Study on affinity profile toward native human and bovine adenosine receptors of a series of 1,8-naphthyridine derivatives

Article abstract of DOI:10.1021/jm030977p

A new series of 1,8-naphthyridine derivatives (29-44 and 46-52) bearing various substituents in different positions on the heterocyclic nucleus were synthesized in order to analyze the effects produced on the affinity toward the bovine adenosine receptors

Full text of DOI:10.1021/jm030977p

J . Med. Chem. 2004, 47, 3019-3031
3019
Stu d y on Affin ity P r ofile tow a r d Na tive Hu m a n a n d Bovin e Ad en osin e
Recep tor s of a Ser ies of 1,8-Na p h th yr id in e Der iva tives
Pier Luigi Ferrarini,*,† Laura Betti,‡ Tiziana Cavallini,† Gino Giannaccini,‡ Antonio Lucacchini,‡
Clementina Manera,† Adriano Martinelli,† Gabriella Ortore,† Giuseppe Saccomanni,† and Tiziano Tuccinardi†
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6, 56126 Pisa, Italy, and Dipartimento di Psichiatria,
Neurobiologia, Farmacologia e Biotecnologie, Universita` di Pisa, via Bonanno 6, 56126 Pisa, Italy
Received J uly 29, 2003
A new series of 1,8-naphthyridine derivatives (29-44 and 46-52) bearing various substituents
in different positions on the heterocyclic nucleus were synthesized in order to analyze the effects
produced on the affinity toward the bovine adenosine receptors. These derivatives represent
an extension of our previous work on this class of compounds with high affinity toward A1
adenosine receptors.19 The results of radioligand binding assays indicate that a large number
of the 1,8-naphthyridine derivatives proved to be A1 selective, with a high affinity toward bovine
adenosine receptors in the low nanomolar range, and one (29) in the subnanomolar range.
Furthermore, the new series of 1,8-naphthyridine derivatives (29-44 and 46-52), together
with the analogous derivatives 1-28 previously studied,19 were tested to evaluate their affinity
toward human cortical A1 receptors and human striatal A2A receptors. The results indicate
that all the 1,8-naphthyridine compounds generally possess a higher affinity toward the bovine
A1 receptor compared with the human A1 receptor. As regards the affinity toward the A2A bovine
receptor, only a few compounds possess a moderate affinity, which for some compounds
remained approximately the same toward the A2A human receptor. A molecular modeling study
of the docking of the 1,8-naphthyridine compounds with both the bovine and the human A1
adenosine receptors was carried out with the aim of explaining the marked decrease in the
affinity toward human A1 adenosine receptors in comparison with bovine A1 adenosine receptors.
This study indicated that the structural differences, albeit small, of the active sites of the two
receptors make differences in the dimensions of the site and this influenced the ability of the
title compounds to interact with the two A1 receptors.
In tr od u ction
heterocyclic compounds have been reported to possess
an antagonistic activity at adenosine receptors, includ-
ing xanthine, adenines, 7-deazaadenines, 7-deaza-8-
azapurines,7-12 pyrazolo[3,4-c]quinolines,13 pyrazolo[1,5-
a]pyridines,14 triazoloquinoxaline,15 triazoloquinazoline
(e.g. CGS 15943), pyrazolotriazolopyrimidine,16,17 and
triazinobenzimidazolones.18 Recently19 we have under-
taken a systematic research program involving the
synthesis and testing of a series of 1,8-naphthyridine
derivatives (1-28; see Table 1) bearing a phenyl group
at position 2 and various substituents at positions 4 and
7, to evaluate their affinity for the bovine A1, A2A, and
A3 adenosine receptor subtypes. In binding to bovine
brain cortical membranes, most of the compounds
showed an affinity for A1 receptors in the low nanomolar
range and two (15 and 16) in the subnanomolar range
with an interesting degree of A1 versus A2A and A3
selectivity. Moreover, the two most potent and selective
derivatives in the binding assays, 16 and 21, were also
shown to be full antagonists toward A1 receptors.
Adenosine receptors from different species show a
good amino acid sequence homology (82-93%), the only
exception being the A3 subtype which only exhibits 74%
primary sequence homology between rat and human or
sheep.20-22 Although there is only little difference in the
A1 receptor sequence of different species,11 some species
differences in agonist binding have been reported.23 The
bovine A1 receptor has an affinity for agonist and
antagonist ligands that is 10-fold higher than that of
Adenosine, which is formed from the purine base
adenine and the ribose moiety, is a ubiquitous neuro-
modulator in both the periphery and the central nervous
system. Part of the biological activity of adenosine occurs
through the activation of specific cell membrane recep-
tors belonging to the extensive family of G-protein-
coupled receptors.1,2 Currently, four adenosine receptors
have been cloned and characterized pharmacologically,
namely A1, A2A, A2B, and A3. The adenosine receptors
are associated with different second messenger sys-
tems: A1 and A3 mediate adenylate cyclase inhibition,
whereas A2A and A2B stimulate adenylate cyclase activ-
ity by controlling intracellular cyclic AMP levels.1 The
discovery of adenosine receptor subtypes opened up new
avenues for potential drug treatment of a variety of
conditions such as asthma, neurodegenerative disorders,
psychosis and anxiety, chronic inflammatory diseases,
and many other physiopathological states that are
believed to be associated with changes in adenosine
levels.3-6 Consequently, selective and potent agonists
or antagonists at the human receptor subtypes are
needed for therapeutic intervention.
In the past few years, a variety of different classes of
* To whom correspondence should be addressed. E-mail: ferrarini@
farm.unipi.it; fax: +39-050-40517.
† Dipartimento di Scienze Farmaceutiche, Universita` di Pisa.
‡ Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie, Universita` di Pisa.
10.1021/jm030977p CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/11/2004

