Functionalized congeners of A3 adenosine receptor-selective nucleosides containing a bicyclo[3.1.0]hexane ring system

Article abstract of DOI:10.1021/jm900426g

(N)-Methanocarba nucleosides containing bicyclo[3.1.0]hexane replacement of the ribose ring previously demonstrated selectivity as A₃ adenosine receptor (AR) agonists (5′-uronamides) or antagonists (5′-truncated) . Here, these two series were m

Full text of DOI:10.1021/jm900426g

7580 J. Med. Chem. 2009, 52, 7580–7592
DOI: 10.1021/jm900426g
Functionalized Congeners of A3 Adenosine Receptor-Selective Nucleosides Containing a
Bicyclo[3.1.0]hexane Ring System†
Dilip K. Tosh,‡ Moshe Chinn,‡ Andrei A. Ivanov,‡,§ Athena M. Klutz,‡ Zhan-Guo Gao,‡ and Kenneth A. Jacobson*,‡
‡Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, Maryland 20892, and §Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road,
Rollins Research Center, Atlanta, Georgia 30322
Received April 2, 2009
(N)-Methanocarba nucleosides containing bicyclo[3.1.0]hexane replacement of the ribose ring pre-
viously demonstrated selectivity as A3 adenosine receptor (AR) agonists (50-uronamides) or antagonists
(50-truncated). Here, these two series were modified in parallel at the adenine C2 position. N6-3-
Chlorobenzyl-50-N-methyluronamides derivatives with functionalized 2-alkynyl chains of varying
length terminating in a reactive carboxylate, ester, or amine group were full, potent human A3AR
agonists. Flexibility of chain substitution allowed the conjugation with a fluorescent cyanine dye (Cy5)
and biotin, resulting in binding Ki values of 17 and 36 nM, respectively. The distal end of the chain was
predicted by homology modeling to bind at the A3AR extracellular regions. Corresponding L-nucleo-
sides were nearly inactive in AR binding. In the 50-truncated nucleoside series, 2-Cl analogues were more
potent at A3AR than 2-H and 2-F, functional efficacy in adenylate cyclase inhibition varied, and
introduction of a 2-alkynyl chain greatly reduced affinity. SAR parallels between the two series lost
stringency at distal positions. The most potent and selective novel compounds were amine congener 15
(Ki=2.1 nM) and truncated partial agonist 22 (Ki=4.9 nM).
Introduction
to maintain A3AR selectivity in both human and murine
species, while most of the non-purine antagonists are selective
for the human (h) but not rat (r) A3AR. Modification of the
ribose moiety of adenosine derivatives, and to a lesser extent
N6 and C2 substituents, has been shown to reduce agonist
efficacy and thus be useful for converting A3AR agonists into
antagonists.12,13
Selective agonist and antagonist ligands of the A3 adenosine
receptor (ARa), a G-protein-coupled receptor (GPCR), have
been reported.1 A3AR antagonists are proposed for the treat-
ment of cancer, inflammation, glaucoma, and asthma.2-5
A3AR agonists are already in clinical trials for liver cancer,
rheumatoid arthritis, psoriasis, and dry eye disease,6,7 with
additional envisioned applications for bowel inflammation,
ischemia, and other autoimmune inflammatory disorders.8-10
Selective A3AR antagonists encompass both non-purine
heterocyclic antagonists1,11 and nucleoside12,13 antagonists.
Nucleoside-derived A3AR antagonists have the advantage of
a species-independent pharmacological profile;4 i.e., they tend
Both the binding and selectivity of adenosine derivatives as
prototypical A3AR agonists can be substantially increased by
replacing the flexible ribose scaffold with a rigid bicyclo[3.1.0]
hexane ring system resulting in a class of carbocyclic nucleo-
sides related to 1 (Chart 1).14-16 These (N)-methanocarba
adenosine agonists have been found to possess high potency
and enhanced selectivity for the A3AR,11 and an agonist of
this class (MRS3558, 1a) has been shown to display antiar-
thritic activity and protection in a model of lung ischemia.17,18
The N6-3-iodobenzyl derivative 1b (MRS1898) was recently
radioiodinated to form an A3AR agonist radioligand that can
be used selectively in murine and in hA3AR binding studies.19
We describe in this work two new series of selective A3AR
ligands that extend recent reports, i.e., agonists and antago-
nists of the (N)-methanocarba family, derived through largely
parallel modifications at the adenine C2 position.20,21 Thus,
the new agonists contained a 50-N-methyluronamide group,
and this amide group was removed entirely in the truncated
series. The principle of lowering the relative efficacy of full
agonists by removing a flexible 50-amide group to produce, in
some cases, hA3AR antagonists was shown for 40-thionucleo-
sides by Jeong et al. and for (N)-methanocarba analogues
by Melman et al. (i.e., 2a-c).13,20 The stereospecificity of
binding to the ARs was also probed with the preparation of
† Contributed to mark the 100th anniversary of the Division of
Medicinal Chemistry of the American Chemical Society.
*To whom correspondence should be addressed. Address: Molecular
Recognition Section, Bldg 8A, Rm B1A-19, NIH, NIDDK, LBC,
Bethesda, MD 20892-0810. Phone: 301-496-9024. Fax: 301-480-8422.
E-mail: kajacobs@helix.nih.gov.
a Abbreviations:AR, adenosinereceptor;cAMP, adenosine30,50-cyclic
phosphate; CCPA, 2-chloro-N6-cyclopentyladenosine; CHO, Chinese
hamster ovary; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-50-N-methyl-
carboxamidoadenosine; DMEM, Dulbecco’s modified Eagle’s medium;
EDTA, ethylenediaminetetraacetic acid; GPCR, G-protein-coupled re-
ceptor; HATU, 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate methanaminium; HEK, human embryonic
kidney; I-AB-MECA, 2-[p-(2-carboxyethyl)phenylethylamino]-50-N-eth-
ylcarboxamidoadenosine; NECA, 50-N-ethylcarboxamidoadenosine; DI-
PEA, diisopropylethylamine; DCM, dichloromethane; DMF, N,N-di-
methylformamide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfo-
nic acid; HRMS, high resolution mass spectroscopy; PET, positron
emission tomography; TEA, triethylamine; TLC, thin layer chromatog-
raphy.
r
pubs.acs.org/jmc
Published on Web 06/08/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7581
Chart 1. (N)-Methanocarba Derivatives of Adenosine as A3AR-Selective Agonists (1a, 1b) and Antagonists (2a, 2b)a
a General formulas I and II represent two parallel structural series included in the present study. The agonist corresponding to X=Cl, n=3, m=2,
R=H in series II was already reported.21
corresponding L-nucleosides. In both series, the conforma-
tionally restricted (N)-methanocarba scaffold was retained,
and most of the analogues contained a substituted 6-(benzyl-
amino) moiety. The most structurally diverse modifications
were made at the adenine C2 position, with the addition
of extended alkynyl chains terminating in a carboxylic,
ester, or amino group, following a functionalized congener
approach.22,23 This terminal functionalized group was remote
from the minimal pharmacophore required for recognition at
the primary binding site of the receptor. The intended appli-
cation of the functional groups is for covalent coupling
to various other moieties, including spectroscopic reporter
groups, without losing biological activity.
of amine congeners. The biotin conjugate 17a was prepared
by a HATU coupling with biotin, and the extended chain
analogue 17b was prepared from the corresponding biotin-ε-
aminocaproyl active ester. The fluorescent derivative 18
similarly was prepared by treating 11 with the active ester
of a chain-derivatized Cy5 cyanine dye.27
Selected L-enantiomers 19-21 of the 50-N-methylurona-
mide methanocarba agonist series were also prepared, as
shown in Scheme 2. The isopropylidene intermediate 31 was
prepared from D-ribose as previously reported. A Mitsunobu
condensation21 with 2-iodo-6-chloropurine provided 32,
which was first converted to the N6-(3-chlorobenzyl) deriva-
tive 33 and then treated with excess methylamine to yield 34.
This 2-iodo intermediate was subjected to a Sonogashira
coupling25 with the corresponding alkyne esters to provide
compounds 35-37 of varying alkynyl chain length. Esters 35
and 37 were aminolyzed with ethylenediamine followed by
hydrolysis of the acetonide group with 10% TFA to provide
compounds 20 and 21. In the case of compound 36, the 20,30-
isopropylidene group was first deprotected, and hydrolysis
of the ester afforded the carboxylic acid derivative 19.
Scheme 3 shows the new synthetic route used to prepare
the truncated (N)-methanocarba derivatives 22-26. While
the previous study of truncated (N)-methanocarba nucleo-
sides as antagonists of the hA3AR depended on a low
yielding decarboxyation step, an alternative simple synthetic
approach was designed. The protected cyclopentenone 38
was stereoselectively reduced to the corresponding alcohol
39,28 which was then subjected to cyclopropanation to yield
the glycosyl donor 40 for the truncated nucleosides and
therefore converged on the previously reported route.20
Compound 40 was subjected to a Mitsunobu condensation20
with the appropriate C2 substituted 6-chloropurine deriva-
tive to give the 2-H (41) and 2-halopurine (42, 43) derivatives.
