Synthetic studies on novel Syk inhibitors. Part 1: Synthesis and structure-activity relationships of pyrimidine-5-carboxamide derivatives

Article abstract of DOI:10.1016/j.bmc.2005.05.033

Spleen tyrosine kinase (Syk) is a non-receptor-type tyrosine kinase which mediates diverse responses in haematopoietic cells. Therefore, Syk is an attractive therapeutic target, and in a study of Syk inhibitors as potentially new therapeutic agents, we di

Full text of DOI:10.1016/j.bmc.2005.05.033

Bioorganic & Medicinal Chemistry 13 (2005) 4936–4951
Synthetic studies on novel Syk inhibitors. Part 1:
Synthesis and structure–activity relationships
of pyrimidine-5-carboxamide derivatives
Hiroyuki Hisamichi,a,* Ryo Naito,a Akira Toyoshima,b Noriyuki Kawano,a
Atsushi Ichikawa,a Akiko Orita,a Masaya Orita,a Noritaka Hamada,a
Makoto Takeuchi,a Mitsuaki Ohtaa and Shin-ichi Tsukamotoa
aInstitute for Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
bDepartment of Clinical Development, Astellas Pharma Inc., 3-17-1 Hasune, Itabashi, Tokyo 174-8612, Japan
Received 3 March 2005; revised 17 May 2005; accepted 17 May 2005
Abstract—Spleen tyrosine kinase (Syk) is a non-receptor-type tyrosine kinase which mediates diverse responses in haematopoietic
cells. Therefore, Syk is an attractive therapeutic target, and in a study of Syk inhibitors as potentially new therapeutic agents, we
discovered the 4-anilinopyrimidine-5-carboxamides. Enzyme screening indicated that an aminoethylamino moiety at the 2-position
of the pyrimidine ring was important for Syk inhibitory activity, and an investigation of the substituents at the 4-position revealed
that an anilino moiety substituted at the meta position was preferred. These compounds showed high selectivity for Syk, compared
to other kinases, such as ZAP-70, c-Src, and PKC, and exhibited good inhibitory activities against 5-HT release from RBL-cells.
Among them, compound 9a inhibited the passive cutaneous anaphylaxis reaction in mice, with an ID50 of 13 mg/kg following sub-
cutaneous administration. These results suggest that our compounds are worthy of further evaluation as new anti-allergic agents.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
derivatives have been carried out.6,7 Recently, many
types of PTK inhibitors have been reported, and work
in this area has prospered. However, the main focus of
this work has been inhibitors of receptor-type PTKs,
and only a few studies have reported on non-receptor-
type PTK inhibitors.
Protein tyrosine kinases (PTKs) are well known to be
important in cellular signal transduction, and regulate
cellular activation, proliferation, differentiation, and
mitogenesis.1 The PTKs can be classified into two
types:2 receptor-type PTKs, such as platelet-derived
growth factor receptor (PDGFR), epidermal growth
factor receptor (EGFR), and fibroblast growth factor
(FGFR), all of which contain an extracellular ligand-
binding domain; and non-receptor-type PTKs, such as
c-Src, Lck, ZAP-70, and Syk, which have an intracellu-
lar location. Since abnormal activation of PTKs has
been related to a number of diseases, PTK inhibitors
are expected to have potential therapeutic utility.
Among the non-receptor PTKs, spleen tyrosine kinase
(Syk) is a 72 kDa cytoplasmic PTK that is expressed
in B cells, mast cells, macrophages, and platelets.8 This
kinase is recruited to receptors, such as the B cell antigen
receptor or FceRI and plays an important role in signal
transduction pathways in these cells.9–11 Therefore, inhi-
bition of Syk may result in the suppression of functions
of these cells and be effective in the treatment of allergic
or immunological disorders. Indeed, several series of a
Syk inhibitor have been reported and it has been shown
that antigen-induced responses are prevented by the
inhibition of phosphorylation and activation of Syk.12
Several natural product PTK inhibitors, including
Erbstatin,3 Lavendustin A,4 and Piceatannol,5 have
been reported previously, and many studies of their
To discover a novel Syk inhibitor, we carried out high-
throughput screening using our chemical library and
discovered the 4-anilino-2-(2-aminoethylamino)pyrimi-
dine-5-carboxamide derivative 9a as a member of a
Keywords: Tyrosine kinase; Syk; ZAP-70; Pyrimidine.
*
Corresponding author. Tel.: +81 0 29 863 6741; fax: +81 0 29 852
2971; e-mail: hiroyuki.hisamichi@jp.astellas.com
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.033

H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
4937
novel class of Syk inhibitors. In this paper, we report
the synthesis and structure–activity relationships of
pyrimidine-5-carboxamide derivatives as selective Syk
inhibitors, as well as the pharmacological profile of com-
pound 9a.
dine derivatives 9. In the case of reaction with diamines,
such as ethylenediamine, about 10 equiv of diamines
were used to ensure monosubstitution.
Commercially available ethyl-4-chloro-2-methylsulfa-
nylpyrimidine-5-carboxylate 10 was also used as a start-
ing material, especially for the preparation of
4-alkylamino derivatives, because the reaction of alkyl-
amines, such as methylamine with 5 gave 2,4-dialkyl-
amino derivatives as the major product (Scheme 3).
The 4-alkylaminopyrimidine-5-carboxamides were ob-
tained from 10 in a manner similar to that shown in
Scheme 2, whereas the substitution of 2-methylsulfanyl
groups by amino groups required a higher temperature,
in contrast to the displacement of 2-benzotriazol-1-oxy
groups.
2. Chemistry
As shown in Scheme 1, compound 4a, which has no sub-
stituent at the 2-position, was synthesized starting from
ethyl-4-chloropyrimidine-5-carboxylate 1a.13 The reac-
tion of 1a with 3-trifluoromethylaniline in toluene gave
4-anilinopyrimidine derivative 2a. Hydrolysis of the car-
boethoxy group by 1 M NaOH afforded carboxylic acid
3a, and condensation of the carboxylic acid with
aqueous ammonia in the presence of 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole gave the pyrimidine-5-car-
boxamide derivative 4a. The 2-methyl derivative 4b
and 2-phenyl derivative 4c were prepared from appro-
priate 4-chloropyrimidine derivatives (1b and 1c)14 by
substitution with 3-trifluoromethylaniline, followed by
hydrolysis and amidation.
The 5-ester derivative 15 was prepared by a substitution
reaction of 6a with ethylenediamine and subsequent
hydrolysis afforded the 5-carboxylic acid derivative 16
(see Scheme 4).
The chemical structures of the synthesized compounds
were confirmed from spectroscopic data (1H NMR,
mass spectrometry) and elemental analysis.
Most of the pyrimidine-5-carboxamide derivatives with
an amino group at the 2-position were prepared in five
steps from ethyl 2,4-dichloropyrimidine-5-carboxylate
5.15 The synthetic route is shown in Scheme 2. The reac-
tion of 5 with the appropriate anilines in MeCN or tetra-
hydrofuran (THF) gave the 4-anilinopyrimidine
derivatives 6. The substitution reaction proceeded selec-
tively at the 4-position, and 2-anilino or 2,4-dianilino
derivatives were not obtained. Hydrolysis of the carbo-
ethoxy group by 1 M NaOH provided the correspond-
ing carboxylic acids 7. Condensation with aqueous
ammonia in the presence of EDCI and 1-hydroxybenzo-
triazole (HOBt) gave the corresponding pyrimidine-5-
carboxamide derivatives 8. Interestingly, displacement
of the 2-chloro group to form a benzotriazol-1-yloxy
group took place under these conditions and 2-chloro
derivatives were obtained only in trace amounts. How-
ever, it was found that the benzotriazol-1-yloxy group
served as a leaving group in the next reaction; displace-
ment of this group by an amino group proceeded easily
at room temperature and afforded the 2-aminopyrimi-
3. Results and discussion
The compounds were evaluated for their ability to inhi-
bit tyrosine phosphorylation of Band-3 peptide16 by Syk
and ZAP-70. These data are shown in Tables 1 and 2.
Compounds showing potency in these assays were fur-
ther investigated for their enzyme selectivity for Syk,
compared to c-Src and several PKCs, and selected com-
pounds were also evaluated for their inhibition of sero-
tonin (5-HT) release in rat basophilic leukemia (RBL)
cells.17
The 2-ethylenediaminopyrimidine derivative 9a, which
was discovered by high-throughput screening, exhibited
good Syk inhibitory activity, with an IC50 value of
0.041 lM. In the first series of compounds, the effect
of the 2-ethylenediamino group on Syk inhibitory activ-
ity was investigated. Compound 4a, which has no substi-
tuent at the 2-position, did not show any Syk inhibitory
Scheme 1. Reagents: (a) ArNH2, iPr2NEt, MeCN; (b) 1 M NaOH, MeOH; (c) aq NH3, EDCI, HOBt, ClCH2CH2Cl or DMF; (d) R2R3NH, MeCN;
(e) 4 N HCl/AcOEt, EtOH.

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H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
Scheme 2. Reagents: (a) R4NH2, iPr2NEt, MeCN; (b) 1 M NaOH, MeOH; (c) R5R6NH, EDCI, HOBt, ClCH2CH2Cl or DMF; (d) R2NH2, MeCN.
activity. Similarly, a lack of potency was observed for
the 2-methyl and 2-phenyl derivatives, 4b and 4c. Com-
pound 14b, bearing a hydroxy group, was also less active
than 9a. For the N-methyl derivative 9d, the activity was
4-fold less potent compared with 9a, and the N,N-di-
methyl derivative 9e was less active than N-methyl
derivative. Furthermore, introduction of a tertiary ami-
no group at the 2-position strongly reduced the activity
(9f), and conversion of the ethylenediamino group into a
piperazinyl group (9g) also resulted in a large reduction
in potency. These data suggest that not only the terminal
primary amine but also the secondary amine at the 2-po-
sition of the pyrimidine ring is necessary for Syk inhib-
itory activity. The chain length of the diamino moiety at
the 2-position has a strong influence on the Syk inhibi-
tory activity; a marked difference was observed with a
2- or 4-carbon chain length (9a and 9b) compared to a
3-carbon chain length (14a).
derivative 9h, the 30-Me derivative 9j was about 10-fold
more potent than 9h. Based on the effectiveness of the
30-substituent, we then synthesized and evaluated a ser-
ies of compounds with meta substitution. Compounds
with small alkyl groups at the 30-position exhibited high
potency, with 30-alkyl derivatives being slightly more
potent than 30-alkoxy derivatives. Enlarging the substit-
uents at the 30-position decreased the activities of the 30-
alkyl derivatives. The 30-bromo derivative 9r was the
most potent molecule in this series. On the other hand,
14c and 14d, which have substituents other than an ani-
lino group at the 4-position of the pyrimidine ring,
showed no activity. These structure–activity relation-
ships associated with the anilino group are similar to
those reported for other tyrosine kinase inhibitors, such
as anilinoquinazoline derivatives18 and anilinopyrrolo-
pyrimidine derivatives,19 which are reported to be com-
petitors for ATP binding. These results suggest that our
pyrimidine–carboxamide derivatives may interact with
the ATP-binding site in a manner similar to other kinase
inhibitors.20
Subsequently, several 4-anilinopyrimidine-5-carboxam-
ide derivatives were evaluated to examine the effect of
varying the substituent in the anilino group. Although
the 20-Me and 40-Me derivatives (9i and 9k) showed
inhibitory activities similar to those of the unsubstituted
Finally, the effect of the 5-carboxamide group was
examined. The 5-carboxylate 15 and 5-carboxylic acid

