Synthesis and evaluation of 2-{[2-(4-hydroxyphenyl)-ethyl]amino}pyrimidine-5-carboxamide derivatives as novel STAT6 inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.10.015

The STAT6 (signal transducers and activators of transcription 6) protein is activated by interleukin (IL)-4 and IL-13, and plays an important role in T-helper cell 2 (Th2) differentiation. STAT6 might therefore be an excellent therapeutic target for various allergic conditions, including asthma and atopic diseases. We synthesized a series of 2-{[2-(4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide derivatives and evaluated their STAT6 inhibitory activities. Among these compounds, 4-(benzylamino)-2-{[2-(3-chloro-4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide (2t, AS1517499) showed potent STAT6 inhibition with an IC₅₀ value of 21 nM, and also inhibited IL-4-induced Th2 differentiation of mouse spleen T cells with an IC₅₀ value of 2.3 nM and without influencing T-helper cell 1 (Th1) differentiation induced by IL-12.

Full text of DOI:10.1016/j.bmc.2006.10.015

Bioorganic & Medicinal Chemistry 15 (2007) 1044–1055
Synthesis and evaluation of 2-{[2-(4-hydroxyphenyl)-
ethyl]amino}pyrimidine-5-carboxamide derivatives
as novel STAT6 inhibitors
a,
a
b,ꢀ
b
*
Shinya Nagashima, Masaki Yokota, Ei-ichi Nakai, Sadao Kuromitsu,
b
a
a,ꢁ
a
Keiko Ohga, Makoto Takeuchi, Shin-ichi Tsukamoto and Mitsuaki Ohta
a
Institute for Drug Discovery Research, Astellas Pharm Inc., 2-1-6, Kashima, Yodogawa-ku, Osaka 532-8514, Japan
b
Institute for Drug Discovery Research, 21, Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
Received 21 September 2006; revised 10 October 2006; accepted 11 October 2006
Available online 13 October 2006
Abstract—The STAT6 (signal transducers and activators of transcription 6) protein is activated by interleukin (IL)-4 and IL-13, and
plays an important role in T-helper cell 2 (Th2) differentiation. STAT6 might therefore be an excellent therapeutic target for various
allergic conditions, including asthma and atopic diseases. We synthesized a series of 2-{[2-(4-hydroxyphenyl)ethyl]amino}pyrimi-
dine-5-carboxamide derivatives and evaluated their STAT6 inhibitory activities. Among these compounds, 4-(benzylamino)-2-
{[2-(3-chloro-4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide (2t, AS1517499) showed potent STAT6 inhibition with an
IC50 value of 21 nM, and also inhibited IL-4-induced Th2 differentiation of mouse spleen T cells with an IC50 value of 2.3 nM
and without influencing T-helper cell 1 (Th1) differentiation induced by IL-12.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
responsible for allergic diseases; therefore, the balance
of the Th1/Th2 response is closely associated with hu-
man health and disease. Recent studies have shown
+
CD4 T helper (Th) cells are classified into two subsets
that are referred to as Th1 and Th2. Th1 cells produce
2
1
that Th2 cytokines contribute to allergic inflammatory
responses by regulating immunoglobulin E (IgE) pro-
duction and the functions of eosinophils and mast cells.
In particular, IL-4 is thought to be a critical factor for
Th2 differentiation and is known to regulate both IgE
interferon-c (IFN-c), interleukin (IL)-2, and tumor
necrosis factor b, and enhance cellular immunity for
elimination of intracellular pathogens. In contrast, Th2
cells produce Th2 cytokines, such as IL-4, IL-5, IL-10,
and IL-13, and are involved in the development of
humoral immunity for protection against extracellular
pathogens. The important physiologic functions of
Th1 and Th2 cells are mutually regulated and the im-
mune system maintains a proper balance between Th1
and Th2 responses. Several diseases are thought to be
caused by imbalance of these responses: a chronically
ongoing Th1 response may result in inflammatory tissue
damage, whereas an excessive Th2 response may be
3
production by B cells and mast cell function.
STAT6 (signal transducers and activators of transcrip-
tion 6) is a member of the STAT family of transcription
factors and is specifically activated by IL-4 and IL-13.
The importance of STAT6 in Th2 differentiation has
been established using STAT6-deficient mice, in which
T cells fail to differentiate into Th2 cells in response to
4
IL-4 and B cells are unable to produce IgE. Since many
studies indicate that Th2 cytokines and IgE are major
5
players in allergic diseases, STAT6 might be an excel-
Keywords: Signal transducers and activators of transcription 6; Aller-
gic disease; Interleukin-4; Th2 differentiation.
lent therapeutic target for the treatment of various aller-
gic conditions, including asthma and atopic diseases.
However, although a few STAT6 inhibitors such as
*
Corresponding author. Tel.: +81 6 6390 1286; fax: +81 6 6340
414; e-mail: shinya.nagashima@jp.astellas.com
5
6
ꢀ
TMC-264 have been reported, no compounds have
Present address: Institute for Drug Discovery Research, 5, Toukou-
dai, Tsukuba, Ibaraki 300-2698, Japan.
been investigated in clinical trials (Fig. 1). Therefore,
to find novel STAT6 inhibitors, we performed high-
throughput screening of our chemical libraries using a
ꢁ
Present address: Department of Corporate Strategy, 2-3-11, Nihonb-
ashi-Honcho, Chuo-ku, Tokyo 103-8411, Japan.
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.015

S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
1045
7
Cl
O
sized according to the procedure of Hisamichi et al.
The displacement reaction of 2-benzotriazo-l-yloxy-4-
[(3-methylphenyl)amino]pyrimidine-5-carboxamide 1 with
phenylalkylamines was carried out at 80 ꢁC in N-meth-
ylpyrrolidinone (NMP) and afforded 4-[(3-methylphe-
nyl)amino]pyrimidine-5-carboxamide derivatives 2 with
phenylalkylamino groups at C-2 of the pyrimidine ring.
HO
Me
OMe
O
MeO
OH O
Figure 1. The Structure of TMC-264.
The synthesis of various N-alkylamide analogues of 2a is
described in Scheme 2. Treatment of 2-chloropyrimi-
dine-5-carboxylic acid 3 with tyramine, followed by
condensation with the corresponding amines in the pres-
ence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCÆHCl) and 1-hydroxybenzotriazole
STAT6 reporter assay; this led to the identification of
-{[2-(4-hydroxyphenyl)ethyl]amino}-4-[(3-methylphe-
nyl)amino] pyrimidine-5-carboxamide (2a) as a STAT6
2
7
inhibitor. Herein, we describe the synthesis of a series of
2
-{[2-(4-hydroxyphenyl)ethyl]amino}pyrimidine-5-carbox-
amide derivatives, their structure–activity relationships
SARs) for STAT6 inhibition, and the effect of one
(HOBt), resulted in formation of 2m–p.
(
derivative on Th1/Th2 differentiation in mouse spleen
T cells.
The synthetic route of analogues of 2a with various
substituents at C-4 of the pyrimidine ring is outlined in
Scheme 3. Treatment of ethyl 2,4-dichloropyrimidine-
5
-carboxylate 5 with sodium thiomethoxide (NaSMe) in
2
. Chemistry
tetrahydrofuran (THF) at ꢀ10 ꢁC in the presence of ben-
zyltriethylammonium chloride (BnEt NCl) gave ethyl
3
As shown in Scheme 1, 4-[(3-methylphenyl)amino]py-
rimidine-5-carboxamide derivatives 2a–l were synthe-
2-chloro-4-methylsulfanylpyrimidine-5-carboxylate 6.
This displacement reaction proceeded very slowly below
ꢀ
20 ꢁC, whereas ethyl 2,4-dimethylsulfanylpyrimidine-
-carboxylate was obtained as the major product above
5
0
ꢁC. Reaction of 6 with tyramine proceeded selectively
at C-2 of the pyrimidine ring to give 4-methylsulfanylpyr-
imidine derivative 7. Hydrolysis of the ethoxycarbonyl
group of 7 by 1 M NaOH and condensation using aque-
ous ammonia gave pyrimidine-5-carboxamide derivative
9. The methylsulfanyl group of 9 was carefully oxidized
with m-chloroperbenzoic acid (m-CPBA) to afford meth-
ylsulfinyl derivative 10. In the case of using excess
amounts of m-CPBA gave several unknown products
and desirable methylsulfonyl derivative was not
obtained in acceptable yield. Finally, displacement of
the methylsulfinyl group of 10 with cyclohexylamine, ben-
zylamine or aniline furnished compounds 2q, 2r or 2s,
respectively.
NH O
N
NH O
N
a
2
3
N
NH2
R
R
N
N
N
N
NH2
1
O
R
(CH2)n
N
H
1
2a
l
1
2
3
No.
R
R
R
n
2
2
2
2
2
2
2
2
2
a
b
c
d
e
f
g
h
i
j
HO
H
MeO
HO
HO
H
H
H
H
H
H
HO
H
Me
H
H
H
H
H
H
HO
H
H
H
H
H
2
2
2
1
3
2
2
2
2
2
2
2
H
HO
HO
HO
HO
HO
MeO
F
An alternative synthetic route for analogues of 2a is de-
scribed in Scheme 4. 4-Benzylamino-2-methylsulfanyl-
pyrimidine derivatives 14 and 15, which were readily
prepared from commercially available 2-methylsulfanyl-
pyrimidine derivative 11 according to the procedure in
2
2
2
k
l
Cl
Br
Scheme 1. Reagents: (a) phenylalkylamine (see text and Section 5),
NMP.
7
the literature, were converted into the corresponding
a
NH
N
b
NH O
N
NH
4
HO
CO H
HO
R
5
CO H
N
2
N
N
R
2
N
N
N
Cl
N
H
H
3
4
2
m p
4
5
No.
