Quinazolindione derivatives as potent 5-HT3A receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2009.04.029

5-HT_3A receptor antagonists have been used mainly for the treatment of nausea and vomiting. These days, the antagonists are of special interest due to their therapeutic potential to treat other diseases such as depression, psychotic disorder, d

Full text of DOI:10.1016/j.bmc.2009.04.029

Bioorganic & Medicinal Chemistry 17 (2009) 4793–4796
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Quinazolindione derivatives as potent 5-HT3A receptor antagonists
Byung-Hwan Lee a, Min Jung Choi b, Mi Na Jo b, Hee Jeong Seo b, Seung-Yeol Nah a, Yong Seo Cho b,
Ghilsoo Nam b, Ae Nim Pae b, Hyewhon Rhim b, , , Hyunah Choo b, ,
*
*
a Department of Physiology, College of Veterinary Medicine and Bio/Molecular Informatics Center, Konkuk University, Seoul 110-460, Republic of Korea
b Center for Chemoinformatics Research, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
5-HT3A receptor antagonists have been used mainly for the treatment of nausea and vomiting. These days,
the antagonists are of special interest due to their therapeutic potential to treat other diseases such as
depression, psychotic disorder, drug abuse, and irritable bowel syndrome. To discover novel 5-HT3A
receptor antagonists, we screened our in-house small molecule library, resulting in identifying the quin-
azolindione derivatives as potent 5-HT3A receptor antagonists. For the purpose of structure–activity rela-
tionship study, 24 quinazolindione analogues were biologically evaluated against 5-HT3A receptor.
Among those, KKHT10612 shows the best antagonistic effect against 5-HT3A receptor with an IC50 value
Received 10 March 2009
Revised 14 April 2009
Accepted 15 April 2009
Available online 19 April 2009
Keywords:
5-HT3A Receptor
Antagonist
Quinazolindione derivatives
MDL72222
of 0.8
lM which is comparable with that of the reference compound, MDL72222, and selectivity over
T-type calcium channel as well.
Ó 2009 Elsevier Ltd. All rights reserved.
Calcium channel
Serotonin
Bemesetron
1. Introduction
The physiological and pathological roles of 5-HT3 receptors have
been suggested to be involved in pain transmission, vomiting, mood
disorders, drug abuse, anxiety, and alcohol dependence.7,8 Specific
5-HT3 receptor antagonists (ondansetron, granisetron, tropisetron,
and bemesetron) are effective in control of chemotherapy related
nausea and vomiting (Fig. 1). Although these drugs are the first-line
treatment choice as antiemetic agents for many cancer patients,
they can cause adverse effects such as headache, constipation, diar-
rhea, asthenia, and somnolence.9,10 Therefore, the investigation of
new 5-HT3 receptor antagonists is still needed to provide a wide
selection range of long-term usable antiemetic agents.
The 5-hydroxytryptamine (5-HT) receptors are divided into se-
ven distinct subfamilies (5-HT1 to 5-HT7) by their structure and
operational system. Among the subfamilies of 5-HT receptors, the
5-HT3 receptor is the only ligand-gated ion channel (LGIC), and
all other 5-HT receptors are G-protein-coupled receptors (GPCRs).1
5-HT3 Receptors are cationic ion channels that produce a transient
inward current upon activation by serotonin or its agonists and
share structural similarity with other LGICs such as GABAA, glycine,
and nicotinic acetylcholine receptors.2,3
After the 5-HT3A subunit was first cloned,4 four additional sub-
units (5-HT3B, 5-HT3C, 5-HT3D, and 5-HT3E) have been added to the
5-HT3 receptor family.5,6 5-HT3A, 5-HT3B, and 5-HT3C subunits exist
in both the central nervous system (CNS) and peripheral nervous
system (PNS), whereas 5-HT3D and 5-HT3E subunits exist only in
peripheral tissue.6 The 5-HT3 receptor consists of a pentamer,
and each subunit includes a large N-terminal extracellular domain,
four hydrophobic transmembrane domains (M1–M4) and a short
C-terminal domain. The 5-HT3 receptor exists either as a 5-HT3A
homomer or a heteromer of 5-HT3A and 5-HT3B, and in both cases
the functional activity of the 5-HT3 receptor is provided by the
common 5-HT3A subtype. All other subunit subtypes must hetero-
pentamerize with 5-HT3A subunits to form functional channels.4,7
There have been a lot of efforts to search for new scaffolds active
against the 5-HT3 receptor, especially 5-HT3A receptor, which is a
basic component of homomeric or heteromeric pentamers. To dis-
cover a new 5-HT3 receptor antagonist, an in-house small molecule
library was screened against the 5-HT3A receptor, which was previ-
ously designed and synthesized to discover T-type calcium channel
blockers.11 Some quinazolindione derivatives show potent antago-
nistic activities against 5-HT3A receptor. Herein, we report the bio-
logical evaluation of novel 5-HT3 receptor antagonists.
