2-Dialkynyl derivatives of (N)-methanocarba nucleosides: 'Clickable' A3 adenosine receptor-selective agonists

Article abstract of DOI:10.1016/j.bmc.2009.12.018

We modified a series of (N)-methanocarba nucleoside 5′-uronamides to contain dialkyne groups on an extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A₃ adenosine receptor (AR) agonists. The proximal alk

Full text of DOI:10.1016/j.bmc.2009.12.018

Bioorganic & Medicinal Chemistry 18 (2010) 508–517
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-Dialkynyl derivatives of (N)-methanocarba nucleosides: ‘Clickable’ A3
adenosine receptor-selective agonists
Dilip K. Tosh a, Moshe Chinn a, Lena S. Yoo a, Dong Wook Kang b, Hans Luecke b, Zhan-Guo Gao a,
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,
Bldg. 8A, Rm. B1A-19, NIH, NIDDK, LBC, Bethesda, MD 20892, United States
b Gene Regulation Group, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health,
Bethesda, MD 20892, United States
a r t i c l e i n f o
a b s t r a c t
We modified a series of (N)-methanocarba nucleoside 50-uronamides to contain dialkyne groups on an
extended adenine C2 substituent, as synthetic intermediates leading to potent and selective A3 adenosine
receptor (AR) agonists. The proximal alkyne was intended to promote receptor recognition, and the distal
alkyne reacted with azides to form triazole derivatives (click cycloaddition). Click chemistry was utilized
to couple an octadiynyl A3AR agonist to azido-containing fluorescent, chemically reactive, biotinylated,
and other moieties with retention of selective binding to the A3AR. A bifunctional thiol-reactive crosslink-
ing reagent was introduced. The most potent and selective novel compound was a 1-adamantyl deriva-
tive (Ki 6.5 nM), although some of the click products had Ki values in the range of 200–400 nM. Other
potent, selective derivatives (Ki at A3AR in nM) were intended as possible receptor affinity labels: 3-
Article history:
Received 30 September 2009
Revised 2 December 2009
Accepted 5 December 2009
Available online 11 December 2009
Keywords:
G protein-coupled receptor
Purines
Azide
nitro-4-fluorophenyl (10.6), a-bromophenacyl (9.6), thiol-reactive isothiazolone (102), and arylisothiocy-
Structure–activity relationship
Radioligand binding
anate (37.5) derivatives. The maximal functional effects in inhibition of forskolin-stimulated cAMP were
measured, indicating that this class of click adducts varied from partial to full A3AR agonist compared to
other widely used agonists. Thus, this strategy provides a general chemical approach to linking potent
and selective A3AR agonists to reporter groups of diverse structure and to carrier moieties.
Published by Elsevier Ltd.
1. Introduction
The crystallographic structure of the human (h) A2AAR has been
determined, but a directly determined structure is lacking for the
The A3 adenosine receptor (AR), a G protein-coupled receptor
(GPCR), is found in myocytes, astrocytes, neurons, neutrophils,
eosinophils, and other cell types.1 Both A3AR agonists and antago-
nists are proposed for the treatment of cancer and inflammatory
diseases.2–6 An antiischemic effect of A3AR agonists also suggests
their use in protection of skeletal muscle and cardiac muscle.7
A3AR.8 We recently used the coordinates of the A2AAR structure as
a template for homology modeling of the A3AR and have predicted
specific interactions of the ligand with the receptor protein.9
Hypotheses for agonist binding at the A3AR have been supported
using site-directed mutagenesis and reengineering of the putative
binding site to recognize tailored nucleoside ligands (neoceptors).10
Thus, structural insights into recognition in the A3AR binding site
have been gained even in the absence of an X-ray structure. The 2
and 6 positions of the adenine ring are most amenable to chain
derivatization without sterically interfering in the binding process.
Abbreviations: AR, adenosine receptor; cAMP, adenosine 30,50-cyclic phosphate;
CHO, Chinese hamster ovary; Cl-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-50-N-meth-
ylcarboxamidoadenosine; DMEM, Dulbecco’s modified Eagle’s medium; EDC, 1-
ethyl-3-(3-dimethylaminopropyl) carbodiimide; EDTA, ethylenediaminetetraacetic
Molecular probes for the A3AR containing
a rigid bicy-
clo[3.1.0]hexane ring system of the North conformation ((N)-
methanocarba) in place of the freely twisting ribose moiety were
recently reported.9,11,12 Amide-linked fluorescent 1 and biotin-con-
taining probes 2 and 3 of high A3AR affinity and selectivity were
designed using a functionalized congener approach based on step-
wise chain extension designed to preserve or enhance receptor
affinity (Chart 1).13
acid; GPCR,
G protein-coupled receptor; HATU, 2-(1H-7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium; HEK, human
embryonic kidney; HOBT, 1-hydroxybenzotriazole; I-AB-MECA, N6-(3-iodo-4-ami-
nobenzyl)-50-N-methylcarboxamidoadenosine; IB-MECA, N6-(3-iodobenzyl)-50-N-
methylcarboxamido-adenosine; NECA, 50-N-ethylcarboxamidoadenosine; DMF,
N,N-dimethylformamide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; HRMS, high resolution mass spectroscopy; TBTA, tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl] amine; TEA, triethylamine; TLC, thin layer chromatography.
* Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480 8422.
The present study introduces 2-dialkynyl groups into selective
A3AR ligands of the structural class of the (N)-methanocarba-
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.12.018

D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
509
50ꢀN-methyluronamides. The proximal alkyne was intended to
promote receptor recognition.9 Earlier studies established the
enhancing effect in recognition at both A2A and A3ARs of introduc-
ing an alkynyl group adjacent to the purine C2.14 The distal alkyne
was shown to react selectively with alkyl or aryl azides by click
cycloaddition to form triazole derivatives of the general structure
4.15 Furthermore, an optimized N6-(3-chlorobenzyl) group, which
favors A3AR selectivity in human, mouse, and rat, was included
in the nucleoside structures.12 Fluorescent, chemically reactive,
biotinylated, and other moieties were incorporated by this chemi-
cal route, leading to selective A3AR agonists.
ity of two regioisomers, we obtained only one isomer. Based on the
regiochemistry of a single click reaction, the reaction on the inter-
nal alkyne moiety was assumed to occur from its less hindered
face. Use of the Cu(I)-stabilizing catalyst tris[(1-benzyl-1H-1,2,3-
triazol-4-yl)methyl] amine (TBTA)19 in this reaction provided the
desired monosubstituted analogue 26.
Compounds 15 and 17 were intended as models compounds for
the amide coupling of amine functionalized congeners such as 16
to alkyl carboxylic carriers. Compound 18 was included in order
to probe steric tolerance at the distal position.
The fluorescent squaraine-rotaxane derivative 22 was prepared
from a commercial reactive azide, of undisclosed structure, which
fluoresces strongly at 680 nm.20,21 Such rotaxane derivatives have
the following advantages over other small fluorophores: improved
chemical and photochemical stability, sharp absorption and emis-
sion bands, and stability over the pH range from 2 to 12. They are
typically much brighter in fluorescence than the Alexa dyes.22 The
elemental composition of this proprietary dye moiety, but not the
full chemical structure, was revealed by the supplier, and the
integrity of the synthetic product 22 was demonstrated by high
resolution mass spectroscopy. The expected molecular weight of
the click product of the 1:1 reaction with dialkyne 9 was obtained.
An isothiazolone azide derivative 32 designed as a bifunctional
linker for thiol reactivity was synthesized as shown in Scheme 3.
