Short and efficient syntheses of analogues of way-100635: New and potent 5-HT1A receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(00)00287-X

Simple syntheses of four new and potent analogues of the 5-HT_1A receptor ligand, WAY-100635 are described, namely the 6- pyridinyl)-fluoro-, the pyrimidine- and the 5-pyridinyl)-bromo-analogues. The first three analogues were obtained by aromat

Full text of DOI:10.1016/S0968-0896(00)00287-X

Bioorganic & Medicinal Chemistry 9 (2001) 695±702
Short and Ecient Syntheses of Analogues of WAY-100635:
New and Potent 5-HT1A Receptor Antagonists
Sandrine Marchais,a,Ã Bartek Nowicki,a Hakan Wikstrom,a Lise T. Brennum,b
Christer Halldinc and Victor W. Piked
aDepartment of Medicinal Chemistry, University Center for Pharmacy, University of Groningen, Antonius Deusinglaan 1,
NL-9713 AV Groningen, The Netherlands
bDepartment of Molecular Pharmacology, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, Denmark
cKarolinska Institutet, Department of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, S-17176 Stockholm, Sweden
dChemistry and Engineering Group, MRC Cyclotron Unit, Imperial College School of Medicine, Hammersmith Hospital,
Ducane Road, London W12 ONN, UK
Received 22 May 2000; accepted 24 October 2000
AbstractÐSimple syntheses of four new and potent analogues of the 5-HT1A receptor ligand, WAY-100635 are described, namely
the 6-(pyridinyl)-bromo-, the 6-(pyridinyl)-¯uoro-, the pyrimidine- and the 5-(pyridinyl)-bromo-analogues. The ®rst three analogues
were obtained by aromatic nucleophilic substitution of the 2,6-dihalogenopyridine (activated or not as an N-oxide) or of the
2-chloropyrimidine with the corresponding amine nucleophile as a key step. The fourth analogue, the 5-(pyridinyl)-bromo-analogue,
was synthesized from the 2-amino-5-bromopyridine via a progressive elongation of the skeleton. The four compounds described are
all full antagonists and show good in vitro binding anities (Ki). # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
sought to develop new analogues of WAY-100635,
halogenated and non-halogenated. The halogenated
analogues of WAY-100635 could give an easy access to
the labelled 18F, 76Br or 123I analogues while the pyr-
imidine analogue labelled with 11C, with its extra nitro-
gen, could lead to radioligands with an enhanced
anity for the receptor. We have synthesized three new
halogenated WAY-analogues, namely: N-(2-(1-(4-(2-
methoxyphenyl)piperazinyl)ethyl))-N-(2-(6-bromo)py-
ridinyl)cyclohexanecarboxamide 2 (6BPWAY), N-(2-(1-
(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-(6-¯uoro)-
pyridinyl) cyclohexanecarboxamide 3 (6FPWAY), N-(2-
(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-(5-bro-
WAY-100635
1
[N-(2-(1-(4-(2-methoxyphenyl)piper-
azinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide] is
a potent and silent antagonist of the 5-HT1A receptor,1,2
a receptor which is strongly implicated in neuro-
psychiatric diseases (e.g. anxiety, depression and schizo-
phrenia).3 The labelling of WAY-100635 with 00C (t1/
2=20.3 min)4 provided the ®rst radioligand for imaging
5-HT1A receptors in the living human brain with posi-
tron emission tomography (PET).5,6 Carbonyl-11C-
labelled WAY-100635 is a very e€ective radioligand6
but it is rapidly metabolised,7 such that its input func-
tion into brain is dicult to determine accurately in the
late stage of a PET experiment. A similarly e€ective
radioligand that could be labelled easily with a longer-
lived radionuclide such as 18F (t1/2=109.7 min) or 76Br
(t1/2=16.1 h) might have considerable advantages for
PET. Also labelling with g-emitting 123I (t1/2=13.2 h)
might deliver a much-needed radioligand for studies
with the more widely used imaging technique, single
photon emission tomography (SPET). Therefore, we
mo)pyridinyl)cyclohexanecarboxamide
4 (5BPWAY)
and a non-halogenated analogue: N-(2-(1-(4-(2-methoxy-
phenyl)piperazinyl)ethyl))-N-(2-pyrimidinyl)cyclohexane
carboxamide 5 (PmWAY) (Fig. 1).
Chemistry
The retrosynthetic analysis of 6BPWAY
2 and
6FPWAY 3 showed that 2-amino-6-halopyridines 8
would be suitable precursors of the intermediates 6 and
7, which could be key compounds of the synthesis
(Scheme 1).
