Selective A3 adenosine receptor antagonists derived from nucleosides containing a bicyclo[3.1.0]hexane ring system

Article abstract of DOI:10.1016/j.bmc.2008.08.007

We have prepared 5′-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleoside series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays at the A₃ AR. The H-bonding ability of a group of 9-riboside derivatives containing a 5′-uronamide moiety was reduced by modification of the NH; however these derivatives did not display the desired activity as selective A₃ AR antagonists, as occurs with 5′-N,N-dimethyluronamides. However, truncated (N)-methanocarba analogues lacking a 4′-hydroxymethyl group were highly potent and selective antagonists of the human A₃ AR. The compounds were synthesized from d-ribose using a reductive free radical decarboxylation of a 5′-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was similar to the published synthesis of (N)-methanocarba A₃AR agonists. Compounds 33b-39b (N⁶-3-halobenzyl and related arylalkyl derivatives) were potent A₃AR antagonists with binding K_i values of 0.7-1.4 nM. In a functional assay of [³⁵S]GTPγS binding, 33b (3-iodobenzyl) completely inhibited stimulation by NECA with a K_B of 8.9 nM. Thus, a highly potent and selective series of A₃AR antagonists has been described.

Full text of DOI:10.1016/j.bmc.2008.08.007

Bioorganic & Medicinal Chemistry 16 (2008) 8546–8556
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Selective A3 adenosine receptor antagonists derived from nucleosides
containing a bicyclo[3.1.0]hexane ring system
Artem Melman a, , Ben Wang a, , Bhalchandra V. Joshi a, , Zhan-Guo Gao a, Sonia de Castro a,
Cara L. Heller a, Soo-Kyung Kim a,c, Lak Shin Jeong b, Kenneth A. Jacobson a,
*
a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Building 8A, Room B1A-19, Bethesda, MD 20892, USA
b Laboratory of Medicinal Chemistry, College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
c Beckman Institute, Materials and Process Simulation Center, California Institute of Technology, Pasadena, CA 91125, USA
a r t i c l e i n f o
a b s t r a c t
We have prepared 50-modified derivatives of adenosine and a corresponding (N)-methanocarba nucleo-
side series containing a bicyclo[3.1.0]hexane ring system in place of the ribose moiety. The compounds
were examined in binding assays at three subtypes of adenosine receptors (ARs) and in functional assays
at the A3 AR. The H-bonding ability of a group of 9-riboside derivatives containing a 50-uronamide moiety
was reduced by modification of the NH; however these derivatives did not display the desired activity as
selective A3 AR antagonists, as occurs with 50-N,N-dimethyluronamides. However, truncated (N)-metha-
nocarba analogues lacking a 40-hydroxymethyl group were highly potent and selective antagonists of the
Article history:
Received 16 June 2008
Revised 1 August 2008
Accepted 4 August 2008
Available online 7 August 2008
Keywords:
G protein-coupled receptor
Purines
Molecular modeling
Structure–activity relationship
Radioligand binding
Adenylate cyclase
human A3 AR. The compounds were synthesized from D-ribose using a reductive free radical decarboxyl-
ation of a 50-carboxy intermediate. A less efficient synthetic approach began with L-ribose, which was
similar to the published synthesis of (N)-methanocarba A3AR agonists. Compounds 33b–39b (N6-3-
halobenzyl and related arylalkyl derivatives) were potent A3AR antagonists with binding Ki values of
0.7–1.4 nM. In a functional assay of [35S]GTP
cS binding, 33b (3-iodobenzyl) completely inhibited stimu-
lation by NECA with a KB of 8.9 nM. Thus, a highly potent and selective series of A3AR antagonists has
been described.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
of competitive tricyclic5 and nucleoside6 antagonists of ARs have
been reported recently. The structure of some of the antagonists
There are four subtypes of receptors for adenosine 1, designated
A1, A2A, A2B, and A3.1 Selective antagonists of the A3 adenosine
receptor (AR) are of potential use for clinical targets, including
the treatment of cancer,2 glaucoma,3 and inflammation.4 A number
may resemble adenosine, while other compounds possess a com-
pletely different structure. An important advantage of nucleo-
side-based A3AR antagonists is their species-independent
activity.7 These antagonists typically possess high affinity at the
human, rat, and mouse A3ARs and therefore may be suitable for
evaluation in small animal models or for further development as
drugs. For example, A3AR antagonists derived from adenosine have
been shown to lower intraocular pressure in a mouse model of
glaucoma.7
We have previously shown that local modifications of the po-
tent AR agonist Cl-IB-MECA 2 (Chart 1) may result in a complete
loss of agonist efficacy, while retaining a strong binding inhibition
constant (Ki).8 These modifications resulted in potent ARs antago-
nists of this type in compounds 3 and 4. The most probable reason
for the drastic change in activity is a disruption of H-bonding at the
50 position either through the introduction of an additional N-
methyl substituent, or enforcement of a conformation of the amide
group at this position that is unable to activate the receptor.9 An-
other approach to adenosine-derived A3AR antagonists involves
Abbreviations used: AIBN, 2,20-azo-bis-isobutyronitrile; AR, adenosine receptor;
CCPA, 2-chloro-N6-cyclopentyladenosine; CHO, Chinese hamster ovary; Cl-IB-
MECA,
2-chloro-N6-(3-iodobenzyl)-50-N-methylcarboxamidoadenosine;
DCC,
dicyclohexylcarbodiimide; DMEM, Dulbecco’s modified Eagle’s medium; EDTA,
ethylenediaminetetraacetic acid; GPCR, G protein-coupled receptor; I-AB-MECA,
2-[p-(2-carboxyethyl)phenyl-ethylamino]-50-N-ethylcarboxamidoadenosine; NECA,
50-N-ethylcarboxamidoadenosine; DIPEA, diisopropylethylamine; DCM, dichloro-
methane; DMF, N,N-dimethylformamide; HEPES, 4-(2-hydroxyethyl)-1-piperazinee-
thanesulfonic acid; HRMS, high resolution mass spectroscopy; MRS1292,
(2R,3R,4S,5S)-2-[N6-3-iodobenzyl)adenos-9’-yl]-7-aza-1-oxa-6-oxospiro[4.4]-nonan-4,
5-diol; PIA, R-N6-(phenylisopropyl)adenosine; TEA, triethylamine; TLC, thin layer
chromatography
* Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480 8422.
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
Present addresses: Nektar Therapeutics, Huntsville, AL 35801, USA (B.V.J.),
Northern Virginia Community College, VA, USA (B.W.), and Clarkson University,
Potsdam, NY, USA (A.M.).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.007

A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
8547
the complete removal of the substituent at position 40. This ap-
2. Results
proach has been successfully implemented in thionucleoside
antagonists of the class represented by compound 6 (Chart 2),
which was derived from 5, the 40-thio analogue of Cl-IB-MECA.10
Both the binding and selectivity of prototypical A3AR agonists
can be substantially increased by replacing the flexible ribose scaf-
fold in the series of compounds related to 2 with a rigid bicy-
clo[3.1.0]hexane ring system. The resulting (N)-methanocarba
adenosine agonists 7–9 have been found to possess high potency
and enhanced selectivity for the A3 receptor,11 and agonists of this
class are currently under development as anti-arthritis drug
candidates.
This study reports on a new series of selective A3AR antagonists
of the (N)-methanocarba family. Design of these compounds in-
volved the disruption of H-bonding at the 40 position through re-
moval of the N-methylcarboxamide function, while retaining the
conformationally restricted (N)-methanocarba scaffold bearing 6-
(arylalkylamino)-2-chloropurine moieties, as in agonists 7–9.
2.1. Chemical synthesis
Prior to probing the structure–activity relationship (SAR) with
the more synthetically challenging (N)-methanocarba system, we
explored alternatives to the N,N-dimethyl group as a means of
obtaining A3AR antagonists in the ribose series similar to com-
pound 3. The corresponding methyl ester 10b (Scheme 1A, pre-
pared from the protected intermediate 10a)10,12 was prepared
using a previously reported synthetic approach. This ester group
was directly aminolyzed using several hydroxylamino nucleo-
philes to provide 11 and 12. Alternately, the ester was hydrolyzed
to yield the carboxylic acid 13. All of these derivatives were in-
tended for testing at ARs, because they have altered H-bonding
ability in relation to 1. Attempts to prepare an active ester from
13 as an intermediate for further amide synthesis were
unsuccessful.
O
O
MeO
O
MeO
HO
O
I
I
N
N
N
N
H3COCO
a
H3COCO
HO
N
NH
N
NH
N
N
Cl
10a
b
Cl
10b
O
R
O
I
N
N
HO
HO
11 R = CH3ONH
12 R = CH3ON(CH3)
13 R = HO
N
NH
N
Cl
O
EtO
O
O
X
N
N
c
d
EtO
O
O
OH
N
NH
O
N
R2
14
15 X = 3-Cl; R2=Cl
16 X = 5-Cl-2-OCH3;R2=Cl
17 X = 3-Cl; R2=SMe
O
O
EtO
R3
HO
X
N
N
N
N
X
HO
e
HO
HO
N
NH
N
NH
N
N
R2
Cl
18 X = 3-Cl; R2=Cl
19 X = 5-Cl-2-OCH3;R2=Cl
20 X = 3-Cl; R2=SMe
21 X = 3-Cl; R3 = (CH3)2N
Scheme 1. Reagents: (a) K2CO3, MeOH, H2O; (b) K2CO3, MeOH, H2O, MeONH, MeONHMe or LiOH, NaOH, H2O; (c) two steps11; (d) TFA, EtOH–water; (e) R3H (amine), EtOH.

