Synthesis and biological evaluation of imidazo[1,2-a]pyridine derivatives as novel PI3 kinase p110α inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.09.047

3-{1-[(4-Fluorophenyl)sulfonyl]-1H-pyrazol-3-yl}-2-methylimidazo[1,2-a]pyridine, 2a, was discovered in our chemical library as a novel p110α inhibitor with an IC₅₀ of 0.67 μM, through screening in a scintillation proximity assay. Optimization o

Full text of DOI:10.1016/j.bmc.2006.09.047

Bioorganic & Medicinal Chemistry 15 (2007) 403–412
Synthesis and biological evaluation of imidazo[1,2-a]pyridine
derivatives as novel PI3 kinase p110a inhibitors
Masahiko Hayakawa,a,* Hiroyuki Kaizawa,a Ken-ichi Kawaguchi,a Noriko Ishikawa,a
Tomonobu Koizumi,a Takahide Ohishi,a Mayumi Yamano,a Minoru Okada,a
Mitsuaki Ohta,a Shin-ichi Tsukamoto,a Florence I. Raynaud,b Michael D. Waterfield,c
Peter Parkerd and Paul Workmanb
aInstitute for Drug Discovery Research, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan
bCancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey SN2 5NG, UK
cLudwig Institute for Cancer Research, Gower Street, London WC1E 6BT, UK
dCancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
Received 1 August 2006; revised 21 September 2006; accepted 22 September 2006
Available online 16 October 2006
Abstract—3-{1-[(4-Fluorophenyl)sulfonyl]-1H-pyrazol-3-yl}-2-methylimidazo[1,2-a]pyridine, 2a, was discovered in our chemical
library as a novel p110a inhibitor with an IC50 of 0.67 lM, through screening in a scintillation proximity assay. Optimization of
the substituents of 2a increased the p110a inhibitory activity by more than 300-fold (2g: IC50 = 0.0018 lM). Further structural mod-
ification of 2g afforded thiazole derivative 12, which has potent p110a inhibitory activity (IC50 of 0.0028 lM) and is highly selective
for p110a over other PI3K isoforms. Compound 12 also inhibited serum-induced cell proliferation of A375 and HeLa cells in vitro
with IC50 values of 0.14 lM and 0.21 lM, respectively, and suppressed tumor growth by 37% in a mouse HeLa xenograft model
when dosed intraperitoneally at 25 mg/kg. These results suggest that selective p110a inhibitors may have potential as cancer
therapeutic agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
include p110a, p110b, and p110d, are activated by recep-
tor tyrosine kinases and are thought to play crucial roles
in cell proliferation via the growth factor-tyrosine kinase
pathway.11 The PIK3CA gene that encodes p110a is
amplified and overexpressed in ovarian and other can-
cers,12,13 and is also frequently mutated in many differ-
ent cancers.14–17 Thus, class Ia PI3Ks, and particularly
p110a, are potential therapeutic targets for cancer, and
inhibitors of these enzymes are expected to be useful
in cancer treatment.
Phosphoinositide 3-kinase (PI3K) is an enzyme that cat-
alyzes phosphorylation of the 3-hydroxyl position of
phosphatidylinositides (PIs), and the resulting 3-phos-
phorylated phospholipids are known to activate the cell
survival kinase PKB/Akt, leading to cell proliferation
and survival.1–4 Negative regulation of PI3K signaling
is mediated by PTEN, a lipid-phosphatase that dephos-
phorylates PI3K products. Loss of PTEN protein or
function has been found in a large number of human
cancers5,6 and mutation of PTEN is one of the most
common mutations in human cancers.7
Reported PI3K inhibitors include the fungal metabolite
wortmannin and the flavonoid-related compound
LY294002. Although wortmannin is a potent PI3K
inhibitor with an IC50 in the low nanomolar range, it
has low in vitro anti-tumor activity, probably due to
instability.18,19 LY294002 is more stable, but it is a rela-
tively weak PI3K inhibitor with an IC50 of 0.63 lM.20
Furthermore, both wortmannin and LY294002 exhibit
no selectivity among PI3K isoforms, and therefore the
discovery of isoform-specific and therapeutically useful
p110a inhibitors is an important goal.21,22 We have
The family of PI3K enzymes8–10 is divided into three
classes: class I, II, and III; and class I is further sub-
classified into classes Ia and Ib. Class Ia PI3Ks, which
Keywords: PI3 kinase; p110a; Inhibitor; Cancer treatment.
Corresponding author. Tel.: +81 029 865 7124; fax: +81 029 847
8313; e-mail: masahiko.hayakawa@jp.astellas.com
*
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.047

404
M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
found that the thieno[3,2-d]pyrimidine derivative 1 is a
O
O
O
O
highly selective p110a inhibitor that potently inhibits tu-
mor cell proliferation in vitro.23 However, the PK profile
of 1 was considered to be insufficient for inhibitory
activity of tumor cell growth in vivo, since the half-life
of 1 was less than 10 min when dosed intraperitoneally
in mice. Therefore, to obtain a potent p110a inhibitor
that is effective in vivo, we have focused our efforts on
structural modification of another type of p110a inhibi-
tor, 2a, which was discovered in our chemical library
and found to inhibit p110a with an IC50 of 0.67 lM.
We now report the synthesis and evaluation of novel
imidazo[1,2-a]pyridine derivatives based on 2a, and
show that these molecules are potent and selective
p110a inhibitors.
MeO
O
N
O
O
H
O
O
O
LY294002
wortmannin
N
N
O
N
N
N
S
N
N
OH
S O
O
F
2. Chemistry
2a
1
As shown in Scheme 1, the sulfonyl pyrazole deriva-
tives 2a–g were prepared from the 1-(imidazo[1,2-
a]pyridin-3-yl)ethanones 3a–d. Condensation of 3a–d
Figure 1. Structure of PI3K inhibitors.
N
N
N
Y
Y
N
Y
a
b
N
X
N
X
X
N
O
N
Me
N
N
S O
H
Ar
O
3a: X = H, Y = Me
3b: X = Cl, Y = Me
3c: X = Br, Y = Me
3d: X = Br, Y = H
4a: X = H, Y = Me
4b: X = Cl, Y = Me
4c: X = Br, Y = Me
4d: X = Br, Y = H
2a: X = H, Y = Me, Ar = 4-fluorophenyl
2b: X = Cl, Y = Me, Ar = 4-fluorophenyl
2c: X = Cl, Y = Me, Ar = 4-nitrophenyl
2d: X = Cl, Y = Me, Ar = 3-nitrophenyl
2e: X = Cl, Y = Me, Ar = 2-methyl-5-nitrophenyl
2f: X = Br, Y = Me, Ar = 2-methyl-5-nitrophenyl
2g: X = Br, Y = H, Ar = 2-methyl-5-nitrophenyl
Scheme 1. Reagents and conditions: (a) (i) Me2NCH(OMe)2, reflux; (ii) H2NNH2 hydrate, EtOH, reflux; (b) substituted benzenesulfonyl chloride,
pyridine.
