Synthesis and biological evaluation of 4-morpholino-2-phenylquinazolines and related derivatives as novel PI3 kinase p110α inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.06.046

A series of 4-morpholino-2-phenylquinazolines and related derivatives were prepared and evaluated as inhibitors of PI3 kinase p110α. In this series, the thieno[3,2-d]pyrimidine derivative 15e showed the strongest inhibitory activity against p110α, with an

Full text of DOI:10.1016/j.bmc.2006.06.046

Bioorganic & Medicinal Chemistry 14 (2006) 6847–6858
Synthesis and biological evaluation of 4-morpholino-2-
phenylquinazolines and related derivatives as novel
PI3 kinase p110a inhibitors
Masahiko Hayakawa,a,* Hiroyuki Kaizawa,a Hiroyuki Moritomo,a Tomonobu Koizumi,a
Takahide Ohishi,a Minoru Okada,a Mitsuaki Ohta,a Shin-ichi Tsukamoto,a Peter Parker,b
Paul Workmanc and Mike Waterfieldd
aInstitute for Drug Discovery Research, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan
bCancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
cCancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SN2 5NG, UK
dLudwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, UK
Received 23 May 2006; revised 19 June 2006; accepted 19 June 2006
Available online 11 July 2006
Abstract—A series of 4-morpholino-2-phenylquinazolines and related derivatives were prepared and evaluated as inhibitors of PI3
kinase p110a. In this series, the thieno[3,2-d]pyrimidine derivative 15e showed the strongest inhibitory activity against p110a, with
an IC50 value of 2.0 nM, and inhibited proliferation of A375 melanoma cells with an IC50 value of 0.58 lM. Moreover, 15e was
found to be selective for p110a over other PI3K isoforms and protein kinases, making it the first example of a selective PI3K
p110a inhibitor.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
expression or function of PTEN occurs in many human
cancers5,6 and mutation of PTEN is one of the most
common mutations in human cancers,7 making PI3Ks
potential therapeutic targets for proliferative disorders
such as cancer.
Phosphoinositide 3-kinase (PI3K) is an enzyme that cat-
alyzes phosphorylation of the 3-hydroxyl position of
phosphatidylinositides (PIs) and is known to regulate
various cellular functions, including cell proliferation
and survival.1–3 The 3-phosphorylated phospholipids
generated by PI3K activity bind to the pleckstrin homol-
ogy (PH) domain of protein kinase B (PKB), causing
translocation of PKB to the cell membrane and subse-
quent phosphorylation of PKB. Phosphorylated PKB
inhibits apoptosis-inducing proteins such as FKHR,
Bad, and caspases, and is thought to play an important
role in cancer progression.4 Negative regulation of PI3K
signaling is mediated by the lipid phosphatase PTEN,
which dephosphorylates products of PI3K. Loss of
The PI3Ks known to date8–10 are divided into classes
I–III, and class I is further subclassified into classes Ia
and Ib. Among these isoforms, class Ia enzymes are
thought to play the most important role in cell prolifer-
ation in response to growth factor-tyrosine kinase path-
way activation.11 The PIK3CA gene, which encodes
PI3K p110a, is amplified and overexpressed in ovarian
and other cancers,12,13 and is also mutated in a spectrum
of cancers.14–17 Thus, class Ia PI3Ks, and particularly
p110a, are potential targets in treatment of cancer,
and their inhibitors are potential cancer therapeutic
agents. To date, reported PI3K inhibitors include the
fungal metabolite wortmannin and the flavonoid-related
compound LY294002 (Fig. 1). Although wortmannin is
a potent PI3K inhibitor with a low nanomolar IC50
value, it has low in vivo anti-tumor activity.18 Moreover,
wortmannin is unstable in solution, probably due to the
Keywords: PI3 kinase; p110a; Inhibitor; Cancer treatment.
Corresponding author. Tel.: +81 29 865 7124; fax: +81 29 847
8313; e-mail: masahiko.hayakawa@jp.astellas.com
*
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.046

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M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
2. Chemistry
O
O
O
O
MeO
O
As shown in Scheme 1, the 6-hydroxy-2-phenylquinazo-
lines 5a–d were prepared from 5-hydroxyanthranilic acid
2. Condensation of 2 with phenylimidate in refluxing
methanol followed by treatment with acetic anhydride
afforded the known 6-acetoxyquinazolinone 3.23 Chlori-
nation of 3 with phosphorus oxychloride followed by
treatment with appropriate amines in refluxing THF
then gave 5a–d. The 6-hydroxy-4-(morpholino)quinazo-
line derivatives with a substituent on the benzene at C2,
6a–g, were also prepared from 5-hydroxyanthranilic
acid 2. Condensation of 2 with the corresponding imi-
dates and subsequent acetylation gave the 6-acetoxyqui-
nazolinones 4a–g. When the imidates were not
commercially available, they were prepared by treat-
ment of the corresponding nitriles with hydrochloric
acid and ethanol. Chlorination of 4a–g and subsequent
reaction with morpholine gave 6a–g. The ester groups
of 6d and 6e were reduced with lithium aluminum
hydride to provide 6h and 6i, and hydrogenation of 6f
and 6g gave 6j and 6k, respectively.
N
O
O
H
O
O
O
LY294002
Wortmannin
O
N
HO
N
N
1
Figure 1. Structure of PI3K inhibitors.
presence of a reactive furan ring.19 LY294002 is more
stable, but is a relatively weak PI3K inhibitor with an
IC50 value of 0.63 lM.20 Furthermore, neither wortman-
nin nor LY294002 exhibits selectivity among PI3K iso-
forms, and the lack of isoform-specific PI3K inhibitors
has made it difficult to understand the biological roles
of individual PI3K isoforms.21,22 We have carried out
high-throughput screening to obtain novel PI3K p110a
inhibitors, and 4-morpholino-2-phenylquinazolin-6-ol 1
was discovered as a p110a inhibitor with an IC50 value
of 1.3 lM. We now report the synthesis and evaluation
of a new class of compounds derived from 1, 4-morpho-
lino-2-phenylquinazolines and related compounds,
which are novel, potent, and highly selective PI3K
p110a inhibitors.
For the synthesis of 10a and 10c, the corresponding
2-substituted phenylimidates were difficult to prepare
from nitriles using the method described above; 10a
and 10c were therefore synthesized from the known
anthranilamide 7b,24 as shown in Scheme 2. Reaction
of 7b with acyl chlorides and cyclization under basic
conditions gave the quinazolinones 8a and 8c, which
were converted to 9a and 9c via demethylation with
hydrobromic acid and subsequent acetylation with
acetic anhydride. Chlorination of 9a and 9c followed
O
N
O
AcO
HO
a
NH
OH
NH2
R
2
3: R = H
4d: R = 3-CO2Me
4e: R = 4-CO2Me
4f: R = 3-NO2
4a: R = 3-OAc
4b: R = 3-OMe
4c: R = 3-F
4g: R = 4-NO2
NR'2
N
b
HO
N
R
5: R = H
6: NR'2 = morpholino
5a: NR'2 = piperidino
6a: R = 3-OH
5b: NR'2 = 4-hydroxypiperidino
5c: NR'2 = 4-oxopiperidino
5d: NR'2 = 2-methoxyethylamino
6b: R = 3-OMe
6c: R = 3-F
c
c
d
d
6d: R = 3-CO2Me
6e: R = 4-CO2Me
6f: R = 3-NO2
6g: R = 4-NO2
6h: R = 3-CH2OH
6i: R = 4-CH2OH
6j: R = 3-NH2
6k: R = 4-NH2
Scheme 1. Reagents and conditions: (a) (i) ethyl arylimidate hydrochloride, MeONa, MeOH, reflux; (ii) Ac2O, AcONa, reflux; (b) (i) POCl3, reflux;
(ii) R2NH, THF, reflux; (c) LiAlH4, THF; (d) H2, 10% Pd–C.

M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
6849
O
O
N
O
5
5
5
6
7
6
7
6
7
NH2
NH2
NH
b
a
NH
R
R
R
N
R'
R'
9a: R = 6-OAc; R' = 2-OAc
9b: R = 6-OAc; R' = 4-OAc
9c: R = 6-OAc; R' = 2-NO2
9e: R = 5-OAc; R' = H
8a: R = 6-OMe; R' = 2-OMe
7a: R = 5-OMe
7b: R = 6-OMe
7c: R = 7-OMe
8b: R = 6-OMe; R' = 4-OMe
8c: R = 6-OMe; R' = 2-NO2
8e: R = 5-OMe; R' = H
8f: R = 7-OMe; R' = H
9f: R = 7-OAc; R' = H
O
N
N
5
c
6
7
R
N
R'
10: R = OH
10a: R = 6-OH; R' = 2-OH
10b: R = 6-OH; R' = 4-OH
10c: R = 6-OH; R' = 2-NO2
10d: R = 6-OH; R' = 2-NH2
10e: R = 5-OH; R' = H
d
10f: R = 7-OH; R' = H
Scheme 2. Reagents and conditions: (a) (i) ArCOCl, Et3N, THF; (ii) 2 N NaOH, MeOH, reflux; (b) (i) 48% HBr, AcOH; (ii) Ac2O, AcONa, reflux;
(c) (i) POCl3, reflux; (ii) morpholine, reflux; (d) H2, 10% Pd–C.