8548
A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
In the (N)-methanocarba series, the intermediate 20,30-isopro-
In contrast to the low-yielding hydrogenation of 26, silyl ether
31 was smoothly deprotected with TBAF. The resultant alcohol
27 was converted into a key dichloropurine derivative 32 through
a Mitsonobu reaction (Scheme 3).13 The dichloropurine derivative
32 reacted with an excess of the corresponding primary amine to
give the N6 substituted and 20,30-isopropylidene protected deriva-
tives 33a–39a, followed by acid catalyzed deprotection to give
the N6-3-halobenzyl and related arylmethyl derivatives 33b–39b.
pylidene protected derivatives 15–17 were prepared from the
key intermediate 14 as described in the literature (Scheme
1B).11,13 The key intermediate 14 was in turn prepared according
to our recent modified procedure.13b The isopropylidene group in
15–17 was conveniently removed by using aqueous TFA in EtOH
to afford the requisite 40-ester methanocarba derivatives 18–20
in good yields. Attempts to react the ester group of either 15–17
or 18–20 with dimethylamine to prepare dimethylamides similar
to 3 in the (N)-methanocarba series, that is, 21, were unsuccessful.
Our initial synthetic approach (Scheme 2) toward (N)-metha-
nocarba derivatives lacking the 40 substituent was based on our
previously published synthesis of (N)-methanocarba agonists from
3. Pharmacological activity
The 3-chloro- and 5-chloro-2-methoxy derivatives, 8 and 9,
respectively, of compound 7 were used for comparison in the bio-
logical assays (Table 1). Binding assays were carried out using stan-
dard radioligands and membrane preparations from Chinese
hamster ovary (CHO) cells (A1 and A3) or HEK293 cells (A2A) stably
expressing a hAR subtype (Table 1). Functional effects at the A3AR
were determined in assays of adenylate cyclase or guanine nucle-
otide binding.22,23 Replacement of the amide NH of 2 with an isos-
teric, but non-H-bond donating, ester oxygen in 10b resulted in a
one-order of magnitude loss of affinity at the A1 and A3ARs, and
no change at the A2AAR. Insertion of an O between the NH and
the methyl group in 11 reduced the A3AR affinity by 62-fold. Both
10b and 11 lost nearly all efficacy as A3AR agonists in relation to
full agonist 2. This confirmed previous findings that the efficacy
of adenosine derivatives in activating the A3AR was highly depen-
dent on structural factors in the region of the 40 carbon. The N-
methylation of derivative 12 had no effect on the A3AR affinity,
while completely abolishing activation of the receptor. Unfortu-
nately, compound 12 was not a suitable lead as an A3AR antagonist,
because it was only 4-fold selective in comparison to the A1AR.
However, this compound could be a useful pharmacological probe
of mixed selectivity for the A1AR and A3AR. Compound 12 was
L
-ribose 22.13 It involved protection of a key alcohol in 14 as a ben-
zyl ether followed by hydrolysis of the ester group in 24 with a
subsequent free-radical reductive decarboxylation of 25 using the
methodology of Barton,14 leading to conversion into the desired
compounds by previously described methodology.13 The decarbox-
ylation of 25 to yield 26 was performed using a one-pot modifica-
tion,15 which involved reaction with a mixture of tributyltin
hydride, 2,20-dithiopyridine-1,10-dioxide, tributylphosphine, and a
radical initiator. We found that under these conditions preparation
of benzyl ether 26 could be achieved, although the reaction yield
was variable and difficult to reproduce. Another problem was the
difficulty of deprotection of benzyl ether 26 by hydrogenation,
which gave alcohol 27 in poor yield.
These synthetic problems prompted us to evaluate an alterna-
tive synthetic approach (Scheme 3). It involved the synthesis of
alcohol 29 from inexpensive D-ribose using the same methodol-
ogy.13 Alcohol 29 was protected with TBDPS-Cl followed by alka-
line hydrolysis, thus providing acid 30.
Reductive decarboxylation of acid 30 was initially carried out
through a sequential one-pot procedure involving a DCC-medi-
ated coupling of the acid with 2-mercaptopyridine N-oxide at
room temperature followed by reductive free radical decarboxyl-
ation with tributyltin hydride at 80ꢀC.14a However, the resultant
ether 31 was difficult to separate from toxic and foul-smelling
tributyltin-containing byproducts. A modified approach for the
reductive decarboxylation used non-toxic tris(trimethyl-
silyl)silane as a hydrogen donor16 and produced the silyl ether
31 in 40% yield. To the best of our knowledge, this is the first
application of tris(trimethylsilyl)silane for reductive decarboxyl-
ation, and this reaction may possess a considerable preparative
potential.
tested in a functional assay at the A1AR ([35S]GTP
cS binding in
A1AR-expressing CHO cells) and found to lack the ability to activate
the receptor (data not shown). Therefore, compound 12 appears to
be a mixed A1/A3 AR antagonist. Compound 13, derived by sapon-
ification of 10a, was nearly inactive at the A1 and A2AARs and a very
weak agonist at the A3AR. Thus, a negative charge in the region of
the 40 carbon appears to be highly detrimental toward interaction
with ARs.
The (N)-methanocarba agonists 7–9 were already reported to be
potent and selective at the A3AR and fully efficacious.11 Replace-
ment of the amide NH of 8 or 9 with an ester oxygen resulted in
O
O
a
b
HOCH
O
OH
OH
2
EtO
HO
EtO
OX
O
O
HO
O
O
22 L-ribose
23
14 X=H
c
X=Bn
24
d
N
O
S
O
O
2
H
H
O
f
HO
21%
e
OH
OBn
OBn
O
O
O
O
27
26
25
Scheme 2. Reagents and condition: (a) Seven steps13; (b) CF3SO3H (cat.), DCM; (c) BnCl, NaH, THF; d) NaOH/H2O/MeOH, reflux; (e) PBu3, AIBN, Bu3SnH, toluene; (f) H2/Pd,
MeOH.

A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
8549
O
O
a
b, c
HOCH2
HO
O
OH
OH
Et O
HO
HO
TBDPSO
O
O
O
O
28 D-ribose
29
30
d,e
H
H
H
f
HO
TBDPSO
OH
O
O
O
O
O
O
27
31
g
H
H
O
H
h
i
N
N
N
N
N
N
O
HO
R
R
O
O
HO
N
Cl
N
NH
N
NH
N
N
N
Cl
Cl
33a-39a
33 X =
34 X = Cl
35 X = Br
Cl
32
33b-39b
R =
Me O
X
I
37
OMe
OMe
HO
36
38
39
Scheme 3. Reagents (a) Seven steps13; (b) TBDPS-Cl, imidazole, DMF; (c) NaOH, H2O, MeOH, reflux; (d) 2-mercaptopyridine N-oxide, DCC, toluene; (e) (Me3Si)3SiH, AIBN,
toluene; (f) Bu4NF, THF; (g) 2,6-dichloropurine, PPh3, DIAD, THF; (h) RNH2, EtOH; (i) TFA/H2O/MeOH.
greatly reduced affinity at the three AR subtypes, and only partial
activation of the A3AR was observed. Thus, this modification is un-
likely to provide an entry into selective A3AR antagonists. Replace-
ment of the 2-chloro of 18 with methylthio, which was tolerated in
the ribose series of A3AR agonists,6b resulted in 20 and failed to in-
crease the affinity. However, compound 20 completely lacked the
ability to activate the A3AR and thus appears to be a weak
antagonist.
A3AR. Compound 38b, a demethylated analogue of 37b, was
slightly less potent in binding to the A3AR.
Compounds 33b–39b were examined for the ability to stimu-
late [35S]GTP
cS binding in membranes of CHO cells expressing
the human A3AR. None of the truncated (N)-methanocarba deriva-
tives induced substantial (>10%) activation of the A3AR. In the same
functional assay applied to detect antagonism, a representative
analogue 33b completely inhibited stimulation by 1 lM NECA
Compounds 33b–35b (3-halobenzyl) in the (N)-methanocarba
series were potent A3 AR ligands with binding Ki values of 0.7–
1.4 nM. Compound 35b (3-bromobenzyl analogue) proved to be
the most potent A3AR ligand of this series in binding with a Ki value
of 0.73 nM, and it displayed high selectivity (2400-fold and 2190-
fold in comparison to the A1 and A2AAR, respectively). The most
A3AR selective compound was the 3-chloro analogue 34b with
2900-fold and 4250-fold selectivity in comparison to the A1 and
(50-N-ethylcarboxamidoadenosine) with an IC50 of 29.8 nM
(Fig. 1). Schild analysis of the right shifts by 33b of the response
curves in the inhibition of adenylate cyclase by NECA provided a
KB value of 8.9 nM. Thus, truncated (N)-methanocarba derivatives
displayed A3AR antagonist properties.
