Potent and orally bioavailable CCR4 antagonists: Synthesis and structure-activity relationship study of 2-aminoquinazolines

Article abstract of DOI:10.1016/j.bmc.2008.11.020

Starting with a series of CC chemokine receptor-4 (CCR4) antagonists developed in a previous study, the potency was improved by replacing the pyrrolidine moiety of N-(4-chlorophenyl)-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidin-1-yl)quinazolin-4-amine 2 with a 3-(hydroxymethyl)piperidine. The resulting compound (1′-{4-[(4-chlorophenyl)amino]-6,7-dimethoxyquinazolin-2-yl}-1,4′-bipiperidin-3-yl)methanol 8ic was a strong inhibitor of human/mouse chemotaxis. Oral administration of 8ic showed anti-inflammatory activity in a murine model of acute dermatitis (oxazolone-induced contact hypersensitivity test) in a dose-dependent manner.

Full text of DOI:10.1016/j.bmc.2008.11.020

Bioorganic & Medicinal Chemistry 17 (2009) 64–73
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Potent and orally bioavailable CCR4 antagonists: Synthesis and
structure–activity relationship study of 2-aminoquinazolines
*
Kazuhiro Yokoyama , Noriko Ishikawa, Susumu Igarashi, Noriyuki Kawano, Naoyuki Masuda,
Wataru Hamaguchi, Shingo Yamasaki, Yohei Koganemaru, Kazuyuki Hattori, Takahiro Miyazaki,
Shin-ichi Ogino, Yuzo Matsumoto, Makoto Takeuchi, Mitsuaki Ohta
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba-shi, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Starting with a series of CC chemokine receptor-4 (CCR4) antagonists developed in a previous study, the
potency was improved by replacing the pyrrolidine moiety of N-(4-chlorophenyl)-6,7-dimethoxy-2-(4-
pyrrolidin-1-ylpiperidin-1-yl)quinazolin-4-amine 2 with a 3-(hydroxymethyl)piperidine. The resulting
compound (10-{4-[(4-chlorophenyl)amino]-6,7-dimethoxyquinazolin-2-yl}-1,40-bipiperidin-3-yl)metha-
nol 8ic was a strong inhibitor of human/mouse chemotaxis. Oral administration of 8ic showed anti-
inflammatory activity in a murine model of acute dermatitis (oxazolone-induced contact hypersensitivity
test) in a dose-dependent manner.
Received 16 September 2008
Revised 7 November 2008
Accepted 8 November 2008
Available online 17 November 2008
Keywords:
Chemokine receptor 4 (CCR4) antagonists
2-Aminoquinazolines
Ó 2008 Elsevier Ltd. All rights reserved.
Inflammatory disease
1. Introduction
phenyl)-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidin-1-yl)quinaz-
olin-4-amine 2, which had a low intrinsic clearance value (human
Chemokines are relatively small (ꢀ8 to 14 kDa), mostly basic
proteins that are involved in various physiological and patho-
logical processes.1,2 CC chemokine receptor 4 (CCR4) was originally
cloned from T-lymphocyte and thymus cells, where it is highly
expressed.3–5 Almost all skin-homing cells expressing the cutane-
ous lymphocyte antigen in blood carry CCR4.6 Thymus and
activation-regulated chemokine (TARC, CCL17) as well as macro-
phage-derived chemokine (MDC, CCL22) are CC chemokines that
are both highly specific biological ligands for CCR4.7,8 In vivo stud-
ies of CCL17 and CCL22 antibodies have indicated their utility in
preventing several immunological responses.9–14 Therefore, the
use of CCR4 antagonists would be a novel, therapeutic method of
intervention for disease in which CCR4 participates.
liver microsomes), although the functional inhibition of chemotaxis
in mice remained moderate.16 The potency of these compounds, 1a,
1b, and 2, was evident after subcutaneous administration in the
murine oxazolone-induced contact hypersensitivity test, a known
model of acute skin inflammation. When these compounds were
administered orally, however, very little inhibition was observed
(Table 5). In this paper, further efforts to circumvent these problems
via synthesis are described. In the previous report, the metabolic
liability of the cycloheptylamine substituent was identified as a fac-
tor that might affect the high clearance of compound 1a in human
liver microsomes. The previous structure–activity relationship
(SAR) study on compound 2 also suggested that the introduction
of an aminoalcohol at the 4-position of the 2-piperidine ring
produced a favorable effect on the inhibition of human chemotaxis.
Based on these findings, a series of compounds was synthesized
where the cycloheptylamine and piperidine of 2 were replaced with
various substituents.
In a previous manuscript,15 N-cycloheptyl-6,7-dimethoxy-2-(4-
pyrrolidin-1-ylpiperidin-1-yl)quinazolin-4-amine 1a was identi-
fied as a potent CCR4 antagonist with potent in vitro (GTPcS
binding) activity (Fig. 1). Optimization of compound 1a led to the
discovery of 2-(1,40-bipiperidin-10-yl)-N-cycloheptyl-6,7-dimeth-
oxyquinazolin-4-amine 1b, which potently inhibited functional
chemotaxis in mice (IC50 = 39 nM), although the compound also
had a high intrinsic clearance value (human liver microsomes).
Additional optimization to identify a CCR4 antagonist with im-
proved metabolic stability led to the discovery of N-(4-chloro-
2. Chemistry
Novel aminoalcohol 5 was prepared from tert-butyl 4-oxopi-
peridine-1-carboxylate 3 in 2 or 3 steps, as summarized in Scheme
1. The reductive amination of compound 3 with an appropriate
amine afforded the corresponding N-Boc-protected diamines 4a,
4c, and 4e. The O-methylated compounds 4b and 4d were obtained
from 4a and 4c, respectively, by treatment with sodium hydride
* Corresponding author. Tel.: +81 29 863 6773; fax: +81 29 854 1519.
E-mail address: kazuhiro.yokoyama@jp.astellas.com (K. Yokoyama).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.11.020

K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
65
HN
N
MeO
MeO
N
Cl
Cl
N
X1
N
HN
1a
MeO
MeO
MeO
N
N
N
N
MeO
N
N
R
HN
N
N
N
X2
MeO
MeO
2
N
N
N
1b
Figure 1. CCR4 inhibitors 1a, 1b, 2 discovered previously, and 2-aminoquinazolines evaluated in this article.
The preparation of 2-aminoquinazolines 8 is summarized in
Scheme 2. Selective substitutions at the 4-position of quinazoline
6 with various amines proceeded smoothly to yield the corre-
sponding 4-amino-2-chloro-quinazolines 7a–h, even though this
procedure did not work well with the reaction of 4-chloroaniline.
The coupling of 4-chloroaniline and quinazoline 6 occurred in the
mixture of aqueous hydrogen chloride and ethanol to yield an
inseparable mixture of the desired 4-amino-2-chloro-quinazoline
7i and 2,4-disubstituted quilazoline (ca. 9–10:1), which was
brought to the next reaction. Introduction of the 4-chlorophenyl
moiety was achieved under Suzuki-coupling conditions to yield
7j. Couplings of 4-chlorobenzamide/4-chlorosulfonamide with 6
were performed under basic (NaH/DMF and t-BuOK/DMF, respec-
tively) conditions to afford the corresponding benzamide 7k and
sulfonamide 7l. Aroylation of 6 with 4-chlorobenzaldehyde pro-
ceeded via treatment with NaH/1,3-dimethylimidazolium iodide
in refluxing dioxane to yield benzoylquinazoline 7m.17 Subsequent
substitutions at the 2-position of quinazolines 7a–m with piperi-
dines readily proceeded in the presence of base (Hunig’s base or
DBU) to yield the corresponding 2-aminoquinazolines 8a–m. Ben-
zylalcohol 8n was prepared by the reduction of the ketone 8m.
O
O
i
iii
HN
O
N
O
N
R1
R1
N
O
3
4
5
R2
R1=
R1=
N
OH
O
4a : R2=OH
4e
ii
ii
4b : R2=OMe
4c : R2=CH2OH
4d : R2=CH2OMe
Scheme 1. Reagents and conditions: (i) Amines, 10% Pd–C, MeOH; (ii) NaH, MeI,
DMF; (iii) 4-M HCl, AcOEt.
and methyl iodide. Deprotection of the Boc group of compounds
4a–e proceeded readily under acidic conditions to yield the desired
amines 5a–e.
O
Cl
N
R1
R1
8m : R1=
i
ii
O
O
O
O
O
O
N
N
N
Cl
Cl
iii
OH
Cl
N
Cl
N
N
8n : R1=
R2
8
6
7
H
N
O
N
7e : R1=
7f : R1=
7g : R1=
7h : R1=
7i : R1=
7j : R1=
7k : R1=
7l : R1=
7a : R1=
7b : R1=
7c : R1=
7d : R1=
N
7m : R1=
H
H
Cl
Cl
Cl
Cl
H
N
O
N
H
Cl
Cl
O
S
Me
N
O
O
N
H
N
H
Cl
Cl
H
N
O
Cl
N
H
S
N
O
Cl
Scheme 2. Reagents and conditions: (i) For 7a–h, amines, Hunig’s base, DMF; For 7i, 4-chloroaniline, 1-M HCl, EtOH; For 7j, 4-chlorophenyl boronic acid, Pd(PPh3)4, Na2CO3
aq., toluene; For 7k, 4-chlorobenzene amide, NaH, DMF; For 7l, 4-chlorobenzene sulfoneamide, t-BuOK, DMF; For 7m, 4-chlorobenzaldehyde, 1,3-dimethylimidazolium
iodide, NaH, dioxane; (ii) 4R1-piperidine, n-BuOH, Hunig’s base or DBU; (iii) NaBH4, MeOH.

