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

Article abstract of DOI:10.1248/cpb.50.1073

We synthesized various 6-nitroquinazolines by modifying the structure of compound 1 and evaluated their inhibitory activities toward both TNF-α production and T cell proliferation responses. The presence of the unsubstituted piperazine ring at the C(7)-position was required for both inhibitory activities. In this series of compounds, 5d and 5f, containing the 4-fluorophenyl and 3,4-difluorophenyl moiety, respectively, at the C(4)-position, showed the suppressing effects toward both responses with low cell growth inhibition. Furthermore, the oral administration of these compounds mentioned above at doses of 30 and 100 mg/kg also resulted in significant inhibition of TNF-α production induced by LPS in vivo.

Full text of DOI:10.1248/cpb.50.1073

August 2002
Chem. Pharm. Bull. 50(8) 1073—1080 (2002)
1073
Structure–Activity Relationships of 6-Nitroquinazolines: Dual-Acting
a
Compounds with Inhibitory Activities toward both TNF- Production
and T Cell Proliferation
Masanori TOBE, Yoshiaki ISOBE, Hideyuki TOMIZAWA, Takahiro NAGASAKI, Fumihiro OBARA,
Mitsuhiro MATSUMOTO, and Hideya HAYASHI*
Pharmaceuticals & Biotechnology Laboratory, Japan Energy Corporation; Toda-shi, Saitama 335–8502, Japan.
Received March 29, 2002; accepted May 24, 2002
We synthesized various 6-nitroquinazolines by modifying the structure of compound 1 and evaluated their
inhibitory activities toward both TNF-a production and T cell proliferation responses. The presence of the un-
substituted piperazine ring at the C(7)-position was required for both inhibitory activities. In this series of com-
pounds, 5d and 5f, containing the 4-fluorophenyl and 3,4-difluorophenyl moiety, respectively, at the C(4)-posi-
tion, showed the suppressing effects toward both responses with low cell growth inhibition. Furthermore, the oral
administration of these compounds mentioned above at doses of 30 and 100 mg/kg also resulted in significant in-
hibition of TNF-a production induced by LPS in vivo.
Key words TNF-a production; T cell proliferation; 6-nitroquinazoline
Tumor necrosis factor a (TNF-a), which is mainly pro- give rise to several side effects such as infection and osteo-
duced by activated monocytes and macrophages,1) plays an porosis. Therefore, there is a great need for new nonsteroidal
important role in the protective immune response of the host compounds that have the same efficacy as steroids but with-
against bacterial and viral infections. For example, TNF-a is out the side effects.
an essential substance for granuloma formation and the con-
Recently, we reported that nonsteroidal compound 1 (Fig.
trol of bacterial dissemination in experimental tuberculosis in 1) showed inhibitory activities toward both TNF-a produc-
mice.2,3) Additionally, TNF-a added to infected cells inhibits tion and T cell proliferation.22) In this study, we examined
the replication of both DNA and RNA viruses.4,5)
further the structure–activity relationships of 6-nitroquinazo-
On the other hand, TNF-a is a key cytokine in the inflam- lines by generating new drugs made on the basis of 1 by
matory cascade6); and elevated TNF-a levels in serum are as- placing various substituents on the phenyl ring at the C(4)-
sociated with autoimmune diseases such as rheumatoid position, replacing a piperazine at the C(7)-position with
arthritis (RA),7,8) septic shock,9) Crohn’s disease,10) and mul- other heterocycles, such as a morpholine and piperidines,
tiple sclerosis.11—14) The best example for this evidence is the and varying the methylene chain length at the C(4)-position.
dramatic reduction in disease activity observed in RA15,16) In addition, we also evaluated the inhibitory effects of several
and Crohn’s disease17) after treatment of such patients with compounds on TNF-a production in vivo.
anti-TNF-a chimera antibody (RemicadeTM). Accordingly,
Chemistry The general synthetic pathway to the 4,7-di-
anti-TNF-a therapy may be a hopeful treatment for these dis- substituted-6-nitroquinazolines is shown in Chart 1. The key
eases. In addition, autoimmune diseases are considered to be intermediate 4,7-dichloro-6-nitroquinazoline (3) was pre-
caused by abnormalities of T cell immune responses. In par- pared by the chlorination of 7-chloro-6-nitro-4-quinazolone
ticular, the activation of T cells (especially Th1) may be in- (2)23) with thionyl chloride in good yield. Compound 3 was
volved in a possible pathogenesis by which inflammation is subsequently treated with the corresponding amines to give
accelerated in these diseases.18—21) Therefore, the correction
of the abnormal immune responses mediated by T cell is also
regarded as a possible approach for treatment of these dis-
eases.
In view of these reports, we concluded that ideal anti-au-
toimmune disease agents should possess inhibitory activities
toward both TNF-a production and T cell proliferation. Al-
though glucocorticoids show the inhibition toward both of
these activities, the long-term use of them is well known to
Fig. 1. Design of 6-Nitroquinazolines
Chart 1. General Procedure for 4,7-Di-substituted-6-nitroquinazolines
To whom correspondence should be addressed. e-mail: hhayashi @j-energy.co.jp
© 2002 Pharmaceutical Society of Japan

1074
Vol. 50, No. 8
Chart 2. Synthesis of Compounds 5l—n
Table 1. Inhibition of TNF-a Production and T Cell Proliferation by Compounds 5a—n
IC50 (mM)
% Inhibition
Cytotoxicityc)
Compound
R1
R2
R3
n
TNF-aa)
Con Ab)
1
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
H
H
F
H
H
F
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
F
H
H
H
H
H
H
H
H
Cl
Cl
H
H
F
F
F
H
CF3
NO2
CN
NHAc
CO2Et
CO2H
CONH2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0.8
0.7
0.5
0.4
0.4
0.5
0.4
0.9
2.4
1.1
2
9.6
4.5
3
4.5
3.2
6.3
3.7
48.5
46.3
3.3
3.6
4.3
3.8
4.2
2.5
17.7
20.1
2.3
1.4
25.6
1.5
2.4
0.8
7.8
Ͼ10
Ͼ10
5.5
Ͼ10
Ͼ10
0.005
Ͼ10
2.5
Ͼ10
Ͼ10
0.01
1.7
100
Dexamethasone
a) IC50 for inhibition of TNF-a production from human PBMCs stimulated by LPS. b) IC50 for inhibition of Con A-induced proliferation of mouse spleen cells. c) Inhi-
bition of the growth of human PBMCs stimulated by LPS at 10 mM test compound.
4-substituted-7-chloro-6-nitroquinazolines (4). Reaction of monia gave 12, which was subsequently deprotected the Boc
these compounds with amines, such as piperazines and group to give the amide 5n.
piperidines, in the presence of Hunig’s base provided com-
pounds 5a—k, 6a—i, 7a—c, and 8a—l.
Results and Discussion
Compounds 5l—n were prepared by following the reaction
The compounds listed in Tables 1—4 were evaluated for
sequences described in Chart 2. Compound 9 was substituted their inhibitory activities toward both TNF-a production and
with N-Boc-piperazine to give the key intermediate N-Boc- T cell proliferation. The potency of the inhibition of TNF-a
protected ester 10. The N-Boc deprotection using 4 N HCl in production was measured by ELISA using human peripheral
dioxane afforded 5l in 65% yield. Compound 10 was hy- blood mononuclear cells (PBMCs) stimulated by LPS, as
drolyzed to give 11, which was then deprotected the Boc previously reported.24,25) The cell growth inhibition of human
group to provide 5m. Treatment of 11 with EDC (N-ethyl- PBMCs was measured by the MTS [3-(4,5-dimethylthiazol-
NЈ-[3-(dimethylamino)propyl] carbodiimide), and HOBt (1- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
hydroxybenzotriazole) followed by the additon of 28% am- tetrazolium, inner salt] assay.26) Suppression of concanavalin

August 2002
1075
Table 2. Inhibition of TNF-a Production and T Cell Proliferation by Compounds 6a—i
IC50 (mM)
% Inhibition
Compound
R1
R2
R3
R4
TNF-aa)
Con Ab)
Cytotoxicityc)
6a
6b
6c
6d
6e
6f
6g
6h
H
H
H
MeO
H
H
H
H
Cl
H
H
MeO
H
H
MeO
H
Me
MeO
H
H
MeO
MeO
H
H
H
H
MeO
MeO
1.1
0.9
1.7
1.2
5.2
7.5
0.08
1.5
0.7
0.01
1.9
2.1
5.6
7.2
7.6
50.4
52.5
16.1
10.2
9.2
11.8
51.1
8.1
Ͼ10
2.7
–OCH2O–
–OCH2O–
–OCH2O–
MeO
H
6
3.8
0.005
6i
10
100
Dexamethasone
a—c) See corresponding footnotes in Table 1.
