Synthesis and binding properties of novel selective 5-HT3 receptor ligands

Article abstract of DOI:10.1016/j.bmc.2004.04.043

This work reports on the synthesis and affinities for the 5-HT₃ versus the 5-HT₄ receptor of new piperazinyl-substituted thienopyrimidine derivatives 20-45 with a view to identify potent and selective ligands for the 5-HT₃ receptor. Some of the new compounds show good affinity for the 5-HT₃ receptor and, notably, do not display any affinity for the 5-HT₄ receptor. 4-(4-Methyl-1-piperazinyl)-2- methylthio-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine 31 exhibits the highest affinity for the 5-HT₃ receptor (K_i=33nM) and behaves as noncompetitive antagonist.

Full text of DOI:10.1016/j.bmc.2004.04.043

Bioorganic & Medicinal Chemistry 12 (2004) 3891–3901
Synthesis and binding properties of novel selective
5-HT3 receptor ligands
Maria Modica,a,* Giuseppe Romeo,a Luisa Materia,a Filippo Russo,a Alfredo Cagnotto,b
b
Tiziana Mennini, Robert Gaspar, George Falkay and Ferenc Fulop
c
c
d
ꢀ
ꢀ ꢀ
€
€
aDipartimento di Scienze Farmaceutiche, Universitꢁa di Catania, viale A. Doria 6, 95125 Catania, Italy
bIstituto di Ricerche Farmacologiche ‘Mario Negri’, via Eritrea 62, 20157 Milano, Italy
cDepartment of Pharmacodynamics and Biopharmacy, University of Szeged, H-6701 Szeged, POB 121, Hungary
dInstitute of Pharmaceutical Chemistry, University of Szeged, H-6701 Szeged, POB 121, Hungary
Received 30 May 2003; accepted 30 April 2004
Available online 2 June 2004
Abstract—This work reports on the synthesis and affinities for the 5-HT3 versus the 5-HT4 receptor of new piperazinyl-substituted
thienopyrimidine derivatives 20–45 with a view to identify potent and selective ligands for the 5-HT3 receptor. Some of the new
compounds show good affinity for the 5-HT3 receptor and, notably, do not display any affinity for the 5-HT4 receptor. 4-(4-Methyl-
1-piperazinyl)-2-methylthio-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine 31 exhibits the highest affinity for the 5-HT3
receptor (Ki ¼ 33 nM) and behaves as noncompetitive antagonist.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
choline.5 Moreover, 5-HT3 receptors are involved in the
peripheral control of acethylcholine release of the distal
colon.6
The serotonin neurotransmitter is involved in a number
of different physiological functions through its interac-
tion with 14 types of receptors.1 These are G-protein-
coupled receptors, with the exception of the 5-HT3,
which is a ligand gated-ion channel receptor. 5-HT3
receptor antagonists, such as ondansetron, granisetron,
or tropisetron,2 are used as antiemetic drugs to prevent
vomiting associated with chemotherapy or radiation-
induced emesis, but literature studies3 indicate that they
could possess numerous other potential therapeutic
applications in the control of pain or in the treatment of
psychosis, memory impairment, depression, anxiety,
schizophrenia, and drugs abuse. On the other hand, little
is known about the therapeutic potential of 5-HT3
receptor agonists although some potent and selective
ligands with full agonistic properties4 were recently re-
ported. It has been suggested that stimulation of the 5-
HT3 receptor modulates, in the central nervous system,
the release of dopamine, cholecystokinin, and acetyl-
In recent years, we have been engaged in the preparation
of piperazinyl-substituted thieno[2,3-d]pyrimidin-4(3H)-
one derivatives as 5-HT3 receptor ligands.7 These com-
pounds possess the three key pharmacophoric elements
(an aromatic moiety, a hydrogen-bond acceptor and a
basic amino group) required for interaction with the 5-
HT3 receptor and are structurally related to quipazine
(Fig. 1), a potent ligand for the 5-HT3 receptor.8 Among
these thieno[2,3-d]pyrimidine derivatives, 3-amino-5,6-
dimethyl-2-[4-(1-phenylmethyl)-1-piperazinyl]thieno[2,3-
d]pyrimidin-4(3H)-one A (Fig. 1) exhibited the highest
affinity and selectivity for the 5-HT3 receptor (5-HT3:
Ki ¼ 3:92 nM; 5-HT4: not active), behaving as a full
agonist in the Bezold–Jarisch reflex assay.7 This result
induced us to continue research in this field with the aim
to obtain more potent and selective ligands for the 5-
HT3 receptor. The present work reports on the synthesis
and 5-HT3 receptor-binding properties of a series of new
derivatives B9;10 (Fig. 1), which can be regarded as
structural analogues of compound A. The new mole-
cules maintain the thieno[2,3-d]pyrimidine scaffold as in
A and exhibit the following structural variations: (i) the
piperazine moiety at 4-position of the pyrimidine
Keywords: 5-HT3 receptor; Ligands; Thieno[2,3-d]pyrimidine deriva-
tives.
* Corresponding author. Tel.: +39-095-7384010; fax: +39-095-222239;
e-mail: mmodica@mbox.unict.it
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.043

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M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
R4
N
N
N
N
H
N
N
R1
Quipazine
N
R2
S
R3
B
O
H3C
R1 , R2 = CH3 , CH3 ; -(CH2)3- ;
-CH(COOC2H5)CH2CH2- ;
-CH(COOC2H5)CH2CH2CH2- .
R3 = H , SCH3 .
NH2
N
H3C
S
N
N
R4 = CH2C6H5 , C6H4OCH3(o) , C6H5 , pyrimidyl , CH3 , H .
N
A
CH2C6H5
Figure 1.
nucleus in place of the carbonyl group; (ii) in some
compounds, 5- and 6-positions of the bicyclic system
bear methyl groups (as in A); in others, a trimethylene or
tetramethylene chain forms a third condensed ring,
which, in some cases, is substituted with an ethoxycar-
bonyl group; (iii) 2-position of the pyrimidine is un-
substituted or bears a methylthio group.
piperazines. The proposed structures for 3–12, 14–17,
and 20–45 were confirmed by elemental analyses, and by
the IR and H NMR data (see Experimental section).
1
3. Pharmacology
The groups on N-4 of the piperazine ring (e.g., benzyl,
2-methoxyphenyl, phenyl, 2-pyrimidyl, methyl, and
hydrogen) were the same as in a previous work.7 They
were examined, together with the other structural
modifications, with the aim to obtain more information
on the structure–activity relationships in this new series.
The title compounds 20–45 were tested in in vitro
binding assays to evaluate their affinity for the 5-HT3
and the 5-HT4 receptors, using [3H]LY 278584 or
[3H]GR113808 as radioligand, respectively. Binding
data, reported in Table 1, are expressed as Ki values.
Moreover, in order to evaluate the putative agonistic or
antagonistic properties of new molecules, two of them
(31 and 33) were tested in an in vitro functional assay on
isolated guinea pig distal colon.17 Results are reported in
Figure 2 and Table 2.