N
N
N
b
a
N
N
Cl
N
Cl
CO2Me
Cl
O
H
N
CO2Me
H
3f
6
5
N
N
N
Cl
c
d
N
Cl
O2N
N
N
S
O
H
O
7
8
Scheme 2. Reagents and conditions: (a) K2CO3, (EtO)2P(O)CH2CO2Me, DMF; (b) NaH, TosMIC, DMSO/Et2O; (c) (i) KOH, MeOH/H2O;
(ii) 2-aminoethanol, D; (d) NaH, 2-methyl-5-nitrobenzenesulfonyl chloride, THF.

M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
405
with 1,1-dimethoxy-N,N-dimethylmethanamine and
cyclization of the resulting enamines with hydrazine
hydrate gave pyrazole derivatives 4a–d. Sulfonylation
of 4a–d with the appropriate arylsulfonyl chlorides
afforded 2a–g.
treated with sulfonyl chloride to provide the desired pyr-
role derivative 8.
The thiazole derivatives 11 and 12 were synthesized as
shown in Scheme 3. The ketone 3e was converted to
the a-bromoketone 9 via bromination under acidic con-
ditions. Reaction of 9 with ammonium dithiocarbamate
in methanol followed by cyclization in acetic acid gave
the thiol 10. A subsequent coupling reaction of 10 with
a diazonium salt afforded sulfide derivative 11,26 which
was oxidized with 30% H2O2 in acetic acid to give the
desired sulfone derivative 12.
The pyrrole derivative 8 was synthesized from the alde-
hyde 3f, as shown in Scheme 2. Horner–Emmons olefin-
ation of 3f gave the ester 5, which was treated with
TosMIC in the presence of NaH to give the cyclized
product 6.24 Hydrolysis and subsequent decarboxylation
by heating in 2-aminoethanol afforded 7,25 which was
N
N
b
N
N
a
Cl
N
N
Cl
Cl
N
S
O
O
Br
Me
HS
9
3e
10
N
N
N
N
Cl
Cl
c
d
N
N
S
S
O2N
O2N
S
S
O
O
11
12
Scheme 3. Reagents and conditions: (a) Br2, HBr/AcOH; (b) (i) NH4H2NCS2, MeOH; (ii) AcOH, reflux; (c) NaH, DMSO, then 2-methyl-5-
nitrobenzenediazonium tetrafluoroborate; (d) 30% H2O2, AcOH, D.
N
N
N
N
N
Cl
Cl
N
b
a
Cl
N
N
S
O2N
S
O2N
N
S
S
X
HS
CO2Et
13
CH2OH
10
14a: X = S
14b: X = SO
c
N
N
N
N
Cl
O2N
Cl
d
N
N
S
S
O2N
S
S
O
O
CH2OH
14b
15
Scheme 4. Reagents and conditions: (a) ethyl 2-fluoro-4-nitrobenzoate, NaH, DMF, D; (b) DIBAL-H, CH2Cl2; (c) m-CPBA, CH2Cl2; (d) (i) MsCl,
Et3N, THF; (ii) NaBH4, DMSO.

406
M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
The sulfoxide derivatives 14b and 15 were prepared from
the thiol 10, as shown in Scheme 4. Displacement of the
fluorine in ethyl 2-fluoro-4-nitrobenzoate with 10 and
subsequent reduction of the ester group with DIBAL-
H gave the sulfide derivative 14a. Oxidation of 14a with
m-CPBA gave sulfoxide 14b, and mesylation and subse-
quent reduction of 14b with NaBH4 in DMSO gave
15.27
Scheme 5. Cyclization of commercially available 16a
and 16b with 3-chloropentane-2,4-dione in refluxing
EtOH gave the 2-methylimidazo[1,2-a]pyridines 3b and
3c. Compound 3d and 3e, which have no substituent
at C2, were synthesized by condensation of 16b and
16a with dimethylformamide dimethylacetal followed
by treatment with bromoacetone.28 Cyclization of 16a
with bromomalonaldehyde gave the aldehyde 3f.
The ketones 3b–e and the aldehyde 3f, which were used
as starting materials, were synthesized as depicted in
3. Results and discussion
The lead compound, 2a, had an IC50 of 0.67 lM for
inhibition of p110a in an enzymatic scintillation proxim-
ity assay (SPA). Under our assay conditions, LY294002
inhibited p110a with an IC50 of 0.63 lM, which was
comparable with that of 2a.
N
NH2
a
Me
N
N
X
X
O
Me
16a: X = Cl
16b: X = Br
3b: X = Cl
3c: X = Br
As shown in Table 1, inhibitory activity against p110a
was retained with introduction of a chloro group into
the imidazopyridine ring of 2a at C6 (2b: IC50
=
N
N
0.76 lM). Replacement of the electron-withdrawing
fluorine in 2b with a nitro group resulted in a loss of
p110a activity (2c: IC50 > 30 lM), while the 3-nitro
derivative 2d exhibited 2-fold more potent activity than
2a and 2b. A dramatic increase in p110a inhibitory
activity was achieved by modification of the benzene
ring of 2d: compound 2e, which has a methyl group
on the phenyl ring of 2d, was 100-fold more potent
against p110a (2e: IC50 = 0.0028 lM) and also showed
potent inhibitory activity against A375 cell proliferation
(IC50 = 0.83 lM). This suggests that interactions of the
methyl group and the oxygen of the sulfone play an
extremely important role in p110a inhibition. The
inhibitory activities against p110a and cell proliferation
were retained with substitution of the chloro group on
the imidazopyridine of 2e with a bromo group (2f:
IC50 against p110a = 0.0031 lM; IC50 against A375
NH2
N
b
X
X
O
Me
16a: X = Cl
16b: X = Br
3e: X = Cl
3d: X = Br
N
N
NH2
N
c
X
X
O
H
3f: X = Cl
16a: X = Cl
Scheme 5. Reagents and conditions: (a) 3-chloropentane-2,4-dione,
EtOH, reflux; (b) (i) Me2NCH(OMe)2, reflux; (ii) bromoacetone,
MeOH; (c) BrCH(CHO)2, MeCN, D.
Table 1. Inhibition of p110a activity of 3-(1H-pyrazol-3-yl)imidazo[1,2-a]pyridine derivatives
N
R'
N
R
X
N
Ar S O
O
c
Compound
R
R0
X
Ar
IC50 (lM)
p110a
A375
LY294002a
0.63
0.67
8.4
23
2aa
2ba
2ca
2da
2ea
2fa
2ga
8b
H
Me
Me
Me
Me
Me
Me
H
N
4-Fluorophenyl
4-Fluorophenyl
Cl
Cl
Cl
Cl
Br
Br
Cl
N
0.76
>30
NT
NT
NT
0.83
0.73
0.17
12
N
4-Nitrophenyl
3-Nitrophenyl
N
0.28
N
2-Methyl-5-nitrophenyl
2-Methyl-5-nitrophenyl
2-Methyl-5-nitrophenyl
2-Methyl-5-nitrophenyl
0.0028
0.0031
0.0018
0.10
N
N
CH
H
a Free base.
b HCl salt.
c IC50 values represent means of at least two separate determinations with typical variations of less than 20% both for p110a enzyme and A375 cell
proliferation assays.

