Synthesis and biological evaluation of novel 1-deoxy-1-[6-[((hetero) arylcarbonyl)hydrazino]-9H-purin-9-yl]-N-ethyl-β-D-ribofuranuronamide derivatives as useful templates for the development of A2B adenosine receptor agonists

Article abstract of DOI:10.1021/jm061170a

The lack of molecules endowed with selective and potent agonistic activity toward the hA_2B adenosine receptors has limited the studies on this pharmacological target and consequently the evaluation of its therapeutic potential. We report the de

Full text of DOI:10.1021/jm061170a

374
J. Med. Chem. 2007, 50, 374-380
Synthesis and Biological Evaluation of Novel 1-Deoxy-1-[6-[((hetero)arylcarbonyl)hydrazino]-
9H-purin-9-yl]-N-ethyl-â-D-ribofuranuronamide Derivatives as Useful Templates for the
Development of A2B Adenosine Receptor Agonists
Pier Giovanni Baraldi,*,† Delia Preti,† Mojgan Aghazadeh Tabrizi,† Francesca Fruttarolo,† Romeo Romagnoli,†
Maria Dora Carrion,† Luisa Carlota Lopez Cara,† Allan R. Moorman,‡ Katia Varani,§ and Pier Andrea Borea§
Dipartimento di Scienze Farmaceutiche and Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, UniVersita` di Ferrara,
44100 Ferrara Italy, King Pharmaceutical Research and DeVelopment, Inc., 4000 CentreGreen Way, Suite 300, Cary, North Carolina 27513
ReceiVed October 10, 2006
The lack of molecules endowed with selective and potent agonistic activity toward the hA2B adenosine
receptors has limited the studies on this pharmacological target and consequently the evaluation of its
therapeutic potential. We report the design and the synthesis of the first potent (EC50 in the nanomolar
range) and selective hA2B adenosine receptor agonists consisting of 1-deoxy-1-[6-[((hetero)arylcarbonyl)-
hydrazino]-9H-purin-9-yl]-N-ethyl-â-D-ribofuranuronamide derivatives. The concurrent effect of 6-substitution
of the purine nucleus with a ((hetero)arylcarbonyl)hydrazino function and a 2-chloro substitution has been
investigated in such NECA derivatives.
