New pyridobenzoxazepine derivatives derived from 5-(4-methylpiperazin-1-yl) -8-chloro-pyrido[2,3-b ][1,5]benzoxazepine (JL13): Chemical synthesis and pharmacological evaluation

Article abstract of DOI:10.1021/jm2013419

A series of new pyridobenzoxazepine derivatives with various heterocyclic amine side chains were synthesized to explore two main parameters related to the distal basic nitrogen. These compounds were tested for their affinity for dopamine D_2L and D₄, serotonin 5-HT_1A and 5-HT_2A, and adrenergic α_2A receptors in comparison with 5-(4-methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine, JL13 (1), and other diarylazepine derivatives. In terms of multireceptor target strategy, 2 and 5 present the most promising in vitro binding profile. Bulky, polar, and more flexible side chains are not favorable in this context. Compounds 2 and 5 were tested in adult rats to evaluate their long-term effects on dopamine and serotonin receptors density in different brain areas. Similar to 1 and other second-generation antipsychotic drugs, repeated treatment with 2 significantly increased D₁ and D₄ receptors in nucleus accumbens and caudate putamen and D₂ receptors in medial prefrontal cortex and hippocampus, while 5 significantly increased D₂ and D ₄ receptors in nucleus accumbens. In addition, 2 increased 5-HT _1A and decreased 5-HT_2A receptors in cerebral cortex. In contrast, 5 did not alter levels of any 5-HT receptor subtype in any brain region examined. These results encourage further development of 2 as a novel second-generation antipsychotic agent.

Full text of DOI:10.1021/jm2013419

Article
pubs.acs.org/jmc
New Pyridobenzoxazepine Derivatives Derived from 5-(4-
Methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]benzoxazepine
(JL13): Chemical Synthesis and Pharmacological Evaluation
Jean-Franco
Melissa Resimont,† and Frank I. Tarazi‡
̧
is Liegeois,*,† Marine Deville,† Sebastien Dilly,† Cedric Lamy,† Floriane Mangin,†
́ ́ ́
́
́
†Laboratory of Medicinal Chemistry, Drug Research Center, University of Lieg
Belgium
̀ ̂ ̀
e, avenue de l’Hopital 1 (B36), B-4000 Liege 1,
‡Department of Psychiatry and Neuroscience Program, Harvard Medical School and McLean Hospital, Boston, Massachusetts,
United States
S
* Supporting Information
ABSTRACT: A series of new pyridobenzoxazepine derivatives with various
heterocyclic amine side chains were synthesized to explore two main
parameters related to the distal basic nitrogen. These compounds were tested
for their affinity for dopamine D2L and D4, serotonin 5-HT1A and 5-HT2A, and
adrenergic α2A receptors in comparison with 5-(4-methylpiperazin-1-yl)-8-
chloro-pyrido[2,3-b][1,5]benzoxazepine, JL13 (1), and other diarylazepine
derivatives. In terms of multireceptor target strategy, 2 and 5 present the most
promising in vitro binding profile. Bulky, polar, and more flexible side chains
are not favorable in this context. Compounds 2 and 5 were tested in adult rats
to evaluate their long-term effects on dopamine and serotonin receptors density in different brain areas. Similar to 1 and other
second-generation antipsychotic drugs, repeated treatment with 2 significantly increased D1 and D4 receptors in nucleus
accumbens and caudate putamen and D2 receptors in medial prefrontal cortex and hippocampus, while 5 significantly increased
D2 and D4 receptors in nucleus accumbens. In addition, 2 increased 5-HT1A and decreased 5-HT2A receptors in cerebral cortex.
In contrast, 5 did not alter levels of any 5-HT receptor subtype in any brain region examined. These results encourage further
development of 2 as a novel second-generation antipsychotic agent.
produces comparable effect to SGAs on dopamine release in
the prefrontal cortex,14 significantly reduces the conditioned
avoidance response in rats,15 and reverses catalepsy induced by
haloperidol in rats.16 Thus, selective or mixed ligands for these
receptors (5-HT1A and D4 receptors and α2A-adrenoceptors)
would be of great interest. Multireceptor target strategy was put
forward as an innovative approach for discovering new drug
candidates.9,17,18
5-(4-Methylpiperazin-1-yl)-8-chloro-pyrido[2,3-b][1,5]-
benzoxazepine, JL13 (Figure 1), tested as fumarate in the
studies reported by our group (see below), is a clozapine-like
compound that shares with clozapine its high affinity for 5-
HT2A over D2 receptors.19 Neurochemical studies reported that
JL13 (1), like clozapine and other SGAs, selectively increased
dopamine (DA) levels in prefrontal cortex in a dose-dependent
fashion without altering concentrations of dopamine or its
metabolites in striatum.20 Behavioral studies suggested that 1
might possess antipsychotic activity, evidenced by its ability to
reverse amphetamine-induced disruption of prepulse inhib-
ition21 and antagonize apomorphine-induced climbing. In
addition, 1 also appears to exhibit a benign neurological
INTRODUCTION
■
The hypothesis of higher affinity for serotonin 5-HT2A
receptors versus dopamine D2 receptors1 led to the develop-
ment of the second generation of antipsychotic drugs (SGAs).
Other receptors such as dopamine D4 receptors2−6 or more
recently glutamate receptors7,8 were targets for extensive
pharmaceutical development programs. Importantly, the
beneficial combination of several neuronal interactions among
different neurotransmitter systems have been raised by in vivo
microdialysis and radioligand binding studies.9 Thus, for
instance, serotonin 5-HT1A partial agonism is likely involved
in the ability of clozapine and other SGAs to increase dopamine
release in several important brain areas such as hippocampus10
and prefrontal cortex.11,12 The highest density of 5-HT1A
receptors in rat brain is found in the hippocampus followed
by the medial prefrontal cortex. The interaction of dissimilar
SGAs with 5-HT1A receptors leading to the modulation of
external dopamine concentrations has been proposed to be
beneficial for improving negative symptoms and cognitive
deficits in patients with schizophrenia.13 Alternatively, α2-
adrenergic receptor antagonism has also been proposed as an
explanation for the unique antipsychotic properties of
clozapine. Therefore, the combination of a D2 antagonist
such as raclopride and an α2-antagonist such as idazoxan also
Received: October 7, 2011
Published: January 23, 2012
© 2012 American Chemical Society
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In the present work, we were interested in preparing several
compounds related to 1 by changing the nature and the size of
the lateral piperazine side chain in order to explore two main
parameters related to the pharmacophoric distal nitrogen,
namely, the ionization and the spatial position. Bulky, aromatic,
more flexible, and also polar groups were selected for this
purpose. Otherwise, in the diarylazepine series, N-dealkylated
analogues are usually produced by metabolism and sometimes
used in therapy like amoxapine. Thus, we have synthesized the
N-dealkylated analogue of 1. These compounds were tested to
evaluate the impact on the affinity for several receptors involved
in the mechanism of action of SGAs. Moreover, two
compounds showing an interesting in vitro binding profile
were also tested in long-term studies in rats to evaluate their
effects on the density of dopamine and serotonin receptors in
different brain areas, and the results were compared with those
of 1 from previous studies.24,25
CHEMISTRY
Figure 1. Chemical structure of reference diarylazepine derivatives.
■
Two chemical pathways were followed for preparing target
compounds (Scheme 1). Procedure 1 was used for preparing
oxazepine analogues30 and was the best choice for preparing the
new analogues with a hydroxyl side chain (6, 7). In this
approach, the lactam reacted with phosphorus oxychloride to
give the iminochloride. This reactive entity is immediately used
in the next step to give the appropriate amidine by reaction
with an excess of the appropriate amine. Procedure 2 was
advantageously used for the synthesis of pyridobenzodiazepine
analogues31,32 according to a modified Fryer amidine syn-
thesis33 and was also followed for preparing other oxazepine
analogues (1−5, 8−11) in this work due to a reduced reaction
time. Indeed, the appropriate lactam was reacted with an excess
of amine and titanium tetrachloride in refluxing toluene. All
profile, evidenced by its lack of ability to antagonize
amphetamine-induced stereotypy or produce catalepsy in
rats,22 as well as its low incidence of adverse side effects in
non-human primates.23 In more recent studies, the long-term
effects of 1 on DA (D1, D2, D3, and D4) receptors and
serotonin (5-HT1A and 5-HT2A) receptors subtypes in rat
forebrain regions was examined, and it was found that this
compound induced regionally selective changes in tissue levels
of specific DA and 5-HT receptor subtypes mimicking those
observed with clozapine and other SGAs.24 The long-term
effects of 1 on glutamatergic receptors (NMDA, AMPA) were
also investigated, and the compound had comparable effects to
SGAs.25
Interestingly, from a chemical point of view, pyridobenzox-
azepine structure allowed maintenance of similar physicochem-
ical properties, such as lipophilicity, to clozapine but also led to
less sensitivity to oxidative phenomena.26 Indeed, clozapine has
a low oxidation potential leading to a high sensitivity to
oxidative conditions.26−28 More recently, another consequence
of this sensitivity to oxidation has been reported. Unlike
oxazepine analogues (loxapine, 1), the clozapine nitrenium ions
generated in oxidative conditions possess a lower affinity for D2
receptors.29
1
target compounds were subjected to H and 13C NMR and
elemental analysis before biological testing as criteria for their
chemical structure or purity.
