Discovery of N-{5-[3-(3-hydroxypiperidin-1-yl)-1,2,4-oxadiazol-5-yl]-4- methyl-1,3-thiazol-2-yl}acetamide (TASP0415914) as an orally potent phosphoinositide 3-kinase γ inhibitor for the treatment of inflammatory diseases

Article abstract of DOI:10.1016/j.bmc.2013.10.042

Class I phosphoinositide 3-kinases (PI3Ks), particularly PI3Kγ, have become attractive drug targets for inflammatory and autoimmune disorders such as rheumatoid arthritis. Herein, we describe the synthesis and the structure-activity relationships (SAR) of a series of 2-amino-5-oxadiazolyl thiazoles, culminating in the identification of 8j (TASP0415914), an orally potent inhibitor of phosphoinositide 3-kinase γ (PI3Kγ). TASP0415914 demonstrated good potency in a cell-based assay and, furthermore, exhibited in vivo efficacy in a collagen induced arthritis (CIA) model in mice after oral administration.

Full text of DOI:10.1016/j.bmc.2013.10.042

Bioorganic & Medicinal Chemistry 21 (2013) 7578–7583
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of N-{5-[3-(3-hydroxypiperidin-1-yl)-1,2,
4
(
c
-oxadiazol-5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide
TASP0415914) as an orally potent phosphoinositide 3-kinase
inhibitor for the treatment of inflammatory diseases
Yusuke Oka a, , Tetsuya Yabuuchi , Takahiro Oi , Shoichi Kuroda , Yasuyuki Fujii , Hidenori Ohtake ,
⇑
a
a
a
b
b
b
b
b
b
a
c
Tomoyuki Inoue , Shunichi Wakahara , Kayo Kimura , Kiyoko Fujita , Mayumi Endo , Kyoko Taguchi ,
a
Yoshinori Sekiguchi
a
Medicinal Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-Cho, Kita-Ku, Saitama-Shi, Saitama 331-9530, Japan
Molecular Function and Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-Cho, Kita-Ku, Saitama-Shi, Saitama 331-9530, Japan
Pharmacokinetics and Metabolism Drug Safety and Pharmacokinetics Laboratories, Taisho Pharmaceutical Co., Ltd, 1-403, Yoshino-Cho, Kita-Ku,
b
c
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:
Class I phosphoinositide 3-kinases (PI3Ks), particularly PI3Kc, have become attractive drug targets for
inflammatory and autoimmune disorders such as rheumatoid arthritis. Herein, we describe the synthesis
and the structure–activity relationships (SAR) of a series of 2-amino-5-oxadiazolyl thiazoles, culminating
Received 5 October 2013
Revised 25 October 2013
Accepted 26 October 2013
Available online 7 November 2013
in the identification of 8j (TASP0415914), an orally potent inhibitor of phosphoinositide 3-kinase
PI3K ). TASP0415914 demonstrated good potency in a cell-based assay and, furthermore, exhibited
in vivo efficacy in a collagen induced arthritis (CIA) model in mice after oral administration.
Ó 2013 Elsevier Ltd. All rights reserved.
c
(
c
Keywords:
Phosphoinositide 3-kinase
-Amino-5-oxadiazolyl thiazole
The Ames test
Collagen induced arthritis (CIA) model
c
2
3
1
. Introduction
diseases. In contrast, Class IB PI3Ks contain only p110c as a cata-
lytic subunit, which is mostly activated by seven-transmembrane
Phosphoinositide 3-kinases (PI3Ks), a family of enzymes that
G-protein-coupled receptors (GPCRs) via its regulatory subunit
4
,5
catalyze the phosphorylation of the 3-hydroxyl position of the ino-
sitol ring, are activated by a wide variety of different stimuli
including growth factors, inflammatory mediators, hormones, neu-
rotransmitters, immunoglobulins, and antigens.1 PI3Ks are in-
volved in the regulation of numerous biological processes,
including cell growth, differentiation, survival, proliferation,
p101 and G-protein b
ited to hematopoietic and endothelial cells. PI3K
have shown that PI3K is essential for T-cell and B-cell develop-
ment and functionalities, and PI3K is also required for neutrophil
and mast cell migration as well as mast cell degranulation. These
results suggest that PI3K may be a valuable therapeutic target for
inflammatory disorders, such as rheumatoid arthritis.
As part of our efforts to identify potent PI3K inhibitors with
c
subunits. The expression of PI3K
c is lim-
c
knockout mice
c
c
2
c
2
6–10
migration, and metabolism. The most extensively investigated
are the Class I PI3Ks, which are subdivided into class IA and class
c
IB. Class IA PI3Ks contain p110
subunits, and these subunits are activated during tyrosine kinase
receptor signaling. PI3K and PI3Kb are ubiquitously expressed,
and mice lacking PI3K or PI3Kb die during embryonic develop-
ment. The deregulation of class IA PI3Ks is involved in the patho-
genesis of various diseases, thereby representing attractive
targets for oncology, inflammatory, metabolic and cardiovascular
a
, p110b, and p110d as catalytic
drug-like properties that are suitable for oral administration, we
recently reported the 2-amino-5-oxazolyl thiazole derivative 1.11
Although 2-acetylaminothiazole was a good hinge binder for PI3Ks,
one of the concerns regarding 2-aminothiazole derivatives is a po-
tential risk of mutagens. Indeed, a de-acetylated analogue of 1 was
positive in the Ames fluctuation test (2), although 1 itself was neg-
ative in the Ames test. Based on these findings, we sought to mit-
igate the mutagenic risk by introducing an electron-withdrawing
a
a
substituent or by replacing the oxazole ring with more
p-elec-
⇑
Corresponding author. Tel.: +81 48 669 3107; fax: +81 48 652 7254.