The 2-I derivative 44 was prepared in a similar manner and
was substituted with 3-chlorobenzylamine at the 6 position
to give 45, which serves as a precursor for 2-alkynyl deriva-
tives in the truncated series. This route afforded the isopro-
pylidene-protected truncated nucleosides 46-49, which were
subsequently deprotected to provide compounds 22-25 for
biological testing.
Results
Chemical Synthesis. In this study, we have elaborated and
extended functionalization at the C2 position of previously
reported (N)-methanocarba nucleoside derivatives, which
were shown to be selective A3AR agonists15,16,21 and antago-
nists.20 The series of 50-N-methyluronamides (Chart 1, II) are
similar to the reported agonists 1, and the series of truncated
nucleosides (I) are similar to the reported antagonists 2.
Here, these two series were modified in parallel at the adenine
C2 position.
The synthetic route to the 50-N-methyluronamide (N)-
methanocarba derivatives 5-16 is shown in Scheme 1A.
The synthesis of the 20,30-protected intermediate 27, contain-
ing an N6-(3-chlorobenzyl) group, from L-ribose was pre-
viously reported.24 This 2-iodo intermediate was then
subjected to Sonogashira coupling25 with the corresponding
alkyne esters. The products 28-30 of varying alkynyl chain
length were deprotected to liberate the 20,30-hydroxyl groups
to provide nucleosides 8-10, followed by hydrolysis of the
methyl ester to afford agonists 5-7. Alternatively, com-
pounds 28-30 were aminolyzed using the appropriate alkyl-
diamine in excess, and the product amines were deprotected
to yield the amine functionalized agonists 11-16 of varying
chain length.
The amino and carboxylic derivatives contained chemi-
cally functionalized, extended alkynyl chains to serve as
functionalized congeners for conjugation to other biologi-
cally active moieties or to carriers.23,26 Several representative
biologically active conjugates for the detection and char-
acterization of the A3AR were prepared as shown in
Scheme 1B. Biotin conjugates of varying length 17a and
17b were synthesized from the amine 11, which was the
analogue having the shortest chain in the homologous series
A truncated 2-iodo intermediate 45 was subjected to
Sonogashira coupling25 with methyl pentynate to give com-
pound 50, which upon amination with ethylenediamine and
subsequent acid hydrolysis provided the truncated nucleo-
side 26 containing an amine functionalized chain.
Pharmacological Activity. Several previously reported 2-
chloro-(N)-methanocarba agonists (1a, 1b, 3, 4, 6, 9, and 12)

7582 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Tosh et al.
Scheme 1a
a (A) (i) HCꢀC(CH2)nCOOMe, Pd(PPh3)2Cl2, CuI, Et3N, DMF, room temp; (ii) 10% TFA, MeOH, 70 °C; (iii) appropriate alkyldiamine, MeOH,
room temp; (iv) KOH, MeOH, room temp. (B) (v) For 17a: biotin, HATU, DIEA, DMF, room temp. For 17b: biotin-ε-aminocaproyl N-succinimidyl
ester, Et3N, DMF, room temp. (vi) N-succinimidyl ester of chain-derivatized Cy5 cyanine dye, bicarbonate buffer, DMF.
and antagonists (2a-c) were used for comparison in the
biological assays (Table 1). Binding assays at three hAR
subtypes were carried out using standard radioligands29-31
and membrane preparations from Chinese hamster ovary
(CHO) cells (A1 and A3) or human embryonic kidney
(HEK293) cells (A2A) stably expressing a hAR subtype
(Table 1).32,33 Since activity within the class of (N)-metha-
nocarba nucleosides was previously noted to be very weak or
absent at the hA2BAR,16 we did not include this receptor in
the screening protocol. Functional data were determined in
an assay of adenylate cyclase (A3AR-induced inhibition).34
Functional data for selected compounds in a functional
assay consisting of A3AR-induced stimulation of guanine
nucleotide binding are reported in Table 1.35
Compounds 5-15 in the (N)-methanocarba 50-N-methyl-
uronamide series (series A), which had functionalized
2-alkynyl chains terminating in a carboxylic acid, ester, or
amino group, were potent A3AR ligands. These derivatives
having charged, polar, and chemically reactive groups at the
end of a chain were intended for structural probing of distal
regions of the ligand binding site of the receptor and for the
design of specialized high-affinity ligands, such as fluores-
cent probes.
reported compound 6,21 which had three methylenes in the
chain. The corresponding ester derivative 921 was also the
most potent in the series of homologous esters 8-10.
A shorter chain carboxylic acid derivative 5 was considerably
less potent than other members of the series in binding to the
hA3AR.
The carboxylic acid derivatives were then extended by
amide linkage to diaminoalkyl groups. In the series of
resulting terminal amino derivatives 11-13, the length of
the carboxyl alkyl chain was varied, and in the series of 11
and 14 and 15 the length of the diaminoalkyl chain was
varied. While 12 displayed an atypically high A1AR affinity
(Ki=454 nM) and 13 an atypically high A2AAR affinity (Ki=
270 nM), most of the members of these series were highly
selective for the A3AR. The Ki values at the hA3AR of the
primary amino derivatives 11-15 of varying chain length
were in the range of 2-3 nM. The most potent and selective
novel member of this series, an amine derivative 15 (Figure 1,
Table 1), was 360 and >5000-fold selective in binding to the
hA3AR in comparison to hA1 and A2AARs, respectively.
Compound 16, containing two free amino groups in the
chain, one primary and another secondary, was slightly
reduced in affinity at the A3AR with a Ki value of 15.4 nM.
As functionalized congeners, these long chain deriva-
tives were intended for conjugation to carrier moieties for
The optimal chain length for A3AR affinity among
carboxylic acid derivatives occurred with the previously

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7583
Scheme 2a
a (i) 2-Iodo-6-chloropurine, Ph3P, DIAD, THF, room temp; (ii) 3-chlorobenzylamine, Et3N, MeOH, room temp; (iii) 10% methylamine, MeOH,
room temp; (iv) HCꢀC(CH2)nCOOMe, Pd(PPh3)2Cl2, CuI, Et3N, DMF, room temp; (v) KOH, MeOH; (vi) ethylenediamine, MeOH, room temp;
(vii) 10% TFA, MeOH, 70 °C.
receptor detection and characterization, in a manner that did
not prevent receptor recognition.22,23 A biotin conjugate 17b
derived from amine 11 in the series of 50-N-methyluronamide
derivatives displayed a Ki value at the hA3AR of 58 nM
(Figure 1) and selectivity of >170-fold and 66-fold in
comparison to the A1 and A2AAR, respectively. A shorter
analogue 17a lacking the ε-aminocaproyl spacer chain was
slightly more potent in hA3AR binding with a Ki value of
36 nM. A fluorescent conjugate 18 containing the sulfonated
carbocyanine dye Cy527 was potent and selective in A3AR
binding. The fluorescent excitation and emission maxima of
18 in aqueous medium, 646 and 660 nm, respectively, were
characteristic of Cy5 dye conjugates.
Various compounds were examined for the ability to
inhibit the production of adenosine 30,50-cyclic phosphate
(cAMP) in membranes of CHO cells expressing the hA3AR
(Table 1).34 Inhibition by 10 μM NECA (51, 50-N-ethylcar-
boxamidoadenosine) was set at 100% relative efficacy.
The (N)-methanocarba 50-N-methyluronamide derivatives
(series A) were consistently full agonists. Full concentration
response curves for compounds 17a and 18 provided IC50
values in hA3AR-mediated inhibition of adenylate cyclase
of 2.50(0.42 and 1.09(0.28 nM, respectively (Figure 2A).
Thus, this series provided many new potent and selective
A3AR agonists, and alkynyl chain derivatization at the C2
position appears to be an effective strategy for covalent
attaching sterically bulky groups while maintaining A3AR
recognition and activation.
A series of L-nucleosides was prepared as enantiomers of
members of the potent agonist functionalized congeners
(series B). Compounds 19, 20, and 21 were the enantiomers
of the potent A3AR agonists 5, 11, and 13, respectively. We
confirmed that at submicromolar concentrations no sub-
stantial AR binding activity was seen with these L-enantio-
mers, confirming stereoselectivity of binding at the ARs. At
10 μM, 19-21 were weak or inactive in binding at the A1 and
A2AARs, and at the A3AR they tended to be slightly more
potent in binding. These L-nucleosides induced partial acti-
vation of the A3AR at 10 μM, as confirmed in the cAMP
functional assay in which compounds 20 and 21 activated to
the degree of 26% and 52%, respectively. Nevertheless, these
L-nucleosides can be used at nanomolar concentrations in
pharmacological studies as inactive control compounds of
similar physicochemical properties, by analogy to the use of
inactive enantiomers of other GPCRs.36 At these concentra-
tions, interactions with ARs were lacking. In the truncated
40-thionucleoside class of A3AR antagonists, stereospecifi-
city of binding was similarly demonstrated.45
The effects of modification at the C2 position were ex-
plored in the truncated series C. In the 50-N-methylurona-
mide derivatives (series A), even the presence of a long chain
at the C2 position did not interfere with the binding and
activation of the A3AR. However, in the series of nucleosides
that were truncated at the 40-carbon, variability in both
binding affinity and efficacy at the hA3AR was observed,
depending on the nature of the C2 substitution. Compounds

7584 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Tosh et al.
