A novel Syk family kinase inhibitor: Design, synthesis, and structure-activity relationship of 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmc.2008.06.017

Splenic tyrosine kinase (Syk) family kinases, which are members of the protein tyrosine kinase family, play crucial roles in immune responses, with Syk participating in B-cell activation and the zeta-associated protein 70 kDa (ZAP-70) kinase being involved in T-cell activation. Therefore, Syk family kinase inhibitors are candidate therapeutic agents for the treatment of various allergic disorders and autoimmune diseases. We designed 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives as Syk family kinase inhibitors, based on literature reports and structure-based drug design. These derivatives showed significant Syk inhibitory activities, with ZAP-70 inhibition. Representative compounds 10d and 11 not only exhibited strong inhibition of both Syk and ZAP-70 kinase but also suppressed IL-2 production by peripheral blood mononuclear cells and whole blood.

Full text of DOI:10.1016/j.bmc.2008.06.017

Bioorganic & Medicinal Chemistry 16 (2008) 7347–7357
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel Syk family kinase inhibitor: Design, synthesis, and structure–activity
relationship of 1,2,4-triazolo[4,3-c]pyrimidine and
1,2,4-triazolo[1,5-c]pyrimidine derivatives
*
Akihito Hirabayashi , Harunobu Mukaiyama, Hiroaki Kobayashi, Hiroaki Shiohara, Satoko Nakayama,
Motoyasu Ozawa, Keiji Miyazawa, Keiko Misawa, Hideki Ohnota, Masayuki Isaji
Central Research Laboratory, Kissei Pharmaceutical Company Ltd, 4365-1 Kashiwabara, Hotaka, Azumino-City, Nagano-Pref 399-8304, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Splenic tyrosine kinase (Syk) family kinases, which are members of the protein tyrosine kinase family,
play crucial roles in immune responses, with Syk participating in B-cell activation and the zeta-associated
protein 70 kDa (ZAP-70) kinase being involved in T-cell activation. Therefore, Syk family kinase inhibitors
are candidate therapeutic agents for the treatment of various allergic disorders and autoimmune dis-
eases. We designed 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine derivatives as
Syk family kinase inhibitors, based on literature reports and structure-based drug design. These deriva-
tives showed significant Syk inhibitory activities, with ZAP-70 inhibition. Representative compounds 10d
and 11 not only exhibited strong inhibition of both Syk and ZAP-70 kinase but also suppressed IL-2 pro-
duction by peripheral blood mononuclear cells and whole blood.
Received 9 April 2008
Revised 10 June 2008
Accepted 11 June 2008
Available online 14 June 2008
Keywords:
Protein tyrosine kinases (PTKs)
Syk
ZAP-70
Ó 2008 Elsevier Ltd. All rights reserved.
Syk and/or ZAP-70 kinase inhibitor
1. Introduction
1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine
derivatives as Syk family kinase inhibitors.
The splenic tyrosine kinase (Syk) family kinases are cytosolic
non-receptor tyrosine kinases, represented by Syk and zeta-associ-
ated protein 70 kDa (ZAP-70).1,2 Syk is present in platelets, B lym-
phocytes, mast cell, basophils, neutrophils, dendritic cells,
macrophages, and monocytes. Syk kinase has functional roles in
the signal transduction mediated by immunoreceptor tyrosine-
based activation motifs expressed in various hematopoietic cells.
ZAP-70 is found in T lymphocytes and natural killer cells. Syk fam-
ily kinases play important roles in immune responses, and are in-
volved in inflammatory and autoimmune diseases.3–5 Recently, it
has been reported that several Syk inhibitors are effective in the
treatment of inflammation and autoimmune diseases.6–10 Thus,
Syk family tyrosine kinase inhibitors are candidate therapeutic
agents for the treatment of various allergic and autoimmune
disorders.
In our search for protein tyrosine kinase inhibitors, we discov-
ered the following compounds, based on literature reports and
structure-based drug design (SBDD): 1,2,4-triazolo[4,3-c]pyrimi-
dine and 1,2,4-triazolo[1,5-c]pyrimidine.11 These compounds
showed highly potent inhibitory activities for Syk and/or ZAP-70
kinase. In the present study, we describe the discovery process
and investigate the structure–activity relationships (SARs) of
2. Chemistry
To prepare the 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazol-
o[1,5-c]pyrimidine derivatives, we selected 5-cyano-2,4,6-trichlo-
ropyrimidine 4 as the starting material and used the synthetic
route shown in Scheme 1. Substitution of compound 4 with 3,5-
dimethoxyaniline in THF at ꢀ78 °C gave the 6-anilinopyrimidine
derivative 5, along with the undesired 4-anilinopyridine derivative.
These compounds were easily separated by crystallization using
appropriate solvents. Compound 5 was reacted with various
amines to afford 6a–g. Protection of the hydroxyl group of 6b with
tert-butyldimethylsilyl (TBS) group gave 6c. Each compound was
treated with hydrazine in THF, and the corresponding intermediate
was heated with trimethylorthoformate to afford 1,2,4-triazolo-
[4,3-c]pyrimidine derivatives 8a–f.12 Hydrolysis of the nitrile
group using 30% H2O2 and 5 M NaOH generated the carbamoyl
derivatives 9a–f. Furthermore, removal of the Boc groups from
compounds 9c–f was achieved by treatment with 4 M HCl–AcOEt,
to give compounds 10a–d.
Synthesis of 1,2,4-triazolo[1,5-c]pyrimidine derivative 11 was
readily achieved by the Dimroth rearrangement under the heating
conditions illustrated in Scheme 2.13 The 2-phenyl-1,2,4-triazolo
[4,3-c]pyrimidine derivative 13a and 2-phenyl-1,2,4-triazolo[1,5-c]
pyrimidine derivatives 14a–c were synthesized according to the
* Corresponding author. Tel.: +81 263 82 8820; fax: +81 263 82 8827.
E-mail address: akihito_hirabayashi@pharm.kissei.co.jp (A. Hirabayashi).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.017

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A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
Scheme 1. Reagents: (a) 3,5-dimethoxyaniline, i-Pr2NEt, THF; (b) RNH2, i-Pr2NEt, THF; (c) TBSCl, imidazole, DMF; (d) NH2NH2H2O, THF; (e) HC(OMe)3; (f) 30% H2O2, 5 M
NaOH, DMSO, EtOH; (g) 1 M TBAF, THF; (h) 4 M HCl–AcOEt.
deprotection of the Boc group of 12a under acidic conditions. Dim-
roth rearrangement products were synthesized in accordance with
the following method. Removal of the Boc group from 12a–c was
achieved by treating under acidic conditions, and the crude com-
pounds were heated in DMF, to give 2-phenyl-1,2,4-triazolo[1,5-
c]pyrimidine derivatives 14a–c. The chemical structures of the
synthesized compounds were determined by spectroscopy (1H
NMR, mass spectrometry) and elemental analysis.
Scheme 2. Reagent and condition: (a) DMF, 100 °C.
3. Results and discussion
method shown in Scheme 3. Compound 6g was treated with
hydrazine monohydrate in THF and was mixed with the corre-
sponding benzaldehyde derivatives in MeCN. These mixtures were
hydrolyzed with 30% H2O2 and 5 M NaOH, and the resulting car-
bamoyl products were treated with diisopropyl azodicarboxylate
(DIAD) in MeCN, to provide the 3-phenyl-1,2,4-triazolo[4,3-
c]pyrimidine derivatives 12a–c. Compound 13a was prepared by
3.1. Design of novel Syk family tyrosine kinase inhibitors
Initially, we sought lead compounds based on a combination of
literature reports and SBDD. At that time, there was little structural
information available on human ZAP-7014 and Syk.15 A comparison
of the primary amino acid sequence of the ZAP-70 kinase domain
with Lck revealed approximately 34% homology, and a comparison

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
7349
Scheme 3. Reagents and condition: (a) NH2NH2H2O, THF; (b) ArCHO, MeCN; (c) DIAD, Ph3P, MeCN; (d) 30% H2O2, 5 M NaOH, DMSO, EtOH; (e) 4 M HCl–AcOEt; (f) DMF, 100 °C.
