A novel class of highly potent and selective A1 adenosine antagonists: Structure-affinity profile of a series of 1,8-naphthyridine derivatives

Article abstract of DOI:10.1021/jm990321p

A series of 1,8-naphthyridine derivatives (12-36), bearing a phenyl group in position 2 and various substituents in positions 4 and 7, were synthesized in an attempt to obtain potent, selective antagonists for the A₁ adenosine receptor subtype.

Full text of DOI:10.1021/jm990321p

2814
J . Med. Chem. 2000, 43, 2814-2823
A Novel Cla ss of High ly P oten t a n d Selective A1 Ad en osin e An ta gon ists:
Str u ctu r e-Affin ity P r ofile of a Ser ies of 1,8-Na p h th yr id in e Der iva tives
Pier Luigi Ferrarini,*,† Claudio Mori,† Clementina Manera,† Adriano Martinelli,† Filippo Mori,†
Giuseppe Saccomanni,† Pier Luigi Barili,‡ Laura Betti,# Gino Giannaccini,# Letizia Trincavelli,# and
Antonio Lucacchini#
Dipartimento di Scienze Farmaceutiche, Universita` di Pisa, via Bonanno 6, 56126 Pisa, Italy, Dipartimento di Bioorganica e
Biofarmacia, Universita` di Pisa, via Bonanno 33, 56126 Pisa, Italy, and Dipartimento di Psichiatria, Neurobiologia,
Farmacologia e Biotecnologie, Universita` di Pisa, sez. via Bonanno 6, 56126 Pisa, Italy
Received J une 22, 1999
A series of 1,8-naphthyridine derivatives (12-36), bearing a phenyl group in position 2 and
various substituents in positions 4 and 7, were synthesized in an attempt to obtain potent,
selective antagonists for the A1 adenosine receptor subtype. The compounds were tested to
evaluate their affinity for A1 compared with A2A and A3 adenosine receptor subtypes. In binding
studies in bovine brain cortical membranes, most of the compounds showed an affinity for A1
receptors in the low nanomolar range and two in the subnanomolar range with an interesting
degree of A1 versus A2A and A3 selectivity. Comparison of the 4-substituted derivatives indicated
that 4-OH substitution, with a 4-quinoid structure, causes an increase in the A1 and A2A affinity
and generally also in A1 selectivity. The kind of substitution in position 7 can greatly modulate
the affinity: the most interesting substituents in this position seemed to be electron-
withdrawing groups; in particular the 7-chloronaphthyridine 25d showed a remarkable
selectivity (A2A/A1 ratio of 670, A3/A1 ratio of 14 000) associated with a higher A1 affinity (Ki )
0.15 nM). NMR studies on these compounds 12-36 indicated that the 4-OH-substituted ones
prefer the tautomer in which the oxygen in position 4 is in the quinoid form and the nitrogen
in position 1 is protonated. Theoretical calculations are in agreement with the NMR data.
In tr od u ction
isoquinoline (X ) CH), and quinazoline (X ) N) 9,19 have
been found to be potent and selective adenosine A1, A2A
,
Adenosine exhibits a wide variety of physiological
actions, including central nervous system depression,
lowering of blood pressure, and inhibition of lipolysis
and platelet aggregation, which are mediated by mem-
brane receptors. Four distinct subtypes of adenosine
receptors, A1, A2A, A2B, and A3, have been identified and
cloned for several species including humans. The ad-
enosine receptors are associated with a variety of
second-messenger systems. Among these, the A1 and A3
receptor subtypes are coupled to inhibition of adenylyl
cyclase, inhibition or stimulation of phosphoinositol
turnover, and activation of guanylyl cyclase, whereas
the activation of A2A and A2B receptor subtypes stimu-
lates adenylyl cyclase.1-9
or A3 antagonists (Figure 1). Recently two novel classes
of adenosine receptor antagonists, 1,8-naphthyridines
10 and pyridopyrimidines 11, have also been identified
(Figure 1). These compounds exhibit affinities for A1 and
A2A of rat brain in the micromolar range.20
In light of these considerations, we have now under-
taken a systematic research program involving the
preparation and testing of a variety of 1,8-naphthyridine
derivatives with different substituents in the 1-, 2-, 4-,
and 7-positions, to study the influence of these substit-
uents on the binding affinity toward the A1, A2A, and
A3 receptors.
Ch em istr y
Intensive efforts have been made over the past few
years with the aim of synthesizing novel compounds,
either xanthine or non-xanthine, and highly selective
ligands as agonists or antagonists have been developed.
A number of classes of tri- and bicyclic non-xanthine
antagonists have been reported. Among these, some
pyrazolotriazolopyrimidine 1,10-12 triazoloquinazoline
2,13,14 and furobenzopyranone 315 derivatives, pyran-
opyrazoles 4,16 tetrahydrobenzothiophenones 5,17,18 fla-
vonoid derivatives 6,15,18 and other bicyclic compounds,
including dipyridamole 7,15 pyridopyrimidinone 8,15
The compounds described in this study are shown in
Tables 1-3, and their synthetic methods are outlined
in Schemes 1-4. Alkylation in position 1 of 1221 was
achieved by means of EtI and KOH in a hydro-alcoholic
solution to give 13 (Scheme 1). When the 4-hydroxy-7-
methyl-2-phenyl-1,8-naphthyridine 1221 was refluxed in
toluene with Lawesson’s reagent, the corresponding
mercapto derivative 14 was obtained. Treatment of the
4-chloronaphthyridine 15, prepared with POCl3 as
described in the literature,21 with MeONa in MeOH
under reflux led to the methoxy derivative 16. Com-
pound 17 was obtained when 1521 was allowed to react
with PhONa in DMF at reflux temperature (Scheme 1).
Selective reduction of the 4-azido derivative 18,21
obtained by reaction of 15 with NaN3 as described in
the literature,21 was performed in MeOH, in the pres-
* To whom correspondence should be addressed. Tel: +39-050-
500209. Fax: +39-050-40517. E-mail: ferrarini@farm.unipi.it.
† Dipartimento di Scienze Farmaceutiche.
‡ Dipartimento di Bioorganica e Biofarmacia.
#
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Bio-
tecnologie.
10.1021/jm990321p CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/08/2000

1,8-Naphthyridine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2815
Sch em e 2
Sch em e 3
F igu r e 1. Bi- and tricyclic substances that have an affinity
at A1, A2A, and A3 adenosine receptors.
Sch em e 1
was then alkylated with MeI and KOH in a hydro-
alcoholic solution to give 31, which by demethylation
performed with 48% hydrobromic acid in acetic acid
gave 32 (Scheme 4).
ence of Pd/C as a catalyst, to give the 4-amino derivative
19 (Scheme 2). Reaction of 4-chloro-7-methyl-2-phenyl-
1,8-naphthyridine 1521 with an excess of the appropriate
amine in a sealed tube at 140 °C gave 20-22 (Scheme
2). The base 23 was then obtained by alkaline hydrolysis
of compound 22 (Scheme 2). The isomerization of 4H-
pyrido[1,2-a]pyrimidinones 24a -e22 to the correspond-
ing 1,8-naphthyridines 25a -e was carried out in
All the compounds synthesized were characterized by
1
elemental analysis, IR, and H NMR. Some compounds
were also characterized by 13C NMR.
