2-MeS-β,γ-CCl2-ATP is a potent agent for reducing intraocular pressure

Article abstract of DOI:10.1021/jm100030u

Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydrolyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37 °C. In human blood serum, analogues 8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t_1/2 1 h-30 days). Analogues 8-14 were agonists of the P2Y₁ receptor (EC₅₀ 0.57-9.54 μM). Ocular administration of most analogues into rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC₅₀ 95.5 nM). Analogue 9 was more effective at reducing IOP than several common glaucoma drugs and represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.

Full text of DOI:10.1021/jm100030u

J. Med. Chem. 2010, 53, 3305–3319 3305
DOI: 10.1021/jm100030u
2-MeS-β,γ-CCl2-ATP is a Potent Agent for Reducing Intraocular Pressure†
§
§
Shay Eliahu,‡ Alba Martın-Gil,§ Marı
a Jesus Perez de Lara, Jesus Pintor, Jean Camden, Gary A. Weisman,
ꢀ ꢀ
´ ´
ꢀ
Joanna Lecka, Jean Sevigny, and Bilha Fischer*
^
^
,‡
‡
§
´
Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel, Dept. Bioquımica, E.U. Optica, Universidad Complutense de
Madrid, c/Arcos de Jalon s/n, E-28037 Madrid, Spain, Biochemistry Department, 540E Bond Life Sciences Center, University of
Missouri, Columbia, Missouri 65211, and ^Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier
Universitaire de Queꢀbec, Universiteꢀ Laval, Queꢀbec, QC, Canada
Received January 11, 2010
Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of
P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydro-
lyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely
resistant to hydrolysis by alkaline phosphatase over 30 min at 37 ꢀC. In human blood serum, analogues
8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h,
respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t1/2 1 h-30 days). Analogues 8-14
were agonists of the P2Y1 receptor (EC50 0.57-9.54 μM). Ocular administration of most analogues into
rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC50 95.5 nM). Analogue 9 was more
effective at reducing IOP than several common glaucoma drugs and represents a promising alternative
to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac
problems.
Introduction
reduces aqueous humor outflow in bovines. Other studies
have reported the presence of P2Y1 and P2Y2 receptors in
bovine TM cells and P2Y1, P2Y4, and P2Y11 receptors in a
human TM cell line.13
Extracellular nucleotides that activate G protein-coupled
P2Y receptors (P2YRs) are attractive pharmaceutical targets
due to their ability to modulate various functions in many
tissues and organs under normal and pathophysiological
conditions.1-5 Extracellular nucleotides and dinucleotides
have been shown to play a role in ocular physiology and
pathophysiology6 and have been suggested as therapeutic
agents for dry eye, retinal detachment, and glaucoma.7
Ocular hypertension, the most common cause of glaucoma,
is a target for agents that reduce intraocular pressure (IOPa).8
When topically applied to New Zealand white rabbits, some
nucleotides produce an increase in IOP while others decrease
IOP. IOP can be reduced by administration of ATP,9 adeno-
sine tetraphosphate (Ap4),10 and diadenosine tetraphosphate
(Ap4A).11 In contrast, diadenosine triphosphate (Ap3A) and
diadenosine pentaphosphate (Ap5A) can increase IOP.9,11
Receptors for extracellular nucleotides, including P2Y1,
P2Y2, and P2Y4, have been identified in trabecular meshwork
cells (TM), an area of tissue in the eye that is responsible for
draining the aqueous humor.12 Among these P2YR subtypes,
activation of the P2Y1R by the selective agonist 2-MeS-ADP
The therapeutic potential of endogenous nucleotides for the
treatment of glaucoma is limited because they are degraded by
extracellular enzymes, which reduces their potency, efficacy,
and duration of action. In addition, although nucleotides are
chemically stable in a pH range of 4-11,14 they are rapidly
degraded at more acidic or basic pH. Nucleotides are hydro-
lyzed enzymatically by the ecto-nucleoside triphosphate dipho-
sphohydrolase family of ectonucleotidases (i.e., e-NTPDase
and alkaline phosphatases)15 and ecto-nucleotide pyropho-
sphatases/phosphodiesterases, (i.e., e-NPPs).16,17 Therefore,
there is a need for the identification of enzymatically and
chemically stable nucleotide scaffolds that can be used to
develop selective and potent P2YR agonists.
A few attempts to improve the stability of nucleotides have
been reported,18-26 including the use of phosphate bioisosters
of nucleotides such as phosphonate,27,28 phosphoramide,29
and boranophosphate15,20,27,30,31 analogues. Previously, we
developed a series of β,γ-methylene-ATP, analogue 1, and
β,γ-methylene-ATP(R-borano) derivatives, analogues 2, 3
(Figure 1).27 We showed that replacing a β,γ-bridging oxygen
in ATP with a methylene group conferred a significant resis-
tance to enzymatic and chemical (i.e., at pH 1.4 and 37 ꢀC)
hydrolysis.27 We also showed that replacing the PR nonbridging
oxygen by a borano isoster decreased enzymatic hydrolysis
of the analogues by human blood serum, NTPDase1,2,3,8,
NPP1,3, and alkaline phosphatase.27
†U.S. Provisional application no. 61/282,133 was filed on 22 December,
2009.
*To whom correspondence should be addressed. Phone: 972-3-
5318303. Fax: 972-3-6354907. E-mail: bfischer@mail.biu.ac.il.
aAbbreviations: IOP, intraocular pressure; P2R, P2 receptor;
E-NTPDase, ecto-nucleosidetriphosphate diphosphohydrolase;E-NPPs,
ecto-nucleotide pyrophosphatases; MPLC, medium pressure liquid chro-
matography; TEAA, triethylammonium acetate; HRMS-MALDI, high
resolution mass spectrometry matrix-assisted laser desorption/ionization;
MS-ESI, mass spectrometry-electron spray ionization; CDI, carbonyl
diimidazole; DCC, dicyclohexylcarbodiimide; TMP, trimethylphosphate;
AP, alkaline phosphatase.
Analogues 1-3 also were evaluated as agonists of the
P2Y1R.27 The most effective P2Y1R agonist was analogue 1,
with an EC50 of 80 nM. Yet, although analogues 2 and 3 were
r
2010 American Chemical Society
Published on Web 03/25/2010
pubs.acs.org/jmc

3306 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Eliahu et al.
Figure 1. Structures of previously tested nucleotides.
Figure 2. Structures of nucleotides tested in this study.
weak P2Y1R agonists (EC50 of 17.2 μM for analogue 3B,
whereas analogues 2A, 2B, and 3A were inactive),27 while the
structurally related analogue 4A (Figure 1) was a potent
P2Y1R agonist with an EC50 of 2.6 nM.15
The decreased activity of analogues 1, 2A, 2B, 3A, and 3B,
as compared to analogue 4A, is possibly related to the elevated
pKa of the terminal phosphate in analogue 4A, as compared to
phosphate (pKa 8.4 vs 6.5, respectively).32,33 Under physiolo-
gical conditions, i.e., pH 7.4, 91% of ATP is ionized, whereas
the β,γ-methylene analogues of ATP are only 9% ionized in
solution. Analogues 1-3 are 90% protonated at Pγ at pH 7.4,
which likely prevents significant electrostatic interactions with
positively charged amino residues in the P2Y1R.34,35
The electronegativity of a dihalogenated methylene group
(e.g., CF2, CCl2) lowers the pKa of phosphonate from 8.4 to
6.7-7.0, making it closer to the pKa of phosphate (i.e., 6.5).36,37
Indeed, nucleotide analogues with a β,γ-dihalomethylene
group were reported to be promising inhibitors of HIV-1
reverse transcriptase,30,37 agonists of P2X2/3 receptors,38 and
potent antagonists of the P2Y12 receptor.39,40
The current study describes the synthesis of several nucleo-
side-50-dihalomethylene-di- and triphosphate analogues (i.e.,
analogues 8-14, Figure 2) and characterizes their activity at the
P2Y1R, their chemical stability at acidic pH, and their hydro-
lytic stability with alkaline phosphatase, NTPDase1,2,3,8, and
NPP1,3, and human blood serum. Furthermore, the potency,
efficacy, and duration of action of analogues 8-14 have been
evaluated for reduction of IOP in vivo. In comparison to
several commercially available antiglaucoma drugs, analogues
1and 9were found to be promising and novel candidates for the
treatment of ocular hypertension and glaucoma through acti-
vation of P2 receptors (P2R).
yield.27 Here, we used this synthetic procedure to prepare the
β,γ-CF2/CCl2-2-MeS-ATP analogues 8 and 9 (Scheme 1). To
ensure the selective phosphorylation of 2-MeS-adenosine at the
50-OH, we used 20,30-methoxy-methylidene-2-MeS-adenosine,
analogue 15, as the starting material. Analogue 15 was first
treated with POCl3 in trimethylphosphate (TMP) in the pre-
sence of proton sponge at 0 ꢀC for 1-3 h, followed by treatment
with bis(tributylammonium) dihalomethylene-diphosphonate
and tributylamine at 0 ꢀC for 40 min to 1.5 h. Finally, hydrolysis
of the cyclic intermediates, analogues 17 and 18, in
0.5 M TEAB and deprotection of the methoxymethylidene
groups generated β,γ-CF2-2-MeS-ATP, analogue 8, at a 46%
overall yield, and β,γ-CCl2-2-MeS-ATP, analogue 9, at a 10%
overall yield, respectively. The formation of analogues 8 and 9
was confirmed by the presence of three typical signals in 31P
NMR, as described previously:43 3.24 (m, Pγ), -4.89 (m, Pβ),
and -10.61 (d, PR) ppm for analogue 8, and 8.15 (d, Pγ), 1.04
(dd, Pβ), and -10.22 (d, PR) ppm for analogue 9.
The preparation of R-borano-β,γ-CF2-2-MeS-ATP, analo-
gue 10, R-borano-β,γ-CCl2-ATP, analogue 11, and R-borano-
β,γ-CCl2-2-MeS-ATP, analogue 12, required the protection
of the 20- and 30-hydroxyl groups in analogues 15 and 21 by
methoxy-methylidene groups, which remained throughout
the entire synthesis until removal in the last step (Scheme 2).
In the first synthetic step, we used PCl3, which was rapidly and
completely consumed in less than 30 min to form analogues 22
and 23. Next, dihalogenbisphosphonate salt was added and
the reactions progressed for 25-60 min at 0 ꢀC, followed by
addition of BH3 SMe2 at 0 ꢀC and stirring for 1 h at RT to
3
obtain intermediates 27-29. Finally, hydrolysis of the cyclic
intermediates in 0.5 M TEAB produced analogues 30-32.
The latter analogues were deprotected to generate analogues
10, 11, and 12 at 21%, 18%, and 7% overall yields, respec-
tively, after LC. The identity and purity of analogues 10-12
were established by 1H and 31P NMR, ESI, or MALDI
negative mass spectrometry, and HPLC using two solvent
systems. 31P NMR spectra of analogues 10-12 showed a
Results
Synthesis of Analogues 8-14. ATP analogues in which the
β,γ-bridging oxygen is substituted by a methylene group are
conventionally prepared via the activation of the 50-phos-
phate of nucleoside-50-monophosphate (NMP) to form a
phosphoryl donor, followed by a reaction with methylene
bisphosphonate salt (phosphoryl acceptor). Phosphoryl do-
nors were prepared by activation of NMP with carbonyl
diimidazole (CDI),41 trifluoroaceticanhydride, and N-methy-
limidazole42 or dicyclohexylcarbodiimide (DCC),33 followed
by condensation with methylene bisphosphonic acid or its
salt.
1
typical PR signal as a multiplet at about 80 ppm. H NMR
spectra showed borane hydrogen atoms as a very broad signal
at about 0.4 ppm. Because of the chiral center at PR, each
analogue was obtained as a pair of diastereoisomers in a 1:1
ratio. In both the 1H and 31P NMR spectra, there was a slight
difference between the chemical shifts for the two diastereo-
isomers of each analogue. These isomers were well separated
by reverse-phase HPLC with about a 1-2 min difference in
their retention times with the A isomer eluting before the B
isomer.
Recently, we reported a facile three-step one-pot synthesis
of β,γ-methylene-2-MeS-ATP, analogue 1, at a 35% overall