H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
4939
Scheme 3. Reagents: (a) EtONa, EtOH; (b) POCl3; (c) 3-aminobenztrifluoride, iPr2NEt, MeCN; (d) 1 M NaOH, MeOH; (e) aq NH3, EDCI, HOBt,
ClCH2CH2Cl.
Scheme 4. Reagents: (a) ethylenediamine, MeCN; (b) 1 M NaOH, MeOH.
16 were both inactive. Furthermore, N-methyl carbox-
amide derivative 14e and N,N-dimethyl carboxamide
derivative 14f were more than 100 times less potent than
the primary carboxamide 9a. The requirement of an
interaction of the NH2 moiety of the primary carboxam-
ide with the receptor and steric limitations at this posi-
tion in derivatives 14e and 14f may explain these weak
activities. Because the 4-N-methyl-anilino and 4-phen-
ylsulfanyl derivatives showed poor inhibitory activities
(not shown in this paper), hydrogen bond formation
between the 5-carboxamide and the NH group at the
4-position may be important for activity. The overall
geometry of PTK catalytic domains may make inhibitor
planarity a key factor for improved interaction of some
classes of PTK inhibitors.21 The carboxamide deriva-
tives can form a pseudo-six-membered ring through for-
mation of a hydrogen bond, resulting in a planar
conformation, and X-ray crystallographic analysis of
compound 9q provided support for this proposal
(Fig. 1).
reported,22 and therefore we used the published X-ray
structure of Lck as a template, since the Lck catalytic
domain has high homology to the Syk kinase domain
(Fig. 2).1 Having built the 3D model of the Syk kinase
domain, we assumed that the binding mode of our com-
pounds to the domain was as shown in Figure 3. In this
model, the 4-amino group and 5-carboxamide group act
as a hydrogen bond donor and acceptor, respectively, to
enhance binding at the ATP binding site. Specifically,
the N–H of the 5-carboxamide group forms a hydrogen
bond to the carbonyl group of Glu 449 and the carbonyl
group of the inhibitor interacts with the N–H group of
Ala 451. Furthermore, a third strong interaction with
the enzyme is obtained by hydrogen bonding between
the N–H of the 4-anilino group and the carbonyl group
of Ala 451, while the 2-ethylenediamino group seems to
mimic the triphosphate group of ATP. These inter-
actions lead to a potent Syk inhibitory activity of our
compounds.
Some of the compounds, which showed Syk inhibitory
activities with IC50 values below 0.1 lM, were tested
for their selectivity for Syk, compared to other kinases.
Excellent selectivity was observed for all compounds,
To investigate the binding mode of our compounds, we
built a three-dimensional (3D) model of the catalytic
domain of Syk. The X-ray structure of Syk had not been

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H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
Table 1. Syk and ZAP-70 inhibitory activities of 4-(3-trifluoromethyl-
anilino)pyrimidine derivatives
No.
R2
IC50 (lM)a
ZAP-70
Syk
4a
4b
4c
H
Me
>5
>5
>5
>5
>5
Ph
>5
9a
9b
9d
9e
H2N(CH2)2NH
H2N(CH2)4NH
MeHN(CH2)2NH
Me2N(CH2)2NH
Me2N(CH2)2NMe
Piperazin-1-yl
H2N(CH2)3NH
HO(CH2)2NH
0.041
11.2
10.5
0.047
0.21
0.75
>10
>10
>10
>5
9f
>10
>5
0.23
0.35
9g
14a
14b
>10
>10
a IC50 values were determined in duplicate.
Table 2. Syk and ZAP-70 inhibitory activities of 2-(2-aminoethyl-
amino)pyrimidine derivatives
Figure 1. X-ray crystal structure of 9q.
No.
R1
R3
IC (lM)a
50
Syk
ZAP-70
9a
9h
9i
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONHMe
CONMe2
CO2Et
3-CF3–Ph
Ph
0.041
0.28
11.9
13.2
assay, which may be due to lack of penetration into
the cells.
2-Me–Ph
3-Me–Ph
4-Me–Ph
3-NC–Ph
3-Et–Ph
3-iPr–Ph
3-MeO–Ph
3-EtO–Ph
3-iPrO–Ph
3-Br–Ph
Me
0.23
0.03
>30
9j
5.2
6.5
8.4
Finally, to investigate the anti-allergic activity of this
series of compounds in vivo, compound 9a was evalu-
ated for its ability to inhibit the passive cutaneous ana-
phylaxis (PCA) reaction in mice. Compound 9a was
subcutaneously administered to mice 30 min before anti-
gen challenge, and the effect was evaluated by measuring
the amount of pigment in the blueing region. Com-
pound 9a inhibited the anaphylaxis reaction dose-depen-
dently with an ID50 value of 13.2 mg/kg, as shown in
Figure 4.
9k
9l
0.15
0.11
9m
9n
9o
9p
9q
9r
0.053
0.092
0.066
0.081
0.072
0.023
>10
>10
11.2
>10
>10
4.9
14c
14d
14e
14f
15
16
>10
>10
>10
>10
>10
Cyclohexyl
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
3-CF3–Ph
>10
>10
>10
>5.0
>5.0
>5.0
>5.0
4. Conclusion
CO2H
In conclusion, we have discovered a series of anilinopyr-
imidine derivatives as Syk inhibitors and investigated
the structure–activity relationships of these derivatives.
An N–H group at the 2-position of the pyrimidine ring
is necessary for Syk inhibitory activity, with ethylenedi-
amino groups being the most favorable substituent.
With regard to substituents at the 4-position, anilino
groups were effective and formation of a hydrogen bond
as shown in Table 3. These compounds also inhibited
5-HT release with IC50 values of 0.2–2.0 lM. Among
them, compound 9r showed the most potent activity,
with an IC50 value of 0.27 lM. Although 30-alkoxy
derivatives showed potent Syk inhibitory activities,
these compounds were less effective in the cellular

H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
4941
Figure 2. Sequence alignment of Syk (323–600) with Lck (231–501). In the sequences, an asterisk (*) indicates an identical amino acid.
Figure 3. Binding model of 9a to Syk.
Table 3. In vitro selectivity of Syk inhibition over other tyrosine kinases and 5-HT release inhibitory activity
No.
IC50 (lM)a
5-HT release inhibition
IC50 (lM)a
Syk
Itk
Btk
PKCe
PKCb2
9a
9b
9j
0.041
0.047
0.03
22.6
4.0
29.3
ntb
nt
15.5
2.9
9.6
nt
5.1
1.5
6
11
16
0.46
1.45
0.73
0.3
13
10
9m
9n
9o
9p
9q
9r
0.053
0.092
0.066
0.081
0.072
0.023
6.4
7.9
9
nt
9.5
19
0.45
2.0
28.0
23.1
nt
8.7
10.7
nt
8.5
nt
27
nt
0.93
1.60
0.27
nt
nt
2.8
0.69
a IC50 values were determined in duplicate.
b Not tested.

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H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
5.1.2. Ethyl-2-methyl-4-(3-trifluoromethylanilino)pyrimi-
dine-5-carboxylate (2b). Compound 2b was prepared
from ethyl-4-chloro-2-methylpyrimidine-5-carboxylate
(1b) and m-aminobenztrifluoride in 97% yield as a yel-
low amorphous solid similarly: 1H NMR (CDCl3) d
1.46 (3H, t, J = 7.2 Hz), 2.92 (3H, s), 4.51 (2H, q,
J = 7.2 Hz), 7.58–7.65 (2H, m), 7.80–7.87 (1H, m), 7.99
(1H, s), 8.98 (1H, s), 11.27 (1H, br s); FAB MS m/e
(M+ H)+ 326.
5.1.3. Ethyl-2-phenyl-4-(3-trifluoromethylanilino)pyrimi-
dine-5-carboxylate hydrochloride (2c). Compound 2c
was prepared from ethyl-4-chloro-2-phenylpyrimidine-
5-carboxylate (1c) and m-aminobenztrifluoride in 69%
yield as a pale yellow amorphous solid similarly: 1H
NMR (DMSO-d6) d 1.39 (3H, t, J = 7.4 Hz), 4.42 (2H,
q, J = 7.4 Hz), 7.48–7.63 (4H, m), 7.67 (1H, d,
J = 7.6 Hz), 7.89 (1H, d, J = 8.8 Hz), 8.35 (2H, d,
J = 7.6 Hz), 8.52 (1H, s), 9.07 (1H, s), 10.38 (1H, br s);
FAB MS m/e (M+H)+ 388.
Figure 4. Effect of 9a on the passive cutaneous anaphylaxis reaction in
mice.
with the 5-carboxamide group may be very important
for maintenance of inhibitor planarity, which will in-
crease activity. All potent compounds showed high
selectivity for Syk compared to other kinases and inhib-
ited 5-HT release from RBL cells. Furthermore, one of
the compounds was found to inhibit the PCA reaction
in mice. These results suggest that potent Syk inhibitors
may be useful for the treatment of allergic disorders.
The results of further optimization and evaluation of
this series of compounds will be reported elsewhere.
5.1.4. 4-(3-Trifluoromethylanilino)pyrimidine-5-carbox-
ylic acid (3a). A solution of 3a (0.22 g, 0.71 mmol) in
EtOH and 1 M NaOH was heated at 50 ꢁC for 1 h.
The solution was acidified with 1 M HCl and cooled
to give a solid, which was collected by filtration and
washed well with water and n-hexane to give 0.15 g of
3a as a pale yellow amorphous powder: 1H NMR
((DMSO-d6) d 7.49 (1H, d, J = 7.8 Hz), 7.62 (1H, t,
J = 8.3 Hz), 7.92 (1H, br d, J = 8.3 Hz), 8.25 (1H, s),
8.83 (1H, s), 8.95 (1H, s), 10.68 (1H, br s); FAB MS
m/e (M+H)+ 284.
5. Experimental
5.1. Chemistry
5.1.5. 2-Methyl-4-(3-trifluoromethylanilino)pyrimidine-5-
carboxylic acid (3b). Compound 3b was prepared in 77%
yield as a pale yellow amorphous solid similarly: 1H
NMR (DMSO-d6) 2.58 (3H, s), 7.46 (1H, d, J = 7.8
Hz), 7.60 (1H, t, J = 7.8 Hz), 7.88 (1H, br d, J = 8.3
Hz), 8.32 (1H, s), 8.85 (1H, s), 10.77 (1H, br s); FAB
MS m/e (M+H)+ 298.
1H NMR spectra were measured with a JEOL EX400
(400 MHz) or GX500 (500 MHz) spectrometer; chemi-
cal shifts are expressed in d units using tetramethylsilane
as the standard (NMR peak description: s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; and br, broad
peak). Some compounds with an amino group at the 2-
1
position showed broad peaks in the H NMR spectra,
probably because of the presence of a conformational
isomer. Therefore, the 1H NMR spectra of selected com-
pounds were measured at 80 ꢁC to confirm the structure.
Mass spectra were recorded with a Hitachi M-80 or a
JEOL JMS-DX300 spectrometer. Column chromato-
graphy was carried out on silica gel (Wakogel C-200).
Unless otherwise noted, all reagents and solvents ob-
tained from commercial suppliers were used without fur-
ther purification.
5.1.6. 2-Phenyl-4-(3-trifluoromethylanilino)pyrimidine-5-
carboxylic acid (3c). Compound 3c was prepared in 95%
yield as a colorless amorphous solid similarly: 1H NMR
(DMSO-d6) 7.48–7.61 (4H, m), 7.64 (1H, t, J = 8.3 Hz),
7.84 (1H, br d, J = 8.3 Hz), 8.31–8.39 (2H, m), 8.60 (1H,
s), 9.04 (1H, s), 11.28 (1H, br s); FAB MS m/e (M+H)+
360.
5.1.7. 4-(3-Trifluoromethylanilino)pyrimidine-5-carbox-
amide (4a). To a solution of 4a (0.15 g, 0.53 mmol) in
dimethylformamide (6 mL) were added 1-(3-dimethyl-
5.1.1. Ethyl-4-(3-trifluoromethylanilino) pyrimidine-5-
carboxylate (2a). A mixture of ethyl-4-chloropyrimi-
dine-5-carboxylate 1a (680 mg, 3.6 mmol) and 3-trifluo-
romethylaniline (0.45 m, 3.6 mmol) in toluene (10 mL)
was heated with reflux for 8 h. The mixture was concen-
trated and the residue was chromatographed on silica
gel with elution using CHCl3, to give 240 mg of 2a
aminopropyl)-3-ethylcarbodiimide
hydrochloride
(EDCI) (0.11 g, 18 mmol) and HOBt (0.8 g, 18 mmol).
After stirring for 4 h at room temperature, NH4OH
(1 mL) was added and stirred for 14 h at room temper-
ature. The solution was washed with water, satd NaCl,
dried over anhydrous Na2SO4, and concentrated. The
residue was chromatographed on silica gel with elution
using CHCl3–MeOH, to give 0.14 g of 4a (94%) as a
white powder: mp 201–203 ꢁC (EtOH); 1H NMR
(DMSO-d6) 5.98 (2H, br s), 7.39 (1H, d, J = 8.0 Hz),
7.49 (1H, t, J = 8.0 Hz), 7.86 (1H, br d, J = 8.6 Hz),
1
(21%) as a yellow amorphous solid: H NMR (CDCl3)
d 1.45 (3H, t, J = 7.2 Hz), 4.45 (2H, q, J = 7.2 Hz),
7.40 (1H, d, J = 7.5 Hz), 7.50 (1H, t, J = 7.5 Hz), 7.88
(1H, d, J = 7.5 Hz), 8.06 (1H, s), 8.82 (1H, s), 9.04
(1H, s), 10.48 (1H, br s); FAB MS m/e (M+H)+ 312.

