Synthesis and receptor binding of new thieno[2,3-d]-pyrimidines as selective ligands of 5-HT3 receptors

Article abstract of DOI:10.1002/ardp.200700205

With the aim to develop new potent and selective ligands of 5-HT ₃-type serotonin receptors and to acquire more information on their structure-affinity relationships, new thieno[2,3-d]pyrimidine derivatives 32-39 were synthesized and their binding to 5-HT₃ versus 5-HT₄ receptors was studied. Some of these new compounds exhibit good affinity for cortical 5-HT₃ receptors, but not for 5-HT₄ receptors. Among these derivatives, 6-ethyl-4-(4-methyl-1-piperazinyl)-2-(methylthio)- thieno[2,3-d]pyrimidine 32 is the most potent ligand (K_i = 67 nM); it behaves as a competitive antagonist of the 5-HT₃ receptor function in the guinea pig colon. Its binding interactions with 5-HT_3A receptors were analysed by using receptor modelling and comparative docking.

Full text of DOI:10.1002/ardp.200700205

Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
M. N. Modica et al.
333
Full Paper
Synthesis and Receptor Binding of New Thieno[2,3-d]-
pyrimidines as Selective Ligands of 5-HT3 Receptors
Maria N. Modica1, Giuseppe Romeo1, Loredana Salerno1, Valeria Pittalꢀ1, Maria A. Siracusa1,
Ilario Mereghetti2, Alfredo Cagnotto2, Tiziana Mennini2, Rꢁbert Gꢂspꢂr3, Adrienn Gꢂl3,
George Falkay3, Mꢂrta Palkꢁ4, Gꢂbor Maksay5, and Ferenc Fꢃlꢄp4
1 Dipartimento di Scienze Farmaceutiche, Universitꢀ di Catania, Catania, Italy
2 Istituto di Ricerche Farmacologiche “Mario Negri”, Milano, Italy
3 Department of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged, Hungary
4 Institute of Pharmaceutical Chemistry, University of Szeged, Szeged, Hungary
5 Department of Molecular Pharmacology, Institute of Biomolecular Chemistry, Chemical Research Centre,
Budapest, Hungary
With the aim to develop new potent and selective ligands of 5-HT3-type serotonin receptors and
to acquire more information on their structure-affinity relationships, new thieno[2,3-d]pyrimi-
dine derivatives 32–39 were synthesized and their binding to 5-HT3 versus 5-HT4 receptors was
studied. Some of these new compounds exhibit good affinity for cortical 5-HT3 receptors, but not
for 5-HT4 receptors. Among these derivatives, 6-ethyl-4-(4-methyl-1-piperazinyl)-2-(methylthio)-
thieno[2,3-d]pyrimidine 32 is the most potent ligand (Ki = 67 nM); it behaves as a competitive
antagonist of the 5-HT3 receptor function in the guinea pig colon. Its binding interactions with
5-HT3A receptors were analysed by using receptor modelling and comparative docking.
Keywords: Antagonists / 5-HT3 Receptors / Receptor docking / Thieno[2,3-d]pyrimidines /
Received: October 8, 2007; accepted: January 21, 2008
DOI 10.1002/ardp.200700205
choline (nACh), A-type c-aminobutyric acid (GABAA) and
glycine (Gly) receptors [2]. Their subunits form pentamers
encircling an ion channel. Most 5-HT3Rs are composed of
subunits A and B [3]. Recently, 5-HT3C, 5-HT3D and 5-HT3E
subunits were cloned from human samples, but their
functional roles are not yet known [4, 5]. 5-HT3Rs can be
presynaptic and postsynaptic, and are located both in the
CNS and peripherally [2]. In the CNS, 5-HT3Rs have been
localized in the area postrema, nucleus tractus solitarii,
nucleus caudatus, nucleus accumbens, amygdala, hippo-
campus, entorhinal, frontal and cingulate cortex, and
the dorsal horn ganglia. In the periphery, they are found
in autonomic neurons and in neurons of the sensory and
enteric nervous systems, where they are involved in eme-
sis, nociception and gut motility [6]. The 5-HT3Rs modu-
late the release of neurotransmitters and neuropeptides
such as dopamine, cholecystokinin, acetylcholine, GABA,
substance P and 5-HT itself [6].
Introduction
Serotonin (5-hydroxytryptamine, 5-HT) is a biogenic
monoamine with a variety of functions in the peripheral
and central nervous systems (CNS). It is involved in many
physiological and pathophysiological processes, such as
depression, anxiety, sleep, the circadian rhythm, aggres-
sion, feeding and sexual behaviour, schizophrenia, buli-
mia, anorexia and asthma [1].
The various biological effects of 5-HT are mediated
through different 5-HT receptors and their signal trans-
duction pathways. There are seven classes of serotonin
receptors (5-HT1R to 5-HT7R) and some of them are further
divided into several subtypes [2]. They are coupled to G
proteins, except for the 5-HT type-3 receptor (5-HT3R),
which is a member of the cysteine (Cys)-loop family of
ligand-gated ion channels as well as the nicotinic acetyl-
The 5-HT3Rs have gained considerable attention
because of the clinical use of 5-HT3R antagonists (5-
HT3RAs)suchasondansetron, granisetronandtropisetron
Correspondence: Maria N. Modica, Dipartimento di Scienze Farmaceu-
tiche, Universitꢀ di Catania, Viale A. Doria 6, 95125 Catania, Italy.
E-mail: mmodica@unict.it
Fax: +39 095 222239
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334
M. N. Modica et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
in the treatment of chemotherapy- and radiotherapy-
induced nausea and vomiting and also in post-operative
nausea and vomiting [7]. Moreover, a number of preclini-
cal studies suggest that 5-HT3RAs can be used in the treat-
ment of various CNS disorders, such as anxiety, depres-
sion, schizophrenia, drug and alcohol abuse, chronic
fatigue, withdrawal and age-associated memory impair-
ments, pain (fibromyalgia and migraine) [6], and gastroen-
teric motility disorders such as diarrhoea accompanying
the irritable bowel syndrome [8]. Recent data suggest that
5-HT3RAs are also effective in the treatment of rheumatic
diseases such as rheumatoid arthritis, tendinopathies,
periarthropathies and myofascial pain [9].
The therapeutic potential of 5-HT3 agonists is less well
known; it was recently reported that they could be useful
to treat or prevent neurodegenerative diseases such as
ischaemic stroke, Alzheimer's disease, diabetic periph-
eral neuropathy, multiple sclerosis, amyotrophic lateral
sclerosis, traumatic brain injury and spinal cord injury,
Huntington's disease or Parkinson's disease [10].
Figure 1. Structures of published and new thieno[2,3-d]pyrimi-
dines.
midine system were modified. In particular, alkyl moi-
eties such as ethyl, n-propyl and n-butyl were attached to
the 6-position, or a bulky tetrahydropyridine-substituted
system was condensed at the 5- and 6-positions. This lat-
ter substitution has been done assuming a possible addi-
tional interaction between the lipophilic tetrahydropyri-
dine-substituted system and the receptor counterpart
which could be able to tolerate bulkier ligands.
In some of the new compounds, a methyl group linked
to N4 of the piperazine ring was inserted on the basis of
well known literature data [16, 17, 22].
Moreover, the binding interactions of 5-HT3R-selective
ligands 2 and 32 were analysed via receptor modelling
and comparative docking.
In view of the wide-ranging involvement of 5-HT3Rs in
various physiological and pathological processes, a better
understanding of the full therapeutic potential of their
agonists and antagonists appears to be necessary.
