Structure-activity relationship studies of imidazo[1,2-c]pyrimidine derivatives as potent and orally effective Syk family kinases inhibitors

Article abstract of DOI:10.1016/j.bmc.2008.09.015

Spleen tyrosine kinase (Syk) and zeta-associated protein kinase of 70 kDa (ZAP-70) are members of the Syk family and non-receptor-type protein tyrosine kinases, which play crucial roles in B- and T-cell activation. Therefore, a Syk family tyrosine kinases inhibitor would be a useful therapeutic agent for the treatment of various allergic disorders and autoimmune diseases. Previously, we reported that 1,2,4-triazolo[4,3-c]pyrimidine derivative 1 and 1,2,4-triazolo[1,5-c]pyrimidine derivative 2 showed strong inhibitory activities against Syk family kinases. These compounds also exhibited high-level suppression of IL-2 in cellular assays. However, their oral efficacies were poor in a mouse model of IL-2 production. To improve oral effectiveness, we investigated a new series of Syk family kinases inhibitors. We found that imidazo[1,2-c]pyrimidine derivatives potently inhibited the Syk family kinases. Among these agents, compound 9f not only showed strong inhibitory activities against Syk and ZAP-70 kinases in vitro, but its oral administration resulted in the in vivo suppression of both the passive cutaneous anaphylaxis reaction and Concanavalin A-induced IL-2 production in a mouse model.

Full text of DOI:10.1016/j.bmc.2008.09.015

Bioorganic & Medicinal Chemistry 16 (2008) 9247–9260
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationship studies of imidazo[1,2-c]pyrimidine
derivatives as potent and orally effective Syk family kinases inhibitors
*
Akihito Hirabayashi , Harunobu Mukaiyama, Hiroaki Kobayashi, Hiroaki Shiohara, Satoko Nakayama,
Motoyasu Ozawa, Eiichi Tsuji, 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:
Received 1 July 2008
Revised 1 September 2008
Accepted 5 September 2008
Available online 9 September 2008
Spleen tyrosine kinase (Syk) and zeta-associated protein kinase of 70 kDa (ZAP-70) are members of the
Syk family and non-receptor-type protein tyrosine kinases, which play crucial roles in B- and T-cell acti-
vation. Therefore, a Syk family tyrosine kinases inhibitor would be a useful therapeutic agent for the
treatment of various allergic disorders and autoimmune diseases. Previously, we reported that 1,2,4-
triazolo[4,3-c]pyrimidine derivative 1 and 1,2,4-triazolo[1,5-c]pyrimidine derivative 2 showed strong
inhibitory activities against Syk family kinases. These compounds also exhibited high-level suppression
of IL-2 in cellular assays. However, their oral efficacies were poor in a mouse model of IL-2 production.
To improve oral effectiveness, we investigated a new series of Syk family kinases inhibitors. We found
that imidazo[1,2-c]pyrimidine derivatives potently inhibited the Syk family kinases. Among these agents,
compound 9f not only showed strong inhibitory activities against Syk and ZAP-70 kinases in vitro, but its
oral administration resulted in the in vivo suppression of both the passive cutaneous anaphylaxis reac-
tion and Concanavalin A-induced IL-2 production in a mouse model.
Keywords:
Protein tyrosine kinases (PTKs)
Syk family kinases
Syk
ZAP-70
Autoimmune diseases
Syk and/or ZAP-70 kinase inhibitor
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Syk family kinase inhibition in vitro, these derivatives exhibited
poor oral effectiveness in suppressing IL-2 production in a mouse
Protein tyrosine kinases (PTKs) are important enzymes in cell
signal transduction.1 Syk family kinases comprise the cytosolic
non-receptor tyrosine classes of Syk and ZAP-70.2,3 Specifically,
Syk is present in platelets, B lymphocytes, mast cells, basophils,
neutrophils, dendritic cells, macrophages, and monocytes. Syk ki-
nases also possess functional roles in immunoreceptor tyrosine-
based activation motif (ITAM)-mediated signal transduction,
which is expressed in various hematopoietic cell types. In contrast,
ZAP-70 expression is restricted to T lymphocytes and natural killer
(NK) cells. Syk family kinases play crucial roles in immune re-
sponses and are implicated in inflammatory and autoimmune dis-
eases.4–6 Furthermore, several Syk inhibitors were recently
reported to be effective in the treatment of inflammatory and auto-
immune diseases.7–11 Therefore, Syk family tyrosine kinase inhibi-
tors are candidate therapeutic agents for the treatment of various
allergic and autoimmune disorders.
model (data not shown). We examined the physicochemical prop-
erties of compounds 1 and 2 and found that these derivatives
exhibited low Caco-2 permeability and poor solubility in water.
As these parameters are correlated with oral bioavailability,13,14
we designed new imidazo[1,2-c]pyrimidine derivatives. Analyses
of these new imidazo[1,2-c]pyrimidine derivatives indicated that
compound 9f possessed not only excellent in vitro inhibitory
activity against Syk family kinases, but also oral efficacy in sup-
pressing both the passive cutaneous anaphylaxis (PCA) reaction
and Concanavalin (Con)A-induced IL-2 production in mice. In
the present paper, we outline the development of this imi-
dazo[1,2-c]pyrimidine derivative as a new Syk family kinase
inhibitor.
2. Chemistry
In the course of our studies aimed at developing a tyrosine ki-
nase inhibitor,12 we discovered that 1,2,4-triazolo[4,3-c]pyrimi-
dine derivative 1 and 1,2,4-triazolo[1,5-c]pyrimidine derivative 2
showed strong inhibitory activities against Syk and ZAP-70 ki-
nases in a cellular assay. However, despite the high potency of
As shown in Scheme 1, imidazo[1,2-c]pyrimidine derivatives
were synthesized from 4,6-dichloro-2-methylsulfanylpyrimidine-
5-carbonitrile 315 in a four-step conversion. Compound 3 was
treated with various aniline derivatives and Hünig’s base in tetra-
hydrofuran (THF), to produce 4-anilinopyrimidine derivatives 4a–
f. The reaction of compounds 4a–f with aqueous ammonia solu-
tion in N,N-dimethylformamide (DMF) generated 6-aminopyrimi-
dine derivatives 5a–f. Hydrolysis of the nitrile groups of 5a–f
* 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.2008.09.015

9248
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
Scheme 1. Reagents: (a) RNH2, i-Pr2NEt, THF; (b) NH4OH, DMF; (c) 30% H2O2, 5 M NaOH, DMSO, EtOH; (d) ClCH2CHO, DMF.
under basic conditions afforded the corresponding carboxamides
6a–f. The imidazo[1,2-c]pyrimidine derivatives 7a–f were synthe-
sized by treating carboxamide derivatives 6a–f with chloroacetal-
dehyde in DMF. Compounds 8a–g and 9a–f were synthesized by
substitution reactions of the appropriate commercially available
amines in DMF or N-methylpyrrolidone (NMP), as shown in
Scheme 2.
3. Binding model
Due to the relatively high degrees of structural homology be-
tween Lck and ZAP-70 (or Syk) at their ATP-binding sites, we con-
structed a ZAP-70 homology model based on the previously
published crystal structures of the activated Lck kinase domain
(PDB code 1QPD)16,17 using the full automatic modeling system
(FAMS).18 The binding modes of several compounds were exam-
ined using the ADAM software.19
Scheme 3 summarizes the synthesis of 5-amino-2-phenylimi-
dazo[1,2-c]pyrimidine-8-carboxamide derivatives 15a–c. Com-
pound 4f was reacted with (R)-(ꢀ)-2-phenylglycinol to give the
alcohol derivative 10. Construction of 2,3-dihydroimidazo[1,2-
c]pyrimidine core 11 was achieved by treating compound 10 with
phosphorus oxychloride. Conversion of compound 11 to com-
pounds 12a–c was achieved by introducing various mono-Boc-pro-
tected diamino derivatives. Hydrolysis of the nitrile groups in 12a–
c under basic conditions yielded compounds 13a–c. The absolute
configuration of 13a was confirmed by X-ray crystallography, as
shown in Figure 1. The configuration of another diastereomer,
13b, was determined based on X-ray crystallography of 13a. The
palladium-catalyzed oxidation of 2-phenyl-2,3-dihydroimi-
dazo[1,2-c]pyrimidine derivatives 13a–c afforded 2-phenylimi-
dazo[1,2-c]pyrimidine derivatives 14a–c. Finally, deprotection of
the Boc group by treatment with hydrochloric acid or TFA of deriv-
atives 14a–c gave compounds 15a–c. The chemical structures of
the compounds thus prepared were determined from spectro-
scopic data (1H NMR, mass spectrometry) and elemental analyses.
4. Results and discussion
4.1. SAR for Syk and ZAP-70 kinases
The Syk and ZAP-70 kinase inhibitory activities of the com-
pounds synthesized in the present study were evaluated by cou-
pled spectrophotometric enzyme assays (Tables 1–5).
1,2,4-Triazolo[4,3-c]pyrimidine derivative 1 and 1,2,4-triazol-
o[1,5-c]pyrimidine derivative 2 showed good inhibitory activities
against Syk and ZAP-70, being almost equivalent to the activities
of staurosporine. However, these compounds were not orally effec-
tive in suppressing IL-2 production in the mouse model. In the
analyses of the physicochemical properties of 1,2,4-triazolo[4,3-
c]pyrimidine derivative
derivative 2, these compounds showed low Caco-2 permeability
and poor solubility in water (Table 1). Compounds with high polar
1 and 1,2,4-triazolo[1,5-c]pyrimidine

