Structure-activity relationships in a series of 8-substituted xanthines as A1-adenosine receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(00)00271-6

A series of 8-substituted xanthines were synthesized and their affinity in vitro towards A₁, A_2A-adenosine receptors was evaluated by radioligand receptor binding assays. All compounds showed a greater affinity and selectivity toward

Full text of DOI:10.1016/S0968-0896(00)00271-6

Bioorganic & Medicinal Chemistry 9 (2001) 575±583
Structure±Activity Relationships in a Series of 8-Substituted
Xanthines as A1-Adenosine Receptor Antagonists
Giovannella Strappaghetti,a,* Stefano Corsano,a Roberta Barbaro,a Gino Giannaccinib
and Laura Bettib
a
Á
Dipartimento di Chimica e Tecnologia del Farmaco, Universita di Perugia, Via del Liceo 1, 06123Perugia, Italy
b
Á
Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita di Pisa, Via Bonanno 6, 56126 Pisa, Italy
Received 28 April 2000; accepted 25September 2000
AbstractÐA series of 8-substituted xanthines were synthesized and their anity in vitro towards A1, A2A-adenosine receptors was
evaluated by radioligand receptor binding assays. All compounds showed a greater anity and selectivity towards the A1-adenosine
receptor than theophylline. The compounds in which the n-proyl group is in 1-position of the xanthine nucleus and the pyridazinone
system in 8-position is linked through a chain of two or four carbon atoms, showed the highest anity and selectivity. # 2001
Elsevier Science Ltd. All rights reserved.
Introduction
containing a pyridazinone ring which showed a good a-
nity towards A1-receptor, particularly the compound 114
has higher anity (Ki=0.85 mM) towards this receptor.
Adenosine receptors are involved in many peripheral and
central regulatory mechanisms, including vasodilatation1
vasoconstriction in the kidney,2 inhibition of lypolysis and
insulin release,3 and moderation of cerebral ischemia.4
Four subtypes of adenosine receptors (A1, A2A, A2B, and
A3) have been identi®ed, both pharmacologically and
through cloning techniques.5,6
Therefore to obtain further information on the role played
by this pyridazinone ring in the interaction between the
ligand and the receptor, we have synthesized a series of
xanthine derivatives substituted at 8-position, in which the
pyridazinone group was linked through a linker of one,
two, three or four carbon atoms. In order to obtain more
information on structure±activity relationships, we have
synthesized compounds in which the methyl group in
1-position of the theophylline nucleus was substituted
by a n-propyl group.
All adenosine receptor agonists already studied are
derivatives of adenosine, while there are several classes
of adenosine antagonists, but the more studied is the
xanthine class. However they are virtually non-selective
antagonists and have weak anity for A1 and A2a
adenosine receptors.7,8 It is therefore unclear to what
extent the antiasthmatic, diuretic, respiratory stimulant,
central stimulant, cardiac stimulant, and analgesic
adjuvant activities of xanthines re¯ect interactions at A1
or A2 receptors or at some other site. Studies of structure±
activity relationships of xanthines reveal that the intro-
duction of an alkyl group at 1- and 3-position markedly
increases anity for both adenosine receptors sub-
types.9,10 On the other hand, either 8-aryl or 8-cycloalkyl
group increases the anity towards A1-receptor.11
Recently we have synthesized xanthine derivatives sub-
stituted at the N7 position12,13 or C8 and with fragment
Chemistry
The synthesis of xanthine 5±22 was performed accord-
ing to the general method summarized in Scheme 1,
using the standard procedure of Traube:15 acylation of
*Corresponding author. Tel.: +39-75-585-5136; e-mail: noemi@
unipg.it
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00271-6

576
G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
of the pyridazinone ring. While a decrease in anity
and selectivity was found when the chlorine atom was
substituted with a phenyl group (compounds 9±12), or
with a hydrogen atom (compounds 13±14). Replace-
ment of the 1-methyl substituent of theophylline with a
n-propyl group has a marked e€ect on the A1 receptor
(compounds 15±22), in particular compounds 16, 18
and 21 show the highest selectivity.
In conclusion, these xanthine derivatives show a higher
anity and selectivity towards the A1 receptor than
theophylline and ca€eine. The replacement of the
1-methyl substituent of theophylline with n-propyl group
con®rms the increase of the anity towards the A1- and
A2A-adenosine receptors. A chlorine atom or a phenyl
group in 6-position of the pyridazinone ring and a n-
propyl group in 1-position in the xanthine nucleus are
important for the selectivity. Moreover the length of the
alkyl chain, a spacer between the two major constituents of
the molecule, can in¯uence the anity and the selectivity.
Several of these theophylline analogues may prove use-
ful for the investigation of the signi®cance of A1 and A2
receptors in various physiological processes.
Experimental Protocols
Biological methods
Scheme 1. (i) DCC/methanol; (ii) NaOH 2.5% Á/HCl 5%.
Anity of the new xanthine derivatives 5±22 towards
A1 and A2A adenosine receptors were evaluated using
radioligand binding technique.
the appropriate 5,6-diamino-1,3-dialkyl-uracil with the
corresponding carboxylic acid in methanol and DCC,
followed by treatment with a solution of sodium
hydroxide and acidi®cation by HCl, gave the corre-
sponding xanthine derivatives. The preparation of the
5,6-diamino-1-methyl-3-propyl-uracil (2a) was carried
out starting from 6-amino-1-methyl-uracil by alkylation
in 3-position (corresponding to 1-position of xanthine)
with alkyl bromides or iodides in the presence of sodium
hydroxide as base16 followed by nitrosation in 5-posi-
tion and reduction to 5,6-diaminouracil.
Competition assays for A1 and A2A adenosine receptors
were determined in bovine brain cortical membranes,
and bovine striatal membranes, respectively.
N6-(cyclohexyl)-adenosine ([3H]CHA), was used as A1
ligand, and 2-[p-(2-carboxyethyl)-phenylethyl]amino]-50-
(N-ethylcarbamoyl)adenosine ([3H]CGS 21680) as A2A
ligand. The biological results expressed in Ki are repor-
ted in Table 3.
A1 receptor binding
Results and Discussion
Bovine cerebral cortex was homogenized in 10 volumes
ice-cold 0.25M sucrose containing 5mM EDTA and
protease inhibitors (0.1 mM of phenylmethylsulfonil-
¯uoride (PMSF), 200 mg/mL bacitracine, and 160 mg/mL
benzamidine) in an ultra-turrax homogenizer. The homo-
genate was centrifuged at 1000g for 10min at 4 ꢀC and the
supernatant again centrifuged at 46,000g for 20 min at
4 ꢀC. The resulting pellet was suspended in 10 volumes of
ice-cold 50 mM Tris±HCl bu€er at pH 7.7 containing
1 mM EDTA, 5mM MgCl 2 and protease inhibitors
(bu€er T1). It was then homogenized and centrifuged at
46,000g for 20 min at 4 ꢀC.
The 8-substituted theophylline analogues (compounds 5±
22) were tested in radioligand binding assays for anity to
A1 and A2A adenosine receptors in bovine brain cortical,
and bovine striatal membranes, respectively. CHA was
used as A1 ligand and CGS21680 as A2A ligand.
Theophylline and ca€eine were used as reference com-
pounds.