3020 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Ferrarini et al.
Ta ble 1. Affinity of 1,8-Naphthyridine Derivatives in Radioligand Binding Assays at Bovine Brain A1, A2A and Human Brain A1 and
A2A Receptorsa,b
Ki (nM)
compd
R1
R2
R3
bA1
bA2A
bA2A/bA1
hA1
hA2A
hA2A/hA1
1.3
1
2
3
4
5
6
7
8
9
Ph
OH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
5.3 ( 0.6
450 ( 92
80 ( 7
1000 ( 110
6000 ( 600
810 ( 87
>10000
17 ( 4
550 ( 57
28 ( 3
1200 ( 130
8700 ( 750
5.3 ( 0.4
11 ( 4
460 ( 38
> 10000
1250 ( 230
>10000
87
>22
16
430 ( 35
>10000
550 ( 47
>10000
230 ( 20
>10000
>10000
>10000
>10000
210 ( 15
>10000
490 ( 45
>10000
>10000
440 ( 45
40 ( 5
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
SH
Cl
2000 ( 150
>10000
0.12
>10
OCH3
OPh
N3
>10000
>2
>10000
>10000
>12
8800 ( 850
>10000
>1.1
0.10
0.88
>10000
NH2
420 ( 29
7000 ( 630
830 ( 79
>10000
25
13
30
>8
>1
119
43
94
670
41
2000 ( 170
>10000
N(CH3)2
cyclohexylNH CH3
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
38
39
40
41
560 ( 52
>10000
>10000
c
Cep
Pipz
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
CH3
CH3
CH3
CH3
Br
d
>10000
p-FPh
o-FPh
Ph
630 ( 65
470 ( 42
66 ( 5
3900 ( 400
490 ( 45
360 ( 35
300 ( 27
0.11
0.081
0.28
1.5
1.9
1.5
< 0.48
0.58
0.25
0.70 ( 0.05
0.15 ( 0.01
4.1 ( 0.6
16 ( 4
100 ( 9
450 ( 42
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Cl
F
100 ( 15
170 ( 35
>10000
1400 ( 150 2700 ( 25
1300 ( 120 2000 ( 180
>625
73
>1920
875
OPh
OEt
26 ( 4
1900 ( 170
>10000
>10000
4800 ( 450
5.2 ( 0.7
1.6 ( 0.2
>10000
2600 ( 270 1500 ( 130
OCH3
OH
1400 ( 140
>10000
2000 ( 180
>10000
490 ( 50
>10000
>10000
6500 ( 620
>10000
640 ( 57
4100 ( 400
1300 ( 280
4900 ( 340
6900 ( 700
9.9 ( 0.8
100 ( 11
5.3 ( 0.5
0.56 ( 0.03 2300 ( 210
7.2 ( 0.7
70 ( 6
>10000
>8
>2
>10000
>10000
>10000
< 0.65
Ph
CH3
CH3
CH3
NH2
>10000
>1
>10000
p-NO2Ph
Ph
OH
460 ( 37
1800 ( 150
5900 ( 420
46
3100 ( 280
>10000
0.21
< 0.41
2.6
1.3
>1.3
0.77
NHNH2
OH
18
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
1110
4110
>1390
36
3400 ( 350 9000 ( 870
OH
OH
OH
OH
OH
OH
OEt
N(CH3)2
980 ( 90
7500 ( 720
1300 ( 110
>10000
Pipe
>10000
2500 ( 230
>10000
cyclohexylNH
Morphf
Cepc
2200 ( 200 1700 ( 160
160 ( 15
>10000
>10000
50 ( 4
>63
>10000
>10000
>10000
>10000
>10000
>10000
970 ( 90
3800 ( 370
1400 ( 150
>10000
>10000
210 ( 20
110 ( 10
>10000
Pipzd
Br
>10000
2900 ( 250
58
1.5
1000 ( 110
>10000
0.97
< 0.38
1.5
4700 ( 450 7200 ( 650
OEt
OEt
48 ( 5
290 ( 30
340 ( 35
380 ( 35
6.8 ( 0.5
1.0 ( 0.2
15 ( 4
>10000
>10000
>10000
>10000
500 ( 47
2300 ( 220
580 ( 60
230 ( 20
>10000
>10000
>10000
>10000
>10000
900 ( 85
>10000
200 ( 17
2.0 ( 0.1
208
>34
>29
>26
73
2300
39
10
930 ( 90
>10000
Ph
Ph
Ph
OEt
OH
SCH3
OH
CH3CONH
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
OH
>10000
>10000
< 0.021
0.12
0.8
1.5
0.32
p-NH2C6H5
p-AcNHC6H5 OH
m-NO2C6H5
m-NH2C6H5
CH2Ph
H
910 ( 90
42
43
44
46
47
48
49
50
2000 ( 200 1600 ( 150
2200 ( 200 3400 ( 350
4000 ( 380 1300 ( 110
OH
OH
OH
OH
NH2
OH
OH
23 ( 3
1900 ( 200
7000 ( 710
>10000
>5.3
>1.4
>10000
>10000
>10000
>10000
>10000
>10000
1800 ( 170
>10000
>10000
7500 ( 730
< 0.18
H
CH3
CH3
>10000
NH2
CH3
NH2
>10000
< 0.75
0.66
>2.4
21.5
51
52
CH2CH2CH3 OH
CH2CH2CH3 OH
48 ( 6
19
>48
800
1500 ( 140 1000 ( 110
210 ( 20
0.25 ( 0.02
357 ( 35
4100 ( 400
6.7 ( 0.5
463 ( 45
>10000
144
3.25 ( 0.2
DPCPX
SCH58261
0.0056
0.0070
a
Inhibition of specific [3H]CHA binding to bovine and human brain cortical membranes expressed as Ki ( SEM (n ) 3) in nM. b Inhibition
of specific [3H]CGS21680 binding to bovine and human striatal membranes expressed as Ki ( SEM (n ) 3) in nM. c Cep )
d
ethylcarbethoxypiperazinyl. Pipz ) piperazinyl. e Pip ) piperidinyl. f Morph ) morpholinyl.
rat and human receptors; in the bovine receptor, the
typical A1 receptor rank order of potency (R)-PIA >
NECA > (S)-PIA is partially altered in that it has a
specifically reduced binding affinity for the 5′-substi-
tuted adenosine analogues compared with rat and
human receptors.24 Furthermore, N6-substituted ad-
enosine derivatives, such as (R)-PIA, are more potent
at bovine than at human or rat A1 receptors. This
phenomenon has been called the “phenyl effect” and is
the strongest at the bovine A1 receptor.23
Recently, human recombinant adenosine receptors
expressed in mammalian cell lines (CHO, HEK) have
often been found application in the screening of new
ligands.25-30 Reevaluation of compounds at all four

Affinity of 1,8-Naphthyridines toward Adenosine Receptors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3021
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (i) EtOH, KOH, EtI, heated in sealed
tube at 80 °C, 72 h.
a
Reagents and conditions: (i) amine, heated in a sealed tube
Sch em e 3a
at 140 °C, 48 h; (ii) EtOH, NaOH, reflux, 5 h; (iii) DMSO, NaH,
EtI, heated at 80 °C, 24 h.
human cloned adenosine receptor subtypes has shown
that ligands which had been considered to be A1-
selective may not be selective for human A1 adenosine
receptors, in some cases due to species differences (e.g.
a lower affinity for human than for rat receptors), or
due to a high affinity of the compounds for the new A3
or A2B adenosine receptors.31,32
a
Reagents and conditions: (i) acetic anhydride, heated at 100
°C, 4.5 h.
Sch em e 4a
Although methods of purification and characterization
of the native human adenosine A1 and A2A receptors
have been reported in the literature,33,34 no binding tests
using these receptors have so far been published for new
synthetic compounds structurally different from clas-
sical ligands of adenosine receptors.
a
Reagents and conditions: (i) MeOH, Na, CH3I, 2 h.
In light of these considerations, we established two
aims: (i) extending and completing the analysis of the
effects produced on the affinity and the selectivity
toward the bovine A1 adenosine receptor (bA1AR) by the
substitution of the 1,8-naphthyridine nucleus at the 1,
2, 4, and 7 positions, synthesizing for this purpose a new
series of compounds which were analogues of those
previously studied; (ii) both for this new series of
compounds and for the 1,8-naphthyridine derivatives
previously reported,19 evaluating the binding activity at
human A1, A2A, and A3 receptors, using human cortical
(hA1AR) and striatal (hA2AAR) membranes and human
cloned receptors (hA3AR).
1). Reaction of compounds 15 with EtI and KOH in a
hydro-alcoholic solution gave 36-38 (Scheme 2).
The 7-acetylamino derivative 3935 was prepared by
reaction of the 7-amino derivative 2835 with Ac2O
(Scheme 3). The reaction of the 4-mercaptonaphthyri-
dine 319 with MeONa and CH3I in MeOH at room
temperature afforded the corresponding methylthio
derivative 40 (Scheme 4). The catalytic reduction of the
p-nitrophenyl derivative 2636 was performed in AcOH,
in the presence of Pd/C as a catalyst, to give the
p-aminophenylnaphthyridine 41, which by the reaction
with Ac2O gave the corresponding acetamido derivative
42 (Scheme 5). The nitration of 2-phenylnaphthyridine
137 carried out with potassium nitrate in concentrated
H2SO4 gave the m-nitrophenyl derivative 43, which by
catalytic reduction in the presence of Pd/C as a catalyst
was converted to the corresponding m-aminophenyl-
naphthyridine 44 (Scheme 6). The condensation of
2-amino-6-methylpyridine with ethyl 4-phenylacetoac-
etate, prepared as described in the literature,38 in
polyphosphoric acid at 100 °C gave the 4H-pyrido-[1,2-
a]pyrimidin-4-one (45), which was converted to the
2-benzyl-1,8-naphthyridine derivative 46 by reflux in
Dowtherm A (Scheme 7).
Finally, a molecular modeling study of the interaction
of the 1,8-naphthyridine compounds with both the
bovine and the human A1 adenosine receptors was
carried out.
Ch em istr y
The compounds described in this study are shown in
Tables 1, and their methods of synthesis are outlined
in Schemes 1-7. Reaction of 7-bromo-4-hydroxy-2-
phenyl-1,8-naphthyridine 1519 with an excess of the
appropriate amine in a sealed tube at 140 °C gave 29-
33 (Scheme 1). The piperazine derivative 34 was
obtained by alkaline hydrolysis of compound 33. When
the naphthyridine 15 was treated with NaH and EtI in
DMSO, the 4-ethoxy derivative 35 was obtained. (Scheme
All the compounds synthesized were characterized by
1
elemental analysis, IR, and H NMR.

