Structure-activity relationship studies of 5-benzylaminoimidazo[1,2-c]pyrimidine-8-carboxamide derivatives as potent, highly selective ZAP-70 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.10.070

Zeta-associated protein, 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family kinase, is normally expressed on T cells and natural killer cells and plays a crucial role in activation of the T cell immunoresponse. Thus, selective ZAP-70 inhibitors might

Full text of DOI:10.1016/j.bmc.2008.10.070

Bioorganic & Medicinal Chemistry 17 (2009) 284–294
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Bioorganic & Medicinal Chemistry
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Structure–activity relationship studies of 5-benzylaminoimidazo[1,2-c]
pyrimidine-8-carboxamide derivatives as potent, highly selective ZAP-70
kinase inhibitors
*
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, 4365-1 Kashiwabara, Hotaka, Azumino, Nagano Prefecture 399-8304, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Zeta-associated protein, 70 kDa (ZAP-70), a spleen tyrosine kinase (Syk) family kinase, is normally
expressed on T cells and natural killer cells and plays a crucial role in activation of the T cell immunore-
sponse. Thus, selective ZAP-70 inhibitors might be useful not only for treating autoimmune diseases, but
also for suppressing organ transplant rejection. In our recent study on the synthesis of Syk family kinase
inhibitors, we discovered that novel imidazo[1,2-c]pyrimidine-8-carboxamide derivatives possessed
potent ZAP-70 inhibitory activity with good selectivity for ZAP-70 over other kinases. In particular, com-
pound 26 showed excellent ZAP-70 kinase inhibition and high selectivity for ZAP-70 over structurally
related Syk. The discovery of a potent, highly selective ZAP-70 inhibitor would contribute a new thera-
peutic tool for autoimmune diseases and organ transplant medication.
Received 19 September 2008
Revised 30 October 2008
Accepted 31 October 2008
Available online 5 November 2008
Keywords:
Syk family kinase
Syk
ZAP-70
Ó 2008 Elsevier Ltd. All rights reserved.
T cell immunoresponse
Immunosuppressant
Selective ZAP-70 inhibitor
1. Introduction
ZAP-70 takes part in T cell activation without a pathway involving
calcineurin.
Cyclosporin A 11 and tacrolimus (FK506) 22 are the main immu-
nosuppressants used to prevent organ transplant rejection (Fig. 1).
Both drugs, however, have severe side effects on the renal, nervous,
and gastrointestinal systems.3 Therefore, novel immunosuppres-
sants without adverse effects are being sought for use in transplan-
tation. Zeta-associated protein, 70 kDa (ZAP-70), a spleen tyrosine
kinase (Syk) family kinase, is a non-receptor type protein tyrosine
kinase.4 ZAP-70 is expressed mainly in T cells and natural killer
cells and its activation dominates downstream signal transduction
and induces the mobilization of cellular calcium ions, IL-2 produc-
tion, and T cell proliferation.5,6 Therefore, ZAP-70 plays an impor-
tant role in T cell signal transduction.7,8 Arpaia et al. reported
that peripheral CD4+ T cells from patients who were ZAP-70-defi-
cient exhibited markedly reduced tyrosine phosphorylation and
failed to produce IL-2.9 In addition, signal transduction through T
cell receptors, such as for the mobilization of cellular calcium ions
and production of IL-2, is not observed in a ZAP-70-deficient cell
line.10 Moreover, cyclosporin A 1 and tacrolimus 2 cause severe
side effects on the kidney, and the major factor responsible is
thought to be the inhibition of calcineurin in the kidney. However,
Previously, we developed a Syk family kinase inhibitor for treat-
ing various allergic and autoimmune disorders.11 However, disrup-
tion of the Syk gene in knockout mice leads to embryonic
hemorrhage and death, in addition to the loss of immune receptor
signaling. In addition, the Syk mutation impaired the differentia-
tion of B cells by disrupting signaling from the pre-B cell antigen
receptor complex and preventing the clonal expansion and muta-
tion of pre-B cells.12 Thus, we postulated that a selective ZAP-70
inhibitor would be useful not only for suppressing organ transplant
rejection, but also for treating autoimmune diseases. Few reports
exist on selective ZAP-70 inhibitors. In 1999, the ARIAD Research
Group reported potent, selective SH2 inhibitors of the tyrosine
kinase ZAP-70, such as compound 3 (Table 1), which had moderate
ZAP-70 inhibitory activity (IC50 = 4.0
the closely structurally related Syk (IC50 > 500
Research Group reported that 4-pyridin-5-yl-2-(3,4,5-trimethoxy-
phenylamino)pyrimidine derivative was potent, selective
non-peptide ZAP-70 inhibitor that had excellent inhibitory activity
l
M) with high selectivity over
l
M).13 The Celltech
4
a
for ZAP-70 (IC50 = 0.011 lM) and was highly selective for ZAP-70
over PKC, Lck, EGFr, and csk.14 However, the selectivity of com-
pound 4 over closely related Syk was not described. We found that
imidazo[1,2-c]pyrimidine-8-carboxamide derivative 9 was a novel,
selective ZAP-70 inhibitor in our research into protein tyrosine
* 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.2008.10.070

A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
285
Figure 1. Structures of the immunosuppressants cyclosporin A 1 and tacrolimus (FK506) 2.
Table 1
highly selective ZAP-70 inhibitor as a novel immunosuppressant
for use in organ transplantation with no unfavorable side effects.
ZAP-70 inhibitory activity of published compounds
2. Chemistry
The general synthetic route for imidazo[1,2-c]pyrimidine is
shown in Scheme 1. To prepare imidazo[1,2-c]pyrimidine deriva-
tives, we started from commercial 4,6-dichloro-2-methylsulfanyl-
pyrimidine-5-carbonitrile 5. The 4-anilinopyrimidine derivative 6
was prepared by treating it with 3,5-dimethoxyaniline in tetra-
hydrofuran (THF) with i-Pr2NEt. The reaction of compound 6
with aqueous ammonia in N,N-dimethylformamide (DMF) gave
6-aminopyrimidine derivative 7. Hydrolysis of the nitrile group
of 7 under basic conditions provided the corresponding carbox-
amide derivative 8. Then, 7-phenylamino-imidazo[1,2-c]-pyrimi-
Compound
IC50 (lM)
ZAP-70
Syk
Src
Grb2
3
4
9
4.013
>50013
NDb
28213
NDb
NTc
>50013
NDb
0.01114
3.0a
>10a
NTc
dine-8-carboxamide
9 was constructed by treatment with
a
IC50 values are for the inhibition of intracellular ZAP-70 or Syk and were
determined in duplicate.
chloroacetaldehyde in DMF under suitable thermal conditions.
Finally, 5-amino-imidazo[1,2-c]pyrimidine-8-carboxamide deriva-
tives 10a–t were synthesized by substitution with the corre-
sponding commercial amines in DMF or N-methyl-2-pyrrolidone
(NMP). In addition, compounds 10t, 11, 12, 15, 20–22, 26, and 27
were synthesized following the method shown in Scheme 2.
Compound 10t was treated with 4-carbometoxybenzylamine,
and hydrolysis of the methyl ester group of 10t afforded the cor-
b
ND, no data available.
NT, not tested.
c
kinase inhibitors.15 This compound moderately inhibited ZAP-70
and had less of an inhibitory effect on closely related Syk. Therefore,
we decided to optimize compound 9 to find an improved selective
ZAP-70 inhibitor. Here, we describe the development a potent,
Scheme 1. Reagents and condition: (a) 3,5-dimethoxyaniline, i-Pr2NEt, THF; (b) NH4OH, DMF; (c) 30% H2O2, 5 M NaOH, DMSO, EtOH; (d) ClCH2CHO, DMF; (e) commercial
amines, i-Pr2NEt, DMF or NMP.

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A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
Scheme 2. Reagents and condition: (a) 4-MeOOCC6H4CH2NH2, i-Pr2NEt, NMP; (b) 2 M NaOH, EtOH; (c) CDI, THF; NH4OH; (d) LiAlH4, THF; 4 M HCl–EtOAc; (e) 9, i-Pr2NEt,
NMP; (f) tBuOC(NH)CCl3, BF3ꢀEt2O, THF; (g) potassium phthalimide, DMF; (h) NH2NH2 H2O, EtOH; (i) TFA, CH2Cl2; (j) (Boc)2NH, NaH, THF; (k) 4 M HCl–EtOAc.
responding carboxylic acid 11. Carboxamide derivative 12 was
prepared by the condensation of carboxylic acid 11 and ammonia
with 1,10-carbonyldiimidazole (CDI). Compound 15 was synthe-
sized by treatment with 14, which was prepared from 4-cyano-
benzaldehyde 13 via lithium aluminum hydride reduction.
Compound 20 was synthesized from tert-butyl 4-aminomethyl-
phenylacetate 19, which was synthesized from commercial
4-bromomethylphenylacetic acid 16 in three steps. The hydroly-
sis of the tert-butyl ester group in compound 20 under acidic
conditions afforded compound 21. Compound 22 was prepared
in the same manner as 12. Compound 26 was prepared from 9
by substitution with ethyl (E)-4-aminomethylcinnamate 25,
which was synthesized from commercial 23 in two steps. Hydro-
lysis of the ethoxycarbonyl group of 26 under basic conditions
afforded the corresponding carboxylic acid 27. The chemical
structures of these compounds were confirmed by 1H NMR, mass
spectroscopy, and element analysis.
3. Results and discussion
3.1. SAR for ZAP-70 kinase and Syk kinase
The Syk and ZAP-70 inhibitory activity of our synthesized com-
pounds were evaluated using a coupled spectrophotometric en-
zyme assay (Tables 1–3).
As noted previously, we discovered that the imidazo[1,2-
c]pyrimidine-8-carboxamide derivative 9 was a ZAP-70-selective
inhibitor. This compound had moderate ZAP-70 inhibition
(IC50 = 3.0
lM) and did not inhibit closely related Syk (IC50 >
10 M), as shown in Table 1. In our research on Syk family kinase
l
inhibitors, we found out that introducing substituents at the C5
position of the imidazo[1,2-c]pyrimidine skeleton enhanced the
ZAP-70 inhibitory effect. Therefore, we investigated the effects of
substituents at C5 position of the imidazo[1,2-c]pyrimidine
framework.

