Discovery of potent CCR4 antagonists: Synthesis and structure-activity relationship study of 2,4-diaminoquinazolines

Article abstract of DOI:10.1016/j.bmc.2008.05.036

A new series of quinazolines that function as CCR4 antagonists were discovered during the screening of our corporate compound libraries. Subsequent compound optimization elucidated the structure-activity relationships and led the identification of 2-(1,4′-bipiperidine-1′-yl)-N-cycloheptyl-6,7-dimethoxyquinazolin-4-amine 14a, which showed potent inhibition in the [³⁵S]GTPγS-binding assay (IC₅₀ = 18 nM). This compound also inhibited the chemotaxis of human and mouse CCR4-expressing cells (IC₅₀ = 140 nM, 39 nM).

Full text of DOI:10.1016/j.bmc.2008.05.036

Bioorganic & Medicinal Chemistry 16 (2008) 7021–7032
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of potent CCR4 antagonists: Synthesis and structure–activity
relationship study of 2,4-diaminoquinazolines
*
Kazuhiro Yokoyama , Noriko Ishikawa, Susumu Igarashi, Noriyuki Kawano, 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:
A new series of quinazolines that function as CCR4 antagonists were discovered during the screening of
our corporate compound libraries. Subsequent compound optimization elucidated the structure–activity
relationships and led the identification of 2-(1,40-bipiperidine-10-yl)-N-cycloheptyl-6,7-dimethoxyqui-
Received 27 February 2008
Revised 13 May 2008
Accepted 15 May 2008
Available online 20 May 2008
nazolin-4-amine 14a, which showed potent inhibition in the [35S]GTP
cS-binding assay (IC50 = 18 nM).
This compound also inhibited the chemotaxis of human and mouse CCR4-expressing cells (IC50 = 140 nM,
39 nM).
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Chemokine receptor 4 (CCR4)
Antagonists
2,4-Diaminoquinazolines
Inflammatory disease
1. Introduction
inflammatory signals.7 The involvement of CCR4 and its ligands
CCL17 and CCL22 is indicated in a wide range of diseases, including
Chemokines are a group of structurally related chemoattractant
cytokine molecules that regulate leukocyte trafficking. They are
relatively small (ꢀ8–14 kDa) and consist of mostly basic proteins
that are involved in various physiological and pathological pro-
cesses, such as immune response, viral infection, angiogenesis/
angiostasis, and more fundamentally organogenesis and homeo-
stasis.1 They are classified based on the position of the first two
of four conserved cysteines, and there are two main subclasses,
asthma,8 atopic dermatitis,9 psoriasis,10 rheumatoid arthritis,11 and
chronic inflammatory bowel disease.12In vivo studies of CCL17
and CCL22 antibodies have indicated their usefulness for prevent-
ing several immunological responses.13 Therefore, therapy using
CCR4 antagonists would be a novel intervention method for dis-
eases in which CCR4 participates.
Several small molecule CCR4 antagonists have been reported
in the literature (Fig. 1).14 During the screening of our corporate
CXC (or
a) and CC (or b) chemokines. Chemokines exert their bio-
compound libraries using the [35S]GTP S-binding assay,15 2,4-dia-
c
logical activity through interactions with a subset of G-coupled se-
ven transmembrane receptors. These are named according to the
type of chemokines they interact with, that is, CXC receptors
(CXCRs) and CC receptors (CCRs).2
mino 6,7-dimethoxyquinazoline derivative 5 was located in a qui-
nazoline-based diversity library (Fig. 2) and was found to have
potent inhibitory activity, and thus designated as the lead
compound.
CC chemokine receptor 4 (CCR4) was originally cloned from
T-lymphocytes and the thymus, where CCR4 is highly expressed.3
CCR4 was reported to be expressed predominantly on Th2-lym-
phocytes,4 but recent studies revealed that CCR4 is also widely ex-
pressed on monocytes, macrophages, dendritic cells, and natural
killer cells.5 Thymus and activation-regulated chemokine (TARC,
CCL17) as well as macrophage-derived chemokine (MDC, CCL22)
are CC chemokines, and are both highly specific biological ligands
for CCR4.6 These two chemokines are constitutive chemokines
that are responsible for normal leucocyte trafficking, but are also
inducible chemokines that recruit leukocytes in response to
2. Combinatorial synthesis and the initial SAR study
The lead compound 5 was synthesized from 2,4-dichloro-6,7-
dimethoxyquinazoline using
a two-step, selective, sequential
substitution of amines.16 An additional quinazoline-templated
combinatorial library was prepared to obtain the initial SAR of
the lead compound 5. Numerous commercially available alkyl-
amines (anilines were not employed due to their poor reactivity)
were introduced at the 2- and 4-positions of 6,7-dimethoxyquinaz-
oline derivatives, and the synthesized compounds were evaluated
in the [35S] GTP
the concentration of 1
tion of quinazoline (Table 1), the cycloalkylamines were found to
c
S-binding assay and [121I]CCL22-binding assay at
lM. Among the substituents at the 4-posi-
* 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.2008.05.036

7022
K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
F
F
Cl
HN
Cl
HN
Cl
S
NC
N
O
Me
S O
N
N
N
N
HN
O
N
Me
N
S
N
N
N
N
H
N
N
Me
N
N
H
Me
O
4
1
2
3
Figure 1. Small molecule CCR4 antagonists.
Table 1
R1
N
CCR4 inhibitory activities of 4-substituted quinazoline derivatives
HN
N
HN
R2
R3
R1
MeO
MeO
N
R5
MeO
N
N
N
N
R4
N
MeO
N
N
5
N
Figure 2. Discovery of compound 5 from quinazoline-based diversity library.
Compound
R1
[
35S]GTP
c
S
l
[
125I]CCL22
inh.% at 1
inh.% at 1
Ma
l
Ma
yield potent activity, while cycloheptylamine was the most potent.
In general, a subtle change in the 4-substituents resulted in a con-
siderable loss of potency. Only cycloalkylamines caused more than
HN Ph
C-001
C-002
10
<0
Ph
50% inhibition at the 1
l
M concentration; all other alkylamines
7
4
HN
inhibited less than 50%. Exploration of substituents at the 2-posi-
tion included the testing of diamines such as piperazine and 4-
aminosubstituted piperidine, which were required to yield potent
activity (Table 2). In contrast to the SAR for the 4-position substit-
uents, however, a subtle stereochemical change in the 2-position
substituents did not affect the potency, and several types of the
diamine substituents were found to have potent activity. Based
on these data, further optimization of the quinazoline derivatives
was performed using 4-cycloheptylamine analogues.
HN Me
C-003
C-004
3
<0
17
18
HN
HN
C-005
71 (IC50 = 480 nM)
54 (IC50 = 1900 nM)
C-006 (5)
C-007
93
11
100
<0
HN
HN
HN
tBu
3. Chemistry
The novel amines 8 and 10 were synthesized as summarized
in Scheme 1. Diamine 8 was prepared via reductive amination
of the protected piperidone 6 with the 4-hydroxypiperidine,
followed by the fluorination of the alcohol group using DAST,
and subsequent deprotection of the benzyloxycarbonyl group.
Compound 10a was also prepared from the piperidone 6 by
reductive amination with cyclopentylamine, followed by depro-
tection of the protecting group. Compound 10b was synthesized
by methylation of the secondary amine 9a and subsequent
deprotection.
The preparation of 2,4-diaminoquinazolines 14 is summarized
in Scheme 2. The anthranic acid 11 was obtained commercially
or prepared in-house (see Section 6). The reaction of 11 with urea,
followed by treatment with phosphorous oxychloride under reflux,
provided 2,4-dichloroquinazolines 12. Selective substitution of the
chlorine at the 4-position with cycloheptylamine yielded the corre-
sponding 4-amino-2-chloro-quinazolines 13, which was subse-
quently coupled with commercially available amines or prepared
amines 8 and 10 at the 2-position under reflux in isopropanol to
yield 2,4-diaminoquinazolines 14.
C-008
C-009
71 (IC50 = 200 nM)
54 (IC50 = 1200 nM)
16
16
NH
HN
O
O
N
OEt
Ph
C-010
10
6
HN
HN
N
C-011
C-012
C-013
4
<0
<0
19
Me Me
N
<0
8
N
O
N
C-014
<0
2
a
See Section 6, pharmacology.
N-Cycloheptyl-2-(1-cyclohexylpiperidine-4-yl)-6,7-dimethoxy-
quinazolin-4-amine 18 was prepared as shown in Scheme 3.
The condensation of 1-[(benzyloxy)carbonyl]piperidine-4-car-
boxylic acid 15 with 2-amino-4, 5-dimethoxybenzonitrile,
followed by treatment with H2O2 under reflux, yielded the
cyclized product 16. Treatment of 16 with phosphorous oxy-
chloride under reflux, followed by the substitution of cyclohep-
tylamine, provided 4-aminoquinazolines 17. The removal of the
benzyloxycarbonyl (CBz) group was readily accomplished by
hydrogenolysis to yield the secondary amine, which was alkyl-
ated with iodocyclohexane in the presence of sodium carbonate
to yield compound 18.

K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
7023
Table 2
firmed by chemotaxis assay. The results are summarized in Tables
3–5.
