Synthesis and in vitro and in vivo functional studies of Ortho- substituted phenylpiperazine and N-substituted 4-N-(o- methoxyphenyl)aminopiperidine analogues of WAY100635

Article abstract of DOI:10.1021/jm991088y

WAY100635 (2), N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2- pyridinyl)cyclohexane-carboxamide, is a silent serotonin 5-HT(1A) antagonist, which is now widely used to study the 5-HT(1A) receptor both in vivo and in vitro. In this paper, we describe

Full text of DOI:10.1021/jm991088y

432
J . Med. Chem. 2000, 43, 432-439
Syn th esis a n d in Vitr o a n d in Vivo F u n ction a l Stu d ies of Or th o-Su bstitu ted
P h en ylp ip er a zin e a n d N-Su bstitu ted 4-N-(o-Meth oxyp h en yl)a m in op ip er id in e
An a logu es of WAY100635
Margue´rite M. Mensonides-Harsema,*,§ Yi Liao,§ Henning Bo¨ttcher,# Gerd D. Bartoszyk,# Hartmunt E. Greiner,#
J u¨rgen Harting,# Peter de Boer,‡ and Håkan V. Wikstro¨m§
Department of Medicinal Chemistry, University of Groningen, A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands,
Preclinical Pharmaceutical Research, Merck KGaA, D-64271 Darmstadt, Germany, and Pharma Bio-Research International
B.V., NL-9470 AE Zuidlaren, The Netherlands
Received May 31, 1999
WAY100635 (2), N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane-
carboxamide, is a silent serotonin 5-HT1A antagonist, which is now widely used to study the
5-HT1A receptor both in vivo and in vitro. In this paper, we describe the synthesis and in vitro
(5-HT1A affinity and pA2 values at guinea pig ileum strips) and in vivo (hypothermia and
ultrasonic vocalization) pharmacology at the serotonin 5-HT1A receptor of several closely related
analogues of 2. Test compounds 12 and 14, in which the arylpiperazine moiety of 2 has been
replaced by an arylaminopiperidine moiety, showed no affinity or antagonistic activity at the
5-HT1A receptor. Substitution of the o-methoxy group of 2 by larger fluoroalkoxy or sulfonyloxy
substituents did not alter the in vitro or in vivo pharmacology to any great extent; in vivo both
the fluoropropyl analogue 5 and the triflate analogue 7 are equipotent to WAY100635 itself.
The O-desmethyl analogue 3 proved to be the most potent antagonist at the serotonin 5-HT1A
postsynaptic receptor sites in this series.
In tr od u ction
logical changes that can easily be quantified in several
in vitro and in vivo assays. In rats, the administration
of serotonin 5-HT1A agonists results in a rectal temper-
ature decrease,15 a characteristic set of motor changes
collectively known as the serotonin motor syndrome16
(both models for postsynaptic activation), inhibition of
synthesis rate of serotonin 5-HT in the terminal areas,17
inhibition of ultrasonic vocalization,18,19 and inhibition
of neuronal firing activity20 (all models for presynaptic
activation).
The serotonin (5-hydroxytryptamine, 5-HT) receptor
family consists of seven distinct classes (serotonin
5-HT1-7). All serotonin receptors belong to the G-
protein-coupled receptor (GPCR) superfamily, bar the
serotonin 5-HT3 receptor, which is a ligand-gated ion
channel.1 The serotonin 5-HT1 receptors, based on
considerations of structural features, functional phar-
macology, and secondary messenger coupling systems,
have been further divided into subtypes. They have been
Positron emission tomography (PET), using effective
positron-emitting radioligands, provides the means to
visualize 5-HT1A receptor densities in living subjects and
to monitor inhibition of receptor binding with unlabeled
ligands (both endogenous and exogenous).21-23 Agonists,
however, are limited in their use for imaging in vivo,
possibly because they bind to the high-affinity agonist
state of the receptor only, which quickly reverts to a low-
affinity state and a fast dissociation of radioligand.24-26
Antagonists bind to receptors in both their high- and
low-affinity states. WAY100635 (N-(2-(4-(2-methoxy-
phenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)cyclohexane-
carboxamide, 2) (Chart 1) is known to be one of the first
potent, silent, and selective antagonists for serotonin
5-HT1A receptors at both somatodendritic and postsyn-
aptic receptor sites.27-29 The [11C-carbonyl]-labeled O-des-
methyl analogue 3 of WAY100635 has proven to be a
successful radioligand for studies of central serotonin
5-HT1A receptors in rat, monkey, and human brain
using PET.30
classified as serotonin 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E
,
and 5-HT1F receptors and are all negatively coupled to
adenylyl cyclase.2-5
The serotonin 5-HT1A receptors are present through-
out the whole body, including the central nervous
system (CNS). In the CNS, the serotonin 5-HT1A recep-
tors are located both presynaptically on the cell body of
the serotonergic neurons in the raphe´ nucleus - where
they control the firing of the serotonin 5-HT neuron via
autoinhibition - as well as postsynaptically in the
projection areas such as the dentate gyrus of the
hippocampus (CA1 region), frontal cortex and neocortex,
amygdala, and lateral septum.6-11 These brain areas
have frequently been associated with mood control, and
the serotonin 5-HT1A receptor is generally considered
to be an important target for the treatment of mood
disorders such as anxiety and depression.12-14 Activa-
tion of serotonin 5-HT1A receptors with agonists, like
8-OH-DPAT (1) (Chart 1), leads to a number of physio-
We have synthesized several ortho-substituted phen-
ylpiperazine and N-substituted 4-N-(o-methoxyphenyl)-
aminopiperidine analogues of 2 and have determined
their affinity and selectivity at the serotonin 5-HT1A
* To whom correspondence should be addressed. Phone: +31-50-
363 3302. Fax: +31-50-363 6908. E-mail: m.m.mensonides@farm.rug.nl.
§ University of Groningen.
#
Merck KGaA.
‡ Pharma Bio-Research International B.V.
