Novel inhibitors of nucleoside triphosphate diphosphohydrolases: Chemical synthesis and biochemical and pharmacological characterizations

Article abstract of DOI:10.1021/jm000020b

To elucidate the physiological role played by nucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.6.1.5), adenine nucleotide analogues, modified on the purine ring, hay e been synthesized and tested as potential inhibitors. Resistance of ATp analogues to hydrolysis and their potency as NTPDase inhibitors were evaluated. For this purpose, a particulate fraction isolated from bovine spleen was used as the enzyme source. Among the synthesized analogues; 8-thiobutyladenosine 5'-triphosphate (8-BuS-ATP) was found to be the most effective nonhydrolyzable competitive inhibitor, with an estimated K(i) of 10 μM. This nonhydrolyzable analogue did not exert any P2X-receptor-mediated effect on endothelium-denuded blood vessels, from the guinea pig mesenteric bed. In agreement with this observation, infusion of the analogue did not cause any significant blood pressure variations of the precontracted vessel. Because in previous studies on isolated turkey erythrocytes and rat astrocytes 8-BuS-ATP was not able to trigger any P2Y₁- receptor-mediated effect, it therefore appears that this NTPDase inhibitor does not interfere with purinergic receptors.

Full text of DOI:10.1021/jm000020b

J . Med. Chem. 2000, 43, 2239-2247
2239
Novel In h ibitor s of Nu cleosid e Tr ip h osp h a te Dip h osp h oh yd r ola ses: Ch em ica l
Syn th esis a n d Bioch em ica l a n d P h a r m a cologica l Ch a r a cter iza tion s
Fernand-Pierre Gendron,† Efrat Halbfinger,‡ Bilha Fischer,‡ Martine Duval,§ Pe´dro D’Orle´ans-J uste,§ and
Adrien R. Beaudoin*,†
De´partment de Biologie, Universite´ de Sherbrooke, Sherbrooke, QC J 1K 2R1, Canada, Department of Chemistry,
Gonda-Goldschmied Center, Bar-Ilan University, Ramat-Gan 52900, Israel, and Institute de Pharmacologie de Sherbrooke,
Universite´ de Sherbrooke, Sherbrooke, QC J 1H 5N4, Canada
Received J anuary 19, 2000
To elucidate the physiological role played by nucleoside triphosphate diphosphohydrolase
(NTPDase; EC 3.6.1.5), adenine nucleotide analogues, modified on the purine ring, have been
synthesized and tested as potential inhibitors. Resistance of ATP analogues to hydrolysis and
their potency as NTPDase inhibitors were evaluated. For this purpose, a particulate fraction
isolated from bovine spleen was used as the enzyme source. Among the synthesized analogues,
8-thiobutyladenosine 5′-triphosphate (8-BuS-ATP) was found to be the most effective nonhy-
drolyzable competitive inhibitor, with an estimated Ki of 10 µM. This nonhydrolyzable analogue
did not exert any P2X-receptor-mediated effect on endothelium-denuded blood vessels, from
the guinea pig mesenteric bed. In agreement with this observation, infusion of the analogue
did not cause any significant blood pressure variations of the precontracted vessel. Because in
previous studies on isolated turkey erythrocytes and rat astrocytes 8-BuS-ATP was not able
to trigger any P2Y1-receptor-mediated effect, it therefore appears that this NTPDase inhibitor
does not interfere with purinergic receptors.
In tr od u ction
nucleoside monophosphate derivative.20-24 Several NT-
PDases have been recently sequenced and their encod-
ing gene shows that it corresponds to CD39, a marker
of activated lymphocytes.25-27 NTPDase assays on tis-
sues of “knock out” and “wild-type” mice have demon-
strated the major role of this enzyme in the extracellular
metabolism of purine nucleotides.28 Two main types of
NTPDase have been found in rat, pig, bovine, and
human tissues (78 and 54 kDa).20,24,29-30 In pig and
bovine organs NTPDase, the 78 kDa isoform is pre-
dominant with one exception, the pancreas, where the
54 kDa type is the main type, as previously shown.24
The latter is a truncated fragment of the 78 kDa
isoform.24 Recent data show that the other fragment (27
kDa) is still associated with the cellular membrane.
In the past decade, evidence has grown in support of
the concept that extracellular ATP and its dephospho-
rylated metabolites are involved in cell signaling in the
different physiological systems of vertebrates.1-5 The
biological effects elicited by extracellular purine nucleo-
tides are determined by their rate of release in the
extracellular medium, the activity of ectonucleotidases,
and their binding affinity for ATP-receptors (P2-Rs).
ATP and ADP are released by both exocytotic and
nonexocytotic mechanisms, which are still poorly
defined.6-8 These nucleotides and the pyrimidine nucle-
otide UTP exert their effects through purinergic type-2
receptors (P2) activation. Receptors of this class are
designated either P2Y for those coupled to G-protein or
P2X for those which are ligand-gated ion channels.1,9-13
Adenosine, the end product of ATP dephosphorylation,
can interact with P1-purinoceptors (G-protein coupled).
It often elicits a cellular response antagonistic to the
one triggered by P2 receptor activation.1,14
Extracellular concentrations of ATP and its metabo-
lites are modulated primarily by ectonucleotidases,
namely ecto-nucleotide triphosphate diphosphohydro-
lase (NTPDase; EC 3.6.1.5), ecto-nucleotide pyrophos-
phatase/phosphodiesterase (E-NPP; no EC) and 5′ nu-
cleotidase (EC 3.1.3.5).15-19 NTPDase, formerly known
as ATP diphosphohydrolase (ATPDase), catalyses the
sequential hydrolysis of â- and γ-phosphate of nucleo-
side di- and triphosphate, producing the corresponding
The rapid breakdown of ATP to adenosine is a
variable generally ignored in purinoceptor studies.18,31-33
This can be partially circumvented either by the use of
nonhydrolyzable ATP analogues or by introducing spe-
cific inhibitors of ectonucleotidases, provided that the
latter are not P2R-ligands. In this respect, we have
previously shown that P2-R agonists, modified on the
phosphate chain e.g., ADP-â-S, R,â-CH2-ATP, â,γ-CH2-
ATP, and â,γ-imido-ATP, 1a -d (Chart 1), are not
hydrolyzed by NTPDase while acting as NTPDase
inhibitors.34 On one hand various 2-thioether adenine
nucleotides exhibit relative enzymatic stability regard-
ing dephosphorylation by ectonucleotidases in various
preparations: bovine “brain membrane”, chick skeletal
muscle derived myotubes, and C6 glioma cell.35 On the
other hand, these compounds are potent P2Y-R ligands
whereas noactivitywas observed with P2X-Rsubtypes.36-39
Likewise, 2-thioether-ATP-R-S derivatives, 3, which are
potent insulin secretagogues acting through P2-Rs,
exhibited relative stability in the presence of the 54 kDa
* Corresponding author. Tel: (819) 821-7946. Fax: (819) 821-8049.
