Optimization of (arylpiperazinylbutyl)oxindoles exhibiting selective 5-HT7 receptor antagonist activity

Article abstract of DOI:10.1021/jm200547z

A series of (arylpiperazinylbutyl)oxindoles as highly potent 5-HT ₇ receptor antagonists has been studied for their selectivity toward the 5-HT_1A receptor and α₁-adrenoceptor. Several derivatives exhibited high 5-HT_7<

Full text of DOI:10.1021/jm200547z

ARTICLE
pubs.acs.org/jmc
Optimization of (Arylpiperazinylbutyl)oxindoles Exhibiting Selective
5-HT7 Receptor Antagonist Activity
,‡
§
§
‡
§
‡
§ ꢀ
Balꢀazs Volk,* Istvꢀan Gacsꢀalyi, Katalin Pallagi, Lꢀaszlꢀo Poszꢀavꢀacz, Ildikꢀo Gy€on€os, Eva Szabꢀo, Tibor Bakꢀo,
Michael Spedding,^ Gyula Simig,‡ and Gꢀabor Szꢀenꢀasi*,§
‡Chemical Research Division, EGIS Pharmaceuticals Plc., P.O. Box 100, Budapest, H-1475 Hungary
§Preclinical Research Division, EGIS Pharmaceuticals Plc., P.O. Box 100, Budapest, H-1475 Hungary
^Les Laboratoires Servier, 22 rue Garnier, 92578 Neuilly-sur-Seine, France
S Supporting Information
b
ABSTRACT: A series of (arylpiperazinylbutyl)oxindoles as
highly potent 5-HT7 receptor antagonists has been studied
for their selectivity toward the 5-HT1A receptor and α1-adreno-
ceptor. Several derivatives exhibited high 5-HT7/5-HT1A selec-
tivity, and the key structural factors for reducing undesired α1-
adrenergic receptor binding have also been identified. Rapid
metabolism, a common problem within this family of compounds, could be circumvented with appropriate substitution patterns on
the oxindole carbocycle. Contrary to expectations, none of the compounds produced an antidepressant-like action in the forced
swimming test in mice despite sufficiently high brain concentrations. On the other hand, certain analogues showed significant
anxiolytic activity in two different animal models: the Vogel conflict drinking test in rats and the lightꢀdark test in mice.
’ INTRODUCTION
toward other 5-HT receptor subtypes and a series of other
receptors. A representative of the family 3-{4-[4-(4-chlorophenyl)-
piperazin-1-yl]butyl}-3-ethyl-6-fluoro-1,3-dihydro-2H-indol-
2-one (1a, Table 1) exhibited selective 5-HT7 receptor antago-
nist activity. Nevertheless, this derivative was not effective in two
different anxiolytic tests: the conflict drinking (Vogel) test10 and
the lightꢀdark test.11 On the other hand, we have demonstrated
that 2a (Table 1) showed outstanding anxiolytic efficacy in both
tests, although it had lower 5-HT7 receptor affinity and it was less
selective toward the 5-HT2A receptor than 1a. As the microsomal
metabolic stability of the two compounds is poor, in case of 2a
one can not exclude the formation of an active metabolite, which
is responsible for the anxiolytic effect. Thus, in the present study
we aimed to evaluate also the structureꢀmetabolic stability
relationship of the synthesized compounds. Furthermore, we
also assayed the affinity of our new chemical entities (NCEs) for
the α1-adrenoceptor (α1-AR) as a potential source of serious
cardiovascular side effects, because compounds with an arylpi-
perazine moiety can likely bind to this receptor.12 Lack of α1-AR
affinity is also important, as blockade of this receptor can
diminish the efficacy of antidepressant drugs.13,14
In recent decades, an increasing demand has emerged for the
discovery of new types of anxiolytics and antidepressants. The
therapy of these psychiatric diseases is dominated by benzodia-
zepine-type anxiolytics and selective serotonin (5-hydroxytrypt-
amine, 5-HT) reuptakeinhibitor (SSRI) antidepressants. However,
both classes of drugs possess several drawbacks. Benzodiazepines
have sedative, hypnotic, muscle relaxant, and amnesic effects, and
their abuse and dependence potential is high, while 5-HT uptake
inhibitors produce a therapeutic effect only after several weeks of
treatment.
One of the possible alternatives to benzodiazepines and SSRIs
is to apply 5-HT7 receptor ligands for the treatment of anxiety,
stress, and depression. The 5-HT7 receptor was the last addition
to the mammalian 5-HT receptor family.1,2 This receptor belongs
to the group of G-protein coupled receptors that are positively
coupled to adenylate cyclase. Several pieces of experimental
evidence support the role of the 5-HT7 receptor in the pathome-
chanism of depression,3 sleep disorders,3d,4 stress, and anxiety,3e,5
and furthermore in learning and memory deficiencies.4a,6 Con-
tinuous interest in 5-HT7 receptor ligands is best demonstrated by
the high number of papers and reviews4a,7 dealing with this topic.
In a recent publication8 we have demonstrated the pharma-
cological efficacy of (arylpiperazinylbutyl)oxindoles as 5-HT7
ligands. The structureꢀactivity relationship studies performed
led us to compounds that exhibited subnanomolar binding to the
5-HT7 receptor, combined with good selectivity toward the
structurally related9 5-HT1A receptor. All compounds tested
proved to be 5-HT7 receptor antagonists. The selectivity of some
compounds with high 5-HT7 receptor affinity was evaluated
’ CHEMISTRY
Synthesis of 3,3-disubstituted oxindole derivatives was carried
out in most cases via the synthetic route described in our previous
paper (1, Scheme 1, route A).8 Isatin (3, X = H) is commercially
available, and the substituted isatins (3, X = 5-F, 6-F) were
Received: May 3, 2011
Published: August 22, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
Table 1. Representative Receptor Affinity Data and Anxiolytic Activity of Two Oxindole-Type 5-HT7 Receptor Antagonists
ARTICLE
5-HT7
5-HT1A
5-HT2A
conflict drinking testb
lightꢀdark testc
MED (mg/kg ip)
a
a
a
compound
R
X
Ki (nM)
Ki (nM)
Ki (nM)
MED (mg/kg ip)
1a
2a
Et
H
F
0.79
7.0
1610
1800
19.4
17.5
>20
>10
<1
H
<2.5
a Receptors and radioligands used in binding assays and data analysis: see Experimental Section. b A modification of the Vogel10 method was used for the
conflict drinking test in rats, drugs were administered intraperitoneally. For details, see Experimental Section. c Testing of drugs in the lightꢀdark test was
carried out in mice as described by Costall11 after intraperitoneal administration of the drugs. For details, see Experimental Section.
Scheme 1a
a Reagents and conditions: (a) EtOH, RaꢀNi, 15 bar H2, 180ꢀ210 ꢀC, 3ꢀ5 h, 71ꢀ91%; (b) BuLi, Br-(CH2)4-Cl, THF, ꢀ78 ꢀC to rt, 4 h, 86ꢀ95%;
(c) chlorination at the 5-position: SO2Cl2, glacial AcOH, 16ꢀ18 ꢀC, 2 h, 86ꢀ93%; 7-chlorination or 5,7-dichlorination: SO2Cl2, glacial AcOH, 60 ꢀC,
3 h, 65ꢀ79%; (d) HNR2R3, Na2CO3, in melt, 180 ꢀC, 1ꢀ2 h, 39ꢀ81%; (e) HO-(CH2)4-OH, RaꢀNi, 15 bar H2, 190 ꢀC, 4ꢀ5 h, 75ꢀ81%;
(f) CH3SO2Cl, Et3N, THF, ꢀ78 ꢀC to rt, 1ꢀ2 h, 81ꢀ93%; (g) HNR2R3, Na2CO3, in melt, 120 ꢀC, 1 h, 41ꢀ82%.
synthesized, starting from anilines, by the classical Sandmeyer
procedure.15 The one-pot reductive alkylation of these starting
materials (with alcohols, in the presence of Raney nickel) to the
corresponding 3-alkyloxindoles (4) was performed by the meth-
od elaborated in our laboratory.16 Selective C(3)-alkylation of
compounds 4 was carried out using 1-bromo-4-chlorobutane
after deprotonation with BuLi,17 resulting in 3-ethyl-3-(4-chloro-
butyl)oxindole intermediates 5. Chlorination of compounds 5
afforded the corresponding mono- and dichloro derivatives 6.
Reaction of 5a and 5c with sulfuryl chloride (3 equiv) in glacial
acetic acid at 16ꢀ18 ꢀC yielded selectively the 5-chloro deriva-
tives (6a and 6b), while at 60 ꢀC the 7-position was also affected;
thus, 7-chloro-5-fluoro (5b f 6d), 5,7-dichloro (5a f 6c), or
5,7-dichloro-6-fluoro (5c f 6e) analogues were obtained.
Finally, replacement of the side-chain chlorine atom with various
secondary amines in melt led to the target compounds 1. When
necessary, the product was purified by column chromatography.
In cases where it did not result in a solid compound, the oily
products were converted into their hydrochloride salts.