CO2Me
NH
O
N
CO2Me
NH2
b
a
Ar
Ar
NH
Ar
OMe
OMe
O
11a-e
12a-e
13a-e
O
N
O
N
c
NH
d
Ar
HCl
OH
OAc
N
Ar
N
14a-e
15a-e
N
N
Ar = a:
b:
c:
S
d:
e:
N
Scheme 3. Reagents and conditions: (a) ArCOCl, Et3N, THF; (b) (i) 28% aq NH3, MeOH; (ii) 2 N NaOH, MeOH, reflux; (c) (i) 48% HBr, AcOH,
reflux; (ii) Ac2O, AcONa, reflux; (d) (i) POCl3, reflux; (ii) morpholine, reflux; (iii) 4 N HCl/AcOEt.
by reaction with morpholine gave 10a and10c, respec-
tively. Compounds 10b, 10e, and 10f were also prepared
from the appropriate methoxyanthranilamides 7a–c,
and 10c was hydrogenated to provide 10d.
cyclization with aqueous sodium hydroxide provided
13a–e, and demethylation of 13a–e, acetylation, chlori-
nation, and subsequent substitution with morpholine
afforded the desired compounds 15a–e, respectively.
The quinazoline derivative 15a, the pyridopyrimidine
derivatives 15b–d, and the thienopyrimidine derivative
15e were prepared as shown in Scheme 3. Acylation of
the amino groups of 11a–e with 3-methoxybenzoyl
chloride provided 12a–e, respectively. Treatment of
12a–e with aqueous ammonium hydroxide followed by
3. Results and discussion
The lead compound 1 had an IC50 value of 1.3 lM for
inhibition of p110a in an enzymatic scintillation proxim-
ity assay (SPA), which was performed using purified

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M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
Table 2. Inhibition of p110a activity by 4-morpholin-4-yl-2-phenyl-
quinazolin-6-ols
bovine p110a. In the SPA method, LY294002 inhibited
p110a with an IC50 value of 0.63 lM, which is consistent
with the IC50 value reported using the conventional
TLC method.20
O
N
HO
N
2
As shown in Table 1, the morpholino group at C4 of 1 is
essential for p110a inhibitory activity; a substantial
decrease in activity was observed with compound 5a,
the analogue of 1 with piperidine at C4. The importance
of the morpholino group is further shown by the
decreased activities of the 4-hydroxypiperidine and
4-piperidone derivatives, 5b and 5c, and compound 5d,
which has a 2-methoxyethylamino group at C4, showed
approximately10-fold lower activitythan 1. Interestingly,
similar SARs for the morpholino group of LY294002
have been reported;20 replacement of the morpholine of
LY294002 with cyclic amines including piperidine and
4-hydroxypiperidine causes a dramatic decrease in the
efficacy of these compounds against PI3K. This result
suggests that the morpholine groups of 1 and LY294002
occupy the same region when bound to PI3K enzymes.
3
R
4
N
a
Compound
R
IC50 (lM)
1
H
2-OH
1.3
2.9
10a
6a
10b
6b
6c
3-OH
4-OH
0.075
2.8
3-OMe
3-F
0.60
1.8
6h
6i
3-CH2OH
4-CH2OH
2-NH2
3-NH2
4-NH2
0.10
3.7
10d
6j
3.9
0.44
0.93
6k
a IC50 values are means of at least two separate determinations with
typical variations of less than 20%.
The effects on p110a inhibitory activity of introducing
substituents onto the phenyl ring at C2 of 1 are shown
in Table 2. While the 2- and 4-hydroxy derivatives,
10a and 10b, were about 2-fold less active than 1, the
3-hydroxy derivative 6a (IC50 value of 0.075 lM) was
about 15-fold more potent than 1. Replacement of the
3-hydroxy group of 6a with a 3-methoxy group or a
3-fluoro group resulted in a decrease in activity (IC50
values of 0.60 and 1.8 lM for 6b and 6c, respectively).
The 4-hydroxymethyl derivative 6i had decreased activity
in comparison with 1 (IC50 value of 3.7 lM), while the
3-hydroxymethyl derivative 6h had an increased activity
(IC50 value of 0.10 lM). With respect to the amino
derivatives, the positional trend was similar to those seen
for the hydroxy and hydroxymethyl derivatives; that is,
the 3-amino derivative 6j was more active than the
2- and 4-amino substituted derivatives, 10d and 6k.
The effect of varying the position of the hydroxy group
on the quinazoline ring of 1 was also investigated, as
shown in Table 3. The 7-hydroxy derivative 10f was
about 8-fold less active than 1, and a further decrease
in activity was observed with the 5-hydroxy derivative
10e. The 6-methoxy analogue 10g was only slightly less
potent than 1, and surprisingly removal of the
6-hydroxy group of 1 resulted in only a 10-fold decrease
in activity (10h: IC50 value of 14 lM), while removal of
the 6-hydroxy group of 6a retained activity (15a: IC50
value of 0.056 lM). These data suggest that the
6-hydroxy group is not essential for p110a inhibitory
activity in analogues with a 3-hydroxyphenyl group at
C2 of quinazoline ring.
Table 1. Inhibition of p110a activity by 4-amino-substituted 6-
hydroxy-2-phenylquinazolines
NR'2
HO
N
N
a
Compound
NR02
O
IC50 (lM)
1
1.3
N
Table 3. Inhibition of p110a activity by 4-morpholin-4-yl-2-phenyl-
quinazolines
5a
5b
>60
12
O
N
N
5
OH
6
N
R
R'
7
N
N
O
a
Compound
R
R0
IC50 (lM)
5c
21
1
6a
6-OH
6-OH
5-OH
7-OH
6-OMe
H
H
1.3
0.075
>30
9.8
N
3-OH
H
10e
10f
10g
10h
15a
MeO
5d
15
NH
H
H
2.1
H
3-OH
14
H
0.056
LY294002
0.63
a IC50 values are means of at least two separate determinations with
typical variations of less than 20%.
a IC50 values are means of at least two separate determinations with
typical variations of less than 20%.

M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
6851
Finally, the effects of exchanging the quinazoline ring of
15e
3
15a with other heterocycles are listed in Table 4. The
activities of these derivatives against A375 tumor cell
proliferation were also evaluated. Regarding the pyrido-
pyrimidine derivatives, the pyrido[3,2-d]pyrimidine 15b,
the pyrido[4,3-d]pyrimidine 15c, and the pyrido[3,4-d]-
pyrimidine 15d all suppressed both p110a activity and
cell proliferation from 2- to 4-fold more than 15a; these
results suggest that the position of the nitrogen atom on
the pyridine ring is not a crucial determinant of p110a
inhibitory activity. Replacement of the quinazoline ring
with thieno[3,2-d]pyrimidine afforded 15e, which
showed the highest activity in this series in both the en-
zyme (IC50 value of 0.0020 lM) and cellular (IC50 value
of 0.58 lM) assays.
0.3
30 (μM)
pPKB
PKB
Figure 2. Effect of 15e on fetal bovine serum-mediated PKB phos-
phorylation in A375 cells. Cells were grown to confluence in 12-well
plates and growth-arrested in serum-free medium overnight. The cells
were then exposed to the indicated concentrations of 15e for 1 h and
subsequently stimulated with fetal bovine serum (FBS) for 15 min. The
cell lysate was subjected to SDS–PAGE and Western blot analysis.
To determine whether 15e inhibits intracellular PI3K in
A375 cells, the effect of 15e on fetal bovine serum-in-
duced PKB phosphorylation was evaluated. As shown
in Figure 2, serum-induced PKB phosphorylation was
suppressed by 15e with an IC50 value of 0.3–3 lM,
which is consistent with the IC50 value in the cell prolif-
eration assay. This result indicates that 15e suppresses
A375 cell proliferation by inhibition of intracellular
PI3K.
Table 5. Inhibition of PI3K isoforms by LY294002 and 15e
a
Compound
IC50 (lM)
p110a
p110b
p110c
PI3K C2b
LY294002
15e
0.63
0.0020
0.34
0.016
1.6
0.66
2.1
0.22
a IC50 values are means of at least two separate determinations with
typical variations of less than 20%.
To check selectivity for p110a, the most active com-
pound 15e and LY294002 were evaluated against
other PI3K isoforms (Table 5). LY294002 exhibited
no selectivity for p110a over p110b, a class Ia PI3K,
and only showed 3- to 4-fold selectivities over p110c
(class Ib) and PI3K C2b (class II). On the other hand,
15e showed about 10-fold selectivity for p110a over
p110b and was >100-fold more selective for p110a
versus p110c and PI3K C2b, showing that 15e is a
p110a inhibitor with greater isoform selectivity than
LY294002. Next, we investigated the selectivity of
15e for p110a over other protein kinases, including
PKA, KDR, PKCa, and cyclin E/CDK2. The inhibi-
tory activities against these protein kinases were 91,
3.4, 466, and 28 lM, respectively, indicating a greater
than 1000-fold selectivity of 15e for p110a over these
kinases.
Table 4. Inhibition of p110a activity and A375 cell proliferation by
3-(4-morpholin 4-ylquinazolin-2-yl)phenol and related heterocycles
O
N
N
Ar
OH
N
a
Compound
Ar
IC50 (lM)
p110a
A375
5.7
4. Conclusion
We have reported the SARs for p110a inhibition by a
series of 4-morpholino-2-phenylquinazolines and related
analogues. The lead compound 1, which was discovered
in our chemical library, is a novel p110a inhibitor with
an IC50 value of 1.3 lM. The morpholino group of 1
is essential for p110a inhibitory activity. Introduction
of a 3-hydroxy group on the phenyl group of 1 resulted
in significantly greater p110a inhibitory activity.
