Discovery of a novel 5-HT3 antagonist/5-HT1A agonist 3-amino-5,6,7,8-tetrahydro-2-{4-[4-(quinolin-2-yl)piperazin-1-yl]butyl} quinazolin-4(3 H)-one (TZB-30878) as an orally bioavailable agent for irritable bowel syndrome

Article abstract of DOI:10.1021/jm1002292

We have prepared a series of quinazolinone derivatives linked with piperazinylquinoline for the treatment of irritable bowel syndrome (IBS). Using pharmacophore analysis, we designed and synthesized compounds which bind to both serotonin receptor subtype 1A (5-HT_1A) and subtype 3 (5-HT ₃). Quinazolinone derivatives with a sulfur atom in the linker showed high affinity in in vitro assays, but low in vivo activity. Focusing on the linker to improve the pharmacokinetic profile, the sulfur atom in the linker was replaced with a methylene group. Further optimization led to the discovery of compound 17m (TZB-30878) (J. Pharmacol. Exp. Ther. 2007, 322, 1315 -1323, Patent WO2005082887 (A1), 2005), a novel 5-HT_1A agonist/5-HT₃ antagonist in the 3-aminoquinazolinone series. In in vivo functional assays, 17m dose dependently inhibited the Bezold-Jarisch reflex and induced 5-HT _1A-mediated behaviors, and in an IBS animal model, 17m significantly inhibited stress-induced defecation. Pretreatment by WAY-100635 (5-HT _1A antagonist) significantly attenuated but did not abolish the inhibitory effects of 17m. These results suggested that 17m exerted inhibitory effects via both 5-HT_1A agonistic and 5-HT₃ antagonistic activities and that 17m would be useful as a therapeutic agent for IBS.

Full text of DOI:10.1021/jm1002292

J. Med. Chem. 2010, 53, 7549–7563 7549
DOI: 10.1021/jm1002292
Discovery of a Novel 5-HT3 Antagonist/5-HT1A Agonist 3-Amino-5,6,7,8-tetrahydro-
2-{4-[4-(quinolin-2-yl)piperazin-1-yl]butyl}quinazolin-4(3H)-one (TZB-30878) as an
Orally Bioavailable Agent for Irritable Bowel Syndrome
Akira Asagarasu,*,† Teruaki Matsui,† Hiroyuki Hayashi,† Satoru Tamaoki,‡ Yukinao Yamauchi,‡ Kouichi Minato,§ and
Michitaka Sato†
§
†Synthetic Research Department, ‡Pharmacological Research Department, and Pharmacokinetics Research Department,
ASKA Pharmaceutical Co., Ltd., 5-36-1, Shimosakunobe, Takatsu-ku, Kawasaki 213-8522, Japan
Received February 22, 2010
We have prepared a series of quinazolinone derivatives linked with piperazinylquinoline for the treat-
ment of irritable bowel syndrome (IBS). Using pharmacophore analysis, we designed and synthesized
compounds which bind to both serotonin receptor subtype 1A (5-HT1A) and subtype 3 (5-HT3). Quinazo-
linone derivatives with a sulfur atom in the linker showed high affinity in in vitro assays, but low in vivo
activity. Focusing on the linker to improve the pharmacokinetic profile, the sulfur atom in the linker was
replaced with a methylene group. Further optimization led to the discovery of compound 17m (TZB-30878)
(J. Pharmacol. Exp. Ther. 2007, 322, 1315-1323, Patent WO2005082887 (A1), 2005), a novel 5-HT1A
agonist/5-HT3 antagonist in the 3-aminoquinazolinone series. In in vivo functional assays, 17m dose
dependently inhibited the Bezold-Jarisch reflex and induced 5-HT1A-mediated behaviors, and in an IBS
animal model, 17m significantly inhibited stress-induced defecation. Pretreatment by WAY-100635 (5-HT1A
antagonist) significantly attenuated but did not abolish the inhibitory effects of 17m. These results suggested
that 17m exerted inhibitory effects via both 5-HT1A agonistic and 5-HT3 antagonistic activities and that 17m
would be useful as a therapeutic agent for IBS.
Introduction
receptor subtype 1A (5-HT1A) agonists7 (Figure 1). 2 is used
for the treatment of stress-induced dyspeptic ulcers, and its
mechanism of action has been attributed to an antianxiety
activity through 5-HT1A agonism.
Irritable bowel syndrome (IBSa) is a disease of which the
main symptoms are evacuation abnormalities including diar-
rhea, constipation, or bellyache, and IBS is not caused by an
intestinal organic lesion.1,2 This disease develops as a result of
a mutual association of intestinal motion disorder, viscero-
sensory anaphylaxis, and psychological and social factors.3,4
Indeed, antimotility and anxiolytic agents have been used for
the treatment of this disorder.
Serotonin receptor subtype 3 (5-HT3) in intestinal tissue
plays a role in intestinal contraction, secretion of intestinal
juice, peristalsis, and content transport; thus, diarrheal symp-
toms can be improved by administration of 5-HT3 antago-
nists. Alosetron (3) and Ondansetron (4) are selective 5-HT3
antagonists5 useful for diarrhea-predominant IBS patients6
(Figure 1).
In a previous paper, we reported the synthesis of a com-
pound which acts as both a 5-HT1A agonist and 5-HT3
antagonist, with the goal being to find a single compound that
acts on both receptors as a treatment for IBS.8 Compounds in
this series showed both 5-HT1A agonist and 5-HT3 antagonist
activity both in vitro and in vivo. From this lead, further
structure-activity relationship analysis as well as the design
and synthesis of compounds based on our pharmacophore
analysis led to the identification of a dual action compound
suitable for IBS therapy. In this paper, the design and synthesis
of such compounds acting on both 5-HT1A and 5-HT3 recep-
tors and their in vitro and in vivo activities are presented.
Because psychological and social factors are recognized as
one of the causes of IBS, the administration of benzodiazepine
antianxiety agents has been investigated for IBS therapy.
8-OH-DPAT (1) and Buspirone (2) are well-known serotonin
Superposition of Pharmacophores
To design compounds with dual receptor affinity, we ana-
lyzed many structures of 5-HT1A agonists and 5-HT3 antago-
nists and extracted pharmacophoric elements (Figure 2). For
example, from 5-HT1A agonist 2, we extracted a pharmaco-
phore that includes an aromatic ring as the basic template, a
hydrogen bond acceptor, a basic nitrogen a certain constant
distance from the aromatic ring or a hydrogen-bond acceptor,
and a bulky hydrophobic group linked by a spacer from the
basic nitrogen. Similarly, from the 5-HT3 antagonist Quipa-
zine (5),9,10 we extracted a pharmacophore that includes an
aromatic ring as the basic template similar to the 5-HT1A
*To whom correspondence should be addressed. Phone: þ81-44-812-
8635. Fax: þ81-44-833-5310. E-mail: asagarasu-a@aska-pharma.co.jp.
Address: Akira Asagarasu Synthetic Research Department, ASKA
Pharmaceutical Co., Ltd., 5-36-1 Shimosakunobe, Takatsu-ku, Kawasaki
213-8522, Japan.
a Abbreviations: IBS, irritable bowel syndrome; 5-HT1A, serotonin
receptor subtype 1A; 5-HT3, serotonin receptor subtype 3; 8-OH-
DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; LLR, lower lip retrac-
tion; FBP, flat body posture; ΔT, change in rectal temperature; BJ reflex,
Bezold-Jarisch reflex.
r
2010 American Chemical Society
Published on Web 10/08/2010
pubs.acs.org/jmc

7550 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Asagarasu et al.
agonist, a hydrogen bond acceptor (nitrogen) in the aromatic
ring, and a basic nitrogen a certain constant distance from the
aromatic ring or a hydrogen-bond acceptor.
Because someofthesecompounds have a commonelement,
an aryl piperazine, in their structure, we superimposed
these pharmacophores at the aryl piperazine. We modeled
a pharmacophorewhich couldgeneratecompounds withboth
5-HT1A and 5-HT3 receptor binding as follows: (1) an aro-
matic ring as the basic template, (2) a hydrogen bond acceptor
(nitrogen) in the aromatic ring, (3) a basic nitrogen that exists
a certain constant distance from the aromatic ring, and (4) a
bulky hydrophobic group linked by a spacer from the basic
nitrogen. On the basis of this analysis, we designed and
synthesized compounds having the features described above
which would bind to both 5-HT receptor subtypes.
Synthesis of Pyrimidinone Derivatives Linked with Aryl Piperazine
Initially, we synthesized pyrimidinone derivatives linked
with an aryl piperazine by a thioether (Scheme 1).11 Starting
from the anthranilate analogue (6), reaction with thiophos-
gene gave the isothiocyanate (7). Treatment of 7 with hydra-
zine followed by refluxing in KOH/EtOH provided the
potassium salt of the pyrimidinone with an amino group at
the 3-position (9). To obtain derivatives without an amino
group at the 3-position (11), the anthranilate analogue was
treated with ammonium thiocyanate and benzoyl chloride,
followed by heating in KOH/EtOH. Aryl piperazines were
synthesized from aryl chloride and piperazine by heating at
140 °C in ethylene glycol.8 These aryl piperazines were treated
with 1-bromo-3-chloropropane to give alkyl chloride deriva-
tives (13). The potassium salts (9 and 11) were coupled with 13
Figure 1. Structure of 5-HT1A agonists and 5-HT3 antagonists.
Figure 2. Pharmacophore analyses of compounds that bind to 5-HT1A or 5-HT3 and development of a hybrid pharmacophore model for
compounds that bind to both 5-HT1A and 5-HT3 receptors.
Scheme 1. Synthesis of Pyrimidinone Derivatives Linked with Aryl Piperazine by a Thioether

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7551
Scheme 2. Synthesis of Pyrimidinone Derivatives Linked with Aryl Piperazine by Methylene Groups
Scheme 3. Synthesis of Pyrimidinone Derivatives Linked with Aryl Piperazine by a Rigid Linker
by heating to give the target compounds (14). Tetrahydropyr-
idine derivative 14r was synthesized from 14p by deprotection
of the Boc group and acetylation.
N-Boc derivative analogous to the synthesis of 14r. Deamination
of 17m was conducted with NaNO2 in acetic acid to give 18a.
Synthesis of compounds with the conformationally restricted
linker was as follows (Scheme 3). Ethyl cis-4-aminocyclohex-
anecarboxylate, which was prepared from cis-4-aminocyclo-
hexanecarboxylic acid, was treated with N-benzyl-bis(2-chloro-
ethyl)amine to prepare the piperazine derivative (20). After the
deprotection, the piperazine derivative (21) was treated with
2-chloroquinoline to give the N-substituted 5 derivative (22).
Hydrolyzing the ester to the carboxylic acid (23) and coupling
with a anthranilate analogue by PCl3 in pyridine gave com-
pound 24. After the hydrolysis of 24, heterocyclization of 25
with hydrazine gave the rigid cis-form linker compound (26a)
To improve in vivo activities, we synthesized the pyrimidi-
none derivatives linked with an aryl piperazine by a methylene
spacer (Scheme 2). Treatment of the anthranilate analogue (6)
with a haloalkyl acid chloride gave the alkyl halide derivative (15).
The alkyl halide intermediates were reacted with aryl piper-
azine followed by heating with hydrazine to give the 3-ami-
nopyrimidinone derivatives (17-19), which had a methylene
spacer. Protected ketal derivative (17r) was deprotected and
reduced to give the 6-hydroxytetrahydroquinazolinone (17t).
The N-acetylated compound (17w) was synthesized from the

7552 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Asagarasu et al.
Table 1. Preliminary Analysis of Binding Inhibition Activity for Lead
Compound Aryl Piperazine Substitution
with a quinoline moiety, had affinity to both receptors, it
became our new lead compound.
Lead Optimization
For optimization studies, the structure of the lead com-
pound, 14b, could be divided into three parts: aryl piper-
azine, linker, and bulky hydrophobic region. Since it was
reported in several papers that a length of four atoms is
suitable for the linker,14,15 the linker was initially fixed to this
length and compound modification focused on the other two
parts of the lead structure. For the aryl piperazine, because
this part must carry the 5-HT3 receptor affinity, we first
checked the affinity of the aryl piperazine units to the 5-HT3
receptor (data not shown), and those aryl piperazines which
had a high affinity were then linked with a bulky hydro-
phobic unit (Table 1). Compound 14c, with a 4-methylqui-
nolylpiperazine unit having a high affinity for the 5-HT3
receptor, had high affinities for both receptors, the same as
compound 14b. On the other hand, compounds with a
heterocyclic ring other than quinoline, e.g. benzothiazole
(14e) or 3-phenylquinoxaline (14f), resulted in low affinities
to both receptors even though the parent aryl piperazine
moieties had high affinity to 5-HT3. Interestingly, the com-
pound with tetrahydroquinoline (14d) showed high affinity
for 5-HT1A but greatly decreased the affinity to 5-HT3. From
these results, we decided to use unsubstituted or 4-methyl
substituted quinoline as the optimized aryl piperazine por-
tion of the structure.
Next, we studied the bulky hydrophobic region and the
amino group at the pyrimidinone 3-position (Table 2). The
amino group was found to have an important role in receptor
binding as compounds without it had low affinities to 5-HT1A
(14i and 14j). Introduction of a polar group into the bulky
hydrophobic region greatly improved affinity to 5-HT1A
(14q); however, this strategy did not result in high affinity to
the 5-HT3 receptor. Thus, introduction of a polar group into
this part of the structure was not suitable for our purpose. The
compound 14r, the acetylated secondary amine derivative of
14q, had a high affinity to 5-HT1A receptor while somewhat
maintaining affinity to the 5-HT3 receptor. Fused pyridines in
the structure (14n and 14o) had high 5-HT1A affinity and
moderate affinity to the 5-HT3 receptor.
and a small amount of the rigid trans-form linker compound
26b to which 26a was isomerized.
