Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. Part 1

Article abstract of DOI:10.1016/S0960-894X(00)00290-0

6-Nitroquipazine has been known as one of the most potent and selective inhibitors of serotonin transporter in vitro and in vivo. Nine derivatives of 6-nitroquipazine were synthesized and tested for their potential abilities to displace [³H]citalopram binding to the rat cortical membranes. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00290-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1559±1562
Syntheses and Binding Anities of 6-Nitroquipazine Analogues
for Serotonin Transporter. Part 1
Byoung Se Lee,a Soyoung Chu,a Byung Chul Lee,a Dae Yoon Chi,a,*
Yearn Seong Choe,b Kyung Ja Jeongc and Changbae Jinc
aDepartment of Chemistry, Inha University, 253 Yonghyun-Dong, Nam-Ku, Inchon, 402-751, South Korea
bDepartment of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University, School of Medicine,
50 Ilwon-Dong, Kangnam-Ku, Seoul, 135-710, South Korea
cBioanalysis & Biotransformation Research Center, Korea Institute of Science and Technology,
PO Box 131, Cheongryang, Seoul, 130-650, South Korea
Received 21 March 2000; accepted 8 May 2000
AbstractÐ6-Nitroquipazine has been known as one of the most potent and selective inhibitors of serotonin transporter in vitro and in
vivo. Nine derivatives of 6-nitroquipazine were synthesized and tested for their potential abilities to displace [3H]citalopram binding
to the rat cortical membranes. # 2000 Elsevier Science Ltd. All rights reserved.
Serotonin (5-hydoxytryptamine or 5-HT) is a neuro-
transmitter considered to be related to depression and
other psychiatric diseases. 5-HT reuptake sites in the
mammalian brain have been studied extensively with
radiotracers such as [3H]imipramine, [3H]paroxetine, and
[3H]citalopram. 6-Nitroquipazine (1, 6-NQ) has been
known as one of the most potent and selective antago-
nists for serotonin transporter (SERT) in vitro1,2 and in
vivo,3,4 showing higher potency (Ki=0.17 nM) than
paroxetine (Ki=0.58 nM) or citalopram (Ki=1.50 nM)
for 5-HT reuptake site. Imaging of brain using radio-
pharmaceuticals is, therefore, expected to be a useful tool
for understanding of dysfunction of psychiatric diseases as
well as pathological studies of neurotransmission of 5-HT.
synthesis of 6-nitroquipazine in three steps, in overall 82%
yield,8 only few structure modi®cation of 6-nitroquipazine
has been reported.9 This regioselective synthesis enabled
us to modify the structure of 6-NQ and to synthesize the
compounds we designed. In this paper, we reported the
synthesis of several 6-NQ derivatives and the measurement
of their biological activities for 5-HT transporter to ®nd
out structure±activity relationship of 6-NQ derivatives.
Chemistry
Nine new quipazine derivatives were prepared according
to our synthetic route recently reported.8 In Scheme 1, 2-
chloro-6-nitroquinoline (11) was obtained from hydro-
carbostyril9 (12). Compounds 2±5 were prepared by simple
displacement reactions with various corresponding amines.
The ®rst step of our regioseletive synthetic route of 6-NQ
resulted in addition of a nitro group. Dinitro compound
13 was also obtained in harsher nitration condition
(Scheme 2). Rest steps are the same as the synthesis of
6-NQ.
Although Mathis et al. reported syntheses of 5-
[
as a SPECT and a PET imaging agent, respectively,5 7
their synthetic methods of quipazine analogues were too
inecient and tricky to give target molecules in good
yield. Until we recently reported the regioselective
123I]iodo- (1a) and 5-[76Br]bromo-6-nitroquipazine (1b)
To check the electronic e€ects of a nitro group on C6
postion, three 6-haloquipazines were prepared according
to Scheme 3. Halogenation of hydrocarbostyril under-
went only on C6 position and the rest stepsÐchlorination
followed by aromatization and aromatic substitution
reactionÐare the same as the synthesis of 6-NQ. As an
*Corresponding author. Tel.: +82-32-860-7686: fax: +82-32-867-
5604; e-mail: dychi@inha.ac.kr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00290-0

