N6-[(Hetero)aryl/(cyclo)alkyl-carbamoyl-methoxy-phenyl]-(2-chloro)-5′-N-ethylcarboxamido-adenosines: The first example of adenosine-related structures with potent agonist activity at the human A2B adenosine receptor

Article abstract of DOI:10.1016/j.bmc.2007.01.055

A new series of N⁶-[(hetero)aryl/(cyclo)alkyl-carbamoyl-methoxy-phenyl]-(2-chloro)-5′-N-ethylcarboxamido-adenosines (24-43) has been synthesised and tested in binding assays at hA₁, hA_2A and hA₃ adenosine receptors, and in a functional assay at the hA_2B subtype. The examined compounds displayed high potency in activating A_2B receptors with good selectivity versus A_2A subtypes. The introduction of an unsubstituted 4-[(phenylcarbamoyl)-methoxy]-phenyl chain at the N⁶ position of 5′-N-ethylcarboxamido-adenosine led us to the recognition of compound 24 as a full agonist displaying the highest efficacy of the series (EC₅₀ hA_2B = 7.3 nM). These compounds represent the first report about adenosine-related structures capable of activating hA_2B subtype in the low nanomolar range.

Full text of DOI:10.1016/j.bmc.2007.01.055

Bioorganic & Medicinal Chemistry 15 (2007) 2514–2527
N6-[(Hetero)aryl/(cyclo)alkyl-carbamoyl-methoxy-phenyl]-(2-
chloro)-50-N-ethylcarboxamido-adenosines: The first example
of adenosine-related structures with potent agonist activity
at the human A2B adenosine receptor
Pier Giovanni Baraldi,a,* Delia Preti,a Mojgan Aghazadeh Tabrizi,a Francesca Fruttarolo,a
Giulia Saponaro,a Stefania Baraldi,a Romeo Romagnoli,a Allan R. Moorman,c
Stefania Gessi,b Katia Varanib and Pier Andrea Boreab
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Ferrara, 44100 Ferrara, Italy
b
`
Dipartimento di Medicina Clinica e Sperimentale-sezione di Farmacologia, Universita di Ferrara, 44100 Ferrara, Italy
cKing Pharmaceutical Research and Development, Inc., 4000 CentreGreen Way, Suite 300, Cary, NC 27513, USA
Received 21 December 2006; revised 22 January 2007; accepted 31 January 2007
Available online 2 February 2007
Abstract—A new series of N6-[(hetero)aryl/(cyclo)alkyl-carbamoyl-methoxy-phenyl]-(2-chloro)-50-N-ethylcarboxamido-adenosines
(24–43) has been synthesised and tested in binding assays at hA1, hA2A and hA3 adenosine receptors, and in a functional assay
at the hA2B subtype. The examined compounds displayed high potency in activating A2B receptors with good selectivity versus
A2A subtypes. The introduction of an unsubstituted 4-[(phenylcarbamoyl)-methoxy]-phenyl chain at the N6 position of 50-N-ethyl-
carboxamido-adenosine led us to the recognition of compound 24 as a full agonist displaying the highest efficacy of the series (EC50
hA2B = 7.3 nM). These compounds represent the first report about adenosine-related structures capable of activating hA2B subtype
in the low nanomolar range.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
identified for each subtype. In particular, A2B receptors
are associated with stimulation of adenylate cyclase and
activation of phospholipase C through the coupling to
Gs and Gq/11 proteins, respectively. A2B receptors have
been found on practically every cell in most species and
their sequences are highly similar across species.3 A role
of A2B adenosine receptors in regulation of vascular
tone, neurosecretion and neurotransmission, intestinal
tone and smooth muscle growth has been suggested.3
RT-PCR studies revealed their highest expression in cae-
cum, large intestine and urinary bladder, but lower lev-
els in brain, spinal cord, lung and vas deferens.4 Recent
evidence of a probable involvement of A2B adenosine
receptors in tumur growth and development has been
reported.5 The expression of A2B receptors is regulated
in T cell activation events, thus indicating that A2B
receptors are involved in the regulation of signalling
processes occurring in human lymphocytes by adeno-
sine.6 There is growing evidence that adenosine plays a
role in asthma and chronic obstructive pulmonary dis-
ease (COPD).7 It has been recently demonstrated that
adenosine stimulates production of IL-4 and IL-13 in
Adenosine is a ubiquitous nucleoside involved in impor-
tant biochemical processes, such as energy transfer
(ATP and ADP), as well as in signal transduction as cyc-
lic adenosine monophosphate (cAMP), and as a build-
ing block for some biologically significant molecules
such as NAD (nicotinamide-adenine-dinucleotide),
FAD (flavin-adenine-dinucleotide), SAM (S-adenosyl-
L-methionine), DNA and RNA. Four adenosine recep-
tors, classified as A1, A2A, A2B and A3, have been cloned
from many mammalian and some non-mammalian spe-
cies.1,2 The adenosine receptors have extensive tissue dis-
tribution and can be co-expressed in the same cell type.
All the adenosine receptor subclasses belong to the
family of cell membrane G protein-coupled receptors.
Different signal transduction mechanisms have been
Keywords: Adenosine; A2B receptors; Agonists; N6-substituted-NECA.
*
Corresponding author. Tel.: +39 0532 291 293; fax: 39 0532 291
296; e-mail addresses: pgb@ifeuniv.unife.it; brlpgv@unife.it
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.055

P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
2515
mast cells. This effect seems to be mediated via A2B
adenosine receptors.8
the substituent at the 8-position, as the selective struc-
tural element for the design of potent A2B antagonists,
has been recently provided by our group.17
The treatment of asthma with selective A2B adenosine
receptor antagonists is a promising therapeutic aim.9
The use of A2B adenosine receptor agonists for the treat-
ment of atherosclerosis, septic shock10 and impotence3
has been investigated. However, the lack of selective
pharmacological tools, in particular, the lack of selective
A2B receptor agonists, has certainly determined the gen-
eral scarceness of information about the physiological
functions of this potential therapeutic target.
The strategy here reported to obtain new A2B selective
NECA derivatives has been furthermore suggested by
SAR studies which would indicate the N6 a useful posi-
tion for A2B binding site recognition. A docking study
related to a small collection of known A2B agonists high-
lighted the interaction of the exocyclic amino group at
the 6-position of NECA with a residue of asparagine
254 belonging to the VI transmembrane receptor helix.18
We have recently confirmed the importance of the
N6-position for A2B adenosine receptor recognition,
by the synthesis of a series of 1-deoxy-1-[6-[((hetero)
aryl-carbonyl)-hydrazino]-9H-purin-9-yl]-N-ethyl-b-D-
ribofuranuronamide and 1-deoxy-1-[2-chloro-6-[((het-
ero)aryl-carbonyl)-hydrazino]-9H-purin-9-yl]-N-ethyl-b-
D-ribofuranuronamide derivatives which have been
found to be the first examples of both potent and selec-
tive A2B adenosine receptor agonists.19
Up to now, several non-selective adenosine analogues
with poor affinity have been identified and employed
for the characterization of the A2B adenosine receptor,
which has been classified as a low-affinity adenosine
receptor.10,11 A leading relevance has been ascribed for
years to some nucleoside ligands such as NECA (50-N-
ethylcarboxamido-adenosine) and (S)-PHPNECA((S)-
2-phenylhydroxypropynylNECA)10 which show the
capability to activate the A2B receptor with EC50 values
in the micromolar range based on the measurement of
receptor-stimulated adenylate cyclase activity.10,12 An
innovative series of 2-amino-pyridine-3,5-dicarbonitrile
derivatives has been recently reported as the first exam-
ple of high-potency non-adenosine full and partial
agonists of the human A2B receptor.13,14
In light of this, and thanks to the identification of a ver-
satile synthetic route which permitted us to functionalize
the N6-position of the known non-selective adenosine
agonists NECA20,21 and 2-Cl-NECA (2-chloro-50-N-
ethylcarboxamido-adenosine),22 we designed a series of
adenosine analogues bearing a (substituted) phenylcar-
bamoyl-methoxy-phenyl chain at the N6-NECA posi-
tion. Our attempt was to evaluate the possibility of
identifying a molecular hybrid obtained by the molecu-
lar combination of the nucleoside core responsible for
receptor activation with the structural element able to
grant A2B selectivity to the cited series of xanthine
derivatives.16
For the design of new nucleoside A2B adenosine receptor
agonists, we considered the possibility to exploit some
information deriving from the structural analysis of
known A2B adenosine receptor antagonists. In this field,
several compounds with high affinity and selectivity
have been identified among structures based upon a
xanthine core,15 bearing different substituents at the
1-, 3- and 8-positions. Kim et al.16 reported that a
(substituted)phenylcarbamoyl-methoxy-phenyl chain at
the 8-position of a series of 1,3-dipropyl-xanthines selec-
tively addresses the antagonists’ affinity to the adenosine
A2B receptor. Further evidence of the important role of
We have then synthesised a new series of N6-[(substitut-
ed)phenyl/cycloalkyl/benzyl/heteroaryl-carbamoyl-meth-
oxy-phenyl]-50-N-ethylcarboxamido-adenosine
2-chloro-adenosine derivatives (24–43; Fig. 1, Table 1).
and
The N6 mono-substitution demonstrated to be well
NH
O
2
O
H
N
N
N
N
O
NH
H
N
N
N
N
X (H, Cl)
O
N
O
O
OH OH
1
NECA or 2-Cl-NECA
O
O
R'
N
H
HN
N
N
N
N
H
X (H, Cl)
N
O
O
OH OH
24-43
Figure 1. Design of new N6-substituted-NECA derivatives as A2B adenosine receptor agonists based on a hybridization approach between known
selective A2B AR antagonists and non-selective ARs agonist.

2516
P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
Table 1. Structures and physicochemical parameters of the synthesised N6-substituted-NECA derivatives
O
R'
N
H
O
HN
N
N
N
N
H
N
R
O
O
OH OH
Compound
R
R0
Mp (ꢁC)
MW
Formula
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Ph
145–146
274–275
279–280
272
533.54
551.19
567.98
612.43
659.43
601.53
563.56
593.59
593.59
577.55
589.26
618.26
534.20
547.56
577.23
539.58
567.98
598.01
582.01
574.03
C26H27N7O6
C26H26FN7O6
C26H26ClN7O6
C26H26BrN7O6
C26H26IN7O6
C27H26F3N7O6
C27H29N7O7
C28H31N7O8
C28H31N7O8
C27H27N7O8
C30H35N7O6
C30H34N8O7
C25H26N8O6
C27H29N7O6
C28H31N7O7
C26H33N7O6
C26H26ClN7O6
C27H28ClN7O7
C27H28ClN7O6
C26H32ClN7O6
4-F–Ph
4-Cl–Ph
4-Br–Ph
4-I–Ph
2-CF3–Ph
284–285
222
4-OCH3–Ph
249–250
240
3,4-(OCH3)2–Ph
3,5-(OCH3)2–Ph
3,4-OCH2O–Ph
4-tert-Butyl–Ph
4-N-Morpholino–Ph
4-Pyridyl
219–220
270–272
155–156
274
145
Benzyl
129–130
115–116
211–212
261–262
264–265
235–236
275–276
4-OCH3–benzyl
Cyclohexyl
Ph
4-OCH3–Ph
Benzyl
Cyclohexyl
tolerated by the A2B receptor in a series of adenosine
and 50-N-ethyl-carboxamidoadenosine derivatives previ-
ously reported.23 The literature in the field of A2B ago-
nists also indicated that a second possible site of
modification was the 2-position of the purine nucleus.
Compounds such as (S)-PHPNECA and 2-alkynyl-50-
N-alkylcarboxamidoadenosines10,12 have been synthe-
sised and identified as quite potent agonists at the A2B
receptors. We chose to introduce in some of the N6-
substituted-NECA derivatives here reported, a chlorine
atom at the 2-position of the purine moiety.
pylidene-NECA)24 and 20,30-O-isopropylidene-2-chloro-
50-N-ethylcarboxamidoadenosine (20,30-O-isopropyl-
idene-2-chloro-NECA)22 were quite effectively converted
into the corresponding 6-iodo derivatives (2 and 3) by
treatment with diiodomethane and isopentylnitrite, as
reported by Nair (yield 60%).19,25 Intermediates 2 and 3
proved to be useful key substrates for the subsequent
substitution reactions with the appropriate 2-(4-amino-
phenoxy)-N-substituted-acetamide 76–91, which were
performed in a steel bomb at 100 ꢁC for 7–8 h (yield
20–50%) to provide intermediates 4–23. Despite the dras-
tic reaction condition, in no case did we observe the by-
products arising from substitution of the 2-chloro atom.
Protected N6-substituted nucleoside derivatives 4–23
were stirred for about 3 h at room temperature in a 1:1
mixture of water and trifluoroacetic acid to give unpro-
tected final compounds 24–43 in nearly quantitative
yield.
All of the synthesised compounds were evaluated in
radioligand binding assays to define their affinities for
human A1, A2A and A3 adenosine receptors (Table 2).
Efficacy of the compounds at the hA2B adenosine recep-
tors was assessed via functional tests, based on the esti-
mation of cyclic AMP levels in stably transfected CHO
cells. In light of our biological evaluation, we can report
the identification of the first series of adenosine ana-
logues endowed with potent agonist activity for the
human A2B adenosine receptor with EC50 values ranging
from 7.3 to 360 nM.
2-(4-Amino-phenoxy)-N-substituted-acetamides 76–91
were synthesised according to the procedure described
in Scheme 2. Chloroacetyl chloride was reacted with
(substituted)phenyl/heteroaryl/(substituted)benzyl/cyclo-
alkyl-amines to give the corresponding 2-chloro-N-
substituted-acetamides 44–59.26 Subsequent alkylation
of 4-nitro-phenol with intermediates 44–59 furnished
the 2-(4-nitro-phenoxy)-N-substituted-acetamides 60–
75 which were efficiently converted into the desired
2-(4-amino-phenoxy)-N-substituted-acetamides 76–91
by reduction with NaBH4 and 10% Pd/C.
2. Chemistry
Scheme 1 depicts the synthetic strategy adopted to
prepare the target compounds 24–43. 20,30-O-isopropyl-
idene-50-N-ethylcarboxamidoadenosine (20,30-O-isopro-