CCR4 inhibitory activities of 2-substituted quinazoline derivatives
The parent compound 5 showed potent inhibition in the
[
35S]GTP
cS-binding assay (IC50 = 19 nM, Table 3). This compound
HN
also inhibited the chemotaxis of human and mouse CCR4-express-
ing cells (IC50 = 200 nM, 130 nM). The cytotoxicity of this com-
pound
MeO
N
MeO
N
R1
Sa inh.%
5
was also evaluated. The results showed that the
M, see Section 6) was far from the
minimum cytotoxic dose (3
l
Compound
R1
[
35S]GTP
c
[
125I]CCL22 inh.%
at 1
concentration that inhibited chemotaxis, which indicates that the
observed inhibition was due to receptor antagonism, not cytotox-
icity. To improve the potency of this compound, the substituents
at the 4-position of the piperidine were first examined. From the
results of combinatorial study mentioned above it was concluded
that an aryl or alkyl substituent, like phenyl or cyclohexyl, would
result in a significant loss of potency. In addition, the nitrogen
atom of the pyrrolidine in compound 5 appeared to be important
at 1
l
Ma
l
Ma
N
C-015
7
11
N
N
Me
C-016
C-017
65 (IC50 = 260 nM)
12
77 (IC50 = 730 nM)
15
N
Me
Me
for potent activity in the [35S]GTP
of the pyrrolidine with a piperidine did not significantly change
35S]GTP
S-binding or the human chemotactic factor, but there
cS-binding assay. Replacement
N
N
C-018
C-019
C-020
<0
17
1
3
OH
[
c
was a threefold increase in the inhibition of mouse cell chemotaxis
compared to the parent compound 5. Compounds 5 and 14a were
further evaluated by determining the [125I]CCL22-binding inhibi-
tion. Both showed potent activity that was almost equivalent.
The introduction of a fluoro substituent at the 4-position of the
piperidine (14b) in compound 14a also did not result in any signif-
NH2
<0
<0
O
N
N
Ph
C-021 (14a)
C-022
92
1
100
18
icant loss in the [35S]GTP
cS-binding assay or the inhibition of hu-
N
N
man cell chemotaxis, but a 15-fold loss of potency was observed
in the inhibition of mouse chemotaxis compared to compound
14a. These findings indicate that the trend in SAR seemed to differ
between human and mouse cell chemotaxis; mouse cell chemo-
taxis was very sensitive to subtle changes in this part of the tem-
plate. Cyclopentylamine (14c) and N-methylcyclopentylamine
(14d) were also hindered in this position. These substituents were
tolerated, but their presence resulted in slight decreases in the
inhibition of mouse cell chemotaxis.
N
H
N
H
N
N
C-023
35
16
O
C-024
C-025
8
8
N
H
N
N
N
N
13
<0
More significant changes were then introduced to the right-
hand portion of the structure (Table 4). A shift in the position
of the nitrogen attached to piperidine at the 4-position was
speculated to affect inhibitory activity, but the results of pipera-
zine derivative 14e were almost comparable to those of all three
parent compound assays. This result showed that the positional
shift of this basic nitrogen did not affect its potent inhibitory
activity. The effect of the other piperazine nitrogen attached to
the quinazoline core was also investigated. Removal of this
nitrogen (20) produced a 10-fold loss of potency in the binding
assay, which clearly showed that this nitrogen needs to be in
its original position.
Finally, the SAR of the methoxy substituents at the 6- and 7-
positions of quinazoline was investigated (Table 5). While removal
of both of these methoxy groups (14h) resulted in significant loss
of potency, the 6-methoxy analogue (14f) showed only a slight de-
crease of potency, and the 7-methoxy analogue (14g) maintained
its activity compared to the parent compound 5 in all three assays.
These results indicate that the 7-methoxy substituent is more
important than 6-methoxy substituent for activity.
N
Me
Me
N
N
C-026
C-027
68 (IC50 = 220 nM)
7
68 (IC50 = 660 nM)
7
Me
Ph
C-028
C-029
<0
<0
8
6
O
Me
N
H
Me
C-030
<0
4
N
H
N
H
Ph
C-031
C-032
C-033
13
0
<0
<0
<0
<0
S
N
H
Ph
Ph
N
H
16
<0
N
Me
C-034
5. Conclusion
a
See Section 6, pharmacology.
The screening of a corporate library of compounds yielded a new
series of potent competitive CCR4 antagonists, which were then
subjected to chemical optimization. Among this series, compound
4. Results and discussion
14a yielded the most potent inhibition in the [35S]GTP
cS-binding
assay. This compound also potently inhibited the chemotaxis of hu-
man and mouse CCR4-expressing cells. The most interesting part of
the SAR study was the stereochemical tolerance at the 4-position of
the piperidine, and the increase in potency caused by the conversion
The CCR4 antagonist activities of the synthesized compounds
were determined by measuring the degree to which human
CCL22-derived [35S]GTP
cS was prevented from binding to the
receptor. The CCR4-antagonism of these compounds was also con-

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K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
O
O
HN
O
N
O
N
iii
iii
i, ii
iv
N
N
O
F
6
7
8
F
O
HN
O
N
6
N
R1
N
R1
v
9a: R1=H
10a: R1=H
9b: R1=Me
10b: R1=Me
Scheme 1. Reagents and conditions: (i) 4-hydroxypiperidine, Ti(Oi-Pr)4, 80 °C, then NaBH4, EtOH; (ii) DAST, CH2Cl2; (iii) 10% Pd–C, EtOH; (iv) cyclopentylamine, NaBH(OAc)3,
AcOH; (v) 36%HCHO aq, HCO2H, 70 °C.
Cl
HN
HN
N
R1
R2
CO2H
NH2
R1
R2
R1
R2
R1
R2
iii
iv
i, ii
N
N
N
R2
N
Cl
N
Cl
N
R1
13
11
12
14
Scheme 2. Reagents and conditions: (i) urea, 200 °C; (ii) POCl3; (iii) cycloheptylamine, DMF; (iv) amines, n-BuOH, reflux.
Table 3
O
CCR4 inhibitory activities of 4-substituted piperidine derivatives
MeO
O
NH
i, ii
iii, iv
HO
MeO
N
N
O
N
N
O
HN
N
O
O
16
15
MeO
MeO
N
N
R1
HN
HN
v, vi
MeO
MeO
MeO
MeO
N
Compound R1
[
(
35S]GTP
M)
c
Sa Chemotaxis Chemotaxis
[
(
125I]CCL22a
M)
N
N
N
l
(l
M)
(
l
M)
l
N
O
18
humana
mousea
17
O
N
5
0.019
0.018
0.032
0.20
0.13
0.070
0.073
NT
Scheme 3. Reagents and conditions: (i) oxalylchloride, CH2Cl2, DMF (drops), then
2-amino-4,5-dimethoxybenzonitrile, pyridine; (ii) NaOH, 30%H2O2 aq, EtOH, reflux;
(iii) POCl3, reflux; (iv) cycloheptylamine, DMF; (v) 10% Pd–C, MeOH; (vi) iodocy-
clohexane, Na2CO3, DMF.
N
N
14a
14b
0.14
0.31
0.039
0.60
F
of pyrrolidine to piperidine. Therefore, it was shown that intensive
synthetic conversions can lead to additional improvement of po-
tency. Further developments related to the optimization of this ser-
ies will be reported in due course.
14c
0.013
0.018
0.10
0.12
0.29
0.35
NT
NT
N
H
14d
6. Experimental
6.1. Chemistry
N
Me
a
See Section 6, pharmacology.
In general, reagents and solvents were used as purchased
without further purification. Melting points were determined
with a Yanaco MP-500D melting point apparatus and left uncor-
rected. 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 the internal stan-
dard (NMR description key: s = singlet, d = doublet, t = triplet,
m = multiplet, and br = broad peak). Mass spectra were recorded
on a JEOL JMS-LX2000 spectrometer. High-resolution mass spec-
tra were recorded on a Waters QTOF Premier spectrometer; the
results were within 0.2 mDa of the theoretical values. The ele-
mental analyses were performed with a Yanaco MT-5 microana-
lyzer (C,H,N) and Yokogawa IC-7000S ion chromatographic
analyzer (halogens); the results were within 0.4% of the theo-
retical values.
6.1.1. N-Cycloheptyl-6,7-dimethoxy-2-(4-pyrrolidine-1-
ylpiperidine-1-yl)quinazolin-4-amine (5)
4-Pyrrolidin-1-ylpiperidine (930 mg, 6.0 mmol) was added to a
solution of 2-chloro-N-cycloheptyl-6,7-dimethoxyquinazolin-4-
amine 13a (1.01 g, 3.0 mmol) in n-BuOH (10 mL), and the mixture
was stirred under reflux for 3 h. The reaction mixture was cooled to
room temperature and concentrated in vacuo. The residue in chlo-
roform was washed with brine, dried over sodium sulfate, filtered,
and evaporated in vacuo. The residue was purified using column
chromatography (chloroform/MeOH/NH4OH) to yield N-cyclohep-
tyl-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidine-1-yl)quinazolin-
4-amine 5 (500 mg, 37%).
Mp (dec) 205–206 °C (Et2O); MS (FAB+) m/z 454 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d 1.25–1.36 (2H, m), 1.45–1.75 (14H,

K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
7025
Table 4
CCR4 inhibitory activities of 2-substituted quinazoline derivatives
HN
N
MeO
MeO
N
R2
Compound
R2
[
35S]GTP
cS (l
M)a
Chemotaxis (
l
M) humana
Chemotaxis (
l
M) mousea
[ (lM)
125I]CCL22a
N
N
N
14e
0.055
0.23
0.21
NT
20
0.51
12% at 1
0.20
l
M
NT
NT
5
0.019
0.13
0.070
a
See Section 6, pharmacology.
