Synthesis and structure-activity relationships of 2-hydrazinyladenosine derivatives as A2A adenosine receptor ligands

Article abstract of DOI:10.1016/j.bmc.2012.11.021

A series of 2-hydrazinyladenosine derivatives was synthesized and investigated in radioligand binding studies for their affinity at the adenosine receptor subtypes with the goal to obtain potent and A_2AAR selective agonists and to explore the structure-activity relationships of this class of compounds at A_2AAR. Modifications included introduction of a second sugar moiety at position 2 of adenosine to form new bis-sugar nucleosides and/or modifications of the 2-position linker in different ways. The performed modifications were found to produce compounds with relatively high A _2AAR affinity and very high selectivity toward A_2AAR. The most potent bis-sugar nucleoside was obtained with the D-galactose derivative 16 which exhibited a K_i value of 329 nM at A_2AAR with marked selectivity against the other AR subtypes. In another set of compounds, compound 3 was modified via replacement of its cyclic structure with mono- and disubstituted phenyl moieties and the resulting hydrazones 10-14 were found to have low nanomolar affinity for A_2AAR. In addition to 3, compounds 10, 11 and 13 have been identified as the most potent compounds in the present series with K_i values of 16.1, 24.4, and 12.0 nM, respectively, at rat A_2AAR. Species differences were tested and found to exist in different rates. Functional properties of the most potent compounds 10, 11, 13 and 16 were assessed showing that the compounds acted as agonists at A _2AAR.

Full text of DOI:10.1016/j.bmc.2012.11.021

Bioorganic & Medicinal Chemistry 21 (2013) 436–447
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationships of 2-hydrazinyladenosine
derivatives as A2A adenosine receptor ligands
⇑
,
Ali El-Tayeb a,b, , Sabrina Gollos c
a Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
b Department of Pharmaceutical Medicinal Chemistry, Faculty of Pharmacy, University of Al-Azhar, Assiut Branch, 71524 Assiut, Egypt
c Isotope Laboratory, PharmaCenter Bonn, Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 2-hydrazinyladenosine derivatives was synthesized and investigated in radioligand binding
studies for their affinity at the adenosine receptor subtypes with the goal to obtain potent and A2AAR
selective agonists and to explore the structure–activity relationships of this class of compounds at
A2AAR. Modifications included introduction of a second sugar moiety at position 2 of adenosine to form
new bis-sugar nucleosides and/or modifications of the 2-position linker in different ways. The performed
modifications were found to produce compounds with relatively high A2AAR affinity and very high selec-
Received 10 September 2012
Revised 7 November 2012
Accepted 12 November 2012
Available online 27 November 2012
Keywords:
Adenosine A2A receptor ligand
A2A agonist
Bis-sugar nucleoside
2-Hydrazinyladenosine derivative
tivity toward A2AAR. The most potent bis-sugar nucleoside was obtained with the D-galactose derivative
16 which exhibited a Ki value of 329 nM at A2AAR with marked selectivity against the other AR subtypes.
In another set of compounds, compound 3 was modified via replacement of its cyclic structure with
mono- and disubstituted phenyl moieties and the resulting hydrazones 10–14 were found to have low
nanomolar affinity for A2AAR. In addition to 3, compounds 10, 11 and 13 have been identified as the most
potent compounds in the present series with Ki values of 16.1, 24.4, and 12.0 nM, respectively, at rat
A2AAR. Species differences were tested and found to exist in different rates. Functional properties of
the most potent compounds 10, 11, 13 and 16 were assessed showing that the compounds acted as ago-
nists at A2AAR.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and pathophysiological processes through its action on cell-surface
G protein-coupled adenosine receptors.1–3 Four adenosine receptor
Nucleoside adenosine (1, Fig. 1) is a naturally occurring extra-
cellular purine nucleoside, which modulates multiple physiological
(AR) subtypes have been cloned and characterized, A1, A2A, A2B and
A3. The A1 and A3 receptor subtypes are Gi-coupled receptors, acti-
vation of these receptors leads to inhibition of adenylate cyclase,
while activation of A2A and A2B receptor subtypes stimulate the
adenylate cyclase through Gs protein. In addition, coupling to other
second messenger systems has been described.1–3 A1AR are highly
expressed in the brain (cortex, cerebellum, and hippocampus), dor-
sal horn of spinal cord, eye, adrenal gland and atria. A2AAR are
highly expressed in spleen, thymus, leukocytes, blood platelets,
striatopallidal GABAergic neurons, and olfactory bulb.1,2 In the
brain, A2AAR are expressed in high density in the striatum, while
in the periphery A2AAR are highly expressed in the intestinal
mucosa, enteric neurons, hepatocytes and a variety of immune
cells. In the intestine, A2AAR are expressed in the jejunum, ileum,
and cecum.1,2 A2BAR are highly expressed in cecum, colon, bladder
and in intermediate or low levels in other regions, for example,
lung, blood vessels, mast cells and ovary. A3AR are highly
expressed in testis, mast cells, lung, and spleen.1,2 The crystal
structure of the human A2A receptors has been solved via number
of successive studies in the last few years. The structure of A2AAR
Abbreviations: AR, adenosine receptor(s); binodenoson, 2-((cyclohexylmethyl-
ene)hydrazino)adenosine; BSA, bovine serum albumin; CADO, 2-chloroadenosine;
[3H]CCPA, [3H]2-chloro-N6-cyclopentyladenosine; CHO, Chinese hamster ovary;
[3H]CGS21680, [3H]((2-(4-(2-carboxyethyl)phenyl)ethylamino)-50-N-ethylcarbox-
amido-adenosine; CPA, N6-cyclopentyladenosine; DMSO, dimethylsulfoxide; DPCPX,
8-cyclopentyl-1,3-dipropylxanthine; ESI, electrospray ionization; h, human; HEK,
human embryonic kidney; NECA, 50-(N-ethylcarboxamido)adenosine; R-PIA, (R)-N6-
phenylisopropyladenosine; PSB, Pharmaceutical Science Bonn; [3H]PSB-603, [3H]8-
(4-(4-(4-chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine; PSB-0777,
4-(2-(6-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-
2-yl)-9H-purin-2-ylthio)ethyl)benzene sulfonic acid; r, rat; SAR, structure–activity
relationships; TLC, thin layer chromatography; TRIS, tris(hydroxymethyl)amino-
methane; vs., versus.
Corresponding author. Present address: Pharmazeutisches Institut, Pharmaze-
utische Chemie I, An der Immenburg 4, D-53121 Bonn, Germany. Tel.: +49 228 73
2564; fax: +49 228 73 2567.
E-mail addresses: a.el-tayeb@uni-bonn.de, ali72eg@yahoo.com (A. El-Tayeb).
Permanent address: Department of Pharmaceutical Medicinal Chemistry, Faculty
of Pharmacy, Al-Azhar University, Assiut Branch, 71524 Assiut, Egypt. Tel.: +2 088
2312191; fax: +2 088 2181191.
⇑
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.021

