Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. Part 2: 4-Substituted 6-nitroquipazines

Article abstract of DOI:10.1016/S0960-894X(02)00028-8

Eleven 4-substituted derivatives of 6-nitroquipazine were synthesized and evaluated for their abilities to displace [³H]citalopram binding to the rat cortical synaptic membranes. Among them, 4-chloro-6-nitroquipazine was shown to possess the hi

Full text of DOI:10.1016/S0960-894X(02)00028-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 811–815
Syntheses and Binding Affinities of 6-Nitroquipazine Analogues
y
for Serotonin Transporter. Part 2: 4-Substituted
6
-Nitroquipazines
a
a
a
a,
Byoung Se Lee, Soyoung Chu, Bon-Su Lee, Dae Yoon Chi, *
b
b
Yun Seon Song and Changbae Jin
aDepartment of Chemistry, Inha University, 253 Yonghyundong, Namgu, Inchon 402-751, Republic of Korea
bBioanalysis & Biotransformation Research Center, Korea Institute of Science and Technology, PO Box 131,
Cheongryang, Seoul 130-650, Republic of Korea
Received 6 September 2001; accepted 29 December 2001
Abstract—Eleven 4-substituted derivatives of 6-nitroquipazine were synthesized and evaluated for their abilities to displace
3
[
highest binding affinity (Ki=0.03 nM) which was approximately 6 times higher than that of 6-nitroquipazine (K
H]citalopram binding to the rat cortical synaptic membranes. Among them, 4-chloro-6-nitroquipazine was shown to possess the
=0.17 nM) itself. In
i
this paper, we describe the syntheses of 4-substituted 6-nitroquipazine derivatives, the results of corresponding biological evaluation
and the SAR study. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
derivatives such as 5-iodo-6-nitroquipazine (2) have
1
1ꢀ15
the detailed SAR studies on them
Serotonin (5-hydroxytryptamine or 5-HT) has been
known as a neurotransmitter associated with many
psychiatric diseases including depression, anxiety,
obsessive-compulsive disorder, psychosis, eating dis-
order, and dependence in human brain.2 It could not
only bind to seven receptor subclasses but also be reup-
taken into presynaptic neurons through its transporter,
which plays an important role in modulation of sero-
tonin concentrations in neuronal synapses. Therefore,
inhibition of serotonin transporter results in increased
concentration of serotonin in the synaptic cleft. Sero-
tonin transporter (SERT) is an integral protein con-
been reported,
have not been performed. Previously, we reported not
1
6
only a novel synthetic route of 6-NQ itself but also
syntheses and in vitro biological results of several 6-NQ
,3
1
derivatives toward SERT. Moreover, during efforts for
1
8
the syntheses of several analogues, 3-(3-[ F]fluoro-
propyl)-6-nitroquipazine (3, K =0.32 nM) was prepared
as a positron emission tomography (PET) imaging
i
1
7
agent. The biodistribution and the metabolic decom-
position rate of 3 were investigated in mice brain.
Through these previous studies, we found that the sub-
stitution of several alkyl groups at C3 position would be
tolerated, while a nitro group at C6 position and a
piperazine group at C2 position play a critically impor-
tant role in retaining the strong binding affinity for
SERT. In this paper, 4-substituted 6-NQ derivatives
were synthesized and tested for their potential abilities
sisted of 12 transmembrane domains with the NH - and
2
COOH- terminal domains in the cytoplasm.
4
,5
It has been reported that 6-nitroquipazine (1, 6-NQ) has
higher binding affinity for SERT than other selective
serotonin reuptake inhibitors (SSRIs, i.e., citalopram,
fluoxetine, fluvoxamine, paroxetine and sertraline)
approved by the Food and Drug Administration as
3
to displace [ H]citalopram binding to the rat cortical
synaptic membranes.
6ꢀ10
antidepressants.
Although a few number of 6-NQ
*Corresponding author. Tel.:+82-32-860-7686; fax:+82-32-867-5604;
e-mail: dychi@inha.ac.kr
y
See ref 1.
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00028-8