RESULTS
■
In Vitro Binding Experiments. The affinity of these
original compounds on D4, 5-HT1A, 5-HT2A, and α2A receptors
is reported in Tables 1 and 2. The screening evaluation of all
original compounds indicated that they have low affinity at D2L
receptors, evidenced by their inability to show significant
a
Scheme 1
a
Reagents and conditions: (i) POCl3, reflux; (ii) amine A, toluene, reflux; (iii) amine B, anisole, TiCl4, toluene, reflux. Amine A: R = piperazine, 1-
phenylpiperazine, 1-benzylpiperazine, 1-(2-phenylethyl)piperazine, 1-methyl-4-aminopiperidine, 1-benzyl-4-aminopiperidine, 1-(2-aminoethyl)-
piperidine, or 4-(2-aminoethyl)morpholine. Amine B: R = 1-(2-hydroxyethyl)piperazine or 1-[2-(2-hydroxyethoxy)ethyl]piperazine.
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Table 1. In Vitro Binding Evaluation of New
Table 2. In Vitro Binding Evaluation of New
Pyridobenzoxazepine Analogues with Modified Group on
the Distal Basic Nitrogen of the Piperazine in Comparison
Pyridobenzoxazepine Analogues with Piperidine or More
Flexible Side Chains in Comparison with Reference
a
a
with Reference Compounds
Compounds
a
Binding affinity is expressed in Ki, mean SD, n ≥ 3 (except for
amoxapine n = 2), or in percentage of displacement at 10−6 M.
Table 3. Physicochemical Parameters of
Pyridobenzoxazepine Derivatives and Reference
Compounds
a
Binding affinity is expressed in Ki, mean SD, n ≥ 3 (except for
a
amoxapine n = 2), or in percentage of displacement at 10−6 M.
compounds
pKa
log kI′AM
2
8.16
6.51
7.03
7.38
6.8
2.68
4.16
4.45
4.34
2.46
2.55
2.98
4.63
2.63
3.33
2.95
3.55
3.23
3.17
2.84
percentage of displacement at 10−6 M, and they were not tested
further (data not shown).
4
5
Impact of the Nature of the Side Chain on the Affinity for
D4 Receptors. In comparison with 1, the affinity for D4
receptors is reduced when the compound is dealkylated (2).
Similarly, this is observed with amoxapine versus loxapine. The
affinity is increased 2-fold when the methyl group is replaced by
a phenethyl side chain (5). The benzyl analogue (4) is less
potent on these receptor sites. The phenyl analogue (3) has no
affinity, while the cyclohexyl derivative (6) presents a weak
affinity. All other substitutions gave compounds with low or no
significant affinity for these sites. Otherwise, compounds with a
polar side chain (7, 8) and quetiapine and compounds with a
more flexible side chain (9−12) possess less affinity in
comparison with reference compounds.
6
7
8
6.24
8.88
8.11
6.24
8.86
7.48
7.51
7.52
8.36
6.41
9
10
11
12
1
clozapine
loxapine
amoxapine
quetiapine
Predicted by the SPARC online calculator.58
a
Impact of the Nature of the Side Chain on the Affinity for
5-HT1A Receptors. Compound 2 has a higher affinity for these
sites than the parent compound 1. This is also observed with
amoxapine and loxapine. The affinity is also increased when the
side chain is a benzyl (4), phenethyl (5), or cyclohexyl (6)
group. The phenethyl analogue (5) possesses a higher affinity
for these sites than 1 and other reference compounds.
Compounds with a phenyl ring (3), a polar side chain (7, 8),
or a more flexible side chain (11, 12) have no significant affinity
for these sites.
parent compound 1. In the piperazine series, the cyclohexyl (6)
and the phenethyl (5) analogues also possess a similar affinity
to 2. Quetiapine has also an affinity close to these molecules.
Other reference compounds possess a higher affinity for these
sites. In the piperidine series, N-methyl (9) and N-benzyl (10)
analogues have an affinity close to that of 1. Compounds with a
phenyl ring (3), a polar side chain (7, 8), or a more flexible side
chain (11, 12) have no significant affinity for these sites.
Impact of the Nature of the Side Chain on the Affinity for
α2A Receptors. Compound 1 has an affinity similar to that of
Impact of the Nature of the Side Chain on the Affinity for
5-HT2A Receptors. Compound 2 has a higher affinity than the
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a
Table 4. D1 Receptor Binding in Rats after 4 Weeks of Daily Injections of Compounds 2 and 5
b
brain region
cerebral cortex
controls
2
5
1
medial-prefrontal
39.8 2.4 (100)
26.3 1.2 (100)
154.4 7.7 (100)
38.6 1.2 (97)
25.1 2.6 (95)
39.2 2.9 (98)
23.7 1.7 (90)
156.0 1.8 (101)
(107)
(95)
dorsolateral frontal
nucleus accumbens
198.0 6.5 (128)*
(126)*
caudate putamen
medial
149.4 8.3 (100)
150.4 6.8 (100)
18.4 3.5 (100)
28.3 3.1 (100)
195.8 6.9 (131)*
199.1 7.7 (132)*
19.4 1.8 (105)
27.1 2.5 (96)
152.2 1.6 (102)
146.0 4.6 (97)
21.0 2.5 (114)
25.3 1.1 (89)
(124)*
(122)*
(106)
(97)
lateral
hippocampus
entorhinal cortex
a
Data are mean SEM values (N = 8 rats/group) for binding in fmol/mg tissue and (% of control given in parentheses) following daily ip injections
b
of vehicle, compounds 2 or 5 for 28 days, with significant differences from controls indicated in bold (∗ indicates p < 0.001). Data (% of control)
for 1 (10 mg/(kg·d)) in adult animals reported previously24 and shown for comparison.
a
Table 5. D2 Receptor Binding in Rats after 4 Weeks of Daily Injections of Compounds 2 and 5
b
brain region
cerebral cortex
controls
2
5
1
medial-prefrontal
25.1 1.8 (100)
20.2 1.7 (100)
100.7 7.2 (100)
35.4 0.6 (141)*
20.9 0.8 (103)
25.2 0.7 (100)
21.6 0.9 (107)
153.5 4.0 (152)*
(132)*
(93)
dorsolateral frontal
nucleus accumbens
141.0 8.9 (140)*
(108)
caudate putamen
medial
143.3 8.1 (100)
198.8 8.5 (100)
44.9 2.9 (100)
22.5 4.3 (100)
157.9 6.2 (110)
195.3 7.0 (98)
60.0 1.5 (134)*
21.5 3.6 (96)
156.4 4.8 (109)
202.9 3.9 (102)
46.3 2.7 (103)
23.8 2.5 (106)
(106)
(103)
(119)*
(110)
lateral
hippocampus
entorhinal cortex
a
Data are mean SEM values (N = 8 rats/group) for binding in fmol/mg tissue (% of control is given in parentheses) following daily ip injections
b
of vehicle or compounds 2 or 5 for 28 days, with significant differences from controls indicated in bold (∗ indicates p < 0.05). Data (% of control)
for 1 (10 mg/(kg·d)) in adult animals reported previously24 and shown for comparison.