66
K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
Table 3
3. Results and discussion
The effect of the linker between the 4-chlorobenzene and the quinazoline
The CCR4 antagonist activities of the synthesized compounds
were determined by measuring the degree to which human
Cl
CCL22-derived [35S]GTP
cS was prevented from binding to the
X
MeO
receptor. The CCR4-antagonism of these compounds was also con-
firmed using the chemotaxis assay. The results are summarized in
Tables 1–3.
N
MeO
N
N
N
OH
We first explored the substituents at the 4-position of piperi-
dine (Table 1). The 3-hydroxypiperidine analogue 8ia was slightly
less active than the parent compounds 1a, 1b, and 2 in the
Compound
X
[
35S]GTP
c
S
Chemotaxis
Chemotaxis
[
35S]GTP
cS-binding assay, but was a potent chemotactic factor
IC50
(lM)
IC50
(
l
M)
IC50 (lM)
Humana
Humana
Mousea
8a
8b
8c
8d
8j
8k
8l
8m
8n
8ic
2
–NHCH2–
–NHCH2CH2–
–N(CH3)CH2CH2–
–NHCH2CH2CH2–
none
–NHCO–
–NHSO2–
–CO–
0.087
0.061
23% at 1
0.18
0.45
43% at 1
NTb
0.14
0.39
NTb
NTb
NTb
NTb
NTb
NTb
NTb
0.058
0.13
Table 1
lM
CCR4 inhibitory activities of 4-substituted piperidine derivatives
l
l
M
l
M
M
NTb
Cl
>1
lM
NTb
NTb
HN
11% at 1
5% at 1
l
NTb
MeO
N
42% at 1
M
NTb
–CHOH–
–NH–
7% at 1
0.019
l
M
NTb
MeO
N
N
R1
0.023
0.11
N
0.024
X
a
See Section 6.2, pharmacology.
Not tested.
b
Compound
X
R1
[
35S]GTP
c
S
Chemotaxis
Chemotaxis
IC50
(
l
M)
IC50
(
l
M)
IC50 (lM)
Humana
Humana
Mousea
8ia
8ib
8ic
8id
8ie
1a
–CH2–
–CH2–
–CH2–
–CH2–
–O–
–OH
–OMe
–CH2OH
–CH2OMe
–CH2OH
0.048
0.10
0.019
0.052
0.10
0.019
0.018
0.024
0.085
0.51
0.023
0.064
0.62
0.20
0.14
0.11
0.43
0.42
0.058
0.23
50% at 1
0.13
for human cells. O-Methylation of the hydroxyl group deteriorated
its activity in the [35S]GTP
S-binding and human chemotaxis as-
says (8ib). The results of the 3-(hydroxymethyl)piperidine ana-
c
logue 8ic were comparable to those of the parent compounds in
lM
the [35S]GTP
cS-binding and mouse chemotaxis assays, and also
1b
2
0.039
0.13
showed more potent activity than the parent compounds in the
human chemotaxis assay. O-Methylation of the hydroxyl group
of 8ic resulted in the loss of potency in all three assays (8id). This
indicates that the hydroxyl group at this position is important,
especially for the inhibition of chemotaxis. Introduction of oxygen
into the piperidine ring resulted in significant loss in all three as-
says (8ie).
a
See Section 6.2, pharmacology.
Table 2
CCR4 inhibitory activities of 4-substituted quinazoline derivatives
With the finding of this most promising 3-(hydroxymethyl)-
piperidine moiety, the focus of the modification was shifted to
the substituent at the 4-position of compound 8ic (Table 2). First,
the cycloheptylamine moieties were introduced to verify that the
SAR discovered in a previous report15 would be applicable. The
results obtained with compound 8e were almost comparable to
those with 8ic, and indicated that the SAR at this position would
be similar to that of the derivatives with a 4-(1-pyrrolidinyl)-piper-
idine moiety at the 2-position of the quinazoline core. The
introduction of tetrahydro-2H-pyran-4-amine (8f) and tetrahy-
dro-2H-thiopyran-4-amine 1,1-dioxide (8g) resulted in a complete
loss of potency. These results clearly showed that the hydrophobic
moieties are needed for potent activity. Another more rigid 5-chlo-
roindoline substituent was also introduced (8h), but it was not
effective.
The effect of the linker between the 4-chlorobenzene and the
quinazoline was investigated last (Table 3). One to three methy-
lene linkers were introduced between benzene and nitrogen, but
these modifications (8a, 8b, 8d) did not result in improved po-
tency. N-Methylation of the nitrogen atom to the corresponding
N-methyl derivative (8c) led to considerable loss of potency. Re-
moval of the linker (8j) or replacement of the nitrogen with an
amide (8k), sulfonamide (8l), ketone (8m), or hydroxymethyl
(8n) all reduced the potency. These findings indicated that an ami-
no, NH linker is necessary for optimal potency.
R2
MeO
N
MeO
N
N
N
OH
Compound R2
[
(
35S]GTP
c
S IC50 Chemotaxis
Chemotaxis
l
M) Humana
IC50
(
l
M)
IC50
(
l
M)
Humana
Mousea
8e
0.030
0.10
0.019
HN
O
8f
4% at 1
0% at 1
lM
NTb
NTb
NTb
NTb
HN
HN
O
S
8g
lM
O
Cl
8h
40% at 1
0.019
lM
NTb
NTb
N
8ic
0.023
0.058
a
Before the evaluation of in vivo pharmacology, the metabolic
stabilities of 8e and 8ic in human liver microsomes were further
See Section 6.2, pharmacology.
Not tested.
b

K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
67
Table 4
The intrinsic clearance values of compounds 1a, 1b, 2, 8e, and 8ic
300
Compound
Intrinsic clearance values (ml/min/kg)
po,100 mg/kg
po, 30 mg/kg
200
100
0
1a
1b
2
8e
8ic
6600
17377
2532
9449
5047
0
4
8
12
16
20
24
Table 5
Time (hr)
The inhibitory effects of 1a, b, 2, 8e, and 8ic in vivo
Compound
Oxazolone-induced contact hypersensitivity
Figure 2. Pharmacokinetic profile of compound 8ic.
Dose
% Inhibition of ear swellinga
1a
30 mg/kg s.c., sidb
100 mg/kg, p.o., bidc
30 mg/kg, p.o., bidc
30 mg/kg, s.c., bidc
30 mg/kg, p.o., bidc
30 mg/kg, p.o., bidc
30 mg/kg, s.c., sidb
10 mg/kg, p.o., sidb
30 mg/kg, p.o., sidb
100 mg/kg, p.o., sidb
39**,d
8
Table 6
1b
2
2
Pharmacokinetic parameters of compound 8ic in plasma after a single oral admin-
istration to mice
43**,d
2
Parameter
Dose (mg/kg)
30
8e
8ic
11
44**
,d
100
13
Tmax (h)
2
2
17
Cmax (ng/mL)
AUC0?24h (ng/h/mL)
139
1014
262
1749
39**,d
a
See Section 6.2, pharmacology.
Administered once daily.
Administered twice daily.
b
c
availability of the compounds compared to the piperidinopyrroli-
dine moiety.
d
p < 0.01 versus vehicle, Dunnett’s multiple range test.
The plasma concentration of compound 8ic was also measured
after oral administration of 30 mg/kg and 100 mg/kg for PK-PD
correlation (Fig. 2 and Table 6). The maximum plasma concentra-
tion (Cmax) reached 139 ng/mL (30 mg/kg dose) and 262 ng/mL
(100 mg/kg dose), which are both well above the mouse chemo-
taxis IC50 value (30 ng/mL). A good relationship between the
maximum plasma concentration and the rate of ear swelling inhi-
bition was also noted. These results suggested that the level of
concentration of the compound obtained by the 100 mg/kg dose
is required to obtain a potent activity in the acute dermatitis
model.
characterized to verify the effect of the hydroxymethyl piperidine
moiety of these compounds on the metabolic factor (Table 4).
Interestingly, introduction of the hydroxymethyl group to the
piperidine ring improved the clearance value (8e, CLint
=
9499 mL/h/kg) of the compound 1b (CLint = 17,377 mL/h/kg). The
clearance value of compound 8e was further improved by replac-
ing the cycloheptyl ring with the 4-chrolobenzene ring (8ic,
CLint = 5,047 mL/h/kg). These data suggest that the hydroxymethyl
group somewhat impedes the metabolism of the piperidine ring.
Although the hydroxymethylpiperidine moiety is slightly more
vulnerable than the pyrrolidine moiety (8e vs 1a and 8ic vs 2),
compounds 8ic and 2 are thought to be metabolically stable
enough, and these clearance values are comparable to those of sev-
eral commercial drugs.18
5. Conclusion
Starting with CCR4 antagonists 1a, 1b, and 2 (reported previ-
ously), potency was improved by replacing the pyrrolidine moiety
of 2 with a 3-(hydroxymethyl)piperidine. The resulting compound
(8ic) was a strong inhibitor of human (IC50 = 23 nM) and mouse
(IC50 = 58 nM) cell chemotaxis. Also, 8ic was found to be an orally
bioavailable inhibitor, and its oral administration resulted in dose-
dependent anti-inflammatory activity in a murine model of acute
dermatitis. The potency obtained at the 100 mg/kg dose was almost
comparable to those of other anti-inflammatory drugs, including
steroids. These results indicated that this quinazoline-based CCR4
antagonist has the potential to treat inflammatory skin diseases.
Further efforts to improve the pharmacokinetic parameters and
potency related to this series will be reported in due course.
