Structure-activity relationships of 6-fluoroquinazolines: Dual-Acting compounds with inhibitory activities toward both TNF-α production and T cell proliferation

Article abstract of DOI:10.1016/S0968-0896(02)00338-3

We synthesized various 6-fluoro-7-(1-piperazino)quinazolines based on the structure of 1 and evaluated their inhibitory activities toward both TNF-α production and T cell proliferation responses. Among these compounds, 7a, having the 3,4-(methylenedioxy)phenyl moiety at the C(4)-position of the quinazoline ring, showed both inhibitory activities. Furthermore, the oral treatment with 7a exhibited an anti-inflammatory effect in rats with adjuvant arthritis as well as an inhibitory activity toward LPS-induced TNF-α production.

Full text of DOI:10.1016/S0968-0896(02)00338-3

Bioorganic & Medicinal Chemistry 11 (2003) 609–616
Structure–Activity Relationships of 6-Fluoroquinazolines: Dual-
Acting Compounds with Inhibitory Activities Toward Both TNF-ꢀ
Production and T Cell Proliferation
Masanori Tobe, Yoshiaki Isobe, Hideyuki Tomizawa, Takahiro Nagasaki,
Fumihiro Obara and Hideya Hayashi*
Pharmaceuticals and Biotechnology Laboratory, Japan Energy Corporation, Toda-shi, Saitama 335-8502, Japan
Received 18July 2002; accepted 22 July 2002
Abstract—We synthesized various 6-fluoro-7-(1-piperazino)quinazolines based on the structure of 1 and evaluated their inhibitory
activities toward both TNF-a production and T cell proliferation responses. Among these compounds, 7a, having the
3,4-(methylenedioxy)phenyl moiety at the C(4)-position of the quinazoline ring, showed both inhibitory activities. Furthermore,
the oral treatment with 7a exhibited an anti-inflammatory effect in rats with adjuvant arthritis as well as an inhibitory activity
toward LPS-induced TNF-a production.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
immune responses as well as to inhibit TNF-a activ-
ity. Glucocorticoids show inhibitory activities toward
both TNF-a production and T cell proliferation. How-
ever, the long-term use of steroids is well known to give
rise to several side effects including infection and
osteoporosis.
The demonstration of dichotomy between Th1 and
Th2 cells represents one of the most important
advances in immunology. T cells within the rheuma-
toid arthritis (RA) synovium show mainly a Th1
pattern of cytokine production.1 Therefore, Th1 cells
play an important role in the pathogenesis of RA,
and their activation has been shown to be a possible
mechanism by which inflammation is enhanced in
RA.2
In a previous paper, we demonstrated that 6-nitro-
quinazoline derivative 1 exhibited inhibitory activities
toward both TNF-a production and T cell prolifera-
tion; however, compound 1 did not show an oral
activity on TNF-a production, presumably due to its
poor bioavailability.5 In order to find novel quinazo-
line derivatives exhibiting oral activities, we per-
formed further modifications starting from 1 by
focusing on the replacement of the nitro group with
the fluoro group, which is an electron-withdrawing
group similar to the nitro one, at the C(6)-position of
the quinazoline ring.
Among the proinflammatory cytokines, tumor necro-
sis factor-a (TNF-a) has been shown to play an
essential role in the disease process of RA.3 The
blockade of TNF-a by anti-TNF-a antibody (Remi-
cadeTM) and soluble TNF-a receptor (EnbrelTM) has
been proved to be dramatically effective in improving
both subjective and objective findings in RA
patients.4
This paper describes the results of our study on the
structure–activity relationship of 6-fluoroquinazolines,
which was conducted on the basis of the replacement of
the phenyl group at the C(4)-position of 1 with other
heteroaryl groups and alteration of the methylene chain
length (Fig. 1). In addition, we report the anti-inflam-
matory effect of a selected compound on a rat model of
adjuvant arthritis.
Based on the above information, we speculated that
it is necessary for anti-rheumatic agents to possess
the ability to block abnormal
T
cell-mediated
*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)00338-3

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M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 609–616
Figure 1. Design of 6-fluoro-7-(1-piperazino)quinazolines.
Regioselective displacement of the 7-fluorine atom in 2
with 1-formylpiperazine gave compound 3. The struc-
ture of 3 was established on the basis of the long-range
correlations observed in the COLOC spectrum of 3: H-5
(d 8.14, d, JHÀF=12.2 Hz) correlated with C-4 (d 160.8,
s), whereas H-8( d 7.45, d, JHÀF=6.2 Hz) did not.
Table 1. Inhibition of TNF-a production and T cell proliferation by
compounds 7a–7d
Chlorination of 3 with phosphorus oxychloride gave 4,
which was treated with the corresponding amines to
provide 5 in low yields (3–8%) through two steps. The
reason for this poor yield was due to the production of
many by-products. Deprotection of the N-formyl group
of 5 using 5 N NaOH–EtOH led to the 7-(1-piper-
azino)quinazolines (Method A).
Compd
R6
n
IC50 (mM)
TNF-aa
Con Ab
MTSc
1
7a
7b
7c
NO2
F
F
F
F
1
1
0d
2
0.082.1
0.5
4.7
2.1
3.2
7.2
5.1
7.8
6.2
8.4
>30
>30
>30
>30
>30
0.04
From the above-mentioned result, we considered that
Method A was a rather troublesome procedure. There-
fore, we selected alternatively a more convenient and
facile procedure for the synthesis of the 7-(1-piper-
azino)quinazolines (Method B). The key intermediate 6
was prepared by the chlorination of 2 with thionyl
chloride. Compound 6 was subsequently treated with
the corresponding amines to give 4-substituted-6,7-
difluoroquinazolines. Reaction of these compounds
with piperazine in the presence of Hunig’s base pro-
vided the objective compounds (7a–7d and 8a–8j) in
good yield. Regioselectivity in Method B was confirmed
by another route of synthesis, Method A. The same
compounds were afforded by Methods A and B in sev-
eral cases.
