A novel structural class of potent inhibitors of NF-κB activation: Structure-activity relationships and biological effects of 6-aminoquinazoline derivatives

Article abstract of DOI:10.1016/S0968-0896(03)00438-3

In this study, we have investigated the roles of substituents on the terminal phenyl ring at the C(4)-position of the quinazoline core to complete the structure-activity relationships (SARs) of our NF-κB activation inhibitors. Among them, compound 12j aff

Full text of DOI:10.1016/S0968-0896(03)00438-3

Bioorganic & Medicinal Chemistry 11 (2003) 3869–3878
A Novel Structural Class of Potent Inhibitors of NF-ꢀB
Activation: Structure–Activity Relationships and Biological
Effects of 6-Aminoquinazoline Derivatives
Masanori Tobe, Yoshiaki Isobe, Hideyuki Tomizawa, Takahiro Nagasaki,
Hirotada Takahashi and Hideya Hayashi*
Research Division, Sumitomo Pharmaceuticals Co., Ltd., 1-98 Kasugade Naka 3-Chome,
Konohana-ku, Osaka 554-0022, Japan
Received 1 May 2003; accepted 23 June 2003
Abstract—In this study, we have investigated the roles of substituents on the terminal phenyl ring at the C(4)-position of the quin-
azoline core to complete the structure–activity relationships (SARs) of our NF-kB activation inhibitors. Among them, compound
12j afforded highly potent inhibitory activity toward NF-kB transcriptional activation with IC50 value of 2 nM, along with an
excellent in vivo efficacy by reducing the edema formation seen in carrageenin-induced inflammation of the rat hind paw.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
regulatory mediators that then leads to inflammatory
diseases, such as septic shock, psoriasis, asthma, and
rheumatoid arthritis.2
Nuclear factor kappa B (NF-kB) is composed of two
subunits and is normally sequestered in the cytoplasm
through being associated with an IkB protein. When the
cell is exposed to activation signals, such as endotoxin
or TNF-a binding to cell surface receptors, the IkB
protein is phosphorylated, then ubiquinated and broken
down in proteosomes. After being freed from associ-
ation with IkB, the NF-kB complex moves to the
nucleus where it binds to specific sequences in the pro-
moter/enhancer regions of genes.1
The key role that NF-kB plays in controlling the
expression of multiple inflammatory and immune genes
involved in above mentioned diseases makes this factor
a central and favorable target for therapeutic interven-
tion of diseases. From this view point, an inhibitor of
NF-kB activation would be expected to possess a
therapeutic potential in the treatment of inflammatory
diseases.3
Under baseline conditions, in the healthy human, NF-
kB functions in regulating the expression of genes
involved in normal immunologic responses, such as
generation of antibody light chains and other immu-
noregulatory molecules. Rapid mobilization of an acute
inflammatory response is necessary for defense against
external biologic assaults to the organism, such as bac-
terial infection. However, excessive activation of NF-kB
results in enhanced expression of pro-inflammatory
cytokines, such as TNF-a, and IL-1b, and immuno-
To our knowledge, several inhibitors of NF-kB tran-
scriptional activation have been previously reported,
MG-132 (1),4 an indan derivative (2),5 or BAY 11-7085
(3),6 which are illustrated in Figure 1. Recently, we
reported the identification of a series of 6-amino-
quinazoline derivatives represented by compounds 4a–
4e as the novel structural class of NF-kB activation
inhibitors (Fig. 2).7 Our initial structure–activity
relationship (SAR) studies revealed that the following
key elements were required for enhanced inhibitory
activity toward NF-kB activation: (1) a quinazoline ring
system, (2) a basic nitrogen as well as a smaller
substituents at the C(6)-position, and (3) an ethylene
chain as a spacer between the quinazoline ring and the
phenyl one at the C(4)-position.
*Corresponding author. Tel.: +81-6-6466-5189; fax: +81-6-6466-
5287; e-mail: hayashih@sumitomopharm.co.jp
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00438-3

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M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
Figure 1. Several inhibitors of NF-kB-mediated transcriptional activation: 1, MG-132; 2, indan derivative; 3, BAY 11-7085.
Figure 2. Structures of 6-amino-4-phenethylaminoquinazolines.
In the present study, we investigated further attempts to
refine the basic framework by replacing the phenyl ring,
which was connected with an ethylene chain at the C(4)-
position, with the cyclohexyl ring and several hetero-
cyclic rings, and by placing various substituents on the
phenyl ring (Fig. 2). Extensive optimization of these
new quinazoline derivatives led us to identify potent NF-
kB activation inhibitors, with nanomolar IC50’s toward
NF-kB transcriptional activation in human Jurkat cells.
Furthermore, selected analogues, such as 12d and 12j,
demonstrated excellent in vivo efficacy by reducing the
edema formation seen in carrageenin-induced inflam-
mation of the rat hind paw. Herein, we wish to describe
the SAR revealed on the way to the discovery of these
highly potent NF-kB activation inhibitors.
thranilic acid (5) with 28% ammonia in the presence of
1-ethyl-3-[(dimethylamino)propyl]carbodiimide (WSCD)
and 1-hydroxybenzotriazole (HOBt) followed by cycli-
zation with trimethyl orthoformate gave the 6-nitro-4-
quinazolone (7). Subsequently, chlorination of the 4-
quinazolone (7) with thionyl chloride gave the corre-
sponding 4-chloroquinazoline (8). Reaction of 8 with
appropriate phenethylamines in the presence of triethy-
lamine provided the corresponding 6-nitro-4-phenethy-
laminoquinazolines (9). Reduction of 9 with iron dust
gave the target compounds 10a–10e, 11a–11f, i, j, and
12a–12k.
Procedures for the preparation of 11g and 11h are
shown in Scheme 2. The acid (14) was obtained by
saponification of the methyl ester (13). Treatment of 14
with WSCD and HOBt followed by the addition of the
corresponding amines gave the amides 15 and 16.
Compounds 15 and 16 underwent iron reduction to
give the target 6-aminoquinazolines 11g and 11h,
respectively.
Chemistry
The general synthetic pathway to the 6-aminoquinazo-
lines is shown in Scheme 1. Condensation of 5-nitroan-
Scheme 1. General procedure for 4-substituted-6-aminoquinazolines. Reagents and conditions: (a) 28% aqueous ammonia solution, WSCD, HOBt,
DMF, 4 h; (b) HC(OMe)3, 12 N HCl, 1 h; (c) SOCl2, DMF, reflux, 3 h; (d) phenethylamines, triethylamine, i-PrOH, 2–3 h; (e) Fe, glacial acetic acid,
EtOH–H2O, reflux, 30min.