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
9249
Scheme 2. Reagents: (a) Commercially available amines, i-Pr2NEt, DMF or NMP; (b) cis-1,2-cyclohexyldiamine, DMF.
surface areas (PSAs) tend to show low Caco-2 permeability and
poor oral bioavailability.20,21 We found that the PSA values of com-
pounds 1–2 were comparatively high (141 for compound 1 and
139 for compound 2). We designed imidazo[1,2-c]pyrimidine ana-
logs of 1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-
c]pyrimidine derivatives with lower PSA values by reducing the
number of nitrogen atom. The results of our previous studies on
1,2,4-triazolo[4,3-c]pyrimidine and 1,2,4-triazolo[1,5-c]pyrimidine
derivatives12 indicate that imidazo[1,2-c]pyrimidine derivatives
retain similar pharmacophores with triazolopyrimidine analogs.
Therefore, 5-aminoimidazo[1,2-c]pyrimidine derivatives would
be expected to show strong inhibitory activities against Syk family
kinases. We investigated the effects of substituents at the 5-posi-
tion in the imidazo[1,2-c]pyrimidine skeleton (Table 2).
lent kinase inhibitory activity. This stereochemical preference was
consistent with that found in the preceding triazole derivatives 1
and 2.12b These findings indicate that the amino group in the cyclo-
hexyl ring plays a crucial role in Syk and ZAP-70 inhibition in com-
parison to other substituents, such as the methyl and hydroxyl
groups. Moreover, the configuration of the amino group in the
cyclohexyl ring has an impact inhibitory activity against Syk family
kinases.
Subsequently, we investigated the effects of varying substitu-
tions at the 7-anilino groups of the imidazo[1,2-c]pyrimidines (Ta-
ble 3). The 2-methoxy derivative 9a showed no inhibitory effects
on the Syk or ZAP-70 kinase (IC50 > 10 lM for both Syk and ZAP-
70). The 3-methoxy derivative 9b and 4-methyoxy derivative 9c
showed strong inhibitory effects against Syk kinase (IC50 = 0.030
The simple amino derivative 8a showed weak inhibitory activ-
ities against Syk and ZAP-70 kinase. For compound 8b, the inhibi-
tory effects on both Syk and ZAP-70 were slightly improved by the
introduction of a hydroxyethylamino group, in comparison to com-
pound 8a. The 2-aminoethylamino analog 8c exhibited enhanced
Syk kinase inhibition, whereas 8c showed only slightly improved
ZAP-70 inhibitory activity. Both the Syk and ZAP-70 inhibitory
activities were reduced in the 3-aminopropylamino derivative
8d. We investigated several cyclohexylamino derivatives based
on their abilities to fit in the sugar pocket. The 2-meth-
ylcyclohexylamino derivative 8e exhibited moderate inhibitory
activities against Syk and ZAP-70. Compound 8f, which possesses
a 2-hydroxycyclohexylamine at the 5-position, showed enhanced
inhibition of both Syk and ZAP-70 in comparison to 8e. Next, we
examined the cyclohexyldiamino derivatives, which show excel-
lent inhibitory effects on Syk family kinases in the 1,2,4-triazol-
and 0.092
lM for 9b and 9c, respectively) although they showed
weak inhibitory activities against the ZAP-70 kinase (IC50 > 1.0
lM
for both 9b and 9c) in comparison to Syk inhibition. The results for
compounds 9b and 9c indicate that substitution in the meta posi-
tion is superior to that in the para position. Therefore, we focused
on the meta substituent and investigated 3,5-di-substituted deriv-
atives. Compounds 9d–f showed excellent inhibitory effects
against Syk family kinases, especially the Syk kinase. Among these
three derivatives, compound 9f exhibited the greatest inhibitory
potency against Syk family kinases (IC50 = 0.006 lM and 0.23 lM
for Syk and ZAP-70, respectively). These findings indicate that sub-
stituents on the 7-anilino group influence inhibitory activity
against Syk family kinase inhibition. Substitution at the meta and
para positions is desirable for kinase inhibition. Substitution at
the meta position was most favorable and di-substitution at the
meta position in the 7-anilino-group contributed to a greater
improvement in inhibition potency against Syk family kinases, as
compared to a mono-substituent analog. Specifically, the 3,5-dime-
thoxy group was the most effective substituent for Syk family ki-
nases inhibition as compared to 3,5-difluoro or 3,5-dimethyl
o[4,3-c]pyrimidine
state.12b
The
trans-cyclohexyldiamino
derivative 8g showed weak inhibitory activity against Syk family
kinases and had completely lost the ZAP-70 kinase inhibitory activ-
ity. In contrast, cis-cyclohexyldiamino derivative 9f showed excel-

9250
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
Scheme 3. Reagents: (a) (R)-(ꢀ)-2-Phenylglycinol, i-Pr2NEt, DMF; (b) POCl3; (c) diamine derivatives, DMF; Boc2O, CH2Cl2; (d) 30% H2O2, 5 M NaOH, DMSO, EtOH; (e) O2, 10%
Pd–C, cyclohexene, EtOH, DMSO; (f) 4 M HCl–EtOAc; (g) TFA, CH2Cl2.
derivatives. Examination of the binding mode between ZAP-70 and
compound 9f revealed that the N–H group and the carbonyl group
of 8-carbamoyl interacts with the carbonyl group of Glu415 and
the N–H group of Ala417, respectively; the N–H group of aniline
forms a hydrogen bond with the carbonyl group of Ala417; the
imidazole core of compound 9f occupies the gate keeper residue22
When examining the binding mode in the ZAP-70 homology
model, we found a comparatively large space around the imidaz-
ole unit. In addition, Syk family kinase inhibition was increased
by the introduction of a phenyl group at the 2-position.12 To
improve further the inhibitory activities against Syk and ZAP-70
kinases, we examined 2-phenylimidazo[1,2-c]pyrimidine-8-car-
boxamide derivatives (Table 4). The inhibitory effects of com-
pound 15b on ZAP-70 kinase were improved by introducing a
to form a CH–
the 7-anilino group of compound 9f on the lipophilic plug22 forms
a CH– interaction with the methylene groups of Leu344 and
p interaction with the methyl group of Val352; and
p
phenyl group at the 2-position (IC50 = 0.041
Syk inhibitory activity was somewhat reduced (IC50 = 0.028
compared to that of 9f (IC50 = 0.006 M). In contrast, compound
lM). However, the
Gly420. Moreover, the cyclohexyldiamino group at the 5-position
of the imidazo[1,2-c]pyrimidine core may contribute to the effec-
tive occupation of the sugar pocket (Fig. 2).22 Conversely, substitu-
tion at the ortho position was undesirable. Several studies have
reported that the conformation of the ortho-substituted dipheny-
laniline derivatives show twisting between the two aromatic
rings.23 We anticipated that twisting conformation between the
lM)
l
15a, which is a diastereomer of compound 15b, showed de-
creased potency for Syk family kinase inhibition. The 2-methyl-
propane-1,2-diamine derivative 15c showed moderate inhibitory
activities against Syk and ZAP-70 kinases, as compared to the
cyclohexyldiamino derivative 15b. A full large space was pro-
duced around the imidazole core by the introduction of a phenyl
group (Fig. 3), such that this modification contributed to the
inhibitory effect on ZAP-70. Contrary to our expectations, this
conversion did not yield an inhibitory effect on Syk.
two aromatic rings broke CH–p interaction with the methylene
groups of Leu344 and Gly420 shown in Figure 2. This phenomenon
may be responsible for the loss of Syk family kinase inhibitory
activity of compound 9a.

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
9251
Table 2
Syk and ZAP-70 inhibitory activities of imidazo[1,2-c]pyrimidines
a
Compound
R
IC50 (lM)
Syk
ZAP-70
8a
8b
8c
8d
NH2
7.1
4.3
0.23
1.5
10.0
1.9
3.5
NHCH2CH2OH
NHCH2CH2NH2
NHCH2CH2CH2NH2
12.4
8e
1.0
3.3
1.8
HN
8f
0.72
1.2
HN
OH
Figure 1. X-ray crystal structure of 13a.
8g
>10
0.23
HN
4.2. Cellular assays and in vivo efficacy in several mouse models
NH2
Compounds 9d–f and 15b showed excellent kinase inhibitory
activities. Therefore, we investigated their effects on IL-2 produc-
tion in peripheral blood mononuclear cells (PBMCs) and a whole
blood (WB) assay system, to evaluate the inhibitory effects on
Syk family kinases. The results are summarized in Table 5.
Although compounds 9d and 9e showed good Syk kinase inhibitory
activities, they were less potent than staurosporine in both the
PBMCs and WB assay. The 2-phenylimidazo[1,2-c]pyrimidine
derivative 15b was also less active than staurosporine in the cellu-
lar assay and in the WB system. In contrast, compound 9f showed
excellent inhibition in both the PBMCs and the WB assay. The IL-2-
suppressive effect of compound 9f was almost equivalent to that of
staurosporine in our assay system. We then investigated the phys-
icochemical properties of 9f and 15b. The Caco-2 permeability of
compound 9f (3.33 ꢁ 10–6 cm/s) was superior to that of compound
15b (0.43 ꢁ 10–6 cm/s). We postulate that the higher Caco-2 per-
meability of 9f contributes to its greater cellular potency in com-
parison with 15b.
9f
0.006
HN
NH2
a
The IC50 values were determined in duplicate.
Finally, we evaluated the in vivo effects of optimized Syk family
kinase inhibitor 9f on both the PCA reaction and ConA-induced IL-2
production in mice. Compound 9f was administered orally to mice
and showed a dose-dependent inhibitory effect on the anaphylaxis
reaction, as shown in Figure 4. Moreover, compound 9f, which was
administered orally to mice, suppressed IL-2 production in a dose-
dependent manner, as shown inFigure 5. These results indicate that
compound 9f, which strongly inhibits Syk and moderately inhibits
ZAP-70, could improve diseases that involve both Syk and ZAP-70.
The triazolopyrimidine derivative 1 did not suppress IL-2 produc-
tion in mouse model (data not shown). We investigated the physi-
cochemical properties and plasma concentrations of compounds 1
Table 1
Physicochemical properties of triazopyrimidine derivatives
Caco2/Papp (10ꢀ6 cm/s)
IC50
(
lM)
a
Compound
Solubility in water (
l
g/mL)
Syk
ZAP-70
PBMC
WB
Staurosporine
1
2
NTb
351
42
NTb
0.01
0.33
0.003
0.009
0.004
0.053
0.12
0.33
0.016
0.046
0.076
0.10
0.44
0.48
a
The IC50 values were determined in duplicate.
NT, not tested.
b