3022 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Ferrarini et al.
Sch em e 5a
Ta ble 2. Affinity of Several Representative 1,8-Naphthyridine
Derivatives in Antagonist Radioligand Binding Assays at A1
Receptors in Bovine and Human Brain
A1 Ki (nM)a
compd
bovine
human
10
15
16
22
29
42
50
51
39.0 ( 5 (28 ( 3)
1.60 ( 0.5 (0.70 ( 0.05)
1.62 ( 0.7 (0.15 ( 0.01)
>10000
448 ( 52 (560 ( 52)
336 ( 27 (0.70 ( 0.05)
670 ( 58 (300 ( 27)
>10000
6.02 ( 0.6 (0.56 ( 0.03)
1.67 ( 0.5 (1 ( 0.2)
>10000
1370 ( 130 (980 ( 90)
1460 ( 150 (2000 ( 200)
>10000
61.5 ( 5 (48 ( 6)
1447 ( 130 (1500 ( 140)
a
Displacement of [3H]DPCPX from bovine and human brain
cortical membranes expressed as Ki ( SEM (n ) 3). In parentheses
are reported the value of Ki obtained using the agonist [3H]CHA.
a
Reagents and conditions: (i) H2, Pd/C, 3h; (ii) acetic anhydride,
determined by displacement experiment using the
antagonist
reflux, 5 h.
[3H]-1,3-dipropyl-8-cyclopentylxanthine
Sch em e 6a
(DPCPX). These data, plus the receptor affinities for the
antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX)
and 5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4,3-
e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261), expressed
as inhibition constants (Ki, nM), are summarized in
Table 1 and Table 2. Finally, the affinity of some 1,8-
naphthyridine derivatives (3, 8, 14, 15, 40, and 41) was
determined by measuring their ability to displace the
binding of [125I]AB-MECA from human cloned receptors
(A3).
For a better understanding of the biological data, it
needs to be pointed out that the numbering of the 1,8-
naphthyridine nucleus used is the same as that reported
in Table 1.
a
Reagents and conditions: (i) H2SO4, KNO3, 30 minutes; (ii)
H2, Pd/C, 3 h.
A1 Recep tor s. Bovin e A1 Recep tor (bA1AR). The
results reported in Table 1 for the new compounds 29-
44 and 46-5239-41 show that, as regards the structural
modifications introduced at the position 2 (R1) of the
heterocyclic nucleus, the lack of any substituent, or
replacing of the phenyl group in this position with a
methyl group or an n-propyl group reduces the receptor
affinity, as can be seen from a comparison of compounds
4739 (Ki ) 7000 nM) and 5140(Ki ) 48 nM) versus 1 (Ki
) 5.3 nM), a comparison of compound 5035,41 (Ki > 10000
nM) and 5235 (Ki ) 210 nM) versus 28 (Ki ) 5.3 nM)
and finally that of compound 4839 (Ki > 10000 nM)
versus 8 (Ki ) 17 nM). The introduction of a methylenic
group between the naphthyridine nucleus and the
phenyl group induces a decrease in the affinity, as can
be seen from a comparison of compound 46 (Ki ) 1900
nM) versus 1 (Ki ) 5.3 nM). Furthermore, the introduc-
tion of a substituent on the phenyl group (41-44)
maintains an affinity in the nanomolar range (Ki ) 1.0-
23 nM), similar to that of the analogous compound 1,
unsubstituted on the phenyl group (Ki ) 5.3 nM). In
the case of compound 42 (Ki ) 1.0 nM) the affinity is
higher than that of 1 (Ki ) 5.3 nM).
Sch em e 7a
a
Reagents and conditions: (i) PPA, heated at 100 °C, 4 h; (ii)
Dowtherm A, heated at 220 °C, 5 h.
Resu lts a n d Discu ssion
The affinities of 1,8-naphthyridine derivatives 1-44
and 46-5239-41 were determined by measuring their
ability to displace the specific binding of agonist [3H]-
N6-(cyclohexyl)-adenosine ([3H]CHA) and [3H]2-{[[p-(2-
carboxyethyl)-phenyl]ethyl]amino}-5′-(N-ethylcarbamoyl)-
adenosine ([3H]CGS21680) from bovine (29-44 and 46-
5239-41) and human (1-44 and 46-5239-41) cortical (A1)
and striatal (A2A) membranes, respectively.33,34,42,43
Moreover, for some compounds (10, 15, 16, 22, 29,
42, 50,35,41 5140), the affinity for A1 receptors was also
As regards the structural modifications at the position
4 (R2) of the 1,8-naphthyridine nucleus, the substitution
of the mercapto group (3, Ki ) 80 nM) with a methylthio
group was found to produce a decrease in the affinity
(40, Ki ) 380 nM). Analogously, the substitution of the
hydroxy group in the same position with the ethoxy
group leads to compounds exhibiting a decrease in
affinity, as is clear from a comparison of compound 35
(Ki ) 50 nM) versus 15 (Ki ) 0.70 nM) and compound

Affinity of 1,8-Naphthyridines toward Adenosine Receptors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3023
38 (Ki ) 290 nM) versus 20 (Ki ) 5.2 nM). The low
affinity of compounds 35 and 38 might also be justified
by the fact that the tautomeric structure, in which the
oxygen in position 4 is in the quinoid form and the
nitrogen in position 1 is protonated, is not possible for
these compounds.19 This quinoid structure was found
to be preferred for the 4-OH substituted compounds and
proved to be very important for the affinity at the bA1-
AR.19
AR (16, Ki ) 300 nM; 29, Ki ) 980 nM), with a decrease
in the affinity of 2000 and 1750 times, respectively.
To establish whether the reduction in the affinity of
the 1,8-naphthyridine derivatives studied for human
tissue compared with bovine tissue might reflect a
difference in the coupling between receptors and G
proteins of different species,44 we performed competition
experiments for certain representative compounds (10,
15, 16, 22, 29, 42, 50,35,41 and 5140), using the selective
antagonist [3H]DPCPX as the radioligand. The results
shown in Table 2 revealed, also in this case, a different
level of affinity for bovine and human tissue, confirming
the loss of affinity for human tissue compared with
bovine tissue, as had been found by using the agonist
radioligand, [3H]CHA. Only derivatives 16 and 29 were
found to be about 10 times less potent on bovine tissue,
using the antagonist instead of the agonist as the
radioligand, probably due to a difference in G protein
coupling. However, the large differences in affinity
between the two species remain incomprehensible,
seeing that they cannot be completely explained by the
different type of interaction between receptors and G
proteins. The discrepancies between the two species
might be due to the primary amino acid sequence of the
receptor: it is possible that the variation in this
sequence might modulate the affinity of the ligand and
determine differences in the pharmacological profile of
the different species.1,11
As regards the structural modifications at the position
7 (R3) of the 1,8-naphthyridine nucleus, the substitution
of the methyl group with secondary amines, like N(CH3)2
or piperidine, was found to lead to compounds 29 and
30, respectively, with a remarkable affinity at the bA1-
AR (29, Ki ) 0.56 nM, 30, Ki ) 7.2 nM). The 7-cyclo-
hexylamino derivative 31 showed an appreciable affinity
(Ki ) 70 nM). On the contrary, the substitution at the
same position with a cyclic amine bearing another
heteroatom on the ring, like morpholine, caused a
decrease in the affinity at the bA1AR (32, Ki ) 160 nM),
which in some cases was remarkable, as for the ethoxy-
carbonylpiperazine (33) and piperazine groups (34), with
a Ki > 10000 nM. Furthermore, the substitution of the
methyl group with an acetamido group produced a
decrease in the affinity, as can be seen from a compari-
son of compound 39 (Ki ) 340 nM) versus 1 (Ki ) 5.3
nM).
As regards the compounds which possess a quinoid
structure in position 4 or 7, the N8-ethyl-substituted
derivative 36, with the quinoid structure at the position
7, showed a low affinity toward the bA1AR (Ki ) 4700
nM), similar to that of compound 23 (Ki ) 1300 nM)
previously studied.19 The 4,7-dihydroxy derivative 49,41
which possesses the quinoid structure with the N8
protonated (as demonstrated on the basis of theoretical
calculations for the analogous compound 2219) showed
a very low affinity (Ki > 10000 nM). Finally, the N1-
ethyl-substituted compound 37 showed an intermediate
affinity Ki ) 48 nM. This last result confirms the
importance for the affinity at the bA1AR of the tautomer
in which the oxygen in position 4 is in the quinoid form
and the nitrogen in position 1 is protonated.19
All compounds exhibit a poor affinity at the native
hA1AR with a remarkable decrease in affinity in com-
parison with the affinity at the bA1AR.
A2A Recep tor s. Bovin e A2A Recep tor (bA2AAR).
The data in Table 1 show that the new 1,8-naphthy-
ridines 29-44 and 46-5239-41 possess a very poor
affinity at the bA2AAR, like the previous series. Only
four derivatives (41, 43, 44, and 5140) possess an
intermediate affinity, with a Ki of 230-1000 nM. Five
derivatives (29, 31, 35, 36, and 42) showed a moderate
affinity in the order of a micromolar concentration (Ki
) 2300-7200 nM), whereas other compounds were
completely ineffective.
Na tive Hu m a n A2A Recep tor (h A2AAR). The most
effective compound toward the hA2AAR was 14, with a
Ki ) 40 nM. A few other compounds (1, 3, 8, 10, 13, 15,
16, 21, 26, 35, 40, and 41) showed an average affinity
(Ki ) 970-100 nM). The rest of the compounds pos-
sessed a very low affinity (Ki values > 1000 nM).
The results show that some of compounds 29-44 and
46-52 possess interesting affinity at the bA1AR, in
some cases in the low nanomolar range, with a different
degree of selectivity at this receptor. In particular
compounds 29, 30, 41, and 42 exhibit a remarkable
affinity with Ki < 10 nM.
The results of the binding studies indicate that the
affinity at the hA2AAR is not very different from that
Na t ive Hu m a n A1 R ecep t or (h A1AR ). To obtain
information about the differences between the affinity
versus bovine receptors and versus native human recep-
tors, the new series of 1,8-naphthyridine derivatives
29-44, 46-5239-41 and the derivatives 1-28 previously
studied19 were tested to evaluate their affinity toward
human cortical A1 receptors. The results shown in Table
1 indicate that all compounds showed a poor affinity at
the native hA1AR. Only compounds 1, 10, 14-16 pos-
sessed an intermediate affinity (Ki ) 560-300 nM).
Moreover, it is evident that all the compounds exhibit
a remarkable decrease in affinity, ranging from 10 to
2000 times, in comparison with the affinity at the bA1-
AR. In particular, compounds 16 and 29, which showed
a high affinity at the bA1AR (16, Ki ) 0.15 nM; 29, Ki )
0.56 nM), possess an intermediate affinity at the hA1-
at the bA2AAR. Indeed, the affinity at the native hA2A
-
AR decreased a little for some compounds, for other
compounds it remained approximately the same, whereas
for a few compounds there was a little increase. In
particular, the 4-methylthio derivative 40, which was
found to be inactive toward the bA2AAR (Ki > 10000
nM), showed an appreciable affinity toward the hA2A
-
AR (Ki ) 210 nM), with the highest increase in affinity
compared with the affinity at bovine receptors. Fur-
thermore, the 2-o-fluorophenyl-substituted derivative 14
showed the highest affinity at human receptors, with a
Ki value of 40 nM and a high increase in comparison
with the affinity at bovine receptors (Ki ) 470 nM).
The most compounds possess a low affinity both at
the hA2AAR and at the bA2AAR. Furthermore, the