of the ATP-binding site between ZAP-70 and Lck revealed approx-
imately 57% homology.16 Given the relatively high degree of
homology at the ATP-binding site between Lck and ZAP-70 (or
Syk), we constructed a ZAP-70 homology model, based on the pub-
lished crystal structure of the activated Lck kinase domain,16,17
using FAMS.18 The binding properties of several compounds de-
rived from the model were examined using the ADAM software.19
Initially, we adopted the well-known tyrosine kinase inhibitor
staurosporine 1,20 which showed strong inhibitory activities for
both Syk and ZAP-70 kinase in our evaluation (Table 1). Stauro-
sporine 1 also demonstrated strong suppression of IL-2 production
by peripheral blood mononuclear cells (PBMCs) and by whole
blood (Table 5), as well as in an in vivo murine model (data not
shown). Several research groups have reported Syk kinase inhibi-
tors.21 Researchers at Astellas Pharmaceutical have reported that
the potent and selective low-molecular-weight Syk kinase inhibi-
tor 2 is highly potent in vitro and efficacious against the anaphy-
laxis reaction in vivo.9 We compared staurosporine 1 with the
4-anilinopyrimidine-5-carboxamide derivative 2 in the ZAP-70
receptor model, to identify structural differences in their inhibition
profiles. The binding modes of the ATP-binding site on stauro-
sporine 1 and compound 2 are shown in Figure 1.22,9 We examined
the regions surrounding the adenosine pockets, namely sites A, B,
and C.14 After the binding of staurosporine 1, sites A and B formed
of site B formed a CH-p interaction with the methylene groups of
Leu344 and Gly420 (Fig. 1B). Site C corresponded to a sugar pocket
based on the crystal structures of ATP-containing analogues. In the
binding mode, staurosporine 1 effectively occupied all the A, B, and
C sites, whereas compound 2 was shared at sites B and C and did
not occupy site A. The inhibitory activity of compound 2 for ZAP-
70 was extremely low compared with that for Syk kinase. We
hypothesized that the occupation of all the A, B, and C sites was
required to inhibit both the Syk and ZAP-70 kinases via hydro-
gen-bonding interactions and CH-p interactions. Based on this
hypothesis, we designed the virtual 1,2,4-triazolo[4,3-c]pyrimidine
derivative 3 (Fig. 1). Examination of the binding mode between
ZAP-70 and compound 3 revealed that (1) the N–H group and car-
bonyl group of the 8-carbamoyl group interacted with the carbonyl
group of Glu415 and the N–H group of Ala417, respectively; (2) the
N–H group of the aniline formed a hydrogen bond with the car-
bonyl group of Ala417, similar to compound 2; (3) the triazole core
of compound 3 occupied site A to form a CH-
methyl group of Val352; and (4) the 7-anilino group of compound
3 on site B formed a CH- interaction with the methylene groups of
p interaction with the
p
Leu344 and Gly420. Regarding substituents on the 7-anilino group,
several literature reports have pointed out that meta substituents
were favorable for Syk inhibition.9,24,25 Thus, we investigated pri-
marily meta-substituted benzene derivatives. Moreover, it was
conceivable that the cycloalkyldiamino group at the 5-position of
1,2,4-triazolo[4,3-c]pyrimidine core contributed to effective occu-
pation of site C, similar to the cyclohexane core of staurosporine
1. In fact, the cyclohexyldiamino derivative showed excellent Syk
inhibitory activity.26 Based on these considerations, we synthe-
sized 1,2,4-triazolo[4,3-c]pyrimidine derivatives such as virtual
compound 3 and 1,2,4-triazolo[1,5-c]pyrimidine derivatives, and
studied their SARs.
a CH-
or methyl groups. Thus, the indole skeleton at site A formed a CH-
p
interaction23 between the aromatic ring and the methylene
p
interaction with the methyl group of Val352, and the phenyl group
Table 1
Inhibitory effects of staurosporine 1 and compound 2 on Syk family kinases
3.2. SARs for Syk and ZAP-70 kinase inhibition
The Syk and ZAP-70 inhibitory activities of our synthesized
compounds were evaluated using a coupled spectrophotometric
enzyme assay (Tables 1–4). Potent compounds were further inves-
tigated for enzyme selectivity compared with other tyrosine
kinases (Lck and PKCbII) (Table 5).
We investigated the inhibitory effects of various substituents at
the 5-position of the 1,2,4-triazolo[4,3-c]pyrimidine core on Syk
Compound
IC50 (lM)
Syk
ZAP-70
family kinases (Table 2). Compound 9a, which possesses
a
1
2
0.009a
0.041b
0.053a
11.2b
hydroxyethylamino group at the 5-position, showed weak inhibi-
tory activities for Syk and ZAP-70. Similarly, low inhibitory activi-
ties for both Syk and ZAP-70 were observed with compound 9b, in
which tert-butyl glycine is substituted at the 5-position. The 5-
aminoethylenediamino derivative 10a exhibited highly potent
a
Results from in-house evaluations. The IC50 values for inhibition of Syk and
ZAP-70 kinases were determined in duplicate.
b
The results are presented in the following Ref. 9.

7350
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
Figure 1. (A) Proposed docking modes of staurosporine 1, compound 2, and compound 3 to the ATP-binding site of the ZAP-70 kinase. (B) Proposed docking mode of
staurosporine 1 to the ZAP-70 kinase complex. (C) Proposed docking mode of compound 10d to the ZAP-70 kinase complex.
Table 3
inhibition of Syk (IC50 = 0.009
ZAP-70 (IC50 = 8.3
lM) but was less potent at inhibiting
Inhibition of Syk and ZAP-70 activities by 1,2,4-triazolo[1,5-c]pyrimidine derivatives
l
M). This result was similar to the outcome ob-
served previously for compound 2.9 Compound 10b, which is
substituted with a dimethyl group at the approximate terminal
primary amine at the C5 position in 10a, showed lower inhibition
of Syk kinase (IC50 = 0.15
10c, which bears a 3-propylenediamino group, had significantly
lower inhibitory activities for Syk and ZAP-70 (IC50 = 7.7 M for
Syk, IC50 = 50 M for ZAP-70). Compound 10d, in which a cis-cyclo-
lM), as compared with 10a. Compound
l
l
hexyldiamino group is introduced at the 5-position of the 1,2,4-
triazolo[4,3-c]pyrimidine core, indicated excellent Syk kinase
Compound
R
IC50 (l
M)a
Syk
ZAP-70
11
H
Ph
0.004
0.083
0.12
0.33
0.11
0.12
0.097
Table 2
14a
14b
14c
Inhibition of Syk and ZAP-70 activities by 1,2,4-triazolo[4,3-c]pyrimidine derivatives
3-MeOC6H4
2-ClC6H4
0.050
a
The IC50 values were determined in duplicate.
Table 4
Suppression of IL-2 production in PBMCs and WB, by Syk family kinase inhibitors
Compound
X
Y
R
IC50
ZAP-70
(l
M)a
Compound
R1
R2
IC50
(
l
M)a
ZAP-70
Syk
PBMCs
WB
0.44
Syk
10d
11
14a
14c
N
CH
N
N
–
H
0.009
0.004
0.083
0.050
0.12
0.33
0.11
0.097
0.046
0.076
0.21
9a
9b
10a
10b
10c
NHCH2CH2OH
NHCH2CO2tBu
NHCH2CH2NH2
NHCH2C(Me)2NH2
NHCH2CH2CH2NH2
H
H
H
H
H
6.1
6.4
9.0
8.3
1.1
50
CH
CH
CH
0.48
2.0
79.1
0.009
0.15
7.7
Ph
2-ClC6H4
N
1.76
NTb
a
The IC50 values were determined in duplicate.
NT, not tested.
b
10d
H
0.009
15.2
0.12
26
HN
NH2
inhibition (IC50 = 0.009
lM) and improved ZAP-70 inhibitory activ-
ity (IC50 = 0.12 M), as compared with compound 10a. These find-
l
ings indicate that an amino group introduced at the 5-position of
the 1,2,4-triazolo[4,3-c]pyrimidine skeleton has an important im-
pact on Syk family kinase inhibition. A specific length of the con-
necting diamino group is also crucial for kinase inhibition.
13a
Ph
HN
NH2
a
The IC50 values were determined in duplicate.