A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
287
Table 2
amine at the 5-position, had improved inhibition of ZAP-70
(IC50 = 0.40 M) compared to compound 9 and retained good
selectivity against Syk kinase (IC50 = 4.3 M). The N–Me deriva-
tive 10e had weaker inhibitory activity against ZAP-70
(IC50 = 5.5 M) than compound 9. The introduction of a phen-
ethyl group at the C5 position (10f) also decreased the ZAP-70
inhibitory activity (IC50 = 5.1 M). To examine the effect of sub-
ZAP-70 and Syk inhibitory activity of imidazo[1,2-c]pyrimidine derivatives substi-
tuted with various amino groups attached to the C-2 position
l
l
l
l
stituents on the benzyl position of 10d, we synthesized optically
active 1-phenethylamine derivatives. The (R)-1-phenethylamine
derivative 10g had the same ZAP-70 inhibitory activity
a
Compound
R
IC50
(
lM)
(IC50 = 0.41
l
M) as 10d, with extremely good selectivity against
M). In contrast, the (S)-1-phenethyl-
ZAP-70
Syk
ZAP-70 over Syk (IC50 > 10
l
9
SMe
NH2
3.0
10.0
1.9
3.5
0.40
5.5
5.1
0.41
0.93
>10
7.1
4.3
0.23
4.3
13.4
4.8
amine derivative 10h showed attenuation of both the ZAP-70
inhibitory activity and selectivity against Syk. These findings
indicate that the N–H group at the 5-postion of imidazo[1,2-
c]pyrimidine is critical for ZAP-70 inhibition. Moreover, the ben-
zylamino group was preferable for both ZAP-70 inhibition and
selectivity against Syk. Extension of the methylene group be-
tween the N–H group and the benzene ring was not favorable
for ZAP-70 kinase inhibition, but had no influence on Syk inhibi-
tion. Introduction of a methyl group at the benzyl position in
compound 10d was effective for ZAP-70 inhibition. The configu-
ration at this position influenced the ZAP-70 inhibitory activity;
in particular, the R-isomer was favorable in terms of both ZAP-
70 inhibition and selectivity against Syk.
Next, we evaluated the effects of substituents on the benzene
ring in the benzylamino group, to improve of ZAP-70 inhibitory
activity while retaining selectivity against Syk kinase; the results
are summarized in Table 3. The 2-methoxy derivative 10l and
3-methoxy derivative 10k had almost the same or inferior inhib-
itory activity, while the 4-methoxy derivative 10j had superior
ZAP-70 inhibitory potency and selectivity against Syk kinase
compared to 10d. The 4-fluoro derivative 10i had the same po-
tency as the 4-methoxy derivative 10j. Compared to compound
10d, the 3,5-difluorobenzyl derivative 10m had less inhibitory
activity for ZAP-70, while the ZAP-70 inhibitory activity of 10n
was enhanced by introducing a 2,4-diflurobenzyl group. Regard-
ing replacement of the phenyl group with a heteroaromatic
group, the 4-pyridyl derivative 10o and benzimidazolyl derivative
10q showed improved ZAP-70 inhibitory activity, while the 2-
furanyl derivative 10p showed weak inhibition against ZAP-70
compared to compound 10d. These results also indicated that
substitution at the ortho or para-position affected the ZAP-70
inhibitory activity, and substitution at the para-position had the
most promising ZAP-70 inhibitory effect. Incorporating a hetero-
aromatic group at the 5-position also improved the ZAP-70 kinase
inhibition.
10a
10b
10c
10d
10e
10f
10g
10h
NHCH2CH2OH
NHCH2CH2NH2
NHCH2Ph
N(Me)CH2Ph
NHCH2CH2Ph
(R)-NHCH(Me)Ph
(S)-NHCH(Me)Ph
>10
3.3
a
The IC50 values were determined in duplicate.
Initially, we synthesized NH-linked derivatives by incorporat-
ing various amine units; the results are shown in Table 2. Com-
pound 10a had weak and similar inhibitory potency for ZAP-70
and Syk kinase. Compound 10b, with a hydroxyethylamino
group at the 5-position of the imidazo[1,2-c]pyrimidine core,
had slightly improved ZAP-70 and Syk inhibitory activity com-
pared with 10a. The 5-ethylenediamino derivative 10c had al-
most the same potency of ZAP-70 inhibition, but enhanced Syk
inhibitory activity (IC50 = 3.5 lM for ZAP-70, 0.23 lM for Syk)
in comparison with 10a. Compound 10d, which had benzyl-
Table 3
ZAP-70 and Syk inhibitory activity of 2-benzylaminoimidazo[1,2-c]pyrimidine deriv-
atives substituted with various benzylamino groups
a
Compound
Ar
IC50
ZAP-70
(lM)
Syk
10d
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
10t
11
12
15
20
21
22
26
27
Ph
4-FC6H4
4-MeOC6H4
3-MeOC6H4
0.40
0.22
0.23
0.53
0.34
0.91
0.15
0.25
0.92
0.20
0.15
0.19
0.42
0.18
0.66
0.69
3.20
0.24
0.48
0.088
0.047
4.3
1.9
11.2
6.7
5.8
9.8
3.7
2.3
2.5
As substituents in the para-position were promising in terms of
both the ZAP-70 inhibitory activity and selectivity against Syk
kinase, we concentrated our efforts on preparing para-substituted
benzylamino derivatives at the 5-position of the imidazo[1,2-c]
pyrimidine skeleton. The sulfonamide 10r and sulfone derivative
10s showed improved ZAP-70 inhibitory activity along with good
selectivity against Syk compared to 10d. The methoxycarbonyl
derivative 10t, carboxamide derivative 12, and 4-hydroxymethyl
derivative 15 had lower ZAP-70 kinase inhibitory potency com-
pared to 10d. However, compound 11 incorporating a carboxyl
group at the para-position had better ZAP-70 inhibition than com-
pound 10d. The inhibitory activity of tert-butoxycarbonylmethyl-
phenyl derivative 20 against ZAP-70 was attenuated. The
phenylacetic acid derivative 21 and phenylacetamide derivative
22 had similar or slightly improved inhibitory activity against
ZAP-70 compared to 10d. Finally, compounds 26 and 27 with an
acrylate moiety in the para-position had superior ZAP-70 inhibi-
tory activity compared to other modifications. Compound 26
2-MeOC6H4
3,5-Di-FC6H3
2,4-Di-FC6H3
4-Pyridyl
2-Furanyl
2-Benzimidazoyl
4-C6H4SO2NH2
4-C6H4SO2Me
4-C6H4CO2Me
4-C6H4CO2H
4-C6H4CONH2
4-C6H4CH2OH
4-C6H4CH2CO2tBu
4-C6H4CH2CO2H
4-C6H4CH2CONH2
(E)-4-C6H4CH@CHACO2Et
(E)-4-C6H4CH@CHACO2H
9.8
>10
11.3
4.8
8.1
8.7
8.5
>10
0.76
1.9
>10
0.69
a
The IC50 values were determined in duplicate.
showed good inhibitory potency for ZAP-70 (IC50 = 0.088 lM) and

288
A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
Table 4
shown in Figure 2. These hydrogen-bonded interactions and the
CH– interaction could play a crucial role in the ZAP-70 inhibitory
Selectivity profiles of the synthesized compounds
p
a
activity. Next, we tried to determine the reason for the selectivity
over Syk using the docking mode of Syk and compound 10d. How-
ever, we could not identify any factors explaining the ZAP-70-spe-
cific inhibition over Syk. Nevertheless, we believe that interactions
between ZAP-70 and compound 10d at the characteristic Pro421-
Leu422-His423-Lys424 motif in ZAP-70 contribute to the ZAP-70-
specific inhibition over Syk based on the results of SARs studies.
Next, we examined the docking mode between ZAP-70 and com-
pound 26 or 27, as shown in Figures 3 and 4, respectively. The ben-
Compound
IC50
(
l
M)
ZAP-70 Syk
Lck
PKCb2 EGFR Src ERK-1 JNK 1 p38a
10d
26
27
0.40
4.3
>12.5
0.69 >10
>25
NTb
>12.5 >10
>10
NTb
>10
NTb NTb
>10 >10
NTb
>10
90%c
NTb
>10
>10
0.088
0.047
89%c >10 >10
a
The IC50 values were determined in duplicate.
NT, not tested.
b
c
% Inhibition of control values at 10ꢁ5 M concentration.
zene ring of the 5-benzylamino group created a CH–
with Pro421, similar to 10d; moreover, the acrylate group of com-
pound 26 formed a CH– interaction between the ethenyl group
and methylene group in Arg465 and the CH– interaction between
ethyl group of the ester and guanidyl group of Arg465 (Fig. 3).22 We
believe that these CH– interactions are responsible for enhancing
p interaction
excellent selectivity against Syk (IC50 > 10 lM). Based on these
p
findings, sulfonamide and sulfone groups are better substituents
for ZAP-70 inhibition and Syk selectivity compared to other sub-
stituents. Extension of the methylene group, such as in 20, 21,
and 22, might be unfavorable for ZAP-70 inhibition, while the
p
p
the ZAP-70 inhibitory activity of compound 26. For the docking
mode of the ZAP-70-compound 27 complex, the carboxyl group
of compound 27 produced a hydrogen bond with His423 (Fig. 4).
Similarly, the carboxyl group of compound 27 produced a hydro-
gen bond with Asn457 in Syk (Fig. 5). Since compound 27 had en-
hanced inhibitory activity for both ZAP-70 and Syk, these results
suggest that hydrogen-bonded interactions between the carboxyl
group and His423 in ZAP-70 and Asn457 in Syk improve the kinase
inhibitory activity. We believe that these findings support the
docking mode between compounds 26 and 27 and ZAP-70.