M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
Table 3. Inhibition of PI3K isoforms by LY294002 and 12
407
cells = 0.73 lM). Removal of the methyl group at C2 on
the imidazopyridine ring further increased inhibitory
activities against p110a and cell proliferation (2g: IC50
against the enzyme = 0.0018 lM; IC50 against cell
growth = 0.17 lM). Notably, the pyrrole derivative 8
was much less potent than the pyrazole derivatives 2e
or 2g, indicating that a nitrogen at the 2-position on
the pyrazole ring is crucial for p110a inhibitory activity.
c
Compound
IC50 (lM)
p110a
p110b p110c
PI3K C2b
LY294002a
12b
0.63
0.0028
0.34
0.17
1.6
0.23
2.1
0.10
a Free base.
b HCl salt.
c IC50 values represent means of at least two separate determinations
with typical variations of less than 20%.
The pyrazole derivatives, including 2e, 2f, and 2g, were
extremely potent p110a inhibitors, as described above;
however, they were found to be unstable in solution,
in which they degraded into the desulfonylated pyrazole
and benzenesulfonic acid. Furthermore, they were not
effective in vivo in xenograft models. Thus, our efforts
shifted to obtaining more stable thiazole derivatives
such as 11, 12, and 15, which have a carbon-sulfone
linkage instead of a nitrogen-sulfone linkage.
700
600
500
400
300
200
100
0
Vehicle
Compound 12, 25 mg/kg
The results for the thiazole derivatives are shown in
Table 2. The sulfide derivative 11 was approximately
45-fold less potent than the pyrazole derivative 2g, and
the sulfoxide 15 was about 2-fold more potent than
the sulfide 11. A further increase in p110a activity was
observed for the sulfone derivative 12, which exhibited
almost the same potencies to 2g in the enzyme (IC50 of
0.0028 lM) and cellular (IC50 of 0.14 lM) assays. This
result suggests that interactions with the oxygen of the
sulfone and the methyl group on the benzene ring are
also important for exerting potent p110a inhibitory
activity in thiazole derivatives. The sulfoxide derivative
14b, which has a hydroxymethyl group, also showed
more potent p110a inhibitory activity (IC50 of
0.020 lM) than the corresponding methyl derivative
15, however it did not show cellular activity.
0
7
14
Time (days)
Figure 2. HeLa (human cervical cancer cell line) tumor growth during
treatment with compound 12 (HCl salt). Compound 12 (25 mg/kg) was
intraperitoneally administered daily for 2 weeks to HeLa xenograft
nude mice. Compound 12 was suspended in 20% hydroxypropyl-b-
cyclodextrin/saline. Tumor growth was suppressed by 37% at 2 weeks
after the start of treatment with 12 compared with vehicle alone
(p = 0.060)
showed no selectivity between p110a and p110b (class
Ia) and only 3- to 4-fold selectivity for p110a over
p110c (class Ib) and PI3K C2b (class II). In contrast,
12 showed about 60-fold selectivity for p110a
(IC50 = 0.0028 lM) over p110b (IC50 = 0.17 lM) and
was approximately 80-fold and 35-fold more selective
for p110a versus p110c (IC50 = 0.23 lM) and PI3K
C2b (IC50 = 0.10 lM), respectively. These data show
that 12 is a highly isoform-selective p110a inhibitor
(Fig. 1).
Compound 12 was evaluated further, since it was the
most potent p110a inhibitor in thiazole derivatives. To
check selectivity for p110a, 12 and LY294002 were eval-
uated against other PI3K isoforms (Table 3). LY294002
Table 2. Inhibition of p110a activity of 3-(1,3-thiazol-4-yl)imi-
dazo[1,2-a]pyridine derivatives
N
Finally, the in vivo activity of 12 on tumor growth was
evaluated in mice carrying a HeLa tumor xenograft. In
HeLa cells, 12 showed antiproliferative activity in vitro
(IC50 = 0.21 lM) comparable to that observed in A375
cells. Furthermore, the half-life of 12, as measured by
HPLC/MS/MS, was 2.6 h when administered intraperi-
toneally in mice, and 12 suppressed tumor growth by
37% in HeLa xenograft mice when dosed intraperitone-
ally at 25 mg/kg once daily for 2 weeks (Fig. 2). No sig-
nificant weight loss was observed in these mice.
N
R
N
S
O2N
X
R'
Compounda
R
–X–
R0
IC50 (lM)
b
p110a
A375
11
15
Cl
Cl
Cl
Cl
–S–
Me
Me
Me
0.082
0.031
0.0028
0.020
3.37
0.27
0.14
19
–SO–
–SO2–
–SO–
12
14b
CH2OH
4. Conclusion
a HCl salt.
b IC50 values represent means of at least two separate determinations
with typical variations of less than 20% both for p110a enzyme and
A375 cell proliferation assays.
Structure–activity relationships for PI3K p110a inhibi-
tion were examined in a series of imidazo[1,2-a]pyridine
compounds, among which the thiazole derivative 12

408
M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
showed potent p110a inhibitory activity and strong
selectivity for p110a over other PI3K isoforms. Com-
pound 12 also inhibited tumor cell growth both in vitro
and in vivo, suggesting that PI3K p110a is a potential
target in cancer treatment.
J = 9.3 Hz), 8.31–8.37 (2H, m), 8.45–8.51 (2H, m),
8.80 (1H, d, J = 2.9 Hz), 9.07–9.10 (1H, m); FAB
MS m/e (M+H)+ 418; Anal. Calcd for C17H12-
N5O4SCl: C, 48.87; H, 2.89; N, 16.76; S, 7.67; Cl,
8.48. Found: C, 48.90; H, 2.81; N, 16.76; S, 7.65; Cl,
8.45.
5. Experimental
5.1. Chemistry
5.1.4. 6-Chloro-2-methyl-3-{1-[(3-nitrophenyl)sulfonyl]-
1H-pyrazol-3-yl}imidazo[1,2-a]pyridine (2d). Compound
2d was prepared from 4b and 3-nitrobenzenesulfonyl
chloride according to the same procedure as that of 2a.
Compound 2d was obtained as a colorless solid (41%
1H NMR spectra were recorded on a JEOL EX400 or
GX500 spectrometer; chemical shifts are expressed in d
units using tetramethylsilane as the standard (in the
description of the NMR signals, s, singlet; d, doublet;
t, triplet; q, quartet; m, multiplet; and br, broad peak).
Mass spectra were recorded on a Hitachi M-80 or
JEOL JMS-DX300 spectrometer. Silica gel column
chromatography was performed with Wakogel C-200
or Merck Silica gel 60.