3024 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Ferrarini et al.
F igu r e 1. DPCPX docked into the hA1AR (left) and bA1AR (right) binding site. Interatomic distances between H-bonded atoms
are reported in blue; carbon-carbon distances evidencing lipophilic interactions are reported in red. All distances are in Angstroms.
affinity at the hA2AAR is not very different from that
at the bA2AAR.
Figure 1 illustrates DPCPX docking into both the site
of the hA1AR (on the left) and the bA1AR (on the right)
and shows that DPCPX interacts at a similar distance
with Ser94 and His251 through its hydroxylic functions
by means of H-bonds which appear to be shorter and
therefore stronger in the hA1AR (d ) 2.9 and 3.0 Å) than
the bA1AR (d ) 3.1 Å). However, a series of lipophilic
interactions due to Leu90, Ile95, Leu250, Ala273, and
Ile274 are able to better stabilize DPCPX in the bA1-
AR: the cyclophentyl group interacts with Ala273 in
hA1AR (d ) 4.1 Å) and Ile274 (d)4.4 Å), while it
interacts with Ala273 (d ) 4.0 Å) and Leu90 (d ) 4.0
Å) in bA1AR; moreover, in bA1AR the two n-propyl
chains are able to interact with Ile95 (d ) 3.9 Å) and
Leu250 (d ) 4.0 Å), while in hA1AR there is only the
interaction with Ile95 (d ) 4.3 Å).
A3 Recep tor s. As the analogous 1,8-naphthyridine
derivatives previously studied19 were ineffective at
bovine A3 receptors, we considered it sufficient to
evaluate the affinity toward bovine A3 receptors only
for some new compounds (29, 30, 41, and 42). The
binding assay showed that these compounds presented
very low inhibition percentages at a concentration of 10
µM, with the result that the corresponding Ki values
were not calculated.
For the compounds which proved to be most effective
at the native hA2AAR (3, 8, 14, 15, 40, and 41), the
affinity at the hA3AR was also evaluated using cloned
receptors, but all the tested compounds provided to be
ineffective.
Figure 2 shows that the fairly limited structural
differences that exist in the transmembranal regions of
the two receptors hA1AR and bA1AR (only seven resi-
dues) are, however, able to induce a clear difference in
the 3D arrangement of the seven helices in the two
models (the RMSD calculated on the backbone is 3.79
Å). An important point in determining the conforma-
tional differences in the models of the hA1AR and the
bA1AR could be the replacement of the Met82 residue
of the hA1AR with Lys82 in the bA1AR, which induces
a different arrangement of the interhelix H-bonds and
therefore helices TM2 and TM3 are closer in the case
of the hA1AR. The RMSD between the R carbons of helix
2 and the corresponding R carbons of helix 3 is 9.8 Å in
the case of the hA1AR and 10.5 Å in the case of the bA1-
AR.
The different arrangement of these two helices also
modifies the rest of the structure, and, as a consequence,
the binding pocket appears to be narrower in the bA1-
AR than in the hA1AR. This fact could be the reason
for the interaction differences shown in Figure 1.
Some 1,8-naphthyridine derivatives were then docked
into the two receptor models; the compounds selected
for this purpose were 16, 22, 28, 29, 50, 51, and 52,
which possessed different Ki values toward the hA1AR
and the bA1AR. The docking procedure was carried out
by taking into account the site-directed mutagenesis
Molecu la r Mod elin g
With the aim of explaining the marked lowering of
the affinity toward the native hA1AR in comparison with
the bA1AR, a molecular modeling study was carried out
in order to evaluate theoretically the interaction of the
1,8-naphthyridine derivatives with the two A1 adenosine
receptors from two species.
The model of the hA1AR, made up of seven helices,
was constructed by following a homology procedure in
which the crystallographic structure of bovine rhodop-
sin45 was used as a template; the model thus obtained
was then optimized so as to interact suitably with the
specific ligands CPA and DPCPX on the basis of the
available site-directed mutagenesis data.46 These data
indicated that the residues Thr91, Ser94, His278, and
Thr277 were important for the activity of the agonists,
and the residues Ser94 and His251 were fundamental
for the affinity of the antagonists. Therefore, the start-
ing geometries of the complexes between the hA1AR and
the agonist CPA and subsequently the antagonist
DPCPX were arranged in such a manner that the
ligands could favorably interact with the appropriate
residues. The computational procedures are fully de-
scribed in the Experimental Section.
The model of the bA1AR was constructed on the basis
of the hA1AR model, following a similar procedure.