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3307
Scheme 1a
a Reaction conditions for analogue 8: (a) trimethylphosphate, POCl3, proton sponge, 0 ꢀC, 3 h; (b) 0.5 M bis(tributylammonium)difluoromethylene
diphosphonate in dry DMF, Bu3N, 0 ꢀC, 1.5 h; (c) 0.5 M TEAB, pH 7, RT, 1 h; (d) (1) 18% HCl, pH 2.3, RT, 3 h, (2) 24% NH4OH, pH 9, RT, 45 min.
Reaction conditions for analogue 9: (a) trimethylphosphate, POCl3, proton sponge, 0 ꢀC, 1 h; (b) 1 M bis(tributylammonium)dichloromethylene
diphosphonate in dry DMF, Bu3N, 0 ꢀC, 40 min, and then RT, 5 min; (c) 0.5 M TEAB, pH 7, RT, 1 h; (d) as described for analogue 8.
2-MeS-ADP, 7, is a selective and highly potent P2Y1R
agonist. Yet, this agonist suffers from low chemical and
enzymatic stability.44 Therefore, we replaced the R,β-brid-
ging oxygen with a dichloro- or difluoromethylene group in
an attempt to generate potent P2Y1R agonists with increased
chemical and metabolic stabilities (i.e., analogues 13 and 14).
The R,β-dihalomethylene-2-MeS-ADP analogues 13 and 14
were prepared as previously reported (Scheme 3).45 Specifi-
cally, the 50-OH of the 20,30-protected 2-MeS-adenosine
analogues 15 and 33 were activated with tosyl chloride
followed by coupling with tris(tetra-n-butylammonium) dihalo
diphosphonate salt. Finally, removal of the protecting group
provided the R,β-dihalomethylene-2-MeS-ADP derivatives,
analogues 13 and 14 at 34% and 18% overall yields, respec-
tively, after LC/MPLC. The relatively high yield of analogue
13 may be due to the higher nucleophility of the difluoro
diphosphonate salt, as compared to analogue 14. In addition
to analogue 13, a small amount of the dinucleotide, analogue
38, (4% yield) was isolated, due to coupling of analogue 36
with analogue 34. Interestingly, the corresponding dichloro
analogue was not detected under the identical reaction
conditions.
Hydrolytic Stability of Analogues 8-14. To explore the
potential of analogues 8-14 as drug candidates, we first
evaluated their hydrolytic stability. Previously, we investi-
gated the effect of a β,γ-methylene group on the hydrolytic
stability of analogues 1-3 at acidic pH, which mimics the
acidity of gastric juice (i.e., pH 1.4 at 37 ꢀC).27 2-MeS-β,γ-
CH2-ATP, analogue 1, R-B,β,γ-CH2-ATP, analogue 2, and
2-MeS-R-B,β,γ-CH2-ATP, analogue 3, were found to be
highly stable at pH 1.4 with t1/2 values for hydrolysis of 65,
19, and 14.5 h, respectively. Although we intend to adminis-
ter these analogues topically, a higher stability of analogues
8-14 at acidic pH, as compared to analogues 1-3, may
suggest a wider range of therapeutic applications.
The hydrolysis of 2-MeS-β,γ-CCl2-ATP, analogue 9, at
pH 1.4 and 37 ꢀC was monitored by HPLC over 216 h
(Figure 3A), and the hydrolysis rate was determined by
integration of the HPLC peaks with time to fit a pseudo-
first-order exponential decay rate equation (Figure 3B),
yielding a half-life of ∼65 h. Analogue 9 was degraded to
2-MeS-AMP and 2-MeS-adenosine.
The hydrolysis of 2-MeS-R-B-β,γ-CF2-ATP, analogue
10 (B isomer) was similarly determined, yielding a half-life
of ∼8 h (Figure 3C). The half-lives of analogues 8, 11
(isomer A), and 12 (isomer B) were 25, 6.7, and 0.7 h,
respectively (data not shown). 2-MeS-R,β-dichloromethy-
lene-ADP, analogue 14, was highly stable at pH 1.4,
with only 15% hydrolysis of the starting material after
6 days. 2-MeS-R,β-difluoromethylene-ADP, analogue 13,
was highly stable, with 61% of the starting material
remaining after 30 days with the remainder degraded to
2-MeS-adenosine.
Resistance of Analogues 8-14 to Degradation by Alkaline
Phosphatase. Alkaline phosphatase is a hydrolase that re-
moves phosphate groups from nucleotides, thereby regulating
extracellular nucleotide concentrations in vivo. AP usually
catalyzes the hydrolysisof phosphomonoesters yieldingPi and
the corresponding alcohol.46
Therefore, we evaluated the effect of R,β/β,γ-dihalomethy-
lene groups in analogues 8-14 on the resistance to hydrolysis by
alkaline phosphatase, as compared to ATP, ADP and analo-
gues 1-3. Results indicated that AP degraded 96% of ATP after
3 h (t1/2 = 1.4 h). Likewise, ADP was completely degraded by
AP after 3 h, whereas 2-MeS-ADP was 87% degraded after 3 h.
Yet, analogues 8-14 were completely resistant to hydrolysis by

3308 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Eliahu et al.
Scheme 2a
a Reaction conditions for analogue 10: (a) trimethylphosphate, PCl3, proton sponge, 0 ꢀC, 30 min; (b) 0.5 M bis(tributylammonium)dichloro-
methylene diphosphonate in dry DMF, Bu3N, 0 ꢀC, 1 h; (c) 2 M BH3 SMe in THF, 0 ꢀC, 5 min, and then RT, 60 min; (d) 0.5 M TEAB, pH 7, RT, 1 h;
3
(e) (1) 18% HCl, pH 2.3, RT, 3 h, (2) 24% NH4OH, pH 9, RT, 45 min. Reaction conditions for analogue 11: (a) trimethylphosphate, PCl3, proton
sponge, 0 ꢀC, 30 min; (b) 0.5 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu3N, 0 ꢀC, 25 min; (c) 2 M BH3 SMe2 in THF,
3
0 ꢀC, 5 min and then RT, 25 min; (d) 0.5 M TEAB, pH 7, RT, 45 min; (e) as described for analogue 10. Reaction conditions for analogue 12:
(a) trimethylphosphate, PCl3, proton sponge, 0 ꢀC, 45 min; (b) 0.5 M bis(tributylammonium)difluoromethylene diphosphonate in dry DMF, Bu3N,
0 ꢀC, 50 min; (c) 2 M BH3 SMe in THF, 0 ꢀC, 5 min then RT, 60 min; (d) and (e) as described for analogue 10.
3
AP under the same conditions (i.e., 0.2 mg analogue/77.5 μL in
the presence of 12.5 units of AP; data not shown).
As compared to ATP, which is hydrolyzed to ADP and
AMP with a half-life of 4.9 h (Figure 4A), analogue 8 was
hydrolyzed to the corresponding nucleoside 50-monopho-
sphate with a half-life of 12.4 h, whereas analogue 9 was only
15% degraded over 24 h (Figure 4B). Furthermore, replace-
ment of the PR nonbridging oxygen in analogues 10-12 with
a BH3 group endowed complete resistance to hydrolysis in
human blood serum over 24 h (data not shown). Similarly,
replacement of the PR-Pβ bridging oxygen in 2-MeS-ADP
with CF2/CCl2 produced analogues 13 and 14 that were
completely resistant to hydrolysis in human blood serum
over 24 h, as compared to ADP (t1/2 = 1.4 h) or 2-MeS-ADP
(t1/2 = 23.5 h) (data not shown).
Resistance of Analogues 8-14 to Hydrolysis in Human
Blood Serum. Nucleotides and their analogues undergo depho-
sphorylation by enzymes in physiological systems.47-50 Blood
serum contains such enzymes and, therefore, provides a good
model system for assessing the metabolic stability of extra-
cellular nucleotides. Previous studies have used human blood
serum51 to demonstrate the metabolic stability of phosphonate
modified nucleotide analogues.30,37 Similarly, we have shown
that in human blood serum the half-lives of β,γ-CH2-ATP and
R-B,β,γ-CH2-ATP, analogues 1-3, were increased by 3.5-
20-fold, as compared to ATP.27
Here, we investigated the effect of incorporation of a β,γ-
dihalomethylene group in analogues 8-14 on the metabolic
stability of these compounds in human blood serum at 37 ꢀC.
Analogues 8-14 are Poor Substrates for ecto-50-Nucleoti-
dases. NTPDase1,2,3,8 and NPP1,3 are the principal enzymes
that metabolize extracellular nucleotides. In comparison to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3309
Scheme 3a
a Reaction conditions for analogue 15: (a) CH2Cl2, DMAP, TsCl, RT, 12 h; (b) tetra-(n-butylammonium)difluoromethylene diphosphonate in dry
DMF, RT, 72 h; (c) (1) 18% HCl, pH 2.3, RT, 3 h; (2) 24% NH4OH, pH 9, RT, 45 min. Reaction conditions for analogue 33: (a) CH2Cl2, DMAP, TsCl,
RT, 12 h; (b) tetra-(n-butylammonium) dichloromethylene diphosphonate in dry DMF, RT, 72 h; (c) TFA, argon bubbling, RT, 10 min.
ATP (Table 1), analogues 8-14 were barely hydrolyzed
by NTPDase1,2,3,8 (<5%) or NPP1,3 (<10%).
EC50 values of 0.57-9.54 μM (Table 2). Replacement of the
nonbridging oxygen with BH3 in 2-MeS-R-borano-β,γ-di-
chloromethylene-ATP, analogue 12A (A isomer), produced
the most potent P2Y1R agonist in this series of analogues
(EC50 = 0.57 μM), whereas the B isomer (analogue 12B) was
2-fold less potent (EC50 = 1.20 μM). Analogues 11A and
11B that lacked a 2-MeS group were inactive at the P2Y1R.
Replacement of the nonbridging oxygen in 2-MeS-β,γ-
difluoromethylene-ATP, analogue 8, with BH3 to generate
analogue 10B resulted in a 3-fold reduction in P2Y1R activity
(EC50 = 0.76 μM for analogue 8 vs EC50 = 2.13 μM for
analogue 10B), whereas the A isomer (analogue 10A) had no
activity. Although the R,β-dihalomethylene substitution in
analogues 13 and 14 decreased the rate of degradation by
ecto-nucleotidases (Table 1), as compared to 2-MeS-ADP,
analogues 13 and 14 were weak P2Y1R agonists with EC50
values of 0.98 and 3.1 μM, respectively (Table 2). None of the
analogues tested (8-14) antagonized the effect of equimolar
concentrations of 2-MeS-ADP on P2Y1R activation in
1321N1 cell transfectants (data not shown).
Activities of Analogues 8-14 at the P2Y1 Receptor. Earlier,
we reported that 2-MeS-β,γ-methylene-ATP was a potent
and selective P2Y1 receptor (P2Y1R) agonist.27 Here, we
examined whether a β,γ-dihalomethylene substitution in
analogues 8-14 would enhance agonist potency at the
P2Y1R due to the reduced pKa of the terminal phospho-
nate (∼7), as compared to the pKa of 8.452 for the corres-
ponding methylene derivatives. Because analogues 8-14
should be 90% ionized under physiological conditions
(vs 9% ionization for analogue 1), we expected that nega-
tively charged phosphonates in analogues 8-14 would
increase binding to the P2Y1R. Accordingly, the activities
of analogues 8-14 were determined at the turkey P2Y1R
heterologously expressed in human 1321N1 astrocytoma
cells that are devoid of endogenous P2Y receptors.53
P2Y1R activities were evaluated by monitoring increases in
the intracellular calcium concentration, [Ca2þ]i, induced by
the analogues, as compared to 2-MeS-ADP (EC50 0.0025 μM
at the P2Y1R).
Effect of Nucleotide Analogues on IOP. Nucleotides are
present in the aqueous humor,11 although their action has
not been fully elucidated due to the multiple P2R subtypes
Surprisingly, analogues 8-14 were found to be weaker
agonists of the P2Y1R than 2-MeS-ADP, analogue 7, with