2. Chemistry
Compounds 20–45 were prepared according to Scheme
1. Derivatives 3 and 4 were obtained by refluxing in
acetone the corresponding b-amino esters of 4,5-disub-
stituted-thiophenes 1 and 211 with benzoyl isothiocya-
nate, commercially available or prepared in situ. When
refluxed in an ethanolic potassium hydroxide solution, 3
and 4 gave the respective monopotassium salts of the
4. Results and discussion
The results of the binding tests, presented in Table 1,
demonstrate that many of the title compounds (20, 24–
27, 31–33, 38, 39, and 44) possess good affinities for the
5-HT3 receptor and high selectivity over the 5-HT4
receptor. In fact, none of the new derivatives displays
measurable affinity for the latter receptor.
5,6-disubstituted-2-thioxothieno[2,3-d]pyrimidinones
5
and 6. On acidification of the potassium salts 5 and 6
with concentrated hydrochloric acid, the corresponding
thioxo compounds 8 and 9 were obtained, which con-
firmed the structures of salts 5 and 6. The 2-methylthio
derivatives 10–12 were obtained by reaction of the
potassium salts 5–712 with methyl iodide in water at
room temperature. The 4-chloro derivatives 14–18 were
obtained by heating the corresponding 4-oxo derivatives
10–12 and 1313 with an excess of phosphorus oxychlo-
ride, according to a literature method.14 The 4-(1-pip-
erazinyl)thieno[2,3-d]pyrimidine derivatives 20–45 were
synthesized by refluxing in ethanol the 4-chloro deriva-
tives 14–17, 18,15 and 1916 with piperazine or substituted
In this new series of derivatives the presence of the third
unsubstituted cyclopentane ring fused with the thi-
eno[2,3-d]pyrimidine system leads to the compounds
with the best affinities (31 and 33, Ki ¼ 33 and 70 nM,
respectively); the affinity is lower when two methyl
groups or a condensed ethoxycarbonyl cyclopentane are
present at 5- and 6-positions of the thieno[2,3-d]pyrim-
idine system (20, 24, 26, 38, and 39, Ki ¼ 723, 216, 114,
202, and 249 nM, respectively).

M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
3893
R1
R1
COOEt
NH2
COOEt
R1
R2
R3
a
-
1, 3, 5, 8, 10, 14 -CH(COOC2H5)CH2CH2
2, 4, 6, 9, 11, 15 -CH(COOC2H5)CH2CH2CH2
SCH3
SCH3
SCH3
H
SCH3
H
R2
NHCSNHCOC6H5
R2
S
S
-
-
7, 12, 16
-(CH2)3
3, 4
b
1, 2
-
17
18
19
-(CH2)3
CH3
CH3
CH3
CH3
R1
R1
R1
O
O
O
NH
NH
NH
c
R2
R2
d
R2
S
S- K+
S
S
N
5-7
N
S
N
SCH3
H
10-12
8, 9
e
R4
N
R1
O
N
Cl
NH
N
f
e
R1
N
R2
S
S
R3
N
N
R2
14-19
13
S
R3
N
20-45
Scheme 1. Reagents and conditions: (a) SCNCOC6H5, acetone, reflux; (b) KOH, EtOH, reflux; (c) HCl, H2O, rt; (d) CH3I, H2O, rt; (e) POCl3,
150 ꢁC; (f) unsubstituted or substituted piperazine, EtOH, K2CO3, reflux.
The best substituents on N-4 of the piperazine ring for
the affinity at the 5-HT3 receptor are a hydrogen and a
methyl group (24, 26, 31, 33, 38, and 39, Ki ¼ 216, 114,
33, 70, 202, and 249 nM, respectively), while the benzyl-
piperazine-substituted derivatives 20 and 27 display
decreased affinities (Ki ¼ 723and 421 nM, respectively).
The corresponding analogues 34 and 40, which bear an
ethoxycarbonyl group on a fused cyclopentane and a
cyclohexane ring at positions R1 and R2 of the thio-
phene nucleus, do not exhibit any measurable affinity
for this receptor.
which display lower affinities (Ki ¼ 341 and 221 nM,
respectively). Thus, the presence of the methylthio group
seems to be important for the affinity, but its mode of
interaction with the binding site is not clear.
This new series of thieno[2,3-d]pyrimidine derivatives
possesses the key elements of the three-component
pharmacophoric model proposed for the interaction of
5-HT3 ligands structurally related to quipazine with the
5-HT3 receptor-binding site:18 a charge-assisted hydro-
gen bond (N-4 of the piperazine nucleus), a hydrogen-
bond interaction (the nitrogen of the pyrimidine
system), and an aromatic interaction (the thiophene
nucleus). The location of the piperazine nucleus in a
different position in comparison with quipazine and our
previous derivatives7 could explain the lower affinities of
these new compounds.
The introduction on compounds 31 and 33 of an eth-
oxycarbonyl group on the cyclopentane ring, as in 38
and 39, is detrimental for affinity (Ki ¼ 202 and 249 nM,
respectively).
When the cyclopentane ring in 38 and 39 is enlarged to
a cyclohexane ring, as in 44 and 45, a notable drop in
affinity (Ki ¼ 1791 nM and not active, respectively) is
seen.
In this new series of derivatives, compounds with the
best affinities are characterized by an unsubstituted
piperazine or a methyl-substituted piperazine, a benzyl-
substituted piperazine leading to compounds with lower
affinity. This trend is peculiar for this series of deriva-
tives because, in the quipazine-like ligands,19 the intro-
duction of a benzyl moiety leads to compounds with
higher affinity for the 5-HT3 receptor with respect to the
unsubstituted piperazine derivatives.
Substitution on N-4 of the piperazine ring with a 2-
methoxyphenyl (21, 28, 35, and 41), a phenyl (22, 29, 36,
and 42) or a 2-pyrimidyl nucleus (23, 30, 37, and 43)
leads to compounds without affinity for the 5-HT3
receptor; this behavior was earlier reported for 3-amino-
5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one analogues7
in which the piperazine nucleus was at 2-position of the
pyrimidine system.
The presence of two methyl groups at 5- and 6-positions
of the thieno[2,3-d]pyrimidine system in this new series
of derivatives furnishes compounds with good affinity,
which is increased when a third cyclopentane ring is
fused with the thieno[2,3-d]pyrimidine system. On
Removal of the methylthio group at 2-position of the
pyrimidine nucleus in 24 and 31 leads to 25 and 32,

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M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
Table 1. Binding properties of compounds 20–45 on 5-HT3 and 5-HT4 serotonin receptors
R4
N
N
N
R1
N
R2
S
SCH3
Ki (nM) ( SD)
Compd
R1
R2
R4
5-HT3
[3H]LY 278584
Rat cortex
5-HT4
[3H]GR 113808
Guinea pig striatum
20
21
22
23
24
25a
26
27
28
29
30
31
32a
33
34
35
36
37
38
39
40
41
42
43
44
45
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2C6H5
C6H4OCH3(o)
C6H5
723 109
N.A.b
N.A. b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
N.A.b
79 10
N.A.b
2-Pyrimidyl
CH3
CH3
N.A.b
216 22
341 45.74
114 16
421 102
N.A.b
N.A.b
N.A.b
H
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
CH2C6H5
C6H4OCH3(o)
C6H5
2-Pyrimidyl
CH3
CH3
33
221 27.67
5
H
70 7
N.A.b
–CH(COOC2H5)CH2CH2–
–CH(COOC2H5)CH2CH2–
–CH(COOC2H5)CH2CH2–
–CH(COOC2H5)CH2CH2–
–CH(COOC2H5)CH2CH2–
–CH(COOC2H5)CH2CH2–
–CH(COOC2H5)CH2CH2CH2–
–CH(COOC2H5)CH2CH2CH2–
–CH(COOC2H5)CH2CH2CH2–
–CH(COOC2H5)CH2CH2CH2–
–CH(COOC2H5)CH2CH2CH2–
–CH(COOC2H5)CH2CH2CH2–
Serotonin
CH2C6H5
C6H4OCH3(o)
C6H5
N.A.b
N.A.b
2-Pyrimidyl
CH3
N.A.b
202 32
249 82
N.A.b
H
CH2C6H5
C6H4OCH3(o)
C6H5
N.A.b
N.A.b
2-Pyrimidyl
CH3
N.A.b
1791 187
N.A.b
354 97
H
a The methylthio group is replaced by a hydrogen.
b <50% inhibition at 10ꢀ5 M.