E-mail: abeaudoi @ courrier.usherb.ca.
† De´partment de Biologie, Universite´ de Sherbrooke.
‡ Bar-Ilan University.
§ Institute de Pharmacologie de Sherbrooke, Universite´ de Sher-
brooke.
10.1021/jm000020b CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/11/2000

2240 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
Ch a r t 1. Structures of Different ATP Analogues
Gendron et al.
NTPDase.40 We have also shown that some 2-thioether
adenosine-5′-monophosphorothioate (2-thioether-AMP-
S), 4, derivatives exert an inhibitory effect on the same
enzyme.40 In a previous study we have also shown that
etheno-ATP derivatives, 5, were hydrolyzed at 47% (5a )
and 22% (5b), the rate of ATP. From this study it was
concluded that the relative stability of these molecules
could be attributed either to the bulky aryletheno
moiety, which turns these derivatives into poor sub-
strates, or might indicate that a free N1, N6 region is
required for H bonding.41
NTPDase inhibitor that does not interfere with pu-
rinoceptors. Indeed, this inhibitor binds neither the P2Y
nor the P2X receptor subtypes tested so far. In this
work, resistance to hydrolysis and inhibitory properties
of new ATP analogues, bearing a substitution at either
position C2 or C8 of the purine ring, were evaluated.
Kinetic studies carried out with a NTPDase particulate
preparation put in evidence a new family of ATP
analogues that act as NTPDase inhibitors.
Resu lts
A specific NTPDase inhibitor could potentially be a
valuable tool to amplify the biological effects induced
by endogenous ATP and ADP and to define the role of
NTPDase in the extracellular metabolism of these
purines. Ideally, this inhibitor should not interact with
either P2Y or P2X receptor subtypes. For this purpose
we synthesized 8-substituted ATP derivatives which we
have recently identified as very poor P2Y-R ligands in
turkey erythrocyte membranes and rat astrocytes.39
These derivatives included 8-butyl thioether-, 8-butyl-
amino-, and 8-butyl ether-ATP derivatives, 6-8.
In this paper we present what appears to be the first
Selection a n d Syn th esis of ATP An a logu es for
Eva lu a tion a s NTP Da se In h ibitor s. Compounds
2a -d and 6-8 were prepared as described before.39 On
the basis of the promising hydrolytic stability of com-
pounds 6-8, a new series of 8-thioether ATP analogues
were synthesized for structure activity relationship
(SAR) studies (Scheme 1). The desired derivatives, 6a -
e, were obtained in three steps from 8-Br-adenosine in
good yields. 8-Mercaptoadenosine, 10, was obtained in
a quantitative yield from 8-Br-adenosine upon treat-
ment with 10 equiv of NaSH in wet DMF at 100 °C
overnight. The corresponding dry sodium thiolate salt,

Novel Inhibitors of NTPDases
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2241
Sch em e 1. Synthesis of 8-Thioether-ATP Derivativesa
and 1.65 µmol of Pi released/min/mg of protein, respec-
tively (Table 1). With ADP as the substrate, the
calculated apparent Km and Vmax were 33 µM and 1.3
µmol of Pi released/min/mg of protein (Table 1), respec-
tively. These Km values are comparable to those previ-
ously reported.34,42 As summarized in Table 1, ATP,
2-BuS-ATP (2a ), 2-BuO-ATP (2d ), 2-BuNH-ATP (2c),
8-Br-ATP, 8-EtS-ATP (6c), 8-BuNH-ATP (7), and 8-BuO-
ATP (8) show about the same affinities for the enzyme,
their Km values ranging between 12 and 36 µM. In
contrast, the estimated Vmax for these C2-substituted
analogues are about one-half the value obtained with
ATP. With the C8-substituted analogues, 8-Br-ATP,
8-EtS-ATP (6c), 8-BuNH-ATP (7), and 8-BuO-ATP (8),
Vmax values were estimated at 38%, 18%, 17%, and 12%
the value obtained with ATP, respectively. As for the
ADP analogue, 2-BuS-ADP (2b), apparent Km and Vmax
were estimated at 63 ( 14 µM and 0.94 ( 0.1 µmol of
Pi released/min/mg of protein, respectively (Table 1).
a
(a) 10 equiv NaSH, wet DMF, 100 °C overnight, 100% yield.
(b) (1) compound 10, MeOH, 0.25 M NaOH, RT, freeze-drying, (2)
alkyl bromide, DMF, 60 °C overnight, 83-93% yield; (c) (1) POCl3,
Proton Sponge, (2) P2O7H2(Bu3NH+)2, (3) 0.2 M TEAB.
In h ibition by C-8-Su bstitu ted An a logu es. Inhibi-
tion and Ki of nonhydrolyzable analogues, 6a ,b,d ,e, were
investigated with ATP as the substrate at two concen-
trations (three in the case of 8-BuS-ATP, 6e). Figures
S1 and S2 (see the Supporting Information) represent
rates of ATP catalysis in the presence of the different
inhibitors. With 8-BuS-ATP (6e), the Lineweaver-Burk
linear representation shows intersection on the ordinate
axis, indicative of competitive inhibition (Figure S1A,
Supporting Information).43 Dixon replots of the data
(Figure S1B, Supporting Information) allowed us to
estimate a Ki of 10 ( 2 µM (Table 1). The same protocol
was applied to determine Ki and the type of inhibitions
for 8-CH2tBuS-ATP (6b), (Figure S2A, Supporting In-
formation), 8-cycloheptylS-ATP (6a ) (Figure 3B), and
8-hexylS-ATP (6d ) (Figure S3, Supporting Information),
which were estimated at 45, 31, and 16 µM, respectively
(Table 1). In contrast, to analogues 6a and 6b (Figure
S2, Supporting Information), which produced a mixed
type of inhibition, 8-hexylS-ATP (6d ) behaves as a
noncompetitive inhibitor (Figure S3, Supporting Infor-
mation).
obtained upon dissolution of 10 in MeOH/0.25 M NaOH
and subsequent freeze-drying, was treated with the
appropriate alkyl bromide in DMF at 60 °C to yield
compounds 11 in high yields. Finally these compounds
were 5′-triphosphorylated,39 to give nucleotides 6 in
reasonable yields.