3310 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Eliahu et al.
Figure 4. Hydrolysis of ATP and analogues 1, 8, and 9 in human
blood serum (180 μL) and RPMI-1640 medium (540 μL) over 24 h at
37 ꢀC, as monitored by HPLC. (A) Hydrolysis of 0.25 mM ATP and
production of ADP and AMP. (B) Degradation of analogues 1, 8,
and 9 in human blood serum.
Table 1. Relative Hydrolysis [%] of Analogues 8-14 by Human ecto-
Nucleotidasesa
analogues
human ecto-
nucleotidases
8
9
10A 10B 11A 11B 12A 12B 13
14
NTPDase1 2.6 0.5
NTPDase2 1.1 0.5
1.0 0.4 5.5 3.2 0.7 ND 1.3 ND
1.8 0.9 1.3 1.4 1.5 ND 2.4 1.7
NTPDase3 2.6 ND 4.4 ND 2.6 3.1 0.3 ND ND ND
NTPDase8 5.2 ND 1.7 ND 4.4 4.0 ND 1.8 0.1 ND
NPP1
NPP3
6.2 5.8 4.0 3.4 5.3 3.3 0.7 4.0 3.9 3.3
0.5 1.9 10.0 3.2 ND ND 5.0 ND ND 1.8
a The ATP and ADP analogues 8-14 (100 μM) were used as sub-
strates of the ecto-nucleotidases identified in the left column. The
relative hydrolysis rates vs 100 μM ATP (set at 100%) were: 403 ( 40,
1006 ( 60, 533 ( 42, 229 ( 20 [nmol Pi min-1 mg protein-1] for
3
3
NTPDase1,2,3,8, respectively, and 619 and 245 [nmol nucl min-1 mg
3
3
protein-1] for NPP1,3, respectively. ND = not detectable.
Figure 3. Rates of hydrolysis of analogues 9 and 10B at pH 1.4 at
37 ꢀC, as monitored by HPLC. Hydrolysis of 7.5 mM analogue 9 or
10B in KCl/HCl buffer at pH 1.4 and 37 ꢀC was recorded for 5 days at
7-17 h intervals. (A) HPLC chromatograms of analogue 9 and
hydrolysates at t = 6, 52, 216 h. (B) Kinetics of acidic hydrolysis of
analogue 9 (t1/2 = 65 h) and time-dependent formation of degradation
products. (C) Kinetics of acidic hydrolysis of analogue 10B (t1/2 = 8h).
identified in intraocular tissues that are bathed by the aqueous
humor.54 Studies suggest that IOP is regulated by G-protein
coupled P2Y1Rs in trabecular meshwork cells that control
the evacuation of the aqueous humor12 and ligand-gated ion
channel P2X2 receptors located on parasympathetic nerve

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3311
Table 2. Activity of Analogues 8-14 at the P2Y1R, i.e., EC50 (μM)
terminals innervating the ciliary bodies.55 P2X receptor activa-
tion potentiates the release of acetylcholine, which induces
contraction of ciliary muscle to open the trabecular meshwork
and reduce IOP.56 In contrast, activation of G-protein coupled
P2Y2 receptors increases IOP.56 The current study examined
the effect of analogues 8-14, as compared to 2-MeS-β,γ-
methylene-ATP (analogue 1), on reduction of IOP in normo-
tensive rabbits to identify novel and potent candidates for the
treatment of ocular hypertension.56 For all the single dose
experiments, 10 μL of a 100 μM solution of tested compounds
was instilled.
Values for Analogue-Induced Increases in [Ca2þ
]
i
analogue
no.
analogue
no.
EC50
EC50
7
0.0025
0.76
9.54
nr
11B
12A
12B
13
nra
8
0.57
1.20
0.98
3.10
9
10A
10B
11A
2.13
nr
14
a nr = no response.
Figure 5. (A) Effect of analogues 1, 8-9, and 11-14 (100 μM) on rabbit IOP measured over 8 h vs control: (1) an equal volume of saline
was administered to the contralateral eye; (2) an equal volume of saline was administered to other animals. IOP was measured twice before
the application of any drug and was nearly identical in all animals. (B) Time course for the effects of analogues 1, 9, 13, and 14 (100 μM) on
rabbit IOP over 8 h vs control. Values are the means ( SEM of results from 10 independent experiments. (C) Time-course for the effects of
analogues 11B, 12A, and 12B (100 μM) on rabbit IOP over 8 h. Values are the means ( SEM of results from eight independent
experiments. (D) Dose-response curves for the maximal effects of analogues 1, 9, 13, and 14 (100 μM) on rabbit IOP. Values are the means
( SEM of results from eight independent experiments. (E) Maximal effects of analogues 1 and 9 (100 μM, 10 μL) on rabbit IOP, as
compared to latanoprost (0.005%), dorzolamide hydrochloride (2%), and timolol maleate (0.5%) applied in 40 μL, as compared to
control. (F) Duration of the effects of analogues 1 and 9 (100 μM, 10 μL), as compared to latanoprost (0.005%), dorzolamide
hydrochloride (2%), and timolol maleate (0.5%) applied in 40 μL, as compared to control. The duration of the effect, termed mean-time
effect, was calculated as the time between 50% maximal decrease in IOP after drug administration and a 50% recovery of the initial IOP
value.75

3312 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Eliahu et al.
Analogues 8 and 11A increased IOP (Figure 5A), whereas
analogues 1, 9, 11B, 12A/B, 13, and 14 reduced IOP
(Figure 5A-C). Analogues 1 and 9 reduced IOP by ∼32%
(at an instilled volume of 10 μL of a 100 μM solution) and
were more effective than Ap4A and Ap4.10,11 On the basis of
concentration response curves, analogues 1, 9, 13, and 14
exhibited the strongest hypotensive effects with pD2 values
of 4.5 ( 0.7, 7.0 ( 0.7, 5.1 ( 0.3, and 4.5 ( 0.4, respectively
(all assays were performed by instilling 10 μL of 1 nM-
100 μM analogue solutions). The rank order of potency
for reduction of IOP was 9 > 13 > 1 > 14, with EC50
values of 95.5 nM, and 7.9, 30.2, and 31.6 μM, respectively
(Figure 5D). In comparison to other agents shown to reduce
IOP, analogues 1 and 9 were more effective than latanoprost
(a prostglandin analogue) and dorzolamide hydrochloride
(a carbonic anhydrase inhibitor) and equally effective as
timolol maleate (a β-blocker) (all commercial drugs were
tested at a fixed volume of 40 μL) (Figure 5E). The duration
effect of the reduction in IOP induced by the nucleotides was
comparable to the commercially available compounds. The
reduction in IOP induced by analogues 1 and 9 persisted for
3.5 and 4.5 h, respectively, whereas latanoprost was effective
for 5.5 h (Figure 5F).
for enzymatic dephosphorylation. Likewise, analogues
10-14 were not enzymatically hydrolyzed due to the repla-
cement of the nonbridging oxygen with a borano group.
Previously, we found that 2-MeS-R-borano-ATP (A-iso-
mer), analogue 4, was susceptible to hydrolysis by alka-
line phosphatase, resulting in R-borano-2-MeS-AMP and
2-MeS-R-borano-ADP.27 For example, alkaline phospha-
tase hydrolyzed 60% of 2-MeS-R-borano-ATP (A-isomer)
within 12 min at 37 ꢀC, and only traces of 2-MeS-R-borano-
ATP remained after 100 min. The substitution of the Pβ,
Pγ-bridging CH2 group with a CX2 group as in analogues 10
and 12 rendered these analogues completely resistant to
hydrolysis by alkaline phosphatase over 30 min at 37 ꢀC.
Furthermore, analogues 8-14 were highly resistant to hydro-
lysis by NTPDases1,2,3,8, and NPP1,3 (less than 5% hydro-
lysis, as compared to ATP). In particular, analogue 12B was
completely resistant to hydrolysis by NTPDase1,2,3 and NPP3.
The extremely low rate of enzymatic hydrolysis of these
analogues is related primarily to the presence of a phosphonate
moiety at Pβ, Pγ (analogues 8-12) or PR, Pβ (analogues 13, 14).
Bulky Cl atoms at CX2 further increase the enzymatic stability
of the analogues, as compared to F atoms (e.g., greater stability
for analogue 9 vs analogue 8). The further addition of a borano
modification at PR of analogue 9 yielded analogue 12, the most
stable analogue synthesized, indicating that NTPDase and
NPP are not tolerant to steric hindrance at PR.
Discussion
Chemical Stability of Analogues 8-14. Previously, we
analyzed the chemical stability of analogues 1-3 at gastric
juice pH of 1.4 (t1/2 65, 19, and 14.5 h, respectively).27 Here,
we determined whether a β,γ-dihalomethylene group in-
creases the chemical stability of these nucleotide analogues
as compared to the presence of a β,γ-methylene group.
We found that analogues 8 and 9 exhibited high stability at
pH 1.4 with half-lives of 25 and 65 h, respectively, compar-
able to the t1/2 values of the methylene analogues 1-3.
Hydrolytic cleavage of the PR-O-Pβ bond in both analo-
gues 8 and 9 results in formation of 2-MeS-AMP and β,γ-
dihalomethylene diphosphonate. We hypothesize that the
lower chemical stability of analogue 8 as compared to 9 may be
due to the higher electronegativity of fluorine vs chlorine,
resulting in a better leaving group in β,γ-difluoromethylene
diphosphonate. Furthermore, we postulate that accessibility of
a water molecule for nucleophilic attack on PR or Pβ is more
restricted for analogue 9 than analogue 8 due to the steric
Activity of Analogues 8-14 at the P2Y1R. Although
analogues 8-14 were shown to be resistant to enzymatic
hydrolysis, they were less potent agonists of the P2Y1R than
the more rapidly hydrolyzed 2-MeS-ADP.
Whereas the activity of analogue 1 at the P2Y1R was
20-fold lower than 2-MeS-ADP (EC50 = 0.0025 μM),27 the
activities of the corresponding β,γ-dihalomethylene analo-
gues 8 and 9 were 300- to 3800-fold lower than 2-MeS-ADP,
respectively.
Apparently, the presence of a thiomethyl group at the
adenine C-2 position is essential for P2Y1R activity because
analogues 11A and 11B were inactive. Yet, any halogen
substitution at the phosphonate carbon was not tolerated
by the P2Y1R.
Considering the rank order of agonist potency obtained at
the P2Y1R, analogue 1 > 8 > 9, we believe that the major
parameter determining the affinity of this series of P2Y1R
agonists is the steric constraints of the ligand binding site of
the P2Y1R rather than the pKa values of the analogues’
phosphonate moieties.52,59 The smaller size of the β,γ-
methylene group presumably allows tighter receptor binding
than the larger β,γ-dihalomethylene group, consistent with
the EC50 values at the P2Y1R obtained for analogues 13 and
14 (Table 2).
Effect of Nucleotide Analogues on IOP. In the current
study, we demonstrate the ability of analogues 1, 9, 11B, 12A,
12B, 13, and 14 to reduce IOP in male New Zealand white
rabbits, whereas analogues 8 and 11A increased IOP. The
two compounds exhibiting the strongest hypotensive effect
(maximal hypotensive effect of about 32% reduction) were
analogues 1 and 9 (EC50 values of 30.2 μM and 95.5 nM,
respectively), equally or more effective than hypotensive
drugs currently on the market (Figure 5E). This is a sig-
nificant finding because nucleotide analogues activate dif-
ferent receptors than hypotensive drugs commonly used for
treatment of glaucoma and may represent alternatives for
patients unable to use the currently approved drugs. Side
effects are common with current glaucoma medications.
hindrance of the larger chlorine atom resulting in a longer t1/2
.
Analogues 10-12 were less stable at pH 1.4 (t1/2 = 8.0, 6.7,
and 0.7 h, respectively), as compared to analogues 8 and 9, due
to the low stability of the P-B bond under acidic conditions.57,58
Analogues 13 and 14 were significantly more stable at pH
1.4, as compared to analogues 8 and 9, respectively. This may
be related to differences in the stability of the partial negative
charge on the 50-phosphonate (PR) and 50-oxygen atoms
of analogues 8/9 and analogues 13/14, respectively, after
hydrolysis, i.e., greater stability of a phosphate vs phospho-
nate group resulting in more extensive hydrolysis and shorter
t1/2 values for analogues 8/9 vs 13/14.
Enzymatic Stability of Analogues 8-14. Previously, we
reported on the stability of analogue 1 vs ATP in human
blood serum (t1/2 = 12.1 vs 3.6 h, respectively),27 whereas
here we found that analogue 9 was even more stable,
exhibiting only 15% degradation after 24 h in human blood
serum vs ATP (t1/2 = 4.9 h). We postulate that the enhanced
stability of analogue 9 is probably due to steric effects. The
bulky chlorine atoms cause analogue 9 to be a weak substrate