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
3871
Scheme 2. Synthesis of the 6-aminoquinazolines, 11g and 11h. Reagents and conditions: (a) 5 N NaOH, EtOH, 2 h; (b) 28% aqueous ammonia
solution or 40% Me2NH aqueous solution, WSCD, HOBt, DMF, 3–5 h; (c) Fe, glacial acetic acid, EtOH–H2O, reflux, 30min.
Results and Discussion
Table 1. Inhibition of NF-kB activation and TNF-a production by
compounds 10a–10e
The above quinazoline derivatives were synthesized and
evaluated for their inhibitory activities toward NF-kB
transcriptional activation in the luciferase reporter gene
assays featuring phorbol 12-myristate-13-acetate (PMA)
plus phytohemagglutin (PHA) stimulation of human
Jurkat T cells. Reporter gene assays were performed as
described in the Experimental. Inhibition of LPS-
induced TNF-a production was conducted in a manner
similar to that described previously using mouse spleno-
cytes.7 Cytotoxicity toward both human Jurkat cells
and murine splenocytes was measured by using the
MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay.
The results are summarized in Tables 1–3.
Compd
4a
R
NF-kBa Toxicityb TNF-ac Toxicityd
IC50 (nM) TC50 (nM) IC50 (nM) TC50 (nM)
311 >10,000
685 >10,000
1426 >10,000
3240 >10,000
10a
10b
8271 >10,000 >10,000 >10,000
With the aim to confirm the importance of the phenyl
ring at the C(4)-position in 4a, we prepared compounds
10a–10e by replacing the phenyl ring with the cyclohexyl
ring and several heterocycles, respectively. As shown in
Table 1, the cyclohexyl (10a) showed a 2-fold loss in the
inhibitory activities toward both NF-kB activation and
TNF-a production as compared with the phenyl ring
(4a). Moreover, the pyridyl (10b) showed a 27-fold loss
in the inhibition of NF-kB activation as well as led to
decreased inhibitory activity toward TNF-a production.
The morpholino (10c), the piperidino (10d), and pyrro-
lidino (10e) also exhibited decreased or diminished the
activities. These results suggest that the phenyl ring
would be preferable for the inhibitory activities toward
both NF-kB activation and TNF-a production.
10c
>10,000 >10,000
>10,000 >10,000
nt
nt
nt
nt
10d
10e
3694 >10,000 >10,000 >10,000
MG-132 (1)
715
1141
809
1893
nt, not tested.
aIC50 for the inhibition of NF-kB activation in human Jurkat cells
transfected with pNFkB-Luc.
bTC50 for the growth inhibition of human Jurkat cells.
cIC50 for the inhibition of TNF-a production from murine splenocytes
stimulated with LPS.
We next focused on the substitution studies on the
phenyl ring at the C(4)-position in more detail. As
shown in Table 2, the role of the para-chloro-
phenethylamine substitution pattern at the 4-position
dTC50 for the growth inhibition of mouse splenocytes stimulated with
LPS.

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M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
Table 2. Inhibition of NF-kB activation and TNF-a production by compounds 11a–11j
Compd
R1
R2
R3
NF-kBa
Toxicityb
TNF-ac
Toxicityd
IC50 (nM)
TC50 (nM)
IC50 (nM)
TC50 (nM)
11a
11b
4b
11c
11d
4d
11e
11f
11g
11h
11i
Cl
H
H
MeO
H
H
H
H
H
H
H
Cl
H
H
MeO
H
H
H
H
H
H
H
Cl
H
H
MeO
Br
254
389
176
155
210
90
207
574
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1141
275
243
142
312
366
279
128
510
nt
nt
nt
nt
809
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1893
F
CONH2
CONMe2
NHAc
SO2NH2
7333
2594
5979
>10,000
715
H
H
H
H
11j
MG-132 (1)
nt, not tested.
aÀdSee corresponding footnotes in Table 1.
was studied by comparison with the corresponding
meta- and ortho-analogues (4b vs 11a and 11b). Moving
the chloro atom from a para- to a ortho- or a meta-
position decreased the inhibitory activities toward both
NF-kB activation and TNF-a production. Moreover,
the same tendency was observed when the position of
the methoxy group on the phenyl ring was changed
from a para- to an ortho- or a meta-position (4d vs 11c
and 11d).
kB activation.7 Among these compounds, 4e showed the
most potent inhibitory activity toward both NF-kB
activation and TNF-a production. With these data in
mind, we focused on the bulky phenoxy analogue and
synthesized the related aryloxy or heteroaryloxy com-
pounds (12a–12c) listed in Table 3. The benzyloxy (12a),
which was added to 1 methylene unit in 4e, resulted in
an IC50 value comparable to that of 4e for the inhibitory
activity. As shown by the pyridylmethoxy (12b), intro-
duction of a nitrogen atom within the phenyl ring in
12a, displayed similar inhibitory activity toward NF-kB
activation over 12a, whereas this compound exhibited a
4-fold loss in TNF-a inhibitory activity. Replacement of
the monocyclic rings with the condensed ring provided
the quinolinylmethoxy (12c) which led to decreased
both inhibitory activities. Furthermore, substitution of
the phenoxy group in 4e with simplified substituents,
keeping an electron rich feature constant, afforded the
allyloxy (12d) which retained good inhibitory activity.
Although the propargyloxy (12e) somewhat reduced
NF-kB inhibitory activity, the high potency shown by
the allyloxy group prompted us to further investigate
such simplified alkoxy analogues in more detail (Table 3).
On the basis of these data, we performed a para-sub-
stitution study on the phenyl ring at the C(4)-position of
the quinazoline ring. The bromo atom (11e) was com-
parable to the chloro atom (4b) in their inhibitory
activities toward NF-kB activation, whereas the fluorine
atom (11f) resulted in a 3-fold loss in activity compared
with 4b. The same trend was observed when these com-
pounds were evaluated for TNF-a inhibitory activities.
The introduction of polar, hydrogen bonding groups
(11g–11j) had less potent inhibitory activity on NF-kB
activation. In particular, the sulfonamide (11j), which is
an electron-withdrawing substituent similar to the
fluorine atom (11f), led to a substantial decrease in
activity. From these results, it was found that placement
of a hydrophilic group as well as an electron-with-
drawing one on the phenyl ring was unfavorable for
increasing activity.
The ethoxy (12f) exhibited a 4.5- and 16-fold improve-
ment of the inhibitory activities toward NF-kB activa-
tion and TNF-a production, respectively, compared
with the methoxy (4d). The n-propoxy (12g) showed a
potent inhibitory activity toward NF-kB activation
similar to 12f, whereas the iso-propyl (12h) displayed a
loss of its inhibitory activity. From these results, we
considered the hypothesis that the enhancement of
lipophilicity would lead to an active compound.