7d
Rolipram
Dexamethasone
3
0.5
0.01
>10
0.005
aIC50 for inhibition of TNF-a production from human PBMCs sti-
mulated by LPS.
bIC50 for inhibition of Con A-induced proliferation of mouse spleen
cells.
cIC50 for the growth inhibition of human PBMCs stimulated by LPS.
d0 means the anilino derivative.
Chemistry
The 6-fluoro-7-(1-piperazino)quinazolines described in
this study are listed in Tables 1 and 2, and the general
synthesis of the compounds is shown in Scheme 1.
Scheme 1. Synthesis of compounds 7a–7d and 8a–8j. Reagents and conditions: (a) 1-formylpiperazine, N,N-diisopropylethylamine, i-PrOH, 70 ꢀC,
6 h; (b) phosphorus oxychloride, 100 ꢀC, 30 min; (c) RNH2, triethylamine, EtOH, reflux, 4–8h; (d) 5 N NaOH, EtOH, reflux, 3–4 h; (e) SOCl 2, DMF,
reflux, 2 h; (f) RNH2, triethylamine, i-PrOH, 6–10 h; (g) piperazine, N,N-diisopropylethylamine, n-BuOH, 110 ꢀC, 30–40 h.

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 609–616
Table 2. Inhibition of TNF-a production and T cell proliferation by
compounds 8a–8j
611
Results and Discussion
The compounds listed in Tables 1 and 2 were evaluated
for their abilities to inhibit both TNF-a production and
T cell proliferation, as previously reported.5 Also inclu-
ded in Tables 1 and 2 are the results of cytotoxicity
experiments using the MTS assay in human PBMCs.6
In general, some compounds having the nitro group are
not preferable for a drug candidate because of the pos-
sibility that the nitro group might change into the
mutagenetic nitroso one in vivo. Thus, our initial efforts
focused on investigating the effects of the replacement
of the nitro group with the fluorine atom at the C(6)-
position in 1 (Table 1). The inhibitory activities of the 6-
fluoro derivative (7a) were several fold weaker than
those of the 6-nitro one (1); however, the oral bioavail-
ability (46%) of 7a was much greater than that of 1
(1%). Moreover, 7a showed reduced cell growth inhibi-
tion in human PBMCs stimulated by LPS. On the basis
of these data, we performed the structure–activity rela-
tionship study by using the 6-fluoroquinazolines.
Compound
R
IC50 (mM)
Con Ab
5.1
TNF-aa
MTSc
7a
0.5
>30
8a
8b
8c
8d
8e
8f
1.2
2.2
1.3
6.5
2.6
11.2
4.4
3.7
2.5
8.2
8.1
>30
>30
27.2
>30
>30
>30
>30
>30
>30
6.2
To explore the effects of varying the length of the linker
between the quinazoline and the phenyl group at the
C(4)-position, we prepared compounds 7b–7d (Table 1).
The maximum inhibitory activities toward TNF-a pro-
duction and T cell proliferation were obtained when the
spacer was the methylene chain (7a).
>10
>10
>10
>10
>10
8
Next, we investigated the structure–activity relationship
at the C(4)-position by replacing the 3,4-methylene-
dioxyphenyl group with several heteroaryl substituents
(Table 2). The benzyl analogues (8a–8c) resulted in a 2–
4-fold loss in TNF-a inhibitory activity as compared
with the activity of 7a. The pyridyl (8d–8g), the furyl
(8h), and the thienyl (8i) analogues also exhibited
decreased or diminished activities. The naphthyl analo-
gue (8j) potently inhibited TNF-a production with an
IC50 value of 0.8 mM, while this analogue showed cell
growth inhibition (IC50=6.4 mM).
8g
8h
8i
We selected compound 7a from the results of the above-
mentioned in vitro study and evaluated its activity on
TNF-a production in vivo (Table 3).5 Also included in
Table 3 are the results of the pharmacokinetic study on
compounds 1 and 7a conducted on male rats. Orally
administered compound 7a showed inhibitory activity
toward LPS-induced TNF-a production, with an ED50
of 28mg/kg, whereas compound 1 exhibited low oral
activity in this model. Compound 7a showed a plasma
concentration of over 1.0 mM, and had 46% bioavail-
8j
0.84.86.4
Rolipram
Dexamethasone
0.5
0.01
>10
>30
0.005
0.04
aIC50 for inhibition of TNF-a production from human PBMCs sti-
mulated by LPS.
bIC50 for inhibition of Con A-induced proliferation of mouse spleen
cells.
cIC50 for the growth inhibition of human PBMCs stimulated by LPS.
Table 3. Effects of 1 and 7a on LPS-induced TNF-a production in mice, and pharmacokinetic profile in rats
Compd
ED50 or % of TNF-a
Pharmacokinetic profileb
inhibition (mg/kg/po)a
Cmax (mM)
Tmax (h)
AUC (mM min)
F (%)
1
7a
18% @ 30
281.4
0.04
3
2
324
40
46
1
aED50 value was determined from dose–response curve of TNF-a inhibition. Compounds were evaluated as their corresponding hydrochroride salts,
and administered orally to mice 0.5 h prior to the LPS challenge.
bCompounds were examined for pharmacokinetics when orally administered to rats in water (30 mg/kg).

612
M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 609–616
ability after oral administration in rats. On the other
hand, the plasma level of was very low
(Cmax=0.04 mM), and its oral bioavailability was poor
(1%). Therefore, we considered that the improved oral
bioavailability of 7a compared with that of 1 reflects its
greater inhibitory activity on TNF-a production in vivo.
inhibitors. In addition, 7a did not show any inhibitory
activity toward cyclooxygenase-1 (À5%), cyclooxygen-
ase-2 (2%), constitutive NO synthase (À7%), or indu-
cible NO synthase (À7%); the binding affinity for the
adenosine A2A receptor was zero at 10 mM in vitro.13
Therefore, we are currently investigating some mechan-
istic targets of 7a other than the above-mentioned
molecular targets in more detail.