Replacement of the quinazoline ring with a thieno[3,2-
d]pyrimidine ring resulted in 15e, which inhibited
p110a with an IC50 value of 2.0 nM and suppressed
A375 tumor cell proliferation with a submicromolar
IC50. Furthermore, 15e is a highly selective inhibitor
for p110a among other kinases, making it the first exam-
ple of a p110a-selective PI3K inhibitor. Other types of
selective p110a inhibitors will be reported in future
publications.
15a
15b
15c
15d
15e
0.056
N
N
0.027
0.013
0.019
0.0020
3.1
3.5
2.8
0.58
N
S
a IC50 values are means of at least two separate determinations with
typical variations of less than 20% for both the p110a enzyme assay
and the A375 cell proliferation assay.

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M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
5. Experimental
5.1.4. Methyl 3-[6-(acetyloxy)-4-oxo-3,4-dihydroquinazo-
lin-2-yl]benzoate (4d). HCl gas was bubbled through a
solution of methyl 3-cyanobenzoate26 (4.9 g, 30 mmol)
in CHCl3 (50 mL) and EtOH (3mL) for 15 min with
cooling below 10 ꢁC. After standing at 5 ꢁC for 17h,
the reaction mixture was concentrated and the resulting
solid was washed with Et2O to give methyl 3-[eth-
oxy(imino)methyl]benzoate hydrochloride as a colorless
5.1. Chemistry
1H NMR spectra were measured with a JEOL EX400 or
GX500 spectrometer; chemical shifts are expressed in d
units using tetramethylsilane as the standard (in NMR
description, s, singlet; d, doublet; t, triplet; q, quartet,
m, multiplet, and br, broad peak). Mass spectra were
recorded with a Hitachi M-80 or JEOL JMS-DX300
spectrometer. Silica gel column chromatography was
performed by Wakogel C-200 or Merck Silica gel 60.
1
solid (3.2 g, 43%); H NMR (DMSO-d6) d 1.50 (3H, t,
J = 6.9 Hz), 3.92 (3H, s), 4.65 (2H, q, J = 6.9 Hz),
7.76–7.85 (1H, m), 7.29–8.42 (2H, m), 8.55–8.60 (1H,
m); FAB-MS m/e (MH)+ 208.
5.1.1.
3-[6-(Acetyloxy)-4-oxo-3,4-dihydroquinazolin-2-
Compound 4d was prepared from methyl 3-[eth-
oxy(imino)methyl]benzoate hydrochloride and 2 accord-
ing to the same procedure as that for 4a. Compound 4d
yl]phenyl acetate (4a). HCl gas was bubbled through a
solution of 3-hydroxybenzonitrile (3.6 g, 30 mmol) in a
mixture of CHCl3 (50 mL), EtOH (3 mL), and dioxane
(10 mL) with cooling below 10 ꢁC for 15 min. After
standing at 5 ꢁC for 18 h, the resulting solid was collect-
ed and washed with Et2O to give ethyl 3-hydroxyben-
zenecarboximidoate hydrochloride as a light pink solid
(5.3 g, 87%); 1H NMR (DMSO-d6) d 1.47 (3H, t,
J = 6.9 Hz), 4.63 (2H, q, J = 6.9 Hz), 7.20–7.28 (1H,
m), 7.39–7.47 (2H, m), 7.54–7.60 (1H, m), 10.35 (1H,
br s), 11.71 (1H, br s); FAB-MS m/e (MH)+ 166.
1
was obtained as a colorless solid (32% yield); H NMR
(DMSO-d6) d 2.33 (3H, s), 3.92 (3H, s), 7.60–7.76 (2H,
m), 7.81–7.90 (2H, m), 8.11–8.20 (1H, m), 8.38–8.45
(1H, m), 8.75–8.79 (1H, m), 12.82 (1H, br s); FAB-MS
m/e (MH)+ 339.
5.1.5. Methyl 4-[6-(acetyloxy)-4-oxo-3,4-dihydroquinazo-
lin-2-yl]benzoate (4e). To a solution of methyl 4-cya-
nobenzoate (4.8 g, 30 mmol) in EtOH (12 mL) was
added 4 N HCl/AcOEt. After standing at room temper-
ature for 23 h, Et2O was added to the reaction mixture
and the resulting solid was collected to give methyl
4-[ethoxy(imino)methyl]benzoate hydrochloride as a
colorless solid (4.8 g, 66%); 1H NMR (DMSO-d6) d
1.49 (3H, t, J = 6.9 Hz), 3.91 (3H, s), 4.63 (2H, q,
J = 6.9 Hz), 8.13–8.25 (4H, m); FAB-MS m/e (MH)+
208.
A mixture of ethyl 3-hydroxybenzenecarboximidoate
hydrochloride (5.3 g, 26 mmol), 2 (3.1 g, 20 mmol),
and NaOMe (1.1 g, 20 mmol) in MeOH (100 mL) was
refluxed for 0.5 h. After cooling to room temperature,
the reaction mixture was poured into water, and the
resulting solid was collected by filtration, and washed
with CHCl3 to afford crude 6-hydroxy-2-(3-hydroxyphe-
nyl)quinazolin-4(3H)-one. To the obtained gray solid,
NaOAc (120 mg) and Ac2O (36 mL) were added and
the reaction mixture was heated at reflux for 0.5 h. After
cooling to room temperature, the resulting precipitate
was collected by filtration and washed with MeOH to
give 4a (1.2 g, 18%) as a colorless solid; 1H NMR
(DMSO-d6) d 2.33 (6H, s), 7.33–7.41 (1H, m), 7.55–
7.66 (2H, m), 7.76–7.90 (2H, m), 7.94–8.00 (1H, m),
8.04–8.12 (1H, m), 12.65 (1H, br s); FAB-MS m/e
(MH)+ 339.
Compound 4e was prepared from methyl 4-[eth-
oxy(imino)methyl]benzoate hydrochloride and 2 accord-
ing to the same procedure as that for 4a. Compound 4e
1
was obtained as a colorless solid (46% yield); H NMR
(DMSO-d6) d 2.33 (3H, s), 3.91 (3H, s), 7.60–7.68 (1H,
m), 7.79–7.91 (2H, m), 8.07–8.14 (2H, m), 8.27–8.35
(2H, m), 12.76 (1H, br s); FAB-MS m/e (MH)+ 339.
5.1.6. 2-(3-Nitrophenyl)-4-oxo-3,4-dihydroquinazolin-6-yl
acetate (4f). Compound 4f was prepared from ethyl
3-nitrobenzenecarboximidoate hydrochloride27,28 and 2
according to the same procedure as that for 4a. Com-
pound 4a was obtained as a colorless solid (43% yield);
1H NMR (DMSO-d6) d 2.34 (3H, s), 7.61–7.69 (1H, m),
7.81–7.91 (2H, m), 8.39–8.47 (1H, m), 8.57–8.65 (1H,
m), 8.99–9.04 (1H, m), 12.94 (1H, br s); FAB-MS m/e
(MH)+ 326.
5.1.2. 2-(3-Methoxyphenyl)-4-oxo-3,4-dihydroquinazolin-
6-yl acetate (4b). Compound 4c was prepared from ethyl
3-methoxybenzenecarboximidoate hydrochloride25 and
2 according to the same procedure as that for 4a. Com-
pound 4b was obtained as a colorless solid (29% yield);
1H NMR (DMSO-d6) d 2.33 (3H, s), 3.87 (3H, s), 7.12–
7.20 (1H, m), 7.47 (1H, t, J = 7.8 Hz), 7.58–7.65 (1H,
m), 7.72–7.89 (4H, m), 12.61 (1H, br s); FAB-MS m/e
(MH)+ 311.
5.1.7. 2-(4-Nitrophenyl)-4-oxo-3,4-dihydroquinazolin-6-yl
acetate (4g). Compound 4g was prepared from ethyl
4-nitrobenzenecarboximidoate hydrochloride28 and 2
according to the same procedure as that for 4a. Com-
pound 4g was obtained as a pale yellow solid (44%
5.1.3. 2-(3-Fluorophenyl)-4-oxo-3,4-dihydroquinazolin-6-
yl acetate (4c). Compound 4c was prepared from ethyl
3-fluorobenzenecarboximidoate hydrochloride25 and 2
according to the same procedure as that for 4a. Com-
pound 4c was obtained as a colorless solid (13%
yield); 1H NMR (DMSO-d6) d 2.33 (3H, s), 7.40–
7.50 (1H, m), 7.55–7.67 (2H, m), 7.78–7.90 (2H, m),
7.96–8.09 (2H, m), 12.68 (1H, br s); FAB-MS m/e
(MH)+ 299.
1
yield); H NMR (DMSO-d6) d 2.34 (3H, s), 7.62–7.70
(1H, m), 7.81–7.92 (2H, m), 8.40 (4H, s), 12.90 (1H, br
s); FAB-MS m/e (MH)+ 326.
