Synthesis and spasmolytic action of 2-substituted thienopyrimidin-4-one derivatives

Article abstract of DOI:10.1211/0022357021778871

In the search for novel compounds to treat disorders of smooth muscle function, efforts have focused on some 2-substituted thieno[2,3-d]pyrimidin-4-one derivatives that show interesting spasmolytic action. Our laboratories have developed a new series of quaternary salts of 2-substituted thieno[2,3-d]pyrimidin-4-one and thieno[3,2-d]pyrimidin-4-one isomers with therapeutic potential. These substances were prepared starting from simple derivatives of thiophene. Their spasmolytic activity was evaluated on transmurally stimulated guinea-pig ileum. The most active compounds (IC50 1.12-2.71 μM) 7f-7h, 12d and 12f had the terminal piperidino nucleus in the thioalkyl chain and lacked two methyl groups in the thiophene ring. Their relaxant activity on the isolated ileum was potential (approx. 20-25%) by phosphodiesterase inhibitors. Compounds 7f-h, 12d and 12f were less effective in inhibiting contractions of the guinea-pig ileum induced by acetylcholine (IC50 26.7-41.4 μM) or histamine (IC50 41.5-63.4 μM) and had a moderate binding activity to muscarinic receptors in membrane homogenates from the rat heart (M₂ sites; pK_i values between 5.55±0.08 and 5.14±0.12; n = 3) and submaxillary gland (M₃ sites; pK_i values between 6.15±0.07 and 5.76±0.08; n = 3). Action involving soluble guanylyl cyclase or any potential binding to guinea-pig ventricular L-type calcium channels was not considered likely. It is concluded that at least two different mechanisms of action contribute to their spasmolytic activity.

Full text of DOI:10.1211/0022357021778871

Journal of
JPP 2002, 54: 717–728
# 2002 The Authors
Received May 30, 2001
Accepted November 26, 2001
ISSN 0022-3573
Synthesis and spasmolytic action of 2-substituted
thienopyrimidin-4-one derivatives
Natale Alfredo Santagati, Orazio Prezzavento, Ennio Bousquet,
Giuseppe Ronsisvalle and Santi Spampinato
Abstract
In the search for novel compounds to treat disorders of smooth muscle function, efforts have
focused on some 2-substituted thieno[2,3-d]pyrimidin-4-onederivatives that show interesting
spasmolytic action. Our laboratories have developed a new series of quaternary salts of 2-
substituted thieno[2,3-d]pyrimidin-4-oneand thieno[3,2-d]pyrimidin-4-oneisomers with thera-
peuticpotential. These substances were preparedstarting from simple derivativesof thiophene.
Their spasmolytic activity was evaluatedon transmurally stimulatedguinea-pigileum. The most
.
.
lM) 7f–7h, 12d and 12f had the terminal piperidino nucleus
active compounds (IC50 1 12–2 71
in the thioalkyl chain and lacked two methyl groups in the thiophene ring. Their relaxant
activity on the isolated ileum was potentiated (approx. 20–25%) by phosphodiesterase
inhibitors. Compounds 7f–h, 12d and 12f were less effective in inhibiting contractions of the
.
. lM) or histamine (IC50 41 5–63 4
.
. lM)
guinea-pig ileum induced by acetylcholine (IC50 26 7–41 4
and had a moderate binding activity to muscarinic receptors in membrane homogenates from
.
.
.
.
the rat heart (M2 sites; pKi values between 5 55³0 08 and 5 14³0 12; n ¯ 3) and submaxillary
.
.
.
.
gland (M3 sites; pKi values between 6 15³0 07 and 5 76³0 08; n ¯ 3). Action involving soluble
guanylylcyclase or any potential binding to guinea-pig ventricular L-type calcium channels was
not considered likely.It is concluded that atleast two different mechanismsof action contribute
to their spasmolytic activity.
Introduction
Several drugs with diŒerent mechanisms of action are employed for the treatment
of gastrointestinal and biliary spasms (for review see RuoΠet al 1991; De Ponti &
Malagelada 1998). Among others, antimuscarinics (Ehlert et al 1999; Wallis &
Napier 1999), calcium antagonists (Elorriaga et al 1996) and antispasmodic agents
acting through diŒerent mechanisms (Parker et al 1987; Nardi et al 1993; Santicioli
et al 1999) have been widely investigated. We previously reported the synthesis and
spasmolytic properties of a series of compounds containing a thieno[2,3-d]pyri-
midin-4-one system corresponding to structure A (Russo et al 1990) (Figure 1). The
relaxant action of these derivatives on intestinal smooth muscle prompted us to
extend our research to investigate the pharmacological mechanism underlying their
activity. The choice of the 2-(N-diethylamino)ethylthio-3-phenyl-5,6-dimethyl-
thieno[2,3-d]pyrimidin-4-one methyl iodide (1) as lead compound was made
because, among the previously reported derivatives, it was shown to have the best
relaxant activity on intestinal smooth muscle in-vivo and in-vitro. On the basis of
the structural features of the described compounds and in order to obtain more
Department of Pharmaceutical
Sciences, University of Catania,
Viale A. Doria 6, 95125 Catania,
Italy
Natale Alfredo Santagati, Orazio
Prezzavento, Ennio Bousquet,
Giuseppe Ronsisvalle
Department of Pharmacology,
University of Bologna, Via
Irnerio 48, 40126 Bologna, Italy
Santi Spampinato
Correspondence: N. A.
Santagati, Dipartimento di
Scienze Farmaceutiche,
Universita di Catania Viale A.
Doria, 6, 95125 Catania, Italy.
E-mail: santagat!mbox.unict.it
717

