Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives as potent, orally active and S1P3-sparing S1P1 agonists

Article abstract of DOI:10.1016/j.bmcl.2012.03.067

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P₃-sparing S1P₁ agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC₅₀ value of 3.4 nM for human S1P₁, and over 5800-fold selectivity against S1P₃. In rat on host versus graft reaction (HvGR), the ID₅₀ value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.

Full text of DOI:10.1016/j.bmcl.2012.03.067

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3083–3088
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives
as potent, orally active and S1P3-sparing S1P1 agonists
Masayoshi Asano a, Tsuyoshi Nakamura a, Yukiko Sekiguchi a, Yumiko Mizuno a, Takahiro Yamaguchi a,
Kazuhiko Tamaki a, Takaichi Shimozato b, Hiromi Doi-Komuro b, Takashi Kagari b, Wataru Tomisato b,
Ryotaku Inoue b, Hiroshi Yuita c, Keiko Oguchi-Oshima c, Reina Kaneko d, Futoshi Nara e, Yumi Kawase e,
Noriko Masubuchi f, Shintaro Nakayama f, Tetsufumi Koga g, Eiko Namba g, Hatsumi Nasu g,
Takahide Nishi a,
⇑
a Lead Discovery & Optimization Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
b Frontier Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Oncology Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
d Global Project Management Department, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
e Cardiovascular Metabolics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
f Drug Metabolism & Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
g Biological Research Laboratories, Daiichi Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P3-sparing S1P1 agonist.
Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo
models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decom-
position. As provisions for unpredictable issues, a series of alternative compounds were synthesized on
the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC50 value
of 3.4 nM for human S1P1, and over 5800-fold selectivity against S1P3. In rat on host versus graft reaction
(HvGR), the ID50 value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is
also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 28 January 2012
Revised 10 March 2012
Accepted 16 March 2012
Available online 23 March 2012
Keywords:
S1P1
Agonist
Lymphocyte
HvGR
Sphingosine-1-phosphate (S1P) (1, Fig. 1) is a bioactive sphingo-
lipid that plays a role in a wide range of physiological processes
such as cell differentiation, morphogenesis and motility, through
its interaction with the five-membered S1P family (S1P1–S1P5) of
G-protein coupled receptors (GPCRs).1 Among these five receptors,
in particular, S1P1 modulators have recently been focused upon as
a suppressant of autoimmunity by affecting lymphocyte traffick-
ing, through a rapid progress of studies on FTY720 (fingolimod)
(2).2
The systemic administration of FTY720 induces a dose–respon-
sive lowering of circulating lymphocytes and its immunosuppres-
sive actions have been reported to result from the active
phosphate ester metabolite, FTY720-P,3 which is an agonist of
S1P1,3,4,5 but not of S1P2. Studies on both FTY720 and S1P receptors
has also revealed that the agonism of S1P1 alone is sufficient to
control lymphocyte recirculation.4 On the other hand, S1P3 is
implicated in bradycardia as reported in rodents.5 Recent studies
suggested that the removal of the S1P3 agonism is insufficient to
exclude the cardiovascular side effect.6a However, a great deal of
research efforts has been focused on the exploration of S1P3-spar-
ing S1P1 agonists,6 with the aim of reducing potential side effects,
NH2
NH2
OH
OH
CH3(CH2)12
O
O OH
OH
P
OH
CH3(CH2)7
2 (fingolimod, FTY720)
1 (S1P)
O N
N
O
S
CO2H
N
3
⇑
Corresponding author. Tel.: +81 3 3492 3131; fax: +81 3 5436 8563.
E-mail address: nishi.takahide.xw@daiichisankyo.co.jp (T. Nishi).
Figure 1. Structures of S1P, fingolimod (FTY720) and compound 3.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.067

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M. Asano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3083–3088
while preserving the impressive efficacy as demonstrated in the
clinical trials of FTY720 for the treatment of transplant rejection7
and remitting relapsing multiple sclerosis.8
The synthetic routes are shown in Scheme 1. In order to acquire
diverse SAR information, 4-ethylthiophene 5 was set as a common
intermediate, which was prepared from commercially available 4-
bromo-2-thiophencarbaldehyde 4 in three steps; reduction, TBS-
protection, and Ni-catalyzed cross coupling reaction with EtMgBr.
