New selective and potent 5-HT(1B/1D) antagonists: Chemistry and pharmacological evaluation of N-piperazinylphenyl biphenylcarboxamides and biphenylsulfonamides

Article abstract of DOI:10.1021/jm990397l

A series of new analogues of N-[4-methoxy-3-(4-methylpiperazin-1- yl)phenyl] 2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carboxamide (1; GR127935) as potent and selective 5-HT(1B/1D) antagonists were synthesized and evaluated pharmacologically. Their receptor binding profiles were comparable to that of 1. The 1,3,4-oxadiazole isomer 2 and the 4'- aminocarbonyl and 4'-amidinyl analogues (9 and 10) of 1 had higher affinities at the rat 5-HT(1B) receptor (IC₅₀ = 0.93, 1.3, and 0.5 nM, respectively) and calf 5-HT(1D) receptor (IC₅₀ = 37, 10, and 3 nM, respectively) than did 1 (1.6 and 52 nM for rat 5-HT(1B) and calf 5-HT(1D) receptors, respectively). In the functional in vitro testing of 5-HT(1B/1D) antagonistic properties, 2, 9, 10, 11b (Odemethylated derivative of 2), 13a (O-methylsulfonyl analogue of 2), and 16 (which differs from 2 with a sulfonamide linker) showed more pronounced effects in the K⁺-induced 5-HT release in the cortex of guinea pig than did 1 and 3 (SB224289). Compounds 2, 9, and 10 were equally potent as 1 in rabbit saphenous vein model (pA₂ > 9). A biochemical study of 2 with in vivo microdialysis in the rat brain showed that it is capable of augmenting citalopram (a selective serotonin reuptake inhibitor, SSRI) induced 5-HT release in rat ventral hippocampus, while preventing the decrease in acetylcholine release elicited by citalopram administration. The molecular structure of 2 was determined by single-crystal X-ray analysis. The log P and log D values of these compounds were calculated. This study contributes to the SAR study of N-piperazinylphenyl biphenylcarboxamides as selective and potent 5-HT(1B/1D) antagonists.

Full text of DOI:10.1021/jm990397l

J . Med. Chem. 2000, 43, 517-525
517
Brief Articles
New Selective a n d P oten t 5-HT1B/1D An ta gon ists: Ch em istr y a n d
P h a r m a cologica l Eva lu a tion of N-P ip er a zin ylp h en yl Bip h en ylca r boxa m id es a n d
Bip h en ylsu lfon a m id es
Yi Liao,*,† Henning Bo¨ttcher,‡ J u¨rgen Harting,‡ Hartmut Greiner,‡ Christoph van Amsterdam,‡
Thomas Cremers,† Staffan Sundell,§ J oachim Ma¨rz,‡ Wilfried Rautenberg,‡ and Ha˚kan Wikstro¨m†
Department of Medicinal Chemistry, Center for Pharmacy, University of Groningen, A. Deusinglaan 1, NL-9713 AV Groningen,
The Netherlands, Preclinical Pharmaceutical Research, Merck KGaA, D-64271 Darmstadt, Germany, and Department of
Medical Biochemistry, University of Go¨teborg, P.O. Box 440, SE-405 30 Go¨teborg, Sweden
Received August 4, 1999
A series of new analogues of N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl] 2′-methyl-4′-(5-
methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-carboxamide (1; GR127935) as potent and selective
5-HT1B/1D antagonists were synthesized and evaluated pharmacologically. Their receptor binding
profiles were comparable to that of 1. The 1,3,4-oxadiazole isomer 2 and the 4′-aminocarbonyl
and 4′-amidinyl analogues (9 and 10) of 1 had higher affinities at the rat 5-HT1B receptor (IC50
) 0.93, 1.3, and 0.5 nM, respectively) and calf 5-HT1D receptor (IC50 ) 37, 10, and 3 nM,
respectively) than did 1 (1.6 and 52 nM for rat 5-HT1B and calf 5-HT1D receptors, respectively).
In the functional in vitro testing of 5-HT1B/1D antagonistic properties, 2, 9, 10, 11b (O-
demethylated derivative of 2), 13a (O-methylsulfonyl analogue of 2), and 16 (which differs
from 2 with a sulfonamide linker) showed more pronounced effects in the K+-induced 5-HT
release in the cortex of guinea pig than did 1 and 3 (SB224289). Compounds 2, 9, and 10 were
equally potent as 1 in rabbit saphenous vein model (pA2 > 9). A biochemical study of 2 with in
vivo microdialysis in the rat brain showed that it is capable of augmenting citalopram (a
selective serotonin reuptake inhibitor, SSRI) induced 5-HT release in rat ventral hippocampus,
while preventing the decrease in acetylcholine release elicited by citalopram administration.
The molecular structure of 2 was determined by single-crystal X-ray analysis. The log P and
log D values of these compounds were calculated. This study contributes to the SAR study of
N-piperazinylphenyl biphenylcarboxamides as selective and potent 5-HT1B/1D antagonists.
In tr od u ction
constituent.6,7 The 5-HT1B receptor is concentrated in
the basal ganglia, striatum, and frontal cortex. It was
reported that the terminal autoreceptor controlling
5-HT release in the rat, guinea pig, and human is of
the 5-HT1B type.4 Low levels of the 5-HT1D receptor
mRNA are found in several areas of the rat brain, in
particular in the dorsal raphe, which suggests that it
may function as an 5-HT autoreceptor.4 Nevertheless,
the 5-HT1B/1D receptors belong to the family of seven
transmembrane G-protein-coupled receptors (both nega-
tively coupled to adenylate cyclase) and mediate sero-
tonergic neurotransmission although their precise func-
tion remains to be determined.5,8,9
The serotonin 5-HT1B receptor was first found in rat
brain and the 5-HT1D receptor in bovine caudate nucleus.1
Molecular biological studies demonstrated that the
human and rabbit 5-HT1D receptors are encoded by a
family of two distinct genes, termed initially 5-HT1DR
and 5-HT1Dâ but now 5-HT1D and 5-HT1B, respectively.2
The rat 5-HT1B (r5-HT1B) receptor and human 5-HT1B
(h5-HT1B) receptor were found to be species homologues
with a single amino acid modification and pharmaco-
logically still different as demonstrated.3 The 5-HT1D
receptor has 63% overall structural homology with the
5-HT1B receptor.4 Unlike the 5-HT1B receptor subtype,
the pharmacology of the cloned 5-HT1D receptor subtype
seems conserved among various mammal species such
as the human, guinea pig, and rat.5 However, the bovine
5-HT1D receptor is not yet cloned, and the 5-HT1D
receptor in calf caudate membranes was reported to be
a mixture of bovine 5-HT1D receptor and the homologous
bovine 5-HT1B receptor with the latter as the major
The tricyclic antidepressants and the selective sero-
tonin reuptake inhibitors (SSRIs) are believed to in-
crease serotonin transmission by blocking reuptake of
the released 5-HT. The clinical effect of these classical
drugs only becomes apparent after chronic treatment
(2-3 weeks). This delayed onset of therapeutic action
of SSRIs is thought to be due to the time required to
desensitize somatodendritic 5-HT1A and terminal 5-HT1B
autoreceptors. Therefore, the blockade of terminal 5-HT1B
receptors by selective antagonists has been proposed8,10
as a new approach for more efficient and/or fast-acting
* Corresponding author: Dr. Yi Liao. Tel: +31-50-3633302. Fax:
+31-50-3636908. E-mail: y.liao@farm.rug.nl.
