Specific targeting of peripheral serotonin 5-HT3 receptors. Synthesis, biological investigation, and structure-activity relationships

Article abstract of DOI:10.1021/jm900018b

The synthesis and the biological characterization of novel highly selective pyrroloquinoxaline 5-HT₃ receptor (5-HT₃R) ligands are described. In functional and in vivo biological studies the novel quinoxalines modulated cardiac param

Full text of DOI:10.1021/jm900018b

3548
J. Med. Chem. 2009, 52, 3548–3562
Specific Targeting of Peripheral Serotonin 5-HT3 Receptors. Synthesis, Biological Investigation,
and Structure-Activity Relationships
Elena Morelli,|, Sandra Gemma,|,§ Roberta Budriesi,# Giuseppe Campiani,*,|,§ Ettore Novellino,|, Caterina Fattorusso,|,†
Bruno Catalanotti,|,† Salvatore Sanna Coccone,|,§ Sindu Ros,|,§ Giuseppe Borrelli,|,§ Vinod Kumar,|,§ Marco Persico,|,†
Isabella Fiorini,|,§ Vito Nacci,|,§ Pierfranco Ioan,# Alberto Chiarini,# Michel Hamon,‡ Alfredo Cagnotto,|,∞ Tiziana Mennini,|,∞
Claudia Fracasso,|,∞ Milena Colovic,|,∞ Silvio Caccia,|,∞ and Stefania Butini|,§
European Research Centre for Drug DiscoVery and DeVelopment, Banchi di Sotto 55, 53100 Siena, Italy, Dipartimento Farmaco Chimico
Tecnologico, UniVersita` di Siena, Via Aldo Moro 53100 Siena, Italy, Dipartimento di Chimica delle Sostanze Naturali (DCSN) e Dipartimento
di Chimica Farmaceutica e Tossicologica (DCFT), UniVersita` di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy,
Dipartimento di Scienze Farmaceutiche, UniVersita` di Bologna, Via Belmeloro 6, 40126 Bologna, Italy, Neurobiologie Cellulaire et
Functionnelle, Istitut National de la Sante´ et de la Recherche Medicale (INSERM) U. 677, Faculte` de Medicine Pitie-Salpetriere 91, BouleVard
de l’Hopital, 75634 Paris, Cedex 13, France, and Istituto di Ricercha Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy
ReceiVed September 25, 2008
The synthesis and the biological characterization of novel highly selective pyrroloquinoxaline 5-HT3 receptor
(5-HT3R) ligands are described. In functional and in vivo biological studies the novel quinoxalines modulated
cardiac parameters by direct interaction with myocardial 5-HT3Rs. The potent 5-HT3R ligands 4h and 4n
modulate chronotropy (right atrium) but not inotropy (left atrium) at the cardiac level, being antagonist and
partial agonist, respectively. Preliminary pharmacokinetic studies indicate that (S)-4n and 4a, representatives
of the novel 5-HT3R ligands, possess poor blood-brain barrier permeability, being the prototypes of
peripherally acting 5-HT3R modulators endowed with a clear-cut pharmacological activity at the cardiac
level. The unique properties of 4h and 4n, compared to their previously described centrally active N-methyl
analogue 5a, are mainly due to the hydrophilic groups at the distal piperazine nitrogen. These analogues
represent novel pharmacological tools for investigating the role of peripheral 5-HT3R in the modulation of
cardiac parameters.
Introduction
5-HT3A: the short and the long isoforms.7 Expression of
splice variants and subunits is different in the central nervous
system (CNS) and in peripheral tissues.8 The presence of
intraspecies differences would have a great impact, not least
because selective interaction with individual 5-HT3R subtypes
may offer increased therapeutic benefits.
Serotonin (5-HTa) is a neurotransmitter involved in many
biological and pathological processes. Among the seven major
families of 5-HT receptors only the 5-HT3 receptor (5-HT3R)
is a pentameric ligand-gated ion channel, and it belongs to
the Cys-loop family.1 The 5-HT3R has attracted much
attention as a therapeutic target, since its antagonists are
effective in preventing emesis induced by treatment with
chemotherapeutic drugs. On the contrary, the potential
therapeutic relevance of 5-HT3R agonists is still unclear.2
Many studies indicate heterogeneous properties for this
receptor which could be explained on the basis of different
subunit composition. To date, five subunits, 5-HT3A-E,3
have been identified and cloned showing differences in both
the amino terminal and the transmembrane region.4-6 These
subunits are coassembled in homomeric or heteromeric
receptor subtypes differing in localization and functions.4
Furthermore, there is evidence of two splice variants of
The role of 5-HT3R in peripheral tissues has not been widely
investigated. Modulation of cardiac function by 5-HT through
5-HT3R is highly intriguing, and development of peripheral
5-HT3R ligands (agonists or antagonists) still remains a chal-
lenge and might lead to novel cardiomodulators. The cardio-
vascular effects of 5-HT are mediated by a direct interaction
with three main types of receptors, the so-called 5-HT1-like,
5-HT2R, and 5-HT3R, and by modulation of the cholinergic
neurotransmission at the cardiac level.9 5-HT can produce a
tachycardic effect mediated by activation of 5-HT3R, with
positive inotropic and chronotropic effects, mimicked by the
agonist quipazine (1, Chart 1) and by 2-Me-5-HT (2).10
Recently, the cardiovascular profile of a 5-HT3R agonist (YM-
31636, 3) was reported. However, positive chronotropic effects
of 3 were accompanied by potent agonist activity on intestinal
receptors (5-HT4R, colon water secretion) and on neuronal
5-HT3Rs.11
* To whom correspondence should be addressed. Phone: 0039-0577-
234172. Fax: 0039-0577-234333. E-mail: campiani@unisi.it.
|
European Research Centre for Drug Discovery and Development.
DCFT, Universita` di Napoli.
Universita` di Siena.
Universita` di Bologna.
DCSN, Universita` di Napoli.
Istitut National de la Sante´ et de la Recherche Medicale.
Istituto di Ricerche Farmacologiche Mario Negri.
§
#
In order to exploit the intriguing therapeutic potential of
modulating peripheral 5-HT3R, the aim of the present study
was the development of novel and selective 5-HT3R ligands
targeting peripheral cardiac receptors possibly lacking be-
havioral effects. Because of the lack of structural information
relative to Cys-loop receptors that could drive the design
strategy of peripheral 5-HT3R modulators, we exploited our
previous experience in the field.12,13 Accordingly, we decided
of achieving “peripheral” selectivity by modulating the
†
‡
∞
a Abbreviations: 5-HT, serotonin; 5-HT3R, 5-HT3 receptor; CNS, central
nervous system; BBB, blood-brain barrier; von B-J, von Bezold-Jarish;
CDI, N,N-carbonyldiimidazole; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
MM, molecular mechanics; PBG, 1-phenylbiguanide; IS, internal standard;
DIPEA, N,N-diisopropylethylamine; DCM, dichloromethane; THF, tetrahy-
drofuran; N,N-DMF, N,N-dimethylformamide; CV, coefficients of variation;
PSS, physiological salt solution; DMSO, dimethyl sufoxide.
10.1021/jm900018b CCC: $40.75
2009 American Chemical Society
Published on Web 05/08/2009

Targeting of Serotonin 5-HT3 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3549
Chart 1. Reference and Title Compounds
In addition, brain to plasma concentrations of 4n and 4a were
measured after systemic injection.
Chemistry
As shown in Schemes 1-3, the synthesis of compounds 4a-o
was accomplished starting from our previously described
piperazinylpyrroloquinoxalines 10a-c by standard functional
group transformations.13 Intermediates 11a-d (Scheme 1) were
obtained by N-alkylation of the 10a-c piperazine N-4, using
suitable 2-bromoacetyl esters. These latter represent key inter-
mediates for the synthesis of 4a,b,d,e,j,m. The free acids 4a,b
(Scheme 1) were obtained by catalytic hydrogenation of the
corresponding benzyl esters 11c,d. From 10a, treatment with
2-bromoacetonitrile provided the intermediate 12 that, after
exposure to potassium hydroxide, afforded 4c. Ethyl esters 11a,b
were treated with hydroxylamine to afford the corresponding
hydroxyacetamides 4d,e. Alkylation of 10a with 2-bromophe-
nylacetic acid ethyl ester afforded an ester intermediate that was
hydrolyzed to obtain 4f or reduced to give 4g. Treatment of 4a
with methanesulfonamide in the presence of N,N-carbonyldi-
imidazole (CDI) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
furnished 4j (Scheme 2). Exposure of intermediate 12 (obtained
form 10a) to hydroxylamine or sodium azide afforded 4h and
4k, respectively. For the synthesis of 4i and 4l, compound 10a
was heated in the presence of N-formyl-2-chloroacetamidohy-
drazone (4i) or N-carbomethoxy-2-chloroacetamidohydrazone
(4l).16 Compound 11a was converted into 4m through a two
step procedure involving the synthesis of a hydrazido intermedi-
ate obtained by exposure of 11a to hydrazine, followed by
cyclization with CDI.
Chart 2. Potent 5-HT3 Receptor Ligands Previously Described
For the synthesis of 4n (Scheme 3) the introduction of the
alkyl chain was performed by using (R)- or (S)-3-[(tert-
butoxycarbonyl)amino]oxetan-2-one, in turn prepared from (R)-
or (S)-serine. This latter acted as chiral electrophilic alanine
cation equivalent that reacted with the nucleophiles and provided
optically pure alanine derivatives.17 Compound 4o was synthe-
sized by a coupling reaction between 10a and N-Boc-serine in
the presence of 1-hydroxybenzotriazole. The resulting interme-
diate (S)-13 was deprotected by exposure to trifluoroacetic acid
to afford (S)-4o in good overall yield.
physical-chemical properties of our previously developed
arylpiperazine 5-HT3R selective ligands, thus preventing their
crossing of the blood-brain barrier (BBB). With this aim,
we designed pyrroloquinoxalines (4) characterized by dif-
ferent pKa values and endowed with high affinity and
selectivity for 5-HT3R. Most of the quipazine-like 5-HT3R
ligands so far reported by our group (5-7)12,13 and by others
(814 and 9,15 Chart 2) were characterized by different
substituents at the piperazine N-4 position and at the
polycyclic system. Thus, we report herein the development
of novel pyrroloquinoxalines that combine specific substit-
uents at the benzo-fused system (7-F, 9-Me, 7-OH) to key
“acidic” (4a-f,h-n, Table 1) or polar (4g and 4o, Table 1)
functions at piperazine the N-4 position. We also explored
the effect of carboxyl bioisosterism. The selected functional
groups differ in their charge distribution, in the number and
the position of their H-bonding groups, and in steric
hindrance. Explored functions are represented by carboxylic
group (4a and 4b), amide (4c), hydroxamic acid (4d and 4e),
N-hydroxyimidamide (4h), alcoholic (4g), sulfonylamide (4j),
heterocyclic azoles (4i, 4k, and 4l), oxadiazolone (4m),
R-amino acid (4n). Their 5-HT3R occupancy was evaluated
by in vitro binding (Table 1). A set of potent and selective
5-HT3R ligands were used in functional studies for assessing
their capability of modulating cardiac parameters and were
tested in vivo on the von Bezold-Jarish (von B-J) reflex test.
Quipazine derivative 15 (Scheme 4) was easily obtained from
1 by alkylation (14) followed by saponification (15).
Biological Assays. The 5-HT3R affinity of the new com-
pounds was assessed in binding studies given in Table 1. A
subset of compounds were further examined for their selectivity
toward serotonin (5-HT1AR, 5-HT2AR, and 5-HT4R), adrenergic
(R1 and R2), dopamine (DA1 and DA2), and muscarinic receptors
(Table 2). The tested compounds did not show affinity up to
10 µM, except compounds (S)- and (R)-4n, which showed a
submicromolar affinity for 5-HT1AR with no stereoselectivity
of interaction.
5-HT3R efficacy was measured by functional assays in guinea
pig myocardium and aortic strips (Table 3). In vivo studies were
performed on several new compounds on the von B-J reflex
test (Table 4). Additionally, brain and plasma concentrations
were simultaneously measured for a subset of ligands after
systemic injection (Figure 4).
Molecular Modeling. In order to define the pharmacophoric
features of our new series of arylpiperazine ligands, we
performed a 3D structure-activity relationship (SAR) analysis
taking into account previously reported pharmacophoric and
receptor models for 5-HT3R14,18-37 (Table 1 of Supporting
Information (SI) and Figure 1 of SI). For this goal, compounds
4a-o (Table 1) were subjected to computational studies in order

