1-, 3- and 8-substituted-9-deazaxanthines as potent and selective antagonists at the human A2B adenosine receptor

Article abstract of DOI:10.1016/j.bmc.2008.01.002

A large series of piperazin-, piperidin- and tetrahydroisoquinolinamides of 4-(1,3-dialkyl-9-deazaxanthin-8-yl)phenoxyacetic acid were prepared through conventional or multiple parallel syntheses and evaluated for their binding affinity at the recombinant human adenosine receptors, chiefly at the hA_2B and hA_2A receptor subtypes. Several ligands endowed with high binding affinity at hA_2B receptors, excellent selectivity over hA_2A and hA₃ and a significant, but lower, selectivity over hA₁ were identified. Among them, piperazinamide derivatives 23 and 52, and piperidinamide derivative 69 proved highly potent at hA_2B (K_i = 11, 2 and 5.5 nM, respectively) and selective towards hA_2A (hA_2A/hA_2B SI = 912, 159 and 630, respectively), hA₃ (hA₃/hA_2B SI = > 100, 3090 and >180, respectively) and hA₁ (hA₁/hA_2B SI = > 100, 44 and 120, respectively), SI being the selectivity index. A number of selected ligands tested in functional assays in vitro showed very interesting antagonist activities and efficacies at both A_2A and A_2B receptor subtypes, with pA₂ values close to the corresponding pK_is. Structure-affinity and structure-selectivity relationships suggested that the binding potency at the hA_2B receptor may be increased by lipophilic substituents at the N4-position of piperazinamides and that an ortho-methoxy substituent at the 8-phenyl ring and alkyl groups at N1 larger than the ones at N3, in the 9-deazaxanthine ring, may strongly enhance the hA_2A/hA_2B SI.

Full text of DOI:10.1016/j.bmc.2008.01.002

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2852–2869
1-, 3- and 8-substituted-9-deazaxanthines as potent
and selective antagonists at the human A2B adenosine receptor
Angela Stefanachi,a Jose Manuel Brea,c Maria Isabel Cadavid,c Nuria B. Centeno,d
Cristina Esteve,e Maria Isabel Loza,c Ana Martinez,b Rosa Nieto,b Enrique Ravina,b
˜
Ferran Sanz,d Victor Segarra,e Eddy Sotelo,b Bernat Vidale and Angelo Carottia,*
a
`
Dipartimento Farmaco-chimico, Universita di Bari, via Orabona 4, I-70125 Bari, Italy
bDepartamento de Quimica Organica, Universidade de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
cDepartamento de Farmacologia, Universidade de Santiago de Compostela, E-15782 Santiago de Compostela, Spain
d
`
`
Unitat de Recerca en Informatica Biomedica (GRIB), IMIM-UPF, C/Dr. Aiguader 80, E-08003 Barcelona, Spain
`
´
Almirall Research Center, Laboratorios Almirall S.A., Ctra. Laurea Miro 408, E-08980 St. Feliu de Llobregat, Barcelona, Spain
e
Received 7 September 2007; revised 20 December 2007; accepted 4 January 2008
Available online 10 January 2008
Abstract—A large series of piperazin-, piperidin- and tetrahydroisoquinolinamides of 4-(1,3-dialkyl-9-deazaxanthin-8-yl)phenoxy-
acetic acid were prepared through conventional or multiple parallel syntheses and evaluated for their binding affinity at the recom-
binant human adenosine receptors, chiefly at the hA2B and hA2A receptor subtypes. Several ligands endowed with high binding
affinity at hA2B receptors, excellent selectivity over hA2A and hA3 and a significant, but lower, selectivity over hA1 were identified.
Among them, piperazinamide derivatives 23 and 52, and piperidinamide derivative 69 proved highly potent at hA2B (Ki = 11, 2 and
5.5 nM, respectively) and selective towards hA2A (hA2A/hA2B SI = 912, 159 and 630, respectively), hA3 (hA3/hA2B SI = > 100, 3090
and >180, respectively) and hA1 (hA1/hA2B SI = > 100, 44 and 120, respectively), SI being the selectivity index. A number of selected
ligands tested in functional assays in vitro showed very interesting antagonist activities and efficacies at both A2A and A2B receptor
subtypes, with pA2 values close to the corresponding pKis. Structure–affinity and structure–selectivity relationships suggested that
the binding potency at the hA2B receptor may be increased by lipophilic substituents at the N4-position of piperazinamides and that
an ortho-methoxy substituent at the 8-phenyl ring and alkyl groups at N1 larger than the ones at N3, in the 9-deazaxanthine ring,
may strongly enhance the hA2A/hA2B SI.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
biological and pharmacological profiles. Among the
four AdoR subtypes, A2B receptor has been recently
the object of an intense medicinal chemistry research
since growing evidence indicated that its antagonists,
and agonists as well, may have a variety of potential
therapeutic applications.5,6 Indeed it has been shown
that the activation of A2B receptor subtypes may lead
to: (i) an increase of intracellular calcium concentration
and chloride ion secretion in intestinal cells (thus caus-
ing gastrointestinal irritations),7 (ii) an increased forma-
tion and release of a cerebral interleukin, IL-6, which
has been associated with dementias and Alzheimer’s dis-
ease (AD),8 (iii) an increased production of hepatic glu-
cose,9 (iv) a stimulation of angiogenesis in endothelial
cells,10 (v) an induction of apoptosis in arterial smooth
muscle cells11 and (vi) an over-stimulation of mast cells,
thereby inducing hypersensitive disorders, hay fever and
atopic eczema.12–16 Selective A2B receptor antagonists
may therefore play a role in important pathologies such
Adenosine, a naturally occurring nucleoside, modulates
several important physiological and pathological events
by increasing the ratio of oxygen supply on demand,
protecting against ischaemic injuries, triggering anti-
inflammatory responses and promoting angiogenesis.1–3
Four diverse G-protein-coupled adenosine receptor
(AdoR) subtypes—A1, A2A, A2B and A3—have been
identified and characterized at the biological, pharmaco-
logical and physiological level.4 They differ in amino
acid sequence, tissue distribution, effector coupling and
Keywords:
1,3-Dialkyl-6-substituted-1H-pyrrolo[3,2-d]pyrimidine-2,
4(3H,5H)-diones; 9-Deazaxanthines; Adenosine receptor antagonists;
hA2B and hA2A binding affinities; hA2A/hA2B selectivity; Structure–
affinity relationships; Structure–selectivity relationships.
*
Corresponding author. Tel.: +39 080 5442782; fax: +39 080
5442230; e-mail: carotti@farmchim.uniba.it
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.002

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2853
as gastrointestinal (i.e., diarrhoea),17 neurological (i.e.,
R1= or R3
R2= or R4
R5= or R6
Y=
Alkyl
R5
4
2
R6
O
AD and dementia) and hypersensitive disorders (i.e.,
asthma), and in diabetes,9 atherosclerosis,18 restenosis18
and cancer.11 Our main interest in this challenging sce-
nario was the development of selective A2B antagonists
as potential antiasthmatic agents.
R
H
N
R
N
Y
R1
H, Me,
N
O
O
O
N
H, Alkyl, (Hetero)aryl
H, OMe
R3
I
Chart 2. General structure and main structural variations of 9-dAX
AdoR ligands.
The rational of our research was based on the observa-
tion that the bronchodilating activity of theophylline,
and its structural analogue enprofilline, may be ascribed
to a selective, albeit small, antagonism at the A2B
AdoRs.19 These findings prompted several groups to de-
sign and test a large number of xanthine derivatives in
the search for new, more potent and A2B-selective li-
gands.20–24 Indeed, besides the low active and A2B selec-
tive 1 and 2, in the last few years very potent and highly
A2B-selective xanthines,25–29 such as XAC (3),30
MRS1754 (4),31 5,31 8,32 9,33 10,34 and 9-dAXs such as
6 and 735 (Chart 1), have been discovered.
logical evaluation of 9-dAX derivatives bearing a phenyl
or benzyl oxyacetamido group on the para position of
the 8-phenyl ring (see general structure I in Chart 2)
has been recently published.35 Our investigation led to
the discovery of ligands endowed with sub-micromolar
to low nanomolar binding affinity at hA2B receptors,
good selectivity over hA2A and hA3 and a relatively poor
selectivity over hA1. Good antagonistic potencies and
efficacies, with pA2 values close to the corresponding
pKis, were observed in functional in vitro assays per-
formed on a selected series of compounds.35 para-
Bromoanilides of 4-(1,3-dimethyl-9-deazaxanthin-8-yl)
phenoxyacetic acid proved to be the most interesting
leads, showing outstanding hA2B affinity (pKi = 8.58
and 8.46, respectively) and a high selectivity index
(SI = KihAx/KihA2B) over hA2A, hA3 and hA1 (741 >
1000, 11.7 and 1412, 3090, 9.3 for compounds 6 and 7,
respectively, in Chart 1).
Since 9-deazaxanthines (9-dAXs) have been only rarely
studied, especially for their antagonistic activity at the
A
2Breceptor,22,24 we began a systematic study address-
ing first an easy, efficient and expeditious synthetic path-
way to fully functionalized 9-dAXs followed by a
thorough investigation of their biological and pharma-
cological properties.35 Our goal was to discover potent
(Ki < 50 nM) antagonists of human A2B receptor
(hA2B) with at least 100-fold selectivity over hA2A and,
possibly, over the other two AdoR subtypes, namely
hA1 and hA3. A first account of this study dealing more
specifically with the synthesis, biological and pharmaco-
As a continuation of our ongoing research in the field we
designed, synthesized and evaluated at AdoRs a new
series of 9-dAXs, which differs from previously reported
O
O
H
H
N
Y
R1
R1
N
N
N
OCH2COR
Z
N
O
N
R3
O
N
R3
3, XAC: R1= R3= CH2CH2CH3; Z= N; Y= H; R = NH(CH2)2NH2
4, MRS1754: R1= R3= CH2CH2CH3; Z= N; Y= H; R= NHC6H4-4-CN
5, R1= R3= CH2CH2CH3; Z= N; Y= H; R = NHC6H4-4-I
6, R1= R3= CH3; Z= CH; Y= H; R= NHC6H4-4-Br
1, Theophylline, R1= R3= CH3
2, Enprofilline, R1= H, R3= CH2CH2CH3
7, R1= R3= CH3; Z= CH; Y= OMe; R= NHC6H4-4-Br
O
H
N
N
N
N
H
N
N
O
N
N
O
O
O
8
O
O
N
H
N
O
OCH3
N
N
N
O
O
9, CVT-5440
Cl
O
N
S
H
N
O
N
S
N
N
O
O
10
Chart 1. Xanthines and 9-dAX derivatives with antiasthmatic (1 and 2) and selective A2B AdoR antagonistic activities (3–10).