As can be seen from the data in Table 3, these com-
pounds are more potent than theophylline and ca€eine
showing more anity and selectivity towards A1 recep-
tor. In the series of compounds 5±8, a gradual increase
in anity and selectivity was observed by promoting the
polymethylene chain length from one to four carbon
atoms and when a chlorine atom is present in 6-position
The pellet was dispersed in 5volumes of fresh T bu€er
1
and incubated with adenosine deaminase (2 UI/mL) at
37 ꢀC for 30 min, then recentrifuged at 46,000g for
20 min at 4 ꢀC.

G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
577
The resulting pellet was frozen at À80 ꢀC until the time
The binding reaction was terminated by ®ltering through
Whatman GF/C glass ®ber ®lters under suction and
washing twice with 5mL ice-cold Tris-bu€er. The ®lters
were placed in scintillation vials and 4 mL Gold MN
Cocktail-Packard solvent scintillation ¯uid was added.
The radioactivity was counted with an Packard 1600
TR scintillation counter. Speci®c binding was obtained
by subtracting non-speci®c binding from total binding
and was approximated to 85±90% of the total binding.
of assay.
The pellet was suspended in 20 volumes of ice-cold T1
bu€er and A1 binding assay was performed in triplicate
by incubating at 25 ꢀC for 120 min or 0 ꢀC for 150 min in
0.5mL T 1 bu€er containing aliquots of the membrane
fraction (0.2±0.3 mg protein) and 1.3 nM[3H]CHA in the
absence or presence of unlabelled 15 mM R-PIA.
A2A receptor binding
Table 1. Chemical data of the amide derivatives 4a±4t
Striatum was dissected from bovine brain and the tissue
was homogenized in 20 volumes of ice-cold 50 mM
Tris±HCl bu€er at pH 7.4 containing 10 mM MgCl2,
1 mM EDTA, and protease inhibitors as reported above
(bu€er T2). The homogenate was centrifuged at 46,000g
for 10 min at 4 ꢀC. The pellet was then suspended in 20
volumes of Tris±HCl bu€er (T2) containing adenosine
deaminase (2 UI/mL) and incubated for 30 min at 37 ꢀC.
The resulting pellet was diluted in 20 volumes of 50 mM
Compound
n
R
R1
Yield (%)
mp (ꢀC)
4a
4b
4c
4d
4e
4f
4g
4h
4i
1
2
3
4
1
2
3
4
2
3
1
2
3
4
1
2
3
4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
Cl
Cl
Cl
Cl
Ph
Ph
Ph
Ph
H
80
80
75>325
>325
>325
Tris±HCl bu€er at pH 7.5containing 10 mM MgCl
and used in the binding assay.
2
±218 65215
70
287±290
>325
>325
Binding assay was performed in triplicate, by incubating
aliquots of the membrane fraction (0.2±0.3 mg protein)
in Tris±HCl at pH 7.5, with approximately 5 nM [3H]
CGS 21680 in a ®nal volume of 0.5mL. Incubation was
60
80
±228 85225
70
60
90
90
30
60
60
70
40
60
290±293
195±198
235±238
218±221
232±235
215±218
255±258
286±289
225±228
132±135
4l
H
4m
4n
4o
4p
4q
4r
4s
4t
Cl
Cl
Cl
Cl
Ph
Ph
Ph
Ph
Table 3. Anity of 8-substituted xanthines as antagonists at A1 and
A2A adenosine receptors
Table 2. Chemical data of the compounds 5±22
a
b
Compound
n
R1
R
Ki A1
(mM)
Ki A2A
(mM)
Ratio Kic
A2A/A1
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
1
2
3
4
1
2
3
4
2
3
1
2
3
4
1
2
3
4
Cl
Cl
Cl
Cl
Ph
Ph
Ph
Ph
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
>100
14.7
8.8
>100
1.0
3.6
3.8
56.0
0.5
9.4
9.2
8.0
4.8
1.7
10.8
212.0
26.5
53.0
33.6
21.0
30.0
57.6
0.37
53.8
6.1
10.8
2.12
12.8
12.7
9.2
0.47
1.2
0.19
2.27
0.76
0.81
0.38
>100
Compound
n
R
R1
Yield
(%)
mp
(ꢀC)
Recryst.
solvent
17.0
60.4
22.4
H
5
6
7
8
1
2
3
4
1
2
3
4
2
3
1
2
3
4
1
2
3
4
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
C3H7
Cl
Cl
Cl
Cl
Ph
Ph
Ph
Ph
H
25>325 EtOH
25303±306
Cl
Cl
Cl
Cl
Ph
Ph
Ph
Ph
>100
>100
31.8
EtOH
50
60
50
250±253
241±244
>325
EtOH/EtOAc
EtOH/H2O
EtOH/H2O
13.4570.8
23.8
17.523.0
61.0
9.99
9
10.5
10
11
12
13
14
15
16
17
18
19
20
21
22
35318±321
EtOH
30
30
60
293±296
280±283
305±309
EtOH
EtOH/H2O
EtOH
75.0
26.3
1.5
Theophylline
Ca€eine
18.0
40.0
22.0
45.0
H
25273±276
EtOH/EtOAc
1.02
Cl
Cl
Cl
Cl
Ph
Ph
Ph
Ph
60
60
50
40
>325EtOH/H
2O
EtOH/H2O
EtOH/EtOAc
EtOH/EtOAc
EtOH
264±267
208±210
194±197
284±287
aA1 binding was measured as inhibition of [3H]-CHA binding as described
in the Experimental protocols. The Ki values are means Æ SEM of four
separate assays, each performed in triplicate.
70
bA2A binding was measured as inhibition of [3H]-CGS 21860 binding as
described in the Experimental protocols.
9±2256225
70
30
EtOH
224±227
116±119
EtOH/EtOAc
EtOH
cThe Ki values are means Æ SEM of four separate assays, each performed
in triplicate.

578
G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
carried out at 25 ꢀC for 90 min. Non-speci®c binding
was de®ned in the presence of 50mM NECA. The binding
reaction was concluded by ®ltraction through Whatman
GF/C glass ®ber ®lters under reduced pressure. Filters
were washed four times with 5mL aliquots of ice-cold
bu€er and placed in scintillation vials.
H-pyrid.), 7.20 (d, J=9 Hz, 1H, H-pyrid.), 13.0 (s, 1H,
NH-xanthine).
Anal. calcd for C12H11ClN6O3: C 44.65; H 3.40; N
26.00; found: C 44.77; H 3.50; N 25.90.
1,3-Dimethyl-8-{2-[6-chloro-pyridazin-3(2H)-one-2-yl]-
ethyl-}xanthine (6). From N1-(4-amino-1,3-dimethyl-
uracil - 5yl)- 3 - [6 - chloro - pyridazin - 3(2H) - one - 2 - yl] -
propanamide (4b). 1H NMR-400 MHz, T=350K-
(DMSO-d6) d 3.10 (t, J=6 Hz, 2H, CH2), 3.25(s, 3H,
CH3), 3.70 (s, 3H, CH3), 4.25(t, J=6 Hz, 2H, CH2), 6.90
(d, J=9 Hz, 1H, H-pyrid.), 7.15(d, J=9 Hz, 1H, H-
pyrid.), 13.0 (s, 1H, NH-xanthine). Anal. calcd for
C13H13ClN6O3: C 46.36; H 3.86; N 24.96; found: C 46.01;
H 3.5 8; N 24.5 0.
Speci®c binding was obtained by subtracting non-spe-
ci®c binding from total binding and approximated to
85±90% of the total binding. The recepto-bound radio-
activity was measured as described above.
Compounds were dissolved in ethanol or DMSO (bu€er/
concentration of 2%) and added to the assay mixture.
Blank experiment were carried out to determine the e€ect
of the solvent on binding.
