standard in
case of 1H and 13C NMR spectra (for spectra in DMSO-d6: d 1H:
2.51, d 13C: 39.7, in D2O: 1H 4.65). Melting points were measured
on a Büchi 510 melting point apparatus and are uncorrected.
CHN elemental analyses were determined at the Institute of Inor-
ganic Chemistry, University of Würzburg, Germany, using a Carlo
Erba Elemental Analyzer. The reactions were monitored and the
purity of the compounds was checked by thin layer chromatogra-
phy (TLC) using aluminum sheets with Silica Gel 60 F254 (Merck).
As solvent systems dichloromethane/methanol (9:1), or for all
phosphates and phosphonates, isopropanol/NH3/H2O (6:3:1) were
used.
O
O
N
O
N
H
N
H
O
H
O
N
N
O
P
O
P
O
N
O
P
n O
O
P
-
O
O
O
-
( )
O
O
O
-
O
O
-
O
(
)
n
III
-
(
)
O
n
m
-
-
I
II
m
m
11a, c-d
12a, c-d
5b-d
9a, b
6d
10a, b
a
Compd
Structure
n
m
IC50 (lM) (% inhibition SEM)
UTP
5b
5c
5d
6d
—
I
I
I
I
II
II
II
II
III
III
III
III
III
III
—
2
3
4
4
1
2
1
2
1
3
4
1
3
4
—
2
2
2
1
1
1
3
3
1
1
1
3
3
3
1.51 0.60
ꢂ500 (52 1%)
92
1
166
6
ꢂ500 (55 1%)
9a
9b
>500 (16 1%)
338
>500 (16 3%)
264
2
10a
10b
11a
11c
11d
12a
12c
12d
4
>500 (22 7%)
6500 (60 3%)
267b
A GradiFrac system (Pharmacia-Biotech) with a high-load pump
P50 and Sephadex DEAE A25 (Pharmacia) was used for anion ex-
change chromatography. Preparative HPLC was performed using
a Knauer instrument (HPLC pump 64) on a Eurospher column
6500 (60 7%)
596 0c
ꢂ500 (50 1%)
a
n = 2–5, each in quadruplicate.
n = 1.
UTP = 3 lM.
b
c
(100, C18: 7
lm, 20 mm) applying the software package Euro-
chrom 2000. The purity control of the synthesized nucleotides

5076
R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
3000
2500
2000
1500
1000
500
3000
2500
2000
1500
1000
500
0
0
10-6
10-5
10-4
10-3
10-6
10-5
10-4
10-3
[5c], M
[5d], M
Figure 2. Typical concentration–inhibition curves for compounds 5c and 5d. Data points represent mean SEM (n = 4).
was performed by capillary electrophoresis using a Beckmann P/
ACE system with a 5500 P/ACE diode array detector. The purity
of the nucleotides was examined in a second chromatography sys-
tem by dissolving 1 mg/ml of compound in H2O/MeOH = 1:1, con-
30 min of stirring under ice-cooling 15 ml of 1 M triethylammo-
nium hydrogen carbonate (TEAB, pH 7.5) was added to the reaction
mixture. After 1 h the solvent was removed under high vacuum at
40 °C and the residue was further purified by ion exchange chro-
matography and subsequently by preparative reversed phase
HPLC.
taining 2 mM ammonium acetate. A sample of 10
into an HPLC instrument (Agilent 1100) using a Phenomenex Luna
C18 column. Elution was performed with a gradient of water:
methanol (containing 2 mM ammonium acetate) from 90:10 to
0:100 for 30 min at a flow rate of 250 l/min, starting the gradient
ll was injected
3l
5.5. General procedure for the preparation of 1-(2-
hydroxyethoxymethyl) uracil (8a) and 1-(3-
hydroxypropoxymethyl)uracil (8b)
l
after 10 min. UV absorption was detected from 190 to 400 nm
using a diode array detector. The demineralized water used for
chromatography was obtained from a Purelab-plus instrument
Uracil (1.12 g, 10 mmol) was suspended in 15 ml of absolute
acetonitrile, bis(trimethylsilyl)acetamide (5.4 ml, 22 mmol) was
added, and the reaction mixture was stirred at rt for 3 h while pro-
tecting it from light until a clear solution was obtained. Then KI
(1.7 g, 10 mmol) and trimethylsilyl chloride (1.38 ml, 10 mmol)
were added, and after 15 min of stirring, 10 mmol of 1,3-dioxolane,
or 1,3-dioxane, respectively, were added. The reaction mixture was
stirred for 24 h at rt and then quenched by the addition of 20 ml of
methanol and subsequent neutralization with 4 g of sodium bicar-
bonate. After filtration of the solid residue, the filtrate was evapo-
rated under reduced pressure and the obtained residue was
dissolved in 3 ml of methanol, applied to a silica gel-column and
eluted with chloroform/methanol (95:5). The products were then
recrystallized from isopropanol to yield pure products.