3550 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Morelli et al.
Table 1. 5-HT3R Binding Affinities (Ki, nM), Tautomers, Percentage of Ionic Forms, Clusters, and Single pKa Values of Compounds 1, 4a-o, 5a,b,
6a,b, and 15

Targeting of Serotonin 5-HT3 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3551
Table 1. Continued
a Percentage of the ionic forms derived from apparent pKa values: A ) anionic; N ) neutral; P ) protonated; ZW ) zwitterionic (ACD/Labs V10.0,
Toronto, Canada). b Ki in nM. Each value is the mean ( SEM of three determinations and represent the concentration giving half-maximal inhibition of
[3H]LY278584 binding to rat cortical homogenate. c ACD/Labs V10.0, Toronto, Canada. d The ionizable function is specified in parentheses by the position
of the heteroatom with respect to N-4. e Ionization of 7-OH substituent. f From ref 12. g From ref 13.
Scheme 1. Synthesis of Compounds 4a-g
to analyze their conformational and electronic behavior. Dif-
ferent calculation methods were used (see Experimental Section
for details) for properly describing the 4a-o extended π system
and piperazine N-4 substituents (in equilibrium among different
ionizable tautomeric forms). First, 4a-o tautomers were mod-
eled in their most abundant ionic form at pH 7.2, according to
calculated pKa values (Table 1). The conformational space was
then sampled by means of a simulated annealing procedure

3552 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Scheme 2. Synthesis of Compounds 4h-m
Morelli et al.
Scheme 3. Synthesis of Compounds 4n-o
Scheme 4. Synthesis of Compound 15
formational energy. In order to widen structure selection for
successive semiempirical (AM1) geometry optimization, MM
conformers’ energy was also evaluated by performing a single
point (1-SCF) AM1 calculation. The MM global minimum
energy conformer of each compound and the 1-SCF lowest
energy conformer of each family were selected for successive
full geometry optimization by using AM1 semiempirical method
(performed in vacuum). Finally, Coulson and ESP partial
charges of AM1 conformers were calculated either with AM1
or with MNDO semiempirical methods.
Fully optimized AM1 conformers were resubjected to struc-
tural classification into families in the frame of previously
reported pharmacophoric models of 5-HT3R ligands.14,18 These
latter conformers are characterized by the presence of a total
positive net charge on the molecule and by the correct
orientation of three key pharmacophoric groups: (i) a protonated
nitrogen, (ii) an H-bond acceptor (carbonyl oxygen or hetero-
followed by molecular mechanics (MM) geometry optimization
using the dielectric constant of water (ε ) 80r) to simulate the
conformational behavior in aqueous solution. Resulting con-
formers were grouped into families according to geometric
features (see Experimental Section) and ranked by their con-

Targeting of Serotonin 5-HT3 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3553
Table 2. Binding Profile of Selected Compounds on a Panel of Neurotransmitter Receptors
a
Ki ((SEM), nM
b
c
d
e
f
g
h
compd
5-HT1A
5-HT2A
5-HT4
R1NA
R2NA
DA1
DA2
muscarinici
4h
>10000
446(24)
480(80)
>10000
1584 (180)
138(14)
>10000
>10000
5692(1120)
>10000
>10000
794(99)
124(45)
501(76)
1258(156)
>10000
>10000
>10000
5750(1004)
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
919(133)
>10000
>10000
>10000
>10000
(S)-4n
(R)-4n
15
1
5aj
>10000
6ak
5-HTk
1.6(0.2)
100(8)
a Ki in nM, derived from IC50 according to the method of Cheng and Prosoff. Values in parentheses are the mean ( SEM of three determinations.
b [3H]8-OHDPAT, rat hippocampus. c [3H]ketanserin, rat cortex. d [3H]GR113808, guinea pig striatum. e [3H]prazosin, rat cortex. f [3H]RX 821002, rat cortex.
h
i
g [3H]SCH-23390, rat striatum. [3H]spiperone, rat striatum. [3H]QNB, rat cortex. j From ref 12. k From ref 13.
Table 3. Agonist Potency in Guinea Pig Left and Right Atria and Aortic Strips and Antagonist Activity in Guinea Pig Right Atrium
positive inotropya
positive chronotropyb
contractility on aortic stripsc
antagonist activityd
e
e
e
g
compd
5-HT
PBG
tropisetron
2
4a
4c
4h
4i
(S)-4n
5a
pD2 ( SE
Rf
pD2 ( SE
5.78 ( 0.09
Rf
1h
pD2 ( SE
4.19 ( 0.09
R f
1h
pIC50 ( SE
6.85 ( 0.12
4.31 ( 0.09
inactivei
1h
1h
inactivei
inactivei
7.52 ( 0.08j
5.51 ( 0.19
1h
5.06 ( 0.05
1h
inactivei
inactivei
inactivei
inactivei
inactivei
inactivei
inactivei
inactivei
5.72 ( 0.13
4.95 ( 0.19
5.24 ( 0.14
5.07 ( 0.07
inactivei
inactivei
inactivei
inactivei
inactivei
inactivei
inactivei
5.98 ( 0.10
6.79 ( 0.09
0.81k
0.60k
inactivei
a Positive inotropic potency in guinea pig left atrium driver at 1 Hz. b Positive chronotropic potency in guinea pig spontaneously beating right atrium.
Pretreatment ranged from 170 to 200 beats/min. c Positive contractility potency in guinea pig aortic strips. d Antagonist activity value in guinea pig spontaneously
beating right atrium. The agonist was 5-HT. e pD2 ) -log EC50. EC50 values are calculated from log concentration-response curves (Probit analysis according
to Litchfield and Wilcoxon48). pD2 values are expressed as the mean ( SE of at least four independent experiments. f R ) intrinsic activity of compound
relative to 5-HT. g Each pIC50 value of noncompetitive antagonism was obtained from inhibition of compounds at three different concentrations. pIC50
)
-log IC50. IC50 value, calculated from log concentration-response curves (Probit analysis according to Litchfield and Wilcoxon48 with n ) 4-6), is the
concentration of the noncompetitive antagonist that inhibits 50% of maximum response to agonist. The values are expressed as the mean ( SE of five
independent experiments performed. h Full agonist. i Inactive ) no significant variation (p < 0.05) from the control at the maximum concentration tested
(100 µM). j pA2 ( SE values were calculated from Schild plots,51 constrained to slope -1.0.52 pA2 is the positive value of the intercept of the line derived
by plotting log(DR - 1) vs log [antagonist]. The log(DR - 1) was calculated from at least three different antagonist concentrations, and each concentration
was tested from four to six times. Dose-ratio (DR) values represent the ratio of the potency of the agonist 5-HT (EC50) in the presence of the antagonist and
in its absence. Parallelism of concentrationscurves was checked by linear regression, and the slope was tested for significance (p < 0.05). k Partial agonist.
Table 4. Functional Behavior of Compounds 4a, 4c, 4d, 4h, and 5a
von Bezold-Jarishreflex test
form at physiological pH (Table 1), was not included in this
classification.
As revealed by the analysis of all conformers resulting from
MM (data not shown) and AM1 geometry optimization (global
minima reported in Table 2 of SI and Table 3 of SI) geometry
optimization, our calculations resulted in coherent conforma-
tional trends. Geometrical parameters selected for organizing
conformers into families were the following: (i) the distance
between pyrroloquinoxaline N-1 and piperazine N-4 (d1) and
the height (h) of the piperazine N-4 with respect to the plane of
the aromatic system, which are related to the conformation of
the piperazine ring; (ii) the distance between the exchangeable
hydrogen and the closest heteroatom (dN4-X2, Table 2 of SI; dN4-
Hγ/δ, Table 3 of SI), demonstrating the presence (or not) of an
intramolecular H-bond; (iii) the distances between the exchange-
able hydrogen and the pyrroloquinoaxaline system (dN1-H4, dX1-
H4, Table 2 of SI; dN1-Hγ/δ, dX1-Hγ/δ, Table 3 of SI), indicative of
piperazine rotation with respect to the pyrroloquinoxaline
system. AM1 calculations resulted in two possible piperazine
ring conformers: pseudoaxial chair (h > 1.0 Å) or pseudoequa-
torial chair (h < 1.0 Å). Piperazine ring pseudoaxial conforma-
tion was energetically favored in all compounds bearing a
protonated piperazine N-4 (cluster A, Table 2 of SI), as well as
for most of the structures belonging to cluster B (Table 3 of
SI) with the exception of 4i tautomer 2 and of all tautomers of
4l and 4o. MM indicated the sp2 nature of the conjugated
proximal nitrogen, thus with piperazine resulting in a pseu-
compd
behavior
ID50,a µg/kg iv
4a
4c
4d
4h
5ab
agonist
antagonist
antagonist
partial agonist
antagonist
36 (ED50)
75
50
30
600
a The ID50 value is the test drug concentration that produces a 50%
inhibition of the maximal effect induced by PBG. b From ref 12.
cyclic nitrogen), and (iii) an aromatic moiety (Table 1 of SI).
Indeed the protonated nitrogen is involved in cation-π and/or
charge assisted hydrogen bond, while different protein residues
characterized by H-bond donor/acceptor groups assist the
binding. The aromatic system of the ligand establishes π-π
polarized interactions with several aromatic residues present
along the receptor binding site (Figure 1 of SI).
Finally, on the basis of single pKa values of the piperazine
N-4 substituents (Table 1), compounds 4a-o were grouped in
two clusters: cluster A including compounds 4a, 4b, 4f, 4k,
and 4m (R′ single pKa < 7), cluster B including compounds
4c-e, 4g-i, 4l, (S)-4n, (R)-4n, and 4o (R′ single pKa > 7).
Compounds in which R′ was characterized by more than one
single pKa value were clustered according to the resulting overall
acidity, taking in to account R′ internal proton exchange.
Compound 4j, characterized by the presence of 100% anionic

