Discovery of quinazolines as a novel structural class of potent inhibitors of NF-κB activation

Article abstract of DOI:10.1016/S0968-0896(02)00440-6

We disclose here a new structural class of low-molecular-weight inhibitors of NF-κB activation that were designed and synthesized by starting from quinazoline derivative 6a. Structure-activity relationship (SAR) studies based on 6a elucidated the structural requirements essential for the inhibitory activity toward NF-κB transcriptional activation, and led to the identification of the 6-amino-4-phenethylaminoquinazoline skeleton as the basic framework. In this series of compounds, 11q, containing the 4-phenoxyphenethyl moiety at the C(4)-position, showed strong inhibitory effects on both NF-κB transcriptional activation and TNF-α production. Furthermore, 11q exhibited an anti-inflammatory effect on carrageenin-induced paw edema in rats.

Full text of DOI:10.1016/S0968-0896(02)00440-6

Bioorganic & Medicinal Chemistry 11 (2003) 383–391
Discovery of Quinazolines as a Novel Structural Class of Potent
Inhibitors of NF-ꢀB Activation
Masanori Tobe, Yoshiaki Isobe, Hideyuki Tomizawa, Takahiro Nagasaki,
Hirotada Takahashi, Tominaga Fukazawa and Hideya Hayashi*
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, Toda-shi, Saitama 335-8502, Japan
Received 7 August 2002; accepted 10 September 2002
Abstract—We disclose here a new structural class of low-molecular-weight inhibitors of NF-kB activation that were designed and
synthesized by starting from quinazoline derivative 6a. Structure–activity relationship (SAR) studies based on 6a elucidated the
structural requirements essential for the inhibitory activity toward NF-kB transcriptional activation, and led to the identification of
the 6-amino-4-phenethylaminoquinazoline skeleton as the basic framework. In this series of compounds, 11q, containing the
4-phenoxyphenethyl moiety at the C(4)-position, showed strong inhibitory effects on both NF-kB transcriptional activation and
TNF-a production. Furthermore, 11q exhibited an anti-inflammatory effect on carrageenin-induced paw edema in rats.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
or IkB-e. However, a large variety of inflammatory
conditions, such as inflammatory cytokines, bacterial
and viral infections, and certain chemical agents, induce
NF-kB activity.5 NF-kB activation involves signaled
phosphorylation, ubiquitination, and proteolysis of
IkB-a.4 Liberated NF-kB migrates to the nucleus, where
it stimulates the transcription of its target gene. The
activation of NF-kB initiates both extracellular and
intracellular regulatory events that result in autoregula-
tion of the inflammatory cascade through modulation
of NF-kB activation. The inhibition of NF-kB activa-
tion would lead to suppression of proinflammatory
cytokines levels and be beneficial for the treatment of
the above-mentioned diseases.6
Autoimmune diseases and allergies, such as rheumatoid
arthritis, septic shock, transplant rejection, asthma, and
psoriasis, are considered to be caused by abnormalities
of T cell immune responses.1 In particular, the activa-
tion of T cells initiates a network of events that results
in the overproduction of certain transcription factors
and proinflammatory cytokines.2 Transcription factors
are DNA-binding proteins that regulate the production
of proinflammatory cytokines as well as that of a variety
of other cellular regulators.
Nuclear factor-kB (NF-kB) is a pivotal transcription
factor that functions to enhance the transcription of
proinflammatory cytokines including TNF-a, IL-1b, IL-
6, and IL-8, which are thought to be important in the
generation of acute inflammatory responses.3 Activation
and regulation of NF-kB are tightly controlled by
members of a family of inhibitory proteins referred to as
IkB. Several IkB proteins have been identified, including
IkB-a, IkB-b, IkB-g, IkB-e, and Bcl-3.4
Several disclosures have reported some compounds with
the inhibitory activities toward NF-kB-mediated tran-
scriptional activation. Low-molecular-weight com-
pounds, such as MG-132 (1),7 BAY 11-7085 (2),8 and an
indan derivative (3),9 as well as natural products, such
as caffeic acid phenylethyl ester (4)10 and the sesqui-
terpene lactone helenalin (5),11 have been shown to
inhibit NF-kB activation (Fig. 1).
In resting cells, NF-kB is sequestered in the cytoplasm
through its interaction with its inhibitor, IkB-a, IkB-b
In order to find a new structural class of NF-kB activa-
tion inhibitors, we conducted a reporter gene-based
screening featuring phorbol 12-myristate-13-acetate
(PMA) plus phytohemagglutin (PHA) stimulation of
human Jurkat T cells transfected with the reporter
*Corresponding author. Tel.: +81-6-6466-5189; fax: +81-6-6466-
5483; e-mail: hayashih@sumitomopharm.co.jp
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00440-6

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M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
Figure 1. Several inhibitors of NF-kB-mediated transcriptional activation. (1) MG-132; (2) BAY 11–7085; (3) indan derivative; (4) caffeic acid
phenylethyl ester; (5) helenalin.
DNAhaving the binding sequence for NF- kB and the
luciferase gene.12 This led to the identification of the
quinazoline 6a as a lead compound (IC50=2381 nM).
To date, no one has reported quinazoline derivatives as
NF-kB activation inhibitors. This situation prompted us
to investigate structure–activity relationship (SAR) of
quinazoline derivatives as inhibitors of NF-kB activa-
tion. In this paper, we describe the results of our pre-
liminary SAR studies on the quinazolines, conducted on
the basis of the lead compound, 6a.
ammonia, gave the corresponding 2-aminobenzamides
(8). The benzamide (8) was cyclized to the 4-quin-
azolone (9) by using trimethyl orthoformate. Subse-
quently, chlorination of the 4-quinazolones (9) with
thionyl chloride gave the corresponding 4-chloro-
quinazolines (10). Reaction of these compounds with
phenethylamines in the presence of triethylamine
provided compounds 11a–11d, f, i, and k. Catalytic
hydrogenation of 11d gave the 6-hydroxyquinazoline,
11e. Compound 11f underwent iron dust reduction to
provide the 6-aminoquinazoline, 11g. Acetylation of
11g with acetic anhydride in pyridine gave 11h.
Compounds 11j, l, m–q were also prepared by the
same procedure used for 11g.
Chemistry
Procedures for the preparation of 6a–6h having the
6–6 condensed-ring systems are shown in Scheme 1.