Preliminary Studies to Find a Lead Compound
Compound affinity for the 5-HT1A receptor was calculated
as percent inhibition of [3H]-1 binding to the 5-HT1A receptor
in a CHO cell membrane sample expressing human 5-HT1A
receptors.8,12,13 Compound affinity for the 5-HT3 receptor
was calculated as percent inhibition of [3H]BRL-43694 binding
in HEK-293 cell membrane sample expressing human 5-HT3
receptors. Initially, compounds were designed using a phar-
macophore model, and after synthesis, compound screening
and preliminary structure-activity relationship analysis were
used to verify the pharmacophore model.
M. Modica et al. reported some compounds which bind to
the 5-HT1A receptor,11 and a subset of these compounds de-
monstrated an additional weak binding to the 5-HT3 receptor.
Onthebasisofthisdata, wesynthesizeda seriesof compounds
which incorporated a thienopyrimidinone unit.
For secondary screening, two in vitro functional tests, a
[35S]GTPγS binding assay for 5-HT1A agonistic activity, and a
guinea pig ileum contraction inhibition assay for 5-HT3
antagonistic activity were carried out for those compounds
with high affinities to both receptors (Table 6 and 7).8,12,13 In
the [35S]GTPγS binding assay, Emax of compounds 14l and 14r
in this series had about 90% and were confirmed to be full
5-HT1A agonists. In the contraction inhibition assay, these
compound showed 90-100% inhibition at 1 μmol/L, confirm-
ing that they were 5-HT3 antagonists. In particular, 14r showed
excellent results in these in vitro functional assays. In a tertiary
screening, compound 14r was studied in two other functional
examinations in rats: measurement of 5-HT1A agonistic activity
(lower lip retraction, LLR; flat body posture, FBP; change in
rectal temperature, ΔT) and 5-HT3 antagonistic activity (inhi-
bition of the Bezold-Jarisch (B-J) reflex caused by 5-HT)
(Table 9 and 10).8,12,13 Compound 14r showed disappointing
results with respect to 5-HT1A agonist activity; therefore, we
measured the compound total concentrations in the blood and
brain (Table 8).16 The compound total concentration in the
brain was found to be too low to have an effect. Thus, to
Compound 14a, with a pyridine substituted piperazine unit,
showed high affinity exclusively at the 5-HT1A receptor and
not the 5-HT3 receptor (Table 1). We next examined quinoline
or phenanthridine as the aryl piperazine substitution because
the parent moieties’ affinity for the 5-HT3 receptor could be
expected to carry over into the more complex derivatives.
Indeed, 5 and phenanthridinylpiperazine showed high affinity
for the 5-HT3 receptor (data not shown), and introduction of
a bulky hydrophobic group and linker (which were necessary
for 5-HT1A affinity) resulted in high affinity for the 5-HT1A
receptor (14b and 14g). Interestingly, compared to compound
14b with a quinoline, the phenanthridine moiety in com-
pound 14g greatly decreased the affinity to 5-HT3 and to a
lesser degree to 5-HT1A. From these data, we modified the
pharmacophore model such that neither the substituents nor
the fused aryl ring were too large. Because compound 14b,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7553
Table 2. Optimization of the Lead Compound Bulky Hydrophobic and Amino Groups
Table 3. Optimization of the Bulky Hydrophobic Portion of Methylene Linked Compounds

7554 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Asagarasu et al.
Table 4. IC50 Value of Tetrahydroquinazolinone Derivatives with
Methylene-Type Linker
improve the concentration of these compounds in the brain, we
examined the effect of decreasing the number of heteroatoms in
the structure.
Improvement of Compound Penetration into Brain and
Affinity to Both Receptors
To reduce the number of heteroatoms, we chose to replace
the sulfur atom linker with a methylene group because
we believed that the aryl piperazine and pyrimidinone amino
groups were necessary for receptor binding while the sulfur
atominthe linker would have no influence on binding affinity.
Wefixed the aryl piperazine to5 andpreparedderivatives with
a methylene linker that showed very high inhibition activities
(Table 3). Compound 17b, with a fused benzene ring instead
ofthiophene, showedhighaffinities tobothreceptors. Mostof
the compounds with a halogen substituted benzene ring had
high affinities for the 5-HT1A receptor but did not exceed
compound 17b in binding affinity to 5-HT3. Substitution of a
methyl group on the 6-position of the quinazolinone showed
almost equal affinity for the unsubstituted quinazolinone
(compound 17k versus 17b). Compound 17m, with a tetra-
hydroquinazolinone, showed high affinities to both receptors
as much as 17b. As introduction of substituents on the tetra-
hydroquinazolinone (17n and 17o) did not increase affinity
and resizing the ring (17p and 17q) resulted in a decrease in the
affinity to 5-HT3 receptor, the size of the tetrahydroquinazoline
appeared to be optimal for affinity to both receptors. Com-
pound 17t that had a hydroxy group showed high affinities to
both receptors and N-acetylated tetrahydropyridine derivative
17w maintained high affinity for 5-HT1A and increased affinity
for 5-HT3 compared with thioether type compound 14r.
For the quinolinepart, compounds that hada substituent at
the 3-position had a low affinity to both receptors (18c-g,
Table4) although the parent aryl piperazine moietyhad a high
affinity to the 5-HT3 receptor. Thus we reconfirmed that
substitution at the 3-position of the quinoline was unsuitable
5-HT1A IC50 5-HT3 IC50
compd no. R1
R2 R3 R4
X
(nM)
1.4
6.2
<10
(nM)
17m
18a
18b
18c
18d
18e
18f
H
H
H
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
H
NH2
H
8.9
H
H
24.6
59.1
H
CH3
CH3
H
NH2
NH2
NH2
NH2
NH2
NH2
NH2
96.0
64.6
62.3
>100
CH3 CH3
-(CH2)3-
-(CH2)4-
>100
>100
>100
>100
18g
18h
18i
OH
H
H
H
H
96.4
59.3
46.6
52.6
>100
55.2
H
OH NH2
Table 5. Optimization of the Methylene Linker Length
compd no.
n
5-HT1A IC50 (nM)
5-HT3 IC50 (nM)
19a
19b
19c
17m
19d
1
2
3
4
5
>100
>100
90.4
1.4
86.6
>100
>100
8.9
7.6
>100
Table 9. 5-HT1A Agonistic Activity in Vivo; 5-HT1A Receptor-
Mediated Behavior and Hypothermia in Ratsa,b
14rc
17b
17m
18b
NEd
Table 6. 5-HT1A Agonistic Activity in Vitro; [35S]GTPγS Binding
Assay
LLR
FBP
ΔT
1.0
1.0
1.8
1.8
1.5
1.8
14l
14r
17b
17m
18b
-0.1
-1.9
-1.4
EC50 (nM)
Emax (%)
182
2.5
84.8
30.7
94.7
22.3
99.8
13.7
93.5
a Dose: 10 mg/kg, ip. b LLR = lower lip retraction, FBP = flat body
posture and ΔT = change in rectal temperature. c Dose: 10 mg/kg, iv.
d No effect.
90.3
Table 7. 5-HT3 Antagonistic Activity in Vitro; Guinea Pig Ileum
Contraction Inhibition Assaya
Table 10. 5-HT3 Antagonistic Activities in Vivo; Inhibition of the
Bezold-Jarisch Reflex Caused by 5-HTa
14b
14l
14r
17b
17m
18b
100
97.1
14r
17b
17m
18bb
1 μmol/L
0.1 μmol/L
41.9
NDb
95.1
80.0
97.6
79.9
90.1
-23.2
96.8
70.4
inhibit %
77.0
21.9
64.0
64.5
a Results are listed for inhibition % of control. b Not determined.
a Dose: 10 μg/kg, iv. b Dose: 100 μg/kg, iv.
Table 8. Total Concentrations of Compound in Brain and Blooda,b
2 h
4 h
8 h
compd
no.
brain conc
(nM)
blood conc
(nM)
brain/blood
ratio
brain conc
(nM)
blood conc
(nM)
brain/blood
ratio
brain conc
(nM)
blood conc
(nM)
brain/blood
ratio
14l
63.1 ( 12.1
18.7 ( 3.61
685 ( 268
160 ( 42.3
15.1 ( 4.36
21.2 ( 4.25
56.0 ( 5.41 0.332 ( 0.036 12.9 ( 1.79
97.4 ( 34.3
66.5 ( 32.2
51.0 ( 7.00 0.294 ( 0.071 19.5 ( 2.85
2.99 ( 0.261 32.2 ( 6.32
10.6 ( 2.28
36.1 ( 1.02 0.357 ( 0.044 2.96 ( 1.32
55.5 ( 6.88
16.8 ( 6.29
55.8 ( 12.9 0.355 ( 0.040 13.4 ( 1.12
3.05 ( 0.151 13.0 ( 5.55
5.17 ( 2.42
8.98 ( 4.66 0.338 ( 0.003
28.3 ( 18.5
9.97 ( 4.74
43.7 ( 4.02 0.307 ( 0.004
2.53 ( 0.092
14r
17b
17m
17w
7.11 ( 1.38
367 ( 68.4
6.59 ( 0.643
204 ( 105
7.65 ( 1.28
6.54 ( 2.83
2.56 ( 0.488 70.2 ( 11.0
4.40 ( 0.873 56.4 ( 12.7
a Each value represents the mean ( SD of three rats. b Dose: 3 mg/kg, po.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7555
Figure 3. The effects of 17m, tandospirone, and 3 on stress-induced defecation in rats.12 The upper bodies of lightly anesthetized rats were
wrapped with adhesive tape, and the animals were returned to the observation cages. The number of feces dropped on the tray was counted 1 h
after the wrapping. Vehicle or test drugs were administered orally 1 h before the restraint; n = 8 per group. Values represent mean ((SD). $$$
P < 0.001 versus normal (N); Wilcoxon test. **, P < 0.01, ***, P < 0.001 versus vehicle (V); nonparametric Dunnett’s multiple comparison
test. Normal group represents animals without wrapping.
Figure 4. The influence of 5-HT1A antagonist, WAY-100635, on the inhibitory effect of 17m in stress-induced defecation.12 Vehicle or 17m was
injected intraperitoneally 20 min following subcutaneous injection with saline (containing diluted hydrochloric acid) or WAY-100635. Five
minutes later, lightly anesthetized rats were wrapped for 1 h and the number of feces was counted. n = 8 per group. Values represent mean
((SD). $$$ P < 0.001 versus normal (N); Wilcoxon test. **, P < 0.01, ***, P < 0.001 versus vehicle (V); nonparametric Dunnett’s multiple
comparison test. # P < 0.05, ### P < 0.001 between indicated two groups; Wilcoxon test. NS means nonsignificant. Normal group represents
animals without wrapping.
for binding to the 5-HT1A receptor as well as the 5-HT3
receptor. Deaminated compound (18a) showed somewhat
lower affinity than 17m for 5-HT1A, and 4-methylquinoline
derivative (18b) had moderate affinity for 5-HT3. Compounds
18h and 18i with a hydroxyl group at R3 or R4 had greatly
17b and 17m had significantly increased concentrations both in
the blood and brain (versus 14l), with compound 17b having a
very high concentration in brain compared to 17m. The con-
version from S to CH2 in the linker improved the compound
pharmacokinetics without decreasing the affinity to either
receptor.
decreased affinity to 5-HT1A
.
Optimization of the linker length was carried out (Table 5).
Affinity to the 5-HT1A receptor was maintained to some
degree at n = 5 (19d), but the affinity to both receptors was
maximized for n = 4 (17m).
In secondary screens, compounds with high affinities to
bothreceptors in the methylene linker class were subjected toin
vitro functional examinations ([35S]GTPγS binding assay and
a guinea pig ileum contraction inhibition assay, Table 6 and 7).
In the [35S]GTPγS binding assay, every compound in this
series (17b, 17m, and 18b) was confirmed to be a full 5-HT1A
agonist, and for the contraction inhibition assay, almost every
compound showed 90-100% inhibition at 1 μmol/L, confirm-
ing that they were 5-HT3 antagonists.
Compounds 17b and 17m were found to have excellent in vivo
activity (Tables 9 and 10). In the B-J reflex assay, compound
17m inhibited bradycardia at a very low dosage. Compound 18b
had no effect on LLR, FBP, and ΔT. Though compound 18b
had affinity to 5-HT1A and agonistic activity in vitro, the
5-HT1A agonistic action was not seen in vivo. Because its B-J
reflection was about one order weaker than 17m, we thought
that its blood level would be low and the concentration in brain
also would be low along with it (concentration of 18b in brain
and blood was not measured). Therefore, it seemed that 5-HT1A
agonistic effect was not seen.
As compounds 17b and 17m showed excellent results, we
checked their selectivity to other receptors (Table 11).17,18 17m
had a low affinity to these other receptors including the R1
receptor, but17b, an aromatized type of 17m, hadsomedegree
Total concentrations of compounds 17b, 17m, and 17w in
the blood and brain were measured (Table 8).16 Compounds

7556 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Table 11. In Vitro Binding (IC50a) to Other Receptors
Asagarasu et al.
b
c
compd no.
rat R1
rat R2
hD2
rat 5-HT2
gp 5-HT4
17b
17m
63% at 0.1 μM
>0.1
42% at 0.1 μM
>0.1
NDd
>0.1
78% at 0.1 μM
≈0.1
ND
>1
a IC50; μM. b h = human. c gp = guinea pig. d Not determined.
Table 12. In Vivo Pharmacokinetic Data for Compound 17m in Rat
cis-cyclohexane locked linker (bended shape) shows some
affinity for 5-HT3 and has decreased affinity for 5-HT1A com-
pared with 26b. Thus, we believe that 17m acts selectively on
the 5-HT receptor subtypes via two different conformations.
Tmax
dose (hr)
AUC0-t
(ng h/mL)
T1/2
(h)
Cmax
(ng/mL)
MRT
(h)
F
(%)
3
705
poa
0.5
2.054
241.488
3.100 25.5
a Dose: 10 mg/kg, po.
