2-thioether 5'-O-(1-thiotriphosphate)adenosine derivatives as new insulin secretagogues acting through P2Y-receptors

Article abstract of DOI:10.1021/jm990158y

P2-Receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were identified also on pancreatic B cells and are involved in insulin secretion. Therefore, novel P2Y-R ligands, 2-thioether 5'-O-phosphorothioate adenosine derivatives

Full text of DOI:10.1021/jm990158y

3636
J . Med. Chem. 1999, 42, 3636-3646
2-Th ioeth er 5′-O-(1-Th iotr ip h osp h a te)a d en osin e Der iva tives a s New In su lin
Secr eta gogu es Actin g th r ou gh P 2Y-Recep tor s
Bilha Fischer,*,† Ana Chulkin,† J ose L. Boyer,‡ Kendall T. Harden,‡ Fernand-Pierre Gendron,§
Adrien R. Beaudoin,§ J eannie Chapal, Dominique Hillaire-Buys, and Pierre Petit
Department of Chemistry, Gonda-Goldschmied Medical Research Center, Bar-Ilan University, Ramat-Gan 52900, Israel,
Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599,
Biology Sciences, University of Sherbrooke, J 1K 2R1 Canada, and Laboratory of Pharmacology,
Research Unit UPRES EA 1677, School of Medicine, University of Montpellier I, 34060 Montpellier Cedex 1, France
Received April 1, 1999
P2-Receptors (P2-Rs) represent significant targets for novel drug development. P2-Rs were
identified also on pancreatic B cells and are involved in insulin secretion. Therefore, novel
P2Y-R ligands, 2-thioether 5′-O-phosphorothioate adenosine derivatives (2-RS-ATP-R-S), were
synthesized as potential insulin secretagogues. An efficient synthesis of these nucleotides and
a facile method for separation of the chiral products are described. The enzymatic stability of
the compounds toward pig pancreas type I ATPDase was evaluated. The rate of hydrolysis of
2-hexylthio-5′-O-(1-thiotriphosphate)adenosine (2-hexylthio-ATP-R-S) isomers by ATPDase was
28% of that of ATP. Some 2-thioether 5′-(monophosphorothioate)adenosine derivatives (2-RS-
AMP-S) exerted an inhibitory effect on ATPDase. The apparent affinity of the compounds to
P2Y1-R was determined by measurement of P2Y-R-promoted phospholipase C activity in turkey
erythrocyte membranes. 2-RS-ATP-R-S derivatives were agonists, stimulating the production
of inositol phosphates with K0.5 values in the nanomolar range. 2-RS-AMP-S derivatives were
full agonists, although 2 orders of magnitude less potent. All the compounds were more potent
than ATP. The effect on insulin secretion and pancreatic flow rate was evaluated on isolated
and perfused rat pancreas. A high increase, up to 500%, in glucose-induced insulin secretion
was due to addition of 2-hexylthio-ATP-R-S in the nanomolar concentration range, which
represents 100-fold enhancement of activity relative to ATP. 2-Hexylthio-AMP-S was 2.5 orders
of magnitude less effective.
In tr od u ction
in a large increase in potency (2-5 orders of magnitude)
toward P2Y1-R compared with ATP.8-10 For instance,
some 2-thioether ATP derivatives stimulated the pro-
duction of inositol phosphates in turkey erythrocyte
membranes, through the activation of P2Y1-R, with K0.5
values in the nanomolar range.8a Other 2-thioether ATP
derivatives inhibited c-AMP accumulation in C6 rat
glioma cells with EC50 values in the picomolar range.10
Some 2-thioether AMP derivatives proved to be full
agonists, whereas AMP was practically inactive.8 More-
over, a long C2 thioether substitution on ATP rendered
the molecule relatively stable to enzymatic hydrolysis11
and also affected the receptor subtype selectivity, mak-
ing these derivatives active mainly at the P2Y-R
subtypes.8a,9
P2-Receptors (P2-Rs) are membrane proteins that
lead to inhibitory or excitatory effects upon binding
ADP, ATP, or, in some subtypes, UTP.1 A distinction
was made between G-protein-coupled receptors and
ligand-gated ion-channel receptors as the basis of the
separation of P2-R into two broad classes: P2Y and P2X,
respectively.2 P2-Rs represent significant targets for
novel drug development for a variety of pathophysi-
ological conditions.3 Moreover, the large heterogeneity
of P2-R subtypes in different tissues opens the possibil-
ity of developing selective organ- or tissue-specific P2-
R-targeted drugs.
P2-Rs have been characterized on pancreatic B cells4
including P2Y-R subtypes.5 Their activation by extra-
cellular ATP results in stimulation of insulin secretion.
Various selective ligands have been shown to increase
insulin secretion and decrease glycemia in vivo.6 There-
fore, P2Y-R agonists may be considered as novel insulin-
releasing compounds, with potential for the treatment
of type 2 diabetes (non-insulin-dependent diabetes,
NIDDM).7
The relative metabolic stability of nucleoside-5′-phos-
phorothioates is well-documented. For instance, AMP-S
is relatively resistant to enzymatic transformations
by adenylate deaminase, adenylate kinase, and 5′-
nucleotidase,12,13 and ATP-R-S diastereoisomers exhibit
selective metabolic stability.14 In addition to their
relative metabolic stability, phosphorothioates are
also characterized by their higher acidity relative to
phosphates.12,15-17 Since electrostatic interactions of the
ATP phosphate chain with positively charged amino
acid residues at the P2Y-R binding site are likely a
major determinant for binding,18 it is expected that the
higher acidity of nucleoside-phosphorothioates will en-
hance binding affinity to P2Y-R. Indeed, ATP-R-S, Rp
In previous studies, we showed that long thioether
chain substitutions at the C2 position of ATP resulted
* Address correspondence to Bilha Fischer at Bar-Ilan University.
Fax: 972-3-5351250. Tel: 972-3-5318303. E-mail: bfischer@mail.biu.ac.il.
† Bar-Ilan University.
‡ University of North Carolina.
§ University of Sherbrooke.
University of Montpellier I.
10.1021/jm990158y CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/17/1999

Adenosine Derivatives as Insulin Secretagogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3637
Sch em e 1. Synthesis of 2-Thioether 5′-O-(Phosphorothioate)adenosine Derivatives
isomer, a P2Y-R selective ligand, was 50 times more
potent than ATP at inducing relaxation of guinea pig
taenia coli.19
treated with NaOH/MeOH, and the isolated sodium
thiolate salt was selectively S-alkylated with an alkyl
bromide in DMF. The selection of the side chains at C2
is based on our previous results.8-10 For the preparation
of the corresponding 5′-phosphorothioate-nucleosides,
the resulting 2-thioether adenosine derivatives 2 were
used with no further 2′,3′-OH protection.
This paper describes the synthesis and functional
investigation of novel insulin secretagogues which am-
plify glucose-stimulated insulin secretion from pancre-
atic B cells by binding to P2Y-Rs in the membrane of
these cells. We report here a highly efficient, rapid
synthesis and diastereomeric separation of 2-thioether
5′-O-(1-thiotriphosphate)adenosine-nucleotide (2-RS-
ATP-R-S) derivatives and their corresponding mono-
phosphorothioate (2-RS-AMP-S) analogues. The high
potency of these compounds as P2Y1-R ligands in turkey
erythrocytes and as insulin secretagogues in rat pan-
creas is described, as well as their relative enzymatic
stability.