Scheme 3a
a (i) NaBH4, CeCl3 7H2O, MeOH, 0 °C; (ii) Et2Zn, CH2I2, CH2Cl2, room temp; (iii) 2-substituted-6-chloropurine, Ph3P, DIAD, THF, room temp;
3
(iv) 3-iodo- or 3-chlorobenzylamine or cyclopentylamine, Et3N, MeOH, room temp; (v) 10% TFA, MeOH, 70 °C; (vi) HCꢀC(CH2)2COOMe,
Pd(PPh3)2Cl2, CuI, Et3N, DMF, room temp; (vii) ethylenediamine, MeOH, room temp.
22-24, lacking a functionalized chain at the C2 position,
were potent and selective A3AR ligands with binding Ki
values of 5-11 nM. Removal of the 2-Cl substitution in
N6-benzyl-type 40-truncated derivatives (e.g., 22, 23) or its
replacement with 2-F (e.g., 24) maintained A3AR selectivity
but reduced the A3AR affinity by 5- to 10-fold. Replacement
of 2-Cl with 2-H either had no effect (cf. 2a and 22) on affinity
at the A2AAR or moderately reduced it (cf. 2c and 23), and
affinity at the A1AR was moderately increased. The most
A3AR selective truncated compound was the 2-unsubsti-
tuted N6-(3-chlorobenzyl) derivative 22 with 330-fold and
940-fold selectivity in comparison to the A1 and A2AAR,
respectively.
An amine functionalized congener 26 in the truncated
series, with a Ki value of 403 nM, proved to be weaker in
binding to the A3AR than those analogues with less sterically
bulky substitution at the C2 position. However, it never-
theless displayed moderate selectivity of >20-fold in com-
parison to both the A1 and A2AARs. This derivative was
analogous to compound 11 in the 50-N-methyluronamide
series, which was 160-fold more potent in binding to the
A3AR.
substitution of the (N)-methanocarba nucleosides preserved
the AR selectivity profile upon truncation better than an
N6-cyclopentyl group.
Selected compounds in the present series were tested in
binding at the rA3AR. The Ki values (nM) were determined
to be the following: 6, 66.5 ( 3.9; 15, 8.68 ( 0.85; 18, 9.62 (
1.50; 22, 20.3 ( 2.0. Thus, the high A3AR affinity was
maintained in a murine species.
The A3AR efficacy in the cAMP functional assay varied
greatly in this series of truncated (N)-methanocarba deriva-
tives. Compounds 2b and 2c, which were reported as A3AR
antagonists based on an assay of [35S]GTPγS binding,20 were
partial agonists in the assay of adenylate cyclase (Figure 2B).
Both partial (e.g., 22) and nearly full agonism (e.g., 23 and
25) were observed for the novel truncated nucleosides in
the cyclase assay. Replacement of the 2-Cl of the truncated
analogues 2b with hydrogen in 22 resulted in a 58% relative
efficacy, which was comparable to 46% for 2b. The 2-H 23
and 2-F 24 N6-(3-iodobenzyl) analogues displayed relative
efficacy of 81% and 19%, respectively. Compound 25 in-
hibited the production of cAMP via the hA3AR to the same
extent as 51 and was therefore a full agonist as judged by
adenylate cyclase inhibition.
A 50-truncated N6-cyclopentyl derivative 25, by analogy to
compound 4,37 was intended to display enhanced affinity at
both A1 and A3ARs but not at the A2AAR. However, the loss
of the 50-N-methyluronamide group reduced affinity at A1
and A3ARs by 6- and 32-fold, respectively. Thus, N6-benzyl
A homology model of the hA3AR based on the A2AAR X-
ray structure38,39 was used to study the putative interactions
between the chain-functionalized agonist 15 and the A3AR.
The binding mode of 15 obtained after InducedFit docking

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7585
Table 1. Potency of a Series of (N)-Methanocarba Adenosine Derivatives at Three Subtypes of Human ARs and the Functional Efficacy at the A3AR
structure
affinity (Ki, nM) or % inhibitiona
compd
R1
R2
A1
A2A
A3
% efficacy,b A3
Series A
1ac
1b
3c
Cl
Cl
3-Cl-Bn
3-I-Bn
3-I-Bn
260 ( 60h
136 ( 22d,h
700 ( 270h
18.3 ( 6.3h
(17 ( 4%)
14,900 ( 3500h
(11 ( 5%)
(41 ( 0%)
482 ( 23h
(20 ( 7%)
2320 ( 520
454 ( 44h
(47 ( 3%)
(31 ( 9%)
(30 ( 2%)
(33 ( 2%)
(1 ( 1%)
2300 ( 100
784 ( 97d
6200 ( 100
3250 ( 300
(24 ( 12%)
(43%)
0.29 ( 0.04
1.5 ( 0.2c
2.4 ( 0.5
103 ( 7
100c
100
H
Cl
4c
cyclopentyl
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3-Cl-Bn
3.7 ( 0.9
101
106 ( 18
ND
5
CꢀC(CH2)2COOH
CꢀC(CH2)3COOH
CꢀC(CH2)4COOH
CꢀC(CH2)2COOCH3
CꢀC(CH2)3COOCH3
CꢀC(CH2)4COOCH3
61.1 ( 35.8
2.38 ( 0.56
12.4 ( 1.8
5.5 ( 0.3
1.17 ( 0.27
11.1 ( 2.3
2.5 ( 0.5
2.17 ( 0.51
3.4 ( 0.7
3.1 ( 0.4
6c,e
7
(38 ( 2%)
(43 ( 5%)
(49%)
74.4 ( 5.1
90.0 ( 6.2
ND
8
9c
10
11
12c
13
14
15
16
17a
17b
18
3540 ( 1370
550 ( 83
(81%)
85.0 ( 10.2
96.9 ( 6.8
ND
CꢀC(CH2)2CONH(CH2)2NH2
CꢀC(CH2)3CONH(CH2)2NH2
CꢀC(CH2)4CONH(CH2)2NH2
277 ( 33
100 ( 2
97.9 ( 18.4
102 ( 13
87.6 ( 21.2
84.5 ( 12.0
107 ( 18
94.4 ( 9.6
CꢀC(CH2)2CONH(CH2)3NH2
979 ( 181
766 ( 109
890 ( 110
(51 ( 2%)
(47 ( 11%)
4730 ( 1020
CꢀC(CH2)2CONH(CH2)4NH2
2.1 ( 0.4
CꢀC(CH2)2CO[NH(CH2)2]2NH2
CꢀC(CH2)2CONH(CH2)2NH-biotin
CꢀC(CH2)2CONH(CH2)2NH-CO(CH2)5NH-biotin
CꢀC(CH2)2CONH(CH2)2NH-CO-(CH2)5Cy5g
15.4 ( 4.4
36.4 ( 5.6
57.7 ( 16.2
17.2 ( 3.1
(12 ( 4%)
(36 ( 3%)
Series B
3-Cl-Bn
3-Cl-Bn
19
20
21
CꢀC(CH2)3COOH
(0%)
(33 ( 7%)h
(9 ( 2%)
(8 ( 1%)
(5 ( 1%)
5270 ( 1090
(30 ( 1%)
4630 ( 1060
ND
25.6 ( 10.9
52.0 ( 9.4
CꢀC(CH2)2CONH(CH2)2NH2
CꢀC(CH2)4CONH(CH2)2NH2
3-Cl-Bn
(36 ( 5%)h
Series C
3-Cl-Bn
3-Br-Bn
3-I-Bn
2ad
2bd,e
2cd,e
22
Cl
Cl
3070 ( 1500h
1760 ( 1010h
3040 ( 610h
1600 ( 150
839 ( 68
513 ( 4
109 ( 16
(15 ( 2%)
4510 ( 910
1600 ( 480
1080 ( 310
4520 ( 830
2330 ( 680
4000 ( 780
1640 ( 360
(35 ( 6%)
1.06 ( 0.36
0.73 ( 0.30
1.44 ( 0.60
4.9 ( 0.7
11.0 ( 2.2
10.7 ( 0.9
120 ( 31
2.9 ( 3.7f
46 ( 4,5.8 ( 0.8f
44 ( 6, 1.0 ( 3.2f
58.3 ( 8.0
Cl
H
3-Cl-Bn
3-I-Bn
3-I-Bn
23
24
H
80.8 ( 19.0
19.0 ( 3.0
95.1 ( 4.6
F
Cl
25
26
cyclopentyl
3-Cl-Bn
CꢀC(CH2)2CONH(CH2)2NH2
404 ( 67
0.9 ( 8.5
a All experiments were done on CHO or HEK293 (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]51, [3H]54, or [125I]53, respectively), unless
otherwise noted. A percent in parentheses refers to inhibition of radioligand binding at 10 μM. b Unless otherwise noted, the efficacy at the human A3AR
was determined by inhibition of forskolin-stimulated cAMP production in AR-transfected CHO cells. At a concentration of 10 μM, in comparison to the
maximal effect of 51 (=100%) at 10 μM. Data are expressed as mean ( standard error (n = 3). ND, not determined. c Values from Melman et al.; Lee et
al.; Tchilibon et al.15,16,21 d Values from Melman et al.20 e 2b, MRS5147; 2c, MRS5127; 6, MRS5151.21,33 f A3AR functional assay consisted of
stimulation of [35S]GTPγS binding at 10 μM, expressed as a percentage of the full effect induced by 10 μM 51 (100 ( 5%). g Structure given in Scheme 1B.
h A1AR binding determined using [3H]52.
revealed the following binding mode of the ligand (Figure 3).