Concerning the selectivity over Lck, some studies have reported
that the gatekeeper pocket contributes to ligand selectivity in pro-
tein kinase.20,23 Looking at the homology model, the gatekeeper
residue in ZAP-70 is Met414, and its side chain makes a narrow
gatekeeper pocket. Conversely, the gatekeeper residue in Lck is
Thr316, which enlarges the gatekeeper pocket in comparison to
ZAP-70. However, based on the docking mode of compound 10d,
it was difficult to explain the selectivity profile for ZAP-70 and
introduction of a
a desirable modification for ZAP-70 inhibitory activity.
p-electron unit, such as an acrylate group, was
3.2. Discussion of the kinase selectivity profiles using docking
mode
The kinase selectivity profiles of representative compounds
10d, 26, and 27 were assayed against an extended panel of tyrosine
and serine/threonine kinases; the results are summarized in Table
4. Syk and ZAP-70 were found to possess high structural homology.
Nevertheless, these compounds showed excellent ZAP-70 kinase
inhibition and high selectivity for ZAP-70 over Syk. We considered
the reason for the enzyme selectivity profile of our synthesized
compounds using a ZAP-70 homology model. We constructed a
ZAP-70 homology model based on the published crystal structure
of an activated Lck kinase domain16,17 using FAMS,18 and the bind-
TM
ing modes of 10d, 26, and 27 were examined using ADAM soft-
ware.19 In the docking mode for compound 10d, the N–H group
and carbonyl group of the 8-carbamoyl group interacted with the
carbonyl group of Glu415 and the N–H group of Ala417, respec-
tively, and the N–H group of aniline formed a hydrogen bond with
the carbonyl group of Ala417. The imidazole ring of the imi-
dazo[1,2-c]pyrimidine skeleton was located close to a hydrophobic
pocket, the so-called gatekeeper pocket,20 and effectively formed a
CH–
p
interaction21 with Val352. The 3,5-dimethoxyphenyl group
interaction with the meth-
at the C7-position also created a CH–
p
ylene groups of both Leu344 and Gly420. One of the motifs charac-
terizing ZAP-70 is Pro421-Leu422-His423-Lys424, whereas in Syk,
this motif is Pro455-Leu456-Asn457-Lys458.20 Looking at the
docking model of ZAP-70 and the compound 10d complex, a dis-
tinct pocket was surrounded by Pro421, His423, Lys424, and
Arg465. We were able to observe the CH–p interaction between
the benzene ring of the C5-benzylamino group and Pro421, as
Figure 3. Docking mode of compound 26 with ZAP-70.
Figure 2. Docking mode of the ZAP-70 homology model and compound 10d.

A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
289
evaluated our selective ZAP-70 inhibitors in cellular or in vivo
assays, and plan to investigate the in vivo efficacy of the IL-2-sup-
pressing effect of compound 26 and its derivatives further. In addi-
tion, we will explore the reason for the specificity of the
compounds we discovered for the inhibition of ZAP-70 over Syk.
The results of these evaluations will be published in due course.
We anticipate that compound 26 or a derivative will prove to be
a new useful and effective immunosuppressant and that our dis-
covery of a novel ZAP-70-selective inhibitor will contribute to
the success of medical transplantation.
Figure 4. Docking mode of compound 27 with ZAP-70.
5. Experimental
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
patterns were shown as the following abbreviations: br, broad; d,
doublet; m, multiplet; s, singlet; t, triplet; and 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
Figure 5. Docking mode of compound 27 with Syk.
Lck on this site. Looking at the docking mode of compound 10d
with Lck, no specific interaction occurred between the benzyl-
amino residue at the 5-position of 10d and the sugar pocket sur-
rounding Gly322, Ser323, Asp326, and Leu371 in Lck (Fig. 6). In
contrast, the benzylamino residue of 10d made a good fit in the
sugar pocket consisting of Pro421-Leu422-His423-Lys424 and
Arg465 in ZAP-70. This characteristic pocket would contribute to
the ZAP-70-specific inhibition of 10d over Lck.
APS Daisogel IR-60 (particle size 25–40
lM) from Daiso Co., Ltd.
All reagents and solvent were commercially available unless other-
wise indicated.
5.1.1. 4-Chloro-6-(3,5-dimethoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (6)
4. Conclusions
To a mixture of 4,6-dichloro-2-methylsulfanylpyrimidine-5-
carbonitrile
5 (6.08 g, 27.6 mmol) and 3,5-dimethoxyaniline
We synthesized various 5-benzylaminoimidazo[1,2-c]pyrimi-
dine-8-carboxamide derivatives and succeeded in discovering a
potent, highly selective ZAP-70 inhibitor. Compound 26 had excel-
(4.32 g, 28,2 mmol) in THF (130 mL) was added diisopropylethyl-
amine (5.05 mL, 29.0 mmol) at 0 °C and the mixture was stirred
overnight at room temperature. Resulting precipitates were col-
lected by suction filtration, washed with water and dried under
reduced pressure to give 8.91 g of 6 (91%) as a pale yellowish solid:
1H NMR (DMSO-d6) d: 2.55 (3H, s), 3.80 (6H, s), 6.33 (1H, t,
J = 2.0 Hz), 6.76 (2H, d, J = 2.0 Hz), 7.15 (1H, br s); MS m/z: 355
(M+H)+.
lent inhibitory activity for ZAP-70 (IC50 = 0.088
tivity over other tyrosine kinases, especially the structurally
similar Syk kinase (IC50 > 10 M). As far as we know, no other
lM) and high selec-
l
compound is reported to have excellent ZAP-70 inhibitory activity
and highly selectivity for ZAP-70 kinase over Syk. We have not yet
Figure 6. Docking mode of compound 10d with ZAP-70 or Lck. The left side shows the complex of 10d and ZAP-70. The right side shows the complex of 10d with Lck.

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A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
5.1.2. 4-Amino-6-(3,5-dimethoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (7)
s); MS m/z: 373 (M+H)+. Anal. Calcd for: C17H20N6O4: C, 54.83; H,
5.41; N, 22.57. Found: C, 54.56; H, 5.43; N, 22.43.
To a suspension of 6 (8.60 g, 25.5 mmol) in DMF (52 mL) was
added 28% ammonia solution (14 mL) and the mixture was heated
for 2 h at 60 °C. After cooled to room temperature, the mixture was
poured into water. Resulting precipitates were collected by suction
filtration, and washed with water to give 8.12 g of 7 (quant.) as
white solid: 1H NMR (DMSO-d6) d: 2.40 (3H, s), 3.71 (6H, s),
6.20–6.25 (1H, m), 6.85–6.90 (2H, m), 7.41 (2H, br s), 9.08 (1H,s);
MS m/z: 318 (M+H)+.
5.1.7. 5-(2-Aminoethylamino)-7-(3,5-dimethoxyphenylamino)-
imidazo[1,2-c]pyrimidine-8-carboxamide (10c)
The title compound was prepared from 9 and 2-aminoethyl-
amine in the same manner as described 10a, and obtained as white
solid (46%): mp 221–223 °C (MeOH–THF); IR (KBr) 1668, 1595,
1502, 1478, 1372 cmꢁ1
;
1H NMR (DMSO-d6) d: 2.85 (2H, t,
J = 6.0 Hz), 3.25–3.45 (3H, br), 3.56 (2H, t, J = 6.0 Hz), 3.76 (6H, s),
6.17 (1H, t, J = 2.0 Hz), 6.89 (2H, d, J = 2.0 Hz), 7.34 (1H, d,
J = 1.5 Hz), 7.41 (1H, d, J = 3.0 Hz), 7.90 (1H, d, J = 1.5 Hz), 9.54
(1H, d, J = 3.0 Hz), 12.51 (1H, s); MS m/z: 372 (M+H)+. Anal. Calcd
for: C17H21N7O3ꢀC5H9NO: C, 56.16; H, 6.43; N, 23.81. Found: C,
55.57; H, 6.35; N, 23.66.
5.1.3. 4-Amino-6-(3,5-dimethoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carboxamide (8)
To a stirred suspension of compound 7 in DMSO (80 mL) and
EtOH (80 mL) were added reagent 5 M NaOH (25.2 mL,
0.126 mol) solution and 30% H2O2 solution (14.3 mL, 0.126 mol)
at 0 °C and the mixture was stirred for 30 min at room tempera-
ture. The mixture was added to 2 M HCl (63 mL) solution at 0 °C
and resulting precipitates were collected by suction filtration and
washed with water. This precipitates were suspended in EtOAc
and refluxed for 1 h. After cooling to room temperature, resulting
precipitates were collected by suction filtration, and washed with
EtOAc to give 3.04 g of 8 (36%) as white solid: mp 232–235 °C;
1H NMR (DMSO-d6) d: 2.44 (3H, s), 3.72 (6H, s), 6.10–6.20 (1H,
m), 6.80–6.90 (4H, m), 7.54 (2H, br s), 9.87 (1H, s); MS m/z: 336
(M+H)+. Anal. Calcd for C14H17N5O3Sꢀ0.5H2O: C, 48.83; H, 5.27; N,
20.34. Found: C, 48.63; H, 4.95; N, 20.31.
5.1.8. 5-Benzylamino-7-(3,5-dimethoxyphenylamino)-imidazo-
[1,2-c]pyrimidine-8-carboxamide (10d)
To a suspension of compound 9 (0.120 g, 0.334 mmol) in NMP
(1.2 mL) was added benzylamine (0.182 mL, 1.67 mmol) and the
mixture was heated for 3 h at 90 °C. Water was added and ex-
tracted with EtOAc. Organic phase was washed with 1 M HCl solu-
tion, water, and brine, successively; dried over MgSO4, filtered; and
concentrated in vacuo. Resulting residue was purified by column
chromatography on silica gel with hexane/EtOAc = 1:2 and recrys-
tallized from EtOH–EtOAc to give 0.053 g of 10d (38%) as pale yel-
lowish solid: mp 244–246 °C (EtOH–EtOAc); IR (KBr) 1599, 1554,
1502, 1480, 1154 cmꢁ1 1H NMR (DMSO-d6) d: 3.62 (6H, s), 4.83
;
5.1.4. 7-(3,5-Dimethoxyphenylamino)-5-methylsulfanylimidazo-
[1,2-c]pyrimidine-8-carboxamide (9)
(2H, s), 6.14 (1H, t, J = 2.2 Hz), 6.81 (2H, d, J = 2.2 Hz), 7.20–7.45
(7H, m), 7.90–7.95 (1H, m), 8.90–9.00 (1H, br s), 9.50–9.60 (1H,
m), 12.48 (1H, br s); MS m/z: 419 (M+H)+. Anal. Calcd for
C22H22N6O3: C, 63.15; H, 5.30; N, 20.08. Found: C, 63.17; H, 5.30;
N, 20.02.