Affinity of 1,8-Naphthyridines toward Adenosine Receptors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3025
F igu r e 2. Superimposition of the complexes of DPCPX with hA1AR (blue) and bA1AR (orange); the seven not conserved residues
of the seven transmembranal helices are shown.
Ta ble 3. Interaction Energies (kcal/mol) of hA1AR and bA1AR
Models and Selected Ligands
con-
con-
compd A1bARa formerb A1hARa formerb selectivityc ∆Ed
DPCPX -30.2
-
-26.7
-24.0
-25.2
-22.2
-26.3
-24.1
-21.0
-21.9
-
27
2000
-
640
1750
-
3.5
5.7
-
5.1
4.4
-
16
22
28
29
50
51
52
-29.7
-27.4
-27.3
-30.7
-21.1
-22.6
-23.0
B
B
B
B
B
A
B
B
B
B
B
B
B
B
31
20
1.6
1.1
F igu r e 3. The two possible geometries for the interaction of
1,8-naphthyridine derivatives with A1 adenosine receptors.
a
Sum of the nonbonded terms of the interaction between the
b
atoms of the receptor model and the atoms of the ligand. Ar-
rangement energetically preferred by the ligand for interaction
with the receptor model (see Figure 4). c Selectivity of the ligand
computed as the ratio between the Ki for hA1AR and Ki for bA1AR;
the selectivity is not reported in the case of inactive compounds.
data, as for DPCPX. Therefore, the selected compounds
were initially placed in the receptor sites so that they
could interact favorably with Ser94 and His251.
The groups of compounds considered capable of
interacting at the same time with these two residues
were the substituent in position 4 and the nitrogen in
position 8, which possess suitable chemical character-
istics and a suitable spatial arrangement. Therefore, the
two interaction geometries A and B, shown in Figure
3, are to be taken into consideration: in the first one,
A, the substituent in position 4 gives an H bond with
Ser94 and the nitrogen in position 8 gives an H-bond
with His251; in the other one, B, the same groups of
the selected compounds give H-bonds with the same
residues, but in an inverse manner.
d
Energy difference between the interaction energy of the ligand
with hA1AR minus the interaction energy with bA1AR; the energy
difference is not reported in the case of inactive compounds.
molecular mechanics steric energy referring to the
interaction between the atoms of the receptor model and
the atoms of the ligand.
All active compounds prefer arrangement B when
they are fitted into both the hA1AR and the bA1AR. The
differences in the interaction energy between the two
arrangements, A and B, range from 2 to 6 kcal/mol.
The relative values of the interaction energy are
reported in Table 3 and indicate that all active ligands
have a more favorable interaction with the model of the
bA1AR, and this is in agreement with the greater
affinity of the considered ligands toward the bA1AR with
respect to the hA1AR; a correlation between these values
and the selectivity ratio can also be observed.
The complexes thus obtained (two for each compound
and for each receptor model) were then optimized by
means of molecular dynamic simulations followed by
energy minimization with the AMBER force field; the
computational procedure is fully described in the Ex-
perimental Section.
The antagonist-receptor interaction energies were
calculated as the sum of the nonbonded terms of the
In Figure 4 the complex between compound 16, the
most selective one, and the two models of the hA1AR

3026 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Ferrarini et al.
F igu r e 4. Compound 16 docked into the hA1AR (left) and bA1AR (right) binding sites. The volumes of the cavities between 16
and the two receptors are shown.
and bA1AR receptors is shown. Figure 4 also reports the
volumes of the cavities between the ligand and the
receptors and is thus able to show the difference in the
dimensions of the two binding sites, which could be quite
important in determining the selectivity of the 1,8-
naphthyridine derivatives considered.
The compounds considered here are less bulky than
classic A1AR antagonists and therefore they can interact
more strongly with the sterically restricted site in the
bA1AR, while they cannot occupy so efficiently the larger
site of the hA1AR, as already indicated by the interac-
tion energy values reported in Table 3.
In Figure 5 the details of the interaction of compounds
16 and 29 with the human and bovine A1AR are
presented. In the case of the complex with the bA1AR,
these antagonists are able to give H-bonds with Ser94
and His251 and at the same time their phenyl substitu-
ent in position 2 is able to give a hydrophobic interaction
with Ile270 and other residues that make up a pocket
(Tyr271, Ala273, Ile274). The pocket in the bA1AR is
able to optimally accept the phenyl ring linked in
position 2 of the naphthyridine system, and therefore
this structural feature of the bA1AR binding site could
explain the higher activity found for compounds that
possess only an unsubstituted phenyl ring in position 2
of the naphthyridine system such as, for example, 16
and 29. In fact, the presence of substituents on this
phenyl ring, as, for example, in 13, 26, and 43 and the
replacement of this phenyl ring with a less bulky group,
as in 50, 51, and 52, induces a decrease in the affinity.
Moreover, the insertion of a spacer between the naph-
thyridine system and this phenyl ring, as in 46, induces
the almost complete loss of affinity.
A1 and A2A receptors and human cloned A3 receptors of
this new series of compounds and of the 1,8-naphthy-
ridine derivatives previously reported,19 was assessed.
Results showed that also a large number of the new
1,8-naphthyridine derivatives proved to be A1 bovine
adenosine receptor selective with a high affinity toward
the same receptor. In particular, compounds 29 and 42
showed a very high selectivity (A2A/A1 ratio 4110 and
2300, respectively), higher than that of the compounds
previously studied.19 Furthermore, it is possible to
assume that the highest affinity at the bA1AR requires
a phenyl group in position 2, a quinoid oxygen in
position 4 with the nitrogen in position 1 protonated,
and a lipophilic group or an aliphatic amine without a
large steric hindrance in position 7a.
As regards the affinity toward the bA2AAR, only a few
compounds possess a moderate affinity, which for some
compounds remained approximately the same as toward
the native hA2AAR. In some cases, on the contrary, the
affinity toward the hA2AAR was found to be higher than
both the affinity toward the bA2AAR and toward the hA1-
AR. All the tested compounds proved to be totally
inactive toward the cloned hA3AR.
As regards the affinity toward the hA1AR, all the 1,8-
naphthyridine derivatives generally lost their affinity
to an extent that in some cases is truly considerable
(more than 1000 times) and decidedly higher than that
reported in the literature for agonist and antagonist
ligands.23,24 To verify whether this great species selec-
tivity could be explained in terms of a difference
between the binding site of the native hA1AR and the
bA1AR, these two sites were modeled, taking into
account the available mutagenesis data. The interaction
energy values of the 1,8-naphthyridine derivatives with
the two receptor models were in agreement with their
affinities and therefore with the observed species se-
lectivity; the better interaction with the bA1AR seems
to be due to the smaller size of the binding site of this
receptor, which allows a particularly good interaction
with these 1,8-naphthyridine derivatives. In fact, the
molecules of these ligands have a lower hindrance with
respect to the classic A1AR antagonists such as DPCPX,
and therefore they can better occupy this site. Figure 6
shows that the volume of DPCPX is larger than that of
16 and that it is mainly due to the precence of the freely
rotatable n-propyl chains in the structure of DPCPX.
Other differences in the structure of the hA1AR and
the bA1AR, and in particular in the loop regions, could
In the case of the hA1AR, the H-bonds and the
hydrophobic interaction are weaker due to the larger
dimensions of the site; moreover, in this receptor, Ile270
is substituted by the less hydrophobic Thr270. In
particular, the lipophilic pocket in the hA1AR is larger
and the presence of a phenyl ring linked in position 2
is less important for the affinity.
Con clu sion s
On the basis of the results obtained in a previous
paper,19 a new series of 1,8-naphthyridine derivatives
(29-44 and 46-52), with various substituents in dif-
ferent positions of the heterocyclic nucleus, were tested
to evaluate their affinity toward bovine A1 and A2A
receptors. Furthermore, the affinity at native human

Affinity of 1,8-Naphthyridines toward Adenosine Receptors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3027
F igu r e 5. Compounds 16 (up) and 29 (down) docked into the hA1AR (left) and bA1AR (right) binding sites.
1
°C (crystallized from toluene); MS m/z 265 (M+). H NMR: δ
9.00 (brs, OH), 8.33 (d, 1H, H5), 7.50 (m, 5H, Ar), 6.56 (d, 1H,
H6), 6.44 (s, 1H, H3), 3.15 (s, 6H, N(CH3)2). Anal. (C16H15N3O)
C, H, N. 4-Hyd r oxy-2-p h en yl-7-p ip er id in yl-1,8-n a p h th y-
r id in e (30): 0.240 g, yield 65%; mp 247-249 °C (crystallized
provide further reasons for the species selectivity, but
the results of the molecular modeling study suggest that
in the case of the 1,8-naphthyridine derivatives, their
interaction with the intrahelical binding site of the two
receptors should be surely responsible for this selectiv-
ity.
from toluene); MS m/z 305 (M+). H NMR: δ 8.95 (brs, OH),
1
8.27 (d, 1H, H5), 7.46 (m, 5H, Ar), 6.63 (d, 1H, H6), 6.40 (s,
1H, H3), 3.63 (m, 4H, piperidine), 1.63 (m, 6H, piperidine).
Anal. (C20H22N3O) C, H, N. 7-Cycloh exyla m in o-4-h yd r oxy-
2-p h en yl-1,8-n a p h th yr id in e (31): 0.120 g, yield 31%; mp
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Kofler
hot stage apparatus and are uncorrected. IR spectra in Nujol
mulls were recorded on an ATI Mattson Genesis Series FTIR
156-159 °C (crystallized from toluene); MS m/z 319 (M+). H
1
NMR: δ 9.20 (brs, OH), 8.21 (d, 1H, H5), 7.47 (m, 5H, Ar),
6.42 (s, 1H, H3), 6.33 (d, 1H, H6), 4.95 (brs, NH), 3.75 (m, 1H,
cyclohexylamine), 2.15-1.74 (m, 10H, cyclohexylamine). Anal.
(C20H21N3O) C, H, N. 4-Hyd r oxy-7-m or p h olin yl-2-p h en yl-
1,8-n a p h th yr id in e (32): 0.271 g, yield 74%; mp 258-261 °C
(crystallized from toluene); MS m/z 307 (M+). 1H NMR: δ 8.68
(brs, OH), 8.34 (d, 1H, H5), 7.50 (m, 5H, Ar), 6.62 (d, 1H, H6),
6.43, (s, 1H, H3), 3.71 (m, 8H, morpholine). Anal. (C18H17N3O2)
C, H, N. 7-(4-Ca r b et h oxyp ip er a zin -1-yl)-4-h yd r oxy-2-
p h en yl-1,8-n a p h th yr id in e (33): 0.315 g, yield 70%; mp
1
spectrometer. H NMR spectra were recorded with a Bruker
AC-200 spectrometer in δ units from TMS as an internal
standard. Mass spectra were performed with a Hewlett-
Packard MS/System 5988. Elemental analyses (C, H, N) were
within (0.4% of the theoretical values and were performed
on a Carlo Erba elemental analyzer model 1106 apparatus.
Gen er a l P r oced u r e for th e P r ep a r a tion of 7-Su bsti-
tu ted -4-h yd r oxy-2-p h en yl-1,8-n a p h th yr id in e 29-33. A
mixture of 7-bromonaphthyridine 1519 (0.300 g, 1.2 mmol) and
an excess of the suitable amine (2 mL) was heated at 140 °C
in a sealed tube for 48 h. The reaction mixture was treated
with H2O, and the solid was collected by filtration to obtain
the title compounds. 7-Dim eth yla m in o-4-h yd r oxy-2-p h en -
yl-1,8-n a p h th yr id in e (29): 0.230 g, yield 72%; mp 257-260
237-239 °C (crystallized from toluene); MS m/z 378 (M+). H
1
NMR: δ 8.65 (brs, OH), 8.33 (d, 1H, H5), 7.50 (m, 5H, Ar),
6.53 (d, 1H, H6), 6.42, (s, 1H, H3), 4.16 (q, 2H, CH2), 3.64 (m,
8H, piperazine), 1.28 (t, 3H, CH3). Anal. (C21H22N4O3) C, H,
N.