718
N. Alfredo Santagati et al
Figure 1 Structure of analogues of thieno[2,3-d]pyrimidin-4-one.
active derivatives, we performed the following modi- 60 F264 aluminium sheets (Merck) with visualization
®cations: (i) substitution of the diethylamino group under UV light. Melting points were determined in open
with the piperidine ring; (ii) insertion of a substituent capillary tubes with a Buchi melting point apparatus,
para to the aromatic ring; (iii) removal of the methyl and are uncorrected. IR spectra were obtained using a
groups on the thiophene nucleus ; and (iv) substitution 1600 FT-IR Perkin-Elmer spectrophotometer. Nuclear
of the thieno[2,3-d]pyrimidin-4-one nucleus with the magnetic resonance (1H NMR) spectra were recorded
corresponding thieno[3,2-d]pyrimidin-4-one isomers.
with a Varian Inova 200 spectrometer. Elemental analy-
The spasmolytic activity of these novel derivatives ses (C, H, N, S) were determined on a Carlo Erba Model
was evaluated on transmurally stimulated guinea-pig 1106 elemental analyser and were within 0.4% of the
ileum. The most promising compounds were evaluated theoretical values.
more extensively to check the involvement of phos-
phodiesterases of the soluble guanylyl cyclase or of the
General procedure for the preparation of ethyl
2( [(phenyl)amino]carbonothioyl amino)-4,5-
dimethyl-thiophene-3-carboxylate (4a–b), methyl
2( [(phenyl)amino]carbonothioyl amino)thiophene-
3-carboxylate (4c–e) and methyl
3( [(phenyl)amino]carbonothioyl amino)thiophene-
2-carboxylate (9a–c)
muscarinic receptors. We also measured their binding
activity to muscarinic receptors in membrane homo-
genates from the rat heart (M2 sites) and submaxillary
gland (M3 sites), or any potential binding to guinea-pig
ventricular L-type calcium channels.
Derivatives 7a±l were synthesized by the action of the
appropriate arylisothiocyanate derivative on starting
The appropriate arylisothiocyanate (0.015 mol) was
added, dropwise, to a hot and stirred solution of thio-
phene amino ester derivative 2, 3 or 8 (0.013 mol) in
ethanol (40 mL) The mixture was then re¯ uxed for a
reaction time of between 0.5 and 4 h. When the mixture
was cooled, the crystals were separated by ®ltration.
After washing with ethanol, the ®nal compounds were
recrystallized from ethanol.
amines 2 and 3 to give the thiourea derivatives 4a±e.
Cyclization of the thiourea derivatives yielded the
2-mercapto-thienopyrimidin-4-one compounds 5a±e.
Base-catalysed alkylation of these thienopyrimidin-4-
one derivatives with the appropriate 2-chloroethyl
amine gave compounds 6a±l. Quaternization of the
bases with methyl iodide provided the ®nal compounds
7a±l (Figure 2). The corresponding thieno[3,2-d]pyrimi-
din-4-one isomers 12a±f were synthesized by the same
procedure, starting from the methyl 3-aminothiophene-
2-carboxylate 8 (Figure 3). Compound 2 was prepared
as reported by Gewald et al (1966). Methyl 2-amino-3-
thiophenecarboxylate (3) and methyl 3-amino-2-thio-
phenecarboxylate (8) are commercially available.
Ethyl 2-( ² [ ( 4 - chlorophenyl ) amino ] carbonothioyl´
amino)-4,5-dimethyl-thiophene-3-carboxylate (4a)
(80% ) mp 192±194°C; IR (KBr) 1660. Reaction time:
2 h. Analysis: calc. for C16H17ClN2O2S2.
Ethyl 4,5-dimethyl-2-( ² [ ( 4 - methylphenyl ) amino]
carbonothioyl´amino)thiophene-3-carboxylate (4b)
(80% ) mp 161±162°C; IR (KBr) 1660. Reaction time:
3 h. Analysis: calc. for C17H20N2O2S2.
Materials and Methods
Chemistry
Methyl2-[ (anilinocarbonothioyl )amino]thiophene-3 -
Reagents used for synthesis were purchased from Sigma- carboxylate (4c)
Aldrich (Milan, Italy) unless otherwise speci®ed. Thin- (40% ) mp 165±167°C; IR (KBr) 1660. Reaction time:
layer chromatography was done on pre-coated silica gel 4 h. Analysis: calc. for C13H12N2O2S2.

719
2-Substituted thienopyrimidin-4-one derivatives
m
m
HCl;
Figure 2 Synthesis of compounds 7a±l. Reagents and conditions: a. S¯C¯NC6H4R4, C2H5OH, re¯ ux; b. i. 2
m
c. ClC2H4N(C2H5)2 or 1-(2-chloroethyl)piperidine, 2 NaOH; d. CH3I, dioxane.
KOH, ii. 3
Methyl 2-(² [ ( 4 - chlorophenyl )amino ] carbonothioyl´ Methyl2-(²[ (4 - methylphenyl )amino ]carbonothioyl´
amino)thiophene-3-carboxylate (4d) amino)thiophene-3-carboxylate (4e)
(52% ) mp 190±192°C; IR (KBr) 1660. Reaction time: (46% ) mp 186±189°C; IR (KBr) 1660. Reaction time:
2.5 h. Analysis: calc. for C11H13ClN2O2S2. 3 h. Analysis: calc. for C14H14N2O2S2.

720
N. Alfredo Santagati et al
m
Figure 3 Synthesis of compounds 12a±f. Reagents and conditions: a. S¯C¯NC6H4R4, C2H5OH, re¯ ux; b. i. 2 KOH, ii. HCl;
m
c. ClC2H4N(C2H5)2 or 1-(2-chloroethyl)piperidine, 2 NaOH; d. CH3I, dioxane.
Methyl3-[ (anilinocarbonothioyl )amino]thiophene-2 - Methyl3-( ²[ ( 4 - methylphenyl )amino ]carbonothioyl´
carboxylate (9a)
amino)thiophene-2-carboxylate (9c)
(85% ); mp 260±262°C; IR (KBr) 1660. Reaction time: (88% ); mp 249±251°C; IR (KBr) 1660. Reaction time:
3 h. Analysis: calc. for C12H8N2OS2.
0.5 h. Analysis: calc. for C13H10N2OS2.
Methyl 3-( ² [ ( 4 - chlorophenyl)amino ] carbonothioyl´
amino)thiophene-2-carboxylate (9b)
(80% ); mp 282±285°C; IR (KBr) 1660. Reaction time:
2 h. Analysis: calc. for C12H7ClN2OS2.