5.1.8. 2-Phenyl-4-piperidin-1-ylquinazolin-6-ol (5a). A
mixture of piperidine (3 mL) and 4-chloro-2-phenylqui-

M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
6853
nazolin-6-yl acetate23 (600 mg, 2.0 mmol) in THF
(10 mL) was refluxed for 3 h. After concentration in vac-
uo, the resulting residue was dissolved in CHCl3, washed
with water and brine, dried over anhydrous MgSO4, and
evaporated. The residue was chromatographed on silica
gel eluting with hexane/AcOEt (3:1) and crystallized
from AcOEt to give 5a (565 mg, 89%) as a colorless
mixture was evaporated and dried in vacuo. The residue
was dissolved in CHCl3, washed with saturated NaH-
CO3, dried over anhydrous Na2SO4, and concentrated
in vacuo. Morpholine (20 mL) was added to the result-
ing residue in toluene (40 mL) and the reaction mixture
was refluxed for 14 h. After evaporation, the residue was
chromatographed on silica gel eluting with CHCl3/
MeOH (15:1) and recrystallized from CHCl3/MeOH/
hexane to give 6a (601 mg, 51%) as a pale yellow solid:
mp 280–284 ꢁC; 1H NMR (DMSO-d6) d 3.64–3.72
(4H, m), 3.82–3.91 (4H, m), 6.83–6.89 (1H, m), 7.25–
7.31 (2H, m), 7.37 (1H, dd, J = 2.4, 8.7 Hz), 7.79 (1H,
d, J = 9.3 Hz), 7.87–7.93 (2H, m), 9.49 (1H, s), 10.12
(1H, s); FAB-MS m/e (MH)+ 324; Anal. Calcd for
C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C,
66.50; H, 5.25; N, 12.97.
solid: mp 260–262 ꢁC; 1H NMR (DMSO-d6)
d
1.65–1.85 (6H, m), 3.60–3.75 (4H, m), 7.23 (1H, d,
J = 2.4 Hz), 7.36 (1H, dd, J = 2.4, 9.3 Hz), 7.41–7.53
(3H, m), 7.77 (1H, d, J = 9.3 Hz), 8.40–8.50 (2H, m),
10.08 (1H, s); FAB-MS m/e (MH)+ 306; Anal. Calcd
for C19H19N3OÆ0.6H2O: C, 72.18; H, 6.44; N, 13.29.
Found: C, 72.23; H, 6.21; N, 13.00.
5.1.9. 4-(4-Hydroxypiperidin-1-yl)-2-phenylquinazolin-6-
ol (5b). Compound 5b was prepared from 4-chloro-2-
phenylquinazolin-6-yl acetate and 4-hydroxypiperidine
according to the same procedure as that for 5a. Com-
pound 5b was obtained as a colorless solid (59% yield):
5.1.13. 2-(3-Methoxyphenyl)-4-morpholin-4-ylquinazolin-
6-ol (6b). Compound 6b was prepared from 4b according
to the same procedure as that for 6a. Compound 6b was
obtained as a colorless solid (59% yield): mp 226–228 ꢁC
1
mp 249–250 ꢁC (AcOEt/hexane); H NMR (DMSO-d6)
1
d 1.55–1.75 (2H, m), 1.90–2.05 (2H, m), 3.20–3.40 (2H,
m), 3.75–3.90 (1H, m), 3.95–4.15 (2H, m), 4.81 (1H, d,
J = 4.4 Hz), 7.24 (1H, d, J = 2.4 Hz), 7.36 (1H, dd,
J = 2.4, 9.6 Hz), 7.42–7.53 (3H, m), 7.77 (1H, d,
J = 9.6 Hz), 8.40–8.50 (2H, m), 10.08 (1H, s); FAB-MS
m/e (MH)+ 322; Anal. Calcd for C19H19N3O2: C, 71.01;
H, 5.96; N, 13.07. Found: C, 71.03; H, 5.97; N, 13.03.
(CHCl3/MeOH/hexane); H NMR (DMSO-d6) d 3.66–
3.71 (4H, m), 3.83–3.88 (7H, m), 7.02–7.08 (1H, m),
7.26 (1H, d, J = 2.4 Hz), 7.35–7.45 (2H, m), 7.81 (1H,
d, J = 9.3 Hz), 7.98–8.01 (1H, m), 8.02–8.07 (1H, m),
10.13 (1H, s); FAB-MS m/e (MH)+ 338; Anal. Calcd
for C19H19N3O3: C, 67.64; H, 5.68; N, 12.45. Found:
C, 67.33; H, 5.65; N, 12.39.
5.1.10. 1-(6-Hydroxy-2-phenylquinazolin-4-yl)piperidin-
4-one (5c). To a solution of piperidin-4-one hydrochlo-
ride (600 mg, 4.4 mmol) and 4-chloro-2-phenylquinazo-
lin-6-yl acetate (650 mg, 2.2 mmol) in DMF (10 mL)
was added NaHCO3 (600 mg, 7.1 mmol) and the reac-
tion mixture was heated at 55 ꢁC for 8 h. After the reac-
tion mixture was concentrated in vacuo, the residue was
diluted with AcOEt and washed with water and brine.
The organic layer was dried over MgSO4 and concen-
trated. The resulting solid was recrystallized from
AcOEt/hexane to give 5c (340 mg, 48%) as a colorless
solid: mp 258–262 ꢁC (dec); 1H NMR (DMSO-d6) d
2.62–2.70 (4H, m), 4.02–4.10 (4H, m), 7.31–7.55 (5H,
m), 7.82 (1H, d, J = 8.8 Hz), 8.42–8.50 (2H, m), 10.14
(1H, s); FAB-MS m/e (MH)+ 318; Anal. Calcd for
C19H17N3O2: C, 71.46; H, 5.37; N, 13.16. Found: C,
71.24; H, 5.45; N, 12.89.
5.1.14. 2-(3-Fluorophenyl)-4-morpholin-4-ylquinazolin-6-
ol (6c). Compound 6c was prepared from 4c according
to the same procedure as that for 6a. Compound 6c
was obtained as a colorless solid (51% yield): mp 270–
1
274 ꢁC (CHCl3/MeOH/hexane); H NMR (DMSO-d6)
d 3.67–3.75 (4H, m), 3.82–3.90 (4H, m), 7.26–7.35 (2H,
m), 7.41 (1H, dd, J = 2.4, 8.8 Hz), 7.51–7.59 (1H, m),
7.83 (1H, d, J = 8.8 Hz), 8.12–8.18 (1H, m), 8.26–8.33
(1H, m), 10.20 (1H, s); FAB-MS m/e (MH)+ 326; Anal.
Calcd for C18H16N3O2F: C, 66.45; H, 4.96; N, 12.92; F,
5.84. Found: C, 66.39; H, 4.96; N, 12.87; F, 5.95.
5.1.15. Methyl 3-(6-hydroxy-4-morpholin-4-ylquinazolin-
2-yl)benzoate (6d). Compound 6d was prepared from 4d
according to the same procedure as that for 6a. Com-
pound 6d was obtained as a pale brown solid (35% yield):
mp 184–186 ꢁC (CHCl3/MeOH/Et2O/hexane); 1H NMR
(DMSO-d6) d 3.67–3.75 (4H, m), 3.83–3.95 (7H, m), 7.28
(1H, d, J = 2.4 Hz), 7.41 (1H, dd, J = 1.5, 8.3), 7.66 (1H,
t, J = 7.8 Hz), 7.86 (1H, d, J = 9.3 Hz), 8.05 (1H, d,
J = 7.9 Hz), 8.70 (1H, d, J = 7.8 Hz), 9.00–9.06 (1H,
m), 10.19 (1H, s); FAB-MS m/e (MH)+ 366; Anal. Calcd
for C20H19N3O4: C, 65.74; H, 5.24; N, 11.50. Found: C,
65.56; H, 5.31; N, 11.62.
5.1.11. 4-[(2-Methoxyethyl)amino]-2-phenylquinazolin-6-ol
(5d). Compound 5d was prepared from 4-chloro-2-phe-
nylquinazolin-6-yl acetate and (2-methoxyethyl)amine
according to the same procedure as that for 5a. Com-
pound 5d was obtained as a colorless solid (400 mg,
62%): mp 212–215 ꢁC (AcOEt/hexane); 1H NMR
(DMSO-d6) d 3.31 (3H, s), 3.67 (2H, t, J = 5.8 Hz),
3.78–3.86 (2H, m), 7.30–7.51 (5H, m), 7.65 (1H, d,
J = 8.8 Hz), 8.04 (1H, br), 8.40–8.50 (2H, m), 9.88
(1H, s); FAB-MS m/e (MH)+ 296; Anal. Calcd for
C17H17N3O2: C, 69.14; H, 5.80; N, 14.23. Found: C,
69.20; H, 5.89; N, 14.28.
5.1.16. Methyl 4-(6-hydroxy-4-morpholin-4-ylquinazolin-
2-yl)benzoate (6e). Compound 6e was prepared from 4e
according to the same procedure as that for 6a. Com-
pound 6e was obtained as a colorless solid (34% yield):
mp 239–241 ꢁC (CHCl3/MeOH/Et2O); 1H NMR
(DMSO-d6) d 3.68–3.74 (4H, m), 3.84–3.92 (7H, m),
7.28 (1H, d, J = 2.9 Hz), 7.41 (1H, dd, J = 2.4, 9.3),
7.84 (1H, d, J = 9.2 Hz), 8.05–8.11 (2H, m), 8.53–8.59
(2H, d, J = 7.8 Hz), 10.23 (1H, s); FAB-MS m/e
5.1.12. 2-(3-Hydroxyphenyl)-4-morpholin-4-ylquinazolin-
6-ol (6a). Compound 4a (1.2 g, 3.6 mmol) in phosphorus
oxychloride (15 mL) was refluxed for 1 h. The reaction

6854
M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
(MH)+ 366; Anal. Calcd for C20H19N3O4: C, 65.74; H,
5.24; N, 11.50. Found: C, 65.63; H, 5.18; N, 11.67.