Chlorination of 4-quinazolone with phosphorus oxy-
chloride followed by the reaction of the corresponding
amines provided the quinazolines 6a–6e. The quinoline
(6f), the isoquinoline (6g), and the phthalazine (6h) were
also prepared by the same procedures used for the
quinazolines.
Results and Discussion
To screen compounds for their inhibitory potency
toward NF-kB transcriptional activation, we set up a
luciferase reporter gene assay using human Jurkat cells.
Compounds were also evaluated for their inhibitory
activities toward TNF-a production by using murine
splenocytes stimulated with LPS. Cytotoxicity toward
both human Jurkat cells and murine splenocytes was
measured by using the MTS assay. The results obtained
by using these tools to drive the SAR efforts are shown
in Tables 1 and 2. Among NF-kB activation inhibitors,
The general synthetic pathway to the phenethylamino-
quinazolines is shown in Scheme 2. Treatment of the
appropriate anthranilic acids (7) with EDC (N-ethyl-N0-
[3-(dimethylamino)propyl] carbodiimide) and HOBt (1-
hydroxybenzotriazole), followed by the addition of 28%
Scheme 1. Synthesis of compounds 6a–6h. Reagents and conditions: (a) phosphorus oxychloride, 100 ꢀC, 2–3 h.; (b) RNH2, triethylamine, i-PrOH,
reflux, 5–10 h.

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
385
Scheme 2. Synthesis of the 4-phenethylaminoquinazolines, 11a–11q. Reagents and conditions: (a) 28% aqueous ammonia solution, EDCꢁ HCl,
HOBt, DMF, 3–5 h; (b) HC(OMe)3, 12 N HCl, 1–3 h; (c) SOCl2, DMF, reflux, 2–3 h; (d) phenethylamines, triethylamine, i-PrOH, 1–2 h; (e) H2, Pd–
C, MeOH, 5 h; (f) Fe, glacial acetic acid, EtOH–H2O, reflux, 30 min; (g) Ac2O, pyridine, DMF, 50 ꢀC, 2 h.
Table 1. Inhibition of NF-kB activation and TNF-a production by
compounds 6a–6h
tion of the quinazoline (6b–6e) (Table 1). The
maximum suppressing effect on NF-kB transcriptional
activation was obtained when the spacer was the ethyl-
ene chain (6c). The optimal length was also confirmed
to be 2 methylene units (6c) toward TNF-a inhibitory
activity.
To establish the role of the quinazoline ring, we studied
a set of the quinazoline and related heteroaromatic
NF-kBa Toxicityb TNF-ac Toxicityd
derivatives. The quinoline, 6f, was 2-fold less active than
the quinazoline 6c; and the isoquinoline, 6g, or the
phthalazine, 6h, showed severe loss of their inhibitory
activities toward both NF-kB activation and TNF-a
production. These results indicate that, according to the
numbering of the quinazoline ring, the 1-nitrogen atom
is essential to exhibit both inhibitory activities and the
combination of 1- and 3-nitrogen atoms is more effec-
tive than that of 1- and 2-nitrogen atom. Therefore, we
considered the quinazoline core to be the most favor-
able in this SAR study.
Compd
X
Y
Z n R IC50 (nM) TC50 (nM) IC50 (nM) TC50 (nM)
6a
6b
6c
6d
6e
6f
6g
N CH N 3 H
N CH N 3 Cl
N CH N 2 Cl
N CH N 1 Cl
2381 >10,000
1401 >10,000
630 >10,000
7273 >10,000
3220 >10,000
398 >10,000
1832 >10,000 >10,000 >10,000
N CH N 0 Cl >10,000 >10,000 >10,000 >10,000
CH CH N 2 Cl 1392 >10,000 1643 5103
N CH CH 2 Cl >10,000 >10,000 >10,000 >10,000
N CH 2 Cl >10,000 >10,000 >10,000 >10,000
715 1141 809 1893
6h
MG-132
N
aIC50 for the inhibition of NFkB 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.
Next, we investigated the effect of C(6)- and C(7)-sub-
stituents on the quinazoline ring (Table 2). Among the
C(6)-substituents, compounds 11a–c and e were equi-
potent or 2-fold more potent than 6c in their inhibitory
activity toward NF-kB activation; whereas the benzy-
loxy (11d) and the nitro (11f) showed great loss of this
inhibitory activity. Furthermore, as shown by 11g,
introduction of the amino group at the C(6)-position
afforded a 3–4 fold increase in the inhibitory activities
toward both NF-kB activation and TNF-a production
over 6c, whereas the 7-amino analogue (11l) displayed a
loss of both inhibitory activities. Acetamidation (11h)
resulted in a 26-fold loss in NF-kB inhibitory activity
compared with 11g. Additional introduction of a chloro
group at the 7-position of 11g also gave a less potent
compound (11j). This trend was also observed in the
case of 11i. These results suggest that the presence of a
basic nitrogen as well as a smaller substituent at the
C(6)-position of the quinazoline ring was necessary to
inhibit both NF-kB transcriptional activation and
TNF-a production.
dTC50 for the growth inhibition of mouse splenocytes stimulated with
LPS.
MG-132 (1) has been shown to be a proteasome inhi-
bitor blocking NF-kB transcriptional activation in Hela
cells stimulated by TNF-a (IC50=3 mM).7 The indan
derivative (3) was also reported to inhibit NF-kB tran-
scriptional activation, in A549 cells stimulated with
TNF-a or IL-1b (TNF-a: IC50=89 nM; IL-1b:
IC50=51 nM).9 We found both compounds to block the
activation of NF-kB transcriptional factor in our assay
system (MG-132: IC50=715 nM; 3: IC50=38 nM), and
so we selected MG-132 and 3 as the positive controls in
our system.