(5h), the nitro (5i), the cyano (5j), and the ester (5l) ana-
logues) were not improved toward either response. The intro-
duction of polar, hydrogen bonding groups also diminished
both inhibitory activities (5k, m, and n). These results sug-
gest that the placement of a hydrophilic group on the phenyl
group was not suitable to exert either inhibitory activity.
Secondly, we investigated the effect of an electron-donat-
ing moiety on the phenyl group (Table 2). Based on the fluo-
rine substitution study, the same tendency was observed
when the position of the methoxy group on the phenyl group
was changed from para to ortho or meta. That is, the para
substitution (6b) was suitable for maintaining both inhibitory
activities. The 3,4-dimethoxy (6e) and the 3,4,5-trimethoxy
(6f) analogues decreased both inhibitory activities. However,
the replacement the 3,4-dimethoxy group with the 3,4-meth-
ylenedioxy one (6g) led to a 10-fold increase in the TNF-a
inhibitory activity as compared with that of 1. The compari-
son between 6f and 6h also showed that the substitution with
Table 3. Inhibition of TNF-a Production and T Cell Proliferation by
Compounds 7a—c
IC50 (mM)
% Inhibition
Cytotoxicityc)
Compound
n
TNF-aa)
Con Ab)
7a
6g
7b
7c
0d)
1
2
3.3
0.08
0.7
5.1
2.7
4.2
6.4
45.1
51.1
48.5
10.2
3
1.1
a—c) See corresponding footnotes in Table 1. d) 0 means the anilino derivative.
A (Con A) induced proliferation of mouse spleen cells was the 3,4-methylenedioxy group was more preferable than that
determined by the MTS assay according to the previously de- with the 3,4-dimethoxy group for the TNF-a inhibitory ac-
scribed method.27,28)
tivity. These results indicate that the substituents on the
At first, comparing the results for 1 and 5a, we found that phenyl group must be sterically compact to show a potent
these compounds had nearly equal inhibitory activity toward TNF-a inhibitory activity.
TNF-a production and T cell proliferation. On the basis of
Thirdly, we explored the effects of varying the length of
this data, we performed a substitution study on the phenyl the linker between the quinazoline and the phenyl group at
group at the C(4)-position by using the benzyl type deriva- the C(4)-position of the quinazoline (Table 3). The maximum
tive. As shown in Table 1, the introduction of a fluorine atom potency of TNF-a inhibitory activity was obtained when the
on the phenyl group (5b—f) resulted in an approximately 2- spacer was the methylene chain (6g). The optimal length was
fold greater potency against TNF-a inhibitory activity as also confirmed to be 1 methylene unit (6g) for suppressing T
compared with the potency of 1. The change in the position cell proliferation.
of the fluorine atom from para (5d) to ortho (5b) or meta
Finally, to further refine the framework at the C(7)-posi-
(5c) reduced the inhibitory activities toward T cell prolifera- tion, we investigated the effects of the C-methylpiperazines
tion, whereas the substitution pattern (ortho, meta, para) had and the replacement of the piperazine ring with other hetero-
no affect on the TNF-a inhibitory activity. It should be noted cycles (Table 4). The 3-methylpiperazine analogues (8a, b)
that 5d showed reduced cell growth inhibition; i.e., the cell resulted in a 4—5-fold loss in TNF-a inhibitory activity as
growth inhibition of compound 1 was 48.5% at 10 mM, compared with the activity of 6g. The 3,5-dimethypiperazine
whereas that of 5d was only 4.3%. The activities of com- (8c) led to dramatically decreased inhibitory activities toward
pounds having an electron-withdrawing moiety in the para TNF-a production and T cell proliferation. These results
position of the phenyl group (such as the trifluoromethyl suggest that the presence of the simple piperazine ring at the

1076
Vol. 50, No. 8
Table 4. Inhibition of TNF-a Production and T Cell Proliferation by Compounds 8a—l
IC40 (mM)
% Inhibition
Compound
X
R1
R2
TNF-aa)
Con Ab)
Cytotoxicityc)
6g
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
NH
NH
NH
NH
NCO2Et
O
H
H
H
H
0.08
0.4
0.3
2.7
3.6
3.5
51.1
49.3
47.4
5
1.8
8.7
1.5
6.8
10
19.7
12.3
11.5
5.9
3(S)-Me
3(R)-Me
cis-3,5-dimethyl
Ͼ10
Ͼ10
6.9
Ͼ10
7.1
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ10
0.01
Ͼ10
Ͼ10
Ͼ10
Ͼ10
Ͼ10
8.2
Ͼ10
Ͼ10
Ͼ10
Ͼ10
0.005
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2
CHOH
CHCH2OH
CHCO2Et
CHCO2H
CH2
H
H
OH
CH2OH
8l
CH2
Dexamethasone
100
a—c) See corresponding footnotes in Table 1.
active in spite of its highly inhibitory activity in vitro. These
results suggest the poor oral bioavailability of 6g. Addition-
ally, 5d and 6g also showed potent inhibitory activities
against B cell proliferation (5d, IC50ϭ0.3 mM; 6g, IC50ϭ
0.3 mM). This property suggests possible suppression of au-
toantibody production, which would make these compounds
desirable drug candidates for RA. Therefore, to develop a
novel compound exhibiting more potency and orally
bioavailability than 6g, we are planning further chemical
modification of 6g.
Conclusions
A number of 6-nitroquinazolines were synthesized and
evaluated for their inhibitory activities toward both TNF-a
production and T cell proliferation. We determined that the
best framework at the C(7)-position was the simple piper-
azine ring. Furthermore, in this series of compounds, we
found that the introduction of a fluorine atom on the phenyl
group resulted in reduced cell toxicity as well as in improved
inhibitory activities. Especially, 5d and 5f exhibited almost
the same inhibitory activity toward both TNF-a production
and T cell proliferation in vitro, and inhibited the LPS-in-
duced TNF-a production when given by oral administration.
Although 6g exerted the most potent activity against TNF-a
production (IC50ϭ0.08 mM), it failed to inhibit this response
by oral administration, probably due to its poor oral bioavail-
ability. At present, we are continuing our study to obtain
even more potent and orally active quinazoline derivatives.
Fig. 2. Effects of 5d, 5f, and 6g on LPS-Induced TNF-a Production in
Mice
Compounds 5d, 5f, and 6g were evaluated as their corresponding hydrochloride salts.
The results are expressed as the meanϮS.E.M. of 5 mice per group. *** pϽ0.001 ver-
sus vehicle control (Dunnett’s test). Veh., vehicle control; Dex., dexamethasone.
C(7)-position was necessary to show both inhibitory activi-
ties. Compound 8d, in which the NH group of the piperazine
ring was blocked by the ethoxycarbonyl group, had notably
decreased inhibitory activities toward both parameters.
Moreover, other heterocycle analogues (8e—l) (morpholine
and piperidine), which are lacking in a basic nitrogen, also
showed loss of both inhibitory activities. The above-men-
tioned results support the possibility of the interaction be-
tween the distal piperazine nitrogen and some target mole-
cules which we have not identified yet.
Experimental
Chemistry All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Melting points were
measured with a BÜCHI 535 melting point apparatus and were uncorrected.
