Discovery and optimization of a series of 2-aminothiazole-oxazoles as potent phosphoinositide 3-kinase γ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.10.028

A novel series of 2-aminothiazole-oxazoles was designed and synthesized as part of efforts to develop potent phosphoinositide 3-kinase γ (PI3Kγ) inhibitors. The modification of a high-throughput screening hit, compound 1, resulted in the identification of compounds 10 and 15, which displayed potent inhibitory activities in enzyme-based and cell-based assays.

Full text of DOI:10.1016/j.bmcl.2012.10.028

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7534–7538
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery and optimization of a series of 2-aminothiazole-oxazoles
as potent phosphoinositide 3-kinase
c inhibitors
Yusuke Oka a, , Tetsuya Yabuuchi a, Yasuyuki Fujii b, Hidenori Ohtake b, Shunichi Wakahara b,
⇑
Kayo Matsumoto b, Mayumi Endo a, Yunoshin Tamura a, Yoshinori Sekiguchi a
a Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-Cho, Kita-Ku, Saitama-Shi, Saitama 331-9530, Japan
b Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-Cho, Kita-Ku, Saitama-Shi, Saitama 331-9530, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 2-aminothiazole-oxazoles was designed and synthesized as part of efforts to develop
Received 11 July 2012
Revised 2 October 2012
Accepted 5 October 2012
Available online 12 October 2012
potent phosphoinositide 3-kinase
hit, compound 1, resulted in the identification of compounds 10 and 15, which displayed potent inhibi-
tory activities in enzyme-based and cell-based assays.
c (PI3Kc) inhibitors. The modification of a high-throughput screening
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Phosphoinositide 3-kinase
PI3Kc inhibitor
2-Aminothiazole-oxazoles
S
N
The phosphoinositide 3-kinase family (PI3Ks) of lipid kinases is
involved in a diverse set of cellular functions, including cell growth,
proliferation, motility, differentiation, glucose transport, survival
intracellular trafficking, and membrane ruffling.1 PI3Ks can be cat-
egorized as class I, II, or III molecules, depending on their subunit
structure, regulation, and substrate selectivity. Class I PI3Ks are
subdivided into class IA and class IB. Class IA PI3Ks contain
O
OH
N
N
H
S
N
H
O
1
PI3Kγ IC50 = 5 nM
Figure 1. Structure and inhibitory activity of the initial hit compound 1.
p110
are activated in tyrosine kinase receptor signaling. Class IB PI3Ks
contain only p110 as a catalytic subunit, which is mostly acti-
vated by seven-transmembrane G-protein-coupled receptors
(GPCRs) via the regulatory subunit p101 and the G-protein b sub-
units.2,3 While PI3K
and PI3Kb are ubiquitously expressed, the
expressions of PI3K and PI3Kd are mainly restricted to the hema-
topoietic system. Genetic studies show that PI3K plays a crucial
a, p110b, and p110d as catalytic subunits, and these subunits
c
c
a
c
c
role in mediating leukocyte chemotaxis as well as mast cell
degranulation, making it a potentially interesting target for auto-
immune and inflammatory diseases such as psoriasis and rheuma-
toid arthritis.4–10 Herein, we describe our initial efforts in this field,
which led to the discovery and optimization of a series of 2-amino-
thiazole-oxazoles as potent PI3Kc inhibitors.
We conducted a high-throughput screening (HTS) and identi-
fied compound 1 as an initial hit (Fig. 1). In response to this result,
which had a promising enzyme activity (IC50 = 5 nM), compound 1
was docked into a molecular model that was constructed based on
the X-ray structure of PI3Kc
co-crystallized with its inhibitor.11,12
As shown in Figure 2, we speculated that nitrogen atoms in the
Figure 2. Predicted binding model of HTS hit compound 1 (orange) bound to the
ATP site of PI3Kc
(PDB code:2CHZ).11 The protein surface is colored according to the
residue type (magenta, hydrophilic; light green, lipophilic; white, neutral). Oxygen,
nitrogen and sulfur atoms are shown in the structure in red, blue, and yellow,
respectively. Each hydrogen bond is shown by dotted lines (light blue).
⇑
Corresponding author. Tel.: +81 48 669 3064; fax: +81 48 652 7254.