A different route was applied for the synthesis of the 3-mono-
substituted derivatives (2, Scheme 1, route B). Isatins (3) were re-
ductively hydroxybutylated with butane-1,4-diol in the presence
of RaꢀNi to afford 3-(4-hydroxybutyl)oxindoles (7).16 Mesylation
of compounds 7 with methanesulfonyl chloride to derivatives 8,
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Journal of Medicinal Chemistry
ARTICLE
Scheme 2a
a Reagents and conditions: (a) SO2Cl2, reflux, 4 h, 80%; (b) RaꢀNi, 20 bar H2, rt, 18 h, 79%; (c) 1-(4-chlorophenyl)piperazine, Na2CO3, in melt,
120ꢀ180 ꢀC, 1ꢀ2 h; (d) 1-(4-chlorophenyl)piperazine, acetonitrile, evaporation, then rt, 2 h, 47%.
followed by treatment with the appropriate secondary amines,
led to the desired compounds 2. Finally, the crude products were
purified by column chromatography, and, if necessary, they were
converted into their hydrochloride or oxalate salts.
introduction of one or more halogen atoms. The effect of
halogenation was studied both on the oxindole carbocycle and
the aromatic ring of the basic group, so that we could determine
how electronegative halogen atoms affect the receptor binding
affinities. Binding of the compounds was evaluated to a series of
5-HT and other receptors. Undesired 5-HT1A receptor affinity
had already been a crucial element in our previous study for the
evaluation of the receptor profile of the compound family.
Because blockade or activation of α1A-ARs can cause cardiovas-
cular side effects, α1A-AR affinity was also measured for all
compounds.
A different pathway had now to be elaborated for the pre-
paration of 3-monosubstituted 5,7-dichloro derivative 11 (Scheme2).
It is known from the literature18 that chlorination (SO2Cl2) or
bromination (Br2, NBS) of 3-unsubstituted or 3-monosubsti-
tuted oxindoles leads primarily to halogenation at position 3,
while aromatic substitution at C(5) and especially at C(7)
necessitates harsher conditions. Thus, selective direct 5,7-di-
chlorination of 8a to 10 could not be expected. Therefore, mesyl
ester 8a was 3,5,7-trichlorinated, resulting in compound 9. Since
selective removal of the 3-chloro substituent using catalytic
hydrogenation could only be expected under mild conditions,
Raney nickel was used as catalyst in THF. Preparation of 10
was accomplished in 79% yield. Coupling of mesylate 10 with 1-
(4-chlorophenyl)piperazine was first attempted in melt, in the
presence of Na2CO3. Although we have synthesized all other
derivatives using this methodology, we surprisingly found that in
this particular case, a multicomponent reaction mixture was ob-
tained. HPLC-MS analysis of the mixture showed that the desired
compound 11 was only a minor component. Finally, a modified
version of the coupling reaction led to 11. The acetonitrile solution
of 10 and 1-(4-chlorophenyl)piperazine was evaporated, and the
thus obtained intimate mixture was stirred at ambient temperature
for 2 h. After chromatographic purification, 11 was obtained in
47% yield.
The pharmacological action of compounds demonstrating
high 5-HT7 receptor affinity and an acceptable selectivity profile
was evaluated in two anxiolytic tests, the conflict drinking test in
rats10 and lightꢀdark test in mice,11 and in a model assessing the
depression-like behavior, the forced swimming test in mice.20ꢀ22
Oxindoles withpiperazine basicgroup aresummarizedinTable2,
and derivatives with different basic groups are shown in Table 3.
5-HT7 Receptor Binding. Data show that most of the com-
pounds synthesized were very potent 5-HT7 receptor ligands
(Tables 2 and 3). Not surprisingly, receptor affinities could be
influenced by various substitution patterns of the phenylpiper-
azine moiety. Compounds having a CF3, OMe, F, or Cl sub-
stituent on the phenyl group showed 5-HT7 receptor Ki values
ranging from 0.38 to 107 nM. The position of substituents also
affected the 5-HT7 receptor affinity. As demonstrated in our
previous publication,8 among methoxyphenyl analogues, the
position of the substituent changed the binding significantly, in
an order of meta > ortho . para. These results were in accordance
with the ones published recently for a structurally related
family of compounds.23 However, in the case of fluoro com-
pounds, the above postulated trend failed, the para-substituted
compound (1o) proved to be a more potent 5-HT7 receptor
ligand than the meta-substituted analogue (1n). Concerning the
chloro-substituted analogues, both 3-chloro- (2d,e, 1dꢀg) and
4-chlorophenylpiperazines (1hꢀm, 2a, 2f,g) show high affinity
for the 5-HT7 receptor. In our hands, the 3,4-dichloro analogue
(1r) was similarly potent, contrary to the expectations based on
literature data.24
’ RESULTS AND DISCUSSION
The above results encouraged us to synthesize further deriva-
tives and to study the structureꢀactivity relationship of this
family of compounds more in detail. In the course of our previous
work,8,19 compounds unsubstituted at the oxindole nitrogen
atom and containing a tetramethylene spacer between the ox-
indole skeleton and the basic nitrogen atom proved to be the most
potent and most selective 5-HT7 receptor ligands, so further
investigations, disclosed in the present paper, were performed
with such derivatives. Starting from the simplest representative
of this family, 3-[4-(4-phenylpiperazin-1-yl)butyl]-1,3-dihydro-
2H-indol-2-one (2b, Table 2), systematic modifications were
performed: (i) variation of the basic group, (ii) introduction of an
alkyl substituent at position 3 of the oxindole ring, (iii)
A halogen substituent on the oxindole carbocycle did not
substantially change the 5-HT7 receptor affinities, as 5-chloro,
5-fluoro,25 and 6-fluoro derivatives had similar Ki values to the
unsubstituted derivatives. Na et al. drew the same conclusion
regarding the effect of fluorine substitution on 5-HT7 receptor
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Journal of Medicinal Chemistry
ARTICLE
Table 2. Oxindoles with Phenylpiperazine-Type Basic Groups
a
a
5-HT7
5-HT1A
α1-ARa
compound
V
W
X
Y
Z
R
Ki (nM)
Ki (nM) or % at 10ꢀ7 M
Ki (nM)
2b
2c
1b
1c
2d
1d
2e
1e
1f
H
H
H
H
F
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
H
H
H
H
H
H
Et
Et
H
Et
H
Et
Et
Et
H
H
H
Et
Et
Et
Et
Et
Et
H
Et
Et
H
Et
Et
Et
Et
16
47
243 nM
154 nM
9.8 nM
30 nM
66 nM
46 nM
118 nM
111 nM
16%
22
20
H
20-MeO
20-Cl
30-Cl
30-Cl
30-Cl
30-Cl
30-Cl
30-Cl
40-Cl
40-Cl
40-Cl
40-Cl
40-Cl
40-Cl
40-Cl
40-Cl
40-Cl
40-Cl
40-Cl
30-F
H
H
H
H
H
H
F
5.4
53
27
H
64
H
0.49
0.40
6.7
2.1
9.07
72
26
H
31
H
12.5
46
H
H
F
H
Cl
Cl
H
F
117
97
1g
2a
2f
H
F
680 nM
1800 nM
0%
H
H
H
F
7.0
40
42
H
116
55
2g
1h
1i
H
H
H
F
25
6%
H
H
H
H
F
0.38
2.81
1.1
0.79
10
11%
93
H
9%
190
175
215
191
110
757
380
ND
14
1j
H
Cl
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
0%
1a
1k
1l
H
9%
H
F
0%
H
H
H
F
9.5
107
96
5%
NDb
11
1m
1n
2h
1o
1p
1q
1r
H
H
10%
H
H
H
H
H
H
H
11
73 nM
10%
40-F
40-F
30-CF3
20-Cl
30-Cl
H
5.0
0.43
5.1
139
0.60
H
10%
39
H
36 nM
11%
ND
380
125
40-Cl
40-Cl
11%
a Receptors and radioligands used in binding assays and data analysis: see Experimental Section. b Not determined.
affinity in quinazolinone-type agents.24 However, when studying
compounds di- (e.g., 1f, 1l) or especially trihalogenated (1g, 1m)
on the aromatic ring of the oxindole skeleton, a clear tendency of
diminished 5-HT7 receptor potency could be observed.
Selectivity toward the 5-HT1A Receptor. The examination
performed by Wilcox et al. demonstrated close similarities
between the 5-HT7 and 5-HT1A receptor binding sites that can
explain difficulties in developing selective ligands for either of the
above receptors.9 This finding is well exemplified by “long-chain
arylpiperazines”,26 an intensively investigated family of seroto-
nergic compounds, which, besides being 5-HT7 receptor agents,
have long been known to be strong 5-HT1A receptor ligands as
well, as reported by Leopoldo et al. and other research groups.7a,27
We now report our efforts in identifying a new series of 5-HT7
receptor ligands of arylpiperazine-type with good selectivity
toward the 5-HT1A receptor.
action as exemplified by buspirone, a marketed 5-HT1A receptor
partial agonist.28 On the contrary, inhibition of the 5-HT1A
receptor is anxiogenic in animal experiments, and anxiolytic
effects of some compounds, depending on the mechanism of
action, can be blocked by WAY 100635, a 5-HT1A receptor
antagonist.29 The picture is further complicated by results dem-
onstrating that the anxiolytic effect of 5-HT1A receptor ligand
anxiolytic drugs was modified by acute stress in mice, and
adrenalectomy turned the anxiolytic effect of 8-OH-DPAT to an
anxiogenic effect in rats.30 Therefore, the results produced by
5-HT1A receptor ligands in animal experiments may not be easily
extrapolated to humans.
Selectivity of various 3-(arylpiperazinylalkyl)oxindoles (type
1, 2) toward the 5-HT1A receptor was investigated in detail in our
recent paper.8 Previous studies revealed that the substitution
pattern of the phenylpiperazine ring strongly influenced the
5-HT1A receptor affinity.31 Therefore, we also aimed at varying
the substituents on the aromatic ring of the basic group.