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A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
Table 3
Syk and ZAP-70 inhibitory activities of imidazo[1,2-c]pyrimidines
a
Compound
Ar
IC50 (lM)
Syk
ZAP-70
9a
9b
9c
9d
9e
9f
2-MeOC6H4
3-MeOC6H4
4-MeOC6H4
3,5-Di-FC6H3
3,5-Di-MeC6H3
3,5-Di-MeOC6H3
>10
>10
1.7
4.1
0.73
0.48
0.23
0.030
0.092
0.030
0.006
0.006
a
The IC50 values were determined in duplicate.
Table 4
Figure 2. Docking mode of compound 9f with ZAP-70.
Syk and ZAP-70 kinase inhibitory activities of 2-phenylimidazolo[1,2-c]pyrimidine
derivatives
in accordance with our initial hypothesis that Caco-2 permeability,
PSA, and solubility in water affect oral efficacy. Therefore, we con-
sider that improvements in its physicochemical properties contrib-
ute to the in vivo efficacy of compound 9f.
5. Conclusions
We demonstrate that novel imidazo[1,2-c]pyrimidine deriva-
tives are highly potent and orally effective Syk family kinase inhib-
itors. Among these agents, optimized compound 9f is superior to
1,2,4-triazolo[4,3-c]pyrimidine derivative 1 with regard to physi-
cochemical profile. Compound 9f showed not only excellent
in vitro kinase inhibitory activity, but also effectively suppressed
both the PCA reaction and IL-2 production in a mouse model. We
anticipate that the discovery of orally active inhibitors of Syk fam-
ily kinases will contribute to the treatment of various allergic and
immunologic disorders. We intend to continue the optimization
and evaluation of these derivatives and will report further develop-
ments in due course.
a
Compound
R1
IC50
(l
M)
Syk
ZAP-70
0.60
15a
15b
0.38
HN
HN
NH2
NH2
0.028
0.84
0.041
0.16
15c
NHCH2C(Me)2NH2
The IC50 values were determined in duplicate.
a
6. Experimental
6.1. Chemistry
Table 5
Syk and ZAP-70 kinase inhibitory activities and the suppression of IL-2 production in
PBMCs and a WB assay
Melting points were taken on a Yanako MP-3S Micromelting
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,
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 60 F254 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
a
Compound
IC50
ZAP-70
(lM)
Syk
PBMC
WB
Staurosporine
9d
9e
9f
15b
0.003
0.030
0.006
0.006
0.028
0.053
0.73
0.48
0.23
0.041
0.016
0.15
0.20
0.058
0.20
0.10
3.0
1.0
0.28
2.0
a
The IC50 values were determined in duplicate.
and 9f, as summarized in Tables 6 and 7. Triazolopyrimidine deriv-
ative 1 showed low Caco-2 permeability and poor solubility in
water. In contrast, imidazopyrimidine derivative 9f showed higher
Caco-2 permeability and better solubility in water than compound
1. Furthermore, the PSA value for compound 9f was lower than that
for compound 1 (Table 6). With regard to their pharmacokinetic
profiles, the plasma concentration of compound 9f increased in a
dose-dependent manner, while compound 1 was scarcely detected
in the plasma after oral administration (Table 7). These results are
APS Daisogel IR-60 (particle size 25–40 lM) from Daiso Co., Ltd.
All reagents and solvents were commercially available unless
otherwise indicated.

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
9253
Figure 3. Docking mode of compound 15b with ZAP-70.
Figure 5. Suppressive effect of compound 9f on ConA-induced IL-2 production in
mice.
Figure 4. Suppressive effect of compound 9f on the passive cutaneous anaphylaxis
(PCA) reaction.
6.1.2. 4-Chloro-6-(3-methoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (4b)
6.1.1. 4-Chloro-6-(2-methoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (4a)
The title compound was prepared from 3 and m-anisidine in the
same manner as described for 4a, and was obtained as white solid
(92%): mp 234–235 °C (MeOH–THF); IR (KBr) 2223, 1616, 1555,
To a mixture of 4,6-dichloro-2-methylsulfanylpyrimidine-5-
carbonitrile
3 (0.660 g, 3.00 mmol) and o-anisidine (0.377 g,
3.06 mmol) in THF (10 mL) was added diisopropylethylamine
(0.549 mL, 3.15 mmol) at 0 °C and the mixture was stirred over-
night at room temperature. Water was added to the mixture and
resulting precipitates were collected by suction filtration. The pre-
cipitates were washed with water and dried in vacuo to give 4a
(0.920 g, quant.) as white solid: mp 190–192 °C (MeOH–THF); IR
1464, 1404, 1261 cmꢀ1 1H NMR (DMSO-d6) d: 2.42 (3H, s), 3.75
;
(3H, s), 6.78 (1H, dd, J = 7.0, 1.0 Hz), 7.14–7.19 (2H, m), 7.28 (1H,
t, J = 8.0 Hz), 10.13 (1H, br s); MS m/z: 307 (M+H)+. Anal. Calcd
for C13H11ClN4OS: C, 50.90; H, 3.61; N, 18.26. Found: C, 50.80; H,
3.58; N, 18.21.
(KBr) 2214, 1615, 1549, 1463, 1423, 1355, 1261, 1116 cmꢀ1
;
1H
6.1.3. 4-Chloro-6-(4-methoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (4c)
The title compound was prepared from 3 and p-anisidine in the
same manner as described for 4a, and was obtained as white solid
(94%): mp 195–197 °C (MeOH–THF); IR (KBr) 2219, 1617, 1561,
NMR (DMSO-d6) d: 2.32 (3H, s), 3.79 (3H, s), 6.95–7.05 (1H, m),
7.11 (1H, d, J = 7.5 Hz), 7.27–7.32 (1H, m), 7.42 (1H, d, J = 7.5 Hz),
9.76 (1H, br s); MS m/z: 307 (M+H)+. Anal. Calcd for C13H11ClN4OS:
C, 50.90; H, 3.61; N, 18.26. Found: C, 50.71; H, 3.56; N, 18.11.

9254
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
Table 6
(1.10 mL, 5.50 mmol) and 30% H2O2 solution (0.625 mL,
5.50 mmol) at 0 °C and the mixture was stirred for 30 min at
room temperature. The mixture was poured into water (ca.
100 mL) and resulting precipitates were collected by suction fil-
tration. The precipitates were washed with water and were sus-
pended in EtOAc. The mixture was heated at 110 °C for 1 h and
resulted precipitates were collected by glass filter to give 6a
(0.105 g, 31%) as white solid: mp 285–287 °C (MeOH–THF); IR
Physicochemical properties and cellular potencies of compounds 1 and 9f
a
Compound Solubility in
PSA Caco2/Papp
(10ꢀ6 cm/s)
IC50
(l
M)
water (lg/mL)
Syk
ZAP-70 PBMC WB
1
9f
351
930
141 0.01
128 3.33
0.009 0.12
0.006 0.23
0.046 0.44
0.058 0.28
a
The IC50 values were determined in duplicate.
(KBr) 1653, 1589, 1575, 1539, 1533, 1375, 1298, 1272 cmꢀ1 1H
;
NMR (DMSO-d6) d: 2.44 (3H, s), 3.84 (3H, s), 6.76 (2H, br s),
6.87–7.04 (3H, m), 7.55 (2H, br s), 8.09 (1H, d, J = 8.0 Hz), 10.16
(1H, s); MS m/z: 306 (M+H)+. Anal. Calcd for C13H15N5O2Sꢂ0.2-
CH4O: C, 50.85; H, 5.11; N, 22.46. Found: C, 50.85; H, 4.90; N,
22.11.
Table 7
Plasma concentrations of compounds 1 and 9f after oral administration
Applied dose (mg/kg)
Plasma concentration (ng/mL)
1
9f
3
NDa
28.4
26.5
45.2
40
6.1.8. 4-Amino-6-(3-methoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carboxamide (6b)
10
30
100
191
770
4031
The title compound was prepared from 4b in the same manner
as described for 6a, and was obtained as white solid (48%): mp
a
ND, not detected.
219–221 °C (MeOH–THF); IR (KBr) 1576, 1539, 1517, 1300 cmꢀ1
;
1H NMR (DMSO-d6) d: 2.43 (3H, s), 3.73 (3H, s), 6.52–6.60 (1H,
m), 6.85 (2H, br s), 7.00–7.06 (1H, m), 7.14–7.21 (1H, m), 7.31–
7.36 (1H, m), 7.55 (2H, br s), 9.88 (1H, br s); MS m/z: 306
(M+H)+. Anal. Calcd for C13H15N5O2Sꢂ0.3H2O: C, 50.24; H, 5.06; N,
22.54. Found: C, 50.34; H, 4.77; N, 22.35.
1512, 1466, 1437, 1250 cmꢀ1 1H NMR (DMSO-d6) d: 2.38 (3H, s),
;
3.76 (3H, s), 6.94 (2H, d, J = 9.0 Hz), 7.41 (2H, d, J = 9.0 Hz), 10.08
(1H, br s); MS m/z: 307 (M+H)+. Anal. Calcd for C13H11ClN4OS: C,
50.90; H, 3.61; N, 18.26. Found: C, 50.92; H, 3.63; N, 18.26.
6.1.9. 4-Amino-6-(4-methoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carboxamide (6c)
6.1.4. 4-Chloro-6-(3,5-difluorophenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (4d)
The title compound was prepared from 4c in the same manner
as described for 6a, and was obtained as white solid (46%): mp
246–248 °C (MeOH–THF); IR (KBr) 1576, 1521, 1506, 1300,
The title compound was prepared from 3 and 3,5-difluoroani-
line in the same manner as described for 4a, and was obtained as
white solid (92%): mp 203–204 °C (MeOH–THF); IR (KBr) 2221,
1250 cmꢀ1 1H NMR (DMSO-d6) d: 2.37 (3H, s), 3.72 (3H, s),
;
1630, 1562, 1410, 1123 cmꢀ1
;
1H NMR (DMSO-d6) d: 2.47 (3H, s),
6.65–6.90 (4H, m), 7.30–7.55 (4H, m), 9.69 (1H, br s); MS m/z:
306 (M+H)+. Anal. Calcd for C13H15N5O2Sꢂ0.42CH4O: C, 50.52; H,
5.27; N, 21.97. Found: C, 50.12; H, 4.88; N, 21.82.
7.02–7.10 (1H, m), 7.41 (2H, dd, J = 8.5 Hz, 2.0 Hz), 10.38 (1H, br
s); MS m/z: 313 (M+H)+. Anal. Calcd for C12H7ClF2N4Sꢂ0.2H2O: C,
45.56; H, 2.36; N, 17.71. Found: C, 45.60; H, 2.26; N, 17.68.
6.1.10. 4-Amino-6-(3,5-difluorophenylamino)-2-methylsulfanyl-
pyrimidine-5-carboxamide (6d)
6.1.5. 4-Chloro-6-(3,5-dimethylphenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (4e)
The title compound was prepared from 4d in the same manner
as described for 6a, and was obtained as white solid (50%): mp
274–276 °C (MeOH–THF); IR (KBr) 1586, 1576, 1538, 1525, 1477,
The title compound was prepared from 3 and 3,5-dimethylani-
line in the same manner as described for 4a, and was obtained as
white solid (98%): mp 180–182 °C (MeOH–THF); IR (KBr) 2223,
1297, 1182 cmꢀ1 1H NMR (DMSO-d6) d: 2.42 (3H, s), 6.60–7.18
;
1589, 1559,1540,1405 cmꢀ1
;
1H NMR (DMSO-d6) d: 2.26 (6H, s),
(3H, m), 7.20–7.90 (4H, m), 9.90 (1H, br s); MS m/z: 312 (M+H)+.
Anal. Calcd for C12H11F2N5OSꢂ0.72H2O 0.1CH4O: C, 44.37; H, 3.95;
N, 21.38. Found: C, 44.77; H, 3.55; N, 20.98.
2.41 (3H, s), 6.84 (1H, br s), 7.18 (2H, br s), 10.07 (1H, br s); MS
m/z: 305 (M+H)+. Anal. Calcd for C14H13ClN4S: C, 55.17; H, 4.30;
N, 18.38. Found: C, 54.95; H, 4.26; N, 18.24.
6.1.11. 4-Amino-6-(3,5-dimethylphenylamino)-2-methylsulfanyl-
pyrimidine-5-carboxamide (6e)
6.1.6. 4-Chloro-6-(3,5-dimethoxylphenylamino)-2-methylsulfanyl-
pyrimidine-5-carbonitrile (4f)
The title compound was prepared from 4e in the same manner
as described for 6a, and was obtained as white solid (40%): mp
The title compound was prepared from 3 and 3,5-dimethoxyan-
iline in the same manner as described for 4a, and was obtained as
white solid (91%): mp 214–216 °C (MeOH–THF); IR (KBr) 2225,
260–262 °C (MeOH–THF); IR (KBr) 1575, 1538, 1520, 1293 cmꢀ1
;
1H NMR (DMSO-d6) d: 2.23 (6H, s), 2.43 (3H, s), 6.64 (1H, br s),
6.81 (2H, br s), 7.24 (2H, br s), 7.54 (2H, br s), 9.88 (1H, s); MS
m/z: 304 (M+H)+. Anal. Calcd for C14H17N5OSꢂ0.3H2O: C, 54.46; H,
5.74; N, 22.68. Found: C, 54.73; H, 5.51; N, 22.69.
1584, 1540, 1481, 1404, 1158 cmꢀ1 1H NMR (DMSO-d6) d: 2.45
;
(3H, s), 3.73 (6H, s), 6.35 (1H, t, J = 2.0 Hz), 6.83 (2H, d,
J = 2.0 Hz), 10.05 (1H, br s); MS m/z: 355 (M+H)+. Anal. Calcd for
C14H13ClN4O2S: C, 49.93; H, 3.89; N, 16.64. Found: C, 49.93; H,
3.99; N, 16.49.
6.1.12. 4-Amino-6-(3,5-dimethoxyphenylamino)-2-methylsulf-
anylpyrimidine-5-carboxamide (6f)
6.1.7. 4-Amino-6-(2-methoxyphenylamino)-2-methylsulfanyl-
pyrimidine-5-carboxamide (6a)
The title compound was prepared from 4f in the same manner
as described for 6a, and was obtained as white solid (46%): mp
232–234 °C (MeOH–THF); IR (KBr) 1583, 1529, 1482, 1471,
To a stirred suspension of 4a (0.337 g, 1.10 mmol) in DMF
(2.4 mL) was added 28% ammonia solution (0.6 mL) and the mix-
ture was heated for 4 h at 60 °C. The mixture was poured into
water and resulting residue was collected by suction filtration
to give 5a as white solid. To a suspension of crude 5a in DMSO
(2.5 mL) and EtOH (2.5 mL) were added 5 M NaOH solution
1151 cmꢀ1 1H NMR (DMSO-d6) d: 2.44 (3H, s), 3.72 (6H, s), 6.15
;
(1H, t, J = 2.0 Hz), 6.83 (2H, d, J = 2.0 Hz), 6.85 (2H, s), 7.56 (2H,
s), 9.87 (1H, s); MS m/z: 336 (M+H)+. Anal. Calcd for
C14H17N5O3Sꢂ0.5H2O: C, 48.83; H, 5.27; N, 20.34. Found: C, 48.63;
H, 4.95; N, 20.31.

