Synthesis and pharmacological characterization of a new benzoxazole derivative as a potent 5-HT3 receptor agonist

Article abstract of DOI:10.1016/S0968-0896(03)00243-8

N-(2-Benzoxazol-2-yl-ethyl)-guanidine hydrochloride (10) was synthesized and pharmacologically tested. This compound showed high affinity for the 5-HT₃ receptor (K_i=0.77 nM) and potently triggered the von Bezold-Jarisch reflex (BJR) in rats with an ED₅₀=0.52 μg/kg iv and intrinsic activity next to 1 (i.a.=0.94). This stimulant effect was abolished by pretreatment with the 5-HT₃ receptor antagonist granisetron and was subject to a rapid and pronounced tachyphylaxis, due to desensitization of the peripheric cardiac 5-HT₃ receptor. Consequently, 10 acts as an in vivo 5-HT₃ antagonist inhibiting the BJR responses evoked by submaximal doses of 5-HT with an ID₅₀=5.8 μg/kg iv.

Full text of DOI:10.1016/S0968-0896(03)00243-8

Bioorganic & Medicinal Chemistry 11 (2003) 2709–2714
Synthesis and Pharmacological Characterization of a New
Benzoxazole Derivative as a Potent 5-HT3 Receptor Agonist
Pedro Luis Lopez-Tudanca, Luis Labeaga, Ana Innerarity, Luisa Alonso-Cires,
Ines Tapia, Ramon Mosquera and Aurelio Orjales*
´
Department of Research, FAES FARMA, S.A., Maximo Aguirre 14, 48940 Leioa, Vizcaya, Spain
Received 17 January 2003; accepted 4 April 2003
Abstract—N-(2-Benzoxazol-2-yl-ethyl)-guanidine hydrochloride (10) was synthesized and pharmacologically tested. This com-
pound showed high affinity for the 5-HT3 receptor (Ki=0.77 nM) and potently triggered the von Bezold–Jarisch reflex (BJR) in rats
with an ED50=0.52 mg/kg iv and intrinsic activity next to 1 (i.a.=0.94). This stimulant effect was abolished by pretreatment with
the 5-HT3 receptor antagonist granisetron and was subject to a rapid and pronounced tachyphylaxis, due to desensitization of the
peripheric cardiac 5-HT3 receptor. Consequently, 10 acts as an in vivo 5-HT3 antagonist inhibiting the BJR responses evoked by
submaximal doses of 5-HT with an ID50=5.8 mg/kg iv.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
21007 (6),14 5-chloro-7-methyl-2-(4-methyl-1-homo-
piperazinyl)benzoxazole (7),15 9-methyl-4-(4-methyl-
piperazin-1-yl)pyrrolo[1,2-a]-quinoxaline (8),16 3,4,5-
trichloro-phenylguanidine (3,4,5-trichloro-PG, 9)17
(Chart 1).
In recent years much research effort has been made in
the field of serotonin (5-HT) receptors. Until now seven
families of 5-HT receptor have been recognised1 and
only the 5-HT3 receptor belongs to the ligand-gate
cation channel family.2,3 The high affinity, selectivity
and low incidence of side-effects of the 5-HT3 receptor
ligands have caused a huge interest in this receptor due
to their potential therapeutic applications in a number
of areas: emesis, anxiety, psychotic disorders, drug
abuse, depression, migraine, pain, irritable bowel
syndrome.4À7
Numerous selective antagonists among 5-HT3 receptor
ligands are currently available and they are of high
therapeutic interest in the prevention and treatment of
emesis associated with anticancer chemotherapy8,9 and
the prophylaxis of postoperative nausea and vomiting.10
However, relatively little attention has been paid to the
5-HT3 receptor agonists although several potent and
selective agonists have also been identified such as
2-methyl-5-HT
(m-CPBG, 2),11 2,3,5-trichloro-phenylbiguanide (2,3,5-
trichloro-PBG, 3),11 YM 31636 (4),12 SR 57227 (5),13
(1),
meta-chlorophenylbiguanide
S
*Corresponding author. Tel.: +34-944818300; fax: +34-944818309;
e-mail: aorjales@faes.es
Chart 1.