Introduction
of A2B receptors on tumor cell surfaces.7 Because of the
widespread distribution of A2B adenosine receptors and the
involvement of this receptor subtype in important (patho)-
physiological processes both in peripheral tissues and in the
central nervous system, many efforts have been carried out in
order to identify potent and selective A2B ligands endowed with
noteworthy therapeutic potential. Treatment of asthma with
selective A2B adenosine receptor antagonists has been, up to
now, the most interesting therapeutic goal.8 However, several
remarkable therapeutic applications have been proposed for the
employment of A2B receptor agonists. It has been shown that
activation of A2B is related to an inhibition of fibroblasts9 and
smooth muscle proliferation. Therefore, A2B agonists have been
suggested for the treatment of cardiac diseases such as hyper-
plasia consequent to hypertension, heart attacks, and arterio-
sclerosis.10,11 Since interaction of adenosine with A2B receptors
inhibits production of the proinflammatory cytokine TNFR in
monocytes,12 A2B agonists have been proposed for treatment
of septic shock. Moreover, A2B agonists may be useful for the
treatment of cystic fibrosis13 and impotence,14 as antidiarrhoeal
drugs,15 and as coronary dilatatory agents.16
Adenosine, a naturally occurring nucleoside, is well-known
to be involved in a large variety of physiological and patho-
physiological processes that are modulated through the interac-
tion of the endogenous ligand with specific cell membrane
G-protein-coupled receptors classified into four subtypes, A1,
A2A, A2B, and A3.1 Of these, the A2B subtype has been identified
on the basis of functional assays on rat brain slices by Daly et
al.,2 and subsequently the existence of this pharmacological
target has been confirmed by receptor cloning experiments
conducted in various species such as rat and human.3 The A2B
receptor, described as a low-affinity subtype, shows a well-
preserved interspecies sequence with 85% identity between
human and mouse and 95% identity between rat and mouse.4
Quantitative tissue distribution of the A2B adenosine receptors
is so far unknown because of the lack of potent radioligands
endowed with sufficient receptor selectivity. Determination of
receptor-coding mRNA levels furnished important information
about A2B tissue distribution,4 assuming that high mRNA levels
should correspond to high receptor protein expression. On this
basis, high concentrations of adenosine A2B receptors have been
suggested in caecum, large intestine, and urinary bladder, while
a lower expression level has been suggested in lung, blood
vessels, eye, and mast cells. Adipose tissue, adrenal gland, brain,
kidney, liver, ovary, and pituitary gland are thought to have a
very low concentration of A2B adenosine receptor.5 Recently it
has been demonstrated that activation of A2B receptors in
primary cultures of mouse cortical astrocytes leads to an increase
of glycogen synthesis through the modulation of gene expres-
sion,6 suggesting that adenosine probably exerts a fundamental
role in brain energy metabolism. A study by Zeng and
co-workers highlighted the positive effect of adenosine on the
release of angiogenic factors (IL-8) from the glioblastoma cell
line U87MG, which seems to be correlated to an overexpression
Identification of potent and selective A2B adenosine receptor
agonists has been an ambitious goal for years in order to
characterize the potential physiological role of A2B receptors,
especially in tissues where all four adenosine receptor subtypes
are coexpressed. From a pharmacological point of view, the
lack of highly selective agents has so far hampered efforts to
better characterize the adenosine A2B receptor subtype and
consequently to fully define its therapeutic potential.17 5′-N-
Ethylcarboxamidoadenosine (NECA, 1, Figure 1) has been
considered one of the most useful ligands at the A2B receptor
subtype18-20 (EC50 ) 160 nM, Table 2), although it shows high
affinity toward all other adenosine receptors (Ki from binding
assays in the low nanomolar range; see Table 2). In a recent
patent application by Rosentreter et al., a series of substituted
2-thio-4-aryl-3,5-dicyano-6-aminopyrimidine derivatives were
claimed to behave as potent non-nucleosidic agonists for
adenosine receptors.21 An extension of this work22 led to the
identification of both partial (2-amino-4-(4-hydroxyphenyl)-6-
(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile, hA1
* To whom correspondence should be addressed. Phone: +39-0532-
291293. Fax: +39-0532-291296. E-mail: baraldi@unife.it.
† Dipartimento di Scienze Farmaceutiche, Universita` di Ferrara.
‡ King Pharmaceutical Research and Development, Inc.
§ Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farma-
cologia, Universita` di Ferrara.
10.1021/jm061170a CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/22/2006

Ribofuranuronamide DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 375
Figure 1. Representative structural correlation among 1-deoxy-1-[6-[((hetero)arylcarbonyl)hydrazino]-9H-purin-9-yl]-N-ethyl-â-D-ribofuranuronamides
and N6-arylcarbamoyl/carboxamido NECA analogues previously reported.
Ki ) 2.6 nM, hA2A Ki ) 28 nM, hA2B EC50 ) 12 nM, hA3
Ki ) 538 nM) and full (2-amino-4-(3-hydroxyphenyl)-6-
(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile,
hA1 Ki ) 4.4 nM, hA2A Ki ) 21 nM, hA2B EC50 ) 10 nM, hA3
Ki ) 104 nM) nonselective agonists at the A2B adenosine
receptor.
Scheme 1a
A few years ago, we reported the synthesis and the biological
activity of a series of N6-arylcarbamoyl and N6-carboxamido
derivatives of adenosine-5′-N-ethyluronamide (NECA) as A1
and A3 adenosine receptor agonists.23-25 From the binding data
we obtained, we observed that the carboxamido derivatives
(general structure 2, Figure 1) generally behaved as low selective
A1 receptors ligands, while some N6-(substituted phenylcar-
bamoyl) derivatives (general structure 3, Figure 1) were found
to have affinity at rat A3 receptors in the low nanomolar range
with different degrees of selectivity versus A1 and A2A adenosine
receptors. These results suggested that small modifications of
the chain at the 6-position of the purine nucleus are able to
produce significant changes in the selectivity pattern of such
compounds. In particular, it appeared that the presence of an
amide vs a urea functionality at the 6-position was generally
detrimental in terms of affinity at rat A3 receptors. Considering
its fundamental importance for the modulation of both affinity
and selectivity, we decided to further investigate the N6-position,
synthesising a new series of N6-functionalized 5′-N-ethylcar-
boxamidoadenosine analogues. According to the principles of
bioisosterism, we replaced the (hetero)arylurea function of the
reported A3 agonists with the isomeric (hetero)arylcarbonylhy-
drazino moiety and evaluated the effect on the binding and
functional profile of the synthesized compounds (general
structure 4, Figure 1). This spacer is able to provide flexibility
to the N6-chain, being at the same time rich in potential hydrogen
bond anchoring sites. The coexisting effect of substitution at
the 2-position of the purine with a chlorine atom has been also
evaluated. Surprisingly, the new class of 1-deoxy-1-[6-[((hetero)-
arylcarbonyl)hydrazino]-9H-purin-9-yl]-N-ethyl-â-D-ribofuranu-
ronamide and 1-deoxy-1-[2-chloro-6-[((hetero)arylcarbonyl)-
hydrazino]-9H-purin-9-yl]-N-ethyl-â-D-ribofuranuronamidederivatives
has been found to be the first examples of both potent and
selective A2B adenosine receptor agonists. Some N6-substituted
adenosine analogues containing cyclic hydrazines at the C6
position have in the past been conceived as the aza isosteres of
a Reagents: (a) CH2I2, isopentylnitrite, 85 °C, 1 h; (b) TEA, substituted
(hetero)arylhydrazides, autoclave, 90-100 °C, 7-8 h (for the 2-chloro
derivative 8), or 120 °C, ON (for the 2-unsubstituted derivative 7); (c) TFA/
H2O, 1:1, room temp, 3 h.
the known A1 receptor agonist CPA (N-cyclopentyladenosine),
and their A2A/A1 selectivity was estimated by means of
radioligand binding assays.26,27 These studies allowed the
detection of interesting molecules exhibiting a high level of
selectivity at the A1 receptor versus A2A receptor. Unfortunately
no data were furnished with respect to the A2B subtype. At any
rate, with the exception of compound 20b, none of the newly
reported compounds were found to exert significant affinity at
the A1 adenosine receptor subtype.
