High affinity and selectivity of [[(arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives for the 5-HT1(A) receptor. Synthesis and structure - Affinity relationships

Article abstract of DOI:10.1016/S0223-5234(00)00175-6

In this work we report the affinity of new thienopyrimidinones for 5- HT₁(A)Rs and the selectivity versus α(1A)Rs. The 3-amino-2-[[3-[4-(2- methoxyphenyl)-1-piperazinyl]propyl]thio]-6-ethyl-thieno[2,3-d]pyrimidin- 4(3H)-one 27 is the most potent and selective (Ki 0.19 nM, selectivity 115). Compound 31 with the N4 piperazine orthonitrophenyl nucleus instead of the orthomethoxyphenyl also shows a good affinity and selectivity (Ki 1.46 nM, selectivity 84). The results of derivatives 28, 29 and 30 (Ki 3.28, 12.59 and 4.38 nM; selectivity 24, 4 and 5, respectively), which have, respectively, an ethyl, an allyl and an acetylamino group instead of an N3 amino group, indicate the importance of this last group for the interaction with 5- HT(1A)R. Comparison of the results for the superior homologue 53 (Ki 3.72 nM, selectivity 51) and the inferior homologue 52 (5-HT(1A) Ki 1 499 nM, α₁A Ki NA) of 2-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-6,7-dimethyl-8H- [1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidiN-8-one 57 (Ki 23 nM, selectivity 5) shows how important the length of the chain binding the two heterocyclic systems is in the interaction with 5-HT(1A)Rs and α₁ARs. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00175-6

Eur. J. Med. Chem. 35 (2000) 677−689
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001756/FLA
Original article
High affinity and selectivity of [[(arylpiperazinyl)alkyl]thio]thieno[2,3-
d]pyrimidinone derivatives for the 5-HT1A receptor.
Synthesis and structure–affinity relationships
Maria Modicaa, Maria Santagatia*, Filippo Russoa, Carlo Selvagginib, Alfredo Cagnottob,
Tiziana Menninib
aDipartimento di Scienze Farmaceutiche, Università di Catania, Viale A. Doria 6, 95125 Catania, Italy
bIstituto di Ricerche Farmacologiche “Mario Negri”, Via Eritrea 62, 20157 Milan, Italy
Received 15 November 1999; revised and accepted 3 February 2000
Abstract – In this work we report the affinity of new thienopyrimidinones for 5-HT1ARs and the selectivity versus α1ARs. The
3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]thio]-6-ethyl-thieno[2,3-d]pyrimidin-4(3H)-one 27 is the most potent and selective
(Ki 0.19 nM, selectivity 115). Compound 31 with the N4 piperazine orthonitrophenyl nucleus instead of the orthomethoxyphenyl also shows
a good affinity and selectivity (Ki 1.46 nM, selectivity 84). The results of derivatives 28, 29 and 30 (Ki 3.28, 12.59 and 4.38 nM; selectivity
24, 4 and 5, respectively), which have, respectively, an ethyl, an allyl and an acetylamino group instead of an N3 amino group, indicate the
importance of this last group for the interaction with 5-HT1AR. Comparison of the results for the superior homologue 53 (Ki 3.72 nM,
selectivity 51) and the inferior homologue 52 (5-HT1A Ki 1 499 nM, α1A Ki NA) of 2-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-6,7-
dimethyl-8H-[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one 57 (Ki 23 nM, selectivity 5) shows how important the length of the chain
binding the two heterocyclic systems is in the interaction with 5-HT1ARs and α1ARs. © 2000 Éditions scientifiques et médicales Elsevier SAS
5-HT1A receptor / ligands / thienopyrimidinones
1. Introduction
[1]. Radioligand binding assays showed compound 1 as
the derivative with the highest affinity and compound 2 as
the one with the best selectivity (Ki 0.16 and 3.72 nM;
selectivity 39 and 117, respectively) (figure 1). The present
paper reports the synthesis and in vitro assays for the
5-HT1AR and the α1AR of new compounds 3 and 4
(figure 1), whose structures and structural variations were
referred to compound 1, chosen as the lead.
In a recent paper [1] we described new arylpiperazi-
nylalkylthiothienopyrimidinone derivatives prepared and
tested with the specific aim of developing ligands with
higher affinity and selectivity for the 5-HT1A receptors
rather than the α1-adrenoreceptors. Selective ligands for
the 5-HT1AR versus the α1AR might help clarify the
structural requirements distinguishing the two receptors,
which are representative members of the same G protein
superfamily, showing some common features in their
binding sites. Moreover antianxiety and antidepressant
arylpiperazine compounds such as buspirone or NAN-
190 show high affinity for the 5-HT1AR but also act on
α1AR, therefore selective 5-HT1AR ligands might help
explain the role of 5-HT1AR in anxiety and depression
The aim is that of obtaining compounds with still high
affinity and with an improved selectivity for the 5-HT1AR
versus the α1AR and also of acquiring further structure-
–affinity relationships.
Our interest is mainly addressed to the non-
pharmacophoric part because the interaction of the
arylpiperazine pharmacophore with the 5-HT1AR is well
known [2]. With reference to the type 3 compounds,
modifications performed on lead 1 refer in particular to: i)
the variation of substituents 5 and 6 of the thienopyrimi-
dine nucleus; the substitution of the thienopyrimidine
* Correspondence and reprints: msantaga@mbox.unict.it
Abbreviations: 5-HT1ARs: 5-HT1A receptors; α1ARs: α1A
receptors; SD: standard deviation

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M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
Figure 1. 1 and 2 are the most
interesting compounds, already re-
ported in our previous work [1] and
3 and 4 are the general structures of
new compounds (n = 2, is published
[1]).
system with other systems such as the pyridopyrimidine,
pyrazolopyrimidine and the indolopyrimidine ones; ii)
the substitution of the amino group in position 3 of the
pyrimidine ring with the ethyl, allyl, and acetylamino
groups, to verify and clarify which structural features of
the N3 substituents affect the 5-HT1A ligand–receptor
interaction and iii) the substitution on the N4 piperazine
ring of the orthomethoxyphenyl nucleus with the orthoni-
trophenyl, bioisoster of the N4 pyrimidine nucleus, present
in the most selective compound 2 of the previous work
[1]. Moreover, believing that the higher molecule rigidity
might privilege structures with higher affinity and selec-
tivity, the influence of the chain length on the thiadiazo-
lothienopyrimidine system (compounds 4, figure 1) was
studied.
2. Chemistry
Compounds 6–32 were prepared according to figure 2.
The potassium salts of the 2-thioxothieno[2,3-
d]pyrimidine derivatives 13–18 [1, 3, 4] reacted at reflux
in ethanol with the appropriate chloroalkyl compounds
23–25 [5] to give the respective 2-(alkylthio)thieno[2,3-