P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
2517
Table 2. Binding affinities and functional parameters of the synthesised N6-substituted-NECA derivatives at the human A1, A2A, A2B and A3
adenosine receptors expressed in CHO cells
Compound
[3H]CHA bindinga hA1
CHO cells Ki (nM)
[3H]CGS21680 bindingb hA2A
CHO cells Ki (nM)
cAMP assayc hA2B CHO
cells EC50 (nM)
[
125I]ABMECA bindingd hA3
CHO cells Ki (nM)
NECA
S-PHPNECA
18.2 2.1
2.110
12.4 2.7
2.010
155 12
22010
35.7 3.3
0.7510
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
8.5 0.8
2.3 0.2
3.1 0.3
3.5 0.4
5.2 0.5
31.6 3.3
4.7 0.4
7.7 0.5
15.3 2.2
8.4 0.9
18.6 2.1
77.5 4.6
11.2 1.3
20.4 2.1
28.3 3.7
16.2 2.4
30.5 3.3
50.4 3.5
22.6 2.4
30.8 3.6
>1000 (45%)
>1000 (48%)
>1000 (35%)
>1000 (26%)
>1000 (28%)
>1000 (1%)
>1000 (42%)
>1000 (30%)
>1000 (13%)
>1000 (5%)
>1000 (1%)
>1000 (1%)
>1000 (37%)
>1000 (49%)
>1000 (1%)
>1000 (40%)
>1000 (36%)
>1000 (27%)
>1000 (49%)
>1000 (46%)
7.3 0.6
15.2 2.1
12.3 1.4
10.5 1.2
30.2 2.8
107 10
32.4 3.3
45.4 5.3
53.6 5.4
35.5 2.7
16.4 2.1
300 35
32.3 2.4
150 17
240 23
315 34
42.6 4.2
360 32
175 14
302 16
38.4 3.7
72.3 7.4
34.2 3.7
36.4 3.7
85.2 8.3
115 12
25.3 2.6
53.2 5.4
78.6 7.5
81.4 8.3
40.2 3.9
320 35
42.3 4.7
82.7 8.9
65.4 6.8
72.5 7.5
107 10
101
9
75.7 7.4
95.8 9.6
a Displacement of specific [3H]CHA binding at human A1 receptors expressed in CHO cells (n = 3–6).
b Displacement of specific [3H]CGS21680 binding at human A2A receptors expressed in CHO cells (n = 3–6).
c cAMP assay in CHO cells expressing human A2B adenosine receptors IC50 (nM).
d Displacement of specific [125I]ABMECA binding at human A3 receptors expressed in CHO cells (n = 3–6). Data are expressed as geometric means
with 95% confidence limits. In parentheses is indicated the percentage of displacement of the examined compounds.
O
O
I
NH2
N
R
ii
i
Cl
Cl
N
N
N
N
Cl
O
N
H
N
R NH2
H
N
H
N
N
R'
R'
N
O
O
44-59
O
O
i
2 R = H
3 R = Cl
O
O
O
O
O
O
iii
O
R
R
N
N
H
H
H2N
O2N
Protected
O
60-75
76-91
R
O
NECA or 2-Cl-NECA
ii
N
H
Scheme 2. Reagents and conditions: (i) K2CO3, CH2Cl2, reflux, 4 h;
(ii) 4-NO2-phenol, K2CO3, acetone, reflux, 12 h; (iii) NaBH4, 10%
Pd/C, CH3OH, H2O, rt, 3 h.
NH2
O
O
76-91
R
O
N
H
R
O
N
H
NH
NH
CHO cells using as radioligands [3H]-CHA, [3H]-CGS
21680 and [125I]-AB-MECA, respectively. The com-
pounds were also evaluated in a functional assay,17
measuring their capacity to modulate cAMP levels in
CHO cells expressing hA2B receptors.
N
N
N
N
N
H
N
N
R'
N
H
N
O
N
R'
iii
O
O
O
O
O
OH OH
Structures, chemical properties and biological data of
the synthesised compounds are listed in Tables 1 and
2. Different kinds of substitutions have been considered
at the nitrogen of the acetamide chain at the N6-position
of NECA: unsubstituted phenyl (24), phenyl bearing
weak electron-withdrawing functions at the ortho (29)
and para position (25–28), strong electron-donating sub-
stituents at several positions (30–33), hindered lipophilic
(34) and hydrophilic moieties (35). The effect of the
replacement of the phenyl nucleus with a pyridine (36),
a benzyl moiety (37), a 4-OCH3-benzyl (38) and a
24-43
4-23
Scheme 1. Reagents and conditions: (i) CH2I2, isopentylnitrite, 80 ꢁC,
1 h; (ii) TEA, EtOH, steel bomb, 100–110 ꢁC, 12 h; (iii) TFA/H2O 1:1,
rt, 3 h.
3. Results and discussion
Competition binding experiments27 were performed to
evaluate the affinity of the synthesised compounds
9b–26b to hA1, hA2A and hA3 receptors expressed in

2518
P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
cycloalkyl group (39) has also been investigated. A chlo-
rine atom at the 2-position of the purine nucleus has also
been introduced (compounds 40–43).
100
80
60
40
20
0
NECA
28
In the absence of binding data on the adenosine A2B
receptor, caused by the substantial lack of a useful
radiolabelled agonist, we deduced a selectivity profile
in view of the ratio of Ki (A1, A2A and A3) to EC50
(A2B).
26
24
34
All the synthesised compounds showed the capacity to
bind to the adenosine A1 receptor and to activate the
adenosine A2B receptor in the low nanomolar range,
displaying at the same time scarce affinity (Ki > 1lM)
towards A2A adenosine subtypes and a relevant capabil-
ity to bind A3 receptors. Considering the binding and
functional profile of NECA (see Table 2) and (S)-
PHPNECA (Ki hA1 = 2.1 nM; Ki hA2A = 2.0 nM;
EC50 hA2B = 220 nM; Ki hA3 = 0.75 nM)10 which are
among the most potent adenosine-like A2B adenosine
receptor agonists reported thus far, the molecules de-
scribed here represent a remarkable advance in this field,
albeit the selectivity profile must be undoubtedly
improved.
-11
-10
-9
-8
-7
-6
-5
-4
log [drug] (M)
Figure 2. Stimulation curves representing the capability of the
examined adenosine agonists to stimulate cAMP production in
CHOhA2B cells.
38, EC50 hA2B = 240 nM), or the complete saturation
of the 6-carbon cycle (cyclohexyl derivative 39, EC50
hA2B = 315 nM), led to a severe reduction in the capa-
bility to activate A2B receptors. The presence of a chlo-
rine atom at the 2-position had little or a slightly
detrimental effect in terms of A2B receptor activation,
as emerged from the comparison of the biological data
of the 2-chloro derivatives 40–43 with the corresponding
2-unsubstituted 24, 30, 37 and 39 compounds.
Substitution at the para-position of the phenyl ring with
a halogen atom led to a substantial loss of activity in
comparison with the unsubstituted phenyl derivative
24 (EC50 hA2B = 7.3 nM). Among the 4-halogenated-
phenyl derivatives, the 4-iodo 28 (EC50 hA2B = 30.2 nM)
showed to be less potent than the 4-fluoro 25 (EC50
hA2B = 15.2 nM), the 4-chloro 26 (EC50 hA2B = 12.3
nM) and the 4-bromo 27 (EC50 hA2B = 10.5 nM) ana-
logues in activating the A2B adenosine receptor. The
same behaviour has been observed by introducing func-
tions with reverse electronic effects, such as the 4-meth-
oxy group, which led us to the identification of
compound 30 (EC50 hA2B = 32.4 nM). The bis-methoxy
derivatives 31 (EC50 hA2B = 45.4 nM) and 32 (EC50
hA2B = 53.6 nM) were slightly less potent than the 4-
methoxy counterpart 30. Derivative33 displayed the
same potency of the 4-methoxy-phenyl (30). The pres-
ence of the trifluoromethyl function at the 2-position
of the phenyl ring was quite detrimental for the A2B
receptor activation potency (29, EC50 hA2B = 107 nM).
Increasing the steric hindrance around the para-position
by the introduction of the branched and lipophilic tert-
butyl chain in compound 34 permitted us to obtain a
very potent agonist for the adenosine A2B receptor with
an EC50 value comparable to that of the unsubstituted-
phenyl derivative 24. Replacement of the tert-butyl
substituent with the sterically demanding but more polar
4-morpholino group (35, EC50 hA2B = 300 nM) leads to
poor activity, suggesting a possible lipophilic interaction
of the terminal side of the N6-phenylcarbamoyl residue
with the A2B receptor binding site.
Unfortunately, despite being very potent ligands for A2B
adenosine receptor, the reported series of N6-[substitut-
ed-carbamoyl-methoxy-phenyl]-(2-chloro)-50-N-ethylcarb-
oxamido-adenosines exhibited very low, or no, selectivity
versus the A1 and A3 adenosine receptor subtypes. Most
of the examined molecules, in fact, preferentially bound
to the A1 receptor, with Ki binding values ranging from
2.3 to 77 nM. This experimental observation can be
explained in light of the literature indicating that A1
adenosine receptor selectivity is enhanced by monosub-
stitution of the exocyclic amino group at the 6-position
of adenosine with bulky cycloalkyl or arylalkyl substitu-
ents.28 A lower, but significant, affinity for the A3
receptor was observed. The most selective compounds
versus A3 adenosine subtype were the unsubstituted
phenyl derivative (24) and the 4-halo-phenyl derivatives
(25–28).
Figure 2 reports the dose–response curves of NECA and
compounds 24, 26, 28 and 34 in hA2BCHO cells showing
that the designed molecules behave as full A2B agonists.
4. Conclusions
In the present study, we described the affinity at the
human adenosine receptors A1, A2A and A3 and the effi-
cacy at the human A2B receptor of a new series of N6-
[(hetero)aryl/(cyclo)alkyl-carbamoyl-methoxy-phenyl]-
The replacement of the phenyl with the 4-pyridyl moiety
resulted in a 4-fold decrease in the potency (36, EC50
hA2B = 32.3 nM). Further modifications of the phenyl
ring, such as the insertion of a methylene group between
the acetamide nitrogen and the phenyl ring (benzyl 37,
EC50 hA2B = 150 nM, and 4-OCH3-benzyl derivatives
(2-chloro)-50-N-ethylcarboxamido-adenosines.
These
molecules can be considered the first example of adeno-
sine-related structures able to exert very high potency
(EC50 values from functional assay in the low nanomo-
lar range) in activating the A2B adenosine subtype. The