To a stirred solution of 8 (2.90 g, 8.65 mmol) in DMF (30 mL)
was added 40% chloroacetaldehyde (5.1 mL, 25.9 mmol) solution
and the mixture was heated for 5 h at 60 °C. After cooling to room
temperature, resulting precipitates were collected by suction filtra-
tion and washed with EtOAc to give 2.40 g of 9 (80%) as yellow so-
lid: mp 232–233 °C (THF); 1H NMR (DMSO-d6) d: 2.80 (3H, s), 3.76
(6H, s), 6.20–6.30 (1H, m), 6.75–6.85 (2H, m), 7.45–7.55 (1H, m),
7.70–7.75 (1H, m), 7.85–7.90 (1H, m), 9.60–9.65 (1H, br), 12.25–
12.30 (1H, br); MS m/z: 360 (M+H)+. Anal. Calcd for C16H17N5O3S:
C, 53.47; H, 4.77; N, 19.49. Found: C, 53.45; H, 4.82; N, 19.54.
5.1.9. 5-(N-Benzylmethylamino)-7-(3,5-dimethoxyphenylamino)-
imidazo[1,2-c]pyrimidine-8-carboxamide (10e)
The title compound was prepared from 9 and N-benzylmethyl-
amine in the same manner as described 10d, and obtained as pale
yellowish crystal (38%): mp 215–216 °C (EtOH–EtOAc); IR (KBr)
1594, 1505, 1410, 1155 cmꢁ1 1H NMR (DMSO-d6) d: 3.21 (3H, s),
;
3.67 (6H, s), 4.91 (2H, s), 6.15 (1H, t, J = 2.0 Hz), 6.79 (2H, d,
J = 2.0 Hz), 7.28–7.42 (6H, m), 7.55 (1H, d, J = 3.0 Hz), 7.62 (1H, d,
J = 2.0 Hz), 9.69 (1H, d, J = 3.0 Hz), 12.42 (1H, br s); MS m/z: 433
(M+H)+. Anal. Calcd for C23H24N6O3: C, 63.88; H, 5.59; N, 19.43.
Found: C, 63.71; H, 5.51; N, 19.51.
5.1.5. 5-Amino-7-(3,5-dimethoxyphenylamino)-imidazo[1,2-c]-
pyrimidine-8-carboxamide (10a)
A mixture of 9 (0.150 g, 0.417 mmol) and 28% ammonia solu-
tion (0.5 mL) in NMP (2 mL) was heated overnight at 100 °C. The
mixture was poured into water and extracted with EtOAc. The or-
ganic phase was washed with water and brine, sequentially, dried
over anhydrous MgSO4, and evaporated in vacuo. Resulting residue
was purified by column chromatography on silica gel with hexane/
EtOAc = 1:2 and recrystallized from THF to give 0.049 g of 10a
(36%) as pale green crystal: mp 274–276 °C (THF); IR (KBr) 1595,
5.1.10. 7-(3,5-Dimethoxyphenylamino)-5-phenethylamino-
imidazo[1,2-c]pyrimidine-8-carboxamide (10f)
The title compound was prepared from 9 and phenethylamine
in the same manner as described 10d, and obtained as pale yellow-
ish crystal as yellowish solid (27%): mp 158–160 °C (THF); IR (KBr)
1508, 1481, and 1150 cmꢁ1
;
1H NMR (DMSO-d6) d: 3.76 (6H, s),
1599, 1558, 1500, 1150 cmꢁ1 1H NMR (DMSO-d6) d: 2.97 (2H, t,
;
6.16 (1H, s), 6.86 (2H, s), 7.33 (1H, s), 7.35 (1H, br s), 7.79 (1H, s),
8.14 (2H, br s), 9.51 (1H, br s), 12.4 (1H, s); MS m/z: 329 (M+H)+.
Anal. Calcd for C15H16N6O3: C, 54.87; H, 4.91; N, 25.60. Found: C,
54.88; H, 5.09; N, 25.21.
J = 7.0 Hz), 3.67 (6H, s), 3.72–3.80 (2H, m), 6.19 (1H, t, J = 2.0 Hz),
6.85 (2H, d, J = 2.0 Hz), 7.20–7.32 (5H, m), 7.35 (1H, d, J = 2.5 Hz),
7.43 (1H, d, J = 3.5 Hz), 7.86 (1H, d, J = 2.5 Hz), 8.54 (1H, t,
J = 5.0 Hz), 9.53 (1H, d, J = 3.5 Hz), 12.48 (1H, br s); MS m/z: 433
(M+H)+. Anal. Calcd for C23H24N6O3ꢀC5H9NO: C, 63.26; H, 6.26; N,
18.44. Found: C, 63.19; H, 6.28; N, 18.32.
5.1.6. 7-(3,5-Dimethoxyphenylamino)-5-(2-hydroxyethylamino)-
imidazo[1,2-c]pyrimidine-8-carboxamide (10b)
The title compound was prepared from 9 and 2-hydroxyethyl-
amine in the same manner as described 10a, and obtained as white
solid (13%): mp 261–263 °C (MeOH–THF); IR (KBr) 1616, 1593,
5.1.11. 7-(3,5-Dimethoxyphenylamino)-5-((R)-1-phenylethyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10g)
The title compound was prepared from 9 and (R)-phenylethyl-
amine in the same manner as described 10d, and obtained as pale
yellowish crystal as pale yellowish solid (24%): mp 99–101 °C
1550, 1507 cmꢁ1 1H NMR (DMSO-d6) d: 3.61–3.70 (4H, m), 3.75
;
(6H, s), 4.90 (1H, t, J = 5.0 Hz), 6.16 (1H, t, J = 2.0 Hz), 6.86 (2H, d,
J = 2.0 Hz), 7.34 (1H, d, J = 1.5 Hz), 7.41 (1H, d, J = 3.0 Hz), 7.93
(1H, d, J = 1.5 Hz), 8.46 (1H, s), 9.55 (1H, d, J = 3.0 Hz), 12.52 (1H,
(EtOAc); ½a 2D8
ꢂ
+178.0° (c 0.30, MeOH); IR (KBr) 1599, 1558, 1497,
1457, 1151 cmꢁ1
;
1H NMR (DMSO-d6) d: 1.63 (3H, d, J = 7.0 Hz),

A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
291
3.73 (6H, br s), 5.50 (1H, quint, J = 7.0 Hz), 6.22 (1H, t, J = 2.0 Hz),
6.79 (2H, d, J = 2.0 Hz), 7.21–7.35 (3H, m), 7.38 (1H, d, J = 2.0 Hz),
7.40–7.50 (3H, m), 8.09 (1H, d, J = 2.0 Hz), 8.64 (1H, d, J = 2.0 Hz),
9.55 (1H, br s), 12.43 (1H, s); MS m/z: 433 (M+H)+. Anal. Calcd
for C23H24N6O3ꢀ0.36H2O: C, 62.93; H, 5.67; N, 19.14. Found: C,
63.38; H, 5.67; N, 18.64.
IR (KBr) 1598, 1558, 1492, 1147 cmꢁ1
;
1H NMR (DMSO-d6) d:
3.62 (6H, s), 3.82 (3H, s), 4.79 (2H, s), 6.11 (1H, t, J = 2.0 Hz), 6.77
(2H, d, J = 2.0 Hz), 6.89 (1H, t, J = 7.0 Hz), 7.24 (1H, d, J = 8.0 Hz),
7.20-7.30 (2H, m), 7.37 (1H, d, J = 2.0 Hz), 7.44 (1H, br s), 7.96
(1H, s), 8.73 (1H, br s), 9.56 (1H br s), 12.51 (1H, s); MS m/z: 449
(M+H)+. Anal. Calcd for C23H24N6O4: C, 61.60; H, 5.39; N, 18.74.
Found: C, 61.38; H, 5.29; N, 18.54.
5.1.12. 7-(3,5-Dimethoxyphenylamino)-5-((S)-1-phenylethyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10h)
5.1.17. 5-(3,5-Difluorobenzylamino)-7-(3,5-dimethoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10m)
The title compound was prepared from 9 and (S)-phenylethyl-
amine in the same manner as described for 10d, and obtained as
pale yellowish crystal as pale yellowish solid (18%): mp 99–
The title compound was prepared from 9 and 3,5-difluoroben-
zylamine in the same manner as described 10d, and obtained as pale
yellowish solid (35%): mp 252–253 °C (MeOH– EtOAc); IR (KBr)
101 °C (EtOAc); ½a D28
ꢂ
–185.2° (c 0.31, MeOH); IR (KBr) 1599,
1559, 1497, 1457, 1152 cmꢁ1
;
1H NMR (DMSO-d6) d: 1.63 (3H, d,
1599, 1505, 1456, 1149 cmꢁ1 1H NMR (DMSO-d6) d: 3.66 (6H, s),
;
J = 7.0 Hz), 3.73 (6H, br s), 5.49 (1H, quint, J = 7.0 Hz), 6.21 (1H, t,
J = 2.0 Hz), 6.78 (2H, d, J = 2.0 Hz), 7.21-7.35 (3H, m), 7.37 (1H, d,
J = 2.0 Hz), 7.40–7.50 (3H, m), 8.08 (1H, d, J = 2.0 Hz), 8.64 (1H, d,
J = 2.0 Hz), 9.54 (1H, br s), 12.42 (1H, s); MS m/z: 433 (M+H)+. Anal.
Calcd for C23H24N6O3ꢀ0.5H2O: C, 62.57; H, 5.71; N, 19.03. Found: C,
63.01; H, 5.66; N, 18.66.
