Novel and selective partial agonists of 5-HT3 receptors. 2. Synthesis and biological evaluation of piperazinopyridopyrrolopyrazines, piperazinopyrroloquinoxalines, and piperazinopyridopyrroloquinoxalines

Article abstract of DOI:10.1021/jm960501o

In continuation of our previous work on piperazinopyrrolothienopyrazine derivatives, three series of piperazinopyridopyrrolopyrazines, piperazinopyrroloquinoxalines, and piperazinopyridopyrroloquinoxalines were prepared and evaluated as 5-HT₃ receptor ligands. The chemical modifications performed within these new series led to structure-activity relationships regarding both high affinity and selectivity for the 5-HT₃ receptors that are in agreement with those established previously for the pyrrolothienopyrazine series. The best compound (8a) obtained in these new series is in the picomolar range of affinity for 5-HT₃ receptors with a selectivity higher than 10⁶. Four of the high-affinity 5-HT₃ ligands (8a, 15a,b, and 16d) were selected in both the pyridopyrrolopyrazine and the pyrroloquinoxaline series and were characterized in vitro and in vivo as agonists or partial agonists. Compound 8a was also evaluated in the light/dark test where it showed potential anxiolytic-like activity at very low doses per os.

Full text of DOI:10.1021/jm960501o

1808
J . Med. Chem. 1997, 40, 1808-1819
Novel a n d Selective P a r tia l Agon ists of 5-HT3 Recep tor s. 2. Syn th esis a n d
Biologica l Eva lu a tion of P ip er a zin op yr id op yr r olop yr a zin es,
P ip er a zin op yr r oloqu in oxa lin es, a n d P ip er a zin op yr id op yr r oloqu in oxa lin es
Herve´ Prunier,† Sylvain Rault,*,† J ean-Charles Lancelot,† Max Robba,† Pierre Renard,‡ Philippe Delagrange,§
Bruno Pfeiffer,‡ Daniel-Henri Caignard,‡ Rene´ Misslin,| Be´atrice Guardiola-Lemaitre,§ and Michel Hamon
Centre d’Etudes et de Recherche sur le Me´dicament de Normandie, Universite´ de Caen, 1, rue Vaube´nard,
14032 Caen Cedex, France, ADIR, 1, rue Carle He´bert, 92415 Courbevoie Cedex, France, IRI Servier, 6, place des Ple´iades,
92415 Courbevoie Cedex, France, Laboratoire de Psychophysiologie, 7 rue de l’Universite´, 67000 Strasbourg, France, and
INSERM U288, CHU Pitie´-Salpe´trie`re, 75634 Paris Cedex 13, France
Received J uly 11, 1996X
In continuation of our previous work on piperazinopyrrolothienopyrazine derivatives, three
series of piperazinopyridopyrrolopyrazines, piperazinopyrroloquinoxalines, and piperazino-
pyridopyrroloquinoxalines were prepared and evaluated as 5-HT3 receptor ligands. The
chemical modifications performed within these new series led to structure-activity relationships
regarding both high affinity and selectivity for the 5-HT3 receptors that are in agreement with
those established previously for the pyrrolothienopyrazine series. The best compound (8a )
obtained in these new series is in the picomolar range of affinity for 5-HT3 receptors with a
selectivity higher than 106. Four of the high-affinity 5-HT3 ligands (8a , 15a ,b, and 16d ) were
selected in both the pyridopyrrolopyrazine and the pyrroloquinoxaline series and were
characterized in vitro and in vivo as agonists or partial agonists. Compound 8a was also
evaluated in the light/dark test where it showed potential anxiolytic-like activity at very low
doses per os.
In tr od u ction
frequently mentioned agonists are 2-methyl-5-hydroxy-
tryptamine, (m-chlorophenyl)biguanide (mCPBG),10,11
and quipazine.12-15
Serotonin (5-hydroxytryptamine, 5-HT) and its recep-
tor subtypes as well as its physiological implications
have been the matter of well-documented general
reviews.1-3 Among the 5-HT receptor subtypes, special
attention has been paid to the 5-HT3 receptors4 and the
numerous potential therapeutic applications of their
antagonists: treatment of emesis associated with anti-
cancer chemotherapy,5 pain, memory impairment, drug
addiction, and psychosis.6 Highly selective and high-
affinity 5-HT3 antagonists have been identified and
developed, such as granisetron,7 ondansetron,8 and
tropisetron.9
NH2
CH2
CH2
CH3
NH2
Cl
NH
NH
HO
C
C
N
H
N
H
N
H
2-methyl-5-hydroxytryptamine
mCPBG
N
N
NH
quipazine
O
CH3
O
C
Recently we reported the synthesis,16 the receptor-
binding profile, and the in vitro and in vivo pharmaco-
logical evaluation of two series of tricyclic piperazino-
pyrrolothienopyrazine (PPTP) derivatives which have
in common with quipazine a polycyclic aromatic moiety
linked to a substituted piperazine via a “pseudo-
amidinic” bond.
NH
N
CH3
N
N
N
N
N
CH3
CH3
granisetron
ondansetron
O
C
O
N
CH3
R1
N
N
N
N
S
N
H
R1
R2
S
N
N
tropisetron
R2
NR
NR
PPTP
Less is known concerning selective and potent 5-HT3
agonists and their therapeutic potential. The most
This work on PPTP derivatives led us to obtain,
among others, 5-(4-benzylpiperazino)pyrrolo[1,2-a]thieno-
[3,2-e]pyrazine (2), which proved to be a very high-
affinity and selective 5-HT3 receptor partial agonist,
with potent anxiolytic-like activity in the light/dark
test.16
† Universite´ de Caen.
‡ ADIR.
§ IRI Servier.
| Universite´ de Strasbourg.
INSERM U288.
X Abstract published in Advance ACS Abstracts, May 1, 1997.
S0022-2623(96)00501-8 CCC: $14.00 © 1997 American Chemical Society

Novel and Selective Partial Agonists of 5-HT3 Receptors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1809
Sch em e 1a
N
S
RO
N
NH2
NO2
Cl
N
NH2
NO2
i
N
N
N
23d
23b: R = CH3
23c: R = C6H5CH2
2
In continuation of our previous studies on PPTP, we
now report our work on piperazinopyridopyrrolopyr-
azines (PPPP), piperazinopyrroloquinoxalines (PPQ),
and some piperazinopyridopyrroloquinoxalines (PPPQ).
R1
N
NH2
NO2
R1
N
N
iii
iv
25a-c,e
25d
ii
NO2
R2
R2
23a-e
24a-e
R1
N
N
N
R1
R2
N
N
R1
N
N
R1
N
N
v
vi
N
N
N
R2
NR
NR
NH2
N
H
O
R2
R2
PPPP
PPQ
25a-e
26a-e
N
N
R1
N
N
N
R1
N
N
N
vii
N
N
Cl
NR
R2
N
R2
R
PPPQ
6-13
27a-e
Chemical modifications via substitution of the PPPP
and PPQ skeletons were systematically carried out in
order to compare the structure-activity relationships
(SARs) for good affinity and high selectivity for 5-HT3
receptors with those previously established from the
PPTP series.17
As for the PPTP derivatives, the influence of the
substitution on the nature of the interaction with the
receptor (agonist, partial agonist, antagonist) was also
determined for the most active compounds.
viii
5
13
a
(i) RONa, ROH, ∆; (ii) DMTHF, ∆; (iii) Raney nickel, N2H4,
H2O, EtOH, ∆; (iv) FeSO4, 7H2O, EtOH, ∆ (v) COCl2, toluene, ∆;
(vi) POCl3, pyridine, ∆; (vii) N-substituted piperazine, K2CO3,
DMF, ∆; (viii) NaOH, CH3OH, H2O, ∆.
Sch em e 2a
N
N
NH2
Cl
N
ii
i
N
N
N
Cl
28
29
14
Ch em istr y
N
a
(i) DMTHF, ∆; (ii) N-benzylpiperazine, K2CO3, DMF, ∆.
After the chemistry of the pyrrolothieno-
pyrazines,16,18-25 we now report the syntheses of the
PPPP, the PPQ, and the PPPQ series.26 Some pyrrolo-
[1,2-a]quinoxalines are described in the literature,27 in
particular the 4-(4-methylpiperazino)-7-(trifluorometh-
yl)pyrrolo[1,2-a]quinoxaline.28
yielded the monosubstituted piperazine 5. 3-Amino-2-
chloropyridine (28) gave compound 14 after formation
of the pyrrole ring and subsequent substitution with the
N-benzylpiperazine (Scheme 2).
In order to obtain the pyrroloquinoxalines (Scheme
3), methyl anthranylate (30) reacted with DMTHF35,36
and the same catalyst as above for the pyridines. After
saponification, the acid was converted to the unstable
acyl azide which rearranged and cyclized directly into
lactam 36a . Acid 32 treated by TEA and then by ethyl
chloroformate gave in situ the carbonic mixed anhydride
which reacted with sodium azide. After extraction with
Et2O, the product underwent a spontaneous rearrange-
ment into lactam 36a during evaporation of the solvent.
The last two steps were similar to those of the PPPP
series. We studied some substitutions on the 7- or
8-position of the pyrroloquinoxaline. The first part of
the synthesis was changed. The raw materials were the
substituted 2-nitroanilines 33b-e. The Clauson-Kaas
reaction of anilines 33b-e in acetic acid gave the
pyrrolic derivatives 34b-e,29,30 which were reduced
either with iron, acetic acid, and hydrochloric acid for
34b,c or with Raney nickel and hydrazine hydrate for
34d ,e to provide anilines 35b-e. Subsequent cycliza-
tion with phosgene gave lactams 36b-e, which were
converted to pyrroloquinoxalines 15b-17 as previously
Treatment of 6-chloropyridine 23d with an alkoxide
gave the 6-alkoxypyridines 23b,c (Scheme 1). The
reaction of 2-aminopyridines 23a -e with 2,5-dimethoxy-
tetrahydrofuran (DMTHF) either in 1,4-dioxane with
4-chloropyridine hydrochloride as catalyst or in acetic
acid yielded the pyrrolic compounds 24a -e.29,30 The
Be´champ’s reduction31,32 of the nitro group was then
carried out to give 3-amino-2-pyrrolopyridine (25a ), but
because a better yield was achieved with Raney nickel
(92% instead of 68%), the latter method was chosen for
25a -c,e. For 25d , the presence of the chlorine atom
would not allow the same hydrogenation, so iron(II)
sulfate was used because of its good selectivity.33 The
cyclization was then possible between the NH2 and the
CR of the pyrrole ring by reacting 25a -e with phosgene
in a 20% toluenic solution to give the lactams 26a -e,
which were subsequently chlorinated with phosphoryl
chloride to obtain the chloropyrazines 27a -e.34 Finally,
a nucleophilic substitution with an appropriate piper-
azine furnished the pyridopyrrolopyrazines 6-13. The
deprotection of the carbamate function of 13 by NaOH