Selectivity toward the 5-HT1A receptor was required, although
stimulation of this serotonin receptor may elicit an anxiolytic
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ARTICLE
Table 3. Oxindoles with Basic Groups Other Than
Phenylpiperazines
toward the 5-HT1A receptor (cp., e.g., 2aꢀ2b, 2bꢀh), contrary
to the literature data.31a 2,4-Dichloro (1q) and 3,4-dichloro (1r)
derivatives maintained the high selectivity of the 4-monochloro
analogue (1h) toward the 5-HT1A receptor, although in the
former case with lower 5-HT7 receptor affinity. A similarly low
5-HT1A receptor binding caused by 2,3-dichlorination of the
phenylpiperazine ring was also observed by Wustrow et al.31a
Alterations in receptor bindings caused by replacement of the
phenylpiperazine moiety with other basic groups were also
studied. Although 2-pyridylpiperazine derivatives were found
by other researchers to exhibit strong 5-HT1A receptor binding,31,36
our results demonstrated that the 5-HT7 and 5-HT1A receptor
affinities of the 2-pyridylpiperazine analogues were very similar
to those of the corresponding phenylpiperazine analogues (cp.,
2bꢀ2i). When the piperazine moiety was replaced with the
analogously substituted 1,2,3,6-tetrahydropyridine, the 5-HT1A
receptor affinity did not decrease and the 5-HT7/5-HT1A
selectivity did not improve either (cp., 1oꢀ1t, 1eꢀ1v).
Selectivity toward the α1-Adrenoceptor. Inhibitors of the
α1-AR play an important role in the treatment of benign prostatic
hyperplasia (BPH)37 and hypertension.38 Arylpiperazines are
one of the most studied classes of molecules exhibiting affinity for
the α1-AR.12 However, it was our aim to synthesize compounds
that exhibit good selectivity not only toward the 5-HT1A receptor
but also toward the α1-AR, thereby decreasing the risk of
cardiovascular side effects. Low affinity for the α1-AR is impor-
tant to avoid the potential hypotensive or hypertensive effects of
compounds due to blockade or activation of the α1-AR in the
vascular bed, respectively. Another reason to avoid α1-AR affinity
of our NCEs was to prevent interference with the assumed
antidepressant-like effect of 5-HT7 receptor antagonists via
inhibition of central α1-ARs. It has been shown for some
compounds such as lamotrigine13 and chlorpheniramine14 that
their antidepressant-like effect was antagonized by α1-AR in-
hibitors or potentiated by α1-AR activation in the tail suspension
or forced swimming tests in mice. On the other hand, the
antidepressant-like effect of imipramine was not altered by the
α1-AR antagonist prazosin in the same animal experiments.14
Therefore, we set a minimum of 50-fold selectivity ratio between
the 5-HT7 receptor and the α1-AR affinities for selecting
compounds for further development.
Data in Tables 2 and 3 demonstrate that the majority of our
derivatives, as expected, are also α1-AR ligands. The unsubsti-
tuted phenylpiperazine derivatives (2b,c) and the 4-fluorophenyl
compounds (1o, 1t,u, 2h) possessed high α1-AR binding.
Similarly, 3-chlorophenylpiperazine derivatives (1dꢀg, 2d,e),
which otherwise had strong 5-HT7 receptor affinity, and dis-
played partial selectivity toward the 5-HT1A receptor, had
stronger α1-AR than 5-HT1A receptor binding.
Although literature results showed that 2-methoxy substitu-
tion on the phenylpiperazine aromatic ring decreased the α1-AR
binding of compounds,32a other studies of Perrone et al.31b,39 and
other research groups36 demonstrated, in accordance with our
results with 1b, that these derivatives had high affinity not only
for the 5-HT7 and 5-HT1A receptors but also for the α1-AR.
2-Pyridylpiperazine derivatives (1s, 2i,j), known from the litera-
ture as moderate α1-AR ligands,36 also exhibited strong α1-AR
affinity in our hands.
a Receptors and radioligands used in binding assays and data analysis: see
Experimental Section. b Not determined.
According to the data in Tables 2 and 3, selectivity toward the
5-HT1A receptor was acceptable in most cases. Compounds 2b
and 2c containing an unsubstituted phenylpiperazine moiety
showed some selectivity, in accordance with the results of other
research groups.32 Derivative 1b exhibited a rather strong
5-HT1A receptor binding, due to the presence of the 2-methoxy-
phenylpiperazine moiety, which is a key fragment in many 5-HT1A
receptor ligands.32a,33 In our previous paper8 we demonstrated
that both the 3- and 4-methoxyphenyl derivatives had an affinity
for the 5-HT1A receptor weaker than that of the 2-methoxy
congener.34 Concerning the 3-trifluoromethyl moiety on the phenyl-
piperazine basic group, the literature data are contradictory: this
substituent favors 5-HT1A receptor binding in certain families of
compounds,35 while one paper reports low 5-HT1A receptor
affinity of these derivatives.31a Our observations supported the
former finding, i.e., strong 5-HT1A receptor affinity and poor
5-HT7/5-HT1A selectivity, independently of whether the 3-CF3
substituent was on a phenylpiperazine (1p) or on an aryl-1,2,3,6-
tetrahydropyridine (2k,l) moiety.
Bojarski et al. found that 3-chlorophenylpiperazine derivatives
had strong affinity for the 5-HT1A receptor in two different
compound families.32b,35b According to our measurements,
5-HT1A receptor bindingof3-chlorophenylpiperazine compounds
unsubstituted on the oxindole skeleton was moderate (1d, 2d),
and it could be further reduced with appropriate substitution
patterns (1eꢀg). When changing the position of the chlorine
atom to position 4, 5-HT1A receptor affinity decreased drama-
tically, while the 5-HT7 receptor binding was maintained. The
4-chloro and 4-fluoro substituents on the phenylpiperazine ring
positively influenced both 5-HT7 receptor binding and selectivity
In terms of low α1-AR binding, the 3-trifluoromethylphenyl-
1,2,3,6-tetrahydropyridine basic group in 2k and 2l proved to be
advantageous.40,41 However, these derivatives showed very strong
affinity for the 5-HT1A receptor, as discussed above. On the other
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Table 4. Microsomal Metabolic Stability and in Vivo Anxiolytic and Antidepressant Activity of Some Selective (upper portion of
table) and Nonselective (lower portion of table) Compounds
a
5-HT1A
conflict
forced
α1-ARa
ratio of
MF%b MF%b
drinking testc
lightꢀdark testd
swimming teste
a
5-HT7
Ki (nM) or
ratio of
compound Ki (nM) % at 10ꢀ7 M 5-HT1A vs 5-HT7 Ki (nM) α1 vs 5-HT7
rat
human MED (mg/kg) ip MED (mg/kg) ip MED (mg/kg) ip
1a
1d
1h
1i
0.79
0.40
0.38
2.81
1.1
9%
.100
115
215
31
272
78
9
4
29
31
34
36
23
34
29
39
NDf
>10
10
>10
>20
>10
>10
ND
<1
>10
>10
>10
>10
>10
>10
>10
>10
46 nM
11%
9%
.100
>100
.100
.100
.100
135
93
245
68
9
190
175
39
17
21
8
ND
>20
10
1j
0%
159
91
1o
1r
2d
0.43
0.60
0.49
10%
11%
66 nM
125
26
208
53
13
9
>20
<5
ND
>10
1f
9.07
16%
0%
>100
>100
>100
ND
117
191
110
380
380
42
13
19
12
4
16
18
76
72
17
10
28
46
10
53
76
28
52
44
ND
ND
5
ND
ND
1
ND
ND
>30
>30
ND
ND
ND
1k
1l
10
9.5
96
139
7.0
40
5%
1m
1q
2a
2f
10%
11%
1800 nM
0%
>20
ND
>10
ND
<1
ND
3
257
6
<2.5
ND
>100
116
3
ND
a Receptors and radioligands used in binding assays and data analysis: see Experimental Section. b In vitro microsomal metabolic stability. For the
protocol of its determination, see Experimental Section. c A modified version of the Vogel10 method was used for the conflict drinking test in rats, drugs
were administered intraperitoneally. For details, see Experimental Section. d Testing of drugs in the lightꢀdark test was carried out in mice as described
by Costall11 after intraperitoneal administration of the drugs. For details, see Experimental Section. e The forced swimming test of compounds
administered intraperitoneally was carried out in male NMRI mice according to the method of Lucki et al.20 For details, see Experimental Section. f Not
determined.
hand, 4-chlorophenylpiperazine derivatives exhibited substan-
tially lower α1-AR binding than the corresponding 3-chloro
analogues (cp., 1eꢀ1i, 1dꢀ1h). When the 3-unsubstituted and
the 3-ethyl series are compared, it can be stated that the presence
of the 3-ethyl substituent is favorable (cp., e.g., 1hꢀ2a). Halogen
substituents at positions 5, 6, or 7 of the oxindole carbocycle did
not significantly influence the α1-AR affinity. Taking into con-
sideration all the compounds in Tables 2 and 3, there are only a
few, namely 4-chlorophenylpiperazine compounds 1a, 1h, and
1r, that were more than 100-fold selective toward both the
5-HT1A receptor and α1-AR.
