Novel antagonists of serotonin-4 receptors: Synthesis and biological evaluation of pyrrolothienopyrazines

Article abstract of DOI:10.1016/j.bmc.2008.11.045

Based on the definition of a 5-HT₄ receptor antagonist pharmacophore, a series of pyrrolo[1,2-a]thieno[3,2-e] and pyrrolo[1,2-a]thieno[2,3-e] pyrazine derivatives were designed, prepared, and evaluated to determine the properties necessary for high-affinity binding to 5-HT₄ receptors. The compounds were synthesized by substituting the chlorine atom of the pyrazine ring with various N-alkyl-4-piperidinylmethanolates. They were evaluated in binding assays with [³H]GR113808 (1) as the 5-HT₄ receptor radioligand. The affinity values (K_i or inhibition percentages) were affected by both the substituent on the aromatic ring and the substituent on the lateral piperidine chain. A methyl group on the tricyclic ring produced a marked increase in affinity while an N-propyl or N-butyl group gave compounds with nanomolar affinities. Among the most potent ligands, 34d was selected for further pharmacological studies and evaluated in vivo. This compound acts as an antagonist/weak partial agonist in COS-7 cells stably expressing the 5-HT_4(a) receptor and is of great interest as a peripheral antinociceptive agent.

Full text of DOI:10.1016/j.bmc.2008.11.045

Bioorganic & Medicinal Chemistry 17 (2009) 2607–2622
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel antagonists of serotonin-4 receptors: Synthesis and biological
evaluation of pyrrolothienopyrazines
Stéphane Lemaître a, Alban Lepailleur a, Ronan Bureau a, Sabrina Butt-Gueulle a,
Véronique Lelong-Boulouard a, , Pascal Duchatelle a,à, Michel Boulouard a,à, Aline Dumuis b,
Cyril Daveu a, Frank Lezoualc’h c, Bruno Pfeiffer d, François Dauphin a,à, Sylvain Rault a,
*
a Centre d’Etudes et de Recherche sur le Médicament de Normandie, Université de Caen, 1 rue Vaubénard, 14032 Caen Cedex, France
b UPR 9023, CCIPE, 141 rue de la Cardonille, 34094 Montpellier Cedex 5, France
c INSERM U769, University Paris XI, IFR141, 5 rue JB Clément, 92296 Châtenay-Malabry, France
d Les Laboratoires Servier 1, rue Carle Hebert 92415 Courbevoie Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 July 2008
Revised 13 November 2008
Accepted 15 November 2008
Available online 24 November 2008
Based on the definition of a 5-HT4 receptor antagonist pharmacophore, a series of pyrrolo[1,2-a]thie-
no[3,2-e] and pyrrolo[1,2-a]thieno[2,3-e] pyrazine derivatives were designed, prepared, and evaluated
to determine the properties necessary for high-affinity binding to 5-HT4 receptors. The compounds were
synthesized by substituting the chlorine atom of the pyrazine ring with various N-alkyl-4-piperidinylme-
thanolates. They were evaluated in binding assays with [3H]GR113808 (1) as the 5-HT4 receptor radioli-
gand. The affinity values (Ki or inhibition percentages) were affected by both the substituent on the
aromatic ring and the substituent on the lateral piperidine chain. A methyl group on the tricyclic ring pro-
duced a marked increase in affinity while an N-propyl or N-butyl group gave compounds with nanomolar
affinities. Among the most potent ligands, 34d was selected for further pharmacological studies and eval-
uated in vivo. This compound acts as an antagonist/weak partial agonist in COS-7 cells stably expressing
the 5-HT4(a) receptor and is of great interest as a peripheral antinociceptive agent.
Keywords:
5-HT4 ligands
Antagonists
Pharmacophore
Pyrrolo[1,2-a]thieno[3,2-e] pyrazine
Pyrrolo[1,2-a]thieno[2,3-e] pyrazine
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tionally, though with some inconsistencies,23 the observations that
5-HT4 receptor antagonists such as 1-methyl-1H-indole-3-carbox-
In the last decade, much effort has been directed toward under-
standing the functions1 of the various receptor subtypes of the
neurotransmitter serotonin (5-hydroxytryptamine, 5-HT), with
emphasis on the most recently discovered binding sites, that is,
the 5-HT4, 5-HT5, 5-HT6 and 5-HT7 receptors.2–7 Among these recep-
tors, which are linked to stimulation of cAMP production, the 5-HT4
subtypes have sparked the interest of scientists and physicians8 be-
cause of their functional and physiological significance. Indeed, 5-
HT4 receptors have been demonstrated to be modulators of neuro-
transmitter release in various neuronal populations in the central
nervous system, including basalocortical cholinergic,9,10 striatal
dopaminergic11,12 and hippocampal serotoninergic13 cells. In paral-
lel, the 5-HT4 receptor has been implicated in cognitive perfor-
mance,14–22 making it a potential therapeutic target for treatment
of the cognitive deficits associated with Alzheimer’s disease. Addi-
ylic acid 1-(2-methanesulfonylamino-ethyl)-piperidin-4-ylmethyl
ester GR113808 (1), 1-butyl-4-piperidinylmethyl 8-amino-7-
chloro-1,4-benzodioxan-5-carboxylate SB204070 (2), and N-[(1-
butyl-4-piperidinyl)methyl]-3,4-dihydro-2H-[1,3]-oxazino[3,2-a]-
indole-10-carboxamide SB207266 (3) have shown anxiolytic-like
action in various models of anxiety in the rat,24,25 suggest another
possible therapeutic application of 5-HT4 receptor ligands.
Considering the distribution (e.g., atrium, gut) and the roles of 5-
HT4 peripheral receptors, various cardiac or gastrointestinal effects
can also be anticipated for selective ligands.26–29 Indeed, 5-HT4
receptor partial agonists such as tegaserod (4) and prucalopride
(5)30 are being developed for the management of irritable bowel
syndrome. However, there exist only a limited number of high-
affinity ligands selective for 5-HT4 receptors31 (Chart 1). When we
began this work, very little was known about structure–activity
relationships (SAR) of the 5-HT4 receptor ligands. Among the 5-
HT4 receptor antagonists (Chart 2), tropisetron32 (6) was the first
compound available; it exhibits low affinity for 5-HT4 receptors
(pKi = 6.5) and high affinity for 5-HT3 receptors (pKi = 10). The
second generation of ligands consisted of compounds such as
endo-8-methyl-8-azabicyclo[3.2.1]oct-3-y1-2,3-dihydro-6-meth-
oxy-2-oxo-1H-benzimidazole-1-carboxylate DAU6285 (7) possess-
* Corresponding author. Tel.: +33 2 31 93 41 69; fax: +33 2 31 93 11 88.
E-mail address: sylvain.rault@unicaen.fr (S. Rault).
Present address: Cognition et impulsivité, EA3917, Université de Caen, 14032
Caen.
à
Present address: GMPC, EA4259, Université de Caen, 5 rue vaubénard, 14032
Caen.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.045

2608
S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
NH2
O
(CH2)2NHSO2CH3
O
O
O
N
HO
Cl
O
N
N
H
N
CH3
nBu
H2N
O
5-HT
GR113808
pKi (5-HT3) < 5 ; pKi (5-HT4) =9.8
SB204070
pKi (5-HT3) =6.8 ; pKi (5-HT4) = 10.8
1
2
NH
H
N
O
H
N
(CH2)3CH3
N
H
(CH2)4CH3
N
O
O
N
OMe
N
MeO
Cl
N
H
O
N
N
H
H2N
SB207266
Tegaserod
Prucalopride
pKi (5-HT3) < 6 ; pKi (5-HT4) =9.3
pKi (5-HT3) < 5 ; pKi (5-HT4) = 8.4
pKi (5-HT3) = 5.4 ; pKi (5-HT4) = 8.6
3
4
5
Chart 1. 5-HT and selective 5-HT4 ligands.
NCH3
O
O
O
N
MeO
N
CH2CH3
O
NCH3
H
N
Cl
CH2CH3
O
O
N
H
N
H
H2N
OMe
DAU 6285
pKi (5-HT3) = 6.5 ; pKi (5-HT4) = 7.2
SDZ 205557
pKi (5-HT3) = 6.9 ; pKi (5-HT4) = 8.2
Tropisetron
pKi (5-HT3) = 8.55 ; pKi (5-HT4) = 6.7
7
8
6
O
O
Cl
Cl
O
N
N
N
H
H2N
OMe
NH
N
RS 23597
pKi (5-HT3) = 5.7 ; pKi (5-HT4) = 7.5
SC 53606
pKi (5-HT3) = 6.6 ; pKi (5-HT4) = 8.85
9
10
Chart 2. Evolution of 5-HT4 ligands.
ing an equivalent affinity for 5-HT3 and 5-HT4 receptors.31 For com-
pounds of the third generation, such as 2-diethylaminoethyl-[2-
methoxy-4-amino-5-chloro] benzoate SDZ205557 (8), (1-S,8-S)-
N-[(hexahydro-1H-pyrrolizin-1-yl)methyl]-6-chloroimidazo[1,2-
a]pyridine-8-carboxamide33,34 SC53606 (9), or 2-piperidinopropyl
4-amino-5-chloro-2-methoxybenzoate35,36 RS23597 (10), the
selectivity ratio toward 5-HT4 was greatly improved compared to
5-HT3 receptors, but these compounds remained insufficiently
selective toward other receptors to be used as references. Finally,
compounds 1 and 2 emerged as the first selective and high-affinity
5-HT4 receptor antagonists37,38 (e.g., for 1, pKi = 9.5 toward 5-HT4
receptors; pKi < 6 toward 5-HT3 receptors).
The search for potent and selective 5-HT ligands has been ongo-
ing in our laboratory for several years. The 5-HT3 receptor ligands
have caused a huge interest in this receptor due to their potential
therapeutic applications in a number of areas: emesis, anxiety,
psychotic disorders, drug abuse, depression, migraine, pain, irrita-
ble bowel syndrome.39 We previously reported the synthesis,
receptor binding profiles, and in vitro and in vivo pharmacological
evaluation of several tricyclic series of piperazinopyrrolothieno-
pyrazine, piperazinopyrido-pyrrolopyrazine, piperazinopyrrolo-
quinoxaline and piperazinopyridopyrroloquinoxaline derivatives
having high affinity, good selectivity, and partial agonist activity
toward 5-HT3 receptors.40,41 A 3D-QSAR study led to a precise def-
inition of the pharmacophore for these partial 5-HT3 agonists42
(Fig. 1). The hypothesis that these compounds possess several
characteristics in common with the 5-HT3 antagonists and also
with 5-hydroxytryptamine itself, in terms of functional groups,
was validated.
Furthermore, we developed a definition of a pharmacophore for
the 5-HT4 receptor antagonists (Fig. 2) by considering a 3D-QSAR
study starting from 15 antagonists described in the literature.43
In light of these two studies, and after a comparison between the
two pharmacophores (partial 5-HT3 agonists and 5-HT4 antago-
nists), we formulated the hypothesis that it should be possible to
transform a 5-HT3 ligand into a 5-HT4 ligand. This was the basis
of our study of pharmacomodulation43 starting from 5-(4-ben-
zylpiperazin-1-yl)pyrrolo[1,2-a]thieno[3,2-e]pyrazine39 S21007 (11).
We also successfully used this pharmacophore to design
benzo[h][1,6]naphthyridine and azepino[3,2-c]quinoline deriva-

S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
2609
e]pyrazines 34a–k are shown in Tables 1–4. The general synthetic
procedures used for their preparation are illustrated in Scheme 1.
These compounds were obtained40 from 5-chloropyrrolo[1,2-a]thi-
eno[3,2-e]pyrazine (28), 5-chloropyrrolo[1,2-a]thieno[2,3-e]pyra-
zine
(29),
and
1-methyl-5-chloropyrrolo[1,2-a]thieno[2,3-
e]pyrazine (30) by nucleophilic substitution of the chlorine atom
by aminoalcohols. The chloroimines 28–30 were obtained in a
five-step pathway starting from the methyl 2-amino-3-thiophene-
carboxylate (13), methyl 3-amino-2-thiophenecarboxylate (14)
and methyl 4-methyl-3-amino-2-thiophenecarboxylate (15).
Treatment of compounds 13–15 with 2,5-dimethoxytetrahydrofu-
ran and a catalytic amount of 4-chloropyridinium hydrochloride in
boiling 1,4-dioxane gave the pyrrole compounds 16–18. The car-
boxylic acids 19–21 obtained by alkaline hydrolysis of 16–18 were
then converted into their carbonyl azides 22–24 by treatment with
ethyl chloroformate and sodium azide in acetone/acetonitrile. A
Curtius rearrangement of the azides 22–24 in boiling orthodichlo-
robenzene and subsequent cyclization gave the pyrazinones 25–
27. The chloroimine derivatives 28–30 were obtained in boiling
phosphoryl chloride.
Figure 1. Pharmacophore for 5-HT3 partial agonists (HBA PP: projected point (PP)
of hydrogen bond acceptor (HBA)).
Finally, nucleophilic substitution of the chlorine atom was car-
ried out using aminoalcoholates prepared by treatment of the cor-
responding alcohols with sodium hydride in boiling toluene.
Schemes 2 and 3 illustrate the synthesis of the aminoalcohols using
well-known chemistry.
2.2. Pharmacomodulations
Exact agreement (in terms of distances and chemical features)
between two elements of the pharmacophores (aromatic ring
and HBA) allowed us to modify mostly the lateral side chain of
the tricyclic group. However, some modifications of the tricyclic
platform were also carried out. More than fifty compounds were
synthesized and screened for their affinity toward the 5-HT4 recep-
tor (Tables 1–4).
Figure 2. Pharmacophore for 5-HT4 antagonists (HBA: hydrogen bond acceptor).
2.2.1. Modification of the lateral side chain
Withregard tothebasicaminomoietyandin relationtoour phar-
macophore, the presence of one methylene unit (Table 2 compared
to Table 1) bound to a piperidine ring gave the best ligands toward
5-HT4 receptors (32f and 32g). The agreement between the new
derivatives and the 5-HT4 receptor pharmacophore was optimum
in terms of the orientation of the vector associated with the hydro-
gen bond acceptor (Fig. 3) and the dihedral angle (preferential value
of 0°) associated with the linker component (imino-ether group) for
these derivatives. Indeed, this last conformation fits the conforma-
tion imposed by ester or amide groups for other 5-HT4 receptor
antagonists (Fig. 3). Accordingly, increasing(n = 2, 3, 4)or decreasing
(n = 0) the length of the methylene unit (see Ref. 45 for initial data)
did not improve the affinity (Table 1). As also described in the 5-
HT4 receptorpharmacophore, ahydrophobicgrouparoundthedistal
nitrogen was confirmed to be a favorable feature (Fig. 3). Interest-
ingly, starting from the tricyclic feature associated with 11 (8.85
(pIC50 for 5-HT3 receptor) and <5 (pIC50 for 5-HT4 receptor)), the
selectivity between 5-HT3 and 5-HT4 receptors was reversed for
two derivatives 32f and 32h (see Table 5). Moreover, derivatives
with the lateral chain (31q, 31r, 31t, 31u) closer to benzamide46 10
showed nearly the same affinity values as their corresponding com-
pound, as expected if we consider the pharmacophore.
HN
N
N
S
N
O
N
N
N
C3H7
CH2Ph
12
11
Chart 3. 5-HT3 towards 5-HT4 ligands.
tives44 (12) by considering the bioisosteric replacement of the ester
function [the hydrogen bond acceptor (HBA) component of the 5-
HT4 pharmacophore] by a cyclic iminoether (see Chart 3).
We are now reporting in this paper the most recent results we
obtained for the pharmacomodulation of the pyrrolothienopyr-
azine core by considering the 5-HT4 receptor antagonist pharmaco-
phore as the starting point. On the basis of the potential
therapeutic interest associated to selective 5-HT4 receptor ligands
but also to bipotent 5-HT3/5-HT4 receptor ligands, the 5-HT3/5-HT4
receptor selectivity of our best derivatives will be assessed.
2. Results and discussion
2.1. Chemistry
2.2.2. Modification of the tricyclic ring
Addition of a hydrophobic fragment (methyl group) to the tricy-
clic ring led to better agreement (fit value of 8.56 for 32g compared
to 9.64 for 34d) between the tricyclic ring and the characteristics of
the pharmacophore (Fig. 3). This was confirmed by a large increase
in affinity (see Table 3). However, the selectivity profile for 5-HT
The new 5-substituted pyrrolo[1,2-a]thieno[3,2-e]pyrazines
31a–u, 32a–32n, 5-substituted pyrrolo[1,2-a]thieno[2,3-e]pyra-
zines 33a–f and 1-methyl-5-substituted pyrrolo[1,2-a]thieno[2,3-

