Synthesis and biological evaluation of sulfonylhydrazone-substituted imidazo[1,2-a]pyridines as novel PI3 kinase p110α inhibitors

Article abstract of DOI:10.1016/j.bmc.2007.05.070

We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110α inhibitor; however, although 4 is a potent inhibitor of p110α enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo

Full text of DOI:10.1016/j.bmc.2007.05.070

Bioorganic & Medicinal Chemistry 15 (2007) 5837–5844
Synthesis and biological evaluation of sulfonylhydrazone-
substituted imidazo[1,2-a]pyridines as novel
PI3 kinase p110a inhibitors
Masahiko Hayakawa,a,* Ken-ichi Kawaguchi,a Hiroyuki Kaizawa,a Tomonobu Koizumi,a
Takahide Ohishi,a Mayumi Yamano,a Minoru Okada,a Mitsuaki Ohta,a
Shin-ichi Tsukamoto,a Florence I. Raynaud,b Peter Parker,c Paul Workmanb
and Michael D. Waterfieldd
aDrug Discovery Research, Astellas Pharma Inc., 5-2-3 Tokodai, Tsukuba, Ibaraki 300-2698, Japan
bCancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey SN2 5NG, UK
cCancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
dLudwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, UK
Received 16 March 2007; revised 29 May 2007; accepted 31 May 2007
Available online 6 June 2007
Abstract—We have previously reported the imidazo[1,2-a]pyridine derivative 4 as a novel p110a inhibitor; however, although 4 is a
potent inhibitor of p110a enzymatic activity and tumor cell proliferation in vitro, it is unstable in solution and ineffective in vivo. To
increase stability the pyrazole of 4 was replaced with a hydrazone and a moderately potent p110a inhibitor 7a was obtained. Sub-
sequent optimization of 7a afforded exceptionally potent p110a inhibitors, including 8c and 8h, with IC50 values of 0.30 nM and
0.26 nM, respectively; to the best of our knowledge, these compounds are the most potent PI3K p110a inhibitors reported to date.
Compound 8c was also stable in solution and exhibited significant anti-tumor effectiveness in vivo.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
reported. Among the PI3K isoforms, PI3K p110a
expression correlates most strongly with cancer progres-
sion, since amplification11,12 and frequent mutation13–16
of the PIK3CA gene that encodes PI3K p110a have been
observed in several cancers.
Phosphoinositide 3-kinases (PI3Ks) have emerged as po-
tential targets for anti-cancer therapy.1–3 PI3K signaling
is negatively regulated by PTEN, which is one of the
most commonly mutated proteins in human cancers,4–6
suggesting that inhibitors of PI3Ks have potential as
anti-cancer agents. PI3Ks are divided into three major
classes based on their primary structure and mechanism
of activation: classes I, II, and III.7–10 Class I PI3Ks are
further divided into class Ia enzymes: p110a, p110b, and
p110d, which are activated by tyrosine kinase receptors;
and the class Ib enzyme p110c, which is activated by a G
protein-coupled receptor. The class II PI3Ks C2a, C2b,
and C2c are characterized by the presence of a C2 do-
main at the C terminus. Regarding class III PI3Ks, the
mechanism of activation is still not understood, but at
least two different complexes of this protein have been
Examples of non-isoform-specific PI3K inhibitors in-
clude wortmannin and LY294002, and we have reported
several series of potent and isoform-specific PI3K p110a
inhibitors represented by 1, 2, and 4 (Fig. 1).17–19 Com-
pound 4 contains an imidazo[1,2-a]pyridine ring and
was derived from lead compound 3, which was discov-
ered in high-throughput screening. Although 4 has
excellent in vitro potency as a p110a inhibitor, it is
unstable in solution and ineffective in vivo. The instabil-
ity is probably due to cleavage of the pyrazole–sulfone
linkage, and therefore we focused our efforts on replace-
ment of the pyrazole ring of 4 with other substituents.
Introduction of hydrazones gave a novel series of
p110a inhibitors, and here we report the synthesis and
evaluation of a new series of hydrazone-containing
PI3K p110a inhibitors that are exceptionally potent,
selective, and effective in vivo.
Keywords: PI3 kinase; p110a; Inhibitor; Anti-cancer agent.
Corresponding author. Tel.: +81 29 865 7124; fax: +81 29 847
8313; e-mail: masahiko.hayakawa@jp.astellas.com
*
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.070

5838
Me
M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 5837–5844
O
N
which were subjected to methylation using MeI to give
O
O
O
O
8a and 8b, respectively. Imidazo[1,2-a]pyridine deriva-
tives 8c–j were synthesized as shown in Scheme 2. Alde-
hydes 10a–g were prepared from the 2-aminopyridines
9a–g by cyclization with bromomalonaldehyde.18 8c
and 8d were synthesized from aldehydes 10a and 10b
by the same method as that for synthesis of 8a and 8b.