M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
679
Figure 2. General synthetic procedure for compounds 26–32. Reagents and conditions: (a) CSCl2, NaHCO3, CHCl3/H2O; (b)
.
N2H4 H2O, allylamine or acethydrazine, CH2Cl2, r.t.; (c) KOH, EtOH, reflux; (d) HCl, H2O, r.t.; (e) Cl(CH2)3Br, K2CO3, DMF, r.t.;
(f) EtOH, reflux.
d]pyrimidinones 26–32. The new potassium salts 13, 14
and 17 were obtained by heating of derivatives 9, 10 and
12 in an ethanolic potassium hydroxide solution. Com-
pounds 9 and 10 were synthesized from the isothiocyan-
ates 6 and 7 [6] and hydrazine hydrate and compounds 11
and 12 from isothiocyanate 8 and allylamine and acethy-
drazine, respectively, in dichloromethane at room tem-
perature. Analytical and spectral data of compound 11
were like those of the known compound synthesized with
another procedure [3].
Acidification of an aqueous solution of the potassium
salts 13, 14 and 17 gave the thioxo compounds 19–21,
whose structures were substantiated by elemental analy-
ses and IR spectra. Isothiocyanate 6 was obtained by
reaction of diethyl ester of 5-amino-3-methyl-2,4-
thiophenedicarboxylic acid 5 with thiophosgene in a
solution of chloroform/water. The unknown piperazine 24
was obtained by reaction of 1-(2-nitrophenyl)piperazine
[7] and 1-bromo-3-chloropropane in dimethylformamide
in the presence of potassium carbonate.
The arylpiperazinylalkylthiopyrimidinones 42–45 were
prepared by condensation between chloroalkylpiperazine
23 [5] and the monopotassium salts of 3-amino-2-
thioxopyrido[2,3-d]pyrimidin-4(1H)-one 37, of 5-amino-
1,5,6,7-tetrahydro-1-methyl 38 and 5-amino-1,5,6,7-
tetrahydro-1-(phenyl-methyl)-6-thioxo-4H-pyrazolo[3,4-
d]pyrimidin-4-one 39 [8], and of 3-amino-1,2,3,5-
tetrahydro-2-thioxo-4H-pyrimido[5,4-b]indol-4-one 40
[9] (figure 3). The unknown salts 37 and 38 were prepared
from isothiocyanates 33 [10] and 35 [11] and hydrazine
hydrate in dichloromethane at room temperature, and
subsequent treatment with an ethanolic potassium hy-
droxide solution of the thioxoderivative 34 and of the
(hydrazinothioxomethyl)amino derivative 36. Acidifica-
tion of an aqueous solution of the salt 38 gave the thioxo

680
M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
.
Figure 3. General synthetic procedure for compounds 42–45. Reagents and conditions: (a) N2H4 H2O, CH2Cl2, r.t.; (b) KOH, EtOH,
reflux; (c) HCl, H2O, r.t.; (d) 1-(3-chloropropyl)-4-(2-methoxyphenyl)piperazine 23, EtOH, reflux.
compound 41, whose structure was substantiated by
elemental analysis and IR spectrum (figure 3).
methoxyphenyl)piperazine to give compounds 52 and 53
(figure 5). The proposed structures of compounds 26–32,
42–45, 49, 52 and 53 were confirmed by elemental
analyses (tables I and II) and by spectroscopic IR data
and 1H-NMR spectra of some representative samples (see
the experimental protocols).
Treatment of the amino ester 46 [12] with chlorov-
aleryl chloride gave the chloroderivative 47, which re-
acted with 1-(2-methoxyphenyl)piperazine to give com-
pound 48; lastly, by reaction between derivative 48 and
hydrazine monohydrate at reflux in ethanol was synthe-
sized the alkylpyrimidinone 49 (figure 4).
3. Pharmacology
Monopotassium salt 18 with 2-chloroacetylchloride
and 4-chlorobutyrylchloride gave the 2-chloromethyl and
the 2-chloropropyl-thiadiazolothienopyrimidinones 50 and
51, respectively, which reacted at 140 °C with 1-(2-
The compounds 26–32, 42–45, 49, 52 and 53 were
evaluated for in vitro affinity on 5-HT1A and α1A
Figure 4. Synthesis of arylpiperazinylalkylthieno[2,3-d]pyrimidine 49. Reagents and conditions: (a) ClCO(CH2)4Cl, CHCl3, reflux;
.
(b) 1-(2-methoxyphenyl)piperazine, K2CO3, DMF, reflux; (c) N2H4 H2O, EtOH, reflux.

M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
681
Figure 5. Synthesis of [1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidinones 52 and 53. Reagents and conditions: (a) ClCO(CH2)nCl,
CH3SO3H, P2O5; (b) 1-(2-methoxyphenyl)piperazine.
dopyrimido and the quinazoline [1] moieties. Changes in
this system affect affinity and selectivity. In fact, the only
compound in the whole 26–32, 42–45, 49, 52 and 53
series with the same affinity of the lead 1 is 27 (Ki
0.19 nM), where a hydrogen and an ethyl replace the two
methyls in positions 5 and 6. As for compound 26, where
there is a methyl in position 5 and an ethoxycarbonyl in
position 6, the affinity is decreased (Ki 1.36 nM); except
for the pyrido derivative 42 (Ki 0.38 nM), the same
applies to 43, 44 and 45, where the pyrazole and indole
heterocycles are used in place of thiophene (Ki 0.82, 2.4
and 3.72 nM). The selectivity of compounds 26, 42 and
especially of compound 27 is better than the lead 1
(selectivity 53, 72, 115 and 39, respectively). These data
and that of the quinazoline derivative 58 [1] (Ki 0.27 nM,
selectivity 12) show how, in the 5-HT1AR ligand interac-
tion, steric factors play a role on the non-pharmacophoric
part condensed with the pyrimidinone moiety rather than
receptors by radioligand binding assays. For comparison
data for lead 1 and compounds 2 and 54–58 previously
published are included [1]. The results are shown in
tables III and IV.
Table V reports the affinity (Ki) of some structures (26,
27, 31, 42, 49 and 53; representative of those most active
on the 5-HT1AR and selective towards the α1AR, com-
pound 1 was also included for comparison) for other
5-HT receptor subtypes and D1 and D2 dopaminergic
receptors.
4. Results and discussion
In general, binding assays show that the thienopyrimi-
dine system, with adequate substituents, is the most
favourable structure for affinity towards the 5-HT1ARs,
when compared to other systems, except for the pyri-
Table I. Physical and chemical properties of compounds 26–32 and 49.
Compound
R1
R2
R3
M.p. °C
Recryst. solv.
% yield
Formula
26
27
28
29
Me
H
COOC2H5
C2H5
Me
NH2
NH2
C2H5
143–144
112–114
103–105
104–105
210–212
153–155
108–110
136–138
EtOH 22
EtOH 40
cyclohexane 22
EtOH 40
EtOH/H2O 31
EtOH 21
EtOH 24
C24H31N5O4S2
C22H29N5O2S2
C24H32N4O2S2
C25H32N4O2S2
C24H31N5O3S2
C21H26N6O3S2
C13H20N4OS2
C23H31N5O2S
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
CH2CH=CH2
NHCOCH3
NH2
30
31a
32b
49c
NH2
NH2
Me
EtOH 52
a The orthomethoxy is replaced by a orthonitro group; b The phenylpiperazine system is replaced by an N(CH3)2 group; c The S in the alkyl
chain in this case is substituted by a methylene group.