Chemistry
The synthetic strategy employed for the preparation of target
compounds 9b-26b is depicted in Scheme 1. 2′,3′-O-Isopro-

376 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Baraldi et al.
Table 1. Structures and Physicochemical Parameters of the Synthesized NECA Derivatives 9b-26b
compd
R
R′
mp (°C)
MW
formula
9b
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
phenyl
174-175
188-189
170
233-234
171
427.16
461.86
428.40
417.38
496.27
431.4
476.40
447.47
461.86
451.82
481.91
481.91
467.89
467.89
481.91
450.84
466.11
528.91
C19H21N7O5
C19H20 ClN7O5
C18H20 N8O5
C17H19 N7O6
C17H18Br N7O6
C18H21 N7O6
C17H20 N10O7
C18H21 N7O5S
C19H20ClN7O5
C17H18ClN7O6
C18H20ClN7O5S
C18H20 ClN7O5S
C17H18 ClN7O5S
C17H18 ClN7O5S
C18H20 ClN7O5S
C17H19ClN8O5
C17H19ClN8O6
C22H21ClN8O6
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
20b
21b
22b
23b
24b
25b
26b
4-chlorophenyl
3-pyridyl
2-furyl
5-bromofuran-2-yl
5-methylfuran-2-yl
1-methyl-4-nitro-1H-imidazol-2-yl
5-methylthiophen-2-yl
phenyl
154-155
169-170
153-154
253-254
269-270
156
153-154
249-250
189-190
160-161
230-231
182-183
169-170
2-furyl
5-methylthiophen-2-yl
(thiophen-2-yl)methyl
thiophen-3-yl
thiophen-2-yl
3-methylthiophen-2-yl
1H-pyrrol-2-yl
5-methylisoxazol-3-yl
5-phenylisoxazol-3-yl
pylidene-5′-N-ethylcarboxamidoadenosine (5)28 and 2′,3′-O-
isopropylidene-2-chloro-5′-N-ethylcarboxamidoadenosine (6)
were quite efficiently converted into the corresponding 6-iodo
derivatives 7 and 8 by treatment with diiodomethane and
isopentyl nitrite as reported by Nair (yield 60%).26,29 Intermedi-
ates 7 and 8 proved to be useful key substrates for the
subsequent substitution reactions with the appropriate substituted
(hetero)arylcarboxylic acid hydrazides, which were performed
in a steal bomb at 90-100 °C for 7-8 h in the case of the
2-chloro derivative 8 or at 120 °C overnight in the case of the
2-unsubstituted 7 to furnish derivative 9a-26a (yield 20-80%).
The 2-chloro intermediate 8 was shown to be visibly more
reactive then the corresponding 2-dehalogenated intermediate
7 toward the SNAr reaction with the employed hydrazides,
requiring milder reaction conditions and reduced reaction times.
This is reflected in the differences observed in the reaction
yields, which are about 20-30% for derivative 7 compared to
70-80% for 2-chloro intermediate 8. Despite the drastic reaction
conditions (steel bomb, 100-120 °C), in no case did we observe
the byproducts deriving from the substitution of the 2-chloro
atom by the hydrazide nucleophilic species. The (hetero)-
arylcarboxylic acid hydrazides of our interest were found to be
commercially available or readily synthesized from the corre-
sponding carboxylic acids or carboxylic acid ethyl esters,
according to well-known procedures.30
assays,32 measuring their capacity to modulate cAMP levels in
CHO cells expressing hA2B receptors. Structures, chemical
properties, and biological data of the synthesized compounds
are listed in Tables 1 and 2.
The series has been developed introducing different aromatic
nuclei on the N6-hydrazide chain. We have chosen phenyl (9b,
17b), 4-chlorophenyl (10b), a six-membered heterocycle (py-
ridine) (11b), and several five-membered heterocycles, such as
furan (12-14b, 18b), thiophene (16b, 19-23b), imidazole
(15b), pyrrole (24b), and isoxazole (25-26b). Replacement of
the hydrogen at the 2-position of the purine ring with a chlorine
atom has also been evaluated.
From the analysis of binding and functional data reported in
Table 2 it is apparent that of the synthesized compounds 9b-
26b some of the heteroarylcarbonylhydrazino derivatives here
described show considerable potency in activating A2B adenosine
receptors, with EC50 values ranging from 82 to 450 nM. The
most innovative finding rests in the analysis of the selectivity
information emerging from the comparison between affinity and
functional data related to the four adenosine receptors subtypes.
