A novel insulin secretagogue based on a dinucleoside polyphosphate scaffold

Article abstract of DOI:10.1021/jm901621h

Dinucleoside polyphosphates exert their physiological effects via P2 receptors (P2Rs). They are attractive drug candidates, as they offer better stability and specificity compared to nucleotides, the most common P2 receptor ligands. The activation of pancreatic P2Y receptors by nucleotides increases insulin secretion. Therefore, in the current study, dinucleoside polyphosphate analogues (di-(2-MeS)-adenosine-5′,5 -P¹,P ⁴,α,β-methylene-tetraphosphate), 8, (di-(2-MeS)-adenosine- 5′,5 -P¹,P⁴, β, γ-methylene- tetraphosphate), 9, and di-(2-MeS)-adenosine-5′,5 -P ¹,P³, α, β-methylene triphosphate, 10, were developed as potential insulin secretagogues. Analogues 8 and 9 were found to be agonists of the P2Y₁R with EC50 values of 0.42 and 0.46 μM, respectively, whereas analogue 10 had no activity. Analogues 8-10 were found to be completely resistant to hydrolysis by alkaline phosphatase over 3 h at 37 °C. Analogue 8 also was found to be 2.5-fold more stable in human blood serum than ATP, with a half-life of 12.1 h. Analogue 8 administration in rats caused a decrease in a blood glucose load from 155 mg/dL to ca. 100 mg/dL and increased blood insulin levels 4-fold as compared to basal levels. In addition, analogue 8 reduced a blood glucose load to normal values (80-110 mg/dL), unlike the commonly prescribed glibenclamide, which reduced glucose levels below normal values (60 mg/dL). These findings suggest that analogue 8 may prove to be an effective and safe treatment for type 2 diabetes.

Full text of DOI:10.1021/jm901621h

2
472 J. Med. Chem. 2010, 53, 2472–2481
DOI: 10.1021/jm901621h
1
A Novel Insulin Secretagogue Based on a Dinucleoside Polyphosphate Scaffold
†
‡
§
§
Shay Eliahu, Haim M. Barr, Jean Camden, Gary A. Weisman, and Bilha Fischer*
,†
†
‡
Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel, BioLineRx Ltd., 23 Hillel Street, Jerusalem, 91450,
§
Israel, and Biochemistry Department, 540E Bond Life Sciences Center, University of Missouri-Columbia, Columbia, Missouri 65211
Received November 3, 2009
Dinucleoside polyphosphates exert their physiological effects via P2 receptors (P2Rs). They are attrac-
tive drug candidates, as they offer better stability and specificity compared to nucleotides, the most
common P2 receptor ligands. The activation of pancreatic P2Y receptors by nucleotides increases insulin
secretion. Therefore, in the current study, dinucleoside polyphosphate analogues (di-(2-MeS)-adeno-
0
00
1
4
0
00
1
4
sine-5 ,5 -P ,P ,R,β-methylene-tetraphosphate), 8, (di-(2-MeS)-adenosine-5 ,5 -P ,P ,β,γ-methylene-
0
oped as potential insulin secretagogues. Analogues 8 and 9 were found to be agonists of the P2Y R with
00
1
3
tetraphosphate), 9, and di-(2-MeS)-adenosine-5 ,5 -P ,P ,R,β-methylene triphosphate, 10, were devel-
1
EC50 values of 0.42 and 0.46 μM, respectively, whereas analogue 10 had no activity. Analogues 8-10
were found to be completely resistant to hydrolysis by alkaline phosphatase over 3 h at 37 °C. Analogue
8
also was found to be 2.5-fold more stable in human blood serum than ATP, with a half-life of 12.1 h.
Analogue 8 administration in rats caused a decrease in a blood glucose load from 155 mg/dL to ca.
00 mg/dL and increased blood insulin levels 4-fold as compared to basal levels. In addition, analogue 8
1
reduced a blood glucose load to normal values (80-110 mg/dL), unlike the commonly prescribed
glibenclamide, which reduced glucose levels below normal values (60 mg/dL). These findings suggest
that analogue 8 may prove to be an effective and safe treatment for type 2 diabetes.
Introduction
concentrations, the difficulty of achieving normal glycemia in
a significant number of patients, the 5-10% per year second-
ary failure rate of adequate glycemic control, and possible
Diabetes is one of the most prevalent chronic diseases in the
2
Western world, affecting up to 5% of the population. It is a
heterogeneous group of disorders characterized by a chronic
hyperglycemia with additional abnormalities in lipid and
protein metabolism. The hyperglycemia results from defects
6
,7
negative effects on the cardiovascular system. A growing
a
body of evidence suggests that P2 receptors (P2R ) for extra-
cellular nucleotides may be involved in the regulation of
3
glucose homeostasis. Pancreatic β-cells express a variety of
P2 receptors, including the P2Y R subtype. P2 receptors on
in insulin secretion, insulin action, or a combination of both.
8
1
In addition to its chronic metabolic abnormalities, diabetes is
associated with long-term complications involving various
organs, especially the eyes, nerves, blood vessels, heart, and
kidney, which may result in blindness, amputations, cardio-
vascular disease, and end stage renal disease. The develop-
ment of diabetic complications appears to be related to the
chronic elevation of blood glucose. There is no current cure
for diabetes, however, effective glycemic control can lower the
incidence of diabetic complications and reduce their severity.
Thus, improvement of insulin secretion is a major therapeutic
goal. Approximately half of the patients with type 2 diabetes
aretreatedwithvariousoralagents, a considerable proportion
pancreatic β-cells respond to adenosine triphosphate (ATP)
coreleased with insulin during glucose-induced exocytosis of
9
secretory granules. In response to ATP, P2Y receptor activa-
tion has been shown to enhance glucose-induced insulin
release from β-cells, in part dueto increases in cyclic adenosine
monophosphate (cAMP) levelsand protein kinase A activity.
In human pancreatic islets and isolated insulin-secreting cells,
9
2
þ
extracellular ATP also increases the cytoplasmic free Ca
2þ
10
concentration, [Ca ] . A role for P2Y receptor activation
i
1
has been implicated in the maintenance of glucose homeo-
1
1
stasis and insulin secretion in mice.
Thus, the use of P2Y receptor-selective ligands may rep-
4
,5
of them with agents that stimulate insulin secretion. The
choice of insulin secretagogues is limited to the sulfonylureas
and related compounds (“glinides”), which elicit insulin secre-
tion by binding to a regulatory subunit of membrane ATP-
1
resent a novel and effective approach for regulating glucose
homeostasis in type 2 diabetes via increased insulin secretion
from pancreatic β-cells.
6
sensitive potassium channel, inducing its closure.
Sulfonylureas have several undesired effects in addition to
possible long-term adverse effects on their specific target, the
pancreatic β-cell. These side effects include the risk of hypo-
glycemia due to stimulation of insulin secretion at low glucose
a
Abbreviations: P2R, P2 receptor; ATP, adenosine triphosphate;
ADP, adenosine diphosphate; cAMP, cyclic adenosine monophosphate;
0
Np N , dinucleoside polyphosphate; e-NTPDase, ecto-nucleoside tri-
n
phosphate diphosphohydrolase; e-NPPs, ecto-nucleotide pyrophospha-
tases; MPLC, medium pressure liquid chromatography; TEAA,
triethylammonium acetate; CDI, carbonyldiimidazole; HRMS MALDI,
high resolution mass spectrometry matrix-assisted laser desorption/
ionization; MS-ESI, electron spray ionization mass spectrometry.
*To whom correspondence should be addressed. Phone: 972-3-
5318303. Fax: 972-3-6354907. E-mail: bfischer@mail.biu.ac.il.
r
pubs.acs.org/jmc
Published on Web 02/22/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2473
1
Figure 1. P2Y receptor agonists.
Figure 2. A series of novel dinucleoside polyphosphate analogues.