H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
4943
8.07 (1H, s), 8.65 (1H, s), 8.79 (1H, s), 11.00 (1H, br s);
FAB MS m/e (M+H)+ 283, Anal. Calcd for C12H9N4O-
F3Æ0.3H2O: C, 50.11; H, 3.36; N, 19.48; F, 19.82. Found:
C, 50.06; H, 3.41; N, 19.10; F, 20.22.
5.1.13. Ethyl-2-chloro-4-(3-methylanilino)pyrimidine-5-
carboxylate (6d). Compound 6d was prepared from
ethyl-2,4-dichloropyrimidine-5-carboxylate (5) and m-to-
luidine in 82% yield as a colorless amorphous powder
1
similarly: H NMR (CDCl3) d 1.43 (3H, t, J = 7.0 Hz),
5.1.8. 2-Methyl-4-(3-trifluoromethylanilino)pyrimidine-5-
carboxamide (4b). Compound 4b was prepared in 58%
yield as a pale yellow solid similarly: mp 254–255 ꢁC
(H2O–MeOH); 1H NMR (DMSO-d6) 2.67 (3H, s),
7.60 (1H, d, J = 7.9 Hz), 7.67 (1H, t, J = 7.8 Hz), 7.93
(1H, d, J = 8.3 Hz), 8.19 (2H, br s), 8.76 (1H, s), 9.11
(1H, s), 12.15 (1H, br s); FAB MS m/e (M+H)+ 297.
Anal. Calcd for C13H11N4OF3ÆHCl: C, 46.93; H, 3.64;
N, 16.84; Cl, 10.66; F, 17.13. Found: C, 46.85; H,
3.62; N, 16.88; Cl, 10.57; F, 17.31.
2.38 (3H, s), 4.42 (2H, q, J = 7.0 Hz), 6.99 (1H, d,
J = 7.6 Hz), 7.26–7.30 (1H, m), 7.40 (1H, s), 7.53–7.55
(1H, m), 8.85 (1H, s), 10.39 (1H, br s); FAB MS m/e
(M+H)+ 292.
5.1.14. Ethyl-2-chloro-4-(4-methylanilino)pyrimidine-5-
carboxylate (6e). Compound 6e was prepared from
ethyl-2,4-dichloropyrimidine-5-carboxylate (5) and p-to-
luidine in 77% yield as a pale yellow amorphous powder
1
similarly: H NMR (CDCl3) d 1.43 (3H, t, J = 7.2 Hz),
2.35 (3H, s), 4.42 (2H, q, J = 7.2 Hz), 7.19 (2H, d,
J = 8.4 Hz), 7.52 (2H, d, J = 8.4 Hz), 8.80 (1H, s),
10.35 (1H, br s); FAB MS m/e (M+H)+ 292.
5.1.9. 2-Phenyl-4-(3-trifluoromethylanilino)pyrimidine-5-
carboxamide (4c). Compound 4c was prepared in 57%
yield as a pale yellow solid similarly: mp 233–235 ꢁC
(EtOH); 1H NMR (DMSO-d6) 5.90 (2H, br s), 7.39
(1H, d, J = 7.8 Hz), 7.47–7.55 (4H, m), 7.76 (1H, br
d, J = 7.5 Hz), 8.32–8.47 (2H, m), 8.49 (1H, s), 8.74
(1H, s), 11.08 (1H, br s); FAB MS m/e (M+H)+ 359.
Anal. Calcd for C18H13N4OF3: C, 60.34; H, 3.66;
N, 15.64; F, 15.91. Found: C, 60.09; H, 3.76; N,
15.68; F, 16.06.
5.1.15.
Ethyl-2-chloro-4-(3-cyanoanilino)pyrimidine-5-
carboxylate (6f). Compound 6f was prepared from
ethyl-2, 4-dichloropyrimidine-5-carboxylate (5) and 3-
aminobenzonitrile in 95% yield as a colorless amor-
1
phous powder similarly: H NMR (CDCl3) d 1.45 (3H,
t, J = 7.3 Hz), 4.45 (2H, q, J = 7.3 Hz), 7.44–7.52 (2H,
m), 7.86–7.89 (1H, m), 8.08–8.12 (1H, m), 8.89 (1H, s),
10.62 (1H, br s); FAB MS m/e (M+H)+ 303.
5.1.10. Ethyl-2-chloro-4-(3-trifluoromethylanilino)pyrim-
idine-5-carboxylate (6a). A mixture of ethyl-2,4-dichlo-
ropyrimidine-5-carboxylate (4.86 g, 22.0 mmol), 3-amino-
benztrifluoride (3.0 mL, 24.2 mmol), and diisopropyl-
ethylamine (3.82 mL, 22.0 mmol) in CH3CN (50 mL)
was heated with reflux for 1 h. The mixture was diluted
with AcOEt and washed successively with water, satu-
rated aqueous NaCl, dried over Na2SO4, and concen-
trated. The residue was filtered and washed with
hexane to give 6.66 g of 6a (88%) as a pale yellow amor-
phous solid: 1H NMR (CDCl3) d 1.37 (3H, t,
J = 7.5 Hz), 4.40 (2H, q, J = 7.5 Hz), 7.55 (1H, d,
J = 7.5 Hz), 7.65 (1H, t, J = 7.5 Hz), 7.87 (1H, d,
J = 7.5 Hz), 8.13 (1H, s), 8.84 (1H, s), 10.38 (1H, br s);
FAB MS m/e (M+H)+ 346.
5.1.16.
Ethyl-2-chloro-4-(3-ethylanilino)pyrimidine-5-
carboxylate (6g). Compound 6g was prepared from
ethyl-2,4-dichloropyrimidine-5-carboxylate (5) and m-
ethylaniline in 61% yield as a pale yellow amorphous
powder similarly: 1H NMR (CDCl3) d 1.36 (3H, t,
J = 7.4 Hz), 1.42 (3H, t, J = 7.4 Hz), 2.67 (2H, q,
J = 7.4 Hz), 4.42 (2H, q, J = 7.4 Hz), 7.01 (1H, br d,
J = 8.3 Hz), 7.29 (1H, t, J = 7.8 Hz), 7.42 (1H, s), 7.55
(1H, dd, J = 8.3, 1.4 Hz), 8.80 (1H, s), 10.39 (1H, br
s); FAB MS m/e (M+H)+ 306.
5.1.17. Ethyl-2-chloro-4-(3-isopropylanilino)pyrimidine-5-
carboxylate (6h). Compound 6h was prepared from
ethyl-2,4-dichloropyrimidine-5-carboxylate (5) and
m-isopropylaniline in 45% yield as a pale yellow amor-
1
The following analogues were prepared in a similar
manner.
phous powder similarly: H NMR (CDCl3) d 1.28 (6H,
d, J = 6.8 Hz), 1.44 (3H, t, J = 7.3 Hz), 2.93 (1H, qq,
J = 6.8 Hz), 4.43 (2H, q, J = 7.3 Hz), 7.05 (1H, br d,
J = 7.8 Hz), 7.32 (1H, t, J = 7.8 Hz), 7.45–7.48 (1H,
m), 7.54–7.58 (1H, m), 8.82 (1H, s), 10.40 (1H, br s);
FAB MS m/e (M+H)+ 320.
5.1.11. Ethyl-4-anilino-2-chloropyrimidine-5-carboxylate
(6b). Compound 6b was prepared from ethyl-2,4-dichlo-
ropyrimidine-5-carboxylate (5) and aniline in 81% yield
as a colorless amorphous solid similarly: 1H NMR
(CDCl3) d 1.44 (3H, t, J = 7.4 Hz), 4.43 (2H, q,
J = 7.4 Hz), 7.18 (1H, t, J = 7.5 Hz), 7.40 (1H, t,
J = 7.5 Hz), 7.67 (1H, d, J = 7.5 Hz), 8.83 (1H, s),
10.45 (1H, br s); FAB MS m/e (M+H)+ 278.
5.1.18. Ethyl-2-chloro-4-(3-methoxyanilino)pyrimidine-
5-carboxylate (6i). Compound 6i was prepared from
ethyl-2, 4-dichloropyrimidine-5-carboxylate (5) and
m-methoxyaniline in 87% yield as a colorless amorphous
powder similarly: 1H NMR (CDCl3) d 1.43 (3H, t,
J = 7.3 Hz), 3.85 (3H, s), 4.43 (2H, q, J = 7.3 Hz), 6.73
(1H, ddd, J = 8.3, 2.4, 1.0 Hz), 7.15–7.19 (1H, m),
7.26–7.30 (1H, m), 7.43 (1H, t, J = 2.4 Hz), 8.83 (1H,
s), 10.45 (1H, br s); FAB MS m/e (M+H)+ 308.
5.1.12. Ethyl-2-chloro-4-(2-methylanilino)pyrimidine-5-
carboxylate (6c). Compound 6c was prepared from
ethyl-2,4-dichloropyrimidine-5-carboxylate (5) and o-to-
luidine in 86% yield as a pale gray amorphous powder
1
similarly: H NMR (CDCl3) d 1.43 (3H, t, J = 7.4 Hz),
2.35 (3H, s), 4.43 (2H, q, J = 7.4 Hz), 7.11–7.15 (1H,
m), 7.20–7.29 (2H, m), 7.98 (1H, d, J = 8.3 Hz), 8.80
(1H, s), 10.28 (1H, br s); FAB MS m/e (M+H)+ 292.
5.1.19. Ethyl-2-chloro-4-(3-ethoxyanilino)pyrimidine-5-
carboxylate (6j). Compound 6j was prepared from
ethyl-2, 4-dichloropyrimidine-5-carboxylate (5) and