Table 5
CCR4 inhibitory activities of 6,7-substituted quinazoline derivatives
HN
N
R3
R4
N
N
N
Compound
R3
R4
[
35S]GTP
c
S (
l
M)a
Chemotaxis (
l
M) humana
Chemotaxis (
l
M) mousea
[ (lM)
125I]CCL22a
14f
14g
14h
5
MeO–
H–
H
H
MeO–
H
0.031
0.013
0.20
0.41
0.12
1.4
0.80
0.22
NT
NT
NT
NT
0.070
MeO–
MeO–
0.019
0.20
0.13
a
See Section 6, pharmacology.
m), 1.82–1.89 (2H, m), 1.93–2.02 (2H, m), 2.12–2.26 (1H, m), 2.47–
2.53 (2H, m), 2.81–2.95 (2H, m), 3.25–3.40 (2H, m), 3.80 (3H, s),
3.81 (3H, s), 4.13–4.24 (1H, m), 4.54–4.64 (2H, m), 6.71 (1H, s),
7.24 (1H, d, J = 7.2 Hz), 9.45 (1H, s). Anal. Calcd for C26H39N5O2:
C, 68.84; H, 8.67; N, 15.44. Found: C, 68.61; H, 8.92; N, 15.26.
concentrated in vacuo. The residue was purified via column
chromatography (chloroform/MeOH) to yield benzyl 4-fluoro-
1,40-bipiperidine-10-carboxylate 7 (1.11 g, 3.47 mol, 75%).
MS (ESI+) m/z 321 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d
1.23–1.36 (2H, m), 1.58–1.72 (4H, m), 1.74–1.90 (2H, m),
2.32–2.49 (5H, m), 2.69–2.86 (2H, m), 3.96–4.06 (2H, m),
4.52–4.72 (1H, m), 5.01 (2H, s), 7.28–7.40 (5H, m).
6.1.2. 4-Fluoro-1,40-bipiperidine (8)
The mixture of 4-hydroxypiperidine 6 (1.01 g, 10 mmol), benzyl
4-oxo-1-piperidine carboxylate (2.33 g, 10 mmol), and Ti(Oi-Pr)4
(5.69 g, 20 mmol) was stirred at 80 °C for 5 h. The reaction mixture
was cooled on ice. Ethanol (100 mL) and sodium borohydride
(757 mg, 20 mmol) were added, and the resulting mixture was stir-
red at room temperature for 1 h. The organic layer was concen-
trated in vacuo. The residue was partitioned between chloroform
and 1 M-NaOH aqueous solution, and an insoluble solid was re-
moved by filtration on Celite. The organic layer was then washed
with brine, dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified via silica gel column chromatography
(chloroform/MeOH) to yield benzyl 4-hydroxy-1,40-bipiperidine-
10-carboxylate (1.46 g, 4.6 mol, 46%).
Palladium on carbon (10%,100 mg) was added to a solution of
benzyl 4-fluoro-1,40-bipiperidine-10-carboxylate7 (1.11 g) in
ethanol (30 mL). The resulting mixture was stirred at room
temperature under 1 atm of hydrogen gas for 6 h, and then fil-
tered through a pad of Celite. The filtrate was concentrated in va-
cuo to yield 4-fluoro-1,40-bipiperidine 8 (469 mg) as a colorless
oil, which was used in a subsequent reaction without further
purification.
6.1.3. Benzyl 4-(cyclopentylamino)piperidine-1-carboxylate
(9a)
Benzyl 4-oxo-1-piperidine carboxylate 6 (3.5g, 15 mmol) was
added to a solution of cyclopentylamine (850 mg, 10 mmol) in ace-
tic acid (100 mL). The resulting mixture was stirred at room tem-
perature for 1 h. Sodium acetoxyborohydride (5.3 g, 25 mmol)
was added to the reaction mixture, and the mixture was stirred
at room temperature for 16 h. The reaction mixture was concen-
trated in vacuo. Potassium carbonate aqueous solution was added
to the residue, and the resulting mixture was extracted with chlo-
roform. The organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied via column chromatography (chloroform/MeOH/ NH4OH) to
A solution of benzyl 4-hydroxy-1,40-bipiperidine-10-carboxylate
dissolved in methylenechloride (20 mL) was added slowly to a
solution of DAST [(diethylamino)sulfur trifluoride] in methylene-
chloride (20 mL) over 1 h at 4 °C. The resulting mixture was stirred
at room temperature for 4 h. The reaction was carefully quenched
with saturated sodium bicarbonate aqueous solution, while main-
taining the temperature of the reaction mixture under 15 °C. The
reaction mixture was extracted with chloroform, and the organic
layer was washed with brine, dried over Na2SO4, filtered, and

7026
K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
yield benzyl 4-(cyclopentylamino)piperidine-1-carboxylate 9a
(2.15 g, 7.11 mol, 71%).
6.1.8. 2,4-Dichloro-7-methoxyquinazoline (12c)
Palladium on carbon (10%, 400 mg) was added to the solution of
4-methoxy-2-nitrobenzoic acid (4.02 g, 20.4 mmol) in ethanol
(100 mL). The resulting mixture was stirred at room temperature
under 1 atm of hydrogen gas for 1 h, and then filtered through a
pad of Celite. The filtrate was concentrated in vacuo to yield crude
2-amino-4-methoxybenzoic acid 11b (3.18 g) as a grayish solid,
which was used in a subsequent reaction without further purifica-
tion. Compound 12c (660 mg, 14% from 4-methoxy-2-nitrobenzoic
acid) was prepared from crude 11b (3.17 g) using a procedure sim-
ilar to that described for the synthesis of 12b.
MS (ESI+) m/z 303 [M+H]+. 1H NMR (400 MHz, CDCl3) d 1.22–
1.30 (4H, m), 1.47–1.58 (2H, m), 1.63–1.73 (2H, m), 1.82–1.88
(4H, m), 2.63–2.70 (1H, m), 2.83–2.89 (2H, m), 3.16–3.23 (1H,
m), 4.12 (2H, br), 5.12 (2H, s), 7.28–7.38 (5H, m).
6.1.4. Benzyl 4-[cyclopentyl(methyl)amino]piperidine-1-
carboxylate (9b)
A mixture of benzyl 4-(cyclopentylamino)piperidine-1-carboxyl-
ate 9a (364 mg, 1.2 mmol), 36% formaldehyde aqueous solution
(1 mL), and formic acid (2 mL) was stirred at 70 °C for 1 h. Formalde-
hyde aqueous solution (36%, 2mL) and formic acid (1 mL) were added
to the reaction mixture, which was then stirred at 90 °C for 18 h. The
reaction mixture was concentrated in vacuo. Potassium carbonate
aqueous solution was added to the residue, and the resulting mixture
was extracted with chloroform. The organic layer was washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The res-
idue was purified via column chromatography (chloroform/MeOH/
NH4OH) to yield benzyl 4-[cyclopentyl(methyl)amino]piperidine-1-
carboxylate 9b (369 mg, 1.17 mol, 97%).
1H NMR (300 MHz, DMSO-d6) d 4.00 (3H, s), 7.44–7.52 (2H, m),
8.18 (1H, d, J = 9.2 Hz).
6.1.9. 2-Chloro-N-cycloheptyl-6,7-dimethoxyquinazolin-4-
amine (13a)
Cycloheptylamine (2.02 g, 17.9 mmol) was added to a solution
of 2,4-dichloro-6,7-dimethoxyquinazoline 12a (2.11 g, 8.14 mmol)
in DMF (20 mL), and the resulting mixture was stirred at room
temperature for 2 h. Water was added to the reaction mixture,
and the resulting solution was extracted with ethyl acetate. The or-
ganic layer was washed with 5% citric acid aqueous solution and
brine, dried over sodium sulfate, filtered, and evaporated in vacuo.
The residue was washed with diethyl ether to yield 2-chloro-N-
cycloheptyl-6,7-dimethoxyquinazolin-4-amine 13a as a colorless
solid (2.15 g, 79%).
MS (ESI+) m/z 317 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d 1.25–
1.40 (4H, m), 1.43–1.53 (2H, m), 1.54–1.66 (4H, m), 1.70–1.80 (2H,
m), 2.09 (3H, s, br), 2.75 (2H, m, br), 2.89 (1H, m, br), 3.26–3.30 (1H,
m), 4.01–4.10 (2H, br), 5.06 (2H, s), 7.28–7.40 (5H, m).
6.1.5. N-Cyclopentylpiperidine-4-amine (10a)
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).
Compounds 13b–d were prepared using procedures similar to
those described for the synthesis of 13a.
Palladium on carbon (10%, 50 mg) was added to a solution
of benzyl 4-(cyclopentylamino)piperidine-1-carboxylate 9a
(493 mg) in ethanol (10 mL). The resulting mixture was stirred at
room temperature under 1 atm of hydrogen gas for 8 h, and then
filtered through a pad of Celite. The filtrate was concentrated in va-
cuo to yield crude N-cyclopentylpiperidine-4-amine 10a (279 mg)
as a colorless oil, which was used in a subsequent reaction without
further purification.
6.1.10. 2-Chloro-N-cycloheptyl-6-methoxyquinazolin-4-amine
(13b)
13b (1.13 g, 58%) was obtained as a colorless solid from 2,4-di-
chloro-6-methoxyquinazoline 12b (1.46 g, 6.4 mmol).
MS (ESI+) m/z 306 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d 1.45–
1.76 (10H, m), 1.92–2.02 (2H, m), 3.90 (3H, s), 4.26–4.33 (1H, m),
7.41 (1H, dd, J = 9.2 Hz, 2.8 Hz), 7.54 (1H, d, J = 8.8 Hz), 7.75 (1H,
d, J = 2.4 Hz), 8.19 (1H, d, J = 7.6 Hz).
