5-Substituted UTP derivatives as P2Y2 receptor agonists

Article abstract of DOI:10.1016/S0223-5234(99)00211-1

A series of 5-alkyl-substituted UTP derivatives, which had been synthesized previously with a moderate degree of purity, was resynthesized, purified, and characterized. Synthetic and purification procedures were optimized. New spectroscopic data, including ¹³C- and ³¹P NMR data, are presented. Phosphorylation reactions yielded a number of side products, such as the 2'-, 3'-, and 5'-monophosphates, the 2',3'-cyclic monophosphates, and the 2',3'-cyclic phosphates of the 5'-triphosphates. Furthermore, raw products were contaminated with inorganic phosphates, including cyclometatriphosphate, phosphate, and pyrophosphate. The uracil nucleotides were investigated for their potency to increase intracellular calcium concentrations by stimulation of P2Y₂ receptors (P2Y₂R) on NG108- 15 cells, a mouse neuroblastoma x glioma cell line, and in human basal epithelial airway cells, including a cystic fibrosis (CF/T43) cell line. UTP exhibited EC₅₀ values of ca. 1 μM (in NG108-15 cells) and of 0.1 μM (in CF/T43 cells), respectively. 5-Substituted UTP derivatives were agonists at the P2Y₂R, but were less potent than UTP. 5-Ethyl-UTP, for example, exhibited an EC₅₀ value of 99 μM at P2Y₂R of NG108-15 cells and proved to be a full agonist. With increasing volume of the 5- substituent of UTP derivatives, P2Y₂ activity decreased.

Full text of DOI:10.1016/S0223-5234(99)00211-1

Eur. J. Med. Chem. 34 (1999) 809−824
© 1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
809
Original article
5-Substituted UTP derivatives as P2Y2 receptor agonists#
Bernd H.A. Knoblaucha,d, Christa E. Müllera,c*, Leif Järlebarkd, Grace Lawokod, Thomas Kottkeb,
Martin A. Wikströme, Edith Heilbronnd
aInstitut für Pharmazie und Lebensmittelchemie, Pharmazeutische Chemie,
Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
bInstitut für Anorganische Chemie, Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany
cUniversität Bonn, Pharmazeutisches Institut, Pharmazeutische Chemie Poppelsdorf, Germany
dStockholm University, Department of Neurochemistry and Neurotoxicology,
Arrhenius Laboratories of Natural Sciences, Stockholm, Sweden
eKarolinska Institute, Nobel Institute for Neurophysiology, Department of Neuroscience, Stockholm, Sweden
(Received 8 February 1999; revised 30 April 1999; accepted 6 May 1999)
Abstract – A series of 5-alkyl-substituted UTP derivatives, which had been synthesized previously with a moderate degree of purity, was
resynthesized, purified, and characterized. Synthetic and purification procedures were optimized. New spectroscopic data, including 13C- and
31P NMR data, are presented. Phosphorylation reactions yielded a number of side products, such as the 2≠-, 3≠-, and 5≠-monophosphates, the
2≠,3≠-cyclic monophosphates, and the 2≠,3≠-cyclic phosphates of the 5≠-triphosphates. Furthermore, raw products were contaminated with
inorganic phosphates, including cyclometatriphosphate, phosphate, and pyrophosphate. The uracil nucleotides were investigated for their
potency to increase intracellular calcium concentrations by stimulation of P2Y2 receptors (P2Y2R) on NG108–15 cells, a mouse
neuroblastoma × glioma cell line, and in human basal epithelial airway cells, including a cystic fibrosis (CF/T43) cell line. UTP exhibited EC50
values of ca. 1 µM (in NG108–15 cells) and of 0.1 µM (in CF/T43 cells), respectively. 5-Substituted UTP derivatives were agonists at the
P2Y2R, but were less potent than UTP. 5-Ethyl-UTP, for example, exhibited an EC50 value of 99 µM at P2Y2R of NG108–15 cells and proved
to be a full agonist. With increasing volume of the 5-substituent of UTP derivatives, P2Y2 activity decreased. © 1999 Éditions scientifiques
et médicales Elsevier SAS
P2Y2 receptor agonists / P2U receptor agonists / UTP analogues / pyrimidine nucleotides / cystic fibrosis
1. Introduction
tors, or P1-receptors (P1R), and the purine and pyrimi-
dine nucleotide receptors or P2-receptors (P2R) [1, 2].
The growing family of known P2 receptors comprises
subfamilies of ionotropic (P2X) and metabotropic (P2Y)
receptors. While P2XR subtypes appear to be activated
exclusively by adenine nucleotides, subtypes of P2YR
exist, including P2Y2, P2Y4, P2Y6, at which uracil
nucleotides, namely UTP or UDP, show activity and may
act as physiological agonists. The P2Y2R is a ubiqui-
tously expressed UTP-sensitive P2YR subtype, also
known as P2UR [3]. ATP and UTP are equipotent as
agonists at the P2Y2R. It has recently been found that the
impaired chloride transport in the bronchi of patients
suffering from cystic fibrosis can be bypassed by stimu-
lation of a P2Y2R to activate an alternative chloride
channel [4]. UTP is currently under development as a
Membrane receptors for endogenous nucleotides, such
as ATP and UTP (figure 1) belong to the purine/
pyrimidine receptor family. Purine receptors are sub-
divided into two separate families, the adenosine recep-
# Preliminary results were presented at the Joint Meeting of the
German and Swiss Pharmaceutical Societies in Zürich, Swit-
zerland, 1997, and at the International Symposium on Adeno-
sine and Adenine Nucleotides in Ferrara, Italy (abstract in Drug
Dev. Res. 43 (1998) 34).
* Correspondence and reprints
Dr Christa Müller, Pharmazeutisches Institut der Universität
Bonn, Pharmazeutische Chemie Poppelsdorf, Kreuzbergweg
26, D-53115 Bonn.