NMR Sp ectr oscop y
Dowtherm A under reflux conditions as previously
In this series of 1,8-naphthyridine derivatives, com-
pounds 12-23, 25-30, and 33-36 present different
heteroatoms, O, N, or S, in the 4-position of the nucleus,
whereas compounds 30 and 31 present an oxygen atom
in the 5-position. As the chemical shift of Hx (H3 or H6)
also depends on the heteroatom present in the 4- or
5-position, we examined the three homogeneous classes
of products separately.
In case of the 4-amino derivatives, compounds 20, 22,
and 23 show the chemical shift of Hx ranging at δ 7.20-
7.63, and compounds 19, 21, and 35 show the chemical
shift of Hx ranging at δ 6.90-7.15. In the case of the
mercapto derivative 14, the chemical shift of Hx is at δ
7.53.
21,23
reported by us
(Scheme 3).
Dehalogenation of 25c with Raney Ni in refluxing
dioxane led to 4-hydroxy-2-phenyl-1,8-naphthyridine 26
(Scheme 4). Treatment of the 7-bromo derivative 25c
with EtONa in absolute EtOH or MeONa in MeOH
under reflux gave the 7-ethoxynaphthyridine 28 or
7-methoxynaphthyridine 29, respectively (Scheme 4).
The 7-phenoxy derivative 27 was synthesized by reac-
tion of 25c with PhONa in DMF at 80 °C. Demethyla-
tion of 29 was performed with 48% hydrobromic acid in
acetic acid to give the dihydroxynaphthyridine 30, which
was also prepared, in low yield, by alkaline hydrolysis
of 25c with 5 N NaOH. The dihydroxynaphthyridine 30

2816 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ferrarini et al.
Ta ble 1. Model Compounds of 30 and 36:a Relative Energy Values (kcal/mol) of the Six Possible Tautomers Calculated through
Different Methods
X
method
6-31G*
O
10.20
7.62
6.3
21.12
21.58
17.5
0.75
1.33
3.0
5.53
7.61
8.6
0.00
0.00
0.0
22.47
21.55
17.5
O
O
6-31G*/MP2
PM3
O
NH
NH
PM3/6-31G*
6-31G*
PM3
9.27
11.24
4.03
22.13
17.79
10.67
0.00
0.00
0.00
5.71
21.29
16.25
1.16
16.35
7.36
22.36
39.05
24.75
a
For the sake of clarity we use the same ring numeration for the model compounds as for the corresponding unsimplified compounds
which possess a phenyl ring linked in position 2.
Sch em e 4
F igu r e 2. Tautomeric forms of 4-hydroxy-1,8-naphthyridine
derivatives.
1
and 7.50 (3H) due to the phenyl group. The H NMR
spectra of compounds 17 and 30-32 exhibit a chemical
shift of Hx and of the phenyl group analogous to those
1
of 16. All the H NMR spectra of the other compounds
(12, 25-29, 34, 36) show a singlet due to Hx ranging at
δ 6.12-6.48 and two multiplets due to the phenyl group
ranging at δ 7.29-8.11 and 7.59-8.34, respectively; this
behavior is close to that of compound 13. In light of these
results the tautomeric form B (Figure 2) was assigned
to compounds 12, 25-29, 34, and 36, whereas the
tautomeric form A was assigned to compounds 30 and
32.
The 13C NMR spectra of compounds 12-14, 16, 25a ,
and 29-31 show a chemical shift due to Cx (C3 or C6),
confirming the above results obtained with 1H NMR.
In fact, the 13C NMR spectrum of quinoid compound 13
shows a signal due to Cx at δ 112.38, whereas the
4-methoxy derivatives 16 and 31 show the Cx signal at
δ 98.46 and 97.63, respectively. The 13C NMR spectra
of compounds 12, 25a , and 29, with the Cx signal
ranging at δ 108.50-108.99, close to that of compound
13, confirmed the quinoid structure B of these com-
pounds. Moreover, for compound 30 the phenolic struc-
ture A was also confirmed by 13C NMR, because the Cx
signal at δ 101.13 is close to that of compound 31.
Ta ble 2. RHF 6-31G* SCF Relative Energies of the Tautomers
of the Model Compounds of 12, 25d , 29, 30, and 36
R
Th eor etica l Ca lcu la tion s
CH3
Cl
OCH3
OH
10.96
10.29
9.35
14.78
18.98
22.54
21.12a
17.79a
0.00
0.00
0.00
0.75a
0.00a
Compounds 12, 25-30, 32, 34, and 36, which possess
an OH group capable of lacking a proton in position 4
of the 1,8-naphthyridine system, can exist in three
tautomeric forms, as shown in Figure 2; in the case of
compounds 30 and 36, in which also the substituent in
position 7 is a group able to lack a proton (OH in 30
and NH2 in 36), the number of possible tautomers rises
to six (Table 1). NMR studies indicated that these
compounds prefer the quinoid form but were not able
to specify which tautomer was preferred. Therefore
theoretical quantum mechanics calculations were per-
formed in order to study the tautomeric equilibrium in
detail and, in addition, to explore the possibility that
this equilibrium could furnish an explanation for the
10.20a
11.24a
NH2
a
See also Table 1.
To explain the affinity of 4- or 5-oxy derivatives (12,
13, 16, 17, 25-32, 34, 36) at the adenosine receptors,
it was necessary to establish their structure, which was
1
determined on the basis of their NMR spectra. The H
NMR spectrum of the quinoid compound 13 shows a
singlet due to Hx at δ 5.95 and a narrow multiplet due
to a phenyl group at δ 7.54, whereas the 1H NMR
spectrum of the aromatic compound 16 shows a singlet
due to Hx at δ 7.56 and two multiplets at δ 8.30 (2H)

1,8-Naphthyridine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2817
Ta ble 3. Affinity of 1,8-Naphthyridine Derivatives in Radioligand Binding Assays at Bovine Brain A1, A2A, and Rat Testis A3
Receptorsa-c
Ki (nM)
selectivity
A3/A1
a
b
c,d
compd
A1
A2A
A3
A2A/A1
A3/A2A
19
12
5.3 ( 0.6
450 ( 92
80 ( 7
1000 ( 110
6000 ( 600
810 ( 87
460 ( 38
8800 ( 740
87
>22
16
1660
>22
>125
>10
>2
13
>10000
>10000
14
1250 ( 230
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>8
15
>10000
>10
16
>10000
>10000
>10000
>2
17
>12
>12
18
>10000
19
17 ( 4
550 ( 57
28 ( 3
1200 ( 130
8700 ( 750
5.3 ( 0.4
11( 4
420 ( 29
25
13
30
>590
>18
>24
>1
>12
20
7000 ( 630
830 ( 79
21
>360
>8
22
>10000
>10000
>8
23
>1
>1
25a
25b
25c
25d
25e
26
630( 65
6100 ( 510
119
43
1150
418
10
10
32
21
>59
>0.3
>5
470 ( 42
66 ( 5
4600 ( 480
2100 ( 170
2100 ( 180
0.70 ( 0.05
0.15 ( 0.01
4.1 ( 0.6
16 ( 4
94
670
41
3000
14000
>2440
188
>385
>1920
>6250
100 ( 15
170 ( 35
>10000
>10000
3000 ( 290
>10000
>625
27
26( 4
1900 ( 170
>10000
1400 ( 140
73
28
5.2 ( 0.7
1.6 ( 0.2
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>1920
875
29
>7
30
>10000
>10000
31
1300 ( 280
4900 ( 340
6900 ( 700
9.9 ( 0.8
100 ( 11
5.3 ( 0.5
1.3 ( 0.1
0.3 ( 0.14
0.6 ( 0.2
12 ( 4
>10000
>10000
>10000
>8
>2
>8
>2
>1
32
33
>1
34
460 ( 37
46
>1010
>100
>1890
19
>22
>5
>2
35
1800 ( 150
5900 ( 420
750 ( 65
390 ( 81
750 ( 84
1800 ( 580
16 ( 3
18
36
1110
577
1300
1250
150
CHA
CPA
(R)-PIA
(S)-PIA
NECA
XAC
25 ( 1
0.03
0.07
0.07
0.13
3.1
26 ( 7e
53 ( 4e
87
88
241 ( 12e
20
14 ( 4
1.3 ( 0.1
49 ( 3e
1.1
48
3.5
63 ( 6
418 ( 80
323
6.7
a
b
Inhibition of specific [3H]CHA binding to bovine brain cortical membranes expressed as Ki ( SEM (n ) 3) in nM. Inhibition of
specific [3H]CGS21680 binding to bovine striatal membranes expressed as Ki ( SEM (n ) 3) in nM. c Inhibition of specific [3H](R)-PIA
d
binding to rat testis membranes in the presence of 150 nM DPCPX expressed as Ki ( SEM (n ) 3) in nM. Similar results were obtained
in A3 bovine testis membranes. e Ref 33. Each value represents the average of three experiments.
