Synthesis and c-Src inhibitory activity of imidazo[1,5-a]pyrazine derivatives as an agent for treatment of acute ischemic stroke

Article abstract of DOI:10.1016/j.bmc.2006.10.041

We synthesized and evaluated a series of C-5 substituted imidazo[1,5-a]pyrazine derivatives to identify potent c-Src inhibitors as potential therapeutic agents for acute ischemic stroke. Among these compounds, compound 14c·HCl demonstrated remarkable cent

Full text of DOI:10.1016/j.bmc.2006.10.041

Bioorganic & Medicinal Chemistry 15 (2007) 868–885
Synthesis and c-Src inhibitory activity of imidazo[1,5-a]pyrazine
derivatives as an agent for treatment of acute ischemic stroke
Harunobu Mukaiyama,* Toshihiro Nishimura, Satoko Kobayashi,
Tomonaga Ozawa, Noboru Kamada, Yoshimitsu Komatsu, Shinji Kikuchi,
Hideki Oonota and Hiroshi Kusama
Central Research Laboratory, Kissei Pharmaceutical Company Ltd, 4365-1, Azumino-city, Nagano 399-8304, Japan
Received 25 August 2006; revised 18 October 2006; accepted 20 October 2006
Available online 24 October 2006
Abstract—We synthesized and evaluated a series of C-5 substituted imidazo[1,5-a]pyrazine derivatives to identify potent c-Src inhib-
itors as potential therapeutic agents for acute ischemic stroke. Among these compounds, compound 14cÆHCl demonstrated remark-
able central nervous system (CNS) penetration and significant neuroprotective efficacy in vivo in rat models.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
itor selectively active against tyrosine kinase c-Src would
hold promise as an effective therapeutic medication
against stroke.
The protein tyrosine kinase c-Src is prototypical of the
eight members of the kinase family found in vertebrates,
namely, c-Src, Fyn, Yes, Fgr, Hck, Lyn, Lck, and Blk.
Widely found in mammalian cells, c-Src has particularly
high concentrations in the brain, platelets, and osteo-
clasts,1 acting as a common signal mediator to a broad
spectrum of physiological responses.2 In particular,
c-Src regulates the activity of the N-methyl-D-aspartate
(NMDA) receptor, which induces a large and prolonged
Ca2+ influx into the neurons after cerebral ischemia3,4
that culminates in neuronal damage. The c-Src kinase
also plays a predominant role in cytokine release and
superoxide production in neutrophils,5,6 in addition to
mediating the signaling events in response to vascular
endothelial growth factor (VEGF),7 which modulates
vascular permeability and contributes to cerebral
edema.8,9
Mice lacking pp60c-Src show both a reduction in infarct
volumes and VEGF-mediated vascular permeability
after brain ischemia.10 Additionally, treatment of wild-
type mice with PP1, a c-Src selective inhibitor (Fig. 1),
reduces infarct size and decreases edema.10–12 An inhib-
Keywords: c-Src; Brain ischemia; Imidazo[1,5-a]pyrazine; Central ne-
rvous system (CNS) penetration; CH–p interaction.
*
Corresponding author. Tel.: +81 263 82 8820; fax: +81 263 82
8827; e-mail: harunobu_mukaiyama@pharm.kissei.co.jp
Figure 1. Structures of the Src family of kinase inhibitors.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.041

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
869
We explore the activity of an exciting new compound,
compound 14cÆHCl, which has better CNS penetration
and c-Src inhibitory activity compared to BMS-
279700, in addition to significant neuroprotective
efficacy in vivo in rats. We describe the synthesis and
structure–activity relationship (SAR) of the imi-
dazo[1,5-a]pyrazine derivatives in detail, and report the
pharmacological profile and biological evaluation of
compound 14cÆHCl.
Figure 2.
2. Chemistry
2.1. Synthesis of the C-5 aryl imidazo[1,5-a]pyrazine
derivatives
Several kinase inhibitors in the Src family, such as
PP1,12 CGP-77675 (c-Src inhibitors),13 and BMS-
279700 (Lck inhibitor),14 act at the catalytic site (see
Fig. 1). We evaluated whether these kinase inhibitors
could reduce cerebral infarct volume after middle cere-
bral artery (MCA) occlusion in rats. PP1 reduced brain
injury with cerebral infarct volume (23%, data not
shown) 24 h after MCA occlusion. BMS-279700, an
effective inhibitor of Lck, has both excellent in vivo
anti-inflammatory effects in mice and superior c-Src
inhibitory activity compared to PP1. However, it has
poor central nervous system (CNS) penetration (brain/
plasma = 0.6, Table 5). Consequently, we sought to im-
prove CNS penetration by designing and synthesizing
compounds based on the core structure of BMS-
279700, resulting in a series of C-5 substituted imi-
dazo[1,5-a]pyrazine derivatives, represented by the
general formula 1 (Fig. 2).
The C-5 aryl imidazo[1,5-a]pyrazine derivatives 6a–j
were prepared as shown in Scheme 1. The pyrazine-2-
one derivatives (3a and b) were prepared by regioselec-
tive condensation of the phenylglyoxal derivatives
(2a and b) with glycinamide hydrochloride.15 After
protection of the nitrogen in the pyrazine ring with
p-methoxybenzyl chloride (PMB-Cl), the imidazo[1,5-
a]pyrazine core 4a and b was synthesized via condensa-
tion with tosylmethyl isocyanide (Tos-MIC) according
to the procedure of Chen et al.16 Cleavage of the p-meth-
oxybenzyl group with TfOH/TFA and treatment of 5a
and b with POCl3 yielded the 8-chloroimidazo[1,5-a]pyr-
azine derivatives. Finally, the chloride at the 8-position
was substituted with various anilines in the presence of
sodium bis(trimethylsilyl)amide (NaHMDS) to produce
the C-5 aryl imidazo[1,5-a]pyrazine derivatives 6a–j.
Scheme 1. Reagents: (a) NaOH, glycinamide hydrochloride; (b) PMB-Cl, NaH, n-Bu4N+Iꢀ; (c) Tos-MIC, NaH; (d) TfOH, TFA, anisol; (e) POCl3,
cat. DMF; (f) aniline analogues, NaHMDS.

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H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
Scheme 2. Reagents: (a) NaNO2; (b) PMB-Cl, NaH, n-Bu4N+Iꢀ; (c) Tos-MIC, NaH; (d) TfOH, TFA, anisol; (e) POCl3, (cat.)DMF; (f) aniline
analogues, NaHMDS; (g) Aryl-B(OH)2, Pd(II)(dppf)2Cl2, K2CO3; (h) 5-n-Bu3Sn-thiophen-2-CHO, (PPh3)2PdCl2; (i) n-BuLi; (j) NaOH; (k) 1 M
Me2NH in THF, WSCÆHCl, HOBt, NEt3; (l) amines, Ti(Oi-Pr)4, NaBH4.
Scheme 3. Reagents: (a) 1 M BBr3; (b) aminoalcohol derivatives,
DEAD, PPh3.
Select phenyl groups were introduced at position C-5 as
described above. We also introduced various aryl groups
at the C-5 position by Suzuki- or Stille-coupling of the
5-bromo intermediate 10, as shown in Scheme 2.
Compound 7 was converted to pyrazine-2-one (8) with
sodium nitrite.17 The major intermediate 10 was pre-
pared from pyrazine-2-one (8) in five steps, as noted
above. Reaction of intermediate 10 with aryl boronic
acid derivatives in the presence of Pd(dppf)2Cl2 provid-
ed the C-5 aryl substituted imidazo[1,5-a]pyrazine deriv-
atives 12a–g, j, and k. N,N-Dimethyl amide 12i was
prepared by hydrolysis of the ester 12g, followed by con-
densation with dimethylamine. Treatment of intermedi-
Scheme 4. Reagents: (a) 2-bromoacetophenone derivatives; (b) imid-
azole; (c) POCl3, cat. DMF; (d) aniline analogues, NaHMDS.
ate 10 with 5-tributylstannylthiophen-2-carbaldehyde
produced 12l; reductive amination formed the amine
derivatives 13a and b. The methyl groups were removed
from compounds 6h, 12a, and Mitsunobu-coupling with
several amino-alcohols produced derivatives 14a–e with
2-bromoacetophenone derivatives produced imidazo-
pyrazinium salt derivatives (16a and b). The benzyl
groups were deprotected with imidazole to yield C-6 aryl
substituted imidazo[1,5-a]pyrazine-8-one derivatives 17a
and b. Subsequently, the C-6 aryl substituted imi-
dazo[1,5-a]pyrazine derivatives 18a–c were prepared
through coupling reactions with anilines via the chlo-
roimidate derivatives.
various polar functionalities, as shown in Scheme 3.
2.2. Synthesis of the C-6 aryl substituted imidazo[1,5-a]
pyrazine
Synthesis of the derivatives with C-6 substituents is illus-
trated in Scheme 4.18 Treatment of imidazole (15) with

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
871
3. Binding model
The SAR (Table 1) around the aniline phenyl ring at the
C-5 position of the imidazo[1,5-a]pyrazine nucleus was
evaluated for compounds 6a–j. Modifying the substitu-
ents (R1–R3) on the aniline phenyl ring led to a dramatic
change in inhibitory activity against c-Src kinase. The
IC50 for the derivatives with unsubstituted aniline phen-
yl rings 6b and j were 5.5 and 3.7 lM, respectively. The
ortho-methyl group 6i significantly enhanced c-Src
inhibitory activity. Furthermore, the 2,6-dimethyl com-
pounds 6a and h showed greater inhibitory activity than
the ortho-methyl compound 6i. The methyl group could
be replaced by chloride without any loss of activity (6a
vs 6c). However, smaller substituents, such as fluoride
(6d), or bulkier methoxy groups (6f) decreased activity.
Addition of a substituent to the 4-position of the aniline
phenyl ring 6e (R3 = Me) did not alter activity, whereas
N-methyl substitution in 6g showed less activity. This
suggests that aniline N–H plays an important inhibitory
role.
We examined the binding model of compound 6a to the
c-Src kinase domain (PDB code. 1YOL)19 using a dock-
ing program (Quanta/CharmTM),20 which is analogous to
the X-ray crystal structure of the complex between 6a
and Lck kinase.21 As previously reported,22–24 com-
pound 6a interacted with several amino acid residues
to enhance its binding affinity with the ATP-binding site
(Fig. 4). For example, a hydrogen bond formed between
the amino group of the aniline and the hydroxyl group
(acceptor) of Thr 340; the N-2 nitrogen (acceptor) of
imidazo[1,5-a]pyrazine interacted with the backbone
NH (donor) of Met 343; and there was a strong hydro-
gen bonding interaction between C-1-H (donor) of imi-
dazo[1,5-a]pyrazine and the backbone CO (acceptor) of
Glu 341. Furthermore, the aniline phenyl ring formed a
CH–p interaction25 with the side chains of Thr 340 and
Lys 297 in the hydrophobic pocket. In the deep cleft,
CH–p interactions formed between the imidazo[1,5-
a]pyrazine core and the side chains of Val 283 and Leu
395, as well as between the C-5 phenyl ring and the side
chains of Leu 275.
We used a binding model and conformation analysis to
analyze the importance of the two substituents on the
aniline phenyl ring. In the binding model of 6a to the
c-Src kinase domain, the amino group of the aniline
formed a hydrogen bond with the side-chain hydroxyl
group of Thr 340 (Fig. 4); this interaction is highly con-
served in the tyrosine kinase family. The model revealed
that the 2,6-dimethyl substituted aniline phenyl ring is
about 90ꢁ to the plane of the imidazo[1,5-a]pyrazine
nucleus, suggesting CH–p interactions between the ani-
line phenyl ring and hydrogen atoms of both the meth-
ylene and methyl moieties (Lys 297 and Thr 340,
respectively). The model also suggested that a twisted
conformation, formed by repulsion of the methyl group
4. Results and discussion
4.1. SAR for the c-Src kinase and c-Src cellular assays
The c-Src inhibitory activity of imidazo[1,5-a]pyrazine
derivatives was evaluated by both coupled spectrophoto-
metric enzyme assay and enzyme-linked immunosorbent
assay (ELISA), using COS7 cells as an intracellular assay
(Tables 1–4).
Table 1. The c-Src inhibitory activity of imidazo[1,5-a]pyrazine derivatives substituted with various aniline groups attached to C-8
R1
R4
N
R3
N
N
N
R2
R
Compound
R
R1
R2
R3
R4
c-Src inhibition
Enzyme
a
IC50 (lM)
ELISA
% at 1 lMb
6a
6b
6c
6d
6e
6f
H
Me
H
Me
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
0.013
5.5
10.6
0
H
H
Cl
F
Cl
F
0.042
0.490
0.038
0.693
1000
0.015
0.260
3.7
25.6
0
H
H
Me
Me
H
Me
MeO
H
0.2
0
NTc
12.5
22.7
NTc
H
6g
6h
6i
H
MeO
MeO
MeO
Me
Me
H
Me
H
6j
H
a IC50 values for inhibition of c-Src were determined in duplicate.
b Inhibition (%) of intracellular c-Src kinase with 1 lM each compound; determined in duplicate.
c NT means not tested.