† University of Groningen.
‡ Merck KGaA.
§ University of Go¨teborg.
10.1021/jm990397l CCC: $19.00 © 2000 American Chemical Society
Published on Web 01/20/2000

518 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Ch a r t 1. Structures of the Reference Compounds
Brief Articles
An additional reason for these differences in results of
1 might be the difference of its affinity to the 5-HT1D in
addition to the 5-HT1B receptors. As the 5-HT1D recep-
tors are thought to be located in dorsal raphe nucleus,
pharmacological interference with these receptors might
explain the discrepancies described above. Combined
systemic administration of 1 with sertraline (a SSRI),
however, resulted in a pronounced, long-lasting 5-HT
increase in guinea pig hypothalamus, indicative of the
synergistic effect of this combined pharmacological
intervention.21
In this study, we present the chemistry and pharma-
cological evaluation of a series of new structurally
modified analogues of GR127935 (1) as selective and
potent 5-HT1B/1D or 5-HT1B antagonists.
Ch em istr y
The syntheses of the tested compounds are outlined
in Schemes 1 and 2. The precursors 8a ,b,22 compound
1, and its 1,3,4-oxadiazole isomer 2 were synthesized
according to the method reported by Glaxo researchers13
(i.e. steps a-c in Scheme 1). Incidentally, the application
of Suzuki reaction of 8a and 7 in a refluxing solution of
N,N-dimethylformamide (DMF) instead of 1,2-dimethox-
yethane (DME) in the presence of aqueous sodium
carbonate (step d in Scheme 1) gave a mixture of 1 and
9, which was isolated and characterized to be the
partially hydrolyzed byproduct of the 1,2,4-oxadiazole
moiety of 1. It was found that the extensive refluxing
treatment under these reaction conditions could bring
out almost exclusively 9. No partial hydrolysis of the
1,3,4-oxadiazole moiety of 2 was observed correspond-
ingly.
Hydrogenation of 1 with Raney nickel water slurry
in a mixture of methanol and acetic acid (step e in
Scheme 1) generated successfully 10, which has an
amidino group instead of the 1,2,4-oxadiazole moiety of
1. O-Demethylations of 1 and 2 were carried out by
treatment with aluminum chloride in ethylmercaptan
and CH2Cl2 at room temperature, affording 11a ,b,
respectively, which were further converted to their
sulfonyloxy derivatives (13a ,b) or the O-alkylated ana-
logue 13c under the conditions shown in Scheme 1 (step
f, g, or h).
Compound 16, which differs from 2 with a sulfona-
mide linker, was synthesized in the manner outlined
in Scheme 2. Compound 4b reacted with 5 to give 14,
which was converted to its boronic acid derivative 15
and further coupled with 8b by Suzuki reaction, afford-
ing 16 in good total yield.
Attempts of obtaining crystals of 1 and 2 suitable for
X-ray studies were successful in the case of 2, where
its crystals grew by slow evaporation from ethanol.
Subsequently, the single-crystal X-ray analysis revealed
the molecular conformation of 2 (Figure 1), thus permit-
ting the molecular appearance of GR127935-like ben-
zanilide derivatives to be inferred. The basic features
of the molecule 2 are as follows: (i) The molecule has
an elongated conformation due to a trans configuration
at the central amide bond. The torsion angles around
the C1-N22 and N22-C23 bonds are 179° and 176°,
respectively. This makes the amide linker and the
phenyl ring bearing the piperazine ring approximately
coplanar. (ii) The piperazine ring is in a chair conforma-
antidepressant drugs, since the acute blockade of these
5-HT autoreceptors will in theory immediately mimic
their desensitization. The combination of a SSRI with
a 5-HT1B antagonist may thus shorten the onset of the
antidepressant effect in depressed patients.9,11 Recent
studies also suggested that the supersensitive 5-HT1B/1D
receptors may be involved in the pathophysiology of
obsessive compulsive disorders (OCD) and the selective
5-HT1B/1D antagonists may also be potential drugs for
its treatment.12
Until recently, GR127935 (1; Chart 1) was reported
to be the first selective and potent 5-HT1B/1D antago-
nist,13 which was later described as a “non-silent”
antagonist (partial agonist) at human cloned 5-HT1B
receptors.9,14 Following the discovery of 1, many
GR127935-like derivatives and other compounds were
patented or reported subsequently as potential 5-HT1B/1D
receptor agents.7,9,11,15,16 The subtle structural modifica-
tion of 1 generated 5-HT1B/1D agents, which range from
partial agonists to full antagonists or inverse agonists.
SB224289 (3; Chart 1), for instance, which has the same
oxadiazole biphenylcarboxamido moiety of 1 on one side
with a spiro moiety on the other side of the molecule,
has been reported to be a selective h5-HT1B inverse
agonist. This compound is a potent antagonist of ter-
minal 5-HT autoreceptor function both in vitro and in
vivo.15,17 Microdialysis studies have yielded controver-
sial results concerning the ability of 1 to increase
synaptic 5-HT levels. Local perfusion of 1 into the
guinea pig frontal cortex increased 5-HT release, whereas
the 5-HT release was decreased after systemic
administration.9,18-20 It is unlikely that one can explain
the above microdialysis data due only to the choice of
the terminal area of those measurements. A reason for
this discrepancy might be that 1 is a partial agonist and
not a full antagonist at the 5-HT1B receptor. This is
supported by the failure of the selective 5-HT1B inverse
agonist 3 to effect 5-HT release in the frontal cortex.17

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 519
Sch em e 1a
a
Reagents and conditions: (a) triethylamine, CH2Cl2 or THF, 0 °C to rt, overnight; (b) B(O-i-Pr)3, n-BuLi, -100 to -78 °C, 3 h, then
-78 °C to rt, overnight; (c) Pd(PPd3)4, Na2CO3, DME-H2O (1:1), reflux 24 h; (d) Pd(PPh3)4, Na2CO3, DMF-H2O (1:1), reflux 24 h; (e) H2,
Raney-Ni, H2O, MeOH-AcOH; (f) AlCl3, EtSH-CH2Cl2, rt, 4-6 h; (g) triethylamine, CH2Cl2, -78 to 0 °C, 2 h, MeSO2Cl (12a ) or (CF3SO2)2O
(12b); (h) CsCO3, CH3CN, reflux 3 h, FCH2CH2Br (12c).
tion. The torsion angle of the C25-N31 bond (C24-C25-
N31-C32) connecting the piperazine ring to the benzene
ring is 15°. (iii) The torsion angles around the linking
bonds N22-C1-C3-C4, C5-C6-C9-C14, and C13-C12-
C16-N20 of the linear arranged biphenyl rings and the
1,3,4-oxadiazole ring are -45°, -48°, and 9°, respec-
tively. Other detailed information about the molecular
geometry is given as Supporting Information.
