Pyrrolizidine esters and amides as 5-HT4 receptor agonists and antagonists

Article abstract of DOI:10.1021/jm0509501

A series of pyrrolizidine esters, amides, and ureas was prepared and tested for 5-HT₄ and 5-HT₃ receptor binding, 5-HT₄ receptor agonism in the rat tunica muscularis mucosae (TMM) assay, and for 5-HT₃ receptor-mediated functional antagonism in the Bezold-Jarisch reflex assay. Several pyrrolizidine derivatives were identified with high affinity for the 5-HT₄ receptor, including benzamide 12a (SC-53116), a potent and selective 5-HT₄ partial agonist that exhibits efficacy in promoting antral contractions and activity in promoting gastric emptying in canine models. Also discovered were 5-HT₄ receptor antagonists, including imidazopyridine amide 12h (SC-53606), which is a potent and selective 5-HT₄ receptor antagonist with a pA₂ value of 8.13 in the rat TMM assay. N-Methyl indole ester 13d was identified as a potent 5-HT ₄ antagonist with a pA₂ value of 8.93. High selectivity was observed for these pyrrolizidine derivatives versus other monoamine receptors, including 5-HT₁, 5-HT₂, D₁, D ₂, α₁, α₂, and β receptors. 2006 American Chemical Society.

Full text of DOI:10.1021/jm0509501

J. Med. Chem. 2006, 49, 1125-1139
1125
Pyrrolizidine Esters and Amides as 5-HT4 Receptor Agonists and Antagonists
Daniel P. Becker,* Daniel L. Flynn,† Alan E. Moormann, Roger Nosal, Clara I. Villamil,‡ Richard Loeffler, Gary W. Gullikson,
Chafiq Moummi, and Dai-C. Yang
Department of Medicinal Chemistry and Department of Pharmacology, Pfizer, 4901 Searle Parkway, Skokie, Illinois 60077
ReceiVed September 23, 2005
A series of pyrrolizidine esters, amides, and ureas was prepared and tested for 5-HT4 and 5-HT3 receptor
binding, 5-HT4 receptor agonism in the rat tunica muscularis mucosae (TMM) assay, and for 5-HT3 receptor-
mediated functional antagonism in the Bezold-Jarisch reflex assay. Several pyrrolizidine derivatives were
identified with high affinity for the 5-HT4 receptor, including benzamide 12a (SC-53116), a potent and
selective 5-HT4 partial agonist that exhibits efficacy in promoting antral contractions and activity in promoting
gastric emptying in canine models. Also discovered were 5-HT4 receptor antagonists, including imidazopyr-
idine amide 12h (SC-53606), which is a potent and selective 5-HT4 receptor antagonist with a pA2 value of
8.13 in the rat TMM assay. N-Methyl indole ester 13d was identified as a potent 5-HT4 antagonist with a
pA2 value of 8.93. High selectivity was observed for these pyrrolizidine derivatives versus other monoamine
receptors, including 5-HT1, 5-HT2, D1, D2, R1, R2, and â receptors.
Introduction
Serotonin (5-hydroxytryptamine, 5-HT) functions as both a
hormone and a neurotransmitter, controlling a host of central
and peripheral effects in mammalian systems, and is noteworthy
in its diversity of receptors and subtypes.1 Given the diversity
and ubiquitous nature of serotonin receptors, it is imperative to
prepare potent and selective 5-HT ligands to enable pharma-
cological studies of the various receptor subtypes and to serve
as drugs to treat diseases that have an etiology in serotonin
receptor imbalance.
The 5-HT4 receptor was discovered by Baxter et al.2 and by
Dumuis et al.3 in the gut and brain, respectively, and is expressed
in a wide variety of tissues, including brain, heart, bladder, gut,
and kidney4,5 Initial demonstration that the gastrointestinal
prokinetic6 benzamides cisapride (1) and renzapride (2) (Figure
1) enhance contractile activity at neuronal 5-HT4 receptors in
the guinea pig ileum was made by Craig and Clarke.7 It was
later demonstrated by Dumuis and Bockaert that 5-HT4 receptors
mediate the relaxation of smooth muscle of the inner muscularis
mucosae of rat esophagus8 and also the cholinergic stimulation
of the ascending colon of the guinea pig.9 New tools including
fluorescent antagonists have recently been developed for the
study of 5-HT4 receptors,10 and an excellent review of the 5-HT4
receptor and key ligands was recently published.11
Figure 1. 5-HT4 agonists and antagonists.
plexus.14 Compound 1 had been marketed for motility disor-
ders15 but was withdrawn due to QT prolongation.16,17 The
5-HT4 partial agonist tegaserod (compound 3, SDZ HTF 919)
was approved in 2002 for the treatment of constipation-
predominant IBS.18-20 Tegaserod shows a clear effect on the
total colonic transit time in healthy subjects, and a significant
improvement in patients with constipation-predominant IBS in
a phase III trial.21 Compound 4, prucalopride,22 accelerates
colonic transit in healthy subjects but without modification of
either gastric emptying or small bowel transit.23 In patients with
severe constipation, the benzamide prucalopride elicited a dose-
dependent effect on acceleration of the overall transit time.24
We have pursued the exploration of various conformationally
constrained bicyclic and tricyclic amines as ligands for the
serotonin 5-HT4 receptor to treat gastrointestinal motility
disorders. Our focus has been on receptor selectivity to avoid
potential side effects, and we were particularly cognizant of
Selective ligands for the 5-HT4 receptor show promise in the
treatment of a wide variety of diseases, including irritable bowel
syndrome (IBS) and other gastrointestinal motility disorders,
urinary incontinence, atrial arrhythmia,11,12 and cognitive dis-
orders.13 Gastrointestinal motility disorders are a collection of
syndromes that are characterized by a hypofunctional bowel,
abnormal motility patterns and transit, and painful gut wall
distension. The mechanisms thought to be common to many of
these motility disorders is the malfunction of enteric nervous
system control of peristalsis at the level of the myenteric
* To whom correspondence should be addressed. Loyola University, 6525
North Sheridan, Chicago, IL 60626. Phone: 773-508-3089. Fax: 847-730-
3181. E-mail dbecke3@luc.edu.
† Current address: Deciphera Pharmaceuticals Inc., 1505 Wakarusa
Drive, Lawrence, KS 66047. E-mail: dflynn@deciphera.com.
‡ Current address: Abbott Labs, 100 Abbott Park Road, Abbott Park,
IL 60064-6099. E-mail clara.villamil@abbott.com.
10.1021/jm0509501 CCC: $33.50 © 2006 American Chemical Society
Published on Web 01/10/2006

1126 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Becker et al.
Scheme 1. Preparation of (-)-Trachelanthamidine 9 per Nagao
Scheme 5. Preparation of Imidazol[1,2-a]pyridine-8-carboxylic
Acid 17 and 6-Chloroimidazo[1,2-a]pyridine-8-carboxylic Acid
et al.38
19
Scheme 2. Preparation of Pyrrolizidine Amides 12a-i
Scheme 6. Preparation of 6-Chloro-3-methylimidazo[1,2-a]pyri-
dine-8-carboxylic Acid 21
Scheme 3. Preparation of Pyrrolizidine Esters 13a-h
H-bond of the 2-methoxybenzamides. This approach led to the
potent 5-HT4 receptor antagonist 12h.33 We also explored both
amides and esters of the pyrrolizidine amine moiety with indole
and indazole aromatic moieties, inspired by the potent 5-HT3
receptor antagonists tropisetron34 and granisetron,34,35 particu-
larly considering that tropisetron was shown to be the first
surmountable antagonist of 5-HT4 receptors,3 and Buchheit et
al. demonstrated that the ester derivative of metoclopramide,
SDZ 205-557, is a potent antagonist of 5-HT4 receptors.36
We were aware of the toxicity of the pyrrolizidine alkaloid
natural products37 and considered the fact that toxicity among
pyrrolizidine alkaloids varies dramatically with differences in
chemical structure. Certain pyrrolizidine natural products can
form highly reactive pyrrole intermediates upon metabolism by
CYP3A4, which are responsible for their nascent toxicity. A
ring nucleus containing a double bond at the 1,2-position is
considered to be essential for toxic effects of the alkaloid, along
with additional hydroxyl groups around the nucleus. On the basis
of this premise of structure-specificity we decided to explore
the pyrrolizidine nucleus for incorporation into novel 5-HT4
receptor agonists and antagonists. This paper details our work
employing the pyrrolizidine scaffold resulting in the potent
5-HT4 receptor agonist 12a, as well as 5-HT4 receptor antagonist
12h and other potent 5-HT4 antagonists.
Scheme 4. Preparation of Benzimidazolone 15a and Oxindole
15b
avoiding the dopamine D2 receptor, as D2 antagonism is
responsible for extrapyramidal side effects and hyperprolactine-
mia observed with the marketed benzamide metoclopramide.25
We were also vigilant of other monoamine receptors, including
the 5-HT2 receptor, which is potently inhibited by compound
1.26 We were attracted to the pyrrolizidine azabicyclo[3.3.0]-
octane ring system and reasoned that this fairly rigid bicyclic
amine could provide a fruitful scaffold for preparing potent and
selective serotonin 5-HT4 agonists. A similar approach was used
by King in the discovery of renzapride (2), which was designed
to mimic the higher energy conformers of azabicyclic quino-
lizidine benzamides of their earlier studies27 and in particular
was designed to lock in the cis form of the ring junction of the
azabicycle and reduce the steric bulk extending outward around
the basic nitrogen. The Beecham group then also employed a
pyrrolizidine scaffold.28,29 More recently, the 5-HT4 receptor
agonist meso-pyrrolizidine 5 (SK-951) has been described,
which is a potent gastrointestinal prokinetic agent in rats and
dogs.30,31 Our own successful approach employing the pyr-
rolizidine moiety led to the potent 5-HT4 receptor partial agonist
12a32 by attaching the pyrrolizidine to the traditional aromatic
moiety of prokinetic benzamides 1 and 2. In addition, we
explored the attachment of other aromatic amides to the
pyrrolizidine bicyclic amine, seeking to mimic the intramolecular
Chemistry
The (S,S)-carbinol (-)-trachelanthamidine 9 was prepared by
a diastereoselective alkylation of the chiral tin enolate of 3-(4-
chlorobutyryl)-4(S)-isopropyl-1,3-thiazolidine-2-thione 7 with
5-acetoxy-2-pyrrolidinone as elegantly described by Nagao et
al.38 and summarized in Scheme 1. We analyzed the Mosher
ester of 9 by 19F NMR to confirm the enantiomeric purity, which
showed the material to be g99.1% ee. Aminomethylpyrrolizi-
dine 11 was prepared by the Mistunobu reaction of 9 with
phthalimide in the presence of triphenylphosphine and diethyl-
azodicarboxylate to afford phthalimide derivative 10, and the
phthalimide moiety was removed with hydrazine to afford 11
(Scheme 2). Coupling of the amine with an aryl carboxylic acid

5-HT4 Receptor Agonists and Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1127
Table 1. Pyrrolizidine 5-HT4 Agonists
Figure 2. 5-HT4 receptor-mediated relaxation of rat tunica muscularis mucosa by 5-HT, 12a (SC-53116), and ent-12a (SC-53117).
was then typically accomplished with 1,1′-carbonyl diimidazole
(CDI) to give the requisite pyrrolizidine amides 12. Esters of 9
were prepared by treating the imidazolide derived from the
appropriate aryl carboxylic acid and CDI with the sodium salt
of alcohol 9 to afford esters 13 (Scheme 3). The enantiomer of
aminomethylpyrrolizidine 11 and its derivatives (in particular,