Compounds 8e–i were synthesized from the suitable
aldehydes 10c–g by condensation with methylhydrazine
followed by sulfonylation to give the desired compounds
8e–i. The carboxamide derivative 8j was prepared from
the corresponding ester 8i by hydrolysis and subsequent
condensation with ammonia. The commercially unavail-
able 2-aminopyridine 9f was synthesized by dehydration
of 12 with TFAA, as shown in Scheme 3. Compounds
7a–b and 8a–j were all obtained as E isomers. The ste-
reochemistry of each of the compounds was determined
by the NMR (NOE) and X-ray analysis.
MeO
O
N
S
N
O
O
O
H
O
OH
N
O
Wortmannin
LY294002
1
N
O
N
N
N
Me
Me
N
Br
O2N
N
O
N
N
N
S O
O
N
S O
O
N
OH
N
F
Me
4
2
3
Figure 1. Structures of PI3K inhibitors.
2. Chemistry
As shown in Scheme 1, the 6-bromoimidazo[1,2-a]pyri-
dine derivatives 7a, 7b, 8a, and 8b were prepared from
ketones 5a and 5b. Condensation of 5a and 5b with
hydrazine hydrate in refluxing ethanol, followed by
treatment with 2-methyl-5-nitrobenzenesulfonyl chlo-
ride, afforded sulfonylhydrazone derivatives 7a and 7b,
3. Results and discussion
Compound 4 is a potent p110a inhibitor in vitro with an
IC50 of 3.1 nM, but is unstable in solution and does not
inhibit tumor growth in vivo. Since the instability is
N
N
R
N
R
N
N
N
R
Br
Br
R
a
N
b
c
N
Br
Me
Me
Me
Br
N
NH
N
N
S O
O
Me
Me
NH2
O2N
Me
O2N
N
O
S O
O
Me
5a: R = Me
5b: R = H
6a: R = Me
6b: R = H
7a: R = Me
7b: R = H
8a: R = Me
8b: R = H
Scheme 1. Reagents and conditions: (a) H2NNH2 hydrate, EtOH, reflux; (b) 2-methyl-5-nitrobenzenesulfonyl chloride, pyridine; (c) MeI, NaH, DMF.
N
N
N
N
N
NH2
X
X
c
d
N
X
N
N
N
X
H
Me
N
O2N
O2N
N
N
NH2
S O
O
S O
O
9a: X = Br
9b: X = Cl
9f: X = CN
9g: X = CO2Et
Me
Me
9c: X = F
9d: X = CF3
6c: X = Br
6d: X = Cl
7c: X = Br
7d: X = Cl
8c: X = Br
8d: X = Cl
9e: X = Me
b
a
N
N
N
N
N
X
e
c
N
X
N
X
Me
O2N
N
NHMe
N
H
O
S O
O
Me
8e: X = F
8f: X = CF3
11a: X = F
11b: X = CF3
10a: X = Br
10b: X = Cl
10c: X = F
8g: X = Me
8h: X = CN
11c: X = Me
11d: X = CN
10d: X = CF3
10e: X = Me
10f: X = CN
8i: X = CO Et
11e: X = CO2Et
2
f
8j: X = CONH
2
10g: X = CO Et
2
Scheme 2. Reagents and conditions: (a) BrCH(CHO)2, MeCN, D; (b) H2NNH2 hydrate, EtOH, reflux; (c) 2-methyl-5-nitrobenzenesulfonyl chloride,
pyridine; (d) MeI, NaH, DMF; (e) H2NNHMe, EtOH, D; (f) i—LiOH, EtOH, ii—CDI, NH4OH.

M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 5837–5844
5839
NH2
tion of A375 human melanoma cells, with an IC50 of
0.058 lM.
NH2
a
N
N
NC
H2NOC
9f
12
Next, the bromo group of 8c at the C6 position of the
imidazo[1,2-a]pyridine ring was replaced with other sub-
stituents (Table 2). Introduction of fluoro and chloro as
less bulky halogens gave reduced potency against p110a
compared to the bromo derivative 8c, with a 2.5-fold
and an 18-fold drop in potency, respectively. Trifluo-
romethyl, methyl, ethyl ester, and carboxamide deriva-
tives 8f, g, i, and j were also less potent than 8c, but
the cyano derivative 8h was an extremely potent p110a
inhibitor (IC50: 0.26 nM) and also a potent inhibitor of
proliferation of A375 human melanoma cells (IC50:
0.033 lM). In contrast to the large differences in p110a
inhibitory activities among 8c–h in cell-free assays, their
inhibitory activities in the cell-based assay were rela-
tively similar, with IC50s ranging from 0.033 to
0.18 lM. The trifluoromethyl derivative 8f was 54-fold
less potent than 8h in the enzymatic assay, whereas in
the cell-based assay 8f was only 2.7-fold less potent than
8h, suggesting that 8f may have improved cell perme-
ability compared to 8h. It is possible that the cell activity
may be due in part to effects on mTOR and DNA-PK,
as reported for 8c.20 This requires further mechanistic
studies.