Of the examined molecules, the ones showing the capability to
activate A2B adenosine receptors were inactive at the hA3AR
(Ki > 5000) and showed high nanomolar to micromolar affinity
at the A1 and A2A subtypes (Ki varying from 700 to 5000 nM).
Thus, we have identified, to the best of our knowledge, the first
examples of A2B adenosine receptor agonists endowed with good
selectivity.
Protected N6-substituted nucleoside derivatives 9a-26a were
stirred for about 3 h at room temperature in a 1:1 mixture of
water and trifluoroacetic acid to give unprotected final com-
pounds 9b-26b in a nearly quantitative yield.
Figure 2 reports the dose response curves of NECA and
compound 12b in hA2B CHO cells showing that this new ligand
is a full A2B agonist. Similar behavior is observed for the other
examined compounds.
Biological Activity. Results and Discussion
Competition binding experiments31 were performed to evalu-
ate the affinity of the synthesized compounds 9b-26b to hA1,
hA2A, and hA3 receptors expressed in CHO cells using as
radioligands [3H]CHA, [3H]CGS 21680, and [125I]AB-MECA,
respectively. The compounds were also evaluated in functional
Cristalli and co-workers have explored the SAR of 2-substi-
tuted 5′-uronamide adenosine derivatives, such as S-PHPNECA
((S)-2-phenylhydroxypropynyl-NECA), as potent but nonselec-
tive agonists at the A2BAR.33-35 Comparable marks have been
attained with the cited 2-thio-4-aryl-3,5-dicyano-6-aminopyri-

Ribofuranuronamide DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 377
Table 2. Binding, Functional Data, and RM Values of the Synthesized NECA Derivatives 9b-26ba
[3H]CHA binding
[3H]CGS21680 binding
cAMP assay
[
125I]AB-MECA binding
compd
hA1 CHO Ki (nM)
hA2A CHO Ki (nM)
hA2B CHO EC50 (nM)
hA3 CHO Ki (nM)
RM (0)
1 (NECA)
9b
18.3 ( 2.5
>5000 (35%)
>5000 (5%)
>5000 (11%)
1050 ( 132
780 ( 34
12.5 ( 2.8
160 ( 20
34.6 ( 3.3
0.94 ( 0.09
1.65 ( 0.15
2.10 ( 0.18
0.62 ( 0.05
0.84 ( 0.09
1.45 ( 0.13
1.05 ( 0.09
0.35 ( 0.04
1.52 ( 0.14
2.21 ( 0.18
1.37 ( 0.11
2.02 ( 0.20
2.15 ( 0.22
1.89 ( 0.19
1.84 ( 0.16
2.26 ( 0.24
1.54 ( 0.16
0.86 ( 0.09
2.41 ( 0.25
>5000 (18%)
>5000 (8%)
>5000 (7%)
1550 ( 165
1200 ( 135
1600 ( 147
>5000 (12%)
2100 ( 185
>5000 (40%)
4950 ( 356
4100 ( 390
633 ( 60
>5000 (10%)
>5000 (6%)
>5000 (9%)
82 ( 10
>5000 (8%)
>5000 (20%)
>5000 (17%)
>5000 (23%)
>5000 (13%)
>5000 (15%)
>5000 (11%)
>5000 (19%)
>5000 (45%)
>5000 (26%)
>5000 (17%)
25 ( 3
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
20b
21b
22b
23b
24b
25b
26b
369 ( 42
227 ( 18
700 ( 25
>5000 (7%)
1100 ( 124
>5000 (37%)
3500 ( 275
2600 ( 194
62 ( 4
933 ( 76
737 ( 46
1600 ( 140
610 ( 32
>5000 (7%)
>5000 (5%)
>5000 (14%)
273 ( 12
>5000 (5%)
210 ( 13
175 ( 20
603 ( 31
3300 ( 315
1700 ( 180
3800 ( 305
3200 ( 330
>5000 (3%)
>5000 (2%)
450 ( 29
>5000 (18%)
>5000 (12%)
>5000 (9%)
>5000 (11%)
>5000 (16%)
>5000 (15%)
200 ( 20
340 ( 35
359 ( 36
>5000 (21%)
>5000 (24%)
a The data are expressed as the mean ( SEM. The percentages in parentheses indicate the % of displacement of the new tested compounds in the binding
experiments or the % of stimulation of cAMP levels in functional experiments.
activity of about 3-fold (14b, hA2B EC50 ) 227 nM) in
comparison with the unsubstituted derivative 12b, while the
introduction of bromine atom determined a 4-fold loss of activity
(13b, hA2B EC50 ) 369 nM). The unsubstituted thiophene
derivative 22b showed Ki and EC50 values similar to those of
the 5-methylthiophene analogue 19b, while the substitution at
the 3-position with a methyl group was able to slightly affect
the results of the functional assay (compound 23b, hA2B
EC50 ) 340 nM). This preliminary SAR investigation seems to
suggest that the steric hindrance at the N6 chain could play a
primary role for the receptor-ligand interaction. A loss of
activity has in fact been observed for those derivatives in which
the heterocycle has been expanded from five to six members
Figure 2. Dose response curve of NECA and 12b on cAMP assays in
or when a small substituent, such as a bromine atom or a methyl
group, has been introduced into five-membered rings.
