New N6- or N(9)-hydroxyalkyl substituted 8-azaadenines or adenines as effective A1 adenosine receptor ligands.

Article abstract of DOI:10.1016/S0223-5234(03)00147-8

In this paper we describe synthesis and biological assays of some A(1) ligands more water-soluble than the effective, but very lipophilic, 8-azaadenines and adenines discovered in the past and obtained introducing on N(6) or N(9) substituent a hydroxy group. Five of the new N(6)-hydroxyalkyl- and N(6)-hydroxycycloalkyl-2-phenyl-9-benzyl-8-azaadenines showed very high affinity (Ki<40 nM) and selectivity for A(1) adenosine receptors. Among the 2-phenyl-9-(2-hydroxy-3-alkyl)-8-azaadenines or adenines prepared, the compounds with the higher A(1) affinity and selectivity resulted 2-phenyl-9-(2-hydroxy-3-propyl)-N(6)-cyclopentyl- and cyclohexyl-8-azaadenine with Ki 2.2+/-0.2 nM and 2.8+/-0.3 nM respectively. From the point of view of water-solubility, 2-phenyl-9-(2-hydroxy-3-propyl)-8-azaadenine was the most interesting compound, having a CLogP of 1.066991 and a water-solubility of 1.2 mg mL(-1).

Full text of DOI:10.1016/S0223-5234(03)00147-8

European Journal of Medicinal Chemistry 38 (2003) 801ꢁ810
/
www.elsevier.com/locate/ejmech
Original article
New N6- or N(9)-hydroxyalkyl substituted 8-azaadenines or adenines
as effective A1 adenosine receptor ligandsꢀ
Giuliana Biagi a, Irene Giorgi a,*, Michele Leonardi a, Oreste Livi a, Federica Pacchini a,
Valerio Scartoni a, Barbara Costa b, Antonio Lucacchini b
a Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6, I-56126 Pisa, Italy
b Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, via Bonanno 6, I-56126 Pisa, Italy
Received 22 April 2003; received in revised form 7 July 2003; accepted 10 July 2003
Abstract
In this paper we describe synthesis and biological assays of some A1 ligands more water-soluble than the effective, but very
lipophilic, 8-azaadenines and adenines discovered in the past and obtained introducing on N6 or N(9) substituent a hydroxy group.
Five of the new N6-hydroxyalkyl- and N6-hydroxycycloalkyl-2-phenyl-9-benzyl-8-azaadenines showed very high affinity (KiB
40
nM) and selectivity for A1 adenosine receptors. Among the 2-phenyl-9-(2-hydroxy-3-alkyl)-8-azaadenines or adenines prepared, the
/
compounds with the higher A1 affinity and selectivity resulted 2-phenyl-9-(2-hydroxy-3-propyl)-N6-cyclopentyl- and cyclohexyl-8-
azaadenine with Ki 2.29
/
0.2 nM and 2.890.3 nM respectively. From the point of view of water-solubility, 2-phenyl-9-(2-hydroxy-3-
/
propyl)-8-azaadenine was the most interesting compound, having a CLogP of 1.066991 and a water-solubility of 1.2 mg mLꢀ1
.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: N6-hydroxyalkyl-2-phenyl-9-benzyl-8-azaadenines;
azaadenines; A1 adenosine receptor ligands
2-phenyl-9-(2-hydroxy-3-alkyl)adenines;
2-phenyl-9-(2-hydroxy-3-alkyl)-8-
1. Introduction
adenosine receptor ligands are very interesting; hun-
dreds of agonists and antagonists have been assayed to
obtain some compounds useful in therapy. Regarding
A1 antagonists, some of these are studied as novel
potassium sparing diuretics with kidney-protection
properties, in dementia such as Alzheimer’s disease, in
cardiac therapy and in kidney disease [2].
In the last years we have synthesised a great number
of A1 adenosine receptor ligands varying the substituent
groups in the positions 3, 6 and 9 of 8-azaadenine or
adenine nucleus and some of these have been very
effective, having a Ki in the range of nanomolar units
A1 receptors are widely distributed in the central
nervous system and in peripheral tissue and mediate
diverse biological effects. For example the role of
adenosine as neuroprotective agent during hypoxia
and ischemic conditions seems to be mediate by the A1
receptors; these receptors have been implicated in
sedative, anticonvulsant, anxiolitic effects. A1 receptors
mediate cardiac depression through negative chrono-
tropic, dromotropic and inotropic effects. In the kidney,
activation of A1 receptors causes vasoconstriction,
inhibition of renine secretion, diuresis, natriuresis and
others effects [1]. A great number of other physiological
effects have been demonstrated so the studies on A1
[3ꢁ8]. All these compounds were characterised by a high
/
lipophilicity being the substituents on the bicyclic
nucleus, such as alkyl, cycloalkyl or alkyl-aromatic
groups, hydrophobic. This can make the compounds
nearly water-insoluble, causing the determination of
activity in the in vitro binding assays to be uncertain
in some cases. Low water-solubility also leads to a low
bioavailability of the compounds so they are unsuitable
for in vivo studies.
ꢀ A part of this work was presented at the 12th Camerino-
NordwijKherout Symposium: Trends in Drug Research, Camerino,
5ꢁ/9 September 1999.