4. In vivo pharmacology
Since several compounds (1a, 1b, 2, 8e, 8ic) were identified as
potent inhibitors of mouse chemotaxis, their efficacy was evalu-
ated in vivo (murine oxazolone-induced contact hypersensitivity
test, Table 5). Compounds 1a, 2, and 8ic were administered subcu-
taneously to mice previously sensitized to oxazolone. Potent inhi-
bition (39–44%) of ear swelling was observed at the 30 mg/kg level
(one or two doses). These results were almost comparable to those
of anti-inflammatories (steroids and non-steroidal anti-inflamma-
tory drugs) reported in the literature.19 Compounds 1a and 2,
which have a piperidinopyrrolidine moiety at the 2-position of
the quinazoline core, were also administered orally, but very little
inhibition was observed. Given that these compounds are effica-
cious when administered subcutaneously, their poor oral bioavail-
ability is probably due to poor intestinal absorption. In contrast,
oral administration of compounds 8e and 8ie, which have a bipipe-
ridinylmethanol moiety, yielded more favorable results than that
of compounds 1a and 2, and oral administration of 8ic at a
100 mg/kg dose was found to be almost as efficacious as the
30 mg/kg subcutaneous dose. These results indicated that the
bipiperidinylmethanol moiety had a good effect on the oral bio-
6. Experimental
6.1. Chemistry
In general, reagents and solvents were used as purchased with-
out further purification. Melting points were determined with a
Yanaco MP-500D melting point apparatus and left uncorrected.
1H NMR spectra were recorded on a JEOL JNM-LA300 or a JEOL
JNM-EX400 spectrometer. Chemical shifts were expressed in
d(ppm) values with tetramethylsilane as an internal standard

68
K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
(NMR description: s, singlet; d, doublet; t, triplet; m, multiplet; and
br, broad peak). Mass spectra were recorded on a JEOL JMS-LX2000
spectrometer. The elemental analyses were performed with a
Yanaco MT-5 microanalyzer (C, H, N) and Yokogawa IC-7000S ion
chromatographic analyzer (halogens) and were within 0.4% of
the theoretical values.
mixture was stirred at room temperature for 3 days. The reaction
mixture was concentrated in vacuo, and the residue was washed
with ethyl acetate to yield 1,40-bipiperidin-3-ol hydrochloride salt
5a (973 mg, 3.8 mmol, 2 steps, 68%) as a colorless solid.
MS (FAB+) m/z 185 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.27–1.36 (1H, m), 1.58–1.69 (1H, m), 1.84–2.30 (6H, m), 2.55–
2.63 (1H, m), 2.81–2.90 (2H, m), 2.99–3.17 (1H, m), 3.22–3.28
(2H, m), 3.38–3.50 (3H, m), 3.98–4.08 (1H, m), 9.14 (1H, br), 9.33
(1H, br), 11.38 (1H, s, br).
6.1.1. tert-Butyl 3-hydroxy-1,40-bipiperidine-10-carboxylate (4a)
3-Hydroxypiperidine (2.02 g, 20 mmol) and 10% palladium on
carbon (1.0 g) was added to the solution of tert-butyl 4-oxopiperi-
dine-1-carboxylate (3.98 g, 20 mmol) in MeOH (100 ml), and the
mixture was stirred at room temperature under 1 atm of hydrogen
gas for 18 h, and then filtered through a pad of Celite. The filtrate
was concentrated to yield crude tert-butyl 3-hydroxy-1,40-bipiper-
idine-10-carboxylate 4a (5.75 g) as a yellow oil.
Compounds 5b–d were prepared using procedures similar to
those described for the synthesis of 5a.
6.1.6. 3-Methoxy-1,40-bipiperidine (5b)
The hydrochloride salt of 5b (1.38 g, 5.1 mmol, 77%) was ob-
tained as a colorless solid from tert-butyl 3-methoxy-1,40-bipiper-
idine-10-carboxylate 4b (1.98 g).
6.1.2. tert-Butyl 3-methoxy-1,40-bipiperidine-10-carboxylate (4b)
NaH (60% in oil, 1.11 g, 27.8 mmol) was added to the solution
of crude 4a (4.15 g) in DMF (70 ml), and the mixture was stirred
at room temperature for 30 min. Methyl iodide (1.56 ml,
25.1 mmol) was added to the reaction mixture, and the mixture
was stirred at room temperature for 2 h. The reaction mixture
was quenched by adding ice water, and the resulting mixture
was extracted with ethyl acetate. The organic layer was washed
with brine, dried over sodium sulfate, filtered, and evaporated in
vacuo. The residue was purified via column chromatography
(chloroform/MeOH/NH4OH) to yield tert-butyl 3-methoxy-1,40-
bipiperidine-10-carboxylate 4b (1.98 g, 6.63 mmol, 2 steps, 46%)
as a yellow oil.
MS (FAB+) m/z 199 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.23–1.1.35 (1H, m), 1.55–1.72 (1H, m), 1.89–2.34 (6H, m), 2.64–
2.72 (1H, m), 2.87–2.90 (3H, m), 2.97–3.17 (1H, m), 3.23–3.3.35
(1H, m), 3.31 (3H, s), 3.38–3.49 (5H, m), 3.72–3.77 (1H, m), 9.34–
9.37 (1H, br), 9.50 (1H, s, br), 11.50 (1H, s, br).
6.1.7. 1,40-Bipiperidin-3-ylmethanol (5c)
The hydrochloride salt of 5c (5.08 g, 94%) was obtained as a pale
yellow solid from tert-butyl 3-(hydroxymethyl)-1,40-bipiperidine-
10-carboxylate 4c (6.23 g).
1H NMR (400 MHz, CD3OD) d: 1.20–1.34 (1H, m), 1.70–1.77 (1H,
m), 1.80–2.07 (4H, m), 2.10–2.25 (1H, m), 2.28–2.40 (2H, m), 2.74–
2.82 (1H, m), 2.87–2.96 (1H, m), 3.00–3.09 (2H, m), 3.23 (3H, s),
3.27–3.33 (2H, m), 3.45–3.54 (5H, m).
MS (FAB+) m/z 299 [M+H]+. 1H NMR (400 MHz, CDCl3) d: 1.20–
1.29 (1H, m), 1.46 (9H, s), 1.69–1.78 (4H, m), 1.92–1.96 (1H, m),
2.13–2.26 (2H, m), 2.40–2.48 (1H, m), 2.66–2.68 (3H, m), 2.93–
2.95 (1H, m), 3.00–3.12 (1H, m), 3.25–3.31 (1H, m), 3.36 (3H, s),
3.70–3.83 (1H, m), 4.14 (2H, br).
6.1.8. 3-(Methoxymethyl)-1,40-bipiperidine (5d)
Compound 5d (662 mg, 3.12 mmol, 75%) was obtained as a pale
yellow oil from tert-butyl 3-(methoxymethyl)-1,40-bipiperidine-10-
carboxylate 4d (1.3 g, 4.2 mmol).
6.1.3. tert-Butyl 3-(hydroxymethyl)-1,40-bipiperidine-10-carbo-
xylate (4c)
1H NMR (400 MHz, CDCl3) d: 1.20–1.34 (1H, m), 1.70–1.77 (1H,
m), 1.80–2.07 (4H, m), 2.10–2.25 (1H, m), 2.28–2.40 (2H, m), 2.74–
2.82 (1H, m), 2.87–2.96 (1H, m), 3.00–3.09 (2H, m), 3.23 (3H, s),
3.27–3.33 (2H, m), 3.45–3.54 (5H, m).
3-Piperidinemethanol (4.43 g, 38 mmol) and 10% palladium on
carbon (700 mg) was added to the solution of tert-butyl 4-oxo-1-
piperidine carboxylate (7.66 g, 38 mmol) in MeOH (200 ml). The
mixture was stirred at room temperature under 1 atm of hydrogen
gas for 2 days, and then filtered through a pad of Celite. The filtrate
was concentrated in vacuo. The residue was purified via column
chromatography (chloroform/MeOH/NH4OH) to yield tert-butyl
3-(hydroxymethyl)-1,40-bipiperidine-10-carboxylate 4c (9.98 g,
81%) as a pale yellow oil.
6.1.9. 2-Chloro-N-(4-chlorobenzyl)-6,7-dimethoxyquinazolin-
4-amine (7a)
4-Chlorobenzylamine (547 mg, 3.80 mmol) was added to a
solution of 2,4-dichloro-6,7-dimethoxyquinazoline
6 (500 mg,
1.93 mmol) in DMF (70 ml). The resulting mixture was stirred at
room temperature for 29 h, and then concentrated in vacuo. 1-M
NaOH aqueous solution (2 ml) was added to the residue, and the
resulting mixture was extracted with chloroform. The organic layer
was washed with brine, dried over sodium sulfate, filtered, and
evaporated in vacuo. The residue was purified via column chroma-
tography (hexane/ethyl acetate) to yield 2-chloro-N-(4-chloroben-
zyl)-6,7-dimethoxyquinazolin-4-amine 7a (762 mg, quant.) as a
white solid.
MS (FAB+) m/z 299 [M+H]+. 1H NMR (400 MHz, CDCl3) d: 1.17–
1.26 (1H, m), 1.45 (9H, s), 1.51–1.62 (2H, m), 1.65–1.84 (6H, m),
2.23–2.27 (1H, m), 2.34–2.47 (2H, m), 2.64–2.67 (3H, m), 2.85
(1H, m), 3.54–3.58 (1H, m), 3.67–3.71 (1H, m), 4.14 (2H, s, br).