6.1.6. N-Cyclopentyl-N-methylpiperidine-4-amine (10b)
Palladium on carbon (10%, 40 mg) was added to a solution of
benzyl 4-[cyclopentyl(methyl)amino]piperidine-1-carboxylate 9b
(369 mg, 1.17 mol) in ethanol (10 mL). The resulting mixture
was stirred at room temperature under 1 atm of hydrogen
gas for 8 h, and then filtered through a pad of Celite. The fil-
trate was concentrated in vacuo to yield crude N-cyclopentyl-
6.1.11. 2-Chloro-N-cycloheptyl-7-methoxyquinazolin-4-amine
(13c)
N-methylpiperidine-4-amine 10b (187 mg) as
a
white solid,
13c (590 mg, 89%) was obtained as a colorless solid from 2,4-di-
chloro-6-methoxyquinazoline 12c (500 mg, 2.18 mmol).
MS (ESI+) m/z 306 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d 1.42–
1.76 (10H, m), 1.88–1.98 (2H, m), 3.87 (3H, s), 4.20–4.31 (1H, m),
7.02 (1H, d, J = 2.4 Hz), 7.11 (1H, dd, J = 9.2, 2.8 Hz), 8.18 (1H, d,
J = 8.0 Hz), 8.26 (1H, d, J = 9.6 Hz).
which was used in
purification.
a subsequent reaction without further
6.1.7. 2,4-Dichloro-6-methoxyquinazoline (12b)
A mixture of 2-amino-5-methoxybenzoic acid 11a (5.13 g,
31 mmol) and urea (9.22 g, 153 mmol) was stirred at 200 °C for
1 h. The resulting mixture was treated with 2 M-NaOH aqueous
solution, and the insoluble material was removed by filtration.
The filtrate was acidified with concd HCl aqueous solution, and
the resulting precipitate was filtered and washed with EtOH/H2O
(1:1) to yield crude 6-methoxyquinazoline-2,4-diol (3.93 g) as a
grayish solid. POCl3 (50 mL) was added to this crude compound
and stirred under reflux for 6 h. The reaction mixture was concen-
trated in vacuo, and the resulting residue was added to a solution
of potassium carbonate aqueous solution. The resulting mixture
was then extracted with chloroform, and the organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. This residue was purified via column chromatography
(chloroform/MeOH) to yield 2,4-dichloro-6-methoxyquinazoline
12b (2.81 g, 40% from 11a).
6.1.12. 2-Chloro-N-cycloheptylquinazolin-4-amine (13d)
Crude 13d was obtained from 2, 4-dichloroquinazoline15
(1.50 g, 7.54 mmol) using a procedure similar to that described
for the synthesis of 13a. This crude solid was used in a subsequent
reaction without further purification.
Compounds 14a–h were prepared using procedures similar as
described for the synthesis of 5.
6.1.13. 2-(1,40-Bipiperidine-10-yl)-N-cycloheptyl-6,7-
dimethoxyquinazolin-4-amine (14a)
14a (310 mg, 22%) was obtained as a yellowish solid from 2-
chloro-N-cycloheptyl-6,7-dimethoxyquinazolin-4-amine 13a (1.02 g,
3.0 mmol), and 4-piperidinopiperidine (1.14 g, 6.1 mmol).
Mp (dec) 184–186 °C (Et2O); MS (FAB+) m/z 468 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d 1.25–1.40 (4H, m), 1.42–1.80 (16H,
m), 1.92–2.02 (2H, m), 2.40–2.55 (3H, m), 2.65–2.77 (2H, m),
1H NMR (400 MHz, DMSO-d6) d 4.00 (3H, s), 7.49 (1H, d,
J = 2.4 Hz), 7.80–7.83 (1H, m), 7.99 (1H, d, J = 9.2 Hz).

K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
7027
3.27–3.35 (2H, m), 3.806 (3H, s), 3.813 (3H, s), 4.12–4.24 (1H, m),
4.74–4.84 (2H, m), 6.71 (1H, s), 7.24 (1H, d, J = 7.6 Hz), 7.45 (1H,
s). Anal. Calcd for C26H39N5O2.0.5H2O: C, 68.04; H, 8.88; N, 14.69.
Found: C, 68.27; H, 9.13; N, 14.68.
Mp (dec) 203–204 °C (Et2O); MS (FAB+) m/z 468 [M+H]+. 1H NMR
(400 MHz, DMSO-d6) d 1.04–1.17 (1H, m), 1.20–1.33 (2H, m), 1.37–
1.69 (10H, m), 1.70–1.88 (6H, m), 1.90–2.00 (2H, m), 2.07–2.19
(2H, m), 3.52–3.65 (4H, m), 3.72–3.84 (2H, m), 3.87 (3H, s), 3.90
(3H, s), 4.26–4.38 (1H, m), 4.82–4.94 (2H, m), 7.70 (1H, s), 7.97
(1H, s), 9.21 (1H, d, J = 6.8 Hz), 11.53 (1H, s), 12.90 (1H, s). Anal. Calcd
for C27H41N5O2ꢁ2HClꢁ1.6H2O: C, 56.95; H, 8.18; N, 12.30; Cl, 12.45.
Found: C, 56.81; H, 8.30; N, 12.21; Cl, 12.80.
6.1.14. N-Cycloheptyl-2-(4-fluoro-1,40-bipiperidine-10-yl)-6,7-
dimethoxyquinazolin-4-amine (14b)
14b (682 mg, 94%) was obtained as a white solid from 2-chloro-
N-cycloheptyl-6,7-dimethoxyquinazolin-4-amine 13a (504 mg,
1.5 mmol), Hunig’s base (233 mg), and crude 4-fluoro-1,40-bipiper-
idine 8 (279 mg). 14b (682 mg) was treated with 4 M-HCl-dioxane,
and the mixture was concentrated. The residue was recrystallized
from isopropanol to yield the hydrochloride salt (440 mg, 0.79
mmol, 56%) as a white solid.
6.1.18. N-Cycloheptyl-6-methoxy-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazolin-4-amine-N-cycloheptyl-6-
methoxy-2-(4-pyrrolidin-1-ylpiperidine-1-yl)quinazolin-4-
amine (14f)
14f (480 mg, 57%) was obtained as a colorless solid from 2-
chloro-N-cycloheptyl-6-methoxyquinazolin-4-amine 13b (610 mg,
2 mmol) and 4-pyrrolidin-1-ylpiperidine (620 mg). 14f (450 mg)
was treated with 4 M-HCl-ethylacetate and the mixture was con-
centrated. The resulting residue was washed with Et2O to yield
the hydrochloride salt (500 mg, 95%) as a colorless solid.
Mp (dec) 198–199 °C (Et2O); MS (ESI+) m/z 424 [M+H]+. 1H NMR
(400 MHz, DMSO-d6) d 1.46–1.69 (6H, m), 1.70–2.02 (13H, m),
2.18–2.28 (2H, m), 3.02–3.22 (4H, m), 3.42–3.52 (2H, m), 3.88
(3H, s), 4.26–4.38 (1H, m), 4.78–4.88 (2H, m), 7.41–7.48 (1H, m),
7.92–8.01 (2H, br), 9.25 (1H, s), 11.39 (1H, s), 12.49 (1H, s). Anal.
Calcd for C25H37N5Oꢁ2HCl H2O: C, 58.36; H, 8.03; N, 13.61; Cl,
13.78. Found: C, 58.52; H, 8.30; N, 13.72; Cl, 13.43.
Mp (dec) 225–226 °C (i-PrOH); MS (FAB+) m/z 486 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d 1.44–1.68 (6H, m), 1.71–1.90 (6H,
m), 1.92–2.02 (2H, m), 2.03–2.40 (6H, m), 3.04–3.20 (4H,
m), 3.35–3.50 (2H, m), 3.52–3.62 (1H, m), 3.87 (3H, s), 3.89 (3H,
s), 4.26–4.32 (1H, m), 4.82–4.90 (2H, m), 4.93–5.10 (1H, m),
7.62–7.70 (1H, m), 7.90 (1H, s), 8.99–9.10 (1H, m), 11.28 (1H, m),
12.42–12.56 (1H, m). Anal. Calcd for C27H40FN5O2ꢁ2HClꢁ1.7H2Oꢁ
0.1C3H8O: C, 55.09; H, 7.82; N, 11.77; Cl, 11.91; F, 3.19. Found: C,
54.99; H, 7.88; N, 11.75; Cl, 12.05; F, 3.19.
6.1.15. N-Cycloheptyl-2-[4-(cyclopentylamino)piperidine-1-yl]-
6,7-dimethoxyquinazolin-4-amine (14c)
14c (660 mg, 87%) was obtained from 2-chloro-N-cycloheptyl-
6,7-dimethoxyquinazolin-4-amine 13a (548 mg, 1.63 mmol), Hu-
nig’s base (253 mg), and crude N-cyclopentylpiperidine-4-amine
10a (275 mg). 14c (660 mg) was treated with 4 M-HCl-dioxane,
and the mixture was concentrated. The resulting residue was
washed with Et2O to yield the hydrochloride salt (660 mg, 87%)
as a white solid.
6.1.19. N-Cycloheptyl-7-methoxy-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazolin-4-amine (14g)
14g(470 mg, 94%) wasobtainedas a colorlessamorphousfrom2-
chloro-N-cycloheptyl-7-methoxyquinazolin-4-amine 13c (360 mg,
1.18 mmol) and 4-pyrrolidin-1-ylpiperidine (360 mg). 14g
(470 mg) was treated with 4 M-HCl-ethylacetate and the mixture
was concentrated. The resulting residue was washed with Et2O to
yield the hydrochloride salt (370 mg, 67%) as a yellowish solid.