E-mail address: y-oka@so.taisho.co.jp (Y. Oka).
tron-deficient heterocycles, even though de-acetylated analogue
was not the major metabolite. These chemical modifications led
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.042

Y. Oka et al. / Bioorg. Med. Chem. 21 (2013) 7578–7583
7579
O
N
N
N
N
N
N
O
O
S
N
S
O
N
H
N
H
1
3
3
nM
14nM
negative
negative
O
N
N
S
8
O
H N
S
6
N
H N
2
2
2
4
8 nM
PI3Kγ IC50 values
results of the Ames test
1 nM
negative
positive
Figure 1. PI3K
c
inhibitory activities and the results of the Ames test for 2-amino-5-oxazolyl thiazole (1 and 2) and 2-amino-5-oxadiazolyl thiazole (3 and 4).
OH
O
OH
O
O
N
O
O
a
d
N
N
b
N
c
O
N
N
N
O
S
R1
S
S
N
H
S
H N
N
H
H N
2
2
5
6
7
8a-8m
OH
R1
N
R1
NH2
9a-9m
10a-10m
Scheme 1. Reagents and conditions: (a) 1 M NaOH, EtOH, rt, 97%; (b) acetyl chloride, pyridine, THF, rt, 91%; (c) 10a–m, EDCꢀHCl, HOBtꢀH
2
O, DMF, rt then 100 °C; (d) 50%
hydroxylamine solution, EtOH, reflux.
O
O
OH
O
NC
O
a
b, c
O
N
O
N
N
O
O
1
1
12
9j
HO
O
N
O
N
N
N
N
N
O
O
d
O
e
f
O
OH
H
2
N
N
S
S
N
N
N
N
N
H
H
O
O
1
0j
13
8j
Scheme 2. Reagents and conditions: (a) acetyl chloride, pyridine, rt; (b) 4 M HCl–AcOEt, AcOEt, rt; (c) cyanogen bromide, K
hydroxylamine solution, EtOH, reflux; (e) 7, EDCꢀHCl, HOBtꢀH
2
3 3
CO , CH CN, rt, 79% (three steps); (d) 50%
2
O, DMF, rt then 100 °C; (f) 2 M NaOH, THF–EtOH, rt, 10% (three steps).
to the identification of 2-amino-5-oxadiazolyl thiazole 3 which
was an equally potent PI3K inhibitor compared with 1. In addi-
tion, both 3 and its de-acetylated analogue 4 were negative in
the Ames fluctuation test up to 1000 and 2000 g/mL (at the same
concentration of 2), respectively (Fig. 1). These results prompted us
to further derivatize this lead compound. Herein, we report the
synthesis and the structure–activity relationship study of a series
of 2-amino-5-oxadiazolyl thiazoles with the goal of identifying or-
hydroxylamidine 10, prepared by the treatment of nitrile 9 with
50% hydroxylamine solution in ethanol, and the subsequent ring
closure reaction yielded oxadiazole 8. The nitrile intermediates 9
were prepared from commercially available carboxylic acid via
condensation and dehydration or the nucleophilic addition of so-
c
l
1
2
dium cyanide to alkyl halide. Nitrogen atom linked analogue 8j
was synthesized as shown in Scheme 2. Cyanamide 9j was readily
prepared from 1-Boc-3-hydroxylpiperidine 11 in three steps as fol-
lows: (1) acetylation of 11 with acetyl chloride, (2) removal of the
Boc group of 12 under acidic conditions (4 M HCl), and (3) cyana-
tion of the resulting secondary amine of 12 using cyanogen bro-
mide. Treatment of 9j with 50% hydroxylamine, followed by
coupling of 10j with 7 and thermal ring closure of the coupling
product, yielded 13. Removal of the acetyl group furnished the de-
ally potent PI3K
. Chemistry
The general route for the preparation of 2-amino-5-oxadiazolyl
c inhibitors.
2
thiazole derivatives is outlined in Scheme 1.
1
3
sired product 8j. Compound 8m was obtained in a manner sim-
ilar to that described above using morpholine as a starting
material.
Commercially available aminothiazole ester 5 was hydrolyzed
to the corresponding carboxylic acid 6, followed by treatment
with acetyl chloride to give acetamide 7. Coupling of 7 with

7
580
Y. Oka et al. / Bioorg. Med. Chem. 21 (2013) 7578–7583
Table 2
3
. Results and discussion
Metabolic stabilities in rat/human liver microsomes and hepatocytes and systemic
exposure level after the oral administration of selected compounds in rats
The series of 2-amino-5-oxadiazolyl thiazoles (3, 8a–m) were
Compd
Metabolic stability
Plasma exposure after poa
initially screened against the PI3Kc inhibition assay and com-
MSb (h/r)
Hepatocytec (h/r)
Cmax (ng/mL)
AUCd (ng h/mL)
pounds with an IC50 value of less than 50 nM in the enzyme assay
were then used to measure their ability to inhibit C5a-mediated
PKB/Akt phosphorylation in Raw-264 murine macrophages.
As shown in Table 1, lipophilic substituents on the oxadiazole
ring such as tert-butyl (3) and trifluoroethyl (8a) were well toler-
ated and resulted in equally potent Akt inhibitory activities. In con-
trast, the introduction of a hydroxyl group as a polar functional
e
e
8
8g
8i
a
79.1/72.9
102.1/94.0
94.1/91.5
96.9/97.1
NT /NT
345
38
7800
3920
1180
92
10900
10800
9.3/11.3
35.2/20.9
75.7/62.1
8j
a
Dosed po at 30 mg/kg as a 0.5% methyl cellulose (MC) solution to male SD rats.
Percentage remaining after 15 min of incubation with human/rat liver
b
1
microsomes.
moiety into the lipophilic substituent (R ) resulted in 4 to 11-fold
c
Percentage remaining after 1 h of incubation with human/rat hepatocytes.