Proton NMR spectra were recorded on a JEOL GSX270 FT NMR spectrom-
eter. Chemical shifts were given in d values (ppm) using tetramethylsilane as
an internal standard, and following abbreviations were used: sϭsinglet,
We selected 3 compounds (5d, f, 6g) from the results of
above in vitro study to further evaluate their activities toward
TNF-a production in vivo (Fig. 2). By oral administration,
both 5d and 5f showed almost the same inhibitory activity
against LPS-induced TNF-a production, whereas 6g was less

August 2002
1077
1
salt as a pale yellow powder (102 mg, 94% yield). mp 251 °C (Dec.). H-
dϭdoublet, tϭtriplet, qϭquartet, mϭmultiplet, bsϭbroad singlet, and
ddϭdouble doublet. Time-of-flight mass spectrometry (TOF-MS) was
recorded on a KOMPACT MALDI III spectrometer. Optical rotations were
determined by using a JASCO DIP-370 digital polarimeter. Elemental analy-
ses 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). Following abbreviations
are used for solvents: DMF (N,N-dimethylformamide), AcOEt (ethyl ac-
etate).
NMR (DMSO-d6) d: 10.80 (1H, bs), 9.39 (2H, bs), 9.29 (1H, s), 8.86 (1H,
s), 7.49—7.44 (3H, m), 7.23—7.16 (2H, m), 4.90 (2H, d, Jϭ5.4 Hz), 3.42—
3.26 (8H, m); Anal. Calcd for C19H20ClFN6O2·2.5H2O: C, 49.19; H, 4.89;
N, 18.12. Found: C, 48.82; H, 4.78; N, 18.05.
4-(2,4-Difluorobenzylamino)-6-nitro-7-(1-piperazino)quinazoline (5e)
Yellow solid (33% yield for 3 steps from 2); mp, 182—184 °C; H-NMR
(CDCl3) d: 8.65 (1H, s), 8.21 (1H, s), 7.50—7.41 (1H, m), 7.33 (1H, s),
6.90—6.84 (2H, m), 5.97 (1H, t, Jϭ4.1 Hz), 4.88 (2H, d, Jϭ4.1 Hz), 3.13—
3.10 (4H, m), 3.06—3.03 (4H, m); TOF-MS m/z: 401 (MϩH)ϩ; Anal. Calcd
for C19H18F2N6O2: C, 57.00; H, 4.53; N, 20.99. Found: C, 56.83; H, 4.83; N,
20.80.
1
General Procedure for Preparation of 4,7-Di-substituted-6-nitro-
quinazolines. 4-Benzylamino-6-nitro-7-(1-piperazino)quinazoline (5g)
A suspension of 7-chloro-6-nitro-4-quinazolone (2)23) (250 mg, 1.11 mmol)
in thionyl chloride (6.0 ml) containing 1 drop of N,N-dimethylformamide
(DMF) was refluxed for 2 h to give a clear solution. Excess thionyl chloride
was removed under reduced pressure to provide crude 4,7-dichloro-6-nitro-
quinazoline (3), which was used directly. To a mixture of 3 and triethylamine
(185 ml, 1.33 mmol) in i-PrOH (12 ml) was added benzylamine (145 ml,
1.33 mmol). The resulting mixture was stirred at ambient temperature and
concentrated in vacuo, and partitioned between CH2Cl2 and 5% aqueous cit-
ric 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, v/v). The light yellow solid was filtered to give 4-benzylamino-7-
chloro-6-nitroquinazoline (269 mg, 77% yield). Next, to a mixture of the
above compound (150 mg, 0.48 mmol) and N,NЈ-diisopropylethylamine
(502 ml, 2.88 mmol) in n-BuOH (9.5 ml) was added piperazine (248 mg,
2.88 mmol). The reaction was stirred at 110 °C for 10 h under a nitrogen at-
mosphere. The reaction mixture was cooled to ambient temperature, and
then concentrated under reduced pressure. The brown residue was parti-
tioned 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, dried over Na2SO4. The so-
lution was evaporated in vacuo, and the yellow residue was suspended in
CH2Cl2–hexane (1 : 1, v/v) until a solid formed. The light yellow solid was
filtered to give 5g (130 mg, 74% yield). mp 189—191 °C. 1H-NMR (CDCl3)
d: 8.66 (1H, s), 8.20 (1H, s), 7.41—7.36 (5H, m), 7.33 (1H, s), 5.92 (1H, t,
Jϭ4.9 Hz), 4.87—4.85 (2H, m), 3.13—3.10 (4H, m), 3.06—3.03 (4H, m).
TOF-MS m/z: 365 (MϩH)ϩ. Anal. Calcd for C19H20N6O2·0.1H2O: C, 62.31;
H, 5.53; N, 22.95. Found: C, 62.17; H, 5.59; N, 22.84.
4-(3,4-Difluorobenzylamino)-6-nitro-7-(1-piperazino)quinazoline (5f)
Yellow solid (26% yield for 3 steps from 2); mp, 203—205 °C; H-NMR
1
(CDCl3) d: 8.64 (1H, s), 8.24 (1H, s), 7.34 (1H, s), 7.24—7.12 (3H, m), 6.04
(1H, t, Jϭ3.8 Hz), 4.84 (2H, d, Jϭ3.8 Hz), 3.14—3.11 (4H, m), 3.06—3.03
(4H, m); TOF-MS m/z: 401 (MϩH)ϩ; Anal. Calcd for C19H18F2N6O2: C,
57.00; H, 4.53; N, 20.99. Found: C, 56.92; H, 4.56; N, 21.18.
Similarly to the procedure described for 5d, compound 5f was converted
to its hydrochloride salt (92% yield); mp, 248 °C (Dec.); 1H-NMR (DMSO-
d6) d: 10.63 (1H, bs), 9.27—9.25 (3H, m), 8.82 (1H, s), 7.54—7.28 (4H, m),
4.88 (2H, d, Jϭ5.1 Hz), 3.41—3.26 (8H, m); Anal. Calcd for
C19H19ClF2N6O2·2.2H2O: C, 47.89; H, 4.48; N, 17.64. Found: C, 48.00; H,
4.27; N, 17.33.
6-Nitro-7-(1-piperazino)-4-(4-trifluoromethylbenzylamino)quinazo-
line (5h) Yellow solid (35% yield for 3 steps from 2); mp, 215—217 °C;
1H-NMR (CDCl3) d: 8.64 (1H, s), 8.25 (1H, s), 7.63 (2H, d, Jϭ8.4 Hz), 7.51
(2H, d, Jϭ8.4 Hz), 7.35 (1H, s), 6.06 (1H, t, Jϭ4.6 Hz), 4.95 (2H, d,
Jϭ4.6 Hz), 3.14—3.11 (4H, m), 3.06—3.03 (4H, m); TOF-MS m/z: 433
(MϩH)ϩ; Anal. Calcd for C20H19F3N6O2·0.2H2O: C, 55.09; H, 4.44; N,
19.28. Found: C, 54.90; H, 4.52; N, 19.43.
6-Nitro-4-(4-nitrobenzylamino)-7-(1-piperazino)quinazoline (5i) Yel-
low solid (18% yield for 3 steps from 2); mp, 182—184 °C; 1H-NMR
(CDCl3) d: 8.62 (1H, s), 8.35 (1H, s), 8.20 (2H, d, Jϭ8.6 Hz), 7.55 (2H, d,
Jϭ8.6 Hz), 7.34 (1H, s), 6.49 (1H, t, Jϭ5.9 Hz), 5.00 (2H, d, Jϭ5.9 Hz),
3.13—3.10 (4H, m), 3.06—3.03 (4H, m); TOF-MS m/z: 410 (MϩH)ϩ; Anal.
Calcd for C19H19N7O4·0.9H2O: C, 53.62; H, 4.71; N, 23.04. Found: C,
53.76; H, 5.10; N, 22.98.
