1,3-Dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines as potent A2B adenosine receptor antagonists: Design, synthesis, structure-affinity and structure-selectivity relationships

Article abstract of DOI:10.1016/j.bmc.2008.09.067

A number of 1,3-dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines were prepared and evaluated as ligands of recombinant human adenosine receptors (hARs). Several 1,3-dipropyl derivatives endowed with nanomolar binding affinity at hA_2B receptors, but poor selectivity over hA_2A, hA₁ and hA₃ AR subtypes were identified. A comparison with the corresponding 7-OH- and 7,9-unsubstituted-deazaxanthines revealed that 9-OH-9-deazaxanthines are more potent hA_2B ligands with lower partition coefficients and higher water solubility compared to the other two congeneric classes of deazaxanthines. An optimization of the para-substituent of the 8-phenyl ring of 9-OH-9-deazaxanthines led to the discovery of compound 38, which exhibited outstanding hA_2B affinity (Ki = 1.0 nM), good selectivity over hA_2A, hA₁ and hA₃ (selectivity indices = 100, 79 and 1290, respectively) and excellent antagonist potency in a functional assay on rat A_2B (pA_2B = 9.33).

Full text of DOI:10.1016/j.bmc.2008.09.067

Bioorganic & Medicinal Chemistry 16 (2008) 9780–9789
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,3-Dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines as potent A2B
adenosine receptor antagonists: Design, synthesis, structure–affinity
and structure–selectivity relationships
Angela Stefanachi a, Orazio Nicolotti a, Francesco Leonetti a, Saverio Cellamare a, Francesco Campagna a,
Maria Isabel Loza b, Jose Manuel Brea b, Fernando Mazza c, Enrico Gavuzzo d,z, Angelo Carotti a,
*
a Dipartimento Farmaco-chimico, Università degli Studi di Bari, via Orabona 4, I-70125 Bari, Italy
b Departamento de Farmacologia, Universidade de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
c Dipartimento di Chimica, Ingegneria Chimica e dei Materiali, Università dell’Aquila, I-67100 L’Aquila, Italy
d CNR, Montelibretti, I-00016 Monterotondo Scalo (Rome), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2008
Revised 19 September 2008
Accepted 26 September 2008
Available online 30 September 2008
A number of 1,3-dialkyl-8-(hetero)aryl-9-OH-9-deazaxanthines were prepared and evaluated as ligands
of recombinant human adenosine receptors (hARs). Several 1,3-dipropyl derivatives endowed with nano-
molar binding affinity at hA2B receptors, but poor selectivity over hA2A, hA1 and hA3 AR subtypes were
identified. A comparison with the corresponding 7-OH- and 7,9-unsubstituted-deazaxanthines revealed
that 9-OH-9-deazaxanthines are more potent hA2B ligands with lower partition coefficients and higher
water solubility compared to the other two congeneric classes of deazaxanthines. An optimization of
the para-substituent of the 8-phenyl ring of 9-OH-9-deazaxanthines led to the discovery of compound
38, which exhibited outstanding hA2B affinity (Ki = 1.0 nM), good selectivity over hA2A, hA1 and hA3 (selec-
tivity indices = 100, 79 and 1290, respectively) and excellent antagonist potency in a functional assay on
rat A2B (pA2B = 9.33).
Keywords:
1,3-Dialkyl-8-(hetero)aryl-9-OH-9-
deazaxanthines
Adenosine receptor antagonists
Structure–affinity relationships
Structure–selectivity relationships
X-ray crystallography
Ó 2008 Published by Elsevier Ltd.
1. Introduction
several groups to design and test a large number of xanthine deriv-
atives in search for new, more potent and A2B-selective ligands.20–
Adenosine is an endogenous purine nucleoside that modulates
several important physiological processes1–3 by triggering four di-
23 Indeed, in the last few years very potent and highly A2B-selective
xanthines have been discovered.24–28 Some of them, namely XAC
(1),29 MRS1754 (2),30 3,30 6,31 7,32 and 8,33,34 are reported in Chart 1.
Within this line of research, we recently reported the design,
synthesis, and structure–activity (SAR) and structure–selectivity
relationship (SSR) studies of a large series of 9-deazaxanthines
(9-dAXs), a less exploited class of AR ligands, leading to the discov-
ery of very potent and selective A2B antagonists (e.g., 4 and 5 in
Chart 1).35,36
A direct comparison of the molecular electrostatic potentials
(MEPs) of xanthine and 9-dAX model compounds indicated in
the latter the lack of the deepest MEP minimum observed near
N-9 in xanthines.37 As a consequence, the ability of 9-dAXs to form
hydrogen bonds (HBs) and/or participate in other electrostatic (po-
lar) interactions was strongly diminished. In fact, despite their
excellent affinity and selectivity profiles, 9-dAXs displayed lower
water solubility, higher partition coefficients and poorer pharma-
cokinetic properties compared to xanthines which hampered their
preclinical development as antiasthmatic drugs.25 To improve the
pharmacokinetic properties of 9-dAXs, it was deemed necessary
to design new and more polar analogues, possibly through an eas-
ily accessible synthetic pathway.
verse G-protein-coupled adenosine receptors (ARs), named A1, A2A
,
A2B and A3.4 ARs differ in amino acid sequence, tissue distribution,
effector coupling, and biological and pharmacological profiles.
Among AR ligands, A2B selective receptor antagonists have recently
been the object of intense medicinal chemistry research since grow-
ing evidence has indicated a variety of potential therapeutic applica-
tions5–16 at the gastrointestinal (e.g., diarrhea)7 and neurological
levels (e.g., Alzheimer’s disease and dementia),8 in hypersensitive
disorders (e.g., asthma),12–16 diabetes,9 atherosclerosis,17 resteno-
sis,18 and cancer.11 In this challenging scenariowe focusedour atten-
tion on the discovery of selective A2B antagonists as potential
antiasthmatic agents. The rationale of this investigation stems from
the observation that the bronchodilating activity of two xanthinic
drugs, thatis, theophyllineandenprofiline,mightbeduetoselective,
albeit small, antagonism at the A2B AR.19 These findings prompted
* Corresponding author. Tel.: +39 080 5442782; fax: +39 080 5442230.
E-mail address: carotti@farmchim.uniba.it (A. Carotti).
Deceased.
z
0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.09.067

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
9781
Yoneda et al.40,41 under more severe experimental conditions (in
refluxing DMF for a few hours). Therefore, we decided to carry
out an X-ray diffractometric study to unequivocally assign the 9-
OH-9-dAX structure to the isolated compound 32 (Table 1). Fig-
ure 1 shows a stereo view of the X-ray crystallographic structure
of 32 that was identified as a 9-OH-9-dAX derivative. The dihedral
angle between the mean planes of the 9-dAX and the 8-phenyl
rings was nearly 25°. This conformation was a compromise be-
O
H
N
Y
R1
N
OCH2COR
Z
O
N
R3
1, XAC: R1= R3= CH2CH2CH3; Z= N; Y= H; R = NH(CH2)2NH2
2, MRS1754: R1= R3= CH2CH2CH3; Z= N; Y= H; R= NHC6H4-4-CN
3, R1= R3= CH2CH2CH3; Z= N; Y= H; R = NHC6H4-4-I
4, R1= R3= CH3; Z= CH; Y= H; R= NHC6H4-4-Br
tween electron delocalization on the entire
p system and steric
5, R1= R3= CH3; Z= CH; Y= OMe; R= NHC6H4-4-Br
hindrance among facing non-bonded atoms of the two close ring
systems. Both propyl chains were in trans conformation and pro-
truded out of the plane of the heterocyclic system on the same
side. The 9-hydroxyl hydrogen laid outside the pyrrole ring plane
but on the opposite side of the propyl chains.