The 20- and 30-hydroxyl groups were located in proximity to
Ser271 (7.42) and His272 (7.43) and could form H-bonds
with these residues. The oxygen atom of the 50-N-methylcar-
boxamido moiety was involved in H-bonding with the side
chain hydroxyl group of Thr94 (3.36). Both N6-amino group
and the N7 atom of the adenine ring formed H-bonds with
Asn250 (6.55). A π-π interaction was observed between the
adenine ring of 15 and Phe168 (EL2). The ligand-receptor
interactions observed in the present model were in good
agreement with the data of site-directed mutagenesis and
with our previously published models of ARs, including the
studies of AR agonists docked to the A2AAR crystal struc-
ture.38,40 In the model obtained, the 3-chlorobenzyl ring was
located in the hydrophobic pocket formed by Val169 (EL2),
Met172 (EL2), Met174 (EL2), Met177 (5.38), and Ile253(EL3).
The long chain substituent at the C2 position of 15 was
oriented toward the extracellular part of the receptor, which
is a prerequisite in functionalized congeners.23 A similar
orientation of the 2-propynyl fragment of 15 was previ-
ously suggested for C2 substituted agonists of the A2B
receptor.41,42 The carbonyl oxygen of the amide group of

7586 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Tosh et al.
Figure 1. Inhibition of radioligand binding by (N)-methanocarba
nucleoside analogues in membranes of CHO cells expressing the
human A3AR. Inhibition curves are shown for the agonist analo-
gues containing a 50-N-methyluronamide group and compounds 15
(amine functionalized congener), 17b (biotinylated probe), and 18
(fluoresecent probe) and for the truncated partial agonist 22. All of
the analogues shown were highly selective for the A3AR in compar-
ison to the A1 and A2AARs.
Figure 3. Binding mode of amine congener 15 in the 50-N-methyl-
uronamide series obtained after InducedFit docking to the A3AR
homology model built based on the crystal structure of the A2AAR.
(N)-methanocarba nucleosides, in cases with substituted
N6-benzyl groups and small substituents present at the ade-
nine C2 position. This parallel behavior was not maintained in
a derivative bearing a chain at the C2 position.
The replacement of the adenine 2-Cl with H in the series of
substituted N6-benzyl-(N)-methanocarbaagonists containing
the 50-CONHCH3 group was previously shown to have only
minor effects on AR binding.16 Specifically, it moderately
reduced affinity at the A1 and A2AARs without a significant
reduction at the A3AR (cf. 1a and 3). In the present 50-N-
methyluronamide series with more elaborate modification at
the C2 position, the preservation of potent and selective
binding to the hA3AR was demonstrated.
The flexibility of chain substitution at C2 allowed the
conjugation with sterically bulky moieties. This distal region
of the nucleosides was predicted by homology modeling to be
at A3AR extracellular regions and consequently free from the
steric constraints present at the pharmacophore binding
region. Thus, this series of highly potent and selective A3AR
agonist functionalized congeners is suitable for conjugation to
prosthetic groups for radiolabeling, fluorescent detection,
avidin complexation, and polymeric coupling with retention
of biological activity. The functionalized congener approach
was already applied to 1,4-dihydropyridine derivatives as
antagonists of the hA3AR.26
Specifically, agonists in this series were conjugated with
biotin and a fluorescent dye with retention of high A3AR
affinity and selectivity. The fluorescent conjugate 18 was a
highly potent, full agonist with a Ki values at the hA3AR and
the rA3AR of 17.2 and 9.6 nM, respectively. The fluorescent
dye Cy5, which was incorporated covalently in 18, has favor-
able properties for use in biological systems including near-
infrared imaging, with excitation and emission maxima at
649 and 670 nm, respectively, and with a relatively high
quantum yield of 0.28.27 Another fluorescent agonist of the
hA3AR having lower affinity than 18 was used in fluorescence
correlation spectroscopy to demonstrate that the receptor
Figure 2. Functional agonism by the tested in an assay of adenylate
cyclase in membranes of CHO cells expressing the hA3AR. Each
experiment was repeated three times. (A) Activity of the 50-N-
methyluronamide (N)-methanocarba analogues 17a and 18. The
EC50 values of 17a and 18 were 2.50 ( 0.42 and 1.09 ( 0.28 nM,
respectively. (B) Activity of the truncated (N)-methanocarba ana-
logues 2b and 2c at the hA3AR. The full agonist 51 was included for
comparison (representing 100% efficacy). The percent relative
efficacy of 2b and 2c was 46 ( 4% and 44 ( 6%, respectively. The
EC50 values of 2b and 2c were 4.2(0.6 and 12(1 nM, respectively.
the C2 substituent of 15 was H-bonded to the side chain
NH2-group of Gln167 (EL2), and consequently the NH-
group of the C2 substituent of 15 was oriented toward the
hydroxyl group of Tyr265 (7.36) (Figure 3). The terminal
amine of the ligand interacted with negatively charged
Glu258 (EL3), and it was also H-bonded to the backbone
oxygen atom of Val259 (EL3).
Discussion
In the present work and in a previous study,20 parallel
patterns of SAR in receptor binding were observed in the
two series of 50-CONHCH3 (full agonist) and truncated

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7587
exists in heterogeneous microdomains of individual living
cells.43
agonists, partial agonists, and antagonists. Furthermore, in
the 50-N-methyluronamide series but not in the truncated
series, the functionalized congener approach was found to
maintain potency and selectivity at the hA3AR and therefore
provides an entry into high affinity molecular probes. Thus,
the binding and activation profiles of the truncated analogues
indicated the biological interdependence of substitution at the
C2, 50, and N6 positions. Potent agonist ligands containing
biotin and fluorescent dye moieties should be useful in future
pharmacological studies of the localization, association, reg-
ulation, and internalization of the A3AR and might provide
an alternative to the use of radioligands for this receptor.
In our previous study of truncated (N)-methanocarba
nucleosides,20 the docking of two representative structures
in a rhodopsin-based homology model of the hA3AR indi-
cated that both (N)-methanocarba agonists and nucleoside-
derived antagonists were able to bind in a very similar mode
in the receptor binding site, which is common to other
A3AR-selective nucleosides, such as the prototypical agonist
2-chloro-N6-(3-iodobenzyl)-50-N-methylcarboxamidoade-
nosine (Cl-IB-MECA).20 We have shown that the SAR
parallels between these two series were less stringent with
respect to distal modification, e.g., when a long functionalized
chain was attached at the C2 position. Thus, in the truncated
series the attachment of a functionalized amino chain at the
C2 position greatly reduced A3AR affinity. Therefore, among
the 2-alkynyl nucleosides in this study, only those containing
a 50-N-methyluronamide group, i.e., full agonists, were suita-
ble as functionalized congeners.
Truncation of the 50-CONHCH3 moiety of A3AR agonists
in the (N)-methanocarba class was again demonstrated as
a means of reducing the relative efficacy of A3AR-selective
agonists to produce partial agonism, as indicated in the
cyclase assay. A previous study of 2-triazolo derivatives of
adenosine demonstrated modulation of the relative efficacy
at the A3AR by structural changes at the 2 position.12c In the
truncated series, the remaining efficacy at the hA3AR, as was
evident in a cAMP functional assay, ranged from small (e.g.,
24 and 26) to substantial (e.g., 23).