2610
S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
Table 1
Table 2
Binding properties of pyrrolo[1,2-a]thieno[2,3-e]pyrazine derivatives (n = 0, 2 and 3)
Binding properties of pyrrolo[1,2-a]thieno[2,3-e]pyrazine derivatives (n = 1)
N
N
N
N
S
S
OCH2R
O(CH2)nR
No.
R
% Inhibition 5-HT4
pKi (n = 3)
n
No.
R
% Inhibition 5-HT4
pKi (n = 3)
10ꢀ6/10ꢀ8
M
10ꢀ6/10ꢀ8
53/11
M
0
0
31a
31b
ND
ND
32a
90/18
ND
N
N
H
26/0
NH
32b
32c
91/0
82/0
ND
ND
N
N
0
0
31c
31d
67/10
50/1
ND
ND
C3H7
NC3H7
NC4H9
C4H9
NH
2
2
2
31e
31f
31g
–NH2
–NHCH3
–NCH3(CH2Ph)
0/0
7/9
20/2
ND
ND
ND
32d
32e
32f
74/8
ND
2
2
31h
31i
60/7
73/5
ND
N
100/0
84/42
100/97
100/31
99/55
100/38
56/2
7.23 ( 0.21)
7.72 ( 0.10)
7.79 ( 0.44)
7.68 ( 0.10)
7.59 ( 0.11)
7.74 ( 0.37)
ND
NCH3
NC2H5
NC3H7
NC4H9
NC5H11
N
6.38 ( 0.10)
N
2
2
2
2
2
31j
18/12
38/0
ND
ND
ND
ND
ND
N
N
N
N
N
O
32g
32h
32i
31k
31l
NH
72/0
NC3H7
NC4H9
NCH2Ph
31m
31n
66/12
13/0
32j
2
2
31o
31p
90/5
72/0
6.21 ( 0.22)
32k
32l
NCH(CH3)CH2CH3
NCH2CH=CH2
NCH2CH2CH=CH2
NCH2Ph
N
H
95/32
100/23
100/0
8.06 ( 0.61)
7.43 ( 0.31)
7.20 ( 0.13)
ND
N
C3H7
N
32m
32n
3
3
31q
31r
100/15
100/30
7.13 ( 0.50)
7.28 ( 0.42)
N
3
4
31s
31t
75/14
ND
N
NH
receptors (see Table 5) appeared to be different 34d, compared to
32f and 32h. In contradiction to the previous observations, 34d
showed higher affinity toward 5-HT3 receptors and a different
selectivity profile for 5-HT2 receptor subtypes (Table 5). The first
observation points out the importance of this additional hydropho-
bic group for binding 5-HT3 receptors. Indeed, for several benzam-
–NEt2
100/34
7.79 ( 0.27)
4
31u
100/16
7.24 ( 0.34)
N

S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
2611
Table 3
Table 4
Binding properties of pyrrolo[1,2-a]thieno[3,2-e]pyrazine derivatives
Binding properties of 1-methylpyrrolo[1,2-a]thieno[3,2-e]pyrazine derivatives
H3C
N
N
S
S
N
OCH2R
N
OR
No.
R
% Inhibition 5-HT4
pKi (n = 3)
No.
R
% Inhibition 5-HT4
pKi (n = 3)
10ꢀ6/10ꢀ8
100/26
M
10ꢀ6/10ꢀ8
100/20
M
34a
7.89 ( 0.38)
ND
33a
7.42 ( 0.37)
(CH2)3
N
NCH3
34b
34c
34d
34e
34f
34g
34h
34i
100/13
100/30
100/78
100/75
100/20
100/30
100/18
100/26
100/28
100/11
NCH3
33b
33c
33d
33e
33f
92/0
ND
NC2H5
8.02 ( 0.30)
8.76 ( 0.40)
8.27 ( 0.41)
ND
NC2H5
100/54
100/56
100/13
99/49
7.93 ( 0.02)
7.68 ( 0.31)
7.31 ( 0.45)
7.53 ( 0.28)
NC3H7
NC3H7
NC4H9
NC4H9
NC5H11
NCH2CH=CH2
NC5H11
8.19 ( 0.23)
ND
N
ide derivatives with 5-HT3 affinity, a chlorine atom on the phenyl
ring was found to be important to achieve good potency.46 Starting
from our pharmacophore, this new methyl group fit the attributes
of the chlorine atom of the benzamide derivatives (Fig. 3). For the
5-HT2 receptor, the study of Micheli et al., showing the importance
of three hydrophobic features for a 5-HT2C pharmacophore, pro-
vides some key points concerning this second observation.47
NCH(CH3)CH2CH3
NCH2CH=CH2
NCH2CH2CH=CH2
NCH2Ph
8.07 ( 0.13)
7.95 ( 0.29)
ND
2.3. Pharmacology results
34j
The 5-HT4 agonist or antagonist properties of compound 34d,
which exhibits the highest affinity of our present series, were
determined by measuring the production of cAMP in COS-7 cells
expressing the mouse 5-HT4(a) receptor.48,49 In vivo studies were
also conducted, first to evaluate any gross behavioral effects and
acute toxicity.50 Second, in view of the role of 5-HT4 receptors in
learning and memory,18–20 the ability of 34d to modulate such cen-
tral action was studied by testing its effect on spontaneous alterna-
tion21 (a model of working-memory) in mice treated with
scopolamine. Third, the action profile of 34d was investigated by
studying its analgesic potential using both the writhing test51,52
(peripheral analgesic activity) and the hot plate method53 (central
analgesic activity) on mice, since recent work described the poten-
tial of 5-HT4 receptor ligands to dampen nociceptive responses in
different models of pain in the mouse.54,55 Finally, based on the
affinity of compound 34d for 5-HT3 receptors, the effect of com-
pound 34d on 5-HT evoked currents was evaluated in NG108-15
cells known to express these receptor channels.
34k
receptor antagonistic effect of 34d amounted to ꢁ80% of the 5-
HT response, with an affinity of 10ꢀ7 M (IC50) (Fig. 4).
Preliminary toxicological and pharmacological screening of com-
pound 34d (Tables 5 and 6) showed its major effect to be hypoactiv-
ity of mice at subtoxic doses (at <1 approximate LD50) and a LD50
4
value at 200 mg/kg. Subsequent pharmacological studies were then
performed at a maximum dose around 2 mg/kg (1/100 of LD50).
At the doses tested (see Section 4), compound 34d had neither a
per se effect on spontaneous alternation performance in the
mouse, nor action on the scopolamine-induced deficit in this mod-
el of immediate working memory (data not shown). In the writhing
test (Table 7), 34d exhibited powerful antinociceptive activities at
very low doses and revealed an activity profile close to those of the
5-HT4 receptor antagonists GR 125487 and GR 113808. In the same
range of doses, no effect could be detected in the hot plate test
(data not shown). In electrophysiological studies, when applied
by itself for 1 min prior to application of the agonist, compound
34d failed to elicit 5-HT3 receptor current. Compound 34d blunted
The nature of the interaction between compound 34d and the
5-HT4 receptor was studied in vitro using COS-7 cells. In the ab-
sence of 5-HT, compound 34d produced a slight increase in cAMP
levels, which corresponds to ꢁ20% of the 5-HT response and there-
fore demonstrates a weak partial agonist effect for this compound
(Fig. 4). Compound 34d also reversed the increase in production of
cAMP in COS-7 cells elicited by 5-HT (10ꢀ7 M). The specific 5-HT4
the ability of serotonin (50 lM) to stimulate inward 5-HT3 cur-

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S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
NH2
N
N
ii
-
i
Ar
Ar
Ar
CO2CH3
CO2CH3
CO2H
13-15
16-18
19-21
N
N
ii
-
iv
-
v
-
Ar
Ar
CON3
N
H
O
22-24
25-27
H3C
N
N
N
N
vi,vii
S
Ar
Ar
Ar =
S
S
Cl
OR
28-30
31a-u ; 32a-32n ; 33a-f ; 34a-k
Scheme 1. Synthesis of thienopyrrolopyrazines. Reagents and conditions: (i) 2,5-dimethoxytetrahydrofuran, 1,4-dioxane, 4-chloropyridinium hydrochloride, reflux, 3 h;
yield range: 90%; (ii) NaOH, H2O, CH3OH, reflux, 4 h; yield range: 80%; (iii) TEA, ClCO2Et, NaN3, Me2CO, CH3CN, 0 °C, 1.5 h; yield range: 75%; (iv) o-dichlorobenzene, reflux,
5 min; yield range: 85%; (v) POCl3, reflux, 5 h; yield range: 80%; (vi) ROH, NaH, toluene, reflux, 2–8 h; (vii) i-PrOH, oxalic or fumaric or hydrochloric acid, reflux, 1 h.
i
HO
(CH2)n NRR'
HO
(CH2)n Cl
n = 2-4
,
,
O
,
NH
R,R' = Et
,
ii
HO(CH2)2
N
NH
HO(CH2)2
N
N
R''
R'' = Pr, Bu, Bn
Scheme 2. Synthesis of hydroxyalkyl piperazine derivatives. Reagents and condi-
tions: (i) RR0NH, K2CO3, NaI, EtOH, reflux, 12 h; (ii) K2CO3, R00I or R00Br, EtOH, reflux,
8 h.
Figure 3. Superimposition of 34d (in red) and 2 (in green) with 5-HT4 pharmaco-
phore, highlighting the correspondence between chlorine atom of 2 and methyl
group of 34d.
i
HOH2C
R
HO2C
NH
NH
demonstrated that it is possible to transform 5-HT3 ligands into
5-HT4 ligands by the means of a slight pharmacomodulation. Thus,
these compounds allowed us to validate the 5-HT4 receptor antag-
onist pharmacophore and provided some insights into the selectiv-
ity toward 5-HT3/5-HT4 receptors. However, we have not yet found
all the structural features in our series which could lead to high
selectivity toward 5-HT receptors. Experimental in vivo and
in vitro data revealed the antagonistic character of the best 5-
HT4 derivatives. Among these, 34d proved to be of great interest
as a peripheral antinociceptive agent. Complementary studies are
underway to evaluate the therapeutic potential of this compound.
ii
HOH2C
N
Scheme 3. Synthesis of 4-hydroxymethyl piperidine derivatives. Reagents and
conditions: (i) LiAlH4, THF, reflux; (ii) RI or RBr, EtOH, reflux.
rents. The dose–response relationship (Fig. 5) shows a clear antag-
onistic effect of 34d (IC50 = 3.85 ꢂ 10ꢀ7 M) on 5-HT-induced cur-
rents. Taken together, these data suggest a clear peripheral
profile for 34d. Considering its high activity in a visceral pain mod-
el, this compound could be a lead in the search for new peripheral
analgesics with molecular targets different from classical nonste-
roidal anti-inflammatory drugs and, thus, potentially devoid of
gastrointestinal side effects.
4. Experimental
4.1. Chemistry
Each compound was characterized by elemental analysis, IR
spectra and 1H and 13C NMR spectra. 1H NMR and 13C NMR spectra
were recorded on a JEOL Lambda 400 MHz spectrometer. The val-
ues of chemical shifts (d) are reported in parts per million (ppm)
while coupling constants are given in Hertz (Hz) (these data are re-
3. Conclusion
We described a new family of compounds, pyrrolothienopyr-
azine derivatives, with high affinity for 5-HT4 receptors. We have

S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
2613
Table 5
Binding selectivity of 34d, 32f and 32h
Receptor
Radioligand/species
[3H]8-OH-DPAT/human recombinant
34d pIC50 (M)
32f pIC50 (M)
32h pIC50 (M)
5-HT1A
5.3
<4
5.28
5-HT1B
5-HT1D
5-HT2A
5-HT2B
5-HT2C
5-HT3
5-HT4
5-HT5A
[3H]5-OH-tryptamine/rat cerebral cortex
[3H]5-OH-tryptamine/rat recombinant
[3H]ketanserin/human recombinant
[3H]mesulergine/human recombinant
[3H]mesulergine/human recombinant
[3H]granisetron/human recombinant
[3H]1/guinea pig
<5
5.34
<5
6.55
7.14
8.16
9.17
<5
5.09
8.01
5.25
<6
6.78
7.28
4.53
<5
7.72
7.67
[3H]LSD/human recombinant
5-HT6
5-HT7
[3H]LSD/human recombinant
<5
<6
<5
<6
<5
<6
[3H]LSD/human recombinant
Adrenergic a1A
Adrenergic a1B
Adrenergic a1D
Adrenergic a2A
Adrenergic a2B
Adrenergic a2C
[3H]prazosin/human recombinant
[3H]prazosin/human recombinant
[3H]prazosin/human recombinant
[3H]RX821002/human recombinant
[3H]RX821002/human recombinant
[3H]RX821002/human recombinant
7.13
6.84
6.69
6.05
6.02
6.13
Table 7
Antinociceptive activity of tested compounds and references in the mouse writhing
test after intraperitoneal administration
Compound
Number of stretchesa
0
0.01 mg/kg
0.1 mg/kg
1 mg/kg
5 mg/kg
30 mg/kg
8.3**
34d
GR125487
1
Aspirin
Piroxicam
26.1
23.4
25.3
24.2
23
17.2*
19.1
21.1
12.5**
13.2*
14.8*
7.6***
12.9*
14.3*
2.2**
Statistical significance between control and treated groups (ANOVA + PLSD of
Fischer: *p < 0.05; **p < 0.01; ***p < 0.001).
Groups of 8 mice (20–24 g) were injected by ip with 10 mL/kg of 0.6% aqueous
acetic acid. The mice were placed in an observation beaker and the number of
stretches per animal was counted during a 10-min period starting 10 min after
acetic acid treatment. Tested and reference compounds were administered 15 min
before the acetic acid solution.
a
Number of stretches induced by a 0.6% acetic acid solution.
ported only for the compounds tested in the pharmacological
study). Melting points were determined on a Köfler bank. IR spec-
tra were taken with a Genesis Series FTIR spectrophotometer.
The general procedure (Scheme 1) for the preparation of 28 and
29 is described in the publication of Rault et al.40 The preparation
of compound 30 has followed the same procedure by starting from
3-amino-4-methylthiophene-2-carboxylic acid methyl ester.56 The
general procedure for the preparation of the aminoalcohols de-
scribed in Scheme 2 used well-known chemistry. For Scheme 3,
the publication of Sorensen et al.57 described the preparation of
2-(-1-alkyl-piperidin-4-yl)-alcohol starting from N-alkylpiperidin-
ecarboxylic esters.58
Figure 4. Agonist and antagonist activity of compound 34d (31d in the graph) on
cAMP production by COS-7 cells. (A) Upper curve: the agonist activity was
evaluated by measuring intracellular cAMP levels in response to increasing
concentrations of compound 34d for 10 min at 37 °C in COS-7 cells expressing 5-
HT4(a) receptor at a density of 560 67 fmol/mg protein. The results are expressed
as a percentage of maximum 5-HT stimulation over basal activity. Basal conversion
of [3H]ATP to [3H]cAMP was equal to 0.1 0.08%. (B) Lower curve: the antagonist
activity was evaluated on the same transfected COS-7 cells by the capacity of
4.1.1. 5-(1-Azabicyclo[2.2.2]oct-3-yloxy)pyrrolo[1,2-a]thieno-
[3,2-e]pyrazine fumarate hemi-hydrate salt (31a)
This procedure illustrates the general method of preparation of
compounds 31a–u, 32a–32n, 33a–f and 34a–k. Sodium hydride
(60% in oil, 1.15 g, 28.7 mmol) was portion wise added to a solution
of 3-quinuclidinol (1.2 g, 9.43 mmol) in 30 mL of toluene; the
compound 34d to reverse cAMP produced by 1
lM 5-HT and taken as 100% for
10 min at 37 °C. At 1 M 5-HT, the percent conversion of [3H]ATP to [3H]cAMP was
l
equal to 0.87 0.12%. The results are means of four independent experiments.
Table 6
Pharmacological and toxicological properties of 34d
Compound
Doses (mg/kg)
LD50 (mg/kg)
Symptoms (subtoxic doses)
Symptoms (toxic doses)
34d
100–200–300
25
5
200
—
—
Hypoactivity, relaxation, passivity
Hyperactivity, irritability, stereotypy
Hypoactivity, passivity, ptosis
Convulsions
—
—
Methylphenidate
Perphenazine