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H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
Table 2. The c-Src inhibitory activity of C-5 aryl substituted imidazo[1,5-a]pyrazine derivatives (Part I)
Types of Aryl substituent
HN
N
N
N
S
N
Aryl
R1
R2
B
A
C
Compound
Type
R1
R2
c-Src inhibition
Enzyme
IC50 (lM)
ELISA
% at 1 lMb
a
6a
6h
A
A
—
A
A
A
A
A
A
A
A
B
H
MeO
H
H
0.013
0.015
3.3
10.6
12.5
NTc
18.9
0
11
12a
H
CN
MeO
H
0.011
0.235
0.023
0.042
0.235
0.019
0.235
0.052
0.108
0.012
0.096
12b
12c
MeO
F
MeO
H
16.4
0.20
7.0
12d
12e
CF3
NMe2
COOH
H
12f
12h
H
25.7
12.0
0.0
H
12i
12j
40-CONMe2
H
H
H
0
12k
BMS-279700
C
—
22.4
NTc
a IC50 values for inhibition of c-Src were determined in duplicate.
b Inhibition (%) of intracellular c-Src kinase with 1 lM each compound; determined in duplicate.
c NT means not tested.
Table 3. The c-Src inhibitory activity of C-6 aryl substituted imidazo[1,5-a]pyrazine derivatives
R3
HN
N
N
N R4
R2
R
Compound
R
R2
R3
R4
c-Src inhibition
Enzyme
IC50 (lM)
ELISA
% at 1 lMb
a
18a
18b
18c
H
F
F
OMe
H
Cl
Cl
Me
Me
Me
0.059
0.145
0.403
0
5.8
7.0
H
Me
a IC50 values for inhibition of c-Src were determined in duplicate.
b Inhibition (%) of intracellular c-Src kinase with 1 lM each compound; determined in duplicate.
on the aniline phenyl ring, was responsible for increased
potency.
N–Ph bond was performed in 30ꢁ increments (Fig. 3a
and b). There are two planar conformers to the plane
of imidazo[1,5-a]pyrazine (Fig. 3 top). The torsion angles
(h1) are 0ꢁ for conformer A and 180ꢁ for conformer B.
The binding model indicated that the position of con-
former A (h1 = 0ꢁ) was clearly unfavorable to hydrogen
bonding with amino acids in the hinge region. With
respect to conformer B (h1 = 180ꢁ), the 2,6-dimethyl
analogue was markedly stable when the torsion angle
(h2) was 90ꢁ. Conversely, an energy difference (DE = 2.9
kcal/mol) between the two suggests the o-methyl
For conformation analysis, we calculated the conforma-
tional energy of each of the 2,6-dimethyl, o-monomethyl,
and unsubstituted anilines bound to imidazo[1,5-a]pyra-
zine in accordance with the method of Snow et al.26 Each
conformational energy value was calculated using ab
initio quantum mechanics (Hartree–Fock method with
a 6-31G**) implemented in SPARTANTM.27 Conforma-
tional analysis of the torsion angle (h2) around the

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
873
Table 4. The c-Src inhibitory activity of C-5 aryl substituted imidazo[1,5-a]pyrazine derivatives (Part II)
NH
N
N
N
R-Aryl
Compound
Aryl
R
c-Src inhibition
ELISA
Enzyme
IC50 (lM)
a
b
IC50 (lM)
% at 1 lMc
2'
1'
3'
4'
14a
14b
14c
14d
14e
13a
13b
0.037
0.015
0.020
0.019
0.055
0.305
0.052
0.70
0.95
0.53
0.74
1.00
2.30
0.82
57.3
51.6
91.6
59.9
49.1
28.7
57.6
4' O (CH2)2
4' O (CH2)2
N
N
O
NMe
NMe
4' O CH2
3' O CH2
3' O (CH2)2
N
HCl
2' S
3'
5'
4'
5'
CH2 N
NMe
HCl
5'
CH2 N
a IC50 values for inhibition of c-Src were determined in duplicate.
b IC50 values for inhibition of intracellular c-Src were determined in duplicate.
c Inhibition (%) of intracellular c-Src kinase at 1 lM compound; determined in duplicate.
analogue favors a planar conformation (h2 = 0ꢁ) over a
perpendicular one (h2 = 90ꢁ). Furthermore, the unsubsti-
tuted analogue was more stable (DE = 4.6 kcal/mol) in
the planar conformations than the twisted conformation.
ethylamino 12f, or an electron-rich aryl ring (thienyl
group) 12k, did not adversely affect inhibition. In
contrast, introducing an electron-withdrawing group to
the phenyl ring considerably reduced activity against
c-Src (12b, d, e, and h). Similarly, electron-deficient aryl
groups, such as the 4-pyridinyl group 12j, showed
decreased activity. These results underscore the impor-
tance of electron-rich functional groups in c-Src inhibi-
tion. Indeed, the C-6 substituted compounds (Table 3)
were less potent than the C-5 aryl ring compounds
(18c vs 12d), suggesting that C-5 aryl groups interact
more readily with the cleft region of the c-Src enzyme,
forming CH–p interactions with Leu 275 (Fig. 4).
Moreover, compounds with electron-rich aryl groups,
such as 6h and 12a, showed superior inhibition of
BMS-279700 in the enzyme assay. Thus, the electron-
rich ring of the C-5 imidazo[1,5-a]pyrazine seems to
afford greater CH–p interactions with the cleft region
than BMS-279700.
These results strongly suggest that 2,6-dimethyl aniline
adopts a conformation that is perpendicular to the plane
of imidazo[1,5-a]pyrazine. Hence, the 2,6-disubstituent
may play an important role in stabilizing the binding
conformation with CH–p interactions. Conversely, the
low inhibitory activity of the monomethyl and unsubsti-
tuted analogues could be due to the loss of hydrogen
bonds and CH–p interactions. Our findings agree well
with similar studies by Snow26 and Chen.14
Based on these results, we optimized the aryl ring at the
C-5 position of the imidazo[1,5-a]pyrazine by adding
highly favorable 2,6-dimethyl groups to the aniline
phenyl ring. Table 2 lists the results of those analogues.
Introducing a phenyl ring at the C-5 position of the imi-
dazo[1,5-a]pyrazine clearly enhanced inhibitory activity
compared to non-substituted compounds (6a vs 11).
Several compounds were evaluated to examine the ef-
fects of varying substituents on the phenyl ring at the
C-5 position. Adding electron-donating substituents,
such as 30- and/or 40-methoxy (6h, 12a, and c), N,N-dim-
Although the compounds listed in Tables 1–3 were
active against the enzyme in vitro, none of them demon-
strated a significant level of intracellular c-Src inhibition
(IC50 < 1 lM). Therefore, we focused on enhancing the
compound’s solubility to improve cell penetration in
the cellular assay (ELISA). The binding model (Fig. 4)

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H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
Figure 3. Energy versus torsion angle (h2) of the aniline phenyl ring H3–N3–C4–C5 moiety. The illustration at the top compares the two conformers.
The energy in each conformer was computed using ab initio quantum mechanics (Hartree–Fock 6-31G**) implemented in SPARTAMTM
relative to the minimum energy for each compound.
.
a Each DE is
indicated that the 3- and/or 4-substituents on the phenyl
ring at the C-5 position were exposed to the solvent side
of the cleft in the c-Src kinase, creating the possibility for
additional substitution (see Table 4 for the results of
those derivatives). The carboxyl group 12h showed poor
inhibition in both the enzyme and cell assays. Replacing
the carboxyl group 12h with an amide group 12i lowered
c-Src inhibition, probably due to electrostatic repulsion
with Asp 350. We then selectively introduced cyclic ter-
tiary amine analogues at the 30- or 40-position to improve
CNS penetration.28,29 The pyrrolidine derivatives (14b
and e) were shown to be more effective inhibitors of c-
Src cellular activity than the methoxy derivatives (6h
and 12a). Moreover, introducing the piperidine deriva-
tive at the 30- or 40-position (14c and d) produced the best
results in both the enzyme and cell assays. The addition
of a pyrrolidine derivative to the 5-position of thienyl
group 13a did not alter the activity in comparison to
compound 12k, whereas the piperazinyl derivative 13b
enhanced the inhibition activity in the cellular assay.
4.2. Compound concentrations in the brain and plasma
The concentrations of compounds 14cÆHCl and 14dÆHCl
in the brain and plasma were determined to estimate
whether these compounds would have sufficient expo-
sure levels in the brain to treat acute ischemic stroke.
Each compound was administered at a dose of 3 mg/
kg/h by intravenous infusion, and concentration levels
were measured after 3 h (results shown in Table 5). Sub-
stitution of the cyclic tertiary amino group at the 40-po-
sition of 14cÆHCl significantly enhanced CNS
penetration compared to the substituent at the 30-posi-
tion of 14dÆHCl, with brain/plasma ratios of 1.1 and
0.3, respectively. In addition, compound 14b also
showed a significant brain/plasma ratio (1.7), but 14e
had poor CNS penetration. The pharmacokinetic profile
of compound 14cÆHCl justified advanced efficacy studies
in vivo. Table 6 shows the rat PK parameters of com-
pound 14cÆHCl. Compound 14cÆHCl was also highly
soluble (>5 mg/mL) in aqueous solution.

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
875
4.3. The selectivity profile of compound 14cÆHCl against
other kinase families
Selectivity against other kinase families is important to
circumvent undesirable side effects. Compound 14cÆHCl
for c-Src kinase was evaluated against various protein
kinases (Table 7). Poor inhibition was observed with
EGFR, Zap70, syk, PKCb2, and KDR. However, the
inhibition of Lck was comparable to that of c-Src due
to high structural homology. These assays suggest that
compound 14cÆHCl is more selective toward the Src
family of kinases compared to other kinase families.
4.4. Neuroprotective effect in the rat MCA occlusion
model
The neuroprotective effect of compound 14cÆHCl was
examined using the photochemically induced middle
cerebral artery thrombosis (PIT) model in rats, reported
by K. Umemura et al.30 Compound 14cÆHCl was deliv-
ered by intravenous infusions for 6 h after MCA occlu-
sion to evaluate its effect on infarct size following stroke.
Infarct volumes were significantly reduced with inhibi-
tion, by 20% at 3 mg/kg/h to a maximum of 29% at
10 mg/kg/h (Figs. 5 and 6).
5. Conclusion
We designed and synthesized several novel C5-selective
substituted imidazo[1,5-a]pyrazine derivatives (6a–j,
12a–k) as potent c-Src inhibitors. SAR and binding
model analyses indicated that a twisted conformation
Figure 4. Views of 6a in relationship to the cleft region, and binding
orientations of 6a to the ATP-binding site of c-Src kinase.
Table 5. CNS penetration of select c-Src kinase inhibitors in ratsa
Compoundb
Plasmac C3 h (ng/mL)
Brainc C3 h (ng/g)
Ratio (brain/plasma)
14cÆHCl
14dÆHCl
14b
457 33
520 50
368 42
242 15
139 35
501 47
133 31
631 90
229 17
81 35
1.1 0.2
0.3 0.1
1.7 0.1
0.9 0.0
0.6 0.3
14e
BMS-279700
a Results are shown as means SE (n = 3).
b Each compound was administered at a dose of 3 mg/kg/h by intravenous infusion.
c Concentration levels were measured after 3 h.
Table 6. Pharmacokinetic parameters of c-Src inhibitor 14cÆHCl in ratsa
Pharmacokinetic parameters (iv (3 mg/kg))
AUCb (lM h)
t1/2 (min)
Cl (mL/min/kg)
91
Vss (L/kg)
Compound 14cÆHCl
1.25
63
7.64
a Results are shown as a mean value.
b The area under the curves represents 0 h to infinity.
Table 7. Selectivity profile of compound 14cÆHCl for various kinases
Kinase
c-Src
Lck
Syk
Zap
>10
PKCb2
EGFR
2.730
KDR
>10
a
IC50 (lM)
0.020
0.008
>10
>10
a IC50 values for inhibition of each kinase were determined in duplicate.

