Synthesis and pharmacological properties of novel hydrophilic 5-HT 4 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2010.10.011

Serotonin (5-hydroxytryptamine, 5-HT) is an important signalling molecule in the human body. The 5-HT₄ serotonin receptor, coupled to the G protein G_s, plays important physiological and pathophysiological roles in the heart, urinary

Full text of DOI:10.1016/j.bmc.2010.10.011

Bioorganic & Medicinal Chemistry 18 (2010) 8600–8613
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological properties of novel hydrophilic 5-HT4
receptor antagonists
Bjarne Brudeli a, , Lise Román Moltzau b, , Kjetil Wessel Andressen b, Kurt A. Krobert b,
Jo Klaveness a,c, Finn Olav Levy b,
⇑
a Drug Discovery Laboratory AS, Oslo Research Park, Oslo, Norway
b Department of Pharmacology and Center for Heart Failure Research, Faculty of Medicine, University of Oslo and Oslo University Hospital, PO Box 1057 Blindern, N-0316 Oslo, Norway
c Department of Medicinal Chemistry, School of Pharmacy, University of Oslo, N-0316 Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Serotonin (5-hydroxytryptamine, 5-HT) is an important signalling molecule in the human body. The 5-
HT4 serotonin receptor, coupled to the G protein Gs, plays important physiological and pathophysiological
roles in the heart, urinary bladder, gastrointestinal tract and the adrenal gland. Both 5-HT4 antagonists
and agonists have been developed in the aim to treat diseases in these organs. 5-HT4 agonists might have
beneficial effects in the central nervous system (CNS) and therefore, 5-HT4 antagonists might cause CNS
side effects. In this study, we have developed new amphoteric 5-HT4 antagonists. A series of cyclic indole
amide derivatives possessing an oxazine ring and a piperidine alkane carboxylic acid side chain and the
corresponding prodrug esters were synthesized and their binding to 5-HT4 receptors and antagonist
properties were evaluated. In addition, an indole ester without the oxazine ring and the corresponding
indole amide derivatives were also tested. Octanol–water distribution (Log DOct7.4) was tested for some
of the synthesized ligands. The main structure–affinity characteristics of the 5-HT4 compounds tested
were that the prodrug esters show higher affinity than their corresponding free acids, indole esters show
higher affinity than the corresponding amides and ligands containing the oxazine ring in the indole skel-
eton show higher affinity than indole derivatives not containing the ring. One representative prodrug
ester and its corresponding free acid were tested for binding on a panel of receptors and showed pre-
served selectivity for the 5-HT4 receptor. These new molecules may be useful to target peripheral 5-
HT4 receptors.
Received 23 November 2009
Revised 22 September 2010
Accepted 6 October 2010
Available online 12 November 2010
Keywords:
5-HT
5-Hydroxytryptamine
Serotonin
5-HT4 antagonist
Hydrophilic
Indole amide
Indole ester
Prodrug esters
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
syndrome (IBS) patients, whereas SB207266 (piboserod), an antag-
onist of 5-HT4 receptors was effective in the preliminary clinical
Serotonin (5-HT) is an important signalling molecule in the hu-
man body and has important effects both as a centrally acting neu-
rotransmitter and a locally acting signalling molecule. The 14
different 5-HT receptors, divided into seven subfamilies (5-HT1–
7), are all G-protein-coupled receptors, except the 5-HT3 receptor
which is a ligand-gated ion channel. The 5-HT4 receptor stimulates
adenylyl cyclase (AC) through Gs.1 There are at least 10 splice vari-
studies in patients with diarrhoea-predominant IBS.9 In the heart
atrium, 5-HT4 antagonists may prevent atrial fibrillation as they
block arrhythmic contractions induced by 5-HT.10 In addition, a
5-HT4-mediated inotropic response to serotonin was observed in
the ventricle of porcine and failing human hearts11 and both in-
farcted and failing rat hearts, but was absent in normal rat hearts.12
In vivo cardiac function and ex vivo myocardial function improved
in rats with heart failure treated for 6 weeks with the 5-HT4 recep-
tor antagonist SB207266 (piboserod),13 in accordance with a pro-
posed beneficial role of 5-HT4 receptor antagonists in the
treatment of heart failure.14 This was supported by a recently con-
cluded phase II clinical trial of 24-week treatment of heart failure
patients with SB207266, which showed increased left ventricular
ejection fraction in the patients treated with SB207266 compared
to placebo.15 The 5-HT4 receptor mediates contractile effects in
the urinary bladder. In clinical studies with the 5-HT4 agonists
metoclopramide and cisapride facilitation of urinary bladder emp-
tying and occasional urinary incontinence was observed.16 The
ants of the human 5-HT4 receptor (5-HT4(a–i)
, (hb) and (n)) with very
similar pharmacological properties.2–8
In addition to the central nervous system (CNS), 5-HT4 receptors
are found in peripheral organs like the gastrointestinal tract, heart
and urinary bladder, and may be implicated in diseases in these or-
gans. 5-HT4 agonists or partial agonists (e.g. cisapride, tegaserod)
have shown effects in constipation-predominant irritable bowel
⇑
Corresponding author. Tel.: +47 22840237; fax: +47 22840202.
E-mail address: f.o.levy@medisin.uio.no (F.O. Levy).
These authors contributed equally.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.10.011

B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
8601
acetone at reflux in the presence of potassium carbonate. The in-
dole-3-carboxoyl chloride was prepared from the corresponding
acid with oxalyl chloride in tetrahydrofuran according to Gaster
et al.21 The coupling of intermediate 2 with indole-3-carboxoyl
chloride was achieved in a mixture of dichloromethane and tetra-
hydrofuran in the presence of triethylamine at room temperature.
The trichloroethyl ester 3 was isolated after purification by column
chromatography in 26% yield. Deprotection to the free acid 4 was
achieved with zinc in a mixture of aqueous phosphate buffer and
tetrahydrofuran in 84% yield.
H-bond acceptor
Basic N
6.8 Å
O
3.4-3.7 Å
N
Liphophilic moiety
O
8.0-8.2 Å
Aromatic ring
H-bond acceptor
Figure 1. A proposed pharmacophore for the 5-HT4 receptor (adapted from
Target compounds
7 and 8 were prepared according to
Clark).20
Scheme 2. 1-Benzyl-4-aminomethylpiperidine22 was coupled with
indole-3-carboxoyl chloride in a mixture of dichloromethane and
tetrahydrofuran in the presence of triethylamine at room temper-
ature. The intermediate 5 was isolated after purification by column
chromatography in 29% yield. The benzyl protected amine 5 was
deprotected to intermediate 6 with hydrazine monohydrate in eth-
anol at reflux in 93% yield. The indole amide 7 was prepared by N-
alkylation of 6 with ethyl 4-bromobutyrate in acetone at reflux in
the presence of potassium carbonate in 75% yield. The free acid 8
was prepared by hydrolysis of the ethyl ester 7 with potassium
hydroxide in a mixture of water and methanol at reflux in 61%
yield.
Synthesis of target compounds 9 and 26 are outlined in
Scheme 3. The prodrug esters 10–23 (Table 2) were all prepared
by N-alkylation of 3,4-dihydro-N-[[4-piperidinyl]methyl]-2H-
[1,3]oxazino[3,2-a]indole-10-carboxamide23 (6) with different
halo alkyl esters in acetone at reflux in the presence of potassium
carbonate. The compounds were purified with column chromatog-
raphy in 50–80% yield.
selective 5-HT4 antagonist SB207266 antagonized the ability of 5-
HT to enhance contraction in isolated human bladder strips.17
Since 5-HT4 receptors are involved in aldosterone secretion by
the adrenal gland in man,18 5-HT4 antagonists might be useful in
treatment of hyperaldosteronism, which may cause harmful car-
diovascular effects.
In the central nervous system (CNS), 5-HT4 receptors are in-
volved in the release of neurotransmitters such as acetylcholine
(ACh), 5-HT and dopamine, and 5-HT4 agonists may improve mem-
ory, cognitive and learning functions.16 It is therefore possible that
5-HT4 antagonists which access the CNS would have undesirable
side effects.
Another problem in drug development is a possible QT prolon-
gation due to human ether-a-go-go-related gene (hERG) potassium
channel binding in the heart. Incorporation of a carboxylic acid
group on benzamidine compounds with high hERG binding re-
duced this binding dramatically.19 Since incorporating a carboxylic
acid group in a 5-HT4 antagonist molecule would thus potentially
both reduce CNS penetration due to reduced lipophilicity and also
minimise hERG binding, we decided to test whether such 5-HT4
antagonists could be made without compromising 5-HT4 receptor
binding affinity, antagonist activity and selectivity.
Adhering to the structural requirements of 5-HT4 antagonists
(an aromatic ring, at least one hydrogen bond acceptor, a basic
nitrogen and a hydrophobic group, see pharmacophore model in
Fig. 1),20 we synthesized 27 indole-derivatives and tested their
binding affinity and antagonist activity at the 5-HT4 receptor.
Although the prodrug esters show better potencies than their cor-
responding free acids, the results demonstrate that this approach is
feasible and may be useful in development of antagonists targeting
5-HT4 receptors outside the CNS.
Table 1
Structures, pKi-values from binding and pKb-values from adenylyl cyclase assay of
GR113808 and compounds 4, 7 and 8
R'
N
X
O
N
R
Compound
GR113808
X
O
R
R’
pKi SEMa
pKb SEMb
NHSO2Me
OH
Me
10.13 0.07 9.24 0.06
9.09 0.16 8.16 0.08
4
7
8
O
H
H
H
2. Chemistry
O
O
CH3
All of the new derivatives synthesized and tested had minor dif-
ferences from potent and well known 5-HT4 antagonists. Figure 2
shows known 5-HT4 antagonists.
The new amphoteric compounds 4, 8, 26–32 and the prodrugs
7, 9–25 are shown in Tables 1 and 2.
NH
NH
6.97 0.05 5.84 0.21
5.44 0.01 4.62 0.26
O
OH
O
The target compound 4 was prepared according to Scheme 1.
The piperidine intermediate 2 was prepared by N-alkylation of 4-
piperidinemethanol with 2,2,2-trichloroethyl 4-bromobutyrate in
a
n = 3, except for GR113808 where n = 5.
n = 2, except for GR113808, where n = 12. Where n = 2, the values are given as
b
pKb half-range.
NHSO2Me
NHSO2Me
N
N
N
H
O
N
O
O
O
O
O
O
F
O
O
OMe
N
N
N
N
Me
Me
O
SB207266
SB204070
GR113808
GR125487
Figure 2. Known 5-HT4 receptor antagonists.