A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
437
NH2
N
NH2
NH2
N
N
N
N
N
N
N
N
H
N
O
N
HO
HO
N
N
N
N
H
O
O
O
OH
OH
OH
OH
OH
OH
1 Adenosine
2 NECA
3 Binodenoson
rA2AAR Ki = 15 nM20
hA2AAR Ki = 20 nM3
hA2AAR Ki = 270 nM3
O
O
S
NH2
NH2
O
OH
N
N
N
OH
N
H
N
O
HO
N
N
N
N
N
H
S
O
O
OH OH
OH OH
4 CGS-21680
5 PSB-0777
rA2AAR Ki = 18 nM20
hA2AAR Ki = 27 nM3
rA2AAR Ki = 44.4 nM13
hA2AAR Ki = 360 nM13
Figure 1. Structures of selected potent and selective A2AAR agonists.
have been solved in complex with the conformationally selective
agonists, which would be preferably dephosphorylated in inflamed
tissues with high expression of ecto-50-nucleotidase.12a,b This is
thought to allow a separation of anti-inflammatory and hypoten-
sive effects since the concentration of the active drug, the A2AAR
agonist, would only be achieved locally at the sites of inflammation
with high ecto-50-nucleotidase activity.12a,b In a further study and
to avoid the vasodilatory side effects we have developed local
highly polar, perorally non-absorbable A2A-selective agonists as a
local therapy, for example, of inflammatory bowel disease, avoid-
ing the hypotensive effects of centrally acting anti-inflammatory
A
2AAR agonist, that capable of receptor stabilization in a specific
active-state configuration, 2-(3-(1-(pyridin-2-yl)piperidin-4-yl)
ureido)ethyl-6-N-(2,2-diphenylethyl)-50-N-ethylcarboxamidoade-
nosine-2-carboxamide (UK-432097)4 and with the A2AAR antago-
nist
4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]
triazin-5-ylamino]ethyl)phenol (ZM241385),5 in 2.7, and 2.6 Å
resolution, respectively. In addition, X-ray structures of the
A
2AAR bound to its endogenous agonist adenosine (1, Fig. 1, 3 Å
resolution), and the synthetic analogue 50-(N-ethylcarboxami-
do)adenosine (NECA, 2, Fig. 1, 2.6 Å resolution) have recently been
obtained showing that the ribose residue of agonists occupies a
distinct hydrophilic binding pocket from that occupied by antago-
nists. The agonists induce motion of the helices 3, 5 and 7 and the
antagonists prevent conformational change of the receptor.6
Due to the wide distribution of the AR subtypes, selective AR
agonists are recommended for the treatment of a variety of patho-
logical conditions. After more than three decades of research,
selective agonists and antagonists for almost all four adenosine
receptor subtypes are available.7 A considerable number of syn-
thetic selective AR agonists have been discovered and clinically
evaluated. For example, the A1AR agonist GW493838 was investi-
gated for the treatment of peripheral neuropathic pain in patients
with postherpetic neuralgia or peripheral nerve injury.3,7 The A3AR
agonist CF101 was designed for the treatment of patients suffering
from rheumatoid arthritis.3,7 Regadenoson, an A2AAR agonist, has
been approved in 2008 by the food and drug administration
(FDA) for stress testing in conjunction with myocardial perfusion
imaging because of its vasodilatory effects via its activation of
A
2AAR agonists.13
Most of the potent and selective A2AAR agonists are adenosine
derivatives modified in the 2-position of the adenine moiety by
introducing bulky substituents and/or in the 50-position of the
riboside of the adenosine scaffold as shown in Figure 1. N6,2-Disub-
stitution and substitution in position 8 may also lead to potent
A2AAR agonists. Introduction of N-ethylcarboxamide moiety at
the 50-position (e.g., NECA, 2, Fig. 1) or a tetrazole ring at the 50-po-
sition of the ribose ring9a represents the most structural modifica-
tions at the ribose moiety of the adenosine scaffold. At position 2 of
the purine moiety, variations include the introduction of large cyc-
lic substituents (aromatic or cycloalkyl) or a simple long lipophilic
alkyl chain, and in most cases a three-membered linker between
the large cyclic substituents and the purine moiety of the adeno-
sine scaffold often containing an electron-rich function (e.g., O,
NH, triple bond) attached to the purine ring, for example, Binode-
noson (3), CGS-21680 (4) and PSB-0777 (5) as shown in Figure 1.
However, substitution at 2-position of purine moiety represents
the essential substitution site and the shared common structural
features in all known potent and selective A2AAR agonists.3,7,9b–18
The recently published crystal structures of A2AAR showed the
importance of the extracellular loops, for example, EL3 of A2AAR
in the binding with the substituents in the 2-position of the purine
moiety.4–6,19 Therefore, in the present study, our goal was to ex-
plore the structure–activity relationships (SAR) of A2AAR agonists
modified at 2-position of the purine moiety of the adenosine scaf-
fold via synthesis of some 2-substituted adenosine derivatives and
evaluation of their affinity at A2AAR as well as at the other adeno-
sine receptor subtypes.
A
2AAR.3,7
Adenosine A2A receptors mediate the potent immunosuppressive
and hypotensive effects of adenosine.3,8 One of the main therapeutic
potentials of A2AAR agonists is due to their anti-inflammatory and
immunosuppressive effects. On the other hand, A2AAR agonists
are potent vasodilators and are applied as diagnostics for pharma-
cologic stress testing in the heart. Further potential therapeutic
applications of A2AAR agonists are the treatment of psychosis and
9–11
´
Huntingtons disease.
Activation of the A2AAR on a variety of
inflammatory cell types leads to anti-inflammatory effects. How-
ever, the systemic use of A2AAR agonists as anti-inflammatory
drugs is limited by their potent hypotensive activity due to activa-
tion of A2AAR expressed in heart and blood vessels.1,8 Recently,
As starting structure for modifications, we have selected 2-
((cyclohexylmethylene)hydrazino)adenosine (Binodenoson, Cor-
Vue™, 3, Fig. 1). Binodenoson (3) has been identified as a potent
and selective A2AAR agonist and has been clinically evaluated to
we have developed phosphorylated adenosine-derived
A2AAR

438
A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
be used as a stress agent in conjunction with myocardial perfusion
imaging.21,22 To achieve our goals, we planned to resynthesize and
evaluate nucleoside 3 at A2AAR, and then to replace the cyclohexyl
moiety of 3 with different mono- or disubstituted aromatic moie-
ties. As common feature in the most known potent A2AAR agonists
(Fig. 1) a three atom linker between the purine moiety and the cyc-
lic structure was observed and seems to be required for A2AAR
activity. In this study our goal was to explore the SAR of this linker
via its extension by one more atom, to remove the hydrazone dou-
ble bond and to introduce hydrogen bond acceptors in the linker
via introduction of urea and thiourea moieties within the linker.
Removal of the hydrogen bond donor at the nitrogen atom of the
2-hydrazinoadenosine via introduction of methyl moiety was also
designed. In another set of compounds, our goal was to introduce
monosaccharide pentose and hexose moieties (aldose or ketose)
as a second sugar moiety at the 2-position of adenosine scaffold
to replace the cyclohexyl moiety of 3 to form bis-sugar nucleosides.
Formation of such bis-sugar nucleosides connected to the adenine
moiety via different lengths and possessing multiple hydroxyl
groups distributed with different stereogenic centers will give
more information about the receptor ligand interaction. For exam-
ple, the binding mode, the possibility of hydrogen bond formation
with A2AAR and the sterogenic requirements which might be in-
volved in the agonist receptor interaction. In addition to SAR explo-
ration, formation of the bis-sugar nucleosides with multiple
hydroxyl groups will markedly increase the hydrophilicity of the
produced compounds and accordingly, the resulting highly polar
and less absorbable A2AAR agonists can work locally as anti-inflam-
matory, for example, for the treatment of inflammatory bowel
disease, avoiding the hypotensive effects of centrally acting
anti-inflammatory A2AAR agonists.13 Finally, the effect of the new
modifications on the A2AAR as well as on the other AR subtypes
were evaluated and reported in the present study.
corresponding hydrazones 3, 10–24 in high yields (Scheme 1).
The nucleoside hydrazones 3, 10–11, 13–14 were precipitated
from methanol after cooling while compound 12 was further puri-
fied using silica gel column chromatography. The bis-sugar nucleo-
sides 15–24 were purified by high performance liquid
chromatography (HPLC) on reverse phase C18 material in order
to remove the traces of the unreacted sugars.
N-Phenylhydrazinecarboxamide and N-phenylhydrazinecar-
bothioamide moieties were introduced into the 2-position of
adenosine via reaction of 9 with phenylisocyanate or phenylisothi-
ocyanate in methanol under mild condition to give the desired
products 25 and 26, respectively, in high yields as shown in
Scheme 2. Compounds 25 and 26 were precipitated from the
reaction mixture, washed with methanol and crystalized by addi-
tion of ethyl acetate and excess n-hexane without further need
for chromatographic purifications.
The structures of the synthesized nucleosides were confirmed
by 1H- and 13C NMR spectroscopy, in addition to HPLC analysis
coupled to electrospray ionization mass spectrometry (LC/ESI-
MS) performed in both, positive and negative mode. The purity
of the products was generally P95%.
3. Biological activity
The synthesized 2-hydrazinyladenosine derivatives were
in vitro investigated in radioligand binding studies at the AR sub-
types. The binding studies were performed at rat brain cortical
membranes for A1AR using [3H]CCPA as a radioligand, at rat brain
striatal A2AAR using the agonist radioligand [3H]CGS21680 and at
membrane preparations of human recombinant A3AR expressed
in Chinese hamster ovary (CHO) cells using [3H]NECA as a radioli-
gand. Two nucleosides were additionally tested at human recombi-
nant A1 and A2A receptors using [3H]CCPA and [3H]CGS21680 as
radioligands, respectively, in order to investigate the potential spe-
cies differences. Bis-sugar nucleosides 15–24 as well as nucleoside
3 were additionally investigated at human recombinant A2BAR
using the antagonist radioligand [3H]PSB-603 because the agonist
radioligand for A2BAR does not exist. Functional properties of the
most potent compounds 10, 11, 13 and 16 were assessed by aden-
ylate cyclase assays measuring cAMP accumulation in human CHO
cells stably expressing the human A2AAR. The test results of all syn-
thesized nucleosides at the four adenosine receptor subtypes are
depicted in Table 1.
2. Results and discussion
2.1. Syntheses
2-Chloroadenosine (7) was synthesized starting from guanosine
(6) in four steps according to published procedures23–26 with few
modifications, later it was bought from commercial sources. At
first, protection of the 20-, 30- and 50-hydroxyl groups of guanosine
(6) was achieved via acetylation using acetic anhydride in acetoni-
trile under mild conditions to give 20,30,50-acetylguanosine
(6a).23,24 The 6-chloro-20,30,50-acetylguanosine derivative (6b)
was obtained via nucleophilic displacement reaction of 20,30,50-
acetylguanosine with phosphorus oxychloride at 100 °C to give
the product in a good yield.25 2,6-Dichloro-20,30,50-acetyl adenosine
nucleoside (6c) was obtained via diazotization/chloro-dediazonia-
tion of 6-chloro-20,30,50-acetylguanosine according to published
procedures25,26 which was used as such for the next reaction steps
after preliminary purification and without further structure eluci-
dation. An N6-amino function was introduced and 20-, 30- and 50-
acetyl groups were removed in one reaction step using saturated
ammonia in ethanol solution to afford the 2-chloroadenosine
nucleoside 7 in low yields (20%) after reaction time of 3 days as
shown in Scheme 1. For detailed reaction procedures, NMR charac-
terization and LCMS data of the intermediate compounds see Sup-
plementary data.
3.1. Adenosine receptor affinity
In general, the determined Ki values in the present series of
compounds confirm the previous observations and the reported
SAR at A2AAR in which the 2-substituted adenosine derivatives
possess a higher affinity toward the A2AAR than the other AR sub-
types. This affinity is strongly depending on the nature of the sub-
stituent, which should be in accordance with the general SAR
requirements at A2AAR, for example, presence of cyclic structure
or long chain separated from the adenine moiety of the position-
2 of the adenosine scaffold via a linker consists of mostly three
atoms. Introduction of a second sugar moiety linked to position 2
of adenosine via a hydrazone linker to form a bis-sugar nucleoside
structure with different sugar lengths and distinguished stereogen-
ic centers (nucleosides 15–24) generally led to compounds with a
relatively higher affinity toward A2AAR than the other AR subtypes
as shown in Table 1 (see also Supplementary Fig. S1). However, the
affinities still lower than that of the parent compound 3 (Fig. 1),
probably due to a lack of the cyclic structure observed at the most
potent A2AAR agonists. The rank order of potency of bis-sugar
2-Chloroadenosine (7) was treated with hydrazine21,27 or meth-
ylhydrazine to give the corresponding 2-hydrazinoadenosine (8)
and 2-N-methylhydrazinoadenosine (9), respectively. Refluxing of
8 or 9 with aromatic aldehydes, aldose monosaccharides (
cose, -galactose, -mannose and -ribose) or ketose monosaccha-
ride ( -fructose) in methanol for several hours afforded the
D-glu-
D
D
D
D
nucleosides 15–19 at A2AAR was 16 (D-galactose; 329 nM) > 15