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H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
position increased cellular inhibitory potency and pro-
vided good CNS penetration. Among these compounds,
compound 14cÆHCl was highly soluble in aqueous solu-
tion, and significantly reduced infarct volume after brain
ischemia. This compound holds promise as a new ther-
apeutic modality for acute ischemic stroke.
6. Experimental
6.1. Chemistry
Melting points were taken on a Yanako MP-3S Micro
melting point apparatus and are uncorrected. Infrared
spectra were measured on a Nicolet 510 FT-IR spec-
trophotometer and are reported in reciprocal centime-
ters. Proton NMR spectra were recorded at 400 or
500 MHz with a Bruker AMX 400 or DRX 500
instrument, and chemical shifts are reported in parts
per million (d) downfield from tetramethylsilane as
the internal standard. The peak patterns are 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 a Thermo Quest
FINNIGAN AQA electrospray ionization mass spec-
trometer. Elemental analyses were performed on an
Elementar Vario EL analyzer (C, H, and N). The ana-
lytical results obtained were within 0.4% of the the-
oretical values unless otherwise stated. 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-pres-
sure liquid column chromatography (MPLC) was per-
formed on silica gel 60 N (particle size 40–50 lm) from
Kanto Chemical Co., Inc. or APS Daisogel IR-60
(particle size 25–40 lm) from Daiso Co., Ltd. All re-
agents and solvents were commercially available unless
otherwise indicated.
Figure 5. Representative images of six 2-mm-thick slices of rat brain
24 h after MCA occlusion. Compound 14cÆHCl or saline was admin-
istered as a continuous infusion for 6 h. The sections were incubated in
1% TTC. The infarct area appears as a white region. (n = 6–12) Top:
compound 14cÆHCl (10 mg/kg/h); bottom: saline.
6.1.1. 5-Phenylpyrazine-2(1H)-one (3a). To a suspension
of a-aminoacetamide hydrochloride (15 g, 0.14 mol) in
MeOH (125 mL)–water (31 mL) was added 12.5 M
aqueous NaOH (16.5 mL, 0.20 mol) solution at
ꢀ30 ꢁC, and then
a solution of NaOH (5.43 g,
0.14 mol) in MeOH (61 mL) was added to the mixture.
A solution of 2a in MeOH (111 mL) was added to the
mixture at ꢀ20 ꢁC, and then the resulting suspension
was stirred for 2 h at same temperature, and the stirring
continued for 1 h at room temperature. After cooling
with ice water, the mixture was acidified with AcOH.
Collection of the resulting precipitates by filtration gave
17.0 g of 3a (73%) as a pale yellow solid: 1H NMR
(DMSO-d6) d 7.25–7.35 (1H, m), 7.40–7.45 (2H, m),
7.85–7.90 (2H, m), 8.07 (1H, br s), 8.12 (1H, d,
J = 1.3 Hz), 12.51 (1H, br s).
Figure 6. Neuroprotective effects of compound 14cÆHCl (intravenous
infusion, 6 h) in an MCA occlusion model. **p < 0.01, *<0.05 versus
control (Dunnett-type test; n = 6–12).
is stabilized by the 2,6-dimethyl substituents, and the
C-5 electron-rich aryl groups play an important role in
enhancing inhibition. We also found that the 30- or
40-methoxy substituted compounds 6h and 12a had
excellent inhibitory activity. Furthermore, introduction
of tertiary amines 14a–e on the phenyl ring at the C-5
6.1.2. 5-(4-Methoxyphenyl)pyrazine-2(1H)-one (3b). This
title compound was prepared from 2b in the same man-
ner as described above, and obtained as a pale yellow
solid (61%): 1H NMR (DMSO-d6) d 6.98 (2H, d,
J = 9.1 Hz), 7.80 (2H, d, J = 9.1 Hz), 7.99 (1H, br s),
8.08 (1H, d, J = 1.3 Hz), 12.32 (1H, br s).

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
877
6.1.3. 7-(4-Methoxybenzyl)-5-phenylimidazo[1,5-a]pyra-
zin-8(7H)-one (4a). To a suspension of NaH (60% w/w
dispersion in mineral oil, 2.69 g, 67.3 mmol) in DMF
added trifluoromethanesulfonic acid (31 mL, 0.35 mol)
at 0 ꢁC. The mixture was stirred for 30 min at room
temperature, and stirring continued for 6 h at 40 ꢁC.
The solvent was evaporated in vacuo, and the residue
was poured into saturated aqueous NaHCO3 solution.
Collection of the resulting precipitates by filtration gave
3.49 g (57%) of 5a as an off-white solid: mp 290–291 ꢁC
(56 mL) was added
a suspension of 3a (10.1 g,
58.5 mmol) in DMF (28 mL) and THF (28 mL) at
0 ꢁC. After stirring for 30 min, 4-methoxybenzyl chlo-
ride (8.7 mL, 64 mmol) and tetrabutylammonium iodide
(2.2 g, 5.8 mmol) were added to the reaction mixture.
The mixture was stirred for 12 h at room temperature
and stirring continued for 1 h at 70 ꢁC. The reaction
mixture was quenched with 10% aqueous NH4Cl solu-
tion and extracted with EtOAc. The organic layer was
washed with brine, dried over anhydrous MgSO4, and
evaporated in vacuo to give 9.3 g (54%) of 1-(4-meth-
(MeOH); IR (KBr) 3119, 2904, 1666, 1350, 928 cmꢀ1
;
1H NMR (DMSO-d6) d 6.87 (1H, s), 7.65–7.90 (5H,
m), 8.07 (1H, s), 8.31 (1H, s), 11.14 (1H, br s); MS
m/z: 212 (M+H)+. Anal. Calcd for C12H9N3OÆ0.2H2O:
C, 67.09; H, 4.41; N, 19.56. Found: C, 67.41; H, 4.11;
N, 19.55.
oxybenzyl)-5-phenylpyrazine-2(1H)-one as
solid: mp 111–112 ꢁC (EtOAc/diisopropylether); IR
(KBr) 3052, 1653, 1606, 1596, 1515 cmꢀ1 1H NMR
(DMSO-d6) d 3.72 (3H, s), 5.08 (2H, s), 6.91 (2H, d,
J = 8.8 Hz), 7.30–7.35 (1H, m), 7.35–7.50 (4H, m), 7.84
(2H, dd, J = 8.4, 1.1 Hz), 8.15 (1H, d, J = 1.3 Hz), 8.49
(1H, d, J = 1.3 Hz); MS m/z: 293 (M+H)+.
a
brown
6.1.6. 5-(4-Methoxyphenyl)imidazo[1,5-a]pyrazin-8(7H)-
one (5b). This title compound was prepared from 4b
in the same manner described above, and obtained as
a white solid (63%): mp 268–269 ꢁC; IR (KBr) 3041,
;
1652, 1515, 1255, 823 cmꢀ1 1H NMR (DMSO-d6)
;
d 3.83 (3H, s), 6.59 (1H, d, J = 5.7 Hz), 7.09 (2H,
d, J = 8.8 Hz), 7.56 (2H, d, J = 8.8 Hz), 7.86 (1H,
s), 8.05 (1H, s), 10.88 (1H, br s); MS m/z: 242
(M+H)+. Anal. Calcd for C13H11N3O2Æ0.25H2O: C,
63.54; H, 4.72; N, 17.10. Found: C, 63.87; H, 4.45;
N, 17.02.
To a suspension of 60% NaH (2.4 g, 60.2 mmol) in dry
THF (30 mL) was added a solution of 1-(4-methoxyben-
zyl)-5-phenylpyrazine-2(1H)-one (8.0 g, 27.4 mmol) and
tosylmethyl isocyanide (5.9 g, 30.1 mmol) in THF
(34 mL) at 0 ꢁC. The mixture was stirred for 30 min at
same temperature, and stirring continued for 1 h at
room temperature. Water (300 mL) was poured into
the resulting suspension and collection of the precipi-
tates gave 9.90 g (100%) of 4a as a pale brown solid:
6.1.7. N-(2,6-Dimethylphenyl)-5-phenylimidazo[1,5-a]
pyrazine-8-amine (6a). A suspension of 5a (107 mg,
0.51 mmol) in phosphorus oxychloride (1.2 mL) was
stirred for 1.5 h at reflux. The mixture was evaporated
in vacuo to give 116 mg of 8-chloro-5-phenylimi-
dazo[1,5-a]pyrazine as a crude mixture. To a suspension
of 8-chloro-5-phenylimidazo[1,5-a]pyrazine and 2,6-
dimethylaniline (0.16 mL, 1.36 mmol) in THF (1.0 mL)
was added sodium bis(trimethylsilyl)amide (NaHMDS,
1 M in THF; 3.0 mL) at room temperature under an ar-
gon atmosphere, and the mixture was stirred for 2 h at
60 ꢁC. A saturated aqueous NH4Cl was added to the
stirring suspension, and the resulting suspension extract-
ed with EtOAc. The organic layer was washed with
brine, dried over anhydrous MgSO4, and evaporated
in vacuo. Purification of the residue by flash column
chromatography on APS (eluent: hexane/EtOAc, 1:2)
gave 75 mg (47%) of 6a as an off white solid: mp 196–
197 ꢁC (CH3CN); IR (KBr) 3133, 3039, 1516, 1435,
1
mp 111–112 ꢁC; IR (KBr) 1642, 1515, 1251 cmꢀ1; H
NMR (DMSO-d6) d 3.72 (3H, s), 5.00 (2H, s), 6.90
(2H, d, J = 8.8 Hz), 7.12 (1H, s), 7.37 (2H, d,
J = 8.8 Hz), 7.50–7.70 (5H, m), 7.90 (1H, s), 8.13 (1H,
s); MS m/z: 332 (M+H)+.
6.1.4. 7-(4-Methoxybenzyl)-5-(4-methoxyphenyl)imidazo
[1,5-a]pyrazin-8(7H)-one (4b). 1-(4-Methoxybenzyl)-5-
(4-methoxyphenyl)pyrazine-2(1H)-one was prepared
from 3b in the same manner described above, and
obtained as a pale brown solid (57%): mp 104–105 ꢁC;
IR (KBr) 3056, 1647, 1612, 1515, 1250, 838 cmꢀ1
;
1H NMR (DMSO-d6) d 3.72 (3H, s), 3.78 (3H, s),
5.06 (2H, s), 6.91 (2H, d, J = 8.8 Hz), 7.00 (2H, d,
J = 8.8 Hz), 7.40 (2H, d, J = 8.8 Hz), 7.76 (2H, d, J =
8.8 Hz), 8.11 (1H, d, J = 1.3 Hz), 8.34 (1H, d, J =
1.3 Hz); MS m/z: 323 (M+H)+.
763 cmꢀ1 1H NMR (DMSO-d6) d 2.19 (6H, s), 6.97
;
(1H, s), 7.15 (3H, s), 7.40–7.60 (3H, m), 7.67 (2H, d,
J = 8.0 Hz), 8.06 (1H, br s), 8.33 (1H, s), 9.05 (1H, s);
MS m/z: 315 (M+H)+. Anal. Calcd for C20H18N4Æ0.1-
H2O: C, 75.97; H, 5.80; N, 17.72. Found: C, 76.16; H,
5.86; N, 17.34.
Compound 4b was prepared from 1-(4-methoxybenzyl)-
5-(4-methoxyphenyl)pyrazine-2(1H)-one in the same
manner described above, and obtained as a pale yellow
solid (65%): mp 106–107 ꢁC; IR (KBr) 3055, 1646, 1595,
1515, 1249, 828 cmꢀ1; 1H NMR (CDCl3) d 3.79 (3H, s),
3.86 (3H, s), 5.03 (2H, s), 6.29 (1H, s), 6.88 (2H, d,
J = 8.8 Hz), 7.00 (2H, d, J = 8.8 Hz), 7.30 (2H, d, J =
8.8 Hz), 7.38 (2H, d, J = 8.8 Hz), 7.86 (1H, d,
J = 0.9 Hz), 8.05 (1H, d, J = 0.9 Hz); MS m/z: 362
(M+H)+.
6.1.8. N-Phenyl-5-phenylimidazo[1,5-a]pyrazin-8-amine
(6b). This title compound was prepared from 5a in the
same manner as described in 6a, and obtained as a pale
yellow solid (44%): mp 203–204 ꢁC (CH3CN); IR (KBr)
1
3056, 1516, 1496, 1447 cmꢀ1; H NMR (DMSO-d6) d
7.00–7.10 (1H, m), 7.18 (1H, s), 7.30–7.40 (2H, m),
7.45–7.60 (3H, m), 7.65–7.75 (2H, m), 7.90–8.00 (2H,
m), 8.19 (1H, s), 8.36 (1H, s), 9.47 (1H, s); MS m/z:
287 (M+H)+. Anal. Calcd for C18H14N4Æ0.15H2O: C,
74.80; H, 4.99; N, 19.38. Found: C, 75.05; H, 4.94; N,
19.00.
6.1.5. 5-Phenylimidazo[1,5-a]pyrazin-8(7H)-one (5a). To
a solution of 4a (9.62 g, 29.0 mmol) and anisole
(57 mL, 0.52 mol) in trifluoroacetic acid (134 mL) was