1810 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Prunier et al.
Sch em e 3a
Sch em e 5a
N
R1
R2
N
NH2
N
iii
i, ii
i, ii
iii
35a
Cl
N
CO2CH3
CO2R
N
H
O
30
31: R = CH3
32: R = H
41
36a-e
N
N
N
vi
N
F
19
R1
R2
NH2
R1
R2
N
R
a
(i) ClCOCH2Cl, pyridine, dioxane, ∆; (ii) POCl3, toluene, ∆;
(iii) N-(4-fluorobenzyl)piperazine, K2CO3, DMF, ∆.
i, iv
i, v
35b-c
35d-e
NO2
Sch em e 6a
33b-e
34b-e: R = NO2
35b-e: R = NH2
N
NH2
NO2
N
N
R
R1
R2
N
N
R1
R2
N
i, ii
iii
vii
viii
36a-e
Cl
N
N
42
43: R = NO2
44: R= NH2
37a-e
N
15a-17
R
a
(i) DMTHF, ∆; (ii) NaOH, H2O, MeOH, ∆; (iii) TEA, ClCO2Et,
NaN3, Me2CO, MeCN, 0 °C; (iv) Fe, HCl, AcOH, ∆; (v) Raney
nickel, N2H4, H2O, EtOH, ∆; (vi) COCl2, toluene, ∆; (vii) POCl3,
pyridine, ∆; (viii) N-substituted piperazine, K2CO3, DMF, ∆.
N
N
N
N
N
iv
v
20-22
Cl
N
H
O
Sch em e 4a
46
45
a
N
(i) DMTHF, AcOH, ∆; (ii) Raney nickel, N2H4, H2O, EtOH, ∆;
i
ii
(iii) COCl2, toluene, ∆; (iv) POCl3, pyridine, ∆; (v) N-substituted
piperazine, K2CO3, DMF, ∆.
37d
CH3O
N
N
NH
38
Resu lts a n d Discu ssion
N
N
Twenty-eight piperazinopyrido[3,2-e]pyrrolo[1,2-a]-
pyrazines (PPPP), piperazinopyrrolo[1,2-a]quinoxalines
(PPQ), and piperazinopyrido[2,3-h]quinoxalines (PPPQ)
were prepared and evaluated in a prescreening proce-
dure for their affinity for the 5-HT3 receptor and their
selectivity compared to other 5-HT receptor subtypes
(5-HT1A, 5-HT1B, 5-HT2A, and 5-HT2C) (Tables 1-3). In
order to quickly obtain compounds with high affinity
and selectivity, we have transposed some of the SARs
established in our previous work on tricyclic pyrrolo-
thienopyrazine 5-HT3 ligands and then synthesized only
substituted or nonsubstituted piperazino derivatives.
Strict analogue 5 of quipazine was first synthesized
in the PPPP series and proved to be a potent and
relatively selective ligand of the 5-HT3 receptors with
-log IC50 of 8.83 for 5-HT3, 7.19 for 5-HT1A, and 6.82
for 5-HT1B. Both affinity and selectivity were clearly
higher than for its counterpart 1 in the PPTP series.
Once the potentialities of the PPPP series were estab-
lished, chemical modifications were systematically per-
formed on the piperazine and the pyridine moieties
(Table 1). The following became rapidly apparent:
(a) Substitution of the piperazine with a methyl (6,
-log IC50 ) 9.01) results in a slight increase in affinity
without change of the selectivity. In contrast, substitu-
tion with an allyl (7) leads to a very high-affinity and
selective ligand for the 5-HT3 receptors with a -log IC50
of 11.40 and a selectivity greater than 10000-100000,
depending on the receptor subtype.
iii
Br–
CH3O
N
N+
39
N
N
CH3O
N
N
N
(CH2)2
N
18
a
(i) Piperazine, ∆; (ii) Br(CH2)4Br, K2CO3, EtOH, ∆; (iii)
pyrazole, K2CO3, toluene, ∆.
described. From the reaction of excess piperazine with
the chloro compound 37d , it was possible to isolate the
monosubstituted piperazine 38 (Scheme 4), which on
reaction with 1,4-dibromobutane produced ammonium
salt 39.37,38 The latter reacted with pyrazole to furnish
18. Acylation of (o-aminophenyl)pyrrole (35a ) gave
chloroacetamide (40) which afforded 4-(chloromethyl)-
pyrrolo[1,2-a]quinoxaline (41) on treatment with phos-
phoryl chloride in toluene (Scheme 5). Subsequent
nucleophilic substitution with a piperazine furnished
compound 19 with a methylene group between the
pyrazine and the piperazine. The synthesis of PPQ
derivatives 20-22 is very similar to that of the PPPP
(Scheme 6).39 In this case, the formation of the pyrrole
ring was done in acetic acid. All the final compounds
5-22 were isolated as their stable salt form (hydrochlo-
ride, fumarate, or oxalate).
(b) Substitution of the piperazine with an ethoxycar-
bonyl (13, -log IC50 ) 4.55) or a phenyl (12, -log IC50
) 4.56) induces a total loss in 5-HT receptor affinity.

Novel and Selective Partial Agonists of 5-HT3 Receptors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1811
Ta ble 1. Binding Properties (-log IC50) of the Pyrido[3,2-e]pyrrolo[1,2-a]pyrazine Derivatives
(c) As with the allyl, substitution of the piperazine
with a benzyl results in a very high-affinity 5-HT3 ligand
(8, -log IC50 ) 12.09). Affinity for other 5-HT receptor
subtypes is clearly lowered with a selectivity greater
than 1000000, depending on the receptor subtype.
(d) The various substitutions performed on the ben-
zylpiperazine moiety show that substitution with a 3,4-
methylenedioxy (10) or with a 4-fluoro (9a ) results in a
good selectivity but approximately a 1000-fold decrease
in affinity with respect to 8a . However, affinities
remain relatively high with -log IC50 values of 9.10 and
8.58 for 10 and 9a , respectively. In contrast, substitu-
tion with 2,4-dichloro results in a dramatic loss in
affinity (11, -log IC50 ) 5.89).
9b; 2-benzyloxy, 8c; 2-benzylpiperazino, 8d ; 4-methyl,
8e and 9e), in a marked decrease of affinity compared
to the nonsubstituted derivatives 8a and 9a .
(f) Compound 14, which, to some extent, can be
considered as an open monocyclic analogue of 8a , was
found to be 100000 times less active.
In a general manner, these results correlate well with
those previously established with the PPTP series, but
with sometimes much higher affinity and selectivity as
is the case for the compounds 8a (-log IC50 ) 12.09)
and 7 (-log IC50 ) 11.40) which are between 100 and
1000 times more potent than their PPTP analogues 2
and 3.
Except for a few differences, results are the same with
the PPQ derivatives. There again, substitution of the
piperazine with a benzyl leads to a good affinity for the
5-HT3 receptor (15a , -log IC50 ) 9.45) with a very high
(e) All of the substitutions performed on the pyridine
ring of the PPPP skeleton result, regardless of the site
of substitution and the substituent (2-methoxy, 8b and

1812 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Prunier et al.
Ta ble 2. Binding Properties (-log IC50) of the Pyrrolo[1,2-a]quinoxaline Derivatives
Ta ble 3. Binding Properties (-log IC50) of the Pyrido[2,3-h]Pyrrolo[1,2-a]quinoxaline Derivatives
compd
Ra
5-HT1A
5-HT1B
5-HT1D
5-HT2A
5-HT2C
5-HT3
20
21
22
Bn
piperonyl
allyl
4.20 ( 0.03
4.64 ( 0.07
5.34 ( 0.06
4.01 ( 0.09
4.23 ( 0.03
4.71 ( 0.01
<4
<4
<4
<4
7.51 ( 0.03
7.55 ( 0.02
7.71 ( 0.02
4.40 ( 0.12
5.24 ( 0.09
4.70 ( 0.15
4.67 ( 0.02
4.44 ( 0.07
a
Bn ) benzyl.
selectivity (around 10000). This compound is however
430 times less potent than its PPPP direct analogue 8a .
Substitution of the benzylpiperazine with a 4-fluoro
(16a , -log IC50 ) 8.62) results in a slight decrease in
affinity with respect to 15a while substitution with a
3,4-methylenedioxy (17, -log IC50 ) 10.09) increases it.
In agreement with the SARs previously established
in the PPTP series, substitution of the piperazine with
a 4-(pyrazolobutyl) (18) results in an inactive compound
perhaps because of the π-electron density located too
far from the basic nitrogen atom of the piperazine.
Substitution on the benzene part of the pyrroloquinoxa-
line skeleton with an 8-chloro (15c) clearly decreases
the affinity. Introduction of a 7-chloro, 7-methyl, or
7-methoxy substituent has no effect (15b) or only
moderate deleterious incidence (16b and 16d ,e).
Three compounds (20-22) have also been prepared
and evaluated in the PPPQ series; these show -log IC50
values of 7.51, 7.55, 7.71, respectively. All three proved
to be less active than their strict analogues in the PPQ
and PPPP series.
In conclusion to this preliminary evaluation, the
chemical modifications achieved within these novel
three series led us to confirm the SARs previously

