Synthesis and pharmacology of isoquinuclidine derivatives as 5-HT3 ligands

Article abstract of DOI:10.1016/S0960-894X(01)00693-X

A series of 4-amino-5-chloro-2-methoxybenzoates and benzamides containing the 5- and 6-isoquinuclidinyl system was synthesised and evaluated for binding to 5-HT₃, 5-HT₄ and D₂ receptors. In general, the isoquinuclidine derivatives at the 5-position have shown to be more potent as 5-HT₃ ligands but they also possess 5-HT₄ and D₂ properties. However, the results show that the derivatives at the 6-position afforded the most promising compounds in terms of both receptor affinity and selectivity.

Full text of DOI:10.1016/S0960-894X(01)00693-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 189–192
Synthesis and Pharmacology of Isoquinuclidine
Derivatives as 5-HT3 Ligands
Isabel Iriepa,a,* Francisco J. Villasante,a Enrique Galvez,a Luis Labeaga,b
Ana Innerarityb and Aurelio Orjalesb
a
´
Departamento de Quımica Organica, Universidad de Alcala, Ctra. Madrid-Barcelona Km. 33,600,
´
28871 Alcala de Henares, Madrid, Spain
´
´
Departamento de Investigacion, FAES, S.A., Maximo Aguirre 14, 48940 Leioa, Vizcaya, Spain
b
´
´
Received 10 July 2001; revised 17 October 2001; accepted 19 October 2001
Abstract—A series of 4-amino-5-chloro-2-methoxybenzoates and benzamides containing the 5- and 6-isoquinuclidinyl system was
synthesised and evaluated for binding to 5-HT3, 5-HT4 and D2 receptors. In general, the isoquinuclidine derivatives at the 5-posi-
tion have shown to be more potent as 5-HT3 ligands but they also possess 5-HT4 and D2 properties. However, the results show that
the derivatives at the 6-position afforded the most promising compounds in terms of both receptor affinity and selectivity. # 2002
Elsevier Science Ltd. All rights reserved.
A number of potent 5-HT3 antagonists have been
reported and shown to be effective in the control of
cancer chemotherapy-induced emesis.1 It was clear from
the earliest discovery that there were probably three
pharmacophore elements required for high affinity: an
aromatic ring at an appropriate distance from a car-
bonyl or bioisoster connected to a basic centre. It can be
assumed that the basic centre binds in its protonated
form. Different QSAR and modelling studies have been
made to delineate this pharmacophore and to determine
angles and distances.2
To our knowledge, only five compounds with this sys-
tem have been reported as 5-HT3 antagonists and no
systematical synthesis is described.3 We also synthesised
the equivalent esters 5, 6 and 11, 12 due to the fact that
it can be possible to convert the agonist/partial agonist
5-HT4 properties of the benzamides into antagonists
when the amidic function is replaced by an ester func-
tion. The first published indication that this can be done
came from the finding that the ester equivalent of
metoclopramide, SDZ 205-557, had antagonist activity
in a number of pharmacological models.1
There are many variations referring to the aromatic
component, the linkers and the basic side chain. Our
own interest came from the observation that metoclo-
pramide (Fig. 1) had three major identified pharmaco-
logical activities: dopamine antagonism, 5-HT3 receptor
antagonism, and gastric motility stimulant properties
(5-HT4 agonism).1 We decided to synthesise a series of
amides to investigate the effect that the replacement of
the diethylaminoethyl chain by a 2-azabicyclo[2.2.2]-
octane (isoquinuclidine) system could exert in their
pharmacological properties.
In this paper, we compare the esters and the amides in
terms of both receptor affinity and selectivity for the D2,
5-HT3 and 5-HT4 receptors.
Furthermore, the isoquinuclidine system has not been
much used as basic side chain in such a class of ligands.
*Corresponding author. Tel.: +34-91-885-4651; fax: +34-91-885-
4686; e-mail: isabel.iriepa@uah.es
Figure 1. Metoclopramide, SDZ 205-557 and the general structure for
compounds 5, 6, 11, 12 and 17–20.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00693-X

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I. Iriepa et al. / Bioorg. Med. Chem. Lett. 12 (2002) 189–192
Chemistry
al.8 where nBuLi as base was used instead of DBU.