3554 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Morelli et al.
doequatorial position (Figure 2 of SI and Table 5 of SI). Taken
together, these results suggested that the intramolecular attraction
between exchangeable hydrogens and N-1 may affect piperazine
conformation of 4a-o. This was confirmed by the analysis of
related distance values (i.e., dN1-H4, cluster A; dN1-Hγ/δ, cluster
B). Interestingly, MM calculations simulating an aqueous
environment (ε ) 80r) and semiempirical AM1 calculations
(performed in vacuum) revealed that the piperazine N-4 and R′
(Figure 2 of SI and Table 5 of SI) establish an intramolecular
H-bond, in agreement with calculated pKa values (Table 1).
Structure-Activity Relationship Studies. Discussion. Be-
cause of the nature of R′, the new derivatives 4a-o presented
piperazine N-4 single pKa values 3-4 orders of magnitude lower
than those of the reference compounds 1, 5a, 6a, and 6b (Table
1), in line with the reduced electron density at piperazine N-4
(e.g., AM1 ESP partial charge at piperazine N-4: 5a (-0.10)
vs 4a (-0.02), Table 4 of SI). Accordingly, the calculated
distribution of ionic species at physiological pH showed that
most of the compounds are present in the neutral or in the
zwitterionic form, depending on the single pKa values of R′ and
piperazine N-4 (Table 1). Despite the lack of a total net positive
charge on the molecule (with the exception of 4o) and the
presence of acidic functional groups at piperazine N-4, com-
pounds 4a-o showed 5-HT3R binding affinities from the low
to the high nanomolar range (Table 1). The presence of a net
negative charge on the molecule (which implies the lack of a
key pharmacophoric element for 5-HT3R ligands) is detrimental
for 5-HT3R affinity, 4j being the least potent of the series (4j,
100% anionic at pH 7.2, Ki ) 246 nM, Table 1).
Compounds belonging to cluster A (R′ single pKa < 7, Table
1) are in equilibrium between their anionic and zwitterionic
forms at pH 7.2 (Table 1 of the main text and Table 2 of SI).
The almost identical single pKa values of the amino and carboxyl
functions of 4a and 4b (Table 1) allowed us to speculate about
the presence of a rapid proton exchange equilibrium between
the amino and the carboxyl groups. As demonstrated by the
structural comparison depicted in Figure 3 of SI and by the
distances reported in Table 2 of SI, either in the zwitterionic or
in the neutral form, the exchangeable hydrogen is sufficiently
close to the piperazine N-4 to fulfill the 5-HT3R pharmacophore.
Accordingly, although presenting a percentage of anionic form
at pH 7.2 (Table 1), 4a and 4b showed low nanomolar 5-HT3R
affinity (4a, Ki ) 11.7 nM; 4b, Ki ) 2.1 nM). The higher affinity
of 4b with respect to 4a is due to the 7-OH substituent on the
pyrroloquinoxaline ring system, which could (i) mimic the
hydroxyl group of serotonin (Figure 1 of SI), thus acting as an
additional H-bond donor/acceptor, or (ii) drive alternative
binding modes of the anionic form of 4b. Compounds (()-4f
and 4k, although fulfilling 5-HT3R pharmacophoric requirements
when in the zwitterionic form (Table 2 of SI), are mostly present
in the anionic form (Table 1), consequently showing lower
5-HT3R affinity with respect to 4a and 4b (4f, Ki ) 44 nM; 4k,
Ki ) 65 nM). The extra volume occupied by the phenyl
substituent of (()-4f could play a role in receptor affinity (see
discussion below; Figure 4 of SI). In the case of 4m, only the
tautomer 1 was calculated to be mostly (55%) present in the
zwitterionic form while 100% of tautomer 2 was anionic at
physiological pH (Table 1). Moreover, despite what occurred
for 4a, 4b, 4f, and 4k, tautomer 1 of 4m is not stabilized by an
intramolecular H-bond with piperazine N-4 (dH4-X2, Table 2 of
SI). Accordingly, 4m was the least potent compound of cluster
A toward 5-HT3R (4m, Ki ) 127 nM). In summary, 5-HT3R
affinity of cluster A mainly correlates with the calculated
percentage of anionic form at physiological pH and with the
Figure 1. Superimposition (aromatic heavy atoms and exchangeable
hydrogen) of the AM1 global minimum conformers of 4b (magenta
carbon atoms) and of 4h tautomer 1 (orange carbon atoms). Nitrogen
atoms are in blue and oxygen atoms in red. Only exchangeable
hydrogens are shown, for the sake of clarity. H-bonds are highlighted
with dashed lines and colored as carbon atoms.
availability of an exchangeable hydrogen in the molecule. These
SARs indicated that to obtain compounds with decreased BBB
permeability and still endowed with high 5-HT3R affinity, a
crucial issue is the “masking” of the acidic function R′ (Table
1) through an internal proton exchange with the basic distal
piperazine nitrogen.
Compounds of cluster B (4c-e, 4g-i, 4l, (S)-4n, (R)-4n, and
4o, Table 1) bearing “mild” acidic piperazine N-4 substituents
(pKa > 7) do not present an anionic form at physiological pH,
with the exception of 4l, and most of them present the neutral
form as the most abundant one. Therefore, their bioactive form
presents piperazine N-4 always deprotonated with the exchange-
able hydrogen located on the R′ substituent heteroatom at the
γ or δ position with respect to piperazine N-4 (Table 1 of the
main text, Table 3 of SI, and Figure 1).
Compounds 4d, 4e, 4h, (S)-4n, (R)-4n, and 4i displayed
5-HT3R affinity in the range of 0.8 and 3.4 nM, 4h being as
potent as the reference compound 5a (Table 1). Accordingly,
they share the hypothesized key pharmacophoric features,
namely, the presence in tautomer 1 of a polarized H-bond
between R′(Hγ) and piperazine N-4 (dN4-Hγ/δ in Table 3 of
SI) and they show the same distances between the exchange-
able hydrogen and the other pharmacophoric groups, i.e., N-1
and the pyrroloquinoxaline system (dN1-Hγ/δ ≈ 6.6 Å and
dX1-Hγ/δ ≈ 8.6 Å, Table 3 of SI; 4h tautomer 1 vs 4b, Figure
1). 4h, the most active compound of the whole series, also
presents tautomer 2 stabilized by an internal H-bond with
R′(Hδ), together with the correct orientation of the pharma-
cophoric groups (dN1-Hγ/δ ) 6.5 Å and dX1-Hγ/δ ) 8.5 Å, Table
3 of SI). Compound 4c also shows nanomolar affinity (Ki )
7.3 nM, Table 1), although being slightly less potent than
the above-mentioned set of compounds. Affinity data of 4c,
presenting an amide moiety, confirmed a correlation between
the 5-HT3R affinity and the acidity of the exchangeable
hydrogen (i.e., single pKa value, Table 1). Furthermore, taking
into account 4n, the similar 5-HT3R affinity shown by the
two enantiomers (Table 1) indicates a receptor tolerance in
accommodating a carboxyl function close to piperazine N-4
(Figure 2), in agreement with what was observed for 4b and
4h (Figures 1 and 2). Apart from 4h, 4l is the only compound
presenting a second tautomer with an internal H-bond
between R′(Hγ) and piperazine N-4 (dN4-Hγ/δ, Table 3 of SI).

Targeting of Serotonin 5-HT3 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3555
Figure 3. Superimposition on pharmacophoric points (aromatic heavy
atoms, including the aryl nitrogen, and exchangeable hydrogen) of the
AM1 global minimum conformers of (R)-4n (pink carbon atoms) and
4o (green carbon atoms). Nitrogen atoms are in blue and oxygen atoms
in red. Only exchangeable hydrogens are shown, for the sake of clarity.
H-bonds are highlighted with dashed lines and colored as carbon atoms.
Figure 2. Superimposition (aromatic heavy atoms and exchangeable
hydrogen) of the AM1 global minimum conformer of (R)-4n (pink
carbon atoms) and a low energy conformer of (S)-4n (cyan carbon
atoms) (∆EAM1 ) 0.6 kcal/mol). Nitrogen atoms are in blue and oxygen
atoms in red. Only exchangeable hydrogens are shown, for the sake of
clarity. H-bonds are highlighted with dashed lines and colored as carbon
atoms.
Nevertheless, its 5-HT3R affinity is lower than that of 4h
(4h Ki ) 0.8 nM vs 4l Ki ) 17 nM, Table 1). This can be
explained considering that 4l, because of the presence of the
carbonyl oxygen on the triazole ring, presents four tautomeric
forms. The availability of an exchangeable hydrogen close
to piperazine N-4 is not preserved in all tautomers as it occurs
for 4h (Table 3 of SI). Furthermore, two out of four 4l
tautomers show a calculated percentage of the anionic form
at physiological pH (Table 1), in agreement with its reduced
affinity with respect to 4h. Compounds (()-4g (Ki ) 79 nM)
and (()-4f (Ki ) 44 nM), characterized by a bulky phenyl
substituent at piperazine N-4, show a nearly comparable
affinity, even though (()-4f was predicted to be mostly
present in the anionic form and (()-4g in the neutral one
(Table 1). The activity of (()-4g compared to that of (()-4f
could be ascribed (i) to the different location of the volume
occupied by the phenyl group caused by intramolecular
H-bond formation (Figure 4 of SI), and/or (ii) to the higher
acidity of the hydrogen-donor group of (()-4f with respect
to the alcoholic group of (()-4g (i.e., single pKa values, Table
1). The serine derivative 4o was much less potent (Ki ) 237
nM), although being the only compound of the whole series
predicted to be mostly (94%) present in the protonated form
at pH 7.2. The lower 5-HT3R affinity of 4o (4o vs 4n) is in
full agreement with the SARs emerging from our analysis.
As a result of the reduced availability of the nitrogen lone
pair (N-4) and of the reduced flexibility of the carbonyl group,
4o does not present an H-bond with R′ (dN4-Hγ/γδ/δ ) 3.8, 3.2
Å, Table 3 of SI) (4o vs 4n, Figure 3). By consequence, 4o
does not present the correct orientation of the exchangeable
hydrogen with respect to the other pharmacophoric groups
(dN1-Hγ/δ ) 8.3, 4.0 Å; dX1-Hγ/δ ) 10.4, 7.0 Å).
Figure 4. Mean plasma concentration-time curves of compound (S)-
4n after ip injection in the mouse. Each value is the mean ( SD of
three mice for the 5 (closed symbol) and 20 mg/kg doses (open symbol).
Compound (S)-4n was generally undetectable in the brain, but in one
animal each, 15 and 30 min after dosing, it reached levels close to the
limit of quantitation of the method (about 0.5 nmol/g).
affinity was found, thus suggesting that polarization plays a role
in interaction with the receptor. On these bases, it can be stated
that, at least in our series of derivatives, the cation-π and/or
salt-bridge interaction proposed for the amine nitrogen in
different receptor interaction models can be replaced with a
polarized H-bond interaction to obtain equally active compounds
with decreased BBB permeability.
Functional in Vitro Assays. Assessment of Cardiovas-
cular Activity of 4a, 4c, 4h, 4i, (S)-4n, and 5a on Isolated
Atria under the Effect of 5-HT. 5-HT induces a positive
chronotropic and inotropic effect in isolated right and left atria
preparations, respectively. This effect is mimicked by 5-HT3R
agonists such as 2, 1-phenylbiguanide (PBG), and 1 (a brain-
penetrating compound).10 We therefore evaluated the effects
produced by a subset of compounds on isolated atrial preparation
after treatment with 5-HT. Modulation of cardiovascular
parameters by compounds 4a, 4c, 4h, 4i, (S)-4n, 5a, and
reference 5-HT3R ligands is reported in Table 3. All tested
compounds are devoid of positive inotropic activity and do not
induce vasoconstriction. While compounds 4a, 4c, 4h, and 4i
In conclusion, the presence of a net positive charge on the
molecule and the protonation at piperazine N-4 are not necessary
for high 5-HT3R affinity (e.g., 4h vs 5a, Table 1). A net negative
charge on the molecule is detrimental for the affinity (4j), while
the presence of an exchangeable hydrogen close to piperazine
N-4 is required. Indeed, the formation of an intramolecular
H-bond between piperazine N-4 and the hydrogen at R′ is related
to high affinity likely because it results in a correct orientation
of the exchangeable pharmacophoric hydrogen. Furthermore, a
correlation between exchangeable hydrogen acidity and 5-HT3R