4.76–4.82 (2H, br), 6.15 (1H, t, J = 2.0 Hz), 6.72 (2H, d, J = 2.0 Hz),
7.06–7.15 (3H, m), 7.36–7.39 (1H, br), 7.43–7.48 (1H, m), 7.86–
7.89 (1H, br), 8.96–9.03 (1H, br), 9.52–9.58 (1H, m), 12.43 (1H, s);
MS m/z: 455 (M+H)+. Anal. Calcd for C22H20F2N6O3ꢀ0.1H2O: C,
57.91; H, 4.46; N, 18.42. Found: C, 57.56; H, 4.36; N, 18.35.
5.1.18. 5-(2,4-Difluorobenzylamino)-7-(3,5-dimethoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10n)
5.1.13. 7-(3,5-Dimethoxyphenylamino)-5-(4-fluorobenzyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10i)
The title compound was prepared from 9 and 2,4-difluoroben-
zylamine in the same manner as described for 10d, and obtained
as pale yellowish solid (35%): mp 242–243 °C (MeOH–EtOAc); IR
The title compound was prepared from 9 and 4-fluorobenzyl-
amine in the same manner as described for 10d, and obtained as
pale yellowish crystal as pale yellowish solid (30%): mp 255–
(KBr) 1602, 1558, 1505, 1158 cmꢁ1 1H NMR (DMSO-d6) d: 3.66
;
257 °C (MeOH: EtOAc); IR (KBr) 1601, 1558, 1502, 1158 cmꢁ1
;
1H
(6H, s), 4.79–4.85 (2H, br), 6.15 (1H, t, J = 2.0 Hz), 6.74 (2H, d,
J = 2.0 Hz), 7.02 (1H, dt, J = 2.0, 9.0 Hz), 7.28 (1H, dt, J = 2.0,
9.0 Hz), 7.37 (1H, d, J = 2.0 Hz), 7.42–7.50 (2H, m), 7.92 (1H, d,
J = 2.0 Hz), 8.84–8.89 (1H, br), 9.53–9.57 (1H, m), 12.43 (1H, s);
MS m/z: 455 (M+H)+. Anal. Calcd for C22H20F2N6O3ꢀ0.1H2O: C,
57.91; H, 4.46; N, 18.42. Found: C, 57.92; H, 4.38; N, 18.65.
NMR (DMSO-d6) d: 3.62 (6H, s), 4.80 (1H, d, J = 5.0 Hz), 6.15 (1H,
t, J = 2.0 Hz), 6.79 (2H, d, J = 2.0 Hz), 7.12–7.19 (2H, m), 7.37 (2H,
d, J = 1.5 Hz), 7.41–7.47 (3H, m), 7.90 (1H, d, J = 1.5 Hz), 7.96 (1H,
t, J = 4.5 Hz), 9.55 (1H, d, J = 3.5 Hz), 12.48 (1H, s); MS m/z: 433
(M+H)+. Anal. Calcd for C22H21FN6O3: C, 60.54; H, 4.85; N, 19.26.
Found: C, 60.41; H, 4.79; N, 19.23.
5.1.19. 7-(3,5-Dimethoxyphenylamino)-5-[(pyridin-4-ylmethyl)-
amino]imidazo[1,2-c]pyrimidine-8-carboxamide (10o)
5.1.14. 7-(3,5-Dimethoxyphenylamino)-5-(4-
methoxybenzylamino)imidazo[1,2-c]pyrimidine-8-
carboxamide (10j)
The title compound was prepared from 9 and pyridin-4-ylmeth-
ylamine in the same manner as described for 10d, and obtained as
white solid (7.7%): mp 255–256 °C (MeOH– EtOAc); IR (KBr) 1600,
The title compound was prepared from 9 and 4-methoxybenzy-
amine in the same manner as described for 10d, and obtained as pale
yellowish crystal as pale yellowish solid (30%): mp 203–204 °C
1551, 1505, 1153 cmꢁ1 1H NMR (DMSO-d6) d: 3.63 (6H, s), 4.80–
;
4.85 (2H, br), 6.13 (1H, t, J = 2.0 Hz), 6.69 (2H, d, J = 2.0 Hz), 7.38
(2H, d, J = 6.0 Hz), 7.39 (1H, d, J = 2.0 Hz), 7.44–7.47 (1H, br), 7.90
(1H, d, J = 2.0 Hz), 8.50 (2H, d, J = 6.0 Hz), 9.03–9.07 (1H, br), 9.54
(1H, d, J = 2.0 Hz), 12.44 (1H, s); MS m/z: 420 (M+H)+. Anal. Calcd
for C21H21N7O3ꢀ0.5H2O: C, 58.87; H, 5.17; N, 22.88. Found: C,
58.97; H, 4.98; N, 22.94.
(THF); IR (KBr) 1599, 1559, 1499, 1153 cmꢁ1 1H NMR (DMSO-d6)
;
d: 3.65 (6H, s), 3.72 (3H, s), 4.75 (2H, d, J = 4.0 Hz), 6.15 (1H, t,
J = 2.0 Hz), 6.84 (2H, d, J = 2.0 Hz), 6.88 (2H, d, J = 9.0 Hz), 7.32 (2H,
d, J = 9.0 Hz), 7.36 (1H, d, J = 2.0 Hz), 7.41-7.46 (1H, m), 7.91 (1H, d,
J = 2.0 Hz), 8.86–8.91 (1H, br), 9.52–9.57 (1H, m), 12.50 (1H, s); MS
m/z: 449 (M+H)+. Anal. Calcd for C23H24N6O4: C, 61.60; H, 5.39; N,
18.74. Found: C, 61.48; H, 5.40; N, 18.69.
5.1.20. 7-(3,5-Dimethoxyphenylamino)-5-[(furan-2-ylmethyl)-
amino]imidazo[1,2-c]pyrimidine-8-carboxamide (10p)
5.1.15. 7-(3,5-Dimethoxyphenylamino)-5-(3-methoxybenzyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10k)
The title compound was prepared from 9 and furan-2-ylmethyl-
amine in the same manner as described for 10d, and obtained as
pale green solid (28%): mp 232–233 °C (MeOH–EtOAc); IR (KBr)
The title compound was prepared from 9 and 3-methoxybenzyl-
amine in the same manner as described for 10d, and obtained as pale
yellowish crystal as pale yellowish solid (33%): mp 216–218 °C
1600, 1550, 1501, 1149 cmꢁ1 1H NMR (DMSO-d6) d: 3.69 (6H, s),
;
4.77–4.83 (2H, br), 6.18 (1H, t, J = 2.0 Hz), 6.41(2H, d, J = 2.0 Hz),
6.86 (2H, d, J = 6.0 Hz), 7.36 (1H, d, J = 2.0 Hz), 7.46 (1H, d,
J = 3.0 Hz), 7.62 (1H, t, J = 2.0 Hz), 7.90 (1H, d, J = 2.0 Hz), 8.92–
8.98 (1H, br), 9.54 (1H, d, J = 3.0 Hz), 12.51 (1H, s); MS m/z: 409
(M+H)+. Anal. Calcd for C20H20N6O4: C, 58.82; H, 4.94; N, 20.58.
Found: C, 58.61; H, 4.86; N, 20.58.
(THF); IR (KBr) 1599, 1559, 1492, 1152 cmꢁ1 1H NMR (DMSO-d6)
;
d: 3.64 (6H, s), 3.67 (3H, s), 4.79 (2H, d, J = 4.5 Hz), 6.15 (1H, t,
J = 2.0 Hz), 6.83 (2H, d, J = 2.0 Hz), 6.95–6.98 (2H, m), 7.24 (2H, t,
J = 7.5 Hz), 7.37 (1H, d, J = 2.0 Hz), 7.42–7.47 (1H, m), 7.92 (1H, d,
J = 2.0 Hz), 8.92–8.98 (1H, br), 9.53–9.58 (1H, m), 12.50 (1H, s); MS
m/z: 449 (M+H)+. Anal. Calcd for C23H24N6O4ꢀ0.2H2O: C, 61.11; H,
5.44; N, 18.59. Found: C, 61.14; H, 5.37; N, 18.63.
5.1.21. 5-[(1H-Benzoimidazol-2-ylmethyl)amino]-7-(3,5-dimeth-
oxyphenylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (10q)
The title compound was prepared from 9 and 1H-benzoimidazol-
2-ylmethylamine in the same manner as described for 10d, and
obtained as pale green solid (7.8%): mp 265 °C (dec) (MeOH–EtOAc);
IR (KBr) 1609, 1559, 1506, 1458, 1155 cmꢁ1; 1H NMR (DMSO-d6) d:
3.53 (6H, s), 5.08 (2H, d, J = 5.0 Hz), 6.07 (1H, t, J = 2.0 Hz), 6.73
5.1.16. 7-(3,5-Dimethoxyphenylamino)-5-(2-methoxybenzyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10l)
The title compound was prepared from 9 and 2-methoxyben-
zylamine in the same manner as described for 10d, and obtained
as pale yellowish solid (33%): mp 246–248 °C (MeOH–Toluene);

292
A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
(2H, d, J = 2.0 Hz), 7.12–7.18 (2H, m), 7.39-7.42 (2H, m), 7.49 (1H, d,
J = 3.0 Hz), 7.56–7.58 (1H, m), 7.91 (1H, t, J = 2.0 Hz), 9.11 (1H, t,
J = 5.0 Hz), 9.59 (1H, d, J = 3.0 Hz), 12.38 (1H, s), 12.50 (1H, s); MS
m/z: 459 (M+H)+. Anal. Calcd for C23H22N8O3: C, 60.25; H, 4.84; N,
24.44. Found: C, 59.92; H, 4.79; N, 24.50.
at room temperature. To the mixture was added 28% ammonia
solution (0.3 mL) and stirred for 4 h at room temperature. Volatiles
were evaporated in vacuo and resulting residue was purified by
column chromatography on silica gel with EtOH/EtOAc = 1:30 to
give 0.017 g of 12 (57%) as pale yellowish solid: mp 275 °C (dec);
IR (KBr) 1603, 1570, 1559, 1506, 1150 cmꢁ1 1H NMR (DMSO-d6)
;
5.1.22. 7-(3,5-Dimethoxyphenylamino)-5-(4-sulfamoylbenzyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (10r)
d: 3.63 (6H, s), 4.86 (2H, d, J = 6.0 Hz), 6.14 (1H, t, J = 2.0 Hz), 6.78
(2H, d, J = 2.0 Hz), 7.33, (1H, br s), 7.38 (1H, d, J = 1.5 Hz), 7.43–
7.48 (3H, m), 7.82 (2H, d, J = 8.5 Hz), 7.92 (1H, d, J = 1.5 Hz), 7.94
(1H, br s), 9.02 (1H, t, J = 6.0 Hz), 9.54–9.57 (1H, m), 12.49 (1H,
s); MS m/z: 472 (M+H)+. Anal. Calcd for C23H23N7O4ꢀ0.2H2O: C,
59.40; H, 5.07; N, 21.08. Found: C, 59.49; H, 5.11; N, 20.74.