1128 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Table 2. Pyrrolizidine Amide and Urea 5-HT4 Antagonists
Becker et al.
cosal (TMM) tissue,2,39 as this model allows for determination
of full cumulative dose-response curves to agonists. Serotonin
5-HT4 receptor antagonists were also evaluated using the TMM
model. Binding to serotonin 5-HT4 receptors in guinea pig
striatum was measured utilizing [3H]GR113,808 as the ligand.
Binding to serotonin 5-HT3 receptors in rat cortical tissue was
measured using [3H]GR656630. Functional 5-HT3 antagonism
was measured by utilizing the von Bezold-Jarisch reflex
model.40
Pyrrolizidine 5-HT4 agonists are summarized in Table 1. In
the rat TMM assay, the chiral pyrrolizidine benzamide 12a and
its distomer ent-12a had EC50 values of 16.5 ( 3.8 and 323 (
46 nM, respectively. A plot of the TMM activity of 12a and
ent-12a relative to serotonin is shown in Figure 2, demonstrating
that the pyrrolizidine benzamide 12a has comparable potency
to serotonin (16.5 nM vs 9.0 nM for 5-HT) and has 80% of the
efficacy of serotonin in this preparation. The distomer ent-12a
is 36 times less potent than serotonin. The potency of 12a
relative to its enantiomer is reflected in 5-HT4 receptor binding.
Pyrrolizidine 12a has a Ki value of 5.2 nM, while ent-12a
inhibits ligand binding by only 18% at 500 nM (Table 1).
Benzamide 12a is approximately 30 times more potent in
binding to the 5-HT4 receptor relative to its 5-HT3 binding (Ki
) 152 nM). In contrast to the 30× difference in 5-HT4 binding,
12a is only half as potent as ent-12a in 5-HT3 binding. We
previously reported a similar trend with compound 6 and its
enantiomer, where the 5-HT4 receptor showed a greater discern-
ment of the antipodes than the 5-HT3 receptor, although in that
case the eutomer was the same compound for both receptors.41
Functional antagonism of the 5-HT3 receptor is demonstrated
by inhibition of the von Bezold-Jarisch reflex in mice for both
12a and ent-12a (87% at 10 mg/kg for both compounds).
The ethoxy benzamide 12b was prepared in an attempt to
increase 5-HT3 potency, which was successful in boosting 5-HT3
receptor binding by 6-fold (Ki ) 25 nM); however, the
compound was approximately 12 times less potent in the rat
5-HT4 TMM assay, so 5-HT3 receptor binding potency was
boosted at the expense of 5-HT4 potency. The methiodide
quaternary ammonium salt 12c was prepared to probe the
possibility of synthesizing a potent radiolabeled pyrrolizidine
analogue by employing radiolabeled methyl iodide. The qua-
ternary ammonium salt 12c was indeed very potent in binding
to the 5-HT4 receptor (Ki ) 0.35 nM), but despite its greater
potency in binding, 12c was markedly less potent than 12a as
an agonist in the TMM functional assay, presumably due to
the membrane transport limitations of the quaternary salt.
Compound 13a is the ester corresponding to 12a and was the
most potent compound tested in our hands for both 5-HT4
receptor binding (Ki ) 183 pM) and in functional agonism at
the 5-HT4 receptor in the TMM assay (EC50 ) 1.5 nM). It is
actually a partial agonist at the 5-HT4 receptor, with 57%
efficacy as an agonist relative to serotonin. N-Isopropyl benz-
imidazolone urea 15a, which incorporates the benzimidazolone
moiety of BIMU 842 and DAU 6215,43 exhibited potent binding
at the 5-HT4 receptor (Ki ) 7.0 nM) but was 22 times less potent
in 5-HT4 agonism than 12a.
benzamide ent-12a, the enantiomer of 12a) were prepared from
the (R)-enantiomer of thiazolidinethione 7.
Benzimidazolone 15a and oxindole urea 15b were prepared
by treatment of the sodium salt of the corresponding benzimid-
azolone 14a or oxindole 14b with phosgene followed by the
aminomethylpyrrolizidine 11 (Scheme 4). Imidazol[1,2-a]pyr-
idine-8-carboxylic acid 17 was prepared by reacting 2-aminoni-
cotinic acid 16 with chloroacetaldehyde, and 6-chloroimidazo-
[1,2-a]pyridine-8-carboxylic acid 19 was prepared from methyl
2-amino-5-chloronicotinate 18 with chloroacetaldehyde followed
by saponification (Scheme 5). Similarly, 6-chloro-3-methylim-
idazo[1,2-a]pyridine-8-carboxylic acid 21 was prepared by
alkylative esterification of 2-aminonicotinic acid 16 and regio-
selective chlorination with tert-butyl hypochlorite to afford 20,
followed by conversion to the imidazopyridine with 2-bro-
mopropionaldehyde and hydrolysis of the ester to afford
carboxylic acid 21 (Scheme 6). The pyrrolizidine esters and
amides of these imidazopyridines were prepared by the general
methods described in Schemes 2 and 3.
The excellent potency of the pyrrolizidine moiety in 5-HT4
receptor agonism prompted us to consider employing this
scaffold for the preparation of antagonists; indeed, we were
encouraged in this regard by the potent but partial agonism of
benzoate ester 13a. Agonism of the 5-HT4 receptor may be
dependent upon a planar array of the amide bond with the
aromatic ring system, enabled by an intramolecular H-bond
between the amide NH and the methoxy group as for 12a or
Results and Discussion
Initial testing of pyrrolizidine 5-HT4 receptor ligands was
accomplished using the rat esophageal tunica muscularis mu-

5-HT4 Receptor Agonists and Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1129
Table 3. Pyrrolizidine Ester 5-HT4 Antagonists
between the amide NH and the benzimidazolone carbonyl of
15a. The 5-HT4 agonist efficacy of ester 13a is lower (57%),
apparently due to the inability to form this H-bond and the
corresponding conformational mobility.
benzamides, assuming a similar binding mode for the two series
based on modeling considerations. Unsubstituted imidazopyri-
dine 12g was moderately potent (pA2 ) 6.75), whereas addition
of the chlorine in the 6-position boosted potency of compound
12h33 by over an order of magnitude (pA2 ) 8.13). Imidazopyr-
idine 12h exhibited very potent binding to the 5-HT4 receptor
(Ki ) 1.4 nM). Incorporation of a 3-methyl substitutent was
probed in order to augment potency with an additional hydro-
phobic interaction from the synthetically accessible imidazopyr-
idine, but further substitution with the 3-methyl group decreased
the 5-HT4 inhibitory potency of compound 12i (pA2 ) 7.09)
relative to 12h. Oxindole urea 15b, which is structurally related
to benzimidazolone 5-HT4 receptor agonist 15a, is an antagonist
that exhibits a pA2 value of 7.0. As with the imidazopyridines,
we originally hoped that the oxindole carbonyl could participate
in an intramolecular hydrogen bond with the urea NH. Oxindole
15b exhibits potent binding to the 5-HT4 receptor (Ki ) 6.8
nM).
Pyrrolizidine ester 5-HT4 receptor antagonists are included
in Table 3. The esters were inspired by the work of Buchheit et
al., who reported SDZ 205-557, the ester analogue of metoclo-
pramide, as the first potent antagonist of 5-HT4 receptors.36
These compounds all lack agonist activity in the TMM assay
(EC50 > 10 000 nM). On the basis of the inhibitory potency of
6-chloroimidazopyridine amide 12h, we prepared the corre-
sponding ester 13b, which is a moderate 5-HT4 receptor
antagonist (pA2 ) 6.75). Next, we prepared esters incorporating
the indole and indazole aromatic moieties present in tropisetron
and granisetron, respectively. Attachment at the indole 2-position
afforded N-methyl indole-2-carboxylic ester 13c, which was
inactive, but good antagonism was obtained with 3-substituted
Table 2 includes pyrrolizidine amides and ureas that are
functional 5-HT4 antagonists in the rat TMM assay employing
different aromatic groups. All of these compounds were
determined to have EC50 values for agonism in the rat TMM
assay of >10 000 nM. Two of the derivatives tested were
determined to be inactive in the rat TMM antagonism assay at
concentrations of up to 100-1000 nM, including indole
carboxamide 12d and indolizine 12e, which borrows the
indolizine moiety described by Bermudez et al.44 We employed
an imidazopyridine aromatic nucleus with the nitrogen lone pair
orthogonal to the aromatic π-system (compounds 12f-i) point-
ing toward the amide NH in an attempt to mimic the intramo-
lecular hydrogen bond present in the 2-methoxybenzamide
prokinetic 5-HT4 agonists. Additionally, we were intrigued that
the bridgehead nitrogen lone pair is partially delocalized into
the amide carbonyl as in the 4-aminobenzamides 1 and 2,
making the carbonyl oxygen more electron rich and favoring
the coplanarity of the aromatic heterocycle and the amide. The
structural similarity to benzofuran amides 4 and 5 is also
apparent. Nonetheless, we found that these compounds function
as 5-HT4 receptor antagonists. Several imidazopyridines afforded
good 5-HT4 antagonist activity, including 3-ethylimidazopyri-
dine 12f (pA2 ) 6.56). The role of the second nitrogen atom in
the antagonist imidazopyridine 12f relative to inactive indolizine
12e is noteworthy. The 6-chloro substituent was incorporated
in analogue 12h to boost binding on the basis of structural
overlays done with the benzamide moiety of the prokinetic

1130 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Becker et al.
Figure 3. Gastrointestinal response to compound 12a (0.3 mg/kg, iv) in the dog. Contractile responses of the antrum (panel 1), jejunum (panel 2),
ileum (panel 3), and colon (panel 4) before and after 5-HT4 partial agonist 12a is given iv to fasted dogs (n ) 4). A sustained stimulation of antral
and jejunal motility occurs with brief augmentation of ileal and colonic contractions.
an antagonist. 5-Methoxyindole-3-carboxylate ester 13e exhib-
ited moderate inhibitory potency at the 5-HT4 receptor (pA2 )
7.15), and potency was markedly increased for the 5-fluoro
derivative 13f in 5-HT4 inhibitory potency (pA2 ) 8.5) as well
as binding to the 5-HT4 and 5-HT3 receptors (Ki ) 700 pM
and 10 nM, respectively). Indazole ester 13g had good inhibitory
potency (pA2 ) 8.48) and the corresponding N-methyl indazole
derivative 13h had comparable potency (pA2 ) 8.56). These
indazoles exhibited potent subnanomolar binding to the 5-HT4
receptor (Ki ) 800 and 400 pM, respectively) and moderate
binding to the 5-HT3 receptor (Ki ) 135 and 20 nM, respec-
tively). The N-methyl analogue was thus modestly more potent
than the N-H analogue toward both receptors. Both indazoles
were free of binding at 5-HT1, 5-HT2, D1, D2, R1, R2, â1, and
â2 receptors. Thus, the most potent member of the pyrrolizidine
ester 5-HT4 receptor antagonist series summarized in Table 3
Figure 4. Stimulation of gastric antral contractions by 12a (SC-53116),
is N-methyl indole-3-carboxylic ester 13d with a pA2 value of
8.93, which is comparable in potency to the Glaxo antagonist
GR-113808 (pA2 ) 9.39). Indole 13d is a very potent dual
receptor 5-HT4/5-HT3 ligand, with a Ki ) 183 pM at the 5-HT4
receptor and Ki ) 5.0 nM at the 5-HT3 receptor. This
ent-12a (SC-53117), and 1 (cisapride) given in phase 1 of the MMC
cycle as the percentage of maximal activity seen during phase 3 of the
MMC.
indoles and indazoles, particularly with N-methyl indole-3-
carboxylic ester 13d, which exhibited exceptional potency as