No correlation can be established between the biological
activity and the lipophilic character of the molecules. Table 2
reports RM values of the examined compounds showing that
the major part of the new ligands show lipophilicity parameters
ranging from 0.36 to 2.19. The reference compound NECA,
with a RM value of 0.94, is located among the most hydrophilic
studied molecules. The calculated RM value of our most potent
compound 12b was comparable to that of NECA. An evident
index that the lipophilic nature of the aromatic ring is not so
important in influencing the potency of this class of compounds
is derived from a comparison of RM values. For example,
compounds 12b, 11b, and 25b showed comparable RM values
while exerting opposite biological properties.
hA2B CHO cells.
midines,21,22 an interesting example of non-adenosine receptor
partial and full agonists, which can be considered a model for
the development of very potent, but as yet not selective, ligands.
Compounds 9b-11b and 17b, containing six-membered
aromatic rings at the N6-position, did not show any significant
affinity or activity at the four adenosine receptors (Ki and EC50
values of >5000 nM). Comparable results have been ac-
complished with some of the derivatives functionalized with
five-membered heterocycles, such as the imidazole (15b) and
the isoxazole (25b, 26b). Interesting levels of receptor affinity
and selectivity have been otherwise reached as a result of the
introduction of thiophene (16b, 19b-23b), furan (12b-14b,
18b) and pyrrole (24b) moieties. The pyrrole 24b (hA2B
EC50 ) 359 nM) is 2-fold less active than the corresponding
unsubstituted thiophene 22b (hA2B EC50 ) 200 nM) and furan
derivative 18b (hA2B EC50 ) 210 nM). The presence of a
hydrogen bond acceptor (O or S) is therefore preferred to a
hydrogen bond donor (NH). By comparison of the pharmaco-
logical behavior of compounds containing the furan ring, 14b
(hA2B EC50 ) 227 nM) and 18b, with the related thiophene
derivatives 16b (hA2B EC50 ) 273 nM) and 22b, it is possible
to assert that both furan and thiophene are able to exert similar
receptor interactions that are favorable for receptor activation.
Extraordinarily, in our series of compounds, thiophene did not
behave as a bioisoster of the phenyl moiety.
The linkage position of the thiophene ring is also important.
The thiophen-3-yl derivative 21b (hA2B EC50 ) 450 nM) was
2-fold less active than the thiophen-2-yl positional isomer 22b
(hA2B EC50 ) 200 nM).
The introduction of a methylene spacer between the thiophene
and the hydrazide function led us to identify compound 20b.
The examined structural modulation appeared to address the
binding toward human A1 and A3 adenosine receptors subtypes,
decreasing the capability of the molecule to interact with
the hA2B receptor. This confirms our previous results23-25
indicating that small modifications of the N6 chain can lead
to dramatic shifts in the corresponding ligand activity;
compound 22b showed a hA3 Ki value higher than 5µM, while
20b acquires low nanomolar affinity for the same target (hA3,
Ki ) 25 nM).
The effect of introducing several substituents at the 5-position
of the furan nucleus has been evaluated. Introduction of the
small lipophilic methyl group is sufficient to produce a loss of

378 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Baraldi et al.
Cl2/light petroleum, 1:2. Yellow solid; 60% yield; mp 79-80 °C;
1H NMR (200 MHz, DMSO-d6) δ (ppm) 0.49 (t, 3 H, J ) 7.2),
1.32 (s, 3H), 1.52 (s, 3H), 2.70 (m, 2H), 4.50-4.60 (m, 1H), 5.43
(s, 2H), 6.42 (s, 1H), 7.47 (bt, 1H), 8.43 (s, 1H), 8.47 (s, 1H).
1-Deoxy-1-(2-chloro-6-iodo-9H-purin-9-yl)-2,3-O-isopropy-
lidene-N-ethyl-â-D-ribofuranuronamide (8). The product was
purified by column chromatography with silica gel, eluting with a
mixture of EtOAc/light petroleum, 3:7, and crystallizing with a
mixture of EtOAc/Et2O/light petroleum, 1:1:1. White solid; 70%
yield; mp 95 °C; 1H NMR (200 MHz, DMSO-d6) δ (ppm) 0.55 (t,
3 H, J ) 7.2), 1.35 (s, 3H), 1.53 (s, 3H), 2.80 (m, 2H), 4.61 (s,
1H), 5.43 (m, 2H), 6.42 (s, 1H), 7.58 (bt, 1H), 8.77 (s, 1H).