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00243-8

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P. L. Lopez-Tudanca et al. / Bioorg. Med. Chem. 11 (2003) 2709–2714
2710
Chart 2.
Structure–activity relationship studies of the 5-HT3
receptor agonists have generated several pharmaco-
phoric hypotheses17À21 to explain their interactions with
this receptor. From these hypotheses three structural
requirements for the 5-HT3 agonism could be pointed
out: a quaternary ammonium ion, a hydrogen bond
acceptor and an aromatic site.
Some 5-HT3 receptor agonists are able to cross the
blood–brain barrier. This makes them ideal pharmaco-
logical tools to explore functional roles of central and
peripheric 5-HT3 receptors. Nowadays, the main inter-
est on the 5-HT3 receptor agonism is focused on diverse
areas of psychoactivity, particularly depression, and
gastrointestinal disorders.
Scheme 1. Synthesis of compound 10. Reagents: (a) Phthalic anhydride;
(b) 2-aminophenol, polyphosphoric acid, 180 ꢀC; (c) N2H4; (d) 1H-
pyrazolecarboxamidine hydrochloride.
benzoxazole derivative 13 with hydrazine hydrate affor-
ded the free amine 13 which was readily converted into
the benzoxazolguanidine 10 by reaction with 1H-pyra-
zole-1-carboxamidine hydrochloride27 and diisopropyl-
ethylamine in DMF.
As a part of a research project on 5-HT3/5-HT4 receptor
ligands we have synthesized the benzoxazoleguanidine
10 (Chart 2) which has been characterized as a potent
5-HT3 receptor agonist.
Pharmacological Activity
Radioligand binding studies
Compound 10 displays two moieties of interest, a gua-
nidine group found in other ligands,17,22,23 and a ben-
zoxazole fragment related to a selective 5-HT3 receptor
partial agonistic activity.15,18,24 or a weak gut related
antagonism.25 Morain et al.11 have reported a guanidine
derivative 11 (Chart 2), closely related to 10, in which
the guanidine group is directly linked to a substituted
benzoxazole system, and surprisingly it lacks the affinity
for the 5-HT3 receptor. This fact suggests that the dis-
tance between the benzoxazole system and the guani-
dine fragment or the influence of the trifluoromethyl
group placed on the aromatic ring or both play a fun-
damental role to achieve the fitting at the receptor.
However, similar modifications (chain shorthening22 or
introduction of a trifluoromethyl group17) in arylguani-
dines did not produce significant reduction of affinity.
Additional studies to know the structural requirement
for the 5-HT3 agonistic activity are required.
Radioligand binding data for 10 and the reference drugs
are shown in Table 1. Compound 10 binds to the 5-HT3
receptor in rat entorhinal cortex labelled with [3H]-
LY278584 with high affinity (Ki=0.77 nM). Its Ki value
Table 1. Affinity values of compound 10 and other 5-HT3 receptor
ligands
Drugs
Ki (nM)
Radioligand
Tissue
10
Granisetronb
0.77Æ0.09a [3H]-LY278584 Rat entorhinal cortex
1.76Æ0.15a [3H]-LY278584 Rat entorhinal cortex
Ondansetronb 2.00Æ0.30a [3H]-LY278584 Rat entorhinal cortex
Tropisetronb
Lerisetronb
1.55Æ0.10a [3H]-LY278584 Rat entorhinal cortex
0.62Æ0.08a [3H]-LY278584 Rat entorhinal cortex
(S)-Zacoprideb 0.49Æ0.03a [3H]-LY278584 Rat entorhinal cortex
5-HTc
1c
42Æ7d
350Æ30d
13Æ2d
[3H]-BRL43694
[3H]-BRL43694
[3H]-BRL43694
N1E-115 cells
N1E-115 cells
N1E-115 cells
2c
3c
0.44Æ0.09d [3H]-BRL43694
N1E-115 cells
4c
0.21Æ0.05e [3H]-Ramosetron
Cloned human
Rat cerebral cortex
N1E-115 cells
Rat cerebral cortex
Rat cerebral cortex
NG108-15 cells
5c
115Æ31f
[3H]-Zacopride
Results and Discussion
Synthesis
6c
2.8Æ0.3g [3H]-Granisetron
7c
1.1Æ0.1h
0.79Æ0.1i
0.7Æ0.1j
[3H]-GR65630
[3H]-Zacopride
[3H]-GR65630
8c
9c
The preparation of the benzoxazolguanidine 10 was
accomplished by the synthetic pathway illustrated in
Scheme 1 using standard reactions.