Our initial efforts focused on investigating the effects of
varying the methylene chain length between the quina-
zoline and the 4-chlorophenyl group at the C(4)-posi-

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M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
Table 2. Inhibition of NF-kB activation and TNF-a production by 4-phenethylaminoquinazolines 11a–11q
NF-kBa
Toxicityb
TNF-ac
Toxicityd
Compd
R6
R7
X
IC50 (nM)
TC50 (nM)
IC50 (nM)
TC50 (nM)
6c
H
Cl
Br
Me
BnO
HO
NO2
NH2
NHAc
NO2
NH2
H
H
H
H
H
H
H
H
H
H
Cl
Cl
NO2
NH2
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Me
HO
MeO
PhO
630
344
851
730
7340
313
1515
176
4562
9060
429
1765
2202
311
210
3645
90
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>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
398
5478
1763
358
5103
1376
2031
142
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
6775
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
11o
11p
11q
3
6540
>10,000
781
1763
1376
1426
138
1702
279
7
84
809
H
6775
NH2
NH2
NH2
NH2
NH2
>10,000
>10,000
>10,000
>10,000
>10,000
>1000
11
38
715
MG-132
1893
aꢂdSee corresponding footnotes in Table 1.
To improve the inhibitory activity toward NF-kB tran-
scriptional activation, we next performed substitution of
the chloro group on the terminal phenyl ring with sev-
eral other substituents, keeping the 6-amino group con-
stant. Removal of the chloro group from 11g gave 11m,
which caused a slight loss of the inhibitory activity.
Replacing the chloro group with the methyl group (11n)
resulted in an IC50 value comparable to that of 11g for
the inhibitory activity. Substantially decreased potency
was observed when the methyl group in 11n was
replaced with a hydroxyl group (11o). However, the
methoxy (11p) showed a 2-fold higher inhibitory activity
toward NF-kB transcriptional activation over 11g.
Replacement of the methoxy group in 11p with the
phenoxy group gave 11q which displayed a remarkable
improvement in the inhibition of both NF-kB tran-
scriptional activation and TNF-a production compared
with the inhibitory activities of 11g. Moreover, 11q
showed 4- and 12-fold improvement of the inhibitory
activities toward NF-kB activation and TNF-a produc-
tion, respectively, compared with 3. Finally, on the basis
of the above-mentioned in vitro study, we selected
compound 11q and evaluated its anti-inflammatory
effect in a rat carrageenin-induced paw edema model.13
d paw swelling was induced by the injection of carra-
geenin into the right hind paw of rats. The resulting
edema was quantified 2 h later by measuring the
increase in the volume of the inflamed paw. As seen in
Figure 2, compound 11q inhibited edema formation
dose-dependently and showed a more potent suppres-
sing effect than 3 when both were tested at a dose of
1 mg/kg. In this model, the activation of NF-kB has
been reported to play a key role in the development of
inflammatory response.14 Therefore, we consider that
compound 11q had a suppressing effect of the NF-kB
mediated-inflammatory response. We are continuing
further optimization study based on 11q, especially
focusing on the substitution at the terminal phenyl ring
at the C(4)-position of the quinazoline ring. The results
of this detailed SAR study will be reported in the near
future.
Conclusions
Based on the lead compound 6a obtained from random
screening, we identified and synthesized a novel struc-
tural class of NF-kB activation inhibitors. Our SAR
investigation allowed us to identify the 6-amino-4-phe-
nethylaminoquinazoline skeleton as the basic frame-
work and led to the discovery of 11q, whose inhibitory
activity toward NF-kB transcriptional activation repre-
sented a 2 order of magnitude increase relative to that of
6a. Compound 11q was also highly potent in the inhi-
bition of TNF-a production (IC50=7 nM). In addition,
11q demonstrated in vivo efficacy by reducing the edema
formation seen in carrageenin-induced inflammation of
the rat hind paw. Further SAR studies on 6-amino-
quinazolines made on the basis of 11q are ongoing and
will be reported in due course.

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
387
the crude chloro compound and triethylamine (571 mL,
4.10 mmol) in EtOH (15 mL) was added 3-(phenyl)pro-
pylamine (692 mg, 5.12 mmol). The resulting mixture
was stirred at reflux for 5 h and concentrated in vacuo.
The residue was partitioned between CH2Cl2 and 5%
aqueous citric acid solution. The organic layer was
washed with water and brine, and then dried over
Na2SO4. The solution was evaporated in vacuo, and the
residue was triturated with diethyl ether. The white solid
was filtered and dried at 40 ꢀC under high vacuum to
give 6a (309 mg, 57% yield for 2 steps from 4-quinazo-
linol): mp 116–117 ꢀC; H NMR (DMSO-d6) d 8.45 (s,
1
1H), 8.29–8.23 (m, 2H), 7.79–7.65 (m, 2H), 7.53–7.47
(m, 1H), 7.32–7.15 (m, 5H), 3.59–3.52 (m, 2H), 2.69 (t,
J=7.6 Hz, 2H), 2.02–1.91 (m, 2H); MS (TOF) m/z 264
(M + H)+; anal. calcd for C17H17N3: C, 77.54; H, 6.51;
N, 15.96. Found: C, 77.26; H, 6.46; N, 15.71.
Compounds 6b–6h were also prepared by using the
same procedure as for 6a.
4-[3-(4-Chlorophenyl)propylamino]quinazoline (6b).
White solid (49% yield for 2 steps from 4-quinazoli-
Figure 2. Effects of 11q and 3 on carrageenin-induced paw edema in
rats. Compound 11q and 3 were intraperitoneally administered 15 min
prior to the injection of carrageenin. The resulting edema was quanti-
fied 2 h later by measuring the increase in the volume of the inflamed
paw. The results are expressed as the meanꢃSEM of 5 rats per group.
* p <0.05 versus vehicle control (Dunnett’s test).
nol): mp 151–153 ꢀC; H NMR (DMSO-d6) d 8.45 (s,
1
1H), 8.29–8.22 (m, 2H), 7.79–7.65 (m, 2H), 7.53–7.47
(m, 1H), 7.35–7.26 (m, 4H), 3.58–3.51 (m, 2H), 2.68 (t,
J=7.6 Hz, 2H), 2.00–1.89 (m, 2H); MS (TOF) m/z 298
(M + H)+; anal. calcd for C17H16ClN3: C, 68.57; H,
5.42; N, 14.11. Found: C, 68.53; H, 5.42; N, 14.03.