For some years, we have been engaged in the develop-
ment of subtype-selective ligands of 5-HTRs [11–15]. Con-
sidering the various therapeutic implications of 5-HT3R
ligands, we have recently focused our interest on devel-
oping novel 5-HT3R ligands [16, 17]. As suggested by the
literature [18], the three key pharmacophoric elements
(an aromatic/heteroaromatic ring, a hydrogen-bond
acceptor and a basic amino group) required for the inter-
action with 5-HT3Rs, were taken into account for the syn-
thesis of these compounds. They contain as “scaffold” a
thienopyrimidine system selected for its broad range of
biological activities [19], and differ particularly in the
position of the piperazine ring. In one series of com-
pounds, it is linked to the 2-position of the pyrimidine
ring; among these derivatives, the most potent com-
pound 1 (Fig. 1) behaves as an agonist in the Bezold–Jar-
isch reflex assay [16]. In a second series (compounds 2–6),
it is linked to the 4-position of the pyrimidine nucleus;
among them, the most potent compound 2 (Fig. 1, R1,
R2 = –(CH2)3 –, R3 = CH3) behaves as a non-competitive
antagonist in the isolated guinea-pig colon test [17]. The
present work focuses on the acquisition of more informa-
tion on the structure-affinity relationships of these latter
derivatives. A new series of compounds 32–39 [20, 21]
was synthesized (Fig. 1), which maintain the thieno[2,3-
d]pyrimidine backbone and the piperazine ring linked to
the 4-position of the pyrimidine nucleus, while the sub-
stituents at the 5- and 6-positions of the thieno[2,3-d]pyri-
Chemistry
The synthetic procedure adopted for the preparation of
thieno[2,3-d]pyrimidines 32–39 is depicted in Scheme 1.
Aminoesters 8–10 were prepared as described [23–25],
the unknown aminoester 11 was prepared from 4-oxo-1-
(phenylmethyl)-3-piperidinecarboxylic acid methyl ester
7, ethyl cyanoacetate, and sulphur following the proce-
dure of Gewald et al. [23].
Derivatives 12–15 were obtained by refluxing the cor-
responding b-amino esters of 4,5-disubstituted-thio-
phenes 8–11 in acetone with benzoyl isothiocyanate,
commercially available or prepared in situ. Successively,
derivatives 12–15 were heated under reflux in ethanolic
KOH solution to give the corresponding monopotassium
salts of the 5,6-disubstituted-2-thioxothieno[2,3-d]pyrimi-
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Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
New Thienopyrimidines as 5-HT Receptor Ligands
335
Reagents and conditions: (a) CNCH2COOC2H5, sulphur, EtOH, 508C, then diethylamine, 608C, 5 h; (b) SCNCOC6H5, acetone, reflux, 2 h; (c) KOH, absolute EtOH, reflux, 3 h; (d)
HCl, H2O, rt, 30 min; (e) CH3I, H2O, rt, 1.5 h; (f) POCl3, 1508C, 35 min or 2.5 h; (g) 1-methylpiperazine or piperazine, absolute EtOH, reflux, 1 or 3 h.
Scheme 1. Synthesis route of presented compounds 7–39.
dinones 16–19. Acidification with concentrated HCl of and [3H]GR 113808 binding to 5-HT3Rs and 5-HT4 recep-
aqueous solutions of monopotassium salts 16–19 gave tors (5-HT4Rs), respectively, using the rat cortex for 5-
the corresponding thioxo compounds 20–23, which HT3Rs and the guinea pig striatum for 5-HT4Rs. 5-HT and
were useful to confirm the structures of salts 16–19. tropisetron were used as reference substances. The bind-
Reaction of the potassium salts 16–19 with CH3I in water ing data reported in Table 1 are expressed as Ki values.
at room temperature furnished the 2-methylthio deriv-
Compound 32, which displayed the best affinity, was
atives 24–27, which were then converted into 4-chloro additionally tested in a functional assay in vitro, using
derivatives 28–31 by heating with an excess of POCl3. the isolated guinea pig distal colon, in order to evaluate
4-(1-Piperazinyl)thieno[2,3-d]pyrimidine derivatives 32–
its putative agonistic or antagonistic properties (Fig. 2
39 were finally synthesised by refluxing the 4-chloro and Table 2) [26].
derivatives 28–31 with piperazine or 1-methylpiperazine
in ethanol. The proposed structures for compounds 8–39
were confirmed via elemental analyses and the IR and
1H-NMR spectra (see the Experimental, Section 5).
Structure-affinity relationships of receptor binding
The results of the binding tests, presented in Table 1,
demonstrate that compounds 32–39 (except for 38)
exhibit appreciable affinities for 5-HT3Rs and high selec-
tivity relative to the 5-HT4Rs, since none of the new deriv-
atives display significant affinity for the latter.
Results and discussion
The new thienopyrimidines 32–39 were synthesized to
explore how the affinity for 5-HT3Rs is affected by alkyl
substituents at the 6-position of the thienopyrimidine
Pharmacology
Compounds 32–39 were tested in binding assays in vitro
to determine their displacing potencies on [3H]LY 278584
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Table 1. Chemical structures of new and published thieno[2,3-d]pyrimidines and their binding affinities to 5-HT3Rs.
Compd.
Kia) (nM) (l SD)
R1
R2
R3
5-HT3
5-HT4
2b)
3b)
4b)
5b)
6b)
32
33
34
35
36
37
38
39
–(CH2)3 –
–(CH2)3 –
–(CH2)3 –
CH3
H
4-OCH3C6H4
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
33 l 5
70 l 7
nac)
nac)
nac)
nac)
nac)
nac)
nac)
nac)
nac)
nac)
nac)
nac)
nac)
nac)
–CH2CH2CH2CH(COOC2H5)–
–CH2CH2CH2CH(COOC2H5)–
1791 l 187
nac)
C2H5
C2H5
n-C3H7
n-C3H7
n-C4H9
n-C4H9
H
H
H
H
H
H
67 l 7
124 l 14
298 l 28
572 l 86
644 l 44
1060 l 104
nac)
3679 l 327
354 l 97
6.58 l 0.72
–CH2N(CH2C6H5)CH2CH(COOCH3)–
–CH2N(CH2C6H5)CH2CH(COOCH3)–
5-HT
79 l 10
4357 l 653
tropisetron
a)
Each value is the mean l SD of the data from three separate experiments.
These data are included for the sake of clarity [16, 17].
Not active, percentage of inhibition of specific binding a50% at 10– 5 M.
b)
c)
Table 2. Potencies (EC50) and maximum effects (Emax) of 2-Me-
5-HT in the presence of 10 nM tropisetron (trop) and compound
32 in increasing the contractions of guinea pig colon in vitro.
stituent at the 6-position, from ethyl, 32 and 33, to
n-propyl, 34 and 35, and to n-butyl derivatives 36 and 37
(Ki = 67, 124, 298, 572, 644 and 1060 nM, respectively).
The ethyl moiety seems to fit better into the receptor
binding pocket than the more bulky n-propyl and n-butyl
groups. Condensation of a tetrahydropyridine-substi-
tuted ring at the 5- and 6-positions of the thieno[2,3-d]pyr-
imidine system leads to compounds exhibiting a com-
plete loss, e.g. 38, or a considerable decrease in affinity,
39 with Ki = 3679 nM. With regard to the substitution on
the piperazine nucleus, the trend was that the displacing
potencies of 4-methylpiperazine derivatives 32, 34 and 36
were about twice as high (Ki = 67, 298 and 644 nM, respec-
tively) as those of the corresponding unsubstituted piper-
EC50 (M) l SEM
Emax (%) l SEMa)
2-Me-5-HT
9.1610-6 l 0.9610– 6
65.4 l 13.6
2-Me-5-HT + trop 60.8610– 6 l 14.1610– 6b) 66.1 l 32.6c)
2-Me-5-HT + 32
22.1610– 6 l 7.2610– 6d) 63.2 l 11.4c)
The level of significance is indicated next to the values as com-
pared with 2-Me-5-HT.
a)
SEM, standard error of the mean.
p a 0.01.
Not significant.
b)
c)
d)
p a 0.05.
system and by a bulky tetrahydropyridine-substituted azine derivatives 33, 35 and 37 (Ki = 124, 572 and
system condensed to the thiophene nucleus.
