Novel and highly potent 5-HT3 receptor agonists based on a pyrroloquinoxaline structure

Article abstract of DOI:10.1021/jm970376w

The synthesis and the biological evaluation of a series of novel pyrroloquinoxaline derivatives are described. In binding studies several compounds proved to be potent and selective 5-HT₃ receptor ligands. The most active pyrroloquinoxalines, 1

Full text of DOI:10.1021/jm970376w

3670
J . Med. Chem. 1997, 40, 3670-3678
Novel a n d High ly P oten t 5-HT3 Recep tor Agon ists Ba sed on a
P yr r oloqu in oxa lin e Str u ctu r e†
Giuseppe Campiani,‡ Andrea Cappelli,‡ Vito Nacci,*,‡ Maurizio Anzini,‡ Salvatore Vomero,‡ Michel Hamon,§
Alfredo Cagnotto,| Claudia Fracasso,| Chiara Uboldi,| Silvio Caccia,| Silvana Consolo,| and Tiziana Mennini|
Dipartimento Farmaco Chimico Tecnologico, Universita´ di Siena, Banchi di Sotto 55, 53100 Siena, Italy,
Neurobiologie Cellulaire et Functionnelle, Istitut National de la Sante´ et de la Recherche Medicale (INSERM) U.288,
Faculte´ de Medicine Pitie´-Salpetriere 91, boulevard del’ Hopital, 75634 Paris, Cedex 13, France,
and Istituto di Ricerche Farmacologiche “Mario Negri”, via Eritrea 62, 20127 Milano, Italy
Received J une 9, 1997X
The synthesis and the biological evaluation of a series of novel pyrroloquinoxaline derivatives
are described. In binding studies several compounds proved to be potent and selective 5-HT3
receptor ligands. The most active pyrroloquinoxalines, 11d and 11e, showed a subnanomolar
affinity for 5-HT3 receptor and were able to functionally discriminate the central and peripheral
5-HT3 receptors, being agonists and antagonists, respectively. In functional studies ([14C]-
guanidinium accumulation test in NG 108-15 cells, in vitro) most of the synthesized compounds
showed clear-cut 5-HT3 agonist properties. In in vivo studies on the von Bezold-J arisch reflex
test (a peripheral interaction model) the behavior of the tested compounds ranged from agonist
to antagonist, while clear agonist properties were obtained with 12a on cortical acetylcholine
release in freely moving rats. Pharmacokinetic studies with 11e and 12c indicate that the
compounds easily cross the blood-brain barrier (BBB) after systemic administration with a
brain/plasma ratio of 17.5 and 37.5, respectively. Thus compounds 11e and 12c represent the
most potent central 5-HT3 agonists identified to date that are able to cross the blood-brain
barrier.
Ch a r t 1
In tr od u ction
Serotonin (5-hydroxytryptamine, 5-HT) is a neu-
rotransmitter involved in many biological processes at
both the central and peripheral level, and its receptors
are classified into several subtypes. Among the differ-
ent subtypes, the 5-HT3 receptor has attracted consider-
able interest in the last decade, especially with the
synthesis of potent and selective antagonists, such as
granisetron and ondansetron. On the contrary, few and
poorly selective agonists have been developed: among
them the 2-methyl-5-HT, 1 (Chart 1), is an agonist
rapidly metabolized in vivo and (likewise phenylbigu-
anide derivatives) is unable to cross the blood-brain
barrier (BBB).1 Thus its usefulness is largely restricted
to in vitro models. Quipazine, 2, has been reported to
behave as a 5-HT3 receptor antagonist in peripheral
models,2 but subsequent studies revealed its 5-HT3
agonism in [14C]guanidinium accumulation in NG 108-
15 cells.3 This pharmacological property, taken together
with the fact that the 5-HT3 receptors on NG 108-15
cells and on rat cerebral cortex show common pharma-
cological and physiochemical properties,4a makes qui-
pazine a useful lead structure4b for designing more
potent and selective central 5-HT3 agonists. The po-
tential therapeutic role of 5-HT3 receptor agonists is
based on their modulation of acetylcholine release in
vivo,5 which makes these compounds of interest for the
treatment of neurodegenerative and neuropsychiatric
disorders in which cholinergic neurons are affected.
Consequently, there is increasing and considerable
interest in the development of potent and selective
5-HT3 receptor agonists. In order to design novel 5-HT3
receptor agonists, the structure of quipazine has been
modified by the introduction of the lipophilic pyrrole
ring on the quinoline system. However, this structural
alteration could lead to compounds interacting with
other 5-HT receptor subtypes (e.g. 5-HT1B), as in the
case of CGS 12066B (3).6 We previously described a
novel synthetic approach to pyrroloquinoxaline deriva-
tives7 (CGS 12066B 3 and related analogues), and later
preliminary and unpublished studies revealed that the
electron-withdrawing and bulky trifluoromethyl group
of 3 was deleterious for 5-HT3 receptor affinity, while
its des-CF3 analogue showed a subnanomolar affinity
for the same 5-HT receptor subtype. These results
stimulated our interest in the development of a series
of compounds with potent and selective central 5-HT3
* To whom correspondence should be addressed.
† In honor of Professor Raffaele Giuliano’s 85th birthday.
‡ Dipartimento Farmaco Chimico Tecnologico, Universita´ di Siena.
§ INSERM, Paris.
| Istituto di Ricerche Farmacologiche “Mario Negri”, Milano.
X Abstract published in Advance ACS Abstracts, September 15, 1997.
S0022-2623(97)00376-2 CCC: $14.00 © 1997 American Chemical Society

Novel and Highly Potent 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3671
Sch em e 1
while the intermediate 2-chloroquinoxaline (10) was
prepared according to a literature procedure.11a The
appropriate anilines (5a ,b) bearing a fluorine atom at
position 2 as a leaving group were chosen as starting
materials. These latter were subjected to the Clauson-
Kaas reaction to give the corresponding arylpyrroles
6a ,b, which were transformed into 1-aryl-2-cyanopyr-
roles 7a ,b through a one-pot sequence involving formi-
lation (replacing POCl3 with oxalyl chloride7), oximation,
and dehydration. The procedure also furnished small
amounts of the isomeric 3-nitriles. The nitriles 7a ,b
were successively cyclized to the pyrrolo[1,2-a]quinox-
alinones 8a ,b by treatment with potassium hydroxide
in ethylene glycol at 145 °C. This one-pot transforma-
tion of nitriles into lactams involves selective hydrolysis
to amides, which are able to carry out intramolecular
displacement of aromatic fluorine. By treatment with
phosphoryl chloride, 8a ,b were transformed into the
4-chloro derivatives 9a ,b. The chloro derivatives 9a -e
and 10 were then reacted with excess of N-methylpip-
erazine to give 11a -e, while the piperazine derivatives
13a -h were synthesized by alkylation with the ap-
propriate alkyl halides of 12a -d , in turn prepared by
reaction of 9b-e with an excess of piperazine. Phisical
and chemical data of the described compounds are
reported in Table 1.