10.1021/jm991088y CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/13/2000

Synthesis/ Functional Studies of WAY100635 Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 433
Ch a r t 1
Sch em e 1a
receptor vs the serotonin 5-HT1B/1D receptor, which is
also located both presynaptically on the nerve terminals
of the serotonergic neurons as well as postsynaptically.
In addition, the in vitro antagonistic properties (pA2
values) and the ability to counteract, in vivo, the 8-OH-
DPAT-induced inhibition of ultrasonic vocalization and
hypothermia in rats were measured for test compounds
displaying a high affinity and selectivity for the sero-
tonin 5-HT1A receptors.
Ch em istr y
WAY100635 (2) was prepared as previously de-
scribed.27 The O-demethylation of 2 could not be effected
with refluxing HBr (48%), with or without acetic acid
(partial conversion), or by employing BBr3 in CH2Cl2
(no conversion). The reaction was, however, carried out
smoothly and in high yield upon treatment with 6 equiv
of AlCl3 refluxing in freshly distilled benzene under a
nitrogen atmosphere, yielding N-[2-[4-(2-hydroxyphen-
yl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecar-
boxamide (3).31 Derivatization of the hydroxyl group of
compound 3 was accomplished either with the appropri-
ate alkyl bromides in the presence of Cs2CO3, yielding
N-[2-[4-(2-(2-fluoroethoxy)phenyl)-1-piperazinyl]ethyl]-
N-(2-pyridinyl)cyclohexanecarboxamide (4) and N-[2-[4-
(2-(3-fluoropropoxy)phenyl)-1-piperazinyl]ethyl]-N-(2-
pyridinyl)cyclohexanecarboxamide (5), or with methane-
sulfonyl chloride in the presence of triethylamine,
yielding N-[2-[4-[2-[methanesulfonoxyl]phenyl]-1-pipera-
zinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide (6),
or with N-phenyltrifluoromethanesulfonimide using
phase-transfer conditions with 10% NaOH(aq) and
CH2Cl2, in the presence of the phase-transfer catalyst
tetrabutylammonium iodide, yielding N-[2-[4-[2-[(trifluo-
romethyl)sulfonoxyl]phenyl]-1-piperazinyl]ethyl]-N-(2-
pyridinyl)cyclohexanecarboxamide (7) (Scheme 1).
For the synthesis of 4-N-(o-methoxyphenyl)piperidine
analogues, 2-methoxyaniline was coupled to 1-benzyl-
4-piperidone in refluxing toluene in the presence of
p-toluenesulfonic acid, hydrogenated using Adam’s cata-
lyst, and debenzylated by applying H2 (60 psi) in the
presence of palladium hydroxide on carbon in a mixture
of MeOH/THF, yielding 4-N-(2-methoxyphenyl)aminopi-
peridine (8). This intermediate was then coupled to
2-chloro-N-pyridin-2-ylacetamide27 in refluxing aceto-
nitrile in the presence of triethylamine, potassium
carbonate, and tetrabutylammonium iodide, yielding
2-[4-N-(o-methoxyphenyl)aminopiperidinyl]-N-pyridin-
2-ylacetamide (9). N-Methylation of compound 9 was
achieved with reductive amination in a Parr shaker
using formaldehyde in EtOH in the presence of pal-
ladium hydroxide (20%) on carbon, yielding 2-[4-N-(o-
methoxyphenyl)-N-methylaminopiperidinyl]-N-pyridin-
2-ylacetamide (10). The acetamide was reduced in ether
by using DIBAL-H, yielding 2-[4-N-(o-methoxyphenyl)-
N-methylaminopiperidinyl]-N-pyridin-2-ylamine (11),
a
Reagents: (a) aluminum chloride, benzene, ∆; (b) fluoroethyl
bromide/fluoropropyl bromide, cesium carbonate, acetonitrile, ∆
(4/5); methanesulfonyl chloride, triethylamine, CH2Cl2, rt (6);
N-phenyltrifluoromethane sulfonimide, tetrabutylammonium io-
dide, 10% NaOH(aq)/CH2Cl2, rt (7).
which was subsequently coupled to cyclohexanecarboxy-
lic acid chloride in CH2Cl2 in the presence of triethy-
lamine yielding 2-[4-N-(o-methoxyphenyl)-N-methylami-
nopiperidinyl]-N-(2-pyridinyl)cyclohexanecarboxamide
(12). Reduction of intermediate 9 in ether by using
DIBAL-H yielded 2-[4-N-(o-methoxyphenyl)aminopi-
peridinyl]-N-pyridin-2-ylamine (13). Reacting compound
13 with cyclohexanecarboxylic acid chloride in CH2Cl2
in the presence of triethylamine yielded [4-N-(o-meth-
oxyphenyl)-N-cyclohexanecarboxamidopiperidinyl]-N-(2-
pyridinyl)cyclohexanecarboxamide (14) (Scheme 2).
P h a r m a cology
In Vitr o Bin d in g. The abilities of the new com-
pounds to displace the radioligands [3H]-8-OH-DPAT (5-
HT1A) and [3H]-5-HT (5-HT1B/1D) were assessed (Table
1).46-48
p A2 Va lu es. The ability of the new compounds to
antagonize the 8-OH-DPAT-induced contractions of a
strip of guinea pig ileum tissue was measured (Table
1).38,51
Ultr a son ic Voca liza tion . The ability of the new
compounds to antagonize the 8-OH-DPAT-induced in-
hibition of footshock-evoked ultrasonic vocalization in
rats was determined (Table 2).18,19,45
Hyp oth er m ia . The ability of the new compounds to
counteract the 8-OH-DPAT-induced hypothermia in rats
was also measured (Table 3).16
Resu lts a n d Discu ssion
Structure-activity relationships (SARs) were deter-
mined from affinity values obtained using radioligand
binding for both the 5-HT1A and 5-HT1B/1D autoreceptors
(Table 1). The affinity of the phenylpiperazine ana-
logues, in which the methoxy group of WAY100635 (2)
has been replaced by a hydroxy (3), a fluoroalkoxy (4,
5), a methanesulfonoxy (6), or the even stronger electron-

434 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Mensonides-Harsema et al.