Novel and Selective Partial Agonists of 5-HT3 Receptors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1813
Ta ble 4. In Vitro 5-HT3 Agonist Activity on the
[
Ta ble 5. In Vivo 5-HT3 Activity in the Von Bezold-J arisch
Reflex in Rats
14C]Guanidinium Accumulation in NG 108-15 Cells
compd
EC50 (nM)a
agonist activitya
antagonist activityb
15a
15b
16d
8a
18 ( 3
52 ( 3
dose
compd (µg/kg iv)
bradycardia
dose
inhibition of
(%)a
(µg/kg iv) bradycardia (%)b
104 ( 14
32 ( 4
15a
15b
16d
8a
60
120
120
120
120
54 ( 4.5
0
0
59 ( 7
40 ( 6
60
80 ( 3.5
IC50: 31.2 µg/kg
IC50: 75.4 µg/kg
40 ( 4
2
11 ( 3
293 ( 26
5-HT
120
60
a
2
42 ( 3
Compounds were tested in triplicate for each concentration.
Each value is the mean ( SEM of three separate determinations.
a
Decrease in heart rate, as a percentage of the baseline control
b
value (prior to any treatment). Percent of inhibition or IC50 of
bradycardia induced by 5-HT (30 µg/kg iv). The reported values
are the means ( SEM of four to seven independent determina-
tions.
established with the PPTP derivatives. Extremely
potent and selective 5-HT3 ligands in the picomolar
range of affinity were obtained in the PPPP series (8a
and 7) which, of all the heteropolycyclic skeletons
investigated, gives the best results.
Ta ble 6. Active Doses (µg/kg) in the Mouse Light/Dark Test
after per os Administration
time in the lit boxa
10 100 1000 0.1
transitionsb
10 100 1000
P h a r m a cology. Among the high-affinity and selec-
tive 5-HT3 ligands described above, four compounds, 8a ,
15a ,b, and 16d , have been selected for characterization
of their activity at 5-HT3 receptors. Their agonist and/
or antagonist properties have been determined in two
different models generally used for 5-HT3 receptor
ligands: in vitro on the [14C]guanidinium influx into NG
108-15 cells in the presence of substance P15 and in vivo
on the Von Bezold-J arisch reflex7,9 in anesthetized rats.
The most potent 5-HT3 compound, 8a , has been chosen
for behavioral studies in the mouse light/dark test in
order to detect possible anxiolytic-like activity.40,41 In
this test, 5-HT3 antagonists42 and partial agonists16 are
active at very low doses.
compd 0.1
1
1
8a
2
+++ +++
++ +++ +++ +++ +++
+
++
0
+++
+
+
++
++ ++
0
++
+
+
a
Significant increase in time spent in the lit compartment.
b
Significant increase in the number of transitions between the
lit and dark compartments. Statistical significance between
control group and treated group after a combined analysis of
variance and a Bonferroni’s posteriori t-test: +++, p < 0.0001;
++, p < 0.01; + p < 0.05; 0, p g 0.05.
from 5 min to 15 min. In the Von Bezold-J arisch reflex,
15b and 16d behave like antagonists whereas 15a and
8a are partial agonists like compound 2.
The differences in binding affinities between com-
pounds 8a , 15a ,b, or 16d are not reflected in either the
[14C]guanidinium influx model or the Von Bezold-
J arisch reflex. These differences could be explained
either by the existence of variations in the pharmaco-
logical properties of 5-HT3 receptors from one model to
another43 or by different affinity states of the 5-HT3
receptor.44-46
The most potent 5-HT3 compound, 8a , was studied
in the light/dark test. Mice were treated per os with
8a in a range of doses from 0.1 µg/kg to 1 mg/kg.
Compound 8a increased the time spent in the aversive
compartment and the transitions between the two
compartments (Table 6). This effect was significant for
both parameters at 0.1, 1, 10, and 100 µg/kg. In
comparison, compound 2 which is a partial 5-HT3
agonist16 has a similar effect. Both compounds 8a and
2 which were characterized as agonists in vitro in the
[14C]guanidinium uptake test and partial agonists in
vivo in the Von Bezold-J arisch reflex assay presented
an anxiolytic-like activity in the light/dark test.
The anxiolytic-like activity of the partial agonists can
be explained either by a long-lasting receptor desensi-
tization46 or by an antagonist activity on 5-HT3 receptors
in some tissues.
P h a r m a cology Resu lts. Compounds 8a , 15a ,b, and
16d were studied in vitro on NG 108-15 cells. In the
presence of substance P (10 µM), the four compounds
increased the uptake of [14C]guanidinium into the cells.
The EC50 values ranged from 18 to 104 nM (Table 4),
indicating that these compounds are more potent than
5-HT (EC50 ) 293 nM). However, the maximum in-
crease of uptake (=+150%) due to 8a , 15a ,b, and 16d
was similar to that observed with 5-HT. Compound 16d
was also able to reduce the stimulatory effect of 5-HT
(1 µM) on the uptake of [14C]guanidinium, with a
maximal effect of -25% at 3 nM. In this model,
compounds 8a and 15a ,b act as 5-HT3 agonists like 2,
and 16d is a partial agonist.
The activity of these compounds was also tested in
vivo on the Von Bezold-J arisch reflex. In this model,
5-HT administration (30 µg/kg iv) induces a rapid and
transient decrease in heart rate which represents the
Von Bezold-J arisch reflex. Of the four compounds
studied in this model, only two of them, 8a and 15a ,
were able to trigger this reflex. The bradycardic effects
(=-55% reduction in heart rate) of 8a at 120 µg/kg iv
or 15a at 60 µg/kg iv were similar to those of 5-HT at
30 µg/kg iv. In order to determine whether these
compounds were able to antagonize the Von Bezold-
J arisch reflex induced by 5-HT (30 µg/kg iv), rats were
pretreated with these compounds 5 min before 5-HT
injection. All of them have presented some antagonist
activity (Table 5) with a total prevention of the 5-HT-
induced bradycardia at 120 µg/kg iv for 15b and 16d .
Nevertheless, this antagonist effect is very transient.
For example, the reduction in 5-HT evoked bradycardia
shifts from -100% to only -60% when the time interval
between the pretreatment with 15b (120 µg/kg iv) and
the subsequent injection of 5-HT (30 µg/kg iv) increased
Con clu sion
As previously achieved with the tricyclic piperazino-
pyrrolothienopyrazine derivatives,16 the chemical modi-
fications made in the piperazinopyridopyrrolopyrazine
and piperazinopyrroloquinoxaline series have enabled
us to obtain very potent and selective ligands for the
5-HT3 receptors. The SARs established with these new
compounds are in agreement with those previously
inferred from the pyrrolothienopyrazine series. There

1814 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Prunier et al.
>264 °C (60% MeCN, 40% i-PrOH). 1H-NMR (DMSO-d6): δ
3.42 (m, 4H, H piperazine), 3.98 (s, 3H, CH3), 4.43 (m, 9H,
CH2, H piperazine, NH+), 6.93 (m, 2H, H7, H8), 7.43, 7.63 (m,
6H, C6H5, H3), 8.30 (m, 2H, H4, H9). IR: 3360 (m, NH+), 1610
(s, CdN) cm-1. Anal. (C22H26Cl3N5O) C, H, Cl, N.
again, in vivo and in vitro data have shown the agonist
or partial agonist character of the best 5-HT3 deriva-
tives. Among these, 8a proved to be of great interest
as a potential anxiolytic agent.
2-(Be n zyloxy)-6-(4-b e n zylp ip e r a zin o)p yr id o[3,2-e]-
p yr r olo[1,2-a ]p yr a zin e Mon ofu m a r a te (8c). 27c (1.0 g, 3
mmol) reacted with the method described for 6 until obtain-
ment of the base of 8c. The latter was dissolved in acetone
(60 mL). Fumaric acid (1.1 equiv) was added, and the mixture
was heated at 40 °C for 15 min. After cooling, the precipitate
was filtered and dried. This gave 8c as a white powder (0.88
g, 48% yield). Mp: 208 °C (MeCN). 1H-NMR (DMSO-d6): δ
2.17 (m, 2H, H piperazine), 2.63 (m, 2H, H piperazine), 3.40
(m, 4H, H piperazine), 4.20 (s, 2H, CH2), 5.27 (s, 2H, CH2O),
6.27 (s, 2H, CHdCH), 6.47, 6.60 (m, 3H, H3, H7, H8), 7.00 (m,
10H, C6H5, C6H5), 7.40 (m, 2H, NH+, OH), 7.47 (d, 1H, H4),
7.80 (q, 1H, H9). IR: 3080 (s, NH+), 1670 (s, CdO), 1575 (m,
CdN) cm-1. Anal. (C32H31N5O5) C, H, N.
Exp er im en ta l Section
Ch em istr y. Every compound was characterized by elemen-
tal analysis, IR spectra, and H-NMR spectra; these data are
1
reported only for the compounds tested in the pharmacological
study. IR spectra were recorded on a Philips PU 9716 infrared
spectrometer using KBr pellets; the frequencies are expressed
1
in cm-1. The H-NMR spectra were obtained on a Varian EM
90 spectrometer, with Me4Si as the internal standard and
DMSO-d6 as the solvent; the chemical shifts are reported in
ppm of Me4Si in δ units, and the coupling constants are in
hertz. The IR and 1H-NMR spectra were consistent with
assigned structures. Elementary analyses were within (0.4%
of the theoretical values.
2,6-Bis(4-ben zylp iper a zin o)p yr ido[3,2-e]pyr r olo[1,2-a ]-
p yr a zin e Tetr a h yd r och lor id e (8d ). 27d (1.3 g, 5.5 mmol)
was dissolved in DMF (30 mL) in the presence of N-benzyl-
piperazine (2.12 g, 12 mmol) and K2CO3 (1.89 g, 13 mmol).
The mixture was then treated as described for the synthesis
of 6. This gave 8d as a yellow powder (1.37 g, 37% yield).
Mp: 210 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.26 (m, 20H,
H piperazine, NH+), 4.14 (s, 2H, CH2), 4.36 (s, 2H, CH2), 6.90
(t, 1H, H8), 7.15 (t, 1H, H7), 7.40, 7.68 (m, 11H, C6H5, C6H5,
H3), 7.95 (d, 1H, H4), 8.23 (t, 1H, H9). IR: 3350 (s, NH+), 1570
(m, CdN) cm-1. Anal. (C32H39Cl4N7) C, H, Cl, N.
6-(4-Ben zylp ip er a zin o)-4-m eth ylp yr id o[3,2-e]p yr r olo-
[1,2-a ]p yr a zin e Dih yd r och lor id e (8e). 27e (2.0 g, 9.2
mmol) reacted under the same conditions as those described
to obtain 6. This gave 8e as a yellow powder (3.00 g, 75%
yield). Mp: 240 °C (60% MeCN, 40% i-PrOH). 1H-NMR
(DMSO-d6): δ 2.60 (s, 3H, CH3), 3.40 (m, 4H, H piperazine),
4.28 (m, 6H, CH2, H piperazine), 6.83 (q, 1H, H8), 7.10 (q, 1H,
H7), 7.30 (d, 1H, H3), 7.46, 7.67 (m, 5H, C6H5), 8.18 (d, 1H,
H2), 8.30 (q, 1H, H9), 11.93 (m, 2H, NH+). IR: 3400 (s, NH+),
1610 (m, CdN) cm-1. Anal. (C22H25Cl2N5) C, H, Cl, N.
6-[4-(4-F lu or oben zyl)p ip er a zin o]p yr id o[3,2-e]p yr r olo-
[1,2-a ]p yr a zin e Tr ih yd r och lor id e (9a ). 27a (2.0 g, 9.8
mmol) reacted under the same conditions as those used to
obtain 6. This gave 9a as a yellow powder (2.93 g, 63% yield).
Mp: 202 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.40 (m, 4H, H
piperazine), 4.43 (m, 6H, CH2, H piperazine), 5.10 (m, 3H,
NH+), 6.93 (t, 1H, H8), 7.20, 7.43, 7.76 (m, 6H, C6H4, H3, H7),
8.40 (m, 3H, H2, H4, H9). IR: 2580, 2560 (s, NH+), 1620 (s,
CdN) cm-1. Anal. (C21H23Cl3FN5) C, H, Cl, N.
6-[4-(4-Flu or oben zyl)piper azin o]-2-m eth oxypyr ido[3,2-
e]p yr r olo[1,2-a ]p yr a zin e Dih yd r och lor id e (9b). 27b (2.0
g, 8 mmol) reacted under the same conditions as those used
to obtain 6. This gave 9b as a yellow powder (1.25 g, 31%
yield). Mp: 210 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.45 (m,
4H, H piperazine), 3.95 (s, 3H, CH3), 4.47 (m, 4H, H piper-
azine), 4.85 (m, 4H, CH2, NH+), 6.78, 6.91 (m, 2H, H7, H8),
7.25, 7.37, 7.75 (m, 5H, C6H4, H3), 8.40 (m, 2H, H4, H9). IR:
3380 (s, NH+), 1610 (s, CdN) cm-1. Anal. (C22H24Cl2FN5O)
C, H, Cl, N.
6-[4-(4-F lu or oben zyl)p ip er a zin o]-4-m eth ylp yr id o[3,2-
e]p yr r olo[1,2-a ]p yr a zin e Dih yd r och lor id e (9e). 27e (2.0
g, 9.2 mmol) reacted under the same conditions as those used
to obtain 6. This gave 9e as a yellow powder (3.34 g, 81%
yield). Mp: 228 °C (MeCN). 1H-NMR (DMSO-d6): δ 2.47 (s,
3H, CH3), 3.21, 3.35, 3.65 (m, 6H, H piperazine), 4.36 (s, 2H,
CH2), 4.50 (m, 2H, H piperazine), 5.46 (m, 2H, NH+), 6.80 (q,
1H, H8), 7.07 (q, 1H, H7), 7.27 (m, 3H, H3, C6H2), 7.73 (m, 2H,
C6H2), 8.13 (d, 1H, H2), 8.27 (q, 1H, H9). IR: 3440 (s, NH+),
1590 (m, CdN) cm-1. Anal. (C22H24Cl2FN5) C, H, Cl, N.
6-[4-[3,4-(Met h ylen ed ioxy)ben zyl]p ip er a zin o]p yr id o-
[3,2-e]p yr r olo[1,2-a ]p yr a zin e Tr ih yd r och lor id e (10). 27a
(2.0 g, 9.8 mmol) reacted under the same conditions as those
described to obtain 6. This gave 10 as a white powder (2.26
g, 46% yield). Mp: 220 °C (MeCN). 1H-NMR (DMSO-d6): δ
3.43 (m, 4H, H piperazine), 3.98 (m, 2H, H piperazine), 4.33
(s, 2H, CH2-Ar), 4.65 (m, 2H, H piperazine), 5.27 (m, 3H, NH+),
6-P ip er a zin op yr id o[3,2-e]p yr r olo[1,2-a ]p yr a zin e Di-
h yd r och lor id e (5). 13 (2.0 g, 9.9 mmol) was dissolved in
CH3OH (50 mL) with aqueous sodium hydroxide at 40% (50
mL). The solution was heated at 60 °C for 4 h. The methanol
was eliminated under reduced pressure. The precipitate in
water was extracted with Et2O. The organic layer was washed
and concentrated under vacuum to furnish the base of 5. The
latter was dissolved in i-PrOH (50 mL) and then salified with
37% HCl (3 mL). After stirring, the precipitate was filtered,
washed with i-PrOH, and dried. This gave 5 as a white
powder (0.54 g, 32% yield). Mp: 265 °C (MeCN). 1H-NMR
(DMSO-d6): δ 3.33 (m, 4H, H piperazine), 4.17 (m, 4H, H
piperazine), 4.63 (m, 2H, NH2+), 6.93 (q, 1H, H8, J 7,8 ) 4.2 Hz,
J 8,9 ) 3.0 Hz), 7.37 (q, 1H, H7, J 7,9 ) 1.2 Hz), 7,43 (q, 1H, H3,
J 2,3 ) 4.5 Hz, J 3,4 ) 7.5 Hz), 8.27, 8.38 (m, 3H, H2, H4, H9),
9.73 (m, 1H, NH+). IR: 3440 (m, NH2+), 2760 (s, NH+), 1630
(s, CdN) cm-1. Anal. (C14H17Cl2N5) C, H, Cl, N.
6-(4-Me t h ylp ip e r a zin o)p yr id o[3,2-e]p yr r olo[1,2-a ]-
p yr a zin e Tr ih yd r och lor id e (6). 27a (2.0 g, 9.8 mmol) was
added in DMF (30 mL) with N-methylpiperazine (0.99 g, 9.8
mmol) and K2CO3 (1.63 g, 11.8 mmol). The solution was
heated at 130 °C for 3 h and, after cooling, was added to 100
mL of stirred water. The suspension was extracted with Et2O.
After usual treatments, the oil obtained was converted into
its hydrochloride salt. The base was dissolved in i-PrOH with
heating, and an excess of HCl (3.5 equiv) was added. After
30 min of stirring at 20 °C, the precipitate was filtered. This
gave 6 as a white powder (1.02 g, 27% yield). Mp: >265 °C
(MeCN). 1H-NMR (DMSO-d6): δ 2.81 (s, 3H, CH3), 3.47 (m,
4H, H piperazine), 3.83 (m, 2H, H piperazine), 4.66 (m, 2H, H
piperazine), 6.10 (m, 3H, NH+), 6.93 (q, 1H, H8, J 7,8 ) 4.2 Hz,
J 8,9 ) 3.0 Hz), 7.37 (q, 1H, H7, J 7,9 ) 1.5 Hz), 7.43 (q, 1H, H3,
J 2,3 ) 4.8 Hz, J 3,4 ) 8.1 Hz), 8.27, 8.37 (m, 3H, H2, H4, H9, J 2,4
) 1.5 Hz). IR: 3380 (s, NH+), 3120, 2970 (m, CH), 1625 (s,
CdN) cm-1. Anal. (C15H20Cl3N5) C, H, Cl, N.
6-(4-Allylp ip er a zin o)p yr id o[3,2-e]p yr r olo[1,2-a ]p yr -
a zin e Tr ih yd r och lor id e (7). 27a (2.0 g, 9.8 mmol) reacted
under the same conditions as those described to obtain 6. This
gave 7 as a beige powder (0.91 g, 23% yield). Mp: 250 °C
(MeCN). 1H-NMR (DMSO-d6): δ 3.43 (m, 4H, H piperazine),
3.90 (d, 2H, N-CH2), 4.20 (m, 7H, H piperazine, NH+), 5.56 (d,
2H, CH2), 6.00 (m, 1H, CH), 6.96 (t, 1H, H8), 7.50 (m, 2H, H3,
H7), 8.43 (m, 3H, H2, H4, H9). IR: 2340, 2530 (s, NH+), 1620
(s, CdN) cm-1. Anal. (C17H22Cl3N5) C, H, Cl, N.
6-(4-Be n zylp ip e r a zin o)p yr id o[3,2-e]p yr r olo[1,2-a ]-
p yr a zin e Tr ih yd r och lor id e (8a ). 27a (2.0 g, 9.8 mmol)
reacted under the same conditions as those described to obtain
6. This gave 8a as a white powder (3.65 g, 81% yield). Mp:
186 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.43 (m, 4H, H
piperazine), 4.16, 4.43 (m, 6H, CH2, H piperazine), 5.03 (m,
3H, NH+), 6.96 (t, 1H, H8), 7.43, 7.70 (m, 7H, C6H5, H3, H7),
8.40 (m, 3H, H2, H4, H9). IR: 2800, 2540, 2470, 2300 (m, NH+),
1600 (s, CdN) cm-1. Anal. (C21H24Cl3N5) C, H, Cl, N.
6-(4-Ben zylpiper azin o)-2-m eth oxypyr ido[3,2-e]pyr r olo-
[1,2-a ]p yr a zin e Tr ih yd r och lor id e (8b). 27b (0.7 g, 4 mmol)
reacted under the same conditions as those described to obtain
6. This gave 8b as a white powder (0.70 g, 40% yield). Mp:

Novel and Selective Partial Agonists of 5-HT3 Receptors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1815
6.03 (s, 2H, CH2O), 6.97, 7.33 (m, 6H, C6H3, H3, H7, H8), 8,40
gave 16a as a white powder (2.28 g, 49% yield). Mp: 182 °C
(MeCN). 1H-NMR (DMSO-d6): δ 3.43 (m, 4H, H piperazine),
4.20 (m, 4H, H piperazine), 4.40 (s, 2H, CH2), 4.46 (m, 3H,
NH+), 7.00 (q, 1H, H2), 7.36, 7.73 (m, 7H, C6H4, H3, H7, H8),
8.20 (m, 2H, H6, H9), 8.60 (q, 1H, H1). IR: 2880, 2660, 2560
(m, NH+), 1580 (s, CdN) cm-1. Anal. (C22H24Cl3FN4) C, H,
Cl, N.
7-Ch lor o-4-[4-(4-flu or oben zyl)p ip er a zin o]p yr r olo[1,2-
a ]qu in oxa lin e Tr ih yd r och lor id e (16b). 37b (2.0 g, 8.4
mmol) reacted under the same conditions as those used to
obtain 6. This gave 16b as a white powder (3.04 g, 71% yield).
Mp: 212 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.33 (m, 4H, H
piperazine), 4.00 (m, 2H, H piperazine), 4.40 (s, 2H, CH2), 4.65
(m, 2H, H piperazine), 5.00 (m, 3H, NH+), 6.93 (q, 1H, H2),
7.33 (m, 1H, H3), 7.33, 7.76 (m, 4H, C6H4), 7.33, 7.76, 8.13 (m,
3H, H6, H8, H9), 8.56 (q, 1H, H1). IR: 2720, 2620 (m, NH+),
1600 (s, CdN) cm-1. Anal. (C22H23Cl4FN4) C, H, Cl, N.
4-[4-(4-F lu or ob en zyl)p ip er a zin o]-7-m et h oxyp yr r olo-
[1,2-a ]qu in oxa lin e Dih yd r och lor id e (16d ). 37d (2.0 g, 8.6
mmol) reacted under the same conditions as those used to
obtain 6. This gave 16d as a white powder (2.80 g, 70% yield).
Mp: 220 °C (70% MeCN, 30% n-PrOH). 1H-NMR (DMSO-
d6): δ 3.43 (m, 4H, H piperazine), 3.81 (s, 3H, CH3), 4.12, 4.43,
4.69 (m, 8H, H piperazine, CH2, NH+), 6.93 (t, 1H, H2), 7.02
(d, 1H, H8), 7.27 (t, 2H, C6H2), 7.40 (t, 1H, H3), 7.78 (m, 3H,
C6H2, H6), 8.10 (d, 1H, H9), 8.47 (t, 1H, H1). IR: 2700, 2600
(s, NH+), 1600 (s, CdN) cm-1. Anal. (C23H25Cl2FN4O) C, H,
Cl, N.
4-[4-(4-F lu or oben zyl)p ip er a zin o]-7-m eth ylp yr r olo[1,2-
a ]qu in oxa lin e Tr ih yd r och lor id e (16e). 37e (2.0 g, 9.2
mmol) reacted under the same conditions as those described
to obtain 6. This gave 16e as a white powder (2.55 g, 57%
yield). Mp: 206 °C (MeCN). 1H-NMR (DMSO-d6): δ 2.40 (s,
3H, CH3), 3.40, 4.10, 4.40 (m, 10H, CH2), 4.66 (m, 3H, NH+),
7.00 (q, 1H, H2), 7.20 (q, 1H, H3), 7.33, 7.73 (m, 4H, C6H4),
7.33, 7.96, 8.13 (m, 3H, H6, H8, H9), 8.55 (q, 1H, H1). IR: 3340,
2600, 2540 (s, NH+), 1605 (s, CdN) cm-1. Anal. (C23H26Cl3-
FN4) C, H, Cl, N.
(m, 3H, H2, H4, H9). IR: 3400 (s, NH+), 1605 (s, CdN) cm-1
.
Anal. (C22H24Cl3N5O2) C, H, Cl, N.
6-[4-(2,4-Dich lor ob e n zyl)p ip e r a zin o]p yr id o[3,2-e]-
p yr r olo[1,2-a ]p yr a zin e Dih yd r och lor id e (11). 27a (2.0 g,
9.8 mmol) reacted under the same conditions as those de-
scribed to obtain 6. This gave 11 as a white powder (2.80 g,
60% yield). Mp: 220 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.45
(m, 8H, H piperazine), 3.93, 4.25 (m, 2H, NH+), 4.51 (s, 2H,
CH2), 6.89 (q, 1H, H8, J 8,9 ) 3.9 Hz), 7.22 (d, 1H, H7, J 7,8 ) 3.0
Hz), 7.44 (q, 1H, H3, J 2,3 ) 4.7 Hz, J 3,4 ) 7.9 Hz), 7.54 (q, 1H,
H5′, J 5′,6′ ) 8.4 Hz), 7.70 (d, 1H, H3′, J 3′,5′ ) 2.2 Hz), 8.11 (m,
2H, H4, H6′), 8.34 (m, 2H, H2, H9). IR: 3440 (s, NH+), 1605 (s,
CdN) cm-1. Anal. (C21H21Cl4N5) C, H, Cl, N.
6-(4-P h e n ylp ip e r a zin o)p yr id o[3,2-e]p yr r olo[1,2-a ]-
p yr a zin e Tr ih yd r och lor id e (12). 27a (2.0 g, 9.8 mmol)
reacted under the same conditions as those described to obtain
6. This gave 12 as a white powder (0.90 g, 20% yield). Mp:
180 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.61 (m, 4H, H
piperazine), 4.40 (m, 4H, H piperazine), 6.97 (q, 1H, H8, J 7,8
3.0 Hz, J 8,9 ) 4.2 Hz), 7.23 (m, 10H, C6H5, H3, H7, NH+), 8.37
(q, 1H, H9, J 7,9 ) 1.2 Hz), 8.43 (q, 1H, H2, J 2,3 ) 2.7 Hz, J 2,4
)
)
1.2 Hz), 8.63 (q, 1H, H4, J 3,4 ) 8.4 Hz). IR: 3510 (s, NH+),
1620 (s, CdN) cm-1. Anal. (C20H22Cl3N5) C, H, Cl, N.
6-[4-(Eth oxyca r bon yl)piper a zin o]p yr ido[3,2-e]pyr r olo-
[1,2-a ]p yr a zin e Dih yd r och lor id e (13). 27a (2.0 g, 9.8
mmol) reacted under the same conditions as those described
to obtain 6. This gave 13 as a white powder (2.60 g, 66% yield).
Mp: 196 °C (MeCN). 1H-NMR (DMSO-d6): δ 1.25 (t, 3H, CH3,
J Et ) 7.5 Hz), 3.40 (m, 4H, H piperazine), 4.14 (m, 6H, CH2, H
piperazine), 4.80 (m, 2H, NH+), 7.00 (q, 1H, H8), 7.55 (m, 2H,
H3, H7), 8.47 (m, 3H, H2, H4, H9). IR: 3500, 3440 (s, NH+),
1690 (s, CO), 1620 (s, CdN) cm-1. Anal. (C17H21Cl2N5O2) C,
H, Cl, N.
2-(4-Ben zylp ip er a zin o)-3-p yr r olop yr id in e Tr ih yd r o-
ch lor id e (14). 29 (2.0 g, 11 mmol) reacted under the same
conditions as those described to obtain 6. This gave 14 as a
white powder (0.70 g, 21% yield). Mp: 232 °C (MeCN). 1H-
NMR (DMSO-d6): δ 3.00 (m, 8H, H piperazine), 4.17 (s, 2H,
CH2), 5.07 (m, 3H, NH+), 6.07 (t, 2H, H3′, H4′), 6.90 (m, 3H,
H5, H2′, H5′), 7.23, 7.50 (m, 6H, C6H5, H4), 8.02 (dd, 1H, H6).
IR: 2580 (s, NH+), 1610 (s, CdN) cm-1. Anal. (C20H25Cl3N4)
C, H, Cl, N.
4-(4-Ben zylp ip er a zin o)p yr r olo[1,2-a ]qu in oxa lin e Tr i-
h yd r och lor id e (15a ). 37a (2.0 g, 10 mmol) reacted under
the same conditions as those described to obtain 6. This gave
15a as a white powder (2.52 g, 56% yield). Mp: 182 °C
(MeCN). 1H-NMR (DMSO-d6): δ 3.43 (m, 4H, H piperazine),
4.20, 4.67 (m, 4H, H piperazine), 4.43 (s, 2H, CH2), 4.70 (m,
3H, NH+), 6.98 (q, 1H, H2), 7.47, 7.63 (m, 8H, C6H5, H3, H7,
H8), 8.17 (m, 2H, H6, H9), 8.61 (q, 1H, H1). IR: 2880, 2650,
2550 (m, NH+), 1590 (s, CdN) cm-1. Anal. (C22H25Cl3N4) C,
H, Cl, N.
4-(4-Ben zylp ip er a zin o)-7-ch lor op yr r olo[1,2-a ]qu in oxa -
lin e Dih yd r och lor id e (15b). 37b (2.0 g, 8.4 mmol) reacted
under the same conditions as those described for the produc-
tion of 6. This gave 15b as a white powder (2.67 g, 70% yield).
Mp: 228 °C (50% MeCN, 50% i-PrOH). 1H-NMR (DMSO-d6):
δ 3.36 (m, 4H, H piperazine), 4.06 (m, 2H, H piperazine), 4.43
(s, 2H, CH2), 4.83 (m, 4H, H piperazine, NH+), 6.93 (q, 1H,
H2), 7.33, 7.60, 8.13 (m, 9H, C6H5, H3, H6, H8, H9), 8.53 (q, 1H,
H1). IR: 3400, 2700, 2600 (s, NH+), 1600 (s, CdN) cm-1. Anal.
(C22H23Cl3N4) C, H, Cl, N.
4-(4-Ben zylp ip er a zin o)-8-ch lor op yr r olo[1,2-a ]qu in oxa -
lin e Tr ih yd r och lor id e (15c). 37c (2.0 g, 8.4 mmol) reacted
under the same conditions as those described to synthesize 6.
This gave 15c as a white powder (1.52 g, 37% yield). Mp: 210
°C (MeCN). 1H-NMR (DMSO-d6): δ 3.44 (m, 4H, H piper-
azine), 4.05, 4.69 (m, 7H, H piperazine, NH+), 4.45 (s, 2H, CH2),
6.92 (q, 1H, H2), 7.34, 7.42 (m, 5H, C6H3, H3, H7), 7.66 (m, 2H,
C6H2), 8.03 (d, 1H, H6), 8.26 (s, 1H, H9), 8.51 (q, 1H, H1). IR:
2700, 2600 (s, NH+), 1595 (s, CdN) cm-1. Anal. (C22H24Cl4N4)
C, H, Cl, N.
4-[4-[3,4-(Met h ylen ed ioxy)ben zyl]p ip er a zin o]-7-ch lo-
r op yr r olo[1,2-a ]qu in oxa lin e Tr ih yd r och lor id e (17). 37b
(2.0 g, 8.4 mmol) reacted under the same conditions as those
described to obtain 6. This gave 17 as a white powder (3.0 g,
67% yield). Mp: 208 °C (MeCN). 1H-NMR (DMSO-d6): δ 3.36
(m, 4H, H piperazine), 4.00, 4.30 (m, 6H, H piperazine, CH2),
4.90 (m, 3H, NH+), 6.00 (s, 2H, CH2O), 6.96, 7.36 (m, 5H, H2,
H3, C6H3), 7.36, 8.13 (m, 3H, H6, H8, H9), 8.50 (q, 1H, H1). IR:
2700, 2600 (m, NH+), 1600 (s, CdN) cm-1
. Anal. (C23H24-
Cl4N4O2) C, H, Cl, N.
7-Meth oxy-4-[4-(4-pyr azolobu t-1-yl)piper azin o]pyr r olo-
[1,2-a ]q u in oxa lin e Oxa la t e (18). 39 (1.0 g, 2.3 mmol),
pyrazole (0.16 g, 2.3 mmol), K2CO3 (0.4 g, 2.9 mmol), and
dibenzo-18-crown-6 (10 mg) in toluene (20 mL) were heated
at 100 °C for 22 h. The hot suspension was filtered and then
concentrated under reduced pressure. The residue was dis-
solved in i-PrOH (50 mL), oxalic acid (0.17 g) was added, and
the mixture was heated at 50 °C for 15 min. After cooling,
the precipitate was filtered and dried. This gave 18 as a white
powder (0.7 g, 59% yield). Mp: 178 °C (MeCN). 1H-NMR
(DMSO-d6): δ 1.80 (m, 4H, (CH2)2,3), 3.10, 3.30 (m, 6H, CH2),
3.80 (s, 3H, CH3), 3.90, 4.10 (m, 6H, CH2), 6.20 (m, 1H, H4
pyrazole), 6.90 (m, 4H, H2, H3, H6, H8), 7.40 (d, 1H, H pyrazole),
7.70 (d, 1H, H pyrazole), 8.00 (d, 1H, H9), 8.20 (t, 1H, H1), 10.20
(m, 2H, NH+, OH). IR: 3360 (s, OH), 1710 (s, CdO), 1600 (s,
CdN) cm-1. Anal. (C25H30N6O5) C, H, N.
4-[[4-(4-F lu or oben zyl)p ip er a zin o]m eth yl]p yr r olo[1,2-
a ]qu in oxa lin e Tr ih yd r och lor id e Mon oh yd r a te (19). 41
(2.0 g, 9.2 mmol) reacted under the same conditions as those
described to obtain 6. This gave 19 as a yellow powder (1.90
g, 41% yield). Mp: 196 °C (MeCN). 1H-NMR (DMSO-d6): δ
3.44 (m, 8H, H piperazine), 3.57 (m, 5H, NH+, H2O), 4.42 (s,
2H, CH2-Ar), 4.73 (s, 2H, CH2), 7.09 (t, 1H, H2), 7.26 (t, 2H,
C6H2), 7.56 (m, 1H, H3), 7.56, 7.68 (m, 2H, H7, H8), 7.75 (q,
2H, C6H2), 8.12, 8.38 (m, 2H, H6, H9), 8.72 (t, 1H, H1). IR:
3380 (s, NH+), 3070, 2900 (m, CH), 1610 (s, CdN) cm-1. Anal.
(C23H28Cl3FN4O) C, H, Cl, N.
4-[4-(4-F lu or oben zyl)p ip er a zin o]p yr r olo[1,2-a ]qu in ox-
a lin e Tr ih yd r och lor id e (16a ). 37a (2.0 g, 10 mmol) reacted
under the same conditions as those described to obtain 6. This

