Novel quinazolinone derivatives as 5-HT7 receptor ligands

Article abstract of DOI:10.1016/j.bmc.2007.11.049

5-HT₇ receptor antagonists generated antidepressant-like effects in animal model and the involvement of the 5-HT₇ receptor in other pathophysiological mechanisms such as thermoregulation, learning and memory, and sleep has been highl

Full text of DOI:10.1016/j.bmc.2007.11.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2570–2578
Novel quinazolinone derivatives as 5-HT7 receptor ligands
Yong Ho Na,a,c Sung Ho Hong,a,c Jung Hyang Lee,a Woo-Kyu Park,b Du-Jong Baek,c
Hun Yeong Koh,d Yong Seo Cho,a Hyunah Chooa,*, and Ae Nim Paea,*,
aLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
bPharmaceutical Screening Research Team, Korea Research Institute of Chemical Technology, Daejon 305-343, Republic of Korea
cDepartment of Chemistry, College of Natural Sciences, Sangmyung University, Seoul 110-743, Republic of Korea
dDepartment of Chemistry, Inha University, Nam-gu, Incheon 402-751, Republic of Korea
Received 5 September 2007; revised 16 November 2007; accepted 17 November 2007
Available online 22 November 2007
Abstract—5-HT7 receptor antagonists generated antidepressant-like effects in animal model and the involvement of the 5-HT7 recep-
tor in other pathophysiological mechanisms such as thermoregulation, learning and memory, and sleep has been highlighted by var-
ious studies. As one of our efforts to discover a new type of 5-HT7 receptor antagonists, we here report on the synthesis and binding
affinities to the 5-HT7 receptor of the quinazolinone library 1, which was designed with various substituents (X, Y, R1, and R2) on
the aromatic rings and different carbon chain length. Total 85 compounds of the quinazolinone library 1 were synthesized and the
binding affinities of all the synthesized compounds were obtained by radioligand binding assay for the 5-HT7 receptor. Among the
85 compounds, 24 compounds show very good binding affinities with IC50 values below 100 nM. Mainly the compounds with IC50
values below 100 nM have o-OMe or o-OEt as R2 substituent. The compound with the best binding affinity is 1-68 of which the IC50
value is 12 nM. In in vivo animal study, some synthesized compounds really have the antidepressant activity in the forced swimming
test in mice.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
in hippocampal neurons possibly results in an effect that
can be of importance for hippocampal function and
mood regulation. Due to the availability of selective
antagonists and knockout mice, recent studies suggest
that the 5-HT7 receptor is involved in thermoregulation,
circadian rhythm, learning and memory, hippocampal
signaling, sleep, and endocrine regulation. It is unclear
how receptor blockade could lead to an antidepressant
effect. However, the direct actions of antidepressants on
the 5-HT7 receptor and the reversal of sleep disturbances
were observed in depressed patients after the receptor
blockade, which leads to suggest that a 5-HT7 receptor
antagonist may be sufficient to treat depression.3
Serotonin (5-hydroxytryptamine, 5-HT) is a major neu-
rotransmitter that plays important roles in physiological
and pathophysiological processes such as memory, ther-
moregulation, sleep, depression, and so on.1 There are
seven types of 5-HT receptors, 5-HT1–5-HT7, which
are redivided to 14 subtypes. Among the 14 subtypes
of 5-HT receptors, the 5-HT7 receptor is the most
recently cloned in 1993.2 The 5-HT7 receptor is found
in the brain, mainly in the hypothalamus, thalamus,
hippocampus, and cortex.3 The 5-HT7 receptor is a G
protein-coupled receptor (GPCR) protein and found
to stimulate cAMP production and activate the
extracellular signal-regulated kinase (ERK) through a
mechanism that is dependent on a Ras monomeric
GTPase. The stimulation of ERK by the 5-HT7 receptor
Up to date, there have not been many 5-HT7 receptor
antagonists developed.4,5 DR-4004 and DR-4485 by
Meiji Seika6 and SB-269970 and SB-656104 by Glaxo-
SmithKline7 are being developed for the treatment of
depression and sleep disorders (Fig. 1). Due to the phe-
nolic hydroxyl group, SB-269970 is metabolized by
Phase II metabolic mechanism and excreted from the
body.8 To overcome the defect, studies were carried
out on replacing the phenolic moiety in SB-269970 with
metabolically more stable bioisosteres, resulting in the
introduction of SB-656104.7b
Keywords: Quinazolinone derivatives; Small molecule library; 5-HT7
receptor; 5-HT7 receptor antagonist.
*
Corresponding authors. Tel.: +82 2 958 5157 (H.C.); tel.: +82 2 958
5185 (A.N.P.); fax: +82 958 5189; e-mail addresses:
hchoo@kist.re.kr; anpae@kist.re.kr
These authors contributed equally to this work.
2
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.049

Y. H. Na et al. / Bioorg. Med. Chem. 16 (2008) 2570–2578
2571
N
O
N
N
N
O
N
O
H
N
H
DR-4004
DR-4365
O
N
S
O
N
Cl
N
H
S
HO
N
N
O
O
O
SB-269970
SB-656104
Figure 1. Selective 5-HT7 receptor antagonists.
In order to find 5-HT7 receptor antagonists, our in-
house small molecule library, which consists of com-
pounds with a quinazolinone or a sulfonamide as core
structure, was assayed against the 5-HT7 receptor.