The reported preparations of 5′-monothiophosphate-
nucleosides involved treatment of a suitably protected
nucleoside with various phosphorothioate reagents,21a,c,22
phosphitylation and subsequent addition of sulfur,21b or,
alternatively, a direct thiophosphorylation of unpro-
tected nucleosides using thiophosphoryl chloride in
trimethyl phosphate.12,15 Conventional phosphorylation
methods of nonprotected nucleosides require the use of
trimethyl phosphate (TMP) as a solvent,23,24 since it
dissolves both polar nucleosides and nonpolar phospho-
rylating reagents. In our hands, however, applying these
conditions12 to thiophosphorylated 2-RS-adenosine de-
rivatives 2 did not lead to the expected 5′-phosphoro-
thioate nucleoside in good yield but rather to a mixture
Resu lts
Liga n d Syn th esis. Scheme 1 outlines the synthesis
of 2-RS-ATP-R-S nucleotides and their lower homo-
logues, starting at 2-SH-adenosine, 1.20 The latter was

3638 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Fischer et al.
of 2-RS-AMP and 2-RS-AMP-S in a ratio of 3:2. More-
over, when these conditions were applied to the syn-
thesis of 2-RS-ATP-R-S derivatives, inseparable mix-
tures of phosphorylated and thiophosphorylated nu-
cleosides (e.g., 2-RS-AMP, 4, 2-RS-ATP, and 2-RS-ATP-
R-S(Rp+Sp), 5) formed. The phosphate side products
might stem from a reaction of thiophosphoryl chloride
with TMP. Monitoring the reaction by FAB spectroscopy
revealed species where the sulfur atom was exchanged
with an oxygen. Thus, when P(S)Cl3 and PO(OMe)3, in
a 1:48 ratio, as in the reaction mixture, were added to
the nucleoside, only a ′regular′ phosphate product (2-
RS-AMP), was observed after 5 min at 0 °C. The
phosphorothioate product 4 was identified as a minor
product (phosphate:phosphorothioate ratio 5:1) only
after 20 min.
phorothioate. These diastereoisomers are almost indis-
tinguishable by 1H NMR, except for the H-8 signal
which appears at 8.46 and 8.53 ppm for the two
diastereoisomers. The latter difference is due to the
proximity of the phosphorothioate sulfur atom to H-8
in one of the isomers (in the anti conformation, which
is expected for 2-RS-ATP derivatives). A larger differ-
ence between the two diastereomers is observed in the
31P NMR spectrum, where the PR appears as two
doublets at 44.56 and 44.21 ppm, J ) 13.5 Hz.
Many enzymes show a strong preference for one of
the diastereomers of ATP-R-S.16 A similar preference
was observed for GTP-R-S in activating various G-
proteins.33b,c It was expected, therefore, that such ste-
reoselectivity would prevail with the P2Y-Rs as well.
Therefore, diastereoisomers of 5 were separated on a
reverse-phase HPLC column applying a linear gradient
of triethylammonium acetate (TEAA):CH3CN. Separa-
tion was improved upon addition of 0.01% (w/w) MgCl2.
This solvent system is a better alternative to the
reported phosphate or Tris-HCl buffers34 which cannot
be removed by freeze-drying. The separated diastere-
oisomers are designated A and B, e.g., 5a A and 5a B,
and their absolute configuration has not been yet
established. A isomer is the one with a shorter retention
time. Any A isomer is of the same chirality in all
analogues, based on correlation of retention time with
1H NMR data for H-8.
Alternative solvents such as acetonitrile,25 m-cresol,
nitrobenzene,26 or trimethyl thiophosphate27 were con-
sidered. However, no product was obtained in suspen-
sions with these solvents. Apparently, the dissolution
of the nucleoside is essential for the occurrence of the
reaction.
Dry pyridine was found to be a superior alternative
to TMP. Not only does it dissolve both the thiophos-
phorylation reagent and the polar nucleoside 2 and
lead to the product in a quantitative yield, but it also
speeds up the reaction, which is completed within
15 min, instead of 12 h, when using TMP. This effect
is attributed to formation of a reactive species: (S)P-
(pyridinium)Cl2+Cl-, 6. This species was observed, as
the only new signal, in the 31P NMR spectrum taken
10 min after the addition of excess dry pyridine to P(S)-
Cl3 at 20 °C. The signal for the reagent appeared at 44
ppm and shifted to 56 ppm upon pyridine addition. After
10 min at 20 °C, the ratio of (S)P(pyridinium)Cl2+Cl-:
P(S)Cl3 was 1:3. Moreover, the basic environment,
produced by pyridine and the addition of 1,8-bis-
(dimethylamino)naphthalene (proton sponge), prevents
the desulfurization process typical of phosphorothioate
diesters at very low or high pH.28a,29
Some of the newly synthesized derivatives were
evaluated biochemically as described below. Only 4a ,
5a A, and 5a B which were the most promising P2Y1-R
agonists were evaluated in all the biochemical assays.
En zym a tic Sta bility tow a r d Typ e I ATP Da se.
Analysis of the potency of the new compounds as P2Y-R
ligands may be complicated by the presence of ecto-
nucleotidases in the preparation. The relative potency
of some P2-R ligands is related to their resistance to
hydrolysis.35 Therefore, an evaluation of the enzymatic
stability of the new compounds is an essential step in
the process of the development of P2-R ligands as
potential leads.
Although a nonprotected nucleoside is used, phospho-
rylation takes place selectively at the 5′-position as
Up until recently it was believed that ATP was
converted to adenosine by ecto-ATPase, ecto-ADPase,
and 5′-nucleotidase.36 However, in many organs and
tissues including endothelial, smooth muscle, and pan-
creatic cells,37 ATP-diphosphorohydrolase, ATPDase
(EC 3.6.1.5), is probably responsible for most of the
hydrolysis of extracellular triphospho- and diphospho-
nucleosides.35 ATPDase hydrolyzes pyrophosphate bonds
of nucleoside di- and triphosphates in the presence of
1
evidenced in the H NMR spectrum. This selectivity is
achieved when the reaction is carried out at 0 °C. At
higher temperature, e.g., at 6 °C, the 2′- and 3′-
hydroxyls also undergo thiophosphorylation, in addition
to the 5′-OH.
2-Thioether 5′-O-(1-thiotriphosphate)adenosine-nucle-
otides were prepared in a one-pot synthesis and were
obtained as the only product in high yield (Scheme 1).
These conditions were superior to reported syntheses
of ATP-R-S.30,31 A short reaction time with pyrophos-
phate and neutral pH at the hydrolysis step are impera-
tive for obtaining the product in quantitative yield. This
procedure prevents the formation of side products such
as nucleoside-monophosphorothioate, ′regular′ triphos-
phate, tetraphosphate, pentaphosphate, ADP-R-S, and
cyclic phosphorothioates. DMF plays an essential role
in the second synthetic step. When the pyrophosphate
salt was dissolved in pyridine instead of DMF, product
5 was not obtained. Apparently, DMF forms a Vilsmeier
type intermediate, effective in the condensation reaction
of pyrophosphate with 3.32
Ca2+ or Mg2+ 38
.