Protein estimation was based on a reported method,17
after solubilization with 0.75N sodium hydroxide, using
bovine serum albumin as standard.
1,3-Dimethyl-8-{3-[6-chloro-pyridazin-3(2H)-one-2-yl]-
propyl-}xanthine (7). From N1-(4-amino-1,3-dimethyl-
uracil-5-yl)-4-[6-chloro-pyridazin-3(2H)-one-2-yl]-buta-
namide (4c). 1H NMR-400 MHz-(DMSO-d6) d 2.10±
2.30 (m, 2H, CH2), 2.80 (t, J=6 Hz, 2H, CH2), 3.20 (s,
3H, CH3), 3.60 (s, 3H, CH3), 4.00 (t, J=6 Hz, 2H, CH2)
6.90 (d, J=9 Hz, 1H, H-pyrid.), 7.15(d, J=9 Hz, 1H, H-
pyrid.), 13.0 (s, 1H, NH-xanthine). Anal. calcd for C14
H15ClN6O3: C 47.93; H 4.27; N 23.96; found: C 48.20;
H 4.62; N 23.57.
The concentration of tested compound that produce
50% inhibition of speci®c [3H]CHA or [3H]CGS 21680
binding (IC50) was determined by log-probit analysis
with seven concentrations of the displacer, each per-
formed in triplicate. Inhibition constants (Ki) were cal-
culated according the equation of Cheng and Pruso€;18
Ki=IC50/([L]/Kd), where [L] is the ligand concentration
and Kd its dissociation constant.
1,3-Dimethyl-8-{4-[6-chloro-pyridazin-3(2H)-one-2-yl]-
butyl-}xanthine (8). From N1-(4-amino-1,3-dimethyl-
uracil-5-yl)-5-[6-chloro-pyridazin-3(2H)-one-2-yl]-pen-
Kd of [3H]CHA binding to cortex membranes was 1.2
nM and Kd of [3H]CGS 21680 binding to striatal mem-
branes was 10 nM.
1
tanamide (4d). H NMR-200 MHz-(DMSO-d6) d 1.50±
1.60 (m, 4H, 2CH2), 2.60±2.80 (m, 2H, CH2), 3.15(s, 3H,
CH3), 3.60 (s, 3H, CH3), 3.90±4.10 (m, 2H, CH2), 7.00 (d,
J=9 Hz, 1H, H-pyrid.), 7.50 (d, J=9 Hz, 1H, H-pyrid.),
13.0 (s, 1H, NH-xanthine). Anal. calcd for C15H17
ClN6O3: C 49.38; H 4.7; N 23.04; found: C 49.70; H 5.10;
N 22.82.
Experimental
Melting points were determined using a Ko¯er hot-stage
apparatus and are uncorrected. The NMR spectra were
recorded with a Bruker AC 200 MHz or 400 MHz
instrument in the solvent indicated below. The chemical
shift values (ppm) are relative to tetramethylsilane as
internal standard. Elemental analyses are within Æ0.4%
of theoretical values. Precoated Kiesegel 60 F254 plates
(Merck) were used for TLC.
1,3-Dimethyl-8-{2-[6-phenyl-pyridazin-3(2H)-one-2-yl]-
methyl-}xanthine (9). From N1-(4-amino-1,3-dimethyl-
uracil-5-yl)-2-[6-phenyl-pyridazin-3(2H)-one-2-yl]-acetamide
(4e). 1H NMR-200MHz (DMSO-d6) d 3.10 (s, 3H, CH3),
3.40 (s, 3H, CH3), 5.40 (s, 2H, CH2), 7.10 (d, J=9 Hz,
1H, H-pyrid.), 7.20±7.40 (m, 3H, H-arom.), 7.70±7.80 (m,
2H, H-arom.), 8.00 (d, J=9 Hz, 1H, H-pirid.), 13.5(s,
1H, NH-xanthine). Anal. calcd for C18 H16N6O3: C 59.50;
H 4.40; N 23.14; found: C 59.85; H 4.62; N 23.57.
General method for preparation of the compounds 5±22
and the chemical data are reported in Table 2.
1,3-Dimethyl-8-{2-[6-phenyl-pyridazin-3(2H)-one-2-yl]-
ethyl-}xanthine (10). From N1-(4-amino-1,3-dimethyl-
4-amino-uracil-5-yl)-3-[6-phenyl-pyridazin-3(2H)-one-2-
yl]-propanamide (4f). 1H NMR-400 MHz, T=350K-
(DMSO-d6) d 3.10±3.30 (m, 5H, CH2, CH3), 3.40 (s, 3H,
CH3), 4.50 (t, J=6 Hz, 2H, CH2), 7.00 (d, J=9 Hz, 1H,
H-pyrid.), 7.30 (s, 3H, H-arom.), 7.70±7.80 (m, 2H, H-
arom.), 7.90 (d, J=9 Hz, 1H, H-pirid.), 12.9 (s, 1H,
NH-xanthine). Anal. calcd for C19H18N6O3: C 60.31; H
4.76; N 22.20; found: C 59,94; H 4.63; N 22.03.
1,3-Dimethyl-8-{1-[6-chloro-pyridazin-3(2H)-one-2-yl]-
methyl-}xanthine (5). A mixture (0.42 g, 1.2 mmol) N1-
(4-amino-1,3-dimethyl-uracil-5-yl)-2-[6-chloro-pyridazin-
3(2H)-one-2-yl]-acetamide (4a) in 15±20 mL of solution
of sodium hydroxide 2.5% was re¯uxed under stirring
for about 2 h. After cooling the mixture was made acid
by the addition of HCl 5% to pH 3±4 and the pre-
cipitate was ®ltered o€, and crystallized from ethanol. A
white solid was obtained (yield: 25%); mp: >325 ꢀC. 1H
NMR-200 MHz-(DMSO-d6) d 3.20 (s, 3H, CH3), 3.70
(s, 3H, CH3), 5.25 (s, 2H, CH2), 6.95(d, J=9 Hz, 1H,
1,3-Dimethyl-8-{3-[6-phenyl-pyridazin-3(2H)-one-2-yl]-
propyl-}xanthine (11). By N1-(4-amino-1,3-dimethyl-
uracil-5-yl)-4-[6-phenyl-pyridazin-3(2H)-one-2-yl]-

G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
579
butanamide (4g). 1H NMR-400 MHz-(DMSO-d6) d
2.10±2.30 (m, 2H, CH2), 2.80 (t, J=6 Hz, 2H, CH2), 3.20
(s, 3H, CH3), 3.40 (s, 3H, CH3), 4.20 (t, J=6 Hz, 2H,
CH2), 7.00 (d, J=9 Hz, 1H, H-pyrid.), 7.40±7.50
(m, 3H, H-arom.), 7.70±7.80 (m, 2H, H-arom.), 8.00
(d, J=9 Hz, 1H, H-pyrid.), 13.0 (s, 1H, NH-xanthine).
Anal. calcd for C20H20N6O3: C 61.22; H 5.10; N 21.42;
found: C 61.01; H 4.74; N 21.37.
Anal. calcd for C15H17ClN6O3: C 49.50; H 4.60; N
23.10; found: C 49.70; H 4.79; N 22.97.