878
H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
6.1.9. N-(2,6-Dichlorophenyl)-5-phenylimidazo[1,5-a]pyr-
azin-8-amine (6c). This title compound was prepared
from 5a in the same manner as described in 6a, and
obtained as a white solid (46%): mp 266–267 ꢁC
(6H, s), 3.82 (3H, s), 6.88 (1H, s), 7.05–7.15 (2H, m),
7.14 (3H, s), 7.55–7.65 (2H, m), 8.26 (1H, s), 8.97 (1H,
s); MS m/z: 345 (M+H)+. Anal. Calcd for
C21H20N4OÆ0.25H2O: C, 72.29; H, 5.92; N, 16.06.
Found: C, 72.25; H, 5.82; N, 15.89.
(CH3CN); IR (KBr) cmꢀ1: 3139, 1635, 1130, 769 cmꢀ1
;
1H NMR (DMSO-d6) d 7.00 (1H, s), 7.35–7.75 (8H,
m), 8.07 (1H, s), 8.37 (1H, s), 9.61 (1H, s); MS m/z (rel-
ative intensity): 357 (0.67), 355 (M+H)+, 320 (0.41).
Anal. Calcd for C18H12Cl2N4: C, 60.86; H, 3.41; N,
15.77. Found: C, 60.92; H, 3.40; N, 15.54.
6.1.15. N-[5-(4-Methoxyphenyl)imidazo[1,5-a]pyrazin-8-
yl]-o-tolylamine (6i). This title compound was prepared
from 5b in the same manner as described in 6a, and
obtained as a pale yellow solid (53%): mp 183–184 ꢁC
(CH3CN); IR (KBr) 3041, 1607, 1516, 1457, 749 cmꢀ1
;
6.1.10. N-(2,6-Difluorophenyl)-5-phenylimidazo[1,5-a]
pyrazin-8-amine (6d). This title compound was prepared
from 5a in the same manner as described in 6a, and ob-
tained as a white solid (48%): mp 256–257 ꢁC (CH3CN);
1H NMR (DMSO-d6) d 2.26 (3H, s), 3.84 (3H, s), 6.90
(1H, s), 7.12 (2H, d, J = 8.8 Hz), 7.25–7.45 (4H, m),
7.61 (2H, d, J = 8.8 Hz), 8.13 (1H, br s), 8.39 (1H, s),
9.95 (1H, br s); MS m/z: 331 (M+H)+. Anal. Calcd for
C20H18N4O: C, 72.71; H, 5.49; N, 16.96. Found: C,
72.39; H, 5.43; N, 16.93.
IR (KBr) 3141, 3048, 1623, 1517, 1470, 1001, 769 cmꢀ1
;
1H NMR (DMSO-d6) d 7.03 (1H, s), 7.15–7.30 (2H, m),
7.30–7.45 (1H, m), 7.45–7.60 (3H, m), 7.65–7.75 (2H,
m), 8.08 (1H, s), 8.37 (1H, s), 9.43 (1H, s). MS m/z:
323 (M+H)+. Anal. Calcd for C18H12F2N4: C, 67.08;
H, 3.75; N, 17.38. Found: C, 67.01; H, 3.64; N, 17.24.
6.1.16. N-[5-(4-Methoxyphenyl)imidazo[1,5-a]pyrazin-8-
yl]phenylamine (6j). This title compound was prepared
from 5b in the same manner as described in 6a, and
obtained as an off white solid (52%): mp 182–183 ꢁC
(CH3CN); IR (KBr) 3312, 3134, 1609, 1516, 1444,
6.1.11. N-(5-Phenylimidazo[1,5-a]pyrazin-8-yl)-2,4,6-trim-
ethylphenylamine (6e). This title compound was pre-
pared from 5a in the same manner as described in 6a,
and obtained as a pale brown solid (43%): mp 197–
198 ꢁC (CH3CN); IR (KBr) 3129, 1538, 1432,
1
749 cmꢀ1; H NMR (DMSO-d6) d 3.84 (3H, s), 7.00–
7.15 (4H, m), 7.30–7.40 (2H, m), 7.64 (2H, d,
J = 8.5 Hz), 7.92 (2H, d, J = 8.5 Hz), 8.21 (1H, s), 8.35
(1H, s), 9.55 (1H, br s); MS m/z: 317 (M+H)+. Anal.
Calcd for C19H16N4O: C, 72.13; H, 5.10; N, 17.71.
Found: C, 72.05; H, 5.02; N, 17.48.
765 cmꢀ1 1H NMR (DMSO-d6) d 2.14 (6H, s), 2.28
;
(3H, s), 6.95 (3H, s), 7.40–7.50 (1H, m), 7.50–7.60
(2H, m), 7.60–7.70 (2H, m), 8.32 (1H, s), 8.96 (1H, s);
MS m/z: 329 (M+H)+. Anal. Calcd for C21H20N4Æ0.5-
H2O: C, 74.75; H, 6.27; N, 16.60. Found: C, 74.89; H,
6.04; N, 16.21.
6.1.17. 5-Bromopyrazin-2(1H)-one (8). To a solution of 7
(50.5 g, 0.29 mol) in 50% aqueous acetic acid (450 mL)
and dioxane (50 mL) was added a solution of sodium
nitrite (22.0 g, 0.32 mol) in 50% aqueous acetic acid
(20 mL) at 4 ꢁC, and the mixture was stirred for 10 min
at same temperature. The mixture was neutralized with
5 M NaOH and extracted with EtOAc. The organic layer
was washed with brine, dried over anhydrous MgSO4,
and removed in vacuo. The residue was recrystallized
from EtOAc to give 29.8 g (59%) of 8 as a pale brown sol-
6.1.12. N-(2-Methoxy-6-methylphenyl)-5-phenylimidazo
[1,5-a]pyrazin-8-amine (6f). This title compound was
prepared from 5a in the same manner as described in
6a, and obtained as a pale brown solid (20%): mp
118–119 ꢁC (CH3CN); IR (KBr) 3134, 2964, 1506,
1
1085, 764 cmꢀ1; H NMR (DMSO-d6) d 2.17 (3H, s),
1
3.72 (3H, s), 6.85–7.00 (3H, m), 7.15–7.25 (1H, m),
7.40–7.50 (1H, m), 7.50–7.60 (2H, m), 7.60–7.70 (2H,
m), 8.31 (1H, s), 8.88 (1H, s); MS m/z: 331 (M+H)+.
Anal. Calcd for C20H18N4O: C, 72.71; H, 5.49; N,
19.96. Found: C, 72.51; H, 5.40; N, 16.88.
id: H NMR (DMSO-d6) d 7.92 (1H, s), 8.09 (1H, s),
12.22 (1H, br s).
6.1.18. 5-Bromoimidazo[1,5-a]pyrazin-8(7H)-one (9). To
a suspension of 60% NaH (6.41 g, 0.16 mol) in DMF
(75 mL) was added
a suspension of 8 (24.4 g,
6.1.13. N-Methyl-N-phenyl-5-phenylimidazo[1,5-a]pyra-
zin-8-amine (6g). This title compound was prepared
from 5a in the same manner as described in 6a, and
obtained as a pale brown solid (43%): mp 114–115 ꢁC
0.14 mol) in DMF (75 mL) and THF (75 mL) under
ice cooling, and the mixture was stirred for 30 min
at same temperature. A solution of 4-methoxybenzyl
chloride (20.8 mL, 0.15 mol) in THF (75 mL) was add-
ed to the mixture, which was followed by addition of
tetrabutylammonium iodide (5.14 g, 13.9 mmol). The
mixture was stirred for 12 h at room temperature
and stirring continued for 1 h at 70 ꢁC. After cooling
with ice water, the mixture was quenched with 10%
aqueous NH4Cl and extracted with EtOAc. The
organic layer was washed with brine, dried over
anhydrous MgSO4, and evaporated in vacuo. Purifica-
tion of the residue by MPLC on silica gel (eluent:
hexane/EtOAc = 2:1) gave 26.3 g (64%) of 5-bromo-1-
(4-methoxybenzyl)pyrazin-2(1H)-one as an amorphous
(CH3CN); IR (KBr) 3130, 1495, 1394, 701 cmꢀ1 1H
;
NMR (DMSO-d6) d 3.51 (3H, s), 5.65–5.75 (1H, m),
7.23 (1H, s), 7.40–7.60 (8H, m), 7.60–7.70 (2H, m),
8.15–8.20 (1H, m); MS m/z: 301 (M+H)+. Anal. Calcd
for C19H16N4Æ0.25H2O: C, 74.85; H, 5.46; N, 18.38.
Found: C, 75.07; H, 5.28; N, 18.11.
6.1.14. N-(2,6-Dimethylphenyl)-5-(4-methoxyphenyl)imi-
dazo[1,5-a]pyrazin-8-amine (6h). This title compound
was prepared from 5b in the same manner as described
in 6a, and obtained as an off white solid (83%): mp
190–191 ꢁC (CH3CN); IR (KBr) 3134, 3041, 1607,
1
oil: IR (KBr) 3052, 1654, 1577, 1513, 1250 cmꢀ1; H
1515, 1437, 769 cmꢀ1
;
1H NMR (DMSO-d6) d 2.18
NMR (DMSO-d6) d 3.73 (3H, s), 4.97 (2H, s), 6,92