We compared selected compounds in the ability to stimu-
late the A3AR using multiple functional criteria. When sti-
mulation of [35S]GTPγS binding was used as a criterion of
receptor activation, truncated (N)-methanocarba nucleosides
(e.g., 2a-c) reported by Melman et al.20 did not induce a
substantial activation of the A3AR, with <10% of the full
agonism. However, in the current study using a different
functional assay that measured inhibition of the production
of cAMP to detect antagonism, considerable activity in the
truncated series was seen. The efficacy in the cyclase assay
generally exceeded the efficacy in [35S]GTPγS binding. Other
truncated nucleosides in the 40-thio series were A3AR antago-
nists in both [35S]GTPγS and cAMP assays.44 Recently,
truncated nucleosides in the 40-oxo series were demonstrated
to be A3AR antagonists in the cAMP assay.45
In the truncated series, removal of the 2-Cl substitution or
replacement with 2-F in 24 moderately reduced the A3AR
affinity but retained high selectivity. The introduction of
fluorine in 24 was intended for possible radiofluorination as
a means of preparing tracers for in vivo PET imaging of the
A3AR.47 One member of the previously reported truncated
(N)-methanocarba nucleoside series 2b, (1R,2R,3S,4R,5S)-
4-(6-(3-bromobenzylamino)-2-chloro-9H-purin-9-yl)bicyclo-
[3.1.0]hexane-2,3-diol (MRS5147), was recently labeled with
the short-lived positron-emitter 76Br to form a selective
hA3AR radioligand of high affinity for use in positron emis-
sion tomography (PET). This ligand was intended for imaging
of the A3AR in murine as well as primate species, because high
affinity binding was maintained at the rA3AR. Binding of
these truncated (N)-methanocarba nucleosides at the A3AR
was competitive,20 and therefore, the relatedderivatives in this
study should be useful as receptor probes.
Experimental Section
Chemical Synthesis. Materials and Instrumentation. D-Ri-
bose, L-ribose, and other reagents and solvents were purchased
from Sigma-Aldrich (St. Louis, MO). Alcohol derivative 31 was
prepared as reported.201H NMR spectra were obtained with a
Varian Gemini 300 spectrometer using CDCl3 and CD3OD as
solvents. Chemical shifts are expressed in δ values (ppm) with
tetramethylsilane (δ 0.00) for CDCl3 and water (δ 3.30) for
CD3OD. TLC analysis was carried out on aluminum sheets
precoated with silica gel F254 (0.2 mm) from Aldrich. All target
compounds are g95% pure as determined by HPLC. HPLC
mobile phases consisted of the following: for system A, linear
gradient solvent system of CH3CN/triethyl ammonium acetate
from 5/95 to 60/40 in 20 min and flow rate of 1.0 mL/min; for
ystem B, linear gradient solvent system of CH3CN/tetrabutyl
ammonium phosphate from 20/80 to 60/40 in 20 min and
flow rate of 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 column. High resolution mass spectroscopic
(HRMS) measurements were performed on a proteomics opti-
mized Q-TOF-2 (Micromass-Waters) using external calibration
with polyalanine, unless otherwise noted. Observed mass ac-
curacies are those expected on the basis of known performance
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.
(10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(4-meth-
oxycarbonyl-1-butynyl)-9-yl]-20,30-O-isopropylidenebicyclo[3.1.0]-
hexane-10-carboxylic Acid N-Methylamide (28). To a solution
of compound 27 (52 mg, 0.087 mmol) in anhydrous DMF
(1.5 mL), PdCl2(PPh3)2 (12 mg, 0.017 mmol), CuI (2 mg,
0.010 mmol), methyl-ω-pentynate (39 mg, 0.347 mmol), and
then triethylamine (0.12 mL, 0.86 mmol) were added. The
reaction mixture was stirred at room temperature overnight.
Solvent was evaporated under vacuum, and the residue was
purified on flash silica gel column chromatography (CH2Cl2/
MeOH=30:1) to give compound 28 (40 mg, 78%) as a foamy
syrup. 1H NMR (CD3OD, 300 MHz) δ 8.16 (s, 1H), 7.42-7.44
(m, 4H), 5.81 (d, J=6.7 Hz, 1H), 5.02 (s, 1H), 4.82 (m, 2H), 3.78
(s, 3H), 2.85 (s, 3H), 2.72-281 (m, 5H), 2.09-2.14 (m, 1H), 1.55
(s, 3H), 1.41 (t, J=5.1 Hz, 1H), 1.29 (s, 3H), 0.82-0.96 (m, 1H).
HRMS calculated for C29H32ClN6O5 (M + H)+: 579.2105;
found 579.2123.
(10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(4-(β-ami-
noethylaminocarbonyl)-1-butynyl)-9-yl]-20,30-dihydroxybicyclo
[3.1.0]hexane-10-carboxylic Acid N-Methylamide (11). To a solu-
tion of compound 28 (20 mg, 0.034 mmol) in methanol (0.3 mL),
ethylenediamine (1.5 mL) was added. The mixture was stirred
overnight at room temperature. Solvent was evaporated, and
the residue was roughly purified on flash silica gel column
chromatography. The aminated product was dissolved in
methanol (1.5 mL) and 10% trifluoroacetic acid (1.5 mL)
In conclusion, two highly potent and selective series of
nucleoside ligands containing the (N)-methanocarba ring
system have been synthesized and characterized as A3AR

7588 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Tosh et al.
and heated at 70 °C for 15 h. Solvent was evaporated and the
residue was purified on flash silica gel column chromatography
(CH2Cl2/MeOH/NH4OH = 10:1:0.1) to give compound 11
1.96-2.13 (m, 5H), 1.15-1.16 (m, 14H), 0.74-0.88 (m, 1H).
C43H57ClN11O7S (M+H)+: 906.3852; found 906.3878.
(10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(N-cya-
nine(β-aminoethylaminocarbonyl)-1-butynyl)-9-yl]-20,30-dihydrox-
ybicyclo[3.1.0]hexane-10-carboxylic Acid N-Methylamide (18).
To a solution of compound 11 (1.69 mg, 0.0029 mmol) in
DMF (0.3 mL), Cy5 fluorescent dye (2.36 mg, 0.0029 mmol)
and bicarbonate buffer (60 μL) were added. The mixture was
stirred at room temperature overnight. The reaction mixture
was covered with aluminum foil in order to protect from light.
Solvent was evaporated and the residue was purified on flash
silica gel column chromatography (CH2Cl2/MeOH/NH4OH=
3:1:0.1) to give compound 18 (3.2 mg, 89%) as a dark-blue
syrup. 1H NMR (CD3OD, 300 MHz) δ 8.21-8.42 (m, 1H), 8.08
(s, 1H), 7.85-7.93 (m, 2H), 7.29-7.46 (m, 6H), 6.28-6.45 (m,
1H), 5.07 (d, J=6.9 Hz, 1H), 4.84-4.86 (m, 2H), 3.97-4.20 (m,
3H), 3.53 (t, J=6.0 Hz, 2H), 3.12 (t, J=6.0 Hz, 2H), 2.89 (s, 3H),
2.49 (t, J=7.5 Hz, 2H), 2.52-2.64 (m, 2H), 2.01-2.17 (m, 2H),
1.56-1.89 (m, 9H), 1.22-1.49 (m, 10H), 0.86-0.96 (m, 1H).
HRMS calculated for C60H68ClIN10 O11S2 (M+): 1203.4199;
found 1203.4175.
1
(15 mg, 79%). H NMR (CD3OD, 300 MHz) δ 8.09 (s, 1H),
7.39 (s, 1H), 7.27-7.32 (m, 3H), 4.99 (d, J=6.6 Hz, 1H), 4.78-
4.91 (m, 2H), 3.97 (d, J=6.6 Hz, 1H), 3.33-3.36 (m, 4H), 2.87
(s, 3H), 2.75-2.80 (m, 4H), 2.53 (t, J=7.2 Hz, 2H), 2.59-3.41
(m, 1H), 1.88 (t, J=4.5 Hz, 1H), 1.34-1.43 (m, 1H). HRMS
calculated for C27H31ClN6O2Na (M+Na)+: 589.2068; found
589.2054.
(10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(4-meth-
oxycarbonyl)-1-butynyl)-9-yl]-20,30-dihydroxybicyclo[3.1.0]hex-
ane-10-carboxylic Acid N-Methylamide (8). A solution of com-
pound 28 (67 mg, 0.115 mmol) in methanol (3 mL) and 10%
triflromethanesulfonic acid (2 mL) was heated at 70 °C over-
night. Solvent was evaporated and the residue was purified on
flash silica gel column chromatography (CH2Cl2/MeOH=20:1)
to give compound 8 (46 mg, 75%). 1H NMR (CD3OD, 300
MHz) δ 8.13 (s, 1H), 7.40 (s, 1H), 7.22-7.45 (m, 3H), 5.10 (d, J=
6.3 Hz, 1H), 4.80-4.86 (m, 2H), 4.03 (d, J = 6.6 Hz, 1H), 3.63
(s, 3H), 2.86 (s, 3H), 2.76-2.85 (m, 2H), 2.46-2.58 (m, 2H),
2.05-2.10 (m, 1H), 1.77-1.83 (m, 1H), 1.35-1.40 (m, 1H),
0.82-0.96 (m, 1H). HRMS calculated for C26H28ClN6O5 (M+
H)+: 539.1731; found 539.1743.