2614
S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
0.86 (t, J = 7.0 Hz, 3H), 1.55 (sext., J = 7.2 Hz, 2H), 1.93 (m, 2H),
2.13 (m, 2H), 2.61 (t, J = 7.0 Hz, 2H), 2.75 (m, 2H), 3.0 (m, 2H),
5.37 (m, 1H), 6.54 (s, 2H), 6.84 (m, 1H), 6.93 (m, 1H), 7.27 (d,
J = 5.5 Hz, 1H), 7.42 (d, J = 5.5 Hz, 1H), 7.86 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 11.5, 18.3, 28.8, 49.1, 58.1, 68.8, 104.6,
113.6, 116.0, 117.9, 118.5, 123.8, 124.0, 134.8, 135.7, 153.6,
167.4. IR: 3436 (s, NH+), 2968, 1726 (s, C@O), 1583 cmꢀ1. Anal.
(C21H25N3O5S) C, H, N.
4.1.4. 5-[(1-Butylpiperidin-4-yl)hydroxy]pyrrolo[1,2-a]thieno-
[3,2-e]pyrazine fumarate salt (31d)
Compound 31d was prepared from 0.66 g (3.2 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1 g (6.4 mmol) of 1-bu-
tyl-4-hydroxypiperidine and 0.76 g (19.2 mmol) of sodium
hydride. The reaction mixture was refluxed for 4 h. Salification
with 0.37 g (3.2 mmol) of fumaric acid gave 0.6 g (42%) of 31d as
a yellow powder. Mp: 178 °C. 1H NMR (400 MHz, DMSO-d6): d
0.87 (t, J = 7.2 Hz, 3H), 1.29 (sext., J = 7.2 Hz, 2H), 1.52 (m, 2H),
1.92 (m, 2H), 2.13 (m, 2H), 2.64 (m, 2H), 2.74 (m, 2H), 3.0 (m,
2H), 5.37 (m, 1H), 6.54 (s, 2H), 6.84 (m, 1H), 6.93 (m, 1H), 7.27
(d, J = 5.5 Hz, 1H), 7.42 (d, J = 5.5 Hz, 1H), 7.87 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 13.8, 19.9, 27.1, 29.0, 49.3, 56.3, 68.8,
104.6, 113.6, 116.1, 117.9, 118.5, 123.8, 124.0, 134.7, 135.7,
Figure 5. Antagonist activity of compound 34d (31d in the graph) on 5-HT evoked
currents of NG108-15 cells expressing 5-HT3 receptor. Insert shows 5-HT induced
current before (CTRL) and after application of compound 34d at 3.10–7 M. The
straight line represents 0 current level and the calibration bars 50 pA and 5 s.
153.7, 167.2. IR: 3433 (s, NH+), 2963, 1723 (s, C@O), 1582 cmꢀ1
.
mixture was stirred at 80 °C for 1 h. 5-Chloropyrrolo[1,2-a]thie-
no[3,2-e]pyrazine 28 (1.64 g, 7.86 mmol) was added and stirred
at reflux for 5 h. The reaction mixture was diluted with water
and extracted with Et2O, and the extract was washed with water
and dried over MgSO4. The solvent was evaporated under vacuum
and the residue was dissolved in i-PrOH (15 mL) and then salified
with fumaric acid (0.81 g, 7.86 mmol). After stirring at 80 °C for
1 h, the precipitate was filtered, washed with Et2O and dried. This
gave compound 31a as a white powder (1.07 g, 33% yield). Mp:
174 °C. 1H NMR (400 MHz, DMSO-d6): d 1.72 (m, 1H), 1.88 (m,
2H), 2.15 (m, 1H), 2.43 (m, 1H), 3.08 (m, 1H), 3.08–3.15 (m, 4H),
3.66 (dd, J = 8.5 Hz, J = 13.5 Hz, 1H), 5.45 (m, 1H), 6.51 (s, 2H),
6.88 (dd , J = 2.7 Hz, J = 3.8 Hz, 1H), 6.99 (d , J = 3.8 Hz, 1H), 7.28
(d, J = 5.6 Hz, 1H), 7.45 (d, J = 5.6 Hz, 1H), 7.91 (d, J = 2.7 Hz, 1H),
9.9 (M, 2H). 13C NMR (400 MHz, DMSO-d6): d 17.5, 21.2, 24.4,
44.6, 45.4, 53.2, 69.5, 104.6, 113.7, 116.2, 117.9, 118.3, 123.9,
124.1, 134.9, 135.5, 153.6, 167.8. IR: 3430 (s, NH+), 3095, 1698 (s,
C@O), 1464, 1407 cmꢀ1. Anal. (C20H22N3O11/2S) C, H, N.
Anal. (C22H27N3O5S) C, H, N.
4.1.5. 5-(2-Aminoethyloxy)pyrrolo[1,2-a]thieno[3,2-e]pyrazine
oxalate salt (31e)
Compound 31e was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.58 g (9.6 mmol) of etha-
nolamine and 1.15 g (28.7 mmol) of sodium hydride. The reaction
mixture was refluxed for 5 h. Salification with 0.43 g (4.8 mmol)
of oxalic acid gave 1.1 g (70%) of 31e as a beige powder. Mp:
216 °C. 1H NMR (400 MHz, DMSO-d6): d 3.32 (t, J = 4.4 Hz, 2H),
4.62 (t, J = 4.4 Hz, 2H), 5.85 (M, 2H), 6.87 (m, 1H), 7.07 (m, 1H),
7.30 (d, J = 5.5 Hz, 1H), 7.44 (d, J = 5.5 Hz, 1H), 7.91 (m, 1H). 13C
NMR (400 MHz, DMSO-d6): d 38.4, 62.7, 105.3, 113.9, 116.4,
118.2, 118.4, 124.1, 124.4, 135.6, 154.3, 164.8. IR: 3218 (s, NH+),
2972, 1729 (s, C@O), 1521 cmꢀ1. Anal. (C13H13N3O5S) C, H, N.
4.1.6. 5-[(2-Methylamino)ethyloxy]pyrrolo[1,2-a]thieno[3,2-
e]pyrazine fumarate salt (31f)
Compound 31f was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.72 g (9.6 mmol) of 2-
methylaminoethanol and 1.15 g (28.8 mmol) of sodium hydride.
The reaction mixture was refluxed for 7 h. Salification with 0.55 g
(4.8 mmol) of fumaric acid gave 1.35 g (78%) of 31f as a white pow-
der. Mp: 200 °C. 1H NMR (400 MHz, DMSO-d6) d 2.60(s, 3H), 3.34 (t,
J = 5.2 Hz, 2H), 4.66 (t, J = 5.2 Hz, 2H), 6.46 (s, 2H), 6.85 (m, 1H),
7.04 (m, 1H), 7.30 (d, J = 5.5 Hz, 1H), 7.40 (M, 2H), 7.43 (d,
J = 5.5 Hz, 1H), 7.89 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d
33.0, 47.2, 62.8, 105.0, 113.6, 116.1, 118.0, 118.2, 123.9, 124.2,
135.0, 135.5, 154.1, 167.8. IR: 3440 (s, NH+), 2953, 1690 (s, C@O),
1522 cmꢀ1. Anal. (C16H17N3O5S) C, H, N.
4.1.2. 5-(Piperidin-4-yl)oxy]pyrrolo[1,2-a]thieno[3,2-e]
pyrazine dichlorhydrate salt (31b)
Compound 31b was prepared from 2 g (9.6 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.97 g (9.6 mmol) of 4-
hydroxypiperidine and 1.14 g (28.7 mmol) of sodium hydride.
The reaction mixture was refluxed for 5 h. Salification with 3 mL
of chlorhydric acid (10 N) gave 1.50 g (45%) of 31b as a yellow
powder. Mp: 218 °C. 1H NMR (400 MHz, DMSO-d6): d 2.20 (m,
4H), 3.20 (m, 4H), 4.30 (m, 2H), 5.50 (m, 1H), 6.80 (m, 1H), 7.0
(m, 1H), 7.25 (d, J = 5.5 Hz, 1H), 7.40 (d, J = 5.5 Hz, 1H), 7.80 (m,
1H), 9.30 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 48.9, 57.8,
69.5, 104.7, 113.5, 115.9, 117.7, 118.3, 123.7, 123.9, 135.5, 153.2.
IR: 3060 (s, NH+), 1590 (s, C@O), 1440, 1320, 1020 cmꢀ1. Anal.
(C14H17 Cl2N3OS) C, H, N.
4.1.7. 5-[(2-Benzyl-2-methylamino)ethyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine oxalate salt (31g)
Compound 31g was prepared from 0.5 g (2.4 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.79 g (4.8 mmol) of 2-
benzyl-2-methylaminoethanol and 0.57 g (14.4 mmol) of sodium
hydride. The reaction mixture was refluxed for 8 h. Salification
with 0.21 g (2.4 mmol) of oxalic acid gave 0.54 g (53%) of 31g as
a white powder. Mp: 202 °C. 1H NMR (400 MHz, DMSO-d6): d
2.60 (s, 3H), 3.26 (m, 2H), 4.05 (m, 2H), 4.75 (m, 2H), 6.85 (m,
1H), 7.2–7.5 (M, 7H), 7.82 (m, 1H), 7.94 (M, 2H). 13C NMR
(400 MHz, DMSO-d6): d 40.8, 54.0, 60.0, 61.7, 104.3, 113.4, 115.7,
4.1.3. 5-[(1-Propylpiperidin-4-yl)oxy]pyrrolo[1,2-a]thieno[3,2-
e]pyrazine fumarate salt (31c)
Compound 31c was prepared from 0.73 g (3.5 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1 g (7 mmol) of 1-pro-
pyl-4-hydroxypiperidine and 0.84 g (21 mmol) of sodium
hydride. The reaction mixture was refluxed for 5 h. Salification
with 0.41 g (3.5 mmol) of fumaric acid gave 0.95 g (63%) of 31c
as a beige powder. Mp: 146 °C. 1H NMR (400 MHz, DMSO-d6): d

