Methanocarba modification of uracil and adenine nucleotides: High potency of northern ring conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but not P2Y6 receptors

Article abstract of DOI:10.1021/jm010369e

The potency of nucleotide antagonists at P2Y₁ receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba an

Full text of DOI:10.1021/jm010369e

208
J . Med. Chem. 2002, 45, 208-218
Meth a n oca r ba Mod ifica tion of Ur a cil a n d Ad en in e Nu cleotid es: High P oten cy
of Nor th er n Rin g Con for m a tion a t P 2Y1, P 2Y2, P 2Y4, a n d P 2Y11 bu t Not P 2Y6
Recep tor s
Hak Sung Kim,† R. Gnana Ravi,† Victor E. Marquez,‡ Savitri Maddileti,§ Anna-Karin Wihlborg,| David Erlinge,|
Malin Malmsjo¨,| J ose´ L. Boyer,§ T. Kendall Harden,§ and Kenneth A. J acobson*,†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, Laboratory of Medicinal Chemistry,
Center for Cancer Research, National Cancer Institute at Frederick, Frederick, Maryland 21702, Department of Pharmacology,
University of North Carolina, School of Medicine, Chapel Hill, North Carolina 27599-7365, and Department of Cardiology,
Lund University Hospital, SE-221 85 Lund, Sweden
Received August 6, 2001
The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose
moiety with a constrained carbocyclic ring (Nandanan, et al. J . Med. Chem. 2000, 43, 829-
842). We have now synthesized ring-constrained methanocarba analogues (in which a fused
cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the
endogenous activators of P2Y receptors. Methanocarba-adenosine 5′-triphosphate (ATP) was
fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational
cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment
with â-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was
carried out at the 5′-hydroxyl group through a multistep process: Reaction with phosphora-
midite followed by oxidation provided the 5′-monophosphates, which then were treated with
1,1′-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the
analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1,
P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured.
At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-
fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-
methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-
uridine 5′-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors
and also activated P2Y4 receptors with an EC50 of 85 nM. (N)-Methanocarba-uridine 5′-
diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methano-
carba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery.
The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC50
)
6.1 ( 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6
receptor-mediated contractile response. Our results suggest that new nucleotide agonists may
be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4,
and P2Y11 receptors.
In tr od u ction
P2 receptors, which are activated by purine and/or
pyrimidine nucleotides, consist of two families:
cellular calcium and activation of protein kinase C. The
P2Y11 receptor activates adenylyl cyclase in addition to
the activation of PLC,9 and G protein-coupled P2Y
receptors in C6 glioma cells10 and the recently cloned
P2Y12 receptor in platelets11 inhibit adenylyl cyclase.
G
protein-coupled receptors termed P2Y, of which six
mammalian subtypes have been cloned, and ligand-
gated cation channels termed P2X, of which seven
mammalian subtypes have been cloned.1-3 The nomen-
clature of P2 receptors and their various ligand speci-
ficities has been reviewed.4-7 Uracil nucleotides activate
P2Y2, P2Y4, and P2Y6 subtypes. Adenine nucleotides are
required for the activation of P2Y1 and P2Y11 subtypes.
Activation of P2Y receptors generally results in the
stimulation of phospholipase C (PLC), which generates
inositol phosphates and diacylglycerol from phosphatid-
yl inositol(4,5)bisphosphate,8 leading to a rise in intra-
The P2Y1 receptor is present in the heart, skeletal,
and various smooth muscles, prostate, ovary, and
brain.12 A selective P2Y1 receptor agonist may have
potential as an antihypertensive or antidiabetic agent.13,14
The P2Y2 receptor is associated with the ion secretory
actions of uridine 5′-triphosphate (UTP) and adenosine
5′-triphosphate (ATP) in the mucosa of the nasal and
tracheal epithelia and is a target in therapeutics of
pulmonary diseases, e.g., cystic fibrosis15,16 and oph-
thalmic diseases.17 The P2Y4 and P2Y6 receptors may
have a role in regulation of blood flow18 and have been
implicated in the gastrointestinal responses to UTP and
uridine 5′-diphosphate (UDP).16 The P2Y11 receptor
subtype is present in the immune system, and the P2Y12
receptor subtype plays a key role in ADP action in
* To whom correspondence should be addressed. Tel: (301)496-9024.
Fax: (301)480-8422. E-mail: kajacobs@helix.nih.gov.
† National Institutes of Health.
‡ National Cancer Institute.
§ University of North Carolina, School of Medicine.
| Lund University Hospital.
10.1021/jm010369e CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/22/2001

Methanocarba Modification of Nucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 209
Ta ble 1. Potencies for Methanocarba Analogues and the Corresponding Naturally Occurring Nucleotides in the Activation of
Phospholipase C
Northern
a , methanocarba
b, ribose
EC50 (µM) of (N)-methanocarba
EC50 (µM) of corresponding
compd
analogue
ATP
subtype
analoguea
ribose nucleotidea,b
3a
tP2Y1
hP2Y1
hP2Y2
hP2Y4
hP2Y11
tP2Y1
hP2Y2
hP2Y4
hP2Y6
hP2Y11
hP2Y6
hP2Y6
0.014 ( 0.003
0.052 ( 0.022
0.091 ( 0.005
32% at 10 µM
34.5 ( 4.7% at 10 µM
NEd
0.0159 ( 0.007
0.085 ( 0.005
NE
2.8 ( 0.7
1.5 ( 0.2
0.085 ( 0.012
c
17.25 ( 2.80
NE
4a
5a
AMP
UTP
0.008 ( 0.002
0.049 ( 0.010
NE
NE
NE
6a
7a
UDP
UMP
0.015 ( 0.004
NE
Southern
EC50 (µM) of (S)-methanocarba
EC50 (µM) of corresponding
compd
analogue
ATP
subtype
analoguea
ribose nucleotidea,b
8a
tP2Y1
hP2Y1
hP2Y2
hP2Y4
hP2Y11
tP2Y1
3.5 ( 0.66
7.2 ( 1.0
3.7 ( 0.5
NE
NE
NE
2.8 ( 0.7
1.5 ( 0.2
0.085 ( 0.012
c
17.25 ( 2.80
NE
9a
AMP
a
b
d
Average ( SEM. Values from refs 32, 33, 38, and 39. c Antagonist effect.37 NE means “no effect at 100 µM”.
platelets. Currently, there are few selective agonists or
antagonists at these subtypes.7 While numerous ad-
enine nucleotide ligands have been explored at the P2Y1
receptor, few uracil nucleotides have been reported as
P2Y receptor agonists.15,18
tors, the ribose-containing antagonist 2-chloro-2′-deoxy-
N6-methyladenosine-3′,5′-bisphosphate, 1, has been sub-
stituted with the (N)-methanocarba modification to yield
(1R,2S,4S,5S)-1-[(phosphato)methyl]-4-(2-chloro-6-
methylaminopurin-9-yl)bicyclo[3.1.0]hexane-2-phos-
phate, 2.20 Both 1 and 2 are selective antagonists of the
turkey erythrocyte P2Y1 receptor, with 2 (IC50 52 nM)
approximately 4-fold more potent than 1.
We wished to determine if the preference for the (N)
conformation applies to subtypes other than P2Y1
receptors and also whether this preference seen for
bisphosphate antagonist derivatives is generalizable to
5′-triphosphate agonists and to pyrimidine as well as
purine nucleotides. Here, we describe the synthesis and
biological characterization in several species of the
methanocarba analogues of adenine and uracil nucle-
otides. For direct comparison, the adenine nucleotide
analogues have been locked in both the (N) ring and
the (S) ring conformations. In general, the constrained
(N) conformation of the pseudoribose ring increased
agonist potencies at P2Y1 receptors and preserved
potencies at P2Y2, P2Y4, and P2Y11 receptors but not
at the UDP-activated P2Y6 receptor. The corresponding
(S) isomer of methanocarba-ATP displayed greatly
reduced potency at P2Y1, P2Y2, and P2Y11 receptors.