ÃCorresponding author. Tel.: +31-50-363-3302; fax: +31-50-363-
6908; e-mail: marchais@farm.rug.nl
0968-0896/00/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00287-X

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S. Marchais et al. / Bioorg. Med. Chem. 9(2001) 695±702
at elevated temperature (200 ꢀC) and yields are low. Hence,
direct halogenation of 2-aminopyridine does not seem to
be a very convenient method to use in order to obtain the
2-amino-6-bromo- and 2-amino-6-¯uoropyridine.
The amination reaction of a 2-halogenopyridine11 can
be performed via the Chichibabin reaction12 or by
reaction with aqueous ammonia13 at high temperature
under pressure and for a long period. The yields are
low. The Curtius rearrangement is a well-known reac-
tion for introducing an amino group into a pyridine
ring, starting from a picolinic acid.14 However, it is
sometimes dicult to have the carboxyl group and the
other substituents in the desired ring positions. Hence,
from the literature, it appeared dicult to prepare e-
ciently brominated or ¯uorinated compounds 8 in a
reasonably high yield.
Therefore, we decided to look for a method that would
be better adapted to the direct synthesis of compounds 6
and 7. An easy method to synthesize 2-amino-6-halo-
pyridines is aromatic nucleophilic substitution (SNAr)
of 2,6-dihalopyridines, in which one of the halogens is
substituted by a suitable amine nucleophile.
The amine nucleophile, 4-(2-aminoethyl)-1-(2-methoxy-
phenyl)piperazine 9 was prepared by reaction of 1-(2-
methoxyphenyl)piperazine and bromoacetonitrile fol-
lowed by reduction with lithium aluminium hydride.15
The amine 9 was heated with the 2,6-dibromopyridine
and triethylamine.16 No reaction occurred. However,
when the nitrogen of the pyridine ring was converted
into its N-oxide form, the electronic density in the ring
was reduced and the aromatic nucleophilic substitution
was facilitated.16 This kind of reactivity has already
been described by Katritkzy17 a long time ago and has
been recently recon®rmed for the 2-chloropyridine N-
oxide by Lee et al.18 We, therefore, decided to use 2,6-
dibromopyridine N-oxide, as starting material for the
synthesis of compounds 6 and 7 (Scheme 2).
Figure 1.
Halogenation of the pyridine ring is not equally easy to
achieve at each position of the ring. The 2-amino-
pyridine is easily halogenated in positions ortho and
para (positions 3 and 5) to the amino group due to its
electronic e€ects. Polyhalogenated derivatives are often
obtained. Selective monobromination in position 5 of
2-aminopyridine can be achieved with tetrabutyl-
ammonium tribromide8 but no monobromination at
position 6 has ever been described. In general, ¯uoro-
pyridines are prepared from aminopyridines by diazoti-
sation in hydro¯uoroboric acid.9 The yields are often
low and the ¯uorine replaces the aminogroup. Success-
ful substitution of chlorine by ¯uorine has been repor-
ted.10 However, the reaction has to be kept for a long time
The nitrogen of the 2,6-dibromopyridine was oxidized
with hydrogen peroxide (30% in water) in tri¯uoro-
Scheme 1. Retrosynthesis of 6BPWAY and 6FPWAY.

S. Marchais et al. / Bioorg. Med. Chem. 9(2001) 695±702
697
acetic acid.19 Reaction of the nucleophile 9 with the
2,6-dibromopyridine N-oxide 10 in n-butanol gave the
desired compound 11 in 40% yield. No disubstitution of
10 was noticed. The N-oxide 11 was reduced to the pyri-
dine 6 with iron in acetic acid at 100 ꢀC for 2 h. Reaction
of 6 with the cyclohexanecarbonyl chloride in dichloro-
methane then gave the desired compound 2 in 75% yield.
The synthesis of pyrimidine-WAY 5 is also feasible via
the nucleophilic aromatic substitution of the 2-chloro-
pyrimidine with the amine nucleophile 9 (Scheme 3).
Re¯ux of the 2-chloropyrimidine with the amino-
precursor 9 in absolute ethanol for 7 h gave the expected
compound 12 with 50% yield. Reaction of 12 with the
cyclohexanecarbonyl chloride in dichloromethane then
gave the desired compound 5 in 91% yield in a two-step
synthesis.