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B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
Table 2
Structures, pKi-values from competition of [3H]GR113808 binding and pKb-values from antagonism of 5-HT-stimulated adenylyl cyclase activity of SB207266 and compounds 9–
32
R
N
H
O
N
O
N
Compound
SB207266
R
pKi SEMa
pKb SEMb
CH3
10.28 0.15
9.26 0.08
O
CH3
9
10
11
12
13
14
15
—
—
O
O
—
—
CH3
O
O
O
CH3
9.99 0.17
9.43 0.18
O
—
—
CH3
O
O
CH3
9.97 0.16
9.71 0.11
9.47 0.16
9.13 0.12
9.33 0.06
9.89 0.12
O
O
CH3
O
O
CH3
O
O
16
—
—
CH3
O
H3C
CH3
O
17
18
9.12 0.05
9.63 0.11
9.01 0.20
9.47 0.29
O
CH3
H3C
CH3
O
O
CH3
H3C
CH3
O
19
20
10.27 0.14
8.90 0.10
9.89 0.16
8.64 0.18
O
CH3
O
OEt
P
OEt
O
21
22
23
24
10.37 0.20
11.84 0.52
9.85 0.25
9.47 0.43
9.85 0.15
10.18 0.20
10.16 0.22
9.07 0.18
O
O
O
O
O
O
CH3
O
CH3
CH3
O
O

B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
8603
Table 2 (continued)
Compound
R
pKi SEMa
pKb SEMb
CH3
O
O
25
10.19 0.25
9.83 0.28
CH3
CH3
O
O
OH
O
26
27
28
29
30
31
8.55 0.04
8.95 0.16
8.70 0.08
8.69 0.13
8.81 0.15
8.61 0.02
7.91 0.25
8.08 0.19
7.96 0.02
8.10 0.13
7.94 0.06
8.97 0.18
O
OH
OH
O
O
OH
OH
O
OH
O
OH
32
8.30 0.15
7.25 0.07
O
a
n = 3–6.
b
n = 2–3, except for SB207266 where n = 13. Where n = 2, the values are given as pKb half-range.
b
a
OCH2Cl3
OCH2Cl3
OH
Br
N
Br
HO
O
O
O
2
1
O
OH
CH2CCl3
N
3
N
O
O
O
O
O
O
d
c
4
N
H
N
H
Scheme 1. Reagents and conditions: (a) HOCH2CCl3, pTsOH, toluene, reflux; (b) 4-piperidinemethanol, K2CO3, acetone, reflux; (c) indole-3-carboxoyl chloride, NEt3,
tetrahydrofuran/dichloromethane, room temperature; (d) zinc dust, 1 M KH2PO3/tetrahydrofuran, room temperature.
The amphoteric drugs 26–32 (Table 2) were prepared by hydro-
lysis of the corresponding esters 10–23 with potassium hydroxide
in a mixture of water and methanol in 60–70% yield.
GR113808, SB207266 and compounds 4 and 7–32 are summarized
in Tables 1 and 2.
The double esters 24 and 25 were prepared by alkylating the
free acids 32 and 30, respectively, with chloromethyl pivalate in
dimethylformamide at reflux in the presence of caesium carbonate
as outlined in Scheme 4. The compounds were purified with col-
umn chromatography in 59–66% yield.
4. Results and discussion
The results from the binding experiments (Tables 1 and 2) dem-
onstrate that many of the amphoteric compounds and their corre-
sponding ester prodrugs possess moderate to high affinity for the
5-HT4 receptor.
3. Pharmacology
The amphoteric indole-3-carbonyl ester 4 has excellent recep-
tor binding (pKi 9.09 0.16, pKb 8.16 0.08). The binding data for
the corresponding amide 8 reveals much lower affinity measured
in both binding and AC assays (pKi 5.44 0.01, pKb 4.62 0.26).
Switching from an ester to an amide seems to have a detrimental
effect on receptor binding (Fig. 3). This has also been shown for
tropisetron, a ligand with affinity for both 5-HT3 and 5-HT4 recep-
tors, where replacement of the ester with an amide linkage de-
creased the 5-HT4 receptor affinity.25 Other reports suggest that
5-HT4 naphthalene derivatives with an ester linkage display higher
Using membranes from HEK293 cells stably expressing the hu-
man 5-HT4(b) receptor,24 competitive binding and concentration-
dependent inhibition of 5-HT-stimulated adenylyl cyclase activity
was studied for GR113808, SB207266 and compounds 4, 7, 8, 11,
13–15 and 17–32 to determine binding affinities and 5-HT4 antag-
onist properties of the compounds. pKi-values (ꢀLog Ki) from com-
petition of [3H]GR113808 binding and pKb-values (ꢀLog Kb) from
antagonism of 5-HT-stimulated adenylyl cyclase (AC) activity of

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B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
N
H
NH
H
N
O
Cl
O
N
O
a
b
N
H
N
H
N
H
5
6
O
OH
Et
N
N
8
H
N
H
N
O
O
O
O
c
d
N
H
N
H
7
Scheme 2. Reagents and conditions: (a) 1-benzyl-4-aminomethylpiperidine, NEt3, dichloromethane, room temperature; (b) NH2NH2ꢁH2O, Pd–C 10%, EtOH, reflux; (c) ethyl 4-
bromobutyrate, K2CO3, acetone, reflux; (d) KOH, H2O/MeOH, reflux.
O
OH
NH
CH3
N
N
H
N
H
N
H
N
O
9
O
O
O
O
a
b
O
O
O
N
N
N
26
Scheme 3. Reagents and conditions: (a) methyl 2-bromoacetate, K2CO3, acetone, reflux; (b) KOH, H2O/MeOH, reflux.
CH3
O
OH
O
CH3
CH3
N
N
H
N
H
N
O
O
O
O
O
a
O
O
N
N
30
25
Scheme 4. Reagents and conditions: (a) chloromethyl pivalate, Cs2CO3, DMF, reflux.
affinities than their corresponding amides, and that this might be a
result of conformational differences.26 However, due to the possi-
ble lability of ester compounds in vivo, amide was preferred as lin-
ker between the aromatic indole and piperidine ring systems for
compounds 9–32.
The introduction of a [3,2-a] oxazino ring on indole amides 7
(pKi 6.97 0.05, pKb 5.84 0.21) and 8 (pKi 5.44 0.01, pKb
4.62 0.26) increases receptor binding. The corresponding [3,2-a]
oxazino amides 11 (pKi 9.99 0.17, pKb 9.43 0.18) and 28 (pKi
8.70 0.08, pKb 7.96 0.02) show considerably higher binding
affinity (Table 2, Fig. 4). These results might be explained by the
pharmacophore model, where both the carbonyl group and the
oxygen in the oxazino ring can act as hydrogen bond acceptors,
but also because of a more favourable planar conformation for
the oxazino derivatives.
The binding and AC data for the amphoteric compounds 26–31
reveal that they are promising candidates as antagonists for the 5-
HT4 receptor. Binding affinities determined from binding and AC
assays range from pKi 8.55 0.04 to 8.95 0.16 and pKb
7.91 0.25 to 8.97 0.18, respectively. The pharmacophore model
predicts a requirement of a hydrophobic group attached to the ba-
sic nitrogen in the piperidine ring. We therefore predicted increas-
ing affinity to the receptor with increasing length and
hydrophobicity of the alkyl chain attaching the hydrophilic group.
However, the binding affinities do not seem to increase or decrease
with increasing length of the side chain. The same lack of trend can
be seen for the corresponding carboxylic esters 11 and 13–15. Fur-
ther molecular modelling studies are needed to investigate the role
of the hydrophobic pocket in receptor–ligand interaction of the
new ligands. Previous studies have shown the importance of the
hydrophobic pocket in receptor binding.27,28 Also the need for
voluminous substituents to obtain selectivity for the 5-HT4 recep-
tor over 5-HT3 has previously been reported.29 Further synthetic
and receptor binding studies are in progress to evaluate new
amphoteric 5-HT4 antagonists comprising different voluminous
substituents with acidic moieties.
Amphoteric compounds in general have low bioavailability.30
This reduced bioavailability is most likely caused by a lack of lipo-
philicity. The corresponding ester prodrugs to the amphoteric com-
pounds were therefore prepared and since these compounds are
expected to have some biological activity the receptor binding
and AC activity were determined. The prodrug esters seem to dis-
play better potencies than their corresponding free acids. The piv-
olate ester 25 (pKi 10.19 0.25, pKb 9.83 0.28), the benzyl ester
23 (pKi 9.85 0.25, pKb 10.16 0.22), the tert-butyl ester 19 (pKi
10.27 0.14, pKb 9.89 0.16) and the methyl ester 13 (pKi
9.97 0.16, pKb 9.13 0.12) show better binding affinity than the
corresponding free acid 30 (pKi 8.81 0.15, pKb 7.94 0.06). The
same characteristic is seen for the ethyl ester 11 (pKi 9.99 0.17,
pKb 9.43 0.18) and the methyl ester 15 (pKi 9.47 0.16, pKb
9.89 0.12) compared to their corresponding free acids 28 (pKi
8.70 0.08, pKb 7.96 0.02) and 31 (pKi 8.61 0.02, pKb