5-HT4 Receptor Agonists and Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1131
Figure 5. Enhancement of gastric emptying of radiolabeled solid meals by 12a (SC-53116), ent-12a (SC-53117), and 1 (cisapride).
Table 4. Comparison of Enantiomers 12a and ent-12a with 1 and 2
times less potent than 12a in promoting gastric antral contrac-
tions with an ED50 of 0.45 mg/kg iv. A maximally effective
dose of pyrrolizidine 12a induced antral contractions that were
approximately 60% of the maximum phase 3 contractions,
compared to 54% for compound 1 and 69% for compound 2
(not shown).
assay
1
2
12a ent-12a
5-HT4 agonism rat TMM
(EC50, nM)
antral contractility in fasted dog 0.048 0.015
(ED50, mg/kg, iv)
solid gastric emptying
(ED50, mg/kg, iv)
55 ( 8 98 ( 14
17 ( 4 323 ( 46
0.010 0.45
0.001 1.0
0.03 active, but full
dose-response
not performed
In vivo gastrointestinal prokinetic efficacy was also deter-
mined in a functional model for gastroparesis employing
γ-scintigraphy. Since 5-HT4 agonists enhance solid gastric
emptying largely by increasing the force of contraction of the
gastric antrum, the canine antral contractile response was used
to select compounds for the gastroparesis model. In the
gastroparesis model, an R-adrenergic agonist (2-methyl-3-[(2E)-
pyrrolidin-2-ylideneamino]phenol) inhibits food-induced gastric
antral and duodenal contractions, thus mimicking motor abnor-
malities characteristic of clinical dysmotilities.45 Enhancement
of gastric emptying in the gastroparesis model is demonstrated
by pyrrolizidines 12a and ent-12a and benzamide 1 (Figure 5).
Pyrrolizidine benzamide 12a was shown to be approximately
30 times more potent than benzamide 1 in enhancing gastric
emptying (ED50 ) 0.001 mg/kg vs an ED50 ) 0.03 mg/kg for
compound 1. As expected, distomer ent-12a was less potent
than both benzamide 1 and pyrrolizidine 12a, with an ED50 of
1.0 mg/kg.
pyrrolizidine ester is free of binding at 5-HT1, 5-HT2, D1, D2,
R1, R2, â1, and â2 receptors (Ki > 10 µM).
In general, the esters are more potent as 5-HT4 antagonists
than the corresponding amides, although the present work only
allows for two direct comparators. Specifically, N-methyl indole
ester 13d is more potent than the N-methyl indole carboxamide
12d. In contrast, the 6-chloroimidazopyridine amide 12h is more
potent than the corresponding ester 13b.
In vivo 5-HT4 agonism was examined in conscious dogs by
recording contractile activity of the distal region of the stomach
(antrum), as well as along other portions of the gastrointestinal
tract. Test compounds were intravenously administered to fasted
animals during phase I (a period of quiescence that lasts about
50-80 min) of the interdigestive migrating motor complex
(MMC). The maximal contractile activity that occurred during
phase III of the MMC was used to normalize the motility
response to the compound. Pyrrolizidine amide 12a stimulated
sustained antral and jejunal contractile activity as well as a brief
augmentation of ileal and colonic contractions (Figure 3).
The antral responses to intravenous dosing of 12a, ent-12a,
and 1 in the canine contractility model are plotted in Figure 4.
Pyrrolizidine benzamide 12a exhibited an ED50 of 0.010 mg/
kg iv, whereas benzamide 1 was 5-fold less potent with an ED50
of 0.056 mg/kg iv. Distomer ent-12a was approximately 45
5-HT4 agonism data is summarized in Table 4 along with
canine antral contractility and canine solid gastric emptying for
the pyrrolizidine benzamides 12a and ent-12a and the benz-
amide 1. Pyrrolizidine 12a is the most potent compound in vitro,
as well as in vivo, in both canine motility models. Compound
12a has an ED50 in the fasted dog antral contractility model of
0.010 mg/kg, iv, and exhibits an ED50 of 0.001 mg/kg, iv in
the canine solid gastric emptying model. Benzamide 1 has an
Table 5. Receptor Profiling of Compounds: EC50 or Ki
Ki (nM)
EC50 (nM):
5-HT4
compd
agonism
5-HT4
5-HT1
5-HT2
5-HT3
D1
D2
227
>10K
>10K
>10K
R1
30
>10K
>10K
>10K
R2
â
1
54.7
16.5
>10K
51
17
>1K
>10K
>10K
>10K
6.1
134
152
259
1.2
1700
4500
>10K
>10K
>10K
>10K
12a
12h
6
5.2
1.4
29
>10K
>10K
>10K
>10K
>10K
>10K
>10K
>10K
>10K

1132 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Becker et al.
was then stirred for 1 h at 0 °C and then 16 h at room temperature.
Concentration gave a residue which was purified on silica gel
eluting with MeOH (saturated with NH3)/CHCl3 (4/96 and then
6/94) to afford the desired phthalimide 10 (2.12 g, 82%) as a pale
yellow crystalline solid: mp 72-73 °C; IR (MIR) 1763, 1706.5
cm-1; [R]D ) -2.5°; [R]365 ) -13.3° (c ) 0.285 g/dL in
ED50 of 55 nM in the rat TMM assay, whereas benzamide 2
exhibits an ED50 of 98 nM. Compound 2 is also more potent
than benzamide 1 in the antral contractility model, with an ED50
of 0.015 mg/kg iv relative to 0.048 mg/kg iv for compound 1.
Pyrrolizidine derivatives of the present series are highly
selective for the 5-HT4 receptor. Pyrrolizidine 12a is very potent
as a partial agonist at the 5-HT4 receptor and in binding to the
5-HT4 receptor (Ki ) 5.2 nM) (Table 5). Compound 12a exhibits
moderate binding at the 5-HT3 receptor (Ki ) 152 nM), but no
detectible binding (Ki > 10 000 nM) at any of the other
monoamine receptors tested (5-HT1, 5-HT2, D1, D2, R1, R2, and
â receptors). Imidazopyridine 12h, as reported previously,33
is a very potent antagonist at the 5-HT4 receptor (Ki ) 1.4 nM;
pA2 ) 8.13) with excellent selectivity versus the other mono-
amine receptors tested (Ki > 10 µM). Standards included
for comparison are compound 6, which we have reported to
be a potent and selective dual 5-HT4 agonist and 5-HT3 re-
ceptor antagonist, and compound 1, which shows potent
inhibition of R1 receptors (Ki ) 30 nM) and 5-HT2 receptors
(6.1 nM).
In summary, incorporation of the pyrrolizidine scaffold has
resulted in the discovery of potent and selective ligands for the
5-HT4 receptor, including the potent and selective 5-HT4 agonist
12a, with excellent efficacy in canine antral motility and gastric
emptying models. Toxicological profiling of 12a revealed that
the compound is active in the Ames assay after S9 activation,
so the compound was not pursued as a clinical candidate. It is
interesting to note that metabolic activation giving rise to
mutagenicity appears to be enantiospecific, as the enantiomer
ent-12a is not mutagenic with or without S9 activation. The
potency and efficacy of this series has prompted analogue work
to avoid metabolic activation by bridgehead-methyl substitu-
tion.46 It is worth noting in this context that pyrrolizidine 5 is
substituted at the bridgehead carbon, which would not permit
the formation of a pyrrole-containing metabolite. Nevertheless,
pyrrolizidine 12a represents a useful pharmacological tool.47-50
Work employing the pyrrolizidine scaffold also resulted in the
discovery of imidazopyridine 12h, which is a potent and
selective 5-HT4 receptor antagonist,33 as well as the potent
N-methylindole 5-HT4 receptor antagonist 13d. Related work
in these labs on conformationally constrained amines also led
to the discovery of azanoradamantane 6, which is a potent,
nonmutagenic, and selective dual 5-HT4 agonist/5-HT3 antago-
nist.41,51,52
1
chloroform); H NMR (300 MHz, CDCl3) δ 7.86 (2H, m), 7.73
(2H, m), 3.76 (2H, d, J ) 7.1 Hz), 3.28 (1H, m), 3.21 (1H, m),
2.94 (1H, dt, J ) 10.2, 5.9 Hz), 2.51 (2H, m), 2.12 (1H, m), 1.99
(1H, m), 1.90-1.63 (3H, m), 1.43 (1H, m); 13C NMR (75 MHz,
CDCl3) δ 167.8, 133.5, 131.5, 122.7, 67.7, 54.6, 53.9, 44.9, 40.3,
31.6, 31.0, 25.8. HRMS calcd for C16H18N2O2 270.1369, found
270.1369. Anal. Calcd for C16H18N2O2·0.4H2O: C, 69.24; H, 6.83;
N, 10.09. Found: C, 69.12; H, 6.56; N, 9.94.
(1S,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethylamine Dihy-
drochloride (11). To a solution of phthalimide 10 (4.11 g, 15.2
mmol) in ethanol (60 mL) was added hydrazine hydrate (3.70 g,
74 mmol) and the reaction was stirred for 15 h at room temperature.
The resulting suspension was concentrated to give a residue. To
the residue was added 4 N KOH (50 mL, presaturated with NaCl)
and the mixture was extracted with chloroform (15 × 20 mL). The
combined extracts were dried over sodium sulfate and concentrated
to give the title amine 11 (2.13 g, 100%) as a yellow semisolid,
which may be stored under argon at -30°, if necessary, before use
in the subsequent step: 1H NMR (300 MHz, CDCl3) δ 3.13 (2H,
m), 2.95 (1H, m), 2.74 (2H, m), 2.56 (2H, m), 2.03 (1H, m), 1.98-
1.63 (4H, m), 1.62-1.43 (2H, m). Dihydrochloride salt of 11: 1H
NMR (300 MHz, MeOD-d4) δ 3.97 (1H, m), 3.79 (1H, m), 3.46
(1H, m), 3.29-3.03 (3H, m), 2.51-2.34 (2H, m), 2.32-1.78 (5H,
m); 13C NMR (75 MHz, MeOD-d4) δ 70.4, 54.4, 53.9, 42.5, 40.7,
29.8, 29.4, 24.3; HRMS MH+ calcd for C8H16N3 141.1392, found
141.1394.
4-Amino-5-chloro-N-[(1S-cis-hexahydro-1H-pyrrolizin-1-yl)-
methyl]-2-methoxybenzenecarboxamide, Hydrochloride (12a,
SC-53116). To a solution of 4-acetamido-5-chloro-2-methoxyben-
zoic acid (3.70 g, 15.2 mmol) in DMF (15 mL, freshly distilled
under high vacuum) was added 1,1′-carbonyldiimidazole (2.46 g,
15.2 mmol), which resulted in a vigorous gas evolution. After 40
min at room temperature a solution of the free base amine 11 (2.13
g, 15.2 mmol) in DMF (6 mL) was added and reaction was stirred
for 40 h at room temperature. Concentration gave a yellow oil which
was treated with 15% K2CO3 (65 mL) and extracted with
chloroform (4×). The combined organic extracts were washed
successively with water (2×) and brine and dried over sodium
sulfate to give a pale yellow solid (5.9 g). Purification on silica gel
(170 g) eluting with MeOH (saturated with NH3)/CHCl3 (15/85)
gave 4-(acetylamino)-N-[(7aS)-hexahydro-1H-pyrrolizin-1-ylmethyl]-
5-chloro-2-methoxybenzamide (4.89 g, 88%) as a colorless solid
after drying for 1 h at 100 °C: mp 131-132 °C; [R]D ) -8.8° (c
1
) 1.2 g/dL); IR (MIR) 3407, 3310, 1696, 1656 cm-1; H NMR
Experimental Section
(300 MHz, CDCl3) δ 8.33 (1H, br s), 8.16 (1H, s), 8.12 (1H, s),
7.98 (1H, t, J ) 5 Hz), 3.95 (3H, s), 3.47 (2H, br s), 3.19 (2H, m),
(-)-Trachelanthamidine (9). The (S,S)-carbinol (-)-trachelan-
thamidine 9 was prepared by a diastereoselective alkylation of the
chiral tin enolate of 3-(4-chlorobutyryl)-4(S)-isopropyl-1,3-thiazo-
lidine-2-thione 7 with 5-acetoxy-2-pyrrolidinone and subsequent
reduction with lithium aluminum hydride as described by Nagao
et al.38 to afford 9 (3.12 g, 45.5%) as a colorless oil: [R]D ) +9.2
(c ) 0.195 g/dL in CHCl3, 10 cm); [R]365 ) -63.1 (c ) 0.195
g/dL in CHCl3, 10 cm). The Mosher ester was prepared by treatment
of the alcohol with the Mosher acid chloride [from (S)-(-)-R-
methoxy-R-(trifluoromethyl)phenylacetic acid and thionyl chloride]
and pyridine. 19F NMR analysis of the peak at -172.17 ppm and
the absence of the peak at -172.29 ppm from the diastereomeric
standard showed that the material had g99.6% diastereomeric
purity, or g99.1% ee.
2-[(1S,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethyl]-1H-isoin-
dole-1,3(2H)-dione (10). To a solution of alcohol 3 (1.44 g, 10.2
mmol) in dry THF (45 mL) were added triphenylphosphine (5.35
g, 20.4 mmol) and phthalimide (3.00 g, 20.4 mmol). The solution
was then cooled to 0 °C and diethylazodicarboxylate (3.21 mL,
3.55 g, 10.4 mmol) was added dropwise over 15 min. The reaction
2.94 (1H, m), 2.56 (2H, m), 2.28 (3H, s), 2.19-1.47 (7H, m); 13
C
NMR (75 MHz, CDCl3) δ 168.5, 163.2, 155.9, 135.0, 131.3, 117.0,
114.2, 104.2, 68.0, 55.8, 54.3, 53.9, 45.2, 42.4, 31.3, 31.2, 25.5,
24.2; HRMS calcd for C18H24N3O3Cl 365.1506, found 365.1514.
Anal. Calcd for C18H24N3O3Cl: C, 59.09, H, 6.61, N, 11.48, Cl,
9.69. Found: C, 58.76; H, 6.68; N, 11.42; N, 10.02.
A solution of the acetamide (4.88 g, 13.3 mmol) in ethanol (670
mL) was treated with potassium hydroxide (4.49 g, 80 mmol) and
heated under reflux for 2 h. Concentration gave a residue to which
was added water (150 mL), and the resulting mixture was extracted
with chloroform (4×). The combined organic extracts were washed
successively with water (2×) and brine and dried over sodium
sulfate. Concentration gave the desired benzamide as a solid (4.21
g, 98%) which was purified on silica gel eluting with MeOH
(saturated with NH3)/CHCl3 (12/88 and then 16/84) to afford the
pure free base of 12a (3.86 g, 90%) as a colorless solid: mp 165.5-
166.5 °C; [R]D ) -10.6°; [R]365 ) -36.7° (c ) 0.330 g/dL in
chloroform); 1H NMR (300 MHz, CDCl3) δ 8.11 (1H, s), 7.80 (1H,
t, J ) 6 Hz), 6.30 (1H, s), 4.40 (2H, s), 3.90 (3H, s), 3.47 (2H, m),