6.1.4. tert-Butyl 3-(methoxymethyl)-1,40-bipiperidine-10-carbo-
xylate (4d)
Compound 4d (1.3 g, 4.2 mmol, 84%) was obtained as a colorless
oil from tert-butyl 3-(hydroxymethyl)-1,40-bipiperidine-10-carbox-
ylate 4c (1.49 g, 5 mmol) using procedures similar to those de-
scribed for the synthesis of 4b.
MS (ESI+) m/z 364 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
3.88 (3H, s), 3.97 (3H, s), 4.73 (2H, d, J = 5.7 Hz), 7.10 (1H, s),
7.40 (4H, br s), 7.67 (1H, s), 7.95 (1H, s), 8.31 (1H, s), 8.88 (1H,
t, J = 5.7 Hz).
Compounds 7b–h were prepared using procedures similar to
those described for the synthesis of 7a.
MS (FAB+) m/z 313 [M+H]+. 1H NMR (400 MHz, CDCl3) d: 0.92–
1.00 (1H, m), 1.37–1.44 (1H, m), 1.45 (9H, s), 1.51–1.59 (1H, m),
1.61–1.77 (5H, m), 1.84–1.94 (2H, m), 2.11–2.17 (1H, m), 2.37–
2.44 (1H, m), 2.63–2.70 (2H, m), 2.79–2.82 (1H, m), 2.92–2.94
(1H, m), 3.18–3.30 (2H, m), 3.31 (3H, s), 4.13 (2H, s, br).
6.1.10. 2-Chloro-N-[2-(4-chlorophenyl)ethyl]-6,7-dimetho-
xyquinazolin-4-amine (7b)
Compound 7b (955 mg, quant.) was obtained as a white amor-
phous from 2,4-dichloro-6,7-dimethoxyquinazoline 6 (500 mg,
1.93 mmol), 4-chlorophenethylamine (300 mg, 1.93 mmol) and
Hunig’s base (300 mg, 2.32 mmol).
6.1.5. 1,40-Bipiperidin-3-ol (5a)
4-M HCl ethyl acetate solution (10 ml) was added to the solu-
tion of crude 4a (1.6 g) in ethyl acetate (20 ml), and the resulting

K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
69
MS (FAB+) m/z 378 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
2.94–2.98 (2H, m), 3.67–3.72 (2H, m), 3.87 (3H, s), 3.89 (3H,s),
7.08 (1H, s), 7.30 (1H, d, J = 8.2 Hz), 7.36 (1H, d, J = 8.2 Hz), 7.59
(1H, s), 7.95 (1H, s), 8.43 (1H, t, J = 5.2 Hz).
6.1.17. 2-Chloro-N-(4-chlorophenyl)-6,7-dimethoxyquinazolin-
4-amine (7i)
4-Chloroaniline (2.55 g, 20 mmol) and 1-M HCl aqueous solu-
tion (15 ml) was added to the solution of 2,4-dichloro-6,7-dimeth-
oxyquinazoline (5.18 g, 20 mmol) in EtOH (150 ml), and the
resulting mixture was stirred at 60 °C for 10 h. The reaction mix-
ture was neutralized with 1-M NaOH aqueous solution, and the
resulting precipitate was filtered to yield crude 7i (6.92 g), which
was used in subsequent reaction without further purification.
6.1.11. 2-Chloro-N-[2-(4-chlorophenyl)ethyl]-6,7-dimethoxy-N-
methylquinazolin-4-amine (7c)
Compound 7c (239 mg, 32%) was obtained as a grayish solid
from 2,4-dichloro-6,7-dimethoxyquinazoline
6 (500 mg, 1.93
mmol), 2-(4-chlorophenyl)-N-methylethanamine (327 mg, 1.93
mmol) and Hunig’s base (301 mg, 2.32 mmol).
6.1.18. 2-Chloro-4-(4-chlorophenyl)-6,7-dimethoxyquinazoline
(7j)
MS (FAB+) m/z 392 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
3.01–3.05 (2H, m), 3.38 (3H, s), 3.86 (3H, s), 3.89–3.90 (2H, m),
3.91 (3H, s), 7.11 (1H, s), 7.31–7.36 (5H, m).
4-Chlorophenyl boronic acid (810 mg, 5.2 mmol) was added to
the solution of 2,4-dichloro-6,7-dimethoxyquinazoline (1.04 g,
4 mmol) in toluene (8 ml), 1-M Na2CO3 aqueous solution (4 ml)
and Pd(PPh3)4 (230 mg, 0.2 mmol), and the resulting mixture was
stirred under reflux for 6 h. Ethyl acetate and water was added to
the reaction mixture. The resulting precipitate was filtered and
washed with ethyl acetate to yield crude 2-chloro-4-(4-chloro-
phenyl)-6,7-dimethoxyquinazoline 7j (950 mg), which was used
in subsequent reaction without further purification.
6.1.12. 2-Chloro-N-[3-(4-chlorophenyl)propyl]-6,7-dimetho-
xyquinazolin-4-amine (7d)
Compound 7d (414 mg, 68%) was obtained as a yellowish solid
from 2,4-dichloro-6,7-dimethoxyquinazoline
6 (400 mg, 1.54
mmol), 3-(4-chlorophenyl)propan-1-amine (262 mg, 1.54 mmol)
and Hunig’s base (240 mg, 1.85 mmol).
MS (FAB+) m/z 392 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.92–2.00 (2H, m), 2.63 (2H, t, J = 7.6 Hz) 3.48–3.53 (2H, m),
3.886 (3H, s), 3.889 (3H,s), 7.07 (1H, s), 7.28–7.34 (4H, m), 7.60
(1H, s), 8.32 (1H, t, J = 5.2 Hz).
6.1.19. 4-Chloro-N-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-
benzamide (7k)
NaH (101 mg, 2.31 mmol) was added to the solution of 4-chloro-
benzamide (300 mg, 1.93 mmol) in DMF (20 ml), and the mixture
was stirred at room temperature for 30 min. The solution of 2,4-di-
chloro-6,7-dimethoxyquinazoline (500 mg, 1.93 mmol) in DMF
(20 ml) was added to the reaction mixture, and the resulting mixture
was stirred at 80 °C for 8 h. The reaction mixture was concentrated
in vacuo. Water was added to the residue, and the resulting mixture
was extracted with chloroform. The organic layer was washed with
brine, dried over sodium sulfate, filtered, and evaporated in vacuo.
The residue was washed with chloroform–acetonitrile solution
to yield 4-chloro-N-(2-chloro-6,7-dimethoxyquinazolin-4-yl)benz-
amide 7k (238 mg, 0.63 mmol, 33%) as a white solid.
6.1.13. 2-Chloro-N-cycloheptyl-6,7-dimethoxyquinazolin-4-
amine (7e)
Compound 7e (2.15 g, 79%) was obtained as a colorless solid
from 2,4-dichloro-6,7-dimethoxyquinazoline 6 (2.11 g, 8.14 mmol)
and cycloheptylamine (2.02 g, 17.9 mmol).
MS (ESI+) m/z 336 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.44–1.76 (10H, m), 1.92–2.01 (2H, m), 3.88 (3H, s), 3.90 (3H, s),
4.22–4.33 (1H, m), 7.05 (1H, s), 7.66 (1H, s), 7.98 (1H, d, J = 9.5 Hz).
6.1.14. 2-Chloro-6,7-dimethoxy-N-(tetrahydro-2H-pyran-4-
yl)quinazolin-4-amine (7f)
MS (ESI+) m/z 378 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d: 3.83
(3H, s), 3.88 (3H, s), 6.98 (1H, s), 7.56 (1H, br s), 7.50 (2H, d,
J = 8.8 Hz), 7.97(2H, d, J = 8.8 Hz).
Compound 7f (114 mg, 0.35 mmol, 23%) was obtained as a yel-
low solid from 2,4-dichloro-6,7-dimethoxyquinazoline 6 (389 mg,
1.5 mmol), tetrahydro-2H-pyran-4-amine20 (152 mg, 1.5 mmol)
and Hunig’s base (0.52 ml).
6.1.20. 4-Chloro-N-(2-chloro-6,7-dimethoxyquinazolin-4-yl)-
benzenesulfonamide (7l)
MS (ESI+) m/z 324 [M+H]+. 1H NMR (400 MHz, CDCl3) d: 1.60–
1.70 (2H, m), 2.13–2.16 (2H, m), 3.58–3.65 (2H, m), 3.98 (3H, s),
4.02 (3H, s), 4.04–4.07 (2H, m), 4.49–4.52 (1H, m), 5.33 (1H, s,
br), 6.80 (1H, s), 7.15 (1H, s).
Potassium tert-butoxide (238 mg, 2.12 mmol) was added to the
solution of 4-chlorobenzesulfonamide (370 mg, 1.93 mmol) in
DMF (20 ml), and the mixture was stirred at room temperature
for 30 min. The solution of 2,4-dichloro-6,7-dimethoxyquinazoline
(500 mg, 1.93 mmol) in DMF (10 ml) was added to the reaction
mixture, and the resulting mixture was stirred at 80 °C for 20 h.
The reaction mixture was concentrated in vacuo. Water was added
to the residue, and the resulting mixture was extracted with chlo-
roform. The organic layer was washed with brine, dried over so-
dium sulfate, filtered, and evaporated in vacuo. The residue was
washed with chloroform–acetonitrile solution to yield 4-chloro-
N-(2-chloro-6,7-dimethoxyquinazolin-4-yl)benzenesulfonamide
7l (154 mg, 0.37 mmol, 19%) as a white solid.