810
Figure 1. Structures of standard nucleotides.

811
Figure 2. Preparation of 5-substituted UTP derivatives.
novel therapeutic agent for the symptomatic treatment of
cystic fibrosis.
respectively. Derivatives with longer side chains dis-
solved faster in HMDS due to their higher lipophilicity as
compared to those with smaller substituents. After com-
plete dissolution, which indicated completion of the
reaction, silylated uracil derivatives 7a–e were character-
ized by their 1H and 13C NMR spectra (table I). Conden-
sation with 1-O-acetyl-2,3,5-tri-O-benzoyl-â-D-ribofura-
nose in the presence of tin(IV) chloride yielded
nucleosides 8a–e, essentially as described [9]. For 5-ethyl
derivative 7b, as an example, different reaction condi-
tions were investigated. As solvents, 1,2-dichloroethane
or acetonitrile were used. As Lewis acid catalysts tin(IV)
chloride, or trimethylsilyltriflate, were applied. All differ-
ent combinations gave very good yields of 8b (80–90%).
In 1,2-dichloroethane as a solvent, traces of N3-
nucleoside were formed, which could be removed by
silica gel column chromatography. Trimethylsilyltriflate
was found not to be superior to tin(IV) chloride.
Only few P2Y2R agonists are known so far and
structure-activity relationships for P2Y2R agonists are
virtually unknown [5, 6]. In the present study we synthe-
sized a series of 5-substituted UTP derivatives. We
selected UTP as a lead structure to develop P2Y2R
ligands, rather than ATP, because of the higher selectivity
of UTP versus other P2R subtypes, e.g., P2XR. In
addition, degradation products of ATP and analogues
(adenosine and analogues) may interact with P1R (ad-
enosine receptors), but not those of UTP and ana-
logues [7]. The synthesized nucleotides were investigated
in vitro for their potency to increase intracellular calcium
concentration ([Ca2+]i) caused by a P2Y2R-mediated
stimulation of phospholipase C [8].
2. Chemistry
Hydrolysis was performed using a saturated aqueous
sodium hydrogencarbonate solution. In our hands, potas-
sium hydrogencarbonate showed no advantage over the
sodium salt, in contrast to results reported by Szemo et
al. [9]. Filtration over silica gel efficiently removed
tin(IV) oxide hydrate formed during the reaction. The
5-Alkyl-substituted UTP derivatives 10a–e were syn-
thesized according to published procedures with modifi-
cations (figure 2) [9–12]. Uracil derivatives 6a–e were
silylated using hexamethyldisilazane (HMDS) in the
presence of trimethylsilylchloride, or ammonium sulfate,

812
Table I. 13C-NMR data of silylated uracil derivatives (in CDCl3) δ (ppm).
Compound
O-Si(CH3)3
C2
C4
C5
C6
R5
7a
7b
7c
7d
7e
0.32
0.29
0.26
0.29
–0.41
168.15
167.84
167.87
167.39
167.26
161.78
161.69
161.66
167.39
161.08
112.57
118.33
116.75
122.30
116.08
158.87
157.90
158.63
158.63
157.83
12.20
13.42, 20.30
13.67, 22.27, 28.91
21.63, 26.24
13.08, 21.54, 25.91, 30.76
benzoylated nucleosides 8a–e were deprotected via trans-
esterification using methanolic sodium methylate solu-
tion. The formed benzoic acid methyl ester was conve-
niently removed by extraction with diethyl ether and
subsequent lyophilization to yield the nucleosides 9a–e.
A recently described direct method for nucleoside syn-
thesis [13], which does not require protection of the
sugar, was investigated for the preparation of the 5-propyl
derivative 10c, but was not successful in our hands. At
low temperatures no reaction occurred and the starting
material was recovered. At temperatures over 80 °C only
degradation was observed.
Nucleotides 10a–e were prepared from nucleosides
9a–e according to published procedures with slight modifi-
cations [9–12]. The lyophilized nucleosides were dis-
solved in dry trimethylphosphate and reacted with phos-
phorus oxychloride, with or without the addition of
1,8-bis(dimethylamino)naphthalene (‘proton sponge’).
The reactive intermediate phosphorodichloridate was
coupled with a mixture of one equivalent of tri-n-
butylamine and a five-fold excess of tri-n-butyl-
ammonium diphosphate in dimethylformamide to yield
10a–e (figure 2).
Compound 8d showed a nucleosidic torsion angle (O3-
C5-N1-C1) of –133.22°. Thus, the nucleoside crystallized
in the anti-conformation despite bulky 5- and 5≠-
substituents. The torsion angle γ about the exocyclic
C8–C9 bond (figure 3) may be described as +sc (gauche/
gauche). A vibrational disorder of the isopropyl side
chain could be seen, probably facilitated by the low data
collection temperature of 173 K. As an interesting fea-
ture, the sugar puckering in 8d deviated from the confor-
mation observed in unprotected 5-methyluridine [14],
which adopted a C3≠-endo conformation, while 8d
showed a sugar puckering that can be described as
C1≠-exo.
The synthesized nucleotides were purified by anion
exchange chromatography using diethylaminoethyl-
(DEAE-) Sephadex A25. The triethylammonium salts
were converted to the corresponding sodium salts by
dissolution in absolute methanol and subsequent precipi-
tation by the addition of sodium iodide in acetone. The
sodium salts were obtained as amorphous powders and
therefore better to handle for biological tests than the
triethylammonium salts, which were obtained as oily,
transparent liquids.
Nucleotides were characterized by 13C and 31P NMR
spectroscopy and plasma desorption mass spectrometry.
Purity was determined by HPLC/UV and 31P NMR
spectroscopy (tables III and IV). The combination of both
methods proved to be important for purity control (ta-
ble IV).
Nucleosides 8a–e and 9a–e were characterized by their
1H NMR spectra, which were in accordance with pub-
lished data [9]. In addition, 13C NMR spectra were
recorded (tables II and III).
Crystals suitable for X-ray crystallography could be
obtained from nucleosides 8b and 8d. The structure of
nucleoside 8d is shown in figure 3 (see also table VI).
Table II. 13C-NMR data of protected nucleosides (in DMSO-d6), δ (ppm).
Compound C5≠ C3≠ C2≠ C4≠ C1≠ Caromat.
benzoyl
C5
C6
C2
C4
R5
8a
8b
8c
8d
8e
63.7 70.7 73.1 78.8 88.5 128.4, 128.5, 128.7, 129.2, 164.4, 164.6, 165.5
129.3, 133.5, 133.7
63.6 70.6 73.0 78.8 88.8 128.4, 128.6, 128.7, 129.2, 164.5, 164.6, 165.5
129.3, 133.5, 133.8
63.6 70.7 72.9 78.8 88.4 128.4, 128.5, 128.6, 129.3, 164.5, 164.6, 165.5
133.5, 133.6, 133.7
63.7 70.6 73.0 78.8 89.4 128.4, 128.6, 128.7, 129.2, 164.5, 164.6, 165.5
129.3, 133.5, 133.8
110.1 136.9 150.9 162.9 11.9
115.7 136.7 150.2 163.2 12.9, 19.5
114.1 137.0 150.2 163.2 13.3, 21.1, 28.1
119.9 136.1 150.0 162.9 21.0, 21.1, 25.5
114.4 137.1 150.3 163.3 13.6, 21.7, 25.9, 30.2
63.8 70.7 73.0 78.9 88.6 128.4, 128.5, 128.7, 129.2, 164.6, 164.7, 165.5
129.3, 133.5, 133.7