3556 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Morelli et al.
show antagonist activity, (S)-4n potently modulates chronotropy,
as well as 5-HT. In particular 4a, 4c, 4h, and 4i were
noncompetitive antagonists of the effect induced by 5-HT on
cardiac 5-HT3R (pIC50 ) 5.72, 4.95, 5.24, and 5.07, respectively,
Table 3), while tropisetron was found to be a competitive
antagonist of 5-HT3R (pA2 ) 7.52). Compound (S)-4n was a
partial agonist (R ) 0.81) 1.6- and 8.3-fold more potent than
5-HT and 2, respectively (pD2 ) 5.98, 5.78, and 5.06,
respectively, Table 3). These results indicate that (S)-4n
specifically acts at the right atrium through 5-HT3Rs. Moreover,
compound 5a showed a weak partial agonist activity (R ) 0.60)
at the right atrium level. Finally the peculiar cardiac activity of
(S)-4n and 5a, eliciting an effect only at the right atrium level,
might be due to selective action on a subpopulation of 5-HT3Rs.
Further investigations are needed in order to assess this
important issue.
5-HT3R with high affinity and is brain-permeable. In this test,
with an ID50 of 600 µg/kg (iv), compound 5a exerted antagonist
activity, even though it is less potent than 4c and 4d.
The partial agonist 4h, at a dose of 50 µg/kg (iv), induced a
transient bradycardia that was about 60% of that due to PBG
(40 µg/kg, iv). In addition to this agonist effect, 4h also exerted
an antagonist action, since it can prevent PBG-induced brady-
cardia. In this case the effect of 4h was clearly dose-dependent,
with half-blockade at 30 µg/kg when this drug was injected 5
min before PBG. The behavior of (S)-4n was difficult to
interpret. In fact after iv injection at a dose of 100 µg/kg, in
contrast to PBG, (S)-4n on its own did not alter heart rate. A
significant reduction in PBG-evoked bradycardia was found by
pretreating the rats with (S)-4n, but the effect was not time-
dependent and not reproducible in all rats (2 out of 7).
Brain-to-Plasma Distribution Studies. Mice were given
compound (S)-4n ip and were sacrificed at various times
thereafter for obtaining basic information on the concentrations
achieved in brain after doses of potential pharmacological
interest and for obtaining their relationship with plasma
concentrations. Figure 4 shows the mean plasma concentration-
time curves of (S)-4n after 5 and 20 mg/kg. The compound
rapidly reached the systemic circulation with maximal plasma
concentrations (2 and 12 µM at 5 and 20 mg/kg, respectively)
already 15 min after dosing (i.e., the first sampling time). At
the lower dose the assay was not sensitive enough for following
plasma concentrations for more than 30 min. This rapid decay
probably approximates the distribution phase of the compound. In
fact, a parallel distribution phase was also observed at 20 mg/kg,
but this was followed by a slower decay, between 30 and 180 min
after dosing. The elimination half-life (0.693/ꢀ) associated with
this phase averaged 53 min, this being the result of the high
clearance in conjunction with a relatively high volume of distribu-
tion of this compound (Cl ) dose F/AUC ) 90 (mL/min)/kg; Vd/F
) C1/ꢀ ) 6 L/kg, assuming complete absorption of (S)-4n from
the ip site). This pharmacokinetic behavior is consistent with that
of other pyrroloquinoxalines and structurally related compounds
such as arylpiperazines and their derivatives.12,13,39
In previous work, we evaluated the ability of pyrroloqui-
noxaline and arylpiperazine derivatives to cross the BBB in vivo
in rodents. Most of them rapidly entered the rat brain, achieving
whole brain concentrations higher than in plasma, although with
differences in the brain-to-plasma partition value.12,13,39 This
is explained by their lipophilicity, which allows free and rapid
diffusion across the BBB.40 An exception was a pyrroloqui-
noxaline derivative with a hydrophilic (carboxylic) group at the
piperazine ring (compound 7b as the internal standard (IS) of
the present HPLC procedure) for which the brain-to-plasma
distribution ratio averaged only 0.1 after 5 mg/kg in rats.13
Compound (S)-4n was generally undetectable in mouse brain
after ip doses, suggesting poor BBB permeability. However,
after 20 mg/kg it reached brain concentrations close to the limits
of quantitation of the analytical procedure (about 0.5 µM
assuming 1 g of tissue equivalent to 1 mL of water) in only
two animals (n ) 2) within 15-30 min of dosing. In these cases,
plasma concentrations averaged 10 µM.
Functional in Vivo Assays. von Bezold-Jarish Reflex
Test. In vivo investigations on the 5-HT3R-dependent von B-J
reflex in urethane-anesthetized rats (1.6 g/kg intraperitoneal
(ip))38 (Table 4) supported a different behavior for the tested
quinoxalines, with respect to that observed in the in vitro assays
performed on isolated atria preparation. The results ranged from
the agonist properties of 4a and the partial agonist properties
of 4h to the antagonist properties of 4c,d, and 5a. The von B-J
reflex is a cardiovascular reflex that has been demonstrated to
involve the activation of central 5-HT receptors. However, it
could also be mediated by stimulation of 5-HT3Rs located in
the cardiopulmonary area, in particular on sensory vagal nerve
endings in the heart, atrium, and ventricles. Substances experi-
mentally used to elicit the von B-J reflex include veratridine,
5-HT, and PBG, a selective 5-HT3R agonist. PBG caused dose-
dependent fall of heart rate and arterial pressure when injected
into the left or right atrium of unanesthetized animals. PBG
acts as an agonist through pharmacologically specific 5-HT3Rs.
So, PBG (agonist) and zacopride (antagonist) were tested as
reference 5-HT3R ligands on von B-J reflex. PBG (40 µg/kg,
iv) produced a marked transient bradycardia (64% decrease in
heart rate within the first 30 s after injection). Its effects are
prevented by pretreatment with zacopride (1 µg/kg, iv, 5 min
before PBG administration). Therefore, each tested compound
was injected iv 5, 15, 30 min prior PBG to assess their potential
antagonist effect.
When compound 4a, an agonist at 5-HT3Rs, was administered
alone, it mediated the von B-J reflex with an efficacy close to
that of PBG (ED50 value of 36 µg/kg (iv)), producing a transient
bradycardia similar to that observed after the iv injection of 40
µg/kg of PBG. At the same dose, 4a only exerted marginal
effects on the bradycardia evoked by PBG, indicating negligible
antagonist properties at cardiac 5-HT3Rs.
Compound 4c dose-dependently inhibited PBG-induced brady-
cardia with an ID50 value of 75 µg/kg (iv). This pyrroloqui-
noxaline was unable, at 50 µg/kg (iv), to affect heart rate in
urethane-anesthetized rats. However, pretreatment with 50 µg/
kg (iv) of 4c significantly attenuated the bradycardia elicited
by subsequent injection of PBG (40 µg/kg, iv). The effect
persisted to the same extent (-35% to -45%) for at least 30
min after administration of 4c. These data led to the conclusion
that 4c is an antagonist at cardiac 5-HT3Rs, even though its
potency is lower than that of zacopride (100% prevention at 1
µg/kg, iv). The hydroxamic acid derivative 4d, a potent 5-HT3R
ligand, also exerted a dose-dependent antagonist action, since
it could prevent PBG-induced bradycardia, with an ID50 value
of 50 µg/kg (iv). We also characterized 5a at the cardiac level.13
Compound 5a was used as a reference drug because it binds
Preliminary brain-to-plasma distribution studies were also
performed for compound 4a. Analogously to compound 7b,13
4a mean plasma concentrations exceeded those in brain.
Conclusions
This work led to the discovery of potent and selective 5-HT3R
ligands with poor BBB permeability due to their unique
structural features. The intrinsic pharmacological properties and