1060 nM, respectively). The 4-methyl group might inter-
All the alkyl derivatives 32–37 possess noteworthy dis- act with a hydrophobic accessory site. Regarding the sub-
stitution on the piperazine ring, the new series of com-
from 67 nM to 1060 nM. Among them, 6-ethyl-4-(4- pounds display a general trend similar to that reported
methyl-1-piperazinyl)-2-(methylthio)thieno[2,3-d]pyrimi-
for derivatives 2 and 3, where the 4-methyl derivative 2 is
dine 32 demonstrated the highest affinity (Ki = 67 nM). As about twice as potent as the unsubstituted 3.
Although tetrahydropyridine derivatives 38 and 39 are
37 gradually decreases with the length of the alkyl sub- the least potent ligands in the series, the unsubstituted
placing potencies on 5-HT3R binding, with Ki ranging
a general trend, the binding affinity of compounds 32–
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New Thienopyrimidines as 5-HT Receptor Ligands
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piperazine derivative 39 still revealed a measurable affin-
ity for 5-HT3Rs in the micromolar range (Ki = 3679 nM),
whereas the methylpiperazine derivative 38 had none.
This finding is not in accordance with the trend shown
by this series of derivatives (32–37) and with that of com-
pounds 2 and 3.
Moreover, the affinity displayed by compound 39 indi-
cates that although it contains a bulky phenylmethyl
substituent linked at the 7-position of the tetrahydropyr-
ido[49,39:4,5]thieno[2,3-d]pyrimidine system, it can still
interact with the receptor binding site. Comparison of
the binding properties of compounds 32 and 2 show that
32 has a slightly lower affinity than that of 2 (Table 1).
Thus, a 5-membered unsubstituted ring fused to the
thieno[2,3-d]pyrimidine system, or an ethyl moiety
linked to the 6-position of the same nucleus, appears to
be optimal for strong interactions with the 5-HT3R bind-
ing site.
Figure 2. The effects of 10 nM tropisetron (0) and compound 32
(f) in increasing the contractions elicited by a selective 5-HT3R
agonist, 2-Me-5-HT (9), in the isolated guinea pig colon in vitro.
The effects of 2-Me-5-HT were expressed as the percentage
increases in contractions as compared with the basal colon
activity (n = 6).
Functional properties
Compound 32, the new derivative with the highest affin-
ity for 5-HT3Rs, was also tested to evaluate its functional
properties in vitro concerning the increase of the contrac-
tions of the isolated guinea pig colon. Tropisetron, a well-
known 5-HT3R antagonist, was used as a reference com-
pound. In the isolated guinea pig colon, 10 nM tropise-
tron shifted the dose-response curve of the selective
5-HT3R agonist 2-methyl-5-hydroxytryptamine (2-Me-5-HT)
to the right, suggesting competitive antagonism (Fig. 2,
Table 2). Compound 32 (10 nM) affected the 2-Me-5-HT-
induced contractions similarly to tropisetron, but the
right shift of the curve turned out to be smaller (Fig. 2,
Table 2). Nevertheless, both compounds shifted the curve
of 2-Me-5-HT to the right, but because of the high maxi-
mum concentration of 2-Me-5-HT alone (10–4 M), the maxi-
mum values in the presence of the compounds were
impossible to measure in the isolated organ bath studies.
For this reason, the maximum and the EC50 values were
predicted via computer program. These findings can be
reconciledwith compound 32 beingacompetitive antago-
nist of 5-HT3Rs, butaless potent one thantropisetron.
The homology model of 5-HT3ARs based on the struc-
ture of nACh receptors with an agonist-free (empty) bind-
ing cavity [27] is an improvement compared to previous
models involving an acetylcholine-binding protein [28]
where, according to an emerging consensus, bound ago-
nists “pull in” loop C to close the binding cavity. In con-
trast, the open binding cavity can accommodate
5-HT3RAs in more docking positions [27]. Consequently,
the docking of 2 to 5-HT3ARs resulted in three predomi-
nant positions in the interface of the subunits under
loop C (Fig. 3) in the binding cavity where 5-HT3RAs [28]
and agonists [29] can be docked and bound. How can we
decide which docking position is relevant in binding?
Comparative docking of active, 2 and 32, versus less
active and inactive, 4, 5, 6 and 38 analogues in displace-
ment of the [3H]LY 278584 and [3H]zacopride bindings to
the 5-HT3Rs of the rat cortex were used to distinguish
probable binding positions from docking artefacts. Over-
lapping docking positions of active and inactive deriv-
atives were considered artefacts, while the similar dock-
ings of the active ones support similar binding modes
associated with high affinity. The light grey structure of
compound 2 (next to 1. in Fig. 3) with best energy was in
juxtaposition with the best docking of compound 32 (not
shown for clarity), and it represents the most probable
binding mode with its aromatic rings close to tyrosines
Y143 and Y153 and the piperazine ring close to aspara-
gines N128 and Y234. The two other dockings (medium
grey (2.) and dark grey (3.) in Fig. 3) were in juxtaposition
with the identical parts of the best docking positions of
the less active and inactive compounds 4, 5, 6 and 38, and
5-HT3AR modelling
Receptor modelling was used to localize the binding sites
of selective ligands 2 and 32 on 5-HT3Rs. This was based
on the building of a homology model of 3A–type 5-HT
receptors (5-HT3ARs), obtained from the electron micro-
scopic structure of the nACh receptor with agonist-free
binding cavities at 4 ꢀ resolution [27]. This model, used
for the comparative docking of potent versus inactive
ligands, could elucidate possible binding interactions of
compound 2 with 5-HT3ARs.
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M. N. Modica et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
the binding affinity of [3H]granisetron for recombinant
5-HT3ARs [27]. This supports the view that compounds 2,
32 and granisetron bind in the same binding cavity of
5-HT3ARs.
Receptor modelling and binding studies were perform-
ed with homomeric 5-HT3ARs and in the brain, respec-
tively, while the functional test was carried out in the
guinea pig colon probably containing peripheral-type
heteromeric 5-HT3ABRs too. In fact, recent studies suggest
that mainly homomeric 5-HT3ARs are present in the CNS,
whereas both heteromeric 5-HT3ABRs and homomeric
5-HT3ARs exist in the periphery [32].
Consequently, receptor modelling can be correlated
with binding data to 5-HT3Rs in brain rather than with
the peripheral functional assay.
This work was supported by grants from the Italian MIUR
(Ministero dell'Universitꢀ e della Ricerca), the Universitꢀ di
Catania, and the Hungarian Science Research Fund (OTKA
K62203). We are grateful to Dr. Zsolt Bikꢁdi for his contribu-
tion to the receptor modelling.
Figure 3. Three major docking positions of compound 2 with
best energies and highest docking frequencies in the interface of
two subunits of agonist-free 5-HT3ARs under loop C. The num-
bers (1. light grey, 2. medium grey, 3. dark grey) show the rank
order of docking energies and frequencies. Amino acids which
are crucial in the binding of granisetron to 5-HT3ARs are indi-
cated [27].
The authors have declared no conflict of interest.
Conclusion
they were therefore considered artefacts. The docking
vicinity of the distal piperazinyl tertiary amino group of
compounds 2 and 32 to N128 is in agreement with the
docking vicinity of the analogous granatane amino
group of granisetron, a 5-HT3R antagonist (Ki = 0.20 nM)
[30], to N128 [27]. Moreover, mutations of the homolo-
gous asparagine (N102 in a1 Gly receptors) support the
vicinity of N102 to the tropanic amino group of tropise-
tron in Gly receptor antagonism [31]. This asparagine res-
idue is conserved in (Cys)-loop receptors and it can be
associated with the requirement of the basic tertiary
amino groups in the structure-affinity relationships for
antagonist binding to 5-HT3Rs.
In conclusion, a new series of thieno[2,3-d]pyrimidine
derivatives has been prepared as ligands for 5-HT3Rs.
