A multivalent approach to the design and discovery of orally efficacious 5-HT4 receptor agonists

Article abstract of DOI:10.1021/jm900881j

5-HT₄ receptor agonists such as tegaserod have demonstrated efficacy in the treatment of constipation predominant irritable bowel syndrome (IBS-C), a highly prevalent disorder characterized by chronic constipation and impairment of intestinal p

Full text of DOI:10.1021/jm900881j

5330 J. Med. Chem. 2009, 52, 5330–5343
DOI: 10.1021/jm900881j
A Multivalent Approach to the Design and Discovery of Orally Efficacious 5-HT4 Receptor Agonists
R. Murray McKinnell,*,† Scott R. Armstrong,‡ David T. Beattie,‡ Seok-Ki Choi,† Paul R. Fatheree,† Roland A. L. Gendron,†
Adam Goldblum,† Patrick P. Humphrey,† Daniel D. Long,† Daniel G. Marquess,† J. P. Shaw,§ Jacqueline A. M. Smith,
S. Derek Turner,† and Ross G. Vickery
†Department of Medicinal Chemistry and ‡Department of Pharmacology and §Department of Drug Metabolism and Pharmacokinetics and
Department of Molecular and Cellular Biology, Theravance, Inc., 901 Gateway Boulevard, South San Francisco, California 94080
Received June 16, 2009
5-HT4 receptor agonists such as tegaserod have demonstrated efficacy in the treatment of constipation
predominant irritable bowel syndrome (IBS-C), a highly prevalent disorder characterized by chronic
constipation and impairment of intestinal propulsion, abdominal bloating, and pain. The 5-HT4
receptor binding site can accommodate functionally and sterically diverse groups attached to the amine
nitrogen atom of common ligands, occupying what may be termed a “secondary” binding site. Using a
multivalent approach to lead discovery, we have investigated how varying the position and nature of the
secondary binding group can be used as a strategy to achieve the desired 5-HT4 agonist pharmacological
profile. During this study, we discovered the ability of amine-based secondary binding groups to impart
exceptional gains in the binding affinity, selectivity, and functional potency of 5-HT4 agonists.
Optimization of the leads generated by this approach afforded compound 26, a selective, orally
efficacious 5-HT4 agonist for the potential treatment of gastrointestinal motility-related disorders.
Introduction
ileum and colon.13 Subsequently, the role of 5-HT4 agonist
activity in promoting gastrointestinal motility has become
more fully appreciated. For example, activation of 5-HT4
receptors on motorneurons and interneurons within the gut
wall is associated with facilitation of cholinergic and nona-
drenergic, noncholinergic neurotransmission. Release of acet-
ylcholine, substance P, and calcitonin gene-related peptide
results in the coordinated propulsion of contents along the
gastrointestinal tract.14 Additionally, 5-HT4 receptors are
expressed on smooth muscle cells; their activation results in,
for example, esophageal relaxation in rats15 and inhibition of
human colonic circular muscle contractile activity.16 Further-
more, activation of 5-HT4 receptors on enteric neurons or
enterocytes promotes fluid secretion in the gastrointestinal
tract in a variety of species, including humans.17
Serotonin (5-hydroxytryptamine, 5-HTa) mediates its di-
verse pharmacological functions via activation of at least
seven 5-HT receptor families (5-HT1-5-HT7, with multiple
subtypes for the majority), differentiated on the basis of their
location, structure, function, and transductional pathways.1,2
In mammals, approximately 95% of 5-HT in the body is
localized in the gastrointestinal tract, primarily in enterochro-
maffin cells. The 5-HT4 receptor was discovered in cultured
mouse embryonic colliculi neuronal cells,3 and its expression
has since been demonstrated in a range of human tissues,
including the brain,4 gut,5 heart,6 adrenal cortex,7 and blad-
der.8 The 5-HT4 receptor belongs to the superfamily of seven-
transmembrane G protein-coupled receptors (GPCRs),
which, together with its potential role in many central and
peripherally mediated disorders (irritable bowel syndrome,9
gastroparesis,10 Alzheimer’s disease,11arrhythmia12), has
made it an attractive target for drug discovery.
Consequently, much effort has been devoted to the dis-
covery and development of selective 5-HT4 receptor agonists
for the treatment of chronic constipation and constipation-
predominant irritable bowel syndrome (IBS-C), a highly
prevalent disorder characterized by impairment of intestinal
propulsion, abdominal bloating, and discomfort.18 Cisapride,
a 5-HT4 receptor agonist (albeit with significant activity at
other 5-HT receptor subtypes), was frequently prescribed for
this indication but was withdrawn in 2000 following its
association with an increased risk of mortality as a conse-
Of relevance to the treatment of gastrointestinal disorders
was the initial observation by Craig and Clark that the
chloroaniline class of 5-HT4 agonists such as Renzapride
(Figure 1) activates the peristaltic reflex of the guinea pig
*To whom correspondence should be addressed. Phone: þ1-650-808-
6039. Fax: þ1-650-808-6120. E-mail: mmckinnell@theravance.com.
a Abbreviations: IBS-C, constipation predominant irritable bowel
syndrome; 5-HT, 5-hydroxytryptamine; GPCR, G-protein coupled
ꢀ
quence of inhibition of the human ether-a-go-go-related gene
(hERG) potassium ion channel.19 Prucalopride, a highly
selective 5-HT4 agonist, was shown in several clinical trials
to significantly increase the frequency of bowel movements,
improve stool consistency, and accelerate colonic transit in
constipated patients.10,20 Prucalopride was recently licensed
by Movetis from Janssen with the objective of gaining regis-
tration in Europe.21 Tegaserod, an indole-based 5-HT4
ꢀ
receptor; hERG, human ether-a-go-go-related gene; HEK, human
embryonic kidney; GR113808, 1-methyl-1H-indole-3-carboxylic acid
1-(2-methanesulfonylamino-ethyl)-piperidin-4-ylmethyl ester; GR65630,
3-(4-methyl-1H-imidazol-5-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone;
cAMP, cyclic adenosine monophosphate; SAR, structure-activity re-
lationship; CHO, Chinese hamster ovary; RLM, rat liver microsome;
AUC, area under the curve; F, oral bioavailability.
r
pubs.acs.org/jmc
Published on Web 08/10/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5331
Figure 1. 5-HT4 agonists.
receptor agonist, was approved in the United States for IBS-C
and chronic constipation and has been shown to reduce total
colonic transit time in healthy subjects22 as well as improving
multiple symptoms in patients with IBS-C.23 Tegaserod has
an oral bioavailability of 10% in humans,24 is dosed twice-
daily,25 andissubjecttoa significantnegativefoodeffect onits
oral absorption.23 Tegaserod also lacks selectivity for the
5-HT4 receptor, having significant affinity for a number of
Figure 2. 5-HT4 agonist pharmacophore.
of 5-HT4 agonist primary binding groups,35,36 relatively few
studies have focused on variation of the secondary binding
group as a means to achieve the desired pharmacokinetic and
pharmacological profile for an orally bioavailable prokinetic
agent.37,38 In light of this, a multivalent approach to the lead
discovery efforts was adopted in which functionally diverse
secondarybindinggroupswere attachedvia linkersof variable
length to selected 5-HT4 agonist primary binding groups, with
the goal of enhancing their potency, oral pharmacokinetics,
and selectivity over the 5-HT3 receptor (5-HT3 antagonism is
known to reduce gastrointestinal motility in man and may
partially attenuate the clinical benefit of 5-HT4 agonists).39 In
addition, secondary binding groups, which resulted in mini-
mal inhibition of the hERG potassium ion channel, were
targeted, as this would potentially minimize the risk of the
cardiac arrhythmia issues associated with cisapride.
other 5-HT receptor subtypes (i.e., 5-HT1D, 5-HT2A, 5-HT2B
,
and 5-HT7).26 Recently, tegaserod was removed from the U.S.
market on the basis of a possible association with ischemic
cardiovascular events.27
Herein, we describe the application of a multivalent ap-
proach to the discovery and optimization of potent, selective,
and orally bioavailable 5-HT4 agonists for the treatment of
gastrointestinal disorders characterized by reduced motility.
Chemical Strategy
Multivalent interactions have been defined as those in-
stances where a ligand has two or more exclusive binding
domains that can bind to two or more distinct sites either on
the same receptor or on two distinct receptors.28,29 This mode
of binding can improve the ligands association/dissociation
equilibrium at a particular receptor and also allow the ligand
to better differentiate between two or more closely related
receptors. Thus, a multivalent approach, when applied to the
design of bioactive molecules, can afford compounds with
enhanced potency and selectivity for the desired target. In the
most simple case, a molecule can consist of two distinct
binding groups (“primary” and “secondary”), which bind to
proximal primaryand secondary binding sites, respectively. In
the case of 5-HT4 receptor ligands, the primary binding group
may be regarded as an appropriately substituted aromatic
system connected through a coplanar amide bond to a con-
formationally restricted amine. Suitable aromatic systems
include 4-amino-5-chloro-2-methoxy benzoic acid,30 N-alkyl
quinolones,31 and N-alkyl indazoles,32 among others. Multi-
ple conformationally restricted amines have been employed,
such as tropane,32 quinuclidine,33 and pyrrolizidine,34
Chemistry
Synthesis of Primary Binding Groups. For the synthesis of
primary binding groups 1-3a-d and 5a-d, the requisite
aromatic systems were prepared using literature procedures
and coupled to commercially available BOC-protected dia-
mines using one of several standard amide bond-forming
methods, followed by removal of the BOC group using
trifluoroacetic acid (Scheme 1). In the case of benzimidazo-
lones 4a-d, 1-isopropyl-1,3-dihydro-2H-benzimidazol-
2-one was first converted to an activated carbamate using
p-nitrophenylchloroformate then treated with the appropri-
ate amine (Scheme 2).
