Methanocarba analogues of purine nucleosides as potent and selective adenosine receptor agonists

Article abstract of DOI:10.1021/jm9905965

Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory properties. We report that a carbocyclic modification of the ribose moiety incorporating ring constraints is a general approach for the design of A₁ and

Full text of DOI:10.1021/jm9905965

2196
J . Med. Chem. 2000, 43, 2196-2203
Meth a n oca r ba An a logu es of P u r in e Nu cleosid es a s P oten t a n d Selective
Ad en osin e Recep tor Agon ists
Kenneth A. J acobson,*,† Xiao-duo J i,† An-Hu Li,† Neli Melman,† Maqbool A. Siddiqui,‡ Kye-J ung Shin,‡
Victor E. Marquez,‡ and R. Gnana Ravi†
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and
Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0810, and Laboratory of Medicinal Chemistry,
National Cancer Institute, Bethesda, Maryland 20892-0810
Received December 3, 1999
Adenosine receptor agonists have cardioprotective, cerebroprotective, and antiinflammatory
properties. We report that a carbocyclic modification of the ribose moiety incorporating ring
constraints is a general approach for the design of A1 and A3 receptor agonists having favorable
pharmacodynamic properties. While simple carbocyclic substitution of adenosine agonists
greatly diminishes potency, methanocarba-adenosine analogues have now defined the role of
sugar puckering in stabilizing the active adenosine receptor-bound conformation and thereby
have allowed identification of a favored isomer. In such analogues a fused cyclopropane moiety
constrains the pseudosugar ring of the nucleoside to either a Northern (N) or Southern (S)
conformation, as defined in the pseudorotational cycle. In binding assays at A1, A2A, and A3
receptors, (N)-methanocarba-adenosine was of higher affinity than the (S)-analogue, particularly
at the human A3 receptor (N/S affinity ratio of 150). (N)-Methanocarba analogues of various
N6-substituted adenosine derivatives, including cyclopentyl and 3-iodobenzyl, in which the
parent compounds are potent agonists at either A1 or A3 receptors, respectively, were
synthesized. The N6-cyclopentyl derivatives were A1 receptor-selective and maintained high
efficacy at recombinant human but not rat brain A1 receptors, as indicated by stimulation of
binding of [35S]GTP-γ-S. The (N)-methanocarba-N6-(3-iodobenzyl)adenosine and its 2-chloro
derivative had Ki values of 4.1 and 2.2 nM at A3 receptors, respectively, and were highly selective
partial agonists. Partial agonism combined with high functional potency at A3 receptors (EC50
< 1 nM) may produce tissue selectivity. In conclusion, as for P2Y1 receptors, at least three
adenosine receptors favor the ribose (N)-conformation.
In work designed to develop potent and selective
agents, the structure-activity relationships of adenos-
ine derivatives as ligands (principally agonists) at the
four subtypes of adenosine receptors (A1, A2A, A2B, and
A3) have been explored extensively. Adenosine receptor
agonists1,2 are being studied for their potential use as
antiarrhythmic,3 antinociceptive,4 and antilipolytic5,6
agents (A1 subtype); as cerebroprotective7 and cardio-
protective8 agents (A1 and A3 subtypes); and as hypo-
tensive9 and antipsychotic10 agents (A2A subtype).
In general, for adenosine agonists, numerous modi-
fications of the N6-position with cycloalkyl and other
hydrophobic moieties provide selectivity for A1 receptors,
although the affinities of these N6-substituted adenosine
derivatives (e.g. N6-cyclopentyl) at A3 receptors are often
intermediate between their respective A1 and A2A af-
finities.1 Structurally, few ribose modifications, other
than amide substitution at the 5′-position, are tolerated
in adenosine agonists. An intact furanose moiety is
present in most of the potent adenosine agonists previ-
ously developed. Adenine riboside derivatives are sub-
ject to scission of the glycosidic bond and other pathways
of metabolic degradation in vivo.5 Alternately, carbocy-
clic modifications of the ribose moiety have been intro-
duced in order to design nonglycosylic adenosine ago-
nists and thereby potentially increase selectivity and
biological stability. In previous studies of adenosine
analogues it was found that many adenosine derivatives
having carbocyclic modifications of the ribose ring
(compounds 1-4, 5b) bind to adenosine receptors, but
usually only with reduced affinity.11-16 The simplest of
such 9-cyclopentyladenosine derivatives, aristeromycin,
5b, was reported to have hypotensive properties related
to A2A receptor activation.11 This led to the design of
(1R,2S,3R,5R)-3-[6-amino-2-(phenylamino)-9H-purin-9-
yl]-5-(hydroxymethyl)-1,2-cyclopentanediol (CGS 23321),
1, which also relaxed porcine coronary smooth muscle
through activation of adenosine A2 receptors.12 [1S-
[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methyl-
propyl]amino]-3H-imidazo[4,5-b]pyridin-3-yl]cyclopen-
tanecarboxamide (AMP 579), 2, activated A1 and A2
adenosine receptors and, in the heart, induced coronary
dilation without causing endocardial steal and de-
creased postischemic myocardial infarct size.13 (()-9-
[2R,3R-Dihydroxy-4â-(N-methylcarbamoyl)cyclopent-1â-
yl)]-N6-(3-iodobenzyl)adenine (MRS 582), 3, is 17 700-
fold less potent in binding to rat A3 adenosine receptors
than the corresponding 4′-oxygen analogue, N6-(3-iodo-
benzyl)-5′-N-methylcarboxamidoadenosine (IB-MECA).14
The carbocyclic derivative 9-[(1R,3R)-trans-cyclopentan-
3-ol]adenine hydrochloride (MDL 201,449), 4, is a weak
*Address correspondence to Dr. Kenneth A. J acobson, Chief, Mo-
lecular Recognition Section, Bldg. 8A, Rm. B1A-19, NIH, NIDDK, LBC,
Bethesda, MD 20892-0810. Tel: (301) 496-9024. Fax: (301) 480-8422.