P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
2519
lack of agonists endowed with satisfactory levels of A2B
potency and selectivity has hampered the pharmacolog-
ical characterization of this potential therapeutic target.
Even though non-selective versus the adenosine A1 and
A3 adenosine receptors, the synthesised molecules may
represent a promising device for the development of use-
ful ligands to be employed for pharmacological investi-
gation of the physiological functions mediated by
adenosine through the A2B receptors.
5.2.2. 1-Deoxy-1-[6-{4-[(4-fluoro-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-methyl-
ethylidene)-b-D-ribofuranuronamide (5). The product was
purified by column chromatography on silica gel eluting
with EtOAc: yield 43%; pale yellow solid; mp 120–
1
121 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 0.60 (t,
3H, J = 7.2), 1.35 (s, 3H), 1.54 (s, 3H), 2.78 (m, 2H),
4.56 (s, 1H), 4.67 (s, 2H), 5.44 (m, 2H), 6.40 (s, 1H),
6.96–7.22 (m, 4H), 7.55 (br t, 1H), 7.64–7.82 (m, 4H),
8.29 (s, 1H), 8.42 (s, 1H), 10.00 (s, 1H).
5. Experimental
5.1. Chemistry
5.2.3. 1-Deoxy-1-[6-{4-[(4-chloro-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-methyl-
ethylidene)-b-D-ribofuranuronamide (6). The product was
purified by column chromatography on silica gel eluting
with a gradient from light petroleum/EtOAc 1:1 to
EtOAc: yield 33%; pale yellow solid; mp 122–123 ꢁC;
1H NMR (200 MHz, CDCl3): d (ppm) 0.90 (t, 3H,
J = 7.2), 1.39 (s, 3H), 1.64 (s, 3H), 3.13 (m, 2H), 4.63
(s, 2H), 4.73 (s, 1H), 5.39 (m, 2H), 6.07 (d, 1H,
J = 2.3), 7.01 (m, 3H), 7.34 (d, 2H, J = 8), 7.48 (d, 2H,
J = 8), 7.69 (s, 1H), 7.74 (d, 2H, J = 8), 7.91 (s, 1H),
8.29 (br s, 1H), 8.45 (s, 1H).
Reaction progress and product mixtures were moni-
tored by thin-layer chromatography (TLC) on silica
gel (precoated F254 Merck plates) and visualized with
aqueous potassium permanganate or a methanolic solu-
1
tion of H2SO4. H NMR were determined in CDCl3 or
DMSO-d6 solutions with a Varian VXR 200 spectrom-
eter or a Varian Mercury Plus 400 spectrometer; peak
positions are given in parts per million (d) 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 analy-
ses were performed by the microanalytical laboratory
of Dipartimento di Chimica, University of Ferrara,
and were within 0.4% of the theoretical values for
C, H and N.
5.2.4. 1-Deoxy-1-[6-{4-[(4-bromo-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-meth-
ylethylidene)-b-D-ribofuranuronamide (7). The product
was purified by column chromatography on silica gel
eluting with a gradient from light petroleum/EtOAc 1:1
to light petroleum/EtOAc 3:7: yield 35%; pale yellow sol-
id; mp 113–114 ꢁC; 1H NMR (200 MHz, CDCl3): d
(ppm) 0.91 (t, 3H, J = 7.3), 1.40 (s, 3H), 1.64 (s, 3H),
3.13 (m, 2H), 4.62 (s, 2H), 4.73 (s, 1H), 5.40 (m, 2H),
6.07 (d, 1H, J = 2.3), 7.01 (br m, 3H), 7.50 (m, 4H),
7.74 (m, 3H), 7.91 (s, 1H), 8.29 (br s, 1H), 8.45 (s, 1H).
5.2. General procedure for the preparation of 1-deoxy-1-
[(2-chloro)-6-{4-[(substituted-phenyl/heteroayl/benzyl/
cycloalkylcarbamoyl)-methoxy]-phenylamino}-9H-purin-
9-yl]-N-ethyl-2,3-O-(1-methylethylidene)-b-D-ribofuranu-
ronamide derivatives 4–23
5.2.5. 1-Deoxy-1-[6-{4-[(4-iodo-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-meth-
ylethylidene)-b-D-ribofuranuronamide (8). The product
was purified by column chromatography on silica gel
eluting with a gradient from light petroleum/EtOAc
1:1 to EtOAc: yield 44%; pale yellow solid; mp 130–
A mixture of the 6-iodo derivatives 2 or 319 (0.17 mmol),
TEA (0.21 mmol) and the appropriate 2-(4-amino-phen-
oxy)-N-substituted-acetamide 76–91 (0.21 mmol) in
absolute EtOH (3 mL) was heated in a steel bomb at
100–110 ꢁC overnight. The solvent was evaporated and
the residue partitioned between H2O (10 mL) and
EtOAc (3 · 20 mL). The organic phase was dried over
anhydrous Na2SO4, filtered and the solvent evaporated
under vacuum to give a residue which was purified by
flash chromatography.
1
131 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 0.90 (t,
3H, J = 7.2), 1.39 (s, 3H), 1.64 (s, 3H), 3.13 (m, 2H),
4.61 (s, 2H), 4.73 (s, 1H), 5.39 (br s, 2H), 6.07 (d, 1H,
J = 2.3), 7.03 (m, 3H), 7.40 (d, 2H, J = 8), 7.64–7.66
(m, 5H), 7.91 (s, 1H), 8.27 (br s, 1H), 8.45 (s, 1H).
5.2.6. 1-Deoxy-1-[6-{4-[(2-trifluoromethyl-phenylcarba-
moyl)-methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-
2, 3-O-(1-methylethylidene)- b-D-ribofuranuronamide (9).
The product was purified by column chromatography
on silica gel eluting with a gradient from light petro-
leum/EtOAc 1:1 to EtOAc: yield 35%; white solid; mp
110–112 ꢁC; 1H NMR (200 MHz, CDCl3): d (ppm)
0.91 (t, 3H, J = 7.2), 1.40 (s, 3H), 1.64 (s, 3H), 3.13
(m, 2H), 4.66 (s, 2H), 4.73 (s, 1H), 5.40 (m, 2H), 6.07
(d, 1H, J = 2.3), 7.01 (m, 3H), 7.56–7.76 (m, 5H), 7.91
(s, 1H), 8.41 (d, 2H, J = 8), 8.45 (s, 1H), 8.97 (br s, 1H).
5.2.1. 1-Deoxy-1-[6-{4-[(phenylcarbamoyl)-methoxy]-phe-
nylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-methylethy-
lidene)-b-D-ribofuranuronamide (4). The product was
purified by column chromatography on silica gel eluting
with light petroleum/EtOAc 1:9: yield 34%; pale yellow
1
solid; mp 215 ꢁC; H NMR (200 MHz, DMSO-d6): d
(ppm) 0.60 (t, 3H, J = 7.2), 1.35 (s, 3H), 1.54 (s, 3H),
2.81 (m, 2H), 4.56 (s, 1H), 4.68 (s, 2H), 5.44 (s, 2H),
6.41 (s, 1H), 6.96–7.11 (m, 3H), 7.28–7.36 (m, 2H),
7.55 (br t, 1H), 7.63–7.81 (m, 4H), 8.29 (s, 1H), 8.41
(s, 1H), 9.85 (s, 1H), 10.09 (s, 1H).
5.2.7. 1-Deoxy-1-[6-{4-[(4-methoxy-phenylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-
methylethylidene)-b-D-ribofuranuronamide (10). The