An analogous pathway was considered for the synthesis
of 6FPWAY 3, but it was impossible to isolate the 2,6-
di¯uoropyridine-N-oxide. Therefore, the nucleophilic
substitution was performed directly on the 2,6-di¯uoro-
pyridine in THF at 45 ꢀC for 2 days (Scheme 2). Product
7 was isolated in low yield (10%) but was successfully
acylated with cyclohexanecarbonyl chloride to obtain
6FPWAY 3. The addition of a base like triethylamine
or sodium hydride did not improve the yield of com-
pound 7. When the reaction was performed at higher
temperature, no product 7 was isolated. The 2-¯uoro-
pyridine is unstable at room temperature, it rapidly gives
a pyridyl±pyridinium product.20 Because of the electron-
withdrawing e€ect of the two ¯uorine atoms, the 2,6-
di¯uoropyridine is a quite reactive reagent. It is there-
fore expected to be dicult to obtain a monosubstituted
product only. It has already been described that the
aromatic nucleophilic subtitution of the 2,6-di¯uoro-
pyridine can go even further leading to the formation of
polymers.21 Despite the low yield of the coupling reac-
tion, a new ¯uorinated analogue of WAY-100635 was
obtained in a straightforward two-step synthesis.
The synthesis of 5BPWAY 4 followed a di€erent path-
way. It was easily achieved from the commercially
available 2-amino-5-bromopyridine (Scheme 4). The
acylated product was treated with 1-(2-methoxy-
phenyl)piperazine, and the product amide 13 reduced
with lithium aluminium hydride. Some expected debro-
mination occurred during this step, limiting the yield of
the desired product to 51%. This side reaction can be
avoided by reduction with borane-dimethyl sulphide.
Treated with cyclohexanecarbonyl chloride, the desired
compound 4 was obtained with 60% yield.
Pharmacology
The new halogenated analogues of WAY-100635 2±4
and the pyrimidine-WAY analogue 5 were assessed for
their ability to inhibit the binding of [3H]-5-CT to
human cloned 5-HT1A receptors in vitro. The results of
these binding studies are shown in Table 1.
Scheme 2. Synthesis of 6BPWAY and 6FPWAY.

698
S. Marchais et al. / Bioorg. Med. Chem. 9(2001) 695±702
The intrinsic ecacy of each compound 2±5 was deter-
mined in human cloned 5-HT1A receptors, looking at
the production of cAMP(in the presence of forskolin)
with a concentration up to 10 mM of each compound.
Results and Discussion
Each new analogue of WAY-100635 was found to show
a good binding anity for the 5-HT1A receptor. The Ki
value obtained for 5BPWAY 4 is somewhat higher than
the one of 6BPWAY 2, indicating that the position of
the halogen could interfere in the binding of the com-
pound to the receptor. The Ki value of PmWAY 5
(1.00 nM) is higher than the one of WAY-100635 (0.17
nM) or of the 6-substituted pyridine WAY (0.33 nM and
0.51 nM). The extra-nitrogen of the pyrimidine ring does
not enhance the anity of the compound for the receptor.
The abilities of the new compounds to inhibit the pro-
duction of cAMP(in the presence of forskolin) were
also assessed in human cloned 5-HT1A receptors with
1000 nM of 5-HT. The results of these ecacy assays
are shown in Table 1.
Table 1. In vitro binding anities and intrinsic ecacies
None of these new compounds 2±5 presents any intrin-
sic ecacy, they are all full antagonists of the 5-HT1A
receptor.
Anity Ki (nM)
Ecacy Ki (nM)
WAY-100635
6BPWAY 2
6FPWAY 3
5BPWAY 4
PmWAY 5
0.17
0.51
0.33
6.30
1.00
0.029
4.30
10.0
2.80
0.082
The lipophilicity of each compound has been calculated
as a logP/logD22 value to get insight of their possible
penetration into the brain. The values are shown in
Scheme 3. Synthesis of PmWAY.
Scheme 4. Synthesis of 5BPWAY.

S. Marchais et al. / Bioorg. Med. Chem. 9(2001) 695±702
699
PF 254 pre-coated aluminum sheet (Merck) with a layer
thickness of 0.2 mm (analytical) and on silica gel 60 PF
254 pre-coated glass plate (Merck) with a layer thick-
ness of 1 mm (preparative).
Table 2. Calculated log D values at physiological pH and log Pvalues
Log D at pH=7.40
Log P
WAY-100635
6BPWAY 2
6FPWAY 3
5BPWAY 4
PmWAY 5
2.88
4.14
3.45
4.16
2.30
3.28
4.53
3.85
4.16
2.70
All chemicals used were commercially available (Aldrich,
Acros or Fluka) and were used without puri®cation.