Targeting of Serotonin 5-HT3 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3557
described for 11a. After recrystallization 11b was obtained as yellow
the cardiac modulatory activity of the new compounds was
assessed by means of in vitro (functional preparations) and in
vivo (von B-J reflex test) studies. Because the compounds were
based on a pyrroloquinoxaline skeleton, the unique pharmaco-
logical properties of the novel modulators are mainly due to
the key substituents at the piperazine N-4. In cardiac tissue,
while 5-HT and 2 stimulate inotropic and chronotropic activity
(isolated left and right atria 5-HT3R) and 5-HT, but not 2,
induces vasoconstriction (non-5-HT3R), several of the new
compounds herein reported showed antagonism or partial
agonism of the positive chronotropy (right atrium) induced by
5-HT without affecting inotropy (left atrium). Among these
compounds 4c, 4h, and 4i were shown to be potent antagonists
in functional assays, selective for those 5-HT3Rs localized in
guinea pig right atrium (no vasoconstriction). Compound (S)-
4n was found as a potent cardiomodulator of the chronotropy
parameter with no effects on inotropy, indicating a selective
action on 5-HT3Rs localized at the right atrium. These com-
pounds may pave the way for the future development of new
peripherally acting 5-HT3R subtype-selective cardiomodulators.
In vivo functional evaluation (von B-J reflex) of a subset of
compounds was substantially in line with their in vitro functional
evaluation with the exception of compounds 4a, characterized
by a zwitterionic substituent at the piperazine N-4, which was
an agonist.
1
prisms (mp 217-221 °C, n-hexane). H NMR (CDCl3) δ 1.28 (t,
3H, J ) 7.0 Hz), 2.80 (m, 4H), 2.86 (s, 3H), 3.30 (s, 2H), 3.83 (m,
4H), 4.22 (q, 2H, J ) 7.0 Hz), 6.75 (m, 2H), 7.07 (m, 1H), 7.21
(m, 1H), 7.57 (m, 1H), 8.13 (m, 1H); IR (chloroform) 1732 cm-1
.
[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]acetic
Acid Benzyl Ester (11c). A suspension of 10a (0.200 g, 0.74 mmol),
anhydrous potassium carbonate (0.960 g, 0.74 mmol), and benzyl
bromoacetate (0.170 g, 0.74 mmol) in 2-butanone (33 mL) was
heated under reflux for 1 h under argon atmosphere. The solvent
was evaporated, and the residue was partitioned between water (15
mL) and DCM (30 mL). The organic layer was washed with brine,
dried over sodium sulfate, and evaporated. The residue was purified
by flash chromatography (1:5 DCM/ethyl acetate) to give 11c (0.220
1
g, 72%) as colorless prisms (mp 165-167 °C, ethyl acetate). H
NMR (CDCl3) δ 2.79 (m, 4H), 3.36 (s, 2H), 3.90 (m, 4H), 5.19 (s,
2H), 6.76 (m, 2H), 6.97 (m, 1H), 7.36 (m, 6H), 7.65 (m, 1H), 7.75
(m, 1H); IR (Nujol) 1681, 1622 cm-1
.
[4-(7-Benzyloxypyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-
yl]acetic Acid Benzyl Ester (11d). From 10c, the title compound
was obtained in 77% yield following the procedure described for
11c. After recrystallization, 11d was obtained as pale-yellow prisms
(mp 115-118 °C, ethyl acetate). 1H NMR (CDCl3) δ 2.60 (m, 4H),
3.37 (s, 2H), 3.82 (m, 4H), 5.15 (s, 2H), 5.19 (s, 2H) 6.73 (m, 2H),
6.96 (m, 1H), 7.22-7.48 (m, 11H), 7.63 (m, 1H), 7.72 (m, 1H);
IR (Nujol) 1675, 1628 cm-1
.
7-Fluoro-4-(4-cyanomethylpiperazin-1-yl)pyrrolo[1,2-a]quinoxa-
line (12). A mixture of 10a (0.220 g, 0.83 mmol), anhydrous
potassium carbonate (0.110 g, 0.83 mmol), and bromoacetonitrile
(58 µL, 0.83 mmol) in 2-butanone (35 mL) was heated under reflux
for 3 h under argon atmosphere. The solvent was evaporated, and
the residue was partitioned between water (15 mL) and DCM (30
mL). The organic layer was washed with brine, dried over sodium
sulfate, and concentrated. The residue was purified by flash
chromatography (1:3 DCM/ethyl acetate) to give 12 (0.187 g, 73%)
as colorless prisms after recrystallization (mp 147-149 °C, 1:1 ethyl
acetate/n-hexane). 1H NMR (CDCl3) δ 2.80 (m, 4H), 3.61 (s, 2H),
3.86 (m, 4H), 6.76 (m, 2H), 6.97 (m, 1H), 7.33 (m, 1H), 7.65 (m,
In summary, the design of selective 5-HT3R ligands poorly
penetrating the brain represents a valuable strategy to develop
novel cardiomodulators with minimized central side effects,
being an important step in the progression toward peripherally
acting 5-HT3R ligands for the treatment of a variety of diseases.
Compounds 4a and 4n represent novel pharmacological tools
to investigate the role of putative 5-HT3R subpopulations at the
cardiac level.
Experimental Section
1H), 7.76 (m, 1H); IR (Nujol) 2245 cm-1
.
Reagents were purchased from Sigma-Aldrich and were used as
received. Reaction progress was monitored by TLC using Merck
silica gel 60 F254 (0.040-0.063 mm) with detection by UV. Merck
silica gel 60 (0.040-0.063 mm) was used for flash chromatography.
Melting points were determined using an Electrothermal 8103
apparatus. IR spectra were taken with Perkin-Elmer 398 and FT
[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]acetic
Acid (4a). A solution of 11c (0.230 g, 0.55 mmol) in methanol (5
mL) was hydrogenated at atmospheric pressure over 10% Pd/C for
1 h. The catalyst was removed by filtration over Celite, the filtrate
was evaporated, and the residue was recrystallized from ethyl acetate
to provide 4a (0.161 g, 89%) as colorless prisms (mp 219-221
°C, n-hexane). 1H NMR (DMSO-d6) δ 2.72 (m, 4H), 3.25 (s, 2H),
3.81 (m, 4H), 6.78 (m, 1H), 6.94 (m, 1H), 7.15 (m, 2H), 8.12 (m,
1H), 8.31 (m, 1H), 11.05 (br, 1H); IR (Nujol) 1715 cm-1. Anal.
(C17H17N4O2F) C, H, N.
1
1600 spectrophotometers. H NMR spectra were recorded on a
Bru¨ker 200 spectrometer with TMS as internal standard. Splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet
(q), and broad (br). The value of chemical shifts (δ) are given in
ppm and coupling constants (J) in hertz (Hz). All reactions were
carried out in an argon atmosphere. Elemental analyses were
performed on a Perkin-Elmer 240 °C elemental analyzer, and the
results were within (0.4% of the theoretical values (purity >95%).
Yields refer to purified products and are not optimized.
[4-(7-Hydroxypyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]ace-
tic Acid (4b). From 11d, the title compound was treated with 10%
Pd/C for 12 h and was obtained as described for the preparation of
4a in 85% yield. After recrystallization, 4b was obtained as colorless
1
prisms (mp 140-141 °C, n-hexane). H NMR (DMSO-d6) δ 2.69
[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]acetic
Acid Ethyl Ester (11a). Diisopropylethylamine (DIPEA) (85.0 mg,
0.66 mmol) and ethyl bromoacetate (0.120 g, 0.75 mmol) were
added to a solution of 10a (0.120 g, 0.44 mmol) in acetonitrile (10
mL). The mixture was stirred at room temperature for 12 h. The
solvent was evaporated, and the residue was dissolved in dichlo-
romethane (DCM) and washed with water (10 mL). The organic
phase was dried over sodium sulfate and evaporated. The residue
was purified by flash chromatography (1:1 n-hexane/ethyl acetate)
to give 11a (0.140 g, 89%) as colorless prisms (mp 208-210 °C,
n-hexane). 1H NMR (CDCl3) δ 1.28 (t, 3H, J ) 6.9 Hz), 2.79 (m,
4H), 3.31 (s, 2H), 3.91 (m, 4H), 4.22 (m, 2H), 6.78 (q, 2H, J )
6.9 Hz), 6.92 (m, 1H), 7.33 (m, 1H), 7.65 (m, 1H), 7.78 (m, 1H);
(m, 4H), 3.12 (s, 2H), 3.68 (m, 4H), 6.69 (m, 2H), 6.85 (m, 2H),
7.86 (m, 1H), 8.15 (m, 1H); IR (Nujol) 1705 cm-1. Anal.
(C17H18N4O3) C, H, N.
2-[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)-piperazin-1-yl]ac-
etamide (4c). Finely powdered potassium hydroxide (0.130 g,
2.3 mmol) was added to a stirred solution of 12 (0.200 g, 0.65
mmol) in tert-butyl alcohol (5 mL). The reaction mixture was
heated under reflux for 35 min with vigorous stirring. The
mixture was cooled to room temperature and poured into an
aqueous sodium chloride solution, and the aqueous phase was
extracted with chloroform (3 × 30 mL). The organic layers were
dried over sodium sulfate and concentrated in vacuo. The crude
product was purified by flash chromatography (1:3 ethyl acetate/
chloroform). After recrystallization 4c (0.150 g, 71%) was
obtained as a yellow solid (mp 148-150 °C, n-hexane). 1H NMR
(CD3OD) δ 2.62 (m, 4H), 2.98 (s, 2H), 3.81 (m, 4H), 6.82 (m,
IR (chloroform) 1728 cm-1
.
[4-(9-Methylpyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]ace-
tic Acid Ethyl Ester (11b). From 10b and ethyl bromoacetate, the
title compound was obtained in 88% yield following the procedure