Next, we investigated the effect of an electron-donating
group on the phenyl ring. In our previous paper, we
reported that the electron-donating groups, such as the
methyl (4c), the methoxy (4d), and the phenoxy (4e)
analogues, were found to be potent inhibitors for NF-

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
3873
Table 3. Inhibition of NF-kB activation and TNF-a production by compounds 12a–12k
Compd
4e
R
NF-kBa
IC50 (nM)
11
Toxicityb
TC50 (nM)
>10,000
TNF-ac
IC50 (nM)
7
Toxicityd
TC50 (nM)
>1000
12a
12b
12c
14
12
93
12
40
>10,000
>10,000
>10,000
>10,000
>10,000
9
41
>1000
>1000
>1000
>1000
>1000
100
18
12d
12e
59
4c
4d
12f
12g
12h
12i
12j
12k
Me
MeO
EtO
n-PrO
i-PrO
n-BuO
n-PenO
n-HexO
210
90
21
20
40
11
2
4
715
38
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1141
138
279
17
15
23
4
3
8
809
84
>10,000
>10,000
>1000
>1000
>1000
>1000
>1000
>1000
1893
MG-132 (1)
2
>10,000
>1000
aÀdSee corresponding footnotes in Table 1.
Therefore, in order to increase the lipophilicity, we pre-
pared larger alkoxy analogues, such as n-butoxy (12i),
n-pentyloxy (12j), and n-hexyloxy (12k) analogues. The
n-butoxy (12i) led to a 2-fold increase in the NF-kB
inhibitory activity as compared with that of 12g. Fur-
thermore, compounds 12j and 12k exhibited highly
potent inhibitory activities toward both NF-kB activa-
tion and TNF-a production, respectively. As we expec-
ted, the enhancement of lipophilicity of compounds 12i–
12k compared with that of 4d reflects these greater
inhibitory activities.
comparative test of 4e and the selected compounds
using the same model when these compounds were tes-
ted at a ip dose of 1 mg/kg. The results of the reducing
effects on edema formation for these compounds were
summarized in Figure 3. Compounds 12a, 12d, and 12j
inhibited edema formation and showed more potent
suppressing effects than 2. Suppressing effect of the
benzyloxy analogue (12a) on edema formation was
comparable to that of 4e. The allyloxy analogue (12d)
displayed a more potent suppressing effect than 12a.
Moreover, the n-pentyloxy (12j) afforded low nanomo-
lar IC50’s toward NF-kB activation and TNF-a pro-
duction along with further enhancement of in vivo
activity. It has been reported that the activation of NF-
kB may be involved in a pathogenesis by which inflam-
mation is accelerated in the carrageenin-induced paw
edema model.8 Therefore, we consider that the above-
mentioned compounds have a reducing effect of NF-kB
mediated-inflammatory response.
Finally, we selected three compounds (12a, 12d, and 12j)
from the results of the above-mentioned in vitro study
to further evaluate their anti-inflammatory effects. In
our previous study, compound 4e showed a more potent
suppressing effect on edema formation than the indan
derivative (2) in a rat carrageenin-induced paw edema
model.7 On the basis of these data, we performed the

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M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
BUCHI 535 melting point apparatus and were uncor-
1
rected. H NMR was recorded on a JEOL GSX270FT
NMR spectrometer. Chemical shifts were given in parts
per million (ppm) using tetramethylsilane as the internal
standard for spectra obtained in DMSO-d6 and CDCl3.
TOF MS (time-of-flight mass spectrometry) was recor-
ded on a Kompact MALDI 3 V 4.0.0 spectrometer.
High-resolution mass spectra were obtained on a JEOL
JMS-700 mass spectrometer. Elemental analyses were
performed at the Toray Research Center. Monitoring of
reactions was carried out using Merck 60F 254 silica gel,
glass-supported TLC plates, and visualization with UV
light (254 and 365 nm). Following abbreviations are
used for solvents: AcOH (acetic acid), DMF (N,N-
dimethylformamide), AcOEt (ethyl acetate), i-PrOH (2-
propanol).
6-Amino-4-(4-bromo)phenethylaminoquinazoline (11e). A
suspension of 6-nitro-4-quinazolone (7)7 (300 mg,
1.57 mmol) in thionyl chloride (10mL) containing 1
drop of DMF was heated at reflux for 3 h. After cooling
to ambient temperature, excess thionyl chloride was
removed under reduced pressure to give crude 4-chloro-
6-nitroquinazoline (8), which was used directly. To a
mixture of 8 and triethylamine (262 mL, 1.88 mmol) in
i-PrOH (15 mL) was added (4-bromo)phenethylamine
(292 mL, 1.88 mmol). The resulting mixture was stirred
at ambient temperature for 2 h and then was con-
centrated under reduced pressure. The residue was par-
titioned between CH2Cl2 and 5% aqueous citric acid
solution. The organic layer was washed with 1 N NaOH
and brine, and then dried over Na2SO4. The solution
was concentrated under reduced pressure, and then the
residue was triturated with CH2Cl2/hexane (1:1). The
light yellow solid was filtered to give 4-(4-bromo)phe-
nethylamino-6-nitroquinazoline (9: R1=4-bromophe-
nyl) (373 mg, 64% yield for two steps from 7). Next,
iron powder (60mg) was added to a refluxing solution
of the above 6-nitroquinazoline (100 mg, 0.27 mmol) in
EtOH/H2O (2:1, 12 mL) containing glacial AcOH
(185 mL). The resulting suspension was heated at reflux
with vigorous stirring for 30min, then cooled, basified
with concentrated NH4OH solution and EtOH, and
combined filtrate was evaporated under reduced pres-
sure, diluted with water, and extracted with AcOEt. The
organic layer was dried over Na2SO4 and concentrated
under reduced pressure. The residue was triturated with
AcOEt/hexane (1:1). The light yellow solid was ꢀfiltered
Figure 3. Effects of 12a, 12d, and 12j on carrageenin-induced paw
edema in rats. Test compounds were intraperitoneally administered
15 min prior to the injection of carrageenin. The resulting edema was
quantified 2 h later by measuring the increase in the volume of the
inflamed paw. The results are expressed as the meanÆSEM of five rats
per group. *p <0.05; **p <0.01; ***p <0.001 versus vehicle control
(Dunnett’s test).