Conclusion
Table 13. Binding Inhibition Constants (Ki Values) of Compound 17m
Our initial aim was to search for compounds with both
gastrointestinal motor inhibition and antianxiety effects for the
treatment of diarrhea type IBS through dual 5-HT3 antago-
nist and 5-HT1A agonist activity. By superposition of the
pharmacophores of 5-HT1A agonists and 5-HT3 antagonists
and use of the aryl piperazine moiety as a common structural
feature, we discovered compound 14b, which had affinity to
both receptors. We further optimized this lead compound to
discover quinazolinone derivatives with a sulfur containing
linker. These showed high affinity to both receptors but did
not show in vivo activity. To improve the blood-brain barrier
penetration, we designed a compound which replaces sulfur in
the linker with a methylene group. These compounds showed
high affinity to both receptors, and in addition, the compound
total concentrations in brain and blood were improved. In this
compound series, 17m has a pronounced affinity to both
receptors. 17m showed 5-HT1A agonistic/5-HT3 antagonistic
activity concurrently in in vitro/vivo functional assays. In the
stress-induced defecation IBS model, 17m significantly inhibited
stress-induced defecation. Pretreatment with WAY-100635, a
5-HT1A antagonist, significantly attenuated but did not abol-
ish the inhibitory effect of 17m. These results suggested that
17m exerted an inhibitory effect via both 5-HT1A agonist and
5-HT3 antagonist activities. This compound can be expected
to contribute to the improvement of compliance of taking
medicine and the reduction of patient’s medical cost. From
these results, 17m was selected for advanced evaluation as a
treatment for IBS.
5-HT1A
7.008 ꢀ 10-10
5-HT3
4.720 ꢀ 10-9
Ki (mol/L)
(
(
0.3309 ꢀ 10-10
0.2270 ꢀ 10-9
Table 14. Inhibition Activities of Tetrahydroquinazolinone Derivatives
with a Rigid Linker
of affinity to several receptors. This illustrates that the bulky
hydrophobic region is important for receptor binding selectivity.
The pharmacokinetic data for 17m show it is orally bioavailable
(Table 12). Binding inhibition constants (Ki values) were calcu-
lated (Table 13). Ki values of 17m for human 5-HT1A receptors
were 7.008 ꢀ 10-10 ( 0.3309 ꢀ 10-10 mol/L and for human
5-HT3 receptors were 4.720 ꢀ 10-9 ( 0.2270 ꢀ 10-9 mol/L.
Finally, we examined 17m in the wrap restraint-induced
stress model of IBS (Figure 3).12 Compound 17m reduced the
restraint stress-induced defecation as 3 did. It is thought that
at the doses used in this assay, 17m reduced the defecation at
least partially through 5-HT3 antagonism. It is interesting
that, although 3 only partially inhibited the restraint stress-
induced defecation, 17m completely normalized this process.
Moreover, the effect of 17m was partly inhibited by a 5-HT1A
antagonist, WAY-100635 (Figure 4).12 These results indicate
that stimulation of the 5-HT1A receptors by 17m also reduces
the restraint stress-induced defecation. Taken together, these
data suggest that the suppression of restraint stress-induced
defecation by 17m was the result of contributions from both
5-HT1A agonism and 5-HT3 antagonism.
Many compounds, with an affinity to the 5-HT1A receptor,
generally have a long shape in which an aryl piperazine
derivative is linked to a bulky hydrophobic region such as in
compound 17m. However, 5-HT3 antagonists in general do
not have such a long structure. In considering why 17m dis-
played affinity to both receptors, we postulated that 17m had
an extended conformation when binding to 5-HT1A and had a
bent conformation when binding to 5-HT3. To verify this
hypothesis, we synthesized two conformationally restricted
compounds with a cyclohexane ring linker (Table 14).
Although both rigid linker compounds had decreased
affinity for the 5-HT receptors, compound 26b with a
trans-cyclohexane locked linker (extended shape) has a parti-
cular affinity for the 5-HT1A receptor and it has no affinity for
the 5-HT3 receptor. On the other hand, compound 26a with a
Experimental Section
Unless otherwise noted, all nonaqueous reactions were carried
out under an Ar atmosphere using commercial grade solvents and
reagents. 1H NMR spectra were recorded on a JEOL JNM-ECP
400. Chemical shifts are reported in parts per million (ppm, δ)
relative to tetramethylsilane as an internal standard, using the
following abbreviations: s, singlet; d, doublet; t, triplet; dd, doublet
of doublet; m, multiplet; br, broad. Coupling constants (J) are
reported in hertz (Hz) where relevant. Mass spectrometric analyses
were obtained with a Shimadzu GC/MS QP-5000, with electro-
spray ionization methodology. The purities of the compounds
were examined by HPLC (g95%) using a Waters 2695 HPLC
system.
Synthesis of 5, General Procedure of Synthesis of Aryl Piper-
azine (12). Anhydrous piperazine (4.31 g, 50.0 mmol) was
dissolved in ethylene glycol (30 mL), and 2-chloroquinoline
(818 mg, 5.00 mmol) was added. The mixture was stirred at
140 °C for 2 h. After cooling, saturated aqueous sodium hydro-
gencarbonate solution was added and the system was extracted
with chloroform. The organic layer was dried over anhydrous
magnesium sulfate, and the solvent was evaporated under re-
duced pressure. The residue was purified by silica gel column
chromatography (chloroform:methanol = 2:1) to provide 1.09 g

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7557
1
(100%) of 2-piperazin-1-ylquinoline (5). H NMR (CDCl3) δ:
7.89 (d, J = 9.2 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.59 (dd, J =
1.5, 8.0 Hz, 1H), 7.53 (ddd, J = 1.5, 7.0, 8.4 Hz, 1H), 7.26-7.22
(m, 1H), 6.97 (d, J = 9.2 Hz, 1H), 3.70 (t, J = 5.0 Hz, 4H), 3.01
(t, J = 5.0 Hz, 4H). Mass, m/z: 213 (Mþ), 145 (base).
1.97 (q, J = 7.3 Hz, 1H), 1.86-1.80 (m, 2H), 1.78-1.75 (m, 2H).
Mass, m/z: 484 (Mþ), 161 (base).
3-Amino-2-[3-(4-benzothiazol-2-ylpiperazin-1-yl)propylthio]-
5,6-dimethyl-3H-thieno[2,3-d]pyrimidin-4-one (14e). This com-
pound was synthesized using the same procedure as for 14b
starting with 2-chlorobenzothiazol and ethyl 2-amino-4,5-di-
methylthiophene-3-carboxylate. 1H NMR (CDCl3) δ: 7.61-7.59
(m, 1H), 7.57-7.55 (m, 1H), 7.29 (ddd, J = 1.4, 7.5, 7.9 Hz, 1H),
7.08 (ddd, J = 1.2, 7.5, 7.9 Hz, 1H), 4.77 (s, 2H), 3.68 (t, J = 5.1
Hz, 4H), 3.18 (t, J = 7.4 Hz, 2H), 2.62 (t, J = 5.1 Hz, 4H),
2.56-2.54 (m, 2H), 2.44 (d, J = 0.7 Hz, 3H), 2.36 (d, J = 0.9 Hz,
3H), 2.00-1.94 (m, 2H). Mass, m/z: 486 (Mþ), 128 (base).
3-Amino-5,6-dimethyl-2-[3-[4-(3-phenylquinoxalin-2-yl)pipera-
zin-1-yl]propylthio]-3H-thieno[2,3-d]pyrimidin-4-one (14f). This
compound was synthesized using the same procedure as for 14b
starting withethyl 2-amino-4,5-dimethylthiophene-3-carboxylate
2-[4-(3-Chloropropyl)piperazin-1-yl]quinoline, General Proce-
dure for the Synthesis of Alkylchloride Derivatives (13). Com-
pound 5 (853 mg, 4.00 mmol) was dissolved in acetone (5 mL), and
aqueous sodium hydroxide (160 mg in 5 mL) was added. 1-Bromo-
3-chloropropane (0.5 mL) was added dropwise to the solution,
and stirring was continued overnight at room temperature. The
reaction was diluted with diethyl ether, and the organic layer was
washed with saturated aqueous sodium hydrogencarbonate solu-
tion and dried over anhydrous magnesium sulfate. The solvent
was evaporated under reduced pressure, and the residue was
purified by silica gel column chromatography (chloroform:
methanol = 50:1) to provide 1.10 g (95%) of 13. 1H NMR
(CDCl3) δ: 7.89 (d, J = 9.2 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H),
7.59 (dd, J = 1.4, 8.0Hz, 1H), 7.53 (ddd, J = 1.5, 7.1, 8.5Hz, 1H),
7.22 (ddd, J = 1.1, 6.9, 8.0 Hz, 1H), 6.98 (d, J = 9.2 Hz, 1H), 3.75
(t, J = 5.1 Hz, 4H), 3.61 (t, J = 6.5 Hz, 2H), 2.63-2.43 (m, 6H),
2.04-1.97 (m, 2H). Mass, m/z: 289 (Mþ), 157 (base).
1
and 2-chloro-3-phenylquinoxaline. H NMR (CDCl3) δ: 7.96-
8.01 (m, 2H), 7.84 (dd, J = 1.5, 8.4 Hz, 1H), 7.60 (ddd, J = 1.1,
6.9, 8.1 Hz, 1H), 7.41-7.52 (m, 5H), 4.75 (s, 2H), 3.29-3.37 (m,
4H), 3.14 (t, J = 7.0 Hz, 2H), 2.47-2.53 (m, 4H), 2.43 (s, 3H),
2.35 (s, 3H), 1.91 (q, J = 7.0 Hz, 2H), 1.24 (t, J = 7.0 Hz, 2H).
Mass, m/z: 557 (Mþ), 128 (base).
3-Amino-5,6-dimethyl-2-[3-(4-phenanthridin-6-ylpiperazin-1-yl)-
propylthio]-3H-thieno[2,3-d]pyrimidin-4-one (14g). This compound
was synthesized using the same procedure as for 14b starting with
6-chlorophenanthridine and ethyl 2-amino-4,5-dimethylthio-
phene-3-carboxylate. 1H NMR (CDCl3) δ: 8.55 (d, J = 8.4 Hz,
1H), 8.42 (dd, J = 1.1, 8.1 Hz, 1H), 8.21 (d, J = 7.3 Hz, 1H), 7.92
(dd, J = 1.1, 8.1 Hz, 1H), 7.79-7.75 (m, 1H), 7.63-7.60 (m, 2H),
7.50-7.46 (m, 1H), 3.56 (br s, 4H), 3.21 (t, J = 7.0 Hz, 2H), 2.79
(br s, 4H), 2.64 (t, J = 7.0 Hz, 2H), 2.44 (s, 3H), 2.35 (s, 3H). Mass,
m/z: 530 (Mþ), 207 (base).
3-Amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-5,6,7,
8-tetrahydro-3H-benzo[4,5]thieno[2,3-d]pyrimidin-4-one (14h). This
compound was synthesized using the same procedure as for 14b
starting with ethyl 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophene-
3-carboxylate and 2-chloroquinoline. 1HNMR(CDCl3) δ:7.88(d,
J= 8.7 Hz, 1H), 7.70 (d, J= 7.8 Hz, 1H), 7.59 (dd, J= 1.3, 8.0 Hz,
1H), 7.53 (m, 1H), 7.24-7.20 (m, 1H), 6.99 (d, J = 9.2 Hz, 1H),
4.77 (s, 2H), 3.79 (t, J = 5.1 Hz, 4H), 3.21-3.17 (m, 2H), 2.99-
2.95 (m, 2H), 2.75-2.71 (m, 2H), 2.62 (t, J = 5.1 Hz, 4H), 2.56 (t,
J = 7.0 Hz, 2H), 1.99 (q, J = 7.3 Hz, 2H), 1.91-1.80 (m, 4H).
Mass, m/z: 506 (Mþ), 157 (base).
5,6-Dimethyl-2-[3-(4-quinolin-2-yl-piperazin-1-yl)propylthio]-
3H-thieno[2,3-d]pyrimidin-4-one (14i). This compound was synthe-
sized using the same procedure as for 14b starting with potassium
5,6-dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-2-thiolate,
which was prepared from ethyl 2-amino-4,5-dimethylthiophene-3-
carboxylate. 1HNMR(CDCl3) δ:7.88(d, J=8.8Hz, 1H), 7.70(d,
J= 8.6 Hz, 1H), 7.59 (dd, J= 1.2, 8.0 Hz, 1H), 7.54-7.50 (m, 1H),
7.22 (ddd, J= 1.2, 6.9, 8.0 Hz, 1H), 6.98 (d, J=9.2Hz, 1H), 3.89-
3.80 (m, 4H), 3.29 (t, J = 6.9 Hz, 2H), 2.67 (t, J =4.9Hz, 4H), 2.62
(t, J = 6.8 Hz, 2H), 2.43 (d, J = 0.8 Hz, 3H), 2.43 (d, J = 0.8 Hz,
3H), 2.08-1.98 (m, 2H). Mass, m/z: 465 (Mþ), 157 (base).
2-[3-(4-Quinolin-2-ylpiperazin-1-yl)propylthio]-5,6,7,8-tetrahy-
dro-3H-benzo[4,5]thieno[2,3-d]pyrimidin-4-one (14j). This com-
pound was synthesized using the same procedure as for 14i starting
with ethyl 2-amino-4,5,6,7-tetrahydro-benzo[b]thiophene-3-car-
boxylate. 1H NMR (CDCl3) δ: 7.89 (d, J = 9.0 Hz, 1H), 7.70 (d,
J = 8.0 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.55-7.51 (m, 1H),
7.24-7.18 (m, 1H), 6.99 (d, J = 9.0 Hz, 1H), 3.89 (br s, 4H), 3.30
(t, J = 6.8 Hz, 2H), 3.01-2.92 (m, 2H), 2.78-2.70 (m, 2H), 2.66
(br s, 4H), 2.68-2.58 (m, 2H), 1.89-1.78 (m, 2H), 1.59-1.50 (m,
2H). Mass, m/z: 491 (Mþ), 157 (base).
3-Amino-5,6-dimethyl-2-[3-(4-quinolin-2-ylpiperazin-1-yl)-
propylthio]-3H-thieno[2,3-d]pyrimidin-4-one (14b), General
Procedure for the Synthesis of Thioether Linked Compounds.