Compound 5 was able to be functionalized variously at the C-5 po-
sition and provided multiple thiophene units (6, 10, 14 and 25) for
heteroaromatic cyclization. For the preparation of 1,2-oxazole 9,
we obtained the precursor 8 by performing 1,3-dipolar cycloaddi-
tion with 1-ethynyl-4-phenoxybenzene 7 and nitrile N-oxide gen-
erated from oxime 6. Subsequent deprotection of the TBS group,
chlorination of the resultant primary hydroxyl group, substitution
by methyl azetizine-3-carboxylate, and hydrolysis gave 1,2-oxa-
zole derivative 9. Likewise, the isomeric 13 was synthesized from
corresponding ethynyl thiophene 10 and 4-phenoxybenzonitrile
N-oxide 11 generated from available 4-phenoxybenzaldehyde.
With regard to the preparation of the other heteroaromatics,
namely 1,3,4-oxadiazole 18, 1,3,4-thiadiazole 19, 1,3-oxazole 23,
29 and 1,3-thiazole 24a–k, 30a, b, thiophenyl carboxylic acid 14
was utilized as a second intermediate. In 18 and 19, condensation
of 14 with 4-phenoxyhydrazide 15, followed by treatment of Bur-
gess reagent or Lawesson’s reagent provided 1,3,4-oxadiazole 16 or
1,3,4-thiadiazole 17, respectively. After the juncture process with
the azetizine unit, both 18 and 19 were obtained. 1,3-Oxazole 23
and 1,3-thiazoles 24a–k were synthesized by the condensation of
14 with a variety of separately-prepared 2-aminoacetophenones
20a–k,14 following treatment of Burgess reagent or Lawesson’s re-
agent. For the synthesis of 1,3-oxazole 29 or 1,3-thiazoles 30a, b,
In a previous paper, we disclosed 1,2,4-oxadiazole derivative 3
as a potent S1P1 agonist.9 This compound exhibits high agonistic
activity to human S1P1 (EC50 = 4.0 nM) with good selectivity
against S1P3 (>5000-fold), measured by an [35S]GTP
c-S binding as-
say. In Lewis rats administered a single oral dose of 0.1 or 1 mg/kg
of 3, peripheral blood lymphocyte counts decreased to 27% and
11% of the vehicle-treated control values for the 0.1 and 1 mg/kg
dose levels, respectively, 8 h after compound 3 administration.
The decreased peripheral blood lymphocyte counts were returned
to the vehicle control levels by 24–48 h post-dose. In regards to the
immunosuppressive effect on transplant rejection, compound 3
showed fair efficacy in rat on host versus graft reaction10 (HvGR),
(ID50 = 0.41 mg/kg, orally administered once daily for 4 successive
days from the day of immunization).
However, further studies on 3 revealed that the central 1,2,4-oxa-
diazole ring has a tendency to be unfavorably cleaved by enterobac-
teria in rat and monkey.9b,11 This observation would arouse concern
about broad differences in pharmacokinetics and pharmacodynam-
ics between species or individuals.12,13 Therefore, we started to ex-
plore new structural classes on the basis of compound 3. Herein,
we report the discovery of several potent S1P1 agonists based on
1,3-thiazolyl thiophene and 1,3-thiazolyl pyridine scaffolds.
With the aim of avoiding cleavage of the central ring, we have
initiated the replacement of the central 1,2,4-oxadiazole ring to
other heteroaromatic rings.