721
2-Substituted thienopyrimidin-4-one derivatives
General procedure for the preparation of 3-
(phenyl)-2-mercapto-5,6-dimethylthieno[2,3-
d]pyrimidin-4(3H)-one (5a–b), 2-mercapto-3-
phenylthieno[2,3-d]pyrimidin-4(3H)-one (5c–e)
and 2-mercapto-3-phenylthieno[3,2-
2-Mercapto-3-(4-methylphenyl)thieno[3,2-d]pyrimidin-
4(3H)-one (10c)
(88% ); mp 249±251°C; IR (KBr) 1680; Analysis: calc.
for C13H10N2OS2.
d]pyrimidin-4-(3H)-one (10a–c)
General procedure for the preparation of 3-
(phenyl)-2- [2-(diethylamino)ethyl]thio -5,6-
dimethylthieno[2,3-d]pyrimidin-4(3H)-one
(6a–b, i–l), 2- [2-(diethylamino)ethyl]thio -3-
phenylthieno[2,3-d]pyrimidin-4(3H)-one (6c–h)
and 2- [2-diethylamino)ethyl]thio -3-
Compound of type 4 or 9 (0.015 mol) was added to a
solution of 2 m KOH (100 mL), and the reaction mixture
was stirred under re¯ ux for 1 h until complete dis-
solution. After cooling and acidi®cation with 3 m HCl,
the precipitate obtained was removed by ®ltration and
washed with water (3¬100 mL). It was then recrystal-
lized from ethanol, giving solid as white crystals.
phenylthieno[3,2-d]pyrimidin-4(3H)-one (11a–f)
The suitable chloroethyl tertiary amine hydrochloride
(0.014 mol) in water (40 mL) was added to an aqueous
solution of 2 m NaOH (40 mL) containing the appro-
priate mercapto derivatives (0.014 mol) in water
(100 mL). The reaction mixture was stirred at 50°C for
0.5 h. After cooling, the solid was ®ltered, washed with
water (2¬40 mL)and recrystallized from ethanol giving
the desired product.
3-(4-Chlorophenyl)-2-mercapto-5,6-dimethylthieno[2,
3-d]pyrimidin-4(3H)-one (5a)
(85% ); mp 293±295°C; IR (KBr) 1670; Analysis: calc.
for C14H11ClN2OS2.
2-Mercapto-5,6-dimethyl-3-(4-methylphenyl)-thieno[2,
3-d]pyrimidin-4(3H)-one (5b)
(79% ); mp 269±271°C; IR (KBr) 1670; Analysis: calc.
for C15H14N2OS2.
3-(4-Chlorophenyl)-2-²[2-(diethylamino)ethyl]thio´-5,
6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (6a)
(85% ) mp 113±116°C; Analysis: calc. for C20H24
ClN3OS2.
2-Mercapto-3-phenylthieno[2,3-d]pyrimidin-4(3H)-
one (5c)
(89% ); mp 279±282°C; IR (KBr) 1670; Analysis: calc.
for C12H8N2OS2.
2-²[2-(diethylamino)ethyl]thio´-5,6-dimethyl-3-(4-
methylphenyl)thieno [2,3-d]pyrimidin-4(3H)-one (6b)
(80% ); mp 97±100°C; Analysis: calc. for C21H27N3OS2.
3-(4-Chlorophenyl)-2-mercaptothieno[2,3-d]pyrimidin-
4(3H)-one (5d)
(90% ); mp 288±290°C; IR (KBr) 1670; Analysis: calc.
for C12H7ClN2OS2.
2-²[2-(diethylamino)ethyl]thio´-3-phenylthieno[2,3-
d]pyrimidin-4(3H)-one (6c)
(66% ); mp 72±74°C; Analysis: calc. for C18H21N3OS2.
2-Mercapto-3-(4-methylphenyl)thieno[2,3-d]pyrimidin-
4(3H)-one (5e)
3-(4-Chlorophenyl)-2-²[2-(diethylamino)ethyl]thio´
thieno[2,3-d]pyrimidin-4(3H)-one (6d)
(90% ); mp 130±133°C; Analysis: calc. for C18H20
ClN3OS2.
(90% ); mp 285±287°C; IR (KBr) 1670; Analysis: calc.
for C13H10N2OS2.
2-Mercapto-3-phenylthieno[3,2-d]pyrimidin-4-(3H)-
one (10a)
2-²[2-(Diethylamino)ethyl]thio´-3-(4-methylphenyl)
thieno[2,3-d]pyrimidin-4(3H)-one (6e)
(85% ); mp 260±262°C; IR (KBr) 1680; Analysis: calc.
for C12H8N2OS2.
(77% ); mp 100±102°C;Analysis: calc. for C19H23N3OS2.
3-(4-Chlorophenyl)-2-mercaptothieno[3,2-d]pyrimidin-
4(3H)-one (10b)
3-Phenyl-2-[(2-piperidin-1-ylethyl)thio]thieno[2,3-d]
(80% ); mp 282±285°C; IR (KBr) 1680; Analysis: calc. pyrimidin-4(3H)-one (6f )
for C12H7ClN2OS2. (78% ); mp 130±132°C;Analysis: calc. for C19H21N3OS2.

