Pyrroloquinoxaline derivatives as high-affinity and selective 5-HT3 receptor agonists: Synthesis, further structure-activity relationships, and biological studies

Article abstract of DOI:10.1021/jm990151g

The synthesis, pharmacological evaluation, and structure-activity relationships (SARs) of a series of novel pyrroloquinoxalines and heteroaromatic-related derivatives are described. The new pyrroloquinoxaline- related ligands were tested in rat cortex, a tissue expressing high density of 5-HT₃ receptors, and on NG108-15 cells and exhibited IC₅₀ values in the low nanomolar or subnanomolar range, as measured by the inhibition of [³H]zacopride binding. The SAR studies detailed herein delineated a number of structural features required for improving affinity. Some of the ligands were employed as 'molecular yardsticks' to probe the spatial dimensions of the lipophilic pockets L1, L2, and L3 in the 5-HT₃ receptor cleft, while the 7-OH pyrroloquinoxaline analogue was designed to investigate hydrogen bonding with a putative receptor site H1 possibly interacting with the serotonin hydroxy group. The most active pyrroloquinoxaline derivatives showed subnanomolar affinity for the 5-HT₃ receptor. In functional studies ([¹⁴C]guanidinium accumulation test in NG108-15 hybrid cells, in vitro) most of the tested compounds showed clear-cut 5-HT₃ agonist properties, while some others were found to be partial agonists. Several heteroaromatic systems, bearing N-substituted piperazine moieties, have been explored with respect to 5-HT₃ affinity, and novel structural leads for the development of potent and selective central 5-HT₃ receptor agonists have been identified. Preliminary pharmacokinetic studies indicate that these compounds easily cross the blood brain barrier (BBB) after systemic administration with a brain/plasma ratio between 2 and 20, unless they bear a highly hydrophilic group on the piperazine ring. None of the tested compounds showed in vivo anxiolytic-like activity, but potential analgesic-like properties have been possibly disclosed for this new class of 5-HT₃ receptor agonists.

Full text of DOI:10.1021/jm990151g

4362
J . Med. Chem. 1999, 42, 4362-4379
P yr r oloqu in oxa lin e Der iva tives a s High -Affin ity a n d Selective 5-HT3 Recep tor
Agon ists: Syn th esis, F u r th er Str u ctu r e-Activity Rela tion sh ip s, a n d Biologica l
Stu d ies
Giuseppe Campiani,*,§ Elena Morelli,† Sandra Gemma,† Vito Nacci,† Stefania Butini,† Michel Hamon,‡
Ettore Novellino, Giovanni Greco, Alfredo Cagnotto,∇ Mara Goegan,∇ Luigi Cervo,∇ Fabio Dalla Valle,∇
Claudia Fracasso,∇ Silvio Caccia,∇ and Tiziana Mennini∇
Dipartimento di Scienze Farmaceutiche, Facolta′ di Farmacia, Universita′ degli Studi di Salerno, via Ponte Don Melillo,
84084 Fisciano, Salerno, Italy, Dipartimento Farmaco Chimico Tecnologico, Facolta′ di Farmacia, Universita′ degli Studi di
Siena, via Aldo Moro, 53100 Siena, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, Facolta′ di Farmacia,
Universita′ di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy, Istituto di Ricerche Farmacologiche
“Mario Negri”, via Eritrea 62, 20157 Milano, Italy, and Neurobiologie Cellulaire et Functionnelle, Istitut National de la
Sante′ et de la Recherche Medicale (INSERM) U. 288, Faculte′ de Medicine Pitie′-Salpetriere 91, boulevard de l’Hopital,
75634 Paris Cedex 13, France
Received March 30, 1999
The synthesis, pharmacological evaluation, and structure-activity relationships (SARs) of a
series of novel pyrroloquinoxalines and heteroaromatic-related derivatives are described. The
new pyrroloquinoxaline-related ligands were tested in rat cortex, a tissue expressing high
density of 5-HT3 receptors, and on NG108-15 cells and exhibited IC50 values in the low
nanomolar or subnanomolar range, as measured by the inhibition of [3H]zacopride binding.
The SAR studies detailed herein delineated a number of structural features required for
improving affinity. Some of the ligands were employed as “molecular yardsticks” to probe the
spatial dimensions of the lipophilic pockets L1, L2, and L3 in the 5-HT3 receptor cleft, while
the 7-OH pyrroloquinoxaline analogue was designed to investigate hydrogen bonding with a
putative receptor site H1 possibly interacting with the serotonin hydroxy group. The most active
pyrroloquinoxaline derivatives showed subnanomolar affinity for the 5-HT3 receptor. In
functional studies ([14C]guanidinium accumulation test in NG108-15 hybrid cells, in vitro) most
of the tested compounds showed clear-cut 5-HT3 agonist properties, while some others were
found to be partial agonists. Several heteroaromatic systems, bearing N-substituted piperazine
moieties, have been explored with respect to 5-HT3 affinity, and novel structural leads for the
development of potent and selective central 5-HT3 receptor agonists have been identified.
Preliminary pharmacokinetic studies indicate that these compounds easily cross the blood-
brain barrier (BBB) after systemic administration with a brain/plasma ratio between 2 and
20, unless they bear a highly hydrophilic group on the piperazine ring. None of the tested
compounds showed in vivo anxiolytic-like activity, but potential analgesic-like properties have
been possibly disclosed for this new class of 5-HT3 receptor agonists.
In tr od u ction
potential treatment of neurodegenerative and neurop-
sychiatric disorders. The agonist activities of 2-meth-
ylserotonin (2-Me-5-HT) (1) and quipazine (2) are well-
documented, but unlike 2-Me-5-HT, quipazine is able
to cross the blood-brain barrier (BBB).7 Based on the
structure of quipazine, we have recently designed a new
class of high-affinity 5-HT3 receptor ligands.6a Along
with high affinity for the 5-HT3 receptor, these qui-
pazine-related ligands behaved as agonists in [14C]-
guanidinium accumulation test and in cortical acetyl-
choline release in freely moving rats.6a Quinoxalines
3a ,b6a are the representative compounds of this series.
Other high-affinity 5-HT3 receptor ligands, structurally
related to quipazine, have been recently described by
Castan and co-workers (4),8 while Rault and co-workers9
reported new 5-HT3 partial agonists (5) (Chart 1).
Serotonin (5-HT) is one of the most attractive targets
in medicinal chemistry since this neurotransmitter is
involved in many biological and pathological processes.1
5-HT receptors are classified into several subtypes.2
Among these, the 5-HT3 receptor has attracted much
attention because of the effectiveness of its antagonists
in preventing emesis induced by treatment with che-
motherapeutic drugs.3 Furthermore, numerous potential
therapeutic applications of 5-HT3 antagonists have been
claimed.4 On the contrary, the information on 5-HT3
receptor agonists and their potential specific therapeutic
relevance is scarce, although it has been shown that
5-HT3 receptor agonists may modulate central acetyl-
choline release5,6a making them of interest for the
Because of the increasing interest in the development
of potent and selective 5-HT3 receptor agonists, we
decided to expand our structure-activity relationship
(SAR) studies of the quipazine-related class of ligands
in order to optimize their 5-HT3 binding affinity and
selectivity. The present article details the synthesis of
* To whom correspondence should be addressed. Tel: 0577-234331.
Fax: 0577-234333. E-mail: campiani@unisi.it.
§ Universita′ degli Studi di Salerno.
† Universita′ degli Studi di Siena.
Universita′ degli Studi di Napoli “Federico II”.
∇ Istituto di Ricerche Farmacologiche “Mario Negri”.
‡ INSERM, Paris.
10.1021/jm990151g CCC: $18.00 © 1999 American Chemical Society
Published on Web 09/24/1999

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4363
Ch a r t 1
Sch em e 1
novel pyrroloquinoxalines and pyrroloquinoxaline-re-
lated analogues and their SARs for rat cortical 5-HT3
receptor affinity associated with variation of the sub-
stituents on the heterocyclic system. A subset of ligands
with high affinity in brain was further tested for their
ability to inhibit [3H]zacopride binding in NG108-15
hybrid cells, and all were found to have potencies
comparable to those in rat cortex. From analysis of the
SARs relative to the newly investigated pyrroloquinoxa-
line analogues, a pharmacophore model, schematically
represented in Chart 2, could be defined. Accordingly,
a hydrogen bond acceptor/donor function (H1) and three
lipophilic pockets (L1, L2, and L3) within the receptor
binding site are available to our ligands. The 5-HT3
receptor intrinsic efficacy of selected derivatives was
assessed in vitro on 5-HT3 receptor-dependent [14C]-
guanidinium uptake in NG108-15 hybrid cells. Ad-
ditionally, brain and plasma concentrations were si-
multaneously measured for a subset of ligands after
systemic injection, and in vivo studies were performed
in order to evaluate potential anxiolytic-like or analgesic-
like properties of the tested compounds.
lines, pyrrolopyrazine, imidazolinopyrazine, and pyri-
dopyrrolopyrazines 20a -jj has been accomplished as
described in Schemes 1-6. The key lactam intermedi-
ates 10a -g have been synthesized following the path-
ways reported in Schemes 1-3, while the lactams
10h -o were obtained following already known proce-
dures described in refs 6 and 9-11 (see Table 1). As
described in Scheme 1, starting from the commercially
available 2-nitroanilines 6a -e, by a Clauson-Kaas
reaction, the 1-(2-nitroaryl)pyrroles were obtained (7a -
e). Next, the nitro groups were reduced, by exposure to
tin(II) chloride, and the 1-(2-aminoaryl)pyrroles 9a -c,e
were isolated. In the case of 7d , the free hydroxy
function was protected to 8, and this benzyloxy analogue
was treated with tin(II) chloride, obtaining 9d . Then
10a -e were prepared by reaction of amines 9a -e with
triphosgene in high yield.
The lactam 10f was in turn prepared as described in
Scheme 2. Starting from 11, the amino group was
protected by a Cbz function by using benzyl chlorofor-
mate (Cbz-Cl) (12), and the nitro group was treated with
tin(II) chloride. The corresponding pyridine 13 was
Ch em istr y
The synthesis of the new pyrroloquinoxalines, imida-
zoquinoxalines, imidazopyridopyrazines, pyrazoquinazo-
Ch a r t 2a
a
H1 is a hydrogen bond acceptor/donor site on the receptor protein. L1, L2, and L3 are lipophilic pockets in the protein at the binding
site.

4364 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
Sch em e 2
Sch em e 5
Sch em e 6
Sch em e 3
Sch em e 4
Following a similar approach, the lactam intermedi-
ate 10g was synthesized. Starting from 15, after intro-
duction of the imidazole ring10a (16) and reduction of
the nitro group with tin(II) chloride (17), the cyclization
step was perfomed by using N,N-carbonyldiimidazole
(CDI) (10g).
Successively, the N-4-substituted piperazine ring was
introduced following a standard method6a as described
in Scheme 4. Lactams 10a -o were transformed into the
4-chloro derivatives,6a,11a and the latter were treated
with piperazine to obtain 19a -c,g or with N-alkylpip-
erazines to obtain 20a -t,v,w ,ee-gg. Some of the
desired compounds were synthesized by alkylation of
the corresponding 19a -h (20u ,x-d d ).11b
subjected to the Clauson-Kaas reaction (14), and the
cyclization was performed using triphosgene (10f). Dur-
ing this cyclization step the deprotection occurred.
The imidazopyrazine derivative 20n was prepared as
reported in Scheme 5. Commercially available dichlo-

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4365
Ta ble 1. Physical and Chemical Data for Compounds 7-10 and 12-17
compd
V
W
R
X
Y
Z
yielda (%)
mp (°C)
recryst solvent
formula
anal.b
7a
7b
7c
7d
7e
8
9a
9b
9c
4,5-diMe
6-Me
4-CN
4-OH
4,5-diCl
76
68
57
89
72
98
95
76
71
87
81
86
79
75
79
82
73
51
60-61
103-104
91-92
hexanes
hexanes
EtOAc
EtOH
C12H12N2O2
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
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
11H10N2O2
11H7N3O2
10H8N2O3
10H6Cl2N2O2
17H14N2O3
12H14N2
157-158
68-69
hexanes
EtOAc
EtOAc
hexanes
hexanes
EtOAc
hexanes
EtOAc
EtOAc
EtOAc
EtOAc
EtOAc
EtOAc
EtOAc
69-70
4,5-diMe
6-Me
83-85
46-47
11H12N2
11H9N3
4-CN
4-OBn
4,5-diCl
7,8-diMe
9-Me
7-CN
7-OBn
7,8-diCl
H
101-103
135-136
145-146
9d
9e
17H16N2O
10H8Cl2N2
13H12N2O
12H10N2O
12H7N3O
18H14N2O2
11H6Cl2N2O
10H7N3O
10a
10b
10c
10d
10e
10f
10g
10h c
10ic
10jd
10k e
10lf
10m e
10n g
10og
12
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
CH
CH
CH
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
CH
CH
CH
CH
CH
CH
N
>300
280 dec
280 dec
272-273
>300
>250
>250
7-F
7-Me
H
10H6 FN3O
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
H
H
H
7-F
9-F
N
C
N
N
N
C
N
C
C
C
N
N
N
CH
CH
78
94
42
48
77
C
C
C
13H11N3O4
13H13N3O2
17H15N3O2
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
13
14
16
17
148-149
benzene
hexanes
hexanes
cyclohexane
96-98
83-84
C9H6FN3O2
C9H8FN3
127-128
a
b
Yields refer to isolated and purified materials. All the compounds were analyzed within (0.4% of the theoretical values. c Reference
d
g
9. Reference 10c. e Reference 10a. f Reference 10b. Reference 6.
ropyrazine 21 was reacted with 2-aminoethanol to give
22,12a and this was treated with thionyl chloride to
obtain 23 as hydrochloride salt. The 4-chloro derivative
23 was then heated with N-methylpiperazine to obtain
20n .
Compounds bearing highly hydrophilic moieties at
N-4 of the piperazine ring were obtained following
Scheme 6. Starting from 19d the benzyloxycarbonylm-
ethyl function at N-4 was introduced by using benzyl
bromoacetate (24). Next, the benzyl group was removed
by catalytic hydrogenation (20ii). The reaction per-
formed by using bromoacetonitrile led to 20h h , which
was transformed into the 5-tetrazolylmethyl analogue
20jj by using sodium azide.12b
3 and 4), analogues in which the fused aromatic system
is missing, were investigated to evaluate the role of the
benzo-fused system in the binding to the 5-HT3 receptor.
Both compounds were found to be at least 4 orders of
magnitude less active than 3b or 20b, indicating that
the aromatic ring plays a key role in the interaction with
the receptor protein.
(ii) Su b st it u t ion s on t h e Ben zo-F u sed R in g of
Qu in oxa lin es. The 7,8-dimethyl (19a , 20a ), 7-methyl
(19c, 20g), and 9-methyl (19b, 20b) substituted pyr-
roloquinoxalines have been synthesized and tested to
probe the spatial dimension of the lipophilic pocket L1
within the receptor cleft. As shown in Table 3, the drop
in affinity of analogues 19a and 20a with respect to the
7-monoalkylated analogues 19b and 20g indicates that
the steric hindrance of a methyl group at C-8 is
detrimental to affinity (Chart 2). In contrast, the
introduction of a methyl group at C-9 leads to a high-
affinity ligand (20b ) with subnanomolar affinity
(IC50 ) 0.79 nM), similar to that of 3a .6a On NG108-15
cells 20b proves to be extremely efficacious in displacing
the [3H]zacopride from the 5-HT3 receptor. Replacement
of the methyl groups at C-7 and C-8 of 20a with chlorine
atoms (20e) or introduction at position 7 of a cyano
group (20c) caused a large drop in affinity, the IC50s
being equal to 97 and 104 nM, respectively. The
introduction of a hydroxy group at C-7 (20f), intended
to mimic the hydroxy group of serotonin (5-HT), confers
Resu lts a n d Discu ssion
1. 5-HT3 Bin d in g SAR Stu d ies.13a The affinities of
the new pyrroloquinoxaline-related analogues 19a -c,g
and 20a -jj for 5-HT3 receptors in rat cortex homogenate
are illustrated in Tables 3-5, while Table 6 reports the
ability of selected compounds to inhibit [3H]zacopride
binding in rat cortex and NG108-15 cells. Binding data
of quipazine and 3b are also included. Table 7 lists the
affinities of selected compounds for different 5-HT
receptor subtypes. The results reported in these tables
are summarized as follows.
(i) Key Role of th e Ben zo-F u sed System on 5-HT3
Receptor Affin ity (L1 Site). Compounds 20h ,n (Tables