(<0.055 lS/cm).
The synthesis of compounds 2a–d,28–30 7a,c–d35 and 8a43 has
previously been described. Compounds 9a,44 10a,45,46 11a,d,47
11c48 and 12c47 have also been reported in the literature but with-
out physicochemical or spectral data.
5.2. General procedure for the preparation of phosphonic acid
diethyl ester derivatives 3b–d
Compound 2b, c, or d (200 mg) was dissolved in 10 ml triethyl
phosphite and the reaction mixture was refluxed for 3 h. After the
reaction was completed, the solvent was distilled off and the resi-
due was dried under vacuum. Coevaporation of the sticky residue
with absolute ethanol for several times afforded compounds 3b–
d as waxy substances.
5.6. General procedure for the preparation of 1-(2-
hydroxyethoxymethyl)uracil monophosphate (9a) and 1-(3-
hydroxypropoxymethyl)uracil monophosphate (9b)
5.3. General procedure for the preparation of phosphonic acid
derivatives 4b–d
Compound 8a or b (0.5 mmol) was dissolved in 2.5 ml of tri-
methyl phosphate (dried over molecular sieve). The reaction was
cooled to 0–4 °C and to it 0.15 g (0.75 mmol) of 1,8-bis(dimethyl-
amino)naphthaline were added while keeping the reaction mixture
under argon. After 15 min of stirring, 0.075 ml (0.8 mmol) POCl3
was added and stirring was continued for 1 h at 0–4 °C. Hydrolysis
was achieved by addition of 15 ml of 1 M TEAB (pH 7.5) to the reac-
tion mixture. Trimethyl phosphate was removed by extraction
with t-butylmethyl ether (three times) and the aqueous phases
were combined and dried under high vacuum at 40 °C until dry-
ness. The residue was dissolved in 2 ml of 1 M TEAB and applied
to a DEAE-A25 Sephadex column for purification.
Compound 3b, c, or d (150 mg) was dissolved in 10 ml of abso-
lute acetonitrile at rt, a 5–6-fold excess of trimethylsilyl bromide
was added and the reaction mixture was stirred overnight. To
the resulting solution 10 ml of methanol/water (5:1) was added
dropwise followed by stirring for a few minutes. Then the solvent
was removed under reduced pressure, and the resulting yellow oily
product was dissolved in 5 ml of water and subsequently lyophi-
lized. The product was recrystallized from isopropanol to yield
pure compounds as solid colorless materials.
5.4. General procedure for preparation of the diphosphoryl
phosphonate derivatives 5b–d
5.7. General procedure for the preparation of 1-(2-
hydroxyethoxymethyl)uracil triphosphate (10a) and 1-(3-
hydroxypropoxymethyl)uracil triphosphate (10b)
Compound 4b, c, or d (0.4 mmol) was dissolved in 15 ml DMF
followed by the addition of 0.15 ml tributylamine. Carbonyldiimid-
azole (300 mg, 2 mmol) was added and the reaction mixture was
stirred at rt for 12 h while protecting it from light. Tributylammo-
nium diphosphate solution in DMF (2 ml of 0.5 M, 3 equiv) and
0.5 ml tributylamine were added to the reaction mixture. After
Acyclic nucleoside analog 8a or 8b (0.5 mmol) was dissolved in
2.5 ml trimethyl phosphate (dried over molecular sieve) under ar-
gon. The reaction mixture was cooled down to 0–4 °C and 0.15 g

R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
5077
(0.75 mmol) 1,8-bis(dimethylamino)naphthaline was added upon
stirring. After 15 min, POCl3 (0.075 ml, 0.8 mmol) was added to
the reaction mixture. After stirring for 1 h, a mixture of 0.3 ml
(2 mmol) tributylamine and 2.5 ml (1.25 mmol) 0.5 M tributylam-
monium diphosphate in DMF was added and the stirring was con-
tinued for another 30 min at 0–4 °C. Hydrolysis was achieved by
addition of 15 ml of 0.5 M TEAB (pH 7.5). Trimethyl phosphate
and proton sponge were removed from the reaction mixture by
extraction with t-butylmethyl ether. The aqueous phase was dried
under high vacuum at 40 °C and the crude nucleotides were puri-
fied using ion exchange chromatography followed by preparative
HPLC.
and injected into an RP-HPLC column (Knauer 20 mm ID, Euro-
spher-100, C18, 7 , 25 cm). The column was eluted with a solvent
l
gradient of 0–5% of acetonitrile in 50 mM aqueous ammonium for-
mate solution for 20–30 min at a flow rate of 15 ml/min (10 MPa).
The UV absorption was detected at 254 nm. Fractions were col-
lected and appropriate fractions pooled, diluted with water and
lyophilized several times to remove the remaining buffer yielding
the final pure nucleotides as white powders.