2854
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
R5
R6
2R4
N R6
R5
O
2R4
O
H
N
R
H
N
R
OZ
O
Y
Y
N
H
R1
R1
N
N
O
O
O
Methods A-D
O
N
R3
O
N
R3
(I)
Z = H (II), Et (III)
Scheme 1. Reagents: method A (Z = Et): NaCN/dioxane, reflux/sealed tube. Method B (Z = H): isobutyl chloroformate/NMM/THF. Method C
(Z = H): EDC/OHBt/triethylamine. Method D (Z = H): EDC/OHBt/polymer bound morpholine.
ligands mainly in the NR5R6 moieties, which now con-
sist of heterocyclic amines (i.e., piperazines, piperidines
and tetrahydroisoquinolines).
agonist to the right in a parallel way without depression
of their maximum, which is the typical behavior of com-
petitive antagonists. A representative example (ligand
17) is shown in Figure 1b. The antagonist activity and
efficacy of the tested ligands are collected in Table 4.
Furthermore, antagonistic activity and efficacy at hA2A
and hA2B receptors were measured for some selected li-
gands by means of cAMP assays on HEK-293 cells,
endogenously over expressing hA2B receptors, and over
CHO cells transfected with hA2A receptors. All the
tested compounds concentration-dependently displaced
the curves of the NECA AdoR agonist to the right in
a parallel way without depression of their maximum,
which is the typical behavior of competitive antagonists.
A representative example (ligand 17) is shown in
Figure 1c.
2. Chemistry
The synthesis of 9-dAX (1,3-dialkyl-8-substituted-1H-
pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-diones) acid and
ester intermediates (II and III) was performed according
to our previously published procedures.35 The synthetic
strategies (see Scheme 1) to obtain cyclic amides I, re-
ported in Tables 1–3, include the use of conventional
syntheses (methods A–C) as well as a procedure adapted
to medium-throughput parallel synthesis (method D).
The first amidation procedure (Scheme 1, method A) im-
plies the reaction of the 9-dAX esters with an excess of
the appropriate amine and a catalytic amount of sodium
cyanide in dioxane at reflux or under appropriate heat-
ing in a sealed tube. In a second procedure (method
B), the carboxylic acids were converted into mixed anhy-
dride intermediates, by treatment with isobutyl chloro-
formate and N-methylmorpholine (NMM), and then
reacted with the proper amines. The other amidation
methods involved direct condensation of carboxylic
acids and the suitable amines in polar aprotic solvents
(DMF, tetrahydrofuran) in the presence of an organic
base (triethylamine or polymer supported morpholine)
4. Results and discussion
The chemical structures of all the examined novel li-
gands are reported in Tables 1–3 along with their bind-
ing affinities at the indicated AdoR subtype. It must be
remembered that the main goal of our research is the
discovery of new, highly active hA2B antagonists en-
dowed with a good hA2A/hA2B selectivity index and
therefore only when ligands with these characteristics
were identified the affinities for the other two AdoR sub-
types, hA1 and hA3, were measured. According to this
strategy, the biological assays were performed up to
the determination of the Ki only for those ligands found
most promising in the initial biological screening. As a
consequence, binding affinities at the hA2B and hA2A
AdoR were firstly measured as a percentage of radioli-
gand displacement at 0.1 (or 1) lM concentration and
only when indicative of a high hA2B AdoR affinity and
likely high hA2A/hA2B SI; the Kis were determined at
both AdoR subtypes. For this reason, only the percent-
age of radioligand displacement at the indicated concen-
trations is reported for a few ligands in Tables 1–3.
and
1-[3-dimethylaminopropyl]-3-ethylcarbodiimide
hydrochloride (EDC) and 1-hydroxybenzotriazole
(OHBt) (methods C and D) as coupling reagents. Mole-
cules with chiral centres were isolated and tested as race-
mic mixtures.
3. 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
AdoRs. All the active compounds showed competition
concentration–response curves of specific radioligand
binding against increasing concentrations of ligands,
the slopes not being significantly different from unity
at the 5% level of statistical significance. A representa-
tive binding competition curve is shown in Figure 1a
for ligand 17. Antagonistic activity and efficacy were
measured for a few selected ligands by means of isolated
organs assay at both guinea pig A2B and rat A2A AdoR
subtypes. All the tested compounds concentration-
dependently displaced the curves of the NECA AdoR
For sake of clarity, the discussion of the structure–affin-
ity (SAFIR) and structure–selectivity relationships
(SSR) is given here separately for each class of ligands,
according to the structural classification reported in Ta-
bles 1–3, where examined ligands are ordered within
structural types on the basis of their decreasing hA2B
affinity expressed as pKis. The percentage of radioligand
displacement at 0.1 or 1 lM is reported for low active
compounds or compounds whose low solubility pre-
vented the determination of their Ki. Selectivity is re-
ferred to the hA2B AdoR and is expressed as selectivity

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2855
Table 1. Chemical structures and binding affinitiesa at the hA2B AdoR of 1,3-dialkyl-9-dAX piperazinamides 11–66
R5
N
2 R4
O
R6
H
R
Y
R1
N
N
O
O
O
N
R3
Compound
R1/R3
R2/R4
NR5R6
Y
pKihA2B
pKihA2A
hA2A/
hA2B SI
pKi A1
pKi A3
11
CH3/CH3
CH3/H
N
N
H
H
8.74
8.55
6.41
213
nd
nd
nd
N
N
12
CH3/CH3
H/H
6.81
55
nd
Cl
Cl
13
14
CH3/CH3
CH3/CH3
H/H
H/H
H
H
8.47
8.46
6.40
6.94
117
nd
nd
nd
nd
N
N
N
N
N
N
33.1
Br
Cl
N
S
15
CH3/CH3
H/H
H
8.44
6.74
50.1
nd
nd
nd
N
16
17
CH3/CH3
CH3/CH3
H/H
H/H
CF3
H
H
8.38
8.28
6.88
6.97
31.6
20.4
nd
N
N
6.30
20% at
1 lM
N
N
N
18
19
CH3/CH3
CH3/CH3
H/H
H/H
H
H
8.20
8.20
6.45
6.45
56
56
nd
nd
nd
nd
Cl
CN
N
N
N
Cl
Cl
N
N
20
CH3/CH3
H/H
H
8.15
21% at
1 lM
nd
nd
nd
21
22
23
CH3/CH3
CH3/CH3
CH3/CH3
CH3/H
H/H
Br
H
H
H
8.09
8.02
7.96
6.57
6.49
5.00
26
33.8
912
nd
nd
nd
nd
N
N
N
N
F
Cl
N
S
CH3/CH3
8% at
1 lM
18% at
1 lM
N
N
(continued on next page)

2856
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
Table 1 (continued)
Compound
R1/R3
R2/R4
H/H
NR5R6
Y
H
pKihA2B
pKihA2A
hA2A
hA2B SI
/
pKi A1
pKi A3
24
CH3/CH3
CH3/CH3
OCH3
7.88
6.77
12.9
nd
nd
N
N
N
N
25
26
27
28
29
H/H
H
7.87
7.69
7.56
7.50
7.49
6.38
6.45
6.21
6.76
5.15
30.9
17.4
22
nd
nd
nd
nd
nd
N
N
N
N
N
N
CH3/CH3
CH3/CH3
CH3/CH3
CH3/CH3
CH3/H
H/H
H
nd
N
OH
H
nd
N
CH3/CH3
H/H
H
5.49
218.8
nd
N
o-OCH3
6.27
9% at
1 lM
N
N
N
N
30
31
CH3/CH3
CH3/CH3
H/H
H/H
H
H
7.29
7.27
5.63
5.81
45.7
28.8
nd
nd
n.d
nd
H3CO
N
32
33
CH3/CH3
CH3/CH3
H/H
H/H
m-OCH3
7.24
7.23
5.84
6.15
25.1
12
nd
nd
nd
nd
N
N
N
N
H
N
N
N
34
CH3/CH3
CH3/H
H
7.15
6.13
6.10
10.5
nd
nd
Cl
35
36
CH3/CH3
CH3/CH3
H/H
H/H
N
N
H
6.76
6.32
4.05
nd
nd
nd
nd
N
Cl
N
o-OCH3
4% at
—
1 lM
OCH3
37
38
CH3/CH3
Pr/Pr
H/H
H/H
o-OCH3
30% at
0.1 lM
1% at
0.1 lM
—
nd
nd
nd
nd
N
N
N
N
H
8.82
6.94
75.9
Cl