This served as the precursor for the adenosine derivative 19. The
synthesis of isothiazolone derivatives from 3-benzoylpropionic
acid was reported by Tsolomitis and Sandris.23 An amide coupling
reaction between 27 and p-toluidine formed 4-oxo-4-phenyl-N-p-
tolyl-butyramide 28, which was treated with an excess of thionyl
chloride to give 5-benzoyl-2-p-tolylisothiazol-3-one 29. Deben-
zoylation of 29 afforded 2-p-tolylisothiazol-3-one 30 in the pres-
ence of 10% aqueous sodium hydroxide in benzene, along with
dithietane derivatives as by-products. Compound 30 was trans-
formed with N-bromosuccinimide in the presence of a catalytic
amount of benzoyl peroxide in carbon tetrachloride to the bromo-
methyl derivative 31, which upon treatment with sodium azide
afforded 2-(4-azidomethylphenyl)isothiazol-3-one 32.
2. Results
2.1. Chemical synthesis
We have introduced terminal alkynyl groups on the C2 position
substituents of (N)-methanocarba nucleoside derivatives to serve
as cross-linking sites on the A3AR agonists, such as for the intro-
duction of reporter groups (R1 in general structure 4 of Chart 1).
Prior to introduction of a terminal alkyne for coupling of the
nucleosides to reporter groups, the structure activity relationship
(SAR) of this series of N6-(3-chlorobenzyl)-(N)-methanocarba-
50ꢀN-methyluronamide derivatives was explored in detail.9,11
Thus, here we started with a nucleoside pharmacophore that was
already optimized for activation of the A3AR. Only the adenine
C2 position was structurally modified, and all modifications were
intended for click chemistry.
The synthetic route to the small molecular 50-N-methylurona-
mide (N)-methanocarba 2-alkynyl triazole-containing derivatives
10–23 is shown in Scheme 1. The synthesis of the 20,30-protected
dialkynyl intermediates 6 and 7 was performed using a Sonoga-
shira coupling16 on the corresponding 2-iodo intermediate 5. After
deprotection of the 20,30-hydroxyl groups to provide nucleosides 8
and 9, compounds 10–23 were obtained using the Cu(I)-catalyzed
2+3 cyclization reaction of the terminal acetylene group with an
appropriate azide.15 The reactions were generally selective for
the terminal alkyne, but reaction with 4-isothiocyanatophenylaz-
ide 24 initially produced the disubstituted product 25 as the major
product isolated (Scheme 2). It is well known that copper-cata-
lyzed click reactions direct the formation of only one regioisomer,
that is, the 1,4-regioisomer is formed (out of the two possibili-
ties).17,18 In the case of compound 25, although there is a possibil-
2.2. Quantification of pharmacological activity
Binding assays at three hAR subtypes were carried out on the al-
kyne and triazole derivatives using standard radioligands24–26 and
membrane preparations from Chinese hamster ovary (CHO) cells
Cl
NHCH2
N
SO3H
N
O
C
O
S
N
CH3NHC
N
(CH2)4
(CH2)2 CONH (CH2)2
R
CH3
CH3
NH
HN
N
OH
OH
CH3CH2
2 R=NH
O
3 R=NHCO(CH2)5NH
Cl
Cl
NHCH2
N
NHCH2
N
N
N
O
O
+
N
N
N
CH3
H
N
O
CH3NHC
N
(CH2)5
(CH2)2 CONH (CH2)2
C
CH3
(CH2)n
N
R1
CH3NHC
N
N
N
OH
OH
1
OH
-
HO
SO3
4 R1 = aryl or alkyl containing reporter groups
n = 3 - 4
Chart 1. Molecular probes in the (N)-methanocarba series of adenosine A3AR agonists that were previously reported (1–3)9 and a general formula (4) for triazole derivatives
prepared in this study.

510
D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
Cl
Cl
Cl
NHCH2
NHCH2
N
NHCH2
N
N
N
N
N
N
N
O
O
O
N
CH3NHC
N
CH3NHC
I
N
(CH2)n
CH3NHC
(CH2)n
N
i
ii
L-Ribose
O
O
OH
HO
O
O
6 n = 3
7 n = 4
8 n = 3
9 n = 4
Cl
5
NHCH2
N
N
N
O
iii
CH3NHC
N
(CH2)n
N
R
N
N
10 n = 3
OH
HO
11 - 16, 18 - 23 n = 4
R =
n = 4, unless noted
O
NO2
19
O
C
15
10
23
(CH2)6
NH
H
N
n = 3
11
CH2
N
S
(CH2)2
CH3
F
O
n = 4
16
17
12
O
-
S
CO2
NH2
(CH2)4 NH2
(CH2)4
(CH2)6
X
C
iv
13
NH
O
C
HN
+
H2N
O
H
NH2
CO2H
20 X = NH
(CH2)4
N
CH3
-
-
SO3
(CH3CH2)3NH+
SO3
O
(CH3CH2)3NH+
21 X = CONH(CH2)2[O(CH2)2] -NH
4
18
14
COCH2Br
22 Squaraine-rotaxane derivative
Scheme 1. Use of click reactions to couple a terminal alkynyl groups present on a functionalized chain of an adenosine analogue to various azido moieties. Reagents and
conditions: (A) (i) HC„C(CH2)nC„CH (n = 3, 4), Pd(PPh3)4, CuI, Et3N, DMF, rt; (ii) 10% TFA, MeOH, 70 °C; (iii) appropriate azide, CuSO4ꢁ5H2O, sodium ascorbate, TBTA, THF/H2O
or t-BuOH/H2O, rt; (iv) acetic acid N-hydroxysuccinimide ester, DMF, rt.
Cl
NHCH2
N
N
O
CH3NHC
N
N
(CH2)4
N
NCS
N
N
N
N
Cl
i
N
OH
HO
NHCH2
N
NCS
N
N
25
O
Cl
CH3NHC
N
+
N3
NCS
(CH2)4
ii
NHCH2
OH
N
N
HO
N
N
O
24
9
CH3NHC
(CH2)4
N
NCS
N
N
OH
HO
26
Scheme 2. The unusual example of click reaction occurring at both alkynyl groups present on a functionalized chain of an adenosine analogue. When the catalyst TBTA was
used, only the monosubstituted product 26 was isolated. Reagents and conditions: (i) CuSO4ꢁ5H2O, sodium ascorbate, THF/H2O, rt; (ii) CuSO4ꢁ5H2O, sodium ascorbate, TBTA, t-
BuOH/H2O, rt.
(A1 and A3) or human embryonic kidney (HEK293) cells (A2A) sta-
bly expressing a hAR subtype (Table 1).27,28 The previously re-
ported molecular probes (1–3) were used for comparison in the
biological assays.
The two homologous dialkyne intermediates 8 and 9 were equi-
potent, with Ki values in A3AR binding of 24–29 nM. The selectivity
of both intermediates in comparison to the A1 and A2AARs was
>400 and roughly 300-fold, respectively. Following the click reac-
tion with either aryl or alkyl azides, considerable affinity and selec-
tivity at the A3AR were preserved. Thus, the main pharmacophore
maintained its receptor recognition function in the triazole-ex-
tended series. The 3-nitro-4-fluorophenyl adducts 10 and 11 were

D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
511
H
N
Bz
S
Bz
Bz
OH
ii
i
iii
N
O
O
O
27
28
29
Br
N3
S
S
S
v
iv
N
N
N
O
O
O
30
31
32
Scheme 3. Synthesis of isothiazolone derivative 32 to serve as a thiol-reactive crosslinker. Reagents and conditions: (i) p-toluidine, EDC, HOBT, Et3N, 1,4-dioxane, rt, 16 h; (ii)
thionyl chloride, rt, 16 h; (iii) 10% aqueous NaOH, benzene, rt, 3 d; (iv) N-bromosuccinimide, benzoyl peroxide, carbon tetrachloride, reflux, 2 h; (v) sodium azide, DMF.