3.1. A3AR molecular modeling: Docking of nucleoside agonist
and antagonists
A
2AAR, respectively. The SAR of substitution of the N6-benzyl group
further showed that dimethoxy substitution (37b), fusion of the
phenyl ring to a second ring (36b), and extension by one carbon
(i.e., in the rotationally constrained 2-phenylcyclopropyl analogue,
39b) were all tolerated with nanomolar binding affinity at the
The previous docking study showed that the binding site of a
full agonist (Cl-IB-MECA 2) in the A3AR was located in the upper
transmembrane domain (TM) near EL2 (second extracellular loop)
shown in Figure 2A.17 In the present study, the nucleoside antago-

8550
A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
R1
N
R
R1,R2
H, H
Activity at A3AR
agonist
N
1 adenosine
2 Cl-IB-MECA
3 MRS3771
4 MRS542
CH2OH
NHR2
agonist
CONHMe Cl,3-iodobenzyl
CONMe2 Cl,3-iodobenzyl
O
R
N
N
antagonist
antagonist
Cl,3-iodobenzyl
HO
OH
1 - 4
CH2OH
Chart 1. Adenine 9-riboside derivatives as prototypical ligands for the A3AR.
O
Cl
N
N
N
NH
H
I
R
N
N
HO
S
R
N
N
HO
N
NH
N
HO
OH
R=CONHMe A3 agonist
R=H A3 antagonist
7 R = 3-I MRS1898
8 R = 3-Cl MRS3558
9 R = 5-Cl-2-OCH3
Cl
5
6
Chart 2. 40-Thio and (N)-methanocarba derivatives of adenosine as later generation ligands for the A3AR.
nist 33b shown in Figure 2B was initially docked to the A3AR model
based on the structure with Cl-IB-MECA 2 bound, followed by min-
imization and molecular dynamics, and the complexes were com-
pared. These two A3AR selective ligands were exactly overlapped in
the binding site with respect to the phenyl ring of the N6 substitu-
ent and the ribose ring, as depicted in Figure 2C. Common binding
sites were conserved: (1) The NH of the side-chain of Q167 in EL2
formed H-bonds with the 20-hydroxyl group; (2) The 30-hydroxyl
group interacted with the side chain of H2727.43 and the backbone
of S2717.42. However, different additional interactions were evi-
dent for agonist 2 and antagonist 33b. In the agonist binding do-
mains, additional interactions were present at the site of helical
bending of TM6 (due to a Pro residue) in proximity to TM7. The
additional interactions were shown in the interaction of the ribose
40O with T943.36, the 50-carbonyl group with S2727.42, and the 50-
terminal methyl group with the hydrophobic side-chain of
F2396.44 in the conserved FxxxWxP motif of Class A GPCRs.
The docking complex of compound 33b shown in Figure 2B
indicated the importance for agonism of binding at the helical
bending region of TM6 in proximity to TM7. Compound 33b, an
(N)-methanocarba analogue of Cl-IB-MECA 2 lacking the 50-group,
bound to the A3AR with a similar binding affinity to 2 but did not
induce activation. The docking result showed a loss of the typical
interaction of the 50-position with T3.36, S7.42, and F6.44. The
analysis of binding energy showed losses of 1.3 kcal/mol,
3.6 kcal/mol, and 2.8 kcal/mol related to binding in TM3, TM6,
and TM7, respectively, compared to the agonist 2. However, the
N6-(3-iodobenzyl) moiety formed the same hydrophobic interac-
tion with F168 in EL2 with the similar binding energy in TM5
(0.04 kcal/mol energy difference). The EL2, which interacted with
the common 20-hydroxyl group, revealed a more favorable energy
of interaction, by 0.5 kcal/mol, for the antagonist 33b. Since this
docking result is based on the structure of the meta-I state of rho-
dopsin, in which a different rotamer of W6.48 was revealed follow-
ing an anti-clockwise rotation from the extracellular perspective,
the docking with the ground-state structure, the inverse-agonist
preferred form, was more energetically favorable in the antagonist
33b-bound structure. However, the agonist 2 displayed a severe
steric clash at W6.48 and failed to form an optimum interaction
in the ground state.
As reported previously,8,9 the flexibility and the additional H-
bonding of the 50-substituent correlated with putative conforma-
tional changes of the receptor associated with the movement of
W6.48 upon activation. Selective A3AR agonists, such as Cl-IB-
MECA 2 and its 40-thio analogue 5, have been successfully trans-
formed into antagonists selective for the A3AR by appending an
additional N-methyl group on the 50-uronamide position or by
reducing the flexibility through cyclization of the 50 substituent.8
The 50-CO group of those antagonists was oriented perpendicular
to the ribose ring and could form a H-bond with W6.48, thus block-
ing the shift of W6.48 side-chain during the conformational
change. Thus, the 50-cyclized uronamide MRS1292 and the 50-
N,N-dimethyluronamide 3 did not adopt the typical receptor-
bound agonist conformation because of a locked conformation
and a different energetically favored geometry, respectively.8,17
The current mode of docking of a new nucleoside antagonist 33b
also supports the view that the additional binding domains present
in the full agonist 2 but absent in 33b are associated with receptor
activation. Thus, nucleoside ligands have spatially distinct regions
to fulfill separate roles in binding and activation processes.
4. Discussion
The truncation of the 50-CONHCH3 of A3AR-selective agonists
proved to be the most useful means of converting agonists into pure
antagonists. This reduces both the H-bond donating and accepting
abilities of the ribose or ribose-like ring. Other means of diminishing
the H-bonding ability through methylation, etc. tended to reduce
the potency and or selectivity, or allow residual efficacy to remain,
which was evident in assays of adenylate cyclase.
The AR binding affinities of the five agonists (i.e., 50-CONHCH3
derivatives) reported by Tchilibon et al.11 were compared to those
of the corresponding truncated compounds 33b–35b, 37b, and
39b. The mean affinities (nM) at the human A2A and A3ARs were
comparable in the two series (A2AAR: 3800 for the agonists, 2400
for the truncated analogues; A3AR: 1.22 for the agonists, 0.87 for

A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
8551
Table 1
Potency of a series of (N)-methanocarba adenosine derivatives at three subtypes of human ARs and the functional efficacy at the A3AR
NH R2
N
NH R2
N
NH R2
N
N
N
N
R3
R3
R3
N
N
N
N
N
N
R1CO
R1CO
O
HO OH
HO OH
HO OH
C
B
A
Compound
Structure
Affinity (Ki, nM) or % inhibitiona
A2A A3
% Efficacyb
A3
R1
R3
R2
A1
Series A
2c
CH3NH
(CH3)2N
CH3O
CH3ONH
CH3O(CH3)N
HO
Cl
Cl
Cl
Cl
Cl
Cl
3-I-Phenyl-CH2
3-I-Phenyl-CH2
3-I-Phenyl-CH2
3-I-Phenyl-CH2
3-I-Phenyl-CH2
3-I-Phenyl-CH2
222 22
5360 2470
>10,000
4770 240
1670 350
(52 3%)
(4 4%)
1.4 0.3
100
0
11
4
0
42
3d,e
10b
11
5870 930
1600 130
870 180
29.0 4.9
12.4 2.4
86.2 8.4
70.2 6.8
(58 3%)
12
13
252
8
(18 5%)
Series B
7
CH3NH
CH3NH
CH3NH
C2H5O
C2H5O
C2H5O
Cl
Cl
Cl
Cl
Cl
SCH3
3-I-Phenyl-CH2
136 22d
260 60
240 50
4820 680
1010 120
6630 120
784 97d
2300 100
1200 100
(19%)
(37%)
(20%)
1.5 0.2c
0.29 0.04
1.5 0.0
310 66
196 33
100c
103
107 15
54
39
0
8c
9c
18
19
20
3-Cl-Phenyl-CH2
5-Cl-2-MeOPh-CH2
3-Cl-Phenyl-CH2
5-Cl-2-MeOPh-CH2
3-Cl-Phenyl-CH2
7
1010 130
Series C
33be
34b
—
—
—
—
—
—
—
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3-I-Phenyl-CH2
3-Cl-Phenyl-CH2
3040 610
3070 1500
1760 1010
1120 640
3000 1260
1110 300
1790 1430
1080 310
4510 910
1600 480
1530 350
2620 730
6870 1440
2010 890
1.44 0.60
1.06 0.36
0.73 0.30
1.42 0.12
1.58 0.56
4.06 0.35
1.30 0.39
1.0 3.2f
2.9 3.7f
5.8 0.8f
3.1 0.3f
4.6 3.8f
0.4 1.3f
9.7 4.1f
35be
36b
3-Br-Phenyl-CH2
1-Naphthyl-CH2
2,5-diMeO-Ph-CH2
2-OH-5-MeO-Ph-CH2
trans-2-Ph-cyclopropyl
37b
38b
39b
a
All experiments were done on CHO or HEK (A2A only) cells stably expressing one of four subtypes of human ARs. The binding affinity for A1, A2A, and A3ARs was expressed
as Ki values (n = 3–5), and was determined by using agonist radioligands ([3H]CCPA or [3H]R-PIA; [3H]CGS21680; or [125I]I-AB-MECA; respectively). A percent in parentheses
refers to inhibition of radioligand binding at 10
l
M.
b
Unless noted, the efficacy at the human A3AR was determined by inhibition of forskolin-stimulated cyclic AMP production in AR-transfected CHO cells, as described in the
text. At a concentration of 10
l
M, in comparison to the maximal effect of a full agonist NECA at 10
l
M. Data are expressed as mean standard error (n = 3).
c
Values from Ref. 11.