Area under the plasma concentration–time curve from 0 to 8 h.
NT = not tested.
decrease in the enzyme inhibitory activities (8b–d). Elongation of
the ethylene spacer between the oxadiazole ring and the hydroxyl
group, however, slightly improved the potency (8b vs 8e). Interest-
ingly, the incorporation of a geminal dimethyl group into the 2-po-
sition of the hydroxypropyl side-chain (8g) enhanced the enzyme
inhibitory activity, while the installation to the 1- or 3-position
did not increase the potency (8f and 8h). The introduction of a
methyl moiety at the 3-position of the hydroxypropyl group of
d
e
methylether (8k), cyclic ether (8l) and morpholine (8m) deriva-
tives were more than nine-fold less potent than 8g. Most com-
pounds with an IC50 value of less than 50 nM in the enzyme
inhibition assay exhibited a good inhibitory activity for the phos-
phorylation of Akt with an IC50 value of less than 300 nM.
8
g was tolerable in terms of the potency, including the phosphor-
ylation of Akt (8i). The 3-hydroxypiperidine analogue (8j) with a
conformationally restricted secondary alcohol retained good
potencies in both enzyme and cell-based assays. By contrast,
The metabolic stabilities of these cellular potent inhibitors were
then further investigated in rat/human liver microsomes and hepa-
tocytes, and their exposure levels were measured after oral admin-
istration in rats at a dose of 30 mg/kg (Table 2).
Table 1
The in vitro metabolic stability of 8a in rat/human liver micro-
somes was lower than those of the others, while 8g resulted in the
lowest plasma exposure level in rats, probably because of its poor
metabolic stability in hepatocytes. On the other hand, 8i and 8j
exhibited plasma exposure levels more than 10-fold higher than
those of 8a and 8g. The reason why 8j was more stable in hepato-
cytes than 8g and 8i is probably because the hydroxyl group is
insusceptible to oxidative metabolism for its steric hindrance and
Structure–activity relationships of compounds 3 and 8a–m
O
N
N
O
S
N
R1
N
H
Compd
R1
PI2K
14
c IC50 (nM)
a
Akt IC50 (nM)
a
1
4
rigid conformation.
Further ADME evaluation of 8j revealed that it showed no CYP
inhibition up to 10 M for CYP1A2, 2C9, 2C19, 2D6 and 3A4. In
*
231a
3
l
F
F
F
278b
NTc
8
8
8
a
b
c
*
28
addition, 8j and its de-acetylated analogue were negative in the
Ames fluctuation test and the Ames test, respectively. Based on
the above data, 8j was firstly chosen for further in vivo evaluation.
The efficacy of 8j was evaluated in a mouse collagen-induced
arthritis (CIA) model, an autoimmune disease model that shares
pathogenic similarities with human rheumatoid arthritis.1 Mice
*
158
52
OH
OH
*
*
NTc
5a
8
d
68
NTc
OH
*
*
NTc
NTc
8
8
e
f
OH
OH
121
62
8
g
*
*
7.2
191
NTc
OH
OH
8
h
203
*
OH
8
i
17
155
OH
8
8
8
j
29
68
57
294
NTc
558
*
N
N
k
l
*
*
O
O
Figure 2. Suppression of the development of acute collagen-induced arthritis by 8j.
Collagen-primed DBA/1 mice (n = 9 mice/group) were treated orally with the
vehicle (n = 10 mice) or the indicated dosages of 8j twice daily from days 0 to 14
and the resulting arthritis scores were monitored for indications of disease
progression. Data are represented as the mean ± SEM (n = 9 or 10). ns: no significant
*
NTc
8
m
O
100
a
b
c
The IC50 values represent the mean of at least two independent experiments.
Data from one experiment (n = 1).
NT = not tested.
⁄
⁄⁄
differences, p < 0.05, p < 0.01, Steel’s test, relative to vehicle-treated group.

Y. Oka et al. / Bioorg. Med. Chem. 21 (2013) 7578–7583
7581
were orally treated with 8j twice daily for 14 consecutive days and
the mixture were removed in vacuo, water was poured into the
residue, and the suspension was stirred for 1 h. The resulting pre-
cipitate was collected by filtration, washed with water and dried in
vacuo to yield 7 (585 mg, 91%) as a colorless powder. 1H NMR
their clinical arthritis severity score was monitored.1 As a result,
5b
8
j suppressed the progression of the disease compared with a vehi-
cle control, at 30 and 100 mg/kg in a dose-dependent manner
Fig. 2).
(
(200 MHz, DMSO-d
6
) d 2.15 (s, 3H), 2.52 (s, 3H), 12.35 (br s, 1H);
+
+
ꢂ
MS (ESI): m/z 201 [M+H] , 223 [M+Na] , 199 [MꢂH] .
4
. Conclusion
5
.1.3. tert-Butyl 3-(acetyloxy)piperidine-1-carboxylate (12)
To solution of 1-Boc-3-hydroxylpiperidine 11 (2.50 g,
2.4 mmol) in pyridine (15.0 mL) was added acetic anhydride
a
In summary, we have performed a synthesis and a structure–
1
activity relationship study of a novel series of 2-amino-5-oxadiaz-
olyl thiazoles as potent PI3K inhibitors and have discovered that
(
11.7 mL, 124 mmol) at 0 °C, and the reaction mixture was stirred
c
for 4 h at room temperature. The mixture was diluted with water
in an ice bath and extracted with ethyl acetate twice. The com-
bined organic layer was washed with saturated aqueous sodium
hydrogen carbonate followed by brine, dried over anhydrous mag-
the central oxadiazole was efficacious for mitigating a mutagenic
risk, compared with an oxazole ring. Further optimization culmi-
nated in the identification of 8j (TASP0415914), which exhibited
good potency in a cell-based assay and in vivo efficacy using a ther-
apeutic CIA mouse model after oral administration. TASP0415914
nesium sulfate, and concentrated in vacuo to yield crude 12
1
(
3.33 g) as a colorless oil. H NMR (200 MHz, CDCl
3
) d 1.45 (s,
is a promising lead as an orally potent PI3K
of further modifications will be reported in due course.
c inhibitor. The results
9
4
H), 1.48ꢂ1.92 (m, 4H), 2.05 (s, 3H), 3.24ꢂ3.55 (m, 4H),
+
.71ꢂ4.84 (m, 1H); MS (ESI): m/z 266 [M+Na] .