Sch em e 2a
a
Reagents: (a) i. p-TsOH, toluene, ∆, ii. H2 (50 psi), PtO2, toluene, 60 °C, iii. H2 (60 psi), 20% Pd(OH)2/C, MeOH/THF, 60 °C; (b)
2-chloro-N-pyridin-2-ylacetamide, K2CO3, tetrabutylammonium iodide, acetonitrile, ∆; (c) HCHO, H2 (60 psi), 20% Pd(OH)2/C, EtOH, 60
°C; (d) DIBAL-H, Et2O, 0 °C to rt; (e) cyclohexanecarboxylic acid chloride, TEA, CH2Cl2, 0 °C to rt.
Ta ble 1. In Vitro Affinities at 5-HT1A and 5-HT1B/1D Receptors
(IC50 in nM) and pA2 Valuesa
Ta ble 2. Inhibition of Ultrasonic Vocalization after
Administration of 8-OH-DPAT (0.1 mg/kg sc) Alone or in
Combination with WAY100635 (2) or Test Compounds 3-7 and
14 (0.3 mg/kg sc)
b
c
compd
5-HT1A
5-HT1B/1D
pA2
2
3
4
5
6
7
12
14
0.37 ( 0.02
2.10 ( 0.52
1.55 ( 0.61
6.05 ( 0.18
7.25 ( 2.71
36.0 ( 13.5
1290 ( 1330
500 ( 266
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
8.7
8.0
8.5
8.3
8.6
7.7
<7
treatment (n ) 4-8)
vocalization time (s ( SEM)
control
8-OH-DPAT
2
3
4
5
6
7
148 ( 11
4.9 ( 4.1
158 ( 11*
166 ( 11*
164 ( 8*
171 ( 4*
167 ( 3*
146 ( 12*
7.4 ( 5.8§
<7
vs 8-OH-DPAT (guinea pig ileum). [3H]-8-OH-DPAT (rat
a
b
hippocampus). c [3H]-5-HT (calf striatum).
14
*p < 0.05 vs 8-OH-DPAT. § p < 0.05 vs control.
withdrawing trifluoromethanesulfonoxy group (7), for
the serotonin 5-HT1A receptor remains in the nanomolar
range. None of the test compounds showed affinity for
the serotonin 5-HT1B receptor. The aminopiperidines 12
and 14 showed no affinity for the serotonin 5-HT1A
receptor (Table 1). These binding results suggest that
the serotonin 5-HT1A receptor site can accommodate
larger substituents than the methoxy group, also with
electron-withdrawing properties, in the ortho-position
of the phenyl ring. Thus, the fluoroalkyl analogues offer
the opportunity to introduce 18F, resulting in radioli-
gands with a much longer half-life than 11C (t1/2 of 109
min vs 20 min, respectively).32,33 The aryl triflate, which
is known to make the phenyl ring less prone to meta-
bolic degradation, can serve as an alternative to aro-
matic methoxy substituents, present in several com-
pounds with CNS activity, improving their oral availa-
bility.34-37 Replacement of the rather flexible piperazine
ring with an aminopiperidine leads to a total loss of
affinity for the serotonin 5-HT1A receptor. This might
be due to the fact that the basic nitrogen of the
piperazine has been moved away from the aromatic ring
with one extra carbon bond (ca. 1.5 Å).
In vitro functional data were obtained using the
guinea pig ileum assay, in which the ability of the test
compounds (2-7, 12, and 14) to antagonize the 8-OH-
DPAT-induced contractions of a strip of ileum tissue was
measured (Table 1).38 The data obtained with this model

Synthesis/ Functional Studies of WAY100635 Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 435
Ta ble 3. Effects of WAY100635 (2) and Test Compounds 3-7
on 8-OH-DPAT (1 mg/kg sc)-Induced Hypothermia (n ) 4)
To summarize, a small series of structural analogues
of WAY100635 (2) have been synthesized and their in
vitro and in vivo pharmacology has been evaluated.
From the pharmacological results, it may be concluded
that the arylaminopiperidine moiety is not able to act
as a bioisostere for the arylpiperazine that is present
in WAY100635 (2). Compounds 12 and 14 showed no
serotonin 5-HT1A receptor affinity or activity, neither
in vitro nor in vivo. The substituent in the ortho-position
of the phenylpiperazine, on the other hand, can be both
smaller and larger than the original methoxy substitu-
ent of WAY100635 (2) and have strong electron-
withdrawing properties. In the 8-OH-DPAT-induced
hypothermia assay, WAY100635 (2) is equipotent to the
fluoropropyl analogue 5 (a possible [18F]PET ligand)32
and the triflate analogue 7. However, the O-desmethyl
analogue 3, which has shown to be a very promising
radioligand in PET studies,30 is the functionally most
potent serotonin 5-HT1A antagonist at the postsynaptic
receptor sites.
decrease in body temperature (oC)
compd
0.1 (µmol/kg)
1.0 (µmol/kg)
10 (µmol/kg)
2
3
4
5
6
7
1.5 ( 0.2
0.6 ( 0.0*
1.8 ( 0.1
2.4 ( 0.1
1.1 ( 0.3
1.5 ( 0.2
0.0 ( 0.1
2.0 ( 0.1
0.9 ( 0.1*
0.2 ( 0.1*
1.1 ( 0.1
0.6 ( 0.1*
0.5 ( 0.2*
0.0 ( 0.1*
0.9 ( 0.0*
0.3 ( 0.1*
0.4 ( 0.2*
0.5 ( 0.2*
0.4 ( 0.0*
0.3 ( 0.1*
control
8-OH-DPAT
*p < 0.05 vs 8-OH-DPAT.
show that compounds 4-6 are potent antagonists, while
the aminopiperidine analogues 12 and 14 failed to
antagonize the 8-OH-DPAT-induced contractions.
In vivo functional data of the test compounds were
obtained using the rat ultrasonic vocalization model
(Table 2) and the rat hypothermia model (Table 3).