812
B. S. Lee et al. / Bioorg. Med. Chem. Lett. 12 (2002) 811–815
ꢁ
ꢁ
Scheme 1. Reagents and conditions: (a) diethylmalonate, 150 C, 12 h; (b) PPA, 120 C, 3 h; (c) POCl
ꢁ
3 3 3
, reflux, 6 h; (d) POBr , CH CN, reflux, 3 h;
ꢁ
(
e) 1-piperazinecarboxaldehyde, DMF, 60 C, 3 h; (f) 4 M H
2
SO
4
, THF, 80 C, 3 h.
Chemistry
The key intermediate required to synthesize 4-sub-
stituted 6-NQ is either 2,4-dichloro- (6) or 2,4-dibromo-
6
-nitroquinoline (7), which was prepared in three steps
from 4-nitroaniline as shown in Scheme 1. N-(4-
Nitrophenyl)malonamic acid ethyl ester (4) was syn-
thesized by the reaction of 4-nitroaniline and 10 equiv
ꢁ
of neat diethylmalonate at 150 C for 12 h, isolated by
column chromatography after removal of excess mal-
onate by distillation. The ring formation of 4 by heating
with polyphosphoric acid gave 4-hydroxy-6-nitro-
Scheme 2. Reagents and conditions: (a) 1-piperazinecarboxaldehyde,
ꢁ
2
(1H)quinolinone (5) in 36% yield. This polar and
DMF, 120 C, 3 h; (b) bis(tri-n-butyltin), Pd(PPh
3
)
4
, dioxane, reflux,
insoluble compound was purified by dissolving in
aqueous NaOH (1N) solution and supernatant sub-
stance was filtered out. The filtrate was acidified by
addition of aqueous H SO (4 M) solution until
3 4
12 h; (c) NaI, H PO , dichloramine T, EtOH, rt, 30 min; (d) 4 M
ꢁ
2 4
H SO , THF, 80 C, 3 h.
2
4
slightly acidic to give pale brown precipitate 5. 2,4-
Dichloro-6-nitroquinoline (6) was obtained by chlor-
ination of 5 with phosphorus oxychloride in 61% yield.
Likewise, refluxing of 5 with phosphorus oxybromide in
acetonitrile gave 2,4-dibromo-6-nitroquinoline (7) in 56%
yield. 4-Chloro- (8) and 4-bromo-6-nitroquipazine (9)
were synthesized by the reaction of corresponding 2,4-
dihalo-6-nitroquinolines and 1.5 equiv of 1-piper-
ꢁ
azinecarboxaldehyde in DMF at 60 C for 3 h, followed
by deformylation of N-formyl moiety in 4 M H SO at
2
4
ꢁ
0 C for 3 h.
8
N-Formyl-4-bromo-6-nitroquipazine (10) obtained by
the reaction of 2,4-dibromoquinoline and 1-piper-
azinecarboxaldehyde was used for the introduction of
several substituents including iodine at C4 position. As
shown in Scheme 2, the palladium catalyzed coupling
reaction of heteroaryl bromide (10) and bis(tri-n-butyl-
tin) gave N-formyl-4-tributylstannanyl-6-nitroquipazine
Scheme 3. Reagents and conditions: (a) Pd(PPh
Bu) , Br or I), dioxane, reflux, 2–32 h; (d) 4 M H
3
)
SO
4
, R-Y (Y=Sn(n-
ꢁ
3
2
4
, THF, 80 C, 3
bromo-6-nitroquipazine (10) and tributyl(vinyl)tin, allyl-
tributyltin and 2-tributylstannanylfuran, respectively.
(11) in 58% yield. The tributyltin moiety was sub-
stituted with iodine under oxidative condition to give N-
formyl-4-iodo-6-nitroquipazine, which was hydrolyzed
using aqueous H SO (4 M) to form 4-iodo-6-nitroqui-
After Stille coupling reaction, the toxic by-product, tri-
butyltin halide was quenched by addition of 10% aqu-
eous KF solution into reaction mixture, which was
stirred for 3 h. The resulting insoluble substances, white
tributyltin polymer and palladium metal were removed
by filtration with Celite.
2
4
pazine (12).
Various 4-substituted 6-NQs were synthesized by Stille
coupling reaction under the similar condition (Scheme
3
). Compounds 14, 16, 17, and 19 were prepared by the
The nucleophilic aromatic substitution reaction of 20
with pyrrolidine at C4 position was carried out by
heating at 120 C for 3 h. Deprotection of N-formyl
reaction of N-formyl-4-tributylstannanyl-6-nitroquipa-
zine with 2-bromopropene, iodobenzene, benzylbromide
and 2-bromothiophene, respectively. Compounds 13, 15,
and 18 were obtained by the reaction of N-formyl-4-
ꢁ
moiety under acidic condition with 4 M H SO afforded
6-nitro-4-(pyrrolidin-1-yl)quipazine (21) (Scheme 4).
2
4