The log P and log D values of this series of compounds
were calculated for indicating their lipophilicity and
solubility (Table 1). Compounds 3 and 13b,c have log P
values greater than 5 (6.29, 6.06, and 5.33, respectively),
implicating high lipophilicity and thus alerting the
likely absorption problems as drugs for oral administra-
tion.23 Compound 1 has a log P value (4.90) approaching
5. In comparison with them, 2, 9-11, 13a , and 16 have
smaller log P values and thus are less lipophilic, with
13a being optimal (log P ) 3.38). The calculated log D
values, which are pH-dependent, are more realistic to
predict solubility and permeability of a drug. We need:
log D (at pH ) 7.4) > -0.80 for good absorption in the
intestine and slow elimination and in an optimal range
of 0.00-3.00 for good absorption and good penetration
through the blood-brain barrier. Generally, a compound
with a log D value greater than 4 could have solubility
problems and often shows a high plasma protein binding
(>98%). This leads to low brain concentration of a
drug.32 As can be seen, log D values for 1, 3, and 13b,c
are greater than 4 and that of 2, 11b, 13a , and 16 are
improved (<4). Compounds 11b and 13a have the most
favorable log D values (3.12 and 2.96, respectively) in
this respect. Interestingly, 10 with a basic amidine
moiety has a log D value (-0.35) approaching the
opposite territory due to its high pKa value (11.98) and
is expected to have very good solubility although its
ability to penetrate the blood-brain barrier needs to be
examined experimentally.

520 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Brief Articles
F igu r e 1. Molecular structure of compound 2, showing 50% probability thermal ellipsoids for the non-H atoms and the atom-
numbering scheme.
Sch em e 2a
receptor (IC50 ) 57 and 23 nM, respectively), however,
these were relatively lower in comparison with their
high affinities at the 5-HT1B/1D receptors. Other test
compounds had virtually low or no affinities (IC50 > 700
nM) at the 5-HT1A receptor.
In the literature, 1 has been reported to display only
low affinities at other CNS receptors, like 5-HT2, R1, and
dopamine DA receptors.13,15 In testing of the selected
compounds at other CNS receptors, 2, 9, 10, and 16 were
found to have weak and thus negligible affinities at the
5-HT2A, 5-HT2C, R1, R2, and hD2 receptors (data not
presented) as compared to their affinities at the r5-HT1B
and c5-HT1D receptors.
In Vit r o F u n ct ion a l St u d y. For determining the
5-HT1B/1D antagonistic activity, the inhibition of con-
tractile responses elicited by sumatriptan in rabbit
saphenous vein (RSV), challenged by test compound,
was measured. Compounds 1, 2, 9, 10, 11b, and 13a ,c
all showed significant effects (pA2 values > 8.0) as
potent 5-HT1B/1D antagonists. Among them, 2, 9, and
10 are equally potent as 1 (pA2 >9.1). Interestingly, 11a ,
16, and the reported inverse 5-HT1B agonist 3 showed
insignificant effects (pA2 < 7.4) in this model. Consider-
ing the binding profiles of these compounds, it appeared
that the potent 5-HT1B/1D ligand had a much more
pronounced effect than did the selective 5-HT1B ligand
in this model.
In testing of [3H]5-HT release to determine the
5-HT1B/1D antagonistic properties, the effect on the
sumatriptan-induced inhibition of K+-evoked [3H]5-HT
release from guinea pig cortex slices by test compound
was measured. Compound 1 only slightly increased the
5-HT release (114% of the control level), and 3 increased
the 5-HT release to 134%. Correspondingly, 2, 9, 10,
11b, 13a , and 16 showed much more pronounced effects
on the 5-HT release (in a range of 147-166%) with 13a
being the most potent one. Interestingly, not only the
potent 5-HT1B/1D ligands such as 2, 9, and 10 but also
compounds such as 11b, 13a , and 16 with low affinities
at the 5-HT1D receptor showed such pronounced effects
on the 5-HT release.
a
Reagents and conditions: (a) triethylamine, CH2Cl2 or THF,
0 °C to rt, overnight; (b) B(O-i-Pr)3, n-BuLi, -100 to -78 °C, 3 h,
then -78 °C to rt, overnight; (c) Pd(PPd3)4, Na2CO3, DME-H2O
(1:1), reflux 24 h.
P h a r m a cologica l Resu lts a n d Discu ssion
In Vitr o Recep tor Bin d in g Assa ys. The compounds
were tested in binding assays at the 5-HT1B receptor in
rat frontal cortex (r5-HT1B), 5-HT1D receptor in calf
striatum membranes (c5-HT1D), and 5-HT1A receptor in
rat hippocampus membranes. As shown in Table 1, 1
had affinities (IC50 values) of 52 and 1.6 nM at the
5-HT1D and 5-HT1B receptors, respectively, in our testing
system, while 3 showed high affinity only at the 5-HT1B
receptor (IC50 ) 2 nM). Compounds 2, 9, and 10 were
found to be more potent than 1 at the 5-HT1B and
5-HT1D receptors, with 10 being the most potent ligand
to date (IC50 ) 0.5 and 3 nM for 5-HT1B and 5-HT1D
receptors, respectively). Compounds 11a ,b and 13c had
decreased affinities at the 5-HT1D receptor (IC50 > 400
nM) but comparable affinities at the 5-HT1B receptor
(IC50 ) 7.3, 6.3, and 10 nM, respectively) to that of 1.
Compounds 13a ,b and 16 lost affinities at the 5-HT1D
receptor (IC50 > 700 nM), but 13a and 16 still retained
good affinities at the 5-HT1B receptor (IC50 ) 70 and 60
nM, respectively).