Conformational considerations of both the receptors
and their ligands are important in structure-based drug
design. The ribose rings of purine and pyrimidine
nucleotides can adopt a range of conformations, al-
though the P2 receptors, which they activate, likely
prefer specific conformations. Therefore, constraining
the riboselike ring of nucleotide analogues would help
establish these parameters, and we hypothesize that an
analogue in which the ribose has been replaced with a
carbocyclic ring locked in a preferred conformation may
display unique potency and selectivity. Thus, we have
adopted the methanocarba approach,19 in which fused
cyclopropane and cyclopentane rings replace the ribose
moiety. Two structural variations, depending on the
position of the cyclopropane ring, restrict the ring
pucker, i.e., hold the riboselike ring (pseudosugar) in
either a Northern (N, 2′-exo) or a Southern (S, 2′-endo)
envelope conformation, as defined in the pseudorota-
tional cycle. For two classes of purine ligands, i.e.,
adenosine bisphosphate derivatives binding at P2Y1
receptors20 and adenosine derivatives binding at A1 and
A3 receptors,21 a preference for the (N)-methanocarba
isomer already has been demonstrated. At P2Y1 recep-
Resu lts
Ch em ica l Syn th esis. Methanocarbocyclic analogues
of naturally occuring adenine and uracil nucleotides
(Table 1) were synthesized containing fused cyclopro-
pane and cyclopentane rings that fixed the pseudoribose
moiety in a rigid (N) or (S) envelope conformation.
Nucleotide analogues having either the (N)-methano-
carba modification,19,20 3a -7a , or the (S)-methanocarba
modification,22 8a and 9a , were prepared in the am-
monium salt form according to the methods shown in
Schemes 1-4 and tested biologically as agonists at

210 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Kim et al.
Ta ble 2. Synthetic Data for Nucleotide Derivatives, Including Structural Verification Using High-Resolution Mass Spectroscopy and
Purity Verification Using HPLC
HPLC (rt; min)a
FAB (M - H+)
system 1
system 2
yield
(%)e
no.
formula
calcd
found
(purity) (%)
(purity) (%)
3a
4a
5a
6a
7a
8a
9a
C12H18N5O12P3
516.0087
356.0760
492.9814
413.0151
333.0488
356.0760
516.0087
516.0085
356.0765
492.9832
413.0151
333.0506
356.0765
516.0085
4.289 (98)
4.178 (99)
1.737 (99)
2.130 (98)
3.235 (99)
5.057 (97)
5.709 (97)
17.009d (98)
8.145d (98)
6.764b (96)
4.531c (97)
3.466c (98)
11.091d (99)
17.517d (97)
14
65
22
24
58
60
30
C
C
C
C
C
C
12H16N5O6P
11H17N2O14P3
11H16N2O11P2
11H15N2O8P
12H16N5O6P
12H18N5O12P3
a
Purity of each derivative was g95%, as determined using HPLC with two different mobile phases. Purity of all compounds in solvent
1 and compounds 5a , 6a , and 7a in solvent 2 was checked using an SMT OD-5-60 RP-C18 analytical column (250 × 4.6 mm; Separation
Methods Technologies, Inc., Newark, DE) in two solvent systems. Compounds 3a , 4a , 8a , and 9a were checked in solvent 2 using a
Phenomenex Luna 5µ C18(2) analytical column (250 × 4.6 mm). All gradients were completed in 20 min. System 1: gradient of 0.1 M
b
TEAA/CH3CN from 95/5 to 40/60. System 2: as specified below. Gradient of 5 mM TBAP/CH3CN from 80/20 to 40/60. c Gradient of 0.1
d
M TEAB/CH3CN from 100/0 to 90/10. Gradient of 5 mM TBAP/CH3CN from 95/5 to 40/60. e The percent yields refer to overall yield for
the entire phosphorylation sequence. f 3a , MRS2340; 5a , MRS 2341.
Sch em e 1. General Method for Stepwise Phosphorylation of (N)-Methanocarba Nucleosides: Formation of Di- and
Triphosphates via the Phosphorimidazolidate Intermediate, IIa
a
Reagents: (i) 1,1′-Carbonyldiimidazole, DMF, room temperature, 6 h and then 5% aqueous triethylamine, room temperature, 2 h. (ii)
Tributylammonium phosphate, DMF, room temperature, 2 days, 43%. (iii) Tributylammonium pyrophosphate, DMF, 3 days, 6-40%.
various P2Y receptors (Table 1). Identity was confirmed
yldiimidazole24,25 with a nucleoside 5′-monophosphate,
I, followed by the addition of either phosphate or
pyrophosphate. By this method, the monophosphate
analogues were cleanly converted to either di- or tri-
phosphate, III or IV, respectively, via a phosphorimi-
dazolidate intermediate, II. Typically, the monophos-
phate, I, was treated with an excess of carbonyldiimi-
dazole in N,N-dimethylformamide (DMF) and stirred for
4-6 h. To hydrolyze the cyclic carbonate24 that formed
at the 2′,3′-dihydroxy groups, a 5% triethylamine solu-
tion in MeOH:water (1/1) was added to the reaction
mixture, and stirring was continued for 2 h. HPLC
monitoring of this reaction step indicated that the
phosphorimidazolidate, II, was stable to mild base
treatment. This intermediate, II, was treated with
phosphoric acid tributylammonium salt or with pyro-
phosphoric acid tributylammonium salt to give the
diphosphate, III, or triphosphate, IV, respectively.
The (N)-methanocarba analogues of ATP and adenos-
ine 5′-monophosphate (AMP) were prepared by this
approach (Scheme 2). The (N)-methanocarba-adenosine
precursors were prepared by the general approaches of
1
using nuclear magnetic resonance (NMR, H and 31P)
and high-resolution mass spectrometry, and purity was
demonstrated using high-pressure liquid chromatogra-
phy (HPLC) in two different solvent systems (Table 2).
Several methods of 5′-phosphorylation of the (N)-
methanocarba nucleoside were compared. A standard
method using sequential reaction with phosphorus
oxychloride and pyrophosphate to provide nucleoside 5′-
triphosphate analogues23 proved problematic. Several
attempts to obtain the (N)-methanocarba-UTP by varia-
tions of this one-pot method were unsuccessful. We have
evidence that the one-pot method in the (N) series leads
to the formation of cyclic-3′,5′-nucleotides (unpublished,
V. Marquez). This is due to the close proximity between
the 3′-OH and the 5′-OH that can be achieved only in
the fixed (N) envelope conformation. On the other hand,
these two hydroxyl groups never come close to one
another in the antipodal (S) conformers. Therefore, the
multistep elongation of a 5′-monophosphate group to di-
and triphosphates was pursued. The general method
adopted (Scheme 1) consisted of reaction of 1,1′-carbon-

Methanocarba Modification of Nucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 211
Sch em e 2. Synthesis of (N)-Methanocarba Adenosine
5′-Phosphate Derivativesa
Sch em e 3. Synthesis of (N)-Methanocarba-uridine
5′-Monophosphate, 7a , and 5′-Di- and Triphosphates, 6a
and 5a a
a
Reagents: (i) (a) EtOCHdCHC(dO)NCO, benzene, room tem-
perature, 10 min, 89%; (b) 2 N H2SO4, MeOH, reflux, 30 min, 67%.
(ii) Pd black, HCO2H, methanol, 74%. (iii) p-Toluene sulfonic acid,
acetone, room temperature, 4 h, 86%. (iv) Di-t-butyl N,N-dieth-
ylphosphoramidite, tetrazole, THF, room temperature, 20 min and
then m-CPBA, -78 °C, room temperature, 79%. (v) DOWEX 50 ×
8-200, methanol, 60-70 °C, 86%. (vi) (1) 1,1′-Carbonyldiimidazole,
(2) triethylamine/MeOH, (3) tributylammonium phosphate or
pyrophosphate, DMF, 2-3 days, 41% (6a ), 38% (5a ).
a
Reagents: (i) DEAD, Ph3P, THF (ref 40). (ii) NH3/i-PrOH, 95%.