Experimental
Chemistry
4-(4-Chlorophenethylamino)quinazoline (6c). White solid
(39% yield for 2 steps from 4-quinazolinol): mp 192–
General. All reagents and solvents were obtained from
commercial suppliers and were used without further
purification. Melting points were measured with a
BUCHI 535 melting point apparatus and were uncor-
194 ꢀC; H NMR (DMSO-d6) d 8.48 (s, 1H), 8.36 (t,
1
J=5.4 Hz, 1H), 8.19 (d, J=7.8 Hz, 1H), 7.79–7.66 (m,
2H), 7.53–7.47 (m, 1H), 7.37–7.27 (m, 4H), 3.75 (dt,
J=5.4, 7.3 Hz, 2H), 2.97 (t, J=7.3 Hz, 2H); MS (TOF)
m/z 284 (M + H)+; anal. calcd for C16H14ClN3: C,
67.72; H, 4.97; N, 14.81. Found: C, 67.54; H, 4.98; N,
15.20.
1
rected. H NMR was recorded on a Jeol GSX270 FT
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.
TOFMS (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. Wakogel
C-200 (Wako; 70–150 mm) was used for column chro-
matography. Monitoring of reactions was carried out
using Merck 60 F254 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).
4-(4-Chlorobenzylamino)quinazoline (6d). White solid
(52% yield for 2 steps from 4-quinazolinol): mp 206–
208 ꢀC; H NMR (DMSO-d6) d 8.88 (t, J=5.9 Hz, 1H),
1
8.45 (s, 1H), 8.29 (d, J=7.8 Hz, 1H), 7.82–7.69 (m, 2H),
7.57–7.51 (m, 1H), 7.37 (s, 4H), 4.77 (d, J=5.9 Hz, 2H);
MS (TOF) m/z 270 (M + H)+; anal. calcd for
ꢁ
C15H12ClN3 0.1H2O: C, 66.35; H, 4.49; N, 15.48.
Found: C, 66.01; H, 4.54; N, 15.78.
4-(4-Chlorophenylamino)quinazoline (6e). White solid
(45% yield for 2 steps from 4-quinazolinol): mp 163–
ꢀ
1
164 C; H NMR (DMSO-d6) d 9.89 (br s, 1H), 8.63 (s,
1H), 8.56 (d, J=8.1 Hz, 1H), 7.97–7.92 (m, 2H), 7.89–
7.80 (m, 2H), 7.69–7.63 (m, 1H), 7.49–7.43 (m, 2H); MS
4-[3-(Phenyl)propylamino]quinazoline (6a). Asuspension
of 4-quinazolinol (300 mg, 2.05 mmol) in phosphorus
oxychloride (6.0 mL) was heated at 100 ꢀC for 2 h, when
a clear solution was obtained. The reaction mixture was
cooled to ambient temperature, and then concentrated
under reduced pressure. The residue was partitioned
between CH2Cl2 and 5% aqueous NaHCO3 solution.
The organic layer was washed with water and brine,
dried over Na2SO4. The solution was concentrated
under reduced pressure to provide crude 4-chloro-
quinazoline, which was used directly. To a mixture of
(TOF) m/z 256 (M
ꢁ
+
H)+; anal. calcd for
C14H10ClN3 0.1H2O: C, 65.30; H, 3.95; N, 16.32.
Found: C, 65.16; H, 4.05; N, 16.30.
4-(4-Chlorophenethylamino)quinoline (6f). White solid
(58% yield for 2 steps from 4-quinolinol): mp 158–
160 ꢀC; 1H NMR (DMSO-d6) d 8.39 (d, J=5.4 Hz, 1H),
8.17 (d, J=7.8 Hz, 1H), 7.77 (d, J=7.8 Hz, 1H), 7.63–
7.57 (m, 1H), 7.44–7.38 (m, 1H), 7.35 (s, 4H), 7.22 (t,

388
M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
J=5.4 Hz, 1H), 6.53 (t, J=5.4 Hz, 1H), 3.50 (dt, J=5.4,
7.3 Hz, 2H), 2.98 (t, J=7.3 Hz, 2H); MS (TOF) m/z 283
(M + H)+; anal. calcd for C17H15ClN2: C, 72.21; H,
5.35; N, 9.91. Found: C, 72.00; H, 5.34; N, 9.78.
and concentrated in vacuo. The residue was partitioned
between CH2Cl2 and 5% aqueous citric acid solution.
The organic layer was washed successively with 1 N
NaOH, water, and brine, and then dried over Na2SO4.
The solution was concentrated under reduced pressure
and the residue was triturated with CH2Cl2/hexane
(1:4). The light yellow solid was filtered and dried at
40 ꢀC under high vacuum to give 11f (1.43 g, 83% yield
for 2 steps from 6-nitro-4-quinazolone): mp 232–234 ꢀC;
1H NMR (DMSO-d6) d 9.33 (d, J=2.4 Hz, 1H), 9.08 (br
s, 1H), 8.61 (s, 1H), 8.49–8.45 (m, 1H), 7.85–7.81 (m,
1H), 7.37–7.28 (m, 4H), 3.82–3.77 (m, 2H), 2.99 (t,
J=7.3 Hz, 2H); MS (TOF) m/z 329 (M + H)+; anal.
calcd for C16H13ClN4O2: C, 58.45; H, 3.99; N, 17.04.
Found: C, 58.29; H, 3.99; N, 16.95.
1-(4-Chlorophenethylamino)isoquinoline (6g). White
solid (18% yield for 2 steps from isocarbostyril): mp
127–128 ꢀC; H NMR (CDCl3) d 9.21 (br s, 1H), 8.73
1
(d, J=7.8 Hz, 1H), 7.79 (d, J=6.8 Hz, 1H), 7.70–7.66
(m, 2H), 7.62 (d, J=6.8 Hz, 1H), 7.19 (d, J=8.1 Hz,
2H), 6.96 (d, J=8.1 Hz, 2H), 6.91 (d, J=6.8 Hz, 1H),
4.15–4.07 (m, 2H), 3.09 (t, J=7.3 Hz, 2H); MS (TOF)
m/z
283
(M
+
H)+;
anal.
calcd
for
ꢁ
C17H15ClN2 0.6H2O: C, 69.55; H, 5.36; N, 9.54. Found:
C, 69.62; H, 5.28; N, 9.45.
1-(4-Chlorophenethylamino)phthalazine (6h). White solid
(42% yield for 2 steps from 1(2H)-phthalazinone): mp
6-Chloro-4-(4-chlorophenethylamino)quinazoline (11a).