Treatment of those esters with HCl produced the trityl
group cleavage and the compounds could be obtained
as hydrochloride salts. To synthesise the free base,
treatment with a Na2CO3 solution was necessary.
The key intermediates in the synthesis of esters 5, 6 and
11, 12 were, of course, the corresponding alcohols 3, 4, 9
and 10. Two basic approaches were utilised and are
shown in Scheme 1. The synthesis of alcohols 3 and 4
was accomplished by reduction with lithium aluminium
hydride of 2-carbethoxy-2-azabicyclo[2.2.2]oct-6-one (2)
which was prepared by the method of Krow et al.4 This
method involves the addition of phenylselenenyl chlo-
ride to 2-carbethoxy-2-azabicyclo[2.2.2]oct-5-ene (1)
followed by dehydrohalogenation and hydrolysis of the
derived vinyl selenide to give regioselectively the above
mentioned compound. Reduction of this compound
gave two different alcohols 3 and 4 in a ratio of 40/60
based on NMR data. The alcohols 9 and 10 were
obtained by the procedure described by Krow,5 using
again the 2-carbethoxy-2-azabicyclo[2.2.2]oct-5-ene (1)
but in this case an oxymercuration-demercuration pro-
cedure was applied. The addition of mercuric acetate to
the alkene, followed by the reduction of the mercuric
intermediate with NaBH4, gave an epimeric mixture of 5-
syn- and 5-anti-2-ethoxycarbonyl-2-azabicyclo[2.2.2]-
octanols (7, 8) in a ratio of 50/50 based on NMR data.
Finally, the alcohols 9 and 10 can be obtained by
reduction of this epimeric mixture with LiAlH4.
Once alcohols have been synthesised the easiest proce-
dure to obtain the amines is by reduction of the azides
which can be obtained from the alcohols through mesi-
lates. In the case of compounds 15 and 16, alcohols 7
and 8 were used as starting materials since the reduction
of the azide and the carbethoxy group can be accom-
plished at the same time, this approach would avoid one
reaction step (Scheme 2).
Finally, by reaction of the corresponding amines with
the 4-amino-5-chloro-2-methoxybenzoic acid using
ethyl chloroformate in presence of triethylamine, the
amides 17–20 were obtained.9 The epimeric mixtures
were separated by column chromatography on silica-
gel.
Pharmacology
Even though all the compounds are racemic a pre-
liminary pharmacological evaluation was carried out on
compounds 5, 6, 11, 12 and 17–20 using granisetron,
lerisetron and tropisetron as reference compounds
(Table 1).10 Compounds 6, 11, 12 and 20 were found to
show high affinity for the 5-HT3 receptor. The esters
derivatives 11 and 12 in spite of being the most potent
5-HT3 ligands are not selective because they showed
also 5-HT4 properties.
The tritylated benzoate derivatives were synthesised in a
straightforward manner by esterifying the corre-
sponding mixture of alcohols in the presence of DBU
with 5-chloro-4-(tritylamino)-2-methoxybenzoic acid
imidazolide previously synthesised.6,7 The resulting
residue was chromatographed on silica gel with the
appropriate solvent system to separate each of the epi-
mers of the corresponding esters. This procedure gave
slightly better yields than the one used by Fernandez et
The amides 19 and 20 are dopaminergic ligands, as it
had been published by Blaney et al.,11 showing the latter
compound 5-HT3 properties.
Finally, compound 6 can be identified as a moderately
potent and selective 5-HT3 receptor ligand.
Scheme 1. (a) ClSePh, CH2Cl2, À78 ꢀC and then 15 h at rt; (b) DBU,
140 ꢀC for 15 h; (c) HCl (20%), dioxan, 65 ꢀC for 48 h; (d) LiAlH4,
THF, reflux for 24 h; (e) 5-chloro-2-methoxy-4-(tritylamino)benzoic
acid imidazolide, DBU, THF, reflux for 24 h; (f) HCl, CHCl3, rt, 12 h;
Scheme 2. (a) Et3N, CH2Cl2, 0 ꢀC, then ClSO2CH3, rt for 1.5 h; (b)
NaN3, N-methyl-2-pirrolidone, 150 ꢀC for 1 h; (c) LiAlH4, THF, reflux
for 5 h; (d) 4-amino-5-chloro-2-methoxybenzoic acid, ethyl chloro-
formate, Et3N, CH2Cl2, rt for 12 h.