Indeed, structural analogues of ATP with high stability and
selectivity have been shown to increase insulin secretion
Furthermore, analogue 3A was shown to be a very potent
insulin secretagogue in isolated rat pancreas, in a glucose-
1
2
through activation of P2Y receptors in isolated rat pancreas
1
dependent manner, being active only at stimulating glucose
20,21
3
and human islets. Moreover, stable analogues of ATP and
adenosine diphosphate (ADP) have been shown to increase
concentrations,
cing the risk of hypoglycemia. The maximal efficacy of
as opposed to sulfonylureas, thereby redu-
1
4,15
insulin secretion in vivo.
Likewise, a substitution at the
analogue 3A was 9-fold above basal secretion, with an EC
2
5
0
0
adenine C-2 position and modifications of the polyphosphate
chain of ATP have been reported to enhance insulin secre-
of 28.1 nM. Moreover, analogue 3A exhibited high stability
atphysiological andgastric pH at 37°C, withhalf-lives of 1395
and 5.9 h, respectively. In addition, analogue 3A was only
1
tion. We have reported that a C-2-thioether substitution on
6
19
ATP and substitution of the nonbridging oxygen at P by a
R
weakly hydrolyzed by spleen ecto-nucleoside triphosphate
diphosphohydrolase (NTPDase), with only 5% degradation
sulfur atom, such as 2-benzylthio-ATP-R-S, 1 (A and B
1
7
18
19
isomers), or 2-hexylthio-ATP-R-S, 2 (A and B isomers)
after 7 min at 37 °C as compared to ATP. Yet, analogue 3A
was hydrolyzed almost completely by alkaline phosphatase
(Figure 1), generated highly specific, potent, and stable
agonists of the P2Y receptor (e.g., EC values were 17 and
2
2
over 100 min at 37 °C. Attempts to improve the stability of
3 by synthesizing the corresponding methylene analogues 4-6
(Figure 1), resulted in compounds highly stable to alkaline
1
50
2
1 nM for 2-hexylthio-ATP-R-S, 2, A and B isomers, res-
18
pectively). Furthermore, these ATP derivatives increased
insulin secretion from rat pancreas by 100-fold, as compared
to ATP.
phosphatase, yet these analogues were weak P2Y R ago-
1
1
8
22
nists.
Recently, we found that R-borano-2-MeS-ATP, 3, A iso-
Our preliminary findings, on one hand, and the side-effects
of sulfonylurea-based insulin secretagogues, especially the
risk of hypoglycemia, on the other hand, encouraged us to
develop alternative and safe drug candidates acting via a
mer (Figure 1), is a potent and selective agonist of the P2Y R,
with an EC50 of 2.6 nM in HEK 293 cells overexpressing the
1
1
9
receptor (compared with an EC50 of 0.2 μM for ATP).

2
474 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Eliahu et al.
a
Scheme 1
a
Reaction conditions: (a) CH
2
Cl
2
, DMAP, TsCl, RT, 12 h. (b) Analogue 13: tetra-(n-butylammonium)methylenediphosphonate in dry DMF, RT,
48 h. (c) Analogue 14: tetra-(n-butylammonium)pyrophosphate in dry DMF, RT, 48 h. (d) 18% HCl, pH 2.3, RT for 3 h followed by 24% NH
4
OH, pH 9,
RT for 45 min. (e) CDI, DMF, RT, 3 h; f) MgCl , RT, 12 h.
2
novel mechanism. Specifically, we targeted the enhancement
of insulin secretion via the activation of the P2Y R expressed
in pancreatic β-cells instead of the ATP-sensitive K -channel
activated by sulfonylureas.
Inaddition, weutilizeda different scaffold thanATP for the
design of insulin secretagogues, namely a dinucleoside poly-
phosphate scaffold.
The enhanced metabolic stability offered by methylene
3
0
bioisosters of dinucleoside polyphosphates encouraged us
to design a novel series of stable compounds that act as insulin
secretagogues via P2YR activation. Here, we report on the
synthesis and characterization of dinucleoside polyphosphate
analogues 8-10 (Figure 2), their resistance to hydrolysis by
1
þ
alkaline phosphatase, their activity at the P2Y R, the stability
1
Dinucleoside polyphosphates, such as Ap A and Ap A,
3
of 8 (the most promising analogue) in human blood serum,
and the modulation of blood glucose and insulin levels in vivo
by analogue 8.
4
2
3-27
induce physiological effects via activation of P2 receptors.
These compounds are more stable and specific than endo-
genous nucleotide agonists of P2Rs and, therefore, make
2
attractive drug candidates. The stability of dinucleoside
8
Results
polyphosphates can be further improved. Thus, the Ap A
4
Because dinucleoside polyphosphates generally show en-
hanced enzymatic stability, as compared to nucleotides, and
analogues Ap(CH )pp(CH )pA, Ap(CH )pppA, and App-
2
2
2
2
bind with high affinity to P2Y receptors, we designed ana-
8
(
CH )ppA, 7A-C (Figure 1), were synthesized with a methy-
2
lene group replacing a bridging oxygen in the phosphate
2
chain, thereby enhancing enzymatic stability.
logues 8-10 as potential P2Y R agonists based on dinucleo-
1
9
side tri- or tetra-phosphate scaffolds. To further enhance

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2475
stability, we replaced the R,β or β,γ-bridging oxygen with a
methylene group. To confer agonist potency and selectivity at
the P2Y R, we included a methylthio group at the adenine C2
1
3
1
position. Thismodificationis basedonourpreviousfindings
showing that incorporation of a MeS group at the C2 position
of adenine nucleotides or nucleotide-methylene isosters, in-
2
2
duces selectivity at the P2Y R.
1
Synthesis of Analogues 8-10. Dinucleoside polypho-
sphates are conventionally prepared via the activation of
0
the 5 terminal phosphate of a nucleotide with carbonyl
3
2
diimidazole (CDI), thereby forming a phosphoryl donor
P-donor) that reacts with a nonactivated nucleotide (i.e.,
(
phosphoryl (P-acceptor)). We attempted two different app-
roaches to synthesize analogues 8-10. In the first approach,
analogue 15 was activated with CDI to generate the
3
3
P-donor and analogue 17 was the P-acceptor (Scheme 1).
In the second approach, CDI-activated analogue 17 was the
P-donor and analogue 15 was the P-acceptor. The activation
of 15 and 17 with CDI was monitored by TLC, which showed
that analogue 15 reacted completely with CDI after 3 h,
whereas the activation of analogue 17 was incomplete over
this time period, likely due to the low nucleophilicity of
the phosphonate in analogue 17. To overcome the low
nucleophilicity of the phosphonate group in analogue 17
Figure 3. Agonist potency profile of compounds 8, 9, and 2-MeS-
2þ
ADP for the increase in [Ca
expressing recombinant P2Y receptors. Cells loaded with Fura-2
i
] in 1321N1 astrocytoma cells
1
as described in Experimental Section were exposed to 8, 9, and
-MeS-ADP at the indicated concentrations. Each value shown is a
2
mean ( SE of three experiments.
(Scheme 1), MgCl2 was added to the reactions because
phosphoroimidazolide was reported to be most reactive in
evaluated for in vivo efficacy in the regulation of glucose
homeostasis using an oral glucose tolerance test (OGTT) in
rats. Blood glucose levels of overnight-fasted Wistar Han
male rats rose quickly after oral administration of a glucose
challenge. However, this increase in blood glucose levels was
significantly reduced when the animals were administered
analogue 8 (2.5 mg/kg body weight) or glibenclamide (1 mg/kg
3
0
the presence of divalent metal ions.
Briefly, the synthesis of analogues 8-10 involved the
activation of the 2-MeS-ADP(Bu NH) or 2-MeS-AMP-
3
2
((octyl) NH) salts with CDI in dry DMF at RT for 3 h,
followed by the addition of nonactivated nucleotides, i.e., the
3 2
35
bis/tris(Bu NH) salts of R, β-methylene-2-MeS-ADP, 17, or
body weight), a well-known sulfonylurea insulin secretagogue
3
β,γ-methylene-2-MeS-ATP (Scheme 1, above, Schemes 1 and
(Figure 4A). When rats were exposed to analogue 4, there was a
reduction in blood glucose only after 150 min to ca. 85 mg/dL.