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2857
Table 1 (continued)
Compound
R1/R3
R2/R4
H/H
NR5R6
Y
H
H
pKihA2B
8.38
pKihA2A
6.76
hA2A
hA2B SI
/
pKi A1
nd
pKi A3
39
40
Pr/Pr
Pr/Pr
N
N
CF3
41.7
nd
nd
N
N
H/H
8.32
6.79
33.8
41.7
nd
41
Pr/Pr
H/H
H
8.21
6.59
nd
nd
N
N
N
N
N
N
42
43
Pr/Pr
Pr/Pr
CH3/H
H/H
H
H
8.18
8.16
6.62
7.42
36.3
5.5
nd
nd
nd
nd
Cl
Cl
N
N
N
44
45
Pr/Pr
H/H
H/H
H
H
8.12
7.98
7.13
6.09
9.8
nd
nd
nd
nd
Cl
Cl
S
N
Pr/Pr2
77.6
N
N
N
N
N
N
O
46
47
Pr/Pr
Pr/Pr
H/H
H
H
7.69
7.61
6.81
6.58
7.6
nd
nd
nd
nd
O
CH3/CH3
10.7
O
O
48
Pr/Pr
H/H
H
7.41
6.82
3.9
nd
nd
N
N
N
N
N
N
49
50
Pr/Pr
Pr/Pr
H/H
H/H
H
H
7.29
7.00
5.8
30.9
nd
nd
nd
nd
N
11% at
0.1 lM
nd
N
Cl
S
N
51
52
CH3/Pr
CH3/Pr
CH3/CH3
H/H
H
H
8.98
8.69
9% at
1 lM
nd
nd
nd
N
N
N
CF3
6.49
158.5
7.05
5.65
N
(continued on next page)

2858
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
Table 1 (continued)
Compound R1/R3
R2/R4
NR5R6
Y
H
pKihA2B
pKihA2A
hA2A/
hA2B SI
pKi A1 pKi A3
53
54
55
CH3/Pr
CH3/Pr
CH3/CH3
N
N
8.38
6.25
134.9
nd
nd
nd
nd
nd
nd
N
Cl
Cl
N
N
N
H/H
H/H
H
H
8.37
8.31
6.19
6.40
151.4
CH3OCH2
CH2/CH3
OCH2CH2
81
N
N
S
N
N
N
N
56
57
58
CH3/Pr
CH3/Pr
H/H
H/H
H/H
H
H
H
8.24
8.22
8.13
6.20
106
79
nd
nd
nd
nd
nd
nd
Cl
6.32
Cl
(CH3)2CH
CH2/Pr
15% at 1 lM
nd
N
N
N
59
Cypropyl-
CH2/cypro-
pyl-CH2
H/H
H
7.93
7.31
4.2
nd
nd
N
N
60
61
Pr/CH3
CH3/Pr
H/H
H/H
N
N
H
H
7.88
7.83
6.50
5.96
24
nd
nd
nd
nd
Cl
74.1
N
62
63
CH3/Pr
H/H
H/H
F
H
H
7.82
7.59
6.33
6.16
30.9
26.9
nd
nd
nd
nd
N
N
N
CH3CH2/
CH3CH2
N
64
65
CH3/Pr
Pr/CH3
H/H
H/H
N
H
H
7.59
7.16
6.15
6.65
27.5
3.2
nd
nd
nd
nd
N
Cl
N
N
N
66
CF3CH2/
CF3CH2
H/H
H
32% at 0.1 lM 3% at 0.1 lM nd
nd
nd
N
a Affinity values are expressed as pKi or as % of inhibition at the indicated concentration, SEMs were always lower than 10%.

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2859
Table 2. Chemical structures and binding affinitiesa at the hA2B AdoR of 1,3-dialkyl-9-dAX piperidinamides 67–81
R5
N
R4
R2
O
R6
O
H
Y
R1
N
N
O
O
N
R3
Compound
R1/R3
R2/R4
H/H
NR5R6
Y
pKihA2B
pKihA2A
hA2A/
hA2BSI
pKi A1
pKi A3
67
CH3/Pr
N
H
H
8.59
5.99
398
nd
nd
nd
OH
N
68
69
CH3/CH3
H/H
H/H
8.54
8.25
6.49
5.45
112.2
nd
Cl
CH3/Pr
H
630
6.17
4% at
1 lM
N
H3CO
70
71
CH3/Pr
CH3/Pr
H/H
H/H
H
H
8.21
8.04
6.39
6.4
66.1
43.7
nd
nd
nd
nd
N
N
OCH3
72
CH3/CH3
H/H
H
8.02
28% at
1 lM
nd
nd
nd
N
N
O
73
CH3/CH3
H/H
H
7.95
6.52
26.9
nd
nd
F
74
75
CH3/CH3
H/H
H/H
H
H
7.91
7.87
6.10
5.94
64.6
85.1
nd
nd
nd
nd
N
N
OH
Pr/Pr
76
77
Pr/Pr
Pr/Pr
H/H
H/H
H
H
7.68
7.67
6.23
5.98
28.2
40.7
nd
nd
nd
nd
N
N
(continued on next page)

2860
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
Table 2 (continued)
Compound
R1/R3
R2/R4
H/H
NR5R6
Y
pKihA2B
pKihA2A
hA2A/
hA2BSI
pKi A1
pKi A3
OCH3
78
CH3/CH3
CH3/CH3
m-OCH3
7.51
6.06
28.18
nd
nd
N
79
80
H/H
o-OCH3
6.05
3% at
1 lM
—
nd
nd
nd
nd
N
N
CH3/Pr
CH3/CH3
H
35% at
0.1 lM
11% at
0.1 lM
nd
81
CH3/CH3
CH3/CH3
H
34% at
0.1 lM
8% at
0.1 lM
nd
nd
nd
N
a Affinity values are expressed as pKi or as % of inhibition at the indicated concentration, SEMs were always lower than 10%.
index (SI), that is, the affinity ratio KihAx/KihA2B. For a
more immediate and efficient analysis of the variation of
both affinity and selectivity, the binding data are pre-
sented graphically as a plot of pKihA2A (Y-axis) versus
pKi hA2B (X-axis) using the same scale and range for
both axes (square plot). Thus, on the diagonal of this
plot (Y = X) lie compounds with equal affinities at both
receptors, whereas highly hA2B selective compounds will
be seen far below the diagonal. The distance of their pKi
values from the diagonal is a direct measure of the de-
gree of selectivity. Ligands targeted in the present
work—those endowed with both high hA2B affinity
and high hA2A/hA2B SI—will be therefore located in
the lower right-hand corner of the plots. Whenever pos-
sible, a comparative analysis was conducted among sim-
ilarly substituted ligands belonging to different classes.
a number of examined ligands (lower right corner of
the plot) were potent and strongly selective hA2B recep-
tor ligands. Indeed, both high pKis (>7.45) and hA2A
/
hA2B SI > 50 were observed for many diM (i.e., 11–13,
15, 18, 19, 23 and 29), diA (i.e., 51–57 and 61)and two
diP derivatives (i.e., 38 and 45). The highest hA2A
/
hA2B SIs were shown by diM piperazinamide derivatives
11, 29 and 23 (SI = 213, 219 and 912, respectively). The
two most selective ligands 29 and 23 were tested also at
the hA1 and hA3 AdoRs. Unfortunately, the low solubil-
ity of compound 23 prevented the determination of its
Ki at both AdoRs. Nonetheless, the percentage of its
radioligand displacement at hA1 and hA3 AdoRs (8%
and 18%, respectively, at 1 lM) surely indicated high
hA1/hA2B and hA3/hA2B SIs. Affinity data of 29 at
hA1(pKi = 6.27) and hA3 (9% displacement at 1 lM)
pointed out a low hA1/hA2B SI (SI = 17) and a presumed
much higher hA3/hA2B SI, in full agreement with our
previous findings.35 The introduction of an ortho-meth-
oxy group in the 8-phenyl ring of different ligands gave
contrasting results. For lead compound 17 the hA1/hA2B
SI increased by one order of magnitude (from 20.4 to
219 in compound 29), as a result of a much stronger de-
crease of hA2A than hA2B affinity (pKi from 6.97 to 5.15
and from 8.28 to 7.49, respectively). The same structural
modification, that recently drove us to the identification
of compound 7 (Chart 1), one of the most potent and
hA2B-selective antagonists discovered so far, resulted in-
stead in a dramatic decrease of both hA2B and hA2A
affinities for the N4-para-methoxyphenyl- and N4-
para-chlorophenyl-piperazinamides (24 vs 37 and 13 vs
36). This unexpectedly high diminution of hA2B affinity
may be due to negative steric effects at the para position
of the N4-phenyl ring that may occur only when the
phenyl ring at position 8 likely assumes a different con-
formation because of the presence of the ortho-methoxy
group. Indeed, a conformational analysis indicated for
the most stable conformers of the phenyl and ortho-
4.1. 1,3-Dialkyl-8-[4-phenoxy(N1-piperazinyl)acetam-
ido]-9-deazaxanthines
A large array of 1,3-dialkyl-8-substituted-9-dAXs con-
taining a piperazinyl NR5R6 group was prepared and
evaluated for their binding affinity at hA2B and hA2A
AdoRs (Table 1). Three different subclasses, constituted
by 1,3-dimethyl (diM, 27 members), 1,3-dipropyl (diP,
13 members) and 1,3-dialkyl (diA, 16 members, contain-
ing different alkyl groups at positions 1 and 3) deriva-
tives, are listed separately in Table 1 for a better
evaluation of the SAFIRs and SSRs.
pKi values ranging from 5.00 to 7.42 and from 6.32 to
8.98 were measured for hA2A and hA2B AdoR affinities,
respectively. Their distribution and relationship can be
seen in the colour square plot of pKihA2B versus pKi
hA2A of Figure 2 where diM, diP and diA derivatives
are indicated in blue, pink and yellow, respectively. A
simple visual inspection of the plot revealed that hA2B
and hA2A binding affinities are not correlated and that

