New spiro-piperidines as 5-HT2B receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.06.068

A functional screening highlighted a series of spiro-piperidines as 5-HT_2B receptor antagonists. Preliminary structure-activity relationship has been explored driving to potent antagonists (IC₅₀ = 1 nM) and indicating directions for

Full text of DOI:10.1016/j.bmcl.2006.06.068

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4830–4833
New spiro-piperidines as 5-HT2B receptor antagonists
*
´
ˆ
Hugues Bienayme, Laurent Chene, Serge Grisoni, Antonio Grondin, El-Bachir Kaloun,
´
Stephane Poigny, Houcine Rahali and Eric Tam
Urogene, Hopital Saint-Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France
Received 2 June 2006; revised 20 June 2006; accepted 20 June 2006
Available online 17 July 2006
Abstract—A functional screening highlighted a series of spiro-piperidines as 5-HT2B receptor antagonists. Preliminary structure–
activity relationship has been explored driving to potent antagonists (IC50 = 1 nM) and indicating directions for further
explorations.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Serotonin (5-hydroxytryptamine, 5-HT) is a well-known
monoamine neurotransmitter, hormone, and mitogenic
factor which mediates a wide range of physiological
activities in different cells by binding to multiple recep-
tor subtypes. With the exception of the 5-HT3 which is
a ligand-gated ion channel, all of these receptors are
known to be G-protein coupled receptors (GPCRs).
selective antagonists at the human receptor. We present
here the identification of a new series of 5-HT2B
antagonists.
Hit identification was realized by screening a set of 1250
small molecules selected for their structural relevance.16
The functional test performed corresponds to the inhibi-
tion of calcium release after activation of 5-HT2B recep-
tor by an agonist (a-methyl-5-hydroxytryptamine) in
CHO cells.17 A series of spiro-piperidine compounds
exhibited potent antagonistic effects, 1 and 2 (Fig. 2)
showing IC50 of 95 and 30 nM, respectively.
The 5-HT2B receptor was first identified in rat stomach
fundus.1,2 Although originally termed 5-HT2F or
5-HT1-like receptor,3 it was reclassified as 5-HT2B to be-
come the third member of the 5-HT2 receptor family.4–7
5-HT2B receptors are widely expressed in peripheral
tissues of various species. 5-HT2B receptor mRNA or
protein immunoreactivity has been found throughout
the gastrointestinal tract including smooth muscle of
the stomach fundus, esophagus, small intestine, and
colon. The receptor has also been found in the placenta,
uterus, lung, and prostate, and 5-HT2B receptor antago-
nist activities were reported in several diseases such as
prostatic cancer,8 gastrointestinal diseases, migraine,9
CNS disorders, urinary incontinence and bladder
dysfunction,10 and pulmonary hypertension.11 Regulat-
ing 5-HT2B/5-HT interaction appears to be a promising
approach in the development of small-molecule drugs.
In this study, all the spiro compounds were either
purchased commercially or prepared using a 3-step
synthesis (Scheme 1). According to the literature, the
formation of 20-hydroxychalcone involved the Claisen–
Schmidt condensation of a 2-hydroxybenzaldehyde I
with an acetophenone II in the presence of base as
catalyst.18,19 Subsequently, hydrazine hydrate was
NH
OMe
N
N
NH2
H
OMe
Cl
N
LY-266097
Among many 5-HT2B antagonists, SB204741,12,13
LY266097,14 and RS12744515 (Fig. 1) are described as
H
F
O
S
N
NH
N
RS-127445
N
Keywords: HTR2B antagonists; G-protein coupled receptors; Spiro-
piperidines; Pyrazoline.
SB-204741
Figure 1. 5-HT2B receptor ligands.
*
Corresponding author. Fax: +0437472381; e-mail: serge.grisoni@
pierre-fabre-urologie.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.06.068

´
H. Bienayme et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4830–4833
4831
Table 1. Biological assay results for compounds 1–24
F
N
N
Ar
N
X
N
N
N
Br
N
N
O
N
Y
O
O
N
Compounda Ar
X
Y
Activityb
c
IC50 (nM)
1
2
1
2
4-Pyridyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
95
Figure 2. Lead compounds.