The title compound was prepared from 9 and 4-sulfamoylben-
zylamine in the same manner as described for 10d, and obtained
as pale green solid (45%): mp 265–267 °C (MeOH–THF); IR (KBr)
1624, 1590, 1540, 1151 cmꢁ1 1H NMR (DMSO-d6) d: 3.63 (6H, s),
;
4.87 (2H, br s), 6.14 (1H, t, J = 2.0 Hz), 6.76 (2H, d, J = 2.0 Hz),
7.32 (2H, br s), 7.37–7.39 (1H, br s), 7.56 (2H, d, J = 8.0 Hz), 7.76
(2H, d, J = 8.0 Hz), 7.89–7.92 (1H, br s), 9.01–9.07 (1H, br), 9.53–
9.57 (1H, m), 12.38 (1H, s), 12.47 (1H, s); MS m/z: 498 (M+H)+.
Anal. Calcd for C22H23N7O5Sꢀ1.0H2O: C, 51.25; H, 4.89; N, 19.02.
Found: C, 51.58; H, 4.58; N, 18.95.
5.1.27. (4-Aminomethyl)benzyl alcohol hydrochloride (14)
To
a suspension of lithium aluminum hydride (0.399 g,
10.5 mmol) in THF (5 mL) was added 4-Formylbenzonitrile 13
(0.918 g, 7.00 mmol) in THF at 0 °C and stirred for 2 h at 60 °C.
To the mixture water (0.4 mL), 2 M NaOH (0.4 mL) and water
(1.2 mL) were added, successively, and resulting precipitates were
filtered off. The filtrate was dried over Na2SO4, and evaporated in
vacuo. After being cooled at 0 °C, 4 M HCl in EtOAc (1.75 mL,
1 equiv) was added to the mixture. After being stirred for 15 min,
resulting precipitates were collected by suction filtration, and
dried under reduced pressure to give 0.914 g of 4-aminomethylb-
enzylalcohol hydrochloride 14 (75%) as pale yellowish solid: 1H
NMR (DMSO-d6) d: 3.95 (2H, s), 4.50 (2H, s), 5.23 (1H, br s), 7.33
(2H, t, J = 8.0 Hz), 7.39 (2H, d, J = 8.0 Hz), 7.50–8.30 (3H, br).
5.1.23. 7-(3,5-Dimethoxyphenylamino)-5-(4-methanesulfonyl-
benzylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (10s)
The title compound was prepared from 9 and 4-methanesulfo-
nylbenzylamine in the same manner as described for 10d, and ob-
tained as pale green solid (54%): mp 266–268 °C (MeOH–THF); IR
(KBr) 1604, 1499, 1480, 1145 cmꢁ1 1H NMR (DMSO-d6) d: 3.18
;
(3H, s), 3.63 (6H, s), 4.90 (2H, br s), 6.15 (1H, t, J = 2.0 Hz), 6.72
(2H, d, J = 2.0 Hz), 7.39 (1H, d, J = 1.5 Hz), 7.46 (1H, d, J = 3.5 Hz),
7.65 (2H, d, J = 8.5 Hz), 7.88 (2H, d, J = 8.5 Hz), 7.90 (1H, d,
J = 1.5 Hz), 9.05–9.09 (1H, br), 9.54 (1H, d, J = 3.5 Hz), 12.45(1H,
s); MS m/z: 497 (M+H)+. Anal. Calcd for C23H24N6O5Sꢀ0.2H2O: C,
55.23; H, 4.84; N, 16.63. Found: C, 55.02; H, 4.84; N, 16.65.
5.1.28. 7-(3,5-Dimethoxyphenylamino)-5-(4-hydroxymethyl-
benzylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (15)
The title compound was prepared from 9 and 14 in the same
manner as described 10d, and obtained as yellowish solid (58%):
mp 209–211 °C (MeOH–THF); IR (KBr) 3393, 1595, 1558, 1506,
5.1.24. Methyl 4-{[8-Carbamoyl-7-(3,5-dimethoxyphenylamino)-
imidazo[1,2-c]pyrimidin-5-ylamino]-methyl}benzoate (10t)
The title compound was prepared from 9 and methyl 4-amin-
omethylbenzoate in the same manner as described for 10d, and ob-
tained as pale green solid (42%): mp 234–235 °C (MeOH–EtOAc); IR
1147 cmꢁ1 1H NMR (DMSO-d6) d: 3.64 (6H, s), 4.46 (2H, d,
;
J = 5.5 Hz), 4.80 (2H, d, J = 5.5 Hz), 5.14 (1H, t, J = 5.5 Hz), 6.14
(1H, t, J = 2.0 Hz), 6.83 (2H, d, J = 2.0 Hz), 7.26 (2H, d, J = 8.0 Hz),
7.33–7.38 (3H, m), 7.40–7.46 (1H, m), 7.92 (1H, br s), 8.96 (1H, t,
J = 5.5 Hz), 9.52–9.56 (1H, m), 12.50 (1H, s); MS m/z: 449 (M+H)+.
Anal. Calcd for C23H24N6O4ꢀ1.0H2O: C, 59.22; H, 5.62; N, 18.02.
Found: C, 59.04; H, 5.55; N, 17.81.
(KBr) 1600, 1599, 1506, 1283, 1147 cmꢁ1 1H NMR (DMSO-d6) d:
;
3.62 (6H, s), 3.83 (3H, s), 4.88 (2H, br s), 6.14 (1H, t, J = 2.0 Hz),
6.73 (2H, d, J = 2.0 Hz), 7.38 (1H, d, J = 2.0 Hz), 7.45 (1H, d,
J = 3.5 Hz), 7.53 (2H, d, J = 8.5 Hz), 7.90–7.94 (3H, m), 9.04–9.08
(1H, br), 9.54 (1H, d, J = 3.5 Hz), 12.45 (1H, s); MS m/z: 477
(M+H)+. Anal. Calcd for C24H24N6O5ꢀ0.5H2O: C, 59.37; H, 5.19; N,
17.31. Found: C, 59.73; H, 5.10; N, 17.53.
5.1.29. tert-Butyl 4-bromomethylphenylacetate (17)
To a mixture of 4-bromomethylphenylacetic acid 16 (1.37 g,
6.00 mmol) and tert-butyl 2,2,2-trichloroacetimidate (2.15 mL,
12.00 mmol) in THF (20 mL) was added boron trifluoride etherate
(1.52 mL, 12.00 mmol) at room temperature and the mixture was
stirred overnight at room temperature. Saturated NaHCO3 solution
was added to the mixture and extracted with EtOAc. The organic
phase was washed with brine, dried over anhydrous MgSO4, and
evaporated in vacuo. Resulting residue was purified by column
chromatography on silica gel with hexane/EtOAc = 5:1 to give 17
as colorless oil: 1H NMR (CDCl3) d:1.44 (9H, s), 3.52 (2H, s), 4.49
(2H, s), 7.24 (2H, d, J = 8.0 Hz), 7.34 (2H, d, J = 8.0 Hz).
5.1.25. 4-{[8-Carbamoyl-7-(3,5-dimethoxyphenylamino)-
imidazo[1,2-c]pyrimidin-5-ylamino]methyl}benzoic acid (11)
To a mixture of 10t (0.066 g, 0.139 mmol) in MeOH (2 mL) and
THF (2 mL) was added 2 M NaOH solution (0.695 mL, 1.39 mmol)
and stirred overnight at room temperature at 60 °C. To the mixture
was added 2 M HCl solution (0.70 mL) at 0 °C, resulting precipitates
were collected by suction filtration, and washed with water. The
precipitates were recrystallized from MeOH–THF to give 0.042 g
of 11 (66%) as flesh colored solid: mp 289 °C (dec) (MeOH–THF);
IR (KBr) 1599, 1544, 1502, 1151 cmꢁ1
;
1H NMR (DMSO-d6) d:
5.1.30. tert-Butyl [4-(1,3-Dioxo-1,3-dihydroisoindol-2-
ylmethyl)phenyl]acetate (18)
3.62 (6H, s), 4.88 (2H, d, J = 5.0 Hz), 6.14 (1H, br s), 6.74 (2H, d,
J = 2.0 Hz), 7.39 (1H, s), 7.46 (1H, br s), 7.50 (2H, d, J = 8.0 Hz),
7.89 (2H, d, J = 8.0 Hz), 7.92 (1H, br s), 9.06 (1H, t, J = 5.0 Hz),
9.55 (1H, br s), 12.46 (1H, s), 12.92 (1H, br s); MS m/z: 463
(M+H)+. Anal. Calcd for C23H22N6O5ꢀ0.4H2O: C, 58.81; H, 4.89; N,
17.89. Found: C, 58.80; H, 4.81; N, 17.53
A mixture of tert-butyl 4-bromomethylphenylacetate 17 (1.43 g,
5.01 mmol) and phthalimide potassium salt (1.02 g, 5.51 mmol) in
DMF (15 mL) was stirred overnight at room temperature. Water
was added to the mixture and extracted with EtOAc. The orgainc
phase was washed with water and brine, sequentially, dried over
anhydrous MgSO4, and evaporated in vacuo. Resulting residue was
purified by column chromatography on silica gel with hexane/
EtOAc = 4:1 to give 1.31 g of 18 (74%) as white solid: 1H NMR (CDCl3)
5.1.26. 5-(4-Carbamoylbenzylamino)-7-(3,5-dimethoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxmide (12)
A mixture of 11 (0.030 g, 0.0649 mmol) and 1,10-carbonyldiim-
idazole (0.011 g, 0.0703 mmol) in THF (1 mL) was stirred overnight
d:
1.42 (9H, s), 3.48 (2H, s), 4.82 (2H, s), 7.21 (2H, d, J = 8.5 Hz), 7.39
(2H, d, J = 8.5 Hz), 7.65–7.73 (2H, m), 7.82–7.87 (2H, m).