(g) Hg(Aco)2, THF–H2O(1:1), rt for 24 h, then 3 M NaOH, NaBH
(in 3 M NaOH).
4

I. Iriepa et al. / Bioorg. Med. Chem. Lett. 12 (2002) 189–192
191
Table 1. Binding affinities at the 5-HT3, 5-HT4 and D2 receptors for
compounds 5–6, 11–12 and 17–20
aminobenzoate derivatives, which were separated by column
chromatography on silica gel. For the 6-isoquinuclidinyl deri-
vatives a mixture of CHCl3/CH3OH (95/5) was used as eluent
to obtain the 2-methyl-2-azabicyclo[2.2.2]octan-6-syn-yl 5-
chloro-2-methoxy-4-tritylaminobenzoate (37%) as the first-
eluting epimer and the 2-methyl-2-azabicyclo[2.2.2]octan-6-
anti-yl 5-chloro-2-methoxy-4-tritylaminobenzoate (35%) as
the second one. For the 5-isoquinuclidinyl derivatives a mix-
ture of CHCl3/CH3OH (95/5) was employed to obtain the 2-
methyl-2-azabicyclo[2.2.2]octan-5-anti-yl 5-chloro-2-methoxy-
4-tritylaminobenzoate (43%) as the first-eluting epimer. Con-
tinued elution gave the 2-methyl-2-azabicyclo[2.2.2]octan-5-
syn-yl 5-chloro-2-methoxy-4-tritylaminobenzoate (16%).
Concentrated HCl (2 equiv) was added to a solution of the
tritylated ester (1 equiv) in CHCl3. The reaction was stirred for
12 h and then concentrated under reduced pressure to dryness.
The mixture was taken up with water, basified with Na2CO3
and then extracted with CHCl3. The organic layer was dried
over Na2SO4 and the solvent removed to afford a solid which
was purified by column chromatography on silica gel to
obtain the corresponding non-tritylated benzoate. In com-
pounds 5 and 6, CHCl3/CH3OH (9/1) was employed as eluent
while CHCl3/CH3OH (95/5) was used for compounds 11 and
Compd
Binding 5-HT3
IC50 (mM)a
Binding 5-HT4
IC50 (mM)a
Binding D2
IC50 (mM)a
5
6
11
12
17
18
19
191.8 (Æ27.3)b
13.79 (Æ1.81)b
11.17 (Æ0.85)b
5.19 (Æ0.65)b
ꢁ1
>1
ꢁ1
ꢁ1
ꢁ1
240.3 (Æ30.6)b
ꢁ1
82.55 (Æ7.92)b
ꢁ1
ꢁ1
1
327.2 (Æ32.8)b
1
1
ꢁ1
842.2 (Æ47.1)b
130.3 (Æ10.8)b
>1
1
20
17.26 (Æ2.25)b
1.76 (Æ0.15)b
0.62 (Æ0.04)b
1.55 (Æ0.10)b
ꢁ1
>1
>1
125.1b
Granisetron
Lerisetron
Tropisetron
>1
>1
aValues are means of three experiments, standard deviation is given in
parentheses.
bKi value (nM).
As far as 5-HT3 affinity is concerned, the following
conclusions can be drawn:
12. 5: Yield: 61%; mp 126 ꢀC (decomp); H NMR (CDCl3) d
1
7.85 (s, 1H, Har), 6.27 (s, 1H, Har), 4.93 (m, 1H, H-6), 4.44
(brs, 2H, NH2), 3.83 (s, 3H, OCH3), 2.99 (m, 1H, H-3n), 2.86
(m, 1H, H-1), 2.62 (m, 1H, H-3x), 2.49 (s, 3H, NCH3), 2.17
(m, 1H, H-5a), 2.04 (m, 1H, H-7s), 1.83 (m, 1H, H-4), 1.74 (m,
1H, H-5s), 1.62 (m, 1H, H-8s), 1.51 (m, 1H, H-7a), 1.44 (m,
1H, H8a). Anal. calcd for C16H21ClN2O3: C, 59.16; H, 6.52;
N, 8.63; found: C, 59.22; H, 6.58; N, 8.64. 6: Yield: 70%; mp
a. Compounds containing the ester linkage (5, 6, 11
and 12) are more potent than those containing the
amide one (17–20).
b. Anti steroisomers (amides and esters) were found
to be more potent than syn steroisomers.