3028 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Ferrarini et al.
1
(EtOH); MS m/z 294 (M+). H NMR: δ 8.01 (m, 2H, Ar), 7.97
(d, 1H, H4), 7.44 (m, 3H, Ar), 7.03 (s, 1H, H6), 6.61 (d, 1H, H3),
4.66 (q, 2H, CH2), 4.29 (q, 2H, CH2), 1.48 (m, 6H, CH3). Anal.
(C18H18N2O2) C, H, N. 37: (0.061 g, yield 13%): mp 158-159
1
°C (petroleum ether 100-140 °C); MS m/z 294(M+). H NMR
δ 8.53 (d, 1H, H5), 7.43 (m, 5H, Ar), 6.72 (d, 1H, H6), 6.19 (s,
1H, H3), 4.45 (q, 2H, CH2), 4.23 (q, 2H, CH2), 1.34 (m, 6H, CH3).
Anal. (C18H18N2O2) C, H, N. 38: (0.033 g, yield 7%): mp 176-
1
178 °C (EtOH); MS m/z 294 (M+). H NMR δ 8.60 (d, 1H, H5),
7.63 (m, 5H, Ar), 7.26 (d, 1H, H6), 7.04 (s, 1H, H3), 4.67 (m,
4H, CH2), 1.56 (m, 6H, CH3). Anal. (C18H18N2O2) C, H, N.
7-Acet a m id o-4-h yd r oxy-2-p h en yl-1,8-n a p h t h yr id in e
(39). A mixture of 7-aminonaphthyridine 2835 (0.200 g, 0.84
mmol) and acetic anhydride (78 mL) was heated at 100 °C for
4.5 h. After cooling, the solid obtained was collected by
filtration, washed with H2O, purified by flash chromatography
(ethyl acetate:petroleum ether, 10:1), and recrystallized from
EtOH to give 39 (0.085 g, yield 31%): mp >320 °C MS m/z
1
279 (M+). H NMR: δ 11.80 (brs, OH), 10.61 (brs, NH), 8.40
(d, 1H, H5), 8.05 (d, 1H, H6), 7.78 (m, 2H, Ar), 7.56 (m, 3H,
Ar), 6.31, (s, 1H, H3), 2.19 (s, 3H, CH3). Anal. (C16H13N3O2) C,
H, N.
7-Meth yl-4-m eth ylth io-2-ph en yl-1,8-n aph th yr idin e (40).
Mercaptonaphthyridine 319 (0.250 g, 0.99 mmol) was added
to a solution of sodium (0.027 g, 1.2 g atom) in anhydrous
methanol (10 mL), and the mixture was stirred at room
temperature for 2 h. Methyl iodide (0.081 g, 0.52 mmol) was
then added. After 16 h, the solvent was removed under reduced
pressure to obtain a residue which was treated with H2O and
then the mixture was extracted with chloroform. The organic
solution was dried (MgSO4) and evaporated to dryness under
reduced pressure. The crude solid was purified by crystalliza-
tion from cyclohexane to give 40 (0.112 g, yield 42%): mp 135-
F igu r e 6. Molecular volumes of DPCPX (up) and 16 (down).
4-Hyd r oxy-2-p h en yl-7-(p ip er a zin -1-yl)-1,8-n a p h th yr i-
d in e (34). A suspension of carbethoxypiperazinylnaphthy-
ridine 33 (0.400 g, 1.05 mmol) in EtOH (10 mL) and 10%
aqueous NaOH (25 mL) was refluxed for 5 h. The organic
solvent was evaporated from the reaction mixture under
reduced pressure, and the pH was adjusted to 8 with 3 N
aqueous HCl. The suspension obtained was extracted three
times with chloroform, and the combined extracts were dried
(MgSO4) and evaporated under reduced pressure. The solid
residue was purified by crystallization from toluene to obtain
34 (0.075 g, yield 23%): mp 239-241 °C; MS m/z 306 (M+).
1H NMR: δ 10.22 (brs, 1H exch.), 9.31 (brs, 1H exch.), 8.05
(d, 1H, H5), 7.75 (m, 2H, Ar), 7.48 (m, 3H, Ar), 6.82 (d, 1H,
H6), 6.13, (s, 1H, H3), 3.27 (m, 8H, piperazine). Anal. (C18H18N4O)
C, H, N.
7-Br om o-4-eth oxy-2-ph en yl-1,8-n aph th yr idin e (35). NaH
(0.100 g, 2.17 mmol, 50% in mineral oil) was added to a
solution of 7-bromonaphthyridine 1519 (0.516 g, 1.70 mmol)
in anhydrous DMSO (10 mL), and the mixture was stirred for
1 h at room temperature. Ethyl iodide (0.20 mL), 2.50 mmol)
was added to the suspension obtained, and the mixture was
heated at 80 °C for 24 h. The reaction mixture was treated
with H2O and filtered. The residual solid was crystallized from
petroleum ether 100°-140°C to obtain 35 (0.180 g, yield 32%):
mp 182-184 °C; MS m/z 329 (M+). 1H NMR: δ 8.31 (d, 1H,
H5), 8.15 (m, 2H, Ar), 7.47 (m, 4H, Ar + H6), 7.23, (s, 1H, H3),
4.31 (q, 2H, CH2), 1.60 (t, 3H, CH3). Anal. (C16H13BrN2O) C,
H, N.
1
138 °C; MS m/z 266 (M+). H NMR: δ 8.36 (d, 1H, H5), 8.32
(m, 2H, Ar), 7.64 (s, 1H, H3), 7.53 (m, 3H, Ar), 7.35 (d, 1H,
H6), 2.84 (s, 3H, CH3), 2.73 (s, 3H, CH3). Anal. (C16H14N2S) C,
H, N.
2-(4-Am in op h en yl)-4-h yd r oxy-7-m eth yl-1,8-n a p h th yr i-
d in e (41). A solution of 4-nitrophenylnaphthyridine 2636 (0.20
g, 0.71 mmol) in glacial acetic acid (45 mL) was submitted to
hydrogenation in the presence of 10% Pd/C (0.02 g) at room
pressure and temperature for 3 h. The catalyst was filtered
off, and the solvent was evaporated to dryness under reduced
pressure to give a residual solid which was crystallized from
H2O to obtain 41 (0.100 g, yield 60%): mp 130-134 °C; MS
m/z 251 (M+). 1H NMR: δ 11.83 (brs, OH) 8.28 (d, 1H, H5),
7.57 (d, 2H, Ar), 7.23 (d, 1H, H6), 6.33 (d, 2H, Ar), 6.24 (s, 1H,
H3), 5.71 (brs, NH2), 2.59 (s, 3H, CH3). Anal. (C15H13N3O) C,
H, N.
2-(4-Aceta m id op h en yl)-4-h yd r oxy-7-m eth yl-1,8-n a p h -
th yr id in e (42). A mixture of 4-aminophenylnaphthyridine 41
(0.40 g, 1.59 mmol) and acetic anhydride (4.0 mL) was refluxed
for 5 h. After cooling, the solid was collected by filtration,
washed with H2O, and crystallized from DMF to obtain 42
1
(0.15 g, yield 33%): mp > 320 °C MS m/z 293 (M+). H NMR:
δ 12.10 and 10.20 (2 brs, NH and OH), 8.32 (d, 1H, H5), 7.80
(d, 2H, Ar), 7.70 (d, 2H, Ar), 7.27 (d, 1H, H6), 6.34 (s, 1H, H3),
2.60 (s, 3H, CH3), 2.08 (s, 3H, CH3). Anal. (C17H15N3O2) C, H,
N.
4-Hyd r oxy-7-m eth yl-2-(3-n itr op h en yl)-1,8-n a p h th yr i-
d in e (43). Potassium nitrate (0.215 g, 2.12 mmol) was added
portionwise to an ice-cooled solution of 2-phenylnaphthyridine
137 (0.50 g, 2.12 mmol) in concentrated sulfuric acid (6.5 mL).
The reaction mixture was stirred at room temperature for 30
min and then treated with crushed ice and concentrated
ammonium hydroxide at pH 8. The solid precipitate was
collected by filtration, washed with H2O, and purified by
crystallization from EtOH to obtain 43 (0.10 g, yield 17%): mp
306-310 °C MS m/z 281 (M+). 1H NMR: δ 8.67 (s, 1H, Ar),
8.36 (m, 3H, Ar + H5), 7.82 (m, 1H, Ar), 7.33 (d, 1H, H6), 6.50
(s, 1H, H3), 2.62 (s, 3H, CH3). Anal. (C15H11N3O3) C, H, N.
5-E t h oxy-1-et h yl-7-p h en yl- (36), 7-et h oxy-1-et h yl-2-
p h en yl- (37), a n d 4,7-d ieth oxy-2-p h en yl-1,8-n a p h th yr i-
d in e (38). A suspension of bromonaphthyridine 1519 (0.500 g,
1.60 mmol) and ethyl iodide (1.95 mL, 2.4 mmol) in EtOH (3.7
mL) and 10% aqueous KOH (7.5 mL) was heated at 100 °C in
a sealed tube for 72 h. The reaction mixture was evaporated
to dryness under reduced pressure to obtain a residue which
was treated with H2O. The pH was adjusted to 8 with
concentrated ammonium hydroxide, and then the mixture was
extracted with chloroform. The organic solution was dried
(MgSO4), and the solvent was removed under reduced pressure
to give an oily mixture of products 36, 37, and 38. Fraction-
ation of the mixture by flash chromatography on silica gel
eluting with ethyl acetate:petroleum ether, 4:1, provided the
title compounds. 36: (0.037 g, yield 8%): mp 149-150 °C
2-(3-Am in op h en yl)-4-h yd r oxy-7-m eth yl-1,8-n a p h th yr i-
d in e (44). A solution of 3-nitrophenylnaphthyridine 43 (0.18
g, 0.64 mmol) in glacial acetic acid (40 mL) was submitted to