B. S. Lee et al. / Bioorg. Med. Chem. Lett. 12 (2002) 811–815
813
As mentioned above, the bulkiness of substituents is
responsible for decreasing the binding affinities of the
rest 10 6-NQs. In contrast to 4-chloro-6-NQ (8), for
example, 4-benzyl-6-NQ (17) substituted with the
bulkiest group has the worst inhibition constant
(
(
K =126.86 nM) and much lower binding affinity
i
approximately more than 4000-fold) than 8. The sen-
ꢁ
Scheme 4. Reagents and conditions: (a) pyrrolidine, DMF, 120 C, 3
ꢁ
sitivity of SERT to the size of substituents indicates
that C4 position of 6-NQs is very close to the residue of
SERT in the binding site, so there exists steric repulsion
between the substituent on C4 position and the binding
pocket of SERT. The fact that chlorine is larger than
hydrogen but result in better binding affinity could be
explained by an electronic factor. By changing from
chlorine to other bulkier halogens, the binding affinities
for the rat cortical SERT drops by the factor of one
order: K value of 4-bromo-6-NQ (9)=0.37 nM, K
h; (b) 4 M H
2
SO
4
, THF, 80 C, 3 h.
Binding Studies
1
According to the method of our previous study, using
crude synaptic membranes prepared from the cerebral
cortex of male Sprague–Dawley rats, competition
binding assays were performed to measure the con-
centrations of test compounds which inhibited the spe-
1
8
i
i
value of 4-iodo-6-NQ (10)=1.73 nM. The binding affi-
nity of 4-bromo-6-NQ (9) for 5-HT uptake site was
obtained by Hashimoto et al. and showed similar
cific binding by 50% (IC
3
values) using 1nM
0
5
[
compounds between 10
H]citalopram and 11 concentrations of the unlabelled
ꢀ
11
ꢀ5
21
and 10
M. Nonspecific
trend.
binding was defined as that determined in the presence
of 10 mM fluoxetine. IC50 values were determined from
the competition binding data using computer-assisted
curve fitting with GraphPad Prism 3.0 program. Inhibi-
Second, in addition to the aspect of steric hindrance, the
electronic environments of the binding pocket should
also be taken into account. When compound 18 was
compared with compound 19, we found that there was a
significant difference between them in spite of their
similar bulkiness. Based on the fact that the furanyl
group having oxygen atom with ability to induce
hydrogen bond interaction resulted in higher binding
affinity than compound 19, it could be expected that
hydrogen bond donor to ligand might be existed in the
binding pocket. Although the steric effect of substituents
is superior to electronic effect, the hydrogen bond inter-
action with the residue of SERT would compensate, to
some extent, for the drawback resulted from steric
repulsion.
tion binding constant (K ) values were subsequently
i
calculated from IC values using the Cheng–Prusoff
0
5
1
9
equation. Table 1illustrates the structures and the in
vitro binding affinities of eleven 4-substituted deriva-
tives of 6-NQ for the 5-HT transporter, including 6-NQ,
fluoxetine, and paroxetine used as reference compounds.
Discussion
N-Formylated 4-halo-6-NQs 10 and 20 were synthesized
from 4-nitroaniline and diethylmalonate in four steps as
shown in Scheme 1. We synthesized new eleven 6-NQ
derivatives including 4-chloro-6-NQ (8). Eight 6-NQ