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2861
Table 3. Chemical structures and binding affinitiesa at the hA2B AdoR of 1,3-dialkyl-9-dAX tetrahydroisoquinolinamides 82–90
R5
N
R4
R2
O
R6
O
H
Y
R1
N
N
O
N
R3
O
Compound
R1/R3
R2/R4
H/H
NR5R6
Y
H
pKihA2B
pKihA2A
hA2A
hA2B SI
/
pKi A1
pKi A3
N
N
N
N
N
82
Pr/Pr
8.64
7.03
40.7
nd
nd
83
84
85
86
Pr/Pr
CH3/H
H/H
H
H
H
H
8.33
8.14
8.06
8.02
6.8
33.9
138
49
nd
nd
nd
CH3/Pr
CH3/CH3
6.00
6.37
6.62
nd
H/H
6.44
nd
15% at 1 lM
Cypropyl-CH2/
cypropyl-CH2
H/H
25
nd
N
N
87
88
CH3/Pr
H/H
H/H
H
H
7.84
7.77
6.68
14.5
nd
nd
nd
nd
CH3OCH2CH2/
CH3OCH2CH2
20% at
1 lM
nd
N
N
89
90
CH3/CH3
CH3/CH3
H/H
H/H
o-OCH3
m-OCH3
7.36
6.77
4.92
5.17
275.4
39
6.57
nd
15% at 1 lM
nd
a Affinity values are expressed as pKi or as % of inhibition at the indicated concentration, SEMs were always lower than 10%.
methoxy phenyl congeners diverse N7C8C10C20(OMe)
dihedral angles (nearly 30ꢁ and 0ꢁ, respectively). Very
interestingly, the highly hA2B selective ligand 11 was en-
dowed also with an outstanding hA2B affinity
(Ki = 2 nM).
vs 47/28; 54 vs 44; 56 vs 45/15 and 61 vs 43/18). The
same observation holds for the 1-isobutyl-3-methly
derivative 58 which is much more selective than the cor-
responding diM derivative 17 (hA2A/hA2B SI > 400 vs
20). These results are consistent with literature data indi-
cating higher hA2A/hA2B SIs for xanthines bearing alkyl
substituents at position 1 larger than those at position
3.21 Notably, several highly hA2B selective ligands (i.e.,
compounds 15, 23, 45, 51 and 56) presented a 5-
chloro-2-benzothiazolyl substituent at the N4 position
of the piperazine ring which may be likely engaged in
diM congeners generally showed hA2A/hA2B SIs close to
those of the corresponding diP congeners (12 versus 38;
17 versus 40; 16 versus 39 and 28 versus 47), whereas 1-
propyl-3-methyl derivatives displayed SIs higher than
the corresponding diP/diM derivatives (52 vs 39/16; 53

2862
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
a
b
c
15000
10000
5000
0
75
50
25
0
4500
3500
2500
1500
500
-25
-11 -10 -9
-8
-7
-6
-5
-4
-8
-7
-6
-5
-4
-3
-10 -9
-8
-7
-6
-5
-4
-3
Log [NECA] (M)
log [NECA] (M)
Log [Antagonist] (M)
Figure 1. (a) Binding competition experiments at cloned hA2B AdoR of compound 17 (j). Non-specific binding was measured with 10ꢀ3 M NECA
(m). The assay was performed in duplicate. (b) Functional assay with compound 17 at A2B receptors in smooth muscle of guinea pig thoracic aorta.
Cumulative concentration–response curves to NECA in the absence (j) and in the presence (m) of 17, 0.1 lM. (c) Functional assay with compound
17 at hA2B receptors expressed in HEK-293 cells. Cumulative concentration–response curves to NECA in the absence (j) and in the presence (m) of
17, 30 nM.
deed the most hA2B-selective ligand obtained in this
Table 4. Binding affinities and antagonist potency at A2B and A2A
work. In other cases an opposite effect resulted from
AdoRs of selected 9-dAXsa
C-dimethylation: compounds 28 and 47 showed SI val-
b
c
Compound
pKihA2B
pKihA2A
pA2A2B
pA2A2A
ues of 5 and 11, respectively, which were much lower
than those observed for the corresponding monomethyl
(26 and 42, respectively) and non-methylated congeners
(17 and 40, respectively).
17
47
62
69
85
8.28
7.61
7.82
8.25
8.06
6.97
6.58
6.33
5.45
6.37
8.00
7.50
7.90
8.00
7.89
6.80
6.40
6.63
5.10
6.30
Other interesting insights can be inferred from the SA-
FIR study of piperazinamides in Table 1 that was lim-
ited to the hA2B affinity since affinity data at hA2A
presented a much lower variance. As observed previ-
ously in anilide and benzylamide series (Chart 2, struc-
ture I, R5 = H, R6 = Ph or Bn), lipophilicity of the
NR5R6 fragment plays a crucial role in the hA2B-recep-
tor binding as demonstrated by the higher affinity ob-
served moving from the N4-methyl (35) to the -phenyl
(17) and -benzyl (31) piperazine derivatives. The key role
of lipophilicity of the N4-substituent was confirmed also
by the lower hA2B affinities found for the more hydro-
philic 2-pyrimidinyl (25) and 2-pyridyl (33) diM deriva-
tives and the 2-pyridazinyl diP derivative (50) compared
to the corresponding N4-phenyl leads 17 and 40, respec-
tively. It is worth to note that heterocyclic rings substi-
tuted with lipophilic chloro substituents recovered a
good hA2B binding affinity (see the dichloropyridyl
derivatives20 and 44). The introduction of one or two
methyl groups on the oxycetamido bridge led to a dim-
inution of hA2B affinity in both the diM and diP series,
being the gem-dimethyl derivatives always less active
than the corresponding monomethyl congeners, as re-
sulted from the following rank of hA2B affinity:
14 > 21; 17 > 26 > 28; 40 > 42 > 47; 18 > 34. The de-
creased hA2B affinity of carbamate (46 and 48) and ben-
zydryl (49) diP derivatives compared to the N4-phenyl
lead 40 may be ascribed to negative steric effects. The
SAFIR at hA2B was assessed more thoroughly by intro-
ducing several substituents on the ortho, meta and para
positions of the N4-phenyl ring of diM, diP and 1-pro-
pyl-3-methyl lead compounds 17, 40 and 64, respec-
tively. The most explored class was that of diM
congeners. Substituents at the phenyl ring were chosen
to properly sample the physicochemical domain of their
steric, lipophilic and electronic properties. Once again,
the substituent lipophilicity was a crucial determinant
a pKi and pA2 values represent means of at least three independent
experiments with a SEM always lower than 10%.
b Smooth muscle of guinea pig thoracic aorta.
c Smooth muscle of male Sprague–Dawley rat aorta.
selective binding interactions with hA2A and hA2B bind-
ing sites. The effect of C-methylation at the alpha posi-
tion of the oxyacetamido bridge on hA2B AdoR
affinity and selectivity was investigated with the synthe-
sis and biological evaluation of a small series of mono-
and dimethyl derivatives. While for most mono-C-
methyl derivatives hA2A/hA2B SIs remained close to
those of the starting methylene congeners (14 vs 21, 17
vs 26, 40 vs 42) a dramatic increase of SI (from 50 to
912) resulted from the C-dimethylation of the N4-chlo-
robenzothiazolyl derivative 15. Compound 23 was in-
dimethyl
8.5
dipropyl
dialkyl
7.5
38
6.5
11
54
45
5.5
23
4.5
4.5
5.5
6.5
7.5
8.5
pKi A2B
Figure 2. Affinity–selectivity plot for 1,3-dipropyl-9-dAX piperazina-
mides 11–66 (see Table 1).