General Procedure for the Preparation of Compounds 9a-
26a. A mixture of 7 or 8 (0.17 mmol), TEA (30 µL, 0.21 mmol),
and the appropriate (hetero)arylhydrazide (0.21 mmol) in absolute
EtOH (3 mL) was heated in a steal bomb at 90-100 °C for 7-8
h in the case of the 2-chloro derivative 8 or at 120 °C overnight in
the case of the 2-unsubstituted 7. The solvent was evaporated, and
the residue was suspended with EtOAc. The organic layer was
washed with H2O (2 × 20 mL) and dried with anhydrous Na2SO4.
After filtration, the solvent was evaporated under vacuum, and the
products were purified by crystallization or by column chroma-
tography on silica gel.
The presence of the chlorine atom at the 2-position of the
purine nucleus does not seem to affect the ability of the tested
compounds to activate hA2BAR, as is clear from the comparison
of chlorinated derivatives 18b (hA2B EC50 ) 210 nM) and 19b
(hA2B EC50 ) 175 nM) with the corresponding nonchlorinated
12b (hA2B EC50 ) 82 nM) and 16b (hA2B EC50 ) 273 nM).
Even though the chlorine atom at the 2-position did not allow
us to improve the pharmacological profile, most of the
synthesized compounds show this structural element for chemi-
cal reasons, in light of the relevant improvement in the
substitution reaction yields.
Conclusions
In conclusion, we have designed and synthesized a new class
of nucleoside adenosine ligands structurally related to NECA
that appear to be the first example of potent and rather selective
A
2B adenosine receptor agonists. Compound 1-deoxy-1-{6-[N′-
(furan-2-carbonyl)hydrazino]-9H-purin-9-yl}-N-ethyl-â-D-ribo-
furanuronamide (12b, hA1,hA2A Ki > 1000 nM; hA2B EC50
)
82 nM, hA3 Ki > 5000 nM) was the most potent of the series,
and it was confirmed to be a full agonist in a functional assay
based on the measurement of its capacity to modulate cAMP
levels in CHO cells expressing the hA2B receptor. The examined
molecules can be considered valuable tools for the design and
development of new and even more selective and potent ligands.
Furthermore, this study could provide useful foundations for
the attainment of a detailed pharmacological and physiological
characterization of the adenosine A2B receptor. A potent and
selective radiolabeled agonist at the hA2B adenosine receptor is
thus far unavailable; only antagonist radioligands have been
identified with the aim to perform binding studies at the hA2B
receptor subtype.36 The present report can contribute to the
identification of the first useful agonist radioligand for the
characterization of the human A2B adenosine receptor.
1-Deoxy-1-[6-(N′-benzoylhydrazino)-9H-purin-9-yl]-2,3-O-iso-
propylidene-N-ethyl-â-D-ribofuranuronamide (9a). The product
was purified by crystallization with a mixture of Et2O/petroluem
ether 1:2. Pale-yellow solid; 25% yield; mp 124-125 °C; 1H NMR
(200 MHz, DMSO-d6) δ (ppm) 0.64 (t, 3H, J ) 7.2), 1.32 (s, 3H),
1.52 (s, 3H), 2.83 (bm, 2H), 4.53 (bs, 1H), 5.38 (bs, 2H), 6.35 (bs,
1H), 7.20 (bm, 1H), 7.49-7.57 (m, 3H), 7.90-7.95 (m,2H), 8.21
(s, 1H), 8.40(bs, 1H), 10.00-11.00 (bs, 2H).
1-Deoxy-1-{6-[N′-(4-chlorobenzoyl)hydrazino]-9H-purin-9-
yl}-2,3-O-isopropylidene-N-ethyl-â-D-ribofuranuronamide (10a).
The product was purified by column chromatography on silica gel,
eluting with a mixture of EtOAc/petroleum ether, 1:1. White solid;
1
20% yield; mp 116-117 °C; H NMR (200 MHz, DMSO-d6) δ
(ppm) 0.80 (t, 3H, J ) 7.2), 1.32 (s, 3H), 1.54 (s, 3H), 3.04 (m,
2H), 4.57 (s, 1H), 5.30 (s, 2H), 6.17 (s, 1H), 7.18 (bt, 1H), 7.38 (d,
2H, J ) 8), 7.92 (d, 2H, J ) 8), 8.01 (s, 1H), 8.26 (s, 1H), 9.30
(bs, 1H), 10.56 (bs, 1H).
Experimental Section
Chemistry. Reaction progress and product mixtures were
monitored by thin-layer chromatography (TLC) on silica gel
(precoated F254 Merck plates) and visualized with aqueous potas-
General Procedure for the Preparation of Compounds 9b-
26b. The appropriate protected derivative, 9a-26a (0.6 mmol), was
dissolved in a mixture of trifluoroacetic acid/H2O, 1:1 (4 mL), and
the solution was stirred at room temperature for 3 h. The solvents
were evaporated to dryness, and the residue was suspended with
EtOAc. The organic layer was washed with H2O (2 × 15 mL) and
dried with Na2SO4, and after filtration, the solvent was evaporated.