2,6-Dibromopyridine-N-oxide19 10 and of 4-(2-amino-
ethyl)-1-(2-methoxyphenyl)piperazine16 9 were prepared
according to described procedures.
Table 2. They are all in a good range; it is expected that
they can pass the blood-brain barrier and, if labelled
with a positron-emitter, can then be studied with PET.
1-(2-Methoxyphenyl)-4-(2-(2-(6-bromo)aminopyridinyl-
N-oxide)ethyl)piperazine (11). To 2,6-dibromopyridine-
N-oxide 10 (2.34 g, 9.25 mmol) in n-butanol (40 mL) was
added 4-(2-aminoethyl)-1-(2-methoxyphenyl)piperazine
6BPWAY 2, 6FPWAY 3 and PmWAY 5, from their
respective binding pro®les, look interesting for PET
studies. They can be labelled with 11C and, in the case of
3, with 18F. 6BPWAY 2 has also been transformed to
the 6-(pyridinyl)-stannate analogue in order to prepare
123IPWAY, a new radioligand which could be studied
with SPET.
9
(2.18 g, 9.25 mmol) and triethylamine (1.29 mL,
9.25 mmol). The reaction mixture was heated at 120 ꢀC
for 20 h. The solvent was evaporated and the residue
chromatographed on silica (CH2Cl2/MeOH 95/5) to
a€ord 1.50 g (40% yield) of 11 as a brown oil. IR (neat):
1590, 1499, 1453, 1240 cmÀ1 1H NMR (200 MHz,
;
5BPWAY 4 can be labelled with 11C but the labelling
with 18F will be dicult. The electron distribution of the
pyridine ring is not favorable for the displacement of
the bromine atom for a 18F at position 5 of the ring.
CDCl3): d 2.74 (m, 6H), 3.08 (br s, 4H), 3.36 (m, 2H),
3.80 (s, 3H), 6.52 (d, J=8 Hz, 1H), 6.99 (m, 5H), 7.37
(br s, 1H); 13C NMR (50 MHz, CDCl3) d 37.55, 48.90,
51.65, 53.85, 54.65, 102.20, 109.60, 114.35, 116.85,
119.50, 126.15, 150.70; MS (EI): m/z 390±392 (M±O),
218, 205, 190, 162.
Conclusion
1-(2-Methoxyphenyl)-4-(2-(2-(6-bromo)aminopyridinyl)-
ethyl)piperazine (6). To 1-(2-methoxyphenyl)-4-(2-(2-
(6-bromo)aminopyridinyl-N-oxide)ethyl)piperazine 11
(1.20 g, 2.90 mmol) in acetic acid (10 mL ) was added Fe
(0.34 g, 6.10 mmol). The mixture was stirred at 100 ꢀC
for 17 h. The acid was evaporated, the residue was
basi®ed with a solution of Na2CO3 (5%) and the pro-
duct extracted with CH2Cl2. The combined organic
layers were dried over MgSO4, ®ltered and concentrated.
The product 6 was puri®ed by crystallization as the
hydrochloride salt and then basi®ed to give 0.98 g of 6
as a brown solid (85% yield). IR (KBr): 3300, 1599,
We have described short and ecient syntheses of four
new analogues of WAY-100635 displaying potent, full
antagonist e€ects on the 5-HT1A receptor. The 6-halo-
pyridine compounds 2 and 3, and the pyrimidine-WAY
analogue 5 have been synthesized via aromatic nucleo-
philic substitution as a key step on the 2,6-dibromopyr-
idine N-oxide, the 2,6-di¯uoropyridine and the 2-
chloropyrimidine, respectively. This reaction gives a
general and easy access to new analogues of WAY-
100635, halogenated or not. The 5-bromopyridine ana-
logue 4 has been prepared via a linear and progressive
elongation of the skeleton, starting from the 2-amino-5-
bromopyridine. These ligands 2, 3, 5 can be labelled
with carbon-11, with ¯uorine-18 or bromine-76. These
new radioligands are now being evaluated in vivo in
PET.
