Synthesis and SAR of 3- and 4-substituted quinolin-2-ones: Discovery of mixed 5-HT1B/5-HT2A receptor antagonists

Article abstract of DOI:10.1016/S0968-0896(01)00118-3

Quinolin-2-ones bearing a heteroaryl-piperazine linked by a two carbon chain at the 3- or 4-position were synthesised and evaluated as mixed 5-HT_1B/5-HT_2A receptor antagonists. Potent mixed antagonists were obtained with thieno[3,2-c]pyridine derivatives. In this series, compound 2.1 (SL 65.0472) proved to be functional antagonist at both the 5-HT_2A receptor (rat in vivo 5-HT-induced hypertension model) and the 5-HT_1B receptor (dog in vitro saphenous vein assay).

Full text of DOI:10.1016/S0968-0896(01)00118-3

Bioorganic & Medicinal Chemistry 9 (2001) 2129–2137
Synthesis and SAR of 3- and 4-Substituted Quinolin-2-ones:
Discovery of Mixed 5-HT1B/5-HT2A Receptor Antagonists
Gary McCort,a,* Christian Hoornaert,a Michel Aletru,a Colombe Denys,a
Olivier Duclos,a Caroline Cadilhac,a Eric Guilpain,a Genevieve Dellac,a Philip Janiak,b
Anne-Marie Galzinb,y, Monique Delahaye,b Frederique Guilbertb
and Stephen O’Connorb
a
´
Department of Cardiovascular/Thrombosis Medicinal Chemistry, Sanofi-Synthelabo Recherche, 1 Avenue Pierre Brossolette,
91385 Chilly-Mazarin Cedex, France
b
´
Department of Cardiovascular/Thrombosis Pharmacology, Sanofi-Synthelabo Recherche, 1 Avenue Pierre Brossolette,
91385 Chilly-Mazarin Cedex, France
Received 5 January 2001; accepted 9 April 2001
Abstract—Quinolin-2-ones bearing a heteroaryl-piperazine linked by a two carbon chain at the 3- or 4-position were synthesised
and evaluated as mixed 5-HT1B/5-HT2A receptor antagonists. Potent mixed antagonists were obtained with thieno[3,2-c]pyridine
derivatives. In this series, compound 2.1 (SL 65.0472) proved to be functional antagonist at both the 5-HT2A receptor (rat in vivo 5-
HT-induced hypertension model) and the 5-HT1B receptor (dog in vitro saphenous vein assay). # 2001 Elsevier Science Ltd. All
rights reserved.
Serotonin (5-HT), a well-known neurotransmitter
and vasoactive agent is stored in platelets in the cardio-
vascular system. Upon platelet activation by endothelial
damage or dysfunction, 5-HT is liberated in the blood
where it exerts deleterious effects such as vasoconstric-
tion and amplification of platelet aggregation. Platelet
aggregation in response to serotonin is mediated by the
activation of 5-HT2A receptors and 5-HT2A antagonists,
such as ketanserin, inhibit this response.1,2 5-HT-
induced vasoconstriction is mediated through two dis-
coronary artery,8À12 human cerebral arteries11,13 and
human pulmonary artery14 is probably due to 5-HT1B
receptor activation. In haemodynamic studies in man,
sumatriptan causes coronary, pulmonary and systemic
vasoconstriction15,16 and has been reported to provoke
cardiac ischaemia.17 Isolated tissue studies and clinical
investigations indicate that contractions of human cor-
onary arteries produced by 5-HT are only partially and
inconsistently blocked by ketanserin because they result
from stimulation of a mixed population of 5-HT2A and
5-HT1B receptors.8,9,18À21
tinct receptor subtypes, the 5-HT2A receptor and a sub-
3
1-like
type previously known as 5-HT
but now
considered likely to be the 5-HT1B receptor (formerly 5-
HT1D beta receptor).4 Hence, a number of blood vessels
have been identified in which sumatriptan, a 5-HT1B/D
receptor agonist, causes vasoconstriction that is resis-
tant to antagonism by ketanserin which has low affinity
for 5-HT1B receptors.5 Molecular biological and phar-
macological analyses demonstrate that sumatriptan-
induced constriction of dog saphenous vein,6,7 human
These findings led us to initiate a program directed
toward the discovery of mixed 5-HT2A/5-HT1B antago-
nists permitting a blockade of the two receptors sub-
types responsible for the adverse cardiovascular effects
of the 5-HT. Our objective was to identify compounds
showing high affinity (ꢀ10 nM) for both 5-HT2A and 5-
HT1B receptors. These compounds should also be orally
active, with good haemodynamic tolerance and poor
cerebral penetration. Backscreening of compounds
from a former 5-HT antagonist program, yielded the
lead compound 1, which was a good 5-HT2A antagonist
and had moderate 5-HT1B antagonistic activity. In this
paper, we describe the synthesis, affinity and in vivo
functional antagonism at the 5-HT2A receptor site an in
*Corresponding author. Tel.: +33-1-6979-7886; fax: +33-1-6979-
7936; e-mail: gary.mccort@sanofi-synthelabo.com
yPresent address: Department of Pharmacology, Internal Medicine,
Sanofi-Synthelabo Recherche, 10 rue des Carrieres, 92504 Rueil-
Malmaison, France.