The thioether side chains varied in length (e.g., from
ethyl to butyl and hexyl, 6c, 6e, 6d ), and in bulkiness
(from ethyl to cycloheptyl and tert-butylmethylene, 6c,
6a , 6b), for evaluation of the effect of these parameters
on the inhibitory potential of these new derivatives. For
the sake of comparison, we also included 8-Br-ATP in
the above series, for studying possible electronic effects
of an electron-withdrawing group at the C8 position on
the affinity to NTPDase.
Hyd r olysis of ATP An a logu es. Hydrolysis rates of
ATP analogues 2a -d and 6-8 as well as 8-Br-ATP were
compared with ATP. As shown in Figure 1, NTPDase
hydrolyzes all the analogues bearing a substitution at
C2 of the purine ring, but the rate was slower than that
obtained with ATP. Hydrolysis rates of 2-BuS-ATP (2a )
and 2-BuS-ADP (2b) were 53% and 50% of that of ATP
hydrolysis, respectively, as compared to 65% and 75%
for 2-BuNH-ATP (2c) and 2-BuO-ATP (2d ), respectively
(Figure 1). In contrast, rates of hydrolysis of C8-
substituted analogues were much lower, some of them
being almost totally resistant. Indeed, 8-EtS-ATP (6c),
8-hexS-ATP (6d ), 8-BuNH-ATP (7), and 8-BuO-ATP (8),
were hydrolyzed at 15%, 11.6%, 23%, and 11% the rate
of ATP, whereas 8-BuS-ATP (6e), 8-CH2tBuS-ATP (6b),
and 8-cycloheptylS-ATP (6a ) were almost completely
resistant to NTPDase hydrolysis, with rates of hydroly-
sis 1,4%, 7.2%, and 6.6% the rate obtained with ATP,
respectively. For comparison, 8-Br-ATP was tested and,
as shown in Figure 1, it was hydrolyzed at about 83%
the rate of ATP. From these data, it appears that both
the position and nature of the substituents are impor-
tant to confer resistance to enzymatic hydrolysis. Thus,
analogues with electron-donating (ED) groups at C8, but
not 8-Br-ATP, are hydrolyzed at a lower rate than their
corresponding C2 analogues.
Eva lu a tion of 8-Bu S-ATP P oten cy a s a P 2X-
Recep tor Agon ist. It is well-known that P2X-R activa-
tion, at the surface of vascular smooth muscle cells,
causes vasoconstriction.1,14 In this respect, 8-BuS-ATP
(6e) was evaluated for P2X-R activity using the model
of the guinea pig mesenteric bed, treated with CHAPS
to remove the endothelium. Response to 200 µM of
noradrenaline was set at 100% (37 mmHg), correspond-
ing to the baseline used for the calculation of perfusion
pressure variations. Intra-arterial bolus administration
of increasing concentrations of 8-BuS-ATP (6e) (0.1-
1000 pmol) did not induce any significant variations of
perfusion pressure (Figure 2A). Effective removal of the
endothelium was shown by a lack of response to NK-1
([Sar9,Met(O2)11]SP), as compared to intact mesentery.
Denuded vessels were still responsive to NaNP (sodium
nitroprussiate) induced vasodilatation (Figure 2C), in-
dicating the integrity of the smooth muscle layer and
thereby confirming that 8-BuS-ATP did not interact
with the P2X purinoceptor associated to these cells.
In flu en ce of 8-Bu S-ATP on th e Va sod ila ta tion
In d u ced by ATP on a n In ta ct Mesen ter ic Bed . It
was shown, in a previous study, that 8-BuS-ATP (6e)
Kin et ic P a r a m et er s of Hyd r olyzed An a logu es.
Apparent Km and Vmax of ATP were estimated at 18 µM

2242 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
Gendron et al.
F igu r e 1. Hydrolysis of ATP and analogues by NTPDase. ATP and analogues were used at a concentration of 100 µM. Hydrolysis
was carried out at 37 °C for 7 min in the presence of 1.9 µg of protein. C2-Substituted analogues (2a -d ) are all hydrolyzed by the
enzyme, whereas C8-substituted analogues (6-8) are more or less resistant to NTPDase hydrolysis. Results are expressed as the
mean ( SEM of n replicates (see figure) carried out in triplicate.
Ta ble 1. Kinetic Parameters of Bovine Spleen NTPDase for ATP, ADP, and Purine Analoguesa
Vmax
substrates
ATP
Km (µM)
(µmol/min/mg of protein)
inhibitors
Ki (µM)
18 ( 1
33 ( 1
36 ( 6
63 ( 14
32 ( 8
28 ( 8
22 ( 5
12 ( 5
20 ( 7
26 ( 5
1.65 ( 0.10
1.30 ( 0.08
0.83 ( 0.05
0.94 ( 0.10
0.99 ( 0.10
0.82 ( 0.09
0.63 ( 0.04
0.30 ( 0.03
0.28 ( 0.03
0.20 ( 0.01
8-cycloheptylS-ATP
8-CH2tBuS-ATP
8-hexylS-ATP
6a
6b
6d
6e
31 ( 2.5
45 ( 2.5
16 ( 2.0
10 ( 2.0
ADP
2-BuS-ATP
2-BuS-ADP
2-BuNH-ATP
2-BuO-ATP2d
8-Br-ATP
8-EtS-ATP
8-BuNH-ATP
8-BuO-ATP
2a
2b
2c
2d
8-BuS-ATP
6c
7
8
a
Experiments were carried out in triplicate and results are expressed as the mean ( SEM of the best fit obtained with GraFit 4
software (Erithacus, UK). Apparent Km and Vmax were estimated from the Eadie and Hofstee representation and Ki values were from
Dixon plots.
did not activate the P2Y1 receptor, in vitro.39 We showed
here that 8-BuS-ATP (7 µM) did not modify the response
of the isolated mesenteric bed to ATP. From these
experiments it appears that this analogue is also devoid
of any agonistic effect on the P2Y-receptor (Figure 2B).