Lead Compound Selection and Further Optimization. For
a better understanding of the structureꢀmetabolic stability
relationship, eight selective 5-HT7 ligands (Table 4, upper
portion) and seven compounds not selective for the α1-AR
(Table 4, lower portion) were then tested for their metabolic
stability in rat and human liver microsomes, in vitro, for predic-
tion of their oral bioavailability. The anxiolytic activity of most
compounds was determined in the conflict drinking (Vogel)
test10 in rats and the lightꢀdark test11 in mice. The antidepres-
sant-like effect of the 5-HT7 receptor ligands was tested using the
forced swimming test (Porsolt test or FST) in mice.3e,22 It was
found that 5 out of 15 compounds showed anxiolytic activity in
the Vogel test. The most active compound, 2a, produced an
effect at 2.5 mg/kg ip, while 1l and 2d as well as 1h and 1o were
effective at 5 mg/kg ip and at 10 mg/kg ip, respectively. Among
these, 1l, 1o and 2a were also effective at low dose (1 mg/kg ip or
lower) in the lightꢀdark test in mice. Except for 1l, the com-
pounds exhibited poor metabolic stability prediction for humans;
thus, these compounds were not suitable for further development,
despite two of them (1h, 1o) having good receptor selectivity.
In terms of bioavailability, serious problems were described in
the literature also for tetrahydrobenzindolinones, a structurally
related family of compounds.42 Halogen substitution somewhat
increased the in vitro metabolic stability; however, the measured
value of bioavailability (18% in rats for compound DR4485) was
still quite low.43 We encountered the same difficulty when
investigating the oxindole family. Microsomal metabolic stability
tests, in vitro, demonstrated that the bioavailability prediction of
most oxindole derivatives for rats and humans was below 20%
and 40%, respectively, making these compounds hardly appro-
priate for further development (Table 4). Interestingly, the
3-unsubstituted derivatives were more stable than the corre-
sponding 3-ethyl analogues (cp., e.g., 1hꢀ2a, 1iꢀ2f). In an
attempt to improve metabolic stability we introduced halogen
substituents at different positions of the molecule. It became
obvious that halogenation of the aromatic ring of the aryl-
piperazine or aryltetrahydropyridine moieties did not result in
stable derivatives, not even in the case of compounds bearing
dichlorinated aromatic rings (1q, 1r). As a next step, the same
approach was applied to the oxindole carbocycle. The introduc-
tion of a fluorine atom at position 5 or 6, or a chlorine atom at
position 5 of the carbocycle, did not improve the stability. The
presence of a substituent at both positions simultaneously
(1f, 1k) also failed to increase metabolic stability, in vitro. On
the other hand, 5,7-dichloro (1l) and 5,7-dichloro-6-fluoro
(1m) substitutions led to metabolically stable compounds,
suggesting that blocking position 7 of the oxindole ring was
the key step for achieving metabolic stability. These two
compounds were selective toward the 5-HT1A receptor, but
only a low (4 to 12-fold) selectivity was observed toward the
α1-AR (Table 4).
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Journal of Medicinal Chemistry
ARTICLE
Table 5. Plasma and Brain Concentrations of 5-HT7 Selective
Compounds after ip Administration at Various Doses in Male
NMRI Mice (n = 3/dose)
not necessarily in contradiction, as these compounds can have
better microsomal metabolic stability in mice than in rats and
humans. Bloodꢀbrain barrier permeability (BBBP), expressed as
the percentage of brain to plasma concentrations, ranged from
about 0.7 to about 8, proving good brain penetration of the
compounds. Molar brain concentrations were above the 5-HT7
Ki values for all compounds even at the low dose tested (1 mg/
kg ip), and, except for 1j, they were a minimum of 100-fold
higher than the individual Ki values at the high dose adminis-
tered (10 mg/kg ip). On the basis of the above results, it can be
stated that free brain concentrations were likely above the level
necessary to produce sufficient 5-HT7 receptor occupancy.
Therefore, the lack of antidepressant effect of our oxindole
derivatives may not be explained with poor metabolic stability,
inappropriate pharmacokinetics, or insufficient bloodꢀbrain
barrier penetration.
dose
plasma concentration brain concentration
BBBP
(%)
(mg/kg ip)
(nM)
(nM)
1a
1d
1h
1i
1
46 ( 9
130 ( 11
177 ( 95
33 ( 3
77 ( 11
77 ( 5
79 ( 4
3
99 ( 3
10
135 ( 67
1
22 ( 13
168 ( 10
272 ( 144
65 ( 47
613 ( 60
244 ( 50
364 ( 14
266 ( 35
3
10
824 ( 523
’ CONCLUSION
1
70 ( 39
245 ( 133
399 ( 82
42 ( 7
141 ( 15
379 ( 78
89 ( 26
87 ( 28
95 ( 4
3
In our earlier study we reported on a new compound family,
(arylpiperazinylbutyl)oxindoles, which proved to be highly potent
5-HT7 receptor antagonists. The compounds were tested routinely
for their 5-HT1A receptor binding, and several selective 5-HT7
receptor agents were identified. Certain analogues (e.g., 2a) also
showed anxiolytic activity in in vivo animal models.
In the present paper we aimed at studying also the selectivity
toward the α1-AR and metabolic stability, by involving new,
structurally optimized derivatives, as well. Moreover, antidepres-
sant activity of selected compounds was now also tested in the
forced swimming test in mice.
10
1
44 ( 31
211 ( 157
271 ( 1
51 ( 19
186 ( 49
342 ( 79
188 ( 53
188 ( 69
177 ( 22
3
10
1j
1
10 ( 2
14 ( 4
8 ( 4
17 ( 1
23 ( 1
172 ( 24
191 ( 53
271 ( 62
3
10
27 ( 19
Our lead compound 2a was not selective toward the α1-AR,
and its metabolic stability was also insufficient. In the present
study, several compounds exhibiting strong affinity for the 5-HT7
receptor were selective toward the 5-HT1A receptor and in some
cases toward the α1-AR. We also successfully identified structural
key elements of metabolic stability within this family. Some of the
selective 5-HT7 receptor ligands were active in one or two anxio-
lytic tests, the Vogel conflict drinking test in rats and the lightꢀ
dark test in mice. Surprisingly, none of the tested compounds
produced an antidepressant-like action in the forced swimming
test in mice.
1o
1r
2d
1
34 ( 23
178 ( 14
168 ( 101
62 ( 34
431 ( 21
364 ( 191
226 ( 37
245 ( 22
233 ( 27
3
10
1
31 ( 19
174 ( 126
861 ( 390
43 ( 29
223 ( 170
1154 ( 537
128 ( 11
117 ( 10
125 ( 10
3
10
The current study greatly advanced the understanding of the
receptor binding and metabolic stability SAR in the (arylpipera-
zinylbutyl)oxindole family. As all compounds are chiral, enantio-
mer separation can be a further step in the hope of improving the
receptor selectivity and anxiolytic efficacy of our compounds.
1
21 ( 18
114 ( 97
744 ( 339
216 ( 173
827 ( 668
1310 ( 171
854 ( 77
615 ( 40
3
10
4311 ( 1924
Published data showed that the majority of 5-HT7 receptor
antagonists with various chemical structures possessed an anti-
depressant effect.44 Surprisingly, despite their high 5-HT7 receptor
affinity, none of the oxindole derivatives tested demonstrated a
significant antidepressant activity. Even compounds with sub-
nanomolar affinity for the 5-HT7 receptor failed to change the
immobility time in the mouse FST.
In order to check further if poor metabolic stability explains
the lack of antidepressant effect of the eight selective high-affinity
5-HT7 receptor ligands (1a, 1d, 1hꢀj, 1o, 1r, 2d), their plasma
and brain concentrations were measured 30 min after ip admin-
istrations at the doses used also in the FST (see results in
Table 5). The plasma and brain concentrations increased dose-
dependently in most cases and varied considerably among the
eight compounds. Poor microsomal bioavailability and relatively
high plasma and brain concentrations of some compounds are
’ EXPERIMENTAL SECTION
Chemistry. All melting points were determined on a B€uchi 535
capillary melting point apparatus and are uncorrected. IR spectra were
obtained on a Bruker Vector 22 FT spectrometer in KBr pellets. 1H and
13C NMR spectra were recorded in CDCl3 or DMSO-d6 on a Varian
Unity Inova 400 (400 and 100 MHz for H and 13C NMR spectra,
1
respectively) or 500 (500 and 125 MHz for 1H and 13C NMR spectra,
respectively) spectrometer using TMS as internal standard. Chemical
shifts (δ) and coupling constants (J) are given in ppm and in hertz (Hz),
respectively. Elemental analyses were performed on a Perkin-Elmer 2400
analyzer. Measured elemental analysis data were in the range of calculated
value ( 0.4%, except where the measured value is given specifically. All
reactions were followed by analytical thin layer chromatography (TLC)
on silica gel 60 F254. Hydrogenation reactions were carried out in an
autoclave (volume: 250 mL), which was equipped with a temperature
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Journal of Medicinal Chemistry
ARTICLE
controller, a manometer (60 bar), a valve for a gas inlet, and a magnetic
stirrer. Degussa’s activated Raney nickel catalyst in water was used as
hydrogenation catalyst. Synthetic procedures, yields, melting point,
spectral data, and elemental analyses of all new compounds are
described below. Purity of new compounds was above 95%, based
on 1H NMR.Compounds 1a,b, 1dꢀf, 1hꢀj, 1o, 1r, 1t,u, 2a, and 2c,d
were already described in our earlier paper,8 although with a smaller
set of biological data.
General Procedure A: Coupling Reaction of the 3-Ethyl-3-(4-chloro-
butyl)oxindole Intermediates (5 or 6) with the Appropriate Secondary
Amines. The melt of the secondary amine (12 mmol) was heated to
180 ꢀC under slow stirring. The appropriate 3-ethyl-3-(4-chlorobutyl)-
oxindole (5 or 6, 12 mmol) and sodium carbonate (1.36 g, 12 mmol)
were added. After 1 h reaction time, the brown melt was cooled to
ambient temperature. Ethyl acetate and water were added, and the layers
were separated. The organic layer was dried over MgSO4 and evapo-
rated. The residual oil or solid was purified by column chromatography
using ethyl acetate as eluent.