Table 1
Potency of a series of (N)-methanocarba adenosine derivatives at three subtypes of human ARs
Cl
Cl
NHCH2
NHCH2
N
N
N
N
N
O
O
N
R1
CH3NHC
CH3NHC
N
N
(CH2)n
N
R2
N
N
OH
OH
HO
HO
1–3, 8, 9, 25
10–23, 26
Compd
Structure
Affinity (Ki, nM) or % inhibitiona
A1
A2A
A3
R1
1b,c
2b,c
3b,c
C„C(CH2)2CONH(CH2)2NH–CO–(CH2)5Cy5
C„C(CH2)2CONH(CH2)2NH–biotin
C„C(CH2)2CONH(CH2)2NH–CO(CH2)5NH–biotin
(36 3%)
(1 1%)
(12 4%)
4730 1020
(51 2%)
(47 11%)
17.2 3.1
36.4 5.6
57.7 16.2
(CH2)4
N
NCS
25e
(4 2%)
(40 3%)
8.8 1.3
N
N
N
N
N
NCS
8
9
C„C(CH2)3C„CH
C„C(CH2)4C„CH
R2
(36 4%)
(31 2%)
4330 500
7040 1430
23.6 3.9
29.4 9.8
n
3
4
4
4
4
4
4
4
4
4
10
11e
12
13
26
14e
15
16
17
18e
3-Nitro-4-fluorophenyl
3-Nitro-4-fluorophenyl
4-Aminophenyl
4-Carboxyphenyl
4-Isothiocyanatophenyl
(10 3%)
(8 4%)
(3 1%)
(39 4%)
6730 280
5490 1150
(40 1%)
26.0 8.2
10.6 3.8
87.1 13.1
180 23
37.5 16.0
9.6 1.3
22.3 1.6
47.0 1.8
89.5 12.6
6.5 0.5
(0 0%)
(2 11%)
(12 3%)
(12 4%)
(13 3%)
(17 1%)
(22 2%)
(28 4%)
4-(
a-Br-phenacyl)
5740 730
2440 320
1630 350
7240 510
3280 700
(CH2)2NHCOCH3
(CH2)4NH2
(CH2)4NHCOCH3
1-Adamantyl
O
19
4
(15 3%)
(49 1%)
102 25
CH2
N
S
20d
21d
22
4
4
4
4
–(CH2)6NH–biotin
(0 0%)
(0 0%)
(0 0%)
(0 0%)
(27 1%)
(19 5%)
(2 1%)
285 54
235 43
239 43
416 45
–(CH2)6CONH(CH2)2[O(CH2)]4NH–biotin
Squaraine-Rotaxane derivative
(CH2)6NHCO-Alexa Fluor
23d
(23 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]36; [3H]37; or [125I]38; respectively), unless noted. A percent in parentheses refers to
inhibition of radioligand binding at 10
l
M.
b
Values from Tosh et al.9
c
Structure given in Chart 1.
Structure given in Scheme 1.
d
e
11, MRS5223; 14, MRS5226; 15, MRS5233; 18, MRS5224; 25, MRS5225.
roughly equipotent to the dialkyne precursors. The A3AR affinity of
the p-substituted phenyl adducts 12, 13, 14, and 26 varied depend-
ing on the aryl substituent. The order of potency depending on the
4 position substitution was: bromoacetyl 14 > isothiocyanate
26 > amino 12 > carboxy 13. Compound 14 displayed a Ki value of
9.6 nM. The disubstituted click product 25 was highly potent in

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D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
Table 2
binding to the A3AR, with a Ki value of 8.8 nM. Thus, in spite of the
added steric bulk, it was surprisingly fourfold more potent than the
monosubstituted isothiocyanate derivative 26.
Maximal efficacy of (N)-methanocarba adenosine derivatives in a functional assay at
the A3AR
Compound
% Inhibition of cAMP formationa at hA3 AR
Among adducts of alkyl azides, a short chain acetamidoalkyl
derivative 15 was moderately potent with a Ki value of 22 nM,
and this ethyl derivative was fourfold more potent than a higher
butyl homologue 17. Compound 15 was 109-fold selective for the
A3AR in comparison to the A2AAR (Fig. 1A). The free aminobutyl
precursor derivative 16 displayed only moderate affinity at the
A3AR. The sterically bulky 1-adamantyl adduct 18 was highly po-
tent in binding with a Ki value of 6.5 nM at the A3AR and 500-fold
selectivity in comparison to the A2AAR (Fig. 1B).
A thiol-reactive derivative 19 displayed only moderate affinity
at the A3AR. The two derivatives of biotin 20 and 21 having un-
branched chains and a fluorescent derivative of Alexa Fluor 488
23 were relatively weak in binding to the A3AR with Ki values in
the range of 200–400 nM. The longer biotin derivative 21 con-
tained a tetraethylene glycol spacer, which apparently did not en-
hance affinity. The rotaxane derivative 22 bound to the A3AR
receptor with a Ki value of 239 nM.
IB-MECA
Cl-IB-MECA
99
97
6
36, NECA
100
1b
94.4 9.6
84.5 12.0
107 18
83.6 7.2
18.3 7.6
60.2 17.0
ND
2b
3b
25
8
9
10
11
12
13
26
14
15
16
17
18
19
20
21
22
23
116 22
44.3 2.4
83.4 13.1
19.5 14.5
109 12
93.6 17.7
64.4 12.4
59.7 19.2
27.8 17.4
75.9 16.5
55.6 12.5
41.8 11.3
111 18
The click products were tested in a functional assay at the A3AR
(inhibition of forskolin-stimulated cAMP production29,30 in A3AR-
expressing CHO cells) as shown in Table 2. The (N)-methanocarba
derivatives displayed A3AR agonist properties with varying degrees
37.8 14.6
of maximal inhibition of cAMP production at 10 lM. In general, the
a
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 M, in comparison to the maximal effect of a full
M. Data are expressed as mean standard error (n = 3). ND, not
degree of inhibition was similar to or less than that of 50-N-ethyl-
l
agonist NECA at 10
l
determined.
b
Values from Tosh et al.9
carboxamidoadenosine (NECA), taken as a reference standard.
Therefore, some of these derivatives, for example, 4-fluoro-3-nitro-
phenyl 11, bromophenacyl 14, acetamidoethyl 15, and rotaxane 22
derivatives, were highly efficacious agonists. Compounds that dis-
played intermediate (50–90%) efficacies were: 9, 13, 16, 17, 19, and
25. Agonists of lower efficacy (<50%) were the short dialkyne deriv-
ative 8 and the aminophenyl 12, isothiocyanate 26, and 1-adaman-
tyl 18 triazole derivatives. The affinity of compounds 20, 21, and 23
was so low that a 10 lM test concentration in the cAMP assay was
not sufficient to ensure full receptor occupancy. The corresponding
efficacy values for two A3AR agonists, N6-(3-iodobenzyl)-50-N-
methylcarboxamidoadenosine (IB-MECA) and its 2-chloro ana-
logue Cl-IB-MECA, which are currently in Phase II clinical trials,
are included for comparison.1 Both are full agonists in this assay.
This series of (N)-methanocarba nucleosides is known to be
much weaker in interaction with the A2BAR than with other sub-
types.12 We have tested selected nucleosides at 10
lM for agonist
activity in the stimulation of cAMP accumulation in CHO cells sta-
bly expressing the human A2BAR. The percent stimulation was:
100% (NECA), 30.2% (11), 19.4%, (15), and 36.5% (18).
3. Discussion
Click chemistry as a means of assembling complex ligands and
for introducing structural diversity is finding increasing application
in biological systems.15 With respect to the ARs, a series of triazole
derivatives of adenosine was previously prepared using click
chemistry, resulting in A3AR selective agonists, partial agonists,
and antagonists.31 The triazole ring was directly attached at the 2
position of adenine, which caused wide variation of the receptor
affinity and relative efficacy of the analogues. However, in the pres-
ent study, the triazole was incorporated at a more distal position of
an elongated and flexible 2-adenine substituent. This resulted in a
greater degree of preservation of the agonist properties in this
Figure 1. Inhibition of radioligand binding by (N)-methanocarba nucleoside
analogues in membranes of CHO cells expressing the human A1 (j) and A2A
(N
)
and A3 (.) ARs. Inhibition curves are shown for the agonist analogues containing a
triazole group, the adamantyl derivative 18, and the amide model compound for
click linkage to carriers 15. Both of the analogues shown were highly selective for
the hA3AR in comparison to the A1 and A2AARs.