Resu lts a n d Discu ssion
1. 5-HT3 Bin d in g SAR Stu d y.11b The affinities of
the new pyrroloquinoxaline derivatives 3, 11a -e, 12a ,c,
and 13a -h for the 5-HT3 receptors in rat cortex homo-
genate are illustrated in Table 2. The binding data
represent the ability of the tested compounds to displace
[3H]BRL 43694 from the receptor protein. Binding data
of quipazine is also included. In Table 3 the affinity of
selected compounds on different 5-HT receptor subtypes
is reported. The results show that the introduction of
a lipophilic pyrrole moiety on the c-edge of the quinoline
system of quipazine led to more potent 5-HT3 receptor
ligands (e.g. 11d ), maintaining the same selectivity
ratios versus the other 5-HT receptor subtypes, as for
quipazine, and showing (11d ) an improved selectivity
vs 5-HT1B and the serotonin uptake site. On the
contrary, the presence of an extra nitrogen atom on the
quipazine structure diminished significantly the recep-
tor affinity (11c). These data are consistent with
hypothesized favorable lipophilic interactions of the
pyrrole portion with the corresponding area in the
receptor protein. Since the introduction of the pyrrole
moiety was revealed to be crucial for 5-HT3 receptor
potency and for selectivity, to further identify structural
requirements capable of modulating the affinity and
selectivity, the SAR study was carried out as a function
of (i) the position of a fluorine atom in the benzo-fused
ring and (ii) the nature and hindrance of the alkyl chain
at position 4 in the piperazine ring.
receptor agonism, based on a pyrroloquinoxaline skel-
eton (4). While this work was in progress, related
compounds have been reported in literature,8 but none
of the quinoxalines and thieno derivatives described
show central full agonist properties. So, in the present
article we report the development of potent central
5-HT3 full agonists. The effect of the introduction of a
fluorine atom in the benzo-fused ring with respect to
the 5-HT3 selectivity has also been explored, and potent
and selective agonists have been developed. The po-
tential 5-HT3 receptor agonist properties were evaluated
in vitro in [14C]guanidinium accumulation test on NG
108-15 cells,3 and in vivo on the von Bezold-J arisch
reflex test9 (these data provide further support to
functional properties of the 5-HT3 receptors being dif-
ferent in cardiac tissue and in NG 108-15 cells) and on
acetylcholine release in frontal cortex of freely moving
rats which is modulated by 5-HT3 receptors.10a In
addition, brain and plasma concentrations of 11e and
12c were simultaneously measured, after systemic
injection, using a newly developed HPLC procedure with
UV detection.
(i) With respect to the unsubstituted pyrroloquinoxa-
line 11d (5-HT3 binding IC50 ) 0.37 nM), the introduc-
tion of a bulky trifluoromethyl group at position 7 (CGS
12066B, 3) plays a key role in the 5-HT receptor subtype
selectivity.12 In fact, compound 3 is devoid of any 5-HT3
receptor affinity, while 11d shows a subnanomolar
Ch em istr y
5-HT3 receptor affinity and an affinity for 5-HT1A
,
The new 4-chloropyrrolo[1,2-a]quinoxalines 9a ,b were
synthesized as shown in Scheme 1, according to the
procedure utilized to prepare already known 9c-e,7
5-HT2A receptors in the higher nanomolar range. On
the other hand, since the trifluoromethyl group is
deleterious for 5-HT3 affinity (11d vs CGS 12066B, 3),

3672 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Ta ble 1. Physical and Chemical Data for Compounds 6-13
Campiani et al.
compd
R
R′
X
% yielda
mp (°C)
recryst/solvent
formula
analysisb
3c
6a
6b
7a
7b
8a
8b
9a
7-CF3
3-F
6-F
3-F
6-F
6-F
9-F
6-F
9-F
H
7-F
7-CF3
6-F
9-F
H
pyrrolo
76
83
54
79
94
80
95
72
C
C
C
C
C
C
C
C
10H7F2N
10H7F2N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
76-77
49
77-78
265-266
273-274
201-202
183-184
hexanes
hexanes
hexanes
EtOAc
EtOAc
hexanes
hexanes
11H6F2N2
11H6F2N2
11H7FN2O
11H7FN2O
11H6ClFN2
11H6ClFN2
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
9b
9cc
9d c
9ec
11a
11b
11c
11d c
11ec
12a
12b
12c
12d
13a
13b
13c
13d
13e
13f
13g
13h
97
85
80
C
C
C
16H17FN4
16H17FN4
13H16N4
C,H,N
C,H,N
C,H,N
108-109
107-108
hexanes
hexanes
H
pyrrolo
pyrrolo
7-F
9-F
H
7-F
7-CF3
9-F
9-F
H
H
H
7-F
7-F
7-CF3
91
94
128-129
147-148
130-131
154-155
48-49
EtOAc
EtOAc
hexanes
hexanes
hexanes
hexanes
C
C
C
15H15FN4
15H16N4
15H15FN4
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C16H15F3N4
n-propyl
allyl
ethyl
n-propyl
allyl
n-propyl
allyl
74
84
87
81
73
78
81
69
C
C
C
C
C
C
C
18H21FN4
18H19FN4
17H20N4
18H22N4
18H20N4
44-45
69-70
53-54
97-98
hexanes
hexanes
pentane
18H21FN4
18H19FN4
allyl
C19H19F3N4
a
b
Yields refer to isolated and purified materials. All the compounds were analyzed within (0.4% of the theoretical values. c Reference
7.
Ta ble 2. 5-HT3 Receptor Affinity and Functional Behavior of Compounds 3, 11a -e, 12a ,c, and 13a -h
effect on [14C]guanidinium
uptake on NG 108-15 cells
von Bezold-J arisch
reflex test
IC50 (nM)a
((SEM)
NAc
9.5 ( 1
0.44 ( 0.05
22 ( 3
compd
R
R′
X
efficacyb
EC50 (nM)
behaviorb
ID50 (µg/kg iv)
3
11a
11b
11c
11d
11e
12a
12c
13a
13b
13c
13d
13e
13f
13g
13h
quipazine
7-CF3
6-F
9-F
H
Me
Me
Me
Me
Me
Me
H
pyrrolo
pyrrolo
pyrrolo
NA
A
A
A
A
A
A
A
A
A
A
A
A
A
A
NA
A
Ant
PA
PA
A
300
120
120
4.7 ( 0.8
6.5 ( 1
510 ( 120
10 ( 3
60-120d
H
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
pyrrolo
0.37 ( 0.06
0.81 ( 0.05
3.9 ( 0.4
5.4 ( 0.8
82 ( 23
28 ( 3
Ant
Ant
Ant
Ant
PA
PA
Ant
PA
PA
PA
PA
Ant
300
600
70
10
300
75
100
240
180
200
100
240
7-F
9-F
7-F
9-F
9-F
H
H
H
7-F
7-F
7-CF3
3.0 ( 0.4
98 ( 25
3.5 ( 0.6
73 ( 15
30 ( 4
H
n-propyl
allyl
ethyl
n-propyl
allyl
n-propyl
allyl
9 ( 0.5
8.0 ( 1
51 ( 3
8.5 ( 2
11 ( 1
8.2 ( 2
88 ( 15
22 ( 2
580 ( 110
76 ( 12
allyl
NAc
6.2 ( 0.7
25 ( 5
a
Each value is the mean ( SEM of three determinations and represent the concentration giving half-maximal inhibition of [3H]BRL43694
binding to rat cortical homogenate. A ) pure agonist. PA ) partial agonist. Ant ) pure antagonist. NA ) not active. c NA at 10 000
nM. ED50
b
d
.
the effect of a smaller fluorine substituent in the benzo-
fused ring was evaluated. The introduction of a fluorine
atom at positions 7 and 9 led to potent 5-HT3 receptor
ligands, with equal affinity for the unsubstituted 11d
(11b and 11e vs 11d ), while a 6-F substituent was found
to decrease the affinity (11a vs 11d ). Furthermore, the
presence of a fluorine atom at position 7 slightly improve
the selectivity: in particular 11e shows a reduced
5-HT2A affinity (11e vs 11d ).
(ii) Among different N-substituents, the methyl group
was found to be optimal for 5-HT3 receptor affinity.