4-(4-Cyanobenzylamino)-6-nitro-7-(1-piperazino)quinazoline (5j)
Yellow solid (23% yield for 3 steps from 2); mp, 207—209 °C; H-NMR
1
(CDCl3) d: 8.61 (1H, s), 8.34 (1H, s), 7.64 (2H, d, Jϭ8.9 Hz), 7.50 (2H, d,
Jϭ8.9 Hz), 7.33 (1H, s), 6.45 (1H, t, Jϭ5.7 Hz), 4.96 (2H, d, Jϭ5.7 Hz),
3.13—3.10 (4H, m), 3.06—3.03 (4H, m); TOF-MS m/z: 390 (MϩH)ϩ; Anal.
Calcd for C20H19N7O3·0.7H2O: C, 59.75; H, 4.94; N, 24.39. Found: C,
59.65; H, 5.08; N, 24.78.
Similarly to the procedure described for 5g, compounds 5a—f and 5h—k
were prepared from 2.
4-(4-Chlorobenzylamino)-6-nitro-7-(1-piperazino)quinazoline (5a)
1
Yellow solid (30% yield for 3 steps from 2); mp, 209—210 °C; H-NMR
(DMSO-d6) d: 9.08 (1H, t, Jϭ5.9 Hz), 8.91 (1H, s), 8.44 (1H, s), 7.38 (4H,
bs), 7.19 (1H, s), 4.74 (2H, d, Jϭ5.9 Hz), 3.00—2.97 (4H, m), 2.83—2.79
(4H, m); TOF-MS m/z: 399 (MϩH)ϩ; Anal. Calcd for C19H19ClN6O2: C,
57.22; H, 4.80; N, 21.07. Found: C, 57.03; H, 4.54; N, 21.24.
4-(4-Acetamidobenzylamino)-6-nitro-7-(1-piperazino)quinazoline
1
(5k) Yellow solid (8% yield for 3 steps from 2); mp, 253—255 °C; H-
NMR (DMSO-d6) d: 9.90 (1H, s), 9.02 (1H, t, Jϭ5.4 Hz), 8.92 (1H, s), 8.45
(1H, s), 7.52 (2H, d, Jϭ8.4 Hz), 7.28 (2H, d, Jϭ8.4 Hz), 7.18 (1H, s), 4.70
(2H, t, Jϭ5.4 Hz), 3.00—2.97 (4H, m), 2.83—2.80 (4H,m), 2.20 (3H, s);
TOF-MS m/z: 422 (MϩH)ϩ; Anal. Calcd for C21H23N7O3·0.5H2O: C, 58.59;
H, 5.50; N, 22.78. Found: C, 58.51; H, 5.56; N, 22.90.
4-(2-Fluorobenzylamino)-6-nitro-7-(1-piperazino)quinazoline (5b)
1
Yellow solid (18% yield for 3 steps from 2); mp, 194—196 °C; H-NMR
(CDCl3) d: 8.65 (1H, s), 8.21 (1H, s), 7.48—7.31 (3H, m), 7.17—7.08 (2H,
m), 5.99 (1H, t, Jϭ3.5 Hz), 4.93 (2H, d, Jϭ3.5 Hz), 3.13—3.10 (4H, m),
3.06—3.03 (4H, m); TOF-MS m/z: 383 (MϩH)ϩ+; Anal. Calcd for
C19H19FN6O2·0.1H2O: C, 59.40; H, 5.01; N, 21.87. Found: C, 59.21; H,
4.97; N, 21.92.
7-[1-(4-tert-Butoxycarbonyl)piperazino]-4-(4-ethoxycarbonylbenzyl-
amino)-6-nitroquinazoline (10) Similarly to the procedure described for
5g, the title compound was prepared from 7-chloro-4-(4-ethoxycarbonylben-
zylamino)-6-nitroquinazoline (9) (300 mg, 0.78 mmol) and 1-(tert-butoxy-
carbonyl)piperazine (433 mg, 2.33 mmol). After purification, 10 was ob-
4-(3-Fluorobenzylamino)-6-nitro-7-(1-piperazino)quinazoline (5c)
1
Yellow solid (11% yield for 3 steps from 2); mp, 191—193 °C; H-NMR
1
tained as a orange solid (270 mg, 65% yield); mp, 201—203 °C; H-NMR
(CDCl3) d: 8.65 (1H, s), 8.25 (1H, s), 7.39—7.30 (2H, m), 7.18—6.99 (3H,
m), 6.05 (1H, t, Jϭ5.4 Hz), 4.89—4.87 (2H, m), 3.14—3.10 (4H, m), 3.06—
3.03 (4H, m); TOF-MS m/z: 383 (MϩH)ϩ; Anal. Calcd for C19H19FN6O2·
0.3H2O: C, 58.85; H, 5.01; N, 21.67. Found: C, 58.71; H, 4.75; N, 21.42.
4-(4-Fluorobenzylamino)-6-nitro-7-(1-piperazino)quinazoline (5d)
(CDCl3) d: 8.34 (1H, s), 7.97 (1H, s), 7.72 (2H, d, Jϭ8.4 Hz), 7.12 (2H, d,
Jϭ8.4 Hz), 7.04 (1H, s), 5.76 (1H, t, Jϭ3.2 Hz), 4.63 (2H, d, Jϭ3.2 Hz),
4.06 (2H, q, Jϭ7.0 Hz), 3.32—3.29 (4H, m), 2.81—2.77 (4H, m), 1.17 (9H,
s), 1.07 (3H, t, Jϭ7.0 Hz); TOF-MS m/z: 537 (MϩH)ϩ; Anal. Calcd for
C27H32N6O6: C, 60.44; H, 6.01 N, 15.66. Found: C, 60.10; H, 6.00; N, 15.37.
4-(4-Ethoxycarbonylbenzylamino)-6-nitro-7-(1-piperazino)quinazo-
line (5l) To a suspension of 10 (100 mg, 0.19 mmol) in 1,4-dioxane
(3.0 ml) was added 4 N HCl–1,4-dioxane (1.3 ml) dropwisely at ambient tem-
perature. The reaction mixture was stirred at the same temperature for 8 h,
and was concentrated under reduced pressure. The residue 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, dried over Na2SO4. The solution was
evaporated under reduced pressure, and the yellow residue was suspended in
CH2Cl2–hexane (1 : 2, v/v). The light yellow solid was filtered to give 5l
1
Yellow solid (50% yield for 3 steps from 2); mp, 216—218 °C; H-NMR
(CDCl3) d: 8.65 (1H, s), 8.21 (1H, s), 7.40—7.33 (3H, m), 7.10—7.03 (2H,
m), 5.92 (1H, t, Jϭ3.8 Hz), 4.84 (2H, d, Jϭ3.8 Hz), 3.13—3.10 (4H, m),
3.06—3.03 (4H, m); TOF-MS m/z: 383 (MϩH)ϩ; Anal. Calcd for
C19H19FN6O2: C, 59.68; H, 5.01; N, 21.98. Found: C, 59.56; H, 5.02; N,
22.30.
Compound 5d was converted to its hydrochloride salt according to the
following procedure: To a suspension of 5d (100 mg, 0.26 mmol) in EtOH
(8 ml) was added 12 N HCl (65 ml). The mixture was stirred at ambient tem-
perature for 4 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
1
(53 mg, 65% yield); mp, 179—181 °C; H-NMR (CDCl3) d: 8.64 (1H, s),

1078
Vol. 50, No. 8
8.25 (1H, s), 8.04 (2H, d, Jϭ8.1 Hz), 7.45 (2H, d, Jϭ8.1 Hz), 7.34 (1H, s), 6.99—6.87 (3H, m), 5.91 (1H, t, Jϭ3.5 Hz), 4.83 (2H, d, Jϭ3.5 Hz), 3.82
6.06 (1H, t, Jϭ3.8 Hz), 4.94 (2H, d, Jϭ3.8 Hz), 4.38 (2H, q, Jϭ7.2 Hz), (3H, s,), 3.14—3.10 (4H, m), 3.06—3.03 (4H, m); TOF-MS m/z: 395
3.14—3.11 (4H, m), 3.06—3.03 (4H, m), 1.39 (3H, t, Jϭ7.2 Hz); TOF-MS (MϩH)ϩ; Anal. Calcd for C20H22N6O3·0.5H2O: C, 59.54; H, 5.62; N, 20.83.
m/z: 437 (MϩH)ϩ; Anal. Calcd for C22H24N6O4: C, 60.54; H, 5.54; N, 19.25.