Some of the new compounds display good affinity for 5-
HT3Rs but not for 5-HT4Rs. Among these derivatives, 6-
ethyl-4-(4-methyl-1-piperazinyl)-2-(methylthio)thieno[2,3-
d]pyrimidine 32 possesses the highest affinity and in an
in vitro functional test behaves as a competitive antago-
nist, like tropisetron. Receptor modelling and compara-
tive docking support the conclusion that compounds 2
and 32 and the reference antagonist granisetron bind to
5-HT3ARs in the same binding cavity. The modelling
results rationalise the structure-affinity data of receptor
binding with the surrounding amino acid residues.
On the basis of the best docking position of 2 in Fig. 3,
the structure-affinity relationships observed for 5-HT3R
binding of compounds 2–6, 32–39 could be explained.
The R3 groups being larger than methyl do not fit well in
the binding cavity because of possible steric clashes
around N128 and Y234. Small R1 alkyl groups or a propyl
chain bridging 5- and 6-positions are also tolerated, while
large groups in these positions are probably sterically
hindered by the wall of the cavity (Y234 in Fig. 3).
Further, docking studies also suggest that amino acid
residues W90, N128, Y143, Y153, W183 and Y234 con-
served in 5-HT3ARs play key roles in binding compound 2
(Fig. 3). Point mutations of these residues deteriorated
Experimental
General methods
Melting points were determined in glass capillary tubes on a Gal-
lenkamp apparatus with an MFB-595 digital thermometer
(Weiss-Gallenkamp, London, UK) and are uncorrected. Elemental
analyses for C, H, N and S were performed on a Fisons-Carlo Erba
EA1108 Elemental Analyzer (Carlo Erba, Milan, Italy) and were
within l 0.4% of the theoretical values. Infrared spectra were
recorded in KBr disks on a Perkin Elmer FT-IR 1600 spectrometer
(Perkin-Elmer, Norwalk, CT, USA). 1H-NMR spectra were recorded
in DMSO-d6 or CDCl3 solution at 200 MHz on a Varian Inova-Unity
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Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
New Thienopyrimidines as 5-HT Receptor Ligands
339
200 spectrometer (Varian Inc., Palo Alto, CA, USA); chemical
shifts are given in d values (ppm), relative to tetramethylsilane
as the internal standard; coupling constants (J) are given in Hz.
Signal multiplicities are characterized as s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet) or br s (broad singlet). The
purities of all the synthesized compounds were checked by TLC
on an aluminium sheet coated with silica gel 60 F254 (Merck, Ger-
many) and visualized by UV (k = 254 and 366 nm). All commer-
cial chemicals and solvents were of reagent grade and were pur-
chased from commercial vendors.
(s, 1H, aromatic), 7.48-8.08 (m, 5H, aromatic), 11.93 (s, 1H, NH),
14.75 (s 1H, NH). Anal. (C18H20N2O3S2) C, H, N, S.
2-[[(Benzoylamino)thioxomethyl]amino]-5-butyl-3-
thiophenecarboxylic acid methyl ester 14
This compound was prepared from amino ester 10 by the same
procedure as described for 12, and was recrystallised from etha-
nol. Yield: 10.97 g (80%); mp. 150–1528C; IR (KBr, selected lines)
cm– 1 3334, 2919, 1694, 1666, 1555, 1523, 1490, 1458, 1225,
1161. 1H-NMR (DMSO-d6) d 0.91 (t, J = 7.2 Hz, 3H, CH2CH2CH2CH3),
1.22–1.42 (m, 2H, CH2CH2CH2CH3), 1.52–1.68 (m, 2H,
CH2CH2CH2CH3), 2.73 (t, J = 7.2 Hz, 2H, CH2CH2CH2CH3), 3.88 (s,
3H, COOCH3), 7.05 (s, 1H, aromatic), 7.50–8.10 (m, 5H, aromatic),
11.94 (s, 1H, NH), 14.75 (s, 1H, NH). Anal. (C18H20N2O3S2) C, H, N, S.
Chemistry
2-Amino-6-(phenylmethyl)-4,5,6,7-tetrahydrothieno[2,3-
c]pyridine-3,4-dicarboxylic acid 3-ethyl 4-methyl diester
11
2-[[(Benzoylamino)thioxomethyl]amino]-6-
(phenylmethyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-
3,4-dicarboxylic acid 3-ethyl 4-methyl diester 15
To a mixture of 4-oxo-1-(phenylmethyl)-3-piperidinecarboxylic
acid methyl ester 7 (16.80 g, 68.00 mmol), ethyl cyanoacetate
(7.70 g, 68.00 mmol) and sulphur (2.20 g, 68.75 mmol) in ethanol
(14 mL), stirred on an oil bath at 508C, diethylamine (7.00 mL)
was added slowly and the mixture was stirred at 608C for 5 h.
After the mixture had been cooled, the precipitate was filtered
off, washed with cold ethanol, dried and recrystallised from etha-
nol to give 11 as a pure solid. Yield: 14.76 g (58%); mp. 193–1948C;
IR (KBr, selected lines) cm– 1 3386, 3289, 1723, 1665, 1580, 1495,
1361, 1278, 1203, 1169. 1H-NMR (CDCl3) d 1.25 (t, J = 7.0 Hz, 3H,
CH2CH3), 2.75 (dd, 2J = 11.8 Hz, 3J = 4.9 Hz, 1H, CHAHBCHCOOCH3),
3.15 (dd,2J = 11.8 Hz,3J = 3.6 Hz, 1H, CHAHBCHCOOCH3), 3.30 (d, J =
This compound was prepared from amino ester 11 by the same
procedure as described for 12, with slight variations: the reflux-
ing time was 3 h and, after cooling, the solvent was removed
under reduced pressure and the resulting solid was collected,
washed with water, dried and recrystallised from ethyl acetate.
Yield: 15.49 g (82%); mp. 172–1748C; IR (KBr, selected lines) cm– 1
3251, 1718, 1709, 1673, 1534, 1423, 1323, 1246, 1181, 707. H-
NMR (DMSO-d6) d 1.22 (t, J = 7.0 Hz, 3H, CH2CH3), 2.60-2.70 (m, 1H,
CHAHBCHCOOCH3), 3.00-3.10 (m, 1H, CHAHBCHCOOCH3), 3.46 (d, J
1
14.6 Hz, 1H, PhCH2NCHCHD), 3.60 (d,
J = 13.1 Hz, 1H,
=
15.1 Hz, 1H, PhCH2NCHCHD), 3.54–3.56 (m, 1H + 3H,
PhCHAHBNCH2), 3.64–3.68 (m, 1H + 3H, PhCH2NCHCHD, COOCH3),
3.73 (d, J = 13.1 Hz, 1H, PhCHAHBNCH2), 3.90–3.95 (m, 1H,
CHCOOCH3), 4.19 (q, J = 7.0 Hz, 2H, CH2CH3), 5.95 (s, 2H, NH2),
7.24–7.36(m, 5H, aromatic). Anal. (C19H22N2O4S)C, H, N, S.
PhCH2NCHCHD, COOCH3), 3.72-3.90 (m, 2H, PhCH2N), 3.97–4.07
(m, 1H, CHCOOCH3), 4.28 (q, J = 7.0 Hz, 2H, CH2CH3), 7.23–8.11
(m, 10H, aromatic), 11.90 (s, 1H, NH), 14.77 (s, 1H, NH). Anal.
(C27H27N3O5S2) C, H, N, S.
2-[[(Benzoylamino)thioxomethyl]amino]-5-ethyl-3-
6-Ethyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-
thiophenecarboxylic acid ethyl ester 12
one 20
Benzoyl chloride (4.08 mL, 35.13 mmol) was added under stir-
ring to a solution of NH4NCS (3.11 g, 40.86 mmol) in anhydrous
acetone (28 mL). The mixture was heated at reflux under stirring
for 5 min. A solution of the ethyl ester 8 (7.00 g, 35.13 mmol) in
anhydrous acetone (70 mL) was then added and the mixture was
stirred under reflux for 2 h. After the mixture had been cooled, a
small amount of solvent was evaporated off under reduced pres-
sure, and the solid obtained was collected, washed with water,
dried and recrystallised from ethanol to give 12 as a pure solid.