NR R
2
2
2
2
m
NHMe
NHEt
NH Pr
n
o
p
i
NMe
2
4
5
i
2
Scheme 2. Reagents: (a) tyramine, NMP; (b) EDCÆHCl, HOBt, R R NH, DMF, Pr NEt.

1046
S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
SMe
CO Et
Cl
N
SMe
HO
CO Et
a
CO Et
b
2
c
2
N
2
N
N
N
H
N
Cl
Cl
N
5
6
7
SMe
SMe
HO
HO
CONH2
N
e
HO
CO H
2
d
N
N
H
N
N
H
N
8
9
2
6
R
O
Me
NH
N
S
N
CONH2
HO
CONH2
f
N
N
N
H
N
H
q
s
1
0
6
No.
R
2
2
q
r
Cyclohexyl
Ph-CH
2
2s
Ph
i
NCl, THF; (b) tyramine, Pr
Scheme 3. Reagents: (a) NaSMe, BnEt
(
3
2
NEt, NMP; (c) 1 M NaOH, MeOH; (d) EDCÆHCl, HOBt, NH
4
OH, DMF;
6
e) m-CPBA, NMP; (f) R NH
i
, Pr
2
2
NEt, NMP.
Cl
NH
N
c
CO Et
a
b
NH
N
2
CO Et
2
N
N
CO H
2
N
S
N
S
S
1
1
12
13
d
e
NH O
Cl
NH O
N
NH O
4
4
4
R
R
R
HO
N
N
H
N
N
N
N
H
H
S
N
N
H
S
N
O
O
1
4–15
16–17
2t–u
4
4
4
No
No.
No.
R
1
1
4
5
H
Me
16
17
H
Me
2t
2u
H
Me
i
Scheme 4. Reagents: (a) benzylamine, Pr
2
NEt, MeCN; (b) 1 M NaOH, THF; (c) EDCÆHCl, HOBt, NH
4
OH or MeNH , DMF; (d) m-CPBA, NMP;
2
i
2
(e) 2-(3-chloro-4-hydroxylphenyl)ethylamine, Pr NEt, NMP.
methylsulfonyl derivatives 16 and 17, respectively, by
reaction with m-CPBA. Displacement of the meth-
ylsulfonyl group by 2-(3-chloro-4-hydroxy)phenethyl-
amine proceeded smoothly at 80 ꢁC and gave
compounds 2t and 2u.
enemalonate, gave the 2-{3-[4-(benzyloxy)phenyl]pro-
pyl}-4-hydroxypyrimidine derivative 19. Chlorination
of 19 and subsequent substitution with m-toluidine pro-
vided compound 21. Hydrolysis of the ethoxycarbonyl
group afforded carboxylic acid 22, and condensation
of the carboxylic acid using aqueous ammonia gave
the pyrimidine-5-carboxamide derivative 23. Finally,
deprotection of the benzyl group using hydrogenation
in the presence of palladium on carbon furnished com-
pound 24.
The preparation of 2-[3-(4-hydroxyphenyl)propyl]py-
rimidine derivative 24 is shown in Scheme 5. Conver-
sion of butyronitrile 18 into the corresponding
amidine, followed by condensation with ethoxymethyl-

S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
1047
OH
N
Table 1. STAT6 inhibitory activity of 4-[(3-methylphenyl)amino]py-
rimidine-5-carboxamide derivatives 2a–l and 24
BnO
CO Et
a, b, c
N
2
d
CN
BnO
1
2
9
1
1
2
8
0
NH O
2
3
R
R
3
N
NH2
Cl
N
2
BnO
CO Et
1
4
N
2
NH
N
f
R
(CH )
X
N
e
g
2 n
BnO
BnO
CO Et
N
2
1
R
2
3
R
a
Compound
R
n
X
IC50 (nM)
2a
2b
2c
HO
H
H
H
H
H
H
H
H
HO
H
H
H
H
H
H
2
2
2
1
3
2
2
2
2
2
2
2
2
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
190
b
H
16%
b
MeO
HO
HO
H
H
49%
NH
NH
N
h
b
BnO
CO H
CONH2
2d
H
19%
N
2
N
b
2
2
e
f
H
23%
N
HO
H
3000
2
3
b
2
2
2
2g
H
15%
2
2
2
2
2
2
h
i
HO
HO
HO
HO
HO
HO
Me
130
660
64
MeO
F
Cl
j
NH
N
CONH2
k
l
45
83
HO
N
Br
H
b
4
CH
2
32%
4
a
IC50 values were determined in triplicate in one experiment.
% inhibition at 10 lm.
b
Scheme 5. Reagents: (a) HCl, EtOH–CHCl ; (b) AcONH
3
4
, EtOH; (c)
diethyl ethoxymethylenemalonate, NaOMe, EtOH; (d) POCl
3
, diethy-
NEt, MeCN; (f) 1 M NaOH, EtOH–THF;
OH, DMF; (h) H , 10% Pd–C, MeOH–
i
laniline; (e) m-toluidine, Pr
g) EDCÆHCl, HOBt, NH
THF.
2
(
4
2
halogen at the 3-position (2j–l) showed improved activ-
ity compared to compound 2a. In particular, chloro
derivative 2k was 4-fold more potent than 2a. In con-
trast, methoxy derivative 2i was 3-fold less active than
compound 2a.
3
. Results and discussion
The ability of the compounds to inhibit STAT6 activa-
tion was measured in a STAT6-dependent promoter
reporter assay utilizing FW4 reporter cells, which ex-
press both human IL-4 receptor a and human IL-2
receptor c subunits and were stably transfected with
an IL-4-responsive luciferase reporter plasmid.
Vinogradova et al. have shown that chemical shifts of
hydroxyl groups in substituted phenols correlate with
acidity; compounds with higher chemical shifts are more
8
1
acidic. The H NMR chemical shifts of the hydroxyl
group of 2a and 2h–l are listed in Table 2. The hydroxyl
groups of the halogen derivatives 2j–l resonated at lower
field compared to that of 2a, and the chemical shift of
the hydroxyl group in the methyl derivative 2h was
The activities of derivatives with modifications of the
2
-(4-hydroxyphenyl)ethylamino moiety are shown in
Table 1. 2-[2-(4-Hydroxyphenyl)ethylamino]pyrimidine
derivative 2a, which was found in high-throughput
screening, exhibited a moderate STAT6 inhibitory activ-
ity with an IC50 value of 190 nM. Removal or methyla-
tion of the hydroxyl group resulted in loss of activity (2b
and 2c) and a similar loss of activity was also observed
in the case of conversion of the nitrogen into carbon
at C-2 of the pyrimidine ring (24). These results indicate
that the hydroxyl group and the NH moiety at C-2 of
the pyrimidine ring are essential for STAT6 inhibition.
Compounds 2d and 2e, in which the ethylene linker of
1
Table 2. H NMR chemical shifts of the proton of hydroxyl group and
STAT6 inhibitory activity of 2a and 2h–l
2
R
NH O
N
HO
N
NH2
N
H
2
a was replaced by methylene and propylene, respective-
ly, showed no activity. Transposition of the hydroxyl
group of 2a from the 4-position to the 3-position (2f)
led to a 16-fold decrease in the activity, whereas the 2-
hydroxyl derivative (2g) showed no activity. These re-
sults suggest that the position of the hydroxyl group
has a significant influence on STAT6 inhibitory activity
and that a 2-(4-hydroxyphenyl)ethyl moiety might place
the hydroxyl group in a suitable position to increase
potency. Introduction of a methyl group at the 3-posi-
tion of 2a was well tolerated and compounds with a
2
a
b
Compound
R
d (ppm)
IC50 (nM)
2
2
a
h
H
8.90
8.73
8.39
9.29
9.59
9.67
190
130
660
64
Me
MeO
F
2i
2j
2k
Cl
Br
45
83
2
l
a 1
b
H NMR chemical shifts were measured in DMSO-d . See Section 5.
6
IC50 values were determined in triplicate in one experiment.

1048
S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
similar to that of 2a. On the contrary, the hydroxyl
group of the methoxy derivative 2i resonated at higher
field compared to that of 2a. These data suggest that
the hydroxyl group of halogen derivatives 2j–l, which
showed improved STAT6 inhibitory activity, might be
more acidic than that in 2a, and that the hydroxyl group
in methoxy derivative 2i, which had reduced activity,
might be less acidic than that in 2a. On the basis of these
results, it appears likely that increased acidity of pheno-
lic group is beneficial effect for the STAT6 inhibitory
activity.
Table 4. STAT6 inhibitory activity of 2-{[2-(4-hydroxyphe-
nyl)ethy]lamino}pyrimidine-5-carboxamide derivatives 2a and 2q–u
6
2
R
R
NH O
N
4
HO
R
N
N
H
N
H
2
R
4
R
6
R
a
Compound
IC50 (nM)
2
2
2
2
2
2
a
H
H
H
H
Cl
Cl
H
3-Me–Ph
Ph
Cyclohexyl
190
410
100
88
s
H
q
r
H
H
PhCH
PhCH
PhCH
2
2
2
Next, the effect of the 5-carboxamide group on activity
was assessed. As shown in Table 3, N-methyl amide
t
H
Me
21
30
u
2
m exhibited activity comparable with that of 2a,
a
IC50 values were determined by in triplicate in one experiment.
whereas replacement of the methyl group with larger
substituents, such as ethyl (2m) or isopropyl (2n) groups,
led to a decrease in activity. Furthermore, a greater de-
crease in activity was observed with the N,N-dimethyl
amide derivative 2p. These results suggest that the allow-
able steric bulk around the amide nitrogen might be
small.
1
80
160
IFN-
IL-4
1
1
1
40
20
00
The effects of substituents at C-4 of the pyrimidine ring
on STAT6 inhibitory activity are shown in Table 4.