Values from Ref. 8.
Compounds 3, MRS3771; 33b, MRS5127; 35b, MRS5147.
d
e
f
A3AR functional assay consisted of stimulation of [35S]GTP
cS binding at 10 lM, expressed as a percentage of the full effect induced by 10 lM NECA (100 5%).
the truncated analogues). However, the mean affinities (nM) at the
human A1AR were higher in the agonist series (610) in comparison
to the truncated analogues (2500). Thus, the A3AR selectivity in
comparison to the A1AR tended to be higher in the truncated series.
The relatively close correspondence of the AR binding affinities in
the agonist and truncated antagonist series is consistent with the
mode of overlay of the representative ligands docked in the recep-
tor, that is, the riboside agonist 2 and the truncated (N)-methanoc-
arba antagonist 33b. The moderate hydrophobicity of the
truncated (N)-methanocarba analogues was similar to the corre-
sponding agonist series (e.g., 7–9) and in a favorable range for
in vivo administration. For example, the clogP value of the 3-bro-
mo truncated derivative 35b is 1.96 in comparison to 2.17 for the
3-bromo agonist analogue.
In conclusion, a highly potent and selective series of A3AR
antagonists in the truncated (N)-methanocarba series has been de-
scribed. These analogues, particularly the 3-bromobenzyl 35b and
3-chlorobenzyl 34b and other analogues, can now be examined in
models of glaucoma and other diseases for which modulation of
the A3AR has been proposed to be useful.
Figure 1. Functional antagonism by the 3-iodobenzyl analogue 33b tested in an
assay of guanine nucleotide binding ([35S]GTP
expressing the human A3AR.
cS) in membranes of CHO cells

8552
A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
CH3CN/triethyl ammonium acetate from 5:95 to 60:40 in 20 min,
flow rate 1.0 mL/min; System B: linear gradient solvent system:
CH3CN/tetrabutyl ammonium phosphate from 20:80 to 60:40 in
20 min, flow rate 1.0 mL/min. Low-resolution mass spectrometry
was performed with a JEOL SX102 spectrometer with 6-kV Xe
atoms following desorption from a glycerol matrix or on an Agilent
LC/MS 1100 MSD, with a Waters (Milford, MA) Atlantis C18 col-
umn. High resolution mass spectroscopic (HRMS) measurements
were performed on a proteomics optimized Q-TOF-2 (Micromass-
Waters) using external calibration with polyalanine, unless noted.
Observed mass accuracies are those expected based on known per-
formance of the instrument as well as trends in masses of standard
compounds observed at intervals during the series of measure-
ments. Reported masses are observed masses uncorrected for this
time-dependent drift in mass accuracy.
5.1.2. 5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-
dihydroxy-tetrahydro-furan-2-carboxylic acid methyl ester
(10b)
The methyl ester 10a (0.062 g, 0.1 mmol) was dissolved in
MeOH (5 mL), potassium carbonate (0.028 g, 0.2 mmol) was added,
and the mixture was stirred at room temperature for 10 min. Ace-
tic acid (0.2 mL) was added to neutralize the base, the reaction
mixture was concentrated under reduced pressure and subjected
to preparative thin layer chromatography by using chloroform/
methanol (9:1) as solvent to afford the diol 10b as a colorless solid
(0.023 g, 43%). 1H NMR (CDCl3) d 8.20 (s, 1H), 7.73 (s, 1H), 7.66 (d,
1H, 7.5 Hz), 7.29 (d, 1H, 6.5 Hz), 7.12 (t, 1H, 4.0, 2.4 Hz), 6.05 (d, 1H,
5.1 Hz), 4.70–4.82 (m, 2H),4.55–4.67 (m, 2H), 3.79 (s, 3H), HRMS
calculated for C18H18ClIN5O5 (M+H)+: Exact Mass: 546.0041;
+
found, 546.0042. HPLC: RT 21.5 min (98%) in solvent system A,
10.5 min (99%) in system B.
5.1.3. 5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-
dihydroxy-tetrahydro-furan-2-carboxylic acid methoxy-amide
(11)
The methyl ester 10a (0.031 g, 0.05 mmol) was dissolved in
MeOH (5 mL), potassium carbonate (0.054 g, 0.4 mmol) was added,
and the mixture was stirred at room temperature for 10 min.
Methoxyamine hydrochloride (0.017 g, 0.2 mmol) was added and
the reaction mixture was stirred at room temperature for 30 min.
The reaction mixture was treated with acetic acid (0.3 mL) and
concentrated under reduced pressure. The residue was subjected
to preparative thin layer chromatography by using chloroform/
methanol (8:2) as solvent to afford the diol 11 as a colorless solid
(0.004 g, 16%). 1H NMR (CD3OD) d 8.26 (s, 1H), 7.79 (s, 1H), 7.61 (d,
1H, 6.5 Hz), 7.40 (d, 1H, 6.5 Hz), 7.10 (t, 1H, 4.2, 2.4 Hz), 5.98 (d, 1H,
6.6 Hz), 4.72–4.82 (m, 2H), 4.45–4.67 (m, 2H), 3.78 (s, 3H), HRMS
Figure 2. Docking complexes of A3AR ligands. (A) nucleoside agonist 2, Cl-IB-MECA
in light pink color, (B) nucleoside antagonist 33b in cyan color, and (C) the
superimposition of agonist 2 and antagonist 33b. Intermolecular H-bonding is
indicated with a dotted line. Using Pymol program, all ligands are represented by
stick models and the A3AR are shown in ribbon model with different colors for each
TM (TM1: purple, TM2: blue, TM3: light blue, TM4: green, TM5: yellow, TM6:
orange, TM7: red).
calculated for C18H19ClIN6O5 (M+H)+: 561.0156; found 561.0150.
+
HPLC: RT 19.4 min (98%) in solvent system A, 11.1 min (98%) in
system B.
5. Experimental
5.1.4. 5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-
dihydroxy-tetrahydro-furan-2-carboxylic acid methoxy-
methyl-amide (12)
5.1. Chemical synthesis
5.1.1. Materials and instrumentation
The methyl ester 10a (0.031 g, 0.05 mmol) was dissolved in
MeOH (5 mL), potassium carbonate (0.054 g, 0.4 mmol) was added,
and the mixture was stirred at room temperature for 10 min. N,O-
Dimethylhydroxylamine hydrochloride (0.019 g, 0.2 mmol) was
added and the reaction mixture was stirred at room temperature
for 30 min. The reaction mixture was treated with acetic acid
(0.3 mL) and concentrated under reduced pressure. The residue
was subjected to preparative thin layer chromatography by using
chloroform/methanol (8:2) as solvent to afford the diol 12 as a col-
orless solid (0.004 g, 14%). 1H NMR (CD3OD) d 8.29 (s, 1H), 7.74 (s,
1H), 7.52 (d, 1H, 5.5 Hz), 7.36 (d, 1H, 8.5 Hz), 7.14 (t, 1H, 4.1,
D-ribose, L-ribose, and other reagents and solvents were pur-
chased from Sigma–Aldrich (St. Louis, MO). Alcohol derivative 14
was prepared as reported.10 1H NMR spectra were obtained with
a Varian Gemini 300 spectrometer using CDCl3 and CD3OD as sol-
vents. Chemical shifts are expressed in d values (ppm) using as
standards tetramethylsilane (d 0.00) in CDCl3 and CHD2OD (d
3.30) in CD3OD. TLC analysis was carried out on aluminum sheets
precoated with silica gel F254 (0.2 mm) from Aldrich. HPLC mobile
phases consisted of System A: linear gradient solvent system:

A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
8553
2.5 Hz), 6.04 (d, 1H, 6.4 Hz), 4.62–4.80 (m, 2H), 4.42–4.62 (m, 2H),
nitrogen, and 60% NaH in mineral oil (45.0 mg, 1.10 mmol) was
+
3.58 (s, 3H), 2.68 (s, 3H) HRMS calculated for C19H21ClIN6O5
added. The reaction mixture was stirred at ꢀ70 °C for 5 min and
(M+H)+: 575.0312; found, 575.0310. HPLC: RT 15.2 min (98%) in
solvent system A, 18.5 min (99%) in system B.