O
H
N
N
N
N
H
N
Once the correct chemical structure to our compound was as-
signed, we decided to apply the synthetic approach illustrated in
Scheme 2 to prepare a properly designed series of 1,3-dialkyl-9-
OH-8(hetero)aryl-9-dAXs aiming at the discovery of new, highly po-
tent and selective A2B antagonists with improved pharmacokinetic
properties compared to the corresponding isomeric 7-OH- and 7,9-
unsubstituted- 9-dAXs. During the execution of our work, a patent
reporting one 1,3-dipropyl-9-OH-9dAX, that is, the 8-(p-CN-phenyl)
derivative, along with a series of other heterocyclicAR antagonists to
treat ischemia reperfusion injury, was filed to Biogen. Inc.42
A preliminary comparative modeling study was performed on
two reference molecules, that is, 9-dAX derivative A and 9-OH-9-
dAX derivative B (depicted in Chart 2), to analyze how the different
electronic distributions, HB-making ability and hydrophobic prop-
erties of the two classes of molecules would have resulted in im-
proved pharmacokinetic properties.
N
O
N
O
O
O
6
O
O
N
H
N
CF3
N
N
N
N
O
7
Cl
O
N
S
H
N
O
S
N
N
N
O
O
8
Chart 1. Xanthine and 9-dAX derivatives with potent A2B AR antagonistic activities
(1–8).
2. Chemistry
The synthesis of 8-substituted-9-OH-9-dAXs was first success-
fully improved by applying microwave heating to the reaction
illustrated below in Scheme 3.
9-OH-9-dAXs were obtained in medium-to-high yields, in a
very short time-frame (15 min), in DMF at 200 °C by microwave
irradiation at a maximum power of 400 W.
8-Anilino derivative 24 was obtained by a SnCl2-mediated
reduction of the corresponding nitroderivative 32 whereas 8-anili-
no derivative 23 was synthesized by basic hydrolysis of 7-OH-8-(4-
acetamidophenyl)-9-dAX intermediate.37
8-(p-OH-phenyl)-9-OH-9-dAX 27 was prepared by demethyla-
tion of methoxy derivative 30 with BBr3.
Serendipitously, we found a simple, viable and efficient solution
to our problem.
While performing the first reaction of a two-step synthetic
pathway, outlined in Scheme 1,38 to prepare the 1,3-dipropyl-8-
p-nitrophenyl-9-dAX, instead of the styryl derivative III, unexpect-
edly, a compound with a molecular weight of a hydroxy-9-dAX
was obtained as a unique reaction product.
Even if a careful examination of the analytical and spectral data
suggested a 9-OH-9-dAX derivative as the most likely chemical
structure, an unequivocal and definitive structural assignment
was required because, to the best of our knowledge, 9-OH-9-dAXs
had never been produced under such relatively mild experimental
conditions (Scheme 2). A literature search indicated that 9-OH-9-
dAXs had already been reported by Senda et al.39 as by-products
of the photoreductive cyclization of 5-nitro-6-styril-uracil, and by
8-Substituted-7-OH-9-dAXs 10 and 26, and the novel 9-dAX 25,
were obtained from 1,3-dialkyl-5-nitro-6-styryl uracil by a reduc-
tive cyclization with SnCl2 in DMF at room temperature or under
reflux, respectively, according to our reported method.38
O
O
O
R1
O
NO2
R1
O
NO2
H
H
N
R1
N
N
i
ii
N
O
X
+
N
R3
N
R3
O
N
R3
X
X
9, 12, 16, 19, 22, 25, 28
II
I
III
iii
O
OH
R1
N
N
X
O
N
R3
10, 13, 17, 20, 26, 29
Scheme 1. Reagents and conditions: (i) Piperidine, EtOH reflux; (ii) SnCl2, DMF, reflux; (iii) SnCl2, DMF, r.t.

9782
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
Table 1
Chemical structures and AR binding affinitiesa of 1,3-dialkyl-8-(hetero)aryl-9-dAX derivatives 9–37
O
7
R
1
R
N
N
8
R
O
N
9
3
R
R
R1
R3
R7
R9
R8
Ki hA2B
Ki hA2A
SIb
Ki hA1
Ki hA3
c
c
Compounds
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
OH
H
H
OH
H
H
H
OH
H
H
OH
H
H
OH
H
H
OH
H
H
OH
H
H
H
H
OH
H
H
OH
OH
H
H
OH
H
H
OH
H
H
OH
H
H
OH
H
C6H5
C6H5
C6H5
4–CH3–C6H4
4–CH3–C6H4
4–CH3–C6H4
4–CH3SO2–C6H4
C6H5
C6H5
182
1660
288
457
2239
513
68
229
676
54
199
589
89
8.9
155
19
44
115
11
490
479
24
71
50
6.5
64
23
479
12%
1470
126
8%
2450
692
646
32%
50
631
398
89
162
240
166
48
102
118
199
631
148
324
145
28
468
104
933
408
2.6
nd
5.1
0.28
nd
4.8
10.2
2.8
nd
0.93
3.2
0.66
1
18.2
1,5
8.7
1.1
0.89
10.7
0.41
1.3
6.2
4.6
2.9
4.3
7.3
4.5
2.8
11.7
nd
nd
nd
nd
nd
nd
nd
34
25
83
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
63
240
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
96
30%
182
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
295
7%
nd
nd
nd
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
nC3H7
CH3
C6H5
4–CH3–C6H4
4–CH3–C6H4
4–CH3–C6H4
4–NH2–C6H4
4–NH2–C6H4
4–NH2–C6H4
4–OH–C6H4
4–OH–C6H4
4–OH–C6H4
4–CH3O–C6H4
4–CH3O–C6H4
4–CH3O–C6H4
4–CH3CO–C6H4
4–NO2–C6H4
4–CH3SO2–C6H4
4–CH3SO2–C6H4
2–Furyl
H
OH
OH
OH
OH
OH
OH
OH
OSO3H
H
H
H
H
H
H
nC3H7
nC3H7
nC3H7
4–COOH–C6H4
2–Furyl
338
35
a
Affinity values at the indicated human cloned ARs are expressed as Ki (nM) or as % of inhibition at a 1 lM concentration. SEMs were always lower than 10%.
b
c
SI is the selectivity index defined as the ratio Ki hA2A/Ki hA2B
.
nd stands for not determined.
To improve water solubility of 8-(2-furyl)-9-OH-9-dAX deriva-
uated by using the H2O probe which easily detected the emer-
gence of HB interactions. As expected, the presence of the 9-
hydroxyl group was relevant to engage HB interaction as indi-
cated by the cyan mesh, contoured at a value of ꢀ4.0 kcal/mol,
and surrounding position 9 only in the case of 9-OH-9-dAX refer-
ence molecule B.