1
To further confirm the anti-inflammatory effect of 7a,
we evaluated it using a rat model of adjuvant-induced
arthritis (Fig. 2).7 Compound 7a (30 mg/kg, po) sig-
nificantly reduced the increase in hind paw volume on
day 15. In this model, elevated TNF-a levels and T cell
activation have been observed in serum and joint tis-
sue.8 Moreover, 7a also showed an inhibitory activity
on B cell proliferation (IC50=0.57 mM).9 This pharma-
cological profile suggests that 7a would be desirable for
the therapy of RA.
Conclusions
A series of 6-fluoro-7-(1-piperazino)quinazolines were
synthesized and evaluated for their inhibitory activities
toward both TNF-a production and T cell proliferation.
We found that introduction of a fluorine atom in place
of the nitro group at the C(6)-position resulted in
slightly weaker activity in vitro, but exhibited a good
oral bioavailability. Among them, 7a, having both inhi-
bitory activities in vitro, showed inhibition of TNF-a
production in vivo. Furthermore, 7a (30 mg/kg, po)
exhibited an anti-inflammatory effect in a rat model of
adjuvant arthritis and also had an inhibitory effect on B
cell proliferation. This pharmacological profile of 7a
suggests that this compound would be desirable for use
in the therapy of RA. We are continuing to search for
more potent analogues and to elucidate their mechan-
isms of action.
As the mechanistic target for 7a is unknown, a further
evaluation of its pharmacological characteristics was
undertaken. Nuclear factor-kB (NF-kB) is essential for
the transcriptional regulation of the proinflammatory
cytokines such as TNF-a, IL-1, and IL-6.10 However, 7a
did not influence NF-kB gene expression when tested at
10 mM in the luciferase assay system.11 Inhibition of
phosphodiesterase 4 (PDE 4) is also one of the
mechanisms by which TNF-a production may be
reduced.12 Indeed, 7a showed a moderate inhibitory
activity toward PDE 4 isolated from U937 cells
(IC50=1.6 mM). However, the IC50 of 7a for PDE 4
inhibition was higher than that for TNF-a inhibition in
vitro (cell culture). Although rolipram, which is well
known as a specific PDE 4 inhibitor, inhibited TNF-a
production in our assay systems, it did not suppress T
cell proliferation. These results suggest that the biologi-
cal properties of 7a are different from those of PDE 4
Experimental
Chemistry
General. All reagents and solvents were obtained from
commercial suppliers and were used without further
purification. Melting points were measured with a Buchi
1
535 melting point apparatus and were uncorrected. H
NMR and 13C NMR were recorded on a JEOL
GSX270 FT NMR spectrometer. Chemical shifts are
given in parts per million (ppm) using tetramethylsilane
as the internal standard for spectra obtained in DMSO-
d6 and CF3CO2D. TOF MS (time-of-flight mass spec-
trometry) was recorded on a Kompact MALDI 3 V
4.0.0 spectrometer. High-resolution mass spectra were
obtained on a JEOL JMS-700 mass spectrometer. Ele-
mental analyses were performed at the Toray Research
Center. Wakogel C-200 (Wako; 70–150 mm) was used
for column chromatography. 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).
6,7-Difluoro-4-quinazolone (2). To a mixture of 2-amino-
4,5-difluorobenzamide (13.0 g, 75.5 mmol) in trimethyl
orthoformate (490 mL) was added 12 N HCl (25.2 mL,
302 mmol) dropwise at 0 ꢀC. The reaction was stirred at
ambient temperature for 4 h. The reaction mixture was
concentrated under reduced pressure, and the residue
was poured into 5% aqueous NaHCO3 solution. The
precipitate was collected, washed with MeOH and
water, and dried in air. The white solid was filtered and
dried at 40 ꢀC under high vacuum to give 2 (12.2 g, 89%
Figure 2. Effect of 7a on hind paw swelling in rats with adjuvant-
induced arthritis. Compound 7a (HCl salt, 30 mg/kg) or indomethacin
(1 mg/kg) was orally administered for 15 days after adjuvant injection
into the rat’s right hind paw. Each volume was obtained by compar-
ison between the left paw volume in the arthritic group and that in the
control group. The results are expressed as the meanÆSEM of eight
rats per group. * p<0.05; ** p<0.01; *** p<0.001 versus arthritis
control (Dunnett’s test). *, Arthritis control; *, 7a; ~, indometha-
cin; ~, normal control.

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 609–616
613
yield): 1H NMR (DMSO-d6) d 12.48(br s, 1H, CONH),
8.15 (s, 1H, H-2), 8.03 (dd, JHÀF=10.5, 9.2 Hz, 1H, H-
5), 7.76 (dd, JHÀF=11.3, 7.3 Hz, 1H, H-8); MS (TOF)
m/z 183 (M+H)+. Anal. calcd for C8H4F2N2O: C,
52.76; H, 2.21; N, 15.38. Found: C, 52.80; H, 2.25; N,
15.56.
(TOF) m/z 410 (M+H)+. Anal. calcd for
.
C21H20N5FO3 0.8H2O: C, 59.51; H, 4.95; N, 16.52.
Found: C, 59.42; H, 5.11; N, 16.40.