813
Table III. 13C-NMR data of nucleosides and nucleotides (in DMSO-d6), δ (ppm).
Compound
C5≠
C3≠
C2≠
C4≠
C1≠
C5
C6
C2
C4
R5
3JC4≠-P 2JC5≠-P
9a
9b
9c
9d
9e
60.9
60.8
60.8
60.7
60.8
69.8
69.9
69.8
69.9
69.8
73.4
73.5
73.4
73.8
73.6
84.8
84.8
84.7
84.7
84.8
87.6
87.7
87.8
88.1
87.8
109.2
115.1
113.2
119.4
113.5
136.4
135.8
136.4
134.9
136.4
150.8
150.7
150.6
150.4
150.7
163.8
163.4
163.3
163.0
163.5
12.2
12.9, 19.7
12.9, 21.0, 28.4
21.2, 21.4, 25.4
12.9, 21.7, 25.9, 30.1
10a
10b
10c
10d
10e
66.6
66.7
66.8
67.0
66.9
74.9
74.6
64.8
75.0
74.8
71.5
71.6
71.6
71.8
71.6
85.0
85.1
85.1
85.3
85.1
89.2
89.0
89.2
89.4
89.3
113.2
118.9
117.2
123.5
117.7
138.7
138.2
139.0
138.7
138.9
153.4
153.2
153.3
153.2
153.8
167.8
167.1
167.3
166.9
167.5
14.2
12.1, 21.2
14.4, 22.8, 29.6
22.2, 22.5, 21.2
14.9, 23.1, 27.5, 31.9
9.2
9.2
9.2
9.2
9.2
6.1
4.9
6.1
6.1
6.1
Figure 3. X-ray structure of nucleoside 8d.

814
Table IV. 31P-NMR (in D2O), MS data and HPLC retention times of nucleotides 10a–e, δ (ppm).
31P-NMR
Pâ
MSa
Mole Peak Ion
HPLC (UV; λ =266 nm)
Compound pH
Pα
Pγ
System Ab
System Bc
Retention time (Purity) Retention time (Purity)
10a
10b
10c
10d
10e
5.9
6.5
6.7
7.7
5.8
–10.69 d
–22.56 t
–10.04 d
496.9
511.1
524.9
524.8
539.1
[M – H]–
13.51
21.88
25.97
17.36
31.15
(94 %)
(95 %)
(93 %)
(94 %)
(92 %)
7.56
7.82
8.01
9.40
8.07
(95 %)
(96 %)
(94 %)
(92 %)
(93 %)
J = 19.6 Hz J = 19.5 Hz J = 19.5 Hz
–10.26 d –21.90 t –9.26 d
J = 19.6 Hz J = 19.6 Hz J = 19.5 Hz
–10.38 d –22.30 t –9.05 d
J = 19.6 Hz J = 19.5 Hz J = 19.5 Hz
–10.58 d
J = 20.0
–10.98 d
[M – H]–
[M – H]–
[M – H]–
[M – H]–
–22.03 t
J = 20.2 Hz J = 19.8 Hz
–22.56 t –10.04 d
–7.14 d
J = 20.2 Hz J = 20.0 Hz J = 19.8 Hz
a Negative ion plasma desorption MS. b System A: Nucleosil RP-18 column with eluent A, 0.1 M triethylammonium acetate buffer and eluent
B, acetonitrile (gradient: 0–15%, 30 min). c System B: Macherey-Nagel ET125/4 Nucleosil 4000-7 PEI column with eluent A, 0.01 M
Tris/HCl (pH = 8.4) and eluent B, Tris/HCl (pH = 8.4), 1.0 M NaCl.
Phosphorylation reactions were investigated in some
detail. It had been reported that reaction of nucleosides
with phosphorus oxychloride was somewhat faster in
trimethyl phosphate as compared to triethyl phos-
phate [15]. We investigated the reaction of ethyl deriva-
tive 9b with phosphorus oxychloride under various con-
ditions. Reaction time was always virtually identical in
both solvents. Proton sponge accelerated reaction time
from 7 to 2 h, while pre-heating of reagents before the
addition of phosphorus oxychloride had no effect.
Phosphorylation of 9a–e to obtain triphosphates 10a–e
yielded a number of side products (figure 4). For butyl
derivative 10e, structure elucidation of the major side
products was performed as preparation of 10e yielded the
largest quantity of side products. A fraction isolated at a
concentration of 0.13–0.16 M triethyl ammonium bicar-
bonate (TEAB) buffer showed five spots in thin layer
chromatography on cellulose-coated plates, all of which
contained phosphorus as shown by spraying with a
phosphate-specific reagent. Four of the spots showed UV
absorption at 266 nm (table V). The mass spectra (anionic
mode) confirmed triphosphate 10e as the major product.
Several pH-dependent signals were detected in the 31P
NMR spectrum. According to Cozzone and Jar-
detzky [16], the signals could be assigned to the 2≠-
monophosphate (11), the 3≠-monophosphate (12), the
5≠-monophosphate (13), and the 2≠,3≠-cyclomonophos-
phate (14, figure 5). The identification of the side prod-
ucts is shown in table V.
21.8 Hz, JPγPâ = 22.9 Hz), a doublet at –11.93 ppm (Pα,
JPαPâ = 21.8 Hz), a doublet at –11.39 ppm (Pγ, JPγPâ
=
22.9 Hz), and a singlet at 16.41 ppm. These data indicate
that the compound (15) contains a 2≠,3≠-cyclophosphate
group in addition to the 5≠-triphosphate residue of 10e.
The second side product with a molecular mass of 619.1
is a ring-open analogue of 15, since the 31P NMR
spectrum clearly shows a signal in the range of a
monophosphate in addition to the 5≠-triphosphate struc-
ture. Bisphosphorylation at the 2≠- and 3≠-hydroxyl
groups was not observed. The ratio of formed 10e:15 was
ca. 2:1.
The particularly large amount of side products ob-
served in the preparation of the 5-butyluridine triphos-
phate 10e may be due to steric interaction between the
bulky butyl residue and the 5≠-hydroxyl function in polar
aprotic solvents, which leads to an increased phosphory-
lation of the secondary hydroxyl groups at C2≠ and C3≠.
The preparation of other nucleotides yielded the analo-
gous side products, but in smaller quantities. Use of
proton sponge as reaction accelerator was not the cause
for the formation of 2≠- and 3≠-phosphoric acid esters
and 2≠,3≠-cyclophosphates as side products, as we ob-
served the formation of the same compounds in the
absence of proton sponge.
In addition to nucleotide side products, large amounts
of inorganic phosphates, including cyclometatriphosphate
4–
(P3O93–), phosphate (PO43–), pyrophosphate (P2O7
)
A fraction isolated at 0.43 M TEAB buffer concentra-
tion contained two more side products and a small
amount of 10e. The major side product showed a mass of
600.7. The 31P NMR spectrum in dimethylsulfoxide
and linear triphosphate (P3O105–) were obtained (table V).
The stable cyclometatriphosphate was particularly diffi-
cult to remove by standard chromatographic methods due
to partial coelution with the target UTP derivatives
(figure 4 and table V).
showed four signals, a triplet at –23.87 ppm (Pâ, JPαPâ
=