E-mail: kajacobs@helix.nih.gov.
† National Institutes of Health.
‡ National Cancer Institute.
10.1021/jm9905965 This article not subject to U.S. Copyright. Published 2000 by the American Chemical Society
Published on Web 05/16/2000

Methanocarba Analogues of Purine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2197
Ta ble 1. Affinities of Adenosine (a), Simple Carbocyclic (b), and Methanocarba-adenosine ((N)-conformation, c, and (S)-conformation,
d) Derivatives in Radioligand Binding Assays at Rat A1, Rat A2A, Human A2B, and Human A3 Receptors, Unless Notedg
Ki (nM) or % displacement
a
b
b
c
compound
R2
rA1
rA2A
hA2B
hA3
A1/A3
5a
5b
5c
H
H
H
estd 10d
6260 ( 730
1680 ( 80
15% at 100 µM
1.50 ( 0 51
5.06 ( 0.51
20.0 ( 8.5
25900 ( 1600
69.2 ( 9.8
estd 30d
2150 ( 950
22500 ( 100 (h)e,f
>100000 (h)e,f
857 ( 163
<10% at 100 µM
47300 ( 10600
35 ( 2% at 50 µMf
20 ( 4% at 50 µMf
21200 ( 4300
139000 ( 19000
3570 ( 100
estd 1000 (r)d,e
20000 ( 7900 (r)e
404 ( 70f
100
0.31
4.2
5d (racemic)
H
62500 ( 2900f
274 ( 20240 (r)e
170 ( 51
>1
6a
6c
7a
7b
7c
8a
8c
9a
9c
CP
CP
IB
IB
IB
CP
CP
IB
IB
0.0055
0.030
2.1
6800 ( 1800
17.5 ( 0.5
9.5 ( 1.4 (r)e
1960 ( 370
4.13 ( 1.76
237 (r)e
<10% at 100 µM
601 ( 236
nd
13
17
0.0056
0.019
13
12100 ( 1300
20400 ( 1200
27 ( 7% at 100 µM
5010 ( 1400
1.33 ( 0.19
8.76 ( 0.81
18.5 ( 4.7
605 ( 154
3390 ( 520
38.5 ( 2.0
466 ( 58
1.41 ( 0.17 (r)e
2.24 ( 1.45
141 ( 22
732 ( 207
41000 ( 7000
63
Displacement of specific [3H]R-PIA binding to A1 receptors in rat brain membranes, expressed as Ki ( SEM (n ) 3-5), unless noted.
Displacement of specific [3H]CGS 21680 binding to A2A receptors in rat striatal membranes, expressed as Ki ( SEM (n ) 3-6), and at
a
b
A2B receptors expressed in HEK-293 cells vs [3H]ZM241,385, unless noted. c Displacement of specific [125I]AB-MECA binding at human
d
A3 receptors expressed in HEK cells, in membranes, expressed as Ki ( SEM (n ) 3-4), unless noted. Reference 41. e Ki values were
determined in radioligand binding assays at recombinant human A2A receptors expressed in HEK-293 cells vs [3H]ZM241385 or [125I]AB-
g
MECA binding at rat A3 receptors expressed in CHO cells. f Measured in the absence of ADA. nd, not determined; 6c, MRS 1781; 7c,
g
MRS 1743; 8c, MRS 1761; 9c, MRS 1760. CP ) cyclopentyl; IB ) 3-iodobenzyl.
adenosine agonist that inhibits synthesis of TNFR
(tumor necrosis factor) in murine bone-marrow-derived
macrophages and has therapeutic potential for treat-
ment of inflammatory diseases.15,16
as a complex carbocyclic modification which maintains
a fixed conformation. In methanocarba analogues a
fused cyclopropane ring constrains the accompanying
cyclopentane moiety to mimic the conformation of a rigid
furanose ring held in either a Northern (N) or Southern
(S) conformation. These analogues adopt envelope, as
opposed to twist, conformations. This has allowed us to
focus on the impact of pseudorotation of the ribose
moiety on both receptor affinity and efficacy of adenos-
ine agonists, since often only one isomeric form of
methanocarba adenosine retains high affinity and se-
lectivity at a given binding site.
Resu lts
Ch em ica l Syn th esis. The optically active methano-
carbocyclic adenosine analogues (Table 1), in which a
fused cyclopropane ring constrains the cyclopentane ring
into a rigid (N)-envelope conformation, were synthesized
by the general approach (Scheme 1) of Marquez and co-
workers.18 The (N)-methanocarba analogues of various
N6-substituted adenosine derivatives, including cyclo-
pentyl and 3-iodobenzyl, were prepared. The parent
adenosine analogues are potent agonists at either A1
(cyclopentyl) or A3 (3-iodobenzyl) receptors.1 The N6-(3-
iodobenzyl) substitution is present in the selective A3
agonist IB-MECA and also promotes A3 receptor affinity
in the 5′-hydroxy series.25 2,6-Dichloropurine, 10, was
condensed with the cyclopentyl derivative, 11,18 using
the Mitsunobu reaction, followed by substitution at the
6-position and deprotection to give 8c or 9c. These
intermediates allowed the incorporation, in the adenos-
In the present study we have explored the effects at
adenosine receptors of the “methanocarba” nucleoside
modification introduced by Marquez and co-workers17-24

2198 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
J acobson et al.