differences in the biopharmacological properties of the
4-OH-substituted compounds.
HF level (Table 1: 6-31G*). Moreover, calculations at
the PM3 semiempirical level were performed on both
model compounds of 30 and 36 (Table 1: PM3), and
results showed that this semiempirical method could be
used, even if only for a qualitative evaluation of the
relative stability of the tautomers; on the contrary, the
calculation of the relative energies at the 6-31G* level
on the geometries optimized at the PM3 level does not
give good results (Table 1: PM3/6-31G*).
The results reported in Tables 1 and 2 clearly show
that the 4-OH-substituted compounds prefer the tau-
tomer in which the substituent in position 4 of the
naphthyridine system is in the quinoid form and the
nitrogen in position 1 is protonated. However in the case
of compound 30, the preferred tautomer is the one in
which the oxygen in position 7 is in the quinoid form
and the nitrogen in position 8 is protonated. These
results are thus in agreement with the NMR data and,
moreover, could suggest an explanation for the lower
activity of compound 30 in comparison with the other
4-OH-substituted compounds, because of the preference
of this compound to exist in a different tautomeric form
with respect to the other ones.
These calculations were performed in vacuo at the
Hartree-Fock SCF level by using a 6-31G* basis set,
and a full geometry optimization was carried on for each
tautomer using the Gaussian94W program.29 However
the molecular dimension of the compounds made the
computing time too expensive; therefore the calculations
were made on simplified model compounds in which the
phenyl ring linked at position 2 of the 1,8-naphthyridine
system was substituted by a hydrogen atom; this
approximation should be more reliable than that of
simplifying the calculation by reducing the basis set or
by using semiempirical methods. The compounds taken
into consideration were 12, 25d , 29, and 36, which
possessed a high affinity for the A1 receptor, and 30,
which, on the contrary, is totally inactive. The relative
energies of the three tautomers indicated in Figure 2
are reported for all model compounds considered in
Table 2.
As regards the model compounds of 30 and 36, all six
possible tautomers were considered and the complete
results are reported in Table 1, together with the
relative energy values obtained using different calcula-
tion methods. In particular, for the tautomers of the
model compound of 30, the contribution of the electron
correlation was evaluated at the MP2 level on the RHF/
6-31G* minimized structures (Table 1: 6-31G*/MP2);
results indicated that the electron correlation increases
the stability of the tautomer already preferred at the
Biologica l Eva lu a tion
Ra d ioliga n d Bin d in g Assa ys. The 1,8-naphthyri-
dines 12-36 were tested for their ability to displace the
specific binding of [3H]N6-cyclohexyladenosine ([3H]-
CHA) and [3H]2-[[[p-(2-carboxyethyl)phenyl]ethyl]amino]-
5′-(N-ethylcarbamoyl)adenosine ([3H]CGS21680) to

2818 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ferrarini et al.
bovine cortical (A1) and striatal (A2A) membranes,
respectively.30,31 Affinity for A3 adenosine receptors was
determined in competition assays of [3H](R)-(-)-N6-(2-
phenylisopropyl)adenosine ([3H](R)-PIA) on rat testis
membranes in the presence of the A1-selective antago-
nist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX).32,33
A1, A2A, a n d A3 Ad en osin e Recep tor s. Table 3
reports the results of the A1, A2A, and A3 adenosine
receptors binding assay, expressed as inhibition con-
stants (Ki, nM), for compounds 12-36. All the com-
pounds were more potent at A1 receptors, less potent
at A2A receptors, and substantially inactive at rat A3
receptors. Similar results were obtained in A3 bovine
testis membranes.
Variation of substituents in compounds 12-36 yielded
different A1 affinities. The lowest levels of A1 affinity
were displayed by derivatives 18 and 30 with Ki values
> 10 000 nM. The Ki values of compounds 15, 16, 22,
23, and 31-33 ranged from 8700 to 1000 nM, confirm-
ing the binding data reported for 1,8-naphthyridine
derivatives.20
F igu r e 3. Concentration-dependent reversal of CHA adenylyl
cyclase activity inhibition by 29 (9) and 25d (2) derivatives.
The enzyme activity was assayed at the concentrations of the
antagonists indicated in the presence of 100 nM CHA and 0.1
mM forskolin as described in Biological Methods (Experimen-
tal Section). Each data point is expressed as a percentage of
adenylyl cyclase activity and represents the mean ( SEM of
at least three independent experiments.
Compounds 17 (Ki ) 810 nM), 20 (Ki ) 550 nM), 13
(Ki ) 450 nM), and 35 (Ki ) 100 nM) possessed an
average receptor affinity; instead compounds 14, 21, and
27 showed an appreciable affinity with Ki values rang-
ing from 80 to 26 nM. Compounds 19 (Ki ) 17 nM), 26
(Ki ) 16 nM), 25b (Ki ) 11 nM), and 34 (Ki ) 9.9 nM)
showed a reduction in Ki values, similar to that of (S)-
PIA and NECA. Derivatives 12, 25a ,e, 28, and 36
showed a high affinity with Ki ) 5.3, 5.3, 4.1, 5.2, and
5.3 nM, respectively.
29 and 25d , with IC50 values of 0.47 and 0.034 nM,
respectively (Figure 3). Moreover, in agreement with
radioligand binding data, derivative 25d was 10-fold
more potent than derivative 29 in counteracting the
CHA functional response. 29 (5 nM) and 25d (0.5 nM)
also caused a parallel rightward shift of the CHA
inhibitory concentration-response curve, suggesting
that both compounds showed full antagonist properties
(results not shown).
Compound 29 showed an affinity constant with Ki )
1.6 nM, similar to the N6-substituted A1-selective ago-
nist analogues, CHA (Ki ) 1.3 nM). The compounds
substituted with Br (25c) or Cl (25d ) in the 7-position
show a substantial increase in potency, with Ki values
in the subnanomolar range (25c: Ki ) 0.7 nM; 25d : Ki
) 0.15 nM), similar to, or lower than, that of (R)-PIA
(Ki ) 0.6 nM).