Scheme 3. Reagents: (a) TFAA, K2CO3, then water.
probably due to cleavage of the sulfonyl-pyrazole link-
age, we replaced the pyrazole ring of 4 with other groups
to connect the imidazo[1,2-a]pyridine ring and the 2-
methyl-5-nitrophenylsulfone group. Introduction of a
hydrazone instead of the pyrazole afforded a moderately
potent p110a inhibitor 7a with an IC50 of 0.40 lM, com-
parable with that of LY294002 (Table 1). Our focus then
shifted to increasing the p110a inhibitory activity of 7a
by further structural modification.
First, the methyl groups at R1 or R2 of 7a were removed
(Table 1). Demethylation at R1 on 7a gave 7b as a 20-
fold more potent p110a inhibitor (IC50: 0.017 lM).
The potency of 7b was beyond our expectation, since
the increase in inhibitory activity caused by removal of
the methyl group in the corresponding pyrazole deriva-
tive was only 1.7-fold.18 Removal of the methyl group at
R2 of 7b resulted in retention of p110a inhibitory activ-
ity in 7c (IC50: 0.021 lM).
A methyl group was then introduced at R3 in 7a–c to
give compounds 8a–c. Methylation at R3 of 7a gave
8a, which showed a 2.4-fold increase in p110a inhibitory
activity (IC50: 0.17 lM), but methylation at R3 of 7b re-
sulted in a compound (8b) with decreased p110a inhibi-
tory activity (IC50: 0.081 lM). However, methylation at
R3 of 7c gave an unexpectedly large 70-fold increase in
inhibitory activity over 7c, with compound 8c having
an IC50 of 0.30 nM. Moreover, 8c exhibited highly po-
tent inhibitory activity against serum-induced prolifera-
Table 2. Inhibition of p110a activity by imidazopyridine derivatives
N
N
X
N
N
Me
O2N
S O
O
Me
Compounda
X
IC50 (lM)
p110a
b
A375
Table 1. Inhibition of p110a activity by imidazopyridine derivatives
8c
8d
8e
8f
8g
8h
8i
Br
Cl
0.00030
0.00077
0.0053
0.014
0.058
0.056
0.18
N
R1
F
N
Br
–CF3
Me
0.090
0.12
R2
N
0.0060
0.00026
0.34
R3
O2N
N
–CN
–CO2Et
–CONH2
0.033
9.35
8.96
S O
O
Me
8j
0.78
a HCl salt.
Compounda
R1
R2
R3
IC50 (lM)
c
b IC50 values represent means of at least two separate determinations
with typical variations of less than 20%.
p110a
A375
LY294002
3
0.63
0.67
8.4
23
Table 3. Isoform selectivity of compound 8c against PI3Ks
4
0.0031
0.40
0.73
NTd
12
c
7a
7b
7c
8a
8b
8cb
Me
H
Me
Me
H
H
Compound
IC50 (lM)
H
0.017
0.021
0.17
p110a
p110b
p110c
PI3K C2b
H
H
9.0
Me
H
Me
Me
H
Me
Me
Me
NTd
20
LY294002a
0.63
0.34
0.85
1.6
2.1
0.081
0.00030
8cb
1b
0.00030
0.0025
0.0036
0.040
0.66
0.25
0.10
0.22
0.010
H
0.058
0.016
0.0030
2b
a Free base.
b HCl salt.
a Free base.
b HCl salt.
c IC50 values represent means of at least two separate determinations
with typical variations of less than 20%.
d NT, not tested.
c IC50 values represent means of at least two separate determinations
with typical variations of less than 20%.

5840
M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 5837–5844
Table 4. Inhibition of tumor cell proliferation in vitro by compound 8c
b
Compound
IC50 (lM)
A375 (Melanoma)
HeLa (Cervix)
A549 (Breast)
MCF7 (Breast)
MCF7 ADR-res
8ca
0.058
NTc
NTc
0.051
NTc
NTc
0.069
NTc
NTc
0.019
0.0071
0.0018
0.039
17
8.2
Doxorubicin
Paclitaxel
a HCl salt.
b IC50 values represent means of at least two separate determinations with typical variations of less than 20%.
c NT, not tested.
Compound 8c was evaluated further as a representative
and potent example of this series. First, its selectivity
profile over several PI3K isoforms was investigated (Ta-
ble 3). For comparison, data for the p110a inhibitors 1
and 2, which we identified previously,17,19 are also listed
in Table 3. Compound 8c showed excellent selectivity
for p110a over other PI3K isoforms, while the deriva-
tives with morpholinopyrimidine as a common motif
showed relatively lower isoform selectivity for p110a.