1-Deoxy-1-[6-(N′-benzoylhydrazino)-9H-purin-9-yl]-N-ethyl-
â-D-ribofuranuronamide (9b). The product was purified by
column chromatography on silica gel, eluting with EtOAc. White
1
sium permanganate or a methanolic solution of H2SO4. H NMR
data were determined in CDCl3 or DMSO-d6 solutions with a Varian
VXR 200 spectrometer or a Varian Mercury Plus 400 spectrometer.
Peak positions are given in parts per million (δ) downfield from
tetramethylsilane as internal standard, and J values are given in
hertz. light petroleum refers to the fractions boiling at 40-60 °C.
Melting points were determined on a Buchi-Tottoli instrument and
are uncorrected. Chromatography was performed on Merck 230-
400 mesh silica gel. Organic solutions were dried over anhydrous
sodium sulfate. Elemental analyses were performed by the mi-
croanalytical laboratory of Dipartimento di Chimica, University of
Ferrara, and were within (0.4% of the theoretical values for C, H,
and N.
1
solid; 85% yield; mp 173-174 °C; H NMR (200 MHz, DMSO-
d6) δ (ppm) 1.07 (t, 3H, J ) 7.2), 3.21 (m, 2H), 4.17 (bs, 1H),
4.32 (s, 1H), 4.61 (bm, 1H), 5.61 (bm, 1H), 5.75 (bm, 1H), 6.00
(bm, 1H), 7.56 (m, 3H), 7.94 (m, 2H), 8.34 (s, 1H), 8.45 (bs, 1H),
8.74 (bt, 1H), 10.00 (bs, 1H), 10.60 (bs, 1H). Anal. (C19H21N7O5)
C, H, N.
General Procedure for the Preparation of 1-Deoxy-1-(6-iodo-
9H-purin-9-yl)-2,3-O-isopropylidene-N-ethyl-â-D-ribofuranu-
ronamide (7) and 1-Deoxy-1-(2-chloro-6-iodo-9H-purin-9-yl)-
1-Deoxy-1-[6-(N′-(4-chlorobenzoyl)hydrazino)-9H-purin-9-yl]-
N-ethyl-â-D-ribofuranuronamide (10b). The product was purified
by column chromatography on silica gel, eluting with a mixture of
CH2Cl2/CH3OH, 9.5:0.5. White solid; 80% yield; mp 188-190 °C;
1H NMR (200 MHz, DMSO-d6) δ (ppm) 1.10 (t, 3H, J ) 7.2),
3.17 (m, 2H), 4.02 (bm, 1H), 4.31 (m, 1H), 4.63 (bs, 1H), 4.68
(bm, 2H), 6.00 (d, 1H, J ) 7.3), 7.61 (d, 2H, J ) 8), 7.96 (d, 2H,
J ) 8), 8.34 (s, 1H), 8.51 (bs, 1H), 8.75 (bt, 1H), 10.00 (bs, 1H),
10.80 (bs, 1H). Anal. (C19H20ClN7O5) C, H, N.
Determination of Binding (Ki Values) and Functional Pa-
rameters (EC50 Values). All synthesized compounds have been
tested, by radioligand binding assay, for their affinity to human
A1, A2A, and A3 adenosine receptors and for their potency, in a
cAMP assay, to human A2B subtypes. The expression of the human
A1, A2A, A2B, and A3 receptors in CHO cells has been previously
2,3-O-isopropylidene-N-ethyl-â-D-ribofuranuronamide (8).29
A
suspension of NECA (5) or 2-chloro-NECA (6) (2.85 mmol) in
isopentyl nitrite (8.25 mL) and CH2I2 (21.55 mL) was heated at 85
°C for 1 h. The reagents were evaporated, and the residue was
dissolved with CH2Cl2 (100 mL). The organic phase was washed
with H2O (2 × 50 mL) and dried with anhydrous Na2SO4, and the
solvent was evaporated under vacuum to obtain a crude oil, which
was washed with light petroleum (3 × 20 mL).