Inhibition of ultrasonic vocalization by serotonin 5-HT1A
receptor agonists results from activation of presynaptic
(somatodendritic) receptors, because local (as well as
systemic) injections inhibit firing of raphe´ nuclei neu-
rons and reduce ultrasonic vocalization even after
hippocampal lesions.39-41 WAY100635 (2), as well as
compounds 3-7, did antagonize the 8-OH-DPAT-
induced inhibition of ultrasonic vocalization at 0.3 mg/
kg sc test compound vs 0.1 mg/kg agonist, demonstrat-
ing their ability to act as autoreceptor antagonists in
vivo. This suggests that the arylpiperazine analogues
are all able to cross the blood-brain barrier. The
aminopiperidine 14 was not able to counteract the
agonist-induced inhibition at the doses tested, which is
in line with in vitro data. The decrease in body tem-
perature, as observed in rats treated with serotonin
5-HT1A receptor agonists, is postsynaptically mediated,
because hypothermia is still present following depletion
of endogenous 5-HT.16,42-45 The O-desmethyl arylpi-
perazine analogue 3 proved to be the most potent
functional postsynaptic antagonist in this model. It
significantly antagonized the 8-OH-DPAT-induced hy-
pothermia (3 µmol/kg sc) at the lowest dose tested (0.1
µmol/kg). WAY100635 (2) and the fluoropropyl (5), the
mesylate (6), and the triflate (7) analogues proved to
be equipotent in this assay. They were all able to
significantly block the decrease in body temperature at
1.0 µmol/kg sc. The fluoroethyl analogue 4 was the
weakest antagonist in this postsynaptic serotonin 5-HT1A
activation model. It significantly blocked the decrease
in body temperature at the highest dose tested (10 µmol/
kg sc). The in vivo results show that the in vitro data
obtained are not able to discriminate the most potent
serotonin 5-HT1A receptor antagonist. The subnanomo-
lar affinity of WAY100635 (2) for the serotonin 5-HT1A
receptor binding site combined with the highest pA2
value do not ensure that it is indeed the best serotonin
5-HT1A antagonist. In fact, in vivo in the postsynaptic
8-OH-DPAT-induced hypothermia assay, WAY100635
(2) showed to be equipotent to the triflate analogue 7,
which is, in vitro, the weakest arylpiperazine. This
discrepancy between the in vitro and in vivo data might
be due to differences in pharmacokinetic, pharmacody-
namic, and/or metabolic profiles of the different test
compounds and WAY100635 (2).
Exp er im en ta l Section
Ch em istr y. Melting points were determined with an Elec-
trothermal digital capillary melting point apparatus and are
uncorrected. NMR spectra were acquired on a Varian Gemini-
200 spectrometer at 200 MHz (proton) and 50.3 MHz (carbon);
J values are given in hertz (Hz). CDCl3 was employed as the
solvent unless otherwise stated. Chemical shifts are given in
δ units (ppm) and relative to tetramethylsilane or deuterated
solvent. Infrared spectra were recorded with an ATI-Mattson
spectrometer. Elemental analyses were performed in the labs
of Merck KGaA Darmstadt (D) or in the Microanalytical
Department of the University of Groningen (NL) and were
within 0.4% of theoretical values unless otherwise indicated.
GC/MS (EI and CI) mass spectra were recorded on a Uni-
cam610/Automass 150 GC/MS system (70 mV). HRMS spectra
were performed in the Microanalytical Department of the
University of Groningen (NL) and were recorded on a J EOL
MS Route J MS 600H. For column chromatography silica gel
60 (70-230 mesh) (Merck) was used. Reactions were carried
out under a nitrogen atmosphere, solvents used were distilled
and/or dried by standard techniques immediately prior to use,
and in the case of an aqueous workup, magnesium sulfate
monohydrate was used as the drying agent. WAY100635 (2)
was prepared as previously described.27
N-[2-[4-(2-H yd r oxyp h en yl)-1-p ip er a zin yl]et h yl]-N-(2-
p yr id in yl)cycloh exa n eca r boxa m id e (3). A suspension of
2 (50.0 mg, 118 µmol) and AlCl3 (95.0 mg, 700 µmol) in benzene
(5 mL) was refluxed for 2 h. The reaction was quenched with
H2O (5 mL) and neutralized with solid NaHCO3. The mixture
was extracted with CH2Cl2, (3 × 20 mL) and the combined
organic layers were washed with brine, dried, and concentrated
in vacuo, to yield 45 mg (93%) of a colorless oil, which was
converted to the oxalate salt and recrystallized from i-PrOH
yielding 43 mg (73%) of 3 as white crystals: mp 204-207 °C;
1H NMR δ 8.56-8.53 (dd, J 1 ) 4.96, J 2 ) 1.78, 1 H), 7.84-
7.75 (dt, J 1 ) 7.75, J 2 ) 2.01, 1 H), 7.31-7.23 (m, 2 H), 7.14-
7.03 (m, 2 H), 6.96-6.81 (m, 2 H), 4.03-3.96 (t, J ) 6.93, 2
H), 2.82-2.78 (m, 4 H), 2.67-2.60 (m, 6 H), 2.31-2.18 (m, 1
H), 1.79-1.57 (m, 7 H), 1.26-0.98 (m, 3 H); 13C NMR δ 176.1,
151.5, 149.3, 138.9, 138.0, 126.4, 122.1, 119.9, 114.0, 100.0.
56.1, 53.8, 52.5, 45.1, 42.4, 29.5, 25.6; IR (NaCl) 3299, 1659
cm-1; MS (CI with NH3) m/z 409 (M+); HRMS calcd (obsd) for
C
24H32N4O2: 408.253 (408.253). Anal. (C24H32N4O2‚C2H2O4‚
0.5H2O) C, H, N.