Among the compounds of the small molecule library,
some quinazolinone derivatives were found to show
binding affinities to the 5-HT7 receptor. Based on the
structure of the quinazolinone derivatives, a focused
small molecule library 1 was designed (Fig. 2). In this
paper, we describe the synthesis of the quinazolinone li-
brary 1 and the binding affinities to the 5-HT7 receptor.
acids 4 possessing various arylpiperazine moieties
(Fig. 2). The anthranilic acids 2 contain anthranilic acid
2a, 4-fluoroanthranilic acid 2b, and 5-fluoroanthranilic
acid 2c. The anilines 3 have five different substituents
as R1 such as H, p-F, o-OMe, m-OMe, and p-OMe.
The arylpiperazinylalkanoic acids 4 have 19 different
substituents as R2 which are shown in Figure 2. And
the carboxylic acids 4 have different alkyl chains (n = 0
or 1) like arylpiperazinylpropanoic acids and arylpiper-
azinylbutanoic acids.
Anthranilic acids 2 and anilines 3 are commercially
available, but no arylpiperazinylalkanoic acids 4 are
available. Therefore, arylpiperazinylalkanoic acids 4
were obtained from the commercially available arylpip-
erazines 5 (Scheme 1). The arylpiperazines have substit-
uents as R2. The substituents are H, F, Cl, dichloro,
trifluoromethyl, methyl, nitro, dimethyl, methoxy, eth-
oxy, and acetyl. Each arylpiperazine 5 was treated with
2. Results
2.1. Chemistry
The quinazolinone library 1 is composed of three build-
ing blocks: anthranilic acids 2, anilines 3, and alkanoic
O
R1
X
N
H
N
Y
N
N
n
R2
O
N
1
NH2
HO
X
Y
CO2H
NH2
N
R2
n
O
N
R1
2
3
4
2a X = Y = H
2b X = H, Y = F
2c X = F, Y = H
3a R1 = H
4 n = 0 or n = 1
3b R1 = p-F
3c R1 = o-OMe
R2 = H, o-F, p-F, o-Cl, p-Cl, 2,3-Cl2,
m-CF3, p-Me, p-NO2,
2,3-Me2, 2,4-Me2, 2,5-Me2,
2,6-Me2, 3,4-Me2,
3d R1 = m-OMe
3e R1 = p-OMe
o-OMe, m-OMe, p-OMe, o-OEt,
p-Ac
Figure 2. Three building blocks 2, 3, 4 of the quinazolinone library 1.

2572
Y. H. Na et al. / Bioorg. Med. Chem. 16 (2008) 2570–2578
HN
R2
NaHCO3, EtOH
EtO
N
N
n
R2
EtO
Br
n
O
N
O
6
5
7
59-89%
5% NaOH, Dioxane
20-81%
HO
N
R2
n
O
N
4
Scheme 1. Synthesis of the arylpiperazinylalkanoic acids 4.
ethyl 3-bromopropanoate 6 (or ethyl 4-bromobutano-
ate) and NaHCO3 in EtOH under reflux to give the cor-
responding compound 7 in 59–89% yield. Compounds 7
underwent hydrolysis under basic conditions to afford
the arylpiperazinylalkanoic acids 4 in 20–81% yields.
piperazinylalkanoic acids 4 when treated with EDCI,
HOBt, and N-methylmorpholine (NMM) in methylene
chloride at room temperature to afford the correspond-
ing compounds of the quinazolinone library 1 in 17–85%
yields. Total 85 compounds were synthesized with three
building blocks, each of which has different substituents.
All the synthesized 85 compounds were assigned num-
bers as 1-01–1-85, respectively, as shown in Tables 1–3.
The synthesis of the quinazolinone library 1 was started
from the formation of quinazolinone moiety (Scheme 2).
Quinazolinones 9 were synthesized by an efficient three-
component, one-pot reaction from anthranilic acids 2,
anilines 3, and N-Boc-glycine 8.9 Anthranilic acid 2
and N-Boc-glycine 8 in the presence of the coupling re-
agent (P(OPh)3) generated the intermediate benzoxazi-
none derivative, followed by the addition of aniline 3
to give the corresponding quinazolinone 9 in 47–69%
yields. The reaction is very sensitive to reaction temper-
ature. If the reaction temperature was over 70 ꢁC or be-
low 60 ꢁC, the yield was low. The optimum yield was
obtained between 60 ꢁC and 70 ꢁC. Quinazolinones 9
were deprotected by treating with TFA at room temper-
ature to give the primary amines 10 in 90–96% yields.
Primary amines 5 underwent amide coupling with aryl-
2.2. Binding assay to the 5-HT7 receptor
All the synthesized compounds of quinazolinone library
1 were biologically evaluated to estimate binding affini-
ties to the human recombinant 5-HT7 receptor stably ex-
pressed by HEK293 cell line through [3H]lysergic acid
diethylamide (LSD) binding assay. Among all the bio-
logical data, the results of the quinazolinone library 1
where n = 0 are shown in Table 1. Compound 1-01 with
a phenylpiperazine group shows binding affinity to the
5-HT7 receptor with an IC50 value of 940 nM. Com-
pounds 1-02 and 1-03 with o-Cl and p-Cl as R2 show
binding affinities with IC50 values of 510 nM and
NH2
O
R1
X
Y
CO2H
NH2
P(OPh)3, Pyr.