(iii) Palladium black, HCO2H, methanol, 76%. (iv) Di-t-butyl N,N-
diethylphosphoramidite, tetrazole, THF, room temperature, 6 h
and then m-CPBA, -78 °C, room temperature, 78%. (v) DOWEX
50 × 8-200, methanol, 60-70 °C, 69%. (vi) (1) 1,1′-Carbonyldi-
imidazole, (2) triethylamine/MeOH, (3) tributylammonium pyro-
phosphate, 20%.
methanol gave the 5′-protected (N)-methanocarba-
uracil, 17, in good yield. Removal of the O-benzyl group
with palladium black and formic acid in methanol
afforded the triol, 18. The acetonide of the 2,3-diol, 19,
was prepared, and the primary alcohol was converted
to the di-tert-butyl phosphate, 20.29 Both di-tert-butyl
and acetonide groups were deprotected simultaneously
under acidic conditions, leading to the successful syn-
thesis of monophosphate, 7a .
The (S)-methanocarba analogues of ATP and AMP,
8a and 9a (Scheme 4), respectively, were prepared using
a sequential phosphorus oxychloride method from the
unprotected, racemic (S)-methanocarba-adenosine, 21.22
The monophosphate, 9a , was isolated and converted to
the triphosphate, 8a , using carbonyldiimidazole.
Biologica l Activity. Methanocarba analogues were
tested for agonist activity by measuring P2Y1 receptor-
promoted PLC activity in turkey erythrocyte mem-
branes and by measuring inositol phosphate accumu-
lation in 1321N1 human astrocytoma cells stably
expressing the human P2Y1, P2Y2, P2Y4, P2Y6, or P2Y11
receptors. In each case, full concentration effect curves
were generated, and EC50 values from at least three
separate experiments were determined (Table 1). The
agonist potencies of the corresponding ribose-containing
nucleotides (denoted “b”) were simultaneously deter-
mined for comparison purposes.
Marquez and co-workers19,26,27 or Lee et al.28 6-Chloro-
purine, 10, was condensed with the protected bicyclo-
[3.1.0]hexane derivative, 11,28 using Mitsunobu condi-
tions to give 12, which was then treated with a satu-
rated solution of ammonia in THF to give the corre-
sponding adenosine analogue, 13. Deprotection of a 5′-
benzyl group of 13 was carried out smoothly using Pd
black/formic acid to give 14 and was followed by phos-
phorylation using a phosphoramidite method29 to give
the di-tert-butyl-protected monophosphate, 15. The
monophosphate, 15, was deprotected and either tested
biologically or carried further to the di- and triphos-
phates by the method shown in Scheme 1.
The (N)-methanocarba analogues of UTP (5a ), UDP
(6a ), and UMP (7a ), were also prepared. A synthetic
route to (N)-methanocarba-uridine as a precursor for
uracil nucleotide analogues was devised (Scheme 3).
Several attempts to condense uracil and the pseu-
dosugar moiety by the Mitsunobu reaction gave only a
di-O-alkylated byproduct, likely due to the low solubility
of uracil in tetrahydrofuran (THF). A protected uracil
derivative having greater solubility in THF, 3-benzoyl-
uracil,30 was used as a reactant in the Mitsunobu
reaction; however, an O-alkylated byproduct resulted.
Eventually, formation of the uracil moiety was achieved
in two sequential steps (Scheme 3).31 The amino-
cyclopentane derivative, 16, was converted to an acryl-
oylureido compound (isolated but not shown in Scheme
3) by treatment with â-ethoxyacryloyl cyanate.31 Cy-
clization upon refluxing in 2 N sulfuric acid and
The (N)-methanocarba-ATP analogue, 3a , was 250-
fold more potent than the racemic (S)-methanocarba-
ATP analogue, 8a , and 200-fold more potent than ATP,
3b, at the turkey P2Y1 receptor (Table 1). The (N)-

212 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Kim et al.
Sch em e 4. Synthesis of (S)-Methanocarba Adenosine
5′-Phosphate Derivativesa
a
Reagents: (i) POCl3, trimethyl phosphate, 60%. (ii) 1,1′-
Carbonyldiimidazole, DMF, room temperature, 6 h and then 5%
aqueous triethylamine, room temperature, 2 h. (iii) Tributyl-
ammonium pyrophosphate, DMF, 3 days, 30%.
methanocarba-ATP analogue, 3a , was also more potent
than the corresponding (S)-methanocarba analogue, 8a ,
at the recombinant human P2Y1 and P2Y2 receptors,
by factors of 138- and 41-fold, respectively (Figure 1 and
Table 1). The (N) and (S) isomers of methanocarba-ATP
were full agonists at both the P2Y1 and the P2Y2
receptors. At the human P2Y1 receptor (N)-methano-
carba-ATP, 3a , displaying an EC50 value of 52 nM, was
29-fold more potent than ATP, 3b. In contrast, the (N)-
methanocarba-ATP analogue was equipotent with ATP
at the human P2Y2 receptor and also exhibited a similar
potency to ATP at the P2Y11 receptor. Note that
concentration-effect curves for 3a at the human P2Y11
receptor were only carried out to 10 µM concentration.
Nonetheless, these curves predicted an EC50 for 3a
essentially equivalent to that of ATP (∼20 µM). As is
the case with ATP, (N)-methanocarba-ATP was not an
agonist at the human P2Y4 receptor. The (N)-methano-
carba-AMP analogue, 4a , was not active at the turkey
P2Y1 receptor.
F igu r e 1. Effects of (N)-methanocarba or (S)-methanocarba
modification of ATP on PLC activity in 1321N1 astrocytoma
cells expressing either hP2Y1 (A) or hP2Y2 (B) receptors.
Concentration-dependent stimulation of inositol phosphate
formation by adenine nucleotides (triphosphates), compounds
3a (2), 3b (O), and 8a (1). Membranes from [3H]inositol-
labeled membranes were incubated for 5 min at 30 °C in the
presence of the indicated concentrations of agonist. The data
shown are typical curves for at least three experiments carried
out in duplicate using different membrane preparations.
activation of vascular smooth muscle P2Y6 receptors by
UDP with a small P2Y2,4 receptor-dependent compo-
nent.
Compound 5a was significantly more potent than
UTP in inducing a dilatory response (pEC50 ) 6.1 ( 0.2
for 5a and 5.5 ( 0.2 for 5b, Figure 4). The maximal
dilatory effects (Rmax, as a percentage of the correspond-
ing precontraction with 1 µM norepinephrine) observed
were 70.6 ( 9.5 and 50.0 ( 14.7% for 5a and 5b,
respectively. In the contractile response (Figure 5),
although a response could be observed for UDP, (N)-
methanocarba-UDP, 6a , had no agonist effect at a
concentration of 300 µM. Compound 5a induced con-
tractions but much more weakly than the ribose equiva-
lent, UTP. Contraction induced at an agonist concen-
tration of 100 µM, expressed as percent of the response
to 60 mM potassium, was 17.2 ( 6.6% (n ) 4) for 5a
and 52.6 ( 18.6% (n ) 5) for 5b. A complete dose-
response curve could not be constructed for 5a , due to
limited amounts of the compound. These results are
consistent with selectivities observed at the human P2Y
receptors, in which the (N)-methanocarba modification
precluded activation of P2Y6 receptors.
(N)-Methanocarba analogues of uridine nucleotides
also were synthesized as potential agonists at the uracil-
activated, P2Y2, or uracil selective, P2Y4 and P2Y6,
receptors. The high potency in the (N)-methanocarba
series was maintained or increased at the human P2Y2
and P2Y4 receptor subtypes, where the (N)-methano-
carba-UTP analogue, 5a , was only slightly less potent
than UTP (Table 1 and Figure 2). Like UTP, the (N)-
methanocarba-UTP analogue was inactive at the human
P2Y6 and P2Y11 receptors. Surprisingly, the (N)-metha-
nocarba-UDP analogue, 6a , was inactive at the UDP-
selective human P2Y6 receptor (Figure 3). (N)-Metha-
nocarba-UMP also was without effect at the P2Y6
receptor.