Table V. Analysis of isolated fractions obtained from ion exchange chromatography of crude 10e (compare figure 3).
Fraction
I
II
III
IV
V
VI
80
TEAB concentration
[%]a
0
13–16
31–36
41–44
70–80
Yield [mg] (of 1 820
810
241
250
67
114
33
mg crude product)b
Spectroscopic methods
266
–9.8 (d); –11.0 (d);
–20.8 (s); –22.4 (t)
UV maximum at [nm]
31P-NMR (in D2O)
218c
266
266
266
266
–7.3 (s); 2.4 (s); 3.0
(s); 3.1 (s)
(pH = 7.2)
(s); 2.8 (s); 2.3 (s);
0.4 (s); 0.0 (s)
(pH = 4.5)
19.2 (s); 4.0 (s); 3.7
(s); 3.5 (s); 2.7 (s)
21.8 (s); –5.2 (d);
–9.8 (d);
–20.8 (t)
4.8 (s); 4.2 (s);
2.2(s);
21.8 (s); 4.5. (s); –8.1
(s)d; –10.3 (s)d;
(pH value)
(pH = 6.2)
–7.7– –10.3 (m);
–21.7 (s)d
(pH=8.0)
(pH = 8.0)
–20.0 (s); –21.7 (s) d
(pH = 7.4)
(pH = 9.5)
MS (neg. Mode)
n.d.e
379f
538f
601f, [619]
[540, 619], 700f
760f
Cellulose-TLC (solvent: 2-propanol:NH3(25 %):H2O = 6:3:1)
UV detection at 266
nm
Phosphate detection
3 spots
3 spots
4 spots
1 spots
2 spots
4 spots
5 spots
3 spots
5 spots
5 spots
2 spots
2 spots
(FeCl3/5-sulfosalicylic
acid reagent)
Deduced structure
P3O9
3–
3–
3–
3–
3–
5–
Inorganic phosphate as
determined by 31P-
NMR
PO4
PO4
P3O9
,
PO4
,
PO43–, P3O10
5–
P3O10
4–
P2O7
Nucleotidesg and other
organic compounds
phosphates of 1,8-
bis-(dimethyl)-
ammonium-
11
12
13
14
10e
15
structure could not
be determinedh
structure could not be
determinedi
naphthalene
a 100% corresponds to 1 M TEAB buffer. b Unidentified: 305 mg. c Typical spectrum for aromatic compounds. d Signal not resolved. e n.d. = not determined. f Main
peak. g For structures see figures 2 and 5. h Structure contains a total of 5 phosphate groups but no 2′3′-cyclic phosphate groups. i Structure contains a total of
6 phosphate groups (one of them is a 2′3′-cyclic phosphate group).

816
Figure 4. Separation of crude 10e on DEAE-Sephadex A25 by FPLC.
3. Biological evaluation
ganic phosphates pentasodium triphosphate (Na5P3O10),
trisodium tricyclometaphosphate (Na3P3O9), and sodium
pyrophosphate (Na4P2O7), which could be present as side
products in some of the nucleotides, show any effects. As
previously shown, UTP acted as a potent P2Y2 receptor
agonist, exhibiting an EC50 value of 1.25 µM and a
maximal increase in intracellular calcium of 69% (ta-
ble VIII). The observed potency for UTP is in accordance
with published data [5, 17]. ATP was somewhat less
potent, exhibiting an EC50 value of 17.7 µM, but showed
about the same maximal stimulation as UTP. The lower
activity of ATP may be due to faster enzymatic degrada-
tion of ATP as compared to UTP under test condi-
tions [18, 19]. The ring-open, base-modified ATP ana-
logue ZTP (5) was found to be nearly equipotent to ATP
in our assay system.
In the system described, addition of UDP (2) also
resulted in an increase in intracellular calcium concentra-
tion, with an EC50 value of 3.2 µM (table VIII). Efficacy,
however, was only 61% of that of UTP. UDP has
previously been shown to be inactive at P2Y2R [19, 20],
but it might have been contaminated with UTP, or could
have been phosphorylated enzymatically, and the result-
P2Y2 receptor activity was determined in a mouse
neuroblastoma × glioma hybrid cell line (NG 108-15) by
fluorimetric measurement of the increase in intracellular
calcium concentration caused by stimulation of P2Y2
receptors via activation of phospholipase C. Selected
compounds were additionally tested in a human cystic
fibrosis epithelial airway cell line (CF/T43) and a human
basal epithelial airway cell line (BEA).
4. Results and discussion
4.1. Neuroblastoma × glioma cells (NG108-15 cell line)
The putative physiological P2Y2 receptor agonists
UTP (1) and ATP (4), the ATP analogue ZTP (5), uridine
diphosphate UDP (2) and the corresponding monophos-
phate UMP (3) were investigated at NG108-15 cells for
comparison. Initially, single concentrations (50 and/or
500 µM) of UTP derivatives and standard nucleotides
were used (table VII). Before testing it was shown that
not even at high concentrations (500 µM) did the inor-

817
Table VI. Crystal data of compound 8d.
Empirical formula
Formula weight
Temperature
C33H30N2O9
598.59
173 (2) K
Wavelength
MoKα, 71.073 pm
Crystal system
Space group
orthorhombic
C2221
Unit cell dimensions
a = 1 482.37 (7) pm α = 90 deg.
b = 2 322.16 (14) pm â = 90 deg.
c = 1 780.29 (13) pm γ = 90 deg.
Volume
6.1283 (6) nm3
Z
8
Reflections used for cell refinement
Density (calculated)
Absorption coefficient
F(000)
25
1.298 Mg/m3
0.095 mm-1
2 512
Crystal size
Theta range for data collection
Index ranges
0.3 × 0.2 × 0.2 mm
2.29–25.05 deg.
–17 ≤ h ≤ 17, –27 ≤ k ≤ 27, –21 ≤ l ≤ 21
20 046
5 425 [R(int) = 0.0561]
3 / 0 %
Reflections collected
Independent reflections
No. of standard reflections / decay
Completeness to 2Theta = 25.05
Absorption correction
Refinement method
Treatment of hydrogen atoms
Data / restraints / parameters
Goodness-of-fit on F^2
Final R indices [I > 2sigma(I)]b
R indices (all data)b
Absolute structure parameter
Extinction coefficient
Largest diff. peak and hole
Diffractometer:
99.6 %
N/a
Full-matrix least-squares on F2
mixeda
5 425 / 203 / 436
0.912
R1 = 0.0392, wR2 = 0.0835
R1 = 0.0638, wR2 = 0.0913
–0.5(9)
0.0017(2)
157 and –201 e.nm–3
ENRAF-NONIUS CAD4
SHELXS-93 [28], SHELXL-97 [29]
Program:
a See Experimental. b Definition of R values:
R1 = R Fo|–|Fc /R| and wR2 = Rw F − F 2/Rw F2 1/2; w = 1/ r2 F2
+
0.0494 P 2}; P = Max 0, F2 + 2 F2 /3.
͑ ͒ ͑ ͑ ͒ ͒
o c
2
2
2
͒
ʈ
ʈ
͑
͒
͑
͑
͒
͕
͖
͕
o
c
o
o
ing UTP may have been responsible for the effects
measured. Uridine monophosphate (UMP, 3) exhibited
only very weak activity at high concentrations of 500 µM,
which might, again, be due to contamination by UTP
and/or enzymatic conversion to UTP [18, 19].
4.2. CF/T43 and basal epithelial airway (BEA) cells
Selected compounds were further tested in CF/T43
cells [4], a human epithelial airway cell line containing
the defective chloride channel (CFTR), that causes cystic
fibrosis, and compared with a basal epithelial airway
(BEA) cell line. Results from measurements of P2Y2R-
mediated increase [Ca2+]i were virtually identical in
CF/T43 and BEA cells (tables IX and X). Activity of
nucleotides and maximal increase in [Ca2+]i was higher in
CF/T43 and BEA cells as compared to NG108-15 cells.
ATP was found to be nearly equipotent to UTP in CFT/43
and BEA cells (table IX), while ZTP was somewhat less
potent (table X). 5-Methyl-UTP (10a) showed a dose-
5-Alkyl-substituted UTP derivatives showed lower
activity at P2Y2 receptors than UTP. Activity decreased
with increasing size of the 5-substituent exhibiting the
following rank order of potency: UTP > 5-methyl-UTP >
5-ethyl-UTP > 5-isopropyl-UTP > 5-propyl-UTP >
5-butyl-UTP (table VII). A recorded dose-response curve
for 5-ethyl-UTP (10b) showed this compound to be a full
agonist at P2Y2R of NG108-15 cells exhibiting an EC50
value of 99 µM (table VIII).