In contrast, analogue 8 reduced the glucose load within 45 min
from 155 to 100 mg/dL, whereas glibenclamide reduced glucose
levels to ca. 63 mg/dL, a level lower than normal.
Analogue 8 is a Potent Insulin Secretagogue in Rat. Insulin
secretagogues act by promoting the physiological release of
insulin from intracellular granulae in pancreatic β-cells.
Blood insulin concentrations in male Wistar Han rats admini-
stered a glucose load were markedly increased 5 min after
injection of analogue 8 (2.5 mg/kg body weight) as compared
with saline (Figure 4B). Analogue 8 greatly increased blood
insulin levels, i.e., 4-fold as compared to basal levels.
2
MgCl with stirring at room temperature for 12 h. Analogues
, Supporting Information), respectively, in the presence of
2
8-10 were formed as the exclusive product at good to high
yields (up to 85% after LC separation). The reaction pro-
ceeded equally well with 2-MeS-AMP, 18, or 2-MeS-ADP, 15.
Yet, formation of analogues 8 or 9 (Np N) was more efficient
4
than analogue 10 (Np N). Analogues 8-10 were character-
3
3
1
ized by P NMR spectrometry and high resolution mass
spectrometry. The formation of analogue 8 was confirmed by
four characteristic peaks: δ 19.49 (d, P ), 12.60 (dd, P ), -8.40
R
β
(
9
(
d, P ), and -18.51 (dd, P ) ppm. The symmetrical analogue
γ
δ
was characterized by two peaks at 10.35 (d, P ) and -7.97
β
d, P ) ppm, whereas analogue 10 showed three peaks at
Resistance of Analogues 8-10 to Alkaline Phosphatase.
Alkaline phosphatase (AP) is a hydrolase that catalyzes the
removal of phosphate groups from nucleotides, proteins,
R
1
Supporting Information).
7.13 (d, P ), 9.45 (dd, P ), and -9.93 (d, P ) ppm (Figure 1,
R
β
γ
3
6
Activity of Analogues 8-10 at the P2Y Receptor. The
and alkaloids to generate P
AP catalyzes the stepwise production of P
i
and the corresponding alcohol.
by ATP hydro-
1
activities of analogues 8-10 were examined at the G protein-
i
3
7
lysis to ADP and subsequently AMP. In humans, AP is
coupled P2Y receptor expressed in human 1321N1 astro-
3
1
4
38
cytoma cells that are devoid of endogenous P2Y receptors.
P2Y R activity was evaluated by monitoring increases in
[Ca ]i induced by the analogues in 1321N1 cell transfectants
expressing the P2Y R. Analogues 8 and 9 activated the
present in all tissues.
We found that AP hydrolyzed ATP with a half-life of 1.4 h
at 37 °C. ATP was 96% degraded to ADP (4%) and AMP
(92%) after 3 h, whereas analogues 8-10 remained unhydro-
lyzed by AP over this time course, as measured by HPLC
(Figure 5).
Resistance of Analogue 8 to Hydrolysis in Human Blood
Serum. Blood serum contains ecto-nucleotide pyrophospha-
tases (e-NPPs) and, therefore, provides a good model system
to study the stability of dinucleoside polyphosphate analo-
gues.
1
þ
2
1
P2Y R with EC values of 0.42 and 0.46 μM, respectively,
1
50
as compared to 2-MeS-ADP (EC50 =0.0025 μM), Figure 3.
Surprisingly, analogue 10 had no activity, although the
2
7
related compound Ap A had an EC value of 0.011 μM.
3
50
The different positions of the methylene groups in the phos-
phate chain (either between the R,β or β,γ phosphates) had
practically no effect on the EC50 values of analogues 8 and 9.
Effects of Analogues 4 and 8 on Blood Glucose Levels in
The half-life of several dinucleoside polyphosphates in
human whole blood and plasma has been reported. Gp G
4
2
2
Rat. Analogues 4 (a potent P2Y R agonist ) and 8 were
1

2
476 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Eliahu et al.
Figure 4. Overnight fasting Wistar Han rats (5 per group) were administered (iv) 2.5 mg/kg bodyweight of analogue 4 or analogue 8, 10 min
postglucose challenge. Glibenclamide was administered orally at -30 min. (A) The effect of analogues 4 or 8 (2.5 mg/kg body weight) on blood
glucose levels over time were compared to glibenclamide (1 mg/kg body weight) or saline administration. (B) The effect of analogue 8(2.5 mg/kg body
weight) or saline on blood insulin concentrations over time.
3
9
þ
has a half-life in blood of ∼5 min, and Ap A has half-lives
other than the ATP-sensitive K -channel, which is activated
by sulfonylureas.
4
of 2 and 4.4 min in whole blood and plasma, respectively,
whereas the half-lives of ATP in whole blood and plasma
In the past years, we have attempted to develop novel insulin
secretagogues targeting P2YRs present in pancreatic β-cells that
are activated by extracellular nucleotides to induce insulin
secretion. We focused on designing modified nucleotides as drug
candidates for the treatment of type 2 diabetes. The goal of
the modifications made to both the adenine (i.e., thioether at
C-2) and phosphate moieties (i.e., phosphorothioate or
boranophosphate ) was to improve the potency and selectivity
of the nucleotide at P2Y Rs that mediate insulin secretion and to
4
0
were 3.6 and 2.2 min, respectively.
Analogue 8, the potent P2Y R agonist and insulin
secretagogue, was hydrolyzed by human blood serum to
1
17,18
2
(
-MeS-AMP, 2-MeS-ADP, and R,β-methylene-2-MeS-ADP
Figure 6A) with a half-life of 12.1 h (Figure 6B) as
monitored by HPLC. In contrast, ATP was hydrolyzed
to ADP and AMP with a half-life of 4.9 h under the same
conditions (Figure 6B).
18
19
1
improve the chemical and metabolic stability of these agonists.
Indeed, 2-hexylthio-ATP-R-S, 2, proved to be a stable and
potent insulin secretagogue at isolated and perfused rat
pancreasby increasing insulin secretion100-fold, as compared
Discussion
Sulfonylurea-based insulin secretagogues used for the treat-
ment of type 2 diabetes suffer from various limitations, especi-
ally the risk of hypoglycemia. These limitations justify the
search for novel insulin secretagogues acting via a protein
1
8
to ATP. Yet, this compound induced a transient vasocon-
1
striction at all concentrations tested.
8

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2477
Figure 5. Hydrolysis of analogues 8-10 and ATP by alkaline
phosphatase monitored by HPLC. Hydrolysis of 4 mM nucleotide
by alkaline phosphatase (12.5 units) at 37 °C was monitored for 3 h.
Another promising candidate was 2-thiomethyl-ATP-R-B,
A, which induced insulin secretion 9-fold above the basal
3
20
level with an EC50 of 28 nM in isolated rat pancreas. Yet,
this analogue was hydrolyzed almost completely by AP over
2
2
1
00 min at 37 °C.
Therefore, our approach in the current study was to synthe-
size hydrolysis-resistant P2Y R agonists with high potency
1
and selectivity using a diadenosine polyphosphate scaffold.
Diadenosine polyphosphates are known to be more resistant
to enzyme-induced hydrolysis than the corresponding mono-
2
8
nucleotides. We conferred additional stability to the dia-
denosine polyphosphates by replacing the R,β or β,γ-bridging
oxygen with a methylene group. In addition, we enhanced
2
2
selectivity of the analogues for the P2Y R by modifying the
1
Figure 6. Enzymatic hydrolysis of analogue 8 and ATP in human
blood serum monitored by HPLC. Hydrolysis of 0.25 mM analogue
8 or ATP in human blood serum (180 μL) and RPMI-1640 (540 μL)
at 37 °C was monitored for 24 h. (A) HPLC chromatograms of
analogue 8 hydrolysates at 3, 7, and 24 h. The location of the peaks
was determined in comparison to nucleotide standards. (B) Percen-
tage of analogue 8 and ATP in human blood serum over time
adenine C2-position by addition of a MeS group to form
analogues 8-10 (Figure 2).