5-HT4 Receptor Agonists and Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1133
3.19 (2H, m), 2.98 (1H, m), 2.57 (2h, m), 2.10-1.47 (8H, m); 13
C
toluene (50 mL). The reaction flask was wrapped with foil and
allowed to stand at room temperature for 24 h. Filtration afforded
the methiodide salt 12c (35 mg, 75%) as a pale yellow solid: 1H
NMR (300 MHz, MeOD-d4) δ 7.79 (1H, s), 6.53 (1H, s), 3.95 (3H,
s), 3.88 (1H, dd, J ) 8.1, 3.4 Hz), 3.78-3.63 (3H, m), 3.63-3.56
(2H, m), 3.45 (1H, m), 3.23 (3H, s), 2.52 (1H, m), 2.40-2.13 (4H,
m), 2.12-1.90 (2H, m); 13C NMR (75 MHz, MeOD-d4) δ 167.7,
159.5, 150.5, 133.1, 111.4, 111.1, 98.5, 83.8, 67.2, 66.3, 56.7, 53.5,
47.7, 41.8, 31.3, 29.3, 25.1. Anal. Calcd for C17H25N3O2I: C, 47.45;
H, 5.86; N, 9.77. Found: C, 47.17; H, 5.92; N, 9.54.
N-[(1S,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethyl]-1-methyl-
1H-indole-3-carboxamide (12d). To a solution of N-methylindole-
3-carboxylic acid (1.0 equiv) in DMF at room temperature was
added CDI (1 equiv). After 1 h, a solution of amine 11 (1 equiv)
in DMF was added and the solution stirred for 16 h at room
temperature. Aqueous workup and chromatography on silica gel
afforded the desired indole carboxamide 12d. Anal. Calcd for
C18H23N3O: C, 72.70; H, 7.80; N, 14.13. Found: C, 72.91; H, 7.68;
N, 13.92.
N-exo-(Tetrahydro-1H-pyrrolizin-4(5H)-ylmethyl)-3-ethylin-
dolizine-1-carboxamide (12e). 3-Ethylindolizine-1-carboxylic acid44
(190 mg, 1.0 mmol) was suspended in CHCl3 (2 mL). Oxalyl
chloride (184 uL, 2.1 mmol) and DMF (1 drop) were added, and
the mixture was stirred for 2 h. The reaction mixture was
concentrated in vacuo, azeotroping once with toluene. To the residue
dissolved in CHC13 was added a solution of exo-tetrahydro-1H-
pyrrolizin-4(5H)-methylamine 11 (140 mg, 1.0 mmol) and triethyl-
amine (279 uL, 2.0 mmol) in CHCl3 (2 mL) and the mixture was
stirred for 18 h. The organic solution was washed with 1 N NaOH
and brine, dried over K2CO3, filtered, and concentrated to give a
crude oil. Purification on silica gel eluting with MeOH (saturated
with NH3)/CHCl3 (30/70) gave the desired compound 12e as a very
hydroscopic solid (190 mg, 63%): 1H NMR (400 MHz, MeOD-
d4) δ 8.20 (1H, d, J ) 18 Hz), 8.02 (1H, t, J ) 14 Hz), 7.03-6.97
(3H, m), 6.80-6.75 (1H, m), 4.06-3.99 (1H, m), 3.83-3.75 (1H,
m), 3.67 (2H, d, J ) 6 Hz), 3.46-3.42 (1H, m), 3.25-3.18 (1H,
m), 3.18-3.03 (1H, m), 2.54-2.45 (1H, m), 2.37-2.29 (1H, m),
2.22-2.12 (2H, m), 2.09-1.86 (3H, m); MS calcd for C19H25N3O
311.43, found 311.19. Anal. Calcd for C19H25N3O·2.0H2O: C,
65.68; H, 7.25; N, 12.09. Found: C, 65.79; H, 7.56; N, 12.03.
Attempts to make the HCl salt resulted in a brown oil, so the
compound was tested as the free base.
N-exo-(Tetrahydro-lH-pyrrolizin-4(5H)-ylmethyl)-3-ethylim-
idazo[1,2-a]pyridine-l-carboxamide Monohydrochloride (12f).
3-Ethylimidazo[1,5-a]pyridine-l-carboxylic acid44 (190 mg, 1.0
mmol) was suspended in CHCl3 (2 mL). Oxalyl chloride (184 uL,
2.1 mmol) and DMF (1 drop) were added, and the mixture was
stirred for 2 h. The reaction mixture was concentrated in vacuo,
azeotroping once with toluene. To the residue dissolved in CHCl3
was added a solution of exo-tetrahydro-lH-pyrrolizin-4(5H)-
methylamine 11 (140 mg, 1.0 mmol) and triethylamine (279 uL,
2.0 mmol) in CHCl3 (2 mL) and the mixture stirred for 18 h. The
organic solution was washed with 1 N NaOH and brine, dried over
K2CO3, filtered, and concentrated to give a crude oil which was
chromatographed on silica gel eluting with 5% CH3OH(NH3)/CHCl3
to give 110 mg (35%) of desired compound as the free base. The
HCl salt 12f was prepared in the same manner as for 12a: MS
calcd for C18H24N4O 312, found 312. Anal. Calcd for C18H24N4O·
HCl·0.75H2O: C, 57.91; H, 7.24; N, 15.01; Cl, 12.35. Found: C,
58.15; H, 6.95; N, 14.95; Cl, 12.25.
NMR (75 MHz, CDCl3) δ 164.5, 157.3, 146.9, 132.7, 111.8, 111.2,
97.6, 68.5, 55.9, 54.8, 54.3, 45.7, 42.7, 31.7, 31.6, 25.9. Anal. Calcd
for C16H22N3O2Cl: C, 59.34; H, 5.85; N, 12.98; Cl, 10.95. Found:
C, 59.09; H, 6.71; N, 12.84; Cl, 11.18.
To a suspension of the free base of 12a (3.52 g, 10.87 mmol) in
methanol (10 mL) was added a solution of HCl (10.87 mmol) in
methanol [prepared by the addition of acetyl chloride (0.76 g, 10.9
mmol) to methanol (10 mL)]. The resulting solution was concen-
trated to give a solid which was redissolved in methanol (5 mL)
and added slowly to diethyl ether (3 L) with rapid stirring. The
resulting suspension was stored at 0 °C for 16 h. Filtration gave
the desired hydrochloride salt of 12a (3.63 g, 88%) as a colorless
solid: mp 103-105 °C; [R]D ) -2.6°; [R]365 ) -6.4° (c ) 0.47
1
g/dL in methanol); IR (MIR) 3323, 3194, 1620, 1594 cm-1; H
NMR (300 MHz, MeOD-d4) δ 7.79 (1H, s), 6.52 (1H, s), 3.98 (1H,
m), 3.93 (3H, s), 3.76 (1H, ddd, J ) 3.0, 7.2, 10.8 Hz), 3.53 (2H,
d, J ) 6.3 Hz), 3.20 (1H, m), 3.10 (1H, td, J ) 11.4, 6.0 Hz), 2.39
(1H, m), 2.30-1.79 (6H, m); 13C NMR (75 MHz, MeOD-d4) δ
167.7, 159.6, 133.2, 111.5, 111.2, 98.5, 72.3, 56.6, 55.8, 55.5, 46.5,
31.2, 31.1, 25.8; HRMS calcd for C16H22N3O2Cl 323.1415, found
323,1400. Anal. Calcd for C16H22N3O2Cl·HCl·0.75H2O: C, 51.41;
H, 6.61; N, 11.24; Cl, 18.97. Found: C, 51.58; H, 6.70; N, 10.94;
Cl, 18.57.
Amide Coupling General Procedure. We have found that
benzamide couplings utilizing 4-amino-5-chloro-2-methoxybenzoic
acid generally proceed very well without acetamide protection of
the 4-amino group. According to this general procedure, to a
solution of carboxylic acid (1 equiv) in DMF (freshly opened) is
added 1,1′-carbonyldiimidazole (1 equiv), which results in a
vigorous gas evolution. After 0.5-1 h at room temperature a
solution of the amine 11 in DMF is added and reaction is stirred
for 2 h at room temperature followed by 2 h at 50 °C. Concentration
affords a residue which is treated with 15% K2CO3 and extracted
with chloroform (3×). The combined organic extracts are washed
successively with water (2×) and brine and dried over sodium
sulfate. Concentration affords the desired product, which may be
purified by crystallization or may be pure enough to carry on
directly to the hydrochloride salt formation as described above for
12a. Alternatively, purification on silica gel eluting with MeOH
(saturated with NH3)/CHCl3 (15/85) affords the pure coupled
material as the free base which is then converted to the hydrochlo-
ride salt.
4-Amino-5-chloro-N-[(1R,7aR)-hexahydro-1H-pyrrolizin-1-yl-
methyl]-2-methoxybenzamide (ent-12a, SC-53117). This com-
pound was prepared from (+)-trachelanthamidine, prepared ac-
cording to the method of Nagao et al.38 and continued as above for
the enantiomeric series beginning with 3-(4-chlorobutyryl)-4(R)-
isopropyl-1,3-thiazolidine-2-thione to afford ent-12a: [R]D
)
+0.5°; [R]365 ) +3.3° (c ) 0.183 g/dL in methanol). Anal. Calcd
for C16H22N3O2Cl·1.2HCl·0.8H2O: C, 50.31; H, 6.54; N, 11.00; Cl,
20.42. Found: C, 50.45; H, 6.62; N, 10.67; Cl, 20.40.
4-Amino-5-chloro-2-ethoxy-N-[(1S,7aS)-hexahydro-1H-pyr-
rolizin-1-ylmethyl]benzamide Hydrochloride (12b). To a solution
of 4-amino-5-chloro-2-methoxybenzoic acid (1 equiv) in DMF was
added 1,1′-carbonyldiimidazole (1 equiv). After 1 h at room
temperature a solution of the amine 5 in DMF was added. After
the reaction was complete, aqueous workup as for 12a and
chromatography on silica gel afforded the requisite amide 12b: 1H
NMR (400 MHz, MeOD-d4) δ 7.77 (1H, s), 6.49 (1H, s), 4.17 (2H,
q, J ) 7 Hz), 3.96-3.91 (1H, m), 3.78-3.73 (1H, m), 3.54-3.52
(2H, m), 3.47-3.41 (1H, m), 3.24 to3.17 (2H, m), 3.13-3.06 (1H,
m), 2.43-2.34 (1H, m), 2.29-2.21 (1H, m), 2.19-2.09 (2H, m),
2.07-1.99 (1H, m), 1.96-1.89 (1H, m), 1.86-1.79 (1H, m), 1.48
(3H, t). Anal. Calcd for C17H24N3O2Cl·HCl·0.5H2O: C, 53.27; H,
6.84; N, 10.96; Cl, 18.50. Found: C, 53.14; H, 6.88; N, 10.87; Cl,
20.07.
N-exo-((4S,7rS)-Tetrahydro-1H-pyrrolizin-4(5H)-yl)imidazo-
[1,2-a]pyridine-8-carboxamide (12g). Imidazol[1,2-a]pyridine-8-
carboxylic acid monohydrochloride 17 (198 mg, 1.00 mmol) and
1,1′-carbonyldiimidizole (178 mg, 1.1 mmol) were dissolved in
DMF (5 mL) and stirred for 1 h. A solution of the aminometh-
ylpyrrolizidine 11 (120 mg, 0.85 mmol) in Et3N (560 µL, 4.0 mmol)
was added to the reaction mixture and stirred for 1 h before
concentrating. The residue was partitioned between CHCl3 and 5%
aqueous K2CO3. The organic layer was dried over Na2SO4 and
concentrated. The residue was purified by preparative thin-layer
chromatography eluting with 20/80 MeOH/CHCl3 containing 0.25%
(1S,7aS)-1-{[(4-Amino-5-chloro-2-methoxybenzoyl)amino]-
methyl}-4-methylhexahydro-1H-pyrrolizinium Iodide (12c). To
a solution of the free base of 12a (32.5 mg, 0.10 mmol) in toluene
(5 mL) was added a solution of MeI (12.5 µL, 0.103 mmol) in