D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
513
series than in the previous study, in which some derivatives be-
came antagonists.31 Thus, we have used the azide/alkyne cycload-
dition reaction to easily synthesize a wide range of biologically
active molecules. The click reaction was intended for linking aden-
osine functionalized congeners to other moieties and carriers, such
as reporter groups, chemically reactive groups, and macromolecu-
lar carriers. Other studies of biologically-active small molecules
have used click cycloaddition reactions to incorporate reporter
groups, such as fluorescent dyes and biotin.32,33
from 80:20 to 40:60 in 20 min with a flow rate of 1 mL/min. Peaks
were detected by UV absorption (254 nm) using a diode array detec-
tor. All derivatives tested for biological activity were shown to be at
least 97% pure using this analytical HPLC system.
High-resolution mass measurements were performed on a
Micromass/Waters LCT Premier Electrospray Time of Flight (TOF)
mass spectrometer coupled with a Waters HPLC system. Unless
noted otherwise, reagents and solvents were purchased from Sig-
ma–Aldrich (St. Louis, MO). Solutions of the nucleoside analogues
in DMSO (5 mM) were prepared for biological testing and stored
at ꢀ20 °C.
The small molecule adducts in the present series were not uni-
formly potent as A3AR selective agonists. Rather, only certain
derivatives displayed exceptionally high affinity at this subtype
(Ki <20 nM): 14, 18, and 25. Two different lengths of dialkynyl
chains were compared in binding at the A3AR. The octadiynyl
derivative 9 appeared to be better suited than the shorter homo-
logue 8 for binding to this receptor when coupled to an aryl azide
(cf. 10 and 11). Therefore, the octadiynyl chain was used in subse-
quent derivatives. A study by Seela and co-workers also utilized
octadiynyl-derivatized nucleosides that were incorporated into oli-
gonucleotides for click reactions that took place exclusively at the
distal alkynyl group.34 Compounds 10, 14, 19, and 26 were de-
signed as potential affinity labels of the A3AR, but the chemical
irreversibility of binding was not tested in the present study. The
use of isothiocyanates, 3-isothiazolones, and other electrophilic
derivatives to affinity label biopolymers has been described.13,35
In conclusion, the most potent and selective novel compound
was a 1-adamantyl derivative (Ki 6.5 nM), which suggested the
existence of a hydrophobic binding pocket in this region of the
receptor. Curiously, this compound proved to be a partial agonist
of the A3AR with only 27.8% of the maximal efficacy in comparison
to NECA. Various other click products were in the Ki range of 200–
400 nM. Other potent, selective derivatives (Ki at A3AR in nM) were
intended as possible receptor affinity labels: 3-nitro-4-fluoro-
The squaraine-rotaxane azide derivative SRfluorÒ 680 Azide
was obtained from Molecular Targeting Technologies, Inc. (West
Chester, PA). Alexa Fluor 488 azide and biotin(PEG)4 azide were
purchased from Invitrogen Corp. (Carlsbad, CA). DMEM/F12 med-
ium and 1 M Tris–HCl (pH 7.5) were purchased from Mediatech,
Inc. (Herndon, VA). Unless noted otherwise, reagents and solvents
were purchased from Sigma–Aldrich (St. Louis, MO).
4.1.1. (10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(1,6-
heptadiynyl)-9H -purin-9-yl]-(10S,20R,30S,40S,50S)-40-[6-(3-chloro-
benzylamino)-2-(1,7-octadiynyl)-9H -purin-9-yl]-20,30-O-iso-
propylidenebicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (7)
To a solution of compound 5 (440 mg, 0.73 mmol) in anhydrous
DMF (12 mL), Pd(PPh3)4 (92 mg, 0.08 mmol), CuI (30.5 mg,
0.16 mmol), 1,7-octadiyne (1.0 mL, 8.01 mmol) and then triethyl-
amine (0.22 mL, 1.6 mmol) was added. The reaction mixture was
heated at 60 °C for overnight. Solvent was evaporated under vac-
uum and the residue was purified on flash silica gel column chro-
matography (CH2Cl2/MeOH = 70:1) to give the compound
7
(352 mg, 83%) as foamy syrup. 1H NMR (CD3OD, 300 MHz) d 8.11
(s, 1H), 7.43 (s, 1H), 7.26–7.33 (m, 3H), 5.74 (d, J = 7.2 Hz, 1H),
5.01 (s, 1H), 4.83 (m, 1H), 2.87 (s, 3H), 2.53 (t, J = 6.9 Hz, 2H),
2.34–2.31 (m, 3H), 2.10–2.15 (m, 1H), 1.71–1.84 (m, 4H), 1.54–
1.57 (m, 4H), 1.40 (t, J = 5.4 Hz, 1H), 1.29 (s, 3H). HRMS calcd for
C31H34ClN6O3 (M+H)+: 573.2381; found 573.2397.
phenyl derivative 11 (10.6), a-bromophenacyl 14 (9.6), thiol-reac-
tive isothiazolone 19 (102), and arylisothiocyanate 26 (37.5). The
maximal functional effects in inhibition of forskolin-stimulated
cAMP were measured, indicating that this class of click adducts
varied from partial to full A3AR agonist compared to other widely
used agonists. Thus, this strategy provides a general chemical ap-
proach to linking potent and selective A3AR agonists to reporter
groups of diverse structure and to carrier moieties.
One disadvantage of the standard cycloaddition reaction is that
cuprous ions are required. This reactant might not be compatible
with the full range of GPCR ligands desired to be coupled to carriers
and is certainly not useful in cell systems. To overcome this draw-
back, Bertozzi and colleagues have explored cyclooctyne deriva-
tives that are substrates for ‘copper-free’ click chemistry.36 These
groups provide greater compatibility with living systems and allow
covalent coupling to azido groups on biopolymers and other bio-
molecules. We are currently extending these results to application
of cyclooctynyl derivatives of A3AR agonists to conjugation using
copper-free click chemistry.
4.1.2. 20,30-O-Isopropylidenebicyclo[3.1.0]hexane-10-carboxylic
acid N-methylamide (6)
Compound 6 (81%) was synthesized from 5 following same pro-
cedure as for compound 7. 1H NMR (CD3OD, 300 MHz) d 8.12 (s,
1H), 7.45 (s, 1H), 7.28–7.34 (m, 3H), 5.76 (d, J = 6.9 Hz, 1H), 5.02
(s, 1H), 4.85–4.87 (m, 1H), 2.88 (s, 3H), 2.64 (t, J = 6.9 Hz, 2H),
2.41–2.46 (m, 2H), 2.31 (t, J = 2.7, 1H) 2.12–2.17 (m, 1H), 1.85–
1.94 (m, 2H), 1.56 (m, 4H), 1.40–1.44 (m, 1H). 1.30 (s, 3H). HRMS
calcd for C30H32ClN6O3 (M+H)+: 559.3046; found 559.3085.
4.1.3. (10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(1,6-
heptadiynyl)-9H-purin-9-yl]-20,30-dihydroxybicyclo[3.1.0]-
hexane-10-carboxylic acid N-methylamide (8)
Compound 8 (86%) was synthesized from 6 following same pro-
cedure as for compound 9. 1H NMR (CD3OD, 300 MHz) d 8.07 (s,
1H), 7.42 (s, 1H), 7.25–7.42 (m, 3H), 5.01 (d, J = 6.9 Hz, 1H), 4.84–
4.87 (m, 1H), 3.98 (d, J = 6.6 Hz, 1H), 2.86 (s, 3H), 2.59 (t,
J = 7.2 Hz, 2H), 2.36–2.44 (m, 2H), 2.28 (t, J = 2.4 Hz, 1H), 2.06–
2.10 (m, 1H), 1.79–1.88 (m, 3H), 1.34–1.39 (m, 1H). HRMS calcd
for C27H28ClN6O3 (M+H)+: 519.1911; found 519.1912.