Table 2. Change of effective concentration 50 (EC50) and maximum
effect (Emax) in contraction increasing effect of 2-Me-5-HT in the
presence of 10 nM of tropisetron, compound 31 and 33 in the guinea
pig colon in vitro
140
120
100
80
2-Me-5-HT
2-Me-5-HT + trop
2-Me-5-HT + 31
2-Me-5-HT + 33
EC50 (M) SEMa
Emax (%) SEMa
60
2-Me-5-HT
1.2 · 10ꢀ6 1.6 · 10ꢀ7
145.4 22.0
40
2-Me-5-HT + trop 4.7 · 10ꢀ6 1.1 · 10ꢀ6b 145.8 11.3c
2-Me-5-HT + 31
2-Me-5-HT + 33
1.4 · 10ꢀ6 2.4 · 10ꢀ7c
2.0 · 10ꢀ6 3 .8· 10ꢀ7c
85.7 32.3d
79.5 17.0d
20
0
-5.5
10
-4.5
10
10-7.5
10-7.0
10-6.5
10-6.0
10-5.0
Level of significance is indicated next to the values compared to the 2-
2-Me-5-HT [M]
Me-5-HT values.
a Standard error mean.
b p < 0:01.
Figure 2. The effect of 10 nM of tropisetron (trop), compound 31 and
33 on the contraction increasing action of selective 5-HT3 agonist 2-
Me-5-HT in the isolated guinea pig colon in vitro. The effect of 2-Me-
5-HT was expressed as the percent of contraction increase compared to
basal colon activity (n ¼ 6).
c Not significant.
d p < 0:05.
bonyl group are detrimental for affinity at 5-HT3
receptor, leading to very poor ligands or inactive com-
pounds.
the other hand, the enlargement of the fused ring to
six atoms as well as the introduction of an ethoxycar-

M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
3895
Compounds 31 and 33, which showed the highest
affinities for the 5-HT3 receptor, were also tested in vitro
to evaluate their functional activities. Tropisetron
(trop), a well-known 5-HT3 antagonist, was used as
reference compound. In the isolated guinea pig colon
tropisetron (10 nM) shifted the dose response curve of
the selective 5-HT3 agonist 2-methyl-5-hydroxytrypt-
amine (2-Me-5-HT) to the right, indicating its competi-
tive antagonist properties (Fig. 2, Table 2). Compounds
31 and 33 (10 nM), however, did not cause any shift in
the dose response curve, but elicited a decrease in
maximum effect of 2-Me-5-HT, suggesting their char-
acter as noncompetitive antagonists. This result would
suggest the possibility of an interaction of these com-
pounds with a postulated modulatory binding site at the
5-HT3 receptor.20
recrystallized from ethanol. Yield: 1.25 g (40%); mp
184 ꢁC; IR (KBr, cmꢀ1) 3261, 2971, 2942, 1712, 1681,
1564, 1534, 1334, 1219, 1185, 705. 1H NMR (DMSO-d6)
d 1.16 (t, J ¼ 7 Hz, 3H), 1.26 (t, J ¼ 7 Hz, 3H), 2.25–
2.42 (m, 1H), 2.66–3.06 (m, 3H), 3.92–4.40 (m, 5H),
7.45–8.12 (m, 5H), 11.91 (s, 1H), 14.71 (s, 1H). Anal.
C21H22N2O5S2.
5.2. 2-[(Benzoylaminothioxomethyl)amino]-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3,4-dicarboxylic acid diethyl
ester (4)
This was prepared from the amino ester 2 by the
same procedure as for 3, and was recrystallized from
ethanol. Yield: 2.07 g (64%); mp 170–171 ꢁC; IR (KBr
cmꢀ1) 3248, 2927, 1710, 1683, 1526, 1436, 1330, 1225,
1184, 1028, 708. 1H NMR (DMSO-d6) d 1.17 (t,
J ¼ 7:2 Hz, 3H), 1.28 (t, J ¼ 7:2 Hz, 3H), 1.55–2.18 (m,
4H), 2.61–2.85 (m, 2H), 3.90–4.48 (m, 5H), 7.50–8.18
(m, 5H), 11.90 (s, 1H), 14.74 (s, 1H). Anal. C22H24-
N2O5S2.
In conclusion, a new series of thieno[2,3-d]pyrimidine
derivatives as 5-HT3 receptor ligands has been prepared.
Some compounds, namely 31 and 33, showed good
affinity for the 5-HT3 receptor coupled to a complete
lack of affinity for the 5-HT4 receptor. Moreover, results
of in vitro functional assays indicate that 31 and 33,
unlike tropisetron, could be reported as noncompetitive
antagonists. Further studies to better clarify the phar-
macological profile of 31 and 33 are in progress.
5.3. Monopotassium salt of ethyl 4-oxo-2-thioxo-3,5,6,7-
tetrahydro-4H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-5-
carboxylate (5) and its thioxo derivative (8)
Benzoyl derivative 3 (1 g, 2.24 mmol) was added to a
solution of KOH (0.25 g, 4.45 mmol) in absolute ethanol
(13mL) and the mixture was refluxed during stirring for
3h. The solid was then collected while hot, washed with
hot absolute ethanol and dried to obtain salt 5 (0.60 g,
80%). Potassium salt 5 (0.50 g, 1.49 mmol) was sus-
pended in water (50 mL), acidified with concentrated
hydrochloric acid, and stirred for 10 min at room tem-
perature. The solid was collected, washed with water,
dried, and recrystallized from ethanol. Yield: 0.17 g
(39%); mp 260 ꢁC (dec); IR (KBr cmꢀ1) 3430, 3068,
5. Experimental section
Melting points were determined in open capillary tubes
on a Gallenkamp M.p. apparatus and are uncorrected.
Elemental analyses for C, H, N, and S were performed
on a Fisons-Carlo Erba EA1108 Elemental Analyzer
and were within 0.4% of the theoretical values. The IR
spectra were recorded in KBr disks on a Perkin Elmer
1
1600 Series FT-IR spectrometer. H NMR spectra were
1
2898, 1718, 1668, 1548, 1376, 1202, 1157, 1021, 849. H
recorded in DMSO-d6 solution at 200 MHz on a Varian
Inova-Unity 200 spectrometer; chemical shifts (d) are
reported in ppm, with TMS as internal standard; cou-
pling constants (J) are in Hertz. Signal multiplicities are
denoted by s (singlet), d (doublet), t (triplet), q (quartet),
br s (broad singlet), or m (multiplet). The purity of
compounds was checked by TLC on Merck silica gel 60
F-254 plates. All commercial chemicals were purchased
from Aldrich, Fluka, Merck, and Carlo Erba and were
used without further purification.