Discu ssion
For a better understanding of the biological data, it
needs to be pointed out that the numbering of the 1,8-
naphthyridine nucleus used in this discussion is the
same used in the NMR spectroscopy and theoretical
calculations sections.
Derivatives 13-18, 22, 23, 26, 27, 29-33, and 36
showed a poor affinity at A2A receptors, confirming the
binding data reported for 1,8-naphthyridine deriva-
tives;20 the affinity of compounds 12, 19, 21, 25a ,b,d ,e,
and 34 ranged from 830 to 100 nM. The most effective
compound toward A2A receptors was 25c, which showed
an affinity constant of 66 nM.
The binding assay at adenosine A3 receptors showed
that these compounds, 12-36, presented very low
inhibition percentages at a concentration of 1 µM, with
the result that the corresponding Ki values were not
calculated for the majority of the compounds.
In a recent paper, a series of 1,8-naphthyridines 10
had been found to have a micromolar affinity at A1 and
A
2A adenosine receptors and to be slightly A1-selective.20
With the aim of enhancing both the affinity and
selectivity, we focused our studies on a series of 1,8-
naphthyridine derivatives with the following charac-
teristics: (i) a methyl group in position 7; (ii) a variously
substituted nitrogen function in position 4, present in
several derivatives of types 1, 2, 7, 10, and 11; (iii) a
substituent in position 2 with a lipophilic nature,
through the introduction of a phenyl group, present in
some compounds of types 3, 4, and 6.
F u n ction a l Assa y a t A1 Recep tor s. Two potent,
selective A1 compounds (29: Ki ) 1.6 nM; 25d : Ki )
0.15 nM) were also tested for their ability to reverse the
inhibition of forskolin-stimulated adenylyl cyclase activ-
ity induced by the agonist CHA (100 nM).
In rat cerebral cortex membranes, the A1 adenosine
agonist CHA induced a maximal inhibition of adenylyl
cyclase activity of 15-20% of total activity, under
conditions of stimulation (typically 3-4-fold) in the
presence of 0.1 mM forskolin, with an IC50 value of 1.4
( 0.7 nM.33 The inhibition effect of CHA (100 nM) on
adenylyl cyclase activity was antagonized completely
and in a concentration-dependent manner by derivatives
The results reported in Table 3 for compounds 18-
23 and 35 show that some of these compounds have an
affinity at A1 and A2A receptors in the low nanomolar
range with a different degree of selectivity toward A1
receptors. In particular, compound 19 exhibits a re-
markable affinity at A1 receptors (Ki ) 17 nM), when
compared with compound 1020 (generally >1000-fold)
with a better selectivity at A1 receptors. Introduction
of an SH group (compound 14) in position 4 resulted in
a small decrease in the affinity and selectivity (Ki ) 80
nM, A2A/A1 ratio of 16). Replacement of the SH group
with an OH group (compound 12) led to a better affinity
and selectivity (Ki ) 5.3 nM, A2A/A1 ratio of 87).

1,8-Naphthyridine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2819
We therefore focused our studies on the lead com-
pound 12, designing the following structural modifica-
tions: (i) introduction of a substituent on the phenyl
group; (ii) substitution of the methyl group in position
7 with various different groups. With the introduction
of o-F-, p-F-, and p-NO2-phenyl groups in position 2,
both the affinity and the selectivity remained in the
same nanomolar range when compared with compound
12.
The substitution of the methyl group in position 7
with an electron-withdrawing group generally resulted
in an increase in both the affinity and the selectivity
and gave compounds with a subnanomolar affinity at
the A1 receptor (25c,d ). The substitution of the methyl
group in position 7 with an electron-withdrawing group
led to compounds with a very good affinity at A1
adenosine receptors; the order of the A1 affinity of the
substituents is, in the case of the halogens, Cl > Br >
F and, in the case of the alkoxy derivatives, CH3O >
C2H5O > C6H5O.
14 000). The introduction of a phenyl group in position
2 and removal of the substituent in position 3 resulted
in an increase in the affinity and selectivity compared
with those observed for 1,8-naphthyridine derivatives
10.20 On the basis of NMR data, theoretical calculations,
and biological data, it may be deduced that the presence
of a hydroxy group in position 4, which can lead to a
quinoid structure, seems to be important. The most
favorable substituents in position 7 seem to be, at this
time, electron-withdrawing groups.
The field of 1,8-naphthyridine derivatives deserves to
be further developed, to optimize their affinity at the
A1, A2A, and A3 adenosine receptor subtypes.
Exp er im en ta l Section
A. 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
1
spectrometer. H and 13C 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
These modifications thus generally caused a remark-
able increase in affinity at A1 and A2A receptors (Table
3), especially in the case of the compounds with a
heteroatom in position 4 bearing an acidic hydrogen
atom (12, 14, 19, 21, 25-29, 34-36). To explain these
very good results, we think that a very important factor
for the affinity of these 4-hydroxy-substituted com-
pounds could be the tautomeric equilibrium between
forms A and B (Figure 2): all compounds which exhibit
a remarkable affinity at A1 receptors and an interesting
affinity also at A2A receptors prefer the quinoid tauto-
meric structure B, with the N1 protonated (12, 25-29,
34, 36) rather than the phenolic structure A, as shown
by NMR data and theoretical calculations, whereas
compounds which exhibit a lower affinity, analogous to
that reported for 1,8-naphthyridine derivatives 10,20
cannot exist in form B.
Compounds 30-32 deserve particular attention. Un-
like the compounds with tautomeric form B, these
compounds with the quinoid structure in position 2
showed a decrease in the affinity at both receptor
subtypes. Compounds 31 and 32, with a methyl group
in position 1, showed a low affinity. Compound 30,
which possesses the same quinoid structure as 31 and
32, with the N8 protonated, as demonstrated on the
basis of theoretical calculations, dramatically reduced
the affinity.
Among all the 1,8-naphthyridine derivatives tested,
only six derivatives also possess a moderate affinity at
A3 receptors, in the order of a micromolar concentration,
whereas the others are completely ineffective.
The results of the binding studies indicate that all
the compounds are A1-selective. In particular, com-
pounds 25d (7-Cl), 26 (7-H), 28 (7-EtO), 29 (7-MeO), and
36 (7-NH2) showed a very high selectivity (A2A/A1 ratio
ranging between 625 and 1920). Moreover, the two most
potent and selective derivatives in the binding assays,
25d and 29, were also shown to be full antagonists.
Hewlett-Packard MS/System 5988. Elemental analyses (C,H,N)
were within (0.4% of the theoretical values and were per-
formed on a Carlo Erba elemental analyzer model 1106
apparatus.
7-Meth yl-2-p h en yl-1,8-n a p h th yr id in -4(1H)-on e (12).21
1H NMR: δ 11.18 (brs, OH), 8.34 (d, 1H, H5), 7.84 (m, 2H,
Ar), 7.35 (m, 3H, Ar), 7.27 (d, 1H, H6), 6.36 (s, 1H, H3), 2.59
(s, 3H, CH3). 13C NMR: δ 176.02 (C4), 162.59 (C7), 150.92 (C2),
150.92 (C8a), 134.66 (C5), 133.64 (C1′), 130.53 (C4′), 128.85 (C3′),
127.66 (C2′), 120.12 (C6), 117.25 (C4a), 108.51 (C3), 24.33 (7st).