N-[2-[4-(2-(2-Flu or oeth oxy)ph en yl)-1-piper azin yl]eth yl]-
N-(2-p yr id in yl)cycloh exa n eca r boxa m id e (4). To a suspen-
sion of 3 (0.10 g, 0.25 mmol) and Cs2CO3 (0.60 g, 1.84 mmol)
in acetonitrile (30 mL) was added fluoroethyl bromide (0.05
g, 0.40 mmol). After refluxing for 3 h, the solvent was removed
in vacuo. The residue was redissolved in H2O and extracted

436 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Mensonides-Harsema et al.
with CH2Cl2. The combined organic layers were washed with
brine, dried, and concentrated in vacuo. The oil obtained was
converted to the oxalate salt and recrystallized from EtOH,
yielding 60 mg (45%) of 4 as white crystals: mp 207-209 °C;
1H NMR δ 8.55-8.52 (dd, J 1 ) 5.25, J 2 ) 1.59, 1 H), 7.81-
7.72 (dt, J 1 ) 7.70, J 2 ) 2.06, 1 H), 7.32-7.21 (m, 2 H), 6.99-
6.82 (m, 4 H), 4.88 (t, J ) 4.11, 1 H), 4.64 (t, J ) 4.13, 1 H),
4.31 (t, J ) 4.09, 1 H), 4.17 (t, J ) 4.13, 1 H), 3.99 (t, J ) 6.96,
2 H), 3.10-2.95 (m, 4 H), 2.65-2.58 (m, 6 H), 2.24-2.18 (m, 1
H), 1.89-0.83 (m, 10 H); IR (KBr) 1658; MS (CI with NH3)
m/z 455 (M+); HRMS calcd (obsd) for C26H35N4O2F1: 454.274
(454.274). Anal. (C26H35N4O2F1‚C2H2O4‚0.25H2O) C, H, N.
N-[2-[4-(2-(2-Flu or opr opoxy)ph en yl)-1-piper azin yl]eth -
yl]-N-(2-p yr id in yl)cycloh exa n eca r boxa m id e (5). To a sus-
pension of 3 (0.02 g, 0.05 mmol) and Cs2CO3 (0.08 g, 0.25 mmol)
in acetonitrile (4 mL) was added fluoropropyl bromide (0.08
g, 0.06 mmol). After refluxing for 2 h, the solvent was removed
in vacuo. The residue was redissolved in H2O and extracted
with CH2Cl2. The combined organic layers were washed with
brine, dried, and concentrated in vacuo. The oil obtained was
converted to the oxalate salt and recrystallized from EtOH
yielding 17 mg (62%) of 5 as white crystals (monooxalate
salt): mp 181-183 °C; 1H NMR δ 8.55-8.52 (dd, J 1 ) 5.22, J 2
) 1.61, 1 H), 7.81-7.72 (dt, J 1 ) 7.70, J 2 ) 1.93, 1 H), 7.32-
7.21 (m, 2 H), 7.02-6.84 (m, 4 H), 4.78 (t, J ) 5.82, 1 H), 4.55
(t, J ) 5.80, 1 H), 4.12 (t, J ) 6.63, 1 H), 4.00 (t, J ) 6.96, 1
H), 3.00 (m, 4 H), 2.71-2.58 (m, 6 H), 2.27 (t, J ) 5.96, 1 H),
2.14 (t, J ) 5.96, 1 H), 1.93 (m, 1 H), 1.79-1.52 (m, 7 H), 1.26-
0.97 (m, 3 H); 13C NMR δ 176.2, 156.0, 149.2, 141.5, 138.0,
131.0, 122.7, 122.3, 122.2, 121.3, 118.2, 82.5, 79.3, 63.8, 63.7,
56.2, 53.4, 50.6, 45.1, 42.4, 30.9, 30.5, 29.5, 25.6; IR (KBr) 1656;
MS (CI with NH3) m/z 469 (M+); HRMS calcd (obsd) for
C26H35N4O2F1: 468.290 (468.290). Anal. (C26H35N4O2F1‚C2H2O4‚
0.5H2O) C, H, N.
calcd (obsd) for C25H31N4O4S1F3: 540.201 (540.201). Anal.
(C25H31N4O4S1F3‚C2H2O4) C, H, N.
4-N-(2-Meth oxyp h en yl)a m in op ip er id in e (8). A solution
of o-anisidin (3.00 g, 25.0 mmol) and N-benzyl-4-piperidone
(3.80 g, 20.0 mmol) and p-toluenesulfonic acid (0.10 g) in
toluene (50 mL) was refluxed under Dean-Stark conditions
for 3 h. After cooling to room temperature, PtO2 (0.10 g) was
added and the mixture was hydrogenated in a Parr shaker
with H2 (60 psi) for 2 h at 60 °C. A solution of 25% ammonia-
(aq) (5 mL) was added and the mixture was hydrogenated
further for another 1 h (50 psi) at room temperature. The
reaction mixture was filtered over Celite and the solvent
removed in vacuo. The residue was purified by gradient flash
chromatography (hexane to EtOAc), yielding 2.7 g (46%) of the
intermediate as a brown-yellow oil. For the debenzylation, this
intermediate (2.7 g, 9.1 mmol) was dissolved in THF (10 mL)
and MeOH (190 mL), 20% Pd(OH)2/C (200 mg) was added, and
the mixture was hydrogenated in the Parr shaker with H2 (60
psi) for 2 h at 60 °C. The reaction mixture was filtered over
Celite and the solvent removed in vacuo. The residue was
recrystallized from hexane, yielding 1.7 g (91%) of 8 as white
crystals: mp 107-109 °C; 1H NMR δ 6.89-6.60 (m, 4 H), 3.83
(s, 3 H), 3.39-2.70 (m, 4 H), 2.3-2.08 (m, 2 H), 1.51-1.10 (m,
4 H), 0.88 (m, 1 H); 13C NMR δ 145.3, 135.2, 119.7, 114.8, 108.7,
108.1, 53.9, 48.1, 43.7, 31.9; MS (EIPI) m/z 206 (M+); HRMS
calcd (obsd) for C12H18N2O1: 206.14 (206.14).