The vascular effects of (N)-methanocarba-UTP, 5a ,
and (N)-methanocarba-UDP, 6a , were studied in a
model of the rat mesenteric artery, in which uracil
nucleotides elicit both dilatory and contractile responses
(Figures 4-6).18 The dilatory response is dependent on
the activation of endothelial P2Y2,4 receptors by UTP,
and the contractile response is mainly dependent on
Because (N)-methanocarba-UDP, 6a , did not have any
agonistic (contractile) effects on the P2Y6 receptors in
this preparation, we wanted to examine a possible

Methanocarba Modification of Nucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 213
F igu r e 3. Effects of the (N)-methanocarba modification in
uracil nucleotides (diphosphates) on activation of PLC in
1321N1 astrocytoma cells expressing hP2Y6 receptors, showing
concentration-dependent stimulation of inositol phosphate
formation by compound 6b (b) but not by compound 6a (O).
Membranes from [3H]inositol-labeled membranes were incu-
bated for 5 min at 30 °C in the presence of the indicated
concentrations of nucleotide. The data shown are typical curves
for at least three experiments carried out in duplicate using
different membrane preparations.
F igu r e 4. Vasodilatory responses to UTP (2) and 5a (9) in
the rat mesenteric artery. The uracil nucleotides were added
after P2X receptor desensitization with 10 µM R,â-meATP.
Relaxation is expressed as a percentage of an initial contrac-
tion induced by 1 µM norepinephrine. Data are shown as
means ( sem of 5-7 vessel segments.
F igu r e 2. Effects of the (N)-methanocarba modification in
uracil nucleotides (triphosphates) on activation of PLC in
1321N1 astrocytoma cells expressing either hP2Y2 (A) or
hP2Y4 (B) receptors, showing concentration-dependent stimu-
lation of inositol phosphate formation by compounds 5a (O)
and 5b (b). Membranes from [3H]inositol-labeled membranes
were incubated for 5 min at 30 °C in the presence of the
indicated concentrations of agonist. The data shown are typical
curves for at least three experiments carried out in duplicate
using different membrane preparations.
antagonistic effect. (N)-Methanocarba-UDP, 6a , was
demonstrated to lack any antagonistic effects at the
UDP-mediated contraction. In this preparation, UDP at
100 µM produced a 33.9 (7.1% contraction (n ) 4) in
the absence of 6a and a 34.0 (2.4% contraction (n ) 4)
following pretreatment with 6a (Figure 6).
Discu ssion
Our previous work with bisphosphate adenosine
derivatives as P2Y1 receptor antagonists revealed that
high binding affinity at the P2Y1 receptor was retained
in nonnucleotide bisphosphate analogues.20 Indeed,
replacement of the ribose moiety with a constrained
carbocyclic ring, e.g., 2, enhanced P2Y1 receptor affinity.
Here, we have synthesized ring-constrained methano-
carba analogues of ATP and UTP and illustrated that
these derivatives exhibit as high or even higher potency
for activation of certain P2Y receptors than the corre-
sponding natural nucleotide. As was observed with
F igu r e 5. Vasocontractile responses to uracil nucleotides UTP
(2) and 5a (9) in the rat mesenteric artery. The uracil
nucleotides were added after P2X receptor desensitization with
10 µM R,â-meATP. L-NAME (0.1 mM) was present to inhibit
the release of NO. Data are shown as means ( sem of 2-4
vessel segments, except for the highest concentration of 5a ,
where n ) 1.
bisphosphate antagonists of the P2Y1 receptor, the
presence of a fused cyclopropane moiety constraining a
cyclopentane in a pseudorotational (N) conformation
produced an agonist at adenine and uridine nucleotide-

214 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Kim et al.
methanocarba-UDP, 6a , did not exhibit any contractile
effect involving both P2Y2,4 and P2Y6 receptor-mediated
components.18 A likely explanation for the inactivity of
(N)-methanocarba-UDP, 6a , is that it does not possess
any agonist activity at the rat P2Y6 receptor.
In previous experiments,18 we found that the stable
UDP analogue, â-thio-UDP, was more potent in the
contractile response than a stable UTP analogue, al-
though the reverse was true for the natural pyrimidines
(UTP was more potent than UDP). Our interpretation
was that the P2Y6 receptor is a more important vaso-
constrictor than P2Y2,4 and that UTP mediates its
contraction mainly after degradation to UDP.
In the present study, (N)-methanocarba-UTP, 5a , may
be degraded to (N)-methanocarba-UDP, 6a , although it
may be more stable than UTP due to the methanocarba
substitution. The lack of contractile effect of (N)-
methanocarba-UDP, 6a , can explain the low potency for
(N)-methanocarba-UTP, 5a , as a vasoconstrictor, as
compared to its activity as a vasodilator through P2Y2,4
receptors. Differences in the expression of P2Y2 and
P2Y4 receptors at endothelium vs smooth muscle could
also contribute to this phenomenon.
F igu r e 6. Vasocontractile responses to UDP (4), 6a (0), and
UDP in the presence of 10 µM 6a (]) in the rat mesenteric
artery. The uracil nucleotides were added after P2X receptor
desensitization with 10 µM R,â-meATP. L-NAME (0.1 mM)
was present to inhibit the release of NO. Data are shown as
means ( sem of 2-4 vessel segments.
activated P2Y receptors that was much more potent
than the corresponding molecule constrained in the (S)
conformation.
The methanocarba modification, originally applied by
Marquez and co-workers to antiviral nucleosides, fixes
the ring pucker conformation in carbocyclic analogues
by virtue of a bridging cyclopropane ring.26,27,31 The (N)
conformation of the pseudorotational cycle was clearly
favored in binding of the methanocarba-ATP analogue
in the turkey and human P2Y1 receptors and the hP2Y2
receptor. The difference in fit in the binding pocket
observed with the (N) and (S) conformers of ATP was
∼300-fold at the P2Y1 receptor (turkey) and 41-fold at
the hP2Y2 receptor. The potency enhancement in (N)-
methanocarba-ATP vs its ribose equivalent was 350-
fold at the P2Y1 receptor (turkey) and was insignificant
at the hP2Y2 receptor. In another study (in preparation),
we have extended this finding of preference for the (N)
conformers at the P2Y1 receptor to molecules that also
possess other functional groups, e.g., 2-MeS or N6-CH3
substitution, known to increase binding affinity and
selectivity for P2Y receptor agonists.
The observation that the (N)-methanocarba analogue
of UDP was inactive at the UDP-activated hP2Y6
receptor was very surprising given the activity of (N)-
methanocarba analogues at the P2Y1 and P2Y2 recep-
tors and the fact that (N)-methanocarba-UTP was
essentially equipotent with UTP at the hP2Y4 receptor.
Therefore, the binding pocket of the P2Y6 receptor must
markedly differ from that of at least three of its family
members. As such, it will be important to extend
analyses of activities of potential agonists in the (N) vs
(S) conformations to studies of all members of the
human P2Y receptor family.
Thus, it appears that (N)-methanocarba-UDP, 6a , was
neither an agonist nor an antagonist at the rat P2Y6
receptor, and the weak contractile effect of (N)-metha-
nocarba-UTP, 5a , was from action at P2Y2,4 receptors
alone. While UDP and its more stable analogue â-thio-
UDP can be used to define action at P2Y6 receptors,18
it will be useful to have a molecule such as (N)-
methanocarba-UTP, 5a , that exhibits the opposite se-
lectivity, i.e., active at P2Y2,4 receptors but inactive,
itself or through a metabolite, at P2Y6 receptors.
In conclusion, we have established that constraining
the riboselike ring as methanocarba analogues is an
important principle for producing P2Y receptor subtype
selectivity, similar to our previous demonstration of this
phenomenon for P2Y1 receptor antagonists. These find-
ings promise to be useful in both defining the micro-
scopic determinants of the binding sites on these
receptors and in the design of novel pharmacological
probes and/or therapeutic agents. We observed that the
(N) ring conformation was associated with increased
agonist potency at P2Y1 receptors and preserved agonist
potency at P2Y2, P2Y4, and P2Y11 but not P2Y6 recep-
tors. This suggests that new, nucleotide agonists may
be designed on the basis of the (N) conformation that
favors P2Y receptor subtype selectivity.