Conclusions
In this study we have investigated the roles of sub-
stituents on the terminal phenyl ring at the C(4)-posi-
tion of the quinazoline ring to complete the SAR of our
NF-kB activation inhibitors. Since replacement of the
phenyl ring with the cyclohexyl ring and heterocycles
(10a–10e) reduced activity, we established that the best
basic framework was the 6-amino-4-phenethylamino-
quinazoline skeleton. The para substitution pattern on
the phenyl ring at the C(4)-position was suitable for
potent inhibitory activities toward both NF-kB activa-
tion and TNF-a production. Introduction of the
hydrophilic groups (11g–11j) on the phenyl ring reduced
the activity, whereas that of the larger alkoxy groups
(12i–12k) was found to be optimal for potent activity. In
particular, the n-pentyloxy (12j) afforded highly potent
inhibitory activities toward both NF-kB activation and
TNF-a production, with IC50 values of 2 and 3 nM,
respectively, along with an excellent in vivo efficacy by
reducing the edema formation seen in carrageenin-
induced inflammation of the rat hind paw. Therefore,
the representative compound 12j is expected to be a
candidate drug for use in the therapy of various inflam-
matory diseases. The details of pharmacological studies
on compound 12j and related compounds are currently
underway.
1
to give (11e) (30mg, 32% yield): mp 112–114 C; H
NMR (DMSO-d6) d 8.21 (s, 1H), 7.76 (t, J=5.3 Hz,
1H), 7.47 (d, J=8.4 Hz, 2H), 7.41 (d, J=8.6 Hz, 1H),
7.22 (d, J=8.4 Hz, 2H), 7.12 (dd, J=2.4, 8.6 Hz, 1H),
6.99 (d, J=2.4 Hz, 1H), 5.41 (br s, 2H), 3.68 (dt, J=5.3,
7.2 Hz, 2H), 2.92 (t, J=7.2 Hz, 2H); MS (TOF) m/z 343
+
.
(M+H) . Anal. calcd for C16H15BrN4 0.5H2O: C,
54.56; H, 4.58; N, 15.91. Found: C, 54.66; H, 4.59; N,
15.67.
Experimental
Chemistry
6-Amino-4-[2-(cyclohexyl)ethylamino]quinazoline (10a).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 10a was obtained as a light yellow solid (39%
General. All reagents and solvents were obtained from
commercial suppliers and were used without further
purification. Melting points were measured with a
yield for three steps from 7): mp 130–132 ꢀC; H NMR
1

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
3875
(DMSO-d6) d 9.12 (t, J=5.0Hz, 1H), 8.51 (s, 1H), 7.54
(d, J=8.6 Hz, 1H), 7.27 (dd, J=2.2, 8.6 Hz, 1H), 7.21
(d, J=2.2 Hz, 1H), 5.89 (br s, 2H), 3.67–3.60(m, 2H),
1.77–1.51 (m, 7H), 1.37–1.10 (m, 4H), 0.99–0.86 (m,
2H); HR-FABMS m/z (M+H)+; calcd for C16H22N4:
271.1923. Found: 271.1919.
from 7. After purification, 10e was obtained as a white
solid (53% yield for four steps from 7): mp 162–164 ꢀC;
1H NMR (DMSO-d6) d 10.21 (t, J=5.3 Hz, 1H), 8.74 (s,
1H), 7.80(d, J=8.9 Hz, 1H), 7.59 (d, J=1.9 Hz, 1H),
7.49 (dd, J=8.9, 1.9 Hz, 1H), 4.06–4.04 (m, 2H), 3.72–
3.65 (m, 2H), 3.56–3.40(m, 2H), 3.11–3.0(m, 2H),
2.03–1.87 (m, 4H); MS (TOF) m/z 258 (M+H)+. Anal.
.
6-Amino-4-[2-(3-pyridyl)ethylamino]quinazoline hydro-
chloride (10b). Similarly to the procedure described for
11e, 4-[2-(3-pyridyl)ethylamino]-6-nitroquinazoline (9:
R1=3-pyridyl) was prepared starting from 7. A mixture
of the above 6-nitroquinazoline (150mg, 0.51 mmol)
and 5% palladium on carbon (15 mg) in MeOH (20mL)
containing 12 N HCl (55 mL) was stirred under H2
atmosphere. After 5 h at ambient temperature, the cat-
alyst was filtered off over a pad of Celite, and the pad
was washed with MeOH. The combined filtrates were
concentrated under reduced pressure to give crude
compound, which was used directly. To a mixture of the
crude compound in EtOH (5 mL) was added 12 N HCl
(64 mL). The mixture was stirred at ambient temperature
for 3 h, and then concentrated under reduced pressure.
The residue was triturated with diethyl ether, and the
precipitated solid was collected by filtration. The
obtained solid was dried in vacuo to give the hydro-
chloride salt as a pale yellow powder (153 mg, 38% yield
calcd for C14H20ClN5 5.2H2O: C, 43.40; H, 6.56; N,
18.07. Found: C, 43.34; H, 6.35; N, 17.79.
6-Amino - 4 - (2 - chloro)phenethylaminoquinazoline (11a).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 11a was obtained as a light yellow solid (28%
yield for three steps from 7): mp 178–180 ꢀC; H NMR
1
(DMSO-d6) d 8.30(s, 1H), 8.20(t, J=4.9 Hz, 1H), 7.46–
7.40(m, 2H), 7.36–7.33 (m, 1H), 7.30–7.24 (m, 2H), 7.17
(dd, J=2.3, 9.0Hz, 1H), 7.05 (d, J=2.3 Hz, 1H), 5.55
(br s, 2H), 3.76 (dt, J=4.9, 7.2 Hz, 2H), 3.09 (t,
J=7.2 Hz, 2H); MS (TOF) m/z 299 (M+H)+. Anal.
.
calcd for C16H15ClN4 0.7H2O: C, 61.72; H, 5.08; N,
17.79. Found: C, 62.02; H, 5.09; N, 17.72.
6-Amino - 4 - (3 - chloro)phenethylaminoquinazoline (11b).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 11b was obtained as a light yellow solid (53%
for 4 steps from 7): mp 277–278 ꢀC; H NMR (DMSO-
1
yield for three steps from 7): mp 101–103 ꢀC; H NMR
1
d6) d 9.94 (t, J=5.4 Hz, 1H), 8.93 (s, 1H), 8.81 (d,
J=5.4 Hz, 1H), 8.65 (s, 1H), 8.52 (d, J=8.1 Hz, 1H),
8.03–8.00 (m, 1H), 7.71 (d, J=8.6 Hz, 1H), 7.42–7.32
(m, 2H), 4.05–3.98 (m, 2H), 3.25 (t, J=6.3 Hz, 2H); MS
(TOF) m/z 266 (M+H)+. Anal. calcd for
(DMSO-d6) d 8.21 (s, 1H), 7.78 (t, J=5.3 Hz, 1H), 7.41
(d, J=8.9 Hz, 1H), 7.33–7.20(m, 4H), 7.12 (dd, J=2.4,
8.9 Hz, 1H), 7.00 (d, J=2.4 Hz, 1H), 5.42 (br s, 2H),
3.71 (dt, J=5.3, 7.2 Hz, 2H), 2.96 (t, J=7.2 Hz, 2H);
MS (TOF) m/z 299 (M+H)+. Anal. calcd for
.