* Correspondence and reprints:.
E-mail address: igiorgi@farm.unipi.it (I. Giorgi).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(03)00147-8

802
G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ810
/
The design of reasonably water-soluble A1 adenosine
A1 adenosine receptors, with Ki 28 and 2.8 nM,
respectively [17]; also their N6-cyclopentyl derivatives
were good ligands with Ki 5.5 and 4.3 nM respectively.
In these compounds a hydroxy function is bound to a
long alkylic chain in the 9 position of adenine or 8-
azaadenine nucleus. Given that a shorter chain could
decrease the lipophilicity of these compounds, we
decided to prepare and assay also the analogues 9-(2-
hydroxy-3-heptyl), 9-(2-hydroxy-3-pentyl) and 9-(2-hy-
droxy-3-propyl) substituted, to verify if these com-
pounds maintained high affinity for A1 receptors.
receptor antagonists has become a very important
objective in the development of this class of ligands.
In these last years A1 selective ligands with increased
water-solubility have been obtained, including xanthine
derivatives, such as 8-(1-aminocyclopentyl)-1,3-dipro-
pylxanthine I [9] (Fig. 1), non-xanthine derivatives,
like N6-hydroxy-endonorbornyl-8-(N-methyl-N-isopro-
pylamino)-9-methyladenine II [10] or FR 166124 III [11]
(Fig. 1), a 2-phenylpyrazolo[1,5-a]pyridine derivative,
developed by Kuroda and co-workers. Some authors
[12,13] have introduced various polar substituents at C-2
and C-4 position of pyrrolo[2,3-d]pyrimidine or pyri-
mido[4,5-b]indole structure to improve water-solubility
of some interesting A1 ligands synthesised in the past by
Muller and co-workers [14]. Recently other authors have
presented the pyrrolo[2,3-d]pirimidinamide IV (Fig. 1)
as a potent and relatively good water-soluble A1
2. Chemistry
The synthetic route to prepare compounds 3ꢁ9 (Fig.
/
2) employed a known two-step reaction in the presence
of sodium ethoxide: the first step was the 1,3 dipolar
addition reaction of benzylazide and cyanoacetamide to
antagonist (CLogPꢂ1.5) [15]. Therefore we planned
/
to obtain some A1 ligands more water-soluble than the
active compounds discovered in the past by introducing
on the molecules a polar function. Our first attempt has
been reported in a recent paper [16] where we described
synthesis of new 2-(2?-phenyl-9?-benzyl-8?-azapurin-6?-
ylamino)-carboxylic acid methylesters: the carbonylox-
ymethyl group could assure the molecules a better
water-solubility but the compounds demonstrated only
low capability of binding to the receptors.
With the same aim, in this paper we describe synthesis
and biological activity as A1 adenosine receptor ligands
of some compounds obtained by introducing polar N6-
hydroxyalkyl- or N6-hydroxycycloalkyl groups on 2-
phenyl-9-benzyl-8-azaadenines. Recently we have de-
scribed erythro-2-phenyl-9-(2-hydroxy-3-nonyl)adenine
and its 8-aza analogue which showed high affinity for
give
1-benzyl-4-carbamoyl-5-amino-1H-1,2,3-triazole
which was not isolated; then, in the same flask, ethyl
benzoate was added to obtain the 2-phenyl-9-benzyl-8-
azahypoxanthine 1 [18] by annulation reaction. The
reaction of 1 with phosphorus oxychloride afforded 6-
chloro-8-azapurine 2 which, by nucleophilic displace-
ment of chlorine atom operated by suitable amines [4],
gave the N6-substituted-8-azaadenines 3ꢁ
5-Amino-4,6-dichloro-2-phenylpyrimidine 10 was
used for synthesis of compounds 20ꢁ37 (Fig. 3); it was
/
9.
/
obtained by a modification of a known method [19]
from 5-nitro-4,6-dichloro-2-phenylpyrimidine [20] by
reduction with iron powder in a mixture of acetic acid,
water and THF.
Substitution of a chlorine atom in compound 10 with
the suitable 3-aminoalkan-2-ol gave 5-amino-4-(2-hy-
droxy-3-alkyl)-6-chloro-2-phenylpyrimidines 11ꢁ13. 3-
/
Aminopropan-2-ol is a commercial product (Fluka); 3-
aminopentan-2-ol and 3-aminoheptan-2-ol were ob-
tained from (DL)-2-amino-1-butanoic acid and (DL)-
2-amino-1-hexanoic acid respectively, by a Dakingꢁ
/
West reaction followed by a reduction with NaBH4 as
described by Schaeffer and Schwender to obtain 2-
amino-nonan-1-ol [21]. Gas-chromatography analysis of
the acetyl derivatives showed an erythroꢁthreo ratio of
/
80:20; the diastereoisomers were not separated. Cyclisa-
tion of 11, 12 and 13 with an acid solution of NaNO2
gave the 8-azaadenines 14ꢁ
triethyl orthoformate in the presence of catalytic 12N
HCl [23] gave the corresponding adenines 17ꢁ19.