818
Figure 5. Structures of identified side products in the phosphorylation of 9e (preparation of 10e).
dependent increase in [Ca2+]i and appeared to be nearly
equipotent to ATP and UTP (table X), while 5-ethyl-UTP
(10b, 10–100 nM) only slightly increased [Ca2+]i in BEA
cells (table X).
The higher potency of nucleotides in epithelial airway
cells as compared to NG108-15 cells may reflect species
differences (human, mouse). Differences in enzyme pat-
tern and enzymatic activity of nucleotidases and phos-
phatases may also contribute to the differences mea-
sured [18, 19, 21]. A further factor, which has to be taken
into account, may be differences in techniques used for
the measurements. Thus, NG108-15 cells were used in
Table VII. Effects of nucleotides on P2Y2 receptor-mediated increase in intracellular calcium concentration in NG108-15 cells, results from
testing of single concentrations of compounds.
Compounda
Percent increase in [Ca2+]i ± SEM (number (n) of independent experiments)
50 µM concentration of test compound
500 µM concentration of test compound
1
2
3
4
5
10a
UTP
UDP
UMP
ATP
ZTP
5-Methyl-UTP
69.3 ± 8.2 (n = 10)
42.4 ± 4.5 (n = 4)
n.d.c
44.6 ± 5.9 (n = 3)
54.3 ± 13.5 (n = 2)
n.d.
(179.0 ± 26.9)b (n = 5)
31.3 (n = 1)
10.6 ± 1.3 (n = 2)
67.2 ± 17.9 (n = 2)
n.d.
n.d.
52.0 ± 29.7 (n = 2) at 100 µM
28.9 ± 7.8 (n = 3)
0 (n = 2)
n.d.
n.d.
10b
10c
10d
10e
5-Ethyl-UTP
71.7 ± 12.0 (n = 3)
38.6 ± 5.3 (n = 3)
46.2 ± 25.0 (n = 2)
25.0 ± 0.7 (n = 3)
5-Propyl-UTP
5-Isopropyl-UTP
5-Butyl-UTP
a For structures see figures 1 and 2. b High values are due to lysis of cells observed at a concentration of 500 µM of UTP. c n.d. = not
determined.

819
Table VIII. Effects of nucleotides on P2Y2 receptor-mediated increase in intracellular calcium concentration in NG108-15 cells, results from
dose-response curves.
Compound
EC50 [µM]a
Maximal effect in % of max. UTP effect (= 100 %)
(95 % confidence intervals)
(number (n) of independent experiments)
1
2
4
5
UTP
UDP
ATP
ZTP
1.25 (0.11–14)
3.21 (0.28–36)
17.7 (5.6–56)
17.1b
100 ± 12 (n = 10)
61 ± 7 (n = 4)
95 ± 12 (n = 3)
78 ± 19 (n = 2)
104 ± 17 (n = 3)
10b
5-Ethyl-UTP
99 (20–480)
a Results from three independent experiments unless otherwise noted. b Single dose-effect curve.
suspension while CF/T43 and BEA cells were used as
attached cell layers in culture dishes (see Experimental
protocol). It has been shown that stressing of cells, e.g,.
by agitation, may result in a massive release of endog-
enous nucleotides [22], a fact which clearly affects the
test results obtained with exogenously applied nucle-
otides. Such variations in EC50 values for nucleotides,
e.g., UTP and ATP, at P2Y2R in different test systems
have been described [5]. In the present study, degradation
of compounds was not investigated, though enzymatic
and chemical hydrolysis undoubtedly play a role [21].
Calcium measurements were, however, performed very
fast, within seconds up to a few minutes, and therefore,
only moderate degradation will have occurred. It had
been shown that in airway epithelial cells, where strong
UTP degradation is observed, after 5 min, about 70% of
intact UTP is still present [21].
4.3. Structure-activity relationships
Table IX. Effects of standard nucleotides on P2Y2 receptor-
mediated increase in intracellular calcium concentration in CF/T43
cells and basal epithelial airway (BEA) cells, results from dose-
response curves.
The P2Y2R is unique in that it is stimulated by purine
(ATP) as well as pyrimidine nucleotides (UTP). We
confirmed previous findings [5, 20, 22, 23] that UTP (1)
and ATP (4) activate P2Y2R in low micromolar to
submicromolar concentrations (EC50 of UTP: 1.25 µM at
NG108-15 cells, 0.10 µM at CF/T43 cells, and 0.16 µM
at BEA cells; EC50 of ATP: ca. 18 µM at NG108-15 cells,
and ca. 0.2 µM at CF/T43 and BEA cells, see tables VIII
and IX). Efficacy was similar for both physiological
agonists. A ring-open analogue of ATP, ZTP (5) exhibited
similar potency as ATP indicating that purine base modi-
fication was possible without loss of activity. Truncation
Compound
Cell line
EC50 [µM]
(95 % confidence intervals)a
1 UTP
4 ATP
CF/T43cells
BEA cells
CF/T43cells
BEA cells
0.10 (0.01–0.93)
0.16 (0.005–5.6)
0.17 (0.01–2.9)
0.20b
a Determined in 3–4 separate experiments unless otherwise noted.
b Two separate experiments.
Table X. Effects of nucleotides on P2Y2 receptor-mediated increase in intracellular calcium concentration in CF/T43 cells and BEA cells,
results from testing of single concentrations of compounds
Compound
Cell line
Percent increase in [Ca2+]i ± SEM
(number of independent experiments)
10 nM concentration of test compound
100 nM concentration of test compound
1 UTP
CF/T43cells
BEA cells
CF/T43cells
BEA cells
CF/T43cells
BEA cells
CF/T43cells
BEA cells
108 ± 80 (n = 3)
110 ± 49 (n = 3)
103 ± 20 (n = 3)
199 ± 94 (n = 3)
36 ± 29 (n = 2)
80 ± 35 (n = 2)
155 ± 46 (n = 2)
113 ± 32 (n = 2)
39 ± 11 (n = 2)
17 ± 24 (n = 2)
197 ± 58 (n = 3)
173 ± 113 (n = 3)
266 ± 94 (n = 3)
208 ± 42 (n = 3)
117 ± 55 (n = 2)
138 ± 22 (n = 2)
269 ± 24 (n = 2)
312 ± 15 (n = 2)
43 ± 26 (n = 2)
41 ± 7 (n = 2)
4 ATP
5 ZTP
10a 5-Methyl-UTP
10b 5-Ethyl-UTP
CF/T43cells
BEA cells