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M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
Table II. Physical and chemical properties of compounds 42–45, 52 and 53.
Compound
R
n
3
M.p., °C
174–176
Recryst. solv. % yield Formula
42
EtOH 21
C21H26N6O2S
43
44
3
3
155–157
69–71
EtOH 39
C20H27N7O2S
EtOH 35
C26H31N7O2S
45
52
3
1
192–193
183–185
EtOH/dioxane 40
EtOH 55
C24H28N6O2S
C21H23N5O2S2
53
3
144–154
EtOH 59
C23H27N5O2S2
electronic factors, towards which the α1AR seem to be
more sensitive.
the region near the R3 position of the ligands, several
negatively charged O and N atoms are present which may
act as hydrogen bond acceptors.
The binding tests of compounds 28–30, with the N3
ethyl, allyl and acetylamino groups, indicate the favour-
able effect of the N3 amino group on the affinity and
selectivity towards the 5-HT1AR; these three compounds
show less affinity and selectivity (Ki 3.28, 12.59 and
4.38 nM, selectivity 24, 4 and 5, respectively) than the
lead 1 and the compound 27.
Molecular modelling approaches through the utiliza-
tion of 3-D-QSAR methodologies PARM (Pseudo Atomic
Receptor Model), HASL (Hypothetical Active Site Lat-
tice) and CoMFA (Comparative Molecular Field Analy-
sis) upon 23 molecules belonging to the previous work
[1], were found to provide predictive 5-HT1A receptor
models [13, 14]. The PARM receptor model shows that at
A hydrogen bond interaction or even an electrostatic
interaction might be supposed, between our N3 substitu-
ents and the 5-HT1A receptor site; such an interaction
might also be influenced by steric factors (Guccione S. et
al., 1999 QSAR, Gordon Research Conference, Tilton,
New Hampshire) and is at its maximum level in the
amino group, while it decreases in the sequence methyl
55, ethyl 28, acetylamino 30, allyl 29, hydrogen 54, and
aniline 56 groups (Ki 1.64, 3.28, 4.38, 12.59, 13.14 and
272 nM, selectivity 14, 24, 5, 4, 2.6 and 0.10, respec-
tively). With reference to the α1AR, the results of these
compounds are less explicit; however, an electrostatic
interaction at the R3 region seems not as important and

M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
683
Table III. Affinities of compounds 1, 2, 26–32, 49, 54–56 and of two reference compounds.
K (nM)(± SD)a
α1-Ac
selectivityd
b
Compound
R1
R2
R3
5-HT1A
1e
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
COOC2H5
C2H5
Me
NH2
NH2
NH2
NH2
C2H5
0.16 ± 0.01
3.72 ± 0.27
1.36 ± 0.2
0.19 ± 0.05
3.28 ± 0.27
12.59 ± 7.66
4.38 ± 1.09
1.46 ± 0.2
2951 ± 816
0.32 ± 0.02
13.14 ± 1.09
1.64 ± 0.08
272 ± 9.3
6.26 ± 0.85
434 ± 83
39
117
53
115
24
4
5
84
/
56
2.6
14
0.10
2e, f
26
72.08 ± 21.4
21.96 ± 6.16
79.8 ± 28
51.48 ± 8.5
20.59 ± 5.1
123 ± 13
27
28
29
Me
Me
Me
Me
Me
Me
Me
Me
CH2CH=CH2
NHCOCH3
NH2
30
31g
32i
NH2
NH2
H
Me
NAh
49l
18.02 ± 4.2
34.32 ± 4.29
23 ± 4.3
54e
55e
56e
NHC6H5
27 ± 4.3
Serotonin
Prazosin
1.47 ± 0.1
3.86 ± 0.68
c
a Ki values (nM) are followed by SD. b Affinity at [3H]-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)-labelled 5-HT1A sites.
Affinity at [3H]prazosin-labelled α1-adrenergic sites. d Selectivity for 5-HT1A over α1-adrenergic sites: calculated as Ki (α1A)/Ki (5-HT1A).
e Previously published data [1], here reported for comparison as Ki. f The orthomethoxyphenyl is replaced by a pyrimidine nucleus. g The
orthomethoxy is replaced by an orthonitro group. h < 50% inhibition at 10–5 M. i The phenylpiperazine system is replaced by an N(CH3)2
group. l The S in the alkyl chain is substituted by a methylene group.
the steric features of the N3 substituents are less condi-
tioning, as indicated in the α1AR 3-D-QSAR models [13,
14].
shows a very small decrease in affinity and a slight
improvement in selectivity (Ki 0.32 nM; selectivity 56).
In the arylpiperazinylalkylthiadiazolothienopyrimi-
dinone series, the compound 53 shows a greater affinity
and selectivity towards the 5-HT1AR (Ki 3.72 nM, selec-
tivity 51) than the inferior homologue 57 (Ki 23 nM;
selectivity 5); compound 52, with only one methylene,
has a significant fall in affinity for both the 5-HT1AR (Ki
1 499 nM) and the α1AR (Ki NA). Consequently, it
seems that in these compounds, a chain length of three
carbon atoms is optimum for 5-HT1AR affinity and also
for selectivity. These three products display not only a
lower, but also a less differentiated affinity for α1AR. In
fact, the affinity for the 5-HT1AR as a function of the
methylene linker length decreases in the order 3 > 2 >> 1,
while for the α1AR, the affinity decreases in the order
2 = 3 >> 1. Moreover these data confirm that the presence
of the N3 amino group improves the interaction with the
5-HT1AR binding site.
Compound 31, which has the N4 orthonitrophenyl
nucleus in place of the orthomethoxyphenyl, shows an
affinity towards the 5-HT1AR lower than that of lead, but
a good increase in selectivity (Ki 1.46 nM, selectivity
84). Instead, the few data known in the literature [15, 16]
report that the ortho substitutions in the N4 phenyl ring
by electron withdrawing groups are favourable to both
receptors. In our previous study [1], the most selective
compound was the N4 pyrimido derivative 2 (Ki 3.72 nM,
selectivity 117). These results suggest that, in such
structures, the steric and/or electronic properties of the
N4 piperazine substituent may affect the interaction of the
ligand, especially with α1AR.
In compound 32, the substitution of the arylpiperazine
nucleus with the dimethylamine group leads to a loss of
affinity towards both receptors (5-HT1A Ki 2 951 nM and
α1A Ki NA). This confirms that the arylpiperazine
nucleus is the fundamental pharmacophore.
The only substitution in the thioalkyl chain of sulphur
with a methylene is not influential; in fact, compound 49
Compounds 26, 27, 31, 42, 49 and 53 show good
selectivity towards 5-HT1B, 5-HT2A, 5-HT2C, 5-HT3 and
D1 receptors, with the exception of 53 which has an
affinity for the 5-HT1B and the 5-HT2A receptors 24 and