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
9255
6.1.13. 7-(2-Methoxyphenylamino)-5-methylsulfanylimidazo-
[1,2-c]pyrimidine-8-carboxamide (7a)
232–233 °C (MeOH–THF); IR (KBr) 1658, 1578, 1480, 1236,
1157 cmꢀ1 1H NMR (DMSO-d6) d: 2.80 (3H, s), 3.76 (6H, s), 6.25
;
To a solution of 6a (0.095 g, 0.311 mmol) in DMF (1 mL) was
added 40% chloroacetaldehyde solution (0.183 mL, 0.933 mmol)
and the mixture was heated for 6 h at 60 °C. Resulting precipitates
were collected by glass filter and washed with water to give 7a
(0.050 g, 49%) as yellowish solid. mp 244–246 °C (MeOH–THF);
(1H, t, J = 2.0 Hz), 6.80 (2H, d, J = 2.0 Hz), 7.51 (1H, d, J = 1.5 Hz),
7.74 (1H, d, J = 1.5 Hz), 7.90 (1H, d, J = 3.0 Hz), 9.63 (1H, d,
J = 3.0 Hz), 12.32 (1H, s); 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.
IR (KBr) 1659, 1610, 1572, 1480, 1312, 1233 cmꢀ1 1H NMR
;
(DMSO-d6) d: 2.77 (3H, s), 3.87 (3H, s), 6.94–6.99 (1H, m), 7.02–
7.09 (2H, m), 7.49 (1H, d, J = 2.0 Hz), 7.67–7.72 (1H, br), 7.70 (1H,
d, J = 2.0 Hz), 8.22 (1H, dd, J = 8.0, 2.0 Hz), 9.56 (1H, d, J = 2.5 Hz),
12.24 (1H, s); MS m/z: 330 (M+H)+. Anal. Calcd for
C15H15N5O2Sꢂ0.2H2O: C, 54.10; H, 4.66; N, 21.03. Found: C, 54.21;
H, 4.60; N, 20.93.
6.1.19. 5-Amino-7-(3,5-dimethoxyphenylamino)imidazo[1,2-
c]pyrimidine-8-carboxamide (8a)
A mixture of 7f (0.150 g, 0.417 mmol) and 28% ammonia solu-
tion (0.5 mL) in NMP (1 mL) was heated overnight at 100 °C. The
mixture was poured into water and extracted with EtOAc. Organic
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 hex-
ane/EtOAc = 1:2 to give 8a (0.049 g, 38%) as pale green crystal:
mp 274–276 °C (MeOH–THF); IR (KBr) 1595, 1508, 1481,
6.1.14. 7-(3-Methoxyphenylamino)-5-methylsulfanylimidazo-
[1,2-c]pyrimidine-8-carboxamide (7b)
The title compound was prepared from 6b in the same manner
as described for 7a, and was obtained as white solid (66%): mp
190–192 °C (MeOH–THF); IR (KBr) 1657, 1595, 1573, 1480, 1465,
1150 cmꢀ1
;
1H NMR (DMSO-d6) d: 3.76 (6H, s), 6.17 (1H, t,
J = 2.0 Hz), 6.86 (2H, d, J = 2.0 Hz), 7.33 (1H, d, J = 2.0 Hz), 7.39
(1H, d, J = 3.5 Hz), 7.80 (1H, d, J = 2.0 Hz), 8.17 (2H, br s), 9.52
(1H, d, J = 3.5 Hz), 12.40 (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.
1310, 1237, 1158 cmꢀ1 1H NMR (DMSO-d6) d: 2.78 (3H, s), 3.77
;
(3H, s), 6.66 (1H, dd, J = 8.0, 2.0 Hz), 7.11 (1H, d, J = 8.0 Hz), 7.20–
7.30 (2H, m), 7.51 (1H, d, J = 1.5 Hz), 7.73 (1H, d, J = 1.5 Hz), 7.88
(1H, br s), 9.61 (1H, br s), 12.29 (1H, s); MS m/z: 330 (M+H)+. Anal.
Calcd for C15H15N5O2S: C, 54.70; H, 4.59; N, 21.26. Found: C, 54.63;
H, 4.56; N, 21.07.
6.1.20. 7-(3,5-Dimethoxyphenylamino)-5-(2-hydroxyethyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (8b)
6.1.15. 7-(4-Methoxyphenylamino)-5-methylsulfanylimidazo-
[1,2-c]pyrimidine-8-carboxamide (7c)
The title compound was prepared from 7f and 2-hydroxyeth-
ylamine in the same manner as described for 8a, and was ob-
tained as white solid (13%): mp 261–263 °C (MeOH–THF); IR
The title compound was prepared from 6c in the same man-
ner as described for 7a, and was obtained as white solid (70%):
mp 221–223 °C (MeOH–THF); IR (KBr) 1607, 1576, 1508, 1487,
;
(KBr) 1616, 1593, 1550, 1507 cmꢀ1 1H NMR (DMSO-d6) d:
;
1363, 1261 cmꢀ1 1H NMR (DMSO-d6) d: 2.65–2.71 (3H, m),
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, 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.
3.70–3.77 (3H, m), 6.90–6.98 (2H, m), 7.46–7.53 (3H, m),
7.68–7.71 (1H, m), 7.80 (1H, br s), 9.55 (1H, br s), 11.95–
12.05 (1H, m); MS m/z: 330 (M+H)+. Anal. Calcd for
C15H15N5O2S: C, 54.70; H, 4.59; N, 21.26. Found: C, 54.57; H,
4.56; N, 21.16.
6.1.16. 7-(3,5-Difluorophenylamino)-5-methylsulfanylimidazo-
[1,2-c]pyrimidine-8-carboxamide (7d)
6.1.21. 5-(2-Aminoethylamino)-7-(3,5-dimethoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (8c)
The title compound was prepared from 6d in the same manner
as described for 7a, and was obtained as white solid (80%): mp
295–297 °C (MeOH–THF); IR (KBr) 1657, 1608, 1587, 1476, 1363,
The title compound was prepared from 7f and 2-aminoethyl-
amine in the same manner as described for 8a, and was obtained
as white solid (46%): mp 221–223 °C (MeOH–THF); IR (KBr)
1285, 1235, 1146 cmꢀ1 1H NMR (DMSO-d6) d: 2.81 (3H, s), 6.87–
;
1668, 1595, 1502, 1478, 1372 cmꢀ1 1H NMR (DMSO-d6) d: 2.85
;
6.92 (1H, m), 7.40–7.45 (2H, m), 7.56 (1H, d, J = 1.5 Hz), 7.80 (1H,
d, J = 1.5 Hz), 8.03 (1H, d, J = 3.0 Hz), 9.63 (1H, d, J = 3.0 Hz), 12.50
(1H, s); MS m/z: 336 (M+H)+. Anal. Calcd for C14H11F2N5OS: C,
50.14; H, 3.31; N, 20.88. Found: C, 49.79; H, 3.28; N, 20.68.
(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 C17H21N7O3ꢂ1.0C5H9NO: C, 56.16; H, 6.43; N, 23.81. Found: C,
55.57; H, 6.35; N, 23.66.
6.1.17. 7-(3,5-Dimethylphenylamino)-5-methylsulfanylimidazo-
[1,2-c]pyrimidine-8-carboxamide (7e)
The title compound was prepared from 6e in the same man-
ner as described for 7a, and was obtained as white solid (65%):
mp 252–254 °C (MeOH–THF); IR (KBr) 1652, 1613, 1582, 1480,
6.1.22. 5-(3-Aminopropylamino)-7-(3,5-dimethoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (8d)
1324, 1236, 1096 cmꢀ1
;
1H NMR (DMSO-d6) d: 2.27 (6H, s),
The title compound was prepared from 7f and 1,3-diaminopro-
pane in the same manner as described for 8a, and was obtained as
white solid (24%): mp 241–242 °C (MeOH–THF); IR (KBr) 3390,
2.79 (3H, s), 6.72 (1H, s), 7.27 (2H, s), 7.49 (1H, d, J = 2.0 Hz),
7.72 (1H, d, J = 2.0 Hz), 7.86 (1H, d, J = 3.0 Hz), 9.60 (1H, d,
J = 3.0 Hz), 12.20 (1H, s); MS m/z: 328 (M+H)+. Anal. Calcd for
C16H17N5OS: C, 58.70; H, 5.23; N, 21.39. Found: C, 58.65; H,
5.21; N, 21.30.
3282, 1691, 1594, 1553, 1503, 1480, 1458, 1239, 1149 cmꢀ1 1H
;
NMR (DMSO-d6) d: 1.82–1.86 (2H, m), 3.20 (2H, q, J = 7.5 Hz),
3.50–3.70 (2H, m), 3.75 (6H, s), 6.18 (1H, t, J = 2.0 Hz), 6.86 (2H,
d, J = 2.0 Hz), 7.34 (1H, d, J = 2.0 Hz), 7.40 (1H, d, J = 4.5 Hz), 7.85
(1H, d, J = 2.0 Hz), 7.95–8.05 (2H, m), 8.31 (1H, br s), 9.53 (1H, d,
J = 4.5 Hz), 12.49 (1H, s); MS m/z: 386 (M+H)+. Anal. Calcd for
C18H23N7O3ꢂ1.0CH4O: C, 54.66; H, 5.52; N, 23.49. Found: C, 54.79;
H, 5.69; N, 23.31.
6.1.18. 7-(3,5-Dimethoxylphenylamino)-5-methylsulfanyl-
imidazo[1,2-c]pyrimidine-8-carboxamide (7f)
The title compound was prepared from 6f in the same manner
as described for 7a, and was obtained as white solid (65%): mp