B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
8605
A 300
A
300
250
200
150
100
50
250
200
150
SB207266
100
50
0
7
8
SB207266
11
28
4
8
0
-13 -12 -11 -10
-9
-8
-7
-6
-5
-4
-13
-12
-11
-10
-9
-8
-7
-6
-5
-4
B 100
B
100
80
60
40
20
0
80
60
40
20
0
SB207266
7
SB207266
8
4
8
11
28
-12 -11 -10
-9
-8
-7
-6
-5
-4
-3
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
Log [compound] (M)
Log [compound] (M)
Figure 3. Competition binding curves (0.3 nM [3H]GR113808 present) (panel A)
and concentration–response curves of the inhibition of 5-HT (1 M)-stimulated
adenylyl cyclase (AC) activity (panel B) with an ester (ligand 4) and the
corresponding amide (ligand 8). The data shown in panels A and B are from one
Figure 4. Competition binding curves (0.3 nM [3H]GR113808 present) (panel A)
and concentration–response curves of the inhibition of 5-HT (1 M)-stimulated
adenylyl cyclase (AC) activity (panel B) of ligands 7 and 8 not containing the
oxazino ring in indole position, and their respective corresponding drugs
l
l
2
representative experiment. The basal and the maximum 5-HT (1
l
M)-stimulated
containing the oxazino ring, ligands 11 and 28. Panel A: one representative
AC activity in panel B was 25 and 148 pmol cAMP/mg protein/min, respectively.
experiment, panel B: data compiled from three representative experiments. The
basal and the maximum 5-HT (1 lM)-stimulated AC activities in panel B was 21–27
and 127–149 pmol cAMP/mg protein/min, respectively.
8.97 0.18), respectively. This seems to be a trend as all the esters
show higher pKi and pKb values than their corresponding free acids
(where this was tested) (Table 2, Fig. 5).
whereas compounds with good CNS penetration will have an opti-
mal Log DOct7.4 value of approximately 2.33 The logarithmic distri-
bution coefficients for amphoteric compounds 26–31 were
experimentally determined to range from ꢀ1.1 to ꢀ1.3, indicating
low CNS penetration, whereas the distribution coefficient of the
corresponding prodrug esters 13, 24 and SB207266 were deter-
mined to 2.2–2.3 indicating potentially good CNS penetration
(Table 3).
Cisapride, a partial 5-HT4 agonist used in treatment of gastro-
oesophageal reflux, has been withdrawn from the market or is
now used with more restricted indications because of prolongation
of QT intervals.34 Due to structural similarities between 5-HT4 ago-
nists and antagonists it was decided to determine the effect of
compounds 22 and 29 on hERG (human ether-a-go-go related
gene) tail current. Compounds that inhibit hERG current have been
shown to prolong the cardiac action potential and hence QT inter-
val in man.35 The benzyl ester prodrug 22 and the free carboxylic
The amphoteric compounds 26–32 may be derivatized to pro-
drug esters to obtain sufficient bioavailability. These prodrug com-
pounds could be either metabolized by first pass metabolism in the
liver or be hydrolysed in plasma to free acid. Further investigations
are necessary to address the degree of liver metabolism of the new
ligands, but the plasma stability of the corresponding prodrugs of
compound 28 were evaluated by incubating the ethyl ester 11,
benzyl ester 21 and tert-butyl ester 18 in bovine plasma. The con-
centration of the prodrugs and degradation products were fol-
lowed by HPLC. The benzyl ester 21 and ethyl ester 11 were fully
metabolized to the free acid 28 in bovine plasma after 10 h, and
the half-life calculated to be approximately 20 and 30 min, respec-
tively. The tert-butyl ester 18 was only metabolized to a minor ex-
tent, and the incubation time of 10 h was insufficient to obtain any
half-life value. This is in agreement with earlier studies, where tert-
butyl esters have been shown to be stable against hydrolysis in
plasma.31 However, further in vivo studies of 11, 21 and 28 are nec-
essary to obtain more precise bioavailability data.
acid 29, both tested at a concentration of 10 lM, reduced the hERG
tail current to 6.6% 1.4% and 93.5% 2.5%, respectively. Assuming
an inhibition curve of the hERG tail current with a Hill coefficient
Different methods have been used to predict the ability of a
compound to reach the central nervous system. One of the most
important factors used to explain the brain penetration is the par-
tition coefficient (P) between octanol and water.32 For ionizable
compounds both the pKa (acid dissociation constant) values and
the partition coefficient is important, and the distribution coeffi-
cient between octanol and an aqueous buffer of pH 7.4 (DOct7.4) is
therefore commonly used. According to literature, when Log DOct7.4
is smaller than zero, brain penetration is expected to be negligible,
of one this would predict
a
ꢀlog IC50 of 6.15 0.10 and
3.84 0.18 for compounds 22 and 29, respectively. Despite the
small number of test compounds, incorporation of a free carboxylic
acid group seems to be important in preventing hERG blockade.
This supports data shown earlier by others.19
To reduce undesired side effects, it is important that the com-
pounds are selective for the 5-HT4 receptor. The free carboxylic
acid compound 30 and the corresponding ester 13 were tested
for binding to a panel of other receptors than the 5-HT4 receptor;

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B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
C
A
B
300
300
250
200
150
100
50
250
200
150
GR113808
GR113808
SB207266
SB206277
100
50
0
15
31
17
26
11
18
21
28
0
-14 -13 -12 -11 -10 -9
-8
-7
-6
-5
-4
-13 -12 -11 -10
-9
-8
-7
-6
-5
-4
D
100
80
60
40
20
0
100
80
60
40
20
0
SB207266
SB207266
15
31
17
26
11
18
21
28
-12 -11 -10
-9
-8
-7
-6
-5
-4
-3
-12 -11 -10
-9
-8
-7
-6
-5
-4
-3
Log [compound] (M)
Log [compound] (M)
Figure 5. Competition binding curves (0.3 nM [3H]GR113808 present) (panels A and C) and concentration–response curves of the inhibition of 5-HT (1
lM)-stimulated
adenylyl cyclase activity (panels B and D) of the esters 15 and 17 and their respective corresponding free acids, ligands 31 and 26 (panels A and B) and of the free acid 28 and
its corresponding esters 11, 18 and 21 (panels C and D). Panels A and C: one representative experiment, panels B and D: data compiled from three and four representative
experiments, respectively. The basal AC activities in panels B and D were 17–34 and 21–25 pmol cAMP/mg protein/min and the maximum 5-HT (1
activities were 90–158 and 125–154 pmol cAMP/mg protein/min, respectively.
lM)-stimulated AC
the serotonin receptors 5-HT1A 1B 1D 1E 2A 2B 2C 3 5A 6 7
,
,
,
,
,
,
, , , , , the adrener-
5-HT4 antagonists in this study possess higher pKi-values (compe-
gic receptors a1A 1B 1D 2A 2B 2C, b1,2,3, the dopamine receptors
,
,
,
a
,
,
tition with [3H]GR113808) than pKb-values (competition with 5-
HT).
D1,2,3,4,5, histamine receptors H1,2,3,4, muscarinic acetylcholine
receptors M1,2,3,4,5, the opioid receptors DOR, MOR and KOR, Sigma
1 and 2, the benzodiazepine binding site, the GABAA receptor
(muscimol site) and the monoamine transporters DAT (dopamine
transporter) and SERT (serotonin transporter). The free carboxylic
acid compound 30 did not show any significant binding (pKi >5)
to the selected receptors. The corresponding ester 13 showed bind-
ing to some of the selected receptors, but with low affinity (sum-
marized in Table 4). Both compounds 13 and 30 show preserved
selectivity for the 5-HT4 receptor as the affinity for the 5-HT4
receptor is at least 3 log units higher than for the other receptors
tested, for example compound 13 shows a pKi of 6.5 for the 5-
HT2B receptor compared to 9.97 0.16 for the 5-HT4 receptor.
For all the drugs, except ligands 15, 23 and 31, the pKi-values
calculated from binding assays are higher than the pKb-values cal-
culated from adenylyl cyclase assays. These values are usually ex-
pected to be similar. Figure 6 shows the correlation between pKi-
values determined in binding assays and pKb-values from adenylyl
cyclase assays. By linear regression the slope was determined to be
0.99 0.08 (r2 = 0.86). As the slope is close to 1, the difference be-
tween pKi and pKb seems to be a methodological issue and not rep-
resent differences in the ability of the 5-HT4 antagonists to bind to
the receptor versus block an agonist activity. Recently, Mikami
et al. demonstrated a lower binding affinity for 5-HT4 antagonists
when using [3H]5-HT than when using [3H]GR113808 in binding
assays at the 5-HT4(d) receptor. The opposite was demonstrated
with agonists, whereas partial agonists showed similar binding
affinities with both radioligands.36 If this is also true for the
5-HT4(b) receptor used in this study, this might explain why the
In conclusion, a new series of amphoteric 3,4-dihydro-N-[[4-
piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide
derivatives 26–32 have been prepared and show promising bind-
ing affinities and potencies as novel 5-HT4 antagonists. The loga-
rithmic octanol/buffer distribution coefficients of the amphoteric
compounds may predict reduced CNS distribution. Additionally
the hydrophilic characteristics of the free carboxylic acids might
also reduce binding to the hERG potassium channel. In summary
the hydrophilic properties may predict reduced probability of
CNS side effects as well as reduced probability of hERG channel
blockade. To obtain acceptable bioavailability it may be necessary
to give the amphoteric derivatives as a prodrug ester 9–25. The re-
sults from the binding studies of the corresponding esters showed
higher receptor binding than the corresponding free acid. Further
studies with amphoteric 5-HT4 antagonists to optimise the phar-
macological binding profile and to better clarify bioavailability
are in progress.
5. Experimental section
5.1. Pharmacology
5.1.1. Membrane preparation
The development of human embryonic kidney (HEK293) cell
lines stably expressing human 5-HT4(b) receptors was described
and published previously.24 The stably transfected HEK293 cells
were cultured in 150 mm cell culture dishes. The cells were