4366 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
Ta ble 2. Physical and Chemical Data for Compounds 18a -o, 19a -h , 20a -jj, and 22-24
yielda
(%)
recryst
compd
V
W
R
X
Y
Z
R′
mp (°C)
solventb
formula
C13H11ClN2
anal.c
18a
18b
18c
18d
18e
18f
18g
18h d
18id
18j
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
CH
CH
CH
7,8-diMe
9-Me
7-CN
7-OBn
7,8-diCl
H
7-F
7-Me
H
N
N
N
N
N
N
N
N
N
N
N
C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
C
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
C
C
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
N
91
83
72
79
80
82
84
139-140
145-146
140-141
113-114
206-207
163-164
178-179
A/B
A/B
A/B
B/C
C
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C
C
C
C
C
C
12H9ClN2
12H6ClN2
18H13ClN2O
11H5Cl3N2
10H6ClN3
10H5FClN3
A/B
A/B
69
72
77
81
112-113
188-189
122-123
214-215
B
C7H7ClN2
C,H,N
C,H,N
C,H,N
C,H,N
18k
18l
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
H
H
H
7-F
A/B
A/B
A/B
C
C
10H6ClN3
10H6ClN3
N
N
18m
18n e
18oe
19a
19b
19c
19d e
19ee
19fe
19g
19h d
20a
20b
20c
20d
20e
20f
20g
20h
20i
20j
20k
20l
20m
20n
20o
20p
20q
20r
C9H5ClN4
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
9-F
7,8-diMe
9-Me
7-Me
9-F
7-F
H
H
H
H
H
80
84
82
127-129
124-125
128-130
C
A/B
A/B
C
C
C
17H20N4
16H18N4
16H18N4
C,H,N
C,H,N
C,H,N
H
H
H
H
H
H
73
107-108
A
C
14H15N5
C,H,N
7,8-diMe
9-Me
7-CN
7-OBn
7,8-diCl
7-OH
7-Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
87
72
67
84
65
78
83
71
68
87
56
83
60
48
85
89
75
67
78
83
58
87
82
39
44
46
43
36
46
35
85
88
82
70
88
70
133-134
146-147
159-161
150-152
148-149
164-165
151-153
103-105
97-98
A/B
A/B
A/B
A/B
A/B
A
A/B
A
A/B
A/B
A/B
A
C18 H22N4
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
C,H,N
C
C
C
C
17H20N4
17H17N5
23H24N4O
16H16Cl2N4
C16H18N4O
17H20N4
C12H18N4
C
CH
CH
CH
CH
N
CH
CH
CH
N
H
7-F
H
H
H
C
C
C
C
C
C
15H17N5
15H16FN5
15H17N5
15H17N5
14H16N6
11H17N5
N
N
CH
N
N
126-127
91-92
89-91
108-109
93-95
CH
A
A
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
N
7-Me
7,8-diMe
9-Me
7,8-diCl
7-F
9-F
9-F
H
H
9-F
H
7-F
H
9-Me
7-F
7-F
9-F
7-F
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
2-naphthylmethyl
CH2Ph
Ph
2-thienylmethyl
2-thienylmethyl
2-thienylmethyl
2-thienylmethyl
2-Br-5-thienylmethyl
2-furylmethyl
2-Br-5-furylmethyl
CH2CH2OH
132-134
122-123
119-120
147-148
153-154
149-151
128-130
118-119
163-165
113-114
117-118
122-123
144-146
108-110
133-134
99-100
A/B
A/B
A/B
A/B
A/B
A/B
A/B
A/B
C
A/B
A/B
A/B
A/B
A/B
A/B
A/B
A
C23H24N4
C24H26N4
C23H24N4
C22H20Cl2N4
C22H21FN4
C22H21FN4
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
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
20s
20t
20u
20v
20w
20x
20y
20z
20a a
20bb
20cc
20d d
20ee
20ff
20gg
20h h
20ii
20jj
22f
C
26H23FN4
C21H21N5
N
C
C
C
C
20H19N5
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
20H19FN4S
20H20N4S
20H19FN4S
C19 H19N5S
C
C
C
21H21BrN4S
20H19FN4O
18H18FBrN4O
123-124
166-168
149-151
133-134
217-218
233-234
C17 H19FN4O
C17 H19FN4O
C15H18N6O
C17 H16 FN5
C17H17FN4O2
CH2CH2OH
CH2CH2OH
CH2CN
CH2CO2H
A
A
A
A
H
9-F
9-F
9-F
CH
CH
CH
CH
CH
CH
5-tetrazolylmethyl
A
C17H17 FN8
23
24
62
75
274-275
74-75
A/B
A/B
C6H7Cl2N3
24H23FN4O2
C,H,N
C,H,N
C
a
b
Yields refer to isolated and purified materials. A ) EtOAc; B ) hexanes; C ) benzene. c All the compounds were analyzed within
(0.4% of the theoretical values. Reference 9. e Reference 6. f Reference 12a.
d
subnanomolar affinity for the central 5-HT3 receptor,
similar to that of 3b and 20b (IC50s ) 0.81 and 0.79
nM, respectively) which lack the hydroxy function. The
nature of the interaction involving the substituent at

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4367
Ta ble 3. 5-HT3 Receptor Binding Affinities for Compounds 19a -c,g and 20a -h ,l,o-d d and Functional Behavior of Selected
Compounds
effect on [14C]guanidinium
uptake on NG108-15 cells
IC50 (nM)a
((SEM)
compd
19a
19b
19c
19g
20a
20b
20c
20d
20e
20f
20g
X
Y
R
R′
efficacyb
PA
EC50 (nM)c
9.5
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
7,8-diMe
9-Me
7-Me
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
113 ( 28
1.13 ( 0.3
16.4 ( 4.1
3.1 ( 0.3
30 ( 8
0.79 ( 0.1
104 ( 17
225 ( 52
97 ( 22
H
7,8-diMe
9-Me
7-CN
7-OBn
7,8-diCl
7-OH
A
3.8
0.67 ( 0.09
18 ( 7
A
A
1.6
14.0
7-Me
20h
20l
20o
20p
20q
20r
20s
20t
20u
20v
20w
20x
20y
1930 ( 315
2.4 ( 0.5
24 ( 9
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
N
H
Me
PA
1.7
CH
CH
CH
CH
CH
CH
CH
N
7-Me
7,8-diMe
9-Me
7,8-diCl
7-F
9-F
9-F
H
H
9-F
H
7-F
H
9-Me
7-F
7-F
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
2-naphthylmethyl
CH2Ph
Ph
2-thienylmethyl
2-thienylmethyl
2-thienylmethyl
2-thienylmethyl
2-Br-5-thienylmethyl
2-furylmethyl
2-Br-5-furylmethyl
44 ( 9
8.8 ( 0.9
118 ( 19
13 ( 3
A
12.0
4.2
PA
61 ( 18
780 ( 148
2.2 ( 0.5
NA
PA
4.2
N
CH
CH
CH
CH
CH
CH
CH
60 ( 13
22 ( 5
20z
12 ( 2
20a a
20bb
20cc
20d d
2
3.6 ( 0.5
104 ( 24
11 ( 3
CH
CH
CH
PA
6.6
148 ( 43
3.8 ( 0.4
0.81 ( 0.05
1.0 ( 0.3
2.3 ( 0.8
6.80 ( 1.4
97 ( 30
PA
A
1.0
3.0
3bd
5a e
5be
PA
32f
GR38032
MDL72222
a
Each value is the mean ( SEM of three determinations and represents the concentration giving half-maximal inhibition of [3H]zacopride
binding to rat cortical homogenate. A ) pure agonist; PA ) partial agonist. c EC50 values are the mean of two independent experiments.
b
Reference 6. e 5a ,b have been previously reported by Rault et al.9 They have been resynthesized and used as reference compounds.
d
Binding data are those obtained following our protocol. f Reference 9. NA ) not active.
the 7-position remains elusive since a ligand/receptor
neutral-neutral hydrogen bond contributes to binding
affinity from 15-fold to 0.13b In our pharmacophore
model, the role of hydrogen bond acceptor/donor as-
signed to the 7-OH group of 20f relied on the hypothesis
that this hydroxy group mimics the one of 5-HT at the
binding site. Overall, analogues 20b,f represent two of
the most potent and selective 5-HT3 receptor ligands
reported to date. Transformation of the hydroxy group
at C-7 of 20f into a benzyloxy function (20d ) caused a
significant decrease of affinity, due to unfavorable steric
interactions.
(iii) Rep la cem en t of th e Meth yl Gr ou p a t N-4 by
π-Electr on -Rich System s (L2 Site). In general, af-
finity is owered by replacement of the methyl group at
N-4 of the piperazine ring with a benzyl group. This
trend can be seen in Table 3 by comparison of the
affinity values of several pair of analogues: 3b/20s, 20a /
20p , 20b/20q, and 20g/20o. Conversely, for the pyrido-
fused pair 20l/20v (see the following section) a similar
affinity has been found. Substitution of the benzyl by
isosteric heteroaryl groups leads to less active analogues
(20x-z,bb,cc,d d vs 20q,s,t). Among the 2-thienylm-
ethyl derivatives synthesized, 20a a shows the better
binding profile (Table 3).
Replacement of the benzyl group of 20t with a bulkier
2-naphthylmethyl group gave an inactive analogue
(20u ) due to unfavorable steric interactions (Chart 2).
Furthermore, a phenyl substituent directly linked to the
N-4 of the piperazine ring leads to an inactive analogue
(20w ), probably due to the reduced basicity of the N-4
piperazine nitrogen and to unfavorable shape effects.
(iv) P yr id o-F u sed ver su s Ben zo-F u sed An a -
logu es: Effect on 5-HT3 Recep tor Affin ity. The
presence of an extra nitrogen (pyridopyrrolopyrazines)
on the benzo-fused ring of our quinoxalines is slightly
unfavorable for affinity (Table 3). In fact, while 3a
showed an IC50 equal to 0.37 nM, the pyrido-fused
analogue 20l is 8-fold less potent at the 5-HT3 receptor.
Though in the series of pyrido-fused analogues (19g,

4368 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
Ta ble 4. Comparison of the 5-HT3 Receptor Binding Affinity of Compounds 20i-n Characterized by Different Fused Five-Membered
Rings and Functional Behavior of Compounds 20i-l
effect on [14C]guanidinium
uptake on NG108-15 cells
compd
R
V
W
X
Y
Z
IC50 (nM)a ((SEM)
efficacyb
EC50 (nM)c
3bd
0.81 ( 0.05
1.0 ( 0.3
3.8 ( 0.5
5.2 ( 0.4
1.6 ( 0.1
2.4 ( 0.5
6.6 ( 1.0
3650 ( 320
A
3.0
5a e
20i
20j
20k
20l
20m
20n
H
7-F
H
H
H
CH
CH
CH
CH
N
CH
CH
CH
N
N
N
C
N
N
C
C
N
C
C
N
N
N
CH
N
A
A
A
PA
15.0
7.2
2.7
1.7
CH
a
Each value is the mean ( SEM of three determinations and represents the concentration giving half-maximal inhibition of [3H]zacopride
binding to rat cortical homogenate. A ) pure agonist; PA ) partial agonist. c EC50 values are the mean of two independent experiments.
b
Reference 6. e 5a has been previously reported by Rault et al.9 This analogue has been resynthesized and used as a reference compound.
d
Binding data is that obtained following our protocol.
Ta ble 5. Comparison of the 5-HT3 Receptor Binding Affinity of Compounds 20ee-jj Bearing Hydrophilic Substituents on the
Piperazine Ring and Functional Behavior for Compounds 20ee,ff
effect on [14C]guanidinium
uptake on NG108-15 cells
compd
R
X
Y
R′
IC50 (nM)a ((SEM)
efficacyb
EC50 (nM)c
3bd
20ee
20ff
20gg
20h h
20ii
0.81 ( 0.05
8.2 ( 2.4
2.6 ( 0.3
38 ( 7
3800 ( 300
920 ( 170
690 ( 115
A
PA
A
3.0
54.0
2.4
9-F
7-F
H
9-F
9-F
9-F
CH
CH
N
CH
CH
CH
CH
CH
N
CH
CH
CH
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2CN
CH2CO2H
5-tetrazolylmethyl
20jj
a
Each value is the mean ( SEM of three determinations and represents the concentration giving half-maximal inhibition of [3H]zacopride
binding to rat cortical homogenate. A ) pure agonist; PA ) partial agonist. c EC50 values are the mean of two independent experiments.
Reference 6.
b
d
20l,v,a a ) the 5-HT3 binding data are virtually identical,
in the low nanomolar range, a different selectivity
profile is shown by compounds 20l,v versus the different
5-HT receptor subtypes (see Selectivity section and
Table 7). The new 5-HT3 receptor ligands 19g and 20l,v
represent three novel pyridopyrrolopyrazines, bearing
a hydrogen, a methyl group, and a benzyl group,
respectively, on the piperazine ring and are isomers of
those pyridopyrrolopyrazines recently described by Rault
and co-workers (5a ,b).9b To compare, in the same
experimental conditions, compounds 20l,v with those
described by Rault and co-workers we decided to resyn-
thesize 5a ,b, for which -log IC50s of 9.01 and 12.09,
respectively, were reported. In our hands (Table 3) the
affinity of 20l,v overlaps the values of their isosters
5a ,b. It is to be noticed that our 5-HT3 binding affinity
for compound 5b (rat cortex and NG108-15 cells, [3H]-
zacopride) does not confirm the one (IC50 ) 0.81 pM)
reported by Rault’s group (NG108-15 cells, [3H]grani-
setron9b). We have no explanation for this apparent
discrepancy, since binding values for 5-HT3 receptors
obtained in rat cortex with our compounds and 5b are
similar to those obtained in NG108-15 cells (Table 6).
We also replaced the benzyl group of the Rault com-
pound 5b by a 2-thienylmethyl moiety (20a a ). This
derivative showed an affinity (IC50 ) 3.6 nM) compa-
rable to those of 20v and 5b (5-HT3 binding IC50 ) 2.2
and 2.3 nM, respectively), with a selectivity profile
similar to that of 20v.
(v) Effect of Differ en t F u sed F ive-Mem ber ed
Rin gs (L3 Site). With respect to unsubstituted pyr-
roloquinoxalines (3a , IC50 ) 0.37 nM)6a the introduction
of an extra basic nitrogen on the pyrrole ring (20i,j)
caused a 6-fold decrease of affinity, although it remains
in the low nanomolar range (see Table 4). Replacement
of the imidazole ring of 20i with a pyrazole (20k ) led to
a more potent 5-HT3 receptor ligand, whose affinity
overlaps those of unsubstituted pyrroloquinoxalines,
suggesting that the basicity of the nitrogen could be
slightly unfavorable for receptor interaction (20k vs
20i). Combination of a pyrido-fused ring with an imid-
azole system, in place of the pyrrole ring of 5a , leads to
20m whose affinity appears slightly lower than those
of 5a and 20i.