2-[4-N-(o-Meth oxyph en yl)am in opiper idin yl]-N-pyr idin -
2-yla ceta m id e (9). A suspension of compound 8 (0.25 g, 1.20
mmol), 2-chloro-N-pyridin-2-ylacetamide (0.20 g, 1.17 mmol),
triethylamine (2 mL), K2CO3 (0.10 g, 0.72 mmol), and tetrabu-
tylammonium iodide (0.05 g) was refluxed for 18 h, after which
the solvent was removed in vacuo. The residue was purified
by flash chromatography (hexane with 20% EtOAc to pure
EtOAc), yielding 270 mg (66%) of a brown-yellow oil. The oil
was redissolved in CH2Cl2, converted to the HCl salt, and
N-[2-[4-[2-[Meth a n esu lfon oxyl]p h en yl]-1-p ip er a zin yl]-
eth yl]-N-(2-p yr id in yl)cycloh exa n eca r boxa m id e (6). To a
cooled solution (-78 °C) of 3 (0.05 g, 0.12 mmol) and triethyl-
amine (10 µL) in CH2Cl2 (2 mL) was slowly added methane-
sulfonyl chloride (0.02 g, 0.18 mmol). The reaction was allowed
to warm to room temperature, after which it was stirred for
another 6 h. The mixture was quenched with H2O and
extracted with CH2Cl2. The combined organic layers were
washed with brine, dried, and concentrated in vacuo. The
residue was purified by chromatography (CH2Cl2 with 3%
MeOH), converted to the oxalate salt, and recrystallized from
EtOH to yield 30 mg (43%) of 6 as white crystals: mp 234-
1
recrystallized from MeOH/Et2O: mp 156 °C; H NMR δ 9.65
(b, 1 H), 8.32-8.22 (m, 2 H), 7.73-7.63 (m, 1 H), 7.06 (m, 1
H), 6.85-6.59 (m, 4 H), 4.15 (b, 1 H), 3.83 (s, 3 H), 3.35 (m, 1
H), 3.16 (s, 2 H), 2.87 (m, 2 H)2.40 (m, 2 H), 2.10 (m, 2 H),
1.60 (m, 2 H); 13C NMR δ 169.0, 105.9, 147.9, 146.8, 132.2,
136.6, 121.0, 119.7, 116.3, 113.6, 110.2, 109.5, 62.0, 55.2, 52.8,
48.5, 32.3; MS (EI) m/z 340 (M+); HRMS calcd (obsd) for
C
19H24N3O2: 340.19 (340.19).
2-[4-N-(o-Meth oxyph en yl)-N-m eth ylam in opiper idin yl]-
N-p yr id in -2-yla ceta m id e (10). A mixture of compound 9
(0.50 g, 1.47 mmol), formaldehyde (10 mL), and 20% Pd(OH)2/C
(0.10 g) in EtOH (100 mL) was hydrogenated with H2 (60 psi)
for 18 h at 60 °C. The residue was purified by gradient flash
chromatography (CH2Cl2 with 10-20% EtOH), yielding 400
mg (77%) of a viscous brown oil. The oil was redissolved in
1
237 °C; H NMR δ 8.55-8.52 (dd, J 1 ) 5.49, J 2 ) 1.93, 1 H),
7.82-7.73 (dt, J 1 ) 7.71, J 2 ) 2.00, 1 H), 7.32-7.20 (m, 4 H),
7.08-7.00 (m, 2 H), 3.98 (t, J ) 6.89, 2 H), 3.15 (s, 3 H), 3.02-
2.98 (m, 4 H) 2.65-2.58 (m, 6 H), 2.32-2.15 (m, 1 H), 1.77-
0.96 (m, 10 H); 13C NMR δ 176.3, 155.8, 149.3, 144.7, 142.9,
138.1, 128.2, 124.6, 123.3, 122.2, 122.1, 119.8, 56.1, 53.4, 51.0,
45.0, 42.3, 38.3, 29.5, 25.5; IR (KBr) 1657, 1366, 1157; HRMS
1
Et2O and converted to the HCl salt: mp 180 °C; H NMR δ
9.60 (b, 1 H), 8.32-8.21 (m, 2 H), 7.69-7.65 (m, 1 H), 7.06-
6.85 (m, 5 H), 3.84 (s, 3 H), 3.22 (m, 1 H), 3.11 (s, 2 H), 2.97
(m, 2 H), 2.74 (s, 3 H), 2.24 (m, 2 H), 1.82 (m, 2 H), 1.76 (m, 2
H); 13C NMR δ 169.5, 153.1, 150.9, 147.8, 140.9, 138.2, 122.8,
121.3, 120.4, 119.7, 113.8, 111.1, 62.1, 58.4, 55.2, 53.9, 34.2,
28.2; MS (EIPI) m/z 354 (M+); HRMS calcd (obsd) for
calcd (obsd) for
C25H34N4O4S1: 486.230 (486.230). Anal.
(C25H34N4O4S1‚C2H2O4‚0.5H2O) C, H, N.
N-[2-[4-[2-[(Tr iflu or om et h yl)su lfon oxyl]p h en yl]-1-p i-
per azin yl]eth yl]-N-(2-pyr idin yl)cycloh exan ecar boxam ide
(7). To a suspension of 3 (0.10 g, 0.20 mmol) and tetrabutyl-
ammonium iodide (10 mol %) in CH2Cl2 (3.0 mL) and a 10%
solution of NaOH(aq) (1.5 mL) was added N-phenyltrifluo-
romethanesulfonimide (0.36 g, 1.0 mmol). The reaction was
stirred at room temperature for 36 h, after which it was
quenched with H2O (10 mL) and extracted with CH2Cl2. The
combined organic layers were washed with brine, dried, and
concentrated in vacuo. The residue was purified by flash
chromatography (CH2Cl2 with 5% MeOH), converted to the
oxalate salt, and recrystallized from EtOH to yield 46 mg (36%)
of 7 as white crystals: mp 182-184 °C; 1H NMR δ 8.55-8.52
C
20H26N3O2: 354.21 (354.21).