B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
8607
Table 3
Harvester (Packard Instrument Co.). The membranes were washed
6–8 times with cold (4 °C) washing buffer (50 mM Tris–HCl, pH 7.0
Log DOct7.4 values of 5-HT4 antagonists
R
at 20 °C, 2 mM MgCl2). Filters were dried and 20 ll MicroScint
scintillation fluid (Packard Instrument Co.) were added to each
N
H
N
O
well. The filters were counted in a Top-Count Liquid scintillation
counter (Packard). In radioligand binding assays,
a cell line
expressing the 5-HT4(b) receptors at high levels (5000 fmol/mg pro-
tein) was used. In all experiments, GR113808 or SB207266 were
used as controls.
O
N
Compound
SB207266
R
Log DOct7.4
5.1.3. Adenylyl cyclase assay
The adenylyl cyclase activity was measured by determining the
CH3
2.2
conversion of [
cell line expressing the h5-HT4(b) receptor. Adenylyl cyclase activ-
ity was measured in 10 l of crude membrane preparations in a fi-
nal volume of 50 l in the presence of 0.1 mM [
32P]-ATP, 1 mM
cAMP, 1 mM EDTA, 1 mM [3H]cAMP (ca 10,000 cpm/assay), 4 mM
MgCl2, 1 mM IBMX (3-isobutyl-1-methylxanthine), 20 M GTP
a-
32P]-ATP to [32P]cyclic AMP in membranes from a
O
CH3
O
13
2.2
l
O
l
a-
CH3
O
24
2.3
CH3
CH3
l
and an ATP-regenerating system consisting of 20 mM creatine
phosphate, 0.2 mg/ml creatine phosphokinase and 40 U/ml myoki-
nase. Ligands were used in 21 different concentration in the range
O
O
OH
26
27
28
29
30
31
ꢀ1.1
ꢀ1.3
ꢀ1.2
ꢀ1.3
M to 9.5 ꢂ 10ꢀ13—9.5 ꢂ 10ꢀ11 M inhibiting the adenylyl
O
of 1–100
l
O
cyclase activity stimulated by 1 lM 5-HT. Incubations were at
32 °C for 20 min. The reaction products were separated by sequen-
tial chromatography on Dowex 50 cation exchanger and on neutral
aluminium oxide (Alumina) columns and cAMP formed quantified
as originally described by Salomon et al.37 with minor modifica-
tions.38 In adenylyl cyclase assay a cell line expressing the 5-
HT4(b) receptor at low levels (400 fmol/mg protein) were used. In
all experiments, GR113808 or SB207266 were used as controls.
OH
OH
O
O
OH
OH
ꢀ1.3
5.1.4. Analysis of binding and adenylyl cyclase data
Binding and adenylyl cyclase data were analysed by non-linear
regression using MicrosoftÒ Excel with the Solver add-in, using the
below equations.
O
OH
a
O
a
Micelle formation. Log DOct7.4 value not determined.
5.1.4.1. Competitive binding assay.
The data were fit to the
equation:
washed twice with 10 ml ice cold Hanks Balanced Salt Solution
(HBSS) and scraped with a rubber scraper in 10 ml HBSS. The cells
were collected by centrifugation at 800g for 5 min at 4 °C. After
centrifugation the cells were resuspended in 1 ml ice cold STE
(27% sucrose (w/w), 50 mM Tris–HCl, pH 7.5 at 20 °C, 5 mM EDTA)
and homogenised with Ultra-Turrax T8 (IKA Labortechnik) 30 s
chilled in ice-water. The homogenate was centrifuged for 20 min
at 27,000g at 4 °C. The supernatant was discarded and the pellet
resuspended in 1 ml ice cold TE (50 mM Tris–HCl pH 7.5 at 20 °C,
1 mM EDTA) using a cold Dounce glass-glass homogeniser, 10
strokes with a tight-fitting pestle, aliquoted and snap-frozen in li-
quid N2 and stored at ꢀ70 °C until use for assays. The membranes
were re-homogenised in a cold Dounce glass-glass homogeniser
before each assay. In radioligand binding assays and adenylyl cy-
clase assays, cell lines expressing the 5-HT4(b) receptor at high
and low levels, respectively, were used.24
Ki ¼ ðIC50Þ=ð1 þ ðL=KdÞÞ
Ki is the affinity constant of the unlabelled competitor for the recep-
tor. IC50 is the concentration of unlabelled ligand that inhibits the
binding of the radioactive ligand by 50%. L is the concentration of
the radiolabelled ligand used. Kd is the affinity constant for the
radiolabelled ligand.
5.1.4.2. Activation of adenylyl cyclase.
equation:
The data were fit to the
Y ¼ a þ ðb ꢀ aÞx=ðc þ xÞ
where a is basal adenylyl cyclase activity, b is maximal adenylyl cy-
clase activity stimulated by the agonist, c is EC50 (agonist concentra-
tion at 50% of maximal response) and x is the concentration of
agonist.
5.1.2. Radioligand binding assay for the 5-HT4 receptor
The radioligand binding assays were performed in 96-well,
round bottom microtiter plates with the total reaction volumes
5.1.4.3. Inhibition of 5-HT-evoked adenylyl cyclase stimula-
tion.
The data were fit to the equation:
of 50–200 l
l, containing the 5-HT4 specific ligand [3H]GR113808
Y ¼ a þ ðb ꢀ aÞ=ð1 þ x=cÞ
(0.3 nM) in the presence of 21 different concentrations of cold li-
gand in the range of 1–100
50 mM Tris–HCl (pH 7.5 at 20 °C), 1 mM EDTA, 5 mM EGTA,
l
M to 9.5 ꢂ 10ꢀ13—9.5 ꢂ 10ꢀ11 M,
where a is basal adenylyl cyclase activity in the presence of saturat-
ing concentrations of antagonist, b is maximal adenylyl cyclase
activity stimulated by the agonist in the absence of antagonist, c
is the IC50 of the antagonist (antagonist concentration when it
inhibits 50% of the maximal response of the agonist) and x is the
concentration of antagonist.
2 mM MgCl2, 1 mM ascorbic acid, 0.1% BSA, 100 lM GTP and mem-
branes. The plates were incubated at room temperature for 1 h and
the membranes harvested onto MultiscreenÒ FC filter (Millipore)
presoaked in 0.3% polyethyleneimine, with
a Packard Cell

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B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
Table 4
pKi determined for ligands 13 and 30
R
N
H
O
N
O
N
pKi ligand 13
pKi ligand 30
O
OH
Receptor
pKi (reference substance)
CH3
R =
R =
O
O
Serotonin receptors
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT2A
5-HT2B
5-HT2C
5-HT3
<5
<5
5.5
<5
5.2
6.5
<5
5.6
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
8.5 (ergotamine)
7.8 (chlorpromazine)
8.7 (methysergide)
8.7 (zacopride)
5-HT5A
5-HT6
5-HT7
Adrenergic receptors
a1A
a1B
a1D
a2A
a2B
a2C
b1
b2
<5
<5
<5
<5
5.9
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
8.1 (yohimbine)
b3
Dopamine receptors
D1
D2
D3
D4
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
D5
Histamine receptors
H1
H2
H3
H4
5.9
5.5
<5
<5
<5
<5
<5
8.5 (chlorpheniramine)
6.5 (cimetidine)
<5
Muscarinic acetylcholine receptors
M1
M2
M3
M4
<5
5.6
<5
<5
<5
<5
<5
<5
<5
<5
8.6 (atropine)
M5
Opioid receptors
DOR
KOR
MOR
Sigma1
<5
<5
<5
6.4
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
8.7 (haloperidol)
Sigma2
BDZ site
GABAA receptor (muscimol site)
Monoamine transporters
DAT
SERT
<5
<5
<5
<5
The Kd values of the ligands were calculated using the equation:
Kd ¼ að1=ð1 þ x=bÞÞ
5.1.6. Selectivity testing on other receptors than the 5-HT4
receptor
Receptor binding profiles and pKi determinations were gener-
ously provided by the National Institute of Mental Health’s Psycho-
Where a is the IC50 value for the antagonist, b is the EC50 value of
the agonist and x is the concentration of agonist.39
active Drug Screening Program, Contract
# HHSN-271-2008-
00025-C (NIMH PDSP). The NIMH PDSP is directed by Bryan L. Roth
MD, Ph.D. at the University of North Carolina at Chapel Hill and
Project Officer Jamie Driscol at NIMH, Bethesda, MD, USA. For
experimental details please refer to the PDSP web site http://
pdsp.med.unc.edu/ and click on ‘Binding Assay’.
5.1.5. Protein measurement
The protein concentrations in the membrane preparations were
measured with the Micro BCA Protein Assay Reagent Kit (Uptima)
using bovine serum albumin (BSA) as standard.

B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
8609
12
10
8
5.4. Chemistry
1H NMR spectra were recorded on a Bruker Spectrospin Avance
200 or 300 MHz spectrometer and the corresponding 13C NMR
spectra at 50 and 75 MHz, respectively. Chemical shifts are re-
ported in parts per million relative to internal tetramethylsilane.
Coupling constants (J) are reported in hertz (Hz). The following
abbreviations are used to describe peak patterns when appropri-
ate: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet),
br (broad). Electron spray mass spectra were recorded at Vitas
AS, Oslo, Norway. GC analyses were carried out on a Shimadzu
GC-14A gas chromatograph equipped with a flame-ionization
6
Slope=0.99 0.08
r2=0.86
4
detector and a 30 m ꢂ 0.25
lm i.d. glass capillary column coated
6
8
10
12
Binding (pKi)
with poly(dimethylsiloxane), at a flow rate of nitrogen of 40 ml/
min. The column temperature was ranged from 80 °C to 200 °C at
8 °C/min. The temperatures of the injector and detector were
250 °C. Analytical thin layer chromatography (TLC) was run on
Merck silica gel plates (Kieselgel 60 F-254) with detection with
UV-light or iodine. For flash chromatography, Fluka silica gel type
60 (size 200–400 mesh) was used. All solvents and reagents were
of analytical or reagent grade and were obtained from commercial
sources.
Figure 6. Correlation between the estimated affinities from competition binding
(pKi) and the inhibition of 5-HT-stimulated adenylyl cyclase (AC) activity (pKb).
Linear regression analysis produced a slope of 0.99 0.08 (r2 = 0.86).
5.1.7. Radiochemicals
[3H]GR113808 (81–84 Ci/mmol),
[a-
32P]ATP (400–3000 Ci/
The following known intermediates and reference compounds
mmol) and [2,8-3H]cAMP (42 Ci/mmol) were from GE Healthcare
(Little Chalfont, Buckinghamshire, UK).
were
synthesized
according
to
literature
procedures:
1-benzyl-4-aminomethylpiperidine,22 methyl 2-(3-chloropropoxy)-
indole-3-carboxylate, methyl 3,4-dihydro-2H-[1,3]oxazino[3,2-a]-
indole-10-carboxylate, 3,4-dihydro-N-[[1-(phenylmethyl)-4-pipe-
ridinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carboxamide,
3,4-dihydro-N-[[4-piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]-
indole-10-carboxamide.23
5.1.8. Drugs
5-Hydroxytryptamine hydrochloride (5-HT, serotonin) was
from Sigma–Aldrich (St. Louis, MO, USA). GR113808 (1-methyl-
1H-indole-3-carboxylic acid, [1-[2-[(methylsulfonyl)amino]ethyl]-
4-piperidinyl]methyl ester) maleate was from Tocris (Avonmouth,
UK). The other compounds tested were synthesized by Drug Dis-
covery Laboratory AS (DDL) (Oslo, Norway).
Spectral data of all described compounds were consistent with
the proposed structures.
5.4.1. 2,2,2-Trichloroethyl 4-bromobutyrate (1)
2,2,2-Trichloroethanol (14.94 g, 0.10 mol) was added to a stir-
red solution of 4-bromobutyric acid (3.34 g, 20.0 mmol) and p-tol-
uenesulfonic acid monohydrate (7.60 g, 40.0 mmol) in toluene
(50 ml). The reaction mixture was heated to reflux with a Dean–
Stark trap attached for 6 h. Water was removed continuously.
The reaction mixture was cooled to room temperature and concen-
trated in vacuo. The mixture was added CH2Cl2 (75 ml) and washed
with H2O (3 ꢂ 25 ml). The organic layer was dried over Na2SO4, fil-
tered and evaporated in vacuo to leave an oil. The residue was dis-
tilled to leave the title compound as a colourless oil (4.77 g, 79.9%)
(bp 100 °C at 0.5 mmHg). 1H NMR (CDCl3): d 4.74 (s, 2H), 3.48 (t,
2H), 2.65 (t, 2H), 2.21–2.13 (m, 2H).
5.1.9. Effect on hERG tail current
The experimental work was carried out at Huntingdon Life Sci-
ences (Cambridgeshire, England). The test compounds were tested
in an in vitro system of HEK293 cells stably transfected with hERG
ion channel cDNA. Using the patch-clamp technique, peak hERG
tail current amplitude was measured prior to and following expo-
sure to a nominal concentration of 10 lM of the compounds, vehi-
cle (0.1% DMSO) and 50 nM terfenadine as control.
5.2. Log DOct7.4. measurements
The 5-HT4 prodrugs and corresponding amphoteric drugs (5 mg
of each compound), were added to a mixture of aqueous 1 M Tris
buffer with pH 7.4 (1.0 ml) and n-octanol (1.0 ml) at ambient tem-
perature. The mixture was vigorously shaken, allowed to reach
equilibrium for 3 days at room temperature before 0.1 ml of the
aqueous and n-octanol solution was removed. The samples were
diluted and the ligand concentration in the two solutions deter-
mined by HPLC.
5.4.2. 2,2,2-Trichloroethyl 4-[1-hydroxymethylperidinyl]buty-
rate (2)
Trichloroethyl ester 1 (4.47 g, 15.0 mmol) was added to a stirred
suspension of 4-piperidinemethanol (1.72 g, 15.0 mmol) and
K2CO3 (4.14 g, 30.0 mmol) in acetone (100 ml). The reaction mix-
ture was heated at reflux for 3 h, cooled to room temperature
and filtered. The filtrate was concentrated in vacuo, the residue
added CH2Cl2 (75 ml) and washed with brine (25 ml) and H2O
(2 ꢂ 25 ml). The organic layer was dried over Na2SO4, filtered and
evaporated in vacuo to leave the title compound as a viscous oil
(4.70 g, 94.1%). 1H NMR (CDCl3): d 4.74 (s, 2H), 3.50 (d, 2H), 2.92
(d, 2H), 2.52–2.35 (m, 4H), 1.97–1.70 (m, 7H), 1.52–1.45 (m, 1H),
1.32–1.23 (m, 2H).
5.3. Metabolic stability in bovine plasma
The tested compounds were added to bovine plasma (Sigma–
Aldrich) and incubated at 37 °C. The initial concentration was
40
lM. At different time intervals, 0.25 ml of plasma was removed
and 10
ll H3PO4 added to remove any drug–protein complex.
CH3CN (0.25 ml) was added to the plasma samples and the mix-
tures centrifuged at 12,000 rpm (mini Spin plus, Eppendorf AG,
Hamburg, Germany) for 3 min. The supernatant was removed
and the concentration of the compound that remained was deter-
mined by HPLC.
5.4.3. 2,2,2-Trichloroethyl 4-[1H-indole-3-carboxylate]-1-meth-
ylpiperidinylbutanoate (3)
Oxalyl chloride (1.84 ml, 20.7 mmol) and DMF (one drop) was
added to a stirred suspension of indole-3-carboxylic acid (2.90 g,
18.0 mmol) in CH2Cl2 (75 ml) and stirred at room temperature