Yield: 11.40 g (89%); mp. 148–1518C; IR (KBr, selected lines) cm– 1
3280, 2970, 1691, 1563, 1527, 1464, 1232, 1193, 1152, 675. 1H-
NMR (DMSO-d6) d 1.24 (t, J = 7.6 Hz, 3H, CH2CH3), 1.33 (t, J = 7.0 Hz,
3H, CH2CH3), 2.75 (q, J = 7.6 Hz, 2H, CH2CH3), 4.35 (q, J = 7.0 Hz,
2H, CH2CH3), 7.05 (s, 1H, aromatic), 7.50–8.08 (m, 5H, aromatic),
11.92 (s, 1H, NH), 14.74 (s, 1H, NH). Anal. (C17H18N2O3S2) C, H, N, S.
Benzoyl derivative 12 (10.28 g, 28.36 mmol) was added to a solu-
tion of KOH (3.18 g, 56.68 mmol) in absolute ethanol (62 mL)
and the mixture was refluxed under stirring for 3 h. After the
mixture had been cooled, a small amount of solvent was evapo-
rated under reduced pressure, and the solid obtained was col-
lected, washed with absolute ethanol and dried to give salt 16
(2.03 g, 28%). A suspension in water (10 mL) of potassium salt 16
(0.40 g, 1.60 mmol) was acidified with concentrated HCl and
stirred for 30 min at room temperature. The solid obtained was
collected, washed with water, dried and recrystallised from etha-
nol to give 20 as a pure solid. Yield: 0.12 g (35%); mp. 280–2828C;
IR (KBr, selected lines) cm– 1 3046, 2920, 1668, 1566, 1535, 1466,
1264, 1188, 1127, 842. 1H-NMR (DMSO-d6) d 1.21 (t, J = 7.6 Hz, 3H,
CH3), 2.76 (q, J = 7.6 Hz, 2H, CH2), 6.93 (s, 1H, aromatic), 12.41 (s,
1H, NH), 13.78 (s, 1H, NH). Anal. (C8H8N2OS2) C, H, N, S.
2-[[(Benzoylamino)thioxomethyl]amino]-5-propyl-3-
6-Propyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-
thiophenecarboxylic acid ethyl ester 13
4(1H)-one 21
This compound was prepared from amino ester 9 by the same
procedure as described for 12, and was recrystallised from etha-
nol. Yield: 8.73 g (66%); mp. 148–1508C; IR (KBr, selected lines)
cm– 1 3304, 2956, 1680, 1552, 1525, 1449, 1316, 1223, 1163,
1082.1H-NMR (DMSO-d6) d 0.93 (t, J = 7.2 Hz, 3H, CH2CH2CH3), 1.33
(t, J = 7.0 Hz, 3H, CH2CH3), 1.57–1.70 (m, 2H, CH2CH2CH3), 2.71 (t,
J = 7.2 Hz, 2H, CH2CH2CH3), 4.36 (q, J = 7.2 Hz, 2H, CH2CH3), 7.05
Benzoyl derivative 13 (4.00 g, 10.62 mmol) was added to a solu-
tion of KOH (1.19 g, 21.20 mmol) in absolute ethanol (43 mL)
and the mixture was refluxed under stirring for 4 h. After the
mixture had been cooled, the solid was filtered off, washed with
absolute ethanol and dried to obtain salt 17 (2.60 g, 92%). Potas-
sium salt 17 (1.00 g, 3.78 mmol) was suspended in water (50 mL)
and the suspension was then acidified with concentrated HCl
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340
M. N. Modica et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
and stirred for 30 min at room temperature. The solid obtained
was collected, washed with water, dried and recrystallised from
ethanol to give 21 as a pure solid. Yield: 0.38 g (44%); mp. 248–
2508C; IR (KBr, selected lines) cm– 1 2827, 1677, 1580, 1548, 1265,
1241, 1185, 1128, 886, 752. 1H-NMR (DMSO-d6) d 0.90 (t, J = 7.4 Hz,
3H, CH3), 1.52-1.70 (m, 2H, CH2CH2CH3), 2.71 (t, J = 7.4 Hz, 2H,
CH2CH2CH3), 6.93 (s, 1H, aromatic), 12.40 (s, 1H, NH), 13.38 (s, 1H,
NH). Anal. (C9H10N2OS2) C, H, N, S.
2-(Methylthio)-6-propylthieno[2,3-d]pyrimidin-4(1H)-one
25
This compound was prepared from salt 17 by the same proce-
dure as described for 24, and was recrystallised from ethanol.
Yield: 1.73 g (89%); mp. 174–1768C; IR (KBr, selected lines) cm– 1
1
2958, 1652, 1550, 1481, 1426, 1287, 1190, 1147, 1110, 888. H-
NMR (DMSO-d6) d 0.93 (t, J = 7.2 Hz, 3H, CH3), 1.57-1.76 (m, 2H,
CH2CH2CH3), 2.52 (s, 3H, SCH3), 2.77 (t, J = 7.2 Hz, 2H, CH2CH2CH3),
7.01 (s, 1H, aromatic), 12.71 (s, 1H, NH). Anal. (C10H12N2OS2) C, H,
N, S.
6-Butyl-2-thioxo-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-
one 22
6-Butyl-2-(methylthio)thieno[2,3-d]pyrimidin-4(1H)-one
26
This compound was prepared from salt 18 by the same proce-
dure as described for 24, and was recrystallised from ethanol.
Yield: 1.05 g (51%); mp. 188–1908C; IR (KBr, selected lines) cm– 1
This compound was prepared from benzoyl derivative 14 by the
same procedure as described for 17. Potassium salt 18: yield:
6.79 g (86%). After washing, compound 22 was recrystallised
from ethanol. Yield: 0.22 g (58%); mp. 198–2008C; IR (KBr,
selected lines) cm– 1 3049, 2865, 1678, 1580, 1547, 1268, 1188,
1124, 894, 750. 1H-NMR (DMSO-d6) d 0.88 (t, J = 7.2 Hz, 3H, CH3),
1.21–1.42 (m, 2H, CH2CH2CH2CH3), 1.42-1.65 (m, 2H,
CH2CH2CH2CH3), 2.71 (t, J = 7.2 Hz, 2H, CH2CH2CH2CH3), 6.93 (s,
1H, aromatic), 12.40 (s, 1H, NH), 13.39 (s, 1H, NH). Anal.
(C10H12N2OS2) C, H, N, S.
1
2927, 1652, 1537, 1283, 1185, 1139, 1080, 1012, 882, 760. H-
NMR (DMSO-d6) d 0.90 (t, J = 7.2 Hz, 3H, CH3), 1.22-1.42 (m, 2H,
CH2CH2CH2CH3), 1.50–1.68 (m, 2H, CH2CH2CH2CH3), 2.53 (s, 3H,
SCH3), 2.80 (t, J = 7.2 Hz, 2H, CH2CH2CH2CH3), 7.00 (s, 1H, aro-
matic), 12.70 (s, 1H, NH). Anal. (C11H14N2OS2) C, H, N, S.