A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
293
5.1.31. tert-Butyl (4-{[8-Carbamoyl-7-(3,5-dimethoxyphenyl-
amino)imidazo[1,2-c]pyrimidin-5-ylamino]methyl}phenyl)-
acetate (20)
1H NMR (CDCl3) d: 1.33 (3H, t, J = 7.0 Hz), 1.45 (18H, s), 4.26
(2H, q, J = 7.0 Hz), 4.79 (2H, s), 6.41 (1H, d, J = 16.5 Hz), 7.29
(2H, d, J = 8.0 Hz), 7.48 (2H, d, J = 8.0 Hz), 7.66 (1H, d,
J = 16.5 Hz).
A mixture of 18 (1.25 g, 3.56 mmol) and hydrazine monohy-
drate (0.518 mL, 10.67 mmol) in EtOH (20 mL) was refluxed
overnight. After being cooled to room temperature, resulting
precipitates were filtered off, and the filtrate was concentrated
in vacuo. To the residue was added EtOAc and resulting precip-
itates were filtered off. The filtrate was evaporated in vacuo to
give crude 19 as yellowish oil. The title compound was prepared
from 9 and crude 19 in the same manner as described for 10d,
and obtained as yellowish solid (75%): mp 178–180 °C (EtOAc);
5.1.35. Ethyl (E)-4-aminomethylphenylcinnamate hydrochloride
(25)
To a mixture of 24 (1.60 g, 3.95 mmol) in EtOAc (20 mL) was
added 4 M HCl–EtOAc (9.90 mL, 39.46 mmol) and stirred overnight
at room temperature. Resulting precipitates were collected by suc-
tion filtration and dried under reduced pressure to give 25 (0.645 g,
68%) as white solid: 1H NMR (DMSO-d6) d: 1.26 (3H, t, J = 7.0 Hz),
4.05 (2H, s), 4.20 (2H, t, J = 7.0 Hz), 6.69 (1H, d, J = 16.0 Hz), 7.52
(2H, d, J = 8.5 Hz), 7.66 (1H, d, J = 16.0 Hz), 7.78 (2H, d, J = 8.5 Hz),
8.00–8.80 (3H, br); MS m/z: 206 (M+H)+.
IR (KBr) cmꢁ1 1H NMR (DMSO-d6) d: 1.37 (9H, s), 3.51 (2H, s),
;
3.60 (6H, s), 4.80 (2H, d, J = 4.5 Hz), 6.13 (1H, t, J = 2.0 Hz), 6.80
(2H, d, J = 2.0 Hz), 7.20 (2H, d, J = 8.5 Hz), 7.34 (2H, d,
J = 8.5 Hz), 7.36 (1H, d, J = 1.5 Hz), 7.43–7.46 (1H, m), 7.91 (1H,
d, J = 1.5 Hz), 8.96 (1H, t, J = 4.5 Hz), 9.53–9.57 (1H, m), 12.50
(1H, s); MS m/z: 533 (M+H)+. Anal. Calcd for C28H32N6O5ꢀ1.0H2O:
C, 61.08; H, 6.22; N, 15.26. Found: C, 60.99; H, 6.19; N, 15.04.
5.1.36. Ethyl (E)-(4-{[8-Carbamoyl-7-(3,5-dimethoxyphenyl-
amino)imidazo[1,2-c]pyrimidin-5-ylamino]methyl}phenyl)-
cinnamate (26)
The title compound was prepared from 9 and 25 in the same
manner as described for 10d, and obtained as pale green solid
(37%): mp 172–173 °C (EtOAc–THF); IR (KBr) 1636, 1600, 1559,
5.1.32. (4-{[8-Carbamoyl-7-(3,5-dimethoxyphenylamino)imidazo-
[1,2-c]pyrimidin-5-ylamino]methyl}phenyl)acetic acid (21)
A mixture of compound 20 (0.282 g, 0.530 mmol) and trifluo-
roacetic acid (1.5 mL) in CH2Cl2 (1.5 mL) was stirred for 3 h at
room temperature. Volatiles were evaporated in vacuo and the
residue was dissolved in THF. To the mixture was added NaHCO3
solution and acidified again with 1 M HCl solution. The mixture
was extracted with EtOAc–THF, organic phase was washed with
brine, dried over anhydrous MgSO4, and evaporated in vacuo.
Resulting residue was treated with MeOH to give 21 (0.139 g,
55%) as yellowish solid: mp 266–268 °C (MeOH–THF); IR (KBr)
1507, 1154 cmꢁ1 1H NMR (DMSO-d6) d: 1.25 (3H, t, J = 7.0 Hz),
;
3.63 (6H, s), 3.70-3.90 (2H, br), 4.17 (2H, q, J = 7.0 Hz), 4.84 (2H,
d, J = 5.5 Hz), 6.15 (1H, t, J = 2.0 Hz), 6.61 (1H, d, J = 16.0 Hz), 6.78
(2H, d, J = 2.0 Hz), 7.40 (1H, d, J = 1.5 Hz), 7.43 (2 H, d, J = 8.0 Hz),
7.62 (1H, d, J = 16.0 Hz), 7.68 (2H, d, J = 8.0 Hz), 7.94 (1H, d,
J = 1.5 Hz), 9.06 (1H, br s), 12.25–12.50 (1H, br); MS m/z: 517
(M+H)+. Anal. Calcd for C27H28N6O5ꢀ1.8H2O: C, 59.07; H, 5.80; N,
15.30. Found: C, 59.12; H, 5.48; N, 14.92.
1617, 1591, 1559, 1506, 1146 cmꢁ1
;
1H NMR (DMSO-d6) d:
5.1.37. (E)-(4-{[8-Carbamoyl-7-(3,5-dimethoxyphenylamino)-
imidazo[1,2-c]pyrimidin-5-ylamino]methyl}phenyl)cinnamic
acid (27)
3.53 (2H, s), 3.62 (6H, s), 4.80 (2H, d, J = 5.5 Hz), 6.14 (1H, br
s), 6.81 (2H, d, J = 2.0 Hz), 7.21 (2H, d, J = 8.0 Hz), 7.34 (2H, d,
J = 8.0 Hz), 7.36 (1H, s), 7.43 (1H, d, J = 3.0 Hz), 7.92 (1H, s),
8.94 (1H, t, J = 5.5 Hz), 9.55 (1H, d, J = 3.0 Hz), 12.29 (1H, br s),
12.50 (1H, s); MS m/z: 477 (M+H)+. Anal. Calcd for
C24H24N6O5ꢀ0.4CH4O: C, 59.89; H, 5.27; N, 17.17. Found: C,
60.23; H, 5.20; N, 16.83.
The title compound was prepared from 26 in the same manner
as described for 11, and obtained as pale yellowish solid (85%): mp
262 °C (dec) (MeOH–THF); IR (KBr) 1601, 1499, 1481, 1153 cmꢁ1
;
1H NMR (DMSO-d6) d: 3.64 (6H, s), 3.67–4.10 (2H, br), 4.83 (2H,
d, J = 5.5 Hz), 6.15 (1H, t, J = 2.0 Hz), 6.50 (1H, d, J = 16.0 Hz), 6.78
(2H, d, J = 2.0 Hz), 7.41 (1H, s), 7.43 (2H, d, J = 8.5 Hz), 7.56 (1H,
d, J = 16.0 Hz), 7.65 (2H, d, J = 8.5 Hz), 7.94 (1H, d, J = 1.5 Hz), 9.07
(1H, br s), 11.50–13.00 (2H, br); MS m/z: 489 (M+H)+. Anal. Calcd
for C25H24N6O5ꢀ1.4H2O: C, 58.45; H, 5.26; N, 16.36. Found: C,
58.55; H, 5.10; N, 16.05.
5.1.33. 5-(4-Carbamoylmethylbenzylamino)-7-(3,5-dimethoxy-
phenylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (22)
The title compound was prepared from 21 in the same man-
ner as described for 12, and obtained as yellowish solid (12%):
mp 224–226 °C (MeOH–THF); IR (KBr) 1600, 1558, 1499,
1150 cmꢁ1
;
1H NMR (DMSO-d6) d: 3.30 (2H, s), 3.63 (6H, s),
5.2. Molecular modeling
4.79 (2H, d, J = 5.0 Hz), 6.14 (1H, t, J = 2.0 Hz), 6.82 (2H, d,
J = 2.0 Hz), 6.84 (1H, br s), 7.21 (2H, d, J = 8.5 Hz), 7.32 (2H, d,
J = 8.0 Hz), 7.36 (1H, d, J = 1.5 Hz), 7.40–7.46 (2H, m), 7.92 (1H,
d, J = 1.5 Hz), 8.93 (1H, t, J = 5.0 Hz), 9.54 (1H, d, J = 3.5 Hz),
12.49 (1H, s); MS m/z: 476 (M+H)+. Anal. Calcd for C24H25N7O4:
C, 60.62; H, 5.30; N, 20.62. Found: C, 60.38; H, 5.28; N, 20.23.