A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
439
O
O
OH
N
O
O
N
N
H3C
H3C
O
O
O
O
O
Cl
N
N
N
a
b
NH
NH2
N
NH
NH2
N
O
N
O
N
HO
OH
O
O
O
O
O
O
NH2
H3C
H3C
6
6a
6b
CH3
CH3
c
O
OH
OH
N
N
N
O
N
H3C
O
NH2
O
NH2
O
Cl
N
N
N
d
e
N
N
N
N
N
O
HO
OH
HO
OH
O
O
O
HN
Cl
Cl
8
7
6c
NH2
H3C
CH3
g
f
OH
OH
N
N
N
OH
N
O
NH2
O
NH2
N
N
O
NH2
g
N
N
N
N
N
N
HO
OH
HO
OH
HO
3,10-19
OH
N
N
R
NH2
N
HN
R
9
20-24
H3C
H3C
N
OH
Cl
Cl
Cl
OCH3
OCH3
Cl
3
10
11
12
13
14
Cl
R =
OH OH
OH OH
OH OH
OH OH
OH
OH HO
OH
OH
OH
OH
OH OH
15, 20
OH OH
16, 21
OH OH
17, 22
OH
OH OH
18, 23
19, 24
Scheme 1. Synthesis of nucleosides (7–24), Reagents and conditions: (a) acetic anhydride, 4-dimethylaminopyridine, ethyldimethylamine, acetonitrile, 1 h, rt; (b) POCl3,
tetraethylammonium chloride, N,N-dimethylaniline, 15–30 min, 100 °C; (c) benzyltriethylammonium nitrite, acetylchloride, dichloromethane, 1 h, 0 °C; (d) saturated
ammonia in ethanol solution, 3 days, rt; (e) hydrazine hydrate, rt, 10 h; (f) methylhydrazine, rt, 7 h; (g) R-CHO or R-C@O for compounds 19 and 24, methanol, reflux, 3–12 h.
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
H
N
H
N
H
N
H
N
NH2
HO
HO
HO
N
N
N
N
N
N
b
a
O
O
O
CH3
S
CH3
CH3 O
OH
OH
OH
OH
25
OH
OH
9
26
Scheme 2. Synthesis of nucleosides (25–26), Reagents and conditions: (a) phenylisocyanate, methanol, 2 h, rt; (b) phenylisothiocyanate, methanol, 2 h, rt.
(
(
D
D
-glucose; 42% inhibition) > 19 (
-mannose; 33% inhibition) ꢀ 18 (
D
-fructose; 34% inhibition) > 17
-ribose; 33% inhibition). The
led to relatively lower affinity bis-sugar nucleosides, compare
15–19 versus (vs.) 20–24, this was also observed in compounds
25 and 26. The methylated analogue of the most potent nucleoside
16, compound 21, exhibited only 25% inhibition at A2AAR (Table 1).
Further modifications and experimental evaluations of 16 includ-
ing functional assays and binding studies using human receptors
are aimed in order to have more potent A2AAR agonists while keep-
ing their A2AAR selectivity.
The synthesized bis-sugar nucleosides were screened at A2BAR
using the antagonist radioligand [3H]PSB-603 and the compounds
showed almost low or no affinity at this low affinity AR subtype.
The promising substituent was observed in the case of compounds
D
most potent compound in the present bis-sugar nucleoside series
at A2AAR was obtained with -galactose modification, compound
D
16, which exhibited a Ki value of 329 nM (Fig. 2B). Compound 16
was only 13-fold less potent than 3 but 16 possessed higher selec-
tivity toward the other AR subtypes in comparison with 3. The ste-
reogenic distribution of the hydroxyl groups of the second sugar
moiety seems to be important for activity and selectivity; the fa-
vored stereogenic distribution at A2AAR was obtained with D-gal-
actose sugar. Removal of the hydrogen bond donor at the
nitrogen atom of the 2-hydrazinoadenosine via introduction of a
methyl moiety at the nitrogen atom of the hydrazine structure
19 (D
-fructose; 17% inhibition) ꢀ 21 (D-galactose; 17% inhibition)

Table 1
Adenosine receptor affinities of the synthesized nucleosides 3 and 10–26
NH2
N
NH2
N
NH2
N
N
N
N
N
N
H
N
H
N
R
R
HO
HO
N
HO
N
N
N
N
N
N
H
N
N
O
O
O
CH3
CH3
R
R = O 25
OH
OH
20 24
OH
OH
25 26
-
OH
,
OH
R
26
= S
3 10 19
-
-
Nucleoside
R
A1AR rat brain cortical membranes vs.
[3H]CCPAa
A2AAR rat brain striatal membranes vs.
[3H]CGS21680a
A2BAR human recombinant vs. [3H]PSB-
603b,c,d
A3AR human recombinant vs.
[3H]NECAa
Ki SEM (nM) or (% inhibition SEM at 1
lM), (n = 3)
2 (NECA)
-
-
-
5.1 [3]
15 [20]
20g [3]
1890 [29]
6.2 [28]
13.6f [28]
1800 [3]
289f [3]
4 (CGS21680)
5 (PSB-0777)
18 [20]
>10,000 [29]
>10,000 [13]
114h [30]
>1000 [13]
27g [3]
P10,000 [13]
44.4 [13]
360g [13]
54f [13]
70.8 3.6
169 13f
48,000f [3]
25.5 3.5
81 34g
270g [3]
>10,000
(17 3)
430,000 [3]
52.6 5.1
903 [3]
3 (Binodenoson)
10
108 31
263 53
16.1 1.9
24.4 7.2
n.d.e
n.d.
71.9 4.7
88.8 8.6
Cl
Cl
11
12
Cl
OH
58.0 9.2
91.7 22.9
n.d.
131 25
Cl
65.7 11.6
723 280f
12.0 4.1
13
14
n.d.
n.d.
112 47
687 127g
OCH3
Cl
32.1 2.1
53.7 15.1
51.1 17.5
OCH3
OH OH
>1000
(11 8)
>1000
(42 1)
>10,000
(10 1)
>1000
(3 2)
15
16
17
OH
OH
OH
OH OH
OH OH
>10,000d
(31 2)
>10,000
(6 7)
>10,000d
(38 4)
329 104
OH OH
OH OH
>1000
(34 1)
>1000
(33 1)
>10,000
(12 5)
>1000
(16 2)
OH OH

OH OH
OH
>1000
(24 2)
>1000
(33 5)
>10,000
(14 3)
>1000
(23 11)
OH
18
19
20
21
22
23
24
OH
>1000
(25 2)
>1000
(34 3)
>10,000
(17 5)
>1000
(15 5)
HO
OH
OH OH
OH OH
>1000
(2 4)
>1000
(31 3)
>10,000
(7 5)
>1000
(19 6)
OH
OH OH
OH OH
>1000
(12 2)
>1000
(25 1)
>10,000
(17 1)
>1000
(13 4)
OH
OH
OH OH
OH OH
>1000
(15 4)
>1000
(2 1)
>10,000
(5 10)
>1000
(11 3)
OH OH
OH OH
>1000
(9 3)
>1000
(7 1)
>10,000
(9 8)
>1000
(1 3)
OH
OH
OH
>1000
(17 4)
>1000
(25 2)
>10,000
(6 4)
>1000
(5 2)
HO
OH
OH OH
25
26
O
S
>1000
(3 8)
>1000
(1 11)
>1000
(11 7)
>1000
n.d.
n.d.
>1000
(23 5)
>1000
(16 6)
(36 7)
a
b
c
Agonist radioligand.
Antagonist radioligand.
Affinities of the agonists will be underestimated since an antagonist radioligand was used for the determination.
d
e
f
At 10 lM.
Not determined.
Using human recombinant A1AR vs. [3H]CCPA.
Using human recombinant A2AAR vs. [3H]CGS21680.
g
h
[
125I]I-AB-MECA was used as a radioligand.