a
Table 6. D4 Receptor Binding in Rats after 4 Weeks of Daily Injections of Compounds 2 and 5
b
brain region
cerebral cortex
controls
2
5
1
medial-prefrontal
18.4 0.7 (100)
19.7 0.9 (100)
23.2 3.7 (100)
20.3 0.7 (110)
21.3 0.6 (108)
41.2 3.3 (178)*
17.1 0.8 (93)
20.6 1.5 (105)
52.4 1.2 (225)*
(111)
(108)
(161)*
dorsolateral frontal
nucleus accumbens
caudate putamen
medial
24.1 1.7 (100)
33.0 1.6 (100)
22.7 0.5 (100)
12.2 1.0 (100)
39.9 1.3 (166)*
50.1 1.8 (152)*
26.6 2.8 (117)
12.6 1.3 (103)
21.6 0.7 (89)
28.7 0.5 (87)
23.5 1.5 (104)
13.2 1.2 (108)
(152)*
(140)*
(157)*
(92)
lateral
hippocampus
entorhinal cortex
a
Data are mean SEM values (N = 8 rats/group) for binding in fmol/mg tissue (% of control is given in parentheses) following daily ip injections
b
of vehicle or compounds 2 or 5 for 28 days, with significant differences from controls indicated in bold (∗ indicates p < 0.001). Data (% of control)
for 1 (10 mg/(kg·d)) in adult animals reported previously24 and shown for comparison.
clozapine. The dealkylated analogue (2) has a 4-fold lower
affinity than the parent compound, while phenethyl (5),
cyclohexyl (6), and hydroxyethoxyethyl (8) analogues have a 2-
fold lower affinity. Compounds with a polar side chain (7, 8)
have a low affinity for these sites. 4-Aminopiperidine analogues
(9, 10) have no significant affinity for these sites.
Long-Term Effects of N-Dealkylated Analogue 2 and
N-Phenethyl Analogue 5 on the Density of Dopaminer-
gic and Serotonergic Receptors in Rat Brain. Effects on
Dopaminergic Receptors. Repeated treatment with 2 increased
D1 receptor levels in nucleus accumbens (by 28%), medial
caudate putamen (31%), and lateral caudate putamen (32%)
[all p < 0.001, Table 4]. In contrast, repeated treatment with 5
did not alter levels of D1 receptors in all brain regions examined
(Table 4). Treatment with 2 also increased abundance of D2
receptors in medial prefrontal cortex (41%), nucleus
accumbens (40%), and hippocampus (34%) [p < 0.05, Table
5]. Treatment with 5 selectively increased levels of D2 receptors
in nucleus accumbens (52%) but not in other brain regions
(Table 5). DA D4 receptors were also altered after repeated
treatment with both agents. Compound 2 increased D4
receptors in nucleus accumbens (78%), medial caudate
putamen (66%), and lateral caudate putamen (52%), whereas
5 profoundly increased D4 receptors in nucleus accumbens (by
125%) [all p < 0.05, Table 6].
Effects on Serotonergic Receptors. Long-term administra-
tion of 2 increased 5-HT1A receptors in medial prefrontal cortex
(37%) and dorsolateral frontal cortex (41%) [p < 0.001, Table
7]. In addition, 2 selectively decreased 5-HT2A receptors in
medial prefrontal cortex (by 44%) and dorsolateral frontal
cortex (47%) [p < 0.001, Table 8]. Long-term administration of
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a
Table 7. 5-HT1A Receptor Binding in Rats after 4 Weeks of Daily Injections of Compounds 2 and 5
b
brain region
cerebral cortex
controls
2
5
1
medial-prefrontal
42.2 1.2 (100)
38.2 1.5 (100)
4.0 0.7 (100)
57.9 1.9 (137)*
53.7 1.3 (141)*
3.5 0.8 (88)
44.4 1.4 (105)
42.6 0.9 (112)
3.6 0.5 (90)
(128)*
(125)*
(97)
dorsolateral frontal
nucleus accumbens
caudate putamen
medial
4.5 0.9 (100)
4.3 1.0 (100)
3.9 0.7 (87)
4.1 0.6 (95)
4.0 0.5 (89)
4.2 0.4 (98)
(97)
(98)
lateral
hippocampus
CA1 region
CA3 region
entorhinal cortex
84.5 1.9 (100)
50.5 1.7 (100)
55.8 1.6 (100)
80.6 3.1 (95)
52.1 1.2 (105)
52.6 1.9 (103)
81.0 3.0 (96)
49.4 2.1 (98)
55.6 1.8 (100)
(109)
(105)
(108)
a
Data are mean SEM values (N = 8 rats/group) for binding in fmol/mg tissue (% of control is given in parentheses) following daily ip injections
b
of vehicle or compounds 2 or 5 for 28 days, with significant differences from controls indicated in bold (∗ indicates p < 0.001). Data (% of control)
for 1 (10 mg/(kg·d)) in adult animals reported previously24 and shown for comparison.
a
Table 8. 5-HT2A Receptor Binding in Rats after 4 Weeks of Daily Injections of Compounds 2 and 5
b
brain region
cerebral cortex
controls
2
5
1
medial-prefrontal
67.8 3.0 (100)
66.7 5.2 (100)
20.1 2.7 (100)
38.3 2.6 (56)*
35.6 3.1 (53)*
19.7 2.5 (98)
64.8 3.8 (96)
67.1 2.2 (101)
22.2 3.0 (110)
(62)*
(65)*
(102)
dorsolateral frontal
nucleus accumbens
caudate putamen
medial
24.7 3.1 (100)
25.5 2.9 (100)
21.5 2.8 (87)
27.2 2.5 (107)
23.3 2.0 (94)
26.0 3.6 (102)
(94)
lateral
(106)
hippocampus
CA1 region
CA3 region
entorhinal cortex
44.5 1.6 (100)
27.0 1.5 (100)
32.8 2.5 (100)
42.7 1.1 (96)
24.8 1.3 (92)
29.5 1.0 (90)
44.2 1.9 (99)
25.8 1.6 (96)
27.9 1.3 (85)
(94)
(96)
(94)
a
Data are mean SEM values (N = 8 rats/group) for binding in fmol/mg tissue (% of control is given in parentheses) following daily ip injections
b
of vehicle or compounds 2 or 5 for 28 days, with significant differences from controls indicated in bold (∗ indicates p < 0.001). Data (% of control)
for 1 (10 mg/(kg·d)) in adult animals reported previously24 and shown for comparison.
5 did not alter concentrations of 5-HT1A and 5-HT2A receptors
in all brain regions examined (Tables 7 and 8).
neuronal activity in frontal cortex or limbic brain regions, which
are involved in the pathophysiology of neuropsychiatric
diseases such as depression, schizophrenia, and other idiopathic
psychotic disorders.
DISCUSSION
■
The data obtained with different compounds raise the
importance of the basicity of the distal nitrogen more than a
steric hindrance in its vicinity, but this remark depends on the
receptor examined. Indeed, the phenyl analogue (3) has no
affinity for any receptors, while the compound with a bulky
cyclohexyl group (6) has significant affinity for the receptors
examined. Flexible (9−12) and polar (7, 8) side chains are not
favorable for receptor binding. Nevertheless, the higher distance
of the distal nitrogen in 9 and 10 is tolerated in terms of
interaction with 5-HT2A receptors. In this in vitro binding study,
two compounds, 2 and 5, emerged in the context of
multireceptor target strategy.
Indeed, 5, with a phenethyl side chain, displays an interesting
multireceptor affinity profile with a significant affinity for D4
and particularly 5-HT1A receptors, while its affinity for 5-HT2A
receptors is reduced compared with those of reference
compounds but remained in the same range as that of 1 and
its N-dealkylated analogue 2. This phenethyl side chain is more
hydrophobic because an increase in lipophilicity is observed
(Table 3). Nevertheless, it seems difficult to correlate such
increase of lipophilicity and the higher affinity for some
receptors because 10 has also a higher hydrophobicity but
exhibits lower affinity for D4, 5-HT1A, and α2A receptors.