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M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
Table IV. Affinities of compounds 42–45, 52, 53, 57, and 58.
Compound
R
n
3
Ki (nM) (±SD)
5–HT1A
α1–A
selectivity
72
42
0.38 ± 0.07
27.28 ± 2.5
43
44
3
3
0.82 ± 0.07
16.9 ± 4.29
21
5
2.4 ± 0.16
11.67 ± 2.83
45
52
3
1
3.72 ± 0.38
1 499 ± 365
58.95 ± 11.9
16
/
NAa
53
3
2
3
3.72 ± 0.2
23 ± 3.3
189 ± 28
107 ± 35
3.4 ± 0.7
51
5
57b
58b
0.27 ± 0.02
12
a < 50% inhibition at 10–5 M. b Previously published data [1], here reported for comparison as Ki.
50 times lower, respectively, than for the 5-HT1AR.
Compounds 26, 31, 49 and 53 show the worst selectivity
for the D2 receptor with an affinity about 8–50 times
lower than for 5-HT1AR.
In conclusion, the continuation of our researches on
new potent and selective arylpiperazinylalkylth-
ienopirimidinone derivatives for the 5-HT1AR rather than
the α1AR, gives further information about the structure-
–affinity relationships. The results indicate the impor-
tance of the non-pharmacophoric thienopyrimidine por-
tion for affinity and selectivity towards 5-HT1AR. The
highest affinity and selectivity is obtained with derivative
27, structurally similar to the lead 1, followed by the
isosteric pyridopyrimidinone 42; these compounds also

M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
685
Table V. Ki (nM) values in binding tests of compounds 1, 26, 27, 31, 42, 49 and 53 on 5-HT1A, 5-HT1B, 5-HT2A, 5-HT2C and 5-HT3 receptors
and on D1 and D2 receptors.
Compound
5-HT1A
5-HT1B
5-HT2A
5-HT2C
5-HT3
D1
D2
1a
0.16
1.36
0.19
1.46
0.38
0.32
3.72
385 (2 406)b
316 (232)
720 (3 789)
4 020 (1 472)
1 648 (4 337)
949 (2 966)
188d(24)
170 (1 062)
299 (220)
239 (1 258)
219 (80)
460 (1 210)
200 (625)
382 (50)
405 (2 531)
1 587 (1 167)
1 787 (9 405)
831 (304)
400 (2 500)
NAc
35 (219)
1 397 (1 027)
296 (1 558)
456 (167)
1 175 (3 092)
978 (3 056)
2 591 (338)
27 (169)
49 (36)
56 (295)
105 (38)
115 (303)
19 (59)
26
27
31
42
49
53
257 (1 353)
2 278 (834)
NAc
300 (789)
1 373 (4 291)
NAc
5 427d (708)
NAc
63 (8)
a Previously published data [1], here reported for comparison as Ki. b Values in parentheses are the selectivity for the 5-HT1A receptor vs. the
other serotonin and dopamine receptors calculated as Ki (x)/Ki (5-HT1A). c < 50% inhibition at 10–5 M. For the sake of clarity SD are omitted
since they were always less than 10%. c, d The dose–response curve was better fitted by a two-component model, with the following results:
c
d
site I, Ki 49 nM; site II, Ki > 10 000 nM, proportion site II 34%; site I, Ki 628 nM; site II, Ki > 10 000 nM, proportion site II 49%.
show a remarkable selectivity for the 5-HT1B, 5-HT2A
,
on an EA1108 elemental analyzer Fisons-Carlo Erba
instrument and were within ± 0.4% of the theoretical
values. The purity of the compounds was checked by
thin-layer chromatography (TLC) on Merck silica gel 60
F-254 plates.
5-HT2C, 5-HT3, D1 and D2 receptors. The N4 orthonitro-
phenyl derivative 31 shows a good selectivity for 5-HT1AR
over the α1AR.
The presence in the compounds 28, 29 and 30 of an
ethyl, an allyl and an acetylamino group, instead of the
N3 amino group, causes a decrease in both affinity and
selectivity, showing the importance of the amino group
for the interaction with the 5-HT1A receptor binding site.
The binding data of the thienopyrimidothiadiazole
derivatives 52, 53 and 57 show that the chain length in
these structures affects the affinity for both receptors with
a diversifying modulation, leading to interesting results.
In order to determine the most critical features to
design selective and potent ligands which can improve
and provide the 5-HT1A receptor model, non-ambiguous
and distinct from the α1A receptor one, conformational
analysis and 3-D-QSAR studies about more significant
compounds of this series are in progress.
5.1.1. 5-Isothiocyanato-3-methyl-2,4-
thiophenedicarboxylic acid diethyl ester 6
A solution of amino ester 5 (12.85 g, 50 mmol) in
chloroform (100 mL) was added under stirring to a
mixture of thiophosgene (3.8 mL, 50 mmol), sodium
hydrogencarbonate (6 g, 71.4 mmol), water (50 mL), and
chloroform (200 mL) for 40 min. The mixture was stirred
at room temperature for 1.3 h, then the organic layer was
separated and washed twice with water. The organic layer
was dried over anhydrous sodium sulfate and evaporated
under reduced pressure. The solid was purified by column
chromatography (silica gel, cyclohexane/ethyl acetate
80:20) to give 6 (6 g, 40%); m.p. 76–78 °C; IR (KBr)
2 120 (N=C=S), 1 730 (C=O) cm–1. 1H-NMR (CDCl3) δ
1.26–1.39 (m, 6H, CH2CH3), 2.64 (s, 3H, CH3), 4.20–4.37
(m, 4H, CH2CH3). Anal. (C12H13NO4S2) C, H, N, S.
5. Experimental protocols
5.1. Chemistry
5.1.2. 5-[(Hydrazinothioxomethyl)amino]-3-
methyl-2,4-thiophenedicarboxylic acid diethyl ester 9
A solution of isothiocyanate 6 (2.8 g, 9.36 mmol) in
dichloromethane (30 mL) was added under stirring to a
solution of hydrazine monohydrate (0.47 mL, 9.4 mmol)
in dichloromethane (20 mL). The suspension obtained
was stirred at room temperature for 1 h, and then the solid
was collected by filtration and washed with dichloro-
methane and ethanol. Recrystallization from ethanol/
dioxane gave 9 (0.9 g, 29%); m.p. 210–212 °C (dec); IR
(KBr) 3 360 and 3 210 (NH), 1 715 and 1 660
Melting points were determined in open capillary tubes
on a Gallenkamp melting point apparatus and are uncor-
rected. Infrared spectra were recorded on a Perkin-Elmer
281 spectrometer in KBr (potassium bromide) disks.
1H-NMR spectra were obtained at 200 MHz on a Varian
Inova Unity 200 spectrometer in DMSO-d6 or CDCl3
solution. Chemical shifts (δ) are reported in parts per
million relative to tetramethylsilane (TMS) as an internal
standard; coupling constants (J) are in hertz. Signal
multiplicities are presented by s (singlet), d (doublet), t
(triplet), q (quartet), br s (broad singlet), and m (multip-
let). Elemental analyses for C, H, N, and S were obtained
1
(C=O) cm–1. H-NMR (DMSO-d6) δ 1.25–1.37 (m, 6H,
CH2CH3), 2.68 (s, 3H, CH3), 4.20–4.39 (m, 4H, CH2CH3),