6.1.15. 2-Chloro-N-(1,1-dioxidotetrahydro-2H-thiopyran-4-yl)-
6,7-dimethoxyquinazolin-4-amine (7g)
Compound 7g (147 mg, 0.40 mmol, 26%) was obtained as a yel-
low solid from 2,4-dichloro-6,7-dimethoxyquinazoline 6 (389 mg,
1.5 mmol), tetrahydro-2H-thiopyran-4-amine 1,1-dioxide21 (279
mg, 1.5 mmol) and Hunig’s base (0.52 ml).
MS (ESI+) m/z 372 [M+H]+. 1H NMR (400 MHz, CDCl3) d: 2.30–
2.39 (2H, m), 2.52–2.55 (2H, m), 3.15–3.19 (2H, m), 3.26–3.32
(2H, m), 3.99 (3H, s), 4.02 (3H, s), 4.63–4.66 (1H, m), 5.87 (1H, s,
br), 6.91 (1H, s), 7.16 (1H, s).
MS (ESI+) m/z 414 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d: 3.86
(3H, s), 3.87 (3H, s), 6.98 (1H, s), 7.50 (1H, br s), 7.50 (2H, d,
J = 8.8 Hz), 7.97 (2H, d, J = 8.8 Hz).
6.1.16. 2-Chloro-4-(5-chloro-2,3-dihydro-1H-indol-1-yl)-6,7-
dimethoxyquinazoline (7h)
Compound 7h (1.26 g, 3.4 mmol, 49%)was obtained as a white so-
lid from 2,4-dichloro-6,7-dimethoxyquinazoline 6 (1.8 g, 6.8 mmol),
5-chloroindoline (1.04 g, 6.8 mmol) and Hunig’s base (1.06 g).
MS (ESI+) m/z 376 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d: 3.21
(2H, t, J = 8.0 Hz), 3.85 (3H, s), 3.96 (3H, s), 4.52 (2H, t, J = 8.0 Hz),
7.23 (1H, d, J = 8.4 Hz), 7.26 (1H, s), 7.31 (1H, s), 7.40 (1H, s), 7.41
(1H, t, J = 8.4 Hz)
6.1.21. (2-Chloro-6,7-dimethoxyquinazolin-4-yl)(4-chloro-
phenyl)methanone (7m)
4-Chlorobenzaldehyde (1.2 g, 8.8 mmol) was added to the solu-
tion of 2,4-dichloro-6,7-dimethoxyquinazoline (2.08 g, 8.0 mmol)
in dioxane (50 ml), 1,3-dimethylimidazolium iodide (448 mg,
2.0 mmol) and NaH (55% in oil, 384 mg, 8.8 mmol), and the mix-
ture was stirred under reflux for 3 h. The reaction mixture was

70
K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
poured into ice water, and the resulting mixture was extracted
with chloroform. The organic layer was washed with brine, dried
over sodium sulfate, filtered, and evaporated in vacuo. The residue
was recrystallized from EtOH to yield (2-chloro-6,7-dimethoxyqui-
nazolin-4-yl)(4-chlorophenyl)methanone 7m (1.83 g, 5.0 mmol,
63%) as a yellow solid.
BuOH (20 ml), and the resulting mixture was stirred at 110 °C for
1 day. The reaction mixture was cooled to room temperature and
concentrated in vacuo, and the residue was purified via column
chromatography (chloroform/MeOH/NH4OH) to yield 8c (58 mg,
0.10 mmol, 18%). This compound 8c was treated with 4-M HCl
ethyl acetate solution, and the precipitate was washed with isopro-
panol and ethyl acetate to yield the hydrochloride salt of 8c
(46 mg, 70%) as a white solid.
MS (ESI+) m/z 363 [M+H]+, 1H NMR (400 MHz, DMSO-d6) d: 3.86
(3H, s), 4.04 (3H, s), 7.32 (1H, s), 7.52 (1H, s), 7.67 (2H, d, J = 8.4 Hz),
7.98 (2H, d, J = 8.4 Hz).
MS (FAB+) m/z 554 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.07–1.24 (1H, m), 1.60–2.05 (5H, m), 2.08–2.35 (3H, m), 2.58–
2.70 (1H, m), 2.79–2.92 (1H, m), 3.00–3.07 (2H, m), 3.09–3.20
(2H, m), 3.23–3.45 (4H, m), 3.56 (3H, s), 3.85 (3H, s), 3.91 (3H, s),
3.95–4.05 (2H, m), 4.74–4.90 (2H, br d), 7.30–7.45 (5H, s), 7.78
(1H, s), 10.90 (1H, s), 12.68 (1H, s).
Anal. calcd. for C30H40ClN5O3 2HCl 2.2H2O 0.4NH4Cl: C, 52.37, H,
7.03, N, 10.99, Cl, 17.52. Found: C, 52.49, H, 6.73, N, 11.20, Cl,
17.73.
6.1.22. (10-{4-[(4-Chlorobenzyl)amino]-6,7-dimethoxyquina-
zolin-2-yl}-1,40-bipiperidin-3-yl)methanol (8a)
1,40-Bipiperidin-3-ylmethanol dihydrochloride 5c (476 mg,
1.75 mmol) and Hunig’s base (910 mg, 7.02 mmol) was added to a
solution of the 2-chloro-N-(4-chlorobenzyl)-6,7-dimethoxyquinaz-
olin-4-amine 7a (639 mg) in n-BuOH (20 ml), and the resulting mix-
ture was stirred at 110 °C for 1 day. The reaction mixture was cooled
to room temperature and concentrated in vacuo, and the residue
was purified via column chromatography (chloroform/MeOH/
NH4OH) to yield (10-{4-[(4-chlorobenzyl)amino]-6,7-dimethoxy-
quinazolin-2-yl}-1,40-bipiperidin-3-yl)methanol 8a (561 mg, 1.1
mmol, 61%) as a wine-red amorphous. The residue was treated with
4-M HCl ethyl acetate solution (0.6 ml), and the precipitate
was washed with ethanol to yield the hydrochloride salt of 8a
(259 mg, 28%).
6.1.25. [10-(4-{[3-(4-Chlorophenyl)propyl]amino}-6,7-dimetho-
xyquinazolin-2-yl)-1,40-bipiperidin-3-yl]methanol (8d)
Compound 8d (652 mg, quant.) was obtained as a colorless solid
from 2-chloro-N-[3-(4-chlorophenyl)propyl]-6,7-dimethoxyqui-
nazolin-4-amine 7d (332 mg, 0.85 mmol), 1,40-bipiperidin-3-
ylmethanol dihydrochloride 5c (230 mg, 0.85 mmol) and DBU
(387 mg). This compound 8d was treated with 4-M HCl ethyl ace-
tate solution, and the precipitate was washed with isopropanol,
ethanol and methanol to yield the hydrochloride salt of 8d
(211 mg, 33%) as a white solid.
Mp (dec.) 216–226 °C (EtOH); MS (FAB+) m/z 526 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.11–1.20 (1H, m), 1.69–1.71 (3H,
m), 1.81–1.84 (1H, m), 1.89–1.96 (1H, m), 2.04–2.22 (3H, m),
2.49–2.51 (1H, m), 2.55–2.63 (1H, m), 3.06–3.11 (2H, m), 3.23–
3.39 (4H, m), 3.48–3.51 (1H, m), 3.88 (3H, s), 3.89 (3H, s), 4.75–
4.76 (2H, m), 4.81 (2H, br s), 7.42 (2H, d, J = 8.6 Hz), 7.50 (2H, d,
J = 8.6 Hz), 7.65 (1H, s), 7.98 (1H, s), 10.24 (1H, s), 10.82 (1H, s),
12.54 (1H, s).
Anal. calcd. for C28H36ClN5O3 2HCl 0.8H2O: C, 54.83, H, 6.51, N,
11.42, Cl, 17.34. Found: C, 54.67, H, 6.48, N, 11.47, Cl, 17.57.
Compounds 8b–m were prepared using procedures similar to
those described for the synthesis of 8a.
MS (FAB+) m/z 554 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.10–1.23 (1H, m), 1.60–1.83 (4H, m), 1.83–2.03 (3H, m), 2.09–
2.30 (3H, m), 2.58–2.66 (1H, m), 2.66–2.72 (2H, m), 2.77–2.88
(1H, m), 3.00–3.15 (2H, m), 3.23–3.44 (4H, m), 3.48–3.60 (3H,
m), 3.87 (6H, s), 4.68–4.85 (2H, br d), 7.28 (2H,d, J = 8.4 Hz), 7.35
(2H, d, J = 8.4 Hz), 7.64 (1H, s), 7.87 (1H, s), 9.57 (1H, s), 10.87
(1H, s), 12.44 (1H, s).
Anal. calcd. for C30H40ClN5O3 2HCl 1.5H2O 0.3C3H8O: C, 55.22, H,
7.11, N, 10.42, Cl, 15.82. Found: C, 55.34, H, 7.20, N, 10.50, Cl, 15.82.
6.1.23. 10-(4-{[2-(4-Chlorophenyl)ethyl]amino}-6,7-dimetho-
xyquinazolin-2-yl)-1,40-bipiperidin-3-yl]methanol (8b)
Compound 8b (500 mg, 1.01 mmol, 74%) was obtained as a
colorless amorphous from 2-chloro-N-[2-(4-chlorophenyl)ethyl]-
6,7-dimethoxyquinazolin-4-amine 7b (522 mg, 1.38 mmol),
1,40-bipiperidin-3-ylmethanol dihydrochloride 5c (374 mg, 1.38 mmol)
and Hunig’s base (716 mg). This compound 8b was treated with
4-M HCl ethyl acetate solution (0.6 ml), and the precipitate was
washed with ethanol to yield the hydrochloride salt of 8b
(343 mg, 60%) as a colorless solid.