Mp (dec) 164–166 °C (Et2O); MS (ESI+) m/z 424 [M+H]+. 1H NMR
(400 MHz, DMSO-d6) d 1.42–1.76 (13H, m), 1.78–2.04 (6H, m),
2.06–2.20 (2H, m), 2.85–3.12 (4H, m), 3.40–3.55 (2H, m), 3.83
(3H, s), 4.14–4.28 (1H, m), 4.78–4.92 (2H, m), 6.65–7.10 (2H, br),
8.00–8.20 (1H, s), 10.86 (1H, br), 12.35 (1H, br). Anal. Calcd for
Mp (dec) 248 °C (Et2O); MS (FAB+) m/z 468 [M+H]+. 1H NMR
(400 MHz, DMSO-d6) d 1.40–1.80 (18H, m), 1.95–2.05 (4H, m),
2.20–2.28 (2H, m), 3.15–3.24 (2H, m), 3.58–3.62 (1H, m), 3.88
(6H, s), 4.24–4.36 (1H, m), 4.66–4.76 (2H, m), 7.52 (1H, s),
7.84 (1H, s), 8.96 (1H, br), 9.16 (2H, s), 12.30 (1H, s). Anal.
Calcd for
C
27H41N5O2ꢁ2HClꢁ1.4H2Oꢁ0.4C4H8O2: C, 57.15; H,
8.22; N, 11.65; Cl, 11.80. Found: C, 57.05; H, 8.18; N, 11.65;
Cl, 11.96.
C
25H37N5Oꢁ1.4HClꢁ2H2O: C, 58.80; H, 8.37; N, 13.71; Cl, 9.72.
Found: C, 59.04; H, 8.72; N, 13.79; Cl, 9.48.
6.1.16. N-Cycloheptyl-2-{4-[cyclopentyl(methyl)amino]piper-
idine-1-yl}-6,7-dimethoxyquinazolin-4-amine (14d)
6.1.20. N-Cycloheptyl-2-(4-pyrrolidin-1-ylpiperidine-1-
yl)quinazolin-4-amine (14h)
14d (185 mg, 37%) was obtained from 2-chloro-N-cycloheptyl-
6,7-dimethoxyquinazolin-4-amine 13a (344 mg, 1.03 mmol),
Hunig’s base (160 mg), and crude N-cyclopentyl-N-methylpiperi-
dine-4-amine 10b (187 mg). 14d (185 mg) was treated with
4 M-HCl-dioxane, and the mixture was concentrated. The resulting
residue was washed with Et2O and EtOH to yield the hydrochloride
salt (229 mg) as a white solid.
14h (2.20 g) was obtained from crude 2-chloro-N-cyclohep-
tylquinazolin-4-amine13d, Hunig’s base (1.3 mL), and 4-pyrroli-
din-1-ylpiperidine (1.16 g). 14h (2.20 g) was treated with
4 M-HCl-dioxane, and the mixture was concentrated. The result-
ing residue was recrystallized from Et2O/MeCN to yield the
hydrochloride salt (1.51 g, 3.2 mmol, 43% from 2, 4-dichloroqui-
nazoline) as a slightly orange solid.
Mp (dec) 265–266 °C (EtOH); MS (FAB+) m/z 482 [M+H]+. 1H
NMR (400 MHz, CD3OD) d 1.55–1.94 (17H), 1.95–2.32 (7H, m),
2.79 (3H, s), 3.29–3.31 (1H, m), 3.83–3.87 (2H, m), 3.88 (6H, s),
4.35–4.44 (1H, m), 4.84–4.86 (2H, m), 7.25 (1H, s), 7.70 (1H, s).
Anal. Calcd for C28H43N5O2ꢁ2HClꢁ0.8 H2O: C, 59.10; H, 8.25; N,
12.31; Cl, 12.46. Found: C, 58.71; H, 8.28; N, 12.22; Cl, 12.85.
MS (FAB+) m/z 394 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d
1.44–2.03 (19H, m), 2.18–2.28 (2H, m), 2.98–3.25 (4H, m), 3.44–
3.52 (2H, m), 4.25–4.36 (1H, m), 4.87–4.98 (2H, m), 7.38–7.45
(1H, m), 7.76–7.82 (1H, m), 8.10 (1H, d, J = 8.3 Hz), 8.50 (1H, d,
J = 8.3 Hz), 9.32 (1H, d, J = 7.3 Hz), 11.55 (1H, s), 12.71 (1H, s). Anal.
Calcd for C24H35N5ꢁ2.1ꢁHClꢁ1.8H2O: C, 57.36; H, 8.16; N, 13.94; Cl,
14.81. Found: C, 57.58; H, 8.41; N, 13.96; Cl, 14.57.
6.1.17. N-Cycloheptyl-2-(4-cyclohexylpiperazine-1-yl)-6,7-
dimethoxyquinazolin-4-amine (14e)
14e (780 mg, 67%) was obtained as a colorless solid from
6.1.21. Benzyl 4-[4-(cycloheptylamino)-6,7-
2-chloro-N-cycloheptyl-6,7-dimethoxyquinazolin-4-amine
13a
dimethoxyquinazolin-2-yl]piperidine-1-carboxylate (17)
DMF (3 drops) and oxalyl chloride (1.54 mL, 17.68 mmol) were
added to a solution of 1-[(benzyloxy)carbonyl]piperidine-4-car-
boxylic acid 15 (4.43 g, 16.84 mmol) in CH2Cl2 (50 mL), and the
(840 mg, 5 mmol) and 1-cyclohexylpiperazine (860 mg). 14e
(700 mg) was treated with 4 M-HCl-dioxane, and the mixture
was concentrated. The resulting residue was washed with Et2O to
yield the hydrochloride salt (760 mg, 94%) as a colorless solid.

7028
K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
resulting mixture was stirred at room temperature for 2 h. A solu-
tion of 2-amino-4,5-dimethoxybenzonitrile (3 g, 16.84 mmol) in
pyridine (30 mL) at 4 °C was added to the reaction mixture, and
the resulting mixture was stirred at room temperature for 18 h.
Chloroform was added to the reaction mixture, and the solution
was washed with 1 M HCl aqueous solution and brine, dried over
sodium sulfate, filtered, and evaporated in vacuo. The residue
was washed with diethyl ether to yield benzyl 4-{[(2-cyano-4,
5-dimethoxyphenyl)amino]carbonyl}piperidine-1-carboxylate
(5.6 g, 13.23 mmol, 79%) as a yellow solid. Benzyl 4-{[(2-cyano-4,5-
dimethoxyphenyl) amino]carbonyl}piperidine-1-carboxylate (5.6
g) and 30% H2O2 aqueous solution (31 mL) were added to the mix-
ture of NaOH (529 mg, 13.23 mmol) and EtOH (140 mL). The
resulting mixture was stirred under reflux for 2 h, and the reaction
mixture was concentrated in vacuo. HCl aqueous solution (1 M)
was added to the solution of residue in chloroform, and the organic
layer was washed with brine, dried over sodium sulfate, filtered,
and evaporated in vacuo. The residue was washed with diethyl
ether to yield benzyl 4-(6,7-dimethoxy-4-oxo-3,4-dihydroquinaz-
olin-2-yl)piperidine-1-carboxylate 16 (5.4 g, 12.74 mmol, 96%) as
a pale yellow solid. POCl3 (50 mL) was added to this compound
16 (5.39 g) and stirred under reflux for 1 h. The reaction mixture
was concentrated in vacuo, and potassium carbonate aqueous
solution was added to the residue. The resulting mixture was ex-
tracted with chloroform, and the organic layer was washed with
brine, dried over sodium sulfate, filtered, and evaporated in vacuo.
The residue was purified via column chromatography to yield ben-
zyl 4-(4-chloro-6,7-dimethoxyquinazolin-2-yl)piperidine-1-car-
boxylate (2.45 g, 5.55 mmol, 44%).
m), 1.35–1.70 (10H, m), 1.70–1.88 (6H, m), 1.90–2.02 (2H, m),
2.05–2.45 (6H, m), 3.01–3.23 (3H, m), 3.48–3.58 (2H, m), 3.92
(3H, s), 3.94 (3H, s), 4.45–4.59 (1H, m), 7.42 (1H, s), 8.08 (1H, s),
9.62 (1H, s), 10.50 (1H, s). Anal. Calcd for C28H42N4O2ꢁ2HClꢁ1.6H2O:
C, 59.17; H, 8.37; N, 9.86; Cl, 12.47. Found: C, 58.67; H, 8.35; N,
9.90; Cl, 12.98.
6.1.23. N-Benzyl-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidine-
1-yl)quinazolin-4-amine (C-001)
Benzylamine (21 mg, 0.2 mmol) was added to a solution of 2,4-
dichloro-6,7-dimethoxyquinazoline 12a (52 mg, 0.1 mmol) in DMF
(0.1 mL), and the resulting mixture was stirred at room tempera-
ture for 10 h. Water was added to the reaction mixture, and the
resulting solution was extracted with chloroform. The organic
layer was evaporated in vacuo to yield crude N-benzyl-2-chloro-
6,7-dimethoxyquinazolin-4-amine as an oil, which was used in a
subsequent reaction without further purification. 4-pyrrolidin-1-
ylpiperidine (31 mg, 0.2 mmol) was added to a solution of crude
N-benzyl-2-chloro-6,7-dimethoxyquinazolin-4-amine in n-BuOH
(0.1 mL), and the mixture was stirred under reflux for 10 h. The
reaction mixture was cooled to room temperature and concen-
trated in vacuo. The residue was purified using preparative HPLC
to yield N-benzyl-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidine-
1-yl)quinazolin-4-amine C-001 (30mg, 67%).