Whereas phenyl derivative 2s was about 2-fold less ac-
tive than 2a, cyclohexyl analogue 2q and benzyl ana-
logue 2r were about 2-fold more potent than
compound 2a. In the case of 4-benzylamino derivatives,
introduction of a chloro group at the 3-position also im-
proved the activity; compounds 2t and 2u showed potent
STAT6 inhibitory activity with IC50 values of 21 and
8
0
60
4
0
20
0
3
0 nM, respectively.
0
0.3
1
3
10
30
The effect of the potent STAT6 inhibitor 2t on differen-
tiation of T cells to Th cell subsets was examined. Mouse
spleen T cells stimulated by anti-CD3 antibody in the
presence of IL-12 or IL-4 differentiate into Th1 or Th2
cells, respectively. As shown in Figure 2, in the presence
of IL-12 compound 2t showed no effect on the ability of
spleen T cells to produce IFN-c, which is a marker of
Th1 differentiation. In contrast, in the presence of IL-4
compound 2t inhibited production of IL-4, a marker
2t (nM)
Figure 2. The effect of compound 2t on cytokine production in T cells
from spleen of mice. Squares indicate the amount of IFN-c produced
by cells cultured in the presence of IL-12, which induces differentiation
to Th1 cells, and circles indicate the amount of IL-4 produced by cells
cultured with IL-4, which induces Th2 cells. The cytokine levels in
control cells (DMSO) were as follows (ng/ml): IFN-c = 292 ± 84.0; IL-
4 = 2.68 ± 0.27. Data are shown as means ± SEM expressed as
percentages relative to values from the control cells (n = 6).
Table 3. STAT6 inhibitory activity of 2-{[2-(4-hydroxyphenyl)-
ethy]lamino}-4-[(3-methylphenyl)amino]pyrimidine-5-carboxamide deri-
vatives 2a and 2m–p
9
of Th2 differentiation, with an IC50 value of 2.3 nM.
These results suggest that compound 2t selectively inhib-
its Th2 differentiation without affecting Th1 differentia-
tion; we note that a similar response to IL-4 was
4
observed in spleen T cells from STAT6-deficient mice.
NH O
4
HO
R
5
As far as we are aware, our results provide the first dem-
onstration of a low molecular weight compound that
selectively inhibits Th2 differentiation by regulation of
N
N
R
N
N
H
1
0
the IL-4 signaling cascade.
4
R
5
R
a
Compound
IC50 (nM)
2
2
2
2
2
a
H
H
H
H
H
190
170
m
n
Me
Et
4. Conclusion
380
o
CHMe
Me
2
1100
8200
In conclusion, we have reported a series of 2-{[2-(4-
hydroxyphenyl)ethyl]amino}pyrimidine-5-carboxamide
derivatives as novel STAT6 inhibitors and investigated
p
Me
a
IC50 values were determined by in triplicate in one experiment.

S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
1049
1
the SARs of these derivatives. With regard to substitu-
ents at C-2 of the pyrimidine ring, a 2-(4-hydroxyphe-
nyl)ethylamino moiety gives the best activity, and
introduction of a halogen group at the 3-position of
the phenol moiety led to further improvement of activi-
ty. Concerning the carboxamide moiety, introduction of
a sterically less-hindered alkyl group, such as methyl, is
tolerable. With respect to substituents at C-4 of the
pyrimidine ring, benzylamine and cyclohexylamine were
more favorable than aniline. On the basis of the SAR
studies, we optimized the structure and identified 2t as
the most potent STAT6 inhibitor. Compound 2t was
subsequently shown to inhibit IL-4-induced Th2 differ-
entiation of mouse spleen T cells, but had no influence
on Th1 differentiation induced by IL-12. These results
suggest that compound 2t (AS1517499) might be useful
for treatment of various allergic conditions caused by
excess Th2 responses, such as asthma and atopic diseas-
es. A further investigation of this novel class of STAT6
inhibitors will be reported in the near future.
Mp 178–180 ꢁC; H NMR (DMSO-d , 80 ꢁC) d 2.26
6
(3H, s), 2.75 (2H, t, J = 7.2 Hz), 3.46–3.52 (2H, m),
6.67 (2H, d, J = 8.0 Hz), 6.84 (1H, d, J = 7.6 Hz), 6.99
(2H, d, J = 8.0 Hz), 7.14–7.19 (2H, m), 7.33 (1H, br s),
7.50 (1H, s), 7.53 (1H, s), 8.55 (1H, s), 8.90 (1H, s),
11.42 (1H, s); FAB MS m/e (M+H) 364. Anal. Calcd
for C H N O : C, 66.10; H, 5.82; N, 19.27. Found:
C, 66.09; H, 5.87; N, 19.30.
+
2
0
21
5
2
5.1.2. 4-[(3-Methylphenyl)amino]-2-[(2-phenylethyl)ami-
no]pyrimidine-5-carboxamide (2b). Compound 2b was
prepared from compound 1 and 2-phenylethylamine
in 38% yield as a colorless solid, using a similar ap-
proach to that described for 2a: mp 162–165 ꢁC
(MeOH–THF); H NMR (DMSO-d , 80 ꢁC) d 2.25
(3H, s), 2.85 (2H, t, J = 7.2 Hz), 3.52–3.54 (2H,
m), 6.85 (1H, d, J = 7.2 Hz), 7.15–7.30 (7H, m),
7.46–7.52 (3H, m), 7.83 (1H, br s), 7.51 (2H, br)+,
8.54 (1H, s), 11.42 (1H, s); FAB MS m/e (M+H)
348. Anal. Calcd for C H N O: C, 69.14;
1
6
2
0
21
5
H, 6.09; N, 20.16. Found: C, 68.94; H, 5.78; N,
19.99.
5. Experimental
5.1.3. 2-{[2-(4-Methoxyphenyl)ethyl]amino}-4-[(3-methyl-
5
.1. Chemistry
phenyl)amino]pyrimidine-5-carboxamide (2c). Com-
pound 2c was prepared from compound 1 and 2-(4-
methoxyphenyl)ethylamine in 40% yield as a colorless
solid, using a similar approach to that described for
1
H NMR spectra were measured with a JEOL EX400
400 MHz) or GX500 (500 MHz) spectrometer; chem-
(
1
ical shifts are expressed in d units using tetramethylsi-
lane as the standard (NMR peak description: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
and br, broad peak). Compounds with a phenylalkyla-
mino group at C-2 of the pyrimidine ring showed
2a: mp 171–172 ꢁC (MeOH–THF); H NMR (DMSO-
d , 80 ꢁC) d 2.26 (3H, s), 2.80 (2H, t, J = 7.2 Hz),
6
3.49–3.54 (2H, m), 3.72 (3H, s), 6.82–6.84 (3H, m),
7.16–7.21 (5H, m), 7.33 (1H, br s), 7.49 (2H, br), 8.54
(1H, s), 11.41 (1H, s); FAB MS m/e (M+H) 378. Anal.
+
1
characteristic broad peaks in the H NMR spectra
Calcd for C H N O : C, 66.83; H, 6.14; N, 18.55.
2
2
1
23
5
at room temperature, probably because of the pres-
ence of a conformational isomer. As an example of
typical case, the methyl group of the 3-methylphenyl
moiety at C-4 of the pyrimidine ring and both NH
Found: C, 66.70; H, 5.98; N, 18.50.
5.1.4. 2-[(4-Hydroxybenzyl)amino]-4-[(3-methylphenyl)-
amino]pyrimidine-5-carboxamide (2d). Compound 2d
was prepared from compound 1 and 4-hydroxybenzyl-
amine in 44% yield as a colorless solid, using a similar
protons attached to the pyrimidine ring of compound
2
1
a were observed as broad signal. Therefore, the H
NMR spectra of selected compounds were measured
at 80 ꢁC to confirm the structure. Mass spectra were
recorded with a Hitachi M-80 or a JEOL JMS-
DX300 spectrometer. Drying of organic solutions dur-
ing workup was performed over anhydrous MgSO4.
Column chromatography was carried out on silica
gel (Kieselgel 60). Unless otherwise noted, all reagents
and solvents obtained from commercial suppliers were
used without further purification.
approach to that described for 2a: mp 280–281 ꢁC
1
(MeOH–THF); H NMR (DMSO-d , 80 ꢁC) d 2.26
6
(3H, s), 4.42 (2H, s), 6.68 (2H, d, J = 7.8 Hz), 6.81
(1H, d, J = 6.8 Hz), 7.10–7.19 (4H, m), 7.24–7.95 (4H,
m), 8.56 (1H, s), 9.22 (1H, s), 11.43 (1H, s); FAB MS
+
m/e (M+H) 350. Anal. Calcd for C H N O : C,
1
9
19
5
2
65.32; H, 5.48; N, 20.04. Found: C, 65.29; H, 5.49; N,
20.33.
5.1.5. 2-{[3-(4-Hydroxyphenyl)propyl]amino}-4-[(3-meth-
5
.1.1. 2-{[2-(4-Hydroxyphenyl)ethyl]amino}-4-[(3-methyl-
phenyl)amino]pyrimidine-5-carboxamide (2a). Tyramine
ylphenyl)amino]pyrimidine-5-carboxamide (2e). Com-
pound 2e was prepared from compound 1 and 3-(4-
hydroxyphenyl)propylamine in 45% yield as a pale-yel-
low solid, using a similar approach to that described
7
(5.31 g, 38.8 mmol) was added to a solution of 1
(7.00 g, 19.4 mmol) in NMP (140 mL) and the mixture
1
was stirred for 3 h at 80 ꢁC. The reaction mixture was
then diluted with AcOEt and washed successively with
H O and saturated aqueous NaCl. The organic layer
2
was dried and concentrated in vacuo. The residue
was chromatographed on silica gel with elution using
CHCl –MeOH–NH OH (28%) (500:10:1) to give a
colorless solid, which was recrystallized from
AcOEt–hexane to give 2a (5.30 g, 75%) as a colorless
solid.
for 2a: mp 203–204 ꢁC (MeOH–THF); H NMR
(DMSO-d , 80 ꢁC) d 1.76–1.83 (2H, m), 2.29 (3H, s),
6
2.52–2.56 (2H, m), 3.28–3.30 (2H, m), 6.65 (2H, d,
J = 7.8 Hz), 6.84 (1H, d, J = 7.4 Hz), 6.98 (2H, d,
J = 7.8 Hz), 7.18 (1H, t, J = 7.8 Hz), 7.21–7.83 (5H,
m), 8.55 (1H, s), 9.08 (1H, s), 11.43 (1H, s); FAB MS
3
4
+
m/e (M+H) 378. Anal. Calcd for C H N O : C,
5
2
1
23
2
66.83; H, 6.14; N, 18.55. Found: C, 66.92; H, 6.29; N,
18.49.