Sch em e 1. Synthesis of N6-Substituted
(N)-Methanocarba-adenosine Derivatives Optimized for
Interaction with A1 (CP ) cyclopentyl) or A3 (IB )
3-iodobenzyl) Receptorsa
F igu r e 1. Representative competition curves for inhibition
of binding of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-ade-
nosine-5′-N-methyluronamide) by (N)-methanocarba-adenos-
ine, 5c (circles), and (S)-methanocarba-adenosine, 5d (racemic)
(diamonds), at human A3 receptors expressed in CHO cells at
25 °C, in the presence (solid symbols) or absence (open
symbols) of 2 IU/mL ADA. Structures show ring pucker
envelope conformations.
receptor, binding was carried out using [3H]ZM 241,385
(4-(2-[7-amino-2-{furyl}{1,2,4}triazolo{2,3-a}{1,3,5}-
triazin-5-ylaminoethyl)phenol);27 however, the affinity
was too weak to establish selectivity for a specific
isomer. Affinity of (N)-methanocarba-adenosine, 5c, vs
adenosine, 5a , was particularly enhanced at the A3
receptor subtype (Figure 1), for which the ratio of
affinities of (N)- to (S)-analogues was 150-fold. Although
a relatively poor substrate for adenosine deaminase
(ADA),22 the binding curve for 5c was shifted to the right
in the presence of ADA; therefore, Table 1 reports the
affinity values for 5c and 5d obtained in the absence of
ADA. The (S)-conformer, 5d , as the 2′-deoxy analogue,22
behaved as an even worse substrate of ADA (100-fold
less), which would explain why the curves in the
presence and absence of ADA for 5d are virtually the
same (Figure 1). Aristeromycin, 5b, bound weakly to
adenosine receptors, with slight selectivity for the A2A
subtype. Compound 5c was more potent than aristero-
mycin, 5b, in binding to A1 (4-fold) and A3 (4500-fold)
adenosine receptors.
Compounds 6c and 8c are patterned after A1 receptor-
selective agonists, while compounds 7c and 9c are
patterned after A3 receptor-selective agonists. Com-
pounds 6 and 7 are unsubstituted at the 2-position,
while compounds 8 and 9 contain the potency-enhancing
2-chloro substituent.26 The N6-cyclopentyl (N)-metha-
nocarba derivative, 6c, based on CPA (N6-cyclopentyl-
adenosine), 6a , maintained high selectivity for A1
receptors, although the affinity of 6c at rat A1 receptors
was 3-fold less than for 6a .
a
Reagents: (a) DEAD, Ph3P; (b) MeOH, rt; (c) BCl3; (d) H2/Pd;
(e) 3-iodobenzyl bromide, 50 °C, DMF, 2 days; (f) NH4OH, MeOH,
80 °C, 3 days.
ine series of the (N)-configuration, of the 2-chloro
substitution, of interest for its structure-dependent
affinity-enhancing effects at both A1 and A3 receptors.1,26
The 2-chloro substitution of compound 8c could also be
removed by catalytic reduction to give 6c. An N6-(3-
iodobenzyl) group was introduced in either aristeromy-
cin, 5b, or (N)-methanocarba-adenosine, 5c, by the
Dimroth rearrangement,25 to give 7b (Scheme 2) and
7c (Scheme 1). A sample of the antipodal (S)-methano-
carba-adenosine, 5d , was synthesized in racemic form
by Marquez and co-workers by an intramolecular car-
bene addition reaction.23
Biologica l Activity. A pair of methanocarba ana-
logues of adenosine, 5c18 and 5d ,23 corresponding to (N)-
and (S)-conformations of ribose, were tested in binding
assays (Table 1) at four subtypes of adenosine recep-
tors.25,27 The more synthetically challenging (S)-isomer
(5d ) was available only as the racemate and therefore
was tested as such.23 At rat A1, rat A2A, and human A3
subtypes, the (N)-analogue proved to be of much higher
affinity than the (S)-analogue. At the human A2B
Sch em e 2. Synthesis of an N6-Substituted Aristeromycin Derivative by the Dimroth Rearrangementa
a
Reagents: (a) 3-iodobenzyl bromide, 80 °C, DMF, 3 days; (b) NH4OH, MeOH, 80 °C, 1 h.

Methanocarba Analogues of Purine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2199
Ta ble 2. Effect of Ligands To Stimulate [35S]GTP-γ-S Binding to Membranes of Cells Expressing the Cloned hA1AR or hA3AR or in
Rat Cerebral Cortical Membranes Containing the A1AR
cloned hA1AR
EC50 (nM)a
% maximal
rA1AR
% maximal
cloned hA1AR
EC50 (nM)a
% maximal
ligand
stimulationc
EC50 (nM)a
stimulationc
stimulationc
NECA
6a
6c
7a
7b
7c
8c
9c
nd
nd
155 ( 15
7980 ( 60
>10000
100
100
4.15 ( 0.90
21.5 ( 2.3
43.1 ( 10.4
>10000
218 ( 18
31.2 ( 3.3
142 ( 24
100
102 ( 1
20.3 ( 13.1
100 ( 17
340 ( 98
ndb
940 ( 114
145 ( 35
684 ( 75
100
75 ( 6
95 ( 4
14 ( 2% at 10 µM
91 ( 1
5.16 ( 0.71
>10000
100
5 ( 2% at 10 µM
86 ( 2
14 ( 2% at 10 µM
45.3 ( 6.8
15 ( 5% at 10 µM
22.0 ( 2.8
55 ( 5
96 ( 2
48 ( 3
0.70 ( 0.16
>10000
0.67 ( 0.19
97 ( 1
91 ( 1
a
EC50 for stimulation of basal [35S]GTP-γ-S (0.1 nM) binding by agonists in membranes from transfected CHO cells ((SEM), n )
5-10. Basal and 100% maximal levels of binding correspond to (fmol/mg of protein) 490 ( 60 and 1520 ( 80 (hA1), 340 ( 20 and 1010
( 70 (rA1), and 240 ( 14 and 580 ( 49 (hA3). nd, not determined.
b
In one series it was possible to compare ribose,
cyclopentyl, and (N)-methanocarba derivatives having
the same N6-substitution. The N6-(3-iodobenzyl) (N)-
methanocarba derivative, 7c, with a Ki value of 4.1 nM,
was 2.3-fold more potent at A3 receptors than the ribose-
containing parent, 7a . Thus, the selectivity of 7c for
human A3 versus rat A1 receptors was 17-fold. The
aristeromycin analogue, 7b, was relatively weak in
binding to adenosine receptors.