aAll values are the meanÆSEM of data from 2–3 independent experi-
ments performed in triplicate in our laboratory.
b5-HT3 receptor antagonist.
c5-HT3 receptor agonist. Ki values were previously reported and are
The starting material was b-alanine which was con-
verted into the corresponding phthalimide derivative 12
with phthalic anhydride in refluxing toluene.26 The
benzoxazole ring was subsequently obtained by cyclo-
condensation of the phthalimide acid derivative 12 and
2-aminophenol with polyphosphoric acid at 180 ꢀC.
Finally, deprotection of the amine in the phthalimido-
included only for comparison.
dSee ref 11.
eSee ref 12.
fSee ref 13.
gSee ref 14.
hSee ref 15.
iSee ref 16.
jSee ref 17.

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P. L. Lopez-Tudanca et al. / Bioorg. Med. Chem. 11 (2003) 2709–2714
2711
is comparable to those of granisetron (Ki=1.76 nM),
ondansetron (Ki=2.00 nM), tropisetron (Ki=1.55 nM),
lerisetron (Ki=0.62 nM) and (S)-zacopride (Ki=0.49
nM), all of them potent serotonin 5-HT3 receptor
antagonists. Also, the affinity shown by 10 was similar
to the most potent 5-HT3 agonists described up to date
character with an ED50=0.52 mg/kg iv and an i.a.=0.94
not significantly different from the unity, was observed.
The calculated ED50 values indicate that 10 is about 35
times more potent than 5-HT to induce the BJR in
anaesthetized rats.
such as 4 (Ki=0.21 nM),12 3 (Ki=0.44 nM),11
(Ki=0.79 nM)16 and 9 (Ki=0.70 nM).17
8
The potency of 10 to induce the BJR in urethane-
anaesthetised rats results similar or slightly superior to 2
(ED50 from 0.7 to 1 mg/kg iv)28 and clearly superior to 1
(ED50 from 5 to 27 mg/kg iv).13 Agonists 5 and 6 with
ED50 values of 8 mg/kg iv and higher than 120 mg/kg iv
respectively13,14 are less potent than 10.
On the other hand, 10 did not show significant affinity
for the serotonin 5-HT4 and dopamine D2 receptors at 1
mM concentration (data not shown).
Recently, Whalen et al.29 have reported functional evi-
dence for tachyphylaxis in BJR responses induced in
conscious rats by five consecutive iv injections of 100
mg/kg of two structurally different 5-HT3 receptor ago-
nists 1 and 2. To determine whether the agonistic effect
of 10 is subjected to tachyphylaxis, a similar experiment
using anaesthetized rats was carried out.