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3313
Thus, β-blockers (e.g., timolol maleate) can cause bradycar-
dia and hypertension60 and are not suitable for patients
with cardiac problems or asthma, obstructive pulmonary
disease, and corneal dystrophy.61 Cholinergic agents (e.g.,
pilocarpine) produce fixed pupils and induce myopia and
cataracts,62-64 whereas prostaglandins (e.g., latanoprost)
cause eyelash growth, iris pigmentation,65 and muscle and
joint pain.60 The development of stable and potent nucleo-
tide analogues should expand the limited repertoire of drugs
currently used for the treatment of glaucoma.
We found no correlation between the potency of nucleo-
tide analogues at the P2Y1R and their ability to reduce IOP
because analogues 1 and 9 activated the P2Y1R with EC50
values in the nanomolar and micromolar range, respectively,
yet analogue 9 was more potent than analogue 1 at redu-
cing IOP. These findings may imply that these analogues
reduce IOP not solely via activation of the P2Y1R, as
previously described,55 but via activation of other P2 recep-
tors. Interestingly, replacing the β,γ-bridging oxygen with a
β,γ-difluoromethylene group increases IOP (analogue 8),
whereas substitution with β,γ-dichloromethylene generates
an analogue that is strongly hypotensive (analogue 9). Like-
wise, replacement of the PR nonbridging oxygen with BH3
results in a significant reduction in IOP (except for analogue
11A), as does a thiomethyl substitution at the C-2 position in
the adenine ring.
High resolution mass spectra were recorded on an AutoSpec-E
FISION VG mass spectrometer by chemical ionization. Nucleo-
tides were analyzed under ESI (electron spray ionization) condi-
tions on a Q-TOF microinstrument (Waters, UK). Primary puri-
fication of the nucleotides was achieved on a LC (Isco UA-6)
system using a column of Sephadex DEAE-A25, swollen in
1 M NaHCO3 at 4 ꢀC for 1 day. The resin was washed with
deionized water before use. The LC separation was monitored
by UV detection at 280 nm. Final purification of the nucleo-
tides and separation of diastereomers were achieved on a HPLC
system (Elite Lachrom, Merck-Hitachi) using a semipreparative
reverse-phase column (Gemini 5u C-18 110A 250 mm ꢀ 10.00 mm;
5 μm; Phenomenex, Torrance, CA). The purity of the nucleotides
was evaluated with an analytical reverse-phase column system
(Gemini 5u C-18 110A, 150 mm ꢀ 4.60 mm; 5 μm; Phenomenex,
Torrance, CA) using two solvent systems: solvent system I, (A)
100 mM triethylammonium acetate (TEAA), pH 7:(B) CH3CN;
solvent system II, (A) 0.01 M KH2PO4, pH = 4.5:(B) CH3CN.
The details of the solvent system gradients used for the separation
of each product are given below. The purity of the nucleotides
was generally g92%. All commercial reagents were used without
further purification unless otherwise noted. All reactants in
moisture-sensitive reactions were dried overnight in a vacuum
oven. RPMI (Roswell Park Memorial Institute) 1640 medium
was obtained from Sigma-Aldrich. 20,30-O-Methoxymethylidene-
adenosine and 2-MeS-adenosine were prepared, as previously
described.15,66 20,30-O-Methoxymethylidene-2-MeS-adenosine was
purified with a MPLC system (Biotage, Kungsgatan, Uppsala,
Sweden) using a silica gel (25þM) column and the following
gradient scheme: 3 column volumes (CV) of 100:0 (A) CHCl3:(B)
EtOH, 5 CV of a gradient from 100:0 to 90:10 A:B, and 4 CV of
90:10 A:B at a flow rate of 12.5 mL/min. pH measurements were
performed with an Orion microcombination pH electrode and a
Hanna Instruments pH meter. TEAB was prepared according to
the literature.67 Bis(tributylammonium)difluoromethylene diphos-
phonate salt was purchased from RI Chemical Inc. For prepara-
tion of human blood serum, whole blood taken from healthy
volunteers was obtained from a blood bank (Tel-Hashomer Hos-
pital, Israel). Blood was stored for 12 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.
Preparation of Bis(tributylammonium)dichloromethylene Diphos-
phonate Salt. A Hþ Dowex column was used for ion exchange
chromatography. First, 30 mL of Dowex was placed in a column
with cotton wool at the bottom, and then the column was washed
with 10% NaOH (150 mL) until the pH of the effluent was basic.
Then the column was washed with distilled water until the pH of
the effluent reached neutral. Then the column was washed with
10% HCl (300 mL), followed by distilled water until the effluent
reached acidic and neutral pH, respectively. A flask containing
Bu3N (2 equiv) in EtOH was placed in an ice bath under the
column and stirred.
The disodium form of dichloromethylene diphosphonate salt
was dissolved in distilled water, poured onto the column, and the
column was washed with distilled water until the pH of the
effluent was neutral. The effluent was dropped into the Bu3N/
EtOH solution. The final solution of bis(tributylammonium)-
dichloromethylene diphosphonate salt was then freeze-dried.
Preparation of 2-MeS-adenosine-50-O-triphosphate-β,γ-methy-
lene-dihalogen Derivatives, 8 and 9. 1,8-Bis(dimethylamino)naph-
thalene (156 mg, 0.72mmol, 2 equiv) was added at 0 ꢀC to 20,30-O-
methoxymethylidene-2-MeS-adenosine, analogue 15, (130 mg,
0.36 mmol) in trimethylphosphate (2.5 mL) in a flame-dried two-
neck flaskunder N2, andthe reaction was stirredfor 20 minuntil a
clear solution was attained. POCl3 (66 μL, 0.72 mmol, 2 equiv)
was added at 0 ꢀC. The solution was stirred at 0 ꢀC for 1 h. A
0.5 M solution of bis(tributylammonium)dihalomethylene diphos-
phonate salt (660 mg, 1.13 mmol, 3 equiv) in dry DMF (1.4 mL)
and tributylamine (360 μL, 1.46 mmol, 4 equiv) was added at
0 ꢀC, and the reaction mixture was stirred for 90 min. A 0.5 M
The enhanced activity of analogue 9 for reduction of IOP,
as compared to analogues 11-14, is possibly linked to the
significantly enhanced chemical and metabolic stability of
analogue 9 vs analogues 11-14.
Summary
We synthesized chemically and enzymatically stable ana-
logues 8-14 based on ADP and ATP scaffolds modified by
R,β/β,γ-dihalomethylene groups within the phosphate chain.
These analogues wereagonistsatthe P2Y1R. Analogues 1 and
9, in particular, exhibited the ability to reduce IOP in normo-
tensive rabbits, with EC50 values of 30.2 μM and 95.5 nM,
respectively. Moreover, theseanalogues weremore effective at
reducing IOP than several commonly prescribed glaucoma
drugs (e.g., latanoprost and dorzolamide hydrochloride). In
addition, the duration of the hypotensive effect induced by
analogue 9 was comparable to latanoprost, dorzolamide
hydrochloride, and timolol maleate. We conclude that com-
pound 9 is an attractive drug candidate for the treatment of
glaucoma. Furthermore, although slightly less efficacious
than timolol maleate, compound 9 represents a promising
alternative to timolol maleate, as the latter is a β-blocker and
cannot be used for the treatment of patients suffering from
asthma, bronchitis or cardiovascular problems.
Experimental Section
General. All air- and moisture-sensitive reactions were carried
out in flame-dried, N2-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 by UV light. Compounds were
characterized by nuclear magnetic resonance using Bruker AC-
1
200, DPX-300, or DMX-600 spectrometers. H NMR spectra
were measured at 200, 300, or 600 MHz. Nucleotides were
characterized also by 31P NMR in D2O, using 85% H3PO4,
and 19F NMR using trifluorochloromethane as an external
reference on Bruker AC-200 and DMX-600 spectrometers.