4-Fluorophenyl
4-Fluorophenyl
Phenyl
Br
H
30
3
<10d
50–100
92
4
H
O
O
R1
R1
R2
R2
5
Phenyl
Br
H
a
+
O
6
4-Chlorophenyl
4-Bromophenyl
4-Chlorophenyl
2-Chlorophenyl
2-Chlorophenyl
280
426
OH
OH
7
H
8
Br
H
100–500
753
856
I
II
III
R2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Br
Br
Br
2-Methoxyphenyl
4-Methoxyphenyl
5000
5000
427
R1
HN
N
R1
R2
N
b
c
N
2-Difluoromethoxyphenyl Br
4-Difluoromethoxyphenyl Br
175
542
O
OH
N
R3
3,4-Dichlorophenyl
3,4-Dimethoxyphenyl
3-Pyridyl
Br
Br
H
5000
na
IV
V
Scheme 1. Reagents and conditions: (a) KOH, aqueous ethanol, 16 h,
rt; (b) NH2NH2, MeOH, rt; (c) N-substituted piperidin-4-one, neat,
50 ꢁC.
2-Furyl
2-Thienyl
Br
Br
Br
Br
H
5000
5000
>10000
5000
1-Naphthyl
2-Naphthyl
4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
MeO 46
MeO 1470
MeO 1281
added to the chalcone III in methanol and furnished the
corresponding pyrazoline IV in good yield.19 Finally,
the spiro compound V was obtained by heating the pyr-
azoline with a N-substituted piperdin-4-one at 50 ꢁC.
H
H
a Racemates were used.
b Inhibition of calcium release generated by a-methyl-5-HT (Ref. 17).
c na, not active. Average value from one experiment in duplicate.
d 90% inhibition at 10 nM.
The 5-HT2B antagonist activity for compounds 1–24 is
shown in Table 1. The most potent compound is the
4-fluorophenyl 3 with an IC50 below 10 nM. Generally,
both the hydrogen (3, 4, 7, and 9) and bromine (2, 5,
8, and 10) substitutions at the position X had similar
potency on the target. On the contrary, when the hydro-
gen in position Y (3, 6, and 7) was replaced with elec-
tron-rich methoxy group (22, 23, and 24), a markedly
reduced activity was observed.
3-phenyl substitution was acceptable but does not seem
to be essential. Replacing the methoxy group of 11 or 12
with a difluoromethoxy group, 13 and 14 showed a
10- to 20-fold increase in biological activity. These
results further showed the importance of the 4-fluoro
substitution pattern for 5-HT2B receptor affinity. From
all these results, both 4-fluorophenyl and 4-pyridyl
groups exhibited a high affinity with 5-HT2B receptor.
For the aromatic group (Ar), the inhibition was
decreased by using bulkier groups such as naphthyls
(20 and 21) or with heteroaryl groups such as 2-furyl
(18) and 2-thienyl (19). Interestingly, compared to the
lead compound 1, which has an IC50 = 95 nM, the
3-pyridyl analog (17) completely lost its activity. The
effect of varying the pattern on the phenyl group was
explored. Among the 4-halophenyl compounds, 4-fluoro
substitution was the preferred one (comparing 3, 6, and
7, also 22, 23, and 24 activities). In addition, shifting the
halogen from the 4-position (6 and 8) to the 2-position
(9 and 10) resulted in approximately 2-fold loss in activ-
ity. Introduction of an electron-rich methoxy group to
the 4-position of the phenyl ring led to a decrease in
potency. Compounds without any substitution (4 and
5) did not show any improvement compared to the hit
compounds. The 3,4-dichlorophenyl (15) and 3,4-
dimethoxyphenyl (16) compounds retained similar activ-
ity when compared to their mono-4-substituted analogs
(8 and 12). These results disclosed that the
The modification of the spiro-piperidine moiety (R4 and
R5) was investigated. Replacement by groups such as
dimethyl (25), cyclohexyl (26), 4-methylcyclohexyl (27),
and tetrahydropyranyl (28) resulted in the complete loss
of activity (Table 2).