In the present study, we evaluated the resistance of
2-thioether adenosine-5′-phosphorothioate derivatives
toward hydrolysis by semipurified preparation of pig
pancreas type I ATPDase.38a The enzymatic stability of
the novel phosphorothioate analogues, bearing modifi-
cations on both the purine and the phosphate chain, was
also compared with analogues bearing only one modi-
fication on either the purine or the phosphate chain.
All phosphorothioate analogues tested interacted with
the pig pancreas type I ATPDase. Thus, the hydrolysis
rate of 2-hexylthio-ATP-R-S 5a A and 5a B isomers by
the pancreas type I ATPDase was 28% of that of ATP
and about 50% and 35% of that of ATP-R-S and
2-RS-ATP-R-S derivatives 5 are obtained as a mixture
of two diastereoisomers due to chirality of the R-phos-

Adenosine Derivatives as Insulin Secretagogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3639
F igu r e 1. Hydrolysis rate of ATP and its analogues by type I ATPDase. Assays were carried out for 10 min at 37 °C in the
presence of 1.9 µg of proteins. ATP and analogues were used at 100 µM concentration: 2-hexylthio-AMP-S (4a ); 2-benzylthio-
AMP-S (4c); 2-hexylthio-ATP-R-S-A isomer (5a A); 2-hexylthio-ATP-R-S-B isomer (5a B); ATP-R-S Sp isomer (ATP-S); and
2-butylthio-ATP (2-BuS-ATP).39 5a A and 5a B isomers were hydrolyzed at 28% the rate of ATP, whereas the monophosphorothioate
derivatives were not hydrolyzed by ATPDase. All the experiments were carried out in triplicate, and results are expressed as the
mean ( SEM.
2-butylthio-ATP (2-BuS-ATP),39 respectively (Figure 1).
This result suggests that both the C2 hexylthio sub-
stituent and the phosphorothioate moiety play an
important role in protecting ATP analogues against
ATPDase hydrolysis. Estimated apparent Km and Vmax
for 2-hexylthio-ATP-R-S (5a A) were 41 µM and 0.23
µmol Pi/min/mg protein, respectively (Figure 2). 2-Hexyl-
thio-ATP-R-S, 5a B, exhibited low affinity for ATPDase
as shown by a slow rate of hydrolysis. At concentrations
as high as 100 µM, no saturation of the enzyme was
observed (Figure 2A). The apparent Km and Vmax for
ATP-R-S, in which the C2 hexylthio substituent is
missing, were 18 µM and 0.45 µmol Pi/min/mg protein,
respectively (Figure 3A). 2-Butylthio-ATP (Figure 3B),
in which there is a phosphate instead of phosphorothio-
ate chain, exhibited apparent Km and Vmax of 18 µM and
0.27 µmol Pi/min/mg protein, respectively. For compari-
son purposes, the estimated apparent Km and Vmax for
ATP (data not shown) were 9 µM and 0.5 µmol Pi/min/
mg of protein, respectively.
As expected, the monophosphate derivatives 2-hexyl-
thio-AMP-S, 4a , and 2-benzylthio-AMP-S, 4c, were not
hydrolyzed by type I ATPDase (Figure 1). In the case
of 4a , there was a substantial inorganic phosphorus
background (Figure 1). The latter was a weak competi-
tive inhibitor of ATPDase, with an estimated Ki of 75
µM (Figure 4). 4c and AMP-S, which were stable in the
assay medium (Figure 1), exerted an effect on ATPDase
that could be interpreted as a mixed type of inhibition
(Figures 5 and 6).
nucleotides prepared in this study were tested for their
ability to stimulate phospholipase C activity through
activation of the P2Y1-R in turkey erythrocyte mem-
branes. The substitution of a nonbridging oxygen on the
R-phosphate of ATP analogues by sulfur results in
chirality, and both diastereoisomers of 2-hexylthio-ATP-
R-S, 5a A and 5a B, produced a marked concentration-
dependent activation of phospholipase C activity with
EC50 values of 17 ( 3 and 21 ( 7 nM for 5a A and 5a B,
respectively (Figure 7A and Table 1). These values are
almost identical to that of the corresponding triphos-
phate analogue 2-hexylthio-ATP (10 ( 4 nM).8a As
illustrated in Figure 7A,B, the potency for activation of
P2Y1-Rs by the two diastereoisomers, either 5a A and
5a B or 5bA and 5bB, was identical, suggesting that the
absolute configuration of PR is not important in confer-
ring high-affinity binding of the ATP molecule with the
receptor. In contrast to 2-hexylthio-ATP-R-S derivatives,
5a , which are equipotent with 2-thioether ATP ana-
logues, 2-hexylthio-AMP-S, 4a , was 46-fold less potent
than 2-hexylthio-AMP, EC50 2790 ( 980 and 60 ( 9 nM,
respectively8b (Figure 7A). However, 2-RS-AMP-S de-
rivatives are still P2Y1-R agonists, whereas the parent
compounds AMP and AMP-S were ineffective in stimu-
lating this receptor (not shown). These data confirm our
earlier observations that 2-thioether substitution of the
adenine base results in compounds of high potency for
P2Y1-Rs.8 In contrast to 2-hexylthio-ATP-R-S, 5a , and
2-hexylthio-AMP-S, 4a , the 2-p-nitrophenethylthio sub-
stitution in 5bA and 5bB resulted in 42-74-fold de-
creases in potency for activation of P2Y1-Rs (Figure 7B).
The ineffectiveness of 2-p-nitrophenethylthio substitu-
tion in producing potent P2Y1-R ligands is also reflected
in the low potency of 2-p-nitrophenethylthio-AMP. This
compound has an EC50 value of 3000 nM compared with
E va lu a t ion of t h e New Com p ou n d s a s P 2Y1-R
Liga n d s. Previous work has demonstrated that turkey
erythrocytes are a valuable model system for the study
and development of agonists and antagonists of P2Y1-
Rs.40 The 5′-phosphorothioate analogues of adenine

3640 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Fischer et al.
F igu r e 2. 5a A and 5a B isomer hydrolysis by pig pancreas
type I ATPDase. 5a A isomer ([) and 5a B isomer (9) were used
at 100 µM concentration. (A) Hydrolysis rate of 5a A and 5a B
isomers. The enzyme was not saturated, even at 100 µM B
isomer. (B) Woolf-Augustinson-Hofstee representation of
5a A isomer hydrolysis by ATPDase. The apparent Km and Vmax
were 41 µM and 0.23 µmol Pi/min/mg protein, respectively.
All the experiments were carried out in triplicate.
F igu r e 3. Woolf-Augustinson-Hofstee representation of the
hydrolysis of ATP-R-S (panel A) and 2-BuS-ATP39 (panel B)
by type I ATPDase. (A) The apparent Km and Vmax for ATP-
R-S were 18 µM and 0.45 µmol Pi/min/mg protein, respectively.
(B) The apparent Km and Vmax for 2-BuS-ATP were 18 µM and
0.27 µmol Pi/min/mg protein, respectively. All the experiments
were carried out in triplicate.
60 nM of the corresponding 2-hexylthio-AMP8 (Table 1).