722
N. Alfredo Santagati et al
General procedure to obtain quaternary
derivatives 7a–l and 12a–f
3-(4-Chlorophenyl)-2-[(2-piperidin-1-ylethyl)thio]
thieno[2,3-d]pyrimidin-4(3H)-one (6g)
(95% ); mp 140±142°C; Analysis: calc. for C19H20
ClN3OS2.
CH3I (0.05 mol) was added to a stirred solution of the
suitable tertiary amine (0.05 mol) in dioxane (25 mL)
and the reaction mixture was re¯ uxed for 0.5 h. The
precipitate was ®ltered, washed with (C2H5)2O
(3¬10 mL) and recrystallized from ethanol, giving the
desired ®nal product.
3-(4-Methylphenyl)-2-[(2-piperidin-1-ylethyl)thio]
thieno[2,3-d]pyrimidin-4(3H)-one (6h)
(65% ); mp 127±129°C;Analysis: calc. for C20H23N3OS2.
2 - ²[ 3 - ( 4 - Chlorophenyl)- 5,6- dimethyl- 4 - oxo - 3,4 -
dihydrothieno[2,3-d]pyrimidin-2-yl]thio´-N,N-diethyl-
N-methylethanaminium iodide (7a)
3-(4-Chlorophenyl)-5,6-dimethyl-2-[(2-piperidin-1-
ylethyl)thio]thieno[2,3-d]pyrimidin-4(3H)-one (6i)
(85% ); mp 157±160°C; Analysis: calc. for C21H24
ClN3OS2.
1
(50% ); mp 194±197°C; H NMR (DMSO-d6) d, ppm:
1.16±1.35 (m, 6H, CH3), 2.33 (s, 3H, CH3), 2.37 (s, 3H,
CH3), 3.18±3.43 (m, 11H), 7.41±7.71 (m, 4H, Ar-H);
Analysis: calc. for C21H27ClIN3OS2.
5-6-Dimethyl-3-(4-methylphenyl)-2-[(2-piperidin-1-
ylethyl)thio]thieno[2,3-d]pyrimidin-4(3H)-one (6l)
(90% ); mp 122±124°C;Analysis: calc. for C22H27N3OS2.
2 - ²[ 5,6- Dimethyl - 3 - ( 4 - methylphenyl )- 4 - oxo - 3,4-
dihydrothieno[2,3-d]pyrimidin-2-yl]thio´-N,N-diethyl-
N-methylethanaminium iodide (7b)
2-²[(2-Diethylamino)ethyl]thio´-3-phenylthieno[3,2-d]
pyrimidin-4(3H)-one (11a)
1
(75% ); mp 154±157°C; H NMR (DMSO-d6) d, ppm:
1.18±1.35 (m, 6H, CH3), 2.33 (s, 3H, CH3), 2.37 (s, 3H,
CH3), 2.39 (s, 3H, CH3), 2.95 (s, 3H, CH3), 3.26±3.50
(m, 8H), 7.15±7.40 (m, 4H, Ar-H); Analysis: calc. for
C22H30IN3OS2.
(82% ); mp 85±87°C; Analysis: calc. for C18H21N3OS2.
3-(4-Chlorophenyl)-2-²[(2-diethylamino)ethyl[thio´
thieno[3,2-d]pyrimidin-4(3H)-one (11b)
(69% ); mp 268±271°C; Analysis: calc. for C18H20
ClN3OS2.
N,N - Diethyl - N - methyl - 2 - ² [ 4 - oxo - 3 - phenyl- 3,4 -
dihydrothieno[2,3-d]pyrimidin2-yl]thio´ethanaminium
iodide (7c)
1
(73% ); mp 207±209°C; H NMR (DMSO-d6) d, ppm:
2-²[(2-Diethylamino)ethyl]thio´-3-(4-methylphenyl)
thieno[3,2-d]pyrimidin-4(3H)-one (11c)
1.19±1.38 (m, 6H, CH3), 2.97 (s, 3H, CH3), 3.26±3.50 (m,
8H), 7.36±7.63 (m, 7H, thiophene H, Ar-H); Analysis:
calc. for C19H24IN3OS2.
(92% ); mp 143±145°C;Analysis: calc. for C19H23N3OS2.
3-Phenyl-2-[(2-piperidin-1-ylethyl)thio]thieno[3,2-d]
pyrimidin-4(3H)-one (11d)
2 - ² [ 3 - ( 4 - Chlorophenyl ) - 4 - oxo - 3,4 - dihydro-
thieno [ 2 , 3 - d ] pyrimidin - 2 - yl ]thio ´ - N,N - di-
ethyl - N -methylethanaminium iodide (7d)
(85% ); mp 153±155°C;Analysis: calc. for C19H21N3OS2.
1
(90% ); mp 94±95°C; H NMR (DMSO-d6) d, ppm:
1.17±1.36 (m, 6H, CH3), 2.97 (s, 3H, CH3), 3.26±3.50 (m,
8H), 7.39 (d, H, thiophene H), 7.44±7.71 (m, 5H, thio-
phene H, Ar-H); Analysis: calc. for C19H23ClIN3OS2.
3-(4-Chlorophenyl)-2-[(2-piperidin-1-ylethyl)thio]
thieno[3,2-d]pyrimidin-4(3H)-one (11e)
(90% ); mp 145±148°C; Analysis: calc. for C19H20
ClN3OS2.
N,N-Diethyl-N-methyl-2-²[3-(4-methylphenyl)-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidin-2-yl]thio´ethanami-
3-(4-Methylphenyl)-2-[(2-piperidin-1-ylethyl)thio] nium iodide (7e)
thieno[3,2-d]pyrimidin-4(3H)-one (11f ) (75% ); mp 198±200°C; H NMR (DMSO-d6) d, ppm:
(90% ); mp 121±123°C;Analysis: calc. for C20H23N3OS2. 1.18±1.37 (m, 6H, CH3), 2.39 (s, 3H, CH3), 2.97 (s, 3H,
1