The difference in hydrophobic properties between molecules A
and B may be well appreciated from MIF calculations with the DRY
hydrophobic probe (data not shown). Taken together, these com-
putational data do suggest for 9-OH-9-dAXs higher HB-making
propensities, hydrophilicity and most likely, water solubility than
9-unsubstituted-9-dAXs (Fig. 2).
tive 35, its sulfuric ester 37 was prepared by an esterification reac-
tion with chlorosulfonic acid in pyridine.43
Finally, 8-p-oxyacetamido-9-OH-9-dAX derivatives 38 and 40
were prepared from the corresponding p-oxyacetic ester 42 by a
reaction with p-bromoaniline and 4-N-benzyl-piperazine, respec-
tively, using NaCN as catalyst, as indicated in Scheme 4.35
3. Molecular modeling
To evaluate the main differences between some molecular
properties of 9-dAXs and 9-OH-9-dAXs a computational study
was carried out on reference molecules A and B (Chart 2).
For the same purpose, water solubilities, and HPLC capacity fac-
tors as experimental descriptors of lipophilicity, were measured for
selected 9-dAX and 9-OH-9-dAX derivatives (see Table 3).
4. Biochemical and pharmacological assays
Compounds were assayed for their ability to displace [3H]-
DPCPX, [3H]-ZM241385, [3H]-DPCPX and [3H]-NECA from cloned
human A1, A2A, A2B and A3 ARs as described in our previous pa-
pers.35–37 All the active compounds showed competition concentra-
tion–response curves of specific radioligand binding against
increasing concentrations of ligands, the slopes not being signifi-
cantly different from unity at the 5% level of statistical significance
(Fig. 3).
Antagonistic activity was measured for 38 by means of isolated
organs assays at both rat colon A2B and rat aorta A2A AR subtypes.
This compound concentration-dependently displaced the curves of
the AR agonist NECA to the right in a parallel way without depres-
The calculation of the MEP around position 9 returned for mol-
ecule B lower MEP values than the bound threshold equal to
1.06 kcal/mol observed for 9-dAX A. Along with MEPs, other rele-
vant molecular interaction fields (MIFs) were calculated within
GRID.44 Unlike other traditional approaches, GRID has the advan-
tage of considering the entire system immersed in water with a
bulk dielectric constant ranging from 80, in the aqueous environ-
ment surrounding the target, to 4, in the deep center of globular
macromolecular targets; this allows GRID to more realistically
feature biological interactions. In this regard, the occurrence of
putative HB interactions of reference molecules A and B was eval-

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
9783
Table 2
Chemical structures and hAR binding affinitiesa of 1,3-dipropyl-8-p-oxyacetamido-9-dAX derivatives 38–41
Compound
Chemical structure
Ki hA2B
Ki hA2A
SIb
Ki hA1
Ki hA3
O
O
H
N
N
O
HN
Br
38c
1.0
100
100
79
1290
O
N
OH
O
N
O
H
N
N
O
HN
Br
39
2.2
31
14
21
199
O
O
N
O
N
H
N
N
O
40
2.6
92
35
224
891
O
N
OH
O
N
O
N
H
N
N
O
41
6.2
257
42
43
5%
O
N
a
Affinity values at the indicated human cloned ARs are expressed as Ki (nM) or as % of inhibition at a 1 lmol concentration. SEMs were always lower than 10%.
b
c
SI is the selectivity index defined as the ratio Ki hA2A/Ki hA2B
.
In functional assays, compound 38 exhibited the following antagonist potency: pA2B (rat) = 9.33 0.28, pA2A (rat) = 6.65 0.30.
Figure 1. Stereo-view of the X-ray crystallographic structure of 32.
O
O
N
O
H
N
NO2
H
NO2
N
N
N
i
NO2
+
O
O
N
O
O
N
OH
NO2
NO2
III
I
32
Scheme 2. Reagents and conditions: (i) Piperidine, EtOH reflux.

9784
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
Table 3
O
N
O
N
H
N
Experimental and estimated physicochemical properties of selected 9-dAXs
H
N
N
logPb
logPc
Measured
solubilityd
Estimated
solubilitye
(lg/ml)
a
Compound
logk60
N
(lg/ml)
O
O
OH
19
21
22
24
28
30
40
41
0.97
0.83
1.21
0.96
0.71
0.51
0.29
0.93
4.07
3.04
4.53
3.50
3.30
2.26
2.54
3.79
4.47
3.88
4.97
4.38
3.80
3.21
3.50
3.14
6
30
6
30
28
2.
8
2
8
5
10
97
5.4
55
38
290
9
A
B
Chart 2. 9-dAX and 9-OH-9-dAX reference molecules.
120 10
10
2
2
0.7
1.8
O
H
O
NO2
R1
O
R1
O
a
Isocratic RP-HPLC capacity factors determined on a C8 column, by using 60/40
methanol/water mixture as the mobile phase.45
N
N
N
i
R8
R8CHO
b
+
Computed by using ACD/Labs software (v. 11.00).46
N
R3
N
Computed by using Bioloom software (v.1.5).47
c
R3
OH
Pr
d
Measured solubility in a 50-mM Tris/HCl, pH 7.4 buffer by a turbidimetric
method.48
R1=R3= Me
,
e
Computed by using ACD/Labs software (v. 11.00).46
R1=Pr and R3= Me
R8= Aryl or Heteroaryl
11, 14, 15, 18, 21, 30-36, 42
Scheme 3. Reagents and conditions: (i) Piperidine, DMF, 400 W, 200 °C, 15 min.
sion of their maximum, compatible with a competitive antagonism
(Fig. 4).
5. Results and discussion
Chemical structures of the examined ligands are reported in Ta-
bles 1 and 2 along with their binding affinities at the indicated AR
subtypes. It must be kept in mind that the main goal of our re-
search was the discovery of new and highly potent hA2B antago-
nists endowed with good selectivity over hA2A, measured by the
selectivity index (SI), which is the ratio Ki hA2A/Ki hA2B. Accord-
ingly, binding affinities at both the hA2B and hA2A ARs were first
measured and only when good hA2B AR affinity and SI were found,
the affinities at hA1 and hA3 ARs were determined.
In addition, binding affinities at all four ARs were measured also
for lead compounds 16 and 18 for a more sound evaluation of the
SSRs. In Table 1, only the percentage of radioligand displacement at
1 lM was reported for low-active compounds or for the ones
whose low solubility in the assay medium prevented the determi-
Figure 2. GRID MIF contoured at ꢀ4.0 kcal/mol using the H2O probe for reference
molecules A and B.
125
100
75
50
25
0
nation of their Ki.
After structural assignment, 1,3-dipropyl-8-p-nitrophenyl-9-
OH-9-dAX 32 was assayed at both hA2B and hA2A ARs. A good affin-
ity at hA2B (Ki = 50 nM) and a lower affinity at hA2A ARs, resulting in
a SI equal to 2.9, were measured. Bearing in mind the SARs and
-12 -11 -10
-9
-8
-7
-6
-5
log [Antagonist] (M)
SSRs previously developed for a large series of 9-dAXs,35–37
a
Figure 3. Binding competition experiments at cloned hA2B receptors. Concentra-
tion–response curve for compound 38. Values represent the mean SEM (vertical
bars) of two independent experiments.
number of 1,3-dialkyl-8-(hetero)aryl-9- and -7-OH-9-dAXs were
synthesized and tested together with their corresponding
7,9-unsubstituted-9-dAX analogues. 8-Aryl-1,3-dimethyl-9-dAXs
9–14 were envisaged as our initial target compounds.