A mixture of potassium 3-amino-5,6-dimethyl-4-oxo-3,4-dihy-
drothieno[2,3-d]pyrimidine-2-thiolate (9) (80 mg, 0.30 mmol),
prepared from ethyl 2-amino-4,5-dimethylthiophene-3-carboxy-
late, and 2-[4-(3-chloropropyl)piperazin-1-yl]quinoline (104 mg,
0.36 mmol) inethanol(5 mL) wereheated torefluxfor 4.5 h. After
cooling the reaction mixture, chloroform was added, followed
by washing with saturated brine. The organic layer was dried
over anhydrous magnesium sulfate, and the solvent was evapo-
rated under reduced pressure. The residue was purified by silica
gel column chromatography (chloroform:methanol = 100:1) to
provide 72 mg (50%) of 14b. 1H NMR (CDCl3) δ: 7.89 (d, J =
8.7 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H),
7.54-7.52 (m, 1H), 7.23-7.20 (m, 1H), 6.99 (d, J = 9.2 Hz, 1H),
4.77 (s, 2H), 3.79 (t, J = 5.1 Hz, 4H), 3.19 (t, J = 7.3 Hz, 2H),
2.62 (t, J =5.1 Hz, 4H), 2.56 (t, J = 7.0Hz, 2H), 2.45(s, 3H), 2.36
(s, 3H), 2.00 (q, J = 7.3 Hz, 2H). Mass, m/z: 480 (Mþ), 157 (base).
3-Amino-5,6-dimethyl-2-[3-(4-pyridin-2-ylpiperazin-1-yl)propyl-
thio]-3H-thieno[2,3-d]pyrimidin-4-one (14a). This compound was
synthesized using the same procedure as for 14b starting with
pyridyl piperazine and ethyl 2-amino-4,5-dimethylthiophene-3-
carboxylate. 1H NMR (CDCl3) δ: 8.19 (ddd, J = 0.6, 1.9, 4.9 Hz,
1H), 7.48 (ddd, J = 1.9, 7.2, 8.9 Hz, 1H), 6.65 (d, J = 8.5 Hz, 1H),
6.62 (ddd, J = 0.8, 4.9, 7.1 Hz, 1H), 4.77 (s, 2H), 3.57 (t, J = 5.0
Hz, 4H), 3.18 (t, J = 7.1 Hz, 2H), 2.59 (t, J = 5.1 Hz, 4H), 2.54
(t, J = 7.0 Hz, 2H), 2.44 (s, 3H), 2.36 (s, 3H), 1.98 (q, J = 7.2 Hz,
2H). Mass, m/z: 430 (Mþ), 107 (base).
3-Amino-5,6-dimethyl-2-[3-[4-(4-methylquinolin-2-yl)piperazin-
1-yl]propylthio]-3H-thieno[2,3-d]pyrimidin-4-one (14c). This com-
pound was synthesized using the same procedure as for 14b
startingwithethyl 2-amino-4,5-dimethylthiophene-3-carboxylate
1
and 2-chlorolepidine. H NMR (CDCl3) δ: 7.77 (dd, J = 1.1,
8.1 Hz, 1H), 7.71 (d, J = 7.7 Hz, 1H), 7.53 (ddd, J = 1.5, 7.0, 8.4
Hz, 1H), 7.25-7.22 (m, 1H), 6.84 (d, J = 3.7 Hz, 1H), 4.78 (s,
2H), 3.78 (t, J = 5.1 Hz, 4H), 3.20-3.17 (m, 2H), 2.62-2.60 (m,
4H), 2.60 (s, 3H), 2.58-2.54 (m, 2H), 2.44 (d, J = 0.7 Hz, 3H),
2.36 (d, J = 0.7 Hz, 3H), 1.99 (q, J = 7.3 Hz, 2H). Mass, m/z: 494
(Mþ), 171 (base).
3-Amino-5,6-dimethyl-2-[3-[4-(5,6,7,8-tetrahydroquinolin-2-yl)-
piperazin-1-yl]propylthio]-3H-thieno[2,3-d]pyrimidin-4-one (14d).
This compound was synthesized using the same procedure as for
14b starting with ethyl 2-amino-4,5-dimethylthiophene-3-carboxy-
late and 2-chloro-5,6,7,8-tetrahydroquinoline. 1H NMR (CDCl3)
δ: 7.17 (d, J = 8.4 Hz, 1H), 6.43 (d, J = 8.4 Hz, 1H), 4.77 (s, 2H),
3.50 (t, J = 5.1 Hz, 4H), 3.17 (t, J = 7.3 Hz, 2H), 2.75 (t, J = 5.6
Hz, 2H), 2.63-2.52 (m, 8H), 2.44 (s, 3H), 2.35 (d, J = 7.0 Hz, 3H),
3-Amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-3H-qui-
nazolin-4-one (14k). This compound was synthesized using the same
procedure as for 14b starting with anthranilic acid ethyl ester and
2-chloroquinoline. 1HNMR(CDCl3) δ:8.22-8.19 (m, 1H), 7.89 (d,
J = 9.2 Hz, 1H), 7.73-7.69 (m, 1H), 7.61-7.51 (m, 3H), 7.41-7.36
(m, 1H), 7.24-7.20 (m, 1H), 6.99 (d, J = 9.2 Hz, 1H), 4.82 (s, 2H),

7558 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Asagarasu et al.
3.79 (t, J = 4.8 Hz, 4H), 3.26 (t, J = 7.3 Hz, 2H), 2.65-2.58 (m,
6H), 2.05 (q, J = 7.0 Hz, 2H). Mass, m/z: 446 (Mþ), 157 (base).
3-Amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-5,6,7,
8-tetrahydro-3H-quinazolin-4-one (14l). This compound was
synthesized using the same procedure as for 14b starting with
ethyl 2-aminocyclohex-1-ene-1-carboxylate and 2-chloroquino-
line. 1H NMR (CDCl3) δ: 7.89 (d, J = 9.0 Hz, 1H), 7.70 (d, J =
8.3 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 7.53 (ddd, J = 1.6, 7.0, 8.7
Hz, 1H), 7.24-7.20 (m, 1H), 6.98 (d, J = 8.3 Hz, 1H), 4.80 (s,
2H), 3.77 (t, J = 4.8 Hz, 4H), 3.14 (t, J = 7.4 Hz, 4H), 2.62-2.60
(m, 4H), 2.58-2.45 (m, 6H), 1.99-1.95 (m, 2H), 1.80-1.72 (m,
4H). Mass, m/z: 450 (Mþ), 157 (base).
3-Amino-7-nitro-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-
3H-quinazolin-4-one (14m). This compound was synthesized using
the same procedure as for 14b starting with 7-nitroanthranilic acid
and 2-chloroquinoline. 1H NMR (DMSO-d6) δ: 8.29 (d, J = 8.9
Hz, 1H), 8.23 (d, J = 2.2 Hz, 1H), 8.12 (dd, J = 2.2, 8.9 Hz, 1H),
8.02 (d, J = 9.2 Hz, 1H), 7.67 (d, J = 7.2 Hz, 1H), 7.56-7.40 (m,
2H), 7.23-7.18 (m, 2H), 5.84 (s, 2H), 3.72 (t, J =4.8Hz, 4H), 3.17
(t, J = 7.3 Hz, 2H), 2.55-2.48 (m, 6H), 1.93 (t, J = 7.3 Hz, 2H).
Mass, m/z: 491 (Mþ), 157 (base).
3-Amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-3H-
pyrido[3,2-d]pyrimidin-4-one (14n). This compound was synthe-
sized using the same procedure as for 14b starting with 3-amino-
pyridine-2-carboxylic acid methyl ester and 2-chloroquinoline.
1H NMR (CDCl3) δ: 8.92 (dd, J = 1.8, 4.4 Hz, 1H), 8.55 (dd, J =
1.8, 8.1 Hz, 1H), 7.88 (d, J = 8.8 Hz, 1H), 7.70 (d, J = 8.4 Hz,
1H), 7.59 (dd, J = 1.1, 8.1 Hz, 1H), 7.53 (ddd, J = 1.5, 7.0, 8.4
Hz, 1H), 7.37-7.34 (m, 1H), 7.24-7.20 (m, 1H), 6.98 (d, J = 9.1
Hz, 1H), 4.84 (s, 2H), 3.78 (t, J = 5.1 Hz, 4H), 3.38 (t, J = 7.3 Hz,
2H), 2.64-2.59 (m, 6H), 2.07 (q, J = 7.3 Hz, 2H). Mass, m/z: 447
(Mþ), 157 (base).
and the reaction was stirred for 30 min followed by concentration
under reduced pressure. The residue was purified by silica gel
column chromatography (chloroform:methanol = 25:1) to pro-
vide 150 mg (83%) of 14r. 1H NMR (CDCl3) δ: 7.99 (d, J = 8.9
Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H), 7.55-
7.51 (m, 1H), 7.24-7.21 (m, 1H), 6.99 (d, J = 9.2 Hz, 1H), 4.78
(br s, 4H), 3.79 (t, J = 4.6 Hz, 4H), 3.75 (d, J = 5.8 Hz, 2H),
3.22-3.19 (m, 2H), 3.15-3.07 (m, 2H), 2.67 (t, J = 4.6 Hz, 4H),
2.58-2.55 (m, 2H), 2.21 (s, 3H), 2.04-1.96 (m, 2H). Mass, m/z:
549 (Mþ), 157 (base).
Synthesis of 3-Amino-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-
5,6,7,8-tetrahydro-3H-quinazolin-4-one (17m). General Procedure
for the Synthesis of Methylene Linker Derivatives. Step 1. Synthesis
of Ethyl 2-(5-Bromopentanoylamino)cyclohex-1-enecarboxylate
(15). To an ice cooled mixture of 42.3 g of ethyl 2-aminocyclo-
hex-1-enecarboxylate (6, 250 mmol) and 40.0 g of pyridine (506
mmol) in 150 mL of tetrahydrofuran, 5-bromovaleryl chloride
(54.9 g, 275 mmol) was added dropwise. After stirring the reaction
mixture overnight at room temperature, ethyl acetate was added,
followed by washing sequentially with saturated aqueous sodium
hydrogencarbonate solution, 10% aqueous citric acid, and satu-
rated brine. The product was dried over anhydrous magnesium
sulfate, and the solvent was evaporated under reduced pressure.
The residue was purified by silica gel column chromatography
(n-hexane:ethyl acetate = 8:1), to provide 76.6 g (92%) of 15. 1H
NMR (CDCl3) δ:11.62 (br s, 1H), 4.22-4.09 (m, 2H), 3.42 (t, J =
6.9 Hz, 2H), 2.97-2.94 (m, 2H), 2.34 (t, J = 7.0 Hz, 2H), 2.32-
2.23 (m, 2H), 1.94-1.88 (m, 2H), 1.85-1.79 (m, 2H), 1.65-1.56
(m, 4H), 1.30 (t, J = 7.0 Hz, 3H). Mass, m/z: 333 (Mþ), 55 (base).
Step 2. Synthesis of Ethyl 2-[5-(4-Quinolin-2-ylpiperazin-1-
yl)pentanoylamino]cyclohex-1-enecarboxylate (16). 15 (66.5 g,
200 mmol), 46.9 g of 2-piperazin-1-ylquinoline (220 mmol), and
22.3 g of triethylamine (220 mmol) were dissolved in 350 mL of
toluene, and the mixture was heated at reflux overnight. The
solvent was evaporated under reduced pressure, and ethyl acetate
was added to the residue, followed by washing with saturated
aqueous sodium hydrogencarbonate solution. The organic layer
was dried overanhydrous magnesium sulfate, andthe solvent was
evaporated under reduced pressure. Purification of the residue by
silica gel column chromatography (n-hexane:ethyl acetate:metha-
nol = 1:6:0.2) provided 79.8 g (86%) of 16. 1H NMR (CDCl3)
δ:11.61 (br s, 1H), 7.87 (d, J = 9.2 Hz, 1H), 7.69 (d, J = 8.5 Hz,
1H), 7.58-7.56 (m, 1H), 7.51 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 7.20
(ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 6.97 (d, J = 9.2 Hz, 1H), 4.16 (q,
J = 6.9 Hz, 2H), 3.74 (t, J = 5.0Hz, 4H), 2.97 (t, J = 5.0 Hz, 2H),
2.56 (t, J = 5.0 Hz, 4H), 2.43-2.90 (m, 6H), 1.74-1.70 (m, 2H),
1.68 (m, 4H), 1.28 (t, J = 6.9 Hz, 2H). Mass, m/z: 464 (Mþ), 157
(base).
3-Amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-3H-
pyrido[2,3-d]pyrimidin-4-one (14o). This compound was synthe-
sized using the same procedure as for 14b starting with 2-amino-
nicotinic acid ethyl ester and 2-chloroquinoline. 1H NMR (CDCl3)
δ: 8.77 (dd, J = 1.5, 4.4 Hz, 1H), 7.92 (dd, J = 1.5, 8.4 Hz, 1H),
7.89 (d, J = 9.2 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.64-7.59 (m,
2H), 7.54 (ddd, J = 1.5, 7.0, 8.4 Hz, 1H), 7.25-7.21 (m, 1H), 6.99
(d, J = 9.2 Hz, 1H), 4.94 (s, 2H), 3.79 (t, J = 5.1 Hz, 4H), 3.27 (t,
J = 7.3 Hz, 2H), 2.64 (t, J = 5.1 Hz, 4H), 2.60 (t, J = 7.3 Hz, 2H),
2.05 (q, J = 7.3 Hz, 2H). Mass, m/z: 447 (Mþ), 157 (base).