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00118-3

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G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
vitro functional antagonism at the 5-HT1B receptor site
of ligands with the basic structure of compounds 2 and
3 (Chart 1). These compounds were obtained during the
optimization process of 4-[2-(4-naphtalen-1-yl)-ethyl]-
1H-quinolin-2-one (1) which ultimately led to the dis-
covery of compound 2.1 (SL 65.0472), currently
evaluated in clinical trials.
activity was obtained with 4-substituted quinolinones
(2) and 3-substituted 3,4-dihydroquinolinones (3). Ana-
logues of 2 (Table 1) were readily prepared by N-alkyl-
ation of heteroaryl piperazines (10) with different 4-(2-
chloroethyl)-quinolinones (9) (Scheme 1).22À25 The
heteroaryl piperazines (10) were prepared by published
procedures or modifications thereof.25À29
The substituent R2 at the 1-position of the quinolinone
can either be introduced on the starting aniline (4) or at
the end of the synthesis (20 or 30, R2¼H), by phase
transfer alkylation (Scheme 2).
Chemistry
Early structure–activity relationships (SARs) around
the lead naphtalene (1) showed that most potent 5-HT1B
Synthesis of 3-substituted 3,4-dihydroquinolinone ana-
logues (3) was accomplished by N-alkylation of hetero-
aryl-piperazines (10), with different 3-(2-chloroethyl)-
3,4-dihydroquinolinone derivatives (15) or alternatively
by reductive amination of the 3-acetaldehyde compounds
(13). The 3-(2-hydroxyethyl)-3,4-dihydroquinolinones
(12) were synthesized by reductive cyclization of (ortho-
nitrophenyl)methylenebutyrolactones (11).26 Their sub-
sequent transformation to compound 3 is shown in
Scheme 3 (Table 1).
Pharmacology
The in vitro affinity for 5-HT2A receptors of the com-
pounds reported in Table 2 (1–2.l) was determined by
displacement of [3H]spiroperidol from rat cerebral cor-
tex membranes.30 Concentrations required to inhibit
50% of radioligand specific binding (IC50) were deter-
mined through three or four independent experiments
Chart 1.
Scheme 1. Reagents and conditions: (a) CH3CO2H, room temperature; (b) H2SO4 or CH3SO3H, 80–100 ꢁC; (c) SOCl2, CH3OH, reflux; (d) NaBH4,
THF, reflux; (e) SOCl2, CHCl3, reflux (f); 10, K2CO3, KI, CH3CN/DMF, 70 ꢁC.
Scheme 2. N-alkylation of quinolines 2.

G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
2131
using different concentrations of the test compound.
Specific binding defined in the Experimental represented
more than 75% of total binding. The obtained IC50 nM
values are listed in Table 2.
demonstrating the usefulness of a double 5-HT2A/5-
HT1B blockade.
Results and Discussion
In addition, the in vivo 5-HT2A functional antagonistic
activity was measured as the ability to blockserotonin-
induced hypertension in anesthetized pithed rats
(RS2A).The ID50 is defined as the antagonist dose
required to reduce induced pressor response by a half. 5-
HT1B functional antagonistic activity was evaluated as
the ability of the test compounds to blocksumatriptan-
induced constriction of dog saphenous veins in vitro.7,31
The pharmacological profile of lead compound 2.l was
further characterized by various functional assays
Table 2 shows that in the 4-substituted quinolinone
series 2, 5-HT1B activity can be enhanced by introducing
a nitrogen atom (2.a) into the napthylpiperazine moiety
of compound 1. Substitution on the isoquinoline part of
2.a gave no advantages. Isosteric replacement for a
thienopyridine derivative (2.b) provided an equipotent
compound. The introduction of a fluorine atom at the 7
position of quinolinone 2.c increased both 5-HT2A and
5-HT1B potency. N-Methylation of 2.c shows that the
Scheme 3. Reagents and conditions: (a) H2, Pd/C, THF, AcOH, room temperature; (b) Dess–Martin periodinane, CH2Cl2, room temperature; (c)
10, Ti(OiPr)4, NaBH3CN, EtOH/CH2Cl2, room temperature; (d) TBDMSi-Cl, imidazole, DMF, room temperature; (e) NaH, THF, room tem-
perature; (f) R2-Hal, THF, room temperature; (g) (nBu)4NF, THF, room temperature; (h) SOCl2, CHCl3, reflux; (i) 10, K2CO3, CH3CN, reflux.
