Structure-activity relationships of quinazoline derivatives: Dual-acting compounds with inhibitory activities toward both TNF-α production and T cell proliferation

Article abstract of DOI:10.1016/S0960-894X(00)00718-6

We synthesized 4-chlorophenethylaminoquinazoline derivatives and evaluated their inhibitory activities toward both TNF-α production and T cell proliferation responses. Compound 2f, containing a piperazine ring at the C(7)-position of the quinazoline ring, exhibited more potent inhibitory activities toward both than the lead compound 1a. A smaller N-substituent in the piperazine ring was required for inhibition of TNF-α production.

Full text of DOI:10.1016/S0960-894X(00)00718-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 545±548
Structure±Activity Relationships of Quinazoline Derivatives:
Dual-Acting Compounds with Inhibitory Activities Toward
Both TNF-ꢀ Production and T Cell Proliferation
Masanori Tobe, Yoshiaki Isobe, Hideyuki Tomizawa, Mitsuhiro Matsumoto,
Fumihiro Obara, Takahiro Nagasaki and Hideya Hayashi*
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, Toda-shi, Saitama 335-8502, Japan
Received 4October 2000; accepted 8 December 2000
AbstractÐWe synthesized 4-chlorophenethylaminoquinazoline derivatives and evaluated their inhibitory activities toward both
TNF-a production and T cell proliferation responses. Compound 2f, containing a piperazine ring at the C(7)-position of the qui-
nazoline ring, exhibited more potent inhibitory activities toward both than the lead compound 1a. A smaller N-substituent in the
piperazine ring was required for inhibition of TNF-a production. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
essential for anti-rheumatic agents to have the property
of the correction of T cell mediated abnormal immune
responses accompanied with TNF-a inhibitory activity.
Glucocorticoids show the inhibitory activities toward
both TNF-a production and T cell proliferation, but
long-time use of glucocorticoids is limited because of its
many side e€ects, such as atrophia cutis and infection.
Rheumatoid arthritis (RA) is considered to be char-
acterized by joint destruction as a result of chronic
synovial in¯ammation, and many reseachers have
described the importance of the T cell mediated auto-
immune response in the initiation and maintenance of
this in¯ammation.1 In particular, T cell (especially Th1)
proliferation has been shown to be a possible mechanism
by which in¯ammation is enhanced in RA.2
Therefore, nonsteroidal agents having inhibitory activities
toward both TNF-a production and T cell proliferation
would be expected to be e€ective in the treatment of
RA. To develop this type of nonsteroidal compound, we
conducted a random screening and found lead compound
1a (Fig. 1). In this study, we describe and discuss the SARs
of 4-chlorophenethylaminoquinazoline derivatives as a
dual inhibitor.
Tumor necrosis factor-a3 (TNF-a), among a variety of
cytokines, predominantly functions as the principal
mediator in RA. TNF-a is mainly produced by activated
macrophages/monocytes and induces the production of
several key in¯ammatory cytokines such as IL-1b, IL-6,
and GM-CSF,4 and thereby enhances in¯ammation.
Recently, TNF soluble receptor (EnbrelTM)5 and TNF
antibody (RemicadeTM)6 have been reported to have an
excellent ecacy in RA patients. However, these bio-
pharmaceuticals are very expensive and dicult to
administer orally. On the other hand, it has been
reported that many low-molecular-weight compounds
have an inhibitory activity toward the production of
TNF-a.7 Among them, pentoxifylline has shown a weak
ecacy to RA patients under the recent clinical trial.8
According to this evidence, we considered that it is
Chemistry and Biology
The compounds described in this study are listed in
Tables 1 and 2, and the methods used for their synthesis
are outlined in Schemes 1 and 2. Compounds 1a±1t were
prepared from the corresponding 2-aminobenzamides as
shown in Scheme 1. Catalytic hydrogenation of 1a, 1k
and 1m gave 1f, 1j and 1n, respectively. Compounds 1g
and 1h were obtained by the alkylation of 1f with the
corresponding alkyl halides. Formylation of 1f with
acetic anhydride in formic acid a€orded 1i. The reaction
of 1s with sodium methoxide provided 1t.
*Corresponding author. Tel.: +81-48-433-2194; fax: +81-48-433-1605;
e-mail: hhayashi@j-energy.co.jp
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00718-6