Likewise, a 5′-phosphorothioate moiety in 2-p-nitro-
phenethylthio-AMP-S, 4b, does not compensate for the
loss of activity due to this C2 substitution (Figure 7B
and Table 1).
F u n ction a l In vestiga tion s in Isola ted Ra t P a n -
cr ea s. The newly synthesized analogues were evaluated
as insulin secretagogues on isolated and perfused rat
pancreas. Insulin in the pancreatic effluent was mea-
sured by radioimmunoassay. Variations in flow rate,
which reflect the changes in vascular resistance, were
registered throughout the experiment.
When 2-hexylthio-AMP-S was administered, a moder-
ate increase in glucose-induced insulin secretion was
observed only with a concentration of 15 µM, while 1.5
µM concentration was ineffective (Figure 8A). This
response is similar to that previously obtained with
AMP.4a In addition, this compound induced a concentra-
tion-dependent decrease in pancreatic flow rate (Figure
8B). However, 2-hexylthio-ATP-R-S (A isomer) induced
a large biphasic and concentration-dependent increase
in glucose-induced insulin secretion, in a much lower
range of concentrations: 0.05-1.5 µM (Figure 9A). At
all concentrations tested a transient vasoconstriction is
consistently observed (Figure 9B). At the concentrations
of 0.5 and 1.5 µM, the insulin-secreting response is not
significantly different, suggesting that these values
approach the maximal effective concentrations. In ad-
F igu r e 4. Lineweaver-Burk plot representation of the
inhibitory effect of 2-hexylthio-AMP-S, 4a , on ATP hydrolysis
by type I ATPDase. ATP concentration ranged from 10 to 100
µM. Assays were carried out in the presence of 20 µM 4a (9)
or in the absence of ATP analogue ([). 4a exerted an effect on
type I ATPDase that could be interpreted as a competitive type
of inhibition, with an estimated Ki of 75 µM. All the experi-
ments were carried out in triplicate.
dition, the B isomer of 2-hexylthio-ATP-R-S is equipo-
tent to the A isomer (Figure 10). These compounds are
about 100 times more potent than ATP and approxi-
mately as potent as ADP-â-S.5d

Adenosine Derivatives as Insulin Secretagogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3641
F igu r e 5. Lineweaver-Burk plot representation of the
inhibitory effect of 2-benzylthio-AMP-S, 4c, on ATP hydrolysis.
ATP concentration ranged from 10 to 100 µM. Assays were
carried out in the presence of 30 µM 4c (9) or in the absence
of the inhibitor ([). 4c exerted an effect on type I ATPDase
that could be interpreted as a mixed type of inhibition. All the
experiments were carried out in triplicate.
Figu r e 7. Effects of2-RS-5′-(phosphorothioate)adenosine-nucleo-
tides on P2Y1-R-mediated inositol lipid hydrolysis in turkey
erythrocytes. The capacity of the indicated concentrations of
these nucleotides to stimulate phospholipase C activity was
determined as described in Methods: (A) 2-hexylthio-ATP-R-S
A isomer (2), 2-hexylthio-ATP-R-S B isomer (∆), and 2-hexyl-
thio-AMP-S (9); (B) 2-(p-NO2-phenethyl)thio-ATP-R-S A isomer
(2), 2-(p-NO2-phenethyl)thio-ATP-R-S B isomer (∆), and 2-(p-
NO2-phenethyl)thio-AMP-S (9). The data presented are from
a representative experiment repeated at least three times with
different membrane preparations.
Ta ble 1. 2-Thioether 5′-O-(Thiophosphate)adenosine
Derivatives Are Potent P2Y1-R Agonists
compound
2-hexylthio-ATP8a
2-hexylthio-ATP-R-S-A, 5a A
2-hexylthio-ATP-R-S-B, 5a B
2-hexylthio-AMP8a
EC50 (nM)
10 ( 4
17 ( 3
21 ( 7
F igu r e 6. Lineweaver-Burk plot representation of the
inhibitory effect of AMP-S on ATP hydrolysis by type I
ATPDase. ATP concentration ranged from 10 to 100 µM.
Assays were carried out in the presence of 100 µM AMP-S (9)
or in the absence of inhibitor ([). AMP-S exerted an effect on
type I ATPDase that could be interpreted as a mixed type of
inhibition. All the experiments were carried out in triplicate.
60 ( 9
2-hexylthio-AMP-S, 4a
2790 ( 980
12 ( 4
2-p-nitrophenethylthio-ATP8a
2-p-nitrophenethylthio-ATP-R-S-A, 5bA
2-p-nitrophenethylthio-ATP-R-S-B, 5bB
2-p-nitrophenethylthio-AMP8a
2-p-nitrophenethylthio-AMP-S, 4b
ATP
510 ( 120
890 ( 340
3000 ( 1200
2360 ( 980
6353 ( 2464
6275 ( 2900
no effect
Discu ssion
ATP-R-S (Sp isomer)a
The main determinants that control the level of
extracellular nucleotides in vivo are their release pro-
cess from cells and the presence of ectonucleotidases.
The ectoenzymes involved in the conversion of ATP to
adenosine have been reviewed by Zimmermann.41 Ecto-
ATPase, ecto-ATPDase, and the 5′-nucleotidase play a
key role in the extracellular metabolism of ATP and
other nucleotides. The relative importance of ecto-
ATPase and ATPDase may vary from one system to the
other. Picher et al. have shown that some ATP ana-
logues are hydrolyzed whereas others act as inhibitors
of the enzyme.35 From our previous experiments on a
limited number of analogues, it appears that analogues
modified on a phosphate chain, mainly on the â- or
γ-phosphate, were not hydrolyzed whereas those modi-
fied on the other parts of the molecule were hydrolyzed
at different rates.35
AMP-Sa
a
A commercial compound.
In this study, using a semipurified preparation of
zymogen granule of pig pancreas (type I ATPDase), we
have shown that the 5′-O-phosphorothioate moiety of
ATP-R-S did not suppress the catalytic activity of the
enzyme. This substitution increased the apparent Km,
whereas Vmax was not significantly modified. Interest-
ingly, substitution at C2 of ATP-R-S by a thioether
(butylthio or hexylthio) caused a significant reduction
of catalytic activity expressed by an increase in Km and
a decrease in Vmax. Comparison of the results with those
obtained with 2-substituted ATP derivatives suggests
a possible steric hindrance at the level of the catalytic
site. Indeed, the Km of 2-hexylthio-ATP-R-S A isomer,

3642 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Fischer et al.
F igu r e 8. Effects of 2-hexylthio-AMP-S, 4a , at (b) 15 µM and
(2) 1.5 µM on (A) insulin secretion and (B) pancreatic flow rate,
from the isolated perfused pancreas of rat. Each point repre-
sents the mean of four experiments, with SEM shown by
vertical lines.
F igu r e 9. Effects of 2-hexylthio-ATP-R-S, 5a A, on (A) insulin
secretion and (B) pancreatic flow rate, from the isolated
perfused pancreas of rat. Each point represents the mean of
four experiments, with SEM shown by vertical lines.
5a A, is increased by a factor of 2 with no significant
modification of Vmax, compared to 2-butylthio-ATP.
The 2-RS-AMP-S derivatives, which are not hydro-
lyzed by this enzyme, are potential ATPDase inhibitors.