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4369
Ta ble 6. Comparison of Inhibition of [3H]Zacopride Binding in
Rat Brain and NG108-15 Hybrid Cells
Vivo Studies), we can consider 20h h ,jj lead compounds
for the development of specific peripheral 5-HT3 receptor
agonists.
2. Selectivity Stu d ies on a P a n el of 5-HT Recep -
tor Su btyp es. A subset of compounds, chosen among
the most potent 5-HT3 receptor ligands, were further
examined for their selectivity toward 5-HT1A, 5-HT1B
,
5-HT2A, 5-HT2C, and 5-HT4 receptors. Table 7 sum-
marizes the results of the binding data for compounds
19b and 20b ,c,v,a a ,ee. Among these compounds,
20b,v,a a and, to a lesser extent, 19b reveal to be
extremely selective for the 5-HT3 receptor and, in this
regard, much more performant than quipazine.
IC50 (nM)a ((SEM)
rat
cortex
NG108-15
cells
compd
R
X
Y
R′
5a b
20b
20l
H
N
CH Me
1.0 ( 0.3
5.4 ( 0.4
9-Me CH CH Me
0.79 ( 0.1 0.26 ( 0.02
H
H
CH
N
N
Me
2.4 ( 0.5
3.6 ( 0.5
9.3 ( 0.9
6.4 ( 0.5
3. Biologica l Activity.7 In addition to binding stud-
ies a functional assay was carried out for assessing the
potential agonist/antagonist activity of selected pyrrolo-
quinoxaline, pyridopyrrolopyrazine, and imidazoquin-
oxaline derivatives (Tables 3-5). This 5-HT3-dependent
response was the uptake of [14C]guanidinium in the
presence of substance P (SP), which is markedly in-
creased upon the activation of 5-HT3 receptors on the
neuroblastoma-glioma hybrid NG108-15 cells. The most
intriguing result arising from these pharmacological
experiments was that all the tested compounds mim-
icked the effects of 5-HT on the 5-HT3 receptor-depend-
ent accumulation of [14C]guanidinium in NG108-15
hybridoma cells in the low nanomolar concentration
range, indicating that the novel pyrroloquinoxalines and
pyrroloquinoxaline-related derivatives act as potent
agonists in this assay. Similarly to that observed in
binding experiments, the introduction of a methyl group
at position 9 (20b) or a C-7 hydroxy substituent (20f),
coupled with a methyl at N-4, improves the potency
(20b,f vs 19b, 20q,g) in the guanidinium uptake test.
Agonist properties are also dependent upon the nature
of the fused five-membered ring. Accordingly, the pyr-
role confers maximum efficacy (3b), followed by the
pyrazole (20k ) and the imidazole (20i,j). In the pyrido-
fused series of analogues, the same rank order of
magnitude is seen, with 20l 3 times more active than
the benzyl analogue 20v. Intrinsic efficacy of substituted
pyrroloquinoxalines and related analogues in guani-
dinium uptake is further influenced by the nature of
the π-electron-rich system at N-4 of the piperazine ring.
In fact, maximum potency is achieved with the benzyl
analogue 20s, followed by the 2-furylmethyl-substituted
20cc and the 2-thienylmethyl derivative 20z (EC50s )
20a a
CH 2-thienyl-
methyl
a
Each value is the mean ( SEM of three determinations and
represents the concentration giving half-maximal inhibition of
[3H]zacopride binding to rat cortical homogenate or NG108-15
hybrid cells. Compound 5a has been resynthesized and tested
following our binding protocols. 5a has been reported by Rault
b
et al.9b
(vi) E ffect of Hyd r op h ilic Su b st it u en t s on t h e
P ip er a zin e Rin g. In our previous publication6a we
discussed the effect of different alkyl chains at the N-4
of the piperazine ring. In particular, the ethyl chain
derivative of unsubstituted pyrroloquinoxaline showed
an IC50 of 8 nM. As shown in Table 5 we explore herein
the effect of hydrophilic functions at the piperazine ring
on 5-HT3 affinity. Introduction of a hydroxy group on
the â-carbon of the ethyl chain leads to high-affinity
analogues (20ee,ff). In particular, the â-hydroxyethyl-
substituted 7-fluoroquinoxaline 20ff shows an IC50 value
of 2.57 nM, indicating that a hydroxy group is well-
tolerated in the receptor cleft. On the other hand, if the
â-hydroxyethyl chain is combined with a basic nitrogen
on the heteroaromatic system (20gg), affinity decreases.
Replacement of the ethyl chain with a cyanomethyl
group (20ii) leads to a compound devoid of any 5-HT3
receptor affinity, probably due to unfavorable steric
interactions. In contrast, the replacement of the cyano
group with a carboxylic function or with a tetrazolyl
moiety leads to analogues 20h h ,jj with still significant
affinity for the 5-HT3 receptor. By virtue of the residual
affinity of 20h h and especially of 20jj (IC50 ) 690 nM)
for the 5-HT3 receptor and taking into account that the
presence of highly hydrophilic functions (20h h ) do not
allow the brain penetration of these compounds (see In
Ta ble 7. Binding Profile of Selected Compounds on 5-HT Receptor Subtypes
pKia ( SEM
b
d
e
f
g
h
compd
5-HT3
5-HT1A
5.8 ( 0.5
<5
<5
5-HT1B
5-HT2A
5-HT2C
5-HT4
<5
<5
<5
<5
<5
<5
<5
quipazine
19b
20b
8.2 ( 0.9
9.0 ( 2.1
9.1 ( 1.6
8.6 ( 1.8
8.7 ( 3.1
8.5 ( 1.3
8.1 ( 2.4
6.4 ( 1.8b
6.7 ( 0.5
6.6 ( 1.4
6.4 ( 0.6
7.6 ( 1.4
6.1 ( 0.6
6.0 ( 1.8
6.3 ( 1.2
6.5 ( 0.6
6.0 ( 1.7
<5
20l
7.6 ( 1.0
<5
6.1 ( 1.2
<5
<5
5.7 ( 1.8
20v
20a a
20ee
5-HT
methysergide
mesulergine
GR38032
2-Me-5-HT
<5
<5
<5
6.1 ( 2.3
5.8 ( 0.8
6.3 ( 0.3
5.9 ( 0.9
8.7 ( 1.1
5.4 ( 0.5
5.8 ( 1.4
8.6 ( 0.9
6.3 ( 0.8
8.8 ( 1.4
7.5 ( 0.2
7.0 ( 1.2
8.8 ( 1.6
8.0 ( 1.4b
6.7 ( 1.5c
a
Data are pKi values ( SEM of at least three separate experiments performed in triplicate. Ki values were previously derived from the
IC50s according to the method of Cheng and Prusoff.24 [3H]Zacopride. c [3H]BRL 43694. [3H]8-OH-DPAT. e [3H]5-HT. f [3H]Ketanserin.
b
d
[3H]Mesulergine. [3H]GR113808.
g
h

4370 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
kg-1), significantly increased the time spent by the
animals in the white compartment [from 132 ( 9 to
189 ( 11 s, F(3,36) ) 5.1, P < 0.01, Dunnett’s test] as
well as the number of transitions between the black
and white compartments [from 26 ( 2.4 to 38 ( 2,
F(3,36) ) 3.0, P < 0.05, Dunnett’s test], in agreement
with previous findings.15,16
Compounds 3b and 20b, given orally 45 min before
the test at doses ranging from 0.1 to 100 µg kg-1, did
not modify the mice’s behavior in the light/dark explor-
atory test.
The apparent discrepancy between our negative re-
sults and those reported by Prunier et al.9b using
compounds with very similar structural and in vitro
properties on the 5-HT3 receptor could be due either to
the fact that, after oral administration, the compounds
could be differently absorbed and metabolized, thus
giving rise to different brain levels of active product, or
to the fact that the compounds used by Prunier et al.9b
behaved as partial agonists in the Von Bezold-J arisch
reflex assay in vivo, while compound 3b is a full agonist
in this test.6 This latter possibility seems more plausible,
since anxiolytic-like activity has been reported for 5-HT3
antagonists in mice, rats, and cynomologus monkey.17,18
We also evaluated the possible analgesic-like activity
of compounds 3b and 20b, since 5-HT as well as 5-HT3
agonists, like 2-methylserotonin, have been reported to
produce analgesia after intratecal administration in
rats, and this effect was completely abolished by 5-HT3
antagonists such as ICS 205-930, MDL 72222, and
zacopride.19-21
We tested the effect of 10 mg kg-1 i.p. of 3b, a dose
reported to give, 60 min after dosing, brain levels high
enough to fully saturate the 5-HT3 receptor,6 and
comparable dose of 20b on the radiant heat tail flick
test. In this test morphine, 3 mg kg-1 s.c., induced a
clear analgesic effect [FTreatment×Time(4,60) ) 42.7, P <
0.01, two-way ANOVA for repeated measures] at 30 and
60 min posttreatment (11.9 ( 1.1 and 8.6 ( 1.1 s,
respectively, compared to control group: 2.1 ( 0.2 and
2.0 ( 0.2 s, P < 0.01, Tukey’s test).
Analysis of variance for repeated measures found a
significant interaction between treatment and time
[FTreatment×Time(4,84) ) 2.2, P < 0.05, two-way ANOVA
for repeated measures]. Post-hoc comparison by Tukey’s
test revealed that 3b but not 20b slightly but signifi-
cantly increased the latency of rats’ tail flick 60 min
posttreatment (4.6 ( 0.9 s, compared to control group:
2.2 ( 0.2 s, P < 0.05, Tukey’s test).
However, in a second experiment, we failed to show
a clear dose-response effect of 3b [from 3 to 20 mg kg-1
i.p.; FTreatment×Time(4,112) ) 1.5, P > 0.05, two-way
ANOVA for repeated measures]. Therefore further
experiments will clarify whether compound 3b exerts
analgesic-like activity only in a very narrow dose range,
and by using selective 5-HT3 antagonists it will be
studied whether it is related to 5-HT3 receptor activa-
tion.
F igu r e 1. Scatter plot of pEC50 vs pIC50 values relative to
the functional and binding data of the 11 pure agonists
described.
4.2, 6.58, and 10, respectively). Overall, a good correla-
tion of the binding data (cerebral cortex and NG108-15
cells) and of the EC50 values ([14C]guanidinium uptake)
of the tested compounds is achieved in the series of
5-HT3 full agonists. Accordingly, a highly statistically
linear correlation is found between the functional and
5-HT3 binding data expressed as pEC50 and pIC50
values, respectively, of the 11 pure agonists described
(correlation coefficient 0.77, standard deviation 0.26).
A scatter plot of pEC50 vs pIC50 for these 11 compounds
is shown in Figure 1. Interestingly, functional and 5-HT3
binding data were not correlated at all when the full
agonists were merged with the remaining compounds
classified as partial agonists. Since common pharma-
cological and physicochemical properties of 5-HT3 recep-
tors in cerebral rat cortex and NG108-15 cells have been
recently described,14 the [14C]guanidinium uptake test
on NG108-15 cells is considered a reliable pharmaco-
logical model to infer central functional properties of
ligands of the 5-HT3 receptor. Accordingly, the most
active members of this series of 5-HT3 receptor ligands,
which show a methyl atom at position 9 (20b) or a C-7
hydroxy group (20f), which mimicked that one of sero-
tonin, or an unsubstituted pyrido-fused ring (20l), could
be considered among the most potent central 5-HT3
receptor agonists described until to date.
4. In Vivo Stu d ies. i. Assessm en t of P oten tia l
An xiolytic-lik e a n d An a lgesic-lik e P r op er ties. The
compound 5b, with chemical structure, affinity, and full
agonist property on NG108-15 cells9b similar to our
compounds, has been described to have anxiolytic
properties in the light/dark exploratory test in mice.9b
We therefore evaluated the potential anxiolytic-like
properties of comparable oral doses of 3b, previously
characterized as a potent 5-HT3 agonist able to cross
the blood-brain barrier after systemic administration
in rats,6 and of 20b, a newly described central 5-HT3
receptor agonist, sharing with 3b similar affinity and
efficacy for 5-HT3 receptors (IC50 ) 0.79 and 0.81 nM,
respectively) and a good brain-to-plasma distribution
ratio. In the 5 min of light/dark exploratory test,
chlordiazepoxide, given subcutaneously 30 min before
the test at a dose of 10 mg kg-1 (but not 1 and 3 mg
ii. Br a in -to-P la sm a Distr ibu tion Stu d ies. Pre-
liminary brain-to-plasma partition studies were per-
formed with compounds 20b,l,u ,v,ee,h h , given intra-
venously in rats (5 mg/kg). These compounds cover a
wide range of structural characteristics, including dif-
ferent heteroaromatic-fused rings, halogens, and acidic