3-Amino-7-tert-butoxycarbonyl-2-[3-(4-quinolin-2-ylpiperazin-
1-yl)propylthio]-3,5,6,8-tetrahydro-4H-pyrido[40,30:4,5]thieno[2,3-d]-
pyrimidine (14p). This compound was synthesized using the same
procedure as for 14b starting with 2-chloroquinoline and 2-amino-
4,7-dihydro-5H-thieno[2,3-c]pyridine-3,6-dicarboxylic acid 6-tert-
1
butyl ester 3-ethyl ester. H NMR (CDCl3) δ: 7.89 (d, J = 9.2
Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.61-7.58 (m, 1H), 7.53 (ddd,
J = 1.5, 7.0, 8.4 Hz, 1H), 7.24-7.20 (m, 1H), 6.99 (d, J = 9.2 Hz,
1H),4.78(s,2H),4.60(brs, 2H), 3.79(t,J= 5.1 Hz, 4H), 3.73-3.70
(m, 2H), 3.22-3.18 (m, 2H), 3.06 (br s, 2H), 2.62 (t, J = 5.1 Hz,
4H), 2.58-2.54 (m, 2H), 2.00 (q, J = 7.3 Hz, 2H). Mass, m/z: 607
(Mþ), 157 (base).
3-Amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-5,6,7,
8-tetrahydro-3H-pyrido[40,30:4,5]thieno[2,3-d]pyridin-4-one Trihy-
drochloride (14q). 14p (0.10 g, 0.16 mmol) was added to 6 mL of
4N hydrochloric acid dioxane solution and was stirred for 2.5 h.
Evaporation of the solvent under reduced pressure gave 105 mg
(100%) of 14q. 1H NMR (DMSO-d6) δ: 9.82 (br s, 1H), 8.46 (d,
J = 9.5 Hz, 1H), 8.32 (br s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.78 (m,
1H), 7.58 (d, J = 9.2 Hz, 1H), 7.50 (m, 1H), 4.87 (d, J = 8.2 Hz,
2H), 4.33 (br s, 2H), 4.20-3.85 (m, 4H), 3.70 (d, J = 5.4 Hz, 2H),
3.39 (br s, 2H), 3.26 (br s, 2H), 3.17-3.13 (m, 4H), 2.23-2.16 (m,
2H). Mass, m/z: 507 (Mþ), 157 (base).
Step 3. Synthesis of 3-Amino-2-[4-(4-quinolin-2-ylpiperazin-1-
yl)butyl]-5,6,7,8-tetrahydro-3H-quinazolin-4-one (17m). To a so-
lution of 8.0 g of 16 (17.2 mmol) in 120 mL of ethanol, 60 mL of
hydrazine monohydrate was added, followed by stirring under
reflux for 4 h. After evaporation of the solvent under reduced
pressure, the residue was dissolved in chloroform, washed with
saturated aqueous sodium hydrogencarbonate solution and
saturated brine, and dried over anhydrous magnesium sulfate.
Evaporation of the solvent under reduced pressure gave a residue
which was purified by silica gel column chromatography (chlo-
1
roform:methanol = 50:1) to provide 3.8 g (51%) of 17m. H
NMR (CDCl3) δ: 7.87 (d, J = 9.3 Hz, 1H), 7.69 (d, J = 8.5 Hz,
1H), 7.58 (d, J = 7.7 Hz, 1H), 7.52 (ddd, J = 1.5, 6.9, 7.5 Hz,
1H), 7.25-7.19 (m, 1H), 6.97 (d, J = 8.9 Hz, 1H), 4.93 (s, 2H),
3.74 (t, J = 5.0 Hz, 4H), 2.92 (t, J = 7.7 Hz, 2H), 2.58-2.55 (m,
6H), 2.52-2.49 (m, 2H), 2.44 (t, J = 7.3 Hz, 2H), 1.81-1.63 (m,
8H). Mass, m/z: 432 (Mþ), 157 (base).
7-Acetyl-3-amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propylthio]-
5,6,7,8-tetrahydro-3H-pyrido[40,30:4,5]thieno[2,3-d]pyridin-4-one
(14r). 14q (0.20 g, 0.33 mmol) and 133 mg of triethylamine (1.31
mmol) were added to 10 mL of tetrahydrofuran. To the ice cooled
mixture, acetyl chloride (28 mg, 0.35 mmol) was added dropwise
3-Amino-5,6-dimethyl-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-
3H-thieno[2,3-d]pyrimidin-4-one (17a). This compound was synthe-
sized using the same procedure as for 17m starting with ethyl
2-amino-4,5-dimethylthiophene-3-carboxylate. 1H NMR (CDCl3)
δ: 7.88 (d, J = 9.2 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.58 (dd,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7559
J= 1.2, 8.1 Hz, 1H), 7.52 (ddd, J= 1.5, 6.9, 8.5 Hz, 1H), 7.23-7.19
(m, 1H), 6.97 (d, J = 9.2 Hz, 1H), 4.88 (s, 2H), 3.76 (t, J = 5.0 Hz,
4H), 3.04 (t, J = 7.7 Hz, 2H), 2.57 (t, J = 5.0 Hz, 4H), 2.46-
2.44 (m, 2H), 2.46 (d, J = 0.8 Hz, 3H), 2.37 (d, J = 0.8 Hz,
3H), 1.88-1.82 (m, 2H), 1.72-1.66 (m, 2H). Mass, m/z: 462 (Mþ),
157 (base).
3-Amino-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-quinazo-
lin-4-one (17b). This compound was synthesized using the same
procedure as for 17m starting with ethyl anthranilate. 1H NMR
(CDCl3) δ: 8.24 (dd, J = 1.2, 8.1 Hz, 1H), 7.88 (d, J = 8.9 Hz,
1H), 7.73 (ddd, J = 1.6, 6.9, 8.5 Hz, 1H), 7.71-7.65 (m, 2H), 7.59
(dd, J = 1.2, 7.9Hz, 1H), 7.52 (ddd, J = 1.2, 6.9, 8.1Hz, 1H), 7.44
(ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 7.21 (ddd, J = 1.2, 6.4, 8.1 Hz,
1H), 6.97 (d, J = 9.2 Hz, 1H), 4.91 (s, 2H), 3.75 (t, J = 4.8 Hz,
4H), 3.07 (t, J = 7.7 Hz, 2H), 2.58 (t, J = 5.0 Hz, 4H), 2.48 (t, J =
7.3 Hz, 2H), 1.95-1.87 (m, 2H), 1.76-1.69 (m, 2H). Mass, m/z:
428 (Mþ), 157 (base).
3-Amino-5-fluoro-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17c). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-6-
fluorobenzoate. 1H NMR (CDCl3) δ: 7.88 (d, J = 9.2 Hz, 1H),
7.69 (d, J = 8.9 Hz, 1H), 7.66-7.62 (m, 1H), 7.59 (d, J = 8.1 Hz,
1H), 7.54-7.51 (m, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.23-7.19 (m,
1H), 7.11-7.06 (m, 1H), 6.97 (d, J = 8.9 Hz, 1H), 4.86 (s, 2H),
3.76 (br s, 4H), 3.06 (t, J = 7.7 Hz, 2H), 2.59 (br s, 4H), 2.50-2.46
(m, 2H), 1.92-1.87 (m, 2H), 1.72-1.70 (m, 2H). Mass, m/z: 446
(Mþ), 157 (base).
3-Amino-6-fluoro-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17d). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-5-fluor-
obenzoate. 1HNMR(CDCl3) δ:7.89(d, J= 9.2 Hz, 1H), 7.87 (dd,
J = 2.7, 8.5 Hz, 1H), 7.72-7.65 (m, 2H), 7.59 (dd, J = 1.2, 8.1 Hz,
1H), 7.53 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 7.46 (dt, J = 2.7, 8.5 Hz,
1H), 7.22 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 6.98 (d, J = 9.2 Hz, 1H),
4.92 (s, 2H), 3.76 (t, J = 5.0 Hz, 4H), 3.07 (t, J = 7.7 Hz, 2H), 2.59
(t, J = 5.0 Hz, 4H), 2.49 (t, J = 7.7 Hz, 2H), 1.91 (q, J = 7.7 Hz,
2H), 1.73 (q, J = 7.7 Hz, 2H). Mass, m/z: 446 (Mþ), 157 (base).
3-Amino-7-fluoro-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17e). This compound was synthesized using the
same procedure as for 17m starting withmethyl 2-amino-4-fluoro-
benzoate. 1H NMR (CDCl3) δ: 8.25 (dd, J = 6.2, 8.9 Hz, 1H),
7.89 (d, J = 9.2 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.59 (dd, J =
1.2, 8.1 Hz, 1H), 7.53 (ddd, J = 1.5, 6.9, 8.5Hz, 1H), 7.31 (dd, J =
2.7, 9.6 Hz, 1H), 7.22 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 7.16 (dt, J =
2.3, 8.9 Hz, 1H), 6.98 (d, J = 9.2Hz, 1H), 4.89 (s, 2H), 3.76 (t, J =
5.0 Hz, 4H), 3.07 (t, J = 7.7 Hz, 2H), 2.59 (t, J = 5.0Hz, 4H), 2.49
(t, J = 7.7 Hz, 2H), 1.91 (q, J = 7.7 Hz, 2H), 1.73 (q, J = 7.7 Hz,
2H). Mass, m/z: 446 (Mþ), 157 (base).
3-Amino-5-chloro-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17f). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-6-
chlorobenzoate. 1H NMR (CDCl3) δ: 7.88 (d, J = 9.2 Hz, 1H),
7.69 (d, J = 8.5 Hz, 1H), 7.59-7.50 (m, 4H), 7.44 (dd, J = 2.7, 6.6
Hz, 1H), 7.23-7.19 (m, 1H), 6.97 (d, J = 8.9 Hz, 1H), 4.86 (s, 2H),
3.76 (t, J = 5.0 Hz, 4H), 3.05 (t, J = 7.7 Hz, 2H), 2.59 (t, J = 5.0
Hz, 4H), 2.48 (t, J = 7.7 Hz, 2H), 1.89 (q, J = 7.7 Hz, 2H),
1.75-1.69 (m, 2H). Mass, m/z: 462 (Mþ), 157 (base).
3-Amino-6-chloro-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17g). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-5-chloro-
benzoate. 1H NMR (CDCl3) δ: 8.19 (d, J = 2.3 Hz, 1H), 7.87 (d,
J = 9.2 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H), 7.66 (dd, J = 2.3, 8.4
Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.56-7.59 (m, 1H), 7.52 (ddd,
J = 1.5, 6.9, 8.5 Hz, 1H), 7.21 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 6.96
(d, J = 9.2 Hz, 1H), 4.90 (br s, 2H), 3.75 (t, J = 5.0 Hz, 4H), 3.05
(t, J = 7.4 Hz, 2H), 2.58 (t, J = 5.0 Hz, 4H), 2.47 (t, J = 7.4 Hz,
2H),1.89 (q, J = 7.4 Hz, 2H),1.71 (q, J = 7.4 Hz, 2H). Mass, m/z:
462 (Mþ), 446, 157 (base).
same procedure as for 17m starting with methyl 2-amino-4-chloro-
benzoate. 1H NMR (CDCl3) δ: 8.16 (d, J = 8.5 Hz, 1H), 7.88 (d,
J = 8.9 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 1.9 Hz,
1H), 7.58 (d, J = 8.1 Hz, 1H), 7.52 (ddd, J = 1.5, 6.9, 8.1 Hz, 1H),
7.43 (d, J = 1.9 Hz, 1H), 7.38 (d, J = 2.3 Hz, 1H), 7.21 (ddd, J =
1.2, 6.9, 8.1 Hz, 1H), 6.97 (d, J = 9.2 Hz, 1H), 4.89 (s, 2H), 3.76 (t,
J = 5.0 Hz, 4H), 3.06 (t, J = 7.3 Hz, 2H), 2.58 (t, J = 5.0 Hz, 4H),
2.48 (t, J = 7.3 Hz, 2H), 1.90 (q, J = 7.7 Hz, 2H), 1.71 (q, J = 7.3
Hz, 2H). Mass, m/z: 462 (Mþ), 157 (base).
3-Amino-8-chloro-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17i). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-3-
chlorobenzoate. 1H NMR (CDCl3) δ: 8.16 (dd, J = 1.5, 8.1 Hz,
1H), 7.88 (d, J = 9.2 Hz, 1H), 7.81 (dd, J = 1.5, 8.1 Hz, 1H), 7.69
(d, J = 8.5 Hz, 1H), 7.58 (d, J = 7.7 Hz, 1H), 7.52 (ddd, J = 1.5,
6.9, 8.5 Hz, 1H), 7.38-7.34 (m, 1H), 7.23-7.19 (m, 1H), 6.97 (d,
J = 9.2 Hz, 1H), 4.93 (s, 2H), 3.76 (t, J = 5.0 Hz, 4H), 3.12 (t, J =
7.7 Hz, 2H), 2.60 (t, J = 5.0 Hz, 4H), 2.50 (t, J = 7.7 Hz, 2H), 1.95
(q, J = 7.7 Hz, 2H), 1.75 (q, J = 7.7 Hz, 2H). Mass, m/z: 462
(Mþ), 157 (base).
3-Amino-5-methyl-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17j). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-6-methyl-
benzoate. 1H NMR (CDCl3) δ: 8.08 (d, J = 8.1 Hz, 1H), 7.88 (d,
J = 9.2 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.59-7.54 (m, 2H),
7.54-7.50 (m, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.23-7.20 (m, 1H),
6.97 (d, J = 9.2 Hz, 1H), 4.89 (s, 2H), 3.75 (t, J = 5.0 Hz, 4H), 3.07
(t, J = 7.3 Hz, 2H), 2.61 (s, 3H), 2.58 (t, J = 5.0 Hz, 4H), 2.49 (t,
J = 7.7 Hz, 2H), 1.93 (q, J = 7.7 Hz, 2H), 1.77-1.70 (m, 2H).
Mass, m/z: 442 (Mþ), 157 (base).