Indeed, among the various analogues tested, 2-hexyl-
thio-AMP-S, 4a , is a weak competitive inhibitor, with
a Ki in the order of 75 µM, whereas 2-benzylthio-AMP-
S, 4c, and AMP-S show a mixed type of inhibition.
These results indicate that substitution at the C2
position of the purine ring strongly affects hydrolysis
by ATPDase, thus turning these new analogues into
relatively stable P2-R ligands.
The apparent affinity of the compounds to P2Y1-R was
determined by measurement of P2Y-R-promoted phos-
pholipase C activity in turkey erythrocyte membranes.
2-Hexylthio-ATP-R-S isomers, 5a A and 5a B, were found
to be good agonists, stimulating the production of
inositol phosphates with K0.5 values in the nanomolar
range. 2-RS-AMP-S derivatives were also full agonists,
although 2 orders of magnitude less potent. All of the
compounds were more potent than ATP. A difference
of about 0.5 order of magnitude was observed for 5a A
and 5a B diastereoisomers.
F igu r e 10. Concentration-response curves for 2-hexylthio-
ATP-R-S, 5a : ([) A isomer, (]) B isomer.
and 5a B isomers are about 100 times more potent than
ATP4a and approximately as potent as ADP-â-S.5b In the
same preparation, 2-methylthio-ATP has been shown
to be only 45 times more potent than ATP.5a Conse-
quently, these new P2Y-R ligands may be of potential
interest for the development of antidiabetic drugs that
A high increase, up to 500%, in glucose-induced
insulin secretion was due to addition of 2-hexylthio-
ATP-R-S A isomer, 5a A, in the nanomolar concentration
range, to an isolated and perfused rat pancreas. 5a A

Adenosine Derivatives as Insulin Secretagogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3643
temperature for 1 h. The solvent was evaporated under high
vacuum. 2-Thioadenosine sodium salt, obtained as a yellow
solid after freeze-drying, was dissolved in dry DMF (10 mL),
and 1 equiv of alkyl bromide was added. The clear, yellow
reaction mixture was stirred for 2 h at room temperature. TLC
(CHCl3:MeOH, 4:5) indicated that all starting material reacted
and a major product was formed. The solvent was evaporated
under high vacuum, and the residue was washed with MeOH
(10 mL) several times and evaporated, until the residue turned
to a yellow solid. The solid was triturated three times with a
solution of ether:hexane (1:1), and TLC indicated that the
product was pure. The product was dried under high vacuum.
promote insulin release. 2-Hexylthio-AMP-S was less
potent than ATP, showing similar activity as AMP.4a
These results on insulin secretion from rat pancreas
show some discrepancies with those observed on inositol
lipid hydrolysis in turkey erythrocyte membranes. They
suggest that the P2Y receptor subtypes implicated in
these two preparations may be different. On the other
hand, concerning the vascular effects of these new
derivatives of AMP and ATP, it may be hypothesized
that they result from activation of a P2X-R or P2Y-R
subtype sensitive to UTP, as has been previously
observed in the perfused pancreas.42
In summation, 2-thioether 5′-O-(1-thiotriphosphate)-
adenine-nucleotides are highly potent P2Y1-R ligands
in turkey erythrocytes, and they are highly effective
insulin secretagogues active at the nanomolar concen-
tration range. Moreover, these compounds exhibit rela-
tive enzymatic stability regarding type I ATPDase.
However, their poor selectivity for the insulin-secreting
cell, illustrated by their ability to induce vascular
effects, prevents their further use as potential antidia-
betic agents. Therefore, the development of improved
generation, tissue-specific agents is currently being
investigated by us.
2-(6-Am in o-2-h exylsu lfan yl-7H-pu r in -9-yl)-5-(h ydr oxym -
eth yl)tetr a h yd r ofu r a n -3,4-d iol (2a ). 2a was obtained in
95% yield: 1H NMR (DMSO-d6, 300 MHz) δ 8.23 (s, 1H, H-8),
7.34 (brs, 2H, NH2), 5.82 (d, J ) 6 Hz, 1H, H-1′), 4.62 (“t”, J )
5.5 Hz, 1H, H-2′), 4.14 (dd, J ) 5, 3.5 Hz, 1H, H-3′), 3.91 (q, J
) 4 Hz, 1H, H-4′), 3.64 (dd, J ) 12, 4 Hz, 1H, H-5′), 3.52 (dd,
J ) 12,4 Hz, 1H, H-5′), 3.07 (ABq, J ) 6.5, 13 Hz, 2H, CH2-
S), 1.65 (quintet, J ) 7 Hz, 2H, CH2â-S), 1.42 (quintet, J ) 7
Hz, 2H, CH2γ-S), 1.28 (m, 4H, 2 × (CH2)), 0.87 (t, J ) 7 Hz,
3H, CH3); 13C NMR (DMSO-d6, 300 MHz) δ 163.78 (s, C-2),
155.48 (s, C-6), 150.10 (s, C-4), 138.81 (d, C-8), 116.91 (s, C-5),
87.35 (d, C-1′), 85.40 (d, C-4′), 73.15 (d, C-2′), 70.47 (d, C-3′),
61.60 (t, C-5′), 30.86 (t, CH2-S), 30.10 (t, CH2), 29.08 (t, CH2),
27.98 (t, CH2), 22.03 (t, CH2), 13.90 (q, CH3); MS (CI, ammonia)
384 (MH+).
2-(6-Am in o-2-(p -n it r op h en et h yl)su lfa n yl-7H -p u r in -9-
yl)-5-(h yd r oxym eth yl)tetr a h yd r ofu r a n -3,4-d iol (2b). 2b
was obtained in 96% yield: H NMR (DMSO-d6, 300 MHz) δ
Meth od s
1
Ch em istr y. Gen er a l. All air- and moisture-sensitive reac-
tions were carried out in flame-dried, argon-flushed, two-
necked flasks sealed with rubber septa, and the reagents were
introduced with a syringe. TLC analysis was performed on
precoated Merck silica gel plates (60F-254). New compounds
were characterized (and resonances assigned) by proton
nuclear magnetic resonance using Bruker DPX-300, DMX-600,
or AC-200 spectrometers. 1H NMR spectra was recorded in
D2O, and the chemical shifts are reported in ppm relative to
HDO (4.78 ppm) as an internal standard. Phosphorylated
products were isolated as either Na+, H+, or Et3NH+ salts.