Consequently, our efforts were addressed to the
optimization of the N-substitution of the spiro-piperi-
dine scaffold (Table 3). Attachment of an electron-
withdrawing acetyl group to the nitrogen atom led
to the loss of the biological activity (29, 30, and 31).
The bulky benzyl group (comparison between 34 and
42) was also not suitable for the nitrogen substitution,
but the 2-phenylethyl substitution was tolerated
(comparison between 43 and 51). When the N-alkyl
chain evolved from methyl to ethyl and to n-propyl,
the activity was increased by 2-fold for each

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H. Bienayme et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4830–4833
4832
Table 2. Variations at the R4 and R5 positions
F
F
N
N
N
a
N
N
N
N
R4
O
O
O
R5
N
N
45
IC50 = 1.8 nM
52
IC50 = na
Compounda
Activityb
c
IC50 (nM)
R4
R5
Scheme 2. Reagents and conditions: (a) CAN, CH3CN-water (9:1),
16 h.
25
5000
na
homologation (1, 35, and 43). All the n-propyl series
generally provided the best activity (comparison
between 12–42 and 4–44). Concerning the substitution
at the X-position, the halo-substitution gave similar
activities between 47 and 49 (IC50 = 26 to 32 nM)
but the most active one was the compound without
any substitution (X = H). Therefore, 2-(4-fluorophe-
nyl)-1,10b-dihydro-benzo[e]pyrazolo[1,5-c][1,3]oxazine-
5-spiro-40-(10-propylpiperidine) (45)20 was selected for
chiral resolution (one chiral center is present in the
pyrazoline ring). From the racemate mixture, both
enantiomers were separated by chromatography using
a chiral column.21 Each enantiomer exhibited very
different activity. The enantiomer with the shorter
retention time, 45tR1, was the most active with an
IC50 = 1.0 nM, while the second one (longer retention
time, 45tR2 was inactive up to 500 nM. Therefore, the
stereochemistry of the compound was important for
the target affinity.
26
27
na
28
na
O
a Racemates were prepared by heating pyrazoline IV with corre-
sponding ketone at 50 ꢁC.
b Inhibition of calcium release generated by a-methyl-5-HT (Ref. 17).
c na, not active. Average value from one experiment in duplicate.
Table 3. Variations at the R position
Ar
N
X
N
In order to avoid the chirality issue, pyrazole 52 was
synthesized from the pyrazoline 45. Several oxidizing
agents like MnO2, H2O2, and 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ) were attempted but failed.
Finally, the reaction was carried out successfully using
ammonium cerium (IV) nitrate (CAN) and gave 50%
yield of 52 which was found to be inactive (Scheme 2).
O
N
R
Compounda Ar
X
R
Activityb
c
IC50 (nM)
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Phenyl
Br Acetyl
Br Acetyl
Br Acetyl
Br Benzyl
na
na
4-Fluorophenyl
4-Chlorophenyl
2-Chlorophenyl
4-Chlorophenyl
na
na
In summary, a novel series of 5-HT2B receptor ligand
was identified, 2-aryl-1,10b-dihydro-benzo[e]pyrazolo
H
Benzyl
350
na
4-Methoxyphenyl Br Benzyl
[1,5-c][1,3]oxazine-5-spiro-40-(10-alkylpiperidine).
We
4-Pyridyl
4-Pyridyl
Phenyl
H
H
Ethyl
i-Butyl
42
21
found that variations of aromatic substitution (Ar)
and N-substitution of the piperidyl can influence the
activity and allowed to highlight potent antagonists
(IC50 < 10 nM in a functional assay). The most dra-
matic structural modification observed was the re-
quired basic nitrogen of the piperidine ring,
indicating a strong proton acceptor interaction, and
the stereospecificity on the pyrazoline ring which
seems related to a specific protein pocket for the aro-
matic group. The established S.A.R. pattern gave the
opportunity for further optimization. Further studies
are needed to find out the selectivity of 5HT receptor
sub-types for these spiro-piperidines.