C15H16ClN5 4.2H2O: C, 47.73; H, 5.02; N, 18.34.
Found: C, 47.73; H, 5.39; N, 18.56.
.
C16H15ClN4 1.0H2O: C, 60.66; H, 5.41; N, 17.69.
Found: C, 60.34; H, 5.39; N, 17.72.
6-Amino - 4 - [2 - (1 - morpholinyl)ethylamino]quinazoline
hydrochloride (10c). Similarly to the procedure descri-
bed for 10b, the title compound was prepared starting
from 7. After purification, 10c was obtained as a white
solid (61% yield for four steps from 7): mp 221–223 ꢀC;
1H NMR (DMSO-d6) d 10.06 (t, J=5.3 Hz, 1H), 8.70(s,
1H), 7.74 (d, J=8.9 Hz, 1H), 7.40–7.36 (m, 2H), 4.11–3.92
(m, 6H), 3.53–3.37 (m, 6H); MS (TOF) m/z 274 (M+H)+.
6-Amino-4-(2-methoxy)phenethylaminoquinazoline (11c).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 11c was obtained as a light yellow solid (46%
yield for three steps from 7): mp 167–169 ꢀC; H NMR
1
(DMSO-d6) d 8.25 (s, 1H), 7.94 (t, J=5.3 Hz, 1H), 7.42
(d, J=8.6 Hz, 1H), 7.33–7.12 (m, 3H), 7.05 (d,
J=2.4 Hz, 1H), 6.98–6.95 (m, 1H), 6.89–6.83 (m, 1H),
5.47 (br s, 2H), 3.79 (s, 3H), 3.72–3.64 (m, 2H), 2.94 (t,
J=7.3 Hz, 2H); HR-FABMS m/z (M+H)+; calcd for
C17H18N4O: 295.1559. Found: 295.1555.
.
Anal. calcd for C14H20ClN5O 3.5H2O: C, 45.10; H, 6.35;
N, 18.78. Found: C, 45.49; H, 6.56; N, 18.45.
6-Amino - 4 - [2 - (1 - piperidinyl)ethylamino]quinazoline
hydrochloride (10d). Similarly to the procedure descri-
bed for 10b, the title compound was prepared starting
from 7. After purification, 10d was obtained as a white
solid (54% yield for four steps from 7): mp 183–185 ꢀC;
1H NMR (DMSO-d6) d 10.18 (t, J=5.3 Hz, 1H), 8.72 (s,
1H), 7.76 (d, J=8.9 Hz, 1H), 7.52 (d, J=2.2 Hz, 1H),
7.44 (dd, J=8.9, 2.2 Hz, 1H), 4.10–4.08 (m, 2H), 3.59–
3.55 (m, 2H), 3.41–3.35 (m, 2H), 3.02–2.88 (m, 2H),
6-Amino-4-(3-methoxy)phenethylaminoquinazoline (11d).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 11d was obtained as a light yellow solid (42%
yield for three steps from 7): mp 166–168 ꢀC; H NMR
1
(DMSO-d6) d 8.40(t, J=5.7 Hz, 1H), 8.36 (s, 1H), 7.46
(d, J=8.9 Hz, 1H), 7.24–7.17 (m, 2H), 7.08 (d,
J=2.4 Hz, 1H), 6.84–6.75 (m, 3H), 5.63 (br s, 2H), 3.80–
3.71 (m, 2H), 3.67 (s, 3H), 2.94 (t, J=7.3 Hz, 2H); HR-
FABMS m/z (M+H)+; calcd for C17H18N4O:
295.1559. Found: 295.1564.
1.86–1.68 (m, 5H), 1.47–1.32 (m, 1H); MS (TOF) m/z
+
.
272 (M+H) . Anal. calcd for C15H22ClN5 5.0H2O: C,
45.28; H, 6.84; N, 17.60. Found: C, 45.22; H, 6.81; N,
17.56.
6-Amino - 4 - [2 - (1 - pyrrolidinyl)ethylamino]quinazoline
hydrochloride (10e). Similarly to the procedure descri-
bed for 10b, the title compound was prepared starting
6-Amino-4- (4- fluorophenethylamino)quinazoline hydro-
chloride (11f). Similarly to the procedure described for
10b, the title compound was prepared starting from 7.

3876
M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
After purification, 11f was obtained as a white solid
(47% yield for four steps from 7): mp 231–233 ꢀC; H
6-Amino-4-[4-(2 - quinolylmethyloxy)]phenethylamino]
1
quinazoline (12c). Similarly to the procedure described
for 11e, the title compound was prepared starting from
7. After purification, 12c was obtained as a light yellow
solid (44% yield for three steps from 7): mp 211–213 ꢀC;
1H NMR (DMSO-d6) d 9.31 (t, J=4.6 Hz, 1H), 8.55 (s,
1H), 8.41 (d, J=8.4 Hz, 1H), 8.03–7.98 (m, 2H), 7.82–
7.76 (m, 1H), 7.67–7.53 (m, 3H), 7.31–7.27 (m, 1H),
7.21–7.17 (m, 3H), 7.00 (d, J=8.4 Hz, 2H), 5.94 (br s,
2H), 5.34 (s, 2H), 3.84–3.77 (m, 2H), 2.91 (t, J=7.3 Hz,
2H); MS (TOF) m/z 422 (M+H)+. Anal. calcd for
NMR (DMSO-d6) d 9.87 (t, J=5.3 Hz, 1H), 8.66 (s,
1H), 7.67 (d, J=8.9 Hz, 1H), 7.41–7.27 (m, 4H), 7.16–
7.08 (m, 2H), 3.92–3.85 (m, 2H), 3.00 (t, J=7.2 Hz, 2H);
MS (TOF) m/z 283 (M+H)+. Anal. calcd for
.
C16H16ClFN4 2.0H2O: C, 54.16; H, 5.11; N, 15.79.
Found: C, 54.12; H, 5.11; N, 15.79.
4-(4-Acetamido)phenethylamino - 6 - aminoquinazoline
(11i). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 11i was obtained as a white solid (41%
.