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 was extracted from X-ray structure of LCK
(PDBcode. 1QPD). All docking calculations were performed by
using program ADAM.19
5.1.34. Ethyl (E)-4-[[bis[(1,1-dimethylethoxy)carbonyl]amino]-
methyl]cinnamate (24)
5.3. Biology
To a stirred suspension of sodium hydride (0.220 g (60% in
mineral oil), 5.50 mmol) in THF (15 mL) was added ethyl (E)-4-
bromomethylphenylcinnamate 23 (1.35 g, 5.00 mmol). To this
mixture was slowly added a solution of di-tert-butyliminodi-
carboxylate (1.19 g, 5.50 mmol) in THF (10 mL) at 0 °C. After stir-
ring 14 h at room temperature, the reaction mixture was poured
into saturated ammonium chloride solution, and extracted with
EtOAc. The organic phase was washed with brine, dried over
anhydrous MgSO4, and evaporated in vacuo. Resulting residue
was purified by column chromatography on silica gel with hex-
ane/EtOAc = 10:1 ? 3:1 to give 24 (1.61 g, 79%) as white solid:
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 pFASTBAC1 vector of the BAC-TO-BACTM (GIBCO-BRL Inc.)
baculovirus expression system. The transfected virus was ob-
tained by transfecting Sf-9 cell (Invitrogen Inc.) with pFASTBAC1
containing His-tag-fused ZAP kinase domain described above.

294
A. Hirabayashi et al. / Bioorg. Med. Chem. 17 (2009) 284–294
High FiveTM baculocells, which was infected with this transfec-
ted virus, were recovered and these cells were dissolved by
ultrasonication. After soluble fraction being separated by centri-
fuge, 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 extracted His-tag-fused protein of ZAP-70 kinase do-
main by imidazole containing a buffer. A coupled spectrophoto-
metric 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, and started by adding ATP. PK
Acknowledgments
The authors acknowledge Dr. Y. Terao for valuable discussions
and performing syntheses. We also thank Dr. N. Tanaka for provid-
ing valuable suggestions during the preparation of this manuscript.
References and notes
1. Borel, J. F.. Wien Klin. Wochenschr. 2002, 114, 433.
2. Kino, T.; Hatanaka, H.; Hashimoto, M.; Nishiyama, M.; Goto, T.; Okuhara, M.;
Kohsaka, M.; Aoki, H.; Imanaka, H. J. Antibiot. 1987, 40, 1249.
3. Dumont, F. J.; Staruch, M. J.; Koprak, S. L.; Siekierka, J. J.; Lin, C. S.; Harrinson, R.;
Sewell, O.; Kindt, V. M.; Beattie, T. R.; Wyvratt, M.; Sigal, N. H. J. Exp. Med. 1992,
176, 751.
4. Faruki, S.; Geahlen, R. L.; Asai, D. J. J. Cell. Sci. 2000, 113, 2557.
5. (a) Weiss, A. Cell 1993, 73, 209; (b) Weiss, A.; Littman, D. R. Cell 1994, 76, 263;
(c) Chan, A. C.; Shaw, A. S. Curr. Opin. Immunol. 1995, 8, 394; (d) Kurosaki, T.;
Tsukada, S. Immunity 2000, 12, 1.
6. Chu, D. H.; Morita, C. T.; Weiss, A. Immunol. Rev. 1998, 105, 167.
7. Chan, A. C.; Iwashima, M.; Turck, C. W.; Weiss, A. Cell 1992, 71, 649.
8. Isakov, N.; Wange, R. L.; Burgess, W. H.; Watts, J. D.; Aebersold, R.; Samelson, L.
E. J. Exp. Med. 1995, 181, 375.
9. Arpaia, E.; Shahar, H.; Dadi, H.; Cohen, A.; Rolfman, C. M. Cell 1994, 76, 947.
10. Williams, B. L.; Schreiber, K. L.; Zhang, W.; Wange, R. L.; Samelson, L. E.;
Leibson, P. L.; Abraham, R. T. Mol. Cell. Biol. 1998, 18, 1388.
(150
(150
l
l
g/mL), LDH (500
M) were added in large excess. Addition of 100
l
g/mL), PEP (2.5 mM), and NADH
M ZAP-
l
70 optimal peptide substrate (peptide sequence: AEEEIYGE-
FEAKKKK, Sawady, Tokyo), allowed measurement of kinase
activity.
11.
a Hirabayashi, A.; Shiohara, H.; Kobayashi, H.; Terao, Y.; Miyazawa, M.;
Misawa, K. Jpn. Kokai Tokkyo Koho, JP 2004238296.; (b) Hirabayashi, A.;
Mukaiyama, H.; Kobayashi, H.; Shiohara, H.; Nakayama, S.; Ozawa, M.;
Miyazawa, K.; Misawa, K.; Ohnota, H.; Isaji, M. Bioorg. Med. Chem. 2008, 16,
7347.
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
(Novagen Inc.) by PCR amplification from human thymus Mara-
thon-ReadyTM cDNA (CLONTECH Inc). The Syk kinase domain
binding with pET-19b His-tag gene at 50 region was integrated
to pFASTBAC1 vector of the BAC-TO-BACTM baculovirus expres-
sion system (GIBCO-BRL Inc.). The transfected virus was obtained
by transfecting Sf-9 cell (Invitrogen Inc.) with pFASTBAC vector
containing His-tag-fused Syk kinase domain described above.
High FiveTM baculocells, which was infected with this transfected
virus, were recovered and dissolved by ultrasonication. After sol-
uble fraction being separated by centrifuge, the supernatant was
mixed with TALONTM metal affinity resin (CLONTECH Inc.) and
to this resin was adsorbed His-tag-fused protein of Syk kinase do-
main. The resin was washed several times and extracted His-tag-
fused protein of Syk kinase domain by imidazole containing a
buffer. A coupled spectrophotometric assay was used, wherein
ADP generated by Syk kinase was converted to ATP by pyruvate
kinase (PK), with concomitant production of pyruvate from phos-
phoenolpyruvate (PEP). LDH reduces pyruvate to lactate by oxi-
dizing 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 37 °C in 100 mM
HEPES buffer (pH 7.6), containing 40 mM MgCl2 and 10% glycerol,
12. (a) Cheng, A. M.; Rowley, B.; Pao, W.; Hayday, A.; Bolen, J. B.; Pawson, T. Nature
1995, 378, 303; (b) Turner, M.; Mee, P. J.; Costello, P. S.; Williams, O.; Price, A.
A.; Duddy, L. P.; Furlong, M. T.; Geahlen, R. L.; Tybulewicz, V. L. J. Nature 1995,
378, 298; (c) Abtahian, F.; Guerriero, A.; Sebzda, E.; Lu, M.-M.; Zhou, R.; Mocsai,
A.; Myers, E. E.; Huang, B.; Jackson, D. G.; Ferrari, A.; Tybulewicz, V.; Lowell, A.
L.; Lepore, J. J.; Loretzky, G. A.; Kahn, M. Science 2003, 299, 247.
13. Vu, C. B.; Corpuz, E. G.; Merry, T. J.; Pradeepan, S. G.; Bartlett, C.; Bohacek, R. S.;
Botfield, M. C.; Eyermann, C. J.; Lynch, B. A.; MacNeil, I. A.; Ram, M. K.;
Schravendijk, M. R.; Violette, S.; Sawyer, T. K. J. Med. Chem. 1999, 42, 4088.
14. Moffat, D.; Davis, P.; Hutchings, M.; Davis, J.; Berg, D.; Batchelor, M.; Johnson, J.;
O’Connell, J.; Martin, R.; Crabbe, T.; Delgado, J.; Perry, M. Bioorg. Med. Chem.
Lett. 1999, 9, 3351.
15.
a Hirabayashi, A.; Mukaiyama, H.; Shiohara, H.; Kobayashi, H.; Terao, Y.;
Miyazawa, K.; Misawa, K.; Ohnota, H. Jpn. Kokai Tokkyo Koho, JP 2004203748.;
(b) Hirabayashi, A.; Mukaiyama, H.; Kobayashi, H.; Shiohara, H.; Nakayama, S.;
Ozawa, M.; Tsuji, E.; Miyazawa, K.; Misawa, K.; Ohnota, H.; Isaji, M. Bioorg. Med.
Chem. 2008, 16, 9247.
16. Yamaguchi, H.; Hendrickson, W. A. Nature 1996, 384, 484.
17. Zhu, H.; Kim, J. L.; Newcomb, J. R.; Rose, P. E.; Stover, D. R.; Toledo, L. M.; Zhao,
H.; Morgenstern, K. A. Structure 1999, 7, 651.
18. (a) Ogata, K.; Umeyama, H. Struct. Funct. Genet. 1998, 31, 355; (b) Ogata, K.;
Umeyama, H. J. Mol. Graph. Model. 2000, 18, 258.
19. Mizutani, M. Y.; Tomioka, N.; Itai, A. J. Mol. Biol. 1994, 243, 310.
20. (a) Jin, L.; Pluskey, S.; Petrella, E. C.; Cantin, S. M.; Gorga, J. C.; Rynkiewicz, M. J.;
Pandey, P.; Strikler, J. E.; Babine, R. E.; Weaver, D. T.; Seidl, K. J. J. Biol. Chem.
2004, 279, 42818; (b) Atwell, S.; Adams, J. M.; Badger, J.; Buchanan, M. D.; Feil,
I. K.; Froning, K. J.; Gao, X.; Hendle, J.; Keegan, K.; Leon, B. C.; Muller-Diekmann,
H. J.; Nienaber, V. L.; Noland, B. W.; Post, K.; Rajashankar, K. R.; Ramos, A.;
Russell, V. M.; Burley, S.; Buchanan, S. G. J. Biol. Chem. 2004, 279, 55827.
21. (a) Nishio, M.; Hirota, M.; Umezawa, Y. The CH–
p Interaction: Evidence, Nature
and Consequences; Wiley-VCH: Weinheim, 1998; (b) Nishio, M.; Hirota, M.
Tetrahedron 1989, 45, 7201.
22. Brandl, M.; Weiss, M. S.; Jabs, A.; Sühnel, J.; Hilgenfeld, R. J. Mol. Biol. 2001, 307,
357.
and started by adding ATP. PK (150
(2.5 mM), and NADH (200 M) were added in large excess. Addi-
tion of 100 M Syk optimal peptide (peptide sequence: AEEEIY-
lg/mL), LDH (50 lg/mL), PEP
l
l
23. Adams, J. L.; Veal, J.; Shewchuk, L.. In Protein Crystallography in Drug Discovery;
Babine, R. E., Abdel-Meguid, S. S., Eds.; Wiley-VCH: Weinheim, 2004; Vol. 20, p 47.
GEFEAKKKK, Sawady, Tokyo) allowed measurement of kinase
activity.