Attachment of Linkers and Secondary Binding Groups.
Compounds 6a-l and 7a-l were furnished by simple
N-alkylation of compounds 1c and 2a, respectively, using
commercially available alkyl bromides and potassium iodide
in dimethylformamide (method F, Scheme 3). Compounds
that contained amines as secondary binding groups (6m-u,
7m-u, 8a-e, 9a-e, and 10-23) were synthesized by one of
In marked contrast to the majority of 5-HT3 ligands, several
5-HT4 ligands have demonstrated an ability to accommodate
voluminous substituents attached to the amine nitrogen atom,
occupying what may be described as a secondary binding site
(Figure2).34 While much effort has focused on the optimization

5332 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
McKinnell et al.
Scheme 1a
a Reagents and conditions: Method A: (a) CDI, DMF; (b) TFA, DCM. Method B (a) EDC, iPr2NEt, DMF; (b) TFA, DCM. Method C: (a) EDC,
HOBt, iPr2NEt, DMF; (b) TFA, DCM. Method D: (a) SOCl2, NaOH, PhMe, H2O (b) TFA, DCM.
Scheme 2a
treatment with sodium hydroxide underwent ring closure to
form 1-methylsulfonyl-4-(oxiranylmethyl)piperazine. This
was then treated with compound 2a to afford the desired
products (Scheme 4).
Results and Discussion
Selection of Primary Binding Groups. In this multivalent
approach, the first goal was to identify suitable primary
binding groups from which to rapidly access and explore the
putative secondary binding site. As such, a systematic in
vitro evaluation of the relative binding affinity (pKi), func-
a Reagents and conditions: mMethod E: (a) NaH, THF; (b)
p-NO2C4H4OCOCl, THF; (c) TFA, DCM.
Scheme 3a
tional potency (pEC50), and intrinsic activity (IA) of a range
of primary binding groups at the human recombinant 5-HT4
receptor was initiated. The choice of aromatic systems
encompassed chloroaniline (1), quinolone (2), indazole (3),
benzimidazolinone (4), and indole (5) ring systems, coupled
through amide bonds to four representative amines {(3-
amino-8-methyl-8-azabicyclo[3.2.1]octane (a), 4-(aminome-
thyl)piperidine (b), 4-aminopiperidine (c), and 4-(2-amino-
ethyl)piperazine (d)}.
As can be seen from Table 1, a wide range of in vitro
profiles was observed. In general, these simple primary
binding groups had higher binding affinity for the 5-HT3
receptor than the 5-HT4 receptor. Several compounds (3b,
a Reagents and conditions: Method F: (a) Br(CH2)nR, KI, NaHCO3,
DMF, H2O. Method G: (a) R1R2NH, Br(CH2)nBr, KI, NaHCO3,
DMF, H2O. Method H: (a) R1R2NH, Br(CH2)nBr, iPr2NEt, MeOH.
Method I: (a) Br(CH2)nOH, Et3N, MeCN; (b) PySO3, iPr2NEt, DMSO,
CH2Cl2; (c) R1R2NH, NaBH(OAc)3, MeOH.
5c, 5d) were antagonists at the 5-HT4 receptor and were
removed from further consideration. Of the remaining com-
pounds, those with intrinsic activity greater than 90% were
prioritized. Several compounds met these criteria (1c-d, 2a-
c, 3a, 4a-d) and will be the subject of future publications.
This publication will focus on the elaboration compounds 1c
and 2a, which were adequately representative of major
chloroaniline and N-alkylheteroaromatic classes of 5-HT4
agonist.
Exploring the Secondary Binding Site. To rapidly assess the
electronic, steric, and positional preferences of the putative
secondary binding site, we designed a selection of secondary
binding groups which encompassed neutral, acidic, and basic
functionality and which were capable of varying degrees of
hydrogen bonding and aromatic interactions. These were
linked to the secondary amine of the primary binding group
through simple alkyl chains of two, five, and eight carbon
lengths (Table 2), which allowed evaluation of the role of
distance between the primary and secondary binding groups.
three methods. In methods G and H, an equimolar mixture
of the primary binding group and secondary binding group
was reacted with 1 equiv of alkyl dibromide. The desired
products could easily be separated from the homodimeric
byproduct by preparative HPLC. In method I, compounds
1c and 2a were first alkylated with commercially available
bromoalkylalcohols. The primary alcohol was then oxidized
by sulfur trioxide-pyridine complex to the aldehyde, which
then underwent smooth reductive amination using the re-
quired amine and sodium triacetoxyborohydride.
Compounds 24-26 were made by treatment of piperazine
N-methylsulfonamide with racemic or enantiopure epichlor-
ohydrin to form an intermediate chloro-alcohol, which upon

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5333
Scheme 4a
a Reagents and conditions: (a) rac-epichlorohydrin (24) or (R)-epichlorohydrin, (25) or (S)-epichlorohydrin (26), EtOH; (b) NaOH; (c) 2a, rac-
1-methylsulfonyl-4-(oxiranylmethyl)piperazine (24), or (R)-1-methylsulfonyl-4-(oxiranylmethyl)piperazine (25), or (S)-1-methylsulfonyl-4-(oxiranyl-
methyl)piperazine (26), EtOH, 80 °C.
Table 1. 5-HT4 Agonist Primary Binding Groups
a 5-HT4 pKi values were determined using [3H]-GR113808 in HEK-293 cells stably transfected with human 5-HT4(c) receptor cDNA; 5-HT3 pKi values
were determined with [3H]-GR65630 in HEK-293 cells stably transfected with human 5-HT3A receptor cDNA. b Selectivity for the 5-HT4 receptor
subtype with respect to the 5-HT3 receptor subtype was calculated as the ratio Ki(5-HT3A)/Ki(5-HT4(c)). c pEC50 determined using whole-cell cAMP
accumulation studies in HEK-293 cells stably transfected with the human recombinant 5-HT4(c) receptor splice variant. d Maximum compound-evoked
response (minus basal) was expressed as a percentage of the maximum response evoked by 5-HT.
Because selectivity over 5-HT3 was considered essential,
affinity for this receptor was included in the primary screen
together with functional potency and intrinsic activity at the
5-HT4 receptor.
Attaching a simple alkyl as a secondary binding group in
both the chloroaniline and quinolone series (6a-c, 7a-c)
had the expected orthogonal effect on binding affinity for the
5-HT3 and 5-HT4 receptors, with the resultant improvement
in selectivity in favor of 5-HT4, and this effect was of greater
magnitude with the longer C5 and C8 chains. Functional
potencies revealed the first contrasting SAR between the two
series, with the alkyl chain imparting an approximately 10-
fold gain in the chloroanilines 6a-c compared to a 0.5 log
unit reduction observed with the quinolones. Intrinsic activ-
ity was essentially unchanged in both series. A more polar
secondary binding group (alcohols 6d-f, 7d-f) had no sig-
nificant effect on the 5-HT4 pKi or pEC50 relative to the sim-
ple alkyl chain, indicating a lack of significant interactions of

5334 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Table 2. 5-HT4 Agonist Secondary Binding Group Scan
McKinnell et al.
a 5-HT4 pKi values were determined using [3H]-GR113808 in HEK-293 cells stably transfected with human 5-HT4(c) receptor cDNA; 5-HT3 pKi values
were determined with [3H]-GR65630 in HEK-293 cells stably transfected with human 5-HT3A receptor cDNA. b Selectivity for the 5-HT4 receptor
subtype with respect to the 5-HT3 receptor subtype was calculated as the ratio Ki(5-HT3A)/Ki(5-HT4(c)). c pEC50 determined using whole-cell cAMP
accumulation studies in HEK-293 cells stably transfected with the human recombinant 5-HT4(c) receptor splice variant. d Maximum compound-evoked
response (minus basal) was expressed as a percentage of the maximum response evoked by 5-HT.
this group with the secondary binding site. When the steric
bulk of the secondary binding group was increased consider-
ably (phthalimides 6g-i, 7g-i), there was a sharp drop in
binding affinity in both series at the shortest linker length
(6g, 7g), due perhaps to inductive electron withdrawal from
the amine of the primary binding group by the phthalimide
or a limited amount of space available in this region of the
receptor. At longer linker lengths, higher 5-HT4 binding
affinities and potencies were observed only with the chloro-
anilines (6h-i). Acidic secondary binding groups (sulfonic
acids 6j-l, 7j-l), were not well tolerated in either series and
led to a sharp reduction in binding affinity at both 5-HT3 and
the 5-HT4 receptors. Introduction of piperidine as a basic
secondary binding group afforded a significant increase in
the 5-HT4 pKi in both the chloroaniline (6m-o) and quino-
lone (7m-o) series relative to the simple alkyl chain deriva-
tives. This effect was much more pronounced with the C5
and C8 linkers. Functional potency also benefited from the
introduction of this second positively charged group, and for
the first time an improvement in this property in the
quinolone series was observed. Reducing the basicity of the
secondary binding group by replacing the piperidine
(calculated pKa=10.6) with a piperazinesulfonamide (calcu-
lated pKa = 8.8, compounds 6p-r, 7p-r) appeared to

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5335
Figure 3. Effect of linker length on 5-HT4 pKi, pEC50, and selectivity vs 5-HT3.
partially negate this beneficial effect in both the C5 and C8
series, although this could also be due to negative steric
interactions related to the increased bulk of the methyl-
sulfonamide group. However, excellent binding affinity
and potency could be restored by reverting to a bulky,
strongly basic methyltryptamine system (calculated pKa =
9.9, 6s-u, 7s-u), suggesting that the pKa of the secondary
binding group was the key factor in the observed improve-
ments and that the positively charged secondary binding
group was highly preferred in this region of the receptor.