1
yield). Mp: 217–218 ꢁC (pyridine); H NMR (DMSO-
d6) d: 2.54 (3H, s), 7.13 (1H, d, J = 2.9 Hz), 7.45 (1H,
dd, J = 2.0, 9.3 Hz), 7.66 (1H, d, J = 9.3 Hz), 8.02 (1H,
t, J = 8.3 Hz), 8.49–8.54 (1H, m), 8.60–8.65 (1H, m),
8.73–8.76 (1H, m), 8.83 (1H, d, J = 3.0 Hz), 9.09–9.12
(1H, m); FAB MS m/e (M+H)+ 418; Anal. Calcd for
C17H12N5O4SCl: C, 48.87; H, 2.89; N, 16.76; S, 7.67; Cl,
8.48. Found: C, 48.89; H, 2.70; N, 16.80; S, 7.67; Cl, 8.45.
5.1.1. 3-{1-[(4-Fluorophenyl)sulfonyl]-1H-pyrazol-3-yl}-
2-methylimidazo[1,2-a] pyridine (2a). To a suspension
of 2-methyl-3-(1H-pyrazol-3-yl)imidazo[1,2-a]pyridine
4a (1.8 g, 9.7 mmol) in pyridine (7.1 mL, 88 mmol) was
5.1.5. 6-Chloro-2-methyl-3-{1-[(2-methyl-5-nitrophenyl)-
sulfonyl]-1H-pyrazol-3-yl}imidazo[1,2-a]pyridine (2e).
Compound 2e was prepared from 4b and 2-methyl-5-
nitrobenzenesulfonyl chloride according to the same
procedure as that of 2a. Compound 2e was obtained
as a yellow solid (34% yield). Mp: 207–208 ꢁC (Et2O);
1H NMR (DMSO-d6) d: 2.55 (3H, s), 2.76 (3H, s),
7.13 (1H, d, J = 2.9 Hz), 7.38–7.43 (1H, m), 7.61–7.66
(1H, m), 7.84 (1H, d, J = 8.8 Hz), 8.54 (1H, dd,
J = 2.4, 8.3 Hz), 8.83 (1H, d, J = 2.5 Hz), 8.90 (1H, d,
J = 2.9 Hz), 8.94–8.98 (1H, m); FAB MS m/e (M+H)+
432; Anal. Calcd for C18H14N5O4SCl: C, 50.06; H,
3.27; N, 16.22; S, 7.43; Cl, 8.21. Found: C, 50.07; H,
3.27; N, 16.07; S, 7.54; Cl, 8.19.
added
4-fluorobenzenesulfonyl
chloride
(1.9 g,
9.7 mmol). After stirring at reflux for 1 h, the reaction
mixture was evaporated, diluted with brine, and extract-
ed with CHCl3. The organic layer was dried over MgSO4
and evaporated. The residue was chromatographed on
silica gel eluting with CHCl3/MeOH (100:1–50:1), and
the obtained solid was washed with a mixture of CHCl3
and hexane to give 2a (2.4 g, 78%) as a colorless solid.
1
Mp: 159–160 ꢁC; H NMR (DMSO-d6) d: 2.55 (3H, s),
7.06 (1H, d, J = 3.0 Hz), 7.13 (1H, dd, J = 1.0, 6.8 Hz),
7.36–7.42 (1H, m), 7.51–7.64 (3H, m), 8.18–8.25 (2H,
m), 8.70 (1H, d, J = 3.0 Hz), 9.10–9.16 (1H, m); FAB
MS m/e (M+H)+ 357; Anal. Calcd for C17H13N4O2SF:
C, 57.29; H, 3.68; N, 15.72; S, 9.00; F, 5.33. Found: C,
56.96; H, 3.68; N, 15.68; S, 9.00; F, 5.37.
5.1.6. 6-Bromo-2-methyl-3-{1-[(2-methyl-5-nitrophenyl)-
sulfonyl]-1H-pyrazol-3-yl} imidazo[1,2-a]pyridine (2f).
Compound 2f was prepared from 4c and 2-methyl-5-
nitrobenzenesulfonyl chloride according to the same
procedure as that of 2a. Compound 2f was obtained
as a yellow solid (12% yield). Mp: 202–203 ꢁC (Et2O);
1H NMR (DMSO-d6) d: 2.55 (3H, s), 2.76 (3H, s),
7.13 (1H, d, J = 2.9 Hz), 7.47 (1H, dd, J = 1.9, 9.3 Hz),
7.58 (1H, d, J = 9.3 Hz), 7.85 (1H, d, J = 8.3 Hz), 8.55
(1H, dd, J = 2.4, 8.3 Hz), 8.83 (1H, d, J = 2.5 Hz), 8.89
(1H, d, J = 2.9 Hz), 9.06 (1H, d, J = 2.5 Hz); FAB MS
m/e (M+H)+ 476, 478; Anal. Calcd for C18H14N5O4SBr:
C, 45.39; H, 2.96; N, 14.70; S, 6.73; Br, 16.78. Found: C,
45.27; H, 2.84; N, 14.67; S, 6.82; Br, 16.65.
5.1.2. 6-Chloro-3-{1-[(4-fluorophenyl)sulfonyl]-1H-pyra-
zol-3-yl}-2-methylimidazo[1,2-a]pyridine (2b). Compound
2b was prepared from 4b and 4-fluorobenzenesulfonyl
chloride according to the same procedure as that of
2a. Compound 2b was obtained as a colorless solid
1
(84% yield). Mp: 195–196 ꢁC (CHCl3/Et2O); H NMR
(DMSO-d6) d: 2.58 (3H, s), 7.09 (1H, d, J = 2.9 Hz),
7.44 (1H, dd, J = 2.0, 9.8 Hz), 7.54–7.68 (3H, m),
8.14–8.22 (2H, m), 8.73 (1H, d, J = 2.9 Hz), 9.06–9.09
(1H, m); FAB MS m/e (M+H)+ 391; Anal. Calcd for
C17H12N4O2SFCl: C, 52.25; H, 3.09; N, 14.34; S, 8.20;
Cl, 9.07, F, 4.86. Found: C, 51.88; H, 2.98; N, 14.25;
S, 8.20; Cl, 9.05, F, 4.73.