The integrity of the endothelium was assessed and
confirmed with NK-1 (100 pmol) and NaNP (3 nmol)
(Figure 2C).
otidases to a certain extent.34 Recently, we demonstrated
that ecto-NTPDase (EC 3.6.1.5) was involved in the
hydrolysis of some purine nucleotide analogues, e.g.,
2-thioether-ATP-R-S derivatives, 2-Cl-ATP, and 2-CH3S-
ATP.34,40 In contrast, 2-thioether-AMP-S (4) was re-
sistant to hydrolysis and was an inhibitor of NTPDase.40
To reduce potential artifacts resulting from the solu-
bilization of NTPDase by detergents, a particulate
fraction was used. It is important to mention that there
was no other detectable level of ATPase or ADPase, in
the preparation, than those attributable to NTPDase.
Also, possible alkaline phosphatase activity was inhib-
ited by tetramisole added to our assays. In view of
finding a specific and potent NTPDase inhibitor, in the
present study we examined two series of ATP analogues
substituted at positions C2 and C8, respectively. First,
we tested their resistance to NTPDase hydrolysis and
it clearly appeared that analogues substituted with
electron-donating groups at C8 were more resistant to
hydrolysis than the corresponding C2-substituted ana-
logues. However, a significant level of hydrolysis was
found with 8-Br-ATP, 8-BuNH-ATP (7), 8-BuO-ATP (8),
and 8-EtS-ATP (6c), whereas 8-cycloheptylS-ATP (6a ),
8-CH2tBuS-ATP (6b), 8-hexylS-ATP (6d ), and 8-BuS-
ATP (6e), were resistant to hydrolysis by NTPDase. It
Discu ssion
Extracellular purines, namely ATP and ADP, are
involved in many physiological processes.1-5 These
molecules are rapidly dephosphorylated by ectonucleo-
tidases, mainly by the ecto-nucleotide triphosphate
diphosphohydrolase.16-19,44 To define the role played by
extracellular nucleotides and to develop drugs based on
P2-R ligands, it is desirable to block ectonucleotidases
activity or alternatively to use nonhydrolyzable ana-
logues of these nucleotides as agonists.37,45-46 In previ-
ous studies, several analogues of ATP were found to be
hydrolyzed by ectonucleotidases.32,47-49 However, P2-
purinoceptors agonists modified on the phosphate chain,
such as ADP-â-S, R,â-, or â,γ-methylene-ATP, and â,γ-
imido-ATP (1a -d ), were generally resistant to hydroly-
sis by NTPDase and were also inhibitors of ectonucle-

Novel Inhibitors of NTPDases
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2243
tolerated by the tentative P2Y1-R binding site.39,50,51 In
contrast, the NTPDase active site can accommodate
8-thioether-ATP analogues, and even derivatives bear-
ing large or bulky substituents (8-CH2tBuS-ATP, and
8-cycloheptylS-ATP, and 8-hexylS-ATP), probably in
their expected syn conformation. This conformation is
likely unfavorable for catalytic activity, namely, the
orientation of the triphosphate chain in the syn confor-
mation is probably shifted away from the catalytic
amino acid residues.
A)
Having established that these 8-thioether-ATP de-
rivatives were not significantly hydrolyzed, we then
investigated their potency as NTPDase inhibitors. One
striking feature is that the four derivatives, 6a ,b,d ,e
are good inhibitors. Again the affinity (Ki) falls in the
same range of affinity as that of ATP and ADP, with
one exception, 8-CH2tBuS-ATP, which has a slightly
higher Ki. However, even if these four analogues all
display efficient NTPDase inhibition, we consider 8-BuS-
ATP, 6e, as the inhibitor of choice, because it exhibits
competitive inhibition with a Ki lower than those
measured for compounds 6a ,b,d , which are less stable
analogues toward NTPDase hydrolysis. The fact that
this inhibitor interacts specifically with the binding site
of the enzyme potentially reduce the risk of interference
with other ATP-binding enzymes or receptors. 8-CH2-
tBuS-ATP (6b) and 8-cycloheptylS-ATP (6a ) showed
mixed types of inhibition, thereby complicating the
interpretation of their interaction with NTPDase. The
mixed type of inhibition obtained with these two inhibi-
tors may be explained by a new theoretical mechanism
derived from a mechanism proposed by Westley.52 We
know that the enzyme could exist in either a monomer,
dimer, and tetramer.53,54 Polymerization would modify
the protein in such a way to unmask the hidden
catalytic site. In the case mentioned above, the inhibitor
would favor a dissociation of the polymer and on the
other hand would also interact with the catalytic site,
thereby explaining the mixed type of inhibition. In
agreement with this hypothesis, recent studies of Kir-
ley53 and Guidotti54 have shown that polymerization
may be an important modulator of NTPDase catalytic
activity. In this context, an inhibitor that favors a
dissociation of the enzyme polymers would reduce its
activity while competing at the same time with ATP for
the catalytic site of either the polymeric form of the
NTPDase or for the catalytic site of subunits. This new
theoretical model could also explain the characteristic
sensitivity of the NTPDase to detergents. In the case of
the analogue 6d , it gives rise to noncompetitive inhibi-
tion, thereby suggesting that its inhibitory effects are
the results of an interaction with another part of the
enzyme distinct from the catalytic site. From these
results, the catalytic site of NTPDase would apparently
tolerate long and bulky substituents at the C8 position
and also tolerate nucleotides in syn and anti conforma-
tions. The compounds with the same electronic nature
of the modified purine ring have the same affinity for
the catalytic site, since thioether, amino and ether-ATP
derivatives show similar apparent affinity (Km or Ki) for
the enzyme. Moreover, we could hypothesize that there
is a hydrophobic pocket of limited volume which inter-
acts favorably with the hydrophobic side chain at C8
position. Furthermore, the sulfur atom is capable of
B)
C)
F igu r e 2. Purinergic activity of 8-BuS-ATP (6e). Effect of
8-BuS-ATP (0.1-1000 pmol) on denuded mesenteric bed of
guinea pig. Results are expressed as a percentage of the control
(control ) 37 mmHg). No variations of perfusion pressure were
measured. Results are the mean ( SEM of at least three
experiments. Effect of 8-BuS-ATP on the relaxing effect of ATP
on intact mesenteric bed. ATP (0.1-10000 pmol) in the
presence (open bars) or in the absence (control, closed bars) of
7 µM of 8-BuS-ATP. Results are expressed as a percentage of
vasodilatation measured from a precontracted vessel (200 µM
of noradrenaline). No significant difference. Results are the
mean ( SEM of at least three experiments. Vascular responses
with or without endothelium. Endothelium integrity was
tested with 100 pmol of NK-1 (closed bar) and blood vessels
responsiveness was evaluated by 3 nmol of NaNP (open bar).