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
7351
Table 5
inhibitory activities for Syk and/or ZAP-70 kinase (i.e., compounds
10d, 11, 14a, and 14c) were tested for suppressive effects on IL-2
production (Table 4). In PBMCs, compounds 10d and 11 demon-
strated strong suppression of IL-2 production, whereas the 2-phe-
nyl derivatives 14a and 14c were less potent in this regard. In WB,
compound 14a showed decreased suppressive potency for IL-2
production. We considered that the higher molecular weights of
14a and 14c as compared with theose of compounds 10d and 11
Summary of the effects of the Syk family kinase inhibitors
Compound
IC50
Lck
(l
M)a
PKCbII
Syk
ZAP-70
PBMC
WB
1
10d
11
0.003
0.009
0.004
0.053
0.12
0.33
0.018
0.18
0.59
0.004
0.68
2.66
0.016
0.046
0.076
0.10
0.44
0.48
a
The IC50 values were determined in duplicate.
or the weaker solubility in water of 14a (1.2
lg/mL) as compared
with that of compounds 10d (351 g/mL) and 11 (42
l
lg/mL) influ-
enced the cellular activities. Compounds 10d and 11 showed excel-
lent suppression of IL-2 production in WB. Compounds 10d and 11
demonstrated almost equal potencies for IL-2 suppression to
staurosporine 1 in the cellular assay system.
3.4. In vitro selectivity of Syk family kinase inhibition over Lck
and PKCbII
We assessed the selectivity of the actions of compounds 10d,
11, and staurosporine 1 (Table 5). Staurosporine 1 strongly inhib-
ited not only Syk family kinases but also Lck and PKCbII. On the
other hand, compounds 10d and 11 showed potent inhibitory
activities against Syk family kinases, but less potent inhibitory
activities against Lck and PKCbII than staurosporine 1. Whereas
staurosporine 1 formed a hydrogen bond with Ser323 in Lck, no
interaction occurred between compound 10d and Ser323 in Lck
(Fig. 3A). This interaction may be responsible for the Syk family
selectivity of compound 3, as compared with that of staurosporine
1. Regarding the selectivity of compound 10d for PKCbII, we as-
sumed that the hydrogen bonding between the N–H group and
Asp470 was important. The N–H in the methylamino group on
the tetrahydropyran core of staurosporine 1 interacted with the
carboxyl group in Asp 470, whereas there was no interaction be-
tween the NH2 group in compound 10d and the carboxyl group
in Asp470 (Fig. 3B).
Figure 2. Proposed docking mode of compound 14a to the ZAP-70 kinase complex.
Furthermore, incorporation of the cyclohexyldiamino group was
very effective at inhibiting Syk family kinases. In particular, ZAP-
70 inhibition was strongly enhanced by this modification. Dimroth
rearrangement derivative 1,2,4-triazolo[1,5-c]pyrimidine (deriva-
tive 11) showed strong inhibition of Syk family kinases
(IC50 = 0.004 lM for Syk; IC50 = 0.33 lM for ZAP-70; Table 3), sim-
ilar to compound 10d. As they showed almost identical inhibitory
profiles, compounds 10d and 11 were considered to have similar
pharmacophores. We consider that these results are in accordance
with our binding mode hypothesis.
We also studied the effect of substitutions at the 3-position in
the 1,2,4-triazolo[4,3-c]pyrimidine core and the 2-position in the
1,2,4-triazolo[1,5-c]pyrimidine core. Compound 13a, with a phenyl
group at the 3-position in the 1,2,4-triazolo[4,3-c]pyrimidine skel-
eton, showed complete loss of ability to inhibit Syk and ZAP-70 ki-
nases (Table 2). Compounds that incorporated the phenyl group at
the 2-position in the 1,2,4-triazolo[1,5-c]pyrimidine core were also
evaluated (Table 3). The inhibitory activity of compound 14a for
4. Conclusions
We have discovered novel Syk family kinase inhibitors based on
literature reports and SBDD and ZAP-70 homology models
constructed from the published Lck crystal structure. The 1,2,4-
triazolo[4,3-c]pyrimidine derivative 10d and its regioisomer
1,2,4-triazolo[1,5-c]pyrimidine derivative 11 exhibited strong
kinase inhibitory activities. Moreover, the potencies of the IL-2-
suppressive effects of 10d and 11 on PBMCs and WB were almost
equivalent to that of staurosporine 1. Concerning Syk family kinase
inhibition, similar selectivity profiles were observed in stauro-
sporine 1 and compounds 10d and 11. However, compounds 10d
and 11 had higher selectivity than staurosporine 1 for Lck and
PKCbII. As these derivatives possess promising in vitro potencies,
we intend to investigate their in vivo efficacies, the results of which
will be reported in due course.
ZAP-70 kinase was slightly improved (IC50 = 0.11
to our expectations, substitution of the phenyl group decreased
Syk kinase inhibition (IC50 = 0.083 M). Compounds 14b and 14c
lM). Contrary
l
exhibited Syk family kinase inhibitory activities similar to that of
compound 14a (see Fig. 1C).
An examination of the binding profile of compound 13a, as pre-
dicted by the ADAM19 software, revealed a narrow space around
the 3-position in the 1,2,4-triazolo[4,3-c]pyrimidine core and a
phenyl group at the 3-position that might interact with Lys369
and/or Asp479. This repulsion would abolish the Syk family kinase
inhibitory activity of compound 13a. Regarding the binding of 14a
(Fig. 2) to ZAP-70, there is a comparatively large space at the 2-po-
sition in the 1,2,4-triazolo[1,5-c]pyrimidine skeleton. The phenyl
group of compounds 14a–c fitted well into this space. As these
derivatives effectively occupied sites A, B, and C (Fig. 1), we antic-
ipated that the Syk kinase inhibitory profile of 14a–c would resem-
ble that of staurosporine 1.
5. Experimental procedures
5.1. Chemistry
Melting points were taken on a Yanako MP-3S Micro melting
point apparatus and were uncorrected. Infrared spectra were mea-
sured on a Nicolet 510 FT-IR spectrophotometer and were reported
in reciprocal centimeters. Proton NMR spectra were recorded at
400 or 500 MHz with a Brucker AMX 400 or DRX 500 instrument,
and chemical shifts were reported in parts per million (d) down-
field from tetramethylsilane as internal standard. The peak pat-
terns were shown as the following abbreviations: br, broad; d,
3.3. IL-2 production
IL-2 production levels of PBMCs and WB were assayed by en-
zyme-linked immunosorbent assay (ELISA). Compounds with high

7352
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
Figure 3. (A) Proposed docking mode of staurosporine 1, compound 10d, and Lck kinase complex. (B) Proposed docking modes of staurosporine 1, compound 10d, and PKCbII
kinase complex.
doublet; m, multiplet; s, singlet; t, triplet; q, quartet. The mass
spectra (MS) were carried out with Thermo Quest FINNIGAN AQA
electrospray ionization mass spectrometer. Elemental analyses
were performed on an Elementar Vario EL analyzer (C, H, and N).
Silica gel 60F254 precoated plates on glass from Merck KgaA or
aminopropyl silica gel (APS) precoated NH plates from Fuji Silysia
Chemical Ltd were used for thin layer chromatography (TLC). Flash
or medium-pressure liquid chromatography (MPLC) was per-
formed on silica gel BW-350 from Fuji Silysia Chemical Ltd or
ambient temperature. Saturated ammonium chloride solution
was added to the mixture and extracted with AcOEt. The organic
layer was washed with brine, dried over anhydrous MgSO4, and
evaporated in vacuo to give 6a (0.546 g, 98%) as white solid: mp
213–214 °C (THF); 1H NMR (CDCl3) d: 1.40–1.50 (9H, m), 3.78–
3.83 (6H, m), 4.07–4.15 (2H, m), 5.82–6.02 (1H, m), 6.28–6.32
(1H, m), 6.73–6.78 (2H, m), 6.92–7.08 (1H, m); Anal. Calcd for
C19H22ClN5O4ꢁ0.2H2O: C, 53.89; H, 5.33; N, 16.53. Found: C,
54.10; H, 5.41; N, 16.19.
APS Daisogel IR-60 (particle size 25–40 lM) from Daiso Co., Ltd.
All reagents and solvents were commercially available unless
otherwise indicated.