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
879
(2H, d, J = 8.8 Hz), 7.34 (2H, d, J = 8.8 Hz), 7.88 (1H,
s), 8.18 (1H, s).
phous: IR (KBr) 3201, 1624, 1519, 1465, 1209,
772 cmꢀ1 1H NMR (DMSO-d6) d 2.14 (6H, s), 6.91
;
(1H, d, J = 4.7 Hz), 7.10–7.15 (3H, m), 7.66 (1H, d,
J = 4.7 Hz), 7.90 (1H, br s), 8.35 (1H, s), 8.91 (1H, s);
MS m/z: 239 (M + H)+. Anal. Calcd for C14H14N4: C,
70.57; H, 5.92; N, 14.96. Found: C, 70.62; H, 5.89; N,
14.93.
To a suspension of 60% NaH (7.9 g, 0.20 mol) in miner-
al oil in dry THF (100 mL) was added a solution of
5-bromo-1-(4-methoxybenzyl)pyrazin-2(1H)-one (26.3 g,
89.1 mmol) and tosylmethyl isocyanide (19.1 g,
98.0 mmol) in THF (160 mL) at 0 ꢁC. The mixture was
stirred for 30 min at same temperature, and stirring con-
tinued for 1 h at room temperature. The resulting mix-
ture was poured into water and extracted with EtOAc.
The organic layer was washed successively with water
and brine, dried over anhydrous MgSO4, and evaporat-
ed in vacuo. Purification of the residue by MPLC on
silica gel (eluent: hexane/EtOAc = 1/1:1/3) gave 23.7 g
(80%) of 5-bromo-7-(4-methoxybenzyl)imidazo[1,5-
a]pyrazin-8(7H)-one as a pale brown solid: mp 150–
6.1.21. N-(2,6-Dimethylphenyl)-5-(3-methoxyphenyl)imi-
dazo[1,5-a]pyrazin-8-amine (12a). A mixture of 10
(506 mg, 1.60 mmol), 3-methoxyphenylboronic acid
(255 mg, 1.67 mmol), 1,10-bis(diphenylphosphino)ferro-
cene palladium(II)dichloride dichloromethane (65.1 mg,
0.08 mmol), and potassium carbonate (309 mg,
2.23 mmol) in DMF (0.5 mL), water (2.5 mL), and tolu-
ene (2.5 mL) was stirred for 15 h at 90 ꢁC. The mixture
was diluted with EtOAc, and the organic layer was sep-
arated, dried over anhydrous MgSO4, and evaporated
in vacuo. Purification of the residue by flash column
chromatography on APS (eluent: hexane/EtOAc = 1:1)
gave 572 mg (100%) of 12a as an off white solid: mp
177–178 ꢁC (CH3CN); IR (KBr) 3141, 2950, 1620,
1
151 ꢁC (MeOH); IR (KBr) 3073, 1669, 1512 cmꢀ1; H
NMR (DMSO-d6) d 3.72 (3H, s), 4.94 (2H, s), 6,90
(2H, d, J = 8.8 Hz), 7.32 (2H, d, J = 8.8 Hz), 7.43 (1H,
s), 7.94 (1H, s), 8.32 (1H, s); MS m/z: 335 (M+H)+.
Anal. Calcd for C14H12BrN3O2: C, 50.32; H, 3.62; N,
12.57. Found: C, 50.36; H, 3.53; N, 12.49.
1539, 1535, 1229, 770 cmꢀ1 1H NMR (DMSO-d6) d
;
2.18 (6H, s), 3.82 (3H, s), 6.99 (1H, s), 7.03 (1H, dd,
J = 8.0, 2.5 Hz), 7.14 (3H, s), 7.15–7.25 (2H, m), 7.44
(1H, t, J = 8.0 Hz), 8.08 (1H, br s), 8.37 (1H, s), 9.05
(1H, s); MS m/z: 345 (M+H)+. Anal. Calcd for
C21H20N4O: C, 73.23; H, 5.85; N, 16.27. Found: C,
72.98; H, 5.81; N, 16.26.
To a solution of 5-bromo-7-(4-methoxybenzyl)imi-
dazo[1,5-a]pyrazin-8(7H)-one (23.2 g, 69.5 mmol) and
anisole (135 mL, 1.25 mol) in trifluoroacetic acid
(321 mL) was added trifluoromethanesulfonic acid
(74 mL, 0.83 mol) at 0 ꢁC. The mixture was stirred for
30 min at room temperature, and stirring continued
for 1 h at 40 ꢁC. The solvent was evaporated in vacuo,
and the residue was poured into saturated aqueous
NaHCO3 solution. Collection of the resulting precipi-
tates by filtration gave 12.4 g (83%) of 9 as an off-white
solid: mp 256–257 ꢁC (dec.); IR (KBr) 3021, 2902, 1669,
6.1.22. 4-[8-(2,6-Dimethylphenylamino)imidazo[1,5-a]pyr-
azin-5-yl]benzonitrile (12b). This title compound was
prepared from 10 in the same manner as described in
12a, and obtained as a white solid (47%): mp 138–
139 ꢁC (CH3CN); IR (KBr) 2228, 1617, 1507, 1161,
1
1
1512, 1148 cmꢀ1; H NMR (DMSO-d6) d 7.04 (1H, s),
772 cmꢀ1; H NMR (DMSO-d6) d 2.18 (6H, s), 7.10–
7.92 (1H, d, J = 0.9 Hz), 8.32 (1H, d, J = 0.9 Hz),
11.05 (1H, br s); MS m/z: 215 (M+H)+. Anal. Calcd
for C6H4BrN3O: C, 33.67; H, 1.88; N, 19.63. Found:
C, 33.56; H, 1.76; N, 19.50.
7.20 (4H, m), 7.90 (2H, d, J = 8.5 Hz), 7.97 (2H, d,
J = 8.5 Hz), 8.13 (1H, br s), 8.48 (1H, br s), 9.25 (1H,
br s); MS m/z: 340 (M+H)+. Anal. Calcd for
C21H17N5Æ0.25 H2O: C, 73.34; H, 5.13; N, 20.36. Found:
C, 73.53; H, 5.01; N, 20.15.
6.1.19. N-(5-Bromoimidazo[1,5-a]pyrazin-8-yl)-2,6-dim-
ethylphenylamine (10). This title compound was pre-
pared from 9 in the same manner as described in 6a,
and obtained as an off white solid (33%): mp 151–
152 ꢁC (MeOH); IR (KBr) 3214, 1608, 1513, 1260,
6.1.23. N-[5-(3,4-Dimethoxyphenyl)imidazo[1,5-a]pyra-
zin-8-yl]-2,6-dimethylphenylamine (12c). This title com-
pound was prepared from 10 in the same manner as
described in 12a, and obtained as a white solid (45%):
mp 190–191 ꢁC (CH3CN); IR (KBr) 3124, 2928, 1623,
1
1148 cmꢀ1; H NMR (DMSO-d6) d 2.15 (6H, s), 7.10–
7.20 (4H, m), 8.14 (1H, br s), 8.43 (1H, s), 9.18 (1H,
s); MS m/z: 318 (M+H)+. Anal. Calcd for C14H13BrN4:
C, 53.01; H, 4.13; N, 17.66. Found: C, 53.08; H, 4.07; N,
17.70.
1602, 1505, 1429, 774 cmꢀ1 1H NMR (DMSO-d6) d
;
2.18 (6H, s), 3.81 (3H, s), 3.82 (3H, s), 6.92 (1H, s),
7.09 (1H, d, J = 8.5 Hz), 7.10–7.20 (4H, m), 7.22 (1H,
d, J = 1.9 Hz), 8.05 (1H, br s), 8.37 (1H, s), 9.00 (1H,
s); MS m/z: 375 (M+H)+. Anal. Calcd for C22H22N4O2:
C, 70.57; H, 5.92; N, 14.96. Found: C, 70.62; H, 5.89;
N, 14.93.
6.1.20. N-(2,6-Dimethylphenyl)imidazo[1,5-a]pyrazin-8-
amine (11). To a solution of 10 (237 mg, 0.75 mmol) in
THF (1 mL) was added 2.62 M n-BuLi (2.62 M in n-hex-
ane; 0.6 mL, 1.57 mmol) at ꢀ78 ꢁC. After stirring for
30 min at same temperature, saturated aqueous NH4Cl
was added to the reaction mixture. The resulting mix-
ture was extracted with EtOAc, and the organic layer
was washed with brine, dried over anhydrous MgSO4,
and evaporated in vacuo. Purification of the residue by
flash column chromatography on APS (eluent: hexane/
EtOAc, 1:2) gave 98 mg (55%) of 11 as a colorless amor-
6.1.24. N-(2,6-Dimethylphenyl)-5-(4-fluorophenyl)imidazo
[1,5-a]pyrazin-8-amine (12d). This title compound was
prepared from 10 in the same manner as described in
12a, and obtained as a white solid (84%): mp 174–
175 ꢁC (CH3CN); IR (KBr) 3121, 3044, 1539, 1514,
1433, 1228, 772 cmꢀ1 1H NMR (DMSO-d6) d 2.18
;
(6H, s), 6.94 (1H, s), 7.10–7.20 (3H, m), 7.30–7.40
(2H, m), 7.65–7.75 (2H, m), 8.08 (1H, br s), 8.31 (1H,