5.1.7. 6-Bromo-3-{1-[(2-methyl-5-nitrophenyl)sulfonyl]-
1H-pyrazol-3-yl}imidazo[1,2-a]pyridine (2g). Compound
2g was prepared from 4d and 2-methyl-5-nitro-
benzenesulfonyl chloride according to the same proce-
dure as that of 2a. Compound 2g was obtained as a
colorless solid (16% yield). Mp: 195–196 ꢁC (AcOEt);
1H NMR (DMSO-d6) d: 2.80 (3H, s), 7.32 (1H, d,
J = 2.9 Hz), 7.55 (1H, dd, J = 1.9, 9.3 Hz), 7.73 (1H, d,
J = 9.3 Hz), 7.84 (1H, d, J = 8.7 Hz), 8.35 (1H, s), 8.53
(1H, dd, J = 2.5, 8.8 Hz), 8.80–8.85 (2H, m), 9.20–9.24
(1H, m); FAB MS m/e (M+H)+ 462, 464; Anal. Calcd
for C17H12N5O4SBr: C, 44.17; H, 2.62; N, 15.15; S, 6.94;
5.1.3. 6-Chloro-2-methyl-3-{1-[(4-nitrophenyl)sulfonyl]-
1H-pyrazol-3-yl}imidazo[1,2-a]pyridine (2c). Compound
2c was prepared from 4b and 4-nitrobenzenesulfonyl
chloride according to the same procedure as that of
2a. Compound 2c was obtained as a yellow solid
(56% yield). Mp: 263–264 ꢁC (pyridine); 1H NMR
(DMSO-d6) d: 2.54 (3H, s), 7.15 (1H, d, J = 2.9 Hz),
7.47 (1H, dd, J = 1.9, 9.7 Hz), 7.67 (1H, d,

M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
409
Br, 17.28. Found: C, 44.01; H, 2.58; N, 15.19; S, 6.80; Br,
17.41.
perature, EtOH (30 mL) and hydrazine hydrate
(4.5 mL, 93 mmol) were added and the reaction mixture
was refluxed for 3 h. The mixture was diluted with
water and extracted with CHCl3. The organic layer
was dried over MgSO4 and evaporated. The resulting
solid was washed with Et2O to give 4a (1.75 g, 28%) as
5.1.8. 1-(6-Chloro-2-methylimidazo[1,2-a]pyridin-3-yl)eth-
anone (3b). A mixture of 2-amino-5-chloropyridine 16a
(6.4 g, 50 mmol) and 3-chloropentane-2,4-dione (6.7 g,
50 mmol) in ethanol (30 mL) was refluxed for 10 h.
After evaporation, the residue was chromatographed
on silica gel eluting with CHCl3/MeOH (200:1) to give
1
a brown solid. H NMR (DMSO-d6) d: 2.51 (3H, s),
6.65 (1H, d, J = 2.4 Hz), 6.80–7.10 (1H, m), 7.13–7.40
(1H, m), 7.45–7.65 (1H, m), 7.95 (1H, d, J = 2.4 Hz),
9.00–9.35 (1H, m), 13.18 (1H, br s); FAB MS m/e
(M+H)+ 199.
1
3b (4.4 g, 42%) as a brown solid. H NMR (CDCl3) d:
2.63 (3H, s), 2.79 (3H, s), 7.43 (1H, dd, J = 1.9.
9.3 Hz), 7.58 (1H, d, J = 8.8 Hz), 9.82–9.86 (1H, m);
FAB MS m/e (M+H)+ 209.
5.1.14. 6-Chloro-2-methyl-3-(1H-pyrazol-3-yl)imidazo-
[1,2-a]pyridine (4b). 1-(6-Chloro-2-methylimidazo[1,2-
a]pyridin-3-yl)ethanone3b (4.5 g, 22 mmol) in 1,1-di-
methoxy-N,N-dimethylmethanamine (9 mL, 68 mmol)
was refluxed for 4.5 h. After cooling, MeOH (50 mL)
was added to the mixture and the insoluble matearials
were removed by filtration. The filtrate was concen-
trated, and hydrazine hydrate (2.4 mL, 49 mmol) and
EtOH (25 mL) were added to the resulting residue.
After refluxing for 1 h, the reaction mixture was fil-
tered and the filtrate was allowed to cool. Then, water
(50 mL) was added to it and the resulting precipitates
were collected to give 4b (3.5 g, 68%) as a brown solid.
1H NMR (DMSO-d6) d: 2.56 (3H, s), 6.68–6.72 (1H,
m), 7.33 (1H, dd, J = 1.9, 9.3 Hz), 7.61 (1H, d,
J = 9.8 Hz), 7.99 (1H, s), 9.42–9.47 (1H, m), 13.26
(1H, br s); FAB MS m/e (M+H)+ 233.
5.1.9. 1-(6-Bromo-2-methylimidazo[1,2-a]pyridin-3-yl)eth-
anone (3c). Compound 3c was prepared from 2-amino-5-
bromopyridine 16b according to the same procedure as
that of 3b. Compound 3c was obtained as a brown solid
1
(44% yield). H NMR (DMSO-d6) d: 2.59 (3H, s), 2.72
(3H, s), 7.66–7.76 (2H, m), 9.73–9.76 (1H, m); FAB
MS m/e (M+H)+ 253, 255.
5.1.10. 1-(6-Bromolimidazo[1,2-a]pyridin-3-yl)ethanone
(3d). Compound 16b (50 g, 0.29 mol) in 1,1-dimethoxy-
N,N-dimethylmethanamine (55 g, 0.46 mol) was re-
fluxed for 23 h and then evaporated. To a solution of
the resulting residue in EtOH (500 mL), 90% 1-bro-
moacetone (50 g, 0.33 mmol) was added and the reac-
tion mixture was stirred at rt for 18 h. The resulting
precipitates were collected and washed with MeOH
1
(30 mL) to give 3d (22 g, 32%) as a brown solid. H
5.1.15. 6-Bromo-2-methyl-3-(1H-pyrazol-3-yl)imidazo-
[ 1,2-a]pyridine (4c). Compound 4c was prepared from
3c according to the same procedure as that of 4b. Com-
pound 4c was obtained as a brown solid (54%). 1H
NMR (DMSO-d6) d: 2.56 (3H, s), 6.70 (1H, d,
J = 2.5 Hz), 7.39 (1H, dd, J = 2.0, 9.3 Hz), 7.56 (1H, d,
J = 9.3 Hz), 8.00 (1H, s), 9.53 (1H, s), 13.26 (1H, s);
FAB MS m/e (M+H)+ 276, 278.
NMR (CDCl3) d: 2.59 (3H, s), 7.57 (1H, dd, J = 2.0,
9.8 Hz), 7.66 (1H, J = 8.8 Hz), 8.31 (1H, s), 9.81–9.84
(1H, m); FAB MS m/e (M+H)+ 239, 241.
5.1.11. 1-(6-Chloroimidazo[1,2-a]pyridin-3-yl)ethanone
(3e). Compound 3e was prepared from 16a according
to the same procedure as that of 3d. Compound 3e
1
was obtained as a brown solid (25% yield). H NMR
(DMSO-d6) d: 2.58 (3H, s), 7.66–7.74 (1H, m), 7.78
(1H, d, J = 9.8 Hz), 8.65 (1H, s), 9.52–9.57 (1H, m);
FAB MS m/e (M+H)+ 195.
5.1.16. 6-Bromo-3-(1H-pyrazol-3-yl)imidazo[1,2-a]pyri-
dine (4d). Compound 4d was prepared from 3d accord-
ing to the same procedure as that of 4b. Compound 4d
1
was obtained as a brown solid (34% yield). H NMR
5.1.12.