4. Experimental section
4.1. Chemical synthesis
1H NMR spectra were obtained with a Varian Gemini 300 spec-
trometer. When using D2O was used as a solvent, the chemical
shifts are expressed as relative ppm from HOD (4.80 ppm).
The purity of the final nucleotide derivatives were determined
using a Hewlett–Packard 1100 HPLC equipped with a Zorbax Eclipse
5 mm XDB-C18 analytical column (250 ꢂ 4.6 mm; Agilent Technol-
ogies Inc., Palo Alto, CA), using a linear gradient solvent system:
5 mM TBAP (tetrabutylammonium dihydrogenphosphate)-CH3CN
4.1.4. (10S,20R,30S,40S,50S)-40-[6-(3-Chlorobenzylamino)-2-(1,7-
octadiynyl)- 9H-purin-9-yl]-20,30-dihydroxybicyclo[3.1.0]-
hexane-10-carboxylic acid N-methylamide (9)
To a solution of compound 7 (350 mg, 0.61 mmol) in methanol
(7 mL), 10% trifluoromethane sulfonic acid was added and heated
at 70 °C for 6 h. Solvent was evaporated and the residue was
purified on flash silica gel chromatography (CH2Cl2/MeOH = 40:1)

514
D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
to give the compound 9 (295 mg, 91%) as a syrup. 1H NMR (CD3OD,
300 MHz) d 8.08 (s, 1H), 7.43 (s, 1H), 7.28–7.33 (m, 3H), 5.02 (d,
J = 6.6 Hz, 1H), 4.80–4.82 (M, 1H), 4.00 (dd, J1 = 1.2 Hz, J2 = 5.7 Hz,
1H), 2. 87 (s, 3H), 2.51 (t, J = 6.9 Hz, 1H), 2.22–2.30 (m, 3H), 2.07–
2.10 (m, 1H), 1.86 (t, J = 5.1 Hz, 1H), 1.70–1.81 (m, 4H), 1.35–1.40
(m, 1H). HRMS calcd for C28H30ClN6O3 (M+H)+: 533.2068; found
533.2082.
4H), 5.04 (d, J = 6.6 Hz, 1H), 4.83–4.85 (m, 1H), 3.99 (d, J = 6.6 Hz,
1H), 2.89–2.94 (m, 5H), 2.57 (t, J = 6.6 Hz, 2H), 1.98–2.14 (m, 4H),
1.77–1.90 (m, 4H), 1.38–1.43 (m, 1H). HRMS calcd for
C35H33ClN9O5 (MꢀH)+: 694.2293; found 694.2311.
4.1.9. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-4-(2-(6-(4-(2-
bromoacetyl)phenyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-
purin-9-yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic
acid N-methylamide (14)
Compound 14 (79%) was synthesized from 9 following same
procedure as for compound 11. 1H NMR (CD3OD, 300 MHz) d
8.43 (s, 1H), 8.33 (s, 1H), 8.07–8.15 (m, 4H), 7.21–7.42 (m, 4H),
5.01 (d, J = 6.6 Hz, 1H), 4.77–4.82 (m, 1H), 4.70 (s, 2H), 3.96 (d,
J = 6.9 Hz, 1H), 2.86–2.92 (m, 5H), 2.41 (t, J = 5.1 Hz, 2H), 1.93–
2.10 (m, 4H), 1.74–1.87 (m, 4H), 1.35–1.40 (m, 1H). HRMS calcd
for C36H35ClBrN9O4Na (M+Na)+: 796.1738; found 796.1713.
4.1.5. (1S,2R,3S,4R,5S)-40-(6-(3-Chlorobenzylamino)-2-(6-(1-(4-
fluoro-3-nitrophenyl)-1H-1,2,3-triazol-4-yl)pent-1-ynyl)-9H-
purin-9-yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic
acid N-methylamide (10)
Compound 10 (89%) was synthesized from 8 following same pro-
cedure as for compound 11. 1H NMR (CD3OD, 300 MHz) d 8.52–8.56
(m, 2H), 8.18–8.22 (m, 1H), 8.08 (s, 1H), 7.59–7.65 (m, 1H), 7.41 (s,
1H), 7.25–7.32 (m, 3H), 5.04 (d, J = 5.4 Hz, 1H), 4.80–4.83 (m, 1H),
3.99 (dd, J1 = 0.9 Hz, J2 = 5.7 Hz, 1H), 3.04 (t, J = 7.5 Hz, 2H), 2.87 (s,
3H), 2.60 (t, J = 6.6 Hz, 2H), 2.07–2.17 (m, 3H), 1.86 (t, J = 5.1 Hz,
1H), 1.38–1.45 (m, 2H), 0.89–0.96 (m, 1H). HRMS calcd for
C33H31ClFN10O5 (M+H)+: 701.2151; found 701.2172.
4.1.10. (1S,2R,3S ,4R,5S)-6-(3-Chlorobenzylamino)-(2-(6-(1-(4-
acetamidoethyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-purin-9-
yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (15)
4.1.6. (1S,2R,3S,4R,5S)-40-(6-(3-Chlorobenzylamino)-2-(6-(1-(4-
fluoro-3-nitrophenyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-
purin-9-yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic
acid N-methylamide (11)
To a mixture of compound 9 (34 mg, 0.063 mmol) and 4-fluoro-
3-nitro-phenyl azide (16.2 mg, 0.088 mmol) in in THF/H2O 3:1
Compound 15 (86%) was synthesized from 9 following same
procedure as for compound 11. 1H NMR (CD3OD, 300 MHz) d
8.09 (s, 1H), 7.78 (s, 1H), 7.43 (s, 1H), 7.27–7.33 (m, 3H), 5.03 (d,
J = 5.1 Hz, 1H), 4.83–86 (m, 1H), 4.47 (t, J = 5.7 Hz, 2H), 4.01 (d,
J = 6.6 Hz, 1H), 3.62 (t, J = 5.7 Hz, 2H), 2.87 (s, 3H), 2.79 (t,
J = 7.5 Hz, 2H), 2.53 (t, J = 7.2 Hz, 2H), 2.07–2.10 (m, 1H), 1.85–
1.93 (m, 5H), 1.69–1.74 (m, 2H), 1.36–1.41 (m, 1H). HRMS calcd
for C32H38ClN10O4 (M+H)+: 661.2766; found 661.2751.
(2 mL), was added freshly prepared 1 M sodium ascorbate (51
0.05 mmol) followed by 7.5% aqueous copper sulfate pentahydrate
solution (42 L, 0.012 mmol) and stirred for over night at room tem-
lL,
l
perature. Solvent was evaporated and the residue was purified on
flash silica gel column chromatography (CH2Cl2/MeOH = 45:1) to
give the clicked product 11 (42 mg, 94%) as a syrup. 1H NMR (CD3OD,
300 MHz) d 8.57–8.58 (m, 1H), 8.49 (s, 1H), 8.20–8.25 (m, 1H), 8.09
(s, 1H), 7.60–7.67 (m, 1H), 7.40 (s, 1H), 7.20–7.32 (m, 3H), 5.03 (d,
J = 6.6 Hz, 1H), 4.84–4.87 (m, 1H), 3.99 (d, J = 6.3 Hz, 1H), 2.91 (t,
J = 7.5 Hz, 2H), 2.87 (s, 3H), 2.56 (t, J = 6.9 Hz, 2H), 1.96–2.11 (m,
3H), 1.75–1.87 (m, 3H), 1.31–1.40 (m, 2H). HRMS calcd for
C34H33ClFN10O5 (M+H)+: 715.2308; found 715.2347.