C26H23N5O 3.0H2O: C, 65.67; H, 5.51; N, 14.72.
Found: C, 65.48; H, 5.65; N, 14.37.
yield for three steps from 7): mp 218–220 ꢀC; H NMR
1
(DMSO-d6) d 9.86 (s, 1H), 8.21 (s, 1H), 7.75 (t,
J=5.3 Hz, 1H), 7.48 (d, J=8.4 Hz, 2H), 7.41 (d,
J=8.9 Hz, 1H), 7.17 (d, J=8.4 Hz, 2H), 7.14–7.10(m,
1H), 7.01 (d, J=2.2 Hz, 1H), 5.41 (br s, 2H), 3.66 (dt,
J=5.3, 7.3 Hz, 2H), 2.88 (t, J=7.3 Hz, 2H), 2.02 (s,
3H); MS (TOF) m/z 322 (M+H)+. Anal. calcd for
4-(4-Allyloxy)phenethylamino-6-aminoquinazoline (12d).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 12d was obtained as a light yelloꢀw solid (17%
1
yield for three steps from 7): mp 207–209 C; H NMR
(DMSO-d6) d 9.68 (t, J=5.1 Hz, 1H), 8.63 (s, 1H), 7.59
(d, J=8.9 Hz, 1H), 7.33 (dd, J=2.2, 8.9 Hz, 1H), 7.23
(d, J=2.2 Hz, 1H), 7.17 (d, J=8.6 Hz, 2H), 6.87 (d,
J=8.6 Hz, 2H), 6.09–5.95 (m, 3H), 5.41–5.21 (m, 2H),
4.54–4.51 (m, 2H), 3.88–3.80(m, 2H), 2.92 (t,
J=7.4 Hz, 2H); MS (TOF) m/z 321 (M+H)+. Anal.
.
C18H19N5O 1.7H2O: C, 61.42; H, 5.93; N, 19.90.
Found: C, 61.57; H, 6.15; N, 19.80.
6-Amino - 4 - (4 - sulfonamido)phenethylaminoquinazoline
(11j). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 11j was obtained as a light yellow solid
(15% yield for three steps from 7): mp 233–235 ꢀC;
.
calcd for C19H20N4O 3.5H2O: C, 59.51; H, 6.18; N,
14.61. Found: C, 59.26; H, 6.31; N, 14.71.
1H NMR (DMSO-d6)
d
8.22 (s, 1H), 7.81 (t,
6-Amino- 4- (4 -propargyloxy)phenethylaminoquinazoline
(12e). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 12e was obtained as a light yellow solid
J=5.1 Hz, 1H), 7.74 (d, J=8.4 Hz, 2H), 7.46–7.40(m,
3H), 7.29 (br s, 2H), 7.15–7.11 (m, 1H), 7.00 (d,
J=2.2 Hz, 1H), 5.43 (br s, 2H), 3.72 (dt, J=5.1, 7.0Hz,
2H), 3.03 (t, J=7.0Hz, 2H); MS (TOF) m/z 344
(25% yield for three steps from 7): mp 201–203 ꢀC; H
1
+
.
(M+H) . Anal. calcd for C16H17N5O2S 0.8H2O: C,
53.71; H, 5.01; N, 19.57. Found: C, 53.57; H, 5.17; N,
19.30.
NMR (DMSO-d6) d 9.69 (t, J=5.1 Hz, 1H), 8.63 (s,
1H), 7.61 (d, J=8.9 Hz, 1H), 7.33 (dd, J=2.2, 8.9 Hz,
1H), 7.24–7.18 (m, 3H), 6.91 (d, J=8.4 Hz, 2H), 6.06 (br
s, 2H), 4.75 (d, J=2.2 Hz, 2H), 3.88–3.81 (m, 2H), 3.56
6-Amino - 4 - (4 - benzyloxy)phenethylaminoquinazoline
(12a). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 12a was obtained as a light yellow solid
(t, J=2.2 Hz, 1H), 2.93 (t, J=7.2 Hz, 2H); MS (TOF)
+
.
m/z 319 (M+H) . Anal. calcd for C19H18N4O 3.2H2O:
C, 60.69; H, 5.68; N, 14.90. Found: C, 60.50; H, 5.71; N,
14.97.
ꢀ
1
(17% yield for three steps from 7): mp 208–210 C; H
NMR (DMSO-d6) d 9.66 (t, J=5.0Hz, 1H), 8.63 (s,
1H), 7.59 (d, J=8.9 Hz, 1H), 7.44–7.31 (m, 6H), 7.23–
7.16 (m, 3H), 6.94 (d, J=8.6 Hz, 2H), 6.06 (br s, 2H),
5.06 (s, 2H), 3.87–3.80 (m, 2H), 2.92 (t, J=7.2 Hz, 2H);
MS (TOF) m/z 371 (M+H)+. Anal. calcd for
6-Amino - 4 - (4 - ethoxy)phenethylaminoquinazoline (12f).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 12f was obtained as a light yellow solid (8%
yield for three steps from 7): mp 243–245 ꢀC; H NMR
1
.
C23H22N4O 3.6H2O: C, 63.46; H, 5.93; N, 12.87.
Found: C, 63.71; H, 5.94; N, 12.52.
(DMSO-d6) d 9.69 (t, J=5.0Hz, 1H), 8.64 (s, 1H), 7.58
(d, J=9.2 Hz, 1H), 7.33 (dd, J=1.9, 9.2 Hz, 1H), 7.22
(d, J=1.9 Hz, 1H), 7.16 (d, J=8.6 Hz, 2H), 6.84 (d,
J=8.6 Hz, 2H), 6.07 (br s, 2H), 3.97 (q, J=7.0Hz, 2H),
3.88–3.80(m, 2H), 2.92 (t, J=7.3 Hz, 2H), 1.30(t,
J=7.0Hz, 3H); MS (TOF) m/z 309 (M+H)+. Anal.
6-Amino-4-[4-(2-pyridylmethyloxy)]phenethylamino]qui-
nazoline (12b). Similarly to the procedure described for
11e, the title compound was prepared starting from 7.
After purification, 12b was obtained as a light yellow
solid (16% yield for three steps from 7): mp 173–174 ꢀC;
1H NMR (DMSO-d6) d 9.65 (t, J=5.0Hz, 1H), 8.63 (s,
1H), 8.58–8.56 (m, 1H), 7.86–7.80(m, 1H), 7.58 (d,
J=8.9 Hz, 1H), 7.49 (d, J=8.9 Hz, 1H), 7.36–7.30(m,
2H), 7.22–7.17 (m, 3H), 6.95 (d, J=8.6 Hz, 2H), 6.06 (br
s, 2H), 5.14 (s, 2H), 3.85–3.83 (m, 2H), 2.92 (t,
J=7.2 Hz, 2H); MS (TOF) m/z 372 (M+H)+. Anal.