Results are expressed as a percentage of vasodilatation
measured from a precontracted vessels (200 µM of noradrena-
line). In denuded mesentery there is significant response to
NK-1. Results are the mean of three experiments or more.
is noteworthy that all the C2-substituted molecules
displayed Km in range found for ATP and ADP. This
indicates that affinity for the catalytic site is equivalent
for all these analogues. Hence, the position and nature
of the substituent is clearly important for resistance to
the catalytic activity of the enzyme.
Hydrophobic interactions and H-bonds of the C2
substituent appear to be important determinants for
P2Y-R ligand affinity. The conformational preference of
the ligands in solution, determined by NMR experi-
ments, may explain in part the differences in P2Y-R
potency between the 2- and 8-substituted compounds.
All 2-substituted derivatives possess an anti conforma-
tion, whereas the 8-ether and thioether analogues are
in the syn conformation.39 The latter are apparently not

2244 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
Gendron et al.
phosphate (TBAP) in methanol and (B) 60 mM ammonium
phosphate and 5 mM TBAP in 90% water/10% methanol,
applying a gradient of 25% A to 75% A in 20 min.
H-bonding with nearby H-bond donors in the catalytic
site of NTPDase. These factors may explain the differ-
ences between 8-Br-ATP, 8-BuNH-ATP (7), 8-BuO-ATP
(8), and 8-BuS-ATP (6e). Studies are currently carried
out to map the NTPDase binding site and to identify
the major binding determinants of this enzyme.
8-Mer ca p toa d en osin e (10). NaSH (0.8 g, 10 equiv) was
added to a solution of 8-bromoadenosine (0.5 g, 1.44 mmol) in
DMF (7 mL). The mixture was warmed to 100 °C and a few
drops of water were added to improve solubility. The mixture
was stirred at 100 °C overnight. The solvent was evaporated
under high vacuum and the residue was coevaporated repeat-
edly with MeOH, until the residue turned into a solid. The
residue was dissolved in water and neutralized with NaOH.
After freeze-drying, the product was purified on a silica gel
column (CHCl3:MeOH 10:1). The product was obtained as a
yellowish powder (100% yield, mp 169-170 °C). 1H NMR (CD3-
OD, 200 MHz) 8.09 (s, 1H, H-2), 6.65 (d, J ) 7 Hz, 1H, H-1′),
5.01 (dd, J ) 7, 5.5 Hz,1H, H-2′), 4.39 (dd, J ) 5.5, 2.5 Hz,
1H, H-3′), 4.13 (q, J ) 2.5 Hz, 1H, H-4′), 3.87 (dd, J ) 12.5,
2.5 Hz, 1H, H-5′), 3.71 (dd, J ) 12.5, 3 Hz, 1H, H-5′). 13C NMR
(CD3OD, 300 MHz) δ 167.88 (C-6), 151.92 (C-2), 148.12 (C-4),
The second aspect considered in this study was the
interference with purinoceptors. Recent studies have
indicated that 8-BuS-ATP was a poor agonist for P2Y1
receptor. In rat astrocytes this compound has no effect
on the [Ca2+]i level, whereas the corresponding 2-sub-
stituted ATP analogues potently increased the [Ca2+
]
i
level. Similar results were obtained with turkey eryth-
rocytes membranes.39 Since this molecule qualifies as
a potent inhibitor, we tested its influence in the isolated
mesenteric bed of the guinea pig for potential P2X and
P2Y purinergic effects. Isolated guinea pig mesenteric
artery and vein have been shown to respond to ATP via
a P2X-purinoceptor located at the surface of the smooth
muscle cells.55,56 This latter purinoceptor was sensitive
to R,â-CH2-ATP (1b), a P2X agonist.57,58 CHAPS was
used to remove the blood vessel endothelium, which
gives rise to endothelium denuded vessels. Nonfunc-
tional endothelium has been shown by a lack of response
to 100 pmol of NK-1.59 Our data show that 8-BuS-ATP
was not able to initiate any P2X-R effect at concentra-
tions up to 1000 pmol, indicating that it does not
interact with P2X-R. We also evaluated the effect of an
infusion of 8-BuS-ATP on the vasodilatory response
induced by administration of increasing ATP concentra-
tions (0.1-10 000 pmol), in intact mesenteric bed. Even
in the presence of 7 µM 8-BuS-ATP, the response to ATP
was unmodified, confirming that 8-BuS-ATP did not
interact with P2Y-R.
147.88 (C-8), 107.00 (C-5), 88.62 (C-1′), 85.59 (C-4′), 70.70 (C-
+
2′), 70.62 (C-3′), 62.13 (C-5′). MS (CI/NH3): m/z 317 M + NH4
.