6.1.26. {10-[4-(Cycloheptylamino)-6,7-dimethoxyquinazolin-2-
yl]-1,40-bipiperidin-3-yl}methanol (8e)
Compound 8e (370 mg, 0.74 mmol, 27%) was obtained as a yel-
low solid from 2-chloro-N-cycloheptyl-6,7-dimethoxyquinazolin-
4-amine 7e (940 mg, 2.8 mmol) and 1,40-bipiperidin-3-ylmethanol
dihydrochloride 5c (1.11 g, 5.6 mmol). This compound 8e was trea-
ted with 4-M HCl ethyl acetate solution, and the precipitate was
washed with ethyl acetate to yield the hydrochloride salt of 8e
(300 mg) as a colorless solid.
Mp (dec.) 197–199 °C (AcOEt); MS (FAB+) m/z 498 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.08–1.22 (1H, s), 1.44–1.86 (14H,
m), 1.90–2.04 (3H, m), 2.07–2.20 (1H, m), 2.22–2.34 (2H, m),
2.57–2.69 (1H, m), 2.78–2.92 (1H, m), 3.06–3.20 (2H, m),
3.22–3.44 (4H, m), 3.48–3.60 (1H, m), 3.87 (3H, s), 3.89 (3H, s),
4.24–4.34 (1H, m), 4.29 (1H, br), 4.80–4.90 (2H, br), 7.65 (1H, s),
7.89 (1H, s), 9.01 (1H, d, J = 6.0 Hz), 10.86 (1H, s), 12.45 (1H, s).
Anal. calcd. for C28H43N5O3 2HCl 1.5H2O: C, 56.27, H, 8.10, N,
11.72, Cl, 11.86. Found: C, 56.31, H, 8.23, N, 11.83, Cl, 12.07.
Mp (dec.) 220–221 °C (EtOH); MS (FAB-) m/z 538 [MꢁH]ꢁ. 1H
NMR (400 MHz, DMSO-d6) d: 1.12–1.20 (1H, m), 1.69–2.04 (5H,
m), 2.14 (1H, m), 2.28–2.30 (2H, m), 2.61–2.70 (1H, m), 2.84–
2.87 (1H, m), 2.98–3.01 (2H, m), 3.11–3.18 (2H, m), 3.24–3.45
(4H, m), 3.49–3.60 (1H, m), 3.72–3.82 (2H, m), 3.87 (3H, s), 3.88
(3H, s), 4.80–4.90 (2H, br d), 7.30 (2H,d, J = 8.3 Hz), 7.37 (2H, d,
J = 8.3 Hz), 7.66 (1H, s), 7.85 (1H, s), 9.65 (1H, s), 10.85 (1H, s),
12.50 (1H, s).
Anal. calcd. for C29H38ClN5O3 2HCl 0.8H2O: C, 55.51, H, 6.68, N,
11.16, Cl, 16.95. Found: C, 55.57, H, 6.63, N, 11.19, Cl, 16.92.
6.1.27. {10-[6,7-Dimethoxy-4-(tetrahydro-2H-pyran-4-ylamino)-
quinazolin-2-yl]-1,40-bipiperidin-3-yl}methanol (8f)
6.1.24. [10-(4-{[2-(4-Chlorophenyl)ethyl](methyl)amino}-6,7-
dimethoxyquinazolin-2-yl)-1,40-bipiperidin-3-yl]methanol
dihydrochloride (8c)
Compound 8f (90 mg, 0.19 mmol, 53%) was obtained as a
yellow solid from 2-chloro-6,7-dimethoxy-N-(tetrahydro-2H-pyr-
an-4-yl)quinazolin-4-amine 7f (112 mg, 0.35 mmol), 1,40-bipiperi-
din-3-ylmethanol 5c (99 mg, 0.38 mmol) and DBU (0.19 ml). This
compound 8f was treated with 4-M HCl dioxane solution, and
the precipitate was washed with acetonitrile to yield the hydro-
chloride salt of 8f (50 mg, 45%) as a brown amorphous solid.
1,40-Bipiperidin-3-ylmethanol dihydrochloride 5c (158 mg,
0.58 mmol) and potassium carbonate (242 mg) was added to a
solution of the 2-chloro-N-[2-(4-chlorophenyl)ethyl]-6,7-dime-
thoxy-N-methylquinazolin-4-amine 7c (229 mg, 0.58 mmol) in n-

K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
71
MS (FAB+) m/z 486 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.08–1.24 (1H, s), 1.63–1.98 (9H, m), 2.08–2.21 (1H, br, s), 2.23–
2.34 (2H, m), 2.57–2.70 (1H, m), 2.78–2.92 (1H, m), 3.09–3.20
(2H, m), 3.22–3.60 (7H, m), 3.88 (3H, s), 3.89 (3H, s), 3.93–4.01
(2H, m), 4.32–4.46 (1H, m), 4.78–4.92 (2H, br), 7.66 (1H, s), 7.93
(1H, s), 9.12 (1H, d, J = 7.2 Hz), 10.86 (1H, s), 12.45 (1H, s).
Anal. calcd. for C28H43N5O3 2HCl 2.6H2O: C, 51.58, H, 7.69, N,
11.57, Cl, 11.71. Found: C, 51.58, H, 7.82, N, 11.74, Cl, 11.71.
oxyquinazolin-4-amine hydrochloride 7i (387 mg), 3-methoxy-
1,40-bipiperidine dihydrochloride 5b (271 mg, 1.0 mmol) and
DBU (609 mg). This compound 8ib (450 mg) was treated with 4-
M HCl ethyl acetate solution, and the precipitate was recrystallized
from ethanol to yield the hydrochloride salt of 8ib (243 mg, 47%) as
a colorless solid.
Mp (dec.) 213–223 °C (EtOH); MS (FAB+) m/z 512 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.23–2.30 (8H, m), 2.58–2.70 (1H,
m), 2.90–3.18 (3H, m), 3.30 (3H, s), 3.40–3.55 (3H, m), 3.66–3.76
(1H, m), 3.91 (3H, s), 3.94 (3H, s), 4.69–4.85 (2H, br), 7.50–7.56
(2H, m), 7.71–7.78 (3H, m), 8.19 (1H, s), 11.02–11.20 (2H, br).
Anal. calcd. for C27H34ClN5O3 2HCl 2H2O: C, 52.22, H, 6.49, N,
11.28, Cl, 17.13. Found: C, 52.14, H, 6.56, N, 11.30, Cl, 17.02.
6.1.28. (10-{4-[(1,1-Dioxidotetrahydro-2H-thiopyran-4-yl)-
amino]-6,7-dimethoxyquinazolin-2-yl}-1,40-bipiperidin-3-
yl)-methanol (8g)
Compound 8g (125 mg, 0.23 mmol, 60%) was obtained as a yel-
low solid from 2-chloro-N-(1,1-dioxidotetrahydro-2H-thiopyran-
4-yl)-6,7-dimethoxyquinazolin-4-amine 7g (145 mg, 0.39 mmol),
1,40-bipiperidin-3-ylmethanol 5c (112 mg, 0.43 mmol) and DBU
(0.20 ml). This compound 8g was treated with 4-M HCl dioxane
solution, and the precipitate was washed with acetonitrile to yield
the hydrochloride salt of 8g (115 mg, 75%) as a beige solid.
MS (FAB+) m/z 534 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.08–1.24 (1H, s), 1.65–2.00 (5H, m), 2.08–2.40 (7H, m), 2.58–
2.70 (1H, m), 2.75–2.92 (1H, m), 3.07–3.62 (11H, m), 3.88 (6H, s),
4.58–4.68 (1H, m), 4.84–5.00 (2H, br), 7.65 (1H, s), 7.85 (1H, s),
9.13 (1H, d, J = 7.6 Hz), 10.84 (1H, s), 12.51 (1H, s).
6.1.32. (10-{4-[(4-Chlorophenyl)amino]-6,7-dimethoxyquina-
zolin-2-yl}-1,40-bipiperidin-3-yl)methanol (8ic)
Compound 8ic (390 mg, 66%) was obtained as a colorless
amorphous from 2-chloro-N-(4-chlorophenyl)-6,7-dimethoxyqui-
nazolin-4-amine hydrochloride 7i (550 mg), 1,40-bipiperidin-3-
ylmethanol 5c (230 mg, 1.16 mmol) and Hunig’s base (220 mg,
1.74 mmol). This compound 8ic was treated with 4-M HCl ethyl
acetate solution, and the precipitate was recrystallized from Et2O
to yield the hydrochloride salt of 8ic (330 mg, 0.56 mmol, 74%) as a
colorless solid.
Anal. calcd. for C26H39N5O5S 2.1HCl 2.6H2O: C, 47.52, H, 7.10, N,
10.67, Cl, 11.33, S, 4.88. Found: C, 47.77, H, 7.41, N, 10.88, Cl, 11.43,
S, 4.53.
Mp (dec.) 221–223 °C (Et2O); MS (FAB+) m/z 512 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.10–1.22 (1H, s), 1.60–2.00 (5H,
m), 2.05–2.30 (3H, m), 2.58–2.69 (1H, m), 2.78–2.91 (1H, m),
3.04–3.18 (2H, m), 3.22–3.58 (5H, m), 3.90 (3H, s), 3.94 (3H, s),
4.68–4.84 (2H, br), 7.53 (2H, d, J = 8.8 Hz), 7.70–7.78 (3H, m),
8.19 (1H, s), 10.78 (1H, s), 11.06 (1H, s), 12.86 (1H, s).