820
of the triphosphate chain in UTP to UDP (2) and further
to UMP (3), led to a decrease or loss in activity as
reported [5, 20, 22], suggesting that the four negative
charges are important for electrostatic interactions with
the receptor protein. Only few synthetic UTP analogues
have previously been investigated as P2Y2R ligands [5, 6,
21, 22]. Exchange of the bridging â-γ-oxygen atom in the
triphosphate chain by NH, CH2, or CF2, respectively, in
order to enhance stability towards nuceotidases, have led
to a decrease in activity [23]. The only substitution
tolerated in the phosphate chain was the replacement of a
γ-oxygen atom by sulfur, as in UTPγS [21]. The com-
pound was nearly as potent as UTP itself. A series of UTP
derivatives in which the oxygen in the 4-position of the
uracil moiety was replaced by various substituents, in-
cluding (alkyl)amino, (alkyl)thio or alkoxy, had been
investigated, but all of the derivatives were less potent
than UTP [6].
A 5-substituted UTP derivative that had been investi-
gated was 5-bromo-UTP. It was found to be less potent
than UTP or ATP at P2Y2R [22]. The present study shows
that alkyl substituents in the 5-position of UTP are not
tolerated by the receptors either. Introduction of an ethyl
group in the 5-position of UTP, for example, decreased
activity at P2Y2R of NG108-15 cells ca. 80-fold (from an
EC50 for UTP of 1.25 µM to 99 µM for 5-ethyl-UTP
10b). With increasing volume of the 5-substitutent P2Y2
activity was decreased (table VII). Since both polar
5-substituents, such as bromo, and non-polar, especially
bulky alkyl substituents, led to decreased activity, it can
be concluded, that steric reasons, i.e., lacking bulk
tolerance of the receptor in that area, are responsible for
this effect.
6. Experimental protocols
6.1. Chemistry
Melting points were determined with a Büchi 530
apparatus and are uncorrected. Nuclear magnetic reso-
nance spectra were determined using a Bruker AC-200
spectrometer (1H: 200 MHz, 13C: 50.3 MHz). Chemical
shifts are given in ppm downfield from tetramethylsilane
as internal standard. 31P-NMR spectra were recorded on
a Bruker AMX 400 spectrometer at 161 MHz with H3PO4
as an internal standard. UV spectra were recorded with a
Perkin Elmer Lambda 12 spectrometer. HPLC was per-
formed using the following equipment: Pharmacia 2249
gradient pump, 2141 variable wavelength monitor set to
266 nm, and a 2221 integrator. Purity controls were
performed on two systems: system A: Nucleosil RP-18
column with eluent (A) 0.1 M triethylammonium acetate
buffer, and eluent (B) acetonitrile/H2O (gradient: 0–15%,
30 min). System B: Macherey-Nagel ET125/4 Nucleosil
4000-7 PEI column with eluent (A) 0.01 M Tris/HCl (pH
8.4) and eluent (B) 0.01 M Tris/HCl (pH 8.4), 1.0 M
NaCl. Negative ion plasma desorption mass spectra were
obtained on Applied Biosystem BIO-ION 20 plasma
desorption mass spectrometer, using 252Cf as source of
fission fragments. Nucleotides were purified on a Phar-
macia High Load system using a triethyl ammonium
hydrogen carbonate buffer gradient (0–80%). Thin layer
chromatography was performed on silica gel F254
(Merck), and in some cases on cellulose TLC plates
(Merck). The following solvent systems were used for
development of the TLC:
(S1) dichloromethane:methanol = 9:1 for compounds
8a–e;
(S2) dichloromethane:methanol = 3:1 for compounds
9a–e;
(S3) 2-propanol:NH4OH:water = 6:3:1 for compounds
10a–e, 11–15.
5. Conclusion
Methods and conditions for the preparation and puri-
fication of 5-substituted UTP derivatives were investi-
gated and optimized. New spectroscopic data on UTP
derivatives are presented, including 13C and 31P NMR
data. 5-Substituted UTP derivatives were found to be full
agonists at P2Y2 receptors of NG108-15 cells, and basal
epithelial airway cells without or with defective CFTR
channel (CF/T43 cell line). Potency of 5-substituted UTP
derivatives decreased with increasing volume of the
5-substitutent.
It is planned to investigate the potency of 5-substituted
UTP derivatives at other P2Y receptor subtypes, such as
P2Y4 or P2Y6. The presented SAR of UTP derivatives
may contribute to the design of selective ligands for
subtypes of the uracil nucleotide-sensitive P2Y receptors.
Phosphate spraying agent (0.1% FeCl3 x 6 H2O; 7%
5≠-sulfosalicylic acid in 75% ethanol) according to Wade
and Morgan [24] was used for identification of organic
and inorganic phosphates. RF-values of phosphates were
compared to data published by Ebel [25].
6.1.1. General procedure for silylation
A suspension of 5 mmol of uracil derivatives 6a–e in
20 mL of 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and
1 mL of trimethylsilyl chloride, or a few crystals of
(NH4)2SO4 respectively, was refluxed until a clear solu-
tion was obtained. The solution was allowed to cool, and
excess HMDS was removed under reduced pressure. The
silylated uracil derivatives 7a–e were kept under nitro-
gen. A small sample of the oily sirup was dissolved in