8-(Th iocycloh ep tyl)a d en osin e (11a ). A suspension of
8-mercaptoadenosine (75 mg, 0.25 mmol) in MeOH (2 mL) was
dissolved in 0.25 M NaOH (1 mL). The clear, yellow solution
was stirred at room temperature for 1 h. After freeze-drying,
the thiolate sodium salt, obtained as a yellowish solid, was
dissolved in dry DMF (2 mL) and bromocycloheptane (38 µL,
1.1 equiv) was added. The solution was stirred overnight under
nitrogen at 60 °C. The solvent was evaporated under high
vacuum and the yellow residue was coevaporated repeatedly
with MeOH, until the residue turned into a yellow solid. The
solid was triturated with petroleumether/ether 1:1, and then
separated on a silica gel column (CHCl3:MeOH 20:1). Product
11a was obtained as a white solid in 83% yield (82 mg) after
evaporation and drying, mp 205-6 °C. 1H NMR (DMSO-d6,
200 MHz): 8.07 (s, 1H, H-2), 7.36 (br s, 2H, NH2), 5.84 (d, J )
7 Hz, 1H, H-1′), 5.00 (dd, J ) 7, 5 Hz, 1H, H-2′), 4.16 (dd, J )
5, 2 Hz, 1H, H-3′), 4.07-3.91 (m, 2H, H-4′ & SCH), 3.68 (dd,
J ) 12, 4 Hz, 1H, H-5′), 3.51 (dd, J ) 12.5, 4 Hz, 1H, H-5′),
2.19-1.93 (m, 2H), 1.84-1.42 (m, 11H). 13C NMR (DMSO-d6,
300 MHz): δ 154.80 (C-6), 151.51 (C-2), 150.03 (C-4), 147.94
(C-8), 119.75 (C-5), 88.96 (C-1′), 86.64 (C-4′), 71.31 (C-2′), 71.08
(C-3′), 62.27 (C-5′), 48.42 (SCH), 34.51 (CH2), 34.14 (CH2),
27.77 (CH2), 27.76 (CH2), 25.14 (CH2), 25.04 (CH2). FAB
(positive): m/z 396 MH+.
In conclusion, our study demonstrates the potential
of a new class of ATP analogues as inhibitors (C8
substituted) of this novel family of proteins (NTPDases)
that play a key role in the extracellular metabolism of
nucleotides in the different physiological systems of
vertebrates.
8-(Th io-2,2-d im eth ylp r op yl)a d en osin e (11b). The com-
pound was prepared as described for 8-(thiocycloheptyl)ad-
enosine and obtained in 79% yield (73 mg) as a yellowish solid,
mp 141-2 °C. 1H NMR (CD3OD, 200 MHz) δ 8.06 (s, 1H, H-2),
6.02 (d, J ) 7 Hz, 1H, H-1′), 4.99 (dd, J ) 7, 5 Hz, 1H, H-2′),
4.33 (dd, J ) 5, 2 Hz, 1H, H-3′), 4.17 (“q”, J ) 2 Hz, 1H, H-4′),
3.88 (dd, J ) 12.5, 2.5 Hz, 1H, H-5′), 3.72 (dd, J ) 12.5, 3 Hz,
1H, H-5′), 3.42 and 3.35 (AB, J ) 7 Hz, 2H, SCH), 1.09 (s, 9H,
SCH2(CH3)3). 13C NMR (CD3OD, 300 MHz) 155.92 (C-6), 152.91
(C-4), 152.11 (C-2), 151.69 (C-8), 110.64 (C-5), 91.19 (C-1′),
88.89 (C-4′), 74.13 (C-2′), 73.20 (C-3′), 64.17 (C-5′), 47.69
(SCH2), 29.07 (3C, SCH2(CH3)3). MS (CI/NH3) m/z: 368 (M-
H)+.
Exp er im en ta l Section
Ch em istr y. Gen er a l. New compounds were characterized
by proton and carbon nuclear magnetic resonance using a
Bruker AC-200 or DPX-300 spectrometer. The chemical shifts
are reported in ppm relative to TMS as an internal standard.
Nucleotides were characterized also by 31P NMR in D2O using
85% H3PO4 as an external reference on a Bruker AC-200
spectrometer. Mass spectra were recorded on an AutoSpec-E-
FISION VG high-resolution mass spectrometer. Nucleotides
were characterized by FAB (fast atom bombardment) and high-
resolution FAB using a glycerol matrix under FAB negative
conditions on an AutoSpec-E-FISION VG high-resolution mass
spectrometer. Separation of the newly synthesized nucleotides
was achieved on an LC (Isco UA-6) using DEAE A-25 Sepha-
8-(Th ioeth yl)a d en osin e (11c). A suspension of 8-mercap-
toadenosine (270 mg, 0.9 mmol, in 7 mL MeOH) was dissolved
in 0.25 M NaOH (3.6 mL). The clear, yellow solution was
stirred at room temperature for 1 h. After freeze-drying, the
thiolate sodium salt, obtained as a yellowish solid, was
dissolved in dry DMF (3 mL) and bromoethane (101 µL, 1.5
equiv) was added. The solution was stirred under nitrogen at
room temperature for 3 h. The solvent was evaporated under
high vacuum and the yellow residue was coevaporated repeat-
edly with MeOH, until the residue turned into a yellow solid.
The solid was separated on a silica gel column (CHCl3:MeOH
15:1). Product 11c was obtained as a white solid in 93% yield
after evaporation and drying (273 mg), mp 176 °C. 1H NMR
(DMSO-d6, 200 MHz): 8.05 (s, 1H, H-2), 7.31 (br s, 2H, NH2),
5.76 (d, J ) 7 Hz, 1H, H-1′), 5.66 (dd, J ) 8.5, 3.5 Hz, 1H,
-
dex (HCO3 form) anion exchanger as described below. Final
purification was done on an HPLC (Merck-Hitachi) system
using a semipreparative LiChroCART LiChrospher 60 RP-
select B column (1 × 25 cm, Merck KgaA) and a linear gradient
of 0.1 M triethylammonium acetate buffer (TEAA, pH 7.5) and
methanol (see below) at 6 mL/min flow rate. For analytical
purposes, a LiChroCART LiChrospher 60 RP-select B column
(250 mm × 4.6 mm, Merck KGaA) was used by applying the
same gradient as above at 1 mL/min flow rate. The purity of
the nucleotides described below was evaluated on an analytical
column in two different solvent systems. One solvent system
(I) was 0.1 TEAA/CH3OH, 80:20 to 20:80 in 20 min. The second
solvent system (II) was (A) 5 mM tetrabutylammonium

Novel Inhibitors of NTPDases
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2245
OH-5′), 5.42 (d, J ) 6 Hz, 1H, OH-2′), 5.21 (d, J ) 4 Hz, 1H,
OH-3′), 4.99 (br q, J ) 6 Hz, 1H, H-2′), 4.15 (br s, 1H, H-3′),
3.96 (br s, 1H, H-4′), 3.68 (dt, J ) 12, 3.5 Hz, 1H, H-5′), 3.60-
3.44 (m, 1H, H-5′), 3.43-3.14 (m, 1H, SCH2CH3), 1.36 (t, J )
7 Hz, 3H, SCH2CH3). 13C NMR (DMSO-d6, 300 MHz): 154.49
(C-6), 151.30 (C-2), 150.40 (C-4), 148.53 (C-8), 119.66 (C-5),
88.86 (C-1′), 86.63(C-4′), 71.29 (C-2′), 70.01 (C-3′), 62.24 (C-
5′), 26.77 (SCH2CH3), 14.85(SCH2CH3). MS (CI/CH4): m/z 328
MH+; High-resolution MS: calcd for C12H18N5O4S 328.1079,
found 328.1069.