E-mail address: yusuke.oka@po.rd.taisho.co.jp (Y. Oka).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.028

Y. Oka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7534–7538
7535
S
N
N
N
O
O
OH
OH
N
O
N
H
N
H
S
S
N
H
N
H
O
O
1
2
PI3Kγ IC50 = 12 nM
Figure 3. Modification of central heterocycle and 2-aminothiazole-oxazole derivative 2.
aminothiazole formed known hydrogen bonds to the hinge Val882
and that the benzoic acid moiety interacted with Lys807 and
Lys833 via hydrogen bonds.
Thus, our design strategy prioritized the incorporation of the
benzoic acid pharmacophore (B ring in Fig. 2) into acetylamino-
thiazole (A ring in Fig. 2) through central heterocycles to generate
The ester of 5 was hydrolyzed to the corresponding carboxylic
acid 2. Deprotection of the acetyl groups of 11 and 13 yielded
the desired products 12 and 14, respectively. In the second ap-
proach, reduction of the azide moiety of 4 under acidic condition
yielded 17, which was coupled with an appropriate carboxylic
acid, followed by dehydration of 18–20 to yield target com-
pounds 23, 24, and the intermediate 21. Compound 22 was syn-
thesized in a manner similar to the method described for the
synthesis of 2. Finally, in the third approach, 17 was converted
to 2-mercapto-1,3-oxazole 25, which served as a versatile inter-
mediate for the preparation of several analogues, as shown in
Scheme 1. Compound 25 was coupled with 3-iodopyridine to
yield 26. Chlorination of 25 with phosphorous oxychloride
yielded 2-chlorooxazole 27. Coupling or SNAr displacement of
27 furnished the desired products 28–31, respectively. Methyla-
tion of 25 yielded 32, which was converted to 33 via oxidation.
As shown, the synthetic availability allowed the rapid exploration
of the SAR outlined below.
novel molecules with the expectation of a high affinity for PI3Kc.
For this purpose, we synthesized various kinds of central heterocy-
cles (data not shown) and identified oxazole derivative 2, with an
IC50 value of 12 nM (Fig. 3). This molecule appeared to be a poten-
tial lead for further optimization.
The general synthesis of 2-aminothiazole-oxazole derivatives is
illustrated in Scheme 1.
The first synthetic approach was as follows. Bromoacetylthiaz-
ole 3 was reacted with sodium azide to yield 4. The following
construction of the oxazole ring with acid chloride or isothiocya-
nate (as appropriate) using aza-Wittig reaction13 provided target
compounds 6–10, 15, and 16 and intermediates 5, 11, and 13.
H
N
H
N
H
N
*
*
*
R1
=
Cl
N
Cl
N
HBr
6
7
8
N3
Br
N
N
a
N
b
O
O
N
O
9
10:
:
R
R
1 = Me
S
O
R1
S
1 = CH2CF3
O
N
H
S
N
H
O
N
H
O
O
11: R1 = CH2OAc
H
N
c
*
3
4
1
OEt
1 = CH2OH
12:
R
5
2
: R
=
13: R1 = C(Me)2OAc
c
H
N
c
1
1 = C(Me)2OH
: R
=
14:
15:
R
R
*
OH
1 = tert-Bu
16: R1 = OMe
2HCl
O
NH2
NH
d
N
N
N
O
e
N
f
O
c
O
R1
4
S
S
O
N
N
H
R1
S
O
O
N
H
H
O
O
O
17
*
OMe
OH
21 : R1=
*
OMe
1
18
: R =
22 : R1=
*
*
*
1
g
19
20
: R =
N
23 : R1=
: R1 = 3-pyridyl
N
24 : R1 = 3-pyridyl
N
N
N
O
N
N
h
N
O
N
N
i
O
j
O
N
S
O
O
S
N
SH
S
O
S
S
O
R1
N
H
Cl
N
N
H
H
H
26
25
27
OH
O
k
*
O
R1=
N
N
N
N
*
*
*
28
29
30
31
O
N
N
N
N
O
O
l
S
N
S
O
O
N
S
H
S
H
O
32
33
Scheme 1. Synthesis of 2-aminothiazole-oxazole derivatives. Reagents and conditions: (a) NaN3, TEA, MeOH, rt, 90%; (b) R1COCl, PPh3, THF or dioxane, rt, or R1NCS, PPh3,
dioxane, 100 °C; (c) aq KOH, THF–MeOH, 60 °C; (d) H2, 10% Pd/C, c HCl, MeOH, rt, 75%; (e) R1COOH, PyBOPÒ, TEA, DMF, rt, or R1COCl, TEA, THF–CHCl3, rt; (f) POCl3, reflux, or
Burgess reagent, THF, 80 °C; (g) CS2, aq Na2CO3, EtOH, 80 °C, 57%; (h) 3-iodopyridine, NaOt-Bu, CuI, 1,10-phenanthroline, DMF, 100 °C, 56%; (i) POCl3, 120 °C, 52%; (j) 3-
hydroxypyridine, K2CO3, DMSO, 100 °C, 26%, or amine, THF, 80 °C; (k) MeI, K2CO3, DMF, rt, 45%; (l) H2O2, Mo7O24(NH4)6Á4H2O, THF–EtOH, rt, 62%.