1050
S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
1
5
.1.6. 2-{[2-(3-Hydroxyphenyl)ethyl]amino}-4-[(3-methyl-
phenyl)amino]pyrimidine-5-carboxamide (2f). Compound
f was prepared from compound 1 and 2-(3-hydroxy-
described for 2a: mp 208–209 ꢁC (MeOH–THF);
H
NMR (DMSO-d , 80 ꢁC) d 2.26 (3H, s), 2.76 (2H, t,
J = 7.2 Hz), 3.48–3.53 (2H, m), 6.79–6.85 (3H, m), 6.93
(1H, d, J = 8.4 Hz), 7.15 (1H, d, J = 8.0 Hz), 7.21 (1H,
br s), 7.33 (1H, br s), 7.49 (2H, br), 8.54 (1H, s), 9.29
(1H, s), 11.41 (1H, s); FAB MS m/e (M+H) 382. Anal.
Calcd for C H FN O : C, 62.98; H, 5.29; F, 4.98; N,
18.36. Found: C, 62.91; H, 5.30; F, 5.07; N, 18.36.
6
2
phenyl)ethylamine hydrochloride with diisopropylethyl-
amine in 26% yield as a colorless solid, using a similar
+
approach to that described for 2a: mp 192–193 ꢁC
1
(
(
MeOH–THF); H NMR (DMSO-d , 80 ꢁC) d 2.26
6
20 20
5
2
3H, s), 2.78 (2H, t, J = 6.8 Hz), 3.50–3.55 (2H, m),
6
.59–6.64 (3H, m), 6.83 (1H, d, J = 8.0 Hz), 7.06 (1H,
t, J = 7.6 Hz), 7.17 (1H, t, J = 7.6 Hz), 7.21 (1H, br s),
.34 (1H, br s), 7.49–7.53 (2H, m), 8.55 (1H, s), 9.01
5.1.11. 2-{[2-(3-Chloro-4-hydroxyphenyl)ethyl]amino}-4-
[(3-methylphenyl)amino]pyrimidine-5-carboxamide (2k).
Compound 2k was prepared from compound 1 and
2-(3-chloro-4-hydroxyphenyl)ethylamine in 55% yield
7
+
(1H, s), 11.42 (1H, s); FAB MS m/e (M+H) 364. Anal.
Calcd for C H N O : C, 66.10; H, 5.82; N, 19.27.
2
0
21
5
2
Found: C, 66.18; H, 5.92; N, 19.53.
as a colorless solid, using a similar approach to that de-
scribed for 2a: mp 176–177 ꢁC (MeOH–AcOEt);
1
H
5
.1.7. 2-{[2-(2-Hydroxyphenyl)ethyl]amino}-4-[(3-methyl-
NMR (DMSO-d , 80 ꢁC) d 2.26 (3H, s), 2.76 (2H, t,
6
phenyl)amino]pyrimidine-5-carboxamide (2g). Com-
pound 2g was prepared from compound 1 and 2-(2-
hydroxyphenyl)ethylamine in 46% yield as a pale-yellow
solid, using a similar approach to that described for 2a:
J = 7.2 Hz), 3.47–3.52 (2H, m), 6.84–6.87 (2H, m), 6.95
(1H, d, J = 8.4 Hz), 7.14–7.19 (2H, m), 7.26 (1H, br s),
7.35 (1H, br s), 7.49 (2H, br), 8.54 (1H, s), 9.59 (1H,
+
s), 11.43 (1H, s); FAB MS m/e (M+H) 398. Anal. Calcd
for C H ClN O : C, 60.38; H, 5.07; Cl, 8.91; N, 17.60.
Found: C, 66.00; H, 5.07; Cl, 8.76; N, 17.67.
1
mp 255–257 ꢁC (EtOH–THF); H NMR (DMSO-d6,
20
20
5
2
8
3
0 ꢁC) d 2.25 (3H, s), 2.83 (2H, t, J = 7.2 Hz), 3.51–
.56 (2H, m), 6.70 (1H, t, J = 7.6 Hz), 6.78–6.83 (2H,
m), 6.99–7.06 (2H, m), 7.13–7.19 (2H, m), 7.33 (1H, br
s), 7.51 (2H, br), 8.54 (1H, s), 9.12 (1H, s), 11.42 (1H,
s); FAB MS m/e (M+H) 364. Anal. Calcd for
5.1.12. 2-{[2-(3-Bromo-4-hydroxyphenyl)ethyl]amino}-4-
[(3-methylphenyl)amino]pyrimidine-5-carboxamide (2l).
Compound 2l was prepared from compound 1 and 2-
(3-bromo-4-hydroxyphenyl)ethylamine in 27% yield as
+
C H N O : C, 66.10; H, 5.82; N, 19.27. Found: C,
2
0
21
5
2
6
5.95; H, 5.96; N, 19.27.
a colorless solid, using a similar approach to that de-
scribed for 2a: mp 179–180 ꢁC (EtOH–THF);
1
H
5
.1.8. 2-{[2-(4-Hydroxy-3-methylphenyl)ethyl]amino}-4-
NMR (DMSO-d , 80 ꢁC) d 2.27 (3H, s), 2.76 (2H, t,
6
[
(3-methylphenyl)amino]pyrimidine-5-carboxamide (2h).
Compound 2h was prepared from compound 1 and 2-
4-hydroxy-3-methylphenyl)ethylamine in 16% yield as
a colorless solid, using a similar approach to that de-
J = 7.2 Hz), 3.47–3.52 (2H, m), 6.84 (1H, d,
J = 8.4 Hz), 6.99 (1H, d, J = 8.0 Hz), 7.17–7.30 (4H,
m), 7.49 (2H, br), 8.54 (1H, s), 9.67 (1H, s), 11.40 (1H,
(
+
s); FAB MS m/e (M) 442. Anal. Calcd for
C H BrN O : C, 54.31; H, 4.56; Br, 18.07; N, 15.83.
Found: C, 54.33; H, 4.45; Br, 17.80; N, 15.82.
1
scribed for 2a: mp 209–211 ꢁC (MeOH–THF);
H
2
0
20
5
2
NMR (DMSO-d , 80 ꢁC) d 2.08 (3H, s), 2.26 (3H, s),
6
2
.71 (2H, t, J = 7.2 Hz), 3.46–3.59 (2H, m), 6.66 (1H,
d, J = 8.0 Hz), 6.80–6.85 (3H, m), 7.14–7.18 (2H, m),
.33 (1H, br s), 7.50 (2H, br), 8.54 (1H, s), 8.73 (1H,
5.1.13. 2-{[2-(4-Hydroxyphenyl)ethyl]amino}-4-[(3-meth-
ylphenyl)amino]pyrimidine-5-carboxylic acid (4). Tyra-
7
+
s), 11.42 (1H, s); FAB MS m/e (M+H) 378. Anal. Calcd
for C H N O : C, 66.83; H, 6.14; N, 18.55. Found: C,
6
mine
diisopropylethylamine (5.26 mL, 30.2 mmol) were add-
hydrochloride
(2.39 g,
13.7 mmol)
and
2
1
23
5
2
7
6.60; H, 6.16; N, 18.57.
ed to a solution of 3 (1.81 g, 6.86 mmol) in NMP
(
15 mL) and the mixture was stirred for 14 h at 80 ꢁC.
5
4
.1.9. 2-{[2-(4-Hydroxy-3-methoxyphenyl)ethyl]amino}-
-[(3-methylphenyl)amino]pyrimidine-5-carboxamide (2i).
The reaction mixture was then diluted with H O
2
(150 mL) and the resulting solid was collected by filtra-
tion, triturated with THF–MeOH, and collected by fil-
tration to give 4 (1.23 g, 49%), which was used in the
Compound 2i was prepared from compound 1 and 2-(4-
hydroxy-3-methoxylphenyl)ethylamine in 25% yield as a
1
colorless solid, using a similar approach to that de-
H
next reaction without further purification. H NMR
(DMSO-d ) d 2.25–2.32 (3 H, br), 2.70–2.75 (2H, m),
1
scribed for 2a: mp 182–183 ꢁC (MeOH–THF);
6
NMR (DMSO-d , 80 ꢁC) d 2.25 (3H, s), 2.76 (2H, t,
3.43–3.48 (2H, m), 6.67 (2H, d, J = 8.2 Hz), 6.86–7.04
(3H, m), 7.20 (1H, t, J = 7.6 Hz), 7.49–7.57 (2H, m),
7.65–7.85 (1H, m), 8.53–8.62 (1H, br), 9.18 (1H, s),
10.49–10.56 (1H, br), 12.18 (1H, br); FAB MS m/e
6
J = 7.2 Hz), 3.50–3.55 (2H, m), 3.71 (3H, s), 6.60 (1H,
d, J = 7.6 Hz), 6.67 (1H, d, J = 8.0 Hz), 6.75 (1H, s),
6
(
.83 (1H, d, J = 7.2 Hz), 7.15 (1H, t, J = 8.0 Hz), 7.20
1H, br s), 7.33 (1H, br s), 7.50 (2H, br), 8.39 (1H, s),
+
(M+H) 365.