Shaver et al. reported that diadenosine tetraphosphate
(Ap A) has a similar activity and specificity to ATP at P2Y R,
in 1321N1 human astrocytoma cells. Likewise, diadenosine
4
1
triphosphate (Ap A), a structural analogue of ADP, is a potent
3
(
hydrolysis t1/2 =12.1 and 4.9 h, respectively).
2
7
P2Y R agonist.
1
We found that the methylene isoster of Ap A, compound
3
0, was completely inactive at the P2Y R, whereas either the
1
secretagogues. In contrast to analogue 8 (EC50 420 nM at the
P2Y R), analogue 4 (EC 80 nM at the P2Y R) lowered
1
1
50
1
R,β- or β,γ-CH isosters of Ap A, 8 and 9, respectively, were
2
4
blood glucose concentration only after 150 min to ca. 85 mg/dL
Figure 4A).
moderate P2Y R agonists. The methylene substitution redu-
ces the potencies of analogues 8 and 9 (EC50 ∼ 0.4 μM vs
1
(
0
.025 μM for 2-MeS-ADP), as compared to the parent
27
Summary
compound Ap A (EC 0.011 μM vs 0.014 μM for ADP).
4
50
Yet, both analogues 8 and 9 are structurally similar to 2-MeS-
β,γ-CH -ATP, analogue 4, which we have shown to be a
The diadenosine polyphosphate analogue 8 (i.e., di-(2-MeS)-
4
0
00
1
adenosine5 ,5 -P ,P ,R,β-methylene-tetraphosphate) was shown
to be a P2Y R agonist with moderate affinity (i.e., EC50 0.42 μM).
2
potent P2Y R agonist (EC 80 nM vs 4 nM for 2-MeS-
1
1
50
2
ADP). Analogues 8 and 9 activated the P2Y R with EC
2
Analogue 8 is resistant to enzymatic hydrolysis by AP and is
only slowly hydrolyzed in human serum, as compared to ATP,
which is a prerequisite for P2YR agonist-based drug candi-
dates. Furthermore, analogue 8 increased insulin release in
Wistar Han rats in response to a glucose load, as compared
to the saline-treated control, and subsequently blood glucose
levels decreased to normal in comparison to subnormal levels
induced by glibenclamide administration, a common therapy
for type 2 diabetes. Therefore, analogue 8 is an attractive drug
candidate for the treatment of type 2 diabetes in that it may
promote insulin secretion without hypoglycemia.
1
50
values of 0.42 and 0.46 μM, respectively, suggesting that the
position of the methylene group in the polyphosphate chain
has no significant effect on agonist affinity for the P2Y R.
1
Although analogue 8 has a moderate affinity for the P2Y R,
1
it significantly enhances insulin secretion compared to a control
(saline administration; Figure 4B). Consequently, analogue 8
administration reduces a glucose load to normal levels, unlike
glibenclamide, which promotes subnormal levels (Figure 4A).
No correlation was observed between the potency of ana-
logues 4 and 8 atthe P2Y R andtheirin vivo activityasinsulin
1

2
478 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Eliahu et al.
þ
4þ
3
) , and β,γ-methylene-
-MeS-ATP(NH4 )4 salts, respectively. All salts were passed
Experimental Section
ADP(Et
2
3
NH )
2
, 2-MeS-ADP(NH
þ
General. All air- and moisture-sensitive reactions were carried
out in flame-dried, nitrogen-flushed, two-neck flasks sealed with
rubber septa, and the reagents were introduced with a syringe.
Progress of reactions was monitored by TLC on precoated
Merck silica gel plates (60F-254). Visualization of reactants
and products was accomplished with UV light. Nucleotides and
þ
through a column of activated Dowex 50WX-8 200 mesh, H
form. The column eluates were collected in ice-cooled flasks
containing 2 equiv of tributylamine in EtOH. The resulting
solutions were freeze-dried and stored at -20 °C until use.
Bis(tributylammonium) 2-MeS-ADP salt, 15 (110 mg, 0.15
mmol), was dissolved in dry DMF (3 mL) and CDI (121 mg, 0.75
mmol) was added. The resulting solution was stirred at RT for
h. Dry MeOH (61 μL, 9 mmol) was added. After 30 min, a
solution of bis(tributylammonium) R,β-methylene-2-MeS-
ADP, 17 (112 mg, 0.15 mmol), in dry DMF (3 mL) and MgCl
113 mg, 8 equiv) was added. A clear solution was attained after
1
dinucleoside polyphosphates were characterized by H NMR
3
1
spectrometry at 200 or 300 MHz or by P NMR spectrometry in
D O, using 85% H PO as an external reference on a Bruker
3
2
3
4
AC-200 or DPX-300 spectrometer. High resolution mass spec-
tra of nucleotides and dinucleoside polyphosphates were recor-
ded on an AutoSpec-E FISION VG mass spectrometer using
ESI (electron spray ionization) and a Q-TOF microinstrument
(Waters, UK). Primary purification of the dinucleoside poly-
phosphates was achieved on a LC (Isco UA-6) system using a
2
(
a few minutes. The resulting solution was stirred at RT over-
night. Deionized water was added, and the resulting mixture was
freeze-dried. The resulting solid residue was dissolved in deio-
nized water and separated on an activated Sephadex DEAE-
column of Sephadex DEAE-A25 swollen in 1 M NaHCO at
3
A25 column. A buffer gradient of water (1 L) to 0.5 M NH H-
4
4
°C for 1 day. The resin was washed with deionized water before
use. The LC separation was monitored by UV detection at
80 nm. Final purification of the dinucleoside polyphosphates
CO
freeze-dried, and excess NH
3
(1 L) was applied. The relevant fractions were collected,
HCO was removed by repeated
4
3
2
freeze-drying cycles with deionized water to obtain the product
as a white powder. The final dinucleoside polyphosphate puri-
fication was achieved with a semipreparative reverse-phase
was achieved by HPLC (Elite Lachrom, Merck-Hitachi) using a
semipreparative reverse-phase column (Gemini 5u C-18 110A,
2
50 mm ꢀ 10.00 mm; 5 μm; Phenomenex, Torrance, CA). The
3
column and isocratic elution [CH CN/100 mM triethylammo-
purity of the dinucleoside polyphosphates was evaluated by
analytical reverse-phase column chromatography (Gemini 5u
C-18 110A, 150 mmꢀ4.60 mm; 5 μm) with two solvent systems:
solvent system I, (A) 100 mM triethylammonium acetate
nium acetate (TEAA), pH 7] at a flow rate of 5 mL/min. The
fraction containing the product was freeze-dried. The excess
buffer was removed by repeated freeze-drying cycles, with the
solid residue dissolved each time in deionized water. The triethyl-
ammonium counterions were finally exchanged for Na by
passing the pure dinucleoside polyphosphate analogue through
a Sephadex-CM C-25 (Na form) column. Analogue 8 was
obtained at a 75% yield (113 mg) after LC. Analogue 9 was
obtained from 2-MeS-AMP(NH4 ) , 18 (39 mg, 0.09 mmol),
2
and β,γ-methylene-2-MeS-ATP(NH )
at a 85% yield (78 mg) after LC. Analogue 10 was obtained from
-MeS-AMP(NH )
-MeS-ADP(Et NH ) , 17 (80 mg, 0.18 mmol), at a 51% yield
83 mg) after LC. The identity and purity of the products were
established by H and P NMR spectrometry, MALDI mass
spectrometry, and HPLC with two solvent systems.