Exp er im en ta l Section
Ch em ica l Syn th esis. Nucleosides and synthetic reagents
were purchased from Sigma Chemical Co. (St. Louis, MO) and
Aldrich (Milwaukee, WI). 2,6-Dichloropurine was obtained
from Sigma. Compound 12 was synthesized in our laboratory
as described.28,32
The relative activities observed with the methano-
carba analogues of UTP and UDP apparently are
retained at rat uracil nucleotide-activated P2Y recep-
tors. (N)-Methanocarba-UTP, 5a , was more potent than
UTP as a vasodilator in the rat mesenteric artery
containing intact endothelium, which may be due to
greater resistance of (N)-methanocarba-UTP, 5a , to
ectonucleotidase activity or to higher P2Y2,4 receptor
potency. However, (N)-methanocarba-UTP, 5a , was less
potent than UTP for inducing vascular contraction in
endothelium-denuded rat mesenteric artery, and (N)-
1H NMR spectra were obtained with a Varian Gemini-300
spectrometer (300 MHz) using D2O and CDCl3, CD3OD as a
solvent. 31P NMR spectra were recorded at room temperature
by use of a Varian XL-300 spectrometer (121.42 MHz);
orthophosphoric acid (85%) was used as an external standard.
Purity of all compounds submitted to biological testing was
checked using a Hewlett-Packard 1090 HPLC apparatus
equipped with a C18 reverse phase analytical column (250 ×
4.6 mm, as specified in Table 2) in two solvent systems. Peaks
were detected by UV absorption using a diode array detector.
All derivatives showed more than 95% purity in the HPLC
systems.

Methanocarba Modification of Nucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 215
Low-resolution CI-NH3 (chemical ionization) mass spectra
were carried out with Finnigan 4600 mass spectrometer and
high-resolution EI (electron impact) mass spectrometry with
a VG7070F mass spectrometer at 6 kV. High-resolution FAB
(fast atom bombardment) mass spectrometry was performed
with a J EOL SX102 spectrometer using 6 kV Xe atoms
following desorption from a glycerol matrix. Purification of the
nucleotide analogues for biological testing was carried out on
(diethylamino)ethyl (DEAE)-A25 Sephadex columns as de-
scribed above.
0.25 mmol) in trimethyl phosphate was added POCl3 at 0 °C,
and the mixture was stirred for 40 min. Triethylammonium
bicarbonate buffer (1.0 M, pH 8.5, 2 mL) was added to the
reaction mixture followed by the addition of 2 mL of water.
The reaction mixture was lyophilized, and the residue obtained
was purified on a Sephadex column eluting with 0.5 M
ammonium bicarbonate:water (100 mL:100 mL) to obtain
0.015 g (60%) of the monophosphate, 9a . 1H NMR (D2O): δ
8.33 (s, 1H), 8.23 (s, 1H), 4.16-4.08 (m, 3H), 3.57 (d, J ) 10.44
Hz, 1H), 2.40-2.42 (m, 1H), 1.98-1.94 (m, 1H), 1.88-1.82 (m,
1H), 1.46-1.40 (m, 1H). 31P NMR (D2O): δ 0.80.
Syn th esis of Meth a n oca r ba ATP An a logu es. (1′S,2′R,-
3′S,4′R,5′S)-4-(6-Am in o-9H-p u r in -9-yl)-1-[tr ip h osp h or yl-
oxym eth yl]bicyclo[3.1.0]h exa n e-2,3-d iol (3a ). A mixture
of 4a (0.05 g, 0.089 mmol) and 1,1′-carbonyldiimidazole (0.086
g, 0.534 mmol) in 2 mL of anhydrous DMF was stirred at room
temperature for 8 h. The reaction mixture was treated with
5% Et3N in MeOH and stirred for 2 h. Solvent was removed
under reduced pressure, and the residue was dried in high
vacuum. To this residue was added tributylammonium pyro-
phosphate (0.4 g, 0.89 mmol) suspended in 2 mL of anhydrous
DMF. To this mixture was added 0.1 mL of tributylamine and
stirred at room temperature for 3 days. Triethylammonium
bicarbonate (3 mL) was added, and the solvent was removed.
The residue obtained was purified by a Sephadex column using
200 mL of H2O and 200 mL of 0.5 M NH4HCO3 solution, which
furnished 0.010 g of triphosphate 3a (20%). 1H NMR (D2O): δ
8.50 (s, 1H), 4.73 (s, 1H), 4.58 (dd, 1H, J ) 4.7, 11.0 Hz), 4.02
(d, 1H, J ) 6.6 Hz), 3.87 (dd, 1H, J ) 4.7, 11.0 Hz), 1.96-1.91
(m, 1H), 1.58-1.52 (m, 1H), 1.06-0.98 (m, 1H). 31P NMR
(D2O): δ d (-10.27, -10.43), d (-10.67, -10.84), bs -22.61.
Gen er a l P h osp h or yla tion P r oced u r e. Syn th esis of (N)-
Meth a n oca r ba -a d en osin e Der iva tive (1′S,2′R,3′S,4′R,5′S)-
4-(6-Am in o-9H -p u r in -9-yl)-1-[(p h en ylm et h oxy)m et h yl]-
b icyclo[3.1.0]h exa n e-2,3-(O-isop r op ylid in e) (13). To a
solution of 12 (0.2 g, 0.47 mmol) in i-PrOH (3 mL) was added
NH3 (2 M solution in iPrOH, 5 mL, 10 mmol), and the reaction
mixture was heated at 90 °C in a closed tube for 15 h for
complete reaction. The resulting mixture was concentrated
under reduced pressure, and the residue obtained was purified
by flash chromatography using 9/1 CHCl3/MeOH to furnish
1
0.182 g of 13 (95%). H NMR (CDCl3): δ 8.35 (s, 1H), 8.30 (s,
1H), 7.37 (s, 5H), 5.87 (bs, 2H), 5.31 (d, 1H, J ) 6.8 Hz), 5.13
(s, 1H), 4.64 (qAB, 2H, J ) 11.7, 20.5 Hz), 4.51 (d, 1H, J ) 6.8
Hz), 3.97 (d, 1H, J ) 10.7 Hz), 3.35 (d, 1H, J ) 10.7 Hz), 1.72-
1.62 (m, 1H), 1.55 (s, 3H), 1.32-1.26 (m, 1H), 1.23 (s, 3H),
1.00-0.93 (m, 1H).
(1′S ,2′R ,3′S ,4′R ,5′S )-4-(6-Am in o -9H -p u r in -9-y l)-1-
[h yd r oxym eth yl]bicyclo[3.1.0]h exa n e-2,3-(O-isop r op yli-
d in e) (14). To a solution of 13 (0.182 g, 0.45 mmol) in 5%
formic acid in MeOH (15 mL) at room temperature was added
0.15 g of palladium black and stirred at the same temperature
for 24 h. The resulting mixture was filtered through a plug of
Celite and concentrated under reduced pressure. The crude
product obtained was purified by flash chromatography using
9/1 CHCl3/MeOH to furnish 0.12 g of 14 (76%).1H NMR
(CDCl3): δ 8.35 (s, 1H), 7.83 (s, 1H), 5.94 (bs, 2H), 5.61 (d,
1H, J ) 7.2 Hz), 4.79 (s, 1H), 4.66 (d, 1H, J ) 7.2 Hz), 4.31 (d,
1H, J ) 11.5 Hz), 3.26 (d, 1H, J ) 11.5 Hz), 1.9-1.7 (m, 1H),
1.55 (s, 3H), 1.25 (s, 3H), 1.22-1.10 (m, 1H), 1.10-0.94 (m,
1H).
(()-5-(6-Am in o-9H -p u r in -9-yl)-1-[t r ip h osp h or yloxy-
m eth yl]bicyclo[3.1.0]h exa n e-2,3-d iol (8a ). Synthesis of 8a
from 9a (0.007 g, 0.02 mmol), 1,1′-carbonyldiimidazole (0.016
g, 0.1 mmol) and tributylammonium pyrophosphate (0.055 g,
0.12 mmol), was carried out by the same general procedure
as for 3a to furnish 0.003 mg (30%). 1H NMR (D2O): δ 8.33 (s,
1H), 8.23 (s, 1H), 4.42-4.02 (m, 3H), 3.20 (d, J ) 7.41 Hz,
1H), 2.50-2.64 (m, 1H), 2.02-1.75 (m, 1H), 1.58-1.35 (m, 1H),
1.30-1.22 (m, 1H). 31P NMR (D2O): δ -5.70, -10.47, -21.16.