6.1.29. {10-[4-(5-Chloro-2,3-dihydro-1H-indol-1-yl)-6,7-dimeth-
oxyquinazolin-2-yl]-1,40-bipiperidin-3-yl}methanol (8h)
8h (476 mg, 0.88 mmol, 71%) was obtained from 2-chloro-4-(5-
chloro-2,3-dihydro-1H-indol-1-yl)-6,7-dimethoxyquinazoline 7h
(468 mg, 1.2 mmol), 1,40-bipiperidin-3-ylmethanol dihydrochlo-
ride 5c (337 mg, 1.2 mmol) and DBU (568 mg). This compound
8h (438 mg) was treated with 4-M HCl dioxane solution, and
the precipitate was washed with methanol to yield the hydro-
chloride salt of 8h (176 mg, 35%) as a white solid.
Anal. calcd. for C27H34N5O3Cl 2HCl 2H2O: C, 52.22, H, 6.49, N,
11.28, Cl, 17.13. Found: C, 52.38, H, 6.50, N, 11.23, Cl, 16.75.
6.1.33. N-(4-Chlorophenyl)-6,7-dimethoxy-2-[3-(methoxy-
methyl)-1,40-bipiperidin-10-yl]quinazolin-4-amine (8id)
Compound 8id (525 mg, 77%) was obtained as a yellow oil from
crude 2-chloro-N-(4-chlorophenyl)-6,7-dimethoxyquinazolin-4-amine
hydrochloride 7i (452 mg), 3-(methoxymethyl)-1,40-bipiperidine
MS (FAB+) m/z 538 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.08–1.22 (1H, m), 1.62–2.00 (5H, m), 2.04–2.18 (1H, m), 2.19–
2.32 (2H, m), 2.58–2.70 (1H, m), 2.79–2.90 (1H, m), 3.07–3.60
(9H, m), 3.86 (3H, s), 3.94 (3H, s), 4.55–4.90 (4H, m), 7.25–7.50
(3H, m), 7.72 (2H, br, s), 10.70 (1H, br, s), 13.05 (1H, br, s).
Anal. calcd. for C29H36ClN5O3 2HCl 3H2O: C, 52.37, H, 6.67, N,
10.53, Cl, 15.99. Found: C, 52.50, H, 6.69, N, 10.45, Cl, 15.75.
5d (328 mg, 1.54 mmol) and Hunig’s base (268 ll, 1.54 mmol). This
compound 8id was treated with 4-M HCl dioxane solution, and the
precipitate was recrystallized from ethanol to yield the hydrochlo-
ride salt of 8id (460 mg, 78%) as a white solid.
Mp (dec.) 229–230 °C (EtOH); MS (FAB+) m/z 526 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.12–1.25 (1H, s), 1.64–2.01 (5H,
m), 2.17–2.39 (3H, m), 2.63–2.74 (1H, m), 2.79–2.91 (1H, m),
3.06–3.21 (2H, m), 3.23 (3H, s), 3.32–3.58 (5H, m), 3.90 (3H, s),
3.94 (3H, s), 4.70–4.83 (2H, m), 7.52 (2H, d, J = 8.4 Hz), 7.72–7.80
(3H, m), 8.20 (1H, s), 10.9 (1H, s), 11.1 (1H, s), 12.9 (1H, s).
Anal. calcd. for C28H36N5O3 2HCl H2O: C, 54.51, H, 6.53, N, 11.35,
Cl, 17.24. Found: C, 54.31, H, 6.16, N, 11.29, Cl, 17.17.
6.1.30. 10-{4-[(4-Chlorophenyl)amino]-6,7-dimethoxyquina-
zolin-2-yl}-1,40-bipiperidin-3-ol (8ia)
Compound8ia(329 mg, 0.66 mmol,66%)wasobtainedasabrown
solid from crude 2-chloro-N-(4-chlorophenyl)-6,7-dimethoxyqui-
nazolin-4-amine hydrochloride 7i (350 mg) and 1,40-bipiperidin-
3-ol dihydrochloride 5a (257 mg, 1.0 mmol). This compound 8ia
(325 mg) was treated with 4-M HCl ethyl acetate solution, and the
precipitate was recrystallized from ethanol to yield the hydrochlo-
ride salt of 8ia (254 mg, 68%) as a colorless solid.
6.1.34. [4-(1-{4-[(4-Chlorophenyl)amino]-6,7-dimethoxy-
quinazolin-2-yl}piperidin-4-yl)morpholin-2-yl]methanol (8ie)
Crude (4-piperidin-4-ylmorpholin-2-yl)methanol 5e (1.64 g)
was obtained as a colorless solid from tert-butyl 4-oxopiperidine-
1-carboxylate (940 mg, 4.7 mmol) and morpholin-2-ylmethanol
(550 mg, 4.7 mmol) using procedures similar to those described
for the syntheses of 4a and 5a. The hydrochloride salt of 8ie
(59 mg, 9%) was obtained as a yellow solid from crude 2-chloro-
N-(4-chlorophenyl)-6,7-dimethoxyquinazolin-4-amine hydrochlo-
ride 7i (387 mg), crude (4-piperidin-4-ylmorpholin-2-yl)methanol
dihydrochloride salt 5e (410 mg) and DBU (0.45 ml, 3 mmol) using
procedures similar to those described for the synthesis of 8id.
MS (FAB+) m/z 514 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.76–1.84 (2H, m), 2.23–2.32 (2H, m), 2.78–2.89 (1H, m),
2.96–3.18 (3H, m), 3.24–3.60 (6H, m), 3.91 (3H, s), 3.94 (3H, s),
Mp (dec.) 250–260 °C (EtOH); MS (FAB+) m/z 498 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.24–2.30 (8H, m), 2.52–2.87 (1H,
m), 3.00–3.20 (3H, m), 3.40–3.55 (3H, m), 3.66–3.76 (1H, m),
3.89 (3H, s), 3.94 (3H, s), 4.68–4.88 (2H, br), 7.52 (2H, d,
J = 8.8 Hz), 7.73–7.80 (3H, m), 8.18–8.28 (1H, br), 11.11 (2H, br).
Anal. calcd. for C26H32ClN5O3 2.1HCl H2O: C, 52.70, H, 6.14, N,
11.82, Cl, 18.55. Found: C, 52.88, H, 6.34, N, 11.97, Cl, 18.31.
6.1.31. N-(4-chlorophenyl)-6,7-dimethoxy-2-(3-methoxy-1,40-
bipiperidin-10-yl)quinazolin-4-amine (8ib)
Compound 8ib (452 mg, 0.88 mmol, 88%) was obtained as a
brown solid from crude 2-chloro-N-(4-chlorophenyl)-6,7-dimeth-

72
K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
3.95–4.05 (2H, m), 4.67–4.78 (2H, m), 7.54 (2H, d, J = 8.8 Hz), 7.70
(1H, s), 7.73 (2H, d, J = 6.8 Hz), 8.17 (1H, s), 11.03 (1H, s), 11.65 (1H,
s), 12.79 (1H, s).
Anal. calcd. for C26H32ClN5O4 2HCl 3H2O: C, 48.72, H, 6.29, N,
10.93, Cl, 16.59. Found: C, 48.92, H, 6.24, N, 11.06, Cl, 16.58.
6.1.38. (4-Chlorophenyl){2-[3-(hydroxymethyl)-1,40-bipiper-
idin-10-yl]-6,7-dimethoxyquinazolin-4-yl}methanone (8m)
Compound 8m (321 mg, 0.61 mmol, 67%) was obtained as a
orange oil from (2-chloro-6,7-dimethoxyquinazolin-4-yl)(4-chlo-
rophenyl) methanone 7m (331 mg, 0.91 mmol), 1,40-bipiperidin-
3-ylmethanol 5c (297 mg, 1.1 mmol) and Hunig’s base (0.57 ml,
3.27 mmol). This compound 8m was treated with 4-M HCl ethyl
acetate solution, and the precipitate was recrystallized from iso-
propanol to yield the hydrochloride salt of 8m (162 mg, 44%) as a
yellow solid.
6.1.35. {10-[4-(4-Chlorophenyl)-6,7-dimethoxyquinazolin-2-yl]-
1,40-bipiperidin-3-yl}methanol (8j)
Compound 8j (390 mg, 0.78 mmol, 68%) was obtained as a yel-
low solid from 2-chloro-4-(4-chlorophenyl)-6,7-dimethoxyquinaz-
oline 7j (390 mg, 1.16 mmol), 1,40-bipiperidin-3-ylmethanol 5c
(380 mg, 1.4 mmol) and DBU (880 mg, 5.80 mmol). This compound
8j was treated with 4-M HCl ethyl acetate solution, and the precip-
itate was washed with Et2O to yield the hydrochloride salt of 8j
(390 mg, 88%) as a yellowish solid.
Mp (dec.) 180–181 °C (iPrOH); MS (FAB+) m/z 525 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.06–1.20 (1H, s), 1.60–1.95 (5H,
m), 2.05–2.25 (3H, m), 2.58–2.68 (1H, m), 2.76–2.88 (1H, m),
2.89–3.04 (2H, m), 3.22–3.29 (1H, m), 3.32–3.55 (4H, m), 3.96
(6H, s), 4.72–4.90 (2H, br), 7.06 (1H, s), 7.19 (1H, s), 7.67 (2H, d,
J = 8.4 Hz), 7.98 (2H, d, J = 8.8 Hz), 10.47 (1H, br).