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2863
of a high hA2B affinity. Compounds bearing lipophilic
substituents in meta and para positions were more active
than lead compounds in all the three series (i.e., 12–14,
16 > 17; 38, 39 > 40 and 52, 57, 61, 62 > 64). It is worth
noting that the lipophilic effect depends on the substitu-
ent position on the phenyl ring: meta-chloro derivatives
were found to be highly potent in all three series,
whereas ortho-chloro derivatives 18 and 34 proved less
active than lead compound 17, most likely for a negative
steric effect at the ortho position. The same consider-
ation hold for the ortho-methoxy derivative 30 which
showed an affinity much lower than both the corre-
sponding para-congener 24 and lead compound17. The
introduction of hydrophilic para-substituents such as
methoxy, hydroxyl and cyano produced a decrease of
hA2B affinity (lead compound 17 vs 19, 24 and 27,
respectively); this lent support to the hypothesis that
favourable lipophilic interactions took place at the N4
region of the piperazine ring and in particular, in the
accessible regions around the N4-phenyl ring. A further
proof to this hypothesis came from the observation that
in the diM and diP series the highest hA2B affinity was
displayed by the highly lipophilic 3-chlorophenyl deriv-
atives 12 and 38 (Ki = 3 and 1.5 nM, respectively)
whereas in the diA series the chorobenzothiazolyl deriv-
ative 51 yielded the most potent hA2B AdoR ligand
found in the present work (Ki = 1 nM).
dimethyl
dipropyl
dialkyl
8.5
7.5
6.5
5.5
4.5
69
4.5
5.5
6.5
7.5
8.5
pKiA2B
Figure 3. Affinity–selectivity plot for 1,3-dipropyl-9-dAX piperidina-
mides 67–81 (see Table 2).
than the corresponding diM (72) and diP (77) congeners;
a similar ranking was observed for the 4-phenyl piperaz-
inamide leads (i.e., 67 > 74 > 76). These findings were
consistent with the higher SIs observed for the 1-propyl,
3-methyl derivatives in the piperazine series. The intro-
duction of an ortho-methoxy group on the 8-phenyl ring
of 67 yielded a dramatic decrease, greater than two or-
ders of magnitude, of hA2B affinity in compound 79 as
already seen for compound 36 in the piperazine series.
The same explanation proposed for compound 36 (i.e.,
a conformational change leading to unfavourable steric
interactions at position 4 of the piperidine/piperazine
ring) may be advocated for ligand 79.
4.2. 1,3-Dialkyl-8-[4-phenoxy(N-piperidinyl)acetamido]-
9-deazaxanthines
Fifteen 1,3-dialkyl-9-dAX derivatives bearing a 4-substi-
tuted piperidine as the NR5R6 moiety (structure I, Chart
2) were prepared and evaluated for their affinity at hA2B
and hA2A AdoRs. The new ligands were designed to bet-
ter explore the receptor space around position 4 of the
piperidine ring placing one or two substituents at C4.
It should be emphasized that the spatial domain ex-
plored by 4-aryl substituents in piperidine and in piper-
azine derivatives does not overlap because aryl groups
are linked to atoms with different geometry (i.e., a tetra-
hedral carbon [C4] and an almost planar nitrogen atom
[N4]).
Of particular interest were compounds 67 and 69 which
showed outstanding hA2B affinities (Ki = 2.5 and
5.5 nM, respectively) and selectivities (hA2A/hA2B
SI = 398 and 630, respectively). Binding affinities of
compound 69 were measured also at hA1 and hA3
AdoRs resulting in a pKi of 6.17 for the former and
4% radioligand displacement at 1 lM for the latter.
These values indicated a good hA1/hA2B SI (120) and,
presumably, also a much higher hA3/hA2B SI.
Considering the higher selectivity observed for 1-propyl-
3-methylpiperazinamides compared to similarly substi-
tuted 1,3-diM and 1,3-diP congeners, most of the struc-
tural modifications at C4 were carried out on 1-propyl-
3-methyl derivatives.
4.3. 1,3-Dialkyl-8-[4-phenoxy(N-1,2,3,4-tetrahydroiso-
quinoline)acetamido]-9-deazaxanthines
A small series of 9-dAX derivatives bearing tetrahydro-
isoquinoline as the NR5R6 moiety (structure I, Chart 2)
and new R1 and R3 alkyl (or functionalized alkyl) sub-
stituents were prepared and evaluated for their affinity
at hA2B and hA2A AdoRs. Ligands with good hA2B
affinity (pKi 6.77–8.64) and hA2A/hA2B selectivity in-
dexes were obtained (Table 3). The plot of pKihA2B ver-
sus pKihA2A in Figure 4 showed again no correlation
between the two sets of pKis and allowed clear identifi-
cation of a number of ligands with high SIs.
Piperidinamide ligands with both good hA2B affinity
(pKi from 7.51 to 8.54) and hA2A/hA2B SI were obtained
(Table 2). The plot of pKihA2Bversus pKihA2A (Fig. 3)
demonstrated that no correlation exists between the
two sets of pKis and that a large number of ligands dis-
played elevated SIs. Unfortunately, it was not possible
to determine the SI of 4,4-diphenyl derivatives 69 and
72 because their poor solubility in the assay medium
prevented the measure of pKi at hA2A. Nonetheless,
their very low percentage of radioligand displacements
at 1 lM allowed a safe rank of measured and roughly
estimated hA2B/hA2A SIs. The SI of 1-propyl,3-methyl-
diphenylpiperidinamide 69 proved significantly greater
Among lead compounds 82, 84 and 85, 3-methyl,1-pro-
pyl derivative 84 displayed the highest hA2A/hA2B selec-
tivity ratio (SI = 138) in accordance with previous data

2864
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
9
8.5
8
In expectation of the results of an ongoing 3D QSAR
dimethyl
dipropyl
dialkyl
study on the entire array of data reported herein and
in a previous account of our work.35 Figure 5 may assist
in interpreting the SAFIR and SSR of 8-ortho-methoxy-
phenyl-9-dAX derivatives.
7.5
7
4.4. Antagonist activity and efficacy of selected 9-dAX
AdoR ligands at hA2A and hA2B AdoR subtypes
82
6.5
6
A number of ligands (i.e., 17, 47, 62, 69 and 85) were se-
lected to measure their antagonistic potency through
functional assays on isolated organs at cloned hA2B
and hA2A AdoR subtypes. A representative example of
this experiment is shown for compound 17 in Figure
1b. The pA2 values at both receptor subtypes are re-
ported in Table 4 along with the corresponding pKi val-
ues from binding assays. pA2 values very close to the
corresponding pKis were obtained. Taken together,
our findings indicate that binding affinity parallels
antagonist potency and therefore binding and functional
processes can be considered strictly related.
89
5.5
5
4.5
4.5
5.5
6.5
7.5
8.5
pKi A2B
Figure 4. Affinity–selectivity plot for 1,3-dipropyl-9-dAX tetrahydro-
isoquinolinamides 82–90 (see Table 3).
from piperazin- and piperidinamide series. Another
highly hA2B selective compound was obtained by intro-
ducing an ortho-methoxy group on the 8-phenyl ring of
lead compound 85. Indeed, in ligand 89 the hA2A/hA2B
SI jumped from 49 to 275 as a consequence of a decrease
in hA2A affinity that was significantly greater than in
hA2B (from 6.37 to 4.92 vs 8.06 to 7.36, respectively).
In all probability, the conformational change induced
by the introduction of methoxy group may place the tet-
rahydroisoquinoline ring, or preferably the fused benzo
moiety, of 89 in an accessible region of the hA2B-binding
site analogously to what has been observed for the phe-
nyl ring of anilide. 35NR5R6 moieties with a different
topology, such as N4-/C4-phenyl piperazines/piperi-
dines, in compounds 29 and79, respectively, might im-
pact inaccessible regions of the hA2B binding site
resulting in a very low binding affinity. Plausible fa-
voured and disfavoured binding topologies for the
NR5R6 moieties of 8-ortho-methoxyphenyl derivatives
are represented in a very schematic way in Figure 5,
where accessible and inaccessible molecular fragments
are coloured in green and red, respectively.
5. Conclusions
Careful design, synthesis and biological evaluation of a
large series of novel 9-dAXs at the AdoR subtypes en-
abled the identification of an elevated number of ligands
with outstanding hA2B affinity (pKi > 8) and a good
hA2A/hA2B SI (>100). Among the compounds tested at
the four AdoR subtypes, piperazine derivatives 23 and
52 as well as the piperidine derivative 69 resulted partic-
ularly interesting in terms of both high hA2B affinity
(Ki = 11, 2 and 5.5 nM, respectively) and selectivity to-
wards hA2A (SI hA2A/hA2B = 912, 159 and 630, respec-
tively), hA3 (hA3/hA2B SI > 100, 3090 and >180) and
hA1 (hA1/hA2B SI > 100, 44 and 120, respectively).
Remarkably, in the present work the hA1/hA2B SI of
some 9-dAXs was significantly improved compared to
the anilide derivatives (Chart 2, structure I,
NR5R6 = NHAr) reported in our previous paper.35
A number of selected ligands (Table 4), tested in func-
tional assays in vitro, showed very interesting antagonist
activities and efficacies at both A2A and A2B AdoR sub-
types, consistent with their affinities measured in the
binding assays.
The present study allowed us to identify the key molec-
ular determinants of high hA2B affinity and, more
importantly, high hA2B selectivity. The most salient fea-
tures emerging from the SSR and SAFIR can be sum-
marized as follows: (i) 1-propyl-3-methyl substitution
significantly increased the hA2A/hA2BSIs compared to
1,3-diM and 1,3-diP analogues; (ii) the introduction of
an ortho-methoxy substituent in the 8-phenyl ring affor-
ded highly hA2B selective ligands but strictly depending
upon the nature, size and topology of the NR5R6 sub-
stituents; (iii) lipophilic substituents at the N4 position
of piperazinamides as well as lipophilic groups at the
meta and para positions of the N4-phenyl ring signifi-
cantly augmented hA2B affinity; and (iv) the introduc-
tion of one or two methyl groups on the oxyacetamido
Figure 5. Molecular overlap of the 8-substituents in compounds 29, 79
and 89.