2-[4-N-(o-Meth oxyph en yl)-N-m eth ylam in opiper idin yl]-
N-p yr id in -2-yla m in e (11). To an ice-cooled solution of com-
pound 10 (0.30 g, 0.85 mmol) in Et2O (15 mL) was added
dropwise a 1.0 M solution of DIBAL-H in toluene (6 mL). The
mixture was stirred for 18 h at room temperature after which
H2O (3 drops) and acetone (1 mL) were added. The solvent
was removed in vacuo. The residue was purified by gradient
flash chromatography (EtOAc, THF, and then CH2Cl2 with
10-25% EtOH and 2% aq ammonia), yielding 260 mg (90%)
of 11 as a yellow oil. The oil was redissolved in Et2O and
1
(dd, J 1 ) 5.55, J 2 ) 2.04, 1 H), 7.82-7.73 (dt, J 1 ) 7.72, J 2
)
converted to the HCl salt: mp 165 °C; H NMR δ 8.09 (m, 1
1.91, 1 H), 7.36-7.25 (m, 4 H), 7.22-7.03 (m, 2 H), 4.04-3.97
(t, J ) 6.89, 2 H), 3.04-2.94 (m, 4 H), 2.76-2.64 (m, 6 H),
2.31-2.20 (m, 1 H), 1.88-1.46 (m, 7 H), 1.27-0.96 (m, 3 H);
IR (KBr) 1665, 1414, 1207 cm-1; MS (EI) m/z 540 (M+); HRMS
H), 7.41 (m, 1 H), 7.02-6.81 (m, 4 H), 6.57-6.37 (m, 2 H), 5.13
(b, 1 H), 3.84 (s, 3 H), 3.32 (m, 1 H), 3.18-2.99 (m, 2 H), 2.73
(s, 3 H), 2.57 (m, 2 H), 1.97-1.73 (m, 8 H); 13C NMR δ 153.5,
148.0, 137.2, 137.1, 122.6, 121.3, 120.4, 120.3, 112.5, 111.1,

Synthesis/ Functional Studies of WAY100635 Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 437
106.8, 59.2, 56.4, 55.2, 53.1, 38.6, 33.9, 28.1; MS (EIPI) m/z
DPAT (rat frontal cortex).46,47 For the 5-HT1B/D receptor binding
assays, membranes prepared from calf striatum were used
with 1.7 nM [3H]-5-HT in the presence of 100 nM 8-OH-DPAT
and 100 nM mesulergine to mask the 5-HT1A and 5-HT2C
binding sites.48
340 (M+); HRMS calcd (obsd) for C20H28N3O1: 340.23 (340.23).
2-[4-N-(o-Meth oxyph en yl)-N-m eth ylam in opiper idin yl]-
N-(2-p yr id in yl)cycloh exa n eca r boxa m id e (12). To an ice-
cooled solution of 11 (0.17 g, 0.50 mmol) and triethylamine (2
mL) in CH2Cl2 (10 mL) was added dropwise a solution of
cyclohexanecarboxylic acid chloride (1 mL, 7.5 mmol) in CH2-
Cl2 (10 mL). The reaction mixture was stirred at room
temperature for 2 h, after which the solvent was removed in
vacuo. The residue was purified by gradient flash chromatog-
raphy (CH2Cl2 with 5-15% EtOH), yielding 140 mg (62%) of
12 as a yellow oil. The oil was redissolved in Et2O and
3. Gu in ea P ig Ileu m Assa y for Deter m in a tion of p A2
Va lu e.38 The animals were stunned and exsanguinated. After
removal of the ileum, segments of about 1 cm were dissected
10 cm from the ileoceacal junction. After clearance of the
mesenteric connective tissue and removal of the luminal
contents, each ileal segment was mounted between platinum
stimulation electrodes in a 50-mL, two-chambered organ bath
with an internal circulation. The segments were bathed in a
modified Krebs Henseleit solution (in mmol: NaCl 118;
NaHCO3 25; KH2PO4 1.19; CaCl2 1.25; KCl 4.7; MgSO4 0.6;
glucose 10). Ketanserin (0.3 µmol) and tropisetron (3 µmol)
were added to block 5-HT2, 5-HT3, and 5-HT4 receptors. The
solution, maintained at 35 °C, was gassed and continuously
mixed by bubbling with 95% O2/5% CO2. Contractions were
recorded isometrically under a load of 1 g. After an equilibra-
tion for 20 min the preparations were stimulated continuously
with square-wave pulses (0.05 Hz, 1 ms, 40 V) until the
contractions reached a constant level. The preparations were
washed and then stimulated with trains of 6 stimulations at
intervals of 7 min, until the mean responses were comparable.
Cumulative concentration-response curves were constructed
with an exposure period of 7 min with 8-OH-DPAT, followed
by 6 electrical stimulations, after which the next higher
concentration would be applied. Controls received the corre-
sponding volume of the vehicle only. For examining antago-
nistic activity, the compounds were applied at fixed concen-
trations 60 min before starting the cumulative concentration-
response curve of the agonist. Responses were measured as
an increase in the isometric tension and expressed as a
percentage of the mean effect of the last 6 stimulations before
the start of the experiment. Because the 5-HT1A agonist 8-OH-
DPAT decreases the electrically induced contractions maxi-
mally by about 40%, EC20 values were determined graphically
form the mean effect of 4-6 experiments. Apparent pA2 values
were calculated according to MacKay: pA2 ) -log[antagonist]
+ (DR - 1), where DR is the ratio of EC20 values in the
presence and absence of the antagonist.51
4. 8-OH-DP AT-In d u ced Hyp oth er m ia .16 Animals were
treated with 1 mg/kg 8-OH-DPAT 30 min prior to measure-
ment; test compounds/saline were administered 45 min prior
to measurement. The core temperature was determined by
insertion of a digital temperature probe (CMA/150 temperature
controller, CMA/Microdialysis Sweden) 4-5 cm into the rectum
for 60 s until a stable reading was obtained, to the nearest
1/10 °C. For each independent experiment new control and
8-OH-DPAT studies were conducted and changes in temper-
ature due to test compounds were assessed using a one-way
analysis of variance (ANOVA) with post hoc analysis carried
out using Dunnett’s t-tests against their own control data.
5. Ultr a son ic Voca liza tion .18,19,45 Ultrasonic vocalization
was measured in a sound-attenuated test chamber (W 24 cm,
L 22 cm, H 22 cm) with a grid floor for delivery of footshocks
(scrambled chock of 1.8 mA for 0.3 s; shocker Getra BN 2002).