2. Results and discussion
2.1. Chemistry
* Corresponding authors. Tel.: +82 2 958 5923; fax: +82 2 958 5189 (H.R.);
tel.: +82 2 958 5157; fax: +82 2 958 5189 (H.C.)
The quinazolindione analogues were synthesized starting from
dimethyl 2-aminoterephthlate 1 in total 4 or 5 steps in previously
reported method (Schemes 1 and 2).11 The compound 1 was
E-mail addresses: hrhim@kist.re.kr (H. Rhim), hchoo@kist.re.kr (H. Choo).
These co-corresponding authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.029

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B.-H. Lee et al. / Bioorg. Med. Chem. 17 (2009) 4793–4796
H
N
O
O
O
O
O
O
N
N
N
N
N
N
Cl
N
N
N
H
Cl
ondansetron
granisetron
tropisetron
bemesetron
Figure 1. Specific 5-HT3 receptor antagonists.
O
O
CO2Me
NH2
R1
R1
N
R1NCO
N
TEA, dioxane
10% NaOH
dioxane
MeO2C
MeO2C
N
H
O
HO2C
N
H
O
1
2
3
triphosgene
toluene
10% NaOH
dioxane
CO2Me
NCO
CO2Me
O
R1NH2
TEA, dioxane
MeO2C
MeO2C
N
HN R1
H
4
5
Scheme 1. Synthesis of the key intermediate 3.11
treated with R1-substituted isocyanates and TEA in 1,4-dioxane to
give the corresponding quinazolindiones 2 in 41–90% yields. Com-
pounds 2, thus obtained, were hydrolyzed to the corresponding
key intermediates 3 in 90–99% yields. When R1 was an alkyl group,
on the other hand, the compound 1 was converted to the isocya-
nate 4, which was treated with alkyl amines and TEA in 1,4-diox-
ane to give the corresponding ureas 5 in 37–61% yields. The
compounds 5 underwent cyclization to afford the key intermedi-
ates 3 in 53–98% yields (Scheme 1).
The key intermediate 3 was converted to the corresponding acyl
chloride, which was treated with various R2-substituted alkyl
amines in DCM to give the desired products in 13–60% yields
(Scheme 2).
Thus, 24 quinazolindione analogues (KKHT compounds) which
have three variants such as R1 (morpholinyl, pyrrolidinyl, piperidinyl,
2-methylpiperidinyl or 2-ethylpiperidinyl), R2 (p-methoxyphenyl,
p-fluorophenyl, o-fluorobenzyl, m-fluorobenzyl, p-fluorobenzyl,
o-chlorobenzyl, p-chlorobenzyl, cyclohexyl, propyl or methyl), and
n (1 or 2) were prepared.
of 24 compounds show high efficacies with maximum% inhibition
(Vmax) values of over 96%. Among those, the most active compound
is KKHT10612 (Table 1, entry 12) with an IC50 value of 0.8 lM and
a hill coefficient (nH) of 1.7 which are comparable with those of
MDL72222 (bemesetron).
2.3. Discussion
5-HT3A Receptor antagonists have been used mainly for the
treatment of nausea and vomiting. These days, the antagonists
are of special interest due to their therapeutic potential to treat
other diseases such as depression, psychotic disorder, drug abuse,
irritable bowel syndrome and so on. Therefore, there has been
much attention paid to discover novel 5-HT3A receptor antago-
nists. The quinazolindiones, which were obtained during screen-
ing of our in-house small molecule library against 5-HT3A
receptor, are a novel scaffold among the 5-HT3A receptor antago-
nists. The structure–activity relationship study of the quinazolin-
dione derivatives has been done with three variants such as R1, R2
and n (Table 1). When n was fixed as 1 (entries 1–19), the com-
pounds with a piperidinyl group as R1 (entries 5–17) show more
potent activities against 5-HT3A receptor than the compounds
with a morpholinyl group or pyrrolidinyl group (entries 1–4).
