Selective nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) inhibitors: Nucleotide mimetics derived from uridine-5′-carboxamide

Article abstract of DOI:10.1021/jm800175e

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, subtypes 1, 2, 3, 8 of NTPDases) dephosphorylate nucleoside tri- and diphosphates to the corresponding di- and monophosphates. In the present study we synthesized adenine and uracil nucleotide

Full text of DOI:10.1021/jm800175e

4518
J. Med. Chem. 2008, 51, 4518–4528
Selective Nucleoside Triphosphate Diphosphohydrolase-2 (NTPDase2) Inhibitors: Nucleotide
Mimetics Derived from Uridine-5′-carboxamide†
Andreas Brunschweiger,§ Jamshed Iqbal,§ Frank Umbach,§ Anja B. Scheiff,§ Mercedes N. Munkonda,# Jean Se´vigny,#
Aileen F. Knowles,‡ and Christa E Mu¨ller*,§
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, Pharmaceutical Sciences Bonn (PSB), UniVersity of Bonn, An der
Immenburg 4, D-53121 Bonn, Germany, Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier UniVersitaire de Que´bec,
UniVersite´ LaVal, Que´bec, QC, Canada, and Department of Chemistry and Biochemistry, San Diego State UniVersity,
San Diego, California 92182-1030
ReceiVed February 19, 2008
Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, subtypes 1, 2, 3, 8 of NTPDases)
dephosphorylate nucleoside tri- and diphosphates to the corresponding di- and monophosphates. In the present
study we synthesized adenine and uracil nucleotide mimetics, in which the phosphate residues were replaced
by phosphonic acid esters attached to the nucleoside at the 5′-position by amide linkers. Among the synthesized
uridine derivatives, we identified the first potent and selective inhibitors of human NTPDase2. The most
potent compound was 19a (PSB-6426), which was a competitive inhibitor of NTPDase2 exhibiting a Ki
value of 8.2 µM and selectivity versus other NTPDases. It was inactive toward uracil nucleotide-activated
P2Y2, P2Y4, and P2Y6 receptor subtypes. Compound 19a was chemically and metabolically highly stable.
In contrast to the few known (unselective) NTPDase inhibitors, 19a is an uncharged molecule and may be
perorally bioavailable. NTPDase2 inhibitors have potential as novel cardioprotective drugs for the treatment
of stroke and for cancer therapy.
Introduction
NTPDase1 and 2, showing an about 2- to 3-fold preference for
nucleoside triphosphates.
Extracellular nucleotides, such as ATP, ADP, UTP, and UDP
act as extracellular signaling molecules by activating purine and/
or pyrimidine P2 receptors.1,2 The activation of these receptors
is controlled by various ecto-enzymes that dephosphorylate
nucleotides.3–6 An important family of specific nucleoside tri-
and diphosphate metabolizing enzymes is the ecto-nucleoside
triphosphate diphosphohydrolase (E-NTPDase), whose members
haveformerlyalsobeenknownasecto-ATPasesorecto-apyrases.3,4,7
By their membrane topology and specific catalytic properties,
four of the eight members of the NTPDase family appear to be
responsible for most of the hydrolysis of nucleotides at the cell
surface, namely, NTPDase1 (CD39), NTPDase2 (CD39L1),
NTPDase3 (CD39L3) and NTPDase8.4 These NTPDase mem-
bers are cell surface proteins anchored in the plasma membrane
with their active site orientated toward the extracellular space,
while the remaining members (NTPDases 4, 5, 6, and 7) are
mostly intracellular enzymes. NTPDase5 and 6 may be secreted
into the extracellular space as soluble enzymes.3 NTPDases
differentially catalyze the sequential hydrolysis of the ꢀ- and
γ-phosphate of adenosine and uridine nucleoside di- and
triphosphates,8 the physiological ligands of P2 receptors:
NTPDase1 hydrolyzes nucleoside tri- and diphosphates similarly
well, yielding the monophosphates (AMP, UMP) as products,
while NTPDase2 has a clear preference for nucleoside triph-
osphates and therefore yields ADP and UDP as the main
products. NTPDase3 and 8 are functional intermediates between
The isolated action of NTPDase2 provides P2Y1, P2Y12,
P2Y13 (ADP), and P2Y6 (UDP) receptor agonists9,10 while
terminating the activation of P2X receptors (ATP), as well as
P2Y2 (UTP, ATP), P2Y4 (UTP), and P2Y11 (ATP) receptors.11
NTPDase1 is the major physiological NTPDase member show-
ing the broadest distribution and the highest expression levels
in most tissues.4 The distribution of NTPDase2 appears to be
more restricted. In the vasculature it is expressed in the
adventitia of arteries and on subendothelial cells of veins.10
Upon vessel injury, large amounts of ATP are secreted in the
extracellular environment which, when exposed to NTPDase2,
are efficiently converted to platelet-activating ADP which
activates P2Y1 and P2Y12 receptors on thrombocytes.10 In
addition, ADP potently inhibits ecto-5′-nucleotidase,12–14 thereby
preventing the extracellular formation of adenosine and imped-
ing adenosine receptor-effected vasodilation and antithrombotic
properties.15–17 El Omar et al. observed that patients with
a Abbreviations: Boc, tert-butyloxycarbonyl; 8-Bu-S-ATP, 8-thiobutyl-
adenosine 5′-triphosphate; CE, capillary electrophoresis; CDCl3, deuterated
chloroform; CNS, central nervous system; DIPEA, diisopropylethylamine;
DMEM, Dulbecco’s modified Eagle medium; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; DMSO-d6, deuterated dimethyl sulfoxide; D2O,
deuterated water; DPBS, Dulbecco’s phosphate-buffered saline; FBS, fetal
bovine serum; EI, electron impact; ESI, electrospray ionization; HBTU,
2-(1H-benzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HCTU,
2-(6-chloro-1H-benzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophos-
phate; HEPES, N-[2-hydroxyethyl]piperazine-N′-2-ethansulfonic acid; HPLC,
high performance liquid chromatography; HRMS, high resolution mass
spectrometry; LC-MS, liquid chromatography-mass spectrometry; LOD,
limit of detection; MeOD-d4, deuterated methanol; MeOH, methanol; NMR,
nuclear magnetic resonance; NPP, nucleoside pyrophosphatase; (E)-
NTPDase, (ecto)-nucleoside 5′-triphosphate diphosphohydrolase; PMSF,
phenylmethanesulfonyl fluoride; POM, polyoxometalate; PPADS, pyridox-
alphosphate-6-azophenyl-2′,4′-disulfonic acid; PyBOP, benzotriazol-1-yl-
oxytrispyrrolidinophosphonium hexafluorophosphate; RB-2, reactive blue
2; SAR(s), structure-activity relationship(s); S/N, signal-to-noise; TEMPO,
2,2,6,6-tetramethyl-1-piperidinyl oxide; THF, tetrahydrofurane; TLC, thin
layer chromatography.
†
Preliminary results were presented at the 8th International Symposium
on Adenosine and Adenine Nucleotides in Ferrara, 2006 (Abstract published
in Purinergic Signalling 2006, 2, 170).
* To whom correspondence should be addressed. Phone: +49-228-73-
2301. Fax: +49-228-73-2567. E-mail: christa.mueller@unibonn.de.
§
University of Bonn.
Universite´ Laval.
San Diego State University.
#
‡
10.1021/jm800175e CCC: $40.75
2008 American Chemical Society
Published on Web 07/17/2008

NTPDase2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4519
Figure 1. Structures of standard ectonucleotidase inhibitors.
coronary artery disease had an altered ATP/ADP ratio of
hydrolysis in favor of ATP hydrolysis.18 The ratio of ADP to
ATP hydrolysis appears to play a vital role in the control of
hemostasis, since even a small alteration of the balance may
lead to higher platelet reactivity. A selective inhibitor for
NTPDase2 may therefore exhibit cardioprotective properties.
In the central nervous system (CNSa) NTPDase2 is found
on astrocytes.19 During cerebral ischemia, extracellular ATP
concentrations rise. ATP is first dephosphorylated to ADP by
astrocytic NTPDase2 and then dephosphorylated to AMP by
other ectonucleotidases. The transiently accumulating ADP, a
potent inhibitor of ecto-5′-nucleotidase,12 may delay further
dephosphorylation of AMP to cytoprotective adenosine by ecto-
5′-nucleotidase. Furthermore, activation of P2Y2 receptors by
ATP may also have protective effects in glial cells.20 Thus,
NTPDase2 inhibition may be useful for the treatment of
ischemic conditions in brain, e.g., for the treatment of stroke.
Recently, Zimmermann and colleagues identified NTPDase2 as
a novel marker for certain progenitor cells in the adult and
developing mouse brain, indicating a role in neurogenesis.21
High NTPDase2 expression was also found in human hepato-
ma22 and Walker 256 tumor cells23 and may be implicated in
the stimulation of cancer progression.
ine 5′-triphosphate (8-Bu-S-ATP, 2),26 and some polyoxomet-
alates (POMs)31 have been shown to exhibit enzyme inhibitory
potential without significantly affecting nucleotide receptors.