9256
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
6.1.23. 7-(3,5-Dimethoxyphenylamino)-5-(2-methylcyclo-
hexylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (8e)
The title compound was prepared from 7f and 2-methylcyclo-
hexylamine in the same manner as described for 8a, and was ob-
tained as white solid (22%): mp 187–188 °C (MeOH–THF); IR
(KBr) 3404, 2931, 1595, 1497, 1480, 1378, 1291, 1239, 1203,
6.1.28. cis-5-(2-Aminocyclohexylamino)-7-(4-methoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (9c)
The title compound was prepared from 7c in the same manner
as described 8a, and obtained as white solid (8.3%): mp 213–214 °C
(MeOH–THF); IR (KBr) 1601, 1549, 1506, 1387, 1294, 1237 cmꢀ1
;
1H NMR (DMSO-d6) d 1.25–1.93 (8H, m), 3.26–3.38 (4H, m), 3.74
(3H, s), 3.98–4.05 (1H, m), 6.89 (2H, d, J = 9.0 Hz), 7.29 (1H, d,
J = 1.5 Hz), 7.32 (1H, d, J = 3.5 Hz), 7.50 (2H, d, J = 9.0 Hz), 8.06
(1H, d, J = 1.5 Hz), 9.47 (1H, d, J = 3.5 Hz), 12.14 (1H, s); MS m/z:
396 (M+H)+. Anal. Calcd for C20H25N7O2 0.3H2O: C, 59.92; H,
6.44; N, 24.46. Found: C, 60.21; H, 6.41; N, 24.13.
1151, 1067 cmꢀ1 1H NMR (DMSO-d6) d: 0.92 (3H, d, J = 8.0 Hz),
;
1.08–2.01 (9H, m), 3.74–3.84 (1H, m), 3.76 (6H, s), 6.21 (1H, t,
J = 2.0 Hz), 6.83 (2H, d, J = 2.0 Hz), 7.34 (1H, d, J = 1.5 Hz), 7.36
(1H, d, J = 4.5 Hz), 7.60–8.17 (2H, m), 9.52 (1H, d, J = 4.5 Hz),
12.41 (1H, s); MS m/z: 425 (M+H)+. Anal. Calcd for C22H28N6O3:
C, 62.25; H, 6.65; N, 19.80. Found: C, 61.87; H, 6.61; N, 19.65.
6.1.29. cis-5-(2-Aminocyclohexylamino)-7-(3,5-difluorophenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (9d)
6.1.24. cis-7-(3,5-Dimethoxyphenylamino)-5-(2-hydroxycyclo-
hexylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (8f)
The title compound was prepared from 7f and cis-2-hydrox-
ycyclohexylamine in the same manner as described for 8a, and
was obtained as white solid (22%): mp 261–262 °C (MeOH–THF);
IR (KBr) 3405, 2935, 1594, 1544, 1502, 1476, 1378, 1240,
The title compound was prepared from 7d in the same manner
as described for 8a, and was obtained as white solid (21%): mp
240–241 °C (MeOH–THF); IR (KBr) 1601, 1501, 1475, 1449, 1377,
;
1238, 1145 cmꢀ1 1H NMR (DMSO-d6) d: 1.28–1.98 (8H, m),
3.20–3.45 (3H, br), 3.36–3.41 (1H, m), 4.05–4.11 (1H, m), 6.75–
6.84 (1H, m), 7.36 (1H, d, J = 1.5 Hz), 7.37–7.41 (2H, m), 7.54 (1H,
d, J = 3.0 Hz), 8.15 (1H, d, J = 1.5 Hz), 9.56 (1H, d, J = 3.0 Hz), 12.74
(1H, s); MS m/z: 402 (M+H)+. Anal. Calcd for C19H21F2N7Oꢂ0.2CH4O:
C, 56.55; H, 5.39; N, 24.04. Found: C, 56.53; H, 5.30; N, 23.66.
1150 cmꢀ1 1H NMR (DMSO-d6) d:1.25–2.00 (8 H, m), 3.75 (6H,
;
s), 4.03–4.15 (2H, m), 4.80 (1H, d, J = 5.5 Hz), 6.16 (1H, t,
J = 2.0 Hz), 6.79 (2H, d, J = 2.0 Hz), 7.31 (1H, d, J = 2.0 Hz), 7.37
(1H, d, J = 4.5 Hz), 7.85 (1H, d, J = 9.5 Hz), 8.14 (1H, d, J = 2.0 Hz),
9.54 (1H, d, J = 4.5 Hz), 12.37 (1H, s); MS m/z: 427 (M+H)+. Anal.
Calcd for C21H26N6O4ꢂ0.2H2O: C, 58.64; H, 6.19; N, 19.54. Found:
C, 58.96; H, 6.23; N, 19.19.
6.1.30. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethyl-
phenylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (9e)
The title compound was prepared from 7e in the same manner
as described for 8a, and was obtained as white solid (16%): mp
188–189 °C (MeOH–THF); IR (KBr) 1616, 1558, 1539, 1499,
6.1.25. trans-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (8g)
The title compound was prepared from 7f and trans-cyclo-
hexyldiamine in the same manner as described for 8a, and was
obtained as white solid (37%): mp 213–215 °C (MeOH–THF); IR
1375 cmꢀ1 1H NMR (DMSO-d6) d: 1.22–1.94 (8H, m), 2.28 (6H,
;
s), 3.26–3.38 (4H, m), 4.11–4.16 (1H, m), 6.66 (1H, s), 7.23 (2H,
s), 7.31 (1H, s), 7.37 (1H, d, J = 3.0 Hz), 8.10 (1H, s), 9.54 (1H, d,
J = 3.0 Hz), 12.35 (1H, s); MS m/z: 394 (M+H)+. Anal. Calcd for
C21H27N7Oꢂ0.3CH4O: C, 63.47; H, 7.05; N, 24.32. Found: C, 63.64;
H, 6.91; N, 24.05.
(KBr) 1598, 1499, 1457, 1240, 1147 cmꢀ1 1H NMR (DMSO-d6)
;
d: 1.05–1.41 (4H, m), 1.65–2.10 (4H, m), 2.71–2.78 (1H, m),
3.30–3.35 (3H, br), 3.75–3.83 (1H, m), 3.76 (6H, s), 6.20 (1H, t,
J = 2.0 Hz), 6.84 (2H, d, J = 2.0 Hz), 7.33 (1H, d, J = 1.5 Hz), 7.38
(1H, d, J = 3.5 Hz), 7.96 (1H, d, J = 1.5 Hz), 9.54 (1H, d,
J = 3.5 Hz), 12.41 (1H, s); MS m/z: 426 (M+H)+. Anal. Calcd for
C21H27N7O3ꢂ0.8H2O: C, 57.34; H, 6.55; N, 22.29. Found: C,
57.34; H, 6.25; N, 21.92.
6.1.31. cis-5-(2-Aminocyclohexylamino)-7-(3,5-dimethoxyl-
phenylamino)imidazo[1,2-c]pyrimidine-8-carboxamide (9f)
The title compound was prepared from 7f in the same manner
as described for 8a, and was obtained as white solid (37%): mp
198–200 °C (MeOH–THF); IR (KBr) 1596, 1540, 1497, 1240,
6.1.26. cis-5-(2-Aminocyclohexylamino)-7-(2-methoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (9a)
1154 cmꢀ1 1H NMR (DMSO-d6) d: 1.26–1.96 (8H, m), 3.26–3.40
;
(4H, m), 3.75 (6H, s), 4.11–4.18 (1H, m), 6.21 (1H, s), 6.80 (2H, d,
J = 2.0 Hz), 7.33 (1H, s), 7.40 (1H, s), 8.11 (1H, s), 9.54 (1H, s),
12.38 (1H, s); MS m/z: 426 (M+H)+. Anal. Calcd for
C21H27N7O3ꢂ1.0H2O: C, 56.87; H, 6.59; N, 22.11. Found: C, 56.76;
H, 6.30; N, 21.87.
The title compound was prepared from 7a in the same manner
as described for 8a, and was obtained as white solid (16%): mp
212–214 °C (MeOH–THF); IR (KBr) 1608, 1502, 1480, 1382,
1233 cmꢀ1 1H NMR (DMSO-d6) d: 1.30–1.95 (8H, m), 3.26–3.34
;
(3H, br), 3.35–3.38 (1H, m), 3.85 (3H, s), 4.06–4.13 (1H, m), 6.90
(1H, dt, J = 8.0, 1.5 Hz), 6.97 (1H, dt, J = 8.0, 1.5 Hz), 7.30 (1H, dd,
J = 8.0, 1.5 Hz), 7.19 (1H, d, J = 3.5 Hz), 7.32 (1H, d, J = 2.0 Hz),
8.07 (1H, d, J = 2.0 Hz), 8.38 (1H, dd, J = 8.0, 1.5 Hz), 9.49 (1H, d,
J = 3.5 Hz), 12.35 (1H, s); MS m/z: 396 (M+H)+. Anal. Calcd for
C20H25N7O2ꢂ0.7H2O: 58.87; H, 6.52; N, 24.03. Found: C, 59.16; H,
6.40; N, 23.63.
6.1.32. 4-(3,5-Dimethoxyphenylamino)-6-((R)-2-hydroxy-1-phe-
nylethylamino)-2-methylsulfanylpyrimidine-5-carbonitrile (10)
A mixture of compound 4f (0.914 g, 2.71 mmol), (R)-(ꢀ)-2-
amino-2-phenylethanol (0.391 g, 2.85 mmol) and diisopropyleth-
ylamine (0.520 mL, 2.99 mmol) in DMF (10 mL) was stirred at
50 °C for 3 h. Water was added to the mixture and extracted with
EtOAc. The organic phase was washed with water and brine,
sequentially, dried over anhydrous MgSO4, and evaporated in va-
cuo. Resulting residue was purified by column chromatography
on silica gel with hexane/EtOAc = 1:1 to give 10 (0.876 g, 74%)
as yellowish solid: mp 141–144 °C (MeOH); a2D8 ꢀ34.78° (c 0.23,
6.1.27. cis-5-(2-Aminocyclohexylamino)-7-(3-methoxyphenyl-
amino)imidazo[1,2-c]pyrimidine-8-carboxamide (9b)
The title compound was prepared from 7b in the same manner
as described 8a, and obtained as white solid (26%): mp 188–190 °C
(MeOH–THF); IR (KBr) 1598, 1497, 1374, 1238, 1150 cmꢀ1
;
1H
MeOH); IR (KBr) 2195, 1591, 1560, 1487, 1437, 1155 cmꢀ1 1H
;
NMR (DMSO-d6) d: 1.26–1.97 (8H, m), 3.25–3.38 (4H, m), 3.77
(3H, s), 4.07–4.15 (1H, m), 6.61 (1H, d, J = 8.0 Hz), 7.15 (1H, d,
J = 7.5 Hz), 7.21 (1H, t, J = 7.5 Hz), 7.26 (1H, s), 7.32 (1H, br s),
7.39 (1H, br s), 8.10 (1H, s), 9.53 (1H, s), 12.39 (1H, s); MS m/z:
396 (M+H)+. Anal. Calcd for C20H25N7O2 0.7H2O: C, 58.87; H,
6.52; N, 24.03. Found: C, 59.19; H, 6.42; N, 23.65.
NMR (DMSO-d6) d: 2.31 (3H, s), 3.65–3.84 (2H, m), 3.70 (6H, s),
5.04 (1H, t, J = 6.0 Hz), 5.21–5.28 (1H, m), 6.21 (1H, t, J = 2.0 Hz),
6.82 (2H, d, J = 2.0 Hz), 7.20–7.40 (5H, m), 7.70 (1H, d,
J = 7.0 Hz), 9.14 (1H, s); MS m/z: 438 (M+H)+. Anal. Calcd for
C22H23N5O3Sꢂ0.1H2O: C, 60.14; H, 5.32; N, 15.94. Found: C,
59.92; H, 5.26; N, 15.91.