723
2-Substituted thienopyrimidin-4-one derivatives
CH3), 3.27±3.50 (m, 8H), 7.25±7.42 (m, 5H, thiophene N,N-Diethyl-N-methyl-2-[²4oxo-3-phenyl-3,4-dihydro-
H, Ar-H). 7.54 (d, H, thiophene H); Analysis: calc. for thieno[3,2-d]pyrimidin-2-yl]thio´ethanamoniumiodide
C20H26IN3OS2.
(12a)
1
(90% ); mp 181±182°C; H NMR (DMSO-d6) d, ppm:
1.21±1.35 (m, 6H, CH3), 2.98 (s, 3H, CH3), 3.28±3.46 (m,
8H), 7.36 (d, 1H, thiophene H), 7.41±7.68 (m, 4H, Ar-
H) 8.27 (d, 1H, thiophene H); Analysis: calc. for
C19H24IN3OS2.
1-Methyl-1-²2-[(4-oxo-3-phenyl-3,4-dihydrothieno[2,
3 -d]pyrimidin-2-yl)thio]ethyl´piperidinium iodide (7f)
(95% ); mp 250±252°C; Analysis: 1H NMR (DMSO-d6)
d, ppm: 1.47±1.62 (m, 2H, piperidine H), 1.68±1.93 (m,
4H, piperidine H), 3.09 (s, 3H, CH3), 3.27±3.60 (m, 8H),
7.30±7.68 (m, 7H, thiophene H, Ar-H); Analysis: calc.
for C20H24IN3OS2.
2-²[3-(4-Chlorophenyl)-4oxo-3,4-dihydrothieno[3,2-d]
pyrimidin-2-yl]thio´N,N-diethyl-N-methylethanamo-
nium iodide (12b)
1
(80% ); mp 214±216°C; H NMR (DMSO-d6) d, ppm:
2-²[(3(4-Chlorophenyl)-4-oxo-3,4-dihydrothieno[2,3- 1.21±1.34 (m, 6H, CH3), 2.97 (s, 3H, CH3), 3.28±3.46 (m,
d ] pyrimidin - 2 - yl ] thio ´ ethyl - piperidinium iodide 8H), 7.33 (d, 1H, thiophene H), 7.42±7.71 (m, 4H, Ar-
(7g)
H), 8.23 (d, 1H, thiophene H); Analysis: calc. for
1
(95% ); mp 292±294°C; H NMR (DMSO-d6) d, ppm: C19H23ClIN3OS2.
1.47±1.61 (m, 2H, piperidine H), 1.71±1.93 (m, 4H,
piperidine H), 3.09 (s, 3H, CH3), 3.23±3.61 (m, 8H),
N,N-Diethyl-N-methyl-2-[²3-(4-methylphenyl)-4oxo-
7.32±7.71 (m, 6H, thiophene H, Ar-H); Analysis: calc.
for C20H23ClIN3OS2.
3,4-dihydrothieno[3,2-d]pyrimidin-2-yl]thio´ethanamo-
nium iodide (12c)
1
(95% ); mp 219±220°C; H NMR (DMSO-d6) d, ppm:
1.18±1.38 (m, 6H, CH3), 2.39 (s, 3H, CH3), 2.99 (s, 3H,
CH3), 3.28±3.48 (m, 8H), 7.26±7.43 (m, 5H, thiophene
H, Ar-H) 8.26 (d, 1H, thiophene H); Analysis: calc. for
C20H26IN3OS2.
1-Methyl-1-(2-²[(3(4-methylphenyl)-4-oxo-3,4-dihy-
drothieno[2,3-d]pyrimidin-2-yl]thio´ethyl)piperidinium
iodide (7h)
1
(95% ); mp 235±237°C; H NMR (DMSO-d6) d, ppm:
1.46±1.63 (m, 2H, piperidine H), 1.71±1.93 (m, 4H,
piperidine H), 2.39 (s, 3H, CH3), 3.08 (s, 3H, CH3),
3.26±3.61 (m, 8H), 7.22±7.58 (m, 6H, thiophene H, Ar-
H); Analysis: calc. for C21H26IN3OS2.
1-Methyl-1²2-[(4-oxo-3-phenyl-3,4-dihydrothieno[3,2-
d]pyrimidin-2-yl)thio]ethyl´piperidinium iodide (12d)
(90% ); mp 243±245°C; H NMR (DMSO-d6) d, ppm:
1
1.46±1.61 (m, 2H, piperidine H), 1.73±1.92 (m, 4H,
piperidine H), 3.10 (s, 3H, CH3), 3.28±3.59 (m, 8H),
7.37±7.63 (m, 5H, thiophene H, Ar-H), 8.26 (d, 1H,
thiophene H); Analysis: calc. for C20H24IN3OS2.
2 - ² [ 3 - ( 4 - Chlorophenyl)- 5,6- dimethyl- 4 - oxo -3,4 -
dihydrothieno[ 2,3-d ]pyrimidin- 2 -yl ]thio ´ethyl )- 1 -
methylpiperidinium iodide (7i)
1
(80% ); mp 132±134°C; H NMR (DMSO-d6) d, ppm:
1.47±1.61 (m, 2H, piperidine H), 1.70±1.92 (m, 4H,
piperidine H), 2.33 (s, 3H, CH3), 2.37 (s, 3H, CH3), 3.07
(s, 3H, CH3), 3.18±3.52 (m, 8H), 7.43±7.70 (m, 4H, Ar-
H); Analysis: calc. for C22H27ClIN3OS2.
1-2-²[3-(4-Chlorophenyl)-4-oxo-3,4-dihydrothieno[3,
2-d ]pyrimidin-2-yl]thio´ ethyl-1-methylpiperidinium
iodide (12e)
1
(75% ); mp 221±223°C; H NMR (DMSO-d6) d, ppm:
1.47±1.61 (m, 2H, piperidine H), 1.73±1.92 (m, 4H,
piperidine H), 3.10 (s, 3H, CH3), 3.30±3.58 (m, 8H), 7.38
(d, 1H, thiophene H), 7.48±7.72 (m, 4H, Ar-H), 8.26 (d,
1H, thiophene H); Analysis: calc. for C20H23ClIN3OS2.
2 - ² [ 5,6- Dimethyl - 3 - ( 4 - methylphenyl)- 4 - oxo -3,4 -
dihydrothieno[ 2,3-d ]pyrimidin- 2 -yl ]thio ´ethyl )- 1 -
methylpiperidinium iodide (7l)
1
(80% ); mp 231±233°C; H NMR (DMSO-d6) d, ppm:
1.47±1.61 (m, 2H, piperidine H), 1.72±1.90 (m, 4H, 1-Methyl-1-2-²[3-(4-methylphenyl)-4-oxo-3,4-di-
piperidine H), 2.33 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.39 hydrothieno [ 3,2 - d ] pyrimidin - 2 - yl ] thio ´ ethyl
(s, 3H, CH3), 3.06 (s, 3H, CH3), 3.20±3.52 (m, 8H), piperidinium iodide (12f)
1
7.22±7.41 (m, 4H, Ar-H); Analysis: calc. for (95% ); mp 231±233°C; H NMR (DMSO-d6) d, ppm:
C23H30IN3OS2.
1.47±1.61 (m, 2H, piperidine H), 1.72±1.92 (m, 4H,