Removal of the methyl group led to slightly less potent
pyrroloquinoxaline derivatives (12a ,c vs 11b,e), while
increasing the hindrance with larger lipophilic chains

Novel and Highly Potent 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3673
Ta ble 3. Binding Profile of Compounds 11d ,e and 12a ,c on 5-HT Receptor Subtypes
IC50 (nM)a ((SEM)
d
e
f
g
h
i
compd
R
R′
5-HT3
5-HT1A
5-HT1B
5-HT2A
5-HT2C
5-HT4
5-HTuptake
quipazine
6.2 ( 0.7
2700 ( 235
(435)
89 ( 7
(240)
138 ( 14
(170)
268 ( 46
(69)
380 ( 31
(61)
232 ( 24
(627)
371 ( 58
(458)
106 ( 13
(27)
1400 ( 138
(226)
43 ( 18
(116)
124 ( 45
(153)
761 ( 237
(135)
489 ( 278
(90)
2350 ( 352
3.1 ( 0.4
470 ( 42
>10000
(>1613)
>10000
(>27027)
>10000
(>12345)
>10000
(>2564)
>10000
(>1852)
147 ( 25
204 ( 27
(33)
214 ( 34
(578)
232 ( 20
(286)
1129 ( 295
(289)
(76)
11d
11e
12a
12c
H
Me
Me
H
0.37 ( 0.06b
0.81 ( 0.05b
3.9 ( 0.4b
780 ( 320
(2108)
413 ( 134
(510)
784 ( 132
(201)
2803 ( 694
(519)
42 ( 1
7-F
9-F
7-F
H
5.4 ( 0.8b
151 ( 24
(28)
2.6 ( 0.4
167 ( 23
(31)
4.6 ( 0.5
203 ( 61
(37)
369 ( 80
5-HT
727 ( 200c
methysergide
mesulergine
GR 38032
2-Me-5-HT
2.13 ( 0.38
11 ( 2c
779 ( 171b
a
The concentration of the tested compounds that inhibited the radiolabelled ligand binding to rat cortex homogenate by 50% (IC50
)
was determined by dose-inhibition curves with six concentrations of the displacers, each performed in triplicate. Each value in parentheses
is the selectivity ratio calculated as the ratio between the IC50 value for the indicated 5-HT receptor subtype over the IC50 value for 5-HT3
b
receptor. Values are the mean ( SE of at least three separate experiments performed in triplicate. [3H]BRL 43694. c [3H]Zacopride.
d
g
h
i
[3H]8-OH-DPAT. e [3H]5-HT. f [3H]Ketanserin. [3H]Mesulergine. [3H]GR 113808. [3H]Citalopram.
such as ethyl, propyl, and allyl gave a progressive drop
in 5-HT3 receptor affinity, despite Rault’s observations
on analogous pyrrolothienopyrazines.8b
the 5-HT3 receptor-dependent von Bezold-J arisch reflex
in urethane-anesthetized rats9 proved a different be-
havior for the tested compounds ranging from the
agonist properties of 11c to the full antagonist proper-
ties of 11d ,e, 12a ,c, and 13c,h through the partial
agonist properties of compounds 11a ,b and 13a ,b,d -
g.
2. Biologica l Activity. In addition to binding
studies, two functional assays were carried out for
assessing the potential agonist/antagonist activity of the
whole set of pyrroloquinoxaline derivatives. These two
different 5-HT3-dependent responses were the uptake
of [14C]guanidinium, which is markedly increased upon
the activation of 5-HT3 receptors on the neuroblastoma-
glioma hybrid NG 108-15 cells, and the von Bezold-
J arish reflex, which corresponds to a deep but transient
bradycardia triggered by the stimulation of cardiac
5-HT3 receptors. The most intriguing result risen up
from these pharmacological experiments was that most
of the described compounds mimicked the effects of
5-HT on the 5-HT3 receptor-dependent accumulation of
[14C]guanidinium in NG 108-15 hybridoma cells3 up to
concentrations in the low nanomolar range, indicating
that the novel pyrroloquinoxaline derivatives acted as
potent agonists in this assay. Similarly to that observed
in binding experiments, the introduction of a pyrrole
ring at the c-edge of the quinoline system of a quipazine
improved the potency (11d ) in guanidinium uptake test,
while the introduction of a nitrogen atom drammatically
reduced the potency in this functional test (11c). It is
noteworthy that the most active members of this series
of central 5-HT3 receptor agonists show a fluorine atom
at position 7 (11e and 12c), and they appear to be the
most potent agonists, in this test, to have been described
until to date. Furthermore a good correlation of the
binding data (cerebral cortex) and of the EC50 values
([14C]guanidinium uptake) was found in this series of
central 5-HT3 agonists, whereas the poor correlation
shown by 11d might be due to the complex kinetics of
its interaction with 5-HT3 receptors in NG 108-15 cells
(data not shown). On the other hand investigations on
Accordingly, compound 11c (agonist) (at 60-120 µg/
kg, iv) produced a transient bradycardia which was as
pronunced as that observed after the iv injection of 40
µg/kg of phenylbiguanide. At the same doses, 11c only
exerted marginal effects on the bradycardia due to the
latter drug, indicating that its potential antagonist
properties at cardiac 5-HT3 receptor are most negligible.
On the contrary, a slight bradycardia was observed in
rats which had been treated with 10-100 µg/kg iv of
compound 12c alone. However, this effect did not
exhibit a clear dependence on the dose. In contrast, the
inhibition by 12c, representative of the antagonists, of
phenylbiguanide-induced bradicardia was clearly dose-
dependent with an ID50 value close to 10 µg/kg iv. These
data led to the conclusion that 12c is a potent antagonist
at cardiac 5-HT3 receptors. In addition, it had a rather
long duration of action since a reduction in the cardiac
response to phenylbiguanide was still observed 1 h after
administration of 100 µg/kg iv of 12c.
Quinoxaline 11a , representative of the partial agonist
compounds, at the dose of 60 µg/kg iv, induces a
transient bradycardia which is about 65% of that due
to 40 µg/kg iv of the potent 5-HT3 receptor agonist
phenylbiguanide. In addition to this agonist effect, 11a
also exerts an antagonist action since it can prevent
phenylbiguanide-induced bradycardia. In this case the
effect of 11a is clearly dose-dependent, with half-
blockade at 0.12 mg/kg iv, and maximal blockade at 0.4
mg/kg iv when this drug is injected 5 min before

3674 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Campiani et al.
F igu r e 2. Relationship between the 11e and 12c dose and
mean cortical concentrations at 60 min after dosing. Each
point is the mean ((SD) of four animals for 11e (b) and 12c
(O).
or 250 µg sc did not affect by itself the ACh content of
frontal cortex perfusates during the entire 200-min
collection period, but at 250 µg icv, it completely
prevented the lowering effect of compound 12a . At the
lower dose 30 µg icv, ondansetron elicited only a partial
antagonism.
2. Br a in Distr ibu tion Stu d ies. The previously
known 5-HT3 agonists 2-Me-5-HT and phenylbiguanide
derivatives do not cross the BBB, thus making difficult
their use by systemic route administration. In order
to demonstrate the brain accumulation of our com-
pounds, we studied the brain and plasma concentrations
of two representative compounds of our series, 11e and
12c. Previous kinetic studies with arylpiperazines
indicated that in the rat most of these compounds are
rapidly adsorbed and eliminated with half-lives of about
1 h. Therefore in these preliminary studies the brain-
to-plasma partition ratio was investigated 1 h after
dosing, when presumably pseudoequilibrium will be
achieved for both the investigated compounds. As
quipazine and structurally related compounds,13 these
new pyrroloquinoxaline derivatives rapidly enter the rat
brain, achieving concentrations higher than in the
plasma. Figure 2 illustrates the relationships between
the dose and the mean cortical concentrations of 11e
and 12c measured 60 min after dosing. The mean brain
concentration of the two compounds increased from the
lowest to the highest dose, although the proportion was
better for 11e (r2 ) 0.91) than for 12c (r2 ) 0.73). The
mean brain concentrations of 12c were approximately
4 times those of 11e, although there was variability in
brain (and plasma) concentrations of both compounds,
but particularly with 12c at the higher doses. At 10
mg/kg, for instance, the coefficient of variation (CV) was
36% compared to a 16% CV for 11e brain concentrations
(0.98 ( 0.16 µg/g) at the same dose. With both com-
pounds the brain drug concentrations were a linear
function of plasma drug concentrations (Figure 3), with
12c having a larger brain to plasma ratio (brain
concentration ) 38.5-0.01 plasma concentration; r2 )
0.95) than 11e (brain concentration ) 18.5-0.04 plasma
concentration; r2 ) 0.89). There was no evidence of
preferential concentration of the two compounds in any
F igu r e 1. Effect of 12a and ondansetron, alone or together,
on ACh release in rat frontal cortex. Ondansetron at the dose
of 60 µg sc (A) or 250 µg sc (B) was administered after collection
of four base-line fractions, 20-min each fraction. 12a (30 nmol
icv per side) was given 20 min after ondansetron. For each
time point, data are means ( SEM (vertical bars) (n ) 4),
expressed as picomoles of ACh released/20 min. Split-plot
ANOVA followed by Turkey’s test for unconfounded means
revealed a significant difference in ACh release between 12a
(in A and B) group, and the [ondansetron + 12a ] group (in A)
with the veichle group (*P < 0.05; *P < 0.01), and a significant
interaction between [12a ] vs [ondansetron + 12a ] groups.
phenylbiguanide. So, we concluded that 11a is a mixed
agonist-antagonist at peripheral 5-HT3 receptors.