8610
B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
for 2 h, then concentrated in vacuo to leave the acid chloride as a
yellow solid. This was dissolved in a mixture of CH2Cl2 (30 ml)
and THF (10 ml) and added dropwise (30 min) to a stirred solution
of trichloroethyl ester 2 (4.98 g, 15.0 mmol) and NEt3 (1.82 g,
18.0 mmol) in CH2Cl2 (30 ml). The reaction mixture was stirred
at room temperature overnight, treated with brine (25 ml) and
10% aqueous NaHCO3 solution (25 ml). The organic layer was dried
over Na2SO4, filtered and evaporated in vacuo. The residue was
separated with flash chromatography (EtOAc) to leave the title
compound as a pale yellow solid (1.83 g, 25.6%). Conversion to
the hydrochloride salt was effected using ethereal HCl. 1H NMR
(CDCl3): d 9.02 (br s, 1H), 8.22–8.18 (m, 1H), 7.92 (d, 1H), 7.48–
7.41 (m, 1H), 7.35–7.28 (m, 2H), 4.77 (s, 2H), 4.24 (d, 2H), 3.03
(d, 2H), 2.59–2.44 (q, 5H), 2.13–1.85 (m, 7H), 1.60–1.43 (m, 2H).
13C NMR (CDCl3): d 171.7, 165.5, 136.2, 131.5, 125.7, 122.8,
121.7, 121.0, 111.7, 108.0, 94.8, 73.7, 67.9, 57.5, 53.1, 35.4, 31.7,
28.8, 21.8. MS (ES): 477.1 [M+H]+.
5.4.7. Ethyl 4-[(1H-indole-3-carbonyl)amino]-1-metylpiperidi-
nylbutanoate (7)
Ethyl 4-bromobutyrate (0.81 g, 4.1 mmol) was added to a stir-
red suspension of N-[4-piperidinyl]methyl]indole-3-carboxamide
(1.07 g, 4.1 mmol) and K2CO3 (2.29 g, 16.5 mmol) in acetone
(50 ml) and heated to reflux for 24 h. The mixture was cooled to
room temperature and filtered. The filtrate was evaporated in va-
cuo and the residue added CH2Cl2 (75 ml) and washed with H2O
(3 ꢂ 25 ml). The organic layer was dried over Na2SO4, filtered and
evaporated in vacuo. The residue was separated with flash chroma-
tography (CH2Cl2/MeOH (9: 1) to leave the title compound as a yel-
low solid (1.15 g, 75.2%). 1H NMR (CDCl3): d 9.70 (br s, 1H), 8.00–
7.95 (m, 1H), 7.78 (d, J = 2.8 Hz, 1H), 7.49–7.44 (m, 1H), 7.30–
7.25 (m, 2H), 6.33 (t, J = 5.8 Hz, 1H), 4.14 (q, 2H), 3.40 (t, 2H),
2.97 (d, 2H), 2.44–2.31 (m, 4H), 1.99–1.76 (m, 7H), 1.43–1.24 (m,
5H), 13C NMR (75 MHz, CDCl3):d 173.4, 165.7, 136.4, 128.1, 124.7,
122.7, 121.4, 119.8, 112.1, 60.3, 57.8, 53.2, 44.8, 36.1, 32.2, 29.7,
21.9, 14.2. HRSM (TOF MS ES+) for C19H24N3O4 [M+H]+: calcd:
372.2282; found: 372.2294.
5.4.4. 4-[1H-Indole-3-carboxylate]-1-methylpiperidinylbutanoic
acid (4)
5.4.8. General procedure for the alkali hydrolysis of ester to free
acid
5.4.8.1. 4-[(1H-Indole-3-carbonyl)amino]-1-metylpiperidinylb-
Zn-powder (0.66 g, 10.0 mmol) was added to a stirred solution
of trichloroethyl ester 3 (0.48 g, 1.0 mmol) in THF (25 ml) and
aqueous 1 M KH2PO4 (5 ml). The resulting mixture was stirred at
room temperature for 24 h, and then filtered through a pad of kies-
elguhr and the filtrate evaporated in vacuo. The residue was sepa-
rated with flash chromatography (EtOAc/MeOH 2:1) to leave the
title compound as a white solid (0.29 g, 84.2%). Conversion to the
hydrochloride salt was effected using ethereal HCl. 1H NMR
(DMSO-d6): d 11.98 (s, 1H), 8.08–7.97 (m, 2H), 7.47 (d, 1H), 7.20–
7.17 (m, 2H), 4.11 (d, 2H), 2.96 (d, 2H), 2.50–2.37 (m, 4H), 2.05
(t, 2H), 1.77–1.66 (m, 6H), 1.42–1.35 (m, 2H). 13C NMR (DMSO-
d6): d 171.7, 165.5, 136.2, 131.5, 125.7, 122.8, 121.7, 121.0, 111.7,
108.0, 94.8, 73.7, 67.9, 57.5, 53.1, 35.4, 31.7, 28.8, 21.8. HRSM
(TOF MS ES+) for C19H24N2O4 [M+H]+: calcd: 345.1809; found:
345.1822.
utanoic acid (8).
Ethyl 4-[(1H-indole-3-carbonyl)amino]-1-
metylpiperidinylbutanoate (7) (0.45 g, 1.20 mmol) was added to
a mixture of 2 M aqueous NaOH solution (1.2 ml) and MeOH
(5 ml) and heated to reflux for 2 h. The reaction mixture was
cooled to room temperature, concentrated in vacuo and dropwise
added 10% aqueous HCl to pH 2. The precipitate was filtered off,
washed with water and dried in vacuo to leave the title compound
as a white crystalline solid (0.21 g, 61.1%). 1H NMR (300 MHz,
DMSO-d6): d 11.63 (s, 1H), 8.13 (d, 1H), 8.05 (d, 1H), 7.97 (t, 1H),
7.41 (d, 1H), 7.15–7.05 (m, 2H), 3.14 (t, 2H), 3.02 (d, 2H), 2.50 (t,
2H), 2.26 (t, 2H), 2.17 (t, 2H), 1.75–1.53 (m, 5H), 1.31–1.21 (m,
2H). 13C NMR (75 MHz, DMSO-d6): d 174.4, 164.6, 136.0, 127.5,
126.1, 121.6, 120.9, 120.1, 111.7, 110.5, 57.0, 52.2, 43.6, 35.5,
33.4, 28.7, 20.9. HRSM (TOF MS ES+) for C19H25N3O3 [M+H]+: calcd:
344.1969; found: 344.1989.
5.4.5. N-[1-Benzyl-4-piperidinyl]methyl indole-3-carboxamide
(5)
Indole-3-carboxoyl chloride (5.56 g, 30.95 mmol) dissolved in a
mixture of CH2Cl2 (50 ml) and THF (20 ml) was added dropwise to
5.4.9. General procedure for the synthesis of 3,4-dihydro-N-[[4-
piperidinyl]methyl]-2H-[1,3]oxazino[3,2-a]indole-10-carbo-
xamide derivatives 9–23
a
solution of 1-benzyl-4-aminomethylpiperidine (6.33 g,
5.4.9.1. Methyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
30.95 mmol) and NEt3 (3.13 g, 30.95 mol) in CH2Cl2 (50 ml). The
reaction mixture was stirred at room temperature overnight, and
then treated with brine (25 ml) and 10% aqueous NaHCO3 solution
(25 ml). The organic layer was dried over Na2SO4, filtered and
evaporated in vacuo. The residue was separated with flash chroma-
tography (EtOAc) to leave the title compound as a pale yellow solid
(3.17 g, 29.6%). 1H NMR (CDCl3): d 9.96 (s, 1H), 7.97–7.95 (m, 1H),
7.67 (d, J = 2.8 Hz, 1H), 7.44–7.22 (m, 8H), 6.24 (t, J = 5.9 Hz, 1H),
3.51 (s, 2H), 3.40 (t, J = 5.9 Hz, 2H), 2.92 (br d, 2H), 1.98 (t,
J = 10.9 Hz, 2H), 1.78–1.67 (m, 3H), 1.44–1.30 (m, 2H).
10-carbonyl)amino]-1-methylpiperidinylacetate (9).
Methyl
bromoacetate (0.50 g, 3.3 mmol) was added to a stirred suspension
of N-(4-piperidylmethyl) 3,4-dihydro-2H-[1,3]oxazino[3,2-a]in-
dole-10-carboxamide (1.05 g, 3.0 mmol) and K2CO3 (1.65 g,
12.0 mmol) in acetone (30 ml) and heated to reflux for 24 h. The
mixture was cooled to room temperature and filtered. The filtrate
was evaporated in vacuo and the residue added CH2Cl2 (50 ml) and
washed with H2O (3 ꢂ 25 ml). The organic layer was dried over
Na2SO4, filtered and evaporated in vacuo. The residue was sepa-
rated with flash chromatography (CH2Cl2/MeOH (9:1) to leave
the title compound as a yellow oil (0.62 g, 54.2%). 1H NMR (CDCl3):
d 8.32 (d, J = 7.5 Hz, 1H), 7.28–7.06 (m, 3H), 6.54 (t, J = 5.9 Hz, 1H),
4.51 (t, J = 5.2 Hz, 2H), 4.06 (t, J = 6.2 Hz, 2H), 3.72 (s, 3H), 3.34 (t,
J = 6.2 Hz, 2H), 3.23 (s, 2H), 2.98–2.94 (m, 2H), 2.37–2.30 (m, 2H),
2.22–2.15 (m, 2H), 1.79–1.63 (m, 3H), 1.51–1.39 (m, 2H). 13C
NMR CDCl3): d 170.8, 164.7, 149.1, 131.0, 125.5, 122.0, 120.9,
120.5, 107.4, 89.1, 66.7, 59.7, 53.3, 51.6, 44.2, 38.8, 35.7, 29.7,
5.4.6. N-[4-Piperidinyl]methyl indole-3-carboxamide (6)
Hydrazine monohydrate (0.36 ml) followed by 10% palladium
on activated charcoal (M-type, 0.40 g) was added to a stirred solu-
tion of N-[1-(benzyl)-4-piperidinyl]methyl]indole-3-carboxamide
(1.74 g, 5.0 mmol) in EtOH (20 ml) and heated to reflux for 2 h.
The reaction mixture was cooled to room temperature and filtered
through a pad of kieselguhr. The filtrate was evaporated in vacuo to
leave the title compound as a white solid (1.18 g, 92.7%). 1H NMR
(DMSO-d6): d 11.56 (br s, 1H), 8.15–8.12 (m, 1H), 8.03 (s, 1H),
7.85 (t, J = 5.7 Hz, 1H), 7.43–7.40 (m, 1H), 7.15–7.08 (m, 2H), 3.12
(t, J = 6.0 Hz, 2H), 2.94–2.90 (m, 2H), 2.55–2.49 (m, 1H), 2.4–2.36
(m, 2H), 1.64–1.60 (m, 3H), 1.06–1.01 (m, 2H).
21.1. HRSM (TOF MS ES+) for
386.2074; found: 386.2086.
C
21H27N3O4 [M+H]+: calcd:
5.4.9.2. Methyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-
carbonyl)amino]-1-methylpiperidinylpropanoate (10). Yield:
0.72 g (60.3%). 1H NMR (CDCl3):
8.32 (d, J = 7.5 Hz, 1H),
d