5.10.2.1. [3-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)propyl]-
phosphonic acid diethyl ester (3b). 1H NMR (300 MHz, DMSO-
d6) d 1.23 (t, 3H, J = 7.0 Hz), 1.66–1.81 (m, 4H), 3.71 (t, 2H,
J = 6.21 Hz), 3.91–4.03 (m, 2H), 5.56 (dd, 1H, J5,6 = 7. 9 Hz,
J5,3 = 2.2 Hz), 7.64 (d, 1H, J = 7.9 Hz), 11.23 (s, 1H). 31P NMR
5.8. Ludwig method36 for the preparation of 1-(
x-
hydroxyalkyl)uracil monophosphates (11a,c–d) and 1-(-
(120 MHz, DMSO-d6) d 30.97 (s, P ). 13C NMR (125 MHz, DMSO-
a
hydroxyalkyl)uracil triphosphates (12a,c–d)
d6)
d 16.44 (d, J = 6.1 Hz), 21.87 (d, J = 140.4 Hz), 22.01 (d,
J = 4.57 Hz), 48.05 (d, J = 19.9 Hz), 61.23 (d, J = 6.1 Hz), 101.19,
1-(
x
-Hydroxyalkyl)uracil derivative 7a, c, or d (0.5 mmol) was
145.85, 151.16, 163.96.
dissolved, at 0–4 °C, in 2.5 ml trimethylphosphate (dried over
molecular sieve) under argon and 0.15 g (0.75 mmol) of 1,8-
bis(dimethylamino)naphthaline was added. After 15 min,
0.075 ml (0.8 mmol) POCl3 was added. After 1 h of stirring, a mix-
ture of 0.3 ml (2 mmol) tributylamine and 2.5 ml (1.25 mmol) of a
0.5 M solution of tributylammonium diphosphate in DMF was
added and the stirring was continued for another 30 min at 0–
4 °C. Hydrolysis was achieved by addition of 15 ml of 0.5 M TEAB
solution (pH 7.5). Trimethyl phosphate and proton sponge were re-
moved from the reaction mixture by extraction with t-butylmethyl
ether. The aqueous phase was dried under high vacuum at 40 °C
and the crude nucleotides were subsequently purified by ion ex-
change chromatography to separate mono- and triphosphates fol-
lowed by further purification with preparative HPLC.
5.10.2.2. [4-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)butyl]-
phosphonic acid diethyl ester (3c). 1H NMR (300 MHz, DMSO-
d6) d 1.22 (t, 3H, J = 7.0 Hz), 1.41–1.84 (m, 6H), 3.66 (t, 2H,
J = 6.58 Hz), 3.89–4.01 (m, 2H), 5.54 (dd, 1H, J5,6 = 7. 8 Hz,
J5,3 = 2.3 Hz), 7.65 (d, 1H, J = 7.9 Hz), 11.23 (s, 1H). 31P NMR
(120 MHz, DMSO-d6) d 31.67 (s, P ). 13C NMR (125 MHz, DMSO-
a
d6)
d 16.44 (d, J = 6.1 Hz), 19.20 (d, J = 6.1 Hz), 24.19 (d,
J = 138.9 Hz), 29.25 (d, J = 16.8 Hz), 46.98, 61.02 (d, J = 6.1 Hz),
101.01, 145.79, 151.13, 163.87.
5.10.2.3. [5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)pentyl]-
phosphonic acid diethyl ester (3d). 1H NMR (300 MHz, DMSO-
d6) d 1.22 (t, 3H, J = 7.0 Hz), 1.29–1.79 (m, 8H), 3.64 (t, 2H,
J = 7.1 Hz), 3.89–4.04 (m, 2H), 5.54 (dd, 1H, J5,6 = 7. 9 Hz,
J5,3 = 2.2 Hz), 7.65 (d, 1H, J = 7.9 Hz), 11.21 (s, 1H). 31P NMR
5.9. Alternative procedure for the synthesis of 1-(4-
hydroxybutyl)uracil triphosphate (12c) according to the
Ludwig–Eckstein method37
(120 MHz, DMSO-d6) d 31.89 (s, P ). 13C NMR (125 MHz, DMSO-
a
d6)
d 16.47 (d, J = 6.1 Hz), 21.95 (d, J = 4.6 Hz), 24.49 (d,
J = 138.9 Hz), 26.68 (d, J = 16.8 Hz), 28.12, 60.49 (d, J = 6.1 Hz),
1-Hydroxybutyluracil 7c (30 mg, 0.16 mmol) was dissolved in
3 ml of dry DMF at rt under argon. Then, 0.175 ml (0.17 mmol) of
a freshly prepared 1 M solution of 2-chloro-4H-1,2,3-dioxaphosph-
orine-4-one in dry dioxane was added. After 15 min of stirring,
0.5 ml of a 0.5 M tri-n-butylammonium diphosphate solution in
DMF was added and stirring was continued for an additional
30 min followed by the addition of an aqueous solution of I2. After
1 h, the reaction was hydrolyzed by addition of 15 ml of 0.5 M
TEAB (pH 7.5) and the product was isolated as described above.