X
NHBoc
HO2C
+
+
N
47-69%
NHBoc
Y
N
R1
2
3
8
9
TFA, 90-96%
O
R1
X
Y
O
N
R1
N
H
N
EDCI, HOBt, NMM, 17-85%
X
N
N
N
n
HO
R2
N
n
NH2
R2
O
N
Y
O
N
4
1
10
Scheme 2. Synthesis of the quinazolinone library 1.

Y. H. Na et al. / Bioorg. Med. Chem. 16 (2008) 2570–2578
2573
Table 1. Binding affinities of the quinazolinone library 1 (n = 0) to the
5-HT7 receptor
Table 2. Binding affinities of the quinazolinone library 1 (n = 1,
R1 = H) to the 5-HT7 receptor
O
R1
O
R1
X
N
H
X
N
H
N
Y
N
N
N
n
R2
Y
N
N
n
R2
O
N
O
N
1
1
R1
R2
IC50 (nM)
Compound
n
X
Y
R1
R2
IC50 (nM)
Compound
n
X
Y
1-01
1-02
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
940
510
1-20
1-21
1-22
1-23
1-24
1-25
1-26
1-27
1-28
1-29
1-30
1-31
1-32
1-33
1-34
1-35
1-36
1-37
1-38
1-39
1-40
1-41
1-42
1-43
1-44
1-45
1-46
1-47
1-48
1-49
1-50
1-51
1-52
1-53
1-54
1-55
1-56
1-57
1-58
1-59
1-60
1-61
1-62
1-63
1-64
1-65
1-66
1-67
1-68
1-69
1-70
Methiothepin
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
650
400
2400
130
110
450
160
460
690
420
200
21
H
o-Cl
p-Cl
o-F
p-F
o-Cl
1-03
1-04
H
370
730
H
p-Me
1-05
1-06
H
2,3-Me2
2,4-Me2
3,4-Me2
o-OMe
m-OMe
p-OMe
o-OEt
p-NO2
p-Ac
95
640
m-Cl
p-Cl
H
1-07
1-08
H
1100
80
3,4-Cl2
2,3-Me2
2,4-Me2
2,5-Me2
3,4-Me2
o-OMe
m-OMe
p-OMe
o-OEt
m-CF3
p-Ac
H
1-09
1-10
H
1400
710
H
1-11
1-12
H
45
H
>10,000
9000
680
1-13
1-14
H
>10,000
2000
26
p-F
p-F
p-F
p-F
p-F
p-F
H
1-15
1-16
2,4-Me2
2,6-Me2
p-OMe
o-OEt
p-NO2
500
500
350
>10,000
930
770
770
52
1-17
1-18
2500
1300
7300
3.1
H
o-F
1-19
Methiothepin
F
F
p-F
o-Cl
F
F
m-Cl
p-Cl
190
2200
450
75
370 nM, respectively. Among compounds 1-04–1-07
with methyl or dimethyl groups as R2, compound 1-05
shows very potent binding affinity with an IC50 value of
95 nM. Among compounds 1-08, 1-09, and 1-10 with o-,
m-, and p-OMe as R2, 1-08 shows very good binding affin-
ity to the 5-HT7 receptor with an IC50 value of 80 nM,
while 1-09 shows only marginal binding affinity with an
IC50 value of 1400 nM. Compound 1-11 with o-OEt as
R2 shows the best binding affinity to the 5-HT7 receptor
with an IC50 value of 45 nM. In the case of p-NO2 and
p-Ac as R2, compounds 1-12 and 1-13 show no binding
affinity. Compounds 1-14–1-19, where p-fluoro substitu-
ent was added to the phenyl ring possessing an R1 substi-
tuent, show moderate to marginal binding affinities with
IC50 values between 500 nM and 7300 nM.
F
F
3,4-Cl2
2,3-Me2
2,4-Me2
2,5-Me2
3,4-Me2
o-OMe
m-OMe
p-OMe
o-OEt
m-CF3
p-Ac
F
F
940
1500
590
85
F
F
F
F
>10,000
1100
19
F
F
F
92
F
680
470
1000
420
200
130
1200
270
55
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
o-F
F
F
p-F
o-Cl
F
F
m-Cl
p-Cl
F
After obtaining the binding affinities of quinazolinone
derivatives 1-01–1-19, we synthesized compounds with
a longer carbon chain such as n = 1 and compounds
with fluorine substitution (X = F or Y = F) to determine
how the carbon chain length and the electronegative
fluorine affect the binding affinity to the 5-HT7 receptor.
Due to the high electronegativity and the small size, the
fluorine substituent is widely used in medicinal chemis-
try.10 Total 51 compounds from 1-20 to 1-70 were syn-
thesized and biologically evaluated to obtain binding
affinities to the 5-HT7 receptor. The results are shown
in Table 2. Compound 1-20 with no substituent in
aromatic rings shows binding affinity with an IC50 value
F
3,4-Cl2
2,3-Me2
2,4-Me2
2,5-Me2
3,4-Me2
o-OMe
m-OMe
p-OMe
o-OEt
m-CF3
p-Ac
F
F
620
200
260
120
920
350
12
F
F
F
F
F
F
F
74
F
590
3.1

2574
Y. H. Na et al. / Bioorg. Med. Chem. 16 (2008) 2570–2578
m-CF3 as R2 show also very good binding affinities with
IC50 values of 92 nM and 74 nM, respectively. The bind-
ing affinities of compounds 1-53 and 1-70 with p-Ac are
much improved compared with compounds 1-13 and 1-
36.