.
calcd for C18H20N4O 2.1H2O: C, 62.45; H, 6.43; N,
16.18. Found: C, 62.36; H, 6.13; N, 16.30.
6-Amino - 4 - (4 - n - propoxy)phenethylaminoquinazoline
(12g). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 12g was obtained as a light yellow solid
(20% yield for three steps from 7): mp 127–129 ꢀC; H
1
.
calcd for C22H21N5O 3.2H2O: C, 61.58; H, 5.68; N,
16.32. Found: C, 61.72; H, 5.59; N, 16.03.
NMR (DMSO-d6) d 8.24 (s, 1H), 7.88 (t, J=5.1 Hz,
1H), 7.42 (d, J=8.9 Hz, 1H), 7.17–7.12 (m, 3H), 7.03 (d,

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
3877
J=2.2 Hz, 1H), 6.84 (d, J=8.6 Hz, 2H), 5.45 (br s, 2H),
3.88 (t, J=6.6 Hz, 2H), 3.70–3.63 (m, 2H), 2.87 (t,
J=7.4 Hz, 2H), 1.74–1.64 (m, 2H), 0.96 (t, J=7.6 Hz,
3H); MS (TOF) m/z 323 (M+H)+. Anal. calcd for
C19H22N4O 0.8H2O: C, 67.75; H, 6.82; N, 16.63.
Found: C, 67.63; H, 6.84; N, 16.39.
4-(4-Carboxy)phenethylamino - 6 - nitroquinazoline (14).
Similarly to the procedure described for 11e, 4-(4-meth-
oxycarbonyl)phenethylamino - 6 - nitroquinazoline (13)
was prepared starting from 7. A solution of 13 (1.30g,
3.69 mmol) in EtOH (34 mL) containing 5 N NaOH
(3.4 mL) was stirred at ambient temperature for 2 h. The
reaction mixture was neutralized with 6 N HCl and
concentrated under reduced pressure. The residue was
triturated with MeOH–H2O (1:1, v/v), and then the
yellow solid was filtered to give 14 (1.05 g, 84% yield);
.
6-Amino - 4 - (4 - iso - propoxy)phenethylaminoquinazoline
(12h). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 12h was obtained as a light yellow solid
mp, 290–292 ꢀC; H NMR (DMSO-d6) d 12.82 (br s,
1
(13% yield for three steps from 7): mp 213–215 ꢀC; H
1H), 9.33 (d, J=2.4 Hz, 1H), 9.12 (t, J=5.4 Hz, 1H),
8.63 (s, 1H), 8.48 (dd, J=2.4, 9.2 Hz, 1H), 7.89–7.82 (m,
3H), 7.40(d, J=8.4 Hz, 2H), 3.84 (dt, J=5.4, 7.3 Hz,
2H), 3.07 (t, J=7.3 Hz, 2H); HR-FABMS m/z
(M+H)+; calcd for C17H14N4O4: 339.1093. Found:
339.1096.
1
NMR (DMSO-d6) d 9.46 (t, J=5.0Hz, 1H), 8.58 (s,
1H), 7.57 (d, J=8.9 Hz, 1H), 7.30(dd, J=2.2, 8.9 Hz,
1H), 7.20(d, J=2.2 Hz, 1H), 7.15 (d, J=8.6 Hz, 2H),
6.83 (d, J=8.6 Hz, 2H), 5.99 (br s, 2H), 4.59–4.50(m,
1H), 3.85–3.78 (m, 2H), 2.90(t, J=7.4 Hz, 2H), 1.23 (d,
J=5.9 Hz, 6H); MS (TOF) m/z 323 (M+H)+. Anal.
.
calcd for C19H22N4O 3.0H2O: C, 60.62; H, 6.69; N,
14.88. Found: C, 60.81; H, 6.62; N, 14.97.
6-Amino - 4 - (4 - carboxamido)phenethylaminoquinazoline
(11g). To a solution of 14 (150mg, 0.44 mmol) and
HOBt (65 mg, 0.48 mmol) in DMF (9 mL) was
added WSCD (92 mg, 0.48 mmol). The mixture was
stirred at ambient temperature for 1 h. The resulting
solution was cooled to 0 ꢀC, then 28% ammonia
solution was added and temperature was allowed to
rise to ambient temperature. After 1 h, the reaction
mixture was poured into CH2Cl2/5% aqueous NaHCO3
solution (1:1), and then the precipitated solid was
collected by filtration. The obtained solid was
washed with water, and then dried in vacuo to give
15 as a pale yellow solid (134 mg, 91% yield): 1H
NMR (DMSO-d6) d 9.34 (d, J=2.4 Hz, 1H), 9.12 (t,
J=5.3 Hz, 1H), 8.63 (s, 1H), 8.48 (dd, J=2.4, 9.2 Hz,
1H), 7.91 (br s, 1H), 7.85–7.80(m, 3H), 7.35 (d,
J=8.1 Hz, 2H), 7.30(br s, 1H), 3.83 (dt, J=5.3, 7.2 Hz,
2H), 3.05 (t, J=7.3 Hz, 2H); MS (TOF) m/z 338
(M+H)+.
6-Amino-4-(4-n-butoxy)phenethylaminoquinazoline (12i).
Similarly to the procedure described for 11e, the title
compound was prepared starting from 7. After puri-
fication, 12i was obtained as a light yellow solid (24%
ꢀ
1
yield for three steps from 7): mp 177–179 C; H NMR
(DMSO-d6) d 8.46 (br s, 1H), 8.37 (s, 1H), 7.47 (d,
J=8.9 Hz, 1H), 7.22–7.09 (m, 4H), 6.84 (d, J=8.6 Hz,
2H), 5.65 (br s, 2H), 3.91 (t, J=6.5 Hz, 2H), 3.75–3.68
(m, 2H), 2.88 (t, J=7.3 Hz, 2H), 1.72–1.62 (m, 2H),
1.49–1.35 (m, 2H), 0.92 (t, J=7.3 Hz, 3H); MS (TOF)
+
.
m/z 337 (M+H) . Anal. calcd for C20H24N4O 3.0H2O:
C, 61.52; H, 7.74; N, 14.35. Found: C, 61.71; H, 7.70; N,
14.62.