686
M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
7.83 (br s, 2H, NH), 9.53 (br s, 1H, NH), 10.07 (s, 1H,
NH). Anal. (C12H17N3O4S2) C, H, N, S.
5.1.7. Monopotassium salt of 3-amino-
2,3-dihydro-6-ethyl-2-thioxothieno[2,3-d]-
pyrimidin-4(1H)-one 14 and its 2-thioxo derivative 20
These compounds were obtained like compounds 13
and 19. 14: yield: 3.1 g, 86%. 20: recrystallized from
ethanol, yield: 0.4 g, 61%; m.p. 231–233 °C (dec); IR
(KBr) 3 320 and 3 160 (NH), 1 650 (C=O) cm–1. Anal.
(C8H9N3OS2) C, H, N, S.
5.1.3. 2-[(Hydrazinothioxomethyl)amino]-5-
ethyl-3-thiophenecarboxylic acid ethyl ester 10
Compound 10 was prepared in the same manner of 9
from isothiocyanate 7 and hydrazine monohydrate. Re-
crystallized from ethanol, yield: 1.2 g, 47%; m.p.
198–199 °C; IR (KBr) 3 360, 3 200 and 3 140 (NH),
1 665 (C=O) cm–1. Anal. (C10H15N3O2S2) C, H, N, S.
5.1.8. Monopotassium salt of 3-acetylamino-
2,3-dihydro-5,6-dimethyl-2-thioxothieno[2,3-d]-
pyrimidin-4(1H)-one 17 and its 2-thioxo derivative 21
These compounds were obtained like compounds 13
and 19. 17: yield: 1.44 g, 34%. 21: recrystallized from
ethanol, yield: 0.35 g, 46%; m.p. > 275 °C (dec); IR
(KBr) 3 220 and 3 200 (NH), 1 725 and 1 670
(C=O) cm–1. Anal. (C10H11N3O2S2) C, H, N, S.
5.1.4. 2-[[(2-Propenylamino)thioxomethyl]amino]-
4,5-dimethyl-3-thiophenecarboxylic acid ethyl ester 11
Compound 11 was prepared in the same manner of 9
from isothiocyanate 8 and allylamine. Recrystallized
from ethanol, yield: 0.9 g, 32%; m.p. 132–134 °C; IR
(KBr) 3 210 (NH), 1 660 (C=O) cm–1. Anal.
(C13H18N2O2S2) C, H, N, S.
5.1.9. 1-(3-Chloropropyl)-4-(2-nitrophenyl)piperazine 24
A mixture of 1-(2-nitrophenyl)piperazine 22 (1.54 g,
7.44 mmol),
1-bromo-3-chloropropane
(0.9 mL,
9.1 mmol) and of potassium carbonate (1.23 g, 8.9 mmol)
was stirred at room temperature for 22 h in dimethylfor-
mamide (10 mL). The residue was removed by filtration
and the solvent evaporated under reduced pressure to
yield 24 (1 g, 47%) as a yellow oil, that was purified by
column chromatography (silica gel, ethyl acetate). An
analytical sample of the free base was obtained as
hydrochloride salt with HCl-saturated methanol, dilution
with diethyl ether and recrystallization from absolute
ethanol; m.p. 197–199 °C (dec). 1H-NMR (CDCl3) δ
2.50 (m, 2H, CH2CH2CH2), 3.05–3.25 (m, 6H, CH2N
and piperazine H), 3.55–3.85 (m, 6H, piperazine H and
CH2Cl), 7.18–7.84 (m, 4H, ArH), 12.97 (br s, 1H, NH+).
Anal. (C13H18ClN3O2·HCl·0.5 H2O) C, H, N.
5.1.5. 2-[[(Acetylamino)thioxomethyl]amino]-4,5-
dimethyl-3-thiophenecarboxylic acid ethyl ester 12
Compound 12 was prepared from isothiocyanate 8 and
acethydrazine in the same condition of 9. The solution
was stirred at room temperature for 1 h, then the solvent
was removed under reduced pressure, the solid was
collected with a small amount of ethanol and dried.
Recrystallized from ethanol, yield: 1.21 g, 41%; m.p.
199–201 °C; IR (KBr) 3 340 and 3 160 (NH), 1 660
broad (C=O) cm–1. Anal. (C12H17N3O3S2) C, H, N, S.
5.1.6. Monopotassium salt of 3-amino-6-carbethoxy-
2,3-dihydro-5-methyl-2-thioxothieno[2,3-d]-
pyrimidin-4(1H)-one 13 and its 2-thioxo derivative 19
5.1.10. General procedure for compounds 26–32
To a solution of potassium hydroxide (0.76 g,
13.5 mmol) in absolute ethanol (55 mL) compound 9
(4.47 g, 13.5 mmol) was added, and the mixture was
refluxed under stirring for 1 h. The suspension was
filtered while hot and the solid washed with hot absolute
ethanol to give 13 (3.6 g, 82%). A suspension of potas-
sium salt 13 (1.08 g, 3.34 mmol) in water (50 mL) was
acidified with concentrated hydrochloric acid and stirred
at room temperature for 30 min. The solid was collected
by filtration and washed with water. Recrystallization
from ethanol gave 19 (0.4 g, 42%); m.p. 234–236 °C
(dec); IR (KBr) 3 240 (NH), 1 705 and 1 640 (C=O) cm–1.
1H-NMR (DMSO-d6) δ 1.28 (t, J = 7.2 Hz, 3H, CH2CH3),
2.74 (s, 3H, CH3), 4.27 (q, J = 7.2 Hz, 2H, CH2CH3).
Anal. (C10H11N3O3S2) C, H, N, S.
A mixture of potassium salts 13–18 (3.6 mmol) and
1-(3-chloropropyl)-4-(2-methoxy or 2-nitrophenyl)-
piperazine or 3-dimethylamino-propylchloride hydrochlo-
ride 23–25 (4.32 mmol) was stirred at reflux for 6 h in
ethanol (15 mL). After the suspension was cooled, the
residue was collected, the solvent was removed under
reduced pressure, and the solid collected with a small
amount of ethanol and recrystallized. For product 28 the
solid was collected with diethyl ether, compound 29, after
the suspension was cooled, was obtained as a solid. For
compounds 30 and 32, after cooling, was obtained a
solution, that gave the products by dilution with water, for
32 the refluxing time was 2 h (figure 2 and table I). The
1
IR and H-NMR spectra of the following compound are
reported as a representative sample.