However, these compounds are highly polar and cannot be
perorally bioavailable. Furthermore, we have recently found that
the frequently applied ARL67156 is a weak competitive inhibitor
of NPP1 and, NTPDase1 and 3 and does not inhibit NPP3,
NTPDase2, or NTPDase8 very well.30,37 Thus far, no selective
inhibitors for NTPDase2 have been described.
The present study was aimed at developing selective NTPD-
ase inhibitors, which should not exhibit affinity for P2 receptors
and, in contrast to phosphoric acid ester derivatives, were not
to be dephosphorylated by ecto-nucleotidases or phosphatases.
Furthermore, our goal was to obtain neutral molecules (not
negatively charged at physiologic pH value of 7.4) in order to
allow for peroral application. Our target structures were nucle-
otide mimetics 19-23 derived from ATP and UTP, the
substrates of NTPDases, in which the triphosphate group was
replaced by a phosphonic acid diester residue (I-III) linked
via amide bonds and a linker group to the nucleoside core
(Figure 2).
Syntheses
NTPDase2 inhibitors could therefore modulate P1 and P2
receptor activation in a site- and event-specific manner. From
a medicinal chemistry point of view this concept might be
advantageous in comparison with the development of direct P2
receptor agonists because it has turned out to be difficult to
develop metabolically stable and selective P2 agonists and it
has been impossible to design uncharged, neutral molecules that
can activate P2 receptors.24
So far, only a few NTPDase inhibitors have been described
(Figure 1).25–32 However, reactive blue 2 (3), pyridoxalphos-
phate-6-azophenyl-2′,4′-disulfonic acid (PPADS, 4), and suramin
(5) are also antagonists of many P2 receptors.32–35 Only the
ATP analogue ARL67156 (FPL67156, N6-diethyl-ꢀ,γ-dibro-
momethylene-ATP, 1),36 the ATP derivative 8-thiobutyladenos-
For the preparation of the target compounds 19-23 (Figure
2), a convergent synthetic strategy was applied (see Schemes
1–3). The commercially available nucleosides adenosine (6a)
and uridine (6b) were protected at the 2′- and 3′-hydroxyl groups
(7a,b), and the 5′-hydroxyl group was subsequently oxidized
to the corresponding 5′-carboxylic acid function (8a,b) (Scheme
1). Coupling of the nucleoside-5′-carboxylic acids 8a,b with
amino-substituted phosphonic acid diethyl ester derivatives
16-18 (Scheme 2) followed by deprotection of the ribose
moiety yielded the final products 19-23 (Scheme 3).
Selective derivatization of nucleosides in the 5′-position
usually requires protection of the 2′- and 3′-hydroxyl groups.
Frequently, the nucleosides are treated with acetone to yield

4520 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Brunschweiger et al.
(Boc-) protected amino acids (see Scheme 2). For the condensa-
tion reaction, Wieland’s mixed anhydride method was employed
using isobutyl chloroformate as an activating agent and N-
methylmorpholine to deprotonate the carboxylic acid. The
activation of the carboxylic acid was performed in dry tetrahy-
drofurane.42 The commercially available Boc-protected amino
acids glycine (9a), ꢀ-alanine (9b), and γ-aminobutyric acid (9c)
were thus coupled to p-aminobenzylphosphonic acid diethyl
ester (10), aminomethylphosphonic acid diethyl ester (11), or
2-aminoethylphosphonic acid ester (12), respectively, yielding
amides 13-15. Deprotection with hydrogen chloride in dry
dioxane yielded products 16-18 (Scheme 2). Yields ranged
between 60% and 90%, calculated over the two steps of amide
coupling and Boc-deprotection.
Figure 2. Target structures: adenine and uracil nucleotide mimetics.
The crucial step in the synthetic pathway was the formation
of the nucleoside 5′-carboxylic acid amides (see Scheme 3).
The carboxylic acid functions of nucleoside 5′-carboxylic acids
are sterically hindered and therefore possess a very different
reactivity than, for example, the carboxylic acid functions of
amino acids. Initial attempts to react uridine-5′-carboxylic acid
8b with compounds 16a-c using standard coupling reagents,
including carbodiimides (1-ethyl-3-(3′-dimethylaminopropyl)-
carbodiimide hydrochloride, dicyclohexylcarbodiimide), or isobu-
tyl chloroformate,42 were unsuccessful. Diphenylphosphoryl-
azide, previously applied in the synthesis of uridine-5′-carboxylic
acid amides, resulted in low yields.43 Therefore, we examined
a series of coupling reagents based on 1-hydroxybenzotriazole,
which have been described in the literature as powerful tools
for amide synthesis.44 Among these, benzotriazol-1-yl-oxy-
trispyrrolidinophosphonium hexafluorophosphate (PyBOP) had
been successfully applied to the preparation of adenosine-5′
-carboxamides.45,46 In addition to PyBOP, we also used 2-(1H-
benzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HBTU) and 2-(6-chloro-1H-benzotriazolyl)-1,1,3,3-tetramethy-
luronium hexafluorophosphate (HCTU). The three reagents gave
very different results depending on the starting compounds that
were to be coupled. The best coupling reagent for each reaction
was different mainly depending on the chain length of the
alkylamine moiety; it did not depend on the nature of the
nucleoside-5′-carboxylic acid (adenine or uracil derivative). If
the alkyl chain consisted of only one methylene unit (n ) 1:
16a, 17a, 18a), HCTU turned out to be the coupling reagent of
choice. Longer alkyl chains consisting of two or three methylene
groups (16b,c; 17b,c) required the use of HBTU or PyBOP (see
Experimental Section). The amide formation had to be per-
formed in the presence of a base. Three commonly used bases
were compared for the synthesis of 19a and 19c: triethylamine,
N-methylmorpholine, and diisopropylethylamine (DIPEA). Of
these, only diisopropylethylamine resulted in satisfactory yields.
The amide coupling reactions involving PyBOP were previously
reported to proceed within 15 min;45 however, we found that
stirring overnight gave the highest yields. The order of the
addition of starting compounds and reagents to the solvent was
important for the success of the reaction; the uridine-derived
carboxylic acid 8b was codissolved with the appropriate
coupling reagent and hydroxybenzotriazole in dimethylforma-
mide, followed by the addition of diisopropylamine, which led
to the activation of the carboxylic acid. Then the appropriate
amine derivative was added. In contrast, the adenosine-derived
carboxylic acid 8a had to be dissolved separately in a mixture
of dimethylformamide and diisopropylamine, and a solution of
the appropriate coupling reagent and hydroxybenzotriazole in
dimethylformamide was subsequently added for activation,
followed by the appropriate amine derivative (Scheme 3).
Scheme 1. Synthesis of 2′,3′-Protected Nucleoside-5′-Carboxylic
Acidsa
a Reagents and conditions: (a) p-methoxybenzaldehyde, ZnCl2, dry THF,
room temp, 48 h; (b) TEMPO, bis(acetoxy)iodobenzene, H2O/MeCN )
1:1, room temp, 3 h.
the 2′,3′-acetonides.38 In our hands, however, the hydrolytic
cleavage of the acetonide group required concentrations of at
least 50% of trifluoroacetic acid in water, which resulted in
substantial degradation of the target compounds. We obtained
a complex mixture of products, presumably because of hydroly-
sis of acid-sensitive phosphonic acid ester and nucleosidic bonds,
especially in the case of the uridine derivatives. As an
alternative, a protecting group that would be cleavable by
catalytic hydrogenation such as a benzylidene group was
considered. However, hydrogenolysis of this protective group
with 10% Pd/C requires long reaction times. Johnson and
Widlanski observed hydrogenation of the 5,6-double bond of
the uracil ring under these conditions as an undesired major
side reaction.39,40 Hydrogenolysis might also not be compatible
with the benzylphosphonate moiety III (Figure 2). Therefore,
we selected an alternative protecting group and reacted the
nucleosides with p-methoxybenzaldehyde, yielding the acetal-
protected compounds 7a,b in high yields (see Scheme 1).38 The
p-methoxybenzylidene protecting group could be easily cleaved
off later on under very mild conditions using 5% trifluoroacetic
acid in dichloromethane. Under these conditions, no degradation
of the nucleosides was observed (see Scheme 3).
Subsequent oxidation of the 5′-hydroxymethylene group of
7a,b to carboxylic acids 8a,b was performed in analogy to a
published procedure using catalytic amounts of 2,2,6,6-tetra-
methyl-1-piperidinyl oxide (TEMPO) and stoichiometric amounts
of bis(acetoxy)iodobenzene (Scheme 1).41 The 2′,3′-p-meth-
oxybenzylidene protecting group turned out to be fully compat-
ible with the reaction conditions, although it is somewhat bulkier
and less stable than the 2′,3′-isopropylidene protecting group
employed in the literature procedure.41 High yields of products
8a (72%) and 8b (78%) were obtained.