3314 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Eliahu et al.
solution of TEAB (15 mL) was added at RT, and the reac-
tion mixture was stirred for 60 min and then freeze-dried. The
residue was dissolved in water and treated with 18% HCl until the
pH was 2.3, and then the mixture was stirred for 3 h at RT.
Finally, the mixture was treated with 24% NH4OH solution, and
the pH was adjusted to 9. The solution was stirred for 45 min at
RT and then freeze-dried. The resulting residue was dissolved
in deionized water (100 mL) and extracted with diethyl ether (2 ꢀ
50 mL) and then chloroform (50 mL). The aqueous phase was
freeze-dried. The resulting residue was separated on an activated
Sephadex DEAE-A25 column (0-0.5 M NH4HCO3; total
volume 2 L). The relevant fractions were collected, freeze-dried,
and excess NH4HCO3 was removed by repeated freeze-drying
with deionized water to yield the product as a white powder. The
product was purified by LC, yielding analogue 8 at a 46% yield
(110 mg) and analogue 9 at a 10% yield (19.6 mg). Finally, the
nucleotide triethylammonium counterions were exchanged for
Naþ ions by passing analogues 8 and 9 through a Sephadex-CM
C-25 Naþ-form column. The spectral data for analogues 8 and 9
were consistent with the literature.43
Characterization of 2-MeS-adenosine-50-O-(r-boranotriphos-
phate)-β,γ-CF2 (10A). Retention time on a semipreparative
column: 12.78 min. 1H NMR (D2O; 600 MHz): δ 8.43 (s; H-8;
1H), 6.13 (d; J = 5.40 Hz; H-10; 1H), 4.80 (t; J = 5.40 Hz; H-20;
1H), 4.65 (m; H-30; 1H), 4.36 (m; H-40; 1H), 4.31 (m; H-50; 1H),
4.13 (m; H-500; 1H), 2.58 (s; CH3; 3H), and 0.45 (m; BH3; 3H)
ppm. 31P NMR (D2O; 243 MHz): δ 83.50 (m; PR-BH3), 4.71 (m;
Pγ), and -2.25 (m; Pβ) ppm. 19F NMR (D2O 188 MHz): δ
-115.38 (t, J = 79.27) ppm. MS-ESI m/z: 584 (M-). TLC
(NH4OH:H2O:2-propanol 2:8:11), Rf = 0.12. Purity data ob-
tained on an analytical column: retention time: 7.82 min (95.3%
purity) using solvent system I with a gradient from 85:15 to 50:50
A:B over 15 min at a flow rate of 1 mL/min. Retention time:
4.89 min (92.8% purity) using solvent system II with a gradient
from 85:15 to 50:50 A:B over 20 min at a flow rate of 1 mL/min.
Characterization of 2-MeS-adenosine-50-O-(r-boranotriphos-
phate)-β,γ-CF2 (10B isomer). Retention time on a semiprepara-
1
tive column: 14.75 min. H NMR (D2O; 600 MHz): δ 8.39 (s;
H-8; 1H), 6.12 (d; J = 5.40 Hz; H-10; 1H), 4.80 (t; J = 5.40 Hz;
H-20; 1H), 4.57 (m; H-30; 1H), 4.37 (m; H-40; 1H), 4.26 (m; H-50;
1H), 4.19 (m; H-500; 1H), 2.58 (s; CH3; 3H), and 0.48 (m; BH3;
3H) ppm. 31P NMR (D2O; 243 MHz): δ 84.80 (m; PR-BH3), 4.73
(m; Pγ), and -2.30 (m; Pβ) ppm. 19F NMR (D2O 188 MHz): δ
-115.40 (t, J = 79.27) ppm. MS-ESI m/z: 584 (M-). TLC (NH4-
OH:H2O:2-propanol 2:8:11), Rf = 0.12. Purity data obtained
on an analytical column: retention time: 8.17 min (87.0% purity)
using solvent system I with a gradient from 85:15 to 50:50 A:B
over 15 min at a flow rate of 1 mL/min. Retention time: 3.08 min
(94.95% purity) using solvent system II with a gradient from
70:30 to 50:50 A:B over 10 min at a flow rate of 1 mL/min.
Separation of Adenosine-50-O-(r-boranotriphosphate)-β,γ-
CCl2 (11). The separation of diastereomers 11A and 11B was
accomplished using a semipreparative reverse-phase Gemini 5u
column (C-18 110A; 250 mm ꢀ 10.00 mm; 5 μm) and isocratic
elution with 91:9 (A) 100 mM TEAA, pH 7:(B) MeOH at a flow
rate of 5 mL/min. Fractions containing purified isomers [Rt=
8.37 min (11A isomer); 10.87 min (11B isomer)] were collected
and freeze-dried. Excess buffer was removed by repeated freeze-
drying cycles with the solid residue dissolved each time in
deionized water. Diastereoisomers 11A and 11B were obtained
at 18% overall yield (14 mg) after LC separation.
Preparation of Adenosine-50-O-(r-boranotriphosphate)-β,γ-
methylene-dihalogen Derivatives, 10-12. 20,30-O-Methoxy-
methylidene adenosine derivatives (analogues 14 or 20; 50 mg,
0.14 mmol) were dissolved in trimethylphosphate (0.7 mL) in a
flame-dried two-neck flask under N2. 1,8-Bis(dimethylamino)-
naphthalene (60 mg, 0.28 mmol, 2 equiv) was added at 0 ꢀC, and
the reaction was stirred for 20 min until a clear solution was
attained. PCl3 (23 μL, 0.28 mmol, 2 equiv) was added at 0 ꢀC,
and a white solid precipitated. The suspension was stirred at 0 ꢀC
for 45 min. Then, a 0.5 M solution of bis(tributylammonium)
dihalomethylene diphosphonate salt (245 mg, 0.42 mmol,
3 equiv) in dry DMF (0.6 mL) and tributylamine (134 μL,
0.56 mmol, 4 equiv) was added at 0 ꢀC, and the reaction mixture
was stirred for 50 min. A 2 M solution of BH3 SMe2 complex in
3
THF (0.7 mL, 2.81 mmol, 10 equiv) was added at 0 ꢀC, and the
reaction mixture turned clear. The solution was stirred for 5 min
at 0 ꢀC and then for 60 min at RT. Finally, a 0.5 M TEAB
solution (15 mL) was added at RT, and the mixture was stirred
for 60 min and then freeze-dried. The residue was dissolved in
water, treated with 18% HCl until the pH was 2.3, and then the
mixture was stirred for 3 h at RT. Finally, the mixture was
treated with 24% NH4OH, and the pH was adjusted to 9. The
solution was stirred for 45 min at RT and then freeze-dried. The
resulting residue was dissolved in deionized water (100 mL)
and extracted with diethyl ether (2 ꢀ 30 mL) and chloroform
(30 mL). The aqueous phase was freeze-dried. The resulting
residue was separated on an activated Sephadex DEAE-A25
column (0-0.5 M NH4HCO3; total volume 2 L). The relevant
fractions were collected, freeze-dried, and excess NH4HCO3 was
removed by repeated freeze-drying cycles with deionized water
to obtain the product as a white powder. The product was
purified by LC yielding analogue 10 at a 21% yield (13 mg),
analogue 11 at a 18% yield (14 mg), and analogue 12 at a 7.2%
yield (18.6 mg). The diastereomers of analogues 10, 11, and 12
were separated on a HPLC column, under the conditions
described below. Finally, the purified diasteroisomers were
passed through a Sephadex-CM C-25 Naþ-form column to
exchange triethylammonium ions for Naþ.
Characterization of Adenosine-50-O-(r-boranotriphosphate)-
β,γ-CCl2 (11A). Retention time on a semipreparative column:
8.37 min. 1H NMR (D2O; 300 MHz): δ 8.51 (s; H-8; 1H), 8.14 (s;
H-2; 1H), 6.04 (d; J = 5.7 Hz; H-10; 1H), 4.78 (H-20 and H-30
signals are hidden by the water signal), 4.30 (m; H-50; 2H), 4.05
(m; H-40; 1H), and 0.37 (m; BH3; 3H) ppm. 31P NMR (D2O;
243 MHz): δ 83.80 (m; PR-BH3), 9.10 (d; J = 19.14 Hz; Pγ), and
2.02 (m; Pβ) ppm. MS-ESI m/z: 570 (M-). HRMS-FAB
(negative) m/z: calculated for C11H18BN5O11Na2P3: 546.0104;
found: 546.0104. TLC (NH4OH:H2O:2-propanol 2:8:11), Rf =
0.3. Purity data obtained on an analytical column: retention
time: 6.88 min (98% purity) using solvent system I with a
gradient from 95:5 to 70:30 A:B over 10 min at a flow rate of
1 mL/min. Retention time: 1.55 min (98% purity) using solvent
system II with a gradient from 85:15 to 70:30 A:B over 10 min at
a flow rate of 1 mL/min.
Characterization of Adenosine-50-O-(r-boranotriphosphate)-
β,γ-CCl2 (11B). Retention time on a semipreparative column:
10.87 min. 1H NMR (D2O; 300 MHz): δ 8.49 (s; H-8; 1H), 8.14
(s; H-2; 1H), 6.04 (d; J = 5.7 Hz; H-10; 1H), 4.78 (H-20 signal is
hidden by the water signal), 4.47 (m; H-30; 1H), 4.23 (m; H-50;
H-500; 2H), 4.15 (m; H-40; 1H), and 0.37 (m; BH3; 3H) ppm. 31P
NMR (D2O; 243 MHz): δ 84.58 (m; PR-BH3), 9.14 (d; J =
18.46 Hz; Pγ), and 2.11 (m; Pβ) ppm. MS-ESI m/z: 570 (M-).
TLC (NH4OH:H2O:2-propanol 2:8:11), Rf = 0.3. Purity data
obtained on an analytical column: retention time: 7.62 min
(97% purity) using solvent system I with a gradient from 95:5
to 70:30 A:B over 10 min at a flow rate of 1 mL/min. Retention
time: 1.32 min (98% purity) using solvent system II with a
Separation of 2-MeS-adenosine-50-O-(r-boranotriphosphate)-
β,γ-CF2 (10A and 10B). The separation of analogue 10 diaster-
eoisomers, 10A and 10B, was accomplished using a semipre-
parative reverse-phase Gemini 5u column and isocratic elution
with 80:20 (A) 100 mM TEAA, pH 7:(B) MeOH at a flow rate
of 5 mL/min. Fractions containing purified isomers [Rt = 12.78
min (10A isomer); 14.75 min (10B isomer)] were collected and
freeze-dried. Excess buffer was removed by repeated freeze-
drying cycles, with the solid residue dissolved each time in
deionized water. Diastereoisomers 10A and 10B were obtained
at 21% overall yield (18.6 mg) after LC separation.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3315
gradient from 95:5 to 70:30 A:B over 10 min at a flow rate of
1 mL/min.
1.09 mmol, 4 equiv) in CH2Cl2 (2 mL) and a solution of TsCl
(156 mg, 0.82 mmol, 3 equiv) in CH2Cl2 (0.5 mL) were added to a
solution of 20,30-O-isopropylidene adenosine, analogue 33 (97 mg;
0.27 mmol), in CH2Cl2 (1 mL) in a flame-dried two-neck flask
under N2 at RT, and the reaction mixture was stirred for 2 h.
CH2Cl2 (50 mL) was added to the reaction mixture, which was
then extracted with saturated NaHCO3 solution (3 ꢀ 30 mL). The
organic phase was treated with Na2SO4 and filtered. The solvent
was removed under reduced pressure, and the residue was sepa-
rated using a MPLC system with a silica gel (25þM) column and
the following gradient scheme: 3 column volumes (CV) of 100:0
(A) CH2Cl2:(B) EtOH, 5 CV of a gradient from 100:0 to 90:10 A:
B, and 4 CV of 90:10 A:B at a flow rate of 25 mL/min. The relevant
fractions were collected, and the solvent was removed under
reduced pressure, yielding analogue 35 at a 51% yield (71 mg)
as a white solid. 1H NMR (CDCl3; 300 MHz): δ 7.90 (s; H-8; 1H),
7.67 (d, J = 8.40, 2H), 6.05 (s; H-10; 1H), 5.35 (m; H-20; 1H), 5.00
(m, H-30; 1H), 4.50 (m, H-40; 1H), and 4.25 (m, H-50; H-500; 2H).
MS-ESI m/z:508(MHþ).MS-ESIþ m/z: 508 (MHþ).TLC(CHCl3:
EtOH 95:5), Rf = 0.79.
Separation of 2-MeS-adenosine-50-O-(r-boranotriphosphate)-
β,γ-CCl2 (12A and 12B). The separation of diastereoisomers
12A and 12B was accomplished using a semipreparative reverse-
phase Gemini 5u column (C-18 110A; 250 mm ꢀ 10.00 mm;
5 μm) and isocratic elution with 82:18 (A) 100 mM TEAA, pH
7:(B) MeOH at a flow rate of 5 mL/min. Fractions containing
purified isomers [Rt = 9.33 min (12A isomer); 10.61 min (12B
isomer)] were collected and freeze-dried. Excess buffer was
removed by repeated freeze-drying cycles with the solid residue
dissolved each time in deionized water. Diastereoisomers 12A
and 12B were obtained at a 7.2% overall yield (13 mg) after LC
separation.
Characterization of 2-MeS-adenosine-50-O-(r-boranotriphos-
phate)-β,γ-CCl2 (12A). Retention time on a semipreparative
1
column: 9.33 min. H NMR (D2O; 300 MHz): δ 8.34 (s; H-8;
1H), 6.04 (d; J = 5.10 Hz; H-10; 1H), 4.78 (H-20 and H-30 signals
are hidden by the water signal), 4.30 (m; H-40; 1H), 4.05 (m; H-50;
H-500; 2H), 2.49 (s; CH3; 3H), and 0.42 (m; BH3; 3H) ppm. 31P
NMR (D2O; 243 MHz): δ 83.40 (m; PR-BH3), 9.28 (d; J =
17.32 Hz; Pγ),and2.65(m;Pβ) ppm. MS-ESI+ m/z:548(M-). TLC
(NH4OH:H2O:2-propanol 2:8:11), Rf = 0.15. Purity data ob-
tained on an analytical column: retention time: 7.96 min (90.4%
purity) using solvent system I with a gradient from 95:5 to 65:35
A:B over 13 min at a flow rate of 1 mL/min. Retention time:
2.36 min (87.0% purity) using solvent system II with a gradient
from 95:5 to 60:40 A:B over 10 min at a flow rate of 1 mL/min.
Characterization of 2-MeS-adenosine-50-O-(r-boranotriphos-
phate)-β,γ-CCl2 (12B). Retention time on a semipreparative
column: 10.61 min. 1H NMR (D2O; 300 MHz): δ 8.32 (s; H-8;
1H), 6.02 (d; J = 5.40 Hz; H-10; 1H), 4.78 (H-20 and H-30 signals
are hidden by the water signal), 4.28 (m; H-40; 1H), 4.18 (m; H-50;
H-500; 2H), 2.48 (s; CH3; 3H), and 0.40 (m; BH3; 3H) ppm. 31P
NMR (D2O; 243 MHz): δ 84.49 (m; PR-BH3), 9.17 (d; J = 19.16
Hz; Pγ), and 2.35 (m; Pβ) ppm. MS-ESI m/z: 548 (M-). TLC
(NH4OH:H2O:2-propanol 2:8:11), Rf = 0.15. Purity data ob-
tained on an analytical column: retention time: 3.87 min (92.0%
purity) using solvent system I with a gradient from 85:15 to
60:40 A:B over 10 min at a flow rate of 1 mL/min. Retention time:
4.11 min (91.0% purity) using solvent system II with a gradient
from 90:10 to 60:40 A:B over 10 min at a flow rate of 1 mL/min.
Preparation and Characterization of 20,30-O-Methoxymethy-
lidene-50-O-tosyl-2-MeS-adenosine, 34. A solution of 4-dimethy-
laminopyridine (132 mg, 1.07 mmol, 4 equiv) in CH2Cl2 (1 mL)
and a solution of TsCl (130 mg, 0.67 mmol, 2.5 equiv) in CH2Cl2
(1 mL) were added to a suspension of 20,30-O-methoxymethyli-
dene-2-MeS-adenosine, analogue 15 (95 mg; 0.27 mmol), in
CH2Cl2 (3 mL) in a flame-dried two-neck flask under N2 at
RT. The suspension turned clear, and the reaction mixture was
stirred for 2 h at RT. CH2Cl2 (30 mL) was added to the reaction
mixture, which was then extracted with saturated NaHCO3
solution (3 ꢀ 30 mL). The organic phase was treated with
Na2SO4 and filtered. The solvent was removed under reduced
pressure, and the residue was separated using a MPLC system
with a silica (25þM) column and the following gradient scheme:
3 column volumes (CV) of 100:0 (A) CH2Cl2:(B) EtOH, 5 CV of
a gradient from 100:0 to 90:10 A:B, and 4 CV of 90:10 A:B at a
flow rate of 25 mL/min. The relevant fractions were collected,
and the solvent was removed under reduced pressure, yielding
analogue 34 at a 85% yield (115 mg) as a white solid. 1H NMR
(CDCl3; 200 MHz): δ 7.83 (s; H-8; 2H), 7.69, 7.23 (2 m, 2H),
6.34, 6.29 (2 br s, NH2; 2H), 6.17, 6.05 (2d; J = 2.60 Hz; H-10;
1H), 6.07, 6.01 (2s, CH-Ome; 1H), 5.56, 5.53 (2dd; J = 2.60 Hz;
H-20; 1H, J = 7.20 Hz; H-20; 1H), 5.21, 5.06 (2dd, J = 7.20 Hz;
H-30; 1H, J = 3.20 Hz; H-30; 1H), 4.66, 4.52 (2 m, H-40; 1H), and
4.31 (m, H-50; H-500; 4H) ppm. MS-ESI-m/z: 584 (M-). TLC
(CHCl3:EtOH, 9:1), Rf = 0.64.
Preparation and Characterization of 2-MeS-adenosine-50-O-
diphosphate-r,β-difluoromethylene, 13. 20,30-O-Methoxymethy-
lidene-50-O-tosyl-2-MeS-adenosine, analogue 34 (90 mg; 0.17
mmol), was dissolved in dry DMF (0.4 mL) in a flame-dried two-
neck flask under N2. A solution of tris(tetra-n-butylammo-
nium)difluoro methylenediphosphonate45 (0.28 mmol; 1.5 equiv)
in dry DMF (0.3 mL) was added at RT, and the reaction was
stirred for 72 h. Deionized water (20 mL) was added, and the
reaction was treated with 18% HCl until the pH was 2.3, and then
the mixture was stirred for 3 h at RT. Then, the mixture was
treated with 24% NH4OH, and the pH was adjusted to 9. The
solution was stirred for 45 min at RT and then freeze-dried. The
resulting residue was applied to an activated Sephadex DEAE-
A25 column (0-0.3 M NH4HCO3; total volume of 2 L). The
relevant fractions were collected, freeze-dried, and excess
NH4HCO3 was removed by repeated freeze-drying with deio-
nized water to yield analogue 13 as a white powder. The residue
was separated using a MPLC system with 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.
Finally, triethylammonium ions were exchanged for Naþ by
passing the pure analogue 13 through a Sephadex-CM C-25
Naþ-form column. Analogue 13 was obtained at a 34% yield
(61 mg) after MPLC separation. 1H NMR (D2O; 300 MHz): δ
8.18 (s; H-8; 1H), 5.93 (d; J = 5.40 Hz; H-10; 1H), 4.78 (H-20 and
H-30 signals are hidden by the water signal), 4.43 (m; H-40; 1H),
4.19 (m; H-50; 1H), 4.15 (m; H-500; 1H), and 2.37 (s; CH3; 3H)
ppm. 31P NMR (D2O; 81 MHz): δ 4.48 (m; PR, Pβ) ppm. 19F
NMR (D2O 188 MHz): δ 119.16 (dd, J1 = 84.72 Hz, J2 = 80.95
Hz) ppm. MS-ESI m/z: 506 (M-). HRMS-FAB (negative) m/z:
calculated for C12H15F2N5O9Na3P2S1: 573.9721; found: 573.966.
TLC (NH4OH:H2O:2-propanol 2:8:11), Rf = 0.5. Purity data
obtainedonan analytical column:retention time: 6.85 min(100%
purity) using solvent system I with a gradient from 85:15 to 50:50
A:B over 15 min at a flow rate of 1 mL/min. Retention time:
5.17 min (99.94% purity) using solvent system II with a gradient
from 85:15 to 50:50 A:B over 18 min at a flow rate of 1 mL/min.
Characterization of 2-MeS-adenosine, 50,5000-[P,P0-Difluoro-
1
methylene bisphosphonate], 38. H NMR (D2O; 300 MHz): δ
8.18 (s; H-8; 1H), 5.93 (d; J = 5.40 Hz; H-10; 1H), 4.78 (H-20 and
H-30 signals are hidden by the water signal), 4.43 (m; H-40; 1H),
4.19 (m; H-50, 1H), 4.15 (m; H-500; 1H), and 2.37 (s; CH3; 3H)
ppm. 31P NMR (D2O; 81 MHz): δ 4.48 (m; PR, Pβ) ppm. 19F
NMR (D2O 188 MHz): δ 119.16 (dd, J1 = 84.72 Hz, J2 =
80.95 Hz) ppm. MS-ESI m/z: 506 (M-). HRMS-FAB (negative)
m/z: calculated for C23H28F2N10O12Na3P2S2: 869.0460; found:
869.056. TLC (NH4OH:H2O:2-propanol 2:8:11), Rf = 0.5.
Purity data obtained on an analytical column: retention time:
6.85 min (100.0% purity) using solvent system I with a gradient
Preparation and Characterization of 20,30-O-Isopropylidene-
50-O-tosyl-2-MeS-adenosine, 35. Asolutionof4-DMAP (134mg,