3-Methyl-1-propyl-8-{3-[6-chloro-pyridazin-3(2H)-one-2-
yl]-propyl-}xanthine (17). From N1-(4-amino-3-methyl-
1-propyl-uracil-5-yl)-4-[6-chloro-pyridazin-3(2H)-one-2-
1
yl]-butanamide (4o). H NMR-200 MHz (DMSO-d6) d
0.80 (t, J=7.4 Hz, 3H, CH3), 1.50 (quint., J=7.4 Hz,
2H, CH2), 2.00±2.20 (m, 2H, CH2), 2.70±2.80 (m, 2H,
CH2), 3.20 (s, 3H, CH3), 3.80 (t, J=7.4 Hz, 2H, CH2),
4.20 (t, J=7.0 Hz, 2H, CH2), 7.00 (d, J=9 Hz, 1H, H-
pyrid.), 7.60 (d, J=9 Hz, 1H, H-pyrid.), 13.0 (s, 1H,
NH-xanthine). Anal. calcd for C16H19ClN6O3: C
50.86; H 5.03; N 22.25; found: C 50.20; H 5.62; N
22.63.
1,3-Dimethyl-8-{4-[6-phenyl-pyridazin-3(2H)-one-2-yl]-
butyl-}xanthine (12). From N1-(4-amino-1,3-dimethyl-
uracil-5-yl)-5-[6-phenyl-pyridazin-3(2H)-one-2-yl]-pen-
1
tanamide (4h). H NMR-200 MHz-(DMSO-d6) d 1.65±
1.75(m, 4H, 2CH ), 2,60±2.70 (m, 2H, CH2), 2.80 (t,
2
J=6 Hz, 2H, CH2), 3.20 (s, 3H, CH3), 3.40 (s, 3H,
CH3), 4.20 (m, 2H, CH2), 7.00 (d, J=9 Hz, 1H,
H-pyrid.), 7.40±7.50 (m, 3H, H-arom.), 7.70±7.80 (m, 2H,
H-arom.), 8.00 (d, J=9 Hz, 1H, H-pyrid.), 13.0 (s, 1H,
NH-xanthine). Anal. calcd for C21H22N6O3: C 62.06; H
5.40; N 20.68; found: C 61.92; H 5.57; N 20.24.
3-Methyl-1-propyl-8-{4-[6-chloro-pyridazin-3(2H)-one-2-
yl]-butyl-}xanthine (18). From N1-(4-amino-3-methyl-1-
propyl-uracil-5-yl)-5-[6-chloro-pyridazin-3(2H)-one-2-yl]-
pentanamide (4p). 1H NMR-200 MHz (DMSO-d6) d
0.80 (t, J=7.4 Hz, 3H, CH3), 1.50 (quint., J=7.4 Hz, 2H,
CH2), 1.60±1.80 (m, 6H, 3CH2), 2.70±2.80 (m, 2H, CH2),
3.20 (s, 3H, CH3), 3.80 (t, J=7.4 Hz, 2H, CH2), 4.00±
4.10 (m, 2H, CH2), 7.00 (d, J=9 Hz, 1H, H-pyrid.), 7.60
(d, J=9 Hz, 1H, H-pyrid.), 13.0 (s, 1H, NH-xanthine).
Anal. calcd for C17H21ClN6O3: C 51.97; H 5.35; N
21.40; found: C 51.20; H 5.47; N 20.95.
1,3-Dimethyl-8-{2-[pyridazin-3(2H)-one-2-yl]-ethyl-}xan-
thine (13). From N1-(4-amino-1,3-dimethyl-uracil-5-yl)-
3-[pyridazin-3(2H)-one-2-yl]-propanamide (4i). 1H NMR-
400 MHz-(DMSO-d6) d 3.10 (t, J=6 Hz, 2H, CH2), 3.25
(s, 3H, CH3), 3.40 (s, 3H, CH3), 4.45(t, J=6 Hz, 2H,
CH2), 6.90 (dd, J=9 and 2 Hz, 1H, H-pyrid.), 7.45(m,
1H, H-pyrid.), 7.90 (dd, J=9 and 5Hz, 1H, H-pyrid.),
13.2 (s, 1H, NH-xanthine). Anal. calcd for C13H14N6O3:
C 51.65; H 4.63; N 27.81; found: C 51.31; H 4.42; N
27.45.
3-Methyl-1-propyl-8-{1-[6-phenyl-pyridazin-3(2H)-one-2-
yl]-methyl-}xanthine (19). From N1-(4-amino-3-methyl-
1-propyl-uracil-5-yl)-2-[6-phenyl-pyridazin-3(2H)-one-2-
yl]-acetamide (4q). 1H NMR-200 MHz (DMSO-d6) d
0.80 (t, J=7.4 Hz, 3H, CH3), 1.50 (quint., J=7.4 Hz, 2H,
CH2), 3.30 (s, 3H, CH3), 3.85(t, J=7.4 Hz, 2H, CH2),
5.50 (s, 2H, CH2), 7.10 (d, J=9 Hz, 1H, H-pyrid.), 7.40±
7.50 (m, 3H, H-arom.), 7.80±7.90 (m, 2H, H-arom.), 8.10
(d, J=9 Hz, 1H, H-pyrid.), 13.60 (s, 1H, NH-xanthine).
Anal. calcd for C20H20N6O3: C 61.22; H 5.10; N 21.40;
found: C 61.40; H 4.95; N 20.95.
1,3-Dimethyl-8-{3-[pyridazin-3(2H)-one-2-yl]-propyl-}xan-
thine (14). From N1-(4-amino-1,3-dimethyl-4-amino-
uracil-5-yl)-4-[pyridazin-3(2H)-one-2-yl]-butanamide (4l).
1H NMR-400 MHz, T=350K-(DMSO-d6) d 2.10±2.40
(m, 2H, CH2), 2.75(t, J=6 Hz, 2H, CH2), 3.25(s, 3H,
CH3), 3.45(s, 3H, CH 3), 4.2 (t, J=6 Hz, 2H, CH2), 6.80±
6.90 (m, 1H, H-pyrid.), 7.35±7.45 (m, 1H, H-pyrid.), 7.80±
7.90 (m, 1H, H-pyrid.), 13.0 (s, 1H, NH-xanthine). Anal.
calcd for C14H16N6O3: C 53.16; H 5.06; N 26.58; found:
C 53.01; H 5.38; N 26.37.
3-Methyl-1-propyl-8-{2-[6-phenyl-pyridazin-3(2H)-one-2-
yl]-ethyl-}xanthine (20). From N1-(4-amino-3-methyl-1-
propyl-uracil-5-yl)-3-[6-phenyl-pyridazin-3(2H)-one-2-yl]-
propanamide (4r). 1H NMR-200 MHz (DMSO-d6) d 0.80
(t, J=7.4Hz, 3H, CH3), 1.50 (quint. J=7.4 Hz, 2H,
CH2), 3.10±3.20 (m, 2H, CH2), 3.30 (s, 3H, CH3), 3.85(t,
J=7.4 Hz, 2H, CH2), 4.50 (t, J=7.0 Hz, 2H, CH2), 7.10
(d, J=9 Hz, 1H, H-pyrid.), 7.20±7.30 (m, 3H, H-arom.),
7.70±7.80 (m, 2H, H-arom.), 8.00 (d, J=9Hz, 1H, H-
pyrid.), 13.30 (s, 1H, NH-xanthine). Anal. calcd for
C21H22N6O3: C 62.00; H 5.40; N 20.68; found: C 61.73; H
5 .79; N 20.32.