100.95, 145.82, 151.10, 163.90.
5.10.2.4. [3-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)propyl]-
phosphonic acid (4b). 1H NMR (300 MHz, DMSO-d6) d 1.41–1.58
(m, 2H), 1.65–1.87 (m, 2H), 3.70 (t, 2H, J = 6.67 Hz), 5.56 (d, 1H,
J = 7. 7 Hz), 6.20 (s, 2H), 7.66 (d, 1H, J = 7.9 Hz), 11.24 (s, 1H). 31P
NMR (120 MHz, DMSO-d6) d 25.66 (s, P ). 13C NMR (125 MHz,
a
DMSO-d6) d 22.70 (d, J = 4.6 Hz), 24.65 (d, J = 138.9 Hz), 48.22 (d,
J = 18.3 Hz), 101.01, 145.92, 151.10, 163.93. Calcd for C7H11N2O5P
(234.159 g/mol): C, 35.91; H, 4.74; N 11.96. Found: C, 35.52; H,
4.83; N 11.74.
5.10. Purification of nucleotides
5.10.1. Ion exchange chromatography
5.10.2.5. [4-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)butyl]-
phosphonic acid (4c). 1H NMR (300 MHz, DMSO-d6) d 1.41–1.68
(m, 6H), 3.66 (t, 2H, J = 6.9 Hz), 5.55 (d, 1H, J = 7. 7 Hz), 7.66 (s,
2H), 7.65 (d, 1H, J = 7.7 Hz), 11.23 (s, 1H). 31P NMR (120 MHz,
The crude nucleotides were purified by ion exchange chroma-
tography on a GradiFrac system (Pharmacia-Biotech) with the
high-load pump P50 with an XK 26 mm/20 cm length column
(Pharmacia) using Sephadex DEAE A-25 gel HCO3ꢁ-form swelled
in a 1 M solution of TEAB at 4 °C. After equilibration of the column
with deionized water, the crude product was dissolved in 2 ml of
aqueous triethylammonium hydrogencarbonate solution. The col-
umn was washed with deionized water, followed by a solvent gra-
dient of 0–500 mM TEAB using approximately 1000 ml of solvent
to elute the triphosphates at a flow rate of 1 ml/min. Fractions
were collected and appropriate fractions pooled, diluted in water,
and lyophilized.
DMSO-d6) d 26.51 (s, P ). 13C NMR (125 MHz, DMSO-d6) d 19.88
a
(d, J = 4.6 Hz), 27.27 (d, J = 137.3 Hz), 29.56 (d, J = 16.8 Hz),
47.19, 100.98, 145.79, 151.10, 163.89. Calcd for C8H13N2O5P
(248.18 g/mol): C, 38.68; H, 5.24; N, 11.28. Found: C, 38.51; H,
5.35; N, 10.91.
5.10.2.6. [5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)pentyl]-
phosphonic acid (4d). 1H NMR (300 MHz, DMSO-d6) d 1.24–1.64
(m, 8H), 3.63 (t, 2H, J = 7.1 Hz), 5.504 (d, 1H, J = 7. 1 Hz), 7.05 (s,
2H), 7.64 (d, 1H, J = 7.9 Hz), 11.21 (s, 1H). 31P NMR (120 MHz,
5.10.2. Preparative HPLC
DMSO-d6) d 27.08 (s, P ). 13C NMR (125 MHz, DMSO-d6) d 22.58
a
Lyophilized nucleotides obtained after ion exchange chroma-
tography purification were dissolved in 5 ml of deionized water
(d, J = 4.6 Hz), 26.70 (d, J = 15.3 Hz), 27.51 (d, J = 137.3 Hz), 28.30,
47.46, 101.80, 145.82, 151.10, 163.93. Calcd for C9H15N2O5P

5078
R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
(262.20 g/mol): C, 41.23; H, 5.77; N, 10.68. Found: C, 40.87; H,
5.74; N 10.34.
(m, 6H), 3.54–3.65 (m, 4H), 5.60 (d, 1H, J = 7. 9 Hz), 7.43 (d, 1H,
J = 7.9 Hz). 31P NMR (120 MHz, D2O) d 0.1 (s, P ).
a
5.10.2.7. Diphosphoric 3-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)propylphosphonic anhydride (5b). 1H NMR (300 MHz, D2O) d
1.72–1.91 (m, 4H), 3.79 (t, 2H, J = 7.0 Hz), 5.78 (d, 1H, J = 7. 7 Hz),
7.66 (d, 1H, J = 7.7 Hz). 31P NMR (120 MHz, D2O) d ꢁ22.8 (t, Pb,
J = 21.6 Hz), ꢁ7.7 (d, P , J = 20.4 Hz), 17.8 (d, P , J = 24.1 Hz).