3558 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Morelli et al.
1H), 6.96 (m, 1H), 7.12 (m, 2H), 7.29 (m, 2H); IR (Nujol) 1655
cm-1. Anal. (C17H18N5OF) C, H, N.
[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]hydroxy-
acetamidine (4h). Compound 12 (0.100 g, 0.32 mmol), hydroxy-
lamine hydrochloride (44.0 mg, 0.64 mmol), and sodium carbonate
(67.0 mg, 0.64 mmol) were dissolved in ethanol (6.5 mL) and heated
under reflux for 24 h. After filtration of inorganic salts, the solvent
was evaporated under reduced pressure. The product was purified
by flash chromatography (9:1 DCM/methanol) to provide, after
recrystallization, 4h (68.0 mg, 55%) as colorless prisms (mp
155-157 °C, n-hexane). 1H NMR (DMSO-d6) δ 2.51 (s, 2H), 3.10
(m, 4H), 3.78 (m, 4H), 5.30 (br, 2H), 6.70-7.30 (m, 5H), 8.10
(m,1H), 9.00 (br, 1H); IR (Nujol) 3225, 765 cm-1. Anal.
(C17H19N6OF) C, H, N.
[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]hydroxy-
acetamide (4d). A solution of potassium hydroxide (0.180 g, 3.21
mmol) in methanol (0.86 mL) was added to a solution of
hydroxylamine hydrochloride (0.130 g, 2.0 mmol) in methanol (0.80
mL). The resulting suspension was stirred at 0 °C for 1 h. Afterward
it was filtered into a solution of compound 11a (0.100 g, 0.28 mmol)
in methanol (0.53 mL). The reaction mixture was stirred at room
temperature for 15 min and concentrated to dryness. The residue
was dissolved in water (1.1 mL), and the aqueous phase was
acidified to pH 5 with 1 N hydrochloric acid and extracted with
ethyl acetate (3 × 20 mL). The organic layer was washed with
water, dried over sodium sulfate, and evaporated. The crude product
was purified by flash chromatography (ethyl acetate) to give, after
recrystallization, 4d (56 mg, 59%) as colorless prisms (mp 159-160
°C, 1:1 ethyl acetate/n-hexane). 1H NMR (CD3OD) δ 2.66 (m, 4H),
3.21 (s, 2H), 3.78 (m, 4H), 6.68 (m, 1H), 6.81 (m, 1H), 6.93 (m,
1H), 7.19 (m, 1H), 7.81 (m, 1H), 7.93 (m,1H); IR (Nujol) 1668,
1635, 1468, 1443 cm-1. Anal. (C17H18N5O2F) C, H, N.
7-Fluoro-4-[4-(4H-[1,2,4]triazol-3-ylmethyl)-piperazin-1-yl]pyr-
rolo[1,2-a]quinoxaline (4i). A mixture of 10a (0.200 g, 0.93 mmol),
anhydrous potassium carbonate (0.250 g, 1.86 mmol), and N-formyl-
2-chloroacetamidohydrazone17 (0.188 g, 1.39 mmol) in anhydrous
N,N-dimethylformamide (N,N-DMF, 8 mL) was heated to 60 °C
for 3 h and to 130 °C for the next 12 h. The reaction mixture was
cooled to room temperature and diluted with ethyl acetate. The
organic layer was washed with water, dried over sodium sulfate,
and evaporated. The resulting brown oil was purified by flash
chromatography (7:3 ethyl acetate/petroleum ether) to afford, after
recrystallization, 4i (0.530 g, 81%) as colorless prisms (mp
[4-(9-Methylpyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]hy-
droxyacetamide (4e). From 11b, the title compound was prepared
in 62% yield following the procedure described for 4d. After
recrystallization, 4e was obtained as a yellow prisms (mp 165-166
1
209-210 °C, ethyl acetate). H NMR (CD3OD) δ 3.28 (m, 4H),
4.25 (m, 4H), 4.37 (s, 2H), 6.81 (m, 2H), 7.18 (m, 1H), 7.46 (m,
1H), 7.76 (m, 1H), 7.81 (m, 1H), 8.22 (m, 1H); IR (Nujol) 1679
cm-1. Anal. (C18H18N7F) C, H, N.
1
°C, 1:1 ethyl acetate/n-hexane). H NMR (DMSO-d6) δ 2.62 (m,
4H), 2.82 (s, 3H), 2.95 (s, 2H), 3.65 (m, 4H), 6.79 (m, 1H), 6.90
(m, 1H), 7.12 (m, 1H), 7.21 (m, 1H), 7.42 (m, 1H), 8.25 (m, 1H),
8.80 (br, 1H), 10.49 (br, 1H); IR (Nujol) 1659, 1639, 1471, 1447
cm-1. Anal. (C18H21N5O2) C, H, N.
7-Fluoro-4-[4-(methanesulfonamidoacetyl)piperazin-1-yl]pyr-
rolo[1,2-a]quinoxaline (4j). A solution of 4a (0.100 g, 0.30 mmol)
in dry THF (0.6 mL) was added dropwise to a stirred solution of
CDI (50.0 mg, 0.30 mmol) in dry THF (0.6 mL). The reaction
mixture was stirred at room temperature for 30 min. Afterward it
was heated under reflux for further 30 min and finally was allowed
to cool to room temperature. Methanesulfonamide (29.0 mg, 0.30
mmol) was added in one portion to the reaction mixture which was
stirred for 10 min before a solution of DBU (45.0 mg, 0.30 mmol)
in dry THF (0.5 mL) was added dropwise. The mixture was stirred
for a further 12 h and then concentrated to dryness. The residue
was taken up with water (10 mL) and extracted with ethyl acetate
(3 × 10 mL). The organic layer was washed with water, dried over
sodium sulfate, and concentrated. Purification by flash chromatog-
raphy (9:1 DCM/methanol) afforded, after recrystallization, com-
pound 4j (27.0 mg, 67%) as colorless prisms (mp 187-189 °C,
n-hexane). 1H NMR (CDCl3) δ 2.83 (m, 4H), 3.11 (s, 3H), 3.27 (s,
2H), 3.98 (m, 4H), 6.82 (m, 2H), 7.03 (m, 1H), 7.46 (m, 1H), 7.71
(m, 1H), 7.82 (m, 1H); IR (KBr) 1631, 1083 cm-1. Anal.
(C18H20N5O3SF) C, H, N.
[4-(9-Methylpyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]phenyl-
acetic Acid (4f). From 10b and 1-bromophenylacetic acid ethyl ester,
[4-(9-methylpyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]phenyl ace-
tic acid ethyl ester was prepared in 80% yield following the
procedure described for 11a and, after recrystallization, was
1
obtained as yellow prisms (mp 209-211 °C, n-hexane). H NMR
(CDCl3) δ 1.30 (t, 3H, J ) 7.0 Hz), 2.59 (m, 2H), 2.63 (m, 2H),
2.82 (s, 3H), 3.59 (m, 4H), 3.88 (m, 1H), 4.25 (q, 2H, J ) 7.0 Hz),
6.78 (m, 1H), 6.82 (m, 1H), 7.00-7.42 (m, 6H), 7.49 (m, 2H),
8.24 (m, 1H). The above compound (0.150 g, 0.35 mmol) was
dissolved in tetrahydrofuran (THF, 2.8 mL), and while the mixture
was being stirred, a solution of lithium hydroxide (25.0 mg, 1.0
mmol) in water (1.9 mL) was added dropwise. The reaction mixture
was heated under reflux for 3 h. After this time, THF was removed
under reduced pressure and the residue was treated with water,
acidified, and extracted with ethyl acetate (3 × 10 mL). The organic
phase was dried over sodium sulfate and evaporated. The residue
was purified by flash chromatography (3:1 ethyl acetate/methanol)
to give, after recrystallization, 4f (0.133 g, 89%) as pale-yellow
7-Fluoro-4-[4-(1H-tetrazol-5-yl)methylpiperazin-1-yl]pyrrolo[1,2-
a]quinoxaline (4k). A mixture of 12 (0.100 g, 0.32 mmol),
piperidine hydrochloride (97.0 mg, 0.80 mmol), and sodium azide
(52.0 mg, 0.80 mmol) in anhydrous N,N-DMF (1 mL) was heated
to 115 °C for 16 h. The reaction mixture was diluted with aqueous
ammonium chloride (10 mL) and extracted with ethyl acetate (3 ×
15 mL). The organic layers were washed with brine, dried over
sodium sulfate, and concentrated. The residue was purified by flash
chromatography (7:3 DCM/methanol) to give, after recrystallization,
4k (28.0 mg, 69%) as pale-yellow prisms (mp 233-234 °C,
1
prisms (mp 225 °C dec, n-hexane). H NMR (DMSO-d6) δ 2.53
(m, 2H), 2.66 (m, 2H), 2.80 (s, 3H), 3.63 (m, 4H), 3.85 (m, 1H),
6.74 (m, 1H), 6.86 (m, 1H), 7.09-7.37 (m, 6H), 7.46 (m, 2H),
8.22 (m, 1H), 10.09 (br, 1H); IR (Nujol) 1733 cm-1. Anal.
(C24H24N4O2) C, H, N.
2-[4-(9-Methylpyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]-2-
phenylethanol (4g). Lithium borohydride (2.0 M solution in THF,
460 µL, 0.94 mmol) was added dropwise to a solution of [4-(9-
methylpyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]phenyl acetic
acid ethyl ester (0.100 g, 0.23 mmol) in dry THF (2.5 mL). The
reaction mixture was heated under reflux for 12 h and quenched
with methanol and 1 N hydrochloric acid. Evaporation of reaction
mixture gave a yellow oil which was treated with water and
extracted with ethyl acetate. The organic phase was dried over
sodium sulfate and evaporated to give an oil which was purified
by flash chromatography (20:1 ethyl acetate/methanol). Compound
4g (70.0 mg, 79%) was obtained, after recrystallization, as yellow
prisms (mp 122-123 °C, n-hexane). 1H NMR (CDCl3) δ 2.58 (m,
2H), 2.82 (m, 6H), 3.76 (m, 6H), 4.05 (m, 1H), 6.69 (m, 2H), 7.08
(m, 1H), 7.23 (m, 3H), 7.34 (m, 3H), 7.53 (m, 1H), 8.11 (m, 1H);
IR (chloroform): 1063 cm-1. Anal. (C24H26N4O) C, H, N.
1
n-hexane). H NMR (DMSO-d6) δ 2.70 (m, 4H), 3.74 (m, 4H),
3.88 (s, 2H), 6.81 (m, 1H), 6.96 (m, 1H), 7.19 (m, 2H), 8.17 (m,
2H), 8.32 (m, 1H); IR (Nujol) 3211-2405, 1628 cm-1. Anal.
(C17H17N8F) C, H, N.
7-Fluoro-4-[4-(2,3-dihydro-4H-3-oxo-1,2,4-triazol-5-yl)meth-
ylpiperazin-1-yl]pyrrolo[1,2-a]quinoxaline (4l). A mixture of 10a
(0.500 g, 1.85 mmol), N-carbomethoxy-2-chloroacetamidrazone16
(0.300 g, 1.850 mmol), and potassium carbonate (0.500 g, 3.700
mmol) in N,N-DMF (10 mL) was stirred at 70 °C for 18 h and at
140 °C for 1 h. After cooling to room temperature, the material
was partitioned between ethyl acetate (15 mL) and water (5 mL).
The organic layer was washed with water, dried over sodium sulfate,
and concentrated. The residue was purified by flash chromatography
(95:5 ethyl acetate/methanol) and recrystallized from ethyl acetate