N
O
N
O
d, e
f
g - k
N
OTBS
HO
PhO
PhO
S
PhO
PhO
S
S
S
6
CO2H
CO2H
N
N
OTBS
OTBS
8
9
N O
N O
Br
a - c
l, m, n
o
g - k
OTBS
OTBS
S
CHO
S
S
S
12
13
4
5
10
N N
X
N N
X
p
q, r
g - k
OTBS
or
q, s
HO2C
PhO
S
PhO
S
14
S
CO2H
N
OTBS
OTBS
16 X = O
18 X = O
17
19
X = S
X = S
t, r
or
t, s
R2
R1
N
R2
R1
N
g - k
X
u - z
X
S
S
CO2H
N
21 X = O
23 X = O
22a-k X = S
24a-k X = S
R2
R1
N
a1, a2, r R2
g - k
N
OH
HCl
H2N
X
X
S
R1
S
S
or
CO2H
O
N
OTBS
a1, a2, s
27
29
25
X = O
X = O
28a, b X = S
30a, b X = S
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH. (b) TBSCl, imidazole (quant., 2 steps). (c) NiCl2(dppp), EtMgBr (89%). (d) n-BuLi, DMF (79%). (e) NH2OH–HCl, Et3N,
CH2Cl2, MeOH (96%). (f) NCS, pyridine, CHCl3, then 1-ethynyl-4-phenoxybenzene (7), Et3N, CHCl3 (73%). (g) TBAF, THF. (h) SOCl2, toluene. (i) Methyl azetidine-3-carboxylate
HCl, i-Pr2NEt, MeCN (66–97%, 3 steps). (j) aq. NaOH, EtOH (60–92%). (k) Oxalic acid (9 and 13 were solidified as oxalates). (l) n-BuLi, THF, then I2 (72%). (m)
Trimethylsilylacetylene, CuI, Pd(PPh3)4, Et3N (75%). (n) K2CO3, MeOH (75%). (o) 4-Phenoxybenzonitrile N-oxide (11), Et3N, CHCl3 (59%). (p) n-BuLi, THF then CO2 (73%). (q) 4-
Phenoxybenzhydrazide (15), DCC (46%). (r) Burgess reagent, Et3N, MeCN, 80 °C (61–93%). (s) Lawesson’s reagent, pyridine, toluene, 100 °C (70–97%). (t) EDCI, HOBt, various
aminoacetophenones (20a–k), Et3N, CHCl3 (57–81%). (u) MeNHOMe, EDCI, HOBt, Et3N, CH2Cl2 (52%). (v) MeLi, Et2O (89%). (w) NaHMDS, TMSCl, Et3N, THF. (x) NBS, THF. (y)
NaN(CHO)2, MeCN (3 steps, 59%). (z) HCl, EtOH (99%). (a1) EDCI, HOBt, benzoic acids (26a, b), Et3N, CH2Cl2. (a2) TBSCl, imidazole, DMF (75–92%, 2 steps).

M. Asano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3083–3088
3085
SMe
N
SMe
N
SO2Me
N
O
OMe
OMe
a
b
c, d, e
f
NC
NC
NC
OMe
OMe
Me2N
OMe
OTIPS
OTIPS
OTIPS
O
OMe
31
32
33
34
35
i
j, k
l - p
g, h
CO2H
R2
R1
R2
R1
N
S
N
OHC
HO2C
N
N
N
N
N
S
N
N
OTIPS
OTIPS
36
37
38a, b
39a, b
q, r
H2N
O
s - v
l - p
CO2H
R2
R1
R2
R1
w, x, k
N
N
HCl
N
O
N
N
OTBS
S
S
OTIPS
OH
40
41
42a-c
43a-c
Scheme 2. Reagents and conditions: (a) N,N-Dimethylformamide dimethylacetal, MeCN, reflux. (b) 2-Cyanothioacetamide, MeONa then MeI (2 steps, 61%). (c) cat. DDQ, H2O–
MeCN (80%). (d) NaBH4. (e) TIPSCl, imidazole (2 steps, 100%). (f) m-CPBA, EtOH (91%). (g) EtMgBr, Et2O (98%). (h) DIBAL, toluene (98%). (i) NaClO2, KH2PO4, 2-methyl-2-butene
(100%). (j) EDCI, HOBt, aminoacetophenones (20c, d), Et3N, CHCl3 (60–79%). (k) Lawesson’s reagent, pyridine, toluene, 100 °C (79–95%). (l) TBAF, THF. (m) SOCl2, toluene. (n)
Methyl azetidine-3-carboxylate HCl, i-Pr2NEt, MeCN (66–82%). (o) aq NaOH, EtOH. (p) Oxalic acid (54–83%, 2 steps). (q) MeLi. (r) PDC (2 steps, 85%). (s) NaHMDS, TMSCl, Et3N,
THF. (t) NBS, THF. (u) NaN(CHO)2, MeCN (3 steps, 42%). (v) HCl, EtOH (96%) (w) EDCI, HOBt, various benzoic acids (26b, c, d), Et3N, CHCl3. (x) TBSCl, imidazole, DMF (71–80%, 2
steps).