8-(Th io-n -h exyl)a d en osin e (11d ). The compound was
prepared as described for 8-(thioethyl)adenosine and obtained
in 91% yield (314 mg) as a white solid, mp 169-171 °C. 1H
NMR (DMSO-d6, 200 MHz): δ 8.05 (s, 1H, H-2), 7.29 (br s,
2H, NH2), 5.77 (d, J ) 7 Hz, 1H, H-1′), 5.67 (dd, J ) 9, 3.5 Hz,
1H, OH-5′), 5.42 (d, J ) 6 Hz, 1H, OH-2′), 5.21 (d, J ) 4 Hz,
1H, OH-3′), 4.99 (“q”, J ) 6 Hz, 1H, H-2′), 4.15 (br s, 1H, H-3′),
3.96 (br s, 1H, H-4′), 3.67 (dt, J ) 12, 3.5 Hz, 1H, H-5′), 3.60-
3.43 (m, 1H, H-5′), 3.42-3.18 (m, 1H, SCH2), 1.69 (quint, J )
7 Hz, 2H, SCH2CH2), 1.50-1.18 (m, 6H, CH2CH2CH2CH3), 0.86
(t, J ) 7 Hz, 3H, CH3). 13C NMR (DMSO-d6, 300 MHz): 154.55
(C-6), 151.27 (C-2), 150.38 (C-4), 148.71 (C-8), 119.61 (C-5),
88.85 (C-1′), 86.63(C-4′), 71.28 (C-2′), 71.02 (C-3′), 62.23 (C-
5′), 32.37 (SCH2), 30.70 (CH2), 28.80 (CH2), 27.71 (CH2), 21.99
(CH2), 13.88 (CH3). MS (CI/CH4): m/z 384 MH+. High-resolu-
tion MS: calcd for C16H26N5O4S 384.1705, found 384.1696.
282 nm. tR: 15.25 min (>97% purity) using solvent system I,
15.52 min (>97% purity) using solvent system II.
En zym ology. Rea gen ts a n d Solu tion s. ATP, tetramisole,
malachite green, bovine serum albumin fraction V (BSA),
CHAPS, sodium nitroprussiate (NaNP), (-)-arterenol bitar-
trate (noradrenaline), heparin, and indomethacin were obtain
from Sigma Chemical Co. (St. Louis, MO). ADP was obtained
from Roche (Laval, QC, Canada), and Bradford reagent was
purchased from Bio-Rad Laboratories (Mississauga, Ontario,
Canada). [Sar9,Met(O2)11]SP (NK-1) was synthesized by Dr.
W. Neugebauer from the Universite´ de Sherbrooke. Prepara-
tions of Krebs and phosphate-buffered saline (PBS) were as
followed: Krebs solution, 5.5 mM glucose, 117.5 mM NaCl,
1.2 mM MgSO4, 1.2 mM KH2PO4, 4.7 Mm KCl, 2.5 Mm CaCl2,
25 Mm NaHCO3, pH 7.4; PBS, 137 mM NaCl, 3 mM KCl, 10
mM Na2HPO4 and 1.7 mM KH2PO4, pH 7.4. All the other
reagents were of analytical grade and bought from Sigma
Chemical Co. (St. Louis, MO).
En zym a tic Assa ys. Isola tion of P a r ticu la te F r a ction s.
Bovine spleens were obtained from a local slaughterhouse, and
isolation of the particulate fraction was carried out as previ-
ously described.23
NTP Da se Assa ys. Enzyme activity was routinely measured
by the release of inorganic phosphorus with the malachite
green colorimetric assay.60 Resistance to hydrolysis was mea-
sured at 37 °C in 1 mL of the following incubation medium:
8mM CaCl2, 5 mM tetramisole, 50 mM Tris base, 50 mM
imidazole, buffered at pH 7.6, and 100 µM of either ATP or
its analogues. Apparent Km and Vmax values for ATP, ADP,
and each of the hydrolyzable purine nucleotide analogues were
derived from Eadie and Hofstee plots, with substrate concen-
trations ranging between 10 and 300 µM for ATP and ADP,
and between 15 and 100 µM for the analogues, unless stated
otherwise. In both cases the reaction was started by the
addition of 1.9 µg of the enzyme preparation and stopped after
7 min with 250 µL of the malachite green reagent. Apparent
Ki values for nonhydrolyzable purine nucleotide analogues
were derived from Dixon replots, using inhibitor concentrations
ranging from 0 to 100 µM. Reactions were performed in the
same incubation buffer, as previously described, and were
started by the addition of nonsaturating ATP concentrations.
Protein concentration was determined with the Bradford
microplate assay using bovine serum albumin as a standard
of reference.61
Nu cleosid e 5′-Tr ip h osp h or yla tion . Nucleosides 11a -d
were 5′-triphosphorylated according to a literature procedure.38
8-(Th iocycloh ep tyl)a d en osin e 5′-tr ip h osp h a te (6a ) was
obtained in 60% yield (79 mg). Final separation was achieved
via HPLC by applying a linear gradient of TEAA/CH3OH 70:
1
30 to 20:80 in 20 min (6 mL/min), tR 9.53 min. H NMR (D2O,
200 MHz): δ 8.17 (s, 1H, H-2), 6.09 (d, J ) 6 Hz, 1H, H-1′),
5.15 (t, J ) 6 Hz, 1H, H-2′), 4.62-4.51 (m, 1H, H-3′), 4.37-
4.14 (m, 3H, H-4′ & H-5′), 3.93-3.75 (m, 1H, SCH), 2.15-1.90
(m, 2H),1.80-1.36 (m, 11H). 31P NMR (D2O, 200 MHz, pH 9)
δ -5.34 (d), -10.37 (d), -21.32 (t). UV: λmax 282 nm. HRFAB:
calcd for C17H27N5O13P3S 634.0539, found 634.0540. tR: 14.99
min (95% purity) using solvent system I, 13.19 min (97%
purity) using solvent system II.