The triphosphate mimetic moieties 16-18 were synthesized
by connecting amino-functionalized phosphonic acid diethyl
ester derivatives via an amide bond with tert-butyloxycarbonyl-

NTPDase2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4521
Scheme 2. Synthesis of ω-Aminoalkylcarboxamidoalkyl- and ω-Aminoalkylcarboxamido-p-benzyl-Substituted Phosphonic Acid Diethyl
Estersa
a Reagents and conditions: (a) dry THF, N-methylmorpholine, ClCO2CH2CH(CH3)2, -25 °C to room temp, 3 h; (b) 4 N HCl in dry dioxane, room temp,
2h.
Scheme 3. Synthesis of Nucleoside-5′-Carboxamides and Deprotection of the 2′,3′-Positiona
a Reagents and conditions: (a) (i) coupling reagent (PyBOP, HCTU, or HBTU; see Experimental Section), diisopropylethylamine, DMF, room temp, 12 h,
(ii) silica gel flash chromatography, CH2Cl2/MeOH ) 40:1; (b) (i) 3-5% TFA, CH2Cl2, H2O, 2 h, room temp, (ii) C-18 RP-HPLC, gradient of MeOH/H2O
) 25:75 to 100:0.
After deprotection of the ribose moiety using 5% trifluoro-
acetic acid in dichloromethane, the final products 19-23 were
purified by reversed phase HPLC on a C-18 column and
subsequently dried by lyophilization (see Scheme 3). Final
products were isolated in yields ranging between 30% and 70%,
calculated over the two steps of amide coupling and deprotection
of the 2′,3′-position.
treatment. Initial screening was performed at a concentration
of 1 mM of test compounds. For active compounds, full
concentration-inhibition curves were determined. For com-
pound 19a, the type of NTPDase inhibition was investigated
by determining enzyme kinetics in the presence of different
concentrations of inhibitor, and a competitive mechanism of
inhibition was found for NTPDase2. Ki values were calculated
from IC50 values using the Cheng-Prusoff equation and the
following Km values for the substrate ATP: NTPDase1, 17 µM;
NTPDase2, 70 µM; NTPDase3, 75 µM; NTPDase8, 81 µM.8,47
In order to investigate the selectivity of the compounds on
NTPDases versus P2 receptors, the effects of these compounds
at the uracil nucleotide-activated P2 receptor subtypes P2Y2,
P2Y4, and P2Y6 were also investigated. These subtypes were
selected because the most potent NTPDase inhibitors of the
present series were uracil rather than adenine nucleotide
mimetics. Astrocytoma cell lines expressing the human P2Y2,
the human P2Y4, or the rat P2Y6 receptor were used.48 Inhibition
of agonist- (UTP for P2Y2, P2Y4; UDP for P2Y6) induced
stimulation of intracellular calcium release of the phospholipase
C coupled receptors was measured using the calcium fluorophore
Fura-2 applied as its acetoxymethyl ester (Fura-2 AM).49–51 In
Biological Activity
The nucleotide mimetics were investigated as inhibitors of
the four human NTPDase isoenzymes, NTPDases 1, 2, 3, 8.
NTPDases were expressed in COS-7 cells or HEK 293 cells,
and enzyme-containing membrane preparations were used. The
assays were performed in reaction vials in the presence of ATP
(400 µM) as a substrate. Product formation (AMP or ADP) was
subsequently analyzed by capillary electrophoresis (CE) with
UV detection.30 The recent development of easy and powerful
CE-based nanoscale NTPDase inhibition assays in our group30
was crucial for the current identification and characterization
of NTPDase2 inhibitors. It allowed high-throughput screening
due to the direct and fast separation of the substrates from the
reaction products within a few minutes without sample pre-

4522 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Brunschweiger et al.
Table 1. Inhibitory Potency of Synthesized Nucleotide Mimetics at Human NTPDases
Ki ( SEM [µM] (or inhibition at 1 mM)
compd
R
n
NTPDase1
NTPDase2
NTPDase3
NTPDase8
19a PSB-6426
19b
19c
20a
20b
20c
21a
22a
22b
22c
23a
23b
23c
suramin
RB2
PPADS
ARL67156
I
I
I
II
II
II
III
I
I
I
II
II
II
1
2
3
1
2
3
1
1
2
3
1
2
3
(50%)
55.2 ( 2.6
182 ( 24.3
786 ( 33
(0%)
(45%)
(0%)
(0%)
8.2 ( 2.1
173 ( 17.3
210 ( 25.3
71.7 ( 13.5
167 ( 21.3
29.2 ( 2.7
116 ( 24.3
(0%)
(0%)
(40%)
(48%)
(0%)
(0%)
(0%)
(22%)
(0%)
(0%)
(0%)
(25%)
(0%)
(0%)
12.7a
1.10a
3.0a
(0%)
(0%)
242 ( 39.3
(0%)
(0%)
(0%)
(0%)
(0%)
154 ( 37
161 ( 24
(0%)
1440 ( 64
213 ( 34
(15%)
(0%)
255 ( 25.3
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
300a
65.4a
>100b
>100b
ndc
20.0a
24.2a
46.0a
44.2a
27.0a
(50%)a
112.1a
>100d
a Inhibition values measured at rat enzymes.30 b Inhibition measured at human enzyme.32 c nd ) not determined. d Inhibiton measured at human enzyme.37
this test system, test compounds with antagonistic as well as
those with agonistic activity at the P2Y receptor subtypes can
be detected, since preincubation with either agonists or antago-
nists will lead to an inhibition of ligand-induced receptor
activation. Antagonists will block the receptors, while prein-
cubation with agonists will lead to receptor desensitization.52
We have shown agonist-induced desensitization for all inves-
tigated receptors under the applied conditions (unpublished data).
Selected compounds were also investigated directly for poten-
tially agonistic activity.
NTPDase3 and 8. The inhibitory potency was strongly depend-
ent on the spacer length. The benzylphosphonates of the uracil
series showed decreasing inhibitory potency with increasing
spacer length, especially with NTPDase2. The most potent
compound of the present series was 19a, a benzylphosphonate
with a small methylene spacer, showing a Ki value for
NTPDase2 of 8.2 µM. The compound is selective for NTPDase2
compared to NTPDase1, NTPDase3, and NTPDase8. The
homologues with a longer spacer were 21-fold (ethylene, 19b)
and 26-fold (propylene, 19c) less potent than 19a with NTPD-
ase2. A comparison of 19a with analogues that contain the same
spacer length but different phosphonate groups shows that the
benzylphosphonate is superior to methylenephosphonate (20a)
or ethylenephosphonate (21a) residues. Both analogues are
9-fold (20a) or 14-fold (21a) less potent at inhibiting NTPDase2.
However, when the larger spacer (propylene) was combined with
the smaller methylenephosphonate moiety (compound 20c),
potency at NTPDase2 was enhanced (Ki ) 29.2 µM). These
results indicate that the distance between the terminal phos-
phonate moiety and the nucleoside plays an important role.
Selected dose-response curves of potent inhibitors are shown
in Figure 3. Compound 19a, the most potent inhibitor of the
present series with a Ki value of 8.2 µM at NTPDase2 was
further investigated with regard to the mechanism of enzyme
inhibition. Since the compounds are substrate analogues, it is
likely that they bind competitively to the nucleotide binding
site in the NTPDases. Since they do not have phosphate ester
bonds, they cannot be cleaved by the enzymes in contrast to
the natural substrates. The kinetics of NTPDase2 were deter-
mined in the absence and in the prescence of 19a (5 and 10
µM). The Vmax was not affected by different concentrations of
inhibitor, while the Km value increased with increasing inhibitor
concentration, indicating a competitive mechanism of inhibition.
The resulting Lineweaver-Burk plot is shown in Figure 4.
Structure-Activity Relationships
NTPDase Inhibition. The investigated nucleotide mimetics
can be divided into two structural groups: uracil derivatives
(19-21) and adenine derivatives (22, 23). The compounds
contain a 5′-amide linkage and a spacer unit of different length
(one to three methylene units) connected via another amide
linkage with a polar benzylphosphonate (I), methylenephos-
phonate (II), or ethylenephosphonate (III) moiety, respectively.
Table 1 shows that adenine derivatives were generally less potent
than the corresponding uracil derivatives (compare 19a/22a,
19b/22b, 20a/23a, 20b/23b, 20c/23c). This was surprising, since
ATP is a better substrate of NTPDases than UTP.8 There was
one exception, the benzylphosphonate derivatives 19c/22c
containing a long (propylene) spacer. These compounds were
weak, nonselective NTPDase inhibitors with Ki values ranging
between 161 and 255 µM with only small differences between
the uracil (19c) and the adenine derivative (22c). However, the
adenine derivative, 22c, was inactive against NTPDase3 at the
highest concentration tested (1 mM), whereas the uracil deriva-
tive, 19c, was inhibitory. The benzylphosphonates were the most
potent NTPDase inhibitors of the present series. This was true
for NTPDase1 and 2, while most of the investigated compounds
were inactive or showed only low inhibitory activity with

NTPDase2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4523
Figure 3. Concentration-inhibition curves of selected inhibitors at NTPDase2. The enzyme assays were performed in a final volume of 100 µL
using a substrate (ATP) concentration of 400 µM (Km ) 70 µM). Product formation was determined by capillary electrophoresis as described in
the Experimental Section. Data points represent the mean ( SEM from two separate experiments each performed in triplicate. Determined IC50
values were 42 µM (19a), 101 µM (20c), and 374 µM (20a). Calculated Ki values were 8.2 µM (19a), 29.2 µM (20c), and 71.7 µM (20a).