2520
P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
product was purified by column chromatography on sil-
ica gel eluting with a gradient from light petroleum/
EtOAc 1:1 to light petroleum/EtOAc 3:7: yield 40%;
pale yellow solid; mp 139–140 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 0.91 (t, 3H, J = 7.2), 1.39
(s, 3H), 1.64 (s, 3H), 3.13 (m, 2H), 3.81 (s, 3H), 4.62
(s, 2H), 4.73 (s, 1H), 5.40 (m, 2H), 6.07 (d, 1H,
J = 2.3), 6.89 (d, 2H, J = 8), 7.03 (m, 3H), 7.49 (d, 2H,
J = 8), 7.71 (m, 3H), 7.90 (s, 1H), 8.20 (br s, 1H), 8.45
(s, 1H).
5.2.12. 1-Deoxy-1-[6-{4-[(4-morpholin-4-yl-phenylcarba-
moyl)-methoxy]- phenylamino}-9H-purin-9-yl]-N-ethyl-
2,3-O-(1-methylethylidene)-b-D-ribofuranuronamide (15).
The product was purified by column chromatography
on silica gel eluting with light petroleum/EtOAc 2:8:
yield 25%; pale yellow solid; mp 190 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 0.90 (t, 3H, J = 7.2), 1.39
(s, 3H), 1.63 (s, 3H), 3.08–3.15 (m, 6H), 3.83–3.88 (m,
4H), 4.61 (s, 2H), 4.72 (m, 1H), 5.38 (m, 2H), 6.07 (d,
1H, J = 2.4), 6.88–6.92 (d, 2H, J = 9.0), 7.00–7.05 (d,
2H, J = 9), 7.46–7.50 (d, 2H, J = 8.8), 7.65–7.74 (m,
3H), 7.90 (s, 1H), 8.16 (br s, 1H), 8.44 (s, 1H).
5.2.8. 1-Deoxy-1-[6-{4-[(3,4-dimethoxy-phenylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-
methylethylidene)-b-D-ribofuranuronamide (11). The
product was purified by column chromatography on sil-
ica gel eluting with a gradient from light petroleum/
EtOAc 2:3 to light petroleum/EtOAc 1:4: yield 30%;
pale yellow solid; mp 117 ꢁC; 1H NMR (200 MHz,
CDCl3): d (ppm) 0.91 (t, 3H, J = 7.2), 1.40 (s, 3H),
1.64 (s, 3H), 3.13 (m, 2H), 3.88 (s, 3H), 3.92 (s, 3H),
4.62 (s, 2H), 4.73 (s, 1H), 5.40 (m, 2H), 6.07 (d, 1H,
J = 2.3), 6.82–7.07 (m, 4H), 7.36 (d, 2H, 2.3), 7.73 (m,
3H), 7.91 (s, 1H), 8.20 (br s, 1H), 8.45 (s, 1H).
5.2.13. 1-Deoxy-1-[6-{4-[(pyridin-4-yl-carbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-meth-
ylethylidene)-b-D-ribofuranuronamide (16). The product
was purified by column chromatography on silica gel
eluting with EtOAc: yield 35%; pale yellow solid; mp
1
150 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 0.91 (t,
3H, J = 7.2), 1.39 (s, 3H), 1.65 (s, 3H), 3.18 (m, 2H),
4.60 (s, 2H), 4.75 (s, 1H), 5.38 (m, 2H), 6.09 (d, 1H,
J = 2.3), 7.04 (m, 3H), 7.20 (d, 2H, J = 9.0), 7.40 (d,
2H, J = 9.0), 7.55 (d, 2H, J = 9.0), 7.69 (s, 1H), 7.94
(s, 1H), 8.26 (br s, 1H), 8.45 (s, 1H).
5.2.9.
1-Deoxy-1-[6-{4-[(3,5-dimethoxy-phenylcarba-
moyl)-methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-
2,3-O-(1-methylethylidene)-b-D-ribofuranuronamide (12).
The product was purified by column chromatography
on silica gel eluting with a gradient from light petro-
leum/EtOAc 1:1 to EtOAc: yield 25%; white solid; mp
121–122 ꢁC; 1H NMR (200 MHz, CDCl3): d (ppm)
0.91 (t, 3H, J = 7.2), 1.40 (s, 3H), 1.64 (s, 3H), 3.13
(m, 2H), 3.80 (s, 6H), 4.61 (s, 2H), 4.73 (s, 1H), 5.40
(m, 2H), 6.07 (d, 1H, J = 2.3), 6.28 (m, 1H), 6.83 (m,
2H), 7.01–7.06 (m, 3H), 7.71 (s, 2H), 7.75 (s, 1H), 7.91
(s, 1H), 8.23 (br s, 1H), 8.45 (s, 1H).
5.2.14. 1-Deoxy-1-[6-{4-[(benzylcarbamoyl)-methoxy]-
phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-methyl-
ethylidene)-b-D-ribofuranuronamide (17). The product
was purified by column chromatography on silica gel
eluting with EtOAc: yield 37%; pale yellow solid; mp
1
118 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm) 0.60
(t, 3H, J = 7.2), 1.35 (s, 3H), 1.54 (s, 3H), 2.82 (m,
2H), 4.35 (d, 2H, J = 6.2), 4.55 (m, 3H), 5.44 (s, 2H),
6.41 (s, 1H), 6.95 (m, 2H), 7.21–7.35 (m, 5H), 7.54 (br
t, 1H), 7.78 (m, 2H), 8.29 (s, 1H), 8.41 (s, 1H), 8.65
(br t, 1H), 9.84 (br t, 1H).
5.2.10.
1-Deoxy-1-[6-{4-[(Benzo[1,3]dioxol-5-ylcarba-
5.2.15. 1-Deoxy-1-[6-{4-[(4-methoxy-benzylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-
methylethylidene)-b-D-ribofuranuronamide (18). The
product was purified by column chromatography on sil-
ica gel eluting with EtOAc: yield 38%; pale yellow solid;
moyl)-methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-
2,3-O-(1-methylethylidene)- b-D-ribofuranuronamide (13).
The product was purified by column chromatography
on silica gel eluting with a gradient from light petro-
leum/EtOAc 1:1 to EtOAc: yield 48%; white solid; mp
1
mp 117 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm)
1
145 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 0.90 (t,
0.61 (t, 3H, J = 7.2), 1.35 (s, 3H), 1.54 (s, 3H), 2.78
(m, 2H), 3.71 (s, 3H), 4.27 (br s, 2H), 4.51 (s, 2H),
4.56 (s, 1H), 5.44 (s, 2H), 6.41 (s, 1H), 6.83–6.97 (m,
4H), 7.18 (d, 2H, J = 8.4), 7.55 (br t, 1H), 7.75–7.81
(m, 2H), 8.30 (s, 1H), 8.41 (s, 1H), 8.57 (br t, 1H),
9.80 (br s, 1H).
3H, J = 7.2), 1.39 (s, 3H), 1.64 (s, 3H), 3.13 (m, 2H),
4.61 (s, 2H), 4.73 (s, 1H), 5.39 (m, 2H), 5.97 (s, 2H),
6.07 (d, 1H, J = 2.3), 6.75–6.91 (m, 2H), 7.30 (m, 3H),
7.31 (d, 1H, J = 2), 7.70 (s, 2H), 7.75 (s, 1H), 7.91 (s,
1H), 8.19 (br s, 1H), 8.45 (s, 1H).
5.2.11. 1-Deoxy-1-[6-{4-[(4-tert-butyl-phenylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-
methylethylidene)-b-D-ribofuranuronamide (14). The
product was purified by column chromatography on
silica gel eluting with a gradient from light petro-
leum/EtOAc 1:4 to EtOAc: yield 32%; white solid;
mp 130 ꢁC; 1H NMR (200 MHz, DMSO): d (ppm)
0.61 (t, 3H, J = 7.4), 1.26 (s, 9H), 1.35 (s, 3H), 1.54
(s, 3H), 2.80 (m, 2H), 4.56 (s, 1H), 4.66 (s, 2H), 5.44
(s, 2H), 6.41 (s, 1H), 6.98 (d, 2H, J = 9.4), 7.33 (d,
2H, J = 8.6), 7.55 (d, 2H, J = 8.6), 7.78 (d, 2H,
J = 9.0), 8.29 (s, 1H), 8.41 (s, 1H), 9.84 (s, 1H), 10.01
(s, 1H).
5.2.16. 1-Deoxy-1-[6-{4-[(cyclohexylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-meth-
ylethylidene)-b-D-ribofuranuronamide (19). The product
was purified by column chromatography on silica gel
eluting with light petroleum/EtOAc 2:8: yield 45%; pale
yellow solid; mp 132–135 ꢁC; 1H NMR (200 MHz,
DMSO-d6): d (ppm) 0.61 (t, 3H, J = 7.2), 1.20–1.30
(br m, 6H), 1.35 (s, 3H), 1.54 (s, 3H), 1.68-1.73 (br
m, 4H), 2.78 (m, 2H), 3.60 (br s, 1H), 4.42 (s, 2H),
4.56 (s, 1H), 5.44 (s, 2H), 6.41 (s, 1H), 6.93 (d, 2H,
J = 9.4), 7.55 (br t, 1H), 7.77 (d, 2H, J = 9.2), 7.86
(d, 1H, J = 8.00), 8.29 (s, 1H), 8.41 (s, 1H), 9.82
(s, 1H).

P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
2521
5.2.17. 1-Deoxy-1-[2-chloro-6-{4-[(phenylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-
methylethylidene)-b-D-ribofuranuronamide (20). The
product was purified by column chromatography on sil-
ica gel eluting with a gradient from light petroleum/
EtOAc 7:3 to EtOAc: yield 47%; white solid; mp 148–
5.3.1. 1-Deoxy-1-[6-{4-[(phenylcarbamoyl)-methoxy]-phe-
nylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribofuranuron-
amide (24). The product was purified by column
chromatography on silica gel eluting with EtOAc/
CH3OH 9:1: yield 63%; white solid; mp 145–146 ꢁC;
1H NMR (200 MHz, DMSO-d6): d (ppm) 1.09 (t, 3H,
J = 7.2), 3.20 (m, 2H), 4.16 (m, 1H), 4.33 (s, 1H), 4.64
(m, 3H) 5.63 (d, 1H, J = 6.4), 5.79 (d, 1H, J = 4.4),
6.03 (d, 1H, J = 7.2), 6.98–7.13 (m, 3H), 7.29–7.37 (m,
2H), 7.66 (d, 2H, J = 8.00), 7.80 (d, 2H, J = 8.00), 8.39
(s, 1H), 8.58 (s, 1H), 8.79 (br t, 1H), 9.96 (s, 1H),
10.11 (s, 1H). Anal. (C26H27N7O6) C, H, N.
1
150 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm) 0.66
(t, 3H, J = 7.2), 1.35 (s, 3H), 1.54 (s, 3H), 2.84 (m,
2H), 4.56 (s, 1H), 4.70 (s, 2H), 5.39 (s, 2H), 6.35 (s,
1H), 6.99–7.11 (m, 3H), 7.28–7.36 (m, 2H), 7.55–7.71
(m, 5H), 8.39 (s, 1H), 10.09 (s, 1H), 10.30 (br s, 1H).
5.2.18. 1-Deoxy-1-[2-chloro-6-{4-[(4-methoxy-phenylcar-
bamoyl)-methoxy]- phenylamino}-9H-purin-9-yl]-N-ethyl-
2,3-O-(1-methylethylidene)-b-D-ribofuranuronamide (21).
The product was purified by column chromatography on
silica gel eluting with EtOAc: yield 44%; pale yellow solid;
mp 83–84 ꢁC; 1H NMR (200 MHz, DMSO-d6): d (ppm)
0.67 (t, 3H, J = 7.2), 1.35 (s, 3H), 1.55 (s, 3H), 2.85 (m,
2H), 3.70 (s, 3H), 4.52 (s, 1H), 4.73 (s, 2H), 5.41 (m,
2H), 6.09 (d, 1H, J = 2.3), 6.75–6.98 (m, 4H), 7.33–7.45
(m, 4H), 7.70 (s, 1H), 7.91 (br t, 1H), 8.38 (br s, 1H),
10.02 (s, 1H).
5.3.2. 1-Deoxy-1-[6-{4-[(4-fluoro-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribofuranu-
ronamide (25). The product was purified by column
chromatography on silica gel eluting with EtOAc/
CH3OH 9:1: yield 85%; white solid; mp 274–275 ꢁC;
1H NMR (200 MHz, DMSO-d6): d (ppm) 1.08 (t, 3H,
J = 7.0), 3.23 (m, 2H), 4.16 (m, 1H), 4.33 (m, 1H),
4.63 (m, 3H), 5.63 (d, 1H, J = 6.4), 5.80 (d, 1H,
J = 4.00), 6.03 (d, 1H, J = 7.2), 7.00 (d, 2H, J = 9),
7.13–7.23 (m, 2H), 7.65–7.84 (m, 4H), 8.39 (s, 1H),
8.58 (s, 1H), 8.78 (br t, 1H), 10.00 (s, 1H). Anal.
(C26H26FN7O6) C, H, N.
5.2.19. 1-Deoxy-1-[2-chloro-6-{4-[(benzylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-methyl-
ethylidene)-b-D-ribofuranuronamide (22). The product
was purified by column chromatography on silica gel elut-
ing with light petroleum/EtOAc 1:4: yield 43%; pale yel-
low solid; yield 53%; pale yellow solid; mp 121–122 ꢁC;
1H NMR (200 MHz, DMSO-d6): d (ppm) 0.66 (t, 3H,
J = 7.2), 1.35 (s, 3H), 1.54 (s, 3H), 2.84 (m, 2H), 4.35 (d,
2H, J = 6.0), 4.55 (s, 3H), 5.40 (s, 2H), 6.36 (s, 1H), 6.98
(d, 2H, J = 8.00), 7.26–7.35 (m, 5H), 7.57–7.70 (m, 3H),
8.40 (s, 1H), 8.66 (br t, 1H), 10.27 (s, 1H).
5.3.3. 1-Deoxy-1-[6-{4-[(4-chloro-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribofuranu-
ronamide (26). The product was purified by column
chromatography on silica gel eluting with a mixture of
CH2Cl2/CH31OH 9.5:0.5: yield 80%; white solid; mp
279–280ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm)
1.17 (t, 3H, J = 7.2), 3.35 (m, 2H), 4.25 (m, 1H), 4.44
(s, 1H), 4.64 (m, 3H), 5.44 (m, 1H), 5.71 (m, 1H), 6.04
(d, 1H, J = 7.4), 7.10 (d, 2H, J = 8), 7.28 (d, 2H, J = 8),
7.70 (d, 2H, J = 8), 7.83 (d, 2H, J = 8), 8.32 (s, 1H),
8.36 (s, 1H), 8.99 (br t, 1H), 9.72 (s, 1H), 10.01 (s, 1H).
Anal. (C26H26ClN7O6) C, H, N.
5.2.20. 1-Deoxy-1-[2-chloro-6-{4-[(cyclohexylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-2,3-O-(1-
methylethylidene)-b-D-ribofuranuronamide (23). The prod-
uct was purified by column chromatography on silica gel
eluting with light petroleum/EtOAc 1:4:yield 43%;paleyel-
5.3.4. 1-Deoxy-1-[6-{4-[(4-bromo-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribofuranu-
ronamide (27). The product was purified by column chro-
matography on silica gel eluting with a mixture of
CH2Cl2/CH3OH 9.5:0.5: yield 45%; white solid; mp
1
low solid; mp 120–121 ꢁC; H NMR (200 MHz, DMSO-
d6): d (ppm) 0.66 (t, 3H, J = 7.2), 1.13–1.25 (br m, 6H),
1.35 (s, 3H), 1.54 (s, 3H), 1.68 (br m, 4H), 2.85 (m, 2H),
3.60 (br s, 1H), 4.44 (s, 2H), 4.57 (s, 1H), 5.39 (s, 2H),
6.35 (s, 1H), 6.95 (d, 2H, J = 9.0), 7.60 (br t, 1H), 7.67 (d,
2H, J = 9.2), 7.90 (d, 1H), 8.39 (s, 1H), 10.25 (s, 1H).
1
272 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm) 1.08
(t, 3H, J = 7.2), 3.21 (m, 2H), 4.17 (br m, 1H), 4.32 (m,
1H), 4.63 (br m, 1H), 4.69 (s, 2H), 5.60 (d, 1H,
J = 6.4), 5.77 (d, 1H, J = 4.3), 6.02 (d, 1H, J = 7.4),
7.00 (d, 2H, J = 8), 7.50 (d, 2H, J = 8), 7.65 (d, 2H,
J = 8), 7.80 (d, 2H, J = 8), 8.38 (s, 1H), 8.57 (s, 1H),
8.78 (br t, 1H), 9.95 (s, 1H), 10.25 (s, 1H). Anal.
(C26H26BrN7O6) C, H, N.
5.3. General procedure for the preparation of 1-deoxy-1-
[(2-chloro)-6-{4-[(substituted-phenyl/heteroayl/benzyl/cyclo-
alkylcarbamoyl)-methoxy]-phenylamino}-9H-purin-9-yl]-
N-ethyl-b-D-ribofuranuronamide derivatives 24–43
The appropriate isopropylidene derivative 4–23
(0.6 mmol) was dissolved in a mixture of trifluoroacetic
acid and water 1:1 (4 mL) and the resulting solution was
stirred at room temperature for 3 h. The solvents were
evaporated at reduced pressure to give a residue which
was suspended with water (15 mL) and extracted with
EtOAc (3 · 15 mL). The organic layer was dried over
Na2SO4, filtered and the solvent evaporated under vac-
uum. Purification by flash chromatography afforded
the desired unprotected compounds 24–43.
5.3.5. 1-Deoxy-1-[6-{4-[(4-iodo-phenylcarbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribofuranu-
ronamide (28). The product was purified by column
chromatography on silica gel eluting with a mixture of
CH2Cl2/CH3OH 9.5:0.5: yield 70%; white solid; mp
1
284-285ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm)
1.22 (t, 3H, J = 7.2), 3.35 (m, 2H), 4.31 (m, 1H), 4.51
(s, 1H), 4.62 (s, 2H), 4.74 (br m, 1H), 5.60 (br s, 1H),
5.70 (br s, 1H), 6.01 (d, 1H, J = 7.4), 7.02 (d, 2H,
J = 8), 7.49–7.63 (m, 4H), 7.83 (d, 2H, J = 8), 8.15 (s,