Found: C, 59.70; H, 5.63; N, 20.50.
Found: C, 60.17; H, 5.53; N, 19.20.
4-(2-Methoxybenzylamino)-6-nitro-7-(1-piperazino)quinazoline (6d)
4-[[7-[1-(4-tert-Butoxycarbonyl)piperazino]-6-nitro-4-quinazolinyl]- Yellow solid (26% yield for 3 steps from 2); mp, 94—96 °C; 1H-NMR
aminomethyl]benzoic Acid (11) A solution of 10 (270 mg, 0.50 mmol) in (DMSO-d6) d: 8.97 (1H, s), 8.89 (1H, t, Jϭ5.4 Hz), 8.42 (1H, s), 7.28—7.18
EtOH (20 ml) containing 5 N NaOH (500ml) was stirred at ambient tempera-
(3H, m), 7.01 (1H, d, Jϭ8.4 Hz), 6.90—6.85 (1H, m), 4.70 (2H, d,
Jϭ5.4 Hz), 3.84 (3H, s), 3.00—2.97 (4H, m), 2.83—2.80 (4H, m); TOF-MS
ture for 10 h. The reaction mixture was neutralized with 6 N HCl and concen-
trated under reduced pressure. The residue was partitioned between AcOEt m/z: 395 (MϩH)ϩ; Anal. Calcd for C20H22N6O3·2.5H2O: C, 54.66; H, 5.62;
and 5% aqueous citric acid solution. The organic layer was washed with N, 19.12. Found: C, 54.68; H, 5.66; N, 19.01.
water and brine, dried over Na2SO4. The solution was evaporated in vacuo,
and the yellow residue was triturated with AcOEt–hexane (5 : 1, v/v). The (6e) Yellow solid (33% yield for 3 steps from 2); mp, 229—231 °C; H-
4-(3,4-Dimethoxybenzylamino)-6-nitro-7-(1-piperazino)quinazoline
1
yellow solid was filtered to give 11 (212 mg, 83% yield); mp, 258—260 °C;
NMR (CDCl3) d: 8.66 (1H, s), 8.20 (1H, s), 7.33 (1H, s), 6.97—6.86 (3H,
1H-NMR (DMSO-d6) d: 12.87 (1H, bs), 9.21 (1H, t, Jϭ5.8 Hz), 9.00 (1H, m), 5.90 (1H, t, Jϭ5.4 Hz), 4.78 (2H, d, Jϭ5.4 Hz), 3.89 (3H, s), 3.88 (3H,
s), 8.46 (1H, s), 7.90 (2H, d, Jϭ8.1 Hz), 7.46 (2H, d, Jϭ8.1 Hz), 7.28 (1H, s), 3.13—3.10 (4H, m), 3.06—3.03 (4H, m); TOF-MS m/z: 425 (MϩH)ϩ;
s), 4.83 (2H, d, Jϭ5.8 Hz), 3.47—3.45 (4H, m), 3.09—3.07 (4H, m), 1.43 Anal. Calcd for C21H24N6O4·0.7H2O: C, 57.71; H, 5.70; N, 19.23. Found: C,
(9H, s); TOF-MS m/z: 509 (MϩH)ϩ; Anal. Calcd for C25H28N6O6: C, 59.05;
57.61; H, 5.88; N, 19.51.
H, 5.55; N, 16.53. Found: C, 58.96; H, 5.56; N, 16.39.
4-[[6-Nitro-7-(1-piperazino)-4-quinazolinyl]aminomethyl]benzoic (6f) Yellow solid (25% yield for 3 steps from 2); mp, 204—206 °C; H-
6-Nitro-7-(1-piperazino)-4-(3,4,5-trimethoxybenzylamino)quinazoline
1
Acid (5m) Similarly to the procedure described for 5l, the title compound
NMR (CDCl3) d: 8.67 (1H, s), 8.25 (1H, s), 7.33 (1H, s), 6.61 (2H, s), 5.98
was prepared from the N-Boc-protected acid 11 (70 mg, 0.14 mmol). After (1H, t, Jϭ4.9 Hz), 4.77 (2H, d, Jϭ4.9 Hz), 3.86 (6H, s), 3.85 (3H, s), 3.14—
purification, 5m was obtained as a yellow solid (50 mg, 89% yield); mp, 3.11 (4H, m), 3.07—3.04 (4H, m); TOF-MS m/z: 455 (MϩH)ϩ; Anal. Calcd
266 °C (Dec.); 1H-NMR (DMSO-d6) d: 9.16 (1H, t, Jϭ3.8 Hz), 8.94 (1H, s), for C22H26N6O5·0.2H2O: C, 57.68; H, 5.76; N, 18.35. Found: C, 57.87; H,
8.44 (1H, s), 7.89 (2H, d, Jϭ8.1 Hz), 7.44 (2H, d, Jϭ8.1 Hz), 7.21 (1H, s), 5.94; N, 18.11.
4.82 (2H, d, Jϭ3.8 Hz), 3.02—3.00 (4H, m), 2.86—2.84 (4H, m); TOF-MS
m/z: 409 (MϩH)ϩ; Anal. Calcd for C20H20N6O4·1.3H2O: C, 55.63; H, 4.97; line (6g) Yellow solid (72% yield for 3 steps from 2); mp, 217—219 °C;
N, 19.46. Found: C, 55.59; H, 5.18; N, 19.37.
1H-NMR (CDCl3) d: 8.65 (1H, s), 8.19 (1H, s), 7.33 (1H, s), 6.88—6.79
4-(3,4-Methylenedioxy)benzylamino-6-nitro-7-(1-piperazino)quinazo-
4-[[7-[1-(4-tert-Butoxycarbonyl)piperazino]-6-nitro-4-quinazolinyl]- (3H, m), 5.97 (2H, s), 5.87 (1H, t, Jϭ3.2 Hz), 4.76 (2H, d, Jϭ3.2 Hz),
aminomethyl]benzamide (12) To a solution of 11 (200 mg, 0.39 mmol) 3.12—3.10 (4H, m), 3.06—3.04 (4H, m); TOF-MS m/z: 409 (MϩH)ϩ; Anal.
and HOBt (64 mg, 0.47 mmol) in DMF (8 ml) was added EDC·HCl (90 mg, Calcd for C20H20N6O4·0.1H2O: C, 58.56; H, 4.94; N, 20.49. Found: C,
0.47 mmol). The mixture was stirred at ambient temperature for 1 h. The re- 58.67; H, 4.92; N, 20.50.
sulting solution was cooled to 0 °C, then 28% ammonia solution was added
Similarly to the procedure described for 5d, compound 6g was converted
and temperature was allowed to rise to ambient temperature. After 8 h, the to its hydrochloride salt (89% yield); mp, 255 °C (Dec.); 1H-NMR (DMSO-
reaction mixture was concentrated under reduced pressure, and the residue d6) d: 10.52 (1H, bs), 9.23 (3H, bs), 8.82 (1H, s), 7.41 (1H, s), 7.00 (1H, s),
was partitioned CH2Cl2 and 5% aqueous NaHCO3 solution. The organic 6.89 (2H, s), 6.00 (2H, s), 4.80 (2H, d, Jϭ5.4 Hz), 3.40—3.26 (8H, m);
layer was washed with water and brine, and then dried over Na2SO4. The so- Anal. Calcd for C20H21ClN6O4·2.5H2O: C, 49.03; H, 4.83; N, 17.15. Found:
lution was evaporated in vacuo, and the residue was purified by column C, 49.04; H, 4.78; N, 17.01.