1134 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Becker et al.
NH4OH. The filtrate was concentrated and the residue was dissolved
in CHCl3, filtered through Celite, and concentrated to yield the
imidazopyridine amide (144 mg, 43%) as an oil. This residue was
converted to the pyrrolizidine hydrochloride salt 12g using a mixture
of acetyl chloride/MeOH: IR 1579, 1659, 1697, 3049 and 3325
cm-1. Anal. Calcd for C16H20N4O·2.0H2O·2.2HCl·0.25CHCl3: C,
46.12; H, 6.25; N, 13.24. Found: C, 46.22; H, 6.41; N, 13.43.
N-exo-((4S,7rS)-Tetrahydro-1H-pyrrolizin-4(5H)-yl)-6-chloro-
imidazo[1,2-a]pyridine-8-carboxamide Dihydrochloride (12h,
SC-53606). 6-Chloroimidazo[1,2-a]pyridine-8-carboxylic acid mono-
hydrochloride 19 (1.39 g; 6.0 mmol) and 1,1′-carbonyldiimidizole
(972 mg, 6.0 mmol) were dissolved in DMF (10 mL) and stirred
for 1 h. A solution of aminomethylpyrrolizidine 11 (800 mg, 5.7
mmol) in Et3N (2.5 mL) was added to the reaction mixture and
stirred for 1 h before concentrating. The residue was partitioned
between CHCl3 and 5% aqueous K2CO3. The organic layer was
dried over Na2SO4 and concentrated. The residue was purified by
preparative thin-layer chromatography eluting with 20/80 MeOH/
CHCl3 with 0.25% NH4OH. The filtrate was concentrated and the
residue was dissolved in CHCl3, filtered through Celite, and
concentrated to yield the amide (1.42 g) as an oil. This residue
was converted to the HCl salt using a mixture of acetyl chloride/
MeOH to afford 12h: IR 1652 cm-1; 1H NMR (400 MHz, MeOD-
d4) δ 8.83 (1H, d, J ) 2 Hz), 7.99 (1H, d, J ) 2 Hz), 7.96 (1H, d,
J ) 1.3 Hz), 7.70 (1H, 1.3 Hz), 4.05-3.98 (1H, m), 3.82-3.75
(1H, m), 3.689 (2H, d, J ) 6 Hz), 3.49-3.42 (1H, m), 3.25-3.18
(1H, m), 3.16-3.08 (1H, m), 2.54-2.45 (1H, m), 2.37-2.29 (1H,
m), 2.22-2.12 (2H, m), 2.09 to1.86 (3H, m). Anal.calcd for C16H19-
ClN4O‚H2O·HCl: C, 51.48; H, 5.94; N, 15.01; Cl, 19.00. Found:
C, 51.32; H, 5.64; N, 14.88; Cl, 18.86.
N-exo-((4S,7rS)-Tetrahydro-1H-pyrrolizin-4(5H)-yl)-6-chloro-
imidazo[1,2-a]pyridine-3-methyl-8-carboxamide (12i). A suspen-
sion of 2-aminonicotinic acid 16 (5.0 g; 0.036 mol) and K2CO3
(5.0 g; 0.36 mol) in DMF (50 mL) was heated to reflux, and then
the solution was cooled to ambient temperature. Iodomethane (5.1
g, 2.2 mL, 0.036 mol) was then added to the mixture and the
solution was stirred for 18 h. The mixture was filtered and
concentrated. The residue was filtered through a pad of silica,
eluting with 5/95 EtOH/CH2Cl2 containing 0.1% NH4OH. The
resulting solution was concentrated and the residue was suspended
in Et2O, filtered, and concentrated to yield methyl 2-aminonicotinate
(3.2 g).
The methyl 2-aminonicotinate (800 mg, 0.00525 mol) was
dissolved in MeOH (15 mL). HCl gas was passed over the solution
until the solution was acidic (pH 2) and then the solution was
concentrated. The residue was dissolved in MeOH (15 mL), tert-
butylhypochlorite (570 mg, 5.25 mmol) was added to the reaction
mixture and it was stirred until the yellow color dissipated.
Additional tert-butyl hypochlorite was added until TLC (5/95 EtOH/
CH2Cl2 containing 0.1% NH4OH) indicated that the starting material
was consumed. The reaction mixture was concentrated and the
residue was partitioned between CH2Cl2 and 5% aqueous NaHCO3.
The organic layer was washed with 5% aqueous sodium thiosulfate,
dried over MgSO4, and concentrated. The solid residue was
suspended in 1:1 CH2Cl2/hexane, filtered, washed with hexane, and
suction dried to yield methyl 2-amino-5-chloronicotinate 20 (250
mg, 26%): mp 139-40 °C. Anal. Calcd for C7H7ClN2O2: C, 45.60;
H, 3.78; N, 15.01; Cl, 19.00. Found: C, 44.72; H, 3.75; N, 15.00;
Cl, 19.20.
The methyl 6-chloroimidazo[1,2-a]pyridine-3-methyl-8-carboxy-
late (5.0 g; 0.022 mol) was suspended in 6 N HCl (50 mL) and
heated to reflux for 2 h. The reaction mixture was concentrated to
near dryness. The residue was suspended in acetone and the solid
filtered and washed with acetone to yield 6-chloro-3-methylimidazo-
[1,2-a]pyridine-8-carboxylic acid 21 (4.9 g) as the HCl salt: IR
1704 and 3175 cm-1; MS MH+ calcd for C9H7ClN2O2 211, found
211. Anal. Calcd for C9H7ClN2O2·HCl: C, 43.75; H, 3.26; N, 11.34;
Cl, 28.70. Found: C, 43.54; H, 3.16; N, 11.26; Cl, 28.74.
To a solution of 6-chloro-3-methylimidazo[1,2-a]pyridine-8-
carboxylic acid 21 (247 mg, 1.00 mmol) in DMF (5 mL) was added
1,1′-carbonyldiimidizole (178 mg, 0.011 mol) and the solution
stirred for 1 h. Aminomethylpyrrolizidine 11 (120 mg, 0.85 mmol)
and Et3N (560 µL, 4.0 mmol) were added to the reaction mixture
and the solution was stirred for 1 h before concentrating. The residue
was partitioned between CHCl3 and 5% aqueous K2CO3. The
organic layer was dried over Na2SO4 and concentrated. The residue
was purified by preparative thin-layer chromatography eluting with
20/80 MeOH/CHCl3 containing 0.25% NH4OH. The filtrate was
concentrated and the residue was dissolved in CHCl3, filtered
through Celite, and concentrated to yield an oil (144 mg, 43%).
This residue was converted to the HCl salt of the desired
imidazopyridine amide 12i using a mixture of acetyl chloride/
MeOH: 1H NMR (400 MHz, MeOD-d4) δ 9.10 (1H, d, J ) 2.5
Hz), 8.56 (1H, d, J ) 2.5 Hz), 7.89 (1H, d, J ) 1 Hz), 4.08-4.03
(1H, m), 3.81-3.75 (1H, m), 3.63 (2H, d, J ) 6 Hz), 3.51-3.45
(1H, m), 3.22-3.19 (1H, m), 3.16-3.09 (1H, m), 2.68 (3H, d, J )
1 Hz), 2.52-2.46 (1H, m), 2.35-2.27 (1H, m), 2.25-2.14 (2H,
m), 2.09-2.03 (1H, m), 1.99-1.87 (2H, m). Anal. Calcd for
C7H8N2O2: C, 55.26; H, 5.30; N, 18.41. Found: C, 54.90; H, 5.36;
N, 18.26. Anal. Calcd for C19H26N4O2·1.25H2O·2.2HCl·0.25MeOH:
C, 46.55; H, 6.18; N, 12.41; Cl, 25.12. Found: C, 46.87; H, 5.94;
N, 12.47; Cl, 25.34.
(1R,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethyl 4-Amino-5-
chloro-2-methoxybenzoate Hydrochloride (13a). To solid NaH
(52 mg of 60% dispersion, washed with hexane, 31 mg NaH) was
added a solution of alcohol 9 (185 mg, 1.31 mmol) in DMF (2
mL) at 0 °C. After the effervescence was complete (0.5 h) this
sodium alcoholate was added to the imidazolide of 4-amino-5-
chloro-2-methoxybenzoic acid (330 mg, 1.31 mmol) at 0 °C. [The
imidazolide was prepared by the addition of CDI (656 mg, 4.05
mmol) to 4-amino-5-chloro-2-methoxybenzoic acid (800 mg, 3.97
mmol); aqueous workup and recrystallization from ether/CHCl3
afforded the requisite imidazolide (780 mg, 78%) as colorless
crystals, mp 122-122.5 °C.] The reaction was allowed to warm to
room temperature over 2 h. Water was added and the mixture
extracted with CHCl3. The combined organic extracts were washed
with water (2×) and brine and dried over Na2SO4. Concentration
gave a solid (362 mg) which was purified by chromatography on
silica gel eluting with i-PrOH(NH3)/CHCl3 to afford the free base
ester (129 mg, 30%). To a solution of the free base (117 mg, 0.36
mmol) in MeOH (1.5 mL) was added HCl/MeOH [prepared by
the addition of acetyl chloride (23 uL, 25 mg, 0.36 mmol) to MeOH
(1.5 mL)] to afford the desired monohydrochloride salt of ester
1
13a (98 mg, 72%) as a beige solid: mp 191-193 °C; H NMR
(300 MHz, MeOD-d4) δ 7.73 (1H, s), 6.48 (1H, s), 4.37 (1H, dd,
J ) 11.3, 5.4 Hz), 4.28 (1H, dd, J ) 11.3, 6.6 Hz), 4.06 (1H, m),
3.82 (3H, s), 3.78 (1h, m), 3.48 (1H, m), 3.29-3.10 (2H, m), 2.54
(1H, m), 2.38-1.85 (6H, m); 13C NMR (75 MHz, MeOD-d4) δ
166.3, 162.0, 151.6, 134.0, 110.4, 108.0, 98.8, 71.8, 64.9, 56.3,
55.9, 55.6, 45.3, 31.0, 30.1, 25.6; HRMS calcd for C16H21N2O3Cl
325.1319, found 325.1298. Anal. Calcd for C16H21N2O3Cl·HCl·
0.25H2O: C, 52.54; H, 6.20; N, 7.66; Cl, 19.39. Found: C, 52.44;
H, 6.06; N, 7.66; Cl, 19.18.
Methyl 2-amino-5-chloronicotinate 20 (7.0 g, 0.045 mol) and
2-bromopropionaldehyde (15.8 g, 0.116 mol) were combined in
EtOH (100 mL) and refluxed until TLC (5/95 EtOH/toluene
containing 0.1% NH4OH) indicated that the starting material was
consumed. The reaction mixture was concentrated, and the residue
was triturated with acetone and then partitioned between CH2Cl2
and 5% aqueous K2CO3. The organic layer was dried over MgSO4
and concentrated. The residue was triturated with Et2O to yield
methyl 6-chloro-3-methylimidazo[1,2-a]pyridine-8-carboxylate (5.0
g, 50%) as a solid: IR 1720 cm-1; MS MH+ calcd for C10H9ClN2O2
225, found 225. Anal. Calcd for C10H9ClN2O2: C, 53.47; H, 4.04;
N, 12.47; Cl, 15.78. Found: C, 53.14; H, 4.06; N, 12.37; Cl, 16.03.
(1R,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethyl 6-Chloroim-
idazo[1,2-a]pyridine-8-carboxylate (13b). To NaH (91 mg of 60%,
2.30 mmol, washed with hexane) was added a solution of alcohol
9 (162 mg, 1.15 mmol) in DMF (2 mL) at 0 °C. After 0.5 h the
solution of this sodium salt was added to the imidazolide of
imidazopyridine carboxylic acid [prepared by adding added CDI
(186 mg, 1.15 mmol) to a solution of 6-chloroimidazo[1,2-a]-