Compounds 14ꢁ19 were immediately used for the next
reaction without purification because they easily decom-
pose. Treatment of 14ꢁ19 with the suitable amines gave
the N6-substituted 2-phenyl-9-(2-hydroxy-3-propyl)-8-
azaadenines 20ꢁ28 and adenines 29ꢁ37.
/
16 [22]. Cyclisation with
/
/
/
Fig. 1. Some water-soluble A1 adenosine receptor antagonists re-
ported in the literature.
/
/

G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ
/810
803
Fig. 2. Synthetic route for the preparation of compounds 3ꢁ9.
/
3. Biological evaluation
for affinity at A1 and A2A adenosine receptors in bovine
brain cortical membranes and bovine striatum respec-
tively. In Tables 1 and 2 the results of A1 adenosine
receptor binding are reported; in the A2 adenosine
Substituted 8-azaadenines 3ꢁ
/
9 and 20ꢁ28 and ade-
/
nines 29ꢁ37 were tested in radioligand binding assays
/
Fig. 3. Synthetic route for the preparation of compounds 11ꢁ37.
/

804
G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ810
/
Table 1
Biological results for affinity towards A1 adenosine receptors and calculated partition coefficients (CLogP) of the compounds 3ꢁ
/
9 and the
compounds AꢁE assayed in the past [6,8]
/
substituent on N6 increases affinity of the unsubstituted
compound; biological results of this work showed that
compounds bearing on the N6-aliphatic substituent a
hydroxy function have affinity very similar to the
corresponding dehydroxy compounds (see in Table 1:
3 versus A; 4 and 5 versus B; 6 and 7 versus C and D; 8
and 9 versus E) and, in some cases, better. So we can
deduce that a hydroxy function is well tolerated by the
receptor in the lipophilic pocket in front of N6 of
adenosine. It is worth noting that the steric relation
between the nitrogen atom and the hydroxy group is
trans-diequatorial in the case of 2?-hydroxycyclohexyl
substituted compound 9. We can also see that, moving
OH from positions 2? to 3? (compounds 4 and 5) or from
2? to 4? (compounds 8 and 9), Ki does not vary
significantly. It could mean that this pocket is quite
large and permits different types of interactions. In a
recent paper [24], the study of a model of A1 adenosine
receptor suggested that this protein could modify the
side chain orientation of some aminoacids in the binding
site so that they give rise to the best interaction with the
ligand. The results of the present work can support this
receptor binding assays, all the compounds showed
inhibition values B60% at 1 mM, so were considered
inactive.
/
4. Results and discussion
4.1. Structureꢁactivity relationships
Five of the N6-hydroxyalkyl substituted compounds
/
(3, 4, 5, 8, 9) showed very high affinity (KiB40 nM) and
selectivity for A1 adenosine receptors and this fact
allowed us to make some evaluations about structureꢁ
/
/
activity relationships, also comparing the affinity of
these compounds with that of the corresponding dehy-
droxy ones obtained in the past and indicated by capital
letters (see Table 1). In our compounds, when the
substituent in the 9-position is a benzyl group, an
aromatic substituent in N6 is not allowed. This is
suggested by the very low affinity of compounds 6, 7,
C and D. On the contrary, it is known that an aliphatic

G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ
/810
805
Table 2
Biological results for affinity towards A1 adenosine receptors and calculated partition coefficients (CLogP) of the compounds 20ꢁ
/37 and the
compounds Fꢁ
/
M assayed in the past [9]
Compd
R
H
R1
H
Ki
269
CLogP
Compd
R
R1
Ki
CLogP
20
21
22
F
/2
1.06991
1.90791
2.96591
4.13142
3.36836
4.20636
5.26436
6.43401
3.92736
4.76536
5.82336
6.9931
29
30
31
I
H
H
2639
899
1889
289
229
/
23
1.17742
2.01542
3.07342
4.02391
3.48001
4.31801
5.37601
6.32236
4.03901
4.87701
5.93501
6.88138
a
C2H5
H
C2H5
n-C4H9
n-C6H13
H
H
/8
n-C4H9
n-C6H13
H
H
1229
2.89
2.29
259
/
10
0.3
0.2
2
H
/
15
2 b
2
b
H
/
H
/
23
24
25
G
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
/
32
33
34
L
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclohexyl
cyclohexyl
cyclohexyl
cyclohexyl
/
C2H5
/
C2H5
n-C4H9
n-C6H13
H
539
779
/5
n-C4H9
n-C6H13
H
239
/
3
/6
4.39
2.89
389
/
0.5 b
0.3
24
5.59
259
1259
659
1169
/
0.5
2 b
13
6
26
27
28
H
/
35
36
37
M
/
C2H5
n-C4H9
n-C6H13
/
C2H5
n-C4H9
n-C6H13
/
319
/
4
/
1189
/
12 b
/
10 b
a
Inhibition: 53% 10 mM.
See reference [9].
b
interesting hypothesis which allowed us to see receptors
not as a rigid structure but as a versatile part of the
cellular membrane.