4944
H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
m-ethoxyaniline as a pale yellow amorphous powder.
This compound was used for next reaction without
purification.
(DMSO-d6) d 2.31 (3H, s), 7.22 (2H, d, J = 8.3 Hz),
7.50 (2H, d, J = 8.3 Hz), 8.75 (1H, s), 10.49 (1H, br s);
FAB MS m/e (MꢀH)ꢀ 262.
5.1.20. Ethyl-2-chloro-4-(3-isopropoxyanilino)pyrimidine-
5-carboxylate (6k). Compound 6k was prepared from
ethyl-2,4-dichloropyrimidine-5-carboxylate (5) and m-
isopropoxyaniline in 66% yield as a pale yellow amor-
5.1.27. 2-Chloro-4-(3-cyanoanilino)pyrimidine-5-carbox-
ylic acid (7f). Compound 7f was prepared in 100% yield
1
as a colorless amorphous powder similarly: H NMR
(DMSO) d 7.49–7.52 (1H, m), 7.57 (1H, t, J = 7.8 Hz),
7.82–7.85 (1H, m), 8.23–8.24 (1H, m), 8.64 (1H, s),
14.10 (1H, br s); FAB MS m/e (MꢀH)ꢀ 273.
1
phous powder similarly: H NMR (CDCl3) d 1.38 (6H,
d, J = 6.3 Hz), 1.43 (3H, t, J = 7.3 Hz), 4.43 (2H, q,
J = 7.3 Hz), 4.58 (1H, qq, J = 6.3 Hz), 6.69–6.73 (1H,
m), 7.09–7.14 (1H, m), 7.25 (1H, t, J = 7.8 Hz), 7.42
(1H, t, J = 2.0 Hz), 8.82 (1H, s), 10.42 (1H, br s); FAB
MS m/e (M+H)+ 336.
5.1.28. 2-Chloro-4-(3-ethylanilino)pyrimidine-5-carbox-
ylic acid (7g). Compound 7g was prepared in 98% yield
as a pale yellow amorphous powder similarly: 1H NMR
(DMSO-d6) 1.21 (3H, t, J = 7.8 Hz), 2.63 (2H, q,
J = 7.8 Hz), 7.05 (1H, br d, J = 7.8 Hz), 7.32 (1H, t,
J = 7.8 Hz), 7.43 (1H, br s), 7.52 (1H, br d,
J = 7.8 Hz), 8.77 (1H, s), 10.56 (1H, br s); FAB MS
m/e (M+H)+ 278.
5.1.21. Ethyl-4-(3-bromoanilino)-2-chloropyrimidine-5-
carboxylate (6l). Compound 6l was prepared from
ethyl-2, 4-dichloropyrimidine-5-carboxylate (5) and m-
bromoaniline in 100% yield as a pale yellow amorphous
powder similarly: 1H NMR (CDCl3) d 1.36 (3H, t,
J = 7.4 Hz), 4.39 (2H, q, J = 7.4 Hz), 7.32–7.42 (2H,
m), 7.57–7.62 (1H, m), 7.98 (1H, s), 8.83 (1H, s), 10.27
(1H, br s); FAB MS m/e (M+H)+ 356, 358.
5.1.29. 2-Chloro-4-(3-isopropylanilino)pyrimidine-5-car-
boxylic acid (7h). Compound 7h was prepared in 100%
1
yield as a pale yellow amorphous powder similarly: H
NMR (DMSO-d6): d 1.23 (6H, d, J = 6.8 Hz), 2.93
(1H, qq, J = 6.8 Hz), 7.07 (1H, br d, J = 7.8 Hz), 7.32
(1H, t, J = 7.4 Hz), 7.47–7.52 (2H, m), 8.76 (1H, s),
10.69 (1H, br s); FAB MS m/e (M+H)+ 292.
5.1.22. 2-Chloro-4-(3-trifluoromethylanilino)pyrimidine-
5-carboxylic acid (7a). A solution of 6a (6.22 g, 18 mmol)
in MeOH and 1 M NaOH was heated at 50 ꢁC for 1 h.
The solution was acidified with 1 M HCl and cooled to
give a solid, which was collected by filtration and washed
well with water and n-hexane to give 5.48 g of 7a as a col-
5.1.30. 2-Chloro-4-(3-methoxyanilino)pyrimidine-5-car-
boxylic acid (7i). Compound 7i was prepared in 72%
yield as a colorless amorphous powder similarly: 1H
NMR (DMSO-d6) d 3.77 (3H, s), 6.67 (1H, br d,
J = 8.0 Hz), 7.16 (1H, d, J = 8.0 Hz), 7.27 (1H, t,
J = 8.0 Hz), 7.42 (1H, br s), 8.66 (1H, s), 12.93 (1H, br
s); FAB MS m/e (MꢀH)ꢀ 278.
1
orless amorphous powder: H NMR (DMSO-d6) d 7.54
(1H, d, J = 7.5 Hz), 7.65 (1H, t, J = 7.5 Hz), 7.88 (1H,
br d, J = 8.4 Hz), 8.15 (1H, s), 8.82 (1H, s), 10.72 (1H,
br s); FAB MS m/e (M+H)+ 318.
The following analogues were prepared in a similar
manner.
5.1.31. 2-Chloro-4-(3-ethoxyanilino)pyrimidine-5-carbox-
ylic acid (7j). Compound 7j was prepared in 61% yield as
a yellow amorphous powder similarly: 1H NMR
(DMSO-d6) d 1.35 (3H, t, J = 6.8 Hz), 4.05 (2H, q,
J = 6.8 Hz), 6.75 (1H, dd, J = 8.3, 2.4 Hz), 7.13 (1H,
br d, J = 8.3 Hz), 7.29 (1H, t, J = 8.3 Hz), 7.33–7.36
(1H, m), 8.78 (1H, s), 10.56 (1H, br s); FAB MS m/e
(M+H)+ 294.
5.1.23. 4-Anilino-2-chloropyrimidine-5-carboxylic acid
(7b). Compound 7b was prepared in 79% yield as a pale
yellow amorphous powder similarly: 1H NMR (DMSO-
d6) d 7.16–7.24 (1H, m), 7.33–7.46 (2H, m), 7.61–7.67
(1H, m), 8.78 (1H, s), 10.57 (1H, br s); FAB MS m/e
(MꢀH)ꢀ 248.
5.1.24. 2-Chloro-4-(2-methylanilino)pyrimidine-5-carbox-
ylic acid (7c). Compound 7c was prepared in 99% yield
5.1.32. 2-Chloro-4-(3-isopropoxyanilino)pyrimidine-5-car-
boxylic acid (7k). Compound 7k was prepared in 97%
1
as a pale ivory amorphous powder similarly: H NMR
1
yield as a pale yellow amorphous powder similarly: H
(DMSO-d6) d 2.27 (3H, s), 7.14–7.18 (1H, m), 7.27–
7.38 (2H, m), 7.81 (1H, d, J = 7.9 Hz), 8.77 (1H, s),
10.44 (1H, br s); FAB MS m/e (MꢀH)ꢀ 262.
NMR (DMSO-d6) d 1.30 (6H, d, J = 6.3 Hz), 4.61
(1H, qq, J = 6.3 Hz), 6.77 (1H, dd, J = 8.3, 2.4 Hz),
7.05–7.09 (1H, m), 7.27 (1H, t, J = 8.3 Hz), 7.39 (1H,
m), 8.75 (1H, s), 11.07 (1H, br s); FAB MS m/e
(M+H)+ 308.
5.1.25. 2-Chloro-4-(3-methylanilino)pyrimidine-5-carbox-
ylic acid (7d). Compound 7d was prepared in 99% yield
as a pale yellow amorphous powder similarly: 1H NMR
(DMSO-d6) d 2.29 (3H, s), 6.89 (1H, d, J = 6.9 Hz),
7.22–7.25 (1H, m), 7.37 (1H, s), 7.54 (1H, d,
J = 6.9 Hz), 8.68 (1H, s), 12.82 (1H, br s); FAB MS
m/e (M+H)+ 264.
5.1.33. 4-(3-Bromoanilino)-2-chloropyrimidine-5-carbox-
ylic acid (7l). Compound 7l was prepared in 100% yield
as a pale yellow amorphous powder similarly: 1H NMR
(DMSO-d6) d 7.36–7.39 (2H, m), 7.58–7.62 (1H, m), 8.00
(1H, br s), 8.80 (1H, s), 10.64 (1H, br s); FAB MS m/e
(MꢀH)ꢀ 326, 328.
5.1.26. 2-Chloro-4-(4-methylanilino)pyrimidine-5-carbox-
ylic acid (7e). Compound 7e was prepared in 100% yield
as an ivory amorphous powder similarly: 1H NMR
5.1.34. 2-(1H-Benzotriazol-1-yloxy)-4-(3-trifluoromethyl-
anilino)pyrimidine-5-carboxamide (8a). To a suspension