Syn th esis of P r otected (N)-Meth a n oca r ba -AMP De-
r iva tive. (1′S,2′R,3′S,4′R,5′S)-4-(6-Am in o-9H-p u r in -9-yl)-
1-[(d i-ter t-bu tylp h osp h a to)m eth yl]bicyclo[3.1.0]h exa n e-
2,3-(O-isop r op ylid in e) (15). To a stirred solution of di-tert-
butyl diethylphosphoramidite and 14 (0.040 g, 0.138 mmol)
in 4 mL of anhydrous THF, tetrazole (0.029 g, 0.42 mmol) was
added, and the mixture was stirred for 6 h at room tempera-
ture. The reaction mixture was cooled to -78 °C, and a solution
of MCPBA (70% max, 0.04 g) in CH2Cl2 (2 mL) was added.
The resulting mixture was warmed to room temperature, and
the solvent was removed under reduced pressure. The residue
obtained was purified by preparative thin-layer chromatog-
raphy (9/1 CHCl3/MeOH), which furnished 0.055 g (78%) of
the di-tert-phosphate ester 15. 1H NMR (CDCl3): δ 8.37 (s,
1H), 8.19 (s, 1H), 6.46 (bs, 2H), 5.31 (d, 1H, J ) 6.6 Hz), 5.12
(s, 1H), 4.6-4.42 (m, 2H), 3.92-3.76 (m, 1H), 1.84-1.72 (m,
1H), 1.55 (s, 3H), 1.51 (s, 9H), 1.50 (s, 9H), 1.34-1.26 (m, 1H),
1.23 (s, 3H), 1.12-1.00 (m, 1H).
(1′S ,2′R ,3′S ,4′R ,5′S )-4-(6-Am in o -9H -p u r in -9-y l)-1-
[ph osph or yloxym eth yl]bicyclo[3.1.0]h exan e-2,3-diol (4a).
Compound 15 (0.05 g, 0.097 mmol) was heated at 80 °C with
DOWEX-50-80-200 resin (0.200 g) in 1/1 MeOH/H2O (10 mL)
for 1.5 h. The resin was removed by filtration, and the filtrate
was concentrated under reduced pressure. The residue was
purified by a Sephadex column using 200 mL of H2O and 200
mL of 0.5 M NH4HCO3 solution, which furnished 0.05 g of 4a
as triethylammonium salt (69%). 1H NMR (D2O): δ 8.54 (s,
1H), 8.17 (s, 1H), 4.31 (dd, 1H, J ) 4.8, 11.3 Hz), 4.01 (d, 1H,
J ) 6.5 Hz), 3.62 (dd, 1H, J ) 4.3, 11.3 Hz), 1.86-1.80 (m,
1H), 1.52-1.44 (m, 1H), 1.00-0.92 (m, 1H). 31P NMR (D2O):
δ 0.98.
(1′S,2′R,3′S,4′R,5′S)-4-(2,4(H,3H)-Dioxop yr im id in -1-yl)-
1-[(p h en ylm et h oxy)m et h yl]b icyclo[3.1.0]h exa n e-2,3-d i-
ol (17). A solution of â-ethoxyacryloyl cyanate (0.7 mmol) in
anhydrous benzene (2 mL) was added rapidly to a stirred
solution of amine 16 (105 mg, 0.363 mmol) in anhydrous
benzene (1 mL) at room temperature. The reaction mixture
was stirred at the same temperature for 10 min, diluted with
ethyl acetate (30 mL), and treated with saturated aqueous
sodium bicarbonate (10 mL). After the solution was stirred for
20 min at room temperature, the organic layer was separated,
washed with brine (10 mL), dried (Na2SO4), filtered, and
concentrated in vacuo. The residue was purified by flash
chromatography using 1/2 hexane/ethyl acetate to furnish 126
1
mg of an intermediate (89%) as a syrup. H NMR (CDCl3): δ
8.97 (s, 1H), 8.89 (d, 1H, J ) 7.2 Hz), 7.64 (d, 1H, J ) 12.2
Hz), 7.20-7.41 (m, 5H), 5.30 (d, 1H, J ) 12.2 Hz), 5.05 (dd,
1H, J ) 1.0, 7.2 Hz), 4.58 (s, 2H), 4.55 (dd, 1H, J ) 1.0, 7.3
Hz), 4.19 (d, 1H, J ) 7.3 Hz), 3.97 (q, 2H, J ) 7.1 Hz), 3.63 (d,
1H, J ) 10.4 Hz), 3.40 (d, 1H, J ) 10.4 Hz), 1.49 (s, 3H), 1.45
(dddd, 1H, J ) 1.2, 5.5, 8.0 Hz), 1.35 (t, 3H, J ) 7.1 Hz), 1.24
(s, 3H), 1.05 (t, 1H, J ) 5.1 Hz), 0.78 (ddd, 1H, J ) 1.2, 5.5,
9.2 Hz).
The acryloylureidohexane (130 mg, 0.302 mmol) was re-
fluxed in 2 N H2SO4 (8 mL). After it was refluxed for 30 min,
methanol (5 mL) was added to completely dissolve the sub-
strate and form a homogeneous solution. After the solution
was stirred for 10 min at reflux temperature, the methanol
was removed by purging with an N2 stream with heating at
120 °C. The residue (a clear aqueous solution) was refluxed
for an additional 30 min. The resulting mixture was cooled to
room temperature and neutralized with solid sodium bicarbon-
ate until no further gas evolution was detected. The mixture
was lyophilized, and the residue was extracted with a mixed
solvent (chloroform/methanol ) 1:1, 20 mL × 3) and filtered
(()-5-(6-Am in o-9H-pu r in -9-yl)-1-[ph osph or yloxym eth yl]-
bicyclo[3.1.0]h exa n e-2,3-d iol (9a ). To a solution of com-
pound 21 (0.015 g, 0.05 mmol) and Proton Sponge (0.053 g,

216 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1
Kim et al.
through a plug of silica gel to remove all inorganic salts. The
filtrate was concentrated in vacuo and purified by flash
chromatography using 6/1 CHCl3/MeOH to furnish 70 mg of
nium salt: 1H NMR (D2O): δ 8.04 (d, 1H, J ) 6.0 Hz), 5.93 (d,
1H, J ) 6.0 Hz), 4.75 (s, 1H), 4.68 (m, 1H), 4.38 (m, 1H), 3.98
(d, 1H, J ) 4.7 Hz), 3.62 (m, 1H), 3.20 (q, 2H, J ) 7.3 Hz),
1.58 (m, 1H), 1.37 (m, 1H), 1.28 (t, 3H, J ) 7.3 Hz), 0.92 (m,
1H). CIMS (ammonia) m/e: 487 (M+ + 1), 272 (M+ + NH4).
As a free acid: 1H NMR (D2O): δ 8.04 (d, 1H, J ) 8.0 Hz),
5.92 (d, 1H, J ) 8.0 Hz), 4.75 (s, 1H), 4.66 (d, 1H, J ) 6.8 Hz),
4.42 (m, 1H), 3.98 (d, 1H, J ) 6.5 Hz), 3.62 (m, 1H), 1.60 (m,
1H), 1.38 (m, 1H), 0.92 (m, 1H). High-resolution MS (negative-
ion FAB) calcd for C11H14N2O8P [M2- + H+]-, 333.0488; found,
333.0506. HPLC: 3.235 min (99%) in solvent system A, 3.466
min (98%) in system C.
1
17 (67.3%) as a syrup. H NMR (CD3OD): δ 8.18 (d, 1H, J )
8.1 Hz), 7.28-7.45 (m, 5H), 5.22 (d, 1H, J ) 8.1 Hz), 4.77 (s,
1H), 4.60 (d, 1H, J ) 11.1 Hz), 4.59 (dd, 1H, J ) 1.5, 6.9 Hz),
4.53 (d, 1H, J ) 11.1 Hz), 4.22 (d, 1H, J ) 10.2 Hz), 3.73 (dd,
1H, J ) 1.4, 6.9 Hz), 3.21 (d, 1H, J ) 10.0 Hz), 1.49 (dd, 1H,
J ) 4.0, 5.2 Hz), 1.37 (ddd, 1H, J ) 1.6, 4.0, 8.8 Hz), 0.73 (ddd,
1H, J ) 1.8, 5.5, 8.8 Hz). MS (m/e): 344 (M+). CIMS (ammonia)
m/e: 345 (M+ + 1).