2522
P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
1H), 8.38 (s, 1H), 9.08 (br t, 1H), 9.41 (s, 1H), 9.68 (s,
1H). Anal. (C26H26IN7O6) C, H, N.
1H), 4.35 (s, 1H), 4.59 (m, 3H), 5.48 (m, 1H), 5.71 (m,
1H), 5.93 (s, 2H), 6.02 (d, 1H, J = 7.4), 6.74 (d, 1H,
J = 8), 6.92–7.05 (m, 3H), 7.31 (m, 1H), 7.81 (d, 2H,
J = 8), 8.32 (s, 1H), 8.41 (s, 1H), 8.86 (br t, 1H), 9.81
(s, 1H), 9.85 (s, 1H). Anal. (C27H27N7O8) C, H, N.
5.3.6. 1-Deoxy-1-[6-{4-[(2-trifluoromethyl-phenylcarba-
moyl)-methoxy]- phenylamino}-9H-purin-9-yl]-N-ethyl-b-
D-ribofuranuronamide (29). The product was purified by
column chromatography on silica gel eluting with a mix-
ture of CH21Cl2/CH3OH 9.5:0.5: yield 55%; white solid;
mp 222ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm)
1.20 (t, 3H, J = 7.2), 3.38 (m, 2H), 4.26 (m, 1H), 4.46
(s, 1H), 4.70 (br s, 3H), 5.40 (m, 1H), 5.68 (m, 1H),
6.05 (d, 1H, J = 7.4), 7.02 (d, 2H, J = 8), 7.40 (m, 1H),
7.61–7.71 (m, 2H), 7.89 (d, 2H, J = 8), 8.10 (d, 1H,
J = 8), 8.28 (s, 1H), 8.37 (s, 1H), 9.01 (br t, 1H), 9.25
(s, 1H), 9.70 (s, 1H). Anal. (C27H26F3N7O6) C, H, N.
5.3.11. 1-Deoxy-1-[6-{4-[(4-tert-butyl-phenylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribo-
furanuronamide (34). The product was purified by col-
umn chromatography on silica gel eluting with
EtOAc/CH3OH 9.5:0.5: yield 78%; white solid; mp
155–156 ꢁC; 1H NMR (200 MHz, CDCl3): d (ppm)
1.08 (t, 3H, J = 7.2), 1.26 (s, 9H), 3.20 (m, 2H), 4.20
(m, 1H), 4.32 (m, 1H), 4.67 (br m, 3H), 5.60 (br d,
1H), 5.80 (br d, 1H), 6.02 (d, 1H, J = 7.2), 6.99 (d,
2H, J = 9.0), 7.33 (d, 2H, J = 8.4), 7.56 (d, 2H,
J = 8.8), 7.81 (d, 2H, J = 9.0), 8.39 (s, 1H), 8.57 (s,
1H), 8.80 (br t, 1H), 9.89 (s, 1H), 10.10 (s, 1H). Anal.
(C30H35N7O6) C, H, N.
5.3.7. 1-Deoxy-1-[6-{4-[(4-methoxy-phenylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribo-
furanuronamide (30). The product was purified by
column chromatography on silica gel eluting with a
mixture of CH2Cl2/CH3OH 9.5:0.5: yield 65%; white
solid; mp 249–250 ꢁC; 1H NMR (200 MHz, DMSO-
d6): d (ppm) 1.11 (t, 3H, J = 7.2), 3.21 (m, 2H), 3.72
(s, 3H), 4.17 (br t, 1H), 4.34 (m, 1H), 4.58-4.67 (m,
3H), 5.49 (d, 1H, J = 6.4), 5.71 (d, 1H, J = 4.3), 6.01
(d, 1H, J = 7.4), 6.84 (d, 2H, J = 8), 6.96 (d, 2H,
J = 8), 7.53 (d, 2H, J = 8), 7.80 (d, 2H, J = 8), 8.33 (s,
1H), 8.44 (s, 1H), 8.84 (br t, 1H), 9.82 (br s, 2H). Anal.
(C27H29N7O7) C, H, N.
5.3.12. 1-Deoxy-1-[6-{4-[(4-morpholin-4-yl-phenylcarba-
moyl)-methoxy]- phenylamino}-9H-purin-9-yl]-N-ethyl-b-
D-ribofuranuronamide (35). The product was purified by
column chromatography on silica gel eluting with
EtOAc/CH3OH 9:1: yield 50%; pale yellow solid; mp
1
274 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm) 1.08
(t, 3H, J = 7.4), 3.02–3.07 (m, 4H), 3.20 (m, 2H), 3.70–
3.75 (m, 4H), 4.20 (m, 1H), 4.32 (m, 1H), 4.64 (br m,
3H), 5.60 (br m, 1H), 5.80 (br m, 1H), 6.03 (d, 1H,
J = 7.2), 6.90 (d, 2H, J = 8.8), 6.99 (d, 2H, J = 8.8),
7.51 (d, 2H, J = 9.2), 7.81 (d, 2H, J = 9.0), 8.39 (s,
1H), 8.57 (s, 1H), 8.80 (br m, 1H), 9.90 (br s, 1H),
10.00 (br s, 1H). Anal. (C30H34N8O7) C, H, N.
5.3.8. 1-Deoxy-1-[6-{4-[(3,4-dimethoxy-phenylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-b-D-ribofuranu-
ronamide (31). The product was purified by column chro-
matography on silica gel eluting with a mixture of
CH2Cl2/CH3OH 9.5:0.5: yield 60%; white solid; mp
5.3.13. 1-Deoxy-1-[6-{4-[(pyridin-4-yl-carbamoyl)-meth-
oxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribofuranu-
ronamide (36). The product was purified by crystallization
from EtOH: yield 90%; pale yellow solid; mp 145 ꢁC; 1H
NMR (200 MHz, DMSO-d6): d (ppm) 1.08 (t, 3H,
J = 7.4), 3.20 (m, 2H), 4.16 (m, 1H), 4.33 (m, 1H), 4.62
(m, 1H), 4.76 (s, 2H), 5.60 (br m, 1H), 5.80 (br m, 1H),
6.02 (d, 1H, J = 7.6), 6.99 (d, 2H, J = 9), 7.69–7.84 (m,
4H), 8.39 (s, 1H), 8.48 (m, 2H), 8.58 (s, 1H), 8.77 (br t,
1H), 10.00 (s, 1H), 10.60 (s, 1H). Anal. (C25H26N8O6)
C, H, N.
1
240 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm) 1.08
(t, 3H, J = 7.2), 3.21 (m, 2H), 3.71 (s, 3H), 3.72 (s, 3H),
4.17 (br m, 1H), 4.32 (m, 1H), 4.65 (br s, 3H), 5.60 (d,
1H, J = 6.4), 5.78 (d, 1H, J = 4.3), 6.02 (d, 1H, J = 7.4),
6.87–7.02 (m, 3H), 7.18 (m, 1H), 7.34 (m, 1H), 7.80 (d,
2H, J = 8), 8.39 (s, 1H), 8.57 (s, 1H), 8.78 (br t, 1H),
9.95 (s, 1H), 10.25 (s, 1H). Anal. (C28H31N7O8) C, H, N.
5.3.9. 1-Deoxy-1-[6-{4-[(3,5-dimethoxy-phenylcarba-
moyl)-methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-
D-ribofuranuronamide (32). The product was purified by
column chromatography on silica gel eluting with a
mixture of CH2Cl2/CH3OH 9.5:0.5: yield 50%; white
solid; mp 219–220ꢁC; 1H NMR (200 MHz, DMSO-
d6): d (ppm) 1.20 (t, 3H, J = 7.2), 3.35 (m, 2H), 3.76
(s, 6H), 4.28 (m, 1H), 4.48 (s, 1H), 4.61 (s, 2H), 4.73
(m, 1H), 5.33 (m, 1H), 5.65 (m, 1H), 6.03 (d, 1H,
J = 7.4), 6.20 (m,1H), 6.93–7.02 (m, 4H), 7.82 (d, 2H,
J = 8), 8.21 (s, 1H), 8.36 (s, 1H), 9.04 (br t, 1H), 9.57
(s, 1H), 9.62 (s, 1H). Anal. (C28H31N7O8) C, H, N.
5.3.14. 1-Deoxy-1-[6-{4-[(benzylcarbamoyl)-methoxy]-
phenylamino}-9H-purin- 9-yl]-N-b-D-ribofuranuronamide
(37). The product was purified by column chromatogra-
phy on silica gel eluting with EtOAc/CH3OH 9:1: yield
82%; white; mp 129–130 ꢁC; 1H NMR (200 MHz,
DMSO-d6): d (ppm) 1.08 (t, 3H, J = 7.2), 3.21 (m,
2H), 4.18 (br t, 1H), 4.36 (br d, 3H), 4.54 (s, 2H), 4.62
(m, 1H), 5.61 (d, 1H), 5.79 (d, 1H), 6.05 (d, 1H), 6.96
(d, 2H, J = 8.00), 7.28 (m, 5H), 7.80 (d, 2H, J = 8.00),
8.39 (s, 1H), 8.57 (s, 1H), 8.65 (br t, 1H), 8.8 (br t,
1H), 9.94 (s,1H). Anal. (C27H29N7O6) C, H, N.
5.3.10. 1-Deoxy-1-[6-{4-[(Benzo[1,3]dioxol-5-ylcarba-
moyl)-methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-
D-ribofuranuronamide (33). The product was purified by
column chromatography on silica gel eluting with a mix-
ture of CH2Cl2/CH3OH 9.5:0.5: yield 75%; white solid;
mp 270–272 ꢁC; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 1.11 (t, 3H, J = 7.2), 3.25 (m, 2H), 4.17 (m,
5.3.15. 1-Deoxy-1-[6-{4-[(4-methoxy-benzylcarbamoyl)-
methoxy]-phenylamino}- 9H-purin-9-yl]-N-ethyl-b-D-ribo-
furanuronamide (38). The product was purified by column
chromatography on silica gel eluting with EtOAc/CH3OH
1
9:1: yield 80%; white solid; mp 115–116 ꢁC; H NMR