Chemical shifts of Et3NH+ counterion are 3.18 ppm (q, J )
7.2 Hz, CH2) and 1.26 ppm (t, J ) 7.2 Hz, CH3). The number
of equivalents of triethylammonium counterions was defined
by integration of their peaks, in the 1H NMR spectrum, relative
to those of the nucleotide. Nucleotides were characterized also
by 31P NMR in D2O, using 85% H3PO4 as an external reference,
on an AC-200 spectrometer at pH 8. All final products were
characterized on an AutoSpec-E FISION VG high-resolution
mass spectrometer by chemical ionization and high-resolution
mass spectrometry. Nucleotides were desorbed from a glycerol
matrix under FAB (fast atom bombardment) conditions in low
and high resolution. Primary purification of the nucleotides
was achieved on an LC (Isco UA-6) system using a Sephadex
DEAE-A 25 column, which was swelled in 1 M NaHCO3 in
the cold for 1 day. The resin was washed with deionized water
before use. Separation was monitored by UV, detection at 280
nm. A buffer gradient of 0-0.8 or 0-0.9 M NH4HCO3 (350 mL
of H2O:350 mL of buffer) was applied. Final purification and
separation of diastereoisomers, in the cases of chiral products,
8.31 (s, 1H, H-8), 8.20 (AA′XX′, J ) 8 Hz, 2H, Ar), 7.65 (AA′XX′,
J ) 8 Hz, 2H, Ar), 7.42 (brs, 2H, NH2), 5.91 (d, J ) 6 Hz, 1H,
H-1′), 5.53 (d, J ) 6 Hz, 1H, OH(H-2′)), 5.26 (d, J ) 5 Hz, 1H,
OH (H-3′)), 4.60 (dd, J ) 6, 5.5 Hz, 1H, H-2′), 4.14 (dd, J ) 5,
3.5 Hz, 1H, H-3′), 3.95 (q, J ) 4.5 Hz, 1H, H-4′), 3.56 (dd, J )
12, 5 Hz, 1H, H-5′), 3.56 (dd, J ) 12, 5 Hz, 1H, H-5′), 3.50
(CH2-S under the water signal), 3.13 (t, J ) 7 Hz, 2H, CH2-
Ar); 13C NMR (DMSO-d6, 200 MHz) δ 163.12 (s, C-2), 155. 52
(s, C-6), 150.20 (s, C-4), 148.75 (s, C-NO2), 146.05 (s, Carom
-
CH2), 138.44 (d, C-8), 130.03 (d, CHortho to NO2), 123.37 (d,
CHmeta to NO2), 116.82 (s, C-5), 87.04 (d, C-1′), 85.83 (d, C-4′),
73.49 (d, C-3′), 70.42 (d, C-2′), 61.43 (t, C-5′), 35.31 (t, CH2-
Carom), 31.06 (t, CH2-S); FAB (negative) 449 (MH+).
2-(6-Am in o-2-b e n zylsu lfa n yl-7H -p u r in -9-yl)-5-(h y-
d r oxym eth yl)tetr a h yd r ofu r a n -3,4-d iol (2c). 2c was ob-
tained in 97% yield: 1H NMR (DMSO-d6, 200 MHz) δ 8.25 (s,
1H, H-8), 7.42 (m, 4H, CHortho+NH2), 7.29(m, 3H, 2Hmeta+Hpara),
5.89 (d, J ) 6 Hz, 1H, H-1′), 4.59 (dd, J ) 6, 5 Hz, 1H, H-2′),
4.48 (s,1H, CH2), 4.14 (dd, J ) 5, 3.5 Hz, 1H, H-3′), 3.93 (q, J
) 4 Hz, 1H, H-4′), 3.51(AB dq J ) 12, 4 Hz, 2H, H-5′); 13C
NMR (DMSO-d6, 300 MHz) δ 163.18 (s, C-2), 155.49 (s, C-6),
150.10 (s, C-4), 138.64 (d, C-8 + s, Carom-CH2), 129.65 (d, Cmeta),
128.30 (d, Cortho), 126.83 (d, Cpara), 116.91 (s, C-5), 87.24 (s,
C-1′), 85.43 (C-4′), 73.50 (s, C-2′), 70.39 (s, C-3′), 61.44 (t, C-5′),
34.35 (t, CH2-S); HRMS (DCI, CH4) calcd for C17H19N5O4S
(MH+) 390.1235, found 390.1230.
Th iop h osp h or yla tion Rea ction . Gen er a l P r oced u r e.
Nucleosides were dried for 2 days in a vacuum oven (40-50
°C). Anhydrous solvents (pyridine, DMF) were used. Thio-
phosphoryl chloride was distilled and kept under argon.
were done on
a HPLC (Merck-Hitachi) system using a
semipreparative reverse-phase Nucleotide/Nucleoside 7U col-
umn (1 × 25 cm; Alltech Associates, Deerfield, IL) in different
solvent systems for each product. The purity of all newly
synthezised nucleotides was evaluated on an analytical column
in different solvent systems. Peaks were detected by UV
absorption using a diode array detector. 2RS-AMP-S and 2RS-
ATP-R-S derivatives were generally >95% pure by HPLC.
Minute contaminants are not nucleotides, as shown by their
UV spectrum.
2-Th ioeth er Ad en osin e Der iva tives. Gen er a l Non -
a qu eou s Alk yla tion P r oced u r e. A suspension of 2-SH-
adenosine20 (0.1 g, 0.335 mmol, in 10 mL of MeOH) was
dissolved in 0.25 M NaOH (1.25 mL) and stirred at room
2-(6-Am in o-2-h exylsu lfan yl-7H-pu r in -9-yl)-5-(m on oth io-
p h osp h a te-m eth yl)tetr a h yd r ofu r a n -3,4-d iol (4a ). A sus-
pension of 2-(hexylthio)adenosine, 2a (0.1 g, 0.2 mmol), in dry
pyridine was heated for several minutes until a clear solution
was attained and then cooled to room temperature. Proton
sponge was added (0.12 g, 2 equiv), and the solution was
stirred for 10 min at 0 °C. Thiophosphoryl chloride was added
dropwise (40 µL, 2 equiv, 0.4 mmol). TLC taken after 15 min
(2-propanol:H2O:NH4OH, 16.5:5:1) indicated the complete
disappearance of starting material and the formation of a
single polar product (Rf ) 0.48). After 15 min at 0 °C, a solution
of 0.2 M TEAB (25 mL) was added; the clear solution was
stirred at room temperature for 45 min and then freeze-dried.

3644 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Fischer et al.
The semisolid residue, dissolved in a minimal volume of water/
pyridine, was chromatographed at room temperature on an
activated Sephadex DEAE-A25 column. A buffer gradient of
0-0.8 M NH4HCO3 was applied, and about 100 10-mL frac-
tions were collected, then the column was washed with 2 M
NH4HCO3. The relevant fractions were pooled and freeze-dried
three times to yield the product as a white solid (87% yield).
Final separation was achieved on a semipreparative HPLC
column applying a linear gradient of 0.1 M TEAA (pH 7.5)/
CH3CN, 80:20 to 50:50, in 20 min (flow rate 5 mL/min).
H-4′), 4.30 (m, 2H, H-5′), 3.21 (m, 2H, CH2-S), 1.75 (quintet,
J ) 7 Hz, 2H, CH2â-S), 1.43 (quintet, J ) 7 Hz, 2H, CH2γ-S),
(2 × (CH2) is hidden by the counterion peak), 0.87 (t, J ) 7
Hz, 3H, CH3); 31P NMR (A isomer) (D2O, 200 MHz, pH 8) δ
45.90 (d, J ) 32 Hz), -5.16 (d, J ) 23 Hz), -20.08 (dd, J )
32, 23 Hz); 31P NMR (B isomer) (D2O, 200 MHz, pH 8) δ 45.70
(d, J ) 32 Hz), -4.91 (d, J ) 23 Hz), -19.87 (dd, J ) 32, 23
Hz); FAB (negative) 660 (MW + 2H+ + Na+), 682 (MW + H+
+ 2Na+), 704 (MW + 3Na+); HRFAB calcd for C16H24N5O12P3S2
660.0130, found 660.0230; UV λmax 278 nm.