1
1506, 1444 cmÀ1; H NMR (300 MHz, CDCl3): d 2.75
(br s, 6H), 3.12 (br s, 4H), 3.38 (br s, 2H), 3.81 (s, 3H),
6.31 (d, J=8 Hz, 1H), 6.65 (d, J=7 Hz, 1H), 6.80±
6.96 (m, 4H), 7.18 (t, J=8 Hz, 1H); 13C NMR
(50 MHz, CDCl3): d 36.90, 49.00, 51.55, 53.85, 55.00,
103.25, 109.65, 113.95, 116.70, 119.50, 121.50, 137.90;
MS (EI): m/z 309±392 (M), 218, 205, 190, 162; HRMS
(EI) calcd for C20H25BrN4O: 390.1055, observed:
390.1045. Anal. calcd for C20H25BrN4O.1/3 H2O: C,
54.41, H, 6.00, N, 14.10. Found: C, 54.43; H, 5.62; N,
13.93.
Experimental
Melting points were determined on an Electrothermal
digital melting point apparatus and are uncorrected. IR
spectra were obtained on an ATI-Mattson spectro-
meter. NMR spectra were obtained on a Varian
Gemini, 300 MHz or 200 MHz instrument. Chemical
shifts are reported in d value (ppm) relative to the deut-
erated solvent as internal standard. Mass spectra (ES)
were recorded on a Sciex API 3000 mass spectrometer
or on a Unicam 610/Automass 150 GC±MS system (EI).
Elemental analyses were performed by the Micro-
analytical Laboratory, University of Groningen and are
within Æ0.4% of the theoretical values. Thin-layer
chromatography (TLC) was performed on silica gel 60
N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-(6-
bromo)pyridinyl) cyclohexanecarboxamide (2) 6BPWAY.
To 1-(2-methoxyphenyl)-4-(2-(2-(6-bromo)aminopyridin-
yl)ethyl)piperazine 6 (0.77 g, 1.96 mmol) in CH2Cl2
(25 mL) was added cyclohexanecarbonyl chloride
(0.39 mL, 2.95 mmol, 1.5 equiv) and triethylamine
(0.41 mL, 2.95 mmol, 1.5 equiv). The mixture was stirred
at room temperature under N2 for 17 h. The reaction
mixture was quenched with 10% Na2CO3 and extracted
with CH2Cl2. The combined organic layers were dried

700
S. Marchais et al. / Bioorg. Med. Chem. 9(2001) 695±702
over MgSO4, ®ltered and concentrated. Column
chromatography on silica (CH2Cl2/MeOH 98/2 and
then with hexane/ethyl acetate 5/5) provided 0.74 g of 2
as a pure yellow-red oil (75% yield). IR (neat): 1664,
(M), 218, 205, 190, 162; HRMS (EI) calcd for
C27H35FN4O2: 440.2587, observed: 440.2592.
1-(2-Methoxyphenyl)-4-(2-(2-aminopyrimidinyl)ethyl)piper-
azine (12). To 2-chloropyrimidine (0.10 g, 0.88 mmol) in
absolute ethanol (10 mL) was added 4-(2-aminoethyl)-1-
(2-methoxyphenyl)piperazine 9 (0.21 g, 0.88 mmol). The
reaction mixture was heated at re¯ux for 7 h. The sol-
vent was evaporated, the residue dissolved in water and
extracted with CH2Cl2. The combined organic layers
were dried over MgSO4, ®ltered and concentrated. Col-
umn chromatography on silica (CH2Cl2/MeOH 95/5
and 90/10) provided 137 mg of the desired product 12 as
a yellow oil (50% yield). IR (neat): 3365, 1589, 1500,
1
1499, 1433 cmÀ1; H NMR (200 MHz, CDCl3): d 1.03±
1.19 (m, 3H), 1.50±1.81 (m, 8H), 2.35 (m, 1H), 2.56±2.63
(m, 5H), 2.94 (s, 4H), 3.81 (s, 3H), 3.99 (t, J=4 Hz, 2H),
6.80±6.96 (m, 4H), 7.26±7.36 (m, 2H), 7.55 (t, J=8 Hz,
1H); 13C NMR (50 MHz, CDCl3): d 24.05, 28.15, 41.15,
43.50, 49.05, 51.85, 53.80, 55.00, 109.65, 116.55, 118.50,
119.40, 121.35, 124.15, 138.25, 138.60, 139.70, 150.70,
154.30, 174.95; MS (ES): m/z 501±503 (M+H)+, 457,
391±393; HRMS (EI) calcd for C27H35BrN4O2:
500.1786, observed: 500.1763.