3-Methyl-1-propyl-8-{1-[6-chloro-pyridazin-3(2H)-one-2-
yl]-methyl-}xanthine (15). From N1-(4-amino-3-methyl-
1-propyl-uracil-5-yl)-2-[6-chloro-pyridazin-3(2H)-one-2-
yl]-acetamide (4m). 1H NMR-200 MHz (DMSO-d6) d 0.80
(t, J=7.4Hz, 3H, CH3), 1.50 (quint., J=7.4Hz, 2H,
CH2), 3.30 (s, 3H, CH3), 3.85(t, J=7.4 Hz, 2H, CH2),
5.30 (s, 2H, CH2), 7.00 (d, J=9 Hz, 1H, H-pyrid.), 7.60 (d,
J=9 Hz, 1H, H-pyrid.), 13.0 (s, 1H, NH-xanthine).
Anal. calcd for C14H15ClN6O3: C 48.00; H 4.20; N
24.10; found: C 48.20; H 4.62; N 24.57.
3-Methyl-1-propyl-8-{3-[6-phenyl-pyridazin-3(2H)-one-2-
yl]-propyl-}xanthine (21). From N1-(4-amino-3-methyl-1
-propyl-uracil-5-yl)-4-[6-phenyl-pyridazin-3(2H)-one-2-yl]-
3-Methyl-1-propyl-8-{2-[6-chloro-pyridazin-3(2H)-one-2-
yl]-ethyl-}xanthine (16). From N1-(4-amino-3-methyl-1-
propyl-uracil-5-yl)-3-[6-chloro-pyridazin-3(2H)-one-2-yl]-
propanamide (4n). 1H NMR-200 MHz (DMSO-d6) d
0.80 (t, J=7.4 Hz, 3H, CH3), 1.50 (quint., J=7.4 Hz,
2H, CH2), 3.05(t, J=7.0 Hz, 2H, CH2), 3.20 (s, 3H,
CH3), 3.80 (t, J=7.4 Hz, 2H, CH2), 4.30 (t, J=7.0 Hz,
2H, CH2), 7.00 (d, J=9 Hz, 1H, H-pyrid.), 7.60 (d,
J=9 Hz, 1H, H-pyrid.), 13.0 (s, 1H, NH-xanthine).
1
butanamide (4s). H NMR-200 MHz (DMSO-d6) d 0.80
(t, J=7.4 Hz, 3H, CH3), 1.50 (quint., J=7.4 Hz, 2H,
CH2), 2.70±2.80 (m, 4H, 2CH2), 3.30 (s, 3H, CH3), 3.85
(t, J=7.4 Hz, 2H, CH2), 4.20 (t, J=7.0 Hz, 2H, CH2),
7.10 (d, J=9 Hz, 1H, H-pyrid.), 7.40±7.50 (m, 3H, H-
arom.), 7.80±7.90 (m, 2H, H-arom.), 8.00 (d, J=9 Hz,
1H, H-pyrid.), 13.20 (s, 1H, NH-xanthine). Anal. calcd

580
G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
for C22H24N6O3: C 62.85 ; H 5 .7; N 20.00; found: C 62.5 5 ; N1-(4-amino-1,3-dimethyl-uracil-5-yl)-2-[6-phenyl-pyrida-
H 6.02; N 19.60.
zin-3(2H)-one-2-yl]-acetamide (4e). From reaction of 2
-[6-phenyl-pyridazin-3(2H)-one-2-yl]-acetic acid (3e), with
1
3-Methyl-1-propyl-8-{4-[6-phenyl-pyridazin-3(2H)-one-2-
yl]-butyl-}xanthine (22). From N1-(4-amino-3-methyl-1-
propyl-uracil-5-yl)-5-[6-phenyl-pyridazin-3(2H)-one-2-yl]-
pentanamide (4t). 1H NMR-200 MHz (DMSO-d6) d 0.80
(t, J=7.4 Hz, 3H, CH3), 1.50 (quint., J=7.4 Hz, 2H,
CH2), 1.60±1.80 (m, 4H, 2CH2), 2.70 (t, J=7.0 Hz, 2H,
CH2), 3.30 (s, 3H, CH3), 3.85(t, J=7.4 Hz, 2H, CH2),
4.20 (t, J=7.0 Hz, 2H, CH2), 7.00 (d, J=9 Hz, 1H, H-
pyrid.), 7.40±7.50 (m, 3H, H-arom.), 7.80±7.90 (m, 2H,
H-arom.), 8.00 (d, J=9 Hz, 1H, H-pyrid.), 13.20 (s, 1H,
NH-xanthine). Anal. calcd for C23H26N6O3: C 63.59; H
5.99; N 19.35; found: C 63.78; H 5.97; N 19.03.
5,6-diamino-1,3-dimethyl-uracil. H NMR (DMSO-d6) d
3.10 (s, 3H, CH3), 3.40 (s, 3H, CH3), 4.85(s, 2H, CH ),
2
6.60 (s, 2H, NH2), 7.10 (d, J=9 Hz, 1H, H-pyrid.), 7.40±
7.50 (m, 3H, H-arom.), 7.70±7.80 (m, 2H, H-arom.), 8.10
(d, J=9 Hz, 1H, H-pyrid.), 9.00 (s, 1H, NHCO).
N1-(4-amino-1,3-dimethyl-uracil-5-yl)-3-[6-phenyl-pyrida-
zin-3(2H)-one-2-y]-propanamide (4f). From reaction of
3-[6-phenyl-pyridazin-3(2H)-one-2-yl]-propanoic acid (3f)
with 5,6-diamino-1,3-dimethyl-uracil. 1H NMR (DMSO-
d6) d 2.75(t, J=6 Hz, 2H, CH2), 3.10 (s, 3H, CH3), 3.40 (s,
3H, CH3), 4.20 (t, J=6 Hz, 2H, CH2), 6.60 (s, 2H, NH2),
7.00 (d, J=9 Hz, 1H, H-pyrid.), 7.40±7.50 (m, 3H, H-
arom.), 7.65(d, J=9 Hz, 1H, H-pyrid.), 7.70±7.80 (m,
1H, H-pyrid.), 8.40 (s, 1H, NHCO).
General method for the preparation of the amide deri-
vatives (compounds 4a±4t) and the chemical data are
reported in Table 1.
N1-(4-amino-1,3-dimethyl-uracil-5-yl)-4-[6-phenyl-pyrida-
zin-3(2H)-one-2-yl]-butanamide (4g). From reaction of 4-
[6-phenyl-pyridazin-3(2H)-one-2-yl]-butanoic acid (3g)
with 5,6-diamino-1,3-dimethyl-uracil. 1H NMR (DMSO-
d6) d 1.90±2.10 (m, 2H, CH2), 2.25(t, J=6 Hz, 2H, CH2),
3.10 (s, 3H, CH3), 3.30 (s, 3H, CH3), 4.15(t, J=6 Hz, 2H,
CH2), 6.60 (s, 2H, NH2), 7.00 (d, J=9 Hz, 1H, H-pyrid.),
7.40±7.50 (m, 3H, H-arom.), 7.80±7.90 (m, 2H, H-arom.),
8.00 (d, J=9 Hz, 1H, H-pyrid.), 8.35(s, 1H, NHCO).