NMR (DMSO-d6) d 1.15 (t, J ¼ 7:2 Hz, 3H), 2.27–2.47
(m, 1H), 2.69–3.04 (m, 3H), 3.90–4.21 (m, 3H), 12.37 (s,
1H), 13.40 (br s, 1H). Anal. C12H12N2O3S2.
5.4. Monopotassium salt of ethyl 4-oxo-2-thioxo-3,4,5,
6,7,8-hexahydro[1]benzothieno[2,3-d]pyrimidine-5-car-
boxylate (6) and its thioxo derivative (9)
Benzoyl derivative 4 (3g, 6.51 mmol) was added to a
solution of KOH (0.77 g, 13.72 mmol) in absolute etha-
nol (30 mL) and the mixture was refluxed during stirring
for 6 h. After cooling, a small amount of the solvent was
removed from the suspension under reduced pressure,
and the solid was then collected, washed with a small
amount of absolute ethanol, and dried to give salt 6
(2.22 g, 98%). A suspension of salt 6 (1 g, 2.87 mmol) in
water (50 mL) was acidified with concentrated hydro-
chloric acid and stirred for 1 h at room temperature. The
solid was collected, washed with water, dried, and
recrystallized from ethanol. Yield: 0.55 g (62%); mp
234–236 ꢁC; IR (KBr cmꢀ1) 3442, 3150, 3052, 1694,
1665, 1546, 1473, 1291, 1199, 1030, 867. 1H NMR
5.1. 2-[(Benzoylaminothioxomethyl)amino]-5,6-dihydro-
4H-cyclopenta[b]thiophene-3,4-dicarboxylic acid diethyl
ester (3)
A mixture of NH4NCS (0.64 g, 8.41 mmol) and benzoyl
chloride (0.82 mL, 7.06 mmol) was refluxed in anhy-
drous acetone (10 mL) for 5 min. A solution of amino-
ester 1 (2 g, 7.06 mmol) in anhydrous acetone (25 mL)
was added to the suspension and the mixture was ref-
luxed during stirring for 1 h. After cooling, the suspen-
sion was concentrated under reduced pressure, and the
precipitate was collected, washed with water, dried, and

3896
M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
(DMSO-d6) d 1.16 (t, J ¼ 7 Hz, 3H), 1.59–2.08 (m,
4H), 2.60–2.81 (m, 2H), 3.85–3.97 (m, 1H), 4.05 (q,
J ¼ 7 Hz, 2H), 12.34 (s, 1H), 13.36 (s, 1H). Anal.
C13H14N2O3S2.
5.9. Ethyl 4-chloro-2-methylthio-5,6,7,8-tetrahydro[1]-
benzothieno[2,3-d]pyrimidine-5-carboxylate (15)
This was prepared from methylthio derivative 11 by the
same procedure as for 14 and was recrystallized from
ethanol. Yield: 0.40 g (72%); mp 106–108 ꢁC; IR (KBr
cmꢀ1) 2932, 1724, 1538, 1475, 1389, 1327, 1275, 1228,
1157, 1121, 839. 1H NMR (DMSO-d6) d 1.16 (t,
J ¼ 7:2 Hz, 3H), 1.47–2.30 (m, 4H), 2.57 (s, 3H), 2.70–
3.03 (m, 2H), 3.95 (q, J ¼ 7:2 Hz, 2H), 4.19–4.30 (m,
1H). Anal. C14H15ClN2O2S2.
5.5. Ethyl 2-methylthio-4-oxo-3,5,6,7-tetrahydro-4H-
cyclopenta[4,5]thieno[2,3-d]pyrimidine-5-carboxylate (10)
Methyl iodide (0.74 mL, 11.83mmol) was added under
stirring to a solution of monopotassium salt 5 (1.33 g,
3.98 mmol) in water (70 mL) and the mixture was stirred
at room temperature for 1 h. The solid was then filtered
off, washed with water, dried, and recrystallized from
ethanol. Yield: 0.75 g (61%); mp 198–200 ꢁC; IR (KBr
cmꢀ1) 3066, 2968, 2940, 2803, 1733, 1652, 1565, 1315,
1178, 1015, 643. 1H NMR (DMSO-d6) d 1.10 (t,
J ¼ 7:2 Hz, 3H), 2.22–2.42 (m, 1H), 2.47 (s, 3H), 2.63–
3.05 (m, 3H), 3.91–4.10 (m, 3H), 12.62 (s, 1H). Anal.
C13H14N2O3S2.
5.10. 4-Chloro-2-methylthio-6,7-dihydro-5H-cyclopenta[4,5]-
thieno[2,3-d]pyrimidine (16)
This was prepared from methylthio derivative 12 by the
same procedure as for 14 and was recrystallized from
ethanol. Yield: 0.32 g (78%); mp 115–116 ꢁC (dec); IR
(KBr cmꢀ1) 2920, 2853, 1554, 1468, 1412, 1301, 1251,
1182, 1141, 1015, 819. 1H NMR (DMSO-d6) d 2.34–2.55
(m, 2H), 2.58 (s, 3H), 2.96–3.12 (m, 4H). Anal.
C10H9ClN2S2.
5.6. Ethyl 2-methylthio-4-oxo-3,4,5,6,7,8-hexahydro-
[1]benzothieno[2,3-d]pyrimidine-5-carboxylate (11)
This was prepared from potassium salt 6 by the same
procedure as for 10 and was recrystallized from ethanol.
5.11. General procedure for the synthesis of 2-methylthio-
(4-substituted-1-piperazinyl)thieno[2,3-d]pyrimidine
derivatives 20–22, 24, 25, 27–29, 31, 32, 34–36, 38, 40–42,
and 44
Yield: 0.67 g (52%); mp 224–226 ꢁC; IR (KBr cmꢀ1
3049, 2981, 1726, 1662, 1546, 1263, 1194, 1062, 1024,
)
1
857, 637. H NMR (DMSO-d6) d 1.16 (t, J ¼ 7:2 Hz,
3H), 1.61–2.15 (m, 4H), 2.53 (s, 3H), 2.67–2.91 (m,
2H), 3.92–4.26 (m, 3H), 12.62 (s, 1H). Anal. C14H16-
N2O3S2.
A mixture of the appropriate chloro derivatives 14-19
(2.05 mmol) and methyl, 2-methoxyphenyl, phenyl, or
benzylpiperazine (4.10 mmol) was refluxed in absolute
ethanol (20 mL) during stirring for 1–2 h. After cooling,
a small amount of the solvent was removed under re-
duced pressure, water was added to the mixture, and
after 1 or 2 days the solid product was collected, washed
with water, dried, and recrystallized. For compound 21,
after cooling, a solid was obtained that was collected,
washed with water, dried, and recrystallized.
5.7. 2-Methylthio-3,5,6,7-tetrahydro-4H-cyclopenta-
[4,5]thieno[2,3-d]pyrimidin-4-one (12)
This was prepared from potassium salt 7 by the same
procedure as for 10 and was recrystallized from ethanol/
dioxane. Yield: 0.37 g (39%); mp 182–183 ꢁC (dec); IR
(KBr cmꢀ1) 2917, 2847, 1665, 1552, 1408, 1304, 1270,
1
1191, 1009, 825, 640. H NMR (DMSO-d6) d 2.42–2.59
(m, 2H), 2.65 (s, 3H), 2.92–3.07 (m, 4H), 12.80 (s, 1H).