Among 2-chloro-substituted derivatives, the (N)-
methanocarba analogue, 8c, was less potent at A1 and
A2A receptors than its parent 2-chloro-N6-cyclopentyl-
adenosine, 8a , and roughly equipotent at A3 receptors.
Thus, 8c was 53-fold selective in binding to rat A1 vs
human A3 receptors. The (N)-methanocarba analogue,
9c, of 2-chloro-N6-(3-iodobenzyl)adenosine, 9a , had Ki
values of 141, 732, and 2.2 nM at A1, A2A, and A3
receptors, respectively. Thus, the 2-chloro group slightly
enhanced affinity at A3 receptors, while reducing affinity
at A1 receptors.
Replacement of ribose with the (N)-methanocarba
moiety best preserved receptor binding affinity at the
A3 subtype, at which differences were small. At A1
receptors, the loss of affinity upon substitution of ribose
with an (N)-methanocarba ring for structures 6-9 was
between 3- and 8-fold. At A2A receptors the loss of
affinity was between 6- and 34-fold.
F igu r e 2. Concentration-response curves for stimulation of
binding of [35S]GTP-γ-S by (A) NECA (]), 7a (b), or 7c (2); or
(B) NECA (4), 7c ([), or 9c (b), in membranes prepared from
CHO cells stably expressing human brain A3 receptors.
The agonist-induced stimulation of binding of guanine
nucleotides to activated G-proteins has been used as a
functional assay for a variety of receptors, including
adenosine receptors.28,29 Binding of [35S]GTP-γ-S was
studied in membranes prepared from CHO (Chinese
hamster ovary) cells stably expressing human A1 or A3
receptors (Table 2). The nonselective adenosine agonist
NECA (5′-N-ethyluronamidoadenosine) caused a con-
centration-dependent increase in the level of the gua-
nine nucleotide bound (Figure 2A). Compound 6c was
highly selective and a full agonist at human A1 but not
rat A1 receptors. Both 7c and 9c stimulated the binding
of [35S]GTP-γ-S (Figure 2B); however, the maximal
stimulation was significantly less than that produced
by either NECA or N6-(3-iodobenzyl)adenosine (Figure
2), 7a , both being full A3 agonists. Compounds 7c and
9c resulted in relative stimulation of [35S]GTP-γ-S
binding of only 45% and 22%, respectively, indicating
that the efficacy of the (N)-methanocarba analogue at
A3 receptors was further reduced upon 2-chloro modi-
fication. The potency of compounds 7c and 9c, indicated
by the EC50 values in this functional assay, was greater
than the potencies of either NECA or compound 7a
(Table 2). Thus, the (N)-methanocarba N6-(3-iodobenzyl)
analogues appear to be highly potent and selective
partial agonists at human A3 receptors.
Discu ssion
Nearly all of the thousands of known adenosine
agonists are derivatives of adenosine.1,2 Although mo-
lecular modeling of adenosine agonists has been carried
out,30 there has been little direct evidence for a confor-
mational preference of the ribose ring in the receptor
binding site. The furanose ring of nucleosides and
nucleotides in solution is known to exist in a rapid,
dynamic equilibrium between a range of Northern (2E
3
3
2
f T2 f E) and opposing Southern (2E f T3 f E)
3
conformations as defined in the pseudorotational
cycle.39,40 For methanocarba analogues, the bicyclo-
[3.1.0]hexane ring can constrain the cyclopentane ring
into an (N)-, 2′-exo (2E) envelope pucker, and an (S)-, 3′
exo (3E) form, as shown in Figure 1. These two extreme
forms of ring pucker usually define biologically active

2200 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
J acobson et al.
conformations.19 The energy difference between (N)- and
(S)-conformations, which is roughly 4 kcal/mol, could
explain the difference between micromolar and nano-
molar binding affinities. We have now applied this
approach to nucleosides binding to P1 (adenosine)
receptors and found that one of the fixed ring-envelope
conformations is highly favored at the receptor binding
site.
A pair of methanocarba-adenosine analogues, 5c and
5d , was prepared to explore the role of sugar puckering
in ligand recognition. The (S)-methanocarba analogue
of adenosine, 5d , was only weakly active in binding to
adenosine receptors, presumably because of an unfavor-
able conformation that decreases receptor binding. In
contrast, the methanocarba analogues constrained in
the (N)-conformation, e.g. 5c-9c, displayed high recep-
tor affinity, particularly at the A3 receptor. In binding
assays at A1, A2A, and A3 receptors, (N)-methanocarba-
adenosine, 5c, proved to be of higher affinity than the
(S)-analogue, 5d , with an N/S affinity ratio of 150 at
the human A3 receptor.
As previously reported14 and confirmed in this study,
the SAR of adenosine agonists indicates that the ribose
ring oxygen may be substituted with carbon; however,
much affinity is lost. As with the N6-(3-iodobenzyl)
aristeromycin derivative, 7b, simple carbocyclic substi-
tution of the ribose moiety of otherwise potent N6-
substituted adenosine agonists greatly diminishes af-
finity.