then allowed to warm to 0 °C. Benzyl bromide (130 ll, 1.10 mmol)
was added to the mixture. The mixture was stirred for 5 min at
0 °C, allowed to warm to room temperature, and then stirred under
nitrogen gas for an additional 16 h. The solvent was removed under
vacuum. The resulting orange oil was dissolved in 50 mL ethyl ace-
tate, washed with 3ꢁ 50 mL water, and dried over Na2SO4. The ex-
tract was concentrated to a pale yellow-orange oil and purified by
column chromatography (silica gel, ethyl acetate/hexanes 1:9) to
provide 24 (220 mg, 66%) as a colorless oil. 1H NMR (CDCl3):
7.42–7.27 (m, 5H), 5.26 (d, 1H, 6.7 Hz), 4.69 (s, 2H), 4.56 (t, 1H,
6.7 Hz), 4.25–4.05 (m, 4H), 2.31–2.29 (m, 1H), 1.85 (t, 1H,
4.5 Hz), 1.59 (s, 3H), 1.54–1.50 (m, 1H), 1.31 (s, 3H), 1.28–1.22
5.1.5. 5-[2-Chloro-6-(3-iodo-benzylamino)-purin-9-yl]-3,4-
dihydroxy-tetrahydro-furan-2-carboxylic acid (13)
The methyl ester 10a (0.062 g, 0.1 mmol) was dissolved in EtOH
(3 mL), lithium hydroxide (0.012 g, 0.5 mmol) was added, and the
mixture was stirred at room temperature for 10 h. Acetic acid
(0.2 mL) was added to neutralize the base, and the reaction mix-
ture was concentrated under reduced pressure. The resulting resi-
due was triturated with ethyl acetate (10 mL). The solid obtained
was filtered and dried to afford acid 13 as a colorless solid
(0.027 g, 53%). 1H NMR (CD3OD) d 8.91 (s, 1H), 7.79 (s, 1H), 7.61
(d, 1H, 7.2 Hz), 7.39 (d, 1H, 7.2 Hz), 7.10 (t, 1H, 5.8 Hz), 6.15 (d,
1H, 6.9 Hz), 4.62–4.80 (m, 2H), 4.35–4.42 (m, 2H). HRMS calculated
for C117H16ClIN5O5+ (M+H)+: 531.9879; found, 531.9910. HPLC: RT
15.2 min (98%) in solvent system A, 18.0 min (95%) in system B.
(m, 4H). HRMS calculated for C19H24NaO5 (M+Na)+: 355.1521;
+
found, 355.1482.
5.1.10. (1S,2R,3S,4S,5S)-2,3-O-(Isopropylidene)-4-O-benzyl-
2,3,4-trihydroxybicyclo[3.1.0]-hexanecarboxylic acid (25)
A
mixture of 1.50 mL of 6 N KOH(aq) and 24 (220 mg,
5.1.6. (10S,20R,30S,40S,50S)-40-[2-Chloro-6-(4-chloro-
0.66 mmol) in a sealed vial was heated to 80 °C and stirred for
6 h. The mixture was cooled to 0 °C and diluted with 10 mL of ice
water. The mixture was neutralized upon the dropwise addition
of 0.50 mL glacial acidic acid. The neutralized solution was ex-
tracted with 3 ꢁ 10 mL ethyl acetate. The extracts were combined
and dried over Na2SO4. The solvent was removed under vacuum
and the resulting carboxylic acid 25 was used without further puri-
fication (200 mg, 99%). 1H NMR (CDCl3) 7.42–7.27 (m, 5H), 5.21 (d,
1H, 6.7 Hz), 4.69 (s, 2H), 4.56 (t, 1H, 6.7 Hz), 4.23 (t, 1H, 5.0 Hz)
2.31–2.40 (m, 1H), 1.91 (t, 1H, 4.5 Hz), 1.58 (s, 3H),ꢀ1.54–1.50 (m,
1H), 1.31 (s, 3H). HRMS calculated for C18H21O7 (M+HCO2)ꢀ:
349.1287; found 349.1287.
benzylamino)-purin-9-yl]-20,30-dihydroxy-
bicyclo[3.1.0]hexane-1-carboxylic acid ethyl ester (18)
Compound 15 (10 mg, 0.02 mmol) was treated with a solution
of trifluoroacetic acid in ethanol (10% TFA, 1.0 mL) and H2O
(0.1 mL). The mixture was heated to 70 °C for 3 h. The solution
was then cooled to room temperature and the solvent was re-
moved under vacuum. The residue obtained was subjected to pre-
parative silica gel column chromatography (CHCl3/MeOH 85:15) to
afford diol (18). (7 mg, 74%). 1H NMR (CDCl3) d 7.70 (s, 1H), 7.83 (d,
1H, 8.0 Hz), 7.26 (d, 1H, 8.5 Hz), 7.14 (t, 1H, 4.2, 2.8 Hz), 5.40–5.48
(m, 1H), 4.95 (s, 2H), 4.1–4.4 (m, 3H), 3.61 (s, 1H), 2.20–2.12 (m,
1H), 1.67–1.63 (m, 1H), 1.26 (t, 3H, 8.5, 6.5 Hz), 0.97–0.93 (m,
1H). HRMS calculated for C21H22Cl2N5O4+ (M+H)+: 478.1043; found
478.1042. HPLC: RT 22.1 min (98%) in solvent system A, 17.5 min
(99%) in system B.
5.1.11. (1R,2R,3S,4S,5S)-2,3-O-(Isopropylidene)-4-O-benzyl-
2,3,4-trihydroxybicyclo-[3.1.0]hexane (26)
Carboxylic acid 25 (200 mg, 0.66 mmol) was dissolved in
15.0 mL of dry toluene. The solution was purged of oxygen by 3 cy-
cles of freeze and thaw under vacuum. 2,20-Dithiopyridine-1,10-
dioxide (199 mg, 0.79 mmol), Bu3SnH (956 mg, 3.30 mmol), and
Bu3P (332 mg, 1.65 mmol) were added to the solution. The reaction
mixture was covered with foil and stirred at room temperature un-
der nitrogen gas for 3 h. Over the course of 3 h, the reaction mix-
ture turned slightly yellow. At the end of 3 h, AIBN (54.0 mg,
0.33 mmol) was added to the mixture. The reaction mixture was
then heated to 90 °C for an additional hour and the yellow color
disappeared. The reaction mixture was cooled to room tempera-
ture. The reaction mixture was diluted with 30 mL H2O and ex-
tracted with 3 ꢁ 20 mL ethyl acetate. The extract was dried over
Na2SO4 and the solvent was removed under vacuum. The crude
oil was purified with silica preparative TLC using 20% ethyl ace-
tate/hexanes to give 26 (85.0 mg, 49%). 1H NMR (CDCl3) 7.42–
7.27 (m, 5H), 4.82 (t, 1H, 6.0 Hz), 4.69 (s, 2H), 4.50 (t, 1H, 6.7 Hz),
5.1.7. (10S,20R,30S,40S,50S)-40-[2-Chloro-6-(2-chloro-5-methoxy-
benzylamino)-purin-9-yl]-2,3-dihydroxy-bicyclo[3.1.0]hexane-
1-carboxylic acid ethyl ester (19)
This was prepared following the same procedure as for com-
pound (18) starting from the isopropylidene derivative 16 to give
19 in 71% yield. 1H NMR (CDCl3) d 7.63 (s, 1H), 7.28 (s, 1H), 7.12
(d, 1H, 2.4 Hz), 6.72(d, 1H, 8.7 Hz), 6.32 (bs, 1H), 5.27 (s, 1H),
4.72 (d, 2H, 27 Hz), 4.0–4.2 (m, 3H), 3.78 (s, 3H), 2.08–2.10 (m,
1H), 1.74–1.81 (m, 1H), 1.51–1.59 (m, 1H), 1.22 (t, 3H, 1.2,
6.9 Hz). HRMS calculated for C22H24Cl2N5O5 (M+H)+: 508.1154;
+
found 508.1163. HPLC: RT 22.6 min (99%) in solvent system A,
21.2 min (100%) in system B.
5.1.8. (10S,20R,30S,40S,50S)-40-[6-(4-Chloro-benzylamino)-2-
methylsulfanyl-purin-9-yl]-2,3-dihydroxy-
bicyclo[3.1.0]hexane-1-carboxylic acid ethyl ester (20)
This was prepared following the same procedure as for com-
pound (18) starting from the isopropylidene derivative 17 to afford
20 in 68% yield. 1H NMR (CDCl3) d 7.60 (s, 1H), 7.35 (s, 1H), 7.18–
7.29 (m, 3H), 6.26 (bs, 1H), 5.31–5.36 (m, 1H), 4.78–4.85 (m, 2H),
4.21–4.29 (m, 3H), 2.52 (s, 3H), 2.20–2.39 (m, 1H), 1.64–1.71 (m,
1H), 1.51–1.59 (m, 1H), 1.26–1.31 (m, 4H). HRMS calculated for
4.30 (t, 1H, 5.0 Hz), 1.70–7.75 (m, 1H), 1.60 (s, 3H), 1.31–1.26 (m,
+
4H), 0.65–0.60 (m, 1H). HRMS calculated for
C16H24NO3
(M+NH4)+: 278.1756; found 278.1752.