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Y. Oka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7534–7538
Table 1
SAR of oxazole derivatives with R1-substitution modifications
N
N
O
R1
S
O
N
H
Compound
R1
PI3K
IC50 (nM)
c
PI3K
IC50 (nM)
a
Selectivity (
a/
c
)
Akt
IC50 (nM)
a
a
a
*
OH
OH
2
22
6
12
328
NTb
NTb
27
>10,000
N
H
O
*
*
223
346
>10,000
NTb
O
Cl
N
H
Cl
NTb
102
1040
431c
*
*
7
8
24
12
N
N
N
H
9
N
H
23
28
95
NTb
NTb
NTb
*
*
N
N
117
1666c
O
S
26
24
16
3
112
46
7
279
59
*
N
15
N
*
a
b
c
The IC50 value represents the mean of at least two independent experiments.
NT = not tested.
Data from one experiment (n = 1).
The series of 2-aminothiazole-oxazoles was screened using an
Compound 24 exhibited high permeability in the PAMPA assay
(Pe 77.3 Â 10À6 cm/s at pH 6.2) as to the cell activity and 24
enzyme assay,14 and the effects of the derivatives on C5a-mediated
PKB/Akt phosphorylation were investigated in Raw-264 murine
had an 15-fold selectivity against PI3Ka. However, 24 had poor
macrophages.15 This assay allowed the potency of PI3K
to be assessed in cells (Table 1).
c
inhibition
physicochemical properties (the solubility of 24 was 0.39 lg/mL
[in water]).
Compound 2 exhibited a good enzyme potency but it did not
exhibit any inhibitory activity against PI3K in cells. The discrep-
To further improve poor physicochemical properties, we at-
tempted to incorporate aliphatic groups to produce potent PI3K
inhibitors with an improved solubility. Aliphatic compounds with
PI3K IC50 values <50 nM in the enzyme assay were tested for
c
c
ancy between enzyme and cellular activities was considered to be
due to the limited cell permeability of 2 (PAMPA permeability of
2 was 1.4 Â 10À6 cm/s at pH 6.2). The replacement of the NH lin-
ker with a carbon-linkage in compound 22 resulted in a loss of
enzyme potency and did not improve the cellular activity. An at-
tempt to remove carboxylic acid (6) to change the cell permeabil-
ity diminished the enzyme potency. However, the conversion
from benzene to pyridine (7) resulted in an increased enzyme
activity and consistent cellular potency. This data showed that
the inhibitory activity could be increased without focusing on
the interaction with Lys807, 833. The elimination of a chlorine
atom (8) improved the potency. Further exploration of the linker
pattern, such as modifications of the nitrogen-linked analogue 8
to a carbon-linked analogue 23 or an oxygen-linked analogue
28, reduced the activity, but the sulfur-linked analogue 26 exhib-
ited a potency equivalent to that of compound 8. Interestingly,
the removal of a linkage atom (24) provided a substantial boost
in enzyme and cellular potencies (IC50 = 3, 59 nM, respectively).
c
the inhibition of phosphorylation of Akt (Table 2).