6-Chloroimidazo[1,2-a]pyridine-3-carbaldehyde
(DMSO-d6) d: 6.81–6.86 (1H, m), 7.42–7.48 (1H, m),
7.68 (1H, d, J = 9.3 Hz), 7.90–7.95 (1H, m), 8.10 (1H,
s), 9.65 (1H, s), 13.16 (1H, br s); FAB MS m/e
(M+H)+ 263, 265.
(3f). To a mixture of 16a (14.5 g, 113 mmol) in MeCN
(260 mL) was added bromomalonaldehyde (17.4 g,
116 mmol) and the mixture was heated at reflux for
3 h. After evaporation, the residue was dissolved with
a mixture of CHCl3 and aqueous NaHCO3. The organic
layer was dried over Na2SO4 and evaporated. The resi-
due was dissolved with EtOH and the insoluble materi-
als were removed by filtration. Then, the filtrate was
evaporated and the residue was washed with water to
give 3f (5.18 g, 31%) as a brown solid. 1H NMR (CDCl3)
d: 7.54 (1H, d, J = 2.0, 9.7 Hz), 7.75 (1H, d, J = 9.7 Hz),
8.33 (1H, s), 9.59 (1H, d, J = 2.0 Hz), 9.96 (1H, s); FAB
MS m/e (M+H)+ 181.
5.1.17. Methyl (2E)-3-(6-chloroimidazo[1,2-a]pyridin-3-
yl)acrylate (5). To a mixture of 3f (5.2 g, 29 mmol) in
DMF (55 mL) were added methyl (diethoxyphospho-
ryl)acetate (7.2 g, 34 mmol) and K2CO3 (5.0 g, 36 mmol)
and the reaction mixture was stirred at 60 ꢁC for 27 h.
After evaporation, the residue was washed with water to
give 5 (3.5 g, 70%) as a brown solid. 1H NMR (DMSO-
d6) d: 3.75 (3H, s), 6.66 (1H, d, J = 15.6 Hz), 7.40–7.48
(1H, m), 7.72 (1H, d, J = 9.2 Hz), 8.09 (1H, d,
J = 15.6 Hz), 8.37 (1H, s), 9.19 (1H, s); FAB MS
m/e (M+H)+ 237.
5.1.13. 2-Methyl-3-(1H-pyrazol-3-yl)imidazo[1,2-a]pyri-
dine (4a). A mixture of 1-(2-methylimidazo[1,2-a]
pyridin-3-yl)ethanone 3a29 (5.4 g, 31 mmol) and 1,1-di-
methoxy-N,N-dimethylmethanamine (9.1 mL, 53 mmol)
was heated at 120 ꢁC for 14 h. After cooling to room tem-
5.1.18. Methyl 4-(6-chloroimidazo[1,2-a]pyridin-3-yl)-
1H-pyrrole-3-carboxylate (6). To a suspension of 60%
NaH (540 mg, 14 mmol) were added dropwose a solution

410
M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
of 5 (2.7 g, 29 mmol) and TosMIC (2.2 g, 11 mmol) in a
mixture of DMSO (32 mL) and Et2O (16 mL). After stir-
ring at room temperature for 1 h, the reaction mixture was
evaporated and the residue was washed with water and
hot MeOH (70 mL) to give 10 as a colorless solid (8.0 g,
1
71%). H NMR (DMSO-d6) d: 7.42–7.50 (2H, m), 7.75
(1H, d, J = 9.7 Hz), 8.02 (1H, s), 8.72 (1H, d,
J = 1.9 Hz), 13.71 (1H, br s); FAB MS m/e (M+H)+ 268.
1
then Et2O to give 6 (2.5 g, 80%) as a brown solid. H
NMR (DMSO-d6) d: 3.57 (3H, s), 7.10–7.18 (1H, m),
7.28 (1H, dd, J = 1.2, 10.0 Hz), 7.50–7.72 (3H, m), 7.99
(1H, s), 11.91 (1H, br s); FAB MS m/e (M+H)+ 276.
5.1.23. 6-Chloro-3-{2-[(2-methyl-5-nitrophenyl)thio]-1,3-
thiazol-4-yl}imidazo[1,2-a]pyridine hydrochloride (11).
To a suspension of 2-methyl-5-nitroaniline (1.0 g,
6.6 mmol) in 6 N HCl (5 mL), a solution of NaNO2
(480 mg, 7.0 mmol) in water (5 mL) was added dropwise
below 0 ꢁC. After stirring for 15 min, a solution of
NaBF4 (1.0 g, 9.1 mmol) in water (5 mL) was added
and the reaction mixture was stirred at room tempera-
ture for 0.5 h. The resulting diazonium salt was collected
and dried under vacuum. To a suspension of 60% NaH
(140 mg, 3.5 mmol) in DMSO (20 mL), 10 (950 mg,
3.5 mmol) was added and stirred at 70 ꢁC for 15 min.
To the resulting red solution, diazonium salt was added
portionwise at room temperature. After stirring for
10 min, CHCl3 and saturated aqueous NaHCO3 were
added to the reaction mixture and the insoluble materi-
als were removed by filtration through a bed of Celite.
After the filtrate was separated, the organic layer was
dried over MgSO4 and evaporated. The obtained residue
was chromatographed on silica gel eluting with hexane/
AcOEt (2:3) to give free base of 11 as a brown oil
(270 mg, 19%). To a solution of free base of 11
(270 mg, 0.67 mmol) was added 4 N HCl/AcOEt
(0.3 mL) and the mixture was evaporated. The residue
was crystallized from EtOH to give hydrochloride salt
of 11 (120 mg, 8%) as a pink solid. Mp: 180–182 ꢁC;
1H NMR (DMSO-d6) d: 2.59 (3H, s), 7.78–7.86 (2H,
m), 7.98 (1H, d, J = 9.8 Hz), 8.32–8.38 (2H, m), 8.52
(1H, d, J = 2.4 Hz), 8.64 (1H, s), 9.14–9.17 (1H, m);
FAB MS m/e (M)+ 402; Anal. Calcd for
C17H11N4O2S2Cl0.95HCl: C, 46.67; H, 2.75; N, 12.81;
S, 14.66; Cl, 15.80. Found: C, 46.34; H, 2.75; N, 12.48;
S, 14.45; Cl, 15.55.
5.1.19.
6-Chloro-3-(1H-pyrrol-3-yl)imidazo[1,2-a]pyri-
dine (7). To a mixture of 6 (800 mg, 2.9 mmol) in a mix-
ture of MeOH (5 mL) and water (5 mL) was added
KOH (1.6 g, 29 mmol). After stirring for 3 h, the mix-
ture was neutralized with 1 N HCl and the insoluble
materials were removed. After evaporation, the residue
was dissolved in 2-aminoethanol (5 mL) and heated at
200 ꢁC for 1 h. After the reaction mixture was allowed
to cool, water was added. The resulting precipitates were
1
collected to give 7 (203 mg, 32%) as a brown solid. H
NMR (DMSO-d6) d: 6.44–6.50 (1H, m), 6.93–6.99
(1H, m), 7.24 (1H, dd, J = 2.0, 9.6 Hz), 7.31–7.37 (1H,
m), 7.59–7.70 (2H, m), 8.48–8.52 (1H, m), 11.26 (1H,
br s); FAB MS m/e (M ꢀ H)ꢀ 216.