(30 mL) was added 10% Pd–C (130 mg). After stirring
in a hydrogen atmosphere at room temperature for
2 h, the reaction mixture was filtered through a pad of
Celite and the filtrate was evaporated. The residue was
recrystallized from MeOH/EtOH to give 6j (571 mg,
73%) as a colorless solid: mp 274–277 ꢁC; 1H NMR
(DMSO-d6) d 3.60–3.70 (4H, m), 3.80–3.90 (4H, m),
5.17 (2H, br s), 6.61–6.70 (1H, m), 7.12 (1H, t,
J = 7.8 Hz), 7.25 (1H, d, J = 2.5 Hz), 7.37 (1H, dd,
J = 2.4, 8.8 Hz), 7.60–7.65 (1H, m), 7.69–7.73 (1H, m),
7.76 (1H, d, J = 9.2 Hz), 10.08 (1H, s); FAB-MS m/e
(MH)+ 323; Anal. Calcd for C18H18N4O2: C, 67.07; H,
5.63; N, 17.38. Found: C, 66.94; H, 5.74; N, 17.32.
5.1.17. 4-Morpholin-4-yl-2-(3-nitrophenyl)quinazolin-6-ol
(6f). Compound 6f was prepared from 4f according to
the same procedure as that for 6a. Compound 6f was
obtained as a yellow solid (52% yield): mp 226–227 ꢁC
(CHCl3/toluene); 1H NMR (DMSO-d6) d 3.69–3.78
(4H, m), 3.83–3.92 (4H, m), 7.29 (1H, d. J = 3.0 Hz),
7.42 (1H, dd, J = 2.4, 8.8 Hz), 7.80 (1H, t, J = 7.8 Hz),
7.87 (1H, d, J = 8.8 Hz), 8.29–8.35 (1H, m), 8.81–8.87
(1H, m), 9.14–9.18 (1H, m), 10.25 (1H, br s); FAB-MS
m/e (MH)+ 353; Anal. Calcd for C18H16N4O4Æ0.2H2O:
C, 60.74; H, 4.64; N, 15.74. Found: C, 60.62; H, 4.46;
N, 15.60.
5.1.22. 2-(4-Aminophenyl)-4-morpholin-4-ylquinazolin-6-
ol (6k). Compound 6k was prepared from 6g according
to the same procedure as that for 6j. Compound 6k was
obtained as a pale yellow solid (26% yield): mp 285 ꢁC
5.1.18. 4-Morpholin-4-yl-2-(4-nitrophenyl)quinazolin-6-ol
(6g). Compound 6g was prepared from 4g according to
the same procedure as that for 6a. Compound 6g was
obtained as a yellow solid (76% yield): mp 266–269 ꢁC
1
(MeOH/CHCl3/hexane); H NMR (DMSO-d6) d 3.57–
3.67 (4H, m), 3.80–3.90 (4H, m), 5.51 (2H, br s), 6.59–
6.66 (2H, m), 7.21 (1H, d, J = 3.0 Hz), 7.31 (1H, dd,
J = 2.4, 8.8 Hz), 7.69 (1H, d, J = 8.8 Hz), 8.11–8.18
(2H, m), 9.95 (1H, s); FAB-MS m/e (MH)+ 323; Anal.
Calcd for C18H18N4O2Æ0.1CHCl3: C, 65.03; H, 5.46; N,
16.76. Found: C, 64.93; H, 5.40; N, 16.85.
1
(CHCl3); H NMR (DMSO-d6) d 3.70–3.78 (4H, m),
3.82–3.90 (4H, m), 7.29 (1H, d. J = 2.4 Hz), 7.43 (1H,
dd, J = 2.4, 8.8 Hz), 7.87 (1H, t, J = 8.8 Hz), 8.32–8.38
(2H, m), 8.63–8.70 (2H, m), 10.29 (1H, br s); FAB-MS
m/e (MH)+ 353; Anal. Calcd for C18H16N4O40.2H2O:
C, 60.74; H, 4.64; N, 15.74. Found: C, 60.76; H, 4.54;
N, 15.86.
5.1.23. 6-Methoxy-2-(2-methoxyphenyl)quinazolin-4(3H)-
one (8a). To an ice-cooled solution of 2-amino-5-meth-
oxybenzamide (7b)24 (570 mg, 3.4 mmol) and Et3N
(720 mg, 7.1 mmol) in THF (10 mL) was added 2-meth-
oxybenzoyl chloride (710 mg, 4.2 mmol). After stirring
for 3h at room temperature, the reaction mixture was
concentrated in vacuo and then MeOH (30 mL) and
2 N NaOH (20 mL) were added to the resulting residue.
The reaction mixture was refluxed for 14 h and acidified
with concentrated HCl. The resulting precipitate was
collected by filtration to give 8a (710 mg, 73%) as a col-
5.1.19. 2-[(3-Hydroxymethyl)phenyl]-4-morpholin-4-ylqui-
a solution of 6d (325 mg,
nazolin-6-ol (6h). To
0.89 mmol) in THF (40 mL) was added LiAlH4
(67 mg, 1.8 mmol) at 0 ꢁC and the reaction mixture
was stirred at the same temperature for 2 h. The reaction
was quenched by the addition of water (0.1 mL), 1 N
NaOH (0.1 mL), and water (0.3 mL). The reaction mix-
ture was dried over anhydrous Na2SO4, filtered through
a pad of silica gel, and concentrated in vacuo. The
resulting solid was recrystallized from THF/hexane to
give 6h (158 mg, 52%) as a colorless solid: mp 204–
206 ꢁC; 1H NMR (DMSO-d6) d 3.65–3.75 (4H, m),
3.82–3.94 (4H, m), 4.60 (2H, d, J = 5.8 Hz), 5.28 (1H,
t, J = 5.8 Hz), 7.27 (1H, d, J = 2.5 Hz), 7.35–7.48 (3H,
m), 7.82 (1H, d, J = 9.3 Hz), 8.29–8.36 (1H, m), 8.40–
8.45 (1H, m), 10.13 (1H, s); FAB-MS m/e (MH)+ 338;
Anal. Calcd for C19H19N3O3: C, 67.64; H, 5.68; N,
12.45. Found: C, 67.60; H, 5.77; N, 12.29.
1
orless solid; H NMR (DMSO-d6) d 3.88 (3H, s), 3.92
(3H, s), 7.13 (1H, t, J = 7.8 Hz), 7.24 (1H, d,
J = 8.3 Hz), 7.49–7.63 (3H, m), 7.72 (1H, dd, J = 1.5,
7.8 Hz), 7.80 (1H, d, J = 8.8 Hz); FAB-MS m/e (MH)+
283.
5.1.24. 6-Methoxy-2-(4-methoxyphenyl)quinazolin-4(3H)-
one (8b). Compound 8b was prepared from 7b and
4-methoxybenzoyl chloride according to the same proce-
dure as that for 8a. Compound 8b was obtained as a col-
1
5.1.20. 2-[(4-Hydroxymethyl)phenyl]-4-morpholin-4-ylqui-
nazolin-6-ol (6i). Compound 6i was prepared from 6e
according to the same procedure as that for 6h. Com-
pound 6i was obtained as a colorless solid (50% yield):
mp 260–263 ꢁC; 1H NMR (DMSO-d6) d 3.64–3.74
(4H, m), 3.80–3.91 (4H, m), 4.58 (2H, d, J = 5.4 Hz),
5.26 (1H, t, J = 5.8 Hz), 7.26 (1H, d, J = 2.9 Hz), 7.38
(1H, dd, J = 2.4, 8.8 Hz), 7.44 (2H, d, J = 8.3 Hz), 7.80
(1H, d, J = 9.3 Hz), 8.37–8.45 (2H, m), 10.11 (1H, s);
FAB-MS m/e (MH)+ 338; Anal. Calcd for
C19H19N3O3Æ0.3H2O: C, 66.58; H, 5.76; N, 12.26.
Found: C, 66.36; H, 5.59; N, 12.10.
orless solid (82% yield); H NMR (DMSO-d6) d 3.85
(3H, s), 3.89 (3H, s), 7.08 (2H, d, J = 8.8 Hz), 7.43
(1H, dd, J = 2.9, 8.8 Hz), 7.53 (1H, d, J = 2.9 Hz), 7.68
(1H, d, J = 8.8 Hz), 8.16 (2H, d, J = 8.8 Hz), 12.38
(1H, br s); FAB-MS m/e (MH)+ 283.
5.1.25. 6-Methoxy-2-(2-nitrophenyl)quinazolin-4(3H)-one
(8c). Compound 8c was prepared from 7b and 2-nitro-
benzoyl chloride according to the same procedure as
that for 8a. Compound 8c was obtained as a pale yellow
1
solid (85% yield); H NMR (DMSO-d6) d 3.91 (3H, s),
7.39–8.00 (6H, m), 8.21 (1H, d, J = 7.8 Hz); FAB-MS
m/e (MH)+ 298.