1
1452 cmÀ1; H NMR (200 MHz, CDCl3): d 2.63±2.72
1-(2-Methoxyphenyl)-4-(2-(2-(6-¯uoro)aminopyridinyl)-
ethyl)piperazine (7). To 2,6-di¯uoropyridine (0.32 g,
2.79 mmol) in dry THF (10 mL) was added 4-(2-amino-
ethyl)-1-(2-methoxyphenyl)piperazine 9 (0.66g, 2.79mmol)
and triethylamine (1.94 mL, 13.9 mmol, 5 equiv). The
reaction mixture was heated at 45 ꢀC for 3.5 h. The sol-
vent was evaporated, the residue dissolved in water and
extracted with CH2Cl2. The combined organic layers
were washed with 10% Na2CO3 and with brine, dried
over MgSO4, ®ltered and concentrated. Puri®cation of
the residue by column chromatography on silica
(CH2Cl2/MeOH 98/2) followed by preparative TLC
plate (CH2Cl2/MeOH 95/5) provided 92 mg (10% yield)
of the pure product 7 (yellow oil). IR (neat): 1590, 1499,
(m, 6H), 3.07 (m, 4H), 3.52 (q, J=6 Hz, J=11 Hz, 2H),
3.83 (s, 3H), 5.85 (br s, 1H), 6.48 (t, J=5 Hz, 1H), 6.81±
6.98 (m, 5H), 8.25 (d, J=4 Hz, 2H); 13C NMR (50
MHz, CDCl3): d 36.40, 49.05, 51.60, 53.80, 55.25,
108.85, 109.60, 109.95, 116.70, 119.45, 121.45, 139.70,
150.70, 156.50, 160.90; MS (EI): m/z 313 (M), 298, 218,
205, 162, 120; HRMS (EI) calcd for C17H23N5O:
313.1902, observed: 313.1890.
N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-
pyrimidinyl)cyclohexane carboxamide (5) PmWAY. To 1
- (2 - methoxyphenyl) - 4 - (2 - (2 - aminopyrimidinyl)ethyl)-
piperazine 12 (0.10 g, 0.32 mmol) in CH2Cl2 (5 mL) was
added cyclohexanecarbonyl chloride (64 mL, 0.48 mmol,
1.5 equiv) and triethylamine (67 mL, 0.48 mmol,
1.5 equiv). The mixture was stirred at room tempera-
ture under N2 for 18 h. The reaction mixture was
quenched with 10% Na2CO3 and extracted with
CH2Cl2. The combined organic layers were dried over
MgSO4, ®ltered and concentrated. Column chromatog-
raphy on silica (CH2Cl2/MeOH 99/1) provided 0.16 g of
5 as a yellow oil (91% yield). IR (neat): 1670, 1565,
1449 cmÀ1 1H NMR (200 MHz, CDCl3): d 2.73 (m,
;
6H), 3.13 (br s, 4H), 3.40 (q, J=5 Hz, J=11 Hz, 2H),
3.86 (s, 3H), 5.42 (br s, 1H), 6.12 (dd, J=2 Hz, J=8 Hz,
1H), 6.24 (dd, J=2 Hz, J=8 Hz, 1H), 6.84±7.01 (m, 4H),
7.44 (dd, J=8 Hz, J=16 Hz, 1H); 13C NMR (50 MHz,
CDCl3): d 36.65, 48.80, 51.60, 53.85, 55.05, 93.45, 94.20,
101.65, 109.65, 116.75, 119.50, 121.60, 139.95, 140.15,
151.00; 19F NMR (50 MHz, CDCl3): d À70.0; MS (EI):
m/z 330 (M), 277, 263, 235, 205, 190, 162; HRMS (EI)
calcd for C20H25FN4O: 330.1856, observed: 330.1861.
1
1499 cmÀ1, H NMR (300 MHz, CDCl3): d 1.02±1.84
(m, 11H), 2.82 (br s, 4H), 2.98 (br s, 5H), 3.79 (s, 3H),
4.24 (br s, 2H), 6.78±7.02 (m, 5H), 8.58 (d, J=5 Hz,
2H); 13C NMR (50 MHz, CDCl3): d 23.65, 24.05, 24.30,
26.85, 27.70, 28.40, 42.30, 44.25, 48.90, 51.70, 53.80,
55.35, 109.60, 115.25, 116.55, 119.40, 121.30, 139.75,
150.65, 156.30, 160.35, 170.50; MS (CI): m/z 424 (M+H)+.
N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-(6-
¯uoro)pyridinyl) cyclohexanecarboxamide (3) 6FPWAY.