Targeting of Serotonin 5-HT3 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3559
to give 4l (0.137 g, 75%) as colorless prisms (mp 169-171 °C,
n-hexane). 1H NMR (DMSO-d6) δ 2.59 (m, 4H), 3.31 (s, 2H), 3.85
(m, 4H), 6.78 (m, 1H), 6.97 (m, 1H), 7.15 (m, 1H), 7.29 (m, 1H),
8.16 (m, 1H), 8.32 (m, 1H), 10.15 (br, 1H), 11.32 (br, 1H); IR
(Nujol) 1698 cm-1. Anal. (C18H18N7OF) C, H, N.
(R)-2-Amino-3-[4-(7-fluoropyrrolo[1,2-a]quinoxalin-4-yl)piper-
azin-1-yl]propionic Acid ((R)-4n). From 10a and N-(tert-butyloxy-
carbonyl)-D-serine ꢀ-lactone17 the title compound was prepared
following the procedure described for (S)-4n and was obtained,
after recrystallization, as colorless prisms. Spectroscopic and
physical data were identical to those of the (S)-enantiomer; [R]D24
-10.5 (c 0.5, H2O). Anal. (C18H20N5O2F) C, H, N.
5-[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)-piperazin-1-ylmethyl]-
3H-[1,3,4]oxadiazol-2-one (4m). Compound 11a (0.100 g, 0.28
mmol) was dissolved in hydrazine hydrate (8.4 mL) and heated to
120 °C for 2 h. After cooling to room temperature, the mixture
was evaporated under reduced pressure. The residue was treated
with water (10 mL) and extracted with ethyl acetate (3 × 15 mL).
The crude product was purified by flash chromatography (9:1 DCM/
methanol) to give, after recrystallization, [4-(7-fluoropyrrolo[1,2-
a]quinoxalin-4-yl)piperazin-1-yl]acetic acid hydrazide (84.0 mg,
(S)-2-Amino-1-[4-(7-fluoropyrrolo[1,2-a]quinoxalin-4-yl)piper-
azin-1-yl]-3-hydroxypropan-1-one ((S)-4o). The Boc-protected prod-
uct (S)-13 (0.120 g, 0.34 mmol) was treated with an excess of
trifluoracetic acid (3 mL), and the resulting solution was stirred at
room temperature for 30 min. Excess of trifluoroacetic acid was
removed under vacuum. The hydrochloride salt was generated by
stirring an aqueous solution of the trifluoroacetate salt with IRA400
Amberlyte resin in its Cl- form during 24 h at room temperature.
The resin was removed by filtration, and the aqueous solution was
washed twice with ethyl acetate. Lyophilization afforded the pure
1
88%) as colorless prisms (mp 165-167 °C, n-hexane). H NMR
(DMSO-d6) δ 2.62 (m, 4H), 3.02 (s, 2H), 3.78 (m, 4H), 4.26 (br,
2H), 6.80-6.98 (m, 2H), 7.12-7.28 (m, 2H), 8.15 (m, 1H), 8.31
(m, 1H), 9.01 (br, 1H); IR (Nujol) 1695, 1598 cm-1. CDI (0.144
g, 0.87 mmol) was added to a solution of the above compound
(0.200 g, 0.58 mmol) and triethylamine (160 µL, 1.15 mmol) in
THF (6 mL), cooled to 0 °C. The reaction mixture was stirred at
room temperature for 12 h. The volatiles were removed in vacuo,
and the residue was dissolved in diethyl ether. The organic phase
was washed with a saturated solution of sodium bicarbonate and
with brine. The organic layer was dried over sodium sulfate and
concentrated in vacuo. The resulting white solid was purified by
flash chromatography (2:1 n-hexane/ethyl acetate) to afford, after
recrystallization, 4m (0.102 g, 48%) as colorless prisms (mp
1
hydrochloride salt in 86% yield (mp 176-177 °C, n-hexane). H
NMR (D2O) δ 4.05 (m, 6H), 4.29 (m, 4H), 4.71 (m, 1H), 7.02 (m,
1H), 7.19 (m, 1H), 7.43 (m, 1H), 7.57 (m, 1H), 7.83 (m, 1H), 8.18
(m, 1H); IR (Nujol) 1698, 1598, 1088 cm-1; [R]D24 -14.3 (c 0.6,
H2O). Anal. (C18H20N5O2F) C, H, N.
(4-Quinolin-2-yl-piperazin-1-yl)acetic Acid Ethyl Ester (14).
DIPEA (0.180 g, 1.400 mmol) and ethyl bromoacetate (0.260 g,
1.50 mmol) were added to a solution of 1 (0.200 g, 0.94 mmol) in
acetonitrile (15 mL). The mixture was stirred at room temperature
for 12 h. The solvent was evaporated, and the residue was dissolved
in DCM and washed with water. The organic phase was dried over
sodium sulfate and evaporated. The residue was purified by flash
chromatography (1:1 n-hexane/ethyl acetate) to give, after recrys-
tallization, compound 14 (0.260 g, 93%) as colorless prisms (mp
205-207 °C, n-hexane). 1H NMR (CDCl3) δ 1.26 (t, 3H, J ) 7.1
Hz), 2.81 (m, 4H), 3.41 (s, 2H), 3.98 (m, 4H), 4.24 (q, 2H, J ) 7.1
Hz), 6.92 (m, 1H), 7.23 (m, 1H), 7.59 (m, 2H), 7.75 (m, 1H), 7.91
1
151-153 °C, n-hexane). H NMR (acetone-d6) δ 2.75 (m, 4H),
3.59 (s, 2H), 3.84 (m, 4H), 6.77 (m, 1H), 6.95 (m, 1H), 7.05 (m,
1H), 7.25 (m, 1H), 8.01 (m, 1H), 8.12 (m, 1H), 11.33 (br, 1H); IR
(Nujol) 1785 cm-1. Anal. (C18H17N6O2F) C, H, N.
(S)-{2-[4-(7-Fluoropyrrolo[1,2-a]quinoxalin-4-yl)piperazin-1-yl]-
1-hydroxymethyl-2-oxoethyl}carbamic Acid tert-Butyl Ester ((S)-
(13)). N-(tert-Butoxycarbonyl)-L-serine (0.231 g, 1.13 mmol),
1-hydroxybenzotriazole (0.152 g, 1.13 mmol), and N-methylmor-
pholine (60 µL, 0.54 mmol) were added to a stirred solution of
10a (0.200 g, 0.75 mmol) in N,N-DMF (6 mL). The resulting
mixture was cooled to 0 °C and treated with 1-ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride (0.115 g, 0.59
mmol). After being stirred at room temperature for 18 h, the mixture
was poured into a saturated solution of sodium bicarbonate and
the aqueous layer was extracted with ethyl acetate (3 × 20 mL).
The organic phase was washed with water and brine, dried over
sodium sulfate, and evaporated. The residue was purified by flash
chromatography (ethyl acetate) to afford, after recrystallization,
compound (S)-13 (0.220 g, 63%) as colorless prisms (mp 182-183
(m, 1H); IR (Nujol) 1728 cm-1
.
(4-Quinolin-2-ylpiperazin-1-yl)acetic Acid (15). A solution of
lithium hydroxide (20.0 mg, 0.60 mmol) in water (3 mL) was added
dropwise to a solution of 14 (0.150 g, 0.50 mmol) in THF (3 mL).
The mixture was stirred at room temperature for 1 h. Then the
mixture was concentrated to dryness and the residue was dissolved
in water (1 mL). The aqueous phase was acidified to pH 5 with 1
N hydrochloric acid and extracted with ethyl acetate (3 × 10 mL).
The organic layer was washed with water, dried over sodium sulfate,
and evaporated. The residue was purified by flash chromatography
(ethyl acetate) to give, after recrystallization, 15 (88.0 mg, 65%)
as colorless prisms (mp 230 °C dec, n-hexane). 1H NMR (DMSO-
d6) δ 2.75 (m, 4H), 3.35 (s, 2H), 3.76 (m, 4H), 7.21 (m, 2H), 7.51
(m, 2H), 7.65 (m, 1H), 7.98 (m, 1H), 9.81 (br, 1H); IR (Nujol)
1702 cm-1. Anal. (C15H17N3O2) C, H, N.
1
°C, n-hexane). H NMR (CDCl3) δ 1.45 (s, 9H), 3.78 (m, 5H),
3.87 (m, 6H), 4.68 (m, 1H), 5.76 (br, 1H), 6.77 (m, 2H), 7.02 (m,
1H), 7.39 (m, 1H), 7.66 (m, 1H), 7.77 (m, 1H); IR (chloroform)
Binding Assays. 5-HT Receptor Subtypes. Binding assays were
performed according to previously reported experimental
procedures.12,13,41
1695, 1093 cm-1
.
(S)-2-Amino-3-[4-(7-fluoropyrrolo[1,2-a]quinoxalin-4-yl)piper-
azin-1-yl]propionic Acid ((S)-4n)). N-(tert-Butyloxycarbonyl)-L-
serine ꢀ-lactone17 (50.0 mg, 0.27 mmol) was added to a vigorously
stirred solution of 10a (0.200 g, 0.82 mmol) in dry N,N-DMF (8
mL). The resulting mixture was stirred at room temperature for
12 h, then was evaporated under reduced pressure, and trifluoracetic
acid (0.68 mL) was added to the residue with ice-cooling. The
resulting mixture was stirred at room temperature for 12 h. Excess
trifluoracetic acid was removed under reduced pressure, and the
residue was applied to a column of Dowex 50WX8400 resin. The
column was washed with 50% aqueous ethanol and eluted with 1
M aqueous pyridine. The ninhydrin-positive fractions of the 1 M
aqueous pyridine eluate were combined and evaporated to dryness.
Treatment of the residue with 1 N hydrochloric acid afforded the
hydrochloride salt of (S)-4n as an off-white solid in 83% yield (mp
278-279 °C, n-hexane). 1H NMR (D2O) δ 3.65 (m, 6H), 4.38 (m,
5H), 7.09 (m, 1H), 7.28 (m, 1H), 7.46 (m, 1H), 7.62 (m, 1H), 7.93
(m, 1H), 8.31 (m, 1H); IR (Nujol) 1486, 1628 cm-1. [R]D24 +9.7 (c
0.6, H2O). Anal. (C18H20N5O2F) C, H, N.
Dopamine, Adrenergic, and Muscarinic Receptors. Binding
assays were performed according to previously reported experi-
mental procedures.42-44
Molecular Modeling. All molecular modeling studies were
performed on SGI Octane2 2XR14000 workstation. The log D
values together with single and apparent pKa of all possible
tautomers of compounds 1, 4a-o, 5a,b, 6a,b, and 15 were
calculated by using the pKa DB program in the LogD Sol suite,
v10.0 (Advanced Chemistry Development Inc., Toronto, Canada).
The percentage of neutral/ionized forms at physiological pH (7.2)
was computed with the Solubility DB program within the same
software suite according to calculated apparent pKa.
All possible tautomers of compounds 4a-o and 15 were built
in the calculated most abundant ionic form at pH 7.2 by using the
Insight2005 Builder module (Accelrys, San Diego, CA). Com-
pounds 4a and 4k were also modeled in the neutral and zwitterionic
form, respectively. On the bases of R′ single pKa values, compounds
4a-o and 15 were clustered in two groups: group A (pKa < 7) and
group B (pKa > 7). Conformational space was sampled through