In comparison to the ribose analogues, the (N)-
methanocarba N6-substituted adenosine agonists were
of comparable affinity at A3 receptors, but less potent
at A1, A2A, and A2B receptors. The (N)-methanocarba-
N6-cyclopentyl derivatives were A1 receptor-selective
and maintained high efficacy at human recombinant but
not rat brain A1 receptors, as indicated by stimulation
of binding of [35S]GTP-γ-S. This may be related to either
species differences or heterogeneity of G proteins, since
the degree of agonist efficacy of a given compound may
be highly dependent on the receptor-associated G
protein.32-34 (N)-Methanocarba-N6-(3-iodobenzyl)ade-
nosine and the 2-chloro derivative had Ki values of 4.1
and 2.2 nM at A3 receptors, respectively, and were
selective partial agonists. As for the ribose parents,
additional 2-chloro substitution was favorable for recep-
tor selectivity. However, unlike the ribose forms, efficacy
was reduced in N6-(3-iodobenzyl) analogues, such that
7c and 9c proved to be partial A3 receptor agonists.
are important for affinity of adenosine analogues, and
the 2′-hydroxyl is particularly important for activation
of the receptor.31 From energy considerations, it is
expected that the rotation about the hydroxymethyl
group is relatively free for both 5c and 5d ; however,
rotation about the pseudoglycosyl bond is more re-
stricted than for conventional nucleosides.19 The ade-
nine moiety is thought to be present in an anti-
conformation in the adenosine receptor binding site.30
The relative contributions of the barrier to pseudogly-
cosyl bond rotation and ring pucker in the recognition
of 5c have not been explored. Franchetti et al.,37 using
a different type of ribose-modified nucleoside analogue,
also found an indication of higher affinity in binding to
A1 receptors for the (N)-conformation. The 2′-C-methyl-
was more potent than the 3′-C-methylribosyl analogue
of adenosine, suggesting that an anti-conformation
having an (N)-puckered furanose ring was preferred at
the receptor.
An additional element associated with the presence
of the bicyclo[3.1.0]hexane itself could play a role.
Indeed, the presence of the fused cyclopropane moiety
has the potential for causing repulsive steric interac-
tions by the presence of the extra “CH2” below the plane
of the ring. Although this property is common to both
rigid pseudorotational antipodes, the distance between
the “CH2” groups from the superimposed pseudosugar
structures is estimated to be approximately 1.7 Å.19
Therefore, this group appears to be shifted more toward
the base in the case of (S)-methanocarba-adenosine, and
it too might contribute to its diminished biological
activity.
For another member of the GPCR superfamily, the
P2Y1 receptor, we recently reported that the ribose (N)-
conformation of adenine nucleotides also appears to be
preferred at the receptor binding site.36 Therefore, at
least some of the P1 and P2 purinoceptors appear to
share a preference for the (N)-conformation. This may
indicate a common motif38 of binding of nucleoside
moieties to a variety of GPCRs. The insights of this
conformational preference may be utilized in simulated
docking of adenosine agonists in a putative receptor
binding site, to design even more potent and selective
agents.
At the binding site of ADA, the (N)-isomer is also
preferred, although the carbocyclic adenosine analogues
are relatively poor substrates.22 N6-Substituted ana-
logues, such as 6c-9c, would not be expected to be
substrates for ADA. In other systems effective discrimi-
nation between conformational antipodes is evident.19,21
For example, HIV reverse transcriptase has shown
almost exclusive preference for the (N)-isomer of AZT,
and only the (N)-isomer of thymidine was demonstrated
to have effective antiherpes activity.19 In recent experi-
ments kinases in general have shown preference for the
opposite conformation (Victor E. Marquez, Staffan
Eriksson, Riad Agbaria, and D. G. J ohns, unpublished
results). This could serve as the basis for distinguishing
the activities of adenosine analogues at receptors and
in metabolic processes.
Depending on the desired biological activity, partial
receptor agonists may be more desirable than full
agonists as therapeutic agents, due to the possibility of
reduced side effects in the former. Partial agonists may
display in vivo specificity for sites at which spare
receptors are present,32 and the drug would therefore
behave with apparent “full” efficacy.31,35 Thus, com-
pounds 7c and 9c, which combine partial agonism and
high functional potency at A3 receptors (EC50 < 1 nM),
should be explored for tissue-selective effects.
At least three of the four adenosine receptors favor
the (N)-conformation of methanocarba-adenosine. Thus,
the biological potency appears to be correlated to ring
puckering, which in turn would influence the orientation
of the hydroxyl groups (Figure 1) and the base within
the receptor binding site. The 2′- and 3′-hydroxyl groups
Since the methanocarba analogues are lacking a
glycosylic bond, it will be important to determine
whether the in vivo half-life of adenosine analogues is
prolonged by this modification. The half-life of 6a , for

Methanocarba Analogues of Purine
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11 2201
followed by decantation of the supernatant ether phase. The
residue was dried and dissolved in methanol (1 mL), and
ammonium hydroxide (0.5 mL) was added. The mixture was
heated at 80 °C in a closed tube for 1 h. Solvent was removed
under vacuum, and the residue obtained was purified by flash
column chromatography using 7/3 chloroform/methanol to
furnish 3.0 mg (47%) of the product, mp. 85 °C. HPLC showed
93% purity with retention times (min) of 17.53 (A) and 2.31
(B). MS (CI): m/z 482 (M+ + 1). HRMS: (FAB): calcd
481.0611, found 481.0610. 1H NMR(CD3OD): δ 1.86-1.96 (m,
1H, 5′CHH), 2.14-2.30 (m, 1H, 5′CHH), 2.38-2.48 (m, 1H,
4′CH), 3.67 (d, 2H, J ) 5.77 Hz, CH2O), 3.96-4.06 (m, 1H,
3′CH), 4.43-4.48 (m, 1H, 2′CH), 4.73-4.82 (m, 1H, 1′CH), 5.26
(s, 2H, ArCH2), 7.12 (t, 1H, J ) 7.82 Hz, ArH), 7.32 (d, 1H, J
) 7.82 Hz, ArH), 7.66 (d, 1H, J ) 7.82 Hz, ArH), 7.73 (s, 1H,
ArH), 8.06 (s, 1H, 2CH). 8.08 (s, 1H, 8CH).
example, is 24 min in whole blood and, following
infusion in conscious rats, only 8.2 min.5 These data are
typical of N6-substituted adenosine agonists, although
the reasons for this short half-life were not established.