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2865
bridge led to a diminution of hA2B affinity in both the
diM and diP series, the gem-dimethyl derivatives being
generally less active than the corresponding mono-
methyl congeners.
doublet), t (triplet), or m (multiplet), br s (broad singlet),
dt (double triplet). Electron impact mass (EI–MS) or
electron spray ionization mass (ESI-MS) were recorded
in a quadrupolar Hewlett-Packard 5988 and in Waters
ZQ 4000 apparatus, respectively. Elemental analyses
were performed on a Eurovector 300 C, H, N analyzer.
Fully purified products had satisfactory C, H, N analy-
ses (within 0.4% of theoretical values). Non-high-
throughput chemical reactions were generally carried
out under an argon atmosphere whereas dry nitrogen
was used in parallel syntheses. Parallel reactions were
performed on a Chemspeed ASW 1000 instrument. In
several instances, the final products obtained by parallel
synthesis were rapidly purified by flash column chroma-
tography up to an acceptable purity (better than 90–95%
by HPLC check) and tested without further purification.
In such a case only ESI/MS or EI/MS spectra and
HPLC retention times were listed (see Supplementary
Material). The HPLC analyses were performed on a
Symmetry C18 (2.1 · 10 mm) column on a Waters
2690 instrument. As detectors, Micromass ZMD for
ES ionization or Waters 996 Diode Array were used.
Diode array chromatograms were processed at
210 nm. The mobile phases were a mixture of formic
acid (0.46 mL), aqueous ammonia 33% (0.115 mL) and
water (1000 mL) (phase A) and a quaternary mixture
of formic acid (0.4 mL), ammonia (0.1 mL), methanol
(500 mL), and acetonitrile (500 mL) (phase B). A gradi-
ent technique was applied going from A 100% to 95% B
in 20 min. The flow rate was 0.4 mL/min. The injection
volume was 5 lL.
The effect on the hA2B affinity of the substitution pattern
at positions 1 and 3 was less clear than that emerging
from hA2B selectivity. In fact, the ranking of hA2B affin-
ity of N4-phenyl leads differed for the piperazinamides
and the tetrahydroisoquinolinamides (the two most
explored series) according to the following decreasing
order of affinity, respectively:
1,3-Dipropyl (40) ꢁ 1,3-di-2-methoxethyl (55) ꢁ 1,3-di-
methyl (17) > 1-isobutyl-3-methyl (58) > 1,3-dicyclopro-
pyl (59) ꢁ 1-methyl,3-propyl (60) > 1,3-diethyl (63) ꢁ
1-propyl,3-methyl (64) > 1,3-ditrifluoroethyl (66) and
1,3-dipropyl (82) > 1-propyl,3-methyl (84) ꢁ 1,3-di-
methyl (85) ꢁ 1,3-dicyclopropyl (86) > 1-methyl,3-propyl
(87) ꢁ 1, 3-di-2-methoxyethyl (88).
However, it is important to note that in both the series
the most and less active ligands carry the same substitu-
ents at 1,3 positions.
In conclusion, taken together, the data reported in our
previous35 and in this companion paper confirmed that
suitably substituted 9-dAXs represent highly potent
and hA2B selective antagonists that may hold great
promise as antiasthmatic agents. Further studies will
be performed to assess off-target activities of our com-
pounds (e.g., HERK and CYP inhibition) and to con-
firm their efficacy as antiasthmatic agents through
in vitro and in vivo assays, such as degranulation of hu-
man isolated mastocytes36 and tests on animal models
such as actively sensitized Brown Norway rats.37
For compounds prepared by conventional organic syn-
thesis (i.e., 17, 22, 24, 25, 30, 31, 33, 35, 36, 40, 41, 67,
73, 74, 76, 79, 82 and 85) besides the spectral data
(i.e.,1H NMR, ESI/MS or EI/MS spectra) also melting
points and microanalyses were determined. The syntheses
of oxyacetic acids II and oxyacetic esters III of 9-deaza-
xanthine derivatives have been already described.35
6. Experimental
High analytical grade chemicals and solvents were from
commercial suppliers. When necessary, solvents were
dried by standard techniques and distilled. After extrac-
tion from aqueous phases, the organic solvents were
dried over anhydrous magnesium or sodium sulfate.
Thin-layer chromatography (TLC) was performed on
aluminium sheets precoated with silica gel 60 F254
(0.2 mm) type E. Merck. Chromatographic spots were
visualized by UV light or Hanessian reagent.38
6.1. General procedures for the amidation of oxyacetic
acids and oxyacetic esters of 9-deazaxanthine derivatives
(see Scheme 1)
The synthesis of the target amides was performed by
using one of the following methods (A–D).
6.1.1. Method A. The reaction took place in a sealed
tube under argon atmosphere. To 0.15 mmol of the ester
III (see Scheme 1) and 16.00 mmol of amines was added
a catalytic amount of sodium cyanide (5 mg). In the case
of liquid amines the reaction mixture was heated at the
boiling temperature of the amine whereas for solid
amines 2 mL of anhydrous dioxane was used as solvent
and the reaction mixture 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 the final product isolated by filtration
and washed with ethyl ether and crystallized from
H2O/MeOH. When no precipitate was formed, the reac-
tion mixture was concentrated under reduced pressure
and the residue purified by flash column chromatogra-
phy on silica gel.
Purification of crude compounds was carried out by
flash column chromatography on silica gel 60 (Kieselgel
0.040–0.063 mm, E. Merck) or by crystallization. Melt-
ing 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 on a 300 MHz Bruker
AMX-300 spectrometer. 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 con-
stant (J values) are given in hertz (Hz). Spin multiplici-
ties are given as: s (singlet), d (doublet), dd (double