5.1.21. 2-(3-Aminophenyl)-4-morpholin-4-ylquinazolin-6-
ol (6j). To a solution of 6f (860 mg, 2.4 mmol) in a mix-
ture of methanol (30 mL), ethanol (30 mL), and THF
5.1.26. 5-Methoxy-2-phenylquinazolin-4(3H)-one (8e).
Compound 8e was prepared from 7a29 and benzoyl

M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
6855
chloride according to the same procedure as that for 8a.
Compound 8e was obtained as a colorless solid (93%
yield): H NMR (DMSO-d6) d 3.89 (3H, s), 7.02 (1H,
(3H, m), 8.15–8.21 (3H, m), 12.60 (1H, br s); FAB-MS
m/e (MH)+ 281.
1
d, J = 8.3 Hz), 7.26 (1H, d, J = 8.3 Hz), 7.50–7.62 (3H,
m), 7.71 (1H, t, J = 8.3 Hz), 8.15–8.20 (2H, m), 12.20
(1H, br s); FAB-MS m/e (MH)+ 253.
5.1.33. 2-(2-Hydroxyphenyl)-4-morpholin-4-ylquinazolin-
6-ol (10a). Compound 10a was prepared from 9a
according to the same procedure described as that for
6a. Compound 9a was obtained as a colorless solid
1
5.1.27. 7-Methoxy-2-phenylquinazolin-4(3H)-one (8f).30
Compound 8f was prepared from 7c31 and benzoyl chlo-
ride according to the same procedure described for 8a.
Compound 8f was obtained as a colorless solid (75%
(49% yield): mp 234–236 ꢁC (CHCl3/Et2O/hexane); H
NMR (DMSO-d6) d 3.75–3.90 (8H, m), 6.90–6.97 (2H,
m), 7.27–7.44 (3H, m), 7.80 (1H, d, J = 8.8 Hz), 8.41
(1H, dd, J = 1.5, 7.8 Hz), 10.23 (1H, s), 14.20 (1H, s);
FAB-MS m/e (MH)+ 324; Anal. Calcd for
C18H17N3O3: C, 66.86; H, 5.30; N, 13.00. Found: C,
66.75; H, 5.43; N, 12.89.
1
yield): H NMR (DMSO-d6) d 3.92 (3H, s), 7.10 (1H,
dd, J = 2.5, 8.8 Hz), 7.19 (1H, d, J = 2.5 Hz), 7.52–7.62
(3H, m), 8.06 (1H, d, J = 8.8 Hz), 8.16–8.21 (2H, m),
12.41 (1H, br s); FAB-MS m/e (MH)+ 253.
5.1.34. 2-(4-Hydroxyphenyl)-4-morpholin-4-ylquinazolin-
6-ol (10b). Compound 10b was prepared from 9b
according to the same procedure as that for 6a. Com-
pound 10b was obtained as a colorless solid (28%
yield): mp 301–303 ꢁC (EtOH/AcOEt); 1H NMR
(DMSO-d6) d 3.60–3.70 (4H, m), 3.80–3.90 (4H, m),
6.83–6.89 (2H, m), 7.23 (1H, d, J = 2.4 Hz), 7.34 (1H,
dd, J = 2.4, 8.8 Hz), 7.73 (1H, d, J = 8.8 Hz), 8.26–
8.32 (2H, m), 9.78 (1H, s), 10.06 (1H, s); FAB-MS
m/e (MH)+ 324; Anal. Calcd for C18H17N3O30.2H2O:
C, 66.13; H, 5.36; N, 12.85. Found: C, 66.23; H,
5.17; N, 12.67.
5.1.28. 2-[6-(Acetyloxy)-4-oxo-3,4-dihydroquinazolin-2-
yl]phenyl acetate (9a). A mixture of 8a (690 mg,
2.4 mmol), 48% HBr (50 mL), and AcOH (50 mL) was
refluxed for 48 h. The reaction mixture was concentrated
in vacuo. Acetic anhydride (30 mL) and NaOAc
(100 mg) were added to the resulting residue and the
reaction mixture was heated at reflux for 0.5 h. After
concentration, the solid obtained was washed with
methanol and Et2O to give 9a (525 mg, 67%) as a color-
less solid; 1H NMR (DMSO-d6) d 2.18 (3H, s), 2.33 (3H,
s), 7.28–7.33 (1H, m), 7.40–7.46 (1H, m), 7.57–7.64 (2H,
m), 7.72 (1H, d, J = 8.8 Hz), 7.80 (1H, dd, J = 1.5,
7.8 Hz), 7.85 (1H, d, J = 2.5 Hz), 12.57 (1H, br s);
FAB-MS m/e (MH)+ 339.
5.1.35. 4-Morpholin-4-yl-2-(2-nitrophenyl)quinazolin-6-ol
(10c). Compound 10c was prepared from 9c according
to the same procedure as that for 6a. Compound 10c
was obtained as a colorless solid (31% yield): mp 228–
231 ꢁC (AcOEt); 1H NMR (DMSO-d6) d 3.58–3.65
(4H, m), 3.75–3.82 (4H, m), 7.27 (1H, d. J = 2.4 Hz),
7.42 (1H, dd, J = 2.4, 8.8 Hz), 7.67 (1H, dt, J = 1.4,
7.8 Hz), 7.75–7.81 (2H, m), 7.86 (1H, dd, J = 1.0,
7.8 Hz), 8.21 (1H, dd, J = 1.5, 7.8 Hz), 10.26 (1H, s);
FAB-MS m/e (M+H)+ 353; Anal. Calcd for
C18H16N4O4: C, 61.36; H, 4.58; N, 15.90. Found: C,
61.30; H, 4.54; N, 15.99.
5.1.29. 4-[6-(Acetyloxy)-4-oxo-3,4-dihydroquinazolin-2-
yl]phenyl acetate (9b). Compound 9b was prepared from
8b according to the same procedure as that for 9a. Com-
pound 9b was obtained as a colorless solid (74% yield);
1H NMR (DMSO-d6) d 2.32 (3H, s), 2.33 (3H, s), 7.30–
7.36 (2H, m), 7.62 (1H, dd, J = 2.9, 8.8 Hz), 7.79 (1H, d,
J = 8.8 Hz), 7.86 (1H, d, J = 2.9 Hz), 8.19–8.25 (2H, m),
12.65 (1H, br s); FAB-MS m/e (MH)+ 339.
5.1.30. 2-(2-Nitropheyl)-4-oxo-3,4-dihydroquinazolin-6-yl
acetate (9c). Compound 9c was prepared from 8c
according to the same procedure as that for 9a. Com-
pound 9c was obtained as a pale yellow solid (74%
5.1.36. 2-(2-Aminophenyl)-4-morpholin-4-ylquinazolin-6-
ol (10d). Compound 10d was prepared from 10e accord-
ing to the same procedure as that for 6j. Compound 10d
was obtained as a colorless solid (19% yield): mp 245–
246 ꢁC (AcOEt); 1H NMR (DMSO-d6) d 3.58–3.70
(4H, m), 3.82–3.90 (4H, m), 6.55–6.63 (1H, m), 6.76
(1H, dd, J = 1.0, 8.3 Hz), 7.08–7.14 (1H, m), 7.22–7.39
(4H, m), 7.78 (1H, d, J = 8.8 Hz), 8.38 (1H, dd,
J = 1.4, 8.3 Hz), 10.08 (1H, s); FAB-MS m/e (MH)+
323; Anal. Calcd for C18H18N4O2Æ0.1AcOEtÆ0.1H2O:
C, 66.37; H, 5.75; N, 16.83. Found: C, 66.37; H, 5.50;
N, 16.58.
1
yield); H NMR (DMSO-d6) d 2.34 (3H, s), 7.63 (1H,
dd, J = 2.9, 8.8 Hz), 7.72 (1H, d, J = 8.7 Hz), 7.80–
7.96 (4H, m), 8.21–8.25 (1H, m); FAB-MS m/e (MH)+
326.
5.1.31. 4-Oxo-2-phenyl-3,4-dihydroquinazolin-5-yl ace-
tate (9e). Compound 9e was prepared from 8e according
to the same procedure as that for 9a. Compound 9e was
obtained as a colorless solid (92% yield); 1H NMR
(DMSO-d6) d 2.34 (3H, s), 7.15–7.20 (1H, m), 7.53–
7.68 (4H, m), 7.83 (1H, t, J = 8.3 Hz), 8.13–8.18 (2H,
m), 12.48 (1H, br s); FAB-MS m/e (MH)+ 281.
5.1.37. 4-Morpholin-4-yl-2-phenylquinazolin-5-ol (10e).
Compound 10e was prepared from 9e according to the
same procedure as that for 6a. Compound 10e was
5.1.32. 4-Oxo-2-phenyl-3,4-dihydroquinazolin-7-yl ace-
tate (9f). Compound 9f was prepared from 8f according
to the same procedure described as that for 9a. Com-
pound 9f was obtained as a colorless solid (74% yield);
1H NMR (DMSO-d6) d 2.34 (3H, s), 7.30 (1H, dd,
J = 2.2, 8.8 Hz), 7.50 (1H, d, J = 1.9Hz), 7.53–7.64
obtained as a colorless solid (12% yield): mp
153–154 ꢁC (AcOEt/hexane); 1H NMR (DMSO-d6) d
3.60–3.70 (4H, m), 3.75–3.83 (4H, m), 6.88 (1H, dd,
J = 1.0, 7.8 Hz), 7.29 (1H, dd, J = 1.0, 8.3 Hz), 7.46–
7.54 (3H, m), 7.58 (1H, t, J = 7.8 Hz), 8.40–8.48 (2H,
m), 10.73 (1H, s); FAB-MS m/e (M+H)+ 308; Anal.