S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
2615
117.5, 118.2, 123.7, 123.8, 127.9, 128.1, 129.6, 134.1, 135.3, 153.9,
4.1.12. 5-[2-(4-Propylpiperazin-1-yl)ethyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (31l)
162.7. IR: 3445 (s, NH+), 2984, 1721 (s, C@O), 1493 cmꢀ1. Anal.
(C21H21N3O5S) C, H, N.
Compound 31l was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.65 g (9.6 mmol) of 1-pro-
pyl-[4-(2-hydroxyethyl)]piperazine and 1.15 g (29 mmol) of
sodium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 1.10 g (9.6 mmol) of fumaric acid gave 2 g (81%) of 31l as
a beige powder. Mp: 216 °C. 1H NMR (400 MHz, DMSO-d6): d 0.83
(m, 3H), 1.51 (m, 2H), 2.5–2.9 (M, 12 H), 4.58 (m, 2H), 6.56 (s, 4H),
6.85 (m, 1H), 6.87 (m, 1H), 7.28 (m, 1H), 7.42 (m, 1H), 7.87 (m, 1H),
9.55 (M, 4H). 13C NMR (400 MHz, DMSO-d6): d 11.1, 17.6, 50.8,
51.1, 55.4, 57.8, 63.1, 104.2, 113.4, 115.9, 117.7, 118.1, 123.7,
123.8, 134.3, 135.5, 154.2, 166.6. IR: 3431 (s, NH+), 2967, 1717 (s,
C@O), 1587 cmꢀ1. Anal. (C26H32N4O9S) C, H, N.
4.1.8. 5-(2-Pyrrolidinoethyloxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (31h)
Compound 31h was prepared from 1.21 g (5.8 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1 g (8.7 mmol) of 1-(2-
hydroxyethyl)pyrrolidine and 1.04 g (26.1 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
0.67 g (5.8 mmol) of fumaric acid gave 1.95 g (83%) of 31h as a
white powder. Mp: 182 °C. 1H NMR (400 MHz, DMSO-d6): d 1.79
(m, 4H), 2.95 (m, 4H), 3.23 (t, J = 5.0 Hz, 2H), 4.66 (t, J = 5.0 Hz,
2H), 6.52 (s, 2H), 6.84 (m, 1H), 6.92 (m, 1H), 7.29 (d, J = 5.5 Hz,
1H), 7.43 (d, J = 5.5 Hz, 1H), 7.88 (m, 1H), 9.5 (M, 2H). 13C NMR
(400 MHz, DMSO-d6): d 23.0, 53.2, 53.6, 63.2, 104.6, 113.6, 116.1,
117.9, 118.3, 123.9, 124.0, 134.6, 135.6, 154.2, 167.2. IR: 3441 (s,
NH+), 3064, 1726 (s, C@O), 1589, 1423 cmꢀ1. Anal. (C19H21N3O5S)
C, H, N.
4.1.13. 5-[2-(4-Butylpiperazin-1-yl)ethyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (31m)
Compound 31m was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.78 g (9.6 mmol) of 1-bu-
tyl-[4-(2-hydroxyethyl)]piperazine and 1.15 g (29 mmol) of
sodium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 1.11 g (9.6 mmol) of fumaric acid gave 2.12 g (75%) of 31m
as a white powder. Mp: 226 °C. 1 H NMR (400 MHz, DMSO-d6): d
0.85 (m, 3H), 1.27 (m, 2H), 1.51 (m, 2H), 2.5–2.9 (m, 12H), 4.58 (m,
2H), 6.59 (s, 4H), 6.83 (m, 1H), 6.85 (m, 1H), 7.25 (m, 1H), 7.40 (m,
1H), 7.86 (m, 1H), 10.73 (M, 4H). 13C NMR (400 MHz, DMSO-d6): d
13.3, 19.5, 26.3, 50.8, 51.2, 55.4, 55.9, 63.1, 104.2, 113.3, 115.6,
117.3, 118.3, 123.7, 123.8, 134.1, 135.6, 154.3, 166.5. IR: 3422 (s,
NH+), 2963, 1716 (s, C@O), 1304 cmꢀ1. Anal. (C27H34N4O9S) C, H, N.
4.1.9. 5-(2-Piperidinoethyloxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (31i)
Compound 31i was prepared from 1.21 g (5.8 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.9 g (6.98 mmol) of 1-
(2-hydroxyethyl)piperidine and 0.84 g (20.9 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
0.67 g (5.8 mmol) of fumaric acid gave 1.40 g (58%) of 31i as a
white powder. Mp: 182 °C. 1H NMR (400 MHz, DMSO-d6): d 1.41
(m, 2H), 1.58 (m, 4H), 2.78 (m, 4H), 3.06 (t, J = 5.4 Hz, 2H), 4.65
(t, J = 5.4 Hz, 2H), 6.54 (s, 2H), 6.84 (dd, J = 2.6 Hz, J = 3.6 Hz, 1H),
6.89 (d, J = 3.6 Hz, 1H), 7.29 (d, J = 5.6 Hz, 1H), 7.43 (d, J = 5.6 Hz,
1H), 7.88 (m, 1H), 8.02 (M, 2H). 13C NMR (400 MHz, DMSO-d6): d
22.8, 24.3, 53.5, 55.9, 62.3, 104.5, 113.7, 116.1, 117.9, 118.3,
123.9, 124.0, 134.5, 135.6, 154.2, 167.0. IR: 3437 (s, NH+), 2934,
1725 (s, C@O), 1605 cmꢀ1. Anal. (C20H23N3O5S) C, H, N.
4.1.14. 5-[2-(4-Benzylpiperazin-1-yl)ethyloxy]pyrrolo[1,2-
a]thieno[3,2-e]pyrazine fumarate salt (31n)
Compound 31n was prepared from 0.7 g (3.3 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.45 g (6.6 mmol) of 1-
benzyl-[4-(2-hydroxyethyl)]piperazine and 0.8 g (19.8 mmol) of
sodium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 0.76 g (6.6 mmol) of fumaric acid gave 0.91 g (44%) of
31n as a yellow powder. Mp: 217 °C. 1H NMR (400 MHz, DMSO-
d6): d 2.5–2.7 (m, 8H), 2.88 (m, 2H), 3.53 (s, 2H), 4.57 (m, 2H),
5.40 (m, 4H), 6.59 (s, 4H), 6.83 (m, 1H), 6.87 (m, 1H), 7.2–7.35
(m, 6H), 7.42 (m, 1H), 7.85 (m, 1H). 13C NMR (400 MHz, DMSO-
d6): d 51.3, 51.8, 55.6, 61.0, 62.8, 104.6, 113.7, 116.1, 117.9,
118.3, 123.9, 124.0, 127.7, 128.4, 129.5, 134.4, 135.7, 136.0,
4.1.10. 5-(2-Morpholinoethyloxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (31j)
Compound 31j was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.94 g (7.18 mmol) of 1-
(2-hydroxyethyl)morpholine and 0.86 g (21.5 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
0.56 g (4.8 mmol) of fumaric acid gave 1.64 g (81%) of 31j as a
white powder. Mp: 178 °C. 1H NMR (400 MHz, DMSO-d6): d 2.56
(t, J = 3.8 Hz, 4H), 2.83 (t, J = 5.4 Hz, 2H), 3.57 (t, J = 3.8 Hz, 4H),
4.58 (t, J = 5.4 Hz, 2H), 6.60 (s, 2H), 6.83 (d, J = 2.80 Hz, 1H), 6.83
(d, J = 2.80 Hz, 1H), 7.27 (d, J = 5.5 Hz, 1H), 7.41 (d, J = 5.5 Hz, 1H),
7.85 (m, 1H), 9.60 (M, 2H). 13C NMR (400 MHz, DMSO-d6): d 53.4,
56.5, 63.2, 66.0, 104.4, 113.6, 116.0, 117.8, 118.4, 123.9, 124.0,
134.1, 135.7, 154.4, 166.2. IR: 3429 (s, NH+), 3011, 1722 (s, C@O),
1592 cmꢀ1. Anal. (C19H21N3O6S) C, H, N.
154.4, 166.7. IR: 3430 (s, NH+), 3062, 1715 (s, C@O), 1586 cmꢀ1
.
Anal. (C30H32N4O9S) C, H, N.
4.1.15. 5-[2-(Piperidin-2-yl)ethyloxy]pyrrolo[1,2-a]thieno[3,2-
e]pyrazine fumarate salt (31o)
Compound 31o was prepared from 1.08 g (5.1 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1 g (7.7 mmol) of 2-(2-
hydroxyethyl)piperidine and 0.94 g (23.1 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
0.6 g (5.1 mmol) of fumaric acid gave 1.72 g (79%) of 31o as a white
powder. Mp: 170 °C. 1H NMR (400 MHz, DMSO-d6): d 1.4–1.8 (m,
5H), 1.92 (m, 1H), 2.02 (m, 1H), 2.21 (m, 1H), 2.75 (m, 1H), 3.15
(m, 2H), 4.58 (t, J = 6.1 Hz, 2H), 5.51 (m, 2H), 6.48 (s, 2H), 6.83
(m, 1H), 6.90 (m, 1H), 7.26 (d, J = 5.4 Hz, 1H), 7.38 (d, J = 5.4 Hz,
1H), 7.78 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 21.7, 22.0,
28.3, 32.4, 43.7, 53.1, 61.9, 104.1, 113.2, 115.4, 117.2, 118.3,
123.6, 123.7, 134.6, 135.5, 154.2, 167.5. IR: 3436 (s, NH+), 2942,
1695 (s, C@O), 1586 cmꢀ1. Anal. (C20H23N3O5S) C, H, N.
4.1.11. 5-(2-Piperazin-1-ylethyloxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (31k)
Compound 31k was prepared from 4 g (19.2 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 5 g (38.4 mmol) of 1-(2-
hydroxyethyl)piperazine and 4.61 g (0.11 mol) of sodium hydride.
The reaction mixture was refluxed for 6 h. Salification with 4.45 g
(38.4 mmol) of fumaric acid gave 7 g (69%) of 31k as a white pow-
der. Mp: 219 °C. 1H NMR (400 MHz, DMSO-d6): d 2.73 (m, 4H), 2.85
(m, 2H) ; 3.05 (m, 4H), 4.56 (m, 2H), 6.55 (s, 4H), 6.83 (m, 1H), 6.87
(m, 1H), 7.28 (m, 1H), 7.41 (m, 1H), 7.85 (m, 1H), 9.20 (M, 4H). 13C
NMR (400 MHz, DMSO-d6): d 42.8, 49.7, 56.0, 63.4, 104.5, 113.7,
116.1, 117.9, 118.4, 123.9, 124.0, 134.8, 135.7, 154.8, 167.3. IR:
4.1.16. 5-[2-(1-Propylpiperidin-2-yl)ethyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (31p)
Compound 31p was prepared from 0.4 g (1.91 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.65 g (3.8 mmol) of 1-
3428 (s, NH+), 3062, 1710 (s, C@O), 1587 cmꢀ1
(C23H26N4O9S) C, H, N.
.
Anal.

2616
S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
propyl-2-(2-hydroxyethyl)piperidine and 0.47 g (11.4 mmol) of so-
dium hydride. The reaction mixture was refluxed for 5 h. Salifica-
tion with 0.24 g (1.91 mmol) of fumaric acid gave 0.6 g (79%) of
31p as a white powder. Mp: 173 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.83 (t, J = 7.0 Hz, 3H), 1.34 (m, 1H), 1.40–1.55 (M, 6H),
1.98 (m, 1H), 2.09 (m, 1H), 2.41 (m, 1H), 2.45 (m, 2H), 2.70 (m,
1H), 2.77 (m, 1H), 2.86 (m, 1H), 3.60 (M, 2 H), 4.49 (m, 2H), 6.50
(s, 2H), 6.85 (m, 2H), 7.26 (d, J = 5.0 Hz, 1H), 7.42 (d, J = 5.0 Hz,
1H), 7.86 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 11.6, 18.3,
21.9, 23.9, 28.5, 28.5, 50.1, 54.0, 56.8, 63.1, 104.2, 113.6, 115.9,
117.8, 118.4, 123.7, 123.9, 134.7, 135.7, 154.5, 167.0. IR: 3454 (s,
NH+), 2935, 1705 (s, C@O), 1610, 1522 cmꢀ1. Anal. (C21H27N3O3S)
C, H, N.
diethyl-(4-hydroxybutyl)amine and 1.15 g (29 mmol) of sodium
hydride. The reaction mixture was refluxed for 6 h. Salification
with 0.43 g (4.8 mmol) of oxalic acid gave 1.47 g (75%) of 31t as
a yellow powder. Mp: 133 °C. 1H NMR (400 MHz, DMSO-d6): d
1.16 (t, J = 7.0 Hz, 6H), 1.8–1.9 (M, 4H), 3.04 (t, J = 6.0 Hz, 2H),
3.06 (q, J = 7.0 Hz, 4H), 4.53 (t, J = 6.0 Hz, 2H), 5.83 (M, 2H), 6.84
(dd, J = 4.0 Hz, J = 2.7 Hz, 1H), 6.90 (dd, J = 4.0 Hz, J = 1.3 Hz, 1 H),
7.27 (d, J = 5.5 Hz, 1 H), 7.40 (d, J = 5.5 Hz, 1H), 7.81 (dd,
J = 2.7 Hz, J = 1.3 Hz, 1H). 13C NMR (400 MHz, DMSO-d6): d 8.5,
20.2, 25.5, 46.2, 50.6, 64.7, 104.0, 113.3, 115.6, 117.4, 118.3,
123.6, 123.7, 135.5, 154.4, 163.7. IR: 3433 (s, NH+), 3016, 1711 (s,
C@O), 1524 cmꢀ1. Anal. (C19H25N3O5S) C, H, N.
4.1.21. 5-(4-Piperidinobutyloxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (31u)
4.1.17. 5-(2-Pyrrolidinopropyloxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (31q)
Compound 31u was prepared from 0.8 g (3.8 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.2 g (7.7 mmol) of 1-
(4-hydroxybutyl)piperidine and 0.92 g (23.1 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 5 h. Salification with
0.44 g (3.8 mmol) of fumaric acid gave 1.4 g (82%) of 31u as a yel-
low powder. Mp: 168 °C. 1H NMR (400 MHz, DMSO-d6): d 1.40 (m,
2H), 1.55 (m, 4H), 1.68 (m, 2H), 1.79 (m, 2H), 2.50–2.60 (m, 6H),
3.99 (m, 2H), 4.50 (m, 2H), 6.47 (s, 2H), 6.83 (m, 1H), 6.87 (m,
1H), 7.29 (d, J = 5.5 Hz, 1H), 7.41 (d, J = 5.5 Hz, 1H), 7.86 (m, 1H).
13C NMR (400 MHz, DMSO-d6): d 21.9, 23.2, 24.4, 26.2, 53.1, 57.1,
65.4, 104.3, 113.6, 116.0, 117.8, 118.4, 123.8, 124.0, 135.0, 135.8,
Compound 31q was prepared from 0.97 g (4.6 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.9 g (6.9 mmol) of 1-(3-
hydroxypropyl)pyrrolidine and 0.83 g (20.7 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
0.54 g (4.6 mmol) of fumaric acid gave 1.56 g (69%) of 31q as a
beige powder. Mp: 149 °C. 1H NMR (400 MHz, DMSO-d6): d 1.83
(m, 4H), 2.13 (quint., J = 6.2 Hz, 2 H), 3.01 (m, 4H), 3.05 (t,
J = 6.2 Hz, 2H), 4.49 (t, J = 6.2 Hz, 2H), 6.49 (s, 2H), 6.84 (dd,
J = 2.7 Hz, J = 3.8 Hz, 1H), 6.92 (d, J = 3.8 Hz, 1H), 7.28 (d,
J = 5.5 Hz, 1H), 7.42 (d, J = 5.5 Hz, 1H), 7.86 (m, 1H), 9.0 (M, 2H).
13C NMR (400 MHz, DMSO-d6): d 22.9, 25.9, 51.4, 52.8, 63.5,
104.5, 113.6, 116.0, 117.9, 118.3, 123.9, 124.0, 134.9, 135.7,
154.5, 167.7. IR: 3435 (s, NH+), 2966, 1721 (s, C@O), 1584,
1299 cmꢀ1. Anal. (C20H23N3O5S) C, H, N.
154.7, 167.6. IR: 3459 (s, NH+), 2941, 1718 (s, C@O), 1585 cmꢀ1
Anal. (C22H27N3O5S) C, H, N.
.
4.1.22. 5-(Piperidin-2-ylmethoxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (32a)
4.1.18. 5-(2-Piperidinopropyloxy)pyrrolo[1,2-a]thieno[3,2-e]-
pyrazine fumarate salt (31r)
Compound 32a was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.83 g (6.6 mmol) of 2-
(hydroxymethyl)piperidine and 0.86 g (20 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
0.56 g (4.8 mmol) of fumaric acid gave 1.45 g (75%) of 32a as a yel-
low powder. Mp: 199 °C. 1H NMR (400 MHz, DMSO-d6): d 1.4–1.9
(m, 6H), 2.91 (m, 1H), 3.33 (m, 1H), 3.51 (m, 1H), 4.50 (m, 2H),
4.60 (m, 2H), 6.50 (s, 4H), 6.85 (m, 1H), 7.12 (m, 1H), 7.29 (d,
J = 5.2 Hz, 1H), 7.42 (d, J = 5.2 Hz, 1H), 7.87 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 21.6, 22.1, 25.1, 43.9, 54.3, 65.9, 105.2,
113.6, 116.1, 117.9, 118.1, 123.9, 124.2, 134.9, 135.4, 154.1,
167.6. IR: 3450 (s, NH+), 2947, 1683, (s, C@O), 1589 cmꢀ1. Anal.
(C19H21N3O5S) C, H, N.
Compound 31r was prepared from 0.95 g (4.6 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1 g (6.9 mmol) of 1-(3-
hydroxypropyl)piperidine and 0.84 g (20.7 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
0.53 g (4.6 mmol) of fumaric acid gave 1.56 g (80%) of 31r as a
beige powder. Mp: 180 °C. 1 H NMR (400 MHz, DMSO-d6): d 1.42
(m, 2H), 1.57 (m, 4H), 2.05 (m, 2H), 2.64 (m, 4H), 2.71 (m, 2H),
4.2 (M, 2H), 4.47 (t, J = 5.4 Hz, 2H), 6.53 (s, 2H), 6.84 (m, 1H),
6.89 (m, 1H), 7.28 (d, J = 4.4 Hz, 1H), 7.42 (d, J = 4.4 Hz, 1H), 7.86
(m, 1H). 13C NMR (400 MHz, DMSO-d6): d 22.6, 23.9, 24.5, 53.2,
53.7, 63.3, 104.5, 113.7, 116.1, 117.9, 118.3, 123.9, 124.0, 134.5,
135.6, 154.2, 167.0. IR: 3432 (s, NH+), 2938, 1720 (s, C@O),
1585 cmꢀ1. Anal. (C21H25N3O5S) C, H, N.
4.1.23. 5-[(1-Propylpiperidin-3-yl)methoxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine oxalate salt (32b)
4.1.19. 5-(2-Piperazin-1-ylpropyloxy)pyrrolo[1,2-a]thieno[3,2-
e]pyrazine fumarate salt (31s)
Compound 32b was prepared from 0.46 g (2.2 mmol) of 5-
chloropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.7 g (4.4 mmol) of
1-propyl-3-(hydroxymethyl)piperidine and 0.53 g (13.2 mmol) of
sodium hydride. The reaction mixture was refluxed for 5 h. Salifi-
cation with 0.2 g (2.2 mmol) of oxalic acid gave 0.69 g (74%) of
32b as a white powder. Mp: 174 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.88 (m, 3H), 1.38 (m, 1H), 1.65–1.85 (M, 5H), 2.43 (m,
1H), 2.80 (m, 2H), 2.96 (m, 2H), 3.38 (m, 1H), 3.54 (m, 1H),
4.37 (m, 2H), 6.85 (m, 1H), 6.97 (m, 1H), 7.28 (m, 1H), 7.42 (m,
1H), 7.87 (m, 1H), 8.13 (M, 2H). 13C NMR (400 MHz, DMSO-d6):
d 10.9, 16.8, 21.7, 24.7, 33.4, 51.6, 53.7, 58.2, 67.0, 104.5, 113.6,
116.0, 117.8, 118.1, 123.9, 124.0, 135.5, 154.4, 164.5. IR: 3435
Compound 31s was prepared from 4 g (19.2 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 5 g (38.4 mmol) of 1-(3-
hydroxypropyl)piperazine and 4.61 g (0.11 mol) of sodium hy-
dride. The reaction mixture was refluxed for 6 h. Salification with
4.45 g (38.4 mmol) of fumaric acid gave 7 g (69%) of 31 s as a white
powder. Mp: 211 °C. 1H NMR (400 MHz, DMSO-d6): d 2.73 (m, 4 H),
2.85 (m, 2H), 3.05 (m, 4H), 4.56 (m, 2H), 6.55 (s, 4H), 6.83 (m, 1H),
6.87 (m, 1H), 7.28 (m, 1H), 7.41 (m, 1H), 7.85 (m, 1H), 9.20 (M, 4H).
13C NMR (400 MHz, DMSO-d6): d 42.8, 49.7, 56.0, 63.4, 104.5,
113.7, 116.1, 117.9, 118.4, 123.9, 124.0, 134.8, 135.7, 154.8,
167.3. IR: 3428 (s, NH+), 3062, 1710 (s, C@O), 1587 cmꢀ1. Anal.
(C24H28N4O9S) C, H, N.
(s, NH+), 2933, 1716 (s, C@O), 1615, 1519 cmꢀ1
.
Anal.
(C20H25N3O5S) C, H, N.
4.1.20. 5-(4-Diethylaminobutyloxy)pyrrolo[1,2-a]thieno[3,2-
e]pyrazine oxalate salt (31t)
4.1.24. 5-[(1-Butylpiperidin-3-yl)methoxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (32c)
Compound 31t was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.39 g (9.6 mmol) of 1-
Compound 32c was prepared from 0.4 g (1.9 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.66 g (3.8 mmol) of 1-bu-