As mentioned above it could be favorable to combine
the potent 5-HT1B/1D antagonist with a 5-HT reuptake
inhibitor. To check if such double properties were
present in this series of compounds, they were tested
for their abilities to inhibit 5-HT reuptake. None of the
The reference compound 1 had low affinity (IC50
425 nM) at the 5-HT1A receptor. Compounds 9 and 10
showed moderate to good affinities at the 5-HT1A
)

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 521
Ta ble 1. Calculated log P and log D Values, Affinities of Compounds at the r5-HT1A, r5-HT1B, and c5-HT1D Receptors and for 5-HT
Reuptake Inhibition, pA2 Values for the Effects in RSV, and 5-HT Level in K+-Induced 5-HT Release in Guinea Pig Cortex
IC50, nMc
compd
log Pa (log Db)
c5-HT1D
r5-HT1B
r5-HT1A
5-HT reuptake
pA2
5-HT release, % of controld
1
2
3
9
10
11a
11b
13a
13b
13c
16
4.90 (4.48)
4.11 (3.69)
6.29 (4.64)
3.60 (3.18)
3.61 (-0.35)
4.33 (3.91)
3.54 (3.12)
3.38 (2.96)
6.06 (5.64)
5.33 (4.91)
4.04 (3.60)
52 ( 13.2
37 ( 7
1.6 ( 0.7
0.93 ( 0.07
2.0 ( 0.0
1.3 ( 0.3
0.5 ( 0.06
7.3 ( 1.3
6.3 ( 0.9
70 ( 20
425 ( 48
>1000
>10000
57 ( 9
23 ( 3
>1000
>100
1000
>1000
4700
>1000
407 ( 70
>1000
4700
9.9
9.1
7.4
10.0
9.6
7.3
114 ( 12
147 ( 4
134 ( 8
162 ( 5
155 ( 11
122 ( 12
147 ( 4
166 ( 9
NTe
>1000
10 ( 0.0
3.0 ( 0.6
467 ( 88
400 ( 100
8.0
>1000
>1000
600 ( 252
700 ( 58
7000
3800
8.3
700 ( 58
10 ( 5
>10000
>1000
NTe
8.3
5000
900
107 ( 13
155 ( 4
60 ( 11
700
>1000
7.0
a
The log P values of the nonionized compounds in octanol/water system were calculated with SRC Logkow 1.50 (Syracus Research
Corp.).33 The log D values (at pH 7.4) of the ionized compounds (in parentheses) were calculated according to the equation: log D )
b
log[10logP + 10(logP +pK -pH)] - log[1 + 10(pK -pH)], in which the pKa values were calculated with pKa/c 1.2 (Pallas 1.2, Compu Drug Chemistry
ion
a
a
Ltd.). c Results are expressed as IC50 values (nM), mean of at least 3 determinations ( SEM, otherwise indicated. For the method
d
description and data calculation, see Experimental Section and refs 24-29. e NT, not tested.
tested compounds showed activities for the functional
5-HT reuptake inhibition, except for 10, which showed
weak inhibitory activity (IC50 ) 407 nM).
In Vivo Micr od ia lysis. Desensitization of the ter-
minal 5-HT1B/1D autoreceptors can be mimicked by co-
administration of a 5-HT1B/1D antagonist with a SSRI.
The augmentation of the SSRI-induced increase in
extracellular 5-HT in terminal area would equal the
changes in serotonergic neurotransmission after chronic
treatment with the SSRIs. This strategy is believed to
induce instantaneous antidepressant effects of a SSRI.
In rat ventral hippocampus, compound 2 alone had no
effect on the 5-HT level, which is likely indicative of
being a silent antagonist. Administration of citalopram
(10 µmol/kg sc) increased the 5-HT level to 300%, and
co-administration of 2 (10 µmol/kg) and citalopram (10
µmol/kg) sc increased the 5-HT level up to 800%.
Compound 2 was indeed capable of augmenting the
increase of the 5-HT level induced by citalopram as a
SSRI (Figure 2). The postsynaptic effect of SSRI treat-
ment became a focus of interest in recent years. In
ventral hippocampus, the 5-HT1A and 5-HT3 receptors
are thought to increase the acetylcholine (ACh) release,
F igu r e 2. Effect of compound 2 on citalopram-induced
increase in hippocampal extracellular 5-HT level (expressed
b
as area under curve, AUC): asaline (sc, n ) 7), compound 2
whereas the 5-HT1B/1D receptors are postulated to
inhibit the ACh release.30,31 Co-administration of 2 and
citalopram also prevented the 5-HT-mediated inhibition
of ACh release elicited by citalopram administration
(Figure 3). This effect is mediated either by blockade of
postsynaptic 5-HT1B/1D receptors (inhibiting ACh re-
lease) or by augmenting extracellular 5-HT, thereby
stimulating other 5-HT receptors to facilitate the ACh
release.
In summary, the 1,3,4-oxadiazole analogue 2 of 1 was
found to be a very potent and selective 5-HT1B/1D
antagonist both in vitro and in vivo. In the 5-HT release
model, it showed a more pronounced effect than 1 and
3. The successful single-crystal X-ray analysis of 2
demonstrated the molecular geometry of GR127935-like
benzanilides, which will allow further modeling or
QSAR studies by docking its 3D structure to the
computational receptor models to better understand the
receptor-antagonist interactions and to direct further
SAR studies.
c
d
(10 µmol/kg sc, n ) 4), citalopram (10 µmol/kg sc, n ) 7), 10
µmol/kg of citalopram and 10 µmol/kg of compound 2 (sc, n )
5); *p < 0.05 (Student’s t-test).
ally not only in the RSV model but also in the 5-HT
release model. Manipulations of the methoxy moiety of
1 or 2 dramatically decreased the c5-HT1D receptor
binding; therefore, 11a ,b and 13a ,c appeared to be
selective at the r5-HT1B receptor. Compounds 11a ,b and
13c displayed the selectivity of affinities at the r5-HT1B
receptor versus the 5-HT1D receptor in calf striatum
membranes to be at least 60-fold. The selective r5-HT1B
ligands 11b and 13a showed very potent antagonistic
effects in the 5-HT release model although they are not
as highly potent as 1, 2, 9, and 10 in the RSV model.
Replacement of the amide linker of 2 by a sulfonamide
linker led to 16 which also has a very pronounced effect
in the 5-HT release model but not in the RSV model
although its affinities at the 5-HT1B/1D receptors de-
creased comparably.
Manipulation of the oxadiazole moiety of 1 led to the
most potent 5-HT1B/1D receptor ligands, 9 and 10, with
very potent 5-HT1B/1D antagonistic properties function-
In conclusion, a series of new biphenyl derivatives
were generated to be selective and even more potent
antagonists at either the r5-HT1B receptor or both the

522 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Brief Articles
8.4 Hz), 7.98 (s, 1 H), 7.93 (dd, 1 H, J ) 1.5, 7.7 Hz), 7.70 (dd,
1 H, J ) 2.2, 8.8 Hz), 7.44 (d, 2 H, J ) 8 Hz), 7.32 (d, 1 H, J
) 8 Hz), 7.25 (d, 1 H, J ) 2.2 Hz), 6.77 (d, 1 H, J ) 9.1 Hz),
3.81 (s, 3 H), 3.60-3.10 (m, 8 H), 2.85 (s, 3 H), 2.67 (s, 3 H),
2.32 (s, 3 H); 13C NMR (CDCl3, 500 MHz, with attached proton
tested) δ 176.4 (no H), 168.0 (no H), 165.5 (no H), 148.5 (no
H), 144.4 (no H), 143.5 (no H), 138.6 (no H), 135.9 (no H), 133.2
(no H), 131.8 (no H), 130.0 (1 H), 129.2 (1 H), 129.1 (2 C, all
with 1 H), 127.4 (2 C, all with 1 H), 125.9 (no H), 124.7 (1 H),
116.8 (1 H), 112.3 (1 H), 111.3 (1 H), 55.6 (3 H), 54.0 (2 C, all
with 2 H), 47.3 (2 C, all with 2 H), 43.6 (3 H), 20.3 (3 H), 12.3
(3 H); MS (CI) m/z 498 (M+ + 1). Anal. (C29H31N5O3‚0.25H2O)
C, H, N.