In the present study, we have synthesized and tested different
analogues of 1 by changing the characteristics of the N-methyl
piperazine side chain. Compound 1 has been extensively
studied by our group and by others.34,35 Recently, different
chemical developments in related series have also been
reported.36−39 Accordingly, we show that the N-dealkylated
analogue of 1, a compound that could originate by
biotransformation, possesses significant affinity for two
important receptors, namely, serotonin 5-HT1A and 5-HT2A
receptors. This molecule like amoxapine has higher affinity for
these sites than its parent compound. The interest of 2 is
reported for other compounds, for example, clozapine/nor-
clozapine,40,41 loxapine/amoxapine,42 quetiapine/desalkylque-
tiapine,43 and zotepine/nor-zotepine.44
The low D2 affinity in this series was also previously
reported30,31 except for some pyridobenzodiazepine ana-
logues.32 This low dopaminergic D2 potential was also found
in apomorphine-induced climbing in mice22 and follows this
sequence in a more recent study. Clozapine and 1 inhibit the
climbing at 10 and 30 mg/kg, while 2 reduced the climbing
value by ∼20% at 30 mg/kg.45
The affinity for 5-HT1A and α2A receptors, combined with
limited D2 antagonism, may be beneficial for modulating
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Figure 2. Binding mode of 5 (C, N, O, Cl, and H atoms in black, blue, red, green, and cyan, respectively) and 10 (C, N, O, Cl, and H atoms in
orange, blue, red, green and cyan, respectively) in a human 5-HT1A receptor model built by homology modeling from the crystal structure of the
turkey β1 adrenergic GPCR (Protein Data Bank entry 2Y03).57 The hydrogen and ionic bonds are indicated by red dots for 5 and yellow dashed
lines for 10.
Another parameter is the position of the distal nitrogen and the
higher degree of freedom in 10 that could not be favorable in
terms of interactions with the receptor site. To evaluate the
impact of this parameter on the binding affinity, molecular
docking studies of 5 and 10 on the 5-HT1A receptor were
carried out (Figure 2). It is clearly shown that the position of
the protonated nitrogen greatly affects interactions with Ser199
and Asp116, two residues known to be important in the
binding process.48,49 Indeed, the hydrogen bond with the
residue Ser199 and the ionic bond with the residue Asp116
appears to be weaker for 10 with a distance of 3.3 and 3.4 Å,
respectively (versus 1.9 and 2.5 Å for 5).
However, in some cases the low affinity observed for some
compounds (7, 8) could be related to a lower pKa (Table 3)
since the protonated nitrogen is a well-known pharmacophore
in several GPCRs establishing an electrostatic interaction with
an acidic residue like an aspartate residue.50,51 Moreover, the
increase of polar character of 7 and 8 (Table 3) can also be
involved.
2 increased the abundance of cortical and hippocampal D2
receptors as well as striatolimbic D4 receptors, while sparing D2
receptors in extrapyramidal brain regions. Compound 2
displayed directional effects on DA receptors similar to those
of 1 (Tables 4−6), except the effects of 2 were more profound
than those of 1 on the same targets. For example, repeated
treatment with 2 increased D2 receptors in medial prefrontal
cortex (by 41%) and hippocampus (by 34%). An equivalent
dose of 1 increased D2 receptors in medial prefrontal cortex by
32% and in hippocampus by 19%. Greater effects of 2 versus 1
were also detected on D1 and D4 receptors. In addition, 2
selectively increased levels of D2 receptors in nucleus
accumbens (by 40%); an effect not shared by 1 (Table 5).
Treatment with 2 produced different and opposite effects on
5-HT receptor subtypes in cerebral cortex. The tested dose
(13.75 mg/kg) of 2 significantly increased concentrations of 5-
HT1A receptors and decreased 5-HT2A receptors in medial
prefrontal and dorsolateral frontal cortex (Tables 7, 8). The
mechanism for modulation of receptor level by 2 remains
unclear and requires further investigation. However, the pattern
of 5-HT1A and 5-HT2A receptor changes after repeated
treatment with 2 is similar to that observed after repeated
treatment with 1 and several other established SGAs, including
clozapine, olanzapine, risperidone, and quetiapine, and not
typical neuroleptics such as haloperidol.46,47 However, the
effects of 2 were more profound than those of 1 on both 5-
HT1A and 5-HT2A receptors in cerebral cortex (Tables 7, 8).
Following the in vitro binding evaluation, 2 and 5 were tested
in a long-term study to assess its effect on the density of several
neurotransmitter receptors in rat brain. This approach is quite
original because it explores the impact on several targets in one
animal and after a few weeks of administration. Most of the
experimental procedures are frequently made following acute
administration.
Thus, the effects of 2 on DA receptors were very similar to
the superior profile of the effective SGA clozapine. Compound
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acetate), and hexane is added. The mixture is carefully concentrated on
Overall, 2-induced changes in DA and 5HT receptor further
encourage the development of this compound as a novel
effective antipsychotic agent with atypical properties, perhaps
superior to 1, for improved treatment of positive symptoms,
negative symptoms, and cognitive deficits reported to occur in
patients diagnosed with schizophrenia. Nonetheless, the link
between the modulation of receptor level by SGAs and their
antipsychotic efficacy requires further study.
After long-term administration, 5 had more distinct effects
on DA and 5-HT receptor subtypes. Repeated treatment with 5
profoundly and selectively increased abundance of D2 and D4
receptors in nucleus accumbens but did not alter DA or 5-HT
receptor subtypes in any other cortical, extrapyramidal, or
limbic brain regions. These findings suggest that 5 may be less
likely to be an effective antipsychotic drug. However, the
unique effects of 5 on D2 and D4 receptors in nucleus
accumbens, a key brain region involved in mediating the
rewarding actions of drugs of abuse,52 suggest that this agent
may play a role in mechanisms of drug addiction and may be
useful to attenuate drug-induced rewarding behaviors. How-
ever, this hypothesis requires further investigation.
In conclusion, chemical modulation of 1 led us to show that
the N-dealkylated analogue 2 possesses a biological activity that
could contribute to the neurochemical effects of 1 in vivo.
Among other chemical modifications, the presence of an N-
phenethyl moiety in the piperazine series is favorable as 5 has
high affinity for D4, 5-HT1A, and 5-HT2A receptors. Moreover,
the profiles of 2 on DA and 5-HT receptors after long-term
administration, which are similar to that of clozapine and 1,
encourage its development as a novel antipsychotic agent for
improved treatment of schizophrenia and other idiopathic
psychotic disorders.
a Buchi-Rotavapor until crystallization. For salt, an equimolar quantity
̈
of the acid is solubilized separately in a minimal volume of the polar
solvent as the base. Both solutions are mixed and diethylether is added
carefully until crystallization. The mixture can be also maintained at
0−4 °C to get crystallization.
General Procedure for the Synthesis of Oxazepine Deriva-
tives with Hydroxyl Group on Side Chain (7, 8). 8-Chloro-6H-
pyrido[2,3-b][1,5]benzoxazepin-5-one (0.01 mol) was heated to reflux
in the presence of an excess of phosphorus oxychloride (20 mL) for 12
h. After that, the excess phosphorus oxychloride was removed under
reduced pressure. Toluene (20 mL) was added to the brown sticky
residue and evaporated under reduced pressure. The crude
iminochloride was used without further purification. It was dissolved
in toluene (20 mL), and an excess of the amine B (∼0.1 mol) was
added. The mixture was heated to reflux for 2−4 h. The solvent was
then evaporated under reduced pressure, and the residue was dissolved
in chloroform (100 mL) and washed with water (2 × 50 mL). The
organic layer was clarified with activated charcoal, dried over
anhydrous MgSO4, and concentrated under reduced pressure. The
residue can be purified by crystallization directly or after liquid/solid
chromatography on Kieselgel 60 using the appropriate solvent.
Lipophilicity Measurement (log kI′AM). The column was an
IAM-PC-DD2 HPLC column (30 × 4.6 mm2 I.D.; particle size 12 μm;
pore diameter 300 Å) from Regis Technologies (Morton Grove, IL,
USA). The LC system consisted of a Merck-Hitachi L-6000 pump, a
Merck-Hitachi D-2000 chromato-integrator, and a Rheodyne 7125
injector module equipped with a 20 μL loop (Rohnert Park, CA,
USA). Column was maintained in a thermostatted water-bath, Tectron
473-100 from Selecta (Barcelona, Spain), and detection was made with
a Merck-Hitachi L-4000 UV detector. Prior to use, mobile phases were
degassed for 15 min in an ultrasonic bath, Branson 5510 (Branson
Ultrasonics Corporation, Danbury, CT, USA). Buffer was adjusted to
the expected pH values with a Radiometer Analytical pH meter, model
ION check 10 (Villeurbanne, France). Each drug was dissolved in 10.0
mL of methanol at a concentration of 200 μg/mL. One milliliter of
this stock solution was then diluted with methanol in a 10 mL flask in
order to obtain a daughter solution of each compound (20 μg/mL).