In vivo experiments
The 5-HT3 receptor agonistic effect of 10 was evaluated in
vivo on the BJR in anaesthetized rats and compared to
that of 5-HT. The obtained value of ED50=18.6 mg/kg iv
for 5-HT was similar to that found in literature.13,28 Like
5-HT, cumulative doses of 10 elicited the BJR, but a bell-
shaped dose–response curve was obtained as shown in
Figure 1. The maximum percentage of bradycardia
induced by the highest dose of 5-HT was estimated as
the 100% effect (about 75–80% of reduction in heart
rate (HR) at 100–333 mg/kg iv). 10 At 1 mg/kg iv showed
65% of the previously defined maximum effect. Thus, a
value of intrinsic activity (i.a.) relative to 5-HT of 0.65
was estimated, suggesting a partial agonistic behaviour
at the 5-HT3 receptor. But at higher doses each cumu-
lative and succesive injection of 10 produced progres-
sively smaller responses, so that at the dose of 33 mg/kg
iv the BJR response was not significant with respect to
the saline-induced effect. This finding also suggests that
the induction of rapid tachyphylaxis may be due to
receptor desensitization. In this case, its initial partial
agonistic potential could have been understimated.
Five cumulative successive submaximal doses of 10 (1
mg/kg iv) and 5-HT (30 mg/kg iv), graphically calculated,
were administered and the induced changes in BJR
responses registered and plotted (Fig. 3). The reductions
in HR caused by the first injection of the compounds
were similar, with percentages of 67% for 10 and 72%
for 5-HT. Contrary to 5-HT, each consecutive admini-
stration of 10 produced progressively smaller responses
and finally the fifth injection was not able to induce a
significant response. This fact suggests that this differ-
ence could be associated with a different interaction of
both agonists with the 5-HT3 binding sites, as already
described for 6, a 5-HT3 agonist structurally unrelated
to 10 but with high affinity for the 5-HT3 receptor.14
Structurally related to 10 is the derivate 7, that has been
reported15 as a 5-HT3 receptor partial agonist in the gut.
This compound does not induce the BJR in the anaes-
thetised rat at the dose that blocks the BJR induced by
5-HT but acts as a 5-HT3 receptor antagonist.
To minimize the desensitizer effect and to check the real
agonistic potential of 10 a non-cumulative dose–
response curve was obtained administering only one
dose to each rat (Fig. 2). In this assay a full agonistic
Figure 1. Cumulative dose-dependent bradycardic effect (BJR) elicited
by 5-HT (1 to 333 mg/kg iv) and compound 10 (0.1 to 33 mg/kg iv) in
anaesthetized rats. The effect is expressed as a percentage of the maximum
obtained by 5-HT. Each point is the meanÆSEM (n=8 rats/drug). The
saline effect band is the effect induced by bolus injection of sterile saline.
Figure 2. Non-cumulative dose-dependent BJR response elicited by
5-HT (1 to 333 mg/kg iv) and compound 10 (0.01 to 33 mg/kg iv) in
anaesthetized rats. The effect is expressed as a percentage of decrease
in HR. Each point is the meanÆSEM (n=5 rats/dose).

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2712
dependent manner the BJR responses evoked by sub-
maximal doses (30 mg/kg iv) of 5-HT (Fig. 4) with an
ID50=5.8 mg/kg iv. This value is in accordance with its
affinity for the 5-HT3 receptor and is lower than those
of 5-HT3 antagonists presented in Table 1.
Conclusions
The presented results indicate that 10 shows high affi-
nity for the 5-HT3 receptor in rat entorhinal cortex
membranes and behaves as a full agonist about 35 times
more potent than 5-HT in functional 5-HT3 receptor-
mediated studies (BJR in anaesthetized rats). The stimu-
lant effect is abolished by granisetron and it is subject to a
rapid tachyphylaxis, probably due to desensitization
mechanisms that induce loss of responsiveness of
cardiopulmonary vagal afferents. Compound 10 is able
to inhibit the 5-HT induced BJR acting as a potent 5-
HT3 antagonist in vivo. Because of such properties it
could be a useful pharmacological tool to explore func-
tional roles and desensitization mechanisms of central
and peripheral 5-HT3 receptors. A receptor binding
profile is underway to investigate its selectivity.
Although 10 exhibits a high affinity for the 5-HT3
receptor, several analogues are being prepared to eluci-
date the structural requirements for the 5-HT3 receptor
agonistic activity and to evaluate their potential activ-
ities in the neuropsychopharmacology and gastro-
intestinal areas.