Similarly to the procedure described for 11f, the title
compound was prepared starting from 2-amino-5-
chlorobenzoic acid. After purification, 11a was obtained
as a white solid (20% yield for 4 steps): mp 192–194 ꢀC;
1H NMR (DMSO-d6) d 8.50 (s, 1H), 8.45 (t, J=5.3 Hz,
1H), 8.37 (d, J=2.2 Hz, 1H), 7.80–7.68 (m, 2H), 7.37–
7.27 (m, 4H), 3.75 (dt, J=5.3, 7.3 Hz, 2H), 2.96 (t,
J=7.3 Hz, 2H); MS (TOF) m/z 318 (M + H)+; anal.
ꢀ
1
154–156 C; H NMR (DMSO-d6) d 8.91 (s, 1H), 8.25–
8.22 (m, 1H), 7.94–7.83 (m, 3H), 7.55 (t, J=5.4 Hz, 1H),
7.37–7.29 (m, 4H), 3.77 (dt, J=5.4, 7.4 Hz, 2H), 3.02 (t,
J=7.4 Hz, 2H); MS (TOF) m/z 284 (M + H)+; anal.
ꢁ
calcd for C16H14ClN3 0.2H2O: C, 66.88; H, 4.98; N,
14.62. Found: C, 66.80; H, 4.98; N, 14.58.
ꢁ
4-(4-Chlorophenethylamino)-6-nitroquinazoline (11f). To
a solution of 2-amino-5-nitrobenzoic acid (10.0 g,
54.9 mmol) and HOBt (8.16 g, 60.4 mmol) in DMF
(275 mL) was added EDCꢁ HCl (11.6 g, 60.4 mmol). The
mixture was stirred at ambient temperature for 2 h. The
resulting solution was cooled to 0 ꢀC, then 28% ammo-
nia solution (5.5 mL) was added and temperature was
allowed to rise to ambient temperature. After 1 h, the
reaction mixture was concentrated under reduced pres-
sure, and the residue was partitioned AcOEt and 5%
aqueous NaHCO3 solution. The organic layer was washed
with water and brine, and then dried over Na2SO4. The
solution was evaporated in vacuo, and the residue was
triturated with AcOEt/hexane (1:1). The pale yellow solid
was filtered and dried at 40ꢀC under high vacuum to give
2-amino-5-nitrobenzamide (8.78 g, 88% yield).
calcd for C16H13Cl2N3 0.2H2O: C, 59.72; H, 4.13; N,
13.06. Found: C, 59.71; H, 4.08; N, 13.04.
6-Bromo-4-(4-chlorophenethylamino)quinazoline (11b).
Similarly to the procedure described for 11f, the title
compound was prepared starting from 2-amino-5-bro-
mobenzoic acid. After purification, 11b was obtained as
a white solid (37% yield for 4 steps): mp 209–211 ꢀC; 1H
NMR (DMSO-d6) d 8.52–8.44 (m, 3H), 7.89 (dd,
J=8.8, 2.0 Hz, 1H), 7.63 (d, J=8.8 Hz, 1H), 7.37–7.27
(m, 4H), 3.78–3.71 (m, 2H), 2.96 (t, J=7.4 Hz, 2H); MS
(TOF) m/z 362 (M
ꢁ
+
H)+; anal. calcd for
C16H13BrClN3 0.1H2O: C, 52.73; H, 3.62; N, 11.53.
Found: C, 52.34; H, 3.71; N, 11.87.
4-(4-Chlorophenethylamino)-6-methylquinazoline (11c).
Similarly to the procedure described for 11f, the title
compound was prepared starting from 2-amino-5-
methylbenzoic acid. After purification, 11c was
obtainedꢀ as a white solid (35% yield for 4 steps): mp
Next, to a mixture of 2-amino-5-nitrobenzamide (8.78 g,
48.5 mmol) in trimethyl orthoformate (70 mL) was
added 12 N HCl (6.0 mL) dropwise at 0 ꢀC. The reaction
mixture was stirred at ambient temperature for 1.5 h, and
then was concentrated under reduced pressure. The resi-
due was triturated with water, and was adjusted to pH 6
with 5 N NaOH. The precipitate was collected, washed
with MeOH and water, and dried in air. The light yellow
solid was filtered and dried at 40ꢀC under high vacuum to
give 6-nitro-4-quinazolone (8.21 g, 89% yield).
1
153–154 C; H NMR (DMSO-d6) d 8.43 (s, 1H), 8.23
(t, J=5.3 Hz, 1H), 8.00 (s, 1H), 7.62–7.56 (m, 2H), 7.37–
7.27 (m, 4H), 3.74 (dt, J=5.3, 7.3 Hz, 2H), 2.96 (t,
J=7.3 Hz, 2H), 2.46 (s, 3H); MS (TOF) m/z 298 (M +
+
ꢁ
H) ; anal. calcd for C17H16ClN3 0.1H2O: C, 68.16; H,
5.42; N, 14.03. Found: C, 68.03; H, 5.37; N, 13.75.
6-Benzyloxy-4-(4-chlorophenethylamino)quinazoline (11d).
Similarly to the procedure described for 11f, the title
compound was prepared starting from 2-amino-5-ben-
zyloxybenzoic acid. After purification, 11d was obtained
as a white solid (28% yield for 4 steps): mp 182–184 ꢀC;
1H NMR (DMSO-d6) d 8.40 (s, 1H), 8.20 (t, J=5.4 Hz,
1H), 7.79 (d, J=2.4 Hz, 1H), 7.64 (d, J=9.2 Hz, 1H),
7.54–7.28 (m, 10H), 5.19 (s, 2H), 3.76 (dt, J=5.4,
7.4 Hz, 2H), 2.98 (t, J=7.4 Hz, 2H); HR-FABMS m/z
(M + H)+; calcd for C23H20ClN3O: 390.1373. Found:
390.1354.