+
.54 (1H, s), 11.42 (1H, s); FAB MS m/e (M+H) 384.
8
Anal. Calcd for C H N O : C, 64.11; H, 5.89; N,
5.1.14. 2-{[2-(4-Hydroxyphenyl)ethyl]amino}-N-methyl-4-
[(3-methylphenyl)amino]pyrimidine-5-carboxamide (2m).
EDCÆHCl (223 mg, 1.16 mmol) and HOBt (157 mg,
1.16 mmol) were added to a suspension of 4 (352 mg,
0.97 mmol) in DMF (4 mL). After stirring for 10 min
at room temperature, methylamine hydrochloride
2
1
23
5
3
1
7.80. Found: C, 64.18; H, 5.87; N, 17.77.
5
.1.10. 2-{[2-(3-Fluoro-4-hydroxyphenyl)ethyl]amino}-4-
[
(3-methylphenyl)amino]pyrimidine-5-carboxamide (2j).
Compound 2j was prepared from compound 1 and
-(3-fluoro-4-hydroxyphenyl)ethylamine in 16% yield
as a pale-yellow solid, using a similar approach to that
2
(79 mg,
1.16 mmol)
and
(0.38 mL, 2.13 mmol) were added and the mixture was
diisopropylethylamine

S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
1051
stirred for 16 h at room temperature. The reaction mix-
ture was diluted with AcOEt and washed successively
with H O and saturated aqueous NaCl. The organic
5.1.18. Ethyl 2-chloro-4-(methylsulfanyl)pyrimidine-5-
carboxylate (6). NaSMe (4.12 g, 58.9 mmol) and ben-
zyltriethylammonium chloride (128 mg, 0.56 mmol)
were added to a solution of ethyl 2,4-dichloropyrimi-
dine-5-carboxylate (5, 12.4 g, 56.1 mmol) in THF
(180 mL) and the mixture was stirred at ꢀ10 ꢁC for
2
layer was dried and concentrated in vacuo, and the res-
idue was chromatographed on silica gel with elution
using CHCl –MeOH–NH OH (28%) (300:10:1) to give
3
4
1
2
m (154 mg, 42%) as a brown amorphous solid. H
3 h. The reaction mixture was then diluted with Et O
2
NMR (DMSO-d , 80 ꢁC) d 2.26 (3H, s), 2.73–2.77
and ice water, and the resulting solid was collected by fil-
tration to give 6 (7.0 g, 54%), which was used in the next
6
(
5H, m), 3.46–3.52 (2H, m), 6.67 (2H, d, J = 8.4 Hz),
1
6
7
.84 (1H, d, J = 7.2 Hz), 6.99 (2H, d, J = 8.4 Hz),
.14–7.18 (2H, m), 7.51 (2H, br), 8.13 (1H, br s), 8.48
reaction without further purification.
H
NMR
(DMSO-d ) d 1.33 (3H, t, J = 7.1 Hz), 2.50 (3H, s),
6
(
1H, s), 8.90 (1H, s), 11.30 (1H, s); FAB MS m/e
4.35 (2H, q, J = 7.1 Hz), 8.90 (1H, s). FAB MS m/e
(M+H) 233.
+
+
(
M+H) 378. Anal. Calcd for C H N O Æ0.2H O: C,
2
1
23
5
2
2
6
1
6.19; H, 6.19; N, 18.38. Found: C, 66.28; H, 6.17; N,
8.28.
5.1.19. Ethyl 2-{[2-(4-hydroxyphenyl)ethyl]amino}-4-
(methylsulfanyl)pyrimidine-5-carboxylate (7). Tyramine
5
.1.15. N-Ethyl-2-{[2-(4-hydroxyphenyl)ethyl]amino}-4-
hydrochloride (5.34 g, 7.55 mmol) and diisopropylethyl-
amine (11.2 mL, 64.2 mmol) were added to a solution of
6 (6.49 g, 30.7 mmol) in NMP (60 mL) and the mixture
was stirred for 16 h at 80 ꢁC. The reaction mixture was
then diluted with AcOEt (600 mL) and washed succes-
[
(3-methylphenyl)amino]pyrimidine-5-carboxamide (2n).
Compound 2n was prepared from compound 4 and
0% aqueous ethylamine by a procedure similar to that
7
described for 2m, excluding diisopropylethylamine. 2n
was obtained in 56% yield as a colorless solid: mp
sively with H O and saturated aqueous NaCl. The
2
1
1
8
(
79–180 ꢁC (AcOEt–hexane); H NMR (DMSO-d6,
0 ꢁC) d 1.13 (3 H, t, J = 7.2 Hz), 2.26 (3 H, s), 2.74
2H, t, J = 7.2 Hz), 3.24–3.31 (2H, m), 3.46–3.51 (2H,
organic layer was dried and concentrated, and the
resulting solid was triturated with MeCN to give 7
(6.42 g, 69%), which was used in the next reaction with-
1
m), 6.66 (2H, d, J = 8.4 Hz), 6.83 (1H, d, J = 7.2 Hz),
.98 (2H, d, J = 8.4 Hz), 7.14–7.18 (2H, m), 7.50 (2H,
br), 8.16 (1H, br s), 8.50 (1H, s), 8.91 (1H, s), 11.31
out further purification. H NMR (DMSO-d ) d 1.28
6
6
(3H, t, J = 7.1 Hz), 2.37 (1H, s, SCH ), 2.43 (2H, s,
SCH ), 2.72–2.77 (2H, m), 3.48–3.55 (2H, m), 4.22
3
3
+
(1H, s); FAB MS m/e (M+H) 392. Anal. Calcd for
C H N O : C, 67.50; H, 6.44; N, 17.89. Found: C,
6
(2H, q, J = 7.1 Hz), 6.67 (2H, d, J = 8.2 Hz), 7.01 (2H,
d, J = 8.2 Hz), 8.09 (0.33H, br t, J = 5.8 Hz, NH), 8.13
(0.67 H, br t, J = 5.8 Hz, NH), 8.54 (0.67 H, s, CH at
C-6 of pyrimidine), 8.63 (0.33 H, s, CH at C-6 of pyrim-
2
2
25
5
2
7.39; H, 6.54; N, 18.09.
+
5
.1.16. 2-{[2-(4-Hydroxyphenyl)ethyl]amino}-4-[(3-meth-
idine), 9.17 (1H, s). FAB MS m/e (M+H) 334.
ylphenyl)amino]-N-(iso-propyl)pyrimidine-5-carboxamide
2o). Compound 2o was prepared from compound 4 and
iso-propylamine by a procedure that of described for
m, excluding diisopropylethylamine. Compound 2o
was obtained in 53% yield as a colorless solid: mp
(
5.1.20.
2-{[2-(4-Hydroxyphenyl)ethyl]amino}-4-(meth-
ylsulfanyl)pyrimidine-5-carboxylic acid (8). One molar
of NaOH (90 mL) was added to a solution of 7
(12.0 g, 36.0 mmol) in MeOH (180 mL) and the mixture
was refluxed for an hour. One molar of HCl (90 mL)
was added and the resulting solid was collected by filtra-
2
1
1
96–197 ꢁC (AcOEt –hexane); H NMR (DMSO-d6,
0 ꢁC) d 1.16 (6H, d, J = 6.8 Hz), 2.26 (3H, s), 2.75
8
(
2H, t, J = 7.2 Hz), 3.44–3.51 (2H, m), 4.07–4.14 (2H,
tion and washed successively with H O and MeCN–
2
m), 6.67 (2H, d, J = 8.4 Hz), 6.84 (1H, d, J = 7.2 Hz),
.99 (2H, d, J = 8.4 Hz), 7.14–7.18 (2H, m), 7.52 (2H,
br), 8.16 (1H, br s), 8.50 (1H, s), 8.91 (1H, s), 11.33
Et O (1:1) to give 8 (10.7 g, 97%), which was used in
2
1
6
the next reaction without further purification.
H
NMR (DMSO-d ) d 2.35 (1H, s, SCH ), 2.41 (2H, s,
6
3
+
(
C H N O : C, 68.13; H, 6.71; N, 17.27. Found: C,
1H, s); FAB MS m/e (M+H) 406. Anal. Calcd for
SCH ), 2.70–2.77 (2H, m), 3.48–3.54 (2H, m), 6.67
3
(2H, d, J = 8.0 Hz), 7.02 (2H, d, J = 8.0 Hz), 7.90
(0.33 H, br t, J = 5.8 Hz, NH), 8.02 (0.67 H, br t,
J = 5.8 Hz, NH), 8.50 (0.67 H, s, CH at C-6 of pyrimi-
dine), 8.59 (0.33 H, s, CH at C-6 of pyrimidine), 9.18
2
3
27
5
2
6
8.25; H, 6.94; N, 17.37.
5
.1.17. 2-{[2-(4-hydroxyphenyl)ethyl]amino}-N,N-dimeth-
+
yl-4-[(3-methylphenyl)amino]pyrimidine-5-carboxamide
2p). Compound 2p was prepared from compound 4 and
0% aqueous dimethylamine by a procedure that of de-
(1H, br), 12.58 (1H, br). FAB MS m/e (M+H) 306.
(
5
5.1.21.
ylsulfanyl)pyrimidine-5-carboxamide (9).