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M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
Calcd for C18H17N3O2: C, 70.34; H, 5.58; N, 13.67.
Found: C, 70.32; H, 5.47; N, 13.66.
5.1.43. 2-(3-Methoxyphenyl)pyrido[3,2-d]pyrimidin-4(3H)-
one (13b). To a solution of 12b (7.9 g, 28 mmol) in
MeOH (500 mL) was added 28% aqueous NH3
(600 mL). After stirring at room temperature for 16 h,
the reaction mixture was concentrated to one-half of
its initial volume and the resulting solid was collected
to give 3-[(3-methoxybenzoyl)amino]pyridine-2-carbox-
5.1.38. 4-Morpholin-4-yl-2-phenylquinazolin-7-ol (10f).
Compound 10f was prepared from 9f according to the
same procedure as that for 6a. Compound 10f was ob-
tained as a colorless solid (19% yield): mp 245–246 ꢁC
(AcOEt/hexane); 1H NMR (DMSO-d6) d 3.71–3.86
(8H, m), 7.03 (1H, dd, J = 2.4, 8.8 Hz), 7.12 (1H, d,
J = 2.4 Hz), 7.47–7.53 (3H, m), 7.91 (1H, d,
J = 8.8 Hz), 8.42–8.49 (2H, m), 10.49 (1H, s); FAB-MS
m/e (MH)+ 308; Anal. Calcd for C18H17N3O2: C,
70.34; H, 5.58; N, 13.67. Found: C, 70.44; H, 5.49; N,
13.64.
1
amide (6.2 g, 83%): H NMR (DMSO-d6) d 3.32 (3H,
s), 3.86 (3H, s), 7.20–7.28 (1H, m), 7.47–7.58 (3H, m),
7.68 (1H, dd, J = 4.4, 8.6 Hz), 8.13 (1H, br s), 8.38
(1H, dd, J = 1.5, 4.4 Hz), 8.64 (1H, br s), 9.15 (1H, dd,
J = 1.5, 8.6 Hz), 13.35 (1H, br s); FAB-MS m/e (MH)+
272.
To a mixture of 3-[(3-methoxybenzoyl)amino]pyridine-
2-carboxamide (6.2 g, 23 mmol) in 2-PrOH (220 mL)
was added 2 N NaOH (80 mL). After stirring at reflux
for 3 h, the reaction mixture was neutralized with con-
centrated HCl and the resulting precipitate was collected
5.1.39. Methyl 3-[(3-methoxybenzoyl)amino]pyridine-2-
carboxylate (12b). To a mixture of methyl 3-aminopyri-
dine-2-carboxylate (11b)32 (4.9 g, 32 mmol) and Et3N
(5.3 mL, 38 mmol) in CHCl3 (40 mL) was added drop-
wise 3-methoxybenzoyl chloride at 5 ꢁC. After stirring
at room temperature for 2.5 h, the reaction mixture
was diluted with CHCl3 and washed with saturated
NaHCO3 and brine. The organic layer was dried over
Na2SO4 and concentrated in vacuo. The resulting solid
was recrystallized from AcOEt to give 12b (7.9 g, 86%)
1
to give 13b (4.47 g, 77%) as a colorless solid; H NMR
(DMSO-d6) d 3.87 (3H, s), 7.11–7.20 (1H, m), 7.46
(1H, t, J = 8.1), 7.75–7.85 (3H, m), 8.10–8.17 (1H, m),
8.73–8.79 (1H, m), 12.80 (1H, br s); FAB-MS m/e
(MH)+ 254.
1
as a colorless solid; H NMR (CDCl3) d 3.91 (3H, s),
5.1.44. 2-(3-Methoxyphenyl)pyrido[4,3-d]pyrimidin-4(3H)-
one (13c). To a solution of 12c (200 mg, 0.70 mmol) in
MeOH (25 mL) was added 28% aqueous NH3
(30 mL). After stirring at room temperature for 2 h,
the reaction mixture was concentrated in vacuo to give
4.08 (3H, s), 7.11–7.17 (1H, m), 7.41–7.50 (1H, m),
7.53–7.64 (3H, m), 8.44–8.50 (1H, m), 9.33 (1H, dd,
J = 1.5, 8.6 Hz), 11.95 (1H, br s); FAB-MS m/e (MH)+
287.
a
mixture of 13c and uncyclized 4-[(3-meth-
5.1.40. Methyl 4-[(3-methoxybenzoyl)amino]nicotinate
(12c). To a mixture of methyl 4-aminonicotinate
(11c)33 (5.0 g, 33 mmol) and Et3N (5.5 mL, 39 mmol)
in THF (70 mL) was added dropwise 3-methoxybenzoyl
chloride at 0 ꢁC. After stirring at room temperature for
10 min, the reaction mixture was diluted with AcOEt
and water. The aqueous layer was extracted with AcOEt
and the combined organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo to give
oxybenzoyl)amino]nicotinamide. To the obtained crude
mixture, 2-PrOH (6.3 mL) and 2 N NaOH (2.3 mL)
were added and the mixture was heated at reflux for
3 h, then neutralized with 2 N HCl. The resulting precip-
itate was collected and washed with water to give 13c
1
(88 mg, 50%) as a colorless solid; H NMR (DMSO-
d6) d 3.87 (3H, s), 7.15–7.26 (1H, m), 7.38–7.86 (4H,
m), 8.84 (1H, d, J = 5.3 Hz), 9.30 (1H, s), 12.80 (1H,
br s); FAB-MS m/e (M+H)+ 254.
1
12c as a brown solid (8.7 g, 92%); H NMR (CDCl3) d
3.91 (3H, s), 4.02 (3H, s), 7.11–7.17 (1H, m), 7.41–7.49
(1H, m), 7.56–7.63 (2H, m), 8.67 (1H, d, J = 5.9 Hz),
8.81 (1H, d, J = 5.9 Hz), 9.21 (1H, s), 12.10 (1H, br s);
FAB-MS m/e (MH)+ 287.
5.1.45. 2-(3-Methoxyphenyl)pyrido[3,4-d]pyrimidin-4(3H)-
one (13d). Compound 13d was prepared from 12d
according to the same procedure as that for 13c.
Compound 13d was obtained as a colorless solid (67%
1
yield); H NMR (DMSO-d6) d 3.87 (3H, s), 7.01–7.13
5.1.41. Methyl 3-[(3-methoxybenzoyl)amino]isonicotinate
(12d). Compound 12d was prepared from 11d33 accord-
ing to the same procedure as that for 12b. Compound
12d was obtained as a pale yellow solid (75% yield);
1H NMR (CDCl3) d 3.90 (3H, s), 4.02 (3H, s), 7.09–
7.15 (1H, m), 7.41–7.48 (1H, m), 7.55–7.60 (2H, m),
7.85 (1H, d, J = 5.4 Hz), 8.46 (1H, d, J = 4.8 Hz),
10.19 (1H, s), 11.61 (1H, br s); FAB-MS m/e (MH)+ 287.
(1H, m), 7.36–7.46 (1H, m), 7.80–7.98 (3H, m), 8.38–
8.54 (1H, m), 8.93–9.04 (1H, m); FAB-MS m/e (MH)+
254.
5.1.46. 2-(3-Methoxyphenyl)thieno[3,2-d]pyrimidin-4(3H)-
one (13e). Compound 13e was prepared from 12e
according to the same procedure as that for 13b.
Compound 13e was obtained as a colorless solid (91%
1
yield); H NMR (DMSO-d6) d 3.87 (3H, s), 7.10–7.17
5.1.42. Methyl 3-[(3-methoxybenzoyl)amino]thiophene-2-
carboxylate (12e). Compound 12e was prepared from
11e according to the same procedure as that for 12b.
Compound 12e was obtained as a colorless solid (79%
(1H, m), 7.40–7.50 (2H, m), 7.68–7.78 (2H, m), 8.21
(1H, d, J = 5.3 Hz), 12.72 (1H, br s); FAB-MS m/e
(MH)+ 259.
1
yield); H NMR (DMSO-d6) d 3.85 (3H, s), 3.89 (3H,
5.1.47. 3-(4-Oxo-3,4-dihydroquinazolin-2-yl)phenyl ace-
tate (14a). Compound 14a was prepared from 13a23
according to the same procedure as that for 9a. Com-
pound 14a was obtained as a colorless solid (quantita-
s), 7.22–7.29 (1H, m), 7.43–7.59 (3H, m), 8.00 (1H, d,
J = 5.5 Hz), 8.10 (1H, d, J = 5.5 Hz), 10.97 (1H, s);
FAB-MS m/e (MH)+ 292.

M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
6857
1
tive yield); H NMR (DMSO-d6) d 2.33 (3H, s), 7.36–
N, 12.22; Cl, 10.31. Found: C, 62.71; H, 5.16; N,
12.21; Cl, 10.12.