B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
8611
7.28–7.06 (m, 3H), 6.54 (t, J = 5.9 Hz, 1H), 4.51 (t, J = 5.2 Hz, 2H), 4.06
(t, J = 6.2 Hz, 2H), 3.72 (s, 3H), 3.34 (t, J = 6.2 Hz, 2H), 2.98–2.94 (m,
2H), 2.72 (m, 2H), 2.54 (m, 2H), 2.37–2.30 (m, 2H), 2.22–2.15 (m,
2H), 1.79–1.63 (m, 3H), 1.51–1.39 (m, 2H). 13C NMR CDCl3): d
173.2, 165.2, 149.6, 131.5, 126.0, 122.5, 121.4, 121.0, 107.8, 89.5,
67.2, 54.0, 53.6, 52.0, 44.6, 39.3, 36.4, 32.2, 30.1, 21.6. HRSM (TOF
MS ES+) for C22H29N3O4 [M+H]+: calcd: 400.2231; found: 400.2244.
29.0, 27.4, 26.2, 24.8, 21.0. HRSM (TOF MS ES+) for C31H47N3O4
[M+H]+: calcd: 526.3639; found: 526.3639.
5.4.9.8. Methyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinylbenzoate (16).
Yield:
1.04 g (75.1%). 1H NMR (CDCl3): d 8.26 (d, J = 7.5 Hz, 1H), 7.96 (d,
J = 8.1 Hz, 2H), 7.43 (d, J = 8.1 Hz, 2H), 7.16–7.03 (m, 3H), 6.49 (t,
J = 5.9 Hz, 1H), 4.48 (t, J = 5.1 Hz, 2H), 4.03 (t, J = 6.2 Hz, 2H), 3.89
(s, 3H), 3.61 (s, 2H), 3.29 (t, J = 6.2 Hz, 2H), 2.94–2.90 (m, 2H),
2.31–2.28 (m, 2H), 2.14–2.10 (m, 2H), 1.76–1.72 (m, 3H), 1.25–
1.19 (m, 2H). 13C NMR (CDCl3): d 166.8, 164.7, 149.2, 131.0,
129.5, 129.3, 125.5, 122.0, 120.8, 120.6, 107.5, 89.0, 66.8, 62.3,
53.2, 52.4, 52.0, 44.0, 38.8, 35.7, 29.4, 21.1. HRSM (TOF MS ES+)
for C27H31N3O4 [M+H]+: calcd: 462.2387; found: 462.2385.
5.4.9.3. Ethyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-
carbonyl)amino]-1-methylpiperidinylbutanoate (11).
Yield:
0.84 g (65.7%). 1H NMR (CDCl3): d 8.33 (d, J = 7.5 Hz, 1H), 7.30–
7.09 (m, 3H), 6.58 (t, J = 5.9 Hz, 1H), 4.55 (t, J = 5.2 Hz, 2H), 4.20–
4.07 (m, 4H), 3.35 (t, J = 6.1 Hz, 2H), 3.07–3.01 (m, 2H), 2.47–2.32
(m, 6H), 2.09–1.50 (m, 10H), 1.27 (t, J = 7.1 Hz, 3H). 13C NMR
(CDCl3): d 173.2, 164.7, 149.2, 131.0, 125.5, 122.0, 120.9, 120.6,
107.4, 89.0, 66.8, 60.2, 57.6, 53.2, 44.1, 38.9, 35.9, 32.1, 29.4,
21.7, 21.1, 14.1. HRSM (TOF MS ES+) for C24H33N3O4 [M+H]+: calcd:
428.2544; found: 428.2564. Conversion to the hydrochloride salt
was effected with ethereal HCl. The precipitate was collected and
recrystallized from acetone to leave the HCl salt as a white crystal-
line solid.
5.4.9.9. t-Butyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinylacetate (17).
Yield:
0.89 g (69.6%). 1H NMR (CDCl3): d 8.32 (dd, 1H), 7.21–7.05 (m,
3H), 6.53 (t, 1H), 4.50 (t, 2H), 4.04 (t, 2H), 3.33 (t, 2H), 3.11 (s,
2H), 2.96 (br d, 2H), 2.34–2.31 (m, 2H), 2.21–2.12 (m, 2H), 1.77–
1.49 (m, 3H), 1.46–1.40 (m, 11H). 13C NMR (CDCl3): d 169.8,
164.7, 149.1, 131.0, 125.5, 121.9, 120.9, 120.5, 107.4, 89.1, 80.8,
66.7, 60.3, 53.2, 44.2, 38.8, 35.9, 29.9, 28.0, 21.1. HRSM (TOF MS
ES+) for C24H33N3O4 [M+H]+: calcd: 428.2544; found: 428.2556.
5.4.9.4. Methyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinylpentanoate (12).
Yield:
0.79 g (62.3%). 1H NMR (CDCl3): d 8.31 (d, J = 7.5 Hz, 1H), 7.28–7.08
(m, 3H), 6.57 (t, J = 5.9 Hz, 1H), 4.54 (t, J = 5.2 Hz, 2H), 4.09 (t,
J = 6.1 Hz, 2H), 3.67 (s, 3H), 3.34 (t, J = 6.1 Hz, 2H), 3.08–3.04 (m,
2H), 2.49–2.44 (m, 2H), 2.39–2.32 (m, 4H), 2.15–2.08 (m, 2H), 1.84–
1.49 (m, 9H). 13C NMR (CDCl3): d 173.8, 164.8, 149.2, 131.0, 125.5,
122.0, 120.8, 120.6, 107.4, 89.0, 66.8, 58.0, 53.2, 51.4, 44.0, 38.9,
35.6, 33.6, 29.1, 25.6, 22.7, 21.1. HRSM (TOF MS ES+) for C24H33N3O4
[M+H]+: calcd: 428.2544; found: 428.2560.
5.4.9.10. t-Butyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinylbutanoate (18).
Yield:
0.96 g (70.8%). 1H NMR (CDCl3): d 8.33 (d, J = 7.5 Hz, 1H), 7.24–7.10
(m, 3H), 6.57 (t, J = 5.9 Hz, 1H), 4.55 (t, J = 5.1 Hz, 2H), 4.12 (t,
J = 6.2 Hz, 2H), 3.35 (t, J = 6.2 Hz, 2H), 3.05–3.01 (m, 2H), 2.45–
2.35 (m, 4H), 2.26 (t, J = 7.3 Hz, 2H), 2.12–2.04 (m, 2H), 1.87–1.79
(m, 5H), 1.48–1.37 (m, 11H). 13C NMR (75 MHz, CDCl3): d 172.5,
164.7, 149.2, 130.9, 125.4, 121.8, 120.7, 120.4, 88.8, 80.1, 66.7,
57.7, 53.2, 44.1, 38.7, 35.9, 33.2, 29.4, 27.9, 27.8, 21.8, 21.0. HRSM
(TOF MS ES+) for C26H37N3O4 [M+H]+: calcd: 456.2857; found:
456.2855.
5.4.9.5. Methyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinylhexanoate (13).
Yield:
1.11 g (84.5%). 1H NMR (CDCl3): d 8.27 (d, J = 7.5 Hz, 1H), 7.23–7.09
(m, 3H), 6.52 (t, J = 5.9 Hz, 1H), 4.49 (t, J = 5.1 Hz, 2H), 4.03 (t,
J = 6.2 Hz, 2H), 3.62 (s, 3H), 3.30 (t, J = 6.2 Hz, 2H), 3.03–2.99 (m,
2H), 2.42–2.25 (m, 6H), 2.06–2.00 (m, 2H), 1.75–1.29 (m, 10H). 13C
NMR (CDCl3): d 174.1, 164.7, 149.1, 131.0, 125.5, 122.0, 121.0,
120.6, 107.4, 89.1, 66.8, 58.7, 53.5, 51.4, 44.3, 38.9, 36.2, 33.9, 29.8,
27.1, 26.4, 24.7, 21.2. HRSM (TOF MS ES+) for C25H35N3O4 [M+H]+:
calcd: 442.2700; found: 442.2699.
5.4.9.11. t-Butyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinylhexanoate (19).
Yield:
0.99 g (68.3%). 1H NMR (CDCl3): d 8.23 (dd, 1H), 7.19–7.03 (m, 3H),
6.57 (t, J = 6.2 Hz, 1H), 4.51 (t, J = 5.1 Hz, 2H), 4.05 (t, J = 6.2 Hz,
2H), 3.33–3.17 (m, 4H), 2.64–2.54 (m, 2H), 2.33–2.28 (m, 4H), 2.16
(t, J = 7.2 Hz, 2H), 1.83–1.51 (m, 9H), 1.39–1.28 (m, 11H). 13C NMR
(CDCl3): d 172.77, 164.92, 149.47, 131.08, 125.45, 122.06, 120.70,
107.56, 88.82, 80.08, 77.20, 66.93, 57.76, 52.71, 43.61, 38.91, 35.15,
34.69, 28.03, 27.81, 26.55, 24.70, 24.51, 21.13. HRSM (TOF MS ES+)
for C28H41N3O4 [M+H]+: calcd: 484.3170; found: 484.3163.
5.4.9.6. Methyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinyldecanoate (14).
Yield:
1.19 g (80.2%). 1H NMR (CDCl3): d 8.27 (d, J = 7.5 Hz, 1H), 7.24–7.12
(m, 3H), 6.70 (t, J = 5.9 Hz, 1H), 4.60 (t, J = 5.1 Hz, 2H), 4.13 (t,
J = 6.2 Hz, 2H), 3.67 (s, 3H), 3.58–3.51 (m, 2H), 3.35 (t, J = 6.2 Hz,
2H), 2.92–2.87 (m, 2H), 2.68–2.60 (m, 2H), 2.44–2.34 (m, 2H), 2.30
(t, J = 7.5 Hz, 2H), 2.13–1.70 (m, 8H), 1.65–1.56 (m, 2H), 1.29 (br s,
10H). 13C NMR (CDCl3): d 174.2, 165.1, 149.7, 131.1, 125.4, 122.2,
120.8, 120.6, 107.6, 77.2, 67.0, 52.7, 51.4, 43.9, 38.9, 33.9, 33.6,
29.0, 28.9, 28.8, 28.7, 26.7, 24.8, 23.4, 21.1. HRSM (TOF MS ES+) for
5.4.9.12. Diethyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-
10-carbonyl)amino]-1-methylpiperidinylethylphosphonate
(20).
Yield: 1.00 g (70.4%). 1H NMR (CDCl3): d 8.30 (d, J = 7.5 Hz,
1H), 7.23–7.10 (m, 3H), 6.59 (t, J = 5.9 Hz, 1H), 4.56 (t, J = 5.1 Hz,
2H), 4.17–4.06 (m, 6H), 3.35 (t, J = 5.9 Hz, 2H), 3.09 (br d, 2H),
2.86–2.78 (m, 2H), 2.39–2.33 (p, J = 5.5 Hz, 2H), 2.27–2.08 (m,
4H), 1.88–1.60 (m, 3H), 1.58–1.50 (m, 2H), 1.33 (t, J = 7.0 Hz, 6H).
13C NMR (CDCl3): d 163.5, 149.8, 131.5, 125.9, 122.5, 121.3,
121.1, 107.9, 89.4, 67.3, 62.4, 62.3, 53.2, 52.1, 44.3, 39.4, 35.9,
29.3, 21.6, 16.9, 16.8. HRSM (TOF MS ES+) for C24H36N3O5P
[M+H]+: calcd: 478.2465; found: 478.2462.
C
29H43N3O4 [M+H]+: calcd: 498.3326; found: 498.3340. Conversion
to the hydrochloride salt was effected using ethereal HCl.
5.4.9.7. Methyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-
carbonyl)amino]-1-methylpiperidinyldodecanoate (15).
Yield:
1.16 g (73.8%). 1H NMR (CDCl3): d 8.25 (d, J = 7.5 Hz, 1H), 7.17–
7.00 (m, 3H), 6.50 (t, J = 5.9 Hz, 1H), 4.45 (t, J = 5.1 Hz, 2H), 3.98 (t,
J = 6.2 Hz, 2H), 3.61 (s, 3H), 3.28 (t, J = 6.2 Hz, 2H), 3.01–2.98 (m,
2H), 2.39–2.33 (m, 2H), 2.31–2.22 (m, 4H), 2.06–1.98 (m, 2H),
1.77–1.38 (m, 9H), 1.22 (br s, 14H). 13C NMR (CDCl3): d 174.2,
164.7, 149.2, 131.0, 125.4, 121.9, 120.7, 120.5, 107.4, 88.9, 66.7,
58.7, 53.2, 51.3, 50.4, 44.0, 38.8, 35.8, 34.0, 29.4, 29.3, 29.2, 29.1,
5.4.9.13. Benzyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-
carbonyl)amino]-1-methylpiperidinylbutanoate
(21).
Yield:
1.06 g (72.8%). 1H NMR (CDCl3): d 8.29 (dd, 1H), 7.34–7.29 (m, 5H),
7.18–7.06 (m, 3H), 6.53 (t, J = 5.9 Hz, 1H), 5.08 (s, 2H), 4.49 (t,
J = 5.1 Hz, 2H), 4.04 (t, J = 6.2 Hz, 2H), 3.29 (t, J = 6.2 Hz, 2H), 2.96
(br d, 2H), 2.42–2.26 (m, 6H), 2.04–1.96 (m, 2H), 1.91–1.84 (m,