3316 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Eliahu et al.
from 85:15 to 50:50 A:B over 15 min at a flow rate of 1 mL/min.
Retention time: 5.17 min (99.94% purity) using solvent system
II with a gradient from 85:15 to 50:50 A:B over 18 min at a flow
rate of 1 mL/min.
p-nitrophenol from p-nitrophenyl phosphate measured with
a UV-vis spectrophotometer at 405 nm.68 Relative enzyme
activity and resistance of analogues 5-14 to enzymatic hydro-
lysis were determined at 37 ꢀC using a solution of 0.2 mg of
analogue in 77.5 μL of deionized water, 0.1 M Tris-HCl (pH
9.8), and 0.1 M MgCl2 with calf intestine alkaline phosphatase
(Fermentas Inc., Glen Burnie, MD; 10 unit/μL; 1.25 μL; 12.5 u).
After 3 h, the reaction was stopped by incubation of the sample
at 80 ꢀC for 15 min. Each sample was applied to an activated
starta X-AW weak anion exchange cartridge and then freeze-
dried. The residue was purified by HPLC with a Gemini
analytical column (5u C-18 110A; 150 mm ꢀ 4.60 mm) using
the gradient elution system described for the hydrolysis of
analogues in human blood serum at a flow rate of 1 mL/min
(see above). The hydrolysis rates for analogues 5-14 with
alkaline phosphatase were determined by measuring the change
in the integration of the respective HPLC peaks with time.
NTPDase1,2,3,8 (EC 3.6.1.5) Assays. Activity was measured,
as previously described,69 at 37 ꢀC in 0.2 mL of Tris-Ringers
buffer: (in mM) 120 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 25
NaHCO3, 5 glucose, 80 Tris, pH 7.4; (Sigma-Aldrich, Oakville,
ON, Canada). ecto-Nucleotidases were expressed by transiently
transfecting COS-7 cells in 10 cm plates using Lipofectamine
(Invitrogen), as previously described.69 For the preparation of
protein extracts, transfected cells were washed three times with
Tris-saline buffer at 4 ꢀC, collected by scraping in harvesting
buffer (95 mM NaCl, 0.1 mM phenylmethylsulfonyl fluoride
(PMSF) and 45 mM Tris at pH 7.5), and washed twice by 300g
centrifugation for 10 min at 4 ꢀC. Cells were resuspended in
harvesting buffer containing 10 mg/mL aprotinin and sonicated.
Nuclei and cellular debris were discarded by centrifugation at
300g for 10 min at 4 ꢀC, and the supernatant (crude protein
extract) was aliquoted and stored at -80 ꢀC until used for
activity assays. Protein concentration was estimated by the
Bradford microplate assay using bovine serum albumin (BSA)
as a standard.70 NTPDase protein extracts were added to the
reaction mixture and then preincubated at 37 ꢀC for 3 min.
The reaction was initiated by addition of ATP (Sigma-
Aldrich, Oakville, ON, Canada) or analogues 8-14 at a final
concentration of 100 μM, and the reaction was stopped after
20 min with 50 μL of malachite green reagent (Sigma-
Aldrich). The released inorganic phosphate (Pi) was measured
at 630 nm according to Baykov et al.71 The activity obtained
with protein extracts from untransfected cells was subtracted
from the activity obtained with extracts from NTPDase-
transfected cells. The activity of untransfected cell extracts
never exceeded 5% of the activity of extracts from NTPDase-
transfected cells.
NPP1,3 (EC 3.1.4.1; EC 3.6.1.9) Assays. Evaluation of the
activity of human NPP1 and NPP3 with ATP (Sigma-
Aldrich) and analogues 8-14 was performed, as previously
described,72 with some modifications. Reactions were carried
out at 37 ꢀC in 0.2 mL of the following mixture: (in mM)
1 CaCl2, 140 NaCl, 5 KCl, and 50 Tris, pH 8.5 (Sigma-
Aldrich). Human NPP1 or NPP3 extract was added to the
reaction mixture and preincubated at 37 ꢀC for 3 min. The
reaction was initiated by addition of ATP or analogues 8-14
at a final concentration of 100 μM. The reaction was stopped
after 20 min by transferring a 0.1 mL aliquot of the reaction
mixture to 0.125 mL of ice-cold 1 M perchloric acid (Fisher
Scientific, Ottawa, ON, Canada). The samples were centri-
fuged for 5 min at 13000g. Supernatants were neutralized with
1 M KOH (Fisher Scientific) at 4 ꢀC and centrifuged for 5 min
at 13000g. An aliquot of supernatant (20 μL) was separated by
reverse-phase HPLC to evaluate the degradation of ATP and
analogues 8-14 using a SUPELCOSIL LC-18-T column
(15 cm ꢀ 4.6 mm; 3 mm Supelco; Bellefonte, PA) with
a mobile phase composed of 25 mM TBA, 5 mM EDTA,
100 mM KH2PO4/K2HPO4, pH 7.0, and 2% methanol at a
flow rate of 1 mL/min.
Preparation and Characterization of 2-MeS-adenosine,
50,5000-[P,P0-Dichloro-methylene bisphosphonate], 35. 20,30-O-Iso-
propylidene-50-O-tosyl-2-MeS-adenosine, analogue 14 (42 mg;
0.08 mmol), was dissolved in dry DMF (0.2 mL) in a flame-dried
two-neck flask under N2. A solution of tris(tetra-n-butylammo-
nium)dichloro methylenediphosphonate (0.16 mmol; 2 equiv) in
dry DMF (0.3 mL) was added at RT, and the reaction was
stirred for 72 h. Neat TFA was added for 10 min under argon
bubbling and then evaporated under reduced pressure to yield a
yellow solid. The resulting residue was separated on an activated
Sephadex DEAE-A25 column (0-0.3 M NH4HCO3; total
volume of 1.4 L). The relevant fractions were collected, freeze-
dried, and excess NH4HCO3 was removed by repeated freeze-
drying with deionized water to yield analogue 14 as a white
powder. The residue was separated using a HPLC system with a
semipreparative C-18 column and the following gradient
scheme (A) 100 mM TEAA:(B) MeOH with gradients of 85:15
to 75:25 over 10 min, 75:25 to 70:30 over 2 min, and 70:30 over
3 min at a flow rate of 5 mL/min. Retention time: 10.96 min. The
relevant fractions were collected, freeze-dried, and excess TEAA
was removed by repeated freeze-drying with deionized water to
yield analogue 14 as a white powder. Finally, the triethylammo-
nium ions were exchanged for Naþ by passing the pure analogue
14 through a Sephadex-CM C-25 Naþ-form column. Analogue
14 was obtained at a 18% yield (9 mg) after HPLC separation.
Retention time on a semipreparative column: 14.75 min. 1H
NMR (D2O; 600 MHz): δ 8.39 (s; H-8; 1H), 6.09 (d; J = 4.80 Hz;
H-10; 1H), 4.83 (m; H-20; 1H), 4.78 (H-30 signal is hidden by the
water signal), 4.61 (m; H-40; 1H), 4.38 (m; H-50; H-500; 2H), and
2.53 (s; CH3; 3H) ppm. 31P NMR (D2O; 243 MHz): δ 10.88 (d;
J = 15.79 Hz) and 8.70 (d; J = 15.79 Hz) ppm. MS-ESI m/z: 538
(M-). TLC (NH4OH:H2O:2-propanol 2:8:11), Rf = 0.26. Pur-
ity data obtained on an analytical column: retention time:
7.38 min (100% purity) using solvent system I with a gradient
from 85:15 to 50:50 A:B over 10 min at a flow rate of 1 mL/min.
Retention time: 4.38 min (99.9% purity) using solvent system II
with a gradient from 85:15 to 50:50 A:B over 10 min at a flow
rate of 1 mL/min.
Evaluation of the Stability of ADP, ATP, and Analogues 7-14
in Human Blood Serum. The assay mixture containing 40 mM
nucleotide derivative in deionized water (4.5 μL), human blood
serum (180 μL), and RPMI-1640 medium (540 μL) was incu-
bated at 37 ꢀC for 0-24 h and samples were removed 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, centrifuged
for 6 min (13000 rpm, 17000g), and extracted with chloroform
(2 ꢀ 500 μL). The aqueous layer was freeze-dried. Each resulting
residue was applied to an activated starta X-AW weak anion
exchange cartridge and then freeze-dried. The residue was
purified using HPLC with a Gemini analytical column (5u,
C-18, 110A; 150 mm ꢀ 4.60 mm) and gradient elution with
solvent system II at 100:0 A:B over 10 min for analogue 5, 100:0
to 70:30 A:B over 3 min for analogue 6, 90:10 to 80:20 A:B over
10 min, and then 80:20 to 60:40 A:B over 5 min for analogue 7,
85:15 A:B over 10 min for analogue 8, 90:10 to 50:50 A:B over
18 min for analogue 9, 80:20 to 30:70 A:B over 10 min for
analogue 14, and 80:20 to 30:70 A:B over 10 min for analogue 13
or gradient elution with solvent system I at 90:10 to 60:40 A:B
over 12 min for analogues 10A and 10B, 90:10 A:B over 15 min
for analogues 11A and 11B, and 80:20 A:B over 15 min for
analogues 12A and 12B at a flow rate of 1 mL/min. The
hydrolysis rates for the nucleotide analogues in human blood
serum were determined by measuring the change in the integra-
tion of the respective HPLC peaks with time.
Evaluation of the Stability of Analogues 5-14 with Alkaline
Phosphatase. Enzyme activity was determined by the release of