Twenty microliters of these diluted solutions was injected. The flow
rate was maintained at 1 mL/min. Temperature of the column was
fixed at 25 °C, and the ultraviolet absorption wavelength was set at 250
nm. The mobile phases were phosphate buffer (50 mM, pH 7.4)
containing 10−60% methanol, according to the lipophilicity of the
compounds. The increment of methanol between two mobile phases
was set to 10%. All retention factors given represent the mean of three
determinations of each sample solution. Retention times of the test
compounds were transformed into capacity factors (kI′AM) according to
the following equation: kI′AM = (tr − t0)/t0 where tr and t0 are the
retention time of the test compound and a compound that is not
retained by the stationary phase (e.g., methanol) to indicate the dead
time/void volume. Capacity factors were expressed as logarithm (log
kI′AM). The values reported in the text correspond to the extrapolation
at 100% aqueous medium.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined on a Buchi-Tottoli
■
̈
capillary melting point apparatus in open capillary and are uncorrected.
NMR spectra were recorded on a Bruker Avance 500 spectrometer at
500 MHz using compounds as free base dissolved in CDCl3. IR
spectra were performed on a Perkin-Elmer FTIR-1750 spectrometer
using KBr discs. Only significant bands from IR are reported.
Elemental analyses were performed using a Carlo-Erba elemental
analyzer CHNS-O model EA1108, and the results are within 0.4% of
the theoretical values. Based on these evaluations, the purity of key
compounds is assumed to be >95%. All starting materials and reagents
were obtained from Aldrich Chemical Co. or Acros Chemical Co. and
were used without further purification. When necessary separations by
column chromatography were carried out using Merck Kieselgel 60
(230−400 mesh). In the case of 3, the compound crystallized during
the separation process. Concentration and evaporation refer to
removal of volatile materials under reduced pressure (10−15 mmHg
In Vitro Binding Procedures. 5-HT1A Receptor Binding Assays.
CHO cells expressing recombinant human serotonin receptors
subtype 1A were used as membrane preparations (Perkin-
Elmer 6110501). Briefly, incubations were carried out in 50
mM Tris-HCl buffer (pH 7.4) containing 10 mM MgSO4, 0.5
mM EDTA, and 0.1% ascorbic acid. Binding assays were
performed in 540 μL total volume consisting of 500 μL of
diluted membranes, 20 μL of radioligand ([3H]-8-OH-DPAT at
∼0.25 nM), and 20 μL of buffer, unlabeled ligand, or tested
drugs. After 60 min at 27 °C, incubations were terminated by
rapid filtration on Whatman GF/C filters presoaked in 0.3%
polyethylenimine followed by washing two times with ice cold
50 mM Tris-HCl (pH 7.4). Filters were placed in a vial
containing 7.5 mL of Ecoscint A. Radioactivity remaining on
the filter was evaluated by liquid scintillation using a TRI-
CARB 1600TR liquid scintillation analyzer. Nonspecific
binding was estimated in the presence of 10 μM metergoline.
at 30−50 °C) on a Buchi-Rotavapor.
̈
General Procedure for the Synthesis of Oxazepine Deriva-
tives (2−6, 9−12). To a mixture of 0.01 mol of 8-chloro-6H-
pyrido[2,3-b][1,5]benzoxazepin-5-one, 0.1 mol of the appropriate
amine A, and anhydrous toluene (20 mL), a solution of titanium
tetrachloride (1.2 mL) in anisole (5 mL) was added dropwise. The
mixture was heated at reflux for 2−3 h and cooled. 2-Propanol (10
mL), ammonia (3 mL), and Kieselgel 60 (5 g) were added. The
suspension was stirred for 10 min and filtered. The solid was washed
with chloroform. The combined organic layers were extracted with 2 N
aqueous HCl (4 × 200 mL). The acidic phase was then basified with
30% aqueous ammonia solution and extracted with chloroform (4 ×
150 mL). The organic layer, dried over anhydrous MgSO4, was
concentrated under reduced pressure. The solvent for recrystallization
is mentioned in brackets after the melting point. For free base, the
residue is solubilized in a limited volume of dichloromethane (or ethyl
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Affinities were determined at least in duplicate with eight
concentrations in duplicate. A preliminary screening was made
at 10−6 M. Compounds displacing more than 60% specific
radioactivity were further tested for Ki determinations (Kd of
the radioligand is 0.32 nM).
photographic developer and fixative were obtained from
Eastman-Kodak (Rochester, NY).
Drug Treatment and Tissue Preparation. Male Sprague−Dawley
rats (Charles River), initially weighing 200−225 g, were maintained
under controlled artificial daylight (on, 07:00−19:00 h), temperature,
and humidity with free access to standard food and tapwater in a
USDA-inspected, veterinarian-supervised, small-animal research facility
of the Mailman Research Center, with approval by the Institutional
Animal Care and Use Committee (IACUC) of McLean Hospital.
Three groups (N = 8/group) of rats received control vehicle, 2 (13.75
mg/(kg·d)) or 5 (3.87 mg/(kg·d)) by daily (ip) injections for 28 days.
All injections occurred between 9:00 and 10:00 a.m. At the end of
treatment, rats were decapitated; brains were removed, quick-frozen in
isopentane on dry ice, and stored at −80 °C until autoradiographic
analysis. Frozen coronal sections (10 μm) were cut in a cryostat at
−20 °C, mounted on gelatin-coated microscopic slides, and stored at
−80 °C until use. Tissue sections were obtained from caudate
putamen, nucleus accumbens, hippocampal CA1 and CA3 regions,
areas of cerebral cortex including dorsolateral frontal and medial
prefrontal, and entorhinal cortex.
Receptor Autoradiography. Brain sections from all drug- and
vehicle-treated rats were evaluated at the same time in each receptor
assay to minimize experimental variability. Assays were performed
under saturating conditions to allow detection of changes in maximal
binding. Sections were first preincubated for 1 h at room temperature
in assay buffer to minimize the effects of endogenous DA and 5-HT.
D1 Receptors. Rat forebrain sections were incubated for 1 h at room
temperature in the incubating buffer [50 mM Tris-HCl buffer (pH
7.4), 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 1 mM MgCl2]
containing 1 nM [3H]SCH-23390. The incubating buffer also
contained 100 nM ketanserin to block 5-HT2A/2C receptors.
Nonspecific binding was determined with an excess (1 μM) of cis-
flupenthixol. After incubation, slides were washed twice for 5 min in
ice-cold buffer, dipped in ice-cold water, and dried under a stream of
air.24,53
D2 Receptors. Sections were incubated for 1 h at room temperature
in the same incubating buffer described above. For D2 binding, this
buffer also contained 1 nM [3H]nemonapride with 0.5 μM DTG and
0.1 μM pindolol to block sigma (σ1,2) and 5HT1A sites, respectively.
Nonspecific binding was determined with 10 μM S(−)-sulpiride. After
incubation, slides were washed twice for 5 min in ice-cold buffer,
dipped in ice-cold water, and air-dried.24,53
D4 Receptors. Tissue sections were preincubated for 1 h at room
temperature in the D2 assay buffer, and then for 1 h with 1 nM
[3H]nemonapride, 300 nM S(−)-raclopride to occupy D2/D3 sites,
and the other blocking agents (0.5 μM DTG and 0.1 μM pindolol)
used in the D2 assay. Nonspecific binding was determined with 10 μM
S(−)-sulpiride. After incubation, slides were washed twice for 5 min in
ice-cold buffer, dipped in ice-cold water, and air-dried.24,53
5-HT2A Receptor Binding Assays. CHO cells expressing recombi-
nant human serotonin receptor subtype 1A were used as membrane
preparations (Perkin-Elmer 6110501). Briefly, incubations were
carried out in 50 mM Tris-HCl buffer (pH 7.4) containing 4 mM
CaCl2 and 0.1% ascorbic acid. Binding assays were performed in 540
μL total volume consisting of 500 μL of diluted membranes, 20 μL of
radioligand ([3H]-ketanserin at ∼0.6 nM), and 20 μL of buffer,
unlabeled ligand, or tested drugs. After 60 min at 27 °C, incubations
were terminated by rapid filtration on Whatman GF/C filters
presoaked in 0.3% polyethylenimine followed by washing two times
with ice cold 50 mM Tris-HCl (pH 7.4). Filters were placed in a vial
containing 7.5 mL of Ecoscint A. Radioactivity remaining on the filter
was evaluated by liquid scintillation using a TRI-CARB 1600TR liquid
scintillation analyzer. Nonspecific binding was estimated in the
presence of 10 μM mianserin. Affinities were determined at least in
duplicate with eight concentrations in duplicate. A preliminary
screening was made at 10−6 M. Compounds displacing more than
60% specific radioactivity were further tested for Ki determinations (Kd
of the radioligand is 0.95 nM).