7.42 (1H, m), 7.52–7.65 (2H, m), 7.74–7.79 (1H, m),
7.83–7.91 (1H, m), 7.96–8.00 (1H, m), 8.06–8.12 (1H,
m), 8.14–8.20 (1H, m); FAB-MS m/e (MH)+ 281.
5.1.53. 3-(4-Morpholinopyrido[3,2-d]pyrimidin-2-yl)phe-
nol hydrochloride (15b). Compound 15b was prepared
from 14b according to the same procedure described
for 15a. Compound 15b was obtained as a colorless solid
(47% yield): mp 223–228 ꢁC (MeOH); 1H NMR
(DMSO-d6) d 3.80–3.95 (4H, m), 4.73 (4H, br), 7.05–
7.20 (1H, m), 7.43 (1H, t, J = 7.8 Hz), 7.80–7.85 (1H,
m), 7.91 (1H, d, J = 7.8 Hz), 8.00 (1H, dd, J = 4.0,
8.3 Hz), 8.60 (1H, d, J = 7.8 Hz), 8.91 (1H, dd, J = 1.5,
4.4 Hz), 10.02 (1H, br s); FAB-MS m/e (MH)+ 309;
Anal. Calcd for C17H16N4O2ÆHClÆ0.6H2O: C, 57.42; H,
5.16; N, 15.76; Cl, 9.97. Found: C, 57.57; H, 4.99; N,
15.89; Cl, 9.87.
5.1.48.
3-(4-Oxo-3,4-dihydropyrido[3,2-d]pyrimidin-2-
yl)phenyl acetate (14b). Compound 14b was prepared
from 13b according to the same procedure as that for
9a. Compound 14b was obtained as a colorless solid
(93% yield); 1H NMR (DMSO-d6) d 2.33 (3H, s),
7.30–7.36 (1H, m), 7.57 (1H, t, J = 7.9 Hz), 7.76 (1H,
dd, J = 4.2, 8.3 Hz), 7.98–8.17 (3H, m), 8.69–8.74 (1H,
m); FAB-MS m/e (MH)+ 282.
5.1.49.
3-(4-Oxo-3,4-dihydropyrido[4,3-d]pyrimidin-2-
yl)phenyl acetate (14c). Compound 14c was prepared
from 13c according to the same procedure as that for
9a. Compound 22b was obtained as a colorless solid
(93% yield); 1H NMR (DMSO-d6) d 2.33 (3H, s),
7.36–7.43 (1H, m), 7.51–7.65 (2H, m), 7.99–8.04 (1H,
m), 8.10–8.16 (1H, m), 8.76–8.84 (1H, m), 9.27 (1H, s);
FAB-MS m/e (MH)+ 282.
5.1.54. 3-(4-Morpholinopyrido[4,3-d]pyrimidin-2-yl)phe-
nol hydrochloride (15c). Compound 15c was prepared
from 14c according to the same procedure described
for 15a. Compound 15c was obtained as a colorless solid
(16% yield): mp 261–266 ꢁC (MeOH); 1H NMR
(DMSO-d6) d 3.74–3.93 (4H, m), 4.17–4.32 (4H, m),
7.00–7.13 (1H, m), 7.40 (1H, t, J = 7.8 Hz), 7.85–8.10
(3H, m), 8.79 (1H, d, J = 6.4 Hz), 9.51 (1H, s), 9.90
(1H, br); FAB-MS m/e (MH)+ 309; Anal. Calcd for
C17H16N4O2Æ1.1HClÆ0.3H2O: C, 57.70; H, 5.04; N,
15.83; Cl, 11.02. Found: C, 57.74; H, 5.04; N, 15.83;
Cl, 11.03.
5.1.50.
3-(4-Oxo-3,4-dihydropyrido[3,4-d]pyrimidin-2-
yl)phenyl acetate (14d). Compound 14d was prepared
from 13d according to the same procedure as that
for 9a. Compound 14d was obtained as a colorless
solid (98% yield); 1H NMR (DMSO-d6) d 2.33 (3H,
s), 7.26–7.33 (1H, m), 7.51–7.59 (1H, m), 7.86–7.91
(1H, m), 8.02–8.07 (1H, m), 8.15–8.22 (1H, m), 8.49–
8.54 (1H, s), 9.00–9.03 (1H, m); FAB-MS m/e (MH)+
282.
5.1.55. 3-(4-Morpholinopyrido[3,4-d]pyrimidin-2-yl)phe-
nol hydrochloride (15d). Compound 15d was prepared
from 14d according to the same procedure described
for 15a. Compound 15d was obtained as a colorless solid
(26% yield): mp 269–274 ꢁC (MeOH); 1H NMR
(DMSO-d6) d 3.74–3.92 (4H, m), 4.05–4.23 (4H, m),
7.05 (1H, dd, J = 1.9, 8.3 Hz), 7.39 (1H, t, J = 7.8 Hz),
7.85–7.97 (2H, m), 8.07 (1H, d, J = 5.9 Hz), 8.66 (1H,
d, J = 5.9 Hz), 9.44 (1H, s), 9.83 (1H, br); FAB-MS
m/e (MH)+ 309; Anal. Calcd for C17H16N4O2HCl: C,
59.22; H, 4.97; N, 16.25; Cl, 10.28. Found: C, 59.17;
H, 4.95; N, 16.48; Cl, 10.25.
5.1.51.
3-(4-Oxo-3,4-dihydrothieno[3,2-d]pyrimidin-2-
yl)phenyl acetate (14e). Compound 14e was prepared
from 13e according to the same procedure as that for
9a. Compound 14e was obtained as a colorless solid
(89% yield); 1H NMR (DMSO-d6) d 2.32 (3H, s),
7.34–7.39 (1H, m), 7.48 (1H, d, J = 5.3 Hz), 7.55–7.63
(1H, m), 7.91–7.95 (1H, m), 8.02–8.07 (1H, m), 8.24
(1H, d, J = 5.3 Hz); FAB-MS m/e (MH)+ 287.
5.1.52. 3-(4-Morpholin-4-ylquinazolin-2-yl)phenol hydro-
chloride (15a). Compound 14a (3.5 g, 13 mmol) in phos-
phorus oxychloride (31 mL) was refluxed for 3 h and
concentrated in vacuo. The residue was dissolved in
THF (25 mL) and morpholine (25 mL) was added to
it. After stirring at reflux for 1.5 h, the reaction mixture
was diluted with water and extracted with a mixture of
AcOEt and THF. The organic layer was washed with
brine, dried over anhydrous MgSO4, and concentrated
in vacuo. After chromatography on silica gel eluting
with CHCl3/MeOH (96:4), the residue was dissolved in
a mixture of THF (18 mL) and methanol (18 mL).
HCl/AcOEt (4 N, 0.63 mL) was added to the solution
and the mixture was concentrated and recrystallized
from MeOH to give 15a (1.37 g, 31%) as a colorless sol-
5.1.56. 3-(4-Morpholinothieno[3,2-d]pyrimidin-2-yl)phe-
nol hydrochloride (15e). Compound 15e was prepared
from 14e according to the same procedure described
for 15a. Compound 15e was obtained as a colorless solid
(57% yield): mp 207–210 ꢁC (MeOH); 1H NMR
(DMSO-d6) d 3.75–3.92 (4H, m), 4.05–4.28 (4H, m),
7.05 (1H, dd, J = 2.0, 7.9 Hz), 7.40 (1H, t, J = 7.8 Hz),
7.73 (1H, d, J = 5.8 Hz), 7.80 (1H, s), 7.86 (1H, d,
J = 7.8 Hz), 8.51 (1H, d, J = 5.3 Hz), 9.90 (1H, br);
FAB-MS m/e (M+H)+ 314; Anal. Calcd for
C16H15N3O2SÆHCl: C, 54.93; H, 4.61; N, 12.01; S, 9.17;
Cl, 10.13. Found: C, 54.78; H, 4.48; N, 12.02; S, 9.07;
Cl, 10.04.
1
id: mp 214–220 ꢁC; H NMR (DMSO-d6) d 3.75–3.95
5.2. Scintillation proximity assay (SPA) for p110a
(4H, m), 4.15–4.35 (4H, m), 7.10–7.20 (1H, m), 7.40–
7.50 (1H, m), 7.63–7.72 (1H, m), 7.82 (1H, s), 7.90
(1H, d, J = 7.8 Hz), 7.96–8.05 (1H, m), 8.16–8.30
(2H, m), 10.10 (1H, br s); FAB-MS m/e (MH)+ 308;
Anal. Calcd for C18H17N3O2ÆHCl: C, 62.88; H, 5.28;
Bovine p110a was expressed in an Sf9/Baculovirus
system and purified as a GST-fusion protein. The test
compounds dissolved in DMSO (0.5 lL) and p110a en-
zyme were mixed in 25 lL of buffer solution (20 mM

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M. Hayakawa et al. / Bioorg. Med. Chem. 14 (2006) 6847–6858
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1 lg PI (Sigma), 0.125 lCi [c-33P]ATP (Amersham
Pharmacia), and 1 lM non-radiolabeled ATP (Sigma)
were added to the mixture to initiate the reaction. After
the reaction had proceeded at room temperature for
120 min, 0.2 mg of wheat germ agglutinin-coated SPA
beads (Amersham) in 150 lL PBS was added. The mix-
ture was left to stand for 5 min and then centrifuged at
300g for 2 min. The radioactivity was measured using
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means of at least two separate determinations with typ-
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