Br i-Propyl
188
133
>10,000
5000
791
179
15
<10d
1.8
115
26
4-Chlorophenyl
4-Bromophenyl
4-Tolyl
H
H
i-Propyl
i-Propyl
Br n-Propyl
Br n-Propyl
4-Methoxyphenyl Br n-Propyl
4-Bromophenyl
4-Pyridyl
Phenyl
H
H
H
n-Propyl
n-Propyl
n-Propyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
4-Fluorophenyl
2-Chlorophenyl
4-Pyridyl
Me n-Propyl
F
n-Propyl
n-Propyl
Br n-Propyl
Cl
32
29
H
H
n-Propyl
2-Phenylethyl 16
500
a Racemates were used.
References and notes
b Inhibition of calcium release generated by a-methyl-5-HT (Ref. 17).
c na, not active. Average value from one experiment in duplicate.
d 90% inhibition at 10 nM.
1. Clineschmidt, B. V.; Reiss, D. R.; Pettibone, D. J.;
Robinson, J. L. J. Pharmacol. Exp. Ther 1985, 235, 696.

´
H. Bienayme et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4830–4833
4833
2. Baez, M.; Yu, L.; Cohen, M. L. Mol. Pharmacol 1990, 38,
31.
3. Kursar, J. D.; Nelson, D. L.; Wainscott, D. B.; Cohen, M.
L.; Baez, M. Mol. Pharmacol. 1992, 42, 549.
4. Humphrey, P. P. A.; Hartig, P.; Hoyer, D. Trends
Pharmacol. Sci. 1993, 14, 233.
5. Hoyer, D.; Clarke, D. E.; Fozard, J. R.; Hartig, P. R.;
Martin, G. R.; Mylecharane, E. J.; Saxena, P. R.;
Humphrey, P. P. A. Pharmacol. Rev. 1994, 46, 157.
6. Baxter, G.; Kennett, G.; Blaney, F.; Blackburn, T. Trends
Pharmacol. Sci. 1995, 16, 105.
compound followed by the addition of the agonist
(a-methyl-5-HT) at a concentration corresponding to its
EC80 of the day. The luminescence, related to calcium
release, was measured for 30 s following the agonist
addition. Inhibition percentage of the luminescence was
measured and IC50 evaluated for active compounds.
18. Annigeri, A. C.; Siddappa, S. Indian J. Chem., Sect B
1963, 1, 484.
19. Pathak, V. N.; Pathak, R.; Gupta, R.; Oza, C. K. Synth.
Commun. 1997, 27, 1811.
20. Characterization data for compound 45: (mp 130.1–
131.3 ꢁC); 1H NMR (400 MHz, CDCl3): dH (ppm),
7.62–7.58 (m, 2H, CHarom), 7.12–7.08 (m, 1H, CHarom),
7. Hoyer, D.; Martin, G. Neuropharmacology 1997, 36, 419.
ˆ ´
8. Latil, A.; Chene, L.; Grisoni, S.; Bienayme, H. WO Patent
2004061408, 2004.
7.04–6.97 (m, 3H, CHarom), 6.91 (dt, 1H, CHarom
J1 = 1.01 Hz, J2 = 7.58 Hz), 6.77 (dd, 1H, CHarom
J1 = 1.01 Hz, J2 = 8.08 Hz), 5.08 (d, 1H, CHpyrazoline
,
,
,
9. Berger, J.; Flippin, L. A.; Greenhouse, R.; Jaime-Figueroa
S.; Liu, Y.; Miller, A. K.; Putman, D. G.; Weinhardt K.
K.; Zhao, S. WO Patent 9744326, 1997.
10. Audia, J. E.; Baker, S. R.; Ezquerra, J.; Lamas, C.;
Pedregal, C. WO Patent 9624351, 1996.