Figure 3. Time–effect regression lines obtained after five consecutive
intravenous injections of submaximal doses of compound 10 (1 mg/kg),
5-HT (30 mg/kg) or sterile saline (1 mL/kg). The effect is expressed as a
percentage of decrease in HR. Each point is the meanÆSEM (n=6
rats/drug).
Experimental
Chemistry
All starting materials were obtained from commercial
sources. When necessary, solvents were purified and/or
dried by standard procedures. Thin layer chromato-
graphy was performed on 0.2 mm Merck precoated
plates of silica gel 60 F254 and spots were visualised with
UV light or I2. Flash column chromatography was per-
formed on silica gel, particle size 60 A, mesh=230–400
(Merck). High performance liquid chromatograms were
obtained in a Waters LCM-1 apparatus using a C-18
reverse phase Nucleosil 120 column (data not shown).
Melting points were determined in open capillary tubes
on a Buchi SMP-20 apparatus and were uncorrected.
Elemental analyses (C, H and N) were within 0.4% of
the theoretical values. Infrared (IR) spectra (in KBr)
were taken on a Perkin–Elmer 1310 spectrophotometer.
1H- and 13C NMR were recorded on a Bruker AC-200.
1H NMR chemical shifts (d) are reported in parts per
million (ppm) downfield from tetramethylsilane, which
was used as an internal standard. 13C NMR chemical
shifts (d) are reported in ppm relative to the solvent and
converted to the TMS scale, using the value 76.91 for
CDCl3. The structures of all compounds were consistent
with their analytical and spectroscopic data.
Figure 4. Log dose-inhibition regression line obtained with intrave-
nous injections of compound 10, 5 min before of a submaximal dose of
5-HT (30 mg/kg iv) (1 mL/kg). The effect is expressed as a percentage
of inhibition. Each point is the meanÆSEM (n=5 rats/dose).
It should be considered that the performance of 5-HT3
receptor full agonist, partial agonist or antagonist is
conditioned by the type of assay and the nature of the
tissue used. The wide found response diversity could
reflect the existence of subtypes of 5-HT3 receptors,16
suggestion30 that still requires additional studies.
a
To date, all 5-HT3 full and partial agonists, charac-
terized by their responses in the BJR in rats, have in com-
mon that their effects are abolished by a previous
treatment with a 5-HT3 antagonist. In our study, the effect
(65–70% decrease in HR) registered by a submaximal
dose (1 mg/kg iv) of 10 was completely antagonised (95%
of inhibition) by 10 mg/kg iv of granisetron administered 5
min before. This finding corroborates that it acts stimu-
lating the 5-HT3 receptors (data not shown).
The tachyphylaxis induced by 10 suggests that this
compound could also act as an in vivo antagonist. A
pretreatment with 10 was able to inhibit in a dose-
The following compounds were prepared as described in
the literature: 3-(2-phthalimido)propionic acid 1226 and
1H-pyrazole-1-carboxamidine hydrochloride.27

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P. L. Lopez-Tudanca et al. / Bioorg. Med. Chem. 11 (2003) 2709–2714
2713
Preparation of 2-[2-(2-phthalimido)ethyl]benzoxazole 13.
A solution of 3-(2-phthalimido)propionic acid 12 (8.8 g,
40 mmol) and o-aminophenol (4.4 g, 10 mmol) in poly-
phosphoric acid (20 g) was heated at 180 ꢀC for 2 h.