2-{[2-(4-Hydroxyphenyl)ethyl]amino}-4-(meth-
EDCÆHCl
scribed for 2m, excluding diisopropylethylamine. Com-
pound 2p was obtained in 42% yield as a colorless
(8.06 g, 42.0 mmol) and HOBt (5.68 g, 42.0 mmol) were
added to a suspension of 8 (10.7 g, 35.0 mmol) in NMP
(100 mL). After stirring for 2 h at room temperature,
1
solid: mp 172–173 ꢁC (MeOH–THF);
H
NMR
(
DMSO-d , 80 ꢁC) d 2.25 (3H, s), 2.73 (2H, t,
J = 7.2 Hz), 3.01 (6H, s), 3.44–3.50 (2H, m), 6.66 (2H,
d, J = 8.4 Hz), 6.83 (1H, d, J = 7.2 Hz), 6.98 (2H, d,
J = 8.4 Hz), 7.04 (1H, br), 7.15 (1H, t, J = 8.0 Hz),
6
28% NH OH (10.3 mL) was added and the mixture
4
was stirred for 1 h at room temperature. The reaction
mixture was then diluted with H O, and the resulting
2
7
s), 9.25 (1H, br); FAB MS m/e (M+H) 392. Anal.
.45–7.49 (2H, m), 8.05 (1H, s), 8.50 (1H, s), 8.90 (1H,
solid was collected by filtration and washed successively
with H O and MeCN–Et O (1:1) to give 9 (10.5 g, 98%)
as a colorless powder, which was used in the next reac-
+
2
2
Calcd for C H N O : C, 67.50; H, 6.44; N, 17.89.
2
2
2
25
5
1
Found: C, 67.52; H, 6.51; N, 17.83.
tion without further purification. H NMR (DMSO-d )
6

1052
S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
d 2.3–2.37 (3 H, br), 2.71–2.76 (2H, m), 3.47 (2H, br),
.67 (2H, d, J = 8.4 Hz), 7.01 (2H, d, J = 8.4 Hz), 7.06
1H, br), 7.61 (1H, br), 7.73 (1H, br), 8.40–8.45 (1H,
(DMSO-d , 80 ꢁC) d 2.74 (2H, t, J = 8.4 Hz), 3.45–3.51
6
6
(
(2H, m), 6.68 (2H, d, J = 8.4 Hz), 6.99–7.04 (3H, m),
7.11–7.34 (5H, m), 7.67 (2H, d, J = 8.4 Hz), 8.55 (1H+,
s), 8.91 (1H, s), 11.47 (1H, br); FAB MS m/e (M+H)
+
br), 9.17 (1H, s). FAB MS m/e (M+H) 305.
3
5.54; N, 19.84. Found: C, 64.82; H, 5.53; N, 19.75.
50. Anal. Calcd for C H N O Æ0.2H O: C, 64.65; H,
19 19 5 2 2
5
.1.22.
2-{[2-(4-Hydroxyphenyl)ethyl]amino}-4-(meth-
(10). m-CPBA
7.09 g) was added portionwise to a solution of 9
ylsulfinyl)pyrimidine-5-carboxamide
(
(
5.1.26. Ethyl 4-(benzylamino)-2-(methylsulfanyl)pyrimi-
dine-5-carboxylate
10.0 g, 32.9 mmol) in NMP (100 mL) at below 5 ꢁC
(12).
Benzylamine
(3.22 g,
and the mixture was stirred for 1 h. The reaction mixture
was then diluted with H O, and the resulting solid was
30.1 mmol) was added to a solution of ethyl 4-chloro-
2-(methylsulfanyl)pyrimidine-5-carboxylate (11, 7.00 g,
30.1 mmol) and diisopropylethylamine (5.24 mL,
30.1 mmol) in MeCN (70 mL) at 0 ꢁC and the mixture
was stirred for 5 h at room temperature. The reaction
mixture was then diluted with AcOEt and washed suc-
2
collected by filtration and washed successively with
H O, Et O, and MeCN–Et O (1:1) to give 10 (9.68 g,
92%) as a pale-yellow powder, which was used in the
next reaction without further purification. H NMR
2
2
2
1
(
(
(
(
DMSO-d ) d 2.75 (3H, s), 2.78 (2H, br), 3.49–3.58
cessively with H O and saturated aqueous NaCl. The
2
organic layer was dried and concentrated in vacuo to
give 12 (8.75 g, 96%) as a pale-yellow solid, which was
6
2H, m), 6.65–6.69 (2H, m), 7.03–7.09 (2H, m), 7.43
1H, br), 8.00 (1H, br), 8.23 (0.4H, br, NH), 8.45
0.6H, br, NH), 8.70 (0.4 H, s, CH at C-6 of pyrimidine),
used in the next reaction without further purification.
H NMR (CDCl ) d 1.36 (3H, t, J = 7.1 Hz), 2.48 (3H,
s), 4.31 (2H, q, J = 7.1 Hz), 4.76 (2H, d, J = 5.9 Hz),
1
8.78 (0.6H, s, CH at C-6 of pyrimidine), 9.15 (0.4H, s,
OH), 9.18 (0.6H, s, OH). FAB MS m/e (M+H) 321.
3
+
7
.24–7.34 (5H, m), 8.60 (1H, br), 8.65 (1H, s); FAB
MS m/e (M+H) 304.
+
5.1.23. 4-(Cyclohexylamino)-2-{[2-(4-hydroxyphenyl)eth-
yl]amino}pyrimidine-5-carboxamide (2q). Cyclohexyl-
amine (372 mg, 3.75 mmol) was added to a solution of
5.1.27. 4-(Benzylamino)-2-(methylsulfanyl)pyrimidine-5-
carboxylic acid (13). One molar of NaOH (32 mL) was
added to a solution of 12 (8.06 g, 27.3 mmol) in THF
(80 mL) and MeOH (10 mL) and the mixture was stirred
for 5 h at 50 ꢁC. One molar of HCl (32 mL) was added
and the resulting solid was collected by filtration to give
1
(
0 (800 mg, 2.50 mmol) and diisopropylethylamine
0.87 mL, 5.0 mmol) in NMP (8 mL) and the mixture
was stirred for 1 h at 100 ꢁC. The reaction mixture was
then diluted with AcOEt and washed successively with
H O and saturated aqueous NaCl. The organic layer
2
was dried and concentrated in vacuo, and the residue
was chromatographed on silica gel with elution using
CHCl –MeOH (39:1) to give a colorless solid, which
13 (7.35 g, quant.) as a colorless powder, which was used
in the next reaction without further purification.
1
H
NMR (DMSO-d ) d 2.40 (3H, s), 4.71 (2H, d,
3
6
was recrystallized from MeOH–AcOEt to give 2q
(
J = 5.9 Hz), 7.22–7.34 (5H, m), 8.54 (1H, s), 8.88 (1H,
br t, J = 5.9 Hz), 13.26 (1H, br s); FAB MS m/e
1
547 mg, 62%) as a colorless solid. Mp 185–186 ꢁC; H
+
(M+H) 276.
NMR (DMSO-d , 80 ꢁC) d 1.23–1.40 (6H, m), 1.55
6
(
(
1H, br), 1.66–1.70 (2H, m), 1.89–1.91 (2H, m), 2.71
2H, t, J = 7.2 Hz), 3.39–3.40 (2H, m), 3.96 (1H, br),
5.1.28. 4-(Benzylamino)-2-(methylsulfanyl)pyrimidine-5-
carboxamide (14). EDCÆHCl (4.18 g, 21.8 mmol) and
HOBt (2.94 g, 21.8 mmol) were added to a solution
of 13 (5.00 g, 18.2 mmol) in DMF (50 mL). After stir-
ring for 30 min at room temperature, 28% aqueous
ammonia (5.4 mL) was added and the mixture was
stirred for 16 h at room temperature. The reaction
mixture was diluted with AcOEt and washed succes-
sively with water and saturated aqueous NaCl. The
organic layer was dried and concentrated in vacuo
to give 14 (4.89 g, 98%) as a colorless powder, which
6
.67 (2H, d, J = 8.4 Hz), 6.80 (1H, br), 6.99–7.01
3H,m), 8.34 (1H, s), 8.89 (1H, s), 8.96 (1H, br); FAB
(
MS m/e (M+H) 356. Anal. Calcd for C H N O : C,
+
1
9
25
5
2
6
1
4.20; H, 7.09; N, 19.70. Found: C, 64.17; H, 7.42; N,
9.60.
5
.1.24. 4-(Benzylamino)-2-{[2-(4-hydroxyphenyl)ethyl]-
amino}pyrimidine-5-carboxamide (2r). Compound 2r
was prepared from compound 10 and benzylamine in
4
8% yield as a colorless solid, using a similar approach
to that described for 2r: mp 208–209 ꢁC (MeOH–
was used in the next reaction without further purifica-
1
1
THF); H NMR (DMSO-d , 80 ꢁC) d 2.67 (2H, t,
tion. H NMR (DMSO-d ) d 2.40 (3H, s), 4.67 (2H,
6
6
J = 7.6 Hz), 3.38–3.41 (2H, m), 4.63 (2H, d,
J = 5.6 Hz), 6.64 (2H, d, J = 8.4 Hz), 6.84 (1H, br),
d, J = 5.9 Hz), 7.21–7.33 (5H, m), 7.46 (1H, br s),
8.04 (1H, br s), 8.53 (1H, s), 9.48 (1H, br t,
+
J = 5.9 Hz); FAB MS m/e (M+H) 275.
6
m), 7.28–7.31 (5H, m), 8.39 (1H, s), 8.80 (1H, s), 9.31
.93 (2H, d, J = 8.4 Hz), 7.08 (1H, br), 7.22–7.24 (1H,
+
1H, br); FAB MS m/e (M+H) 364. Anal. Calcd for
(
C H N O : C, 66.10; H, 5.82; N, 19.27. Found: C,
5.1.29. 4-(Benzylamino)-N-methyl-2-(methylsulfanyl)py-
rimidine-5-carboxamide (15). Compound 15 was pre-
pared from compound 13 and methylamine
hydrochloride with equimolar of diisopropylamine in
95% yield as a colorless solid, using a similar approach
to that described for 14. Compound 15 was used for
2
0
21
5
2
6
6.13; H, 5.78; N, 19.32.