A
suspension of the above compound (30 mg,
0.07 mmol) in EtOH (3 mL) containing 5 N NaOH
(3 mL) was heated at reflux for 3 h, when a clear solu-
tion was obtained. The reaction mixture was cooled to
ambient temperature, and then concentrated under
reduced pressure. The residue was partitioned between
CH2Cl2 and water. The organic layer was washed with
water and brine, then dried over Na2SO4. The solution
was evaporated in vacuo, and the residue was suspended
in CH2Cl2/hexanes (1:1) until a solid formed. The white
solid was filtered to give 7a (23 mg, 85% yield): mp 192–
6-Fluoro-4-(3,4-methylenedioxybenzylamino)-7-(1-piper-
azino)quinazoline (7a). Example of general method A. To
a mixture of 2 (1.47 g, 8.07 mmol) and N,N-diisopropyl-
ethylamine (7.04 mL, 40.4 mmol) in i-PrOH (20 mL) was
added 1-formylpiperazine (4.61 g, 40.4 mmol). The
reaction was stirred at 70 ꢀC for 6 h under a nitrogen
atmosphere. The reaction mixture was cooled to ambi-
ent temperature, and then concentrated under reduced
pressure. The residue was poured into water, and then
the precipitate was collected, washed with MeOH and
water. The white solid was filtered to give 6-fluoro-7-[1-
(4-formyl)piperazino]-4-quinazolone (3) (1.48g, 66%
ꢀ
1
193 C; H NMR (DMSO-d6) d 8.43 (t, J=5.4 Hz, 1H,
NHCH2), 8.37 (s, 1H, H-2), 8.05 (d, JHÀF=14.0 Hz, 1H,
H-5), 7.08(d, JHÀF=8.4 Hz, 1H, H-8), 6.92 (s, 1H,
ArH), 6.86–6.80 (m, 2H, ArH), 5.97 (s, 2H, OCH2O),
4.64 (d, J=5.4 Hz, 2H, NHCH2), 3.09–3.05 (m, 4H,
piperazinyl methylene), 2.88–2.85 (m, 4H, piperazinyl
methylene); MS (TOF) m/z 382 (M+H)+. Anal. calcd
1
yield): H NMR (CF3CO2D) d 12.31 (s, 1H, CONH),
9.31 (s, 1H, H-2), 8.47 (s, 1H, CHO), 8.14 (d,
JHÀF=12.2 Hz, 1H, H-5), 7.45 (d, JHÀF=6.2 Hz, 1H, H-
8), 4.06–3.97 (m, 4H, piperazinyl methylene), 3.76–3.72
(m, 4H, piperazinyl methylene); 13C NMR (CF3CO2D)
.
for C20H20N5FO2 0.2H2O: C, 62.39; H, 5.29; N, 18.19.
Found: C, 62.33; H, 5.35; N, 18.11.
d
169.6 (s, CHO), 160.8(s, C4), 157.5 (d,
JCÀF=256.7 Hz, C6), 150.3 (s, C2), 150.2 (d,
JCÀF=9.8Hz, C7), 137.1 (s, C9), 116.8 (d,
JCÀF=26.5 Hz, C5), 115.2 (d, JCÀF=9.5 Hz, C10), 109.5
(d, JCÀF=3.7 Hz, C8), 51.4 (d, JCÀF=5.0 Hz, piper-
azine), 50.3 (d, JCÀF=4.2 Hz, piperazine), 49.4 (s,
piperazine), 43.7 (s, piperazine); MS (TOF) m/z 277
(M+H)+.
4-Benzylamino-6-fluoro-7-(1-piperazino)quinazoline (8b).
Example of general method B. A suspension of 2
(250 mg, 1.37 mmol) in thionyl chloride (6 mL) contain-
ing one 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-
chloro-6,7-difluoroquinazoline (6), which was used
directly. To a mixture of the crude chloro compound
and triethylamine (229 mL, 1.64 mmol) in i-PrOH
(12 mL) was added benzylamine (179 mL, 1.64 mmol).
The resulting mixture was stirred at ambient tempera-
ture for 8h 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/hexanes
(1:1). The light yellow solid was filtered to give 4-ben-
zylamino-6,7-difluoroquinazoline (188 mg, 51% yield).
To a mixture of the above compound (150 mg,
0.55 mmol) and N,N-diisopropylethylamine (1.15 mL,
6.60 mmol) in n-BuOH (6 mL) was added piperazine
(569 mg, 6.60 mmol). The reaction was stirred at 110 ꢀC
for 30 h under a nitrogen atmosphere. The reaction
mixture was cooled to ambient temperature, and then
concentrated under reduced pressure. The brown resi-
due was partitioned between CH2Cl2 and 5% aqueous
citric acid solution. The aqueous layer was adjusted to
pH 9 with 5 N NaOH, and extracted with CH2Cl2. The
organic layer was washed with water and brine, then
dried over Na2SO4. The solution was evaporated in
vacuo, and the residue was suspended in CH2Cl2/hex-
anes (1:1) until a solid formed. The white solid was fil-
A suspension of 3 (500 mg, 1.81 mmol) in phosphorus
oxychloride (15 mL) was heated at 100 ꢀC for 30 min,
when a clear solution was obtained. The reaction mix-
ture was cooled to ambient temperature, and then con-
centrated under reduced pressure. The residue was
partitioned between CH2Cl2 and 5% aqueous NaHCO3
solution. The organic layer was washed with water and
brine, then dried over Na2SO4. The solution was con-
centrated under reduced pressure to provide crude 4-
chloro-6-fluoro-7-[1-(4-formyl)piperazino]quinazoline (4),
which was used directly. To a mixture of the crude
chloro compound and triethylamine (379 mL,
2.72 mmol) in EtOH (18mL) was added piperonylamine
(339 mL, 2.72 mmol). The resulting mixture was stirred
at reflux for 2 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 concentrated under reduced pressure and the resi-
due was purified by column chromatography on silica
gel eluting with 1:25 MeOH/CH2Cl2 to provide 37 mg
(5%) of 6-fluoro-7-[1-(4-formyl)piperazino]-4-[(3,4-
methylenedioxy)benzylamino]quinazoline [5: R=(3,4-
methylenedioxy)benzyl]: mp 190–192 ꢀC; 1H NMR
(DMSO-d6) d 8.47 (t, J=5.4 Hz, 1H, NHCH2), 8.39 (s,
1H, H-2), 8.13–8.07 (m, 2H, H-5 and CHO), 7.16 (d,
JHÀF=8.9 Hz, 1H, H-8), 6.92 (s, 1H, ArH), 6.86–6.80
(m, 2H, ArH), 5.97 (s, 2H, OCH2O), 4.65 (d, J=5.4 Hz,
2H, NHCH2), 3.60–3.55 (m, 4H, piperazinyl methyl-
ene), 3.21–3.11 (m, 4H, piperazinyl methylene); MS
ꢀ
1
tered to give 8b (56 mg, 30% yield): mp 213–215 C; H
NMR (DMSO-d6) d 8.51 (t, J=5.9 Hz, 1H, NHCH2),
8.36 (s, 1H, H-2), 8.07 (d, JHÀF=14.9 Hz, 1H, H-5),
7.36–7.20 (m, 5H, ArH), 7.09 (d, JHÀF=8.9 Hz, 1H, H-
8), 4.75 (d, J=5.9 Hz, 2H, NHCH2), 3.09–3.06 (m, 4H,

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M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 609–616
piperazinyl methylene), 2.89–2.85 (m, 4H, piperazinyl
methylene); MS (TOF) m/z 338(M+H) +. Anal. calcd
.