Table 1
SAR of central rings
Ar
O
S
CO2H
-GTPa EC50 (nM)
N
Compound
Ar
c
Rat HvGRb ID50 (mg/kg)
or % inhibition @ 1 mg/kg
hS1P1
4.0
hS1P3
O N
N
3
>20,000
0.41
O N
9
22
15
>20,000
>20,000
2% ( 11)
N O
13
No inhibition
N N
O
N N
S
N
O
N
O
N
S
18
54
25
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
NT
19
No inhibition
NT
23
90
29
300
4.5
35
NT
24a
30a
42% ( 5)
25% ( 9)
N
S
a
EC50 is the mean of two experimental determinations.
Each value of inhibition percentage is the mean of five rats, standard error is given in parentheses (NT = not tested). ID50 is determined by
b
results of at least three different doses.
benzoic acids 26a, b15 were condensed with aminoketone 25 pre-
pared from 14, and the same subsequent processes were
conducted.
In exploratory work, we established that the 6-ethylpyridine
structure can be substituted for 4-ethylthiophene (data not shown).
Therefore, further synthetic efforts were expanded to the 6-ethyl-
pyridine derivatives. The synthetic routes are summarized in
Scheme 2. Starting from 31, the construction of the pyridine struc-
ture via reaction of 32 with 2-cyanothioacetoamide, followed by
some conversions of each functional group, provided pyridylalde-
hyde 36 as an intermediate for 1,3-thiazoles 39a,b and 43a–c. After
administration by sequences similar to those conducted in Scheme
1, 1,3-thiazole derivatives 39a, b and 43a–c15 were obtained.
Initially, we measured the agonist activities against human S1P1
and S1P3 and then evaluated the in vivo immunosuppressive effi-
cacy in rats on HvGR.

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M. Asano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3083–3088
Table 2
SAR of thiophene derivatives
R2
R1
X Y
S
S
CO2H
N
Compound
X
Y
R1
R2
c
-GTPa EC50 (nM)
hS1P3
Rat HvGRb ID50 (mg/kg)
or % inhibition @ 1 mg/kg
hS1P1
3
4.0
4.5
10
>20,000
>20,000
>20,000
>20,000
>20,000
400
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
0.41
24a
24b
24c
24d
24e
24f
24g
24h
24i
24j
24k
30b
C
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
N
N
N
C
PhO
H
H
42% ( 5)
52% ( 2)
0.07
0.16
73% ( 4)
0.53
0.48
54% ( 7)
1.07
0.32
27% ( 7)
0.19
i-PrO
i-PrO
i-PrO
i-PrO
i-PrO
i-PrO
i-PrO
i-Pr
Me
Et
n-Pr
i-Pr
F
3.4
3.4
1.1
1.3
5.8
3.2
3.5
6.0
8.0
Cl
Me
Me
Me
Me
(S)-sec-BuO
(R)-sec-BuO
i-PrO
11
a,b
See Table 1.
The SAR information about the central heteroaromatic ring is
respectively) than 3. Interestingly, installation of the n-propyl
group (24e) largely worsened S1P1/S1P3 selectivity, suggesting a
limitation on the bulkiness of the R2 substituent. We also investi-
gated the effect of halogen atoms exemplified by 24g and 24h,
and the fluoro derivative (24g) showed favorable efficacy on HvGR
(ID50 = 0.48 mg/kg). Subsequently, based on the structure of the
most efficacious compound, 24c, we tried to conduct further min-
ute adjustments on the R1 substituent as illustrated by the isopro-
pyl (24i) and the (S) or (R)-sec-butoxy groups (24j, k). This
scrutinization revealed that the (S)-sec-butoxy group (24j) was also
maintained fair efficacy on HvGR (24j; ID50 = 0.32 mg/kg).
In accordance with the SAR information obtained regarding 1,3-
thiazole derivatives 24a–k, we applied effective substituents to the
isomeric structure as illustrated by 30b. As expected, 30b showed
high efficacy on HvGR, which corresponds to the isomer of 24c.