724
N. Alfredo Santagati et al
piperidine H), 2.40 (s, 3H, CH3), 3.10 (s, 3H, CH3), equilibration, 0.2 mL of the test compounds (dissolved
3.31±3.60 (m, 8H), 7.26±7.42 (m, 5H, thiophene H, Ar- in methanol) was applied during electrical stimulation.
H), 8.25 (d, 1H, thiophene H); Analysis: calc. for Each compound was assayed at four concentrations. In
C21H26IN3OS2.
some experiments, the ileum’s responses to isoprenaline,
histamine and acetylcholine were evaluated; test com-
pounds were applied 2 min before the agonist. All
experiments were repeated at least three times on dif-
ferent strips of ileum from three diŒerent animals. The
data are expressed as mean percentage inhibition³
s.e.m.
Materials
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ),
an inhibitor of soluble guanylyl cyclase (Hebeiss &
Kilbinger 1998) was obtained from ICN Biomedicals
(Milan, Italy). Trequinsin and rolipram, inhibitors of
phosphodiesterases (Souness & Rao 1997), atropine
sulfate, nifedipine, isdradipine, acetylcholine hydro-
chloride, histamine dihydrogen phosphate, isoprenaline
and phenyl-methyl-sulphonyl¯ uoride, were purchased
from Sigma-Aldrich. [3H]isradipine (sp. act. 80 Ci
M2 and M3 muscarinic receptor binding assays
Rats were killed and the hearts were rapidly removed
and weighed. Hearts were minced in 5 vols homogeniz-
ing buŒer (50 mm Tris, 4 mm EDTA pH 7.4, 4°C) using
an Ultraturrax and homogenized with a tissue homo-
genizer (Wheaton ; 10 strokes). The suspension was ®l-
tered through gauze and centrifuged (1000 g, 4°C,
10 min). The supernatant was kept on ice and the pellet
was washed with 2 vols homogenizing buŒer and centri-
fuged as before. The two supernatants were then com-
bined and centrifuged (45000 g, 4°C, 12 min). The pellet
wassuspended in 5 vols assay buŒer (50 mm Tris, 0.5 mm
EDTA pH 7.4, 4°C) and centrifuged as above. The
pellet was washed a second time with 5 vols assay buŒer
and centrifuged as before. After the second wash, a
portion of the suspension was retained for protein assay
(Lowry et al 1951).
1
3
3
-
mmol ) and [ H]N-methylscopolamine ([ H]NMS ; sp.
1
-
act. 79.5 Ci mmol ) were obtained from NEN Life
Science Products (Milan, Italy). All other reagents were
either of analytical grade or of the highest purity
available.
Animals
Male Hartley guinea-pigs (300±400 g), male Sprague±
Dawley rats (250±300 g) and male Swiss mice (20±25 g)
were purchased from Charles River (Calco, Como,
Italy). The animals were kept in air-conditioned rooms
(21³2°C; 60³10% relative humidity)under a constant
12-h light±dark cycle and received standard diet and tap
water. Experiments were carried out in accordance with
the internationally accepted guidelines and all proce-
dures followed the guidelines issued by Animal Care
and Use Committee of the University of Bologna (Bolo-
Submaxillary glands were rapidly removed, weighed,
minced in 10 vols homogenizing buŒer (50 mm Tris,
5 mm EDTA pH 7.4, 4°C), using the Ultraturrax, and
homogenized as described for the hearts. The suspension
was ®ltered through gauze and centrifuged (1000 g, 4°C,
10 min). The supernatant was kept on ice and the pellet
was washed with 2 vols homogenizing buŒer and centri-
fuged as before. The two supernatants were combined
and centrifuged (45000 g, 4°C, 12 min). The pellet was
}
gna, Italy; D. Leg. 116 92).
Isolated guinea-pig ileum
Sections of ileum (approx. 10 cm) from the ileo±caecal suspended in 10 vols assay buŒer and centrifuged as
junction were resected from guinea-pigs and placed in indicated. The pellet was washed a second time with 10
5-mL tissue baths containing modi®ed Tyrode’s solution vols assay buŒer and centrifuged as before. After the
}
kept at 35°C, oxygenated with 95% O2 5% CO2. The second suspension, a portion was retained for protein
Tyrode’s solution had the following composition (mm) assay (Lowry et al 1951). Pellets were stored at ®80°C
137 NaCl;2.7 KCl;1.0 MgCl2 ; 1.8 CaCl2 ; 0.4 NaH2PO4 ; until used.
11.9 NaHCO3 ; 5.5 glucose. The electrode was made of
The detailed methods of the binding assay are pub-
platinum and the intraluminal electrode was used as lished elsewhere (Waelbroeck et al 1986; Melchiorre et
anode. The ileum was stimulated electrically with rec- al 1993). [3H]NMS was used to evaluate binding sites in
tangular pulses of 0.5-ms duration, at the frequency of rat heart homogenates (expressing M2 muscarinic recep-
0.15 Hz and at a high enough voltage to give a maximal tors; Kd 0.32³0.04 nm; Bmax ¯ 77.8³15.3 fmol (mg
1
-
response (70±80 V). Responses were recorded isometri- protein) ) and submaxillary gland homogenates (ex-
cally with an initial tension of 1 g. After 30±60 min pressing M3 muscarinic receptors ; Kd 0.48³0.03 nm;

725
2-Substituted thienopyrimidin-4-one derivatives
1
Bmax ¯ 1102³85 fmol (mg protein) ). Competition trations ranging from 0.05 to 5 nm [3H]isradipine (Kd ¯
-
binding studies were done in incubation buŒer (50 mm 4.3¬10 11). Speci®c binding was determined in the
-
sodium phosphate, pH 7.4; enriched with 2 mm MgCl2 presence of 1 lm nifedipine. Ventricular membranes
and 1% bovine serum albumin). Homogenates (500 lg were incubated at 25°C for 120 min in a ®nal volume of
protein) were incubated for 2 h at 25°C in 1 mL in- 1 mL and incubation was stopped by vacuum ®ltration
}
cubation buŒer and in the indicated concentration of over Whatman GF C ®lters. For competition binding
tracer (0.8 nm). Non-speci®c binding was assessed in the experiments, 0.2 nm isradipine was used. Drugs were
presence of 10 lm atropine sulfate. Binding assays were prepared as stock solutions (10 2 m) in ethanol and
-
}
terminated by ®ltration on Whatman GF C glass-®bre protected from light. Drug dilutions were made in assay
®lters previously soaked in 0.1% poly(ethylenimine) buŒer. Bound radioactivity was measured in a Beckman
and rinsed four times with 5 mL ice-cold 50 mm phos- Ls 1701 scintillation counter and the data were analysed
phate buŒer (pH 7.4).
using the LIGAND program (Munson and Rodbard
Data were analysed using the LIGAND program 1980). At least three independent experiments with
(Munson & Rodbard 1980). DiŒerences in the slopes of assays in duplicate were done for each compound.
the curves were determined by the test of parallelism
described by Tallarida & Murray (1991), and were not
Charcoal meal
"
signi®cantly diŒerent from unity (P 0.05). Hill num-
bers (nH) were not signi®cantly diŒerent from unity
"
(P 0.05). Equilibrium dissociation constants (Ki)were
derived from the Cheng±PrusoŒ equation (Cheng &
Mice were fasted for 24 h. Test compounds, suspended
in 1% carboxymethylcellulose, were administered intra-
1
peritoneally (50 mg kg ) and, 20 min later, 0.2 mL
-
}
}
PrusoŒ 1973), Ki ¯ IC50 (1 - L Kd), where L and Kd
are the concentration and the equilibrium dissociation
constant of [3H]NMS. pKi values are the mean³s.e.m.
of three separate experiments run in duplicate.
charcoal meal (5% charcoal in 1% carboxymethyl-
}
cellulose, w v)was given orally. Mice were killed 30 min
later using sodium pentobarbital (100 mg kg , i.p.) and
1
-
the length of the small intestine traversed by the charcoal
was measured in mm. The mean values of both treated
groups (Xt; n ¯ 6) and the vehicle-treated group (Xc;
n ¯ 6) were calculated, and the percentage inhibition
induced by each compound was evaluated in compari-
Binding to L-type calcium channels
Membranes were prepared according to the method of
Glossmann & Ferry (1985), using guinea-pig heart ven-
tricles. All tissue preparation steps were done at 4°C,
unless otherwise speci®ed. The tissues were ®nely minced
and homogenized with a Polytron homogenizer in 3 vols
ice-cold buŒer solution containing 20 mm NaHCO3 and
0.1 nm phenyl-methyl-sulfonyl¯ uoride, pH 7.4. The
homogenate was diluted 1:7 in the same buŒer solution
and centrifuged at 1500 g for 15 min. The supernatant
was collected and centrifuged at 48000 g for 15 min; the
pellet was washed twice by homogenization under the
conditions already described in 7 vols ice-cold 50 mm
}
son with controls: (Xc ± Xt) Xc ¬100.
Data analysis
Data are expressed as mean³s.e.m. Statistical com-
parisons were done by analysis of variance and post hoc
Dunnett’s multiple comparison test, with diŒerences of
!
P
0.05 being considered signi®cant (GraphPad Soft-
ware, San Diego, CA). The concentrations needed to
produce 50% inhibitory eŒects (IC50) were calculated
from log concentration±response curves (Tallarida &
Murray 1991).
}
Tris HCl, 0.1 mm phenyl-methyl-sulfonyl¯ uoride buf-
fer, pH 7.4, with centrifugation at 48000 g for 15 min.
The ®nal pellet was resuspended in 3 vols of the same
buŒer, and the membrane suspension was divided into
aliquots and stored at ®80°C. On the day of the
Results and Discussion
experiment, aliquots of membranes were thawed, resus- The isolated guinea-pig ileum was used as a functional
}
pended in 50 mm Tris HCl, 1 mm CaCl2 buŒer, pH 7.4 assay to evaluate spasmolytic activity, by measuring the
and used for the binding assay (100 lg protein tube). inhibitory eŒect of compounds on the response to the
}
Protein was determined by the method of Lowry et al transmural electrical stimulation. Electrical stimulation
(1951).
of the ileum induced regular and reproducible contrac-
Binding experiments were done in near-darkness. tions, which were not aŒected by administration of
Saturation binding experiments were done at concen- 0.2 mL methanol (data not shown).