1816 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Prunier et al.
4-(N-su bstitu ted p ip er a zin o)p yr id o[2,3-h ]p yr r olo[1,2-
a ]qu in oxa lin es (20-22). 46 (1.5 g, 6 mmol) reacted with the
wanted amine (6.5 mmol) and K2CO3 (0.98 g, 7 mmol) with
the method described for 6. This gave 20-22: 35-56% yield.
4-(4-Be n zylp ip e r a zin o)p yr id o[2,3-h ]p yr r olo[1,2-a ]-
qu in oxa lin e tr ih yd r och lor id e (20) was obtained as an
orange powder (1.50 g, 50% yield). Mp: 205 °C (60% MeCN,
40% i-PrOH). 1H-NMR (DMSO-d6): δ 3.40 (m, 4H, H piper-
azine), 4.56 (m, 9H, H piperazine, CH2, NH+), 7.00 (t, 1H, H2),
7.16 (t, 1H, H3), 7.43, 7.63 (m, 5H, C6H5), 7.93 (q, 1H, H10),
8.16 (m, 2H, H6, H7), 8.73 (t, 1H, H1), 9.06 (d, 1H, H11), 9.63
was filtered and the filtrate was concentrated under reduced
pressure. This gave 25b as a brown oil (8.21 g, 85% yield).
3-Am in o-6-(ben zyloxy)-2-p yr r olop yr id in e (25c). 24c
(7.8 g, 26 mmol) reacted according to the method described
for 25b with Raney nickel (about 7 g) and N2H4‚H2O (10 mL,
206 mmol). This gave 25c as a brown oil (6.41 g, 91% yield).
3-Am in o-6-ch lor o-2-p yr r olop yr id in e (25d ). 24d (9.27 g,
41 mmol) was dissolved in EtOH (300 mL). FeSO4‚7H2O
(115.25 g, 414 mmol), 10 N HCl (0.5 mL), and water (5 mL)
were successively added. The mixture was heated at 80 °C
for 90 min while the ammonia solution at 30% was added by
little fractions in order to maintain a basic pH. After cooling,
EtOH was evaporated under vacuum. The residue was poured
in water (100 mL) and extracted with Et2O; after the usual
treatments, a first amount of the product was obtained. The
aqueous phase was alcalinized with ammonia solution and
extracted with EtOAc. Finally, this gave 25d as a beige
powder (4.59 g, 57% yield). Mp: 89 °C (80% Et2O, 20%
n-hexane).
5,6-Dih yd r o-2-m eth oxy-6-oxop yr id o[3,2-e]p yr r olo[1,2-
a ]p yr a zin e (26b). 25b (8.21 g, 43 mmol) in toluene (400 mL)
was heated at 100 °C with COCl2 in toluenic solution at 20%
(30 mL, 57 mmol) for 3 h. After elimination of COCl2 in excess
and cooling, the precipitate was filtered and neutralized with
a saturated NaHCO3 solution to give a first amount of 26b.
The toluenic filtrate was concentrated under vacuum, the
residue was triturated with Et2O, and the resulting precipitate
was treated as before. This gave 26b as a whitish powder (6.96
g, 74% yield). Mp: 250 °C.
5,6-Dih yd r o-2-(ben zyloxy)-6-oxop yr id o[3,2-e]p yr r olo-
[1,2-a ]p yr a zin e (26c). 25c (6.4 g, 24 mmol) reacted under
the same conditions as those described to obtain 26b. This
gave 26c as a beige powder (2.73 g, 38% yield). Mp: 264 °C.
5,6-Dih yd r o-2-ch lor o-6-oxop yr id o[3,2-e]p yr r olo[1,2-a ]-
p yr a zin e (26d ). 25d (4.59 g, 23 mmol) reacted under the
same conditions as those described to obtain 26b. This gave
26d as a gray powder (2.60 g, 49% yield). Mp: 270 °C.
6-Ch lor opyr id o[3,2-e]pyr r olo[1,2-a ]pyr a zin e (27a ). 5,6-
Dihydro-6-oxopyrido[3,2-e]pyrrolo[1,2-a]pyrazine (26a ) (9.25 g,
50 mmol) was refluxed in POCl3 (200 mL) with pyridine (10
mL) for 4 h. After cooling, the reactives were eliminated under
vacuum. The residue was carefully dissolved in water at 0
°C, and the resulting solution was alcalinized with 30%
ammonium hydroxide. The precipitate was filtered and
extracted with Et2O and then EtOAc. The organic layers were
washed, treated with animal charcoal and MgSO4, and con-
centrated under reduced pressure to give 27a as a yellow
powder (8.35 g, 82% yield). Mp: 146 °C (MeCN).
(d, 1H, H9). IR: 3400 (s, NH+), 1610 (s, CdN) cm-1
. Anal.
(C25H26Cl3N5) C, H, Cl, N.
4-[4-[3,4-(Met h ylen ed ioxy)b en zyl]p ip er a zin o]p yr id o-
[2,3-h ]p yr r olo[1,2-a ]qu in oxa lin e tr ih yd r och lor id e (21)
was obtained as an orange powder (1.15 g, 35% yield). Mp:
245 °C (50% MeCN, 50% i-PrOH). 1H-NMR (DMSO-d6): δ
3.40 (m, 4H, H piperazine), 4.33 (s, 2H, CH2), 4.73 (m, 7H, H
piperazine, NH+), 6.04 (s, 2H, CH2O), 7.00, 7.16, 7.33 (m, 5H,
C6H3, H2, H3), 7.90 (q, 1H, H10), 8.16 (d, 2H, H6, H7), 8.76 (q,
1H, H1), 9.04 (d, 1H, H11), 9.60 (d, 1H, H9). IR: 3400 (s, NH+),
1615 (s, CdN) cm-1. Anal. (C26H26Cl3N5O2) C, H, Cl, N.
4-(4-Ally lp ip e r a zin o )p y r id o [2,3-h ]p y r r o lo [1,2-a ]-
qu in oxa lin e tr ih yd r och lor id e (22) was obtained as an
orange powder (1.50 g, 56% yield). Mp: 225 °C (60% MeCN,
40%i-PrOH). 1H-NMR (DMSO-d6): δ 3.50 (m, 6H, H piper-
azine),3.83 (d, 2H, CH2), 4.56 (m, 2H, H piperazine), 5.43, 5.53
(m, 6H, CHdCH2, NH+), 7.00 (q, 1H, H2), 7.13 (q, 1H, H3), 7.90
(q, 1H, H10), 8.13 (d, 2H, H6, H7), 8.66 (q, 1H, H1), 9.00 (d, 1H,
H11), 9.47 (d, 1H, H9). IR: 3420 (s, NH+), 1620 (s, CdN) cm-1
.
Anal. (C21H24Cl3N5) C, H, Cl, N.
2-Am in o-6-m eth oxy-3-n itr op yr id in e (23b). 2-Amino-6-
chloro-3-nitropyridine (23d ) (6.2 g, 35 mmol) was added to
MeOH (100mL) in which Na (1.65 g, 71 mmol) was previously
dissolved. The solution was heated at 70 °C for 8 h. MeOH
was eliminated under reduced pressure. The resulting product
was extracted with Et2O and EtOAc. Usual treatments of the
organic layers gave 23b as a yellow powder (4.7 g, 77% yield).
Mp: 172 °C.
2-Am in o-6-(ben zyloxy)-3-n itr op yr id in e (23c). Benzyl
alcohol (7.73 g, 71 mmol) was added to toluene (150 mL) with
Na (1.65 g, 71 mmol). After dissolution, 23d (10.34 g, 59 mmol)
was added and the mixture was heated first carefully for 30
min and then at reflux for 3 h. After cooling, water (50 mL)
was added before elimination of toluene under vacuum.
Extraction of the residual solution with Et2O allowed the
isolation of the product. This gave 23c as an orange powder
(6.73 g, 46% yield). Mp: 134 °C (Et2O).
6-C h lo r o -2-m e t h o x y p y r i d o [3,2-e]p y r r o lo [1,2-a ]-
p yr a zin e (27b). 26b (2.6 g, 12 mmol) reacted under the same
conditions as those described to obtain for 27a . This gave 27b
as a yellow powder (0.94 g, 33% yield). Mp: 163 °C.
2-(Be n zyloxy)-6-ch lor op yr id o[3,2-e]p yr r olo[1,2-a ]-
p yr a zin e (27c). 26c (2.65 g, 9 mmol) reacted under the same
conditions as those described to obtain 27a . This gave 27c as
a brown powder (1.47 g, 52% yield). Mp: 106 °C (EtOAc).
2,6-Dich lor op yr id o[3,2-e]p yr r olo[1,2-a ]p yr a zin e (27d ).
26d (2.6 g, 11 mmol) reacted under the same conditions as
those described to obtain 27a . This gave 27d as a yellow
powder (1.27 g, 45% yield). Mp: 211 °C (EtOAc).
4 -M e t h y l -6 -c h l o r o p y r i d o [3 ,2 -e ]p y r r o l o [1 ,2 -a ]-
p yr a zin e (27e). 5,6-dihydro-4-methyl-6-oxopyrido[3,2-e]pyr-
rolo[1,2-a]pyrazine (26e) (7.8 g, 39 mmol) reacted using the
same procedure as for 27a . This gave 27e as a yellow powder
(5.83 g, 68% yield). Mp: 139 °C (MeCN).
7- or 8-Su bstitu ted 4,5-Dih yd r o-4-oxop yr r olo[1,2-a ]-
qu in oxa lin es (36b-e). These derivatives were prepared with
the same method as that described above to obtain 26b. This
gave 36b-e (64-91% yield).
4,5-Dih yd r o-7-ch lor o-4-oxop yr r olo[1,2-a ]q u in oxa lin e
(36b). 35b (11.9 g, 62 mmol) gave 36b as a gray powder (12.15
g, 90% yield). Mp: >270 °C (MeCN).
6-Meth oxy-3-n itr o-2-p yr r olop yr id in e (24b). DMTHF
(10.45 g, 79 mmol) and 4-chloropyridine hydrochloride (12.0
g, 79 mmol) were stirred for 10 min in dioxane (300 mL). 23b
(11.15 g, 65 mmol) was added, and the suspension was heated
for 4 h. The dioxane was evaporated under reduced pressure,
and the residue was extracted with Et2O. Usual treatments
gave 24b as an orange powder (11.12 g, 76% yield). Mp: 70
°C.
6-(Ben zyloxy)-3-n itr o-2-pyr r olopyr idin e (24c). DMTHF
(8.2 g, 62 mmol) was stirred for 10 min in glacial AcOH (200
mL). After addition of 24b (12.68 g, 51 mmol), the solution
was heated under reflux for 4 h. The acid was eliminated
under reduced pressure. The residue was alcalinized with a
saturated NaHCO3 solution and was extracted with Et2O. This
gave 24c as a red powder (8.48 g, 55% yield). Mp: 130 °C
(Et2O).
6-Ch lor o-3-n itr o-2-p yr r olop yr id in e (24d ). 23d (15.4 g,
88 mmol) reacted with DMTHF (12.9 g, 97 mmol) and
4-chloropyridine hydrochloride (14.64 g, 97 mmol) in dioxane
(300 mL) with the same method as that used to obtain 24b.
This gave 24d as a red powder (10.27 g, 51% yield). Mp: 110
°C.
3-Am in o-6-m eth oxy-2-p yr r olop yr id in e (25b). Raney
nickel (about 8 g) and N2H4‚H2O (2 mL) were added to a
solution of 24b (11.12 g, 50 mmol) in EtOH (300 mL). The
resulting suspension was heated for 5 h while N2H4‚H2O (28
mL) was added drop by drop. After cooling, the suspension
4,5-Dih yd r o-8-ch lor o-4-oxop yr r olo[1,2-a ]q u in oxa lin e
(36c). 35c (20.0 g, 104 mmol) gave 36c as a beige powder
(14.5 g, 64% yield). Mp: >270 °C (MeCN).