P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
2523
(200 MHz, DMSO-d6): d (ppm) 1.08 (t, 3H, J = 7.2), 3.22
(m, 2H), 3.71 (s, 3H), 4.16 (br d, 1H), 4.28 (d, 2H,
J = 5.8), 4.33 (m, 1H), 4.52 (s, 2H), 4.65 (m, 1H), 5.60
(br d, 1H), 5.80 (br d, 1H), 6.03 (d, 1H, J = 7.4), 6.84–
6.98 (m, 4H), 7.18 (d, 2H, J = 8.6), 7.78 (d, 2H, J = 8.4),
8.39 (s, 1H), 8.57 (br m, 2H), 8.78 (br t, 1H), 10.00 (s,
1H). Anal. (C28H31N7O7) C, H, N.
1H NMR (200 MHz, DMSO-d6): d (ppm) 1.02–1.25
(m, 9H), 1.54–1.80 (br m, 4H), 3.21 (m, 2H), 3.61 (br
s, 1H), 4.18 (br s, 1H), 4.33 (s, 1H), 4.44 (s, 2H),
4.59 (br m, 1H), 5.66 (br d, 1H), 5.79 (br d, 1H),
5.97 (d, 1H, J = 7.00), 6.96 (d, 2H, J = 8.8), 7.67 (d,
2H, J = 9.2), 7.88 (d, 1H, J = 8.00), 8.35 (br t, 1H),
8.62 (s, 1H), 10.32 (br s, 1H). Anal. (C26H32ClN7O6)
C, H, N.
5.3.16. 1-Deoxy-1-[6-{4-[(cyclohexylcarbamoyl)-methoxy]-
phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribofuranuro-
namide (39). The product was purified by column chro-
matography on silica gel eluting with EtOAc/CH3OH
5.4. General procedure for the preparation of 2-chloro-N-
substituted-acetamides 44–5926
1
9:1: yield 88%; white solid; mp 211–212 ꢁC; H NMR
Chloroacetyl chloride (33 mmol) was added dropwise to
a mixture of the appropriate amine (28 mmol) and
K2CO3 (33 mmol) in CH2Cl2 (50 mL) at room tempera-
ture. The reaction mixture was refluxed for 4h, then,
after cooling to room temperature, it was slowly poured
into 100 mL of ice water. The aqueous solution was
extracted with CH2Cl2 (2 · 100 mL), the organic phase
was dried over Na2SO4, filtered and the solvent was
evaporated to furnish a solid residue which was purified
(200 MHz, DMSO-d6): d (ppm) 1.02–1.27 (m, 9H),
1.51–1.70 (br m, 4H), 3.16 (m, 2H), 3.59 (br s, 1H),
4.20 (br m, 1H), 4.30 (s, 1H), 4.40 (s, 2H), 4.62 (br m,
1H), 5.58 (d, 1H, J = 6.4), 5.75 (d, 1H, J = 4.2), 5.99
(d, 1H, J = 7.40), 6.90 (d, 2H, J = 8.00), 7.73–7.87 (m,
3H), 8.36 (s, 1H), 8.54 (s, 1H), 8.75 (br t, 1H), 9.90 (s,
1H). Anal. (C26H33N7O6) C, H, N.
5.3.17. 1-Deoxy-1-[2-chloro-6-{4-[(phenylcarbamoyl)-meth-
oxy]-phenylamino}- 9H-purin-9-yl]-N-ethyl-b-D-ribofuranu-
ronamide (40). The product was purified by crystallization
from EtOAc: yield 82%; white solid; mp 261–262 ꢁC; H
NMR (200 MHz, DMSO-d6): d (ppm) 1.05 (t, 3H,
J = 7.2), 3.21 (m, 2H), 4.17 (m, 1H), 4.32 (m, 1H), 4.58
(m, 1H), 4.70 (s, 2H), 5.63 (d, 1H, J = 6.00), 5.75 (d,
1H, J = 4.6), 5.97 (d, 1H, J = 7.2), 7.00–7.12 (m, 3H),
7.29–7.37 (m, 2H), 7.63–7.78 (m, 4H), 8.34 (br t, 1H),
8.62 (s, 1H), 10.10 (s, 1H), 10.36 (s, 1H). Anal.
(C26H26ClN7O6) C, H, N.
by crystallization from
petroleum.
a mixture of Et2O/light
1
5.4.1. 2-Chloro-N-phenyl-acetamide (44). Yield 90%;
white solid; mp 134-137 ꢁC; 1H NMR (200 MHz,
CDCl3): d (ppm) 4.21 (s, 2H), 7.20 (m, 1H), 7.39 (m,
2H), 7.60 (m, 2H), 8.55 (br s, 1H).
5.4.2. 2-Chloro-N-(4-fluoro-phenyl)-acetamide (45). Yield
1
85%; white solid; mp 130–131 ꢁC; H NMR (200 MHz,
CDCl3): d (ppm) 4.19 (s, 2H), 7.01–7.09 (m, 2H), 7.47–
7.54 (m, 2H), 8.21 (br s, 1H).
5.3.18. 1-Deoxy-1-[2-chloro-6-{4-[(4-methoxy-phenylcar-
bamoyl)-methoxy]- phenylamino}-9H-purin-9-yl]-N-ethyl-
b-D-ribofuranuronamide (41). The product was purified
by column chromatography on silica gel eluting with a
mixture EtOAc/CH3OH 9.5:0.5: yield 73%; white solid;
mp 264–265 ꢁC; 1H NMR (200 MHz, DMSO-d6): d
(ppm) 1.05 (t, 3H, J = 7.2), 3.23 (m, 2H), 3.72 (s, 3H),
4.14 (br m, 2H), 4.33 (m, 1H), 4.62 (br m, 2H), 5.60–
5.80 (br m, 2H), 5.97 (d, 1H, J = 6.4), 6.88–7.05 (m,
4H), 7.54–7.73 (m, 4H), 8.34 (br t, 1H), 8.62 (s, 1H),
9.96 (s, 1H), 10.35 (s, 1H). Anal. (C27H28ClN7O7) C,
H, N.
5.4.3. 2-Chloro-N-(4-chloro-phenyl)-acetamide (46).
Yield 60%; pale yellow solid; mp 170–171 ꢁC; 1H
NMR (200 MHz, DMSO-d6): d (ppm) 4.26 (s, 2H),
7.39 (d, 2 H, J = 8), 7.61 (d, 2H, J = 8), 10.45 (br s,
1H).
5.4.4. 2-Chloro-N-(4-bromo-phenyl)-acetamide (47).
Yield 90%; grey solid; mp 180 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 4.19 (s, 2H), 7. 47 (m,
4H), 8.24 (br s, 1H).
5.4.5. 2-Chloro-N-(4-iodo-phenyl)-acetamide (48). Yield
1
5.3.19. 1-Deoxy-1-[2-chloro-6-{4-[(benzylcarbamoyl)-meth-
oxy]-phenylamino}- 9H-purin-9-yl]-N-ethyl-b-D-ribofuranu-
ronamide (42). The product was purified by column
chromatography on silica gel eluting with EtOAc/CH3OH
65%; white solid; mp 194–195 ꢁC; H NMR (200 MHz,
DMSO-d6): d (ppm) 4.25 (s, 2H), 7.42 (d, 2 H, J = 8),
7.68 (d, 2H, J = 8), 10.41 (br s, 1H).
1
9:1: yield 87%; white solid; mp 235–236 ꢁC; H NMR
5.4.6. 2-Chloro-N-(2-trifluoromethyl-phenyl)-acetamide
1
(200 MHz, DMSO-d6): d (ppm) 1.05 (t, 3H, J = 7.2),
3.22 (m, 2H), 4.18 (br s, 1H), 4.35 (m, 3H), 4.56 (s, 3H),
5.68 (br d, 1H), 5.80 (br d, 1H), 5.98 (d, 1H, J = 6),
7.00 (d, 2H, J = 9.2), 7.22–7.32 (m, 5H), 7.70 (d, 2H,
J = 8.8), 8.35 (t, 1H, J = 6.00), 8.68 (m, 2H), 10.35 (s,
1H). Anal. (C27H28ClN7O6) C, H, N.
(49). Yield 80%; white solid; mp 94–95 ꢁC; H NMR
(200 MHz, CDCl3): d (ppm) 4.24 (s, 2H), 7.26–7.68
(m, 3H), 8.22 (d, 1H, J = 8), 8.76 (br s, 1H).
5.4.7. 2-Chloro-N-(4-methoxy-phenyl)-acetamide (50).
Yield 82%; white solid; mp 124 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 3.80 (s, 3H), 4.17 (s, 2H),
6.90 (d, 2H, J = 8), 7.43 (d, 2H, J = 8), 8.17 (br s, 1H).
5.3.20. 1-Deoxy-1-[2-chloro-6-{4-[(cyclohexylcarbamoyl)-
methoxy]-phenylamino}-9H-purin-9-yl]-N-ethyl-b-D-ribo-
furanuronamide (43). The product was purified by col-
umn chromatography on silica gel eluting with EtOAc/
CH3OH 4:1: yield 93%; white solid; mp 275–276 ꢁC;
5.4.8.
2-Chloro-N-(3,4-dimethoxy-phenyl)-acetamide
(51). Yield 73%; white solid; mp 134 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 3.87 (s, 3H), 3.89 (s, 3H),