2866
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
6.1.2. Method B. A solution of the proper carboxylic
acid (II, see Scheme 1) (0.72 mmol) in anhydrous tetra-
hydrofuran (20 mL) under argon atmosphere was
cooled to ꢀ40 ꢁC, and N-methylmorpholine (0.08 mL,
0.72 mmol) and isobutyl chloroformate (0.09 mL,
0.72 mmol) were slowly added. The mixture was stirred
at ꢀ40 ꢁC for 2 h. Then the appropriate amine
(0.72 mmol) was added, and the mixture was stirred
15 min at ꢀ40 ꢁC and then for additional 12 h at room
temperature. The solution was evaporated under re-
duced pressure, and the residue was dissolved in
DCM, washed with a saturated solution of sodium
bicarbonate, water and brine, and then dried (Na2SO4)
and evaporated under reduced pressure. The resulting
crude product was purified by flash column chromatog-
raphy on silica gel or crystallized by H2O/MeOH.
6.1.4.2. 6-(4-{2-[4-(4-Fluorophenyl)piperazin-1-yl]-2-
oxoethoxy}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrim-
idine-2,4(3H,5H)-dione (22). Method A, Yield 50%, mp:
>250 ꢁC, 1H NMR (DMSO-d6): 12.30 (br s, 1H), 7.84 (d,
J = 8.5 Hz, 2H), 7.06–7.02 (m, 6H), 6.63 (s, 1H), 4.95 (s,
2H), 3.64–3.60 (m, 4H), 3.42 (s, 3H), 3.26 (s, 3H), 3.14–
3.10 (m, 2H), 3.09–3.05 (m, 2H). MS (EI) m/z: 491 [M]+,
Anal. Calcd for C26H26FN5O4: C, 63.53; H, 5.33; N,
14.25. Found: C, 63.71; H, 5.54; N, 14.51.
6.1.4.3. 6-(4-{2-[4-(4-Methoxyphenyl)piperazin-1-yl]-
2-oxoethoxy}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]py-
rimidine-2,4(3H,5H)-dione (24). Method A, Yield 28%,
1
mp: >250 ꢁC, H NMR (DMSO-d6): 12.38 (br s, 1H),
7.96 (d, J = 8.8 Hz, 2H), 7.09 (d, J = 9.3 Hz, 2H), 7.05
(d, J = 8.8 Hz, 2H), 6.96 (d, J = 9.3 Hz, 2H), 6.74 (s,
1H), 5.06 (s, 2H), 3.81 (s, 3H), 3.75–3.71 (m, 4H), 3.54
(s, 3H), 3.38 (s, 3H), 3.21–3.17 (m, 2H), 3.13–3.09 (m,
2H), MS (EI) m/z: 503 [M]+ Anal. Calcd for
C27H29N5O5: C, 64.40; H, 5.80; N, 13.91. Found: C,
64.66; H, 5.83; N, 14.06.
6.1.3. Method C. To a mixture of the carboxylic acid (II,
see Scheme 1) (1.24 mmol), N-(3-dimethylaminopropyl)-
N0-ethylcarbodiimide hydrochloride (0.28 g, 1.49 mmol),
1-hydroxybenzotriazole (0.20 g, 1.49 mmol), triethyl-
amine (0.35 mL, 2.48 mmol) and anhydrous DCM
(20 mL) was added the appropriate amine (1.61 mmol)
and the mixture was stirred at room temperature over-
night under argon atmosphere. The resulting solution
was evaporated under reduced pressure, and the residue
was dissolved in DCM and washed with a saturated so-
dium bicarbonate aqueous solution. The organic phase
was separated, washed with water and brine, dried
(Na2SO4) and evaporated under reduced pressure. The
resulting crude was purified by flash column chromatog-
raphy on silica gel or crystallized by H2O/MeOH.
6.1.4.4. 1,3-Dimethyl-6-{4-[2-oxo-2-(4-pyrimidin-2-yl
piperazin-1-yl)ethoxy]phenyl}-1H-pyrrolo[3,2-d]pyrimi-
dine-2,4(3H,5H)-dione (25). Method A, Yield 60%, mp:
1
>250 ꢁC, H NMR (DMSO-d6): 12.27 (br s, 1H), 8.41
(d, J = 4.7 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.03 (d,
J = 8.8 Hz, 2H), 6.69 (t, J = 4.7 Hz, 1H), 6.63 (s, 1H),
4.96 (s, 2H), 3.85–3.81 (m, 2H), 3.78–3.76 (m, 2H),
3.59–3.55 (m, 4H), 3.43 (s, 3H), 3.27 (s, 3H). MS (EI)
m/z: 475 [M]+, Anal. Calcd for C24H25N7O4: C, 60.62;
H, 5.30; N, 20.60. Found: C, 60.82; H, 5.45; N, 20.78.
6.1.4. Method D. To a mixture of the carboxylic acid
(II, see Scheme 1) (0.21 mmol), N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide hydrochloride (0.09 g,
0.23 mmol), 1-hydroxybenzotriazole (0.30 g, 0.23 mmol)
and polymer bound morpholine (0.28 g, 2.75 mmol/g
based on nitrogen analysis) in anhydrous DMF
(4 mL) was added the suitable amine (0.23 mmol), and
the mixture was stirred overnight at room temperature.
To the resulting solution were added macroporous tri-
ethylammonium methylpolystyrene carbonate (0.25 g,
2.8–3.5 mmol/g based on nitrogen elemental analysis)
and Amberlyst 15 (0.65 g) as scavengers, and the sus-
pension was stirred for 2 h (in the case of acidic or basic
final products the corresponding scavenger was not
added). The resulting suspension was filtered and evap-
orated under reduced pressure. The residue was tritu-
rated with a mixture of MeOH/ethyl ether, and the
precipitate was collected by filtration to yield the
desired product in a sufficient purity for biological
testing.
6.1.4.5. 6-(4-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]-
2-oxoethoxy}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]py-
rimidine-2,4(3H,5H)-dione (30). Method A, Yield 60%,
mp: 181–183 ꢁC, 1H NMR (DMSO-d6): 12.21 (br s,
1H), 7.78–7.74 (m, 2H), 6.88–6.84 (m, 6H), 6.52 (s,
1H), 4.88 (s, 2H), 3.76 (s, 3H), 3.60–3.56 (m, 4H), 3.38
(s, 3H), 3.22 (s,3H), 2.51–2.47 (m, 4H). MS (EI) m/z:
503 [M]+, Anal. Calcd for C27H29N5O5: C, 64.40; H,
5.80; N, 13.91. Found: C, 64.56; H, 5.91; N, 14.22.
6.1.4.6. 6-{4-[2-(4-Benzylpiperazin-1-yl)-2-oxoethoxy]
phenyl}-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,
5H)-dione (31). Method A, Yield 40%, mp: 170–172 ꢁC,
1H NMR (DMSO-d6): 12.05 (br s, 1H), 7.64 (d,
J = 8.2 Hz, 2H), 7.14 (s, 5H), 6.78 (d, J = 8.2 Hz, 2H),
6.38 (s, 1H), 4.68 (s, 2H), 3.23–3.17 (m, 6H), 2.26–2.22
(m, 2H), 2.20–2.14 (m, 2H). EI/MS 487 m/z [M]+, Anal.
Calcd for C27H29N5O4: C, 66.51; H, 6.01; N, 14.36.
Found: C, 66.82; H, 6.11; N, 14.65.
6.1.4.7. 1,3-Dimethyl-6-{4-[2-oxo-2-(4-pyridin-2-ylpi-
perazin-1-yl)ethoxy]phenyl}-1H-pyrrolo[3,2-d]pyrimidine-
2,4(3H,5H)-dione (33). Method A, Yield 42%, mp:
>250 ꢁC, H NMR (DMSO-d6): 12.15 (br s, 1H), 8.14
(d, J = 4.5 Hz, 1H), 7.84 (d, J = 7.9 Hz, 2H), 7.59–7.55
(m, 1H), 7.01 (d, J = 7.9 Hz, 2H), 6.90–6.86 (m, 1H),
6.70–6.66 (m, 1H), 6.60 (s, 1H), 4.91 (s, 2H), 3.60–3.26
(m, 14H). MS (EI) m/z: 474 [M]+, Anal. Calcd for
C25H26N6O4: C, 63.28; H, 5.52; N, 17.71. Found: C,
63.31; H, 5.61; N, 17.88.
6.1.4.1. 1,3-Dimethyl-6-{4-[2-oxo-2-(4-phenylpipera-
zin-1-yl)ethoxy]phenyl}-1H-pyrrolo[3,2-d]pyrimidine-2,4
(3H,5H)-dione (17). Method A, Yield 25%, mp: >250 ꢁC,
1H NMR (CDCl3): 11.25 (s, 1H), 7.76 (d, J = 8.7 Hz, 2H),
7.29–7.27 (m, 3H), 7.02 (d, J = 8.7 Hz, 2H), 6.92–6.90
(m,2H), 6.18 (s, 1H), 4.80 (s, 2H), 3.80–3.76 (m, 4H),
3.53 (s, 3H), 3.49 (s, 3H), 3.49–3.45 (m, 4H). MS (EI) m/z:
473 [M]+ Anal. Calcd for C26H27N5O4: C, 65.93; H,
5.75; N, 14.79. Found: C, 66.23; H, 5.96; N, 15.01.
1