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
9257
6.1.33. (R)-7-(3,5-Dimethoxyphenylamino)-5-methylsulfanyl-
2-phenyl-2,3-dihydroimidazo[1,2-c]pyrimidine-8-carbonitrile
(11)
50 °C and was purified by column chromatography on APS with
EtOAc to give 13a (0.332 g, 71%) as white solid: mp 210–211 °C
(EtOAc); a2D8 ꢀ83.08° (c 0.13, MeOH); IR (KBr) 1692, 1630, 1595,
A solution of compound 10 (0.697 g, 1.59 mmol) in phosphorus
oxychloride (6 mL) was stirred at 60 °C for 3 h. The mixture was
poured into ice-cold water and neutralized with NaHCO3 powder.
The mixture was extracted with EtOAc. The organic phase was
wished with water and brine, sequentially, dried over anhydrous
MgSO4, and evaporated in vacuo. The residue was triturated from
Et2O and resulting precipitates were collected by suction filtration
to give 11 (0.622 g, 93%) as yellowish solid: mp 207–209 °C
(MeOH–THF); a2D8 ꢀ76.30° (c 0.54, MeOH); IR (KBr) 2195, 1645,
1529, 1497, 1456, 1202, 1154 cmꢀ1 1H NMR (CDCl3) d: 1.20–
;
1.42 (2H, m), 1.41 (9H, s), 1.52–1.82 (6H, m), 2.18–2.26 (1H, m),
3.44 (1H, dd, J = 9.5, 2.0 Hz), 3.76 (6H, s), 3.84–3.92 (1H, m),
3.98–4.04 (1H, m), 4.25 (1H, t, J = 10.5 Hz), 4.85 (1H, d,
J = 5.5 Hz), 5.20–5.40 (1H, br), 5.43 (1H, dd, J = 10.5, 2.0 Hz), 6.17
(1H, t, J = 2.0 Hz), 6.85 (2H, d, J = 2.0 Hz), 6.84–6.93 (1H, br),
7.26–7.36 (5H, m), 12.56 (1H, s); MS m/z: 604 (M+H)+. Anal. Calcd
for C32H41N7O5: C, 63.29; H, 6.87; N, 16.14. Found: C, 63.04; H,
6.80; N, 16.14.
1588, 1489, 1405, 1205, 1154 cmꢀ1 1H NMR (DMSO-d6) d: 2.47
;
(3H, s), 3.55–3.62 (1H, m), 3.71 (6H, s), 4.36 (1H, t, J = 10.0 Hz),
5.26 (1H, dd, J = 10.0 Hz, 2.5 Hz), 6.26 (1H, t, J = 2.0 Hz), 6.70 (2H,
d, J = 2.0 Hz), 7.25–7.39 (5H, m), 9.43 (1H, s); MS m/z: 420
(M+H)+. Anal. Calcd for C22H21N5O2Sꢂ0.15CH4O: C, 62.70; H, 5.13;
N, 16.50. Found: C, 63.06; H, 5.20; N, 16.14.
6.1.36. tert-Butyl {(1R,2S)-2-[(R)-8-carbamoyl-7-(3,5-
dimethoxyphenylamino)-2-phenyl-2,3-dihydroimidazo[1,2-
c]pyrimidin-5-ylamino]cyclohexyl}carbamate (13b)
The title compound was prepared from 12b in the same
manner as described for 13a, and was obtained as white solid
(64%): mp 248–249 °C (EtOAc); a2D8 ꢀ98.67° (c 0.15, MeOH); IR
6.1.34. tert-Butyl {(1S,2R)-2-[(R)-8-cyano-7-(3,5-dimethoxy-
phenylamino)-2-phenyl-2,3-dihydroimidazo[1,2-c]pyrimidin-
5-ylamino]-cyclohexyl}carbamate (12a) and tert-butyl {(1R,2S)-
2-[(R)-8-cyano-7-(3,5-dimethoxyphenylamino)-2-phenyl-2,3-
dihydroimidazo[1,2-c]pyrimidin-5-
(KBr) 1715, 1623, 1597, 1524, 1496, 1457, 1163 cmꢀ1 1H
;
NMR (CDCl3) d: 1.20–1.41 (2H, m), 1.42 (9H, s), 1.52–1.81 (6H,
m), 2.18–2.25 (1H, m), 3.61–3.68 (1H, m), 3.76 (6H, s), 3.86–
3.98 (2H, m), 4.02 (1H, t, J = 10.5 Hz), 4.79–4.85 (1H, m), 5.23–
5.28 (1H, m), 5.38 (1H, dd, J = 10.5 Hz, 2.0 Hz), 6.17 (1H, t,
J = 2.0 Hz), 6.85 (2H, d, J = 2.0 Hz), 7.26–7.36 (5H, m), 10.03
(1H, s), 12.58 (1H, s); MS m/z: 604 (M+H)+. Anal. Calcd for
C32H41N7O5ꢂ0.5C4H8O2: C, 63.04; H, 7.00; N, 15.13. Found: C,
62.72; H, 6.99; N, 14.90.
ylamino]cyclohexyl}carbamate (12b)
To a suspension of compound 11 (1.260 g, 3.00 mmol) in DMF
(12 mL) was added cis-1,2-diaminocyclohexane (1.80 mL,
15.00 mmol) and the mixture was stirred for 5 h at 50 °C. The mix-
ture was poured into water and extracted with EtOAc. The organic
layer was washed with water and brine, sequentially, dried over
Na2SO4, and evaporated in vacuo. The residue was dissolved in
CH2Cl2 (10 mL) and added di-tert-butyldicarbonate (0.655 g,
3.00 mmol), then stirred overnight at room temperature. The mix-
ture was purified by column chromatography on silica gel with
hexane/EtOAc = 1:4 ? MeOH/EtOAc = 1:50 ? 1:30 to give 12a
(0.454 g, 26%) as white solid and 12b (0.580 g, 33%) as white solid.
Physicochemical data of 12a: mp 163–164 °C (MeOH–THF); aD28
ꢀ65.22° (c 0.23, MeOH); IR (KBr) 2196, 1591, 1538, 1506, 1457,
6.1.37. tert-Butyl {(1S,2R)-2-[8-carbamoyl-7-(3,5-dimethoxy-
phenylamino)-2-phenylimidazo[1,2-c]pyrimidin-5-ylamino]-
cyclohexyl}carbamate (14a)
To a mixture of compound 13a (0.170 g, 0.282 mmol) and
10% Pd–C (56.5% water containing reagent, 0.040 g) in DMSO
(1 mL) and EtOH (1 mL) was added cyclohexene (0.086 mL,
0.845 mmol) and was stirred overnight at 100 °C under oxygen
atmosphere. Catalyst was removed by filtration through Celite
and the filtrate was dissolved in EtOAc. The mixture was washed
with water and brine, sequentially, dried over anhydrous MgSO4,
and evaporated in vacuo. Resulting residue was purified by col-
umn chromatography on silica gel with hexane/EtOAc = 1:1 to
give 14a (0.060 g, 35%) as white solid: mp 224–225 °C (EtOAc);
a2D8 +12.17° (c 0.23, MeOH); IR (KBr) 1685, 1602, 1498, 1458,
1204, 1155 cmꢀ1 1H NMR (CDCl3) d: 1.18–1.39 (2H, m), 1.40
;
(9H, s), 1.41–1.83 (6H, m), 2.08–2.18 (1H, m), 3.45 (1H, dd,
J = 10.0, 2.0 Hz), 3.77 (6H, s), 3.78–3.82 (1H, m), 3.96–4.02 (1H,
m), 4.23 (1H, t, J = 10.0 Hz), 4.83 (1H, d, J = 5.5 Hz), 5.43 (1H, dd,
J = 10.0 Hz, 2.0 Hz), 6.22 (1H, t, J = 2.0 Hz), 6.68 (2H, d, J = 2.0 Hz),
6.92 (1H, s), 7.20-7.25 (1H, m), 7.31–7.36 (4H, m); MS m/z: 586
(M+H)+. Anal. Calcd for C32H39N7O4: C, 65.62; H, 6.71; N, 16.74.
Found: C, 65.39; H, 6.72; N, 16.84.
1236, 1202, 1153 cmꢀ1 1H NMR (CDCl3) d: 1.30–1.55 (2H, m),
;
1.58 (9H, s), 1.60–1.92 (6H, m), 3.79 (6H, s), 4.01–4.17 (2H,
m), 4.98 (1H, d, J = 6.0 Hz), 5.55 (1H, s), 6.18 (1H, t, J = 2.0 Hz),
6.92 (2H, d, J = 2.0 Hz), 7.28–7.34 (1H, m), 7.41 (2H, t,
J = 8.0 Hz), 7.55 (1H, s), 7.87 (2H, d, J = 8.0 Hz), 8.19 (1H, s),
10.10 (1H, s), 12.18 (1H, s); MS m/z: 602 (M+H)+. Anal. Calcd
for C32H39N7O5: C, 63.88; H, 6.53; N, 16.30. Found: C, 63.58;
H, 6.54; N, 16.02.
Physicochemical data of 12b: mp 202–203 °C (MeOH–THF); aD28
ꢀ127.41° (c 0.27, MeOH); IR (KBr) 2195, 1591, 1528, 1506, 1457,
1204, 1153 cmꢀ1 1H NMR (CDCl3) d: 1.42 (9H, s), 1.43–1.82 (8H,
;
m), 2.10–2.18 (1H, m), 3.64–3.69 (1H, m), 3.77 (6H, s), 3.78–3.82
(1H, m), 3.91–3.96 (1H, m), 4.00 (1H, t, J = 10.5 Hz), 4.81 (1H, d,
J = 5.0 Hz), 5.38 (1H, dd, J = 10.5 Hz, 2.0 Hz), 6.22 (1H, t,
J = 2.0 Hz), 6.69 (2H, d, J = 2.0 Hz), 6.92 (1H, s), 7.27–7.38 (5H,
m); MS m/z: 586 (M+H)+. Anal. Calcd for C32H39N7O4ꢂ0.8H2O: C,
64.04; H, 6.82; N, 16.34. Found: C, 64.13; H, 6.82; N, 16.26.
6.1.38. tert-Butyl {(1R,2S)-2-[8-carbamoyl-7-(3,5-dimethoxy-
phenylamino)-2-phenylimidazo[1,2-c]pyrimidin-5-ylamino]-
cyclohexyl}carbamate (14b)
The title compound was prepared from 13b in the same manner
as described for 14a, and was obtained 14b as white solid (33%):
mp 222–224 °C (EtOAc); a2D8 ꢀ25.00° (c 0.20, MeOH); IR (KBr)
6.1.35. tert-Butyl {(1S,2R)-2-[(R)-8-carbamoyl-7-(3,5-
dimethoxyphenylamino)-2-phenyl-2,3-dihydroimidazo[1,2-
c]pyrimidin-5-ylamino]cyclohexyl}carbamate (13a)
1685, 1599, 1497, 1458, 1380, 1235, 1202, 1152 cmꢀ1 1H NMR
;
To a solution of compound 12a (0.454 g, 0.755 mmol) in DMSO
(6 mL) and EtOH (3 mL) was added 5 M NaOH solution (1.09 mL,
5.43 mmol) and 30% H2O2 solution (0.615 mL, 5.43 mmol), then
the mixture was stirred overnight at room temperature. Water
(1 mL) was added and the mixture was adjusted at pH 11 with
1 M HCl solution. The obtained pale yellow precipitate was col-
lected by filtration and washed with H2O. The solid was dried at
(CDCl3) d: 1.29–1.55 (2H, m), 1.58 (9H, s), 1.61–1.92 (6H, m),
3.79 (6H, s), 4.01–4.17 (2H, m), 4.79–4.85 (1H, m), 5.55 (1H, s),
6.18 (1H, t, J = 2.0 Hz), 6.92 (2H, d, J = 2.0 Hz), 7.28–7.34 (1H, m),
7.41 (2H, t, J = 8.0 Hz), 7.55 (1H, s), 7.87 (2H, d, J = 8.0 Hz), 8.17
(1H, s), 10.10 (1H, s), 12.18 (1H, s); MS m/z: 602 (M+H)+. Anal.
Calcd for C32H39N7O5ꢂ0.2H2O: C, 63.49; H, 6.56; N, 16.19. Found:
C, 63.24; H, 6.47; N, 16.03.