546
M. Tobe et al. / Bioorg. Med. Chem. Lett. 11 (2001) 545±548
measured using human peripheral blood mononuclear
cells (PBMCs) stimulated by LPS as previously repor-
ted.10,11 The cytotoxicity for human PBMCs was mea-
sured by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carb-
oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt] assay.12 T cell proliferatrion was determined
by MTS assay using concanavalin A (Con A)-stimulated
mice spleen cells according to the previous report.13,14
Figure 1. Structure of compound 1a.
Synthetic routes of 2a±2m are shown in Scheme 2.
Nitration of 3, followed by removal of the unwanted 8-
nitro isomer by recrystallization from acetic acid, gave
the key intermediate 7-chloro-6-nitro-4-quinazolone
(4).9 Compound 5 was prepared by the chlorination of 4
with phosphorus oxychloride. Subsequently, compound 5
was converted to 2a by selective amination at the 4-posi-
tion of the quinazoline ring. Then, compounds 2b±2m
were obtained from 2a by the displacement of the acti-
vated chlorine with the desired nucleophile such as the
amines or the sodium alkoxides (Method A). This
selectivity described above was con®rmed by another
route of synthesis, method B. The same compounds were
a€orded by method A and method B in several cases.
Results and Discussion
We synthesized various compounds to optimize the
substituents on the quinazoline ring. As shown in Table
1, 6- or 7-substituted quinazoline derivatives (1a, 1b,
1d±1f, 1j, 1m, 1n, 1p) inhibited the T cell proliferation,
but only 1a also showed inhibitory activity toward
TNF-a production. The methyl group (1q) or hydrogen
atom (1b) were preferable as C(2) substituents for
blocking T cell proliferation, but other groups (1r±1t)
were not suitable.
Table 2. Inhibition of TNF-a production and T cell proliferation by
compounds 2a±2m
A series of these compounds was evaluated for their
abilities to inhibit both TNF-a production and T cell
proliferation. Inhibition of TNF-a production was
Table 1. Inhibition of TNF-a production and T cell proliferation by
compounds 1a±1t
IC50 (mM)
Compound
R7
TNF-aa Con Ab MTSc
2a
2b
2c
Cl
EtNH
Me2N
>30
>30
19.48.6
>10
>10
27.3
>30
>30
2d
2e
2f
>30
24.8
0.8
>10
8.3
>30
22.5
12.1
9.8
IC50 (mM)
Compound
R2
R6
R7
TNF-aa Con Ab MTSc
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
NO2
H
C l
B r
M e
NH2
NHMe
NHBn
NHCHO
O H
OBn
H
H
H
F
OMe
H
H
H
H
H
H
H
H
H
H
14.6
>3 0
>3 0
>3 0
>3 0
>30
>30
>30
>30
>3 0
>30
>30
>3 0
>3 0
>30
>30
>3 0
>30
>30
>30
3.2
0 . 5
>1 0
4. 8
0 . 8
2.6
>10
>10
>10
>30
>3 0
>3 0
>3 0
>3 0
>30
>30
>30
>30
1.1
2g
2h
2i
2.1
0.9
1.41.2
>30
>30
>30
22.1
4.3
>10
>10
4.5
>30
>30
1j
1k
1l
H
H
1 . 4>3 0
2j
>10
>10
5 . 6
3 . 4>3 0
nt
1.4>30
0 . 2
>10
1.2
>30
>30
>3 0
Cl
NO2
NH2
F
OMe
H
H
H
H
1m
1n
1o
1p
1q
1r
1s
1t
2k
2l
2m
MeO
EtO
>30
>30
>30
>30
>30
0.5
nt
nt
nt
>30
>30
>30
>30
>30
>30
0.04
>30
Aurano®n
d-Pencillamine
Salazosulfapyridine
Rolipram
M e
Ph
Cl
OMe
>3 0
>30
>30
>30
nt
H
H
H
>10
>10
0.005
>10
Dexamethasone
0.01
nt: not tested.
aIC50 of TNF-a production from human PBMCs stimulated by LPS.
bIC50 of Con A-induced proliferation of mice spleen cells.
cIC50 of the growth in human PBMCs stimulated by LPS.
nt: not tested.
aIC50 of TNF-a production from human PBMCs stimulated by LPS.
bIC50 of Con A-induced proliferation of mice spleen cells.