5
.1.25. 4-Anilino-2-{[2-(4-hydroxyphenyl)ethyl]amino}-
pyrimidine-5-carboxamide (2s). Compound 2s was pre-
pared from compound 10 and aniline in 30% yield as a
pale-yellow solid, using a similar approach to that de-
1
next reaction without further purification. H NMR
(DMSO-d ) d 2.40 (3H, s), 2.74 (3H, d, J = 4.5 Hz),
4.65 (2H, d, J = 5.9 Hz), 7.24–7.36 (5H, m), 8.45 (1H,
6
1
scribed for 2r: mp 248–249 ꢁC (MeOH); H NMR

S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
1053
s), 8.52–8.53 (1H, m), 9.34 (1H, br t, J = 5.9 Hz); FAB
MS m/e (M+H) 289.
C H ClN O Æ0.5H O: C, 59.93; H, 5.51; Cl, 8.42; N,
21 22 5 2 2
16.64. Found: C, 59.64; H, 5.42; Cl, 8.15; N, 16.28.
+
5
.1.30. 4-(Benzylamino)-2-(methylsulfonyl)pyrimidine-5-
5.1.34. Ethyl 2-{3-[4-(benzyloxy)phenyl]propyl}-4-oxo-
1,4-dihydropyrimidine-5-carboxylate (19). Hydrogen
chloride was bubbled through a solution of 18
(1.50 g, 5.97 mmol) in EtOH (30 mL) and CHCl3
(15 mL) for 30 min at below 5 ꢁC. After stirring for
2 h below 5 ꢁC, the reaction mixture was concentrated
carboxamide (16). m-CPBA (1.62 g) was added portion-
wise to a solution of 14 (1.00 g, 3.75 mmol) in NMP
(
3
10 mL) below 5 ꢁC and the mixture was stirred for
h. The reaction mixture was then diluted with CHCl3
and washed successively with saturated aqueous NaH-
CO , 10% aqueous Na SO , H O, and saturated aque-
ous NaCl. The organic layer was dried and
in vacuo. The residue was combined with Et O
3
2
3
2
2
(50 mL) and concentrated in vacuo, and then com-
bined with EtOH (30 mL) and ammonia acetate
(920 mg, 11.9 mmol) before being refluxed for 2 h.
The mixture was again concentrated in vacuo, and a
suspension of the resulting residue in EtOH (50 mL)
was combined with sodium methoxide (1.61 mg,
29.9 mmol) and diethyl ethoxymethylenemalonate
(2.58 g, 11.9 mmol) and stirred for 3 h at room tem-
perature. The reaction mixture was concentrated in
vacuo, and the residue was acidified with 1 M HCl
and extracted with AcOEt. The organic layer was
washed with water and saturated aqueous NaCl,
dried, and concentrated in vacuo. The resulting resi-
due was chromatographed on silica gel with elution
concentrated in vacuo. H O was added to the residue,
2
and the resulting solid was collected by filtration to give
16 (1.02 g, 91%) as a colorless powder, which was used
in the next reaction without further purification.
1
H
NMR (DMSO-d ) d 3.25 (3H, s), 4.70 (2H, d,
6
J = 5.9 Hz), 7.24–7.39 (5H, m), 7.84 (1H, br s), 8.34
(1H, br s), 8.79 (1H, s), 9.67 (1H, br t, J = 5.9 Hz);
FAB MS m/e (M+H) 307.
+
5
.1.31. 4-(Benzylamino)-N-methyl-2-(methylsulfonyl)py-
rimidine-5-carboxamide (17). Compound 17 was pre-
pared from compound 15 in 85% yield as a colorless
powder, using a similar approach to that described
for 16. Compound 17 was used for next reaction
using CHCl –MeOH (40:1) to give 19 (820 mg, 35%)
3
1
without further purification. H NMR (DMSO-d ) d
as an amorphous solid, which was used in the next
step without further purification: H NMR (CDCl3)
6
1
2.77 (3H, d, J = 4.5 Hz), 3.23 (3H, s), 4.68 (2H, d,
J = 5.9 Hz), 7.25–7.38 (5H, m), 8.67 (1H, s), 8.77–8.78
d 1.37 (3H, t, J = 7.1 Hz), 2.07–2.17 (2H, m), 2.66
(2H, t, J = 7.7 Hz), 2.79 (2H, t, J = 7.7 Hz), 4.36
(2H, q, J = 7.1 Hz), 5.02 (2H, s), 6.88 (2H, d,
J = 8.6 Hz), 7.10 (2H, d, J = 8.6 Hz), 7.32–7.44 (5H,
m), 8.72 (1H, s), 12.70 (1H, br s); FAB MS m/e
(
(
1H, m), 9.43 (1H, br t, J = 5.9 Hz); FAB MS m/e
+
M+H) 321.
5.1.32. 4-(Benzylamino)-2-{[2-(3-chloro-4-hydroxyphe-
nyl)ethyl]amino}pyrimidine-5-carboxamide (2t). 2-(3-Chlo-
ro-4-hydroxyphenyl)ethylamine hydrochloride (765 mg,
+
(M+H) 393.
3
2
.68 mmol) was added to a solution of 16 (750 mg,
.45 mmol) and diisopropylamine (1.10 mL, 6.13 mmol)
5.1.35. Ethyl 2-{3-[4-(benzyloxy)phenyl]propyl}-4-chloro-
pyrimidine-5-carboxylate (20). Diethylaniline (0.55 mL,
3.46 mmol) was added to a mixture of 19 (755 mg,
1.92 mmol) and phosphorus trichloride (2 mL) and the
mixture was stirred for 1 h at 90 ꢁC. The reaction mix-
ture was then poured into ice water and the resulting sol-
id was collected by filtration and washed with AcOEt to
give 20 (767 mg, 97%) as a pale brown solid, which was
in NMP (7.5 mL) and the mixture was stirred for 1 h at
100 ꢁC. The reaction mixture was then diluted with
AcOEt and THF, and washed successively with H O
and saturated aqueous NaCl. The organic layer was dried
and concentrated in vacuo, and the residue was
chromatographed on silica gel with elution using
CHCl –MeOH–NH OH (28%) (300:10:1) to give a
2
1
used in the next step without further purification: H
3
4
colorless solid, which was recrystallized from MeOH–
THF to give 2t (280 mg, 29%) as a colorless solid. Mp
NMR (CDCl ) d 1.42 (3H, t, J = 7.3 Hz), 2.10–2.17
3
(2H, m), 2.66 (2H, t, J = 7.8 Hz), 3.00 (2H, t,
J = 7.8 Hz), 4.43 (2H, q, J = 7.3 Hz), 5.04 (2H, s), 6.89
(2H, d, J = 8.3 Hz), 6.94 (2H, d, J = 8.3 Hz), 7.30–7.44
(5H, m), 9.04 (1H, s).
1
1
J = 7.2 Hz), 3.40–3.42 (2H, m), 4.63 (2H, d, J = 6.0 Hz),
51–153 ꢁC; H NMR (DMSO-d , 80 ꢁC) d 2.68 (2H, t,
6
6
7
.83–6.91 (3H, m), 7.10 (2H, br), 7.22–7.24 (1H, m),
.28–7.31 (5H, m), 8.39 (1H, s), 9.33 (1H, br s), 9.57
+
(1H, s); FAB MS m/e (M+H) 398. Anal. Calcd for
C H ClN O : C, 60.38; H, 5.07; Cl, 8.91; N, 17.60.
5.1.36. Ethyl 2-{3-[4-(Benzyloxy)phenyl]propyl}-4-[(3-
(21).
methylphenyl)amino]pyrimidine-5-carboxylate
2
0
20
5
2
Found: C, 60.25; H, 4.97; Cl, 9.04; N, 17.75.
m-Toluidine (206 mg, 1.92 mmol) was added to a solu-
tion of 20 (750 mg, 1.83 mmol) and diisopropylethyl-
amine (0.33 mL, 1.92 mmol) in MeCN (70 mL) and
the mixture was stirred for 2 h at 60 ꢁC. The reaction
mixture was then diluted with AcOEt and washed suc-
5
.1.33. 4-(Benzylamino)-2-{[2-(3-chloro-4-hydroxyphe-
nyl)ethyl]amino}-N-methylpyrimidine-5-carboxamide (2u).
Compound 2u was prepared from compound 2u in 33%
yield as a pale brown amorphous solid, using a similar
cessively with H O and saturated aqueous NaCl. The
2
1
approach to that described for 2t: H NMR(DMSO-
d , 80 ꢁC) d 2.68–2.74 (5H, m), 3.40–3.45 (2H, m), 4.63
organic layer was dried and concentrated in vacuo,
and the residue was chromatographed on silica gel with
elution using hexane–AcOEt (5:1) to give 21 (616 mg,
6
(
2H, d, J = 5.2 Hz), 6.84–6.91 (3H, m), 6.99 (1H, br),
1
7
7
.10 (1H, s), 7.21–7.25 (1H, m), 7.28–7.31 (4H, m),
.99 (1H, br), 8.33 (1H, s), 9.30 (1H, br s), 9.59 (1H,
70%) as an amorphous solid: H NMR (CDCl ) d 1.42
3
(3H, t, J = 7.7 Hz), 1.58 (3H, s), 2.10–2.20 (2H, m),
2.65 (2H, t, J = 7.1 Hz), 2.88 (2H, t, J = 7.5 Hz), 4.40
+
s); FAB MS m/e (M+H) 412. Anal. Calcd for

1054
S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
(
2H, q, J = 7.1 Hz), 5.03 (2H, s), 6.88 (2H, d,
5.2. Biology
J = 8.8 Hz), 6.93–6.95 (1H, m), 7.11 (2H, d,
J = 8.6 Hz), 7.22 (1H, d, J = 7.9 Hz ), 7.31–7.44 (5H,
m), 7.55–7.60 (3H, m), 8.91 (1H, s), 10.25 (1H, s);
5.2.1. Plasmid construction. An IL-4-inducible luciferase
reporter plasmid carrying copies of the CCAAT-enhanc-
er binding protein (C/EBP) binding site and the STAT6
binding site was constructed as previously descri-
+
FAB MS m/e (M+H) 482.