Table 1. Physicochemical properties of 3- and 4-substituted quinolin-2-one derivatives 2 and 3
Compound
Formula
Mp (ꢁC)
Salt
M+H
% Yield (last step)
2.a
2.b
2.c
2.d
2.e
2.f
2.g
2.h
2.i
C24H24N4O
C22H22N4OS
C22H21FN4OS
C23H23FN4OS
C22H22N4O2
C23H22FN5O
C23H22FN5O
C22H21FN6O
C21H20FN5OS
C23H24FN5O
C22H24N4OS
C23H26N4OS
C23H26N4OS
C23H26N4OS
C24H23FN4O3S
C24H24FN5O2S
226–227
215–217
252
None
2 HCl
2 HCl
2 HCl
2 HCl
None
None
None
2 HCl
2 HCl
2 HCl
2 HCl
None
None
2 HCl
2 HCl
385
391
409
423
375
404
404
405
410
406
393
407
407
407
467
466
63
59
42
68
55
46
56
47
54
42
49
64
—
—
95
74
280
220–221
246–248
224
266
194–196
213
260
2.j
3.a
3.b
3.ca
3.db
2.k
2.l
178
52–53
52–53
218–220
269–270
a[a]D +30.0 (c 0.75; CHCl3).
b[a]D À32.3 (c 0.75; CHCl3).

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G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
Table 2. 5-HT2A affinity (IC50 in nM), 5-HT2A antagonistic activity (RS2A; ID50 in mg/kg) and 5-HT1B antagonistic activity (DSV; pA2)a
Compoundb
R1
R2
A
5-HT2A binding
IC50 (nM)
RS2A
(mg/kg)
DSV
(pA2)
1
H
H
H
12
3.8
21
ID50 iv=0.075
ID50 iv=0.015
ID50 iv=0.002
ID50 po=0.021
ID50 po=0.08
N.D.
7.06
7.66
7.69
8.21
8.22
7.28
< 6
< 6
< 6
< 6
< 6
9.08
8.66
8.88
9.23
6.64
8.17
2.a
H
2.b
H
H
2.c
7-F
7-F
7-F
7-F
7-F
7-F
7-F
7-F
H
H
0.2
0.5
1.7
>100
95
2.d
CH3
2.e
H
2.f
H
N.D.
2.g
H
N.D.
2.h
H
>100
16
N.D.
2.i
H
H
N.D.
2.j
3.2
2.8
9.1
2.5
7
N.D.
3.a
H
ID50 po=0.085
(ꢂ)-3.b
(+)-3cc
(À)-3dc
2.k
H
CH3
42% inh @ 0.1 po
ID50 iv=0 .011
H
CH3
4% inh @ 0.1 (po)
ID50 iv=0 .0036
H
CH3
ID50 po=0.1
ID50 iv=0 .021
7-F
7-F
CH2CO2H
CH2CONH2
>100
6
N.D.
2.l
ID50 po=0.031
ID50 iv=0 .0014
aData are the mean of three or four independent determinations.
1
bAll compounds were characterized by H NMR, mass spectroscopy and gave satisfactory CHN analyses.
cSeparated by preparative chiral HPLC.

G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
2133
H-donating property of the amide is not necessary for
activity at either of the 5-HT receptor subtypes.
of 8.58. In comparison, ketanserin had a pA2 value of
7.90 in this tissue. Compound 2.l is also a potent inhi-
bitor of human platelet aggregation induced by 5-
HT+ADP (IC50 49 nM) and by 5-HT+collagen (IC50
48 nM), equivalent to that of ketanserin.35 Compound
2.l demonstrates potent anti-thrombotic activity in the
Folts model of dog coronary artery thrombosis; 10–30
mg/kg iv provides complete protection.33
Among the phenyl bioisosteres tried, it is of interest to
note that besides the thienopyridine moiety, only the
furan derivative 2.e showed high affinity for the 5-HT2A
receptor and reasonable antagonistic activity in the 5-
HT1B DSV assay. The two azaquinoline analogues 2.f
and 2.g and the pyrimidine analogue 2.h were inactive,
and this was a general rule we observed for six-mem-
bered rings fused to the pyridine. Among all the five-
membered heterocycles investigated, only the iso-
thiazole 2.i and the pyrrole 2.j maintained 5-HT2A
activity, but none of them showed any 5-HT1B activity.