1-E t h yl-7-m e t h yl-2-p h e n yl-1,8-n a p h t h yr id in -4(1H )-
on e (13). A solution of hydroxynaphthyridine 12 (0.500 g, 2.12
mmol) and C2H5I (2.5 mL, 31.2 mmol) in 3 N aqueous KOH (5
mL) and C2H5OH (5 mL) was heated in a sealed tube at 100
°C for 24 h. The reaction mixture was treated with H2O, the
pH was adjusted to 8 with concentrated NH4OH, and then the
mixture was extracted with CHCl3. The organic solution was
dried (MgSO4) and the solvent removed under reduced pres-
sure. The crude solid, purified by flash chromatography (ethyl
acetate), followed by crystallization from petroleum ether 100-
140 °C, gave 13 (0.083 g, yield 15%): mp 125-126 °C; MS m/z
1
264 (M+). H NMR: δ 8.41 (d, 1H, H5), 7.54 (m, 5H, Ar), 7.35
(d, 1H, H6), 5.95 (s, 1H, H3), 4.22 (q, 2H, NCH2), 2.62 (s, 3H,
CH3), 1.09 (t, 3H, CH3). 13C NMR: δ 175.71 (C4), 162.05 (C7),
154.54 (C2), 149.39 (C8a), 135.32 (C1′), 135.23 (C5), 129.45 (C4′),
128.69 (C3′), 128.16 (C2′), 120.02 (C6), 118.76 (C4a), 112.38 (C3),
41.28-14.16 (1st), 24.91 (7st). Anal. (C17H16N2O) C, H, N.
4-Mer ca p to-7-m eth yl-2-p h en yl-1,8-n a p h th yr id in e (14).
A solution of hydroxynaphthyridine 12 (0.236 g, 1.0 mmol) and
Lawesson’s reagent (0.242 g, 0.6 mmol) in toluene (40 mL) was
refluxed for 6 h. The solvent was removed under reduced
pressure to obtain the title compound which, purified by
crystallization from EtOH-H2O, gave 14 (0,206 g, yield
1
82%): mp 204-205 °C; MS m/z 252 (M+). H NMR: δ 13.30
(brs SH), 8.83 (d, 1H, H5), 7.89 (m, 2H, Ar), 7.54 (m, 3H, Ar),
7.53 (s, 1H, H3), 7.40 (d, 1H, H6), 2.62 (s, 3H, CH3). 13C NMR:
δ 163.81 (C4), 160.17 (C7), 146.89 (C2), 145.59 (C8a), 137.65 (C5),
132.51 (C1′), 131.01 (C4′), 129.01 (C3′), 128.10 (C2′), 124.51 (C4a),
123.51 (C6), 122.22 (C3), 24.30 (7st). Anal. (C15H12N2S) C, H,
N.
4-Meth oxy-7-m eth yl-2-p h en yl-1,8-n a p h th yr id in e (16).
Chloronaphthyridine 15 (0.254 g, 1.0 mmol) was added to a
solution of sodium (0.046 g, 2 g atom) in anhydrous MeOH
(50 mL), and the mixture was refluxed for 24 h. The resulting
solution was evaporated to dryness under reduced pressure
to obtain a residue which was treated with H2O and 3 N
aqueous HCl to adjust the pH to 8. The solid was then collected
by filtration, washed with H2O and crystallized from toluene-
petroleum ether 60-80 °C to give 16 (0.149 g, yield 59%): mp
Con clu sion s
The modifications carried out on compounds 1020 led
to compounds having a higher affinity, up to the
subnanomolar range (0.15 nM), and a remarkable
selectivity (A2A/A1 ratio of 1920 and A3/A1 ratio of
1
101-102 °C; MS m/z 250 (M+). H NMR: δ 8.36 (d, 1H, H5),
8.30 (m, 2H, Ar), 7.56 (s, 1H, H3), 7.50 (m, 3H, Ar), 7.39 (d,

2820 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Ferrarini et al.
1H, H6), 4.13 (s, 3H, OCH3), 2.66 (s, 3H, CH3). 13C NMR: δ
163.08 (C4), 162.55 (C7), 159.94 (C2), 156.17 (C8a), 138.95 (C1′),
131.03 (C5), 129.91 (C4′), 128.67 (C3′), 127.52 (C2′), 121.62 (C6),
112.28 (C4a), 98.46 (C3), 56.59 (4st), 24.97 (7st). Anal. (C16H14N2O)
C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 4-Hyd r oxy-
7-m eth yl-2-(4-flu or op h en yl)-1,8-n a p h th yr id in e (25a ) a n d
4-H yd r oxy-7-m et h yl-2-(2-flu or op h en yl)-1,8-n a p h t h yr i-
d in e (25b). A solution of appropriate pyridopyrimidinone 24
(1.0 mmol) in Dowtherm A (10 mL) was heated at 240 °C for
4 h. After cooling, the precipitate was collected and washed
with petroleum ether to obtain the title naphthyridines. 25a :
0.20 g, yield 81%; mp 291-293 °C (crystallized from 2-pro-
7-Meth yl-4-p h en oxy-2-p h en yl-1,8-n a p h th yr id in e (17).
NaH (0.170 g, 3.5 mmol, 50% in mineral oil) was added to a
solution of phenol (0.197 g, 2.1 mmol) in anhydrous DMF (10
mL), and the mixture was stirred for 30 min at room temper-
ature. Chloronaphthyridine 15 (0.254 g, 1.0 mmol) was added
to the suspension obtained, and the mixture was heated at 80
°C for 6 h. The reaction mixture was evaporated to dryness
under reduced pressure and the residue was treated with H2O
and filtered. The residual solid was crystallized from petroleum
ether 60-80 °C to obtain 17 (0.240 g, yield 77%): mp 154-
1
panol-water); MS m/z 386 (M+). H NMR: δ 12.30 (brs, OH),
8.34 (d, 1H, H5), 7.98 (m, 2H, Ar), 7.34 (m, 2H, Ar), 7.28 (d,
1H, H6), 6.35 (s, 1H, H3), 2.60 (s, 3H, CH3). 13C NMR: δ 177.15
(C4), 163.43 (C4′), 162.60 (C7), 150.87 (C2), 149.89 (C8a), 134.64
(C5), 130.14 (C2′), 130.05 (C1), 120.16 (C6), 117.18 (C4a), 115.78
(C3′), 108.50 (C3), 24.30 (7st). Anal. (C15H11N2OF) C, H, N.
25b: 0.140 g, yield 55%; mp 256-258 °C (crystallized from
1
toluene); MS m/z 386 (M+). H NMR: δ 11.35 (brs, OH), 8.30
1
155 °C; MS m/z 312 (M+). H NMR: δ 8.52 (d, 1H, H5), 8.05
(d, 1H, H5), 7.75-7.30 (m, 4H, Ar), 7.25 (d, 1H, H6), 6.15 (s,
(m, 2H, Ar), 7.37 (s, 5H, OPh), 7.24 (m, 3H, Ar), 7.22 (d, 1H,
H6), 7.06 (s, 1H, H3), 2.83 (s, 3H, CH3). Anal. (C21H16N2O) C,
H, N.