8612
B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
2H), 1.77–1.55 (m, 3H), 1.44–1.37 (m, 2H). 13C NMR (CDCl3): d 173.1,
164.7, 149.2, 135.8, 131.0, 128.5, 128.1, 125.5, 122.0, 120.8, 120.6,
107.4, 89.0, 66.8, 66.1, 57.6, 53.2, 44.1, 38.8, 35.9, 32.1, 29.4, 21.7,
21.1. HRSM (TOF MS ES+) for C29H35N3O4 [M+H]+: calcd: 490.2700;
found: 490.2694.
5.4.9.18. 4-[(3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-car-
bonyl)amino]-1-methylpiperidinylacetic acid (26). Yield:
(0.29 g, 63.4%). 1H NMR (CD3OD): d 8.09–8.06 (m, 1H), 7.25–7.09
(m, 4H), 4.60 (t, J = 5.1 Hz, 2H), 4.15 (t, J = 6.1 Hz, 2H), 3.65–3.59
(m, 4H), 3.39–3.35 (m, 2H), 2.99 (t, 2H), 2.40–2.33 (m, 2H), 2.04–
1.80 (m, 4H), 1.70–1.57 (m, 2H). 13C NMR (CD3OD): d 169.7,
167.6, 152.0, 132.9, 126.6, 122.8, 121.8, 120.9, 109.2, 89.0, 68.5,
54.0, 44.0, 40.1, 35.5, 28.0, 22.2. HRSM (TOF MS ES+) for
5.4.9.14. Benzyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-
carbonyl)amino]-1-methylpiperidinylpentanoate (22).
Yield:
C
20H25N3O4 [M+H]+: calcd: 372.1918; found: 372.1920.
1.05 g (69.9%). 1H NMR (CDCl3): d 8.26 (d, J = 7.5 Hz, 1H), 7.33–7.28
(m, 5H), 7.20–7.05 (m, 3H), 6.55 (t, J = 5.9 Hz, 1H), 5.07 (s, 2H),
4.51 (t, J = 5.1 Hz, 2H), 4.06 (t, J = 6.2 Hz, 2H), 3.30 (t, J = 6.2 Hz,
2H), 3.10 (br d, 2H), 2.52–2.47 (m, 2H), 2.38–2.28 (m, 4H), 2.19 (t,
2H), 1.83–1.80 (m, 3H), 1.65–1.56 (m, 6H). HRSM (TOF MS ES+) for
5.4.9.19. 4-[(3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-car-
bonyl)amino]-1-methylpiperidinylpropanoic acid (27). Yield:
(0.62 g, 67.1%). 1H NMR (DMSO-d6): d 10.62 (br s, 1H), 8.08–8.03
(m, 1H), 7.32–7.27 (m, 1H), 7.13–7.06 (m, 2H), 6.96 (t, J = 5.9 Hz,
1H), 4.55 (t, J = 5.1 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.34–3.31 (m,
2H), 3.19–3.10 (m, 4H), 2.85–2.76 (m, 4H), 2.27–2.24 (m, 2H),
1.79–1.75 (m, 3H), 1.59–1.51 (m, 2H). 13C NMR (DMSO-d6): d
173.2, 164.7, 149.2, 131.0, 125.5, 122.0, 120.9, 120.6, 107.4, 89.0,
66.8, 60.2, 57.6, 53.2, 50.6, 44.1, 38.9, 35.9, 32.1, 29.4, 21.7, 21.1,
C
30H37N3O4 [M+H]+: calcd: 504.2857; found: 504.2848.
5.4.9.15. Benzyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-
carbonyl)amino]-1-methylpiperidinylhexanoate (23). Yield:
1.09 g (70.5%). 1H NMR (CDCl3): d 8.25 (d, J = 7.5 Hz, 1H), 7.33–7.27
(m, 5H), 7.19–7.06 (m, 3H), 6.56 (t, J = 5.9 Hz, 1H), 5.06 (s, 2H),
4.49 (t, J = 5.1 Hz, 2H), 4.03 (t, J = 6.2 Hz, 2H), 3.29 (t, J = 6.2 Hz,
2H), 3.12 (br d, 2H), 2.52–2.47 (m, 2H), 2.34–2.21 (m, 6H), 1.83–
1.80 (m, 3H), 1.65–1.57 (m, 6H), 1.33–1.26 (m, 2H). 13C NMR (CDCl3):
d 173.24, 164.88, 149.37, 135.88, 131.02, 128.47, 128.11, 125.43,
122.04, 120.72, 120.62, 107.52, 88.83, 66.85, 66.06, 57.93, 52.91,
43.79, 38.87, 35.04, 33.92, 28.31, 26.66, 25.08, 24.44, 21.09. HRSM
(TOF MS ES+) for C31H39N3O4 [M+H]+: calcd: 518.3013; found:
518.3010.
14.1. HRSM (TOF MS ES+) for
386.2074; found: 386.2078.
C
21H27N3O4 [M+H]+: calcd:
5.4.9.20. 4-[(3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-car-
bonyl)amino]-1-methylpiperidinylbutanoic acid (28). Yield:
(0.33 g, 72.9%). 1H NMR (DMSO-d6): d 12.31 (br s, 1H), 10.25 (br
s, 1H), 8.10–8.04 (m, 1H), 7.32–7.26 (m, 1H), 7.14–7.04 (m, 2H),
6.96 (t, J = 5.9 Hz, 1H), 4.59 (t, J = 5.1 Hz, 2H), 4.15 (t, J = 6.1 Hz,
2H), 3.43–3-01 (m, 8H), 2.38–2.28 (m, 4H), 1.97–1.81 (m, 5H),
1.68–1.55 (m, 2H). 13C NMR (DMSO-d6): d 174.3, 164.6, 150.6,
131.8, 126.1, 122.0, 120.7, 120.4, 109.3, 88.7, 67.9, 56.1, 52.4,
43.7, 34.9, 31.5, 27.7, 21.4, 19.7. HRSM (TOF MS ES+) for
5.4.9.16. Pivaloylmethyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-
a]indole-10-carbonyl)amino]-1-methylpiperidinylbenzoate
C
22H29N3O4 [M+H]+: calcd: 400.2231; found: 400.2225.
(24).
NaI (catalytic amount) was added to a stirred suspension of
4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-a]indole-10-carbonyl)-amino]-
1-methylpiperidinylbutanoic acid (28) (0.63 g, 1.30 mmol) and
Cs2CO3 (1.69 g, 5.20 mmol)inDMF (5 ml) andstirred at room temper-
ature for 0.5 h. Chloromethyl pivalate (0.23 g, 1.56 mmol) was added
to the reaction mixture and heated to reflux for 12 h, then cooled to
roomtemperatureand filtered. Thefiltratewasconcentrated invacuo
and the residue separated with flash chromatography (CH2Cl2/MeOH,
9:1) to leave the title compound as an oil. Yield: 0.43 g (59.3%).
Conversion to the hydrochloride salt was effected using ethereal
HCl. 1H NMR (CDCl3): d 8.33 (d, J = 7.5 Hz, 1H), 8.02 (d, J = 7.7 Hz,
2H), 7.48 (br s, 2H), 7.25–7.10 (m, 3H), 6.56 (t, J = 5.9 Hz, 1H), 6.00
(s, 2H), 4.54 (t, J = 5.1 Hz, 2H), 4.12 (t, J = 6.2 Hz, 2H), 3.60 (br s, 2H),
3.35 (t, J = 6.2 Hz, 2H), 2.96–2.89 (m, 2H), 2.41–2.35 (m, 2H), 2.18–
1.40 (m, 7H), 1.24 (s, 9H). 13C NMR (CDCl3): d 166.8, 164.7, 149.2,
131.0, 129.5, 129.3, 125.5, 122.0, 120.8, 120.6, 107.5, 89.0, 66.8,
62.3, 53.2, 52.4, 52.0, 44.0, 38.8, 35.7, 29.4, 21.1. HRSM (TOF MS ES+)
for C32H39N3O6 [M+H]+: calcd: 562.2912; found: 562.2902.
5.4.9.21. 4-[(3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-car-
bonyl)amino]-1-methylpiperidinylpentanoic acid (29). Yield:
0.33 g (69.7%). 1H NMR (DMSO-d6): d 8.04–8.01 (m, 1H), 7.27–
7.25 (m, 1H), 7.09–7.01 (m, 2H), 6.92 (t, J = 5.9 Hz, 1H), 4.56 (t,
J = 5.1 Hz, 2H), 4.10 (t, J = 6.1 Hz, 2H), 3.32–3.29 (m, 2H), 3.22–
3.17 (m, 2H), 2.90–2.85 (m, 2H), 2.80–2.72 (m, 2H), 2.26–2.21
(m, 4H), 1.79–1.46 (m, 10H). 13C NMR (DMSO-d6): d 174.2, 163.7,
149.8, 130.9, 125.2, 121.1, 119.9, 119.5, 108.4, 87.8, 67.1, 61.9,
51.0, 42.7, 34.1, 33.3, 26.5, 25.5, 22.9, 21.9. HRSM (TOF MS ES+)
for C23H31N3O4 [M+H]+: calcd: 414.2384; found: 414.2387.
5.4.9.22. 4-[(3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-car-
bonyl)amino]-1-methylpiperidinylhexanoic acid (30).
Yield:
0.35 g (68.3%). 1H NMR (DMSO-d6): d 8.03–8.01 (m, 1H), 7.26–
7.24 (m, 1H), 7.08–7.01 (m, 2H), 6.91 (t, J = 5.9 Hz, 1H), 4.55 (t,
J = 5.1 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.32–3.28 (m, 2H), 3.21–
3.16 (m, 2H), 2.84–2.80 (m, 2H), 2,51–2.49 (m, 2H), 2.30–2.19
(m, 4H), 1.77–1.44 (m, 9H), 1.30–1.22 (m, 2H). 13C NMR (DMSO-
d6): d 174.4, 163.7, 149.8, 130.9, 125.2, 121.1, 119.8, 119.5, 108.4,
87.8, 67.1, 55.6, 51.1, 48.5, 42.7, 33.5, 26.7, 25.7, 24.0, 23.2, 20.6.
HRSM (TOF MS ES+) for C24H33N3O4 [M+H]+: calcd: 428.2544;
found: 428.25439.
5.4.9.17. Pivaloylmethyl 4-[(3,4-dihydro-2H-[1,3]oxazino[3,2-
a]indole-10-carbonyl)amino]-1-methylpiperidinylhexanoate
(25) was prepared in
24.
a manner similar to compound
Yield: 0.47 g (66.3%). 1H NMR (CDCl3): d 8.23 (d, J = 7.5 Hz,
1H), 7.17–7.01 (m, 3H), 6.53 (t, J = 5.9 Hz, 1H), 5.69 (s, 1H), 4.46
(t, J = 5.1 Hz, 2H), 3.98 (t, J = 6.2 Hz, 2H), 3.28 (t, J = 6.2 Hz, 2H),
3.09 (br d, 2H), 2.51–2.46 (m, 2H), 2.33–2.15 (m, 6H), 1.81–1.53
(m, 9H), 1.31–1.26 (m, 2H), 1.16 (s, 9H). 13C NMR (CDCl3): d
176.9, 171.8, 164.7, 149.2, 130.9, 125.3, 121.8, 120.5, 120.4,
107.4, 88.6, 79.1, 66.7, 57.7, 52.7, 43.6, 38.7, 38.5, 35.1, 33.5,
28.3, 26.7, 26.4, 25.0, 24.0, 20.9. HRSM (TOF MS ES+) for
5.4.9.23. 4-[(3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-car-
bonyl)amino]-1-methylpiperidinyldodecanoic acid (31).
Yield:
0.43 g (70.3%). 1H NMR (DMSO-d6): d 8.05–8.01 (m, 1H), 7.27–7.23
(m, 1H), 7.09–7.01 (m, 2H), 6.88 (t, J = 5.9 Hz, 1H), 4.55 (t,
J = 5.1 Hz, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.24–3.15 (m, 4H), 2.75–2.70
(m, 2H), 2.58–2.54 (m, 2H), 2.28–2.25 (m, 2H), 2.16 (t, 2H), 1.76–
1.44 (m, 9H), 1.23 (br s, 14H). 13C NMR (CDCl3): d 172.5, 171.5,
161.7, 147.7, 128.9, 123.2, 119.1, 117.8, 117.5, 106.4, 85.8, 65.1,
54.1, 49.5, 31.7, 26.8, 26.7, 26.6, 26.5, 26.4, 22.5. HRSM (TOF MS
ES+) for C30H45N3O4 [M+H]+: calcd: 512.3483; found: 512.3476.
C
30H43N3O6 [M+H]+: calcd: 542.3225; found: 542.3227. Conversion
to the hydrochloride salt was effected using ethereal HCl.
Compounds 26–32 were prepared in a manner similar to com-
pound 8.