(10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(4-hydro-
xycarbonyl)-1-butynyl)-9-yl]-20,30-dihydroxybicyclo[3.1.0]hex-
ane-10-carboxylic Acid N-Methylamide (5). To a solution of ester
8 (30 mg, 0.055 mmol) in methanol (1.5 mL), 1 M solution of
potassium hydroxide (1 mL) was added. The mixture was stirred
at room temperature overnight. The reaction mixture was
neutralized with acetic acid, and solvent was evaporated, and
the residue was purified on flash silica gel column chromatog-
raphy (CH2Cl2/MeOH = 10:1) to give compound 5 (23 mg,
80%). 1H NMR (CD3OD, 300 MHz) δ 8.06 (s, 1H), 7.38 (s, 1H),
7.24-7.35 (m, 3H), 5.14 (d, J=5.8 Hz, 1H), 4.79-4.86 (m, 2H),
4.03 (d, J = 6.3 Hz, 1H), 2.87 (s, 3H), 2.42-2.80 (m, 4H), 2.04-
2.32 (m, 1H), 1.82 (t, J=4.8 Hz, 1H), 1.36-1.40 (m, 1H), 0.84-
0.97 (m, 1H). HRMS calculated for C25H26ClN6O5 (M+H)+:
525.9562; found 525.9583.
(10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(N-bioti-
nyl(β-aminoethylaminocarbonyl)-1-butynyl)-9-yl]-20, 30-dihydro-
xybicyclo[3.1.0]hexane-10-carboxylic Acid N-Methylamide (17a).
To a solution of compound 11 (4 mg, 0.007 mmol) in dry DMF
(0.5 mL), biotin (1.89 mg, 0.0077 mmol), HATU (3.2 mg, 0.0084
mmol), and DIEA (1.6 μL, 0.009 mmol) were added. The
mixture was stirred at room temperature overnight. Solvent
was evaporated and the residue was purified on flash silica gel
column chromatography (CH2Cl2/MeOH/NH4OH=5:1:0.1) to
give compound 17a (3.5 mg, 62%). 1H NMR (CD3OD, 300
MHz) δ 8.14 (s, 1H), 7.43 (s, 1H), 7.29-7.39 (m, 3H), 5.07 (d, J=
6.9 Hz, 1H), 4.78-4.86 (m, 2H), 4.48-4.52 (m, 1H), 4.28-4.32
(m, 1H), 4.04 (d, J=6.3 Hz, 1H), 3.70-3.82 (m, 2H), 3.24-3.31
(m, 1H), 3.08-3.23 (m, 1H), 2.91 (s, 3H), 2.70-2.83 (m, 4H),
2.56 (t, J=7.5 Hz, 2H), 2.04-2.15 (m, 2H), 1.89 (t, J=5.1 Hz,
1H), 1.33-1.70 (m, 10H), 0.84-1.02 (m, 1H). HRMS calculated
for C37H46ClN10O6S (M+H)+: 793.3011; found 793.3030.
(10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(N-bioti-
nyl{5-aminopentanyl}(β-aminoethylaminocarbonyl)-1-butynyl)-
9-yl]-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic Acid N-Meth-
ylamide (17b). To a solution of compound 11 (3.4 mg, 0.0059
mmol) in DMF (0.5 mL), sulfo-NHS-LC-biotin (10 mg, 0.017
mmol, Pierce) and 10 μL of triethyl amine were added. The
mixture was stirred overnight at room temperature. Solvent was
evaporated and the residue was purified on flash silica gel
column chromatography (CH2Cl2/MeOH/NH4OH = 7:1:0.1)
to give compound 17b (2.9 mg, 55%). 1H NMR (CD3OD, 300
MHz) δ 7.99 (s, 1H), 7.29 (s, 1H), 7.19-7.32 (m, 3H), 4.93 (d, J=
5.4 Hz, 1H), 4.70-4.80 (m, 2H), 4.36-4.42 (m, 1H), 4.16-4.22
(m, 1H), 3.88 (d, J = 6.9 Hz, 1H), 3.42-3.62 (m, 2H), 3.03-3.15
(m, 4H), 2.89 (s, 3H), 2.56-2.86 (m, 6H), 2.41 (t, J=6.6 Hz, 1H),
(10R,20S,30R,40R,50R)-40-(6-Chloro-2-iodopurin-9-yl)-20,30-iso-
propylidenebicyclo[3.1.0]hexane-10-carboxylic Acid Ethyl Ester
(32). To a solution of triphenylphosphine (0.357 g, 1.361 mmol)
and 6-chloro-2-iodopurine (0.286 g, 1.019 mmol) in dry THF
(4 mL), DIAD (0.26 mL, 1.359 mmol) was added. The mixture
was stirred at room temperature for 10 min. A solution of
compound 31 (0.165 g, 0.681 mmol) in THF (2 mL) was added
to the reaction mixture, and the mixture was further stirred
overnight at room temperature. Solvent was evaporated and the
residue was purified on flash silica gel column chromatography
(hexane/ethyl acetate=3:1) to give the product 32 (0.2 g, 58%)
as a foam. 1H NMR (CDCl3, 300 MHz) δ 1.31-1.37 (m, 4H),
1.56 (s, 6H), 1.77-1.80 (m, 1H), 2.10-2.22 (m, 1H), 4.19-4.27
(m, 2H), 4.77 (d, J=14.2 Hz, 1H), 4.91 (s, 1H), 5.83 (d, J=
15.3 Hz, 1H), 7.97 (s, 1H). HRMS calculated for C17H19ClIN4 O4
(M+H)+: 505.0061; found 505.0073.
(10R,20S,30R,40R,50R)-40-[6-(3-Chlorobenzylamino)-2-iodopu-
rin-9-yl]-20,30-isopropylidenebicyclo[3.1.0]hexane-10-carboxylic
Acid Ethyl Ester (33). To a solution of compound 32 (0.273 g,
0.54 mmol) in anhydrous methanol (5 mL), triethylamine (1 mL,
7.569 mmol) and 3-chlorobenzylamine (0.33 mL, 2.697 mmol)
were added. The mixture was stirred overnight at room tem-
perature. Solvent was evaporated and the residue was purified
on flash silica gel column chromatography (hexane/ethyl ace-
tate=1:1) to give the product 33 (0.255 g, 78%) as a foamy solid.
1H NMR (CDCl3, 300 MHz) δ 7.57 (s, 1H), 7.20-7.25 (m, 4H),
6.03-6.15 (m, 1H), 5.83 (d, J=4.45 Hz, 1H), 5.28 (s, 1H), 4.65-
4.88 (m, 3H), 4.11-4.22 (m, 2H), 2.18-2.23 (m, 1H), 1.22-1.80
(m, 7H), 1.10-1.21 (m, 4H). HRMS calculated for C24H26ClI-
N5O4 (M+H)+: 610.0639; found 610.0649
(10R,20S,30R,40R,50R)-40-[6-(3-Chlorobenzylamino)-2-iodopurin-
9-yl]-20,30-isopropylidenebicyclo[3.1.0]hexane-10-carboxylic Acid-
N-Methylamide (34). To a solution of compound 33 (0.258 g,
0.423 mmol) in methanol (5 mL), 40% methylamine (4 mL) was
added. The mixture was stirred at room temperature for 2 days.
Solvent was evaporated and the residue was purified on flash
silica gel column chromatography (CH2Cl2/MeOH=40:1) to
give the product 34 (0.165 g, 66%) as a foam. 1H NMR (CDCl3,
300 MHz) δ 7.58 (s, 1H), 7.25-7.37 (m, 4H), 6.52 (bs, 1H), 5.65
(d, J=6.9 Hz, 1H), 4.73-4.83 (m, 3H), 2.97 (s, 3H), 2.04-2.07
(m,1H), 1.62-1.82 (m, 1H), 1.55 (s, 3H), 1.22-1.33 (m, 5H).
HRMS calculated for C23H25ClIN6O3 (M+H)+: 595.0643;
found 595.0649.