5.10.2.18. 2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethyl tri-
phosphate (12a). 1H NMR (300 MHz, D2O) d 3.95 (t, 2H, J = 5.0 Hz),
4.08 (m, 2H), 5.71 (d, 1H, J = 7. 9 Hz), 7.61 (d, 1H, J = 7.9 Hz). 31P
NMR (120 MHz, D2O) d ꢁ20.7 (t, Pb, J = 18.5 Hz), ꢁ10.1 (d, P ,
c
J = 19.7 Hz), ꢁ6.5 (d, P , J = 17.2 Hz).
c
a
a
5.10.2.8. Diphosphoric 4-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)butylphosphonic anhydride (5c). 1H NMR (300 MHz, D2O) d
1.52–1.80 (m, 6H), 3.73 (t, 2H, J = 7.3 Hz), 5.74 (d, 1H, J = 7.7 Hz),
7.60 (d, 1H, J = 7.7 Hz). 31P NMR (120 MHz, D2O) d ꢁ23.5 (t, Pb,
J = 24.2 Hz), ꢁ11.2 (d, P , J = 17.8 Hz), 19.7 (d, P , J = 25.4 Hz).
5.10.2.19. 4-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)butyl tri-
phosphate (12c)47 1H NMR (300 MHz, D2O) d 1.24–1.82 (m, 4H),
.
3.81 (t, 2H, J = 7.1 Hz), 4.01 (m, 2H), 5.81 (d, 1H, J = 7.7 Hz), 7.69
(d, 1H, J = 7.7 Hz). 31P NMR (120 MHz, D2O) d ꢁ20.4 (t, Pb,
J = 19.1 Hz), ꢁ11.1 (d, P , J = 19.1 Hz), ꢁ8.4 (d, P , J = 21.6 Hz).
c
a
c
a
5.10.2.9. Diphosphoric 5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)pentylphosphonic anhydride (5d). 1H NMR (300 MHz, D2O) d
1.32–1.76 (m, 8H), 3.71 (t, 2H, J = 7.3 Hz), 5.73 (d, 1H, J = 7. 7 Hz),
7.58 (d, 1H, J = 7.7 Hz). 31P NMR (120 MHz, D2O) d ꢁ23.5 (t, Pb,
J = 23.5 Hz), ꢁ10.9 (d, P , J = 20.4 Hz), 20.6 (d, P , J = 26.7 Hz).
5.10.2.20. 5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)pentyl tri-
phosphate (12d). 1H NMR (300 MHz, D2O) d 0.98–1.38 (m, 6H),
3.43 (t, 2H, J = 7.2 Hz), 3.62 (m, 2H), 5.45 (d, 1H, J = 7. 9 Hz), 7.31
(d, 1H, J = 7.9 Hz). 31P NMR (120 MHz, D2O)
d
ꢁ23.5 (t, Pb,
J = 19.1 Hz), ꢁ11.1 (d, P , J = 17.8 Hz), ꢁ10.9 (d, P , J = 20.4 Hz).
c
a
c
a
5.10.2.10. 1-((3-Hydroxypropoxy)methyl)pyrimidine-
2,4(1H,3H)-dione (8b). 1H NMR (300 MHz, DMSO-d6) d 1.63 (q,
2H, J = 6.4 Hz), 3.41 (t, 2H, J = 6.5 Hz), 3.51 (t, 2H, J = 6.5 Hz), 4.44
(s, 1H), 5.06 (s, 2H), 5.61 (d, 1H, J = 7.8 Hz), 7.69 (d, 1H,
J = 7.8 Hz), 11.31 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d 32.63,
57.65, 65.89, 76.57, 101.79, 145.18, 151.28, 163.86. Elemental Anal.
Calcd for C8H12N2O4 (200.20): C, 48.00; H, 6.04; N, 13.99. Found: C,
47.81; H, 5.99; N, 13.99.
5.11. Chemicals for biological experiments
Uridine-50-triphosphate (UTP), dimethyl sulfoxide (DMSO), con-
stituents of Krebs–Hepes buffer (sodium chloride, potassium chlo-
ride, potassium dihydrogenphosphate, sodium hydrogencarbonate,
anhydrous glucose, sodium hydroxide (for pH adjustment of
Krebs–Hepes buffer to pH 7.4), Hepes (free acid), calcium chloride
and magnesium sulfate), Dulbecco’s modified Eagle’s medium
(DMEM), penicillin–streptomycin and fetal bovine serum for cell
culture were obtained from Sigma (Taufkirchen, Germany). HAT
(hypoxanthine, aminopterin, thymidine) supplement was from
Gibco Life Technologies (Karlsruhe, Germany).
5.10.2.11. 2-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methox-
y)ethyl monophosphate (9a)44 1H NMR (300 MHz, D2O) d 3.48
.