Regarding compounds with the hydroxy group on
N(9) substituent, the new compounds were compared
with the analogues synthesised in the past, indicated by
capital letters in Table 2, having a 2-hydroxy-3-nonyl
chain. We can see in Table 2 that in the case of 2-phenyl-
9-(2-hydroxy-3-alkyl)-8-azaadenines or adenines N6-cy-
considered very important from the point of view of
water-solubility because its CLogP is 1.066991 (P$
1.68), indicating that this compound has a fairly good
solubility in water. In fact, for this compound water-
solubility was assayed and resulted 1.2 mg mLꢀ1 (4.44
mM).
/
5. Conclusion
clopentyl substituted (compounds 23ꢁ
32ꢁ34 versus L) a shorter chain retains a good affinity
for the receptors. The compounds with the higher
affinity were 23 and 26 with Ki 2.290.2 nM and 2.89
0.3 nM respectively; for N6-cyclohexyl substituted
derivatives (compounds 26ꢁ28 versus H and 35ꢁ37
versus M), we can see a general increase in affinity. In
the case of N6-unsubstituted compounds the biological
results showed a general decrease in affinity; never-
theless in this series compound 20 retains a good affinity
/
25 versus G and
Introduction of a hydroxy function on N6 or N(9)
substituents of 2-phenyl-9-benzyl-8-azaadenines and
adenines improves solubility and effectiveness towards
A1 adenosine receptors of these molecules which could
represent a useful tool for pharmacological and biolo-
gical studies.
/
/
/
/
/
6. Experimental protocols
(Ki 2692 nM).
/
6.1. Chemistry
4.2. Water-solubility
For all compounds octanolꢁ
Melting points were determined on a Kofler hot-stage
apparatus and are uncorrected. IR spectra in Nujol
/water partition coeffi-
cient (CLogP) was calculated (Tables 1 and 2) using
ChemDraw Ultra version 6.0.1 program. All the new
compounds showed lower values than the compounds of
the past cited in this paper. Compound 20 which showed
a good affinity for A1 adenosine receptors can be
mulls were recorded on a PerkinꢁElmer Model 1310
/
spectrometer. 1H-NMR spectra were recorded on a
Varian Gemini 200 spectrometer; chemical shifts are
expressed in d units from TMS as an internal standard;
the compounds were dissolved in CDCl3 or DMSO-d6.

806
G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ810
/
Table 3
Reaction and physico-chemical data for compounds 3ꢁ
/
9, 11ꢁ
/
13 and 21ꢁ
/37
Compd.
Yield
Crystallisation solvents
Chromatography eluent
M.p. (8C)
Formula (anal.)
3
4
51
61
62
35
36
30
30
65
68
56
20
52
88
57
48
54
61
44
30
45
60
73
71
62
67
67
68
32
CH2Cl2 ꢁ
/
petroleum ether
198ꢁ
195ꢁ
191ꢁ
205ꢁ
180ꢁ
185ꢁ
180ꢁ
/
200
197
193
206
182
187
181
C19H18N6OH2O (C, H, N)
C20H20N6O (C, H, N)
C20H20N6O (C, H, N)
C25H22N6O (C, H, N)
C26H24N6O (C, H, N)
C23H24N6O (C, H, N)
C23H24N6O (C, H, N)
C13H15ClN4O (C, H, N)
C15H19ClN4O (C, H, N)
C17H23ClN4O (C, H, N)
C13H14N6O (C, H, N)
C15H18N6O (C, H, N)
C17H22N6O (C, H, N)
C18H22N6O (C, H, N)
C20H26N6O (C, H, N)
C22H30N6O (C, H, N)
C19H24N6O (C, H, N)
C21H28N6O (C, H, N)
C23H32N6O (C, H, N)
C14H15N5O (C, H, N)
C16H19N5O (C, H, N)
C18H23N5O (C, H, N)
C19H23N5O (C, H, N)
C21H27N5O (C, H, N)
C23H31N5O (C, H, N)
C20H25N5O (C, H, N)
C22H29N5O (C, H, N)
C24H33N5O (C, H, N)
CHCl3 ꢁpetroleum ether
/
/
5
ethyl acetate
isopropanol
/
6
/
7
EtOHꢁ
EtOHꢁ
EtOH
EtOHꢁ
/
hexane
/
8
/H2O
/
9
/
11
12
13
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
/Et2O
165
oil
CHCl3 100%
CHCl3 ꢁMeOH 95:5
/
oil
EtOHꢁ
EtOH
EtOH
/Et2O
188ꢁ
219
oil
/
190
CH2Cl3 ꢁ/MeOH 96:4
CHCl3 ꢁMeOH 96:4
/
141
oil
CHCl3 100%
CHCl3 100%
oil
155
oil
CHCl3 100%
CHCl3 100%
133
224
171
oil
EtOHꢁ
EtOH
EtOH
/Et2O
CH2Cl3 ꢁ/MeOH 96:4
CHCl3 ꢁMeOH 98:2
/
207
oil
AcOEtꢁ/petroleum ether 1:5
AcOEtꢁ/petroleum ether 1:5
oil
223
153
oil
AcOEtꢁ/petroleum ether 1:5
AcOEtꢁ/petroleum ether 1:5
Mass spectra were performed on a Hewlett-Packard
GC/MS System 5988A. TLC was performed on pre-
coated silica gel F254 plates (Merck). Flash-column
chromatographies were performed using Merck Kiesel-
mmol) and POCl3 (3.0 mL, 32.55 mmol) was heated at
90 8C for 4 h then was evaporated at reduced pressure.