5.1.20. 6-Chloro-3-{1-[(2-methyl-5-nitrophenyl)sulfonyl]-
1H-pyrrol-3-yl}imidazo[1,2-a]pyridine hydrochloride (8).
To a solution of 7 (203 mg, 0.93 mmol) in THF (5 mL)
was added 60% NaH (90 mg, 2.3 mmol) and the mixture
was stirred at room temperature for 0.5 h. Then, after
addition of 2-methyl-5-nitrobenzensulfonyl chloride
(400 mg, 1.7 mmol), the reaction mixture was stirred for
2 h. The mixture was evaporated and chromatographed
on silica gel with eluting CHCl3/MeOH (20/1). The col-
lected fractions were concentrated and the residue was
dissolved with MeOH. 4 N HCl/AcOEt (1 mL) was added
to the solution and then evaporated. The resulting solid
was washed with hot EtOH to give HCl salt of 8
1
(169 mg, 44%) as a colorless solid. Mp: 199–203 ꢁC; H
NMR (DMSO-d6) d: 2.70 (3H, s), 6.99–7.05 (1H, m),
7.75–7.80 (1H, m), 7.82 (1H, d, J = 8.7 Hz), 7.94–8.00
(1H, m), 8.05 (1H, d, J = 9.3 Hz), 8.31 (1H, s), 8.42–8.56
(2H, m), 8.66 (1H, d, J = 2.4 Hz), 8.93 (1H, s); FAB MS
m/e (M+H)+ 417; Anal. Calcd for C18H13N4O4SClHCl:
C, 47.69; H, 3.11; N, 12.36; S, 7.07; Cl, 15.64. Found: C,
47.46; H, 3.09; N, 12.16; S, 7.03; Cl, 15.69.
5.1.24. 6-Chloro-3-{2-[(2-methyl-5-nitrophenyl)sulfonyl]-
1,3- thiazol-4-yl}imidazo[1,2-a]pyridine hydrochloride
(12). To free base of 11 (260 mg, 0.645 mmol), AcOH
(10 mL) and 30% H2O2 (10 mL) were added and the
reaction mixture was stirred at 70 ꢁC for 9.5 h. After
the mixture was diluted with water (50 mL) and CHCl3
(50 mL), the organic layer was washed with 5% Na2S2O3
and saturated aqueous NaHCO3, and dried over
MgSO4. After evaporation, the residue was chromato-
graphed on silica gel with eluting CHCl3/MeOH
(100:1) and the collected fractions were concentrated.
To the solution of the residue in CHCl3 (5 mL) and
MeOH (5 mL), 4 N HCl/AcOEt (0.2 mL) was added
and the mixture was evaporated to give a colorless
solid. The solid was washed with hot MeOH and Et2O
to give HCl salt of 12 (110 mg, 36%) as a colorless
5.1.21. 2-Bromo-1-(6-chloroimidazo[1,2-a]pyridin-3-yl)eth-
anone hydrobromide (9). To a suspension of 3e (10 g,
51 mmol) in 30% HBr/AcOH (100 mL) was added Br2
(9.8 g, 50 mmol) at room temperature. After stirring for
32 h, the precipitates were collected by filtration and
washed with EtOH and Et2O to give 9 (16 g, 90%) as a col-
1
orless solid. H NMR (DMSO-d6) d: 4.65–4.90 (2H, s),
7.90–8.15 (2H, m), 8.90–9.23 (1H, m), 9.55–9.70 (1H, m).
1
5.1.22. 4-(6-Chloroimidazo[1,2-a]pyridin-3-yl)-1,3-thia-
zole-2-thiol (10). To a suspension of 9 (15 g, 42 mmol)
in MeOH (150 mL), ammonium dithiocarbamate (7.0
g, 64 mmol) was added at room temperature. After stir-
ring for 15 min, the solid was collected by filtration. After
the obtained solid in AcOH (40 mL) was refluxed for 1 h,
water (40 mL) was added to the reaction mixture and the
resulting colorless solid was collected and washed with
solid. Mp: 221–223 ꢁC; H NMR (DMSO-d6) d: 2.84
(3H, s), 7.76 (1H, d, J = 1.9, 9.8 Hz), 7.88 (1H, d,
J = 8.8 Hz), 7.94 (1H, d, J = 9.8 Hz), 8.55–8.60 (1H,
m), 8.63 (1H, s), 8.84–8.88 (2H, m), 9.02–9.06 (1H, m);
FAB MS m/e (M)+ 434; Anal. Calcd for C17H11-
N4O4S2ClHCl: C, 43.32; H, 2.57; N, 11.89; S, 13.61;
Cl, 15.04. Found: C, 43.04; H, 2.57; N, 11.78; S, 13.66;
Cl, 15.30.

M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 403–412
411
5.1.25. Ethyl 2-{[4-(6-chloroimidazo[1,2-a]pyridin-3-yl)-
1,3-thiazol-2-yl]thio}-4-nitrobenzoate (13). To a suspen-
sion of 60% NaH (1.8 g, 45 mmol) in DMF (200 mL),
10 (11 g, 41 mmol) was added and the mixture was stir-
red at room temperature for 0.5 h. To the reaction mix-
ture, ethyl 2-fluoro-4-nitrobenzoate (10 g, 47 mmol) was
added. After stirring at 90 ꢁC for 7 h, the reaction mix-
ture was concentrated and dissolved with a mixture of
THF (100 mL) and AcOEt (400 mL). The solution was
washed with brine, dried over MgSO4, and evaporated.
The residue was subjected to silica gel column chroma-
tography (CHCl3) and the obtained solid was washed
with MeOH (100 mL) to give 13 (4.8 g, 25%) as a pale
S, 13.61; Cl, 15.04. Found: C, 43.01; H, 2.48; N,
11.67; S, 13.57; Cl, 14.86.
5.1.28. 6-Chloro-3-{2-[(2-methyl-5-nitrophenyl)sulfinyl]-
1,3-thiazol-4-yl}imidazo[1,2-a]pyridine
hydrochloride
(15). To a suspension of free base of 14b (360 mg,
0.83 mmol) in THF (15 mL) were added Et3N
(300 mg, 3.0 mmol) and methanesulfonyl chloride
(170 mg, 1.5 mmol). After stirring for 0.5 h, DMSO
(15 mL) and NaBH4 (200 mg, 5.3 mmol) were added
to the reaction mixture. After stirring for 0.5 h, the
mixture was evaporated and diluted with AcOEt and
brine. After separation, the organic layer was dried over
MgSO4 and evaporated. The residue was purified with
column chromatography on silica gel (CHCl3/
MeOH = 200:1). The obtained solid was dissolved with
a mixture of CHCl3 (100 mL), THF (50 mL), and
MeOH (50 mL). To the solution was added 4 N HCl/
AcOEt (0.1 mL) and the mixture was evaporated. The
resulting solid was recrystallized from MeOH/Et2O to
give HCl salt of 15 (49 mg, 13%) as a colorless solid.