880
H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
s), 9.07 (1H, s); MS m/z: 333 (M+H)+. Anal. Calcd for
C20H17FN4: C, 72.27; H, 5.16; N, 16.86. Found: C,
72.13; H, 5.12; N, 16.83.
6.1.30. 4-[8-(2,6-Dimethylphenylamino)imidazo[1,5-a]-
pyrazin-5-yl]benzoic acid (12h). To a suspension of 12g
(177 mg, 0.48 mmol) in THF (1 mL) and EtOH (1 mL)
was added 2 M NaOH (0.52 mL, 1.05 mmol), and the
mixture was stirred for 2 h at reflux. After cooling at
room temperature, the mixture was neutralized with
2 M HCl (0.52 mL), extracted with EtOAc, and the
organic layer was washed with brine, dried over anhy-
drous MgSO4, and evaporated in vacuo. The residue
was recrystallized from EtOAc, and collection of the
resulting precipitate gave 165 mg (97%) of 12 h as a
white solid: mp 288–289 ꢁC (dec.); IR (KBr) 3127,
6.1.25. N-(2,6-Dimethylphenyl)-5-(4-trifluoromethylphe-
nyl)imidazo[1,5-a]pyrazin-8-amine (12e). This title com-
pound was prepared from 10 in the same manner as
described in 12a, and obtained as a white solid (99%):
mp 205–206 ꢁC (CH3CN); IR (KBr) 3123, 3040, 1617,
1540, 1327, 1128, 778 cmꢀ1 1H NMR (DMSO-d6) d
;
2.19 (6H, s), 7.09 (1H, s), 7.10–7.20 (3H, m), 7.87 (2H,
d, J = 8.5 Hz), 7.92 (2H, d, J = 8.5 Hz), 8.11 (1H, br
s), 8.47 (1H, s), 9.21 (1H, s); MS m/z: 383 (M+H)+.
Anal. Calcd for C21H17F3N4: C, 65.96; H, 4.48; N,
14.65. Found: C, 66.13; H, 4.47; N, 14.62.
1684, 1540, 1289, 777 cmꢀ1 1H NMR (DMSO-d6) d
;
2.19 (6H, s), 7.09 (1H, s), 7.10–7.20 (3H, m), 7.81 (2H,
d, J = 8.2 Hz), 8.12 (2H, d, J = 8.2 Hz), 8.47 (1H, s),
9.19 (1H, br s), 13.11 (1H, br s); MS m/z: 359
(M+H)+. Anal. Calcd for C21H18N4O2: C, 70.38; H,
5.06; N, 15.63. Found: C, 70.03; H, 5.00; N, 15.55.
6.1.26. N-[5-(4-Dimethylaminophenyl)imidazo[1,5-a]pyr-
azin-8-yl]-2,6-dimethylphenylamine (12f). This title com-
pound was prepared from 10 in the same manner as
described in 12a, and obtained as a white solid (45%):
mp 227–228 ꢁC (CH3CN); IR (KBr) 3154, 1609, 1518,
6.1.31. 4-[8-(2,6-Dimethylphenylamino)imidazo[1,5-a]-
pyrazin-5-yl]-N,N-dimethylbenzamide (12i). A mixture of
12 h (59.0 mg, 0.17 mmol), dimethylamine (1 M in THF;
0.17 mL, 0.17 mmol), 1-(3-dimethylaminopropyl)-3-eth-
1
1196, 772 cmꢀ1; H NMR (DMSO-d6) d 2.18 (6H, s),
2.97 (6H, m), 6.84 (1H, s), 6.84 (2H, d, J = 8.8 Hz),
7.10–7.20 (3H, m), 7.45 (2H, d, J = 8.8 Hz), 7.94 (1H,
br s), 8.26 (1H, s), 8.89 (1H, s); MS m/z: 358 (M+H)+.
Anal. Calcd for C22H23N5: C, 73.92; H, 6.49; N, 19.59.
Found: C, 73.65; H, 6.44; N, 19.44.
ylcarbodiimide
hydrochloride
(EDCl,
38.0 mg,
0.2 mmol), 1-hydroxybenzotriazole (HOBt, 31.0 mg,
0.2 mmol), and triethylamine (69 lL, 0.5 mmol) in THF
(1 mL)wasstirredfor15 hat50 ꢁC. Themixturewasdilut-
ed with EtOAc, and the organic layer was washed succes-
sively with 1 M HCl and brine, dried over anhydrous
MgSO4, and evaporated in vacuo. Purification of the
residue by flash column chromatography on APS (eluent:
hexane/EtOAc = 1:1) gave 52 mg (81%) of 12i as a white
solid: mp 231–232 ꢁC (CH3CN); IR (KBr) 3306, 1613,
1514, 1160, 770 cmꢀ1; 1H NMR (DMSO-d6) d 2.19 (6H,
s), 2.99 (3H, s), 3.01 (3H, s), 7.03 (1H, s), 7.10–7.20 (3H,
m), 7.54 (2H, d, J = 8.2 Hz), 7.73 (2H, d, J = 8.2 Hz),
8.09 (1H, br s), 8.41 (1H, s), 9.10 (1H, s); MS m/z: 386
(M+H)+. Anal. Calcd for C23H23N5O: C, 71.67; H, 6.01;
N, 18.17. Found: C, 71.36; H, 6.03; N, 17.82.
6.1.27. Methyl 4-[8-(2,6-dimethylphenylamino)imidazo
[1,5-a]pyrazin-5-yl]benzoate (12g). This title compound
was prepared from 10 in the same manner as described
in 12a, and obtained as a white solid (56%): mp 202–
203 ꢁC (MeOH); IR (KBr) 3126, 1715, 1539, 1434,
1
1284, 771 cmꢀ1; H NMR (DMSO-d6) d 2.19 (6H, s),
3.89 (3H, s), 7.05–7.20 (4H, m), 7.85 (2H, d,
J = 8.2 Hz), 8.05–8.20 (3H, m), 8,47 (1H, s), 9.21 (1H,
s); MS m/z: 373 (M+H)+. Anal. Calcd for
C22H20N4O2: C, 70.95; H, 5.41; N, 15.04. Found: C,
70.95; H, 5.30; N, 15.26.
6.1.28. N-(2,6-Dimethylphenyl)-5-pyridin-4-ylimidazo[1,5-a]-
pyrazin-8-amine (12j). This title compound was prepared
from 10 in the same manner as described in 12a, and ob-
6.1.32. 5-[8-(2,6-Dimethylphenylamino)imidazo[1,5-a]-
pyrazin-5-yl]thiophene-2-carbaldehyde (12l). A mixture
of 10 (303 mg, 0.96 mmol), 5-tributylstannylthiophen-
2-carbaldehyde (498 mg, 1.24 mmol), and bis(triphenyl-
phosphine)palladium(II) dichloride (67.0 mg, 0.10
mmol) in dioxane (3 mL) was stirred for 15 h at reflux.
The mixture was diluted with EtOAc, and the resulting
suspension was washed with brine, dried over anhydrous
MgSO4, and evaporated in vacuo. Purification of the res-
idue by flash column chromatography on silica gel (elu-
ent: hexane/EtOAc = 1:1) gave 253 mg (76%) of 12l as
a yellow solid: mp 250–251 ꢁC (CH3CN); IR (KBr)
tained as
(CH3CN); IR (KBr) 3193, 3010, 1613, 1596, 1497,
a
yellow solid (30%): mp 246–247 ꢁC
1
1210, 764 cmꢀ1; H NMR (DMSO-d6) d 2.18 (6H, s),
7.05–7.25 (4H, m), 7.65–7.75 (2H, m), 8.14 (1H, br s),
8.57 (1H, s), 8.60–8.70 (2H, m), 9.30 (1H, s); MS m/z:
316 (M+H)+. Anal. Calcd for C19H17N5Æ0.4H2O: C,
70.74; H, 5.56; N, 21.71. Found: C, 71.14; H, 5.58; N,
21.39.
1
6.1.29. N-(2,6-Dimethylphenyl)-5-thiophen-2-ylimidazo
[1,5-a]pyrazin-8-amine (12k). This title compound was
prepared from 10 in the same manner as described in
12a, and obtained as an off white solid (83%): mp
223–224 ꢁC (CH3CN); IR (KBr) 3199, 1621, 1553,
3128, 1659, 1534, 1437, 1060 cmꢀ1; H NMR (DMSO-
d6) d 2.17 (6H, s), 7.10–7.20 (3H, m), 7.42 (1H, s), 7.82
(1H, d, J = 4.1 Hz), 8,12 (1H, d, J = 4.1 Hz), 8.18 (1H,
br s), 8.72 (1H, s), 9.46 (1H, s), 9.95 (1H, s); MS m/z,
349 (M+H)+. Anal. Calcd for C19H16N4OS: C, 65.50;
H, 4.63; N, 16.08. Found: C, 65.34; H, 4.56; N, 16.17.
1519, 1252, 777 cmꢀ1 1H NMR (DMSO-d6) d 2.18
;
(6H, s), 7.10–7.20 (4H, m), 7.45–7.55 (1H, m), 7.70–
7.80 (1H, m), 7.90–8.25 (2H, m), 8.46 (1H, s), 9.05
(1H, s); MS m/z: 321 (M+H)+. Anal. Calcd for
C18H16NP4S: C, 67.47; H, 5.03; N, 17.49. Found: C,
67.35; H, 5.04; N, 17.48.
6.1.33. N-(2,6-Dimethylphenyl)-5-(5-pyrrolidin-1-ylmeth-
ylthiophen-2-yl)imidazo[1,5-a]pyrazin-8-amine hydrochlo-
ride (13a). To a mixture of 12l (62.0 mg, 0.18 mmol) and
pyrrolidine (30.0 lL, 0.35 mmol) in EtOH (0.7 mL) was

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
881
added titanium isopropoxide (0.10 mL, 0.35 mmol).
After stirring for 15 h at room temperature, sodium
borohydride (33.0 mg, 0.88 mmol) was added to the
mixture, and stirring continued for 3 h at room temper-
ature. The mixture was poured into saturated aqueous
NaHCO3 and extracted with EtOAc. The organic layer
was washed with brine, dried over anhydrous MgSO4,
and evaporated in vacuo. Purification of the residue by
flash column chromatography on APS (eluent, EtOAc)
gave 36 mg (51%) of N-(2,6-dimethylphenyl)-5-(5-pyrr-
olidin-1-ylmethylthiophen-2- yl)imidazo[1,5-a]pyrazin-
8-amine as a colorless amorphous oil: 1H NMR
(DMSO-d6) d 1.65–1.75 (4H, m), 2.17 (6H, s), 2.45–
2.60 (4H, m), 3.81 (2H, s), 7.05 (1H, d, J = 3.8 Hz),
7.11 (1H, s), 7.10–7.20 (3H, m), 7.40 (1H, d,
J = 3.8 Hz), 8,10 (1H, br s), 8.56 (1H, s), 9.17 (1H, s);
MS m/z: 404 (M+H)+.
m/z, 331 (M+H)+. Anal. Calcd for C20H18N4OÆ0.2H2O:
C, 71.92; H, 5.55; N, 16.78. Found: C, 72.12; H, 5.58; N,
16.69.
To a mixture of 4-[8-(2,6-dimethylphenylamino)imi-
dazo[1,5-a]pyrazin-5-yl]phenol (71.0 mg, 0.21 mmol),
N-(2-hydroxyethyl)morpholine (104 lL, 0.86 mmol),
and triphenylphosphine (113 mg, 0.43 mmol) in THF
(1 mL) was added 40% diethyl azodicarboxylate solu-
tion in toluene (228 lL) at room temperature, and the
mixture was stirred for 15 h at 50 ꢁC. The mixture was
evaporated in vacuo, and purification of the residue by
flash column chromatography on APS (eluent: hexane/
EtOAc = 2:1) gave 69 mg (72%) of 14a as a white solid:
mp 195–196 ꢁC; IR (KBr) 2955, 1607, 1514, 1250,
771 cmꢀ1 1H NMR (DMSO-d6) d 2.18 (6H, s), 2.72
;
(2H, t, J = 5.7 Hz), 3.55–3.65 (8H, m), 4.16 (2H, t,
J = 5.7 Hz), 6.88 (1H, s), 7.09 (2H, d, J = 8.8 Hz),
7.10–7.20 (3H, m), 7.57 (2H, d, J = 8.8 Hz), 8.05 (1H,
br s), 8.27 (1H, s), 8.99 (1H, s); MS m/z, 444 (M+H)+.
Anal. Calcd for C26H29N5O2: C, 70.41; H, 6.59; N,
15.79. Found: C, 70.27; H, 6.52; N, 15.63.
To a solution of N-(2,6-dimethylphenyl)-5-(5-pyrrolidin-
1-ylmethylthiophen-2- yl)imidazo[1,5-a]pyrazin-8-amine
(24.0 mg, 0.06 mmol) in EtOAc (1 mL) was added 4 M
HCl in EtOAc (33.0 lL, 0.13 mmol) at 4 ꢁC. Collection
of the resulting precipitates by filtration gave 20 mg
(76%) of 13a as a yellow solid: mp 269–270 ꢁC (dec.);
IR (KBr) 3398, 3198, 1617, 1534, 1502, 1208,
6.1.36. N-(2,6-Dimethylphenyl)-5-[4-(2-pyrrolidin-1-yleth-
oxy)phenyl]imidazo[1,5-a]pyrazin-8-amine (14b). This
title compound was prepared from 4-[8-(2,6-dimethyl-
phenylamino)imidazo[1,5-a]pyrazin-5-yl]phenol in the
same manner as described in 14a, and obtained as an
off white solid: mp 222–223 ꢁC; IR (KBr) 3124, 2964,
1
772 cmꢀ1; H NMR (DMSO-d6) d 1.85–2.10 (4H, m),
2.23 (6H, s), 3.05–3.20 (2H, m), 3.40–3.50 (2H, m),
4.60–4.70 (2H, m), 7.10–7.20 (3H, m), 7.47 (1H, d,
J = 3.8 Hz), 7.57 (1H, d, J = 3.8 Hz), 8.19 (1H, br s),
8.60 (1H, s), 9.39 (1H, br s), 10.77 (1H, br s); Anal.
Calcd for C23H25N5SÆHClÆH2O: C, 60.31; H, 6.10; N,
15.29. Found: C, 60.56; H, 6.04; N, 14.95.
1607, 1514, 1251, 770 cmꢀ1 1H NMR (DMSO-d6) d
;
1.65–1.75 (4H, m), 2.18 (6H, s), 2.50–2.60 (4H, m),
2.81 (2H, t, J = 6.0 Hz), 4.13 (2H, t, J = 6.0 Hz), 6.89
(1H, s), 7.08 (2H, d, J = 8.8 Hz), 7.10–7.15 (3H, m),
7.57 (2H, d, J = 8.8 Hz), 8.05 (1H, br s), 8.27 (1H, s),
8.98 (1H, s); Anal. Calcd for C26H29N5OÆ0.25H2O: C,
72.28; H, 6.68; N, 16.21. Found: C, 72.63; H, 6.96; N,
16.03.
6.1.34. N-(2,6-Dimethylphenyl)-5-[5-(4-methylpiperazin-
1-ylmethyl)thiophen-2-yl]imidazo[1,5-a]pyrazin-8-amine
(13b). This title compound was prepared from 12l in the
same manner as described above, and obtained as an off
white solid: mp 186–187 ꢁC (dec.); IR (KBr) 3133, 2940,
1
2806, 1533, 1287, 1163 cmꢀ1; H NMR (DMSO-d6) d
6.1.37. N-(2,6-Dimethylphenyl)-5-[4-(1-methylpiperidin-
3-ylmethoxy)phenyl]imidazo[1,5-a]pyrazin-8-amine (14c).
This title compound was prepared from 4-[8-(2,6-dim-
ethylphenylamino)imidazo[1,5-a]pyrazin-5-yl]phenol in
the same manner as described in 14a, and obtained as
an off white solid: mp 191–192 ꢁC (CH3CN); IR (KBr)
2.15 (3H, s), 2.17 (6H, s), 2.20–2.50 (8H, m), 3.70 (2H,
s), 7.60 (1H, d, J = 3.8 Hz), 7.10–7.20 (4H, m), 7.41
(1H, d, J = 3.8 Hz), 8.09 (1H, br s), 8.54 (1H, s), 9.15
(1H, s); Anal. Calcd for C24H28N6SÆ0.5H2O: C, 65.28;
H, 6.62; N, 19.03. Found: C, 65.56; H, 6.37; N, 18.64.
1
2953, 1606, 1514, 1247, 772 cmꢀ1; H NMR (DMSO-
6.1.35. N-(2,6-Dimethylphenyl)-5-[4-(2-morpholin-4-yleth-
oxy)phenyl]imidazo[1,5-a]pyrazin-8-amine (14a). To a
suspension of 6h (731 mg, 2.12 mmol) in dichlorometh-
ane (CH2Cl2, 5 mL) was added BBr3 (1 M in CH2Cl2;
8.5 mL, 8.50 mmol) at ꢀ78 ꢁC, and the mixture was stir-
red for 5 h. A solution of triethanolamine (3.17 g,
21.2 mmol) in THF (10 mL) was added to the mixture
at 0 ꢁC, and the mixture was stirred for 15 h at reflux.
The mixture was diluted with water and extracted with
EtOAc. The organic layer was washed with brine, dried
over anhydrous MgSO4, and evaporated in vacuo to
give 613 mg (87%) of 4-[8-(2,6-dimethylphenylami-
no)imidazo[1,5-a]pyrazin-5-yl]phenol as a pale brown
solid: mp > 300 ꢁC (CH3CN); IR (KBr) 3408, 2985,
d6) d 1.10–1.15 (1H, m), 1.40–1.55 (1H, m), 1.60–1.85
(3H, m), 1.85–1.95 (1H, m), 1.95–2.10 (1H, m), 2.16
(3H, s), 2.18 (6H, s), 2.55–2.70 (1H, m), 2.75–2.85 (1H,
m), 3.85–4.00 (2H, m), 6.88 (1H, s), 7.07 (2H, d,
J = 8.8 Hz), 7.10–7.20 (3H, m), 7.56 (2H, d,
J = 8.8 Hz), 8.05 (1H, br s), 8.27 (1H, s), 8.99 (1H, s);
MS m/z, 442 (M+H)+. Anal. Calcd for C27H31
N5OÆ0.2H2O: C, 72.85; H, 7.11; N, 15.73. Found: C,
73.02; H, 7.02; N, 15.73.
6.1.38. N-(2,6-Dimethylphenyl)-5-[3-(1-methylpiperidin-
3-ylmethoxy)phenyl]imidazo[1,5-a]pyrazin-8-amine (14d).
3-[8-(2,6-Dimethylphenylamino)imidazo[1,5-a]pyrazin-
5-yl]phenol was prepared from 12a in the same manner
as described in 14a, and obtained as an off white solid:
mp 157–158 ꢁC (MeOH); IR (KBr) 3325, 3050, 1624,
1608, 1517, 1278, 838 cmꢀ1 1H NMR (DMSO-d6) d
;
2.18 (6H, s), 6.85 (1H, s), 6.90 (2H, d, J = 8.5 Hz),
7.10–7.20 (3H, m), 7.45 (2H, d, J = 8.5 Hz), 8.00 (1H,
br s), 8.26 (1H, s), 8.95 (1H, br s), 9.81 (1H, s); MS
1517, 1451, 1221, 775 cmꢀ1 1H NMR (DMSO-d6) d
;
2.18 (6H, s), 6.86 (1H, dd, J = 8.2, 2.2 Hz), 6.94 (1H,