α2A-Adrenoceptor Binding Assays. Sf9 cells expressing human
cloned α2A-adrenoceptors were used as membrane preparations
(Perkin-Elmer 6110113). Briefly, incubations were carried out in 50
mM Tris-HCl buffer (pH 7.4) containing 12.5 mM MgCl2 and 2 mM
EDTA. Binding assays were performed in 540 μL total volume
consisting of 500 μL of diluted membranes, 20 μL of radioligand
([3H]-MK912 at ∼0.7 nM), and 20 μL of buffer, unlabeled ligand, or
tested drugs. After 60 min at 27 °C, incubations were terminated by
rapid filtration on Whatman GF/C filters followed by washing two
times with ice cold 50 mM Tris-HCl (pH 7.4). Filters were placed in a
vial containing 7.5 mL of Ecoscint A. Radioactivity remaining on the
filter was evaluated by liquid scintillation using a TRI-CARB 1600TR
liquid scintillation analyzer. Nonspecific binding was estimated in the
presence of 10 μM WB4101. Affinities were determined at least in
duplicate with eight concentrations in duplicate. A preliminary
screening was made at 10−6 M. Compounds displacing more than
60% specific radioactivity were further tested for Ki determinations (Kd
of the radioligand is 0.75 nM).
D4 Receptor Binding Assays. Sf9 cells expressing human cloned D4
receptors were used as membrane preparations (Sigma D2439).
Briefly, incubations were carried out in 50 mM Tris-HCl buffer (pH
7.4) containing 5 mM MgCl2, 5 mM KCl, 1.5 mM CaCl2, and 5 mM
EDTA. Binding assays were performed in 540 μL total volume
consisting of 500 μL of diluted membranes, 20 μL of radioligand
([3H]-YM-09151-2 at ∼0.2 nM), and 20 μL of buffer, unlabeled
ligand, or tested drugs. After 60 min at 27 °C, incubations were
terminated by rapid filtration on Whatman GF/C filters followed by
washing two times with ice cold 50 mM Tris-HCl (pH 7.4). Filters
were placed in a vial containing 7.5 mL of Ecoscint A. Radioactivity
remaining on the filter was evaluated by liquid scintillation using a
TRI-CARB 1600TR liquid scintillation analyzer. Nonspecific binding
was estimated in the presence of 10 μM clozapine. Affinities were
determined at least in duplicate with eight concentrations in duplicate.
A preliminary screening was made at 10−6 M. Compounds displacing
more than 60% specific radioactivity were further tested for Ki
determinations (Kd of the radioligand is 0.06 nM).
Evaluation of Long-Term Effects of 2 and 5 on DA and 5-HT
Receptor Levels Using Receptor Autoradiography Proce-
dure. Radioligands. The radiochemicals [N-methyl-3H]R(+)-
7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-
benzazepine (SCH-23390, 81 Ci/mmol), R,S( )-[N-
methyl-3H]nemonapride (86 Ci/mmol), and [ethylene-3H]-
ketanserin hydrochloride ([3H]ketanserin; 63.8 Ci/mmol) were
obtained from New England Nuclear-PerkinElmer Inc.
(Boston, MA, USA). Kodak Biomax MR films and D-19
5-HT1A Receptors. Sections were preincubated for 1 h at room
temperature in 50 mM Tris-HCl buffer (pH 7.6) containing ascorbic
acid (0.1%, w/v), CaCl2 (4 mM), and the monoamine oxidase
inhibitor pargyline-HCl (10 μM). Sections were then incubated for
another 1 h at room temperature in buffer containing 2.0 nM [3H]8-
OH-DPAT. Nonspecific binding was defined by 5-HT (1 μM). After
incubation, slides were washed twice for 5 min in ice-cold buffer,
dipped in ice-cold water, and air-dried.47,54
5-HT2A Receptors. Brain sections were preincubated for 1 h at room
temperature in 50 mM Tris-HCl buffer (pH 7.7), then incubated for
another 1 h at room temperature in fresh buffer containing 3.0 nM
[3H]ketanserin. Assays included 1 μM prazosin (to block α1-
adrenoceptors) and 100 nM tetrabenazine (to block a site associated
with monoaminergic nerve terminals). Nonspecific binding was
defined by methysergide (1 μM). After incubation, slides were washed
twice for 30 min in ice-cold buffer, dipped in ice-cold water, and air-
dried.24,47
Autoradiography and Image Analysis. Radiolabeled sections and
calibrated [3H]standards (Amersham, Arlington Heights, IL, USA)
were exposed to Kodak Biomax MR films for 3−6 weeks at 4 °C in
light-tight cassettes, developed, and then fixed. Optical density in brain
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regions of interest was measured with a computerized densitometric
image analyzer (MCID-M4, Imaging Research, St. Catharines, ON,
Canada). Brain regions of interest were outlined, and their optical
density was measured. Optical density was converted to nCi/mg of
tissue with calibrated [3H]standards, and after subtracting nonspecific
from total binding, specific binding was expressed as fmol/mg
tissue.24,47,53
and serotonin2 pKi values. J. Pharmacol. Exp. Ther. 1989, 251, 238−
246.
́
(2) Liegeois, J.-F.; Eyrolles, L.; Bruhwyler, J.; Delarge, J. Dopamine
D4 receptors: A new opportunity for research on schizophrenia. Curr.
Med. Chem. 1998, 5, 77−100.
(3) Tarazi, F. I.; Zhang, K.; Baldessarini, R. J. Dopamine D4
receptors: Beyond schizophrenia. J. Recept. Signal Transduction Res.
2004, 24, 131−147.
(4) Enguehard-Gueiffier, C.; Gueiffier, A. Recent progress in
medicinal chemistry of D4 agonists. Curr. Med. Chem. 2006, 13,
2981−2993.
Statistical Analysis. DA and 5-HT receptor binding data were
analyzed first for overall effects of drug versus vehicle for all receptor
types and brain regions using multiple regression modeling methods.
Density measures were logarithmically transformed to achieve more
Gaussian-like distributions before regression modeling. Model good-
ness-of-fit was checked using partial residual plot methods. Since
individual brain specimens provided receptor-binding data for several
brain regions, resulting in incomplete independence across observa-
tions, we used robust standard error estimates to adjust for this
clustering effect. This estimation method permits relaxation of the
assumption of independence of all observations and requires only that
the observations be independent across specimens.55 Estimates of
interaction effects were employed for post hoc tests of drug effects for
specific receptors and brain regions, with adjustment of p-values
obtained from the regression analyses estimating these multiple
(5) Rondou, P.; Haegeman, G.; Van Craenenbroeck, K. The
dopamine D4 receptor: Biochemical and signalling properties. Cell.
Mol. Life Sci. 2010, 67, 1971−1986.
(6) Lober, S.; Hubner, H.; Tschammer, N.; Gmeiner, P. Recent
̈
̈
advances in the search for D3- and D4-selective drugs: Probes, models
and candidates. Trends Pharmacol. Sci. 2011, 32, 148−157.
(7) Niswender, C. M.; Conn, P. J. Metabotropic glutamate receptors:
Physiology, pharmacology, and disease. Annu. Rev. Pharmacol. Toxicol.
2010, 50, 295−322.
(8) Krystal, J. H.; Mathew, S. J.; D’Souza, D. C.; Garakani, A.;
Gunduz-Bruce, H.; Charney, D. S. Potential psychiatric applications of
metabotropic glutamate receptor agonists and antagonists. CNS Drugs
2010, 24, 669−693.
(9) Roth, B. L.; Sheffler, D. L.; Kroeze, W. K. Magic shotguns versus
magic bullets: Selectively non-selective drugs for mood disorders and
schizophrenia. Nat. Rev. Drug Discovery 2004, 3, 353−259.
(10) Chung, Y.-C.; Li, Z.; Dai, J.; Meltzer, H. Y.; Ichikawa, J.
Clozapine increases both acetylcholine and dopamine release in rat
ventral hippocampus: Role of 5-HT1A receptor agonism. Brain Res.
2004, 1023, 54−63.