S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
2617
tyl-3-(hydroxymethyl)piperidine and 0.46 g (11.4 mmol) of so-
4.1.28. 5-[(1-Propylpiperidin-4-yl)methyloxy]pyrrolo[1,2-a]-
dium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 0.22 g (1.9 mmol) of fumaric acid gave 0.58 g (66%) of
32c as a white powder. Mp: 148 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.85 (t, J = 7.2 Hz, 3H), 1.20 (m, 1H), 1.26 (sext., J = 7.2 Hz,
2H), 1.50 (quint., J = 7.2 Hz, 2H), 1.64 (m, 1H), 1.70–1.78 (m, 2H),
2.24 (m, 1H), 2.31 (m, 2H), 2.60 (m, 2H), 3.01 (m, 1H), 3.18 (m,
1H), 4.33 and 4.38 (dd, J = 10.9 Hz, J = 5.4 Hz, J =, 6.7 Hz, 2H), 6.54
(s, 2H), 6.84 (dd, J = 3.9 Hz, J = 2.6 Hz, 1H), 6.92 (dd, J = 3.9 Hz,
J = 1.2 Hz, 1H), 7.28 (d, J = 5.5 Hz, 1H), 7.42 (d, J = 5.5 Hz, 1H),
7.86 (dd, J = 2.6 Hz, J = 1.2 Hz, 1H). 13C NMR (400 MHz, DMSO-
d6): d 13.7, 19.8, 23.0, 25.6, 26.9, 34.4, 52.6, 55.1, 56.9, 67.7,
104.3, 113.5, 116.0, 117.8, 118.3, 123.8, 123.9, 134.5, 135.6,
154.5, 166.9. IR: 3445 (s, NH+), 2961, 1719 (s, C@O), 1658,
1584 cmꢀ1. Anal. (C23H29N3O5S) C, H, N.
thieno[3,2-e]pyrazine fumarate salt (32g)
Compound 32g was prepared from 2.37 g (11.4 mmol) of 5-
chloropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 2.67 g (19.1 mmol)
of 1-propyl-4-(hydroxymethyl)piperidine and 2.30 g (42.9 mmol)
of sodium hydride. The reaction mixture was refluxed for 3 h. Sali-
fication with 1.32 g (11.4 mmol) of fumaric acid gave 4.03 g (80%)
of 32g as a yellow powder. Mp: 182 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.85 (t, J = 7.3 Hz, 3H), 1.5–1.6 (m, 4H), 1.86 (m, 2H), 1.99 (m,
1H), 2.44 (m, 2H), 2.61 (m, 2H), 3.20 (d, J = 11.5 Hz, 2H), 4.32 (d,
J = 6.1 Hz, 2H), 6.53 (s, 2H), 6.82 (dd , J = 2.7 Hz, J = 3.9 Hz, 1 H),
6.89 (dd , J = 1.0 Hz, J = 3.9 Hz, 1H), 7.28 (d, J = 5.3 Hz, 1H), 7.41
(d, J = 5.3 Hz, 1H), 7.86 (dd , J = 2.7 Hz, J = 1.0 Hz, 1H). 13C NMR
(400 MHz, DMSO-d6): d 11.4, 18.0, 26.7, 38.9, 51.6, 58.3, 69.2,
104.3, 113.5, 116.0, 117.8, 118.3, 123.8, 123.9, 134.8, 135.7,
154.6, 167.3. IR: 3438 (s, NH+), 2927, 1721 (s, C@O), 1583 cmꢀ1
.
4.1.25. 5-[(Piperidin-4-yl)methyloxy]pyrrolo[1,2-a]thieno[3,2-
e]pyrazine fumarate salt (32d)
Anal. (C22H27N3O5S) C, H, N.
Compound 32d was prepared from 1.8 g (8.7 mmol) 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.5 g (13 mmol) of 4-
hydroxymethylpiperidine and 1.56 g (13.2 mmol) of sodium hy-
dride. The reaction mixture was refluxed for 9 h. Salification with
1 g (8.7 mmol) of fumaric acid gave 4.2 g (76%) of 32d as a beige
powder. Mp: 200 °C. 1H NMR (400 MHz, DMSO-d6): d 1.56 (m,
2H), 1.90 (m, 2H), 2.12 (m, 1H), 2.87 (m, 2H), 3.28 (m, 2H), 4.33
(d, J = 5.8 Hz, 2H), 4.55 (M, 2H), 6.44 (s, 2H), 6.83 (m, 1H), 6.90
(m, 1H), 7.27 (d, J = 5.5 Hz, 1H), 7.42 (d, J = 5.5 Hz, 1H), 7.86 (m,
1H). 13C NMR (400 MHz, DMSO-d6): d 25.8, 33.6, 42.9, 69.5,
104.8, 114.0, 116.5, 118.3, 118.7, 124.3, 124.4, 135.8, 136.1,
4.1.29. 5-[(1-Butylpiperidin-4-yl)methyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (32h)
Compound 32h was prepared from 3.04 g (14.6 mmol) of 5-
chloropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 3.7 g (21.9 mmol)
of 1-butyl-4-hydroxymethylpiperidine and 2.64 g (66 mmol) of so-
dium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 1.69 g (14.6 mmol) of fumaric acid gave 4.4 g (66%) of
32h as a beige powder. Mp: 168 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.87 (t, J = 7.0 Hz, 3H), 1.27 (m, 2H), 1.52 (m, 4H), 1.84 (m,
2H), 1.97 (m, 1H), 2.38 (m, 2H), 2.60 (m, 2H), 3.16 (d, J = 9.8 Hz,
2H), 4.32 (d, J = 5.6 Hz, 2H), 6.52 (s, 2 H), 6.83 (m, 1H), 6.90 (m,
1H), 7.28 (d, J = 5.2 Hz, 1H), 7.42 (d, J = 5.2 Hz, 1H), 7.87 (m, 1H),
10.2 (M, 2H). 13C NMR (400 MHz, DMSO-d6): d 13.6, 19.7, 25.9,
26.0, 33.3, 51.0, 55.7, 69.0, 104.4, 113.6, 115.9, 117.8, 118.3,
123.9, 124.0, 135.1, 135.7, 154.6, 168.0. IR: 3426 (s, NH+), 2952,
1711 (s, C@O), 1495, 1425 cmꢀ1. Anal. (C23H29N3O5S) C, H, N.
155.0, 168.8. IR: 3433 (s, NH+), 2942, 1678 (s, C@O), 1522 cmꢀ1
.
Anal. (C19H21N3O5S) C, H, N.
4.1.26. 5-[(1-Methylpiperidin-4-yl)methyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (32e)
Compound 32e was prepared from 0.71 g (3.4 mmol) of 5-
chloropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.58 g (4.1 mmol)
of 1-methyl-4-(hydroxymethyl)piperidine and 0.49 g (12.2 mmol)
of sodium hydride. The reaction mixture was refluxed for 3 h.
Salification with 0.39 g (3.4 mmol) of fumaric acid gave 0.5 g
(34%) of 32e as a beige powder. Mp: 182 °C. 1H NMR (400 MHz,
DMSO-d6): d 1.58 (m, 2H), 1.87 (m, 2H), 1.98 (m, 1H), 2.48 (m,
2H), 2.55 (m, 2H), 3.17 (d, J = 10.7 Hz, 2 H), 4.33 (d, J = 5.8 Hz,
2H), 6.52 (s, 2H), 6.82 (m, 1H), 6.90 (m, 1H), 7.28 (d, J = 5.3 Hz,
1H), 7.41 (d, J = 5.3 Hz, 1H), 7.86 (m, 1H). 13C NMR (400 MHz,
DMSO-d6): d 26.4, 33.0, 43.5, 53.1, 69.0, 104.4, 113.6, 116.0,
117.8, 118.3, 123.8, 124.0, 134.9, 135.7, 154.6, 167.6. IR: 3424
(s, NH+), 2958, 1704 (s, C@O), 1585 cmꢀ1. Anal. (C20H23N3O5S) C,
H, N.
4.1.30. 5-[(1-Pentylpiperidin-4-yl)methyloxy]pyrrolo[1,2-
a]thieno[3,2-e]pyrazine fumarate salt (32i)
Compound 32i was prepared from 0.93 g (4.44 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1 g (5.33 mmol) of 1-
pentyl-4-(hydroxymethyl)piperidine and 0.65 g (16.2 mmol) of so-
dium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 0.51 g (4.44 mmol) of fumaric acid gave 0.5 g (53%) of
32i as a beige powder. Mp: 174 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.86 (t, J = 6.6 Hz, 3H), 1.28 (m, 4H), 1.52 (m, 4H), 1.83 (m,
2H), 1.95 (m, 1H), 2.28 (m, 2H), 2.51 (t, J = 7.3 Hz, 2H), 3.07 (d,
J = 11.5 Hz, 2H), 4.35 (d, J = 6.3 Hz, 2H), 6.56 (s, 2H), 6.82 (d ,
J = 3.4 Hz, 1H), 6.88 (d , J = 3.4 Hz, 1H), 7.26 (d, J = 5.4 Hz, 1H),
7.38 (d, J = 5.4 Hz, 1H), 7.78 (m, 1H), 9.9 (M, 2H). 13C NMR
(400 MHz, DMSO-d6): d 13.5, 21.8, 24.8, 27.4, 28.9, 34.4, 52.1,
57.1, 69.4, 104.3, 113.5, 115.8, 117.7, 118.5, 123.9, 124.0, 134.4,
135.8, 154.8, 166.8. IR: 3431 (s, NH+), 2957, 1715 (s, C@O),
1583 cmꢀ1. Anal. (C24H31N3O5S) C, H, N.
4.1.27. 5-[(1-Ethylpiperidin-4-yl)methyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (32f)
Compound 32f was prepared from 2.06 g (9.9 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.7 g (11.9 mmol) of 1-
ethyl-4-hydroxymethylpiperidine and 1.42 g (35.7 mmol) of so-
dium hydride. The reaction mixture was refluxed for 5 h. Salifica-
tion with 1.14 g (9.9 mmol) of fumaric acid gave 3 g (70%) of 32f
as a white powder. Mp: 162 °C. 1H NMR (400 MHz, DMSO-d6): d
1.14 (t, J = 7.2 Hz, 3H), 1.64 (m, 2H), 1.89 (m, 2H), 2.04 (m, 1H),
2.62 (m, 2H), 2.84 (q, J = 7.2 Hz, 2H), 3.30 (d, J = 11.3 Hz, 2H),
4.33 (d, J = 6.0 Hz, 2H), 6.52 (s, 2H), 6.81 (dd , J = 3.0 Hz, J = 3.5
Hz, 1H), 6.90 (dd , J = 0.8 Hz, J = 3.5 Hz, 1H), 6.95 (M, 2H), 7.27 (d,
J = 5.6 Hz, 1H), 7.41 (d, J = 5.6 Hz, 1H), 7.86 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 9.6, 26.4, 33.4, 50.5, 50.6, 69.0, 104.4,
113.6, 116.0, 117.8, 118.3, 123.8, 124.0, 135.0, 135.7, 154.6,
167.6. IR: 3413 (s, NH+), 2938, 1713 (s, C@O), 1582 cmꢀ1. Anal.
(C21H25N3O5S) C, H, N.
4.1.31. 5-[(1-Isopropylpiperidin-4-yl)methoxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (32j)
Compound 32j was prepared from 0.7 g (3.3 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.79 g (5 mmol) of 1-iso-
propyl-4-hydroxymethylpiperidine and 0.6 g (15 mmol) of
sodium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 0.39 g (3.3 mmol) of fumaric acid gave 0.98 g (72%)of 32j
as a beige powder. Mp: 177 °C. 1H NMR (400 MHz, DMSO-d6): d
1.56 (d, J = 6.2 Hz, 6H), 1.61 (m, 2H), 1.89 (m, 2H), 2.01 (m, 1H),
2.50 (sept., J = 6.2 Hz, 1H), 2.63 (m, 2H), 3.14 (m, 2H), 4.32 (d,
J = 5.5 Hz, 2H), 6.50 (s, 2H), 6.83 (m, 1H), 6.90 (m, 1H), 7.27 (d,
J = 5.3 Hz, 1H), 7.41 (d, J = 5.3 Hz, 1H), 7.87 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 16.9, 26.8, 33.8, 47.1, 55.4, 69.2, 104.3,