821
1
CDCl3 and checked for complete silylation by H NMR
6.1.3. Deprotection of nucleosides
spectroscopy. Yields ranged from 96–98%.
Tribenzoyl nucleoside (8a–e, 0.92 mmol) was dis-
solved in a mixture of 20 mL of absolute methanol and
2.76 mL of 5% methanolic sodium methylate solution.
The solution was stirred for 5–8 h and completion of the
reaction was determined by TLC (dichloromethane:
methanol = 3:1). Neutralisation of the solution was
achieved by adding DOWEX-50 WX-8 ion exchange
resin washed previously with methanol. After filtering the
resin off, 20 mL of water was added and methanol was
evaporated. The benzoic acid methyl ester was extracted
with diethyl ether and the water fraction was lyophilized
to yield the nucleosides 9a–e in 94–98% yield.
1
Selected H-NMR data in CDCl3, δ (ppm):
7d: 0.30 (s, 9H, C-O-Si(CH3)3); 0.32 (s, 9H, C-O-
Si(CH3)3); 1.14 (d, 6H, C5-CH-(CH3)2, J = 6.9Hz); 2.85
(sept., 1H, C5-CH-(CH3)2, J = 6.9Hz); 7.99 (s, 1H,
C6-H).
6.1.2. General procedure for the synthesis of nucleosides
Silylated uracil derivatives 7a–e (5 mmol) under nitro-
gen were dissolved in 5 mL of 1,2-dichloroethane, and
1.30 g (5.5 mmol) of SnCl4 (10% excess) was added with
vigorous stirring. 1-O-Acetyl-2,3,5-tri-O-benzoyl-â-D-
ribofuranose (2.2 g, 0.4 mmol) in 40 mL of 1,2-
dichloroethane was added dropwise to the slightly yel-
lowish solution. The mixture was stirred as indicated
below, completion of reaction was controlled by TLC
(dichloromethane:methanol = 9:1). Reaction times were
between 4–5 h. The reaction solution was poured into a
saturated aqueous NaHCO3 solution (ca. 50 mL), or on
humidified solid NaHCO3 with successive adding of
saturated NaHCO3 solution, respectively, under vigorous
stirring, and then allowed to stand overnight. The suspen-
sion was filtered over silica gel and the gel was washed
twice with 50 mL of dichloromethane and twice with
100 mL of ethyl acetate. The organic phase was sepa-
rated, dried (Na2SO4), filtered over silica gel and evapo-
rated to dryness. The glassy residue was crystallized from
methanol, or ethanol respectively, to yield 8a–e. If
necessary, the removal of unreacted sugar and side
products was achieved by column chromatography on
silica gel using dichloromethane:methanol = 9:1 as elu-
ent. Yields ranged from 81–87%.
1
Selected H-NMR data in DMSO-d6, δ (ppm):
5-n-Propyluridine (9c): 0.84 (t, 3H, C5-CH2-CH2-CH3,
J = 7.3Hz); 1.42 (sext., 2H, C5-CH2-CH2-CH3, J = 7.5
Hz); 2.14 (dt, C5-CH2-CH2-CH3, J1≠2≠ = 7.5 Hz);
3.49–3.68 (m, 2H, C5≠H2); 3.82 (q, 1H, C4≠H, J =
3.46/2.94Hz); 3.96–4.03 (m, 2H, C2≠H, C3≠H); 5.06,
5.33 (br s, 3H, OH (exchangeable)); 5.77 (d, 1H, C1≠H,
J = 5.3 Hz); 7.73 (s, 1H, C6H); 11.24 (br s, 1H, N3-H
(exchangeable)).
6.1.4. Phosphorylation
Synthesis of nucleoside 5≠-triphosphate synthesis was
adapted from literature procedures [10, 26, 27].
6.1.5. Preparation of tri-n-butylammonium diphosphate
Sodium diphosphate decahydrate (6.69 g; 15 mmol)
was dissolved in 150 mL of water (twice distilled).
Excess of Dowex ion exchange resin 50 × 8, 20–50 mesh,
proton form, prewashed several times with water, was
added to the solution of sodium diphosphate, and the
mixture was gently stirred for 60 min. A mixture of
60 mL of ethanol and 7.14 mL of tributylamine in a flask
was placed in an ice-water bath, and the diphosphate
solution was filtered directly into the flask. The resin was
repeatedly washed with water until the filtrate was no
longer acidic. The solvent was then evaporated under
vacuum at 40 °C, yielding a thick, nearly colourless
syrup. This residue was treated twice with 100 mL of
ethanol and then evaporated. The residue was taken up in
40 mL of dimethylformamide (DMF, anhydrous grade,
from Fluka) and evaporated again. This residue was taken
up in 30 mL of anhydrous DMF, yielding 30 mL of 0.5 M
tri-n-butylammonium diphosphate in DMF. The solution
was stored sealed and cooled at 4 °C until used.
1
Selected H-NMR data in DMSO-d6, δ (ppm):
8b: 0.93 (t, 3H, C5-CH2-CH3, J = 7.3 Hz); 2.08 (q, 2H,
CH3, J = 7.3 Hz); 4.64–4.77 (m, 3H, C4≠H, C5≠H2); 5.96
(m, 2 H, C2≠H, C3≠H); 6.21 (d, 1H, C1≠H, J1≠2≠ = 3.8
Hz); 7.39–8.04 (m, 16H, C6-H, Haromat.); 11.48 (br s, 1H,
N1-H, exchangeable).
8c: 0.76 (t, 3H, propyl CH3, J = 7.1 Hz); 1.33 (sext.,
2H, propyl CH3CH2-, J = 7.3Hz); 2.05 (t, 2H, propyl
CH3CH2CH2-, J = 7.2 Hz); 4.61–4.78 (m, 3H, C4≠H,
C5≠H2); 5.90 (m, 2H, C2≠-H, C3≠-H); 6.23 (d, 1H,
C1≠-H, J = 3.7 Hz); 7.38–8.06 (m, 16H, C6H, Haromat);
11.48 (br s, 1H, N3-H, exchangeable).
8d: 1.00 (d, 6H, CH(CH3)2, J = 6.9Hz); 2.85 (sept., 1H,
CH(CH3)2, J = 6.6Hz); 4.65–4.75 (m, 3H, C4≠H,
C5≠H2); 6.00 (d, 2H, C3≠H, C2≠H); 6.25 (d, 1H, C1≠H,
J = 5.1Hz); 7.01 (d, 1H, C6H, J = 0.9Hz); 7.38–8.05 (m,
16H, C6H, Haromat); 11.47 (br s, 1H, N3-H).
6.1.6. Preparation of triethylammoniumhydrogen-
carbonate (TEAB) buffer
A 1 M solution of TEAB was prepared by bubbling
CO2 through a 1 M triethylamine solution in water at
0–4 °C for several hours (pH approx. 7.4–7.6).