726
N. Alfredo Santagati et al
Table 1 EŒect of compounds 1, 7a±l, and 12a±f and isporenaline on
spasmolytic activity induced by electrical stimulation in the isolated
guinea-pig ileum in the presence of either the vehicle or the phospho-
had the terminal piperidino nucleus in the thioalkyl
chain and lacked two methyl groups in the thiophene
ring. The spasmolytic activity displayed by these com-
pounds was not related to the isomer considered or to
the substituent on the thiophene ring. In fact, the
7
-
m
diesterase inhibitors rolipram and trequinsin (10
before test compounds).
; added 30 min
lm
Compound
IC50 (
)
}
spasmolytic activity of the isomeric couples 12d 7f and
12f 7h was comparable. Compound 7i and the lead
}
Vehicle
Rolipram
Trequinsin
compound 1 had lower activity (IC50 ¯ 16.7 and 18.1
lm, respectively). The latter was the only compound,
among those with most spasmolytic activity, with a
diethyl group in the lateral chain. The para substituent
on the aromatic ring (7 g±7i, 12f) did not signi®cantly
in¯ uence the activity.
.
.
.
.
.
.
1
7a
18 72³0 11
13 41³0 04
n.d.
14 3³0 09
n.d.
. .
28 76³0 98
.
.
7b
48 64³1 24
n.d.
n.d.
.
.
7c
7d
33 83³2 1
n.d.
n.d.
n.d.
n.d.
.
.
496 12³22 4
.
.
7e
52 42³0 45
n.d.
n.d.
. .
0 82³0 17
. .
1 25³0 16
Compounds 7a±c, 7e, 7l, 12b±c and 12e were the least
potent of this series, and compounds 7d and 12a were
the less eŒective (Table 1).
.
.
.
.
7f
7g
1 12³0 24
0 73³0 14
.
.
.
.
1 92³0 15
1 03³0 11
.
.
.
.
. .
1 84³0 18
7h
2 35³0 21
1 77³0 10
.
.
.
.
.
.
7i
16 11³0 19
12 5³0 09
n.d.
14 5³0 32
n.d.
The test compounds maintained their spasmolytic
action as long as they were left in contact with the
isolated tissue (2 min), and it was reversible after a
single wash by over¯ ow.
. .
29 16³1 15
7l
.
.
12a
812 43³13 5
n.d.
n.d.
.
.
12b
12c
66 34³0 88
n.d.
n.d.
n.d.
n.d.
.
.
73 43³0 76
.
.
.
.
.
.
12d
1 48³0 11
0 59³0 04
n.d.
0 66³0 13
n.d.
Compounds 1, 7f±i, 12d and 12f, which had strong
spasmolytic activity, were further investigated. Trequin-
.
.
12e
12f
65 80³1 28
.
.
.
.
. .
2 26³0 17
. .
3 971³0 3*
2 71³0 28
2 23³0 04
sin and rolipram (10 7 m), blockers of phosphodiesterase
-
.
.
.
.
Isoprenaline
7 6³0 8
3 4³0 4*
activity (Souness & Rao 1997), potentiated their spas-
molytic activity by approximately 20±25% and were
not active by themselves (data not shown). These selec-
tive inhibitors signi®cantly potentiated the spasmolytic
action of isoprenaline, by approximately 50% (Table
1). Therefore, any inhibition of phosphodiesterase ac-
tivity in the spasmolytic eŒect of the test compounds
Values are the mean³s.e.m. of three diŒerent experiments.n.d. ¯ not
determined.
!
P
*
.
0 01 vs vehicle (Dunnett’s test after analysis of variance).
As shown in Table 1, the most active compounds was less marked than for the potent b-adrenoceptor
!
(IC50 5.0 lm) 7f±h, 12d and 12f were approximately agentisoprenaline. In preliminary experiments, the most
10-times more active than the lead compound 1, and eŒective compounds potentiated the spasmolytic action
Table 2 Spasmolytic activity of compounds 1, 7f±i, 12d and 12f on acetylcholine-or histamine-stimulated
7 m
-
guinea-pig ileum. Test compounds were added 10 min before acetylcholine chloride (10
7 m
) or histamine
-
dihydrogen phosphate (10
).
Compound
Inhibition of acetylcholine-induced
lm
Inhibition of histamine-induced
lm
contractions IC50 (
)
contractions IC50 (
)
.
.
. .
66 4³6 4
1
44 3³3 0
.
.
. .
41 5³3 2
7f
26 7³2 5
.
.
. .
58 7³4 1
7g
32 7³4 9
.
.
. .
7h
7i
31 7³4 1
53 3³2 8
.
.
. .
63 4³5 1
41 4³2 8
.
.
. .
51 0³2 8
12d
30 4³1 8
.
.
.
.
12f
Atropine sulfate
33 7³3 5
59 3³3 4
n.d.
.
.
0 8³0 3*
Values are the mean³s.e.m. of three diŒerent experiments. n.d. ¯ not determined.
!
P
*
.
0 01 vs test compounds (Dunnett’s test after analysis).