General Procedure A/1. In case the product of the chromatographic
purification crystallized upon treatment with diethyl ether, it was tri-
turated in this solvent and filtered, and the solid was recrystallized from
the solvent indicated below and dried in vacuo to give a white solid.
General Procedure A/2. In case the product of the chromatographic
purification did not crystallize upon treatment with diethyl ether, it was
dissolved in diethyl ether (200 mL), the solid residue was removed by
filtration, and a calculated amount (1 equiv) of hydrogen chloride (saturated
solution of HCl gas in diethyl ether) was added dropwise, under vigorous
stirring. The white precipitate was filtered, washed with diethyl ether and
hexane, and dried in vacuo. Where indicated, the obtained hydrochloric
salt was recrystallized from the solvent indicated to give a white solid.
5,7-Dichloro-3-{4-[4-(3-chlorophenyl)piperazin-1-yl]butyl}-3-ethyl-
6-fluoro-1,3-dihydro-2H-indol-2-one Hydrochloride (1g). The title com-
pound was prepared according to the general procedure A/2, starting
from 5,7-dichloro-3-(4-chlorobutyl)-3-ethyl-6-fluorooxindole (6e) and
1-(3-chlorophenyl)piperazine. Yield: 39%, mp 238ꢀ240 ꢀC (isopropyl
alcohol). IR(KBr): 3426, 1723 cmꢀ1. 1H NMR (DMSO-d6, 400 MHz) δ:
0.53 (3H, t, J = 7.3 Hz), 0.90ꢀ0.75 (1H, m), 1.00ꢀ0.90 (1H, m),
1.70ꢀ1.55 (2H, m), 1.90ꢀ1.70 (4H, m), 2.98 (4H, br s), 3.16 (2H, t, J =
12.0 Hz), 3.43 (2H, br s), 3.84 (2H, d, J = 12.6 Hz), 6.86 (1H, dd, J = 1.7,
7.9 Hz), 6.94 (1H, dd, J = 2.3, 8.4 Hz), 7.03 (1H, t, J = 2.0 Hz), 7.25 (1H, t,
J = 8.2 Hz), 7.58 (1H, d, J = 6.8 Hz), 10.9 (1H, br s), 11.3 (1H, s). 13C
NMR (DMSO-d6, 100 MHz) δ: 8.5, 21.3, 23.2, 30.2, 36.4, 45.0, 50.4, 54.6,
55.0, 103.2 (d, J = 22.5 Hz), 113.0 (d, J = 18.3 Hz), 114.3, 115.4, 119.4,
123.6, 129.6 (d, J = 3.8 Hz), 130.8, 134.1, 141.1, 151.0, 152.8 (d, J = 244.5
Hz), 180.6. Anal. (C24H28Cl4FN3O) C, H, N, Cl.
5,7-Dichloro-3-{4-[4-(4-chlorophenyl)piperazin-1-yl]butyl}-3-ethyl-
6-fluoro-1,3-dihydro-2H-indol-2-one (1m). The title compound was
prepared according to the general procedure A/1, starting from 5,7-
dichloro-3-(4-chlorobutyl)-3-ethyl-6-fluorooxindole (5c) and 1-(4-chloro-
phenyl)piperazine. Yield: 61%, mp 102ꢀ103 ꢀC (hexaneꢀethyl acetate).
IR (KBr): 2961, 1736 cmꢀ1. 1H NMR (DMSO-d6, 500 MHz) δ: 0.52
(3H, t, J = 7.4 Hz), 0.86 (1H, m), 0.95 (1H, m), 1.18 (1H, m), 1.29 (1H,
m), 1.74 (2H, m), 1.81 (2H, m), 2.17 (2H, m), 2.38 (t, 4H, J = 4.4 Hz),
3.04 (t, 4H, J = 4.4 Hz), 6.90 (d, 2H, J = 9.1 Hz), 7.21 (d, 2H, J = 9.0 Hz),
7.53 (d, 1H, J = 6.7 Hz), 11.22 (1H, s). 13C NMR (DMSO-d6, 125 MHz)
δ: 8.5, 21.7, 26.3, 30.4, 36.7, 48.1, 52.6, 54.8, 57.2, 103.1 (d, J = 22.5 Hz),
112.8 (d, J = 18.1 Hz), 116.9, 122.4, 123.5, 128.7, 129.9 (d, J = 3.9 Hz),
141.1 (d, J = 2.4 Hz), 150.0, 152.7 (d, J = 244.1 Hz), 180.8. Anal.
(C22H27Cl3FN3O) C, H, N, Cl.
1 h reaction time, the brown melt was cooled to ambient temperature.
Ethyl acetate and water were added, and the layers were separated. The
organic layer was dried over MgSO4 and evaporated. The residual oil or
solid was purified by column chromatography using ethyl acetate as eluent.
General Procedure B/1. In case the product of the chromatographic
purification crystallized upon treatment with diethyl ether, it was
triturated in this solvent and filtered, and the solid was recrystallized
from the solvent indicated below and dried in vacuo to give a white solid.
General Procedure B/2. In case the product of the chromatographic
purification did not crystallize upon treatment with diethyl ether, it was
dissolved in diethyl ether (200 mL), the solid residue was removed by
filtration, and a calculated amount (1 equiv) of hydrogen chloride
(saturated solution of HCl gas in diethyl ether) was added dropwise,
under vigorous stirring. The white precipitate was filtered, washed with
diethyl ether and hexane, and dried in vacuo to give a white solid.
General Procedure B/3. In case the product of the chromatographic
purification did not crystallize upon treatment with diethyl ether and it
did not give a crystalline hydrochloric acid salt in diethyl ether, it was
dissolved in ethyl acetate (100 mL) at 60ꢀ65 ꢀC and the solution of
oxalic acid dihydrate (1 equiv) in ethyl acetate (50 mL) was added
dropwise, keeping the temperature at 60ꢀ65 ꢀC during the addition.
After the addition, the suspension was cooled to ambient temperature,
and the white oxalic acid salt was filtered, washed with ethyl acetate, and
dried in vacuo.
3-[4-(4-Phenylpiperazin-1-yl)butyl]-1,3-dihydro-2H-indol-2-one (2b).
The title compound was prepared according to the general procedure
B/1, starting from 8a and 1-phenylpiperazine. Yield: 61%, mp 111ꢀ
113 ꢀC (hexaneꢀethyl acetate). IR (KBr): 3191, 1705 cmꢀ1. 1H NMR
(DMSO-d6, 500 MHz) δ: 1.31ꢀ1.24 (2H, m), 1.43 (2H, quintet, J =
7.2 Hz), 1.82ꢀ1.79 (1H, m), 1.91ꢀ1.81 (1H, m), 2.25 (2H, t, J = 7.3 Hz),
2.43 (4H, t, J = 5.0 Hz), 3.07 (4H, t, J = 4.9 Hz), 3.42 (1H, t, J = 4.9 Hz),
6.76 (1H, t, J = 7.2 Hz), 6.82 (1H, d, J = 7.7 Hz), 6.90 (2H, dd, J = 1.0,
7.8 Hz), 6.94 (1H, dt, J = 1.0, 7.6 Hz), 7.21ꢀ7.14 (3H, m), 7.24 (1H, d, J =
7.3 Hz), 10.35 (1H, s). 13C NMR (DMSO-d6, 125 MHz) δ: 23.3, 26.4,
29.9, 45.3, 48.3, 52.9, 57.8, 109.3, 115.4, 118.8, 121.3, 124.1, 127.7, 129.0,
129.9, 142.9, 151.2, 179.1. Anal. (C22H27N3O) C, H, N.
3-{4-[(3-Chlorophenyl)piperazin-1-yl]butyl}-6-fluoro-1,3-dihydro-
2H-indol-2-one Oxalate (2e). The title compound was prepared accord-
ing to the general procedure B/3, starting from 8c and 1-(3-chlorophe-
nyl)piperazine. Yield: 41%, mp 215ꢀ218 ꢀC. IR (KBr): 3256, 1712 cmꢀ1
.
1H NMR (DMSO-d6, 400 MHz) δ: 1.29ꢀ1.26 (2H, m), 1.65ꢀ1.58 (2H,
m), 1.89ꢀ1.80 (2H, m), 2.88 (2H, t, J = 7.9 Hz), 3.08 (4H, br s), 3.38
(4H, br s), 3.44 (1H, t, J = 5.4 Hz), 4.67 (2H, br s), 6.65 (1H, dd, J =
2.4, 9.1 Hz), 6.75 (1H, dt, J = 2.4, 9.1 Hz), 6.89 (1H, dd, J = 1.3, 7.8 Hz),
6.94 (1H, dd, J = 1.8, 8.4 Hz), 7.01 (1H, t, J = 2.1 Hz), 7.24 (1H, t, J =
8.2 Hz), 7.29ꢀ7.22 (1H, m), 10.5 (1H, s). 13C NMR (DMSO-d6,100MHz)
δ: 22.7, 24.0, 29.6, 44.6, 45.7, 51.0, 55.8, 97.6 (d, J = 27.1 Hz), 107.4 (d, J =