M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
687
5.1.10.1. 5-Amino-4-oxo-6-[[3-[4-(2-
for 30 min. The solid was collected by filtration and
washed with water. Recrystallization from dioxane gave
41 (0.15 g, 36%); m.p. 262–264 °C (dec); IR (KBr) 3 290
methoxyphenyl)-1-piperazinyl]propyl]thio]-3-methyl-3H-
thieno[2,3-d]pyrimidin-2-carboxylic acid ethyl ester 26
IR (KBr) 3 330 and 3 280 (NH), 1 720 and 1 685
(C=O) cm–1. 1H-NMR (DMSO-d6) δ 1.22 (t, J = 7.2 Hz,
3H, CH2CH3), 1.78 (m, 2H, CH2CH2CH2), 2.38 (t, J =
6.6 Hz, 2H, CH2N), 2.45 (m, 4H, piperazine H), 2.74 (s,
3H, CH3), 2.92 (m, 4H, piperazine H), 3.01 (t, J = 6.6 Hz,
2H, SCH2), 3.70 (s, 3H, OCH3), 4.21 (q, J = 7.2 Hz, 2H,
CH2CH3), 5.68 (s, 2H, NH2, exchanges with D2O),
6.80–6.86 (m, 4H, ArH).
1
and 3 190 (NH), 1 705 (C=O) cm–1. H-NMR (DMSO-
d6) δ 3.88 (s, 3H, CH3), 7.99 (s, 1H, pyrazole H). Anal.
(C6H7N5OS) C, H, N, S.
5.1.14. 3-Amino-2-[[3-[4-(2-methoxyphenyl)-1-pipera-
zinyl]propyl]thio]pyrido[2,3-d]pyrimidin-4(3H)-one 42
To a solution of potassium hydroxide (0.44 g, 7.9 mmol)
in absolute ethanol (32 mL) the 2-thioxoderivative 34
(1.54 g, 7.9 mmol) was added and the mixture was re-
fluxed for 30 min. Then to the hot suspension of potas-
5.1.11. 3-Amino-2,3-dihydro-2-
thioxopyrido[2,3-d]pyrimidin-4(1H)-one 34
sium
salt
37,
1-(3-chloropropyl)-4-(2-methoxy-
To a solution of hydrazine monohydrate (0.53 mL,
10.6 mmol) in dichloromethane (20 mL) was added a
solution of isothiocyanate 33 (2.22 g, 10.67 mmol) in
dichloromethane (20 mL). The mixture was stirred at
room temperature for 1 h, then the solid was collected
and washed with ethanol. Recrystallization from ethanol/
dioxane gave 34 (1.2 g, 58%); m.p. > 310 °C; IR (KBr)
3 300 and 3 200 (NH), 1 715 (C=O) cm–1. 1H-NMR
(DMSO-d6) δ 6.38 (s, 2H, NH2), 7.40 (dd, J = 4.8,
7.8 Hz, 1H, ArH), 8.37 (d, J = 7.8 Hz, 1H, ArH), 8.73 (d,
J = 4.8 Hz, 1H, ArH), 13.60 (s, 1H, NH). Anal.
(C7H6N4OS) C, H, N, S.
phenyl)piperazine 23 (2.55 g, 9.5 mmol) was added and
the mixture was refluxed for 6 h. The solution was
cooled, the solvent was removed under reduced pressure,
the solid collected, and washed with a small amount of
ethanol (table II): IR (KBr) 3 320 and 3 200 (NH), 1 695
(C=O) cm–1. 1H-NMR (DMSO-d6) δ 1.84 (m, 2H,
CH2CH2CH2), 2.43 (m, 6H, CH2N and piperazine H),
2.91 (m, 4H, piperazine H), 3.07 (t, J = 6.8 Hz, 2H,
SCH2), 3.70 (s, 3H, OCH3), 5.74 (s, 2H, NH2), 6.80–6.88
(m, 4H, ArH), 7.38 (dd, J = 4.6, 8 Hz, 1H, ArH), 8.40 (d,
J = 8 Hz, 1H, ArH), 8.81 (d, J = 4.6 Hz, 1H, ArH).
5.1.12. 5-[(Hydrazinothioxomethyl)amino]-1-
5.1.15. General procedure for compounds 43–45
A mixture of potassium salts 38–40 (3.72 mmol) and
1-(3-chloropropyl)-4-(2-methoxyphenyl)piperazine 23
(3.72 mmol) was stirred at reflux for 6 h in ethanol
(15 mL). After the suspension was cooled, the residue
was eliminated by filtration, from the solution by dilution
with water was obtained the desired product 43, that was
collected by filtration and recrystallized. For 44, after the
suspension was cooled, the residue was removed by
filtration and the desired product crystallized from the
solution, while 45 was obtained as a solid, from the
cooled suspension, collected and recrystallized (figure 3
and table II). The IR and 1H-NMR spectra of the
following compound are reported as a representative
sample.
methyl-1H-pyrazolo-4-carboxylic acid ethyl ester 36
A solution of isothiocyanate 35 (1 g, 4.73 mmol) in
dichloromethane (20 mL) was added under stirring to a
solution of hydrazine monohydrate (0.25 mL, 5 mmol) in
dichloromethane (10 mL). The suspension was stirred at
room temperature for 30 min, then the solid was collected
by filtration, washed with dichloromethane and ethanol.
Recrystallization from ethanol gave 36 (0.3 g, 26%); m.p.
191–193 °C (dec); IR (KBr) 3 260 and 3 170 (NH), 1 710
1
(C=O) cm–1. H-NMR (DMSO-d6) δ 1.23 (t, J = 7 Hz,
3H, CH2CH3), 3.65 (s, 3H, CH3), 4.14 (q, J = 7 Hz, 2H,
CH2CH3), 6.43 (br s, 3H, NH), 7.77 (s, 1H, pyrazole H),
9.61 (s, 1H, NH). Anal. (C8H13N5O2S) C, H, N, S.
5.1.13. Monopotassium salt of 5-amino-
1,2,3,5-tetrahydro-1-methyl-6-thioxopyrazolo[3,4-d]-
pyrimidin-4(4H)-one 38 and its 6-thioxo derivative 41
Compound 36 (1.65 g, 6.79 mmol) was added to a
solution of potassium hydroxide (0.38 g, 6.79 mmol) in
absolute ethanol (28 mL)), and the mixture was refluxed
under stirring for 1 h. The suspension was filtered while
hot and the solid washed with hot absolute ethanol to give
38 (1.4 g, 87%). A suspension of potassium salt 38 (0.5 g,
2.11 mmol) in water (50 mL) was acidified with concen-
trated hydrochloric acid and stirred at room temperature
5.1.15.1. 5-Amino-6-[[3-[4-(2-
methoxyphenyl)-1-piperazinyl]propyl]thio]-
1-methyl-pyrazolo[3,4-d]pyrimidin-4(3H)-one 43
IR (KBr) 3 310 and 3 210 (NH), 1 690 (C=O) cm–1.
1H-NMR (DMSO-d6) δ 1.83 (m, 2H, CH2CH2CH2), 2.41
(m, 6H, CH2N and piperazine H), 2.89 (m, 4H, piperazine
H), 3.02 (t, J = 6.8 Hz, 2H, SCH2), 3.69 (s, 3H, OCH3),
3.80 (s, 3H, NCH3), 5.58 (s, 2H, NH2), 6.79–6.88 (m, 4H,
ArH), 7.94 (s, 1H, pyrazole H).