3-Amino-6-methyl-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17k). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-5-methyl-
benzoate. 1H NMR (CDCl3) δ: 8.02 (s, 1H), 7.88 (d, J = 8.9 Hz,
1H), 7.69 (d, J = 8.5 Hz, 1H), 7.59-7.50 (m, 4H), 7.23-7.19 (m,
1H), 6.97 (d, J= 9.2 Hz, 1H), 4.90 (s, 2H), 3.75 (t, J= 5.0 Hz, 4H),
3.06 (t, J = 1.7 Hz, 2H), 2.58 (t, J = 5.0 Hz, 4H), 2.49-2.44 (m,
2H), 2.48 (s, 3H), 1.90 (q, J = 7.7 Hz, 2H), 1.72 (q, J = 7.7 Hz,
2H). Mass, m/z: 442 (Mþ), 157 (base).
3-Amino-8-methyl-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17l). This compound was synthesized using the
same procedure as for 17m starting with methyl 2-amino-3-methyl-
benzoate. 1H NMR (CDCl3) δ: 8.08 (dd, J = 0.8, 8.1 Hz, 1H), 7.88
(d, J = 8.9 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.58 (ddd, J = 1.5,
6.2, 7.7 Hz, 1H), 7.52 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 7.32 (t, J =
7.7 Hz, 1H), 7.23-7.19 (m, 1H), 6.97 (d, J = 9.2 Hz, 1H), 4.88 (s,
2H), 3.76 (t, J = 5.0 Hz, 4H), 3.07 (t, J = 7.7 Hz, 2H), 2.59 (t, J =
5.4 Hz, 4H), 2.50 (t, J = 7.7 Hz, 2H), 1.95 (q, J = 7.3 Hz, 2H),
1.77-1.73 (m, 2H). Mass, m/z: 442 (Mþ), 157 (base).
3-Amino-6-methyl-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-5,
6,7,8-tetrahydro-3H-quinazolin-4-one (17n). This compound was
synthesized using the same procedure as for 17m starting with
ethyl 2-amino-5-methylcyclohex-1-enecarboxylate. 1H NMR
(CDCl3) δ: 7.89 (d, J = 9.2 Hz, 1H), 7.70 (d, J = 8.1 Hz, 1H),
7.59 (dd, J = 1.2, 8.1 Hz, 1H), 7.53 (ddd, J = 1.5, 6.9, 8.5 Hz,
1H), 7.22 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 6.98 (d, J = 9.2 Hz,
1H), 4.95 (s, 2H), 3.76 (t, J = 5.0 Hz, 4H), 2.93 (t, J = 7.7 Hz,
2H), 2.77-2.69 (m, 1H), 2.67-2.60 (m, 2H), 2.58 (t, J = 5.0 Hz,
4H), 2.45 (t, J = 7.3 Hz, 2H), 2.09-1.98 (m, 1H), 1.92-1.62 (m,
6H), 1.45-1.32 (m, 1H), 1.08 (d, J = 6.6 Hz, 3H). Mass, m/z: 446
(Mþ), 157 (base).
3-Amino-7-methyl-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-5,6,
7,8-tetrahydro-3H-quinazolin-4-one (17o). This compound was
synthesized using the same procedure as for 17m starting with ethyl
2-amino-4-methylcyclohex-1-enecarboxylate. 1HNMR(CDCl3) δ:
7.89 (d, J= 9.2 Hz, 1H), 7.70 (d, J= 8.5 Hz, 1H), 7.59 (dd, J=1.2,
8.1 Hz, 1H), 7.53 (ddd, J =1.5, 6.9, 8.5 Hz, 1H), 7.22(ddd, J =1.2,
6.9, 8.1 Hz, 1H), 6.98 (d, J = 9.2 Hz, 1H), 4.95 (s, 2H), 3.76 (t, J =
5.0 Hz, 4H), 2.93 (t, J = 6.9 Hz, 2H), 2.74-2.63 (m, 2H), 2.58 (t,
J= 5.0 Hz, 4H), 2.49-2.37 (m, 3H), 2.28-2.18 (m, 1H), 1.92-1.75
3-Amino-7-chloro-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3H-
quinazolin-4-one (17h). This compound was synthesized using the

7560 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Asagarasu et al.
(m, 4H), 1.72-1.59 (m, 2H), 1.34-1.22 (m, 1H), 1.06 (d, J = 6.6
Hz, 3H). Mass, m/z: 446 (Mþ), 157 (base).
3-Amino-7-tert-butyloxycarbonyl-2-[4-(4-quinolin-2-ylpipera-
zin-1-yl)butyl]-5,6,7,8-tetrahydro-3H-pyrido[40,30:4,5]thieno-[2,3-d]-
pyrimidin-4-one (17u). This compound was synthesized using the
same procedure as for 17m starting with 2-amino-4,7-dihydro-5H-
thieno[2,3-c]pyridine-3,6-dicarboxylic acid 6-tert-butyl ester 3-ethyl
ester. 1H NMR (CDCl3) δ: 7.88 (d, J = 9.2 Hz, 1H), 7.69 (d, J =
8.5 Hz, 1H), 7.56-7.60 (m, 1H), 7.52 (ddd, J = 1.5, 6.9, 8.5 Hz,
1H), 7.21 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 6.97 (d, J = 9.3 Hz, 1H),
4.88 (s, 2H), 4.60 (br s, 2H), 3.68-3.78(m, 6H), 3.00-3.12 (m, 4H),
2.55-2.60 (m, 4H), 2.46 (t, J = 7.0 Hz, 2H), 1.82-1.91 (m, 2H),
1.66-1.73(m, 2H), 1.48(s, 9H). Mass,m/z:589(Mþ), 489, 445, 157
(base).
7-Acetyl-3-amino-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-5,
6,7,8-tetrahydro-3H-pyrido[40,30:4,5]thieno[2,3-d]pyrimidin-4-one
(17w). This compound was synthesized using the same procedure
as for 14r starting with compound 17u. 1H NMR (CDCl3) δ: 7.88
(d, J = 9.2 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.58 (dd, J = 1.1,
8.5 Hz, 1H), 7.52 (ddd, J = 1.1, 6.9, 8.5 Hz, 1H), 7.22 (ddd, J =
1.1, 6.9, 8.5Hz, 1H), 6.97(d, J = 9.2Hz, 1H), 4.90(br s, 2H), 4.79
(s, 2H), 3.72-3.80 (m, 6H), 3.13-3.18 (m, 2H), 3.04 (t, J = 7.0
Hz, 4H), 2.60 (t, J = 5.0 Hz, 4H), 2.48 (t, J = 7.0 Hz, 2H), 2.20 (s,
3H), 1.87 (q, J = 7.0 Hz, 2H), 1.70 (q, J = 7.0 Hz, 2H). Mass,
m/z: 531 (Mþ), 387, 157 (base).
2-[4-(4-Quinolin-2-ylpiperazin-1-yl)butyl]-5,6,7,8-tetrahydro-3H-
quinazolin-4-one (18a). To a ice cold solution of 17m (500 mg, 1.16
mmol) in acetic acid (5 mL), sodium nitrite (88 mg, 1.27 mmol) was
added and then stirred at room temperature for 3 h. After neutra-
lization with saturated aqueous sodium hydrogencarbonate solu-
tion, the solution was extructed with chloroform. Organic layer was
dried over anhydrous magnesium sulfate and evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (chloroform:methanol = 10:1) gave 18a (338 mg,
70%). 1H NMR (CDCl3) δ: 12.27 (br s, 1H), 7.87 (d, J = 9.2 Hz,
1H), 7.68 (d, J = 8.5 Hz, 1H), 7.59-7.57 (m, 1H), 7.54-7.49 (m,
1H), 7.21 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 6.96 (d, J = 9.2 Hz, 1H),
3.80-3.78 (m, 4H), 2.69-2.65 (m, 2H), 2.62-2.57 (m, 6H),
2.50-2.43 (m, 4H), 1.86-1.60 (m, 8H). Mass, m/z: 417 (Mþ),
204, 157 (base).
3-Amino-2-[4-[4-(4-methylquinolin-2-yl)piperazin-1-yl]butyl]-5,
6,7,8-tetrahydro-3H-quinazolin-4-one (18b). This compound was
synthesized using the same procedure as for 17m starting with
ethyl 2-aminocyclohex-1-enecarboxylate and 2-chlorolepizine.
1H NMR (CDCl3) δ: 7.74 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 8.5
Hz, 1H), 7.51 (ddd, J = 1.2, 7.0, 8.1 Hz, 1H), 7.22 (d, J = 8.1 Hz,
1H), 6.82 (s, 1H), 4.94 (s, 2H), 3.74 (t, J = 5.0 Hz, 4H), 2.92 (t,
J = 7.3 Hz, 2H), 2.57 (s, 3H), 2.56-2.54 (m, 6H), 2.53-2.50 (m,
2H), 2.44 (t, J = 7.3 Hz, 2H), 1.82-1.62 (m, 8H). Mass, m/z: 446
(Mþ), 171 (base).
3-Amino-2-[4-[4-(3-methylquinolin-2-yl)piperazin-1-yl]butyl]-5,
6,7,8-tetrahydro-3H-quinazolin-4-one (18c). This compound was
synthesized using the same procedure as for 17m starting with
ethyl 2-aminocyclohex-1-enecarboxylate and 2-chloro-3-methyl-
quinoline. 1H NMR (CDCl3) δ: 7.83 (d, J = 8.5 Hz, 1H), 7.77 (s,
1H), 7.60 (d, J =8.1Hz, 1H), 7.52 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H),
7.31 (ddd, J = 1.1, 6.9, 8.1 Hz, 1H), 4.98 (s, 2H), 3.35-3.33 (m,
4H), 2.93 (t, J = 7.3 Hz, 2H), 2.64 (br s, 4H), 2.59 (t, J = 6.2 Hz,
2H), 2.53-2.50 (m, 2H), 2.47 (t, J = 7.3 Hz, 2H), 2.42 (d, J = 0.8
Hz, 3H), 1.83-1.65 (m, 8H). Mass, m/z: 446 (Mþ), 171 (base).
3-Amino-2-[4-[4-(3,4-dimethylquinolin-2-yl)piperazin-1-yl]butyl]-
5,6,7,8-tetrahydro-3H-quinazolin-4-one (18d). This compound was
synthesized using the same procedure as for 17m starting with ethyl
2-aminocyclohex-1-enecarboxylate and 2-chloro-3,4-dimethylqui-
noline. 1H NMR (CDCl3) δ: 7.87 (d, J = 7.3 Hz, 1H), 7.83 (d, J =
7.3 Hz, 1H), 7.53 (t, J = 8.1 Hz, 1H), 7.37-7.35 (m, 1H), 4.99
(s, 2H), 3.27(brs, 4H), 2.93(t, J= 7.7 Hz, 2H), 2.64 (br s, 4H), 2.59
(t, J = 5.8 Hz, 2H), 2.55 (s, 3H), 2.52 (t, J = 6.2 Hz, 2H), 2.47-
2.46 (m, 2H), 2.37 (s, 3H), 1.81-1.67 (m, 8H). Mass, m/z: 460
(Mþ), 185 (base).
3-Amino-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-5,6,7-tri-
hydro-3H-cyclopenta[d]pyrimidin-4-one (17p). This compound
was synthesizedusing the same procedureas for 17m starting with
ethyl 2-aminocyclopent-1-enecarboxylate. 1H NMR (CDCl3) δ:
7.88 (d, J = 9.3 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 7.7
Hz, 1H), 7.52 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 7.21 (ddd, J = 1.2,
7.0, 8.1Hz, 1H), 6.97 (d, J = 9.2Hz, 1H), 4.97 (s, 2H), 3.75 (t, J =
5.0 Hz, 4H), 2.97 (t, J = 7.7 Hz, 2H), 2.86-2.80 (m, 4H), 2.57 (t,
J = 5.0 Hz, 4H), 2.45 (t, J = 7.5 Hz, 2H), 2.08 (t, J = 7.7Hz, 2H),
1.84-1.78(m, 2H), 1.70-1.61 (m, 2H). Mass, m/z: 418 (Mþ), 402,
157 (base).
3-Amino-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-5,6,7,8,9-
pentahydro-3H-cyclohepta[d]pyrimidin-4-one (17q). This com-
pound was synthesized using the same procedure as for 17m
starting with ethyl 2-aminocyclohept-1-enecarboxylate. 1H
NMR (CDCl3) δ: 7.88 (d, J = 9.3 Hz, 1H), 7.69 (d, J = 8.1 Hz,
1H) 7.58 (d, J = 9.1 Hz, 1H), 7.52 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H),
7.22-7.20 (m, 1H), 6.97 (d, J = 9.3 Hz, 1H), 5.00 (s, 2H), 3.75 (t,
J = 5.0 Hz, 4H), 2.91 (t, J = 7.3 Hz, 2H), 2.75 (t, J = 8.5 Hz, 2H),
2.58 (t, J = 5.0 Hz, 4H), 2.45 (t, J = 7.3 Hz, 2H), 1.83-1.78 (m,
4H), 1.70-1.60 (m, 6H). Mass, m/z: 446 (Mþ), 430, 157 (base).
3-Amino-6,6-ethylenedioxy-2-[4-(4-quinolin-2-ylpiperazin-1-yl)-
butyl]-5,6,7,8-tetrahydro-3H-quinazolin-4-one (17r). This com-
pound was synthesized using the same procedure as for 17m
starting with 2-amino-5,5-ethylenedioxycyclohex-1-enecarboxylic
acid ethyl ester. 1H NMR (CDCl3) δ: 7.89 (d, J = 9.2 Hz, 1H), 7.70
(d, J = 8.1 Hz, 1H), 7.59 (dd, J = 1.2, 7.7 Hz, 1H), 7.53 (ddd, J =
1.5, 6.9, 8.1 Hz, 1H), 7.22 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H), 6.98 (d,
J = 9.2 Hz, 1H), 4.94 (s, 2H), 4.07-3.98 (m, 4H), 3.76 (t, J = 5.0
Hz, 4H), 2.94 (t, J = 7.7 Hz, 2H), 2.85 (t, J = 6.9 Hz, 2H), 2.75 (s,
2H), 2.58 (t, J = 5.0 Hz, 4H), 2.45 (t, J = 7.3 Hz, 2H), 1.96 (t, J =
6.9 Hz, 2H), 1.80 (q, J = 7.7 Hz, 2H), 1.72-1.59 (m, 2H). Mass,
m/z: 490 (Mþ), 157 (base).