for C19H20N5F 0.4H2O: C, 66.22; H, 5.97; N, 20.32.
Found: C, 66.10; H, 6.02; N, 20.10.
6.66 (m, 1H, ArH), 5.95 (s, 2H, OCH2O), 3.48(dt,
J=7.2, 5.4 Hz, 2H, NHCH2), 3.08–3.05 (m, 4H, piper-
azinyl methylene), 2.89–2.86 (m, 4H, piperazinyl meth-
ylene), 2.59 (t, J=7.6 Hz, 2H, ArCH2), 1.89 (tt, J=7.6,
7.2 Hz, 2H, ArCH2CH2); MS (TOF) m/z 410 (M+H)+.
.
Similarly to the procedure described for 8b, compounds
7a–7d, 8a, and 8c–8j were prepared from 2.
Anal. calcd for C22H24N5FO2 0.4H2O: C, 63.42; H,
5.90; N, 16.81. Found: C, 63.29; H, 6.01; N, 16.61.
6-Fluoro-4-(3,4-methylenedioxybenzylamino)-7-(1-piper-
azino)quinazoline hydrochloride (HCl salt of 7a). Com-
pound 7a was obtained as a white solid (48% yield for
three steps from 2), and then was converted to its
hydrochloride salt according to the following proce-
dure: To a suspension of 7a (30 mg, 0.08mmol) in EtOH
(3 mL) was added 12 N HCl (20 mL). The mixture was
stirred at ambient temperature for 3 h, and then con-
centrated under reduced pressure. The residue was tri-
turated 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 white powder
(25 mg, 76% yield): mp 260 ꢀC (dec.); 1H NMR
(DMSO-d6) d 10.38(br s, 1H, N HCH2), 9.27 (br s, 2H,
4-(3,4-Ethylenedioxybenzylamino)-6-fluoro-7-(1-piperazi-
no)quinazoline (8a). White solid (12% yield for three
steps from 2): mp 188–190 ꢀC; H NMR (DMSO-d6) d
1
8.41 (t, J=5.9 Hz, 1H, NHCH2), 8.36 (s, 1H, H-2), 8.05
(d, JHÀF=14.0 Hz, 1H, H-5), 7.07 (d, JHÀF=8.4 Hz, 1H,
H-8), 6.83–6.79 (m, 3H, ArH), 4.61 (d, J=5.9 Hz, 2H,
NHCH2), 4.19 (s, 4H, OCH2CH2O), 3.08–3.05 (m, 4H,
piperazinyl methylene), 2.88–2.85 (m, 4H, piperazinyl
methylene; MS (TOF) m/z 396 (M+H)+. Anal. calcd
.
for C21H22N5FO2 1.5H2O: C, 59.70; H, 5.61; N, 16.58.
Found: C, 59.89; H, 5.97; N, 16.44.
6-Fluoro-4-(4-fluorobenzylamino)-7-(1-piperazino)quina-
zoline (8c). White solid (20% yield for three steps from
2): mp 222–224 ꢀC; 1H NMR (DMSO-d6) d 8.51 (t,
J=5.4 Hz, 1H, NHCH2), 8.36 (s, 1H, H-2), 8.06 (d,
JHÀF=14.6 Hz, 1H, H-5), 7.41–7.36 (m, 2H, ArH),
7.18–7.08 (m, 3H, ArH and H-8), 4.72 (d, J=5.4 Hz,
2H, NHCH2), 3.11–3.07 (m, 4H, piperazinyl methyl-
ene), 2.91–2.88 (m, 4H, piperazinyl methylene); MS
(TOF) m/z 356 (M+H)+. Anal. calcd for
.
NH HCl), 8.83 (s, 1H, H-2), 8.49 (d, JHÀF=14.0 Hz,
1H, H-5), 7.33 (d, JHÀF=7.8Hz, 1H, H-8), 7.00 (s, 1H,
ArH), 6.88 (s, 2H, ArH), 5.99 (s, 2H, OCH2O), 4.80 (d,
J=5.4 Hz, 2H, NHCH2), 3.51–3.36 (m, 8H, piperazinyl
.
methylene). Anal. calcd for C20H21N5ClFO2 2.5H2O: C,
51.89; H, 5.12; N, 15.13. Found: C, 51.95; H, 5.02; N,
15.09.
.
C19H19N5F2 0.3H2O: C, 63.25; H, 5.39; N, 19.41.
Found: C, 63.25; H, 5.31; N, 19.18.