2618
S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
113.6, 116.0, 117.8, 118.3, 123.8, 124.0, 134.9, 135.7, 154.6, 167.4.
J = 5.5 Hz, 1H), 7.85 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d
27.6, 34.4, 52.1, 61.3, 69.6, 104.3, 113.5, 115.9, 117.7, 118.3,
123.8, 124.0, 127.5, 128.3, 129.4, 134.4, 135.7, 136.2, 154.7,
166.7. IR: 3438 (s, NH+), 2951, 1711 (s, C@O), 1585 cmꢀ1. Anal.
(C26H27N3O5S) C, H, N.
IR: 3420 (s, NH+), 2936, 1718 (s, C@O), 1584 cmꢀ1
. Anal.
(C22H27N3O5S) C, H, N.
4.1.32. 5-[(1-Secbutylpiperidin-4-yl)methoxy]pyrrolo[1,2-
a]thieno[3,2-e]pyrazine fumarate salt (32k)
Compound 32k was prepared from 0.7 g (3.3 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.86 g (5 mmol) of 1-sec-
butyl-4-hydroxymethylpiperidine and 0.6 g (15 mmol) of sodium
hydride. The reaction mixture was refluxed for 3 h. Salification
with 0.39 g (3.3 mmol) of fumaric acid gave 1.1 g (71%) of 32k as
a beige powder. Mp: 157 °C. 1H NMR (400 MHz, DMSO-d6): d
0.86 (t, J = 6.9 Hz, 3 H), 1.07 (d, J = 6.1 Hz, 3H), 1.32 (m, 1H), 1.57
(m, 2H), 1.69 (m, 1H), 1.87 (m, 2H), 2.0 (m, 1H), 2.64 (m, 2H),
2.85 (m., 1H), 3.06 (m, 2H), 4.32 (d, J = 5.6 Hz, 2H), 4.55 (M, 2H),
6.52 (s, 2H), 6.83 (m, 1H), 6.89 (m, 1H), 7.28 (d, J = 5.3 Hz, 1H),
7.42 (d, J = 5.3 Hz, 1H), 7.87 (m, 1H). 13C NMR (400 MHz, DMSO-
d6): d 10.9, 12.9, 16.7, 24.2, 34.1, 46.2, 61.2, 69.3, 104.4, 113.6,
116.0, 117.9, 118.3, 123.8, 124.0, 134.9, 135.7, 154.7, 167.4. IR:
4.1.36. 5-[(1-Methylpiperidin-4-yl)methyloxy]pyrrolo[1,2-
a]thieno[2,3-e]pyrazine oxalate salt (33a)
Compound 33a was prepared from 0.3 g (1.4 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[2,3-e]pyrazine 29, 0.28 g (2.2 mmol) of 1-
methyl-4-hydroxymethylpiperidine and 0.26 g (6.5 mmol) of so-
dium hydride. The reaction mixture was refluxed for 8 h. Salifica-
tion with 0.13 g (1.4 mmol) of oxalic acid gave 0.25 g (46%) of
33a as a beige powder. Mp: 144 °C. 1H NMR (400 MHz, DMSO-
d6): d 1.62 (m, 2H), 1.97 (m, 2H), 2.10 (m, 1H), 2.71 (m, 3H), 2.92
(m, 2H), 3.39 (d, J = 10.7 Hz, 2H), 3.90 (M, 2H), 4.36 (d, J = 5.8 Hz,
2H), 6.80 (m, 1H), 6.90 (m, 1H), 7.57 (d, J = 5.3 Hz, 1H), 7.77 (d,
J = 5.3 Hz, 1H), 8.13 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d
26.4, 33.2, 43.8, 53.3, 69.4, 104.1, 112.8, 114.9, 116.4, 118.0,
120.8, 123.5, 138.2, 154.0, 164.1. IR: 3439 (s, NH+), 2935, 1720 (s,
C@O), 1514 cmꢀ1. Anal. (C18H21N3O5S) C, H, N.
3421 (s, NH+), 2938, 1717 (s, C@O), 1584 cmꢀ1
.
Anal.
(C23H29N3O5S) C, H, N.
4.1.33. 5-[(1-Allylpiperidin-4-yl)methyloxy]pyrrolo[1,2-a]-
thieno[3,2-e]pyrazine fumarate salt (32l)
4.1.37. 5-[(1-Ethylpiperidin-4-yl)methyloxy]pyrrolo[1,2-
a]thieno[2,3-e]pyrazine oxalate salt (33b)
Compound 32l was prepared from 2.81 g (13.5 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 2.3 g (14.8 mmol) of 1-
allyl-4-hydroxymethylpiperidine and 1.78 g (44.4 mmol) of so-
dium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 1.56 g (13.5 mmol) of fumaric acid gave 4.74 g (70%) of
32l as a beige powder. Mp: 156 °C. 1H NMR (400 MHz, DMSO-
d6): d 1.52 (m, 2H), 1.84 (m, 2H), 1.94 (m, 1H), 2.36 (m, 2H), 3.11
(d, J = 10.7 Hz, 2H), 3.26 (m, 2H), 4.31 (d, J = 5.3 Hz, 2H), 5.25–
5.32 (m, 2H), 5.87 (m, 1H), 6.56 (s, 2H), 6.81 (m, 1H), 6.88 (m,
1H), 7.27 (d, J = 5.1 Hz, 1H), 7.40 (d, J = 5.1 Hz, 1H), 7.83 (m, 1H),
8.73 (M, 2H). 13C NMR (400 MHz, DMSO-d6): d 26.8, 33.8, 51.4,
59.3, 69.2, 104.3, 113.5, 115.9, 117.8, 118.3, 120.6, 123.8, 124.0,
131.9, 134.7, 135.7, 154.7, 167.3 IR: 3420 (s, NH+), 2961, 1719 (s,
C@O), 1583 cmꢀ1. Anal. (C22H25N3O5S) C, H, N.
Compound 33b was prepared from 0.47 g (1.16 mmol) of 5-
chloropyrrolo[1,2-a]thieno[2,3-e]pyrazine 29, 0.2 g (1.39 mmol)
of 1-ethyl-4-hydroxymethylpiperidine and 0.17 g (4.2 mmol) of
sodium hydride. The reaction mixture was refluxed for 5 h. Salifica-
tion with 0.1 g (1.16 mmol) of oxalic acid gave 0.25 g (49%) of 33b
as a yellow powder. Mp: 152 °C. 1H NMR (400 MHz, DMSO-d6): d
1.21 (t, J = 7.2 Hz, 3H), 1.67 (m, 2H), 2.00 (m, 2H), 2.14 (m, 1H),
2.86 (m, 2H), 3.02 (q, J = 7.2 Hz, 2H), 3.41 (d, J = 11.3 Hz, 2H), 4.20
(M, 2H), 4.39 (d, J = 6.2 Hz, 2H), 6.81 (m, 1H), 6.90 (m, 1H), 7.53 (d,
J = 5.6 Hz, 1H), 7.73 (d, J = 5.6 Hz, 1H), 8.07 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 9.1, 25.6, 32.8, 50.6, 50.7, 68.8, 104.2,
112.8, 114.8, 116.3, 117.9, 120.9, 123.3, 138.1, 153.9, 163.9. IR:
3428 (s, NH+), 2936, 1719 (s, C@O), 1513 cmꢀ1
.
Anal.
(C19H23N3O5S) C, H, N.
4.1.34. 5-[(1-But-3-enyl-piperidin-4-yl-methyloxy]pyrrolo[1,2-
a]thieno[3,2-e]pyrazine fumarate salt (32m)
4.1.38. 5-[(1-Propylpiperidin-4-yl)methyloxy]pyrrolo[1,2-
a]thieno[2,3-e]pyrazine oxalate salt (33c)
Compound 32m was prepared from 0.7 g (3.3 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 0.85 g (5 mmol) of 1-
but-3-enyl-4-hydroxymethylpiperidine and 0.6 g (15 mmol) of so-
dium hydride. The reaction mixture was refluxed for 7 h. Salifica-
tion with 0.39 g (3.3 mmol) of fumaric acid gave 1.1 g (68%) of
32 m as a beige powder. Mp: 148 °C. 1H NMR (400 MHz, DMSO-
d6): d 1.47 (m, 2H), 1.81 (m, 2H), 1.95 (m, 1H), 2.25–2.30 (M,
4H), 2.56 (m., 2H), 3.05 (m., 2H), 4.34 (d, J = 6.3 Hz, 2H), 6.55 (s,
2H), 6.83 (m, 1H), 6.88 (m, 1H), 7.27 (d, J = 5.5 Hz, 1H), 7.40 (d,
J = 5.5 Hz, 1H), 7.81 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d
27.3, 29.7, 34.2, 51.9, 56.3, 69.3, 104.1, 113.3, 115.7, 115.8, 117.5,
118.3, 123.8, 124.0, 134.2, 135.6, 135.9, 154.6, 166.5. IR: 3427 (s,
NH+), 2935, 1717 (s, C@O), 1585 cmꢀ1. Anal. (C23H27N3O5S) C, H, N.
Compound 33c was prepared from 0.8 g (1.91 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[2,3-e]pyrazine 29, 0.4 g (2.86 mmol) of 1-
propyl-4-hydroxymethylpiperidine and 0.46 g (8.6 mmol) of so-
dium hydride. The reaction mixture was refluxed for 5 h. Salifica-
tion with 0.18 g (1.91 mmol) of oxalic acid gave 0.43 g (54%) of
33c as a yellow powder. Mp: 150 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.89 (t, J = 7.2 Hz, 3H), 1.6–1.7 (m, 4H), 1.95 (m, 2 H), 2.14
(m, 1H), 2.9–2.95 (m, 4H), 3.42 (d, J = 11.5 Hz, 2H), 4.36 (d,
J = 6.2 Hz, 2H), 5.46 (M, 2H), 6.80 (m, 1H), 6.90 (m, 1H), 7.54 (d,
J = 5.3 Hz, 1H), 7.74 (d, J = 5.3 Hz, 1H), 8.09 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 11.0, 16.9, 25.3, 32.7, 50.9, 57.2, 68.9,
104.3, 112.9, 114.9, 116.5, 117.8, 121.0, 123.4, 138.0, 153.8,
164.6. IR: 3438 (s, NH+), 2938, 1716 (s, C@O), 1513 cmꢀ1. Anal.
(C20H25N3O5S) C, H, N.
4.1.35. 5-[(1-Benzylpipéridin-4-yl)-méthyloxy]-pyrrolo[1,2-
a]thiéno[3,2-e]pyrazine fumarate salt (32n)
4.1.39. 5-[(1-Butylpiperidin-4-yl)methyloxy]pyrrolo[1,2-a]-
thieno[2,3-e]pyrazine oxalate salt (33d)
Compound 32n was prepared from 1 g (4.8 mmol) of 5-chloro-
pyrrolo[1,2-a]thieno[3,2-e]pyrazine 28, 1.47 g (7.2 mmol) of 1-
benzyl-4-hydroxymethylpiperidine and 0.86 g (21.6 mmol) of so-
dium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 0.56 g (4.8 mmol) of fumaric acid gave 1.75 g (74%) of
32n as a white powder. Mp: 189 °C. 1H NMR (400 MHz, DMSO-
d6): d 1.45 (m, 2H), 1.81 (m, 2H), 1.90 (m, 1H), 2.20 (m, 2H), 2.96
(d, J = 11.5 Hz, 2H), 3.62 (m, 2H), 4.30 (d, J = 6.35 Hz, 2H), 6.59 (s,
2H), 6.82 (m, 1H), 6.88 (m, 1H), 7.25–7.40 (M, 6H), 7.42 (d,
Compound 33d was prepared from 0.6 g (2.92 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[2,3-e]pyrazine 29, 0.6 g (3.5 mmol) of 1-bu-
tyl-4-hydroxymethylpiperidine and 0.42 g (10.5 mmol) of sodium
hydride. The reaction mixture was refluxed for 6 h. Salification
with 0.26 g (2.92 mmol) of oxalic acid gave 0.94 g (62%) of 33d
as a yellow powder. Mp: 190 °C. 1H NMR (400 MHz, DMSO-d6): d
0.96 (t, J = 7.2 Hz, 3H), 1.38 (sext., J = 7.2 Hz, 2H), 1.6–1.8 (m,
4 H), 2.02 (m, 2H), 2.20 (m, 1H), 2.91 (m, 2H), 3.0 (m, 2H), 3.49