chromatography on SiO2 with CH2Cl2–MeOH (98 : 2, v/v) to give 12 as the
4-[(3-Methoxy-4,5-methylenedioxy)benzylamino]-6-nitro-7-(1-piper-
pale yellow solid (157 mg, 78% yield); mp, 251—253 °C; 1H-NMR (DMSO- azino)quinazoline (6h) Yellow solid (49% yield for 3 steps from 2); mp,
1
d6) d: 9.18 (1H, t, Jϭ5.9 Hz), 8.99 (1H, s), 8.46 (1H, s), 7.91 (1H, bs), 7.82
(2H, d, Jϭ8.4 Hz), 7.41 (2H, d, Jϭ8.4 Hz), 7.31 (1H, bs), 7.27 (1H, s), 4.81
197—199 °C; H-NMR (CDCl3) d: 8.65 (1H, s), 8.22 (1H, s), 7.33 (1H, s),
6.59—6.57 (2H, m), 5.98 (2H, s), 5.92 (1H, t, Jϭ5.4 Hz), 4.74 (2H, d,
(2H, d, Jϭ5.9 Hz), 3.49—3.46 (4H, m), 3.09—3.06 (4H, m), 1.43 (9H, s); Jϭ5.4 Hz), 3.91 (3H, s), 3.14—3.10 (4H, m), 3.07—3.04 (4H, m); TOF-MS
TOF-MS m/z: 508 (MϩH)ϩ; Anal. Calcd for C25H29N7O5: C, 58.95; H, 5.76;
N, 19.25. Found: C, 58.89; H, 5.87; N, 18.90.
m/z: 439 (MϩH)ϩ; Anal. Calcd for C21H22N6O5·1.2H2O: C, 54.82; H, 5.08;
N, 18.27. Found: C, 54.92; H, 5.29; N, 18.07.
4-[[6-Nitro-7-(1-piperazino)-4-quinazolinyl]aminomethyl]benzamide
4-[(2-Chloro-4,5-methylenedioxy)benzylamino]-6-nitro-7-(1-piper-
Hydrochloride (5n) To a suspension of 12 (100 mg, 0.20 mmol) in EtOH azino)quinazoline (6i) Yellow solid (43% yield for 3 steps from 2); mp,
1
(2.0 ml) was added 4 N HCl in 1,4-dioxane (2.0 ml) dropwisely at ambient
175—177 °C; H-NMR (CDCl3) d: 8.64 (1H, s), 8.21 (1H, s), 7.32 (1H, s),
temperature for 10 h. The reaction mixture was concentrated under reduced 6.99 (1H, s), 6.88 (1H, s), 6.07 (1H, t, Jϭ5.9 Hz), 5.98 (2H, s), 4.85 (2H, d,
pressure, and the residue was triturated with Et2O. The pale yellow solid was Jϭ5.9 Hz), 3.13—3.10 (4H, m), 3.06—3.03 (4H, m); TOF-MS m/z: 443
filtered to give 5n (76 mg, 87% yield); mp, 263 °C (Dec.); 1H-NMR (MϩH)ϩ; Anal. Calcd for C20H19ClN6O4·0.3H2O: C, 53.59; H, 4.34; N,
(DMSO-d6) d: 10.97 (1H, bs), 9.48 (2H, bs), 9.34 (1H, s), 8.86 (1H, s), 7.97
(1H, bs), 7.86 (2H, d, Jϭ8.4 Hz), 7.52—7.46 (3H, m), 7.36 (1H, bs), 4.96
18.75. Found: C, 53.49; H, 4.36; N, 18.81.
4-(3,4-Methylenedioxyphenyl)amino-6-nitro-7-(1-piperazino)quinazo-
(2H, d, Jϭ5.4 Hz), 3.43—3.26 (8H, m); Anal. Calcd for C20H22ClN7O3· line (7a) Yellow solid (53% yield for 3 steps from 2); mp, 222—223 °C;
4.5H2O: C, 45.76; H, 5.09; N, 18.68. Found: C, 45.81; H, 5.31; N, 18.51.
Compounds 6a—i, 7a—c, 8a—i, 8k, and 8l were also prepared by using
the same procedure as for 5g.
1H-NMR (CDCl3) d: 8.67 (1H, s), 8.39 (1H, s), 7.45 (1H, bs), 7.35 (1H, s),
7.31 (1H, d, Jϭ2.4 Hz), 6.94 (1H, dd, Jϭ8.4, 2.4 Hz), 6.84 (1H, d,
Jϭ8.4 Hz), 6.01 (2H, s), 3.16—3.12 (4H, m), 3.07—3.04 (4H, m); TOF-MS
4-(4-Methylbenzylamino)-6-nitro-7-(1-piperazino)quinazoline
(6a) m/z: 395 (MϩH)ϩ; Anal. Calcd for C19H18N6O4·0.5H2O: C, 56.57; H, 4.62;
1
Yellow solid (32% yield for 3 steps from 2); mp, 227—229 °C; H-NMR N, 20.83. Found: C, 56.70; H, 4.77; N, 21.09.
(CDCl3) d: 8.65 (1H, s), 8.18 (1H, s), 7.32 (1H, s), 7.29 (2H, d, Jϭ8.4 Hz),
4-[2-(3,4-Methylenedioxyphenyl)ethylamino]-6-nitro-7-(1-piper-
7.20 (2H, d, Jϭ8.4 Hz), 5.87 (1H, t, Jϭ3.5 Hz), 4.81 (2H, d, Jϭ3.5 Hz), azino)quinazoline (7b) Yellow solid (30% yield for 3 steps from 2); mp,
1
3.13—3.09 (4H, m), 3.06—3.03 (4H, m), 2.37 (3H, s); TOF-MS m/z: 379 210—212 °C; H-NMR (CDCl3) d: 8.63 (1H, s), 8.10 (1H, s), 7.31 (1H, s),
(MϩH)ϩ; Anal. Calcd for C20H22N6O2: C, 63.48; H, 5.86; N, 22.21. Found:
6.79—6.67 (3H, m), 5.96 (2H, s), 5.79 (1H, t, Jϭ5.7 Hz), 3.92—3.84 (2H,
m), 3.11—3.09 (4H, m), 3.06—3.04 (4H, m), 2.95 (2H, t, Jϭ6.8 Hz); TOF-
C, 63.14; H, 5.81; N, 22.18.
4-(4-Methoxybenzylamino)-6-nitro-7-(1-piperazino)quinazoline (6b) MS m/z: 423 (MϩH)ϩ; Anal. Calcd for C21H22N6O4·0.5H2O: C, 58.64; H,
1
Yellow solid (11% yield for 3 steps from 2); mp, 219—221 °C; H-NMR 5.26; N, 19.48. Found: C, 58.65; H, 5.24; N, 19.37.
(CDCl3) d: 8.66 (1H, s), 8.18 (1H, s), 7.33 (2H, d, Jϭ8.4 Hz), 7.32 (1H, s),
4-[3-(3,4-Methylenedioxyphenyl)propylamino]-6-nitro-7-(1-piper-
6.92 (2H, d, Jϭ8.4 Hz), 5.84 (1H, t, Jϭ4.9 Hz), 4.79—4.77 (2H, m), 3.82 azino)quinazoline (7c) Yellow solid (38% yield for 3 steps from 2); mp,
1
(3H, s), 3.13—3.10 (4H,m), 3.06—3.03 (4H, m); TOF-MS m/z: 395 187—189 °C; H-NMR (CDCl3) d: 8.59 (1H, s), 7.95 (1H, s), 7.29 (1H, s),
(MϩH)ϩ; Anal. Calcd for C20H22N6O3·0.5H2O: C, 59.54; H, 5.62; N, 20.83. 6.75—6.66 (3H, m), 5.92 (2H, s), 5.58 (1H, t, Jϭ5.1 Hz), 3.57—3.67 (2H,
Found: C, 59.67; H, 5.72; N, 20.80.
m), 3.11—3.09 (4H, m), 3.06—3.04 (4H, m), 2.72 (2H, t, Jϭ7.2 Hz), 2.10—
4-(3-Methoxybenzylamino)-6-nitro-7-(1-piperazino)quinazoline (6c) 2.00 (2H, m); TOF-MS m/z: 437 (MϩH)ϩ; Anal. Calcd for C22H24N6O4·
1
Yellow solid (39% yield for 3 steps from 2); mp, 175—177 °C; H-NMR 0.1H2O: C, 60.29; H, 5.54; N, 19.18. Found: C, 60.16; H, 5.61; N, 19.52.