72–75 ꢀC; H NMR (CDCl3) d 7.78 (s, 1H, Har), 6.28 (s, 1H,
1
In summary, the interesting biological profile of com-
pound 6, suggests that it would be promising to study
further the individual enantiomers of these systems and
also to determine the agonistic or antagonistic properties.
Har), 5.24 (m, 1H, H-6s), 4.46 (brs, 2H, NH2), 3.83 (s, 3H,
OCH3), 2.83 (m, 1H, H-1), 2.73 (m, 1H, H-3n), 2.66 (m, 1H,
H-3x), 2.47 (s, 3H, NCH3), 2.23 (m, 1H, H-5s), 1.86 (m, 2H,
H-7a, H-7s), 1.80 (m, 1H, H-4), 1.62 (m, 2H, H-8a, H-8s), 1.57
(m, 1H, H-5a). Anal. found: C, 59.12; H, 6.44; N, 8.73. 11:
Yield; 42%; mp 138–140 ꢀC; H NMR (CDCl3) d 7.82 (s, 1H,
1
Har), 6.26 (s, 1H, Har), 5.00 (m, 1H, H-5), 4.41 (brs, 2H,
NH2), 3.83 (s, 3H, OCH3), 3.03 (m, 1H, H-3n), 2.70 (m, 1H,
H-3x), 2.57 (m, 1H, H-1), 2.39 (s, 3H, NCH3), 2.02 (m, 3H, H-
6a, H-6s, H-7s), 1.91 (m, 1H, H-4), 1.74 (m, 1H, H-8s), 1.64
(m, 1H, H-8a), 1.40 (m, 1H, H-7a). Anal. found: C, 59.33; H,
6.71; N, 8.69. 12: Yield: 32%; mp 148–149 ꢀC; 1H NMR
(CDCl3) d 7.79 (s, 1H, Har), 6.29 (s, 1H, Har), 5.14 (m, 1H, H-
5), 4.78 (brs, 2H, NH2), 3.85 (s, 3H, OCH3), 3.00 (m, 1H, H-
3x), 2.71 (m, 1H, H-1), 2.66 (m, 1H, H-3n), 2.56 (m, 1H, H-
6s), 2.26 (s, 3H, NCH3), 2.10 (m, 1H, H-7s), 2.08 (m, 1H, H-
4), 1.97 (m, 1H, H-8a), 1.65 (m, 2H, H-7a, H-8s), 1.54 (m, 1H,
H-6a). Anal. found: C, 59.29; H, 6.38; N, 8.51.
References and Notes
1. Gaster, L. M.; King, F. D. Med. Res. Rev. 1997, 17, 163,
and references cited therein.
2. Morreale, A.; Iriepa, I.; Galvez, E. Curr. Med. Chem. In
press.
3. (a) Bermudez, J.; Fake, C. S.; Joiner, G. F.; Joiner, K. A.;
King, F. D.; Miner, W. D.; Sanger, G. J. J. Med. Chem. 1990,
33, 1924. (b) Turconi, M.; Nicola, M.; Quintero, M. G.;
Maiocchi, L.; Micheletti, R.; Giraldo, E.; Donetti, A. J. Med.
Chem. 1990, 33, 2101. (c) Swain, C. J.; Baker, R.; Kneen, C.;
Moseley, J.; Saunders, J.; Seward, E. M.; Stevenson, G.; Beer,
M.; Stanton, J.; Watling, K. J. Med. Chem. 1991, 34, 140.
4. Krow, G. R.; Shaw, D. A.; Lynch, B.; Lester, W.; Szcze-
panski, S. W.; Raghavachari, R.; Derome, A. E. J. Org. Chem.
1988, 53, 2258.