Anal. C10H10N2OS2.
5.12. 5,6-Dimethyl-2-methylthio-4-[4-(1-phenylmethyl)-1-
piperazinyl]thieno[2,3-d]pyrimidine (20)
This was recrystallized from ethanol. Yield: 0.28 g
(35%); mp 99–101 ꢁC; IR (KBr cmꢀ1) 2824, 1548, 1498,
1424, 1355, 1250, 1132, 990, 853, 745, 670. H NMR
(DMSO-d6) d 2.32 (s, 3H), 2.37 (s, 3H), 2.46–2.59 (s+m,
3H+4H), 3.29–3.40 (m, 4H), 3.52 (s, 2H), 7.20–7.40 (m,
5H). Anal. C20H24N4S2.
1
5.8. Ethyl 4-chloro-2-methylthio-6,7-dihydro-5H-cyclopenta-
[4,5]thieno[2,3-d]pyrimidine-5-carboxylate (14)
Phosphorus oxychloride (2.5 mL, 26.82 mmol) was
added to 10 (0.50 g, 1.61 mmol) and the suspension was
heated at 150 ꢁC for 40 min during stirring. After cool-
ing, the suspension was poured into cold water and
neutralized with 10% NaOH solution. The sticky residue
was collected after 1 day, washed with water, dried, and
recrystallized from cyclohexane. Yield: 0.10 g (19%); mp
77–79 ꢁC; IR (KBr cmꢀ1) 2968, 2924, 1729, 1558, 1475,
1409, 1265, 1235, 1143, 1047, 825. 1H NMR (DMSO-d6)
d 1.16 (t, J ¼ 7:2 Hz, 3H), 2.41–2.55 (m, 1H), 2.57 (s,
3H), 2.78–3.20 (m, 3H), 4.05–4.29 (m, 3H). Anal.
C13H13ClN2O2S2.
5.13. 5,6-Dimethyl-2-methylthio-4-[4-(2-methoxyphenyl)-
1-piperazinyl]thieno[2,3-d]pyrimidine (21)
This was recrystallized from ethanol. Yield: 0.54 g
(66%); mp 123–125 ꢁC; IR (KBr cmꢀ1) 2816, 1546, 1498,
1451, 1359, 1232, 1128, 1023, 986, 852, 744. H NMR
1
(DMSO-d6) d 2.39 (s, 6H), 2.52 (s, 3H), 3.08–3.20 (m,
4H), 3.43–3.58 (m, 4H), 3.80 (s, 3H), 6.84–7.05 (m, 4H).
Anal. C20H24N4OS2.

M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
3897
5.14. 5,6-Dimethyl-2-methylthio-4-(4-phenyl-1-piperazinyl)-
thieno[2,3-d]pyrimidine (22)
(DMSO-d6) d 2.22 (s, 3H), 2.26–2.53 (m, 9H), 2.86–
3.04 (m, 4H), 3.48–3.58 (m, 4H). Anal. C15H20N4S2Æ3/
2H2O.
This was recrystallized from ethanol. Yield: 0.39 g
(51%); mp 138–140 ꢁC; IR (KBr cmꢀ1) 2817, 1594, 1497,
1523, 1367, 1227, 1134, 983, 848, 761, 693. H NMR
(DMSO-d6) d 2.39 (s, 3H), 2.40 (s, 3H), 2.52 (s, 3H),
3.25–3.38 (m, 4H), 3.42–3.55 (m, 4H), 6.76–7.31 (m,
5H). Anal. C19H22N4S2.
1
5.20. Ethyl 2-methylthio-4-[4-(1-phenylmethyl)-1-piperaz-
inyl]-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrim-
idine-5-carboxylate (34)
This was recrystallized from ethanol. Yield: 0.40 g
(42%); mp 126–128 ꢁC; IR (KBr cmꢀ1) 2932, 2811, 1729,
1550, 1493, 1445, 1318, 1171, 1130, 980, 733. H NMR
(DMSO-d6) d 1.13(t, J ¼ 7 Hz, 3H), 2.30–2.64 (m, 8H),
2.70–3.10 (m, 3H), 3.25–3.70 (s+m, 2H+4H), 4.03 (q,
J ¼ 7 Hz, 2H), 4.22–4.34 (m, 1H) 7.20–7.42 (m, 5H).
Anal. C24H28N4O2S2.
1
5.15. 5,6-Dimethyl-4-(4-methyl-1-piperazinyl)-2-methyl-
thiothieno[2,3-d]pyrimidine (24)
This was recrystallized from ethanol. Yield: 0.11 g
(18%); mp 99–100 ꢁC; IR (KBr cmꢀ1) 2921, 2845, 2792,
1499, 1449, 1409, 1357, 1252, 1136, 992, 856. H NMR
1
(DMSO-d6) d 2.23 (s, 3H), 2.34 (s, 3H), 2.38 (s, 3H),
2.45–2.61 (s+m, 3H+4H), 3.29–3.45 (m, 4H). Anal.
C14H20N4S2.
5.21. Ethyl 4-[4-(2-methoxyphenyl)-1-piperazinyl]-2-methyl-
thio-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-
5-carboxylate (35)
5.16. 2-Methylthio-4-[4-(1-phenylmethyl)-1-piperazinyl]-
6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine
(27)
This was recrystallized from ethanol. Yield: 0.66 g
(67%); mp 128–130 ꢁC; IR (KBr cmꢀ1) 2918, 2828, 1718,
1496, 1449, 1364, 1237, 1142, 1033, 990, 753. H NMR
1
(DMSO-d6) d 1.11 (t, J ¼ 7 Hz, 3H), 2.32–2.60 (m, 4H)
2.70–3.28 (m, 7H), 3.35–3.60 (m, 2H), 3.70–3.90 (m+s,
2H+3H), 4.04 (q, J ¼ 7 Hz, 2H), 4.30–4.42 (m, 1H),
6.83–7.08 (m, 4H). Anal. C24H28N4O3S2.
This was recrystallized from ethanol. Yield: 0.36 g
(44%); mp 105–107 ꢁC; IR (KBr cmꢀ1) 2927, 2814, 2711,
1564, 1445, 1394, 1301, 1170, 911, 742, 699. H NMR
1
(DMSO-d6) d 2.21–2.41 (m, 2H), 2.41–2.59 (s+m,
3H+4H), 2.83–3.00 (m, 4H), 3.40–3.62 (m, 6H), 7.21–
7.40 (m, 5H). Anal. C21H24N4S2.
5.22. Ethyl 2-methylthio-4-(4-phenyl-1-piperazinyl)-6,7-
dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-5-car-
boxylate (36)
5.17. 4-[4-(2-Methoxyphenyl)-1-piperazinyl]-2-methylthio-
6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine
(28)
This was recrystallized from ethanol/water. Yield: 0.20 g
(21%); mp 142–144 ꢁC; IR (KBr cmꢀ1) 2921, 2841, 1728,
1598, 1534, 1496, 1368, 1231, 1155, 987, 757. H NMR
1
This was recrystallized from ethanol. Yield: 0.44 g
(52%); mp 133–135 ꢁC; IR (KBr cmꢀ1) 2915, 2840, 1584,
1446, 1497, 1364, 1230, 1148, 988, 826, 750. H NMR
(DMSO-d6) d 2.28–2.46 (m, 2H), 2.51 (s, 3H), 2.88–3.16
(m, 8H), 3.66–3.76 (m, 4H), 3.80 (s, 3H), 6.81–7.10 (m,
4H). Anal. C21H24N4OS2.