Next, the anti-proliferative effect of 8c was examined
in various cancer cell lines (Table 4). These data showed
that 8c is a potent inhibitor of tumor-cell growth and
has activity against MCF7 ADR-res cells, in which
well-known anti-cancer agents such as doxorubicin
and paclitaxel are ineffective due to overexpression of
P-glycoprotein.21,22
500
400
300
200
100
0
Vehicle
8c, 50mg/kg i.p.
0
7
14
Time (days)
Figure 3. Effect of compound 8c on the growth of HeLa human
cervical tumor xenografts. Compound 8c suspended in 20% hydroxy-
propyl-b-cyclodextrin/saline (50 mg/kg) was intraperitoneally admin-
istered daily for 2 weeks to nude mice carrying a subcutaneous HeLa
xenograft. Error bars show SE.
We first tried to investigate the stability of 8c in aqueous
phosphate buffer (pH 6.8); however, this proved to be
difficult because of the poor solubility and precipitation
of 8c. Therefore, the stability of 8c and 4 was tested in
methanol, as shown in Figure 2; 37% of 4 decomposed
in methanol at 37 ꢁC over 24 h, whereas 8c was com-
pletely stable under the same conditions, probably due
to the absence of the pyrazole–sulfone linkage. After
confirmation of the improved stability of 8c, the
in vivo activity was evaluated in a HeLa human cervical
cancer xenograft model in nude mice. As shown in Fig-
ure 3, 8c dosed intraperitoneally at 50 mg/kg daily for 2
weeks markedly suppressed tumor growth by 62% in
this model, without causing weight loss. The in vivo
efficacy of 8c may be explained partly by the improved
stability as well as the exceptionally potent p110a inhib-
itory activity.
4. Conclusion
We have developed a novel series of PI3K p110a inhib-
itors that are imidazo[1,2-a]pyridines with aryl-
sulfonylhydrazone substituents. Replacement of the
pyrazole of 4 with hydrazone and subsequent optimiza-
tion afforded compounds such as 8c and 8h, which are
the most potent p110a inhibitors reported to date. Com-
pound 8c has excellent selectivity for p110a over other
PI3K isoforms, and is a potent inhibitor of tumor-cell
growth in vitro. Furthermore, 8c is effective in a HeLa
human cervical cancer xenograft model.
100
90
80
5. Experimental
5.1. Chemistry
70
60
1H NMR spectra were measured with a JEOL EX400 or
GX500 spectrometer; chemical shifts are expressed in d
units using tetramethylsilane as the standard (NMR
descriptions: 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 chroma-
4
8c
50
40
0
5
10
15
time, h
20
25
Figure 2. Stability of 4 and 8c in methanol at 37 ꢁC.

M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 5837–5844
5841
tography was performed using Wakogel C-200 or Merck
Silica Gel 60.
5.1.5. N0-[(1E)-1-(6-Bromo-2-methylimidazo[1,2-a]pyridin-
3-yl)ethylidene]-N,2-dimethyl-5-nitrobenzenesulfonohydraz-
ide (8a). Compound 7a (500 mg, 1.07 mmol) and MeI
(198 mg, 1.39 mmol) were added to a suspension of
60% NaH in oil (50 mg) in DMF (5 mL). After stirring
for 10 min, the mixture was diluted with EtOAc and
washed with water and brine. After evaporation, the res-
idue was subjected to silica gel column chromatography
(eluent: CHCl3) and the solid obtained in this procedure
was washed with MeOH to give 8a as a colorless solid
5.1.1. N0-[(1E)-1-(6-Bromo-2-methylimidazo[1,2-a]pyridin-3-
yl)ethylidene]-2-methyl-5-nitrobenzenesulfonohydrazide (7a).
A mixture of 5a18 (2.0 g, 7.9 mmol) and hydrazine hy-
drate (2.0 g, 40 mmol) in EtOH (20 mL) was refluxed
for 2 d and then concentrated. The residue in pyridine
(20 mL) was combined with 2-methyl-5-nitro-
benzenesulfonyl chloride (2.4 g, 10 mmol). After stirring
for 5 h, the mixture was evaporated and dissolved in a
mixture of CHCl3 and water. After separation, the or-
ganic layer was dried over MgSO4 and concentrated.
The residue was crystallized from CHCl3 to give 7a as
a light brown solid (42% yield): mp 128–130 ꢁC; 1H
NMR (DMSO-d6) d 2.39 (3H, s), 2.51 (3H, s), 2.79
(3H, s), 7.40–7.46 (1H, m), 7.51 (1H, d, J = 9.8 Hz),
7.74 (1H, d, J = 8.3 Hz), 8.39 (1H, dd, J = 2.4, 8.3 Hz),
8.76 (1H, d, J = 2.4 Hz), 9.15–9.20 (1H, m), 11.41 (1H,
br s); FAB MS m/e (MH)+ 466, 468; Anal. for
C17H16N5O4SBrÆ0.05CHCl3: Calcd. C, 43.36; H, 3.43;
N, 14.83; S, 6.79; Br, 16.92. Found C, 43.65; H, 3.40;
N, 14.85; S, 6.58; Br, 16.53.