Novel and Selective Partial Agonists of 5-HT3 Receptors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1817
4,5-Dih yd r o-7-m et h oxy-4-oxop yr r olo[1,2-a ]q u in oxa -
lin e (36d ). 35d (20.6 g, 109 mmol) gave 36d as a beige powder
(21.33 g, 91% yield). Mp: 252 °C (MeCN).
4,5-Dih yd r o-7-m e t h yl-4-oxop yr r olo[1,2-a ]q u in oxa -
lin e (36e). 35e (29.4 g, 170 mmol) gave 36e as a beige powder
(25.9 g, 76% yield). Mp: 250 °C (MeCN).
of Sanders-Bush and Breeding.51 Membranes (0.2 mg of
protein/mL) were incubated at 25 °C for 60 min with 1.2 nM
[3H]-N-methylmesulergine and 1 µM spiperone in 50 mM Tris-
HCl buffer, pH 7.4, supplemented with 4 mM CaCl2 and 10
µM pargyline. Nonspecific binding was determined in the
presence of 10 µM mianserin.
7- or 8-Su bstitu ted 4-Ch lor op yr r olo[1,2-a ]qu in oxa -
lin es (37b-e). These compounds were synthesized with the
same method as that described to obtain 27a . This gave
37b-e (53-71% yield).
4,7-Dich lor op yr r olo[1,2-a ]qu in oxa lin e (37b). 36b (11.0
g, 46 mmol) gave 37b as a white powder (8.50 g, 71% yield).
Mp: 204 °C (MeCN).
4,8-Dich lor op yr r olo[1,2-a ]qu in oxa lin e (37c). 36c (13.0
g, 59 mmol) gave 37c as a beige powder (8.10 g, 57% yield).
Mp: 188 °C (MeCN).
4-Ch lor o-7-m et h oxyp yr r olo[1,2-a ]q u in oxa lin e (37d ).
36d (10 g, 46 mmol) gave 37d as a beige powder (7.06 g, 65%
yield). Mp: 132 °C (EtOAc).
5-HT3 receptor binding to NG 108-15 cell membranes was
determined following a slight modification of the procedure of
Hoyer and Neijt.52 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
10-5 M tropisetron.
The affinity of the ligands tested for these receptors was
calculated using LUNDON2 Software and expressed as log
IC50 ( SEM (IC50 ) concentration inhibiting 50% of the specific
binding). The results obtained are reported in Tables 1-3.
[
14C]Gu a n id in iu m In flu x in to NG 108-15 Cells.15 Cells
were grown for 2 days in 35-mm culture dishes with 3 mL of
growth medium.15 Before the experiment was started, the cell
layer was washed twice with 1.5 mL of buffer A (145 mM NaCl,
5.4 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 2.0 mM Na2HPO4,
20 mM glucose, and 20 mM N-(2-hydroxyethyl)piperazine-N′-
2-ethanesulfanic acid (HEPES), the pH being adjusted to 7.4
with NaOH). The incubation (10 min at 37 °C) was then
performed in 1 mL of buffer B (135 mM NaCl, 4.5 mM KCI,
1.8 mM CaCl2, 1.0 mM MgCl2, 2.0 mM Na2HPO4, 20 mM
glucose, 20 mM HEPES, pH 7.4 with NaOH), supplemented
with 10 mM guanidinium chloride, 200-250 nCi (7.40-9.25
kBq) of [14C]guanidinium, 10 µM substance P, and the ap-
propriate drugs.
4-Ch lor o-7-m eth ylp yr r olo[1,2-a ]qu in oxa lin e (37e). 36e
(10.0 g, 50 mmol) gave 37e as a beige powder (5.85 g, 53%
yield). Mp: 158 °C (EtOAc).
7-Meth oxy-4-pip er a zin op yr r olo[1,2-a ]qu in oxa lin e (38).
37d (3.0 g, 13 mmol) was heated with piperazine (14.64 g, 170
mmol) at 160 °C for 4 h. Water (200 mL) was gradually added,
and the mixture was cooled in a crushed ice bath. Extraction
of the suspension with a Et2O/EtOAc mixture (25/75, v/v)
allowed the purification of 38 as a yellow powder (2.36 g, 64%
yield). Mp: 95 °C.
7-Meth oxy-4-(8-a za -5-a zon ia sp ir o[4.5]d eca n -8-yl)p yr -
r olo[1,2-a ]qu in oxa lin e Br om id e (39). 38 (2.0 g, 7 mmol),
1,4-dibromobutane (1.68 g, 8 mmol), K2CO3 (2.34 g, 17 mmol),
and dibenzo-18-crown-6 (30 mg) were dissolved in EtOH (30
mL), and the mixture was heated at 80 °C for 24 h. The hot
mixture was filtered and then concentrated to about one-third.
The precipitate was filtered and washed with anhydrous Et2O.
In the filtrate appeared a new precipitate which was filtered
1 day later. This gave 39 as a white powder (1.46 g, 49% yield).
Mp: >270 °C.
The incubation was stopped by aspiration of the medium,
and the cell layer was washed three times with 1.5 mL of ice-
cold buffer C (same composition as buffer A except that NaCl
was replaced by choline chloride). The cells were then dis-
solved in 0.5 mL of 0.4 N NaOH, and extracts were transferred
to scintillation vials. The culture dishes were rinsed with 0.5
mL of 1 N HCl and 0.5 mL of 0.4 N NaOH, which were mixed
with the first extract for the quantification of radioactivity in
the presence of 10 mL of Aquasol (NEN). For each experiment,
the protein content of a control dish was determined.15
4-Ch lor op yr id o[2,3-h ]p yr r olo[1,2-a ]qu in oxa lin e (46).
45 (9,5 g, 40 mmol) reacted with the same method as that
described to obtain 27a . This gave 46 as a yellow powder (5.3
g, 51% yield). Mp: 210 °C (MeCN).
Bezold -J a r isch Reflex in Ra ts. Male Crl:CD(SD)BR
rats (Charles River) weighing 280-320 g were fasted for 24 h
and then anesthetized with urethane (1.25 g/kg ip). In order
to monitor the Bezold-J arisch reflex (an abrupt dose-related
reduction in heart rate following a rapid iv bolus injection of
5-HT, 30 µg/kg), the carotid artery was cannulated and
connected to a Statham transducer, as described by Richardson
et al.9 Heart rate and blood pressure were monitored by using
the pressure transducer signal and a cardiotachymeter coupler
and recorded onto a Gemini polygraph (Ugo Basile, Italy). Test
compounds were dissolved in saline and administered intra-
venously (0.5 mL/kg) via a cannula inserted into the jugular
vein. To test antagonist activity, compounds were usually
administered iv 5 min before 5-HT (30 µg/kg).
P h a r m a cologica l Met h od s: Bin d in g E xp er im en t s.
5-HT1A receptor binding to bovine frontal cortex and hippo-
campus membranes was determined using a slight modifica-
tion of the method of Hoyer et al.47 Membranes (0.5 mg of
protein/mL) were incubated at 23° C for 40 min with 0.5 nM
[3H]-8-OH-DPAT in 50 mM Tris-HCl buffer, pH 7.4, supple-
mented with 4 mM CaCl2 and 10 µM pargyline. Nonspecific
binding was determined in the presence of 10 µM buspirone.
5-HT1B receptor binding to rat frontal cortex and striatum
membranes was determined using the method of Peroutka48
with slight modifications. Membranes (0.8 mg of protein/mL)
were incubated at 25 °C for 30 min with 2 nM [3H]-5-OH-
tryptamine, 1 µM spiperone, and 50 nM mesulergine in 50 mM
Tris-HCl buffer, pH 7.4, supplemented with 4 mM CaCl2 and
10 µM pargyline. Nonspecific binding was determined in the
presence of 10 µM propranolol.
5-HT1D receptor binding to pig frontal cortex and striatum
membranes was determined following the pocedure of Waeber
et al.49 with slight modifications. Membranes (0.8 mg of
protein/mL) were incubated at 25 °C for 30 min with 2 nM
[3H]-5-OH-tryptamine, 1 µM spiperone, and 50 nM me-
sulergine in 50 mM Tris-HCl buffer, pH 7.4, supplemented
with 4 mM CaCl2 and 10 µM pargyline. Nonspecific binding
was determined in the presence of 10 µM 5-HT.
Ligh t/Da r k Test. The anxiolytic-like activity of the com-
pounds was tested using an unconditioned conflict test, the
light/dark test, behaviorally validated for detecting antianxiety
agents in mice.40,42
In brief, the apparatus consisted of two poly(vinyl chloride)
tools covered by plexiglass. One of these boxes was darkened,
and the other was lightened by a lamp. Mice were placed in
the lit box at the beginning of the test session. The amount
of time spent by mice in the lit box (TLB) and the number of
transitions through the tunnel between the two boxes were
recorded over a 5-min period, after the first entry into the dark
box. A mouse with all four paws in the new box was
considered as having changed box. Tested compounds were
administered per os from 0.1 µg/kg to 1 mg/kg. The lack of
sedative or excitatory effects of the compounds at the tested
doses was previously assessed in a free exploratory test. The
statistical significance of differences between control and
treated groups was ascertained by a combined analysis of
variance and a Bonferroni’s posteriori t-test.
5-HT2A receptor binding to bovine frontal cortex membranes
was determined as described by Leysen et al.50 Membranes
(0.6 mg of protein/mL) were incubated at 37 °C for 30 min with
0.8 nM [3H]ketanserin and 100 nM WB4101 in 50 mM Tris-
HCl buffer, pH 7.4, supplemented with 5 mM MgCl2, 10 mM
NaCl, 0.5 mM EDTA, and 10 µM pargyline. Nonspecific
binding was determined in the presence of 10 µM spiperone.
5-HT2C receptor binding to pig choroid plexus membranes
was determined following a slight adaptation of the protocol