The different behavior observed in the functional
studies suggested that the tested compounds are able
to functionally discriminate between the central and the
peripheral 5-HT3 receptors (e.g. 12c behaved as a potent
full agonist on [14C]guanidinium accumulation test and
as a potent full antagonist in von Bezold-J arisch reflex
test).
In Vivo Stu d ies. 1. Acetylch olin e Relea se. In
previous studies we have shown that 2-Me-5-HT, given
icv, reduces acetylcholine release in rat cortex by a
mechanism which is blocked by ondansetron.10a Thus,
for comparison, we have studied the effect of 12a using
the same treatment schedule as for 2-Me-5-HT. The
5-HT3 receptor agonist 12a (as hydrochloride) reduced
the cortical extracellular ACh content of freely moving
rats (Figure 1A,B). Its inhibitory effect reached its
nadir of 34% below the basal values at 100 min after
its administration. This effect was quantitatively simi-
lar to the effect obtained with 1-2 µmol icv of 2-Me-5-
HT.10a However, for a direct comparison of the in vivo
potencies of the two 5-HT3 agonists, a full response-
dose study is in progress with 12a . Ondansetron, a
selective 5-HT3 serotoninergic antagonist, at 60 µg sc

Novel and Highly Potent 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3675
mL). After the mixture was stirred for 15 min at room
temperature and cooled in a ice-water bath, a solution of 6a
(1.05 g, 5.9 mmol) in anhydrous 1,2-dichloroethane (10 mL)
was slowly added. The reaction mixture was allowed to stir
at room temperature for 4 h, and then a warm solution of
hydroxylamine hydrochloride (0.43 g, 6.2 mmol) and dry
sodium acetate (0.51 g, 6.2 mmol) in anhydrous N,N-dimeth-
ylformamide (10 mL) was slowly added. After the mixture was
refluxed for 20 h, acetic anhydride (2.3 mL, 24.4 mmol) was
added and the solution was refluxed for 4 h. The cooled
mixture was treated with 10% sodium carbonate solution until
pH 8, diluted with water (50 mL), and extracted with ethyl
acetate. The organic layer was washed with brine, dried, and
concentrated. The residue was chromatographed (EtOAc) to
give 0.65 g of 7a as colorless prisms: IR (CHCl3) 2221 cm-1
;
1H NMR (CDCl3) δ 6.42 (m, 1 H), 7.04-7.09 (m, 2 H), 7.17-
7.37 (m, 3 H). Anal. (C11H6F2N2) C, H, N.
F igu r e 3. Relationship between plasma and brain (cortex)
concentrations. Each point represents the plasma and brain
concentrations of 11e (A) and 12c (B) for an individual rat.
1-(2,6-Diflu or op h en yl)-1H-p yr r ole-2-ca r bon itr ile (7b).
Starting from 6b, the title compound was obtained as colorless
prisms: IR (CHCl3) 2220 cm-1; 1H NMR (CDCl3) δ 6.41 (m, 1
H), 7.10 (m, 4 H), 7.45 (m, 1 H). Anal. (C11H6F2N2) C, H, N.
of the brain region examined. One hour after dosing,
the concentrations in hippocampus, striatum, and hy-
pothalamus were comparable to those in cortex (data
not shown).
6-F lu or op yr r olo[1,2-a ]qu in oxa lin -4(5H)-on e (8a ).
A
suspension of 7a (0.2 g, 1.0 mmol) and 85% potassium
hydroxide (0.2 g, 4.0 mmol) in 8 mL of tert-butyl alcohol was
heated at 80 °C for 2 h under argon. The mixture was then
cooled, poured into crushed ice, and extracted with ethyl
acetate. The organic layer was washed with brine, dried, and
concentrated. The residue was chromatographed (ethyl ac-
etate) to afford 0.18 g of 8a as a white solid: IR (Nujol) 1670
Con clu sion s
By appropriate structural modification of the quino-
line system of quipazine, this work has resulted in the
discovery of more potent and selective 5-HT3 receptor
ligands. By virtue of their agonist potencies displayed
in [14C]guanidinium accumulation test, on in vivo
acetylcholine release, and of their ability to cross the
blood-brain barrier, these pyrroloquinoxalines repre-
sent potent pharmacological tools to explore the central
5-HT3 receptor-mediated functions.
1
cm-1; H NMR (DMSO-d6) δ 6.71 (t, 1 H, J ) 3.4 Hz), 7.01-
7.22 (m, 2 H), 7.31 (d, 1 H, J ) 3.9 Hz), 7.44 (d, 1 H, J ) 8.8
Hz), 7.66 (d, 1 H, J ) 2.9 Hz), 8.31 (br s, 1 H). Anal. (C11H7-
FN2O) C, H, N.
9-Flu or opyr r olo[1,2-a ]qu in oxalin -4(5H)-on e (8b). Simi-
larly to 8a , the lactam 8b was prepared starting from 7b, and
using ethylene glycol as solvent. 8b was obtained as a white
solid: IR (Nujol) 1665 cm-1; 1H NMR (DMSO-d6) δ 6.73 (m, 1
H), 7.21 (m, 4 H), 7.33 (m, 1 H), 8.00 (s, 1 H). Anal. (C11H7-
FN2O) C, H, N.
Exp er im en ta l P r oced u r es
4-Ch lor o-6-flu or opyr r olo[1,2-a ]qu in oxalin e (9a). A mix-
ture of 8a (0.3 g, 1.5 mmol) and phosphorus oxychloride (4 mL)
was refluxed for 4 h under argon, cooled, poured into crushed
ice, and extracted with dichloromethane. The organic layers
were washed with brine, dried, and concentrated. The residue
was chromatographed (dichloromethane) and recrystallized to
Melting points were determined using an Electrothermal
8103 apparatus and are uncorrected. IR spectra were taken
with Perkin-Elmer 398 and FT 1600 spectrophotometers. 1H-
NMR spectra were recorded on a Bruker 200 MHz spectrom-
eter with TMS as internal standard; the values of chemical
shifts (δ) are given in ppm and coupling constants (J ) in hertz.
All reactions were carried out in an argon atmosphere.
Progress of the reaction was monitored by TLC on silica gel
plates (Riedel-de-Haen, Art. 37341). Merck silica gel (Kieselgel
60) was used for chromatography (70-230 mesh) and flash
chromatography (230-400 mesh) columns. Extracts were
dried over MgSO4, and solvents were removed under reduced
pressure. Elemental analyses were performed on a Perkin-
Elmer 240C elemental analyzer, and the results are within
(0.4% of the theoretical values, unless otherwise noted. Yields
refer to purified products and are not optimized. Physical data
for compounds 3 and 6-13 are reported in Table 1.
give 0.31 g of 9a as colorless prisms: IR (CHCl3) 3320 cm-1
;
1H NMR (CDCl3) δ 6.93 (m, 1 H), 7.10-7.25 (m, 2 H), 7.43-
7.65 (m, 2 H), 7.95 (d, 1 H, J ) 3.1 Hz). Anal. (C11H6ClFN2)
C, H, N.