Figure 4. Lineweaver-Burk plot for NTPDase2 kinetics in the absence and in the presence of two different concentrations of the inhibitor 19a
(5 and 10 µM).
Interaction with P2Y Receptors. Most of the investigated
nucleotide mimetics did not inhibit or only slightly inhibited
the agonist-induced activation of P2Y2, P2Y4, and P2Y6
receptors at the tested concentration of 100 µM (see Supporting
Information). Thus, the most potent NTPDase2 inhibitors of the
present series, the uracil derivatives 19a and 20c, are selective
for the enzyme versus these P2Y receptor subtypes, which are
activated by uracil nucleotides: P2Y2 by UTP and ATP, P2Y4
by UTP, and P2Y6 by UDP. Three compounds (20a, 20c, 22a)
were evaluated in a broader range of concentrations (0.1, 0.3,
1, 3, 10, 30, 100 µM) at P2Y2 receptors but did not show any
concentration-dependent inhibition of UTP-induced calcium
mobilization. Antagonists, but also agonists, would inhibit UTP-
induced calcium mobilization in this assay, the latter due to
desensitization of the receptors during the preincubation period.
In addition, we showed that the selected compounds (19a, 19b,
20a, 20b, 22b, and 23a) do not or only weakly activate P2Y2
receptors (for details see Supporting Information). In contrast,
known NTPDase2 inhibitors, such as RB2, suramin, and
PPADS, not only are nonselective compared to other NTPDases
but also are equally or more inhibitory for certain P2 receptor
subtypes (see Supporting Information).
Investigation of Chemical and Metabolic Stability. Two
selected phosphonates, the selective NTPDase2 inhibitor 19a
and the moderately potent, nonselective NTPDase inhibitor 19c,
were further investigated for their stability (i) under basic
conditions in 0.001 N aqueous sodium hydroxide solution (pH
11),53 (ii) in artificial gastric fluid at 37 °C,54 and (iii) in rat
liver microsomes55 in order to investigate potential metabolic
degradation by liver enzymes. The experiments were performed
essentially as described previously.53 Samples were incubated
at 37 °C and subsequently analyzed by high performance liquid
chromatography coupled to electrospray ionization mass spec-
troscopy (LC-MS).
Both uridine-derived benzylphosphonates, 19a and 19c, which
contain different spacer lengths between the uridine-derived
moiety and the benzylphosphonate partial structure, showed high
stability in artificial gastric juice even after extended incubation

4524 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Brunschweiger et al.
Scheme 4. Proposed Metabolization of Compounds 19a and 19c by Rat Liver Homogenate
Scheme 5. Amide Hydrolysis of Compound 19a
periods of up to 24 h. No degradation products were detectable
by LC-MS analysis. Similarly, both compounds were highly
stable in sodium hydroxide solution at pH 11 (for details see
Supporting Information), indicating that they may be stable in
the alkaline milieu of the intestine.
expected metabolite 26 was not detectable, probably because
of further degradation.
Conclusions
The synthesis of nucleotide mimetics derived from uridine
5′-carboxylic acid attached via amide bonds to a linker of
variable length to various phosphonic acid diethyl ester moieties
yielded the first potent and selective NTPDase2 inhibitors,
uridine derivatives 19a and 20c. In contrast to nucleotides and
standard NTPDase inhibitors the new compounds are uncharged
at a physiological pH value and may therefore be perorally
bioavailable. The compounds did not interact with the uracil
nucleotide-activated P2 receptor subtypes P2Y2, P2Y4, or P2Y6.
They showed high stability under conditions found in the
gastrointestinal system and upon incubation with rat liver
microsomes. Only a small fraction of 19a was metabolized, the
main pathways presumably being amide hydrolysis and coupling
to glucose as analyzed by LC-MS, while the phosphonic acid
esters remained intact. Compound 19a may become a useful
pharmacological tool for studying NTPDase2 and the potential
of NTPDase2 inhibitors as novel drugs.
The next step was to investigate whether the phosphonates
were susceptible to liver metabolism. It has been described that
short alkyl chain esters of phosphonates are metabolically quite
stable.56,57 Incubation with rat liver microsomes showed that
both compounds, 19a and 19c, were relatively stable toward
liver enzymes. Only a very small percentage (<5%) was
metabolized under the applied conditions (see Experimental
Section), while >95% of the compounds were recovered
unchanged. Both compounds were metabolized to a product with
a molecular mass that was 162 u higher than that of the parent
drug. An increase in the molecular mass of 162 u is atypical
for a phase I reaction, such as oxidation, reduction, or hydrolysis.
It points to a phase II reaction, i.e., a coupling reaction with an
acid, such as glucuronic acid. Glucuronidation of the unsubsti-
tuted ribosyl hydroxyl groups of 19a and 19c is conceivable,
but this would increase the molecular mass by 176 u. One
possible metabolizing phase II reaction that would increase the
molecular weight by 162 u would be coupling with glucose
(Scheme 4).58 Several drugs, e.g., phenobarbital and other
barbituric acid derivatives, have been reported to be conjugated
to glucose in mammals, although this is usually a minor
pathway.58
Candidate enzymes catalyzing this reaction could be phenol
ꢀ-glucosyltransferase (EC 2.4.1.35), arylamine glucosyltrans-
ferase (EC 2.4.1.71), or nicotinate glucosyltransferase (EC
2.4.1.196).58
A second metabolite was detected after incubation of 19a
with rat liver microsomes at 37 °C with a molecular mass of
243 u. This could be explained by a hydrolysis of the amide
bond yielding the aniline derivative 10 (Scheme 5). The same
metabolite was also detected after incubation of 19c. The
Experimental Section
Methods. Chemicals and solvents were obtained from various
producers (Acros, Aldrich, Fluka, Merck, Sigma) and used without
further purification unless otherwise noted. Dichloromethane was
distilled prior to use. The reactions were monitored by thin layer
chromatography (TLC) using aluminum sheets coated with silica
gel 60 F254 (Merck). Column chromatography was carried out on
silica gel 0.040-0.063 mm (Fluka) using a flash chromatography
system (Bu¨chi). Preparative HPLC was performed on a C18 column
(250 mm × 20 mm, particle size 10 µm, Eurospher 100) using a
mixture of MeOH and H2O as eluent at a flow rate of 20 mL/min.
Mass spectra were recorded on an API 2000 mass spectrometer
(electron spray ion source, Applied Biosystems, Darmstadt, Ger-
many) coupled with an HPLC system (Agilent 1100) using a C18
1
column (particle size 3 µm, Phenomenex Luna). H, 31P, and 13C

NTPDase2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4525
NMR spectra were recorded on a 500 MHz spectrometer (Bruker
Avance). CDCl3, DMSO-d6, MeOD-d4, or D2O were used as
solvents as indicated below. 31P NMR spectra were recorded using
aqueous orthophosphoric acid (85%) as an external standard. Shifts
are given in ppm relative to the external standard (31P NMR) or
relative to the remaining protons of the deuterated solvents (1H,
13C). Elemental microanalyses were performed on a VarioEL
apparatus. Yields and analytical data of all synthesized compounds
are given in the Supporting Information.
General Procedure for the Synthesis of Final Products
19a-c, 10a-c, 21a, 22a-c, and 23a-c. Synthesis of 2′,3′-p-
Methoxybenzylideneadenosine-5′-carboxylic Acid Amides. Un-
der an argon atmosphere, 2′,3′-p-methoxybenzylideneadenosine-
5′-carboxylic acid 8a (1 mmol, 399 mg) was dissolved in a mixture
of 2 mL of dry DMF and 0.4 mL of dry diisopropylethylamine at
room temperature. The appropriate coupling reagent (see below)
(1.1 mmol, HBTU 428 mg, HCTU 455 mg, PyBOP 570 mg) and
1-hydroxybenzotriazole (1.1 mmol, 149 mg) was dissolved in 2
mL of dry DMF, and after 1 min compound 16a, 16b, 16c, 17a,
17b, or 17c (2 mmol), dissolved in a mixture of dry DMF (2 mL)
and diisopropylethylamine (0.5 mL), was added via a syringe to
the solution. Vigorous stirring was continued for 24 h at room
temperature. The volatiles were removed in vacuo at 40 °C, and
the residue was purified by silica gel column chromatography using
dichloromethane/methanol (40:1) as eluent. The solvent was
removed by rotary evaporation at 40 °C, and the product was
recrystallized from diethyl ether (20 mL).