Di-(2-MeS)-adenosine 5 ,5 -P ,P ,r,β-Methylene-tetraphos-
phate, 8: Separation and Characterization. Analogue 8 was
purified by HPLC on a semipreparative reverse-phase column
using solvent system I with a gradient from 90:10 to 70:30 A:B
(
KH PO , pH 4.5, and (B) CH
TEAA), pH 7, and (B) CH
3
CN; solvent system II, (A) 0.01 M
CN. The details of the solvent
þ
2
4
3
system gradients used for the separation of each product are
given below. In addition, novel dinucleotides were characterized
by HRMS-ESI (negative). The purity of the dinucleotides was gene-
rally g95%. All commercial reagents were used without further
purification unless otherwise noted. All reactants in moisture-
sensitive reactions were dried overnight in a vacuum oven.
þ
þ
þ
4
4
, 20 (58 mg, 0.09 mmol),
þ
2
2
(
4
2
, 18 (50 mg, 0.18 mmol), and R,β-methylene-
3
0
0
þ
RPMI 1640 medium was purchased from Sigma-Aldrich. 2 ,3 -
O-Methoxymethylidene-2-MeS-adenosine, 11, 2-MeS-AMP,
3
1
9
41
22
1
31
1
8, β,γ-methylene-2-MeS-ATP, 20, R,β-methylene-2-MeS-
42
ADP, 17, and 2-MeS-ADP, 15, were prepared, as previously
described. R,β-Methylene-2-MeS-ADP, 17, was prepared from
0
00
1
4
1
2 (216 mg, 0.42 mmol), using dry DMF (1 mL) and was obtai-
ned at a 88% yield (250 mg, 0.37 mmol) after medium pressure
liquid chromatography (MPLC; Biotage, Kungsgatan, Uppsala,
Sweden). The spectral data for 17 were consistent with the
over 15 min at a flow rate of 5 mL/min. Retention time: 8.58 min.
43
1
literature. 2-MeS-ADP, 15, was prepared from 12 (260 mg,
.51 mmol) using dry DMF (2 mL) and was obtained at a 77%
yield (206 mg, 0.39 mmol) after LC. The spectral data for 15
H NMR (D
2
O; 300 MHz): δ 8.22 (s; H-8; 1H), 8.21 (s; H-8; 1H),
6.00 (d; J=3.30 Hz; H-1 ; 1H), 5.98 (d; J=3.60 Hz; H-1 ; 1H),
0
4.78 (H2 signals are hidden by the water signal), 4.54 (m; H-3 ;
2H), 4.32 (m; H-4 , 2H), 4.22 (m; H-5 ; 1H), 4.16 (m; H-5 ; 1H),
2.52 (s; CH ; 3H), 2.50 (s; CH ; 3H), and 2.44 (t; J=20.70 Hz;
Rβ
CH ; 2H) ppm. P NMR (D O; 81 MHz): δ 19.49 (d; J =
0
0
0
0
4
1
0
0
0
0
00
were consistent with the literature. 2 ,3 -O-Methoxymethyli-
dene-2-MeS-adenosine, 11, was separated by MPLC using a
silica gel (25þM) column and the following gradient scheme:
3
3
3
1
2
2
3
column volumes (CV) of 100:0 (I) CHCl
3
:(II) EtOH, 5 CV of a
R Rβ
7.04 Hz; P ), 12.60 (dd; J = 7.04 Hz, Jγβ = 24.46 Hz; Pβ),
gradient from 100:0 to 90:10 I:II and 4 CV of 90:10 I:II at a flow
rate of 12.5 mL/min and was obtained at a 60% yield. R,β-
Methylene-2-MeS-ADP, 17, was separated by MPLC using a
C-18 (12þM) column and the following gradient scheme: 5 column
volumes (CV) of 100:0 (A) TEAA:(B) MeOH, 7.5 CV of a
gradient from 100:0 to 60:40 A:B and 3 CV of 60:40 A:B at a flow
rate of 12 mL/min. pH measurements were performed with an
Orion microcombination pH electrode and a Hanna Instru-
ments pH meter. For preparation of human blood serum, whole
blood taken from healthy volunteers was obtained from a blood
bank (Tel-Hashomer Hospital, Israel). Blood was stored for
-8.40 (d; J =18.63 Hz; P ), and -18.51 (dd; J =24.46 Hz,
δγ
γδ
δ
βγ
J
γ 4 2
=18.63 Hz; P ) ppm. TLC (NH OH:H O:isopropanol 2:8:11),
R 0.35. HRMS MALDI (negative) m/z C H N Na O P S :
f
23 30 10
4
5
18 4 2
-
þ
calculated: 1036.9620; found: 1036.9614 [M þ (Na ) ]. Puri-
5
fication on an analytical column, retention time: 4.58 min
(98.5% purity), using solvent system I with a gradient from
85:15 to 60:40 A:B over 15 min at a flow rate of 1 mL/min;
retention time: 7.72 min (97.0% purity), using solvent system II
with a gradient from 95:5 to 70:30 A:B over 10 min at a flow rate
of 1 mL/min.
0
00
1
4
Di-(2-MeS)-adenosine 5 ,5 -P ,P ,β,γ-Methylene-tetraphos-
phate, 9: Separation and Characterization. Analogue 9 was
purified by HPLC on a semipreparative reverse-phase column
using solvent system I with a gradient from 80:20 to 74:26 A:B
1
2 h at 4 °C and centrifuged in plastic tubes at 1500g for 15 min
at RT. The serum was separated and stored at -80 °C.
Typical Procedure for the Preparation of Dinucleoside Poly-
phosphates 8-10. 2-MeS-AMP(Oct
2
NH )
þ
3
NH )
2
was prepared from
over 6 min at a flow rate of 5 mL/min. Retention time: 3.45 min.
H NMR (D O; 300 MHz): δ 8.23 (s; H-8; 2H), 6.01 (d; J=5.10
þ
1
-MeS-AMP(NH
)
2
salt. R,β-Methylene-2-MeS-ADP(Bu -
4
3
2
þ
þ
0
0
2
, 2-MeS-ADP(Bu NH )
3
2
, andβ,γ-methylene-2-MeS-ATP-
Hz; H-1 ; 2H), 4.78 (H2 signals are hidden by the water signal),
4.53 (m; H-3 ; 2H), 4.34 (m; H-4 , 2H), 4.23 (m; H-5 , 5 ; 4H),
þ
0 0 0 00
(
Bu
3
NH )
2
salts were prepared from R,β-methylene-2-MeS-

Article
.54 (s; CH
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2479
3
1
2
3
; 6H), and 2.53 (t; J=21.31 Hz; CH
2
; 2H) ppm.
P
(2:25:73, 1 mL), and then freeze-dried. The resulting residue
was analyzed by HPLC on a Gemini analytical column (5μ C-18
110A; 150 mm ꢀ 4.60 mm), using gradient elution with solvent
system I at 90:10 to 70:30 A:B over 12 min at a flow rate of 1 mL/min.
The hydrolysis rates of analogues 8-10 with alkaline phospha-
tase were determined by measuring the change in the integration
of the HPLC peaks for each analogue over time.
NMR (D O; 81 MHz): δ 10.35 (d; J=24.94 Hz; P ), and -7.97
2
β
(
d; J=7.04 Hz, Jγβ =24.94 Hz; P
R
4 2
) ppm. TLC (NH OH:H O:
f
0.35. HRMS MALDI (negative) m/z
isopropanol 2:8:11), R
: calculated: 1036.9610; found: 1036.9614
23 30 5 18 4 2
C H N10Na O P S
4
-
þ
5
[
M
þ (Na ) ]. Purification on an analytical column, retention
time: 3.99 min (94.8% purity), using solvent system I with a
gradient from 85:15 to 60:40 A:B over 15 min at a flow rate of
Evaluation of the Stability of Analogue 8 in Human Blood
Serum. The assay mixture containing 40 mM analogue 8 in
deionized water (4.5 μL), human blood serum (180 μL), and
1 mL/min; retention time: 7.84 min (91.2% purity), using
solvent system II with a gradient from 90:10 to 70:30 A:B over
2
2
1
0 min at a flow rate of 1 mL/min.