1
Retention time: 12.63 min. The product was 98.6% pure: H
2-(6-Am in o-2-(p -n it r op h en et h yl)su lfa n yl-7H -p u r in -9-
yl)-5-(tr ip h osp h o-R-th ioa te-m eth yl)tetr a h yd r ofu r a n -3,4-
d iol (5b). 5b was obtained in 78% yield. Purification of the
product on a semipreparative HPLC column applying a linear
gradient of 0.1 M TEAA (pH 7.5)/CH3CN, 80:20 to 65:35, in
20 min (flow rate 5 mL/min). Final separation of the two
diastereomers was achieved on a semipreparative column
applying a linear gradient of 0.1 M TEAA (pH 7.5)/CH3CN,
80:20 to 75:25, in 20 min. Retention time: 16.09 min for A
isomer, 17.99 min for B isomer. A isomer was 85% pure; B
isomer was >95% pure: 1H NMR (for A & B isomers) (D2O,
600 MHz) δ 8.49 (s, 1H, H-8 for diastereomer A), 8.40 (s, 1H,
H-8 for diastereomer B), 7.97 (AA′XX′, J ) 8 Hz, 2H, Ar), 7.45
(AA′XX′, J ) 8 Hz, 2H, Ar), 6.02 (d, J ) 6 Hz, 1H, H-1′), (H-2′
is hidden by the water signal), 4.58 (m, 1H, H-3′), 4.40 (m,1H,
H-4′), 4.28 (m, 2H, H-5′), 3.52 (m, 4H, CH2-S, CH2-Ar); 31P
NMR (for A & B isomers) (D2O, 200 MHz, pH 8) δ 44.35 (d, J
) 32 Hz), -5.1 (d, J ) 24 Hz), -20.55 (dd, J ) 32, 24 Hz);
NMR (D2O, 200 MHz) δ 8.45 (s, 1H, H-8), 6.13 (d, J ) 6 Hz,
1H, H-1′), (H-2′ is hidden by the water signal), 4.63 (“t”, J )
4 Hz, 1H, H-3′), 4.39 (br. s, 1H, H-4′), 4.06 (m, 2H, H-5′), 3.25
(m, 2H, CH2-S), 1.72 (quintet, J ) 7 Hz, 2H, CH2â-S), 1.44
(quintet, J ) 7 Hz, 2H, CH2γ-S), (2 × (CH2) is hidden by the
counterion signal), 0.84 (t, J ) 7 Hz, 3H, CH3); 31P NMR (D2O,
200 MHz, pH 8) δ 45.70 (s); FAB (negative) 478 (MH+); UV
λmax 278 nm.
2-(6-Am in o-2-(p -n it r op h en et h yl)su lfa n yl-7H -p u r in -9-
yl)-5-(m on oth iop h osp h a te-m eth yl)tetr a h yd r ofu r a n -3,4-
d iol (4b). 4b was obtained in 80% yield. Final purification
was achieved on a semipreparative column applying a linear
gradient of 0.1 M TEAA (pH 10)/CH3CN, 80:20 to 85:25, in 20
min. Retention time: 11.79 min. Product 4b was 98.8% pure:
1H NMR (D2O, 200 MHz) δ 8.48 (s, 1H, H-8), 7.98 (AA′XX′, J
) 8 Hz, 2H, Ar), 7.44 (AA′XX′, J ) 8 Hz, 2H, Ar), 6.03 (d, J )
6 Hz, 1H, H-1′), (H-2′ is hidden by the water signal), 4.53 (t,
J ) 4 Hz, 1H, H-3′), 4.39 (m, 1H, H-4′), 4.05 (m, 2H, H-5′),
3.55 (m, 4H, CH2-S, CH2-Ar); 31P NMR (D2O, 200 MHz, pH
8) δ 43.89 (s); FAB (negative) 543 (MH+), 565 (MW + Na+);
HRFAB calcd for C18H20N6O8PS2 543.0521, found 543.0570;
UV λmax 278 nm.
FAB (negative) 660 (MW + 2H+ + Na+), 682 (MW + H+
+
2Na+), 704 (MW + 3Na+); UV λmax 278 nm.
2-(6-Am in o-2-ben zylsu lfa n yl-7H-p u r in -9-yl)-5-(tr ip h os-
p h o-R-th ioa te-m eth yl)tetr a h yd r ofu r a n -3,4-d iol (5c). 5c
was obtained in 83% yield. Purification of the product on a
semipreparative column applying a linear gradient of 0.1 M
TEAA (pH 7.5) + 0.01% MgCl2/CH3CN, 80:20 to 60:40, in 20
min (flow rate 5 mL/min). Final separation of the two diaster-
eomers was achieved on a semipreparative column applying
a linear gradient of 0.1 M TEAA (pH 7.5)/CH3CN, 80:20 to
55:35, in 20 min. Retention time: 10.08 min for A isomer, 10.59
min for B isomer. The purity of each diastereoisomer was
>96%: 1H NMR (for A & B isomers) (D2O, 300 MHz) δ 8.50
(s, 1H, H-8 for diastereomer A), 8.45 (s, 1H, H-8 for diastere-
omer B), 7.56 (m, 2H, CHortho), 7.35 (m, 3H, 2Hmeta+Hpara), 6.14
(d, J ) 6 Hz, 1H, H-1′), (H-2′ is hidden by the water signal),
4.55 (t, J ) 4 Hz, 1H, H-3′), 4.43 (s, 3H, CH2+ H-4′), 4.27 (m,
2H, H-5′); 31P NMR (D2O, 200 MHz, pH 8) δ 44.10 (d, J ) 32
Hz), -5.52 (d, J ) 22 Hz), -21.99 (t, J ) 27 Hz); FAB
(negative) 643 (MH+); HRFAB calcd C17H19N5O12P3S2 for
643.9841, found 643.9830; UV λmax 278 nm.
Eva lu a tion of En zym a tic Sta bility. ATPDase assays
were carried out with a purified zymogen granule membrane
preparation from pig pancreas.38a This preparation was devoid
of any other nucleotidase activities.43 The enzyme preparation
had a specific activity of about 0.5 µmol Pi/min/mg protein.
Enzyme activity was measured at 37 °C in 1 mL of the
following incubation medium: 8 mM CaCl2, 5 mM tetramisole,
50 mM Tris, and 50 mM imidazole, buffered at pH 8.0. The
reaction was started by the addition of 100 µM nucleotide
substrate or as otherwise indicated. Inorganic phosphorus
release was evaluated by the malachite green method as
described by Baykov et al.44 Protein concentration was deter-
mined by the Bradford microplate assay45 using bovine albu-
min as a standard reference. All the experiments were carried
out in triplicate. The deviation of the triplicate means was
within 5%.