The affinity of 2.l for human 5-HT1B receptors was
assessed on membranes of Hela cells transfected by the
recombinant receptor.36 Compound 2.l inhibited the
specific [3H]-GR125743 binding to the recombinant 5-
HT1B receptor in a competitive and concentration-
dependent manner (Ki=19 nM). Functional 5-HT1B
receptor antagonistic activity was investigated on suma-
triptan-elicited contraction of isolated dog saphenous
vein.7 We have previously demonstrated using molecular
biological techniques that dog saphenous vein contains
the 5-HT1B receptor subtype, which shows a very high
degree of homology with its human counterpart.6
However, no evidence of 5-HT1D receptor was detected.
Sumatriptan caused a concentration-related contraction
which was significantly shifted to the right by 2.l (0.01–
0.1 mM) without reducing its maximal effect. The slope
of the regression line derived from Schild plot analysis
was not significantly different from unity. The pA2 value
was 8.17.34 In comparison, similar results were obtained
with methiothepin, a non-selective 5-HT antagonist, its
pA2 value being 8.43. 2.l also blocked 5-HT-induced
constriction of atherosclerotic human coronary arteries
consistently and potently (pA2 8.8).34
Potent mixed antagonists were also obtained in the 3,4-
dihydroquinolinone series (3) with piperazinyl-
thieno[3,2-c]pyridine; however, here, substitution on the
dihydroquinolinone moiety presented no distinct
advantages. Since the chiral compounds 3.c and 3.d did
not show superior activity over the non-chiral deriva-
tives (2) and due to the fact that the chiral center was
very difficult to control synthetically, the non-chiral 4-
substituted quinolinones were pursued. In order to cir-
cumvent any potential central 5-HT receptor-mediated
effects, we sought to decrease the possible crossing of
the blood brain barrier by the introduction of an acetic
acid substituent at position number 1 (quinolinone 2.c).
However, the carboxylic acid induces a sharp decrease
in activity for both 5-HT receptor subtypes. Introduction
of the non-charged acetamide group (2.l), on the other
hand, resulted in a potent, balanced, mixed antagonist
with limited brain penetration as estimated by the 5-HT-
induced head twitch test (HTA) in rats32 (Table 3).
Finally, the in vivo interaction with the 5-HT1B receptor
was studied on the sumatriptan-induced venoconstric-
tion in anesthetized dogs. Intravenous bolus injection of
sumatriptan produced a significant constriction of the
saphenous vein as measured by a reduction in saphe-
nous vein diameter of ꢃ30%. Compound 2.l antag-
onized this venoconstriction by 90% at 30 mg/kg iv.33 It
should be noted that, at this dose, no adverse effects on
hemodynamics and ECG parameters including mean
arterial pressure, heart rate and PR and QT intervals
were observed. The high potency of 2.l in this in vivo
functional model is consistent with its affinity for the 5-
HT1B receptor.
Compound 2.l, SL 65.0472 inhibited serotonin-induced
hypertension in rats in a dose-dependent manner, with
an ID50 value of 1.4 mg/kg iv and 31 mg/kg po, indicat-
ing it to be a potent antagonist of vascular 5-HT2A
receptors.33 It remains active after oral administration
for 24 h. 2.l also inhibited serotonin-induced vascon-
striction in rabbit aorta.34 Under adrenergic blockade,
aortic rings serotonin caused concentration-dependent
contractions with an EC50 of 1.1 mM (n=10). Com-
pound 2.l (0.01–1 mM) was able to antagonize this 5-
HT2A receptor-mediated response with an apparent pKB
Table 3. Effect of N-1 substitution on CNS penetration of piperazinylthieno[3,2-c]pyridine-quinolinones
Compound
R
HTA
ID50 (mg/kg, po)
RS2A
ID50 (mg/kg, po)
RS2A/HTA
2.c
2.d
2.l
H
CH3
CH2CONH2
0.13
0.03
1.0
0.021
0.08
0.031
6.19
0.38
32.26

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G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
Conclusion
heated to reflux for 16 h. The solvent was removed at
reduced pressure and the residue was redissolved in di-
chloromethane and washed with saturated sodium
hydrogenocarbonate solution. The organic layer was
then washed with water, dried (Na2SO4), filtered and
concentrated. The crude product obtained was chroma-
tographed on silica gel (dichloromethane/methanol,
97:3) to furnish the ester 8 (85–98%).