To more fully understand the positional preference of the
positively charged secondary binding groups, the in vitro
profile of the full set of linker lengths (C2 through C9) was
examined using a representative piperazinesulfonamide
(Figure 3). With the chloroaniline- and quinolone-piperazi-
nesulfonamide systems (6p-r, 8a-e, and 7p-r, 9a-e,
respectively), the 5-HT4 binding affinity generally increased
as the distance between the primary and secondary binding
groups increased, but functional potency remained essen-
tially constant for all linker lengths. With respect to selectiv-
ity over the 5-HT3 receptor, the quinolone-based agonists
were superior to the chloroanilines at all linker lengths except
C6, which had unusually high 5-HT3 binding affinity.
a clear focal point for optimization efforts. Thus, attention
focused on other “drug-likeness” features of these compounds,
specifically permeability in the Caco-2 assay and hERG
potassium ion channel inhibition. In light of the higher pKi,
selectivity, and pEC50 observed with quinolone-based ago-
nists, efforts were concentrated on this series. The set of amine-
based secondary binding groups was expanded to cover a wide
spectrum of calculated pKas (Table 3), and these were attached
via linkers of representative lengths (C3 and C7).
In general, the expanded set of amines followed the SAR
trends described earlier for linker length, with 5-HT4 pKi
increasing as the distance between the primary and secondary
binding groups increased. Functional potency had a more
complex relationship with linker length, at times increasing
(10, 11), remaining unchanged (14, 15), or even declining (18,
19) within a given series as the linker length increased. With
respect to basicity, increasing the calculated pKa of the
tropane and secondary binding group (pKa1 and pKa2, re-
spectively), either by increasing the separation between them
(to avoid inductive electron withdrawal) or employing a more
basic secondary binding group (N-phenylpiperazine 18 vs
morpholine 14), generally afforded an increase in binding
affinity and selectivity. This improvement was maintained
with more basic cyclic amines 20 and 22 although the acyclic
amine 16 did not follow this trend. Functional potency was
independent of the pKa of the secondary binding group, with
excellent potencies being attainable even with relatively low
pKa secondary binding groups (11). Permeability (as mea-
sured by the Caco-2 assay) was low when highly flexible (16,
Lead Optimization
Although at this juncture the ability of the amine-based
secondary binding groups to consistently elevate the in vitro
potency of 5-HT4 agonists to desirable levels had been demon-
strated, the study of the role of linker length had not afforded

5336 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Table 3. Varying the pKa of the Secondary Binding Group
McKinnell et al.
a Calculated pKa of the tropane group. pKa was calculated with the pKalc 3.2 prediction module of Pallas system (CompuDrug Chemistry, Ltd.,
Hungary). b Calculated pKa of the secondary binding group amine c 5-HT4 pKi values were determined using [3H]-GR113808 in HEK-293 cells stably
transfected with human 5-HT4(c) receptor cDNA; 5-HT3 pKi values were determined with [3H]-GR65630 in HEK-293 cells stably transfected with
human 5-HT3A receptor cDNA. d Selectivity for the 5-HT4 receptor subtype with respect to the 5-HT3 receptor subtype was calculated as the ratio Ki(5-
HT3A)/Ki(5-HT4(c)). e pEC50 determined using whole-cell cAMP accumulation studies in HEK-293 cells stably transfected with the human recombinant
5-HT4(c) receptor splice variant. f Maximum compound-evoked response (minus basal) expressed as a percentage of the maximum response evoked by
5-HT. g hERG inhibition determined at a concentration of 3 μM using Chinese hamster ovary (CHO) cells stably expressing hERG potassium channels.
17) or H-bond donating secondary binding groups (10, 11, 22,
23) were employed. However, with cyclic secondary binding
groups, permeability was generally good, even with C7 linkers
(9d, 15) and highly basic amines (18, 20).
The rat in vivo oral pharmacokinetic data for compounds
9a and 9d are shown in Table 4. Although both compounds
had similar permeability in the Caco-2 assay and 9a was less
stable in the rat liver microsome (RLM) assay, the in vivo oral
exposure of 9a was superior to that of the C7 analogue 9d as
well as tegaserod. Thus, optimization efforts were focused on
the C3 linked derivative 9a, and in particular, modifications to
the linker domain, through which improvements in the 5-HT4
receptor binding affinity and reduced hERG inhibition were
sought.
Introduction of a racemic alcohol (compound 24, Table 5)
to the central carbon of the C3 linker provided a convenient
functional group handle with which to explore this domain.
However, this simple modification itself led to a minor
improvement in affinity and a significant reduction in hERG
inhibition. When the single enantiomers 25 and 26 were
isolated, the (S)-enantiomer was found to have significantly
better affinity, selectivity over 5-HT3, and functional potency
relative to the (R)-enantiomer. With respect to selectivity over
other 5-HT receptors, compound 26 displayed less than 50%
Inhibition of the hERG channel was highly dependent on
thenatureof the secondarybinding group, insome cases being
consistently low (prolines 10, 11) or high (phenylpiperazines
18, 19) and having no clear relationship to calculated pKa.
When piperidine was employed as the secondary binding
group, increasingthe linker lengthsuccessfullyreducedhERG
inhibition (20, 21), while in the case of the piperazinesulfona-
mides (9a, 9d), the opposite was true. Overall, hERG inhibi-
tion had a highly unpredictable relationship with linker length
and the nature of the secondary binding group.
On the basis of the promising potency, good in vitro
permeability and moderate hERG inhibition observed with
compounds 9a and 9d, they were advanced into a rat oral
pharmacokinetic study in order to establish the relationship
between their in vitro permeability and metabolism and their
in vivo pharmacokinetics.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5337
Table 4. Oral Pharmacokinetics of Compounds 9a and 9d in Rat
compd
9a
9d
Caco-2 Kp (1 ꢀ 10-6 cm/s)
RLM t1/2 (min)
AUC(0-t) (μg h/mL)a
Cmax (μg/mL)
t1/2 (h)
F%b
3
20
22
11
41
0.183
0.083
0.021
0.034
0.010
0.006
7.9
20.3
1.8
38
20
5
>90
>90
tegaserod
a Pharmacokinetic properties were evaluated in male Sprague-Dawley rats dosed with test compounds via oral gavage (PO) at a dose of 5 mg/kg. n = 3.
b F = oral bioavailability, %.
Table 5. In Vitro Data for Compounds 9a, 24-26
general
procedure
5-HT3 Ki/
5-HT4 Kib
5-HT4
pEC50
Caco-2 Kp
(1 ꢀ 10-6 cm/s)
hERG %
Inhibitione
5-HT4 IAd
c
compd
R
5-HT4 pKia
9a
24
25
26
G
J
H
OH
7.4
7.7
7.3
7.9
2300
2400
1300
7400
8.8
9.1
8.9
9.4
>100
>100
>100
94
20
19
20
14
38
10
6
J
(R)-OH
(S)-OH
J
4
a 5-HT4 pKi values were determined using [3H]-GR113808 in HEK-293 cells stably transfected with human 5-HT4(c) receptor cDNA; 5-HT3 pKi values
were determined with [3H]-GR65630 in HEK-293 cells stably transfected with human 5-HT3A receptor cDNA. b Selectivity for the 5-HT4 receptor
subtype with respect to the 5-HT3 receptor subtype was calculated as the ratio Ki(5-HT3A)/Ki(5-HT4(c)). c pEC50 determined using whole-cell cAMP
accumulation studies in HEK-293 cells stably transfected with the human recombinant 5-HT4(c) receptor splice variant. d Maximum compound-evoked
response (minus basal) was expressed as a percentage of the maximum response evoked by 5-HT. e hERG inhibition determined at a concentration of 3
μM using Chinese hamster ovary (CHO) cells stably expressing hERG potassium channels.