(10R,20S,30R,40R,50R)-40-[6-(3-Chlorobenzylamino)-2-(4-hydro-
xycarbonyl)-1-butynyl)-9-yl]-20,30-dihydroxybicyclo[3.1.0]hex-
ane-10-carboxylic Acid N-Methylamide (19). To a solution of
compound 34 (32 mg, 0.053 mmol) in anhydrous DMF (1.0
mL), PdCl2(PPh3)2 (7 mg, 0.01 mmol), CuI (2 mg, 0.01 mmol),
methyl-ω-hexynate (27 mg, 0.214 mmol), and then triethylamine

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7589
(0.014 mL, 0.10 mmol) were added. The reaction mixture was
stirred at room temperature overnight. Solvent was evaporated
under vacuum, and the residue was roughly purified on flash
silica gel column chromatography. To a solution of the product
in methanol (1 mL), 10% TFA (1 mL) was added, and the
mixture was heated at 70 °C for 7 h. Solvent was evaporated
under vacuum, the crude reaction mixture was further dissolved
in methanol (1 mL), and 1 M solution of KOH (1 mL) was
added, and the mixture was stirred at room temperature over-
night. Reaction mixture was neutralized with acetic acid, solvent
was evaporated under vacuum, and the residue was purified on
flash silica gel column chromatography (CH2Cl2/MeOH=20:1)
column chromatography (CH2Cl2/MeOH=30:1) to give com-
pound 23 (23 mg, 79%). 1H NMR (CD3OD, 300 MHz) δ 8.34
(s, 1H), 8.32 (s, 1H), 7.8 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.42 (d,
J=7.5 Hz, 1H), 7.13 (t, J=7.8 Hz, 1H), 4.90 (s, 2H), 4.72 (t,
J=6.3 Hz, 2H), 3.96 (d, J=6.6 Hz, 1H), 1.96-2.04 (m, 1H),
1.67-1.72 (m, 1H), 1.30-1.34 (m, 1H), 0.75-0.83 (m, 1H).
HRMS calculated for C18H19IlN5O2 (M+H)+: 464.0596; found
464.0584.
(10R,20R,30S,40S,50S)-40-[6-(3-Iodobenzylamino)-2-fluoro-pur-
ine]-20,30-dihydroxybicyclo[3.1.0]hexane (24). To a solution of
compound 42 (10 mg, 0.03 mmol) in anhydrous methanol
(0.5 mL), triethylamine (0.06 mL, 0.42 mmol) and 3-iodobenzy-
lamine (0.02 mL, 0.15 mmol) were added. The mixture was
stirred overnight at room temperature. Solvent was evaporated,
and the residue was roughly purified on flash silica gel column
chromatography. The product was dissolved in methanol
(1 mL) and 10% TFA (0.5 mL), and the reaction mixture was
heated at 70 °C for 6 h. Solvent was evaporated and the residue
was purified on flash silica gel column chromatography (hex-
ane/ ethyl acetae=1:1) to give compound 24 (8 mg, 55%). 1H
NMR (CD3OD, 300 MHz) δ 8.13 (s, 1H), 7.78 (s, 1H), 7.63 (dd,
J=7.8 Hz, 1H), 7.41 (d, J=7.8 Hz, 1H), 7.11 (t, J=7.8 Hz, 1H),
4.76 (s, 1H), 4.71-4.76 (m, 2H), 3.92-3.96 (m, 2H), 1.96-2.01
(m, 1H), 1.64-1.67 (m, 1H), 0.73-0.91 (m, 2H). HRMS calcu-
lated for C18H18FIN5O2 (M+H)+: 482.0473; found 482.0489.
(10R,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(4-meth-
oxycarbonyl-1-butynyl)-9-yl]-20,30-O-isopropylidenebicyclo[3.1.0]-
hexane (50). To a solution of compound 45 (43 mg, 0.08 mmol)
in anhydrous DMF (1.5 mL), PdCl2(PPh3)2 (11 mg, 0.015 mmol),
CuI (1.5 mg, 0.007 mmol), methyl-ω-pentynate (35 mg, 0.31 mmol),
and then triethylamine (0.11 mL, 0.80 mmol) were added. The
reaction mixture was stirred at room temperature overnight.
Solvent was evaporated under vacuum and the residue was
purified on flash silica gel column chromatography (hexane/
ethyl acetate = 1:1) to give compound 50 (30 mg, 74%) as a
foamy syrup. 1H NMR (CDCl3, 300 MHz) 7.98 (s, 1H), 7.32-
7.21 (m, 4H), 6.19 (bs, 1H), 5.31 (t, J = 5.24, 1H), 5.12 (s, 2H),
4.60 (d, J=7.2 Hz, 1H), 3.78 (s, 3H), 2.69-2.84 (m, 4H), 2.06-
2.14 (m, 1H), 1.62-1.72 (m, 1H), 1.56 (s, 3H), 1.25 (s, 3H), 0.88-
1.04 (m, 2H). HRMS calculated for C27H29ClN5O2 (M+H)+:
522.9953; found 522.9967.
1
to give compound 19 (18 mg, 62%) as a colorless powder. H
NMR (CD3OD, 300 MHz) δ 7.97 (s, 1H), 7.30 (s, 1H), 7.14-
7.22 (m, 3H), 4.97 (d, J=6.7 Hz, 1H), 4.73 (brs, 3H), 3.90 (d, J=
6.6 Hz, 1H), 2.75 (s, 3H), 2.42 (t, J = 7.2 Hz, 2H), 2.28 (t, J =
7.2 Hz, 2H), 1.96 (m, 1H), 1.83 (m, 3H), 1.75 (m, 1H), 1.25 (m,
1H). HRMS calculated for C26H28ClN6O5 (M+H)+: 539.1731;
found 539.1724.
(1R,2R,3S,4S,5S)-2,3-O-Isopropylidene-2,3,4-trihydroxybi-
cyclo[3.1.0]hexane (40). To a solution of compound 39 (0.773 g,
4.95 mmol) in dry CH2Cl2 (25 mL), Et2Zn (19.8 mL, 1 M
solution in hexane) was added at 0 °C. The mixture was stirred
for 15 min. After 15 min, CH2I2 (3.2 mL, 39.57 mmol) was added
at the same conditions and then the reaction mixture was stirred
at room temperature overnight. After completion of reaction, it
was quenched with saturated ammonium chloride solution and
the aqueous layer was extracted with CH2Cl2. The combined
organic layer was dried (Na2SO4), filtered, and evaporated. The
residue was purified on flash silica gel column chromatography
(hexane/ethyl acetate=3:1) to give compound 40 (0.710 g, 85%)
as a colorless syrup. 1H NMR (CDCl3, 300 MHz) δ 4.88 (t, J=
6.3 Hz, 1H), 4.46-4.54 (m, 2H), 2.37 (d, J = 9.3 Hz, 1H), 1.83-
1.86 (m, 1H), 1.61-1.66 (m, 1H), 1.55 (s, 3H), 1.29 (s, 3H), 0.95-
0.99 (m, 1H), 0.62-0.68 (m, 1H). HRMS calculated for
C9H15O3 (M+H)+: 171.0943; found 171.0954.
(10R,20R,30S,40S,50S)-40-(6-Chloro-2-iodopurine)-20,30-O-iso-
propylidene-20,30-dihydroxybicyclo[3.1.0]hexane (44). To a solu-
tion of triphenylphosphine (132 mg, 0.5 mmol) and 6-chloro-
2-iodopurine (141 mg, 0.50 mmol) in dry THF (2 mL), DIAD
(0.1 mL, 0.50 mmol) was added. The mixture was stirred at room
temperature for 10 min. A solution of compound 31 (43 mg,
0.252 mmol) in THF (2 mL) was added to the reaction mixture,
and the mixture was further stirred overnight at room tempera-
ture. Solvent was evaporated and the residue was purified on
flash silica gel column chromatography (hexane/ethyl acetate=
(10R,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(4-(β-ami-
noethylaminocarbonyl)-1-butynyl)-9-yl]-20,30-dihydroxybicyclo
[3.1.0]hexane (26). To a solution of compound 50 (25 mg,
0.047 mmol) in methanol (0.4 mL), ethylenediamine (1.5 mL)
was added. The mixture was stirred overnight at room tempera-
ture. Solvent was evaporated, and the residue was roughly
purified on flash silica gel column chromatography. The ami-
nated product was dissolved in methanol (1.5 mL) and 10%
trifluoroacetic acid (1.0 mL), and the mixture was heated at
70 °C for 15 h. Solvent was evaporated and the residue was
purified on flash silica gel column chromatography (CH2Cl2/
1
3:1) to give the product 44 (65 mg, 61%) as a foam. H NMR
(CDCl3, 300 MHz) d 8.07 (s, 1H), 5.37 (t, J = 2.1 Hz, 1H), 4.67
(d, J = 5.7 Hz, 1H), 2.13-2.18 (m, 1H), 1.65-1.69 (m, 2H), 1.27
(s, 3H), 1.26 (s, 3H), 0.96-1.01 (m, 2H). HRMS calculated for
C14H15ClIN4O2 (M+H)+: 432.9927; found 432.9928.
(10R,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-iodopur-
ine]-20,30-O-isopropylidene-20,30-dihydroxybicyclo[3.1.0]hexane
(45). To a solution of compound 44 (0.105 g, 0.243 mmol) in
anhydrous methanol (5 mL), triethylamine (0.4 mL, 3.38 mmol)
and 3-chlorobenzylamine (0.15 mL, 1.21 mmol) were added.