(11) Ichikawa, J.; Ishii, H.; Bonaccorso, S.; Fowler, W. L.; O’Laughlin,
I. A.; Meltzer, H. Y. 5-HT2A and D2 receptor blockade increases
cortical DA release via 5-HT1A receptor activation: A possible
mechanism of atypical antipsychotic-induced cortical dopamine
release. J. Neurochem. 2001, 76, 1521−1531.
(12) Ichikawa, J.; Li, Z.; Dai, J.; Meltzer, H. Y. Atypical antipsychotic
drugs, quetiapine, iloperidone, and melperone, preferentially increase
dopamine and acetylcholine release in rat medial prefrontal cortex:
Role of 5-HT1A receptor agonism. Brain Res. 2002, 956, 349−357.
(13) Roth, B. L.; Hanizavareh, S. M.; Blum, A. E. Serotonin receptors
represent highly favourable molecular targets for cognitive enhance-
ment in schizophrenia and other disorders. Psychopharmacology
(Berlin) 2004, 174, 17−24.
(14) Hertel, P.; Fagerquist, M. V.; Svensson, T. H. Enhanced cortical
dopamine output and antipsychotic-like effects of raclopride by alpha2
adrenoceptor blockade. Science 1999, 286, 105−107.
(15) Jacobson, S. M.; Prus, A. J. Evaluation of the effects of a2-
adrenoceptor antagonism with the D2 receptor antagonist raclopride
on conditioned avoidance responding in rats. Behav. Pharmacol. 2010,
21, 654−659.
́
comparisons by the standard method of Sidak.
56 Data are presented as
means SEM. Comparisons were considered significant at two-tailed
p < 0.05 based on 8 rats per treatment group.
ASSOCIATED CONTENT
* Supporting Information
■
S
Spectroscopic data, elemental analysis results, and melting
points for compounds 2−12. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +32 (0)43 66 43 77. Fax: +32 (0)43 66 43 62. E-mail:JF.
Liegeois@ulg.ac.be.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The technical assistance of Y. Abrassart, P. Fraikin, S.
Counerotte, and J. Widart for IR spectra, 13C NMR
measurements, elemental analyses, and mass spectra, respec-
tively, is gratefully acknowledged. Supported in part by grants
of the F.R.S.-FNRS and the Fonds Spec
́
iaux pour la Recherche
of the University of Liege (Belgium). J.-F.L. is Research
̀
Director of the F.R.S.-FNRS.
ABBREVIATIONS
■
(16) Invernizzi, R. W.; Garavaglia, C.; Samanin, R. The alpha 2-
adrenoceptor antagonist idazoxan reverses catalepsy induced by
haloperidol in rats independent of striatal dopamine release: role of
serotonergic mechanisms. Neuropsychopharmacology 2003, 28, 872−
879.
(17) Morphy, R.; Rankovic, Z. Designed multiple ligands. An
emerging drug discovery paradigm. J. Med. Chem. 2005, 48, 6523−
6543.
SGA, second generation of antipsychotic; DA, dopamine; 5-
HT, 5-hydroxytryptamine; NMDA, N-methyl-D-aspartate;
AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid; GPCR, G-protein coupled receptor; IAM-PC-DD2,
immobilized artificial membrane phosphatidylcholine drug
discovery; kI′AM, capacity factor on immobilized artificial
membrane; CHO cells, Chinese hamster ovary cells; Sf9 cells,
Spodoptera frugiperda cloned cells; Tris, tris(hydroxymethyl)-
aminomethane; EDTA, ethylenediaminetetraacetic acid; DTG,
1,3-di(2-tolyl)guanidine; 8-OH-DPAT, 8-hydroxy-2-
(dipropylamino)tetralin; Kd, apparent equilibrium dissociation
constant; Ki, apparent equilibrium inhibition constant
(18) Marino, M. J.; Knutsen, L. J. S.; Williams, M. Emerging
opportunities for antipsychotic drug discovery in the postgenomic era.
J. Med. Chem. 2008, 51, 1077−1107.
́
(19) Ellenbroek, B. A.; Liegeois, J.-F. JL13, an atypical antipsychotic:
A preclinical review. CNS Drug Rev. 2003, 9, 41−57.
(20) Invernizzi, R. W.; Garavaglia, C.; Samanin, R. JL13, a
pyridobenzoxazepine compound with potential atypical antipsychotic
activity, increases extracellular dopamine in the prefrontal cortex, but
not in the striatum and the nucleus accumbens of rats. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 2000, 361, 298−302.
REFERENCES
■
(1) Meltzer, H. Y.; Matsubara, S.; Lee, J.-C. Classification of typical
and atypical antipsychotic drugs on the basis of dopamine D-1, D-2
1580
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Journal of Medicinal Chemistry
Article
(21) Ellenbroek, B. A.; Lieg
́
eois, J.-F.; Bruhwyler, J.; Cools, A. R. The
clozapine. III. Replacement of the tricyclic nucleus with a bicyclic
template. Aust. J. Chem. 2007, 60, 928−933.
effects of JL13, a pyridobenzoxazepine with potential atypical
antipsychotic activity, in animal models for schizophrenia. J. Pharmacol.
Exp. Ther. 2001, 298, 386−391.
(38) Capuano, B.; Crosby, I. T.; Lloyd, E. J.; Neve, J. E.; Taylor, D. A.
Aminimides as potential CNS acting agents. II. Design, synthesis, and
receptor binding of 4′-arylalkyl aminimide analogues of clozapine as
prospective novel antipsychotics. Aust. J. Chem. 2008, 60, 5−10.
(39) Capuano, B.; Crosby, I. T.; Lloyd, E. J.; Podloucka, A.; Taylor,
D. A. Synthesis and preliminary pharmacological evaluation of 4′-
arylalkyl analogues of clozapine. IV. The effects of aromaticity and
isosteric replacement. Aust. J. Chem. 2008, 60, 930−940.
(22) Bruhwyler, J.; Lieg
Taylor, A.; Meltzer, H. Y.; Delarge, J.; Gec
́
eois, J.-F.; Bergman, J.; Carey, G.; Goudie, A.;
zy, J. JL13, a
́
pyridobenzoxazepine compound with potential atypical antipsychotic
activity: A review of its behavioural properties. Pharmacol. Res. 1997,
36, 255−264.
́ ́
(23) Casey, D. E.; Bruhwyler, J.; Delarge, J.; Geczy, J.; Liegeois, J.-F.
The behavioral effects of acute and chronic JL13, a putative
antipsychotic, in Cebus non-human primates. Psychopharmacology
2001, 157, 228−235.
(40) Li, Z.; Huang, M.; Ichikawa, J.; Dai, J.; Meltzer, H. Y. N-
Desmethylclozapine, a major metabolite of clozapine, increases cortical
acetylcholine and dopamine release in vivo via stimulation of M1
muscarinic receptors. Neuropsychopharmacology 2005, 30, 1986−1995.
(41) Prus, A. J.; Pehrson, A. L.; Philibin, S. D.; Wood, J. T.; Vunck, S.
A.; Porter, J. H. The role of M1 muscarinic cholinergic receptors in the
discriminative stimulus properties of N-desmethylclozapine and the
atypical antipsychotic drug clozapine in rats. Psychopharmacology
(Berlin) 2009, 203, 295−301.
(42) Apiquian, R.; Ulloa, E.; Fresan, A.; Loyzaga, C.; Nicolini, H.;
Kapur, S. Amoxapine shows atypical antipsychotic effects in paptients
with schizophrenia: results from a prospective open-label study.
Schizophr. Res. 2003, 59, 35−39.
(43) Jensen, N. H.; Rodriguiz, R. M.; Caron, M. G.; Wetsel, W. C.;
Rothman, R. B.; Roth, B. L. N-desalkylquetiapine, a potent
norepinephrine reuptake inhibitor and partial 5-HT1A agonist, as a
putative mediator of quetiapine’s antidepressant activity. Neuro-
psychopharmacology 2008, 33, 2303−2312.
(44) Shobo, M.; Kondo, Y.; Yamada, H.; Mihara, T.; Yamamoto, N.;
Katsuoka, M.; Harada, K.; Ni, K.; Matsuoka, N. Norzotepine, a major
metabolite of zotepine, exerts atypical antipsychotic-like and
antidepressant-like actions through its potent inhibition of norepi-
nephrine reuptake. J. Pharmacol. Exp. Ther. 2010, 333, 772−781.