5.1.3. 4-Chloro-6-(3,5-dimethoxyphenylamino)-2-(2-hydroxy-
ethylamino)pyrimidine-5-carbonitrile (6b)
To a stirred suspension of compound 5 (0.138 g, 0.424 mmol) in
THF (10 mL) was added 2-aminoethanol (0.128 mL, 2.12 mmol) at
ꢀ70 °C under argon atmosphere and the mixture was stirred for
2 h at ꢀ20 °C. Water was added to the mixture and extracted with
AcOEt. The organic layer was washed with brine, dried over anhy-
drous MgSO4, and evaporated in vacuo to give 6b (0.147 g, 99%) as
white solid: mp 223–224 °C (THF); 1H NMR (CDCl3) d: 3.30–3.38
(3H, m), 3.46–3.52 (2H, m), 3.71–3.76 (6H, m), 4.66–4.73 (1H,
m), 6.24–6.28 (1H, m), 6.89–6.99 (2H, m), 8.02–8.28 (1H, m); Anal.
Calcd for C15H16ClN5O3: C, 51.51; H, 4.61; N, 20.02. Found: C,
51.31; H, 4.65; N, 19.94.
5.1.1. 2,4-Dichloro-6-(3,5dimethoxyphenylamino)pyrimidine-
5-carbonitrile hydrochloride (5)
To a stirred suspension of 2,4,6-trichloropyrimidine-5-carbo-
nitrile
4 (2.00 g, 9.60 mmol) in THF (50 mL) were added
3,5-dimethoxyaniline (1.47 g, 9.60 mmol) in THF (30 mL) and
diisopropylethylamine (1.84 mL, 10.6 mmol) at ꢀ70 °C under
argon atmosphere for 1 h and stirred for 2 h at 0 °C. Saturated
ammonium chloride solution was added to the mixture and ex-
tracted with AcOEt. Organic phase was washed with brine, dried
over anhydrous MgSO4, and evaporated in vacuo. Tetrahydrofu-
ran was added to the residue and resulting precipitate was
collected by suction. The filtrate was concentrated in vacuo
and crystallized from AcOEt to give 5 (0.800 g, 26%) as pale yel-
lowish solid: 1H NMR (CDCl3) d: 3.82 (6H, s), 6.38 (1H, t,
J = 2.0 Hz), 6.76 (2H, d, J = 2.0 Hz), 7.32 (1H, br s); Anal. Calcd
for C13H10Cl2N4O2ꢁ1.0 HCl: C 43.18; H 3.07; N 15.49. Found: C,
43.03; H, 2.80; N, 15.22.
5.1.4. tert-Butyl N-{2-[4-chloro-5-cyano-6-(3,5-dimethoxy-
phenylamino)pyrimidin-2-ylamino]ethyl}carbamate (6d)
To a stirred suspension of compound 5 (0.358 g, 1.10 mmol) in
THF (15 mL) was added tert-butyl N-(2-aminoethyl)carbamate
(0.873 mL, 8.81 mmol) at ꢀ70 °C under argon atmosphere and
the mixture was stirred for 2 h at ꢀ20 °C. Water was added to
the mixture and extracted with AcOEt. Organic layer was washed
with brine, dried over anhydrous MgSO4, and evaporated in vacuo
to give 6d (0.400 g, 81%) as white solid: mp 223–224 °C (THF); 1H
NMR (CDCl3) d: 1.36 (9H, s), 3.06–3.13 (2H, m), 3.25–3.31 (2H, m),
3.71–3.73 (6H, m), 6.25–6.27 (1H, m), 6.70–6.87 (2H, m), 6.88-6.97
(1H, m), 8.05–8.25 (1H, m), 9.18–9.37 (1H, m); Anal. Calcd for
5.1.2. tert-Butyl [4-chloro-5-cyano-6-(3,5-dimethoxyphenyl-
amino)pyrimidin-2-ylamino]acetate (6a)
To a stirred suspension of compound 5 (0.430 g, 1.32 mmol)
and tert-butyl aminoacetate hydrochloride (0.244 g, 1.45 mmol)
in THF (10 mL) was added diisopropylethylamine in THF (5 mL)
at ꢀ70 °C under argon atmosphere and the mixture was gradu-
ally warmed to room temperature, then stirred overnight at
C20H25ClN6O4: C, 53.51; H, 5.61, N, 18.72. Found: C, 53.27; H,
5.62; N, 18.50.

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
7353
5.1.5. tert-Butyl {2-[4-chloro-5-cyano-6-(3,5-dimethoxyphenyl-
amino)pyrimidin-2-ylamino]-1,1-dimethylethyl}carbamate (6e)
The title compound was prepared from 5 and tert-butyl (2-ami-
no-1,1-dimethylethyl)carbamate in the same manner as described
for 6d, and was obtained as pale yellowish solid (69%): mp 174–
176 °C (AcOEt); 1H NMR (CDCl3) d: 1.23–1.38 (6H, m), 1.40–1.49
(9H, m), 3.63–3.74 (2H, m), 3.78–3.82 (6H, m), 4.52–4.76 (1H,
m), 6.23–6.31 (2H, m), 6.75–7.04 (3H, m); Anal. Calcd for
room temperature, the resulting residue was collected and washed
with Et2O to give 8b (0.278 g, 25%) as white solid: mp 230–231 °C
(THF); 1H NMR (DMSO-d6) d: 1.24 (9H, s), 3.73 (6H, s), 4.14 (2H, d,
J = 5.5 Hz), 6.25 (1H, t, J = 2.5 Hz), 6.76 (2H, d, J = 2.5 Hz), 9.20 (1H,
s), 9.40 (1H, s), 9.52 (1H, t, J = 5.5 Hz); MS m/z: 426 (M+H)+. Anal.
Calcd for C20H23N7O4ꢁ0.7H2O: C, 54.84; H, 5.61; N, 22.38. Found:
C, 55.01; H, 5.33; N, 22.17.
C
22H29ClN6O4: C, 55.40; H, 6.13; N, 17.62. Found: C, 55.12; H,
5.1.10. tert-Butyl {2-[8-cyano-7-(3,5-dimethoxyphenylamino)-
1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]ethyl}carbamate (8c)
The title compound was prepared from 6d in the same manner
as described for 8b, and was obtained as pale white solid (32%): 1H
NMR (DMSO-d6) d: 1.35 (9H, s), 3.11–3.12 (2H, m), 3.70–3.75 (6H,
m), 6.26 (1H, s), 6.68–6.86 (1H, m), 6.87–6.98 (2H, m), 8.00–8.25
(1H, m), 9.16–9.36 (1H, m); MS m/z: 455 (M+H)+.
6.13; N, 17.61.
5.1.6. tert-Butyl {3-[4-chloro-5-cyano-6-(3,5-dimethoxyphenyl-
amino)pyrimidin-2-ylamino]propyl}carbamate (6f)
The title compound was prepared from 5 and tert-butyl (3-ami-
nopropyl)carbamate in the same manner as described for 6d, and
was obtained as pale yellowish solid (40%): mp 183–184 °C
(AcOEt); 1H NMR (CDCl3) d: 1.40–1.47 (9H, m), 1.67–1.80 (2H,
m), 3.17–3.25 (2H, m), 3.45–3.55 (2H, m), 3.77–3.84 (6H, m),
4.70–4.88 (1H, m), 6.16–6.30 (2H, m), 6.78–6.83 (2H, m), 6.90–
7.05 (1H, m); Anal. Calcd for C21H27ClN6O4: C, 54.48; H, 5.88; N,
18.15. Found: C, 54.39; H, 5.87; N, 18.13.
5.1.11. tert-Butyl {2-[8-cyano-7-(3,5-dimethoxyphenylamino)-
1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]-1,1-dimethylethyl}car-
bamate (8d)
The title compound was prepared from 6e in the same manner
as described for 8b, and was obtained as pale yellowish solid (40%):
mp 216–217 °C (THF–MeCN); 1H NMR (DMSO-d6) d: 1.20 (6H, s),
1.32 (9H, s), 3.66 (2H, d, J = 5.0 Hz), 3.73 (6H, s), 6.25–6.30 (1H,
m), 6.35–6.46 (1H, m), 6.80 (2H, d, J = 2.0 Hz), 8.75–8.83 (1H, m),
9.25 (1H, s), 9.35 (1H, s); Anal. Calcd for C23H30N8O4: C, 57.25; H,
6.27; N, 23.22. Found: C, 56.98; H, 6.18; N, 23.17.