The residue was crystallised from chloroformꢁether to
/
give the title compound (0.7 g, 2.18 mmol, 67% yield).
M.p. 184 8C. 1H-NMR (CDCl3) d: 5.94 (s, 2H, N-CH2);
7.38 (m, 3H, arom); 7.57 (m, 5H, arom); 8.59 (m, 2H,
arom).
gel 60 (230ꢁ400 mesh). GLC analyses of compounds 3-
/
aminopentan-2-ol and 3-aminoheptan-2-ol were per-
formed after transformation into the corresponding
acetate derivatives prepared as follows: 10 mg of
compound in anhydrous pyridine (1 mL) was treated
with acetic anhydride (1 mL) at 100 8C for 20 min. An
aliquot of the reaction mixture was gas-chromato-
graphed on a Carlo-Erba mod. 4200 apparatus using a
6.1.3. General procedure to prepare 6-aminosubstituted-
2-phenyl-9-benzyl-8-azaadenines 3ꢁ9
/
In a well-stoppered flask a mixture of 2 (0.2 g, 0.62
mmol), toluene (2 mL), the suitable amine (1 mmol) and
N,N-diethylaniline (0.09 g, 0.63 mmol) was heated at
130 8C for 16 h (72 h for compound 10). Then the
solution was evaporated at reduced pressure, diluted
with chloroform and washed with 10% HCl and water.
After evaporation of the organic layer the residue was
crystallised or chromatographed (see Tables 3 and 4).
glass column (1.5 mꢃ
neopentyl glycol succinate on 80ꢁ
Chromosorb W and a FID detector.
/
2.8 mm) packed with 10%
/100 mesh silanised
Microanalyses (C H N) were carried out on a Carlo
Erba elemental analyser (Model 1106) and were within
9
/
0.4% of the theoretical values. Petroleum-ether corre-
sponds to fraction boiling at 40ꢁ60 8C.
/
6.1.4. 5-Amino-4,6-dichloro-2-phenylpyrimidine (10)
This compound was prepared according to a de-
scribed procedure [17].
6.1.1. 2-Phenyl-9-benzyl-8-azahypoxanthine (1)
This compound was prepared according to a de-
scribed procedure [18].
6.1.5. General procedure for 3-aminopentan-2-one or 3-
aminoheptan-2-one
A mixture of 125 mmol of D,L-2-amino-1-butanoic
6.1.2. 6-Chloro-2-phenyl-9-benzyl-8-azaadenine (2)
A mixture of 2-phenyl-9-benzyl-8-azahypoxanthine
(1.0 g, 3.3 mmol), N,N-diethylaniline (0.8 g, 5.36
acid or D,L-2-amino-1-hexanoic acid, pyridine (66 mL)

G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ
/810
807
Table 4
1H-NMR and mass spectra of synthesised compounds 3ꢁ
/
9, 11ꢁ
/
13 and 21ꢁ37
/
Compound
(solvent)
1H-NMR
MS (m/e)
Aromatic H
Benzylic H
5.76 (s, 2H)
Aliphatic H
3.97 (m, 4H)
Exchang. H
6.78 (1H)
3 (CDCl3)
4 (CDCl3)
5 (CDCl3)
6 (CDCl3)
7 (CDCl3)
8 (CDCl3)
9 (CDCl3)
8.45 (m, 2H); 7.53ꢁ
/
346 (Mꢄ, 3.5); 316 (30);
91 (100)
7.15 (m, 8H)
8.45 (m, 2H); 7.54ꢁ
7.28 (m, 8H)
/
5.76 (s, 2H)
5.80 (s, 2H)
1.33 (d, 3H); 4.01 (m, 2H); 4.19 (m, 1H) 6.53 (1H)
360 (Mꢄ, 8.3); 316 (18);
91 (100)
8.45 (m, 2H); 7.51ꢁ
7.25 (m, 8H)
/
1.94 (m, 2H); 3.67 (m, 2H); 4.01 (m, 2H) 3.53 (1H); 6.72
(1H)
360 (Mꢄ, 2.5); 316 (5);
91 (100)
8.46(m, 2H); 7.49ꢁ
(m, 8H)
8.52 (m, 2H); 7.51ꢁ
7.33 (m, 8H)
/
7.29 5.77 (s, 2H);
3.90 (m, 2H)
4.20 (1H); 6.87
(1H)
316 (23); 287 (15); 91
(100)
5.11 (m, 1H)
/
5.81 (s, 2H);
5.15 (m, 1H)
5.69 (s, 2H)
1.28 (d, 3H); 5.0 (m, 1H)
6.35 (1H); 4.29
(1H)
329 (32); 301 (2); 91
(100)
8.40(m, 2H); 7.42ꢁ
7.20(m, 8H)
/
1.40ꢁ
(m, 2H); 3.74 (m, 1H); 4.35 (m, 1H)