9258
A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
6.1.39. tert-Butyl {2-[8-Carbamoyl-7-(3,5-dimethoxyphenyl-
amino)-2-phenylimidazo[1,2-c]pyrimidin-5-ylamino]-1,1-
dimethyl-ethyl}-carbamate (14c)
give 15c (0.012 g, 73%) as yellowish solid: mp 252–254 °C (EtOAc);
IR (KBr) 1599, 1559, 1507, 1203, 1154 cmꢀ1; 1H NMR (DMSO-d6) d:
1.34 (6H, s), 3.77 (6H, s), 3.78–3.84 (2H, m), 6.24 (1H, s), 6.84 (2H,
s), 7.37 (1H, t, J = 8.0 Hz), 7.49 (2H, t, J = 8.0 Hz), 7.56 (1H, s), 7.87
(2H, d, J = 8.0 Hz), 8.02 (3H, br s), 8.53 (1H, s), 8.61 (1H, br s),
9.63 (1H, br s), 12.46 (1H, s); MS m/z: 476 (M+H)+. Anal. Calcd
for C25H29N7O3ꢂC2HF3O2ꢂ0.1H2O: C, 54.83; H, 5.15; N, 16.58. Found:
C, 54.55; H, 5.39; N, 16.91.
A suspension of compound 11 (0.126 g, 0.300 mmol) and 2-
methylpropane-1,2-diamine (0.157 mL, 1.500 mmol) in DMF
(4 mL) was stirred for 4 h at 60 °C. The mixture was poured into
H2O and extracted with EtOAc. The organic layer was washed
with brine, dried over Na2SO4, and evaporated in vacuo. The res-
idue was dissolved in CH2Cl2 (8 mL) and added di-tertbutyldicar-
bonate (0.066 g, 0.300 mmol) and stirred overnight at room
temperature. The mixture was purified by column chromatogra-
phy on APS with EtOAc ? MeOH/EtOAc = 1:10 to give 12c
(0.094 g, 56%) as yellowish amorphous. The title compound was
prepared from 12c in the same manner as described for 13a
and 14a, and was obtained 14c as white solid (15%, three steps
yield): mp 210–212 °C (EtOAc); IR (KBr) 1617, 1596, 1559,
6.2. Molecular modeling
The 3D coordinates of the ZAP-70 receptor model were con-
structed based on the published crystal structure of an activated
Lck kinase domain16,17 (PDB code: 1QPD) using the program
FAMS.17 The structure of staurosporine was extracted from the
X-ray structure of LCK (PDB code: 1QPD) and manually docked into
the ATP-binding site of the ZAP-70 receptor model using QUAN-
TA200024 (Accelrys Inc., San Diego, CA, USA). All docking calcula-
tions were performed using the program ADAM.18
1457, 1204, 1151 cmꢀ1 1H NMR (CDCl3) d: 1.45 (6H, s), 1.51
;
(9H, s), 2.98 (2H, s), 3.77 (1H, d, J = 5.0 Hz), 3.79 (6H, s), 4.87
(1H, s), 5.58 (1H, s), 6.18 (1H, t, J = 2.0 Hz), 6.97 (1H, d,
J = 2.0 Hz), 7.31–7.35 (1H, m), 7.43 (2H, t, J = 8.0 Hz), 7.61 (1H,
s), 7.90 (2H, d, J = 8.0 Hz), 8.17 (1H, s), 10.10 (1H, s), 12.24 (1H,
s); MS m/z: 576 (M+H)+. Anal. Calcd for C30H37N7O5: C, 62.59;
H, 6.48; N, 17.03. Found: C, 62.29; H, 6.46; N, 16.88.
6.3. Biology
6.3.1. 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 5’ region was integrated
into pFASTBAC1 vector of the BAC-TO-BACTM (Gibco-BRL Inc.) bac-
ulovirus expression system. The transfected virus was obtained
by transfecting Sf-9 cell (Invitrogen Inc.) with pFASTBAC1 con-
taining His-tag-fused ZAP kinase domain described above. High
FiveTM baculocells, which was infected with this transfected virus,
were recovered and these cells were dissolved by ultrasonication.
After soluble fraction being separated by centrifuge, the superna-
tant 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 domain by imidazole contain-
6.1.40. 5-((1R,2S)-2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)-2-phenylimidazo[1,2-c]pyrimidine-8-carbox-
amide (15a)
To a mixture of compound 14a (0.035 mg, 0.0582 mmol) in
MeOH (1 mL) was added 4 M HCl–EtOAc (2 mL). After being stir-
red for 1 h at room temperature, volatiles were evaporated in va-
cuo and to the residue was added EtOAc. Resulting precipitates
were collected by suction filtration and dried under reduced
pressure to give 15a (0.027 mg, 86%) as yellowish solid: mp
279–280 °C (EtOAc); a2D8 +1.90° (c 0.21, DMSO); IR (KBr) 1599,
1500, 1375, 1238, 1203, 1155 cmꢀ1
;
1H NMR (DMSO-d6) d:
1.38–2.06 (8H, m), 3.77 (6H, s), 3.79–3.92 (2H, m), 4.31–4.39
(1H, m), 6.25 (1H, t, J = 2.0 Hz), 6.78 (1H, d, J = 2.0 Hz), 7.36
(1H, t, J = 8.0 Hz), 7.49 (2H, t, J = 8.0 Hz), 7.55 (1H, s), 7.88 (2H,
d, J = 8.0 Hz), 8.02 (4H, br s), 8.70 (1H, s), 9.67 (1H, br s),
12.42 (1H, s); MS m/z: 502 (M+H)+. Anal. Calcd for
C27H31N7O3ꢂ2.0HCl: C, 56.45; H, 5.79; N, 17.07. Found: C,
56.57; H, 5.98; N, 16.80.
ing
a buffer. 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
6.1.41. 5-((1S,2R)-2-Aminocyclohexylamino)-7-(3,5-dimethoxy-
phenylamino)-2-phenylimidazo[1,2-c]pyrimidine-8-carbox-
amide (15b)
The title compound was prepared from 14b in the same manner
as described for 15a, and was obtained 15b as yellowish solid
(54%): mp 280–281 °C (EtOAc); a2D8 +5.22° (c 0.23, DMSO); IR
10% glycerol, and started by adding ATP. PK (150
(500 g/mL), PEP (2.5 mM), and NADH (150 M) were added in
large excess. Addition of 100 M ZAP-70 optimal peptide sub-
lg/mL), LDH
l
l
(KBr) 1653, 1600, 1560, 1507, 1155 cmꢀ1
;
1H NMR (DMSO-d6) d:
l
1.38–2.06 (8H, m), 3.77 (6H, s), 3.78–3.86 (2H, m), 4.34 (1H, br
s), 4.35 (1H, br s), 6.25 (1H, t, J = 2.0 Hz), 6.78 (1H, d, J = 2.0 Hz),
7.36 (1H, t, J = 8.0 Hz), 7.49 (2H, t, J = 8.0 Hz), 7.55 (1H, s), 7.88
(2H, d, J = 8.0 Hz), 8.01 (4H, br s), 8.70 (1H, s), 9.65 (1H, br s),
12.42 (1H, s); MS m/z: 502 (M+H)+. Anal. Calcd for
C27H31N7O3ꢂ2.0HCl: C, 56.45; H, 5.79; N, 17.07. Found: C, 56.34;
H, 5.88; N, 16.83.
strate (peptide sequence: AEEEIYGEFEAKKKK, Sawady, Tokyo), al-
lowed measurement of kinase activity.
6.3.2. 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
Marathon-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 expression
system (Gibco-BRL Inc.). The transfected virus was obtained by
transfecting Sf-9 cell (Invitrogen Inc.) with pFASTBAC vector con-
taining 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 soluble
fraction being separated by centrifuge, the supernatant was
6.1.42. 5-(2-Amino-2-methylpropylamino)-7-(3,5-dimethoxy-
phenylamino)-2-phenylimidazo[1,2-c]pyrimidine-8-carbox-
amide (15c)
To a mixture of compound 14c (0.016 g, 0.028 mmol) in CH2Cl2
(0.5 mL) was added TFA (0.2 mL) and the mixture was stirred for
1 h at room temperature. Volatiles were evaporated in vacuo and
to the residue was added EtOAc. Resulting precipitates were col-
lected by suction filtration and dried under reduced pressure to