Mp (dec.) 171–172 °C (Et2O); MS (FAB+) m/z 497 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.06–1.22 (1H, s), 1.61–2.00 (5H,
m), 2.05–2.30 (3H, m), 2.58–2.70 (1H, m), 2.78–2.91 (1H, m),
3.05–3.15 (2H, m), 3.21–3.75 (5H, m), 3.96 (6H, s), 4.96–5.12
(2H, br), 7.13 (1H, s), 7.56 (1H, s), 7.69 (2H, d, J = 8.3 Hz), 7.87
(2H, d, J = 8.3 Hz), 10.64 (1H, s).
Anal. calcd. for C28H33ClN4O4 2HCl 0.8H2O: C, 54.92, H, 6.02, N,
9.15, Cl, 17.37. Found: C, 55.03, H, 6.03, N, 9.08, Cl, 17.23.
6.1.39. (4-Chlorophenyl){2-[3-(hydroxymethyl)-1,40-bipiperidin-
10-yl]-6,7-dimethoxyquinazolin-4-yl}methanol (8n)
NaBH4 (70 mg, 1.85 mmol) was added to the solution of (4-chlo-
rophenyl){2-[3-(hydroxymethyl)-1,40-bipiperidin-10-yl]-6,7-
dimethoxyquinazolin-4-yl}methadone 8m (807 mg, 1.54 mmol) in
MeOH (15 ml), and the resulting mixture was stirred at room tem-
perature for 2 h. The reaction mixture was quenched with water,
and the resulting mixture was extracted with chloroform. The or-
ganic layer was washed with brine, dried over sodium sulfate, fil-
tered, and evaporated in vacuo. The residue was washed with
Et2O to yield (4-chlorophenyl){2-[3-(hydroxymethyl)-1,40-bipiperi-
din-10-yl]-6,7-dimethoxyquinazolin-4-yl}methanol 8n (663 mg,
1.26 mmol, 82%) as a green solid. This compound 8n (221 mg)
was treated with 4-M HCl ethyl acetate solution, and the precipitate
was recrystallized from isopropanol to yield the hydrochloride salt
of 8n (127 mg, 0.21 mmol, 53%) as a yellow solid.
Anal. calcd. for C27H33N4O3Cl 2HCl 2.5H2O: C, 52.73, H, 6.56, N,
9.11, Cl, 17.29. Found: C, 52.73, H, 6.69, N, 9.15, Cl, 17.45.
6.1.36. 4-chloro-N-{2-[3-(hydroxymethyl)-1,40-bipiperidin-10-
yl]-6,7-dimethoxyquinazolin-4-yl}benzamide (8k)
1,40-Bipiperidin-3-ylmethanol dihydrochloride 5c (136 mg,
0.50 mmol) and Hunig’s base (196 mg) was added to a solution
of the 4-chloro-N-(2-chloro-6,7-dimethoxyquinazolin-4-yl) benz-
amide 7k (190 mg, 0.50 mmol) in n-BuOH (20 ml), and the result-
ing mixture was stirred at 110 °C for 1 day. The reaction mixture
was cooled to room temperature and concentrated in vacuo, and
the residue was purified via column chromatography (chloro-
form/MeOH/NH4OH) to yield 8k (83 mg, 0.15 mmol, 31%) as a
red amorphous. This compound 8k was treated with 4-M HCl ethyl
acetate solution, and the precipitate was washed with isopropanol
to yield the hydrochloride salt of 8k (45 mg, 48%) as a white solid.
MS (FAB+)m/z540[M+H]+. 1H NMR(400 MHz, DMSO-d6) d: 1.12–
1.20 (1H, m), 1.68–1.92 (6H, m), 2.07 (2H, m), 2.73–2.90 (2H, m),
3.23–3.28 (2H, m), 3.35–3.75 (5H, m), 3.88 (3H, s), 3.93 (3H, s),
4.81 (2H, br d), 7.59 (1H, s), 7.61 (2H, d, J = 8.3 Hz), 7.75 (1H, s),
7.86 (2H, d, J = 8.3 Hz), 10.44 (1H, s), 11.41 (1H, s), 12.35 (1H, s).
Anal. calcd. for C28H34ClN5O4 1.9HCl 3.6H2O 0.1NH4Cl: C, 49.49,
H, 6.45, N, 10.51, Cl, 15.65. Found: C, 49.09, H, 6.19, N, 10.82, Cl,
15.36.
Mp (dec.) 204–205 °C (iPrOH); MS (FAB+) m/z 527 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d: 1.10–1.22 (1H, s), 1.63–2.00 (5H,
m), 2.07–2.31 (3H, m), 2.58–2.70 (1H, m), 2.80–2.87 (1H, m),
3.02–3.14 (2H, m), 3.22–3.29 (1H, m), 3.34–3.45 (3H, m), 3.47–
3.58 (1H, m), 3.87 (3H, s), 3.90 (3H, s), 5.00–5.12 (2H, br), 6.31
(1H, s), 7.40 (2H, d, J = 8.4 Hz), 7.45–7.58 (3H, m), 7.63 (1H, s),
10.72 (1H, br).
Anal. calcd. for C28H35ClN4O4 2HCl 2.1H2O: C, 52.73, H, 6.51, N,
8.78, Cl, 16.68. Found: C, 52.80, H, 6.51, N, 8.72, Cl, 16.42.
6.2. Pharmacology
6.1.37. 4-Chloro-N-{2-[3-(hydroxymethyl)-1,40-bipiperidin-10-
yl]-6,7-dimethoxyquinazolin-4-yl}benzenesulfonamide (8l)
Compound 8l (102 mg, 0.18 mmol, 54%) was obtained as a
brown amorphous from 4-chloro-N-(2-chloro-6,7-dimethoxyqui-
nazolin-4-yl)benzenesulfonamide 7l (137 mg, 0.33 mmol), 1,40-
bipiperidin-3-ylmethanol dihydrochloride 5c (90 mg, 0.33 mmol)
and Hunig’s base (129 mg). This compound 8l was treated with
4-M HCl ethyl acetate solution, and the precipitate was washed
with methanol to yield the hydrochloride salt of 8l (27 mg, 20%)
as a white solid.
6.2.1. Human and murine CCR4-expressing cells
Cells from the mouse pre-B cell line B300-19 were cultured in
RPMI 1640 medium containing 10% fetal bovine serum (FBS),
50
l
M 2-mercaptethanol, 100 U/mL penicillin, and 100
lg/mL
carrying
streptomycin. The expression vector pEF-BOS-Neo22
,
full-length human CCR4 cDNA (X85740; GenBank) or mouse
CCR4 cDNA, (X90862; GenBank) was transfected into B300–19
cells via electroporation to isolate stable G418-resistant stable
transformants.
MS (FAB+) m/z 576 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d:
1.14–1.16 (1H, m), 1.41 (2H, br s), 1.70–1.73 (1H, m), 1.86 (2H,
br s), 2.09–2.11 (3H, m), 2.51–2.61 (1H, m), 2.72–2.82 (1H, m),
2.87–3.00 (2H, m), 3.20–3.52 (5H, m), 3.87 (6H, s), 4.49 (2H, d,
J = 12 Hz), 7.39 (1H, s), 7.56 (1H, s), 7.60 (2H, d, J = 8.6 Hz), 7.83
(2H, d, J = 8.6 Hz), 10.49 (1H, s).
6.2.2. [35S]GTP
Human CCR4-expressing cell membranes (1
were incubated at 25 °C for 1.5 h with 150 pM [35S]GTP
Healthcare), 5 mg/mL wheat germ agglutinin SPA beads (GE Health-
care), 2 M GDP, and 3 nM MDC with various concentrations of test
compounds in 200 l of binding buffer [20 mM HEPES–NaOH (pH
cS-binding assay
l
g/well protein)
S (GE
c
l
l
Anal. calcd. for C27H34ClN5O5S 1.9HCl 2.2H2O: C, 47.34, H, 5.93,
N, 10.22, Cl, 15.01, S, 4.68. Found: C, 47.44, H, 6.13, N, 9.91, Cl,
14.84, S, 4.53.
7.05), 100 mM NaCl, 5 mM MgCl2, and 0.2% (w/v) BSA]. Radioactivity
was counted using a TopCount scintillationcounter. Control wells, in
the absence of either test compound (for total counts) or CCL22

K. Yokoyama et al. / Bioorg. Med. Chem. 17 (2009) 64–73
73
(non-specific), were used to calculate the percent of total inhibition
for each set of compounds. Assays were performed in duplicate at
four different concentrations for each test compound, and the value
represents an average of usually two determinations.
helpful support during the preparation of this manuscript. The
authors are also grateful to the staff of the Division of Analytical
Science Laboratories for their assistance with elemental analysis
and spectral measurement.
In our studies, CCL22 was used as the main CCR4 ligand because
it was shown to have higher affinity and efficacy than CCL17 for the
[
References and notes
35S]GTP S-binding reaction and cell chemotaxis.23 In addition, our
c
test compounds were found to inhibit responses induced by CCL22
and CCL17 equally (data not shown).
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Chemotaxis assays were performed using 96-well chemotaxis
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lm pores, polycarbonate fil-
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1 nM mouse CCL22 in the lower wells. The number of cells migrat-
ing to the lower chambers was quantified using a bioluminescent
assay (ATP-Lite; PerkinElmer). Control wells containing no test
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the single dose, the tested compounds were administered only
after the challenge (8 h for 1a and 12h for 8ic). The degree of ear
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and vehicle groups was used to calculate the percent inhibition
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Acknowledgement
The authors express their deep gratitude to Dr. Ryo Naito, Dr.
Hideki Kubota, Ms. Diana Fleig and Ms. Takako Furukawa for their
24. Boyden, S. J. Exp. Med. 1962, 115, 453.