Table 3. Binding affinities of the quinazolinone library 7 (n = 1,
R1 = OMe) to the 5-HT7 receptor
Compound
n
X
Y
R1
R2
IC50 (nM)
1-71
1-72
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
H
H
H
F
H
H
F
o-OMe
o-OMe
o-OMe
o-OMe
o-OMe
m-OMe
m-OMe
m-OMe
m-OMe
m-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
o-OMe
o-OEt
o-OEt
o-OMe
o-OEt
o-OMe
o-OEt
o-OEt
o-OMe
o-OEt
o-OMe
o-OEt
o-OEt
o-OMe
o-OEt
210
29
1-73
1-74
110
79
According to the results of Tables 1 and 2, the com-
pounds with o-OMe or o-OEt as R2 show very good
binding affinities to the 5-HT7 receptor. We decided to
synthesize compounds 1-71–1-85 where R2 is fixed with
o-OMe or o-OEt and R1 is replaced with o-, m-, and p-
OMe instead of hydrogen. The results are shown in Ta-
ble 3. Among 15 compounds, 11 compounds show very
good binding affinities with IC50 values below 100 nM.
The other 4 compounds (1-71, 1-73, 1-79, and 1-84)
show also fairly good binding affinities to the 5-HT7
receptor with IC50 values of 210 nM, 110 nM, 230 nM,
and 190 nM, respectively. Among 15 compounds in Ta-
ble 3, the best compounds are 1-80 and 1-85 with the
same IC50 values of 16 nM.
H
H
H
H
F
1-75
1-76
F
48
98
H
H
H
F
1-77
1-78
55
91
1-79
1-80
H
H
H
H
F
230
16
F
1-81
1-82
H
H
H
F
97
29
1-83
1-84
49
H
H
190
16
1-85
Methiothepin
F
3.1
of 650 nM. In the case of halogen substituents such as
compounds 1-21–1-26, compounds 1-23–1-26 with chol-
orine substituents show better binding affinities to the 5-
HT7 receptor than compounds 1-21 and 1-22 with fluo-
rine substituents. Compound 1-24 with m-Cl as R2
shows good binding affinity with an IC50 value of
110 nM. Compounds 1-26–1-30 with dimethyl substitu-
ents show fairly good binding affinities with IC50 values
between 200 nM and 690 nM. Among compounds 1-31–
1-34 with alkoxy substituents, the binding affinities are
diverse according to the position of substituents. Com-
pound 1-32 with m-OMe as R2 shows no significant
binding affinity up to 10 lM, while compounds 1-31
and 1-34 with o-OMe and o-OEt show best binding
affinities to the 5-HT7 receptor with IC50 values of
21 nM and 26 nM, respectively. Compound 1-33 with
p-OMe shows only marginal binding affinity. Com-
pound 1-35 with m-CF3 shows moderate binding affinity
with an IC50 value of 350 nM and compound 1-36 with
p-Ac shows no significant binding affinity up to 10 lM.
2.3. Selectivities to other neurotransmitter receptors and
antidepressant effects
Compounds with various binding affinities as well as dif-
ferent R1 and R2 groups, such as 1-68, 1-85, 1-83, 1-61,
1-44, 1-52, 1-65, and 1-27 in order of decreasing binding
affinities, were selected to test how selectively those syn-
thesized compounds would bind to the 5-HT7 receptor
and whether they would have antidepressant effects in
animal model, that is, the forced swimming test in
mice.11 The binding affinities of the selected 8 com-
pounds were evaluated against the 5-HT1A, 5-HT2A, 5-
HT2C, and D2 receptors which are also target receptors
for antidepressant effects and/or antipsychotic effects.12
The results are shown in Table 4. Most of the com-
pounds bind more selectively to the 5-HT7 receptor than
other receptors tested such as 5-HT1A, 5-HT2A, 5-HT2C
and D2 receptors.
,
The antidepressant effects of the selected 8 compounds
were tested. The compounds were orally injected at a
dose of 100 mg/kg to mice in the force swimming test
and the total duration of immobility of the mice was
monitored. After administration of compounds 1-68,
1-85, 1-83, 1-52, 1-65, and 1-27, the total duration of
immobility was not reduced and was same as control.
After administration of compounds 1-44 and 1-61, the
total duration of immobility was reduced to 75.9% and
94.4%, respectively (Fig. 3). In the case of Prozacꢂ as
positive control, the duration of immobility was reduced
to 59.5%.