5.1.7. tert-Butyl cis-{2-[4-chloro-5-cyano-6-(3,5-dimethoxy-
phenylamino)pyrimidin-2-ylamino]cyclohexyl}carbamate (6g)
The title compound was prepared from 5 and tert-butyl cis-(2-
aminocyclohexyl)carbamate in the same manner as described for
6d, and was obtained as pale yellowish solid (40%): mp 207–
208 °C (MeOH–AcOEt); 1H NMR (CDCl3) d: 1.16–1.25 (2H, m), 1.33
(9H, s), 1.35-1.76 (6H, m), 3.73 (6H, s), 3.74–4.00 (2H, m), 6.26–
6.30 (1H, m), 6.50–6.60 (1H, m), 6.82–6.90 (2H, m), 7.48–8.20
(1H, m), 9.20–9.45 (1H, m); Anal. Calcd for C24H31ClN6O4: C,
57.31; H, 6.21; N, 16.71. Found: C, 57.26; H, 6.25; N, 16.57.
5.1.12. tert-Butyl {3-[8-cyano-7-(3,5-dimethoxyphenylamino)-
1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]propyl}carbamate (8e)
The title compound was prepared from 6f in the same manner
as described for 8b, and was obtained as pale yellowish solid
(60%): mp 202–203 °C (THF–MeCN); 1H NMR (DMSO-d6) d: 1.34
(9H, s), 1.70–1.78 (2H, m), 2.94–3.02 (2H, m), 3.44 (2H, t,
J = 5.5 Hz), 3.72 (6H, s), 6.23 (1H, t, J = 2.0 Hz), 6.73–6.80 (1H, m),
6.83 (2H, d, J = 2.0 Hz), 8.88–8.94 (1H, m), 9.15 (1H, s), 9.32 (1H,
s); Anal. Calcd for C22H28N8O4ꢁ0.3H2O: C, 55.75; H, 6.08; N, 23.64.
Found: C, 56.02; H, 6.09; N, 23.24.
5.1.8. 5-[2-(tert-Butyldimethylsilanyloxy)ethylamino]-7-(3,5-
dimethoxyphenylamino)-1,2,4-triazolo[4,3-c]pyrimidine-8-
carbonitrile (8a)
To a mixture of compound 6b (0.323 g, 0.923 mmol) in DMF
(5 mL) were added imidazole (0.069 g, 1.02 mmol) and tert-butyl
dimethylchlorosilane (0.153 g, 1.02 mmol) and the mixture was
stirred for 20 min at room temperature. Water was added and
the mixture was extracted with AcOEt. Organic phase was washed
with water and brine, successively, dried over anhydrous MgSO4,
and evaporated in vacuo to give 6c. Crude 6c was dissolved in
THF (10 mL) and hydrazine monohydrate (0.448 mL, 9.23 mmol)
was added at room temperature. After being stirred overnight,
water was added to the mixture and resulting precipitate was col-
lected by suction filtration. Without purification a mixture of this
precipitate in trimethylorthoformate was heated overnight at
100 °C. After being cooled to room temperature, resulting precipi-
tate was collected by suction filtration to give 8a (0.048 mg, 11%)
as white solid: mp 243–245 °C (THF); 1H NMR (DMSO-d6) d:
ꢀ0.06 (6H, s), 0.77 (9H, s), 3.56–3.61 (2H, m), 3.72 (6H, s), 3.75
(2H, d, J = 6.0 Hz), 6.23 (1H, t, J = 2.0 Hz), 6.80 (2H, d, J = 2.0 Hz),
9.03–9.08 (1H, m), 9.18 (1H, s), 9.36 (1H, s); MS m/z: 470
(M+H)+. Anal. Calcd for C22H31N7O3Siꢁ0.4H2O: C, 55.41; H, 6.72;
N, 20.56. Found: C, 55.51; H, 6.60; N, 20.90.
5.1.13. tert-Butyl cis-2-[8-cyano-7-(3,5-dimethoxyphenyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]cyclohexyl}-
carbamate (8f)
The title compound was prepared from 6g in the same
manner as described for 8b, and was obtained as pale yellowish
solid (77%): mp 232–234 °C (THF–MeCN); 1H NMR (DMSO-d6) d:
0.90–0.98 (2H, m), 1.24 (9H, s), 1.30–1.80 (6H, m), 3.73 (6H, s),
3.90–4.05 (1H, m), 4.06–4.14 (1H, m), 6.25–6.29 (1H, m), 6.76–
6.81 (2H, m), 6.87 (1H, d, J = 9.0 Hz), 8.37–8.44 (1H, m), 9.33
(1H, s), 9.39 (1H, s); MS m/z: 509 (M+H)+. Anal. Calcd for
C25H32N8O4: C, 59.04; H, 6.34; N, 22.03. Found: C, 58.93; H,
6.36; N, 21.96.
5.1.14. 7-(3,5-Dimethoxyphenylamino)-5-(2-hydroxyethyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidine-8-carboxamide (9a)
To a solution of 8a (0.040 g, 0.085 mmol) in DMSO (0.4 mL) and
EtOH (0.4 mL) were added 5 M NaOH solution (0.170 mL,
0.852 mmol) and 30% H2O2 solution (0.097 mL, 0.852 mmol), then
the mixture was stirred overnight at room temperature. Water
(1 mL) was added and the mixture was neutralized with 1 M HCl
solution. The obtained pale yellow precipitates were collected by
filtration and washed with H2O. The precipitates were dissolved
in THF and to the mixture was added 1 M tetrabutylammonium
fluoride in THF solution (0.086 mL, 0.085 mmol). After being stir-
red for 2 h, the mixture was purified by column chromatography
on APS with MeOH/AcOEt = 1:20 to give 9a (0.005 g, 16%) as white
solid: mp 280–281 °C (THF); 1H NMR (DMSO-d6) d: 3.11–3.20 (3H,
m), 3.51–3.62 (4H, m), 3.73 (6H, s), 6.02–6.04 (1H, br s), 7.03–7.10
5.1.9. tert-Butyl [8-cyano-7-(3,5-dimethoxyphenylamino)-
1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]acetate (8b)
To a stirred solution of compound 6a (1.10 g, 2.62 mmol) in THF
(10 mL) was added hydrazine monohydrate (1.27 mL, 26.2 mmol)
at room temperature and the mixture was stirred overnight at
ambient temperature. Water was added to the mixture and the
resulting precipitates were collected by suction filtration and dried
in vacuo. The precipitate and trimethylorthoformate (8.53 mL,
80.0 mmol) were heated for 2 days at 90 °C. After being cooled to

7354
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
(2H, br s), 8.61 (2H, br), 12.52 (1H, s); MS m/z: 374 (M+H)+. Anal.
Calcd for C16H19N7O4ꢁ0.3H2O: C, 50.74; H, 5.22; N, 25.88. Found:
C, 50.82; H, 5.26; N, 25.58.
(2H, m), 1.23 (9H, s), 1.26–1.84 (6H, m), 3.76 (6H, s), 4.11–4.20
(2H, m), 6.25 (1H, br s), 6.79 (2H, d, br s), 6.82–6.89 (1H, m),
7.52 (1H, d, J = 3.0 Hz), 8.19–8.27 (1H, m), 8.77 (1H, s), 9.36 (1H,
s), 12.44 (1H, s); MS m/z: 527 (M+H)+. Anal. Calcd for
5.1.15. tert-Butyl [8-carbamoyl-7-(3,5-dimethoxyphenyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]acetate (9b)
To a solution of 8b (0.114 g, 0.268 mmol) in DMSO (1 mL) and
EtOH (1 mL) were added 5 M NaOH solution (0.535 mL,
2.68 mmol), and 30% H2O2 solution (0.305 mL, 2.68 mmol) then
the mixture was stirred overnight at room temperature. Water
(1 mL) was added and the mixture was neutralized with 1M HCl
solution. The obtained pale yellow precipitates were collected by
filtration and washed with H2O. The precipitates were purified by
C
25H34N8O5ꢁ0.4H2O: C, 56.25; H, 6.57; N, 20.99. Found: C, 56.40;
H, 6.46; N, 20.74.