4.1.11. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(6-(1-(4-
aminobutyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-purin-9-yl)-
20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (16)
Compound 16 (73%) was synthesized from 9 following same
procedure as for compound 12. 1H NMR (CD3OD, 300 MHz) d
8.11 (s, 1H), 7.81 (s, 1H), 7.45 (s, 1H), 7.28–7.34 (m, 3H), 5.06 (d,
J = 6.3 Hz, 1H), 4.80–4.83 (m, 1H), 4.42 (t, J = 6.6 Hz, 2H), 4.01 (d,
J = 6.6 Hz, 1H), 2.85–2.88 (m, 5H), 2.79–2.84 (m, 4H), 2.54 (t,
J = 7.2 Hz, 2H), 2.08–2.12 (m, 1H), 1.86–1.99 (m, 5H), 1.66–1.78
(m, 2H), 1.54–1.62 (m, 2H), 1.38–1.43 (m, 1H). HRMS calcd for
C32H40ClN10O3 (M+H)+: 647.2973; found 647.2968.
4.1.7. (1S,2R,3S,4R,5S)-40-(6-(3-Chlorobenzylamino)-2-(6-(1-(4-
amino-phenyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-purin-9-
yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (12)
To a mixture of compound 9 (4.46 mg, 0.008 mmol) and 4-ami-
no-phenylazide (2 mg, 0.011 mmol) in a mixture of t-butanol
(0.5 mL) and water (0.5 mL), was added TBTA (1 mg, 0.001 mmol)
4.1.12. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(6-(1-(4-
acetamidobutyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-purin-9-
yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (17)
and freshly prepared sodium ascorbate (8.3
l
L, 0.008 mmol) fol-
To a solution of compound 16 (1.79 mg, 0.002 mmol) in anhy-
drous DMF (0.5 mL), acetic acid N-hydroxysuccinimide ester
(1 mg, 0.006 mmol) was added and the mixture stirred at room
temperature for overnight. Solvent was evaporated and the residue
was purified in preparative TLC (CH2Cl2/MeOH = 25:1) to give the
lowed by copper sulfate (8.3 L, 0.003 mmol). The reaction mixture
l
was stirred at room temperature for overnight, solvent was evapo-
rated and the residue was purified on flash silica gel column chro-
matography (CH2Cl2/MeOH = 30:1) to give the compound 12
(4 mg, 72%) as a syrup. 1H NMR (CD3OD, 300 MHz) d 8.08 (s, 1H),
8.07 (s, 1H), 7.23–7.41 (m, 6H), 6.74 (d, J = 8.7 Hz, 2H), 5.01 (d,
J = 6.9 Hz, 1H), 4.78–4.82 (m, 1H), 3.96 (d, J = 6.0 Hz, 1H), 2.82–
2.86 (m, 5H), 2.53 (t, J = 6.9 Hz, 2H), 2.06–2.16 (m, 2H), 1.93–1.98
(m, 2H), 1.71–1.87 (m, 4H), 1.29–1.39 (m, 1H). HRMS calcd for
C34H35ClN10O3Na (M+Na)+: 689.2480; found 689.2465.
compound 17 as
a
syrup (1.26 mg, 66%). 1H NMR (CD3OD,
300 MHz) d 8.11 (s, 1H), 7.80 (s, 1H), 7.44 (s, 1H), 7.27–7.33 (m,
3H), 5.04 (d, J = 6.3 Hz, 1H), 4.83–4.85 (m, 1H), 4.39 (t, J = 6.9 Hz,
2H), 3.70 (t, J = 3.9 Hz, 4H), 3.59 (m, 4H), 2.87 (s, 3H), 2.78–2.86
(m, 2H), 2.48–2.57 (m, 2H), 1.93 (s, 3H), 1.85–1.91 (m, 2H), 1.68–
1.79 (m, 2H), 1.39–1.52 (m, 2H), 1.23–1.43 (m, 2H). HRMS calcd
for C34H41ClN10O4Na (M+Na)+: 711.2898; found 711.2917.
4.1.8. (1S,2R,3S,4R,5S)-40-(6-(3-Chlorobenzylamino)-2-(6-(1-(4-
carboxyl-phenyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-purin-9-
yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (13)
Compound 13 (81%) was synthesized from 9 following same
procedure as for compound 12. 1H NMR (CD3OD, 300 MHz) d
8.39 (s, 1H), 8.09–8.14 (m, 3H), 7.81–7.84 (m, 2H), 7.22–7.44 (m,
4.1.13. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(6-(1-
(adamantyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-purin-9-yl)-
20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (18)
Compound 18 (76%) was synthesized from 9 following same
procedure as for compound 11. 1H NMR (CD3OD, 300 MHz) d

D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
515
8.09 (s, 1H), 7.81 (s, 1H), 7.44 (s, 1H), 7.25–7.33 (m, 3H), 5.03 (d,
J = 6.6 Hz, 1H), 4.83–4.85 (m, 1H), 3.98 (d, J = 6.0 Hz, 1H), 2.86 (s,
3H), 2.79 (t, J = 7.2 Hz, 2H), 2.51 (t, J = 6.9 Hz, 2H), 2.16 (s, 9H),
2.06–2.10 (m, 1H), 1.68–1.95 (m, 13H), 1.36–1.40 (m, 1H). HRMS
calcd for C38H45ClN9O3 (M+H)+: 710.3334; found 710.3352.
4.1.18. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(1,6-bis(4-
isothiocyante-phenyl)-1H-bis(1,2,3-triazol-4-yl)hex-1-ynyl)-9H-
purin-9-yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic
acid N-methylamide (25)
To a mixture of compound 9 (17 mg, 0.031 mmol) and 4-azid-
ophenyl isothiocyante (7.8 mg, 0.044 mmol) in in THF/H2O 3:1
(1.2 mL), was added freshly prepared 1 M sodium ascorbate
4.1.14. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(1-(4–3-
oxoisothiazol-2(3H)-yl)benzyl-1H-1,2,3-triazol-4-yl)hex-1-
ynyl)-9H-purin-9-yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-
carboxylic acid N-methylamide (19)
Compound 19 (81%) was synthesized from 9 following same
procedure as for compound 11. 1H NMR (CD3OD, 300 MHz) d
8.56 (d, J = 6.3 Hz, 1H), 8.08 (s, 1H), 7.83 (s, 1H), 7.53–7.56 (m,
2H), 7.39–7.42 (m, 2H), 7.25–7.30 (m, 4H), 6.29 (d, J = 6.3 Hz,
1H), 5.6 (s, 2H), 5.02 (d, J = 6.9 Hz, 1H), 4.80–4.83 (m, 1H), 3.97
(d, J = 6.0 Hz, 1H), 2.86 (s, 3H), 2.80 (t, J = 7.2 Hz, 2H), 2.50 (t,
J = 7.2 Hz, 2H), 2.06–2.10 (m, 1H), 1.84–1.92 (m, 4H), 1.67–1.74
(m, 2H), 1.35–1.40 (m, 1H). HRMS calcd for C38H38ClSN10O4
(M+H)+: 765.2487; found 765.2461.