S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
2619
(d, J = 11.2 Hz, 2H), 4.45 (d, J = 6.4 Hz, 2H), 6.69 (M, 2H), 6.86 (dd,
J = 3.9 Hz, J = 2.4 Hz, 1H), 6.96 (dd, J = 3.9 Hz, J = 1.2 Hz, 1H), 7.59
(d, J = 5.8 Hz, 1H), 7.79 (d, J = 5.2 Hz, 1H), 8.13 (dd, J = 2.4 Hz,
J = 1.2 Hz, 1H). 13C NMR (400 MHz, DMSO-d6): d 13.1, 19.2, 25.3,
25.4, 32.6, 50.9, 55.4, 68.7, 104.0, 112.5, 114.6, 116.1, 117.7,
120.6, 123.1, 137.8, 153.7, 163.7. IR: 3432 (s, NH+), 2954, 1718 (s,
C@O), 1515, 1338 cmꢀ1. Anal. (C21H27N3O5S) C, H, N.
(400 MHz, DMSO-d6): d 1.58 (m, 2H), 1.86 (m, 2H), 1.96 (m, 1H),
2.50 (m, 3H), 2.54 (m, 2H), 2.65 (s, 3H), 3.18 (d, J = 10.8 Hz, 2H),
4.32 (d, J = 5.5 Hz, 2H), 6.53 (s, 2H), 6.80 (m, 1H), 6.92 (m, 1H),
7.16 (s, 1H), 8.09 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 16.7,
26.5, 33.1, 43.7, 53.2, 69.3, 103.9, 112.7, 116.1, 116.2, 118.5,
122.5, 125.5, 134.9, 139.1, 153.8, 167.5. IR: 3450 (s, NH+),
2960,1699 (s, C@O), 1590 cmꢀ1. Anal. (C21H25N3O5S) C, H, N.
4.1.40. 5-[(1-Pentylpiperidin-4-yl)methyloxy]pyrrolo[1,2-a]-
thieno[2,3-e]pyrazine oxalate salt (33e)
4.1.44. 1-Methyl-5-[(1-ethylpiperidin-4-yl)methyloxy]pyrrolo-
[1,2-a]thieno[2,3-e]pyrazine oxalate salt (34c)
Compound 33e was prepared from 0.32 g (1.56 mmol) of 5-
chloropyrrolo[1,2-a]thieno[2,3-e]pyrazine 29, 0.35 g (1.88 mmol)
of 1-pentyl-4-hydroxymethylpiperidine and 0.23 g (5.64 mmol)
of sodium hydride. The reaction mixture was refluxed for 6 h. Sali-
fication with 0.14 g (1.56 mmol) of oxalic acid gave 0.4 g (58%) of
33e as a beige powder. Mp: 219 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.87 (t, J = 6.5 Hz, 3H), 1.2–1.4 (m, 4H), 1.6–1.7 (m, 4H),
1.96 (m, 2H), 2.13 (m, 1H), 2.9–3.05 (m, 4H), 3.44 (d, J = 11.4 Hz,
2H), 4.12 (M, 2H), 4.35 (d, J = 6.3 Hz, 2H), 6.80 (m, 1H), 6.90 (m,
1H), 7.56 (d, J = 5.3 Hz, 1H), 7.76 (d, J = 5.3 Hz, 1H), 8.12 (m, 1H).
13C NMR (400 MHz, DMSO-d6): d 13.7, 21.6, 23.0, 25.5, 28.2, 32.7,
51.0, 55.7, 68.9, 104.3, 112.9, 114.9, 116.5, 117.8, 121.0, 123.4,
137.9, 153.8, 164.4. IR: 3419 (s, NH+), 2951, 1715 (s, C@O),
1514 cmꢀ1. Anal. (C22H29N3O5S) C, H, N.
Compound 34c was prepared from 0.7 g (3.1 mmol) of 5-chloro-
1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 0.67 g (4.7 mmol)
of 1-ethyl-4-hydroxymethylpiperidine and 0.56 g (14.1 mmol) of
sodium hydride. The reaction mixture was refluxed for 7 h. Salifica-
tion with 0.28 g (3.1 mmol) of oxalic acid gave 0.53 g (51%) of 34c
as a white powder. Mp: 161 °C. 1H NMR (400 MHz, DMSO-d6): d
1.11 (t, J = 7.0 Hz, 3H), 1.59 (m, 2H), 1.86 (m, 2H), 1.97 (m, 1H),
2.50 (m, 2H), 2.61 (s, 3H), 2.75 (m, 2H), 3.24 (m, 2H), 4.29 (m,
2H), 5.31 (M, 2H), 6.76 (m, 1H), 6.88 (m, 1H), 7.12 (m, 1H), 8.04
(m, 1H). 13C NMR (400 MHz, DMSO-d6): d 10.0, 16.6, 26.3, 33.6,
50.8, 56.4, 69.3, 103.8, 112.9, 116.0, 116.1, 118.5, 122.4, 125.4,
139.2, 153.7, 166.2. IR: 3440 (s, NH+), 2958, 1705 (s, C@O),
1563 cmꢀ1. Anal. (C20H25N3O5S) C, H, N.
4.1.45. 1-Methyl-5-[(1-propylpiperidin-4-yl)methyloxy]-
4.1.41. 5-[(1-Allylpiperidin-4-yl)methyloxy]pyrrolo[1,2-
a]thieno[2,3-e]pyrazine oxalate salt (33f)
pyrrolo[1,2-a]thieno[2,3-e]pyrazine fumarate salt (34d)
Compound 34d was prepared from 0.7 g (3.2 mmol) of 5-chloro-
1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 1 g (6.4 mmol) of
1-propyl-4-hydroxymethylpiperidine and 0.76 g (19.2 mmol) of so-
dium hydride. The reaction mixture was refluxed for 5 h. Salifica-
tion with 0.36 g (3.2 mmol) of fumaric acid gave 0.48 g (33%) of
34d as a beige powder. Mp: 208 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.85 (t, J = 7.1 Hz, 3H), 1.5–1.6 (m, 4H), 1.86 (m, 2H), 1.97
(m, 1H), 2.42 (m, 2H), 2.60 (m, 2H), 2.65 (s, 3H), 3.16 (m, 2H),
4.10 (M, 2H), 4.31 (d, J = 6.1 Hz, 2H), 6.50 (s, 2H), 6.81 (m, 1H),
6.90 (m, 1H), 7.15 (s, 1H), 8.08 (m, 1H). 13C NMR (400 MHz,
DMSO-d6): d 11.3, 16.3, 18.5, 27.3, 34.3, 52.0, 58.9, 69.5, 103.3,
112.3, 116.0, 116.1, 118.5, 122.2, 125.2, 134.2, 139.1, 153.7, 166.1.
Compound 33f was prepared from 0.7 g (3.36 mmol) of 5-chlo-
ropyrrolo[1,2-a]thieno[2,3-e]pyrazine 29, 0.57 g (3.68 mmol) of 1-
allyl-4-hydroxymethylpiperidine and 0.44 g (11.06 mmol) of so-
dium hydride. The reaction mixture was refluxed for 6 h. Salifica-
tion with 0.3 g (3.36 mmol) of oxalic acid gave 1 g (58%) of 33f as
a beige powder. Mp: 180 °C. 1H NMR (400 MHz, DMSO-d6): d
1.62 (m, 2H), 1.97 (m, 2H), 2.11 (m, 1H), 2.89 (m, 2H), 3.03 (m,
2H), 3.43 (m, 2H), 4.10 (M, 2H), 4.36 (d, J = 6.0 Hz, 2H), 5.05–5.20
(m, 2H), 5.77 (m, 1H), 6.81 (m, 1H), 6.90 (m, 1H), 7.57 (d,
J = 5.8 Hz, 1H), 7.77 (d, J = 5.8 Hz, 1H), 8.12 (m, 1H). 13C NMR
(400 MHz, DMSO-d6): d 27.2, 32.9, 50.9, 59.1, 68.7, 104.5, 113.2,
115.1, 116.5, 118.6, 122.0, 121.5, 123.7, 133.9, 137.9, 154.6,
164.6. IR: 3439 (s, NH+), 2945, 1721 (s, C@O), 1514 cmꢀ1. Anal.
(C20H23N3O5S) C, H, N.
IR: 3430 (s, NH+), 2966, 1700 (s, C@O), 1586 cmꢀ1
(C23H29N3O5S) C, H, N.
. Anal.
4.1.46. 1-Methyl-5-[(1-butylpiperidin-4-yl)-methyloxy]-
4.1.42 1-Methyl-5-(3-piperidinopropyloxy)pyrrolo[1,2-
pyrrolo[1,2-a]thieno[2,3-e]pyrazine fumarate salt (34e)
a]thieno[2,3-e]pyrazine fumarate salt (34a)
Compound 34e was prepared from 0.6 g (2.7 mmol) of 5-
chloro-1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 0.92 g
(5.4 mmol) of 1-butyl-4-hydroxymethylpiperidine and 0.65 g
(16 mmol) of sodium hydride. The reaction mixture was refluxed
for 6 h. Salification with 0.31 g (2.7 mmol) of fumaric acid gave
0.65 g (50%) of 34e as a beige powder. Mp: 203 °C. 1H NMR
Compound 34a was prepared from 0.69 g (3.1 mmol) of 5-
chloro-1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 0.67 g
(4.6 mmol) of 1-(3-hydroxypropyl)piperidine and 0.56 g
(14 mmol) of sodium hydride. The reaction mixture was refluxed
for 6 h. Salification with 0.36 g (3.1 mmol) of fumaric acid gave
0.92 g (67%) of 34a as a beige powder. Mp: 198 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.44 (m, 2H), 1.61 (m, 4H), 2.08 (m, 2H),
2.63 (s, 3H), 2.7–2.9 (M, 6H), 4.45 (m, 2H), 5.34 (M, 2H), 6.50 (s,
2H), 6.80 (m, 1H), 6.91 (m, 1H), 7.14 (s, 1H), 8.07 (m, 1H). 13C
NMR (400 MHz, DMSO-d6): d 16.7, 22.7, 23.9, 24.4, 52.8, 54.0,
64.0, 103.9, 112.7, 116.1, 116.2, 118.6, 122.5, 125.5, 134.8, 139.1,
(400 MHz, DMSO-d6):
d 0.86 (t, J = 7.2 Hz, 3H), 1.26 (sext.,
J = 7.2 Hz, 2H), 1.5–1.6 (m, 4H), 1.84 (m, 2H), 1.95 (m, 1H), 2.36
(m, 2H), 2.60 (m, 2H), 2.64 (s, 3H), 3.17 (d, J = 11.2 Hz, 2H), 3.70
(M, 2H), 4.30 (d, J = 6.1 Hz, 2H), 6.51 (s, 2H), 6.79 (m, 1H), 6.90
(m, 1H), 7.15 (s, 1H), 8.08 (m, 1H). 13C NMR (400 MHz, DMSO-
d6): d 13.3, 16.2, 19.6, 23.5, 27.3, 34.3, 52.0, 56.8, 69.4, 103.4,
112.3, 115.7, 115.8, 118.5, 122.2, 125.1, 133.9, 139.2, 153.7,
166.2. IR: 3433 (s, NH+), 2960, 1699 (s, C@O), 1587 cmꢀ1. Anal.
(C24H31N3O5S) C, H, N.
153.7, 167.3. IR: 3430 (s, NH+), 2944, 1724 (s, C@O), 1589 cmꢀ1
Anal. (C22H27N3O5S) C, H, N.
.
4.1.43. 1-Methyl-5-[(1-methylpiperidin-4-yl)methyloxy]pyrrolo-
[1,2-a]thieno[2,3-e]pyrazine fumarate salt (34b)
4.1.47. 1-Methyl-5-[(1-pentylpiperidin-4-yl)methyloxy]-
pyrrolo[1,2-a]thieno[2,3-e]pyrazine fumarate salt (34f)
Compound 34b was prepared from 0.32 g (1.4 mmol) of 5-
chloro-1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 0.28 g
(2.2 mmol) of 1-methyl-4-hydroxymethylpiperidine and 0.26 g
(6.6 mmol) of sodium hydride. The reaction mixture was refluxed
for 5 h. Salification with 0.24 g (1.4 mmol) of fumaric acid gave
0.35 g (57%) of 34b as a beige powder. Mp: 198 °C. 1H NMR
Compound 34f was prepared from 0.6 g (2.6 mmol) of 5-chloro-
1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 1 g (5.33 mmol)
of 1-pentyl-4-hydroxymethylpiperidine and 0.65 g (16.2 mmol)
of sodium hydride. The reaction mixture was refluxed for 6 h. Sali-
fication with 0.51 g (4.44 mmol) of fumaric acid gave 0.59 g (59%)