M. Tobe et al. / Bioorg. Med. Chem. Lett. 11 (2001) 545±548
547
Scheme 1. Synthesis of compounds 1a±1t. Reagents and conditions: (a) R2CO2Et, EtONa, EtOH, re¯ux or HC(OMe)3, 12 N HCl; (b) phosphorus
oxychloride, 90 ꢀC; (c) 4-chlorophenethylamine, Et3N, i-PrOH; (d) H2, Pd±C, EtOH; (e) MeI, K2CO3, DMF; (f) benzyl bromide, K2CO3, DMF; (g)
HCO2H, Ac2O, 60 ꢀC; (h) MeONa, MeOH, re¯ux.
Scheme 2. Synthesis of compounds 2a±2m. Reagents and conditions: (a) HNO3/H2SO4 (1:1, v/v), 80 ꢀC; (b) phosphorus oxychloride, 90 ꢀC; (c) 4-
chlorophenethylamine, Et3N, i-PrOH; (d) amines, i-Pr2NEt, n-BuOH, re¯ux or sodium alkoxides, alcohols, re¯ux.
Next, we investigated the e€ect of substituents at the
C(7)-position in the 6-nitroquinazolines. The results are
shown in Table 2. Compound 2f, having a piperazine
ring, exhibited inhibitory activities toward both TNF-a
production (IC50=0.8 mM) and T cell proliferation
(IC50=1.1 mM). In addition, this compound did not
show cytotoxicity at 5 mM toward human PBMCs sti-
mulated by LPS. In contrast, both inhibitory activities
of compounds 2c, 2d and 2e containing dimethylamine,
piperidine, and morpholine, respectively, were drasti-
cally decreased. Compound 2h, having an N-methylpi-
perazine ring, showed almost the same activity as 2f.
biological property of compound 2f could be considered
as a novel unique anti-in¯ammatory agent. A substitution
study at the C(4)-position of 2f and elucidation of the
mechanism of action of this compound are underway.
Conclusion
We conducted a study to optimize lead compound 1a,
which showed moderate inhibitory activity toward
TNF-a production and suppression of T cell proliferation.
As a result, we found that compound 2f, containing a
piperazine ring at the C(7)-position of the quinazoline
ring, exhibited more potent inhibitory activities than 1a
toward both parameters. Compound 2f is thus a new
candidate having inhibitory potency toward both TNF-a
production and T cell proliferation. At present, we are now
investigating the SARs of 4-substituted-7-piperazinyl-
quinazolines.
Moreover, compounds 2i±2k, in which the NH group of
the piperazine ring was blocked by bulky substituents,
had diminished activities. These results suggest that not
only the presence of a piperazine ring but also a smaller
N-substituent in piperazine are necessary to inhibit
TNF-a production. Compounds 2l and 2m, containing
a methoxy group and ethoxy one, respectively, at the
C(7)-position did not show an activity toward TNF-a
production.
Acknowledgements
Furthermore, anti-rheumatic drugs such as aurano®n,
d-penicillamine, and salazosulfapyridine were not
e€ective for TNF-a inhibitory activity. Therefore, the
The authors are grateful to Dr. Satoru Misawa and
Mr. Tsutomu Mimoto for useful suggestions and
encouragement during the course of this research.

548
M. Tobe et al. / Bioorg. Med. Chem. Lett. 11 (2001) 545±548
D.; Carson, D. A.; Cottam, H. B. J. Med. Chem. 1999, 42,
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