5-HT4 Receptor Agonists and Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1135
pyridine-8-carboxylic acid 19 (267 mg, 1.15 mmol) in DMF (2
mL) at 0 °C.] After 0.5 h at 0 °C, the solution was allowed to
warm to room temperature over 3 h. Concentration gave a residue
that was quenched with water (20 mL) and extracted with CHCl3
(3×). The combined organic extracts were washed with water (2×)
and brine and dried over Na2SO4. Concentration gave a pale yellow
oil (210 mg) which was purified by recrystallization from EA to
afford the desired pyrrolizidine ester free base (155 mg, 42%) as
yellow crystals: mp 116-117 °C; IR (MIR) 3100, 1690, 1537,
1494, 1307, 1277 cm-1; 1H NMR (300 MHz, CDCl3) δ 8.65 (1H,
d, J ) 1.8 Hz), 7.88 (1h, d, J ) 1.8 Hz), 7.80 (1H, s), 7.65 (1H,
s), 4.46 (2H, m), 3.36 (1H, q, J ) 6.5 Hz), 3.23 (1H, t, J ) 7.6
Hz) 3.00 (1h, dt, J ) 10.2, 5.9 Hz), 2.60 (2H, m), 2.21 (1H, m),
2.14 (1H, m), 1.98 (1H, m), 1.90-1.73 (3H, m), 1.62 (1H, m). To
a solution of this free base (89.1 mg, 0.28 mmol) was added HCl/
MeOH [prepared by the addition of acetyl chloride (17.8 µL, 19.6
mg, 0.279 mmol) to MeOH (1.5 mL)]. After 1 h at room
temperature, the solution was concentrated and the resulting solid
was triturated with EA and then dried to afford the requisite
imidazopyridine ester 13b (92 mg, 92%) as an off-white powder:
mp 192 °C (dec); IR (MIR) 3400, 3093, 2969, 2479, 1721, 1543,
was chromatographed on silica gel (50 g) eluting with MeOH
(saturated with NH3)/CHCl3 (5/95) to give the free base of 13d
(2.03 g, 87%). To a solution of this free base (2.0 g, 6.70 mmol)
in methanol (10 mL) was added methanolic HCl [prepared by the
addition of acetyl chloride (0.48 mL, 6.7 mmol) to methanol (10
mL) at 0 °C]. Concentration gave a foam which was redissolved
in a minimum amount of methanol and added dropwise to diethyl
ether (700 mL) with vigorous stirring. Filtration gave a beige solid
(2.02 g) which contained the desired indole carboxylate as well as
imidazole hydrochloride. This material was recrystallized from
diethyl ether/ethanol to give the title compound 13d (1.19 g, 60%)
as a colorless solid: 1H NMR (400 MHz, MeOD-d4) δ 8.45 (1H,
d, J ) 6 Hz), 8.00 (1H, s), 7.46 (1H, d J ) 6 Hz), 7.3-7.2 (2H,
m), 4.45-4.34 (2H, m), 4.14-4.08 (1H, m), 3.862 (3H, s), 3.83-
3.77 (1H, m), 3.51-3.45 (1H, m), 3.26-3.13 (2H, m), 2.64-2.55
(1H, m), 2.38-2.30 (1H, m), 2.27-2.15 (2H, m), 2.11-1.93 (3H,
m). Anal. Calcd for C18H22N2O2‚HCl: C, 64.61; H, 6.92; N, 8.37;
Cl, 10.59. Found: C, 64.31; H, 7.24; N, 8.65; Cl, 10.95. Purity by
HPLC 99.9%.
(1R,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethyl 5-Methoxy-
1H-indole-3-carboxylate Hydrochloride (13e). To 5-methoxyin-
dole-3-carboxylic acid (203 mg, 1.06 mmol) dissolved in DMF (2.5
mL) was added 1,1′-carbodiimidazole (172 mg, 1.06 mmol) at
ambient temperature. After 1.5 h alcohol 9 (150 mg, 1.06 mmol)
in DMF (0.5 mL) was added and the reaction was stirred for 16 h.
The solvent was concentrated to afford an oil. Purification on silica
gel (20 g) eluting with MeOH (saturated with NH3)/CHCl3 (10/
90) gave the ester as a solid (120 mg, 36%). To a solution of this
free base (110 mg, 0.35 mmol) was added HCl/MeOH [prepared
by the addition of acetyl chloride (50 µL, 0.70 mmol) to MeOH].
After 1 h at room temperature the solution was concentrated and
the resulting solid was triturated with diethyl ether and then dried
to give the desired hydrochloride salt 13e (56 mg, 46%): 1H NMR
(300 MHz, CD3OD) δ 7.95 (s, 1H), 7.54 (d, 1H), 7.32 (d, 1H),
6.87 (dd, 1H), 4.41 (m, 2H), 4.09 (m, 1H), 3.84 (s, 3H), 3.79 (m,
1H), 3.49 (m, 1H), 3.19 (m, 2H), 2.61 (m, 1H), 2.36 (m, 2H), 2.21
(m, 2H), 2.03 (m, 2H). Anal. Calcd C18H22N2O3·HCl·0.33H2O: C,
60.59; H, 6.68; N, 7.85; Cl, 9.94. Found: C, 60.78; H, 6.54; N,
7.81; Cl, 9.69.
(1R,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethyl 5-Fluoro-1H-
indole-3-carboxylate Hydrochloride (13f). To 5-fluoroindole-3-
carboxylic acid (190 mg, 1.06 mmol) dissolved in DMF (2.5 mL)
was added 1,1′-carbodiimidazole (172 mg, 1.06 mmol) at ambient
temperature. After 1.5 h, alcohol 9 (150 mg, 1.06 mmol) in DMF
(0.5 mL) was added and the mixture stirred for 16 h. Solvent was
concentrated to give the desired ester as an oil. Purification on silica
gel (20 g) eluting with MeOH (saturated with NH3)/CHCl3 (10/
90) gave the ester as a solid (170 mg, 53%). To a solution of this
free base (160 mg, 0.53 mmol) was added HCl/MeOH [prepared
by the addition of acetyl chloride (75 µL, 1.06 mmol) to MeOH].
After 1 h at room temperature, the solution was concentrated and
the resulting solid was triturated with diethyl ether and then dried
to give the desired hydrochloride salt 13f (133 mg, 74%): 1H NMR
(300 MHz, CD3OD) δ 8.05 (s, 1H), 7.69 (dd, 1H), 7.42 (dd, 1H),
6.99 (t, 1H), 4.42 (m, 2H), 4.09 (m, 1H), 3.80 (m, 1H), 3.49 (m,
1H), 3.19 (m, 2H), 2.62 (m, 1H), 2.36 (m, 2H), 2.22 (m, 2H), 2.02
(m, 2H). Anal. Calcd C17H19N2O2F·HCl·0.33H2O: C, 59.32; H,
6.03; N, 8.14; Cl, 10.36. Found: C, 59.58; H, 6.13; N, 8.20; Cl,
10.36.
(1S,7aR)-Hexahydro-1H-pyrrolizin-1-ylmethyl 1H-Indazole-
3-carboxylate (13g). To indazole-3-carboxylic acid (172 mg, 1.06
mmol) dissolved in DMF (3 mL) was added 1,1′-carbodiimidazole
(172 mg, 1.06 mmol) at room temperature and the mixture stirred
for 1.5 h, after which time alcohol 9 (150 mg, 1.06 mmol) in DMF
(0.5 mL) was added and the reaction stirred for 16 h. Solvent was
concentrated to give desired ester as an oil. Purification on silica
gel eluting with MeOH (saturated with NH3)/CHCl3 (5/95) gave
the ester as a solid (205 mg, 68%): 1H NMR (300 MHz, CDCl3)
δ 13.08 (s, 1H), 8.14 (d, 1H), 7.62 (d, 1H), 7.36 (t, 1H), 7.22 (t,
1H), 4.49 (m, 2H), 3.54 (q, 1H), 3.39 (m, 1H), 3.11 (quintet, 1H),
2.68 (m, 1H), 2.29 (m, 1H), 2.15 (m, 1H), 2.02 (septet, 1H), 1.85
1
1304, 1282, 1187 cm-1; H NMR (300 MHz, MeOD-d4) δ 8.99
(1H, d, J ) 1.8 Hz), 8.14 (1H, d, J ) 1.4 Hz), 8.07 (1H, d, J ) 1.1
Hz), 7.83 (1H, s), 4.57 (1H, dd, J ) 11.1, 3.5 Hz), 4.48 (1H, dd,
J ) 11.1, 2.3 Hz), 4.29 (1H, td, J ) 8.8, 0.8 Hz), 3.84 (2H, AB
m), 3.55 (1H, m), 3.22 (1H, m), 2.91 (1H, m), 2.63 (1H, m), 2.46-
1.87 (4H, m); 13C NMR (75 MHz, MeOD-d4) δ 164.3, 142.1, 134.7,
133.1, 131.1, 121.2, 119.6, 116.2, 72.1, 70.7, 55.4, 55.2, 42.7, 30.3,
29.2, 24.6; HRMS calcd for C16H18N3O2Cl 321.1040, found
321.1049. Anal. Calcd for C16H18N3O2Cl·HCl·0.25H2O: C, 53.27;
H, 5.45; N, 11.65; Cl, 19.66. Found: C, 53.06; H, 5.36; N, 11.57;
Cl, 19.54.
1-Methyl-1H-indole-2-carboxylic Acid (1R,7aS)-1-(Hexahy-
dropyrrolizin-1-yl)methyl Ester (13c). To indole 2-carboxylic acid
(186 mg, 1.06 mmol) dissolved in DMF (2.5 mL) was added 1,1′-
carbonyldiimidazole (172 mg, 1.06 mmol) at ambient temperature
and the mixture stirred. After 1.5 h, alcohol 3 (150 mg, 1.06 mmol)
in DMF (0.5 mL) was added and the reaction was stirred for 16 h.
Solvent was evaporated to give the desired ester as an oil.
Purification on silica gel eluting with MeOH (saturated with NH3)/
CHCl3 (10/90) gave the ester (170 mg, 53%) as a solid: 1H NMR
(300 MHz, CD3OD) δ 7.68 (m, 1H), 7.37 (d, 2H), 7.35 (d, 1H),
7.16 (t, 1H), 7.10 (s, 1H), 4.64 (dd, 2H), 4.09 (m, 1H), 3.37 (q,
1H), 3.24 (t, 1H), 3.00 (m, 1H), 2.61 (m, 2H), 2.20-1.77 (m, 6H),
1.964 (septet, 1H). To a solution of this free base (150 mg, 0.50
mmol) was added HCl/MeOH [prepared by the addition of acetyl
chloride (72 µL, 1.00 mmol) to MeOH]. After 1 h at room
temperature the solution was concentrated and resulting solid was
triturated with diethyl ether and then dried to give the desired
hydrochloride salt 13c (140 mg, 83%): 1H NMR (300 MHz, CD3-
OD) δ 7.64-7.56 (d, 1H), 7.49 (d, 1H), 7.35 (d, 1H), 7.33 (s, 1H),
7.12 (t, 1H); 4.44 (qq, 2H), 4.10 (m, 1H), 4.05 (s, 3H), 3.82 (m,
1H), 3.49 (m, 1H), 3.26-3.18, (m, 2H); 2.62 (m, 1H), 2.36 (m,
2H), 2.21-2.29 (m, 2H), 1.98-2.02 (m, 2H); HRMS calcd for
C18H22N2O2 298.1673, found 298.1686.
(1R,7aS)-Hexahydro-1H-pyrrolizin-1-ylmethyl 1-Methyl-1H-
indole-3-carboxylate (13d). To N-methylindole-3-carboxylic acid53
(2.0 g, 11 mmol) in DMF (10 mL) was added 1,1′-carbonyldiim-
idazole (1.78 g, 11 mmol) and the solution was stirred at room
temperature for 3 h. Water (20 mL) and ice (30 g) were added,
and the mixture was extracted with chloroform (3×). The combined
organic extracts were washed with brine and dried over magnesium
sulfate. Concentration gave the intermediate imidazolide (2.4 g,
93%), which was used directly.
To a suspension of sodium hydride (312 mg of 60% NaH, 7.8
mmol; washed with hexane) in DMF (5 mL) at 0 °C was added a
solution of alcohol 9 (1.1 g, 7.8 mmol) in DMF (6 mL). The
resulting mixture was stirred at 0 °C for 0.5 h, after which time a
solution of the imidazolide (1.76 g, 7.8 mmol) in DMF (5 mL)
was added. The reaction was allowed to warm to room temperature
for 16 h. The mixture was then concentrated to give a solid which