442
A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
Further modifications based on 3 were obtained via extension of
(A) rA1AR
140
120
100
80
3
the linker between the adenine moiety and the cyclic structure by
one more atom and removal of hydrazone double bond via intro-
duction of urea and thiourea moieties within the linker together
with introduction of methyl residue into the nitrogen atom of 2-
hydazinoadenosine in compounds 25 and 26. The results showed
a complete loss of activity at A1AR and A2AAR compared to the par-
ent compound 3, probably due to the shift of the hydrogen bond
donor between the two adjacent nitrogen atoms of the hydrazine
residue and/or introduction of an unfavorable hydrogen bond
acceptor within the linker. These results proved the importance
of keeping the linker length within three atoms in this class of
compounds. However, compounds 25 and 26 showed somehow
activity at A3AR (Table 1).
10
11
12
13
14
60
40
20
0
10-9
10-8
10-7
10-6
10-5
10-4
[compound], M
(B) rA2AAR
110
80
In the present study, the hydrazone nucleoside 3 was resynthe-
sized and evaluated in radioligand binding studies at the four AR
subtypes and the obtained Ki value of this compound showed in
our hand higher affinity of 3 at A1, A2A and A3AR subtypes exhibiting
70.8, 25.5 and 52.6 nM, respectively (Fig. 2). This is not consistent
with the reported data in which compound 3 showed very low affin-
3
10
11
12
13
14
16
50
ity at A1AR (48 l l
M)3 and relatively low affinity at A3AR (0.903 M).3
20
Compound 3 showed low affinity (17% inhibition) at A2BAR as
shown in Table 1 and in Supplementary Figure 1. Analogues of
hydrazone 3 (compounds 10–14) were synthesized and evaluated
at AR subtypes and the results showed that, as in case of 3, all tested
2-hydrazinyloadenosine derivatives 10–14 possessed very high
affinity at all three AR subtypes. The quantified Ki values were gen-
erally high at A2AAR except in case of compounds 12 and 14, which
showed higher affinity for A1AR with Ki values of 58 and 32.1 nM,
respectively. At A1AR, the rank order of potency was 14
(32.1 nM) > 12 (58.0 nM) P 13 (65.7 nM) P 3 (70.8 nM) > 10
(108 nM) > 11 (263 nM) as shown in Table 1 and Figure 2A.
At A2AAR, replacement of cyclohexyl-moiety of 3 (Ki = 25.5 nM)
by 4-chloro-2-hydroxyphenyl-moiety resulted in a 3.6-fold less
potent compound (compound 12, Ki = 91.7 nM). The difference in
affinity was reduced to about twofold by the replacement of cyclo-
hexyl-moiety with 3-chloro-4-methoxyphenyl-moiety (compound
14, Ki = 53.7 nM). A comparable affinity was achieved by the
replacement with 3,4-dichlorophenyl-moiety in compound 11
with a Ki value of 24.4 nM. The monochloro-substituted phenyl
moiety derivative 10 showed a higher affinity than that of 3 with
a Ki value of 16.1 nM. The most potent compound at A2AAR in
the present series was obtained with compound 13 in which the
cyclohexyl-moiety of 3 was replaced by 4-methoxyphenyl-moiety.
Compound 13 showed about twofold more potency at A2AAR
exhibiting a Ki value of 12.0 nM.
-10
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4
[compound], M
(C) hA3AR
110
3
10
11
12
13
14
90
70
50
30
10
-10
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3
[compound], M
Figure 2. Results of competition curves of potent adenine nucleoside derivatives 3,
10–14, 16 as ligands at A1AR versus [3H]CCPA at rat brain cortical membranes (A),
A
2AAR versus [3H]CGS21680 at rat brain striatal membranes (B) and A3AR versus
[3H]NECA at human recombinant A3AR (C). Data points represent means of three
independent experiments performed in triplicate.
The obtained results from the present short series of com-
pounds 10–14, showed that the monosubstituted phenyl moiety
generally possessed higher affinities than that of disubstituted
moieties. The presence of a chloro-residue in the phenyl moiety
seems to increase the activity at A2AAR. The rank order of potency
at A2AAR was 13 (12.0 nM) > 10 (16.1 nM) > 11 (24.4 nM) P 3
(25.5 nM) > 14 (53.7 nM) > 12 (91.7 nM) as shown in Table 1 and
Figure 2B. At A3AR, the results showed that compound 14 was
the most potent compound with a Ki value of 51 nM, the rank order
of potency at this receptor was 14 (51.1 nM) > 10 (71.9 nM) > 11
(88.8 nM) > 13 (112 nM) > 12 (131 nM) >> 3 (903 nM) see Table 1
and Figure 2C.
The potential species differences were evaluated using selected
potent compounds of this series at A2AAR. Therefore, in addition to
the parent compound 3, compound 13 was tested at human
recombinant A1 and A2A receptors using [3H]CCPA and
[3H]CGS21680 as radioligands, respectively. The results showed
that species differences existed in the case of compound 13 at both
A1 and A2AAR. A sharp decrease in affinity was observed at both
receptor subtypes. At A2AAR, compound 13 showed about 57-fold
but still weak A2BAR ligands (Table 1). The bis-sugar nucleosides 19
and 21 exhibited 17% inhibition at 10 lM at the low affinity A2BAR,
the estimated Ki value of both compounds is expected to be in
micromolar range and probably comparable with that of adenosine
itself at A2BAR (15
into promising starting compounds for the discovery of potent
2BAR agonists, especially if we know that the present data were
l
M).3 These results turn compounds 19 and 21
A
obtained from assays using the antagonist radioligand [3H]PSB-
603 and accordingly the affinities of the A2BAR agonists will be
underestimated, that is, compounds 19 and 21 should show higher
affinities if the agonist radioligand was used which does not exist
so far. Therefore, functional assays were aimed after further opti-
mization of this kind of compounds based on 19 and 21, for exam-
ple, via combined modification with N6-substitution and/or
introduction of N-ethylcarboxamide moiety at 50-position of the
first ribose–sugar moiety which may lead to the discovery of
potent A2BAR agonists. No significant difference in affinity was
observed between aldose and ketose bis-sugar nucleosides at the
four AR subtypes.

A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
443
accumulation in human CHO cells stably expressing the human
2AAR.13 For comparison, the full agonist NECA (2, Fig. 1) was
investigated under the same condition. Concentration–response
curves of the test compounds were obtained showing that 10, 11,
13 and 16 acted as agonists at A2AAR. For NECA (2) an EC50 value
(A) hA1AR
125
100
75
A
3
13
50
of 0.018
lM was determined, while compounds 10, 11, 13 and
25
16 showed EC50 values of 0.493, 1.66, 1.36 and 1.44
lM, respec-
tively, (Fig. 4). The species differences between human and rat
may explain the lower potencies of the test compounds in cAMP
assay than that obtained from the binding assays. The efficacy of
13 was not significantly different from that of NECA (2), indicating
that 13 is full agonists at A2AAR while compounds 10, 11 and 16 are
partial agonists at A2AAR.
0
-25
10-8
10-7
10-6
10-5
10-4
[compound], M
(B) hA2A AR
120
100
80
60
40
20
0
4. Conclusions
3
13
A series of 2-hydrazinyladenosine derivatives based on modifi-
cation of the well-known A2AAR-potent agonist 3 was designed,
synthesized and evaluated for their affinities at different AR sub-
types with the aim of exploring the SAR of this class of compounds
as A2AAR agonists. Modifications included formation of bis-sugar
nucleosides (compounds 15–24), modification of the linker be-
tween the cyclic structure and the adenine moiety via introduction
of methyl group in the nitrogen atom of the hydrazine structure
(e.g., compounds 20–26) and/or modification and extension of
the linker length (compounds 25–26). The synthesized compounds
were pharmacologically evaluated and found to produce com-
pounds with relatively higher A2AAR affinity but still lower than
that of nucleoside 3. The most potent bis-sugar nucleoside at
-20
10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3
[compound], M
Figure 3. Results of competition curves of potent adenine nucleoside derivatives 3
and 13 as ligands at human recombinant A1AR versus [3H]CCPA (A) and at human
recombinant A2AAR versus [3H]CGS21680 (B). Data points represent means of three
independent experiments performed in triplicate.
A2AAR was 16 which exhibited a Ki value of 329 nM with marked
selectivity against the other AR subtypes. Compound 16 represents
a lead compound for synthesizing more potent, highly polar and
selective A2AAR agonists to be used, for example, as a local anti-
inflammatory agent. In another set of compounds, hydrazones 3,
10–14 were synthesized and were found to have low nanomolar
affinity at the A2AAR as well as at the other AR subtypes. Com-
pound 13 has been identified as the most potent compound in
the present series at rA2AAR with a Ki value of 12.0 nM. However,
compounds 3, 10–14 are characterized by the lack of selectivity
against the other AR subtypes. Species differences were tested
and found to exist between human and rat A1 and A2AAR in differ-
ent rates. At A2AAR, compound 3 showed 2.5-fold less potency in
human than that in rat receptors while compound 13 showed
57-fold less potency in human than in rat A2AAR. Functional
properties of the most potent compounds 10, 11, 13 and 16 were
assessed by adenylate cyclase assays measuring cAMP accumula-
tion in human CHO cells stably expressing the human A2AAR and
less potency and exhibited a Ki value of 687 nM at human A2AAR
compared with 12.0 nM at rat A2AAR. The same was observed at
A1AR, compound 13 showed affinities of 723 nM at human and
65.7 nM at rat A1AR (almost 11-fold less potent) as shown in
Figure 3. These species differences were observed with much less
extent in case of compound 3 since the compound showed about
2.5-fold less potency in human than that in rat receptors with Ki
values of 81 nM and 25.5 nM, respectively. Our results of com-
pound 3 at hA2AAR were somehow in the same range of the litera-
ture values (270 nM at hA2AAR)3 and also in agreement with the
literature data obtained from similar reported A2AAR agonists
(e.g., 5, Table 1).13 At hA1AR, compound 3 showed in our hand very
high affinity in comparison to the literature values (48 l
M),3 com-
pound 3 exhibited a Ki value of 169 nM as shown in Table 1 and
Figure 3 (compare also 3 vs. 4 at hA1AR, Table 1).
Functional properties of the most potent compounds 10, 11, 13
and 16 were assessed by adenylate cyclase assays measuring cAMP
the results showed that compound 13 (EC50 = 1.36 0.89
full agonists at 2AAR while compounds 10, 11 and 16
(EC50 = 0.493 0.21, 1.36 0.89 and 1.44 1.04 M, respectively)
lM) is
A
l
120
100
are partial agonists at A2AAR. Further studies will be aimed at
improving the A2AAR affinity and selectivity based on modifica-
tions of bis-sugar nucleoside 16, this will include, for example,
combined modifications at 2- and 50-positions in the adenosine
scaffold.
80
2
10
60
40
20
0
16
13
11
5. Experimental section
10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3
[Compound], M
All commercially available reagents were obtained from various
producers (Acros, Aldrich, Fluka, Merck, and Sigma) and used with-
out further purification. Solvents were used without additional
purification or drying, unless otherwise noted. The reactions were
monitored by thin layer chromatography (TLC) using aluminum
sheets with silica gel 60 F254 (Merck). Column chromatography
was carried out with silica gel 0.060–0.200 mm, pore diameter
Figure 4. Concentration–response curves of 2, 10, 11, 13 and 16 in cAMP
accumulation assays using CHO cells expressing the human A2AAR (n = 3); EC50
0.018 1.22 M (2), 0.493 0.21 M (10), 1.66 0.59 M (11), 1.36 0.89 M (13),
1.44 1.04 M (16). All data represent means SEM of three separate experiments
performed in triplicates.
:
l
l
l
l
l