Methyl (9) and hydroxymethyl (12) derivatives resulted in a
decreased potency, but the trifluoroethyl derivative 10, which
was equivalent to a bulky alkyl group, exhibited an improved po-
tency. Compound 14, which was substituted with two methyl
groups at the benzylic position of 12, also exhibited an increased
potency, and the tert-butyl derivative 15, in which a hydroxyl
substituent was replaced with the methyl moiety of 14, exhibited
an 8-fold improvement in enzyme activity. Membrane permeabil-
ity of 15 was good (PAMPA, Pe 88.4 Â 10À6 cm/s at pH 6.2) and
the cell activity of 15 exhibited an IC50 value of 113 nM. We rea-
soned that filling the empty space near the central heterocycle
using a lipophilic substituent would lead to inhibitors with im-
proved potency. Nitrogen derivatives exhibited modest activities
against PI3K
c. The introduction of polar functional groups, such
as ether (30) or hydroxyl group (31) at the distal position, also

Y. Oka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7534–7538
7537
Table 2
SAR of oxazole derivatives with aliphatic R1 group
N
N
O
S
O
N
H
R1
Compound
R1
PI3K
IC50 (nM)
c
PI3K
IC50 (nM)
a
Selectivity (
a/
c
)
Akt
IC50 (nM)
a
a
a
9
Me
181
NTb
NTb
F
F
F
10
*
10
43
4
88
12
14
OH
OH
161
25
NTb
NTb
NTb
*
*
369c
*
*
15
29
3
15
5
113
NTb
1222c
N
46
*
*
NTb
NTb
599c
237
N
30
31
24
14
O
OH
N
*
*
*
16
32
250
22
NTb
NTb
NTb
O
S
S
322c
33
12
195
16
222
O
O
a
b
c
The IC50 value represents the mean of at least two independent experiments.
NT = not tested.
Data from one experiment (n = 1).
6. Li, Z.; Jiang, H.; Xie, W.; Zhang, Z.; Smrcka, A. V.; Wu, D. Science 2000, 287, 1046.
7. Laffargue, M.; Calvez, R.; Finan, P.; Trifilieff, A.; Barbier, M.; Altruda, F.; Hirsch,
E.; Wymann, M. P. Immunity 2002, 16, 441.
8. Camps, M.; Rückle, T.; Ji, H.; Ardissone, V.; Rintelen, F.; Shaw, J.; Ferrandi, C.;
Chabert, C.; Gillieron, C.; Francon, B.; Martin, T.; Gretener, D.; Perrin, D.; Leroy,
D.; Vitte, P. A.; Hirsch, E.; Wymann, M. P.; Cirillo, R.; Schwarz, M. K.; Rommel, C.
Nat. Med. (NY) 2005, 11, 936.
9. Wymann, M. P.; Bjoerkloef, K.; Calvez, R.; Finan, P.; Thomast, M.; Trifilieff, A.;
Barbier, M.; Altruda, F.; Hirsch, E.; Laffargue, M. Biochem. Soc. Trans. 2003, 31,
275.
10. Vincent, P.; Jasna, K.; David, C.; Dennis, D. C.; Jeffrey, P. S.; Karen, R.; Fabienne,
B.-C.; Delphine, V.; Montserrat, C.; Christian, C.; Corinne, G.; Bernard, F.;
Dominique, P.; Didier, L.; Denise, G.; Anthony, N.; Pierre, A. V.; Susanna, C.;
Christian, R.; Matthias, K. S.; Thomas, R. J. Med. Chem. 2006, 49, 3857.
11. Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.; Goldenberg, D. D.;
Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.;
Williams, R. L.; Shokat, K. M. Cell 2006, 125, 733.
improved the potency. The oxygen derivative 16 showed a re-
duced activity, whereas sulfur derivatives (32 and 33) displayed
potent enzyme activities. However, 33 was unstable under basic
conditions.
As can be seen, 10 and 15 were optimal, and these compounds
tended to be equipotent with 24. Compound 15 was found to have
better physicochemical properties compared with 24 (the solubil-
ity of 15 was 35.20 lg/mL [in water]).