Synthesis of 2′,3′-p-Methoxybenzylideneuridine-5′-carboxy-
lic Acid Amides. Under an argon atmosphere 2′,3′-p-methoxyben-
zylideneuridine-5′-carboxylic acid 8b (1 mmol, 376 mg), the proper
coupling reagent (see experimental part) (1.1 mmol, HBTU 428
mg, HCTU 455 mg, PyBOP 570 mg) and 1-hydroxybenzotriazole
(1.1 mmol, 149 mg) were dissolved in 2 mL of dry DMF at room
temperature. Then diisopropylethylamine (1.1 mmol, 143 mg) and,
after 1 min, compound 16a, 16b, 16c, 17a, 17b, 17c, or 18a (2
mmol), dissolved in a mixture of dry DMF (2 mL) and diisopro-
pylethylamine (0.5 mL) was sequentially added to the solution via
a syringe. Reaction times and workup procedure were the same as
for 2′,3′-p-methoxybenzylideneadenosine-5′-carboxylic acid amides.
The compounds were directly used in the following step.
Deprotection: Removal of the p-Methoxybenzylidene Protect-
ing Group. Deprotection of the ribose moiety was performed by
stirring 2′,3′-p-methoxybenzylidenenucleoside-5′-carboxylic acid
amides (100 mg) in a mixture of dichloromethane (3 mL),
trifluoroacetic acid (0.15 mL), and water (0.1 mL) at room
temperature. After 2 h, the crude product was precipitated by the
addition of diethyl ether (50 mL), and the suspension was stirred
for 1 h. The product was collected by filtration, dissolved in 7 mL
of a mixture of water/methanol (75:25), and purified on a C-18
column by RP-HPLC using a gradient of water/methanol from 75:
25 to 0:100. The final products were isolated by lyophilization.
Yields were determined over two steps, after amide coupling and
removal of the p-methoxybenzylidene group.
2′,3′-p-Methoxybenzylideneadenosine (7a) and 2′,3′-p-Meth-
oxybenzylideneuridine (7b). Introduction of the p-methoxyben-
zylidene protecting group was performed in analogy to a described
procedure.38 Adenosine (5.0 g) or uridine (5.0 g) and ZnCl2 (10.0
g) were suspended in a mixture of 10 mL of dry THF and 20 mL
of p-methoxybenzaldehyde (anisaldehyde). The turbid mixture was
stirred for 2 days, then the product was precipitated by the addition
of 100 mL of diethyl ether, filtered off, and washed with water (3
× 50 mL) and diethyl ether (3 × 50 mL).
2′,3′-p-Methoxybenzylideneadenosine-5′-carboxylic Acid (8a)
and 2′,3′-p-Methoxybenzylideneuridine-5′-carboxylic Acid (8b).
Oxidation was performed according to a published procedure41
starting from 6.0 mmol of 7a (2.31 g) or 7b (2.17 g).
General Procedures for the Synthesis of Amino-Substituted
Phosphonic Acid Ester Derivatives 16a-c, 17a-c, and 18a.
Preparation of Amides 13a-c, 14a-c, and 15a. In a dry vessel,
N-tert-butyloxycarbonylglycine (10 mmol, 1750 mg), or N-tert-
butyloxycarbonyl-ꢀ-alanine (10 mmol, 1890 mg), or N-tert-buty-
loxycarbonyl-γ-aminobutyric acid (10 mmol, 2030 mg) was
dissolved in 10 mL of dry THF and cooled to -25 °C. N-
Methylmorpholine (10 mmol, 1010 mg) and then isobutyl chloro-
formate (10 mmol, 1360 mg) were added under vigorous stirring.
Immediately after the formation of a white precipitate (N-methyl-
morpholine hydrochloride) a solution of p-aminobenzylphosphonic
acid diethyl ester (11 mmol, 2673 mg) in dry THF (10 mL) or a
solution of aminomethylphosphonic acid diethyl ester oxalate (11
mmol, 2830 mg) or aminoethylphosphonic acid diethyl ester oxalate
(11 mmol, 2980 mg) in 11 mL of an aqueous 1 N NaOH solution,
precooled on ice, was added. The resulting mixture was allowed
to warm to room temperature. After 3 h, the volatiles were removed
by rotary evaporation at 40 °C, the residue was dissolved in 10
mL of water, the pH was brought to 1-2 (with 10% aqueous
NaHSO4 solution), and the solution was extracted with ethyl acetate
(3 × 50 mL). The combined organic layers were washed with a
saturated aqueous Na2CO3 solution (3 × 20 mL) and subsequently
with water (3 × 20 mL), dried over Na2SO4, and evaporated to
dryness. The products were directly used for the next step.
4-[2-((2S,3R,4S,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-3,4-dihydroxytetrahydrofurane-2-carboxamido)ethylamido-
]benzylphosphonic Acid Diethyl Ester (19a). Coupling reagent
used in the synthesis was HCTU. Yield over two steps, 310 mg,
Deprotection: Removal of the tert-Butyloxycarbonyl (Boc)
Protective Group. The Boc-protected amides 13a-c, 14a-c, or
15a (3-4 g, the whole amount of the Boc-protected amide except
for a small sample for analytical purposes) were dissolved in 8
mL of a dry 4 N HCl solution in dioxane and stirred for 2 h at
room temperature. The corresponding products 16a-c, 71a-c, and
18a were precipitated as hydrochlorides by the addition of 50 mL
of diethyl ether, filtered off, and thoroughly washed with diethyl
ether (3 × 50 mL).
1
3
57%; mp 159 °C. H NMR (500 MHz, MeOD) δ 8.18 (d, 1H, J
) 7.90 Hz, H-6), 7.58 (d, 2H, 3J ) 8.85 Hz, 2 × CHortho
,
3
4
benzylphosphonate), 7.31 (dd, 2H, J ) 8.80 Hz and J ) 2.80
3
Hz, 2 × CHmeta, benzylphosphonate), 6.02 (d, 1H, J ) 6.30 Hz,
H-1′), 5.78 (d, 1H, 3J ) 8.20 Hz, H-5), 4.51 (d, 1H, 3J ) 3.20 Hz,
3
3
H-4′), 4.47 (dd, 1H, J ) 5.05 Hz and J ) 5.95 Hz, H-2′), 4.41
3
3
(dd, 1H, J ) 3.20 Hz and J ) 5.05 Hz, H-3′), 4.19-4.01 (AB-
system with A d and B d, partially overlapping with 2 × O-CH2,
2H, 2J ) 16.35 Hz, N-CH2, ethylamide), 4.09-4.01 (2q, 4H, 2 ×
O-CH2), 3.25 (d, 2H, 2JH,P ) 21.45 Hz, CH2-P, benzylphospho-
nate), 1.29 (t, 6H, 2 × CH3). 13C NMR (125 MHz, MeOD) δ 173.2
(CdO), 169.5 (CdO), 166.4 (C-4), 153.1 (C-2), 144.3 (C-6), 138.9
(Cpara, benzylphosphonate), 131.7 (2 × CHortho, benzylphosphonate),
p-(Aminomethylcarboxamido)benzylphosphonic Acid Dieth-
yl Ester Hydrochloride (16a). Yield over two steps, 3.0 g, 90%;
mp 197 °C. 1H NMR (500 MHz, DMSO-d6) δ 10.84 (s, 1H,
3
CONH), 8.35 (br s, 3H, NH3+), 7.56 (d, 2H, J ) 8.20 Hz, 2 ×
CHortho, benzylphosphonate), 7.13 (dd, 2H, 3J ) 8.55 Hz and 4J )
2.55 Hz, 2 × CHmeta, benzylphosphonate), 4.02-3.94 (2q, 4H, 2
× O-CH2), 3.77 (br s, 2H, N-CH2, methylcarboxamide), 3.15
2
128.7 (d, JC,P ) 9.4 Hz, Cipso, benzylphosphonate), 121.5 (2 ×
CHmeta, benzylphosphonate), 103.5 (C-5), 92.2 (C-1′), 85.3 (C-4′),
74.9 (C-2′), 74.1 (C-3′), 64.1 and 64.0 (2 × O-CH2), 43.9 (N-CH2,
2
(d, 2H, JH,P ) 21.45 Hz, CH2-P, benzylphosphonate), 1.22 (t,
1
6H, 2 × CH3). 13C NMR (125 MHz, DMSO-d6) δ 164.8 (CdO),
137.0 (Cpara, benzylphosphonate), 130.3 (2 × Cortho, benzylphos-
phonate), 127.6 (d, 2JC,P ) 9.2 Hz, Cipso, benzylphosphonate), 119.2
(2 × Cmeta, benzylphosphonate), 61.5 (2 × OCH2), 41.1 (N-CH2,
ethylamide), 33.4 (d, JC,P ) 137.6 Hz, CH2-P, benzylphospho-
nate), 17.0 and 16.9 (2 × CH3). 31P NMR (202 MHz, MeOD) δ
26.7. LC/ESI-MS: negative mode 539.3 ([M - H]-), positive mode
541.0 ([M + H]+). Anal. (C22H29N4O10P·4.25H2O) C, H, N.