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4371
Ta ble 8. Brain-to-Plasma Partition of Representative
Compounds in the Rat
rium between the brain and peripheral blood. With the
exception of compound 20h h , again mean brain con-
centrations exceeded those in plasma. This was not
surprising as previous studies have shown that lipo-
philic structurally related compounds do concentrate in
brain tissue, achieving concentrations several times
higher than those in blood.6,22a This is probably ex-
plained by their high lipophilicity which leads to the
extensive uptake into lipid-containing tissues. However,
there were differences in the brain-to-plasma ratios of
the various derivatives. The alcoholic derivative 20ee
had the higher brain-to-plasma partition ratio (about
20) followed by compounds 20b (9.5) and 20l (4.9). These
compounds possess calculated log P values22b between
2.8 (20l) and 4.3 (20b). The ratio for the most lipophilic
compounds studied, 20v (log P ) 4.3) and 20u (log P )
7), was 2-3. This is consistent with the observation that
within a series of molecules there is an optimal octanol/
water partition for the brain-to-plasma distribution
ratio. However, for the present series of centrally acting
compounds, the observed brain-to-plasma ratios might
not reflect the true extent of brain uptake since plasma
protein binding has not yet been evaluated.
compda
R
X
R′
brain/plasmab
20b
20l
9-Me
H
CH
N
Me
Me
9.5
4.9
2.8
20u
20v
20ee
20h h
9-F
H
9-F
9-F
CH
N
CH
CH
2-naphthylmethyl
CH2Ph
CH2CH2OH
CH2CO2H
2.1
20.3
0.1
a
b
Compounds were tested as hydrochloride salts. Calculated
as brain-to-plasma AUCt ratio.
Con clu sion s
This work led to the discovery of high-affinity and
selective 5-HT3 receptor ligands. Most of the newly
described compounds show full agonist properties in
functional studies. Binding affinity, selectivity, and
efficacy are dependent upon several structural fea-
tures: 1. the nature of the fused six-membered ring, 2.
the nature of the fused five-membered ring, and 3. the
substituents on the benzo-fused ring and at N-4 of the
piperazine ring. Starting from our lead 3b, we identified
high-affinity ligands endowed with a greatly improved
selectivity for the 5-HT3 receptor, compared to quipazine
(19b, 20b,c,v,a a ). The compounds with pure agonists
properties in functional studies present efficacies well-
correlated to their affinity for the 5-HT3 receptor.
Compounds 20b,f could be considered two of the most
potent 5-HT3 receptor agonists described to date. We
also determined the ability of the new agonists to cross
the blood-brain barrier. The compounds tested differ
in their physicochemical properties, but most are lipo-
philic compounds. Thus, they rapidly enter the brain
and equilibrate between brain and plasma. With the
exception of the polar derivative 20h h , brain concentra-
tions of 20b,l,u ,v,ee are generally higher than plasma
concentrations, although with differences in the brain-
to-blood partition. By virtue of the agonist potencies
displayed in the [14C]guanidinium accumulation test
and of the ability to cross the blood-brain barrier,
pyrroloquinoxalines and pyridopyrrolopyrazines repre-
sent potent pharmacological tools to explore the central
5-HT3 receptor-mediated functions. Accordingly, the
potent agonists 3b and 20b were tested in vivo to assess
anxiolytic-like or analgesic-like properties. No anxio-
lytic-like effect could be detected in the light/dark
exploratory test in mice. However, compound 20b
induces analgesia in rats. Further studies are required
to assess if this effect is related to central 5-HT3 receptor
activation. Furthermore, we have also demostrated that
highly hydrophilic groups at N-4 of the piperazine ring
could be tolerated at the receptor level. In fact, pyrrolo-
quinoxalines bearing a carboxylic or tetrazolyl group
F igu r e 2. Mean plasma and brain concentration-time curves
of selected compounds after intravenous injection in rats. The
dose was 5 mg/kg. Each value is the mean of 2-3 rats.
or hindered fuctions on the piperazine ring. The mean
brain-to-plasma distribution ratios (brain-to-plasma
AUCt ratio) of these compounds are listed in Table 8.
With the exception of compound 20h h (carboxylic func-
tion on the piperazine ring) mean plasma concentrations
were generally low, hardly amounting to about 1 µM at
1 h after dosing (see Figure 2). These concentrations
rapidly declined to levels close to the limits of analytical
procedure within 2 h after injection, except for com-
pound 20u which eliminated more slowly than the other
compounds, although the elimination t1/2 could not be
determined because of the shortage of experimental
points. The different pharmacokinetic behavior of these
derivatives may be the result of differences in clearance
and/or distribution, which could not be evaluated in
these preliminary studies. For all compounds the mean
brain concentrations paralleled those in plasma, within
the time after dosing examined. This suggests that these
compounds rapidly enter the brain and attain equilib-

4372 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
residue was dissolved in dichloromethane. The organic phase
was washed with 1 N HCl and brine, dried, and concentrated.
The residue was chromathographed (dichloromethane and
hexanes, 1/1) to give, after recrystallization, 0.70 g of 8 as
(20h h ,jj) still bind the receptor with a significant
affinity. By virtue of their affinity and of the fact that
20h h and, presumibly, 20jj do not cross the blood-brain
barrier, these analogues represent interesting lead
compounds to develop new peripheral 5-HT3 receptor
agonists.
1
yellow-orange prisms: IR (Nujol) 1530, 1315 cm-1; H NMR
(CDCl3) δ 5.13 (s, 2 H), 6.31 (m, 2 H), 6.71 (m, 2 H), 7.18 (d, 1
H, J ) 2.6 Hz), 7.22 (t, 1 H, J ) 2.37 Hz), 7.33-7.43 (m, 6 H).
Anal. (C17H14N2O3) C, H, N.
Exp er im en ta l P r oced u r es
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
9a -e. This procedure is illustrated for the preparation of 1-(2-
amino-4,5-dimethylphenyl)-1H-pyrrole (9a ). A solution of 7a
(1.0 g, 4.6 mmol) and tin(II) chloride dihydrate (5.36 g, 23.8
mmol) in ethanol (63 mL) was heated to reflux under argon
for 30 min. The mixture was cooled, the pH was adjusted to 8
with saturated sodium bicarbonate solution, and the mixture
was filtered through Celite. The filter cake was washed with
ethanol, and the filtrates were evaporated. The residue was
chromatographed (0.5% methanol in dichloromethane) to give
Melting points were determined using an Electrothermal
8103 apparatus and are uncorrected. IR spectra were taken
with Perkin-Elmer 398 and FT 1600 spectrophotometers. H
1
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 Hz.
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. 2-(Bromo-
methyl)thiophene, 2-(bromomethyl)furan, 2-bromo-5-(bromo-
methyl)thiophene, and 2-bromo-5-(bromomethyl)furan were
prepared starting from 2-methylthiophene and 2-methylfuran
by means of N-bromosuccinimide using catalytic amounts of
azobis(isobutyronitrile) (AIBN).11b Physical data for compounds
7-17 and 18-24 are reported in Tables 1 and 2.
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
7a -e. This procedure is illustrated for the preparation of
1-(4,5-dimethyl-2-nitrophenyl)-1H-pyrrole (7a ). To a solution
of 4,5-dimethyl-2-nitroaniline (6a ) (1.0 g, 6.0 mmol) in glacial
acetic acid (3.9 mL) was slowly added 2,5-dimethoxytetrahy-
drofuran (0.78 mL, 6.0 mmol). The reaction mixture was
refluxed for 1 h, cooled, and evaporated to afford an oily residue
which was chromatographed (20% ethyl acetate in hexanes)
to give 0.97 g of 7a as pale yellow prisms: IR (Nujol) 1599,
1520 cm-1; 1H NMR (CDCl3) δ 2.38 (s, 6H), 6.37 (m, 2H), 6.78
(m, 2H), 7.22 (s,1H), 7.71 (s, 1H). Anal. (C12H12N2O2) C, H, N.
1-(6-Meth yl-2-n itr op h en yl)-1H-p yr r ole (7b). Similarly to
the procedure as described for 7a , the title compound was
prepared starting from 6-methyl-2-nitroaniline (6b). 7b was
obtained as yellow prisms after recrystallization: IR (CHCl3)
1
0.81 g of 9a as red-orange prisms: IR (Nujol) 3400 cm-1; H
NMR (CDCl3) δ 2.18 (s, 6 H), 3.58 (br s, 2 H), 6.36 (m, 2 H),
6.58 (s, 1 H), 6.75 (m, 2 H), 7.03 (m, 1 H). Anal. (C12H14N2) C,
H, N.
1-(2-Am in o-6-m eth ylp h en yl)-1H-p yr r ole (9b). Similarly
to the procedure described for 9a , the title compound was
prepared starting from 7b. After recrystallization 9b was
obtained as a yellowish solid: IR (CHCl3) 3410 cm-1; 1H NMR
(CDCl3) δ 1.88 (s, 3 H), 4.48 (br s, 2 H), 6.27 (m, 2 H), 6.52 (d,
1 H, J ) 7.4 Hz), 6.68 (m, 3 H), 7.03 (t, 1 H, J ) 7.6 Hz). Anal.
(C11H12N2) C, H, N.
1-(2-Am in o-4-cya n op h en yl)-1H-p yr r ole (9c). Starting
from 7c the title compound was obtained following an identical
procedure as for 9a . After recrystallization 9c was obtained
as deep red prisms: IR (CHCl3) 3400, 2240 cm-1 1H NMR
;
(CDCl3) δ 3.98 (br s, 2 H), 6.36 (m, 2 H), 6.82 (m, 2 H), 7.04
(m, 2 H), 7.18 (m, 1 H). Anal. (C11H9N3) C, H, N.
1-(2-Am in o-4-ben zyloxyp h en yl)-1H-p yr r ole (9d ). Simi-
larly to the procedure as described for 9a , the title compound
was prepared starting from 8. 9d was obtained as colorless
prisms after recrystallization: IR (CHCl3) 3405 cm-1; 1H NMR
(CDCl3) δ 3.64 (br s, 2 H), 5.03 (s, 2 H), 6.29 (m, 2 H), 6.38 (m,
2 H), 6.75 (m, 2 H), 7.04 (m, 1 H), 7.28-7.40 (m, 5 H). Anal.
(C17H16N2O) C, H, N.
1-(2-Am in o-4,5-d ich lor op h en yl)-1H-p yr r ole (9e). Start-
ing from 7e the title compound was obtained as described for
9a . After recrystallization 9e was obtained as colorless
prisms: IR (CHCl3) 3400 cm-1; 1H NMR (CDCl3) δ 3.81 (br s,
2 H), 6.35 (m, 2 H), 6.78 (m, 2 H), 6.89 (s, 1 H), 7.23 (s, 1 H).
Anal. (C10H8Cl2N2) C, H, N.
1515 cm-1 1H NMR (CDCl3) δ 2.16 (s, 3 H), 6.35 (m, 2 H),
;
6.66 (m, 2 H), 7.39-7.56 (m, 2 H), 7.67 (d, 1 H, J ) 7.7 Hz).
Anal. (C11H10N2O2) C, H, N.
1-(4-Cyan o-2-n itr oph en yl)-1H-pyr r ole (7c). Starting from
4-cyano-2-nitroaniline (6c) the title compound was obtained
as for 7a . After recrystallization 7c was obtained as red-orange
prisms: IR (CHCl3) 2230, 1340 cm-1; 1H NMR (CDCl3) δ 6.39
(m, 2 H), 6.77 (m 2 H), 7.74 (m, 2 H), 7.86 (m, 1 H). Anal.
(C11H7N3O2) C, H, N.
(3-Nitr op yr id in -2-yl)ca r ba m ic Acid Ben zyl Ester (12).
To a mixture of 2-amino-3-nitropyridine (5.0 g, 36.0 mmol) in
dichloromethane (18 mL) was added a solution of bis(trimeth-
ylsilyl)acetamide (10.5 mL, 43.2 mmol) in dichloromethane (18
mL), and the resulting solution was stirred under argon at
room temperature for 1 h. The reaction mixture was cooled to
0 °C, and a solution of benzyl chloroformate (13.4 mL, 93.6
mmol) in dichloromethane (39 mL) was added dropwise. The
reaction was stirred at room temperature for 25 h. The mixture
was cooled again to 0 °C, quenched with a pH 7 buffer, and
extracted with diethyl ether. The ether extract was washed
with brine, dried, and concentrated. The residue was chro-
matographed (10% ethyl acetate in dichloromethane) to give
1-(4-Hyd r oxy-2-n itr op h en yl)-1H-p yr r ole (7d ). Similarly
to the procedure as described for 7a , the title compound was
prepared starting from 4-hydroxy-2-nitroaniline (6d ). 7d was
obtained as a dark brown solid: IR (CHCl3) 3620, 1580, 1335
1
cm-1; H NMR (CDCl3) δ 5.90 (br s, 1 H), 6.30 (m, 2 H), 6.72
(m, 2 H), 7.08 (dd, 1 H, J ) 2.6, 8.3 Hz), 7.30 (m, 2 H). Anal.
(C10H8N2O3) C, H, N.
7.65 g of 12 as a colorless oil: IR (neat) 3350, 1670, 1559 cm-1
;
1H NMR (CDCl3) δ 5.25 (s, 2 H), 7.17 (dd, 1 H, J ) 4.7, 7.7
Hz), 7.34-7.40 (m, 5 H), 8.46 (dd, 1 H, J ) 1.8, 7.6 Hz), 8.71
(dd, 1 H, J ) 1.7, 4.9 Hz), 9.81 (br s, 1 H). Anal. (C13H11N3O4)
C, H, N.
1-(4,5-Dich lor o-2-n itr op h en yl)-1H-p yr r ole (7e). Starting
from 4,5-dichloro-2-nitroaniline (6e) the title compound was
obtained following a procedure as described for 7a . After
recrystallization, 7e was obtained as colorless prisms: IR
1
(CHCl3) 1510, 1340 cm-1; H NMR (CDCl3) δ 6.37 (m, 2 H),
(3-Am in op yr id in -2-yl)ca r ba m ic Acid Ben zyl Ester (13).
Similarly to the procedure as described for 9a , the title
compound was prepared starting from 12. After recrystalli-
zation 13 was obtained as colorless prisms: IR (CHCl3) 3410,
6.75 (m 2 H), 7.59 (s, 1 H), 7.99 (s, 1 H). Anal. (C10H6Cl2N2O2)
C, H, N.
1-(4-Ben zyloxy-2-n itr op h en yl)-1H-p yr r ole (8). A solu-
tion of 7d (0.5 g, 2.45 mmol) and benzyl chloride (0.42 mL,
3.67 mmol) in DMF (4.28 mL) was treated with K2CO3 (0.67
g, 4.9 mmol), and the resulting mixture was heated at 60 °C
overnight. After cooling, the solvent was evaporated and the
1
1660 cm-1; H NMR (CDCl3) δ 4.26 (br s, 2 H), 5.19 (s, 2 H),
6.94 (dd, 1 H, J ) 4.8, 7.6 Hz), 7.05 (dd, 1 H, J ) 1.6, 7.8 Hz),
7.33-7.42 (m, 6 H), 7.76 (d, 1 H, J ) 3.88 Hz). Anal.
(C13H13N3O2) C, H, N.