444
A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
ca. 6 nm. Mass spectra were recorded on an API 2000 (Applied Bio-
systems, Darmstadt, Germany) mass spectrometer (turbo ion spray
ion source) coupled with an HPLC system (Agilent 1100) using a
5.4. Preparative HPLC purification of 15–24
Crude nucleosides 15–24 were dissolved in 5 mL of deionized
water:methanol (1:1) and injected into an RP-HPLC column
(Knauer 20 mm ID, Eurospher-100 C18). The column was eluted
with a solvent gradient of 0–100% of water/methanol for 30 min
at a flow rate of 5 mL/min. The UV absorption was detected at
254 nm. Fractions were collected and appropriate fractions pooled
and evaporated under vacuum.
Phenomenex Luna 3l
C18 column. 1H- and 13C NMR spectra were
performed on a Bruker Avance 500 MHz spectrometer. DMSO-d6 or
D2O were used as solvents as indicated below. Shifts are given in
ppm relative to the remaining protons of the deuterated solvents
used as internal standard. Purity of the prepared nucleosides was
checked by TLC on silica gel 60 F254 (Merck) aluminum plates,
using dichloromethane/methanol (9:1 or 3:1) or n-butanol/
CH3COOH/H2O = 2:1:1 as the mobile phase. Purity of the final
nucleosides was confirmed by HPLC by dissolving 1 mg/ml in
H2O/MeOH = 1:1, containing 2 mM ammonium acetate. A sample
5.4.1. (2R,3R,4S,5R)-2-(6-Amino-2-(1-methylhydrazinyl)-9H-
purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (9)
1H NMR (500 MHz, DMSO-d6) d 3.17 (s, 3H), 3.50–3.64 (m, 2H),
3.87–3.89 (q, 1H, J = 3.99 Hz), 4.12–4.14 (m, 1H), 4.59–4.62 (q, 1H,
J = 5.56 Hz), 4.66 (s, 2H), 4.93–4.95 (t, 1H, J = 5.83 Hz), 5.08–5.09 (d,
1H, J = 5.04 Hz), 5.29–5.30 (d, 1H, J = 6.30 Hz), 5.74–5.76 (d, 1H,
J = 5.99 Hz), 6.84 (s, 2H), 7.93 (s, 1H). 13C NMR (125 MHz, DMSO-
d6) d 39.40, 61.86, 70.75, 73.10, 85.32, 87.26, 113.36, 137.04,
151.60, 155.80, 160.97. LC/ESI-MS: negative mode 310.1 ([MꢁH]ꢁ),
positive mode 312.3 ([M+H]+).
of 10
a Phenomenex Luna 3
gradient of water/methanol (containing 2 mM ammonium acetate)
from 90:10 to 0:100 for 30 min at a flow rate of 250 L/min, start-
lL was injected into an HPLC instrument (Agilent 1100) using
l
C18 column. Elution was performed with a
l
ing the gradient after 10 min. UV absorption was detected from
220 to 400 nm using a diode array detector. The purity of the prod-
ucts was generally P95%.
Compounds 3,21 8,21,27 10,21 and 13,21 have previously been
synthesized and we provide detailed spectral data in the present
study while the other compounds are new.
5.4.2. (2R,3R,4S,5R)-2-(6-Amino-2-((E)-2-(cyclohexylmethylene)
hydrazinyl)-9H-purin-9-yl)-5-(hydroxymethyl)
tetrahydrofuran-3,4-diol (3)
1H NMR (500 MHz, DMSO-d6) d 1.15–1.26 (m, 5H), 1.60–1.62
(m, 1H), 1.68–1.73 (m, 4H), 2.17–2.21 (m, 1H), 3.52–3.64 (m,
2H), 3.89–3.91 (q, 1H, J = 3.36 Hz), 4.12 (m, 1H), 4.52–4.53 (m,
1H), 5.03–5.09 (m, 2H), 5.36 (m, 1H), 5.74–5.75 (d, 1H,
J = 6.30 Hz), 7.13 (s, 2H), 7.23–7.24 (d, 1H, J = 5.99 Hz), 8.01 (s,
1H), 10.16 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d 24.95, 25.24,
25.64, 30.41, 61.82, 70.84, 73.54, 85.82, 87.08, 114.49, 137.43,
138.09, 148.39, 151.41, 155.41. LC/ESI-MS: negative mode 390.4
([MꢁH]ꢁ), positive mode 392.4 ([M+H]+).
5.1. General procedure for preparation of 2-(methyl)
hydrazinoadenosine derivatives: Synthesis of compounds 8 and
9
Solution of 7 (1.5 g, 5 mmol) in 5 mL of hydrazine hydrate for
synthesis of 8 or methylhydrazine for synthesis of 9 was allowed
to stir over night till disappearance of 7 determined by TLC
(CH2Cl2:MeOH = 3:1). 2-Propanol (50 mL) was added to the reac-
tion mixture and the formed gum was taken in water (100 mL)
and stirred for additional 5 h. The precipitated product was filtered
and washed with water and dried to give the pure product in about
80% overall yield after concentration of the mother liquor and col-
lection of the remaining product.
5.4.3. (2R,3R,4S,5R)-2-(6-Amino-2-((E)-2-(4-chlorobenzylidene)
hydrazinyl)-9H-purin-9-yl)-5-(hydroxymethyl)
tetrahydrofuran-3,4-diol (10)
1H NMR (500 MHz, DMSO-d6) d 3.56–3.71 (m, 2H), 3.94–3.96 (q,
1H, J = 3.04 Hz), 4.18–4.21 (q, 1H, J = 3.15 Hz), 4.65–4.68 (q,
1H, J = 5.25 Hz), 5.08–5.09 (d, 1H, J = 4.41 Hz), 5.20–5.22 (q, 1H,
J = 3.36 Hz), 5.38–5.39 (d, 1H, J = 6.30 Hz), 5.77–5.79 (d, 1H,
J = 6.30 Hz), 7.02 (s, 2H), 7.41–7.43 (d, 2H, J = 8.51 Hz), 7.73–7.75
(d, 2H, J = 8.51 Hz), 8.01 (s, 1H), 8.04 (s, 1H), 10.68 (s, 1H). 13C
NMR (125 MHz, DMSO-d6) d 62.06, 71.15, 73.23, 86.14, 87.90,
115.61, 128.03, 128.72, 132.77, 134.72, 138.07, 138.52, 151.02,
156.31, 156.37. LC/ESI-MS: negative mode 418.0 ([MꢁH]ꢁ), posi-
tive mode 420.1 ([M+H]+).
5.2. General procedure for preparation of 2-(N0-ar(alk)
ylidenehydrazino)adenosines 3, 10–24
A mixture of 8 or 9 (0.5 mmol) and 1.1 equiv of aromatic alde-
hyde, sugar aldehyde or ketone in 30 mL methanol were refluxed
for 3–15 h till the disappearance of the starting material and the
completion of the reaction was determined by TLC using
CH2Cl2:MeOH = 3:1 for compounds 3, 10–14 and using n-buta-
nol:CH3COOH:H2O = 2:1:1 for compounds 15–24. In case of aro-
matic aldehydes, the products were crystallized out on cooling
and filtered off, washed with methanol, and dried to give the pure
products. Products of sugar aldehyde or ketone were precipitated
after concentration under vacuum and the crude product was crys-
tallized by adding ethyl acetate (1 mL) and excess n-hexane. The
products were further purified by reverse phase HPLC chromatog-
raphy to afford the pure product in about 70% overall yield.
5.4.4. (2R,3R,4S,5R)-2-(6-Amino-2-((E)-2-(3,4-
dichlorobenzylidene)hydrazinyl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (11)
1H NMR (500 MHz, DMSO-d6) d 3.58–3.73 (m, 2H), 3.97–3.99 (q,
1H, J = 2.73 Hz), 4.17–4.19 (m, 1H), 4.69–4.73 (q, 1H, J = 5.25 Hz),
5.08–5.09 (d, 1H, J = 4.09 Hz), 5.36–5.37 (d, 1H, J = 6.30 Hz), 5.38–
5.40 (m, 1H), 5.76–5.77 (d, 1H, J = 6.93 Hz), 7.07 (s, 2H), 7.60–
7.62 (m, 2H), 8.01 (d, 2H, J = 2.20 Hz), 8.04 (s, 1H), 10.84 (s, 1H).
13C NMR (125 MHz, DMSO-d6) d 62.20, 71.32, 73.09, 86.39, 88.34,
115.94, 126.73, 127.62, 130.37, 130.79, 131.64, 136.63, 137.17,
138.52, 150.76, 156.13, 156.40. LC/ESI-MS: negative mode 451.94
([MꢁH]ꢁ), positive mode 454.07 ([M+H]+).
5.3. General procedure for preparation of the adenosine
derivatives N-phenylhydrazinecarboxamide (25) and
carbothioamide (26)
To a solution of 9 (1 mmol) in methanol (20 mL), 1.1 equiv of
phenylisocyanate was added for preparation of compound 25, or
phenylisothiocyanate for preparation of compound 26. The reac-
tion mixture was stirred at room temperature for 2 h and the pre-
cipitated compound was filtered off, washed with methanol and
dried to give the pure products in 70% yield.
5.4.5. (2R,3R,4S,5R)-2-(6-Amino-2-((E)-2-(4-chloro-2-
hydroxybenzylidene)hydrazinyl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (12)
1H NMR (500 MHz, DMSO-d6) d 3.54–3.67 (m, 2H), 3.91–3.93 (q,
1H, J = 3.67 Hz), 4.12–4.15 (q, 1H, J = 3.67 Hz), 4.61–4.64 (q, 1H,