We have also synthesized compounds 1-37–1-53 with 7-
fluorine substituents (Y = F) in the quinazolinone ring
moiety and compounds 1-54–1-70 with 6-fluorine sub-
stituents (X = F). Compounds 1-37 and 1-54 with
hydrogens as R2 show moderate binding affinities with
IC50 values of 930 nM and 470 nM, respectively. Among
compounds 1-38–1-43 and 1-55–1-60 with halogen sub-
stituents, compound 1-40 with o-Cl and Y = F shows the
best binding affinity to the 5-HT7 receptor with an IC50
value of 52 nM. On the other hand, compounds 1-44–1-
47 and 1-61–1-64 with dimethyl substituents show very
good to moderate binding affinities with IC50 values be-
tween 55 nM and 1500 nM. The best compounds are 1-
44 and 1-61 with 2,3-dimethyl substituents with IC50 val-
ues of 75 nM and 55 nM, respectively. In the case of the
substituents such as alkoxy groups (compounds 1-48–1-
51 and 1-65–1-68), compounds 1-48, 1-51, 1-65, and 1-68
with o-alkoxy as R2 show very good binding affinities
with IC50 values of 85 nM, 19 nM, 120 nM, and
12 nM, respectively. Compound 1-68 shows the best
binding affinity to the 5-HT7 receptor among all the
synthesized compounds. Compounds 1-52 and 1-69 with
3. Discussion
The binding affinities of all the 85 synthesized com-
pounds were obtained by radioligand [3H]lysergic acid
diethylamide (LSD) binding assay for the 5-HT7 recep-
tor. Among the 85 compounds, 24 compounds show
very good binding affinities with IC50 values below
100 nM. Mainly the compounds with IC50 values below
100 nM have o-OMe or o-OEt as R2 substituent. Except

Y. H. Na et al. / Bioorg. Med. Chem. 16 (2008) 2570–2578
Table 4. Selectivities to 5-HT1A, 5-HT2A, 5-HT2C, and D2 receptors
2575
Compound
5-HT1A
IC50 (nM)
5-HT2A
IC50 (nM)
5-HT2C
IC50 (nM)
D2
IC50 (nM)
5-HT7
SIa
SIa
SIa
SIa
IC50 (nM)
1-27
1-44
1-52
1-61
1-65
1-68
1-83
1-85
500
400
140
220
960
500
370
120
1.1
5.3
1.5
4.0
8.0
42
1100
1700
2.4
23
460
1.0
45
6900
1500
6600
4000
5300
1000
1500
140
15
20
72
73
44
83
31
8.8
460
75
3400
1300
80
620
420
6.7
7.6
>83
>830
43
14
92
55
1.5
1.8
130
78
>10,000
>10,000
2100
220
120
12
1600
3800
>10,000
7.6
7.5
49
16
7900
490
>630
a SI, selectivity index = the binding affinity to the target receptor (in IC50)/the binding affinity to the 5-HT7 (in IC50).
120
of the fluorinated compounds 1-44, 1-52, 1-61, and 1-
69 where R2 is 2,3-dimethyl group and m-CF3. Com-
pounds 1-27 and 1-35 without fluorine substituent have
IC50 values of 460 nM and 350 nM, and the fluorinated
compounds 1-44, 1-52, 1-61, and 1-69 have IC50 values
of 75 nM, 92 nM, 55 nM, and 74 nM. And also, we have
tried to figure out an effect of substitution in the aro-
matic ring containing R1 group. We fixed R2 as o-
OMe or o-OEt and gave substitution of methoxy groups
as R1 at ortho, meta, and para positions. From the re-
sults of the binding assay, there is no effect of the aro-
matic position on the binding affinity to the 5-HT7
receptor. All the compounds with methoxy groups as
R1 show good binding affinities to the 5-HT7 receptor
(Table 3).
100
80
60
40
20
0
vehicle
prozac
1-27
1-44
1-61
Figure 3. Antidepressant effects of Prozacꢂ and synthesized com-
pounds 1-27, 1-44, and 1-61 on immobility in forced swimming test in
mice. Prozacꢂ and the tested compounds (100 mg/kg) were injected
orally (po) 60 min before the testing, and the total duration of
immobility was recorded during the last 5 min of the 6-min testing
period. Values are means SEM.
Compounds 1-27, 1-44, 1-52, 1-61, 1-65, 1-68, 1-83, and
1-85, which show good binding affinities, were selected
to test selectivity to other neurotransmitter receptors
and the in vivo efficacy in the animal model. Most of
the compounds bind more selectively to the 5-HT7
receptor than other receptors tested such as 5-HT1A
,
these substituents, 2,3-dimethyl, o-Cl, and m-CF3 sub-
stituents have a favorable effect on the binding affinity
to the 5-HT7 receptor. Compounds 1-05, 1-40, 1-44, 1-
52, 1-61, and 1-69 with 2,3-dimethyl, o-Cl or m-CF3 sub-
stituent as R2 show binding affinities with IC50 values
between 52 nM and 95 nM. Generally, substituents at
the para position as R2 have an unfavorable effect on
the binding affinity. The compounds with chlorines at
the para position show the least binding affinities among
the compounds with chlorines at ortho, meta, and para
positions. The compounds with other substituents such
as p-NO2 and p-Ac show almost no binding affinity. In
the case of OMe substituents, the compounds with meta
positions at the aromatic ring show no binding affinity.
When the carbon length was changed from n = 0 to
n = 1, the compounds with longer carbon chains show
better binding affinities than those with shorter carbon
chains. Compounds 1-08 and 1-11 with n = 0 show bind-
ing affinities with IC50 values of 80 nM and 45 nM,
while compounds 1-31 and 1-34 with n = 1 with IC50 val-
ues of 21 nM and 26 nM. And other compounds with
n = 1 also have better IC50 values. When fluorine substi-
tution is added to the quinazolinone ring like X = F or
Y = F, the overall binding affinities are equivalent or
better than the binding affinities of the compounds with-
out substitution in the quinazolinone ring (Table 2).