8-(Th io-2,2-d im eth ylp r op yl)a d en osin e 5′-tr ip h osp h a te
(6b) was obtained in 65% yield (77 mg). Final separation was
achieved via HPLC by applying a linear gradient of TEAA/
1
CH3OH 70:30 to 20:80 in 20 min (6 mL/min), tR 7.51 min. H
NMR (D2O, 200 MHz): δ 8.21 (s, 1H, H-2), 6.13 (d, J ) 6 Hz,
1H, H-1′), 5.20 (t, J ) 6 Hz, 1H, H-2′), 4.62 (dd, J ) 6 Hz, 1H,
H-3′), 4.42-4.25 (m, 3H, H-4′ & H-5′), 3.29 and 3.35 (ABq, J
) 12 Hz, 2H, SCH2), 1.06 (s, 3H, SCH2(CH3)3). 31P NMR (D2O,
200 MHz, pH 9) δ -10.23 (d), -10.79 (d), -22.61 (t). UV: λmax
282 nm. HRFAB: calcd for C15H25N5O13P3S 608.0382, found
608.0360. tR: 12.71 min (96% purity) using solvent system I,
11.31 min (95% purity) using solvent system II.
P 2-Recep tor s Assa ys. Su r gica l P r oced u r es. Dunkin-
Hartley guinea pig (300-350 g) of either sex were sacrificed
by cervical dislocation following the Canadian Council on
Animal Care. The guinea pig mesentery was prepared as
described by Berthiaume et al.59 Briefly, the colic and ileocolic
branches of the superior mesenteric artery were tied and the
superior mesenteric artery cannulated (Portex size tube 3FG).
To isolate the mesenteric bed from the intestine, the mesentery
was perfused (2 mL/min, for 5 min) via the mesenteric artery
with a Krebs solution containing heparin (100 U/mL). The
mesentery was then separated by cutting close to the intestine.
A resting period of 60 min was then allowed during which time
the guinea pig mesenteric bed was perfused (2 mL/min) with
a warmed (37 °C) and gassed Krebs solution (95% O2 and 5%
CO2) containing indomethacin (5 µM), as described earlier. In
all the assays, the perfusion pressure was increased to obtain
a flow rate of 6 mL min-1. The response of mesenteric bed,
precontracted with noradrenaline (200 µM) in 0.9% saline
solution, to the different drugs was measured with a pressure
transducer (Statham, model P-23AC) and recorded on a Grass
physiograph (model 79D).
P 2X-Recep tor Assa ys. Guinea pig mesenteric bed was
denuded from its endothelium layer by using 20 mM of CHAPS
in PBS.59 Briefly, the CHAPS solution was infused for 45 s,
followed by a resting period of 30 min. Finally, a second 45 s
infusion of 20 mM CHAPS was carried out. Blood vessels were
then precontracted as earlier described. The efficiency of the
endothelium removal technique was assessed by an intra-
arterial bolus injection of 100 pmol of NK-1 in PBS. Reactivity
of the media layer was confirmed by bolus a injection of 3 nmol
8-(Th ioeth yl)a d en osin e 5′-tr ip h osp h a te (6c) was ob-
tained in 43% yield (84 mg). Final separation was achieved
via HPLC by applying a linear gradient of TEAA/CH3OH 90:
1
10 to 20:80 in 20 min (6 mL/min), tR 8.11 min. H NMR (D2O,
200 MHz): 8.15 (s, 1H, H-2), 6.10 (d, J ) 6.5 Hz, 1H, H-1′),
5.17 (t, J ) 6.5 Hz, 1H, H-2′), 4.65-4.55 (m, 1H, H-3′), 4.40-
4.16 (m, 3H, H-4′ & H-5′), 3.30 and 3.26 (ABq of t, J ) 11.5, 7
Hz, 1H each, SCH2), 1.39 (t, J ) 7 Hz, 3H, CH3). 31P NMR
(D2O, 200 MHz, pH 9) -5.12 (d), -10.31 (d), -20.98 (t). UV:
λmax 282 nm. HRFAB: calcd for C12H18N5O13NaP3S 587.9732,
found 587.9650. tR: 7.16 min (96% purity) using solvent system
I, 3.55 min (94% purity) using solvent system II.
8-(Th io-n -h exyl)a d en osin e 5′-tr ip h osp h a te (6d ) was
obtained in 58% yield (111 mg). Final separation was achieved
via HPLC by applying a linear gradient of TEAA/CH3OH 70:
30 to 20:80 in 20 min (6 mL/min), tR 10.73 min. 1H NMR (D2O,
200 MHz): 8.17 (s, 1H, H-2), 6.01 (d, J ) 6.5 Hz, 1H, H-1′),
5.19 (t, J ) 6.5 Hz, 1H, H-2′), 4.64-4.55 (m, 1H, H-3′), 4.40-
4.14 (m, 3H, H-4′ & H-5′), 3.32 and 3.24 (ABq of t, J ) 14, 7
Hz, 1H each, SCH2), 1.73 (“quint”, J ) 7 Hz, 2H, SCH2CH2),
1.52-1.09 (m, 6H), 0.82 (t, J ) 7 Hz, 3H, CH3); 31P NMR (D2O,
200 MHz, pH 9) -5.12 (d), -10.25 (d), -21.03 (t). UV: λmax

2246 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
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each ATP injection allowed return to baseline, as earlier
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thelium layer was confirmed by injection of NK-1 (100 pmol).
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Purification of the blood vessel ATP diphosphohydrolase, iden-
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Ack n ow led gm en t. This work was supported by the
NSERC of Canada and FCAR. F.-P. Gendron is holder
of a research traineeship from the Heart and Stroke
Foundation of Canada. The authors (B.F. and E.H.)
thank the Marcus Center for Pharmaceutical and
Medicinal Chemistry for financial support.
Su p p or tin g In for m a tion Ava ila ble: Figure S1, the
inhibitory effect of 8-BuS-aTP (6e) on ATP hydrolysis by
NTPDase; Figure S2, Dixon representations of 8-CH2tBuS-
ATP (6b) and 8-cycloheptylS-ATP (6a ) inhibition; and Figure
S3, Dixon representation of 8-thiohexyl-ATP (6d ) inhibition.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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