1-Deoxy-1-(6-iodo-9H-purin-9-yl)-2,3-O-isopropylidene-N-
ethyl-â-D-ribofuranuronamide (7). The product was purified by
column chromatography with silica gel, eluting with a mixture of
CH2Cl2/CH3OH, 9.8:0.2, and crystallizing with a mixture of CH2-

Ribofuranuronamide DeriVatiVes
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 379
described.37 The cells were grown adherently and maintained in
Dulbecco’s modified Eagle’s medium with nutrient mixture F12
(DMEM/F12) without nucleosides, containing 10% fetal calf serum,
penicillin (100 U/mL), streptomycin (100 µg/mL), L-glutamine (2
mM), and Geneticin (G418, 0,2 mg/mL) at 37 °C in 5% CO2/95%
air. Cells were split two or three times weekly at a ratio between
1:5 and 1:20. For membrane preparation the culture medium was
removed and the cells were washed with PBS and scraped off T75
flasks in ice-cold hypotonic buffer (5 mM Tris-HCl, 2 mM EDTA,
pH 7.4). The cell suspension was homogenized and centrifuged
for 30 min at 100000g. The membrane pellet was resuspended in
50 mM Tris-HCl buffer, pH 7.4, and incubated with 2 IU/mL of
adenosine deaminase for 30 min at 37 °C. Then the suspension
was frozen at -80 °C, and the protein concentration was determined
according to a Bio-Rad method38 with bovine albumin as a standard
reference. Binding of 1 nM [3H]CHA to hA1 CHO cells (50 µg of
protein/assay) was performed using 50 mM Tris-HCl buffer, pH
7.4, and at least six to eight different concentrations of agonists
studied for an incubation time of 150 min at 25 °C.39 Nonspecific
binding was determined in the presence of 10 µM CHA and was
about 20% of the total binding. Inhibition binding experiments of
[3H]CGS 21680 to hA2A CHO cells (100 µg of protein/assay) was
performed using 50 mM Tris-HCl buffer, 10 mM MgCl2, pH 7.4,
and at least six to eight different concentrations of agonists studied
for an incubation time of 180 min at 25 °C.40 Nonspecific binding
was determined in the presence of 10 µM CGS 21680 and was
about 25% of the total binding. Binding of [125I]ABMECA to hA3
CHO cells (50 µg of protein/assay) was performed using 0.2 mL
of 50 mM Tris-HCl buffer, 10 mM MgCl2, 1 mM EDTA, pH 7.4,
and at least six to eight different concentrations of tested compounds
for an incubation time of 60 min at 37 °C.31 Nonspecific binding
was determined in the presence of 1 µM ABMECA and was about
20% of total binding. Separation of bound from free radioligand
was performed by rapid filtration through Whatman GF/B filters,
which were washed three times with ice-cold buffer. Filter bound
radioactivity was measured by scintillation spectrometry after
addition of 5 mL of Aquassure. For cell preparation CHO cells
transfected with human A2B receptors were washed with PBS and
diluted trypsine and centrifuged for 10 min at 200g. The pellet
containing the CHO cells (1 × 106 cells/assay) was resuspended
in an incubation mixture of 15 mM NaCl, 0.27 mM KCl, 0.037
mM NaH2PO4, 2 IU/mL adenosine deaminase, and 4-(3-butoxy-
4-methoxybenzyl)-2-imidazolidone (Ro 20-1724) as phosphodi-
esterase inhibitor and preincubated for 10 min in a shaking bath at
37 °C. After the preincubation time at 37 °C the examined ligands
(1 nM to 10 µM) were added to the mixture and incubated for a
further 5 min. The reaction was terminated by the addition of cold
6% thrichloroacetic acid (TCA). The TCA suspension was centri-
fuged at 2000g for 10 min at 4 °C, and the supernatant was extracted
four times with water saturated diethyl ether. The final aqueous
solution was tested for cyclic AMP levels by a competition protein
binding assay.41 Samples of cyclic AMP standard (0-10 pmol) were
added to each test tube containing the incubation buffer (0.1 mM
trizma base , 8.0 mM aminophylline, 6.0 mM 2-mercaptoethanol,
pH 7.4) and [3H]cAMP in a total volume of 0.5 mL. The binding
protein, previously prepared from beef adrenals, was incubated at
4 °C for 150 min, and after the addition of charcoal was centrifuged
at 2000g for 10 min. The clear supernatant was counted in a
Beckman scintillation counter. Inhibitory binding constants, Ki, were
calculated from those of IC50 according to the Cheng and Prusoff
equation,42
Prism, San Diego, CA). All experimental data are expressed as the
mean ( standard error of the mean (SEM) of three or four
independent experiments performed in duplicate.
Determination of RM Values by C18 RP-HPTLC. A reversed-
phase TLC technique for measuring RM values as an expression of
the lipophilic character of molecules was performed. The HPTLC
determinations were carried out on Whatman KC18F plates as
previously described.44 Solvent mixtures of methanol-water buffer
at pH 7.0 were used as mobile phase. The methanol concentration
ranged from 60% to 90%. The test compounds were dissolved in
DMSO, and 1 µL of a 10 µM solution was spotted on the plates.
The developed plates were dried and the spots detected under UV
light (254 nm). RM values were calculated by means of the equation
RM ) log[(1/RF) - 1]
For each RF measurement at least three to four replications were
carried out. For each compound, there was a linear relationship
between the RM values and composition of the mobile phase. RM
values represented the theoretical value at 0% methanol in the
mobile phase, expressed as the mean ( SEM.
Acknowledgment. The authors thank King Pharmaceutical
R&D (4000 CentreGreen Way, Suite 300, Cary, North Carolina
27513) for financial support and Prof. Karl Norbert Klotz
(Wurzburg University, Germany) for supplying hA2B cells and
cDNA for hA1, hA2A, and hA3 receptors.
Supporting Information Available: Detailed experimental
procedures for the synthesis of the reported compounds, elemental
analysis data, and 1H NMR data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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