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4373
1-[2-(Ben zyloxyca r b on yla m in o)p yr id in -3-yl]-1H -p yr -
r ole (14). Starting from 13 the title compound was obtained
following an identical procedure as for 7a , except that the oily
residue was partitioned between saturated NaHCO3 solution
and ethyl acetate, and the organic layer was washed with
brine, dried, and concentrated. After recrystallization, 14 was
recrystallization 10f was obtained as colorless prisms: IR
(Nujol) 3200, 1670 cm-1; 1H NMR (DMSO-d6) δ 6.32 (m, 1 H),
6.74 (m, 1 H), 7.02 (m, 1 H), 7.22 (dd, 1 H, J ) 4.6, 7.9 Hz),
8.23 (dd, 1 H, J ) 1.8, 7.6 Hz), 8.50 (dd, 1 H, J ) 1.7, 4.7 Hz),
11.71 (br s, 1 H). Anal. (C10H7N3O) C, H, N.
7-F lu or oim id a zo[1,2-a ]qu in oxa lin -4(5H)-on e (10g). A
mixture of the amino derivative 17 (0.74 g, 4.18 mmol) and
N,N-carbonyldiimidazole (0.73 g, 4.57 mmol) in 1,2-dichlo-
robenzene (31.7 mL) was heated at reflux under argon for 1.5
h. After cooling to room temperature, the solid was filtered
off and washed with acetone to give, after recrystallization,
0.42 g of 10g as brown prisms: IR (CHCl3) 1660 cm-1; 1H NMR
(DMSO-d6) δ 7.14 (m, 2 H), 7.59 (s, 1 H), 8.15 (m, 1 H), 8.51
(s, 1 H), 11.90 (br s, 1 H). Anal. (C10H6FN3O) 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
18a -g,j-m . This procedure is illustrated for the preparation
of 4-chloro-7,8-dimethylpyrrolo[1,2-a]quinoxaline (18a ). A mix-
ture of 10a (0.30 g, 1.4 mmol), N,N-dimethylaniline (0.35 mL),
and phosphorus oxychloride (4.7 mL) was heated at reflux
under argon for 5 h. The excess POCl3 was removed under
vacuum, and the residue was dissolved in dichloromethane,
washed with brine, dried, and concentrated. The residue was
chromatographed (10% EtOAc in dichloromethane) and re-
crystallized to give 0.28 g of 18a as colorless prisms: IR
(CHCl3) 3320, 1599 cm-1; 1H NMR (CDCl3) δ 2.38 (s, 3 H), 2.45
(s, 3 H), 6.85 (t, 1 H, J ) 3.4 Hz), 7.01 (d, 1 H, J ) 3.8 Hz),
7.59 (s, 1 H), 7.65 (s, 1 H), 7.90 (d, 1 H, J ) 2.9 Hz). Anal.
(C13H11ClN2) C, H, N.
1
obtained as colorless prisms: IR (Nujol) 3360, 1680 cm-1; H
NMR (CDCl3) δ 5.15 (s, 2 H), 6.38 (m, 2 H), 6.80 (m, 2 H), 6.95
(br s, 1 H), 7.13 (dd, 1 H, J ) 4.7, 7.6 Hz), 7.26-7.34 (m, 5 H),
7.58 (dd, 1 H, J ) 1.6, 7.8 Hz), 8.47 (dd, 1 H, J ) 1.7, 4.8 Hz).
Anal. (C17H15N3O2) C, H, N.
1-(4-F lu or o-2-n itr op h en yl)-1H-im id a zole (16). A solu-
tion of 2,5-difluoronitrobenzene 15 (2.0 g, 12.6 mmol) and
imidazole (2.56 g, 37.8 mmol) in dimethyl sulfoxide (23 mL)
was heated to 50 °C under an argon atmosphere for 24 h. The
mixture was then cooled to room temperature, poured into
water, and extracted with ethyl acetate. The organic layer was
washed with brine, dried, and concentrated. The residue was
flash-chromatographed (5% methanol in dichloromethane) to
afford 1.25 g of 16 that after recrystallization (hexanes) was
obtained as yellow-orange prisms: IR (Nujol): 1620, 1549,
1335 cm-1; 1H NMR (CDCl3) δ 7.03 (s, 1 H), 7.20 (s, 1 H), 7.44
(m, 2 H), 7.59 (s, 1 H), 7.73 (dd, 1 H, J ) 2.8, 8.8 Hz). Anal.
(C9H6FN3O2) C, H, N.
1-(2-Am in o-4-flu or op h en yl)-1H-im id a zole (17). Simi-
larly to the procedure as described for 9a , the title compound
was prepared starting from 16. After recrystallization 17 was
1
obtained as yellow prisms: IR (CHCl3) 3400, 1630 cm-1; H
NMR (CDCl3) δ 3.78 (br s, 2 H), 6.48 (m, 2 H), 7.05 (m, 2 H),
4-Ch lor o-9-m eth ylpyr r olo[1,2-a ]qu in oxalin e (18b). Start-
ing from 10b the title compound was obtained following the
synthetic procedure described for 18a . After recrystallization
18b was obtained as pale yellow prisms: IR (CHCl3) 3300,
7.21 (s, 1 H), 7.56 (s, 1 H). Anal. (C9H8FN3) 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
10a -f. This procedure is illustrated for the preparation of 7,8-
dimethylpyrrolo[1,2-a]quinoxalin-4(5H)-one (10a ). To a solu-
tion of 9a (0.81 g, 4.36 mmol) in toluene (9.5 mL) was added
triphosgene (0.43 g, 1.45 mmol). The reaction mixture was
refluxed for 30 min, and nitrogen was bubbled in to drive off
excess of phosgene. The solution was then set aside for 18 h.
The heavy crystalline precipitate was filtered off and washed
with hexanes to give, after recrystallization (EtOAc), 0.79 g
1
1601 cm-1; H NMR (CDCl3) δ 2.87 (s, 3 H), 6.82 (t, 1 H, J )
3.4 Hz), 7.04 (d, 1 H, J ) 3.4 Hz), 7.27 (m, 2 H), 7.72 (m, 1 H),
8.27 (t, 1 H, J ) 2.2 Hz). Anal. (C12H9ClN2) C, H, N.
4-Ch lor o-7-cya n op yr r olo[1,2-a ]qu in oxa lin e (18c). Simi-
larly to the procedure described for 18a , the chloro derivate
18c was prepared starting from 10c. After recrystallization
18c was obtained as colorless prisms: IR (Nujol) 3315, 1600
of 10a as colorless prisms: IR (Nujol) 3280, 1693 cm-1 1H
;
1
cm-1; H NMR (CDCl3) δ 6.99 (m, 1 H), 7.15 (d, 1 H, J ) 3.7
NMR (DMSO-d6) δ 2.22 (s, 3 H), 2.27 (s, 3 H), 6.62 (t, 1 H,
J ) 3.3 Hz), 6.96 (d, 1 H, J ) 3.8 Hz), 7.03 (s, 1 H), 7.82 (s, 1
H), 8.07 (m, 1H), 11.06 (br s, 1 H). Anal. (C13H12N2O) C, H, N.
9-Meth ylpyr r olo[1,2-a ]qu in oxalin -4(5H)-on e (10b). Simi-
larly to the procedure as described for 10a , the title compound
was prepared starting from 9b. After recrystallization, 10b
Hz), 7.75 (dd, 1 H, J ) 1.6, 7.8 Hz), 7.91 (d, 1 H, J ) 8.7 Hz),
8.01 (d, 1 H, J ) 3.0 Hz), 8.19 (d, 1 H, J ) 1.8 Hz). Anal. (C12H6-
ClN2) C, H, N.
7-Ben zyloxy-4-ch lor op yr r olo[1,2-a ]qu in oxa lin e (18d ).
Starting from 10d the title compound was obtained as for 18a .
After recrystallization 18d was obtained as colorless prisms:
IR (CHCl3) 2990, 1605 cm-1; 1H NMR (CDCl3) δ 5.14 (s, 2 H),
6.84 (t, 1 H, J ) 3.3 Hz), 7.01 (d, 1 H, J ) 3.8 Hz), 7.17-7.46
was obtained as colorless prisms: IR (Nujol) 3220, 1660 cm-1
;
1H NMR (DMSO-d6) δ 2.80 (s, 3 H), 6.70 (t, 1 H, J ) 3.4 Hz),
7.04-7.21 (m, 4 H), 8.19 (d, 1 H, J ) 2.7 Hz), 11.26 (br s,
1 H). Anal. (C12H10N2O) C, H, N.
(m, 7 H), 7.73 (d, 1 H, J ) 9.0 Hz), 7.87 (m, 1 H). Anal. (C18H13
ClN2O) C, H, N.
-
7-Cyan opyr r olo[1,2-a ]qu in oxalin -4(5H)-on e (10c). Start-
ing from 9c the title compound was obtained as for 10a . After
recrystallization 10c was obtained as colorless prisms: IR
4,7,8-Tr ich lor op yr r olo[1,2-a ]qu in oxa lin e (18e). Simi-
larly to the procedure described for 18a , the chloro derivate
18e was prepared starting from 10e. After recrystallization
18e was obtained as colorless prisms: IR (Nujol) 3120, 1599
1
(Nujol) 2230, 1655 cm-1; H NMR (DMSO-d6) δ 6.79 (t, 1 H,
1
cm-1; H NMR (CDCl3) δ 6.91 (t, 1 H, J ) 3.4 Hz), 7.06 (d, 1
J ) 3.4 Hz), 7.13 (d, 1 H, J ) 3.9 Hz), 7.61 (d, 1 H, J ) 1.8
Hz), 7.70 (dd, 1 H, J ) 1.6, 7.8 Hz), 8.30 (m, 2 H), 11.53 (br s,
1 H). Anal. (C12H7N3O) C, H, N.
H, J ) 3.7 Hz), 7.87 (d, 1 H, J ) 2.9 Hz), 7.90 (s, 1 H), 7.96 (s,
1 H). Anal. (C11H5Cl3N2) C, H, N.
7-(Ben zyloxy)pyr r olo[1,2-a ]qu in oxalin -4(5H)-on e (10d).
Similarly to the procedure as described for 10a , the title
compound was prepared starting from 9d . After recrystalli-
zation, 10d was obtained as colorless prisms: IR (Nujol) 1665
cm-1; 1H NMR (DMSO-d6) δ 5.14 (s, 2 H), 6.63 (t, 1 H, J ) 2.9
Hz), 6.93 (m, 3 H), 7.32-7.50 (m, 5 H), 7.96 (d, 1 H, J ) 8.5
Hz), 8.09 (m, 1 H), 11.38 (br s, 1 H). Anal. (C18H14N2O2) C, H,
N.
6-Ch lor opyr ido[2,3-e]pyr r olo[1,2-a ]pyr azin e (18f). Simi-
larly to the procedure described for 18a , the chloro derivate
18f was prepared starting from 10f. After recrystallization 18f
was obtained as colorless prisms: IR (CHCl3) 1685, 1600 cm-1
;
1H NMR (CDCl3) δ 6.92 (dd, 1 H, J ) 2.3, 4.1 Hz), 7.08 (t, 1 H,
J ) 2.8 Hz), 7.45 (dd, 1 H, J ) 4.8, 7.8 Hz), 7.99 (d, 1 H, J )
3.2 Hz), 8.16 (dd, 1 H, J ) 1.7, 7.6 Hz), 8.71 (dd, 1 H, J ) 1.8,
4.8 Hz). Anal. (C10H6ClN3) C, H, N.
7,8-Dich lor op yr r olo[1,2-a ]qu in oxa lin -4(5H)-on e (10e).
Starting from 9e the title compound was obtained as for 10a .
After recrystallization, 10e was obtained as colorless prisms:
IR (Nujol) 1685 cm-1; 1H NMR (DMSO-d6) δ 6.34 (t, 1 H, J )
3.4 Hz), 6.86 (d, 1 H, J ) 3.8 Hz), 6.93 (s, 1 H), 7.72 (s, 1 H),
8.02 (m, 1 H), 11.02 (br s, 1 H). Anal. (C11H6Cl2N2O) C, H, N.
5,6-Dih yd r o-6-oxop yr id o[2,3-e]p yr r olo[1,2-a ]p yr a -
zin e (10f). Starting from 14 the title compound was obtained
as for 10a , except that the reaction was refluxed for 1.5 h. After
4-Ch lor o-7-flu or oim idazo[1,2-a ]qu in oxalin e (18g). Start-
ing from 10g the title compound was obtained as for 18a . After
recrystallization 18g was obtained as colorless prisms: IR
(CHCl3) 1599 cm-1; 1H NMR (CDCl3) δ 7.44 (m, 1 H), 7.74 (dd,
1 H, J ) 2.8, 8.8 Hz), 7.85 (m, 2 H), 8.14 (s, 1 H). Anal. (C10H5-
FClN3) C, H, N.
4-Ch lor o-6,7-dih ydr opyr r olo[1,2-a ]pyr azin e (18j). Start-
ing from 10j, following an identical procedure as for 18a , the
title compound was obtained as a colorless thick oil: IR (Nujol)