5
5
. Experimental
5.1.4. 1-Cyanopiperidin-3-yl acetate (9j)
To a solution of 12 (1.00 g, 4.11 mmol) in ethyl acetate (5 mL)
was added 4 M hydrogen chloride in ethyl acetate (5.00 mL,
.1. Chemistry
2
0.0 mmol). The reaction mixture was stirred at room temperature
All commercially available starting materials and reagents were
used without further purification. The reactions were usually mon-
itored using TLC and Merck silica gel 60
for 1 day. After the volatiles of the mixture were removed in vacuo,
chloroform was added to the residue, and the mixture was neutral-
ized with triethylamine (1.72 mL, 12.3 mmol) and evaporated. To a
susupension (5 mL) of the residue in acetonitrile were added
potassium carbonate (0.68 g, 4.93 mmol) and cyanogen bromide
(0.44 g, 4.11 mmol). The reaction mixture was then stirred for 4 h
at room temperature. The precipitate was filtered off, and the fil-
trate was concentrated in vacuo. The residue was purified using sil-
ica gel column chromatography (ethyl acetate/n-hexane) to yield
F254 plates or Fuji Silysia chromatorex NH plates. Column chro-
matography was performed using silica gel Wako Pure Chemical C-
1
2
00 and NH-silica gel Fuji Silysia chromatorex DM1020. H NMR
spectra were recorded at 200, 300 or 600 MHz using a Varian
Instruments Gemini 2000, a Varian Instruments INOVA 300 or a
JEOL ECA 600 with TMS as an internal standard. Proton chemical
shifts were expressed in parts per million (ppm) in the indicated
solvent. Multiplicity was defined as s (singlet), d (doublet), t (trip-
let), q (quartet), dd (double doublet), m (multiplet), br s (broad sin-
glet), br d (broad doublet) or br t (broad triplet). Analytical LC was
performed using an Agilent 1290 Infinity LC (Waters Acquity CSH
1
9j (0.55 g, 79%) as a colorless oil. H NMR (200 MHz, CDCl ) d
3
1.55ꢂ2.05 (m, 4H), 2.11 (s, 3H), 3.07ꢂ3.39 (m, 4H), 4.81ꢂ4.95
+
+
(m, 1H); MS (ESI): m/z 169 [M+H] , 191 [M+Na] .
0
5.1.5. 1-(N -Hydroxycarbamimidoyl)piperidin-3-yl acetate (10j)
C18 column, 1.7
formic acid in acetonitrile gradient; UV detection at 210 and
54 nm). Mass spectra (MS) were recorded on a Micromass Plat-
lm, 2.1 ꢁ 50 mm; 0.1% formic acid in water/0.1%
To a solution of 9j (0.33 g, 1.96 mmol) in ethanol (5 mL) was
added 50% aqueous hydroxylamine solution (0.39 g, 5.88 mmol),
and the reaction mixture was then stirred at room temperature
for 1 h and evaporated in vacuo. The residue was purified using sil-
ica gel column chromatography (methanol/chloroform) to yield
2
form LC mass spectrometer with electrospray ionization (ESI). High
resolution mass spectra (HRMS) were recorded on a Shimadzu
LCMS-IT-TOF mass spectrometer with an electrospray ionization
1
10j (0.23 g, 58%) as a colorless oil. H NMR (200 MHz, CDCl ) d
3
(
ESI)/atmospheric pressure chemical ionization (APCI) dual source.
1.52ꢂ2.09 (m, 7H), 2.87ꢂ3.03 (m, 2H), 3.14ꢂ3.28 (m, 1H),
3.33ꢂ3.46 (m, 1H), 4.35 (br s, 2H), 4.71ꢂ4.90 (m, 1H); MS (ESI):
Elemental analyses were performed using a Perkin–Elmer 2400, a
Yanaco MT-6 or an Elementar vario MICRO cube elemental ana-
lyzer, and the results were within ±0.4% of the calculated values.
+
+
m/z 202 [M+H] , 224 [M+Na] .
5
.1.6. N-{5-[3-(3-Hydroxypiperidin-1-yl)-1,2,4-oxadiazol-5-yl]-
5
.1.1. 2-Amino-4-methyl-1,3-thiazole-5-carboxylic acid (6)
To a solution of ethyl 2-amino-4-methyl-1,3-thiazole-5-carbox-
4-methyl-1,3-thiazol-2-yl}acetamide (8j)
A mixture of 10j (0.23 g, 1.14 mmol), 7 (0.23 g, 1.14 mmol),
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDCꢀHCl) (0.26 g, 1.37 mmol) in dimethylformamide (3 mL) was
stirred at room temperature for 15 h. The reaction mixture was
then allowed to heat at 100 °C for 3 h and was evaporated in vacuo.
The residue was purified using silica gel column chromatography
(methanol/chloroform) to yield 13 (69.0 mg, 17%) as a yellow oil.