(CDCl3) d: 8.66 (1H, s), 8.20 (1H, s), 7.33 (1H, s), 7.31—7.28 (1H, m),
(3S)-4-(3,4-Methylenedioxy)benzylamino-7-[1-(3-methyl)piperazino]-

August 2002
1079
6-nitroquinazoline (8a) Yellow solid (48% yield for 3 steps from 2); mp,
214—216 °C; H-NMR (CDCl3) d: 8.64 (1H, s), 8.20 (1H, s), 7.31 (1H, s),
low residue was suspended in AcOEt–hexane (10 : 7, v/v). The light yellow
1
solid was filtered to give 8j (68 mg, 85% yield); mp, 275 °C (Dec.); 1H-
6.88—6.79 (3H, m), 5.97 (2H, s), 5.91 (1H, bs), 4.75 (2H, d, Jϭ5.4 Hz), NMR (DMSO-d6) d: 12.32 (1H, bs), 9.00 (1H, t, Jϭ5.9 Hz), 8.93 (1H, s),
3.25—3.21 (2H, m), 3.11—3.04 (3H, m), 2.98—2.90 (1H, m), 2.62—2.54
8.46 (1H, s), 7.21 (1H, s), 6.94 (1H, s), 6.88 (1H, s), 6.85 (1H, s), 5.97 (2H,
(1H, m), 1.10 (3H, d, Jϭ6.5 Hz); [a]D20 Ϫ27.8° (cϭ2.0, MeOH); TOF-MS s), 4.65 (2H, d, Jϭ5.9 Hz), 3.25 (2H, bs), 2.95—2.87 (2H, m), 2.45—2.40
m/z: 423 (MϩH)ϩ; Anal. Calcd for C21H22N6O4·0.9H2O: C, 57.50; H, 5.26; (1H, m), 1.99—1.90 (2H, m), 1.73—1.61 (2H, m); TOF-MS m/z: 452
N, 19.16. Found: C, 57.43; H, 5.35; N, 19.14.
(3R)-4-(3,4-Methylenedioxy)benzylamino-7-[1-(3-methyl)piperazino]- C, 58.38; H, 4.73; N, 15.31.
(MϩH)ϩ; Anal. Calcd for C22H21N5O6: C, 58.53; H, 4.69; N, 15.51. Found:
6-nitroquinazoline (8b) Yellow solid (37% yield for 3 steps from 2); mp,
7-(3-Hydroxypiperidino)-4-(3,4-methylenedioxy)benzylamino-6-nitro-
quinazoline (8k) Yellow solid (44% yield for 3 steps from 2); mp, 201—
1
214—216 °C; H-NMR (CDCl3) d: 8.64 (1H, s), 8.20 (1H, s), 7.32 (1H, s),
6.88—6.79 (3H, m), 5.97 (2H, s), 5.89 (1H, bs), 4.75 (2H, d, Jϭ5.4 Hz), 203 °C; 1H-NMR (CDCl3) d: 8.65 (1H, s), 8.23 (1H, s), 7.36 (1H, s), 6.88—
3.25—3.21 (2H, m), 3.11—3.04 (3H, m), 2.98—2.92 (1H, m), 2.62—2.54
6.79 (3H, m), 5.97 (3H, bs), 4.75 (2H, d, Jϭ5.4 Hz), 3.99 (1H, bs), 3.28—
(1H, m), 1.10 (3H, d, Jϭ6.5 Hz); [a]D20 ϩ27.8° (cϭ2.0, MeOH); TOF-MS 3.22 (1H, m), 3.11—3.03 (3H, m), 2.06—1.95 (1H, m), 1.90—1.80 (1H, m),
m/z: 423 (MϩH)ϩ; Anal. Calcd for C21H22N6O4·1.2H2O: C, 56.80; H, 5.27; 1.76—1.67 (3H, m); TOF-MS m/z: 424 (MϩH)ϩ; Anal. Calcd for
N, 18.93. Found: C, 56.89; H, 5.15; N, 18.84.
C21H21N5O5·0.4H2O: C, 58.57; H, 5.01; N, 16.26. Found: C, 58.69; H, 5.01;
7-[1-(cis-3,5-Dimethyl)piperazino]-4-(3,4-methylenedioxy)benzyl- N, 16.11.
amino-6-nitroquinazoline (8c) Yellow solid (44% yield for 3 steps from
2); mp, 208—210 °C; H-NMR (CDCl3) d: 8.64 (1H, s), 8.20 (1H, s), 7.31 6-nitroquinazoline (8l) Yellow solid (52% yield for 3 steps from 2); mp,
(1H, s), 6.88—6.79 (3H, m), 5.97 (2H, s), 5.87 (1H, bs), 4.75 (2H, d, 199—201 °C; H-NMR (CDCl3) d: 8.64 (1H, s), 8.18 (1H, s), 7.35 (1H, s),
7-(3-Hydroxymethylpiperidino)-4-(3,4-methylenedioxy)benzylamino-
1
1
Jϭ5.4 Hz), 3.23—3.09 (4H, m), 2.57—2.49 (2H, m), 1.10 (6H, d,
Jϭ6.5 Hz); TOF-MS m/z: 437 (MϩH)ϩ; Anal. Calcd for C22H24N6O4: C,
60.54; H, 5.54; N, 19.25. Found: C, 60.42; H, 5.58; N, 19.14.
6.88—6.79 (3H, m), 5.97 (2H, s), 5.85 (1H, bs), 4.75 (2H, d, Jϭ5.4 Hz),
3.63—3.58 (2H, m), 3.44—3.40 (1H, m), 3.29—3.24 (1H, m), 2.97—2.76
(2H, m), 2.02—1.73 (4H, m), 1.30—1.27 (2H, m); TOF-MS m/z: 438
(MϩH)ϩ; Anal. Calcd for C22H23N5O5: C, 60.40; H, 5.30; N, 16.01. Found:
7-[1-(4-Ethoxycarbonyl)piperazino]-4-(3,4-methylenedioxy)benzyl-
amino-6-nitroquinazoline (8d) Yellow solid (36% yield for 3 steps from C, 60.35; H, 5.32; N, 15.92.
1
2); mp, 204—206 °C; H-NMR (DMSO-d6) d: 9.05 (1H, t, Jϭ5.1 Hz), 8.98
LPS-induced TNF-a Production in Human PBMCs Inhibition of
(1H, s), 8.48 (1H, s), 7.26 (1H, s), 6.95 (1H, s), 6.85 (2H, s), 5.97 (2H, s), TNF-a production was measured by ELISA using human PBMCs stimu-
4.66 (2H, d, Jϭ5.1 Hz), 4.07 (2H, q, Jϭ7.2 Hz), 3.53—3.50 (4H, m), 3.10— lated by LPS, as previously reported.24,25) Briefly, human PBMCs from
3.07 (4H, m), 1.20 (3H, t, Jϭ7.2 Hz); TOF-MS m/z: 481 (MϩH)ϩ; Anal. healthy volunteers were seeded (3ϫ105 cells/ml RPMI 1640, 10% FCS/well,
Calcd for C23H24N6O6·0.1H2O: C, 57.28; H, 5.04; N, 17.43. Found: C,
57.04; H, 4.99; N, 17.33.
100 U/ml of penicillin, and 100 mg/ml of streptomycin) into 96-well culture
plates, and 100 ml of medium containing 20 ng/ml LPS (Escherichia coli
0111:B4, DIFCO) and test compounds were then added. Cultures were incu-
4-(3,4-Methylenedioxy)benzylamino-7-morpholino-6-nitroquinazoline
1
(8e) Yellow solid (66% yield for 3 steps from 2); mp, 259—261 °C; H- bated at 37 °C for 16 h, and thereafter the supernatants were collected for the
NMR (CDCl3) d: 8.67 (1H, s), 8.22 (1H, s), 7.34 (1H, s), 6.89—6.80 (3H, determination of the TNF-a level by ELISA. The 50% inhibitory concentra-
m), 5.98 (2H, s), 5.86 (1H, t, Jϭ5.4 Hz), 4.76 (2H, d, Jϭ5.4 Hz), 3.90—3.86 tion (IC50) values were calculated by a nonlinear regression method.