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B. S. Lee et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1559±1562
Scheme 1. Reagents and conditions: (a) 2-methylpiperazine (yield, 71%), or N,N0-dimethylethylenediamine (56%), or ethylenediamine (95%), or
homopiperazine (70%), dioxane, re¯ux, 4 h.
Scheme 2. Reagents and conditions: (a) concd H2SO4, concd HNO3, 100 ꢀC, 30 min, 47%; (b) POCl3, DMF, 60 ꢀC, 4 h, 98%; (c) 1-piper-
azinecarboxaldehyde, DMF, rt, 5 min, 70%; (d) 4 M H2SO4, 60 ꢀC, 3 h 30 min, 80%.
Scheme 3. Reagents and conditions: (a) NaXO3/H2O, HX, AcOH, rt, X=Cl, Br, 20 min, 55%, 95%, respectively; (b) ICl, AcOH, rt, 12 h, 91%; (c)
POCl3, DDQ, DMF, rt, 6 h, 87±93%; (d) 1-piperazinecarboxaldehyde, DMF, 80 ꢀC, 3±10 h, 42±57%; (e) 4 M H2SO4, 80 ꢀC, 3±5 h, 57±96%.
alternative route, Sandmeyer reactions from 6-aminoqui-
pazine were examined. Although the reduction of 6-NQ
using 10% palladium on charcoal provided 6-aminoqui-
pazine in high yield, several undesired and inseparable
products were formed in Sandmeyer reaction.
prepared from the rat cerebral cortex.11 Saturation bind-
ing assays were performed to measure the equilibrium
dissociation constant (Kd) and the density of binding
sites (Bmax) using 15 concentrations of [3H]citalopram
between 0.01 and 30 nM. Nonspeci®c binding was
de®ned as that determined in the presence of 10 mM
¯uoxetine. Scatchard analysis of the saturation binding
data indicated a single population of binding sites with a
Kd of 1.12 Æ 0.01 nM and a Bmax of 704 Æ 47 fmol/mg
protein (n=3). Competition binding assays were per-
formed to measure the concentrations of test compounds
which inhibited the speci®c binding by 50% (IC50
3-Bromo-6-NQ was prepared by reaction of 6-nitro-2-
quinolone (16) with sodium bromate in acidic condition,
followed by aromatic substitution reaction with piperazine.
Compound 16 was obtained from 11 by re¯uxing in
acetic acid (Scheme 4).
values) using 1 nM [3H]citalopram and 11 concentra-
Binding studies
11
tions of the unlabelled compounds between 10
and
Binding of [3H]citalopram to the 5-HT transporter was
measured according to the method of Pahkla et al.10
with slight modi®cations using crude synaptic membranes
10 M. IC50 values were determined from the compe-
tition binding data using computer-assisted curve ®tting
with GraphPad Prism 3.0 program. Inhibition binding
5
Scheme 4. Reagents and conditions: (a) AcOH, 100 ꢀC, 10 h, 99%; (b) NaBrO3/H2O, HBr, 100 ꢀC, 4 h, 76%; (c) POCl3, DMF, 100 ꢀC, 30 min, 93%;
(d) 1-piperazinecarboxaldehyde, DMF, 120 ꢀC, 6 h, 97%; (e) 4 M H2SO4, 100 ꢀC, 5 h, 50%; (f) HCl(g), MeOH-ether.

B. S. Lee et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1559±1562
1561
Table 1. Structure and binding data on the 5-HT transporter of the quipazine derivativesa
Compounds
Ki (nM)
Compounds
Ki (nM)
2
22.19 Æ 1.70
8
9
0.91 Æ 0.07
1.68 Æ 0.13
3
8.43 Æ 0.45
4
5
164.30 Æ 4.22
1.90 Æ 0.15
10
1
12.62 Æ 1.44
0.17 Æ 0.03
6
7
312.85 Æ 2.85
6.60 Æ 0.52
Fluoxetine (Prozac)
Paroxetine
22.13 Æ 1.77
0.53 Æ 0.08
aThe values represent mean Æ S.E.M of 3±4 separate experiments done in duplicate.
constant (Ki) values were subsequently calculated from
IC50 values using the Cheng-Pruso€ equation.12 Table 1
illustrates the in vitro binding anities of 2±10 for the
5-HT transporter including 6-NQ, ¯uoxetine, and
paroxetine.
Addition of a nitro group to C8 (compound 6) and a
bromo group to C3 (compound 10), and replacement of a
nitro group on C6 with an iodo, a bromo or a chloro
group (compound 7±9) lead to the wide range of anities.
If the hydrogen-bonding between the heterocyclic nitrogen
atom and a suitable hydrogen bond donor in the SERT is
important, the electronic density of the nitrogen atom in
quinoline ring would greatly be a€ected by variable sub-
stituents.
Discussion
6-NQ has high binding anity (Ki=0.17 nM) for SERT,
while quipazine itself has much lower anity (Ki=63
nM).13 Quipazine and its derivatives have been used for
the study of serotonin receptor. A nitro group at C6
position plays an important role in retaining the strong
binding anity for SERT. To obtain information on
the possible roles of the di€erent substituents on 6-NQ,
the structure of 6-NQ was modi®ed. Cappelli14 and Mor-
reale15 groups recently reported a comparative molecular
®eld analysis (CoMFA) and pharmacological evaluation
of quipazine with subtypes of serotonin receptor. Due to
the diculty of the structural modi®cations of 6-NQ with
remaining 6-nitro group, only few reports of structural
modi®cations of 6-NQ are published. The purpose of this
research is to synthesize and evaluate enough number of
derivatives of 6-NQ to do CoMFA study.
More syntheses of 6-NQ derivatives are being currently
pursued. In addition, more biological studies with some
6-NQ derivatives showing good binding anities are in
progress.
Acknowledgements
This research was supported by the Korea Science and
Engineering Fund (No 97-03-01-01-5-L), and the Inha
Research Fund (1999).
References and Notes
Introduction of a methyl group to piperazine ring drama-
tically decreases the anity by about 2 orders of magni-
tude (compound 2 versus 1). Ring-opened compounds 3
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primary amine 4 has much lower anity implies the
importance of secondary amine for binding. Compound
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interaction of quipazine in the 5-HT3 receptor by Cap-
pelli et al., secondary amine is important for the charge-
assisted interaction between the protonated terminal
piperazine nitrogen atom and a negatively charged car-
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