(26
lL, 0.025 mmol) followed by 7.5% aqueous copper sulfate
pentahydrate solution (21
l
L, 0.006 mmol) and stirred for 2 d at
room temperature. Solvent was evaporated and the residue was
purified on flash silica gel column chromatography (CH2Cl2/
MeOH = 20:1) to give an unusual clicked product 25 (21 mg, 75%)
as a syrup. 1H NMR (CD3OD, 300 MHz) d 8.29 (s, 1H), 8.12 (s,
1H), 7.71 (d, J = 8.7 Hz, 2H), 7.63 (d, J = 9.0 Hz, 2H), 7.50 (d,
J = 6.6 Hz, 2H), 7.41 (s, 1H), 7.23–7.30 (m, 3H), 7.08 (d, J = 6.6 Hz,
2H), 5.02 (d, J = 5.2 Hz, 1H), 4.79–4.81 (m, 1H), 3.97 (d, J = 6.6 Hz,
1H), 2.83–2.91 (m, 5H), 2.55 (t, J = 6.9 Hz, 2H), 1.97–2.09 (m, 4H),
1.74–1.86 (m, 4H), 1.39–1.47 (m, 1H). HRMS calcd for
C41H40ClN14O3S (MꢀCS)+: 843.2817; found 843.2812.
4.1.19. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(6-(1-(4-
isothiocyante-phenyl)-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-
purin-9-yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic
acid N-methylamide (26)
To a mixture of compound 9 (4.22 mg, 0.007 mmol) and 4-
isothiocyante-phenylazide (2 mg, 0.011 mmol) in a mixture of t-
butanol (0.3 mL) and water (0.3 mL), was added TBTA (1 mg,
4.1.15. (1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(6-(1-(6-
(5-(3aS,4S,6aR)-oxohexahydro-1H-thieno[3,4-d]imidazol-4-
yl)pentanamido)hexyl-1H-1,2,3-triazol-4-yl)hex-1-ynyl)-9H-
purin-9-yl)-20,30-dihydroxybicyclo[3.1.0]hexane-10-carboxylic
acid N-methylamide (20)
Compound 20 (73%) was synthesized from 9 and the appropri-
ate azide38 following same procedure as for compound 12. 1H NMR
(CD3OD, 300 MHz) d 8.08 (s, 1H), 7.77 (s, 1H), 7.42 (s, 1H), 7.23–
7.31 (m, 3H), 5.02 (d, J = 6.3 Hz, 1H), 4.83–4.86 (m, 1H), 4.45–
4.49 (m, 1H), 4.26–4.35 (m, 3H), 3.98 (d, J = 7.2 Hz, 1H), 3.65–
3.69 (m, 3H), 3.54–3.57 (m, 3H), 3.03–3.24 (m, 4H), 2.85 (s, 3H),
2.78 (t, J = 7.5 Hz, 2H), 2.51 (t, J = 7.2 Hz, 2H), 2.06–2.28 (m, 6H),
1.81–1.89 (m, 6H), 1.60–1.72 (m, 6H), 1.34–1.46 (m, 2H). HRMS
calcd for C44H57ClN12O5SNa (M+Na)+: 923.3882; found 923.3887.
0.001 mmol) and freshly prepared sodium ascorbate (7.9
lL,
0.007 mmol) followed by copper sulfate (7.9 L, 0.002 mmol).
l
The reaction mixture was stirred at room temperature for over-
night. The starting material and product came in same Rf value
in TLC. Solvent was evaporated and the residue was purified on
flash silica gel column chromatography (CH2Cl2/MeOH = 25:1) to
give the compound 26 (3.8 mg, 69%) as a syrup, no diclicked
product was detected. 1H NMR (CD3OD, 300 MHz) d 8.35 (s,
1H), 8.09 (s, 1H), 7.83 (d, J = 6.9 Hz, 2H), 7.40–7.43 (m, 3H),
7.24–7.32 (m, 3H), 5.02 (d, J = 6.3 Hz, 1H), 4.83–4.85 (m, 1H),
3.97 (d, J = 6.3 Hz, 1H), 2.87–2.92 (m, 5H), 2.55 (t, J = 6.9 Hz,
2H), 1.97–2.13 (m, 4H), 1.87 (t, J = 4.5 Hz, 2H), 1.74–1.83 (m,
2H), 1.38–1.41 (m, 1H). HRMS calcd for C35H34ClN10O3S (M+H)+:
709.2225; found 709.2236.
4.1.16. 1S,2R,3S,4R,5S)-6-(3-Chlorobenzylamino)-(2-(6-(1-(7-
oxo-7-(6-(5-(3aS,4S,6aR)-oxohexahydro-1H-thieno[3,4-
d]imidazol-4-yl)pentanamido)hexylamino)heptyl)-1H-1,2,3-
triazol-4-yl)hex-1-ynyl)-9H-purin-9-yl)-20,30-
dihydroxybicyclo[3.1.0]hexane-10-carboxylic acid N-
methylamide (21)
Compound 21 (82%) was synthesized from 9 following same
procedure as for compound 12. 1H NMR (CD3OD, 300 MHz) d
8.03 (s, 1H), 7.81 (s, 1H), 4.46 (s, 1H), 7.29–7.35 (m, 3H), 5.05 (d,
J = 6.5 Hz, 1H), 4.80–4.85 (m, 1H), 4.42–4.46 (m, 4H), 3.99 (d,
J = 6.9 Hz, 1H), 3.79–3.85 (m, 8H), 3.56–3.62 (m, 8H), 3.15–3.26
(m, 4H), 2.89 (s, 3H), 2.23–2.57 (m, 6H), 1.03–1.91 (bm, 28H).
HRMS calcd for C55H78ClN13O10SNa (M+Na)+: 1170.5302; found
1170.5288.
4.1.20. 4-Oxo-4-phenyl-N-p-tolyl-butyramide (28)
EDC (347 mg, 1.81 mmol) and HOBT (254 mg, 1.88 mmol) were
added to mixture of p-toluidine (129 mg, 1.20 mmol) and 3-ben-
zoylpropionic acid 27 (218 mg, 1.21 mmol) in 1,4-dioxane (3 mL)
at room temperature. It was stirred for 20 min and then added tri-
ethylamine (0.5 mL, 3.59 mmol). The reaction mixture was stirred
for 20 h at room temperature and the solvent was evaporated un-
der vacuum. The residue was purified by flash silica gel column
chromatography (hexane/ethyl acetate = 3:1–2:1) yielded the
compound 28 (252 mg, 78%). 1H NMR (CDCl3, 300 MHz) d 2.30 (s,
3H), 2.80 (t, J = 6.3 Hz, 2H), 3.45 (t, J = 6.3 Hz, 2H), 7.10 (d,
J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.47 (m, 2H), 7.58 (m, 1H),
7.99 (m, 1H).
4.1.17. 2-(6-Amino-3-imino-4,5-disulfonato-3H-xanthen-9-yl)-
5-(6-(4-(6-(6-3-chlorobenzylamino)-9-((1S,2R,3S,4R,5S)-3,4-
dihydroxy-5-(methylcarbamoyl)bicycle[3.1.0]hexane-2-yl)-9H-
purin-2-yl)hex-5-ynyl)-1H-1,2,3-triazol-1-
yl)hexylcarbamoyl)benzoate (23)
Compound 23 (76%) was synthesized from 9 following same
procedure as for compound 12. 1H NMR (CD3OD, 300 MHz) d
8.56 (s, 1H), 8.07(s, 1H), 7.83 (s, 1H), 7.26–7.43 (m, 4H), 7.09–
7.12 (m, 2H), 6. 90 (d, J = 6.9 Hz, 2H), 5.75 (d, J = 6.7 Hz, 1H), 5.57
(d, J = 6.8 Hz, 1H), 4.98 (d, J = 6.6 Hz, 1H), 4.80–4.84 (m, 1H), 4.13
(t, J = 3.6 Hz, 2H), 3.98 (d, J = 7.2 Hz, 1H), 3.69–3.72 (m, 4H),
3.59–3.64 (m, 4H), 2.89 (s, 3H), 2.50–2.55 (m, 4H), 2.19–2.32 (m,
4H), 1.21–1.46 (m, 7H). HRMS calcd for C55H54ClN12O13S2
(M+H)+: 1189.3063; found 1189.3038. Fluorescence (aq pH 7.4):
kex 490 nm, kex 520 nm.