1,3-Dimethyl-9-OH-9-dAXs 11 and 14 elicited sub-micromolar
affinities at hA2B and a low selectivity over hA2A (SI = 5.1 and 4.8,
respectively). The corresponding isomeric 7-OH-9-dAXs 10 and
13 exhibited even higher Kis at hA2B, whereas their low water sol-
ubility and affinity prevented the determination of Ki at the hA2A
O
H
O
H
CONRR'
N
N
COOEt
N
i
N
O
O
O
N
O
N
OH
OH
42
38, 40
Scheme 4. Reagents and conditions: (i) RR0NH, NaCN,
D.

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
9785
ferences, respectively). This trend was experimentally confirmed
by comparing the isocratic RP HPLC capacity factors (k) measured
on a C8 column using a 60/40 methanol/water mixture as the mo-
bile phase. With few exceptions, estimated and measured water
solubility was relatively close, and indeed, a 5- to 10-fold increase
was observed moving from 7,9-unsubstituted to 9-OH-9-dAXs.
These encouraging findings prompted us to prepare two more
hydrophilic 9-OH-9-dAXs, that is, 2-furyl derivative 35 and p-
COOH-phenyl derivative 36. While in the former a significant
improvement of hA2B affinity with respect to isosteric 8-phenyl
derivative 18 was observed, the introduction of the ionized p-
COOH group in the 8-phenyl ring of 18 led to a drop in affinity.
Conversely, highly water-soluble hydrogen sulfate 37 maintained
a good hA2B affinity, comparable to that of its furyl precursor 35,
and even exhibited increased hA2B selectivity (SI = 11.7 from 4.5).
Given that previous data35,36 have indicated an increased SI of
9-dAXs with ligands bearing larger alkyl substituents at position
1 than at position 3, we tried to enhance the SI of the most active
ligand 33 by preparing its 3-methy-1-propyl analogue 34. Unfortu-
nately, only a small increase of the SI was observed (from 4.3 to
7.3), but to the detriment of hA2B affinity (nearly a 10-fold
decrease).
Even in the absence of ligands with a high SI, 1,3-dipropyl-9-
dAX derivatives 16, 17, 18, 33 and 34 were selected for the evalu-
ation of their binding affinities at the hA1 and hA3 ARs. Surprisingly,
the 7,9-unsubstituted lead compound 16 at the hA1 and hA3 ARs
exhibited higher affinities (Ki = 34 and 96 nM, respectively) than
at hA2B (Ki = 229 nM), whereas in the corresponding 9-OH conge-
ner 18 the affinity at hA2B (Ki = 54 nM) remained higher than at
the hA1 and hA3 ARs (Ki = 83 and 182 nM, respectively). The 7-
OH-9-dAX lead compound 17 exhibited an unexpectedly high
affinity at hA1 (Ki = 25 nM) whereas at hA3 a value of Ki > 1000 nM
might be anticipated. Non symmetrically substituted 1-propyl-3-
methyl-9-OH-9-dAX 34 displayed Ki values of 240 and supposedly
>1000 nM at the hA1 and hA3 ARs, respectively, versus a value of Ki
equal to 64 nM observed at hA2B. Finally, 1,3-dipropyl-9-OH-9-dAX
derivative 33 turned out to be the only ligand with a significant, al-
beit small, selectivity for the hA2B receptor subtype yielding Ki val-
ues of 6.5, 28, 63 and 295 nM at the hA2B, hA2A, hA1 and hA3 ARs,
respectively.
100
75
50
25
0
-10 -9
-8
-7
-6
-5
-4
-3
-2
log [NECA] (M)
Figure 4. Isolated organ assays at rat A2B receptors. Cumulative concentration-
response curves to NECA in the absence (j) and in the presence (N) of 30 nM 38.
The assay was performed in duplicate.
AR. The 7,9-unsubstituted 9-dAXs 9 and 12 exhibited a slightly im-
proved hA2B affinity compared to the corresponding 9-OH-9-dAXs
11 and 14 and SI values equal to 2.6 and 0.28, respectively. The lat-
ter value was quite surprising and unusual, since in our previous
studies35–37 similarly substituted 9-dAXs always showed marked
hA2B selectivity.
These preliminary and somewhat discouraging results sug-
gested us to introduce an electron-withdrawing group at the para
position of the 8-phenyl ring since a similar substitution gave a
good hA2B affinity for 1,3-dipropyl-8-p-nitrophenyl-9-OH-9-dAX
32. Very satisfactorily, p-methanesulphonylphenyl derivative 15
resulted as the most potent (Ki = 68 nM) and hA2B-selective
(SI = 10.2) ligand among the first series of targeted 1,3-dimethyl-
9-dAX derivatives.
Guided by these initial findings, a new series of 1,3-dipropyl-8-
(hetero)aryl-9-dAXs was designed, synthesized and tested aiming
at an enhancement of hA2B affinity, hA2B/hA2A selectivity and
molecular hydrophilic properties. To this end, besides compounds
16–21 that are designed to make a direct affinity comparison with
corresponding 1,3-dialkyl congeners 9–15, new ligands bearing
hydrophilic electron-withdrawing and electron-donor substituents
in para position of the 8-phenyl ring were conceived.
Since the above findings did not fulfill our expectations, espe-
cially about selectivity, we synthesized two additional 1,3-dipro-
pyl-9-OH-9-dAXs (38 and 40, Table 2) designed on the basis of
the good hA2B affinity and selectivity observed by us and Jacobson
in p-oxyacetamido-9-deazaxanthine35,36 and xanthine30 deriva-
tives (e.g., 4–5 and 3, respectively, Chart 1). The two novel ligands,
38 and 40, displayed excellent affinity at the hA2B AR subtype
(Ki = 1.0 and 2.6 nM, respectively) and significantly higher selectiv-
ity than that observed for all the other ligands reported in Table 1.
The SIs referred to the Ki ratios hA2A/hA2B, hA1/hA2B and hA3/hA2B
were 100, 79 and 1290 and 35, 86 and 342 for compounds 38
and 40, respectively. Both the affinity and selectivity data of 9-
OH-9-dAXs were significantly improved compared with those
measured for the corresponding 7,9-unsubstituted-9-dAXs 39
and 41 reported for comparison in Table 2. However, the water sol-
ubility of 9-OH-9-dAXs 40, 5-fold higher than that observed for the
corresponding 7,9-unsubstituted-9-dAXs 41, remained quite low.
The substitution of the highly lipophilic benzyl group with smaller
and more hydrophilic alkyl substituents might allow to overcome
this problem.
With the only exception of compound 16, 1,3-dipropyl deriva-
tives 17–21 exhibited a hA2B affinity higher than the corresponding
1,3-dimethyl analogues. However, the SI remained quite low,
reaching a maximum equal to 3.2 for 9-dAX 19. Comparing the
hA2B affinities of the three different classes of 1,3-dipropyl-9-dAXs,
equally substituted at position 8, elicited the following hA2B affin-
ity ranking: 9-OH- > 7,9-unsubstituted- > 7-OH-9-dAXs.