Subsequently, a suspension of 6-nitro-4-quinazolone
(1.00 g, 5.23 mmol) in thionyl chloride (30 mL) contain-
ing 1 drop of DMF was heated at reflux for 3 h to give a
clear solution. Excess thionyl chloride was removed
under reduced pressure to provide crude 4-chloro-6-
nitroquinazoline, which was used directly. To a mixture
of the crude chloro compound and triethylamine
(875 mL, 6.28 mmol) in i-PrOH (50 mL) was added 4-
chlorophenethylamine (878 mL, 6.28 mmol). The result-
ing mixture was stirred at ambient temperature for 2 h

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
389
1
4-(4-Chlorophenethylamino)-7-nitroquinazoline
(11k).
mp 190 ꢀC (Dec.); H NMR (DMSO-d6) d 8.23 (s, 1H),
7.86 (t, J=5.4 Hz, 1H), 7.44–7.26 (m, 5H), 7.13 (dd,
J=2.1, 8.9 Hz, 1H), 7.01 (d, J=2.1 Hz, 1H), 5.45 (br s,
2H), 3.70 (dt, J=5.4, 7.0 Hz, 2H), 2.95 (t, J=7.0 Hz,
2H); MS (TOF) m/z 299 (M + H)+; anal. calcd for
Similarly to the procedure described for 11f, the title
compound was prepared starting from 2-amino-4-
nitrobenzoic acid. After purification, 11k was obtained
as a white solid (43% yield for 4 steps): mp 196–198 ꢀC;
1H NMR (DMSO-d6) d 8.81 (t, J=4.9 Hz, 1H), 8.62 (s,
1H), 8.47 (d, J=9.2 Hz, 1H), 8.41 (d, J=2.2 Hz, 1H),
8.26 (dd, J=2.2, 9.2 Hz, 1H), 7.36–7.27 (m, 4H), 3.78
(dt, J=4.9, 7.2 Hz, 2H), 2.98 (t, J=7.2 Hz, 2H); MS
ꢁ
C16H15ClN4 0.8H2O: C, 61.36; H, 5.09; N, 17.89.
Found: C, 61.67; H, 5.18; N, 17.89.
7-Amino-4-(4-chlorophenethylamino)quinazoline
(11l).
(TOF) m/z 329 (M
+
H)+; anal. calcd for
Similarly to the procedure described for 11j, the title
compound was prepared starting from 11k. After pur-
ification, 11l was obtained as a white solid (85% yield):
mp 228–230 ꢀC; 1H NMR (DMSO-d6) d 9.59 (t,
J=5.4 Hz, 1H), 8.61 (s, 1H), 8.10 (d, J=9.2 Hz, 1H),
7.37–7.27 (m, 4H), 6.95–6.88 (m, 3H), 6.66 (d,
J=1.9 Hz, 1H), 3.84 (dt, J=5.4, 7.2 Hz, 2H), 2.97 (t,
J=7.2 Hz, 2H); MS (TOF) m/z 299 (M + H)+; anal.
C16H13ClN4O2: C, 58.45; H, 3.99; N, 17.04. Found: C,
58.09; H, 4.04; N, 16.95.
7-Chloro-4-(4-chlorophenethylamino)-6-nitroquinazoline
15
(11i). Asuspension of 7-chloro-6-nitro-4-quinazolone
(1.00 g, 4.43 mmol) in thionyl chloride (18 mL) contain-
ing 1 drop of DMF was heated at reflux for 2 h to give a
clear solution. Excess thionyl chloride was removed
under reduced pressure to provide crude 4,7-dichloro-6-
nitroquinazoline, which was used directly. To a mixture
of the crude chloro compound and triethylamine
(742 mL, 5.32 mmol) in i-PrOH (22 mL) was added
4-chlorophenethylamine (745 mL, 5.32 mmol). The
resulting mixture was stirred at ambient temperature
and concentrated in vacuo, and partitioned between
CH2Cl2 and 5% aqueous citric acid solution. The
organic layer was washed successively with 1 N NaOH,
water, and brine, and then dried over Na2SO4. The
solution was concentrated under reduced pressure and
the residue was triturated with CH2Cl2/hexane (1:1).
The light yellow solid was filtered to give 11i (1.34 g,
83% yield): mp 248–250 ꢀC; 1H NMR (CDCl3) d 8.75 (s,
1H), 8.27 (s, 1H), 7.99 (s, 1H), 7.32 (d, J=8.1 Hz, 2H),
7.19 (d, J=8.1 Hz, 2H), 5.91 (t, J=5.5 Hz, 1H), 3.95 (dt,
J=5.5, 7.0 Hz, 2H), 3.03 (t, J=7.0 Hz, 2H); MS (TOF)
m/z 364 (M + H)+; anal. calcd for C16H12Cl2N4O2: C,
52.91; H, 3.33; N, 15.43. Found: C, 53.23; H, 3.48; N,
15.50.
ꢁ
calcd for C16H15ClN4 3H2O: C, 54.47; H, 5.14; N,
15.88. Found: C, 54.39; H, 5.12; N, 15.61.
4-(4-Chlorophenethylamino)-6-hydroxyquinazoline (11e).
Amixture of 11d (148 mg, 0.38 mmol) and 5% palla-
dium on carbon (20 mg) in MeOH (5.0 mL) was stirred
under an atmosphere of H2 gas using a balloon reser-
voir. The reaction mixture was stirred at ambient tem-
perature for 5 h. The catalyst was filtered off over a pad
of Celite, and the pad was washed with MeOH. The
combined filtrate was concentrated under reduced pres-
sure. The residue was triturated with water, and the
precipitate was collected, washed with water, and dried
in air. The solid was filtered and dried at 40 ꢀC under
high vacuum to give 11e (85 mg, 75% yield): mp 188–
190 ꢀC; H NMR (DMSO-d6) d 9.95 (s, 1H), 8.37 (s,
1
1H), 8.17 (br s, 1H), 7.55 (d, J=9.5 Hz, 1H), 7.42 (d,
J=2.4 Hz, 1H), 7.36–7.26 (m, 5H), 3.74–3.69 (m, 2H),
2.96 (t, J=7.3 Hz, 2H); HR-FABMS m/z (M + H)+;
calcd for C16H14ClN3O: 300.0903. Found: 300.0912.
6-Acetamido-4-(4-chlorophenethylamino)quinazoline (11h).
To a solution of 11g (570 mg, 1.91 mmol) in DMF
(19 mL) were added acetic anhydride (270 mL,
2.86 mmol) and pyridine (231 mL, 2.86 mmol). The mix-
ture was stirred at 50 ꢀC for 2 h. The reaction mixture
was concentrated under reduced pressure, and the resi-
due was partitioned AcOEt and water. The organic
layer was washed with brine, and then dried over
Na2SO4. The solution was evaporated in vacuo, and the
residue was triturated with hexane. The white solid was
filtered and dried at 40 ꢀC under high vacuum to give
11h (470 mg, 72% yield): mp 253–255 ꢀC; 1H NMR
(DMSO-d6) d 10.19 (s, 1H), 8.42–8.39 (m, 2H), 8.28 (t,
J=5.3 Hz, 1H) 7.72–7.62 (m, 2H), 7.36–7.27 (m, 4H),
3.73 (dt, J=5.3, 7.3 Hz, 2H), 2.96 (t, J=7.3 Hz, 2H),
2.10 (s, 3H); MS (TOF) m/z 341 (M +H)+; anal. calcd
for C18H17ClN4Oꢁ 0.1H2O: C, 63.10; H, 5.03; N, 16.35.