1
1
5
.1.37. 2-{3-[4-(Benzyloxy)phenyl]propyl}-4-[(3-methyl-
bed. The oligonucleotide containing the C/EBP and
STAT6 binding sites used in the construction of the
phenyl)amino]pyrimidine-5-carboxylic acid (22). One mo-
lar of NaOH (2.7 mL) was added to a solution of 21
0
plasmid had the sequence 5 -TCGAGCGCTGTTGCT
CAATCGACTTCCCAAGAACAGAGCTGTTGCTC
(
640 mg, 1.33 mmol) in EtOH (5 mL) and THF (5 mL),
0
and the mixture was stirred for 16 h at room tempera-
ture. One molar of HCl (2.7 mL) was added and the
resulting solid was collected by filtration and washed suc-
AATCGACTTCCCAAGAACAGAA-3 . The synthe-
sized oligonucleotides were annealed and then ligated
1
2
into the XhoI–BglII site of the pGL2-TATA plasmid.
cessively with MeOH–H O (1:1) to give 22 (595 mg, 99%)
2
as a colorless powder, which was used in the next reac-
tion without further purification: H NMR (DMSO-d )
d 1.99–2.09 (2H, m), 2.27 (3H, s), 2.58 (2H, t,
J = 7.2 Hz), 2.77 (2H, t, J = 7.5 Hz), 5.06 (2H, s), 6.89–
5
.2.2. Cells and cell culture. FW4 is a BAF-B03-derived
1
6
cell line expressing human IL-2Rb, IL-2Rc, and IL-
4
pGL2-CS with a blasticidin resistance gene by electro-
poration, as previously described. The cells were cul-
tured in RPMI1640 medium (Gibco) supplemented
with 10% fetal bovine serum (FBS) and 10% (v/v)
13
R. FW4 cells were obtained by co-transfection of
6
m), 7.32–7.45 (5H, m), 7.55–7.57 (2H, m), 8.81 (1H, s),
.95 (3H, m), 7.10 (2H, d, J = 8.6 Hz), 7.21–7.27 (1H,
12
+
0.60 (1H, br s); FAB MS m/e (M+H) 454.
1
1
4
WEHI-3B conditioned medium as a source of IL-3.
5
.1.38. 2-{3-[4-(Benzyloxy)phenyl]propyl}-4-[(3-methyl-
phenyl)amino]pyrimidine-5-carboxamide (23). EDCÆHCl
292 mg, 1.52 mmol) and HOBt (206 mg, 1.52 mmol)
were added to a solution of 22 (578 mg, 1.27 mmol)
4
(
5.2.3. Luciferase assay. FW4 reporter cells (1 · 10 cells/
0.1 mL RPMI1640 supplemented with 10% (v/v) FBS
and 10% (v/v) WEHI-3B conditioned medium) cultured
in 96-well plates were stimulated with human IL-4 (1 ng/
mL) (Genzyme) for 16 h. The test compounds were add-
ed to each well 30 min before stimulation. The final
DMSO concentrations were 0.1%. The reaction was
stopped by addition of 50 lL of solubilization buffer
in DMF (15 mL). After stirring for 30 min at room
temperature, 28% NH OH (0.74 mL) was added and
4
the mixture was stirred for 16 h at room temperature.
The reaction mixture was then diluted with AcOEt
and washed successively with H O and saturated
2
aqueous NaCl. The organic layer was dried and con-
centrated in vacuo, and the residue was chromato-
graphed on silica gel with elution using CHCl3–
(10 mM Tris–HCl, pH 7.8, 0.5 mM MgCl , 10 mM
dithiothreitol, and 0.1% (v/v) Triton X-100). Luciferase
activities were measured with a ML3000 luminometer
Dynatech Laboratories, Inc.) after addition of 50 lL
of substrate solution (5 mM luciferin, 2 mM coenzyme
A, 2 mM ATP, 0.5 mM MgCl , and 2 mM Mg(OH)
2
MeOH (60:1) to give 23 (452 mg, 79%) as an ivory-
1
(
colored powder: H NMR (DMSO-d ) d 2.04 (2H,
6
quint., J = 7.5 Hz), 2.27 (3H, s), 2.58 (2H, t,
J = 7.5 Hz), 2.75 (2H, t, J = 7.5 Hz), 5.06 (2H, s),
2
2
in 10 mM Tris–HCl, pH 7.8). Relative activities were
calculated as follows: Relative activity (%) = 100 · [rela-
tive light units (RLU) of sample upon stimula-
tion ꢀ RLU for unstimulated sample]/(RLU upon
stimulation ꢀ RLU unstimulated).
6
7
7
.88–6.92 (3H, m), 7.12 (2H, d, J = 8.4 Hz), 7.19–
.24 (1H, m), 7.31–7.45 (5H, m), 7.53–7.55 (2H, m),
.76 (1H, br s), 8.31 (1H, br s), 8.81 (1H, s), 11.30
+
(
1H, s); FAB MS m/e (M+H) 453.
5
.1.39. 2-[3-(4-hydroxyphenyl)propyl]-4-[(3-methylphe-
nyl)amino]pyrimidine-5-carboxamide (24). Palladium
10%) on carbon (100 mg) was added to a solution of
3 (417 mg, 0.92 mmol) in MeOH (4 mL) and THF
6 mL), and the mixture was stirred for 14 h at room
5.2.4. In vitro T cell differentiation. C57BL/6 mice were
purchased from Charles River Laboratories. Spleen T
cells were purified from total spleen cells using a nylon
wool column. For in vitro differentiation assays,
(
2
(
6
temperature under a hydrogen atmosphere. The mixture
was filtered with Celite and the filtrate was concentrated
in vacuo. The residue was chromatographed on silica gel
with elution using CHCl –MeOH (20:1) to give a crude
3
solid, which was recrystallized from MeOH–THF to
give 24 (253 mg, 76%) as a pale-yellow solid: mp 240–
1 · 10 T cells/mL were cultured in RPMI1640 medium
(Gibco) supplemented with 10% fetal bovine serum
ꢀ5
(FBS) and 5 · 10 M 2-mercaptoethanol, stimulated
with 10 lg/mL plate-bound anti-CD3 antibody (Cedar-
lane), and incubated with 10 ng/mL of mouse IL-2 (Pep-
roTech) plus 10 ng/mL of mouse IL-12 (PeproTech)
(Th1) or 10 ng/mL of mouse IL-4 (PeproTech) plus
1 lg/mL of anti-CD28 (Pharmingen) (Th2) for 2 days.
The cells were then cultured in the medium with the
same concentrations of IL-2, IL-12, and IL-4 for anoth-
er 3 days. The test compound was incubated through 5
days. The concentrations of DMSO derived from stock
solution were 0.1%. The cultured cells were washed and
re-stimulated with plate-bound anti-CD3 antibody in
the absence of the test compound, and supernatants
1
2
2
41 ꢁC; H NMR (DMSO-d ) d 1.98–2.06 (2H, m),
6
.28 (3H, s), 2.53 (2H, t, J = 7.3 Hz), 2.74 (2H, t,
J = 7.8 Hz), 6.66 (2H, d, J = 8.6 Hz), 6.89 (1H, d,
J = 7.3 Hz), 6.94 (2H, d, J = 8.3 Hz), 7.22 (1H, t,
J = 7.8 Hz), 7.54–7.56 (2H, m), 7.76 (1H, br s), 8.32
(
1H, br s), 8.81 (1H, s), 9.12 (1H, s), 11.31 (1H, s);
+
FAB MS m/e (M+H)
C H N O : C, 69.59; H, 6.12; N, 15.46. Found: C,
363. Anal. Calcd for
2
1
22
4
2
6
9.51; H, 6.16; N, 15.41.

S. Nagashima et al. / Bioorg. Med. Chem. 15 (2007) 1044–1055
1055
were collected 16 h later. Cytokine ELISAs were per-
formed using antibodies recommended by Pharmingen.
Medina, J. C. Abstracts of Papers, 223rd ACS National
Meeting of the American Chemical Society, Orlando, FL,
2
002, pp 7–11; Washington, DC, 2002; MEDI 080.
7
. (a) Hisamichi, H.; Naito, R.; Toyoshima, A.; Kawano, N.;
Ichikawa, A.; Orita, A.; Orita, M.; Hamada, N.; Takeuchi,
M.; Ohta, M.; Tsukamoto, S. Bioorg. Med. Chem. 2005,
Acknowledgments
1
3, 4936–4951; (b) Hisamichi, H.; Naito, R.; Toyoshima,
The authors are grateful to the staff of the Division of
Analytical Science Laboratories for elemental analysis
and spectral measurements. We thank Dr. Susumu
Igarashi and Dr. Hirokazu Kubota for valuable
comments and help in the preparation of the manuscript.
A.; Kawano, N.; Ichikawa, A.; Orita, A.; Orita, M.;
Hamada, N.; Takeuchi, M.; Ohta, M.; Tsukamoto, S.
Bioorg. Med. Chem. 2005, 13, 6277–6279.
. Vinogradova, S. V.; Vasnev, V. A.; Korshak, V. V.;
Petrovski, P. V.; Mitaishvili, T. I. Russ. Chem. Bull. 1970,
8
9
1
9, 2331–2335.
. Compound 2a showed inhibitory activity of IL-4 produc-
tion with an IC50 value of 14 nM, but no inhibitory
activity of IFN-c production.
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