Biochemical Assays. Membrane Preparation Containing
Expressed Human NTPDase2. The NTPDase2 cDNA cloned from
human small cell lung carcinoma and inserted in a pcDNA3 vector
1
methylcarboxamide), 31.9 (d, JC,P ) 137.8 Hz, CH2-P, ben-
zylphosphonate), 16.4 and 16.3 (2 × CH3). 31P NMR (202 MHz,
DMSO-d6) δ 26.9.

4526 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Brunschweiger et al.
was used to transfect human embryonic kidney (HEK293) cells.
Stably transfected cells were obtained by geneticin selection as
described.22 Membranes were prepared from stably transfected cells
harvested from 10-15 10-cm plates by differential and sucrose
gradient centrifugation as described.22
19a (0, 5, and 10 µM). The curves were analyzed by PRISM 3.0
and transformed to obtain a Lineweaver-Burk plot.
Stability and Metabolism Studies. Preparation of Solutions
and Rat Liver Microsomes. The intestinal environment was
simulated with 0.001 N aqueous sodium hydroxide solution (pH
11). Simulated gastric acid was prepared as previously described:53,54
pepsin (3.20 g), NaCl (2.0 g), and HCl (1 M, 80 mL) were mixed
with distilled water to obtain 1000 mL, and the resulting solution
was stored at 4 °C. Rat liver microsomes were prepared as described
with slight modifications:53,55,61 fresh rat liver (6.5 g) was
homogenized in 30 mL of freshly prepared Dulbecco’s phosphate
buffered saline (DPBS) consisting of 132.5 mg of CaCl2 ·2H2O,
100 mg of MgCl2 ·6H2O, 200 mg of KCl, 200 mg of KH2PO4, 8000
mg of NaCl, and 1500 mg of Na2HPO4 in a total volume of 1000
mL, pH 7.2, and centrifuged at 9000g for 30 min at 4 °C. The
supernatant, which contained the soluble microsomes, was carefully
decanted and stored at -80 °C until used. The protein concentration
was 18 mg/mL as determined by the method of Bradford.60
LC-MS Analyses. HPLC was performed on a C18 column (50
mm × 2 mm, particle size 3 µm, Phenomenex Luna) using a
mixture of H2O (solvent A) and MeOH (solvent B) containing 20
mM of NH4OAc as eluent at a flow rate of 250 µL/min. Mass
spectra were recorded on an API 2000 mass spectrometer (electron
spray ion source, Applied Biosystems, Darmstadt, Germany)
coupled with an HPLC system (Agilent 1100, Bo¨blingen, Germany).
Data were collected and analyzed by Analyst Software, version
1.3.1. The separation was carried out at room temperature by
gradient elution. The elution was started with a mixture of solvent
A and solvent B (90:10, v/v) up to 15 min. Subsequently only
solvent B was used up to 30 min. The limit of detection (LOD),
defined as the lowest analyte concentration with a signal-to-noise
(S/N) ratio of 3, was determined for compounds 19a and 19c to be
approximately 0.1 µg/mL.
Stability in Basic Solution and in Artificial Gastric Acid. Stock
solutions (2 mM) of 19a and 19c in H2O containing 20 mM
NH4OAc were prepared, and 10 µL aliquots were incubated for 2
or 24 h with 990 µL of either 0.001 N aqueous NaOH solution or
simulated gastric acid at 37 °C and subsequently analyzed by
LC-MS. No degradation products were detectable by sensitive
LC-MS analysis.
Metabolism by Rat Liver Microsomes. Compounds 19a and
19c were incubated with rat liver microsomes (6 mg of protein per
vial) at a concentration of 100 µM in a final volume of 1 mL. The
solution contained an NADPH regenerative system consisting of
NADP (0.57 mM), NADH (0.57 mM), isocitrate (6.4 mM),
isocitrate dehydrogenase (0.57 mM), and MgCl2 (23.4 mM) (pH
7.2).62 The samples were incubated for 2 h at 37 °C in a water
bath and subsequently heated at 99 °C for 3 min to stop the
enzymatic reaction. After centrifugation at 14000g, the supernatant
was analyzed by LC-MS.
Cell Transfection with Human NTPDases 1, 3, 8 and
Membrane Preparation. COS-7 cells were transfected in 10 cm
plates using Lipofectamine (Invitrogen), as previously described.59
Briefly, 80-90% confluent cells were incubated for 5 h at 37 °C
in Dulbecco’s modified Eagle medium (DMEM) in the absence of
fetal bovine serum (FBS) with 6 µg of the appropriate plasmid DNA
and 24 µL of Lipofectamine reagent. The reaction was stopped by
the addition of an equal volume of DMEM containing 20% FBS,
and the cells were harvested 40-72 h later. For the preparation of
protein extracts, transfected cells were washed three times with Tris-
saline buffer at 4 °C, collected by scraping in the harvesting buffer
(95 mM NaCl, 0.1 mM phenylmethanesulfonyl fluoride (PMSF)
and 45 mM Tris at pH 7.5), and washed twice by 300g centrifuga-
tion for 10 min at 4 °C. Cells were resuspended in the harvesting
buffer containing 10 mg/mL aprotinin and sonicated. Nucleus and
cellular debris were discarded by centrifugation at 300g for 10 min
at 4 °C, and the supernatant (crude protein extract) was aliquoted
and stored at -80 °C until used for activity assays. The protein
concentration was estimated by the Bradford microplate assay using
bovine serum albumin as a standard.60
Capillary Electrophoresis (CE) Instrumentation. All experi-
ments were carried out using a P/ACE MDQ capillary electro-
phoresis system (Beckman Instruments, Fullerton, CA) equipped
with a UV detection system coupled with a diode array detector
(DAD). Data collection and peak area analysis were performed by
the P/ACE MDQ software 32 KARAT obtained from Beckman
Coulter. The capillary and sample storing unit temperature was kept
constant at 25 °C. The electrophoretic separations were carried out
using an eCAP polyacrylamide-coated fused-silica capillary [(30
cm (20 cm effective length) × 50 µm internal diameter (i.d.) ×
360 µm outside diameter (o.d.), obtained from CS-Chromatographie
(Langerwehe, Germany)]. The separation was performed using an
applied current of -60 µA and a data acquisition rate of 8 Hz.
Analytes were detected using direct UV absorbance at 210 nm.
The capillary was conditioned by rinsing with water for 2 min and
subsequently with buffer (phosphate 50 mM, pH 6.5) for 1 min.
Sample injections were made at the cathodic side of the capillary.
Determination of NTPDase Inhibition. Enzyme inhibition
assays were carried out at 37 °C in a final volume of 100 µL. The
reaction mixture contained 140 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 2 mM CaCl2, and 10 mM Hepes, pH 7.4, and 400 µM
ATP. Different concentrations of inhibitors dissolved in water or
containing 1% DMSO in cases of low water solubility when a stock
solution could not be prepared in water (10 µL) were added, and
the reaction was initiated by the addition of 10 µL of the
appropriately diluted membrane preparations containing human
NTPDase1, NTPDase2, NTPDase3, or NTPDase8. The mixture was
incubated for 10 min, and the reaction was terminated by heating
at 99 °C for 5 min. Aliquots of the reaction mixture (50 µL) were
then diluted 10-fold with water containing UMP (final concentration
20 µM) as an internal standard, transferred to mini-CE vials, and
injected into the CE instrument under the conditions described
above. Each analysis was repeated three times (triplicates) in two
separate experiments. Control experiments were performed using
membrane preparations of cells transfected with the empty plasmid
(pcDNA3). The Cheng-Prusoff equation was used to calculate Ki
values from the IC50 values, determined by the nonlinear curve-
fitting program PRISM 3.0 (GraphPad, San Diego, CA), assuming
a competitive inhibition mechanism. Km values were as follows:
17 µM (NTPDase1), 70 µM (NTPDase2), 75 µM (NTPDase3), and
81 µM (NTPDase8).8,47
Acknowledgment. Karen Schmeling and Sonja Sternberg
are acknowledged for skillful technical assistance. We are
grateful to Dr. Gary Weisman, University of Missouri-Columbia,
and Dr. Petra Hillmann, University of Bonn, for providing
recombinant astrocytoma cell lines expressing P2Y receptor
subtypes. J.I. is grateful for a STIBET scholarship by the
Deutscher Akademischer Austauschdienst (DAAD). A.F.K. was
supported by the California Metabolic Research Foundation. J.S.
was supported by grants from the Canadian Institutes of Health
Research (CIHR) and was the recipient of a New Investigator
award from the CIHR.
Supporting Information Available: Elemental analyses, yields,
and analytical data for final products; yields and analytical data
for 7a and 7b; procedures and test results at P2Y2, P2Y4, and P2Y6
receptors, and HPLC chromatograms and MS spectra of stability
and metabolism studies. This material is available free of charge
via the Internet at http://pubs.acs.org.
For the determination of the mechanism of inhibition, the
enzymatic reaction of NTPDase2 was monitored as described above
using different concentrations of ATP as a substrate (12.5, 25, 50,
125, 250, and 1000 µM) in the absence and presence of the inhibitor

NTPDase2 Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4527
(23) Buffon, A.; Ribeiro, V. B.; Wink, M. R.; Casali, E. A.; Sarkis, J. J.
Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells:
molecular identification, kinetic characterization and biochemical
properties. Life Sci. 2007, 80, 950–958.
(24) Jacobson, K. A.; Jarvis, M. F.; Williams, M. Purine and pyrimidine
(P2) receptors as drug targets. J. Med. Chem. 2002, 45, 4057–4093.
(25) Gendron, F. P.; Halbfinger, E.; Fischer, B.; Beaudoin, A. R. Inhibitors
of NTPDase: key players in the metabolism of extracellular purines.
AdV. Exp. Med. Biol. 2000, 486, 119–123.
(26) Gendron, F. P.; Halbfinger, E.; Fischer, B.; Duval, M.; D’Orleans-
Juste, P.; Beaudoin, A. R. Novel inhibitors of nucleoside triphosphate
diphosphohydrolases: chemical synthesis and biochemical and phar-
macological characterizations. J. Med. Chem. 2000, 43, 2239–2247.
(27) Gendron, F. P.; Benrezzak, O.; Krugh, B. W.; Kong, Q.; Weisman,
G. A.; Beaudoin, A. R. Purine signaling and potential new therapeutic
approach: possible outcomes of NTPDase inhibition. Curr. Drug
Targets 2002, 3, 229–245.
(28) Cechin, S. R.; Schetinger, M. R.; Zanatta, N.; Madruga, C. C.;
Pacholski, I. L.; Flores, D. C.; Bonacorso, H. G.; Martins, M. A. P.;
Morsch, V. M. Inhibitory effect of novel pyrimidines on ATP and
ADP hydrolysis in synaptosomes from rat cerebral cortex. Chem. Res.
Toxicol. 2003, 16, 1433–1439.
(29) Joseph, S. M.; Pifer, M. A.; Przybylski, R. J.; Dubyak, G. R. Methylene
ATP analogs as modulators of extracellular ATP metabolism and
accumulation. Br. J. Pharmacol. 2004, 142, 1002–1014.
(30) Iqbal, J.; Vollmayer, P.; Braun, N.; Zimmermann, H.; Mu¨ller, C. E.
A capillary electrophoresis method for the characterization of ecto-
nucleoside triphosphate diphosphohydrolases (NTPDases) and the
analysis of inhibitors by in-capillary enzymatic microreaction. Puri-
nergic Signalling 2005, 1, 349–358.
(31) Mu¨ller, C. E.; Iqbal, J.; Baqi, Y.; Zimmermann, H.; Rollich, A.;
Stephan, H. Polyoxometalatessa new class of potent ecto-nucleoside
triphosphate diphosphohydrolase (NTPDase) inhibitors. Bioorg. Med.
Chem. Lett. 2006, 16, 5943–5947.
(32) Munkonda, M. N.; Kauffenstein, G.; Kukulski, F.; Le´vesque, S. A.;
Legendre, C.; Pelletier, J.; Lavoie, E. G.; Lecka, J.; Se´vigny, J.
Inhibition of human and mouse plasma membrane bound NTPDases
by P2 receptor antagonists. Biochem. Pharmacol. 2007, 74, 1524–
1534.
(33) Tuluc, F.; Bu¨ltmann, R.; Gla¨nzel, M.; Frahm, A. W.; Starke, K. P2-
receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-
nucleotidases by compounds related to reactive blue 2. Naunyn-
Schmiedeberg’s Arch. Pharmacol 1998, 357, 111–120.
References
(1) (a) Burnstock, G. Physiology and pathophysiology of purinergic
neurotransmission. Physiol. ReV. 2007, 87, 659–797. (b) Burnstock,
G. Purine and pyrimidine receptors. Cell. Mol. Life Sci. 2007, 64,
1471–1483.
(2) Abbracchio, M. P.; Burnstock, G.; Boeynaems, J.; Barnard, E. A.;
Boyer, J. L.; Kennedy, C.; Knight, G. E.; Fumagalli, M.; Gachet, C.;
Jacobson, K. A.; Weisman, G. A. International Union of Pharmacology
LVIII: update on the P2Y G protein-coupled nucleotide receptors: from
molecular mechanisms and pathophysiology to therapy. Pharmacol.
ReV. 2006, 58, 281–341.
(3) Zimmermann, H. Ectonucleotidases: some recent developments and
a note on nomenclature. Drug DeV. Res. 2001, 52, 44–56.
(4) Robson, S. C.; Se´vigny, J.; Zimmermann, H. The E-NTPDase family
of ectonucleotidases: Structure-function relationships and pathophysi-
ological significance. Purinergic Signalling 2006, 2, 409–430.
(5) Stefan, C.; Jansen, S.; Bollen, M. NPP-type ectophosphodiesterases:
unity in diversity. Trends Biochem. Sci. 2005, 30, 542–550.
(6) Colgan, S. P.; Eltzschig, H. K.; Eckle, T.; Thompson, L. F. Physi-
ological roles for ecto-5′-nucleotidase (CD73). Purinergic Signalling
2006, 2, 351–360.
(7) Zimmermann, H. Two novel families of ectonucleotidases: molecular
structures, catalytic properties and a search for function. Trends
Pharmacol. Sci. 1999, 20, 231–236.
(8) Kukulski, F.; Le´vesque, S. A.; Lavoie, E. G.; Lecka, J.; Bigonnesse,
F.; Knowles, A. F.; Robson, S. C.; Kirley, T. L.; Se´vigny, J.
Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3,
and 8. Purinergic Signalling 2005, 1, 193–204.
(9) Alvarado-Castillo, C.; Harden, T. K.; Boyer, J. L. Regulation of P2Y1
receptor-mediated signaling by the ectonucleoside triphosphate diphos-
phohydrolase isozymes NTPDase1 and NTPDase2. Mol. Pharmacol.
2005, 67, 114–122.
(10) Se´vigny, J.; Sundberg, C.; Braun, N.; Guckelberger, O.; Csizmadia,
E.; Qawi, I.; Imai, M.; Zimmermann, H.; Robson, S. C. Differential
catalytic properties and vascular topography of murine nucleoside
triphosphate diphosphohydrolase 1 (NTPDase1) and NTPDase2 have
implications for thromboregulation. Blood 2002, 99, 2801–2809.
(11) Ralevic, V.; Burnstock, G. Receptors for purines and pyrimidines.
Pharmacol. ReV. 1998, 50, 413–492.
(12) Zimmermann, H. 5′-Nucleotidase: molecular structure and functional
aspects. Biochem. J. 1992, 285 (Part 2), 345–365.
(13) Iqbal, J.; Jirovsky, D.; Lee, S. Y.; Zimmermann, H.; Mu¨ller, C. E.
Capillary electrophoresis-based nanoscale assays for monitoring ecto-
5′-nucleotidase activity and inhibition in preparations of recombinant
enzyme and melanoma cell membranes. Anal. Biochem. 2008, 373,
129–140.
(34) Bu¨ltmann, R.; Wittenburg, H.; Pause, B.; Kurz, G.; Nickel, P. Blockade
of P2-purinoceptor subtypes and ecto-nucleotidases by compounds
related to suramin. Naunyn Schmiedeberg’s Arch. Pharmacol. 1996,
354, 498–504.
(35) Chen, B. C.; Lee, C. M.; Lin, W. W. Inhibition of ecto-ATPase by
PPADS, suramin and reactive blue in endothelial cells, C6 glioma
cells and RAW 264.7 macrophages. Br. J. Pharmacol. 1996, 119,
1628–1634.
(14) Stra¨ter, N. Ecto-5′-nucleotidase: structure function relationships.
Purinergic Signalling 2006, 2, 343–350.
(15) Shryock, J. C.; Belardinelli, L. Adenosine and adenosine receptors in
the cardiovascular system: biochemistry, physiology, and pharmacol-
ogy. Am. J. Cardiol 1997, 79, 2–10.
(16) Koszalka, P.; Ozu¨yaman, B.; Huo, Y.; Zernecke, A.; Flo¨gel, U.; Braun,
N.; Buchheiser, A.; Decking, U. K.; Smith, M. L.; Se´vigny, J.; Gear,
A.; Weber, A. A.; Molojavyi, A.; Ding, Z.; Weber, C.; Ley, K.;
Zimmermann, H.; Go¨decke, A.; Schrader, J. Targeted disruption of
cd73/ecto-5′-nucleotidase alters thromboregulation and augments
vascular inflammatory response. Circ. Res. 2004, 95, 814–821.
(17) Kawashima, Y.; Nagasawa, T.; Ninomiya, H. Contribution of ecto-
5′-nucleotidase to the inhibition of platelet aggregation by human
endothelial cells. Blood 2000, 96, 2157–2162.
(18) El Omar, M. M.; Islam, N.; Broekman, M. J.; Drosopoulos, J. H.;
Roa, D. C.; Lorin, J. D.; Sedlis, S. P.; Olson, K. E.; Pulte, E. D.;
Marcus, A. J. The ratio of ADP- to ATP-ectonucleotidase activity is
reduced in patients with coronary artery disease. Thromb. Res. 2005,
116, 199–206.