Afterwards the reaction mixture was allowed to cool
and poured into water (400 mL). This aqueous mixture
was made basic until pH 12 with 50% sodium hydroxide
solution. The obtained precipitate was filtered off,
washed thoroughly with water and dried, yielding the
desired product (8 g, 68.5% yield). Mp 171–4 C. H
NMR (CDCl3): (d) 7.85 (2H, m); 7.75 (2H, m); 7.60
(1H, m); 7.42 (1H, m); 7.31 (2H, m); 4.22 (2H, t, J=7.2
Hz); 3.35 (2H, t, J=7.2 Hz). 13C NMR (CDCl3): (d)
167.8, 163.3, 150.8, 141.0, 134.0, 131.8, 124.7, 124.1,
123.3, 119.6, 110.3, 35.0, 27.7. Anal. calcd for
C17H12N2O3: C, 69.86; H, 4.14; N, 9.58. Found: C, H,
N.
membranes from rat entorhinal cortex, using 15 mg
(original wet weight) of membranes and nM
2
[3H]-LY278584. Nonspecific binding was defined in the
presence of 10 mM 5-HT. The incubation time was 30
min at 25 ꢀC. The affinity for the serotonin 5-HT4
receptor was determined by displacement of
[3H]-GR113808 binding in membranes from guinea-pig
striatum, using 10 mg (original wet weight) of mem-
branes and 0.2 nM [3H]-GR113808. Nonspecific binding
was defined in the presence of 100 mM 5-HT. The incu-
bation time was 30 min at 25 ꢀC. The affinity for the
dopamine D2 receptor was determined by displacement
of [3H]-raclopride binding in membranes from rat stria-
tum, using 5 mg (original wet weight) of membranes
and 1 nM [3H]-raclopride. Nonspecific binding was
defined in the presence of 1 mM (+)-butaclamol. The
incubation time was 60 min at 25 ꢀC. After incubation,
the reaction was stopped by vacuum filtration through
Whatman GF/B presoaked filters (1% poly-
ethylenimine) using a 24 Brandel Cell Harvester. The
filters were counted by a Kontron Betamatic V liquid
scintillation counter in 5 mL of aqueous counting scin-
tillant (Ecoscint-H, National Diagnostic). Data from
binding assays were plotted as log concentration vs
percentage specific bound and analyzed by nonlinear
regression techniques (GraphPad v1.00). The IC50
values were obtained from 2–3 separate competition
experiments with samples in triplicate, using 10–12 dif-
ferent concentrations of drugs. For all of them, Ki
values were calculated from the Cheng and Prusoff
standard equation.34
ꢀ
1
Preparation of 2-benzoxazol-2-yl-ethylamine 14. To a
suspension of 2-[2-(2-phthalimido)ethyl]benzoxazole 13
(6.5 g, 22.2 mmol) in ethanol (50 mL) hydrazine hydrate
(1.2 mL, 24.7 mmol) was added. The mixture was
refluxed for 2 h, allowed to cool, filtered and the
obtained solid washed with ethanol. The filtrate was
concentrated and purified by flash chromatography
(CH2Cl2:MeOH 9:1) to give 2-(2-aminoethyl)benz-
oxazole 14 as an oil (2.3 g, 64% yield). 1H NMR
(CDCl3): (d) 7.58 (1H, m); 7.40 (1H, m); 7.20 (2H, m);
3.15 (2H, t, J=7.0 Hz); 2.95 (2H, t, J=7.0 Hz); 2.10
(2H, br s, D2O exchange). 13C NMR (CDCl3): (d) 165.4,
150.5, 141.1, 124.4, 124.0, 119.4, 110.1, 39.1, 32.5. Anal.
calcd for C9H10N2O: C, 66.65; H, 6.21; N, 17.27.
Found: C, H, N.