(1′S,2′R,3′S,4′R,5′S)-4-(2,4(H,3H)-Dioxop yr im id in -1-yl)-
1-(h ydr oxym eth yl)bicyclo[3.1.0]h exan e-2,3-diol (18). Solid
palladium black (60 mg) was added to a stirred solution of
(1′S,2′R,3′S,4′R,5′S)-4-(2,4(H,3H)-dioxopyrimidin-1-yl)-1-
[(phenylmethoxy)methyl]bicyclo[3.1.0]hexane-2,3-diol (17, 23
mg, 0.0668 mmol) in methanol (4 mL), and 95% formic acid
(1.3 mL) was added. The mixture was stirred at 50 °C for 4 h,
additional formic acid (1.2 mL) was added, and stirring was
continued at 60 °C for 3 h. The resulting mixture was cooled
to room temperature, filtered through a plug of Celite, and
washed with methanol. The filtrate was evaporated, and the
product was purified by silica gel column chromatography
(chloroform/methanol ) 2:1) to give 18 (12.6 mg, 74%) as a
(1′S,2′R,3′S,4′R,5′S)-4-(2,4(H,3H)-Dioxop yr im id in -1-yl)-
1-(d ip h osp h or yloxym et h yl)b icyclo[3.1.0]h exa n e-2,3-d i-
ol, Am m on iu m Sa lt (6a ). Solid 1,1′-carbonyldiimidazole (6.9
mg, 42.5 µmol) was added to a stirred solution of 7a (3.0 mg,
6.89 µmol) in anhydrous DMF (0.5 mL) at room temperature.
After the solution was stirred at room tempreature for 6 h,
the reaction mixture was treated with 5% triethylamine
solution (2.5 mL, in 1/1 water/methanol) and stirred for 2 h.
All volatile materials were removed by purging with nitrogen
stream, and the residue dried in vacuo at room temperature
and dissolved in anhydrous DMF (0.5 mL). To this solution
was successively added tributylamine (30 µL, 126 µmol) and
a solution of bis(tributylammonium) salt of phosphoric acid
(0.1 mL, in DMF). This salt was prepared by mixing tributy-
lamine (128 mg, 0.69 mmol) and phosphoric acid (33.8 mg,
0.345 mmol) in DMF (1 mL). After this solution was stirred
at room temperature for 2 days, 0.2 M triethylammonium
bicarbonate (0.2 mL) was added, and the resulting mixture
was lyophilized. The residue was purified by ion-exchange
column chromatography using a Sephadex-DEAE A-25 resin
with a linear gradient (0.01-1.0 M) of 1.0 M ammonium
bicarbonate as the mobile phase to give 1.3 mg of 6a (40.5%)
1
syrup. H NMR (CD3OD): δ 8.04 (d, 1H, J ) 8.0 Hz), 5.66 (d,
1H, J ) 8.0 Hz), 4.72 (s, 1H), 4.58 (dd, 1H, J ) 1.4, 6.9 Hz),
4.20 (d, 1H, J ) 11.8 Hz), 3.81 (d, 1H, J ) 6.9 Hz), 3.24 (d,
1H, J ) 11.8 Hz), 1.30-1.41 (m, 2H), 0.68 (ddd, 1H, J ) 1.7,
4.4, 8.0 Hz). CIMS (ammonia) m/e: 255 (M+ + 1), 272 (M+
NH4).
+
(1′S ,2′R ,3′S ,4′R ,5′S )-4-(2,4(H ,3H )-Dioxop yr im id in -1-
yl)-1-(h yd r oxym et h yl)b icyclo[3.1.0]h exa n e-2,3-(O-iso-
p r op ylid en e) (19). A mixture of triol 18 (20 mg, 0.0787 mmol)
and p-toluene sulfonic acid monohydrate (50 mg, 0.263 mmol)
and acetone (7 mL) was stirred at ambient temperature for 4
h. Triethylamine (0.5 mL) was added to protect the acetonide
group. All volatile materials were removed in vacuo, and the
residue was purified by preparative thin-layer chromatography
using 4/1 chloroform/methanol to give 20 mg of 19 as an oil
1
as a white solid. H NMR (D2O): δ 8.03 (d, 1H, J ) 8.0 Hz),
5.96 (d, 1H, J ) 8.0 Hz), 4.52 (m, 1H), 4.69-4.91(m, 2H), 3.99
(d, 1H, J ) 6.8 Hz), 3.77 (m, 1H), 1.64 (m, 1H), 1.39 (m, 1H),
0.95 (t, 1H, J ) 7.2 Hz). 31P NMR (D2O): δ -10.81, 9.98. MS
(negative-ion FAB): 414 [M2- + H+]-. High-resolution MS
(negative-ion FAB) calcd for C11H15N2O11P2 [M2- + H+]-,
413.0151; found, 413.0143. HPLC: 2.130 min (98%) in solvent
system A, 4.531 (97%) min in system C.
1
(86%). H NMR (CDCl3): δ 7.39 (d, 1H, J ) 8.0 Hz), 5.74 (d,
1H, J ) 8.0 Hz), 5.32 (dd, 1H, J ) 1.0, 7.3 Hz), 4.75 (d, 1H, J
) 7.3 Hz), 4.31 (s, 1H), 4.18 (d, 1H, J ) 11.4 Hz), 3.31 (d, 1H,
J ) 11.4 Hz), 1.53 (m, 1H), 1.51 (s, 3H), 1.27 (s, 3H), 1.04 (dd,
1H, J ) 4.7, 5.8 Hz), 0.91 (ddd, 1H, J ) 1.2, 5.8, 9.2 Hz). CIMS
(ammonia) m/e: 295 (M+ + 1), 312 (M+ + NH4).
(1′S,2′R,3′S,4′R,5′S)-4-(2,4(H,3H)-Dioxop yr im id in -1-yl)-
1-(t r ip h osp h or ylp h osp h or yloxym et h yl)b icyclo[3.1.0]-
h exa n e-2,3-d iol, Tr ieth yla m in e Sa lt (5a ). Solid carbonyl
diimidazole (26 mg, 0.16 mmol) was added to a stirred solution
of 7a and free acid (9.0 mg, 26.9 µmol) in anhydrous DMF (0.5
mL) at room temperature. After the solution was stirred at
room temperature for 6 h, the reaction mixture was treated
with 5% triethylamine solution (2.5 mL, in water/methanol )
1:1) and stirred for 2 h. All volatile materials were removed
by purging with nitrogen, and the residue was dried in vacuo
at room temperature. The product was purified by ion-
exchange column chromatography using a Sephadex-DEAE
A-25 resin with a linear gradient (0.01-0.5 M) of 0.5 M
triethylammonium bicarbonate as the mobile phase, to give
15 mg of the phosphorimidazolidate monotriethylamine salt,
(1′S,2′R,3′S,4′R,5′S)-4-(2,4(H,3H)-Dioxop yr im id in -1-yl)-
1-[(d i-ter t-bu tylp h osp h a to)m eth yl]bicyclo[3.1.0]h exa n e-
2,3-(O-isop r op ylid en e) (20). Neat di-tert-butyl N,N-dieth-
ylphosphoramidite (57 µL, 0.205 mmol) was added by syringe
to a stirred solution of 19 (20 mg, 0.0680 mmol) in anhydrous
THF (1 mL) at room temperature, followed by an addition of
solid tetrazole (43 mg, 0.614 mmol). After the solution was
stirred at room temperature for 20 min, the reaction mixture
was cooled to -78 °C, and a solution of MCPBA (57 ∼ 85%, 58
mg) in methylene chloride (2 mL) was added rapidly. The
reaction mixture was warmed to 0 °C and stirred for 5 min.
Triethylamine (0.5 mL, 3.59 mmol) was added to maintain a
basic condition in order to avoid cleavage of tert-butyl groups.