1H NMR (400 MHz, CDCl3) d 1.61–1.69 (2H, m), 1.93–2.04 (6H,
m), 2.78–2.96 (6H, m), 3.92 (3H, s), 3.94 (3H, s), 4.65 (2H, d,
J = 5.4 Hz), 4.78 (2H, m), 7.04 (1H, s), 7.20–7.36 (6H, m), 8.10
(1H, br). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
C26H34N5O2: 448.2713. Found: 448.2694.
Benzyl 4-[4-(cycloheptylamino)-6,7-dimethoxyquinazolin-2-
yl]piperidine-1-carboxylate 17 was prepared (1.02 g, 1.96 mmol,
78%) from benzyl 4-(4-chloro-6,7-dimethoxyquinazolin-2-yl)-
piperidine-1-carboxylate (1.1 g, 2.5 mmol) using a procedure
similar to that described for the synthesis of 13a.
Compounds C-002-034 were prepared using procedures similar
to those described for the synthesis of C-001.
6.1.24. 6,7-Dimethoxy-N-(2-phenylethyl)-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazolin-4-amine (C-002)
MS (ESI+) m/z 519 [M+H]+. 1H NMR (400 MHz, DMSO-d6) d 1.42–
1.79 (12H, m), 1.88–2.02 (4H, m), 2.81–2.93 (1H, m), 3.22–3.40
(2H, m), 3.88 (3H, s), 3.89 (3H, s), 4.04–4.12 (2H, m), 4.25–4.38
(1H, m), 5.10 (2H, s), 7.05 (1H, s), 7.29–7.40 (5H, m), 7.66 (1H, s).
27 mg, 58%.
1H NMR (400 MHz, CDCl3) d 1.71–1.80 (2H, m), 1.90–1.98 (6H,
m), 2.05–2.07 (2H, m), 2.76–2.81 (2H, m), 2.94–3.01 (7H, m),
4.76–4.80 (2H, m), 6.99 (1H, s), 7.15 (1H, s), 7.20–7.36 (6H, m),
8.00 (1H, br). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd
for C27H36N5O2: 462.2869. Found: 462.2874.
6.1.22. N-Cycloheptyl-2-(1-cyclohexylpiperidine-4-yl)-6,7-
dimethoxyquinazolin-4-amine (20)
Palladium on carbon (10%, 100 mg) stirred at room temperature
under1 atmof hydrogengas for 4 h was added toa solutionof benzyl
4-[4-(cycloheptylamino)-6,7-dimethoxyquinazolin-2-yl]piper-i-
dine-1-carboxylate 17 (1.02 g, 1.96 mmol) in methanol (20 mL), and
then filtered through a pad of Celite. The filtrate was concentrated in
vacuo to yield crude N-cycloheptyl-6,7-dimethoxy-2-piperidine-4-
ylquinazolin-4-amine (721 mg) as a white solid, which was used in
a subsequent reaction without further purification.
6.1.25. N-Ethyl-6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidine-
1-yl)quinazolin-4-amine (C-003)
9 mg, 23%.
1H NMR (400 MHz, CDCl3 ) d 1.32 (3H, t,
m),
J = 7.1 Hz), 1.46–1.59 (2H,
1.78–1.80 (4H, m), 1.97–2.00 (2H, m), 2.19–2.26 (1H, m), 2.58–
2.70 (4H, m), 2.87–2.94 (2H, m), 3.63 (2H, quartet, J = 7.3 Hz),
3.91 (3H, s), 3.92 (3H, s), 4.82–4.85 (2H, m), 6.74 (1H, s), 6.90
(1H, s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
C21H32N5O2: 386.2556. Found: 386.2547.
Sodium carbonate (239 mg, 2.26 mmol) and iodocyclohexane
(613 mg) were added to the solution of the crude N-cycloheptyl-
6,7-dimethoxy-2-piperidine-4-ylquinazolin-4-amine (721 mg) in
DMF (20 mL). The resulting mixture was stirred at 80 °C for 5 h,
and chloroform was added. The solution was then washed with
brine, dried over sodium sulfate, filtered, and evaporated in vacuo.
The residue was purified via column chromatography (chloroform/
MeOH/NH4OH) to yield N-cycloheptyl-2-(1-cyclohexylpiperidine-
4-yl)-6,7-dimethoxyquinazolin-4-amine 20 (166 mg, 0.36 mmol,
19%) as a yellow oil. HCl ethyl acetate solution (4 M, 0.3 mL) was
added to a solution of this oil 20 (166 mg) in methanol (10 mL).
The resulting solution was stirred at room temperature for 1 h,
and the reaction mixture was concentrated in vacuo. The residue
was recrystallized from isopropanol to yield the hydrochloride salt
(74 mg, 38%) as a white solid.
6.1.26. N-Cyclopentyl-6,7-dimethoxy-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazolin-4-amine (C-004)
23 mg, 54%.
1H NMR (400 MHz, CDCl3) d 1.60–1.85 (8H, m), 1.93–2.10 (4H,
m), 2.07–2.14 (4H, m), 2.86–3.10 (7H, m), 3.92 (3H, s), 3.93 (3H,
s), 4.30–4.35 (1H, m), 4.85–4.88 (2H, m), 7.03 (1H, s), 7.23 (1H,
s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
C24H36N5O2: 426.2869. Found: 426.2855.
6.1.27. N-Cyclohexyl-6,7-dimethoxy-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazolin-4-amine (C-005)
30 mg, 68%.
1H NMR (400 MHz, CDCl3) d 1.20–1.40 (5H, m), 1.68–1.80 (5H,
m), 1.95–2.20 (8H, m), 2.87–3.01 (3H, m), 3.06–3.16 (4H, m),
3.89–4.11 (7H, m), 4.87–4.91 (2H, m), 7.12 (1H, s), 7.23 (1H, s),
Mp (dec) 236–237 °C (i-PrOH); MS (FAB+) m/z 467 [M+H]+. 1H
NMR (400 MHz, DMSO-d6) d 1.04–1.18 (1H, m), 1.20–1.34 (2H,

K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
7029
7.75 (1H, br). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
25H38N5O2: 440.3026. Found: 440.3015.
3.92 (3H, s), 3.98 (3H, s), 4.99–5.08 (2H, m), 6.99 (1H, s), 7.03
C
(1H, s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
C24H36N5O2: 426.2869. Found: 426.2852.
6.1.28. N-(4-tert-Butylcyclohexyl)-6,7-dimethoxy-2-(4-
pyrrolidin-1-ylpiperidine-1-yl)quinazolin-4-amine (C-007)
29 mg, 59%.
6.1.35. 6,7-Dimethoxy-4-morpholin-4-yl-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazoline (C-014)
1H NMR (400 MHz, CDCl3) d 0.88–0.89 (9H, m), 0.96–1.40 (4H,
m), 1.57–1.88 (5H, m), 1.95–2.05 (4H, m), 2.08–2.17 (4H, m),
2.79–3.20 (6H, m), 3.85–4.05 (6H, m), 4.34–4.43 (1H, m), 4.90–
5.06 (2H, m), 6.06–6.07 (1H, m), 6.95 (1H, s), 7.08 (1H, s). High-res-
olution MS (ESI+) m/z [M+H]+. Anal. Calcd for C29H46N5O2:
496.3652. Found: 496.3649.
28 mg, 65%.
1H NMR (400 MHz, CDCl3) d 1.70–1.92 (2H, m), 2.00–2.22
(6H, m), 2.79–2.98 (2H, m), 3.05–3.38 (4H, m), 3.50–3.70
(4H, m), 3.85–3.91 (3H, m), 3.92 (3H, s), 3.98 (3H, s),
4.99–5.08 (2H, m), 6.96 (2H, s). High-resolution MS (ESI+)
m/z [M+H]+. Anal. Calcd for C23H34N5O3: 428.2662. Found:
428.2644.
6.1.29. N-Cyclooctyl-6,7-dimethoxy-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazolin-4-amine (C-008)
6.1.36. N-Cycloheptyl-6,7-dimethoxy-2-piperidine-1-
ylquinazolin-4-amine (C-015)
33 mg, 71%.
1H NMR (400 MHz, CDCl3) d1.50–1.68 (8H, m), 1.70–1.84 (6H, m),
1.88–2.01 (6H, m), 2.05–2.15(2H, m), 2.90–3.20 (6H, m), 3.93 (6H, s),
4.31–4.33 (1H, m), 4.93–4.96(2H, m), 6.83–6.88 (1H, m), 7.15 (1H, s),
7.21 (1H, s), 8.14 (1H, br). High-resolution MS (ESI+) m/z [M+H]+.
Anal. Calcd for C27H42N5O2: 468.3339. Found: 468.3325.
3.5 mg, 9%.
1H NMR (400 MHz, CDCl3) d 1.45–1.80 (14H, m), 1.85–2.20 (4H,
m), 3.77–3.89 (4H, m), 3.92 (3H, s), 3.95 (3H, s), 4.20–4.35 (1H, m),
5.15–5.30 (1H, m), 6.73 (1H, s), 6.94 (1H, s). High-resolution MS
(ESI+) m/z [M+H]+. Anal. Calcd for C22H33N4O2: 385.2604. Found:
385.2609.
6.1.30. 3-{[6,7-Dimethoxy-2-(4-pyrrolidin-1-ylpiperidine-1-
yl)quinazolin-4-yl]amino}azepan-2-one (C-009)
6.1.37. N-Cycloheptyl-2-[4-(dimethylamino)piperidine-1-yl]-
6,7-dimethoxyquinazolin-4-amine (C-016)
3.4 mg, 7%.