00
RPMI-1640 medium (540 μL) was incubated at 37 °C for 0-24 h
and aliquots were withdrawn at 0.5-12 h intervals. Each sample
was then heated to 80 °C for 30 min, treated with CM Sephadex
(1-2 mg), stirred for 2 h using a magnetic stirrer, centrifuged for
6 min (13000 rpm; 17900 rcf), and the aqueous layer was
collected and extracted with chloroform (2 ꢀ 500 μL). The
aqueous layer was freeze-dried and then dissolved in 100 μL of
deionized water. Samples were loaded onto an activated starta
0
1
3
Di-(2-MeS)-adenosine 5 ,5 -P ,P ,r,β-Methylene-triphosphate,
0: Separation and Characterization. Analogue 10 was purified
by HPLC on a semipreparative reverse-phase column using
1
solvent system I with a gradient from 90:10 to 70:30 A:B over
1
1
5 min at a flow rate of 5 mL/min. Retention time: 8.58 min. H
NMR (D O; 300 MHz): δ 8.15 (s; H-8; 2H), 5.96 (d; J=4.50 Hz;
2
0
0
0
H-1 ; 2H), 5.94 (d; J = 4.80 Hz; H-1 ; 2H), 4.68 (m; H-2 ; 2H),
0
0
0
4
.49 (m; H-3 ; 2H), 4.31 (m; H-4 , 2H), 4.24 (m; H-5 ; 2H), 4.15
0
X-AW weak anion exchange cartridge, washed with H O (1 mL)
2
0
(
m; H-5 ; 2H), 2.50 (s; CH ; 3H), 2.48 (s; CH ; 3H), and 2.37
3
and MeOH:H O (1:1, 1 mL), eluted with NH OH:MeOH:H O
2
3
4
2
3
1
(
m; CH ; 4H) ppm. P NMR (D O; 81 MHz): δ 17.13 (d; J
=
(2:25:73, 1 mL), and then freeze-dried. The resulting residue was
analyzed by HPLC on a Gemini analytical column (5u C-18
110A; 150 mm ꢀ 4.60 mm) using gradient elution with solvent
system I at 90:10 to 70:30 A:B over 12 min at a flow rate of 1 mL/min.
The hydrolysis rate of analogue 8 in human blood serum was
determined by measuring the change in the integration of the
HPLC peak for analogue 8 over time.
2
2
βR
8
-
.66 Hz; P
9.93 (d; Jβγ = 25.92 Hz; P
R
), 9.45 (dd; JRβ = 8.66 Hz, Jγβ =25.92 Hz; P
β
), and
O:
0.61. HRMS MALDI (negative) m/z
: calculated: 846.0734; found: 846.0729
M-3-deuterium). Purification on an analytical column, reten-
γ 4 2
) ppm. TLC (NH OH:H
isopropanol 2:8:11), R
C H D N O P S
23 27 3 10 15 3 2
(
f
tion time: 5.20 min (99.7% purity), using solvent system I with a
gradient from 85:15 to 60:40 A:B over 15 min at a flow rate of
Intracellular Calcium Measurements. Human 1321N1 astro-
cytoma cells stably expressing the turkey P2Y receptor were
grown in Dulbecco’s modified Eagle’s medium containing 5%
1
mL/min; retention time: 5.33 min (99.1% purity), using sol-
vent system II with a gradient from 90:10 to 70:30 A:B over
0 min at a flow rate of 1 mL/min.
1
1
(v/v) fetal bovine serum, 100 units/mL penicillin, 100 μg/mL
streptomycin, and 500 μg/mL Geneticin (G-418; Life Techno-
logies, Inc.). Changes in the intracellular free calcium con-
0
0
0
2
A solution of 4-DMAP (132 mg, 1.07 mmol, 4 equiv) in CH Cl
,3 -O-Methoxymethylidene-5 -O-Tosyl-2-MeS-adenosine, 12.
2
2
2þ
(
dry CH
1 mL) and a solution of TsCl (130 mg, 0.67 mmol, 2.5 equiv) in
0
centration, [Ca ]i, were detected by dual-excitation spectro-
fluorometric analysis of cell suspensions loaded with fura-2, as
0
2
Cl
2
(1 mL) were added to a suspension of 2 ,3 -O-
4
5,46
methoxymethylidene 2-MeS-adenosine, 11 (95 mg, 0.27 mmol),
in CH Cl (3 mL) in a flame-dried two-neck flask under N at
RT. The suspension turned clear, and the reaction mixture was
stirred for 2 h at RT. CH Cl (30 mL) was added to the reaction
mixture, which was extracted with saturated NaHCO solution
SO and
described previously.
Cells were treated with the indicated
2
2
2
nucleotide or analogue at 37 °C in 10 mM Hepes-buffered saline
(pH 7.4) containing 1 mM CaCl and 1 mM MgCl , and the
2
2
2
þ
2
2
maximal increase in [Ca ]i was determined at various nucleo-
tide or analogue concentrations to calculate the EC . Concen-
3
5
0
(
3 ꢀ 30 mL). The organic phase was treated with Na
2
4
tration-response data were analyzed with the Prism curve
fitting program (GraphPAD Software, San Diego, CA). Three
experiments were conducted on separate days to evaluate P2Y1
receptor activity.
filtered. The solvent was removed under reduced pressure, and
the residue was separated by MPLC using a silica gel (25þM)
column and the following gradient scheme: 3 column volumes
(
1
2
CV) of 100:0 (A) CH
2
Cl
00:0 to 90:10 A:B and 4 CV of 90:10 A:B at a flow rate of
5 mL/min. The relevant fraction was collected and the solvent
2
:(B) EtOH, 5 CV of a gradient from
In Vivo Study. Animals. All animal studies were performed
by Vetgenerics Ltd. (Rehovot, Israel). Wistar Han male rats,
10-11 weeks old, were supplied by Harlan Laboratories (Israel).
All rats were acclimatized for 6 days prior to commencement of
the study. Rats were inspected daily for health and welfare
throughout the study. The animals were weighed, and those
most uniform in weight were surgically cannulated. Cannulated
rats were examined for approximately 48 h following the sur-
gical cannulation to ensure recovery.
Surgical Cannulation Procedure. Animals were anesthetized
with 2.5% isofluran and 97.5% dry air inhalation. Anesthetized
rats were secured in the supine position, and a 2 cm midline neck
incision was fashioned to access the jugular vein and carotid
artery. A P52 cannula was surgically inserted and fixed in the
jugular vein and flushed with 0.3-0.5 mL of 5% (v/v) hepar-
inized saline after cannulation and also immediately after each
blood collection.
Oral Glucose Tolerance Test (OGTT). The function of each
cannula was checked in the morning of each experiment. Over-
night fasting Wistar Han rats with functional cannulae were
weighed and their peripheral tail vein blood glucose levels were
measured (Abbott Glucometer FreeStyle and/or FreeStyle
Lite), and animals with similar weights and blood glucose levels
were assigned to the same group. Then rats were left untouched
in individual cages for approximately 2 h. Glibenclamide (1 mg/kg
body weight, per os) was administrated at t=-30 min, and then
was removed under reduced pressure to obtain analogue 12 at a
8
δ 7.83 (s; H-8; 2H), 7.69, 7.23 (2 m, 2H), 6.34, 6.29 (2 br s, NH ,
1
5% yield (115 mg) as a white solid. H NMR (CDCl ; 200 MHz):
3
2
0
2
H), 6.17, 6.05 (2d; J = 2.60 Hz; H-1 , 1H), 6.07, 6.01 (2s,
0
CH-OMe, 1H), 5.56, 5.53 (2dd; J = 2.60 Hz; H-2 ; 1H, J =
7
3
0
0
.20 Hz; H-2 , 1H), 5.21, 5.06 (2dd, J =7.20 Hz; H-3 ; 1H, J=
0
0
0
.20 Hz; H-3 , 1H), 4.66, 4.52 (2 m, H-4 , 1H), and 4.31 (m, H5 ,
0
0
-
H5 , 4H) ppm. MS-ESI m/z 584 (M ). TLC (CHCl
0.64.