6-Amino - 4 - (4 - n - pentyloxy)phenethylaminoquinazoline
(12j). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 12j was obtained as a light yellow solid
Similarly to the procedure described for 11e, the title
compound was prepared starting from 15. After puri-
fication, 11g was obtained as a light yellow solid (34%
(32% yield for three steps from 7): mp 238–240 ꢀC; H
1
NMR (DMSO-d6) d 9.71 (t, J=5.1 Hz, 1H), 8.64 (s,
1H), 7.60(d, J=8.9 Hz, 1H), 7.33 (dd, J=2.2, 8.9 Hz,
1H), 7.23 (s, 1H), 7.16 (d, J=8.6 Hz, 2H), 6.84 (d,
J=8.6 Hz, 2H), 6.07 (br s, 2H), 3.93–3.83 (m, 4H), 2.92
(t, J=7.2 Hz, 2H), 1.71–1.66 (m, 2H), 1.36–1.32 (m,
4H), 0.89 (t, J=7.2 Hz, 3H); MS (TOF) m/z 351
ꢀ
1
yield): mp 116–118 C; H NMR (DMSO-d6) d 8.22 (s,
1H), 7.90(br s, 1H), 7.82–7.76 (m, 3H), 7.41 (d,
J=8.6 Hz, 1H), 7.32 (d, J=8.4 Hz, 2H), 7.28 (br s, 1H),
7.12 (dd, J=2.2, 8.6 Hz, 1H), 7.01 (d, J=2.2 Hz, 1H),
5.41 (br s, 2H), 3.76–3.69 (m, 2H), 3.00 (t, J=7.2 Hz,
2H); MS (TOF) m/z 308 (M+H)+. Anal. calcd for
+
.
(M+H) . Anal. calcd for C21H26N4O 3.0H2O: C,
62.35; H, 7.23; N, 13.85. Found: C, 62.23; H, 7.26; N,
13.83.
.
C17H17N5O 0.5H2O: C, 64.54; H, 5.73; N, 22.14.
Found: C, 64.60; H, 5.69; N, 22.01.
6-Amino - 4 - (4 - n - hexyloxy)phenethylaminoquinazoline
(12k). Similarly to the procedure described for 11e, the
title compound was prepared starting from 7. After
purification, 12k was obtained as a light yellow solid
6-Amino - 4 - (4 - N,N - dimethylcarboxamido)phenethyl-
aminoquinazoline (11h). Similarly to the procedure
described for 11g, the title compound was prepared
starting from 14. After purification, 11h was
obtained as a light yellow solid (38% yield for two
(11% yield for three steps from 7): mp 145–147 ꢀC; H
1
steps from 14): mp 294–296 ꢀC; H NMR (DMSO-d6)
1
NMR (DMSO-d6) d 8.27 (s, 1H), 8.03 (t, J=4.9 Hz,
1H), 7.43 (d, J=8.6 Hz, 1H), 7.17–7.13 (m, 3H), 7.05–
7.04 (m, 1H), 6.84 (d, J=8.4 Hz, 2H), 5.50(br s, 2H),
3.91 (t, J=6.5 Hz, 2H), 3.72–3.64 (m, 2H), 2.87 (t,
J=7.3 Hz, 2H), 1.71–1.63 (m, 2H), 1.42–1.26 (m, 6H),
0.87 (t, J=6.8 Hz, 3H); MS (TOF) m/z 365 (M+H)+.
d 8.21 (s, 1H), 7.80(t,
J=5.4 Hz, 1H), 7.41 (d,
J=8.6 Hz, 1H), 7.32 (s, 4H), 7.13 (dd, J=2.2, 8.6 Hz,
1H), 7.01 (d, J=2.2 Hz, 1H), 5.42 (br s, 2H), 3.76–3.69
(m, 2H), 3.01–2.91 (m, 8H); MS (TOF) m/z 337
+
.
(M+H) . Anal. calcd for C19H21N5O 0.5H2O: C,
66.26; H, 6.29; N, 20.33. Found: C, 66.45; H, 6.51; N,
19.97.
.
Anal. calcd for C22H28N4O 0.8H2O: C, 69.74; H, 7.66;
N, 14.79. Found: C, 69.87; H, 7.68; N, 14.75.

3878
M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 3869–3878
Biology
subcutaneously injecting a 1% suspension of carragee-
nin (type l, Sigma) in saline (0.1 mL) into the plantar
surface of the right hind paw. Compounds 2, 4e, 12a,
12d, and 12j were intraperitoneally administered 15 min
prior to the injection of the carrageenin. These com-
pounds were administered as suspensions in 0.5%
hydroxypropyl cellulose. The resulting edema was
quantified 2 h later by measuring the increase in the
volume of the inflamed paw. The ensuing paw swelling
was measured by using a water-displacement plethys-
mograph (Ugo Basile).
NF-ꢀB assay.9 Human Jurkat T cells (Riken, Japan)
were cultured at 37 ꢀC in a 5% CO2 atmosphere in
RPMI1640 containing 10% FCS, 100 U/mL of peni-
cillin, and 100 mg/mL of streptomycin. The cells were
plated in six-well plates (2Â106/well) and transiently
transfected using the SuperFect Transfection Reagent
(QIAGEN) with 1 mg of pNFkB-Luc (PathDetect Cis-
Reporter Plasmid, STRATAGENE). After transfection,
the cells were cultured at 37 ꢀC overnight. They were
then collected, resuspended in fresh medium, and plated
in 96-well plates (2Â105/well). Test compounds were
dissolved in DMSO and added at the appropriate con-
centrations to the 96-well plates containing the cells,
and the plates were then incubated at 37 ꢀC for 1 h. For
induction of transcription, 10ng/mL of PMA and
100 mg/mL of PHA were added to each well, and the
cells were incubated for an additional 6 h at 37 ꢀC. The
culture media were removed, and cell lysis buffer con-
taining luciferase substrate (Bright-Glo Luciferase
Assay System, Promega) was added to each well. The
each portion was transferred to a black 96-well plate,
and then luminescence was immediately measured with
a Packard Topcount (Packard Instruments). The 50%
inhibitory concentration (IC50) values were calculated
by a nonlinear regression method. To measure the
cytotoxicity of test compounds toward Jurkat cells, we
added the compounds to 96-well plates containing non-
transfected cells (2Â105/well), and incubated the plates
at 37 ꢀC for 24 h. Cell viability was measured by using
the MTS assay (Promega).
Acknowledgements
We are grateful for all of the help provided by Dr.
Satoru Misawa. In addition, we would like to express
our thanks to Mr. Toru Negishi, Mr. Toshiyuki Sato,
and Mr. Tominaga Fukazawa for their technical assis-
tance in obtaining the biological data.
References and Notes
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