Affinity of 1,8-Naphthyridines toward Adenosine Receptors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3029
hydrogenation in the presence of 10% Pd/C (0.02 g) at room
pressure and temperature for 3 h. The catalyst was filtered
off, and the solvent was evaporated to dryness under reduced
pressure to give an oily residue, which was treated with H2O
and concentrated ammonium hydroxide to pH 8. The solid
precipitate was collected by filtration, washed with H2O, and
purified by crystallization from EtOH to obtain 44 (0.10 g, yield
62%): mp 239-241 °C MS m/z 251 (M+). 1H NMR: δ 12.07
(brs, OH), 8.31 (d, 1H, H5), 7.27 (d, 1H, H6), 7.15 (t, 1H, Ar),
6.92 (m, 2H, Ar), 6.71 (m, 1H, Ar), 6.19 (s, 1H, H3), 5.31 (brs,
NH2), 2.59 (s, 3H, CH3). Anal. (C15H13N3O) C, H, N.
2-Ben zyl-6-m et h ylp yr id o[1,2-a ]p yr im id in -4(4H )-on e
(45). A mixture of 2-amino-6-methylpyridine (0.90 g, 8.40
mmol) and 4-phenylacetoacetate ethyl ester (2.0 g, 9.7 mmol)
with polyphosphoric acid (30 g) was heated under stirring at
100 °C for 4 h. After cooling, the solution obtained was poured
into crushed ice and treated with concentrated ammonium
hydroxide at pH 5. The suspension obtained was extracted
with chloroform, and the combined extracts were dried (Mg-
SO4) and evaporated under pressure to obtain a tarry residue
which was purified by flash chromatography (ethyl acetate:
petroleum ether, 3:1) to give 45 (0.315 g, yield 15%): mp 117-
120 °C MS m/z 250 (M+). 1H NMR: δ 7.24 (m, 7H, H7 +H9+Ar),
6.48 (m, 1H, H8), 5.95 (s, 1H, H3), 3.83 (s, 2H, CH2), 2.92 (s,
3H, CH3). Anal. (C16H14N2O) C, H, N.
2-Ben zyl-4-h yd r oxy-7-m eth yl-1,8-n a p h th yr id in e (46). A
solution of pyridopyrimidinone 45 (0.130 g, 0.51 mmol) in
Dowtherm A (6.0 mL) was heated at 220 °C for 5 h. After
cooling, the precipitate was collected, washed with petroleum
ether, and crystallized from ethanol to obtain 46 (0.060 g, yield
47%): mp 246-250 °C MS m/z 250 (M+). 1H NMR: δ 11.40
(brs, OH), 8.22 (d, 1H, H5), 7.30 (m, 5H, Ar), 7.18 (d, 1H, H6),
5.88 (s, 1H, H3), 3.92 (s, 2H, CH2), 2.55 (s, 3H, CH3). Anal.
(C16H14N2O) C, H, N.
Biologica l Meth od s. Ma ter ia ls. [3H]-CHA, [3H]-DPCPX,
[3H]-CGS21680, [3H]-(R)-PIA, and [125I]AB-MECA were ob-
tained from PerkinElmer Life Sciences. N6-(Cyclohexyl)ad-
enosine (CHA), N6-(cyclopentyl)adenosine (CPA), (R)-N6-(2-
phenylisopropyl)adenosine [(R)-PIA], (S)-PIA, and 5′-N-(eth-
ylcarboxamido)adenosine (NECA) were purchased from RBI
(Natik, MA). Adenosine deaminase was from Sigma Chemical
Co. (St. Louis, MO). All other reagents were from standard
commercial sources and of the highest grade commercially
available.
EDTA 1 mM, MgCl2 10 mM) and homogenized. Small portions
containing about 40 µg of proteins were incubated with 0.2
nM solution of [125I]AB-MECA (2000 Ci/mmol), 20 µL of a 2
U/mL solution of adenosine deaminase, and, in the case of
nonspecific binding measurements, also 20 µL of a 50 µM
solution of NECA. The resulting mixture was then diluted with
the pH 7.4 buffer to a total volume of 100 µL and incubated at
25 °C for 60 min, after which time the samples were rapidly
filtered under a vacuum through Whatman GF/C filters, which
had been previously treated for 1h at 4 °C with an aqueous
solution (1 g/200 mL of polyethyenimine (PEI). The filters were
then washed three times with 5 mL of cold buffer and then
treated as reported above for the radioactivity measurement.
Compounds were routinely dissolved in DMSO and added
to the assay mixture to make a final volume of 0.5 mL. Blank
experiments were carried out to determine the effect of the
solvent (2%) on binding. At least six different concentrations
spanning 3 orders of magnitude, adjusted approximately for
the IC50 of each compound, were used. IC50 values, computer-
generated using a nonlinear formula on a computer program
(GraphPad, San Diego, CA), were converted to Ki values,
knowing the Kd values of radioligands in these different tissues
and using the Cheng and Prusoff equation.47 The Kd of [3H]-
CHA binding to cortex membranes from bovine and human
was 1.2 nM and 3.5 nM, respectively. The Kd of [3H]DPCPX
binding to cortex membranes from bovine and human was 0.8
nM and 3.9 nM, respectively. The Kd of [3H]CGS 21680 binding
to striatal membranes from bovine and human was 10 nM and
12 nM, respectively. Protein concentration was determined in
accordance with the method of Lowry as modified by Peterson48
using bovine serum albumin as the standard.
Com p u ta tion a l Deta ils. All the molecular mechanics and
molecular Dynamics calculations were performed through the
MACROMODEL program49 by using the AMBER force field.50
The electrostatic charges were those included in the force field
and a distance-dependent dielectric constant of 4.0 was used.
In molecular mechanics minimizations (MM) the minimized
value was the Conjugated Gradient until a convergence value
of 0.1 Kcal/A‚mol; in molecular dynamics simulations (MD)
the temperature was set at 300 °K and the time step was 1 fs.
All graphic manipulations and visualizations were performed
by means of the InsightII51 and WebLabViewe52 programs.
Mod elin g of th e h A1AR. The sequence of the seven helices
was obtained from GPCRDB53 which was aligned on the
crystallographic structure of bovine rhodopsin45 archived in
PDB54 as 1F88. The alignment of the two primary sequences
was obtained through the CLUSTALW program55 by using the
PAM matrix. 1F88 was used as a template for the modeling
of the helices of the hA1AR according to the following proce-
dure:
Bovine brains were obtained from the local slaughterhouse.
Post-mortem human brains were collected at the Department
of Pathological Anatomy, University of Pisa, from subjects with
no past history of neurological or mental disorders and with
no primary or secondary brain diseases. The time between
death and tissue dissection/freezing ranged from 18 to 36 h.
Immediately after removal from the skull, the cortex and
corpus striatus were dissected on ice and frozen in liquid
nitrogen and stored at -80 °C until use.
1. 50 ps of MD followed by MM with a constraint of 10 kcal/
mol on all backbone atoms of the hA1AR forcing them on the
corresponding atoms of 1F88;
2. 50 ps of MD followed by MM with a constraint of 10 kcal/
mol on all C R atoms of the hA1AR forcing them on the
corresponding atoms of 1F88;
3. 50 ps of MD followed by MM with a restraint of 5 kcal/
mol simulating the intramolecular H-bonds that maintain the
structure of each helix plus a constraint of 1kcal/mol main-
taining the C R of the hA1AR in its position;
A1 a n d A2A Recep tor Bin d in g Assa y. Displacement of
[3H]-CHA (39 Ci/mmol) from A1 adenosine receptor in cortical
membranes and [3H]-CGS21680 (45 Ci/mmol) from A2A ad-
enosine receptor in striatal membranes was performed as
previously described.19 Adenosine A1 receptor affinities were
also determined with [3H]-DPCPX as radioligand. The [3H]-
DPCPX (89 Ci/mmol) binding assay was performed in triplicate
by incubating aliquots of the membrane fractions (0.05-0.1
mg of protein) at 25 °C for 120 min in 0.5 mL of 50 mM Tris/
HCl, pH 7.7 containing 1 mM EDTA, 2 mM MgCl2, with
approximately 0.5 nM [3H]-DPCPX. Nonspecific binding was
defined in the presence of 10 µM of (R)-PIA by filtration
through Whatman GF/C glass microfiber filters under suction
and washing twice with 5 mL of ice-cold buffer. The filters
were treated as reported above for the radioactivity measure-
ment.
4. as in the previous step, but reducing the constraint of
the C R to 0.1 kcal/mol.
Mod elin g of th e h A1AR-CP A Com p lex. The selective
agonist CPA was then docked in the receptor model thus
obtained. The docking was performed manually in order to
respect the information given by mutagenesis for the agonist.46
In particular, H-bonds of the ligand with Thr91, Ser94,
Thr277, and His278 were searched together with a hydropho-
bic interaction of the cyplopentyl ring with Leu88. Such
interactions were not possible simultaneously because the
distance between Thr91 (on TM3) and Thr277 (on TM7) was
too large. To make all interactions possible, TM3 was rotated
by 90° and translated by about 2 Å along its axis in the
extracellular direction. The receptor model thus modified
A3 Recep tor Bin d in g Assa y. Displacement of [3H]-(R)-PIA
(75 Ci/mmol) from A3 bovine adenosine receptor was performed
as previously described.19 For hA3AR we used portions (0.20
g) of CHO cell membranes expressing human A3 receptor,
diluted (1:10) in a pH 7.4 buffer solution (Tris-HCl 50 nM,