(DMSO-d6) d 1.05 (t, J ¼ 7 Hz, 3H), 2.30–2.62 (m, 4H),
2.72–2.98 (m, 1H), 2.99–3.28 (m, 4H), 3.32–3.60 (m,
4H), 3.68–3.84 (m, 2H), 4.01 (q, J ¼ 7 Hz, 2H), 4.28–
4.42 (m, 1H), 6.78–7.32 (m, 5H). Anal. C23H26N4O2S2.
1
5.23. Ethyl 4-(4-methyl-1-piperazinyl)-2-methylthio-6,7-
dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-5-car-
boxylate (38)
5.18. 2-Methylthio-4-(4-phenyl-1-piperazinyl)-6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine (29)
This was recrystallized from ethanol. Yield: 0.31 g
(39%); mp 134–136 ꢁC; IR (KBr cmꢀ1) 2975, 2924, 2832,
2789, 1734, 1537, 1491, 1397, 1170, 1136, 986. 1H NMR
(DMSO-d6) d 1.14 (t, J ¼ 7 Hz, 3H), 2.20 (s, 3H), 2.25–
2.58 (m, 8H), 2.71–2.95 (m, 1H), 2.95–3.10 (m, 2H),
3.25–3.41 (m, 2H), 3.54–3.71 (m, 2H), 4.04 (q, J ¼ 7 Hz,
2H), 4.23–4.39 (m, 1H). Anal. C18H24N4O2S2.
This was recrystallized from ethanol. Yield: 0.26 g
(33%); mp 145–146 ꢁC; IR (KBr cmꢀ1) 2923, 2845, 1595,
1492, 1441, 1363, 1232, 1147, 988, 753, 688. H NMR
(DMSO-d6) d 2.28–2.46 (m, 2H), 2.51 (s, 3H), 2.88–3.10
(m, 4H), 3.22–3.33 (m, 4H), 3.62–3.76 (m, 4H), 6.72–
7.35 (m, 5H). Anal. C20H22N4S2.
1
5.19. 4-(4-Methyl-1-piperazinyl)-2-methylthio-6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine (31)
5.24. Ethyl 2-methylthio-4-[4-(1-phenylmethyl)-1-piper-
azinyl]-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine-
5-carboxylate (40)
This was recrystallized from ethanol/water. Yield: 0.17 g
(24%); mp 127–129 ꢁC; IR (KBr cmꢀ1) 2920, 2847, 2794,
1540, 1495, 1449, 1387, 1268, 1143, 992, 824. H NMR
This was recrystallized from ethanol. Yield: 0.80 g
(81%); mp 140–141 ꢁC; IR (KBr cmꢀ1) 2922, 2810, 1729,
1

3898
M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
1
1536, 1499, 1399, 1312, 1171, 1126, 984, 732. H NMR
(DMSO-d6) d 1.12 (t, J ¼ 7:2 Hz, 3H), 1.61–1.88 (m,
3H), 2.18–2.45 (m, 3H), 2.50 (s, 3H), 2.55–2.75 (m, 2H),
2.78–2.90 (m, 2H), 2.97–3.15 (m, 2H), 3.30–3.50 (m,
2H), 3.51 (s, 2H, CH2), 3.82–4.18 (m, 3H), 7.10–7.41 (m,
5H). Anal. C25H30N4O2S2.
5.29. 2-Methylthio-4-[4-(2-pyrimidyl)-1-piperazinyl]-6,7-
dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine (30)
It was prepared from chloro derivative 16 in the same
manner as for 23, but the refluxing time was 3h. The
compound was recrystallized from ethanol. Yield: 0.61 g
(78%); mp 110–111 ꢁC; IR (KBr cmꢀ1) 2846, 1588, 1544,
1
1489, 1440, 1352, 1292, 1253, 1147, 983, 794. H NMR
(DMSO-d6) d 2.29–2.46 (m, 2H), 2.51 (s, 3H), 2.89–3.14
(m, 4H), 3.60–3.68 (m, 4H), 3.84–3.94 (m, 4H), 6.69 (t,
J ¼ 4:6 Hz, 1H), 8.33 (d, J ¼ 4:6 Hz, 2H). Anal.
C18H20N6S2.
5.25. Ethyl 4-[4-(2-methoxyphenyl)-1-piperazinyl]-2-methyl-
thio-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine-5-
carboxylate (41)
This was recrystallized from ethanol. Yield: 0.71 g
(70%); mp 152–154 ꢁC; IR (KBr cmꢀ1) 2930, 2838, 1727,
1536, 1497, 1451, 1372, 1234, 1164, 999, 745. H NMR
(DMSO-d6) d 1.10 (t, J ¼ 7 Hz, 3H), 1.63–1.98 (m, 3H),
2.20–2.35 (m, 1H), 2.54 (s, 3H), 2.80–3.05 (m, 4H), 3.12–
3.31 (m, 4H), 3.45–3.65 (m, 2H), 3.79 (s, 3H), 3.87–4.18
(m, 3H), 6.85–7.05 (m, 4H). Anal. C25H30N4O3S2.
1
5.30. 5,6-Dimethyl-4-(4-methyl-1-piperazinyl)thieno[2,3-
d]pyrimidine (25)
A mixture of chloro derivative 19 (0.48 g, 2.42 mmol)
and 1-methylpiperazine (0.54 mL, 4.87 mmol) was ref-
luxed during stirring for 4 h in absolute ethanol (20 mL).
After cooling, water was added, and the mixture was
extracted with chloroform. The combined organic layer
was dried (Na2SO4) and evaporated under reduced
pressure. After the addition of diethyl ether, the product
solidified, and was recrystallized from ethanol/water.
Yield: 0.33 g (52%); mp 73–75 ꢁC; IR (KBr cmꢀ1) 2920,
2839, 2793, 1533, 1502, 1433, 1366, 1285, 1258, 1137,
5.26. Ethyl 2-methylthio-4-(4-phenyl-1-piperazinyl)-5,6,7,8-
tetrahydro[1]benzothieno[2,3-d]pyrimidine-5-carboxylate
(42)
This was recrystallized from ethanol. Yield: 0.50 g
(52%); mp 128–130 ꢁC; IR (KBr cmꢀ1) 2970, 2837, 1737,
1597, 1499, 1374, 1233, 1168, 984, 760, 690. H NMR
1
977. H NMR (DMSO-d6) d 2.18 (s, 3H), 2.39 (s, 3H),
2.43 (s, 3H), 2.45–2.67 (m, 4H), 3.25–3.39 (m, 4H), 8.47
(s, 1H). Anal. C13H18N4S.
1
(DMSO-d6) d 1.03(t, J ¼ 7:2 Hz, 3H), 1.62–1.98 (m,
3H), 2.20–2.35 (m, 1H), 2.54 (s, 3H), 2.80–2.92 (m, 2H),
3.05–3.30 (m, 4H), 3.30–3.60 (m, 4H), 3.87–4.18 (m,
3H), 6.78–7.32 (m, 5H). Anal. C24H28N4O2S2.