A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
445
J = 5.56 Hz), 5.08–5.10 (m, 2H), 5.34–5.36 (d, 1H, J = 6.30 Hz), 5.79–
5.80 (d, 1H, J = 6.30 Hz), 6.88–6.90 (d, 1H, J = 8.82 Hz), 7.05 (s, 2H),
7.17–7.19 (m, 1H), 7.59–7.60 (d, 1H, J = 2.83 Hz), 8.07 (s, 1H), 8.20
(s, 1H), 10.97 (s, 1H), 11.59 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d
61.85, 70.77, 73.35, 85.74, 87.27, 115.40, 117.97, 121.65, 122.77,
127.61, 128.95, 137.80, 138.69, 151.17, 155.44, 155.89, 156.36.
LC/ESI-MS: negative mode 434.0 ([MꢁH]ꢁ), positive mode 436.3
([M+H]+).
MS: negative mode 458.36 ([MꢁH]ꢁ), positive mode 460.22
([M+H]+).
5.4.11. (2S,3R,4R,E)-5-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)hydrazono)pentane-1,2,3,4-tetraol (18)
1H NMR (500 MHz, DMSO-d6) 3.38–3.67 (m, 7H), 3.96 (d, 1H,
J = 1.57 Hz), 4.08 (m, 1H), 4.25–4.28 (m, 2H), 4.58 (d,
1H, J = 3.46 Hz), 4.63–4.64 (q, 1H, J = 5.25 Hz), 4.72–4.73 (d, 1H,
J = 4.72 Hz), 5.08–5.09 (d, 1H, J = 3.78 Hz), 5.31–5.33 (d, 1H,
J = 6.30 Hz), 5.71–5.73 (d, 1H, J = 7.25 Hz), 5.89 (s, 1H), 6.99 (s,
2H), 7.43–7.45 (d, 1H, J = 5.04 Hz), 7.94 (s, 1H), 10.32 (s, 1H). 13C
NMR (125 MHz, DMSO-d6) d 62.27, 63.31, 71.36, 71.53, 72.38,
72.94, 74.72, 86.33, 88.19, 115.44, 138.05, 143.85, 150.73, 156.42,
156.70. LC/ESI-MS: negative mode 428.23 ([MꢁH]ꢁ), positive mode
430.15 ([M+H]+).
5.4.6. (2R,3R,4S,5R)-2-(6-Amino-2-((E)-2-(4-
methoxybenzylidene)hydrazinyl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (13)
1H NMR (500 MHz, DMSO-d6) d 3.56–3.71 (m, 2H), 3.77 (s,
3H), 3.93–3.95 (q, 1H, J = 2.52 Hz), 4.19 (m, 1H), 4.64–4.68 (q,
1H, J = 5.25 Hz), 5.06–5.07 (d, 1H, J = 4.41 Hz), 5.20–5.22 (q,
1H, J = 3.36 Hz), 5.38–5.40 (d, 1H, J = 6.30 Hz), 5.77–5.78 (d, 1H,
J = 6.30 Hz), 6.93–6.95 (m, 4H), 7.65–7.66 (m, 2H), 7.98 (s, 1H),
8.0 (s, 1H), 10.41 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d 55.31,
62.08, 71.16, 73.26, 86.15, 87.86, 114.20, 115.37, 127.93, 128.40,
137.79, 140.0, 151.12, 156.32, 156.57, 159.76. LC/ESI-MS: negative
mode 413.9 ([MꢁH]ꢁ), positive mode 416.0 ([M+H]+).
5.4.12. (2S,3R,4S,E)-5-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)hydrazono)hexane-1,2,3,4,6-pentaol (19)
1H NMR (500 MHz, DMSO-d6 + D2O) d 3.39–3.42 (m, 2H), 3.51–
3.52 (m, 2H), 3.59–3.61 (m, 2H), 3.90–3.91 (m, 1H), 3.98–3.99 (m,
1H), 4.08–4.09 (m, 2H), 4.40 (m, 1H), 4.59 (m, 1H), 5.70–5.72 (d,
1H, J = 6.93 Hz), 7.98 (s, 1H). LC/ESI-MS: negative mode 458.39
([MꢁH]ꢁ), positive mode 460.22 ([M+H]+).
5.4.7. (2R,3R,4S,5R)-2-(6-Amino-2-((E)-2-(3-chloro-4-
methoxybenzylidene)hydrazinyl)-9H-purin-9-yl)-5-
(hydroxymethyl)tetrahydrofuran-3,4-diol (14)
1H NMR (500 MHz, DMSO-d6) d 3.58–3.73 (m, 2H), 3.87 (s, 3H),
3.97–3.99 (q, 1H, J = 2.73 Hz), 4.16–4.18 (m, 1H), 4.69–4.72 (q, 1H,
J = 5.56 Hz), 5.07 (d, 1H, J = 4.091 Hz), 5.35–5.39 (m, 2H), 5.76–5.77
(d, 1H, J = 6.62 Hz), 7.0 (s, 2H), 7.13–7.15 (d, 1H, J = 8.51 Hz), 7.53–
7.55 (m, 1H), 7.97 (s, 1H), 7.98 (s, 1H), 8.02–8.03 (d, 1H,
J = 1.89 Hz), 10.57 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d 56.33,
62.21, 71.33, 73.08, 86.39, 88.26, 112.77, 115.69, 121.78, 127.11,
127.21, 129.47, 138.22, 138.52, 150.89, 154.71, 156.37, 156.41.
LC/ESI-MS: negative mode 448.0 ([MꢁH]ꢁ), positive mode 450.0
([M+H]+).
5.4.13. (2S,3S,4S,5R,E)-6-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)-2-methylhydrazono)hexane-1,2,3,4,5-pentaol (20)
1H NMR (500 MHz, DMSO-d6) d 3.03–3.10 (m, 2H), 3.19–3.21 (t,
1H, J = 8.82 Hz), 3.27 (s, 3H), 3.35 (m, 2H), 3.41–3.47 (m, 2H), 3.51–
3.56 (m, 2H), 3.61–3.68 (m, 2H), 3.89–3.91 (q, 1H, J = 3.99 Hz),
4.01–4.04 (m, 1H), 4.12 (m, 1H), 4.38 (m, 1H), 4.49–4.50 (m, 1H),
4.89 (m, 1H), 4.95 (m, 1H), 5.17 (m, 1H), 5.40 (m, 1H), 5.75–5.76
(d, 1H, J = 5.76 Hz), 7.0 (m, 2H) 8.21 (s, 1H). 13C NMR (125 MHz,
DMSO-d6) d 38.65, 61.46, 70.41, 72.05, 73.77, 76.85, 78.41, 85.57,
87.21, 92.33, 97.01, 112.64, 139.10, 151.50, 155.06, 159.46,
160.84. LC/ESI-MS: negative mode 472.37 ([MꢁH]ꢁ), positive mode
474.48 ([M+H]+).
5.4.8. (2S,3S,4S,5R,E)-6-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)hydrazono)hexane-1,2,3,4,5-pentaol (15)
1H NMR (500 MHz, DMSO-d6 + D2O) d 3.04–3.08 (m, 2H), 3.13–
3.16 (m, 1H), 3.65–3.39 (m, 1H), 3.41–3.44 (m, 1H), 3.53–3.54 (m,
1H), 3.59–3.69 (m, 2H), 3.90–3.91 (m, 2H), 4.08–4.09 (t, 1H,
J = 4.88 Hz), 4.39–4.41 (t, 1H, J = 5.51 Hz), 5.72–5.73 (d, 1H,
J = 5.99 Hz), 8.22 (s, 1H). LC/ESI-MS: negative mode 458.44
([MꢁH]ꢁ), positive mode 460.31 ([M+H]+).
5.4.14. (2R,3S,4R,5S,E)-6-(2-(6-Amino-9-((2S,3S,4R,5S)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)-2-methylhydrazono)hexane-1,2,3,4,5-pentaol (21)
1H NMR (500 MHz, DMSO-d6) 3.38–3.44 (m, 2H), 3.47 (s, 3H),
3.50–3.57 (m, 3H), 3.64–3.67 (m, 1H), 3.73–3.75 (m, 1H), 3.15–
3.19 (m, 1H), 3.94–3.96 (q, 1H, J = 2.73 Hz), 4.11–4.13 (m, 2H),
4.18–4.19 (m, 1H), 4.37–4.40 (m, 2H), 4.51 (t, 1H, J = 5.20 Hz),
4.57–4.62 (m, 1H), 4.67–4.70 (m, 1H), 5.10–5.11 (d, 1H,
J = 4.09 Hz), 5.19–5.21 (d, 1H, J = 5.99 Hz), 5.35–5.36 (d, 1H,
J = 6.30 Hz), 5.75–5.76 (d, 1H, J = 6.93 Hz), 7.10–7.11 (s, 2H), 8.01
(s, 1H). 13C NMR (125 MHz, DMSO-d6) d 31.34, 62.20, 63.29,
69.38, 70.12, 70.65, 71.30, 72.33, 72.96, 86.12, 88.19, 115.37,
138.79, 141.50, 150.94, 155.97, 156.59. LC/ESI-MS: negative mode
472.31 ([MꢁH]ꢁ), positive mode 474.41 ([M+H]+).
5.4.9. (2S,3R,4S,5R,E)-6-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)hydrazono)hexane-1,2,3,4,5-pentaol (16)
1H NMR (500 MHz, DMSO-d6) 3.37–3.42 (m, 3H), 3.49–3.54 (m,
3H), 3.60–3.72 (m, 2H), 3.95 (q, 1H, J = 1.68 Hz), 4.07–4.13 (m, 4H),
4.38–4.40 (m, 2H), 4.62–4.63 (q, 1H, J = 5.14 Hz), 5.08–5.09 (d, 2H,
J = 4.09 Hz), 5.32 (d, 1H, J = 6.30 Hz), 5.72–5.73 (d, 1H, J = 6.93 Hz),
7.0 (s, 2H), 7.43–7.44 (d, 1H, J = 4.41 Hz), 7.95 (s, 1H), 10.31 (s, 1H).
13C NMR (125 MHz, DMSO-d6) d 62.28, 63.28, 69.40, 70.05, 70.18,
71.32, 71.88, 73.0, 86.30, 88.06, 115.33, 137.97, 145.11, 150.82,
156.35, 156.58. LC/ESI-MS: negative mode 458.38 ([MꢁH]ꢁ), posi-
tive mode 460.22 ([M+H]+).
5.4.15. (2S,3S,4S,5S,E)-6-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)-2-methylhydrazono)hexane-1,2,3,4,5-pentaol (22)
1H NMR (500 MHz, DMSO-d6 + D2O) 3.45 (s, 3H), 3.48–3.54 (m,
3H), 3.59–3.61 (m, 2H), 3.67–3.68 (m, 2H), 3.94–3.96 (q, 1H,
J = 2.73 Hz), 4.10–4.11 (m, 1H), 4.22–4.26 (m, 1H), 4.62–4.65 (m,
1H), 5.75–5.76 (d, 1H, J = 6.62 Hz), 7.12–7.13 (d, 1H, J = 5.35 Hz),
8.01 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d 31.25, 62.22, 64.02,
69.76, 70.70, 71.35, 71.41, 71.65, 72.79, 86.18, 88.24, 115.39,
138.92, 141.65, 150.91, 155.95, 156.85. LC/ESI-MS: negative mode
472.33 ([MꢁH]ꢁ), positive mode 474.42 ([M+H]+).
5.4.10. (2S,3S,4S,5S,E)-6-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)hydrazono)hexane-1,2,3,4,5-pentaol (17)
1H NMR (500 MHz, DMSO-d6 + D2O) 3.21–3.25 (m, 1H), 4.08 (m,
1H), 3.29–3.33 (m, 1H), 3.46–3.60 (m, 3H), 3.88–3.95 (m, 1H),
4.08–4.11 (m, 2H), 4.15–4.20 (m, 1H), 4.38–4.41 (m, 1H), 5.72–
5.74 (m, 1H), 7.54–7.57 (d, 1H, J = 5.67 Hz), 8.28 (s, 1H). LC/ESI-