1H, H3), 2.65 (s, 3H, CH3). Anal. (C15H11N2OF) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 7-Br om o-
(25c), 7-Ch lor o- (25d ), a n d 7-F lu or o-4-h yd r oxy-2-p h en yl-
1,8-n a p h th yr id in e (25e). A solution of appropriate pyridopy-
rimidinone 24 (1.0 mmol) in Dowtherm A (10 mL) was heated
at 220 °C for 10 min. The solution was allowed to rest at room
temperature and after 24 h the precipitate formed was
collected by filtration and washed with petroleum ether to
obtain the title compounds. 25c: 0.27 g, yield 90%; mp 237-
238 °C (crystallized from toluene); MS m/z 300 (M+). 1H
NMR: δ 11.18 (brs, OH), 8.30 (d, 1H, H5), 7.81 (m, 2H, Ar),
7.53 (m, 3H, Ar), 7.50 (d, 1H, H6), 6.43 (s, 1H, H3). Anal.
(C14H9N2OBr) C, H, N. 25d : 0.16 g, yield 61%; mp 293-295
°C (crystallized from DMF-water); MS m/z 256 (M+). 1H
NMR: δ 11.10 (brs, OH), 8.45 (d, 1H, H5), 7.80 (m, 2H, Ar),
7.45 (m, 3H, Ar), 7.40 (d, 1H, H6), 6.42 (s, 1H, H3). Anal.
(C14H9N2OCl) C, H, N. 25e: 0.18 g, yield 76%; mp >300 °C
(crystallized from acetic acid-water); MS m/z 240 (M+). 1H
NMR: δ 11.30 (brs, OH), 8.58 (dd, 1H, H5), 7.76 (m, 2H, Ar),
7.52 (m, 3H, Ar), 7.10 (dd, 1H, H6), 6.42 (s, 1H, H3). Anal.
(C14H9N2OF) C, H, N.
4-Hyd r oxy-2-p h en yl-1,8-n a p h th yr id in e (26). A suspen-
sion of bromonaphthyridine 25c (0.300 g, 1.0 mmol) and
NaHCO3 (0.300 g) in dioxane (15 mL) was heated at 95 °C,
then Raney Ni (3.0 g) was added and the suspension was
refluxed for 3 h. The reaction mixture was filtered to separate
the catalyst, and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography
(ethyl acetate:petroleum ether, 3:1) and recrystallized from
toluene to obtain 26 (0.166 g, yield 75%): mp 225-227 °C;
MS m/z 222 (M+). 1H NMR: δ 11.20 (brs, OH), 8.75 (m, 1H,
H7), 8.45 (dd, 1H, H5), 7.81 (m, 2H, Ar), 7.53 (m, 1H, H6), 7.48
(m, 3H, Ar), 6.38 (s, 1H, H3). Anal. (C14H10N2O) C, H, N.
4-Hydr oxy-7-ph en oxy-2-ph en yl-1,8-n aph th yr idin e (27).
A solution of NaH (0.194 g, 4 mmol, 50% in mineral oil), phenol
(0.376 g, 4.0 mmol) and bromonaphthyridine 25c (0.300 g, 1.0
mmol) in anhydrous DMF (10 mL) was refluxed for 6 h. The
reaction mixture was concentrated under reduced pressure and
H2O was added. The precipitate formed was collected by
filtration, purified by flash chromatography (ethyl acetate:
petroleum ether, 3:1) and recrystallized from ethanol to obtain
27 (0.157 g, yield 50%): mp 245-247 °C; MS m/z 314 (M+).
1H NMR: δ 11.15 (brs, OH), 8.44 (d, 1H, H5), 7.73 (m, 2H,
Ar), 7.46 (s, 5H, OPh), 7.29 (m, 3H, Ar), 6.93 (d, 1H, H6), 6.33
(s, 1H, H3). Anal. (C20H14N2O2) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 7-Eth oxy-
4-h yd r oxy-2-p h en yl-1,8-n a p h th yr id in e (28) a n d 4-Hy-
d r oxy-7-m eth oxy-2-p h en yl-1,8-n a p h th yr id in e (29). Bro-
monaphthyridine 25c (0,300 g, 1.0 mmol) was added to a
solution of sodium (0.230 g, 10 gatom) in anhydrous EtOH or
MeOH (20 mL) and refluxed for 18 h. The reaction mixture
was evaporated to dryness under reduced pressure; the residue
was treated with H2O and 3 N aqueous HCl until pH ) 6.
The solid was collected by filtration and purified by flash
chromatography (ethyl acetate:petroleum ether, 3:1) to obtain
the title compounds. 28: 0.171 g, yield 64%; mp 223-225 °C
(crystallized from toluene); MS m/z 266 (M+). 1H NMR: δ 8.98
4-Am in o-7-m eth yl-2-p h en yl-1,8-n a p h th yr id in e (19). A
solution of azidonaphthyridine 18 (0.261 g, 1.0 mmol) in
anhydrous MeOH (25 mL) was submitted to hydrogenation
in the presence of 10% Pd/C (0.03 g) at room pressure and
temperature for 6 h. The catalyst was filtered off and the
solvent evaporated to dryness under reduced pressure to give
a residue, which was purified by flash chromatography (ethyl
acetate:diethylamine, 10:0.5) and recrystallized from ethyl
acetate to give 19 (0.052 g, yield 22%): mp 268-270 °C; MS
1
m/z 235 (M+). H NMR: δ 8.51 (d, 1H, H5), 8.09 (m, 2H, Ar),
7.50 (m, 3H, Ar), 7.27 (d, 1H, H6), 7.12 (s, 1H, H3), 7.07 (brs,
NH2), 2.62 (s, 3H, CH3). 13C NMR: δ 161.65 (C4), 158.53 (C7),
156.68 (C2), 153.64 (C8a), 139.91 (C1′), 131.91 (C4′), 129.22 (C5),
128.60 (C3′), 126.92 (C2′), 119.48 (C6), 109.97 (C4a), 99.16 (C3),
24.88 (7st). Anal. (C15H13N3) C, H, N.
Gen er a l P r oced u r e for th e P r ep a r a tion of 4-Dim eth yl-
a m in o- (20), 4-Cycloh exyla m in o- (21), a n d 4-(4-Ca r be-
t h oxyp ip er a zin -1-yl)-7-m et h yl-2-p h en yl-1,8-n a p h t h yr i-
d in e (22). A mixture of chloronaphthyridine 15 (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 24 h. The reaction mixture was
treated with H2O, and the solid was collected by filtration to
obtain the title compounds. 20: 0.270 g, yield 86%; mp 147-
149 °C (crystallized from toluene); MS m/z 263 (M+). 1H
NMR: δ 8.22 (d, 1H, H5), 8.16 (m, 2H, Ar), 7.40 (m, 3H, Ar),
7.27 (d, 1H, H6), 7.20 (s, 1H, H3), 3.08 (s, 6H, N(CH3)2), 2.76
(s, 3H, CH3). Anal. (C17H17N3) C, H, N. 21: 0.250 g, yield 66%;
mp 235-237 °C (crystallized from benzene); MS m/z 317 (M+).