The workpresented here has allowed us to combine and
optimize 5-HT1B and 5-HT2A antagonistic activity in
the same molecule, leading to the development of SL
65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno[3,2-c]pyridin-4-
yl)piperazin-1-yl]-1,2-dihydroquinoline-1-acetamide), an
orally active mixed antagonist that is currently under
clinical study.37
General procedure for the synthesis of 4-(2-chloroethyl)-
quinolin-2-(1H)-one (9). To a suspension of the methyl
2-oxo-1,2-dihydroquinolin-4-acetate 8 (12.5 mmol) in
dry tetrahydrofuran (100 mL) and methanol (1 mL) at
room temperature, sodium borohydride (19.0 g, 50
mmol) was added in small portions. Following addition,
the mixture was heated to reflux for 24 h. After cooling
to 5 ꢁC, methanol (3 mL) was added dropwise. The sol-
vents were removed under reduced pressure and the
residue was taken up by a mixture of dichloromethane
(200 mL) and aqueous HCl 1 N (100 mL). The organic
layer was separated, washed with water, dried
(Na2SO4), filtered and concentrated, affording 74–87%
of the 4-(2-hydroxethyl)quinolin-2-(1H)-one.
Experimental
Chemistry
Melting points were determined using open capillary
tubes on a Buchi 530 apparatus and are uncorrected.
MerckKieselgel 60 (230–400 mm) was used for flash
chromatography and Kieselgel 60 F254 silica plates (0.2
mm) were used for TLC. The structures of the com-
pounds were confirmed spectroscopically by proton and
carbon NMR (CDCl3 or DMSO-d6) with TMS as the
internal standard using a Brucker AC-250 instrument,
by IR (KBr) on a Perkin-Elmer 297 spectrophotomer
and by their mass spectra (MS-ES) which were recorded
on a VG Autospec (Fisons Instruments) spectrometer.
The purity of the compounds was analyzed by TLC,
HPLC and elementary analysis. Results obtained were
within ꢂ0.4% of the theoretical values. Reagent grade
chemicals were purchased from Sigma-Aldrich-Fluka.
All solvents were analytical grade and anhydrous reac-
tions were performed in oven-dried glassware under
atmosphere of argon.
To a suspension of the above alcohol (9,95 mmol) in
chloroform (100 mL), pyridine (3 drops), dimethylfor-
mamide (3 drops) and thionyl chloride (3 mL, 41.0
mmol) were added. The mixture was then heated to a
gentle reflux for 4 1=2 h. After cooling to room tem-
perature, water (50 mL) was added dropwise and the
solution was stirred for 30 min The organic phase was
decanted, washed with water, dried (Na2SO4), filtered and
concentrated under reduced pressure yielding 9 (89–97%).
General procedure for the synthesis of 3-(acetoxy)-5-
phenylamino-5-oxo-2-enoic acids (6). To a stirred solu-
tion of the appropriate aniline derivative 4 (53.1 mmol)
in glacial acetic acid (25 mL), 4-acetoxy-2H, 3H-pyrane-
2,6-dione (9.93 g, 58.4 mmol) was added by small por-
tions. Following addition, the reaction mixture was
warmed to 35 ꢁC for 2 h. After cooling at room tem-
perature, the mixture was diluted in ice/water (500 mL).
The resulting precipitate was isolated by filtration,
rinsed with water and vacuum dried to yield the desired
acid 6 (76–93%).
General procedure for the synthesis of 4-[2-[4-heteroaryl-
piperazinyl-1-yl]ethyl] quinolin-2(1H)-ones (2). A mix-
ture of the 4-(2-chloroethyl)quinolin-2(1H)-one 9 (16.4
mmol), the N-substituted piperazine 10 (15.1 mmol),
sodium hydrogenocarbonate (1.6 g, 19.05 mmol) and a
catalytic quantity (100 mg) of potassium iodide in ace-
tonitrile (75 mL) and DMF (5 mL) was heated at 70 ꢁC
for 14–18 h. The solvent was then removed under
reduced pressure and the residue was redissolved in di-
chloromethane. The solution was washed with saturated
sodium hydrogenocarbonate and then with water. The
organic layer was dried (Na2SO4), filtered and con-
centrated. The crude product was purified by flash
chromatography (dichloromethane/methanol/NH4OH
95:4.5:0.5) to obtain the desired 2 (41–74%).
General procedure for the synthesis of 2-oxo-1,2-dihy-
droquinolin-4-acetic acids (7). A suspension of the 3-
(acetoxy)-5-phenylamino-5-oxo-2-enoic acid
6
(107
mmol) in concentrated (95–97%) sulfuric acid (130 mL)
was heated at 90 ꢁC for 90 min After cooling to room
temperature, the solution was poured into a mixture of
ice (500 g) and water (500 mL). The precipitate formed
was isolated by filtration, rinsed with water and
vacuum-dried. The product was triturated in ether
yielding 7 (45–85%).