1136 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
Becker et al.
(m, 4H), 1.67 (s, 1H); HRMS calcd for C16H19N3O2 285.1474, found
285.1500. Anal. Calcd for C16H19N3O2·0.5H2O: C, 65.29; H, 6.85;
N, 14.28. Found: C, 65.39; H, 6.56; N, 14.01. To a solution of
this free base (160 mg, 0.56 mmol) was added HCl/MeOH
[prepared by the addition of acetyl chloride (80 µL, 1.12 mmol) to
MeOH]. After 1 h of stirring at room temperature the solution was
concentrated and the resulting solid was triturated with diethyl ether
and then dried to give the desired hydrochloride salt 13g (137 mg,
76%): 1H NMR (400 MHz, MeOD-d4) δ 8.14 (1H, d, J ) 8.2
Hz), 6.62 (1H, d, 8.5 Hz), 7.46 (1H, t, J ) 8 Hz), 7.32 (1H, t, J )
8 Hz), 4.59-4.49 (2H, m), 4.16 (1H, t, J ) 6 Hz), 3.87-3.82 (1H,
m), 3.54-3.48 (2H, m), 3.27-3.17 (1H, m), 2.73-2.64 (1H, m),
2.43-2.36 (1H, m), 2.32-2.17 (2H, m), 2.10-1.98 (3H, m). HRMS
calcd for C16H19N3O2 285.1496, found 285.1511. Anal. Calcd for
C16H19N3O2·HCl·0.15H2O: C, 59.22; H, 6.31; N, 12.95; Cl, 10.93.
Found: C, 59.13; H, 6.34; N, 12.88; Cl, 11.06.
H2O: C, 57.50; H, 7.32; N, 14.12; Cl, 8.93. Found: C, 57.44; H,
7.42; N, 13.87; Cl, 8.93.
exo-2,3-Dihydro-3,3-dimethyl-N-[(hexahydro-lH-pyrrolizin-
1S-yl)methyl]-2-oxo-1H-indole-1-carboxamide, Hydrate Hydro-
chloride (15b). To sodium hydride (214 mg, 5.6 mmol, washed
twice with hexane) suspended in THF (1 mL) was added 1,3-
dihydro-3,3-dimethyl-2H-indol-2-one55 (226 mg, 1.4 mmol) and the
reaction was stirred for 10 min. The resulting suspension was added
to a solution of 20% phosgene in toluene (5.50 mL, 11.2 mmol) in
THF (5 mL) and the reaction was stirred for 1 h. The reaction
mixture was filtered through Celite and concentrated in vacuo to
give an oil. To a solution of the oil in THF (5 mL) was added a
solution of aminomethylpyrrolizidine 11 (200 mg, 1.4 mmol) and
triethylamine (200 uL, 1.4 mmol) in THF (2 mL) and the reaction
was stirred for 18 h. The solution was then diluted with chloroform,
washed with saturated K2CO3 solution, dried over K2CO3, filtered,
and concentrated in vacuo to give crude desired product as an oil.
Purification on silica gel eluting with 10% CH3OH(NH3)/CHCl3
gave the free base of the benzimidazolone (193 mg, 42%) as a
solid: HRMS calcd for C19H25N3O2 327.1947, found 327.1930.
Anal. Calcd for C19H25N302·0.4H2O: C, 68.19; H, 7.77; N, 12.56.
Found: C, 68.06; H, 7.74; N, 12.44. The free base (180 mg, 0.550
mmol) was converted to the hydrochloride salt by treatment with
methanolic HCl to give the hydrochloride salt 15b (128 mg, 64%)
as a solid: 1H NMR (400 MHz, MeOD-d4) δ 8.10 (1H, d, J ) 2
Hz), 7.33 (1H, d, J ) 2 Hz), 7.28 (1H, t, J ) 2 Hz), 7.19 (1H, t,
J ) 2 Hz), 4.02-3.96 (1H, m), 3.79-3.74 (1H, m), 3.53 (2H, d, J
) 6.4 Hz), 3.49-3.43 (1H, m), 3.24-3.18 (1H, m), 3.15 to3.08
(1H, m), 2.48-2.38 (1H, m), 2.33-2.25 (1H, m), 2.22-2.13 (2H,
m), 2.08-1.02 (1H, m), 1.99-1.92 (1H, m), 1.90-1.83 (1H, m),
1.41 (6H, s); MS calcd for C19H25N3O2 327.1947, found 327.1939.
Anal. Calcd for C19H25N3O2·HCl·0.25H2O: C, 61.95; H, 6.98; N,
11.41; Cl, 9.62. Found: C, 61.54; H, 6.92; N, 11.36; Cl, 9.90.
Imidazol[1,2-a]pyridine-8-carboxylic Acid Monohydrochlo-
ride (17). 2-Aminonicotinic acid 16 (14.1 g; 0.102 mol) and
chloroacetaldehyde [45% aqueous solution] (8.6 g, 100 mmol) were
dissolved in EtOH (100 mL) and heated to reflux. The reaction
was monitored by TLC with 30% MeOH/CH2Cl2/1.0% HOAc, and
additional chloroacetaldehyde was added until the starting material
was consumed. The reaction mixture was concentrated and the solid
filtered, washed with EtOH, and suction dried to yield imidazol-
[1,2-a]pyridine-8-carboxylic acid monohydrochloride 17 (17.5 g,
88%) as a solid: IR 1582, 1651, 1687, 3049 and 3320 cm-1. Anal.
Calcd for C8H6N2O2·HCl: C, 48.38; H, 3.55; N, 14.10; Cl, 17.85.
Found: C, 48.16; H, 3.59; N, 13.95; Cl, 17.50.
6-Chloroimidazo[1,2-a]pyridine-8-carboxylic Acid (19). Meth-
yl 2-amino-5-chloronicotinate 18 (7.0 g; 0.045 mol) and 45%
aqueous chloroacetaldehyde (10.5 g; 0.06 mol) were dissolved in
EtOH (100 mL) and heated to reflux for 3 h. The reaction mixture
was concentrated and the solid filtered, washed with EtOH, and
suction dried to yield methyl 6-chloroimidazo[1,2-a]pyridine-8-
carboxylate (9.5 g, 80%) as a solid. Anal. Calcd for C9H7N2O2·
HCl·H2O C, 40.78; H, 3.80; N, 10.57; Cl, 26.75. Found: C, 40.68;
H, 3.74; N, 10.19; Cl, 26.85.
The methyl 6-chloroimidazo[1,2-a]pyridine-8-carboxylate (9.5
g, 0.036 mol) was combined with 6N HCl (100 mL) and heated to
reflux for 2 h. The reaction mixture was concentrated to near
dryness. The residue was suspended in acetone and the solid filtered
and washed with acetone to yield 6-chloroimidazo[1,2-a]pyridine-
8-carboxylic acid 19 hydrochloride salt (6.3 g, 75.5%) as a solid:
IR 1679 and 3281 cm-1. Anal. Calcd for C8H5N2O2·HCl: C, 41.23;
H, 2.60; N, 12.02; Cl, 30.42. Found: C, 40.95; H, 2.43; N, 12.16;
Cl, 30.67.
(1S,7aR)-Hexahydro-1H-pyrrolizin-1-ylmethyl 1-Methyl-1H-
indazole-3-carboxylate (13h). To N-methylindazole-3-carboxylic
acid (186 mg, 1.06 mmol) dissolved in DMF (2 mL) was added
1,1′-carbodiimidazole (172 mg, 1.06 mmol) at ambient temperature.
After stirring for 1.5 h, the alcohol 9 (150 mg, 1.06 mmol) in DMF
(0.5 mL) was added and the reaction stirred for 16 h. Solvent was
removed in vacuo to give an oil. Purification on silica gel eluting
with MeOH (saturated with NH3)/CHCl3 (10/90) gave the ester as
a solid (196 mg, 62%): 1H NMR (300 MHz, CDCl3) δ 8.20 (d,
1H), 7.47 (d, 2H), 7.34 (t, 1H), 4.49 (m, 2H), 4.18 (S, 3H), 3.37
(q, 1H), 3.21 (m, 1H), 2.99 (m, 1H), 2.59 (m, 2H), 2.37 (m, 1H),
2.16 (m, 1H), 2.16 (m, 1H), 1.99 (septet, 1H), 1.81 (m, 2H), 1.61
(sextet, 1H); HRMS calcd for C17H21N3O2 299.1616, found
285.1632. Anal. Calcd for C17H21N3O2·0.2H2O: C, 67.39; H, 7.12;
N, 13.87. Found: C, 67.54; H, 6.99; N, 13.74. To a solution of
this free base (168 mg, 0.56 mmol) was added HCl/MeOH
[prepared by the addition of acetyl chloride (80 µL, 1.12 mmol) to
MeOH]. After 1 h at room temperature the solution was concen-
trated and the resulting solid was triturated with diethyl ether and
then dried to give the desired hydrochloride salt 13h (174 mg,
1
92%): HRMS calcd C17H21N3O2 299.1649, found 299.1648; H
NMR (400 MHz, CDCl3) δ 8.12 (d, 1H), 7.69 (d, 1H), 7.51 (t,
1H), 4.52 (qq, 2H), 4.16 (S, 3H), 4.13 (m, 1H), 3.84 (m, 1H), 3.50
(m, 1H), 3.22 (m, 2H), 2.69 (m, 1H), 2.40 (m, 1H), 2.22 (m, 5H),
2.05 (m, 1H). Anal. Calcd C17H21N3O2·HCl·0.15H2O: C, 60.31;
H, 6.64; N, 12.41; Cl, 10.47. Found: C, 60.28; H, 6.76; N, 12.40;
Cl, 10.71.
cis-N-[(Hexahydro-1H-pyrrolizin-l-yl)methyl]-2,3-dihydro-3-
(1-methylethyl)-2-oxo-lH-benzimidazole-l-carboxamide (15a). A
dispersion of 60% NaH/mineral oil (80 mg, 0.002 mol) was washed
with hexane and suspended in THF. Solid 1,3-dihydro-l-(l-meth-
ylethyl)-2H-benzimidazol-2-one54 (176 mg, 1.00 mmol) was added
to the suspension. This mixture was stirred for 15 min before being
added to a solution of 2.5 mL (0.004 mol) of 20% phosgene in
toluene/2.5 mL THF. The resulting mixture was filtered through
Celite and concentrated. The residue was dissolved in THF (5.0
mL) and a solution of aminomethylpyrrolizidine 11 (140 mg, 1.0
mmol) was added in Et3N (0.5 mL). This mixture was stirred for
1 h, filtered, and concentrated. The residue was purified by
preparative thin-layer chromatography eluting with 30/70 MeOH/
CHCl3 containing 0.25% NH4OH. The product was rinsed from
the silica with 9/95 NH4OH/MeOH. The filtrate was concentrated
and the residue was dissolved in CHCl3, filtered through Celite,
and concentrated to afford the benzimidazolone 15a (157 mg, 50%).
The product was converted to the HCl salt by dissolving 36 µL of
acetyl chloride in 5.0 mL of MeOH and adding this solution to the
product and then concentrating to dryness: 1H NMR (400 MHz,
MeOD-d4) δ 8.11 (1H, dd, J ) 8.1, 0.8 Hz), 7.32 (1H, dd, J ) 8.1,
0.8 Hz), 7.22 (1H, d of t, J ) 8.1, J ) 1 Hz), 7.13 (1H, d of t, J
) 8.1, J ) 1 Hz), 4.70 (1H, sep, J ) 7 Hz), 4.04-3.96 (1H, m),
3.82-3.74 (1H, m), 3.58-3.55 (2H, m), 3.50-3.42 (1H, m), 3.25-
3.18 (1H, m), 3.16-3.08 (1H, m), 2.49-2.39 (1H, m), 2.36-2.26
(1H, m), 2.25-2.17 (1H, m), 2.11-2.03 (1H, m), 2.01-1.93 (1H,
m), 1.91 to1.82 (1H, m), 1.54 (2H, d, J ) 7 Hz); MS MH+ calcd
for C19H26N4O2 343, found 343. Anal. Calcd for C19H26N4O2·HCl·
(+)-4-Amino-5-chloro-N-(hexahydro-2,5â-methano-1H-3aS,-
3ar,6ar-cyclopenta[c]pyrrol-4r-yl)-2-methoxybenzamide, Mono-
hydrochloride (6, SC-52491). Compound 6 was prepared initially
by synthesis involving resolution as previously described41 and then
via an enantioselective synthesis commencing with an asymmetric
Diels-Alder reaction.52
Serotonin 5-HT3 Receptor Binding. Displacement of [3H]-
GR65630 from brain cortex obtained from male Wistar rats was