2524
P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
4.18 (s, 2H), 6.80–6.98 (m, 2H), 7.27 (m, 1H), 8.17 (br s,
1H).
7.08–7.37 (m, 5H), 7.60 (m, 2H), 8.23 (d, 2H, J = 8),
10.20 (br s, 1H).
5.4.9.
2-Chloro-N-(3,5-dimethoxy-phenyl)-acetamide
5.5.2. N-(4-Fluoro-phenyl)-2-(4-nitro-phenoxy)-acetamide
1
1
(52). Yield 83%; white solid; mp 97–98 ꢁC; H NMR
(200 MHz, CDCl3): d (ppm) 3.77 (s, 6H), 4.14 (s, 2H),
6.23 (m, 1H), 6.85 (m, 2H), 9.48 (br s, 1 H).
(61). Yield 73%; pale yellow solid; mp 159–160 ꢁC; H
NMR (200 MHz, CDCl3): d (ppm) 4.71 (s, 2H), 7.02–
7.12 (m, 4H), 7.51–7.58 (m, 2H), 8.11 (br s, 1H), 8.28
(m, 2H).
5.4.10. N-Benzo[1,3]dioxol-5-yl-2-chloro-acetamide (53).
Yield 74%; white solid; mp 157–158 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 4.18 (s, 2H), 5.97 (s, 2H),
6.74–6.87 (m, 2H), 7.23 (m, 1H), 8.14 (br s, 1H).
5.5.3. N-(4-Chloro-phenyl)-2-(4-nitro-phenoxy)-acetam-
1
ide (62). Yield 65%; pale yellow solid; mp 181 ꢁC; H
NMR (200 MHz, CDCl3): d (ppm) 4.75 (s, 2 H), 7.06
(d, 2H, J = 8), 7.20 (d, 2H, J = 8), 7.58 (d, 2H, J = 8),
8.16 (d, 2H, J = 8), 10.03 (br s, 1H).
5.4.11. 2-Chloro-N-(4-tert-butyl-phenyl)-acetamide (54).
Yield 79%; white solid; mp 87–90 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 1.31 (s, 9H), 4.18 (s, 2H),
7.34–7.48 (m, 4H), 8.18 (br s, 1H).
5.5.4. N-(4-Bromo-phenyl)-2-(4-nitro-phenoxy)-acetam-
ide (63). Yield 70%; pale yellow solid; mp 120–121 ꢁC;
1H NMR (200 MHz, CDCl3): d (ppm) 4.70 (s, 2 H),
7.10 (d, 2H, J = 8), 7. 49 (m, 4H), 8.26 (br s, 1H), 8.30
(d, 2H, J = 8).
5.4.12. 2-Chloro-N-(4-morpholin-4-yl-phenyl)-acetamide
1
(55). Yield 70%; white solid; mp 170–172 ꢁC; H NMR
(200 MHz, CDCl3): d (ppm) 3.07 (br t, 4H), 3.80 (br t,
4H), 4.12 (s, 2H), 6.84 (d, 2H, J = 8.8), 7.37 (d, 2H,
J = 8.2), 8.06 (br s, 1H).
5.5.5. N-(4-Iodo-phenyl)-2-(4-nitro-phenoxy)-acetamide
(64). Yield 62%; pale yellow solid; mp 155 ꢁC; 1H
NMR (200 MHz, DMSO-d6): d (ppm) 4.90 (s, 2H),
7.19 (d, 2H, J = 8), 7.46 (d, 2H, J = 8), 7.68 (d, 2H,
J = 8), 8.23 (d, 2H, J = 8), 10.32 (br s, 1H).
5.4.13. 2-Chloro-N-pyridin-4-yl-acetamide (56). Yield
1
58%; white solid; mp 125–127 ꢁC; H NMR (200 MHz,
CDCl3): d (ppm) 4.19 (s, 2H), 8.03 (d, 2H, J = 7.2),
8.49 (d, 2H, J = 7.00), 11.53 (s, 1H).
5.5.6. 2-(4-Nitro-phenoxy)-N-(2-trifluoromethyl-phenyl)-
acetamide (65). Yield 68%; pale yellow solid; mp 136-
1
5.4.14. N-Benzyl-2-chloro-acetamide (57). Yield 90%;
white solid; mp 95–96 ꢁC; 1H NMR (200 MHz, CDCl3):
d (ppm) 4.04 (s, 2H), 4.43 (d, 2H, J = 6), 6.90 (br s, 1H),
7.26 (m, 5H).
137 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 4.75 (s,
2H), 7.04–7.84 (m, 5H), 8.25–8.40 (m, 3H), 8.79 (br s,
1H).
5.5.7. N-(4-Methoxy-phenyl)-2-(4-nitro-phenoxy)-acet-
amide (66). Yield 85%; pale yellow solid; mp 179–
180 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 3.81 (s,
3H), 4.70 (s, 2H), 6.90 (d, 2H, J = 8), 7.00 (d, 2H,
J = 8), 7.50 (d, 2H, J = 8), 8.05 (br s, 1H), 8.28 (d, 2H,
J = 8).
5.4.15. 2-Bromo-N-(4-methoxy-benzyl)-acetamide (58).
Yield 85%; white solid; H NMR (200 MHz, DMSO-
d6): d (ppm) 3.72 (s, 3H), 3.88 (s, 2H), 4.20 (d, 2H,
J = 5.8), 6.86–6.92 (d, 2H, J = 8.00), 7.16–7.21 (d, 2H,
J = 8.00), 8.70 (br s, 1H).,
1
1
5.4.16. 2-Chloro-N-cyclohexyl-acetamide (59). Yield
5.5.8. N-(3,4-Dimethoxy-phenyl)-2-(4-nitro-phenoxy)-
acetamide (67). Yield 65%; pale yellow solid; mp 145–
146 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 3.88 (s,
3H), 3.90 (s, 3H), 4.70 (s, 2H), 6.82–7.34 (m, 5H), 8.08
(br s, 1H), 8.27 (d, 2H, J = 8).
1
84%; white solid; mp 97–98 ꢁC; H NMR (200 MHz,
CDCl3): d (ppm) 1.10–1.88 (m, 10H), 3.72 (m, 1H),
3.96 (s, 2H), 6.40 (br s, 1H).
1
5.5. General procedure for the preparation of 2-(4-nitro-
phenoxy)-N-substituted-acetamides 60–75
5.5.9. N-(3,5-Dimethoxy-phenyl)-2-(4-nitro-phenoxy)-
acetamide (68). Yield 68%; pale yellow solid; mp
152 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 3.80 (s,
6H), 4.68 (s, 2H), 6.29 (m, 1H), 6.80 (m, 2H), 7.08 (d,
2H, J = 8), 8.06 (br s, 1H), 8.28 (d, 2H, J = 8).
1
A mixture of 4-nitrophenol (10 mmol) and K2CO3
(12 mmol) in anhydrous acetone (60 mL) was stirred at
room temperature for 5 min., then a solution of the
appropriate 2-chloro-N-substituted-acetamide (44–59,
10 mmol) was added dropwise. After 12 h at reflux, the
solvent was evaporated to give a residue which was sus-
pended in H2O (50 mL) and extracted with EtOAc
(3 · 50 mL). After drying over Na2SO4, the organic
layer was filtered and the solvent evaporated to dryness.
The desired products 60–75 were finally isolated as sol-
ids by crystallization from a mixture of EtOAc/Et2O.
5.5.10. N-Benzo[1,3]dioxol-5-yl-2-(4-nitro-phenoxy)-acet-
amide (69). Yield 55%; pale yellow solid; mp 171–
1
172 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 4.76 (s,
2H), 5.91 (s, 2H), 6.69–7.26 (m, 5H), 8.15 (d, 2H,
J = 10), 9.92 (br s, 1H).
5.5.11. N-(4-tert-Butyl-phenyl)-2-(4-nitro-phenoxy)-acet-
amide (70). Yield 76%; pale yellow solid; mp 146–
1
5.5.1. 2-(4-Nitrophenoxy)-N-phenyl-acetamide (60).
Yield 77%; pale yellow solid; mp 170–171 ꢁC; 1H
NMR (200 MHz, DMSO-d6): d (ppm) 4.90 (s, 2H),
148 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 1.31 (s,
9H), 4.70 (s, 2H), 7.10 (m, 2H), 7.35–7.51 (m, 4H),
8.07 (br s, 1H), 8.26 (m, 2H).

P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
2525
5.5.12. N-(4-Morpholin-4-yl-phenyl)-2-(4-nitro-phenoxy)-
acetamide (71). Yield 61%; pale yellow solid; mp 170–
172 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 3.08 (br
t, 4H), 3.81 (br t, 4H), 4.66 (s, 2H), 6.85 (d, 2H,
J = 9.00), 7.06 (m, 2H), 7.42 (m, 2H), 8.19–8.28 (m, 3H).
tography on silica gel eluting with EtOAc/light
petroleum 1:1: yield 80%; white solid; mp 195–196 ꢁC;
1H NMR (200 MHz, DMSO-d6): d (ppm) 3.58 (br s, 2
H), 4.39 (s, 2H), 6.53 (d, 2H, J = 8), 6.68 (d, 2H,
J = 8), 7.15 (d, 2H, J = 8), 7.45 (d, 2H, J = 8), 8.60 (br
s, 1H).
1
5.5.13.
2-(4-Nitro-phenoxy)-N-pyridin-4-yl-acetamide
1
(72). Yield 65%; pale yellow solid; mp 154–156 ꢁC; H
NMR (200 MHz, CDCl3): d (ppm) 4.97 (s, 2H), 7.18
(m, 2H), 7.59 (m, 2H), 8.23 (m, 2H), 8.44 (m, 2H),
10.80 (br s, 1H).
5.6.4. 2-(4-Amino-phenoxy)-N-(4-bromo-phenyl)-acetam-
ide (79). The product was purified by column chroma-
tography on silica gel eluting with EtOAc/light
petroleum 3:2: yield 78%; white solid; mp 189–190 ꢁC;
1H NMR (200 MHz, DMSO-d6): d (ppm) 4.50 (s, 2H),
4.69 (br s, 2H), 6.50 (d, 2H, J = 8), 6.72 (d, 2H, J = 8),
7.50 (d, 2H, J = 8), 7.62 (d, 2H, J = 8), 10.11 (br s, 1H).
5.5.14. N-Benzyl-2-(4-nitro-phenoxy)-acetamide (73).
Yield 85%; pale yellow solid; mp 124–125 ꢁC; 1H
NMR (200 MHz, CDCl3): d (ppm) 4.50 (d, 2H,
J = 6.2), 4.57 (s, 2H), 6.80 (br s, 1H), 6.91–6.98 (m,
2H), 7.19–7.31 (m, 5H), 8.12–8.20 (m, 2H).
5.6.5. 2-(4-Amino-phenoxy)-N-(4-iodo-phenyl)-acetamide
(80). The product was purified by column chromatogra-
phy on silica gel eluting with EtOAc/light petroleum 3:2:
yield 85%; white solid; mp 190–191 ꢁC; 1H NMR
(200 MHz, CDCl3): d (ppm) 3.50 (br s, 2 H), 4.52 (s,
2H), 6.68 (d, 2H, J = 8), 6.81 (d, 2H, J = 8), 7.40 (d,
2H, J = 8), 7.64 (d, 2H, J = 8), 8.30 (b s, 1H).
5.5.15. N-(4-Methoxy-benzyl)-2-(4-nitro-phenoxy)-acet-
amide (74). Yield 68%; pale yellow solid; mp 137–
1
138 ꢁC; H NMR (200 MHz, CDCl3): d (ppm) 3.72 (s,
3H), 4.26 (d, 2H, J = 6.0), 4.72 (s, 2H), 6.86 (m, 2H),
7.11–7.21 (m, 4H), 8.22 (m, 2H), 8.70 (br t, 1H).
5.6.6. 2-(4-Amino-phenoxy)-N-(2-trifluoromethyl-phen-
yl)-acetamide (81). The product was purified by column
chromatography on silica gel eluting with EtOAc/light
petroleum 3:2: yield 69%; white solid; mp 187–188 ꢁC;
1H NMR (200 MHz, CDCl3): d (ppm) 3.50 (br s, 2H),
4.57 (s, 2H), 6.68 (d, 2H, J = 8), 6.83 (d, 2H, J = 8),
7.25-7.65 (m, 3H), 8.40 (d, 1H, J = 10), 8.99 (br s, 1H).
5.5.16.
N-Cyclohexyl-2-(4-nitro-phenoxy)-acetamide
1
(75). Yield 68%; pale yellow solid; mp 119–120 ꢁC; H
NMR (200 MHz, CDCl3): d (ppm) 1.09–1.90 (m,
10H), 3.81 (m, 1H), 4.49 (s, 2H), 6.22 (br d, 1H), 6.92-
6.98 (m, 2H), 8.14–8.22 (m, 2H).
5.6. General procedure for the preparation of 2-(4-amino-
phenoxy)-N-substituted-acetamides 76–9129
5.6.7. 2-(4-Amino-phenoxy)-N-(4-methoxy-phenyl)-acet-
amide (82). The product was purified by crystallization
from CH2Cl2/light petroleum: yield 67%; white solid;
mp 225–228 ꢁC; H NMR (200 MHz, CDCl3): d (ppm)
3.50 (br s, 2H), 3.79 (s, 3H), 4.75 (s, 2H), 6.50 (d, 2H,
J = 8), 6.80 (d, 2H, J = 8), 7.56 (d, 2H, J = 8), 7.99 (br
s, 1H), 8.15 (d, 2H, J = 8).
A solution of the appropriate 2-(4-nitro-phenoxy)-N-
substituted-acetamide (60–75, 1 mmol) in the minimum
amount of CH3OH was added dropwise at 0 ꢁC to a
mixture previously prepared by adding an aqueous solu-
tion (1.5 mL) of NaBH4 (2 mmol) to a suspension of
10% Pd/C (5 mg) in water (1 mL). After stirring at room
temperature for 2–3 h, the reaction mixture was filtered
through Celite and the solvents were removed from the
filtrate under vacuum. The residue was suspended in
water (20 mL) and extracted with CH2Cl2 (3 · 20 mL).
The organic phase was dried on Na2SO4, filtered and
the solvent was removed under vacuum.
1
5.6.8. 2-(4-Amino-phenoxy)-N-(3,4-dimethoxy-phenyl)-
acetamide (83). The product was purified by column
chromatography on silica gel eluting with EtOAc/light
petroleum 3:2: yield 83%; white solid; mp 221–222 ꢁC;
1H NMR (200 MHz, CDCl3): d (ppm) 3.53 (br s, 2H),
3.80 (s, 3H), 3.84 (s, 3H), 4.75 (s, 2 H), 6.50–7.44 (m,
5H), 8.12 (br s, 1H), 8.17 (d, 2H, J = 8).
5.6.1. 2-(4-Amino-phenoxy)-N-phenyl-acetamide (76).
The product was purified by crystallization with
CH2Cl2/light petroleum 1:1: yield 80%; white solid; mp
104–105 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm)
4.50 (s, 2H), 4.68 (br s, 2H), 6.50 (m, 2H), 6.72 (m, 2H),
7.06 (m, 1H), 7.31 (m, 2H), 7.66 (m, 2H), 9.95 (br s, 1H).
5.6.9. 2-(4-Amino-phenoxy)-N-(3,5-dimethoxy-phenyl)-
acetamide (84). The product was purified by column
chromatography on silica gel eluting with EtOAc/light
petroleum 3:2: yield 72%; white solid; mp 215–217 ꢁC;
1H NMR (200 MHz, CDCl3): d (ppm) 3.50 (br s, 2H),
3.79 (s, 6H), 4.51 (s, 2H), 6.27 (m, 1H), 6.65–6.84 (m,
6H), 8.25 (br s, 1H).
1
5.6.2. 2-(4-Amino-phenoxy)-N-(4-fluoro-phenyl)-acetam-
ide (77). The product was purified by crystallization with
Et2O/light petroleum 1:1: yield 85%; pale yellow solid;
5.6.10.
2-(4-Amino-phenoxy)-N-benzo[1,3]dioxol-5-yl-
1
mp 106–107ꢁC; H NMR (200 MHz, CDCl3): d (ppm)
acetamide (85). The product was purified by column
chromatography on silica gel eluting with EtOAc/light
petroleum 1:1: yield 76%; pale yellow solid; mp 210–
211 ꢁC; 1H NMR (200 MHz, DMSO-d6): d (ppm)
4.41 (br s, 4H), 5.92 (s, 2H), 6.50 (d, 2H, J = 8),
6.70 (m, 2H), 7.00 (d, 2H, J = 8), 7.30 (m, 1H), 9.68
(b s, 1H).
3.40 (br s, 2H), 4.52 (s, 2H), 6.63–6.68 (m, 2H), 6.68–
6.83 (m, 2H), 6.99–7.08 (m, 2H), 7.50–7.57 (m, 2H),
8.28 (br s, 1H).
5.6.3. 2-(4-Amino-phenoxy)-N-(4-chloro-phenyl)-acetam-
ide (78). The product was purified by column chroma-