4-Ch lor o-9-flu or op yr r olo[1,2-a ]qu in oxa lin e (9b). Simi-
larly to 9a , the chloro derivative 9b was prepared starting from
8b. 9b was obtained as a white solid: IR (Nujol) 3300 cm-1
;
1H NMR (CDCl3) δ 6.83 (m, 1 H), 7.21-7.33 (m, 2 H), 7.65 (m,
3 H). Anal. (C11H6ClFN2) C, H, N.
6-F lu or o-4-(4-m eth ylp ip er a zin -1-yl)p yr r olo[1,2-a ]qu i-
n oxa lin e (11a ). A mixture of 9a (0.2 g, 0.91 mmol) and
N-methylpiperazine (2 mL) was heated at 130 °C for 2 h, under
argon, cooled, poured into crushed ice, and extracted with ethyl
ether. The combined organic extracts were washed with brine,
dried, and concentrated. The residue was chromatographed
(EtOAc) to give 0.25 g of 11a as a yellow oil: IR (CHCl3) 1599
cm-1; 1H NMR (CDCl3) δ 2.38 (s, 3 H), 2.62 (m, 4 H), 3.90 (m,
4 H), 6.77 (t, 1 H, J ) 3.4 Hz), 6.83 (d, 1 H, J ) 3.7 Hz), 7.02-
7.22 (m, 2 H), 7.50 (d, 1 H, J ) 7.8 Hz), 7.78 (d, 1 H, J ) 2.7
Hz). Anal. (C16H17FN4) C, H, N.
1-(2,3-Diflu or op h en yl)-1H-p yr r ole (6a ). To a solution of
2,3-difluoroaniline 5a (1.0 g, 7.7 mmol) in glacial acetic acid
(5 mL) was slowly added 2,5-dimethoxytetrahydrofuran (1.0
mL, 7.8 mmol). The reaction mixture was refluxed for 1 h,
cooled, and evaporated to afford an oily residue which was
chromatographed (EtOAc) to give 1.05 g of 6a as a colorless
1
oil: IR (CHCl3) 3420 cm-1; H NMR (CDCl3) δ 6.37 (m, 1 H),
7.00-7.24 (m, 5 H). Anal. (C10H7F2N) C, H, N.
9-F lu or o-4-(4-m eth ylp ip er a zin -1-yl)p yr r olo[1,2-a ]qu i-
n oxa lin e (11b). Similarly to the procedure as described for
11a , the title compound was prepared starting from 9b. After
recrystallization 11b was obtained as a white solid: IR
(CHCl3): 1600 cm-1; 1H NMR (CDCl3) δ 2.37 (s, 3 H), 2.61 (m,
1-(2,6-Diflu or op h en yl)-1H-p yr r ole (6b). Similarly to 6a ,
the pyrrole derivative 6b was prepared starting from 2,6-
difluoroaniline 5b. 6b was obtained as a colorless oil: bp 132
1
°C/0.2 mmHg; IR (neat) 3390 cm-1; H NMR (CDCl3) δ 6.36
(m, 2 H), 6.89-7.08 (m, 4 H), 7.20 (m, 1 H). Anal. (C10H7F2N)
C, H, N.
4 H), 3.85 (m, 4 H), 7.07 (m, 5 H), 8.17 (m, 1 H). Anal. (C16H17
FN4) C, H, N.
-
1-(2,3-Diflu or op h en yl)-1H-p yr r ole-2-ca r bon itr ile (7a ).
To a cooled (-5 °C) mixture of anhydrous N,N-dimethylfor-
mamide (0.48 mL, 6.2 mmol) and anhydrous 1,2-dichloroet-
hane (3 mL) was slowly added within 20 min a solution of
oxalyl chloride (0.54 mL, 6.2 mmol) in 1,2-dichloroethane (3
2-(4-Meth ylp ip er a zin -1-yl)qu in oxa lin e (11c). Starting
from 10 the title compound was obtained as for 11a . After
recrystallization 11c was obtained as red-orange prisms. IR
1
(CHCl3) 1609 cm-1; H NMR (CDCl3) δ 2.37 (s, 3 H), 2.57 (m,

3676 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Campiani et al.
4 H), 3.82 (m, 4 H), 7.39 (t, 1 H, J ) 7.3 Hz), 7.57 (t, 1 H, J )
7.3 Hz), 7.68 (d, 1 H, J ) 8.4 Hz), 7.87 (d, 1 H, J ) 8.3 Hz),
8.58 (s, 1 H). Anal. (C13H16N4) C, H, N.
H, J ) 7.3 Hz), 1.48-1.67 (m, 2 H), 2.40 (m, 2 H), 2.64 (t, 4 H,
J ) 5.0 Hz), 3.84 (t, 4 H, J ) 5.0 Hz), 6.77 (m, 2 H), 7.22-7.36
(m, 2 H), 7.64-7.74 (m, 2 H), 7.80 (d, 1 H, J ) 2.8 Hz). Anal.
(C18H22N4) C, H, N.
Gen er a l P r oced u r e for P r ep a r a t ion of Com p ou n d s
12a -d . This procedure is illustrated for the preparation of
4-(4-Allylpiper azin -1-yl)pyr r olo[1,2-a ]qu in oxalin e (13e).
Starting from 12b the title compound was obtained as a pale
yellow oil following a procedure as described for 13a : IR
9-fluoro-4-(piperazin-1-yl)pyrrolo[1,2-a]quinoxaline (12a ).
A
mixture of 9b (1.0 g, 4.5 mmol) and dry piperazine (4.0 g, 46.0
mmol) in ethylene glycol (40 mL) was heated at 140 °C for 2
h under argon. After cooling the mixture was poured into
crushed ice and extracted with chloroform. The organic layers
were washed with brine, dried, and concentrated. The residue
was chromatographed (EtOAc) to give 1.12 g of 12a as colorless
(Nujol) 1599 cm-1 1H NMR (CDCl3) δ 2.66 (t, 4 H, J ) 5.0
;
Hz), 3.09 (d, 2 H, J ) 6.8 Hz), 3.84 (t, 4 H, J ) 4.9 Hz), 5.17-
5.28 (m, 2 H), 5.84-6.03 (m, 1 H), 6.77 (m, 2 H), 7.28 (m, 2
H), 7.70 (m, 2 H), 7.80 (m, 1 H). Anal. (C18H20N4) C, H, N.
7-Flu or o-4-(4-n -pr opylpiper azin -1-yl)pyr r olo[1,2-a ]qu i-
n oxa lin e (13f). Similarly to the procedure as described for
13a , the title compound was prepared starting from 12c and
n-propyl bromide. After recrystallization 13f was obtained as
1
prisms: IR (CHCl3) 3320 cm-1; H NMR (CDCl3) δ 3.08 (t, 4
H, J ) 4.9 Hz), 3.78 (t, 4 H, J ) 4.9 Hz), 6.76 (t, 1 H, J ) 3.8
Hz), 6.83 (d, 1 H, J ) 3.9 Hz), 6.96-7.07 (m, 1 H), 7.15-7.26
(m, 1 H), 7.44 (d, 1 H, J ) 7.8 Hz), 8.18 (d, 1 H, J ) 2.3 Hz).
Anal. (C15H15FN4) C, H, N.
1
colorless prisms: IR (CHCl3) 1599 cm-1; H NMR (CDCl3) δ
0.93 (t, 3 H, J ) 7.4 Hz), 1.55 (m, 2 H), 2.37 (m, 2 H), 2.61 (m,
4 H), 3.85 (m, 4 H), 6.74 (m, 2 H), 6.93 (dt, 1 H, J ) 2.8, 8.5
Hz), 7.30 (dd, 1 H, J ) 2.5, 9.4 Hz), 7.61 (m, 1 H), 7.72 (m, 1
H). Anal. (C18H21FN4) C, H, N.
4-(P iper azin -1-yl)pyr r olo[1,2-a ]qu in oxalin e (12b). Start-
ing from 9c the title compound was obtained following a
procedure as for 12a . After recrystallization 12b was obtained
1
as colorless prisms: IR (CHCl3) 1580 cm-1; H NMR (CDCl3)
4-(4-Allylp ip er a zin -1-yl)-7-flu or op yr r olo[1,2-a ]qu in ox-
a lin e (13g). Similarly to the procedure as described for 13a ,
the title compound was prepared starting from 12c and allyl
bromide. After recrystallization 13g was obtained as colorless
prisms: IR (CHCl3) 1599 cm-1; 1H NMR (CDCl3) δ 2.61 (m, 4
H), 3.08 (d, 2 H, J ) 6.8 Hz), 3.87 (m, 4 H), 5.22 (m, 2 H), 5.91
(m, 1 H), 6.77 (m, 2 H), 6.94 (m, 1 H), 7.31 (dd, 1 H, J ) 2.9,
δ 3.13 (t, 4 H, J ) 5.0 Hz), 3.81 (t, 4 H, J ) 5.1 Hz), 6.77 (m,
2 H), 7.26-7.36 (m, 2 H), 7.65-7.75 (m, 2 H), 7.82 (m, 1 H),
8.20 (d, 1 H, J ) 2.3 Hz). Anal. (C15H16N4) C, H, N.