The results of optimization for the pyridine derivatives are sum-
marized in Table 3. Based on the results of the thiophene deriva-
tives, we installed a combination of favorable substituents to the
benzene ring, as illustrated in 39a and 43a. These compounds
showed an insufficient in vivo immunosuppressive activity but
maintained a good in vitro profile. Slight modification in both the
R1 and R2 substituents revived the in vivo activity to afford the po-
tent pyridine derivatives 39b, 43b, and 43c.
summarized in Table 1. The superior S1P1 agonist activity was ob-
served for 1,3-thiazole 24a (EC50 = 4.5 nM) with sufficient S1P1/
S1P3 selectivity (>4400-fold), which is almost equal to compound
3. In contrast, the isomeric 30a displayed lower but tolerable ago-
nist activity (EC50 = 35 nM) relative to 24a. In a previous report,
Merck’s group also reported a similar tendency between the iso-
mers of 1,3-thiazoles in their scaffold.16 1,2-Oxazoles 9, 13 and
1,3,4-thiadiazole 19 also showed moderate agonist activities.
In rat on HvGR, 1,3-thiazole 24a showed reduced inhibitory
activity (42% inhibition at 1 mg/kg), whose insufficient efficacy
could presumably result from pharmacokinetic issues. Unexpect-
edly, the isomer 30a showed fair efficacy on this model, despite
comparatively lower S1P1 agonist activity than 1,2-oxazoles 9, 13
and 1,3,4-thiadiazole 19.
Hence, based on identified alternative 1,3-thiazole 24 and the
isomeric 30, we conducted further investigation to explore optimal
substituents of the benzene ring adjacent to the 1,3-thiazole.
The SAR dataset is shown in Table 2. In the R1 substituent, the
isopropoxy group was identified as a good alternative to the phen-
oxy group, which led to the improvement of the in vivo efficacy
(comparing 24a with 24b). On the other hand, the R2 substituent
was proved to considerably affect both the in vitro and in vivo as-
pects. Namely, the introduction of alkyl groups, such as methyl
(24c), ethyl (24d), n-propyl (24e), or isopropyl group (24f) into
the R2 position of compound 24b exhibited drastic improvement
for the in vivo efficacy. In particular, 24c and 24d showed more po-
tent inhibitory activity (ID50 = 0.069 mg/kg and 0.16 mg/kg,
The pharmacokinetics of 24c and 39b were evaluated in Lewis
rats and Cynomolgus monkeys. The PK parameters of these ani-
mals after a single oral administration are shown in Table 4. The
thiophene derivative 24c showed remarkable bioavailability
(F = 70.6% in rats, >90% in monkeys) and fair half-life (T1/
Table 3
SAR of pyridine derivatives
R2
R1
CO2H
X Y
S
N
N
Compound
X
Y
R1
R2
c
-GTPa EC50 (nM)
hS1P3
Rat HvGRb ID50 (mg/kg)
or % inhibition @ 1 mg/kg
hS1P1
39a
39b
43a
43b
43c
C
C
N
N
N
N
N
C
C
C
i-PrO
i-PrO
i-PrO
i-Bu
Me
Et
Me
Me
Et
5.5
4.0
2.8
4.1
5.4
>20,000
>20,000
>20,000
>20,000
>20,000
39% ( 9)
0.49
17% ( 9)
0.64
i-Bu
0.74
a,b
See Table 1.

M. Asano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3083–3088
3087
Table 4
Pharmacokinetic parameters and bioavailabilities of 24c and 39b to rat and monkey
Compound
Animals
Dosea (mg/kg)
Cmax
(lg/mL)
Tmax (h)
T1/2 (h)
AUC0–inf
(l
g h/mL)
F (%)
CLb (mL/min/kg)
Vdb (L/kg)
24c
24c
39b
Rat
Monkey
Monkey
3.0
1.0
0.5
0.34
0.63
0.15
5
4
5
11.5
7.2
16.7
5.14
8.69
3.73
70.6
>90
75.4
6.72
2.00
1.75
5.37
0.89
1.46
a
po administration, rat (n = 4), monkey (n = 2).
iv administration, rat (1.0 mg/kg, n = 2), monkey (0.5 mg/kg, n = 2).
b
O
N
N
S
N
O
O
S
S
CO2H
CO2H
N
N
3
24c
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0
24
48
0
24
48
Time (h)
Time (h)
ꢀ
ꢁ
ꢀ
µ
µ
µ
ꢂ
rat (3 M)
monkey (3 M)
broth (3 M)
µ
µ
µ
ꢃ
rat (30 M)
monkey (30 M)
broth (30 M)
ꢁ
Figure 2. Compound stability against enterobacteria from rat cecal contents or monkey feces under anaerobic condition.