1H NMR (400 MHz, CDCl3) d1.46–1.61 (4H, m), 1.65–2.01 (9H, m),
2.05–2.15 (1H, m), 2.22–2.27 (1H, m), 2.38–2.41 (1H, m). 2.55–2.70
(4H, m), 2.88–2.97 (2H, m), 3.20–3.48 (2H, m), 3.95 (3H, s), 3.96 (3H,
s), 4.81–4.86 (3H, m), 6.07–6.10 (1H, m), 6.78–6.79(1H, m), 6.88 (1H,
s), 6.89 (1H, s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
29 mg, 68%.
1H NMR (400 MHz, CDCl3) d 1.44–1.85 (12H m), 1.97–2.14 (4H,
m), 2.56 (3H, s), 2.62 (3H, s), 2.89–3.10 (3H, m), 3.93 (6H, s), 4.10–
4.25 (1H, m), 4.92–5.01 (2H, m), 6.73–6.75 (1H, m), 7.09 (1H, s),
7.14 (1H, s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd
for C24H38N5O2: 428.3026. Found: 428.3009.
C25H37N6O3: 469.2927. Found: 469.2910.
6.1.31. Ethyl 4-{[6,7-dimethoxy-2-(4-pyrrolidin-1-ylpiperidine-
1-yl)quinazolin-4-yl]amino}piperidine-1-carboxylate (C-010)
33 mg, 64%.
6.1.38. N-Cycloheptyl-6,7-dimethoxy-2-(4-methylpiperidine-1-
yl)quinazolin-4-amine (C-017)
6.2 mg, 16%.
1H NMR (400 MHz, CDCl3) d 1.27 (3H, t, J = 6.8 Hz), 1.50–1.65
(2H, m), 1.78–1.90 (2H, m), 1.95–2.20 (8H, m), 2.85–3.20 (8H,
m), 3.92 (3H, s), 3.94 (3H, s), 4.10–4.30 (5H, m), 4.89–4.93 (2H,
m), 6.92–6.94 (1H, m), 7.08 (1H, s), 7.22 (1H, s), 8.58 (1H, br).
High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
C27H41N6O4: 513.3189. Found: 513.3171.
1H NMR (400 MHz, CDCl3) d 0.96 (3H, d, J = 6.4 Hz), 1.12–1.28
(2H, m), 1.40–1.82 (13H, m), 2.02–2.17 (2H, m), 2.82–2.95 (2H,
m), 3.926 (3H, s), 3.933 (3H, s), 4.14–4.25 (1H, m), 4.70–4.80 (2H,
m), 6.05 (1H, br), 6.93 (1H, s), 6.97 (1H, s). High-resolution MS
(ESI+) m/z [M+H]+. Anal. Calcd for C23H35N4O2: 399.2760. Found:
399.2750.
6.1.32. N-(1-Benzylpiperidine-4-yl)-6,7-dimethoxy-2-(4-
pyrrolidin-1-ylpiperidine-1-yl)quinazolin-4-amine (C-011)
39 mg, 73%.
6.1.39. 1-[4-(Cycloheptylamino)-6,7-dimethoxyquinazolin-2-
yl]piperidine-4-ol (C-018)
23 mg, 57%.
1H NMR (400 MHz, CDCl3) d1.74–1.88 (4H, m), 1.95–2.02 (4H, m),
2.05–2.15 (4H, m), 2.31–2.42 (2H, m), 2.75–2.92 (2H, m), 2.95–3.04
(1H, m), 3.06–3.21 (5H, m), 3.72 (2H, s), 3.93 (6H, s), 4.10–4.13 (1H,
m), 4.88–4.91 (2H, m), 6.68–6.69 (1H, m), 7.02 (1H, s), 7.15 (1H, s),
7.30–7.43 (5H, m), 8.59 (1H, br). High-resolution MS (ESI+) m/z
[M+H]+. Anal. Calcd for C31H43N6O2: 531.3448. Found: 531.3432.
1H NMR (400 MHz, CDCl3) d 1.50–1.80 (12H, m), 1.92–2.03 (2H,
m), 2.08–2.18 (2H, m), 3.18–3.30 (2H, m), 3.85–4.00 (7H, m), 4.21–
4.33 (1H, m), 4.50–4.62 (2H, m), 5.03–5.15 (1H, m), 6.71 (1H, s),
6.91 (1H, s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd
for C22H33N4O3: 401.2553. Found: 401.2537.
6.1.40. 1-[4-(Cycloheptylamino)-6,7-dimethoxyquinazolin-2-
yl]piperidine-4-carboxamide (C-019)
6.1.33. 6,7-Dimethoxy-N,N-dimethyl-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazolin-4-amine (C-012)
2 mg, 5%.
18 mg, 47%.
1H NMR (400 MHz, DMSO-d6) d 1.39–1.78 (14H, m), 1.92–
2.03 (2H, m), 2.29–2.39 (1H, m), 2.74–2.86 (2H, m), 3.81
(3H, s), 3.82 (3H s), 4.15–4.23 (1H, m), 4.68–4.77 (2H, m),
6.71–6.74 (2H, br), 7.26 (1H, s), 7.46 (1H, s). High-resolution
MS (ESI+) m/z [M+H]+. Anal. Calcd for C23H34N5O3: 428.2662.
Found: 428.2648.
1H NMR (400 MHz, CDCl3) d 1.79–1.89 (2H, m), 1.95–2.08 (4H,
m), 2.10–2.18 (2H, m), 2.85–2.95 (2H, m), 3.09–3.28 (11H, m),
3.92 (3H, s), 3.99 (3H, s), 5.02–5.05 (2H, m), 7.15 (1H, s), 7.28
(1H, s), 8.53 (1H, br). High-resolution MS (ESI+) m/z [M+H]+. Anal.
Calcd for C21H32N5O2: 386.2556. Found: 386.2546.
6.1.34. 6,7-Dimethoxy-4-piperidine-1-yl-2-(4-pyrrolidin-1-
ylpiperidine-1-yl)quinazoline (C-013)
6.1.41. 2-(4-Benzylpiperidine-1-yl)-N-cycloheptyl-6,7-
dimethoxyquinazolin-4-amine (C-020)
26 mg, 61%.
24 mg, 51%.
1H NMR (400 MHz, CDCl3) d 1.68–1.90 (8H, m), 1.95–2.18 (6H,
m), 2.79–2.95 (2H, m), 3.05–3.30 (5H, m), 3.45–3.65 (4H, m),
1H NMR (400 MHz, CDCl3) d 1.18–1.35 (2H, m), 1.42–1.90
(13H, m), 2.00–2.19 (2H, m), 2.55 (2H, d, J = 6.8 Hz), 2.82–2.96

7030
K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
(2H, m), 3.90 (3H, s), 3.92 (3H, s), 4.07–4.18 (1H, m), 4.68–4.83
(2H, m), 7.01 (1H, s), 7.11–7.38 (6H, m). High-resolution MS
(ESI+) m/z [M+H]+. Anal. Calcd for C29H39N4O2: 475.3073. Found:
475.3059.
(ESI+) m/z [M+H]+. Anal. Calcd for C21H31N4O3: 387.2396. Found:
387.2406.
6.1.49. N4-Cycloheptyl-N2-isopropyl-6,7-
dimethoxyquinazoline-2,4-diamine (C-029)
6.1.42. N4-Cycloheptyl-6,7-dimethoxy-N2-(2-piperidine-1-
ylethyl)quinazoline-2,4-diamine (C-022)
3.1 mg, 9%.
1H NMR (400 MHz, CDCl3) d 1.31 (6H, d, J = 6.3 Hz), 1.50–1.85
(10H, m), 2.05–2.20 (2H, m), 3.94 (3H, s), 3.97 (3H, s), 4.19 (1H,
tt, J = 6.8 Hz), 4.28–4.40 (1H, m), 6.15–6.29 (1H, m), 6.89 (1H, s),
6.96 (1H, s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd
for C20H31N4O2: 359.2447. Found: 359.2442.
9.5 mg, 22%.
1H NMR (400 MHz, CDCl3) d 1.30–1.87 (16H, m), 2.00–2.20 (2H,
m), 2.40–2.93 (6H, m), 3.60–3.76 (2H, m), 4.00 (6H, s), 4.30–4.47
(1H, m), 6.88 (1H, s), 7.03 (1H, br), 7.17 (1H, s). High-resolution
MS (ESI+) m/z [M+H]+. Anal. Calcd for C24H38N5O2: 428.3026.
Found: 428.3015.
6.1.50. N4-Cycloheptyl-N2-cyclopentyl-6,7-
dimethoxyquinazoline-2,4-diamine (C-030)
13 mg, 34%.
6.1.43. N4-Cycloheptyl-6,7-dimethoxy-N2-(3-pyrrolidin-1-
ylpropyl)quinazoline-2,4-diamine (C-023)
1H NMR (400 MHz, CDCl3) d 1.48–1.85 (16H, m), 1.93–2.13 (4H,
m), 3.93 (3H, s), 3.96 (3H, s), 4.24–4.39 (2H, m), 6.92 (1H, s), 7.31
(1H, s). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
13 mg, 30%.
1H NMR (400 MHz, CDCl3) d 1.40–1.80 (10H, m), 1.89–2.18 (8H,
m), 2.98–3.20 (6H, m), 3.45–3.65 (2H, m), 3.94 (3H, s), 3.95 (3H, s),
4.25–4.38 (1H, m), 6.87 (1H, s), 7.05–7.17 (1H, br), 7.21 (1H, s).
High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for
C22H33N4O2: 385.2604. Found: 385.2596.
6.1.51. N2-Benzyl-N4-cycloheptyl-6,7-dimethoxyquinazoline-
2,4-diamine (C-031)
C24H38N5O2: 428.3026. Found: 428.3018.
19 mg, 47%.