2-(6-Am in o-2-b en zylsu lfa n yl-7H -p u r in -9-yl)-5-(m on o-
th iop h osp h a te-m eth yl)tetr a h yd r ofu r a n -3,4-d iol (4c). 4c
was obtained in 84% yield. Final purification was achieved on
a semipreparative column applying a linear gradient of 0.1 M
TEAA (pH 7.5)/CH3CN, 80:20 to 50:50, in 20 min. Retention
time: 9.85 min. Product 4c was >98% pure: 1H NMR (D2O,
200 MHz) δ 8.89 (s, 1H, H-8), 7.85 (m, 2H, CHortho), 7.30 (m,
3H, 2Hmeta+Hpara), 6.10 (d, J ) 6 Hz, 1H, H-1′), (H-2′ is hidden
by the water peak), 4.49 (t, J ) 4 Hz, 1H, H-3′), 4.40 (s, 3H,
CH2+ H-4′), 4.03 (m, 2H, H-5′); 31P NMR (D2O, 200 MHz, pH
8) δ 44.19 (s); FAB (negative) 484 (MH+); HRFAB calcd for
C
17H19N5O6PS2 483.0436, found 483.0509. UV λmax 278 nm.
Gen er a l P r oced u r e for th e P r ep a r a tion of 5′-O-(1-
Th iotr ip h osp h a te)n u cleosid es. 2-(6-Am in o-2-h exylsu lfa -
n yl-7H -p u r in -9-yl)-5-O-(1-t h iot r ip h osp h a t e-m et h yl)t et -
r a h yd r ofu r a n -3,4-d iol (5a ). A suspension of 2-(hexylthio)
adenosine, 2a (0.1 g, 0.2 mmol), in dry pyridine was heated
for several minutes until a clear solution was attained and
then cooled to room temperature. Proton sponge was added
(0.12 g, 2 equiv), and the solution was stirred for 10 min at 0
°C. Thiophosphoryl chloride was added dropwise (40 µL, 2
equiv, 0.4 mmol). After 15 min, a mixture of Bu3N (0.25 mL)
and 1 M (Bu3NH+)2P2O7H2 in DMF8a (2 mL) was added. After
2 min, a solution of 0.2 M TEAB (25 mL) and proton sponge
(2 equiv) was added, and the clear solution was stirred at room
temperature for 45 min in pH 8 and freeze-dried. The semisolid
obtained, dissolved in a minimal volume of water/pyridine, was
chromatographed at room temperature on an activated Sepha-
dex DEAE-A25 column. A buffer gradient of 0-0.9 M NH4-
HCO3 was applied, and 130 10-mL fractions were collected;
then the column was washed with 2 M NH4HCO3. The relevant
fractions were pooled and freeze-dried three times to yield a
white solid (87% yield). Final purification was achieved on a
semipreparative HPLC column applying a linear gradient of
0.1 M TEAA + 0.01% (w/w) MgCl2 (pH 7.5)/CH3CN, 80:20 to
60:40, in 20 min (flow rate 5 mL/min). Retention time: 19.71
min for isomer A and 23.15 min for isomer B. The purity of
each diastereoisomer was >95%: 1H NMR (for A & B isomers)
(D2O, 300 MHz) δ 8.53 (s, 1H, H-8 for diastereomer A), 8.46
(s, 1H, H-8 for diastereomer B), 6.13 (d, J ) 6 Hz, 1H, H-1′),
4.87 (m, 1H, H-2′), 4.64 (t, J ) 4.5 Hz, 1H, H-3′), 4.40 (m, 1H,
Eva lu a tion of th e New Com p ou n d s a s P 2Y-R Liga n d s.
P2Y1-R-promoted stimulation of inositol phosphate formation
by adenine nucleotide analogues was measured in turkey
erythrocyte membranes as previously described.46,47 The EC50
values are the result of three independent experiments carried
out for each compound using different membrane preparations.
Briefly, 1 mL of washed turkey erythrocytes was incubated
with inositol-free DMEM (Gibco, Gaithersburg, MD) with 0.5
mCi of 2-[3H]myoinositol (20 Ci/mmol; American Radiolabeled

Adenosine Derivatives as Insulin Secretagogues
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3645
(5) (a) Bertrand, G.; Chapal, J .; Loubatie`res-Mariani, M. M.; Roye,
M. Evidence for two different P2-purinoceptors on â cell and
pancreatic vascular bed. Br. J . Pharmacol. 1987, 102, 783-787.
(b) Bertrand, G.; Chapal, J .; Puech, R.; Loubatie`res-Mariani, M.
M. Adenosine-5′-O-(2-thiodiphosphate) is a potent agonist at P2
purinoceptors mediating insulin secretion from perfused rat
pancreas. Br. J . Pharmacol. 1991, 102, 627-630. (c) Petit, P.;
Hillaire-Buys, D.; Manteghetti, M.; Debrus, S.; Chapal, J .;
Loubatie`res-Mariani, M. M. Evidence for two different types of
P2 receptors stimulating insulin secretion from pancreatic B cell.
Br. J . Pharmacol. 1998, 125, 1368-1374.
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Chemicals Inc., St. Louis, MO) for 18-24 h in a humidified
atmosphere of 95% air, 5% CO2 at 37 °C. Erythrocyte mem-
branes were prepared by rapid lysis in hypotonic buffer (5 mM
sodium phosphate, pH 7.4, 5 mM MgCl2, 1 mM EGTA) as
described. Phospholipase C activity was measured in 25 µL of
[3H]inositol-labeled membranes (approximately 175 mg of
protein, 200-500 000 cpm/assay) in a medium containing 424
mM CaCl2, 0.91 mM MgSO4, 2 mM EGTA, 115 mM KCl, 5
mM KH2PO4, and 10 mM Hepes, pH 7.0. Assays (200 mL final
volume) contained 1 mM GTPγS and the indicated concentra-
tions of nucleotide analogues. Membranes were incubated at
30 °C for 5 min, and total [3H]inositol phosphates were
quantitated by ion-exchange chromatography as previously
described.46, 47
Da ta An a lysis. Agonist potencies were calculated using a
four-parameter logistic equation and the GraphPad software
package (GraphPad, San Diego, CA). EC50 values (mean (
standard error) represent the concentration at which 50% of
the maximal effect is achieved. All concentration-effect curves
were repeated in at least three separate experiments carried
out with different membrane preparations using duplicate
assays.
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In su lin Relea se fr om Isola ted Ra t P a n cr ea s. The
pancreas was isolated from anesthetized male Wistar rats
(320-350 g) and perfused with a Krebs-bicarbonate buffer
(containing 8.3 mM glucose) at a constant pressure (40-50
cm water). In all the experiments, a 30-min adaptation period
was allowed before taking the first two samples for insulin
assay and flow rate measurement. The new synthetic deriva-
tives, dissolved in a physiological medium containing 0.01 mM
dithiotreitol (DTT), were perfused for 30 min. Samples were
taken every minute during 5 min and then at 8, 10, 15, 20,
and 30 min; each sample was collected during 1 min. Insulin
in the pancreatic effluent was measured by radioimmunoassay.
The variations in the flow rate, which reflect the changes in
vascular resistance, were registered throughout the experi-
ment.
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Ack n ow led gm en t. The Israeli group thanks the
Israeli Ministry of Health for financial support. J .C.,
D.H.B., and P.P. also wish to thank S. Dietz, N. Linck,
and V. Montesinos for technical assistance. J .L.B. and
K.T.H. are indebted to Arvind Mohanram for excellent
technical assistance. This work was supported by US-
PHS Grants GM38213 and HL54889.
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