Table 6. Pharmacokinetics of Compound 26 in Rat and Dog
rat
dog
Cmax (μg/mL)
0.32
compd
RLM t1/2 (min)
AUC(0-t) (μg h/mL)a
Cmax (μg/mL)
t1/2 (h)
F%b
F%
3
26
>90
0.20
0.031
5.2
20
63
a Pharmacokinetic properties were evaluated in male Sprague-Dawley rats dosed with test compounds via oral gavage (PO) at a dose of 5 mg/kg. n = 3.
b F = oral bioavailability, %. c Pharmacokinetic properties were evaluated in male Beagle dogs dosed with test compounds via oral gavage (PO) at a dose of
2 mg/kg. n = 3.
inhibition of 5-HT1A,C,D, 5-HT2A,B,C, 5-HT5A, 5-HT6, and
5-HT7 at a concentration of 10 μM.
myenteric plexus preparation. The potency of 26 (pEC50 =
8.6) was superior to that of 5-HT (pEC50=6.9) and similar to
tegaserod (pEC50=8.8). Compound 26 had an intrinsic acti-
vity greater than that of tegaserod (63% vs 44% of the 5-HT
maximum, respectively). Considering the wealth of literature
demonstrating that 5-HT4 receptor activation results in
contraction of this smooth muscle preparation40,41 and the
presence of antagonists of 5-HT1, 5-HT2 and 5-HT3 recep-
tors, in this study, the observed activity of 26 was concluded
to represent 5-HT4 receptor activation.
The oral pharmacokinetics of 26 in rats and dogs are shown
in Table 6. Introduction of the hydrophilic hydroxyl group
reduced the oral bioavailability in the rat relative to 9a,
perhaps due to lower permeability, although bioavailability
in the dog was excellent.
In Vitro/In Vivo Pharmacology of Compound 26. Com-
pound 26 produced a concentration-dependent contraction
of the guinea pig isolated colon longitudinal muscle/

5338 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
McKinnell et al.
to tegaserod, although tegaserod did not reach statistical
significance at the doses tested.
Conclusions
Applying a multivalent approach to lead discovery led to
conceptualization of the 5-HT4 agonist pharmacophore as
consisting of “primary” and “secondary” binding groups and
investigation of how the secondary binding group could be
used to modulate the profile of fixed primary binding groups.
A diverse selection of secondary binding groups was screened
by linking them to chloroaniline-piperidine 1c and quinolone-
tropane 2a, which demonstrated the distinct ability of amine-
based secondary binding groups to improve the 5-HT4 recep-
tor bindingaffinity andselectivity. A study of the role of linker
length using a representative amine revealed that 5-HT4
binding affinity increased as the linker length was increased,
although functional potency did not improve significantly
after the linker exceeded three carbons in length. Focusing on
the quinolone-tropane series, the influence of the pKa of the
secondary binding group was investigated, and higher pKas
were found to enhance the binding affinities in the C3 linked
compounds. In vitro permeability and hERG inhibition data
were also heavily dependent on the nature of the secondary
binding group. The need to balance potency with satisfactory
oral pharmacokinetics led to a focus on 5-HT4 agonists
containing weakly basic secondary binding groups and short
linkers. Compound 9a was selected for optimization, and
introduction of a chiral alcohol to the linker (compound 26)
resulted in a significant improvement in binding affinity,
selectivity over 5-HT3, and functional potency as well as a
reduction in hERG inhibition. In animal models, 26 produced
a dose-dependent, 5-HT4 receptor-mediated relaxation of the
esophagus in rats and colonic prokinetic activity comparable
to tegaserod in guinea pigs. On the basis of these data,
compound 26 was selected for advanced efficacy and safety
evaluation.
Figure 4. Relaxation of rat esophagus following intravenous and
intraduodenal administration of compound 26.
Figure 5. Guinea pig colonic transit following subcutaneous dos-
ing of compound 26.
Experimental Section
To characterize the in vivo activity of 26, digital sonomi-
crometry was used to monitor 5-HT4 receptor-mediated
relaxation of the rat esophagus.42 This method provided a
novel and sensitive means to demonstrate 5-HT4 receptor
agonist-mediated changes in endogenous esophageal tone
in anesthetized rats. Compound 26 produced a dose-
dependent, 5-HT4 receptor-mediated relaxation of the
esophagus in anesthetized rats, following intravenous or
intraduodenal administration (Figure 4). Following intrave-
nous administration, the rank order of potencies was tega-
serod>26>cisapride. The mean ED50 values for the rela-
xant response mediated by intravenous 26, tegaserod
and cisapride were 16, 10.6, and 142 μg/kg respectively.
Following intraduodenal administration at a dose of
3 mg/kg, the degree of relaxation of the rat esophagus
afforded by 26 was superior to that afforded by tegaserod
and cisapride.
In the guinea pig colonic transit model, 26 (3 mg/kg sc)
produced a statistically significant increase in colonic transit
of carmine red dye relative to vehicle treated animals.43
Following subcutaneous administration, prokinetic activity
was evident when the distance traveled by the dye was
measured 60 min later or when the time for excretion of
the first fecal pellet containing the marker was recorded
(Figure 5). In this assay, 26 appeared to be similar in potency
Chemistry. Unless noted otherwise, starting materials and
solvents were purchased from commercial suppliers and used
without further purification. Reactions were run under nitro-
gen atmosphere, unless noted otherwise. Progress of reaction
mixtures was monitored by analytical high performance liquid
chromatography (HPLC) and mass spectrometry, the details
of which are given below and separately in specific examples of
reactions. Reaction mixtures were worked up as described
specifically in each reaction and routinely purified by prepara-
tive HPLC: a general protocol is described below. Test com-
pounds with >95% purity, assessed by analytical HPLC
(solvent A = 98% water/2%MeCN/1.0 mL/L TFA; solvent
B=90% MeCN/10%water/1.0 mL/L TFA), were submitted
for biological evaluation. Melting points were determined on a
TA Instruments’ Q100 differential scanning calorimeter. Char-
acterization of reaction products was routinely performed by
1H and 13C NMR spectrometry in deuterated solvents
(CD3OD, DMSO-d6) acquired under standard parameters
using a Varian Gemini 2000 instrument (400 or 300 MHz) or
Bruker UltraShieldplus NMR spectrometer (600 MHz). Chiral
purity was determined on a Beckman P/ACE MDQ capillary
electrophoresis system employing a 50 μM ꢀ 60 cm fused silica
capillary and 25 μM triethylammonium phosphate/10% w/v
highly sulfated γ-cyclodextrin (Beckman) buffer system. Mass
spectrometric identification of compounds was performed
using standard electrospray ionization methods (ESMS) with
a Perkin-Elmer SCIEX API 150 EX. High resolution mass

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5339
spectrometry was performed using standard electrospray ioni-
zation methods (ESMS) with an Applied Biosystems/MDS
SCIEX QSTAR.
1-Hydroxybenzotriazole (1 equiv) and N-(3-dimethylamino-
propyl)-N0-ethylcarbodiimide hydrochloride (1 equiv) were
added, and the reaction was allowed to stir overnight at room
temperature. Diisopropylethylamine (3 equiv) was added, and
the reaction was allowed to stir at room temperature for 4 h, at
which point the reaction was still incomplete. A further 1 equiv
of tert-butyl 4-(aminomethyl)piperidinecarboxylate was added
portionwise until the reaction was complete. The solution was
diluted with EtOAc (30 mL) and washed with saturated NH4Cl
(30 mL), followed by 1 M NaOH (30 mL) and then brine
(30 mL). The organics were dried (MgSO4) and the solvent
evaporated to afford 4-{[(5-methoxy-1H-indole-3-carbonyl)-
amino]-methyl}-piperidine-1-carboxylic acid tert-butyl ester
(0.9 g). This compound was dissolved in CH2Cl2 (20 mL) and
cooled to 0 °C. TFA (20 mL) was added dropwise, and the
solution was stirred for 0.5 h. The solvent was evaporated and
the residue triturated with Et2O and filtered to afford a white
solid. This crude material was purified by preparative reversed-
phase HPLC and the pure fractions lyophilized to afford 5b as a
white powder (135 mg, 13%); 93% purity by HPLC (2-90%
General Procedure for Compounds 1a, 1c, 3a, 3c (Method A):
4-Amino-5-chloro-2-methoxy-N-piperidin-4-yl-benzamide TFA
3
(1c). 4-Amino-5-chloro-2-methoxy-benzoic acid (20.1 g, 0.1
mol) was dissolved in DMF (200 mL) and CDI (1 equiv) was
added. The solution was stirred at room temperature for 4 h,
then 4-amino-piperidine-1-carboxylic acid tert-butyl ester
(1 equiv) was added. The reaction was stirred for 16 h at room
temperature, the solvent evaporated, and the residue partitioned
between EtOAc (500 mL) and brine. The organic phase was
separated and washed (2 ꢀ 200 mL 0.2 M H3PO4/brine and 1 M
NaOH/brine). The organic phase was dried (MgSO4), evapo-
rated, and the residue purified by flash chromatography (SiO2)
to afford 4-(4-amino-5-chloro-2-methoxy-benzoylamino)-pi-
peridine-1-carboxylic acid tert-butyl ester (14.6 g). This material
was dissolved in CH2Cl2 (25 mL) and anisole (10 mL), cooled to
0 °C, and TFA (25 mL) was added. After 10 min at 0 °C, the
solution was allowed to warm to room temperature and stirred
for 1 h. The solvent was evaporated, and the residue added to
Et2O (50 mL). The resultant precipitate was filtered and washed
with Et2O (500 mL) to afford a beige solid (12.0 g). This crude
material was purified by preparative reversed-phase HPLC and
the pure fractions lyophilized to afford 1c as a white powder
(1.97 g, 5%); 100% purity by HPLC (2-90% MeCN/H2O over
6 min; tR 2.26 min). 1H NMR (DMSO-d6) δ 1.68 (m, 2H), 2.03
(m, 2H), 3.03 (m, 2H), 3.25 (m, 2H), 3.80 (s, 3H), 3.99 (m, 1H),
5.80 (br s, 2H), 6.48 (s, 1H), 7.60 (s, 1H), 7.76 (d, J=7.2, 1H,),
8.65 (br s, 1H), 8.86 (br s, 1H). 13C NMR (400 MHz, DMSO-d6)