Irrespective of the electron-withdrawing or electron-donor
properties of the para-substituents of the 8-phenyl ring, several
1,3-dipropyl-9-OH-dAX derivatives showed, indeed, very high
hA2B affinities reaching low nanomolar Kis with the p-OH (27)
and p-SO2CH3 (33) congeners (Ki = 11 and 6.5 nM, respectively). It
is worth noting that most para-substituents were strongly hydro-
philic. This fact might thus increase water solubility and lower
the partition coefficient allowing to overcome the most important
pharmacokinetic drawbacks encountered in the vast series of 9-
dAXs prepared recently by us.25,35–37 To support this hypothesis,
water solubility and partition coefficient (P) of a selected number
of 9-OH- and 7,9-unsubstituted-9-dAXs (see Table 3) were esti-
mated by trustable software and experimentally measured by a
turbidimetric assay in DMSO/buffer (pH = 7.4) and reverse-phase
HPLC, respectively.
1,3-Dipropyl-9-OH-9-dAX 38, the most potent and selective
hA2B AR ligand discovered in the present work was tested also
for its antagonistic activity at the A2A and A2B AR subtypes in iso-
lated organ assays in rat. The pA2 values at both receptor subtypes
are reported in Table 2 (footnote c) along with the corresponding Ki
values from binding assays. Full antagonism was observed with
ACD (v. 11.00)46 and Bioloom (v. 1.5)47 predicted quite different
log Ps but, as expected, both software programs indicated lower
log Ps for the 9-OH-9-dAXs (nearly 1 and 0.60 logarithmic unit dif-

9786
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
pA2 values very close to the corresponding pKis for both AR sub-
types: A2B: 9.33 vs 9.00 and A2A: 6.65 vs 7.00.
7.1.1. General procedure for the synthesis of 6-(hetero)aryl-7-
OH-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-diones 11, 14, 15,
18, 21, 30–36, 42
In a microwave reactor, charged with a magnetic stirring bar, 1
mmol of 1,3-dialkyl-5-nitro-6-methyl-uracyl, 1 mmol of (p-substi-
tuted)-benzaldehyde, or 2-furylaldehyde, and piperidine (0.13 ml,
2.5 mmol) were dissolved in 3 ml of DMF. The reaction mixture
was heated by microwave irradiation at 200 °C (5 min of ramping,
400 W maximum power) for 15 min. The mixture was cooled to
room temperature and the solvent was evaporated to dryness un-
der vacuum and the oily residue, treated with absolute EtOH, affor-
ded a solid compound that was filtered and crystallized.
6. Conclusions
The synthesis and comparative analysis of the biological activity at
the AR subtypes of a large array of 9-OH-9-dAX derivatives allowed us
to identify the main structural features for both high hA2B affinity and
hA2B/hA2A selectivity for this new class of AR ligands. A number of 9-
OH-9-dAX derivatives with good hA2B affinity (Ki <50 nM) were ob-
tained, whereas only very few ligands with a value of SI >10 were
found. Among these, compound 38 was particularly interesting, both
intermsofhA2B affinity (Ki = 1.0nM)andselectivityoverhA2A, hA1 and
hA3 (SI = 100, 79 and 1290, respectively).
Interestingly, this highly potent and selective hA2B ligand re-
vealed outstanding antagonist activities in a functional assay on
the A2B AR subtype on rat, with a pA2 = 9.33, a value closely com-
parable to the pKi (9.00) measured in the binding affinity assay
at the corresponding cloned hAR.
7.1.2. 6-(4-Amino-phenyl)-5-hydroxy-1,3-dipropyl-1H-pyrrolo-
[3,2-d]pyrimidine-2,4(3H,5H)-dione 23
N-[4-(5-Hydroxy-2,4-dioxo-1,3-dipropyl-2,3,4,5-tetrahydro-
1H-pyrrolo[3,2-d]pyrimidin-6-yl)-phenyl]-acetamide38
(0.34 g,
0.9 mmol) was dissolved in 5 ml of a: EtOH/1N NaOH (1/1) mixture
and refluxed for 2 h. After cooling the solution was acidified and
the precipitate was collected by filtration and crystallized.
Finally, by preparing a series of hydrophilic 9-OH-9-dAXs with
higher water solubility and lower partition coefficients than the
corresponding 9-dAXs, we were able to potentially improve the
bioavailability of our compounds.
7.1.3. 6-(4-Amino-phenyl)-7-hydroxy-1,3-dipropyl-1H-pyrrolo-
[3,2-d]pyrimidine-2,4(3H,5H)-dione 24
Compound 32 (0.15 g, 0.4 mmol) was dissolved in 5 ml of DMF.
SnCl2 dyhydrate (0.9 g, 4 mmol) was added and the mixture was
stirred at room temperature for 2 h, poured into cold water and
washed with AcOEt. The organic layer was dried on Na2SO4 and
evaporated under vacuum to give an oil that was purified by flash
chromatography (CHCl3/MeOH, 8/2) and then crystallized.
Taken together, our results show that suitably functionalised 9-
OH-9-dAXs represent a class of highly active and hA2B-selective AR
antagonists with promising potential as antiasthmatic agents.
7. Experimental
7.1. Chemistry
7.1.4. 6-(4-Hydroxy-phenyl)-7-hydroxy-1,3-dipropyl-1H-
pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione 27
High analytical grade chemicals and solvents were from com-
mercial suppliers. When necessary, solvents were dried by stan-
dard techniques and distilled. After extraction from aqueous
layers, the organic solvents were dried over anhydrous magnesium
or sodium sulfate. Microwave-assisted reactions were carried out
on a Micro SYNTH (Milestone). Thin-layer chromatography (TLC)
was performed on aluminum sheets precoated with silica gel 60
F254 (0.2 mm) type E. Merck. Chromatographic spots were visual-
ized by UV light or Hanessian reagent.49 Purification of crude com-
pounds was carried out by flash column chromatography on silica
gel 60 (Kieselgel 0.040–0.063 mm, E. Merck) or by crystallization.
Melting points (uncorrected) for fully purified products (see below)
were determined in a glass capillary tube on a Stuart Scientific
electrothermal apparatus SMP3. 1H NMR spectra were recorded
at 300 MHz on a Varian Mercury 300 instrument All the detected
signals were in accordance with the proposed structures. Chemical
shifts (d scale) are reported in parts per million (ppm) relative to
the central peak of the solvent. Coupling constant (J values) are gi-
ven in hertz (Hz). Spin multiplicities are given as: s (singlet), bs
(broad singlet), d (doublet), t (triplet), q (quadruplet) or m (multi-
plet). ESI_MS was performed with an Electrospray interface Ion
Trap Mass spectrometer. (1100 series LC/MSD Trap System Agilent,
Palo Alto, Ca). Elemental analyses were performed on a Eurovector
300 C, H, N analyzer. Fully purified products had satisfactory C, H,
N analyses (within 0.4% of theoretical values). Reaction yields,
crystallization solvent, melting points and spectral data of all the
newly synthesized compounds are reported in Table 4.
Compound 30 (0.12 g, 0.3 mmol) was dissolved in 2 ml of dry
DCM and cooled at 0 °C. Two milliliters of a solution 1M BBr3 in
DCM were added and the reaction was stirred at 0 °C for 1 h. The
reaction mixture was poured into water and the organic layer
washed three times with water, dried on Na2SO4 and evaporated
affording a solid compound that was crystallized.