7-F lu or o-4-(p ip er a zin -1-yl)p yr r olo[1,2-a ]q u in oxa lin e
(12c). Similarly to the procedure as described for 12a , the
title compound was prepared starting from 9d . After recrys-
tallization 12c was obtained as a white solid: IR (CHCl3) 1609
cm-1; 1H NMR (CDCl3) δ 3.06 (m, 4 H), 3.80 (m, 4 H), 6.75 (m,
2 H), 6.95 (dt, 1 H, J ) 2.4, 8.5 Hz), 7.32 (dd, 1 H, J ) 2.4,
10.6 Hz), 7.64 (m, 2 H), 7.76 (d, 1 H, J ) 3.2 Hz). Anal.
(C15H15FN4) C, H, N.
9.9 Hz), 7.63 (m, 1 H), 7.73 (d, 1 H, J ) 2.7 Hz). Anal. (C18H19
FN4) C, H, N.
-
4-(4-Allylp ip er a zin -1-yl)-7-(t r iflu or om et h yl)p yr r olo-
[1,2-a ]qu in oxa lin e (13h ). Similarly to the procedure as
described for 13a , the title compound was prepared starting
from 12d and allyl bromide. After recrystallization 13h was
4-(P iper azin -1-yl)-7-(tr iflu or om eth yl)pyr r olo[2,1-a ]qu i-
n oxa lin e (12d ). Similarly to the procedure as described for
12a , the title compound was prepared starting from 9e. After
recrystallization 12d was obtained as a yellowish solid: IR
obtained as yellow prisms: IR (CHCl3) 1602 cm-1 1H NMR
;
(CDCl3) δ 2.64 (m, 4 H), 3.08 (d, 2 H, J ) 6.5 Hz), 3.93 (m, 4
H), 5.23 (m, 2 H), 5.95 (m, 1 H), 6.80 (m, 2 H), 7.52 (d, 1 H, J
) 8.1 Hz), 7.68 (d, 1 H, J ) 8.4 Hz), 7.84 (m, 1 H), 7.93 (m, 1
H). Anal. (C19H19F3N4) C, H, N.
1
(Nujol) 1600 cm-1; H NMR (CDCl3) δ 3.07 (m, 4 H), 3.87 (m,
4 H), 6.80 (m, 2 H), 7.52 (dd, 1 H, J ) 1.5, 8.7 Hz), 7.68 (d, 1
H, J ) 8.4 Hz), 7.84 (d, 1 H, J ) 2.7 Hz), 7.93 (m, 1 H). Anal.
(C16H15F3N4) C, H, N.
Micr od ia lysis a n d Acetylch olin e Assa y. Under Equith-
esin anesthesia (1% pentobarbital, 4% chloral hydrate; 3 mL/
kg ip), male CD/COBS rats (Charles River, Calco, Italy)
weighing 200-250 g were implanted with AN 69 dialysis fibers
(310 µm o.d.; Dasco Bologna), inserted transversally through
both frontal cortices according to the following coordinates: A,
+0.5 mm from bregma and V, -2.8 mm from the occipital
bone.10b The procedure used to prepare and implant the
dialysis tube was previously described.10c,d At the end of the
release experiments the placement of the dialysis probe was
verified histologically. A polyethylene cannula, 4 mm long,
was inserted into each lateral ventricle for intracerebroven-
tricular (icv) injections of the drug. The coordinates used were
as follows: AP, -1.5 mm; L, (1.5 mm from bregma. The
average in vitro recovery of ACh through three dialysis tubes
was 67.4 ( 1.7% for a probe 8 mm long. On the day after the
implantation, the dialysis tube was perfused at a constant rate
of 2 µL/min with Ringer’s solution (147 mM NaCl, 2.2 mM
CaCl2, and 4.0 mM KCl), containing 5 µM physostigmine, and
adjusted to pH 7.0 with NaOH. The perfusate was discarded
during the first 30-min equilibration period and then collected
at 20-min intervals in small ice-cooled polyethylene test tubes
containing 10 µL of 0.05 mM HCl to prevent ACh hydrolysis.
After an 80-min period of perfusion to allow the ACh output
to reach a steady baseline, saline or drugs were administered
systemically or through chronically implanted cannulae. At
the end of the collection, the perfusate samples were im-
mediately frozen on dry ice and lyophilized. ACh content of
the dialysate was quantified by a specific and sensitive
radioenzymatic method as previously described.10c,d
Gen er a l P r oced u r e for P r ep a r a t ion of Com p ou n d s
13a -h . This procedure is illustrated for the preparation of
9-fluoro-4-(4-n-propylpiperazin-1-yl)pyrrolo[1,2-a ]quinoxa-
line (13a ). A mixture of 12a (0.26 g, 0.96 mmol), anhydrous
potassium carbonate (0.13 g, 0.96 mmol), and n-propyl iodide
(93.5 µL, 0.96 mmol) in ethyl methyl ketone (40 mL) was
heated at reflux for 3 h under argon. The solvent was
evaporated, and the residue was partitioned between water
and dichloromethane. The organic layer was washed with
brine, dried, and concentrated. The residue was chromato-
graphed (EtOAc) to give 0.23 g of 13a as a white solid: IR
(Nujol) 1600 cm-1
;
1H NMR (CDCl3) δ 0.95 (t, 3 H, J ) 7.3
Hz), 1.65 (m, 2 H), 2.45 (m, 2 H), 2.71 (t, 4 H, J ) 4.7 Hz),
3.91 (t, 4 H, J ) 4.9 Hz), 6.76 (t, 1 H, J ) 3.4 Hz), 6.83 (d, 1
H, J ) 3.9 Hz), 7.00 (m, 1 H), 7.20 (m, 1 H), 7.44 (d, 1 H, J )
8.3 Hz), 8.18 (d, 1 H, J ) 2.1 Hz). Anal. (C18H21FN4) C, H, N.
4-(4-Allylp ip er a zin -1-yl)-9-flu or op yr r olo[1,2-a ]qu in ox-
a lin e (13b). Starting from 12a the title compound was
obtained as a colorless oil following a procedure as described
1
for 13a : IR (Nujol) 1590 cm-1; H NMR (CDCl3) δ 2.71 (t, 4
H, J ) 4.9 Hz), 3.14 (d, 2 H, J ) 6.4 Hz), 3.90 (t, 4 H, J ) 4.9
Hz), 5.28 (m, 2 H), 5.90 (m, 1 H), 6.76 (t, 1 H, J ) 3.4 Hz),
6.82 (d, 1 H, J ) 4.0 Hz), 7.00 (m, 1 H), 7.25 (m, 1 H), 7.44 (d,
1 H, J ) 8.4 Hz), 8.18 (d, 1 H, J ) 2.7 Hz). Anal. (C18H19
FN4) C, H, N.
-
4-(4-Eth ylpiper azin -1-yl)pyr r olo[1,2-a ]qu in oxalin e (13c).
Starting from 12b the title compound was obtained as a pale
yellow oil following a procedure as described for 13a : IR
(Nujol) 1590 cm-1 1H NMR (CDCl3) δ 1.15 (t, 3 H, J ) 7.1
;
Hz), 2.51 (q, 2 H, J ) 7.2 Hz), 2.65 (t, 4 H, J ) 5.0 Hz), 3.85
(t, 4 H, J ) 4.9 Hz), 6.77 (m, 2 H), 7.25 (m, 2 H), 7.68 (m, 2 H),
7.81 (d, 1 H, J ) 2.4 Hz). Anal. (C17H20N4) C, H, N.