4374 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
1
1599 cm-1; H NMR (CDCl3) δ 3.59 (m, 2 H), 4.15 (m, 2 H),
layer was washed with brine, dried, and concentrated. The
residue was flash chromatographed (20% EtOAc in dicloro-
methane) to give 0.23 g of 24 as colorless prisms: IR (Nujol)
6.88 (m, 2 H), 6.74 (m, 1 H). Anal. (C7H7ClN2) C, H, N.
4-Ch lor oim id a zo[1,2-a ]qu in oxa lin e (18k ). Similarly to
the procedure described for 18a , the chloro derivate 18k was
prepared starting from 10k . 18k was obtained as colorless
prisms: IR (CHCl3) 3250, 1600 cm-1; 1H NMR (CDCl3) δ 7.23
(s, 1 H), 7.72-7.84 (m, 3 H), 8.06 (m, 1 H), 8.16 (s, 1 H). Anal.
(C10H6ClN3) C, H, N.
6-Ch lor op yr a zolo[1,5-c]qu in a zolin e (18l). Similarly to
the procedure described for 18a , the chloro derivate 18l was
prepared starting from 10l. 18l was obtained as colorless
prisms after recrystallization: IR (CHCl3) 1599 cm-1; 1H NMR
(CDCl3) δ 7.08 (d, 1 H, J ) 1.9 Hz), 7.65 (m, 2 H), 7.90 (d, 1 H,
J ) 8.4 Hz), 8.03 (m, 1 H), 8.13 (d, 1 H, J ) 1.9 Hz). Anal.
(C10H6ClN3) C, H, N.
6-Ch lor oim id a zo[1,2-a ]p yr id o[2,3-e]p yr a zin e (18m ).
Starting from 10m the title compound was obtained as for 18a .
After recrystallization 18m was obtained as colorless prisms:
IR (CHCl3) 1630, 1599 cm-1; 1H NMR (CDCl3) δ 7.61 (dd, 1 H,
J ) 4.7, 7.7 Hz), 7.86 (s, 1 H), 8.37 (dd, 1 H, J ) 1.7, 7.7 Hz),
8.57 (s, 1 H), 8.71 (dd, 1 H J ) 1.5, 7.9 Hz). Anal. (C9H5ClN4)
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
19a -c,g. This procedure is illustrated for the preparation of
7,8-dimethyl-4-(piperazin-1-yl)pyrrolo[1,2-a]quinoxaline (19a ).
A mixture of 18a (1.0 g, 4.34 mmol) and dry piperazine (3.86
g, 44.4 mmol) in ethylene glycol (38 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 (50% dichlorometane in methanol) to
give 1.11 g of 19a as colorless prisms: IR (neat) 3340, 1605
1
1680, 1620 cm-1; H NMR (CDCl3) δ 2.79 (m, 4 H), 3.35 (s, 2
H), 3.87 (m, 4 H), 5.17 (s, 2 H), 6.78 (m, 2 H), 7.01 (m, 1 H),
7.18-7.45 (m, 7 H), 8.16 (m, 1 H). Anal. (C24H23FN4O2) 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
20a -t,v,w . This procedure is illustrated for the preparation
of 7,8-dimethyl-4-(4-methylpiperazin-1-yl)pyrrolo[1,2-a]quin-
oxaline (20a ). A mixture of 18a (0.2 g, 0.87 mmol) and
N-methylpiperazine (2 mL) was heated at 130 °C for 6 h, under
argon. Then the reaction mixture was cooled, poured into
crushed ice, and extracted with EtOAc. The combined organic
extracts were washed with brine, dried, and concentrated. The
residue was flash chromatographed (EtOAc) to give, after
recrystallization, 0.22 g of 20a as colorless prisms: IR (CHCl3)
1600 cm-1; 1H NMR (CDCl3) δ 2.33 (s, 3 H), 2.37 (s, 3 H), 2.38
(s, 3 H), 2.62 (m, 4 H), 3.77 (m, 4 H), 6.72 (m, 2 H), 7.46 (s, 1
H), 7.48 (s, 1 H), 7.75 (t, 1 H, J ) 1.7 Hz). Anal. (C18H22N4) C,
H, N.
9-Me t h yl-4-(4-m e t h ylp ip e r a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20b). Starting from 18b the title compound was
obtained following a similar procedure as described for 20a .
After recrystallization, 20b was obtained as colorless prisms:
1
IR (CHCl3) 1590 cm-1; H NMR (CDCl3) δ 2.35 (s, 3 H), 2.59
(m, 4 H), 2.83 (s, 3 H), 3.76 (m, 4 H), 6.70 (t, 1 H, J ) 2.3 Hz),
6.77 (d, 1 H, J ) 3.8 Hz), 7.04 (d, 1 H, J ) 7.0 Hz), 7.19 (m, 1
H), 7.54 (dd, 1 H, J ) 1.1, 7.8 Hz), 8.23 (m, 1 H). Anal. (C17H20
N4) C, H, N.
-
7-Cya n o-4-(4-m e t h ylp ip e r a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20c). Similarly to the procedure described for
20a , the title compound was prepared starting from 18c. After
recrystallization, 20c was obtained as colorless prisms: IR
(Nujol) 2225, 1600 cm-1; 1H NMR (CDCl3) δ 2.36 (s, 3 H), 2.58
(m, 4 H), 3.89 (m, 4 H), 6.81 (m, 2 H), 7.42 (dd, 1 H, J ) 1.6,
7.8 Hz), 7.70 (d, 1 H, J ) 8.6 Hz), 7.78 (d, 1 H, J ) 2.5 Hz),
7.88 (m, 1 H). Anal. (C17H17N5) C, H, N.
7-Ben zyloxy-4-(4-m eth ylp ip er a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20d ). Starting from 18d the title compound was
obtained following the procedure as described for 20a . After
recrystallization, 20d was obtained as colorless prisms: IR
(Nujol) 1590 cm-1; 1H NMR (CDCl3) δ 2.53 (s, 3 H), 2.88 (m, 4
H), 4.02 (m, 4 H), 5.13 (s, 2 H), 6.72 (m, 2 H), 6.96 (dd, 1 H,
J ) 2.6, 8.3 Hz), 7.31-7.47 (m, 6 H), 7.63 (d, 1 H, J ) 8.9 Hz),
7.74 (d, 1 H, J ) 1.5 Hz). Anal. (C23H24N4O) C, H, N.
cm-1; H NMR (CDCl3) δ 2.32 (s, 3 H), 2.38 (s, 3 H), 2.64 (m,
4 H), 3.78 (m, 4 H), 6.75 (m, 2 H), 7.47 (s, 1 H), 7.48 (s, 1 H),
7.75 (t, 1 H, J ) 1.8 Hz). Anal. (C17H20N4) C, H, N.
9-Meth yl-4-(p ip er a zin -1-yl)p yr r olo[1,2-a ]qu in oxa lin e
(19b). Starting from 18b the title compound was obtained as
for 19a . After recrystallization 19b was obtained as colorless
prisms: IR (CHCl3) 3340, 1601 cm-1; 1H NMR (CDCl3) δ 2.83
(s, 3 H), 3.08 (m, 4 H), 3.71 (m, 4 H), 6.73 (t, 1 H, J ) 3.4 Hz),
6.79 (d, 1 H, J ) 3.7 Hz), 7.08 (m, 1 H), 7.21 (m, 1 H), 7.57 (m,
1 H), 8.13 (d, 1 H, J ) 2.8 Hz); MS m/z 266 (M+), 210, 198
(100), 183, 154. Anal. (C16H18N4) C, H, N.
1
7-Meth yl-4-(p ip er a zin -1-yl)p yr r olo[1,2-a ]qu in oxa lin e
(19c). Similarly to the procedure described for 19a , the title
compound was prepared starting from 18h . After recrystalli-
zation 19c was obtained as a white solid: IR (CHCl3) 3325,
7,8-Dich lor o-4-(4-m eth ylp ip er a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20e). Similarly to the procedure described for
20a , the title compound was prepared starting from 18e. After
recrystallization, 20e was obtained as colorless prisms: IR
(Nujol) 1585 cm-1; 1H NMR (CDCl3) δ 2.35 (s, 3 H), 2.57 (m, 4
1599 cm-1 1H NMR (CDCl3) δ 2.45 (s, 3 H), 2.68 (m, 4 H),
;
3.84 (m, 4 H), 6.75 (m, 2 H), 7.08 (m, 1 H), 7.45 (m, 1 H), 7.56
(d, 1 H, J ) 8.3 Hz), 7.71 (m, 1 H). Anal. (C16H18N4) C, H, N.
6-(P ip e r a zin -1-yl)p yr id o[2,3-e]p yr r olo[1,2-a ]p yr a -
zin e (19g). Similarly to the procedure described for 19a , the
title compound was prepared starting from 18f. After recrys-
tallization 19g was obtained as colorless prisms: IR (CHCl3)
3330, 1685, 1601 cm-1; 1H NMR (CDCl3) δ 3.14 (m, 4 H), 3.84
(m, 4 H), 6.75 (t, 1 H, J ) 3.4 Hz), 6.83 (t, 1 H, J ) 1.9 Hz),
7.26 (dd, 1 H, J ) 4.6, 7.9 Hz), 7.87 (dd, 1 H, J ) 1.8, 7.6 Hz),
8.26 (m, 2 H). Anal. (C14H15N5) C, H, N.
H), 3.85 (m, 4 H), 6.76 (m, 2 H), 7.70 (m, 3 H). Anal. (C16H16
Cl2N4) C, H, N.
-
7-H yd r oxy-4-(4-m et h ylp ip er a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20f). A solution of 20d (0.1 g, 0.27 mmol) in
THF (1 mL) was hydrogenated at atmospheric pressure over
10% Pd-C (1.62 mg) overnight. The catalyst was removed by
filtration, the solvent was evaporated, and the residue was
recrystallized to give 74 mg of 20f as colorless prisms: IR
(Nujol) 3610, 1595 cm-1; 1H NMR (CDCl3) δ 2.32 (s, 3 H), 2.48
(br s, 1 H), 2.59 (m, 4 H), 3.75 (m, 4 H), 6.51 (m, 1 H), 6.70 (m,
2 H), 6.93 (m, 1 H), 7.20 (m, 1 H), 7.48 (m, 1 H). Anal.
(C16H18N4O) C, H, N.
7-Me t h yl-4-(4-m e t h ylp ip e r a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20g). Starting from 18h the title compound was
obtained following the procedure described for 20a . After
recrystallization, 20g was obtained as colorless prisms: IR
(CHCl3) 1600 cm-1; 1H NMR (CDCl3) δ 2.37 (s, 3 H), 2.42 (s, 3
H), 2.61 (m, 4 H), 3.82 (m, 4 H), 6.69-6.75 (m, 2 H), 7.07 (m,
1 H), 7.49 (m, 1 H), 7.58 (d, 1 H, J ) 8.5 Hz), 7.75 (d, 1 H,
J ) 3.2 Hz). Anal. (C17H20N4) C, H, N.
8-Ch lor o-2,3-d ih yd r oim id a zo[1,2-a ]p yr a zin e H yd r o-
ch lor id e (23). A solution of 22 (0.93 g, 5.36 mmol) in thionyl
chloride (2.3 mL) and chloroform (2.9 mL) was heated under
reflux for 2 h. After cooling, the solid was collected and washed
with ethyl ether to give 0.64 g of 23 as a pale yellow solid: IR
(CHCl3) 3380 cm-1
;
1H NMR (DMSO-d6) δ 4.03 (t, 2 H, J )
10.8 Hz), 4.78 (t, 2 H, J ) 10.3 Hz), 7.86 (d, 1 H, J ) 4.0 Hz),
8.39 (d, 1 H, J ) 3.9 Hz), 10.71 (br s, 1 H). Anal. (C6H7Cl2N3)
C, H, N.
r-[4-(9-F lu or op yr r olo[1,2-a ]qu in oxa lin -1-yl)p ip er a zin -
1-yl]a cetic Acid Ben zyl Ester (24). A suspension of 19d (0.2
g, 0.74 mmol), anhydrous potassium carbonate (96 mg, 0.74
mmol), and benzyl bromoacetate (0.17 g, 0.74 mmol) in ethyl
methyl ketone (33 mL) was heated at reflux for 1 h under
argon. The solvent was evaporated, and the residue was
partitioned between water and dichloromethane. The organic
4-(4-Meth ylp ip er a zin -1-yl)-6,7-d ih yd r op yr r olo[1,2-a ]-
p yr a zin e (20h ). Starting from 18j the title compound was
obtained following the procedure described for 20a . After