H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
4945
of 7a (5.41 g, 17 mmol) in dichloroethane (60 mL) were
added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (EDCI) (3.45 g, 18 mmol) and HOBt
(2.43 g, 18 mmol). After stirring for 30 min at room tem-
perature, NH4OH (10 mL) was added and stirred for 1 h
at room temperature. The solution was washed with
water, satd NaCl, dried over anhydrous Na2SO4, and
concentrated. The residue was chromatographed on
silica gel with elution using CHCl3, to give 3.7 g of
8a (52%) as a white amorphous powder: 1H NMR
(DMSO-d6) d 7.19 (1H, t, J = 7.8 Hz), 7.29 (1H, br d,
J = 8.7 Hz), 7.36 (1H, d, J = 7.8 Hz), 7.54 (1H, t,
J = 7.5 Hz), 7.63 (1H, t, J = 7.5 Hz), 7.67 (1H, br s),
7.81 (1H, d, J = 8.4 Hz), 7.96 (1H, br s), 8.19 (1H, d,
J = 8.4 Hz), 8.41 (1H, br s), 8.86 (1H, s), 11.79 (1H, br
s); FAB MS m/e (M+H)+ 416.
J = 8.3 Hz), 7.97 (1H, br s), 8.18 (1H, d, J = 8.3 Hz),
8.42 (1H, br s), 8.88 (1H, s), 11.75 (1H, br s); FAB
MS m/e (MꢀH)ꢀ 371.
5.1.40. 2-(1H-Benzotriazol-1-yloxy)-4-(3-ethylanilino)py-
rimidine-5-carboxamide (8g). Compound 8g was pre-
pared similarly and used for next reaction without
purification.
5.1.41. 2-(1H-Benzotriazol-1-yloxy)-4-(3-isopropylanilino)-
pyrimidine-5-carboxamide (8h). Compound 8h was pre-
pared similarly and used for next reaction without
purification.
5.1.42. 2-(1H-Benzotriazol-1-yloxy)-4-(3-methoxyanilino)-
pyrimidine-5-carboxamide (8i). Compound 8i was pre-
pared in 34% yield as a yellow amorphous powder
similarly: 1H NMR (DMSO-d6) d 3.86 (3H, s), 6.78
(1H, dd, J = 8.6, 2.2 Hz), 7.12–7.16 (1H, m), 7.32 (1H,
t, J = 8.1 Hz), 7.59 (1H, t, J = 7.5 Hz), 7.71 (1H, t,
J = 2.2 Hz), 7.76 (1H, t, J = 7.8 Hz), 7.98–8.00 (3H,
m), 8.52 (1H, d, J = 8.6 Hz), 9.03 (1H, s), 11.54 (1H,
br s); FAB MS m/e (M+H)+ 378.
The following analogues were prepared in a similar
manner.
5.1.35. 4-Anilino-2-(1H-benzotriazol-1-yloxy)pyrimidine-
5-carboxamide (8b). Compound 8b was prepared in 34%
yield as a colorless amorphous powder similarly: 1H
NMR (DMSO-d6) d 6.91–7.00 (5H, m), 7.52–7.60 (1H,
m), 7.62–7.69 (1H, m), 7.82 (1H, d, J = 8.3 Hz), 7.90
(1H, br s), 8.23 (1H, d, J = 8.3 Hz), 8.39 (1H, br s),
8.87 (1H, s), 11.64 (1H, br s); FAB MS m/e (M+H)+ 348.
5.1.43. 2-(1H-Benzotriazol-1-yloxy)-4-(3-ethoxyanilino)-
pyrimidine-5-carboxamide (8j). Compound 8j was pre-
pared in 35% yield as a pale yellow amorphous
1
powder similarly: H NMR (DMSO-d6) d 1.35 (3H, t,
5.1.36. 2-(1H-Benzotriazol-1-yloxy)-4-(2-methylanilino)-
pyrimidine-5-carboxamide (8c). Compound 8c was pre-
pared in 57% yield as an ivory amorphous powder
J = 6.8 Hz), 4.05 (2H, q, J = 6.8 Hz), 6.53–6.60 (2H,
m), 6.80 (1H, t, J = 2.5 Hz), 6.85 (1H, t, J = 7.8 Hz),
7.54 (1H, t, J = 7.3 Hz), 7.64 (1H, t, J = 7.3 Hz), 7.80
(1H, d, J = 8.3 Hz), 7.90 (1H, br s), 8.19 (1H, d,
J = 8.3 Hz), 8.40 (1H, br s), 8.83 (1H, s), 11.53 (1H, br
s); FAB MS m/e (M+H)+ 392.
1
similarly: H NMR (DMSO-d6) d 2.18 (3H, s), 6.59–
6.62 (1H, m), 6.88–6.92 (1H, m), 6.97 (1H, d,
J = 8.3 Hz), 7.12 (1H, d, J = 7.3 Hz), 7.53–7.57 (1H,
m), 7.62–7.66 (1H, m), 7.80 (1H, d, J = 8.3 Hz), 7.88
(1H, br s), 8.20 (1H, d, J = 8.3 Hz), 8.39 (1H, br s),
8.86 (1H, s), 11.59 (1H, br s); FAB MS m/e (M+H)+ 362.
5.1.44. 2-(1H-Benzotriazol-1-yloxy)-4-(3-isopropoxyani-
lino)pyrimidine-5-carboxamide (8k). Compound 8k was
prepared in 55% yield as a pale yellow amorphous pow-
der similarly: 1H NMR (DMSO-d6) d 1.23 (6H, d,
J = 5.8 Hz), 4.50 (1H, qq, J = 5.8 Hz), 6.53 (1H, dd,
J = 7.3, 1.9 Hz), 6.58 (1H, dd, J = 7.3, 2.0 Hz), 6.82–
6.86 (2H, m), 7.51–7.57 (1H, m), 7.61–7.67 (1H, m),
7.80 (1H, d, J = 8.3 Hz), 7.54 (1H, t, J = 7.3 Hz), 7.64
(1H, t, J = 7.3 Hz), 7.80 (1H, d, J = 8.3 Hz), 7.90 (1H,
br s), 8.20 (1H, d, J = 8.3 Hz), 8.37 (1H, br s), 8.80
(1H, s), 11.62 (1H, br s); FAB MS m/e (M+H)+ 406.
5.1.37. 2-(1H-Benzotriazol-1-yloxy)-4-(3-methylanilino)-
pyrimidine-5-carboxamide (8d). Compound 8d was pre-
pared in 50% yield as a pale yellow amorphous
1
powder similarly: H NMR (DMSO-d6) d 2.08 (3H, s),
6.72–6.90 (4H, m), 7.51–7.57 (1H, m), 7.61–7.67 (1H,
m), 7.79–7.83 (1H, m), 7.91 (1H, br s), 8.18–8.23 (1H,
m), 8.40 (1H, br s), 8.87 (1H, s), 11.67 (1H, br s);
FAB MS m/e (M+H)+ 362.
5.1.38. 2-(1H-Benzotriazol-1-yloxy)-4-(4-methylanilino)-
pyrimidine-5-carboxamide (8e). Compound 8e was pre-
pared in 55% yield as a pale yellow amorphous
5.1.45. 2-(1H-Benzotriazol-1-yloxy)-4-(3-bromoanilino)-
pyrimidine-5-carboxamide (8l). Compound 8l was pre-
pared in 80% yield as a pale yellow amorphous
powder similarly: 1H NMR (DMSO-d6) d 6.91–7.20
(2H, m), 7.19 (1H, dt, J = 7.3, 1.4 Hz), 7.49 (1H, t,
J = 1.9 Hz), 7.51–7.57 (1H, m), 7.64 (1H, t,
J = 7.3 Hz), 7.81 (1H, d, J = 8.3 Hz), 7.95 (1H, br s),
8.21 (1H, d, J = 8.3 Hz), 8.41 (1H, br s), 8.86 (1H, s),
11.74 (1H, br s); FAB MS m/e (MꢀH)ꢀ 423, 425.
1
powder similarly: H NMR (DMSO-d6) d 2.19 (3H, s),
6.79 (2H, d, J = 8.3 Hz), 6.83 (2H, d, J = 8.3 Hz), 7.57
(1H, t, J = 7.8 Hz), 7.65 (1H, t, J = 7.8 Hz), 7.81 (1H,
d, J = 8.3 Hz), 7.88 (1H, br s), 8.24 (1H, d,
J = 8.8 Hz), 8.32 (1H, br s), 8.84 (1H, s), 11.56 (1H, br
s); FAB MS m/e (M+H)+ 362.
5.1.39. 2-(1H-Benzotriazol-1-yloxy)-4-(3-cyanoanilino)-
pyrimidine-5-carboxamide (8f). Compound 8f was pre-
pared in 18% yield as a yellow amorphous powder
d 7.17 (1H, t,
J = 7.8 Hz), 7.31–7.34 (1H, m), 7.44–7.46 (1H, m),
7.51–7.55 (1H, m), 7.61–7.65 (2H, m), 7.79 (1H, d,
5.1.46. 2-(2-Aminoethylamino)-4-(3-trifluoromethylanilino)-
pyrimidine-5-carboxamide dihydrochloride (9a). To a sus-
pension of 8a (3.49 g, 8.4 mmol) in CH3CN (50 mL) was
added ethylenediamine (7.35 mL, 110 mmol) and stirred
for 30 min at room temperature. The mixture was di-
luted with water and extracted with AcOEt. The organic
similarly: 1H NMR (DMSO-d6)

4946
H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
layer was dried over anhydrous Na2SO4, and evaporated
to give 2.80 g of 9a (98%) as a colorless solid. 9a was
converted into the hydrochloride and recrystallized from
water–EtOH to give 1.76 g of 9a as a colorless powder:
(0.7H, s), 8.60 (0.7H, s), 8.64 (0.3H, s), 11.78 (0.3H, br
s), 11.94 (0.7H, br s); FAB MS m/e (M+H)+ 369. Anal.
Calcd for C16H19N6OF3Æ0.2H2O: C, 51.67; H, 5.26; N,
22.59; F, 15.32. Found: C, 51.58; H, 5.17; N, 22.66; F,
15.09.
1
mp 270–272 ꢁC (dec.); H NMR (DMSO-d6, 80 ꢁC) d
3.04 (2H, br s), 3.68 (2H, m), 7.08 (1H, br s), 7.48
(1H, br d, J = 7.8 Hz), 7.64 (1H, br t, J = 7.8 Hz),
7.75–8.40 (3H, m), 8.06 (1H, br s), 8.79 (1H, s), 12.09
(1H, br s); FAB MS m/e (M+H)+ 341. Anal. Calcd for
C14H15N6OF3Æ2HCl: C, 40.69; H, 4.15; N, 20.34; Cl,
17.16; F, 13.79. Found: C, 40.69; H, 4.11; N, 20.32;
Cl, 16.87; F, 13.64.
5.1.51. 2-[(2-Dimethylaminoethyl)methylamino]-4-(3-triflu-
oromethylanilino)pyrimidine-5-carboxamide (9f). Com-
pound 9f was prepared from compound 8a and
N,N,N0-trimethylethylenediamine in 51% yield similarly:
1
mp 153–157 ꢁC (AcOEt–hexane); H NMR (DMSO-d6)
d 2.14 (3H, br s), 2.25 (3H, br s), 2.43–2.60 (2H, m), 3.16
(3H, s), 3.71 (0.8H, br s), 3.79 (2H, br s), 7.36 (1H, d,
J = 7.8 Hz), 7.54 (1H, br t, J = 7.8 Hz), 7.60–8.20 (3H,
m), 8.24 (0.4H, br s), 8.62 (0.6H, br s), 8.68 (1H,s),
11.86 (0.4H, br s), 11.90 (0.6H, br s); FAB MS m/e
(M+H)+ 383. Anal. Calcd for C17H21N6OF3Æ0.7H2O:
C, 51.69; H, 5.72; N, 21.28; F, 14.43. Found: C, 51.47;
H, 5.44; N, 21.24; F, 14.75.
The following analogues were prepared similarly.
5.1.47. 2-(4-Aminobutylamino)-4-(3-trifluoromethylanili-
no)pyrimidine-5-carboxamide (9b). Compound 9b was
prepared from compound 8a and 1,4-diaminobutane in
33% yield as a colorless powder similarly: mp 198–
200 ꢁC (AcOEt–EtOH); 1H NMR (DMSO-d6) 1.38
(2H, m), 1.57 (2H, m), 2.52 (2H, m), 3.31 (2H, m),
7.35 (2H, m), 7.50–8.30 (4H, m), 8.60 (0.8H, s), 8.62
(0.2H, s), 11.77 (0.2H, br s), 11.95 (0.8H, br s); FAB
MS m/e (M+H)+. Anal. Calcd for C16H19N6OF3Æ0.25-
C2H6O.1.5H2O: C, 50.38; H, 5.64; N, 21.36; F, 14.49.
Found: C. 50.40; H, 5.66; N, 21.14; F, 14.64.
5.1.52. 2-(Piperazin-1-yl)-4-(3-trifluoromethylanilino)pyr-
imidine-5-carboxamide (9g). Compound 9g was prepared
from compound 8a and piperazine in 59% yield as a col-
orless powder similarly: mp 207–209 ꢁC (AcOEt–hexane);
1H NMR (DMSO-d6) d 2.74 (4H, br s), 3.72 (4H, br s),
7.33–7.45 (1H, m), 7.37 (1H, d, J = 6.8 Hz), 7.55 (1H, t,
J = 8.3 Hz), 7.59 (1H, d, J = 8.3 Hz), 7.99 (1H, br s),
8.46 (1H, s), 8.68 (1H, s), 11.80 (1H, s); FAB MS m/e
(M+H)+ 367. Anal. Calcd for C16H17N6OF3Æ0.2H2O: C,
51.95; H, 4.74; N, 22.72; F, 15.41. Found: C, 52.20; H,
4.71; N, 22.61; F, 15.44.
5.1.48. tert-Butyl-(2-{[5-(aminocarbonyl)-4-(3-trifluoro-
methylanilino)pyrimidin-2-yl]amino}ethyl)methylcarbamate
(9c). Compound 9c was prepared from compound 8a
and tert-butyl (2-aminoethyl)methylcarbamate similarly.
This compound was used for next reaction without
purification.
5.1.53. 2-(2-Aminoethylamino)-4-anilinopyrimidine-5-car-
boxamide (9h). Compound 9h was prepared from com-
pound 8b and ethylenediamine in 46% yield as a
5.1.49. 2-[(2-Methylaminoethyl)amino]-4-(3-trifluorome-
thylanilino)pyrimidine-5-carboxamide (9d). To a suspen-
sion of 9c (0.32 g, 0.7 mmol) in EtOH (10 mL) was
added 4 N HCl/AcOEt (1 mL) and stirred for 1 day at
room temperature. The solution was evaporated and di-
luted with CHCl3–2-PrOH (3:1 by volume). The mixture
was washed with saturated aqueous Na2CO3, H2O,
dried over anhydrous Na2SO4, and evaporated. The res-
idue was chromatographed on silica gel with elution
using CHCl3–MeOH–aq NH3, to give 0.17 g of 9d
(30%) as a colorless solid: mp 194–196 ꢁC (EtOH–
H2O); 1H NMR (DMSO-d6) d 1.74 (1H, br s), 2.28
(3H, s), 2.68 (2H, t, J = 5.9 Hz), 3.39 (2H, q,
J = 5.9 Hz), 7.25–7.45 (2H, m), 7.50–7.57 (2H, m), 7.66
(1H, d, J = 8.3 Hz), 7.80–8.20 (1H, m), 8.54 (1H, s),
8.60 (1H, s), 11.78 (0.3H, br s), 11.94 (0.7H, br s);
FAB MS m/e (M+H)+ 355. Anal. Calcd for
C15H17N6OF3: C, 50.85; H, 4.84; N, 23.72; F, 16.09.
Found: C, 50.59; H, 4.77; N, 23.55; F, 16.37.
1
colorless powder similarly: mp 173–175 ꢁC (EtOH); H
NMR (DMSO-d6, 80 ꢁC) d 2.75 (2H, t, J = 6.0 Hz),
3.33 (2H, q, J = 6.0 Hz), 7.00 (1H, t, J = 7.3 Hz), 7.31
(2H, t, J = 7.3 Hz), 7.70 (2H, d, J = 7.3 Hz), 8.56 (1H,
s), 11.44 (1H, br s); FAB MS m/e (M+H)+ 273. Anal.
Calcd for C13H16N6OÆ0.5H2O: C, 55.50; H, 6.09; N,
29.87. Found: C, 55.61; H, 5.86; N, 29.84.
5.1.54. 2-(2-Aminoethylamino)-4-(2-methylanilino)pyrim-
idine-5-carboxamide (9i). Compound 9i was prepared
from compound 8c and ethylenediamine in 71% yield
as
a
colorless powder similarly: mp185–187 ꢁC
(EtOAc–EtOH); 1H NMR (DMSO-d6, 80 ꢁC) d 1.52
(2H, br s), 2.28 (3H, s), 2.72 (2H, t, J = 6.0 Hz), 3.29
(2H, q, J = 6.0 Hz), 6.94–7.29 (6H, m), 8.28 (1H, d,
J = 7.6 Hz), 8.56 (1H, s), 11.26 (1H, br s); FAB MS
m/e (M+H)+ 287. Anal. Calcd for C14H18N6OÆ0.
25H2O: C, 57.82; H, 6.41; N, 28.90. Found: C, 57.65;
H, 6.19; N, 28.94.
5.1.50. 2-[(2-Dimethylaminoethyl)amino]-4-(3-trifluoro-
methylanilino)pyrimidine-5-carboxamide (9e). Compound
9e was prepared from compound 8a and N,N-dimethy-
lethylenediamine in 71% yield as a colorless powder sim-
ilarly: mp 185–187 ꢁC (AcOEt–hexane); 1H NMR
(DMSO-d6) d 2.15 (6H, s), 2.44 (2H, br t, J = 6.4 Hz),
3.37–3.47 (2H, m), 7.33 (1H, br s), 7.36 (1H, d,
J = 7.4 Hz), 7.45 (1H · 0.7, m), 7.53 (1H, t, J = 7.8 Hz),
7.69 (0.7H, br d, J = 7.8 Hz), 7.58–8.25 (1.9H, m), 8.48
5.1.55. 2-(2-Aminoethylamino)-4-(3-methylanilino)pyrim-
idine-5-carboxamide (9j). Compound 9j was prepared
from compound 8d and ethylenediamine in 33% yield
as
a
colorless powder similarly: mp 185–188 ꢁC
(EtOAc–EtOH); 1H NMR (DMSO-d6, 80 ꢁC): 1.44
(2H, br s), 2.30 (3H, s), 2.75 (2H, t, J = 6.4 Hz), 3.33
(2H, td, J = 6.4, 5.8 Hz), 6.83 (1H, d, J = 7.8 Hz), 7.08
(1H, br s), 7.18 (1H, t, J = 7.8 Hz), 7.29 (2H, br s),