N1-(4-amino-1,3-dimethyl-uracil-5-yl)-2-[6-chloro-pyrida-
zin-3(2H)-one-2-yl]-acetamide (4a). A mixture of (0.37 g,
2.0 mmol) 2-[6-chloro-pyridazin-3(2H)-one-2-yl]-acetic
acid (3a), (0.34 g, 2.4 mmol) 5,6-diamino-1,3-dimethyl-
uracil and (0.49 g, 2.4 mmol) 1,3-dicyclohexylcarbodi-
imide (DCC) in 10 mL of anhydrous methanol was stir-
red at rt for 24 h. After ®ltration a light yellow solid was
obtained washed with CH2Cl2 for eliminate the dicyclo-
hexylurea, (yield: 70%), mp: >325 ꢀC, this compound
was used for the preparation of the corresponding xan-
thine without further puri®cation. 1H NMR (DMSO-d6)
d 3.10 (s, 3H, CH3), 3.40 (s, 3H, CH3), 4.85(s, 2H, CH 2),
6.60 (s, 2H, NH2), 7.10 (d, J=9 Hz, 1H, H-pyrid.), 7.60
(d, J=9 Hz, 1H, H-pyrid.), 8.90 (s, 1H, NHCO).
N1-(4-amino-1,3-dimethyl-4-amino-uracil-5-yl)-5-[6-phenyl-
pyridazin-3(2H)-one-2-yl]-pentanamide (4h). From reac-
tion of 5-[6-phenyl-pyridazin-3(2H)-one-2-yl]-pentanoic
acid (3h) with 5,6-diamino-1,3-dimethyl-uracil. 1H NMR
(DMSO-d6) d 1.70±1.90 (m, 4H, 2CH2), 2.25(t, J=
6 Hz, 2H, CH2), 3.10 (s, 3H, CH3), 3.30 (s, 3H, CH3),
4.15(t, J=6 Hz, 2H, CH2), 6.60 (s, 2H, NH2), 7.00 (d,
J=9 Hz, 1H, H-pyrid.), 7.40±7.50 (m, 3H, H-arom.),
7.80±7.90 (m, 2H, H-arom.), 8.00 (d, J=9 Hz, 1H,
H-pyrid.), 8.30 (s, 1H, NHCO).
N1-(4-amino-1,3-dimethyl-uracil-5yl-)-3-[6-chloro-pyrida-
zin-3(2H)-one-2-yl]-propanamide (4b). From reaction of
3-[6-cloro-pyridazin-3(2H)-one-2-yl]-propanoic acid (3b)
with 5,6-diamino-1,3-dimethyl-uracil. 1H NMR (DMSO-
d6) d 2.75(t, J=6 Hz, 2H, CH2), 3.10 (s, 3H, CH3), 3.40 (s,
3H, CH3), 4.25(t, J=6 Hz, 2H, CH2), 6.60 (s, 2H,
NH2), 7.10 (d, J=9 Hz, 1H, H-pyrid.), 7.60 (d, J=9 Hz,
1H, H-pyrid.), 8.60 (s, 1H, NHCO).
N1-(4-amino-1,3-dimethyl-uracil-5-yl)-3-[pyridazin-3(2H)-
one-2-yl]-propanamide (4i). From reaction of 3-[pyr-
idazin-3(2H)-one-2-yl]-propanoic acid (3i) with 5,6-dia-
1
mino-1,3-dimethyl-uracil. H NMR (DMSO-d6) d 2.70
N1-(-4-amino-1,3-dimethyl-uracil-5-yl)-4-[6-chloro-pyri-
dazin-3(2H)-one-2-yl]-butanamide (4c). From reaction of
4-[6-chloro-pyridazin-3(2H)-one-2-yl]-butanoic acid (3c)
with 5,6-diamino-1,3-dimethyl-uracil.1H NMR (DMSO-
d6) d 1.90±2.10 (m, 2H, CH2), 2.35(t, J=6 Hz, 2H, CH2),
3.10 (s, 3H, CH3), 3.30 (s, 3H, CH3), 4.10 (t, J=
6 Hz, 2H, CH2), 6.60 (s, 2H, NH2), 7.10 (d, J=9 Hz,
1H, H-pyrid.), 7.60 (d, J=9 Hz, 1H, H-pyrid.), 8.60 (s,
1H, NHCO).
(t, J=6 Hz, 2H, CH2), 3.10 (s, 3H, CH3), 3.30 (s, 3H,
CH3), 4.35(t, J=6 Hz, 2H, CH2), 6.60 (s, 2H, NH2),
6.90 (dd, J=9 and 2 Hz, 1H, H-pyrid.), 7.45(dd, J=9
and 5Hz, 1H, H-pyrid.), 7.90±8.00 (m, 1H, H-pyrid.),
8.60 (s, 1H, NHCO).
N1-(4-amino-1,3-dimethyl-uracil-5-yl)-4-[pyridazin-3(2H)-
one-2-yl]-butanamide (4l). From reaction of 4-[pyridazin-
3(2H)-one-2-yl]-butanoic acid (3l) with 5,6-diamino-1,3-
1
dimethyl-uracil. H NMR (DMSO-d6) d 2.10±2.40 (m,
N1-(4-amino-1,3-dimethy-uracil-5-yl)-5-[6-chloro-pyrida-
zin-3(2H)-one-2-yl]-pentanamide (4d). From reaction of
5-[6-chloro-pyridazin-3(2H)-one-2-yl]-pentanoic acid (3d)
with 5,6-diamino-1,3-dimethyl-uracil. 1H NMR (DMSO-
d6) d 1.50±1.80 (m, 4H, 2CH2), 2.25(t, J=6.0Hz, 2H,
CH2), 3.10 (s, 3H, CH3), 3.30 (s, 3H, CH3), 4.00 (t,
J=6 Hz, 2H, CH2), 6.60 (s, 2H, NH2), 7.05(d, J=9 Hz,
1H, H-pyrid.), 7.60 (d, J=9 Hz, 1H, H-pyrid.), 8.30 (s,
1H, NHCO).
2H, CH2), 2.75(t, J=6 Hz, 2H, CH2), 3.20 (s, 3H,
CH3), 3.40 (s, 3H, CH3), 4.20 (t, J=6 Hz, 2H, CH2),
6.50 (s, 2H, NH2), 6.80±6.90 (m, 1H, H-pyrid.), 7.30±
7.40 (m, 1H, H-pyrid.), 7.80±7.90 (m, 1H, H-pyrid.),
8.50 (s, 1H, NHCO).
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-2-[6-chloro-
pyridazin-3(2H)-one-2-yl]-acetamide (4m). From reac-
tion of 2-[6-chloro-pyridazin-3(2H)-one-2-yl]-acetic acid

G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
581
(3a) with 5,6-diamino-3-methyl-1-propyl-uracil. 1H
NMR (DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H, CH3), 1.50±
1.80 (m, 2H, CH2), 3.20 (s, 3H, CH3), 3.65(t, J=7.4 Hz,
2H, CH2), 4.80 (s, 2H, CH2), 6.50 (s, 2H, NH2), 7.00 (d,
J=9 Hz, 1H, H-pyrid.), 7.60 (d, J=9 Hz, 1H, H-pyrid.),
8.85(s, 1H, NHCO).
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-4-[6-phenyl-
pyridazin-3(2H)-one-2-yl]-butanamide (4s). From reac-
tion of 4-[6-phenyl-pyridazin-3(2H)-one-2-yl]-butanoic
1
acid (3g) with 5,6-diamino-3-methyl-1-propyl-uracil. H
NMR-200 MHz (DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H,
CH3), 1.50±1.80 (m, 2H, CH2), 2.80 (m, 4H, 2CH2), 3.25
(s, 3H, CH3), 3.70 (t, J=7.0 Hz, 2H, CH2), 4.10 (t,
J=7.4 Hz, 2H, CH2), 6.50 (s, 2H, NH2), 7.00 (d,
J=9 Hz, 1H, H-pyrid.), 7.40±7.50 (m, 3H, H-arom.),
7.80±7.90 (m, 2H, H-arom.), 8.05(d, J=9 Hz, 1H,
H-pyrid.), 8.35(s, 1H, NHCO).