1
(45% yield): mp 216–220 ꢁC; H NMR (DMSO-d6) d
2.63 (3H, s), 2.66 (3H, s), 2.72 (3H, s), 3.01 (3H, s),
7.56–7.66 (2H, m), 7.83 (1H, s, J = 8.3 Hz), 8.48 (1H,
dd, J = 2.5, 8.8 Hz), 8.53 (1H, d, J = 2.5 Hz), 9.49 (1H,
d, J = 1.0 Hz); FAB MS m/e (MH)+ 480, 482; Anal.
for C18H18N5O4SBr: Calcd. C, 45.01; H, 3.78; N,
14.58; S, 6.68; Br, 16.63. Found: C, 44.90; H, 3.72; N,
14.59; S, 6.60; Br, 16.34.
5.1.6. N0-[(1E)-1-(6-Bromoimidazo[1,2-a]pyridin-3-yl)ethyl-
idene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide (8b).
Compound 8b was prepared from 7b using the same
procedure as that for 8a. Compound 8b was obtained
as a colorless solid (56% yield): mp 216–220 ꢁC
1
5.1.2. N0-[(1E)-1-(6-Bromoimidazo[1,2-a]pyridin-3-yl)eth-
ylidene]-2-methyl-5-nitrobenzenesulfonohydrazide (7b).
Compound 7b was prepared from 5b using the same
procedure as that for 7a. Compound 7b was
obtained as a colorless solid (28% yield): mp 197–
198 ꢁC (MeOH–EtOH); 1H NMR (DMSO-d6) d 2.41
(3H, s), 2.79 (3H, s), 7.51 (1H, d, J = 9.8 Hz), 7.67
(1H, d, J = 9.3 Hz), 7.76 (1H, d, J = 9.8 Hz), 8.19 (1H,
s), 8.42 (1H, dd, J = 2.4, 8.3 Hz), 8.81 (1H, d,
J = 2.0 Hz), 9.24 (1H, s), 11.40 (1H, br s); FAB MS m/e
(MH)+ 452, 454; Anal. for C16H14N5O4SBrÆ0.3C2H5OH:
Calcd. C, 42.50; H, 3.50; N, 14.85; S, 6.84; Br,
17.04. Found C, 42.78; H, 3.42; N, 15.03; S, 6.88; Br,
17.14.
(MeOH–EtOH); H NMR (DMSO-d6) d 2.63 (3H, s),
2.72 (3H, s), 3.02 (3H, s), 7.66 (1H, dd, J = 2.0,
9.8 Hz), 7.79 (1H, d, J = 9.3 Hz), 7.87 (1H, d,
J = 7.8 Hz), 8.48–8.54 (3H, m), 9.34 (1H, d,
J = 1.0 Hz); FAB MS m/e (MH)+ 466, 468; Anal. for
C17H16N5O4SBr: Calcd. C, 43.79; H, 3.46; N, 15.02; S,
6.88; Br, 17.14. Found C, 43.93; H, 3.57; N, 15.13; S,
6.74; Br, 17.13.
5.1.7. N0-[(1E)-(6-Bromoimidazo[1,2-a]pyridin-3-yl)methyl-
ene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide hydro-
chloride (8c). The free base form of 8c was prepared
from 7c using the same procedure as that for 8a (43%
yield). 4 N HCl/EtOAc (0.15 mL) was added to a solu-
tion of the free base of 8c (260 mg, 0.57 mmol) in MeOH
(20 mL) and CHCl3 (20 mL). After evaporation, the so-
lid was recrystallized from MeOH to give 8c (HCl salt)
5.1.3. N0-[(1E)-(6-Bromoimidazo[1,2-a]pyridin-3-yl)meth-
ylene]-2-methyl-5-nitrobenzenesulfonohydrazide (7c). Com-
pound 7c was prepared from 10a using the same
procedure as that for 7a. Compound 7c was obtained as
a colorless solid (19% yield): mp 185–186 ꢁC (MeOH);
1H NMR (DMSO-d6) d 2.77 (3H, s), 7.56 (1H, dd,
J = 2.0, 9.8 Hz), 7.71 (1H, d, J = 9.8 Hz), 7.76 (1H, d,
J = 8.3 Hz), 8.06 (1H, s), 8.31 (1H, s), 8.43 (1H, dd,
J = 2.4, 8.8 Hz), 8.77 (1H, d, J = 2.4 Hz), 9.00 (1H, d,
J = 1.5 Hz), 12.20 (1H, br s); FAB MS m/e (MH)+ 438,
440; Anal. for C15H12N5O4SBr: Calcd. C, 41.11; H,
2.76; N, 15.98; S, 7.32; Br, 18.23. Found C, 40.81; H,
2.71; N, 15.96; S, 7.31; Br, 18.28.