Article
Evaluation of the Chemical Stability of Analogues 8-14. A
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3317
Acknowledgment. Part of this work was supported by
grants from the Canadian Institutes of Health Research
(CIHR) to J.S. In addition, J.S. was a recipient of a New
Investigator award from the CIHR and of a junior 2 scholar-
ship from FRSQ.
nucleotide analogue (1.5 mg) was dissolved in 0.2 M HCl/KCl
buffer (0.8 mL) and the final pH was adjusted to 1.4 using 0.2 M
HCl. Reactions continued at 37 ꢀC for 1-31 days, with samples
taken at 1-24 h intervals. The stabilities of the analogues were
evaluated by HPLC to monitor degradation products using a
Gemini analytical column (5u C-18 110A; 150 mm ꢀ 4.60 mm)
and the gradient elution system described for the hydrolysis of
analogues in human blood serum at a flow rate of 1 mL/min (see
above). The hydrolysis rates of analogues 8-14 at pH 1.4 and
37 ꢀC were determined by measuring the change in the integra-
tion of the respective HPLC peaks with time.
Intracellular Calcium Measurements. Human 1321N1 astro-
cytoma cells stably expressing the turkey P2Y1 receptor were
grown in Dulbecco’s modified Eagle’s medium containing
5% 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-
centration, [Ca2þ]i, were detected by dual-excitation spectro-
fluorometric analysis of suspensions from cells loaded with
fura-2, as previously described.73,74 Cells were treated with
the indicated nucleotide analogue at 37 ꢀC in 10 mM Hepes-
buffered saline (pH 7.4) containing 1 mM CaCl2 and 1 mM
MgCl2, and the maximal increase in [Ca2þ]i was determined at
References
(1) Williams, M.; Jarvis, M. F. Purinergic and pyrimidinergic recep-
tors as potential drug targets. Biochem. Pharmacol. 2000, 59, 1173–
1185.
(2) Guile, S. D.; Ince, F.; Ingall, A. H.; Kindon, N. D.; Meghani, P.;
Mortimore, M. P. The medicinal chemistry of the P2 receptor
family. Prog. Med. Chem. 2001, 38, 115–187.
(3) Fischer, B. Therapeutic applications of ATP-(P2)-receptors ago-
nists and antagonists. Expert Opin. Ther. Pat. 1999, 9, 385–399.
(4) Hillmann, P.; Ko, G.-Y.; Spinrath, A.; Raulf, A.; von Kugelgen, I.;
Wolff, S. C.; Nicholas, R. A.; Kostenis, E.; Holtje, H.-D.; Muller,
C. E. Key Determinants of nucleotide-activated G protein-coupled
P2Y2 receptor function revealed by chemical and pharmacological
experiments, mutagenesis and homology modeling. J. Med. Chem.
2009, 52, 2762–2775.
(5) Burnstock, G.; Verkhratsky, A. Evolutionary origins of the puri-
nergic signalling system. Acta Physiol. 2009, 195, 415–447.
(6) Crooke, A.; Guzman-Aranguez, A.; Peral, A.; Abdurrahman,
M. K. A.; Pintor, J. Nucleotides in ocular secretions: their role in
ocular physiology. Pharmacol. Ther. 2008, 119, 55–73.
(7) 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.
(8) Pintor, J. Adenine nucleotides and dinucleotides as new substances
for the treatment of ocular hypertension and glaucoma. Curr. Opin.
Invest. Drugs 2005, 6, 76–80.
different analogue concentrations to calculate the EC50
.
Concentration-response data were analyzed with the Prism
curve fitting program (GraphPAD Software, San Diego, CA).
Data were obtained from three experiments performed in
triplicate.
Animals. Twenty-four male New Zealand white rabbits (2.5 (
0.5 kg) were kept in individual cages with free access to food and
water on controlled 12 h/12 h light/dark cycles. All the protocols
used adhered to the ARVO Statement for the Use of Animals
in Ophthalmology and Vision Research and also were in
accordance with the European Communities Council Directive
(86/609/EEC).
(9) Peral, A.; Gallar, J.; Pintor, J. Adenine nucleotide effect on
intraocular pressure: involvement of the parasympathetic nervous
system. Exp. Eye Res. 2009, 89, 63–70.
(10) Pintor, J.; Pelaez, T.; Peral, A. Adenosine tetraphosphate, Ap4, a
physiological regulator of intraocular pressure in normotensive
rabbit eyes. J. Pharmacol. Exp. Ther. 2004, 308, 468–473.
(11) Pintor, J.; Peral, A.; Pelaez, T.; Martin, S.; Hoyle, C. H. V. Presence
of diadenosine polyphosphates in the aqueous humor: their effect on
intraocular pressure. J. Pharmacol. Exp. Ther. 2003, 304, 342–348.
(12) Soto, D.; Pintor, J.; Peral, A.; Gual, A.; Gasull, X. Effects of
dinucleoside polyphosphates on trabecular meshwork cells and
aqueous humor outflow facility. J. Pharmacol. Exp. Ther. 2005,
314, 1042–1051.
(13) Crosson, C. E.; Yates, P. W.; Bhat, A. N.; Mukhin, Y. V.; Husain,
S. Evidence for multiple P2Y receptors in trabecular meshwork
cells. J. Pharmacol. Exp. Ther. 2004, 309, 484–489.
(14) El-Tayeb, A.; Qi, A.; Mueller, C. E. Synthesis andstructure-activity
relationships of uracil nucleotide derivatives and analogues as
agonists at human P2Y2, P2Y4, and P2Y6 receptors. J. Med. Chem.
2006, 49, 7076–7087.
IOP Measurements. IOP was measured by means of a Tono-
VET rebound tonometer supplied by Tiolat Oy (Helsinki,
Finland). The application of this tonometer to animals does
not require the use of any anesthetic. For single dose experi-
ments, different analogues were applied unilaterally to the
cornea at a concentration of 100 μM and a fixed volume of
10 μL. The contralateral eye received the same volume of saline
solution (0.9% NaCl, vehicle). Two IOP measurements were
taken before any analogue was instilled. Experiments were
performed following a blinded design where no visible indica-
tion was given to the experimenter as to the nature of the
applied solution. IOP was followed up to 8 h to study the time
course of the effect. Some of the analogues were assayed over
a range of doses from 1 nM to 100 μM to generate dose-
response curves. For these experiments, IOP was measured as
the maximal response obtained with each dose of the analogue.
Dose-response curves were calculated by plotting the IOP
value for a given concentration versus that concentration
(from 1 nM to 100 μM). pD2 values were obtained by fitting
the values to a dose-response curve equation according to
ORIGIN 8.0 software. With the pD2 value, it was possible to
calculate the EC50 by multiplying by -1 and then taking the
antilogarithm. The obtained value is the EC50 expressed in
molar concentration. In all experiments, on any given day, only
a single dose was tested on a single animal, which was washed
out at least 2 days between doses. The commercial hypotensive
agents, Latanoprost (0.005%), dorzolamide hydrochloride
(2%), and timolol maleate (0.5%), were assayed by applying
a volume of 40 μL.
(15) Nahum, V.; Zuendorf, G.; Levesque, S. A.; Beaudoin, A. R.;
Reiser, G.; Fischer, B. Adenosine 50-O-(1-Boranotriphosphate)
derivatives as novel P2Y1 receptor agonists. J. Med. Chem. 2002,
45, 5384–5396.
(16) Grobben, B.; Claes, P.; Roymans, D.; Esmans, E. L.; Van Onckelen,
H.; Slegers, H. Ecto-nucleotide pyrophosphatase modulates the
purinoceptor-mediated signal transduction and is inhibited by pur-
inoceptor antagonists. Br. J. Pharmacol. 2000, 130, 139–145.
(17) Zimmermann, H. Ectonucleotidases: some recent developments
and a note on nomenclature. Drug Dev. Res. 2001, 52, 44–56.
(18) Blackburn, G. M.; Taylor, G. E.; Thatcher, G. R. J.; Prescott, M.;
McLennan, A. G. Synthesis and resistance to enzymic hydrolysis of
stereochemically-defined phosphonate and thiophosphate analogs
of P1,P4-bis(50-adenosyl) tetraphosphate. Nucleic Acids Res. 1987,
15, 6991–7004.
(19) Cusack, N. J.; Hourani, S. M. O.; Loizou, G. D.; Welford, L. A.
Pharmacological effects of isopolar phosphonate analogs of ATP
on P2-purinoceptors in guinea pig tenia coli and urinary bladder.
Br. J. Pharmacol. 1987, 90, 791–795.
(20) He, K.; Hasan, A.; Shaw, B. R. The synthesis of ribonucleoside
50-O-(R-P-boranotriphosphates), separation of diastereomers and
incorporation by RNA polymerases. Nucleic Acids Symp. Ser.
1997, 36, 159.
Statistical Analysis. All data are presented as the mean (
SEM. Significant differences were determined by two-tailed
Student’s t tests. The plotting and fitting of dose-response
curves was carried out with Microcal Origin v.7.0 software
(Microcal Software, USA).
(21) Kowalska, J.; Lewdorowicz, M.; Darzynkiewicz, E.; Jemielity, J.
A simple and rapid synthesis of nucleotide analogues containing a
phosphorothioate moiety at the terminal position of the phosphate
chain. Tetrahedron Lett. 2007, 48, 5475–5479.