1.45ꢁ1.57 (m, 4H); 1.81 (m, 2H); 2.20
(m, 2H); 3.64 (m, 1H); 4.23 (m, 1H)
/
1.62 (m, 4H); 2.10 (m, 2H); 2.30
4.77 (1H); 6.01
(1H)
400 (Mꢄ, 2.1); 302 (9);
273 (19); 91 (100)
400 (Mꢄ, 1); 303 (15);
273 (5); 91 (100)
278 (Mꢄ, 10); 233 (36);
45(100)
8.45 (m, 2H); 7.52ꢁ
/
5.61 (s, 2H)
/
4.68 (1H); 6.68
(1H)
7.26 (m, 8H)
11 (DMSO-d6) 8.16 (m, 2H); 7.40 (m,
1.13 (d, 3H); 3.47 (m, 2H); 3.94 (m, 1H) 4.83 (1H); 5.22
(2H): 6.95 (1H)
3H)
8.25 (m, 2H); 7.46 (m,
3H)
12 (CDCl3)
13 (CDCl3)
20 (CDCl3)
1.05 (t, 3H); 1.21 (d, 3H); 1.64 (m, 2H); 5.13 (1H)
4.09 (m, 1H); 4.35 (m, 1H)
7.94 (m, 2H); 7.52 (m,
3H)
0.89 (t, 3H); 1.20ꢁ1.60 (m, 9H); 3.12
/
(m,1H); 4.34 (m, 1H)
1.13 (d, 3H); 3.32 (d, 2H); 4.45 (m, 1H) 5.20 (1H); 6.08
(2H)
8.37 (m, 2H); 7.49 (m,
3H)
21 (DMSO-d6) 8.15 (m, 2H); 7.59 (m,
270 (Mꢄ, 2); 213 (36);
104 (100)
0.70 (t, 3H); 0.91 (d, 3H); 2.20 (m, 2H); 1.23 (1H)
4.15 (m, 1H); 4.54(m, 1H)
254 (5);104 (45); 45
(100)
3H)
8.36 (m, 3H); 7.48 (m,
2H)
22 (CDCl3)
23 (CDCl3)
24 (CDCl3)
25 (CDCl3)
0.84 (t, 3H); 1.04ꢁ
1H); 4.92 (m, 1H)
1.37 (d, 3H); 1.65ꢁ
1H); 4.62 (m, 1H); 4.84 (m, 2H)
0.83 (t, 3H); 1.05ꢁ2.30 (m, 13H); 4.44
(m, 2H); 4.81 (m, 1H);
0.84 (t, 3H); 0.95ꢁ2.26 (m, 17H); 4.15ꢁ
4.46 (m, 2H); 4.80 (m, 1H)
1.15 (d, 3H); 1.40ꢁ2.00 (m, 10H); 4.28ꢁ
4.50 (m, 4H)
0.83ꢁ
(m, 1H); 4.43 (m,1H); 4.80 (m,1H)
0.84 (t, 3H); 1.22ꢁ2.25 (m, 19H); 4.15ꢁ
6.43 (m, 1H); 5.85 408 (Mꢄ, 2.2); 391 (4);
/2.34 (m, 9H); 4.45 (m, 5.32 (1H); 6.48
326 (Mꢄ, 3); 213 (20);
104 (30); 83 (100)
338 (Mꢄ, 20); 270 (60);
225 (97); 104 (100)
366 (Mꢄ, 2); 149 (27);
41 (100)
(2H)
8.45 (m, 2H); 7.50 (m,
3H);
/
2.23 (m, 8H); 4.45 (m, 5.36 (1H); 6.35
(1H)
8.49 (m, 2H); 7.40
(m,3H);
/
4.80 (1H); 6.43
(1H)
8.42 (m, 2H); 7.49 (m,
3H)
26 (DMSO-d6) 8.42 (m, 2H); 7.51 (m,
/
/
5.71 (1H); 6.34
(1H)
394 (Mꢄ, 2); 322 (11);
104 (31); 45 (100)
352 (Mꢄ, 3); 104 (31);
45 (100)
/
/
5.50 (1H); 8.70
(1H)
3H);
8.40 (m, 2H); 7.50 (m,
3H);
27 (CDCl3)
28 (CDCl3)
/2.30 (m, 18H); 4.31 (m, 1H); 4.31 5.72 (1H); 6.29
380 (Mꢄ, 2); 295 (30);
149 (57); 43 (100)
(1H)
8.42 (m, 2H); 7.48 (m,
3H)
/
/
4.46 (m, 2H); 4.41 (m, 2H); 4.83 (m, 1H) (m, 1H)
1.09 (d, 3H); 3.32 (d, 2H); 4.12 (m, 1H) 4.12 (1H); 7.25
(1H)
104 (61); 41 (100)
269 (Mꢄ, 7.6); 104 (47);
45 (100)
29 (DMSO-d6) 8.38 (m, 2H); 8.07 (s,
1H); 7.46 (m, 3H)
30 (DMSO-d6) 8.33 (m, 2H); 8.16 (s,
1H); 7.43 (m, 3H)
0.67 (t, 3H); 0.94 (d, 3H); 2.12 (m, 2H); 5.21 (1H); 7.26
297 (Mꢄ, 2); 212 (20);
45 (100)
4.16 (m, 2H)
(2H)
1.36 (m, 7H); 2.12 (m, 6.05 (1H); 6.12
(1H)
31 (CDCl3)
32 (CDCl3)
33 (CDCl3)
35 (CDCl3)
36 (CDCl3)
37 (CDCl3)
8.31 (m, 2H); 7.72 (s,
1H); 7.44 (m, 3H)
8.42 (m, 2H); 7.71 (s,
1H); 7.48 (m, 3H)
8.34 (m, 2H); 7.64 (s,
1H); 7.42 (m, 3H)
8.39 (m, 2H); 7.71 (s,
1H); 7.46 (m, 3H)
8.33 (m, 2H); 7.65 (s,
1H); 7.44 (m, 3H)
8.36 (m, 2H); 7.67 (s,
1H); 7.46 (m, 3H)
0.84 (t, 3H); 1.18ꢁ
2H); 4.30 (m, 2H)
/
325 (Mꢄ, 2.4); 212 (40);
45 (100)
1.32 (d, 3H); 1.60ꢁ
/
2.30 (m, 8H); 4.28 (m, 5.84 (2H)
337 (Mꢄ, 28); 269 (100);
211 (99)
3H); 4.80 (m, 1H)
0.80 (m, 3H); 1.33ꢁ
(m, 1H); 4.35 (m, 1H); 4.76 (m, 1H)
/
2.21 (m, 13H); 4.07 5.81 (1H); 6.66
(1H)
5.74 (2H)
365 (Mꢄ, 20); 211(100);