Thus, with R1 fixed with piperidinyl group (entries 5–17), various
R2 substituents gave almost similar IC50 values. Among those,
KKHT10612 (entry 12) with p-methoxybenzyl group at R2 shows
the best inhibition against 5-HT3A receptor with an IC50 value of
2.2. Biological activities
The biological activities of the synthesized quinazolindione ana-
logues were evaluated against 5-HT3A receptor which is stably ex-
pressed in Xenopus oocytes containing cRNA of 5-HT3A receptor. As
a positive control, MDL72222 (bemesetron) was used, which is cur-
rently in phase II clinical trial for treatment of nausea, vomiting
and migraine.12 The biological activities are summarized in Table
0.8 lM, which is comparable with that of the reference com-
1. The range of IC50 values and hill coefficients are 0.8–57.0
and 0.8–5.3, respectively. Most synthesized compounds exhibit po-
tent antagonistic activities against the 5-HT3A receptor, and 18 out
l
M
pound, MDL72222. A methyl subsituent on the piperidine has
no effect on the activity (entry 19), but an ethyl group affects it
negatively (entry 18). On the other hand, when n = 2 (entries
O
O
R1
R1
N
N
1) SOCl2 or (COCl)2
2)
H
N
N
R2
HO2C
N
H
O
N
H
O
NH2
N
n
R2
n
O
6
3
KKHT compounds
Scheme 2. Synthesis of the final products, KKHT compounds.11

B.-H. Lee et al. / Bioorg. Med. Chem. 17 (2009) 4793–4796
4795
Table 1
Antagonistic activities against 5-HT3A receptor
O
R1
N
H
N
N
R2
N
H
O
n
O
KKHT compounds
Entry
Sample code
n
R1
R2
Vmax
34.5 37.1
IC50 (n = 6–7)
nH
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
KKHT10103
KKHT10109
KKHT10118
KKHT10318
KKHT10603
KKHT10606
KKHT10607
KKHT10608
KKHT10609
KKHT10610
KKHT10611
KKHT10612
KKHT10613
KKHT10614
KKHT10615
KKHT10617
KKHT10618
KKHT11018
KKHT11118
KKHT20218
KKHT20713
KKHT20718
KKHT20818
KKHT20918
MDL72222 (bemesetron)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
—
Morpholinyl
Morpholinyl
Morpholinyl
Pyrrolidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
Piperidinyl
2-Ethylpiperidinyl
2-Methylpiperidinyl
Morpholinyl
2-Methylpiperidinyl
2-Methylpiperidinyl
2-Ethylpiperidinyl
Piperidinyl
p-Fluorophenyl
p-Fluorobenzyl
p-Chlorobenzyl
p-Chlorobenzyl
p-Fluorophenyl
p-Methoxyphenyl
o-Fluorobenzyl
m-Fluorobenzyl
p-Fluorobenzyl
o-Methoxybenzyl
m-Methoxybenzyl
p-Methoxybenzyl
Cyclohexyl
57.0 21.9
19.8 1.6
15.8 2.8
7.5 2.5
1.9 0.4
1.6 0.2
2.1 0.6
2.4 0.8
1.9 0.4
1.4 0.2
2.7 0.6
0.8 0.1
2.0 0.3
2.2 0.2
2.2 0.2
2.1 0.2
3.3 0.5
13.6 2.9
2.8 0.2
1.3 0.1
5.9 2.5
11.6 1.4
4.8 0.2
5.7 0.6
0.77 0.16
0.8 0.3
1.2 0.1
1.6 0.4
0.9 0.1
0.9 0.1
1.4 0.2
1.4 0.4
1.0 0.2
1.0 0.2
1.1 0.2
1.3 0.3
1.7 0.2
1.1 0.1
1.3 0.1
1.4 0.1
1.8 0.3
1.2 0.2
1.4 0.3
1.2 0.1
5.3 1.5
1.1 0.4
1.5 0.2
1.4 0.1
1.5 0.2
1.25 0.2
80.0 2.7
78.5 6.2
103.2 12.8
107.9 6.0
100.6 3.6
100.2 9.6
107.5 11.5
104.6 7.5
103.1 5.1
107.4 8.5
96.4 2.9
98.8 4.2
103.7 3.6
103.2 3.3
96.0 3.9
106.4 2.5
104.0 2.5
104.0 2.5
52.5 0.6
86.5 11.5
85.7 4.3
99.5 1.6
98.2 4.5
99.6 7.7
Propyl
Methyl
o-Chlorobenzyl
p-Chlorobenzyl
p-Chlorobenzyl
p-Chlorobenzyl
p-Chlorobenzyl
Cyclohexyl
p-Chlorobenzyl
p-Chlorobenzyl
p-Chlorobenzyl
—
Positive control
—
20–24), the compounds with a morpholinyl group as R1 (entry
20) show very potent activity with an IC50 value of 1.3 M, but
ion channels, pharmacokinetics and therapeutic effect are in
progress.
l
low efficacy with a Vmax value of 52.5%. In other cases with
n = 2 (entries 21–24), the activities were decreased compared
with the congeners with n = 1.