1
as a colorless solid (42% yield): mp 205–208; H NMR
(DMSO-d6) d 2.69 (3H, s), 3.48 (3H, s), 7.76–7.89 (3H,
m), 8.23 (1H, s), 8.33 (1H, s), 8.46 (1H, dd, J = 2.4,
8.3 Hz), 8.74 (1H, d, J = 2.4 Hz), 9.22 (1H, s); FAB
MS m/e (MH)+ 452, 454; Anal. for C16H14N5O4SBrÆH-
ClÆ2H2O: Calcd. C, 36.62; H, 3.65; N, 13.35; S, 6.11;
Br, 15.23; Cl, 6.76. Found C, 36.73; H, 3.27; N, 13.31;
S, 6.11; Br, 15.39; Cl, 6.61.
5.1.8. N0-[(1E)-(6-Chloroimidazo[1,2-a]pyridin-3-yl)methyl-
ene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide hydro-
chloride (8d). The free base form of 8d was prepared
from 7d using the same procedure as that for 8a. 4 N
HCl/EtOAc was added to a solution of the free base
of 8d in EtOH and the mixture was evaporated and
recrystallized from MeOH to give 8d (HCl salt) as a co-
lorless solid (48% overall yield): mp 216–217 ꢁC
(MeOH); 1H NMR (DMSO-d6) d 2.68 (3H, s), 3.50
(3H, s), 7.73–7.78 (1H, dd, J = 2.0, 9.3 Hz), 7.80 (1H,
d, J = 8.3 Hz), 7.88–7.94 (1H, m), 8.26 (1H, s), 8.33
(1H, s), 8.48 (1H, dd, J = 2.4, 8.3 Hz), 8.77 (1H, d,
J = 2.5 Hz), 8.99–9.02 (1H, m); FAB MS m/e (MH)+
5.1.4. N0-[(1E)-(6-Chloroimidazo[1,2-a]pyridin-3-yl)meth-
ylene]-2-methyl-5-nitrobenzenesulfonohydrazide (7d). Com-
pound 7d was prepared from 10b using the same
procedure as that for 7a. Compound 7d was obtained
1
as a colorless solid (60% yield): H NMR (DMSO-d6)
d 2.77 (3H, s), 7.49 (1H, dd, J = 2.0, 9.3 Hz), 7.75
(1H, s), 7.77 (1H, s), 8.08 (1H, s), 8.31 (1H, s), 8.43
(1H, dd, J = 2.4, 8.8 Hz), 8.79 (1H, d, J = 2.4 Hz), 8.82
(1H, d, J = 2.0 Hz), 12.20 (1H, br s); FAB MS m/e
(MH)+ 394.

5842
M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 5837–5844
1
408; Anal. for C16H14N5O4SClÆHClÆ0.8H2O: Calcd. C,
41.69; H, 3.25; N, 15.33; S, 6.94; Cl, 15.38. Found C,
41.89; H, 3.65; N, 15.27; S, 6.99; Cl, 15.46.
238 ꢁC (MeOH); H NMR (DMSO-d6) d 2.68 (3H, s),
3.47 (3H, s), 7.75–7.83 (3H, m), 7.94 (1H, d,
J = 9.3 Hz), 8.22 (1H, s), 8.36 (1H, s), 8.43 (1H, dd,
J = 2.5, 8.3 Hz), 8.71 (1H, d, J = 2.5 Hz), 9.43 (1H, s);
FAB MS m/e (MH)+ 399; Anal. for C17H14N6O4SÆHCl:
Calcd. C, 46.95; H, 3.48; N, 19.33; S, 7.37; Cl, 8.15.
Found C, 46.95; H, 3.34; N, 19.55; S, 7.02; Cl, 8.09.
5.1.9. N0-[(1E)-(6-Fluoroimidazo[1,2-a]pyridin-3-yl)methyl-
ene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide hydro-
chloride (8e). A mixture of 9c (1.0 g, 8.9 mmol) and
bromomalonaldehyde (2.7 g, 18 mmol) in EtOH
(10 mL) was refluxed for 18 h. After evaporation, the
residue was washed with EtOAc and Et2O. A mixture
of the resulting crude aldehyde 10c and methylhydrazine
(0.38 mL, 11 mmol) in EtOH (30 mL) was stirred at
room temperature for 2 h and then heated at 60 ꢁC for
0.5 h. After evaporation, pyridine (13 mL) and 2-
methyl-5-nitrobenzenesulfonyl chloride (2.1 g, 10 mmol)
were added to the resulting crude hydrazone and the
reaction mixture was stirred for 12 h. After evaporation,
the residue was washed with water and EtOH. 4 N HCl/
EtOAc (3 mL) was added to a suspension of the result-
ing solid in EtOH (10 mL). The mixture was concen-
trated and washed with hot EtOH to give 8e (382 mg,
12% yield) as a colorless solid: mp 214–216 ꢁC (MeOH);
1H NMR (DMSO-d6) d 2.67 (3H, s), 3.49 (3H, s), 7.75–
7.85 (2H, m), 7.90–7.99 (1H, m), 8.24–8.29 (1H, m), 8.32
(1H, s), 8.49 (1H, dd, J = 2.5, 8.3 Hz), 8.76–8.83 (1H,
m); FAB MS m/e (MH)+ 392; Anal. for
C16H14N5O4SFHCl: Calcd. C, 44.92; H, 3.53; N,
16.37; S, 7.49; Cl, 8.29; F, 4.44. Found C, 44.77; H,
3.47; N, 16.45; S, 7.49; Cl, 8.28; F, 4.44.