104 (47)
1.32 (d, 3H); 1.40ꢁ
(m, 4H)
0.82 (t, 3H); 1.35ꢁ
(m, 1H); 4.36 (m, 2H)
0.86 (t, 3H); 1.10ꢁ2.30 (m, 19H); 4.15
(m, 3H)
/2.20 (m, 10H); 4.32
351 (Mꢄ, 21); 269 (100);
211 (74)
/2.31 (m, 15H); 4.04
5.73 (1H); 6.68
(1H)
379 (Mꢄ, 7); 211 (29);
41 (100)
/
5.85 (1H); 6.75
(1H)
407 (Mꢄ, 19); 211 (58);
45 (100)
and acetic anhydride (100 mL) was heated at 100 8C for
3 h. After evaporation of the mixture, an oil was
obtained which was partitioned with ethyl ether and
5% NaHCO3. The organic layer was evaporated in
vacuo to give an oil (3-acetamidopentan- or hexan-2-
one) which was treated with concentrated HCl (160 mL)

808
G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ810
/
at 120 8C for 2 h. Evaporation of the mixture gave title
compounds which were crystallised from ethanolꢁethyl
ether. 3-Aminopentan-2-one: m.p. 165 8C; 3-aminohep-
tan-2-one: m.p. 125 8C.
solution of NaNO2 (84 mg, 1.22 mmol) in 3.6 mL of
H2O was added dropwise. The mixture was stirred for 3
h at 0 8C and for 2 h at room temperature, then
evaporated in vacuo to give the crude title compound
which was used without purification for the next
reaction.
/
6.1.6. General procedure for 3-aminopentan-2-ol or 3-
aminoheptan-2-ol
To an iced solution of 3-aminopentan-2-one or 3-
aminoheptan-2-one (18.5 mmol) in MeOH (20 mL),
NaBH4 (0.7 g, 37.0 mmol) was slowly added maintain-
6.1.11. 6-Chloro-9-(2-hydroxy-3-pentyl)-2-phenyl-8-
azapurine (15)
Title compound was obtained as described for 14 and
used without purification for the next reaction.
ing the pH$
/
5ꢁ6 by addition of glacial acetic acid. The
/
reaction mixture was stirred at room temperature for 20
h, then evaporated. The residue was solved in NaOH (10
mL) and the solution was extracted with CHCl3.
Evaporation of the organic layer gave title compounds
as an oil (GLC, NMR), which were used without
purification for the next reactions.
6.1.12. 6-Chloro-9-(2-hydroxy-3-heptyl)-2-phenyl-8-
azapurine (16)
Title compound was obtained as described for 14 and
used without purification for the next reaction.
6.1.13. 6-Chloro-9-(2-hydroxy-3-propyl)-2-phenylpurine
(17)
6.1.7. 5-Amino-6-chloro-4-(2-hydroxy-3-propyl)-2-
phenylpyrimidine (11)
A solution of 11 (0.20 g, 0.72 mmol), triethyl
orthoformate (3 mL) and 12N HCl (0.14 mL) was
stirred at room temperature for 12 h; then the reaction
mixture was evaporated and used without purification
for the next reaction.
A mixture of 10 (0.15 g, 0.63 mmol), erythro-3-
aminopropan-2-ol (0.28 g, 3.73 mmol), N,N-diethylani-
line (0.09 g, 0.63 mmol) in absolute ethanol (5 mL) was
heated at 110 8C for 12 h in a steel bomb. Evaporation
of the mixture gave an oil which was diluted with ethyl
acetate. The solution was washed with 10% HCl, 5%
NaHCO3 and water, then evaporated. The residue was
6.1.14. 6-Chloro-9-(2-hydroxy-3-pentyl)-2-phenylpurine
(18)
Title compound was obtained as described for 17 and
used without purification for the next reaction.
flash-chromatographed using CH2Cl2ꢁ
eluent to give compound 11 (0.115 g, 0.41 mmol) which
was crystallised with ethanolꢁethyl ether (see Tables 3
and 4).