1H NMR: δ 8.15 (m, 2H, Ar), 7.93 (d, 1H, H5), 7.42 (m, 3H,
Ar), 7.13 (d, 1H, H6), 6.90 (s, 1H, H3), 4.90 (brs, NH), 3.65 (m,
1H, cyclohexylamine), 2.74 (s, 3H, CH3), 1.74, 1.35 (m, 10H,
cyclohexylamine). Anal. (C21H23N3) C, H, N. 22: 0.412 g, yield
95%; mp 134-136 °C (crystallized from petroleum ether 100-
1
140 °C); MS m/z 376 (M+). H NMR: δ 8.33 (m, 2H, Ar), 8.30
(d, 1H, H5), 7.56 (m, 3H, Ar), 7.46 (d, 1H, H6), 7.43 (s, 1H, H3),
4.23 (q, 2H, OCH2), 3.83, 3.26 (m, 8H, piperazine), 2.83 (s, 3H,
CH3), 1.33 (t, 3H, CH3). Anal. (C22H24N4O2) C, H, N.
7-Met h yl-2-p h en yl-4-(p ip er a zin -1-yl)-1,8-n a p h t h yr i-
d in e (23). A suspension of carbethoxypiperazinylnaphthy-
ridine 22 (0.364 g, 1.0 mmol) in EtOH (10 mL) and 5 N aqueous
NaOH (10 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 chloro-
form, and the combined extracts were dried (MgSO4) and
evaporated under reduced pressure and recrystallized from
toluene to obtain 23 (0.279 g, yield 92%): mp 187-189 °C. 1H
NMR: δ 10.17 (brs, NH), 8.80 (d, 1H, H5), 8.21 (m, 2H, Ar),
7.71 (d, 1H, H6), 7.63 (s, 1H, H3), 7.62 (m, 3H, Ar), 3.39, 3.69
(m, 8H, piperazine), 2.84 (s, 3H, CH3). 13C NMR: δ 163.13 (C4),
159.09 (C7), 157.63 (C2), 149.69 (C8a), 138.96 (C1′), 134.25 (C5),
131.75 (C4′), 129.02 (C3′), 128.67 (C2′), 122.29 (C6), 113.49 (C4a),
107.05 (C3), 48.38-42.21 (4st), 22.72 (7st). Anal. (C19H2oN4) C,
H, N.

1,8-Naphthyridine Derivatives
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2821
(brs, OH), 8.40 (d, 1H, H5), 7.59 (m, 2H, Ar), 7.46 (m, 3H, Ar),
6.65 (d, 1H, H6), 6.48 (s, 1H, H3), 4.38 (q, 2H, OCH2), 1.39 (t,
3H, CH3). Anal. (C16H14N2O2) C, H, N. 29: 0.130 g, yield 52%;
mp 244-246 °C (crystallized from toluene); MS m/z 252 (M+).
1H NMR: δ 11.80 (brs, OH), 8.31 (d, 1H, H5), 7.80 (m, 2H,
Ar), 7.53 (m, 3H, Ar), 6.81 (d, 1H, H6), 6.35 (s, 1H, H3), 4.00
(s, 3H, OCH3). 13C NMR: δ 176.28 (C4), 165.14 (C7), 150.69
(C2), 150.47 (C8a), 137.17 (C5), 133.85 (C1′), 130.33 (C4′), 128.73
(C3′), 127.70 (C2′), 114.24 (C4a), 108.99 (C3), 108.66 (C6), 63.95
(7st). Anal. (C15H12N2O2) C, H, N.
4,7-Dih ydr oxy-2-ph en yl-1,8-n aph th yr idin e (30). Meth od
a : A solution of bromonaphthyridine 25c (0.300 g, 1.0 mmol)
in 5 N aqueous NaOH (20 mL) was refluxed for 12 h. The
reaction mixture was concentrated and HCl was added until
pH ) 7. The solid obtained, collected by filtration, was
triturated with concentrated HCl, filtered and washed with
H2O to obtain 30 (0.088 g, yield 37%). Meth od b: A solution
of methoxynaphthyridine 29 (0.252 g, 1.0 mmol) and 1 mL of
48% HBr in glacial AcOH (2 mL) was refluxed for 4 h. The
reaction mixture was evaporated to dryness under reduced
pressure and the solid obtained was triturated with (CH3)2-
CO, filtered and purified by crystallization from AcOH to
obtain 30 (0.149 g, yield 63%): mp >300 °C; MS m/z 238 (M+).
1H NMR: δ 8.08 (m, 2H, Ar), 8.02 (d, 1H, H5), 7.50 (m, 3H,
Ar), 7.20 (s, 1H, H3), 6.53 (d, 1H, H6). 13C NMR: δ 162.72 (C4),
162.53 (C7), 157.00 (C2), 151.03 (C8a), 138.17 (C1′), 131.82 (C5),
129.82 (C4′), 128.87 (C3′), 126.81 (C2′), 119.10 (C6), 104.09 (C4a),
101.13 (C3), 28.01 (7st). Anal. (C14H10N2O2) C, H, N.
5-Meth oxy-1-m eth yl-7-p h en yl-1,8-n a p h th yr id in -2(1H)-
on e (31). A suspension of NaH (0.122 g, 2.54 mmol, 50% in
mineral oil), dihydroxynaphthyridine 30 (0.405 g, 1.7 mmol)
in anhydrous DMSO (6 mL) was allowed to react in a sealed
tube. After standing at room temperature for 1 h, CH3I (0.42
mL, 6.8 mmol) was added and the mixture was heated at 90
°C for 24 h. The suspension was treated with H2O and the
precipitate formed was collected by filtration and purified by
crystallization with petroleum ether 100-140 °C to obtain
31: (0.094 g, yield 21%); mp 192-194 °C; MS m/z 266 (M+).
1H NMR: δ 8.25 (m, 2H, Ar), 7.96 (d, 1H, H4), 7.52 (m, 3H,
Ar), 7.45 (s, 1H, H6), 6.55 (d, 1H, H3), 4.09 (s, 3H, OCH3), 3.71
(s, 3H, NCH3). 13C NMR: δ 163.42 (C5), 162.31 (C2), 157.78
(C7), 150.41 (C8a), 138.11 (C1′), 131.25 (C4), 130.07 (C4′), 128.74
(C3′), 127.27 (C2′), 119.83 (C3), 104.21 (C4a), 97.63 (C6), 56.75
(5st), 28.07 (1st). Anal. (C16H14N2O2) C, H, N.
5-Hyd r oxy-1-m eth yl-7-p h en yl-1,8-n a p h th yr id in -2(1H)-
on e (32). A solution of 31 (0.125 g, 0.47 mmol) and 1 mL of
48% HBr in glacial ACOH (1 mL) was refluxed for 4 h. The
mixture was poured over crushed ice and alkalinized with
concentrated NH4OH. The precipitate formed was collected by
filtration, washed with H2O and purified by crystallization
from EtOH to obtain 32 (0.040 g, yield 34%): mp 303-305
°C; MS m/z 252 (M+). 1H NMR: δ 11.23 (brs, OH), 8.00 (m,
2H, Ar), 7.98 (d, 1H, H4), 7.46 (m, 3H, Ar), 7.18 (s, 1H, H6),
6.60 (d, 1H, H3), 3.72 (s, 3H, NCH3). Anal. (C15H12N2O2) C, H,
N.
were purchased from RBI (Natik, MA). Adenosine deaminase
was from Sigma Chemical Co. (St. Louis, MO). Bacitracin,
benzamidine and PMSF were products of Fluka Chemie AG
(Buchs, Switzerland). All other reagents were from standard
commercial sources and of the highest grade commercially
available.