5.1.13. Ethyl 3-((E)-{methyl[(2-methyl-5- nitrophenyl)sul-
fonyl]hydrazono}methyl)imidazo[1,2-a]pyridine-6-carbox-
ylate hydrochloride (8i). Compound 8i was prepared
from 9g using the same procedure as that for 8e. Com-
pound 8i was obtained as a colorless solid (28% yield):
1
mp 174–176 ꢁC (MeOH); H NMR (DMSO-d6) d 1.40
(3H, t, J = 6.8 Hz), 2.71 (3H, s), 3.48 (3H, s), 4.42
(3H, q, J = 6.8 Hz), 7.73 (1H, d, J = 8.3 Hz), 7.94 (1H,
d, J = 9.3 Hz), 8.07 (1H, dd, J = 2.0, 9.6 Hz), 8.30 (1H,
s), 8.34 (1H, s), 8.36 (1H, dd, J = 2.4, 8.8 Hz), 8.79
(1H, d, J = 2.4 Hz), 10.01–10.04 (1H, m); FAB MS m/e
(MH)+ 446; Anal. for C19H19N5O6SÆHCl: Calcd. C,
47.35; H, 4.18; N, 14.53; S, 6.65; Cl, 7.36. Found C,
47.04; H, 4.18; N, 14.39; S, 6.65; Cl, 7.26.
5.1.14. 3-((E)-{Methyl[(2-methyl-5-nitrophenyl)sulfonyl]-
hydrazono}methyl)imidazo[1,2-a]pyridine-6-carboxamide
hydrochloride (8j). A solution of LiOH hydrate (510 mg,
12 mmol) in water (5 mL) was added to a solution of
free base of 8i (2.8 g, 6.3 mmol) in a mixture of EtOH
(30 mL), and water (5 mL). After stirring for 23 h, the
reaction mixture was acidified with 4 N HCl/EtOAc
and then evaporated. The resulting carboxylic acid was
prepared as a suspension in THF (50 mL) and CDI
(2.4 g, 15 mmol) was added. After stirring at room
temperature for 2.5 h, 28% aqueous NH4OH (50 mL)
was added and the reaction mixture was stirred for
2 d. The mixture was concentrated and washed with
hot EtOH suspended in MeOH, acidified with 4 N
HCl/EtOAc (2 mL), evaporated, and washed with
MeOH–EtOH to give 8j as a colorless solid (1.74 g,
5.1.10. N0-[(1E)-(6-Trifluoromethylimidazo[1,2-a]pyridin-3-
yl)methylene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide
hydrochloride (8f). Compound 8f was prepared from 9d
using the same procedure as that for 8e. Compound 8f
was obtained as a colorless solid (11% yield): mp
207–209 ꢁC (MeOH); 1H NMR (DMSO-d6) d 2.67
(3H, s), 3.48 (3H, s), 7.76 (1H, d, J = 8.3 Hz), 7.79–
7.86 (1H, m), 8.00 (1H, d, J = 9.3 Hz), 8.21–8.27 (1H,
m), 8.36 (1H, s), 8.42 (1H, dd, J = 2.4, 8.3 Hz), 8.71
(1H, d, J = 2.5 Hz), 9.62 (1H, s); FAB MS m/e (MH)+
442; Anal. for C17H14N5O4SF3ÆHCl: Calcd. C, 42.73;
H, 3.16; N, 14.66; S, 6.71; Cl, 7.42; F, 11.93. Found C,
42.81; H, 3.03; N, 14.91; S, 6.66; Cl, 7.20; F, 12.04.
1
53% yield): mp 220–223 ꢁC (MeOH–EtOH); H NMR
(DMSO-d6) d 2.68 (3H, s), 3.50 (3H, s), 7.68 (1H, br
s), 7.73 (1H, d, J = 8.3 Hz), 7.98 (1H, d, J = 9.3 Hz),
8.21 (1H, dd, J = 1.0, 9.3 Hz), 8.30 (1H, br s), 8.36
(1H, s), 8.38 (1H, dd, J = 2.5, 8.3 Hz), 8.42 (1H, s),
8.74 (1H, d, J = 2.4 Hz), 9.73 (1H, s); FAB MS m/e
(MH)+ 417; Anal. for C17H16N6O5SÆHClÆ0.2H2O:
Calcd. C, 44.73; H, 3.84; N, 18.41; S, 7.02; Cl, 7.77.