derivatives except 8, 9 and 21 were prepared from a key
tri-n-butylstannane intermediate 11 by Stille coupling.
Compound 21 was obtained by nucleophilic aromatic
substitution of 20 with pyrrolidine.
The last substituent effect is the electronic resonance
effect that influences electron density of quinoline ring.
The chemical shifts of H3 of quinoline in ppm are: 1,
7.01; 8, 7.14; 9, 7.81; 12, 7.96; 13, 7.06; 16, 6.95; 17, 6.77;
18, 7.25; 19, 7.05 and 21, 5.90. With regard to com-
pounds 16, 19 and 21 having similar size of the sub-
stituents and no direct electronic effect to the binding
site, ability of amino group (21) to donate nonpair
electrons to quinoline ring is likely to be superior to the
other two ligands 16, 19. There is, however, little differ-
ence in binding affinities among them.
The results of binding affinities of 10 compounds are
shown in Table 1. The binding affinities of the 10 6-NQ
derivatives are affected by steric hindrance, electronic
inductive effect and electronic resonance effect. First, we
found a propensity that the bulkier substituent was, the
lower binding affinity was. In other words, binding site
of SERT is sensitive to steric hindrance with C4 position
of 6-NQ. According to this propensity, 4-chloro-6-NQ
In summary, we synthesized 11 6-NQ derivatives and
performed in vitro test and SAR study. The substitution
on C4 position of 6-NQ is largely restricted due to steric
repulsion for the binding site. However, a group with
hydrogen bond acceptor would reduce disadvantage
provided by its bulkiness. We also found that the elec-
tron density of quinoline was not an important factor.
The binding affinity of 4-chloro-6-NQ (8) for SERT is
(8) substituted with the smallest group, that is, chlorine
atom showed the highest binding affinity among 11 6-NQ
derivatives. Surprisingly, the K value of 4-chloro-6-NQ
i
(8) was 0.03 nM for the rat cortical SERT. This is
approximately 6-fold higher than that of 6-NQ itself
and the best binding affinity among the SSRIs reported
until now, except for ADAM (2-((2-((dimethylamino)-
so potent in picomolar level (K =30 pM) that this
i
methyl)phenyl)thio)-5-iodoaniline) (K =0.013 nM) syn-
i
compound would be likely to serve as a new lead com-
pound in the development of potent SSRI. Therefore,
more modifications based on 4-chloro-6-NQ (8) are
currently being pursued.
2
0
thesized by Kung et al. Other halogen derivatives
(K value of 4-bromo-6-NQ (9)=0.37 nM, K value of
i
i
4
-iodo-6-NQ (10)=1.73 nM) for the rat cortical SERT.

814
B. S. Lee et al. / Bioorg. Med. Chem. Lett. 12 (2002) 811–815
Table 1. Structures and binding data on the 5-HT transporter of the quipazine derivativesa
Compd
Ki (nM)
Compd
Ki (nM)
8
0.03ꢂ0.01
17
126.86ꢂ14.53
9
0.37ꢂ0.03
1.73ꢂ0.02
1.82ꢂ0.09
18
5.23ꢂ0.53
12
19
67.24ꢂ9.49
1
1
3
4
21
61.12ꢂ17.53
—b
Fluoxetine (Prozac)
22.13ꢂ1.77
15
1.67ꢂ0.08
Paroxetine
0.53ꢂ0.08
16
60.03ꢂ17.17
1
6-Nitroquipazine
0.17ꢂ0.03
a
The values represent mean SEM of 3–4 separate experiments done in duplicate.
Not measured.
b

B. S. Lee et al. / Bioorg. Med. Chem. Lett. 12 (2002) 811–815
815
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This research was supported by the Korea Science and
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