2-(Methylthio)-7-(phenylmethyl)-4-oxo-1,4,5,6,7,8-
hexahydropyrido[49,39:4,5]thieno[2,3-d]pyrimidine-5-
carboxylic acid methyl ester 27
7-(Phenylmethyl)-4-oxo-2-thioxo-1,2,3,4,5,6,7,8-
octahydropyrido[49,39:4,5]thieno[2,3-d]pyrimidine-5-
carboxylic acid methyl ester 23
This compound was prepared from salt 19, by the same proce-
dure as described for 24, and was recrystallised from ethyl ace-
tate. Yield: 2.15 g (66%); mp. 216–2188C dec; IR (KBr, selected
lines) cm– 1 3392, 1741, 1658, 1537, 1495, 1452, 1396, 1305,
1264, 1196. 1H-NMR (DMSO-d6) d 2.45 (s, 3H, SCH3), 2.74 (dd, 2J =
11.6 Hz, 3J = 5.0 Hz, 1H, CHAHBCHCOOCH3), 2.99 (dd, 2J = 11.6 Hz,
3J = 3.5 Hz, 1H, CHAHBCHCOOCH3), 3.47-3.51 (m, 1H + 3H,
PhCH2NCHCHD, COOCH3) 3.60 (d, J = 13.6 Hz, 1H, PhCH2NCHCHD),
3.75-4.02 (m, 1H + 2H, CHCOOCH3, PhCH2N), 7.23–7.44 (m, 5H,
aromatic), 12.59 (br s, 1H, NH). Anal. (C19H19N3O3S2) C, H, N, S.
Benzoyl derivative 15 (3.50 g, 6.51 mmol) was added to a solution
of KOH (0.73 g, 13.03 mmol) in absolute ethanol (30 mL) and the
mixture was refluxed under stirring for 3 h. After the mixture
had been cooled, the solvent was removed under reduced pres-
sure and the solid obtained was collected, washed with a small
amount of absolute ethanol and dried to give salt 19 (2.60 g,
93%). Potassium salt 19 (0.08 g, 0.18 mmol) was poured into
water (10 mL) and the solution was acidified with concentrated
HCl and stirred for 10 min at room temperature. The mixture
was then extracted with ethyl acetate. The organic layer was col-
lected, dried with anhydrous Na2SO4, filtered, and evaporated
under reduced pressure. The crude solid obtained was collected,
washed with diethyl ether, dried and recrystallised from ethanol
to give 23 as a pure solid. Yield: 0.02 g (26%); mp. 174–1788C; IR
(KBr, selected lines) cm– 1 3431, 1693, 1546, 1455, 1368, 1200,
4-Chloro-6-ethyl-2-(methylthio)thieno[2,3-d]pyrimidine 28
A mixture of 2-methylthio derivative 24 (1.20 g, 5.30 mmol) and
POCl3 (6 mL) was heated at 1508C and stirred for 35 min. After
the mixture had been cooled, the solution was poured into cold
water and neutralised with a 10% solution of NaOH. The solution
was then extracted with chloroform. The organic layers were col-
lected, dried with anhydrous Na2SO4 and evaporated under
reduced pressure. The resulting crude oil (1.15 g, 96%) was used
without further purification for the synthesis of compounds 32
and 33.
1144, 1027, 752. 1H-NMR (DMSO-d6)
d 2.60–2.71 (m, 1H,
CHAHBCHCOOCH3), 3.10–3.30 (m, 1H, CHAHBCHCOOCH3), 3.46 (d,
J = 15.1 Hz, 1H, PhCH2NCHCHD), 3.54–3.56 (m, 1H + 3H,
PhCH2NCHCHD, COOCH3), 3.82–4.35 (m, 1H + 2H, CHCOOCH3,
PhCH2N), 7.20–7.57 (m, 5H, aromatic), 12.45 (s, 1H, NH), 13.43 (s,
1H, NH). Anal. (C18H17N3O3S2) C, H, N, S.
6-Ethyl-2-(methylthio)thieno[2,3-d]pyrimidin-4(1H)-one
4-Chloro-2-(methylthio)-6-propylthieno[2,3-d]pyrimidine
24
29
CH3I (1.52 mL, 24.41 mmol) was added to a suspension of monop-
otassium salt 16 (2.03 g, 8.11 mmol) in water (95 mL) and the
mixture was stirred at room temperature for 1.5 h. The solid was
then filtered off, washed with water, dried and recrystallised
from ethanol to give 24 as a pure solid. Yield: 1.50 g (82%); mp.
205–2078C; IR (KBr, selected lines) cm– 1 3056, 2961, 1661, 1563,
1476, 1287, 1188, 1115, 879, 764. 1H-NMR (DMSO-d6) d 1.26 (t, J =
7.4 Hz, 3H, CH3), 2.53 (s, 3H, SCH3), 2.82 (q, J = 7.4 Hz, 2H, CH2),
7.01 (s, 1H, aromatic), 12.70 (s, 1H, NH). Anal. (C9H10N2OS2) C, H,
N, S.
This compound was prepared from 2-methylthio derivative 25
by the same procedure as described for 28, with slight variations:
the solid, obtained after neutralization with a 10% solution of
NaOH, was filtered, washed with water, dried and recrystallised
from ethanol/cyclohexane to give 29 as a pure solid. Yield: 0.48 g
(65%); mp. 82–838C; IR (KBr, selected lines) cm– 1 2952, 1639,
1554, 1478, 1361, 1295, 1233, 1128, 875, 833. 1H-NMR (DMSO-d6)
d 0.95 (t, J = 7.2 Hz, 3H, CH3), 1.60–1.80 (m, 2H, CH2CH2CH3), 2.57
(s, 3H, SCH3), 2.91 (t, J = 7.2 Hz, 2H, CH2CH2CH3), 7.19 (s, 1H, aro-
matic). Anal. (C10H11ClN2S2) C, H, N, S.
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Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
New Thienopyrimidines as 5-HT Receptor Ligands
341
1.22–1.43 (m, 2H, CH2CH2CH2CH3), 1.51-1.70 (m, 2H, CH2CH2-
CH2CH3), 2.21 (s, 3H, NCH3), 2.38–2.50 (m, 4H + 3H, piperazine,
SCH3), 2.81 (t, J = 7.2 Hz, 2H, CH2CH2CH2CH3), 3.75–3.87 (m, 4H,
piperazine), 7.22 (s, 1H, aromatic). Anal. (C16H24N4S2) C, H, N, S.
6-Butyl-4-chloro-2-(methylthio)thieno[2,3-d]pyrimidine 30
This compound was prepared from 2-methylthio derivative 26
by the same procedure as described for 28. The resulting crude
oil (1.23 g, 85%) was used without further purification for the
synthesis of compounds 36 and 37.
4-(4-Methyl-1-piperazinyl)-2-(methylthio)-7-(phenyl-
methyl)-5,6,7,8-tetrahydropyrido[49,39:4,5]thieno[2,3-
4-Chloro-2-(methylthio)-7-(phenylmethyl)-5,6,7,8-
d]pyrimidine-5-carboxylic acid methyl ester 38
tetrahydropyrido[49,39:4,5]thieno[2,3-d]pyrimidine-5-
A mixture of 4-chloro derivative 31 (0.21 g, 0.48 mmol) and 1-
methylpiperazine (0.10 g, 0.99 mmol) in absolute ethanol
(10 mL) was refluxed and stirred for 1 h. After the mixture had
been cooled, the solvent was removed under reduced pressure.
The resulting oil was suspended in water and extracted with
ethyl acetate. The organic layers were collected, dried with anhy-
drous Na2SO4 and evaporated under reduced pressure. The sticky
residue obtained was triturated with diethyl ether, collected,
dried and recrystallised from n-hexane. Yield: 0.060 g (25%); mp.
123–1258C; IR (KBr, selected lines) cm– 1 2926, 2795, 1721, 1529,
1501, 1449, 1403, 1365, 1249, 1151. 1H-NMR (CDCl3) d 2.18 (s, 3H,
NCH3), 2.20–2.39 (m, 2H, piperazine), 2.52 (s, 3H, SCH3), 2.70–
2.90 (m, 1H, CHAHBCHCOOCH3), 2.98–3.18 (m, 2H + 1H, pipera-
zine, CHAHBCHCOOCH3), 3.25–3.45 (m, 4H, piperazine), 3.53 (s,
3H, COOCH3), 3.62-3.83 (m, 2H + 2H, PhCH2NCH2), 3.95–4.10 (m,
1H, CHCOOCH3), 7.20–7.39 (m, 5H, aromatic). Anal.