822
6.1.7. Preparation of nucleotides 10a–e
6.2. Determination of X-ray structure
Lyophilized nucleoside (9a–e, 1 mmol) was dissolved
in 5 mL of trimethyl phosphate (dried over 10 Å molecu-
lar sieve). The mixture was stirred at room temperature
under nitrogen and then cooled to 4 °C. Dry 1,8-
bis(dimethylamino)naphtalene (‘proton sponge’, 0.32 g,
1.5 mmol) was added, followed by 1.3 mmol (0.20 g) of
POCl3, 5 min later. After several hours of stirring at
0–4 °C, tri-n-butylamine (0.1 mL, 0.72 mmol) was added
to the solution followed by 10 mL (5 mmol) of 0.5 M
tri-n-butylammonium diphosphate solution in DMF. After
2–5 min the mixture was poured into 0.5 M cold aqueous
TEAB solution (30 mL, pH 7.5) and stirred at 0–4 °C for
several minutes.
The solutions were allowed to reach room temperature
with stirring and were then left standing for 1 h. Trim-
ethylphosphate was extracted with t-butylmethyl ether
and the aqueous solutions were evaporated and lyo-
philized to yield glassy colourless oils. The reactions
were controlled by TLC using freshly prepared solvent
system S3. TLC plates were dried before UV absorption
was detected and the plates were subsequently sprayed
with phosphate reagent.
Single pure crystals of 8d were obtained by slow
crystallization from ethanol at room temperature over
several weeks. Low temperature X-ray intensity data
were collected on an Enraf-Nonius CAD4 diffractometer
using MoKα radiation. The structure was resolved with
direct methods using SHELXS-97 [28] and refined by
full-matrix least squares iteration against F2, using
SHELXL-97 [29]. All non-hydrogen atoms were refined
anisotropically. The coordinates of hydrogen atoms
bonded to C5, C6, C7, and C8 were freely refined using
isotropic displacement parameters. For all other hydrogen
atoms a riding model was employed in the refinement
with their Uiso constrained to equal 1.2 times the Ueq of
the parent atoms (1.5 times Ueq in the case of the
hydrogen atoms of the methyl groups). Crystallographic
data were deposited at the Cambridge Crystallographic
Data Centre as supplementary publication (deposition
number 120084). Free copies of data are available from:
CCDC; 12 Union Road, Cambridge CB21EZ (fax:
+ 44 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk).
6.3. Biological studies
ATP, UTP, UDP, UMP, and ZTP were purchased from
Sigma Chemical Co. in the highest available purity grade.
Nucleotides were checked for autofluorescence prior to
testing.
6.1.8. Purification of nucleotides
The crude nucleoside 5≠-triphosphates were purified
by ion exchange column chromatography using DEAE-
–
Sephadex A25 (Pharmacia) HCO3 -form swelled in 1 M
NH4HCO3 solution at 4 °C. After equilibrating the col-
umn with deionized water, the crude product was dis-
solved in 5 mM aqueous triethylammonium carbonate
buffer. The column was washed with deionized water,
followed by a solvent gradient of 0–800 mM TEAB in
approximatly 3 000 mL of solvent to elute the triphos-
phates. Fractions were collected and appropriate fractions
pooled, evaporated, diluted in water and lyophilized to
yield a product with a ‘clear glass’ appearance. Purity
was assessed by HPLC/UV and 31P NMR spectroscopy.
Isolated yields: 33% (10a), 24% (10b), 37% (10c), 40%
(10dd), 26% (10e).
6.3.1. NG108-15 cell culture
NG108-15 cells were kindly provided by Berit Färjh,
Dept. of Medical Neurochemistry, Lund University Hospi-
tal, Sweden. NG108-15 cell line (hybrid cell line generated
by fusion of mouse neuroblastoma and C6 glioma cells)
was cultured in tissue culture flasks (75 or 150 cm2). Cells
were grown adherent in tissue culture flasks in a 5% CO2
atmosphere at 37 °C until confluent. The growth medium
consisted of a mixture of 500 mL of Dulbecco’s modified
Eagle medium (DMEM) supplemented with 4.5 g/L of
glucose and pyruvate, 55 mL of foetal calf serum, 10 mL of
HAT (hypoxanthine, aminopterin, thymidine) supplement
(Gibco article No. 15140-114) and 5.5 mL of penicillin/
streptomycin (Gibco article no. 21060-017). Medium was
changed nearly every day depending on cell density as
determined by visual inspection.
6.1.9. Preparation of sodium salts
Nucleotides (50 mmol) were dissolved in absolute
methanol (dried over Mg) and evaporated. This procedure
was repeated twice. The solid was then dissolved in 5 mL
of absolute methanol with stirring. A 1.5-fold excess of a
1 M sodium iodide solution in acetone was added
dropwise. The solution was then diluted with absolute
acetone. The formed precipitate was filtered off and
washed several times with absolute acetone. The white
solid was dried under high vacuum.
6.3.2. Preparation of fura2-loaded cells for measure-
ments
NG108–15 cells were examined under a microscope
and cells were used when confluent. The medium was
removed from the flask, growth medium (10 mL at 37 °C)
was added and the cells were dispersed into a single cell

823
suspension by gentle, brief, repeated pipetting. The cells
were centrifuged and the pellet was resuspended in
Krebs-Ringer-Hepes (KRH) buffer (conc. in mM: NaCl
125; KCl 5.0; MgSO4 1.2; KH2PO4 1.2; CaCl2 2.0;
glucose 6.0 Hepes 25; pH adjusted to 7.4, with NaOH) in
a concentration of 106 cells per mL. The cells were
incubated with 2 µM of fura-2/AM for 45 min at 37 °C in
the dark, spun down (at 1 000 g) and washed twice with
the same amount of KRH buffer as used before.
compound was rapidly added with careful shaking, cul-
ture dishes were re-inserted within 10 s, and changes in
fluorescence were registered until base line levels were
reached again (3–10 min). Then, Ca2+ -ionophore iono-
mycin (10 µM) and subsequently MnCl2 (20 mM) were
added in order to determine maximum and minimum
values of fura-2 fluorescence. Calculations of [Ca2+]i
were made according to Grynkiewicz et al. [31].
6.3.3. Measurement of [Ca2+]i using NG108-15 cells in
suspension
Acknowledgements
[Ca2+]i was determined as described by Lin et al. [30]
measuring fluorescence of fura-2 loaded cells (2 mL of
cell suspension containing 106 cells per experiment,
37 °C) using a Hitachi Model F-2000 dual wavelength
fluorescence spectrophotometer. Test compound (10 µL)
was added. The additon of P2Y2R agonists resulted in a
transient increase in intracellular calcium concentration.
After baseline was reached again, 10 µL of Triton X 100
(10% aqueous solution) was added, followed by the
addition of 10 µL of a saturated solution of EGTA and
10 µL of a saturated solution of Tris base. [Ca2+]i was
calculated as described [31].
We thank Gaby Huhurez for skillful technical assis-
tance. C.E. Müller is grateful for support given by the
Fonds der Chemischen Industrie and the Industrie- und
Handelskammer Würzburg-Schweinfurt. This project
was supported by grants from the Deutscher Akademis-
cher Austauschdienst (DAAD) to B.K. and C.E.M. and
from the Svenska Institutet to E.H.
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