21.7 Hz), 114.2, 115.2, 119.1, 125.4 (d, J = 15.3 Hz), 125.5 (d, J = 7.4 Hz),
130.7, 134.1, 144.5 (d, J = 12.2 Hz), 151.4, 162.1 (d, J = 240.7 Hz), 164.4,
179.4. Anal. (C27H27ClFN3O5) C, H, N, Cl.
3-{4-[4-(4-Chlorophenyl)piperazin-1-yl]butyl}-5-fluoro-1,3-dihydro-
2H-indol-2-one Hydrochloride (2f). The title compound was prepared
according to the general procedure B/2, starting from 8b and 1-(4-
chlorophenyl)piperazine. Yield: 49%, mp 195ꢀ196 ꢀC (ethyl acetateꢀ
ethanol). IR (KBr): 3145, 1712 cmꢀ1. 1H NMR (DMSO-d6, 400 MHz)
δ: 1.32ꢀ1.22 (2H, m), 1.95ꢀ1.71 (4H, m), 3.20ꢀ3.06 (6H, m),
3.53ꢀ3.49 (3H, m), 3.80ꢀ3.76 (2H, m), 6.82 (1H, dd, J = 4.5, 8.5
Hz), 7.04ꢀ6.98 (1H, m), 7.01 (2H, d, J = 9.1 Hz), 7.21 (1H, dd, J = 2.1,
8.6 Hz), 7.28 (2H, d, J = 9.1 Hz), 10.5 (1H, s), 11.1 (1H, br s). 13C NMR
(DMSO-d6, 100 MHz) δ: 22.5, 23.1, 29.3, 42.6, 45.3, 45.4, 45.6 (d, J =
1.9 Hz), 50.5, 55.1, 110.0 (d, J = 8.4 Hz), 112.1 (d, J = 24.4 Hz), 114.0
(d, J = 23.3 Hz), 117.6, 117.7, 123.7, 129.0, 131.6 (d, J = 8.4 Hz), 139.2
(d, J = 1.9 Hz), 148.7, 149.1, 158.1 (d, J = 235.8 Hz), 178.8. Anal.
(C22H26Cl2FN3O) C: calcd, 60.28; found, 59.35; H, N, Cl.
General Procedure B: Coupling Reaction of the Methanesulfony-
loxybutyl Derivative (8) with the Appropriate Secondary Amines. The
melt of the secondary amine (12 mmol) was heated to 120 ꢀC under
slow stirring. The appropriate methanesulfonyloxybutyl derivative 8
(12 mmol) and sodium carbonate (1.36 g, 12 mmol) were added. After
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ARTICLE
5,7-Dichloro-3-(4-chlorobutyl)-3-ethyl-6-fluorooxindole (6e). 3-(4-
Chlorobutyl)-3-ethyl-6-fluorooxindole (5c, 5.39 g, 20 mmol)8 was
dissolved in glacial acetic acid (100 mL), and sulfuryl chloride (8 mL,
100 mmol) was added dropwise. The reaction mixture was heated to
60 ꢀC and stirred at this temperature for 2 h. The reaction mixture was
poured on ice (100 g). At room temperature, it was extracted with
diethyl ether (100 mL). The organic layer was extracted with water (3 ꢁ
50 mL), dried over MgSO4, and evaporated. The residue (7.80 g, 115%)
was triturated in hexane (30 mL) and filtered to give the title compound
as a pale yellow solid (4.40 g, 65%). The product was used without
further purification. An analytical sample was recrystallized from a
mixture of hexane and ethyl acetate. Mp 132ꢀ134 ꢀC. IR (KBr):
250 mL). It was flushed with nitrogen, charged with hydrogen (20 bar)
and stirred at room temperature. After 6 h reaction time, Raney nickel
(5 g, ca. 0.085 mol) was added again and the stirring (under 20 bar H2)
was continued for further 12 h. The mixture was filtered, and the filtrate
was evaporated. The residue was triturated in cold (ca. 5 ꢀC) diethyl ether
to give the title compound as a white solid (7.86 g, 79%). The compound
was used without further purification. An analytical sample was recrys-
tallized from a mixture of hexane and ethyl acetate. Mp 126ꢀ127 ꢀC. IR
1
(KBr): 3389, 1714 cmꢀ1. H NMR (DMSO-d6, 500 MHz) δ: 1.31
(2H, m), 1.66 (2H, m), 1.90 (2H, m), 3.14 (3H, s), 3.65 (1H, t, J =
5.6 Hz), 4.17 (2H, t, J = 6.4 Hz), 7.36 (1H, s), 7.40 (1H, s), 10.95 (1H, s).
13C NMR (DMSO-d6, 125 MHz) δ: 19.8, 27.2, 27.6, 35.4, 44.9, 69.0,
112.7, 121.9, 124.7, 125.8, 131.8, 138.4, 177.1. Anal. (C13H15Cl2NO4S)
C, H, N, Cl, S.
1
3143, 1718 cmꢀ1. H NMR (CDCl3, 400 MHz) δ: 0.66 (3H, t, J =
7.4 Hz), 1.14ꢀ1.00 (1H, m), 1.30ꢀ1.16 (1H, m), 1.80ꢀ1.60 (4H, m),
2.00ꢀ1.86 (2H, m), 3.43 (2H, t, J = 6.6 Hz), 7.05 (1H, dd, J = 0.5, 6.3
Hz), 8.22 (1H, s). 13C NMR (CDCl3, 100 MHz) δ: 8.5, 21.7, 31.0, 32.4,
36.7, 44.3, 55.4, 104.6 (d, J = 22.9 Hz), 115.3 (d, J = 18.7 Hz), 122.8,
128.5 (d, J = 4.2 Hz), 139.0, 153.6 (d, J = 249.1 Hz), 180.4. Anal.
(C14H15Cl3FNO) C, H, N, Cl.
5,7-Dichloro-3-{4-[4-(4-chlorophenyl)piperazin-1-yl]butyl}-1,3-di-
hydro-2H-indol-2-one (11). 4-(5,7-Dichloro-2-oxo-2,3-dihydro-1H-indol-
3-yl)butyl methanesulfonate (10, 1.23 g, 3.5 mmol) and 1-(4-chloro-
phenyl)piperazine (2.07 g, 10.5 mmol) were dissolved in acetonitrile
(20 mL), and the solvent was evaporated. The residual oil was stirred for
2 h at ambient temperature. The crude product was purified by column
chromatography using tolueneꢀethyl acetate (gradient from 50:1 to
4-(6-Fluoro-2-oxo-2,3-dihydro-1H-indol-3-yl)butyl Methanesulfonate
(8c). 6-Fluoro-3-(4-hydroxybutyl)-1,3-dihydro-2H-indol-2-one (7c, 1.11g,
5 mmol)16 and triethylamine (0.83 mL, 6 mmol) were dissolved in di-
chloromethane (25 mL). The reaction mixture was cooled to ca. ꢀ10 ꢀC,
and methanesulfonyl chloride (0.46 mL, 6 mmol), dissolved in dichloro-
methane (10 mL), was added dropwise over a period of 30 min. The
cooling bath was removed, and the reaction was stirred for 1 h at ambient
temperature. The solvent was removed in vacuo at room temperature, and
the residue was taken up in ethyl acetate (30 mL) and extracted with water
(30 mL). The organic layer was dried over MgSO4 and evaporated. The
residual oil was triturated in diisopropyl ether (20 mL) and filtered to give
the title compound as a pale yellow solid (1.26 g, 85%). The product was
used without further purification. An analytical sample was recrystallized
from a mixture of hexane and ethyl acetate to give a white solid. Mp
106ꢀ108 ꢀC. IR (KBr): 3161, 1705 cmꢀ1. 1H NMR (CDCl3, 500 MHz)
δ: 1.51ꢀ1.46 (2H, m), 1.78 (2H, quintet, J = 6.7 Hz), 2.00 (2H, q, J =
8.1 Hz), 2.99 (3H, s), 3.46 (1H, t, J = 5.9 Hz), 4.21 (2H, dt, J = 1.5, 6.5 Hz),
6.68 (1H, dd, J = 2.3, 8.8 Hz), 6.72 (1H, dt, J = 2.3, 8.9 Hz), 7.15 (1H, dd,
J = 5.4, 8.1 Hz), 9.15 (1H, br s). 13C NMR (CDCl3, 125 MHz) δ: 21.6,
28.9, 29.7, 37.3, 45.3, 69.5, 98.6 (d, J = 27.4 Hz), 108.7 (d, J = 22.5 Hz),
124.5 (d, J = 3.0 Hz), 124.9 (d, J = 9.5 Hz), 142.8 (d, J = 11.8 Hz), 162.6 (d,
J = 244.6 Hz), 180.7. Anal. (C13H16FNO4S) C, H, N, S.
4-(3,5,7-Trichloro-2-oxo-2,3-dihydro-1H-indol-3-yl)butyl Methane-
sulfonate (9). 4-(2-Oxo-2,3-dihydro-1H-indol-3-yl)butyl methanesulfo-
nate (8a, 5.0 g, 17.7 mmol) was added in portions into sulfuryl chloride
(20 mL, 0.248 mol) at room temperature, over a period of 10 min. The
reaction mixture was heated to reflux temperature. After 1 h reaction
time, sulfuryl chloride (10 mL, 0.124 mol) was added again, and this
procedure was repeated after 2 h reaction time. After 4 h reaction time,
the mixture was poured on ice (50 g) and extracted with ethyl acetate.
The organic layer was extracted with saturated NaHCO3 solution (2 ꢁ
100 mL), dried over MgSO4, and evaporated. The residual oil was
triturated in diisopropyl ether (30 mL) to give the title compound as a
yellow solid (5.48 g, 80%). The compound was used without further
purification. An analytical sample was recrystallized from a mixture of
hexane and ethyl acetate to give a white solid. Mp 108ꢀ110 ꢀC. IR
(KBr): 3274, 1739 cmꢀ1. 1H NMR (CDCl3, 500 MHz) δ: 1.37 (2H, m),
1.77 (2H, m), 2.29 (2H, m), 3.00 (3H, s), 4.19 (2H, t, J = 6.4 Hz), 7.28
(1H, d, J = 1.8 Hz), 7.33 (d, 1H, J = 1.8 Hz), 8.95 (1H, s). 13C NMR
(CDCl3, 125 MHz) δ: 20.3, 28.5, 37.3, 38.1, 64.8, 68.9, 116.4, 123.3,
129.1, 129.9, 131.8, 136.5, 174.4. Anal. (C13H14Cl3NO4S) C, H, N, Cl, S.