A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
2867
6.1.4.8. 1,3-Dimethyl-6-{4-[2-(4-methylpiperazin-1-yl)-
2-oxoethoxy]phenyl}-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,
5H)-dione (35). Method C, Yield 72%, mp: >250 ꢁC, H
NMR (DMSO-d6): 12.09 (br s, 1H), 7.84 (d, J = 8.7 Hz,
2H), 6.97 (d, J = 8.7 Hz, 2H), 6.57 (s, 1H), 4.88 (s, 2H),
3.42 (s, 3H), 3.27 (s, 3H), 2.53–2.49 (m, 2H), 2.37–2.33
(m, 2H), 2.29–2.25 (m, 2H), 2.15–2.11 (m, 2H). MS
(EI) m/z: 411 [M]+, Anal. Calcd for C21H25N5O4: C,
61.30; H, 6.12; N, 17.02. Found: C, 61.43; H, 6.34; N,
17.14.
J = 8.5, 5.7 Hz, 2H), 7.85 (d, J = 8.6 Hz, 2H), 7.40–7.36
(m, 2H), 7.00 (d, J = 8.6 Hz, 2H), 6.65 (s, 1H), 4.92 (s,
2H), 4.37 (m, 1H), 3.92 (m, 1H), 3.76 (m, 1H), 3.47
(m, 1H), 3.43 (s, 3H), 3.27 (s, 3H), 2.82 (m, 1H), 1.84
(m, 2H), 1.61 (m, 1H), 1.41 (m, 1H), MS (EI) m/z: 518
[M]+, Anal. Calcd for C28H27FN4O5: C, 64.86; H,
5.25; N, 10.80. Found: C, 64.73; H, 4.98; N, 10.43.
1
6.1.4.14. 1,3-Dimethyl-6-{4-[2-oxo-2-(4-phenylpiperi-
din-1-yl)ethoxy]phenyl}-1H-pyrrolo[3,2-d]pyrimidine-2,4
(3H,5H)-dione (74). Method B, Yield 52%, mp: >250 ꢁC,
DMSO-d6: 12.23 (s, 1H), 7.83 (d, J = 8.9 Hz, 2H), 7.31–
7.16 (m, 5H), 6.99 (d, J = 8.9 Hz, 2H), 6.56 (s, 1H), 4.90
(s, 2H), 4.49–4.45 (m, 1H), 3.97–3.93 (m, 1H), 3.40 (s,
3H), 3.24 (s, 3H), 3.17–3.09 (m, 1H) 2.87–2.72 (m,
2H), 1.85–1.76 (m, 2H), 1.67–1.63 (m, 1H), 1.46–1.42
(m, 1H). MS (EI) m/z: 472 [M]+, Anal. Calcd for
C27H28N4O4: C, 68.63; H, 5.97; N, 11.86. Found: C,
68.41; H, 5.75; N, 11.65.
6.1.4.9. 6-(4-{2-[4-(4-Chlorophenyl)piperazin-1-yl]-2-
oxoethoxy}-2-methoxyphenyl)-1,3-dimethyl-1H-pyrrolo-
[3,2-d]pyrimidine-2,4(3H,5H)-dione (36). Method A,
Yield 72%, mp: 191–193 ꢁC, 1H NMR (DMSO-d6):
11.78 (s, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.24 (d,
J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 6.69 (m, 1H)
6.61–6.57 (m, 1H) 6.51 (s, 1H), 4.93 (s, 2H), 3.86 (s,
3H), 3.62–3.58 (m, 4H), 3.39 (s, 3H), 3.23 (s, 3H),
3.22–3.14 (m, 4H). MS (EI) m/z: EI/MS 537 [M]+, Anal.
Calcd for C27H28ClN5O5: C, 60.28; H, 5.25; N, 13.02.
Found: C, 59.89; H, 5.35; N, 13.30.
6.1.4.15. 6-{4-[2-Oxo-2-(4-phenylpiperidin-1-yl)eth-
oxy]phenyl}-1,3-dipropyl-1H-pyrrolo[3,2-d]pyrimidine-2,4
(3H,5H)-dione (76). Method B, Yield 48%, mp: >250 ꢁC,
1H NMR (CDCl3): 10.49 (s, 1H), 7.96–7.61 (m, 2H),
7.32–7.14 (m, 5H), 7.06 (d, J = 8.6 Hz, 2H), 6.18 (s,
1H), 4.79 (s, 2H), 4.29–4.27 (m, 1H), 4.14–4.13 (m,
1H), 4.08–3.91 (m, 4H), 3.21–3.17 (m, 1H) 2.72–2.71
(m, 2H), 2.17–1.68 (m, 10H), 1.03–0.91 (m, 6H). MS
(EI) m/z: 528[M]+, Anal. Calcd for C31H36N4O4: C,
70.43; H, 6.86; N, 10.60. Found: C, 70.23; H, 6.67; N,
10.40.
6.1.4.10. 6-{4-[2-Oxo-2-(4-phenylpiperazin-1-yl)eth-
oxy]phenyl}-1,3-dipropyl-1H-pyrrolo[3,2-d]pyrimidine-2,4
(3H,5H)-dione (40). Method B, Yield 29%, mp: 181–
1
183 ꢁC, H NMR (DMSO-d6): 12.15 (s, 1H), 7.86 (d,
J = 8.8 Hz, 2H), 7.27–7.23 (m, 2H), 7.02–6.98 (m, 4H),
6.85–6.81 (m, 1H), 6.66 (s, 1H), 4.96 (s, 2H), 3.89–3.85
(m, 4H), 3.65–3.61 (m, 4H), 3.23–3.19 (m, 2H), 3.16–
3.12 (m, 2H), 2.54–2.49 (m, 4H), 1.10–0.80 (m, 6H).
MS (EI) m/z: 529 [M]+, Anal. Calcd for C30H35N5O4:
C, 68.03; H, 6.66; N, 13.22. Found: C, 68.44; H, 6.97;
N, 13.43.
6.1.4.16. 6-{2-Methoxy-4-[2-oxo-2-(4-phenylpiperidin-
1-yl)ethoxy]phenyl}-1,3-dimethyl-1H-pyrrolo[3,2-d]pyri-
midine-2,4(3H,5H)-dione (79). Method C, Yield 53%,
1
6.1.4.11. 6-{4-[2-(4-Benzylpiperazin-1-yl)-2-oxoethoxy]
phenyl}-1,3-dipropyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,
mp: 230–232 ꢁC, H NMR (DMSO-d6): 11.79 (s, 1H),
7.70 (d, J = 8.8 Hz, 1H), 7.31–7.15 (m, 5H), 6.69–6.65
(m, 1H), 6.59–6.54 (m, 1H), 6.51 (s, 1H), 4.90 (s, 2H),
4.50–4.45 (m, 1H), 4.00–3.95 (m, 1H), 3.86 (s, 3H),
3.41 (s, 3H), 3.24 (s, 3H), 3.19–3.10 (m, 1H), 2.87–2.52
(m, 1H), 1.88–1.78 (m, 2H), 1.70–1.63 (m, 1H), 1.45–
1.43 (m, 1H). MS (EI) m/z: 502 [M]+, Anal. Calcd for
C28H30N4O5: C, 66.92; H, 6.02; N, 11.15. Found: C,
67.01; H, 6.24; N, 11.36.
1
5H)-dione (41). Method C, Yield 35%, mp: >250 ꢁC, H
NMR (DMSO-d6): 12.20 (s, 1H), 7.81 (d, J = 8.5 Hz,
2H), 7.34 (s, 5H), 6.95 (d, J = 8.5 Hz, 2H), 6.63 (s,
1H), 4.85 (s, 2H), 3.86–3.81 (m, 4H), 3.47 (s, 2H),
3.46–3.43 (m, 4H), 2.39–2.24 (m, 4H), 1.69–1.51 (m,
4H), 0.91–0.81 (m, 6H). MS (EI) m/z: 534 [M]+, Anal.
Calcd for C31H37N5O4: C, 68.49; H, 6.86; N, 12.88.
Found: C, 68.15; H, 6.53; N, 12.53.
6.1.4.17. 6-{4-[2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-
oxoethoxy]phenyl}-1,3-dipropyl-1H-pyrrolo[3,2-d]pyrimi-
dine-2,4(3H,5H)-dione (82). Method B, Yield 30%, mp:
174–176 ꢁC, H NMR (DMSO-d6): 12.01 (s, 1H), 7.62
(d, J = 8.7 Hz, 2H), 6.98 (s, 4H), 6.79 (d, J = 8.7 Hz,
2H), 6.43 (s, 1H), 4.77 (s, 2H), 3.60–3.54 (m, 4H),
3.26–3.22 (m, 2H), 2.67–2.60 (m, 2H), 2.31–2.27 (m,
2H), 1.46–1.37 (m, 4H), 1.10–0.80 (m, 6H). MS (EI)
m/z: 500 [M]+, Anal. Calcd for C29H32N4O4: C, 69.58;
H, 6.45; N, 11.20. Found: C, 69.89; H, 6.38; N, 11.32.
6.1.4.12. 1-Methyl-6-{4-[2-oxo-2-(4-phenylpiperidin-1-
yl)ethoxy]phenyl}-3-propyl-1H-pyrrolo[3,2-d]pyrimidine-
2,4(3H,5H)-dione (67). Method C, Yield 54%, mp: 225–
227 ꢁC, H NMR (DMSO-d6): 12.23 (s, 1H), 7.82 (d,
J = 8.8 Hz, 2H), 7.30–7.50 (m, 5H), 6.99 (d,
J = 8.8 Hz, 2H), 6.59 (s, 1H), 4.90 (s, 2H), 4.48–4.44
(m, 1H), 3.97–3.92 (m, 1H), 3.84 (t, J = 7.2 Hz, 2H),
3.79 (s, 3H), 3.17–3.09 (m, 1H), 2.86–2.54 (m, 3H),
1.93–1.80 (m, 2H), 1.70–1.42 (m, 2H), 0.85 (t,
J = 7.3 Hz, 3H). MS (EI) m/z: 501 [M+H]+, Anal. Calcd
for C28H31N5O4: C, 67.05; H, 6.23; N, 13.96. Found: C,
67.23; H, 6.32; N, 13.80.
1
1
6.1.4.18. 6-{4-[2-(3,4-Dihydroisoquinolin-2(1H)-yl)-2-
oxoethoxy]phenyl}-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimi-
dine-2,4(3H,5H)-dione (85). Method B, Yield 32%, mp:
1
6.1.4.13. 6-(4-{2-[4-(4-Fluorobenzoyl)piperidin-1-yl]-2-
oxoethoxy}phenyl)-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrim-
idine-2,4(3H,5H)-dione (73). Method C, Yield 40%, mp:
>250 ꢁC, H NMR (DMSO-d6): 12.14 (br s, 1H), 7.71
(d, J = 8.5 Hz, 2H), 7.07 (s, 4H), 6.89 (d, J = 8.5 Hz,
2H), 6.50 (s, 1H), 4.86 (s, 2H), 3.58–3.54 (m, 2H),
3.37–3.33 (m, 2H), 3.29 (s, 3H), 3.13 (s, 3H), 2.74–2.72
1
>250 ꢁC, H NMR (DMSO-d6): 12.29 (s, 1H), 8.13 (dd,

2868
A. Stefanachi et al. / Bioorg. Med. Chem. 16 (2008) 2852–2869
(m, 1H), 2.41–2.37 (m, 1H). MS (EI) m/z: 444.2 [M]+,
Anal. Calcd for C25H24N4O4: C, 67.55; H, 5.44; N,
12.60. Found: C, 67.67; H, 5.33; N, 12.43.
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Acknowledgment
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The authors thanks the MIUR (Ministero dell’Univer-
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found, in the online version, at. Supplementary data
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