Major likely factors include 5′-phosphorylation and
renal excretion of uncharged nucleosides, as well as
possible degradation.
In conclusion, we have found that the introduction of
a methano carbocyclic modification of the ribose ring of
purine agonists represents a general approach for the
design of adenosine agonists with favorable pharmaco-
dynamic properties, especially with respect to A1 and
A3 receptors. The present study has identified new
pharmacological probes of A1 and A3 receptors that are
selective and either full or partial agonists and are now
being tested in disease models. The effect of the absence
of the glycosylic bond on pharmacokinetic properties
should now be studied. This approach could be applied
to the development of cardioprotective, cerebroprotec-
tive, and antiinflammatory agents acting through A1
and A3 receptors.24
(1′R,2′R,3′S,4′R,5′S)-1-(H yd r oxym et h yl)-4-{6-[(3-iod o-
p h en ylm et h yl)a m in o]p u r in -9-yl}b icyclo[3.1.0]h exa n e-
2,3-diol (7c). To a solution of 4-(6-aminopurin-9-yl)-1-(hydroxy-
methyl)bicyclo[3.1.0]hexane-2,3-diol (5c, 20 mg, 0.0721 mmol)
in DMF (0.5 mL) was added m-iodobenzyl bromide (64 mg,
0.216 mmol), and the mixture was stirred at 50 °C for 2 days.
DMF was then removed under a stream of N2. Dry ether (1
mL) and 0.5 mL of acetone were added to the resulting syrup
and the syrup solidified. The solvents were removed by
decantation, and again ether was added and removed. The
solid was dried and dissolved in 1 mL of MeOH. NH4OH (1.5
mL) was added, and the mixture was stirred at 80 °C for 3
days. After cooling to room temperature, the solvents were
removed under reduced pressure, and the residue was purified
by preparative TLC (silica 60; 1000 µm; Analtech, Newark,
DE; ethyl acetate-i-PrOH-H2O (8:2:1)) to give 26 mg (73%
Ma ter ia ls a n d Meth od s
Ch em ica l Syn th esis. Nucleosides and synthetic reagents
were purchased from Sigma Chemical Co. (St. Louis, MO) and
Aldrich (St. Louis, MO). 2,6-Dichloropurine was obtained from
Sigma. m-Iodobenzyl bromide was purchased from Aldrich (St.
Louis, MO). Optically active (1′R,2′R,3′S,4′R,5′S)-4-(6-amino-
purin-9-yl)-1-(hydroxymethyl)bicyclo[3.1.0]hexane-2,3-diol (5c)
was synthesized as described.18 4-(6-Aminopurin-9-yl)-1-
(hydroxymethyl)bicyclo[3.1.0]hexane-2,3-diol (11) and racemic
rel-(1′R,2′S,3′R,4′R,5′S)-1-(6-aminopurin-9-yl)-4-(hydroxymethyl)-
bicyclo[3.1.0]hexane-2,3-diol (5d ) were synthesized according
to Marquez and colleagues (manuscript in preparation23).
Compounds 7a and 9a were synthesized as described.25,26
Proton nuclear magnetic resonance spectroscopy was per-
formed on a Varian GEMINI-300 spectrometer, and all spectra
were obtained in CDCl3. Chemical shifts (δ) relative to
tetramethylsilane are given. FAB (fast atom bombardment)
mass was performed with a J EOL SX102 spectrometer using
6 kV Xe atoms. Elemental analysis was performed by Atlantic
Microlab Inc. (Norcross, GA). NMR and mass spectra were
consistent with the assigned structures. The determination of
purity was performed with a Hewlett-Packard 1090 HPLC
system using an SMT OD-5-60 C18 analytical column (250 mm
× 4.6 mm, Separation Methods Technologies, Inc., Newark,
DE) in two different linear gradient solvent systems. One
solvent system (A) was 0.1 M triethylammonium acetate
buffer:CH3CN in ratios of 95:5 to 40:60 for 20 min with flow
rate 1 mL/min. The other (B) was MeOH:CH3CN, 95:5 to 40:
60, in 20 min with flow rate 1 mL/min. Peaks were detected
by UV absorption using a diode array detector.
(1′R,2′R,3′S,4′R,5′S)-4-[6-Cyclop en tyla m in o)p u r in -9-yl]-
1-(h yd r oxym eth yl)bicyclo[3.1.0]h exa n e-2,3-d iol (6c). A
solution of 8c (4 mg, 0.01 mmol) in methanol (0.5 mL) was
hydrogenated at atmospheric pressure over 10% Pd/C (1 mg)
to furnish the product 6c (83% yield). 1H NMR (CD3OD): δ
0.7-0.8 (m, 1H, 6′CHH), 1.46-1.88 (m, 10H, 6′CHH, 5′CH,
4CH2), 2.01-2.20 (m, 1H, NCH), 3.34 (d, 1H, J ) 9.77 Hz,
CH2O), 3.88 (d, 1H, J ) 6.84 Hz, 3′CH), 4.26 (d, 1H, J ) 9.77
Hz, CH2O), 4.66-4.98 (m, 2H, 2′CH, 1′CH), 8.28 (s, 1H, 2CH),
8.5 (s, 1H, 8CH). HRMS (FAB): calcd 346.1879, found 346.1879.
HPLC showed 90% purity with retention times (min) of 11.17
(A) and 2.16 (B).