9 (converted to hydrochloride): mp 286-8 °C (EtOH); IR
(KBr) 3358 (s), 3441-3183 (brs), 1667 (s), 1643 (vs), 1606 (s),
1
1507 (s), 1231 (s) cm-1; H NMR (DMSO-d6, 500 MHz) δ 10.1
(s, 1 H), 7.90 (d, 2 H, J ) 7.7 Hz), 7.72 (s, 1 H), 7.65 (d, 1 H,
J ) 8 Hz), 7.38 (d, 2 H, J ) 7.7 Hz), 7.35 (dd, 1 H, J ) 1.8, 7.7
Hz), 7.26 (s, 1H), 7.18 (d, 1 H, J ) 8 Hz), 6.83 (d, 1 H, J ) 8.8
Hz), 3.65 (s, 3 H), 3.60-2.90 (m, 8 H), 2.67 (s, 3 H), 2.16 (s, 3
H); 13C NMR (CDCl3, 500 MHz, attached proton tested) δ 167.7
(no H), 164.8 (no H), 148.2 (no H), 143.6 (no H), 143.1 (no H),
139.3 (no H), 134.8 (no H), 133.8 (no H), 133.4 (no H), 132.7
(no H), 129.7 (1 H), 129.4 (1 H), 128.9 (1 H), 127.6 (1 H), 125.2
(1 H), 115.1 (1 H), 111.9 (1 H), 111.4 (1 H), 55.7 (3 H), 53.0 (2
C, all with 2 H), 47.4 (2 C, all with 2 H), 42.5 (3 H), 20.3 (3 H);
MS (CI) m/z 459 (M + 1). Anal. (C27H30N4O3‚2HCl‚H2O) C, H,
N.
N-[4-Met h oxy-3-(4-m et h ylp ip er a zin -1-yl)p h en yl] 2′-
Meth yl-4′-(5-m eth yl-1,3,4-oxa d ia zol-2-yl)bip h en yl-4-ca r -
boxa m id e (2). 3-(4′-Bromo-3′-methylphenyl)-5-methyl-1,3,4-
oxadiazole (8b)22 reacted with 7 in the same manner described
above, in either 1,2-dimethoxyethane or N,N-dimethylfora-
mide, affording 2 in 67-72% yield: white crystal, mp 219-22
°C (from EtOAc-EtOH); IR (KBr) 1669 cm-1; 1H NMR (CDCl3)
δ 7.96 (d, 2 H, J ) 8.3 Hz), 7.93 (s, 1 H), 7.80 (m, 2 H), 7.45 (d,
2 H, J ) 8.3 Hz), 7.35 (d, 1 H, J ) 8 Hz), 7.31 (m, 1 H), 7.25
(m, 1 H), 6.85 (d, 1 H, J ) 8.7 Hz), 3.88 (s, 3 H), 3.15 (brs, 4
H), 2.64 (s + brs, 7 H), 2.37 (s, 3 H), 2.34 (s, 3 H); MS (EI) m/z
497 (M+). Anal. (C29H31N5O3‚0.25H2O) C, H, N. The crystals
for X-ray were developed by slow evaporation from EtOH and
collected.
F igu r e 3. Effect of compound 2 on citalopram-induced
decrease in hippocampal extracellular ACh level (expressed
as area under curve, AUC; neostigmine 0.1 M): asaline (sc, n
) 5), bcompound 2 (10 µmol/kg sc, n ) 5), ccitalopram (10 µmol/
kg sc, n ) 6), d10 µmol/kg of citalopram and 10 µmol/kg of
compound 2 (sc, n ) 5); *p < 0.05 (Student’s t-test).
r5-HT1B receptor and the c5-HT1D receptor (mainly
constituting the homologous bovine 5-HT1B receptor),
as compared to GR127935 (1) and SB224289 (3). Several
compounds appear to be interesting for further phar-
macological studies to gain SAR information relevant
to delineating the N-piperazinylphenyl biphenylcar-
boxamides as selective and potent (partial) agonists or
antagonists at the h5-HT1B/1D receptors.
Exp er im en ta l Section
N-[4-Met h oxy-3-(4-m et h ylp ip er a zin -1-yl)p h en yl] 2′-
Meth yl-4′-a m id in ylbip h en yl-4-ca r boxa m id e (10). Com-
pound 1 (200 mg, 0.4 mmol) was dissolved into a mixture of
50 mL of methanol and 10 mL of acetic acid, followed by
addition of 50% Raney-Ni slurry in water (100 mg, 1 mL of
H2O was added additionally). The mixture was hydrogenated
with 4 atm of H2 at room temperature in a Parr shaker
overnight. Filtration through Celite, washing with acetic acid
(10 mL), and evaporation of the filtrate afforded an oily
residue. The residue was dissolved into CH2Cl2 (50 mL),
washed with 2 N aq NaOH (15 mL), dried over MgSO4, filtered,
and evaporated to afford the titled compound which was
solidified from ethyl acetate: 140 mg (77%), mp 159-62 °C;
Ch em istr y. Melting points were determined on an Elec-
trothermal digital melting point apparatus and are uncor-
rected. IR spectra were obtained on a ATI-Mattson spectrom-
eter. 1H and 13C NMR spectra were recorded on Varian Gemini
200 and 500 NMR instruments. Chemical shifts are given in
δ units (ppm) and relative to TMS or deuterated solvent.
Coupling constants (J ) are given in Hz. Mass spectra were
obtained on a Unicam 610/Automass 150 GC-MS system or
on a Finnegan 3300 system. Elemental analyses were per-
formed either in the Analytic Laboratory at Merck KGaA
(Darmstadt) or in the Microanalytic Laboratory of the Uni-
versity of Groningen and were within 0.4% of the theoretical
values. Merck silica gel (Kieselgel 60, 230-400 mesh) was used
for flash chromatography.
1
IR (KBr) 3300, 2935, 2801, 1645, 1607, 1508 cm-1; H NMR
(DMSO-d6) δ 10.10 (brs, 1 H), 9.60 (brs, 2 H), 8.31 (s, 1 H),
8.04 (d, 2 H, J ) 10 Hz), 7.78 (s, 1 H), 7.71 (d, 1 H, J ) 5 Hz),
7.51 (d, 2 H, J ) 10 Hz), 7.46 (m, 1 H), 7.38 (m, 2 H), 6.91 (d,
1 H, J ) 10 Hz), 3.77 (s, 3 H), 3.31 (brs, 4 H), 2.99 (brs, 2 H),
2.47 (brs, 2 H), 2.32 (s, 3 H), 2.23 (s, 3 H); MS (FAB) m/z 458
(M+ + 1). Anal. (C27H31N5O2‚1.5H2O) C, H, N.