(C24H29N5O2S2) C, H, N, S.
carboxylic acid methyl ester 31
This compound was prepared from 2-methylthio derivative 27
by the same procedure as described for 28, with slight variations:
the refluxing time was 2.5 h and, the resulting solid was col-
lected, washed with water, dried and recrystallised from ethyl
acetate to obtain 31 as a pure solid. Yield: 0.42 g (81%); mp. 141–
1448C; IR (KBr, selected lines) cm– 1 1738, 1545, 1476, 1411, 1337,
1271, 1153, 839, 747, 699. 1H-NMR (DMSO-d6) d 2.60 (s, 3H, SCH3),
2
3
2.72 (dd, J = 12.1 Hz, J = 4.5 Hz, 1H, CHAHBCHCOOCH3), 3.25–
3.40 (m, 1H, CHAHBCHCOOCH3), 3.43–3.49 (m, 1H + 3H,
PhCH2NCHCHD, COOCH3), 3.56 (d, J = 13.6 Hz, 1H, PhCH2NCHCHD),
3.63–4.25 (m, 1H + 2H, CHCOOCH3, PhCH2N), 7.30-7.36 (m, 5H,
aromatic). Anal. (C19H18ClN3O2S2) C, H, N, S.
6-Ethyl-4-(4-methyl-1-piperazinyl)-2-
(methylthio)thieno[2,3-d]pyrimidine 32
A mixture of 1-methylpiperazine (0.65 mL, 5.86 mmol) and
4-chloro derivative 28 (0.80 g, 3.27 mmol) was heated under
reflux and stirred for 2.5 h in ethanol (10 mL). After the mixture
had been cooled, the solid was eliminated and the solution was
concentrated under reduced pressure. The resulting crude oil
was purified by flash column chromatography with methanol as
eluent. The homogeneous fractions were evaporated in vacuo to
furnish 32 as pure oil. Yield: 0.85 g (84%); IR (KBr, selected lines)
cm– 1 2929, 2792, 1549, 1511, 1444, 1350, 1298, 1142, 996, 881.
1H-NMR (DMSO-d6) d 1.26 (t, J = 7.6 Hz, 3H, CH2CH3), 2.21 (s, 3H,
NCH3), 2.38–2.50 (m, 4H + 3H, piperazine, SCH3), 2.85 (q, J =
7.6 Hz, 2H, CH2CH3), 3.75–3.85 (m, 4H, piperazine), 7.22 (s, 1H,
6-Ethyl-2-(methylthio)-4-(1-piperazinyl)thieno[2,3-d]-
pyrimidine 33
4-Chloro derivative 28 (0.44 g, 1.79 mmol) was added to a solu-
tion of piperazine (0.31 g, 3.59 mmol) in ethanol (15 mL). The
mixture was heated under reflux, stirred for 3 h and then
cooled. The resulting solid was eliminated by filtration and the
solution was concentrated under reduced pressure. The solid
obtained was collected with diethyl ether, dried and purified by
flash column chromatography with methanol as eluent. The
homogeneous fractions were evaporated in vacuo to afford 33 as
a pure solid. Yield: 0.22 g (42%); mp. 119–1218C; IR (KBr, selected
lines) cm– 1 3278, 2836, 1551, 1514, 1441, 1339, 1268, 1148, 998,
828. 1H-NMR (DMSO-d6) d 1.27 (t, J = 7.4 Hz, 3H, CH2CH3), 2.47 (s,
3H, SCH3), 2.73–2.95 (m, 2H + 4H, CH2CH3, piperazine), 3.70–
3.80 (m, 4H, piperazine), 7.20 (s, 1H, aromatic). Anal.
.
aromatic). Anal. (C14H20N4S2 H2O) C, H, N, S.
4-(4-Methyl-1-piperazinyl)-2-(methylthio)-6-
propylthieno[2,3-d]pyrimidine 34
.
(C13H18N4S2 H2O) C, H, N, S.
This compound was prepared from 4-chloro derivative 29 by the
same procedure as described for 32. The resulting crude oil was
purified by flash column chromatography with methanol/ethyl
acetate (3 : 7, v/v) as eluent. Yield: 0.63 g (60%); IR (KBr, selected
lines) cm– 1 2927, 1552, 1512, 1440, 1348, 1279, 1142, 995, 880,
770. 1H-NMR (DMSO-d6) d 0.94 (t, J = 7.2 Hz, 3H, CH2CH2CH3), 1.55-
1.76 (m, 2H, CH2CH2CH3), 2.22 (s, 3H, NCH3), 2.37–2.49 (m, 4H +
3H, piperazine, SCH3), 2.81 (t, J = 7.2 Hz, 2H, CH2CH2CH3), 3.77-
3.83 (m, 4H, piperazine), 7.24 (s, 1H, aromatic). Anal. (C15H22N4S2)
C, H, N, S.
2-(Methylthio)-4-(1-piperazinyl)-6-propylthieno[2,3-d]-
pyrimidine 35
This compound was prepared from 4-chloro derivative 29 by the
same procedure as described for 33. The crude solid obtained
was purified by flash column chromatography with methanol/
ethyl acetate (3 : 7, v/v) as eluent. Yield: 0.41 g (71%); mp. 93–
958C; IR (KBr, selected lines) cm– 1 2931, 2793, 1550, 1512, 1445,
1348, 1294, 1142, 997, 881. 1H-NMR (DMSO-d6) d 0.94 (t, J = 7.2 Hz,
3H, CH2CH2CH3), 1.53–1.76 (m, 2H, CH2CH2CH3), 2.47 (s, 3H,
SCH3), 2.70-2.90 (m, 4H + 2H, piperazine, CH2CH2CH3), 3.70-3.78
6-Butyl-4-(4-methyl-1-piperazinyl)-2-
(methylthio)thieno[2,3-d]pyrimidine 36
.
(m, 4H, piperazine), 7.21 (s, 1H, aromatic). Anal. (C14H20N4S2 H2O)
C, H, N, S.
This compound was prepared from 4-chloro derivative 30 by the
same procedure as described for 32. The crude oil obtained was
purified by flash column chromatography with methanol/ethyl
acetate (3 : 7, v/v) as eluent. Yield: 0.66 g (60%); IR (KBr, selected
lines) cm– 1 2929, 2852, 2792, 1512, 1443, 1349, 1298, 1142, 997,
885. 1H-NMR (DMSO-d6) d 0.90 (t, J = 7.2 Hz, 3H, CH2CH2CH2CH3),
6-Butyl-2-(methylthio)-4-(1-piperazinyl)thieno[2,3-d]-
pyrimidine 37
This compound was prepared from 4-chloro derivative 30 by the
same procedure as described for 33. The sticky product obtained
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342
M. N. Modica et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
was purified by flash column chromatography with methanol/
ethyl acetate (3 : 7, v/v) as eluent. Yield: 0.11 g (20%); mp. 70–
728C; IR (KBr, selected lines) cm– 1 3268, 2922, 1553, 1509, 1437,
1336, 1143, 992, 878, 805. 1H-NMR (DMSO-d6) d 0.93 (t, J = 7.2 Hz,
3H, CH2CH2CH2CH3), 1.26–1.48 (m, 2H, CH2CH2CH2CH3), 1.55–
1.73 (m, 2H, CH2CH2CH2CH3), 2.49 (s, 3H, SCH3), 2.72–2.95 (m, 2H
+ 4H, piperazine, CH2CH2CH2CH3), 3.68–3.95 (m, 4H, piperazine),
7.24 (s, 1H, aromatic). Anal. (C15H22N4S2) C, H, N, S.
through GF/B filters which were then washed with 12 mL
(463 times) of ice-cold 50 mM Tris HCl, pH 7.4, or 50 mM
Hepes HCl, pH 7.4, using a Brandel M-48R cell harvester (Gai-
thersburg, MD, USA). Dried filters were immersed in vials con-
taining 4 mL of Ultima Gold MV (Packard Instruments, USA) and
counted in a Wallac 1409 liquid scintillation spectrometer (Wal-
lac-PerkinElmer, USA) with a counting efficiency of about 50%.