H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
4947
7.40–7.70 (2H, m), 8.54 (1H, s), 11.39 (1H, br s); FAB
MS m/e (M+H)+ 287. Anal. Calcd for C14H18N6OÆ0.2-
H2O: C, 58.00; H, 6.40; N, 28.99. Found: C, 57.93; H,
6.39; N, 28.84.
7.59 (1H, t, J = 7.5 Hz), 7.71 (1H, t, J = 2.2 Hz), 7.76
(1H, t, J = 7.8 Hz), 7.98–8.00 (3H, m), 8.52 (1H, d,
J = 8.6 Hz), 9.03 (1H, s), 11.54 (1H, br s); FAB MS m/
e (M+H)+ 303. Anal. Calcd for C14H18N6O2Æ0.7H2O:
C, 53.39; H, 6.21; N, 26.68. Found: C, 53.55; H, 6.33;
N, 26.42.
5.1.56. 2-(2-Aminoethylamino)-4-(4-methylanilino)pyrim-
idine-5-carboxamide (9k). Compound 9k was prepared
from compound 8e and ethylenediamine in 92% yield
similarly: mp 191–193 ꢁC (EtOAc–EtOH); 1H NMR
(DMSO-d6, 80 ꢁC) d 1.54 (2H, br s), 2.27 (3H, s), 2.74
(2H, t, J = 6.4 Hz), 3.32 (2H, q, J = 6.4 Hz), 7.04 (1H,
br s), 7.11 (2H, d, J = 8.4 Hz), 7.28 (2H, br s), 7.56
(2H, d, J = 8.4 Hz), 8.53 (1H, s), 11.32 (1H, br s);
FAB MS m/e (M+H)+ 287. Anal. Calcd for
C14H18N6OÆ0.4H2O: C, 57.28; H, 6.46; N, 28.63. Found:
C, 57.46; H, 6.33; N, 28.61.
5.1.61. 2-(2-Aminoethylamino)-4-(3-ethoxyanilino)pyrim-
idine-5-carboxamide (9p). Compound 9p was prepared
from compound 8j and ethylenediamine in 63% yield
as a pale yellow powder similarly: mp 176–178 ꢁC
1
(MeOH); H NMR (DMSO-d6, 80 ꢁC) d 1.33 (3H, t,
J = 6.8 Hz), 1.48 (2H, br s), 2.75 (2H, t, J = 6.4 Hz),
3.34 (2H, q, J = 6.4 Hz), 4.05 (2H, q, J = 6.8 Hz),
6.57 (1H, dd, J = 8.0, 2.0 Hz), 7.05–7.15 (2H, m),
7.18 (1H, t, J = 8.0 Hz), 7.31 (1H, br s), 7.49 (1H, br
s), 8.55 (1H, br s), 11.47 (1H, br s); FAB MS m/e
(M+H)+ 317; Anal. Calcd for C15H20N6O2Æ1.1H2O:
C, 53.59; H, 6.66; N, 25.00. Found: C, 53.63; H,
6.38; N, 24.73.
5.1.57. 2-(2-Aminoethylamino)-4-(3-cyanolanilino)pyrim-
idine-5-carboxamide (9l). Compound 9l was prepared
from compound 8f and ethylenediamine in 66% yield
as a colorless powder similarly: mp 192–194 ꢁC (2-
1
PrOH); H NMR (DMSO-d6, 80 ꢁC) d 1.45 (2H, br s),
5.1.62. 2-(2-Aminoethylamino)-4-(3-isopropoxyanilino)-
pyrimidine-5-carboxamide (9q). Compound 9q was pre-
pared from compound 8k and ethylenediamine in 72%
yield similarly: mp 143–145 ꢁC (MeOH); 1H NMR
(DMSO-d6, 80 ꢁC) d 1.27 (6H, d, J = 6.0 Hz), 1.31
(2H, br s), 2.74 (2H, t, J = 6.4 Hz), 3.33 (2H, q,
J = 6.4 Hz), 4.58 (1H, qq, J = 6.0 Hz), 6.57 (1H, br d,
J = 8.4 Hz), 7.02–7.14 (2H, m), 7.17 (1H, t,
J = 8.4 Hz), 7.22–7.38 (2H, m), 7.40–7.48 (1H, m), 8.54
(1H, s), 11.45 (1H, br s); FAB MS m/e (M+H)+ 331.
Anal. Calcd for C16H22N6O2: C, 58.17; H, 6.71; N,
25.44. Found: C, 58.34; H, 6.52; N, 25.14.
2.76 (2H, t, J = 6.0 Hz), 3.36 (2H, q, J = 6.0 Hz), 7.33
(1H, br s), 7.40–7.41 (3H, m), 7.49 (1H, t, J = 8.0 Hz),
7.81 (1H, d, J = 8.5 Hz), 8.37 (1H, s), 8.60 (1H, s),
11.71 (1H, br s); FAB MS m/e (M+H)+ 298. Anal. Calcd
for C14H15N7O.1. 2H2OÆ0.25C3H7O: C, 53.09; H, 5.78;
N, 29.38. Found: C, 52.96; H, 5.54; N, 29.25.
5.1.58. 2-(2-Aminoethylamino)-4-(3-ethylanilino)pyrim-
idine-5-carboxamide (9m). Compound 9m was prepared
from compound 8g and ethylenediamine in 29% yield
as
a
colorless powder similarly: mp 149–151 ꢁC
1
(MeOH); H NMR (DMSO-d6, 80 ꢁC) d 1.20 (3H, t,
J = 7.6 Hz), 1.49 (2H, br s), 2.61 (2H, q, J = 7.6 Hz),
2.75 (2H, t, J = 6.4 Hz), 3.34 (2H, q, J = 6.4 Hz), 6.86
(1H, d, J = 7.6 Hz), 7.07 (1H, br s), 7.20 (1H, t,
J = 7.6 Hz), 7.29 (1H, br s), 7.44–7.60 (2H, m), 8.54
(1H, s), 11.40 (1H, br s); FAB MS m/e (M+H)+ 301;
Anal. Calcd for C15H20N6OÆ0.5H2O: C, 58.24; H, 6.84;
N, 27.17. Found: C, 58.13; H, 6.76; N, 27.02.
5.1.63. 2-(2-Aminoethylamino)-4-(3-bromoanilino)pyrim-
idine-5-carboxamide (9r). Compound 9r was prepared
from compound 8l and ethylenediamine in 71% yield
similarly: mp 194–196 ꢁC (EtOAc–EtOH); 1H NMR
(DMSO-d6, 80 ꢁC) d 1.37 (2H, br s), 2.76 (2H, t,
J = 6.4 Hz), 3.33 (2H, q, J = 6.4 Hz), 7.15–7.20 (1H,
m), 7.20–7.25 (1H, m), 7.25 (1H, t, J = 6.0 Hz), 7.37
(2H, br s), 7.49 (1H, br s), 8.20 (1H, br s), 8.58 (1H,
s), 11.61 (1H, br s); FAB MS m/e (M+H)+ 351, 353.
Anal. Calcd for C13H15N6OBrÆ0.25H2O: C, 43.90; H,
4.39; N, 23.63; Br, 22.46. Found: C, 43.84; H, 4.18; N,
23.44; Br, 22.19.
5.1.59.
2-(2-Aminoethylamino)-4-(3-isopropylanilino)-
pyrimidine-5-carboxamide (9n). Compound 9n was pre-
pared from compound 8h and ethylenediamine in 9%
yield as a yellow powder similarly: mp 160–162 ꢁC
1
(MeOH); H NMR (DMSO-d6, 80 ꢁC) d 1.22 (6H, d,
J = 6.8 Hz), 1.29 (2H, br s), 2.74 (2H, t, J = 6.4 Hz),
2.89 (1H, qq, J = 6.8 Hz), 3.34 (2H, q, J = 6.4 Hz),
6.80 (1H, d, J = 8.0 Hz), 7.05 (1H, br s), 7.21 (1H, t,
J = 8.0 Hz), 7.29 (1H, br s), 7.42–7.62 (2H, m), 8.54
(1H, s), 11.41 (1H, br s); FAB MS m/e (M+H)+ 315.
Anal. Calcd for C16H22N6OÆ0.5H2O: C, 59.42; H, 7.17;
N, 25.99. Found: C, 59.39; H, 7.39; N, 26.02.
5.1.64. Ethyl-2-methylsulfanyl-4-(3-trifluoromethylani-
lino)pyrimidine-5-carboxylate (11a). Compound 11a
was prepared from ethyl-4-chloro-2-methylsulfanylpyr-
imidine-5-carboxylate (10) and m-aminobenztrifluoride
according to the same procedure as that for 2a in 45%
1
yield as a pale brown amorphous powder similarly: H
NMR (CDCl3) d 1.42 (3H, t, J = 6.9 Hz), 2.57 (3H, s),
4.41 (2H, q, J = 6.9 Hz), 7.39 (1H, br d, J = 7.8 Hz),
7.47 (1H, t, J = 7.8 Hz), 7.65 (1H, br d, J = 7.8 Hz),
8.33 (1H, s), 8.82 (1H, s), 10.62 (1H, br s); FAB MS
m/e (M+H)+ 358.
5.1.60.
2-(2-Aminoethylamino)-4-(3-methoxyanilino)-
pyrimidine-5-carboxamide (9o). Compound 9o was pre-
pared from compound 8i and ethylenediamine in 70%
yield as a pale yellow powder similarly: mp 142–
145 ꢁC (EtOH); 1H NMR (DMSO-d6, 80 ꢁC) d 1.37
(2H, br s), 2.74 (2H, t, J = 6.4 Hz), 3.34 (2H, q,
J = 6.4 Hz), 3.86 (3H, s), 6.78 (1H, dd, J = 8.6,
2.2 Hz), 7.12–7.16 (1H, m), 7.32 (1H, t, J = 8.1 Hz),
5.1.65. Ethyl-4-methylamino-2-methylsulfanylpyrimidine-
5-carboxylate (11b). Compound 11b was prepared from
ethyl-4-chloro-2-methylsulfanylpyrimidine-5-carboxyl-
ate (10) and methylamine in 84% yield as a colorless