5.1.12. (1R,2R,3S,4S,5S)-2,3-O-(Isopropylidene)-2,3,4-
trihydroxybicyclo[3.1.0]hexane (27), Method A
Benzyl ether 26 (50.0 mg, 0.33 mmol) was dissolved in a mix-
ture of 2.0 mL anhydrous methanol and 0.10 mL formic acid. The
solution was stirred in an ice bath and palladium black (8.50 mg,
10% w/w) was added. The solution was carefully purged with
H2(g) and fitted with a balloon filled with H2(g). The reaction mix-
ture was heated to 50 °C and stirred for 16 h. The H2(g) was dis-
C
22H25ClN5O4S+ (M+H)+: 490.1316; found 490.1299. HPLC: RT
23.45 min (97%) in solvent system A, 21.2 min (98%) in system B.
5.1.9. Ethyl (1S,2R,3S,4S,5S)-2,3-O-(isopropylidene)-4-O-benzyl-
2,3,4-trihydroxybicyclo[3.1.0]hexanecarboxylate (24)
Alcohol 14 (240 mg, 1.00 mmol) was dissolved in 5.0 mL of a
DMF/THF (4:1) solution. The solution was cooled to ꢀ70 °C under
charged and the reaction mixture filtered through
syringe filter. The filter was washed with 10 mL of methanol and
a nylon

8554
A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
the filtrate was dried under vacuum. The resulting oil was purified
on a silica preparative TLC plate with 30% ethyl acetate/hexanes as
eluant to give 27 as a homogeneous product (15.0 mg, 27%). 1H
NMR (CDCl3) 4.88 (t, 1H, 4.2 Hz), 4.53 (bs, 1H), 4.48 (t, 1H,
6.7 Hz), 4.30 (t, 1H, 5.0 Hz), 2.40 (br s, 1H), 1.87–1.83 (m, 1H),
1.67–1.63 (m, 1H), 1.55 (s, 3H), 1.29 (s, 3H), 0.99–0.95 (m, 1H),
0.68–0.60 (m, 1H). HRMS calculated for C9H14O3 (M)+: 170.0943;
found, 170.0942.
5.1.16. (1R,2R,3S,4S,5S)-2.3-O-(Isopropylidene)-2,3,4-
trihydroxy-bicyclo[3.1.0]hexane (27), Method B
A 1M solution of tert-butylammonium fluoride in THF (1 mL)
was added to a solution of silyl ether 31 (102 mg, 0.25 mmol)
in THF (1 mL). The reaction mixture was left at 20 °C for 16 h
and evaporated. The residue was diluted with ethyl acetate
(20 mL) and washed with a small amount of brine. The ethyl
acetate solution was dried and evaporated, and the residue
was purified by flash chromatography to afford the title
compound 27 (33 mg, 84%). 1 H NMR and MS are provided under
Method A.
5.1.13. (10R,20R,30S,40S,50S)-40-[(2,6-Dichloropurine)-20,30-O-
(isopropylidene)-20,30-dihydroxybicyclo[3.1.0]hexane (32)
A mixture of triphenyl phosphine (46 mg, 0.176 mmol) and 2,6-
dichloropurine (33 mg, 0.176 mmol) in dry THF (2.0 mL) was trea-
ted with diisopropylazodicarboxylate (36 mg, 0.176 mmol). The
mixture was stirred at room temperature for 20 min. The mixture
was then added to a stirring solution of 27 (15 mg, 0.088 mmol)
dissolved in dry THF (0.50 mL). The mixture was stirred for an
additional 8 h at room temperature. The solvent was evaporated,
and the crude oil was purified with silica preparative TLC in 30%
ethyl acetate/hexanes solution to give 32 (15 mg, 50%). 1H NMR
(CDCl3) 8.15 (s, 1H), 5.38 (t, 1H, 4.2 Hz), 5.06 (s, 1H), 4.67 (d, 1H,
6.7 Hz), 2.20–2.15 (m, 1H), 1.67–1.63 (m, 3H), 1.56 (s, 3H), 1.26
(s, 3H), 1.03–0.97 (m, 3H). HRMS calculated for C14H14Cl2N4O2
(M)+: 340.0494; found, 340.0495.
5.1.17. General procedure for preparation of compounds 33b–
39b
An amine (RNH2 in Scheme 3 and 0.5 mmol) was added to a
solution of 32 (20 mg, 0.06 mmol) in DCM (0.1 mL). The reaction
mixture was stirred at room temperature for 16 h. The solvent
was removed under vacuum, and the residue was separated by
flash chromatography (30–100% ethyl acetate–hexane) to afford
the corresponding 6-alkylaminopurine derivative that was dis-
solved in a mixture of MeOH (4 mL), TFA (0.2 mL), and water
(2 mL). The reaction mixture was stirred at 70 °C for 16 h, and then
evaporated. The residue was evaporated twice with water, and the
residue was purified by flash chromatography (50–100% ethyl
acetate).
5.1.14. (1R,2S,3R,4R,5R)-3,4-O-(Isopropylidene)-2-O-(tert-
butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane-1-
carboxylic acid (30)
5.1.18. (10R,20R,30S,40R,50S)-40-[2-Chloro-6-(3-iodobenzylamino)-
purine]-20,30-O-dihydroxybicyclo-[3.1.0]hexane (33b)
tert-Butyldiphenylsilyl chloride (2.70 g, 10 mmol) and triethyl-
amine (2.0 g, 20 mmol) were added to a solution of alcohol 29
Yield 15 mg (51% 1H NMR (CD3OD), d 8.16 (s, 1H), 7.49 (s, 1H),
7.60 (d, 1H, 8.5 Hz), 7.40 (d, 1H, 8.5 Hz), 7.10 (t, 1H, 8.5 Hz), 4.71 (s,
2H), 3.90 (d, 3.3 Hz, 1H), 3.65 (s, 1H), 2.05–1.95 (m, 1H), 1.67–1.63
(prepared from D
-ribose 28 following the standard procedure,13
1.22 g, 5 mmol) and imidazole (140 mg, 2 mmol) in DMF (3 mL)
while stirring at room temperature. The solution was stirred at
60 °C for 16 h. The reaction mixture was cooled to room temper-
(m, 1H), 1.36 (s, 1H), 1.31–1.27 (m, 1H), 0.95–0.87 (m, 1H), 0.77–
18H18ClIN5O2 (M+H)+:
+
0.75 (m, 1H). HRMS calculated for
C
498.0194; found, 498.0194. HPLC: RT 21.6 min (98%) in solvent
system A, 17.0 min (98%) in system B.
ature and diluted with
a 4:1 ethyl acetate/hexane mixture
(50 mL), washed with water, dried, and solvent was evaporated.
The residue was purified by flash chromatography (0–10% ethyl
acetate–hexane) to give ethyl (1R,2S,3R,4R,5R)-2,3-O-(isopropyli-
dene)-4-O-(tert-butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]-
hexane-1-carboxylate. The compound was dissolved in MeOH
(5 mL) and 2 N aq NaOH (5 mL) was added, and the reaction mix-
ture was refluxed for 2 h. The reaction mixture was neutralized
with NaH2PO4, and extracted with DCM. The combined DCM
solutions were dried and evaporated, and the residue was puri-
fied by flash chromatography to give title compound 30 (1.65 g,
73%). 1H NMR (CDCl3), d 7.72 (d, 4H, J = 7.8 Hz), 7.39 (m, 6H),
5.05 (d, 1H, J = 6.3 Hz), 4.43 (t, 1H, J = 6.0 Hz), 4.08 (t, 1H,
J = 6.6 Hz), 2.26 (m, 1H), 1.97 (s, 3H), 1.56 (s, 3H), 1.52 (m, 1H),
1.21 (s, 3H), 1.08 (s, 9H).
5.1.19. (10R,20R,30S,40R,50S)-40-[2-Chloro-6-(3-chlorobenzylami-
no)purine]-20,30-O-dihydroxybicyclo-[3.1.0]hexane (34b)
Yield 58%. 1H NMR (CD3OD), d 8.16 (br s, 1H), 7.41 (s, 1H), 7.29
(m, 3H), 4.79 (s, 1H), 4.75 (br s, 2H), 4.70 (br t, 1H, J = 5.4 Hz), 3.86
(d, 1H, J = 6.6 Hz), 1.97 (m, 1H), 1.65 (m, 1H), 1.30 (m, 1H), 0.75 (m,
1H). HRMS (ESI MS m/z): calculated for C18H18Cl2N5O2 (M+H)+,
+
406.0832; found, 406.0825. HPLC RT 20.3 min (98%) in solvent sys-
tem A, 15.6 min (98%) in system B.