General procedure for the N-alkylation of 4-[2-piperazinyl-
1-yl]ethyl]quinolin-2(1H)-ones (2.d, 2.k, 2.l). A 0.5 M
solution of the appropriate electrophile (tert-butyl bro-
moacetate, methyl iodide or bromoacetamide; 1.45
mmol) in dry tetrahydrofuran (2.9 mL) was added
dropwise at 0 ꢁC to a suspension of the 4-[2-piperazinyl-
1-yl]ethyl]quinolin-2(1H)-one 2 (1.3 mmol), freshly
ground potassium hydroxide (105 mg, 1.8 mmol) and
tetrabutylammonium bromide (125 mg, 0.4 mmol) in
dry tetrahydrofuran (25 mL). After stirring at 0 ꢁC for
30 min, the temperature was allowed to rise to 20 ꢁC
and stirring was continued for another 6–10 h. The sol-
vent was then removed under reduced pressure and the
General procedure for the synthesis of methyl-2-oxo-1,2-
dihydroquinolin-4-acetates (8). To a stirred suspension
of the 2-oxo-1,2-dihydroquinolin-4-acetic acid 7 (68
mmol) in methanol (250 mL) at room temperature,
thionyl chloride (16 mL, 219 mmol) was added drop-
wise. After the addition, the reaction mixture was

G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
2135
residue is redissolved in dichloromethane. The organic
phase was washed with water, dried (Na2SO4), filtered
and condensed. The crude product was purified by flash
chromatography (AcOEt/MeOH/NH4OH), providing
the N-alkyl quinolines 2 (45–70%).
General procedure for the synthesis of 3-(2-chloroethyl)-
1-methyl-3,4 dihydroquinolin-2(1H)-ones (15). To
a
solution of 14 (13.7 mmol) in dry tetrahydrofuran (80
mL) to which was added 15 mL (15 mmol) of a 1 M
solution of tetrabutylammonium fluoride in THF. The
mixture was stirred at room temperature for 3 h and the
solvent was evaporated. The residue was dissolved in
diethyl ether and the organic solution was washed sev-
eral times with water, dried (MgSO4), filtered and con-
centrated. The alcohol was purified by chromatoflash
(dichloromethane/methanol, 98:2) and isolated in 64–82%
yields.
General procedure for chlorhydrate salts. To a stirred
solution of piperazinylqunolinone 2 (0.7 mmol) in
methanol (5–7 mL) at room temperature was added an
excess of anhydrous HCl/diethyl ether solution 3 N (1
mL). The mixture was stirred at room temperature for
30 min and the precipitate was isolated by filtration,
rinced several times with diethyl ether and vacuum-
dried.
Thionyl chloride (0.7 mL, 9.6 mmol) was added drop-
wise to a solution of the 3-(2-hydroxyethyl)-1-methyl-
3,4-dihydroquinolin-2(1H)-one (6,37 mmol) in chloro-
form (30 mL) at room temperature. After the addition,
the solution was heated at reflux for 1 h. The solvent
was removed under reduced pressure and the crude
product was filtered on a pad of silical gel (CH2Cl2)
yielding the chloro derivative 15 in a 92–97% yield.
General procedure for the synthesis of 3-[2-nitrophenyl)-
methylene]dihydrofuran-2(3H)-ones (11). a-[g-Butyro-
lactonylidene]triphenylphophine (8.3 g, 24 mmol) was
added to a solution of the appropriately-substituted
ortho-nitrobenzaldehyde (27 mmol) in toluene (200 mL)
at room temperature. The solution was heated to reflux
for 6 h then cooled to room temperature overnight. The
precipitate was collected by vacuum filtration, rinsed
with toluene and oven-dried at 50 ꢁC, yielding the
expected olefin 11 (62–88%).
General procedure for the synthesis of 3-[2-[4-heteroaryl-
piperazin-1-yl)ethyl]-1-methyl-3,4-dihydroquinolin-2(1H)-
ones (3). A mixture of 3-(2-chloroethyl)-1-methyl-3,4
dihydroquinolin-2(1H)-one 15 (3.9 mmol), the appro-
priate heteraoarylpiperazine (3.9 mmol) and sodium
bicarbonate (0.65 g, 7.8 mmol) in acetonitrile (20 mL)
was heated at reflux for 7 h. The solvent was then
removed under reduced pressure and the crude product
was purified by flash chromatography (dichloro-
methane/MeOH/NH4OH, 96.5:3:0.5). The free base
obtained was transformed into its chlorhydrate salt with
41–74% combined yields for the two steps.