3
:EtOH 9:1),
R
f
Evaluation of the Stability of Analogues 8-10 with Alkaline
Phosphatase. Alkaline phosphatase activity was determined by
the release of p-nitrophenol from p-nitrophenyl phosphate
measured by a UV-vis spectrophotometer at 405 nm. Rela-
tive enzymatic hydrolysis and resistance to hydrolysis of ana-
logues 8-10 were determined at 37 °C. A solution of 0.2 mg of
the analogue in 77.5 μL deionized water, 0.1 M Tris-HCl, and
4
4
0
.1 M MgCl2 containing calf intestine alkaline phosphatase
(Fermentas Inc., Glen Burnie, MD; 10 unit/μL; 1.25 μL, 12.5 units)
was incubated at 37 °C. The final pH was 9.8. After 3 h, the
reaction was stopped by incubation of the sample at 80 °C for
1
weak anion exchange cartridge, washed with H
5 min. Samples were loaded onto an activated starta X-AW
2
O (1 mL) and
MeOH:H O (1:1, 1 mL), eluted with NH OH:MeOH:H O
2
2
4

2
480 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6
Eliahu et al.
0
a glucose challenge of 2 g/kg body weight in a volume of 3 mL/kg
body weight was orally administered to each rat at t = 0 min.
Administration of analogues 4 or 8 (2.5 mg/kg body weight, iv)
and saline control to rats (5 per group) was performed at t =
5 -triphosphate and related analogs on insulin secretion from the
rat pancreas. Fundam. Clin. Pharmacol. 1997, 11, 537–545.
17) Hillaire-Buys, D.; Shahar, L.; Fischer, B.; Chulkin, A.; Linck, N.;
(
Chapal, J.; Loubatieres-Mariani, M. M.; Petit, P. Pharmacological
evaluation and chemical stability of 2-benzylthioether-5 -O-(1-thio-
0
triphosphate)-adenosine, a new insulin secretagogue acting through
1
0 min. Analogues 4 or 8 or saline were injected intravenously
via an iv catheter in volumes of 1 mL saline/kg body weight.
Blood glucose levels were measured immediately following tail-
vein puncture by placing a blood droplet on a glucometer stick
inserted into a glucometer (Abbott FreeStyle and/or FreeStyle
Lite).
P2Y receptors. Drug Dev. Res. 2001, 53, 33–43.
(18) Fischer, B.; Chulkin, A.; Boyer, J. L.; Harden, K. T.; Gendron,
F.-P.; Beaudoin, A. R.; Chapal, J.; Hillaire-Buys, D.; Petit, P.
2
0
insulin secretagogues acting through P2Y-receptors. J. Med. Chem.
-Thioether 5 -O-(1-thiotriphosphate)adenosine derivatives as new
1
19) Nahum, V.; Zuendorf, G.; Levesque, S. A.; Beaudoin, A. R.;
999, 42, 3636–3646.
For measurements of insulin concentration, blood samples
150 μL) were withdrawn via the jugular vein cannula and
(
(
0
(
Reiser, G.; Fischer, B. Adenosine 5 -O-(1-boranotriphosphate)
derivatives as novel P2Y1 receptor agonists. J. Med. Chem. 2002,
collected in 0.8 mL tubes containing Z serum/Gel (Mini Collect,
Greiner-bio-one, Austria). Blood was allowed to clot at RT for
at least 30 min. Postclotting, the blood was centrifuged (3000g,
4
20) Farret, A.; Filhol, R.; Linck, N.; Manteghetti, M.; Vignon, J.;
5, 5384–5396.
Gross, R.; Petit, P. P2Y receptor mediated modulation of insulin
release by a novel generation of 2-substituted-5 -O-(1-borano-
1
5 min) in a refrigerated centrifuge at 4 °C. Serum was har-
vested, divided into 25 μL aliquots in 0.2 mL flat cap PCR tubes
Neptune, USA cat. no. 3423), and stored frozen at -20 °C for
0
triphosphate)-adenosine analogues. Pharm. Res. 2006, 23, 2665–
2671.
21) Chevassus, H.; Roig, A.; Belloc, C.; Lajoix, A. D.; Broca, C.;
Manteghetti, M.; Petit, P. P2Y receptor activation enhances insulin
release from pancreatic beta-cells by triggering the cyclic AMP/
protein kinase A pathway. Naunyn-Schmiedeberg’s Arch. Pharma-
col. 2002, 366, 464–469.
(
(
5
-7 days pending analysis. Serum insulin concentration was
determined using a Rat/Mouse Insulin kit from Smart Assays,
QBI Laboratories, Nes-Ziona, Israel.
(
22) Eliahu, S. E.; Camden, J.; Lecka, J.; Weisman, G. A.; Sevigny, J.;
Supporting Information Available: Characterization of ana-
31
Gelinas, S.; Fischer, B. Identification of hydrolytically stable and
1
selective P2Y receptor agonists. Eur. J. Med. Chem. 2009, 44,
logues 8-10 by P NMR and syntheses of analogues 9-10.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1
525–1536.
(
23) Hoyle, C. H. V.; Hilderman, R. H.; Pintor, J. J.; Schluter, H.; King,
B. F. Diadenosine polyphosphates as extracellular signal mole-
cules. Drug Dev. Res. 2001, 52, 260–273.
References
(24) Pintor, J.; Peral, A.; Hoyle, C. H. V.; Redick, C.; Douglass, J.;
Sims, I.; Yerxa, B. Effects of diadenosine polyphosphates on tear
secretion in New Zealand white rabbits. J. Pharmacol. Exp. Ther.
(
(
(
1) Fischer, B.; Nahum, V. Dinucleoside poly(borano)phosphate deri-
vatives and uses thereof. U.S. Patent 7,368,439, 2008.
2) Ashcroft, F.; Rorsman, P. Type 2 diabetes mellitus: not quite
exciting enough? Hum. Mol. Genet. 2004, 13, R21–R31.
3) Stumvoll, M.; Goldstein, B. J.; van Haeften, T. W. Type 2 diabetes:
2
002, 300, 291–297.
(
(
(
25) Mediero, A.; Peral, A.; Pintor, J. Dual roles of diadenosine
polyphosphates in corneal epithelial cell migration. Invest.
Ophthalmol. Vis. Sci. 2006, 47, 4500–4506.
principles of pathogenesis and therapy. Lancet 2005, 365, 1333–
26) Guzman-Aranguez, A.; Crooke, A.; Peral, A.; Hoyle, C. H. V.;
Pintor, J. Dinucleoside polyphosphates in the eye: from physiology
to therapeutics. Prog. Retinal Eye Res. 2007, 26, 674–687.
27) Shaver, S. R.; Rideout, J. L.; Pendergast, W.; Douglass, J. G.;
Brown, E. G.; Boyer, J. L.; Patel, R. I.; Redick, C. C.; Jones, A. C.;
Picher, M.; Yerxa, B. R. Structure-activity relationships of dinu-
cleotides: potent and selective agonists of P2Y receptors. Purinergic
Signalling 2005, 1, 183–191.
1346.
(
4) UPDSU Group. Intensive blood-glucose control with sulphony-
lureas or insulin compared with conventional treatment and risk of
complications in patients with type 2 diabetes (UKPDS 33). Lancet
1998, 352, 837-853.
(
(
(
5) Ishida, H. Mechanism of action of antidiabetic sulfonylureas.
Diabetes Front. 1999, 10, 99–104.
6) Lebovitz, H. Oral Antidiabetic Agents; Lea and Febiger: Philadelphia,
(
28) Yerxa, B. R.; Sabater, J. R.; Davis, C. W.; Stutts, M. J.; Lang-Furr,
M.; Picher, M.; Jones, A. C.; Cowlen, M.; Dougherty, R.; Boyer, J.;
Abraham, W. M.; Boucher, R. C. Pharmacology of INS37217
1994; pp 508-529.
7) Leibowitz, G.; Cerasi, E. Sulfonylurea treatment of NIDDM
patients with cardiovascular disease: A mixed blessing? Diabeto-
logia 1996, 39, 503–514.