5.1.20. 5-(2-Aminoethylamino)-7-(3,5-dimethoxyphenyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidine-8-carboxamide (10a)
A mixture of compound 9c in HCl–MeOH was stirred for 1 h at
room temperature. Resulting precipitates were collected by suc-
tion filtration and dried under reduced pressure to give 10a as pale
yellowish solid (40%): mp 234–236 °C; 1H NMR (DMSO-d6) d:
3.19–3.26 (2H, m), 3.64–3.71 (2H, m), 3.76 (6H, s), 6.22 (1H, t,
J = 2.0 Hz), 6.79 (2H, d, J = 2.0 Hz), 8.30 (3H, br s), 8.80 (1H, br s),
9.65 (1H, s), 9.70 (1H, t, J = 5.0 Hz), 12.52 (1H, s); MS m/z: 371
(MꢀH)ꢀ. Anal. Calcd for C16H20N8O3ꢁ3.0HClꢁ2.0H2O: C, 37.11; H,
5.26; N, 21.64. Found: C, 37.35; H, 5.25; N, 21.79.
column
chromatography
on
silica
gel
with
MeOH/
AcOEt = 1:20 ? 1:8 to give 9b as yellowish solid: mp 285 °C
(dec); 1H NMR (DMSO-d6) d: 1.29 (9H, s), 3.77 (6H, s), 4.24 (2H,
s), 6.24 (1H, t, J = 2.5 Hz), 6.75 (2H, d, J = 2.5 Hz), 7.61 (1H, s),
8.76–8.79 (1H, m), 9.21 (1H, s), 9.43 (1H, s), 12.52 (1H, s); MS m/
z: 444 (M+H)+. Anal. Calcd for C20H25N7O5ꢁ 0.3H2O: C, 53.52; H,
5.75; N, 21.84. Found: C, 53.52; H, 5.66; N, 21.50.
5.1.21. 5-(2-Amino-2-methylpropylamino)-7-(3,5-dimethoxy-
phenylamino)-1,2,4-triazolo[4,3-c]pyrimidine-8-carboxamide
(10b)
5.1.16. tert-Butyl {2-[8-carbamoyl-7-(3,5-dimethoxyphenyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]ethyl}car-
bamate (9c)
The title compound was prepared from 9d in the same manner
as described for 10a, and was obtained as pale yellowish solid
(21%): mp 274–275 °C; 1H NMR (DMSO-d6) d: 1.29 (6H, s), 3.72–
3.75 (2H, m), 3.76 (6H, s), 6.29 (1H, t, J = 2.0 Hz), 6.84 (2H, d,
J = 2.0 Hz), 7.67 (1H, br s), 7.87 (3H, br s), 8.44 (1H, s), 8.73 (1H,
br s), 8.81 (1H, t, J = 6.0 Hz), 12.38 (1H, s); MS m/z: 401 (M+H)+.
Anal. Calcd for C18H24N8O3ꢁ1.6HCl: C, 47.12; H, 5.62; N, 24.42.
Found: C, 47.34; H, 5.78; N, 24.20.
To a solution of 8c (0.097 g, 0.213 mmol) in DMSO (1 mL) and
EtOH (1 mL) were added 5 M NaOH solution (0.480 mL,
4.25 mmol) and 30% H2O2 solution (2.13 mL, 4.25 mmol), then
the mixture was stirred overnight at room temperature. Water
(mL) was added and the mixture was neutralized with 1M HCl
solution. The obtained pale yellow precipitates were collected
by filtration and washed with H2O to give 9c (0.072 g, 72%) as
brown solid: 1H NMR (DMSO-d6) d: 1.37 (9H, s), 3.10–3.17
(2H, m), 3.35–3.34 (2H, m), 3.73 (6H, s), 6.20 (1H, t, J = 2.0 Hz),
6.81 (2H, d, J = 2.0 Hz), 6.83–6.89 (2H, m), 7.06–7.09 (1H, br s),
9.15 (1H, br s), 10.62 (1H, br s), 13.05 (1H, br s); MS m/z: 473
(M+H)+.
5.1.22. 5-(3-Aminopropylamino)-7-(3,5-dimethoxyphenyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidine-8-carboxamide (10c)
The title compound was prepared from 9e in the same manner
as described for 10a, and was obtained as pale yellowish solid
(33%): mp 208–210 °C; 1H NMR (DMSO-d6) d: 1.93–2.01 (2H, m),
2.92–3.00 (2H, m), 3.77 (6H, s), 4.16 (2H, t, J = 7.0 Hz), 4.40–4.65
(2H, br s), 6.50 (1H, t, J = 2.0 Hz), 6.63 (2H, d, J = 2.0 Hz), 7.84–
7.98 (1H, m), 8.08 (3H, br s), 9.00 (1H, s), 11.82 (1H, br s), 15.04
(1H, br s); MS m/z: 385 (MꢀH)ꢀ. Anal. Calcd for
C17H22N8O3ꢁ2.0HClꢁ1.8H2O: C, 41.52; H, 5.66; N, 22.79. Found: C,
41.75; H, 5.61; N, 22.76.
5.1.17. tert-Butyl {2-[8-carbamoyl-7-(3,5-dimethoxyphenyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]-1,1-dimethyl-
ethyl}carbamate (9d)
The title compound was prepared from 8d in the same manner
as described for 9b, and was obtained as pale yellowish solid (21%):
mp 207–210 °C (THF); 1H NMR (DMSO-d6) d: 1.27 (6H, s), 1.36 (9H,
s), 1.70–1.78 (2H, m), 3.64–3.70 (2H, m), 3.76 (6H, s), 6.26 (1H, br
s), 6.88 (2H, d, J = 2.0 Hz), 7.64 (1H, br s), 8.65 (1H, br s), 8.72 (1H,
br s), 12.43 (1H, s); MS m/z: 501 (M+H)+. Anal. Calcd for
5.1.23. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxyphe-
nylamino)-1,2,4-triazolo[4,3-c]pyrimidine-8-carboxyamide (10d)
The title compound was prepared from 9f in the same manner
as described for 10a, and was obtained as pale yellowish solid
(33%): mp 208–210 °C; 1H NMR (CD3OD) d: 1.50–2.00 (8H, m),
3.81 (6H, s), 3.94–3.99 (1H, m), 4.30–4.37 (1H, m), 6.39 (1H, t,
J = 2.0 Hz), 6.73 (2H, d, J = 2.0 Hz), 9.47(1H, s); MS m/z: 427
(M+H)+. Anal. Calcd for C20H26N8O3 2.0HCl 2.0H2O: C, 44.86; H,
6.02; N, 20.93. Found: C, 44.63; H, 6.02; N, 21.13.
C
23H32N8O5ꢁ0.2H2O: C, 54.79; H, 6.48; N, 22.22. Found: C, 54.69;
H, 6.39; N, 21.99.
5.1.18. tert-Butyl {3-[8-carbamoyl-7-(3,5-dimethoxyphenyl-
amino)-1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]propyl}car-
bamate (9e)
The title compound was prepared from 8e in the same manner
as described for 9b, and was obtained as pale yellowish solid
(21%): 1H NMR (DMSO-d6) d: 1.45 (9H, s), 1.87–1.95 (2H, m),
3.25–3.33 (2H, m), 3.68–3.75 (2H, m), 3.80 (6H, s), 5.50 (1H, br
s), 6.23 (1H, t, J = 2.0 Hz), 6.93 (2H, d, J = 2.0 Hz), 7.64 (1H, br s),
8.03 (1H, s), 8.65 (1H, br s), 9.08 (1H, br s), 12.20 (1H, s); MS
m/z: 487 (M+H)+.