B. Brudeli et al. / Bioorg. Med. Chem. 18 (2010) 8600–8613
8613
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5.4.9.24. 4-[(3,4-Dihydro-2H-[1,3]oxazino[3,2-a]indole-10-car-
bonyl)amino]-1-methylpiperidinylbenzoic acid (32). Yield:
0.38 g (71.3%). 1H NMR (DMSO-d6): d 8.03–8.00 (m, 1H), 7.88 (d,
2H), 7.40 (d, 2H), 7.26–7.23 (m, 1H), 7.07–7.00 (m, 2H), 6.79 (t,
1H), 4.54 (t, 2H), 4.10 (t, 2H), 3.50 (br s, 2H), 3.15 (d, 2H), 2.84
(d, 2H), 2.27–2.23 (m, 2H), 2.09–2.03 (m, 2H), 1.65–1.52 (m, 3H),
1.31–1.21 (m, 2H). 13C NMR (75 MHz, DMSO-d6): d 164.5, 164.4,
150.5, 131.8, 130.0, 129.6, 126.0, 121.9, 120.7, 120.4, 109.3, 88.8,
88.7, 68.0, 53.4, 36.6, 30.0, 21.4. HRSM (TOF MS ES+) for
C
26H29N3O4 [M+H]+: calcd: 448.2231; found: 448.2234.
6. Conflicts of Interest
16. Langlois, M.; Fischmeister, R. J. Med. Chem. 2003, 46, 319.
17. Darblade, B.; Behr-Roussel, D.; Gorny, D.; Lebret, T.; Benoit, G.; Hieble, J. P.;
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1998, 30, 398.
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Kadambi, V.; Eddy, P.; Sinha, U.; Scarborough, R. M.; Song, Y. Bioorg. Med. Chem.
Lett. 2006, 16, 5507.
20. Clark, R. D. In 5-HT4 Receptors in the Brain and Periphery; Eglen, R. M., Ed.;
Springer: Berlin, Heidelberg, New York, 1998; pp 1–48.
21. Gaster, L. M.; Joiner, G. F.; King, F. D.; Wyman, P. A.; Sutton, J. M.; Bingham, S.;
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22. Contreras, J. M.; Rival, Y. M.; Chayer, S.; Bourguignon, J. J.; Wermuth, C. G. J.
Med. Chem. 1999, 42, 730.
The compounds described in the present paper are described in
WO2005061483 (Klaveness, J.; Levy, F. O.; Brudeli, B.). The poten-
tial use of 5-HT4 antagonists for treatment of heart failure is de-
scribed by a published patent application WO03097065 (Levy, F.
O.). These patent families belong to the Norwegian biotech com-
pany Serodus AS where all the authors are shareholders and FOL
is board member.
Acknowledgements
23. Fedouloff, M.; Hossner, F.; Voyle, M.; Ranson, J.; Powles, J.; Riley, G.; Sanger, G.
Bioorg. Med. Chem. 2001, 9, 2119.
24. Bach, T.; Syversveen, T.; Kvingedal, A. M.; Krobert, K. A.; Brattelid, T.; Kaumann,
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Supported by The Research Council of Norway, The Norwegian
Council on Cardiovascular Diseases, Anders Jahre’s Foundation for
the Promotion of Science, Bio-Medisinsk Innovasjon AS and Sero-
dus AS. The authors wish to thank Trygve Gulbrandsen, Ph.D.,
COO of Serodus AS, for support, advice and discussions. We are
grateful to Bryan Roth, MD, PhD, University of North Carolina at
Chapel Hill and the NIMH Psychoactive Drug Screening Program
(PDSP) for help with the specificity screening.
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