2526
P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
5.6.11. 2-(4-Amino-phenoxy)-N-(4-tert-butyl-phenyl)-
acetamide (86). The product was purified by crystalliza-
tion with Et2O/light petroleum: yield 84%; white solid;
mp 119–120 ꢁC; H NMR (200 MHz, CDCl3): d (ppm)
1.30 (s, 9H), 4.52 (s, 2H), 6.66 (d, 2H, J = 8.8), 6.81
(d, 2H, J = 8.8), 7.35 (d, 2H, J = 8.8), 7.48 (d, 2H,
J = 8.6), 8.30 (br s, 1H).
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 100,000g. The membrane
pellet was resuspended in 50 mM Tris–HCl buffer, pH
7.4, and incubated with 2 IU/mL of adenosine deami-
nase for 30 min at 37 ꢁC. Then the suspension was fro-
zen at ꢀ80 ꢁC and the protein concentration was
determined according to a Bio-Rad method31 with bo-
vine albumin as a standard reference. Binding of 1 nM
[3H]-CHA to hA1CHO cells (50 lg of protein/assay)
was performed using 50 mM Tris–HCl buffer, pH 7.4,
and at least 6–8 different concentrations of agonists
studied for an incubation time of 150 min at 25 ꢁC.32
Non-specific binding was determined in the presence of
10 lM CHA and was about 20% of the total binding.
Inhibition binding experiments of [3H]-CGS 21680 to
hA2ACHO cells (100 lg of protein/assay) were per-
formed using 50 mM Tris–HCl buffer, 10 mM MgCl2,
pH 7.4, and at least 6–8 different concentrations of ago-
nists studied for an incubation time of 180 min at
25 ꢁC.33 Non-specific binding was determined in the
presence of 10 lM CGS 21680 and was about 25%
of the total binding. Binding of [125I]-ABMECA to
hA3CHO cells (50 lg of protein/assay) was performed
using 0.2 mL of 50 mM Tris–HCl buffer, 10 mM MgCl2,
EDTA 1 mM, pH 7.4, and at least 6–8 different concen-
trations of tested compounds for an incubation time of
60 min at 37 ꢁC.27 Non-specific binding was determined
in the presence of 1 lM ABMECA and was about 20%
of total binding. Separation of bound from free radioli-
gand was performed by rapid filtration through What-
man 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.
1
5.6.12. 2-(4-Amino-phenoxy)-N-(4-morpholin-4-yl-phen-
yl)-acetamide (87). The product was purified by crystal-
lization with Et2O/light petroleum 1:1: yield 72%; white
1
solid; mp 188–190 ꢁC; H NMR (200 MHz, CDCl3): d
(ppm) 3.09–3.14 (br m, 4H), 3.83–3.88 (br m, 4H),
4.51 (s, 2H), 6.63–6.68 (m, 2H), 6.79–6.91 (m, 4H),
7.46 (d, 2H, J = 9), 8.19 (br s, 1H).
5.6.13. 2-(4-Amino-phenoxy)-N-pyridin-4-yl-acetamide
(88). The product was purified by crystallization with
EtOAc/Et2O: yield 45%; white solid; mp 148–149 ꢁC;
1H NMR (200 MHz, DMSO-d6): d (ppm) 4.56 (s, 2H),
4.70 (br s, 2H), 6.49–6.55 (m, 2H), 6.69–6.75 (m, 2H),
7.65 (m, 2H), 8.43 (m, 2H), 10.39 (br s, 1H).
5.6.14. 2-(4-Amino-phenoxy)-N-benzyl-acetamide (89).
The product was purified by crystallization from
CH2Cl2/light petroleum: yield 72%; white solid; mp
1
108–111 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm)
4.30–4.35 (m, 4H), 4.66 (br s, 2H), 6.48 (d, 2H,
J = 8.4), 6.68 (d, 2H, J = 8.8) 7.19–7.32 (m, 5H), 8.52
(br t, 1H).
5.6.15. 2-(4-Amino-phenoxy)-N-(4-methoxy-benzyl)-acet-
amide (90). The product was purified by crystallization
with Et2O/light petroleum: yield 73%; white solid; mp
1
103–107 ꢁC; H NMR (200 MHz, DMSO-d6): d (ppm)
3.72 (s, 3H), 4.25 (d, 2H, J = 6.00), 4.34 (s, 2H), 4.65
(br s, 2H), 6.47–6.52 (m, 2H), 6.67–6.71 (m, 2H), 6.87–
6.82 (m, 2H), 7.18–7.14 (m, 2H), 8.46 (br t, 1H).
5.7.2. Cyclic AMP assays. For cell preparation CHO
cells transfected with human A2B receptors were washed
with PBS, diluted trypsin and centrifuged for 10 min at
200 g. The pellet containing the CHO cells (1 · 106 cells/
assay) was resuspended in incubation mixture (mM):
NaCl 15, KCl 0.27, NaH2PO4 0.037, 2 IU/mL adenosine
deaminase and 4-(3-butoxy-4methoxy-benzyl)-2-imidaz-
olidone (Ro 20-1724) as phosphodiesterase inhibitor
and preincubated for 10 min in a shaking bath at
37 ꢁC. After the preincubation time at 37 ꢁC the exam-
ined ligands (1 nM–10 lM) were added to the mixture
and incubated for a further 5 min. The reaction was ter-
minated by the addition of cold 6% trichloroacetic acid
(TCA). The TCA suspension was centrifuged 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.34 Samples of cyclic
AMP standard (0–10 pmol) were added to each test tube
containing the incubation buffer (trizma base 0.1 mM,
aminophylline 8.0 mM, 2-mercaptoethanol 6.0 mM,
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
5.6.16.
2-(4-Amino-phenoxy)-N-cyclohexyl-acetamide
(91). The product was purified by crystallization with
EtOAc/Et2O/light petroleum 1:1:1: yield 55%; white sol-
id; mp 168–169ꢁC ; 1H NMR (200 MHz, CDCl3): d
(ppm) 1.13–1.94 (m, 10H), 3.80 (m, 1H), 4.37 (s, 2H),
6.40 (br s, 1H), 6.61–6.77 (m, 4H).
5.7. Biology experiments
All synthesised compounds have been tested, by radioli-
gand 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.
5.7.1. Binding assays. The expression of the human A1,
A2A, A2B and A3 receptors in CHO cells has been previ-
ously described.30 The cells were grown adherently and
maintained in Dulbecco’s Modified Eagle’s Medium
with nutrient mixture F12 (DMEM/F12) without
nucleosides, containing 10% fotal calf serum, penicillin
(100 U/mL), streptomycin (100 lg/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

P. G. Baraldi et al. / Bioorg. Med. Chem. 15 (2007) 2514–2527
2527
13. Beukers, M. W.; Chang, L. C. W.; von Frijtag Drabbe
Kunzel, J. K.; Mulder-Krieger, T.; Spanjersberg, R. F.;
Brussee, J.; IJzerman, A. P. J. Med. Chem. 2004, 47,
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14. Rosentreter, U.; Kraemer, T.; Shimada, M.; Huebsch, W.;
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PCT Int. Appl. (2003), BAYER AKTIENGESELLS-
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15. Kim, S. A.; Marshall, M. A.; Melman, N.; Kim, H. S.;
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16. Kim, Y-C.; Ji, X.; Melman, N.; Linden, J.; Jacobson, K.
A. J. Med. Chem. 2000, 43, 1165.
17. Baraldi, P. G.; Tabrizi, M. A.; Preti, D.; Bovero, A.;
Romagnoli, R.; Fruttarolo, F.; Zaid, N. A.; Moorman, A.
R.; Varani, K.; Gessi, S.; Merighi, S.; Borea, P. A. J. Med.
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10 min. The clear supernatant was counted in a Beck-
man scintillation counter.
Inhibitory binding constant, Ki, values were calculated
from the IC50 values according to Cheng and Prusoff
equation35 Ki = IC50/(1 + [C*]/KD*), where [C*] is the
concentration of the radioligand and KD* its dissocia-
tion constant. A weighted non-linear least-squares curve
fitting program LIGAND36 was used for computer anal-
ysis of inhibition binding experiments. The EC50 values
obtained in the cyclic AMP assay were calculated by
non-linear regression analysis using the equation for a
sigmoid concentration-response curve (GraphPad
Prism, San Diego, CA, USA). All experimental data
are expressed as means standard error of the mean
(Se mean) of three or four independent experiments
performed in duplicate.
Acknowledgments
The authors thank King Pharmaceutical Research &
Development, 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.
20. Baraldi, P. G.; Cacciari, B.; Pineta de Las Infantas, M. J.;
Romagnoli, R.; Spalluto, G.; Volpini, R.; Costanzi, S.;
Vittori, S.; Cristalli, G.; Melman, N.; Park, K.-S.; Ji, X.;
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