Rosenbach, M.; Shei, G.-J.; Singer, I. I.; Tian, M.; West, S.; White, V.; Xie, J.;
Proia, R. L.; Mandala, S. J. Pharm. Exp. Ther. 2004, 309, 758; (b) Sanna, M. G.; Liao,
J.; Jo, E.; Alfonso, C.; Ahn, M.-Y.; Peterson, M. S.; Webb, B.; Lefebvre, S.; Chun, J.;
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2 = 11.5 h in rats, 7.2 h in monkeys). Meanwhile, the pyridine deriv-
ative 39b exhibited favorable bioavailability (F = 75.4%) and over
twice as long a half-life as 24c in monkeys (T1/2 = 16.7 h).
Finally, we evaluated compound stabilities against enterobacte-
rial decomposition in rats and monkeys.17,18 In order to compare
24c with 3, each compound solution (3.0 and 30 lM) was exposed
6. (a) Hamada, M.; Nakamura, M.; Kiuchi, M.; Marukawa, K.; Tomatsu, A.;
Shimano, K.; Sato, N.; Sugahara, K.; Asayama, M.; Takagi, K.; Adachi, K. J. Med.
Chem. 2010, 53, 3154; (b) Martin, H. B.; Cyrille, L.; Oliver, N. Curr. Top. Med.
Chem. 2011, 11, 726; (c) Pennington, L. D.; Sham, K. K. C.; Pickrell, A. J.;
Harrington, P. E.; Frohn, M. J.; Lanman, B. A.; Reed, A. B.; Croghan, M. D.; Lee, M.
R.; Xu, H.; McElvain, M.; Xu, Y.; Zhang, X.; Fiorino, M.; Horner, M.; Morrison, H.
G.; Arnett, H. A.; Fotsch, C.; Wong, M.; Cee, V. J. ACS Med. Chem. Lett. 2011, 2,
752; (d) Cee, V. J.; Frohn, M.; Lanman, B. A.; Golden, J.; Muller, K.; Neira, S.;
Pickrell, A.; Arnett, H.; Buys, J.; Gore, A.; Fiorino, M.; Horner, M.; Itano, A.; Lee,
M. R.; McElvain, M.; Middleton, S.; Schrag, M.; Rivenzon-Segal, D.; Vargas, H.
M.; Xu, H.; Xu, Y.; Zhang, X.; Siu, J.; Wong, M.; Bürli, W. ACS Med. Chem. Lett.
2011, 2, 107; (e) Bolli, M. H.; Abele, S.; Binkert, C.; Bravo, R.; Buchmann, S.; Bur,
D.; Gatfield, J.; Hess, P.; Kohl, C.; Mangold, C.; Mathys, B.; Menyhart, K.; Müller,
C.; Nayler, O.; Scherz, M.; Schmidt, G.; Sippel, V.; Steiner, B.; Strasser, D.;
Treiber, A.; Weller, T. J. Med. Chem. 2010, 53, 4198; (f) Demont, E. H.; Arpino, S.;
Bit, R. A.; Campbell, C. A.; Deeks, N.; Desai, S.; Dowell, S. J.; Gaskin, P.; Gray, J. R.
J.; Harrison, L. A.; Haynes, A.; Heightman, T. D.; Holmes, D. S.; Humphreys, P. G.;
Kumar, U.; Morse, M. A.; Osborne, G. J.; Panchal, T.; Philpott, K. L.; Taylor, S.;
Watson, R.; Willis, R.; Witherington, J. J. Med. Chem. 2011, 54, 6724.
to broth (control) and the culture fluid of enterobacteria, which
was derived from rat cecal contents or monkey feces and incubated
under anaerobic conditions. The stabilities were estimated by
means of tracing the percentage of each remaining compound
(24 and 48 h after). The results are shown in Figure 2. In contrast
to 3, 1,3-thiazole derivative 24c was found to be stable and re-
mained even 48 h later.
In short, we designed and synthesized a series of 1,3-thiazole
compounds as S1P3-sparing S1P1 agonists. Compound 24c exhib-
ited high S1P1 agonistic activity, S1P1/S1P3 selectivity, inhibitory
activity in rat on HvGR, and good PK profile. This compound also
demonstrated clear endurance to enterobacterial decomposition.