A. Hirabayashi et al. / Bioorg. Med. Chem. 16 (2008) 9247–9260
9259
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,
ous administration of compounds, ConA (20 mg/kg) was intrave-
nously injected. Groups of mice (n = 4) were euthanized at 2 h
following ConA treatment. Plasma was collected and analyzed for
IL-2 levels by ELISA. The ELISA used here are not reported to exhibit
detectable cross-reactivity with the other cytokines.
6.3.6. Single-crystal X-ray diffraction analysis of compound 13a
A
colorless prismatic crystal of compound [13a], [13a
C32H41N7O5] (FW = [603.71]) having approximate dimensions of
0.5 ꢁ 0.2 ꢁ 0.2 mm was mounted on a nylon loop (Hampton Re-
search, Aliso Viejo, CA, USA). All measurements were made on an
R-AXIS V imaging plate detector (Rigaku, Tokyo, Japan) with syn-
chrotron radiation at SPring-8 BL32B2 beam line (Hyogo, Japan).
Cell constants and an orientation matrix for data collection, ob-
tained from a least-squares refinement using the setting angles
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
GEFEAKKKK, Sawady, Tokyo) allowed measurement of kinase
of
25
carefully
centered
reflections
in
the
range
activity.
58.26 < 2h < 60.17°, corresponded to a primitive triclinic cell (P1,
for Z = 1) with dimensions: a = 17.772(3) Å, b = 10.812(3) Å,
c = 10.286(3) Å, V = 1976.2(7) Å 3, the calculated density: 1.22 g/
cm3. The data were collected at a temperature of ꢀ170 1.0 °C at
a maximum resolution of 0.86 Å (k = 0.71 Å). The low-temperature
experiment was performed using Paratone-N-coated rapidly
cooled crystals on the tip of a copper pin. Of the 32,817 reflections
that were collected, 32,452 were unique (Rint = 0.032). Data were
collected and processed using the CrystalClear program (Version.
1.3.5; Rigaku). The structure was solved by direct methods25 and
expanded using Fourier techniques.26 The non-hydrogen atoms
were refined anisotropically. All hydrogen atoms were detected
using difference Fourier techniques, and their positions were fixed
in the course of refinement. The final cycle of full-matrix least-
squares refinement27 was based on 3087 independent reflections
and 440 variable parameters, and converged (largest parameter
shift was 1.69 times its esd) with unweighted and weighted agree-
ment factors of R = 0.066 and Rw = 0.111, respectively. The good-
ness of fit28 was 1.34. The maximum and minimum peaks on the
final difference Fourier map corresponded to 0.30 and ꢀ0.27 eꢀ/
Å3, respectively. Neutral atoms scattering factors were taken from
Cromer and Weber,29 and the values for the mass attenuation coef-
ficients are those of Creagh and Hubbel.30 All calculations were
performed using the teXsan31 crystallographic software package
of the Molecular Structure Corporation (The Woodlands, TX, USA).
6.3.3. Peripheral blood mononuclear cells (PBMCs) and whole
blood (WB)
6.3.3.1. 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 dis-
tilled water and 10ꢁ Hanks’ buffer saline solution (HBSS) to re-
move red blood cells. The cells were resuspended to
a
concentration of 2 ꢁ 106 cells/mL inAIM-V medium (Invitrogen)
containing100 U/mL penicillin and 100 lg/mL streptomycin.
6.3.3.2. Determination of IL-2 production by PBMCs and
WB. PBMCs or WB were cultured with 10 g/mL PHA alone or
l
in 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 immunoassay (ELISA). The ELISA used here
are not reported to exhibit detectable cross-reactivity with the
other cytokines.
6.3.4. Passive cutaneous anaphylaxis (PCA) assays
ICR mice were passively sensitized by subcutaneously injecting
anti-dinitrophenyl (DNP)-coupled IgE under the right ear pinna,
while lightly anesthetizing with ether. After 48 h, each mouse
was challenged by injecting a mixture of DNP-conjugated bovine
Acknowledgments
We are grateful to Dr. K. Hasegawa and the staff of SPring-8
BL32B2 for assistance with X-ray data collection. We thank Dr. Y.
Terao for assistance with the syntheses and helpful discussions,
and Dr. N. Tanaka for valuable suggestions during the preparation
of this manuscript.
serum albumin and 250 lL of 0.5% Evans blue solution via the tail
vein to induce passive cutaneous anaphylaxis. Thirty minutes after
the challenge, the mice were sacrificed to take ears and the amount
of dye from the blueing region was measured. Test compounds or
vehicle alone as a control were orally administered to the mice 1 h
before the antigen challenge. The dye in the tissues was extracted
with acetone and colorimetrically determined at 620 nm. The PCA
reaction (% of vehicle) by the test compound was calculated based
on the following equation. In the formula, A: amount of dye leaked
into the sensitized ear at the time of administration of the vehicle
alone; B: amount of dye leaked into the unsensitized ear at the
time of administration of vehicle alone; C: amount of dye leaked
into the sensitized ear at the time of administration of the com-
pound to be tested. Amount of dye leaked (% of vehi-
cle) = {(A ꢀ B) ꢀ (C ꢀ B)} ꢁ 100/(A ꢀ B).
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P
2
27. Least-squares: function minimized:
wðF2o ꢀ F2c Þ , where w ¼ 1=r2ðF2oÞ ¼
2
2
½
r2c ðFoÞ þ ðpðMaxðFo2Þ þ 2F2c Þ=3Þ ꢃꢀ1
;
rcðF2oÞ ¼ esd
based
on
counting
statistics, P = p-factor (0.05).
P
28. Standard deviation of an observation of full weight:
[
w(|Fo| ꢀ |Fc|)2/
(No ꢀ Nv)]1/2, where No, number of observations; Nv, number of variables.
29. Cromer, D. T.; Waber, J. T.. In In International Tables for X-ray Crystallography;
Kynoch Press: Birmingham, UK, 1974; Vol. IV. Table 2.2A.
30. Creagh, D. C.; Hubbell, J. H.. In In International Tables for Crystallography;
Wilson, A. J. C., Ed.; Kluwer Academic: Boston, MA, 1992; Vol. C, pp 200–206.
Table 4.2.4.3.
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J. Med. Chem. 2002, 45, 2615.
31. teXsan: Crystal Structure Analysis Package. Molecular Structure Corporation
(1995 and 2004).