5-HT4 Receptor Agonists and Antagonists
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3 1137
done by the method of Kilpatrick.56 Cortical membrane preparations
(0.04 mg) were incubated with 0.2 nM [3H]GR65630 in the presence
or absence of graded concentrations test compound for 60 min at
22 °C. A 1 µM solution of tropisetron was used for nonspecific
binding to the membranes. Membranes were filtered, washed three
times, and counted to obtain binding displacement curves and
determine specific binding of [3H]GR65630 to the 5-HT3 receptor.
Serotonin 5-HT4 Receptor Binding. Serotonin 5-HT4 receptor
binding in guinea pig striatum utilizing [3H]GR113,808 was
performed by MDS Pharma Services (formerly Panlabs Taiwan)
according to the method of Grossman and Kilpatrick.57
Selectivity Binding Assays. The following radioligands were
used for receptor profiling studies: [3H]5HT for 5-HT1-like
receptors, [3H]ketanserin for 5-HT2 receptors, [3H]SCH23390 for
D1 receptors, [3H]spiperone for D2 receptors, and [3H]prazosin for
R1-adrenergic receptors.
kg) to a group of three mice. Thirty minutes later, a 5-HT (0.25
mg/kg iv)-induced bradycardia was recorded in pentobarbital-
anesthetized animals. A greater than 50% (>50) reduction in the
bradycardic response relative to vehicle-treated control mice is
considered significant.
Antral Motility in Conscious Fasted Dogs. Gastric antral
contractile activity is stimulated by prokinetic drugs which enhance
gastric emptying of solid food.59 This contractile activity is thought
to enhance gastric emptying by more rapidly reducing food particle
size for passage through the pylorus. The ability of the test
compound to increase the frequency and/or amplitude of the
contractile activity is therefore a measure of GI prokinetic activity
of compounds. Mongrel dogs of either sex weighing 15-25 kg
were surgically implanted with strain gauge force transducers on
the gastric antrum at 6, 4, and 2 cm from the gastroduodenal
junction. Strain gauge transducers were also implanted on the ileum
and proximal colon. Silver monopolar electrodes were implanted
on the jejunum. The dogs were allowed at least 2 weeks to recover
and were trained to stand quietly in Pavlov slings. Dogs were fasted
for 18-24 h prior to each experiment to record a pattern of antral
contractile activity characteristic of the fasted state called the
migrating motor complex (MMC). The period of the MMC cycle
is approximately 90-120 min and consists of 45-60 min of motor
quiescence (phase I), 30-45 min of intermittent activity (phase
II), and 10-15 min of intense contractile activity (phase III). A
control MMC period is recorded prior to compound administration
to obtain the length of the quiescent phase I period. Compound is
given intravenously at the end of phase III of the control MMC
cycle, and the subsequent phase I period is examined for the ability
of the compound to produce contractions. A compound is consid-
ered active if it produces any contractile activity during the normally
quiescent phase I. A motility index of cumulative activity consisting
of frequency and amplitude components is used to quantitatively
describe the total contractile activity of a time period after
compound administration equal to the control MMC period.
Canine Gastric Emptying In Vivo Model. Determination of
the effects of test compounds on gastric emptying of solid meals
in nonsedated dogs was done in separate experiments in an R2-
adrenergic model of gastroparesis as described by Gullikson et
al.45,60 Dogs weighing 15-25 kg were trained to stand quietly in
Pavlov slings for 3-4 h, and consistent control emptying responses
were obtained prior to use in gastric emptying experiments with
the test compounds. The solid meal consisted of two cooked,
scrambled eggs which were divided into 1 cm sized pieces and
mixed with beef stew. Then 1 mCi of Tc-99m sulfur colloid was
incorporated into the eggs prior to cooking. The dogs were fasted
for at least 24 h prior to the study and were fed the solid meal by
intragastric tube. To delay normal gastric emptying, 0.030 mg/kg
of the R2-adrenergic agonist 2-methyl-3-[(2E)-pyrrolidin-2-yl-
ideneamino]phenol was administered immediately following the
meal. The test compounds were given via intravenous injection 45
min prior to feeding.
A Siemens 370 ZLC γ-camera with a high-resolution low-energy
collimator was used to acquire left lateral images during emptying
studies. Acquisition times were 3 min/frame for 180 min of solid
emptying. Disappearance of contents from the stomach region of
interest was plotted over time to obtain emptying curves. The
amount of solid meal remaining in the stomach at the end of each
experiment and the fractional solid emptying rate (% emptied/min)
were calculated from linear regression equations describing solid
emptying. Emptying measurements obtained from one replicate of
each drug treatment were compared to the means of at least three
control responses for the R2-adrenergic agonist in solid emptying
studies.
Ames Mutagenicity Assay. Pyrrolizidine benzamide 12a was
tested for mutagenic activity in a GLP study using the Ames
salmonella/microsome assay with five strains of Salmonella typh-
imurium (TA1535, TA100, TA1538, TA97, and TA98) in the
presence and absence of a rat liver homogenate metabolic activation
system (S9) over test article concentrations ranging from 7.2 to
3600 µg/plate. Significant test-article-related increases of 4 times
In Vitro Functional Assay for Serotonin 5-HT4 Agonism in
the Rat TMM (Tunica Muscularis Mucosae) Assay. Serotonin
5-HT4 agonism was measured in the rat esophagus in vitro
preparation as reported by Baxter et al.2,57 Agonist activity was
determined by utilizing relaxation of carbachol-contracted rat tunica
muscularis mucosae. One 2-cm segment of intrathoracic esophagus
proximal to the diaphragm was removed from male rats weighing
approximately 300 g, and the outer muscle layers were removed.
The inner tunica muscularis mucosa was mounted under 0.2-0.3
g of tension in a tissue bath containing oxygenated Tyrode’s solution
at 37 °C. Cortisterone acetate (30 µM) and fluoxetine (1 µM) were
included in the buffer to prevent uptake of serotonin, as well as
pargyline (10 µM) to inhibit monoamine oxidase. Following a 30-
min equilibrium period, tissues were isometrically contracted with
carbachol (3 µM) to obtain a tonic contraction. A stable plateau
was obtained within 20 min when test compound was added
cumulatively to relax the muscle strip. EC50 values were obtained
for each agonist in tissues from rats.
5-HT4 Receptor Antagonism in the Rat TMM Assay. An-
tagonist activity of compounds at 5-HT4 receptors was determined
in a manner similar to the in vitro agonism activity described by
Gullikson et al.39 Cumulative dose-response curves for agonists
interacting with 5-HT4 receptors of rat TMM were done according
to the method of Baxter et al.2 Male Sprague-Dawley rats (300-
400 g) from Charles River Laboratories (Wilmington, MA) were
asphyxiated with CO2 and the TMM were isolated from 2-cm
segments of rat esophagus obtained orad to the diaphragm. The
TMM was mounted in a 37 °C tissue bath under 2-3 mN of tension
for 60 min before the study. The tissues were suspended in and
washed with continuously oxygenated (95% O2/5% CO2) Tyrode’s
buffer which contained fluoxetine (1 µM) and corticosterone (30
µM) to prevent tissue uptake of 5-HT, methysergide (1 µM) to
block 5-HT1 and 5-HT2 receptors, and pargyline (100 µM) to
prevent oxidation of 5-HT by monoamine oxidase. Selected
concentrations of antagonist were added to the tissue bath 5 min
after TMM were contracted with 3 µM of carbachol. Relaxant
responses to cumulative additions of 5-HT or 5-HT4 agonists (from
10-10 to 10-5 M in half-log increments at 2.5-min intervals) were
started 20 min after addition of the carbachol. Agonist ability to
relax TMM was expressed relative to the maximum relaxant
response by the agonist in the absence of antagonist. EC50 values
were calculated as the concentrations causing 50% of this maximal
effect. EC50 ratios (dose ratios) from the agonist concentration-
response curves in the absence and presence of three increasing
concentrations of antagonist for each tissue were calculated. Dose
ratios from at least four tissues for each of the three antagonist
concentrations were used for Schild plot analysis to determine the
mean pA value ( SEM of the antagonist.58 The pA2 value for single
antagonist concentrations in individual tissues was also calculated
using the method of MacKay: pA2 ) -log(antagonist concentration
[mol/1]) + log(DR - 1). A mean pA2 value ( SEM was calculated
from the individual pA2 values from at least four tissues for a single
concentration of the antagonist.
von Bezold-Jarisch Reflex Assay. According to the method
of Saxena and Lawang,40 the test sample was administered ip (mg/

1138 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 3
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Supporting Information Available: Results from elemental
analysis data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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