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M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
5.1.16. 2-[(Chloropentanoyl)amino]-4,5-
5.1.19. 2-(1-Chloromethyl)-6,7-dimethyl-8H-
[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one 50
dimethyl-3-thiophenecarboxylic acid ethyl ester 47
5-Chlorovalerylchloride (1.3 mL, 10.05 mmol) was
added to a solution of amino ester 46 (2 g, 10.05 mmol)
in chloroform (20 mL) and the solution was refluxed for
4 h. After cooling, the solution was concentrated under
reduced pressure to obtain a dark oil, that, by addition of
a small amount of water and ethanol, yielded a solid that
was collected and washed with diethyl ether. Recrystal-
lization from ethanol gave 47 (1.1 g, 34%); m.p. 82–84 °C;
IR (KBr) 3 250 (NH), 1 660 (C=O) cm–1. 1H-NMR
(DMSO-d6) δ 1.25 (t, J = 7.2 Hz, 3H, CH2CH3),1.67 (m,
4H, CH2CH2CH2CH2), 2.11 (s, 3H, CH3), 2.14 (s, 3H,
CH3), 2.47 (t, J = 7 Hz, 2H, CH2CH2CH2CH2), 3.59 (t,
J = 6.4 Hz, 2H, CH2Cl), 4.22 (q, J = 7.2 Hz, 2H,
CH2CH3), 10.87 (s, 1H, NHCO). Anal. (C14H20ClNO3S)
C, H, N, S.
To a mixture of potassium salt 18 (1.5 g, 5.66 mmol),
P2O5 (0.80 g, 5.66 mmol), and CH3SO3H (1.83 mL) was
added 2-chloroacetylchloride (0.60 mL, 7.43 mmol), and
the mixture was stirred for 4 h at 80–90 °C. The cooled
reaction mixture was poured into cold water and neutral-
ized with a 10% NaOH solution. The product was filtered
and washed with water. Recrystallization from ethanol/
dioxane gave 50 (0.5 g, 31%); m.p. 229–231 °C; IR
1
(KBr) 1 690 (C=O) cm–1. H-NMR (DMSO-d6) δ 2.36
(s, 3H, CH3), 2.42 (s, 3H, CH3), 5.24 (s, 2H, CH2Cl).
Anal. (C10H8ClN3OS2) C, H, N, S.
5.1.20. 2-(3-Chloropropyl)-6,7-dimethyl-8H-
[1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one 51
Compound 51 was prepared in the same condition of
50 from potassium salt 18 and 4-chlorobutyryl chloride.
Recrystallized from ethanol, yield: 0.6 g, 34%; m.p.
161–163 °C; IR (KBr) 1 690 (C=O) cm–1. 1H-NMR
(DMSO-d6) δ 2.22 (m, 2H, CH2CH2CH2), 2.35 (s, 3H,
CH3), 2.41 (s, 3H, CH3), 3.17 (t, J = 7.2 Hz, 2H, CH2),
3.77 (t, J = 6.4 Hz, 2H, CH2Cl). Anal. (C12H12ClN3OS2)
C, H, N, S.
5.1.17. 2-[4-[4-(2-Methoxyphenyl)-1-
piperazinyl]pentanoylamino]-4,5-dimethyl-
3-thiophenecarboxylic acid ethyl ester 48
A mixture of derivative 47 (1.24 g, 3.9 mmol), of
1-(2-methoxyphenyl)piperazine (0.75 g, 3.9 mmol), and
of potassium carbonate (0.54 g, 3.9 mmol) was refluxed
under stirring for 2 h. After cooling, the suspension was
filtered and the solution was extracted with chloroform
and washed with water. The organic layers were dried
over anhydrous sodium sulfate and evaporated under
reduced pressure. Recrystallization from ethanol yielded
48 (0.8 g, 43%); m.p. 101–103 °C; IR (KBr) 3 250 (NH),
5.1.21. 2-[2-[4-(2-Methoxyphenyl)-1-
piperazinyl]methyl]-6,7-dimethyl-8H-[1,3,4]-
thiadiazolo-[3,2-a]thieno[2,3-d]pyrimidin-8-one 52
1
1 650 and 1 690 (C=O) cm–1. H-NMR (DMSO-d6) δ
A mixture of the thiadiazolo derivative 50 (1 g,
3.5 mmol) and of 1-(2-methoxyphenyl)piperazine (3.36 g,
17.5 mmol) was heated at 140 °C on an oil bath for 2 h.
The melted residue was collected with warm ethanol and
recrystallized (table II): IR (KBr) 1 685 (C=O) cm–1.
1H-NMR (DMSO-d6) δ 2.36 (s, 3H, CH3), 2.42 (s, 3H,
CH3), 2.76 (m, 4H, piperazine H), 3.01 (m, 4H, pipera-
zine H), 3.77 (s, 3H, OCH3), 3.97 (s, 2H, CH2), 6.87–6.96
(m, 4H, ArH).
1.24 (t, J = 7 Hz, 3H, CH2CH3), 1.51 (m, 4H,
CH2CH2CH2CH2), 2.12 (s, 3H, CH3), 2.15 (s, 3H, CH3),
2.26 (t, J = 6.8 Hz, 2H, COCH2), 2.43 (m, 6H, CH2N and
piperazine H), 2.85 (m, 4H, piperazine H), 3.69 (s, 3H,
OCH3), 4.21 (q, J = 7 Hz, 2H, CH2CH3), 6.76–6.88 (m,
4H, ArH), 10.90 (s, 1H, NHCO). Anal. (C25H35N3O4S)
C, H, N, S.
5.1.18. 3-Amino-2-[4-[4-(2-
methoxyphenyl)-1-piperazinyl]butyl]-
5,6-dimethyl-thieno[2,3-d]pyrimidin-4(3H)-one 49
A mixture of derivative 48 (1 g, 2.1 mmol) and of
hydrazine hydrate (4 mL, 80 mmol) was refluxed for 12 h
in ethanol (10 mL). After cooling, the product was
collected and washed with ethanol (table II): IR (KBr)
3 280 and 3 110 (NH), 1 670 (C=O) cm–1. 1H-NMR
(DMSO-d6) δ 1.48 (m, 2H, CH2), 1.68 (m, 2H, CH2),
2.27 (s, 3H, CH3), 2.32 (s, 3H, CH3), 2.43 (m, 6H, CH2N
and piperazine H), 2.85 (m, 6H, CH2 and piperazine H),
3.69 (s, 3H, OCH3), 5.66 (s, 2H, NH2), 6.77–6.85 (m, 4H,
ArH).
5.1.22. 2-[3-[4-(2-Methoxyphenyl)-1-
piperazinyl]propyl]-6,7-dimethyl-8H-[1,3,4]-
thiadiazolo-[3,2-a]thieno[2,3-d]pyrimidin-8-one 53
Compound 53 was prepared from thiadiazolo deriva-
tive 51 in the same manner as compound 52 (table II). IR
1
(KBr) 1 680 (C=O) cm–1. H-NMR (DMSO-d6) δ 1.86
(m, 2H, CH2CH2CH2), 2.29 (s, 3H, CH3), 2.35 (s, 3H,
CH3), 2.43 (m, 6H, CH2N and piperazine H), 2.83 (m,
4H, piperazine H), 3.00 (t, J = 7.2 Hz, 2H, CH2CH2CH2),
3.68 (s, 3H, OCH3), 6.72–6.87 (m, 4H, ArH).