1818 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12
Prunier et al.
pyrazine. (Pyrrolothienopyrazines: chemistry of pyrrolo[1,2-a]-
thieno[2,3-e] pyrazine.) J . Heterocycl. Chem. 1986, 23, 17-23.
(23) Effi, Y.; Lancelot, J . C.; Rault, S.; Robba M. Pyrrolothie´no-
pyrazines. IV. Synthe`se de de´rive´s de l’amino-5 pyrrolo[1,2-a]
thie´no[2,3-e]pyrazine. (Pyrrolothienopyrazines. IV. Synthesis of
5-aminopyrrolo[1,2-a]thieno[2,3-e]pyrazine derivatives.) J . Het-
erocycl. Chem. 1987, 24, 431-435.
(24) Rault, S.; Cugnon de Sevricourt, M.; Nguyen-Huy, D.; Robba M.
Pyrrolo[1,2-a]thieno[3,2-e]pyrazines. J . Heterocycl. Chem. 1981,
18, 739-742.
(25) Rault, S.; Lancelot, J . C.; Pilo, J . C.; Robba, M.; Renard, P.;
Guardiola, B.; Adam, G. New pyrrolothienopyrazine compounds,
processes for the preparation thereof and pharmaceutical com-
positions containing them. EP 573360, December 8, 1993.
(26) Robba, M.; Rault, S.; Lancelot, J . C.; Prunier, H.; Guardiola, B.;
Renard, P.; Adam, G. New pyrrolopyrazine compounds, processes
for their preparation and pharmaceutical compositions contain-
ing them. EP 623620, November 9, 1994.
Refer en ces
(1) Martin, G. R.; Humphrey, P. P. A. Classification Review Recep-
tors for 5-hydroxytryptamine: Current Perspectives on Clas-
sification and Nomenclature. Neuropharmacology 1994, 33,
261-273.
(2) Hoyer, D.; Clarke, D. E.; Fozard, J . R.; Hartig, P. R.; Martin, G.
R.; Mylecharane, E. J .; Saxena, P. R.; Humphrey P. P. A. VII
International Union of Pharmacology Classification of Receptors
for 5-Hydroxytryptamine (Serotonin). Pharmacol. Rev. 1994, 46,
157-203.
(3) Zifa, E.; Fillion, G. 5-Hydroxytryptamine Receptors. Pharmacol.
Rev. 1992, 44, 401-458.
(4) Kilpatrick, G. J .; Bunce, K. T.; Tyers, M. B. 5-HT3 Receptors.
Med. Res. Rev. 1990, 10, 441-475.
(5) Aapro, M. S. 5-HT3 receptor antagonists. An overview of their
present status and future potential in cancer therapy-induced
emesis. Drugs 1991, 42, 551-568.
(6) Greenshaw, A. J . Behavioural pharmacology of 5-HT3 receptor
antagonists : a critical update on therapeutic potential. Trends
Pharmacol. Sci. 1993, 14, 265-270.
(7) Sanger, G. J .; Nelson, G. R. Selective and functional 5-hydroxy-
tryptamine receptor antagonism by BRL 43694 (Granisetron).
Eur. J . Pharmacol. 1989, 159, 113-124.
(8) Butler, A.; Hill, J . M.; Ireland, S. J .; J ordan, C. C.; Myers, M. B.
Pharmacological Properties of GR 3032F, a novel antagonist at
5-HT3 receptors. Br. J . Pharmacol. 1988, 94, 387-412.
(9) Richardson, B. P.; Engel, G.; Donatsch, P.; Stadler, P. A.
Identification of serotonin m-receptor subtypes and their specific
blockade by a new class of drugs. Nature 1985, 316, 126-131.
(10) Higgins, G. A.; J oharchi, N.; Sellers E. M. Behavioural Effects
of the 5-Hydroxytryptamine3 Receptor Agonists 1-phenyl bigu-
anide and m-chlorophenylbiguanide in Rats. J . Pharmacol. Exp.
Ther. 1993, 264, 1440-1449.
(27) Nagarajan, K.; Ranga Gao, V.; Venkateswarlu, A. Condensed
heterotricycles: pyrrolo[1,2-a]quinoxalines derivatives. Indian
J . Chem. 1972, 10, 344-350.
(28) Campiani, G.; Nacci, V.; Corelli, F.; Anzini, M. Polycondensed
heterocycles. VII . A convenient synthesis of pyrrolo[1,2-a]-
quinoxaline derivatives by intramolecular aromatic nucleophilic
displacement. Synth. Commun. 1991, 21, 1567-1576.
(29) Clauson-Kaas, N.; Tyle, Z. Preparation of cis and trans 2,5-
dimethoxy-2-(acetamidomethyl)-2,5-dihydrofuran, of cis and trans
2.5-dimethoxy-2-(acetamidomethyl)-tetrahydrofuran and of
1-phenyl-2-(acetamidomethyl)-pyrrole. Acta Chem. Scand. 1952,
6, 667-670.
(30) Elming, N.; Clauson-Kaas, N. The preparation of pyrroles from
furans. Acta Chem. Scand. 1952, 6, 867-874.
(31) Be´champ, A. J . Ann. Chim. Phys. 1854, 42, 186.
(32) Wertheim, E. o-aminobenzenesulfonic acid. Org. Synth. 1949,
II, 16-18.
(33) Smith, L. I.; Opie, J . W. o-Aminobenzaldehyde. Org. Synth. 1955,
III, 56.
(34) Lancelot, J . C.; Rault, S.; Ladure´e, D.; Robba M. Pyrido[3,2-e]-
pyrrolo[1,2-a]pyrazine. Chem. Pharm. Bull. 1985, 33, 2798-
2802.
(35) J osey, A. D.; J enner, E. L. N-functionnally substituted pyrroles.
J . Org. Chem. 1962, 27, 2466-2470.
(36) Raines, S.; Chai, S. Y.; Palopoli, F. P. Mannich reactions.
Synthesis of 4,5-dihydropyrrolo[1,2-a]quinoxalines, 2,3,4,5-tet-
rahydro-1H-pyrrolo[1,2-a][1,4]diazepines and 5,6-dihydro-4H-
pyrrolo[1,2-a][1,4]diazepines. J . Heterocycl. Chem. 1976, 13,
711-716.
(37) Yevich, J . P.; New, J . S.; Smith, D. W.; Lobeck, W. G.; Catt, J .
D.; Minielli, J . L.; Eison, M. S.; Taylor, D. P.; Riblet, L. A.;
Temple, D. L., J r. Synthesis and biological evaluation of 1-(1,2-
benzisothiazol-3-yl)- and (1,2-benzisoxazol-3-yl)piperazine de-
rivatives as potential antipsychotic agents. J . Med. Chem. 1986,
29, 359-369.
(38) Merce´, R. Frigola, J .; Pare´s, J . Process for the preparation of
aryl (or hetaryl)piperazinylbutylazole derivatives. AU 9.211.429;
CA 20.662.468; EP 502.786; ES 2.036.145; FR 2.673.628; J P
93.078.313; NO 9.200.088; US 5.227.486; ZA 9.201.682A.
(39) Lancelot, J . C.; Rault, S.; Nguyen Huy, D.; Robba M. Pyrido-
[3,2-h]pyrrolo[1,2-a]quinoxalines. Chem. Pharm. Bull. 1983, 31,
3160-3167.
(40) Misslin, R.; Belzung, C.; Vogel, E. Behavioural validation of a
light/dark choice procedure for testing anti-anxiety agents.
Behav. Process. 1989, 18, 119-132.
(41) Costall, B.; J ones, B. J .; Kelly, M. E.; Naylor, R. J .; Tomkins, D.
M. Exploration of mice in black and white test box : validation
as a model of anxiety. Pharmacol. Biochem. Behav. 1989, 32,
777-785.
(42) J ones, B. J .; Costall, B.; Domeney, A. M.; Kelly, M. E.; Naylor,
R. J .; Oakley, N. R.; Tyers, M. B. The potential anxiolytic activity
of GR38032F, a 5-HT3 receptor antagonist. Br. J . Pharmacol.
1988, 93, 985-993.
(43) Bonhaus, D. W.; Wong, E. H. F.; Stefanich, E.; Kunysz, E. A.;
Eglen, R. M. Pharmacological characterization of 5-hydroxy-
tryptamine3 receptors in murine brain and ileum using the novel
radioligand [3H]RS-42358-197: evidence for receptor heteroge-
neity.5-HT1B and 5-HT1C binding sites in rat frontal cortex. J .
Neurochem. 1993, 61,1927.
(44) Sepulveda, M. I.; Lummis, S. C. R.; Martin, I. L. The agonist
properties of m-chlorophenylbiguanide and 2-methyl-5-hydroxy-
tryptamine on receptors in N1E-115 neuroblastoma cells. Br.
J . Pharmacol. 1991, 104, 536.
(45) Morain, P. C.; Abraham, C.; Portevin B.; De Nanteuil, G.
Biguanide derivatives: agonist pharmacology at 5-hydroxy-
tryptamine type 3 receptors in vitro. Mol. Pharmacol. 1994, 46,
732.
(11) Kilpatrick, G. J .; Butler, A.; Burridge, J .; Oxford A. W. 1-(m-
chlorophenyl)-biguanide, a potent high affinity 5-HT3 receptor
agonist. Eur. J . Pharmacol. 1990, 182, 193-197.
(12) Barnes, J . M.; Barnes N. M. Differential binding characteristics
of agonists at 5-HT3 receptor recognition sites in NG 108-15
neuroblastoma-glioma cells labelled by [3H]-(S)-zacopride and
[3H] granisetron. Biochem. Pharmacol. 1993, 45, 2155-2158.
(13) Sharif, N. A.; Wong, E. H. F.; Loury, D. N.; Stefanich, E.; Michel,
A. D.; Eglen, R. M.; Whiting R. L. Characteristics of 5-HT3
binding sites in NG 108-15, NCB-20 neuroblastoma cells and
rat cerebral cortex using [3H]-quipazine and [3H]-GR 65630
binding. Br. J . Pharmacol. 1991, 102, 919-925.
(14) Glennon, R. A.; Ismael A. M.; Mc Carthy, B. G.; Peroutka S. J .
Binding of arylpiperazines to 5-HT3 serotonin receptors: results
of a structure-affinity study. Eur. J . Pharmacol. 1989, 168, 387-
392.
(15) Emerit, M. B.; Riad, M.; Fattaccini, C. M.; Hamon M. Charac-
teristics of [14C]Guanidinium Accumulation in NG 108-15 Cells
Exposed to Serotonin 5-HT3 Receptor Ligands and Substance
P. J . Neurochem. 1993, 60, 2059-2061.
(16) Rault, S.; Lancelot, J . C.; Prunier, H.; Robba, M.; Renard, P.;
Delagrange, P.; Pfeiffer, B.; Caignard, D. H.; Guardiola-Lemaˆı-
tre, B.; Hamon M. Novel selective and partial agonists of 5-HT3
receptors. Part 1. Synthesis and biological evaluation of piper-
azinopyrrolothienopyrazines. J . Med. Chem. 1996, 39, 2068-
2080.
(17) Bureau, R.; Lancelot, J . C.; Prunier, H.; Rault, S. Conformational
analysis and 3D QSAR study on novel partial agonists of 5-HT3
receptors. Quant. Struct.-Act. Relat. 1996, 15, 373-381.
(18) Rault, S.; Cugnon de Sevricourt, M.; Robba M. New condensed
triheterocyclic systems: pyrrolothienopyrazines. Heterocycles
1980, 14, 651.
(19) Rault, S.; Effi, Y.; Cugnon de Sevricourt, M.; Lancelot, J . C.;
Robba, M. Pyrrolothie´nopyrazines: synthe`se de la pyrrolo[1,2-
a]thie´no[3,2-e]pyrazine et de la pyrrolo[1,2-a]thie´no[2,3-e]pyr-
azine. (Pyrrolothienopyrazines: synthesis of pyrrolo[1,2-a]thieno-
[3,2-e]pyrazine and pyrrolo[1,2-a]thieno[2,3-e]pyrazine.) J . Het-
erocycl. Chem. 1983, 20, 17-21.
(20) Effi, Y.; Lancelot, J . C.; Rault, S.; Robba M. Pyrrolothie´no-
pyrazines: e´tude de l’oxo-5 pyrrolo[1,2-a]thie´no[2,3-e]pyrazine.
(Pyrrolo[1,2-a]thieno[2,3-e]pyrazines: chemical study of 4,5-
dihydropyrrolo[1,2-a]thieno[2,3-e]pyrazin-5-one.) J . Heterocycl.
Chem. 1983, 20, 913-918.
(21) Rioult, J . P.; Cugnon de Sevricourt, M.; Rault, S.; Robba M.
Pyrrolothie´nopyrazines: e´tude chimique de la pyrrolo[1,2-a]-
thie´no[3,2-e] pyrazine. (Pyrrolothienopyrazines: Chemical study
of pyrrolo[1,2-a]thieno[3,2-e] pyrazine.) J . Heterocycl. Chem.
1984, 21, 1449.
(22) Effi, Y.; Lancelot, J . C.; Rault, S.; Robba M. Pyrrolothie´no-
pyrazines: e´tude chimique de la pyrrolo[1,2-a]thie´no[2,3-e]