6-Fluoro-4-(3,4-methylenedioxyphenyl)amino-7-(1-piper-
azino)quinazoline (7b). White solid (55% yield for three
steps from 2): mp 259–261 ꢀC; H NMR (DMSO-d6) d
6-Fluoro-7-(1-piperazino)-4-(2-pyridylmethylamino)qui-
nazoline (8d). White solid (36% yield for three steps
from 2): mp 154–156 ꢀC; 1H NMR (DMSO-d6) d 8.62 (t,
J=5.9 Hz, 1H, NHCH2), 8.52–8.50 (m, 1H, pyridine),
8.33 (s, 1H, H-2), 8.10 (d, JHÀF=14.0 Hz, 1H, H-5),
7.74–7.68(m, 1H, pyridine), 7.32–7.23 (m, 2H, pyri-
dine), 7.10 (d, JHÀF=8.6 Hz, 1H, H-8), 4.82 (d,
J=5.9 Hz, 2H, NHCH2), 3.10–3.07 (m, 4H, piperazinyl
methylene), 2.89–2.86 (m, 4H, piperazinyl methylene);
HR-FABMS m/z (M+H)+: calcd for C18H19N6F:
339.1733. Found: 339.1723.
1
9.41 (s, 1H, ArNH), 8.46 (s, 1H, H-2), 8.28 (d,
JHÀF=14.6 Hz, 1H, H-5), 7.50 (d, J=2.2 Hz, 1H, ArH),
7.18(dd,
J=8.4, 2.2 Hz, 1H, ArH), 7.14 (d,
JHÀF=9.7 Hz, 1H, H-8), 6.92 (d, J=8.4 Hz, 1H, ArH),
6.03 (s, 2H, OCH2O), 3.13–3.09 (m, 4H, piperazinyl
methylene), 2.90–2.87 (m, 4H, piperazinyl methylene);
+
MS (TOF) m/z 368(M+H)
.
. Anal. calcd for
C19H18N5FO2 0.1H2O: C, 61.81; H, 4.94; N, 18.97.
Found: C, 61.85; H, 4.97; N, 18.76.
6-Fluoro-4-[2-(3,4-methylenedioxyphenyl)ethylamino]-7-
(1-piperazino)quinazoline (7c). White solid (15% yield
for three steps from 2): mp 189–191 ꢀC; 1H NMR
(DMSO-d6) d 8.39 (s, 1H, H-2), 8.03 (t, J=5.9 Hz, 1H,
NHCH2), 8.00 (d, JHÀF=14.6 Hz, 1H, H-5), 7.08(d,
JHÀF=8.4 Hz, 1H, H-8), 6.85–6.80 (m, 2H, ArH), 6.71–
6.67 (m, 1H, ArH), 5.96 (s, 2H, OCH2O), 3.67 (dt,
J=7.0, 5.9 Hz, 2H, NHCH2CH2), 3.13–3.09 (m, 4H,
piperazinyl methylene), 2.94–2.91 (m, 4H, piperazinyl
methylene), 2.85 (t, J=7.3 Hz, 2H, NHCH2CH2); MS
(TOF) m/z 396 (M+H)+. Anal. calcd for
6-Fluoro-7-(1-piperazino)-4-(3-pyridylmethylamino)qui-
nazoline (8e). White solid (15% yield for three steps
from 2): mp 188–190 ꢀC; H NMR (DMSO-d6) d 8.60–
1
8.53 (m, 2H, pyridine and NHCH2), 8.46–8.44 (m, 1H,
pyridine), 8.38 (s, 1H, H-2), 8.05 (d, JHÀF=14.6 Hz, 1H,
H-5), 7.76–7.73 (m, 1H, pyridine), 7.36–7.32 (m, 1H,
pyridine), 7.10 (d, JHÀF=8.1 Hz, 1H, H-8), 4.76 (d,
J=5.9 Hz, 2H, NHCH2), 3.11–3.07 (m, 4H, piperazinyl
methylene), 2.91–2.87 (m, 4H, piperazinyl methylene);
HR-FABMS m/z (M+H)+: calcd for C18H19N6F:
339.1733. Found: 339.1743.
.
C21H22N5FO2 0.7H2O: C, 61.81; H, 5.61; N, 17.16.
Found: C, 62.02; H, 5.63; N, 16.80.
6-Fluoro-7-(1-piperazino)-4-(4-pyridylmethylamino)qui-
nazoline (8f). White ꢀsolid (55% yield for three steps
from 2): mp 226–228 C; 1H NMR (DMSO-d6) d 8.60 (t,
J=5.9 Hz, 1H, NHCH2), 8.49–8.47 (m, 2H, pyridine),
8.34 (s, 1H, H-2), 8.07 (d, JHÀF=14.3 Hz, 1H, H-5), 7.31
(d, J=5.9 Hz, 2H, pyridine), 7.11 (d, JHÀF=8.9 Hz, 1H,
H-8), 4.76 (d, J=5.9 Hz, 2H, NHCH2), 3.10–3.07 (m,
6-Fluoro-4-[3-(3,4-methylenedioxyphenyl)propylamino]-
7-(1-piperazino)quinazoline (7d). White solid (20% yield
for three steps from 2): mp 144–146 ꢀC; 1H NMR
(DMSO-d6) d 8.35 (s, 1H, H-2), 8.01 (d, JHÀF=14.6, Hz,
1H, H-5), 7.90 (t, J=5.4 Hz, 1H, NHCH2), 7.06 (d,
JHÀF=8.4 Hz, 1H, H-8), 6.83–6.79 (m, 2H, ArH), 6.69–

M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 609–616
615
4H, piperazinyl methylene), 2.89–2.86 (m, 4H, piper-
azinyl methylene); HR-FABMS m/z (M+H)+: calcd
for C18H19N6F: 339.1733. Found: 339.1732.
and 100 mL of medium containing 20 ng/mL LPS
(Escherichia coli 0111:B4, Difco) and test compounds
were then added. Cultures were incubated at 37 ꢀC for
16 h, and thereafter the supernatants were collected for
the determination of the TNF-a level by ELISA. The
50% inhibitory concentration (IC50) values were calcu-
lated by a nonlinear regression method.