There is apparent improvement in the binding affinities
5-HT2A, 5-HT2C, and D2 receptors. Compound 1-68
with the best binding affinity to the 5-HT7 receptor
binds 42 to >830 times more selectively to the 5-HT7
receptor than other receptors such as 5-HT1A, 5-HT2A
,
5-HT2C, and D2 receptors (Table 4). The other com-
pounds also bind quite selectively to the 5-HT7 recep-
tors. The eight selected compounds were tested to
detect antidepressant effects in the forced swimming test
in mice. The forced swimming test is one of the most
widely used preclinical tests for detecting antidepressant
activity. It has been well established that the reduction
of immobility time in the forced swimming test depends
mainly on the enhancement of serotonin and catechol-
amine.13 Among the 8 compounds, two compounds 1-
44 and 1-61 show the antidepressant effects. These two
compounds will be further evaluated for the pharmaco-
kinetic profile and metabolic stability.
4. Conclusion
We have described the synthesis of the quinazolinone li-
brary 1 and their binding affinities to the 5-HT7 recep-
tor. Total 85 compounds were synthesized and among
those, 24 compounds show very good binding affinities
with IC50 values below 100 nM. The structure–activity
relationship study on the quinazolinone library 1 indi-

2576
Y. H. Na et al. / Bioorg. Med. Chem. 16 (2008) 2570–2578
cated that among the various substituents as R2, the
compounds with ortho methoxy and the ortho ethoxy
groups show the best binding affinities to the 5-HT7
receptor. We found that the quinazolinone derivatives
1-44 and 1-61 show antidepressant effects in the forced
swimming test as well as selective binding affinities to
the 5-HT7 receptor. The two compounds will be further
evaluated for the pharmacokinetic profile and metabolic
stability. In addition, a small molecule library with more
various substituents will be designed, synthesized, and
biologically evaluated against the 5-HT7 receptor.
1.99–1.92 (m, 2H); 13C NMR (CDCl3, 75.5 MHz) d
177.49, 155.94, 141.90, 128.70, 124.98, 122.97, 116.39,
59.61, 54.69, 51.05, 34.90, 22.77.
5.3. tert-Butyl (4-oxo-3-phenyl-3,4-dihydroquinazolin-2-
yl) methylcarbamate (9)
Triphenyl phosphite (4.8 mL, 18.2 mmol) was added
dropwise to the solution of anthranilic acid 2 (1.00 g,
7.29 mmol) and N-Boc-glycine 8 (1.28 g, 7.29 mmol) in
anhydrous pyridine (4 mL) under nitrogen atmosphere.
The resulting mixture was stirred at 70 ꢁC under nitro-
gen atmosphere. After 2 h, aniline
3
(798 mL,
5. Experimental
8.95 mmol) was added to the reaction mixture. The
resulting mixture was stirred at 70 ꢁC under nitrogen
atmosphere for 4 h. After the reaction completed, the
reaction mixture was concentrated and diluted with
EtOAc (100 mL). The solution was washed with satd
NaHCO3 (2· 50 mL) and dried over MgSO4. After con-
centration, the residue was purified with a mixture of
EtOAc, ether, and hexane (1:1:3) by silica gel column
chromatography to give the product (1.19 g) in 47%
All the commercially available reagents were obtained
from Aldrich and Fluka, and generally used without fur-
ther purification. H NMR and 13C NMR spectra were
1
obtained on Bruker Advance 400 (or 300) spectrometer.
Nuclear magnetic resonance spectra were acquired at
400 (or 300) MHz for 1H, and 100 MHz for 13C
NMR. HR-MS spectra were obtained on a JMS-700
mass spectrometer (Jeol). Analytical thin layer chroma-
tographies (TLC) were carried out on precoated silica
gel plates (Merck Kieselgel 60F254, layer thickness
0.25 mm). Flash column chromatographies were con-
ducted with silica gel grade 230–400 mesh (Merck Kie-
selgel 60 Art 9385).
yield: 1H NMR (CDCl3, 300 MHz)
d 8.30 (d,
J = 7.9 Hz, 1H), 7.81 (m, 2H), 7.52 (m, 4H), 7.29 (m,
2H), 6.05 (br s, 1H), 3.98 (d, J = 3.9 Hz, 2H), 1.47 (s,
9H).
5.4. 2-(Aminomethyl)-3-phenylquinazolin-4(3H)-one (10)
5.1. Ethyl 4-(4-(2-methoxyphenyl)piperazin-1-yl) butano-
ate (7)
Trifluoroacetic acid (2.5 mL) was added dropwise to a
solution of tert-butyl (4-oxo-3-phenyl-3,4-dihydroqui-
nazolin-2-yl) methylcarbamate 9 (550 mg, 1.56 mmol)
in CH2Cl2 (6 mL) at 0 ꢁC and the resulting mixture
was stirred at room temperature for 1 h. After comple-
tion of reaction, the reaction mixture was diluted with
EtOAc (50 mL) and the solution was washed with sat
NaHCO3 (3· 25 mL) and dried over MgSO4. After con-
centration, the product (377 mg) was obtained in 96%
Ethyl 4-bromobutanoate (1.01 g, 5.20 mmol) was added
to the solution of 1-(2-methoxyphenyl)piperazine (5)
(1.0 g, 5.20 mmol) in ethanol (25 mL) at room tempera-
ture and the resulting solution was refluxed under nitro-
gen atmosphere for 6 h. After the completion of
reaction, the mixture was cooled to room temperature.