Dr u gs. All reagents were of analytical grade. Drugs and
reagents used in this study and their sources were as follows:
[3H]acetylcoenzyme-A (2.5-4.3 Ci/mmol) from Amersham
(Bucks, U.K.); physostigmine sulfate (M.W. 324.4), acetylcho-
linesterase (EC 3.1.1.7, type V-S), and choline kinase (EC
2.7.1.32) were purchased from Sigma Chemical Co. (St. Louis,
MO). None of the drugs used interfere with the ACh assay.
4-(4-n -P r op ylp ip e r a zin -1-yl)p yr r olo[1,2-a ]q u in oxa -
lin e (13d ). Starting from 12b the title compound was
obtained as a pale yellow oil following a procedure as described
1
for 13a : IR (Nujol) 1590 cm-1; H NMR (CDCl3) δ 0.94 (t, 3

Novel and Highly Potent 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22 3677
Sta tistica l An a lysis. Statistical analysis was done on the
values calculated as pmol/20 min, not corrected for the in vitro
recovery of the mycrodialysis probe. Analysis of variance
(split-plot design) followed by a multiple comparison test
(Turkey’s test for unconfounded means) was used.
HCl and then 0.5 mL of 0.4 M NaOH, which were added to
the vials. Each mixture (1.5 mL) was supplemented with 10
mL of the scintillation fluid Aquasol (New England Nuclear,
Les Ulis, France) for radioactivity counting at 50% efficiency.
All assays were performed in triplicate.
Under these conditions, [14C]guanidinium accumulation in
NG 108-15 cells was 4-5 times higher in the presence of both
1 mM 5-HT and 10 mM substance P than in their absence
(basal uptake). 5-HT3 receptor antagonists (zacopride, on-
dansetron, tropisetron, etc.) completely prevented the stimu-
latory effect of 5-HT (with substance P) (see ref 3 for details).
von Bezold -J a r isch Reflex. The stimulation of cardiac
5-HT3 receptors is well-known to trigger a transient brady-
cardia known as the von Bezold-J arisch reflex9 in urethane-
anesthetized rats. The ability of drugs to either induce or
prevent this reflex has been used to further assess the 5-HT3
receptor agonist or antagonist properties of the newly synthe-
sized arylpiperazine derivatives. Briefly, adult male Sprague-
Dawley rats (250-300 g body weight, Centre d’Elevage R.
J anvier, Le Genest, France) were anesthetized with urethane
(1.4 g/kg ip), and a tracheotomy was performed to insert an
endotracheal tube. A catheter (0.3 mm internal diameter) was
inserted into the abdominal aorta via the femoral artery in
order to record the arterial pressure and heart rate. A femoral
vein was exposed and cannulated for iv injection of drugs. The
von Bezold-J arisch reflex (which consists of a 60% drop in
heart rate within 10-15 s following the injection of 30 mg/kg
iv of serotonin) was assessed 5, 15, and 30 min after the iv
administration of various doses of each arylpiperazine deriva-
tive. Under these conditions, 10 mg/kg iv of either zacopride,
ondansetron, or tropisetron injected 5 min before 5-HT com-
pletely prevented the bradycardia normally evoked by the
indoleamine.9
Dr u g Ad m in istr a tion a n d P la sm a a n d Br a in Sa m -
p lin g. Male Sprague-Dawley rats (Charles River, Italy)
weighing 175-200 g were used. Procedures involving animals
and their care were conducted in conformity with the institu-
tional guidelines that are in compliance with national and
international laws and policies (ECC Concil Directive 86/609,
OJ L 358, 1, Dec; 12, 1987; NIH Guide for the Care and Use
of Laboratory Animals, NIH publication No. 85-23, 1985).
Animals were injected intraperitoneally with 11e or 12c (as
hydrochloride salts, 2.5, 5, and 10 mg/kg) dissolved in saline
and were killed 1 h thereafter. Blood samples were collected
in heparinized tubes and were centrifuged, and the plasma
was stored at -20 °C. Brains were removed immediately after
exanguination, and brain areas were blotted with paper to
remove excess surface blood and stored at -20 °C until
analysis.
Dr u g An a lysis. Plasma and brain concentrations of the
two piperazine derivatives 11e and 12c were determined by
high-performance liquid chromatography (HPLC) with ultra-
violet (UV) detection at 254 nm.
Briefly, 1 mL of heparin-treated plasma or brain homoge-
nate (0.1 N HCl, 10 mL/g) were basified (pH 9-10) by using 1
N NaOH and extracted twice with 3 mL of n-hexane/isoamylic
alcohol (98:2 v/v), after adding one of the two compounds as
an internal standard. After centrifugation the organic extracts
were transferred to test tubes containing 0.2 mL of 0.01 M
phosphoric acid and were shaken for 15 min. After centrifuga-
tion the organic phase was discarded and the acidic aqueous
phase was injected into the chromatographic column. Separa-
tion was done on a µBondapack C18 column (30 cm × 3.9 mm
i.d., 10 µM particle size), at room temperature. The mobile
phase was 0.01 M KH2PO4:CH3CN:CH3OH (69:28:3, v/v)
delivered isocratically at a flow rate of 1.1 mL/min. The
retention times were 7.8 min for 12c and 9.8 min for 11e.
Daily standard curves containing known concentrations of
the analytes (from 0.01 to 0.25 µg/mL for plasma and from
0.025 to 0.75 µg/mL for brain tissue) were analyzed concur-
rently with each of the unknown samples and quality control
samples. The relationships between the peak height of the
compounds to the internal standard and of the amount of the
compounds added were always linear, with coefficients of
correlation exceeding 0.99. Replicate analysis at two different
concentrations yielded mean coefficients of variation of 5-8%
and 10-15% in plasma and brain tissue for both compounds.
Mea su r em en t of [14C]Gu a n id in iu m Up ta k e in NG 108-
15 Cells. This procedure has been described by Emerit et al.3
as a convenient assay for assessing the agonist/antagonist
activity of drugs acting at 5-HT3 receptors. Thus, 5-HT3
receptor agonists markedly enhance [14C]guanidinium uptake
by these cells, and this response is selectively blocked by 5-HT3
receptor antagonists.3 Briefly, mouse neuroblastoma × rat
glioma hybrid cells of the NG 108-15 clone were grown in
Dulbecco’s modified Eagle’s medium supplemented with the
appropriate nutrients3 for 2 days. The cell layer in each
culture dish (35 mm) was then 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, 20 mM HEPES, pH
adjusted to 7.4 with NaOH), and covered with 1 mL of buffer
B (same composition as buffer A except that [NaCl] was
reduced to 135 mM and 10 mM guanidinium were added)
containing 0.20-0.25 mCi of [14C]guanidinium (s.a. 59 mCi/
mmol, Service des Mole´cules Marque´es at CEA, 91191 Gif-
surYvette, France) and, where indicated, 1 mM 5-HT, 10 mM
substance P, and/or eight different concentrations of each drug
to be tested. After 10 min at 37 °C, the assay 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 dissolved in 0.5 mL of 0.4 M NaOH, and
the resulting extracts were transferred to scintillation vials.
The culture dishes were further rinsed with 0.5 mL of 1 M
In Vitr o Bin d in g Assa ys. Binding assays have been
performed as described in ref 4b.
[3H]BRL 43694 Bin d in g to Ra t Cor tica l Mem br a n es.
Male CRL:CD(SD)BR-COBS rats were killed by decapitation;
their cortex were rapidly removed and stored at -80 °C until
the day of assay. The frozen tissues were homogenized in
about 50 volumes of ice-cold Hepes HCl, 50 mM, pH 7.4 using
an Ultra Turrax TP 1810 homogenizer (2 × 20 s) and
centrifuged at 50000g for 10 min (Beckman model J -21 B
refrigerate centrifuged). The pellet was resuspended in the
same volume of fresh buffer, incubated at 37 °C for 10 min,
and centrifuged again at 50000g for 10 min. The pellet was
then washed once by resuspension in fresh buffer and centri-
fuged as before. The pellet obtained was finally resuspended
in Hepes HCl, 50 mM, pH 7.4 containing 10 µM pargyline.