7.1.5. 6-(2-Furyl)-2,4dioxo-1,3-dipropyl-2,3,4,5-tetrahydro-1H-
pyrrolo[3,2-d]pyrimidin-7-yl hydrogen sulfate 37
Compound 35 (0.044 g, 0.14 mmol) was dissolved in dry pyri-
dine (1 mL) and cooled to 0 °C. To this solution was added chloro-
sulfonic acid (0.163 g, 1.4 mmol) and the mixture allowed to warm
to room temperature overnight. Evaporation afforded a residue
which was treated with 2N HCl giving a precipitate that was crys-
tallized from water.
7.1.6. General procedure for the synthesis of 1,3-dipropyl-9-
OH-8-(4-N-substituted-oxyacetamido-phenyl)-9-dAX 38 and 40
The reaction took place in a sealed tube under argon atmo-
sphere. A catalytic amount of sodium cyanide (1 mg, 0.02 mmol)
was added to ester 42 (0.065 g, 0.15 mmol) (see Scheme 4) and
16 mmol of amines. In the case of benzyl piperazine the reaction
mixture was heated at 150 °C, whereas for 4-Br-aniline 2 mL of
anhydrous dioxane was used as solvent and the reaction heated
at 100 °C. The reactions were monitored by TLC, and when no more
starting material was observed, the mixture was cooled to room
temperature and treated with ethyl ether. The precipitate was iso-
lated by filtration, washed with ethyl ether and crystallized yield-
ing title compounds 38 and 40.
Compounds 9 and 22 have been already described in Ref. 37,
whereas compounds 12, 13, 16, 17, 19, 20, 28 and 29 have been re-
ported in Ref. 38 Compounds 39 and 41 have been described in
Refs. 35 and 36, respectively.
8-Substituted-7-OH-9-dAXs 10 and 26, and the novel 9-dAX 25,
were obtained from 1,3-dialkyl-5-nitro-6-styryl uracil by a reduc-
tive cyclization with SnCl2 in DMF at room temperature or under
reflux, respectively, according to our reported methods.38
7.2. X-ray crystallography
Crystals of 1,3-dipropyl-8-p-nitrophenyl-9-OH-9-dAX deriva-
tive 32 were obtained from methanol by slow evaporation. X-ray

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
9787
Table 4
Reaction yields, crystallization solvents, melting points and spectral data of the newly synthesized 9-dAX derivatives
Compound
Yield (%)
Mp (°C)
1HNMR (DMSO-d6)a
(Molecular formula)
(crystallization
ESI/MS (m/z)
solvent)
10
98
55
51
73
56
58
224–226 (EtOH)
>250 (EtOH)
11.84 (s, 1H), 7.80–7.77 (m, 2H), 7.49–7.41 (m, 3H), 6.38 (s, 1H), 3.61 (s, 3H), 3.22 (s,
3H)
11.79 (s, 1H), 8.32 (s, 1H), 7.95–7.92 (m, 2H), 7.43–7.40 (m, 2H), 7.28–7.25 (m, 1H),
3.60 (s, 3H), 3.21 (s, 3H)
11.75 (s, 1H), 8.28 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 3.60 (s, 3H),
3.23 (s, 3H), 2.30 (s, 3H)
8.45 (bs, 1H), 8.21(d, J = 8.5 Hz, 2H), 7.88 (d, J = 8.5 Hz, 2H), 3.60 (s, 3H), 3.23 (s, 3H),
3.21 (s, 3H)
11.80 (s, 1H), 8.29 (s, 1H), 7.93–7.91 (m, 2H), 7.43–7.38 (m, 2H), 7.28–7.25 (m, 1H),
4.09 (t, J = 7.4 Hz, 2H), 3.84 (t, J = 7.4 Hz, 2H), 1.70–1.48 (m, 4H), 0.89–0.82 (m, 6H)
11.74 (s, 1H), 8.24 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 4.08 (t,
J = 7.1 Hz, 2H), 3.83 (t, J = 6.9 Hz, 2H), 2.30 (s, 3H), 1.72–1.47 (m, 4H), 0.88–0.81 (m,
6H)
(C14H13N3O2) 270 [M–H]ꢀ
(C14H13N3O3) 270 [M–H]ꢀ
(C15H15N3O3) 284 [M–H]ꢀ
(C15H15N3O5S) 348 [M–H]ꢀ
(C18H21N3O3) 326 [M–H]ꢀ.
(C19H23N3O3) 340 [M–H]ꢀ
11 (lit. 39, 40,
41)
14 (lit. 39, 40,
41)
>250° (EtOH)
>250 (EtOH)
15b
18
21
218–220 (EtOH)
192 dec. (EtOH)
23
24
25
26
27
30
51
82
98
98
76
55
>250 (EtOH/Et2O)
200–201 (EtOH/Et2O)
>250 (EtOH)
11.53 (s, 1H), 7.49 (d, J = 8.5 Hz, 2H), 6.59 (d, J = 8.5 Hz, 2H), 6.17 (s, 1H), 5.45 (bs, 2H),
3.85–3.80 (m, 4H), 1.65–1.50 (m, 4H), 0.90–0.82 (m, 6H)
11.42 (s, 1H), 7.97 (s, 1H), 7.61 (d, J = 8.5 Hz, 2H), 6.56 (d, J = 8.8 Hz, 2H), 5.29 (s br,
2H), 4.07 (m, 2H), 3.81 (m, 2H), 1.68–1.50 (m, 4H), 0.88–0.81 (m, 6H)
12.09 (s, 1H), 9.72 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H), 6.54 (s, 1H),
3.83–3.81 (m, 4H), 1.67–1.51 (m, 4H), 0.91–0.82 (m, 6H)
(C18H22N4O3) 341 [M–H]ꢀ
(C18H22N4O3) 341 [M–H]ꢀ
(C18H21N3O3) 326 [M–H]ꢀ
(C18H21N3O4) 342 [M–H]ꢀ
(C18H21N3O4) 342 [M–H]ꢀ
(C19H23N3O4) 356 [M–H]ꢀ
218 dec. (EtOH)
137 dec. (EtOH)
205 dec. (EtOH)
11.65 (s, 1H), 9.78 (s, 1H), 7.62 (d, J = 8.8 Hz, 2H), 6.83 (d, J = 8.8 Hz, 2H), 6.54 (s, 1H),
3.85–3.81 (m, 4H), 1.65–1.51 (m, 4H), 0.91–0.82 (m, 6H)
11.58 (s, 1H), 9.60 (s, 1H), 8.09 (s, 1H), 7.74 (d, J = 8.5 Hz, 2H), 6.78 (d, J = 8.5 Hz, 2H),
4.08 (t, J = 7.2 Hz, 2H), 3.82 (t, J = 7.2 Hz, 2H),1.65–1.52 (m, 4H), 0.87–0.81 (m, 6H)
11.68 (s, 1H), 8.18 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 4.08 (t,
J = 7.2 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 3.76 (s, 3H), 1.66 (q, J = 7.2 Hz, 2H), 1.54 (q,
J = 7.2 Hz, 2H), 0.88–0.81 (m, 6H)
31
32
33
63
68
59
183 dec. (EtOH)
>250 (EtOH)
>250 (EtOH)
11.99 (s, 1H), 8.56 (s, 1H), 8.10 (d, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 2H), 4.10 (t, J = 6.3
Hz, 2H), 3.84 (t, J = 6.3 Hz, 2H), 2.57 (s, 3H), 1.70–1.51 (m, 4H), 0.90–0.82 (m, 6H)
12.16 (s, 1H), 8.78 (s, 1H), 8.28–8.20 (m, 4H), 4.10 (t, J = 7.4 Hz, 2H), 3.85 (t, J = 7.3 Hz,
2H), 1.71–1.54 (m, 4H), 0.90–0.82 (m, 6H).