5.1.20. (10R,20R,30S,40R,50S)-40-[2-Chloro-6-(3-bromobenzylamino)-
purine]-20,30-O-dihydroxybicyclo-[3.1.0]hexane (35b)
Yield 65%. 1H NMR (CD3OD): 8.03 (s, 1H), 7.45 (s, 1H), 7.29
(m, 2H), 7.12 (t, 1H, J = 7.8 Hz), 4.68 (s, 1H), 4.63 (br s, 2H), 4.59
(br t, 1H, J = 5.4 Hz), 3.79 (d, 1H, J = 6.6 Hz), 1.86 (m, 1H),
1.55 (m, 1H), 1.20 (m, 1H), 0.64 (m, 1H). HRMS (ESI MS m/z) calcu-
5.1.15. (1S,2S,3R,4R,5R)-3,4-O-(Isopropylidene)-2-O-(tert-
butyldiphenylsilyl)-2,3,4-trihydroxybicyclo[3.1.0]hexane (31)
A 1M solution of DCC in oxygen-free toluene (0.96 mL) was
added to a solution of acid 30 (363 mg, 0.80 mmol), 2-mercapto-
pyridine N-oxide (112 mg, 0.88 mmol), and AIBN (40 mg,
0.24 mmol) in dry oxygen-free toluene (4 mL). The reaction mix-
ture was stirred for 4 h at 25 °C, tris(trimethylsilyl)silane
(0.50 mL, 1.6 mmol) was added, and the reaction mixture was
heated at 85 °C for 4 h. The reaction mixture was evaporated,
and the residue was separated by flash chromatography (0–10%
ethyl acetate–hexane mixture) to afford the title compound 31
(121 mg, 40%). 1H NMR (CDCl3), d 7.76 (d, 4H, J = 7.8 Hz), 7.39 (m,
6H), 4.66 (t, 1H, J = 6.0 Hz), 4.44 (t, 1H, J = 6.6 Hz), 4.03 (t, 1H,
J = 6.6 Hz), 1.6 (m, 1H), 1.57 (s, 3H), 1.45 (m, 1H), 1.33 (s, 1H),
1.20 (s, 3H), 1.09 (s, 9H), 0.58 (m, 1H).
+
lated for C18H18BrClN5O2 (M+H)+, 450.0327; found 450.0315.
HPLC RT 20.74 min (98%) in solvent system A, 16.1 min (99%) in
system B.
5.1.21. (10R,20R,30S,40R, 50S)-40-[2-Chloro-6-(1-
naphthylamino)purine]-20,30-O-dihydroxybicyclo[3.1.0]hexane
(36b)
Yield 48%. 1H NMR (CD3OD): 8.13 (br d, 2H, J = 7.8 Hz), 7.84
(m, 2H), 7.49 (m, 4 H), 5.21 (s, 1H), 4.79 (br s, 1H), 4.78 (br s,
2H), 4.67 (br t., 1H, J = 5.1 Hz), 3.88 (d, 1H, J = 6.6 Hz), 1.93 (m,
1H), 1.62 (m, 1H), 1.25 (m, 1H), 0.73 (m, 1H). HRMS (ESI MS m/z)
calculated for C22H21ClN5O2 (M+H)+, 422.1378; found 422.1385.
+
HPLC RT 21.5 min (97%) in solvent system A, 17.0 min (98%) in
system B.

A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
8555
5.1.22. (10R,20R,30S,40R,50S)-40-[2-Chloro-6-(2,5-
mented with 10% fetal bovine serum, 100 U/mL penicillin,
100 g/mL streptomycin, 2 mol/mL glutamine, and 800 g/mL
dimethoxybenzylamino)purine]-20,30-O-dihydroxybicyclo-
[3.1.0]hexane (37b)
l
l
l
geneticin. The CHO cells expressing rat A3ARs were cultured in
DMEM and F12 (1:1). Cells were harvested by trypsinization. After
homogenization and suspension, cell membranes were centrifuged
at 500 g for 10 min, and the pellet was re-suspended in 50 mM Tris
HCl buffer (pH 8.0) containing 10 mM MgCl2, 1 mM EDTA, and
0.1 mg/mL CHAPS (3[(3-cholamidopropyl)dimethylammonio]-pro-
panesulfonic acid). The suspension was homogenized with an elec-
tric homogenizer for 10 s, and was then re-centrifuged at 20,000g
for 20 min at 4 °C. The resultant pellets were resuspended in buffer
in the presence of adenosine deaminase (3 U/mL), and the suspen-
sion was stored at ꢀ80 °C until the binding experiments. The pro-
tein concentration was measured using the Bradford assay.18
Yield 44%. 1H NMR (CD3OD): 8.4 (very br s, 1H), 6.95 (s, 1H,
J = 2.7 Hz), 6.89 (d, 1H, J = 9.3 Hz), 6.78 (dd, 1H, J = 2.7, 9.0 Hz),
4.80 (s, 1H), 4.75 (br m, 3H), 3.87 (d, 1H, J = 6.3 Hz), 3.83 (s, 3H),
3.71 (s, 3H), 1.95 (m, 1H), 1.64 (m, 1H), 1.29 (m, 1H), 0.74 (m,
1H). HRMS (ESI MS m/z) calculated for C20H23ClN5O4 (M+H)+,
+
432.1433; found 432.1439. HPLC RT 18.7 min (98%) in solvent sys-
tem A, 16.6 min (98%) in system B.
5.1.23. (10R,20R,30S,40R,50S)-40-[2-Chloro-6-(2-hydroxy-5-
methoxybenzylamino)purine]-20,30-O-dihydroxybicyclo-
[3.1.0]hexane (38b)
Yield 39%. 1H NMR (CD3OD): 8.07 (s, 1H), 6.60–6.82 (m, 3H),
4.69 (s, 1H), 4.59 (br t., 1H, J = 6.0 Hz), 4.56 (br s, 2H), 3.79 (d,
1H, J = 6.6 Hz), 3.61 (s, 3H) 1.86 (m, 1H), 1.55 (m, 1H), 1.20 (m,
6.2.1. Binding assays at the A1 and A2A receptors
For binding to human A1 receptors,19 [3H]R-PIA (N6-[(R)-phe-
nylisopropyl]adenosine, 2 nM) or [3H]CCPA (0.5 nM) was incu-
+
1H), 0.65 (m, 1H). HRMS (ESI MS m/z) calculated for C19H21ClN5O4
(M+H)+, 418.1277; found, 418.1277. HPLC RT 16.0 min (100%) in
solvent system A, 11.0 min (98%) in system B.
bated with membranes (40 lg/tube) from CHO cells stably
expressing human A1 receptors at 25 °C for 60 min in 50 mM
Tris–HCl buffer (pH 7.4; MgCl2, 10 mM) and increasing concentra-
5.1.24. (10R,20R,30S,40R,50S)-40-[2-Chloro-6-(trans-2-
phenylcyclopropylamino)purine]-20,30-O-dihydroxybicyclo-
[3.1.0]hexane (39b)
tions of the test ligand in a total assay volume of 200
cific binding was determined using 10
of CPA (N6-
cyclopentyladenosine). For human A2A receptor binding,20 mem-
ll. Nonspe-
lM
Yield 52%. 1H NMR (CD3OD): 8.16 (very br s, 1H), 7.0–7.48 (m,
5H), 4.79 (s, 1H), 4.68 (br s, 2H), 3.88 (d, 1H, J = 5.7 Hz), 2.17 (m,
1H) 1.97 (m, 1H), 1.65 (m, 1H), 1.29 (m, 2H), 0.74 (m, 1H). HRMS
branes (20 lg/tube) from HEK-293 cells stably expressing human
A2A receptors were incubated with [3H]CGS21680 (2-[p-(2-car-
boxyethyl)phenyl-ethylamino]-50-N-ethylcarboxamido-adenosine,
15 nM) and increasing concentrations of the test ligand at 25 °C for
(ESI MS m/z) calculated for C20H21ClN5O2 (M+H)+, 398.1378;
+
found, 398.1372. HPLC RT 20.3 min (99%) in solvent system A,
15.6 min (98%) in system B.
60 min in 200
ll 50 mM Tri–HCl, pH 7.4, containing 10 mM MgCl2.
NECA (10 M) was used to define nonspecific binding. The reaction
l
was terminated by filtration with GF/B filters.
6. Molecular modeling
6.1. Docking study
6.2.2. Binding assay at the human A3 receptor
For the competitive binding assay, each tube contained 50 lL
membrane suspension (20
(1.0 nM),21 and 25
L of increasing concentrations of the test li-
gands in Tris–HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl2,
and 1 mM EDTA. Nonspecific binding was determined using 10
lg protein), 25 l
L of [125I]I-AB-MECA
l
The homology model of the hA3AR (PDB id: 1OEA) derived using
the X-ray structure of bovine rhodopsin with a 2.8 Å resolution
(PDB id: 1F88) as a template was used for the docking study. The
previously reported A3AR conformation,17 which was bound to
Cl-IB-MECA, 2, was used for the antagonist docking. For the dock-
ing study of compound 33b, a pharmacophore match among
hydrophilic interaction sites, for the 20-OH, 30-OH, and N6H groups,
was performed to obtain an initial geometry. The refinement of the
side chain in the binding site was followed by binding site minimi-
zation with fixed backbone atoms, complex minimization, and
200 ps molecular dynamics.
lM
of Cl-IB-MECA in the buffer. The mixtures were incubated at 37 °C
for 60 min. Binding reactions were terminated by filtration through
Whatman GF/B filters under reduced pressure using a MT-24 cell
harvester (Brandell, Gaithersburgh, MD, USA). Filters were washed
three times with 9 mL ice-cold buffer. Radioactivity was deter-
mined in a Beckman 5500B c-counter. IC50 values were converted
to Ki values as described.25
6.2.3. Cyclic AMP accumulation assay
Atomic charges were recalculated by Mullekin charge using the
Intracellular cyclic AMP levels were measured with a competi-
tive protein binding method.22,23 CHO cells that expressed the re-
combinant human or rat A3AR or the human A1 or A2BAR were
harvested by trypsinization. After centrifugation and resuspended
in medium, cells were planted in 24-well plates in 1.0 mL medium.