Table 2
The antagonistic activities of KKHT compounds against 5-HT3A
receptor were compared with those against T-type calcium chan-
nel (Table 2). It is worth to note that selectivity between 5-HT3A
receptor and T-type calcium channel is achieved depending on
the substituents such as R1, R2 and n. Thus, while all 24 compounds
investigated in this study show moderately to potent antagonistic
activities against 5-HT3A receptor, only 6 compounds are moderate
active against T-type calcium channel with a1G subunit and other
18 compounds show no significant activities. Among the 6 com-
pounds active against both 5-HT3A receptor and T-type calcium
channel, KKHT10608 and KKHT10609 show selective antagonistic
activity against 5-HT3A receptor whereas KKHT10318, 11018 and
20918 are more active against T-type calcium channel. KKHT10618
is the only compound which shows similar activities against
5-HT3A receptor and T-type calcium channel with IC50 values of
Comparison with activities of KKHT compounds against 5-HT3A receptor and T-type
calcium channel
Entry
Sample code
5-HT3A
IC50 in
T-type calcium
11
channel (a1G
)
lM
IC50 in lM
a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
KKHT10103
KKHT10109
KKHT10118
KKHT10318
KKHT10603
KKHT10606
KKHT10607
KKHT10608
KKHT10609
KKHT10610
KKHT10611
KKHT10612
KKHT10613
KKHT10614
KKHT10615
KKHT10617
KKHT10618
KKHT11018
KKHT11118
KKHT20218
KKHT20713
KKHT20718
KKHT20818
KKHT20918
MDL72222 (bemesetron)
57.0 21.9
19.8 1.6
15.8 2.8
7.5 2.5
1.9 0.4
1.6 0.2
2.1 0.6
2.4 0.8
1.9 0.4
1.4 0.2
2.7 0.6
0.8 0.1
2.0 0.3
2.2 0.2
2.2 0.2
2.1 0.2
3.3 0.5
13.6 2.9
2.8 0.2
1.3 0.1
5.9 2.5
11.6 1.4
4.8 0.2
5.7 0.6
0.77 0.16
—
a
—
a
—
2.46 0.22
a
—
a
—
a
—
16.87 2.38
9.23 0.68
a
—
a
—
a
—
a
—
3.3 and 2.08 lM, respectively. Importantly, the most active com-
pound against 5-HT3A receptor, KKHT10612, shows no significant
activity against T-type calcium channel.
a
—
a
—
a
—
2.08 0.36
3.38 0.36
3. Conclusion
a
—
a
—
A focused small molecule library of quinazolindione derivatives
was biologically evaluated to develop novel 5-HT3A receptor antag-
onists. Among 24 compounds, KKHT10612 shows the most potent
antagonistic activity against 5-HT3A receptor and selectivity over
T-type calcium channel. We discovered the quinazolindione com-
pounds as novel 5-HT3A receptor antagonists with a new scaffold.
Further evaluations of KKHT10612 such as selectivity for other
a
—
a
—
a
—
1.95 0.25
Positive control
NDb
a
Not active enough to be tested for IC50 against T-type calcium channel.
Not determined.
b

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B.-H. Lee et al. / Bioorg. Med. Chem. 17 (2009) 4793–4796
milling two concentric wells into the chamber bottom (diameter/
4. Materials and methods
4.1. Synthetic experimental
height: upper well, 8/3 mm; lower well, 6/5 mm) and gluing plastic
mesh (ꢁ0.4 mm grid diameter) onto the bottom of the upper well.
The perfusion inlet (ꢁ1 mm in diameter) was formed through the
wall of the lower well, and a suction tube was placed on the edge
of the upper well. The oocyte was placed on the net that separated
the upper and lower wells, with the net grids serving to keep the
oocyte in place during the electrophysiological recordings. Oocytes
were impaled with two microelectrodes filled with 3 M KCl
All the compounds were synthesized in previously reported
method.11
4.1.1. Spectroscopic data of 3-(4-methoxybenzyl)-N-(3-(2-
piperidin-1-yl)ethyl)-2,4-dioxoquinazoline-7-carboxamide
(KKHT10612)
(0.2–0.7 MX). Recordings were performed in ND96 solution. The
1H NMR (300 MHz, DMSO-d6) d 11.63 (s, 1H), 8.57 (s, 1H), 7.99
(d, J = 8.1 Hz, 1H), 7.57 (d, J = 10.1 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H),
6.85 (d, J = 8.6 Hz, 2H), 5.01 (s, 2H), 3.70 (s, 3H), 3.37-3.34 (m,
2H), 2.44–2.36 (m, 6H), 1.48–1.36 (m, 6H); 13C NMR (75 MHz,
DMSO-d6) d 165.5, 162.0, 158.9, 150.7, 141.2, 139.8, 129.8, 128.1,
121.0, 115.8, 115.1, 114.2, 57.9, 55.5, 54.5, 43.2, 37.6, 26.1, 24.5;
electrophysiological experiments were performed at room temper-
ature using an Oocyte Clamp (OC-725C; Warner Instruments,
Hamden, CT, USA) and stimulation and data acquisition were con-
trolled by pClamp 8 (Axon Instruments, Union City, CA, USA).