As well, the pyridine derivative 39b showed favorable efficacy. Fur-
ther studies on the optimization of these analogues are being con-
ducted at this time.
7. Salvadori, M.; Budde, K.; Charpentier, B.; Klempnauer, J.; Nashan, B.; Pallardo, L.
M.; Eris, J.; Schena, F. P.; Eisenberger, U.; Rostaing, L.; Hmissi, A.; Aradhye, S.
Am. J. Transplant. 2006, 6, 2912.
References and notes
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Nara, F.; Kawase, Y.; Shimozato, T.; Doi, H.; Kagari, T.; Tomisato, W.; Inoue, R.;
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reaction (HvGR) were evaluated by using a popliteal lymph node gain assay in
rats. Spleen cells of WKAH rats were injected into the footpads of LEW rats to
induce an enlargement of the draining popliteal lymph node. Compounds were
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3088
M. Asano et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3083–3088
orally administrated once a day for four days from the day of spleen cell
injection (day 0), and the weight of the popliteal lymph node was measured at
day 4.
15. Preparations for 26a–d are outlined below.
11. Detailed pharmacokinetic information will be reported elsewhere.
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13. 4-Phenoxy benzoic acid (4-PBA) was identified as one of the major metabolites
in rat and monkey plasma after oral administration of 3 (data not shown). 4-
CO2H
PhO
26a
Me
F
CN
Me
CN
Me
CO2H
NaOH
i-PrOH
NaH
i-PrO
i-PrO
26b
Br
Br
i-PrPPh3I
t-BuOK
1) n-BuLi, DMF
2) NaClO2, KH2PO4
2-Me-2-butene
PBA showed weak PPAR
a agonism and a considerably long half-life in those
OHC
animal species (data not shown).
14. Preparations for 20a–k are outlined below.
CO2H
CO2H
H2, Pd-C
X
F
CN
X
CN
NaH
26c
Br
i-PrOH
i-PrO
)
Pd PPh3)4
X = H, F, Cl
CO2H
CO2Me
CO2H
Br2, HNO3
AgNO3
MeLi
O
O
O
Trivinylboroxin
pyridine complex
1) PhMe2NBr3
i-PrI
B
Me
B
NH2
Cs2CO3
2) NaN(CHO)2
3) aq. HCl
i-PrO
HO
i-PrO
Et
CO Me
1) H2, Pd-C
2) NaOH
2
20b-f, g, h
AcCl
AlCl3
B = H, Me, Et, n-Pr, i-Pr, F, Cl
i-PrI, NaH
(R = Et)
R
26d
O
R
AcCl
AlCl3
i-PrI
R
HO
Cs2CO3
(or NaH)
HO
i-PrO
R = n-Pr
i-Pr
(deisopropylated)
R = Et, n-Pr, i-Pr
16. Vachal, P.; Toth, L. M.; Hale, J. J.; Yan, L.; Mills, S. G.; Chrebet, G. L.; Koehane, C.
A.; Hajdu, R.; Milligan, J. A.; Rosenbach, M. J.; Mandala, S. Bioorg. Med. Chem.
Lett. 2006, 16, 3684.
17. Nelson, G. M.; Swank, A. E.; Brooks, L. R.; Bailey, K. C.; George, S. E. Toxicol. Sci.
2001, 60, 232.
18. The stabilities were evaluated by the method of the Ref. 17, with some
modifications. PYF broth was used instead of PYG. The cecal contents and feces
were placed into the prereduced PYF broth instead of prereduced VPI buffer in
the reference.
Br
O
Br
1) MeMgBr
2) EtPPh3Br
Br
O
NMeOMe
PhO
NaHMDS
1) HMTA
2) aq. HCl
1) n-BuLi, AcNMe2
2) H2, (Ph3P)3RhCl
O
O
1) PhMe2NBr3
CN
1) NaH
B
A
NH2
(S) or (R)-
sec-BuOH
2) MeLi
2) NaN(CHO)2
3) aq. HCl
F
A
A = PhO, B = H
20a
20i
A = i-Pr, B = Me
20j
A = (S)-sec-BuO, B = Me
A = (R)-sec-BuO, B = Me
20k