δ 28.7, 42.5, 44.2, 56.4, 98.0, 109.3, 111.0, 131.7, 148.9, 157.7,
163.8. MS m/z: 285.3 (M þ Hþ).
1
MeCN/H2O over 6 min; tR 1.99 min). H NMR (400 MHz,
DMSO-d6) δ 1.32 (m, 2H), 1.80 (m, 2H), 2.85 (m, 2H), 3.15 (t, J=
6.1 Hz, 1H), 3.28 (m, 2H), 3.73 (s, 3H), 6.75 (dd, J=2.5, 8.8, 1H),
7.28 (d,, J=8.8, 1H), 7.65 (d, J=2.5, 1H), 8.01 (m, 1H), 11.51 (d,
J=2.5 1H). 13C NMR (400 MHz, DMSO-d6) δ 27.1, 34.7, 43.6,
43.9, 55.8, 103.2, 110.8, 112.6, 113.1, 127.5, 128.7, 131.8, 154.9,
165.6. MS m/z: 288.4 (M þ Hþ). HRMS calcd for C16H22N3O2,
288.1712; found, 288.1703.
2-Amino-5-chloro-4-methoxy-N-(2-piperazin-1-yl-ethyl)-ben-
zamide TFA (1d). Compound 1d was prepared in the same
3
manner as 5b using 4-amino-5-chloro-2-methoxybenzoic acid
and 4-N-(2-aminoethyl)-1-N-Boc-piperazine; 99% purity by
HPLC (2-30% MeCN/H2O over 4 min; tR 1.32 min). 1H
NMR (400 MHz, DMSO-d6) δ 3.16 (m, 2H), 3.22-3.40 (br s,
General Procedure for Compounds 1b, 3b (Method B): 1-Iso-
propyl-1H-indazole-3-carboxylic Acid (Piperidin-4-ylmethyl)-
amide TFA (1b). 1-Isopropylindazole-3-carboxylic acid (5.0 g,
10H), 3.56 (m, 2H), 3.81 (s, 3H), 6.47 (s, 1H), 7.69 (s, 1H). 13
C
3
24.6 mmol) and tert-butyl 4-(aminomethyl)piperidinecarbo-
xylate (5.2 g, 1 equiv) were dissolved in anhydrous DMF
(30 mL). The solution was stirred in an ice bath and diisopro-
pyethylamine (1.5 equiv) was added, followed by N-(3-dimethyl-
aminopropyl)-N0-ethylcarbodiimide hydrochloride (1.5 equiv)
and washed with an additional 20 mL of DMF). After 10 min,
the ice bath was removed and the reaction was stirred at room
temperature for 16 h. The reaction was quenched with water and
extracted with EtOAc (100 mL). The organic extract was washed
with saturated KHSO4 (100 mL), 1N NaOH (100 mL), and
brine (100 mL) and then dried (MgSO4), filtered, and evapo-
rated to afford 4-{[(1-isopropyl-1H-indazole-3-carbonyl)-
amino]-methyl}-piperidine-1-carboxylic acid tert-butyl ester as
a colorless oil (4.52 g). A dichloromethane (20 mL) solution
of 4-{[(1-isopropyl-1H-indazole-3-carbonyl)-amino]-methyl}-
piperidine-1-carboxylic acid tert-butyl ester (0.484 g, 1.2 mmol)
was cooled in an ice bath. TFA (20 mL) was added dropwise and
stirring continued for one hour. The solution was then evapo-
rated and the residue triturated with Et2O (60 mL) by stirring at
room temperature overnight. The solid was collected by filtra-
tion to give 1b as a white solid (0.47 g); 98% purity by HPLC (2-
90% MeCN/H2O over 6 min; tR 2.72 min). 1H NMR (DMSO-
d6) δ 1.37 (m, 2H), 1.49 (d, J=6.6 Hz, 6H), 1.85 (m, 3H), 2.83 (m,
2H), 3.23 (m, 4H), 5.00 (sept, J=6.6 Hz, 1H), 7.20 (t, J=7.2 Hz,
1H), 7.39 (t, J=6.6 Hz, 1H), 7.73 (d, J=8.6 Hz, 1H), 8.15 (d, J=
8.2 Hz, 1H), 8.35 (t, J=6.3 Hz, 1H), 8.60 (br s, 1H), 8.95 (br s,
1H). 13C NMR (400 MHz, DMSO-d6) δ 22.5, 27.1, 34.5, 43.6,
43.9, 50.9, 110.9, 116.3, 119.3, 122.5, 122.9, 126.9, 137.4, 140.2,
163.0. MS m/z: 301.3 (M þ Hþ). HRMS calcd for C17H24N4O,
301.2028; found, 301.2032.
NMR (400 MHz, DMSO-d6) δ 35.2, 40.8, 49.6, 56.4, 98.0, 109.1,
110.4, 132.4, 149.5, 158.2, 165.1. MS m/z: 313.2 (M þ Hþ).
HRMS calcd for C14H22ClN4O2, 313.1431; found, 313.1433
5-Methoxy-1H-indole-3-carboxylic Acid (8-Aza-bicyclo[3.2.1]-
oct-3-yl)-amide TFA (5a). Compound 5a was prepared in the
3
same manner as 5b using 5-methoxy-1H-indole-3-carboxylic
acid and 4(1S,3R,5R)-3-amino-8-azabicyclo[3.2.1]octane-8-car-
boxylic acid tert-butyl ester; 99% purity by HPLC (2-90%
MeCN/H2O over 6 min; tR 2.06 min). 1H NMR (400 MHz,
DMSO-d6) δ 1.50 (m, 2H), 1.73 (m, 4H), 1.86 (m, 2H), 3.26 (m,
3H), 3.49 (s, 3H), 6.29 (m, 1H), 6.85 (m, 1H), 7.12 (s, 1H), 7.18 (s,
1H), 7.56 (m, 1H), 8.33 (br s, 1H), 8.48 (br s, 1H), 11.13 (s, 1H).
13C NMR (400 MHz, DMSO-d6) δ 25.1, 31.9, 40.4, 53.2, 55.0,
102.2, 110.0, 112.1, 112.5, 126.7, 128.7, 131.1, 154.3, 165.7. MSm/
z: 300.8 (M þ Hþ). HRMS calcd for C17H22N3O2, 300.1712;
found, 300.1721.
5-Methoxy-1H-indole-3-carboxylic Acid Piperidin-4-ylami-
de TFA (5c). Compound 5c was prepared in the same manner
3
as 5b using 5-methoxy-1H-indole-3-carboxylic acid and 4-ami-
no-1-Boc-piperidine; 90% purity by HPLC (2-90% MeCN/
H2O over 6 min; tR 2.23 min). 1H NMR (400 MHz, DMSO-d6) δ
1.69 (m, 2H), 1.98 (m, 2H), 2.99 (m, 2H), 3.33 (m, 2H), 3.72 (s,
3H), 4.02 (m, 1H), 6.75, (dd, J=2.6, 8.8 Hz, 1H), 7.28 (d, J=8.8
Hz, 1H), 7.63 (d, J=2.5 Hz, 1H), 7.86 (d, J=7.2 Hz, 1H), 8.03
(d, J=3.1 Hz, 1H), 8.62-8.74 (br s, 1H), 8.80-8.90 (br s, 1H),
11.52 (s, 1H). 13C NMR (400 MHz, DMSO-d6) δ 29.3, 43.0,
44.2, 55.8, 103.3, 110.5, 112.6, 113.1, 127.7, 129.0, 131.8, 155.0,
165.1. MS m/z: 274.6 (M þ Hþ). HRMS calcd for C15H19N3O2,
274.1566; found, 274.1556.