12.07 (s, 1H), 8.64 (s, 1H), 8.19 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.5 Hz, 2H), 4.10 (t,
J = 7.2 Hz, 2H), 3.84 (t, J = 7.2 Hz, 2H), 3.22 (s, 3H), 1.71–1.51 (m, 4H), 0.89–0.82 (m,
6H)
(C20H23N3O4) 368 [M–H]ꢀ
(C18H20N4O5) 371 [M–H]ꢀ
(C19H23N3O5S): 404 [M–H]ꢀ
34
35
36c
37
38
68
49
65
42
35
>250 (EtOH)
12.06 (s, 1H), 8.68 (s, 1H), 8.20 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H), 3.85 (t,
J = 7.2 Hz, 2H), 3.61 (s, 3H), 3.21 (s, 3H), 1.60–1.54 (m, 2H), 0.86 (t, J = 7.3 Hz, 3H).
11.89 (s, 1H), 8.37 (s, 1H), 7.74 (s, 1H), 6.89–6.88 (m, 1H), 6.60–6.58 (m, 1H), 4.07 (t,
J = 7.2 Hz, 2H), 3.81 (t, J = 7.2 Hz, 2H), 1.69–1.41 (m, 4H), 0.90–0.81 (m, 6H)
11.98 (s, 1H), 8.54 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.93 (d, J = 8.5 Hz, 2H), 4.10 (t,
J = 7.2 Hz, 2H), 3.84 (t, J = 7.2 Hz, 2H), 1.71–1.57 (m, 4H), 0.90–0.82 (m, 6H)
11.89 (s, 1H), 8.88 (bs, 1H), 7.74 (s, 1H), 6.89–6.88 (m, 1H), 6.60–6.58 (m, 1H), 4.07 (t,
J = 7.2 Hz, 2H), 3.81 (t, J = 7.2 Hz, 2H), 1.69–1.41 (m, 4H), 0.90–0.81 (m, 6H)
11.68 (s, 1H), 10.22 (s, 1H), 8.19 (s, 1H), 7.88 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 8.0 Hz, 2H),
7.50 (d, J = 8.0 Hz, 2H), 7.04 (d, J = 8.8 Hz, 2H), 4.73 (s, 2H), 4.08 (m, 2H), 3.83 (m, 2H),
1.67–1.50 (m, 4H), 0.88–0.81 (m, 6H)
11.68 (s, 1H), 8.19 (s, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.31–7.26 (m, 5H), 6.95 (d, J = 8.8
Hz, 2H), 4.84 (s, 2H), 4.08 (t, J = 7.2 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 3.48 (s, 2H), 3.44–
3.55 (m, 4H), 2.30–2.24 (m, 4H), 1.67–1.50 (m, 4H), 0.88–0.81 (m, 6H)
11.69 (s, 1H), 8.19 (s, 1H), 7.86 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 4.80 (s, 2H),
4.15 (q, J = 7.1 Hz, 2H), 4.08 (t, J = 6.9 Hz, 2H), 3.83 (t, J = 7.0 Hz, 2H), 1.70–1.50 (m,
4H), 1,20 (t, J = 7.1 Hz, 3H), 0.91–0.81 (m, 6H)
(C17H19N3O5S) 376 [M–H]ꢀ
(C16H19N3O4) 316 [M–H]ꢀ
(C19H21N3O5) 370 [M–H]ꢀ
(C16H19N3O7S) 396 [M–H]ꢀ
(C26H27BrN4O5) 555 [M–H]ꢀ
>250 (EtOH)
>250 (EtOH)
>250 (H2O)
172 dec. (H2O/MeOH)
40
42
38
46
177 dec. (H2O/MeOH)
183 dec. (EtOH)
(C31H37N5O5) 558 [M–H]ꢀ
(C22H27N3O6) 428 [M–H]ꢀ
a
Heteronuclear protons were detected by exchange with D2O.
N7H proton was undetectable.
COOH proton was undetectable.
b
c
intensity data were collected on
equipped with graphite monochromatized Cu-K
a
Syntex P21 diffractomer
radiation. The
USA) eluting with a mixture of water–methanol (40/60, v/v) as
the mobile phase at a flow rate of 1.0 ml minꢀ1. A solution of KI
in methanol was used for the determination of dead time (t0).
Retention times (tr) were measured in minutes. Logk, calculated
from the capacity factor (k) was taken as the lipophilicity index.
The protocol adopted for measuring aqueous solubility was as
follows. The compound was dissolved in DMSO at a concentration
a
structure was solved by direct methods and refined anysotropical-
ly by using the program package SIR 2002.50 Crystallographic data
(excluding structure factors) have been deposited at the Cambridge
Crystallographic Data Centre and allocated the deposition number
CCDC 693408. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax:
+44 (0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk].
of 1
lg/ml (or 10
l
g/ml for the more soluble compounds). This
l at a time) to 1ml of a 50 mM
solution was added in portion, (2
l
TRIS/HCl pH 7.4 buffer solution at room temperature. Typically a
total of 14 additions were made so that the final volume of DMSO
was well below 5%. The appearance of the precipitate was detected
by an absorbance increase, due to light scattering by particulate
material, in a dedicate diode array UV spectrometer (Agilent
8453). Increased UV absorbance was measured in the 600–
800 nm range. In its simplest implementation, the precipitation
7.3. Partition coefficients and water solubility
Log P values were estimated by using ACD/LogP46 and Bioloom
software.47
The RP-HPLC analyses were performed on a X-TerraÒ C8, 5
lm
3.0 ꢁ 150 mm, chromatographic column (Waters, Milford, MA,

9788
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
Table 5
Experimental conditions used for radioligand binding assays on A1, A2A, A2B, and A3 h.ARs
A1
A2A
A2B
A3
Buffer A
Buffer B
20 mM Hepes, 100 mM
NaCl, 10 mM MgCl2, 2 U/mL
adenosine deaminase
(pH = 7.4)
50 mM Tris–HCl, 1 mM
EDTA, 10 mM MgCl2, 2 U/
mL adenosine deaminase
(pH = 7.4)
50 mM Tris–HCl, 1 mM
EDTA, 5 mM MgCl2, 10
mL bacitracine, 2 U/mL
adenosine deaminase
(pH = 6.5)
50 mM Tris–HCl, 1 mM
EDTA, 5 mM MgCl2,
(pH = 6.5)
50 mM Tris–HCl, 1 mM
EDTA, 5 mM MgCl2, 2 U/mL
adenosine deaminase
(pH = 7.4)
lg/
20 mM Hepes, 100 mM
NaCl, 10 mM MgCl2,
(pH = 7.4)
50 mM Tris–HCl, 1 mM
EDTA, 10 mM MgCl2
(pH = 7.4)
50 mM Tris–HCl (pH = 7.4)
Plate
GF/C
GF/C
GF/B
GF/B
Radioligand
Non-specific binding
Incubation
[3H]DPCPX 2 nM
[3H]ZM241385 3 nM
[3H]DPCPX 25 nM
[3H]NECA 30 nM
10
l
M (R)-PIA
50
lM NECA
1000
25 °C/30 min
lM NECA
100
lM (R)-PIA
25 °C/60 min
25 °C/30 min
25 °C/180 min
point (i.e., the upper aqueous solubility limit), was calculate from a
solution was maintained at 37 °C with aeration by carbogen (95%
CO2 + 5% O2, pH 7.4 0.1). The vessels were cleaned to remove con-
nective tissue, cut into rings, 4 mm in length, and suspended be-
tween stainless steel wires in organ baths containing 20 mL of
Krebs solution, under a basal tension of 2 g (maintained through-
out the experiment). The aorta rings were stabilized for 60 min
in the modified Krebs solution at 37 0.2 °C continuously satu-
rated with carbogen before the start of the assay, and during the
stabilization time they were washed with new Krebs solution at
least three times (15 min each time). All aorta rings were initially
bilinear curve fit in a plot of the absorbance (y axis) versus
DMSO (x axis).