Found: C, 62.89; H, 5.01; N, 16.22.
6-Amino-7-chloro-4-(4-chlorophenethylamino)quinazoline
(11j). Iron powder (92 mg; freshly washed with 1 N HCl
followed by distilled water) was added in portions to a
refluxing solution of 11i (150 mg, 0.41 mmol) in EtOH/
H2O (2:1, 18 mL) containing glacial AcOH (268 mL).
The resulting suspension was heated at reflux with vig-
orous stirring for 30 min, then cooled, basified with
concentrated NH4OH and EtOH, and combined filtrate
was concentrated under reduced pressure, diluted with
water and extracted with AcOEt. The combined organic
extracts were dried over Na2SO4 and concentrated
under reduced pressure. The residue was triturated with
AcOEt and the light yellow solid was filtered to give 11j
(28 mg, 20% yield): mp 172–174 ꢀC; 1H NMR (DMSO-d6)
d 8.31 (s, 1H), 8.26 (t, J=5.3 Hz, 1H), 7.63 (s, 1H), 7.36–
7.26 (m, 5H), 5.72 (br s, 2H), 3.72 (dt, J=5.3, 7.3 Hz, 2H),
2.95 (t, J=7.3 Hz, 2H); MS (TOF) m/z 333 (M + H)+;
ꢁ
anal. calcd for C16H14Cl2N4 0.9H2O: C, 55.00; H, 4.30; N,
16.03. Found: C, 55.08; H, 4.27; N, 15.82.
6-Amino-4-(4-phenoxyphenethylamino)quinazoline (11q).
Asuspension of 6-nitro-4-quinazolone (300 mg,
1.57 mmol) in thiony chloride (10 mL) containing 1 drop
of DMF was refluxed for 3 h until it became clear. The
excess thiony chloride was removed under reduced
pressure. To a mixture of the resulting crude 4-chloro-6-
6-Amino-4-(4-chlorophenethylamino)quinazoline (11g).
Similarly to the procedure described for 11j, the title
compound was prepared starting from 11f. After pur-
ification, 11g was obtained as a white solid (92% yield):

390
M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
ꢁ
nitroquinazoline and triethylamine (262 mL, 1.88 mmol)
in i-PrOH (15 mL) was added 4-phenoxy-
C17H19ClN4 2.0H2O: C, 58.20; H, 6.03; N, 15.97.
Found: C, 58.23; H, 5.80; N, 15.90.
phenethylamine (368 mL, 1.88 mmol). The resulting
mixture was stirred at ambient temperature for 1 h and
concentrated in vacuo. The residue was partitioned
between CH2Cl2 and 5% aqueous citric acid solution.
The organic layer was washed successively with 1 N
NaOH, water, and brine, and then dried over Na2SO4.
The solution was concentrated under reduced pressure
and the residue was triturated with CH2Cl2/hexane
(1:1). The light yellow solid was filtered to give 6-nitro-
4-(4-phenoxyphenethylamino)quinazoline (403 mg, 66%
yield for 2 steps from 6-nitro-4-quinazolone).
6-Amino-4-(4-hydroxyphenethylamino)quinazoline (11o).
Similarly to the procedure described for 11q, the title
compound was prepared starting from 6-nitro-4-quin-
azolone. After purification, 11o was obtained as a white
solid (21% yield for 3 steps from 6-nitro-4-quinazolone):
mp 115–117 ꢀC; H NMR (DMSO-d6) d 9.17 (br s, 1H),
1
8.20 (s, 1H), 7.73 (t, J=5.5 Hz, 1H), 7.40 (d, J=8.9 Hz,
1H), 7.14–7.10 (m, 1H), 7.06–7.01 (m, 3H), 6.68 (d,
J=8.4 Hz, 2H), 5.40 (br s, 2H), 3.63 (dt, J=5.5, 7.4 Hz,
2H), 2.81 (t, J=7.4 Hz, 2H); HR-FABMS m/z (M +
H)+; calcd for C16H16N4O: 281.1402. Found: 281.1410.
Next, iron powder (58 mg; freshly washed with 1 N HCl
followed by distilled water) was added in portions to a
refluxing solution of the above 6-nitroquinazoline
(100 mg, 0.26 mmol) in EtOH/H2O (2:1, 12 mL) con-
taining glacial AcOH (168 mL). The resulting suspension
was heated at reflux with vigorous stirring for 30 min,
then cooled, basified with concentrated NH4OH and
EtOH, and combined filtrate was concentrated under
reduced pressure, diluted with water and extracted with
AcOEt. The combined organic extracts were dried over
Na2SO4 and concentrated under reduced pressure. The
residue was triturated with AcOEt/hexane (1:1). The
light yellow solid was filtered to give 11q (72 mg, 77%
6-Amino-4-(4-methoxyphenethylamino)quinazoline (11p).
Similarly to the procedure described for 11q, the title
compound was prepared starting from 6-nitro-4-quin-
azolone. After purification, 11p was obtained as a white
solid (17% yield for 3 steps from 6-nitro-4-quinazo-
lone): mp 230 ꢀC (Dec.); 1H NMR (DMSO-d6) d 8.23 (s,
1H), 7.85 (t, J=5.1 Hz, 1H), 7.42 (d, J=8.9 Hz, 1H),
7.19–7.11 (m, 3H), 7.03 (d, J=2.2 Hz, 1H), 6.87–6.84
(m, 2H), 5.44 (br s, 2H), 3.71 (s, 3H), 3.68–3.63 (m, 2H),
2.88 (t, J=7.6 Hz, 2H); MS (TOF) m/z 295 (M + H)+.