N-[4-H yd r oxy-3-(4-m et h ylp ip er a zin -1-yl)p h en yl] 2′-
Meth yl-4′-(5-m eth yl-1,2,4-oxa d ia zol-3-yl)bip h en yl-4-ca r -
boxa m id e (11a ). Compound 1 (200 mg, 0.40 mmol) in 3 mL
of EtSH and 2 mL of CH2Cl2 was cooled to 0 °C and treated
with AlCl3 (1.0 g) with stirring overnight at room temperature.
The resulting solution was quenched with ice-water (20 mL),
neutralized with NaHCO3 (powder) until the pH value of the
solution was about 8, and extracted with CH2Cl2 (4 × 30 mL).
The combined organic layers were dried over MgSO4, filtered,
and evaporated under vacuum to give 200 mg of the crude
product, which was purified by flash chromatography (SiO2,
THF with gradually adding small amount of 25% aq NH4OH
N-[4-Met h oxy-3-(4-m et h ylp ip er a zin -1-yl)p h en yl] 2′-
Meth yl-4′-(5-m eth yl-1,2,4-oxa d ia zol-3-yl)bip h en yl-4-ca r -
boxa m id e (1) a n d N-[4-Meth oxy-3-(4-m eth ylp ip er a zin -1-
yl)p h en yl] 2′-Met h yl-4′-a m in oca r b on ylb ip h en yl-4-ca r -
boxa m id e (9). 3-(4′-Bromo-3′-methylphenyl)-5-methyl-1,2,4-
oxadiazole (8a )22 (1.9 g, 7.5 mmol), the boronic acid intermedi-
ate 7 (2.5 g, 6.8 mmol), Na2CO3‚10H2O (6.3 g), N,N-dimeth-
ylformamide (100 mL), and water (100 mL) were combined
and degassed with nitrogen. Tetrakis(triphenylphosphine)-
palladium(0) (160 mg) was added to the solution and the
mixture was heated to reflux for 1 day, evaporated to remove
the solvents, and purified by flash chromatography (CH2Cl2/
EtOH, 9:1 then 4:1, with adding gradually small amount of
25% aq NH4OH), affording 0.6 g (18%) of 1 and 2.0 g (67%) of
9.
1: mp 252-4 °C (EtOAc-EtOH); IR (KBr) 3421, 1663 cm-1
1H NMR (CDCl3, 500 MHz) δ 8.95 (s, 1 H), 8.13 (d, 2 H, J )
;

Brief Articles
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3 523
as eluent) and recrystallization from 1:2 ethyl acetate/hexane
(s, 3 H); MS (EI) m/z 529 (M+); HRMS for C30H32N5O3F: calcd,
to afford 140 mg (73%) of 11a as a fine solid: mp 140-2 °C;
529.2488; found, 529.2471.
IR (KBr) 3296 (brs), 1638 (s), 1504 (s), 1425 (s), 1261 (s) cm-1
;
N-[4-Meth oxy-3-(4-m eth ylpiper azin -1-yl)ph en yl] 4-Br o-
m oben zen esu lfon a m id e (14). A mixture of 4-bromobenze-
nesulfonyl chloride (511 mg, 2.0 mmol), 4-methoxy-3-(N-
methylpiperazinyl)aniline dihydrochloride (588 mg, 2.0 mmol),
10 mL of dry THF, and 2 mL of triethylamine was stirred
overnight at room temperature, filtered to remove the white
precipitate, and evaporated. The residue was purified by flash
chromatography (SiO2, EtOAc and THF as eluents) and
recrystallization from ethyl acetate to afford 0.85 g (96%) of
14 as a white solid: mp 180-2 °C; IR (KBr) 3005, 2946, 2830,
1H NMR (CDCl3) δ 7.95 (m, 5 H), 7.55 (s, 1 H), 7.43 (d, 2 H, J
) 8.3 Hz), 7.30 (m, 1 H), 6.94 (d, 1 H, J ) 8.5 Hz), 2.94 (brs,
4 H), 2.67 (s, 3 H), 2.62 (brs, 4 H), 2.37 (s, 3 H), 2.32 (s, 3 H);
MS (EI) m/z 483 (M+). Anal. (C28H29N5O3‚0.25H2O) C, H, N.
N-[4-Hyd r oxy-3-(4′-m et h ylp ip er a zin -1′-yl)p h en yl] 2′-
m eth yl-4′-(5-m eth yl-1,3,4-oxa d ia zol-2-yl)bip h en yl-4-ca r -
boxa m id e (11b) was prepared from 2 in the same manner
described above for the preparation of 11a . 11b: 85%, white
solid, mp 149-52 °C; IR (KBr) 3504, 3350, 3294, 1646, 1505,
1594, 1574, 1510, 1334, 1153, 998 cm-1; H NMR (CDCl3) δ
1
1
1262, 1241 cm-1; H NMR (CDCl3) δ 8.47 (s, 1 H), 7.96 (d, 2
7.55 (s, 4 H), 6.70 (s, 3 H), 6.50 (s, 1 H), 3.82 (s, 3 H), 2.85 (m,
4 H), 2.60 (m, 4 H), 2.40 (s, 3 H); 13C NMR (CDCl3) δ 151.5,
142.0, 138.9, 132.4, 129.2, 129.0, 127.9, 120.1, 116.3, 111.9,
99.5, 56.0, 55.4, 50.6, 46.4; MS (EI) m/z 440 (M+). Anal.
(C18H22N3SO3Br) C, H, N.
H, J ) 8.3 Hz), 7.93 (s, 1 H), 7.85 (dd, 1 H, J ) 1.4, 7.7 Hz),
7.53 (d, 1 H, J ) 2.3 Hz), 7.38 (d, 2 H, J ) 8.3 Hz), 7.30 (m, 1
H), 6.89 (d, 1 H, J ) 8.6 Hz), 2.96 (m, 4 H), 2.65 (brs, 4 H),
2.61 (s, 3 H), 2.38 (s, 3 H), 2.29 (s, 3 H); MS (EI) m/z 483 (M+,
56), 425 (80), 277 (90), 71 (100). Anal. (C28H29N5O3‚0.5H2O)
C, H, N.
4-{N-[4′-Met h oxy-3′-(4-m et h ylp ip er a zin -1-yl)p h en yl]-
a m in osu lfon yl}ben zen ebor on ic Acid (15). Triisopropyloxy-
boron (2.1 mL, 9 mmol) was added to a solution of 14 (329
mg, 0.75 mmol) in 15 mL of dry THF at -100 °C under
nitrogen. The solution was treated with n-BuLi (3.8 mL in 2.5
M in hexane, 9 mmol) dropwise during 20 min. The resulting
mixture was stirred for 3 h at the temperature in a range of
-100 to -78 °C and then overnight at room temperature,
quenched by adding dropwise 5 mL of water, stirred for 1 h,
and evaporated to dryness under vacuum. The residue was
preadsorbed onto 10 mL of silica gel and purified by flash
chromatography (SiO2, CH2Cl2/EtOH, 9:1 to 7:3 with adding
gradually small amount of 25% aq NH4OH as eluent) to afford
220 mg (73%) of 15 as a white foam, which was used in the
following reaction without further purification.