3318 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Eliahu et al.
(22) Lin, J.; Porter, K. W.; Shaw, B. R. Synthesis and properties of novel
triphosphate analogues: ribonucleoside and deoxyribonucleoside
(R-P-borano, R-P-thio)triphosphates. Nucleosides, Nucleotides
Nucleic Acids 2001, 20, 1019–1023.
(23) Misiura, K.; Szymanowicz, D.; Stec, W. J. Synthesis of nucleoside
R-thiotriphosphates via an oxathiaphospholane approach. Org.
Lett. 2005, 7, 2217–2220.
(24) Romaniuk, P. J.; Eckstein, F. Structure of the metal-nucleotide
complex in the acetate kinase reaction. A study with γ-32P-phos-
phorothioate analogs of ATP. J. Biol. Chem. 1981, 256, 7322–7328.
(25) Stingelin, J.; Bolen, D. W.; Kaiser, E. T. Synthesis, purification,
and characterization of the A and B diastereomers of gamma-32P-
labeled adenosine 50-O-(2-thiotriphosphate). J. Biol. Chem. 1980,
255, 2022–2025.
(26) Tulapurkar, M. E.; Laubinger, W.; Nahum, V.; Fischer, B.; Reiser,
G. Subtype specific internalization of P2Y1 and P2Y2 receptors
induced by novel adenosine 50-O-(1-boranotriphosphate) deriva-
tives. Br. J. Pharmacol. 2004, 142, 869–878.
(27) Eliahu, S. E.; Camden, J.; Lecka, J.; Weisman, G. A.; Sevigny, J.;
Gelinas, S.; Fischer, B. Identification of hydrolytically stable and
selective P2Y1 receptor agonists. Eur. J. Med. Chem. 2009, 44,
1525–1536.
(28) Joseph, S. M.; Pifer, M. A.; Przybylski, R. J.; Dubyak, G. R.
Methylene ATP analogs as modulators of extracellular ATP
metabolism and accumulation. Br. J. Pharmacol. 2004, 142,
1002–1014.
(29) Zhou, Z.; Wang, X.; Li, M.; Sohma, Y.; Zou, X.; Hwang, T.-C.
High affinity ATP/ADP analogues as new tools for studying
CFTR gating. J. Physiol. 2005, 569, 447–457.
(30) Boyle, N. A.; Rajwanshi, V. K.; Prhavc, M.; Wang, G.; Fagan, P.;
Chen, F.; Ewing, G. J.; Brooks, J. L.; Hurd, T.; Leeds, J. M.;
Bruice, T. W.; Cook, P. D. Synthesis of 20,30-dideoxynucleoside
50-R-P-borano-β,γ-(difluoromethylene)triphosphates and their
inhibition of HIV-1 reverse transcriptase. J. Med. Chem. 2005,
48, 2695–2700.
(43) Cusack, N. J.; Hourani, S. M. O.; Loizou, G. D.; Welford, L. A.
Pharmacological effects of isopolar phosphonate analogs of ATP
on P2-purinoceptors in guinea pig tenia coli and urinary bladder.
Br. J. Pharmacol. 1987, 90, 791–795.
(44) Ravi, R. G.; Kim, H. S.; Servos, J.; Zimmermann, H.; Lee, K.;
Maddileti, S.; Boyer, J. L.; Harden, T. K.; Jacobson, K. A. Adenine
nucleotide analogues locked in a northern methanocarba confor-
mation: enhanced stability and potency as P2Y1 receptor agonists.
J. Med. Chem. 2002, 45, 2090–2100.
(45) Davisson, V. J.; Davis, D. R.; Dixit, V. M.; Poulter, C. D. Synthesis
of nucleotide 50-diphosphates from 50-O-tosyl nucleosides. J. Org.
Chem. 1987, 52, 1794–1801.
(46) Hull, W. E.; Halford, S. E.; Gutfreund, H.; Sykes, B. D. Phos-
phorus-31 nuclear magnetic resonance study of alkaline phospha-
tase: the role of inorganic phosphate in limiting the enzyme
turnover rate at alkaline pH. Biochemistry 1976, 15, 1547–1561.
(47) Gendaszewska-Darmach, E.; Maszewska, M.; Zaklos, M.;
Koziolkiewicz, M. Degradation of extracellular nucleotides and
their analogs in HeLa and HUVEC cell cultures. Acta Biochim. Pol.
2003, 50, 973–984.
(48) Bonan, C. D.; Schetinger, M. R. C.; Battastini, A. M. O.; Sarkis,
J. J. F. Ectonucleotidases and synaptic plasticity: implications in
physiological and pathological conditions. Drug Dev. Res. 2001, 52,
57–65.
(49) Schetinger, M. R. C.; Morsch, V. M.; Bonan, C. D.; Wyse, A. T. S.
NTPDase and 50-nucleotidase activities in physiological and dis-
ease conditions: new perspectives for human health. BioFactors
2007, 31, 77–98.
(50) Terkeltaub, R. Physiologic and pathologic functions of the NPP
nucleotide pyrophosphatase/phosphodiesterase family focusing on
NPP1 in calcification. Purinergic Signalling 2006, 2, 371–377.
(51) Arzumanov, A. A.; Semizarov, D. G.; Victorova, L. S.; Dyatkina,
N. B.; Krayevsky, A. A. γ-Phosphate-substituted 20-deoxynucleo-
side 50-triphosphates as substrates for DNA polymerases. J. Biol.
Chem. 1996, 271, 24389–24394.
(31) Barral, K.; Priet, S.; Sire, J.; Neyts, J.; Balzarini, J.; Canard, B.;
Alvarez, K. Synthesis, in vitro antiviral evaluation, and stability
studies of novel R-borano-nucleotide analogues of 9-[2-(phos-
phonomethoxy)ethyl]adenine and (R)-9-[2-(phosphonomethoxy)-
propyl]adenine. J. Med. Chem. 2006, 49, 7799–7806.
(32) Bystrom, C. E.; Pettigrew, D. W.; Remington, S. J.; Branchaud,
B. P. ATP analogs with non-transferable groups in the γ position as
inhibitors of glycerol kinase. Bioorg. Med. Chem. Lett. 1997, 7,
2613–2616.
(33) Myers, T. C.; Nakamura, K.; Flesher, J. W. Phosphonic acid
analogs of nucleoside phosphates. I. The synthesis of 50-adenylyl
methylenediphosphonate, a phosphonic acid analog of adenosine
triphosphate. J. Am. Chem. Soc. 1963, 85, 3292–3295.
(34) Major, D. T.; Fischer, B. Molecular recognition in purinergic
(52) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. The synthesis and
metal binding characteristics of novel, isopolar phosphonate ana-
logs of nucleotides. J. Chem. Soc., Perkin Trans. 1 1984, 1119–1125.
(53) 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.
(54) Pintor, J.; Sanchez-Nogueiro, J.; Irazu, M.; Mediero, A.; Pelaez,
T.; Peral, A. Immunolocalisation of P2Y receptors in the rat eye.
Purinergic Signalling 2004, 1, 83–90.
(55) Markovskaya, A.; Crooke, A.; Guzman-Aranguez, A. I.; Peral, A.;
Ziganshin, A. U.; Pintor, J. Hypotensive effect of UDP on intrao-
cular pressure in rabbits. Eur. J. Pharmacol. 2008, 579, 93–97.
(56) Pintor, J.; Peral, A. Therapeutic potential of nucleotides in the eye.
Drug Dev. Res. 2001, 52, 190–195.
(57) Nahum, V.; Fischer, B. Boranophosphate salts as an excellent
mimic of phosphate salts: Preparation, characterization, and prop-
erties. Eur. J. Inorg. Chem. 2004, 4124–4131.
(58) Li, H.; Hardin, C.; Shaw, B. R. Hydrolysis of Thymidine Borano-
monophosphate and Stepwise Deuterium Substitution of the Bor-
ane Hydrogens. 31P and 11B NMR Studies. J. Am. Chem. Soc. 1996,
118, 6606–6614.
(59) Bystrom, C. E.; Pettigrew, D. W.; Remington, S. J.; Branchaud,
B. P. ATP analogs with non-transferable groups in the γ position as
inhibitors of glycerol kinase. Bioorg. Med. Chem. Lett. 1997, 7,
2613–2616.
receptors. 1.
A comprehensive computational study of the
h-P2Y1-receptor. J. Med. Chem. 2004, 47, 4391–4404.
(35) Major, D. T.; Nahum, V.; Wang, Y.; Reiser, G.; Fischer, B.
Molecular recognition in purinergic receptors. 2. Diastereoselec-
tivity of the h-P2Y1-receptor. J. Med. Chem. 2004, 47, 4405–4416.
(36) Blackburn, G. M.; Kent, D. E.; Kolkmann, F. Three new β,γ-
methylene analogs of adenosine triphosphate. J. Chem. Soc.,
Chem. Commun. 1981, 1188–1190.
(37) Wang, G.; Boyle, N.; Chen, F.; Rajappan, V.; Fagan, P.; Brooks,
J. L.; Hurd, T.; Leeds, J. M.; Rajwanshi, V. K.; Jin, Y.; Prhavc, M.;
Bruice, T. W.; Cook, P. D. Synthesis of AZT 50-triphosphate
mimics and their inhibitory effects on HIV-1 reverse transcriptase.
J. Med. Chem. 2004, 47, 6902–6913.
(38) Spelta, V.; Mekhalfia, A.; Rejman, D.; Thompson, M.; Blackburn,
G. M.; North, R. A. ATP analogues with modified phosphate
chains and their selectivity for rat P2X2 and P2X2/3 receptors.
Br. J. Pharmacol. 2003, 140, 1027–1034.
(39) Ingall, A. H.;Dixon, J.;Bailey, A.; Coombs, M. E.; Cox, D.;McInally,
J. I.; Hunt, S. F.; Kindon, N. D.; Teobald, B. J.; Willis, P. A.;
Humphries, R. G.; Leff, P.; Clegg, J. A.; Smith, J. A.; Tomlinson,
W. Antagonists of the platelet P2T receptor: a novel approach to
antithrombotic therapy. J. Med. Chem. 1999, 42, 213–220.
(40) El-Tayeb, A.; Griessmeier, K. J.; Mueller, C. E. Synthesis and
preliminary evaluation of [3H]PSB-0413, a selective antagonist
radio-ligand for platelet P2Y12 receptors. Bioorg. Med. Chem. Lett.
2005, 15, 5450–5452.
(41) Padyukova, N. S.; Dixon, H. B. F.; Efimtseva, E. V.; Ermolinsky,
B. S.; Mikhailov, S. N.; Karpeisky, M. Y. Synthesis and properties
of novel NTP derivatives. Nucleoides, Nucleotides Nucleic Acids
1999, 18, 1013–1014.
(42) Mohamady, S.; Jakeman, D. L. An improved method for the
synthesis of nucleoside triphosphate analogs. J. Org. Chem. 2005,
70, 10588–10591.
(60) Higginbotham, E. J.; Schuman, J. S.; Goldberg, I.; Gross, R. L.;
VanDenburgh, A. M.; Chen, K.; Whitcup, S. M.; Abelson, M. B.;
Baerveldt, G.; Best, S.; Branch, J. D.; Brandt, J. D.; Brennan, J.;
Brooks, A. M. V.; Butrus, S.; Cacioppo, R.; D’Mellow, G.;
DuBiner, H. B.; Duzman, E.; Forester, R. J.; Frantz, J.; Fried,
W. I.; Gieser, D. K.; Lewis, R. A.; Logan, A.; McGovern, R.;
Mundorf, T.; Nardin, G. F.; Nussdorf, J.; Rait, J.; Shields, R. L.;
Walters, T. R.; Whitsett, J. C.; Wilensky, J. T.; Williams, R.; Beck,
A.; Cantor, L.; Cioffi, G.; Cohen, J. S.; Cooke, D.; Crichton, A.;
Dudley, D. F.; Evans, R.; Greenberg, S.; Gupta, N.; Gurevich, L.;
Kasner, O.; Kellum, D.; Koby, M.; Kwedar, J.; McGarey, D.;
Mikelberg, F.; Noecker, R.; Remis, L. L.; Ritch, R.; Rotberg, M.;
Schenker, H. I.; Sharpe, E.; Sherwood, M. B.; Sokol, J.; Solish, A.;
Whiteside-Michel, J.; Wirostko, B. One-year, randomized study
comparing bimatoprost and timolol in glaucoma and ocular
hypertension. Arch. Ophthalmol. 2002, 120, 1286–1293.
(61) Kaiserman, I.; Fendyur, A.; Vinker, S. Topical beta blockers in
asthmatic patients-is it safe? Curr. Eye Res. 2009, 34, 517–522.
(62) Quigley, H. A. Open-angle glaucoma. N. Engl. J. Med. 1993, 328,
1097–1106.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3319
(63) Hoyng, P. F. J.; Van Beek, L. M. Pharmacological therapy for
glaucoma: a review. Drugs 2000, 59, 411–434.
(64) Alward, W. L. M. Medical management of glaucoma. N. Engl.
(70) Bradford, M. M. A rapid and sensitive method for the quantitation
of microgram quantities of protein utilizingthe principle ofprotein-
dye binding. Anal. Biochem. 1976, 72, 248–254.
J. Med. 1998, 339, 1298–1307.
(71) Baykov, A. A.; Evtushenko, O. A.; Avaeva, S. M. A malachite
green procedure for orthophosphate determination and its use in
alkaline phosphatase-based enzyme immunoassay. Anal. Biochem.
1988, 171, 266–270.
(72) Levesque, S. A.; Lavoie, E. G.; Lecka, J.; Bigonnesse, F.; Sevigny, J.
Specificity of the ecto-ATPase inhibitor ARL 67156 on human and
mouse ectonucleotidases. Br. J. Pharmacol. 2007, 152, 141–150.
(73) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of
calcium indicators with greatly improved fluorescence properties.
J. Biol. Chem. 1985, 260, 3440–3450.
(65) Johnstone, M. A. Hypertrichosis and increased pigmentation of
eyelashes and adjacent hair in the region of the ipsilateral eyelids of
patients treated with unilateral topical latanoprost. Am. J. Ophthal-
mol. 1997, 124, 544–547.
(66) Griffin, B. E.; Jarman, M.; Reese, C. B.; Sulston, J. E. The synthesis
of oligoribonucleotides. II. Methoxymethylidene derivatives of
ribonucleosides and 50-ribonucleotides. Tetrahedron 1967, 23,
2301–2313.
(67) Bachelet, M.; Guibe, M. Preparation of triethylammonium bicar-
bonate. Bull. Soc. Chim. Fr. 1951, 554.
(74) 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
agonist-induced desensitization and sequestration of the P2Y2
nucleotide receptor. Consequences of truncation of the C terminus.
J. Biol. Chem. 1998, 273, 29437–29444.
(75) Morales, M.; Gomez-Cabrero, A.; Peral, A.; Gasull, X.; Pintor, J.
Hypotensive effect of profilin on rabbit intraocular pressure.
Eur. J. Pharmacol. 2007, 567, 145–148.
(68) Brandenberger, H.; Hanson, R. Spectrophotometric determina-
tion of acid and alkaline phosphatases. Helv. Chim. Acta 1953, 36,
900–906.
(69) Kukulski, F.; Levesque, S. A.; Lavoie, E. G.; Lecka, J.; Bigonnesse,
F.; Knowles, A. F.; Robson, S. C.; Kirley, T. L.; Sevigny, J.
Comparative hydrolysis of P2 receptor agonists by NTPDases 1,
2, 3, and 8. Purinergic Signalling 2005, 1, 193–204.