After 24 h, the medium was removed, and cells were washed three
times with 1 mL DMEM, containing 50 mM HEPES, pH 7.4. Cells
were then treated with the agonist NECA and/or test compound
**
DFT/6311G basis set for ligands 2 and 33b. Finally, the complex
structure was minimized using an Amber 7 force field 99 with a
fixed dielectric constant (4.0), until the conjugate gradient reached
0.1 kcal molꢀ1 Åꢀ1. Values of clog P were calculated using Chem-
Draw Ultra 11.0.
6.2. Receptor binding and functional assays
(e.g., 33b) in the presence of rolipram (10
deaminase (3 U/mL). After 45 min, forskolin (10
to the medium, and incubation was continued for an additional
15 min. The reaction was terminated by removing the supernatant,
l
M) and adenosine
[
125I]N6-(4-Amino-3-iodobenzyl)adenosine-50-N-methylurona-
lM) was added
mide (I-AB-MECA; 2000 Ci/mmol), [3H]cyclic AMP (40 Ci/mmol),
and other radioligands were purchased from Perkin–Elmer Life
and Analytical Science (Boston, MA). [3H]CCPA (2-chloro-N6-cyclo-
pentyladenosine) was a custom synthesis product (Perkin-Elmer).
Test compounds were prepared as 5 mM stock solutions in DMSO
and stored frozen.
and cells were lysed upon the addition of 200 lL of 0.1 M ice-cold
HCl. The cell lysate was resuspended and stored at ꢀ20 °C. For
determination of cyclic AMP production, protein kinase A (PKA)
was incubated with [3H]cyclic AMP (2 nM) in K2HPO4/EDTA buffer
Cell culture and membrane preparation: CHO (Chinese hamster
ovary) cells expressing the recombinant human A3AR were cul-
tured in DMEM (Dulbecco’s modified Eagle’s medium) supple-
(K2HPO4, 150 mM; EDTA, 10 mM), 20
30 L 0.1 M HCl or 50 L of cyclic AMP solution (0–16 pmol/
200 L for standard curve). Bound radioactivity was separated by
lL of the cell lysate, and
l
l
l

8556
A. Melman et al. / Bioorg. Med. Chem. 16 (2008) 8546–8556
5. Moro, S.; Deflorian, F.; Bacilieri, M.; Spalluto, G. Curr. Med. Chem. 2006, 13,
639.
6. (a) Joshi, B. V.; Jacobson, K. A. Curr. Topics Med. Chem. 2005, 5, 1275; (b) Kim, H.
O.; Ji, X.-d.; Siddiqi, S. M.; Olah, M. E.; Stiles, G. L.; Jacobson, K. A. J. Med. Chem.
1994, 37, 3614; (c) Gao, Z. G.; Mamedova, L. K.; Chen, P.; Jacobson, K. A.
Biochem. Pharmacol. 2004, 68, 1985.
7. Yang, H.; Avila, M. Y.; Peterson-Yantorno, K.; Coca-Prados, M.; Stone, R. A.;
Jacobson, K. A.; Civan, M. M. Curr. Eye Res. 2005, 30, 747.
8. (a) Gao, Z. G.; Kim, S. K.; Biadatti, T.; Chen, W.; Lee, K.; Barak, D.; Kim, S. G.;
Johnson, C. R.; Jacobson, K. A. J. Med. Chem. 2002, 45, 4471; (b) Gao, Z.-G.; Joshi,
B. V.; Klutz, A. M.; Kim, S.-K.; Lee, H. W.; Kim, H. O.; Jeong, L. S.; Jacobson, K. A.
Bioorg. Med. Chem. Lett. 2006, 16, 596.
9. Kim, S. K.; Jacobson, K. A. J. Chem. Inf. Model. 2007, 47, 1225.
10. Jeong, L. S.; Choe, S. A.; Gunaga, P.; Kim, H. O.; Lee, H. W.; Lee, S. K.; Tosh, D.;
Patel, A.; Palaniappan, K. K.; Gao, Z. G.; Jacobson, K. A.; Moon, H. R. J. Med. Chem.
2007, 50, 3159.
11. (a) Tchilibon, S.; Joshi, B. V.; Kim, S. K.; Duong, H. T.; Gao, Z. G.; Jacobson, K. A. J.
Med. Chem. 2005, 48, 1745; (b) Lee, K.; Ravi, R. G.; Ji, X.-d.; Marquez, V. E.;
Jacobson, K. A. Bioorg. Med. Chem. Lett. 2001, 11, 1333.
rapid filtration through Whatman GF/C filters and washed once
with cold buffer. Bound radioactivity was measured by liquid scin-
tillation spectrometry.
6.2.4. [35S]GTP
35S]GTP
S binding was measured by a variation of the method
described.24 Each assay tube consisted of 200
L buffer containing
50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 1 mM MgCl2, 1 M GDP,
1 mM dithiothreitol, 100 mM NaCl, 3 U/ml ADA, 0.2 nM
35S]GTP
S, 0.004% 3-[(3-cholamidopropyl) dimethylammo-
cS binding assay
[
c
l
l
[
c
nio]propanesulfonate (CHAPS), and 0.5% bovine serum albumin.
Incubations were started upon addition of the membrane suspen-
sion (CHO cells stably expressing either the native human A1AR or
A3AR, 5 lg protein/tube) to the test tubes, and they were carried
out in duplicate for 30 min at 25 °C. The reaction was stopped by
rapid filtration through Whatman GF/B filters, pre-soaked in
50 mM Tris–HCl, 5 mM MgCl2 (pH 7.4) containing 0.02% CHAPS.
The filters were washed twice with 3 mL of the same buffer, and
retained radioactivity was measured using liquid scintillation
12. Kim, H. O.; Hawes, C.; Towers, P.; Jacobson, K. A. J. Label. Comp. Radiopharm.
1996, 38, 547.
13. (a) Joshi, B. V.; Melman, A.; Mackman, R. L.; Jacobson, K. A. Nucleosides
Nucleotides Nucleic Acids 2008, 27, 279; (b) Joshi, B. V.; Moon, H. R.; Fettinger, J.
C.; Marquez, V. E.; Jacobson, K. A. J. Org. Chem. 2005, 70, 439.
14. (a) Barton, D. H. R.; Samadi, M. Tetrahedron 1992, 48, 7083; (b) Gawronska, K.;
Gawronski, J.; Walborsky, H. M. J. Org. Chem. 1991, 56, 2193.
15. Yamaguchi, K.; Kazuta, Y.; Abe, H.; Matsuda, A.; Shuto, S. J. Org. Chem. 2003, 68,
9255.
counting. Nonspecific binding of [35S]GTP
presence of 10 M unlabeled GTP S.
cS was measured in the
l
c
16. Chatgilialoglu, C. Acc. Chem. Res. 1992, 25, 188–194.
17. (a) Kim, S.-K.; Gao, Z.-G.; Van Rompaey, P.; Gross, A. S.; Chen, A.; Van
Calenbergh, S.; Jacobson, K. A. J. Med. Chem. 2003, 46, 4847; (b) Kim, S.-K.; Gao,
Z.-G.; Jeong, L. S.; Jacobson, K. A. J. Mol. Graphics Model 2006, 25, 562.
18. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
19. (a) Schwabe, U.; Trost, T. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1980, 313,
179; (b) Perreira, M.; Jiang, J. K.; Klutz, A. M.; Gao, Z. G.; Shainberg, A.; Lu, C.;
Thomas, C. J.; Jacobson, KA. J. Med. Chem. 2005, 48, 4910.
Acknowledgments
We thank Dr. John Lloyd and Dr. Noel Whittaker (NIDDK) for
mass spectral determinations. This research was supported by
the Intramural Research Program of the NIH, National Institute of
Diabetes and Digestive and Kidney Diseases.
20. Jarvis, M. F.; Schutz, R.; Hutchison, A. J.; Do, E.; Sills, M. A.; Williams, M. J.
Pharmacol. Exp. Ther. 1989, 251, 888–893.
21. Olah, M. E.; Gallo-Rodriguez, C.; Jacobson, K. A.; Stiles, G. L. Mol. Pharmacol.
1994, 45, 978.
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