To the solution of 13 in ethanol–tetrahydrofuran (1:1, 2 mL) was
added 2 M sodium hydroxide solution (1.00 mL) and the reaction
mixture was then stirred for 15 h. The reaction mixture was neu-
tralized by the addition of 2 M aqueous hydrochloric acid at 0 °C
and was extracted with ethyl acetate twice. The combined organic
layer was washed with brine, dried over anhydrous magnesium
sulfate, and concentrated in vacuo. The residue was purified using
silica followed by NH silica gel column chromatography (metha-
nol/chloroform) to yield 8j (36.0 mg, 10%) as a colorless powder:
ylate 5 (3.00 g, 16.1 mmol) in ethanol (100 mL) was added 1 M so-
dium hydroxide solution (50.0 mL) at room temperature, and the
mixture was stirred for 26 h. After the volatiles of the mixture were
removed in vacuo, water and acetic acid were added to the residue
to adjust the pH to 5.0. The resulting precipitate was collected by
filtration, washed with water, and dried in vacuo to yield 6
1
(
2.48 g, 97%) as a colorless powder. H NMR (200 MHz, DMSO-d
6
)
+
ꢂ
d 2.35 (s, 3H), 7.57 (s, 2H); MS (ESI): m/z 159 [M+H] , 157 [MꢂH] .
5
.1.2. 2-(Acetylamino)-4-methyl-1,3-thiazole-5-carboxylic acid
(
7)
To a suspension of 6 (500 mg, 3.20 mmol) in tetrahydrofuran
20 mL) were added pyridine (1.30 mL, 16.0 mmol) and acetyl
(
chloride (0.60 ml, 7.90 mmol) at 0 °C, and the reaction mixture
was stirred for 21 h at room temperature. After the volatiles of

7
582
Y. Oka et al. / Bioorg. Med. Chem. 21 (2013) 7578–7583
1
+
+
mp 257.5ꢂ259.5 °C; H NMR (600 MHz, DMSO-d
6
) d 1.31ꢂ1.41 (m,
12.58 (br s, 1H); MS (ESI): m/z 283 [M+H] , 305 [M+Na] , 281
ꢂ
1
2
H), 1.43ꢂ1.53 (m, 1H), 1.71ꢂ1.79 (m, 1H), 1.83ꢂ1.90 (m, 1H),
[MꢂH] ; HRMS (ESI/APCI Dual) m/z Calcd for
11 14 4 3
C H N O S
+
.19 (s, 3H), 2.61 (s, 3H), 2.83 (dd, J = 12.4, 8.7 Hz, 1H), 2.94ꢂ3.03
[M+Na] , 305.0679; Found 305.0674.
(
m, 1H), 3.51ꢂ3.65 (m, 2H), 3.78 (dd, J = 12.4, 4.1 Hz, 1H), 4.93
+
(
3
d, J = 4.1 Hz, 1H), 12.61 (br s, 1H); MS (ESI): m/z 324 [M+H] ,
5.1.14. N-{5-[3-(4-Hydroxy-2-methylbutan-2-yl)-1,2,4-oxadiazol-
5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (8f)
+
ꢂ
46 [M+Na] , 322 [MꢂH] ; Anal. Calcd for C13
8.28; H, 5.30; N, 21.66. Found: C, 48.24; H, 5.33; N, 21.70.
Compounds 3 and 8a–m were prepared from the corresponding
starting materials in a manner similar to that described above.
17 5 3
H N O S: C,
4
Colorless powder (yield 10%); mp 177.5ꢂ179.5 °C; 1H NMR
3
(600 MHz, CDCl ) d 1.44 (s, 6H), 2.03 (t, J = 6.7 Hz, 2H), 2.30 (s,
3H), 2.69 (s, 3H), 3.75 (t, J = 6.7 Hz, 2H), 9.20 (br s, 1H); MS (ESI):
+
+
ꢂ
m/z 311 [M+H] , 333 [M+Na] , 309 [MꢂH] ; Anal. Calcd for C13
S: C, 50.31; H, 5.85; N, 18.05. Found: C, 50.29; H, 5.86; N,
18.04.
H
18-
5
.1.7. N-[5-(3-tert-Butyl-1,2,4-oxadiazol-5-yl)-4-methyl-1,3-
4 3
N O
thiazol-2-yl]acetamide (3)
Colorless powder (yield 16%); mp 237.5ꢂ239.5 °C; 1H NMR
(
(
200 MHz, CDCl
3
) d 1.41 (s, 9H), 2.30 (s, 3H), 2.72 (s, 3H), 9.11
5.1.15. N-{5-[3-(3-Hydroxy-2,2-dimethylpropyl)-1,2,4-oxadiazol-
5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (8g)
+
+
ꢂ
br s, 1H); MS (ESI): m/z 281 [M+H] , 303 [M+Na] , 279 [MꢂH] ;
Anal. Calcd for C12
H
16
N
4
O
2
S: C, 51.41; H, 5.75; N, 19.98. Found:
Colorless powder (yield 23%); mp 200.0ꢂ203.0 °C; 1H NMR
C, 51.24; H, 5.71; N, 19.97.
3
(200 MHz, CDCl ) d 1.03 (s, 6H), 2.32 (s, 3H), 2.71 (s, 3H), 2.77
(
s, 2H), 2.90 (t, J = 6.8 Hz, 1H), 3.40 (d, J = 6.8 Hz, 2H); MS (ESI):
+
+
ꢂ
5
.1.8. 5-(3-tert-Butyl-1,2,4-oxadiazol-5-yl)-4-methyl-1,3-
m/z 311 [M+H] , 333 [M+Na] , 309 [MꢂH] ; Anal. Calcd for C13-
thiazol-2-amine (4)
18 4 3
H N O
S: C, 50.31; H, 5.85; N, 18.05. Found: C, 50.19; H, 5.83;
Compound 4 was prepared from compound 6, instead of 7, in a
N, 18.08.
manner similar to that described for 8j. Colorless powder (yield
1
4
9
[
5%); mp 194.5ꢂ195.5 °C; H NMR (600 MHz, CDCl
3
) d 1.39 (s,
5.1.16. N-{5-[3-(3-Hydroxy-3-methylbutyl)-1,2,4-oxadiazol-5-yl]-
4-methyl-1,3-thiazol-2-yl}acetamide (8h)
+
H), 2.62 (s, 3H), 5.44 (br s, 2H); MS (ESI): m/z 239 [M+H] , 237
ꢂ
Colorless powder (yield 9%); mp 211.0ꢂ215.0 °C; 1H NMR
MꢂH] ; Anal. Calcd for C10
14 4
H N OS: C, 50.40; H, 5.92; N, 23.51.