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-3-[6-chloro-
pyridazin-3(2H)-one-2-yl]-propanamide (4n). From reac-
tion of 3-[6-cloro-pyridazin-3(2H)-one-2-yl]-propanoic
1
acid (3b) with 5,6-diamino-3-methyl-1-propyl-uracil. H
NMR-200 MHz (DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H,
CH3), 1.50±1.80 (m, 2H, CH2), 2.70±2.80 (m, 2H, CH2),
3.20 (s, 3H, CH3), 3.70 (s, 2H, CH2), 4.20 (t, J=7.4 Hz,
2H, CH2), 6.50 (s, 2H, NH2), 7.00 (d, J=9 Hz, 1H,
H-pyrid.), 7.60 (d, J=9 Hz, 1H, H-pyrid.), 8.50 (s, 1H,
NHCO).
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-5-[6-phenyl-
pyridazin-3(2H)-one-2-yl]-pentanamide (4t). From reac-
tion of 5-[6-phenyl-pyridazin-3(2H)-one-2-yl]-pentanoic
1
acid (3h) with 5,6-diamino-3-methyl-1-propyl-uracil. H
NMR-200 MHz (DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H,
CH3), 1.50±1.70 (m, 2H, CH2), 1.70±1.80 (m, 4H,
2CH2), 2.20 (m, 2H, CH2), 3.30 (s, 3H, CH3), 3.60 (m,
2H, CH2), 4.10 (t, J=7.0 Hz, 2H, CH2), 6.50 (s, 2H,
NH2), 7.00 (d, J=9 Hz, 1H, H-pyrid.), 7.40±7.50 (m,
3H, H-arom.), 7.80±7.90 (m, 2H, H-arom.), 8.05(d,
J=9 Hz, 1H, H-pyrid.), 8.30 (s, 1H, NHCO).
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-4-[6-chloro-
pyridazin-3(2H)-one-2-yl]-butanamide (4o). From reac-
tion of 4-[6-chloro-pyridazin-3(2H)-one-2-yl]-butanoic
1
acid (3c) with 5,6-diamino-3-methyl-1-propyl-uracil. H
NMR-200 MHz (DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H,
CH3), 1.50±1.80 (m, 2H, CH2), 2.00 (t, J=7.0 Hz, 2H,
CH2), 2.20 (t, J=7.4 Hz, 2H, CH2), 3.20 (s, 3H, CH3),
3.70 (t, J=7.4 Hz, 2H, CH2), 4.20 (t, J=7.0 Hz, 2H,
CH2), 6.50 (s, 2H, NH2), 7.00 (d, J=9 Hz, 1H, H-
pyrid.), 7.60 (d, J=9 Hz, 1H, H-pyrid.), 8.40 (s, 1H,
NHCO).
General method for preparation of the acids 3a and 3e
2-(6-Chloro-pyridazin-3(2H)-one-2-yl)-acetic acid (3a).
To a solution of (0.46 g 20 mmol) sodium in 20 mL
absolute ethanol was added (2.6 g 20 mmol) 6-chloro-
pyridazin-3(2H)-one and this mixture was stirred and
re¯uxed for 30 min. After cooling was added dropwise
(3.34 g 20 mmol) ethyl bromoacetate. When the addition
was complete, the reaction was heated under re¯uxed
and stirring for 24 h. At the end the solution was evap-
orated under reduced pressure, the residue was puri®ed
by chromatography silica gel using as eluent a stepwise
gradient of ethanol (0±2%) in CH2Cl2, gave ethyl 2-(6-
chloro-pyridazin-3(2H)-one-2-yl)-acetate (dense oil, yield:
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-5-[6-chloro-
pyridazin-3(2H)-one-2-yl]-pentanamide (4p). From reac-
tion of 5-[6-chloro-pyridazin-3(2H)-one-2-yl]-pentanoic
1
acid (3d) with 5,6-diamino-3-methyl-1-propyl-uracil. H
NMR-200 MHz (DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H,
CH3), 1.50±1.80 (m, 6H, 3CH2), 2.25(t, J=7.0 Hz, 2H,
CH2), 3.20 (s, 3H, CH3), 3.70 (t, J=7.4 Hz, 2H, CH2),
4.00 (t, J=7.0 Hz, 2H, CH2), 6.50 (s, 2H, NH2), 7.00 (d,
J=9 Hz, 1H, H-pyrid.), 7.60 (d, J=9 Hz, 1H, H-pyrid.),
8.30 (s, 1H, NHCO).
1
70%). H NMR (CDCl3) d 1.30 (t, J=7 Hz, 3H, CH3),
4.25(q, J=7 Hz, 2H, CH2), 4.80 (s, 2H, CH2CO), 6.90 (d,
J=9 Hz, 1H, H-pyrid.), 7.20 (d, J=9 Hz, 1H, H-pyrid.).
The ester was treated with hydrochloric acid 5%
(15mL) and the mixture was re¯uxed for 5h, gave the
corresponding acid. This acid was used for the prepara-
tion of amide 4a without further puri®cation.
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-2-[6-phenyl-
pyridazin-3(2H)-one-2-yl]-acetamide (4q). From reaction
of 2-[6-phenyl-pyridazin-3(2H)-one-2-yl]-acetic acid (3e)
with 5,6-diamino-3-methyl-1-propyl-uracil. 1H NMR
(DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H, CH3), 1.50±1.80
(m, 2H, CH2), 3.30 (s, 3H, CH3), 3.85(t, J=7.0 Hz, 2H,
CH2), 4.80 (s, 2H, CH2), 6.50 (s, 2H, NH2), 7.10 (d,
J=9 Hz, 1H, H-pyrid.), 7.40±7.50 (m, 3H, H-arom.),
7.80±7.90 (m, 2H, H-arom.), 8.10 (d, J=9 Hz, 1H,
H-pyrid.), 8.60 (s, 1H, NHCO).
2-(6-Phenyl-pyridazin-3(2H)-one-2-yl)-acetic acid (3e).
Prepared with the same method described above, using 6-
phenyl-3(2H)-pyridazinone. Gave ethyl 2-(6-phenyl-pyr-
idazin-3(2H)-one-2-yl)-acetate (dense oil, yield: 70%).1H
NMR (CDCl3) d1.30 (t, J=7 Hz, 3H, CH3), 4.25(q,
J=7 Hz, 2H, CH2), 5.0 (s, 2H, CH2), 7.0 (d, J=9Hz, 1H,
H-pyrid.), 7.30±7.45(m, 3H, H-arom.), 7.70±7.90 (m, 3H,
H-pyrid., 2H-arom.). This ester subsequently was treated
with HCl 5% and the corresponding acid was used for
preparation of the amide 4e.
N1-(4-amino-3-methyl-1-propyl-uracil-5-yl)-3-[6-phenyl-
pyridazin-3(2H)-one-2-yl]-propanamide (4r). From reac-
tion of 3-[6-phenyl-pyridazin-3(2H)-one-2-yl]-propanoic
1
acid (3f) with 5,6-diamino-3-methyl-1-propyl-uracil. H
NMR-200 MHz (DMSO-d6) d 0.80 (t, J=7.4 Hz, 3H,
CH3), 1.50±1.80 (m, 2H, CH2), 2.80 (t, J=7.4 Hz, 2H,
CH2), 3.25(s, 3H, CH ), 3.80 (t, J=7.0 Hz, 2H, CH2),
4.40 (t, J=7.4 Hz, 2H, CH2), 6.60 (s, 2H, NH2), 7.05(d,
J=9 Hz, 1H, H-pyrid.), 7.40±7.50 (m, 3H, H-arom.),
7.80±7.90 (m, 2H, H-arom.), 8.10 (d, J=9 Hz, 1H,
H-pyrid.), 8.60 (s, 1H, NHCO).