Anal. calcd for C17H18N4Oꢁ 0.7H2O: C, 66.52; H, 6.14;
N, 18.25. Found: C, 66.74; H, 6.27; N, 18.04.
yield): mp 168–170 ꢀC; H NMR (DMSO-d6) d 8.33 (br
1
s, 2H), 7.45 (d, J=8.9 Hz, 1H), 7.40–7.34 (m, 2H), 7.28
(d, J=8.6 Hz, 2H), 7.20–7.07 (m, 3H), 6.98–6.92 (m,
4H), 5.59 (br s, 2H), 3.79–3.72 (m, 2H), 2.95 (t,
J=7.3 Hz, 2H); MS (TOF) m/z 357 (M + H)+; anal.
calcd for C22H20N4Oꢁ 1.0H2O: C, 70.57; H, 5.65; N,
14.96. Found: C, 70.48; H, 5.60; N, 14.87.
Biology
NF-ꢀB assay.12 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 6-well plates (2ꢄ106/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 (2ꢄ105/well). Test compounds were dis-
solved in DMSO and added at the appropriate con-
centrations to the 96-well plates containing the cells, and
the plates were then incubated at 37ꢀC for 1 h. For
induction of transcription, 10ng/mL of PMAand 100 mg/
mL of PHAwere added to each well, and the cells were
incubated for an additional 6 h at 37ꢀC. The culture
media were removed, and cell lysis buffer containing luci-
ferase 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 lumines-
cence was immediately measured with a Packard Top-
count (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 nontransfected cells (2ꢄ105/
well), and incubated the plates at 37ꢀC for 24h. Cell via-
bility was measured by using the MTS assay (Promega).
6-Amino-4-(phenethylamino)quinazoline
hydrochloride
(11m). Similarly to the procedure described for 11q, the
crude compound was prepared starting from 6-nitro-4-
quinazolone. To a mixture of the above compound in
EtOH (14 mL) was added 12 N HCl (66 mL). The mix-
ture was stirred at ambient temperature for 10 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 hydrochloride salt as a pale
yellow powder (63% yield for 4 steps from 6-nitro-4-
quinazolone): mp 223–225 ꢀC; H NMR (DMSO-d6) d
1
9.90 (br s, 1H), 8.67 (s, 1H), 7.67 (d, J=8.6 Hz, 1H),
7.42–7.21 (m, 7H), 3.93–3.86 (m, 2H), 3.00 (t,
J=7.3 Hz, 2H); MS (TOF) m/z 265 (M + H)+; anal.
ꢁ
calcd for C16H17ClN4 2.0H2O: C, 57.06; H, 5.69; N,
16.63. Found: C, 56.97; H, 5.41; N, 16.56.
6-Amino-4-(4-methylphenethylamino)quinazoline hydro-
chloride (11n). Similarly to the procedure described for
11m, the title compound was prepared starting from 6-
nitro-4-quinazolone. After purification, 11n was
obtained as a white solid (54% yield for 4 steps from 6-
nitro-4-quinazolone): mp 240 ꢀC (Dec.); 1H NMR
(DMSO-d6) d 9.88 (t, J=5.1 Hz, 1H), 8.67 (s, 1H), 7.67
(d, J=8.9 Hz, 1H), 7.42–7.35 (m, 2H), 7.17–7.09 (m,
4H), 3.90–3.83 (m, 2H), 2.95 (t, J=7.3 Hz, 2H), 2.26 (s,
3H); MS (TOF) m/z 279 (M + H)+; anal. calcd for
Inhibition of LPS-induced TNF-ꢁ production by murine
splenocytes. Splenocytes were prepared by mechanical
disruption of the spleen from BALB/c mice with a metal

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 383–391
391
sieve, followed by hypoosmotic lysis of red blood cells,
filtration through nylon gauze, and extensive washing
with PBS. The cells were resuspended in RPMI1640
(10% FCS, 100 U/mL of penicillin, and 100 mg/mL of
streptomycin) and plated in 96-well plates (1ꢄ106/well).
Then, cells were cultured in the presence of 3 mg/mL
LPS (Escherichia coli, 0111:B4, DIFCO) and test com-
pounds for 18 h at 37 ꢀC in a 5% CO2 atmosphere. The
culture media were stored at ꢂ20 ꢀC until used for
determination of TNF-a production. The levels of
TNF-a in the culture media were determined by ELISA
(Genzyme TECNE). The 50% inhibitory concentration
(IC50) values were calculated by a nonlinear regression
method. Similarly to the method described for Jurkat
cells, the cell viability for murine splenocytes was mea-
sured by using the MTS assay.
Florquin, S.; Goldman, M. Infect. Immun. 1996, 64, 3443. (d)
Sayegh, M. H.; Turka, L. A. N. Engl. J. Med. 1998, 338, 1813.
(e) Rovira, P.; Mascarell, L.; Truffa-Bachi, P. Curr. Med.
Chem. 2000, 7, 673. (f) Durham, S. R.; Till, S. J.; Corrigan,
C. J. J. Allergy Clin. Immunol. 2000, 106, S221. (g) Djuka-
novic, R. Clin. Exp. Allergy 2000, 30, 46. (h) Prinz, J. C. Clin.
Exp. Dermatol. 1999, 24, 291. (i) Asadullah, K.; Volk, H-.D.;
Sterry, W. Trends Immunol. 2002, 23, 47.
2. (a) Hill, C. S.; Treisman, R. Cell 1995, 80, 199. (b) Karin,
M. Curr. Opin. Cell Biol. 1994, 6, 415.
3. (a) Baeuerle, P. A.; Henkel, T. Ann. Rev. Immunol. 1994,
12, 141. (b) Vossen, A. C. T. M.; Savelkoul, H. F. J. Mediat.
Inflamm. 1994, 3, 403. (c) Baeuerle, P. A.; Baltimore, D. Cell
1996, 87, 13. (d) Blackwell, T. S.; Christman, J. W. Am. J.
Respir. Cell Mol. Biol. 1997, 17, 3. (e) May, M. J.; Ghosh, S.
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Anti-inflammatory effect on carrageenin-induced paw
edema.13 Paw edema was induced in male SD rats (7
weeks old, Charles River Japan Inc.). This was done by
subcutaneously injecting a 1% suspension of carragee-
nin (type 1, Sigma) in saline (0.1 mL) into the plantar
surface of the right hind paw. Compounds 3 and 11q
were intraperitoneally administered 15 min prior to the
injection of the carrageenin. These compounds were
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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. Hideo Omukai for their technical assistance in
obtaining the biological data.
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