727
2-Substituted thienopyrimidin-4-one derivatives
Table 3 A nity estimates, expressed as pKi values, in the rat heart Table 4 EŒect of compounds 1, 7f±i, 12d and 12f on a charcoal meal
(M2) and submaxillary gland (M3) muscarinic receptors subtypes of
compounds 1, 7f±i, 12d and 12f and atropine sulfate.
test in the mouse.
Compound
Inhibition vs control (% )
Compound
PKi
M2
.
.
1
35³3 7
M3
.
7f
55³6 1
.
7g
7h
7i
50³4 3
.
.
.
.
1
5 02³0 12
5 53³0 08
47³3 6
.
.
.
.
.
7f
5 55³0 08
6 15³0 07
37³1 8
.
.
.
.
.
7g
7h
5 50³0 13
5 80³0 05
12d
12f
48³3 1
.
.
.
.
.
5 31³0 12
5 84³0 08
32³1 8
.
.
. .
5 82³0 11
7i
5 26³0 10
.
.
. .
6 01³0 06
. .
5 76³0 08
12d
12f
5 45³0 18
Values are the mean³s.e.m. of six mice.
.
.
5 14³0 12
.
.
. .
9 44³0 09
Atropine sulfate
8 89³0 05
Values are the mean³s.e.m. of three separate experiments performed
in duplicate.
inhibited the transit of a charcoal meal through the
mouse gastrointestinal tract (Table 4). Compounds 7f
and 12d were the most eŒective, reducing transit by
approximately 50% .
of isoprenalime (10 6 m)(compound 7f: from 24.5³2.24
We describe a novel series of 2-substituted thieno-
-
!
to 40.5³3.7, n ¯ 3, P 0.01; compound 7c: from pyrimidin-4-one derivatives, which display spasmolytic
!
24.5³2.24 to 39.5³2.3, n ¯ 3, P 0.01; compound activity through diŒerent mechanisms. The most active
!
12d: from 24.5³2.24 to 38.2³1.9, n ¯ 3, P 0.01).
The selective soluble guanylyl cyclase inhibitor ODQ dino nucleus in the thioalkyl chain and lack the sub-
(10 6 m) (Heibeiss & Kilbinger 1998), which blocked the stituent on the thiophene and aromatic ring, act at least
compounds, 7f and 12d, which have the terminal piperi-
-
}
relaxation evoked by 100 lm sodium nitroprusside in partially as M2 M3 muscarinic receptor antagonists.
the transmurally stimulated guinea-pig ileum, had no The involvement of these receptors in intestinal activity
eŒect on the spasmolytic action of the test compounds is well known (Ehlert et al 1999; Wallis & Napier 1999).
(data not shown). Compounds 1, 7f±i, 12d and 12f, as Spasmolytic activity may also involve diŒerent second
well as atropine sulfate, inhibited contractions induced messengers (Parker et al 1987; Young et al 1996). These
byacetylcholine, atropine being the most eŒective(Table compounds may possibly in¯ uence phosphodiesterase
2). IC50 values of test compounds were signi®cantly activity as well and, by raising cAMP levels, may con-
diŒerent compared with atropine sulfate (Table 2), and tribute to the inhibition of intestinal smooth muscle
these compounds also inhibited histamine-induced con- function (Parker et al 1987). Furthermore, they reduce
tractions of the guinea-pig ileum (Table 2). The potential histamine-induced contractions. Taken together, these
a nity of compounds 1, 7f±i, 12d and 12f towards results suggest that the spasmolytic action of these
muscarinic receptors was explored. We assayed their compounds was, at least partially, post-synaptic and
binding to the muscarinic M2 and M3 receptors, which they have only weak activity on muscarinic receptors.
are found in intestinal smooth muscle (Ehlert et al 1997), Therefore, an atropine-like action cannot be the primary
employing rat heart and submaxillary gland homo- explanation of their spasmolytic eŒect. However, func-
genates. These compounds displayed moderate a nity tional interactions among muscarinc receptors and other
for M3 sites as well as for M2 receptors (Table 3). relaxants such as isporenaline and forskolin, or com-
Compounds 7f and 12d were the most active. In dis- pounds that elicit contractions of the gastrointestinal
placement binding studies on guinea-pig ventricular smooth muscle (such as histamine) have been described
homogenates labelled with [3H]isradipine, these com- (Ehlert et al 1999).
pounds (up to 10 lm) did not bind to L-type calcium
In conclusion, the relaxant eŒects of compounds 1,
channels (data not shown), whereas the calcium channel 7f±i, 12d and 12f on visceral smooth muscle may be the
antagonist nifedipine was eŒective (Ki ¯ 1.9³0.05 nm, result of their interaction with muscarinic receptors,
n ¯ 3). besides being mediated by the cAMP cascade. Spas-
The relaxant activity displayed by the eight selected molytic compounds with a weak antimuscarinic activity
compounds was con®rmed in-vivo. The compounds, that seems to block smooth muscle contraction, through
1
-
administered intraperitoneally atthe dose of 50 mg kg , diŒerent mechanisms, have already been described

728
N. Alfredo Santagati et al
Melchiorre, C., Bolognesi, M. L., Chiarini, A., Minarini, A., Spam-
pinato, S. (1993) Synthesis and biological activity of some methoc-
tramine-related tetramines bearing a 11-acetyl-5,11-dihydro-6H-
(Choo et al 1986; Tonini et al 1987; Abbiati et al 1988;
Evangelista 1999). These drugs are used to treat several
gastrointestinal disorders and cause fewer unpleasant
side-eŒects than the antimuscarinic drugs (De Ponti &
Malagelada 1998; Wallis & Napier 1999). This study
con®rms that some 2-substituted thienopirimidin-4-one
derivatives may serve as novel spasmolytic agents to
treat disorders of smooth muscle function. Further
studies will be necessary to clarify their real therapeutic
potential.
.
pyrido[2,3-b][1 4]-benzodiazepin-6-one moiety as antimuscarinics:
a second generation of high selective M2 muscarinic receptor
J Med Chem
. 36: 3734±3737
antagonists.
.
.
Munson, P. J., Rodbard, D. (1980) Ligand: a versatile computerised
Anal
approach for characterization of ligand binding systems.
Biochem
.
. 107: 220±239
Nardi, D., Leonardi, A., Pennini, R., Tajana, A., Cazzulani, P., Testa,
R. (1993) New basic esters of 2-phenyl-2-methyl-4-oxo-4H-1-ben-
zopyran-8-carboxylic acid endowed with spasmolytic properties.
Arzneim Forsch
.
. 43: 28±34
Parker, I., Ito, Y., Kuriyama, H., Miledi, R. (1987) Beta-adrenergic
agonists and cyclic AMP decrease intracellular resting free-calcium
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}
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