N-[4-Meth ylsu lfon yloxy-3-(4-m eth ylpiper azin -1-yl)ph en -
yl] 2′-Meth yl-4′-(5-m eth yl-1,3,4-oxa d ia zol-2-yl)bip h en yl-
4-ca r boxa m id e (13a ). Compound 11b (40 mg, 0.083 mmol)
in 3 mL of CH2Cl2 and 0.1 mL of triethylamine was treated
with methanesulfonyl chloride (0.06 mL) at -78 °C with
stirring for 1 h (-78 °C to room temperature). To the mixture
were added 0.1 mL of 2 N NaOH and 2 g of SiO2. The mixture
was evaporated and the residue was purified by flash chro-
matography (SiO2, 9:1 CH2Cl2/EtOH with gradually adding
small amount of 25% aq NH4OH as eluent) to afford 40 mg
(86%) of 13a , which was recrystallized from EtOAc-hexane
as a white solid: mp 194-7 °C; IR (KBr) 3420 (brm), 2977 (s),
2941 (s), 2603 (vs), 2496 (vs), 1655 (s), 1476 (s), 1397 (s), 1186
1
(s), 1037 (s) cm-1; H NMR (CDCl3) δ 9.25 (s, 1 H), 8.13 (d, 2
H, J ) 8 Hz), 7.94 (s, 1 H), 7.86 (d, 1 H, J ) 8 Hz), 7.75 (s, 1
H), 7.56 (d, 1 H, J ) 8.3 Hz), 7.41 (d, 2 H, J ) 8.3 Hz), 7.30 (d,
1 H, J ) 8.3 Hz), 7.19 (d, 1 H, J ) 8.3 Hz), 3.20 (brs, 4 H),
3.15 (s, 3 H), 2.80 (brs, 4 H), 2.60 (s, 3 H), 2.49 (s, 3 H), 2.30
(s, 3 H); 13C NMR (CDCl3) δ 165.4, 164.6, 163.6, 144.7, 144.6,
143.7, 138.9, 137.9, 136.2, 133.3, 130.2, 129.3, 128.6, 127.2,
124.2, 123.2, 115.0, 112.2, 54.8, 49.7, 45.0, 38.4, 20.3, 11.1; MS
(EI) m/z 561 (M+); HRMS for C29H31N5SO5: calcd, 561.2045;
found, 561.2038.
N-[4-Tr iflu or om eth ylsu lfon yloxy-3-(4-m eth ylpiper azin -
1-yl)p h en yl] 2′-Meth yl-4′-(5-m eth yl-1,2,4-oxa d ia zol-3-yl)-
bip h en yl-4-ca r boxa m id e (13b). Compound 11a (101 mg,
0.21 mmol) in 10 mL of CH2Cl2 and 0.1 mL of triethylamine
was treated with the solution of Tf2O (0.05 mL) in 2 mL of
CH2Cl2 dropwise at -78 °C. The resulting solution was stirred
overnight at room temperature, quenched with water, and
extracted with CH2Cl2 (3 × 15 mL). The combined organic
layers were dried over MgSO4, filtered, evaporated, and
purified by flash chromatography (SiO2, EtOAc then THF as
eluent) and recrystallization from EtOAc/hexane to afford 80
mg (62%) of 13b: mp 140 °C dec; IR (KBr) 3416 (brm), 1676
(m), 1628 (m), 1595 (s), 1422 (s), 1212 (brs), 1139 (s) cm-1; 1H
NMR (CDCl3) δ 8.10 (s, 1 H), 7.90 (m, 4 H), 7.50 (m, 3 H),
7.35-7.17 (m, 2 H), 6.95 (d, 1 H, J ) 8.6 Hz), 2.95 (m, 4 H),
2.70 (m, 4 H), 2.66 (s, 3 H), 2.40 (s, 3 H), 2.22 (s, 3 H); MS (EI)
m/z 615 (M+, 1.3). Anal. (C29H28N5O5SF3) C, H, N.
N-[4-(2′-F lu or oeth oxy)-3-(4-m eth ylp ip er a zin -1-yl)p h e-
n yl] 2′-Meth yl-4′-(5-m eth yl-1,2,4-oxa d ia zol-3-yl)bip h en yl-
4-ca r boxa m id e (13c). Compound 11a (120 mg, 0.25 mmol),
acetonitrile (15 mL), BrCH2CH2F (52 mg, 0.4 mmol), and
CsCO3 (600 mg, 1.8 mmol) were combined and heated to reflux
for 3 h. The mixture was evaporated, quenched with water
(10 mL), and extracted with CH2Cl2 (3 × 10 mL). The combined
organic layers were dried over MgSO4, filtered, and evapo-
rated. The residue was purified by flash chromatography (SiO2,
10:1 CH2Cl2/EtOH) to afford 94 mg (71%) of 13c: mp 128-30
°C (liquidization); IR (KBr) 3417 (br m), 1654 (m), 1643 (m),
1607 (m), 1507 (s), 1257 (s), 1230 (s) cm-1; 1H NMR (CDCl3) δ
7.99-7.92 (m, 5 H), 7.50-7.20 (m, 5 H), 6.85 (d, 1 H, J ) 8.5
Hz), 4.88 (m, 1 H), 4.62 (m, 1 H), 4.30 (m, 1 H), 4.18 (m, 1 H),
3.20 (brs, 4 H), 2.67 (s, 3 H), 2.60 (m, 4 H), 2.38 (s, 3 H), 2.32
N-[4-Met h oxy-3-(4-m et h ylp ip er a zin -1-yl)p h en yl] 2′-
Meth yl-4′-(5-m eth yl-1,3,4-oxa d ia zol-2-yl)bip h en yl-4-su l-
fon a m id e (16). Intermediate 15 (180 mg, 0.44 mmol) reacted
with 8b (189 mg, 0.75 mmol) in the same manner described
above for the preparation of 2 to afford 200 mg (84%) of 16 as
a white solid, which was recrystallized from ethyl acetate: mp
242-4 °C; IR (KBr) 2984, 2824, 1584, 1503, 1339, 1236, 1166
cm-1; 1H NMR (CDCl3, little CD3OD) δ 7.86 (s, 1 H), 7.80 (dd,
1 H, J ) 1.7, 7.9 Hz), 7.74 (d, 2 H, J ) 8.2 Hz), 7.32 (d, 2 H,
J ) 8.3 Hz), 7.23 (d, 1 H, J ) 8.1 Hz), 6.86 (dd, 1 H, J ) 2.4,
8.6 Hz), 6.68 (d, 1 H, J ) 7.8 Hz), 6.60 (d, 1 H, J ) 2.4 Hz),
3.74 (s, 3 H), 3.27 (brs, 8 H), 2.77 (s, 3 H), 2.56 (s, 3 H), 2.21
(s, 3 H); 13C NMR (CDCl3) δ 164.9, 164.1, 149.9, 145.3, 143.6,
139.3, 138.7, 136.5, 130.5, 130.4, 129.6, 128.9, 127.4, 124.5,
123.3, 118.3, 114.2, 111.9, 55.8, 54.1 (2C), 47.5 (2C), 43.7, 20.4,
11.1; MS (EI) m/z 533 (M+). Anal. (C28H31N5O4S) C, H, N.