5.1.24. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)-1,2,4-triazolo[1,5-c]pyrimidine-8-carboxamide
(11)
A mixture of 10d (0.050 g, 0.108 mmol) in DMF (1 mL) was stir-
red for 2 days at 100 °C. After being cooled to room temperature,
resulting residue was collected by suction filtration and washed
with AcOEt to give 11 (0.025 g, 50%) as white solid. mp 293 °C
(dec); 1H NMR (DMSO-d6) d: 1.30–2.10 (8H, m), 3.77 (6H, s),
4.20–4.35 (1H, m), 6.29 (1H, t, J = 2.0 Hz), 6.74 (2H, d, J = 2.0 Hz),
7.62 (1H, br s), 7.80–8.16 (4H, m), 8.43 (1H, s), 8.80 (1H, br s),
9.58 (1H, s), 12.36 (1H, s); MS m/z: 427 (M+H)+. Anal. Calcd for
5.1.19. tert-Butyl {cis-2-[8-carbamoyl-7-(3,5-dimethoxy-
phenylamino)-1,2,4-triazolo[4,3-c]pyrimidin-5-ylamino]cyclo-
hexyl}carbamate (9f)
The title compound was prepared from 8f in the same manner
as described for 9b, and was obtained as pale yellowish solid
(33%): mp 222–224 °C (THF); 1H NMR (DMSO-d6) d: 0.86–0.96
C
20H26N8O3ꢁ1.0HClꢁ0.2H2O: C, 51.49; H, 5.92; N, 24.02. Found: C,
51.16; H, 5.81; N, 23.65.

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
7355
5.1.25. tert-Butyl cis-{2-[8-carbamoyl-7-(3,5-dimethoxy-
phenylamino)-3-phenyl-1,2,4-triazolo[4,3-c]pyrimidin-5-yl-
amino]cyclohexyl}carbamate (12a)
s), 5.48–5.53 (1H, m), 6.27 (1H, t, J = 2.0 Hz), 6.76 (2H, d,
J = 2.0 Hz), 7.64–7.76 (6H, m), 7.85–7.90 (2H, m), 8.94 (1H, br s),
12.53 (1H, s); MS m/z: 503 (M+H)+. Anal. Calcd for
To a stirred solution of compound 6g (1.00 g, 1.99 mmol) in THF
(8 mL) and EtOH (8 mL) was added hydrazine monohydrate
(0.964 mL, 19.9 mmol) at room temperature and the mixture was
stirred for 3 h at ambient temperature. Water was added to the
mixture and resulting precipitates were collected by suction filtra-
tion and dried in vacuo. A mixture of the precipitates and benzal-
dehyde (0.305 mL, 2.99 mmol) in CH2Cl2 (5 mL) was stirred for 2 h
at room temperature. The mixture was purified by column chro-
matography on APS with AcOEt to give crude product. To this prod-
uct (0.472 g) in DMSO (2.5 mL) and EtOH (2.5 mL) were added 5 M
NaOH solution (1.13 mL, 5.63 mmol) and 30% H2O2 solution
(0.640 mL, 5.63 mmol), then the mixture was stirred overnight at
room temperature. To the mixture was added 2 M HCl solution
(2.85 mL) at 0 °C and resulting residue was collected by suction fil-
tration. This precipitate was washed with water and dried under
reduced pressure. This precipitate (0.100 g) was dissolved in MeCN
(3 mL) and 40% diisopropyl azodicarboxylate in toluene (0.500 mL,
0.993 mmol) was added. After being stirred for 24 h at 110 °C, the
mixture was purified by column chromatography on silica gel with
hexane/AcOEt = 2:3 ? 1:4 and recrystallized from MeOH/AcOEt to
give P1 as white solid.give 12a (0.020 g, 17%) as white solid: mp
214–216 °C (MeOH–AcOEt); 1H NMR (DMSO-d6) d: 0.55–0.74
(2H, m), 1.22 (9H, s), 1.20–1.62 (6H, m), 3.56–3.64 (1H, m), 3.73
(6H, s), 4.40–4.49 (1H, m), 5.50–5.56 (1H, m), 6.19 (1H, s), 6.74–
6.80 (1H, m), 6.79 (2H, d, J = 2.0 Hz), 7.62 (1H, d, J = 3.0 Hz), 7.65–
7.71 (3H, m), 7.81–7.87 (2H, m), 8.95 (1H, s), 12.60 (1H, s); MS m/z:
603 (M+H)+. Anal. Calcd for C31H38N8O5: C, 61.78; H, 6.36; N, 18.59.
Found: C, 61.38; H, 6.31; N, 18.41.
C
26H30N8O3ꢁ2.0HClꢁ0.8H2O: C, 52.94; H, 5.74; N, 19.00. Found: C,
53.10; H, 5.88; N, 18.65.
5.1.29. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)-2-phenyl-1,2,4-triazolo[1,5-c]pyrimidine-8-
carboxamide (14a)
A mixture of 13a (0.030 g, 0.056 mmol) DMF (0.5 mL) and stirred
overnight at 100 °C. Resulting precipitates were collected by suction
filtration to give 14a (0.020 g, 67%) as white solid: mp 260–261 °C
(AcOEt); 1H NMR (DMSO-d6) d: 1.38–2.08 (8H, m), 3.78 (6H, s),
3.80–3.86 (1H, m), 4.27–4.33 (1H, m), 6.30 (1H, t, J = 2.0 Hz), 6.81
(2H, d, J = 2.0 Hz), 7.56–7.60 (3H, m), 7.75 (1H, d, J = 2.5 Hz), 7.86–
8.20 (2H, br), 8.20–8.29 (3H, m), 8.89 (1H, d, J = 2.5 Hz), 12.41 (1H,
s); MS m/z: 503 (M+H)+. Anal. Calcd for C26H30N8O3ꢁ1.5HCl: C,
56.04; H, 5.70; N, 20.10. Found: C, 56.30; H, 5.81; N, 19.78.
5.1.30. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)-2-(3-methoxyphenyl)-1,2,4-triazolo[1,5-c]-
pyrimidine-8-carboxamide (14b)
The title compound was prepared from 12b in the same manner
as described for 13a and 14a, and was obtained as pale yellowish
solid (33%): mp 283–285 °C (AcOEt); 1H NMR (DMSO-d6) d: 1.30–
2.20 (8H, m), 3.77 (6H, s), 3.81–3.90 (2H, m), 3.86 (3H, s),
4.20–4.35 (1H, m), 6.30 (1H, t, J = 2.2 Hz), 6.81 (2H, d, J = 2.2 Hz),
7.10–7.20 (1H, m), 7.49 (1H, t, J = 8.0 Hz), 7.70–8.10 (5H, m),
8.85–8.90 (1H, m), 12.41 (1H, s); MS m/z: 533 (M+H)+. Anal. Calcd
for C27H32N8O4ꢁ6.0HClꢁ4. 0C2H7Nꢁ2.0H2O: C, 43.44; H, 7.29; N,
17.37. Found: C, 43.31; H, 7.44; N, 17.79.27
5.1.26. tert-Butyl cis-2-[8-carbamoyl-7-(3,5-dimethoxyphenyl-
amino)-3-(3-methoxyphenyl)-1,2,4-triazolo[4,3-c]pyrimidin-5-
ylamino]cyclohexylcarbamate (12b)
5.1.31. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)-2-(2-chlorophenyl)-1,2,4-triazolo[1,5-c]pyrim-
idine-8-carboxamide (14c)
The title compound was prepared from 6f in the same manner
as described for 12a, and was obtained as pale yellowish solid
(36%): mp 169–171 °C (AcOEt); 1H NMR (CDCl3) d: 1.35 (9H, br
s), 1.36–1.70 (8H, m), 3.78 (6H, s), 3.84–3.91 (1H, m), 3.89 (3H,
s), 4.26–4.50 (2H, m), 5.60–5.64 (2H, m), 6.22 (1H, t, J = 2.0 Hz),
6.79 (2H, d, J = 2.0 Hz), 7.15–7.23 (3H, m), 7.53 (1H, t, J = 8.0 Hz),
9.33 (1H, br s), 12.25 (1H, br s); MS m/z: 633 (M+H)+. Anal. Calcd
for C32H40N8O6ꢁ0.4H2O: C, 60.06; H, 6.43; N, 17.51. Found: C,
59.95; H, 6.29; N, 17.31.