5.31. 4-(4-Methyl-1-piperazinyl)-6,7-dihydro-5H-cyclopenta-
[4,5]thieno[2,3-d]pyrimidine (32)
5.27. Ethyl 4-(4-methyl-1-piperazinyl)-2-methylthio-
5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine-5-car-
boxylate (44)
It was obtained from chloro derivative 17 by the same
procedure as for 25. Chloro derivative 17 was prepared
from 13 (0.56 g, 2.93mmol) and phosphorus oxychloride
(3mL, 32.18 mmol), the suspension being heated at
150 ꢁC during stirring for 2 h. After cooling, the sus-
pension was poured into cold water and neutralized with
10% NaOH solution. The precipitate was collected,
washed with water, dried, and used for the next step
without further purification; yield: 0.35 g (57%).
This was recrystallized from ethanol/water. Yield: 0.33 g
(40%); mp 129–130 ꢁC; IR (KBr cmꢀ1) 2924, 2837, 1734,
1560, 1529, 1400, 1334, 1172, 988, 857, 772. H NMR
1
(DMSO-d6) d 1.13(t, J ¼ 7:2 Hz, 3H), 1.62–1.94 (m,
3H), 2.15–2.38 (m, 6H), 2.47–2.62 (m, 5H), 2.76–2.88
(m, 2H), 2.95–3.12 (m, 2H), 3.30–3.49 (m, 2H), 3.87–
4.15 (m, 3H). Anal. C19H26N4O2S2.
Compound 32 was recrystallized from ethanol/water.
Yield: 0.30 g (44%); mp 88–91 ꢁC; IR (KBr cmꢀ1) 2936,
2848, 2795, 1545, 1496, 1444, 1363, 1290, 1262, 1142,
1
5.28. 5,6-Dimethyl-2-methylthio-4-[4-(2-pyrimidyl)-1-pip-
erazinyl]thieno[2,3-d]pyrimidine (23)
983. H NMR (DMSO-d6) d 2.10–2.60 (m, 9H), 2.95–
3.18 (m, 4H), 3.40–3.70 (m, 4H), 8.45 (s, 1H). Anal.
C14H18N4SÆ1/2H2O.
A mixture of chloro derivative 18 (0.50 g, 2.04 mmol), 1-
(2-pyrimidyl)piperazine dihydrochloride (0.53g, 2.23
mmol), and potassium carbonate (0.56 g, 4.05 mmol)
was refluxed in absolute ethanol (20 mL) during stirring
for 40 min. After cooling, the precipitate was collected,
washed with water, dried, and recrystallized from etha-
5.32. 5,6-Dimethyl-2-methylthio-4-(1-piperazinyl)thieno-
[2,3-d]pyrimidine (26)
A mixture of chloro derivative 18 (0.60 g, 2.45 mmol)
and piperazine (1.06 g, 12.30 mmol) was refluxed during
stirring for 3h in absolute ethanol (20 mL). After cool-
ing, the solid was eliminated by filtration, and the
solution was diluted with water and extracted with
chloroform, and the combined organic layer was dried
(Na2SO4) and evaporated under reduced pressure. After
nol. Yield: 0.42 g (55%); mp 109–110 ꢁC; IR (KBr cmꢀ1
)
2848, 1585, 1548, 1497, 1446, 1352, 1249, 1139, 976, 860,
1
778. H NMR (DMSO-d6) d 2.40 (s, 6H), 2.51 (s, 3H),
3.39–3.47 (m, 4H), 3.83–3.97 (m, 4H), 6.68 (t,
J ¼ 4:8 Hz, 1H), 8.40 (d, J ¼ 4:8 Hz, 2H). Anal.
C17H20N6S2.

M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
3899
the addition of diethyl ether to the yellow oil, a solid
compound was obtained, which was recrystallized from
ethanol/water. Yield: 0.22 g (27%); mp 119–120 ꢁC; IR
(KBr cmꢀ1) 3302, 2914, 2806, 1502, 1419, 1354, 1255,
1204, 1125, 990, 852. H NMR (DMSO-d6) d 2.33 (s,
3H), 2.37 (s, 3H), 2.50 (s, 3H), 2.78–2.80 (m, 4H), 3.12–
3.38 (m, 4H). Anal. C13H18N4O2S2.
J ¼ 7:2 Hz, 2H), 4.25–4.35 (m, 1H). Anal.
C17H22N4O2S2.
1
5.36. Ethyl 2-methylthio-4-[4-(2-pyrimidyl)-1-piperazinyl]-
5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidine-5-car-
boxylate (43)
A mixture of chloro derivative 15 (0.70 g, 2.04 mmol), 1-
(2-pyrimidyl)piperazine dihydrochloride (0.53g, 2.23
mmol), and potassium carbonate (0.56 g, 4.05 mmol)
was refluxed in absolute ethanol (20 mL) during stirring
for 6 h. After cooling, the precipitate was removed by
filtration and water was added to the solution. The
mixture was then extracted with chloroform, and the
combined organic layer was dried (Na2SO4) and evap-
orated under reduced pressure. The sticky residue was
purified by flash column chromatography (chloroform)
and was solidified by the addition of diethyl ether and
petroleum ether to the yellow oil. Yield: 0.31 g (32%);
mp 114–116 ꢁC; IR (KBr cmꢀ1) 2924, 1719, 1586, 1547,
1480, 1419, 1383, 1358, 1229, 1168, 977. 1H NMR
(DMSO-d6) d 1.03(t, J ¼ 7 Hz, 3H), 1.62–2.00 (m, 3H),
2.18–2.28 (m, 1H), 2.53 (s, 3H), 2.80–2.97 (m, 2H), 3.02–
3.20 (m, 2H), 3.30–3.58 (m, 2H), 3.62–3.80 (m, 2H),
3.90–4.10 (m, 5H), 6.69 (t, J ¼ 4:6 Hz, 1H), 8.33 (d,
J ¼ 4:6 Hz, 2H). Anal. C22H26N6O2S2.
5.33. 2-Methylthio-4-(1-piperazinyl)-6,7-dihydro-5H-cyclo-
penta[4,5]thieno[2,3-d]pyrimidine (33)
A mixture of chloro derivative 16 (0.70 g, 2.73mmol)
and piperazine (1.17 g, 13.58 mmol) was refluxed in
absolute ethanol (20 mL) during stirring for 3h. After
cooling, water was added to the solution to obtain a
solid product, which was recrystallized from ethanol.
Yield: 0.63g (75%); mp 98–100 ꢁC; IR (KBr cmꢀ1) 3293,
2841, 1519, 1500, 1446, 1384, 1289, 1205, 1134, 988, 826.
1H NMR (DMSO-d6) d 2.26–2.44 (m, 2H), 2.49 (s, 3H),
2.76–3.04 (m, 8H), 3.41–3.52 (m, 4H). Anal.
C14H18N4S2.