4-(6-Chloro-pyridin-3-yl)methylamino-6-fluoro-7-(1-piper-
azino)quinazoline (8g). White solid (36% yield for three
steps from 2): mp 164–166 ꢀC; H NMR (DMSO-d6) d
1
8.55 (t, J=5.9 Hz, 1H, NHCH2), 8.42 (d, J=1.9 Hz, 1H,
pyridine), 8.37 (s, 1H, H-2), 8.02 (d, JHÀF=14.9 Hz, 1H,
H-5), 7.84–7.80 (m, 1H, pyridine), 7.46 (d, J=8.4 Hz,
1H, pyridine), 7.10 (d, JHÀF=8.9 Hz, 1H, H-8), 4.74 (d,
J=5.9 Hz, 2H, NHCH2), 3.09–3.06 (m, 4H, piperazinyl
methylene), 2.88–2.85 (m, 4H, piperazinyl methylene);
HR-FABMS m/z (M+H)+: calcd for C18H18N6ClF:
373.1343. Found: 373.1333.
T cell proliferation assay. T cell proliferation was deter-
mined by the MTS assay using Con A-stimulated mouse
spleen cells, as previously described.15 Briefly, male
BALB/c mice, 10 weeks of age, were killed by cervical
dislocation, and their spleens were removed, mashed in
PBS, and filtered through a nylon mesh. The cell sus-
pension was washed twice with RPMI 1640 medium
and resuspended to 8Â106 cells/mL in RPMI 1640
containing 10% FCS, 100 U/mL of penicillin, and
100 mg/mL of streptomycin. Splenocytes (8Â106 cells/
mL) were cultured in 96-well plates in the presence of
Con A (5 mg/mL) with test compounds at 37 ꢀC under
a 5% CO2 atmosphere for 3 days. The MTS assay
was performed by using a commercial kit (Promega),
and formazan dye products were measured by absor-
bance at 490 nm using a microplate reader (Model 450,
Bio-Rad). The 50% inhibitory concentration (IC50)
values were calculated by a nonlinear regression
method.
6-Fluoro-4-furfurylamino-7-(1-piperazino)quinazoline (8h).
White solid (21% yield for three steps from 2): mp
ꢀ
1
122–124 C; H NMR (DMSO-d6) d 8.43–8.40 (m, 2H,
H-2 and NHCH2), 8.06 (d, JHÀF=14.6 Hz, 1H, H-5),
7.58–7.57 (m, 1H, furan), 7.09 (d, JHÀF=8.9 Hz, 1H, H-
8), 6.40–6.38 (m, 1H, furan), 6.31–6.30 (m, 1H, furan),
4.73 (d, J=5.4 Hz, 2H, NHCH2), 3.09–3.06 (m, 4H,
piperazinyl methylene), 2.89–2.85 (m, 4H, piperazinyl
methylene); MS (TOF) m/z 327 (M+H)+. Anal. calcd
.
for C17H18FN5O 1.2H2O: C, 58.51; H, 5.55; N, 20.07.
Found: C, 58.61; H, 5.72; N, 20.09.
6-Fluoro-7-(1-piperazino)-4-[(thiophen-2-yl)methylamino]-
quinazoline (8i). White solid (21% yield for three steps
from 2): mp 202–204 ꢀC; 1H NMR (DMSO-d6) d 8.58 (t,
J=5.9 Hz, 1H, NHCH2), 8.42 (s, 1H, H-2), 8.01 (d,
JHÀF=14.0 Hz, 1H, H-5), 7.37–7.35 (m, 1H, thiophene),
7.11–7.06 (m, 2H, H-8and thiophene), 6.97–6.94 (m,
1H, thiophene), 4.90 (d, J=5.9 Hz, 2H, NHCH2), 3.09–
3.05 (m, 4H, piperazinyl methylene), 2.88–2.85 (m, 4H,
piperazinyl methylene); MS (TOF) m/z 344 (M+H)+.
B cell proliferation assay. Mouse spleen cell prolifera-
tion assay, with proliferation induced by LPS, a well-
known inducer of mouse B cell proliferation, was per-
formed according to the method previously described
with minor modification.9 Briefly, male BALB/c mice,
10 weeks of age, were killed by cervical dislocation, and
their spleens were removed, mashed in PBS, and filtered
through a nylon mesh. The cell suspension was washed
twice with RPMI 1640 medium and resuspended to
8Â105 cells/mL in RPMI 1640 containing 10% FCS,
100 U/mL of penicillin, and 100 mg/mL of streptomycin.
Splenocytes (8Â105 cells/mL) were cultured in 96-well
plates in the presence of LPS (3 mg/mL, serotype
0111:B4, DIFCO) with test compounds at 37 ꢀC under a
5% CO2 atmosphere for 3 days. Next, [3H]thymidine at
50 mCi/mL was added into the medium, and the cells
were incubated for 4 h at 37 ꢀC under a 5% CO2 atmo-
sphere. Then the cells were harvested by trypsinization,
and radioactive thymidine incorporation into DNA was
determined by scintillation counting. The 50% inhibi-
tory concentration (IC50) values were calculated by a
nonlinear regression method.
.
Anal. calcd for C17H18N5FS 0.3H2O: C, 58.53; H, 5.29;
N, 20.08. Found: C, 58.69; H, 5.28; N, 19.96.
6-Fluoro-4-(1-naphthylmethylamino)-7-(1-piperazino)qui-
nazoline (8j). White solid (61% yield for three steps
from 2): mp 222–224 ꢀC; 1H NMR (DMSO-d6) d 8.51 (t,
J=5.1 Hz, 1H, NHCH2), 8.39 (s, 1H, H-2), 8.19–8.16
(m, 1H, ArH), 8.13 (d, JHÀF=14.0 Hz, 1H, H-5), 7.98–
7.95 (m, 1H, ArH), 7.87–7.82 (m, 1H, ArH), 7.59–7.52
(m, 2H, ArH), 7.50–7.42 (m, 2H, ArH), 7.11 (d,
JHÀF=8.6 Hz, 1H, H-8), 5.21 (d, J=5.1 Hz, 2H,
NHCH2), 3.10–3.07 (m, 4H, piperazinyl methylene),
2.90–2.87 (m, 4H, piperazinyl methylene); MS (TOF) m/
+
.
z 388 (M+H) . Anal. calcd for C23H22N5F 0.7H2O: C,
69.05; H, 5.72; N, 17.51. Found: C, 69.06; H, 5.58; N,
17.32.