5-Methoxy-1H-indole-3-carboxylic Acid (2-Piperazin-1-yl-
ethyl)-amide TFA (5d). Compound 5d was prepared in the same
General Procedure for Compounds 1d, 5a-d (Method C): 5-
Methoxy-1H-indole-3-carboxylic Acid (Piperidin-4-ylmethyl)-
amide TFA (5b). 5-Methoxy-1H-indole-3-carboxylic acid
3
manner as 5b using 5-methoxy-1H-indole-3-carboxylic acid and
4-N-(2-aminoethyl)-1-N-Boc-piperazine; 90% purity by HPLC
(2-90% MeCN/H2O over 6 min; tR 2.03 min). 1H NMR (400
MHz, DMSO-d6) δ 3.25 (m, 2H), 3.30-3.50 (br m, 10H), 3.58
3
(0.50 g, 2.6 mmol) and tert-butyl 4-(aminomethyl)piperidinecar-
boxylate (0.60 g, 1.05 equiv) were dissolved in DMF (5 mL).

5340 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
McKinnell et al.
(m, 2H), 3.73 (s, 3H), 6.76 (dd, J=2.5, 8.8 Hz, 1H), 7.30 (d, J=
8.8 Hz, 1H), 7.64 (d, J=2.5 Hz, 1H), 7.96 (d, J=2.9 Hz, 1H), 8.18
(t, J=5.3 Hz, 1H), 11.59 (d, J=2.3 Hz, 1H). 13C NMR (400
MHz, DMSO-d6) δ 34.4, 41.1, 49.1, 55.8, 103.1, 110.3, 112.7,
113.2, 127.4, 129.1, 131.8, 155.1, 166.1. MS m/z: 303.6 (M þ
Hþ). HRMS Calcd for C16H23N4O2, 303.1821; found, 303.1818.
General Procedure for Compounds 2a-d (Method D): 1-Iso-
propyl-2-oxo-1,2-dihydroquinoline-3-carboxylic Acid {(1S,3R,
13C NMR (400 MHz, DMSO-d6) δ 19.9, 29.0, 34.9, 42.8, 44.5,
116.3, 119.3, 120.6, 122.0, 123.4, 132.0, 133.5, 143.9, 162.5,
162.9. MS m/z: 314.6 (M þ Hþ). HRMS calcd for C18H24-
N3O2, 314.1869; found, 314.1864.
General Procedure for Compounds 4a-d (Method E):
N-[(1S,3R,5R)-3-Isopropyl-2-oxo-2,3-dihydrobenzoimidazole-1-
carboxylic Acid (8-Azabicyclo[3.2.1]oct-3-yl)amide TFA (4a).
3
To a suspension of sodium hydride (9.25 g; 1.5 equiv, 60%
dispersion in mineral oil) in dry THF (1 L) in an ice bath was
added 1-isopropyl-1,3-dihydro-2H-benzimidazol-2-one (27.2 g,
154.2 mmol) in THF (50 mL). The mixture was stirred at 0-5 °C
for 0.5 h, and then 4-nitrophenyl chloroformate (34.2 g, 170
mmol) in THF (50 mL) was added. The mixture was stirred at rt
for 16 h. To this solution was added (1S,3R,5R)-3-amino-
8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester
(36.7 g, 1.05 equiv) in THF (50 mL). The mixture was stirred
rt for 12 h and then warmed to 75 °C for 3 h. The mixture
allowed to cool, evaporated, and the residue dissolved in
CH2Cl2 (1 L). The solution was washed with 1 M H3PO4 (500
mL) followed by saturated NaHCO3 (500 mL). The solvent was
evaporated and the intermediate dissolved in CH2Cl2 (200 mL),
cooled to 0 °C, and TFA (200 mL) was added. The mixture was
stirred for 0.5 h at 5 °C and then for 1 h at rt. After evaporation
of the mixture, Et2O (500 mL) was added to the oily residue. The
resulting precipitate was collected, rinsed with Et2O, and dried
in vacuo, to provide 4a as a white solid (47 g, 71% over 2 steps);
98% purity by HPLC (2-90% MeCN/H2O over 6 min; tR=3.05
min). 1H NMR (DMSO-d6) δ 1.45 (d, J=6.8 Hz, 6H), 1.93 (d,
J=15.5 Hz, 2H), 2.05-2.18 (m, 4H), 2.75 (m, 2H), 3.98 (br s,
1H), 4.07 (q, J=6.5 Hz, 1H), 4.69 (sep, J=6.8 Hz, 1H), 7.11 (td,
J=1.1, 7.8 Hz, 1H), 7.18 (td, J=1.4, 7.6 Hz, 1H), 7.40 (d, J=7.4
Hz, 1H), 8.04 (dd, J=1.1, 8.0 Hz, 1H), 8.98 (br s, 1H), 9.12 (br s,
1H) 9.30 (d, J=7.0). 13C NMR (400 MHz, DMSO-d6) δ 20.0,
25.8, 33.5, 40.7, 45.6, 53.7, 110.2, 114.9, 122.5, 124.1, 126.8,
128.1, 151.0, 152.9. MS m/z: 329.6 (M þ Hþ). HRMS calcd for
C18H24N4O2, 329.1978; found, 329.1985.
5R)-8-Azabicyclo[3.2.1]oct-3-yl}amide TFA (2a). To a stirred
3
suspension of 1-isopropyl-2-oxo-1,2-dihydroquinoline-3-car-
boxylic acid (80 g, 0.35 mol) in toluene (600 mL) was added
thionyl chloride (36.6 mL, 1.5 equiv). The mixture was stirred at
95 °C for 2 h then allowed to cool to rt and then added over 0.5 h
to a vigorously stirred biphasic solution of (1S,3R,5R)-3-amino-
8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester
(78.2 g, 1 equiv) and sodium hydroxide (69.2 g, 5 equiv) in
toluene/water (1:1) (1 L) at 0 °C. After 1 h, the layers were
allowed to separate, and the organic phase was concentrated
under reduced pressure. The aqueous phase was washed with
EtOAc (1 L), and the organic extract was used to dissolve the
concentrated organic residue. This solution was washed with 1
M H3PO4 (500 mL), saturated NaHCO3 (500 mL), and brine
(500 mL), dried (MgSO4), filtered, and concentrated under
reduced pressure to afford (1S,3R,5R)-3-[(3-isopropyl-2-oxo-
2,3-dihydro-benzimidazole-1-carbonyl)-amino]-8-aza-bicyclo-
[3.2.1]octane-8-carboxylic acid tert-butyl ester (127.9 g, 84%)
as a yellow solid. This intermediate was dissolved in CH2Cl2
(600 mL) and cooled to 0 °C. TFA (300 mL) was added, and the
reaction mixture was warmed to room temperature and stirred
for 1 h then evaporated. The oily brown residue was then
poured into a vigorously stirred solution of Et2O (3 L) and a
solid precipitate formed immediately. The suspension was
stirred at rt for 16 h and the solid collected by filtration and
washed with Et2O to afford 2a (131.7 g, 86% over two steps) as
a light-yellow solid; 98% purity by HPLC (2-90% MeCN/
H2O over 6 min; tR 3.01 min). 1H NMR (DMSO-d6) δ 1.56 (d,
J=6.8 Hz, 6H), 1.93 (d, J=15.2 Hz, 2H), 2.06-2.31 (m, 6H),
3.32-3.46 (br s, 3H), 4.00 (bs, 2H), 4.16 (m, 1H), 7.34 (t, J=7.6
Hz, 1H), 7.72 (td, J=1.6, 7.2 Hz, 1H), 7.87 (d, J=8.8 Hz, 1H),
8.00 (dd, J=1.6, 8.0 Hz, 1H), 8.70 (br s, 1H), 8.78 (s, 1H), 8.85
(br s, 1H), 10.43 (d, J = 7.2 Hz, 1H). 13C NMR (400 MHz,
DMSO-d6) δ 19.9, 25.9, 33.6, 39.9, 53.8, 116.2, 119.1, 120.6,
121.8, 123.4, 131.9, 133.4, 143.8, 162.5, 162.9. MS m/z: 340.4
(M þ Hþ). HRMS calcd for C20H25N3O2, 340.2025; found,
340.2034.
3-Isopropyl-2-oxo-2,3-dihydro-benzimidazole-1-carboxylic Acid
Piperidin-4-ylamide (4c). Compound 4c was prepared in the same
manner as 4a using 3-isopropyl-2-oxo-2,3-dihydro-benzimida-
zole-1-carboxylic acid and 4-amino-1-Boc-piperidine. 99% purity
1
by HPLC (2-90% MeCN/H2O over 6 min; tR=2.98 min). H
NMR (400 MHz, DMSO-d6) δ 1.45 (d, J=7.1 Hz, 6H), 1.65 (m,
2H), 2.07 (dd, J=3.1, 13.7 Hz, 2H), 3.04 (q, J=11.0 Hz, 2H), 3.28
(d, J=12.9 Hz, 2H), 3.97 (m, 1H), 4.63 (m, 1H), 7.13 (t, J=7.8 Hz,
1H), 7.18 (t, J=7.6 Hz, 1H), 7.40 (d, J=7.4 Hz, 1H), 8.02 (d, J=
7.8 Hz, 1H), 8.64 (br s, 1H), 8.78 (d, J=7.2 Hz, 1H), 8.91 (br s,
1H). 13C NMR (400 MHz, DMSO-d6) δ 20.0, 28.9, 42.7, 45.1,
45.7, 110.2, 114.9, 122.6, 124.2, 126.8, 128.4, 151.0, 152.7. MS m/z:
303.6 (M þ Hþ). HRMS calcd for C16H23N4O2, 303.1821; found,
303.1811.