5.34. Ethyl 2-methylthio-4-[4-(2-pyrimidyl)-1-piperazinyl]-
6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-5-
carboxylate (37)
A mixture of chloro derivative 14 (1.80 g, 5.47 mmol),
(2.60 g,
1-(2-pyrimidyl)piperazine
dihydrochloride
5.37. Ethyl 2-methylthio-4-(1-piperazinyl)-5,6,7,8-tetra-
hydro[1]benzothieno[2,3-d]pyrimidine-5-carboxylate (45)
10.96 mmol), and potassium carbonate (2.55 g,
18.45 mmol) was refluxed in absolute ethanol (50 mL)
during stirring for 1 h. After cooling, the solid was
eliminated by filtration and water was added to the
solution. The precipitated product was filtered off,
washed with water, and recrystallized from ethanol/
water. Yield: 1.50 g (60%); mp 138–140 ꢁC; IR (KBr
cmꢀ1) 2973, 2858, 1725, 1583, 1546, 1486, 1451, 1355,
1254, 1143, 979. 1H NMR (DMSO-d6) d 1.06 (t,
J ¼ 7:2 Hz, 3H), 2.52 (s, 3H), 2.72–3.12 (m, 4H), 3.42–
3.55 (m, 2H), 3.62–3.82 (m, 4H), 3.90–4.15 (m, 4H),
4.32–4.42 (m, 1H), 6.68 (t, J ¼ 4:8 Hz, 1H), 8.41 (d,
J ¼ 4:8 Hz, 2H). Anal. C21H24N6O2S2.
A mixture of chloro derivative 15 (1.30 g, 3.79 mmol)
and piperazine (1.62 g, 18.81 mmol) was refluxed in
absolute ethanol (20 mL) during stirring for 3h. After
cooling, the solid was eliminated by filtration and the
solution was diluted with water. After 2 days, the solid
compound was collected and recrystallized from etha-
nol/water. Yield: 0.30 g (20%); mp 225 ꢁC (dec); IR (KBr
cmꢀ1) 2929, 1735, 1502, 1405, 1368, 1328, 1268, 1157,
988, 850. 1H NMR (DMSO-d6) d 1.11 (t, J ¼ 7 Hz, 3H),
1.62–1.95 (m, 3H), 2.19–2.31 (m, 1H), 2.52 (s, 3H), 2.61–
2.75 (m, 2H), 2.78–3.05 (m, 6H), 3.24–3.43 (m, 2H),
3.80–4.10 (m, 3H). Anal. C18H24N4O2S2.
5.35. Ethyl 2-methylthio-4-(1-piperazinyl)-6,7-dihydro-
5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine-5-carboxylate
(39)
6. In vitro binding assays
A mixture of chloro derivative 14 (0.70 g, 2.13mmol)
and piperazine (0.91 g, 10.56 mmol) was refluxed in
absolute ethanol (10 mL) during stirring for 5 h. After
cooling, the solid was eliminated by filtration, and the
solution was diluted with water and extracted with
chloroform. The combined organic layer was dried
(Na2SO4) and evaporated under reduced pressure. After
the addition of diethyl ether and petroleum ether to the
yellow sticky product, a solid compound was obtained,
which was recrystallized from ethanol/water. Yield:
0.28 g (35%); mp 76–78 ꢁC; IR (KBr cmꢀ1) 3273, 2931,
Male CRL:CD(SD)BR-COBS rats (weighing about
150 g, Charles River, Italy) and male CRL:(HA) BR
albino guinea pigs (weighing about 300 g, Charles River,
Italy) were killed by decapitation; their brains were
rapidly dissected into the appropriate areas (rat cortex
for 5-HT3 and guinea pig striatum for 5-HT4) and stored
at )80 ꢁC until the day of assay.
The tissues were homogenized in 50 vol of ice-cold Tris
HCl, 50 mM, pH 7.4 containing 0.5 mM EDTA, and
10 mM MgSO4 for 5-HT3, or Hepes HCl, 50 mM,
pH 7.4, for 5-HT4, using an Ultra Turrax TP-
1810 homogenizer (2 · 20 s), and homogenates were
1
2827, 1732, 1541, 1494, 1410, 1319, 1235, 1172, 831; H
NMR (DMSO-d6) d 1.14 (t, J ¼ 7:2 Hz, 3H), 2.28–2.58
(m, 4H), 2.63–3.12 (m, 5H), 3.22–3.70 (m, 6H), 4.06 (q,

3900
M. Modica et al. / Bioorg. Med. Chem. 12 (2004) 3891–3901
centrifuged at 50,000g for 10 min (Beckman Avanti J-25
refrigerated centrifuge). Each pellet was resuspended in
the same volume of fresh buffer, incubated at 37 ꢁC for
10 min, and centrifuged again at 50,000g for 10 min. The
pellet was then washed once by resuspension in fresh
buffer and centrifuged as before.
rimetria Ltd. Hungary). The contractile action of 5-HT3
receptor selective agonist 2-Me-5-HT (Sigma, Hungary)
was investigated by cumulative way. The result was
expressed as the percent of contraction increase compared
to basal colon activity. Compounds 31 and 33, and the
specific 5-HT3 receptor antagonist tropisetron (Sigma,
Hungary) were added to the bath 10 min before the
application of 2-Me-5-HT in concentration of 10 nM.
Dose–response curves, the contraction increasing effect,
the changes in EC50 and maximum inhibition, and the
statistical analysis were calculated by Prism 2.0 software
(GraphPad Software, USA).
The pellet obtained was finally resuspended in the
appropriate incubation buffer (Hepes HCl, 50 mM,
pH 7.4, containing 10 lM pargyline for 5-HT4, or Tris
HCl, 50 mM, pH 7.4, containing 10 lM pargyline,
0.5 mM EDTA, 10 mM MgSO4, 0.1% ascorbic acid,
and 140 mM NaCl for 5-HT3) just before the binding
assay.
[3H]LY 27858421 (S.A. 84.0 Ci/mmol Amersham, for 5-
HT3) binding was assayed in a final incubation volume
of 1 mL, consisting of 0.50 mL of tissue (16 mg/sample),
0.50 mL of the [3H]ligand (4 nM), and 0.02 mL of dis-
placing agent or solvent; nonspecific binding was mea-
sured in the presence of 1 lM quipazine. [3H]GR
11380822 (S.A. 84.0 Ci/mmol Amersham, for 5-HT4)
binding was assayed in a final incubation volume of
1.0 mL, consisting of 0.5 mL of tissue (20 mg/sample),
0.5 mL of the [3H]ligand (0.1 nM), and 0.02 mL of dis-
placing agent or solvent; nonspecific binding was mea-
sured in the presence of 10 lM serotonin. Incubation
(30 min at 25 ꢁC for 5-HT3 or 30 min at 37 ꢁC for 5-HT4)
was stopped by rapid filtration under vacuum through
GF/B filters, which were then washed with 12 mL (4 · 3
times) of ice-cold Tris HCl, 50 mM, pH 7.4, or Hepes
HCl, 50 mM, pH 7.4, using a Brandel M-48R cell har-
vester. Dried filters were immersed in vials containing
4 mL of Ultima Gold MV (Packard) and counted in a
Wallac 1409 liquid scintillation spectrometer with a
counting efficiency of about 50%. Drugs were tested in
triplicate at different concentrations (from 10ꢀ5 to
10ꢀ10 M) and dose-inhibition curves were analyzed by
the Allfit23 program to obtain the concentration of
unlabeled drug that caused 50% inhibition of
ligand binding, Ki values were derived from the IC50
values.24
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