(45) Lamy, C. Synthesis, evaluation and psychopharmacological
evaluation of potential antipsychotics. Master Thesis in Pharmaceutical
́
(24) Moran-Gates, T.; Massari, C.; Graulich, A.; Liegeois, J.-F.;
Tarazi, F. I. Long-term effects of novel atypical antipsychotic JL13 on
dopamine and serotonin receptor subtypes. J. Neurosci. Res. 2006, 84,
675−682.
(25) Tarazi, F. I.; Moran-Gates, T.; Gardner, M.; Graulich, A.; Lamy,
́
C.; Liegeois, J.-F. Long-term effects of JL13, a potential atypical
antipsychotic, on ionotropic glutamate receptors. J. Mol. Neurosci.
2007, 32, 192−198.
́
(26) Liegeois, J.-F.; Mouithys-Mickalad, A.; Bruhwyler, J.; Delarge, J.;
Petit, C.; Kauffmann, J.-M.; Lamy, M. JL13, a potential successor to
clozapine is less sensitive to oxidative phenomenons. Biochem. Biophys.
Res. Commun. 1997, 238, 252−255.
́
(27) Liegeois, J.-F.; Rogister, F.; Delarge, J.; Pincemail, J. Peroxidase-
catalysed oxidation of different dibenzazepine derivatives. Arch. Pharm.
(Weinheim) 1995, 328, 109−112.
(28) Lieg
Gec
́
eois, J.-F.; Bruhwyler, J.; Petit, C.; Damas, J.; Delarge, J.;
zy, J.; Kauffmann, J.-M.; Lamy, M.; Meltzer, H. Y.; Mouithys-
́
Mickalad, A. Oxidation sensitivity may be a useful tool for the
detection of the hematotoxic potential of newly developed molecules:
Application to antipsychotic drugs. Arch. Biochem. Biophys. 1999, 370,
126−137.
́
(29) Dilly, S.; Liegeois, J.-F. The formation of nitrenium ion also
influences binding of clozapine on rat D2 dopamine receptors:
docking studies and in vitro binding assays. J. Comput.-Aided Mol. Des.
2011, 25, 163−169.
Sciences, University of Liege, 2005
̀
(46) Florijn, W. J.; Tarazi, F. I.; Creese, I. Dopamine receptor
subtypes: Differential regulation after 8 months treatment with
antipsychotic drugs. J. Pharmacol. Exp. Ther. 2007, 280, 561−569.
(47) Tarazi, F. I.; Zhang, K.; Baldessarini, R. J. Long-term effects of
olanzapine, risperidone, and quetiapine on serotonin 1A, 2A and 2C
receptors in rat forebrain regions. Psychopharmacology 2002, 161,
263−270.
́
(30) Liegeois, J.-F.; Rogister, F.; Bruhwyler, J.; Damas, J.; Nguyen, T.
P.; Inarejos, M. O.; Chleide, E.; Mercier, M.; Delarge, J.
Pyridobenzoxazepine and pyridobenzothiazepine derivatives as
potential central nervous system agents: Synthesis and neurochemical
study. J. Med. Chem. 1994, 37, 519−525.
(31) Lieg
́
eois, J.-F.; Bruhwyler, J.; Damas, J.; Nguyen, T. P.; Chleide,
(48) Ho, B. Y.; Karschin, A.; Branchek, T.; Davidson, N.; Lester, H.
A. The role of conserved aspartate and serine residues in ligand
binding and in function of the 5-HT1A receptor: A site-directed
mutation study. Febs Lett. 1992, 312, 259−262.
(49) Strader, C. D.; Candelore, M. R.; Hill, W. S.; Sigal, I. S.; Dixon,
R. A. F. Identification of two serine residues involved in agonist
activation of the beta-adrenergic receptor. J. Biol. Chem. 1989, 264,
13572−13578.
(50) Hibert, M. F.; Trumpp-Kallmeyer, S.; Bruinvels, A.; Hoflack, J.
Three-dimensional models of neurotransmitter G-binding protein-
coupled receptors. Mol. Pharmacol. 1991, 40, 8−15.
(51) Trumpp-Kallmeyer, S.; Hoflack, J.; Bruinvels, A.; Hibert, M. F.
Modeling of G-protein-coupled receptors: application to dopamine,
adrenaline, serotonin, acetylcholine, and mammalian opsin receptors. J.
Med. Chem. 1992, 35, 3448−3462.
(52) Carelli, R. M. The nucleus accumbens and reward: Neuro-
physiological investigations in behaving animals. Behav. Cogn. Neurosci.
Rev. 2002, 1, 281−296.
(53) Tarazi, F. I.; Zhang, K.; Baldessarini, R. J. Long-term effects of
olanzapine, risperidone, and quetiapine on dopamine receptor types in
regions of rat brain: implications for antipsychotic drug treatment. J.
Pharmacol. Exp. Ther. 2001, 297, 711−717.
(54) Simpson, M. D.; Lubman, D. I.; Slater, P.; Deakin, J. F.
Autoradiography with [3H]8-OH-DPAT reveals increases in 5-HT1A
receptors in ventral prefrontal cortex in schizophrenia. Biol. Psychiatry
1996, 39, 919−928.
E.; Mercier, M.; Rogister, F.; Delarge, J. New pyridobenzodiazepine
derivatives as potential antipsychotics: synthesis and neurochemical
study. J. Med. Chem. 1993, 36, 2107−2114.
(32) Lieg
́
eois, J.-F.; Eyrolles, L.; Carato, P.; Bruhwyler, J.; Gec
́
zy, J.;
Damas, J.; Delarge, J. New pyridobenzodiazepine derivatives as
potentially centrally acting drugs. Modifications on the basic side
chain differentially modulate dopamine (D4, D2L) and serotonin (5-
HT2A) binding. J. Med. Chem. 2002, 45, 5136−5149.
(33) Fryer, R. I.; Earley, J. V.; Field, G. F.; Zally, W.; Sternbach, L. H.
A synthesis of amidines from cyclic amides. J. Org. Chem. 1969, 34,
1143−1145.
(34) Goudie, A. J.; Cole, J. C.; Sumnall, H. R. Olanzapine and JL13
induce cross-tolerance to the clozapine discriminative stimulus in rats.
Behav. Pharmacol. 2007, 18, 9−17.
(35) Capuano, B.; Crosby, I. T.; MacRobb, F. M.; Taylor, D. A.;
Vom, A.; Blessing, W. W. JL13 has clozapine-like actions on
thermoregulatory cutaneous blood flow in rats: involvement of
serotonin 5-HT1A and 5-HT2A receptor mechanisms. Prog. Neuro-
psychopharmacol. Biol. Psychiatry 2010, 34, 136−142.
(36) Capuano, B.; Crosby, I. T.; Lloyd, E. J.; Neve, J. E.; Taylor, D. A.
Aminimides as potential CNS acting agents. I. Design, synthesis, and
receptor binding of 4′-aryl aminimide analogues of clozapine as
prospective novel antipsychotics. Aust. J. Chem. 2007, 60, 673−684.
(37) Capuano, B.; Crosby, I. T.; Lloyd, E. J.; Taylor, D. A. Synthesis
and preliminary pharmacological evaluation of 4′-arylalkyl analogues of
1581
dx.doi.org/10.1021/jm2013419 | J. Med. Chem. 2012, 55, 1572−1582

Journal of Medicinal Chemistry
Article
(55) Greene, W. H. Econometric Analysis, 4th ed.; Prentice Hall:
Upper Saddle River, NJ, 2000; pp 462−465.
(56) Winer, B. J.; Brown, D. R.; Michels, K. M. Statistical Principles in
Experimental Drug Design; 3rd ed.; McGraw Hill: New York, 1991, pp
165−166.
́ ́
(57) Dilly, S.; Scuvee-Moreau, J.; Wouters, J.; Liegeois, J.-F. The 5-
HT1A agonism potential of substituted-piperazine-ethyl-amide deriva-
tives is conserved in the hexyl homologues: Molecular modelling and
pharmacological evaluation. J. Chem. Inf. Model. 2011, 51, 2961−2966.
(58) Hilal, S. H.; El-Shabrawy, Y.; Carreira, L. A.; Karickhoff, S. W.;
Toubar, S. S.; Rizk, M. Estimation of the ionization pK(a) of
pharmaceutical substances using the computer program Sparc. Talanta
1996, 43, 607−619.
1582
dx.doi.org/10.1021/jm2013419 | J. Med. Chem. 2012, 55, 1572−1582