1-Isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic
Acid
(Piperidin-4-ylmethyl)-amide (2b). Compound 2b was prepared
in the same manner as 2a using 1-isopropyl-2-oxo-1,2-dihydro-
quinoline-3-carboxylic acid and tert-butyl 4-(aminomethyl)-
piperidinecarboxylate; 95% purity by HPLC (2-90% MeCN/
H2O over 6 min; tR 2.95 min). 1H NMR (400 MHz, DMSO-d6) δ
1.36 (q, J=10.4 Hz, 2H), 1.55 (d, J=6.8 Hz, 6H), 1.78-1.85 (br
m, 3H), 2.84 (q, J=10.0 Hz, 2H), 3.27 (t, J=6 Hz, 4H), 3.53 (bs,
1H), 7.32 (t, J=7.2 Hz, 1H), 7.70 (t, J=7.6 Hz, 1H), 7.84 (d, J=
8.8 Hz, 1H), 7.95 (d, J=8.0 Hz, 1H), 8.50 (br s, 1H), 8.75 (br s,
1H), 8.86 (bs, 1H), 9.79 (t, J=6.0 Hz, 1H). 13C NMR (400 MHz,
DMSO-d6) δ 19.9, 27.0, 34.4, 43.5, 44.2, 119.3, 120.6, 122.2,
122.3, 123.4, 130.4, 131.9, 133.4, 143.8, 162.5, 163.6. MS m/z:
328.6 (M þ Hþ). HRMS calcd for C19H26N3O2, 328.2025;
found, 328.2013.
3-Isopropyl-2-oxo-2,3-dihydro-benzimidazole-1-carboxylic Acid
(2-Piperazin-1-yl-ethyl)-amide (4d). Compound 4d was prepared
in the same manner as 4a using 3-isopropyl-2-oxo-2,3-dihydro-
benzimidazole-1-carboxylic acid and 4-N-(2-aminoethyl)-1-N-
Boc-piperazine; 99% purity by HPLC (2-90% MeCN/H2O over
6 min; tR=2.62 min). 1H NMR (400 MHz, DMSO-d6) δ 1.45 (d,
J=7.0 Hz, 6H), 3.15 (m, 2H), 3.20-3.40 (br m, 12H), 3.60 (m,
2H), 4.61 (s, J=7.0 Hz, 1H), 7.12 (t, J=7.6 Hz, 1H), 7.18 (t, J=7.8
Hz, 1H), 7.38 (d, J=7.4 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H), 8.92 (t,
J=5.7 Hz, 1H). 13C NMR (400 MHz, DMSO-d6) δ 20.0, 35.5,
41.1, 45.6, 49.2, 56.0, 110.0, 115.0, 122.5, 124.1, 126.8, 128.5, 152.2,
152.4. MS m/z: 332.4 (M þ Hþ). HRMS calcd for C17H25N5O2,
332.2087; found, 332.2103.
1-Isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic
Acid
Piperidin-4-ylamide (2c). Compound 2c was prepared in the
same manner as 2a using 1-isopropyl-2-oxo-1,2-dihydroquino-
line-3-carboxylic acid and 4-amino-1-Boc-piperidine; 98% pur-
ity by HPLC (2-90% MeCN/H2O over 6 min; tR 2.86 min). 1H
NMR (400 MHz, DMSO-d6) δ 1.55 (d, J=6.8 Hz, 6H), 1.68 (q,
J=10.6 Hz, 2H), 2.07 (d, J=10.9 Hz, 2H), 3.05 (t, J=11.9 Hz,
2H), 3.28 (d, J=12.7 Hz, 2H), 4.07 (m, 1H), 4.02 (m, 1H), 7.32 (t,
J=7.4 Hz 1H), 7.70 (t, J=7.4 Hz, 1H), 7.84 (d, J=8.8 Hz, 1H),
7.95 (d, J=6.8 Hz, 1H), 8.75 (s, 1H), 9.80 (d, J=7.2 Hz, 1H).
General Procedure for Compounds 6a-l, 7a-l (Method F): 1-
Isopropyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic Acid {(1R,
3R,5S)-8-[5-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-pentyl]-8-aza-
bicyclo[3.2.1]oct-3-yl}-amide TFA (7h). Compound 2a (200 mg,
3
0.44 mmol) and N-(5-bromopentyl)phthalimide (1.2 equiv) were
dissolved in DMF (3 mL) and H2O (0.5 mL). NaHCO3 (3 equiv)
and KI (1.2 equiv) were added, and the reaction was stirred at

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5341
90 °C for 16 h. The mixture was allowed to cool, then partitioned
between EtOAc (20 mL) and H2O (10 mL). The organic phase
was evaporated and the residue purified by preparative reverse-
phase HPLC (2-90% MeCN/H2O over 50 min) to afford 7h as a
white solid (55 mg, 19%); 97% purity by HPLC (2-90%
solution was stirred at 0 °C for 0.5 h and then diluted with
CH2Cl2 (250 mL). The solution was washed (saturated NaH-
CO3 then brine), dried (MgSO4), and evaporated to afford
4-amino-5-chloro-2-methoxy-N-[1-(7-oxo-heptyl)-piperidin-4-
yl]-benzamide (3.94 g). A portion of this crude intermediate (198
mg, 0.5 mmol) and 1-methanesulfonylpiperazine (1.5 equiv)
were dissolved in MeOH (3 mL). Sodium triacetoxyborohydride
(1.1 equiv) was added, and the solution was stirred at room
temperature for 0.5 h. The solvent was evaporated and the
residue purified by reversed-phase HPLC to afford 8d as a white
solid (62 mg, 16%); 95% purity by HPLC (2-90% MeCN/H2O
over 6 min; tR=2.47 min). 13C NMR (600 MHz, DMSO-d6) δ
23.4, 23.6, 26.0, 26.1, 28.3, 29.3, 35.4, 42.9, 44.7, 50.9, 51.4, 56.0,
56.2, 56.4, 98.0, 109.4, 110.6, 131.8, 149.0, 157.9, 164.1. MS m/z:
544.8 (M þ Hþ). HRMS calcd for C25H43ClN5O4S, 544.2724;
found, 544.2718.
1
MeCN/H2O over 6 min; tR=3.78 min). H NMR (400 MHz,
DMSO-d6) δ 1.30 (m, 2H), 1.56 (d, J=6.8 Hz, 6H), 1.67 (m, 3H),
2.02 (d, J=15.2 Hz, 1H), 2.25 (m, 3H), 2.45 (m, 3H), 2.92 (m,
1H), 3.57 (t, J=6.8 Hz, 2H), 3.98 (br s, 1H.), 4.19 (br m, 6H), 7.33
(t, J=7.2 Hz, 1H), 7.71 (td, J=1.6, 5.6 Hz, 1H), 7.85 (m, 5H),
7.97 (dd, J=1.6, 8.0 Hz, 1H), 8.77 (s, 1H), 9.52 (br m, 1H), 10.44
(d, J=7.2 Hz, 1H). 13C NMR (400 MHz, DMSO-d6) δ 19.9, 23.9,
24.1, 24.4, 28.2, 34.9, 37.8, 39.6, 49.3, 51.4, 61.0, 120.6, 121.1,
121.8, 123.5, 123.7, 131.3, 132.0, 132.2. 133.5, 135.1, 143.8, 162.6,
162.9, 168.7. MS m/z: 555.4 (M þ Hþ). HRMS calcd for
C33H38N4O4, 555.2972; found, 555.2950.
General Procedure for Compounds 7s, 9a, 9c, 9e, 10-13, 16,
22, 23 (Method G): 4-(3-{(1S,3R,5R)-3-[(1-Isopropyl-2-oxo-1,2-
dihydro-quinoline-3-carbonyl)-amino]-8-aza-bicyclo[3.2.1]oct-8-
yl}-propyl)-piperazine-1-carboxylic Acid Ethyl Ester (13). Com-
pound 2a (4.01 g,) and 1-piperazine carboxylic acid ethyl ester (1
equiv) were dissolved in DMF (10 mL). A solution of KI (1
equiv) and NaHCO3 (5 equiv) in water (5 mL) was added
followed by 1,3-dibromopropane (1 equiv). The mixture was
stirred at 85 °C for 16 h, allowed to cool, and then partitioned
between CH2Cl2 (10 mL) and H2O (10 mL). The organic phase
was evaporated, and the residue purified by preparative reverse-
phase HPLC (2-90% MeCN/H2O over 50 min) to afford 13 as a
white solid (2.7 mg, 5%); 98% purity by HPLC (2-90% MeCN/
H2O over 6 min; tR=2.92 min). 1H NMR (400 MHz, DMSO-d6)
δ 1.16 (t, J=7.2 Hz, 3H), 1.56 (d, J=6.9 Hz, 6H), 2.03 (br m,
4H), 2.24 (m, 4H), 2.46 (m, 4H), 2.55-3.80 (br m, 8H), 4.04 (m,
4H), 4.17 (m, 1H), 7.30 (t, 1H, J=7.2), 7.68 (t, 1H, J=7.2), 7.80
(d, 1H, J=8.4), 7.91 (d, 1H, J=7.2), 8.72 (s, 1H), 10.5 (s, 1H).