The mixture was stirred overnight at room temperature. Solvent
was evaporated and the residue was purified on flash silica gel
column chromatography (hexane/ethyl acetate=1:1) to give the
1
MeOH=5:1) to give compound 26 (14 mg, 58%). H NMR
(CD3OD, 300 MHz) δ 8.12 (s, 1H), 7.25-7.39 (m, 4H), 5.97 (s,
1H), 4.70 (t, J=6.3 Hz, 1H), 3.95 (d, J=6.6 Hz, 1H), 3.77 (t, J=
6.3 Hz, 2H), 2.94 (m, 2H), 2.52 (t, J=7.8 Hz, 2H), 1.97-2.01 (m,
1H), 1.68-1.72 (m, 2H), 1.30-1.35 (m, 2H), 0.89-0.91 (m, 2H),
0.78-0.82 (m, 1H). HRMS calculated for C25H29ClN7O3 (M+
H)+: 510.2000; found 510.2020.
UV-Visible Spectra of Biotinyl (17) and Fluorescent (18)
Probes and Fluorescence Spectrum of 18. UV-visible spectra
were measured on an HP diode array spectrophotometer,
model no. 8452A. The concentration of each was 40 μM in
pH 7.5 aqueous buffer (50 mM Tris-HCl containing 10 mM
MgCl2). The parameters for the compounds are as follows: 17a,
1
product 45 (0.096 g, 74%) as a foam. H NMR (CDCl3, 300
MHz) δ 7.63 (s, 1H), 7.37 (s, 1H), 7.27-7.26 (m, 3H), 6.25 (bs,
1H), 5.34 (t, J=5.7 Hz, 1H), 4.94 (s, 2H), 4.64 (d, J=6.9 Hz, 1H),
2.06-2.09 (m, 1H), 1.61-1.69 (m, 2H), 1.54 (s, 3H), 1.26 (s, 3H),
0.88-0.96 (m, 2H). HRMS calculated for C21H22ClIN5O2 (M+
H)+: 538.0497; found 538.0507.
(10R,20R,30S,40S,50S)-40-[6-(3-Iodobenzylamino)purine]-20,30-di-
hydroxybicyclo[3.1.0]hexane (23). A solution of compound 46
(32 mg, 0.063 mmol) in MeOH (2 mL) and 10% trifluoroacetic
acid (1.5 mL) was heated at 70 °C overnight. Solvent was
evaporated and the residue was purified on flash silica gel
λ 276 nm (ε=9890 L/(mol cm)); 17b, λ 276 nm (ε=7400 L/
3
(mol cm)); 18, λ 274 nm (ε = 16 100 L/(mol cm)), λ 650 nm
3
3
(ε=44 900 L/(mol cm)). Fluorescence spectra were measured
3
on a PerkinElmer LS503: excitation λmax =646 nm, emission
λ
max=660 nm.

7590 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Tosh et al.
Receptor Binding and Functional Assays. [3H]2-Chloro-N6-
cyclopentyladenosine (52, [3H]CCPA, 42.6 Ci/mmol) was a
custom synthesis product (Perkin-Elmer). [125I]N6-(4-Amino-
a Bio-Tek Instruments ELx808 Ultra microplate reader at
405 nm.
€
Molecular Modeling. The Prime program of the Schrodinger
3-iodobenzyl)adenosine-50-N-methyluronamide (53,
[
125I]I-
package48 was utilized to build a new molecular model of the
hA3AR. The recently reported model of the complex of the
nonselective AR agonist 51 docked to the crystal structure of the
AB-MECA, 2200 Ci/mmol) and [3H](2-[p-(2-carboxyethyl)-
phenylethylamino]-50-N-ethylcarboxamidoadenosine) (54, [3H]
CGS21680, 40.5 Ci/mmol) were purchased from Perkin-Elmer
Life and Analytical Sciences (Boston, MA). Test compounds
were prepared as 5 mM stock solutions in DMSO and stored
frozen.
A
2AAR was used as a template for modeling of the A3AR.38 The
sequence alignment of the A2A and A3ARs was performed with
Prime, taking into account positions of highly conserved amino
acid residues. The default values of all Prime parameters were
used. The position of full agonist 51 inside the A2A receptor was
taken into account during the modeling. Thus, the resulting
model of the A3AR contained 51 prepositioned inside the
receptor. The Glide program of the Schrodinger package was
used to redock 51 to the A3AR model obtained. The receptor
grid generation was performed for the box with a center in the
centroid of 51 in its initial position. The size of the box was
determined automatically. The extra precision mode (XP) of
Glide was used for the docking. The ligand scaling factor was set
to 1.0. The geometry of the ligand binding site of the complex of
A3AR with 51 docked was optimized. The binding site was
Cell Culture and Membrane Preparation. CHO cells stably
expressing the recombinant hA1, hA3, and rA3Rs and HEK-293
cells stably expressing the hA2AAR were cultured in Dulbecco’s
modified Eagle medium (DMEM) and F12 (1:1) supplemented
with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL
streptomycin, and 2 μmol/mL glutamine. In addition, 500 μg/mL
Geneticin was added to the A2A media, while 800 μg/mL
hygromycin was added to the A1 and A3 media. After being
harvested, cells were homogenized and suspended in PBS. Cells
were then centrifuged at 240g for 5 min, and the pellet was
resuspended in 50 mM Tris-HCl buffer (pH 7.5) containing
10 mM MgCl2. The suspension was homogenized and was then
ultracentrifuged at 14,330 g for 30 min at 4 °C. The resultant
pellets were resuspended in Tris buffer and incubated with
adenosine deaminase (3 units/mL) for 30 min at 37 °C. The
suspension was homogenized with an electric homogenizer for
10 s, pipetted into 1 mL vials, and then stored at -80 °C until the
binding experiments. The protein concentration was measured
using the BCA protein assay kit from Pierce Biotechnology, Inc.
(Rockford, IL).49
˚
defined as 51 and all amino acid residues located within 5 A from
˚
51. All A3AR residues located within 2 A from the binding site
were used as a shell. The following parameters of energy
minimization were used: OPLS2005 force field; water was used
as an implicit solvent; a maximum of 5000 iterations of the
Polak-Ribier conjugate gradient minimization method was
-1
used with a convergence threshold of 0.01 kJ mol-1
.
˚
A
3
3
Another reference nucleoside, 50-N-methylcarboxamidoadeno-
sine, was also docked in this hA3AR homology model (Support-
ing Information).
Binding Assays. Into each tube in the binding assay was added
50 μL of increasing concentrations of the test ligand in Tris-HCl
buffer (50 mM, pH 7.5) containing 10 mM MgCl2, 50 μL of the
appropriate agonist radioligand, and finally 100 μL of mem-
brane suspension. For the A1AR (22 μg of protein/tube) the
radioligand used was [3H]51 (final concentration of 3.5 nM) or
[3H]52 (final concentration of 1.0 nM). For the A2AAR (20 μg/
tube) the radioligand used was [3H]53 (10 nM). For the A3AR
(21 μg/tube) the radioligand used was [125I]54 (0.34 nM). Non-
specific binding was determined using a final concentration of
10 μM 51 diluted with the buffer. The mixtures were incubated
at 25 °C for 60 min in a shaking water bath. Binding reactions
were terminated by filtration through Brandel GF/B filters
under reduced pressure using a M-24 cell harvester (Brandel,
Gaithersburg, MD). Filters were washed three times with 3 mL
of 50 mM ice-cold Tris-HCl buffer (pH 7.5). Filters for A1 and
A2AAR binding were placed in scintillation vials containing
5 mL of Hydrofluor scintillation buffer and counted using a
Perkin-Elmer liquid scintillation analyzer (Tri-Carb 2810TR).
Filters for A3AR binding were counted using a Packard Cobra
II γ-counter. The Ki values were determined using GraphPad
Prism for all assays.
The A3AR model obtained was utilized to study a binding
mode of amine congener 15 using InducedFit docking imple-
€
mented in the Schrodinger package. The grid generation was
performed for the box with a side of 32 A with a center in the
˚
centroid of 51. Thedefaultvalues were usedfor other parameters.
Acknowledgment. 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. Dr. Dilip Tosh thanks Can-Fite Biopharma
for financial support.
Supporting Information Available: Synthetic procedures for
compounds 30, 13-16, 10, 7, 35, 37, 20, 21, 41, 42, 46, 47, 49, 22,
and 25 and their characterization; UV-visible spectra of 17a,
17b, and 18; docking view of a reference agonist in the hA3AR
homology model. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
cAMP Accumulation Assay. Intracellular cAMP levels were
measured with a competitive protein binding method.34 CHO
cells that expressed the recombinant hA3AR 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 of DMEM, containing 50 mM HEPES,
pH 7.4. Cells were then treated with the agonist 51 or test
compound in the presence of rolipram (10 μM) and adeno-
sine deaminase (3 units/mL). After 45 min forskolin (10 μM)
was added to the medium, and incubation was continued for
an additional 15 min. The reaction was terminated by remov-
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of 200 μL of 0.1 M ice-cold HCl. The cell lysate was resus-
pended and stored at -20 °C. For determination of cAMP
production, 100 μL of the HCl solution was used in the Sigma
direct cAMP enzyme immunoassay following the instruc-
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