General procedure for the synthesis of 3-(2-hydroxy-
ethyl)-3,4-dihydroquinolin-2(1H)-ones (12). A mixture of
the 3-[2-nitrophenyl)methylene]dihydrofuran-2(3H)-one
11 (20 mmol) and 5% Pd/C (0.7 g) in dry tetra-
hydrofuran (100 mL) and acetic acid (0.6 mL) was
hydrogenated with H2 (40 psi) at room temperature for
9 h. The mixture was filtered through Celite and the
filtrate was concentrated. The residue was crystallized
from a minimum of dichloromethane, yielding 12 (58–
76%).
General procedure for the synthesis of 2-oxo-1,2,3,4-
tetrahydroquinolin-3-acetaldehydes (13). A solution of
1,1,1-triacetoxy-1,1-dihydro-1,2-benzodioxol-3(1H)-one
(Dess–Martin reagent; 6.4 g, 14 mmol) in dichloro-
methane (100 mL) was added drop wise at room tem-
perature to a stirred solution of the 3-(2-hydroxyethyl)-
3,4-dihydroquinolin-2(1H)-one 12 (12 mmol) in di-
chloromethane (25 mL). After the addition, the mixture
was stirred at room temperature for 20 h. Dichloro-
methane and a saturated sodium hydrogenocarbonate
solution were added. The organic layer was separated
and the aqueous phase was re-extracted twice with di-
chloromethane. The combined organic extracts were
washed with water, dried (MgSO4), filtered and con-
centrated. The crude aldehydes 13 are obtained in 90–
95% yields and are used immediately in the next step.
General procedure for the synthesis of 3-[2-tert butyldime-
thylsilyloxyethyl]-1-methyl-3,4-dihydroquinolin-2(1H)-
ones (14). To a solution of the 3-(2-hydroxyethyl)-3,4-
dihydroquinolin-2(1H)-one 12 (10.84 mmol) and imid-
azole (1.36 g, 20 mmol) in dry DMF (20 mL) at room
temperature was added tert-butyldimethylsilyl chloride
(4.0 g, 26.5 mmol). After the addition, the mixture was
stirred at room temperature for 3 h. The solvent was
removed under reduced pressure and the residue was
redissolved in diethyl ether. The organic solution was
washed with water, dried (MgSO4), filtered and con-
centrated. The compounds thus obtained in a quantita-
tive yield were N-alkylated as follows: sodium hydride
(60% mineral oil dispersion; 0.6 g, 15 mmol) was added
to the above O-protected dihydroquinolin-2(1H)-one
(11.23 mmol) in solution in tetrahydrofuran (100 mL) at
room temperature stirring was continued for 1 h then
methyl iodide (1.6 mL, 25.6 mmol) was added. The
mixture was stirred at room temperature for 20 h and
the solvent was removed under reduced pressure. The
compound was extracted with diethyl ether and the
organic extracts were washed with water, dried
(MgSO4), filtered and concentrated. The crude product
was filtered through a pad of silica gel (CH2Cl2/cylco-
hexane) providing the N-methyl dihydroquinolinones 14
in near quantitative yields.
General procedure for the synthesis of 3-[2-[4-(heteroaryl)-
piperazin-1-yl]ethyl]-3,4-dihydroquinolin-2(1H)-ones (3,
R1=H). To a stirred mixture of the 2-oxo-1,2,3,4-tetra-
hydroquinolin-3-acetaldehyde 13 (4.5 mmol) and the
appropriate N-heteroarylpiperazine (4.8 mmol) in 5 mL
of absolute ethanol at room temperature was added
titanium tetraisopropoxide (2.6 mL ; 9 mmol). After
stirring for 4 h, sodium cyanoborohydride (1.0 g, 18
mmol) was added and the mixture was stirred at room
temperature an additional 20 h. The milky solution was
then diluted with dichloromethane (100 mL) and saturated

2136
G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
ammonium chloride solution (100 mL). After stirring
for 5 min, the mixture was filtered through Celite and
the organic layer of the filtrate was separated. The
aqueous layer was then re-extracted twice with
dichloromethane and the combined organic solutions
were dried (MgSO4), filtered and concentrated. The
compounds 3 where R2=H were purified by flash
chromatography (dichloromethane/methanol, 95:5), and
obtained in 69–94% yields.
recorded just before the serotonin injection. The antag-
onistic effect of drugs was calculated for each dose of
the drug by the percentage of inhibition of the ser-
otonin-induced hypertension:
% inhibition ¼
Â
Ã
1 À ðÁÞMBP
=ÁMBP
ꢀ 100
ðdose xÞ
ðcontrolÞ
where ÁMBP(dose x) was absolute variation in MBP fol-
lowing serotonin injection after the end of the infusion
of the dose ‘x’ of the drug; ÁMBP(control) was the abso-
lute variation in MBP following serotonin injection
before the administration of the first dose of the drug.