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4375
recrystallization, 20h was obtained as a white solid: IR
J ) 7.4 Hz), 7.16-7.39 (m, 6 H), 7.55 (dd, 1 H, J ) 1.1, 7.8
Hz), 8.11 (d, 1 H, J ) 2.7). Anal. (C23H24N4) C, H, N.
1
(CHCl3) 1599 cm-1; H NMR (CDCl3) δ 2.31 (s, 3 H), 2.45 (m,
4 H), 3.50 (m, 4 H), 3.63 (m, 2 H), 4.12 (m, 2 H), 6.73 (m, 1 H),
6.94 (m, 2 H). Anal. (C12H18N4) C, H, N.
7,8-Dich lor o-4-(4-ben zylp ip er a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20r ). Starting from 18e and N-benzylpiperazine
the title compound was obtained following the procedure
described for 20a . After recrystallization, 20r was obtained
as colorless prisms: IR (Nujol) 1595 cm-1; 1H NMR (CDCl3) δ
2.61 (m, 4 H), 3.57 (s, 2 H), 3.84 (m, 4 H), 6.76 (m, 2 H), 7.22-
7.37 (m, 5 H), 7.71 (m, 3 H). Anal. (C22H20Cl2N4) C, H, N.
7-F lu or o-4-(4-b e n zylp ip e r a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20s). Similarly to the procedure described for
20a , the title compound was prepared starting from 18n and
N-benzylpiperazine. After recrystallization, 20s was obtained
as colorless prisms: IR (Nujol) 1610 cm-1; 1H NMR (CDCl3) δ
2.59 (m, 4 H), 3.63 (s, 2 H), 3.85 (m, 4 H), 6.76 (m, 2 H), 6.98
(m, 1 H), 7.18-7.29 (m, 5 H), 7.32 (m, 1 H), 7.65 (m, 1 H),
7.78 (m, 1 H). Anal. (C22H21FN4) C, H, N.
4-(4-Met h ylp ip er a zin -1-yl)im id a zo[1,2-a ]q u in oxa lin e
(20i). Similarly to the procedure described for 20a , the title
compound was prepared starting from 18k . After recrystalli-
zation 20i was obtained as colorless prisms: IR (Nujol) 1625,
1599 cm-1 1H NMR (CDCl3) δ 2.37 (s, 3 H), 2.63 (m, 4 H),
;
4.45 (m, 4 H), 6.61 (d, 1 H, J ) 1.7 Hz), 7.23-7.45 (m, 2 H),
7.70 (dd, 2 H, J ) 2.8, 7.8 Hz), 7.96 (s, 1 H). Anal. (C15H17N5)
C, H, N.
7-F lu or o-4-(4-m e t h ylp ip e r a zin -1-yl)im id a zo[1,2-a ]-
qu in oxa lin e (20j). Starting from 18g the title compound was
obtained following the procedure as for 20a . After recrystal-
lization, 20j was obtained as colorless prisms: IR (CHCl3) 1599
cm-1; 1H NMR (CDCl3) δ 2.35 (s, 3 H), 2.57 (m, 4 H), 4.44 (m,
4 H), 6.95 (m, 1 H), 7.32 (dd, 1 H, J ) 2.8, 8.8 Hz), 7.58 (m, 2
H), 7.89 (s, 1 H). Anal. (C15H16FN5) C, H, N.
9-F lu or o-4-(4-b e n zylp ip e r a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20t). Starting from 18o and N-benzylpiperazine
the title compound was obtained following the procedure as
for 20a . After recrystallization, 20t was obtained as colorlesss
6-(4-Met h ylp ip er a zin -1-yl)p yr a zo[1,5-c]q u in a zolin e
(20k ). Starting from 18l the title compound was obtained
following a procedure described for 20a . After recrystallization
1
prisms: IR (Nujol) 1600 cm-1; H NMR (CDCl3) δ 2.63 (m, 4
20k was obtained as colorless prisms: IR (CHCl3) 1600 cm-1
;
H), 3.58 (s, 2 H), 3.82 (m, 4 H), 6.73 (t, 1 H, J ) 3.4 Hz), 6.80
(d, 1 H, J ) 3.8 Hz), 6.98 (m, 1 H), 7.13-7.44 (m, 7 H), 8.16
(d, 1 H, J ) 2.6 Hz). Anal. (C22H21FN4) C, H, N.
1H NMR (CDCl3) δ 2.38 (s, 3 H), 2.67 (m, 4 H), 4.02 (m, 4 H),
6.90 (d, 1 H, J ) 1.9 Hz), 7.33 (m, 1 H), 7.53 (m 1 H), 7.69 (d,
1 H, J ) 8.0 Hz), 7.90 (dd, 1 H, J ) 1.4, 7.8 Hz), 7.97 (d, 1 H,
J ) 1.9 Hz). Anal. (C15H17N5) C, H, N.
9-Flu or o-4-[4-(2-n aph th ylm eth yl)piper azin -1-yl]pyr r olo-
[1,2-a ]qu in oxa lin e (20u ). Similarly to the procedure as
described for 20a , the title compound was prepared starting
from 18o and 2-(bromomethyl)naphthalene. After recrystal-
lization, 20u was obtained as colorless prisms: IR (Nujol) 1605
cm-1; 1H NMR (CDCl3) δ 2.70 (m, 4 H), 3.73 (s, 2 H), 3.88 (m,
4 H), 6.75 (m, 2 H), 6.98 (m, 1 H), 7.35 (m, 1 H), 7.43-7.68
(m, 4 H), 7.73-7.86 (m, 5 H). Anal. (C26H23FN4) C, H, N.
6-(4-Ben zylp ip er a zin -1-yl)p yr id o[2,3-e]p yr r olo[1,2-a ]-
p yr a zin e (20v). Similarly to the procedure described for 20a ,
the title compound was prepared starting from 18f and
N-benzylpiperazine. After recrystallization 20v was obtained
6-(4-Meth ylp ip er a zin -1-yl)p yr id o[2,3-e]p yr r olo[1,2-a ]-
p yr a zin e (20l). Similarly to the procedure described for 20a ,
the title compound was prepared starting from 18f. After
recrystallization 20l was obtained as colorless prisms: IR
(Nujol) 1601 cm-1; 1H NMR (CDCl3) δ 2.34 (s, 3 H), 2.54 (m, 4
H), 4.02 (m, 4 H), 6.76 (m, 1 H), 6.83 (m, 1 H), 7.12 (dd, 1 H,
J ) 4.7, 7.6 Hz), 7.79 (m, 1 H), 7.95 (dd, 1 H, J ) 1.7, 7.3 Hz),
8.50 (dd, 1 H, J ) 1.9, 4.5 Hz). Anal. (C15H17N5) C, H, N.
6-(4-Meth ylp ip er a zin -1-yl)im id a zo[1,2-a ]p yr id o[2,3-e]-
p yr a zin e (20m ). Similarly to the procedure described for 20a ,
the title compound was prepared starting from 18m . After
recrystallization 20m was obtained as colorless prisms: IR
(Nujol) 1680, 1600 cm-1; 1H NMR (CDCl3) δ 2.34 (s, 3 H), 2.57
(m, 4 H), 4.45 (m, 4 H), 7.34 (dd, 1 H, J ) 4.2, 7.8 Hz), 7.58 (s,
1 H), 7.91 (dd, 1 H, J ) 1.7, 7.5 Hz), 8.26 (dd, 1 H, J ) 1.4, 4.6
Hz), 8.36 (s, 1 H). Anal. (C14H16N6) C, H, N.
2,3-Dih yd r o-8-(4-m eth ylp ip er a zin -1-yl)im id a zo[1,2-a ]-
p yr a zin e (20n ). Similarly to the procedure described for 20a ,
the title compound was prepared starting from 23. After
recrystallization 20n was obtained as colorless prisms: IR
(Nujol) 1630, 1599 cm-1; 1H NMR (CDCl3) δ 2.28 (s, 3 H), 2.46
(m, 4 H), 3.89 (m, 6 H), 6.45 (m, 2 H). Anal. (C11H17N5) C, H,
N.
as colorless prisms: IR (CHCl3) 1670, 1600 cm-1 1H NMR
;
(CDCl3) δ 2.62 (m, 4 H), 3.56 (s, 2 H), 4.01 (m, 4 H), 6.73 (m,
1 H), 6.81 (m, 1 H), 7.11 (dd, 1 H, J ) 4.5, 7.6 Hz), 7.27-7.34
(m, 5 H), 7.77 (m, 1 H), 7.93 (dd, 1 H, J ) 1.8, 7.4 Hz), 8.48
(dd, 1 H, J ) 1.7, 4.7 Hz). Anal. (C21H21N5) C, H, N.
6-(4-P h en ylp ip er a zin -1-yl)p yr id o[2,3-e]p yr r olo[1,2-a ]-
p yr a zin e (20w ). Similarly to the procedure described for 20a ,
the title compound was prepared starting from 18f and
N-phenylpiperazine. After recrystallization, 20w was obtained
as colorless prisms: IR (Nujol) 1599 cm-1; 1H NMR (CDCl3) δ
2.58 (m, 4 H), 4.02 (m, 4 H), 6.76 (t, 1 H, J ) 3.4 Hz), 6.83 (d,
1 H, J ) 3.7 Hz); 7.12 (dd, 1 H, J ) 4.5, 7.9 Hz), 7.32 (m, 5 H),
7.79 (m, 1 H), 7.95 (dd, 1 H, J ) 1.6, 7.8 Hz), 8.50 (dd, 1 H,
J ) 1.7, 4.7 Hz). Anal. (C20H19N5) C, H, N.
7-Me t h yl-4-(4-b e n zylp ip e r a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20o). Similarly to the procedure described for
20a , the title compound was prepared starting from 18h and
N-benzylpiperazine. After recrystallization, 20o was obtained
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
20x-d d . This procedure is illustrated for the preparation of
9-fluoro-4-[4-(2-thienylmethyl)piperazin-1-yl]pyrrolo[1,2-a]-
quinoxaline (20x). A mixture of 19d (0.2 g, 0.74 mmol),
anhydrous potassium carbonate (96 mg, 0.74 mmol), and
2-(bromomethyl)thiophene (0.13 g, 0.74 mmol) in ethyl methyl
ketone (33 mL) was heated at reflux for 1.5 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
flash-chromatographed (30% EtOAc in chloroform) to give 0.1
1
as colorless prisms: IR (CHCl3) 1600 cm-1; H NMR (CDCl3)
δ 2.43 (s, 3 H), 2.65 (m, 4 H), 3.60 (s, 2 H), 3.82 (m, 4 H), 6.73
(m, 2 H), 7.06 (m, 1 H), 7.27-7.37 (m, 5 H), 7.49 (m, 1 H),
7.59 (d, 1 H, J ) 8.2 Hz), 7.74 (d, 1 H, J ) 2.0 Hz). Anal.
(C23H24N4) C, H, N.
7,8-Dim eth yl-4-(4-ben zylp ip er a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20p ). Starting from 18a and N-benzylpipera-
zine the title compound was obtained following the procedure
described for 20a . After recrystallization, 20p was obtained
1
g of 20x as colorless prisms: IR (Nujol) 1590 cm-1; H NMR
1
as colorless prisms: IR (CHCl3) 1590 cm-1; H NMR (CDCl3)
(CDCl3) δ 2.69 (m, 4 H), 3.85 (m, 6 H), 6.74 (t, 1 H, J ) 3.4
Hz), 6.81 (d, 1 H, J ) 3.8 Hz), 6.95-7.05 (m, 3 H), 7.14-7.27
(m, 2 H), 7.43 (d, 1 H, J ) 8.1 Hz), 8.16 (d, 1 H, J ) 2.4 Hz).
Anal. (C20H19FN4S) C, H, N.
δ 2.32 (s, 3 H), 2.37 (s, 3 H), 2.65 (m, 4 H), 3.59 (s, 2 H), 3.76
(m, 4 H), 6.70 (m, 2 H), 7.25-7.36 (m, 5 H), 7.45 (s, 1 H), 7.47
(s, 1 H), 7.73 (t, 1 H, J ) 1.6 Hz). Anal. (C24H26N4) C, H, N.
9-Me t h yl-4-(4-b e n zylp ip e r a zin -1-yl)p yr r olo[1,2-a ]-
qu in oxa lin e (20q). Similarly to the procedure described for
20a , the title compound was prepared starting from 18b and
N-benzylpiperazine. After recrystallization 20q was obtained
4-[4-(2-Th ien ylm et h yl)p ip er a zin -1-yl]p yr r olo[1,2-a ]-
qu in oxa lin e (20y). Similarly to the procedure described for
20x, the title compound was prepared starting from 19f. After
recrystallization, 20y was obtained as colorless prisms: IR
1
1
as colorless prisms: IR (CHCl3) 1620 cm-1; H NMR (CDCl3)
(Nujol) 1585 cm-1; H NMR (CDCl3) δ 2.70 (m, 4 H), 3.84 (m,
δ 2.66 (m, 4 H), 2.85 (s, 3 H), 3.59 (s, 2 H), 3.77 (m, 4 H), 6.70
(t, 1 H, J ) 3.4 Hz), 6.77 (d, 1 H, J ) 3.7 Hz), 7.06 (d, 1 H,
6 H), 6.75 (m, 2 H), 6.96 (m, 2 H), 7.28 (m, 3 H), 7.64-7.80
(m, 3 H). Anal. (C20H20N4S) C, H, N.

4376 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
7-F lu or o-4-[4-(2-th ien ylm eth yl)p ip er a zin -1-yl]p yr r olo-
[1,2-a ]qu in oxa lin e (20z). Starting from 19e the title com-
pound was obtained following a procedure as for 20x. After
recrystallization, 20z was obtained as yellowish prisms: IR
colorless prisms: IR (Nujol) 3595, 1685 cm-1; 1H NMR (CDCl3)
δ 2.25 (br s, 1 H), 2.65 (m, 2 H), 2.72 (m, 4 H), 3.65 (m, 2 H),
4.47 (m, 4 H), 7.35 (dd, 1 H, J ) 4.8, 7.9 Hz), 7.60 (s, 1 H),
7.93 (dd, 1 H, J ) 1.8, 7.8 Hz), 8.29 (dd, 1 H, J ) 1.8, 4.9 Hz),
8.38 (s, 1 H). Anal. (C15H18N6O) C, H, N.
1
(CHCl3) 1595 cm-1; H NMR (CDCl3) δ 2.68 (m, 4 H), 3.81 (s,
2 H), 3.87 (m, 4 H), 6.76 (m, 2 H), 6.95 (m, 3 H), 7.27 (t, 1 H,
J ) 3.8 Hz), 7.34 (d, 1 H, J ) 2.4 Hz), 7.63 (m, 1 H), 7.73 (m,
1 H). Anal. (C20H19FN4S) C, H, N.
9-F lu or o-4-(4-cya n om eth ylp ip er a zin -1-yl)p yr r olo[1,2-
a ]qu in oxa lin e (20h h ). A mixture of 19e (0,22 g, 0.83 mmol),
anhydrous potassium carbonate (0.11 g, 0.83 mmol), and
bromoacetonitrile (57.8 µL, 0.83 mmoL) in ethyl methyl ketone
(35 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 flash-
chromatographed (30% EtOAc in dichloromethane) to give 0.22
g of 20h h as colorless prisms: IR (Nujol) 2245 cm-1; 1H NMR
(CDCl3) δ 2.80 (m, 4 H), 3.60 (s, 2 H), 3.84 (m, 4 H), 6.78 (m,
2 H), 7.04 (dd, 1 H, J ) 8.1, 11.9 Hz), 7.21 (m, 1 H), 7.45 (d, 1
H, J ) 7.9 Hz), 8.19 (m, 1 H). Anal. (C17H16FN5) C, H, N.
r-[4-(9-F lu or op yr r olo[1,2-a ]qu in oxa lin -4-yl)p ip er a zin -
1-yl]a cetic Acid (20ii). A solution of 24 (0.23 g, 0.55 mmol)
in methanol (5 mL) was hydrogenated at atmospheric pressure
over 10% Pd/C (56 mg) for 1 h. The catalyst was removed by
filtration, the solution was evaporated, and the residue was
recrystallized from ethyl acetate to provide 0.16 g of 20ii as
colorless prisms: IR (Nujol) 3360, 1710 cm-1; 1H NMR (DMSO-
d6) δ 2.73 (m, 4 H), 3.13 (s, 2 H), 3.20 (m, 4 H), 6.81 (m, 1 H),
7.01 (m, 1 H), 7.27 (m, 3 H), 8.11 (m, 1 H). Anal. (C17H17FN4O2)
C, H, N.
9-F lu or o-4-[4-(1H-tetr a zol-5-yl)m eth ylp ip er a zin -1-yl]-
p yr r olo[1,2-a ]qu in oxa lin e (20jj). A mixture of 20h h (0.1 g,
0.32 mmol), piperidine hydrochloride (97 mg, 0.8 mmol), and
sodium azide (52 mg, 0.8 mmol) in anhydrous N,N-dimethyl-
formamide (1 mL) was heated at 115 °C for 16 h, under argon.
The reaction mixture was diluted with aqueous NH4Cl and
extracted with EtOAc. The organic layers were washed with
brine, dried, and concentrated. The residue was flash-chro-
matographed (30% methanol in dicloromethane) to give 78 mg
of 20jj as pale yellow prisms: IR (Nujol) 3200-2400, 1625
cm-1; 1H NMR (DMSO-d6) δ 2.72 (m, 4 H), 3.78 (m, 4 H), 4.02
(s, 2 H), 6.86 (t, 1 H, J ) 3.3 Hz), 7.05 (d, 1 H, J ) 4.0 Hz),
7.28 (m, 3 H), 8.17 (m, 1 H). Anal. (C17H17FN8) C, H, N.
In Vitr o Bin d in g Assa ys. Binding assays have been
performed as described in refs 6 and 23.
6-[4-(2-Th ien ylm eth yl)p ip er a zin -1-yl]p yr id o[3,2-e]p yr -
r olo[1,2-a ]p yr a zin e (20a a ). Similarly to the procedure as
described for 20x, the title compound was prepared starting
from 19g. After recrystallization, 20a a was obtained as
colorless prisms: IR (Nujol) 1600 cm-1; 1H NMR (CDCl3) δ 2.67
(m, 4 H), 3.80 (s, 2 H), 3.88 (m, 4 H), 6.73 (t, 1 H, J ) 3.4 Hz),
6.80 (d, 1 H, J ) 3.8 Hz), 6.94 (m, 2 H), 7.22-7.28 (m, 2 H),
7.86 (dd, 1 H, J ) 1.7, 7.8 Hz), 8.24 (m, 2 H). Anal. (C19H19N5S)
C, H, N.
9-Met h yl-4-[4-(2-b r om o-5-t h ien ylm et h yl)p ip er a zin -1-
yl]p yr r olo[1,2-a ]qu in oxa lin e (20bb). Starting from 19b and
2-bromo-5-(bromomethyl)thiophene the title compound was
obtained following a procedure as described for 20x. After
recrystallization, 20bb was obtained as brown prisms: IR
(Nujol) 1615 cm-1; 1H NMR (CDCl3) δ 2.65 (m, 4 H), 2.87 (s, 3
H), 3.60 (s, 2 H), 3.77 (m, 4 H), 6.69 (t, 1 H, J ) 3.4 Hz), 6.74
(d, 1 H, J ) 3.7 Hz), 6.93 (m, 2 H), 7.08 (d, 1 H, J ) 7.4 Hz),
7.28 (m, 1 H), 7.55 (dd, 1 H, J ) 1.1, 7.8 Hz), 8.14 (d, 1 H,
J ) 2.7). Anal. (C21H21BrN4S) C, H, N.
7-F lu or o-4-[4-(2-fu r ylm et h yl)p ip er a zin -1-yl]p yr r olo-
[1,2-a ]qu in oxa lin e (20cc). Similarly to the procedure as
described for 20x, the title compound was prepared starting
from 19e and 2-(bromomethyl)furan. After recrystallization,
20cc was obtained as colorless prisms: IR (Nujol) 1605 cm-1
;
1H NMR (CDCl3) δ 2.65 (m, 4 H), 3.61 (s, 2 H), 3.86 (m, 4 H),
6.23 (m, 1 H), 6.32 (m, 1 H), 6.69-6.76 (m, 2 H), 6.95 (m, 1
H), 7.29 (dd, 1 H, J ) 2.8, 9.8 Hz), 7.38 (m, 1 H), 7.63 (m, 1
H), 7.72 (m, 1 H). Anal. (C20H19FN4O) C, H, N.
7-F lu or o-4-[4-(2-br om o-5-fu r ylm eth yl)p ip er a zin -1-yl]-
p yr r olo[1,2-a ]qu in oxa lin e (20d d ). Starting from 19e and
2-bromo-5-(bromomethyl)furan the title compound was ob-
tained following the procedure as for 20x. After recrystalliza-
tion, 20d d was obtained as a white solid: IR (CHCl3) 1600
cm-1; 1H NMR (CDCl3) δ 2.68 (m, 4 H), 3.63 (s, 2 H), 3.88 (m,
4 H), 6.23 (m, 2 H), 6.73 (m, 2 H), 6.98 (m, 1 H), 7.31 (dd, 1 H,
J ) 2.6, 9.9 Hz), 7.63 (dd, 1 H, J ) 5.2, 8.9 Hz), 7.72 (d, 1 H,
J ) 2.5 Hz). Anal. (C18H18FBrN4O) C, H, N.
1. [3H]Za cop r id e 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 cortices were rapidly removed and stored at -80 °C until
the day of assay. The frozen tissues were homogenized in about
50 vol of ice-cold Tris HCl, 25 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 refrigerated
centrifuge). 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 centrifuged as before. The
pellet obtained was finally resuspended in Tris HCl, 25 mM,
pH 7.4, containing 10 µM pargyline.
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
20ee-gg. This procedure is illustrated for the preparation of
9-fluoro-4-[4-(2-hydroxyethyl)piperazin-1-yl]pyrrolo[1,2-a]-
quinoxaline (20ee). To a solution of 18o (0.15 g, 0.68 mmol)
in ethyl methyl ketone (2 mL) was added 1-(2-hydroxyethyl)-
piperazine (0.18 mL, 1.50 mmol). The reaction mixture was
refluxed for 21 h under argon, cooled, and evaporated to afford
a residue which was partitioned between water and ethyl
acetate. The organic layers were washed with brine, dried, and
concentrated. The residue was flash-chromatographed (10%
methanol in dichloromethane) to give, after recrystallization,
0.18 g of 20ee as colorless prisms: IR (CHCl3) 3590, 1600 cm-1
;
[3H]Zacopride binding was done as described previously7 in
a final incubation volume of 0.5 mL consisting of 0.25 mL of
membrane suspension (10 mg of tissue/sample), 0.25 mL of
[3H]zacopride (s.a. 85 Ci/mmol, final concentration 0.4 nM),
and 10 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 pretreated with 0.5% poly-
(ethylenimmine) 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” pro-
gram6,13 to obtain the concentration of unlabeled drug that
caused 50% inhibition of ligand binding.
2. [3H]Za cop r id e Bin d in g to NG108-15 Cells.14 Neuro-
blastoma-glioma cells (NG108-15) were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
inactivated fetal calf serum and HAT (10 µM hypoxanthine, 5
µM aminopterine, and 16 µM thymine). Cells were cultured
1H NMR (CDCl3) δ 2.43 (br s, 1 H), 2.62 (m, 2 H), 2.69 (m, 4
H), 3.56 (m, 2 H), 3.81 (m, 4 H), 6.73 (t, 1 H, J ) 3.4 Hz), 6.80
(d, 1 H, J ) 3.8 Hz), 7.03 (m, 1 H), 7.19 (m, 1 H), 7.43 (d, 1 H,
J ) 7.9 Hz), 8.16 (m, 1 H). Anal. (C17H19FN4O) C, H, N.
7-F lu or o-4-[4-(2-h yd r oxyeth yl)p ip er a zin -1-yl]p yr r olo-
[1,2-a ]qu in oxa lin e (20ff). Similarly to the procedure de-
scribed for 20ee, the title compound was prepared starting
from 18n . After recrystallization, 20ff was obtained as color-
1
less prisms: IR (CHCl3) 3615 cm-1; H NMR (CDCl3) δ 2.04
(br s, 1 H), 2.66 (m, 2 H), 2.76 (m, 4 H), 3.70 (m, 2 H), 3.88 (m,
4 H), 6.74 (m, 2 H), 6.95 (m, 1 H), 7.31 (dd, 1 H, J ) 2.8, 9.8
Hz), 7.63 (m, 1 H), 7.75 (d, 1 H, J ) 2.6 Hz). Anal. (C17H19
FN4O) C, H, N.
-
6-[4-(2-H yd r oxyet h yl)p ip er a zin -1-yl]im id a zo[1,2-a ]-
p yr id o[2,3-e]p yr a zin e (20gg). Similarly to the procedure
described for 20ee, the title compound was prepared starting
from 18m . After recrystallization, 20gg was obtained as