In vivo. Induction or inhibition of the von Bezold–Jarisch
reflex in rats
Preparation of N-(2-benzoxazol-2-yl-ethyl)-guanidine
hydrochloride 10. 1H-Pyrazole-1-carboxamidine hydro-
chloride (1 g, 6.8 mmol) was added to a solution of
2-benzoxazol-2-yl-ethylamine 14 (1.1 g, 6.7 mmol) and
diisopropylethylamine (4.75 mL, 27 mmol) in DMF (10
mL). The reaction mixture was stirred at room tem-
perature under N2 for 24 h. Subsequently, diethylether (75
mL) was added, the upper layer was decanted and the
remaining oil solidified by treatment with CH2Cl2. The
solid was colected, washed with diethylether, CH2Cl2, and
CHCl3:iPrOH (4:1) ꢀand dried to yield 10 (0.86 g, 52.6%
Adult male Wistar rats weighing 250–320 g fasted for 18
h were used. They were anaesthetized with urethane
(1.50 g/kg ip) and placed on a heating table to maintain
their body temperature at 37 ꢀC. The trachea and right
jugular vein were cannulated to facilitate the respiration
and drug administration, respectively. The carotid
artery was also cannulated and connected to a ISO-
TECTM pressure transducer to record blood pressure
(BP) (Hugo Sachs Elektronik, Freiburg, Germany). The
HR was measured using the BP signal and a cardio-
tachometer coupler and recorded on a Graphtec Line-
arcorder WR3101 (Hugo Sachs Elektronik, Freiburg,
Germany). Both parameters (BP and HR) were also
monitorized and analyzed using a PowerLab/8SP unit
(ADInstruments) connected to WR3101 recorder. The
BJR was evoked by bolus intravenous injection of 5-HT
or 10 dissolved in sterile saline.
1
yield). Mp 183–186 C. H NMR (CDCl3): (d) 7.80 (1H,
m, D2O exchange); 7.65 (2H, m); 7.45 (6H, m, 4H
exchange with D2O); 3.65 (2H, m); 3.20 (2H, t, J=6.5 Hz).
13C NMR (CDCl3): (d) 164.2, 157.2, 150.2, 140.7, 124.9,
124.4, 119.3, 110.6, 37.7, 28.0. Anal. calcd for
C10H12N4O.HCl: C, 49.90; H, 5.44; N, 23.28. Found: C,
H, N.
In agonism studies, a cumulative dose–response curve to
5-HT and 10 was initially constructed on independent
groups of rats. The BJR responses produced by each
injection of the drugs were allowed to subside com-
pletely before another injection was given. Each injec-
tion or dose was given normaly 5–6 min apart. The
intensity of the BJR was expressed in percentage of
bradycardia considering the basal or initial HR value
and the final one obtained after 5-HT or 10 iv adminis-
tration. For each drug a dose–response curve from 6
Pharmacological Methods
In vitro. Binding assays
Bindings to serotonin 5-HT3 and 5-HT4 receptors and
dopamine D2 receptors were determined by radioligand
assays according to previously published procedures.31À33
The affinity for the serotonin 5-HT3 receptor was deter-
mined by displacement of [3H]-LY278584 binding in

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P. L. Lopez-Tudanca et al. / Bioorg. Med. Chem. 11 (2003) 2709–2714
2714
cumulative doses (8 rats/drug) was plotted estimating as
the 100% effect the maximum percentage of brady-
cardia induced by the highest dose of 5-HT. This value
was about 80% of reduction in HR.
8. Lindley, C.; Blower, P. Am. J. Health Syst. Pharm. 2000,
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Lefevre, I. A.; Souilhac, J.; Manara, L.; Emerit, M. B.;
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Subsequently, to minimize a probable desensitizer effect
in the ability of test compound to evoke the BJR,
another set of randomized experiments were evaluated
in a non-cumulative way. Thus, after the initial period
of stabilization of BP and HR parameters only one BJR
response per rat was recorded for each bolus iv injection
of drug. In this experiment the ED50 and i.a. values
from 8 doses of 10 (5 rats/dose) were calculated and
compared with 5-HT.
To determine whether the BJR responses elicited by
5-HT or 10 are subject to rapid tachyphylaxis, sub-
maximal and equiactives doses for each agonist were
selected. Five consecutive and cumulative bolus iv
injections of saline (1 mL/kg), 5-HT (30 mg/kg) or 10 (1
mg/kg) were given every 5 min to rats (6 rats/agonist)
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Acknowledgements
This work was supported in part by the Ministry of
Industry and Energy of Spain (now Ministry of Science
and Technology) and the Department of Industry,
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