P h a r m a cology. 1. In Vitr o Recep tor Bin d in g Assa ys.
Affinities for r5-HT1B receptors were determined in rat frontal
cortex using [125I]iodocynopindolol as radioligand, final con-
centration 0.1 nM.24 The c5-HT1D assays were performed
according to Peroutka et al.,25 using membranes prepared from
calf striatum and 1.7 nM [3H]serotonin in the presence of 100
nM 8-OH-DPAT and 100 nM mesulergine to mask 5-HT1A and
5-HT2C binding sites. Nonspecific binding was determined in
the presence of 10 µM serotonin.25 Inhibition by drugs of the
binding of 0.5 nM [3H]8-OH-DPAT to 5-HT1A receptors in
membranes from rat hippocampus was determined according
to the reported method.26
2. Ra bbit Sa p h en ou s Vein (RSV) Mod el. The inhibition
of the contractile responses elicited by sumatriptan in RSV,
challenged by the test compound, was determined according
to the published method.27 Segments of the isolated RSV were
suspended in organ baths and subjected to two concentration-
response curves to sumatriptan, the second challenge being
performed in the presence of the antagonist or control vehicle
(incubated for 60 min). Apparent pA2 values were calculated
using a single antagonist concentration in at least three
preparations.
3. [3H]5-HT Relea se. The experiments were performed
according to a method described by Ormandy et al.28 Cerebral
cortices were removed from male Dunkin-Hartley guinea pigs,

524 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 3
Brief Articles
(6) Leysen, J . E.; Gommeren, W.; Heylen, L.; Luyten, W.; Van de
Weyer, I.; Vanhoenacker, P.; Haegeman, G.; Schotte, A.; Van
Gompel, P.; Wouters, R.; Lesage, A. S. Alniditan, a new 5-HT1D
agonist and migraine-abortive agent: ligand-binding properties
of human 5-HT1DR, human 5-HT1Dâ, and calf 5-HT1D receptors
investigated with [3H]5-HT and [3H]alniditan. Mol. Pharmacol.
1996, 50, 1567-1580.
cross-chopped into slices, and then incubated 30 min with
[3H]5-HT. Slices were placed into chambers of a superfusion
apparatus and superfused with oxygenated Krebs’s solution.
Superfusate was collected in 4-min periods beginning 60 min
after the slices were placed in the chambers. The slices were
exposed to two periods (4 min) of 30 mM K+ at 68 and 108
min superfusion (S1 and S2, respectively). Sumatriptan at 3
µM (controls) and sumatriptan plus test compounds (30 nM,
300 nM, and 3 µM, respectively) were superfused prior to and
during the S2 stimulation period. The percentage release rate
of each sample was calculated as the radioactivity in that
fraction multiplicated by 100 and divided by the total amount
of radioactivity present in the tissue at the start of the
collection. The K+-stimulated release for S1 and S2 was
calculated as the total overflow during the collection periods
after the onset of the stimulation minus basal outflow. Potas-
sium ion-mediated stimulation of release increased 3H efflux
by a total amount of 7.71 ( 0.69% (n ) 28) of tissue 3H (S1).
The addition of 3 µM sumatriptan to the superfusion buffer
before S2 produced an inhibition of the stimulated release to
4.61 ( 0.42% (n ) 28) of tissue 3H (S2). The effects of drugs
on stimulated release were determined by calculating S2/S1
ratios in control and treated slices, expressing the drug
response as a percent of the control S2/S1 ratio, which was
0.59 ( 0.01 (n ) 28). Results represent the mean ((SEM) of
3-4 determinations. Statistical differences between ratios
were analyzed using ANOVA followed by Dunnett test. Data
of test compound at 300 nM are shown in Table 1.
(7) Berg, S.; Larsson, L.-G.; Renyi, L.; Ross, S. B.; Thorberg, S.-O.;
Thorell-Svantesson, G. (R)-(+)-2-[[[3-(Morpholinomethyl)-2H-
chromen-8-yl]oxy]methyl]morpholine methanesulfonate: a new
selective rat 5-hydroxytryptamine-1B receptor antagonist. J .
Med. Chem. 1998, 41, 1934-1942.
(8) Glennon, R. A.; Westkaemper, R. B. 5-HT1D receptors:
a
serotonin receptor population for the 1990s. Drug News Perspect.
1993, 6, 390-405.
(9) Pauwels, P. J . 5-HT1B/1D antagonists. Gen. Pharmacol. 1997, 29,
293-303.
(10) Briley, M.; Moret, C. Neurological mechanism involved in
antidepressant therapies. Clin. Neuropharmacol. 1993, 16, 387-
400.
(11) Halazy, S.; Lamothe, M.; J orand-Lebrun, C. 5-HT1B/1D antago-
nists and depression. Exp. Opin. Ther. Patents. 1997, 7, 339-
352.
(12) Dolberg, O. T.; Sasson, Y.; Cohen, R.; Zohar, J . The relevance of
behavioural probes in obsessive-compulsive disorders. Eur.
Neuropsychopharmacol. 1995, 5, 161-162.
(13) Clitherow, J . W.; Scopes, D. I. C.; Skingle, M.; J ordan, C. C.;
Feniuk, W.; Campbell, I. B.; Carter, M. C.; Collington, E. W.;
Conner, H. E.; Higgins, G. A.; Beattie, D.; Kelly, H. A.; Mitchell,
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implanted bilaterally into ventral hippocampus of male Wistar
rats (280-340 g) (Harlen Zeist, The Netherlands), perfused
with artificial cerebrospinal fluid (aCSF). Acetylcholine (ACh)
experiments were performed with neostigmine (0.1 M) added
to the aCSF, to obtain detectable amounts of ACh. Serotonin
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cal detection. Postcolumn conversion into hydrogen peroxide
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1,2,4-oxadiazole-3-yl)biphenyl-4-yl]-carbonyl]-2,3,6,7-tetrahydro-
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Ack n ow led gm en t. We are indebted to Merck KGaA
for financial support. SB224289 was kindly given as a
gift from SmithKline Beecham to Merck KGaA CNS
department. The single-crystal X-ray analysis of com-
pound 2 was performed at the University of Go¨teborg.
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