Drugs were tested in triplicate at different concentrations (from
10– 5 to 10– 10 M) and dose-inhibition curves were analysed by the
Allfit program [35] to obtain the concentration of unlabelled
drug that caused 50% inhibition of ligand binding. Ki values
were derived from the IC50 values [36].
2-(Methylthio)-7-(phenylmethyl)-4-(1-piperazinyl)-5,6,7,8-
tetrahydropyrido[49,39:4,5]thieno[2,3-d]pyrimidine-5-
carboxylic acid methyl ester 39
A mixture of 4-chloro derivative 31 (0.16 g, 0.37 mmol) and
piperazine (0.16 g, 1.90 mmol) was refluxed and stirred for 1 h
in absolute ethanol (5 mL). After the mixture had been cooled,
the solvent was removed under reduced pressure to furnish an
oil, which was suspended in water and extracted with diethyl
ether. The organic layers were collected, dried with anhydrous
Na2SO4 and evaporated under reduced pressure to afford 39 as
pure oil. Yield: 0.080 g (46%); IR (KBr, selected lines) cm– 1 2921,
In vitro functional studies on isolated guinea pig
colon
The animals were treated in accordance with the European Com-
munities Council Directives (86/609/ECC) and the Hungarian Act
for the Protection of Animals in Research (XXVIII.tv.32.§). All
experiments involving animal subjects were carried out with
the approval of the Hungarian Ethical Committee for Animal
Research (registration number: IV/1813-1/2002). Crl(HA)BR male
guinea pigs (Charles-River Laboratories, Hungary) were kept at
22 l 38C; the relative humidity was 30–70% and the light/dark
cycle was 12/12 h. They were maintained on a standard guinea
pig pellet diet (Charles-River Laboratories, Hungary) with tap
water available ad libitum. The animals were sacrificed by CO2
inhalation. The distal portion of the colon was removed from a
Hartley guinea pig (400–500 g) starved 24 h before experiments.
The colon was cleaned in Krebs bicarbonate buffer (in mM: NaCl
118.4, KCl 4.7, CaCl2 2.5, NaHCO3 25, MgSO4 1.2, KH2PO4 1.2, glu-
cose 11.7; pH 7.4) at room temperature and cut into 2 cm seg-
ments. The segments were suspended longitudinally in an organ
bath containing Krebs bicarbonate buffer warmed to 378C and
bubbled through with 95% O2/5% CO2. An amount of 1 g of load-
ing tension was applied; the tissues were left to be incubated for
1 h. Isometric contractions were detected with the ISOSYS Data
Acquisition System (Experimetria Ltd., Hungary). The contractile
action of the selective 5-HT3R agonist 2-Me-5-HT (Sigma, Hun-
gary) was investigated in a cumulative way. Results were
expressed as percentage contraction increases compared with
the basal colon activity. Compound 32 and the specific 5-HT3RA
tropisetron (Sigma), 10 nM each were added to the bath 10 min
before the application of 2-Me-5-HT. Dose–response curves were
fitted to the contraction-increasing effects. The EC50 maximum
inhibitions were calculated and statistics were determined by
means of Prism 4.0 software (GraphPad Software, USA), using
the ANOVA Newman-Keuls test.
1
2808, 1735, 1650, 1500, 1449, 1364, 1260, 1156, 986. H-NMR
(CDCl3) d 2.59 (s, 3H, SCH3), 2.78–2.95 (m, 4H, piperazine), 3.10–
3.30 (m, 4H, piperazine), 3.38–3.50 (m, 2H, CH2CHCOOCH3), 3.61
(s, 3H, COOCH3), 3.64–3.68 (m, 2H + 2H, ArCH2NCH2), 4.03–4.15
(m, 1H, CHCOOCH3), 7.24–7.39 (m, 5H, aromatic). Anal.
(C23H27N5O2S2) C, H, N, S.
Binding assays
Male CRL:CD(SD)BR-COBS rats (about 150 g, Charles River Labora-
tories, Italy) and male CRL:(HA) BR albino guinea pigs (about
300 g, Charles River Laboratories, Italy) were killed by decapita-
tion; their brains were rapidly dissected into the various areas
(the rat cortex for 5-HT3Rs and the guinea pig striatum for
5-HT4Rs) and stored at –808C until the day of assay.
Tissues were homogenized in 50 volumes of 50 mM ice-cold
Tris HCl, pH 7.4 containing 0.50 mM EDTA and 10 mM MgSO4
for 5-HT3Rs or 50 mM Hepes HCl, pH 7.4, for 5-HT4Rs, using an
Ultra Turrax TP-1810 homogenizer (2620 s) (Janke &. Kunkel,
Staufen, Germany), and the homogenates were centrifuged at
50000 g for 10 min (Beckman Avanti J-25 refrigerated centri-
fuge; Beckman, USA). Each pellet was resuspended in the same
volume of fresh buffer, incubated at 378C for 10 min and centri-
fuged again at 50000 g for 10 min. The pellet was then washed
once by resuspension in fresh buffer and centrifuged as before.
The pellet obtained was finally resuspended in the appropri-
ate incubation buffer: 50 mM Hepes HCl, pH 7.4, containing
10 lM pargyline for 5-HT4Rs, and 50 mM Tris HCl, pH 7.4, con-
taining 10 lM pargyline, 0.50 mM EDTA, 10 mM MgSO4, 0.1%
ascorbic acid and 140 mM NaCl for 5-HT3Rs.
5-HT3AR modelling
The [3H]LY 278584 [33] (84.0 Ci/mmol Amersham (USA), 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 displacing agent or solvent;
non-specific binding was measured in the presence of 1 lM qui-
pazine. The [3H]GR 113808 [34] (84.0 Ci/mmol, Amersham, for
5-HT4) binding was assayed in a final incubation volume of
1.0 mL, consisting of 0.50 mL of tissue (20 mg/sample), 0.50 mL
of the [3H]ligand (0.1 nM) and 0.02 mL of displacing agent or sol-
vent; non-specific binding was measured in the presence of
10 lM 5-HT. Incubations (30 min at 258C for 5-HT3Rs or 30 min at
378C for 5-HT4Rs) were stopped by rapid filtration under vacuum
The extracellular region of murine 5-HT3ARs was built up on the
basis of the recently published three-dimensional structure of
the nACh receptor [37] (protein data bank entry code 2BG9), as
described [27]. Briefly, multiple sequence alignments were per-
formed by “ClustalW” software. Homology model building of
the extracellular region of murine 5-HT3ARs based on the a and d
subunits of the nACh receptor and its refinement were carried
out using the Jackal protein structure modelling package on a
Silicon Graphics Octane workstation under Irix 6.5 operation
system. The assembled homopentamer was energy-minimized.
The quality of the model was verified using Procheck. Docking
calculations were carried out at the interface of the a-subunit
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Arch. Pharm. Chem. Life Sci. 2008, 341, 333–343
New Thienopyrimidines as 5-HT Receptor Ligands
343
based (“lid-open”) and d-subunit based (“lid-shut”) dimeric part
of the pentamer. AutoDock 3.0 [38] was applied for docking cal-
culations, using the Lamarckian genetic algorithm (LGA) and
the pseudo-Solis and Wets (pSW) methods. Gasteiger–Huckel
partial charges were applied both for ligands and proteins. Sol-
vation parameters were added to the protein coordinate file and
the ligand torsions were defined using the “Addsol” and “Auto-
tors” utilities, respectively. Random starting positions, orienta-
tions and torsions (for flexible bonds) were used for the ligands.
Each docking run consisted of 100 cycles. Final structures with
rmsd a2 ꢀ were considered to belong in the same docking clus-
ter.
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www.archpharm.com