ll of
7.4. Molecular modeling
Reference molecules A and B (Chart 2) were built from the SYB-
YL fragment libraries.51 Geometrical optimization and charge cal-
culation were made by means of a quantum mechanical method
with the PM3 Hamiltonian available in MOPAC 6.0.52 The molecu-
lar electrostatic potential (MEP) was calculated with VEGA-ZZ set-
ting the point density and the probe radius equal to 1000 and 1.4 Å,
respectively.53 By using standard options, molecular interaction
fields (MIFs) were calculated within GRID44 using the H2O and
DRY probes.
exposed to 0.1
and after this the presence of endothelium was confirmed by the
addition of acetylcholine (10 M). Tissues giving less than 25%
lM phenylephrine to elicit a contractile response,
l
relaxation of phenylephrine contraction were discarded. After a
recovery time of 60 min with successive washes with fresh Krebs
solution, a new phenylephrine contraction was elicited and re-
sponses to NECA were measured and used to construct cumulative
relaxant–response curves. After a new recovery time of 60 min
with successive washes with fresh Krebs solution. Antagonists
were incubated for 30 min and then a new phenylephrine contrac-
tion was elicited and responses to NECA were measured and used
to construct cumulative relaxant–response curves.
Isometric contractions were recorded by using Grass FTO3C
force displacement transducers connected to a 7D Grass polygraph,
signals were conducted through a UIM100 module (Biopac sys-
tems) to 16 digital channels to a MP100 data acquisition unit (Bio-
pac systems).
7.5. Biochemistry and pharmacology
7.5.1. Radioligand binding assays
Radioligand binding competition assays were performed
in vitro using A1, A2A, A2B and A3 human receptors expressed in
transfected CHO (hA1), HeLa (hA2A and hA3) and HEK-293 (hA2B
cells. The experimental conditions used are summarized in Table
5. In each instance aliquots of membranes (15 g for A1, 10
for hA2A, 18 g for hA2B and 100 g for hA3) in buffer A (see Table
)
l
lg
l
l
5) were incubated for the specified time at 25 °C with the radioli-
gand (2–35 nM) and six different concentrations (ranging from
0.1 nM to 10
of 200 l. The binding reaction was stopped by rapid filtration in a
multiscreen manifold system (Milipore Iberica, Madrid, Spain). Un-
bound radioligand was removed by washing 4ꢁ with 250 l ice-
cold buffer B for hA1 and hA2A receptors and 6ꢁ 250 l ice-cold
buffer B for hA2B and hA3 receptors (see Table 5). Non specific bind-
ing was determined using a 50–1000 M NECA solution for hA2A
and hA2B receptors and 10–100 M R-PIA solution for hA1 and
hA3. Radioactivity retained on filters was determined by liquid
scintillation counting using Universol (ICN Biochemicals, Inc.).
The binding affinities were determined using [3H]-DPCPX as the
radioligand for A1 and A2B, [3H]-ZM241385 for A2A and [3H]-NECA
for A3. The inhibition constant (Ki) of each compound was calcu-
lated by the Cheng–Prusoff expression Ki = IC50/(1 + (C/KD)54 where
IC50 is the concentration of compound that displaces the binding of
radioligand by 50%, C is the free concentration of radioligand and
KD is the apparent dissociation constant of the radioligand.
l
M) of the test molecule or standard in a final volume
Contractions were processed with Acknowledge 3.0 (Biopac)
and analyzed with Kaleidagraph Software (Synergy Software). Data
were finally represented by using GraphPad Prism 2.1 (GraphPad
Software) by constructing sigmoidal curves representing the loga-
rithm of the concentration of NECA vs maximal effect of the NECA
in the presence and in the absence of the antagonist tested, obtain-
ing values of EC50 (concentration of NECA that exerts the 50% of
maximal effect). Antagonistic potency of the compounds were cal-
culated and expressed as pA2 from this equation:
l
l
l
l
l
0
pA2 ¼ logððEC50 =EC50Þ ꢀ 1Þ=C
0
where EC50 /EC50 is the ratio of concentrations of agonist giving an
equal response (50% of the maximal effect) in the presence and in
the absence, respectively, of a given concentration of the antagonist
(C).56
7.5.3. Isolated organ assays: A2B receptors
Male Wistar–Kyoto rats (weighing 250–300 g) were sacrificed
and distal colon was excised from them.57 Colon was placed into
Tyrode buffer (NaCl 136.9 mM; KCl 2.8 mM; MgSO4ꢂ7H2O 2.1;
CaCl2ꢂ2H2O 1.8 mM, Na2HPO4 0.3 mM; NaHCO3 11.9 mM; Glucose
5.6 mM) and dissected in sections of 10–15 mm in length. Sections
were placed in an organ bath with Tyrode buffer at 37 °C and bub-
7.5.2. Isolated organ assays: A2A receptors
These assays were performed in A2A receptors55 from isolated
aorta of 200–250 g male Sprague–Dawley rats. The aorta was rap-
idly excised and placed in modified Krebs solution of the following
composition (mM): NaCl 118; KCl 4.7; MgSO4.7H2O 1.2;
CaCl2ꢂ2H2O 2.5; KH2PO4 1.18; NaHCO3 25; glucose 11, and the

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 9780–9789
9789
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Sections were equilibrated at a basal tension of 1 g during
60 min, replacing the buffer each 15 min for fresh buffer.
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The Italian authors thank the MIUR (Ministero dell’Istruzione,
dell’Università e della Ricerca Scientifica), Rome (Italy), for finan-
cial support. The Spanish authors thank the Health Institute Carlos
III (RD07/0067/0002) and Xunta de Galicia (07CSA003203PR) for
partial financial support. J.M.B. is a recipient of a financial support
from the Programa Isabel Barreto (Xunta de Galicia).
36. Stefanachi, A.; Brea, J. M.; Cadavid, M. I.; Centeno, N. B.; Esteve, C.; Loza, M. I.;
Martinez, A.; Nieto, R.; Raviña, E.; Sanz, F.; Segarra, V.; Sotelo, E.; Vidal, B.;
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