[3H]BRL 43694 binding was done as previously described5
in a final incubation volume of 1 mL consisting of 0.5 mL of
membrane suspension (20 mg tissue/sample), 0.5 mL of [3H]-
BRL 43694 (s.a. 83.5 Ci/mmol, final concentration 1 nM), and
20 µL of displacing agents or solvent. Incubation (30 min at
25 °C) was stopped by rapid filtration in vacuo (Brandell MR
48R) through GF/B filters which were then washed with 12
mL of cold buffer and counted in a Wallac 1204 betaplate BS
liquid scintillation counter with a counting efficiency of 45%.
Dose-inhibition curves were analyzed by the “Allifit” program11b
to obtain the concentration of unlabeled drug that caused 50%
inhibition of ligand binding.
Ack n ow led gm en t. This work was supported by
grant from MURST (60%) and CNR, Roma.
Refer en ces
(1) Kilpatrick, G. J .; Bunce, K. T.; Tyers, M. B. 5-HT3 Receptors.
Med. Res. Rev. 1990, 10, 441-475 and references cited therein.
(2) (a) Ireland, S. J .; Tyers, M. B. Pharmacological Characterization
of 5-Hydroxytriptamine-Induced Depolarization of the Rat Iso-
lated Vagus Nerve. Br. J . Pharmacol. 1987, 90, 229-238. (b)
Round, A.; Wallis, D. I. Further Studies on the Blockade of 5-HT
Depolarizations of Rabbit Vagal Afferent and Sympathetic
Ganglion Cells by MDL 72222 and Other Antagonists. Neurop-
harmacology 1987, 26, 39-48.
(3) 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, 2059-2067.

3678 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 22
Campiani et al.
(4) (a) Bolanos, F. J .; Schechter, L. E.; Miquel, M. C.; Emerit, M.
B.; Rumigny, J . F.; Hamon, M.; Gozlan, H. Common Pharma-
cological and Physicochemical Properties of 5-HT3 Binding Sites
in the Rat Cerebral Cortex and NG 108-15 Clonal Cells.
Biochem. Pharmacol. 1990, 40, 1541-1550. (b) Anzini, M.;
Cappelli, A.; Vomero, S.; Giorgi, G.; Langer, T.; Hamon, M.;
Merahi, N.; Emerit, B. M.; Cagnotto, A.; Skorupska, M.; Mennini,
T.; Pinto, J . C. Novel Potent and Selective 5-HT3 Receptor
Antagonists Based on the Arylpiperazine Skeleton: Synthesis,
Structure, Biological Activity, and Comparative Molecular Field
Analysis Studies. J . Med. Chem. 1995, 38, 2692-2704.
(5) Consolo, S.; Bertorelli, R.; Russi, G.; Zambelli, M.; Ladinsky, H.
Serotoninergic Facilitation of Acetylcholine Release in Vivo from
Rat Dorsal Hippocampus Via Serotonin 5-HT3 Receptors. J .
Neurochem. 1994, 62, 2254-2261.
(9) Cohen, M. L.; Bloomquist, W.; Gidda, J . S.; Lacefield, W.
Comparison of the 5-HT3 Receptor Antagonist Properties of ICS
205-930, GR38032F and Zacopride. J . Pharmacol. Exp. Ther.
1989, 248, 197-201.
(10) (a) Ladinsky, H.; Borsini, F.; Cortes, R.; Palacios, J . M.; Giorgi,
S.; Russi, G.; Consolo, S. Serotonin-3 Receptors Control Inhibi-
tion of Acetylcholine Release from Frontal Cortex and Facilita-
tion from Dorsal Hippocampus: a Microdialysis Study with
2-Methylserotonin and DAU 6215. Abs. Neurosci. 1993, 19,
724-725. (b) Paxinos, G.; Watson, C. The Rat Brain in Stereo-
taxic Coordinates; Academic Press: Sidney, 1982. (c) Wu, C.-
F.; Bertorelli, R.; Sacconi, M.; Pepeu, G.; Consolo, S. Decrease
of Brain Acetylcholine Release in Aging Freely-Moving Rats
Detected by Microdialysis. Neurobiol. Aging 1988, 9, 357-361.
(d) Consolo, S.; Wu, C.-F.; Fiorentini, F.; Ladinsky, H.; Vezzani,
A. M. Determination of Endogenous Acetylcholine Release in
Freely Moving Rats by Transstriatal Dialysis Coupled to a
Radioenzymatic Assay: Effect of Drugs. J . Neurochem. 1987,
48, 1459-1465.
(6) Neale, R. F.; Fallon, S. L.; Boyar, W. C.; Wasley, J . W. F.; Martin,
L. L.; Stone, G. A.; Glaeser, B. S.; Sinton, C. M.; Williams, M.
Biochemical and Pharmacological Characterization of CGS
12066B a Selective Serotonin-1B Agonist. Eur. J . Pharmacol.
1987, 136, 1-9.
(7) Campiani, G.; Nacci, V.; Corelli, F.; Anzini, M. Polycondensed
Heterocycles. VII. A Convenient Synthesis of Pyrrolo[1,2-a]-
quinoxaline Derivatives by Intramolecular Aromatic Nucleo-
philic Displacement. Synth. Commun. 1991, 21, 1567-1576.
(8) (a) Lancelot, G.; Prunier, H.; Robba, M.; Delagrange, P.; Renard,
P.; Adam, G. Preparation of Pyrrolopyrazines as 5-HT3 Ligands.
Eur. Pat. Appl. EP623,620, 1994; Chem. Abstr. 1995, 122,
105922. (b) Rault, S.; Lancelot, G.; Prunier, H.; Robba, M.;
Renard, P.; Delagrange, P.; Pfeiffer, B.; Caignard, D.; Guardiola-
Lemaitre, B.; Hamon, M. Novel Selective and Partial Agonists
of 5-HT3 Receptors. Part 1. Synthesis and Biological Evaluation
of Piperazinopyrrolothienopyrazines. J . Med. Chem. 1996, 39,
2068-2080. (c) Prunier, H.; Rault, S.; Lancelot, J .-C.; Robba,
M.; Renard, P.; Delagrange, P.; Pfeiffer, B.; Caignard, D.-H.;
Misslin, R.; Guardiola-Lemaitre, B.; Hamon, M. Novel and
Selective Partial Agonists of 5-HT3 Receptors. 2. Synthesis and
Biological Evaluation of Piperazinopyridopyrrolopyrazines, Pip-
erazinopyrroloquinoxalines, and Piperazinopyridopyrroloqui-
noxalines. J . Med. Chem. 1997, 40, 1808-1819.
(11) (a) Makino, K.; Yoshioka, H. Facile Synthesis and Herbicidal
Activities of Novel Fluoroquinoxalines. Heterocycles 1987, 26,
1215-1220. (b) De Lean, K. W.; Munson, P. J .; Rodbard, D.
Simultaneous Analysis of Families of Sigmoidal Curves: Ap-
plication to Bioassay, Radioligand Assay, and Physiological Dose-
Response Curves. Am. J . Physiol. 1978, 235, E97-E102.
(12) Macor, J . E.; Burkhart, C. A.; Heym, J . H.; Ives, J . L.; Lebel, L.
A.; Newman, M. E.; Nielsen, J . A.; Ryan, K.; Schultz, W.;
Torgersen, L. K.; Koe, B. K. 3-(1,2,5,6-Tetrahydropyrid-4-yl)-
pyrrolo[3,2-b]pyrid-5-one: a Potent and Selective Serotonin (5-
HT1B) Agonists and Rotationally Restricted Phenolic Analogue
of 5-Methoxy-3-(1,2,5,6-Tetrahydropyrid-4-yl)indole. J . Med.
Chem. 1990, 33, 2087-2093.
(13) Caccia, S.; Garattini, S. 1-Arylpiperazines as Active Metabolites
of (4-Substituted Aryl-1-piperazinyl)alkyl Heterocyclic Drugs.
Acta Pharmacol. J ugosl. 1990, 40, 441-460.
J M970376W