5. Krow, G. R.; Rodebaugh, R.; Grippi, M. Synth. Commun.
1972, 2, 211.
6. Yang, D.; Soulier, J. L.; Sicsic, S.; Mathe-Allainwat, M.;
Bremont, B.; Croci, T.; Cardamone, R.; Aureggi, G.; Lang-
lois, M. J. Med. Chem. 1997, 40, 608.
7. General procedure for the preparation of 4-amino-5-
chloro-2-methoxybenzoates derivatives 5, 6, 11 and 12: To a
solution of recently prepared 5-chloro-2-methoxy-4-tritylami-
nobenzoic acid imidazolide (1 equiv) in dry THF was added a
solution of the alcohols (epimeric mixture) (1 equiv) in DBU
(1 equiv). The mixture was refluxed for 24 h and concentrated
under pressure to dryness. The residue was taken into CH2Cl2,
washed with water and dried over MgSO4 to obtain a mixture of
the corresponding syn and anti 5-chloro-2-methoxy-4-trityl-
8. Fernandez, M. J.; Huertas, R.; Toledano, M. S.; Galvez,
E.; Cano, F. H.; Fonseca, I.; Sanz-Aparicio, J. J. Phys. Org.
Chem. 1999, 12, 69.
9. General procedure for the preparation of 4-amino-5-
chloro-2-methoxybenzamide derivatives 17–20: An ethyl
chloroformate (1 equiv) solution in anhydrous CH2Cl2 was
slowly added (15 min) over a mixture of Et3N (1 equiv) and
4-amino-5-chloro-2-methoxybenzoic acid (1 equiv) in dry
CH2Cl2. The mixture was stirred at room temperature for
30 min and then a solution of the corresponding amines (1
equiv) in dry CH2Cl2 was added. The stirring was continued
during 12 h and the mixture was basified with 2.5 N NaOH.
After 30 min the organic layer was separated, washed with
brine and dried over Na2CO3. The clear solution was con-
centrated under reduced pressure to give the corresponding
amides mixture which were separated by silica gel chromato-
graphy using the adequate solvents. For the 6-isoquinuclidinyl
derivatives a mixture of CHCl3/CH3OH (ammonia satured)
(97/3) was used to obtain the first-eluting compound 17: Yield:

192
I. Iriepa et al. / Bioorg. Med. Chem. Lett. 12 (2002) 189–192
7%; mp 163–166 ꢀC (decomp); 1H NMR (CDCl3) d 8.10 (s,
1H, Har), 7.98 (m, 1H, NH amide), 6.30 (s, 1H, Har), 4.35
(brs, 2H, NH2), 3.88 (s, 3H, OCH3), 3.75 (m, 1H, H-6a), 3.24
(m, 1H, H5s), 2.70 (m, 1H, H-3n), 2.28 (s, 3H, NCH3), 2.22
(m, 1H, H-3x), 2.12 (m, 2H, H-1, H-5a), 1.85 (m, 1H, H-7s),
1.70–1.40 (m, 4H, H-4, H-7a, H-8a, H-8s). Anal. calcd for
C16H22ClN3O2: C, 59.34; H, 6.85; N, 12.98; found: C, 59.17;
H, 6.63; N, 12.71. Continued elution gave compound 18:
of rat brain (500–600 mg of protein) was incubated at 25 ꢀC for
30 min with 2 nM [3H]-LY278584 in 50 mM Tris–HCl buffer,
pH 7.4, containing 5 mmol/l CaCl2 and 0.1% ascorbate, and
displacer drug was added. Non-specific binding was deter-
mined using 10 mmol/l cold 5-HT. 5-HT4 receptor binding was
determined according to Grossman et al.14 A suspension of
striatum membranes of guinea-pig brain (800–900 mg of pro-
tein) was incubated at 25 ꢀC for 30 min with 2 nM [3H]-GR-
113808 in 50 mM Hepes buffer, pH 7.4, and displacer drug was