(m, 4H), 5.09 (s, 2H), 5.61 (d, 1H, J = 7. 8 Hz), 7.71 (d, 1H,
J = 7.8 Hz). 31P NMR (120 MHz, D2O) d 0.40 (s, P ).
a
5.12. Cell culture
5.10.2.12. 3-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)-
propyl monophosphate (9b). 1H NMR (300 MHz, D2O) d 1.66 (q,
2H, J = 6.3 Hz), 3.47 (t, 2H, J = 6.3 Hz), 3.66 (m, 2H), 4.96 (s, 2H),
5.60 (d, 1H, J = 7. 9 Hz), 7.49 (d, 1H, J = 7.9 Hz). 31P NMR (120 MHz,
NG108-15 (mouse neuroblastoma ꢀ rat glioma) hybrid cells
were cultured as previously described8,10,49 in Dulbecco’s modified
Eagle’s medium (containing 100 lg/ml streptomycin, 100 U/ml
D2O) d 0.25 (s, P ).
penicillin G, 10% fetal bovine serum (Sigma–Aldrich) and HAT
a
(1.0 ꢀ 10ꢁ5 M hypoxanthine, 4.0 ꢀ 10ꢁ8
M
aminopterin, 1.6 ꢀ
5.10.2.13. 2-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)-
10ꢁ6 M thymidine) until cells were 80% confluent. Cells were
ethyl triphosphate (10a)45,46
.
1H NMR (300 MHz, D2O) d 3.48 (m,
incubated at 37 °C with 5% CO2 and 95% relative humidity.
4H), 5.09 (s, 2H), 5.61 (d, 1H, J = 7. 8 Hz), 7.71 (d, 1H, J = 7.8 Hz). 31P
NMR (120 MHz, D2O) d ꢁ23.6 (t, Pb, J = 15.9 Hz), ꢁ11.5 (d, P ,
5.13. Calcium measurements
c
J = 20.3 Hz), ꢁ11.1 (d, P , J = 20.4 Hz).
a
Ca2+ fluorescence measurements were performed using a FLUO-
starÒ plate reader, equipped with one injector (BMG LabTechnolo-
gies, Offenburg, Germany) as previously described.8–10,40 Cells
were loaded for 1 h at 25 °C (continuous shaking at 600 rpm, exclu-
5.10.2.14. 3-((2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)methoxy)-
propyl triphosphate (10b). 1H NMR (300 MHz, D2O) d 1.37 (m,
2H), 3.15 (t, 2H, J = 6.3 Hz), 3.46 (m, 2H), 4.65 (s, 2H), 5.30 (d, 1H,
J = 7. 9 Hz), 7.17 (d, 1H, J = 7.9 Hz). 31P NMR (120 MHz, D2O) d
ꢁ22.4 (t, Pb, J = 15.9 Hz), ꢁ11.4 (d, P , J = 19.1 Hz), ꢁ8.1 (d, P ,
sion of light) with 3 lM of Oregon Green 488 BAPTA-1/AM (Molec-
ular Probes), which had been mixed with a 20% solution of Pluronic
F-127 (Molecular Probes) in DMSO at a ratio of 1:1 immediately
before use. After incubation, cells were rinsed twice with Krebs–
HEPES buffer, diluted and evenly plated into black 96-well plates
(Packard OptiplateTM HTRF-96; Packard BioScience) at a density of
17,000 cells/well. To determine agonistic activity, test compounds
dissolved in buffer were injected sequentially into separate wells
and fluorescence intensity was measured at 520 nm (bandwidth
25 nm) for 60 intervals of 0.4 s each. Excitation wavelength was
at 485 nm (bandwidth 20 nm). Antagonistic potency of test com-
pounds was initially determined by preincubation of the cells with
c
a
J = 14.4 Hz).
5.10.2.15. 2-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)ethylmono-
phosphate (11a).47 1H NMR (300 MHz, D2O) d 3.89 (t, 2H,
J = 4.9 Hz), 4.1 (m, 2H), 5.70 (d, 1H, J = 7. 9 Hz), 7.60 (d, 1H,
J = 7.9 Hz). 31P NMR (120 MHz, D2O) d 1.9 (s, P ).
a
5.10.2.16. 4-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)butyl
monophosphate (11c).48 1H NMR (300 MHz, D2O) d 1.60–1.83
(m, 4H), 3.82 (t, 2H, J = 7.2 Hz), 3.89 (m, 2H), 5.82 (d, 1H, J = 7.
9 Hz), 7.66 (d, 1H, J = 7.9 Hz). 31P NMR (120 MHz, D2O) d 0.7 (s, P ).
two concentrations of test compound (100 and 500
for at least 30 min prior to injection of the agonist UTP (1
The final volume was 200 l. Data points within one assay were
determined in quadruplicate. For compounds that appeared to be
l
M) at 37 °C
a
l
M).
5.10.2.17. 5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)pentyl
l
monophosphate (11d).47 1H NMR (300 MHz, D2O) d 1.15–1.53

R. Sauer et al. / Bioorg. Med. Chem. 17 (2009) 5071–5079
5079
15. Knoblauch, B. H. A.; Müller, C. E.; Järlebark, L.; Lawoko, G.; Kottke, T.;
Wikström, M. A.; Heilbronn, E. Eur. J. Med. Chem. 1999, 34, 809.