3-Amino-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-3,4,5,6,7,
8-hexahydro-4,6-dioxaquinazoline (17s). To 40 mL of 6N hy-
drochloric acid, 2.72 g of 17r (5.54 mmol) was added and the reac-
tion was heated at reflux for an hour. After cooling, the reaction
mixture was neutralized with saturated aqueous sodium hydro-
gencarbonate solution, extracted with methylene chloride, dried
over anhydrous magnesium sulfate, and the solvent was evapo-
rated under reduced pressure. The residue was purified by silica
gel column chromatography (methanol:methylene chloride =
2:23) to provide 1.77 g (71%) of 17r. 1H NMR (CDCl3) δ: 7.89 (d,
J = 9.2 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.60 (dd, J = 1.2, 8.1
Hz, 1H), 7.53 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 7.23 (ddd, J = 1.2,
6.9, 8.1Hz, 1H), 6.98 (d, J = 9.2Hz, 1H), 5.01 (s, 2H), 3.76 (t, J =
5.0 Hz, 4H), 3.39 (s, 2H), 3.06-2.95 (m, 4H), 2.66 (t, J = 7.3 Hz,
2H), 2.59 (t, J = 5.0 Hz, 4H), 2.47 (t, J = 7.3 Hz, 2H), 1.89-1.79
(m, 2H), 1.74-1.64 (m, 2H). Mass, m/z: 446 (Mþ), 157 (base).
3-Amino-6-hydroxy-2-[4-(4-quinolin-2-ylpiperazin-1-yl)butyl]-
5,6,7,8-tetrahydro-3H-quinazolin-4-one (17t). A solution of 1.00 g
of 17s (2.24 mmol) in 25 mL of methanol was added dropwise to an
ice-cooled mixture of 500 mg of sodium borohydride (13.2 mmol)
in 25 mL of methanol. The ice bath was removed, and the reaction
mixture was stirred at room temperature for 3 h, then concentrated
under reduced pressure. The residue was diluted with water,
extracted with methylene chloride, washed with water, dried over
anhydrous magnesium sulfate, and the solvent was evaporated
under reduced pressure. The residue was purified by silica gel
column chromatography (methanol:methylene chloride = 1:9) to
provide 520 mg (52%) of 17t. 1H NMR (CDCl3) δ: 7.88 (d, J = 9.2
Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.53
(ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 7.22 (ddd, J = 1.2, 6.9, 8.1 Hz, 1H),
6.97 (d, J = 9.2 Hz, 1H), 4.97 (s, 2H), 4.24-4.16 (m, 1H), 3.75 (t,
J = 5.0 Hz, 4H), 2.93 (t, J = 7.7 Hz, 2H), 2.88-2.76 (m, 2H),
2.69-2.49 (m, 6H), 2.45 (t, J = 7.3 Hz, 2H), 2.01-1.61 (m, 6H).
Mass, m/z: 448 (Mþ), 157 (base).
3-Amino-2-[4-[4-(2,3-dihydro-1H-cyclopenta[c]quinolin-4-yl)-
piperazin-1-yl]butyl]-5,6,7,8-tetrahydro-3H-quinazolin-4-one (18e).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7561
This compound was synthesized using the same procedure as for
17m starting with ethyl 2-aminocyclohex-1-enecarboxylate and
4-chloro-2,3-dihydro-1H-cyclopenta[c]quinoline. 1H NMR
(CDCl3) δ: 7.80 (d, J = 8.5 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H),
7.53-7.49 (m, 1H), 7.30-7.26 (m, 1H), 4.96 (s, 2H), 3.57 (br s,
4H), 3.17 (t, J = 7.3 Hz, 2H), 3.04 (t, J = 7.3 Hz, 2H), 2.93 (t, J =
7.7 Hz, 2H), 2.60-2.57 (m, 6H), 2.52 (t, J = 6.2 Hz, 2H),
2.47-2.44 (m, 2H), 2.22 (q, J = 7.3 Hz, 2H), 1.82-1.65 (m,
8H). Mass, m/z: 472 (Mþ), 197 (base).
3-Amino-2-[4-[4-(7,8,9,10-tetrahydrophenanthridin-6-yl)pipera-
zin-1-yl]butyl]-5,6,7,8-tetrahydro-3H-quinazolin-4-one (18f). This
compound was synthesized using the same procedure as for 17m
starting with 6-chloro-7,8,9,10-tetrahydrophenanthridine. 1HNMR
(CDCl3) δ: 7.84-7.80 (m, 2H), 7.53 (t, J = 8.1 Hz, 1H), 7.37-7.33
(m, 1H), 4.99 (s, 2H), 3.31 (br s, 4H), 3.10 (t, J = 6.6 Hz, 2H),
2.95-2.91 (m, 2H), 2.76 (t, J = 5.8 Hz, 2H), 2.62 (br s, 4H),
2.60-2.57 (m, 2H), 2.52 (t, J = 6.2 Hz, 2H), 2.49-2.45 (m, 2H),
1.98-1.93 (m, 2H), 1.82-1.65 (m, 10H). Mass, m/z: 486 (Mþ),
211 (base).
9.2 Hz, 1H), 5.57 (s, 2H), 3.70 (t, J = 5.0 Hz, 4H), 3.01-2.98 (m,
2H), 2.61-2.50 (m, 8H), 2.47-2.44 (m, 2H), 2.05 (q, J = 6.9 Hz,
2H), 1.82-1.70 (m, 4H). Mass, m/z: 418 (Mþ), 157 (base).
5-Amino-2-[5-(4-quinolin-2-ylpiperazin-1-yl)pentyl]-5,6,7,8-tet-
rahydro-3H-quinazolin-4-one (19d). This compound was synthe-
sized using the same procedure as for 17m with 6-bromohexanoyl
chlorideinsteadof5-bromovalerylchloride. 1H NMR (CDCl3) δ:
7.87 (d, J = 8.9 Hz, 1H), 7.69(d, J = 8.5 Hz, 1H), 7.58(d, J = 8.2
Hz, 1H), 7,54-7.50 (m, 1H), 7,23-7.19 (m, 1H), 6.97 (d, J = 8.9
Hz, 1H), 4.86 (s, 2H), 3.75 (t, J = 5.0 Hz, 4H), 2.89 (t, J = 7.7 Hz,
2H), 2.60-2.55 (m, 6H), 2.53-2.50 (m, 2H), 2.40 (t, J = 7.7 Hz,
2H), 1.81-1.71 (m, 6H), 1.64-1.57 (m, 2H), 1.50-1.44 (m, 2H).
Mass, m/z: 446 (Mþ), 157 (base).
Ethyl cis-4-(4-Benzylpiperazin-1-yl)cyclohexanecarboxylate (20).
To the ethanol (25 mL) solution of ethyl cis-4-aminocyclohexane
carboxylate hydrochloride (208 mg, 1.00 mmol), N-benzyl-bis(2-
chloroethyl)amine (232 mg, 1.00 mmol), and sodium hydrogen-
carbonate (300 mg, 3.57 mmol) were added and the mixture was
heated at reflux overnight. After concentration, the residue was
purified by silica gel column chromatography (n-hexane:ethyl
3-Amino-2-[4-[4-(3-hydroxyquinolin-2-yl)piperazin-1-yl]butyl]-
5,6,7,8-tetrahydro-3H-quinazolin-4-one (18g). This compound
was synthesized using the same procedure as for 17m starting
with ethyl 2-aminocyclohex-1-enecarboxylate and 2-chloro-3-
1
acetate = 1:2-1:8) to provide 233 mg (68%) of 20. H NMR
(CDCl3) δ: 7.34-7.21 (m, 5H), 4.12 (q, J = 7.3 Hz, 2H), 3.50 (s,
2H), 2.70-2.30 (m, H), 2.30-2.11 (m, 2H), 1.69-1.45 (m, 8H),
1.24 (t, J = 7.3 Hz, 3H). Mass, m/z: 330 (Mþ), 91 (base).
Ethyl cis-4-(4-Quinolin-2-ylpiperazin-1-yl)cyclohexanecarboxylate
(22). To the mixed solution of 20 (920 mg, 2.78 mmol) and ammo-
nium formate (4.40 g, 69.6 mmol) in 100 mL of ethanol, palladium
carbon (10%) (900 mg) was added and the mixture was heated at
reflux for an hour. After filtration, the filtrate was vaporated
under reduced pressure to give a mixture of deprotected piperazine
derivative 21. The residue was dissolved in 2-propanol (100 mL),
and to the solution, triethylamine (6.20 g, 61.2 mmol) and
2-chloroquinoline (9.10 g, 55.6 mmol) were added. The mixture
was heated at reflux overnight. After concentration, the residue
was dissolved with chloroform, washed with saturated aqueous
sodium hydrogencarbonate solution, dried over anhydrous mag-
nesium sulfate, and evaporated under reduced pressure. The
residue was purified by silica gel flush column chromatography
(n-hexane:ethyl acetate = 1:5) to provide 237 mg (23%) of the
N-substituted compound 5 derivative 22.
1
hydroxyquinoline. H NMR (CDCl3) δ: 7.86 (d, J = 8.9 Hz,
1H), 7.62 (d, J = 8.1 Hz, 1H), 7.47 (t, J = 6.9 Hz, 1H), 7.42 (s,
1H), 7.38-7.35 (m, 1H), 4.96 (s, 2H), 3.30 (m, 4H), 2.95-2.91
(m, 2H), 2.67 (br s, 4H), 2.59 (t, J = 6.2 Hz, 2H), 2.54-2.47 (m,
4H), 1.85-1.64 (m, 8H). Mass, m/z: 448 (Mþ), 432, 289, 173 (base).
3-Amino-2-[4-[4-(6-hydroxyquinolin-2-yl)piperazin-1-yl]butyl]-
5,6,7,8-tetrahydro-3H-quinazolin-4-one (18h). This compound
was synthesized using the same procedure as for 17m starting
with ethyl 2-aminocyclohex-1-enecarboxylate and 2-chloro-6-
hydroxyquinoline. 1H NMR (DMSO-d6) δ: 9.36 (br s, 1H), 7.87
(d, J = 9.2 Hz, 1H), 7.44 (d, J = 8.9 Hz, 1H), 7.15 (d, J = 9.2 Hz,
1H), 7.09 (dd, J = 2.7, 9.2 Hz, 1H), 6.96 (d, J = 2.7 Hz, 1H), 5.75
(s, 2H), 3.30 (br s, 4H), 2.85-2.81 (m, 2H), 2.60-2.30 (m, 10H),
1.76-1.54 (m, 8H). Mass, m/z: 289, 173 (base).
3-Amino-2-[4-[4-(7-hydroxyquinolin-2-yl)piperazin-1-yl]butyl]-
5,6,7,8-tetrahydro-3H-quinazolin-4-one (18i). This compound was
synthesized using the same procedure as for 17m starting with
ethyl 2-aminocyclohex-1-enecarboxylate 2-chloro-7-hydroxyqui-
noline. 1H NMR (DMSO-d6) δ: 9.69 (br s, 1H), 7.86 (d, J = 9.2
Hz, 1H), 7.50 (d, J = 8.9 Hz, 1H), 6.95 (d, J = 9.2 Hz, 1H), 6.83
(d, J = 1.9 Hz, 1H), 6.76 (dd, J = 2.3, 8.5 Hz, 1H), 5.75 (s, 2H),
3.62 (br s, 4H), 2.85-2.81 (m, 2H), 2.60-2.30 (m, 10H), 1.76-
1.52 (m, 8H). Mass, m/z: 432, 173 (base).
3-Amino-2-[2-(4-quinolin-2-ylpiperazin-1-yl)methyl]-5,6,7,8-tetra-
hydro-3H-quinazolin-4-one (19a). This compound was synthesized
using the same procedure as for 17m with chloroacetyl chloride
instead of 5-bromovaleryl chloride. 1H NMR (CDCl3) δ: 7.90 (d,
J = 8.9 Hz, 1H), 7.68-7.67 (m, 1H), 7.60 (d, J = 7.7 Hz, 1H),
7.54-7.52 (m, 1H), 7.26-7.24 (m, 1H), 6.96 (d, J = 9.2 Hz, 1H),
6.40 (br s, 2H), 3.73 (br s, 6H), 3.73 (t, J = 5.0 Hz, 4H), 2.64-2.56
(m, 4H), 1.81-1.75 (m, 4H). Mass, m/z: 366 (Mþ), 107 (base).
3-Amino-2-[2-(4-quinolin-2-ylpiperazin-1-yl)ethyl]-5,6,7,8-tet-
rahydro-3H-quinazolin-4-one (19b). This compound was synthe-
sized using the same procedure as for 17 with 3-chloropropionyl
chloride instead of 5-bromovaleryl chloride. 1H NMR (CDCl3)
δ: 7.89 (d, J = 9.2 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.69 (d, J =
8.9 Hz, 1H), 7,52 (ddd, J = 1.5, 6.9, 8.5 Hz, 1H), 7.24-7.20 (m,
1H), 6.96 (d, J = 9.2 Hz, 1H), 5.71 (s, 2H), 3.72 (t, J = 5.0 Hz,
4H), 3.14 (t, J = 6.5 Hz, 2H), 2.86 (t, J = 6.5 Hz, 2H), 2.69 (t,
J = 5.0 Hz, 4H), 2.59 (t, J = 6.2 Hz, 2H), 2.53 (t, J = 6.2 Hz,
2H), 1.81-1.72 (m, 4H). Mass, m/z: 404 (Mþ), 157 (base).