1
yellow solid. H NMR (DMSO-d6) d: 1.29–1.36 (3H,
m), 4.35–4.43 (2H, m), 7.37–7.43 (1H, m), 7.75 (1H, d,
J = 9.2 Hz), 8.16 (1H, d, J = 2.0 Hz), 8.21–8.31 (3H,
m), 8.47 (1H, s), 9.12 (1H, d, J = 1.9 Hz); FAB MS
m/e (M+H)+ 461.
5.1.26. (2-{[4-(6-Chloroimidazo[1,2-a]pyridin-3-yl)-1,3-
thiazol-2-yl]thio}-4-nitrophenyl)methanol hydrochloride
(14a). To a suspension of 13 (4.7 g, 10 mmol) in CH2Cl2
(50 mL) was added dropwise 1 M DIBAL-H/toluene
(50 mL) below 5 ꢁC and the mixture was stirred at room
temperature for 1 h. The reaction was quenched with
MeOH and diluted with saturated aqueous NaHCO3
and CHCl3. After filtration through a bed of Celite,
the organics of the filtrate was dried over MgSO4 and
concentrated. The residue was subjected to column
chromatography on silica gel (CHCl3/MeOH = 100:1)
to give free base of 14a (2.0 g, 47%) as a pale yellow sol-
id. The free base of 14a (250 mg, 0.60 mmol) was dis-
solved with a mixture of MeOH (40 mL) and CHCl3
(40 mL). To the solution was added 4 N HCl/AcOEt
(0.3 mL) and the mixture was evaporated. The resulting
solid was washed with EtOH and recrystallized from
MeOH to give HCl salt of 14a (122 mg, 45%) as a color-
less solid. Mp: 193–194 ꢁC; 1H NMR (DMSO-d6) d: 4.78
(2H, s), 7.83 (1H, dd, J = 2.0. 8.8 Hz), 7.94–8.01 (2H,
m), 8.36 (1H, s), 8.46 (1H, dd, J = 2.5, 8.3 Hz), 8.52
(1H, d, J = 2.4 Hz), 8.63 (1H, s), 9.12–9.16 (1H, m);
FAB MS m/e (M)+ 418; Anal. Calcd for
C17H11N4O3S2ClHCl: C, 44.84; H, 2.66; N, 12.30; S,
14.08; Cl, 15.57. Found: C, 44.90; H, 2.67; N, 12.02; S,
14.12; Cl, 15.48.
1
Mp: 204–206 ꢁC; H NMR (DMSO-d6) d: 2.75 (3H, s),
7.73–7.82 (2H, m), 7.95 (1H, d, J = 9.8 Hz), 8.36 (1H,
dd, J = 2.4, 8.3 Hz), 8.57 (1H, s), 8.61 (1H, d,
J = 2.4 Hz), 8.67 (1H, s), 9.12–9.16 (1H, m); FAB MS
m/e (M+H)+ 419; Anal. Calcd for C17H11N4O3S2ClHCl:
C, 44.84; H, 2.66; N, 12.30; S, 14.08; Cl, 15.57. Found:
C, 44.86; H, 2.76; N, 12.38; S, 14.09; Cl, 15.28.
5.2. Scintillation proximity assay (SPA) for p110a,
p110b, p110c, and PI3K C2b
GST-tagged bovine p110a, GST-tagged human p110b,
His-tagged p110c, and Glu-tagged PI3K C2b were ex-
pressed in an Sf9/Baculovirus system and purified as fu-
sion proteins. The test compounds dissolved in DMSO
(0.5 lL) and each enzyme were mixed in 25 lL of buffer
solution (p110a, b, c assay: 20 mM Tris–HCl (pH 7.4),
160 mM NaCl, 2 mM dithiothreitol, 30 mM MgCl2,
0.4 mM EDTA, and 0.4 mM EGTA; PI3K C2b assay:
20 mM Tris–HCl (pH 7.4), 160 mM NaCl, 2 mM dithi-
othreitol, 5 mM MgCl2, 15 mM CaCl2, and 0.4 mM
EDTA). Then, 25 lL of 5 mM Tris–HCl supplemented
with 1 lg PI (Sigma), 0.125 lCi [c-33P]ATP (Amersham
Pharmacia), and 2 lM non-radiolabeled ATP (Sigma)
was added to the mixture to initiate the reaction. After
allowing the reaction to proceed at room temperature
for 120 min, 0.2 mg of wheat germ agglutinin-coated
SPA beads (Amersham) in 150 lL PBS was added,
and the mixture was left to stand for 5 min and then cen-
trifuged at 300g for 2 min. The radioactivity was mea-
sured using TopCount (Packard). IC50 values are given
as means of at least two separate determinations with
typical variations of less than 20%.
5.1.27. (2-{[4-(6-Chloroimidazo[1,2-a]pyridin-3-yl)-1,3-
thiazol-2-yl]sulfinyl}-4-nitrophenyl)methanol hydrochlo-
ride (14b). To a suspension of free base of 14a
(115 mg, 0.275 mmol) in CH2Cl2 (15 mL) was added
70–75% m-CPBA (180 mg). After stirring for 1 h,
CHCl3 (20 mL) was added and the resulting solid
was collected. The obtained solid was dissolved with
THF (50 mL), MeOH (20 mL), and CHCl3 (20 mL).
To the solution was added 4 N HCl/AcOEt (40 lL)
and the mixture was evaporated. The resulting solid
was recrystallized from MeOH/Et2O to give HCl salt
of 14b (45 mg, 35%) as a colorless solid. mp: 198–
200 ꢁC; 1H NMR (DMSO-d6) d: 4.86–5.00 (2H, m),
7.80–8.00 (3H, m), 8.42–8.50 (1H, m), 8.61 (1H, s),
8.66 (1H, s), 8.78 (1H, d, J = 2.4 Hz), 9.16–9.20
(1H, m); FAB MS m/e (M)+ 434; Anal. Calcd for
C17H11N4O4S2ClHCl: C, 43.32; H, 2.57; N, 11.89;
5.3. In vitro proliferation assays (A375, HeLa cells)
Cells were cultured in DMEM with 10% fetal bovine ser-
um and 1% streptomycin/penicillin. The test compound
in a volume of 1 lL was spotted onto a 96-well culture
plate followed by addition of cells (1 · 104) up to a vol-
ume of 100 lL. After incubation for 46 h, 10 lL of Ala-
mar Blue reagent was added to each well, and after a

412
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Acknowledgments
We thank Drs. K. Matsuda and N. Taniguchi for their
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Research for performing instrumental analysis. This
work was funded in part by Cancer Research UK
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