16. El-Tayeb, A.; Qi, A.; Müller, C. E. J. Med. Chem. 2006, 29, 7076.
17. Fischer, B.; Yefidoff, R.; Major, D. T.; Rutman-Halili, I.; Shneyvays, V.; Zinman,
T.; Jacobson, K. A.; Shainberg, A. J. Med. Chem. 1999, 42, 2685.
18. Kim, H. S.; Barak, D.; Harden, T. K.; Boyer, J. L.; Jacobson, K. A. J. Med. Chem.
2001, 44, 3092.
potent in the initial screening tests (repeated at least once), dose–
response curves were recorded and IC50 values were calculated by
nonlinear regression with a sigmoidal dose–response equation
(variable Hill slope) using the computer program PrismÒ3.0 (Graph
Pad Software, Inc., San Diego, CA, USA).
19. Mishra, R. C.; Ben, D. D.; Lambertucci, C.; Portino, F. R.; Volpini, R.; Vittori, S.;
Cristalli, G. Second Joint Italian-Swiss Meeting in Medicinal Chemistry; September
13–16, 2005, Modena, Italy.
Acknowledgments
20. Houston, D.; Costanzi, S.; Jacobson, K. A.; Harden, T. K. Comb. Chem. High
Throughput Screening 2008, 11, 410.
21. Xu, B.; Stephens, A.; Kirschenheuter, G.; Greslin, A. F.; Cheng, X.; Sennelo, J.;
Cattaneo, M.; Zighetti, M. L.; Chen, A.; Kim, S.-A.; Kim, H. S.; Bischofberger, N.;
Cook, G.; Jacobson, K. A. J. Med. Chem. 2002, 45, 5694.
22. Dolakova, P.; Dracinsky, M.; Masojidkova, M.; Solinova, V.; Kasicka, A. Eur. J.
Med. Chem. 2009, 44, 2408.
23. Holy, A. Curr. Pharm. Des. 2003, 9, 2567.
We would like to thank Dr. Ramat Qurishi for purity determina-
tion of the final products by capillary electrophoresis coupled to
UV detection, and Franziska Streicher for testing some of the com-
pounds. A.E.-T. is grateful for support by the DAAD (STIBET
scholarship).
24. Schaeffer, H. J.; Beauchamp, L.; de Miranda, P.; Elion, G.; Bauer, D. J.; Collins, P.
Nature 1978, 172, 583.
Supplementary data
25. Balzarini, J.; Pannecouque, C.; De Clercq, E.; Aquaro, S.; Perno, C. F.; Egberink,
H.; Holy, A. Antimicrob. Agents Chemother. 2002, 46, 2185.
26. Choo, H.; Beadle, J. R.; Chong, Y.; Trahan, J.; Hostetler, K. Y. Bioorg. Med. Chem.
2007, 15, 1771.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.05.062.
27. Watts, J. K.; Deleavey, G. F.; Damha, M. J. Drug Discovery Today 2008, 13, 842.
28. Tjoeng, F. S.; Kraus, E.; Breitmaier, E.; Jung, G. Chem. Ber. 1976, 109, 2615.
29. Dulcere, J. P.; Baret, N.; Rodriguez, J. Synlett 1995, 923.
30. Nowick, J. S.; Chen, J. S.; Noronha, G. J. Am. Chem. Soc. 1993, 115, 7636.
31. Itahara, T. Bull. Chem. Soc. Jpn. 1997, 70, 2239.
References and notes
1. King, B. F.; Townsend-Nicholson, A.; Burnstock, G. Trends Pharmacol. Sci. 1998,
19, 506.
2. von Kügelgen, I.; Wetter, A. Naunyn Schmiedeberg’s Arch. Pharmacol. 2000, 362,
310.
3. Brunschweiger, A.; Müller, C. E. Curr. Med. Chem. 2006, 13, 289; Müller, C. E.
Curr. Pharm. Des. 2002, 8, 2353.
4. Abbracchio, M. P.; Burnstock, G.; Boeynaems, J. M.; Barnard, E. A.; Boyer, J. L.;
Kennedy, C.; Knight, G. E.; Fumagalli, M.; Gachet, C.; Jacobson, K. A.; Weisman,
G. A. Pharmacol. Rev. 2006, 58, 281.
32. Dezor-Mazur, M.; Kazmierczak, F.; Golankiewicz, K. Heterocycles 1984, 22,
2739.
33. Moffat, J. G. Can. J. Chem. 1964, 42, 599.
34. Cullis, P. M.; Schilling, M. B. J. Chem. Soc., Chem. Commun. 1989, 106.
35. Dezor-Mazur, M.; Koraniak, H.; Langer, J. J.; Golankiewicz, K. J. Chem. Soc.,
Perkin Trans. 2 1989, 1209.