1H NMR (400 MHz, CDCl3) d 1.38–1.72 (10H, m), 1.91–2.03 (2H,
m), 3.92 (3H, s), 3.93 (3H, s), 4.20–4.30 (1H, m), 4.62 (2H, d,
J = 5.9 Hz), 6.88 (1H, s), 7.08 (1H, br), 7.15–7.36 (6H, m). High-res-
olution MS (ESI+) m/z [M+H]+. Anal. Calcd for C24H31N4O2:
407.2447. Found: 407.2433.
6.1.44. 1-(3-{[4-(Cycloheptylamino)-6,7-dimethoxyquinazolin-
2-yl]amino}propyl)pyrrolidin-2-one (C-024)
4.9 mg, 11%.
1H NMR (400 MHz, CDCl3) d 1.49–1.82 (10H, m), 1.86–2.17 (6H,
m), 2.36 (2H, t, J = 8.3 Hz), 3.36–3.55 (4H, m), 3.94 (3H, s), 3.97 (3H,
s), 4.27–4.38 (1H, m), 6.30 (1H, br), 6.91 (2H, s). High-resolution
MS (ESI+) m/z [M+H]+. Anal. Calcd for C24H36N5O3: 442.2818.
Found: 442.2806.
6.1.52. N4-Cycloheptyl-6,7-dimethoxy-N2-(2-
thienylmethyl)quinazoline-2,4-diamine (C-032)
19 mg, 46%.
1H NMR (400 MHz, CDCl3) d 1.40–1.80 (10H, m), 1.98–2.12 (2H,
m), 3.87 (3H, s), 3.90 (3H, s), 4.37–4.40 (1H, m), 4.78 (2H, d,
J = 5.4 Hz), 6.79–6.93 (3H, m), 7.06–7.13 (1H, m), 7.33 (1H, s),
7.56 (1H, br). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd
for C22H29N4O2S: 413.2011. Found: 413.1996.
6.1.45. N-Cycloheptyl-6,7-dimethoxy-2-(4-methylpiperazine-1-
yl)quinazolin-4-amine (C-025)
21 mg, 53%.
1H NMR (400 MHz, CDCl3) d 1.43–1.84 (10H, m), 2.02–2.16 (2H,
m), 2.41 (3H, s), 2.55–2.73 (4H, m), 3.91 (3H, s), 3.92 (3H, s), 3.93–
4.00 (4H, m), 4.08–4.20 (1H, m), 6.99 (1H, s), 7.16 (1H, s). High-res-
olution MS (ESI+) m/z [M+H]+. Anal. Calcd for C22H34N5O2:
400.2713. Found: 400.2698.
6.1.53. N4-Cycloheptyl-6,7-dimethoxy-N2-(2-
phenylethyl)quinazoline-2,4-diamine (C-033)
20 mg, 48%.
6.1.46. N-Cycloheptyl-2-(4-isopropylpiperazine-1-yl)-6,7-
dimethoxyquinazolin-4-amine (C-026)
1H NMR (400 MHz, CDCl3) d 1.52–1.85 (10H, m), 2.07–2.17
(2H, m), 2.91–3.01 (2H, m), 3.65–3.78 (2H, m), 3.95 (3H, s),
3.99 (3H, s), 4.38 (1H, br), 6.84 (1H, s), 6.96 (1H, s). High-resolu-
tion MS (ESI+) m/z [M+H]+. Anal. Calcd for C25H33N4O2: 421.2604.
Found: 421.2597.
22 mg, 51%.
1H NMR (400 MHz, CDCl3) d 1.16 (6H, d, J = 6.4 Hz), 1.46–1.80
(10H, m), 2.00–2.15 (2H, m), 2.70–2.88 (4H, m), 2.95–3.08 (1H,
m), 3.920 (3H, s), 3.924 (3H, s), 3.97–4.08 (4H, m), 4.10–4.22 (1H,
m), 6.70 (1H, br), 6.99 (1H, s), 7.10 (1H, s). High-resolution MS
(ESI+) m/z [M+H]+. Anal. Calcd for C24H38N5O2: 428.3026. Found:
428.3015.
6.1.54. N4-Cycloheptyl-6,7-dimethoxy-N2-methyl-N2-(2-
phenylethyl)quinazoline-2,4-diamine (C-034)
24 mg, 55%.
1H NMR (400 MHz, CDCl3) d 1.45–1.80 (10H, m), 2.05–2.18 (2H,
m), 2.93 (2H, t, J = 7.3 Hz), 3.18 (3H, s), 3.82–4.00 (8H, m), 4.18–
4.29 (1H, m), 6.93 (1H, br), 7.05 (1H, s), 7.12–7.33 (6H, m). High-
resolution MS (ESI+) m/z [M+H]+. Anal. Calcd for C26H35N4O2:
435.2760. Found: 435.2741.
6.1.47. N-Cycloheptyl-6,7-dimethoxy-2-(4-phenylpiperazine-1-
yl)quinazolin-4-amine (C-027)
16 mg, 35%.
1H NMR (400 MHz, CDCl3) d 1.45–1.85 (10H, m), 2.07–2.19 (2H,
m), 3.21–3.34 (4H, m), 3.938 (3H, s), 3.944 (3H, s), 4.02–4.10 (4H,
m), 4.18–4.30 (1H, m), 6.36 (1H, br), 6.85–7.13 (5H, m), 7.25–
7.35 (2H, m). High-resolution MS (ESI+) m/z [M+H]+. Anal. Calcd
for C27H36N5O2: 462.2869. Found: 462.2861.
6.2. Pharmacology
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),
6.1.48. N-Cycloheptyl-6,7-dimethoxy-2-morpholin-4-
ylquinazolin-4-amine (C-028)
50
l
M 2-mercaptethanol, 100 U/mL penicillin, and 100
lg/mL
1.7 mg, 4%.
streptomycin. The expression vector pEF-BOS-Neo17
,
carrying
1H NMR (400 MHz, CDCl3) d 1.50–1.80 (10H, m), 2.07–2.17 (2H,
m), 3.77–3.90 (8H, m), 3.95 (3H, s), 3.96 (3H, s), 4.25–4.33 (1H, m),
5.02–5.10 (1H, m), 6.70 (1H, s), 6.91 (1H, s). High-resolution MS
full-length human CCR4 cDNA (X85740; GenBank) or mouse
CCR4 cDNA, (X90862; GenBank) was transfected into B300-19 cells

K. Yokoyama et al. / Bioorg. Med. Chem. 16 (2008) 7021–7032
7031
via electroporation to isolate stable G418-resistant stable
transformants.
sion (200 ll) was placed in the upper wells, and 3 nM human or
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
compound (for total migrating cells) or CCL22 (non-specific) were
used to calculate the percent of total inhibition for each set of com-
pounds. Assays were performed in triplicate at four different con-
centrations for each test compound.
6.2.2. [125I]CCL22-binding assay
CCR4-receptor-binding assays were performed using a scintilla-
tion proximity assay (SPA). Human CCR4-expressing cells
(2 ꢂ 105 cells/well) were incubated at 25 °C for 2 h with 25 pM
[
125I]CCL22 (PerkinElmer Life Sciences) and 5 mg/mL wheat germ
agglutinin SPA beads (GE Healthcare), with various concentrations
of test compounds in 100 l of binding buffer [50 mM Hepes (pH
l
6.3. Data analysis
7.4), 5 mM MgCl2, 1 mM CaCl2, and 0.1% (w/v) bovine serum albu-
min (BSA)]. Radioactivity was counted using a TopCount scintilla-
tion counter (Packard Biosciences). Control wells free from test
compound (for total counts) or containing excess unlabeled
CCL22 (10 nM, non-specific) were used to calculate the percent
of total inhibition for each set of compounds. Assays were per-
formed in duplicate at four different concentrations for each test
compound, and the value represents the average of two (usually)
determinations.
The concentration causing 50% inhibition (IC50) was determined
by non-linear curve fitting using the SAS system (SAS Institute,
Cary, NC, USA).
Acknowledgments
The authors acknowledge Dr. Ryo Naito and Dr. Hideki Kubota
for their helpful support during the preparation of this manuscript.
We are also grateful to the staff of the Division of Analytical Sci-
ence Laboratories for their help with elemental analysis and spec-
tral measurement.
6.2.3. [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
Healthcare), 2 M GDP, and 3 nM MDC with various concentra-
tions of test compounds in 200 l of binding buffer [20 mM
cS-binding assay
l
g/well protein)
S (GE
c
References and notes
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l
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(for total counts) or CCL22 (non-specific), were used to calculate
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were performed in duplicate at four different concentrations for
each test compound, and the value represents an average of usu-
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say.18 CCR4-expressing cells (5 ꢂ 104 cells/100
l
l/well) were incu-
bated in RPMI 1640 containing 10% FBS, 50
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100 U/mL penicillin, and 100
humidified 5% CO2 atmosphere, and treated with 0.3–10
compound. After 12 h, 10 L of Alamar Blue dye (Biosource) was
l
g/mL streptomycin at 37 °C in a
lM test
l
12. Jo, Y.; Matsumoto, T.; Yada, S.; Fujisawa, K.; Esaki, M.; Onai, N.; Matsushima, K.;
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added to each well. After incubation for 4 h, the plates were read
on a micro plate fluorescence reader (Spectra Max 190, Molecular
Devices) at excitation and emission wavelengths of 570 and
600 nm, respectively. One hundred percent viable control wells
containing no test compound were used to calculate the percent
of total inhibition for each set of compounds. The minimum cyto-
toxic dose was designated as the concentration required to reduce
the cell viability to less than 70%.
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6.2.5. Chemotaxis assay
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chambers (Neuro Probe)19 that had 5-
lm pores, polycarbonate fil-
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cS-
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