(19) Wink, M. R.; Braganhol, E.; Tamajusuku, A. S. K.; Lenz, G.; Zerbini,
L. F.; Libermann, T. A.; Se´vigny, J.; Battastini, A. M. O.; Robson,
S. C. Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/
CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes.
Neuroscience 2006, 138, 421–432.
(20) Chorna, N. E.; Santiago-Perez, L. I.; Erb, L.; Seye, C. I.; Neary, J. T.;
Sun, G. Y.; Weisman, G. A.; Gonzalez, F. A. P2Y2 receptors activate
neuroprotective mechanisms in astrocytic cells. J. Neurochem. 2004,
91, 119–132.
(36) Crack, B. E.; Pollard, C. E.; Beukers, M. W.; Roberts, S. M.; Hunt,
S. F.; Ingall, A. H.; McKechnie, K. C.; IJzerman, A. P.; Leff, P.
Pharmacological and biochemical analysis of FPL 67156, a novel,
selective inhibitor of ecto-ATPase. Br. J. Pharmacol. 1995, 114, 475–
481.
(37) Le´vesque, S. A.; Lavoie, E. G.; Lecka, J.; Bigonnesse, F.; Se´vigny, J.
Specificity of the ecto-ATPase inhibitor ARL 67156 on human and
mouse ectonucleotidases. Br. J. Pharmacol. 2007, 152, 141–150.
(38) Smith, M.; Rammler, D. H.; Goldberg, I. H.; Khorana, H. G.
Polynucleotides. XIV. Specific synthesis of the C3′-C5′ internucle-
otide linkage. Synthesis of uridylyl-(3′ f 5′)-uridine and uridylyl-(3′
f 5′)-adenosine. J. Am. Chem. Soc. 1962, 84, 430–440.
(39) Watkins, B. E.; Kiely, J. S.; Rapoport, H. Synthesis of oligodeoxyri-
bonucleotides using N-(benzyloxycarbonyl)-blocked nucleosides.
J. Am. Chem. Soc. 1982, 104, 5702–5708.
(40) Johnson, D. C.; Widlanski, T. S. Facile deprotection of O-Cbz-
protected nucleosides by hydrogenolysis: an alternative to O-benzyl
ether-protected nucleosides. Org. Lett. 2004, 6, 4643–4646.
(41) Epp, J. B.; Widlanski, T. S. Facile preparation of nucleoside-5′-
carboxylic acids. J. Org. Chem. 1999, 64, 293–295.
(42) Wieland, T. Use of anhydrides of N-acylated amino acids and
derivatives of inorganic acids. Liebigs Ann. Chem. 1951, 572, 190–
194.
(21) Langer, D.; Ikehara, Y.; Takebayashi, H.; Hawkes, R.; Zimmermann,
H. The ectonucleotidases alkaline phosphatase and nucleoside triph-
osphate diphosphohydrolase 2 are associated with subsets of progenitor
cell populations in the mouse embryonic, postnatal and adult neuro-
genic zones. Neuroscience 2007, 150, 863–879.
(22) Knowles, A. F.; Chiang, W.-C. Enzymatic and transcriptional regula-
tion of human ecto-ATPase/E-NTPDase 2. Arch. Biochem. Biophys.
2003, 418, 217–227.
(43) Montgomery, J. A.; Thomas, H. J.; Brockman, R. W.; Wheeler, G. P.
Potential inhibitors of nucleotide biosynthesis. 1. Nitrosoureidonucleo-
sides. J. Med. Chem. 1981, 24, 184–189.
(44) Sabatino, G.; Mulinacci, B.; Alcaro, M. C.; Chelli, M.; Rovero, P.;
Papini, A. M. Assessment of new 6-Cl-HOBt based coupling reagents
for peptide synthesis. Part 1: Coupling efficiency study. Lett. Pept.
Sci. 2003, 9, 119–123.

4528 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Brunschweiger et al.
(45) Uri, A.; Ja¨rlebark, L.; von Ku¨gelgen, I.; Scho¨nberg, T.; Unden,
A.; Heilbronn, E. A new class of compounds, peptide derivatives
of adenosine 5′-carboxylic acid, includes inhibitors of ATP receptor-
mediated responses. Bioorg. Med. Chem. 1994, 2, 1099–1105.
(46) Sak, K.; Uri, A.; Enkvist, E.; Raidaru, G.; Subbi, J.; Kelve, M.; Jarv,
J. Adenosine-derived non-phosphate antagonists for P2Y1 purinocep-
tors. Biochem. Biophys. Res. Commun. 2000, 272, 327–331.
(47) Fausther, M.; Lecka, J.; Kukulski, F.; Le´vesque, S. A.; Pelletier, J.;
Zimmermann, H.; Dranoff, J. A.; Se´vigny, J. Cloning, purification,
and identification of the liver canalicular ecto-ATPase as NTPDase8.
Am. J. Physiol. 2007, 292, G785-G795.
(48) El-Tayeb, A.; Qi, A.; Mu¨ller, C. E. Synthesis and structure-activity
relationships of uracil nucleotide derivatives and analogues as agonists
at human P2Y2, P2Y4, and P2Y6 receptors. J. Med. Chem. 2006, 49,
7076–7087.
(49) Kassack, M. U.; Ho¨fgen, B.; Lehmann, J.; Eckstein, N.; Quillan, J. M.;
Sadee, W. Functional screening of G protein-coupled receptors by
measuring intracellular calcium with a fluorescence microplate reader.
J. Biomol. Screening 2002, 7, 233–246.
(50) Kaulich, M.; Streicher, F.; Mayer, R.; Mu¨ller, I.; Mu¨ller, C. E.
Flavonoidssnovel lead compounds for the development of P2Y2
receptor antagonists. Drug DeV. Res. 2003, 59, 72–81.
(51) Weyler, S.; Baqi, Y.; Hillmann, P.; Kaulich, M.; Hunder, A. M.; Mu¨ller,
I. A.; Mu¨ller, C. E. Combinatorial synthesis of anilinoanthraquinone
derivatives and evaluation as non-nucleotide-derived P2Y2 receptor
antagonists. Bioorg. Med. Chem. Lett. 2008, 18, 223–227.
(52) Brunschweiger, A.; Mu¨ller, C. E. P2 receptors activated by uracil
nucleotidessan update. Curr. Med. Chem. 2006, 13, 289–312.
(53) Yan, L.; Mu¨ller, C. E. Preparation, properties, reactions, and adenosine
receptor affinities of sulfophenylxanthine nitrophenyl esters: toward
the development of sulfonic acid prodrugs with peroral bioavailability.
J. Med. Chem. 2004, 47, 1031–1043.
(54) Guven, K. C.; Ozaydin, F. Stability of sodium cyclamate in simulated
gastric and intestinal media. Pharmazie 1981, 36, 297.
(55) Oliyai, R.; Shaw, J. P.; Sueoka-Lennen, C. M.; Cundy, K. C.; Arimilli,
M. N.; Jones, R. J.; Lee, W. A. Aryl ester prodrugs of cyclic HPMPC.
I: Physicochemical characterization and in vitro biological stability.
Pharm. Res. 1999, 16, 1687–1693.
(56) Krise, J. P.; Stella, V. J. Prodrugs of phosphates, phosphonates, and
phosphinates. AdV. Drug DeliVery ReV. 1996, 19, 287–310.
(57) Serafinowska, H. T.; Ashton, R. J.; Bailey, S.; Harnden, M. R.; Jackson,
S. M.; Sutton, D. Synthesis and in vivo evaluation of prodrugs of
9-[2-(phosphonomethoxy)ethoxy]adenine. J. Med. Chem. 1995, 38,
1372–1379.
(58) Tang, B. K. Drug glucosidation. Pharmacol. Ther. 1990, 46, 53–56.
(59) Kaczmarek, E.; Koziak, K.; Se´vigny, J.; Siegel, J. B.; Anrather, J.;
Beaudoin, A. R.; Bach, F. H.; Robson, S. C. Identification and
characterization of CD39/vascular ATP diphosphohydrolase. J. Biol.
Chem. 1996, 271, 33116–33122.
(60) Bradford, M. M. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-
dye binding. Anal. Biochem. 1976, 72, 248–254.
(61) Fletcher, S. R.; Burkamp, F.; Blurton, P.; Cheng, S. K.; Clarkson, R.;
O’Connor, D.; Spinks, D.; Tudge, M.; van Niel, M. B.; Patel, S.;
Chapman, K.; Marwood, R.; Shepheard, S.; Bentley, G.; Cook, G. P.;
Bristow, L. J.; Castro, J. L.; Hutson, P. H.; MacLeod, A. M.
4-(Phenylsulfonyl)piperidines: novel, selective, and bioavailable
5-HT(2A) receptor antagonists. J. Med. Chem. 2002, 45, 492–503.
(62) De Graaf, I. A.; Van Meijeren, C. E.; Pektas, F.; Koster, H. J.
Comparison of in vitro preparations for semi-quantitative prediction
of in vivo drug metabolism. Drug Metab. Dispos. 2002, 30, 1129–1136.
JM800175E