(4H, m), 3.15—3.12 (4H, m); TOF-MS m/z: 410 (MϩH)ϩ; Anal. Calcd for
C20H19N5O5: C, 58.68; H, 4.68; N, 17.11. Found: C, 58.70; H, 4.82; N,
16.87.
T Cell Proliferation Assay T cell proliferation was determined by the
MTS assay using Con A-stimulated mouse spleen cells, as previously de-
scribed.27,28) Briefly, male BALB/c mice, 10 weeks of age, were sacrificed by
4-(3,4-Methylenedioxy)benzylamino-6-nitro-7-piperidinoquinazoline cervical dislocation; and their spleens were removed, mashed in PBS, and
(8f) Light yellow solid (52% yield for 3 steps from 2); mp, 207—209 °C;
filtered through a nylon mesh. The cell suspension was washed twice with
1H-NMR (DMSO-d6) d: 9.76 (1H, t, Jϭ4.9 Hz), 9.00 (1H, s), 8.63 (1H, s), RPMI 1640 medium and resuspended to 8ϫ106 cells/ml in RPMI 1640 con-
7.23 (1H, s), 6.97 (1H, s), 6.87 (2H, s), 5.98 (2H, s), 4.73 (2H, d, Jϭ4.9 Hz), taining 10% FCS, 100 U/ml of penicillin, and 100 mg/ml of streptomycin.
3.10 (4H, bs), 1.61 (6H, bs); TOF-MS m/z: 408 (MϩH)ϩ; Anal. Calcd for Splenocytes (8ϫ106 cells/ml) were cultured in 96-well plates in the presence
C21H21N5O4·1.2H2O: C, 58.79; H, 5.22; N, 16.32. Found: C, 58.71; H, 5.02;
N, 16.38.
7-(4-Hydroxypiperidino)-4-(3,4-methylenedioxy)benzylamino-6-nitro-
of Con A (5 mg/ml) with test compounds at 37 °C under a 5% CO2 atmos-
phere for 3 d. The MTS assay was performed by using a commercial kit
(Promega), and formazan dye products were measured by absorbance at
quinazoline (8g) Yellow solid (52% yield for 3 steps from 2); mp, 174— 490 nm by using a microplate reader (Model 450, Bio-Rad). The 50% in-
176 °C; 1H-NMR (CDCl3) d: 8.64 (1H, s), 8.22 (1H, s), 7.33 (1H, s), 6.88— hibitory concentration (IC50) values were calculated by a nonlinear regres-
6.79 (3H, m), 5.97—5.93 (3H, m), 4.75 (2H, d, Jϭ4.9 Hz), 3.98—3.92 (1H, sion method.
m), 3.41—3.32 (2H, m), 3.04—2.95 (2H, m), 2.08—2.01 (2H, m), 1.83—
B Cell Proliferation Assay Mouse spleen cell proliferation assay, with
1.71 (2H, m); TOF-MS m/z: 424 (MϩH)ϩ; Anal. Calcd for C21H21N5O5· proliferation induced by LPS, a well-known inducer of mouse B cell prolif-
1.2H2O: C, 56.67; H, 5.03; N, 15.74. Found: C, 56.71; H, 5.18; N, 15.77.
eration, was performed according to the method previously described with
7-(4-Hydroxymethylpiperidino)-4-(3,4-methylenedioxy)benzylamino- minor modification.29) Briefly, male BALB/c mice, 10 weeks of age, were
6-nitroquinazoline (8h) Yellow solid (36% yield for 3 steps from 2); mp, sacrificed by cervical dislocation; and their spleens were removed, mashed in
1
151—153 °C; H-NMR (CDCl3) d: 8.63 (1H, s), 8.21 (1H, s), 7.32 (1H, s),
6.88—6.78 (3H, m), 5.97—5.93 (3H, m), 4.75 (2H, d, Jϭ5.4 Hz), 3.59—
PBS, and filtered through a nylon mesh. The cell suspension was washed
twice with RPMI 1640 medium and resuspended to 8ϫ105 cells/ml in RPMI
3.56 (2H, m), 3.43—3.39 (2H, m), 2.95—2.85 (2H, m), 1.87—1.66 (3H, m), 1640 containing 10% FCS, 100 U/ml of penicillin, and 100 mg/ml of strepto-
1.56—1.41 (3H, m); TOF-MS m/z: 438 (MϩH)ϩ; Anal. Calcd for mycin. Splenocytes (8ϫ105 cells/ml) were cultured in 96-well plates in the
C22H23N5O5·0.8H2O: C, 58.48; H, 5.31; N, 15.50. Found: C, 58.41; H, 5.44;
N, 15.48.
7-(4-Ethoxycarbonylpiperidino)-4-(3,4-methylenedioxy)benzylamino-
presence of LPS (3 mg/ml, serotype 0111:B4, DIFCO) with test compounds
at 37 °C under a 5% CO2 atmosphere for 3 d. Next, [3H] thymidine at
50 mCi/ml was added into the medium, and the cells were incubated for 4 h
6-nitroquinazoline (8i) Yellow solid (63% yield for 3 steps from 2); mp, at 37°C under a 5% CO2 atmosphere. Then the cells were harvested by
1
176—178 °C; H-NMR (CDCl3) d: 8.64 (1H, s), 8.23 (1H, s), 7.36 (1H, s),
trypsinization, and radioactive thymidine incorporation into DNA was deter-
6.88—6.79 (3H, m), 6.05—5.97 (3H, m), 4.76 (2H, d, Jϭ4.1 Hz), 4.17 (2H, mined by scintillation counting. The 50% inhibitory concentration (IC50)
q, Jϭ7.0 Hz), 3.42—3.35 (2H, m), 3.00—2.90 (2H, m), 2.56—2.45 (1H, m),
2.08—1.89 (4H, m), 1.28 (3H, t, Jϭ7.0 Hz); TOF-MS m/z: 480 (MϩH)ϩ;
Anal. Calcd for C24H25N5O6: C, 60.12; H, 5.26; N, 14.61. Found: C, 60.06; compounds against TNF-a production in LPS treated mice were evaluated
H, 5.17; N, 14.43.
values were calculated by a nonlinear regression method.
LPS-Induced TNF-a Production in Mice Inhibitory effects of the test
according to a previously reported method.30) Briefly, BALB/c mice (Charles
1-[4-(3,4-Methylenedioxy)benzylamino-6-nitro-7-quinazolinyl]-4- River Japan Inc.) were used at 10 weeks of age. LPS from Escherichia coli
piperidinecarboxylic Acid (8j) A solution of 8i (85 mg, 0.18 mmol) in (serotype 026:B6) was purchased from Difco Laboratories (Detroit, U.S.A.).
EtOH (5 ml) containing 5 N NaOH (180 ml) was stirred at ambient tempera- A solution of a test compound in water was orally administered to mice 0.5 h
ture for 8 h. The reaction mixture was concentrated under reduced pressure. prior to i.v. injection of the LPS (25 mg/mouse). Blood samples were ob-
The residue was partitioned between AcOEt and 5% aqueous citric acid so- tained 2 h after the LPS injection. Amounts of TNF-a in the blood were de-
lution. The organic layer was washed with water and brine, dried over termined by a specific ELISA kit (Genzyme Techne, U.S.A.).
Na2SO4. The solution was evaporated under reduced pressure, and the yel-

1080
Vol. 50, No. 8
Acknowledgements The authors are grateful to Dr. Satoru Misawa and
Woody J. N., Lancet, 344, 1105—1110 (1994).
Dr. Tsutomu Mimoto for useful suggestions and encouragement during the 16) Maini R. N., Elliott M. J., Brennan F. M., Feldmann M., Clin. Exp. Im-
course of this research.
munol., 101, 207—212 (1995).
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18) Quayle A. J., Chomarat P., Miossec P., Kjeldsen-Kragh J., Førre Ø.,
Natvig J. B., Scand. J. Immunol., 38, 75—82 (1993).
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