4-(5,7-Dichloro-2-oxo-2,3-dihydro-1H-indol-3-yl)butyl Methanesul-
fonate (10). 4-(3,5,7-Trichloro-2-oxo-2,3-dihydro-1H-indol-3-yl)butyl
methanesulfonate (9, 10.96 g, 28 mmol), THF (100 mL), and Raney
nickel (5 g, ca. 0.085 mol) were placed into an autoclave (volume
5:1) as eluent. Yield: 47%, mp 173ꢀ175 ꢀC. IR (KBr): 3148, 1714 cmꢀ1
.
1H NMR (DMSO-d6, 500 MHz) δ: 1.24 (2H, m), 1.42 (2H, m), 1.86
(1H, m), 1.93 (1H, m), 2.26 (2H, m), 2.43 (4H, t, J = 4.8 Hz), 3.08 (4H,
m), 3.63 (1H, t, J = 5.5 Hz), 6.91 (2H, d, J = 9.2 Hz), 7.21 (2H, d, J =
9.2 Hz), 7.33 (1H, d, J = 1.8 Hz), 7.39 (1H, d, J = 1.8 Hz), 10.91 (1H, s).
13C NMR (DMSO-d6, 125 MHz) δ: 22.9, 26.1, 29.2, 46.3, 48.1, 52.6,
57.4, 114.0, 116.8, 122.3, 123.2, 126.0, 127.1, 128.7, 133.3, 139.8, 150.0,
178.5. Anal. (C22H24Cl3N3O) C, H, N, Cl.
Pharmacology. Cell Culture. CHO cells stably expressing the 5-HT7A
receptor were cultured in 1:1 F12:DMEM (Sigma) supplemented with
1% (v/v) penicillinꢀstreptomycin (Sigma), 5% fetal calf serum (Gibco
BRL), and 2 mM L-glutamine (Sigma) at 37 ꢀC in a humidified atmo-
sphere with5%CO2. Cells were seededat30 000 cells per well in a 96-well
plate 24 h before treatment with compounds.
Radioligand Binding Assay and Data Analysis. In the case of 5-HT7
assays, we used cloned human serotonin receptor subtype 7 (h5-HT7)
produced in CHO cells (PerkinElmer), following the recommended
assay conditions using [3H]CT (PerkinElmer) as radioligand. Nonspe-
cific binding was determined in the presence of 25 μM clozapine
(Sigma). Binding assays were terminated by filtration over Whatman
GF/C glass-fiber filter (presoaked in 0.3% polyethylenimine for 30 min)
using a Brandel cell harvester, and bound radioactivity was measured
with a Packard TopCount scintillation spectrometer.
5-HT1A receptor binding assays were performed according to the
method of Peroutka and Snyder45 with minor modifications. 5-HT1A
membrane was prepared from adult rat frontal cortex obtained imme-
diately following decapitation. The wet weight was measured and the
tissue was homogenized (PotterꢀElvehjem) in 50 volumes of 50 mM
Tris-HCl buffer (pH 7.7 at 25 ꢀC). The tissue homogenate was centri-
fuged at 40 000 g for 15 min at 4 ꢀC, and the pellet was resuspended in
the same volume of the above buffer. The homogenate was incubated in
a shaking water bath for 10 min at 37 ꢀC. After a quick chilling on ice, the
homogenate was centrifuged (40 000 g, 15 min, 4 ꢀC), resuspended, and
centrifuged again (40 000 g, 15 min, 4 ꢀC). The membrane was finally
resuspended in 30-fold volume of 50 mM Tris-HCl buffer (pH 7.7). The
incubation buffer contained 50 mM Tris-HCl (pH 7.7), 6.66 mM CaCl2,
16.66 μM pargyline, and 0.166% ascorbic acid. 5-HT1A sites were labeled
with 1.5 nM [3H]8-OH-DPAT (Amersham). Nonspecific binding was
determined in the presence of 10 μM 5-hydroxytryptamineꢀcreatinine
sulfate complex (5-HT, Sigma). The samples were incubated for 45 min
at 25 ꢀC. Following the incubation, the samples were rapidly filtered over
Whatman GF/B glass fiber filter (presoaked in 0.05% polyethylenimine
for 30 min) and washed. Individual filters were inserted into vials,
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Journal of Medicinal Chemistry
ARTICLE
equilibrated for 6 h with Opti-Fluor scintillation fluid (PerkinElmer),
and counted with a Packard TopCount scintillation spectrometer.
α1-AR binding assays were performed in membrane prepared from
adult rat frontal cortex. The tissue was immediately frozen in dry ice and
thenweighed. The tissuewashomogenized(PotterꢀElvehjem) in 50-fold
volume of 50 mM Tris-HCl buffer (pH 7.7 at 25 ꢀC). The tissue
homogenate was centrifuged at 40 000 g for 15 min at 4 ꢀC, and the
pellet was twice resuspended in the same volume of buffer and
centrifuged. The membrane was finally resuspended in 20-fold volume
of 50 mM Tris-HCl buffer (pH 7.7). α1-AR sites were labeled with
0.6 nM [3H]prazosin (Amersham). Nonspecific binding was determined
in the presence of 10 μM prazosin (RBI). The samples were incubated
for 45 min at 25 ꢀC. Following the incubation, the samples were rapidly
filtered over Whatman GF/B glass fiber filter (presoaked in 0.05%
polyethylenimine for 2ꢀ3 h) and washed. Individual filters were inserted
into vials, equilibrated for 4 h with scintillation fluid (Fisons Chemicals),
and counted with a Packard TopCount scintillation spectrometer.
Membranes, radioligands, and varying concentrations of competitive
ligands were incubated in triplicates. Competition binding data were fit
to a single-site binding model using Prism GraphPad for Ki determina-
tion. Ki values were calculated from two displacement curves (nine
concentrations) in triplicate.
Prediction Measurement of Microsomal Metabolic Stability. The
compounds were incubated for 0, 5, 15, 30, and 60 min with rat and
human liver microsomes at 37 ꢀC. Incubation mixtures (the final volume
was 1 mL) consisted of 814 μL of 0.1 mM Tris-HCl buffer (pH 7.4), 100 μL
of 5 mM Mg2+, 36 μL of microsomes (0.33 mg/mL), and 50 μL of 2 μM
substrate (final concentration 100 nM). The test item was added after
dilution of a 1 mM stock solution (DMSO) to 2 μM in 0.1% BSA
solution. Reactions were started by the addition of 100 μL of 20 mM
NADPH and were terminated with ice-cold MeOH. The samples were
centrifuged, and the supernatants were transferred to autosampler vials.
Calibration solutions were prepared in the range of 0.5ꢀ50 nM by
diluting the 100 nM substrate solution with different volumes of BSA/
Tris and MeOH. The assay was performed using a HPLC-MS/MS
system consisting of a Waters Alliance 2795 HPLC and a Quattro Ultima
Platinum mass spectrometer. Appropriate assay methods were devel-
oped for all compounds tested. Ionization was performed in the positive
ion mode, and the drug was monitored in the multiple-reaction
monitoring (MRM) mode. The orifice and ring potentials were opti-
mized to the highest signal for all analytes. Clearance of the compound in
the test system was calculated by linear regression and was expressed as
(mL/min/g protein). Prediction of metabolic stability was calculated,
taking into account of the liver blood flow, liver weight, and body weight
of the species using our own formula.
Measurement of Plasma and Brain Concentrations in Mice. Male
NMRI mice weighing 25ꢀ30 g were treated ip with four compounds
simultaneously. Each dose of the cassette mixtures were administered as
a methyl cellulose (0.4%) suspension (n = 3/dose). 30 min after treat-
ment, blood was taken from the vena cava under isoflurane anesthesia
into Sarstedt (LH/1.3) tubes and centrifuged by a Heraeus Megafuge
1.0 R centrifuge (Kendro Laboratory Products GmbH, Osterode,
Germany) for 10 min (RCF: 1625 ꢁ g) at 4 ꢀC. After decapitation, the
brain was removed and weighed. The plasma and brain tissue was stored
at about ꢀ70 ꢀC until analysis. Upon thawing, the brain tissue was
homogenized with 4-fold volume of distilled water in a potter and mixed.
Calibration curves were prepared by adding variable amounts of test
compound mixtures into drug-free plasma and homogenate of brain
tissue. Both the plasma and homogenized brain tissue were extracted by
mixing in a vortex for 30 min using an acetonitrileꢀdistilled water
mixture (3:1 by volume). The mixtures were centrifuged for 15 min at
15 000 rpm, at 25 ꢀC. After centrifugation, the supernatants were mixed
with an equal amount of the mobile phase. The HPLC-MS/MS system
consisted of a Waters Alliance 2795 HPLC and a Quattro Ultima
Platinum mass spectrometer. Gradient chromatographic separation of
test compounds was achieved with 10 mM ammonium formate, 5%
acetonitrile, and 0.1% trifluoracetic acid in distilled water (mobile phase
A) and acetonitrile and 0.1% trifluoracetic acid (mobile phase B) on a
Purospher STAR RP-8 column (125 mm ꢁ 4 mm, 3 μm particle size). A
linear gradient from 100% A to 100% B was programmed between 0 and
10 min. Appropriate retention windows were set for each test com-
pound. Ionization was performed in the positive ion mode. The dwell
time was set to 200 ms, and the drugs were monitored in the multiple-
reaction monitoring (MRM) mode. The most abundant fragmentation
paths, m/z 384f146 and 384f188, were set for detection of 2d, m/z
412f160 and 412f114 for 1h, m/z 430f178 and 430f192 for 1i,
m/z 446f160 and 446f174 for 1r, m/z 396f160 and 396f174 for
1o, m/z 412f160 and 412f174 for 1d, m/z 430f178 and 430f192
for 1a and m/z 446f194 and 446f208 for 1j, respectively. The orifice
and ring potentials were optimized to the highest signal for the analytes.