The title compound was prepared from 12c in the same manner
as described for 13a and 14a, and was obtained as pale yellowish
solid (8.8%): mp 249–251 °C (AcOEt); 1H NMR (DMSO-d6) d:
1.36–2.05 (8H, m), 3.78 (6H, s), 3.78–3.87 (2H, m), 4.25–4.33
(1H, m), 6.31 (1H, t, J = 2.0 Hz), 6.81 (2H, d, J = 2.0 Hz), 7.53 (1H,
dt, J = 8.0 Hz, 1.0 Hz), 7.59 (1H, dt, J = 8.0 Hz, 2.0 Hz), 7.67 (1H,
dd, J = 8.0 Hz, 1.0 Hz), 7.72 (1H, d, J = 2.5 Hz), 7.95 (1H, dd,
J = 8.0 Hz, 2.0 Hz), 8.28–8.48 (2H, m), 8.78 (1H, d, J = 2.5 Hz),
12.37 (1H, s); MS m/z: 537 (M+H)+. Anal. Calcd for
C
26H29ClN8O3ꢁ4.0HClꢁ2. 0C2H7Nꢁ1.0H2O: C, 45.55; H, 6.24; N,
5.1.27. tert-Butyl cis-2-[8-carbamoyl-3-(2-chlorophenyl)-7-
(3,5-dimethoxyphenylamino)-1,2,4-triazolo[4,3-c]pyrimidin-5-
ylamino]cyclohexylcarbamate (12c)
17.71. Found: C, 45.38; H, 6.42; N, 17.38.27
5.2. Molecular modeling
The title compound was prepared from 6f in the same manner as
described for 12a, and was obtained as pale yellowish solid (12%):
mp 192–194 °C (AcOEt); 1H NMR (CDCl3) d: 1.15–1.35 (6H, m), 1.39
(9H, s), 1.40–1.70 (2H, m), 3.60–3.77 (1H, m), 3.78 (6H, s), 4.20–4.50
(2H, m), 5.15–5.26 (1H, m), 5.63 (1H, br s), 6.22 (1H, t, J = 2.0 Hz),
6.77–6.80 (2H, m), 7.52–7.75 (4H, m), 9.29 (1H, br s), 12.25–12.35
(1H, m); MS m/z: 637 (M+H)+. Anal. Calcd for C31H37ClN8O5: C,
58.44; H, 5.85; N, 17.59. Found: C, 58.11; H, 5.82; N, 17.45.
The 3D coordinates of ZAP-70 receptor model were constructed
based on the published crystal structure of an activated Lck kinase
domain16,17 (PDB code. 1QPD) using program FAMS.18 The struc-
ture of staurosporine 1 was extracted from X-ray structure of
LCK (PDB code. 1QPD) and manually docked into the ATP-binding
site of ZAP-70 receptor model using QUANTA200028 (Accelrys
Inc.: San Diego, CA, U.S.A.). All docking calculations were per-
formed by using program ADAM.19
5.1.28. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)-3-phenyl-1,2,4-triazolo[4,3-c]pyrimidine-8-
carboxamide (13a)
5.3. Biology
A mixture of 12a (0.092 g, 0.153 mmol) in 4 M HCl–AcOEt
(1.14 mL, 4.58 mmol) was stirred overnight at room temperature.
Resulting precipitates were collected by suction filtration and dried
under reduced pressure to give 13a (0.060 g, 72%) as white solid:
mp 210–212 °C (AcOEt); 1H NMR (DMSO-d6) d: 1.14–1.66 (8H,
m), 3.73–3.79 (1H, m), 3.76 (6H, s), 3.90 (1H, br s), 4.36 (1H, br
5.3.1. Intracellular ZAP-70 kinase inhibition assay
The kinase domain of human ZAP-70 kinase (Leu325-Ala619)
was cloned to Ndel and Xhol sites of pET-19b expression vector
(Novagen Inc.) by PCR amplification from human thymus Mara-
thon-ReadyTM cDNA (CLONTECH Inc.). The ZAP-70 kinase domain
binding with pET-19b His-tag gene at 50 region was integrated into

7356
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 7347–7357
pFASTBAC1 vector of the BAC-TO-BACTM (GIBCO BRL Inc.) baculovi-
rus expression system. The transfected virus was obtained by
transfecting Sf-9 cell (Invitrogen Inc.) with pFASTBAC1 containing
His-tag-fused ZAP kinase domain as described above. High FiveTM
baculocells, which were infected with this transfected virus, were
recovered and these cells were dissolved by ultrasonication. After
soluble fraction being separated by centrifuge, the supernatant
was added to TALONTM metal affinity resin (CLONTECH Inc) and
to this resin was adsorbed His-tag-fused protein of ZAP-70 kinase
domain. The resin was washed several times and the His-tag-fused
protein of ZAP-70 kinase domain was extracted by a buffer con-
taining imidazole. A coupled spectrophotometric assay was used
wherein ADP generated by ZAP-70 kinase was converted to ATP
by pyruvate kinase (PK), with concomitant production of pyruvate
from phosphoenolpyruvate (PEP). LDH reduces pyruvate to lactate
by oxidizing NADH. NADH depletion was monitored at 340 nm
using a microplate reader (Spectra Max 250, Molecular Device) at
30 °C for 20 min. Reactions were performed at 30 °C in 100 mM
HEPES buffer (pH 7.6) containing 20 mM MgCl2 and 10% glycerol,
tion of 2ꢂ 106 cells/mL inAIM-V medium (Invitrogen) contain-
ing100 U/mL penicillin and 100 lg/mL streptomycin.
5.3.3.2. Determination of IL-2 Production by PBMCs and
WB. PBMCs or WB were cultured with 10 g/mL PHA alone or in
l
combination with varying concentrations of compounds in 5%
CO2-air humidified atmosphere at 37 °C for 24 h. The cell superna-
tants were then collected and assayed for IL-2 concentrations by
the enzyme-linked immunosorbent assay (ELISA). The ELISA used
here is not reported to exhibit detectable cross-reactivity with
the other cytokines.
Acknowledgments
The authors acknowledge Dr. Yoshihiro Terao for syntheses and
valuable discussions. We also thank Dr. Nobuyuki Tanaka for useful
and kind suggestions during the preparation of this manuscript.
References and notes
and started by adding ATP. PK (150
(2.5 mM), and NADH (150 M) were added in large excess.
Addition of 100 M ZAP-70 optimal peptide substrate (peptide
lg/ml), LDH (500 lg/mL), PEP
l
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5.3.2. Intracellular Syk kinase inhibition assay
The kinase domain of human Syk kinase (Met343–Asn635) was
cloned to Ndel and Xhol sites of pET-19b expression vector (Nova-
gen Inc) by PCR amplification from human thymus Marathon-
ReadyTM cDNA (CLONTECH Inc). The Syk kinase domain binding
with pET-19b His-tag gene at 50 region was integrated to pFAST-
BAC1 vector of the BAC-TO-BACTM baculovirus expression system
(GIBCO BRL Inc.). The transfected virus was obtained by transfect-
ing Sf-9 cell (Invitrogen Inc.) with pFASTBAC vector containing
His-tag-fused Syk kinase domain as described above. High FiveTM
baculocells, which were infected with this transfected virus, were
recovered and dissolved by ultrasonication. After soluble fraction
being separated by centrifuge, the supernatant was mixed with TA-
LONTM metal affinity resin (CLONTECH Inc) and to this resin was
adsorbed His-tag fused protein of Syk kinase domain. The resin
was washed several times and the His-tag-fused protein of Syk ki-
nase domain was extracted by a buffer containing imidazole. A
coupled spectrophotometric assay was used wherein ADP gener-
ated by Syk kinase was converted to ATP by pyruvate kinase
(PK), with concomitant production of pyruvate from phosphoenol-
pyruvate (PEP). LDH reduces pyruvate to lactate by oxidizing
NADH. NADH depletion was monitored at 340 nm using a micro-
plate reader (Spectra Max 250, Molecular Device) at 30 °C for
20 min. Reactions were performed at 37 °C in 100 mM HEPES buf-
fer (pH 7.6) containing 40 mM MgCl2 and 10% glycerol, and started
by adding ATP. PK (150
l
g/mL), LDH (50
l
g/mL), PEP (2.5 mM), and
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using ClustalX.29
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Syk optimal peptide (peptide sequence: AEEEIYGEFEAKKKK, Sawa-
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blood (WB) assay
5.3.3.1. Preparation of PBMCs. Heparinized human peripheral
blood was obtained from healthy donors. PBMCs were isolated
by the Ficoll-Hypaque gradient density method as described previ-
ously. Blood cells were diluted with PBS buffer, and then centri-
fuged in a Ficoll-Hypaque discontinuous gradient at 1500 rpm for
30 min. The PBMC layers were collected and washed with cold
distilled water and 10ꢂ Hanks’ buffer saline solution (HBSS) to
remove red blood cells. The cells were resuspended to a concentra-

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