Sat. NaHCO3 (50 mL) was added to the reaction mix-
ture and the resulting solution was extracted with
CH2Cl2 (3· 50 mL). The combined organic solution
was dried over MgSO4 and concentrated. The residue
was purified with 5% MeOH in CH2Cl2 by silica gel col-
umn chromatography to give the product (1.40 g) in
88% yield: 1H NMR (CDCl3, 300 MHz) d 7.01–6.84
(m, 4H), 4.15 (q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 3.10
(br s, 4H), 2.66 (br s, 4H), 2.50–2.35 (m, 4H), 1.87 (quin-
tet, J = 7.3 Hz, 2H), 1.78 (t, J = 7.1 Hz, 3H).
1
yield: H NMR (CDCl3, 300 MHz) d 8.31 (d, 7.6 Hz,
1H), 7.80–7.75 (m, 2H), 7.57–7.49 (m, 4H), 7.26–7.24
(m, 2H), 3.51 (s, 2H); 13C NMR (CDCl3, 75.5 MHz) d
162.37, 156.59, 147.40, 136.19, 134.86, 130.31, 129.81,
128.43, 127.40, 127.31, 127.16, 121.18, 45.17.
5.5. 4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)-N-((4-oxo-
3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)butanamide
(1-27)
EDCI (106.8 mg, 0.557 mmol) and HOBT (64.0 mg,
0.474 mmol) were added to a solution of 4-(4-(2,3-
5.2. 4-(4-(2-Methoxyphenyl)piperazin-1-yl)butanoic acid
(4)
dimethylphenyl)piperazin-1-yl)
butanoic
acid
4
(124 mg, 0.446 mmol) in anhydrous CH2Cl2 (3 mL) at
room temperature. The resulting mixture was stirred at
room temperature for 2 h. A solution of 2-(amino-
5% NaOH (8.5 mL) was added to the solution of ethyl 4-
(4-(2-methoxyphenyl)piperazin-1-yl)butanoate (7) (1.35 g,
4.41 mmol) in dioxane (3 mL) at room temperature.
The resulting solution was stirred at room temperature
for 2 h. After removing solvent under reduced pressure,
the residue was dissolved in minimum amount of water
and acidified with 6 N HCl solution until pH 6 or 5. The
precipitate was filtered and dried to give the product
(241 mg) in 20% yield: 1H NMR (DMSO-d6,
300 MHz) d 7.04–6.90 (m, 4H), 3.79 (s, 3H), 3.63–3.40
(m, 4H), 3.14–3.09 (m, 6H), 2.35 (t, J = 7.3 Hz, 2H),
methyl)-3-phenylquinazolin-4(3H)-one
10
(70 mg,
0.28 mmol) in CH2Cl2 (1 mL) and NMM (52 mL,
0.47 mmol) were added successively to the reaction mix-
ture at room temperature. The resulting mixture was
stirred for additional 2 h. After reaction was completed,
the reaction mixture was diluted with CH2Cl2 (50 mL)
and washed with aqueous NaHCO3, water, and brine,
successively. The organic layer was dried over MgSO4
and concentrated. The residue was purified with 10%

Y. H. Na et al. / Bioorg. Med. Chem. 16 (2008) 2570–2578
2577
methanol in EtOAc (or 7% methanol in CH2Cl2) by sil-
ica gel column chromatography to give the product
(107 mg) in 75% yield: H NMR (CDCl3, 300 MHz) d
128.28, 126.10, 125.31, 123.52 (d, J = 24.2 Hz), 122.69
(d, J = 8.5 Hz), 116.79, 112.47 (d, J = 23.6 Hz), 57.83,
53.86, 52.32, 42.40, 34.54, 22.69, 34.54, 22.69, 20.93,
14.25; IR (KBr) 3312, 3064, 2939, 2813, 1681, 1649,
1
8.31 (d, J = 6.6 Hz, 1H), 7.80–7.75 (m, 2H), 7.58–7.52
(m, 4H), 7.33 (br s, 1H), 7.28–7.25 (m, 2H), 7.07 (t,
J = 7.1 Hz, 1H), 6.91–6.80 (m, 2H), 4.07 (d,
J = 4.3 Hz, 1H), 2.91 (t, J = 4.7 Hz, 4H), 2.64 (br s,
4H), 2.52 (t, J = 7.0 Hz, 2H), 2.41 (t, J = 7.3 Hz, 2H),
2.27 (s, 3H), 2.21 (s, 3H), 1.95–1.90 (m, 2H); 13C
NMR (CDCl3, 75.5 MHz) d 173.06, 162.17, 152.30,
151.76, 146.87, 138.26, 135.57, 135.05, 131.52, 130.64,
130.27, 128.34, 127.58, 127.25, 126.08, 125.24, 121.38,
116.81, 57.84, 53.88, 52.37, 42.44, 34.57, 22.76, 20.90,
14.22; IR (KBr) 3282, 3062, 2939, 2814, 1681, 1638,
1609, 1590, 1473, 1261, 1012, 774, 695 cmÀ1; HR-MS
(TOF, ES+, M+H) Calcd for C31H36N5O2: 510.2869.
Found: 510.2867.