Calibration curves were calculated from the analyte peak area ratios of
2ꢀ2 parallels of the calibration standards plotted against the nominal
concentration ratios of calibrator sample standard. Quadratic curve
fitting was done using 1/x as weighing factor.
Conflict Drinking (Vogel) Test in Rats. Experiments were performed
in a computer-operated system (LIIKOSYS, Experimetria, Hungary)
consisting of eight test chambers (20 cm ꢁ20 cm ꢁ20 cm Plexiglas
boxes), each of which was equipped with a drinking spout mounted at
appropriate height on the wall of the chamber and a metal grid floor for
delivering electric shocks. Male Wistar rats, weighing 160ꢀ180 g, were
deprived of drinking water for 48 h and fasted for 24 h prior to test. Test
and reference compounds or vehicle were administered ip 30 min prior to
test. All procedures were carried out in a quiet, air-conditioned room
between 07:30 and 13:00 h at an ambient temperature of 23 ꢀC. At the
beginning of the experiment, the animals were placed in the test chamber
where they had free access to drinking water for a 30 s grace period. After
that, electric shocks (600 μA, 0.6 s) were applied through the drinking
spout following every 20 licks during a 5-min test period.10 The number
of punished licks was recordedand storedin a computer. Means ( SEM of
tolerated shocks were calculated in each group, and statistical analysis of the
results was performed by one-way ANOVA, followed by Duncan’s test.
LightꢀDark Test in Mice. Test was performed in a room illuminated
with a 2-lx light source. An animal activity monitor equipped with six
two-compartment automated test chambers (Omnitech, Digiscan,
Model RXYZCM16) was used for all experiments. Each box consisted
of one dark and one lit compartment. Both areas measured 39 cm ꢁ
20 cm ꢁ 29 cm. Access between the two compartments was provided by
an 8 cm ꢁ 8 cm passage way. A 60 W white tungsten light bulb was used
to illuminate the lit area. Interruptions of the 32 infrared beams (16 at
2 cm and 16 at 8 cm height above the box floor) in both compartments
were automatically recorded by the Digiscan analyzer and transmitted to
a computer. Male NMRI mice, weighing 25ꢀ33 g, were used for the test.
Mice were kept in a dark room, treated ip 30 min prior to test and were
placed individually in the center of the lit area. Behavioral activity was
detected for 5 min. Time spent in each area, number of transitions, and
horizontal and vertical activities were recorded.11 Means ( SEM values
were calculated, and the statistical analysis of data was performed by one-
way ANOVA, followed by Duncan’s test.
Forced Swimming Test (Porsolt) in Mice. Adult male NMRI mice
(n = 10ꢀ12/group, weight range: 23ꢀ30 g, Charles River, Hungary)
were used. The animals were introduced into the testing room 1 h before
the test. The compounds were dissolved in 0.9% physiological saline or
were suspended in 0.4% methylcellulose. All drugs were administered ip
30 min prior to the test. Control animals received vehicle only. The test
was carried out according to the method of Lucki et al.20 The animals
were put into 17.7 cm high and 12 cm diameter transparent glass
cylinders filled up with 25 ꢀC water up to 12.5 cm. Four mice were tested
simultaneously. The duration of immobility was measured during the
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last 4 min of the 6 min testing period. Each mouse was judged immobile
when it ceased to struggle and floated motionless in the water, making
only those movements necessary to keep its head above the water
surface. Mean and SEM values and the statistical significance between
groups were determined with one-way ANOVA followed by Dunnett's
posthoc test using GraphPad Software GraphPad Prism 4.0 programs.
5-HT7 receptor antagonist SB 269970 in animal models of anxiety and
depression. Neuropharmacology 2006, 51, 578–586. (f) Guscott, M.;
Bristow, L. J.; Hadingham, K.; Rosahl, T. W.; Beer, M. S.; Stanton, J. A.;
Bromidge, F.; Owens, A. P.; Huscroft, I.; Myers, J.; Rupniak, N. M.;
Patel, S.; Whiting, P. J.; Hutson, P. H.; Fone, K. C.; Biello, S. M.;
Kulagowski, J. J.; McAllister, G. Genetic knockout and pharmacological
blockade studies of the 5-HT7 receptor suggest therapeutic potential in
depression. Neuropharmacology 2005, 48, 492–502.
(4) (a) Lꢀopez-Rodríguez, M. L.; Benhamꢀu, B.; Morcillo, M. J.;
Porras, E.; Lavandera, J. L. Pardo. Serotonin 5-HT7 receptor antagonists.
Curr. Med. Chem. 2004, 4, 203–214. (b) Mullins, U. L.; Gianutsos, G.;
Eison, A. S. Effect of the antidepressants on 5-HT7 receptor regulation in
the rat hypothalamus. Neuropsychopharmacology 1999, 21, 352–367.
(5) Galici, R.; Boggs, J. D.; Miller, K. L.; Bonaventure, P.; Atack, J. R.
Effects of SB-269970, a 5-HT7 receptor antagonist, in mouse models
predictive of antipsychotic-like activity. Behav. Pharmacol. 2008, 19,
153–159.
’ ASSOCIATED CONTENT
S
Supporting Information. Preparation and characteriza-
b
tion of compounds 1c, 1n, 1p, 1s, 1v, 2gꢀm. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*(B.V.) Phone: +36 1 2655874, fax: +36 1 2655613, e-mail: volk.
balazs@egis.hu; (G.S.) phone: +36 1 4014217, fax: +36 1 4014205,
e-mail: szenasi.gabor@egis.hu.
(6) (a) Meneses, A.; Terrꢀon, J. A. Role of 5-HT1A and 5-HT7
receptors in the facilitatory response induced by 8-OH-DPAT on
learning consolidation. Behav. Brain Res. 2001, 21, 21–28. (b) Roth,
B. L.; Craigo, S. C.; Choudharmy, M. S.; Uluer, A.; Monsma, J. F.; Shen,
Y.; Meltzer, Y. H., Jr.; Sibley, R. D. Binding of typical and atypical
antipsychotic agents to 5-hydroxytryptamine-6 and 5-hydroxytrypta-
mine-7 receptors. J. Pharmacol. Exp. Ther. 1994, 268, 1403–1410.
(7) (a) Leopoldo, M. Serotonin7 receptors (5-HT7Rs) and their
ligands. Curr. Med. Chem. 2004, 11, 629–661. (b) Pittalꢁa, V.; Salerno, L.;
Modica, M.; Siracusa, M. A.; Romeo, G. 5-HT7 Receptor ligands: Recent
developments and potential therapeutic applications. Mini-Rev. Med.
Chem. 2007, 7, 945–960. (c) Bojarski, A. J. Pharmacophore models for
metabotropic 5-HT receptor ligands. Curr. Top. Med. Chem. 2006,
6, 2005–2026. (d) Leopoldo, M.; Lacivita, E.; Berardi, F.; Perrone, R.
5-HT7 receptor modulators: a medicinal chemistry survey of recent
patent literature (2004ꢀ2009). Exp. Opin. Ther. Pat. 2010, 20, 739–754.
(e) Mnie-Filali, O.; Lambas-Senas, L.; Zimmer, L.; Haddjeri, N. 5-HT7
receptor antagonists as a new class of antidepressants. Drug News
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’ ACKNOWLEDGMENT
The authors are grateful to Dr. Rita Kapiller-Dezs€ofi for NMR
measurements and helpful discussions. We also thank Ms. Krisztina
Horvꢀath and Mr. Kꢀaroly Lozsi for technical assistance.
’ ABBREVIATIONS USED
5-HT, 5-hydroxytryptamine; SSRI, selective serotonin reuptake
inhibitor; NCE, new chemical entity; AR, adrenoceptor; MED,
minimum effective dose; BuLi, n-butyllithium; equiv, equivalent;
RaꢀNi, Raney nickel; THF, tetrahydrofuran; AcOH, acetic acid;
rt, room temperature; NBS, N-bromosuccinimide; 8-OH-DPAT,
8-hydroxy-2-(di-n-propylamino)tetralin; BPH, benign prostatic
hyperplasia; MF, in vitro microsomal metabolic stability; ip,
intraperitoneal(ly); FST, forced swimming test; BBBP, bloodꢀ
brain barrier permeability; SAR, structureꢀactivity relationship;
DMSO, dimethyl sulfoxide; TMS, tetramethylsilane; CHO, Chi-
nese hamster ovary; F12:DMEM, Dulbecco’s Modified Eagle’s
Medium with Ham’s F-12; 5-CT, 5-carboxytryptamine; cAMP,
cyclic adenosine monophosphate; Tris, tris(hydroxymethyl)amino-
methane; BSA, bovine serum albumin; NADPH, nicotinamide
adenine dinucleotide phosphate;SEM, standard error of the mean;
SNRI, serotoninꢀnorepinephrine reuptake inhibitor
00
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Spedding, M.; Simig, G. (Phenylpiperazinyl-butyl)oxindoles as selective
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