882
H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
s), 6.95–7.05 (1H, m), 7.08 (1H, d, J = 7.6 Hz), 7.10–7.20
(3H, m), 7.32 (1H, t, J = 8.2 Hz), 8.07 (1H, br s), 8.33
(1H, s), 9.05 (1H, s), 9.75 (1H, s); MS m/z, 331
(M+H)+. Anal. Calcd for C20H18N4OÆ0.2H2O: C,
71.92; H, 5.55; N, 16.78. Found: C, 72.24; H, 5.52; N,
16.86.
6.1.42. 6-(3-Methoxyphenyl)imidazo[1,5-a]pyrazin-8(7H)-
one (17a). A mixture of 16a (903 mg, 2.19 mmol) and
imidazole (3.73 g, 54.8 mmol) was stirred for 14 h at
180 ꢁC. After cooling to 100 ꢁC, ice water was added to
the mixture. Collection of the insoluble material by filtra-
tion gave 264 mg (50%) of 17a as a pale brown solid: mp
275–276 ꢁC; IR (KBr) 3100, 1644, 1597 cmꢀ1; 1H NMR
(DMSO-d6) d 3.84 (3H, s), 6.95–7.05 (1H, m), 7.20–7.30
(2H, m), 7.35–7.45 (1H, m), 7.75–7.80 (1H, m), 7.80–7.90
(1H, m), 8.27 (1H, d, J = 0. 6 Hz), 10.99 (1H, s); Anal.
Calcd for C13H11N3O2Æ0.1H2O: C, 64.24; H, 4.64; N,
17.29. Found: C, 64.27; H, 4.60; N, 17.30.
This title compound was prepared from 3-[8-(2,6-dim-
ethylphenylamino)imidazo[1,5-a]pyrazin-5-yl]phenol in
the same manner as described in 14a, and obtained as
an off white solid: mp 173–174 ꢁC (CH3CN); IR (KBr)
3398, 2936, 1543, 1515, 1437, 771 cmꢀ1 1H NMR
;
(DMSO-d6) d 1.00–1.15 (1H, m), 1.40–1.55 (1H, m),
1.55–1.85 (3H, m), 1.85–1.95 (1H, m), 1.95–2.05 (1H,
m), 2.14 (3H, s), 2.19 (6H, s), 2.55–2.65 (1H, m), 2.75–
2.85 (1H, m), 3.85–4.00 (2H, m), 6.98 (1H, s), 7.02
(1H, d, J = 8.2 Hz), 7.10–7.25 (5H, m), 7.42 (1H, t,
J = 8.2 Hz), 8.05 (1H, br s), 8.36 (1H, s), 9.04 (1H, s);
MS m/z, 442 (M+H)+. Anal. Calcd for C27H31N5O: C,
73.44; H, 7.08; N, 15.86. Found: C, 73.09; H, 7.02; N,
15.73.
6.1.43. 6-(4-Fluorophenyl)imidazo[1,5-a]pyrazin-8(7H)-
one (17b). This title compound was prepared from 16b
in the same manner as described above, and obtained
as an off white solid (55%): mp > 300 ꢁC (MeOH); IR
(KBr) 3131, 3069, 1681, 1108 cmꢀ1
;
1H NMR
(DMSO-d6) d 7.30–7.40 (2H, m), 7.65–7.75 (2H, m),
7.75–7.85 (2H, m), 8.28 (1H, d, J = 0.6 Hz), 11.05 (1H,
s); Anal. Calcd for C12H8FN3OÆ0.1H2O: C, 62.39; H,
3.58; N, 18.19. Found: C, 62.44; H, 3.54; N, 18.31.
6.1.39. N-(2,6-Dimethylphenyl)-5-[3-(2-pyrrolidin-1-yleth-
oxy)phenyl]imidazo[1,5-a]pyrazin-8-amine hydrochloride
(14e). This title compound was prepared from 3-[8-
(2,6-dimethylphenylamino)imidazo[1,5-a]pyrazin-5-yl]
phenol in the same manner as described in 14a, and
obtained as an off white solid: mp 282–283 ꢁC
6.1.44. N-(2-Chloro-6-methylphenyl)-6-(3-methoxyphe-
nyl)imidazo[1,5-a]pyrazin-8-amine (18a). The title com-
pound was prepared from 17a in the same manner as
described in 6a, and obtained as an off white solid
(32%): mp 277–278 ꢁC; IR (KBr) 3204, 3115, 2955,
1609, 1580, 1540, 1505, 780 cmꢀ1; 1H NMR (DMSO-d6)
d 2.28 (3H, s), 3.70 (3H, s), 6.80-6.90 (1H, m), 7.20–7.40
(5H, m), 7.40–7.50 (1H, m), 7.86 (1H, br s), 8.40 (1H, s),
8.41 (1H, s), 9.35 (1H, s); MS m/z, 365 (M+H)+. Anal.
Calcd for C20H17ClN4OÆ0.2H2O: C, 65.20; H, 4.76; N,
15.21. Found: C, 65.43; H, 4.61; N, 15.30.
(dec.); IR (KBr) 3412, 3212, 1538, 1507, 1220 cmꢀ1
;
1H NMR (DMSO-d6) d 1.80–2.10 (4H, m), 2.18
(6H, s), 3.00–3.20 (2H, m), 3.50–3.70 (4H, m), 4.41
(2H, t, J = 4.4 Hz), 7.00 (1H, s), 7.05–7.20 (4H, m),
7.25–7.35 (2H, m), 7.48 (1H, t, J = 7.9 Hz), 8.11
(1H, br s), 8.41 (1H, s), 9.13 (1H, br s), 10.28 (1H,
br s); Anal. Calcd for C26H29N5OÆHClÆ0.5H2O: C,
66.02; H, 6.61; N, 14.81. Found: C, 65.94; H, 6.64;
N, 14.68.
6.1.45. N-(2-Chloro-6-methylphenyl)-6-(4-fluorophenyl)-
imidazo[1,5-a]pyrazin-8-amine (18b). The title compound
was prepared from 17b in the same manner as described
in 6a, and obtained as an off white solid: mp 215–216 ꢁC;
IR (KBr) 3184, 3107, 1617, 1545, 1473, 1436, 1223,
6.1.40. 2-Benzyl-6-(3-methoxyphenyl)-8-oxo-7,8-dihydro-
imidazo[1,5-a]pyrazin-2-ium bromide (16a). A mixture of
15 (563 mg, 2.80 mmol) and 2-bromo-30-methoxyace-
tophenone (673 mg, 2.94 mmol) in acetonitrile (5 mL)
and DMF (1.6 mL) was stirred for 14 h at 90 ꢁC. After
cooling to room temperature, collection of the insoluble
material by filtration gave 1.04 g (90%) of 16a as a white
solid: mp 267–268 ꢁC; IR (KBr) 3417, 3100, 1680,
1
776 cmꢀ1; H NMR (DMSO-d6) d 2.27 (3H, s), 7.15–
7.25 (2H, m), 7.25–7.40 (2H, m), 7.40–7.50 (1H, m),
7.65–7.80 (2H, m), 7.84 (1H, br s), 8.36 (1H, s), 8.40
(1H, s), 9.35 (1H, s); MS m/z, 353 (M+H)+. Anal. Calcd
for C19H14ClFN4Æ0.2H2O: C, 64.03; H, 4.07; N, 15.72.
Found: C, 64.35; H, 3.89; N, 15.70.
1
1599 cmꢀ1; H NMR (DMSO-d6) d 3.84 (3H, s), 5.66
(2H, s), 7.05–7.15 (1H, m), 7.20–7.30 (2H, m), 7.35–
7.55 (6H, m), 8.00 (1H, s), 8.85(1H, d, J = 0.7 Hz),
9.66 (1H, d, J = 1.6 Hz), 11.84 (1H, s); Anal. Calcd for
C20H18BrN3O2: C, 58.26; H, 4.40; N, 10.19. Found: C,
58.05; H, 4.40; N, 10.17.
6.1.46. N-(2,6-Dimethylphenyl)-6-(4-fluorophenyl)imidazo-
[1,5-a]pyrazin-8-amine (18c). The title compound was
prepared from 17b in the same manner as described in
6a, and obtained as an off white solid: mp 228–229 ꢁC;
IR (KBr) 3186, 3107, 1616, 1547, 1515, 1223,
1
776 cmꢀ1; H NMR (DMSO-d6) d 2.21 (6H, s), 7.10–
6.1.41. 2-Benzyl-6-(4-fluorophenyl)-8-oxo-7,8-dihydroim-
idazo[1,5-a]pyrazin-2-ium bromide (16b). This title com-
pound was prepared from 15 in the same manner as
described above, and obtained as an off white solid
(37%): mp 277–278 ꢁC; IR (KBr) 3041, 1673, 1520,
7.25 (5H, m), 7.50–7.90 (2H, m), 7.96 (1H, br s), 8.32
(1H, s), 8.38 (1H, s), 9.07 (1H, s); MS m/z, 333
(M+H)+. Anal. Calcd for C20H17FN4Æ0.2H2O: C,
71.50; H, 5.22; N, 16.68. Found: C, 71.57; H, 5.14; N,
16.37.
1
1247, 1120 cmꢀ1; H NMR (DMSO-d6) d 5.66 (2H, s),
7.35–7.55 (7H, m), 7.65–7.80 (2H, m), 7.94 (1H, s),
8.85 (1H, s), 9.68 (1H, s), 11.88 (1H, s); Anal. Calcd
for C19H15BrFN3O: C, 57.02; H, 3.78; N, 10.50. Found:
C, 56.98; H, 3.77; N, 10.56.
6.1.47. N-(2,6-Dimethylphenyl)-5-[4-(1-methylpiperidin-
3-ylmethoxy)phenyl]imidazo[1,5-a]pyrazin-8-amine hydro-
chloride (14cÆHCl). To a solution of 14c (856 mg,
1.94 mmol) in EtOAc (9 mL) was added 4 M HCl in