To 1-(2-methoxyphenyl)-4-(2-(2-(6-¯uoro)aminopyridin-
yl)ethyl)piperazine 7 (50 mg, 0.15 mmol) in CH2Cl2
(5 mL) was added cyclohexanecarbonyl chloride (30 mL,
0.23 mmol, 1.5 equiv) and triethylamine (32 mL, 0.23 mmol,
1.5 equiv). The mixture was stirred at room temperature
under N2 for 17 h. The reaction mixture was quenched
with 10% Na2CO3 and extracted with CH2Cl2. The
combined organic layers were dried over MgSO4, ®ltered
and concentrated. Column chromatography on silica
(CH2Cl2/MeOH 98/2) and preparative TLC plate
(CH2Cl2/acetone 97/3, CH2Cl2/isopropanol 98/2) pro-
vided 22 mg (31% yield) of the pure product 3 (colorless
2-Chloro-N-(2-(5-bromo)pyridinyl)acetamide. To 2-amino-
5-bromopyridine (1.00 g, 5.75 mmol) in CH2Cl2 (50 mL)
was added triethylamine (1.20 mL, 872 mg, 8.62 mmol,
1.5 equiv) and 2-chloroacetyl chloride (0.69 mL, 975 mg,
8.62 mmol, 1.5 equiv) under nitrogen. The mixture was
stirred at room temperature for 17 h and quenched with
25 mL of 10% NaHCO3 solution. The reaction mixture
was extracted with CH2Cl2, the combined organic layers
dried over MgSO4, ®ltered and concentrated. The pro-
duct was puri®ed by crystallization in the mixture
CH2Cl2/MeOH and obtained pure as a white powder
1
oil). IR (KBr): 1667, 1597, 1499, 1447 cmÀ1: H NMR
(200 MHz, CDCl3): 1.03±1.89 (m, 11H), 2.31±2.37 (br s,
1H), 2.61±2.68 (br s, 5H), 3.00 (s, 4H), 3.84 (s, 3H), 3.99
(t, J=7 Hz, 2H), 6.82±7.01 (m, 5H), 7.21 (d, J=8 Hz,
1H), 7.82 (dd, J=8 Hz, J=16 Hz, 1H); 13C NMR
(50 MHz, CDCl3): d 24.05, 28.10, 41.00, 43.30, 48.80,
51.65, 53.80, 54.60, 109.65, 116.60, 117.00, 119.40,
121.45, 139.00, 140.95, 150.50, 163.20, 175.00; 19F
NMR (50 MHz, CDCl3): d À67.75; MS (EI): m/z 440
with 76% yield (1.09 g). mp: 154 ꢀC, IR (KBr): 1671cmÀ1
;
1H NMR (200 MHz, CDCl3): d 4.20 (s, 2H), 7.84 (d,
J=9 Hz, 1H), 8.15 (d, J=9 Hz, 1H), 8.38 (s, 1H), 8.83
(br s, 1H); 13C NMR (50 MHz, CDCl3): d 41.20, 113.55,
121.20, 139.45, 146.50, MS (ES): m/z 249±251
(M+H)+, 173±175; HRMS (EI) calcd for C7H6BrClN2O:
247.9351, observed: 247.9339.

S. Marchais et al. / Bioorg. Med. Chem. 9(2001) 695±702
701
2-(1-(4-(2-Methoxyphenyl)piperazinyl))-N-(2-(5-bromo)-
pyridinyl)acetamide (13). To 1-(2-methoxyphenyl)piper-
azine (0.80 g, 4.15 mmol) and 2-chloro-N-(2-(5-bromo)-
pyridinyl)acetamide (1.04 g, 4.15 mmol) in DMF
(20 mL) was added K2CO3 (1.43 g, 10.4 mmol, 2.5 equiv).
The mixture was stirred at 50 ꢀC for 60 h. The reaction
mixture was quenched with water (25 mL), extracted
with ethyl acetate. The combined organic layers were
dried over MgSO4, ®ltered and concentrated. The crude
was puri®ed by column chromatography on silica with
the mixture hexane/ethyl acetate 7/3 to provide 1.38 g of
13 as a yellow oil (82% yield). IR (neat): 1700, 1500,
(M+H)+; HRMS (EI) calcd for C28H35BrN4O2:
500.1786, observed: 500.1788.
In vitro receptor binding assays
Membranes from HeLa cells expressing human 5-HT1A
receptors are incubated in 50 mM Tris, pH 7.7 with
1.5 nM [3H]5-CT at 37 ꢀC for 15 min. The incubation is
terminated by rapid ®ltration through uni®lter GF/B
®lters. Filters are counted in a Packard Topcounter.