The ID50 was defined as the antagonist dose reducing
the serotonin-induced pressor response by a half.
Pharmacology
5-HT2A binding assay. Radioligand binding assay was
performed following a published procedure.30 Frontal
cortical regions of male Sprague–Dawley rats (Charles
River) were dissected on ice and homogenized (1:20 w/
v) in ice-cold buffer solution (50 nM Tris–HCl, 120 nM
NaCl, 5 mM KCl) and centrifuged at 40,000 g for 10
min The pellet was resuspended in buffer, rehomogen-
ized and recentrifuged. The final pellet was resuspended
in buffer (500 mg/10 mL) containing 10À5 M pargyline.
Canine isolated saphenous vein model of 5-HT1B
antagonism7,31
Saphenous veins were removed from Beagle or Anglo-
poitevin dogs (ECDL, either sex, 25–34 kg) anesthetised
with pentobarbital (35 mg/kg iv). Helical strips
approximately 0.4 cm in width and 0.5 cm in length
were prepared by mounting the vein on metal cannula
and then suspended in 20-mL tissue baths containing
Krebs’ solution of the following millimolar composi-
tion: NaCl 118.0; KCl 4.7; MgCl2 1.2; CaCl2 2.6;
Assays were performed in triplicate in a 1.0 mL volume
containing 5 mg wet weight of tissue and 0.3 nM [3H]
spiroperidol (19 Ci/mmol; New England Nuclear).
Tubes were incubated for 20 min at 37 ꢁC, and the
incubations were terminated by vacuum filtration
through Whatman GF/B filters. The filters were washed
twice with 5 mL of ice-cold Tris–HCl buffer, and the
radioactivity bound to the filters was measured by liquid
scintillation spectrometry. Specific [3H] spiroperidol
binding was defined as the difference between binding in
the absence or the presence of 100 mM 5-HT.
.
NaHCO3 25.0; glucose 11.1; ascorbic acid 0.11; (pH 7.4;
37 ꢁC) and aerated continuously with 95% O2–5% CO2.
The solution also contained atropine 1 mM (to inhibit
muscarinic receptors); mepyramine 1 mM (to inhibit
histamine H1 receptors).
The following studies were conducted in accordance
with ethical guidelines edited by the European Com-
munity (EC directive 86/609), the council of Europe
(Convention ETS 123) and the French Government
(decree of 19.10.87).
Contractile responses were recorded isometrically using
Hugo Sachs model 351 force–displacement transducers
(March Hugstentten, Germany), connected to a Gould
2400S polygraph (Courtaboeuf, France). Optimal
responses were obtained when the tissues were subjected
to an initial tension of 2 g. Data acquisition was per-
formed automatically using the JAD.2 Notocord soft-
ware (Croissy, France) on a PC Compaq. Strips were
allowed to equilibrate for 30 min with several washes
before exposure to a 3 mM noradrenaline challenge to
test the viability of the preparation. Following a 30 min
resting period, the first concentration–response curve to
sumatriptan was obtained by exposing the preparations to
cumulative additions of the compounds until the maximal
response was obtained. Another resting period with several
washes allowed the strips to return to the basal tension.
The antagonist compound was then added to the bath and
left in contact with the tissue for 15 min prior to the second
concentration-response curve to sumatriptan. Responses
were expressed as a percentage of the maximum contrac-
tion obtained with the first sumatriptan curve. pA2 values
were calculated using the Schild plot method.
5-HT2A antagonism in the pithed rat. Rats (Sprague–
Dawley Charles River France were anesthetised with
pentobarbital sodium (60 mg/kg IP), intubated and
pithed. They were mechanically ventilated on a small
rodent ventilator (HarvardTM Apparatus). Polyethylene
cannulas were inserted into the right carotid artery for
blood pressure measurement, and in the femoral vein
for drug infusions. The arterial cannula was connected
to a pressure transducer, and systemic blood pressure
was continuously recorded with the use of data acquisi-
tion software (IOX1, EMKA technologies).
Three to four cumulative doses of the drug were given
intravenously (0.1 mL/min/rat for 5 min) during the
same experiment, so that the rats were their own con-
trols. Serotonin (30 mg/kg/iv bolus) was administered 5
min before the beginning of the infusion of the first dose
of the drug tested, and repeated 5 min after the end of
the infusion of each of the doses of compound.
Acknowledgements
The hypertensive effect of serotonin was calculated for
each injection as the difference between the peak
increase in mean blood pressure (MBP) and the MBP
The authors would like to thank Mrs. Juliana Mersovka
for technical assistance during the preparation of the
manuscript.

G. McCort et al. / Bioorg. Med. Chem. 9 (2001) 2129–2137
2137
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