/
MeOH 97:3 as
/
6.1.15. 6-Chloro-9-(2-hydroxy-3-hexyl)-2-phenylpurine
(19)
Title compound was obtained as described for 17 and
used without purification for the next reaction.
6.1.8. 5-Amino-6-chloro-4-(2-hydroxy-3-pentyl)-2-
phenylpyrimidine (12)
A mixture of 10 (0.80 g, 3.35 mmol), 3-aminopentan-
2-ol (0.517 g, 5.02 mmol), N,N-diethylaniline (0.50 g,
3.35 mmol) in pentanol (7 mL) was heated at 170 8C for
72 h in a steel bomb. Evaporation of the mixture gave an
oil which was flash-chromatographed using CHCl3 as
eluent to give compound 12 (0.70 g, 2.29 mmol, 68%
yield, see Tables 3 and 4).
6.1.16. General procedure to prepare 6-aminosubstituted-
9-(2-hydroxy-3-alkyl)-2-phenyl-8-azapurines 20ꢁ
6-aminosubstituted-9-(2-hydroxy-3-alkyl)-2-
phenylpurines 29ꢁ37
/
28 and
/
A mixture of 6-chloro-9-(2-hydroxy-3-alkyl)-2-phen-
yl-8-azapurine or 6-chloro-9-(2-hydroxy-3-alkyl)-2-phe-
nylpurine (0.27 mmol), 5 mL of anhydrous ethanol
and the suitable amine (1.5 mmol) was heated in a
sealed tube at 120 8C for 24 h. After evaporation, the
residue was diluted with chloroform, washed with 10%
HCl and evaporated. The residue was chromatographed
and/or crystallised to give pure products (see Tables 3
and 4).
6.1.9. 5-Amino-6-chloro-4-(2-hydroxy-3-heptyl)-2-
phenylpyrimidine (13)
A mixture of 10 (0.80 g, 3.35 mmol), 3-aminoheptan-
2-ol (0.65 g, 5.02 mmol), N,N-diethylaniline (0.5 g, 3.35
mmol) in pentanol (7 mL) was heated at 170 8C for 72 h
in a steel bomb. Evaporation of the mixture gave an oil
which was flash-chromatographed using CHCl3ꢁMeOH
/
95:5 as eluent to give compound 13 (0.63 g, 1.88 mmol,
see Tables 3 and 4).
6.2. Water solubility
6.1.10. 6-Chloro-9-(2-hydroxy-3-propyl)-2-phenyl-8-
azapurine (14)
To an ice cold solution of 11 (0.33 g, 1.17 mmol), H2O
(3.6 mL), glacial acetic acid (1.6 mL) and THF (7 mL), a
Bidistilled water (3 mL) was saturated with 10 mg of
compound 20 by heating. After cooling at room
temperature, the undissolved residue was filtered off
and 1 mL of solution was lyophilised to give 1.2 mg,

G. Biagi et al. / European Journal of Medicinal Chemistry 38 (2003) 801ꢁ
/810
809
corresponding to a solubility of 4.44 mM at room
temperature.
inhibition of specific [3H]CHA or [3H]CGS 21680
binding (IC50) were determined from semilog plots of
data from experiments of binding inhibition. The Ki
values were calculated from the IC50 values using the
6.3. Biochemical assays
equation IC50/(L/Kd) [26] ([3H]CHA Kdꢂ
/
10.5 nM and
5 nM);
6.3.1. A1 receptor binding
Lꢂ 1 nM and Lꢂ
/
1.2 nM; [3H]CGS 21680 Kdꢂ
protein estimation was based on the method reported
[27] using bovine serum albumin as standard.
/
/
Bovine cerebral cortex was homogenised in ice-cold
0.32 M sucrose containing protease inhibitors, as
previously described [25]. The homogenate was centri-
fuged at 1000g for 10 min at 4 8C and the supernatant
again centrifuged at 48 000g for 15 min at 4 8C. The
final pellet was dispersed in 10 volumes of fresh buffer,
incubated with adenosine deaminase (2 units mLꢀ1) to
remove endogenous adenosine at 37 8C for 60 min, and
then recentrifuged at 48000g for 15 min at 4 8C. The
pellet was suspended in buffer and used in the binding
assay.
Acknowledgements
This research was supported by Italian MIUR
(Ministero Istruzione Universita` Ricerca).
The [3H]CHA binding assay was performed in
triplicate by incubating aliquots of the membrane
References
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0.5 mL of Trisꢁ
/
0.3 mg of protein) at 25 8C for 45 min in
HCl, pH 7.7, containing 2 mM MgCl2,
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binding was defined in the presence of 50 mM R-PIA.
Binding reactions were terminated by filtration through
Whatman GF/C filters under reduced pressure. Filters
were washed three times with 5 mL of ice-cold buffer
and placed in scintillation vials. Radioactivity was
counted in a 4 mL Beckman Ready-Protein scintillation
cocktail in a scintillation counter.
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/
/
/
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