Membrane currents were recorded at a holding potential of
ꢀ80 mV.
IR (KBr) 3264, 3195, 1712, 1662 cmꢀ1
.
4.2.5. Data analysis
4.2. Biological assay
To obtain concentration–response curves of the effect of drugs
on I5-HT, the peak amplitudes at different concentrations of drugs
were plotted and then fitted to the following Hill equation using
the Origin software (OriginLab Corp, Northampton, MA, USA): Re-
sponse = Vmax ꢀ Vmin/1 + (IC50/[A]nH) + Vmin, where Vmax and Vmin
are maximal and minimal responses, respectively. [A] is concentra-
tion of drugs and nH is the Hill coefficient. IC50 is the concentration
of drugs required to decrease the response by 50%. All values are
presented as means S.E.M.
4.2.1. Materials
The mouse 5-HT3A receptor cDNA was kindly provided by Dr. D.
Julius (University of California San Francisco, CA, USA).
4.2.2. Preparation of Xenopus oocytes
Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor,
MI, USA). Their care and handling were in accordance with the
highest standards of institutional guidelines. For isolation of oo-
cytes, frogs were anesthetized with an aerated solution of 3-amino
benzoic acid ethyl ester. Oocytes were surgically removed and sep-
arated by collagenase treatment followed by agitation for 2 h in a
Ca2+-free medium containing 82.5 mM NaCl, 2 mM KCl, 1 mM
MgCl2, 5 mM HEPES, 2.5 mM sodium pyruvate, 100 units/ml peni-
Acknowledgments
This work was supported by KIST Core-Competence Program
and Brain Research Center of the 21st Century Frontier Research
Program (M103KV010007-08K2201-00710), the Republic of
Korea.
cillin and 100 lg/ml streptomycin. Stage V–VI oocytes were col-
lected and stored in ND96 (96 mM NaCl, 2 mM KCl, 1 mM MgCl2,
1.8 mM CaCl2, and 5 mM HEPES, pH 7.5) supplemented with
0.5 mM theophylline and 50 lg/ml gentamicin. This oocyte-con-
taining solution was maintained at 18 °C with continuous gentle
shaking and changed daily. Electrophysiological experiments with
oocytes were performed within 5–6 days of their isolation.
References and notes
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4.2.3. cRNA preparation of 5-HT3A receptor and microinjection
The 5-HT3A DNA constructs were linearized at the 30 end by SalI
digestion, and run-off transcripts were prepared using the methyl-
ated cap analog, m7G(50)ppp(50)G. For generation of wild-type
cRNAs, recombinant plasmids containing the wild-type 5-HT3A
receptor cDNA were linearized by digestion with appropriate
restriction enzymes. All cRNAs were prepared using T3 RNA poly-
merase and the mMessage mMachine transcription kit (Ambion,
Austin, TX, USA). The final cRNA products were resuspended at
3. Lummis, S. C. Biochem. Soc. Trans. 2004, 32, 535.
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1 lg/l
l with RNase-free water and stored at –80 °C until use.13
The absence of degraded RNA was confirmed by denaturing aga-
rose gel electrophoresis followed by ethidium bromide staining.
40 nl of cRNAs were injected into the animal or vegetal pole of each
oocyte using a 10
Francisco, CA, USA) fitted with a tapered glass pipette tip that
was 15-20
m in diameter (Neuropharm).14
ll VWR microdispenser (VWR Scientific, San
l
14. Lee, B. H.; Lee, J. H.; Lee, S. M.; Jeong, S. M.; Yoon, I. S.; Lee, J. H.; Choi, S. H.; Pyo,
M. K.; Rhim, H.; Kim, H. C.; Jang, C. G.; Lee, B. C.; Park, C. S.; Nah, S. Y.
Neuropharmacology 2007, 52, 1139.
4.2.4. Data recording
A custom-made Plexiglas net chamber was used for two-elec-
trode voltage-clamp recordings. The chamber was constructed by