H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
883
EtOAc (1.93 mL, 3.88 mmol) at 5 ꢁC. Collection of the
resulting precipitates by filtration gave 766mg (83%) of
14cÆHCl as a white solid: mp 187–188 ꢁC (CH3CN–
We used the configuration of the inhibitor in Lck as
the initial structure to build the c-Src model because
both kinases have high sequence homology, particular-
ly within the ATP binding site, and to ensure internal
consistency in the enzymatic assay, among other
reasons.
1
H2O); IR (KBr) 3416, 2954, 1624, 1512, 1250 cmꢀ1; H
NMR (DMSO-d6) d 1.20–1.40 (1H, m), 1.80–1.95 (3H,
m), 2.24 (6H, s), 2.35–2.50 (1H, m), 2.50–2.90 (5H, m),
3.35–3.45 (1H, m), 3.45–3.55 (1H, m), 3.90–3.95 (1H,
m), 4.00–4.10 (1H, m), 6.78 (1H, br s), 7.15 (2H, d,
J = 8.8 Hz), 7.20–7.40 (3H, m), 7.65 (2H, d,
J = 8.8 Hz), 8.54 (1H, br s), 8.82 (1H, br s), 10.74 (1H,
br s), 12.00 (1H, br s); Anal. Calcd for C27H31N5OÆHClÆ-
H2O: C, 65.38; H, 6.91; N, 14.12. Found: C, 65.25; H,
6.79; N, 14.10.
6.4. Biology
6.4.1. Kinase assays. A coupled spectrophotometric
assay was used wherein ADP generated by Src kinase
was converted to ATP by pyruvate kinase (PK), with
concomitant production of pyruvate from phospho-
enolpyruvate (PEP). LDH reduces pyruvate to lactate
by oxidizing NADH. NADH depletion was moni-
tored 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, initiated by adding ATP. PK (100 lg/mL),
LDH (50 lg/mL), PEP (2 mM), and NADH
(140 lM) were also added. Kinase activity was
measured by adding 100 lM Src optimal peptide
substrate (peptide sequence: AEEEIYGEFEAKKKK,
Sawady, Tokyo).
6.1.48. N-(2,6-Dimethylphenyl)-5-[3-(1-methylpiperidin-
3-ylmethoxy)phenyl]imidazo[1,5-a]pyrazin-8-amine hydro-
chloride (14dÆHCl). The title compound was prepared
from 14d in the same manner as described above, and
obtained as an off white solid: mp 188–189 ꢁC
(CH3CN–H2O); IR (KBr) 3421, 2949, 1636, 1587,
1
1472 cmꢀ1; H NMR (CD3OD) d 1.40–1.55 (1H, m),
1.80–2.10 (3H, m), 2.25–2.50 (7H, m), 2.80–3.05
(5H, m), 3.53 (1H, d, J = 11.7 Hz), 3.70 (1H, d,
J = 11.7 Hz), 3.95–4.05 (1H, m), 4.05–4.15 (1H, m),
6.81 (1H, s), 7.15–7.25 (1H, m), 7.25–7.45 (5H, m),
7.54 (1H, t, J = 8.0 Hz), 8.55 (1H, br s), 8.60 (1H, br
s); Anal. Calcd for C27H31N5OÆHClÆH2O: C, 65.38; H,
6.91; N, 14.12. Found: C, 65.29; H, 6.84; N, 13.91.
6.4.2. Intracellular kinase inhibition assay. The kinase
domain of human Src kinase (NM_005417, base# 790-
1650) was cloned by PCR amplification, using the link-
er-containing primers 50-AAACTTAAGCTTCATATG
TCCAAGCCGCAGAC-30 and 50-CTGCAGATATC
CCTAGAAGTAGTCCTCCAGGAA-30. The gene for
the Src kinase domain was integrated between the Hin-
dIII and EcoRV restriction sites within the multiple
cloning site of the pcDNA3.1(+) expression vector.
The vector was transfected into COS7 cells according
to the calcium phosphate method.32 Subsequently,
8.8 lg DNA was mixed with 220 lL of 1 mM Tris–
HCl, 0.1 mM EDTA buffer (pH 8.0), 250 lL 2·
Hepes-buffered saline, and 31 lL of 2 M CaCl2. The
solution was incubated at room temperature for
15 min and then used to resuspend a pellet of 106
COS7 cells. The cell suspension was incubated at room
temperature for 15 min; then, 4.5 mL Dulbecco’s modi-
fied Eagle’s medium supplemented with 10% fetal bo-
vine serum and antibiotics was added, and the solution
was pre-warmed to 37 ꢁC. Cells were seeded into 96-well
multi-well plates with 2 · 104 cells/well/100 lL. A day
after transfection, the culture media were replaced with
media containing fixed concentrations of test com-
pounds. On the third day, the cellular phosphotyrosine
contents were determined using a commercially avail-
able phosphotyrosine ELISA kit (Upstate Biotechnolo-
gy, NY, USA. Cellular Phosphotyrosine ELISA KitTM,
Catalog #17-182). Any basal phosphotyrosine content
unrelated to c-Src activity was screened for using vec-
tor-transfected cells. Intracellular c-Src inhibition was
expressed as the percentage of specific phosphotyrosine
levels in drug-treated cells compared to the levels in con-
trol cells without drug. The toxicity of each chemical
was determined with another replica plate using a color-
imetric MTT assay kit (Chemicon International, CA,
USA. Cat. #CT02).
6.2. X-ray crystallography
A binary complex of purified human Lck protein and
compound 6a was formed by the hanging-drop co-crys-
tallization method. Crystals grew over 7 days to a max-
imum dimension of 0.3 · 0.5 · 0.7 mm. Prior to data
collection, crystals were quickly dipped in 20% (v/v)
glycerol containing a cryosolution and flash-cooled in
liquid nitrogen. X-ray diffraction intensity was mea-
sured under cryogenic conditions using an R-AXIS
IV diffractometer (Rigaku) operating at 50 kV and
108 mA. The data were processed using MOSFLM
Ver.6.2.3.31 The crystal structure diffracted to 2.55 A
˚
resolution and conformed to the space group P3121
with one molecule per asymmetric unit. The structure
of the binary complex was refined and modeled against
the published structure (PDB code: 3LCK)21 in the ab-
sence of ligand, using the CNX program (Accerlys). A
rigid-body rotation–translation refinement was per-
formed to accurately place the model structure into
the new unit cell. Further crystallographic refinement
was done using conjugated-gradient minimization.
Individual B factor refinement was done with CNX,
and model/ligand building was performed using
QUANTA (Accerlys). The final model included 271
residues and 184 water molecules, with an R factor
of 21.6% and an Rf of 28.3%.
6.3. Molecular modeling
The structure of inhibitor 6a was extracted from our
X-ray structure of Lck and manually docked into the
ATP binding site of c-Src (PDB code. 1YOL)19 using
CHARM/QuantaTM.

884
H. Mukaiyama et al. / Bioorg. Med. Chem. 15 (2007) 868–885
6.4.3. Analysis of c-Src inhibitors in the brain and plasma.
To inhibit c-Src kinase in the brain, we first determined
the brain and plasma levels of the test compounds.
Sprague–Dawley rats (300–350 g: n = 3/study) were
anesthetized with 25% urethane (1.1 g/kg, sc), and the
femoral vein was cannulated to deliver the test com-
pounds. Tracheal intubation was performed for artificial
respiration. The animal’s body temperature was main-
tained at 37.5 ꢁC with a heating-pad. Test compounds
were administered by intravenous infusion at 3 mg/kg/
h. Three hours later, blood samples were taken from
the aorta and centrifuged to separate the plasma. Brain
tissue was homogenized with a 3-fold volume of saline.
from the other parts of the brain and cut into six 2-
mm-thick slices using a Brain Matrix (Muromachi Ki-
kai, Japan). Each slice was incubated in 1% tetratrihy-
drochloride (TTC) solution at room temperature for
30 min and then photographed. The area of infarction
was measured for each slice using a computerized image
analysis system (Mac scope, Japan), and the ratio of
infarction size was calculated by dividing the whole area
by the region of cerebral ischemic damage. All animal
studies were approved by the Kissei Pharmaceutical
Co., Ltd. Committee on Ethics of Animal Experimenta-
tion, and special care was taken to prevent animal
suffering.
Plasma and brain tissue homogenate (50 lL) was mixed
with 200 lL of 100% acetonitrile, 100 lL of 0.1% hepta-
fluorobutyric acid, and 200 lL internal standard solu-
tion. After centrifugation at 3300 rpm for 20 min, the
supernatant was removed and transferred to the col-
umn. Samples were quantified using liquid chromatog-
raphy-tandem mass spectrometry (LC–MS/MS). The
LC–MS/MS system consisted of an Alliance HPLC
(Waters 2690) with a SYNERGI MAX-RP 80A column
(4 lm, 50 · 4.6 mm, Phenomenex, Torrance, CA), and a
Sciex API 365 mass spectrometer (Perkin–Elmer Sciex,
Toronto, Canada) equipped with a turbo ion spray
source. The mobile phase consisted of 100% acetonitrile
and 0.1% acetic acid in water (50:50 v/v). The column
was maintained at 50 ꢁC with a constant flow rate of
0.4 mL/min. The data were processed using Mass
Chrom 1.1 software. The standard curves plotted for
each test compound demonstrated good linearity
(coefficient of determination >0.99). The limits of quan-
tification of the compounds in plasma and brain were
50 ng/mL.
Acknowledgments
We thank Dr. Nobuyuki Tanaka and Dr. Hiromu Hara-
da for helpful comments while reviewing the manuscript.
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