1611, 1591, 1545, 1485, 1237, 1013, 754, 696 cmÀ1
;
HR-MS (TOF, ES+, M+H) Calcd for C31H35FN5O2:
528.2775. Found: 528.2778.
5.8. [3H]LSD binding assay to serotonin 5-HT7 receptor
Membranes from stable CHO cell line expressing the hu-
manrecombinant5-HT7 serotoninreceptor(Perkin-Elmer
LifeandAnalyticalSciences, Boston, USA)wereused. For
5-HT7 receptor binding assay, cell membrane, 3 nM
[3H]LSD, and appropriate concentrations of test com-
pounds were added to 0.25 mL of 50 mM Tris–HCl (pH
7.4) buffer containing 10 mM MgCl2 and 0.5 mM EDTA.
The mixture was incubated for 90 min at 27 ꢁC, and the
reaction was terminated by rapid filtration through What-
man GF/C glass fiber filter presoaked in 0.3% polyethyl-
eneimine. The filter was covered with MeltiLex, sealed in
a sample bag followed by drying in the microwave oven,
and counted by MicroBeta Plus (Wallac, Finland). Non-
specific binding was determined in the presence of
0.5 lM methiothepin. Competition binding studies were
carried out with 7–8 varied concentrations of the test com-
pounds run in duplicate tubes, and isotherms from three
assays were calculated by computerized nonlinear regres-
sion analysis (GraphPad Prism Program, San Diego,
USA) to yield inhibition values (IC50).
5.6. 4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)-N-((7-fluoro-
4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)butana-
mide (1-44)
The procedure was same as that of synthesis of the
compound 1-27. 4-(4-(2,3-Dimethylphenyl)piperazin-1-
yl)butanoic acid 4 (115 mg, 0.416 mmol) and 2-(amino-
methyl)-7-fluoro-3-phenylquinazolin-4(3H)-one (70 mg,
0.26 mmol) were used and the product was obtained in
69% yield: 1H NMR (CDCl3, 300 MHz) d 8.33 (dd,
J = 8.7, 6.0 Hz, 1H), 7.63–7.57 (m, 3H), 7.45–7.38 (m,
2H), 7.32–7.22 (m, 3H), 7.11 (t, J = 7.8 Hz, 1H), 6.96–
6.92 (m, 2H), 4.10 (d, J = 4.5 Hz, 2H), 2.96 (t,
J = 4.5 Hz, 4H), 2.70 (br s, 4H), 2.58 (t, J = 6.9 Hz,
2H),2.45 (t, J = 7.5 Hz, 2H), 2.31 (s, 3H), 2.25 (s, 3H),
2.01–1.94 (m, 2H); 13C NMR (CDCl3, 75.5 MHz) d
173.07, 166.98 (d, J = 255 Hz), 161.41, 153.92, 151.68
(d, J = 12.9 Hz), 138.27, 135.38, 131.50, 130.68, 130.43,
130.37, 130.29, 128.29, 126.13, 125.32, 118.11, 116.81,
116.25 (d, J = 23.2 Hz), 112.77 (d, J = 22.1 Hz), 57.85,
53.88, 52.33, 42.52, 34.55, 22.69, 20.93, 14.23; IR
(KBr) 3282, 3062, 2942, 2812, 1683, 1642, 1607, 1590,
1576, 1546, 1485, 1279, 781, 696 cmÀ1; HR-MS (TOF,
ES+, M+H) Calcd for C31H35FN5O2: 528.2775. Found:
528.2776.
5.9. Forced swimming test in mice
The forced swimming test was performed according to
the methods described by Porsolt et al.14 Each mouse
was placed in a 25-cm glass cylinder (10 cm diameter)
containing 15 cm of water maintained at 23 1 ꢁC,
and was forced to swim for 10 min. Twenty-four hours
later, the mouse was replaced into the cylinder and the
total duration of immobility was recorded during the
last 5 min of the 6-min testing period. Mice are judged
immobile when they float in an upright position and
make only small movements to keep their head above
water. Drugs (50 or 100 mg/kg) were suspended in 3%
Tween 80 solution and administered 30 min (ip) or
60 min (po) before the testing.
5.7. 4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)-N-((6-fluoro-
4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)butana-
mide (1-61)
The procedure was same as that of synthesis of the
compound 1-27. 4-(4-(2,3-Dimethylphenyl)piperazin-1-
yl)butanoic acid 4 (115 mg, 0.416 mmol) and 2-(amino-
methyl)-6-fluoro-3-phenylquinazolin-4 (3H)-one (70 mg,
0.26 mmol) were used and the product was obtained in
71% yield: 1H NMR (CDCl3, 300 MHz) d 7.95 (dd,
J = 8.4, 2.7 Hz, 1H), 7.78 (dd, J = 9.0, 4.8 Hz, 1H),
7.63–7.51 (m, 4H), 7.38 (br s, 1H), 7.33–7.29 (m, 2H),
7.11 (t, J = 7.5 Hz, 1H), 6.96–6.91 (m, 2H), 4.10 (d,
J = 3.9 Hz, 2H), 2.96 (t, J = 4.5 Hz, 1H), 2.70 (br s,
4H), 2.76 (t, J = 6.9 Hz, 2H), 2.45 (t, J = 6.9 Hz, 2H),
2.31 (s, 3H), 2.25 (s, 3H), 2.01–1.94 (m, 2H); 13C
NMR (CDCl3, 75.5 MHz) d 173.05, 161.50, 161.46,
161.33 (d, J = 249 Hz), 151.82, 151.70, 143.63, 138.29,
135.43, 131.50, 130.69, 130.37, 129.73 (d, J = 8.0 Hz),
Acknowledgments
This work was supported by Basic Science Program of
Korea Science and Engineering Foundation.
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