Found: C, 50.19; H, 5.82; N, 23.30.
(200 MHz, DMSO-d
6
) d 1.15 (s, 6H), 1.73ꢂ1.82 (m, 2H), 2.20 (s,
3
H), 2.64 (s, 3H), 2.72ꢂ2.81 (m, 2H), 4.37 (s, 1H), 12.66 (br s,
+ +
ꢂ
5
.1.9. N-{4-Methyl-5-[3-(2,2,2-trifluoroethyl)-1,2,4-oxadiazol-5-
1H); MS (ESI): m/z 311 [M+H] , 333 [M+Na] , 309 [MꢂH] ; Anal.
Calcd for C13 O: C, 48.34; H, 6.05; N, 17.35. Found:
Sꢀ0.7H
C, 48.19; H, 5.81; N, 17.41.
yl]-1,3-thiazol-2-yl}acetamide (8a)
H
18
N
4
O
3
2
Colorless powder (yield 34%); 1H NMR (600 MHz, DMSO-d
6
) d
2
1
.21 (s, 3H), 2.66 (s, 3H), 4.11 (q, J = 10.9 Hz, 2H), 12.73 (br s,
+
ꢂ
H); MS (ESI): m/z 307 [M+H] , 305 [MꢂH] ; Anal. Calcd for C10
H
9-
5.1.17. N-{5-[3-(3-Hydroxy-2,2-dimethylbutyl)-1,2,4-oxadiazol-
5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (8i)
F
3
N
4
O
2
S: C, 39.22; H, 2.96; N, 18.29. Found: C, 39.47; H, 3.08; N,
1
8.01.
Colorless powder (yield 10%); mp 183.0–184.0 °C; 1H NMR
(
3
600 MHz, CDCl ) d 0.98 (s, 3H), 1.03 (s, 3H), 1.19 (d, J = 6.4 Hz,
5
1
.1.10. N-{5-[3-(2-Hydroxyethyl)-1,2,4-oxadiazol-5-yl]-4-methyl-
,3-thiazol-2-yl}acetamide (8b)
3H), 2.31 (s, 3H), 2.67 (d, J = 13.7 Hz, 1H), 2.71 (s, 3H), 2.81 (br s,
1H), 2.92 (d, J = 13.7 Hz, 1H), 3.57ꢂ3.60 (m, 1H), 9.39 (br s, 1H);
Colorless powder (yield 20%); mp 229.0ꢂ233.0 °C; 1H NMR
MS (ESI): m/z 325 [M+H] , 347 [M+Na] , 323 [MꢂH] ; Anal. Calcd
+
+
ꢂ
(
200 MHz, DMSO-d
6
)
d
2.20 (s, 3H), 2.65 (s, 3H), 2.86 (t,
for C14
50.39; H, 6.33; N, 16.87.
H
20
N
4
O
3
Sꢀ0.5H
2
O: C, 50.43; H, 6.35; N, 16.80. Found: C,
J = 6.2 Hz, 2H), 3.79 (br t, J = 6.2 Hz, 2H), 4.82 (br s, 1H), 12.70 (br
+
+
ꢂ
s, 1H); MS (ESI): m/z 269 [M+H] , 291 [M+Na] , 267 [MꢂH] ; HRMS
+
(
ESI/APCI Dual) m/z Calcd for C10
H
12
4
N O
3
S [M+Na] , 291.0522;
5.1.18. N-{5-[3-(3-Methoxy-2,2-dimethylpropyl)-1,2,4-oxadiazol-
Found 291.0522.
5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (8k)
1
Colorless powder (yield 6%); H NMR (600 MHz, CDCl
3
) d 1.02
5
.1.11. N-{5-[3-(1-Hydroxy-2-methylpropan-2-yl)-1,2,4-
(s, 6H), 2.30 (s, 3H), 2.72 (s, 3H), 2.76 (s, 2H), 3.19 (s, 2H), 3.38
+
+
oxadiazol-5-yl]-4-methyl-1,3-thiazol-2-yl}acetamide (8c)
(s, 3H), 8.96 (br s, 1H); MS (ESI): m/z 325 [M+H] , 347 [M+Na] ,
Colorless powder (yield 9%); mp 225.0ꢂ227.0 °C; 1H NMR
323 [MꢂH] ; Anal. Calcd for C14
ꢂ
20 4 3
H N O S: C, 51.83; H, 6.21; N,
(
(
(
600 MHz, DMSO-d
d, J = 5.5 Hz, 2H), 4.82 (t, J = 5.5 Hz, 1H), 12.63 (br s, 1H); MS
ESI): m/z 297 [M+H] , 319 [M+Na] , 295 [MꢂH] ; HRMS (ESI/APCI
6
) d 1.28 (s, 6H), 2.20 (s, 3H), 2.64 (s, 3H), 3.54
17.27. Found: C, 51.81; H, 6.27; N, 17.30.
+
+
ꢂ
5.1.19. N-{4-Methyl-5-[3-(tetrahydrofuran-3-yl)-1,2,4-oxadiazol-
5-yl]-1,3-thiazol-2-yl}acetamide (8l)
+
Dual) m/z Calcd for
12 16 4 3
C H N O S [M+H] , 297.1016; Found
2
97.1011.