Purification was accomplished using preparative thin-layer
chromatography 6/1 chloroform/methanol to give 26 mg of 20
1
as a white solid. H NMR (D2O): δ 7.96 (bs, 1H), 7.81 (d, 1H,
J ) 7.9 Hz), 7.33 (bs, 1H), 7.15 (bs, 1H), 5.85 (d, 1H, J ) 7.9
Hz), 4.70 (s, 1H), 4.51 (d, 1H, J ) 6.3 Hz), 4.38 (dd, 1H, J )
5.7, 11.3 Hz), 3.94 (d, 1H, J ) 6.7 Hz), 3.61 (dd, 1H, J ) 5.3,
11.3 Hz), 3.02-3.38 (m, 6H), 1.47 (m, 1H), 1.13-1.44 (m, 10H),
0.85 (m, 1H).
1
(79%) as an oil. H NMR (CDCl3): δ 8.04 (d, 1H, J ) 8.0 Hz),
5.77 (d, 1H, J ) 8.0 Hz), 5.15 (d, 1H, J ) 7.3 Hz), 4.95 (s, 1H),
4.46-4.58 (m, 2H), 3.66 (dd, 1H, J ) 6.7, 11.4 Hz), 1.54 (m,
1H), 1.51 (s, 3H), 1.49 (s, 9H), 1.25 (s, 3H), 0.95 (m, 1H). CIMS
(ammonia) m/e: 487 (M+ + 1).
The phosphorimdazolidate was dissolved in anhydrous DMF
(0.5 mL). To this solution was added solid tributylammonium
pyrophosphate (from Sigma, 36 mg, 0.0790 mmol) and tribut-
ylamine (30 µg, 161 µmol), successively. After the solution was
stirred at room temperature for 3 days, 0.2 M trietylammo-
nium bicarbonate (0.2 mL) was added, and the resulting
mixture was lyophilized. The residue was purified by ion-
exchange column chromatography using a Sephadex-DEAE
A-25 resin with a linear gradient (0.01-1.0 M) of 1.0 M
ammonium bicarbonate as the mobile phase to give 5.7 mg of
(1′S,2′R,3′S,4′R,5′S)-4-(2,4(H,3H)-Dioxop yr im id in -1-yl)-
1-(p h osp h or yloxym et h yl)b icyclo[3.1.0]h exa n e-2,3-d iol,
Tr ieth yla m m on iu m Sa lt (7a ). A mixture of 20 (13 mg,
0.0267 mmol) and DOWEX 50 × 8-200 ion-exchange resin (300
mg) and methanol (4 mL)/water (2 mL) was stirred at 70-80
°C for 12 h. The resulting mixture was filtered and washed
with water (∼5 mL). The filtrate was lyophilized with 0.1 M
triethylammonium bicarbonate to give 10 mg of a triethylam-
monium salt, 7a (86%), as a white solid. As a triethylammo-
1
5a (37.7%) as a white solid. H NMR (D2O): δ 8.01 (d, 1H, J

Methanocarba Modification of Nucleotides
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 1 217
) 8.0 Hz), 5.97 (d, 1H, J ) 8.0 Hz), 4.75 (s, 1H), 4.72 (d, 1H,
J ) 6.8 Hz), 4.54 (m, 1H), 3.99 (d, 1H, J ) 6.7 Hz), 3.80 (m,
methanocarba-UDP, 6a , were not examined as there
are no endothelial P2Y6 receptors in the rat mesenteric
artery.18
1H), 1.20-1.69 (m, 2H), 0.95 (m, 1H). 31P NMR (D2O):
δ
-22.482, d (-10.914, -10.759), -8.979. MS (negative-ion
FAB): 515 (MHNa-), 537 (MHNa2)-. High-resolution MS
(negative-ion FAB) calcd for C11H16N2O14P3 [M2- + H+]-,
492.9814; found, 492.9832. HPLC: 1.737 min (99%) in solvent
system A, 6.764 min (96%) in system B.
Vasocontractile responses of UDP, UTP, (N)-metha-
nocarba-UDP, and (N)-methanocarba-UTP were studied
in the presence of 0.1 mM L-NAME to inhibit the effect
of endothelial P2 receptor-stimulated NO release. The
antagonistic effect of (N)-methanocarba-UTP on UDP
contraction was also assessed.
P h a r m a cologica l An a lyses
P LC Activity. Agonist-promoted stimulation of inos-
itol phosphate formation was measured at human P2Y1,
P2Y2, P2Y4, P2Y6, and P2Y11, receptors stably expressed
in 1321N1 astrocytoma cells as previously described.32,33
Briefly, cells plated in 24 well dishes were labeled with
1 µCi of [3H]myo-inositol/well (20 Ci/mmol; American
Radiolabeled Chemicals, Inc., St. Louis, MO) in inositol-
free Dulbecco’s modified Eagle’s medium (DMEM). [3H]-
Inositol phosphate formation was quantitated the fol-
lowing day during a 10 min incubation at 37 °C in the
presence of 10 mM LiCl. The EC50 values were averaged
from 3 to 8 independently determined concentration-
effect curves for each compound.
The negative logarithm of the drug concentration that
elicited 50% dilation (pEC50) was determined by fitting
the data to the Hill equation, using GraphPad Prism
version 3.02. Rmax refers to maximum relaxation calcu-
lated as a percentage of the corresponding precontrac-
tion with 1 µM norepinephrine. In the contraction
experiments, a plateau phase of the maximum contrac-
tile response was not reached within the agonist con-
centrations interval. Experiments were repeated on 2-7
vessel segments for each concentration of the sub-
stances. Statistical significance was accepted when P
< 0.05, using Student’s t-test for paired data. Values
are presented in the graphs as mean ( SEM.
P2Y1-receptor-promoted activation of PLC was stud-
ied in turkey erythrocyte membranes as we have
described.8,34 Briefly, erythrocytes were incubated in
inositol-free medium (DMEM; Gibco, Gaithersburg, MD)
with 0.5 mCi of 2-[3H]myo-inositol (20 Ci/mmol; Ameri-
can Radiolabeled Chemicals, Inc., St. Louis, MO) for 18-
24 h in a humidified atmosphere of 95% air/5% CO2 at
37 °C. Membranes were prepared and PLC activity was
measured in 25 µL of [3H]inositol-labeled membranes
(approximately 175 µg of protein, 200-500 000 cpm/
assay) in a medium containing 424 µM CaCl2, 0.91 mM
MgSO4, 2 mM EGTA, 115 mM KCl, 5 mM KH2PO4, and
10 mM Hepes, pH 7.0. Assays (200 µL final volume)
contained 1 µM GTPγS and the indicated concentrations
of nucleotide analogues. Membranes were incubated at
30 °C for 5 min, and total [3H]inositol phosphates were
quantified by anion-exchange chromatography as previ-
ously described.8,34
Agonist potencies were calculated using a four pa-
rameter logistic equation and the GraphPad software
package (GraphPad, San Diego, CA). EC50 values
(mean ( standard error (SE)) represent the concentra-
tion at which 50% of the maximal effect is achieved.
Relative efficacies (%) were determined by comparison
with the effect produced by a maximal effective concen-
tration of full agonist in the same experiment. All
concentration-effect curves were repeated in at least
three separate experiments carried out with different
membrane preparations using duplicate or triplicate
assays.
Va cu la r E ffect s a t t h e R a t Mesen t er ic Ar t er y.
UTP, UDP, NG-nitro-L-arginine methyl ester hydrochlo-
ride (L-NAME), R,â-methylene-ATP (R,â-meATP), and
norepinephrine were purchased from Sigma. All sub-
stances were dissolved in saline 0.9%.
Vasodilatory responses to cumulatively added UTP
and (N)-methanocarba-UTP, 5a , were examined in
segments of the rat mesenteric artery using a myograph
by methods previously described.18 Vessel segments
were precontracted with 0.1 µM norepinephrine. The
dilatory responses to both UTP and 5a were studied on
the same vessel segment. The dilatory effects of (N)-
Ack n ow led gm en t. This work was supported by
USPHS grants GM38213 and HL54889. R.G.R. thanks
Gilead Sciences (Foster City, CA) for financial support.
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