added. Non-specific binding was determined using 10 mmol/l
cold 5-HT. D2 receptor binding was determined according to
Kohler et al.15 A suspension of striatum membranes of rat
brain (300–400 mg of protein) was incubated at 25 ꢀC for 60 min
with 1 nM [3H]-raclopride in 50 mM Tris–HCl saline buffer, pH
7.7, containing 10 mM pargyline and 0.01% ascorbate, and dis-
placer drug was added. Non-specific binding was determined
using 1 mM cold (+)-butaclamol. In all binding assays after the
incubation period the reaction was stopped for vacuum filtra-
tion (GF/B glass filters) using a Brandel cell harvester and filter-
retained radioactivity was measured in a liquid scintillation
counter. IC50 and Ki values were calculated using the compu-
ter program EBDA as described by McPherson.16
Yield: 29%; mp 94–96 ꢀC (decomp); H NMR (CDCl3) d 8.08
1
(s, 1H, Har), 7.65 (m, 1H, NH amide) 6.27 (s, 1H, Har), 4.38
(brs, 2H, NH2), 3.86 (s, 3H, OCH3), 3.68 (m, 1H, H-6s), 3.36
(m, 1H, H5a), 2.74 (m, 1H, H-1), 2.52 (m, 1H, H-3n), 2.37 (s,
3H, NCH3), 2.22–2.04 (m, 2H, H-5s, H-7a), 1.98 (m, 1H, H-
3x), 1.84–1.42 (m, 4H, H-4, H-7s, H-8a, H-8s). Anal. found: C,
59.22; H, 6.71; N, 12.80. For the 5-isoquinuclidinyl derivatives
a mixture of CHCl3/CH3OH (ammonia satured) (98/2) was
used to obtain the first-eluting compound 20: Yield: 27%; mp
158–161 ꢀC; H NMR (CDCl3) d 8.08 (s, 1H, Har), 7.85 (m,
1
1H, NH amide), 6.28 (s, 1H, Har), 4.46 (brs, 2H, NH2), 4.26
(m, 1H, H-5s), 3.86 (s, 3H, OCH3), 2.84 (m, 1H, H-3x), 2.70
(m, 1H, H-3n), 2.60 (m, 1H, H-6s), 2.55 (m, 1H, H-1), 2.33 (s,
3H, NCH3), 2.00 (m, 1H, H-7s), 1.91 (brs, 1H, H-4), 1.70 (m,
2H, H-8a, H-8s), 1.50 (m, 1H, H-7a), 1.20 (m, 1H, H-6a).
Anal. found: C, 59.41; H, 6,83; N, 13.11. Continued elution
gave compound 19: Yield: 16%; mp 191–194 ꢀC; 1H NMR
(CDCl3) d 8.06 (m, 2H, Har, NH amide), 6.27 (s, 1H, Har),
4.33 (brs, 2H, NH2), 4.22 (m, 1H, H-5a), 3.89 (s, 3H, OCH3),
2.96 (m, 1H, H-3n), 2.56 (m, 1H, H-1), 2.49 (m, 1H, H-3x),
2.35 (s, 3H, NCH3), 2.04 (m, 1H, H6a), 1.96 (m, 1H, H-7s),
1.86 (m, 1H, H-4), 1.70 (m, 3H, H-6s, H-8a, H-8s), 1.38 (m,
1H, H-7a). Anal. found: C, 59.02; H, 69.02; N, 13.02.
11. Blaney, F. E.; Hadley, M. S.; King, F. D.; Watts, E. A.
European Patent 115,993, 1984; Chem. Abstr. 1985, 102,
24509.
12. Orjales, A.; Mosquera, R.; Labeaga, L.; Rodes, R. J.
Med. Chem. 1997, 40, 586.
13. Wong, D. T.; Robertson, D. W.; Reid, L. R. Eur. J.
Pharmacol. 1989, 166, 107.
14. Grossman, C. J.; Kilpatrick, G. J.; Bunce, K. T. Br. J.
Pharmacol. 1993, 109, 618.
10. The receptor binding assays have been described in detail
elsewhere.12 5-HT3 receptor binding was determined according
to Wong et al.13 A suspension of enthorinal cortex membranes
15. Kohler, C.; Hall, H.; Ogren, S. O.; Gawell, L. Biochem.
Pharmacol. 1985, 34, 2251.
16. McPherson, G. A. Compt. Prog. Biomed. 1983, 17, 107.