J = 9.35 Hz), 3.51–3.45 (m, 1H, CHpyrazoline), 3.23 (dd,
1H, CHpyrazoline, J1 = 1.26 Hz, J2 = 15.92 Hz), 2.84–2.79
(m, 1H, CH), 2.68–2.59 (m, 3H, CH), 2.50 (m, 1H, CH),
2.40–2.36 (m, 3H, CH), 2.11–2.01 (m, 2H, CH2), 1.60–1.51
(m, 2H, CH2), 0.92 (t, 3H, CH3, J = 7.33 Hz); 13C NMR
(100 MHz, CDCl3): d (ppm), 163.1 (d, J = 248.82 Hz),
152.2, 150.7, 129.1, 129.1, 128.2, 128.1, 127.0, 126.4, 123.3,
121.3, 117.5, 115.4, 115.2, 88.1, 60.5, 56.5, 49.6, 49.4, 41.0,
34.8, 32.4, 20.4, 12.1; LC/MS (EI, m/z): (M+l) 380.28; Rf
(10% MeOH in DCM) = 0.52.
´
11. Launay, J. M.; Herve, P.; Peoc’h, K.; Tournois, C.;
Callebert, J.; Nebigil, C. G.; Etienne, N.; Drouet, L.;
Humbert, M.; Simonneau, G.; Maroteaux, L. Nat. Med.
2002, 8, 1129.
12. Bonhaus, D. W.; Bach, C.; Desouza, A.; Salazar, F. H.;
Matsuoka, B. D.; Zuppan, P.; Chan, H. W.; Eglen, R. M.
Br. J. Pharmacol. 1995, 115, 622.
13. Baxter, G. S. Behav. Brain Res. 1996, 73, 149.
14. Audia, J. E.; Evard, D. A.; Murdoch, G. R.; Droste, J. J.;
Nissen, J. S.; Schenck, K. W.; Fludzinski, P.; Lucaites, V.
L.; Nelson, D. L.; Cohen, M. L. J. Med. Chem. 1996, 39,
2773.
21. HPLC experiments were carried out using a Waters HPLC
system equipped with a Quaternary Pump 600 E Multisol-
vent Delivery System, a Waters 996 Photodiode Array
Detector, aWaters 2767 Sample Manager, a make-up
pump Waters Reagent Manager (100% MeOH, flow rate
15. Bonhaus, D. W.; Flippin, L. A.; Greenhouse, R. J.; Jaime,
S.; Rocha, C.; Dawson, M.; Van Natta, K.; Chang, L. K.;
Pulido-Rios, T.; Webber, A.; Leung, E.; Eglen, R. M.;
Martin, G. R. Br. J. Pharmacol. 1999, 127, 1075.
16. The selection has been made creating a chemofilter of
autocorrelative vectors (Oto program) by discriminant
analysis from a set of 167 published selective and not
selective 5-HT2B ligands extracted from Aureus Pharma
database. The confidence of the assay was 92% well
identified compared to 63% in the random case. A virtual
screening was conducted with this filter on an internal
collection of 30,000 compounds driving to the set of 1250
small molecules.
1 ml/min), and a preparative/analytical splitter from
LCPackings with a 1/1000 split. The system was run with
MassLynx 3.5 configured with OpenLynx 3.5 and Frac-
tionLynx 3.5. The chiral stationary phases (CSP) were
Chiralpak AD-H, 5 lm. Analytical and preparative assays
were carried out on 250 · 4.6 mm ID and 250 · 20 mm ID
columns, respectively. Flow rates were 1 ml/min and
19 ml/min, respectively. Samples were dissolved in MeOH
(36 mg/ml). Enantiomers were resolved on a semi-pre-
parative scale. Fraction collections were triggered by the
DAD TIC signal (210–500 nm). The HPLC analytical
assay was performed using mixtures of acetonitrile/2-
propanol/diethylamine (85:15:0.1) as eluent. The retention
17. Screening protocol (Euroscreen): CHO cells expressing
5-HT2B receptors, aequorin, and Ga16 were seeded in
poly-D-lysine coated plates with coelentterazine and fetal
serum albumin. After 15-h incubation at rt, the cell
suspension was incubated for 30 min with the tested
time of the 2 enantiomers was tR1 = 4.2 min and
tR2 = 5.2 min.
tR1
tR2
k10
0.24
k20
a
w1
w2
Rs
4.2
5.2
0.53
2.2
0.30
0.47
1.53