3560 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11
Morelli et al.
200 cycles of simulated annealing (CVFF force field45) by following
our standard protocol. An initial temperature of 1000 K was applied
to the system for 1000 fs with the aim of surmounting torsional
barriers; successively temperature was linearly reduced to 300 K
with a decrement of 0.5 K/fs. Resulting structures were subjected
to energy minimization within the Insight2005 Discover module
(CVFF force field, conjugate gradient algorithm;46 ε ) 80r) until
the maximum rms derivative was less than 0.001 kcal/Å. The
structures were subsequently ranked by their conformational energy
values. Resulting conformers were then subjected to an in depth
structural analysis and accordingly grouped into families. The
following structural parameters were measured: (i) distance between
the protonatable nitrogen (N-4) and the H-bond acceptor group (N-
1); (ii) distance between the center of the aromatic ring (X1) and
N-4; (iii) distance between X1 and N-1; (iv) distance between N-4
and the plane of the aromatic ring; (v) distance between the proton
on N-4 and the closest negatively charged atom on R′ for cluster
A or (vi) distance between the exchangeable hydrogen on R′ and
N-4 for cluster B; (vii) distance between the proton on N-4 and
N-1 for cluster A or (viii) distance between N-1 and the exchange-
able hydrogen on R′ for cluster B; (ix) distance between the proton
on N-4 and X1 for cluster A or (x) distance between X1 and the
exchangeable hydrogen on R′ for cluster B. In particular, parameters
iv-x were used for structural classification into families.
All MM conformers were successively submitted to 1SCF, AM1
energy calculation in the Mopac 6.047a package in Ampac/Mopac
module of Insight2000.1 and ranked by resulting conformational
energies.
5-HT2R. The physiological salt solution (PSS) was buffered at pH
7.4 by saturation with 95% O2-5% CO2 gas, and the temperature
was maintained at 35 °C. Isolated guinea pig heart preparations
were used, spontaneously beating right and left atria driven at 1
Hz. For each preparation, the entire left and right atria were
dissected from the ventricles, cleaned of excess tissue, and hung
vertically in a 15 mL organ bath containing the PSS continuously
bubbled with 95% O2-5% CO2 gas at 35 °C, pH 7.4. The
contractile activity was recorded isometrically by means of a force
transducer (FT 0.3, Grass Instruments Corporation, Quincy, MA)
using Power Lab software (AD-Instruments Pty Ltd., Castle Hill,
Australia). The left atrium was stimulated by rectangular pulses of
0.6-0.8 ms duration and about 50% threshold voltage through two
platinum contact electrodes in the lower holding clamp (Grass S88
stimulator). The right atrium was in spontaneous activity. After the
tissue was beating for several minutes, a length-tension curve was
determined, and the muscle length was maintained at the length
that elicited 90% of maximum contractile force observed at the
optimal length. A stabilization period of 45-60 min was allowed
before the atria were used to test compounds. During the equilibra-
tion period, the bathing solution was changed every 15 min and
the threshold voltage was ascertained for the left atrium. Atrial
muscle preparations were used to examine the inotropic and
chronotropic activity of the compounds (0.1, 0.5, 1, 5, 10, 50, and
100 µM), first dissolved in dimethyl sufoxide (DMSO) and then
diluted with PSS. According to this procedure, the concentration
of DMSO in the bath solution never exceeded 0.3%, a concentration
that did not produce appreciable inotropic and chronotropic effects.
During the construction of cumulative dose-response curves, the
next higher concentration of the compounds was added only after
the preparation reached a steady state.
Guinea Pig Aortic Strips. The thoracic aorta was removed and
placed in Tyrode solution of the following composition (mM): 118
NaCl, 4.75 KCl, 2.54 CaCl2, 1.20 MgSO4, 1.19 KH2PO4, 25
NaHCO3, and 11 glucose equilibrated with 95% O2-5% CO2 gas
at pH 7.4. The vessel was cleaned of extraneous connective tissue.
Two helicoidal strips (10 mm × 1 mm) were cut from each aorta
beginning from the end most proximal to the heart. Vascular strips
were then tied with surgical thread (6-0) and suspended in a
jacketed tissue bath (15 mL) containing aerated PSS at 35 °C. Strips
were secured at one end to a force displacement transducer (FT
0.3, Grass Instruments Corporation) for monitoring changes in
isometric contraction. Aortic strips were subjected to a resting force
of 1 g and washed every 20 min with fresh PSS for 1 h. After the
equilibration period the compounds (0.1, 0.5, 1, 5, 10, 50, and 100
µM) were added cumulatively to the bath allowing for any
contraction for obtaining an equilibrated level of force. Addition
of the drug vehicle had no appreciable effect on induced contraction
(DMSO for all compounds).
Functional Antagonism. The right atrium from guinea pig,
removed and prepared as described above, was equilibrated for 1 h,
and cumulative concentration-response curves to 5-HT (0.001-1.0
µM) were constructed. The concentration of agonist in the organ
bath was increased approximately 5-fold at each step, with each
addition being made only after the response to the previous addition
had attained a maximal level and remained steady. During the
equilibration period the bathing solution was changed every 15 min.
Following incubation with the antagonist for 30 min, a new
concentration-response curve to 5-HT was obtained. Parallel
experiments in which tissues did not receive any antagonist were
run in order to check any variation in sensitivity.
Functional in Vivo Studies. von Bezold-Jarisch Reflex. Adult
male Sprague-Dawley rats (250-300 g body weight, Centre
d’Elevage R. Janvier, Le Genest, France) were anesthetized with
urethane (1.6 g/kg ip), and a tracheotomy was performed to insert
an endotracheal tube. A catheter (0.3 mm internal diameter) was
inserted into the abdominal aorta via the femoral artery in order to
record the arterial pressure and heart rate. A femoral vein was
exposed and cannulated for iv injection of drugs. The von B-J reflex
(which consists of a 64% drop in heart rate within 30 s following
the injection of 40 µg/kg (iv) of PBG) was measured 5, 15, and 30
MM global minima together with the lowest AM1 energy
conformer of each family were selected to be successively subjected
to a full geometry optimization by semiempirical calculations, using
the quantum mechanical method AM1 implemented in the Mopac
2007.47b The EF optimization method (DDMAX ) 0.05) and the
Pulay convergence method (GNORM ) 0.01) have been used. To
reach a full geometry optimization, the criterion for terminating
all optimizations was increased by a factor of 100, using the
keyword PRECISE. ESP charges were computed in addition to
default Coulson atomic charges, by adding the keywords ESP and
STO3G.
The electrostatic potentials of AM1 conformers, including ESP
charges, were also calculated by the MNDO semiempirical method
(1 SCF).
Resulting structures were tabulated, analyzed, and grouped into
families following the same geometric criteria described above.
Functional in Vitro Studies. Compounds were checked at
increasing doses for evaluating the percent decrease of developed
tension on isolated left atrium driven at 1 Hz (positive inotropic
activity), the percent increase in atrial rate on spontaneously beating
right atrium (positive chronotropic activity). Increase in vascular
contraction was evaluated on guinea pig aortic strips. Data were
analyzed by Student’s t-test. The agonistic potency of compounds
was defined as EC50, and IC50 was evaluated from log concentration-
response curves (Probit analysis by Litchfield and Wilcoxon48 or
GraphPad Prism software49,50) in the appropriate pharmacological
preparations. The biological results are expressed as pD2. All data
are presented as mean ( SEM.50
5-HT subtypes blocking activity was assessed by antagonism of
5-HT-induced positive heart rate in guinea pig spontaneously
beating right. Data were analyzed by Student’s t-test. The criterion
for significance was a P < 0.05. IC50 values for noncompetitive
antagonism were calculated from log concentration-response curves
by Probit analysis.48 The biological results are expressed as pIC50.
The competitive antagonist activity of tropisetron was expressed
as pA2. All data are presented as mean values ( SEM.
Guinea Pig Atrial Preparations. Female guinea pigs (300-400
g) were sacrificed by cervical dislocation. After thoracotomy the
heart was immediately removed and washed by perfusion through
the aorta with oxygenated Tyrode solution of the following
composition (mM): 136.9 NaCl, 5.4 KCl, 2.5 CaCl2, 1.0 MgCl2,
0.4 NaH2PO4 ·H2O, 11.9 NaHCO3, and 5.5 glucose. Ketanserin (1
µM) was also added to Tyrode solution to exclude any action at

Targeting of Serotonin 5-HT3 Receptors
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 11 3561
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min after the iv administration of various doses of each tested
compound administered alone (agonist effect) or 5 min before PBG
(antagonist effect). Under these conditions, 1 µg/kg (iv) of zacopride
injected 5 min before PBG completely prevented PBG-evoked
bradycardia.38
Brain-to-Plasma Distribution Studies. Animals and
Treatment. Male CD1 albino mice, 25-30 g (Charles River, Italy),
were used. Procedures involving animals and their care were
conducted in conformity with the institutional guidelines that are
in compliance with national (D.L., No. 116, G.U., Suppl. 40, 18
Febbraio 1992, Circolare No. 8, G.U., 14 Luglio 1994) and
international laws and policies (EEC Council Directive 86/609, 0J
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Animals were injected with 5 and 20 mg/kg (S)-4n (as
hydrochloride salt) and sacrificed by decapitation at various times
thereafter for parallel determination of the compound in plasma
and whole brain. Mixed arteriovenous trunk blood was collected
in heparinized tubes and centrifuged at 3000g for 10 min, and the
plasma was stored at -20 °C. Brain was immediately removed,
blotted with paper for removing excess surface blood, and quickly
frozen in dry ice.
Compound (S)-4n was extracted from plasma and brain homo-
genate by a solid-liquid extraction procedure and quantified by
HPLC with UV detection (240 nm). Briefly, 0.8 mL of 0.01 M
phosphate buffer, pH 7.4, and 50 µL of a solution of 7b (IS)13 in
methanol (5 µg/mL) were added to 0.2 mL of plasma and the
samples were added to Sep-Pac Vac C18 cartridges (contains 100
mg of sorbent), prewetted with 1 mL of methanol and 1 mL of
water. Cartridges were washed with 1 mL of water and 0.2 mL of
methanol. The compound was removed by eluting the cartridges
with 1 mL of methanol, which was evaporated to dryness. The
residue was dissolved in the mobile phase (methanol/0.01 M
KH2PO4 (50:50, v/v) buffered to pH 4.6 with 0.175 M phosphoric
acid) and injected into the HPLC column (µBondpack C18, 30 cm
× 4.6 mm i.d., 10 µm). The retention times were 14 min for
compound (S)-4n and 23 min for the IS.
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for 15 min (at 4 °C). The residue was reconstituted in 1 mL of the
same solution and recentrifuged. The combined supernatants were
processed as described for plasma. The lower limits for quantifica-
tion of (S)-4n in plasma were about 0.25 nmol/mL using 0.2 mL;
in brain the limit was about 0.5 nmol/g, using 1 mL of brain
homogenate or approximately 100 mg of tissue. At these concentra-
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Acknowledgment. The authors thank MIUR (Roma) for
financial support (PRIN).
Supporting Information Available: Tables of pharmacophoric
distances, features, partial charges, structural comparison, calculated
log D values, and elemental analysis results; figures showing
agonists and antagonists and superimpositions. This material is
available free of charge via the Internet at http://pubs.acs.org.
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