Novel and Selective Partial Agonists of 5-HT3 Receptors
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 12 1819
(46) Delagrange, P.; Emerit, M. B.; Merahi, N.; Abraham, C.; Morain,
P.; Rault, S.; Renard, P.; Pfeiffer, B.; Guardiola-Lemaˆıtre, B.;
Hamon, M. Interaction of S 21007 with 5-HT3 receptors. In vitro
and in vivo characterization. Eur. J . Pharmacol., in press.
(47) Hoyer, D.; Engel, G.; Kalkman, H. O. Molecular pharmacology
of 5-HT1 and 5-HT2 recognition sites in rat and pig brain
membranes: radioligand binding studies with [3H]-8-OH-DPAT,
(50) Leysen, J . E.; Niemegeers, C. J . E.; Van Nueten, J . M.; Laduron,
P. M. [3H]-ketanserin (R 41468),
a
selective 3H-ligand for
serotonin 2 receptor binding sites; binding properties, brain
distribution and functional role. Mol. Pharmacol. 1982, 21, 301-
314.
(51) Sanders-Bush, E.; Breeding, M. Putative selective 5-HT2 an-
tagonists block serotonin 5-HT1C receptors in the choroid plexus.
J . Pharmacol. Exp. Ther. 1988, 247, 169-173.
(52) Hoyer, D.; Neijt, H. C. Identification of serotonin 5-HT3 recogni-
tion sites by radioligand binding in NG108-15 neuroblastoma-
glioma cells. Eur. J . Pharmacol. 1987, 143, 291-292.
[
125I]-iodocyanopindolol, [3H]-mesulergine, [3H]-ketanserin. Eur.
J . Pharmacol. 1985, 118, 13-23.
(48) Peroutka, S. J . Pharmacological differentiation and characteriza-
tion of 5-HT1A, 5-HT1B and 5-HT1C binding sites in rat frontal
cortex. J . Neurochem. 1986, 47, 529-540.
(49) Waeber, C.; Schoeffter, P.; Hoyer, D.; Palacios, J . M. The
serotonin 5-HT1D receptor: A progress review. Neurochem. Res.
1990, 15, 567-582.
J M960501O