3030 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Ferrarini et al.
(4) Feoktistov, I.; Polosa, R.; Holgate, S. T.; Biaggioni, I. Adenosine
A2A receptors: a novel therapeutic target in asthma? Trends
Pharmacol. Sci. 1998, 19, 148-153.
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and potential therapeutic applications. Curr. Pharm. Des. 1996,
2, 501-530.
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portunities for future drugs. Bioorg. Med. Chem. 1998, 6, 619-
641.
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chini, A.; Manera, C.; Scartoni, V. Novel 3-Aralkyl-7-(amino-
substituted)-1,2,3-triazolo[4,5-d]pyrimidines with high affinity
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Synthesis and biological evaluation of the enantiomers of the
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allowed all interactions: Thr91 and Ser94 with the nitrogen
atoms of the adenine portion, Thr277 and His278 with the
ribose portion, and the Leu88 with the cyclopentyl ring.
The complex thus obtained was then refined by the following
procedure:
5. 200 ps of MD followed by MM with a constraint of 50
kcal/mol maintaining the interaction between the receptor and
CPA, 10 kcal/mol on all the C R and 5 kcal/mol on all the
intramolecular H-bonds of the helices;
6. As previously, but the constraint on C R was reduced to
1 kcal/mol.
7. As previously, but the constraint on all C R was substi-
tuted with a constraint of 10 kcal/mol on only the fourteen
terminal C R of the receptor model.
8. As previously, but the constraint on the receptor-ligand
interactions was reduced to 20 kcal/mol.
9. As previously, but all constraints maintaining the recep-
tor-ligand interactions were removed, so that CPA was
allowed to relax completely.
At this point CPA was replaced by DPCPX in order to obtain
the model of the hA1AR suitable for interaction with the
antagonists. DPCPX was oriented in accordance with the
mutagenesis data available for the antagonists46 and with the
hypothesis of the N6-C8 superimposition;56 therefore the two
xanthinic oxygens were allowed to form H-bonds with Ser94
and His251 in such a manner that a hydrophobic interaction
between the cyclopentenic ring and Leu88 was observed. The
modeling procedure consisted of three steps very similar to
steps 5-9 described above.
Mod elin g of th e bA1AR a n d of th e Com p lexes bA1AR-
CP A a n d bA1AR-DP CP X. The procedures for the modeling
of the bA1AR model and its complexes with CPA and DPCPX
were the same already used for the hA1AR, but this time the
template was the hA1AR model obtained after the optimization
of the complex with CPA. In this case, due to the high
homology between the hA1AR and the bA1AR, neither transla-
tion nor rotation of helices was required.
Mod elin g of Com p lexes betw een th e h A1AR a n d th e
bA1AR w ith 16, 22, 28, 29, 50, 51, 52. The complexes between
the two models of A1 adenosine receptors with the 1,8-
naphthyridine derivatives were manually constructed on the
basis of the mutagenesis data available for antagonists. The
starting points were the optimized complexes of hA1AR and
bA1AR with DPCPX in which DPCPX was substituted with
compound 16, 22, 28, 29, 50, 51, or 52. The ligands were
considered in their quinoid form, previously found to be largely
preferred19 in the conformation minimized by PM3 calculation.
The ligands were oriented in such a manner that the carbonyl
oxygen in position 4 and the nitrogen in position 8 could accept
an H-bond from Ser94 and His251. Two conformations are
possible, and they are shown in Figure 2. Both these confor-
mations were optimized for each ligand and for each receptor
model in accordance with the procedure already used for the
modeling of the complexes with CPA and DPCPX (steps 5-9).
The ligand-receptor interaction energy was obtained as the
sum of all nonbonded terms of the steric energy (electrostatic
and van der Waals) between the atoms of the ligand and the
atoms of the receptor model calculated by MACROMODEL on
the optimized complexes. The volume of the cavities between
the ligand and the receptor was calculated by means of the
program SURFNET57 and visualized by means of the program
CHIMERA.58
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grants from MURST.
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