446
A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
5.4.16. (2R,3S,4S,E)-5-(2-(6-Amino-9-((2S,3S,4R,5S)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)-2-methylhydrazono)pentane-1,2,3,4-tetraol (23)
1H NMR (500 MHz, DMSO-d6) 3.40–3.43 (m, 2H), 3.46 (s, 3H),
3.50–3.55 (m, 3H), 3.58–3.61 (m, 1H), 3.66–3.69 (m, 1H), 3.86–
3.88 (m, 1H), 3.97 (q, 1H, J = 2.41 Hz), 4.10–4.12 (m, 1H), 4.38–
4.40 (t, 1H, J = 5.04 Hz), 4.67–4.69 (m, 2H), 5.10–5.15 (m, 1H),
5.32–5.44 (m, 2H), 5.74–5.75 (d, 1H, J = 6.93 Hz), 5.85 (s, 1H),
7.07–7.08 (d, 1H, J = 5.35 Hz), 7.13 (s, 2H), 8.0 (s, 1H). 13C NMR
(125 MHz, DMSO-d6) d 31.29, 62.22, 63.29, 71.38, 71.84, 72.52,
72.94, 74.76, 86.22, 88.46, 115.51, 138.98, 140.35, 150.86, 155.93,
156.80. LC/ESI-MS: negative mode 442.40 ([MꢁH]ꢁ), positive mode
444.22 ([M+H]+).
chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine,
73 Ci/mmol)29 and [3H]NECA ([3H]50-(N-ethylcarboxamido)adeno-
sine, 15.3 Ci/mmol),31 were used as A1, A2A, A2B and A3 radioli-
gands, respectively. All other chemical reagents, cell culture
materials and adenosine receptor ligands were obtained from
Sigma.
5.5.2. Receptor-radioligand binding studies
Rat brain cortical membrane preparations were used as a source
for A1 and rat brain striatal membrane preparations as a source for
A
2A receptors as previously described.32,33 Membrane preparations
of CHO cells expressing the human A1, A2B or A3 receptors were
used for hA1, hA2B and hA3 assays.12,13,31,34,35 Membrane prepara-
tion of human embryonic kidney (HEK) cells expressing human
A2A receptors obtained from PerkinElmer (Product No.: RBHA2A-
M400UA), was used for human A2A assays. Stock solutions of the
compounds were prepared in dimethyl sulfoxide (DMSO); the final
concentration of DMSO was 2.5%. The radioligand concentrations
and incubation times (incubation at rt) were as follows: [3H]CCPA:
42.6 Ci/mmol, 1 nM (rat and human A1), incubation for 90 min;
[3H]CGS21680: 41 Ci/mmol 5.0 nM (rat and human A2A), incuba-
tion for 60 min; [3H]NECA: 15.3 Ci/mmol 10.0 nM (human A3),
incubation for 180 min; [3H]PSB-603 73 Ci/mmol 0.3 nM (human
5.4.17. (2S,3R,4S,E)-5-(2-(6-Amino-9-((2R,3R,4S,5R)-3,4-
dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-
2-yl)-2-methylhydrazono)hexane-1,2,3,4,6-pentaol (24)
1H NMR (500 MHz, DMSO-d6 + D2O) d 3.14 (s, 3H), 3.23–3.24
(m, 2H), 3.37–3.93 (m, 1H), 3.47–3.48 (m, 1H), 3.49 (m, 1H),
3.51–3.52 (m, 1H), 3.58–3.63 (m, 2H), 3.79–3.81 (t, 1H,
J = 5.99 Hz), 3.87–3.89 (q, 1H, J = 3.99 Hz), 4.10–4.12 (m, 1H),
4.57–4.59 (t, 1H, J = 5.51 Hz), 5.73 (d, 1H, J = 5.99 Hz), 7.92 (s,
1H). 13C NMR (125 MHz, DMSO-d6) d 39.41, 61.86, 63.13, 64.56,
68.01, 69.33, 70.75, 73.09, 75.90, 82.08, 87.26, 102.13, 113.36,
137.04, 151.60, 155.80, 160.97. LC/ESI-MS: negative mode 472.32
([MꢁH]ꢁ), positive mode 474.33 ([M+H]+).
A
2B), incubation for 75 min. About 50ꢁ125
lg of protein/vial were
used in the assays. Membranes were preincubated for 10–15 min
with 0.12 IU/mL of adenosine deaminase in order to remove
endogenous adenosine. Binding assays were performed essentially
as previously described.12,13,32,33
5.4.18. 2-(6-Amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-2-yl)-2-
methyl-N-phenylhydrazinecarboxamide (25)
Binding assays at A1, A2A and A3 receptors were carried out
using PE-vials in a total volume of 400
TRIS–HCl, pH 7.4) containing 100 L of membrane protein suspen-
sion and 100 L of radioligand solution in the presence of 10 L of
various concentrations of test compound. Nonspecific binding was
determined in the presence of 10 M 2-chloroadenosine (CADO) in
A1AR assays, 50 M NECA in A2AAR assays, or 100
M (R)-N6-phe-
nyl-isopropyladenosine (R-PIA) in A3AR assays. Incubation was ter-
minated by rapid filtration using Brandel 48-channel cell
lL assay buffer (50 mM
1H NMR (500 MHz, DMSO-d6) 3.28 (s, 3H), 3.47–3.62 (m, 2H),
3.85–3.87 (q, 1H, J = 3.99 Hz), 4.11–4.14 (q, 1H, J = 3.99 Hz), 4.57–
4.61 (q, 1H, J = 5.56 Hz), 4.87–4.89 (t, 1H, J = 5.67 Hz), 5.07–5.08
(d, 1H, J = 4.72 Hz), 5.28–5.29 (d, 1H, J = 5.99 Hz), 5.76–5.77 (d,
1H, J = 5.99 Hz), 6.90–6.92 (m, 3H), 7.19–7.22 (m, 2H), 7.47–7.49
(d, 2H, J = 7.88 Hz), 8.01 (s, 1H), 8.18 (s, 1H), 8.70 (s, 1H). 13C
NMR (125 MHz, DMSO-d6) d 39.78, 61.88, 70.71, 73.21, 85.30,
87.08, 114.48, 118.74, 121.72, 128.59, 137.61, 140.09, 151.43,
155.72, 156.01, 160.45. LC/ESI-MS: negative mode 429.18
([MꢁH]ꢁ), positive mode 431.35 ([M+H]+).
l
l
l
l
l
l
a
harvester (Brandel, Gaithersburg, MD) through Whatman GF/B
glass fiber filters. Filters were rinsed three times with 2 mL each
of ice-cold TRIS–HCl buffer (50 mM, pH 7.4) and incubated for 6–
9 h with 2.5 lL of scintillation cocktail (Ready Safe™, Coulter)
5.4.19. 2-(6-Amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-
per well before radioactivity was counted in a liquid scintillation
(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-2-yl)-2-
methyl-N-phenylhydrazinecarbothioamide (26)
counter (Tricarb 2900TR, Canberra Packard). A2BAR binding assays
were carried out in a total volume of 1000
test compound dissolved in 775 L TRIS–HCl buffer (50 mM, pH
7.4), 100 L radioligand solution, and 100 L of membrane suspen-
sion. Nonspecific binding was determined in the presence of 10
lL containing 25 lL of
1H NMR (500 MHz, DMSO-d6) 3.24 (s, 3H), 3.51–3.65 (m, 2H),
3.87–3.89 (q, 1H, J = 3.99 Hz), 4.17–4.18 (q, 1H, J = 3.88 Hz), 4.62–
4.63 (q, 1H, J = 5.25 Hz), 4.81 (m, 1H), 5.07–5.08 (d, 1H,
J = 4.72 Hz), 5.25–5.26 (d, 1H, J = 5.35 Hz), 5.78–5.80 (d, 1H,
J = 5.67 Hz), 7.05 (s, 2H), 7.08–7.11 (m, 1H), 7.26–7.28 (t, 2H,
J = 7.40 Hz), 7.54–7.55 (d, 2H, J = 7.56 Hz), 8.06 (s, 1H), 9.61 (s,
1H), 9.68 (s, 1H). 13C NMR (125 MHz, DMSO-d6) d 39.12, 61.90,
70.64, 73.23, 85.31, 87.19, 115.01, 124.70, 125.56, 127.89, 138.03,
139.55, 151.23, 156.10, 159.84, 179.85. LC/ESI-MS: negative mode
445.13 ([MꢁH]ꢁ), positive mode 447.26 ([M+H]+).
l
l
l
lM
8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The assay was incu-
bated for 75 min at rt. The A2BAR assay was filtered through GF/B
glass fiber filters using a 48-channel cell harvester, and filters were
washed four times with ice-cold TRIS–HCl buffer (50 mM, pH 7.4)
containing 0.1% bovine serum albumin (BSA). Then filters were
transferred to vials and incubated for 9 h with 2.5 mL of scintilla-
tion cocktail (Beckman Coulter). Radioactivity was counted in a li-
quid scintillation counter (Tricarb 2900TR, Canberra Packard) with
a counting efficiency of 53%. Curves were determined using 6ꢁ7
different concentrations of test compounds spanning 3 orders of
magnitude. At least three independent experiments were per-
formed, each in duplicate (human receptors) or triplicate (rat
receptors). Data were analyzed with GraphPad Prism, Version 5.0
(GraphPAD, San Diego, CA, USA). For the calculation of Ki values
by nonlinear regression analysis, the Cheng-Prusoff equation and
KD values of 0.2 nM (rat A1AR) and 0.61 nM (human A1AR) for
[3H]CCPA, 15.9 nM (rat A2AAR) and 26.76 nM (human A2AAR) for
[3H]CGS21680, 0.41 nM (human A2BAR) for [3H]PSB-603 and 6.2
nM (human A3AR) for [3H]NECA were used.12,13,20,29–34
5.5. Adenosine receptor binding assays
5.5.1. Chemicals
Tri(hydroxymethyl)aminomethan (Tris-buffer) was obtained
from Acros Organics (Leverkusen, Germany); DMSO was from Flu-
ka (Switzerland), HCl was from Merck, HAT supplement from Gib-
co. Radioligands were commercially available or custom-labelled
from appropriate precursors as described.12,13 [3H]CCPA ([3H]2-
chloro-N6-cyclopentyladenosine (42.6 Ci/mmol),28 [3H]CGS21680
((2-(4-(2-carboxyethyl)phenyl)ethylamino)-50-N-ethylcarboxami-
do-adenosine, 41 Ci/mmol),20 and [3H]PSB-603 ([3H]8-(4-(4-(4-