3-Amino-2-[3-(4-quinolin-2-ylpiperazin-1-yl)propyl]-5,6,7,8-tet-
rahydro-3H-quinazolin-4-one (19c). This compound was synthe-
sized using the same procedure as for 17m with 4-chlorobutyryl
chloride instead of 5-bromovaleryl chloride. 1H NMR (CDCl3) δ:
7.88 (d, J = 9.2 Hz, 1H), 7.69 (d, J = 8.1 Hz, 1H), 7.58 (d, J =
7.7 Hz, 1H), 7.54-7.50 (m, 1H), 7.24-7.20 (m, 1H), 6.95 (d, J =
cis-4-(4-Quinolin-2-ylpiperazin-1-yl)cyclohexanecarboxylic Acid
(23). To the solution of 22 (0.17 g, 0.46 mmol) in ethanol (1.0 mL),
1 N aqueous sodium hydroxide solution (1.0 mL) was added and
the mixture was stirred at 80 °C for 30 minuites. After cooling and
neutralization with 1N hydrogen chloride solution, the precipitate
was collected, washed with water, and dried to give the carboxylic
acid 23 (137 mg, 87%). 1H NMR (DMSO-d6) δ: 8.02 (d, J = 9.2
Hz, 1H), 7.70-7.68 (m, 1H), 7.56-7.51 (m, 2H), 7.23-7.19 (m,
2H), 3.66 (t, J = 5.0 Hz,4H), 2.60-2.50 (m, 4H), 1.99-1.96 (m,
2H), 1.56-1.47 (m, 8H). Mass, m/z: 339 (Mþ), 157 (base).
2-{[cis-4-(4-Quinolin-2-ylpiperazin-1-yl)cyclohexanecarbonyl]-
amino}cyclohex-1-enecarboxylic Acid Ethyl Ester (24). To the ice-
cold mixture of ethyl 2-aminocyclohex-1-enecarboxylate (6) (0.15 g,
0.88 mmol) in pyridine, phosphorus trichloride (34 mg, 0.25 mmol)
was added. After 15 miniuites, 23 (75 mg, 0.22 mmol) was added at
room temperature to the mixture, and then was stirred at same
temperature for 6 h. Chloroform was added, washed with saturated
aqueous sodium hydrogencarbonate solution, dried over anhy-
drous magnesium sulfate, and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography
(chloroform:methanol = 20:1) to provide 150 mg (quant) of ester
24. 1H NMR (CDCl3) δ: 11.72 (br s, 1H), 7.86 (d, J = 9.1 Hz, 1H),
7.68 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.53-7.49 (m,
1H), 7.22-7.18 (m, 1H), 6.96 (d, J = 9.2 Hz, 1H), 4.21-4.12 (m,
2H), 3.75-3.72 (m, 4H), 3.00-2.97 (m, 2H), 2.65 (t, J = 5.0 Hz,
4H), 2.50-2.45 (m, 1H), 2.32-2.26 (m, 4H), 2.19-2.12 (m, 1H),
1.83-1.75 (m, 2H), 1.70-1.45 (m, 8H), 1.31-1.25 (m, 3H). Mass,
m/z: 490 (Mþ), 157 (base).
2-{[cis-4-(4-Quinolin-2-ylpiperazin-1-yl)cyclohexanecarbonyl]-
amino}cyclohex-1-enecarboxylic Acid (25). To the solution of

7562 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21
Asagarasu et al.
24 (0.10 g, 0.20 mmol) in ethanol (4.0 mL), 1N aqueous sodium
hydroxide solution (4.0 mL) was added and the mixture was
stirred at 60 °C for 1.5 h. After cooling and neutralization with
1N hydrogen chloride solution, the precipitate was collected,
washed with water, and dried to give the carboxylic acid 25
(78 mg, 83%). 1H NMR (DMSO-d6) δ: 11.88 (br s, 1H), 8.02 (d,
J = 9.2 Hz, 1H), 7.70-7.69 (m, 1H), 7.57-7.49 (m, 2H), 7.24-
7.19 (m, 2H), 3.67 (br s, 4H), 2.85-2.84 (m, 2H), 2.63-2.40
(m,4H), 2.25-2.23 (m, 4H), 2.05-1.90 (m, 2H), 1.70-1.54 (m,
10H). Mass, m/z: 462(Mþ), 157(base).
incubation buffer solution B (an aqueous solution of 50 mmol/l
of Tris-hydrochloric acid, 5 mmol/L of magnesium chloride,
and 1 mmol/L of EDTA, with the pH adjusted to 7.5 with 1 N aq
NaOH at 25 °C) and homogenized to provide membrane sample
suspension B. Each test compound was dissolved in DMSO to
give a 270 μmol/L solution and was diluted to a prescribed
concentration with the incubation buffer solution B to provide a
compound solution. A piece of polypropylene tube was charged
with 20 μL of [3H]BRL-43694 (the concentration of [3H]BRL-
43694 had been adjusted in advance to render its concentration
in the reaction mixture to be 0.5 nmol/L) and 20 μL of a com-
pound solution. A further 500 μL of membrane sample suspen-
sion B was added to the tube, followed by incubation at 25 °C for
60 min. The reaction was terminated by rapid filtration of
the reaction mixture using a Brandel cell harvester through a
GF/B filter, which had been previously immersed in a solution
of 0.5% polyethyleneimine in incubation buffer solution B.
The filter was twice washed with about 5 mL of 50 mmol/L of
Tris-hydrochloric acid which had been cooled to 4 °C. Resi-
dual radioactivity on the filter was measured with a liquid
scintillation counter (Aloka Co., LSC-5100), and percent in-
hibition of [3H]BRL-43694 binding to 5-HT3 receptor by each
test compound was calculated. Buffer B or the test drugs, 20 μL,
[3H]BRL-43694 solution, 20 μL, and the human 5-HT3 receptor
membrane preparation, 500 μL (including 1 unit), were placed in
tubes and mixed to prepare reaction mixtures in dupricate or
triplicate.
3-Amino-2-[cis-4-(4-Quinolin-2-yl-piperazin-1-yl)cyclohexyl]-
5,6,7,8-tetrahydro-3H-quinazolin-4-one (26a) and Its Trans
Isomer (26b). To the ice colded solution of 25 (70 mg, 0.15 mmol)
in pyridine (4.0 mL), acetic anhydride (300 mg) was added.The
mixture was warmed to room temperature and stirred for an hour.
Again the mixture was cooled with ice bath, and 2-propanol (10
mL) was added to the mixtre. At the same temperature, hydrazine
hydrate (2 mL) was added to the mixture. The reaction mixture was
warmed to room temperature and stirred for an hour. After
concentration, the residue was purified by silica gel column chro-
matography (chloroform:methanol = 40:1) to provide 39 mg
(56%) of 26a and 17 mg (24%) of 26b.
1
cis-Isomer (26a). H NMR (CDCl3) δ: 7.88 (d, J = 8.9 Hz,
1H), 7.69 (d, J = 8.5 Hz, 1H), 7.58 (dd, J = 1.2, 8.1 Hz,1H),
7.54-7.50 (m, 1H), 7.23-7.19 (m, 1H), 6.98 (d, J = 8.9 Hz, 1H),
4.83 (s, 2H), 3.77-3.74 (m, 4H), 3.44 (br s, 1H), 2.67 (br s, 4H),
2.60 (t, J = 6.2 Hz, 2H), 2.51 (t, J = 6.2 Hz, 2H), 2.35 (br s 1H),
2.11-2.05 (m, 4H), 1.80-1.58 (m, 8H). Mass, m/z: 458(Mþ),
157(base).
5-HT1A Agonist-induced [35S]GTPγS Binding Assays.19
Human 5-HT1A receptor (Cloned Human Serotonin Receptor
Subtype 1A, produced in CHO cells, PerkinElmer, Inc.) was
thawed on ice and diluted with incubation buffer (20 mM
HEPES/3 mM MgCl2/120 mM NaCl, pH 7.4 at 30 °C). The
membranes were incubated with GDP (20 μM) and the test
drugs at a volume of 900 μL for 20 min at 30 °C and then were
placed on ice for 15 min. [35S]GTPγS (100 pM) was added to the
incubation tubes to yield a final volume of 1 mL, and the tubes
were further incubated for 30 min at 30 °C. Incubation was
terminated by filtering the mixtures through GF/B filters using a
Brandel cell harvester. The filters were washed twice with 5 mL
of cold wash buffer (20 mM HEPES/3 mM MgCl2, pH 7.4 at
4 °C). Radioactivity retained on the filters was counted by a
liquid scintillation counter (Aloka Co., LSC-5100).
Inhibition of 5-HT3 Receptor Mediated Contraction in Guinea
Pig Ileum.20 The ileum preparations were suspended in an organ
bath containing Tyrode solution (137 mM NaCl/3 mM KCl/
2 mM CaCl2/1 mM MgCl2/12 mM NaHCO3/0.4 mM NaH2-
PO4/6 mM D-(þ)-glucose), warmed to 37 °C and aerated with a
mixture of 5% CO2 and 95% O2. Isotonic contractions under a
loading tension of 1 g were recorded using an isotonic force
transducer. Experiments were started after stable contractions
induced by 10 μM 2-methyl-5-HT were obtained at least 3 times.
The vehicle (DMSO) or the test drug was added to the organ
bath and the preparations were exposed to the vehicle or test
drug for 20 min. Then 2-methyl-5-HT (10 μM) was added to the
organ bath and the contractions were recorded.
5-HT1A Receptor-mediated Behavior and Hypothermia in Rats.
Rats were acclimated to the test environment for 2 weeks prior to
testing and were conditioned to the test procedures during this
period. On the day of the experiment, rats were acclimated to the
test cage for 1 h. Test compound or vehicle (saline containing
diluted hydrochloric acid) was injected intraperitoneally, and
then 5-HT1A receptor-mediated behavior (lower lip retraction
and flat body posture) was measured in the test cage. Behavioral
responses were measured at 5, 10, 20, and 30 min after adminis-
tration using a 0-3 scale as previously described in the litera-
ture.21 The rectal temperature was recorded before and at 30 min
after administration of the test drug using a thermistoprobe that
was inserted into the rectum 3 cm from the anal orifice. The
difference between the temperatures measured before and after
administration was designated as the index of hypothermia.
trans-Isomer (26b). 1H NMR (CDCl3) δ: 7.88 (d, J = 9.2 Hz,
1H), 7.69 (d, J = 8.5 Hz, 1H), 7.59-7.57 (m,1H), 7.54-7.50 (m,
1H), 7.23-7.19 (m, 1H), 6.98 (d, J = 8.9 Hz, 1H), 4.81 (s, 2H),
3.78-3.75 (m, 4H), 3.29-3.23 (m, 1H), 2.73 (t, J = 5.0 Hz 4H),
2.58-2.55 (m, 2H), 2.52-2.49 (m, 3H), 2.08-1.98 (m, 4H),
1.77-1.60 (m, 8H). Mass, m/z: 458(Mþ), 157(base).
Evaluation of Biological Activity. Measurement of Compound
Affinity to Human 5-HT1A Receptor (in Vitro).8,12,13 A 0.25 mL
(about 50 units) sample of CHO cell membrane expressing
human 5-HT1A receptor (PerkinElmer, Inc.) was added to
24.75 mL of incubation buffer solution A (an aqueous solution
of 50 mmol/l of Tris-hydrochloric acid, 10 mmol/L of magne-
sium sulfate, 0.5 mmol/L of EDTA, and 0.1% ascorbic acid,
with the pH adjusted to 7.4 with 1 N aqueous sodium hydroxide
solution at 27 °C) and was labeled as membrane sample suspen-
sion A. Each test compound was dissolved in DMSO to give a
270 μmol/L solution and was diluted to a prescribed concentra-
tion with the incubation buffer solution A to provide a com-
pound solution. A piece of polypropylene tube was charged with
20 μL of [3H]-1 (the concentration of the [3H]-1 had been
adjusted in advance to render its concentration in the reaction
mixture to be 0.25 nmol/L) and 20 μL of a compound solution. A
further 500 μL of membrane sample suspension A was added to
the tube, followed by incubation at 27 °C for 60 min. The reaction
was terminated by rapid filtration of the reaction mixture using
Brandel cell harvester through a GF/C filter which had been
previously immersed in a solution of 0.3% polyethyleneimine in
incubation buffer solution A. The filter was twice washed with
about 5 mL of 50 mmol/L of Tris-hydrochloric acid which had
been cooled to 4 °C. Residual radioactivity on the filter was mea-
sured with a liquid scintillation counter (Aloka Co., LSC-5100),
and percent inhibition of [3H]-1 binding to 5-HT1A receptor by
each test compound was calculated. Buffer A or the test drugs,
20 μL, [3H]-1 solution, 20 μL, and the human 5-HT1A receptor
membrane preparation, 500 μL, (including 1 unit) were placed in
tubes and mixed to prepare reaction mixtures in duplicate or
triplicate.
Measurement of Compound Affinity to Human 5-HT3 Recep-
tor (in Vitro).8,12,13 A 0.05 mL (about 50 microassay) sample of
HEK-293 cell membrane expressing human 5-HT3 receptor
(purchased from BIOLINKS KK) was added to 24.95 mL of

Article
5-HT-induced Bradycardia (von Bezold-JarischReflex) inRats.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 21 7563
of Irritable Bowel Syndrome (IBS). Chem. Pharm. Bull. 2009,
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The surgical procedures and mean heart rate recordings were
performed as follows: animals were anesthetized with urethane
at a dose of 1.25 g/kg, ip, and then polyethylene cannulas were
inserted into the right common carotid artery and vein to mea-
sure the blood pressure and to administer 5-HT, respectively. The
duodenum was incised, decorticated 2-3 cm from the stomach
and cannulated for intraduodenal administration of the test drugs.
Blood pressure was monitored using a pressure amplifier (AP-
601G; Nihon Kohden Co., Tokyo), and the mean heart rate was
recorded by a tachometer (AT-601G; Nihon Kohden Co., Tokyo)
triggered by blood pressure pulsation. 5-HT was injected intrave-
nously at 300 μg/kg to evoke a transient bradycardia (B-J reflex).
Following recovery to normal blood pressure and heart rate, the
test drugs and vehicle (0.5% Tween 80) were administered into the
duodenum. Thirty minutes later, 5-HT was readministered intra-
venously and the bradycardia was assessed.
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Supporting Information Available: IC50 values of 17m for
receptors and transporters. This material is available free of
charge via the Internet at http://pubs.acs.org.
ꢀ
ꢀ
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