In summary, we have described the lead generation, synthesis,
and initial SAR study for a novel series of 2-aminothiazole-oxaz-
oles. Compounds 10 and 15 which were produced through the
optimization of 2 displayed good enzymatic and cellular activities.
Further modifications of this series to improve
currently in progress.
a/c selectivity are
12. The binding model was examined and visualized using MOE™ (Molecular
Operating Environment) Version 2010.10, Chemical Computing Group:
Montreal, Canada.
Acknowledgment
13. Typical procedure for preparation of 2-aminothiazole-oxazole derivatives (5–
16). To a mixture of 4 (2.94 g, 12.3 mmol) and triphenylphosphine (4.84 g,
18.4 mmol) in dioxane (50 ml) was added pivaloyl chrolide (1.63 g,
13.5 mmol) slowly, stirred at room temperature for 15 h. Sat. NaHCO3 was
added, and the mixture was extracted with AcOEt. The organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography to afford 15
(475 mg, 1.70 mmol) as a colorless powder. 1H NMR (200 MHz, DMSO-d6): d
1.36 (s, 9H) 2.16 (s, 3H) 2.40 (s, 3H) 7.19 (s, 1H) 12.26 (br s, 1H). MS ESI/APCI
Dual m/z 280 [M+H]+.
The authors thank Kyoko Taguchi and Akiko Nozoe for collect-
ing chemical property data.
References and notes
1. Engelman, J. A.; Luo, J.; Cantley, L. C. Nat. Rev. Genet. 2006, 7, 606.
2. Katso, R.; Okkenhaug, K.; Ahmandi, K.; White, S.; Timms, J. Annu. Rev. Cell Dev.
Biol. 2000, 17, 615.
3. Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.; Katso, R.; Driscoll, P.
C.; Woscholski, R.; Parker, P. J.; Waterfield, M. D. Annu. Rev. Biochem. 2001, 70,
535.
4. Sasaki, T.; Irie-Sasaki, J.; Jones, R. G.; Oliveira-dos-Santos, A. J.; Stanford, W. L.;
Bolon, B.; Wakeham, A.; Itie, A.; Bouchard, D.; Kozieradzki, I.; Joza, N.; Mak, T.
W.; Ohashi, P. S.; Suzuki, A.; Penninger, J. M. Science 2000, 287, 1040.
5. Hirsch, E.; Katanaev, V. L.; Garlanda, C.; Azzolino, O.; Pirola, L.; Silengo, L.;
Sozzani, S.; Mantovani, A.; Altruda, F.; Wymann, M. P. Science 2000, 287, 1049.
14. Human PI3K
buffer (20 mM Tris–HCl [pH 7.4], 5 mM MgCl2, 5 mM DTT and 10
Ci
33P]ATP, final concentrations) and lipid vesicles containing 5
PtdIns (Calbiochem) and 25 M of PtdSer (Sigma) (final concentrations) in the
presence of inhibitors or DMSO. The kinase reaction was stopped by the
c
(20 ng, Millipore) was incubated for 2 h at 30 °C with kinase
M ATP/10
M of
l
l
c
[
l
l
addition of 25 mM of EDTA, and the samples were incubated using
a
FlashPlateÒ (a phospholipid 96-well scintillant-coated microplate;
PerkinElmer) with substrate coating buffer (PerkinElmer). After the
incubation step, the wells were washed with PBS. The bound radioactivity
was determined by measuring the counts per second (CPS) using a packard top

7538
Y. Oka et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7534–7538
count microplate scintillation counter (PerkinElmer). A human PI3K
Millipore) assay was performed using the same procedure.
15. C5a-Mediated PKB/Akt phosphorylation in macrophages. Raw264.7, a murine
macrophage-like cell line, was seeded on 6-well plates at a density of 8 Â 106
cells per well and grown for 24 h. After replacing the medium with fresh
a
(20 ng,
serum-free 0.1% BSA medium and incubating for 3 h at 37 °C, the cells were
pretreated with or without various concentration of inhibitors for 30 min and
then stimulated with 5 nM of C5a (R&D Systems) for 5 min at 37 °C. The PKB/
Akt phosphorylation levels were determined using a phospho-Ser-473 Akt-
specific antibody kit (CST), according to the manufacturer’s protocol.