A. El-Tayeb, S. Gollos / Bioorg. Med. Chem. 21 (2013) 436–447
447
5.6. Functional assays
6b, 7 and 8, the results of the initial screening of some tested com-
pounds at A1, A2A, A2B and A3 receptors as well as examples of LC/
ESI-MS purity determination of selected nucleosides (compounds
11, 18, 23, 24, 25 and 26). Supplementary material associated with
this article can be found, in the online version, at http://dx.doi.org/
10.1016/j.bmc.2012.11.021.
5.6.1. Culture of CHO cells
CHO cells expressing the human A2A receptor were stably
grown adherent and maintained in a DMEM F-12, supplemented
with 10% FCS, 100 IU/mL penicillin G, 100
lg/ml streptomycin,
1 mM glutamine and 200 g/ml G 418, containing at 37 °C in 5%
l
CO2. Before recultivation, the cells were washed twice with PBS,
References and notes
trypsinized, and resuspended in new medium and counted.
1. Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Klotz, K. N.; Linden, J.
Pharmacol. Rev. 2001, 53, 527.
2. Klotz, K. N. Naunyn-Schmiedeberg’s Arch. Pharmacol. 2000, 362, 382.
3. Fredholm, B. B.; IJzerman, A. P.; Jacobson, K. A.; Linden, J.; Müller, C. E.
Pharmacol. Rev. 2011, 63, 1.
4. Guillaume, L.; Tony, W.; Patricia, C. E.; Kirstie, B.; Christopher, J. L.; Andrew, G.
W. L.; Christopher, G. T. Nature 2011, 474, 521.
5. Jaakola, V. P.; Griffith, M. T.; Hanson, M. A.; Cherezov, V.; Chien, E. Y.; Lane, J. R.;
IJzerman, A. P.; Stevens, R. C. Science 2008, 322, 1211.
5.6.2. Measurement of cAMP accumulation
The cells were cultured on 24-well (150,000–200,000) over-
night in the culture medium at 37 °C, 5% CO2. After removal of
the culture medium, cells were washed with HBSS buffer (contain-
ing 20 mM HEPES; pH 7.3) and then incubated with HBSS buffer
containing adenosine 1 IU/mL deaminase for 120 min at 37 °C, 5%
CO2. The cells were then preincubated with phosphodiesterase
inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidone (Ro
20–1724), 40 lM, for 15 min. Compounds were added in a volume
of 0.1 mL HBSS buffer and incubations were continued for 15 min.
at 37 °C. The reaction was stopped by removal of the reaction buf-
fer followed by the addition of a hot lysis buffer (500 lL; 90 °C;
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incubated at room temperature for 5 min and freezed at ꢁ20 °C.
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cAMP levels were quantified by incubation of 50
lL each well with
cAMP binding protein prepared from calf adrenal glands (75
lg/
well) and [3H]cAMP (final concentration 3 nM). The plates were
incubated at 4 °C for 60 min and the samples were harvested by fil-
tration through Whatman GF/B filters using a harvester (Brandel
48-channel cell harvester). Each filter was rinsed three times with
1 mL, 50 mM TRIS–HCl, pH 7.4, the filters were punched out into
scintillation vials and counted in a liquid scintillation counter with
2.5 ml Ultima GoldÒ scintillation cocktail. The samples were
counted after 6 h for 1 min. The amount of cAMP was determined
using standard cAMP curves of three independent experiments
each in triplicates.13,36,37
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Ali El-Tayeb planned the experiments, synthesized all com-
pounds, analyzed the structure–activity relationships, and wrote
the manuscript. Sabrina Gollos did the pharmacological testing of
the compounds.
Acknowledgments
28. Klotz, K. N.; Lohse, M. J.; Schwabe, U.; Cristalli, G.; Vittori, S.; Grifantini, M.
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A. El-T. is grateful and expresses his sincere appreciation for
Professor Dr. Christa E. Müller, Pharmaceutical Institute, University
of Bonn, Germany for giving the chance to perform this work in her
laboratory and for her kind help and support. We thank Nicole Flo-
rin for expert technical assistance in performing some of the radi-
oligand binding studies, Sabine Terhart-Krabbe and Annette Reiner
for NMR spectra and Marion Schneider for recording LC/MS
spectra.
34. Klotz, K. N.; Hessling, J.; Hegler, J.; Owman, C.; Kull, B.; Fredholm, B. B.; Lohse,
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Supplementary data
36. Nordstedt, C.; Fredholm, B. B. Anal. Biochem. 1990, 189, 231.
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These data include synthetic procedures, synthetic scheme, 1H
and 13C NMR spectral data and mass spectra of nucleosides 6a,