LPS-induced TNF-ꢀ production in mice. Inhibitory
effects of 1 and 7a against TNF-a production in LPS-
treated mice were evaluated according to a previously
reported method.16 Briefly, BALB/c mice (Charles River
Japan Inc.) were used at 10 weeks of age. LPS from E.
coli (serotype 026:B6) was purchased from Difco
Laboratories (Detroit, USA). A solution of a test com-
pound in water was orally administered to mice 0.5 h
prior to iv injection of the LPS (25 mg/mouse). Blood
samples were obtained 2 h after the LPS injection.
Amounts of TNF-a in the blood were determined by a
specific ELISA kit (Genzyme Techne, USA).
Biology
LPS-induced TNF-ꢀ production in human PBMCs.
Inhibition of TNF-a production was measured by
ELISA using human PBMCs stimulated by LPS, as
previously reported.14 Briefly, human PBMCs from
healthy volunteers were seeded (3 Â 105 cells/mL RPMI
1640, 10% FCS/well, 100 U/mL of penicillin, and
100 mg/mL of streptomycin) into 96-well culture plates,

616
M. Tobe et al. / Bioorg. Med. Chem. 11 (2003) 609–616
Pharmacokinetic analysis. The pharmacokinetic para-
meters of 1 and 7a were studied in rats. Briefly, in the
rat iv or po administration studies, three male SD rats
received either a 10 mg/kg intravenous dose or a 30 mg/
kg oral dose. Blood samples were obtained after dosing
at appropriate times and analyzed by reverse-phase
HPLC. The pharmacokinetic parameters for the com-
riman, G. R.; Maini, R. N. N. Engl. J. Med. 2000, 343, 1594.
(c) Markham, A.; Lamb, H. M. Drugs 2000, 59, 1341. (d)
Weinblatt, M. E.; Kremer, J. M.; Bankhurst, A. D.; Bulpitt,
K. J.; Fleischmann, R. M.; Fox, R. I.; Jackson, C. G.; Lange,
M. L. M.; Burge, D. J. N. Engl. J. Med. 1999, 340, 253. (e)
Moreland, L. W.; Schiff, M. H.; Baumgartner, S. W.; Tindall,
E. A.; Fleischmann, R. M.; Bulpitt, K. J.; Weaver, A. L.;
Keystone, E. C.; Furst, D. E.; Mease, P. J.; Ruderman, E. M.;
Horwitz, D. A.; Arkfeld, D. G.; Garrison, L.; Burge, D. J.;
Blosch, C. M.; Lange, M. L. M.; McDonnell, N. D.; Wein-
blatt, M. E. Ann. Intern. Med. 1999, 130, 478. (f) Bathon,
J. M.; Martin, R. W.; Fleischmann, R. M.; Tesser, J. R.;
Schiff, M. H.; Keystone, E. C.; Genovese, M. C.; Wasko,
M. C.; Moreland, L. W.; Weaver, A. L.; Markenson, J.;
Finck, B. K. N. Engl. J. Med. 2000, 343, 1586.
pounds were estimated by
method.
a non-compartmental
Anti-inflammatory effect on adjuvant arthritis.7 Adju-
vant arthritis was induced in male SD rats (7 weeks old;
Charles River Japan Inc.). This was done by injecting
Freund’s complete adjuvant [a 0.6% suspension of
Mycobacterium butyricum (Difco Labs) in liquid paraf-
fin] (0.1 mL) into the right hind paw. A solution of 7a in
water (30 mg/kg) was orally administered once a day for
14 days. The administration was started just before
sensitization (day 0). The severity of the induced adju-
vant disease was followed by measurement of the
volume of the non-injected left hind paw by using aqu-
eous plethysmography.
5. Tobe, M.; Isobe, Y.; Tomizawa, H.; Nagasaki, T.; Obara, F.;
Matsumoto, M.; Hayashi, H. Chem. Pharm. Bull. 2002, 50, 1073.
6. For the purpose of evaluating the cytotoxicity of the com-
pounds, we assessed cell viability by the MTS assay when we
assayed TNF-a production.
7. Perper, R. J.; Alvarez, B.; Colombo, C.; Schroder, H. Proc.
Soc. Exp. Biol. Med. 1971, 137, 506.
8. (a) Holoshitz, J.; Matitiau, A.; Cohen, I. R. J. Clin. Invest.
1984, 73, 211. (b) Van Eden, W.; Holoshitz, J.; Nevo, Z.;
Frenkel, A.; Klajman, A.; Cohen, I. R. Proc. Natl. Acad. Sci.
U.S.A. 1985, 82, 5117. (c) Lider, O.; Karin, N.; Shinitzky, M.;
Cohen, I. R. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 4577. (d)
Smith-Oliver, T.; Noel, L. S.; Simpson, S. S.; Yarnall, D. P.;
Connolly, K. M. Cytokine 1993, 5, 298. (e) DiMartino, M.;
Wolff, C.; High, W.; Stroup, G.; Hoffman, S.; Laydon, J.; Lee,
J. C.; Bertolini, D.; Galloway, W. A.; Crimmin, M. J.; Davis,
M.; Davies, S. Inflamm. Res. 1997, 46, 211.
9. Mouse spleen cell proliferation assay, with proliferation
induced by LPS, a well-known inducer of mouse B cell pro-
liferation, was performed according to the method previously
described with minor modification; see: Apfel, C.; Bauer, F.;
Crettaz, M.; Forni, L.; Kamber, M.; Kaufmann, F.; LeMotte,
P.; Pirson, W.; Klaus, M. Proc. Natl. Acad. Sci. U.S.A. 1992,
4, 7129.
Acknowledgements
We are grateful for all of the help provided by Dr.
Tsutomu Mimoto and Dr. Satoru Misawa. In addition,
we would like to express our thanks to Mr. Mitsuhiro
Matsumoto, Mr. Toshiyuki Sato, Mr. Toru Negishi,
and Mr. Tominaga Fukazawa for their technical assis-
tance in obtaining the biological data.
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