0 0 0
P1-(uridine 5 )-P4-(2 -deoxycytidine 5 )tetraphosphate, tetraso-
dium salt], a next-generation P2Y2 receptor agonist for the treat-
[
(
8) Coutinho-Silva, R.; Parsons, M.; Robson, T.; Burnstock, G.
Changes in expression of P2 receptors in rat and mouse pancreas
during development and ageing. Cell Tissue Res. 2001, 306, 373–
ment of cystic fibrosis. J. Pharmacol. Exp. Ther. 2002, 302, 871–
80.
8
29) Guranowski, A.; Biryukov, A.; Tarussova, N. B.; Khomutov,
(
383.
R. M.; Jakubowski, H. Phosphonate analogs of diadenosine
5
(
9) Farret, A.; Lugo-Garcia, L.; Galtier, F.; Gross, R.; Petit, P.
Pharmacological interventions that directly stimulate or modulate
insulin secretion from pancreatic beta-cell: Implications for the
treatment of type 2 diabetes. Fundam. Clin. Pharmacol. 2005, 19,
0
tic and eukaryotic enzymes degrading dinucleoside tetraphosphates.
000
,5 -P1,P4-tetraphosphate as substrates or inhibitors of prokaryo-
Biochemistry 1987, 26, 3425–3429.
(
30) Nahum, V.; Tulapurkar, M.; Levesque, S. A.; Sevigny, J.; Reiser,
G.; Fischer, B. Diadenosine and diuridine poly(borano)phosphate
analogs: synthesis, chemical and enzymatic stability, and activity at
P2Y1 and P2Y2 receptors. J. Med. Chem. 2006, 49, 1980–1990.
31) Fischer, B.; Boyer, J. L.; Hoyle, C. H. V.; Ziganshin, A. U.;
Brizzolara, A. L.; Knight, G. E.; Zimmet, J.; Burnstock, G.;
Harden, T. K.; Jacobson, K. A. Identification of potent, selective
P2Y-purinoceptor agonists: structure-activity relationships for
647–656.
(
(
10) Kindmark, H.; Kohler, M.; Nilsson, T.; Arkhammar, P.; Wiechel,
K. L.; Rorsman, P.; Efendic, S.; Berggren, P. O. Measurements of
2
þ
cytoplasmic free Ca concentration in human pancreatic islets and
insulinoma cells. FEBS Lett. 1991, 291, 310–314.
(
11) Leon, C.; Freund, M.; Latchoumanin, O.; Farret, A.; Petit, P.;
Cazenave, J.-P.; Gachet, C. The P2Y1 receptor is involved in the
maintenance of glucose homeostasis and in insulin secretion in
mice. Purinergic Signalling 2005, 1, 145–151.
12) Bertrand, G.; Chapal, J.; Loubatieres-Mariani, M. M.; Roye, M.
Evidence for two different P2-purinoceptors on beta cell and
pancreatic vascular bed. Br. J. Pharmacol. 1987, 91, 783–787.
13) Fernandez-Alvarez, J.; Hillaire-Buys, D.; Loubatieres-Mariani,
M. M.; Gomis, R.; Petit, P. P2 receptor agonists stimulate insulin
release from human pancreatic islets. Pancreas 2001, 22, 69–71.
14) Abracchio, M. P.; Williams, M. In Purinergic and Pyrimidinergic
Signaling; Springer: Berlin, 2001; Vol. 151, pp 377-391.
15) Farret, A.; Vignaud, M.; Dietz, S.; Vignon, J.; Petit, P.; Gross, R.
P2Y purinergic potentiation of glucose-induced insulin secretion
and pancreatic beta-cell metabolism. Diabetes 2004, 53, S63–S66.
16) Chapal, J.; Hillaire-Buys, D.; Bertrand, G.; Pujalte, D.; Petit, P.;
Loubatieres-Mariani, M. M. Comparative effects of adenosine-
0
2-thioether derivatives of adenosine 5 -triphosphate. J. Med.
(
Chem. 1993, 36, 3937–3946.
(32) Zatorski, A.; Goldstein, B. M.; Colby, T. D.; Jones, J. P.; Pankiewicz,
K. W. Potent inhibitors of human inosine monophosphate dehydro-
genase type II. fluorine-substituted analogs of thiazole-4-carboxamide
adenine dinucleotide. J. Med. Chem. 1995, 38, 1098–1105.
(33) Hoard, D. E.; Ott, D. G. Conversion of mono- and oligodeoxyri-
bonucleotides to 5 -triphosphates. J. Am. Chem. Soc. 1965, 87,
(
0
(
(
1785–1788.
(34) Parr, C. E.; Sullivan, D. M.; Paradiso, A. M.; Lazarowski, E. R.;
Burch, L. H.; Olsen, J. C.; Erb, L.; Weisman, G. A.; Boucher, R. C.;
Turner, J. T. Cloning and expression of a human P2U nucleotide
receptor, a target for cystic fibrosis pharmacotherapy. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 3275–3279.
(

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 6 2481
(
35) Ostergard, T.; Degn, K. B.; Gall, M.-A.; Carr, R. D.; Veldhuis,
J. D.; Thomsen, M. K.; Rizza, R. A.; Schmitz, O. The insulin
secretagogues glibenclamide and repaglinide do not influence
growth hormone secretion in humans but stimulate glucagon
secretion during profound insulin deficiency. J. Clin. Endocrinol.
Metab. 2004, 89, 297–302.
36) Hull, W. E.; Halford, S. E.; Gutfreund, H.; Sykes, B. D.
Phosphorus-31 nuclear magnetic resonance study of alkaline
phosphatase: the role of inorganic phosphate in limiting the
enzyme turnover rate at alkaline pH. Biochemistry 1976, 15,
blood ending up as intracellular ATP in the erythrocytes. Eur. J.
Biochem. 1988, 173, 241–245.
(41) Skoblov, M. Y.; Yas’ko, M. V.; Skoblov, Y. S. The preparation of
32P-labeled 2-methylthioadenosine di- and triphosphates. Bioorg.
Khim. 1999, 25, 702–707.
(42) Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. Synthesis
0
0
(
of nucleotide 5 -diphosphates from 5 -O-tosyl nucleosides. J. Org.
Chem. 1987, 52, 1794–1801.
(43) Boeynaems, J.-M.; Goldman, M.; Marteau, F.; Communi, D. Use
of purinergic and pyrimidinergic receptor agonists for dendritic
cell-based immunotherapies. Patent 2006-EP7966 2007020018,
20060811, 2007.
(44) Brandenberger, H.; Hanson, R. Spectrophotometric determination of
acid and alkaline phosphatases. Helv. Chim. Acta 1953, 36, 900–906.
(45) Garrad, R. C.; Otero, M. A.; Erb, L.; Theiss, P. M.; Clarke, L. L.;
Gonzalez, F. A.; Turner, J. T.; Weisman, G. A. Structural basis of
1
547–1561.
(
(
37) Moss, D. W.; Walli, A. K. Intermediates in the hydrolysis of ATP
by human alkaline phosphatase. Biochim. Biophys. Acta, Enzymol.
1969, 191, 476–477.
38) Harada, M.; Udagawa, N.; Fukasawa, K.; Hiraoka, B. Y.; Mogi,
M. Inorganic pyrophosphatase activity of purified bovine pulp
alkaline phosphatase at physiological pH. J. Dent. Res. 1986, 65,
agonist-induced desensitization and sequestration of the P2Y
2
1
25–127.
nucleotide receptor. Consequences of truncation of the C terminus.
J. Biol. Chem. 1998, 273, 29437–29444.
(46) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of
(
39) Grau, V. Union de diguanosin tetrafosfato a cerebro de rata.
Universidad de Extremadura: Badajoz, Spain, 1982.
40) Luethje, J.; Ogilvie, A. Catabolism of Ap4A and Ap3A in whole
blood. The dinucleotides are long-lived signal molecules in the
2
þ
(
Ca indicators with greatly improved fluorescence properties.
J. Biol. Chem. 1985, 260, 3440–3450.