4948
H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
1
amorphous powder similarly: H NMR (CDCl3) d 1.37
(3H, t, J = 7.2 Hz), 2.53 (3H, s), 3.09 (3H, s), 4.33
(2H, q, J = 6.9 Hz), 8.17 (1H, br s), 8.51 (1H, s); FAB
MS m/e (M+H)+ 228.
(1H, s), 8.82 (1H, br d, J = 4.4 Hz), 11.59 (1H, br s);
FAB MS m/e (M+H)+ 343.
5.1.74.
N,N-Dimethyl-2-methylsulfanyl-4-(3-trifluoro-
methylanilino)pyrimidine-5-carboxamide (13e). Com-
pound 13e was prepared from compound 12a and
dimethylaminehydrochloride in 100% yield as a color-
5.1.66. Ethyl-4-cyclohexylamino-2-methylsulfanylpyrimi-
dine-5-carboxylate (11c). Compound 11c was prepared
from ethyl-4-chloro-2-methylsulfanylpyrimidine-5-car-
boxylate (10) and cyclohexylamine. This compound
was used for next reaction without purification.
1
less amorphous powder similarly. H NMR (DMSO-
d6) d 2.43 (3H, s), 3.00 (6H, s), 7.42 (1H, d,
J = 7.8 Hz), 7.57 (1H, t, J = 7.8 Hz), 7.84 (1H, d,
J = 7.8 Hz), 8.21 (1H, s), 8.23 (1H, s), 9.37 (1H, br s);
FAB MS m/e (M+H)+ 357.
5.1.67.
2-Methylsulfanyl-4-(3-trifluoromethylanilino)
pyrimidine-5-carboxylic acid (12a). Compound 12a was
prepared according to the same procedure as that for
3a in 79% yield as a pale brown amorphous powder:
1H NMR (DMSO-d6) d 2.51 (3H, s), 7.48 (1H, d,
J = 8.1 Hz), 7.61 (1H, t, J = 7.8 Hz), 7.79 (1H, d,
J = 8.1 Hz), 8.39 (1H, s), 8.78 (1H, s), 10.72 (1H, br s);
FAB MS m/e (M+H)+ 330.
5.1.75. 2-(3-Aminopropylamino)-4-(3-trifluoromethylani-
lino)pyrimidine-5-carboxamide (14a). Compound 14a
was prepared from compound 13a and 1,3-diaminopro-
pane according to the same procedure as that for 9a in
76% yield as a colorless powder: mp 185–189 ꢁC
1
(AcOEt–hexane); H NMR (DMSO-d6) 1.58–1.72 (2H,
m), 2.58–2.69 (2H, m), 3.30–3.46 (2H, m), 7.35 (1H, d,
J = 7.6 Hz), 7.54 (1H, t, J = 7.6 Hz), 7.25–8.30 (5H,
m), 8.61 (0.8H, s), 8.63 (0.2H, s), 11.78 (0.2H, br s),
11.96 (0.8H, br s); FAB MS m/e (M+H)+. Anal. Calcd
for C15H17N6OF3Æ1.5H2O: C, 47.24; H, 5.29; N, 22.04;
F, 14.95. Found: C, 47.75; H, 5.02; N, 21.61; F, 14.95.
5.1.68. 4-Methylamino-2-methylsulfanylpyrimidine-5-car-
boxylic acid (12b). Compound 12b was prepared in 94%
yield as a colorless amorphous powder similarly: 1H
NMR (DMSO-d6) d 2.98 (3H, s), 8.41 (1H, br s), 8.50
(1H, s); FAB MS m/e (M+H)+ 200.
5.1.69. 4-Cyclohexylamino-2-methylsulfanylpyrimidine-5-
carboxylic acid (12c). Compound 12c was prepared
similarly. This compound was used for next reaction
without purification.
5.1.76. 2-(2-Hydroxyethylamino)-4-(3-trifluoromethylani-
lino)pyrimidine-5-carboxamide (14b). Compound 14b
was prepared from compound 13a and 2-aminoethanol
in 67% yield as a colorless powder similarly: mp 216–
1
219 ꢁC (AcOEt–hexane); H NMR (DMSO-d6) d 3.35–
5.1.70.
2-Methylsulfanyl-4-(3-trifluoromethylanilino)-
3.44 (2H, m), 3.50–3.62 (2H, m), 4.67 (0.7H, br t,
J = 4.8 Hz), 4.70–4.78 (0.3H, m), 7.35 (1H, d,
J = 7.3 Hz), 7.25–8.15 (5.3H, m), 8.53 (0.7H, s), 8.61
(0.7H, s), 8.63 (0.3H, s), 11.79 (0.3H, br s), 11.93
(0.7H, br s); FAB MS m/e (M+H)+ 342. Anal. Calcd
for C14H14N5O2F3Æ0.5H2O: C, 48.00; H, 4.32; N, 19.99;
F, 16.27. Found: C, 47.78; H, 4.27; N, 19.79; F, 16.09.
pyrimidine-5-carboxamide (13a). Compound 13a was
prepared from compound 12a according to the same
procedure as that for 4a in 91% yield as a pale yellow
1
amorphous powder: H NMR (DMSO-d6) d 2.52 (3H,
s), 7.44 (1H, d, J = 6.9 Hz), 7.60 (1H, t, J = 7.5 Hz),
7.72 (1H, d, J = 7.5 Hz), 7.81 (1H, br s), 8.34 (1H, s),
8.42 (1H, s), 8.77 (1H, s), 10.72 (1H, br s); FAB MS
m/e (M+H)+ 329.
5.1.77.
2-(2-Aminoethylamino)-4-methylaminopyrim-
idine-5-carboxamide (14c). Compound 14c was prepared
from compound 13b and ethylenediamine in 28% yield
similarly: mp 136–138 ꢁC (AcOEt); H NMR (DMSO-
d6, 80 ꢁC) d 2.72 (2H, t, J = 6.4 Hz), 2.88 (3H, s), 3.30
(2H, q, J = 6.4 Hz), 6.70 (1H, br s), 6.98 (1H, br s),
8.33 (1H, s), 8.72 (1H, br s); FAB MS m/e (M+H)+
311. Anal. Calcd for C8H14N6OÆ0.75H2O: C, 42.94; H,
6.98; N, 37.56. Found: C, 43.01; H, 6.88; N, 37.56.
5.1.71. 4-Methylamino-2-methylsulfanylpyrimidine-5-car-
boxamide (13b). Compound 13b was prepared in 43%
yield as a colorless amorphous powder similarly: 1H
NMR (DMSO-d6) d 2.49 (3H, s), 2.93 (3H, s), 7.40
(1H, br s), 7.99 (1H, br s), 8.48 (1H, s), 8.92 (1H, br
s); FAB MS m/e (M+H)+ 199.
1
5.1.72. 4-Cyclohexylamino-2-methylsulfanylpyrimidine-5-
carboxamide (13c). Compound 13c was prepared in 72%
as a colorless amorphous powder yield similarly: 1H
NMR (DMSO-d6) d 1.20–1.44 (5H, m), 1.50–1.74 (3H,
m), 1.83–1.95 (2H, m), 2.44 (3H, s), 3.90–4.03 (1H, m),
7.42 (1H, br s), 8.01 (1H, br s), 8.49 (1H, s), 9.13 (1H,
br d, J = 7.6 Hz); FAB MS m/e (M+H)+ 267.
5.1.78. 2-(2-Aminoethylamino)-4-cyclohexylaminopyrim-
idine-5-carboxamide (14d). Compound 14d was prepared
from compound 13c and ethylenediamine in 47% yield
similarly: mp 161–164 ꢁC (EtOH); H NMR (DMSO-
d6) d 1.10–1.72 (8H, m), 1.76–1.95 (2H, m), 2.60–2.70
(2H, m), 3.17–3.27 (2H, m), 3.28–3.36 (2H, m), 3.86–
3.97 (1H, m), 6.88 (1H, br s), 7.06 (1H, br s), 7.32–
7.80 (1H, br s), 8.34 (1H, s), 8.90–9.07 (1H, m); FAB
MS m/e (M+H)+ 279. Anal. Calcd for C13H22N6OÆ0.
5H2O: C, 54.34; H, 8.07; N, 29.25. Found: C, 54.59;
H, 7.97; N, 29.16.
1
5.1.73. N-Methyl-2-methylsulfanyl-4-(3-trifluoromethyl-
anilino)pyrimidine-5-carboxamide (13d). Compound 13d
was prepared from compound 12a and methylamine in
1
97% yield as a colorless amorphous solid similarly: H
NMR (DMSO-d6) d 2.51 (3H, s), 2.82 (3H, d,
J = 4.4 Hz), 7.44 (1H, d, J = 7.9 Hz), 7.59 (1H, t,
J = 7.8 Hz), 7.73 (1H, d, J = 7.8 Hz), 8.42 (1H, s), 8.69
5.1.79. 2-(2-Aminoethylamino)-N-methyl-4-(3-trifluorome-
thylanilino)pyrimidine-5-carboxamide (14e). Compound