Ultrasonic vocalization was recorded (microphone 4004, Bruel
and Kjaer) and processed by an interface (developed at Merck
KGaA) to select 22 ( 4 kHz signals and to digitize the resulting
signals for automatic processing in a personal computer. In a
priming phase, each rat was placed in the test chamber. After
2 min, a series of at most 10 shocks (trials), 1.8 mA for 0.3 s,
separated by 20-s shock-free intervals, was delivered via the
grid floor of the test chamber. In the shock-free intervals the
occurrence of ultrasonic vocalization was automatically re-
corded, and the duration of ultrasonic vocalization was calcu-
lated immediately. The priming session was terminated either
when the rat constantly vocalized at least for 10 s on 3
consecutive trials or after the tenth trial. Rats that did not
respond with ultrasonic vocalization were excluded from the
test. In the actual test performed the next day, each rat
received 5 initial shocks (1.8 mA for 0.3 s, separated by 20-s
1
converted to the HCl salt: mp 130-135 °C; H NMR δ 8.50
(m, 1 H), 7.78 (m, 1 H), 7.26 (m, 2 H), 7.00-6.82 (m, 4 H),
4.11 (m, 2 H), 3.84 (s, 3 H), 3.28 (m, 3 H), 2.90 (m, 2 H), 2.68
(s, 3 H), 2.15-0.93 (m, 17 H). Anal. (C27H38N4O2‚2.4HCl‚
4.6H2O) C, H, N, Cl.
2-[4-N-(o-Meth oxyph en yl)am in opiper idin yl]-N-pyr idin -
2-yla m in e (13). To an ice-cooled solution of 9 (0.27 g, 0.80
mmol) in Et2O (15 mL) was added a 1.0 M solution of DIBAL-H
in toluene (6 mL). The reaction was stirred for 18 h at room
temperature. The excess of DIBAL-H was destroyed with H2O
(3 drops) after which the solvent was removed in vacuo. The
residue was purified by gradient flash chromatography (EtOAc
to THF to CH2Cl2 with 10-25% EtOH and 2% aq ammonia),
yielding 200 mg (77%) of 13 as a yellow oil. The oil was
redissolved in Et2O and converted to the HCl salt: mp 122
1
°C; H NMR δ 8.09 (m, 1 H), 7.26 (m, 1 H), 6.99-6.38 (m, 6
H), 5.16 (b, 1 H), 4.18 (b, 1 H), 3.84 (s, 3 H), 3.33 (m, 3 H),
2.88 (m, 2 H), 2.66 (s, 3 H), 2.33-2.17 (m, 4 H), 1.43 (m, 2 H);
13C NMR δ 158.7, 148.0, 146.7, 137.2, 136.8, 121.1, 116.0,
112.5, 110.1, 109.4, 106.9, 56.2, 55.2, 52.0, 49.3, 38.6, 33.2; MS
(EIPI) m/z 326 (M+); HRMS calcd (obsd) for C20H26N3O2:
326.23 (326.22).
[4-N-(o-Meth oxyp h en yl)-N-cycloh exa n eca r boxa m id o-
piper idin yl]-N-(2-pyr idin yl)cycloh exan ecar boxam ide (14).
To an ice-cooled solution of 13 (0.20 g, 0.62 mmol) and
triethylamine (2 mL) in CH2Cl2 (10 mL) was added dropwise
a solution of cyclohexanecarboxylic acid chloride (2 mL, 15.0
mmol) in CH2Cl2 (10 mL). The reaction mixture was stirred
at room temperature for 2 h, after which the solvent was
removed in vacuo. The residue was purified by gradient flash
chromatography (EtOAc to CH2Cl2 with 10% EtOH), yielding
140 mg (62%) of a yellow oil. The oil was redissolved in Et2O
and converted to the HCl salt: mp 137-140 °C; 1H NMR δ
8.40 (m, 1 H), 7.39 (m, 1 H), 7.26-6.88 (m, 6 H), 4.48 (m, 1
H), 3.81 (m, 2 H), 3.72 (s, 3 H), 2.88 (m, 2 H), 2.41 (m, 2 H),
2.20-1.96 (m, 4 H), 1.74-1.40 (m, 18 H), 1.06-0.81 (6 H); 13
C
NMR δ 176.7, 176.1, 156.4, 155.8, 148.9, 137.8, 131.1, 129.3,
127.7, 122.0, 120.3, 116.0, 57.9, 55.7, 54.9, 52.3, 45.1, 42.1, 42.0,
30.7, 29.6, 29.2, 28.6, 28.5, 25.4, 25.3. Anal. (C33H46N4O3‚
1.9HCl‚3.3H2O) C, H, N, Cl.
Biologica l Assa ys. 1. An im a ls. For the guinea pig ileum
assay male Dunkin Hartley guinea pigs (300-400 g) were
used. The hypothermia experiments were performed using
male Wistar rats (200-300 g). The animals were housed in
groups (four rats per cage) in a temperature- and humidity-
controlled colony room (20 ( 2 °C; 50-60%) on a natural day-
night cycle (light period 6:30-17:00 h). Food and water were
available ad libitum except during the actual experiments.
Testing was done between 10 and 16 h during the light phase
of the day-night cycle. For the ultrasonic vocalization experi-
ments Sprague-Dawley rats (290-390 g) were used. Animals
were housed in groups (two rats per cage) in a temperature-
and humidity-controlled colony room (29 ( 2 °C; 50-60%)
under a natural day-night cycle (light period 6:00-18:00 h)
with food and water ad libitum except during the actual
experiments. Procedures were conducted in accordance with
guidelines published in the NIH Guide for the Care and Use
of Laboratory Animals.
2. Recep tor Bin d in g. Affinities of compounds were deter-
mined using competition binding assays to determine IC50
values at the various receptors. Affinities at the 5-HT1A
receptors were measured as the displacement of [3H]-8-OH-

438 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Mensonides-Harsema et al.
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min. Antagonists or vehicle were given 40 min and 8-OH-
DPAT or vehicle 30 min before the start of the ultrasonic
vocalization test. Means and SEM values were calculated for
each group and compared to the data for the respective placebo
group by one-way analysis of variance (ANOVA) followed by
Dunnett’s t-test.
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