4.1.21. 5-Benzoyl-2-p-tolylisothiazol-3-one (29)
A solution of 4-oxo-4-phenyl-N-p-tolyl-butyramide 28 (252 mg,
0.944 mmol) in thionyl chloride (1 mL) was stirred for 16 h at room
temperature. Excess thionyl chloride was removed under vacuum
and the residue was purified by flash silica gel column chromatog-
raphy (hexane/ethyl acetate = 10:1–4:1) to give the compound 29
(216 mg, 77%). 1H NMR (CDCl3, 300 MHz) d 2.40 (s, 3H), 6.82 (s,
1H), 7.28 (m, 2H), 7.54 (m, 4H), 7.71 (m, 1H), 7.96 (m, 2H). HRMS:
calcd for C17H14NO2S (M+H)+ 296.0745; found 296.0762.

516
D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
4.1.22. 2-p-Tolylisothiazol-3-one (30)
nized with an electric homogenizer for 10 s, pipetted into 1 mL
vials and then stored at ꢀ80 °C until the binding experiments
were conducted. The protein concentration was measured using
the BCA Protein Assay Kit from Pierce Biotechnology, Inc. (Rock-
ford, IL).37
Aqueous sodium hydroxide (4 mL, 10%) was added to the
solution of 5-benzoyl-2-p-tolylisothiazol-3-one 29 (203 mg,
0.678 mmol) in benzene (9 mL), and the mixture was stirred for
3 d at room temperature. The reaction mixture was added water
(20 mL) and extracted with ethyl acetate. The combined organic
layer was dried (MgSO4), filtered and evaporated. The residue
was purified by flash silica gel column chromatography (hexane/
ethyl acetate = 2:1–1:1) and gave the compound 30 (77 mg, 59%).
1H NMR (CDCl3, 300 MHz) d 2.38 (s, 3H), 6.32 (d, J = 6.6 Hz, 1H),
7.25 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 8.14 (d, J = 6.6 Hz,
1H). HRMS: calcd for C10H10NOS (M+H)+ 192.0483; found
192.0494.
4.2.2. Binding assays
Into each tube in the binding assay was added 50
ing concentrations of the test ligand in Tris–HCl buffer (50 mM, pH
7.5) containing 10 mM MgCl2, 50 L of the appropriate agonist
radioligand, and finally 100 L of membrane suspension. For the
A1AR (22
g of protein/tube) the radioligand used was [3H]36 (final
concentration of 3.5 nM). For the A2AAR (20 g/tube) the radioli-
gand used was [3H]37 (10 nM). For the A3AR (21
g/tube) the radi-
oligand used was [125I]38 (0.34 nM). Nonspecific binding was
determined using a final concentration of 10 M unlabeled 36 di-
lL of increas-
l
l
l
l
l
4.1.23. 2-(4-Bromomethylphenyl)isothiazol-3-one (31)
Catalytic amount of benzoyl peroxide (4 mg) was added to a
solution of 2-p-tolylisothiazol-3-one 30 (123 mg, 0.643 mmol),
and N-bromosuccinimide (121 mg, 0.673 mmol) in carbon tetra-
chloride (8 mL), which was refluxed for 2 h and then cooled to
room temperature. Solvent was evaporated under low pressure
and the residue was purified by flash silica gel column chromatog-
raphy (methylene chloride/methanol = 50:1–20:1), afforded the
compound 31 (182 mg, 93%). 1H NMR (CDCl3, 300 MHz) d 4.50 (s,
2H), 6.33 (d, J = 6.3 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.59 (d,
J = 8.4 Hz, 2H), 8.16 (d, J = 6.3 Hz, 1H). HRMS: calcd for C10H9NOSBr
(M+H)+ 269.9588; found 269.9593.
l
luted 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 a 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 c-counter. The Ki values were determined using
GraphPad Prism for all assays.
4.1.24. 2-(4-Azidomethylphenyl)isothiazol-3-one (32)
Sodium azide (6.0 mg, 0.0923 mmol) was added to a solution of 2-
(4-bromomethylphenyl)isothiazol-3-one 31 (18.2 mg, 0.0674 mmol)
in DMF (1 mL) and stirred for 20 h at room temperature. Water
(10 mL) was added and the mixture was extracted with diethyl ether.
The combined organic layer was dried (MgSO4), filtered and evapo-
rated. The residue was purified on flash silica gel column chromatog-
raphy (hexane/ethyl acetate = 4:1–1:1), afforded the desired
compound 32 (11.7 mg, 75%). 1H NMR (CDCl3, 300 MHz) d 4.38 (s,
2H), 6.34 (d, J = 6.3 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.64 (d,
J = 8.4 Hz, 2H), 8.17 (d, J = 6.3 Hz, 1H). HRMS: found 233.0496; calcd
for C10H9N4OS (M+H)+ 233.0497.
4.2.3. Cyclic AMP accumulation assay
Intracellular cyclic AMP levels were measured with a compet-
itive protein binding method.29,30 CHO cells that expressed the re-
combinant human A3AR 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 in the presence of
rolipram (10
lM) and adenosine deaminase (3 units/mL). After
45 min forskolin (10
l
M) was added to the medium, and incuba-
tion was continued for an additional 15 min. The reaction was
terminated by removing the supernatant, and cells were lysed
4.2. Receptor binding and functional assays
upon the addition of 200 lL of 0.1 M ice-cold HCl. The cell lysate
[3H]Adenosine-50-N-methyluronamide (36, [3H]NECA, 42.6 Ci/
mmol) was obtained from Perkin Elmer. [3H](2-[p-(2-Carboxy-
ethyl)phenyl-ethylamino]-50-N-ethylcarboxamido-adenosine) (37,
[3H]CGS21680, 40.5 Ci/mmol) and [125I]N6-(4-amino-3-iodoben-
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 (K2HPO4,
150 mM; EDTA, 10 mM), 20
l
L of the cell lysate, and 30
l
l
L
L
zyl)adenosine-50-N-methyluronamide (38,
[
125I]I-AB-MECA,
0.1 M HCl or 50 L of cyclic AMP solution (0–16 pmol/200
l
2200 Ci/mmol) were purchased from Perkin–Elmer Life and Ana-
lytical Science (Boston, MA). Test compounds were prepared as
5 mM stock solutions in DMSO and stored frozen at ꢀ20 °C.
for standard curve). Bound radioactivity was separated by rapid
filtration through Whatman GF/C filters and washed once with
cold buffer. Bound radioactivity was measured by liquid scintilla-
tion spectrometry.
4.2.1. Cell culture and membrane preparation
CHO cells stably expressing the recombinant hA1 and hA3Rs,
and HEK-293 cells stably expressing the hA2AAR were cultured
in Dulbecco’s modified Eagle medium (DMEM) and F12 (1:1) sup-
plemented with 10% fetal bovine serum, 100 units/mL penicillin,
Acknowledgements
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. We thank Arunkumar
Easwaran, Ph.D., Molecular Targeting Technologies, Inc. West Ches-
ter, PA for helpful discussions.
100
800
l
g/mL streptomycin, and 2
l
mol/mL glutamine. In addition,
lg/mL geneticin was added to the A2A media, while 500
lg/
mL hygromycin was added to the A1 and A3 media. After harvest-
ing, cells were homogenized and suspended in PBS. Cells were
then centrifuged at 240g for 5 min, and the pellet was resus-
pended in 50 mM Tris–HCl buffer (pH 7.5) containing 10 mM
MgCl2. The suspension was homogenized and was then ultra-cen-
trifuged at 14,330g for 30 min at 4 °C. The resultant pellets were
resuspended in Tris buffer, incubated with adenosine deaminase
(3 units/mL) for 30 min at 37 °C. The suspension was homoge-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.12.018.

D. K. Tosh et al. / Bioorg. Med. Chem. 18 (2010) 508–517
517
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