Found C, 44.69; H, 3.72; N, 18.37; S, 6.99; Cl, 7.55.
5.1.11. N,2-Dimethyl-N0-[(1E)-(6-methylimidazo[1,2-a]pyri-
din-3-yl)methylene]-5-nitrobenzenesulfonohydrazide hydro-
chloride (8g). Compound 8g was prepared from 9e
using the same procedure as that for 8e. Compound 8g
was obtained as a colorless solid (14% yield): mp
235–238 ꢁC (MeOH); 1H NMR (DMSO-d6) d 2.37
(3H, s), 2.69 (3H, s), 3.50 (3H, s), 7.79 (1H, d,
J = 8.8 Hz), 7.84 (1H, dd, J = 1.5, 9.3 Hz), 7.95 (1H, d,
J = 9.3 Hz), 8.33 (1H, s), 8.40 (1H, s), 8.45 (1H, dd,
J = 2.5, 8.3 Hz), 8.76 (1H, d, J = 2.9 Hz), 9.04 (1H, s);
FAB MS m/e (MH)+ 388; Anal. for C17H17N5O4SHCl:
Calcd. C, 48.17; H, 4.28; N, 16.52; S, 7.56; Cl, 8.36.
Found C, 47.90; H, 4.18; N, 16.69; S, 7.54; Cl, 8.13.
5.1.15. 2-Amino-5-cyanopyridine (9f). TFAA (18 g,
86 mmol) was slowly added to a mixture of 12 (5.0 g,
37 mmol) and Et3N (15 g, 148 mmol) at 0 ꢁC. After stir-
ring at room temperature for 4 h, the reaction mixture
was evaporated and diluted with brine and EtOAc.
The organic layer was separated, dried over MgSO4,
and evaporated. K2CO3 (5.5 g, 40 mmol), MeOH
(90 mL) and water (30 mL) were added to the resulting
residue and the reaction mixture was stirred overnight
and then evaporated. The residue was dissolved in brine
and EtOAc, and the organic layer was separated, dried
over MgSO4, and concentrated to give 9f (3.5 g, 80%
5.1.12. N0-[(1E)-(6-Cyanoimidazo[1,2-a]pyridin-3-yl)methyl-
ene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide hydro-
chloride (8h). Compound 8h was prepared from 9f
using the same procedure as that for 8e. Compound 8h
was obtained as a colorless solid (9% yield): mp 236–
1
yield) as a brown solid: H NMR (CDCl3) d 5.01 (2H,
br s), 6.50 (1H, d, J = 8.8 Hz), 7.62 (1H, d, J = 2.5,

M. Hayakawa et al. / Bioorg. Med. Chem. 15 (2007) 5837–5844
5843
8.8 Hz), 8.36 (1H, d, J = 2.5 Hz); FAB MS m/e (MH)+
120.
in volume, test compound was administered intraperito-
neally. The tumor volume was calculated by the follow-
ing
formula:
1/2 · (shorter
diameter)2 · (longer
5.1.16. 6-Bromoimidazo[1,2-a]pyridine-3-carbaldehyde (10a).
Bromomalonaldehyde (6.0 g, 40 mmol) was added to a
mixture of 9a (6.0 g, 35 mmol) in MeCN (60 mL) and
the reaction was allowed to proceed at 75 ꢁC for
15 min. After addition of EtOH (10 mL) the mixture
was refluxed for 3 h. Following concentration of the
mixture, the residue was suspended in a mixture of
CHCl3 and water. Insoluble materials were removed
by filtration and then the organic layer was dried over
MgSO4 and evaporated to give 5c (3.3 g, 42% yield) as
a brown solid: 1H NMR (DMSO) d: 7.84–7.89 (2H,
m), 8.56 (1H, s), 9.49 (1H, s), 9.96 (1H, s); FAB MS
m/e (M+H)+ 225, 227.
diameter). Test compound was suspended in 20%
hydroxypropyl-b-cyclodextrin/saline, and doses are gi-
ven as the free base.
Acknowledgments
We thank Dr. N. Taniguchi for his useful advice on the
preparation of the manuscript. We are grateful to Mr.
H. Uebayashi for performing stability tests and mem-
bers of the Division of Analytical Research for spectros-
copy measurements. This work was funded in part by
Cancer Research UK [CUK] Programme Grant C308/
A2187 and Paul Workman is a Cancer Research UK
Life Fellow.
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, 0.4 mM EGTA; PI3K C2b assay:
20 mM Tris–HCl (pH 7.4), 160 mM NaCl, 2 mM dithio-
threitol, 5 mM MgCl2, 15 mM CaCl2, 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.
The mixture was left to stand for 5 min and then centri-
fuged at 300g for 2 min. Radioactivity was measured
using TopCount (Packard). IC50 values represent means
of at least two separate determinations with typical vari-
ations of less than 20%.
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