Pyrroloquinoxalines as 5-HT3 Receptor Agonists
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21 4377
at 37 °C under CO2/air (10/90%) atmosphere and the medium
was replaced every 2 days. The cells were harvested by shaking
and pelleted by centrifugation at 900g for 10 min.
Under these conditions, [14C]guanidinium accumulation in
NG108-15 cells was 4-5 times higher in the presence of both
1 mM 5-HT and 10 mM SP than in their absence (basal
uptake). 5-HT3 receptor antagonists (zacopride, ondansetron,
tropisetron, etc.) completely prevented the stimulatory effect
of 5-HT (with SP) (see ref 7 for details).
Binding assays with NG108-15 cell membranes were per-
formed as described for cortical membranes with little modi-
fication: a final incubation volume of 260 µL consisting of 150
µL of cells suspension (about 420 000 cells/sample in Tris HCl,
50 mM, pH 7.5, containing 25 mM NaCl), 100 µL of [3H]-
zacopride (final concentration 0.4 nM), and 10 µL of displacing
agents or solvent. In addition the cell membranes sample were
incubated for 60 min at 25 °C.
3. Bin d in g Assa ys for 5-HT Recep tor Su btyp es. Binding
assays have been performed as described in ref 6. Briefly, male
CRL:CD(SD)BR-COBS rats and male albino guinea pigs were
killed by decapitation; their brains (rat hippocampus for
5-HT1A, rat striatum for 5-HT1B, rat cortex for for 5-HT2A and
5-HT2C, guinea pig striatum for 5-HT4) were rapidly dissected
into the various areas and stored at -80 °C until the day of
assay.
Tissues were homogenized in about 50 vol of ice-cold
appropriate buffer using an Ultra Turrax TP-1810 homog-
enizer (2 × 20 s), and homogenates were centrifuged at 50000g
for 10 min (Beckman Avanti J -25 centrifuge). Each 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 centrifuged as before. The pellet obtained was
finally resuspended in the appropriate incubation buffer just
before the binding assay.
In Vivo Stu d ies: P oten tia l An xiolytic-lik e a n d An a l-
gesic-lik e P r op er ties a n d Br a in -to-P la sm a Distr ibu tion
Stu d ies. Procedure involving animals and their care was
conducted in conformity with the institutional guidelines that
are in compliance with national laws and policies (D.L. no.
116, G.U., Suppl. 40, Feb 18, 1992; Circolare No. 8, G.U., 1994)
and international laws and policies (EEC Council Directive
86/609, OJ L 358,1, Dec 12, 1987; Guide for the Care and Use
of Laboratory Animals, U.S. National Research Council, 1996).
1. Ligh t/Da r k Exp lor a tor y Test in Mice. Male CD-1 mice
(Charles River, Italy), weighing 20-25 g at the beginning of
the experiments were used. They were housed five per cage
at constant room temperature (21 ( 1 °C) and relative
humidity (60%) under a regular, inverted, light/dark schedule
(light: 10:00 p.m.-10:00 a.m.) with food and water available
ad libitum. Animals were allowed to adapt to laboratory
conditions for at least 3 weeks. Testing was done in the dark
phase between 2:00 and 5:00 p.m. Ten animals per group were
used in the various experiments.
The procedure described by J ones et al.17 is based on the
aversion of the animals to wide brightly lit areas. The
apparatus consisted of a poly(vinyl chloride) open-topped box
(42.5 × 25.5 × 30 cm) divided into a small (17.0 × 25.5 × 30
cm) and a large (25.5 × 25.5 × 30 cm) area by a partition which
had a hole at floor level. The small area was painted black,
while the large compartment was white. The floor of each
compartment was marked in 9-cm squares. The white com-
partment was illuminated with one 60-W incandescent bulb
and the black compartment with a red dark-room incandescent
bulb. Compounds 3b and 20b, as hydrochloride salts, were
dissolved in 0.9% NaCl and given orally 45 min before testing.
Chlordiazepoxide hydrochloride was dissolved in 0.9% NaCl
and administered subcutaneously 30 min before mice were
gently put in the central square of the white compartment and
their behavior videorecorded for 5 min. The time the animals
spent in the white compartment and the number of transitions
between the two compartments were later determinated by
one observer unaware of animal treatments. For “transition”,
it was considered a mouse moving from one compartment to
the other, with all four paws in the new compartment. The
statistical significance of differences between control and
treated groups was analyzed by a one-way ANOVA followed
by a Dunnett’s test.
The following incubation conditions were used. 5-HT1A
:
[3H]8-OH-DPAT (s.a. 127 Ci/mmol; NEN), final concentration
1 nM, 30 min at 25 °C (nonspecific binding: 5-HT 10 µM).
5-HT1B: [3H]5-HT (s.a. 30.0 Ci/mmol; NEN), final concentration
2 nM, 30 min at 25 °C (nonspecific binding: 5-HT 10 µM).
5-HT2A: [3H]ketanserin (s.a. 80.9 Ci/mmol; NEN), final con-
centration 0.7 nM, 15 min at 37 °C (nonspecific binding:
methysergide 1 µM). 5-HT2C: [3H]mesulergine (s.a. 82.0 Ci/
mmol; Amersham), final concentration 1 nM, 30 min at 37 °C
(nonspecific binding: meulergine 10 µM). 5-HT4: [3H]GR
113808 (s.a. 84.0 Ci/mmol; Amersham), final concentration 0.1
nM, 30 min at 37 °C (nonspecific binding: 5-HT 10 µM).
Incubations were stopped by rapid filtration under vacuum
through GF/B filters.
Mea su r em en t of [14C]Gu a n id in iu m Up ta k e in NG108-
15 Cells. This procedure has been described by Emerit et al.7
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 NG108-15 clone were grown in
DMEM 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 was added) containing 0.20-0.25 mCi of [14C]-
guanidinium (s.a. 59 mCi/mmol; Service des Mole´cules Mar-
que´es at CEA, 91191 Gif-surYvette, France) and, where
indicated, 1 mM 5-HT, 10 mM SP, and/or 8 different concen-
trations 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 HCl 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.
2. Ra d ia n t Hea t Ta il F lick Test. Male Sprague-Dawley
rats (CD-COBS, Charles River, Italy) weighing 175-200 g at
the beginning of the study were used. They were housed two
per cage at costant room temperature (21 ( 1 °C) and relative
humidity (60%) under a regular light/dark schedule (light:
7:00 a.m.-7:00 p.m.) with food and water ad libitum. Animals
were allowed to adapt to laboratory conditions for at least 1
week before the experiments and were handled daily during
this period. Testing was done between 2:00 and 5:00 p.m. Eight
rats per group were used.
Compounds 3b and 20b, as hydrochloride salts, were
dissolved in 0.9% NaCl and administered i.p. Morphine
hydrochloride, dissolved in 0.9% NaCl, was administered
subcutaneously. Nociceptive threshold was determined using
an automated tail flick unit (Basile, Comerio, Italy) with the
radiant heat adjusted to attain a mean baseline between 2 and
3 s, and a 15-s cutoff was imposed to avoid tissue damage.
Thirty minutes before drug administration, rats were taken
from their home cage and gently handled on the tail flick unit
to measure the baseline latency. The antinociceptive activity
of the compounds was measured 30, 60, 90, and 120 min after
drug administration. The statistical significance of differences
between control and treated groups was ascertained by a two-
way ANOVA for repeated measures with treatment as between-

4378 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 21
Campiani et al.
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Serotoninergic Facilitation of Acetylcholine Release In Vivo from
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S.; Consolo, S.; Mennini, T. Novel and highly Potent 5-HT3
Receptor Agonists Based on a Pyrroloquinoxaline Structure. J .
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Organic Fluorine Hardly Ever Accepts Hydrogen Bonds. Chem-
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(7) Emerit, M. B.; Riad, M.; Fattaccini, C. M.; Hamon, M. Charac-
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subjects factor and time as within-subjects factor. Post-hoc
comparisons were made with Tukey’s test.
3. 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 CD-COBS rats weighing 175-200 g (Charles
River, Italy) were used. Rats received the test compounds
(dissolved in saline) intravenously at the dose of 5 mg/kg
(compounds have been used as hydrochloride salts) and were
killed by decapitation under deep anesthesia 60, 120, and 180
min after dosing. Blood samples were collected in heparinized
tubes and centrifuged and the plasma was stored at -20 °C.
Brains were rapidly removed, blotted with paper to remove
excess surface blood, and stored at -20 °C until analysis.
4. Dr u g An a lysis. Plasma and brain concentrations of the
test compounds were determined by high-performance liquid
chromatography (HPLC) after a liquid-liquid or solid-liquid
extraction procedure. A chromatographically suitable analogue
with similar extraction characteristics was used as an internal
standard (I.S.) in each assay. The acidic derivative 20h h was
extracted from 1 mL of plasma (buffered to pH 3.5) or 1 mL of
brain homogenate (0.1 M buffer phosphate, pH 3.5/CH3CN,
80:30 v/v, 10 mL/g) with 4 mL of ethyl actetate. The very
lipophilic compound 20u was extracted from plasma (1 mL)
and brain homogenate (0.1 M buffer phosphate/CH3CN, 80:
20, v/v, 10 mL/g) with 4 mL of hexane/ethyl acetate (80:20,
v/v), after adding 1 N NaOH to a final pH of about 10. After
centrifugation the organic extracts were evaporated to dryness
and the residues were dissolved in the mobile phase and
injected in the HPLC column.
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Supelchem LC-18 SPE cartridges (Supelco, Milano, Italy)
were used to clean up plasma and brain samples in the case
of compounds 20b,l,v,ee. Briefly, the cartridges were prewet-
ted with 2 mL of methanol, 2 mL of distilled water, and 2 mL
of 0.1 N KH2PO4, pH 5. Then, after adding the I.S., 1-2 mL
of plasma (diluted to 2 mL with 0.1 M KH2PO4) was added
and the cartridges were washed with 3 mL of KH2PO4, 4 mL
of distilled water, and 2 mL of CH3CN-water (30:70 v/v). The
compound was removed by eluting the cartridges with 3 mL
of 1 M NH4OH in methanol and evaporated to dryness in
vacuo. Brain tissue was homogenized as described for com-
pound 20u and 1-3 mL were centrifuged at 15000g for 15 min.
The supernatant was processed as described for plasma. The
residue was dissolved in the mobile phase and analyzed by
HPLC with UV detection (254 nm).
Separation was done on a Hypersil ODS column (25-cm ×
4.6-mm i.d., 5-µm particle size) (C.P.S. Analitica, Milano, Italy)
at 30 °C, protected by a MPLC New Guard precolumn (Applied
Biosystem, Inc., CA). The mobile phase was 0.01 M KH2PO4:
CH3CN:CH3OH in a ratio (v/v) of 57:40:3 for the analysis of
compound 20u and 70:27:3 for all other compounds. The
retention times were 20 min for 20u and 5.4, 9.8, 12, 13, and
14.5 for 20l, 20v, 20h h , 20ee, and 20b respectively.
The methods had sensitivity limits of 0.03-0.1 and 0.1-
0.3 nmol/mL, depending on the compound and volume of
plasma and brain homogenized analyzed. At these concentra-
tions the coefficients of variation (CV) were between 13% and
18%, and all higher concentrations gave CV below 15% in both
tissues.
Ack n ow led gm en t. This work was supported by a
grant from MURST, Italy. The authors thank Asses-
sorato Ricerca Scientifica, Regione Campania, Italy,
Contr. POP, Sott.prog. 5, Mis. 5.4, Azione 5.4.2.
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