Colorless powder (yield 3%); mp 245.0ꢂ247.0 °C; 1H NMR
(
600 MHz, CDCl
3
) d 2.31 (s, 3H), 2.33ꢂ2.39 (m, 2H), 2.72 (s, 3H),
5
.1.12. N-{5-[3-(2-Hydroxy-2-methylpropyl)-1,2,4-oxadiazol-5-
3.64 (dt, J = 14.7, 7.3 Hz, 1H), 3.94ꢂ4.08 (m, 3H), 4.16ꢂ4.21 (m,
+ +
yl]-4-methyl-1,3-thiazol-2-yl}acetamide (8d)
1H), 9.18 (br s, 1H); MS (ESI): m/z 295 [M+H] , 317 [M+Na] , 293
Colorless powder (yield 9%); mp 213.0ꢂ216.0 °C; 1H NMR
[MꢂH] ; Anal. Calcd for C12
ꢂ
14 4 3
H N O S: C, 48.97; H, 4.79; N, 19.04.
(
200 MHz, CDCl
3
) d 1.35 (s, 6H), 2.32 (s, 3H), 2.72 (s, 3H), 2.99 (s,
Found: C, 49.03; H, 4.73; N, 18.90.
+
2
3
C
H), 3.42 (br s, 1H), 9.68 (br s, 1H); MS (ESI): m/z 297 [M+H] ,
19 [M+Na] , 295 [MꢂH] ; HRMS (ESI/APCI Dual) m/z Calcd for
+
ꢂ
5.1.20. N-{4-Methyl-5-[3-(morpholin-4-yl)-1,2,4-oxadiazol-5-
yl]-1,3-thiazol-2-yl}acetamide (8m)
+
12
H
16
N
4
O
3
S [M+H] , 297.1016; Found 297.1009.
Colorless powder (yield 7%); mp 272.0ꢂ274.0 °C; 1H NMR
5
.1.13. N-{5-[3-(3-Hydroxypropyl)-1,2,4-oxadiazol-5-yl]-4-
(200 MHz, DMSO-d
6
) d 2.19 (s, 3H), 2.62 (s, 3H), 3.30ꢂ3.42
methyl-1,3-thiazol-2-yl}acetamide (8e)
(m, 4H), 3.64ꢂ3.76 (m, 4H), 12.62 (br s, 1H); MS (ESI): m/z
Colorless powder (yield 2%); mp 212.0ꢂ214.0 °C; 1H NMR
310 [M+H] , 332 [M+Na] , 308 [MꢂH] ; Anal. Calcd for C12
S: C, 46.59; H, 4.89; N, 22.64. Found: C, 46.47; H, 4.88; N,
22.35.
+ +
ꢂ
H
15-
(
200 MHz, DMSO-d
6
) d 1.80–1.90 (m, 2H), 2.19 (s, 3H), 2.64 (s,
5 3
N O
3
H), 2.78 (t, J = 6.6 Hz, 2H), 3.44ꢂ3.51 (m, 2H), 4.59 (br s, 1H),

Y. Oka et al. / Bioorg. Med. Chem. 21 (2013) 7578–7583
7583
5
5
.2. Biology
manner using a visual scoring system. Each paw was scored on a
graded scale from 0 to 4: 0, normal paw; 1, mild but definite red-
ness and swelling of the ankle or wrist or apparent redness and
swelling limited to individual digits, regardless of the number of
affected digits; 2, moderate redness and swelling of ankle or wrist;
3, severe redness and swelling of the entire paw including digits; 4,
maximally inflamed limb with involvement of multiple joints. The
four scores were added such that the maximal score per mouse
was 16. Mice were orally dosed with 8j at 30 or 100 mg/kg bid
for 14 days. Disease progression was significantly inhibited by 8j
.2.1. PI3K
Human PI3K
with kinase buffer (20 mM Tris–HCl [pH 7.4], 5 mM MgCl
c
enzyme assay
(20 ng, Millipore) was incubated for 2 h at 30 °C
, 5 mM
P]ATP, final concentrations) and li-
M of PtdIns (Calbiochem) and 25 M of
c
2
33
DTT and 10
pid vesicles containing 5
PtdSer (Sigma) (final concentrations) in the presence of inhibitors
or DMSO. The kinase reaction was stopped by the addition of
lM ATP/10 lCi c[
l
l
2
5 mM of EDTA, and the samples were incubated using a Flash-
Ò
⁄
⁄⁄
Plate (a phospholipid 96-well scintillant-coated microplate; Perk-
inElmer) with Substrate Coating Buffer (PerkinElmer). After the
incubation step, the wells were washed with PBS. The bound radio-
activity was determined by measuring the counts per second (CPS)
compared with the vehicle control ( p < 0.05, p < 0.01, Steel’s test)
at 30 and 100 mg/kg.
5.2.6. Mutagenic activity in the Ames test
using
a
Packard Top Count Microplate Scintillation Counter
To evaluate the mutagenic potential of the compounds, the
Ames test including a microplate version of the Ames fluctuation
test using Salmonella typhimurium strains TA98 and TA100 up to
(
PerkinElmer).
5
.2.2. Akt cellular assay
a maximal concentration of 2000
lg/mL (except for 3, which was
To examine C5a-mediated PKB/Akt phosphorylation in macro-
up to a maximal concentration of 1000
lg/mL because of the solu-
phages, Raw264.7, a murine macrophage-like cell line, was seeded
bility), and the Ames test using S. typhimurium strains TA98, TA100,
and Escherichia coli strain WP2uvrA up to a maximal concentration
of 5000 lg/plate in the presence or absence of rat S9 mix were
6
on 6-well plates at a density of 8 ꢁ 10 cells per well and grown for
2
4 h. After replacing the medium with fresh 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.
performed.