1
yield) of the product (7c). H NMR (CDCl3): δ 0.82 (t, J ) 6.0
Hz, 1 H, 6′CHH), 1.41 (t, J ) 4.8 Hz, 1H, 6′CHH), 1.72 (dd, J
) 8.5, 6.0 Hz, 1H, 5′CH), 3.36 (d, J ) 10.8 Hz, 1H, CH2O),
4.05 (d, J ) 6.9 Hz, 1H, CH2O), 4.33 (m, 1H, 3′CH), 4.80-4.88
(m, 3 H, CH2N, 1′CH), 5.21 (d, J ) 6.9 Hz, 1H, 2′CH), 6.25 (m,
br, 1H, NH), 7.07 (t, J ) 7.8 Hz, 1H, Ar), 7.35 (d, J ) 7.8 Hz,
1H, Ar), 7.61 (d, J ) 7.8 Hz, 1H, Ar), 7.74 (s, 1H), 7.93 (s, 1H,
2CH), 8.33 (s, 1H, 8CH). MS (FAB): m/z 494 (M+ + 1). Anal
CHN.
(1′R,2′R,3′S,4′R,5′S)-4-[2-Ch lor o-6-(cyclop en tyla m in o)-
p u r in -9-yl]-1-(h yd r oxym et h yl)b icyclo[3.1.0]h exa n e-2,3-
d iol (8c). To a solution of 15 (36 mg, 0.076 mmol) in anhydrous
dichloromethane was added BCl3 (1 M solution in dichlo-
romethane, 0.23 mL, 0.23 mmol) at 0 °C. The reaction mixture
was warmed to room temperature and stirred for 10 min. To
this mixture was added methanol (1 mL) followed by am-
monium hydroxide (0.5 mL). The mixture was concentrated
under vacuum, and the residue obtained was purified by flash
column chromatography using 9/1 chloroform/methanol as
eluent to furnish 14 mg of the product 8c (48% yield) as a solid,
mp. 130 °C. HPLC showed 99% purity with retention times
1
(min) of 14.56 (A) and 2.19 (B). H NMR (CDCl3): δ 0.65-0.9
(m, 1H, 6′CHH), 1.1-1.4 (m, 2H, 6′CHH, 5′CH), 1.4-1.9 (m,
8H, 4CH2), 2.0-2.2 (m, 1H, N6CH), 3.34 (d, 1H, J ) 7.2 Hz,
CH2O), 3.97 (d, 1H, J ) 4.6 Hz, 3′CH), 4.25 (d, 1H, J ) 7.2
Hz, CH2O), 4.687 (s, 1H, 1′CH), 5.11 (d, 1H, J ) 4.6, 2′CH),
7.85 (s, 1H, 8CH). HRMS (FAB): calcd 380.1489, found
380.1498.
(1′R,2′R,3′S,4′R,5′S)-4-{2-Ch lor o-6-[(3-iod op h en ylm eth -
yl)a m in o]p u r in -9-yl}-1-(h yd r oxym et h yl)b icyclo[3.1.0]-
h exa n e-2,3-d iol (9c) was synthesized by the same method
as 8c in 53% yield, mp 121 °C. HPLC showed 98% purity with
retention times (min) of 17.59 (A) and 2.17 (B). 1H NMR (CD3-
OD): δ 0.70-0.78 (m, 1H, 6′CHH), 1.50-1.63 (m, 2H, 6′CHH,
5′CH), 3.33 (d, 1H, J ) 11.72 Hz, CH2O), 3.88 (d, 1H, J ) 6.84
Hz, 3′CH), 4.26 (d, 1H, J ) 11.72 Hz, CH2O), 4.71-4.83 (m,
2H, 1′CH, 2′CH), 7.1 (t, 1H, J ) 7.82 Hz, ArH), 7.40 (d, 1H, J
) 7.82 Hz, ArH), 7.61 (d, 1H, 7.82 Hz, ArH), 7.78 (s, 1H, ArH),
8.54 (s, 1H, 8CH). HRMS (FAB): calcd 528.0299, found
528.0295.
(1′R,2′R,3′R,4′R)-2,3-Dih yd r oxy-4-(h yd r oxym et h yl)-1-
(6-(3-iod oben zyla m in o)p u r in -9-yl)cyclop en t a n e (7b). A
mixture of aristeromycin (3.5 mg, 0.013 mmol) and 3-iodoben-
zyl bromide (12 mg, 0.039 mmol) in anhydrous DMF (1 mL)
was heated at 80 °C for 3 days. The solvent was removed under
vacuum, and the excess 3-iodobenzyl bromide was removed
from the reaction mixture by suspending the residue in ether,
(1′R ,2′R ,3′S ,4′R ,5′S )-4-(2,6-D ic h lo r o p u r in -9-y l)-1-
[(ph en ylm eth oxy)m eth yl]bicyclo[3.1.0]h exa n e-2,3-(O-iso-

2202 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 11
J acobson et al.
p r op ylid en e) (12). To a solution of triphenylphosphine (260
mg, 1 mmol) in anhydrous THF (2 mL) was added DEAD
(diethyl azadicarboxylate, 0.16 mL, 1 mmol) dropwise at 0 °C,
and stirring was continued for 20 min. To this solution was
added a solution of 2,6-dichloropurine in THF (4 mL), followed
by the addition of 11 (145 mg, 0.5 mmol) in THF (4 mL). The
reaction mixture was warmed to room temperature, and
stirring was continued for 6 h. The solvent was evaporated
under vacuum, and the residue obtained was purified by flash
chromatography using 7/3 petroleoum ether/ethyl acetate as
eluent to furnish 141 mg of the product (12) (70% yield) as a
gum. 1H NMR (CDCl3): δ 1.0 (m, 1H, 6′CHH), 1.24 (s, 3H,
CH3), 1.27-1.38 (m, 1H, 6′CHH), 1.55 (s, 3H, CH3), 1.62 (dd,
1H, J ) 4.88, 9.77 Hz, 5′CH), 3.34 (d, 1H, J ) 9.77 Hz, CH2O),
3.97 (d, 1H, J ) 9.77 Hz, CH2O), 4.50 (d, 1H, J ) 6.84 Hz,
3′CH), 4.57-4.68 (qAB, 2H, J ) 12.7 Hz, ArCH2), 5.17 (s, 1H,
1′CH), 5.32 (d, 1H, J ) 6.84 Hz, 2′H), 7.2-7.4 (m, 5H, Ar),
8.63 (s, 1H, 8CH).