Non-speci®c binding is determined by including 10 mM
Metergoline.
1
1455 cmÀ1; H NMR (300 MHz, CDCl3): d 2.82 (br s,
4H), 3.72 (br s, 6H), 3.82 (s, 3H), 6.82±6.98 (m, 4H),
7.73 (d, J=8 Hz, 1H), 8.10 (br s, 1H), 8.47 (s, 1H), 9.61
(br s, 1H); MS (ES): m/z 405±407 (M+H)+, 205;
HRMS (EI) calcd for C18H21BrN4O2: 404.0847,
observed: 404.0860.
Ecacy assays
HeLa cells transfected with the human 5-HT1A receptor
are seeded in 96-well plates. After 4±6 days media is
removed and cells are washed once with assay bu€er
(PBS+MgCl2+CaCl2+IBMX). The cells are then
incubated for 20 min with assay bu€er containing for-
skolin and test compound. The incubation is terminated
by aspiration and cAMPproduced during the incuba-
tion is extracted and measured in a RIA based system.
Antagonism is detected by including 1000 nM 5-HT in
the incubation mixture. Compounds which are active on
the 5-HT1A receptor are tested in 10 concentrations to
determine the IC50 value. This value depends on the
anity and concentration of 5-HT present in the assay.
To calculate the value that corresponds to Ki or KD
obtained in binding experiments, the IC50 value is
transformed to Ki by the Cheng-Pruso€ equation:
Ki=IC50/(1+L/KD)), where L and KD refer to con-
centration and anity constant of 5-HT.
1-(2-Methoxyphenyl)-4-(2-(2-(5-bromo)aminopyridinyl)-
ethyl)piperazine. To 2-(1-(4-(2-methoxyphenyl)piper-
azinyl))-N-(2-(5-bromo)pyridinyl)acetamide 13 (1.39 g,
3.42 mmol) in dry THF (40 mL) was added, 0.26 g of
LiAlH4 (6.84 mmol, 2 equiv). The mixture was stirred at
room temperature for 17 h under nitrogen. The reaction
mixture was quenched with water and NaOH 10% and
extracted with ethyl acetate. The combined organic lay-
ers were dried over MgSO4, ®ltered and concentrated.
Column chromatography on silica (CH2Cl2/MeOH 95/5)
provided 0.68 g of the desired compound with 51%
1
yield. IR (neat): 3365, 1592, 1500, 1455 cmÀ1; H NMR
(300 MHz, CDCl3): d 2.85 (br s, 6H), 3.17 (br s, 4H),
3.45 (q, J=5 Hz, J=11 Hz, 2H), 3.80 (s, 3H), 5.62 (br s,
1H), 6.37 (d, J=9 Hz, 1H), 6.80±7.00 (m, 4H), 7.40 (dd,
J=3 Hz, J=9 Hz, 1H), 8.03 (d, J=3 Hz, 1H); 13C NMR
(50 MHz, CDCl3): 36.15, 48.00, 51.65, 53.85, 55.30,
105.60, 108.15, 109.65, 116.90, 119.55, 122.00, 138.10,
138.90, 146.85, 155.40; MS (ES): m/z 391±393
(M+H)+, 199±201; HRMS (EI) calcd for C18H23BrN4O:
390.1055, observed: 390.1049.
The intrinsic activity of each compound was measured
for a concentration up to 10 mM.
Acknowledgements
The authors are grateful to the Human Science Frontier
Program Organization for major grant support for this
project [RG0235/1998-B] and to the COST B3 and B12
programs for facilitating collaborations.
N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-(5-
bromo)pyridinyl) cyclohexanecarboxamide (4) 5BPWAY.
To 1-(2-methoxyphenyl)-4-(2-(2-(5-bromo)aminopyridin-
yl)ethyl)piperazine (0.34 g, 0.87 mmol) in CH2Cl2 (25 mL)
was added cyclohexanecarbonyl chloride (0.17 mL,
1.30 mmol, 1.5 equiv) and triethylamine (0.18 mL,
1.30 mmol, 1.5 equiv). The mixture was stirred at room
temperature under N2 for 17 h. The reaction mixture
was quenched with 10% Na2CO3 and extracted with
CH2Cl2. The combined organic layers were dried over
MgSO4, ®ltered and concentrated. Column chromatog-
raphy on silica (CH2Cl2/MeOH 95/5) and crystal-
lization as an hydrochloric salt provided 0.37 g of the
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1
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