General method for preparation of the acids 3b±3d, 3f±
3h and 3i±3l
3
3-[6-Chloro-pyridazin-3(2H)-one-2-yl]-propanoic acid (3b).
To a solution of (2 g 20 mmol) 6-chloro-pyridazin-
3(2H)-one in 75mL acetone were added (3.2 g 30 mmol)

582
G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
of dry K2CO3 and (4.16 g 30 mmol) of ethyl 3-bromo-
propionate. This mixture was stirred and re¯uxed for
24 h. After the reaction was ®ltered and evaporated
under reduced pressure, the residue was puri®ed by
chromatography silica gel using as eluent a stepwise gra-
dient of ethanol (0±3%) in CH2Cl2, gave ethyl 3-(6-chloro-
pyridazin-3(2H)-one-2-yl)-propionate (dense oil, yield:
70%). H NMR (CDCl3) d H NMR (CDCl3) d 1.30 (t,
J=7Hz, 3H, CH3), 2.85(t, J=6 Hz, 2H, CH2), 4.15(q,
J=7Hz, 2H, CH2), 4.4 (t, J=6 Hz, 2H, CH2); 6.90 (d,
J=9Hz, 1H, H-pyrid.), 7.20 (d, J=9 Hz, 1H, H-pyrid.).
5-[6-Phenyl-pyridazin-3(2H)-one-2-yl]-pentanoic acid (3h).
Prepared with the same method of 3b using ethyl
5-bromovalerate gave ethyl 5-[6-phenyl-pyridazin-3(2H)
one-2yl]-valerate (dense oil, yield: 55%).1H NMR
(CDCl3) d 1.25(t, J=7 Hz, 3H, CH3), 1.70±1.80 (m, 4H,
2CH2), 2.30 (t, J=6 Hz, 2H, CH2), 4.00 (q, J= 7 Hz,
2H, CH2), 4.25(t, J=6 Hz, 2H, CH2), 6.95(d, J=9 Hz,
1H, H-pyrid.), 7.30±7.40 (m, 3H, H-arom.), 7.70 (d,
J=9 Hz, 1H, H-pyrid.), 7.80±7.90 (m, 2H, H-arom.).
This ester was treated with 5% hydrochloric acid, gave
the corresponding acid, which was used for the pre-
paration of amide 4h.
1
1
This ester was treated with hydrochloric acid 5%
(15mL) and the mixture was re¯uxed under stirring for
5h, obtaining the corresponding acid, used without
further puri®cation for the preparation of amide 4b.
3-[Pyridazin-3(2H)-one-2-yl]-propanoic acid (3i). Prepared
with the same method of 3b using ethyl 3-bromo-
propionate gave ethyl 3-[pyridazin-3(2H)-one-2-yl]-pro-
pionate (dense oil, yield: 70%). 1H NMR (CDCl3) d
1.35(t, J=7 Hz, 3H, CH3), 2.85(t, J=6 Hz, 2H, CH2),
4.15(q, J=7 Hz, 2H, CH2), 4.45(t, J=6 Hz, 2H, CH2),
6.90 (dd, J=9 Hz e 2 Hz, 1H, H-pyrid.), 7.20 (dd, J=
9 Hz e 5Hz, 1H, H-pyrid.), 7.70±7.80 (m, 1H, H-pyrid.).
This ester was treated with 5% hydrochloric acid,
obtaining the corresponding acid, used for the prepara-
tion of amide 4i.
4-[6-Chloro-pyridazin-3(2H)-one-2-yl]-butanoic acid (3c).
Prepared with the same method described for 3b starting
ethyl 4-bromobutyrate, gave ethyl 4-[6-chloro-pyridazin-
3(2H)-one-2-yl]-butyrate (dense oil, yield: 75%).1H NMR
(CDCl3) d 1.25(t, J=7 Hz, 3H, CH3), 2.15(q, J=6 Hz,
2H, CH2), 2.40 (t, J=6 Hz, 2H, CH2), 4.05±4.25 (m, 4H,
2CH2); 6.90 (d, J=9 Hz, 1H, H-pyrid.), 7.20 (d, J=
9 Hz, 1H, H-pyrid.), which treated with 5% hydrochlo-
ric acid, gave the corresponding acid, was used for the
preparation of amide 4c.
4-[Pyridazin-3(2H)-one-2-yl]-butanoic acid (3l). Prepared
with the same method of 3b using ethyl 4-bromobuty-
rate gave ethyl 4-[pyridazin-3(2H)-one-2-yl]-butyrate
(dense oil, yield: 95%).1H NMR (CDCl3) d 1.25(t,
J=7 Hz, 3H, CH3), 2.20 (q, J=6 Hz, 2H, CH2), 2.40 (t,
J=6 Hz, 2H, CH2), 4.10±4.30 (m, 4H, 2CH2), 6.90 (dd,
J=9 Hz e 2 Hz, 1H, H-pyrid.), 7.20 (dd, J=9 Hz e 5Hz,
1H, H-pyrid.), 7.70±7.80 (m, 1H, H-pyrid.). This ester
was treated with 5% hydrochloric acid, obtaining the
corresponding acid, used for the preparation of amide
4l.
5-[6-Chloro-pyridazin-3(2H)-one-2-yl]-pentanoic acid (3d).
Prepared with the same method of 3b using ethyl
5-bromovalerate gave ethyl 5-[6-chloro-pyridazin-3(2H)-
one-2-yl]-valerate (dense oil, yield: 75%).1H NMR
(CDCl3) d 1.15(t, J=7 Hz, 3H, CH3), 1.70±1.80 (m, 4H,
2CH2), 2.15(t, J=6 Hz, 2H, CH2), 3.90±4.10 (m, 4H,
2CH2), 6.80 (d, J=9 Hz, 1H, H-pyrid.), 7.10 (d, J=
9 Hz, 1H, H-pyrid.). This ester was treated with 5%
hydrochloric acid, obtaining the corresponding acid,
used for the preparation of amide 4d.
3-[6-Phenyl-pyridazin-3(2H)-one-2-yl]-propanoic acid (3f).
Prepared with the same method of 3b using ethyl
3-bromopropionate gave ethyl 3-[6-phenyl-pyridazin-
Acknowledgements
1
3(2H)-one-2-yl]-propionate (dense oil, yield: 50%). H
Financial support provided by the Ministry of Uni-
versity Scienti®c and Technological Research (MURST)
is acknowledged.
NMR (CDCl3) d 1.25(t, J=7 Hz, 3H, CH3), 2.90 (t,
J=6 Hz, 2H, CH2), 4.10 (q, J=7 Hz, 2H, CH2), 4.50 (t,
J=6 Hz, 2H, CH2), 7.00 (d, J=9 Hz, 1H, H-pyrid.),
7.40±7.50 (m, 3H, H-arom.), 7.65 (d, J=9 Hz, 1H,
H-pyrid.), 7.70±7.80 (m, 2H, H-arom.). This ester was
treated with hydrochloric acid 5%, obtaining the corre-
sponding acid, which was used for the preparation of
amide 4f.
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corresponding acid, used for the preparation of amide 4g.

G. Strappaghetti et al. / Bioorg. Med. Chem. 9 (2001) 575±583
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