(1′R,2′R,3′S,4′R,5′S)-4-[2-Ch lor o-6-(cyclopen tylam in o)pu -
r in -9-yl]-1-[(ph en ylm eth oxy)m eth yl]bicyclo[3.1.0]h exan e-
2,3-(O-isop r op ylid en e) (15). To a solution of 12 (42 mg, 0.105
mmol) in methanol (2 mL) was added cyclopentylamine (52
µL, 0.53 mmol) at room temperature, and stirring was con-
tinued for 6 h for complete reaction. Solvent was removed
under vacuum, and the residue obtained was purified by flash
column chromatography using 7/3 petroleum ether /ethyl
acetate as eluent to furnish 45 mg of the product 15 (90% yield)
as a gum. 1H NMR (CDCl3): δ 0.92-0.96 (m, 1H, 6′CHH),
1.14-1.01 (m, 1H, 6′CHH), 1.23 (s, 3H, CH3), 1.42-1.81 (m,
9H, 5′CH, 4CH2), 1.54 (s, 3H, CH3), 2.08-2.21 (m, 1H, N6CH),
3.44 (d, 1H, J ) 9.76 Hz, CH2O), 3.90 (d, 1H, J ) 9.76 Hz,
CH2O), 4.51 (d, 1H, J ) 6.84 Hz, 3′CH), 4.57-4.67 (qAB, 2H,
J ) 12.7 Hz, ArCH2), 5.04 (s, 1H, 1′CH), 5.32 (d, 1H, J ) 6.84
Hz, 2′CH), 7.2-7.4 (m, 5H, Ar), 8.18 (s, 1H, 8CH).
crude transfected CHO cell membranes (approximately 10 µg
protein/tube) were incubated with 0.5 nM [125I]AB-MECA, 10
mM MgCl2, 2 units/mL adenosine deaminase, and 50 mM Tris-
HCl (pH 7.4) at 25 °C for 60 min. The total volume of the
reaction mixture was 125 µL. Bound and free ligands were
separated by rapid filtration of the reaction mixture through
Whatman GF/B glass filters. The filters were immediately
washed with two 5-mL portions of ice-cold 50 mM Tris-HCl
buffer (pH 7.4). The radioactivity bound to the filters was
determined in a Beckman γ-counter. Specific binding was
defined as the amount of the radioligand bound in the absence
of competing ligand minus the amount of that bound in the
presence of 100 µM NECA. Ki values were calculated using
the Kd for [125I]AB-MECA binding of 0.56 nM.
Deter m in a tion of [35S]GTP -γ-S Bin d in g. [35S]GTP-γ-S
binding was determined by the method of Lorenzen et al.28
The incubation mixture contained in a total volume of 125 µL,
50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM MgCl2, 10 µM
guanosine 5′-diphosphate, 1 mM dithiothreitol, 100 mM NaCl,
0.2 units/mL adenosine deaminase, 0.16 nM [35S]GTP-γ-S, and
0.5% bovine serum albumin. The CHO cell membranes ex-
28
29
pressing A1 or A3 receptors were preincubated with the
above-mentioned assay mixture at 30 °C for 1 h and further
incubated for 1 h after the addition of [35S]GTP-γ-S. Incuba-
tions were terminated by rapid filtration of the samples
through glass fiber filters (Whatman GF/B), followed by two
5-mL washes of the same buffer. After transferring the filters
into a vial containing 3 mL of scintillation cocktail, the
radioactivity was determined in a scintillation counter.
Da ta An a lysis. Analyses of saturation binding assays and
concentration-response curves were carried out using Graph-
Pad Prism (GraphPad Software Inc., San Diego, CA). Com-
parisons between groups were carried out using the unpaired
Student’s t test.
(1′R,2′R,3′S,4′R,5′S)-4-{2-Ch lor o-6-[(3-iod op h en ylm eth -
yl)a m in o]p u r in -9-yl}-1-[(p h en ylm eth oxy)m eth yl]bicyclo-
[3.1.0]h exa n e-2,3-(O-isop r op ylid en e) (16) was synthesized
in 70% yield by the same method as for 15, except using
3-iodobenzylamine hydrochloride and 2 equiv of triethylamine.
1H NMR (CDCl3): δ 0.87-0.91 (m, 1H, 6′CHH), 1.10-1.29 (m,
1H, 6′CHH), 1.17 (s, 3H, CH3), 1.42-1.56 (m, 1H, 5′CH), 1.47
(s, 3H, CH3), 3.37 (d, 1H, J ) 9.77 Hz, CH2O), 3.84 (d, 1H, J
) 9.77 Hz, CH2O), 4.44 (d, 1H, J ) 6.84 Hz, 3′CH), 4.50-4.60
(qAB, 2H, J ) 11.72 Hz, ArCH2), 4.70 (bs, 1H, NH), 4.98 (s,
1H, 1′CH), 5.24 (d, 1H, J ) 6.84 Hz, 2′CH), 7.0 (t, 1H, J )
7.82 Hz, ArH), 7.2-7.34 (m, 6H, ArH), 7.55 (d, 1H, J ) 7.82,
ArH), 7.65 (s, 1H, ArH), 8.08 (s, 1H, 8CH).
Ack n ow led gm en t. We thank Gilead Sciences (Fos-
ter City, CA) for financial support to R.G.R.
Su p p or tin g In for m a tion Ava ila ble: Analysis for 6c,
7b,c, 8c, and 9c. This material is available free of charge via
the Internet at http://pubs.acs.org.
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