References and notes
1
2
.
.
Wymann, M. P.; Pirola, L. Biochim. Biophys. Acta 1998, 1436, 127.
(a) Cantley, L. C. Science 2002, 296, 1655; (b) Wymann, M. P.; Marone, R. Curr.
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Stephens, L. R. Biochem. Soc. Trans. 2006, 34, 647; (d) Engelman, J. A.; Luo, J.;
Cantley, L. C. Nat. Rev. Genet. 2006, 7, 606; (e) Vanhaesebroeck, B.; Guillermet-
Guibert, J.; Graupera, M.; Bilanges, B. Nat. Rev. Mol. Cell Biol. 2010, 11, 329.
Katso, R.; Okkenhaug, K.; Ahmandi, K.; White, S.; Timms, J. Annu. Rev. Cell Dev.
Biol. 2000, 17, 615.
5
.2.3. Metabolic stability in liver microsomes
The test compound (final concentration; 5.0
in the assay buffer containing 1 mg/mL human or rat microsomes,
.5 mM glucose-6-phosphate, 0.16 mM oxidized b-nicotinamide-
lM) was incubated
3
4
.
.
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,
1
adenine dinucleotide phosphate, 0.18 U/mL glucose-6-phosphate
dehydrogenase, 100 mM phosphate buffer and 2.5 mM magnesium
chloride at 37 °C. Concentrations of the test compounds were
determined using LC–MS/MS. Metabolic stability was calculated
as the ratio of the test compound concentrations at 15 min to
535.
5. 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.
6.
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.
0
min of incubation.
7. Li, Z.; Jiang, H.; Xie, W.; Zhang, Z.; Smrcka, A. V.; Wu, D. Science 2000, 287, 1046.
8.
Laffargue, M.; Calvez, R.; Finan, P.; Trifilieff, A.; Barbier, M.; Altruda, F.; Hirsch,
E.; Wymann, M. P. Immunity 2002, 16, 441.
5
.2.4. Metabolic stability in hepatocytes
The test compound (final concentration; 1.0 lM) was incubated
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.
in the assay buffer containing human hepatocytes (Celsis In Vitro
Technologies) or rat hepatocytes (XenoTech, LLC) at a final cell
density of 0.5 million cells/mL at 37 °C. The reactions were termi-
nated by the addition of acetonitrile/methanol (9:1, v/v). Precipi-
tated proteins were removed by centrifugation at 1650g for
1
0. (a) 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. 2005, 11, 936; (b) Pomel, V.; Klicic, J.; Covini, D. D.; Church, D.; Shaw,
J. P.; Roulin, K.; Burgat-Charvillon, F.; Valognes, D.; Camps, M.; Chabert, C.;
Gillieron, C.; Françon, B.; Perrin, D.; Leroy, D.; Gretener, D.; Nichols, A.; Vitte, P.
A.; Carboni, S.; Rommel, C.; Schwarz, M. K.; Rückle, T. J. Med. Chem. 2006, 49,
1
0 min at 4 °C. Concentrations of the test compounds were deter-
3857; (c) Doggrell, S. A. Expert Opin. Ther. Targets 2006, 10, 627; (d) Welker, M.
mined using LC–MS/MS. Metabolic stability was calculated as the
ratio of the test compound concentrations at 1–0 h of incubation.
E.; Kulik, G. Bioorg. Med. Chem. 2013, 21, 4063; (e) Sunose, M.; Bell, K.; Ellard,
K.; Bergamini, G.; Neubauer, G.; Werner, T.; Ramsden, N. Bioorg. Med. Chem.
Lett. 2012, 22, 4613.
5
.2.5. Arthritis score lowering effect studies in collagen-induced
11. Oka, Y.; Yabuuchi, T.; Fujii, Y.; Ohtake, H.; Wakahara, S.; Matsumoto, K.; Endo,
M.; Tamura, Y.; Sekiguchi, Y. Bioorg. Med. Chem. Lett. 2012, 22, 7534.
arthritis (CIA) mouse model
1
1
2. Robertson, G. M. General and Synthetic, Methods; Vol. 16, 195.
3. Robertson, G. M. The yields for cyclization step were not affected with or
without the hydroxyl group protection, in case of 8bꢂj. The low yields of
oxadiazole ring formation might be due to the steric hindrance of methyl group
at the 4-position of the aminothiazole ring, General and Synthetic Methods,
Royal Society of Chemistry, 1994, 16 250.
Arthritis was induced by immunizing male DBA/1 mice with an
initial intradermal injection of Bovine Type II collagen in Freund’s
complete adjuvant. On day 21 after the first immunization, the
mice received an intradermal boost injection (base of the tail) of
1
00
plete adjuvant. LPS (25
ip) 3 days after the intradermal boost injection. The severity of
l
g/100
l
L of Type II collagen emulsified in Freund’s incom-
14. Membrane permeability and solubility of those compounds 8g, 8i, and 8j were
comparable.
lg) was administered intraperitoneally
1
5. (a) Trentham, D. E. Arthritis Rheum. 1982, 25, 911; (b) Fujii, Y.; Hirayama, T.;
Ohtake, H.; Ono, N.; Inoue, T.; Sakurai, T.; Takayama, T.; Matsumoto, K.;
Tsukahara, N.; Hidano, S.; Harima, N.; Nakazawa, K.; Igarashi, Y.; Goitsuka, R. J.
Immunol. 2012, 188, 206.
(
the arthritis symptoms was assessed until 2 weeks after the boos-
ter injection. The severity of arthritis was assessed in a blinded