M. Modica et al. / Eur. J. Med. Chem. 35 (2000) 677–689
689
5.2. Pharmacology
tration 0.2 nM, 15 min, 37 °C (non-specific binding:
(–)-sulpiride 100 µM). α1-Adrenergic: [3H]prazosin (sp
act. 71.8 Ci/mmol, NEN) final concentration 0.2 nM,
30 min, 25 °C (non-specific binding: phentolamine 3 µM).
Incubations were stopped by rapid filtration under
vacuum through GF/B filters which were then washed
with 12 mL (4 × 3 times) of ice-cold 50 mM Tris-HCl
(pH 7.4) or 50 mM Hepes-HCl (pH 7.5) using a Brandel
M-48R apparatus and counted in 4 mL of Filter Count
(Packard) in an LKB 1214 RACKBETA liquid scintilla-
tion spectrometer with counting efficiency about 60%.
Dose inhibition curves were analysed by the ‘Allfit’
program to obtain the concentration of unlabelled drugs
that inhibited ligand binding by 50%. The Ki values were
derived from the IC50 values [18].
5.2.1. In vitro experiments
Binding assays were performed on male
CRL:CD(SD)BR-COBS rats weighing about 150 g. The
animals were killed by decapitation, and their brains were
rapidly dissected (hippocampus for 5-HT1A; striatum for
5-HT1B, D1 and D2; cortex for 5-HT2A, 5-HT3 and
α1-adrenergic receptors), frozen, and stored at –80 °C
until the day of assay. Pig brains were obtained from a
local slaughterhouse, and the cortex (for 5-HT2C) was
rapidly removed and stored at –80 °C until assay.
Tissue was homogenized in about 50 volumes of
ice-cold 50 mM Tris-HCl buffer (pH 7.4) (or 50 mM
Hepes-HCl (pH 7.5) for 5-HT3 receptors) using an Ultra
Turrax TP-1810 (2 × 20 s) and centrifuged at 50 000 g for
10 min (Beckman model J-21B refrigerated centrifuge).
The pellet was resuspended in the same volume of fresh
buffer, incubated at 37 °C for 10 min and centrifuged
again at 50 000 g for 10 min. The pellet was then washed
once by resuspension in fresh buffer and centrifuged as
before. The pellet was then resuspended in the appropri-
ate incubation buffer (50 mM Tris-HCl (pH 7.7) for
5-HT2A receptors, with the addition of 10 µM pargyline
for the other receptors containing 4 mM CaCl2 for
5-HT1A receptors, 0.1% ascorbic acid for α1-adrenergic
receptors, 4 mM CaCl2 and 0.1% ascorbic acid for
5-HT1B and 5-HT2C receptors; 50 mM Hepes-HCl pH 7.4
containing 10 µM pargyline for 5-HT3 receptors, 50 mM
Tris-HCl pH 7.1 containing 10 µM pargyline, 120 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 0.1%
ascorbic acid for D1 and D2 receptors) just before the
binding assay.
References
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Selvaggini C., Salmona M., Mennini T., J. Med. Chem. 40 (1997)
574–585.
[2] López-Rodríguez M.L., Morcillo M.J., Rovat T.K., Fernández E.,
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Binding assays were done as previously described
[17]. Briefly, the following incubation conditions were
used. 5-HT1A: [3H]-8-OH-DPAT (sp act. 157 Ci/mmol,
NEN) final concentration 1 nM, 30 min 25 °C (non-
specific binding: 5-HT 10 µM). 5-HT1B: [3H]-5-HT (sp
act. 14.6 Ci/mmol, NEN) final concentration 2 nM, 30 min
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25 °C (non-specific binding: 5-HT 10 µM). 5-HT2A
:
[13] Santagati M., Doweyko A., Santagati A., Modica M., Guccione S.,
Hongming C., Uccello Barretta G., Balzano F., Proceedings of the
12th European Symposium on QSARs (1998) 185–196.
[3H]ketanserin (sp act. 60.0 Ci/mmol, Amersham) final
concentration 0.7 nM, 15 min, 37 °C (non-specific bind-
ing: methysergide 1 mM). 5-HT2C: [3H]mesulergine (sp
act. 73.0 Ci/mmol, NEN) final concentration 1 nM,
30 min, 37 °C (non-specific binding: mesulergine 10 mM).
5-HT3: [3H]BRL43694 (sp act. 84.8 Ci/mmol, NEN) final
concentration 1 nM, 30 min, 25 °C (non-specific binding:
GR38032 10 µM). D1: [3H]SCH23390 (sp act. 71.1 Ci/
mmol, NEN) final concentration 0.4 nM, 15 min, 37 °C
(non-specific binding: (–)-cis-flupentixol 10 µM). D2:
[3H]spiperone (sp act. 19.0 Ci/mmol, NEN) final concen-
[14] Santagati M., Hongming C., Santagati A., Modica M., Guccione S.,
Russo F., Uccello Barretta G., Balzano F., Proceedings of the 12th
European Symposium on QSARs (1998) 443–450.
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