H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
4949
14e was prepared from compound 13d and ethylenedia-
mine in 80% yield as a pale yellow solid similarly: mp
175–176 ꢁC (EtOH); H NMR (DMSO-d6) d 1.69 (2H,
br s), 2.72 (2H, t, J = 5.9 Hz), 2.77 (3H, d, J = 4.4 Hz),
3.27–3.36 (2H, m), 7.35 (1H, d, J = 7.3 Hz), 7.50–7.70
(2H, m), 8.39 (1H, m), 8.55 (1H, s), 8.59 (1H, s), 11.81
(1H, br s); FAB MS m/e (M+H)+ 355. Anal. Calcd for
C15H17N6OF3Æ0.75H2O: C, 48.98; H, 5.07; N, 22.85; F,
15.49. Found: C, 48.75; H, 4.75; N, 22.70; F, 15.55.
PKCe were purchased from Calbiochem. Biotinylated
substrate peptides were as follows: a peptide from hu-
man Band 3 (MEELQDDYEDMMEENL) for Syk
and ZAP-70 assays; a peptide from human SLP-76
(GEDDGDYESP NEEEE) for Itk and Btk assays. In
the cases of PKCb2 and PKCe , PKC ꢀpseudosubstrateꢁ
peptide (ERMRPR KRQGSVRRRV) was used.
1
Kinase activity was measured by using a scintillation
proximity assay (SPA) system. SPA is a system that
has been developed by Amersham making use of a phe-
nomenon in which scintillation occurs when a molecule
having radioactivity is in proximity to the surface of
plastic beads having a scintillant included therein. These
beads are coated in advance with streptoavidin to which
the biotin moiety of substrate peptide is bound. A 2 lM
portion of DMSO solution of each compound to be
tested was added to each well containing 50 ll of a reac-
tion solution [composition: 20–200 ng of recombinant
kinase, 50 mM Tris–HCl (pH 7–8), 10 mM MgCl2 or
MnCl2, 50 mM NaCl, 1 mM DTT, optimum concentra-
5.1.80.
fluoromethylanilino)pyrimidine-5-carboxamide
2-(2-Aminoethylamino)-N,N-dimethyl-4-(3-tri-
(14f).
Compound 14f was prepared from compound 13e and
ethylenediamine in 58% yield similarly: mp 115–116 ꢁC
1
(AcOEt–hexane); H NMR (DMSO-d6) d 1.48 (2H, br
s), 2.72 (2H, t, J = 5.9 Hz), 3.01 (6H, s), 3.29 (2H, q,
J = 5.9 Hz), 7.34 (1H, d, J = 7.8 Hz), 7.52 (1H, t,
J = 7.9 Hz), 7.70–7.80 (1H, m), 8.10 (1H, s), 8.40–8.50
(1H, m), 9.60 (1H, br s); FAB MS m/e (M+H)+ 369.
Anal. Calcd for C16H19N6OF3Æ0.75H2O: C, 50.32; H,
5.41; N, 22.01; F, 14.92. Found: C, 50.52; H, 5.23; N,
22.22; F, 14.90.
33
tion of the substrate peptide, and 0.1 lCi [cꢀ P]ATP
(10 mCi/mL, Amersham)]. This was prepared in Opti-
plateTM (PACKARD) and allowed to stand at room
temperature for 1 h to effect tyrosine phosphorylation.
The reaction was terminated by adding PBS containing
0.25 mg SPA beads, 50 lM ATP, 5 mM EDTA, and 1%
Triton X-100 in an amount of 150 ll per well. The plate
was sealed, stirred, allowed to stand at room tempera-
ture for 15 min, and then centrifuged at 1500 rpm for
3 min to effect precipitation of the SPA beads. Radio-
activity of each well was measured using TOP COUNT
(PACKARD), and the tyrosine phosphorylation activity
by the kinases was calculated.
5.1.81. Ethyl-2-(2-aminoethylamino)-4-(3-trifluoromethyl-
anilino)pyrimidine-5-carboxylate (15). Compound 15
was prepared from compound 6a and ethylenediamine
in 64% yield similarly: mp 147–151 ꢁC (EtOH); 1H
NMR (DMSO-d6) d 1.34 (3H, t, J = 7.3 Hz), 3.01
(2H, br q, J = 5.4 Hz), 3.53–3.66 (2H, m), 4.34 (2H,
q, J = 7.3 Hz), 7.44 (0.3H, d, J = 7.8 Hz), 7.51 (0.7H,
d, J = 7.8 Hz), 7.58 (0.3H, t, J = 7.8 Hz), 7.65 (0.7H,
t, J = 7.8 Hz), 7.88–8.35 (5H, m), 8.68 (0.3H, s), 8.70
(0.7H, s), 10.39 (0.3H, br s), 10.59 (0.7H, br s); FAB
MS m/e (M+H)+ 370. Anal. Calcd for C16H18N5O2
F3Æ2HCl: C, 43.45; H, 4.56; N, 15.84; Cl, 16.03; F,
12.89. Found: C, 43.33; H, 4.55; N, 15.81; Cl, 16.04;
F, 13.09.
5.2.2. 5-HT release assay. This test was carried out in
accordance with the method reported by Collado-Esco-
bar et al. RBL-2H3 cells were preincubated with tritium-
labeled 5-HT at 37 ꢁC overnight, followed by priming
with dinitrophenyl (DNP)-specific monoclonal IgE anti-
body. The cells were reseeded in 96-well plates and incu-
bated with or without compounds, followed by
stimulation with DNP conjugated with bovine serum
albumin (DNP–BSA, 0.1 lg/mL). Aliquots of culture
supernatant were added to MicroScinti-20 and its radio-
activity was measured.
5.1.82. 2-(2-Aminoethylamino)-4-(3-trifluoromethylani-
lino)pyrimidine-5-carboxylic acid (16). Compound 16
was prepared according to the same procedure as that
for 3 in 72% yield as a colorless solid: mp 248–249 ꢁC
(H2O–EtOH); 1H NMR (DMSO-d6) d 3.07 (2H, m),
3.20–3.50 (2H, m), 3.55 (2H, m), 7.12 (1H, br s), 7.23
(1H, d, J = 7.8 Hz), 7.46 (1H, t, J = 7.8 Hz), 7.50–8.50
(3H, m), 7.74 (1H, m), 8.48 (1H, m), 13.48 (1H, br s);
FAB MS m/e (M+H)+ 342. Anal. Calcd for
C14H14N5O2F3Æ0.2H 2O: C, 48.76; H, 4.21; N, 20.31;
F, 16.53. Found: C, 48.71; H, 4.17; N, 20.41; F, 16.52.
5.2.3. Passive cutaneous anaphylaxis (PCA) assays. ICR
mice were passively sensitized by subcutaneously inject-
ing anti-dinitrophenyl (DNP)-coupled IgE under the
right ear pinna, while lightly anesthetizing with ether.
After 24hr, each mouse was challenged by injecting a
mixture of DNP-conjugated bovine serum albumin
and 200 ll of 0.5% Evans blue solution via the tail vein
to induce passive cutaneous anaphylaxis. Thirty minutes
after the challenge, the mice were sacrificed to take both
ears and the amount of dye from the blueing region was
measured. Test compounds or vehicle alone as a control
were subcutaneously administered to the mice 30 min
before the antigen challenge. The dye in the tissues
was extracted with formamide and colorimetrically
determined at 620 nm. A value obtained by subtracting
the dye content of the left ear from the dye content of
5.2. Biology
5.2.1. Kinase assay. The genes encoding human Syk,
ZAP-70, Itk, and Btk were subcloned into pFastBac1
vector (GIBCO) in which FLAG-tag was incorporated
or pFastBac HT vector (GIBCO). Recombinant baculo-
viruses were obtained by using Bac-to-Bac system
(GIBCO). Sf-9 insect cells (American Type Culture Col-
lection) were infected with the recombinant viruses and
grown in spinner cultures. FLAG- or 6xHis-tagged pro-
teins were partially purified by M2-agarose affinity
gel (SIGMA) or TALON metal affinity resin
(CLONTECH). Recombinant human PKCb2 and

4950
H. Hisamichi et al. / Bioorg. Med. Chem. 13 (2005) 4936–4951
the right ear was used as the amount of dye leaked into
the tissues by the PCA reaction. The PCA inhibition
ratio by the test compound was calculated based on
the following equation. In the formula, CA: amount of
dye leaked into the sensitized right ear at the time
of administration of the vehicle alone, CB: amount of
dye leaked into the unsensitized left ear at the time of
administration of vehicle alone, XA: amount of dye
leaked into the sensitized right ear at the time of admin-
istration of the compound to be tested, and XB: amount
of dye leaked into the unsensitized left ear at the time of
administration of the compound to be tested. Inhibition
ratio (%) = {(CA-CB)-(XA-XB)} · 100/(CA-CB)
Computing Group Inc., Montreal, Quebec), with the
crystal structure of LCK31 (PDB code: 3lck) as a tem-
plate. To remove side-chain close contacts, the obtained
model was finally refined using the Tripos force fieldꢁs
minimization32 in which the backbone of the protein
was held fixed. Docking studies of compound 9a and
AMP-PNP with modeled catalytic domain of Syk were
performed using a program, GOLD.33 The 10 indepen-
dent genetic algorithms (GA) in which a maximum num-
ber of 100,000 GA operations were performed on a
single population of 100 individuals were calculated.
Operator weights for crossover, mutation, and migra-
tion were set to 95, 95, and 10, respectively.
5.2.4. Single-crystal X-ray diffraction analysis of com-
pound 9q. A colorless prismatic crystal of compound 9q
methanolate, C16H22N6O2ÆCH3OH (F.W. = 362.43,
0.50 mm · 0.35 mm · 0.30 mm), was mounted on a glass
fiber. All measurements were made on a Rigaku AFC7R
diffractometer with graphite monochromated CuKa radi-
ation and a rotating anode generator. Cell constants and
an orientation matrix for data collection, obtained from a
least-squares refinement using the setting angles of 25
carefully centered reflections in the range 58.26 <
2h < 60.17 ꢁ, corresponded to a primitive monoclinic cell
Acknowledgments
The authors deeply acknowledge Dr. Nobuaki Tanigu-
chi and Dr. Kiyohiro Samizu for useful discussion and
helpful support for preparing the manuscript, and are
also grateful to the staff of the Division of Analytical
Science Laboratories for the elemental analysis and
spectral measurements.
˚
(P21/n, For Z = 4) with dimensions: a = 17.772(3) A,
3
˚
˚
˚
b = 10.812(3) A, c = 10.286(3) A, V = 1976.2(7) A , the
calculated density: 1.22 g/cm3. The data were collected
at a temperature of 298 1 K using the x-2h scan tech-
nique to a maximum 2h value of 154.5ꢁ.
References and notes
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No decay correction was applied. The linear absorption
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ical absorption correction based on azimuthal scans
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The structure was solved by direct methods23 and ex-
panded using Fourier techniques.24 The non-hydrogen
atoms were refined anisotropically. Hydrogen atoms
were included but not refined. The final cycle of full-
matrix least-squares refinement25 was based on 2225
observed reflections (I > 3.00r(I), 2h < 154.51ꢁ) and 237
variable parameters, and converged (largest parameter
shift was 0.00 times its esd) with unweighted and
weighted agreement factors of: R = 0.066, Rw = 0.111,
respectively. The goodness of fit26 was 1.34. The maxi-
mum and minimum peaks on the finalꢀdifference Fourier
3
˚
map corresponded to 0.22 and ꢀ0.24 e /A , respectively.
Neutral atom scattering factors were taken from Cromer
and Waber.27 The values for the mass attenuation coeffi-
cients are those of Creagh and Hubbel.28 All calculations
were performed using the teXsan29 crystallographic soft-
ware package of Molecular Structure Corporation.
5.2.5. Molecular modeling and docking study. A 3D
model of the catalytic domain of the human Syk has
been constructed using a program, MOE30 (Chemical
13. Bredereck, H.; Effenberger, F.; Schweizer, E. H. Chem.
Ber. 1962, 95, 803–809.

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4951
14. Palanki, M. S. S.; Erdman, P. E.; Manning, A. M.; Ow,
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ATP competitor. Ichikawa, A.; Orita, A.; Hisamichi, H.;
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DIRDIF-94 program system. Technical Report of the
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25. Least-squares: function minimized:
P
2
wðjF oj ꢀ jF cjÞ
where w ¼ 1=½r2ðF oÞꢁ ¼ ½rc2ðF oÞ þ p2F 2o=4ꢁꢀ1
,
esd based on counting statistics, p = p-factor (0.1270).
rc(Fo) =
26. Standard deviation of an observation of unit weight:
P
2
½
wðjF oj ꢀ jF cjÞ ðNo ꢀ NvÞꢁ1=2, where: No = number of
observations, Nv = number of variables.
27. Cromer, D. T.; Waber, J. T. In International Tables for
X-ray Crystallography; The Kynoch Press: Birmingham,
England, 1974; Vol. IV, Table 2.2 A.
28. Creagh, D. C; Hubbell, J. H. In International Tables
for Crystallography; Wilson, A. J. C., Ed.; Kluwer
Academic: Boston, 1992; Vol C, Table 4.2.4.3, 200–
206.
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Chem. 1993, 36, 3015–3020.
22. Recently, the X-ray structure of the unphosphorylated
form of Syk: Atwell, S.; Adams, J. M.; Badger, J.;
Buchanan, M. D.; Feil, I. K.; Froning, K. J.; Gao, X.;
29. teXsan: Crystal Structure Analysis Package, Molecular
Structure Corporation (1985&1999).
30. MOE revision 2003.02; Chemical Computing Group:
Montreal, 2003.
Hendle, J.; Keegan, K.; Leon, B. C.; Muller-Dieckmann,
¨
H. J.; Nienaber, V. L.; Noland, B. W.; Post, K.;
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31. Hubbard, S. R. EMBO J. 1997, 16, 5572–5581.
32. SYBYL revision 6.8, Tripos: St. Louis, 2002
33. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor,
R. J. Mol. Biol. 1997, 267, 727–748.