13C NMR (600 MHz, DMSO-d6) δ 14.7, 19.4, 19.5, 23.7, 34.5,
40.7, 48.5, 51.0, 53.1, 61.0, 61.8, 116.1, 118.0, 120.3, 123.2, 131.7,
133.5, 143.5, 154.8, 162.5, 162.6. MS m/z: 538.4 (M þ Hþ).
HRMS calcd for C30H44N5O4, 538.3393; found, 538.3393.
General Procedure for Compounds 6m, 6o, 6p, 6r, 6s, 7m, 7o,
7p, 7r, 7u, 8a, 8c, 8e, 14, 18-21 (Method H): 1-Isopropyl-2-oxo-
1,2-dihydro-quinoline-3-carboxylic Acid {(1S,3R,5R)-8-[2-(4-
Methanesulfonyl-piperazin-1-yl)-ethyl]-8-aza-bicyclo[3.2.1]oct-
General Procedure for Compounds 24-26 (Method J):
1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic Acid {(1S,
3R,5R)-8-[(S)-2-Hydroxy-3-(4-methanesulfonylpiperazin-1-yl)-
propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide (26). (a). (S)-1-methy-
lsulfonyl-4-(oxiranylmethyl)piperazine. To a stirred solution of
piperazine N-methylsulfonamide (87.3 g, 0.53 mol) in ethanol
(1.3 L) at room temperature was added (S)-epichlorohydrin
(48.0 mL, 1.1 equiv). The reaction mixture was stirred for 18 h,
and the white solid precipitate that formed was collected by
filtration and washed with ethanol to afford (S)-1-chloro-3-(4-
methylsulfonyl-1-piperazinyl)-2-propanol (107.8 g) as a white
solid. This material was dissolved in THF/H2O (4:1, 1500 mL) at
0 °C, and NaOH (22.15 g, 1.15 equiv) was added with vigorous
stirring. The reaction mixture was stirred for 1.5 h, and the layers
were separated. The organic layer was evaporated and the
residue dissolved in CH2Cl2 (1500 mL) and washed with a
mixture of the previously separated aqueous layer and 1 M
NaOH (500 mL). The organic layer was further washed with 1 M
NaOH (500 mL) and brine (500 mL), dried (MgSO4), filtered,
and exaporated to yield a white solid. This solid was recrystal-
lized from EtOAc/hexanes (800 mL) to yield (S)-1-methylsulfo-
nyl-4-(oxiranylmethyl)piperazine as a white solid (43.3 g, 37%).
1H NMR (DMSO-d6) δ 2.22 (m, 1H), 2.45-2.60 (m, 5H), 2.69-
2.75 (m, 2H), 2.87 (s, 3H), 3.02 (m, 1H), 3.11 (m, 4H). MS m/z:
221.3 (M þ H)þ.
(b). 1-Isopropyl-2-oxo-1,2-dihydroquinoline-3-carboxylic Acid
{(1S,3R,5R)-8-[(S)-2-Hydroxy-3-(4-methanesulfonylpiperazin-
1-yl)propyl]-8-azabicyclo[3.2.1]oct-3-yl}amide. Compound 2a
(100 g, 0.3 mol) and (S)-1-methylsulfonyl-4-(oxiranylmethyl)-
piperazine (69.4 g, 1.05 equiv) were mixed in EtOH (980 mL)
and the reaction mixture stirred at 80 °C for 18 h. The reaction
mixture was cooled to room temperature and evaporated. The
foamy solid was suspended in MeCN/H2O (860 mL/940 mL)
and sonicated until it became homogeneous. The solution was
filtered while hot, and the filtrate was allowed to cool to 5 °C.
Crystals were formed and collected by filtration yielded 26 (122
g, 73%) as a white crystalline solid, mp 108 °C; 98% purity by
HPLC (2-40% MeCN/H2O over 6 min; tR=2.84 min); optical
3-yl}-amide TFA (9d). Compound 2a (527 mg, 0.10 mmol) and
3
1-methanesulfonylpiperazine (1 equiv) were dissolved in MeOH
(1 mL). DIPEA (3 equiv) was added, followed by 1,3-dibromo-
propane (1 equiv). The solution was stirred at 65 °C overnight
and then allowed to cool. The solvent was evaporated, and the
residue purified by reverse-phase HPLC to afford 9d as a white
solid (1.2 mg, 2%); 99% purity by HPLC (2-90% MeCN/H2O
over 6 min; tR=2.91 min). 1H NMR (400 MHz, DMSO-d6) δ
1.56 (d, J=7.0 Hz, 6H), 1.98 (m, 2H), 2.20-2.35 (m, 4H), 2.43
(m, 1H), 2.46 (m, 2H), 2.82 (m, 4H), 2.89 (s, 3H), 3.00 (m, 2H),
3.15-3.33 (br m, 6H.), 4.08 (m, 2H), 4.18 (q, J=6.5 Hz, 1H),
7.33 (t, J=7.6 Hz, 1H), 7.71 (td, J=1.8, 7.3 Hz, 1H), 7.87 (d, J=
8.8 Hz 5H), 7.99 (dd, J=1.6, 7.8 Hz, 1H), 8.78 (s, 1H), 10.44 (d,
J=7.2 Hz, 1H). MS m/z: 530.4 (M þ Hþ). HRMS calcd for
C27H40N5O4S, 530.2801; found, 530.2817.
1
purity >99% by chiral CE. H NMR (CD3OD, 400 MHz) δ
(ppm) 1.68 (d, J=6.8 Hz, 6H), 1.72 (br d, J=14.5 Hz, 2H), 2.10
(br s, 4H), 2.28 (m, 2H), 2.36 (m, 1H), 2.42 (m, 1H), 2.47-2.53
(m, 2H), 2.65 (m, 4H), 2.85 (s, 3H), 3.24 (t, J=4.9 Hz, 4H), 3.29
(br s, 1H), 3.36 (br s, 1H), 3.86 (m, 1H), 4.19 (t, J=6.6 Hz, 1H),
5.50 (br s, 1H), 7.34 (t, J=7.2 Hz, 1H), 7.72 (td, J=1.5, 7.2 Hz,
1H), 7.83 (td, J=1.4, 7.6 Hz, 1H), 8.72 (s, 1H). 13C NMR (400
MHz, DMSO-d6) δ 19.9, 26.2, 26.4, 34.2, 36.8, 46.2, 53.3, 57.6,
59.1, 59.5, 62.7, 67.9, 115.9, 120.6, 121.9, 123.3, 131.9, 133.2,
140.0, 143.6, 162.0, 162.8. MS m/z: 560.5 (M þ H)þ. HRMS
calcd for C28H42N5O5S, 560.2907; found, 560.2907.
General Procedure for Compounds 6n, 7n, 6q, 7q, 6t, 6u, 7t, 8b,
8d, 9b, 9d, 15, 17 (Method I): 4-Amino-5-chloro-N-{1-[7-(4-
methanesulfonyl-piperazin-1-yl)-heptyl]-piperidin-4-yl}-2-meth-
oxy-benzamide 2TFA (8d). Compound 1c (5.69 g, 11.1 mmol)
3
and 7-bromo-1-heptanol (1.9 equiv) were dissolved in MeCN
(35 mL). Et3N (3.9 equiv) was added, and the reaction was
stirred at 65 °C for 16 h. The reaction was then diluted with
EtOAc (200 mL), washed (saturated NaHCO3 then brine), and
evaporated to afford 4-amino-5-chloro-N-[1-(7-hydroxy-
heptyl)-piperidin-4-yl]-2-methoxy-benzamide as a clear oil
(4.51 g). The oil was dissolved in CH2Cl2 (70 mL) and DMSO
(25 mL) and cooled to 0 °C. DIPEA (3 equiv) was added,
followed by sulfur trioxide pyridine complex (2.5 equiv). The
Acknowledgment. We greatly appreciate the contributions
of Drs. John Jacobsen, Uwe Klein, and Zhengtian Gu to this
manuscript.

5342 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
McKinnell et al.
Supporting Information Available: Biological assay details,
and characterization data on compounds 6a-u, 7a-u, 8a-e,
9a-e, 10-24. This material is available free of charge via the
Internet at http://pubs.acs.org.
C. Tegaserod, a 5-HT4 receptor partial agonist, accelerates gastric
emptying and gastrointestinal transit in healthy male subjects.
Aliment. Pharmacol. Ther. 2001, 15, 1745–1751.
(23) Muller-Lissner, S. A.; Fumagalli, I.; Bardhan, K. D.; Pace, F.;
Pecher, E.; Nault, B.; Ruegg, P. Tegaserod, a 5-HT4 receptor
partial agonist, relieves symptoms in irritable bowel syndrome
patients with abdominal pain, bloating and constipation. Aliment.
Pharmacol. Ther. 2001, 15, 1655–1666.
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