36. Ludwig, J. Acta Biochim. Biophys. Acad. Sci. Hung. 1981, 16, 131.
37. Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631.
5. Escolar, G.; Heras, M. Drugs Today 2000, 36, 187.
38. Lin, T. A.; Lustig, K. D.; Sportiello, M. G.; Weisman, G. A.; Sun, G. Y. J. Neurochem.
1993, 60, 1115.
6. Ingall, A. H.; Dixon, J.; Bailey, A.; Coombs, M. E.; Cox, D.; McInally, I. J.; Hunt, S.
F.; Kindon, N. D.; Teobald, B. J.; Willis, P. A.; Humphries, R. G.; Leff, P.; Clegg, J.
A.; Smith, J. A.; Tomlinson, W. J. Med. Chem. 1999, 42, 213.
7. Baqi, Y.; Atzler, K.; Köse, M.; Glänzel, M.; Müller, C. E. J. Med. Chem. 2009, in
press. doi:10.1021/jm9003297.
8. Hillmann, P.; Ko, G.-Y.; Spinrath, A.; Raulf, A.; von Kügelgen, I.; Wolff, S. C.;
Nicholas, R. A.; Kostenis, E.; Höltje, H.-D.; Müller, C. E. J. Med. Chem. 2009, 52,
27620.
9. Weyler, S.; Baqi, Y.; Hillman, P.; Kaulich, M.; Hunder, A. M.; Müller, I. A.; Müller,
C. E. Bioorg. Med. Chem. Lett. 2008, 18, 223.
10. Kaulich, M.; Streicher, F.; Mayer, R.; Müller, I.; Müller, C. E. Drug Dev. Res. 2003,
59, 72.
11. Cristalli, G.; Podda, G. M.; Costanzi, S.; Lambertucci, C.; Lecchi, A.; Vittori, S.;
Volpini, R.; Zighetti, M. L.; Cattaneo, M. J. Med. Chem. 2005, 48, 2763.
12. Yerxa, B. R.; Sabater, J. R.; Davis, C. W.; Stutts, M. J.; Lang-Furr, M.; Picher, M.;
Jones, A. C.; Cowlen, M.; Dougherty, R.; Boyer, J.; Abraham, W. M.; Boucher, R.
C. J. Pharmacol. Exp. Ther. 2002, 302, 871.
13. Pendergast, W.; Yerxa, B. R.; Douglass, J. G., 3rd; Shaver, S. R.; Dougherty, R. W.;
Redick, C. C.; Sims, I. F.; Rideout, J. L. Bioorg. Med. Chem. Lett. 2001, 11, 157.
14. Kellerman, D.; Mospan, a. R.; Engels, J.; Schaberg, A.; Gorden, J.; Smiley, L. Pulm.
Pharmacol. Ther. 2008, 21, 600.
39. Sak, K.; Samuel, K.; Kelve, M.; Webb, T. E. Eur. J. Pharmacol. 2001, 415, 127.
40. Brunschweiger, A.; Iqbal, J.; Umbach, F.; Scheiff, A. B.; Munkonda, M. N.;
Sévigny, J.; Knowles, A. F.; Müller, C. E. J. Med. Chem. 2008, 51, 4518.
41. Parr, C. E.; Sullivan, D. M.; Paradiso, A. M.; Lazarowski, E. R.; Burch, L. H.; Olsen,
J. C.; Erb, L.; Weisman, G. A.; Boucher, R. C.; Turner, J. T. Proc. Natl. Acad. Sci.
U.S.A. 1994, 91, 3275.
42. Douglass, J. G.; Patel, R. I.; Yerxa, B. R.; Shaver, S. R.; Watson, P. S.; Bednarski, K.;
Plourde, R.; Redick, C. C.; Brubaker, K.; Jones, A. C.; Boyer, J. L. J. Med. Chem.
2008, 51, 1007.
43. Ubasawa, M.; Takashima, H.; Sekiya, K. Chem. Pharm. Bull. 1995, 43, 142.
44. Rutkowski, M.; Draminski, M. Acta Biochem. Pol. 1991, 38, 449.
45. Gardner, A. F.; Jack, W. E. Nucl. Acid Res. 2002, 30, 601.
46. Cheng, Y. C.; Dutschman, G. E.; Ginger, E.; Bastow, K. F.; Sarngadharan, M. G.;
Ting, R. Y. C. J. Biol. Chem. 1987, 262, 2187.
47. Kritsyn, A. M.; Mikhailov, S. M.; Kolobushkina, L. I.; Padyukova, N. S.; Florentev,
V. L. Izv. Akad. Nauk SSSR, Ser. Khim. 1975, 8, 1846.
48. Baker, B. R.; Jackson, G. D. F.; Chheda, G. B. J. Pharm. Sci. 1965, 54, 1617.
49. Qurishi, R.; Kaulich, M.; Müller, C. E. J. Chromatogr. A 2002, 952, 275.