2620
S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
of 34f as a beige powder. Mp: 206 °C. 1H NMR (400 MHz, DMSO-
d6): d 0.85 (t, J = 6.8 Hz, 3H), 1.2–1.3 (m, 4H), 1.5–1.6 (m, 4H),
1.84 (m, 2H), 1.97 (m, 1H), 2.41 (m, 2H), 2.60 (m, 2H), 2.64 (s,
3H), 3.18 (d, J = 11.4 Hz, 2H), 4.31 (d, J = 6.1 Hz, 2H), 4.80 (M, 2H),
6.52 (s, 2H), 6.78 (m, 1H), 6.90 (m, 1H), 7.14 (s, 1H), 8.07 (m,
1H). 13C NMR (400MHz, DMSO-d6): d 13.8, 16.6, 21.8, 24.2, 26.6,
28.7, 33.8, 51.6, 56.5, 69.4, 103.8, 112.7, 116.1, 116.2, 118.5,
122.4, 125.5, 134.8, 139.1, 153.8, 167.3. IR: 3449 (s, NH+), 2869,
1703 (s, C@O), 1587 cmꢀ1. Anal. (C25H33N3O5S) C, H, N.
as a beige powder. Mp: 198 °C. 1H NMR (400 MHz, DMSO-d6): d 1.50
(m, 2H), 1.82 (m, 2H), 1.93 (m, 1H), 2.3–2.4 (M, 4 H), 2.63–2.67 (M,
5H), 3.14 (d, J = 11.5 Hz, 2H), 4.36 (d, J = 6.2 Hz, 2H), 5–5.1 (m, 2H),
5.76 (m, 1H), 6.54 (s, 2H), 6.79 (m, 1H), 6.89 (m, 1H), 7.21 (s, 1H),
8.13 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 16.6, 27.0, 29.4,
34.0, 51.7, 56.0, 69.5, 103.8, 112.7, 116.1, 116.2, 116.4, 118.5, 122.4,
125.5, 134.6, 135.6, 139.1, 153.8, 167.1. IR: 3433 (s, NH+), 2951,
1888, 1698 (s, C@O), 1588 cmꢀ1. Anal. (C24H29N3O5S) C, H, N.
4.1.52. 1-Methyl-5-[(1-benzylpiperidin-4-yl)methyloxy]-
4.1.48. 1-Methyl-5-[(1-isopropylpiperidin-4-yl)methyloxy]-
pyrrolo[1,2-a]thieno[2,3-e]pyrazine oxalate salt (34g)
pyrrolo[1,2-a]thieno[2,3-e]pyrazine fumarate salt (34k)
Compound 34k was prepared from 0.5 g (2.2 mmol) of 5-
chloro-1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 0.95 g
(3 mmol) of 1-benzyl-4-hydroxymethylpiperidine and 0.55 g
(13.5 mmol) of sodium hydride. The reaction mixture was refluxed
for 8 h. Salification with 0.25 g (2.2 mmol) of fumaric acid gave
0.65 g (58%) of 34k as a beige powder. Mp: 174 °C. 1H NMR
(400 MHz, DMSO-d6): d 1.38 (m, 2H), 1.79 (m, 2H), 1.85 (m, 1H),
2.08 (m, 2H), 2.65 (s, 3H), 2.89 (d, J = 11.1 Hz, 2H), 3.40 (m, 2H),
3.54 (s, 2H), 4.29 (d, J = 6.4 Hz, 2H), 6.59 (s, 2H), 6.80 (m, 1H),
6.89 (m, 1H), 7.15 (s, 1H), 7.25 (m, 1H), 7.25–7.35 (M, 4H), 8.08
(m, 1H). 13C NMR (400 MHz, DMSO-d6): d 16.6, 28.1, 34.8, 52.5,
62.0, 70.0, 103.8, 112.7, 116.0, 116.1, 118.5, 122.4, 125.5, 127.0,
128.1, 129.0, 134.1, 137.6, 139.1, 153.9, 166.2. IR: 3428 (s, NH+),
2949, 1698 (s, C@O), 1589 cmꢀ1. Anal. (C27H29N3O5S) C, H, N.
Compound 34g was prepared from 0.3 g (1.4 mmol) of 5-chloro-
1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 0.46 g (2.9 mmol)
of 1-isopropyl-4-hydroxymethylpiperidine and 0.35 g (8.7 mmol)
of sodium hydride. The reaction mixture was refluxed for 5 h. Sali-
fication with 0.13 g (1.4 mmol) of oxalic acid gave 0.47 g (62%) of
34g as a beige powder. Mp: 130 °C. 1H NMR (400 MHz, DMSO-
d6): d 1.18 (d, J = 6.3 Hz, 6H), 1.66 (m, 2H), 1.92 (m, 2H), 1.99 (m,
1H), 2.58 (m, 1H), 2.59 (s, 3H), 2.92 (m, 2H), 3.32 (m, 2H), 4.16
(m, 2H), 4.28 (m, 2H), 6.76 (m, 1H), 6.87 (m, 1H), 7.11 (s, 1H),
8.03 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 16.3, 16.6, 26.5,
30.7, 48.9, 56.1, 69.6, 103.9, 112.8, 116.0, 116.2, 118.5, 122.5,
125.5, 139.4, 153.7, 164.5. IR: 3435 (s, NH+), 2961, 1715 (s, C@O),
1516 cmꢀ1. Anal. (C21H27N3O5S) C, H, N.
4.1.49. 1-Methyl-5-[(1-secbutylpiperidin-4-yl)methyloxy]-
pyrrolo[1,2-a]thieno[2,3-e]pyrazine oxalate salt (34h)
Compound 34h was prepared from 0.25 g (1.1 mmol) of 5-
4.2. Binding experiments
Binding to native 5-HT4 receptor from guinea pig striatum mem-
branes was determined using a slight modification of the method of
chloro-1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine
30,
1 g
(2.2 mmol) of 1-secbutyl-4-hydroxymethylpiperidine and 0.27 g
(6.7 mmol) of sodium hydride. The reaction mixture was refluxed
for 7 h. Salification with 0.1 g (1.1 mmol) of oxalic acid gave
0.26 g (53%) of 34h as a beige powder. Mp: 163 °C. 1H NMR
(400 MHz, DMSO-d6): d 0.86 (t, J = 6.7 Hz, 3H), 1.14 (d, J = 6.0 Hz,
3H), 1.41 (m, 1H), 1.6–1.7 (m, 3H), 1.91 (m, 2H), 2.11 (m, 1H),
2.59 (s, 3H), 2.94 (m, 2H), 3.09 (m, 1H), 3.26 (m, 2H), 3.74 (M,
2H), 4.28 (d, J = 5.5 Hz, 2H), 6.75 (m, 1H), 6.86 (m, 1H), 7.10 (s,
1H), 8.13 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 10.4, 12.7,
16.6, 23.3, 25.7, 33.8, 51.8, 56.5, 69.3, 103.9, 112.8, 116.2, 116.3,
118.5, 122.5, 125.5, 139.2, 153.7, 164.4. IR: 3421 (s, NH+), 2972,
1716 (s, C@O), 1635 cmꢀ1. Anal. (C22H29N3O5S) C, H, N.
Grossman.59 Membranes (15
lg of protein/mL) were incubated at
37 °C for 30 min with 0.6 nM [3H]GR113808 (Amersham, France)
in 50 mM HEPES buffer (pH 7.4) in the absence or presence of fixed
concentrations of compounds under study. Nonspecific binding was
determined in the presence of 10 lM serotonin.
Selectivity toward 5-HT6 and 5-HT7 binding sites was also deter-
mined for some of the compounds of interest. Fixed concentrations
of compounds under study were incubated with membranes
(14 lg/mL of human 5-HT6 or 38 lg/mL of human 5-HT7 cloned
receptors expressed in sf9 cells, Biosignal Inc) in 50 mM Tris–HCl
buffer containing 10 mM MgSO4, 0.5 mM EDTA and 2 nM [3H]LSD
(NEN, France) for 90 min at 27 °C.60,61 Nonspecific binding was
determined in the presence of 10 and 250 lM clozapine.
4.1.50. 1-Methyl-5-[(1-allylpiperidin-4-yl)methyloxy]-
pyrrolo[1,2-a]thieno[2,3-e]pyrazine fumarate salt (34i)
5-HT3 receptor binding to NG 108-15 cell membranes was deter-
mined following a slight modification of the procedure of Hoyer and
Neijt. Membranes (0.5 mg of protein/mL) were incubated at 25 °C
for 60 min with 1 nM [3H]granisetron in 50 mM Tris-HCl buffer,
pH 7.4, supplemented with 25 mM NaCl. Nonspecific binding was
determined in the presence of 10ꢀ5 M tropisetron.
Compound 34i was prepared from 0.45 g (2.2 mmol) of 5-chloro-
1-methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 0.47 g (3 mmol) of
1-allyl-4-hydroxymethylpiperidine and 0.36 g (9.1 mmol) of so-
dium hydride. The reaction mixture was refluxed for 6 h. Salification
with 0.23 g (2 mmol) of fumaric acid gave 0.66 g (72%) of 34i as a
beige powder. Mp: 153 °C. 1H NMR (400 MHz, DMSO-d6): d 1.43
(m, 2H), 1.82 (m, 2H), 1.89 (m, 1H), 2.19 (m, 2H), 2.66 (s, 3H), 3.01
(m, 2H), 3.12 (m, 2H), 3.50 (M, 2H), 4.30 (d, J = 6.2 Hz, 2H), 5.1–5.3
(m, 2H), 5.84 (m, 1H), 6.58 (s, 2H), 6.81 (m, 1H), 6.90 (m, 1H), 7.15
(s, 1H), 8.08 (m, 1H). 13C NMR (400 MHz, DMSO-d6): d 16.6, 27.6,
34.4, 52.1, 60.3, 69.8, 103.8, 112.7, 116.1, 116.2, 118.5, 122.3,
122.4, 125.5, 133.8, 134.2, 139.1, 153.9, 166.4. IR: 3432 (s, NH+),
2982, 1709 (s, C@O), 1588 cmꢀ1. Anal. (C23H27N3O5S) C, H, N.
Percentages of inhibition of the binding of [3H]GR113808, or
[3H]LSD, were obtained for concentrations of 10ꢀ6 and 10ꢀ8 M of
the ligands tested. Percentages of inhibition of the binding of
[3H]granisetron (5-HT3 receptors) were obtained for concentra-
tions of 10ꢀ6 and 10ꢀ7 M of the ligands tested. For some of these
compounds, affinity constants were calculated from 8- to 10-point
inhibition curves using the EBDA-Ligand software, and expressed
as Ki SEM. The results obtained are given in Tables 1–3.
The other binding tests were carried out at CEREP (see
www.cerep.fr for a complete description of the methodology).
4.1.51. 1-Methyl-5-[(1-but-3-enyl-piperidin-4-yl)methyloxy]-
pyrrolo[1,2-a]thieno[2,3-e]pyrazine fumarate salt (34j)
4.3. Behavioral studies
Compound 34jwas prepared from 0.8 g (3.6 mmol) of 5-chloro-1-
methylpyrrolo[1,2-a]thieno[2,3-e]pyrazine 30, 1.12 g (7.2 mmol) of
1-but-3-enyl-4-hydroxymethylpiperidine and 0.86 g (21.6 mmol)
of sodium hydride. The reaction mixture was refluxed for 5 h. Salifi-
cation with 0.42 g (3.6 mmol) of fumaric acid gave 1.14 g (67%) of 34j
4.3.1. Animals and drug administration
In all studies, male NMRI mice (20–24 g, CER Janvier, France)
were used. All compounds tested were dissolved in saline solution
and administered intraperitoneally (10 mL/kg).

S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
2621
4.3.2. CNS activity and acute toxicity test
significant effects, a post hoc multiple comparison test (PLSD of
Behavioral and neurological changes induced by graded doses
(50, 100, 200, 400 mg/kg) of the tested derivatives were evaluated
in mice, in groups of 4, by a standardized observation technique50
at different times (30 min, 3 and 24 h) after intraperitoneal adminis-
tration. Major changes in behavior (for example, hypo- or hyperac-
tivity, ataxia, tremors, convulsions, etc.) were noted in comparison
to the control group. The approximate LD50 of the compounds were
also calculated through the quantification of deaths after 24 h.
Fisher). p-Values less than 0.05 were considered to be significant.
4.4. Cell biology
4.4.1. Cell culture and transfection
cDNA subcloned into pRK5 was introduced into COS-7 cells by
electroporation.63 Briefly, cells were trypsinized, centrifuged, and
resuspended in electroporating buffer (50 mM K2HPO4, 20 mM
CH3CO2K, 20 mM KOH, 26.7 mM MgSO4, pH 7.4) with 25–
2000 ng of receptor cDNA. The total amount of DNA was kept con-
4.3.3. Spatial working memory
The promnesic activity was evaluated through measurement of
the capacity of the tested compounds to reverse the scopolamine-
induced deficit on spontaneous alternation behavior in the Y maze
test.62 The black wooden maze consisted of three equally spaced
arms (22 cm long, 6.5 cm wide with walls 10 cm high). The mouse
was placed at the end of one of the arms and allowed to move freely
through the maze during a 5 min session while the sequence of arm
entries was recorded by an observer. An arm entry was scored when
all four feet crossed into the arm. An alternation was defined as en-
tries into all three arms on a consecutive occasion. The number of
possible alternations is thus the total number of arm entries minus
two; the percentage of alternation was calculated as (number of ac-
tual alternations/number of possible alternations) ꢂ 100. The per-
centage of alternation of scopolamine-treated mice (1 mg/kg) was
significantly reduced in comparison to control mice (52% vs 66%,
respectively, p = 0.0049; ANOVA and PLSD of Fisher). Compound
34d was tested at 0.3, 1 and 3 mg/kg administered 30 min before
testing. For each dose, four groups were constituted: control (sali-
ne + saline), scopolamine (saline + scopolamine), tested compound
(compound + saline), and association (compound + scopolamine).
In this condition, arecoline (1 mg/kg), used as a pharmacological
reference, significantly reversed the scopolamine-induced deficit
(48% for the scopolamine group vs 64% for the arecoline + scopol-
amine group, p < 0.0001; ANOVA + PLSD of Fisher).
stant at 15
15 min at room temperature, 300
were transferred to a 0.4 cm electroporation cuvette (Bio-Rad,
Heidemannstrabe, Munchem) and pulsed with a Gene pulser appa-
l
g/transfection with wild-type pRK5 vector. After
l
L of cell suspension (107 cells)
ratus (setting 1000 lF, 280 V). Cells were diluted in Dulbecco’s
modified Eagle’s medium (DMEM; 106 cells/mL) containing 10%
dialyzed fetal bovine serum (dFBS) and plated on 15-cm Falcon
Petri dishes or into 12-well clusters at the desired density.
4.4.2. cAMP formation
Intracellular cAMP levels were determined by measuring the
conversion of the [3H]adenine nucleotide precursor [3H]ATP to
[3H]cAMP, as described previously.2 On the sixth day of culture
and before each experiment, neurons were incubated at 37 °C for
2 h with culture medium containing 2
mmol) (Amersham, UK). After 2 h, the cultures were washed and
incubated with 0.75 mM IBMX, 0.1 M forskolin, and test agents
(agonists or antagonists prepared in culture medium), in a volume
of 1 mL, for 5 min at 37 °C. The reaction was stopped by aspiration
of the medium and addition of 1 mL of ice-cold 5% trichloracetic
acid. Cells were loosened with the aid of a rubber scraper and
l
Ci/mL [3H]adenine (24 Ci/
l
100 lL of 5 mM ATP/5 mM cAMP were added to the mixture. Cel-
lular protein was centrifuged at 500 g and the supernatant was
eluted through sequential chromatography on Dowex and alumina
columns, which separated [3H]ATP from [3H]cAMP. We have previ-
4.3.4. Writhing test
ously shown that, in neuronal cultures, 0.1 lM forskolin does not
The test employed was essentially the one described by Hen-
dershot and Forsaith51; however, acetic acid52 rather than phenyl-
quinone was used to elicit stretching. Groups of 8 mice (20–24 g)
were injected ip with 10 mL/kg of 0.6% aqueous acetic acid. The
mice were placed in an observation beaker, and the number of
stretches per animal was counted during a 10 min period starting
10 min after acetic acid treatment. A stretch was defined as a se-
quence of arching of the back, pelvic rotation and hind limb exten-
sion. Tested and reference compounds were administered 15 min
before acetic acid solution.
modify basal cAMP concentrations but increases neurotransmitter
efficacy in cAMP production; and that potency remains
unaffected.64
4.5. Electrophysiological studies
4.5.1. Cell culture and transfection
NG108-15 cells (a kind gift from B. Rouzaire-Dubois) were
maintained in DMEM-HG supplemented with 5% fetal bovine ser-
um, 2% hypoxanthine–aminopterine–thymidine mixture and 1%
antibiotics. Cells to be used in electrophysiological studies were
transferred on polylysine-coated glass pieces when passaged, trea-
ted for 48 h with 2% DMSO diluted in the standard culture medium
followed by at least 24 h in the presence of 1 mM dibutyryl-cAMP.
4.3.5. Hot plate test
The method employed for measuring central analgesic effect
was first described by Woolfe and McDonald.53 Briefly, each mouse
was individually placed on a plate heated to 55 °C and the time un-
til forepaw licking occurred was recorded by a stop-watch. We
measured the reaction times of groups of 10 mice twice before
the injections (mice must react between 4 and 12 s). The com-
pounds were tested at 0.01, 0.1 and 1 mg/kg ip and reaction times
were determined at 15 and 30 min after injection. If an animal did
not respond by 30 s (cutoff time), it was removed from the plate to
avoid tissue damage. Morphine used as a reference at 8 mg/kg
abrogated the avoidance behavior (mean reaction times, 30 s and
26 s at 15 min and 30 min, respectively; p < 0.0001 vs control at
the two times; ANOVA + PLSD of Fisher).
4.5.2. Electrophysiology
Standard whole-cell recording techniques were used. Cells were
bathed in an extracellular solution containing (in mM): NaCl 140,
KCl 3.5, MgCl2 1.3, CaCl2 2.5, HEPES 10, and glucose 11.1. The pH
was adjusted to 7.3. The electrodes used in this study had a resis-
tance of 2–2.5 MX when filled with the following intracelllar solu-
tion (in mM): CsCl 25, CsMeSO3 98, MgCl2 3, HEPES 10, ATP–Na2–
Cs2, GTP–Na3, and EGTA 10. The pH was adjusted to 7.3. When ac-
cess to the cell was gained, the cells were allowed 10 min to equil-
ibrate before experiments were begun. The effects of serotonin and
compound 34d were studied at a membrane potential of ꢀ60 mV.
Drugs were applied by gravity. To prevent desensitization, the ago-
4.3.6. Statistical analyses
All quantitative data were expressed as means SEM and ana-
lyzed using analysis of variance (ANOVA) followed by, in case of
nist (5-HT 50
lM) was applied every 2.5 min. To evaluate its ef-
fects, compound 34d was applied alone for one minute prior to

2622
S. Lemaître et al. / Bioorg. Med. Chem. 17 (2009) 2607–2622
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We thank the CRIHAN (Centre de Ressources Informatiques de
Haute Normandie) and the European Community (FEDER) for the
molecular modeling software. The authors wish to thank Béatrice
Rouzaire Dubois (Laboratoire de Neurobiologie Cellulaire et Molé-
culaire UPR 9040—CNRS, Gif sur Yvette, France) for providing us
with the NG108-15 cells. For the financial support, we thank the
‘‘Conseil Régional de Basse-Normandie”.
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Supplementary data
Supporting information available: Elemental analysis data for
all compounds. This material is available free of charge via the
internet at http://www.elsevier.com. Supplementary data associ-
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