A1 Recep tor Bin d in g Assa y. Bovine brains were obtained
from the local slaughterhouse. The cerebral cortex was dis-
sected from bovine brain and immediately homogenized in 10
volumes of ice-cold 0.32 M sucrose containing protease inhibi-
tors (200 µg/mL bacitracin, 160 µg/mL benzamidine and 20
µg/mL soybean trypsin inhibitor) with an Ultra-Turrax ho-
mogenizer. The homogenate was centrifuged at 1000g for 10
min at 4 °C and the resulting supernatant again centrifuged
at 48000g for 15 min at 4 °C. The pellet was resuspended in
10 volumes of ice-cold buffer A (2 mM MgCl2 and 50 mM Tris/
HCl, pH 7.7) containing protease inhibitors (as above) and
adenosine deaminase (2 units/ml) to remove endogenous
adenosine. After incubation for 30 min at 37 °C, the suspension
was centrifuged at 48000g for 15 min at 4 °C. The final pellet
was stored in aliquots at -80 °C until the time of assay.
The [3H]CHA (39 Ci/mmol) binding assay was performed
in triplicate by incubating aliquots of the membrane fractions
(0.2-0.3 mg of protein) at 25 °C for 45 min in 0.5 mL of buffer
A, with approximately 1 nM [3H]CHA. Nonspecific binding was
defined in the presence of 10 µM of (R)-PIA. Binding reactions
were terminated by filtration through Whatman CG/C glass
microfiber filters under suction, washing twice with 5 mL of
ice-cold buffer, and introduction into scintillation vials. The
radioactivity was counted in 4 mL of scintillation cocktail in a
scintillation counter.
A
2A Recep tor Bin d in g Assa y. The striatum was dissected
from bovine brain and homogenized in 20 volumes of ice-cold
10 mM MgCl2 and 50 mM Tris/HCl buffer, pH 7.4 (buffer B),
containing protease inhibitors and resuspended in 20 volumes
of buffer B containing protease inhibitors (as above) and
adenosine deaminase (2 units/mL). Incubation was carried out
for 30 min at 37 °C. The membrane suspension was recentri-
fuged, and the final pellet was frozen in aliquots at -80 °C
until the time of assay.
Routine [3H]CGS21680 (45 Ci/mmol) binding assays were
performed in triplicate by incubating an aliquot of striatal
membranes (0.2-0.3 mg of protein) in buffer B with ap-
proximately 5 nM [3H]CGS21680 in a final volume of 0.5 mL.
Incubation was carried out at 25 °C for 90 min. Nonspecific
binding was defined in the presence of 10 µM NECA. 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. The radioactivity was
counted as above.
A3 Recep tor Bin d in g Assa y. A3 receptor binding sites
were studied in rat testis membranes. Fresh testicular tissue
from Sprague-Dawley rats was dissected free of epididymis
and membranes were prepared as described.32
Binding of [3H](R)-PIA (37 Ci/mmol) to membranes was
measured in the presence of DPCPX (150 nM) as described.32
Briefly, rat testis membranes (0.1-0.2 mg of protein) and [3H]-
(R)-PIA 4 nM were incubated in 0.5 mL total volume of 50
mM Tris/HCl (pH 7.4), 1 mM EDTA, 10 mM MgCl2 buffer in
the presence of 150 nM 8-cyclopentyl-1,3-dipropylxanthine
(DPCPX) to block A1 adenosine receptors. Nonspecific binding
was determined in the presence of 15 µM (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. The radioactivity was
counted as above.
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 the binding. At least six different concentra-
tions 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
7-Meth yl-2-p h en yl-1,8-n a p h th yr id in e (33).21 1H NMR:
δ 8.44 (d, 1H, H5), 8.30 (m, 2H, Ar), 8.13 (d, 1H, H4), 8.12 (d,
1H, H3), 7.54 (m, 3H, Ar), 7.45 (d, 1H, H6), 2.72 (s, 3H, CH3).
4-Hyd r oxy-7-m eth yl-2-(4-n itr op h en yl)-1,8-n a p h th yr i-
d in e (34).23 1H NMR: δ 11.80 (brs, OH), 8.36 (d, 1H, H5), 8.34
(m, 2H, Ar), 8.11 (m, 2H, Ar), 7.33 (d, 1H, H6), 6.46 (s, 1H,
H3), 2.62 (s, 3H, CH3).
4-Hydr azin o-7-m eth yl-2-ph en yl-1,8-n aph th yr idin e (35).28
1H NMR: δ 8.05 (d, 1H, H5), 8.01 (m, 2H, Ar), 7.40 (m, 3H,
Ar), 7.15 (s, 1H, H3), 7.03 (d, 1H, H6), 3.90 (brs, 3H, NHNH2),
2.62 (s, 3H, CH3).
7-Am in o-2-p h en yl-1,8-n a p h th yr id in -4(1H)-on e (36).24
1H NMR: δ 11.50 (brs, OH), 7.97 (d, 1H, H5), 7.69 (m, 2H,
Ar), 7.48 (m, 3H, Ar), 6.67 (brs, 2H, NH2), 6.46 (d, 1H, H6),
6.12 (s, 1H, H3).
B. Biologica l Meth od s. Ma ter ia ls. [3H](R)-PIA was from
Amersham Corp. (Little Chalfont, Buckinghamshire, U.K.),
while [3H]CHA and [3H]CGS21680 were obtained from NEN
(Boston, MA). CHA, CPA, (R)-PIA, (S)-PIA, NECA and XAC

2822 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 15
values, knowing the Kd values of radioligands in these different
Ferrarini et al.
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Adenosine Receptor Stimulation. In Adenosine and Adenine
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tissues and using the Cheng and Prusoff equation:34 Ki ) IC50
/
(1 + [L]/Kd), where [L] is the ligand concentration and Kd its
dissociation constant. The Kd of [3H]CHA binding to bovine
cortex membranes was 1.2 nM, the Kd of [3H]CGS21680
binding to bovine striatal membranes was 10 nM, and the Kd
of [3H](R)-PIA binding to rat testis membranes was 36 nM.
Protein concentration was determined in accordance with the
method of Lowry as modified by Peterson,35 using bovine
serum albumin as the standard.
Ad en ylyl Cycla se Assa y. The cerebral cortex was obtained
from male Sprague-Dawley rats sacrificed by cervical disloca-
tion. Fresh tissue was suspended in 50 volumes of ice-cold
buffer containing 0.32 M sucrose, 200 µg/mL bacitracin, 160
µg/mL benzamidine, 20 µg/mL trypsin inhibitor, 0.1 mM PMSF
and 10 mM HEPES/NaOH, pH 7.4, and homogenized with 12
strokes of a Teflon homogenizer at 4 °C. The homogenate was
centrifuged at 1000g for 10 min at 4 °C. The supernatant was
collected and centrifuged at 48000g for 30 min at 4 °C. The
pellet was resuspended in 10 volumes of an ice-cold hypotonic
buffer containing all the constituents of the homogenization
buffer except sucrose and incubated with adenosine deaminase
(2 units/ml) for 30 min at 30 °C. The membrane suspension
was centrifuged at 48000g for 15 min at 4 °C. This centrifuga-
tion step was repeated twice. The final pellet was resuspended
in 50 mM HEPES/NaOH, pH 7.4, and used in adenylyl cyclase
assays.
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conversion of [R-32P]ATP to [R-32P]cAMP, using a previously
36
reported method
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